Discovery of a novel antibiotic, Transitmycin, from Streptomyces sp unveils highly efficient activities against tuberculosis and human immunodeficiency virus

Discovery of a novel antibiotic, Transitmycin, from Streptomyces sp unveils highly efficient activities against tuberculosis and human immunodeficiency virus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Discovery of a novel antibiotic, Transitmycin, from Streptomyces sp unveils highly efficient activities against tuberculosis and human immunodeficiency virus Vanaja Kumar, Balagurunathan Ramasamy, Mukesh Doble, Radhakrishnan Manikkam, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5801746/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract HIV is identified as a factor that aggravates tuberculosis disease pathogenesis and its progression to latent TB. While, TB is declared as one of the major causes for AIDS-associated mortality. So there is a dire need for new drugs to combat such ailments that have a synergistic interaction.This has led us to study a novel antibiotic purified from a marine Streptomyces sp isolated from the coral reef ecosystem of South Indian coast. Streptomyces sp. R2 (MTCC 5597; DSM 26035)., isolated from the marine water was grown on agar plates and the crude yellowish orange pigment secreted was extracted using various solvents. The antibiotic, named as Transitmycin, was purified and tested against M. tuberculosis, drug resistant strains, and M. tuberculosis biofilm. The compound was also tested against HIV-1 viruses belonging to six subtypes. Several characterisation tools were used to elucidate the structure of this novel antibiotic. Transitmycin was derivitaised to elucidate the absolute configurations of the amino acids present in it. Tr, unlike actinomycin D, has L-valine in both the rings instead of D-valine (found in the latter). Also, one of the proline in Tr is in D–configuration while it is in L configuration in actinomycin D suggesting that ours is a novel compound and is not reported so far. It exhibits dual activities against the standard H37Rv, 49 drug sensitive clinical isolates, and MtB biofilm as well as standard and 20 clinical isolates of HIV. This is the first paper that reports the isolation of a new antibiotic from marine actinobacteria exhibiting unusual anti-TB and HIV activities which could be exploited further as a lead molecule in the quest for the design of drug with dual activities. Antibiotic Streptomyces sp. Actinomycin Transitmycin Tuberculosis HIV Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Highlights • A novel antibiotic was purified from a marine Streptomyces sp isolated from the coral reef of S. India • Presence of L-valine, not observed in actinomycin D, and one of the proline in D configuration suggest that it is a novel structure not reported before • It exhibits activity against standard MtB strain as well as clinical isolates and drug resistance ones • It exhibits anti-HIV activity against several clinical isolates Introduction Despite, the history of infectious diseases is time immemorial, an urgent need for protection against infectious diseases has one of the major health concerns. Antibiotics have revolutionized the diagnostic factor in various aspects and the drug discovery is considered as a hallmark in human history. However, the concomitant development of resistance against diseases is the most serious consequence [ 1 ]. In 2020, an overall rate of 1.5 million people pretentious from TB. In fact, TB is one of the leading sources of death, wherein occupied the 13th position and moreover the second foremost infective destroyer after COVID-19 (above HIV/AIDS). Since it is an airborne infectious disease caused by M tuberculosis , continues to be a major cause of mortality and morbidity worldwide [ 2 ]. An upsurge of multi drug resistance (MDR) and extensively drug resistance (XDR) is an additional perturbing factor in tuberculosis chemotherapy. Comprehensively, the existing treatment for TB based on half yearly quadruple therapy containing rifampicin, isoniazid, ethambutol and pyrazinamide results the cure rate of 90–95% [ 3 ] for drug-sensitive TB at global level. Nevertheless, this regimen is inadequate to treat MDR and XDR-TB infections, sometimes the second line antibiotics are employed to treat MDR-TB infections for prolonged time whereas XDR infections are almost untreatable. Different TB drugs with unique mode of action are desperately necessary to fight against drug resistant tuberculosis. For example, bedaquiline and delamanid are approved for drug resistant TB, however due to the reported toxicity issues, they may employ only on last resort [ 4 ]. In addition, the second line drugs used to treat MDR-TB with several disadvantages such as less efficacious, more toxic, and more expensive than the first-line drugs [ 5 ]. At presently progressing anti TB drugs should partake essential properties like succeed a short therapy period (sterilizing effect), a simpler regimen, capable of act on MDR/XDR-TB, concurrent treatment of TB/HIV and advanced safety level than the existing drugs [ 6 ]. Alike the human immunodeficiency virus (HIV) leads to destruction of the immune system resulting a condition termed as Acquired Immuno Deficiency Syndrome (AIDS). After its finding AIDS becomes a great risk to humans, reflected to be pandemic and it is the greatest public health crisis in globally. As of 2021, millions of people living with HIV were rescuing antiretroviral therapy [ 7 ]. Several researches have been developed the drugs against HIV disease, such as the growth of many antiretroviral drugs (ARVs), including the atazanavir, ibalizumab, darunavir, abacavir, zidovudine, lamivudine, efavirenz, tenofovir disoproxil fumarate, tenofovir alafenamide, emtricitabine, bictegravir, cabotegravir, rilpivirine etc. nevertheless, the competency of HIV intensive to mutate rapidly leads to the upsurge of drug resistance to existing anti-retrovirals [ 8 ]. Since the finding of penicillin, many of the microbial natural products tends to the new paradigm for novel drug discovery development [ 9 , 10 ]. Over the past 60 years, [ 11 ] the microbial bioprospecting has gained a remarkable quantity of medicinal components, including actinobacteria, the major bacterial phylum group of Gram-positive bacteria with guanine and cytosine (G + C) rich content in their genomic sequence, which are the important prokaryotes in economic and ecological manner. Significantly the existence of actinobacterial multiplicity in several rare ecosystems liable for prolific producers of many biologically active natural compounds with different potential activities [ 12 – 15 ]. Some of the pharmaceutical companies have dramatically declined in the result of novel wide range antibiotics in the past few decades [ 16 ]. Obviously, this critical state has caused in the surplus of such antibiotics with distinct mode of activity targeting the arcade, wherein this precluded the assessment of usual terrestrial sources in particular for actinobacteria and led scientists to pursuit unique habitats on the marine family for novel bioactive molecules [ 12 , 17 , 18 ]. Indeed, actinobacteria are ubiquitous in the marine environment, which are occupying a substantial ecological characteristic in the recycling and production of novel natural products with huge pharmaceutical applications [ 19 ]. Generally, the 16S rRNA gene sequencing has concomitantly employed to identify, classify and quantitation of microbes in multifaceted biological molecules. It is obvious from the 16S rRNA sequencing that marine microbial species as bacteria and archae have an extreme different taxonomy. Even the 16S rRNA sequences of more than ten thousand actinomycetes had been isolated hitherto from marine outsources. The discovery of novel secondary metabolites from marine actinobacteria has just surpassed that of their terrestrial counterparts [ 15 ]. According to the review of Blunt et al., [ 20 ] shown that there are 179 new natural products isolated during 2016, in which the actinobacterial genus Streptomyces remains to be the dominant source. An extensive study on typical marine actinobacterial genera like Salinispora and Verrucosispora produces salinosporamide and abyssomycin respectively, which suggest that actinobacteria improves significant feature towards marine drug discovery research [ 21 ]. Further, marine based antibiotics are more ingenious in combat infections due to the terrestrial bacteria do not have chance to develop resistance against them [ 22 ]. During the microbial bioprospecting process, the actinobacterial extracts isolated from various less-explored ecosystems were screened for anti-TB and anti-HIV activity. The isolation, characterization and potential bioactivities against TB and HIV of an antibiotic obtained from a marine Streptomyces sp. R2 is being reported herewith. Results and Discussion Description of the actinobacterial strain Transitmycin is a depsipeptide (bicyclic) molecule produced by actinobacterial strain R2, which was isolated from the sediment samples from Rameswaram coral reef ecosystem (Lat. 9.2876° N; Long. 79.3129° E), Tamil Nadu, South India using Starch Casein Agar (SCA) supplemented with nalidixic acid (20µg/ml) and nystatin (100µg/ml). Viability of strain was maintained in International Streptomyces Project-2 (ISP2) agar slants, 30% glycerol broth followed by lyophilisation. It was also deposited in Microbial Type Culture Collection (MTCC), India and in DSMZ – German Collection of Microorganisms and Cell Cultures, Germany. (R2 = MTCC5597; DSM26035). Characterization and taxonomy The characterization of a strain is the key element in classification of prokaryotes including actinobacteria [23]. Actinobacterial classification was initially based mainly on morphological and physiological characteristics [24-25]. The onset of chemotaxonomic standards has provided reproducible and reliable data to identify the genera at genus level [26]. This microbial strain R2 under microscopic observation showed the presence of dense aerial and substrate mycelia with long, un-fragmented spore chains with hairy structures (Supplementary Fig. 1a-b). The physiological and biochemical characteristics of the strain R2 are given in Supplementary S-Table 1. Strain R2 showed good growth in various ISP media and utilized a wide range of carbon and nitrogen sources. In addition, good growth was also observed at different physiological conditions. Notably, the extracellular yellow pigment production was greatly influenced by nitrogen substrates, pH, temperature and NaCl. Strain R2 produced lipase and amylase. High sensitivity was observed for most of the antibiotics tested (Supplementary S-Table 1). The cell wall analysis revealed that the strain R2 is rich in LL-2,6-Diaminopimelic acid (L-DAP) and glycine. No diagnostic sugars were found in the cell wall constituents. In our present study, the results of phenotypic characterization and cell wall analysis indicated that the actinobacterial strain R2 belongs to the genus Streptomyces , however it is not adequate for differentiation at species level. Furthermore, the PCR (Polymerase Chain Reaction) amplification of 16S rRNA gene of strain R2 formed around 1400 base pair sequence and the BLAST (Basic Local Alignment Search Tool) analysis have shown 99% similarity to the 16S rRNA gene sequence of Streptomyces variabilis (EU570414) and other closely related species submitted in GenBank. Phylogenetic relationship of the strain R2 and related taxa are given in Supplementary S-Fig. 1c. The 16S rRNA gene sequence of strain R2 has the accession number HQ012501at GenBank, but this gene provides limited resolution for species level identification, since it discloses more extensive genotypic differences. At this stage, the average nucleotide identity (ANI) [27] of all preserved genes between any two genomes shows likely to reform taxonomy, because it also correlates with the best DNA: DNA Hybridization (DDH) values. The most often used standards for species delineation i.e . the 70% DDH [28] which is closely equivalent to 95% ANI (Average Nucleotide Identity) values. Moreover, no organisms have been defined hitherto has shown <98.7% identity in their 16s rRNA gene and shown <95% ANI or 70% DDH. These results supported the substitution of cumbersome DDH and related procedures with simple sequence-based standards. Significantly the metabolic property of transitmycin production had not been reported before from any other meticulously associated Streptomyces species. Based on the aforesaid facts, the actinobacterial strain R2 is identified to be a novel strain of Streptomyces variabilis , but still it showed 99% similarity with its associated proximity neighbors. It is very important to mention here that there is no literature evidence on any commercially available antibiotic, in particular anti-TB and/or anti-HIV compounds from Streptomyces variabilis. Transitmycin production The growth of Streptomyces sp. R2 is simultaneously observed with grey colour aerial mycelia and soluble yellow orange pigment in good yield. The crude compound is extracted well in several solvents namely, methanol, chloroform and dichloromethane, followed by diethyl ether and ethyl acetate (Supplementary Table 2). The extracts in the former solvents were intensely coloured when compared to the extracts from the latter solvents. Salts and debris were present in the extracts while using former solvents, so ultimately ethyl acetate was chosen for extracting the bioactive pigment. The activity of the crude extract was also tested on different strains of M. tuberculosis . More than 95% reduction in Relative Light Units (RLU) was observed through Luciferase Reporter Phage Assay against all three M. tuberculosis strains. When 1.0 L of YEME agar was used for production it yielded 800 mg crude antibiotic extract in ethyl acetate. Major antibiotics reported from actinobacteria are extracellular in nature [29-30]. Industrial production of many antibiotics from Streptomyces is achieved through submerged fermentation process [31]. In contrast, some actinobacterial strains are found to produce antibiotics only on solid media and very little reason is reporeted as to why activity is restricted to solid culture and not observed in submerged cultures. Shomura et al reported [32] that about 1300 out of 6500 actinomycetes showed antimicrobial activity against one or more of the test organisms when tested by agar plug method. In the secondary screening, about 25 (1.9%) of the 1300 strains were found to be non-producers in submerged cultures. So, we conlcude that the reports of antibiotics isolated from only agar cultures of actinomycetes are very rare. According to Mayurama et al . [33] fumaridamycin was detected with much difficulty in submerged cultures, because the mycelium of the producing strain inactivate the antibiotic more readily in liquid than in agar culture. Similarly, Shomura et al [32] demonstrated that the antibiotic produced by Streptomyces halstedii hasshown activity against Gram negative bacteria only in agar dishes, which correlates well with its mycelial morphology. The aerial mycelium was filamentous during antibiotic production in solid cultures, but fragmented in non-producing liquid cultures. Similar to report from Shomura et al . [32] the bioactive pigment production by the Streptomyces strain R2 was observed only in agar culture, where the vegetative mycelium was filamentous. While decreasing the concentration of YEME broth from 2X to 1/10X the mycelia filamentation was found to increase. However, none of the five concentrations of YEME broth produced the pigment (unpublished data). This outcome evidenced that the filamentous mycelial structure does not influence pigment production by the strain R2 in broth while the bioactive pigment production was observed in all the concentrations of YEME agar when filamentous mycelia was formed. In accordance to Ohnishi et al . [34] who reported that 2-aminophenoxazin-3-one containing grixazone A and B, afford yellow pigments during phosphate depletion by Streptomyces griseus . By adopting these strategies, it was concluded that optimizing the medium components may trigger the pigmented bioactive compound production by our isolated Streptomyces sp. R2 in liquid cultures. Transitmycin purification, characterization and structure elucidation The Streptomyces sp. R2 was found to produce 800 mg of crude pigment per litre of yeast extract malt extract medium in agar surface fermentation. The yellow-orange pigment was separated from the crude ethyl acetate extract by TLC and column chromatography and its purity was confirmed by HPLC analysis (SupplementaryFig S13-S17). Primarily the three well separated spots viz. R1, R2 and R3 with R f values of 0.8, 0.6, 0.3, respectively were observed on analytical TLC using EtOAc:MeOH (9.5:0.5) solvent system. In bioassay directed isolation, fraction R1 (named as Transitmycin) showed more than 95% inhibition against M. tuberculosis strain H37Rv in LRP assay. It was isolated as an orange colour amorphous powder with [α] D 25 : -106° ( c = 0.2, MeOH). The R1, R2, R3 in crude extract and as purified compounds R1, R2, R3 showed similar retention time in RP-HPLC chromatograph ( Fig, 1a-d ) .A single peak of transitmycin at a RT of 5.8 minutes confirmed its purity ( Fig. 1c). The chemical structures of these molecules (R1, R2 and R3) were elucidated by UV-Visible, IR, CD, 1 H, 13 C, DEPT 135 NMR, 2D NMR ( 1 H- 1 H COSY, 1 H- 1 H DQF-COSY, 1 H- 1 H TOCSY, 1 H- 13 C HSQC, 1 H- 13 C HMBC, NOSEY, ROESY) and MALDI-TOF-MS, HR-ESIMS, HR-EIMS, HR-LCMS, and 3200 QTRAP-LC/MS/MS analyses and also compared with the previously reported NMR and Mass data of actinomycins (Supplementary Information) [35-57]. Advances in spectroscopic techniques have mainly utilized for compound identification and immensely accelerated the unambiguous representation of compound characterization and structural elucidations. The UV-Vis spectrum of Transitmycin showed a strong absorption band around (λ max 214, 240, 425, 442 nm (in MeOH) (Fig. 1e). The colour and absorption peak in UV-Vis analysis revealed the presence of phenoxazinone chromophore. Singh et al . [58] and Maskey et al . [59] detected the presence of phenoxazinone chromophore in bioactive metabolites from Streptomyces sp. and Actinomadura sp. The absolute configuration of the amino acids were supposed to be identical to that of actinomycin D, as indicated by the negative optical rotation values and the strong cotton effect at about 210 nm in the CD spectra. The CD values of Transitmycin (R1) are: [MeOH, [nm], (mdeg)] λ max (Dε) 195 (+24.0), 210 (-21.5), 241 (+1.7). In the IR spectrum, a strong absorption broad band appears at around 3435 cm -1 , an intense strong peak at 1746 cm -1 and a band around 1099 cm -1 are assignable to be amino (or hydroxyl), lactone ring and alicyclic 6-membered ether type (C-O) groups, respectively. IR data of transitmycin (R1) (KBr cm -1 ), 3435 cm -1 for NH, 2958, 2924 cm -1 (m, -CH str, asym, CH 3 and CH 2 ), 2872 cm -1 , 2853 cm -1 (m, -CH str, sym, CH 3 and CH 2 ),1746 cm -1 (s, C=O str, lactone ring), 1642 cm -1 (s, -C=O str, 2 o amide), 1524, 1503 (m, -NH bend, 2 o amide), 1466 (m, CH bend (scissoring), CH 2 ), 1379 cm -1 (s, -CH bend, isopropyl group), 1268 (s, C-O str, ester), 1194 ( C-O-C of lactone) 1099, 1059, 1017 (s, C-O or C-N), 720, 712, 694, 689 (s, -CH bend, oop, aromatic ring), 909 (w, CH 3 rocking) [48](Fig. 1f). The 1 H and 13 C NMR spectra exhibited the typical features of two (alpha and beta ring) pentapeptido lactone ring attached with phenoxazinone chromophore, i.e., each ring contained four amide carbonyl resonances and one ester carbonyl in one ring (δ C 168.8, 173.4, 173.1, 166.2, 167.4 (α-ring) and 168.9, 173.9, 172.7, 166.4, 167.4 (β-ring), together with phenoxazinone chromophore (101.8 (C-1), 147.3( C-2), 179.0 (C-3), 113.4 (C-4), 144.9 (C-4a), 140.3 (C-5a), 127.6 (C-6), 130.2 (C-7), 126.0 (C-8), 132.1(C-9), 128.4 (C-9a), 14.9 (C-11), 7.6 (C-12), 166.0 (C-13), 165.8 C-14). One of the amino acid proline in the β-ring contained a keto group (208.0 in the 13 C NMR). From 1 H NMR spectrum, NH of amino acids (δ H 8.23, β-L-Valine), (δ H 7.74, α-L-Valine), (δ H 7.69, β-L-Threonine, (δ H 7.2, α-L-Threonine), (δ H 6.55, 2H of β-proline) (δ H 5.93 2H of α-proline) and four N-methyl groups (δ H 2.91, 2.88, 2.89, 2.87 ) (Fig. 2 a-d). In addition, the 1 H NMR spectra of Transitmycin indicated the presence of eight methyl groups arising from four isopropyls. The UV/Vis absorption spectra with maximal absorbance at 240 nm and 442 nm support the presence of an amino phenoxazinone chromophore in its structure. From 1 H- 1 HCOSY and TOCSY experiments, five amino acid systems namely Pro, Thr, Val, N-Methyl Val, and Sar were identified. The assignments of the protonated carbons were obtained from the HSQC spectrum, in combination with inspection of the HMBC spectrum. By comparison of the UV spectrum (λ max 442 nm, in MeOH) of Transitmycin with that of actinomycin series (λ max 440 nm, in MeOH) (Fig. 1e) it was concluded that the former contained an aminophenoxazinone chromophore residue. In 1 H NMR, two ortho coupled protons at 7-H, 8-H; δ H 7.56 and 7.34 corresponding to 1,2,3,4-tetrasubstituted aromatic ring, and two 3H singlet at δ H 2.52 (11-CH 3 ) and 1.95 (12-CH 3 ) of methyl groups in peri-position of the aromatic system were identified. This is the characteristics of phenoxazinone chromophore found in various actinomycins. The result was further confirmed by HMBC correlations between the 7-H (δ H 7.59) and 8-H (δ H 7.34) of the tetra substituted double bond and the carbonyl resonances at δ C 166.0. The carbonyl carbons of Pro, Thr, Sar, Val, and N-methyl Val, were clearly assigned to (δ C 179.0, 174.0, 173.1, 169.02, 198.8, 167.5, 166.5, 166.56, 166.3, 166.1and 165.9) on the basis of the observed correlations between carbonyl groups protons of the same amino acid residue in the HMBC spectrum. All the residues were connected on the basis of DQF-COSY, TOCSY, HMBC, ROESY and NOESY correlations (Fig. 3a-c), thus establishing the amino acid sequences and overall constitution. (Supplementary Fig. S14-65, S-Table 5). The detailed analysis of 1 H- 1 H COSY, 1 H− 1 H DQF-COSY, 1 H− 13 C HMBC, and 1 H− 13 C HSQC, 1 H− 1 H TOCSY, 1 H− 1 H NOESY, 1 H− 1 H ROESY NMR spectra [35-51] (Supplementary Information Fig. S14-65, S-Table S5) and MALDI-TOF-MS (Fig. 4a-m and Fig. 5) and HR-ESIMS, HR-LCMS, ESI-MS, QTRAP LC-MS/MS [52-57] (Fig. 5, S-Table S6), (Supplementary Information Fig. S14-115 , S-Table S7a-b) spectral fragmentation pattern revealed ten amino acids in Transitmycin (Fig. 3d), which is identical to those present in actinomycin X2 [35-57] (2 X MeVal, 2 X Thr, 2 X Sar, 2 X Val, proline and ketoproline). OPro was identified by the ketone moiety (δ C 208.6 ppm) and the altered chemical shifts and coupling patterns of the neighbouring methylene groups (Table 1). Table 1 . 1 H NMR (500 MHz, CDCl 3 )/ 13 C NMR (125 MHz, CDCl 3 ) and 2D NMR (500 MHz, CDCl 3 ) correlation spectral data of Transitmycin (R1) 1 H− 1 H COSY, 1 H− 1 H TOCSY 1 H− 13 C HMBC, 1 H− 1 H ROESY α- ring Position δ C δ H J (Hz) COSY TOCSY HMBC ROSEY Thr 1 168.8 - - - - - - 2 54.6 4.60 d , 10.0 3, 4, -NH 3, 4 1, 3, 13 3, 4, 2-Val, Val-NH 3 74.7 5.13 m 2, 4,- NH 2, 4, 4 2, 4, Val-NH 4 17.0 1.10 d, 6.5 2, 3 2, 3, -NH 2, 3 2, 3, Val-NH NH - 7.29 d, 7.0 2, 3, 4 2, 3, 4 1, 3, 13 2, 4 L-Val 1 173.4 - - - - - - 2 58.7 3.58 dd, 10.5,6.5 3, 4, 5, -NH 3, 4, 5, -NH 1, 5, 1-Thr 3, 4, 5, 2-Pro 3 31.7 2.08 m 2, 4, 5 2, 4, 5 2,5 4, 5, 4-Thr 4 19.0 0.89 d, 7.0 2, 3 2, 3, -NH 2,3,5 2, 3, 5 5 18.9 1.13 d, 6.5 2, 3 2, 3, -NH 2,3,4 2, 3, 2-Val, Val-NH NH - 7.74 d, 5.5 2, 3, 4, 5 2, 3, 4, 5 2, 3, 1-Thr 2, 3, 4, 5, 2- Thr, 3-Thr Pro 1 173.1 - - - - - - 2 56.4 5.93 d, 9.5 3a,3b,4a,4b 3a,3b 4a, 4b 3, 4, 5, 1-Val 3a, 3b, 2a-Sar,2b-Sar, Sar-NMe 3 31.6 2.73 1.84 dd, 17.5,7.0 dd, 19.5, 6.5 3b, 4a, 4b 3a, 4a, 3b, 4a, 4b 3a, 4a, 1, 2, 4 1, 4, 5 3b, 2a-Sar, 2b-Sar 3a, 4a, 4b 4 23.3 2.26 2.01 d, 16.5 d, 8.0 3a, 3b, 4b 3b, 4a 3a, 3b, 4b 3b, 4a 3, 5 3, 5 4b, 5a, 5b 4a, 5a 5a 5b 47.8 3.86 3.67 d, 18.5 d, 18.5 3a, 4a, 4b 3a, 4a, 4b 3a, 4a, 4b 3a, 4a, 4b 4, 1-Val 5b Sar 1 166.2 - - - - - - 2 51.2 2a, 4.70 2b, 3.64 d, 17.5 d, 17.5 2b 2a 2b, NMe 2a, NMe 1, NMe,1-pro 1, NMe,1-Pro 2b, NMe, 2-Pro 4-MeVal, 5-MeVal, NMe N-Me 34.9 2.87 s 2a, 2b 2, 1-Pro 2b, 3-MeVal Me-Val 1 167.4 - - - - - - 2 71.3 2.70 d, 9.0 3, 4, 5 3, 4, 5 1, 3,4,NMe 4, 5 3 26.9 2.62 d, 7.0 2, 4, 5 2, 4, 5 1, 3, 4, 5, NMe 4, 5 4 21.6 0.96 d, 6.0 2, 3 2, 3, 5 2, 3, 5 3 5 19.2 0.72 d, 6.5 2, 3 2, 3, 4 2, 3, 4 3 N-Me 39.2 2.89 s - 2, 3 2, 1-Sar 2, 3, 5, 2a-Sar, 2b-Sar β-ring position δ C δ H J (Hz) COSY TOCSY HMBC NOESY Thr 1 168.9 - - - - - - 2 54.9 4.50 d, 9.0 3,4, -NH 3, 4 1, 3, 14 3, 4, 2-Val, Val-NH 3 74.6 5.23 m 2,4,- NH 2, 4 4 2,4, Val-NH 4 17.6 1.20 d, 6.0 2, 3 2, 3, - NH 2, 3 2,3, Val-NH NH - 7.68 d, 6.0 2, 3, 4 2, 3, 4 1, 3, 14 2, 4 L-Val 1 173.9 - - - - - - 2 57.1 3.71 dd, 10.5, 6.5 3, 4, 5, -NH 3, 4, 5, -NH 1, 3, 5, 1-Thr, 3, 4, 5, 2-Oxo pro 3 31.8 2.20 m 2, 4, 5 2, 4, 5 2, 5 4, 5, 4-Thr 4 19.0 0.86 d, 7.0 2, 3 2, 3, -NH 2, 3, 5 2, 3, 5 5 18.8 1.14 d, 7.0 2, 3 2, 3, -NH 2, 3, 4 2, 3, 2-Val, Val-NH NH 8.23 d, 6.0 2, 3, 4, 5 2, 3, 4, 5 2, 3, 1-Thr 2, 3, 4, 5, 2-Thr, 3-Thr Oxo-Pro 1 172.7 - 2 54.2 6.55 dd, 10.0, 2.0 3, 5 3, 5 3, 1-Val 3, 5, 2a-Sar, 2b-sar-NMe 3 41.8 2.32 2.13 d , 18.5 d, 13.0 2, 5 2, 5 2, 5 2, 5 1 1 2, 5 2, 5 4 208.0 - - - - - - 5 53.7 3.83 3.90 d, 18.5 dd, 18.5, 12.0 2, 3 2, 3 2, 3 2, 3 1, 2 1, 2 2, 3 2, 3 Sar 1 166.4 - - - - - - 2 51.28 2a, 4.54 2b, 3.95 d, 19.5 d, 19.0 2b 2a 2b, NMe 2a, NMe 1, NMe, 1-Pro 1, NMe, 1-Pro 2b, NMe, 2-oxo-pro 4-MeVal , 5-MeVal, NMe N-Me 34.7 2.88 s - 2a, 2b 2, 1-Pro 2b, 2MeVal Me-Val 1 167.4 - - - - - - 2 71.1 2.67 d, 8.5 3, 4, 5 3, 4, 5 1, 3, 4, 5, NMe 4, 5 3 26.8 2.60 d, 8.0 2, 4, 5 2, 4, 5 1, 3, 4, 5, NMe 4, 5 4 21.5 0.93 d, 6.0 2, 3 2, 3, 5 2, 3, 5 3 5 19.1 0.73 d, 6.0 2, 3 2, 3, 4 2, 3, 4 3 N-Me 39.0 2.91 s - 2, 3 2, 1- Sar 2, 5, 2a-Sar, 2b-Sar Chromo phore Position δ C δ H J (Hz) COSY TOCSY HMBC NOESY 1 101.8 - - - - - - 2 147.3 - - - - - - 3 179.0 - - - - - - 4 4a 113.4 144.9 - - - - - - 5 5a - 140.3 - - - - - - 6 127.6 - - - - - - 7 130.2 7.34 d, 8.0 8, 11 8, 11 5a, 9, 11-Me 11 8 126.0 7.56 d, 7.5 7, 11 7, 11 9a, 5a, 13 11 9 9a 132.1 128.4 - - - - - - - 10 - - - - - - - 11 14.9 2.52 s 7, 8 7, 8 5a, 6, 7 7, 8 12 7.6 2.18 s - - 3, 4, 4a - 13 166.0 - - - - - - 14 165.8 - - - - - - The MALDI-TOF-MS spectrum of crude extract of Streptomyces sp. R2 (Fig. 4j-k) and measured, calculated mass of purified compounds Transitmycin (R1), R2, R3 were given in (Fig. 4a-i , Supplementary Figure S70-115). The molecular formula was established as C 62 H 84 N 12 NaO 17 based on Positive HRESI-MS, which showed protonated pseudo molecular ion peak [M+H] + at m/z 1270.7069 (Fig. 4a-m). It also showed intense peaks, due to Na and K adducts, at m/z 1291.8307 [M+Na] + and 1307.8124. [M+K] + respectively (Calcd. for C 62 H 84 N 12 NaO 17 : 1291.5975: Found: 1291.8307 ). Fig. 4a-i Similarly, the MALDI TOF MS spectrum of transitmycin showed intense peak in positive mode at m/z 1293.61316 [M+Na+2H] +3 and at m/z 1309.93062 [M+K] + and in negative mode at m/z 1269.33344 [M-H]- (Fig. 4a-m, Fig. 4-5, Supplementary Information Fig. S115a-b). The compound R2 was isolated as orange red powder and its molecular formula was established as C 62 H 86 N 12 O 16 [M+Na] + by positive HRESIMS. The MALDI TOF molecular ion of R2 showed peak at m/z 1278.95175 [M+Na+H] + and negative ion mode at m/z 1255.38052 [M-H] - For a molecular formula of C 62 H 87 N 12 O 16 Na its molecular weight is calculated as 1278.6261 [M+Na+H] + which matches with 1278.95175, that is similar to that of actinomycin D (Fig. 3e). The compound R3 was obtained as an oranges red powder and the molecular formula of R3 was determined to be C 62 H 87 N 12 O 17 from HR-ESIMS peak at m/z 1271.7159z [M+H] + and when calculated for the molecular formula , C 62 H 87 N 12 O 17 1271.6312, found to be 1271.7159 and 1277.6149 [M-OH-Na] + . The molecular formula of R3 was established as C 62 H 87 N 12 O 17 by positive MALDI-TOF 1294.5888 [M+H+Na] + and negative mode 1268.4852 [M-H] - (Fig. 4f) which is identical to that of actinomycin 0β (Supplementary Information Fig. S70-115 , S-Table S7a-b) [52-57]. The differences between compounds R1, R2, R3 were becaue of the variation in proline at 4 th position. Transitmycin (R1) has keto group in the 4 th position, R2 does not have keto group and compound R3 has hydroxyl group in the 4 th position (Fig. 3a-c). H PLC Analysis of L-FDAA Derivatives of Transitmycin (R1) HPLC experiment was used to determine the absolute configuration of isolated compounds. The absolute configurations of the amino acids were assumed to be identical to that of actinomycin X 2 , as indicated by the negative optical rotation values and the strong cotton effect at about 210 nm in the CD spectra. The assignment of the amino acids was carried out primarily by the analysis of the 1 H- 13 C HSQC and 1 H- 1 H-COSYcorrelations, MALDI-TOF-MS (Fig. 4 a-m, S-Table S6) , QTRAP LC-MS/MS (Supplementary Information Fig. S14-114, S115a-b, S-Table S7a-b ), and completed with the help of HMBC spectrum (Fig. 3a-c). Additionally, a small amount of compound Transitmycin and R3 were hydrolyzed and the free amino acid were analyzed by HPLC, HR-LCMS, HR-ESIMS after chiral derivatization with Marfey’s reagent. Altered proline and sarcocine were not available as reference. Although neither the altered proline and threonine moieties nor N-methylated alanine were available as references, they can be assumed to possess L-configurations due to the fact that the exchange of a single amino acid with its enantiomer leads to significant conformational changes of the respective peptidolactone ring, resulting in reduced biological activities as well as chemical shift deviations that have not been observed. The absolute configuration of the threonine, valine, methyl valine, proline in Transitmycin (R1) was clarified by Marfey’s method applied for the acid hydrolysate of Transitmycin (R1) in comparison with standard amino acid analysis [60-64]. Retention times of the standard N α -(5-fluro-2,4-di-nitrophenyl)-L-alanamide (FDAA) derivatives were as follows: D-threonine, 15.682 min; L-threonine, 13.987 min; D-proline, 17.035 min; L-proline, 16.519 min; D-valine, 21.244 min; L-valine, 19.248 min; D-N-methyl valine, 22.089 min; L-Methyl valine 20.814 min. The chromatogram of the FDAA derivatives of acid hydrolysate Transitmycin (R1) showed peaks corresponding to L-Threonine (14.021 min), L-proline (16.520 min), L-valine (19.252), L-N-methyl valine (21.263 min) were obtained in the hydrolysate. The above same mentioned procedure for compound R3 was also done and similar results was obtained. (Supplementary Information Fig. S116-125, Table S-8a-b) [60-64]. Comparison, with authentic standards, revealed the presence of L-MeVal, L-Thr and D-Val as expected, however the D-Valine is in L-configuration as well as one of the proline is in D-proline instead of L-configuration. (Fig. 6a-c). LC-MS Analysis of L-FDAA Derivatives of Transitmycin (R1) The retention times of the D- and L-FDAA derivatives of standard amino acid , respectively, were as follows: Pro: 14.35, 14.97, min, m/z 366.13 [M +H] + , m/z 363.07 [M-H] + ;Val:19.63,17.52 min, m/z 370.13 [M+H] + , 368.13 [M-H] - ;Thr: 9.90, 13.50 min, m/z 372.07 [M+H] + , 370.13 [M − H] - ; NMeVal: 20.33, 19.23 min, m/z 384.20[M+H] + 382.20 [M –H] - . The L-FDAA was used to derivatize the acid hydrolysates of Transitmycin (R1), R3 and eight standard amino acids (D-Val, L-Val, D-Thr, L-Thr, D-N-MeVal, L-N-MeVal, D-Pro and L-Pro). The reaction with L-FDAA was performed with the same procedure as above. [63-64].The retention times of the L-FDAA derivatives were as follows: D-Pro:15.16 min, L-Pro:14.31 min, m/z 366.13 [M+H] + 363.07 [M−H] - ;D-Val: 19.65 min, L-Val 17.65 min m/z 370.13 [M+H] + 368.07[M−H] − ; D-Thr: 9.90 min, L-Thr, 13.50 min, m/z 372.13[M+H] + 370.13 [M−H] - ; D-N-MeVal 20.33 min, L-N-MeVal 19.25 min, m/z 384.20[M+H] + ,382.20 [M+H] - . The retention time of L-FLDA derivatives of acid hydrolysates Transitmycin (R1) were as follows: L-threonine (13.73 min), L-Proline (14.23 min), D-Proline or keto-proline (15.36 min), L-valine (18.02 min), N-Methyl valine (19.96 min) as illustrated in Fig. 6 a-b. The retention time of L-FLDA derivatives of acid hydrolysates (R3) (Supplementary Information Fig. S-128-161, S-Table S9 a and b) were as follows: to L-threonine (12.45 min) or D-threonine (9.36 min), L-Proline (14.25 min), D-Proline or keto-proline (15.21 min), L-valine (17.49 min), N-Methyl valine (19.93) . Comparison with authentic standards revealed the presence of L-MeVal, L-Thr, L-Proline, L-Valine and one of the Proline as in D-configuration (Fig. 6c). Hence, we named the unusual newly found compound as Transitmycin (Fig. 3d, Table 1& 2), a member of the X-type [35-64]. Table 2. Physico-chemical properties of Transitmycin Transitmycin (R1) Properties TLC Single yellow spot with R f value 0.8 (EtOAc:MeOH (9.5:0.5)) Color and consistency Orange color amorphous powder Yield 200 mg, 20% Melting point (mp) 240-242 o C [α] D 25 -106 o (c = 0.2, MeOH) Solubility Soluble in chloroform, dichloromethane, ethyl acetate, methanol, ethanol, acetonitrile, DMSO and water; Insoluble in Hexane UV (MeOH) λ max (log ε) 214 (3.07), 240 (2.30), 425 (1.44), 442 (1.51) nm CD [MeOH, [nm], (mdeg)]: λ max (Dε) 195 (+11.1), 210 (-21.0), 242 (+4.7) IR (KBr), ν max 3435, 2958, 2924, 2853, 1745, 1642, 1524, 1465, 1379, 1194, 1099, 1059 cm -1 1 H NMR (500MHz) Table 2 13 C NMR (125MHz, CDCl 3 ): 179.0, 174.0, 173.5, 173.17, 169.0, 168.8, 167.5, 167.5, 166.5, 166.3,166.1, 165.9, 144.34, 145.93, 145.04, 140.5, 132.19, 130.3, 129.2, 127.8, 126.1, 113.6, 101.8, 76.7, 74.76, 74.67, 71.4, 71.2, 58.5, 57.2, 56.4, 54.9, 54.7, 54.3, 51.3, 29.6, 29.6, 29.3, 22.6, 21.7, 21.6, 19.2, 19.2, 19.09, 19.06, 18.8, 17.14, 14.11,7.77. HRESI-MS m/z (pos.ions) 656.9243 [M+2H] +2 , 1270.7069 [M+ H] + , 1291.8449 [M + Na] + 1307.9286 [M + K] + C 62 H 84 N 12 O 17 Na [M + Na] + calc. 1291.5975, found. 1291.8449 MALDI-TOF-MS m/z (pos.ions) 1293.61316 [M + Na+2H] + , 1309.93062 [M+K] + m/z (neg.ions) 1269.33344 [M-H] - C 62 H 84 N 12 O 17 Na [M+Na+2H] + calc. 1293.61950, found. 1291.61316 EI-MS: (70 ev) m/z (pos.ions)1348.1437, 1291.4173 [M+Na] + , 1224.7363, 1191.8994, 1023.6241, 886.0243, 743.2058, 614.8185, 347.6111, 202.5464, 138.5079 LCESI-MS m/z (pos.ions) 1291.5995 [M + Na] + C 62 H 84 N 12 O 17 Na [M+Na] + calc. 1291.5975, found. 1291.5995 CHN Anal. calcd for C 62 H 84 N 12 O 17 : C, 58.66; H, 6.67; N, 13.24. Found: C, 59.71; H, 7.28; N, 10.19. Determination of anti TB and anti-HIV activity Activity against planktonic cultures of M. tuberculosis Despite the introduction of new anti TB drugs, emergence of antibiotic resistance among M. tuberculosis strains remains a major challenge in tuberculosis therapy. According to WHO Global Tuberculosis report - 2022, there is a 3.1% increase in incidence of multi drug resistant and rifampicin resistant TB cases from 2020 to 2021[65]. Thus, there is a burning need for more new antitubercular drugs to tackle drug resistant tuberculosis [66]. Since 1940s, secondary metabolites and their associated derivatives have played a key role in anti-TB drugs development. This is best exemplified by an extremely active aminoglycoside, namely streptomycin, the first clinical drug that was made available against TB [67]. In a previous study, Streptocytosines A, Bamitecin and Amitecin isolated from sea water Streptomyces in Japan showed activity against M. smegmatis at 32, 16 and 8 µg/ml concentrations, respectively [68]. In another study, actinomycin X2 and actinomycin D isolated from marine Streptomyces sp. MS449 in China showed activity against M. tuberculosis H37Rv at 1.92mg/ml and 1.77 mg/ml, respectively [69]. In the present study Transitmycin, a novel molecule isolated from a marine Streptomyces sp. R2 picked up from coral reef ecosystem showed activity notably against drug sensitive, multi drug resistant (MDR) and mono resistant strains of M. tuberculosis at concentrations of 5 and 10 µg/ml (Fig. 7). Based on the preliminary experiment on laboratory strain, M. tuberculosis H37Rv, minimum inhibitory concentration for transitmycin was determined on clinical isolates which included 49 drug sensitive strains and 48 drug resistant strains. The drug resistant profile of the clinical isolates are given in (supplementary S-table 12). Out of 97 clinical isolates, MIC at 5 µg/ml was seen in 89 isolates and MIC at 10 µg/ml was seen in 8 isolates for transitmycin based on LRP assay. Activity against M. tuberculosis biofilm Bacteria can persist for extended periods of time inside the biofilm due to their ability to resist the immune system, display increased virulence and become phenotypically more resistant to antibiotics. Moreover, antibiotic concentrations required to control bacteria within a biofilm are estimated to be 100–1,000 fold greater than that is needed to treat planktonic forms. As planktonically-grown M. tuberculosis are unlikely to be entirely representing the bacterial load during human infection, we set out to determine how effective transitmycin can be against M. tuberculosis growing as a biofilm, a bacterial phenotype known to be more resistant to antibiotic treatment [70]. In the present study, M. tuberculosis culture showed biofilm formation in the control wells alone which could be observed by the naked eye. The wells with cells and transitmycin failed to produce biofilm. CFU determined prior to the addition of transitmycin was 1.9 x 10 6 to 2.3 x 10 6 /ml. After 4 days of exposure with the compound, the CFU dropped to 9 x 10 4 to 10 x 10 4 ml. Addition of transitmycin completely killed all the cells at the end of 5 weeks (Table 3). Table – 3. Activity of transitmycin on biofilm formation Culture Zero days 10 5 x CFU/ml After 4 days 10 5 x CFU/ml Biofilm formation after 5 weeks M. tuberculosis alone 1.1 1.8 6.6 3.6 Present M. tuberculosis + Transitmycin (10 µg/ml) 19.0 23.0 0. 9 1.0 0.0 Anti-HIV activity The symbiotic association of TB and HIV poses a challenge to human survival in which HIV complicates the treatment and diagnosis of TB. Besides, HIV–TB patients encounter other unique problems such as cumulative toxicity, immune reconstitution inflammatory syndrome (IRIS), drug-drug interactions, lower plasma drug levels, and the emergence of drug resistance during treatment [71]. The currently available therapy for treating patients co-infected with HIV and TB requires a very high pill load. Therefore, a class of drugs that can be used to treat TB and HIV would be a real breakthrough in TB and AIDS Research. Hence, the present study also evaluated the anti-HIV activity of transitmycin. A dose-dependent reduction was observed in HIV-1 p24 levels in viral culture indicating that transitmycin possessed significant anti-HIV activity. Transitmycin demonstrated good anti-viral activity against the different subtypes of HIV-1 as well as clinical isolates obtained directly from HIV-infected persons. In addition, transitmycin was also active against HIV-1 viruses resistant to nevirapine and AZT (Fig. 8a). The estimated IC 50 value ranged between 0.19 and 0.65µg/ml for the viruses tested. There was a >50% inhibition at a concentration of 0.1µg/ml and 80-95% inhibition at a concentration of1µg/ml in the clinical isolates (Fig. 10b). The compound demonstrated a remarkable inhibitory effect on primary isolates belonging to various subtypes as well as to clinical isolates obtained from HIV-infected individuals. IC 50 values calculated for the various strains tested indicate that transitmycin is a potent inhibitor of HIV-1 under in vitro experimental conditions. Importantly, transitmycin also inhibited drug resistant forms of the virus, in a dose-dependent manner. These findings suggest that transitmycin holds promise as the first potent compound that can be used to treat TB and HIV infections when they occur singly, as well as in combination as HIV/TB co-infection. (Supplementary S-table 13). R1, R2 and R3 intercalates with the genomic DNA of Mycobacterium tuberculosis Actinomycin D is the structurally similar compound for R1, R2 and R3. Actinomycin D and its derivatives are reported to intercalate with the DNA and exhibits fluorescence. So, we tested R1, R2 and R3 for its properties of DNA intercalation and fluorescence. Ethidium bromide 0.5 µg/ml is used as a positive control. The relative fluorescence unit (RFU) of R1, R2 and R3 with the DNA is compared with the RFU of R1, R2 and R3 without DNA. The average RFU of Ethidium bromide was 8561429. From the experiment, it was observed that all the three compounds, R1, R2 and R3 have DNA intercalating property and exhibits fluorescence (Fig. 9). Docking and molecular dynamics studies The concept behind docking was to assess the potential interaction between the transitmycin Fig. 10a or its derivative molecules and various DNA/Protein targets of interest. Through docking studies, we aimed to validate the anticipated interactions, even though comprehensive molecular dynamics studies were restricted, particularly for the entire transitmycin or its derivatives. Nevertheless, the chromophore part Fig. 10b molecular dynamics dynamics displayed enhanced stability, indicating promising outcomes in our research We performed docking and molecular docking experiments with a specific target and three ligands: R1, R2, and R3. Our analysis showed that 1MNV displayed favorable interactions with R2, whereas 3PKE exhibited superior binding with R3 Fig. 10c. The docking score data is presented in Table 4. Docking Score : docking scores were determined using the knowledge-based iterative scoring functions ITScorePP or ITScorePR. A more negative docking score implies a higher likelihood of a binding model. However, it's important to note that the score doesn't represent the true binding affinity, as it hasn't been calibrated against experimental data. Confidence Score: To gauge the likelihood of binding between two molecules, we introduced a confidence score based on docking scores. The formula for the confidence score is: Confidence_score=1.01.0+e0.02×(Docking_Score+150)Confidence_score=1.0+e0.02×(Docking_Score+150)1.0 In this context, when the confidence score surpasses 0.7, it suggests a high probability of binding. Scores between 0.5 and 0.7 indicate a possible binding, whereas scores below 0.5 imply an unlikely binding. Ligand RMSD : Ligand RMSD values were computed by comparing ligands in the docking models with the input or modelled structures. Table 4 . The docking score data of specific target (1MNV, 3PKE, 1MO3, 3IU8) and three ligands: R1, R2, and R3 Name of the Molecule Docking Score Confidence Score Ligand RMSD (A) 1MNV with R1 -489.71 0.9989 22.52 1MNV with R2 -518.80 0.9994 17.61 1MNV with R3 -532.55 0.9995 25.15 1MO3 with R1 -397.33 0.9929 83.36 1MO3 with R2 -424.84 0.9959 83.95 1MO3 with R3 -420.99 0.9956 83.36 3PKE with R1 -391.53 0.9921 52.95 3PKE with R2 -493.89 0.9990 50.96 3PKE with R3 -503.74 0.9992 57.70 3IU8 with R1 -337.77 0.9771 37.59 3IU8 with R2 -418.79 0.9954 47.58 3IU8 with R3 -437.87 0.9969 50.75 We conducted Molecular Dynamics (MD) simulations for the complex 1MNV with R2 and R3. Unfortunately, MD simulations for R2 and R3 couldn't be performed due to the large size of their peptide side chains, preventing accommodation into the minor groove of DNA (1MNV). However, we successfully carried out simulations with the chromophores of R2 and R3. The RMSD of the residues remained within acceptable limits during these simulations. Additionally, MD simulations were conducted for the complex 3PKE with R3. Similar challenges were encountered with the peptide side chains, leading to simulations being performed with the chromophore, as illustrated in Figure 10b. The RMSD values plotted against residues are presented in Figure 10e. The results of the MD simulations indicate the stability of the protein backbone throughout the simulation period. However, it is noteworthy that the simulations faced computational challenges, primarily due to the size of the peptide side chains. Despite these difficulties, the obtained data provides valuable insights into the behavior of the complexes during the simulations. Methods Ethics statement The study commenced on January 15, 2015, and concluded on November 20, 2015. During this period, the screening of the molecule was conducted against clinical isolates that were stored in a de-identified manner. Approval has been expedited for the data collected in this study for the utilization of de-identified stored clinical isolates. Commercial PBMCs were utilized, therefore ethical approval was not necessary. Characterization and taxonomy of Streptomyces sp. R2 Micromorphology of potential Streptomyces sp. R2 was studied by adopting Transplantation Embedding Technique [72]. Briefly, a rectangular trough was dug out of an ISP2 agar plate using sterile knife. Then the spores of Streptomyces strain were inoculated on the edges of the trough under aseptic condition. A sterile cover-slip was placed over the agar, touching the inoculated area on the ISP2 agar plate. The plate was incubated at 28°C for 7-14 days. The cover-slip was aseptically removed using sterile forceps and placed over clean microscopic slide fixing the same using cellophane tape. Micromorphology of Streptomyces sp. R2 was observed under bright field microscope (Olympus) under 10x and 40x magnifications. Spore structure and spore surface morphology were recorded using a scanning electron microscope (JEOL model JSM5600LV). Media and procedures used for determination of cultural characteristics and carbon and nitrogen source utilization were those described originally by Shirling and Gottileb [24]. Effect of pH, temperature, NaCl concentration and anaerobic condition were studied using modified ISP2 medium. Antibiotic susceptibility pattern was determined by disc diffusion method using standard antibiotic discs (Hi media) following the standard protocol [73]. Biomass for cell wall analysis was prepared by growing Streptomyces sp. R2 in shake flasks (120 rpm) containing ISP2 broth at 28 0 C for 5 days. Amino acid and sugar content analyses of whole cell hydrolysates were performed according to the original procedure described by Staneck and Roberts [74]. Molecular characterization and phylogenetic analysis Streptomyces sp. R2 was grown in 50 ml of ISP2 broth at 28 ° C for 48 h. The genomic DNA was extracted using Chromous genomic DNA isolation kit. Polymerase chain reaction (PCR) was performed for the amplification of 16S rRNA gene using the primers 5’ – AGAGTRTGATCMTYGCTWAC – 3’ and 5’ – CGYTAMCTTWTTACGRCT – 3’ on a ABI12720 thermal cycler (Applied Biosystems). The conditions used for thermal cycling were as follows: initial denaturation at 94 0 C for 4 min, followed by 35 cycles consisting of denaturation at 94 0 C for 30 sec, primer annealing at 55 0 C for 30 sec and primer extension at 72 0 C for 2 min, followed by a final extension at 72 0 C for 5 min. The amplified 16s rRNA gene fragment (~ 1.4 kb) was separated by agarose gel electrophoresis and the purified fragment was used for sequencing in an ABI3130 genetic analyser. The nearly complete 16s rRNA gene sequence of strain R2 (1400 nt) was subjected to BLAST comparison against the 16s rRNA sequences given in GenBank/DDBJ/EMBL databases. Phylogenetic analysis was performed using CLUSTAL-W and MEGA version 3.1. Evolutionary distances (Kimura’s two parameter model) [75] and clustering were calculated employing the neighbour-joining method. The topology of phylogenetic tree was evaluated by the bootstrap re-sampling method with 1000 replicates. Production of Transitmycin Hundred microliters of Streptomyces sp. R2 spore suspension was transferred into 10 YEME agar plates and spread using sterile L- rods. The plates were incubated at 28 º C for 10 days. After every 24 hours of fermentation, the mycelial growth was scrapped out and the crude pigment secreted into the agar medium was extracted using equal volume (1:1) of different organic solvents such as n-hexane, dichloromethane, chloroform, ethyl acetate and methanol for 24 hours. The solvent portion was collected and dried at 40 º C using Concentrator plus (Eppendorf) [76]. Anti TB activity of crude extracts were tested against M. tuberculosis H37Rv at 100µg concentration adopting LRP assay [77]. Streptomyces sp. R2 was cultured for 10 days at 28 0 C on YEME agar plates (2000 ml of medium in 100 petriplates of 90 mm diameter) to produce the culture extract in bulk. After the incubation period, the cell material was aseptically removed and discarded after autoclaving. The yellow pigmented antibiotic containing agar medium was cut in to pieces and extracted twice with equal volume (1:1 ratio) of ethyl acetate for 24 hours. Purification of transitmycin Transitmycin was purified by preparative thin layer chromatography (TLC) using Merck silica gel 60 (GF254) pre coated aluminium (6x8 cm size) plates. The extract was separated using different solvents in different proportions. After running, the 200 sheets were kept at room temperature for complete drying of the plate. Spots on TLC were detected through naked eye as well as under UV light (254 and 365 nm). After drying, three major yellow colour spots (R1, R2 and R3) were scrapped, mixed with ethyl acetate and filtered using a funnel fitted with Whatman filter paper. Ethyl acetate was evaporated to dryness under vacuum to obtain the compounds as dry amorphous powder. (Supplementary Fig. S2). All three compounds were tested against M. tuberculosis H37Rv at 100 µg concentration by LRP assay. All three compounds from the ethyl acetate extract was purified using column chromatography packed with neutral alumina using a gradient of 1% methanol/chloroform mixture (CH 3 OH/ CHCl 3 ) as the eluent. Fractions were collected and concentrated under vacuum to obtain pure transitmycin. The product was visualized in a silica gel coated TLC sheet ( Supplementary Fig. S2 and S3). The compound R1 (named as transitmycin) that showed maximum activity was taken for characterization and other studies testing its biological activity. Purity of transitmycin (Fig. 2c). was analysed by HPLC using Shimadzu (Japan) RID-10A gradient high-performance liquid chromatographic instrument, equipped with two LC-20AD pumps controlled by a CBM-10 inter-face module. Refractive index Detector RID 10A (Shimadzu) was used for the peak. Analysis was performed on a Luna 5u C 18 (2) reversed-phase column, 100 (150X4.6mm). The analytical parameters were selected after screening a number of solvent systems and gradient profiles. Separation was achieved using a two-pump gradient program for pump A (0.1% Acetic acid in CH 3 CN) and pump B (0.1% Acetic acid in H 2 O) in a linear gradient of acetonitrile and water from 0:100 to 65:35 in 65 minutes at a flow rate of 2 ml/min. Detection was done at 254 nm, the absorption maxima close to that of majority of the compounds. Injection size for sample was 20 µl. Column temperature was 30°C. ( Supplementary Fig. S4 and S5). Characterization and structure elucidation Colour and consistency of the purified antibiotic was visually observed. Solubility was tested in water, methanol, acetone, ethyl acetate, diethyl ether, dichloromethane, chloroform, and n-hexane by dissolving 1 mg of purified antibiotic. Optical rotations were measured with a Autopol IV Automatic polarimeter, and the [α] D values are given in deg cm 2 g -1 . Melting point was analysed using Mettler Toledo Model FP62 [78]. Ultraviolet (UV) spectrum was determined using Shimadzu UV-1700 series. One milligram of sample was dissolved in 10ml of methanol and the spectra were recorded at wavelength between 190 – 900 nm. The Infrared (IR) spectrum of the purified antibiotic was determined on Perklin Elmer Spectrum One FT-IR. The spectrum was obtained using potassium bromide (KBr) pellet technique in the range of 450 to 4000 cm -1 at a resolution of 1.0 cm -1 . Potassium bromide (AR grade) was dried under vacuum at 100 o C and 100 mg of KBr with 1mg of purified antibiotic was used to prepare KBr pellet. The spectrum was plotted as intensity versus wave number [79]. 1 H and 13 C NMR spectra were recorded on a Bruker Advance 500 NMR spectrometer in CDCl 3 with TMS as internal Standard and with chemical shifts ( δ ) reported in ppm. Two-dimensional 1 H– 1 H COSY, DQF-COSY, NOESY, ROESY, 1 H– 13 C HSQC, HMBC, and spectra were recorded on a Bruker Advance 500 NMR spectrometer. MALDI-TOF MS analyses were performed using an Applied Biosystems ABI4700 TOF mass spectrometer in reflector mode with an accelerating voltage of 20 kV. HRESIMS were measured on a Q-TOF micro mass spectrometer (Waters USA) in positive ion mode with methanol as solvent. QTOF- MS was recorded on an Agilent 6520-QTOF LCMS having an ESI source in positive mode. HPLC Analysis of L-FDAA Derivatives of Transitmycin Transitmycin (3.0 mg) was dissolved in 1 ml of 6NHCl and heated in a sealed glass tube at 110 º C for 24 h. After removing the solvents, the hydrolysate mixture (3 mg) and the amino acid standards (0.5 mg) were separately dissolved in 0.1 mL of water and treated with 0.2 mL of 1% 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide (FDAA) (Marfey’s reagent) in acetone (10 mg/mL in acetone) and 0.04 mL of 1.0 M sodium bicarbonate. The vials were heated at 50 º C for 90 min, and the contents after cooling at room temperature were neutralized with 1N HCl. After degassing, an aliquot of the FDAA derivative was diluted in CH 3 CN, Water (1:1) and analysed by reversed phase HPLC column Luna 5u C 18 (2) 100 (150X4.6mm) and a linear gradient of acetonitrile and water containing 0.05% trifluoroacetic acid from 10:90 to 50:50 in 20 min and then isocratic. The flow rate was adjusted to 1 mL/min and the absorbance detection was at 340 nm. The chromatogram was compared with those of amino acid standards treated in the same conditions [60-61]. LC-MS Analysis of L-FDAA Derivatives of Transitmycin The analysis of the L- and D-FDLA derivatives of Transitmycin was performed on a Waters Acquity UPLC coupled with a Thermo LCQ Deca XP MAX . QTOF- MS was recorded on an Agilent 6520-QTOF LCMS having a ESI source in Positive mode and employing a linear gradient of from 25% to 70% CH 3 CN in 0.01 M formic acid at 0.5 mL/min over 60 min [62-64]. Transitmycin (R1). R1 was obtained as an orange red solid; [α] D 25 -106 o (c 0.2, MeOH); UV (MeOH) λ max (log ε) 214 (3.07), 240 (2.30), 425 (1.44), 442 (1.51) nm; CD [(MeOH),(mdeg)] λ max (Dε) 195 (+11.1), 210 (-21.0), 242 (+4.7) nm; IR (KBr), ν max 3435, 2958, 2924, 2853, 1745, 1642, 1524, 1465, 1379, 1194, 1099, 1059 cm -1 ; HRESI-MS (pos.ions): m/z 1270.7069 [M+2H] + ,1291.8449 [M+Na] + , 1307.9286 [M+K] + 657.3119 [M+2H] +2 ; MALDI-TOF-MS (pos.ions): m/z 1293.07095 [M+Na+2H] + , 1309.93062 [M+K] + MALDI-TOF-MS (neg.ions) 1269.33344 [M-H] - 1 HNMR, 13 NMR and 2DNMR details (Supplementary Fig. S14-52, S-Table 5). Compound (R2). R2 was obtained as a red solid; [α] D 25 : -24 o (c 0.2, MeOH); UV(MeOH) λ max, (log ε) 205(1.25),240(0.63),425 (0.39), 442 (0.42) nm; CD [MeOH, (mdeg)] λ max, (Dε) 195 (+8.8), 210 (-22.0), 240 (+4.3) nm;IR (KBr), ν max 3436, 2961, 2924, 2853, 1744, 1650, 1565, 1415, 1204, 1140, 1045, 1019 cm -1 ; HRESI-MS (pos. ions) :1277.8245 [M + Na] + 1293.8735 [M + K] + m/z 650.3413 [M+2H] +2 ; MALDI-TOF-MS m/z (pos.ions) 1278.95175 [M+Na+H] + ; MALDI-TOF-MS (neg.ions) m/z 1255.38052 [M-H] - , 1 HNMR, 13 NMR and 2D NMR details (Supplementary Fig. S53-63). Compound (R3 ) : R3 was obtained as orange solid; [α] D 25 :-27 o (c 0.2, MeOH); UV (MeOH) λ max (log ε) 206 (1.90), 240 (0.69) 424 (0.191), 442.2 (0.19) nm; CD [MeOH, (mdeg) ] λ max (Dε) 195 (+24.0), 210 (-21.5), 241 (+1.7) nm; IR (KBr), ν max 3415, 2957, 2924, 2853, 1745, 1642, 1583, 1464, 1384, 1193, 1093, 1078 cm -1 ; HRESI-MS (pos.ions):1271.7159 [M +H] + m/z 1277.6149 [M-OH+Na] + ; MALDI-TOF-MS (pos.ions) m/z 1294.5888 [M + Na] + MALDI-TOF-MS (neg. ions) m/z 1268.48582 [M-H] - , 1 HNMR, 13C NMR and 2D NMR details, (Supplementary Fig. S64-69). Determination of anti TB and anti-HIV activity Anti-TB activity was determined by adopting Luciferase Reporter Phage (LRP) assay against the standard laboratory strain, Mycobacterium tuberculosis H 37 Rv, and 97 clinical M. tuberculosis isolates including drug sensitive and cultures exhibiting different drug resistant patterns. Different concentrations (5 – 50 µg/ml) of purified antibiotic – transitmycin were prepared using 10% dimethyl sulfoxide (DMSO). About 50µl of antibiotic solution was added to 350 µl of glycerol 7H9 broth in cryo vials. Effect of DMSO was also tested by adding 50 µl of 10% DMSO instead of the antibiotic. Mycobacterial cell suspension equivalent of 2 McFarland units was prepared from log phase culture and 100 µl of the same was added to all the vials before incubating at 37°C for 72 hours. After incubation, 50 µl of high titre luciferase reporter phage phAE129 and 40 µl of 0.1M CaCl2 were added to test and control vials. All the vials were incubated at 37°C for 4 hours. After incubation 100 µl of suspension from each vial was transferred to a luminometer cuvette. 100 µL of D-luciferin was added and relative light unit (RLU) was measured in a luminometer [77]. Percentage of inhibition was calculated using the RLU of control and test and MIC was determined. [81,82] Activity against M. tuberculosis biofilm Cell suspensions of M. tuberculosis H37Rv were prepared using 7H9 broth. Biofilms of M. tuberculosis were developed on 6 well tissue culture plates by adding 2 ml of Sautons medium (without Tween 80) and inoculating 20 μL of saturated planktonic culture of M. tuberculosis H 37 Rv. The plate was wrapped with parafilm and incubated without shaking at 37°C in humidified conditions for 7 to 14 days. The plate was observed regularly after 7 days for biofilm formation by M. tuberculosis which can be visibly seen. When the biofilm was formed, 10 μg/ml of transitmycin was added to the transitmycin test wells leaving the controls and considered as zero day. The viable counts of tubercle bacilli were determined from the wells on zero day, 4 th day and after 5 weeks and expressed in cfu/ml [80]. Anti-HIV activity Viruses: HIV-1 viruses belonging to subtypes A, B, C, D, E and A/C (Subtype A: 92RW020, Subtype B: JR-FL, Subtype C: 92BR025, Subtype D: 92UG001, Subtype E: 92TH021 and Subtype A/C: 92RW009), were obtained from the NIH AIDS Repository (Germantown, MD, USA). Clinical isolates of HIV-1 were produced in our laboratory by co-culture of HIV-infected peripheral blood mononuclear cells (PBMC) with activated donor PBMC. Testing for anti-HIV activity Anti-HIV activity of Transitmycin was determined using the HIV-1 gag p24 inhibition assay. Initially, testing was performed on a lab-adapted HIV-1 subtype B isolate, HIV-1 IIIB. Donor PBMC were obtained from healthy volunteers after obtaining the approval of the Institutional Ethics Committee of the National Institute for Research in Tuberculosis as well as the informed consent of the participants. Donor PBMC were stimulated with PHA (Phytohemeagglutinin) for 72 hours and incubated with 100 TCID 50 of the virus per 1 x 10 6 cells for 4 h at 37°C. The cells were washed to remove the un-adsorbed virus and plated at a concentration of 10,000 cells/well in a 96-well tissue culture plate. Varying concentrations of Transitmycin (0.01µg/ml, 0.1µg/ml, 1.0µg/ml and 5.0µg/ml) were added to triplicate wells. Control cultures were set up without the addition of the compound. AZT was used as the reference compound. Cultures were maintained for 7 days at 37°C in a CO 2 incubator. On day 7, HIV-1 gag p24 antigen production was determined as an indirect measure of viral replication in the culture supernatants using the Alliance HIV-1 p24 ELISA kit (Perkin Elmer, USA). Testing of Transitmycin against different HIV-1 subtypes Anti-viral activity of Transitmycin was also tested on primary HIV-1 isolates belonging to different subtypes - Subtype A: 92RW020, Subtype B: JR-FL, Subtype C: 92BR025, Subtype D: 92UG001, Subtype E: 92TH021 and Subtype A/C: 92RW009, as well as a Nevirapine resistant and AZT resistant strain, using the method described above. The activity of Transitmycin on different viruses was determined by measuring HIV-1 p24 antigen in 7-day culture supernatants. The IC 50 value (concentration of compound required to inhibit 50% of virus replication) of Transitmysin for the different HIV- 1 subtypes was calculated by fitting a dose response curve using a non-linear regression analysis to generate a sigmoidal three parameter dose response curve (GraphPad Prism, version 6). Activity against clinical strains of HIV Anti-viral activity of Transitmycin was further evaluated on 20 clinical isolates of HIV-1 obtained by co-culture of patient PBMC with PHA-stimulated donor PBMC in the laboratory. For this analysis, only three concentrations (0.01 µg/ml, 0.1 µg/ml and 1µg/ml) of transitmycin was used based on the results of the above experiment. DNA binding and Fluorescence assay 0.5 µg/ml, 1 µg/ml, 2 µg/ml and 5 µg/ml of R1, R2 and R3 were separately added to 100 µl of PBS in triplicates in 96 well plate. Mycobacterium tuberculosis genomic DNA was extracted and 200 ng of DNA is added to each test wells. DNA was not added to the control wells of R1, R2 and R3. For the positive control, 0.5 µg/ml of Ethidium bromide is taken in PBS and 200 ng of DNA is added to it. After 15 mins, the plate is read in Multimode - plate reader, Spinco Biotech, with excitation 546 nm and emission 595 nm. The relative fluorescence unit is recorded. Average of RFU for each concentration of R1, R2 and R3 was taken and plotted as graph. Conclusion Globally, the general public has latent or active tuberculosis, and this is expected to increase in the future, causing the medical system a major challenge. The need of the hour is to identify novel antibiotics to support the current regimens with reduced side effects, duration and cost of treatment. Transitmycin is a novel antibiotic isolated from a novel Streptomyces sp. MTCC from the coral reef soil from Rameshwaram waters in India. It has the unique property of killing latent and active forms of TB bacilli irrespective of the resistance they have towards major antiTB drugs and also sterilising the different resistant and recombinant clades of standard and clinical HIV virus. When brought into the market for human use, transitmycin can strategize treating TB and HIV simultaneously, which could be a major breakthrough, none the less. Of course, preclinical trials and toxicity studies need to be performed before it can be tested on human volunteers which will require sufficient resources. Countries which are burdened with this dual ailment may have to take considerable interest in this direction. Structural medications to the parent compound can further improve its activity. Declarations Acknowledgement Professor Balasubramanian Kalpattu Kuppusami, INSA Senior Scientist, Department of Chemistry, IIT Madras Chennai 600 036, India and Professor Krishna Kumari Gadepalli Narasi, Department of Medicinal Chemistry, Sri Ramachandra University, Chennai 600 116, India, acknowledged for discussion of structure elucidation of Transitmycin. Technical assistance of Dr. S. Balaji and Dr. A. S. Shainaba of National Institute for Research in Tuberculosis is acknowledged in fine tuning and uploading the manuscript. Supplementary materials The comprehensive characterization of the anticipated products have been subjected through UV/Vis, IR, CD, CHNS analyses, whilst, followed by 1 H-NMR 13 C-NMR, 1 H- 1 H COSY, 1 H− 1 H DQF-COSY, 1 H− 13 C HMBC, and 1 H− 13 C HSQC, 1 H− 1 H TOCSY, 1 H− 1 H NOESY, 1 H− 1 H NOESY 2D NMR spectra and MALDI-TOF-MS, HR-ESIMS, HR-LCMS, ESI-MS, QTRAP LC-MS/MS, RP-HPLC and LCMS analyses respectively. The following L-FDAA derivatives of R1, R2, R3 spectral information on all compounds of this article can be found in the online version. Funding Received for this study Potential Tuberculosis Drugs from Marine Actinomycetes, Funded by Department of Science and Technology, New Delhi (November, 2007 to November, 2009). Study to evaluate the baseline anti TB and anti-HIV properties of novel antibiotic transitmycin (Tr) isolated from novel Streptomyces Sp. R2 funded by Indian Council of medical Research (2009-2012). Purity and in vitro efficacy studies on transitmycin funded by Indian Council of Medical Research (2015-2016). Authors’ individual contributions Vanaja Kumar – Principal Investigator, planning, supervision, coordination and data compilation, manuscript writing and editing. Balagurunthan Ramasamy–Facilitating Growing of producer strain, coordination in crude extract preparation as lab head. Mukesh Doble – Purification of compound from crude extract, structure analysis and chemical characterisation and report preparation as lab head. Gandarvakottai Senthilkumar Arumugam – Isolation, purification, characterization, complete structural elucidation and a key role in Innovation Of Transitmycin drug discovery development and manuscript writing and editing. Kannan Damodharan - Isolation, purification, characterization, structural elucidation of Transitmycin compounds and final manuscript drafting. Radhakrishnan Manikkam- Isolation and characterisation of producer strain, biological and biochemical characterisation, testing biological activities, data compilation, manuscript writing. Hanna Luke Elizabeth - Testing crude extract and purified compound on HIV standard and clinical clades. Preparation of write up. K R Uma devi-Investigator, supervision, coordination and data compilation, manuscript writing and editing. Suresh Ganesan – Isolation, purification, characterization, structural elucidation of Transitmycin compounds and final manuscript drafting Azger Dusthackeer- Testing biological activities against latent bacilli and biofilms, preparing the write up. Precilla Lucia – Laboratory work for testing the compound on HIV clades. Shainaba A Saadhali- Testing biological activities against latent bacilli and biofilms, preparing the write up. Shanthi John – laboratory work with respect to growing the producer strain and preparation of crude extract. Poongothai Eswaran - laboratory work with respect to growing the producer strain and preparation of crude extract. Jaleel UCA-Provided the overall conceptualization of theoretical study, and offered guidance throughout the research process and reviewed and edited the manuscript to ensure clarity, consistency, and scientific accuracy. Rakhila M-Conducted the data analysis, molecular docking simulations, and statistical interpretation of the results and edited the manuscript to ensure clarity, consistency, and scientific accuracy. Ayisha Safeeda- Conducted the data analysis, molecular docking simulations, and statistical interpretation of the results and edited the manuscript to ensure clarity, consistency, and scientific accuracy. Satheesh S -was responsible for creating and editing the figures and tables that visually represented the key findings. Selvakumar Nagamiah– supervision of laboratory work on testing biological activities, editing manuscript. 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TB–HIV co-infection: a catastrophic comradeship. Oral Dis.2016; 22:46–52. Xu LH, et al . Actinomycete Systematics – Principles, Methods, and Practice. Science Press: Beijing (2007). Bauer AW, Kirby, WMM. Antibiotic susceptibility testing by a standardized single disc method. American J. Clin. Pathol. 1966; 45:493-496. Staneck JL, Roberts GD. Simplified Approach to Identification of Aerobic Actinomycetes by Thin-Layer Chromatography. Appl. Microbiol.1974; 28 : 226-231. Kimura MA, simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol.1980: 16 : 111–120. Eccleston GP, et al. The occurrence of bioactive micromonosporae in aquatic habitats of the Sunshine Coast in Australia. Mar. Drugs.2008; 6 : 243–261. Radhakrishnan M, et al . Preliminary screening for antibacterial and antimycobacterial activity of actinomycetes from less explored ecosystems. World J. Microbiol. Biotechnol.2010; 26 : 561-566. Harindran J, et al.HA-1-92, A new antifungal antibiotic produced by Streptomyces CDRIL-312: fermentation, isolation, purification and biological activity. World J. Microbiol. Biotechnol.1999; 15 : 425-430. Augustine SK, et al. A non-polyene antifungal antibiotic from Streptomyces albidoflavus PU 23. J. Biosciences . 2005:30201-211. Ojha AK, et al.Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria. Mol. Microbiol .2008; 69 : 164–174. Arumugam, G.S., et al. Significant perspectives on various viral infections targeted antiviral drugs and vaccines including COVID-19 pandemicity. Mol Biomed 3 , 21 (2022). https://doi.org/10.1186/s43556-022-00078-z Mondal R, et al. (2023) In-vivo studies on Transitmycin, a potent Mycobacterium tuberculosis inhibitor. PLoS ONE 18(3): e0282454. https://doi.org/10.1371/journal.pone.0282454. Additional Declarations No competing interests reported. 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1","display":"","copyAsset":false,"role":"figure","size":342353,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative HPLC chromatogram of crude and isolated samples R1, R2 and R3.\u003cstrong\u003e a \u003c/strong\u003eRP HPLC of Chromatogram of crude extract of \u003cem\u003eStreptomyces \u003c/em\u003esp. R2. \u003cstrong\u003eb \u003c/strong\u003eRP HPLC analysis of mixture of R1, R2, R3. \u003cstrong\u003eC\u003c/strong\u003e RP HPLC of Chromatogram of pure Transitmycin R1. \u003cstrong\u003ed\u003c/strong\u003eTransitmycin R1 obtained after column chromatographic purification. \u003cstrong\u003ee\u003c/strong\u003e UV-Vis spectra of Transitmycin R1, R2 and R3. \u003cstrong\u003ef\u003c/strong\u003e IR Spectrum of Transitmycin R1.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5801746/v1/e91dac555d273c4217e6d07f.png"},{"id":74942418,"identity":"afe28b2f-503d-46bc-ac2e-71e2adb96bef","added_by":"auto","created_at":"2025-01-28 14:39:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":416046,"visible":true,"origin":"","legend":"\u003cp\u003eNotable characteristic \u003csup\u003e1\u003c/sup\u003eH NMR spectra of different proline units of Transitmycin as \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003cstrong\u003ea\u003c/strong\u003e R1 (4-oxoproline). \u003cstrong\u003eb\u003c/strong\u003e R2 (proline) and \u003cstrong\u003ec\u003c/strong\u003e R3 (4-OH proline). \u003cstrong\u003ed\u003c/strong\u003e Comparative studies for \u003csup\u003e1\u003c/sup\u003eH NMR (500 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) chemical shift value of NH containing amino acid residue and chromophore motifs of Transitmycin (R1), R2 and R3.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5801746/v1/80f7833cce5a6b9b144b300a.png"},{"id":74941948,"identity":"c1a1dc58-73cd-4783-93aa-35ab38ba2891","added_by":"auto","created_at":"2025-01-28 14:31:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":206438,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Key \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH COSY correlation of Transitmycin (R1). \u003cstrong\u003eb\u003c/strong\u003e Key \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH TOCSY correlation of Transitmycin (R1). \u003cstrong\u003ec\u003c/strong\u003e Key \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC HMBC connectivity for Transitmycin (R1). \u003cstrong\u003ec\u003c/strong\u003e \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH ROESY correlation of Transitmycin (R1). \u003cstrong\u003ed \u003c/strong\u003eChemical structure of isolated compounds Transitmycin (R1). \u003cstrong\u003ee\u003c/strong\u003e R2. \u0026nbsp;\u003cstrong\u003ef\u003c/strong\u003e R3.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5801746/v1/a35c3a367b083c038ac7ac46.png"},{"id":74941952,"identity":"09bdc94e-25cb-4ca1-a73c-7c4486015b60","added_by":"auto","created_at":"2025-01-28 14:31:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":285846,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea-m\u003c/strong\u003e. Comparative analysis of MALDI-TOF-MS for molecular ion peak of Transitmycin (R1), R2, R3. \u003cstrong\u003ea\u003c/strong\u003e R1 (Simulated). \u003cstrong\u003eb\u003c/strong\u003e R2 (Simulated). \u003cstrong\u003ec\u003c/strong\u003e R3 (Simulated). \u003cstrong\u003ed \u003c/strong\u003eR1 (Measured, positive mode. \u003cstrong\u003ee\u003c/strong\u003e R2 (Measured, Positive mode). \u003cstrong\u003ef \u0026nbsp;\u0026nbsp;\u003c/strong\u003eR3 (Measured, Positive mode). \u003cstrong\u003eg\u003c/strong\u003e R1 (Measured, Negative mode). \u003cstrong\u003eh\u003c/strong\u003e R2 (Measured, Negative mode). \u003cstrong\u003ei\u003c/strong\u003e R3 (Measured, Negative mode), MALDI-TOF-MS spectra of crude extract of \u003cem\u003eStreptomyces \u003c/em\u003esp. R2. \u003cstrong\u003ej\u003c/strong\u003e positive mode. \u003cstrong\u003ek\u003c/strong\u003e negative mode \u003cstrong\u003el\u003c/strong\u003e Comparative analysis for HR-ESI-MS and MALDI-TOF-MS of Transitmycin (R1), R2 and R3 on various batches.\u003cstrong\u003e \u003c/strong\u003e\u0026nbsp;\u003cstrong\u003em\u003c/strong\u003e Characteristic retention factor (R\u003csub\u003ef\u003c/sub\u003e) value and corresponding HR-ESI-MS data of purified compounds (Transitmycin (R1), R2 and R3).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5801746/v1/135e4a13b451f8509e559ce0.png"},{"id":74941936,"identity":"25d82356-3202-4eb4-bbfa-2755cbcdd335","added_by":"auto","created_at":"2025-01-28 14:30:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":358003,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Mass spectral fragmentation of Transitmycin (R1) obtained by MALDI-TOF-MS, \u003cstrong\u003eb\u003c/strong\u003e MALDI-TOF-MS spectrum of Transitmycin (R1). \u003cstrong\u003ec\u003c/strong\u003e Mass spectral fragmentation of Transitmycin (R1) obtained by MALDI-TOF-MS, 211- β-Ring-Val-Oxo-Pro, 233- α -Ring-Val-Pro, 294-β-Ring-Oxo-Pro-Sar-NMeVal, 295-Chromophore, 297- β-Ring-Thr-Val-Oxo-Pro, 357-α-Ring-Val-Pro-Sar-MeVal, 371-β-Ring-Val-Oxo-Pro-Sar-MeVal, 412-β-Ring-Val-Oxo-Pro-Sar-NMeVal, 412-α-Ring-Val-Pro-Sar-NMeVal+859 (Y ion), 495-β-Ring-Thr-Val-Oxo-Pro-Sar-NMeVal, 496-α-Ring-Thr-Val-Pro-Sar-NMeVal, 464 α - Ring-Thr-Val-Oxo-Pro-Sar-NMeVal, 746-Y-113 (MeVal), 747- α-Ring-Val-Pro-Sar-NMeVal+Y-MeVal+545.6, 495= β-Ring-Thr-Val-Oxo-Pro-Sar-NMeVal, 495-371=124-23=101 MeVal (β-Ring-Thr-Val-Oxo-Pro-Sar), 371(β-Ring-Thr-Val-Oxo-Pro-Sar), 294 (β-Ring-Thr-Val-Oxo-Pro).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5801746/v1/fad76c60d88d8ba9b6d3e164.png"},{"id":74941940,"identity":"ab2eb0a7-97ff-4807-8045-95da48af3603","added_by":"auto","created_at":"2025-01-28 14:31:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":317818,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eHPLC data analysis for L-FDAA derivatives to hydrolysates of Transitmycin R1 and standard amino acids. \u003cstrong\u003e\u0026nbsp;b \u003c/strong\u003eGraphical representation for RP-HPLC chromatogram of L-FDAA (Marfey’s) derivatives of hydrosylated Transitmycin (R1). \u003cstrong\u003ec\u003c/strong\u003e HR-LCMS data analysis for L-FDAA derivatives to hydrolysates of Transitmycin R1 and standard amino acids.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5801746/v1/8ced97fa5a2ad90197676cec.png"},{"id":74942420,"identity":"05a4c1ba-4103-4e12-9845-1810cd77e7c7","added_by":"auto","created_at":"2025-01-28 14:39:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":279916,"visible":true,"origin":"","legend":"\u003cp\u003eMIC of Transitmycin in micrograms per millilitre for \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e isolates susceptible to anti-TB drugs (n=49) and drug resistant to one or more anti-TB drugs (n=48) as determined by LRP assay. Sensitive strains are represented in blue colour and resistant strains are represented in brown colour.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5801746/v1/a920b0bfb5b2f13f64aca0b9.png"},{"id":74941954,"identity":"08b11544-fa98-423d-b574-1e7730a6831a","added_by":"auto","created_at":"2025-01-28 14:31:00","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":221946,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eActivity of Transitmycin against different HIV-1 clades. Various concentrations of Transitmycin were tested using 100 TCID\u003csub\u003e50\u003c/sub\u003e of virus belonging to 6 different HIV-1 clades as well as two drug-resistant strains. Each concentration was tested in triplicate on all the indicated viruses and mean ± SEM values are shown in the graph. Log\u003csub\u003e10\u003c/sub\u003e -2 in the figure equals 0.01 µg/ml, Log\u003csub\u003e10\u003c/sub\u003e -1 equals 0.1 µg/ml, Log\u003csub\u003e10\u003c/sub\u003e 0 equals 1 µg/ml and Log\u003csub\u003e10\u003c/sub\u003e 1 equals 10 µg/ml. \u003cstrong\u003eb\u003c/strong\u003e Activity of Transitmycin on clinical isolates obtained from HIV-1 infected individuals. Three concentrations of Transitmycin and two concentrations of AZT were tested against 20 clinical isolates and mean \u003cu\u003e+\u003c/u\u003e SD values are shown on the graph. This experiment was performed on two different occasions.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5801746/v1/78cd3e6c45e0402573009c94.png"},{"id":74941947,"identity":"32c29f8c-344a-4af2-9c75-48e3ccf88d6f","added_by":"auto","created_at":"2025-01-28 14:31:00","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":71258,"visible":true,"origin":"","legend":"\u003cp\u003ea) R1, b) R2 and c) R3 binds with the DNA and emit fluorescence in a dose dependent manner. The concentrations of R1, R2 and R3 tested were 0.5 µg/ml, 1 µg/ml, 2 µg/ml and 5 µg/ml. As the concentration increases, the Relative fluorescence unit (RFU) also increases.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-5801746/v1/85cbf198e727f93514050bd9.png"},{"id":74941960,"identity":"c934ca4d-37cd-492e-831b-9fe479b7c378","added_by":"auto","created_at":"2025-01-28 14:31:01","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":206219,"visible":true,"origin":"","legend":"\u003cp\u003ea) Transitmycin structure b) Chromophore part of transitmycin or its derivatives, c) 1 MNV R2 Docking Pose d) 3PKE with R3 Docking pose e) RMSD values of Residues during the simulation Plot (3PKE with R3).\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-5801746/v1/f60c47543924a02e64693c5a.png"},{"id":83845449,"identity":"5f10a6e5-b838-4535-be24-6202f97d2614","added_by":"auto","created_at":"2025-06-03 14:54:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4746889,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5801746/v1/c5d0e9f9-b7ff-4d0f-8266-4c00fa296d6c.pdf"},{"id":74941974,"identity":"b5924282-2c60-4cb8-82af-ef98adb17cea","added_by":"auto","created_at":"2025-01-28 14:31:05","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":80985998,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5801746/v1/04ff5812efa3b997c0a4726b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Discovery of a novel antibiotic, Transitmycin, from Streptomyces sp unveils highly efficient activities against tuberculosis and human immunodeficiency virus","fulltext":[{"header":"Highlights","content":"\u003cp\u003e\u0026bull; A novel antibiotic was purified from a marine Streptomyces sp isolated from the coral reef of S. India\u003c/p\u003e\n\u003cp\u003e\u0026bull; Presence of L-valine, not observed in actinomycin D, and one of the proline in D configuration suggest that it is a novel structure not reported before\u003c/p\u003e\n\u003cp\u003e\u0026bull;\u0026nbsp; It exhibits activity against standard MtB strain as well as clinical isolates and drug resistance ones\u003c/p\u003e\n\u003cp\u003e\u0026bull;\u0026nbsp; It exhibits anti-HIV activity against several clinical isolates\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eDespite, the history of infectious diseases is time immemorial, an urgent need for protection against infectious diseases has one of the major health concerns. Antibiotics have revolutionized the diagnostic factor in various aspects and the drug discovery is considered as a hallmark in human history. However, the concomitant development of resistance against diseases is the most serious consequence [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In 2020, an overall rate of 1.5\u0026nbsp;million people pretentious from TB. In fact, TB is one of the leading sources of death, wherein occupied the 13th position and moreover the second foremost infective destroyer after COVID-19 (above HIV/AIDS). Since it is an airborne infectious disease caused by \u003cem\u003eM tuberculosis\u003c/em\u003e, continues to be a major cause of mortality and morbidity worldwide [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAn upsurge of multi drug resistance (MDR) and extensively drug resistance (XDR) is an additional perturbing factor in tuberculosis chemotherapy. Comprehensively, the existing treatment for TB based on half yearly quadruple therapy containing rifampicin, isoniazid, ethambutol and pyrazinamide results the cure rate of 90\u0026ndash;95% [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] for drug-sensitive TB at global level. Nevertheless, this regimen is inadequate to treat MDR and XDR-TB infections, sometimes the second line antibiotics are employed to treat MDR-TB infections for prolonged time whereas XDR infections are almost untreatable. Different TB drugs with unique mode of action are desperately necessary to fight against drug resistant tuberculosis. For example, bedaquiline and delamanid are approved for drug resistant TB, however due to the reported toxicity issues, they may employ only on last resort [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In addition, the second line drugs used to treat MDR-TB with several disadvantages such as less efficacious, more toxic, and more expensive than the first-line drugs [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. At presently progressing anti TB drugs should partake essential properties like succeed a short therapy period (sterilizing effect), a simpler regimen, capable of act on MDR/XDR-TB, concurrent treatment of TB/HIV and advanced safety level than the existing drugs [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Alike the human immunodeficiency virus (HIV) leads to destruction of the immune system resulting a condition termed as Acquired Immuno Deficiency Syndrome (AIDS). After its finding AIDS becomes a great risk to humans, reflected to be pandemic and it is the greatest public health crisis in globally. As of 2021, millions of people living with HIV were rescuing antiretroviral therapy [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Several researches have been developed the drugs against HIV disease, such as the growth of many antiretroviral drugs (ARVs), including the atazanavir, ibalizumab, darunavir, abacavir, zidovudine, lamivudine, efavirenz, tenofovir disoproxil fumarate, tenofovir alafenamide, emtricitabine, bictegravir, cabotegravir, rilpivirine etc. nevertheless, the competency of HIV intensive to mutate rapidly leads to the upsurge of drug resistance to existing anti-retrovirals [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Since the finding of penicillin, many of the microbial natural products tends to the new paradigm for novel drug discovery development [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOver the past 60 years, [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] the microbial bioprospecting has gained a remarkable quantity of medicinal components, including actinobacteria, the major bacterial phylum group of Gram-positive bacteria with guanine and cytosine (G\u0026thinsp;+\u0026thinsp;C) rich content in their genomic sequence, which are the important prokaryotes in economic and ecological manner. Significantly the existence of actinobacterial multiplicity in several rare ecosystems liable for prolific producers of many biologically active natural compounds with different potential activities [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Some of the pharmaceutical companies have dramatically declined in the result of novel wide range antibiotics in the past few decades [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Obviously, this critical state has caused in the surplus of such antibiotics with distinct mode of activity targeting the arcade, wherein this precluded the assessment of usual terrestrial sources in particular for actinobacteria and led scientists to pursuit unique habitats on the marine family for novel bioactive molecules [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Indeed, actinobacteria are ubiquitous in the marine environment, which are occupying a substantial ecological characteristic in the recycling and production of novel natural products with huge pharmaceutical applications [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGenerally, the 16S rRNA gene sequencing has concomitantly employed to identify, classify and quantitation of microbes in multifaceted biological molecules. It is obvious from the 16S rRNA sequencing that marine microbial species as bacteria and archae have an extreme different taxonomy. Even the 16S rRNA sequences of more than ten thousand actinomycetes had been isolated hitherto from marine outsources. The discovery of novel secondary metabolites from marine actinobacteria has just surpassed that of their terrestrial counterparts [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. According to the review of Blunt et al., [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] shown that there are 179 new natural products isolated during 2016, in which the actinobacterial genus \u003cem\u003eStreptomyces\u003c/em\u003e remains to be the dominant source. An extensive study on typical marine actinobacterial genera like \u003cem\u003eSalinispora\u003c/em\u003e and \u003cem\u003eVerrucosispora\u003c/em\u003e produces salinosporamide and abyssomycin respectively, which suggest that actinobacteria improves significant feature towards marine drug discovery research [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Further, marine based antibiotics are more ingenious in combat infections due to the terrestrial bacteria do not have chance to develop resistance against them [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. During the microbial bioprospecting process, the actinobacterial extracts isolated from various less-explored ecosystems were screened for anti-TB and anti-HIV activity. The isolation, characterization and potential bioactivities against TB and HIV of an antibiotic obtained from a marine \u003cem\u003eStreptomyces\u003c/em\u003e sp. R2 is being reported herewith.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDescription of the actinobacterial strain\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTransitmycin\u003c/em\u003e is a depsipeptide (bicyclic) molecule produced by actinobacterial strain R2, which was isolated from the sediment samples from Rameswaram coral reef ecosystem (Lat. 9.2876\u0026deg; N; Long. 79.3129\u0026deg; E), Tamil Nadu, South India using Starch Casein Agar (SCA) supplemented with nalidixic acid (20\u0026micro;g/ml) and nystatin (100\u0026micro;g/ml). Viability of strain was maintained in International Streptomyces Project-2 (ISP2) agar slants, 30% glycerol broth followed by lyophilisation. It was also deposited in Microbial Type Culture Collection (MTCC), India and in DSMZ \u0026ndash; German Collection of Microorganisms and Cell Cultures, Germany. (R2 = MTCC5597; DSM26035).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCharacterization and taxonomy\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe characterization of a strain is the key element in classification of prokaryotes including actinobacteria [23]. Actinobacterial classification was initially based mainly on morphological and physiological characteristics [24-25]. The onset of chemotaxonomic standards has provided reproducible and reliable data to identify the genera at genus level [26]. This microbial strain R2 under microscopic observation showed the presence of dense aerial and substrate mycelia with long, un-fragmented spore chains with hairy structures (Supplementary Fig. 1a-b). The physiological and biochemical characteristics of the strain R2 are given in Supplementary S-Table 1. Strain R2 showed good growth in various ISP media and utilized a wide range of carbon and nitrogen sources. In addition, good growth was also observed at different physiological conditions. Notably, the extracellular yellow pigment production was greatly influenced by nitrogen substrates, pH, temperature and NaCl. Strain R2 produced lipase and amylase. High sensitivity was observed for most of the antibiotics tested (Supplementary S-Table 1). The cell wall analysis revealed that the strain R2 is rich in LL-2,6-Diaminopimelic acid (L-DAP) and glycine. No diagnostic sugars were found in the cell wall constituents. In our present study, the results of phenotypic characterization and cell wall analysis indicated that the actinobacterial strain R2 belongs to the genus \u003cem\u003eStreptomyces\u003c/em\u003e, however it is not adequate for differentiation at species level.\u003c/p\u003e\n\u003cp\u003eFurthermore, the PCR (Polymerase Chain Reaction) amplification of 16S rRNA gene of strain R2 formed around 1400 base pair sequence and the BLAST (Basic Local Alignment Search Tool) analysis have shown 99% similarity to the 16S rRNA gene sequence of \u003cem\u003eStreptomyces variabilis\u0026nbsp;\u003c/em\u003e(EU570414) and other closely related species submitted in GenBank. Phylogenetic relationship of the strain R2 and related taxa are given in Supplementary S-Fig. 1c.\u003c/p\u003e\n\u003cp\u003eThe 16S rRNA gene sequence of strain R2 has the accession number HQ012501at GenBank, but this gene provides limited resolution for species level identification, since it discloses more extensive genotypic differences. At this stage, the average nucleotide identity (ANI) [27] of all preserved genes between any two genomes shows likely to reform taxonomy, because it also correlates with the best DNA: DNA Hybridization (DDH) values. The most often used standards for species delineation \u003cem\u003ei.e\u003c/em\u003e. the 70% DDH [28] which is closely equivalent to 95% ANI (Average Nucleotide Identity) values. Moreover, no organisms have been defined hitherto has shown \u0026lt;98.7% identity in their 16s rRNA gene and shown \u0026lt;95% ANI or 70% DDH. These results supported the substitution of cumbersome DDH and related procedures with simple sequence-based standards. Significantly the metabolic property of transitmycin production had not been reported before from any other meticulously associated \u003cem\u003eStreptomyces\u003c/em\u003e species. Based on the aforesaid facts, the actinobacterial strain R2 is identified to be a novel strain of \u003cem\u003eStreptomyces variabilis\u003c/em\u003e, but still it showed 99% similarity with its associated proximity neighbors. It is very important to mention here that there is no literature evidence on any commercially available antibiotic, in particular anti-TB and/or anti-HIV compounds from \u003cem\u003eStreptomyces variabilis.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTransitmycin production\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe growth of \u003cem\u003eStreptomyces\u0026nbsp;\u003c/em\u003esp. R2 is simultaneously observed with grey colour aerial mycelia and soluble yellow orange pigment in good yield. The crude compound is extracted well in several solvents namely, methanol, chloroform and dichloromethane, followed by diethyl ether and ethyl acetate (Supplementary Table 2). The extracts in the former solvents were intensely coloured when compared to the extracts from the latter solvents. Salts and debris were present in the extracts while using former solvents, so ultimately ethyl acetate was chosen for extracting the bioactive pigment.\u003c/p\u003e\n\u003cp\u003eThe activity of the crude extract was also tested on different strains of \u003cem\u003eM.\u0026nbsp;\u003c/em\u003e\u003cem\u003etuberculosis\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003eMore than 95% reduction in Relative Light Units (RLU) was observed through Luciferase Reporter Phage Assay against all three \u003cem\u003eM.\u0026nbsp;\u003c/em\u003e\u003cem\u003etuberculosis\u003c/em\u003e strains. When 1.0 L of YEME agar was used for production it yielded 800 mg crude antibiotic extract in ethyl acetate. Major antibiotics reported from actinobacteria are extracellular in nature [29-30]. Industrial production of many antibiotics from \u003cem\u003eStreptomyces\u003c/em\u003e is achieved through submerged fermentation process [31]. In contrast, some actinobacterial strains are found to produce antibiotics only on solid media and very little reason is reporeted as to why activity is restricted to solid culture and not observed in submerged cultures. Shomura \u003cem\u003eet al reported\u0026nbsp;\u003c/em\u003e[32] that about 1300 out of 6500 actinomycetes showed antimicrobial activity against one or more of the test organisms when tested by agar plug method. In the secondary screening, about 25 (1.9%) of the 1300 strains were found to be non-producers in submerged cultures. So, we conlcude that the reports of antibiotics isolated from only agar cultures of actinomycetes are very rare.\u003c/p\u003e\n\u003cp\u003eAccording to Mayurama \u003cem\u003eet al\u003c/em\u003e. [33] fumaridamycin was detected with much difficulty in submerged cultures, because the mycelium of the producing strain inactivate the antibiotic more readily in liquid than in agar culture. Similarly, Shomura\u003cem\u003eet al\u003c/em\u003e [32] demonstrated that the antibiotic produced by \u003cem\u003eStreptomyces halstedii\u0026nbsp;\u003c/em\u003ehasshown activity against Gram negative bacteria only in agar dishes, which correlates well with its mycelial morphology. The aerial mycelium was filamentous during antibiotic production in solid cultures, but fragmented in non-producing liquid cultures. Similar to report from Shomura \u003cem\u003eet al\u003c/em\u003e. [32] the bioactive pigment production by the \u003cem\u003eStreptomyces\u0026nbsp;\u003c/em\u003estrain R2 was observed only in agar culture, where the vegetative mycelium was filamentous. While decreasing the concentration of YEME broth from 2X to 1/10X the mycelia filamentation was found to increase. However, none of the five concentrations of YEME broth produced the pigment (unpublished data). This outcome evidenced that the filamentous mycelial structure does not influence pigment production by the strain R2 in broth while the bioactive pigment production was observed in all the concentrations of YEME agar when filamentous mycelia was formed. In accordance to Ohnishi \u003cem\u003eet al\u003c/em\u003e. [34] who reported that 2-aminophenoxazin-3-one containing grixazone A and B, afford yellow pigments during phosphate depletion by \u003cem\u003eStreptomyces griseus\u003c/em\u003e. By adopting these strategies, it was concluded that optimizing the medium components may trigger the pigmented bioactive compound production by our isolated \u003cem\u003eStreptomyces\u003c/em\u003e sp. R2 in liquid cultures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransitmycin purification, characterization and structure elucidation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eStreptomyces\u003c/em\u003e sp. R2 was found to produce 800 mg of crude pigment per litre of yeast extract malt extract medium in agar surface fermentation. The yellow-orange pigment was separated from the crude ethyl acetate extract by TLC and column chromatography and its purity was confirmed by HPLC analysis (SupplementaryFig S13-S17).\u003c/p\u003e\n\u003cp\u003ePrimarily the three well separated spots \u003cem\u003eviz.\u003c/em\u003e R1, R2 and R3 with R\u003csub\u003ef\u003c/sub\u003e values of 0.8, 0.6, 0.3, respectively were observed on analytical TLC using EtOAc:MeOH (9.5:0.5) solvent system. In bioassay directed isolation, fraction R1 (named as Transitmycin) showed more than 95% inhibition against \u003cem\u003eM. tuberculosis\u0026nbsp;\u003c/em\u003estrain H37Rv in LRP assay. It was isolated as an orange colour amorphous powder with [\u0026alpha;]\u003csub\u003eD\u003c/sub\u003e\u003csup\u003e25\u003c/sup\u003e: -106\u0026deg; (\u003cem\u003ec\u003c/em\u003e = 0.2, MeOH). The R1, R2, R3 in crude extract and as purified compounds R1, R2, R3 showed similar retention time in RP-HPLC chromatograph \u003cstrong\u003e(\u003c/strong\u003eFig, 1a-d\u003cstrong\u003e)\u003c/strong\u003e.A single peak of transitmycin at a RT of 5.8 minutes confirmed its purity \u003cstrong\u003e(\u003c/strong\u003eFig. 1c).\u003c/p\u003e\n\u003cp\u003eThe chemical structures of these molecules (R1, R2 and R3) were elucidated by UV-Visible, IR, CD, \u003csup\u003e1\u003c/sup\u003eH, \u003csup\u003e13\u003c/sup\u003eC, DEPT 135 NMR, 2D NMR (\u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH COSY,\u003csup\u003e\u0026nbsp;1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH DQF-COSY, \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH TOCSY, \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC HSQC, \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC HMBC, NOSEY, ROESY) and MALDI-TOF-MS, HR-ESIMS, HR-EIMS, HR-LCMS, and 3200 QTRAP-LC/MS/MS analyses and also compared with the previously reported NMR and Mass data of actinomycins (Supplementary Information) [35-57]. Advances in spectroscopic techniques have mainly utilized for compound identification and immensely accelerated the unambiguous representation of compound characterization and structural elucidations. The UV-Vis spectrum of Transitmycin showed a strong absorption band around (\u0026lambda;\u003csub\u003emax\u0026nbsp;\u003c/sub\u003e214, 240, 425, 442 nm (in MeOH) (Fig. 1e). The colour and absorption peak in UV-Vis analysis revealed the presence of phenoxazinone chromophore. Singh \u003cem\u003eet al\u003c/em\u003e. [58] and Maskey \u003cem\u003eet al\u003c/em\u003e. [59] detected the presence of phenoxazinone chromophore in bioactive metabolites from \u003cem\u003eStreptomyces\u0026nbsp;\u003c/em\u003esp. and \u003cem\u003eActinomadura\u0026nbsp;\u003c/em\u003esp. The absolute configuration of the amino acids were supposed to be identical to that of actinomycin D, as indicated by the negative optical rotation values and the strong cotton effect at about 210 nm in the CD spectra. The CD values of Transitmycin (R1) are: [MeOH, [nm], (mdeg)] \u0026lambda;\u003csub\u003emax\u003c/sub\u003e(D\u0026epsilon;) 195 (+24.0), 210 (-21.5), 241 (+1.7).\u003c/p\u003e\n\u003cp\u003eIn the IR spectrum, a strong absorption broad band appears at around 3435 cm\u003csup\u003e-1\u003c/sup\u003e, an intense strong peak at 1746 cm\u003csup\u003e-1\u003c/sup\u003e and a band around 1099 cm\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eare assignable to be amino (or hydroxyl), lactone ring and alicyclic 6-membered ether type (C-O) groups, respectively. IR data of transitmycin (R1) (KBr cm\u003csup\u003e-1\u003c/sup\u003e), 3435 cm\u003csup\u003e-1\u003c/sup\u003e for NH, 2958, 2924 cm\u003csup\u003e-1\u003c/sup\u003e (m, -CH str, asym, CH\u003csub\u003e3\u003c/sub\u003e and CH\u003csub\u003e2\u003c/sub\u003e), 2872 cm\u003csup\u003e-1\u003c/sup\u003e, 2853 cm\u003csup\u003e-1\u003c/sup\u003e (m, -CH str, sym, CH\u003csub\u003e3\u003c/sub\u003e and CH\u003csub\u003e2\u003c/sub\u003e),1746 cm\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003e (s, C=O str, lactone ring), 1642 cm\u003csup\u003e-1\u003c/sup\u003e(s, -C=O str, 2\u003csup\u003eo\u003c/sup\u003e amide), 1524, 1503 (m, -NH bend, 2\u003csup\u003eo\u003c/sup\u003e amide), 1466 (m, CH bend (scissoring), CH\u003csub\u003e2\u003c/sub\u003e), 1379 cm\u003csup\u003e-1\u003c/sup\u003e (s, -CH bend, isopropyl group), 1268 (s, C-O str, ester), 1194 ( C-O-C of lactone) 1099, 1059, 1017 (s, C-O or C-N), 720, 712, 694, 689 (s, -CH bend, oop, aromatic ring), 909 (w, CH\u003csub\u003e3\u003c/sub\u003e rocking) [48](Fig. 1f).\u003c/p\u003e\n\u003cp\u003eThe \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR spectra exhibited the typical features of two (alpha and beta ring) pentapeptido lactone ring attached with phenoxazinone chromophore, i.e., each ring contained four amide carbonyl resonances and one ester carbonyl in one ring (\u0026delta;\u003csub\u003eC\u003c/sub\u003e 168.8, 173.4, 173.1, 166.2, 167.4 (\u0026alpha;-ring) and 168.9, 173.9, 172.7, 166.4, 167.4 (\u0026beta;-ring), together with phenoxazinone chromophore (101.8 (C-1), 147.3( C-2), 179.0 (C-3), 113.4 (C-4), 144.9 (C-4a), 140.3 (C-5a), 127.6 (C-6), 130.2 (C-7), 126.0 (C-8), 132.1(C-9), 128.4 (C-9a), 14.9 (C-11), 7.6 (C-12), 166.0 (C-13), 165.8 C-14). One of the amino acid proline in the \u0026beta;-ring contained a keto group (208.0 in the \u003csup\u003e13\u003c/sup\u003eC NMR). From \u003csup\u003e1\u003c/sup\u003eH NMR spectrum, NH of amino acids (\u0026delta;\u003csub\u003eH\u003c/sub\u003e 8.23, \u0026beta;-L-Valine), (\u0026delta;\u003csub\u003eH\u003c/sub\u003e 7.74, \u0026alpha;-L-Valine), (\u0026delta;\u003csub\u003eH\u003c/sub\u003e 7.69, \u0026beta;-L-Threonine, (\u0026delta;\u003csub\u003eH\u0026nbsp;\u003c/sub\u003e7.2, \u0026alpha;-L-Threonine), (\u0026delta;\u003csub\u003eH\u0026nbsp;\u003c/sub\u003e6.55, 2H of \u0026beta;-proline) (\u0026delta;\u003csub\u003eH\u0026nbsp;\u003c/sub\u003e 5.93 2H of \u0026alpha;-proline) and four N-methyl groups (\u0026delta;\u003csub\u003eH\u0026nbsp;\u003c/sub\u003e2.91, 2.88, 2.89, 2.87 ) (Fig. 2 a-d). In addition, the \u003csup\u003e1\u003c/sup\u003eH NMR spectra of Transitmycin indicated the presence of eight methyl groups arising from four isopropyls.\u003c/p\u003e\n\u003cp\u003eThe UV/Vis absorption spectra with maximal absorbance at 240 nm and 442 nm support the presence of an amino phenoxazinone chromophore in its structure. From \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eHCOSY and TOCSY experiments, five amino acid systems namely Pro, Thr, Val, N-Methyl Val, and Sar were identified. The assignments of the protonated carbons were obtained from the HSQC spectrum, in combination with inspection of the HMBC spectrum. By comparison of the UV spectrum \u003cem\u003e(\u0026lambda;\u003csub\u003emax\u003c/sub\u003e\u0026nbsp;\u003c/em\u003e442 nm, in MeOH) of Transitmycin with that of actinomycin series \u003cem\u003e(\u0026lambda;\u003csub\u003emax\u003c/sub\u003e\u0026nbsp;\u003c/em\u003e440 nm, in MeOH) (Fig. 1e) it was concluded that the former contained an aminophenoxazinone chromophore residue. In \u003csup\u003e1\u003c/sup\u003eH NMR, two \u003cem\u003eortho\u0026nbsp;\u003c/em\u003ecoupled protons at 7-H, 8-H; \u0026delta;\u003csub\u003eH\u003c/sub\u003e 7.56 and 7.34 corresponding to 1,2,3,4-tetrasubstituted aromatic ring, and two 3H singlet at \u0026delta;\u003csub\u003eH\u003c/sub\u003e 2.52 (11-CH\u003csub\u003e3\u003c/sub\u003e) and 1.95 (12-CH\u003csub\u003e3\u003c/sub\u003e) of methyl groups in peri-position of the aromatic system were identified. This is the characteristics of phenoxazinone chromophore found in various actinomycins.\u003c/p\u003e\n\u003cp\u003eThe result was further confirmed by HMBC correlations between the 7-H (\u0026delta;\u003csub\u003eH\u003c/sub\u003e 7.59) and 8-H (\u0026delta;\u003csub\u003eH\u003c/sub\u003e 7.34) of the tetra substituted double bond and the carbonyl resonances at \u0026delta;\u003csub\u003eC\u003c/sub\u003e 166.0. The carbonyl carbons of Pro, Thr, Sar, Val, and N-methyl Val, were clearly assigned to (\u0026delta;\u003csub\u003eC\u003c/sub\u003e 179.0, 174.0, 173.1, 169.02, 198.8, 167.5, 166.5, 166.56, 166.3, 166.1and 165.9) on the basis of the observed correlations between carbonyl groups protons of the same amino acid residue in the HMBC spectrum. All the residues were connected on the basis of DQF-COSY, TOCSY, HMBC, ROESY and NOESY correlations (Fig. 3a-c), thus establishing the amino acid sequences and overall constitution.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(Supplementary\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eFig. S14-65, S-Table 5). The detailed analysis of \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH COSY, \u003csup\u003e1\u003c/sup\u003eH\u0026minus;\u003csup\u003e1\u003c/sup\u003eH DQF-COSY, \u003csup\u003e1\u003c/sup\u003eH\u0026minus;\u003csup\u003e13\u003c/sup\u003eC HMBC, and \u003csup\u003e1\u003c/sup\u003eH\u0026minus;\u003csup\u003e13\u003c/sup\u003eC HSQC, \u003csup\u003e1\u003c/sup\u003eH\u0026minus;\u003csup\u003e1\u003c/sup\u003eH \u0026nbsp;TOCSY, \u003csup\u003e1\u003c/sup\u003eH\u0026minus;\u003csup\u003e1\u003c/sup\u003eH NOESY, \u003csup\u003e1\u003c/sup\u003eH\u0026minus;\u003csup\u003e1\u003c/sup\u003eH ROESY NMR spectra [35-51] (Supplementary Information\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eFig. S14-65, S-Table S5) and MALDI-TOF-MS (Fig. 4a-m and Fig. 5)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eHR-ESIMS, HR-LCMS, ESI-MS, QTRAP LC-MS/MS [52-57] (Fig. 5, S-Table S6), (Supplementary Information\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eFig. S14-115\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003eS-Table S7a-b) spectral fragmentation pattern revealed ten amino acids in Transitmycin (Fig. 3d), which is identical to those present in actinomycin X2 [35-57] (2 X MeVal, 2 X Thr, 2 X Sar, 2 X Val, proline and ketoproline). OPro was identified by the ketone moiety (\u0026delta;\u003csub\u003eC\u003c/sub\u003e 208.6 ppm) and the altered chemical shifts and coupling patterns of the neighbouring methylene groups (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e. \u003csup\u003e1\u003c/sup\u003eH NMR (500 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e)/\u003csup\u003e13\u003c/sup\u003eC NMR (125 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) and 2D NMR (500 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) correlation spectral data of Transitmycin (R1)\u003csup\u003e\u0026nbsp;1\u0026nbsp;\u003c/sup\u003eH\u0026minus;\u003csup\u003e1\u0026nbsp;\u003c/sup\u003eH COSY, \u003csup\u003e1\u003c/sup\u003eH\u0026minus;\u003csup\u003e1\u003c/sup\u003eH TOCSY \u003csup\u003e1\u003c/sup\u003eH\u0026minus;\u003csup\u003e13\u003c/sup\u003eC HMBC, \u003csup\u003e1\u003c/sup\u003eH\u0026minus;\u003csup\u003e1\u003c/sup\u003eH ROESY\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"101%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026alpha;- ring\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePosition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026delta;\u003csub\u003eC\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026delta;\u003csub\u003eH\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eJ\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(Hz)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCOSY\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTOCSY\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHMBC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eROSEY\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eThr\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e168.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e54.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e4.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed , 10.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e3, 4, -NH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e3, 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e1, 3, 13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e3, 4, 2-Val, Val-NH\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e74.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e5.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003em\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2, 4,- NH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2, 4,\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e2, 4, Val-NH\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e17.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e1.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed, 6.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2, 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2, 3, -NH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e2, 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e2, 3, Val-NH\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eNH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e7.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed, 7.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2, 3, 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2, 3, 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e1, 3, 13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e2, 4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eL-Val\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e173.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e58.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e3.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003edd, 10.5,6.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e3, 4, 5, -NH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e3, 4, 5, -NH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e1, 5, 1-Thr\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e3, 4, 5, 2-Pro\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e31.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e2.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003em\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2, 4, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2, 4, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e2,5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e4, 5, 4-Thr\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e19.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed, 7.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2, 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2, 3, -NH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e2,3,5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e2, 3, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e18.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e1.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed, 6.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2, 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2, 3, -NH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e2,3,4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e2, 3, 2-Val, Val-NH\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eNH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e7.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed, 5.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2, 3, 4, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2, 3, 4, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e2, 3, 1-Thr\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e2, 3, 4, 5, 2- Thr, 3-Thr\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePro\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e173.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e56.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e5.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed, \u0026nbsp;9.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e3a,3b,4a,4b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e3a,3b 4a, 4b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e3, 4, 5, 1-Val\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e3a, 3b, 2a-Sar,2b-Sar, Sar-NMe\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e31.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e2.73\u003c/p\u003e\n \u003cp\u003e1.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003edd, 17.5,7.0\u003c/p\u003e\n \u003cp\u003edd, 19.5, 6.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e3b, 4a, 4b\u003c/p\u003e\n \u003cp\u003e3a, 4a,\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e3b, 4a, 4b\u003c/p\u003e\n \u003cp\u003e3a, 4a,\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e1, 2, 4\u003c/p\u003e\n \u003cp\u003e1, 4, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e3b, 2a-Sar, 2b-Sar\u003c/p\u003e\n \u003cp\u003e3a, 4a, 4b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e23.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e2.26\u003c/p\u003e\n \u003cp\u003e2.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed, 16.5\u003c/p\u003e\n \u003cp\u003ed, \u0026nbsp;8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e3a, 3b, 4b\u003c/p\u003e\n \u003cp\u003e3b, 4a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e3a, 3b, 4b\u003c/p\u003e\n \u003cp\u003e3b, 4a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e3, 5\u003c/p\u003e\n \u003cp\u003e3, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e4b, 5a, 5b\u003c/p\u003e\n \u003cp\u003e4a, 5a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e5a\u003c/p\u003e\n \u003cp\u003e5b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e47.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e3.86\u003c/p\u003e\n \u003cp\u003e3.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed, 18.5\u003c/p\u003e\n \u003cp\u003ed, 18.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e3a, 4a, 4b\u003c/p\u003e\n \u003cp\u003e3a, 4a, 4b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e3a, 4a, 4b\u003c/p\u003e\n \u003cp\u003e3a, 4a, 4b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e4, 1-Val\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e5b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSar\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e166.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e51.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e2a, 4.70\u003c/p\u003e\n \u003cp\u003e2b, 3.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed, 17.5\u003c/p\u003e\n \u003cp\u003ed, 17.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2b\u003c/p\u003e\n \u003cp\u003e2a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2b, NMe\u003c/p\u003e\n \u003cp\u003e2a, NMe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e1, NMe,1-pro\u003c/p\u003e\n \u003cp\u003e1, NMe,1-Pro\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e2b, NMe, 2-Pro\u003c/p\u003e\n \u003cp\u003e4-MeVal, 5-MeVal, NMe\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eN-Me\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e34.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e2.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003es\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2a, 2b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e2, 1-Pro\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e2b, 3-MeVal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMe-Val\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e167.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e71.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e2.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed, 9.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e3, 4, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e3, 4, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e1, 3,4,NMe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e4, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e26.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e2.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed, 7.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2, 4, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2, 4, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e1, 3, 4, 5, NMe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e4, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e21.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed, 6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2, 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2, 3, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e2, 3, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e19.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed, 6.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2, 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2, 3, 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e2, 3, 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eN-Me\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e39.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e2.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003es\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2, 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e2, 1-Sar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e2, 3, 5, 2a-Sar, 2b-Sar\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"99%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026beta;-ring\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eposition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026delta;\u003csub\u003eC\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026delta;\u003csub\u003eH\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eJ\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(Hz)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCOSY\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTOCSY\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHMBC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNOESY\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eThr\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e168.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e54.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e4.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed, 9.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e3,4, -NH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e3, 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e1, 3, 14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e3, 4, 2-Val, Val-NH\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e74.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e5.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003em\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e2,4,- NH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2, 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e2,4, Val-NH\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e17.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e1.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed, 6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e2, 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2, 3, - NH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2, 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e2,3, Val-NH\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eNH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e7.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed, 6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e2, 3, 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2, 3, 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e1, 3, 14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e2, 4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eL-Val\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e173.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e57.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e3.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003edd, 10.5, 6.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e3, 4, 5, -NH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e3, 4, 5, -NH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e1, 3, 5, 1-Thr,\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e3, 4, 5, 2-Oxo pro\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e31.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e2.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003em\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e2, 4, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2, 4, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e4, 5, 4-Thr\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e19.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed, 7.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e2, 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2, 3, -NH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2, 3, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e2, 3, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e18.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e1.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed, 7.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e2, 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2, 3, -NH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2, 3, 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e2, 3, 2-Val, Val-NH\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eNH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e8.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed, 6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e2, 3, 4, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2, 3, 4, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2, 3, 1-Thr\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e2, 3, 4, 5, 2-Thr, 3-Thr\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOxo-Pro\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e172.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e54.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e6.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003edd, 10.0, 2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e3, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e3, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e3, 1-Val\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e3, 5, 2a-Sar, 2b-sar-NMe\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e41.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e2.32\u003c/p\u003e\n \u003cp\u003e2.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed , 18.5\u003c/p\u003e\n \u003cp\u003ed, 13.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e2, 5\u003c/p\u003e\n \u003cp\u003e2, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2, 5\u003c/p\u003e\n \u003cp\u003e2, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e2, 5\u003c/p\u003e\n \u003cp\u003e2, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e208.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e53.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e3.83\u003c/p\u003e\n \u003cp\u003e3.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed, 18.5\u003c/p\u003e\n \u003cp\u003edd, 18.5, 12.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e2, 3\u003c/p\u003e\n \u003cp\u003e2, 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2, 3\u003c/p\u003e\n \u003cp\u003e2, 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e1, 2\u003c/p\u003e\n \u003cp\u003e1, 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e2, 3\u003c/p\u003e\n \u003cp\u003e2, 3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSar\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e166.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e51.28\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e2a, 4.54\u003c/p\u003e\n \u003cp\u003e2b, 3.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed, 19.5\u003c/p\u003e\n \u003cp\u003ed, 19.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e2b\u003c/p\u003e\n \u003cp\u003e2a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2b, NMe\u003c/p\u003e\n \u003cp\u003e2a, NMe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e1, NMe, 1-Pro\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1, NMe, 1-Pro\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e2b, NMe, 2-oxo-pro\u003c/p\u003e\n \u003cp\u003e4-MeVal , 5-MeVal, NMe\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eN-Me\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e34.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e2.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003es\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2a, 2b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2, 1-Pro\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e2b, 2MeVal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMe-Val\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e167.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e71.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e2.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed, 8.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e3, 4, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e3, 4, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e1, 3, 4, 5, NMe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e4, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e26.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e2.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed, 8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e2, 4, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2, 4, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e1, 3, 4, 5, NMe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e4, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e21.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed, 6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e2, 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2, 3, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2, 3, 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e19.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003ed, 6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e2, 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2, 3, 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2, 3, 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eN-Me\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e39.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e2.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003es\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003e2, 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2, 1- Sar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e2, 5, 2a-Sar, 2b-Sar\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eChromo\u003c/strong\u003e\u003cstrong\u003ephore\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"97%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePosition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026delta;\u003csub\u003eC\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026delta;\u003csub\u003eH\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eJ\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(Hz)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCOSY\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTOCSY\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHMBC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNOESY\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e101.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e147.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e179.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e4a\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e113.4\u003c/p\u003e\n \u003cp\u003e144.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e5a\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003cp\u003e140.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e127.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e130.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e7.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003ed, 8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e8, 11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e8, 11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e5a, 9, 11-Me\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e126.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e7.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003ed, 7.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e7, 11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e7, 11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e9a, 5a, 13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e9\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e9a\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e132.1\u003c/p\u003e\n \u003cp\u003e128.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e11\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e14.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e2.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003es\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e7, 8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e7, 8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e5a, 6, 7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e7, 8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e7.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e2.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003es\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e3, 4, 4a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e166.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e165.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe MALDI-TOF-MS spectrum of crude extract of Streptomyces sp. R2 (Fig. 4j-k) and measured, calculated mass of purified compounds Transitmycin (R1), R2, R3 were given in (Fig. 4a-i\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003eSupplementary Figure S70-115). The molecular formula was established as C\u003csub\u003e62\u003c/sub\u003eH\u003csub\u003e84\u003c/sub\u003eN\u003csub\u003e12\u003c/sub\u003eNaO\u003csub\u003e17\u003c/sub\u003e based on Positive HRESI-MS, which showed protonated pseudo molecular ion peak [M+H] \u003csup\u003e+\u0026nbsp;\u003c/sup\u003eat m/z 1270.7069 (Fig. 4a-m). It also showed intense peaks, due to Na and K adducts, at m/z 1291.8307 [M+Na]\u003csup\u003e+\u003c/sup\u003e and 1307.8124. [M+K]\u003csup\u003e+\u003c/sup\u003e respectively (Calcd. for C\u003csub\u003e62\u003c/sub\u003eH\u003csub\u003e84\u003c/sub\u003eN\u003csub\u003e12\u003c/sub\u003eNaO\u003csub\u003e17\u003c/sub\u003e: 1291.5975: Found: 1291.8307\u003cstrong\u003e).\u0026nbsp;\u003c/strong\u003eFig. 4a-i Similarly, the MALDI TOF MS spectrum of transitmycin showed intense peak in positive mode at \u003cem\u003em/z\u003c/em\u003e 1293.61316 [M+Na+2H] \u003csup\u003e+3\u003c/sup\u003e and at m/z 1309.93062 [M+K]\u003csup\u003e+\u003c/sup\u003e and in negative mode at m/z 1269.33344 [M-H]- (Fig. 4a-m, Fig. 4-5, \u0026nbsp;Supplementary Information\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eFig. S115a-b). The compound R2 was isolated as orange red powder and its molecular formula was established as C\u003csub\u003e62\u003c/sub\u003eH\u003csub\u003e86\u003c/sub\u003eN\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e16\u0026nbsp;\u003c/sub\u003e[M+Na]\u003csup\u003e+\u003c/sup\u003e by positive HRESIMS. The MALDI TOF molecular ion of R2 showed peak at \u003cem\u003em/z\u0026nbsp;\u003c/em\u003e1278.95175 [M+Na+H]\u003csup\u003e+\u003c/sup\u003e and negative ion mode at \u003cem\u003em/z\u0026nbsp;\u003c/em\u003e1255.38052 [M-H]\u003csup\u003e-\u003c/sup\u003e For a molecular formula of C\u003csub\u003e62\u003c/sub\u003eH\u003csub\u003e87\u003c/sub\u003eN\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e16\u003c/sub\u003eNa its molecular weight is calculated as 1278.6261 [M+Na+H]\u003csup\u003e+\u003c/sup\u003e which matches with 1278.95175, that is similar to that of actinomycin D (Fig. 3e). The compound R3 was obtained as an oranges red powder and the molecular formula of R3 was determined to be C\u003csub\u003e62\u003c/sub\u003eH\u003csub\u003e87\u003c/sub\u003eN\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e17\u0026nbsp;\u003c/sub\u003efrom HR-ESIMS peak at \u003cem\u003em/z\u0026nbsp;\u003c/em\u003e1271.7159z [M+H]\u003csup\u003e+\u003c/sup\u003e and when calculated for the molecular formula , C\u003csub\u003e62\u003c/sub\u003eH\u003csub\u003e87\u003c/sub\u003eN\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e17\u003c/sub\u003e 1271.6312, found to be 1271.7159 and 1277.6149 [M-OH-Na]\u003csup\u003e+\u003c/sup\u003e. The molecular formula of R3 was established as C\u003csub\u003e62\u003c/sub\u003eH\u003csub\u003e87\u003c/sub\u003eN\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e17\u003c/sub\u003e by positive MALDI-TOF 1294.5888 [M+H+Na]\u003csup\u003e+\u003c/sup\u003e and negative mode 1268.4852 [M-H]\u003csup\u003e-\u003c/sup\u003e (Fig. 4f) which is identical to that of actinomycin 0\u0026beta; (Supplementary Information\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eFig. S70-115\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003eS-Table S7a-b) [52-57].\u003c/p\u003e\n\u003cp\u003eThe differences between compounds R1, R2, R3 were becaue of the variation in proline at 4\u003csup\u003eth\u003c/sup\u003e position. Transitmycin (R1) has keto group in the 4\u003csup\u003eth\u003c/sup\u003e position, R2 does not have keto group and compound R3 has hydroxyl group in the 4\u003csup\u003eth\u003c/sup\u003e position (Fig. 3a-c).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003cstrong\u003ePLC\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAnalysis of L-FDAA Derivatives of Transitmycin (R1)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHPLC experiment was used to determine the absolute configuration of isolated compounds. The absolute configurations of the amino acids were assumed to be identical to that of actinomycin X\u003csub\u003e2\u003c/sub\u003e, as indicated by the negative optical rotation values and the strong cotton effect at about 210 nm in the CD spectra. The assignment of the amino acids was carried out primarily by the analysis of the \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC HSQC and \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH-COSYcorrelations, MALDI-TOF-MS (Fig. 4 a-m, S-Table S6)\u003cstrong\u003e,\u003c/strong\u003e QTRAP LC-MS/MS (Supplementary Information\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eFig. S14-114, S115a-b, S-Table S7a-b\u003cstrong\u003e),\u003c/strong\u003e and completed with the help of HMBC spectrum (Fig. 3a-c). Additionally, a small amount of compound Transitmycin and R3 were hydrolyzed and the free amino acid were analyzed by HPLC, HR-LCMS, HR-ESIMS after chiral derivatization with Marfey\u0026rsquo;s reagent. Altered proline and sarcocine were not available as reference. Although neither the altered proline and threonine moieties nor N-methylated alanine were available as references, they can be assumed to possess L-configurations due to the fact that the exchange of a single amino acid with its enantiomer leads to significant conformational changes of the respective peptidolactone ring, resulting in reduced biological activities as well as chemical shift deviations that have not been observed. The absolute configuration of the threonine, valine, methyl valine, proline in Transitmycin (R1) was clarified by Marfey\u0026rsquo;s method\u003csup\u003e\u0026nbsp;\u003c/sup\u003eapplied for the acid hydrolysate of Transitmycin (R1) in comparison with standard amino acid analysis [60-64]. Retention times of the standard N\u003csup\u003e\u0026alpha;\u003c/sup\u003e-(5-fluro-2,4-di-nitrophenyl)-L-alanamide (FDAA) derivatives were as follows: D-threonine, 15.682 min; L-threonine, 13.987 min; D-proline, 17.035 min; L-proline, 16.519 min; D-valine, 21.244 min; L-valine, 19.248 min; D-N-methyl valine, 22.089 min; L-Methyl valine 20.814 min. The chromatogram of the FDAA derivatives of acid hydrolysate Transitmycin (R1) showed peaks corresponding to L-Threonine (14.021 min), L-proline (16.520 min), L-valine (19.252), L-N-methyl valine (21.263 min) were obtained in the hydrolysate. The above same mentioned procedure for compound R3 was also done and similar results was obtained. \u0026nbsp;(Supplementary Information Fig. S116-125, Table S-8a-b) [60-64]. Comparison, with authentic standards, revealed the presence of L-MeVal, L-Thr and D-Val as expected, however the D-Valine is in L-configuration as well as one of the proline is in D-proline instead of L-configuration. (Fig. 6a-c).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLC-MS Analysis of L-FDAA Derivatives of Transitmycin (R1)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe retention times of the D- and L-FDAA derivatives of standard amino acid , respectively, were as follows: Pro: 14.35, 14.97, min, \u003cem\u003em/z\u003c/em\u003e 366.13 [M +H]\u003csup\u003e+\u003c/sup\u003e, \u003cem\u003em/z\u003c/em\u003e 363.07 [M-H]\u003csup\u003e+\u003c/sup\u003e;Val:19.63,17.52 min, \u003cem\u003em/z\u0026nbsp;\u003c/em\u003e370.13 [M+H]\u003csup\u003e+\u003c/sup\u003e, 368.13 [M-H]\u003csup\u003e-\u0026nbsp;\u003c/sup\u003e;Thr: 9.90, 13.50 min, \u003cem\u003em/z\u003c/em\u003e372.07 [M+H]\u003csup\u003e+\u003c/sup\u003e, 370.13 [M \u0026minus; H]\u003csup\u003e-\u003c/sup\u003e; NMeVal: 20.33, 19.23 min, \u003cem\u003em/z\u003c/em\u003e 384.20[M+H]\u003csup\u003e+\u003c/sup\u003e382.20 [M \u0026ndash;H]\u003csup\u003e-\u003c/sup\u003e. The L-FDAA was used to derivatize the acid hydrolysates of Transitmycin (R1), R3 and eight standard amino acids (D-Val, L-Val, D-Thr, L-Thr, D-N-MeVal, L-N-MeVal, D-Pro and L-Pro). The reaction with L-FDAA was performed with the same procedure as above. [63-64].The retention times of the L-FDAA derivatives were as follows: D-Pro:15.16 min, L-Pro:14.31 min, \u003cem\u003em/z\u0026nbsp;\u003c/em\u003e366.13 [M+H]\u003csup\u003e+\u0026nbsp;\u003c/sup\u003e363.07 [M\u0026minus;H]\u003csup\u003e-\u003c/sup\u003e;D-Val: 19.65 min, L-Val 17.65 min \u003cem\u003em/z\u003c/em\u003e 370.13 [M+H]\u003csup\u003e+\u003c/sup\u003e368.07[M\u0026minus;H]\u003csup\u003e\u0026minus;\u003c/sup\u003e; D-Thr: 9.90 min, L-Thr, 13.50 min, \u003cem\u003em/z\u003c/em\u003e 372.13[M+H]\u003csup\u003e+\u003c/sup\u003e370.13 [M\u0026minus;H]\u003csup\u003e-\u003c/sup\u003e; D-N-MeVal 20.33 min, L-N-MeVal 19.25 min, \u003cem\u003em/z\u003c/em\u003e 384.20[M+H]\u003csup\u003e+\u003c/sup\u003e,382.20 [M+H]\u003csup\u003e-\u003c/sup\u003e. The retention time of L-FLDA derivatives of acid hydrolysates Transitmycin (R1) were as follows: L-threonine (13.73 min), L-Proline (14.23 min), D-Proline or keto-proline (15.36 min), L-valine (18.02 min), N-Methyl valine (19.96 min) as illustrated in Fig. 6 a-b. The retention time of L-FLDA derivatives of acid hydrolysates (R3) (Supplementary Information Fig. S-128-161, S-Table S9 a and b)\u0026nbsp;were as follows: to L-threonine (12.45 min) or D-threonine (9.36 min), L-Proline (14.25 min), D-Proline or keto-proline (15.21 min), L-valine (17.49 min), N-Methyl valine (19.93)\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eComparison with authentic standards revealed the presence of L-MeVal, L-Thr, L-Proline, L-Valine and one of the Proline as in D-configuration (Fig. 6c). Hence, we named the unusual newly found compound as Transitmycin (Fig. 3d, Table 1\u0026amp; 2), a member of the X-type [35-64].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003ePhysico-chemical properties of Transitmycin\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003eTransitmycin (R1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eProperties\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTLC\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eSingle yellow spot with R\u003csub\u003ef\u003c/sub\u003e value 0.8 (EtOAc:MeOH (9.5:0.5))\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eColor and consistency\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eOrange color amorphous powder\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eYield\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e200 mg, 20%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMelting point (mp)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e240-242\u003csup\u003eo\u003c/sup\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e[\u0026alpha;]\u003csub\u003eD\u003c/sub\u003e\u003csup\u003e25\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e-106 \u003csup\u003eo\u003c/sup\u003e (c = 0.2, MeOH)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSolubility\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eSoluble in chloroform, dichloromethane, ethyl acetate, methanol, ethanol, acetonitrile, DMSO and water; Insoluble in Hexane\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUV\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e(MeOH) \u0026lambda;\u003csub\u003emax\u003c/sub\u003e (log \u0026epsilon;) 214 (3.07), 240 (2.30), 425 (1.44), 442 (1.51) nm\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e[MeOH, [nm], (mdeg)]: \u0026lambda;\u003csub\u003emax\u003c/sub\u003e(D\u0026epsilon;) 195 (+11.1), 210 (-21.0), 242 (+4.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIR (KBr), \u003cem\u003e\u0026nu;\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e3435, 2958, 2924, 2853, 1745, 1642, 1524, 1465, 1379, 1194, 1099, 1059 cm\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003csup\u003e1\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003eH NMR (500MHz)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eTable 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003csup\u003e13\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003eC NMR (125MHz, CDCl\u003csub\u003e3\u003c/sub\u003e):\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e179.0, 174.0, 173.5, 173.17, 169.0, 168.8, 167.5, 167.5, 166.5, 166.3,166.1, 165.9, 144.34, 145.93, 145.04, 140.5, 132.19, 130.3, 129.2, 127.8, 126.1, 113.6, 101.8, 76.7, 74.76, 74.67, 71.4, 71.2, 58.5, 57.2, 56.4, 54.9, 54.7, 54.3, 51.3, 29.6, 29.6, 29.3, 22.6, 21.7, 21.6, 19.2, 19.2, 19.09, 19.06, 18.8, 17.14, 14.11,7.77.\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHRESI-MS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cem\u003em/z\u003c/em\u003e (pos.ions) \u0026nbsp; \u0026nbsp; \u0026nbsp;656.9243 [M+2H]\u003csup\u003e+2\u0026nbsp;\u003c/sup\u003e, 1270.7069 [M+ H]\u003csup\u003e+\u003c/sup\u003e, \u0026nbsp;1291.8449 [M + Na]\u003csup\u003e+\u003c/sup\u003e1307.9286 [M + K]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003eC\u003csub\u003e62\u003c/sub\u003eH\u003csub\u003e84\u003c/sub\u003eN\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e17\u003c/sub\u003eNa [M + Na]\u003csup\u003e\u0026nbsp;+ \u0026nbsp;\u003c/sup\u003e calc. 1291.5975, found. 1291.8449\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMALDI-TOF-MS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cem\u003em/z\u003c/em\u003e (pos.ions) 1293.61316 [M + Na+2H]\u003csup\u003e+\u003c/sup\u003e, 1309.93062 [M+K]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003em/z\u0026nbsp;\u003c/em\u003e(neg.ions) 1269.33344 [M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003eC\u003csub\u003e62\u003c/sub\u003eH\u003csub\u003e84\u003c/sub\u003eN\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e17\u003c/sub\u003eNa [M+Na+2H]\u003csup\u003e+\u003c/sup\u003e calc. 1293.61950, found. 1291.61316\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEI-MS: (70 ev)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cem\u003em/z\u003c/em\u003e(pos.ions)1348.1437, 1291.4173 [M+Na]\u003csup\u003e+\u003c/sup\u003e, 1224.7363, 1191.8994, 1023.6241, 886.0243, 743.2058, 614.8185, 347.6111, 202.5464, 138.5079\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLCESI-MS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cem\u003em/z\u003c/em\u003e (pos.ions) 1291.5995 [M + Na]\u003csup\u003e+\u003c/sup\u003e C\u003csub\u003e62\u003c/sub\u003eH\u003csub\u003e84\u003c/sub\u003eN\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e17\u003c/sub\u003eNa [M+Na]\u003csup\u003e\u0026nbsp;+\u003c/sup\u003e calc. 1291.5975, found. 1291.5995\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCHN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eAnal. calcd for C\u003csub\u003e62\u003c/sub\u003eH\u003csub\u003e84\u003c/sub\u003eN\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e17\u003c/sub\u003e:\u0026nbsp;C, 58.66; H, 6.67; N, 13.24.\u003c/p\u003e\n \u003cp\u003eFound: C, 59.71; H, 7.28; N, 10.19.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of anti TB and anti-HIV activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eActivity against planktonic cultures of M. tuberculosis\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDespite the introduction of new anti TB drugs, emergence of antibiotic resistance among \u003cem\u003eM. tuberculosis\u0026nbsp;\u003c/em\u003estrains remains a major challenge in tuberculosis therapy. According to WHO Global Tuberculosis report - 2022, there is a 3.1% increase in incidence of multi drug resistant and rifampicin resistant TB cases from 2020 to 2021[65]. Thus, there is a burning need for more new antitubercular drugs to tackle drug resistant tuberculosis [66]. Since 1940s, secondary metabolites and their associated derivatives have played a key role in anti-TB drugs development. This is best exemplified by an extremely active aminoglycoside, namely streptomycin, the first clinical drug that was made available against TB [67]. \u0026nbsp;In a previous study, Streptocytosines A, Bamitecin and Amitecin isolated from sea water \u003cem\u003eStreptomyces\u0026nbsp;\u003c/em\u003ein Japan showed activity against \u003cem\u003eM. smegmatis\u003c/em\u003e at 32, 16 and 8 \u0026micro;g/ml concentrations, respectively [68]. In another study, actinomycin X2 and actinomycin D isolated from marine \u003cem\u003eStreptomyces\u003c/em\u003e sp. MS449 in China showed activity against \u003cem\u003eM. tuberculosis\u003c/em\u003e H37Rv at 1.92mg/ml and 1.77 mg/ml, respectively [69]. In the present study Transitmycin, a novel molecule isolated from a marine \u003cem\u003eStreptomyces\u0026nbsp;\u003c/em\u003esp. R2 picked up from coral reef ecosystem showed activity notably against drug sensitive, multi drug resistant (MDR) and mono resistant strains of \u003cem\u003eM. tuberculosis\u003c/em\u003e at concentrations of 5 and 10 \u0026micro;g/ml (Fig. 7). Based on the preliminary experiment on laboratory strain, \u003cem\u003eM. tuberculosis\u003c/em\u003e H37Rv, minimum inhibitory concentration for transitmycin was determined on clinical isolates which included 49 drug sensitive strains and 48 drug resistant strains. The drug resistant profile of the clinical isolates are given in (supplementary S-table 12). Out of 97 clinical isolates, MIC at 5 \u0026micro;g/ml was seen in 89 isolates and MIC at 10 \u0026micro;g/ml was seen in 8 isolates for transitmycin based on LRP assay.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eActivity against M. tuberculosis biofilm\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacteria can persist for extended periods of time inside the biofilm due to their ability to resist the immune system, display increased virulence and become phenotypically more resistant to antibiotics. Moreover, antibiotic concentrations required to control bacteria within a biofilm are estimated to be 100\u0026ndash;1,000 fold greater than that is needed to treat planktonic forms. As planktonically-grown \u003cem\u003eM. tuberculosis\u003c/em\u003e are unlikely to be entirely representing the bacterial load during human infection, we set out to determine how effective transitmycin can be against \u003cem\u003eM. tuberculosis\u0026nbsp;\u003c/em\u003egrowing as a biofilm, a bacterial phenotype known to be more resistant to antibiotic treatment [70]. In the present study, \u003cem\u003eM. tuberculosis\u003c/em\u003e culture showed biofilm formation in the control wells alone which could be observed by the naked eye. The wells with cells and transitmycin failed to produce biofilm. CFU determined prior to the addition of transitmycin was 1.9 x 10\u003csup\u003e6\u003c/sup\u003e to 2.3 x 10\u003csup\u003e6\u003c/sup\u003e/ml. After 4 days of exposure with the compound, the CFU dropped to 9 x 10\u003csup\u003e4\u0026nbsp;\u003c/sup\u003eto 10 x 10\u003csup\u003e4\u0026nbsp;\u003c/sup\u003eml. Addition of transitmycin completely killed all the cells at the end of 5 weeks (Table 3).\u003c/p\u003e\n\u003cp\u003eTable \u0026ndash; 3. Activity of transitmycin on biofilm formation\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCulture\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 127px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eZero days\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e10\u003csup\u003e5\u003c/sup\u003e x CFU/ml\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAfter 4 days\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e10\u003csup\u003e5\u003c/sup\u003e x CFU/ml\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBiofilm formation after 5 weeks\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cem\u003eM. tuberculosis\u0026nbsp;\u003c/em\u003ealone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e6.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e3.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003ePresent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cem\u003eM. tuberculosis\u003c/em\u003e+ Transitmycin\u0026nbsp;\u003cbr\u003e\u0026nbsp;(10 \u0026micro;g/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e19.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e23.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e0. 9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnti-HIV activity\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe symbiotic association of TB and HIV poses a challenge to human survival in which HIV complicates the treatment and diagnosis of TB. Besides, HIV\u0026ndash;TB patients encounter other unique problems such as cumulative toxicity, immune reconstitution inflammatory syndrome (IRIS), drug-drug interactions, lower plasma drug levels, and the emergence of drug resistance during treatment [71]. The currently available therapy for treating patients co-infected with HIV and TB requires a very high pill load. Therefore, a class of drugs that can be used to treat TB and HIV would be a real breakthrough in TB and AIDS Research. Hence, the present study also evaluated the anti-HIV activity of transitmycin. A dose-dependent reduction was observed in HIV-1 p24 levels in viral culture indicating that transitmycin possessed significant anti-HIV activity. Transitmycin demonstrated good anti-viral activity against the different subtypes of HIV-1 as well as clinical isolates obtained directly from HIV-infected persons. In addition, transitmycin was also active against HIV-1 viruses resistant to nevirapine and AZT (Fig. 8a). The estimated IC\u003csub\u003e50\u003c/sub\u003e value ranged between 0.19 and 0.65\u0026micro;g/ml for the viruses tested. There was a \u0026gt;50% inhibition at a concentration of 0.1\u0026micro;g/ml and 80-95% inhibition at a concentration of1\u0026micro;g/ml in the clinical isolates (Fig. 10b). The compound demonstrated a remarkable inhibitory effect on primary isolates belonging to various subtypes as well as to clinical isolates obtained from HIV-infected individuals. IC\u003csub\u003e50\u003c/sub\u003e values calculated for the various strains tested indicate that transitmycin is a potent inhibitor of HIV-1 under \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eexperimental conditions. Importantly, transitmycin also inhibited drug resistant forms of the virus, in a dose-dependent manner. These findings suggest that transitmycin holds promise as the first potent compound that can be used to treat TB and HIV infections when they occur singly, as well as in combination as HIV/TB co-infection. (Supplementary S-table 13).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eR1, R2 and R3 intercalates with the genomic DNA of Mycobacterium tuberculosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eActinomycin D is the structurally similar compound for R1, R2 and R3. Actinomycin D and its derivatives are reported to intercalate with the DNA and exhibits fluorescence. So, we tested R1, R2 and R3 for its properties of DNA intercalation and fluorescence. Ethidium bromide 0.5 \u0026micro;g/ml is used as a positive control. The relative fluorescence unit (RFU) of R1, R2 and R3 with the DNA is compared with the RFU of R1, R2 and R3 without DNA. The average RFU of Ethidium bromide was 8561429. From the experiment, it was observed that all the three compounds, R1, R2 and R3 have DNA intercalating property and exhibits fluorescence (Fig. 9).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDocking and molecular dynamics studies\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe concept behind docking was to assess the potential interaction between the transitmycin \u0026nbsp; Fig. 10a \u0026nbsp; \u0026nbsp;or its derivative molecules and various DNA/Protein targets of interest. Through docking studies, we aimed to validate the anticipated interactions, even though comprehensive molecular dynamics studies were restricted, particularly for the entire transitmycin or its derivatives. Nevertheless, the chromophore part Fig. 10b molecular dynamics dynamics displayed enhanced stability, indicating promising outcomes in our research\u003c/p\u003e\n\u003cp\u003eWe performed docking and molecular docking experiments with a specific target and three ligands: R1, R2, and R3. Our analysis showed that 1MNV displayed favorable interactions with R2, whereas 3PKE exhibited superior binding with R3 Fig. 10c. The docking score data is presented in Table 4.\u003c/p\u003e\n\u003cp\u003eDocking Score\u003cstrong\u003e:\u003c/strong\u003e docking scores were determined using the knowledge-based iterative scoring functions ITScorePP or ITScorePR. A more negative docking score implies a higher likelihood of a binding model. However, it\u0026apos;s important to note that the score doesn\u0026apos;t represent the true binding affinity, as it hasn\u0026apos;t been calibrated against experimental data.\u003c/p\u003e\n\u003cp\u003eConfidence Score: To gauge the likelihood of binding between two molecules, we introduced a confidence score based on docking scores. The formula for the confidence score is:\u003c/p\u003e\n\u003cp\u003eConfidence_score=1.01.0+e0.02\u0026times;(Docking_Score+150)Confidence_score=1.0+e0.02\u0026times;(Docking_Score+150)1.0\u003c/p\u003e\n\u003cp\u003eIn this context, when the confidence score surpasses 0.7, it suggests a high probability of binding. Scores between 0.5 and 0.7 indicate a possible binding, whereas scores below 0.5 imply an unlikely binding.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLigand RMSD\u003cstrong\u003e:\u003c/strong\u003e Ligand RMSD values were computed by comparing ligands in the docking models with the input or modelled structures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u003c/strong\u003e. The docking score data of specific target (1MNV, 3PKE, 1MO3, 3IU8) and three ligands: R1, R2, and R3\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"624\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eName of the Molecule\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDocking Score\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConfidence Score\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLigand RMSD (A)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e1MNV with R1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003e-489.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003e0.9989\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e22.52\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e1MNV with R2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003e-518.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003e0.9994\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e17.61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e1MNV with R3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003e-532.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003e0.9995\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e25.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e1MO3 with R1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003e-397.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003e0.9929\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e83.36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e1MO3 with R2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003e-424.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003e0.9959\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e83.95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e1MO3 with R3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003e-420.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003e0.9956\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e83.36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e3PKE with R1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003e-391.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003e0.9921\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e52.95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e3PKE with R2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003e-493.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003e0.9990\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e50.96\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e3PKE with R3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003e-503.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003e0.9992\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e57.70\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e3IU8 with R1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003e-337.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003e0.9771\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e37.59\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e3IU8 with R2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003e-418.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003e0.9954\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e47.58\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e3IU8 with R3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003e-437.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003e0.9969\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e50.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n\u003c/table\u003e\n\u003cp\u003eWe conducted Molecular Dynamics (MD) simulations for the complex 1MNV with R2 and R3. Unfortunately, MD simulations for R2 and R3 couldn\u0026apos;t be performed due to the large size of their peptide side chains, preventing accommodation into the minor groove of DNA (1MNV). However, we successfully carried out simulations with the chromophores of R2 and R3. The RMSD of the residues remained within acceptable limits during these simulations.\u003c/p\u003e\n\u003cp\u003eAdditionally, MD simulations were conducted for the complex 3PKE with R3. Similar challenges were encountered with the peptide side chains, leading to simulations being performed with the chromophore, as illustrated in Figure\u0026nbsp;10b. The RMSD values plotted against residues are presented in Figure 10e.\u003c/p\u003e\n\u003cp\u003eThe results of the MD simulations indicate the stability of the protein backbone throughout the simulation period. However, it is noteworthy that the simulations faced computational challenges, primarily due to the size of the peptide side chains. Despite these difficulties, the obtained data provides valuable insights into the behavior of the complexes during the simulations.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch3\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe study commenced on January 15, 2015, and concluded on November 20, 2015. During this period, the screening of the molecule was conducted against clinical isolates that were stored in a de-identified manner. Approval has been expedited for the data collected in this study for the utilization of de-identified stored clinical isolates. Commercial PBMCs were utilized, therefore ethical approval was not necessary.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization and taxonomy of \u003cem\u003eStreptomyces \u003c/em\u003esp. R2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicromorphology of potential \u003cem\u003eStreptomyces\u003c/em\u003e sp. R2 was studied by adopting Transplantation Embedding Technique [72]. Briefly, a rectangular trough was dug out of an ISP2 agar plate using sterile knife. Then the spores of \u003cem\u003eStreptomyces\u003c/em\u003e strain were inoculated on the edges of the trough under aseptic condition. A sterile cover-slip was placed over the agar, touching the inoculated area on the ISP2 agar plate. The plate was incubated at 28°C for 7-14 days. The cover-slip was aseptically removed using sterile forceps and placed over clean microscopic slide fixing the same using cellophane tape. Micromorphology of \u003cem\u003eStreptomyces \u003c/em\u003esp. R2 was observed under bright field microscope (Olympus) under 10x and 40x magnifications. Spore structure and spore surface morphology were recorded using a scanning electron microscope (JEOL model JSM5600LV). Media and procedures used for determination of cultural characteristics and carbon and nitrogen source utilization were those described originally by Shirling and Gottileb [24]. Effect of pH, temperature, NaCl concentration and anaerobic condition were studied using modified ISP2 medium. Antibiotic susceptibility pattern was determined by disc diffusion method using standard antibiotic discs (Hi media) following the standard protocol [73]. Biomass for cell wall analysis was prepared by growing \u003cem\u003eStreptomyces\u003c/em\u003e sp. R2 in shake flasks (120 rpm) containing ISP2 broth at 28 \u003csup\u003e0\u003c/sup\u003eC for 5 days. Amino acid and sugar content analyses of whole cell hydrolysates were performed according to the original procedure described by Staneck and Roberts [74].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular characterization and phylogenetic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStreptomyces\u003c/em\u003e sp. R2 was grown in 50 ml of ISP2 broth at 28\u003csup\u003e°\u003c/sup\u003eC for 48 h. The genomic DNA was extracted using Chromous genomic DNA isolation kit. Polymerase chain reaction (PCR) was performed for the amplification of 16S rRNA gene using the primers 5’ – AGAGTRTGATCMTYGCTWAC – 3’ and 5’ – CGYTAMCTTWTTACGRCT – 3’ on a ABI12720 thermal cycler (Applied Biosystems). The conditions used for thermal cycling were as follows: initial denaturation at 94\u003csup\u003e0\u003c/sup\u003eC for 4 min, followed by 35 cycles consisting of denaturation at 94 \u003csup\u003e0\u003c/sup\u003eC for 30 sec, primer annealing at 55\u003csup\u003e0\u003c/sup\u003eC for 30 sec and primer extension at 72\u003csup\u003e0\u003c/sup\u003eC for 2 min, followed by a final extension at 72\u003csup\u003e0\u003c/sup\u003eC for 5 min. The amplified 16s rRNA gene fragment (~ 1.4 kb) was separated by agarose gel electrophoresis and the purified fragment was used for sequencing in an ABI3130 genetic analyser. The nearly complete 16s rRNA gene sequence of strain R2 (1400 nt) was subjected to BLAST comparison against the 16s rRNA sequences given in GenBank/DDBJ/EMBL databases.\u003c/p\u003e\n\u003cp\u003ePhylogenetic analysis was performed using CLUSTAL-W and MEGA version 3.1. Evolutionary distances (Kimura’s two parameter model) [75] and clustering were calculated employing the neighbour-joining method. The topology of phylogenetic tree was evaluated by the bootstrap re-sampling method with 1000 replicates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProduction of Transitmycin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHundred microliters of \u003cem\u003eStreptomyces \u003c/em\u003esp. R2 spore suspension was transferred into 10 YEME agar plates and spread using sterile L- rods. The plates were incubated at 28\u003csup\u003eº\u003c/sup\u003eC for 10 days. After every 24 hours of fermentation, the mycelial growth was scrapped out and the crude pigment secreted into the agar medium was extracted using equal volume (1:1) of different organic solvents such as n-hexane, dichloromethane, chloroform, ethyl acetate and methanol for 24 hours. The solvent portion was collected and dried at 40\u003csup\u003eº\u003c/sup\u003eC using Concentrator plus (Eppendorf) [76]. Anti TB activity of crude extracts were tested against \u003cem\u003eM. tuberculosis\u003c/em\u003e H37Rv at 100µg concentration adopting LRP assay [77].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStreptomyces\u003c/em\u003e sp. R2 was cultured for 10 days at 28\u003csup\u003e0\u003c/sup\u003eC on YEME agar plates (2000 ml of medium in 100 petriplates of 90 mm diameter) to produce the culture extract in bulk. After the incubation period, the cell material was aseptically removed and discarded after autoclaving. The yellow pigmented antibiotic containing agar medium was cut in to pieces and extracted twice with equal volume (1:1 ratio) of ethyl acetate for 24 hours. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePurification of transitmycin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransitmycin was purified by preparative thin layer chromatography (TLC) using Merck silica gel 60 (GF254) pre coated aluminium (6x8 cm size) plates. The extract was separated using different solvents in different proportions. After running, the 200 sheets were kept at room temperature for complete drying of the plate. Spots on TLC were detected through naked eye as well as under UV light (254 and 365 nm). After drying, three major yellow colour spots (R1, R2 and R3) were scrapped, mixed with ethyl acetate and filtered using a funnel fitted with Whatman filter paper. Ethyl acetate was evaporated to dryness under vacuum to obtain the compounds as dry amorphous powder. (Supplementary Fig. S2).\u003c/p\u003e\n\u003cp\u003eAll three compounds were tested against \u003cem\u003eM. tuberculosis\u003c/em\u003e H37Rv at 100 µg concentration by LRP assay. All three compounds from the \u003cstrong\u003eethyl acetate extract was purified using column chromatography packed with neutral alumina \u003c/strong\u003eusing a gradient of 1%\u003cstrong\u003e methanol/chloroform mixture (CH\u003csub\u003e3\u003c/sub\u003eOH/ CHCl\u003csub\u003e3\u003c/sub\u003e) as the eluent. \u003c/strong\u003eFractions were collected and concentrated under vacuum to obtain pure transitmycin. \u003cstrong\u003eThe product was visualized in a silica gel coated TLC sheet\u003c/strong\u003e\u003cstrong\u003e (\u003c/strong\u003eSupplementary Fig. S2 and S3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe compound R1 (named as transitmycin) that showed maximum activity was taken for characterization and other studies testing its biological activity. \u003c/strong\u003ePurity of transitmycin (Fig. 2c). was analysed by HPLC using Shimadzu (Japan) RID-10A gradient high-performance liquid chromatographic instrument, equipped with two LC-20AD pumps controlled by a CBM-10 inter-face module. Refractive index Detector RID 10A (Shimadzu) was used for the peak. Analysis was performed on a Luna 5u C\u003csub\u003e18\u003c/sub\u003e (2) reversed-phase column, 100 (150X4.6mm). The analytical parameters were selected after screening a number of solvent systems and gradient profiles. Separation was achieved using a two-pump gradient program for pump A (0.1% Acetic acid in CH\u003csub\u003e3\u003c/sub\u003eCN) and pump B (0.1% Acetic acid in H\u003csub\u003e2\u003c/sub\u003eO) in a linear gradient of acetonitrile and water from 0:100 to 65:35 in 65 minutes at a flow rate of 2 ml/min. Detection was done at 254 nm, the absorption maxima close to that of majority of the compounds. Injection size for sample was 20 µl. Column temperature was 30°C.\u003cstrong\u003e(\u003c/strong\u003eSupplementary Fig. S4 and S5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization and structure elucidation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eColour and consistency of the purified antibiotic was visually observed. Solubility was tested in water, methanol, acetone, ethyl acetate, diethyl ether, dichloromethane, chloroform, and n-hexane by dissolving 1 mg of purified antibiotic. Optical rotations were measured with a Autopol IV Automatic polarimeter, and the [α]\u003csub\u003eD\u003c/sub\u003e values are given in deg cm\u003csup\u003e2\u003c/sup\u003e g\u003csup\u003e-1\u003c/sup\u003e. Melting point was analysed using Mettler Toledo Model FP62 [78]. Ultraviolet (UV) spectrum was determined using Shimadzu UV-1700 series. One milligram of sample was dissolved in 10ml of methanol and the spectra were recorded at wavelength between 190 – 900 nm. The Infrared (IR) spectrum of the purified antibiotic was determined on Perklin Elmer Spectrum One FT-IR. The spectrum was obtained using potassium bromide (KBr) pellet technique in the range of 450 to 4000 cm\u003csup\u003e-1\u003c/sup\u003e at a resolution of 1.0 cm\u003csup\u003e-1\u003c/sup\u003e. Potassium bromide (AR grade) was dried under vacuum at 100\u003csup\u003eo\u003c/sup\u003eC and 100 mg of KBr with 1mg of purified antibiotic was used to prepare KBr pellet. The spectrum was plotted as intensity versus wave number [79]. \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR spectra were recorded on a Bruker Advance 500 NMR spectrometer in CDCl\u003csub\u003e3\u003c/sub\u003e with TMS as internal Standard and with chemical shifts (\u003cem\u003eδ\u003c/em\u003e) reported in ppm. Two-dimensional \u003csup\u003e1\u003c/sup\u003eH–\u003csup\u003e1\u003c/sup\u003eH COSY, DQF-COSY, NOESY, ROESY, \u003csup\u003e1\u003c/sup\u003eH–\u003csup\u003e13\u003c/sup\u003eC HSQC, HMBC, and spectra were recorded on a Bruker Advance 500 NMR spectrometer. MALDI-TOF MS analyses were performed using an Applied Biosystems ABI4700 TOF mass spectrometer in reflector mode with an accelerating voltage of 20 kV. HRESIMS were measured on a Q-TOF micro mass spectrometer (Waters USA) in positive ion mode with methanol as solvent. QTOF- MS was recorded on an Agilent 6520-QTOF LCMS having an ESI source in positive mode. \u003c/p\u003e\n\u003cp\u003eHPLC \u003cstrong\u003eAnalysis of L-FDAA Derivatives of Transitmycin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransitmycin (3.0 mg) was dissolved in 1 ml of 6NHCl and heated in a sealed glass tube at 110\u003csup\u003eº\u003c/sup\u003eC for 24 h. After removing the solvents, the hydrolysate mixture (3 mg) and the amino acid standards (0.5 mg) were separately dissolved in 0.1 mL of water and treated with 0.2 mL of 1% 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide (FDAA) (Marfey’s reagent) in acetone (10 mg/mL in acetone) and 0.04 mL of 1.0 M sodium bicarbonate. The vials were heated at 50\u003csup\u003eº\u003c/sup\u003eC for 90 min, and the contents after cooling at room temperature were neutralized with 1N HCl. After degassing, an aliquot of the FDAA derivative was diluted in CH\u003csub\u003e3\u003c/sub\u003eCN, Water (1:1) and analysed by reversed phase HPLC column Luna 5u C\u003csub\u003e18\u003c/sub\u003e (2) 100 (150X4.6mm) and a linear gradient of acetonitrile and water containing 0.05% trifluoroacetic acid from 10:90 to 50:50 in 20 min and then isocratic. The flow rate was adjusted to 1 mL/min and the absorbance detection was at 340 nm. The chromatogram was compared with those of amino acid standards treated in the same conditions [60-61].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLC-MS Analysis of L-FDAA Derivatives of Transitmycin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe analysis of the L- and D-FDLA derivatives of Transitmycin was performed on a Waters Acquity UPLC coupled with a Thermo LCQ Deca XP MAX\u003cstrong\u003e. \u003c/strong\u003eQTOF- MS was recorded on an Agilent 6520-QTOF LCMS having a ESI source in Positive mode and employing a linear gradient of from 25% to 70% CH\u003csub\u003e3\u003c/sub\u003eCN in 0.01 M formic acid at 0.5 mL/min over 60 min [62-64]. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransitmycin (R1).\u003c/strong\u003e R1 was obtained as an orange red solid; [α]\u003csub\u003eD\u003c/sub\u003e\u003csup\u003e25\u003c/sup\u003e -106 \u003csup\u003eo\u003c/sup\u003e (c 0.2, MeOH); UV (MeOH) λ \u003csub\u003emax\u003c/sub\u003e (log ε) 214 (3.07), 240 (2.30), 425 (1.44), 442 (1.51) nm; CD [(MeOH),(mdeg)] λ\u003csub\u003emax\u003c/sub\u003e(Dε) 195 (+11.1), 210 (-21.0), 242 (+4.7) nm; IR (KBr), \u003cem\u003eν\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e 3435, 2958, 2924, 2853, 1745, 1642, 1524, 1465, 1379, 1194, 1099, 1059 cm\u003csup\u003e-1\u003c/sup\u003e; HRESI-MS (pos.ions): \u003cem\u003em/z \u003c/em\u003e1270.7069 [M+2H]\u003csup\u003e+\u003c/sup\u003e,1291.8449 [M+Na]\u003csup\u003e+\u003c/sup\u003e, 1307.9286 [M+K]\u003csup\u003e+\u003c/sup\u003e657.3119 [M+2H]\u003csup\u003e+2\u003c/sup\u003e; MALDI-TOF-MS (pos.ions):\u003cem\u003em/z\u003c/em\u003e 1293.07095 [M+Na+2H]\u003csup\u003e+\u003c/sup\u003e, 1309.93062 [M+K]\u003csup\u003e+ \u003c/sup\u003eMALDI-TOF-MS (neg.ions) 1269.33344 [M-H]\u003csup\u003e-\u003c/sup\u003e \u003csup\u003e1\u003c/sup\u003eHNMR, \u003csup\u003e13\u003c/sup\u003eNMR and 2DNMR details (Supplementary Fig. S14-52, S-Table 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompound (R2).\u003c/strong\u003e R2 was obtained as a red solid; [α]\u003csub\u003eD\u003c/sub\u003e\u003csup\u003e25\u003c/sup\u003e: -24\u003csup\u003eo\u003c/sup\u003e (c 0.2, MeOH); UV(MeOH) λ \u003csub\u003emax,\u003c/sub\u003e (log ε) 205(1.25),240(0.63),425 (0.39), 442 (0.42) nm; CD [MeOH, (mdeg)] λ\u003csub\u003emax, \u003c/sub\u003e(Dε) 195 (+8.8), 210 (-22.0), 240 (+4.3) nm;IR (KBr), \u003cem\u003eν\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e3436, 2961, 2924, 2853, 1744, 1650, 1565, 1415, 1204, 1140, 1045, 1019 cm\u003csup\u003e-1\u003c/sup\u003e; HRESI-MS (pos. ions) :1277.8245 [M + Na]\u003csup\u003e+\u003c/sup\u003e1293.8735 [M + K]\u003csup\u003e+\u003c/sup\u003e\u003cem\u003em/z\u003c/em\u003e 650.3413 [M+2H]\u003csup\u003e+2\u003c/sup\u003e; MALDI-TOF-MS \u003cem\u003em/z\u003c/em\u003e (pos.ions) 1278.95175 [M+Na+H]\u003csup\u003e+\u003c/sup\u003e; MALDI-TOF-MS (neg.ions) \u003cem\u003em/z\u003c/em\u003e 1255.38052 [M-H]\u003csup\u003e-\u003c/sup\u003e\u003cem\u003e, \u003c/em\u003e\u003csup\u003e1\u003c/sup\u003eHNMR, \u003csup\u003e13\u003c/sup\u003eNMR and 2D NMR details (Supplementary Fig. S53-63).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompound (R3\u003c/strong\u003e)\u003cstrong\u003e: \u003c/strong\u003eR3 was obtained as orange solid; [α]\u003csub\u003eD\u003c/sub\u003e\u003csup\u003e25\u003c/sup\u003e:-27 \u003csup\u003eo\u003c/sup\u003e (c 0.2, MeOH); UV (MeOH) λ \u003csub\u003emax\u003c/sub\u003e (log ε) 206 (1.90), 240 (0.69) 424 (0.191), 442.2 (0.19) nm; CD [MeOH, (mdeg) ] λ\u003csub\u003emax \u003c/sub\u003e(Dε) 195 (+24.0), 210 (-21.5), 241 (+1.7) nm; IR (KBr), \u003cem\u003eν\u003c/em\u003e\u003csub\u003emax \u003c/sub\u003e3415, 2957, 2924, 2853, 1745, 1642, 1583, 1464, 1384, 1193, 1093, 1078 cm\u003csup\u003e-1\u003c/sup\u003e; HRESI-MS (pos.ions):1271.7159 [M +H]\u003csup\u003e+\u003c/sup\u003e\u003cem\u003e m/z\u003c/em\u003e1277.6149 [M-OH+Na]\u003csup\u003e+\u003c/sup\u003e; MALDI-TOF-MS (pos.ions) \u003cem\u003em/z\u003c/em\u003e 1294.5888 [M + Na]\u003csup\u003e+\u003c/sup\u003e MALDI-TOF-MS (neg. ions) \u003cem\u003em/z\u003c/em\u003e 1268.48582 [M-H]\u003csup\u003e-\u003c/sup\u003e, \u003csup\u003e1\u003c/sup\u003eHNMR, \u003csup\u003e13C\u003c/sup\u003eNMR and 2D NMR details, (Supplementary Fig. S64-69).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of anti TB and anti-HIV activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnti-TB activity was determined by adopting Luciferase Reporter Phage (LRP) assay against the standard laboratory strain, \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e H\u003csub\u003e37\u003c/sub\u003eRv, and 97 clinical \u003cem\u003eM. tuberculosis \u003c/em\u003eisolates including drug sensitive and cultures exhibiting different drug resistant patterns. Different concentrations (5 – 50 µg/ml) of purified antibiotic – transitmycin were prepared using 10% dimethyl sulfoxide (DMSO). About 50µl of antibiotic solution was added to 350 µl of glycerol 7H9 broth in cryo vials. Effect of DMSO was also tested by adding 50 µl of 10% DMSO instead of the antibiotic. Mycobacterial cell suspension equivalent of 2 McFarland units was prepared from log phase culture and 100 µl of the same was added to all the vials before incubating at 37°C for 72 hours. After incubation, 50 µl of high titre luciferase reporter phage phAE129 and 40 µl of 0.1M CaCl2 were added to test and control vials. All the vials were incubated at 37°C for 4 hours. After incubation 100 µl of suspension from each vial was transferred to a luminometer cuvette. 100 µL of D-luciferin was added and relative light unit (RLU) was measured in a luminometer [77]. Percentage of inhibition was calculated using the RLU of control and test and MIC was determined. [81,82]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eActivity against \u003cem\u003eM. tuberculosis\u003c/em\u003e biofilm\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell suspensions of \u003cem\u003eM. \u003c/em\u003e\u003cem\u003etuberculosis\u003c/em\u003e H37Rv were prepared using 7H9 broth. Biofilms of \u003cem\u003eM. tuberculosis\u003c/em\u003e were developed on 6 well tissue culture plates by adding 2 ml of Sautons medium (without Tween 80) and inoculating 20 μL of saturated planktonic culture of \u003cem\u003eM. \u003c/em\u003e\u003cem\u003etuberculosis\u003c/em\u003e H\u003csub\u003e37\u003c/sub\u003eRv. The plate was wrapped with parafilm and incubated without shaking at 37°C in humidified conditions for 7 to 14 days. The plate was observed regularly after 7 days for biofilm formation by \u003cem\u003eM. \u003c/em\u003e\u003cem\u003etuberculosis\u003c/em\u003e which can be visibly seen. When the biofilm was formed, 10 μg/ml of transitmycin was added to the transitmycin test wells leaving the controls and considered as zero day. The viable counts of tubercle bacilli were determined from the wells on zero day, 4\u003csup\u003eth\u003c/sup\u003e day and after 5 weeks and expressed in cfu/ml [80].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnti-HIV activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eViruses: \u003c/strong\u003eHIV-1 viruses belonging to subtypes A, B, C, D, E and A/C (Subtype A: 92RW020, Subtype B: JR-FL, Subtype C: 92BR025, Subtype D: 92UG001, Subtype E: 92TH021 and Subtype A/C: 92RW009), were obtained from the NIH AIDS Repository (Germantown, MD, USA). Clinical isolates of HIV-1 were produced in our laboratory by co-culture of HIV-infected peripheral blood mononuclear cells (PBMC) with activated donor PBMC. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTesting for anti-HIV activity \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnti-HIV activity of Transitmycin was determined using the HIV-1 gag p24 inhibition assay. Initially, testing was performed on a lab-adapted HIV-1 subtype B isolate, HIV-1 IIIB. Donor PBMC were obtained from healthy volunteers after obtaining the approval of the Institutional Ethics Committee of the National Institute for Research in Tuberculosis as well as the informed consent of the participants. Donor PBMC were stimulated with PHA (Phytohemeagglutinin) for 72 hours and incubated with 100 TCID\u003csub\u003e50\u003c/sub\u003e of the virus per 1 x 10\u003csup\u003e6\u003c/sup\u003e cells for 4 h at 37°C. The cells were washed to remove the un-adsorbed virus and plated at a concentration of 10,000 cells/well in a 96-well tissue culture plate. Varying concentrations of Transitmycin (0.01µg/ml, 0.1µg/ml, 1.0µg/ml and 5.0µg/ml) were added to triplicate wells. Control cultures were set up without the addition of the compound. AZT was used as the reference compound. Cultures were maintained for 7 days at 37°C in a CO\u003csub\u003e2 \u003c/sub\u003eincubator. On day 7, HIV-1 gag p24 antigen production was determined as an indirect measure of viral replication in the culture supernatants using the Alliance HIV-1 p24 ELISA kit (Perkin Elmer, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTesting of Transitmycin against different HIV-1 subtypes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnti-viral activity of Transitmycin was also tested on primary HIV-1 isolates belonging to different subtypes - Subtype A: 92RW020, Subtype B: JR-FL, Subtype C: 92BR025, Subtype D: 92UG001, Subtype E: 92TH021 and Subtype A/C: 92RW009, as well as a Nevirapine resistant and AZT resistant strain, using the method described above. The activity of Transitmycin on different viruses was determined by measuring HIV-1 p24 antigen in 7-day culture supernatants. The IC\u003csub\u003e50 \u003c/sub\u003evalue (concentration of compound required to inhibit 50% of virus replication) of Transitmysin for the different HIV- 1 subtypes was calculated by fitting a dose response curve using a non-linear regression analysis to generate a sigmoidal three parameter dose response curve (GraphPad Prism, version 6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eActivity against clinical strains of HIV\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnti-viral activity of Transitmycin was further evaluated on 20 clinical isolates of HIV-1 obtained by co-culture of patient PBMC with PHA-stimulated donor PBMC in the laboratory. For this analysis, only three concentrations (0.01 µg/ml, 0.1 µg/ml and 1µg/ml) of transitmycin was used based on the results of the above experiment. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA binding and Fluorescence assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e0.5 µg/ml, 1 µg/ml, 2 µg/ml and 5 µg/ml of R1, R2 and R3 were separately added to 100 µl of PBS in triplicates in 96 well plate. \u003cem\u003e Mycobacterium tuberculosis\u003c/em\u003e genomic DNA was extracted and 200 ng of DNA is added to each test wells. DNA was not added to the control wells of R1, R2 and R3. For the positive control, 0.5 µg/ml of Ethidium bromide is taken in PBS and 200 ng of DNA is added to it. After 15 mins, the plate is read in Multimode - plate reader, Spinco Biotech, with excitation 546 nm and emission 595 nm. The relative fluorescence unit is recorded. Average of RFU for each concentration of R1, R2 and R3 was taken and plotted as graph.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eGlobally, the general public has latent or active tuberculosis, and this is expected to increase in the future, causing the medical system a major challenge. The need of the hour is to identify novel antibiotics to support the current regimens with reduced side effects, duration and cost of treatment. Transitmycin is a novel antibiotic isolated from a novel \u003cem\u003eStreptomyces\u003c/em\u003e sp. MTCC from the coral reef soil from Rameshwaram waters in India. It has the unique property of killing latent and active forms of TB bacilli irrespective of the resistance they have towards major antiTB drugs and also sterilising the different resistant and recombinant clades of standard and clinical HIV virus. When brought into the market for human use, transitmycin can strategize treating TB and HIV simultaneously, which could be a major breakthrough, none the less. Of course, preclinical trials and toxicity studies need to be performed before it can be tested on human volunteers which will require sufficient resources. Countries which are burdened with this dual ailment may have to take considerable interest in this direction. Structural medications to the parent compound can further improve its activity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProfessor\u0026nbsp;Balasubramanian Kalpattu Kuppusami, INSA Senior Scientist, Department of Chemistry, IIT Madras Chennai 600 036, India and Professor Krishna Kumari Gadepalli Narasi, Department of Medicinal Chemistry, Sri Ramachandra University, Chennai 600 116, India, acknowledged for discussion of structure elucidation of Transitmycin. Technical assistance of Dr. S. Balaji and Dr. A. S. Shainaba of National Institute for Research in Tuberculosis is acknowledged in fine tuning and uploading the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe comprehensive characterization of the anticipated products \u0026nbsp;have been subjected through UV/Vis, IR, CD, CHNS analyses, whilst, followed by \u003csup\u003e1\u003c/sup\u003eH-NMR \u003csup\u003e13\u003c/sup\u003eC-NMR, \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH COSY, \u003csup\u003e1\u003c/sup\u003eH−\u003csup\u003e1\u003c/sup\u003eH DQF-COSY, \u003csup\u003e1\u003c/sup\u003eH−\u003csup\u003e13\u003c/sup\u003eC HMBC, and \u003csup\u003e1\u003c/sup\u003eH−\u003csup\u003e13\u003c/sup\u003eC HSQC, \u003csup\u003e1\u003c/sup\u003eH−\u003csup\u003e1\u003c/sup\u003eH TOCSY, \u003csup\u003e1\u003c/sup\u003eH−\u003csup\u003e1\u003c/sup\u003eH NOESY, \u003csup\u003e1\u003c/sup\u003eH−\u003csup\u003e1\u003c/sup\u003eH NOESY 2D NMR spectra and MALDI-TOF-MS, HR-ESIMS, HR-LCMS, ESI-MS, QTRAP LC-MS/MS, RP-HPLC and LCMS analyses respectively. \u0026nbsp;The following L-FDAA derivatives of R1, R2, R3 spectral information on all compounds of this article can be found in the online version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Received for this study\u003c/strong\u003e\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003ePotential Tuberculosis Drugs from Marine Actinomycetes, Funded by Department of Science and Technology, New Delhi (November, 2007 to November, 2009).\u003c/li\u003e\n \u003cli\u003eStudy to evaluate the baseline anti TB and anti-HIV properties of novel antibiotic transitmycin (Tr) isolated from novel \u003cem\u003eStreptomyces\u003c/em\u003e Sp. R2 funded by Indian Council of medical Research (2009-2012).\u003c/li\u003e\n \u003cli\u003ePurity and in vitro efficacy studies on transitmycin funded by Indian Council of Medical Research (2015-2016).\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ individual contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVanaja Kumar – Principal Investigator, planning, supervision, coordination and data compilation, manuscript writing and editing.\u003c/p\u003e\n\u003cp\u003eBalagurunthan Ramasamy–Facilitating Growing of producer strain, coordination in crude extract preparation as lab head.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMukesh Doble – Purification of compound from crude extract, structure analysis and chemical characterisation and report preparation as lab head.\u003c/p\u003e\n\u003cp\u003eGandarvakottai Senthilkumar Arumugam – Isolation, purification, characterization, complete structural elucidation and a key role in Innovation Of Transitmycin drug discovery development and manuscript writing and editing.\u003c/p\u003e\n\u003cp\u003eKannan Damodharan - Isolation, purification, characterization, structural elucidation of Transitmycin compounds and final manuscript drafting.\u003c/p\u003e\n\u003cp\u003eRadhakrishnan Manikkam- Isolation and characterisation of producer strain, biological and biochemical characterisation, testing biological activities, data compilation, manuscript writing.\u003c/p\u003e\n\u003cp\u003eHanna Luke Elizabeth - Testing crude extract and purified compound on HIV standard and clinical clades. Preparation of write up.\u003c/p\u003e\n\u003cp\u003eK R Uma devi-Investigator, supervision, coordination and data compilation, manuscript writing and editing.\u003c/p\u003e\n\u003cp\u003eSuresh Ganesan – Isolation, purification, characterization, structural elucidation of Transitmycin compounds and final manuscript drafting\u003c/p\u003e\n\u003cp\u003eAzger Dusthackeer- Testing biological activities against latent bacilli and biofilms, preparing the write up.\u003c/p\u003e\n\u003cp\u003ePrecilla Lucia – Laboratory work for testing the compound on HIV clades.\u003c/p\u003e\n\u003cp\u003eShainaba A Saadhali-\u0026nbsp;Testing biological activities against latent bacilli and biofilms, preparing the write up.\u003c/p\u003e\n\u003cp\u003eShanthi John – laboratory work with respect to growing the producer strain and preparation of crude extract.\u003c/p\u003e\n\u003cp\u003ePoongothai Eswaran - laboratory work with respect to growing the producer strain and preparation of crude extract.\u003c/p\u003e\n\u003cp\u003eJaleel UCA-Provided the overall conceptualization of theoretical study, and offered guidance throughout the research process and reviewed and edited the manuscript to ensure clarity, consistency, and scientific accuracy.\u003c/p\u003e\n\u003cp\u003eRakhila M-Conducted the data analysis, molecular docking simulations, and statistical interpretation of the results and edited the manuscript to ensure clarity, consistency, and scientific accuracy.\u003c/p\u003e\n\u003cp\u003eAyisha Safeeda- Conducted the data analysis, molecular docking simulations, and statistical interpretation of the results and edited the manuscript to ensure clarity, consistency, and scientific accuracy.\u003c/p\u003e\n\u003cp\u003eSatheesh S -was responsible for creating and editing the figures and tables that visually represented the key findings.\u003c/p\u003e\n\u003cp\u003eSelvakumar Nagamiah– supervision of laboratory work on testing biological activities, editing manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number: Not applicable.\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbdelmohsen UR, Potential of marine natural products against drug-resistant fungal, viral, and parasitic infections. \u003cem\u003eLancet Infect. 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Microbiol\u003c/em\u003e\u003cem\u003e.2008;\u003c/em\u003e69\u003cstrong\u003e:\u003c/strong\u003e164–174.\u003c/li\u003e\n \u003cli\u003eArumugam, G.S., \u003cem\u003eet al.\u003c/em\u003e Significant perspectives on various viral infections targeted antiviral drugs and vaccines including COVID-19 pandemicity. \u003cem\u003eMol Biomed\u003c/em\u003e\u003cstrong\u003e3\u003c/strong\u003e, 21 (2022). https://doi.org/10.1186/s43556-022-00078-z\u003c/li\u003e\n \u003cli\u003eMondal R, et al. (2023) In-vivo studies on Transitmycin, a potent \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e inhibitor. PLoS ONE 18(3): e0282454. https://doi.org/10.1371/journal.pone.0282454.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Antibiotic, Streptomyces sp., Actinomycin, Transitmycin, Tuberculosis, HIV","lastPublishedDoi":"10.21203/rs.3.rs-5801746/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5801746/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHIV is identified as a factor that aggravates tuberculosis disease pathogenesis and its progression to latent TB. While, TB is declared as one of the major causes for AIDS-associated mortality. So there is a dire need for new drugs to combat such ailments that have a synergistic interaction.This has led us to study a novel antibiotic purified from a marine Streptomyces sp isolated from the coral reef ecosystem of South Indian coast. Streptomyces sp. R2 (MTCC 5597; DSM 26035)., isolated from the marine water was grown on agar plates and the crude yellowish orange pigment secreted was extracted using various solvents. The antibiotic, named as Transitmycin, was purified and tested against M. tuberculosis, drug resistant strains, and M. tuberculosis biofilm. The compound was also tested against HIV-1 viruses belonging to six subtypes. Several characterisation tools were used to elucidate the structure of this novel antibiotic. Transitmycin was derivitaised to elucidate the absolute configurations of the amino acids present in it. Tr, unlike actinomycin D, has L-valine in both the rings instead of D-valine (found in the latter). Also, one of the proline in Tr is in D\u0026ndash;configuration while it is in L configuration in actinomycin D suggesting that ours is a novel compound and is not reported so far. It exhibits dual activities against the standard H37Rv, 49 drug sensitive clinical isolates, and MtB biofilm as well as standard and 20 clinical isolates of HIV. This is the first paper that reports the isolation of a new antibiotic from marine actinobacteria exhibiting unusual anti-TB and HIV activities which could be exploited further as a lead molecule in the quest for the design of drug with dual activities.\u003c/p\u003e","manuscriptTitle":"Discovery of a novel antibiotic, Transitmycin, from Streptomyces sp unveils highly efficient activities against tuberculosis and human immunodeficiency virus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-28 14:30:54","doi":"10.21203/rs.3.rs-5801746/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d46ccdae-b7b7-4a88-8e76-3227f0543dce","owner":[],"postedDate":"January 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-03T14:53:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-28 14:30:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5801746","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5801746","identity":"rs-5801746","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}