{"paper_id":"421e0f33-e92d-45ba-afcb-a9b58ff32902","body_text":"1 \n \nControlled Delivery and Light-Induced \nRelease of Magic Spot Nucleotides in \nEscherichia coli \nChristoph Popp 1, Patrick Moser 1, Xinwei Liu 2, Johannes Freitag 2, Isabel Prucker 1, Robert \nZscherp3, Xuan Wang1, Philipp Klahn3, Hans-Georg Koch4, Gert Bange2, Henning J. Jessen1,* \n1 Faculty of Chemistry and Pharmacy , Institute of Organic Chemistry  & CIBSS, Centre for \nIntegrative Biological Signalling Studies,  Albert-Ludwigs Universität Freiburg, 79104 \nFreiburg, Germany  \n2 Center for Synthetic Microbiology (SYNMIKRO), Marburg University, Marburg, Germany  \n3 Department of Chemistry and Molecular Biology , Division of Organic and Medicinal \nChemistry, University of Gothenburg, 413 90 Gothenburg, Sweden \n4 Faculty of Medicine, Institute for Biochemistry and Molecular Biology, ZBMZ, Albert -\nLudwigs Universität Freiburg, 79104 Freiburg, Germany  \n*corresponding author: henning.jessen@oc.uni -freiburg.de \nAbstract  \nThe “m agic spot nucleotides ” (MSNs) ppGpp and pppGpp (also: (p)ppGpp)  are bacterial \nalarmones central to the conserved stringent response, a stress adaptation mechanism that \nhelps bacteria adapt to stress conditions and hostile environments. Current strategies to \nmanipulate MSN levels rely mainly on genetic or environmental approaches, which are slow \nand lack temporal control. Chemical tools such as photocaged MSN analogues could provide \nsuch temporal control over MSN levels. However, the high negative charge of MSNs prevents \nspontaneous passage through the complex bacterial cell envelope.  Here, we report the \nsynthesis of photocaged, clickable, and isotope -labeled MSN analogues and their delivery \ninto Escherichia coli  comparing different approaches . A cyclodextrin -based synthetic \nnucleotide transporter provides particular advantages.  Upon 400 nm irradiation, these probes \nwere photo -released inside living cells, where we tracked their conversion from pppGpp to \nppGpp by capillary electrophoresis mass spectrometr y and studied their ability to alter growth \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint \n\n2 \n \nin a (p)ppGpp⁰ mutant.  This work provides the first demonstration that highly charged, \nphotocaged MSNs can traverse the bacterial envelope, be photo -released intracellularly, and \nbe metabolically tracked in real time. These probes lay the foundation for spatially and \ntemporally controlled studies of MSN function and of other highly negatively charged \nmetabolites in bacteria.  \nIntroduction  \nWhen bacteria are exposed to stress conditions like temperature change, pH change, \nstarvation, or antibiotics, alarmones like the magic spot nucleotides (MSN s) guanosine \ntetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) accumulate  and induce the \nstringent response  (Figure 1A).[1–5] MSNs help bacteria survive by inducing a dormant -like \nstate until conditions  improve. The extent of stringency is a function of MSN concentration.  \nBeyond stress adaptation, MSNs also act as second messengers during normal growth, where \nbasal levels are essential for regulating transcription, translation, replication, and metabolism \nthrough a wide network of protein interactions .[4,6] \nTo study the concentration-dependent role of MSNs in cellular processes it is useful to be \nable to manipulate their abundance. Currently this can be achieved by genetic modifications, \ntreatment with small molecule drugs or exposure to stress conditions. [7–9] Additionally, it \nwould be highly desirable to study effects of local, constrained pools of these molecules. \nGenetically m odifying organisms is time consuming and often causes secondary effects. In \naddition, MSN-producing enzymes can synthesize both pppGpp and ppGpp , making it \ndifficult to distinguish between different MSN s. To influence concentration levels of single \nMSNs further genetic modifications are needed. [10] An alternative chemical biology approach  \nfor modulation of MSN levels inside bacteria would provide new avenues for complementary \nexperiments and thus enable further  investigations into  the effects of MSN s on bacterial \nphysiology. The aim of this study is to deliver photocaged MSNs  into bacteria l cells  thus \nenabling their  controlled release by light (Figure 1B). Since in vitro data of Escherichia coli  \nand Bacillus subtilis  MSN synthesizing enzymes RelA and SAS1 show that MSN s can \nstimulate their own synthesis , the introduction of small amounts of MSN s into the bacteria \nmight be sufficient to induce high cellular concentrations. [8,11–15] \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint \n\n3 \n \n \nFigure 1. (A) Chemical structure of ppGpp and pppGpp. (B) Overview of our strategy to modulate MSN \nconcentrations inside living cells. Due to charge repulsion from the negatively charged  lipopolysaccharides and \nlipids, MSNs cannot readily pass through the complex bacterial cell envelope. We tested various strategies to deliver \na photocaged MSN analogue into the bacterial cell and release the MSNs by light irradiation. Created in BioRender. \nPopp, C. (2025) https://BioRender.com/wnqejr2 \nThe main  obstacle for this approach is the bacterial cell envelope. In contrast to the lipid \nbilayer of mammalian cells, bacteria possess a much more challenging barrier . In Gram-\nnegative bacteria the cell wall is composed of several peptidoglycan layers surrounded by an \nouter membrane containing phospholipids and lipopolysaccharides. MSNs can carry up to \nseven negative charges that impair translocation through the negatively charged cell \nenvelope.[16,17] Several methods have been developed to transport highly negatively charged  \nsmall molecules like nucleotides into mammalian cells. For example, prodrugs with masked \nnegative charges and/or lipophilic groups, [18–21] positively charged polymers, [22,23] covalently \nlinked cationic groups, [24,25] permeabilization of the plasma membrane with surfactants [26] or \nby physical and mechanical means [27–30]. In bacteria , however, delivery studies of charged \nmetabolites are very limited and only few examples are known. It is possible to transport non-\nnatural nucleotides into bacterial cells by heterologously expressing importers from other \nspecies.[31,32] However, this approach is limited to several non -natural nucleotides and needs \ngenetic modification of the organism. Carlson and co-workers reported the development of a \ncationic polymer, promoting internalization of an adenosine triphosphate derived chemical \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint \n\n4 \n \nprobe into E. coli and B. subtilis.[33] They proposed that the cationic polymers act as general \npermeabilization reagents that promote the entry of various molecules into the cytosol.  Kraus \nand coworkers used a cyclodextrin based synthetic nucleotide transporter (SNT).[34] It consists \nof a per -6-amino-β-cyclodextrin coupled to a peptide consisting of eight aminocaproic \nacid/arginine pairs. The cyclodextrin binds the nucleotide while the peptide disrupts the \nmembrane to facilitate cellular delivery. They postulate that the bound nucleotide is released \nin the cytosol d ue to replacement by endogenous nucleotides. Successful delivery of a \nfluorescently modified deoxyuridine triphosphate into live Mycobacterium smegmatis and E. \ncoli was shown with this approach , yet the study was  mainly focused on delivery into \nmammalian cells.[34] \nOther highly negatively charged molecule that are routinely transported into bacterial cells \nare nucleic acids . Transformation is usually done by electroporation or by heat shock of \nchemically competent cells. [35–38] However, it is also possible to use other methods such as \ncationic polymers and peptides .[39,40] For mammalian cells several advanced methods have \nbeen developed, allowing targeted delivery of nucleic acids in vivo .[41] It has to be noted \nthough that for many applications of nucleic acid delivery, it is sufficient to deliver very small \ncopy numbers, as these will be amplified or are catalytically active. However, i n case of \nnucleotide transport, high concentrations are desirable.  \nFor less polar or cationic substances several further methods have been developed to \nfacilitate delivery.[42,43] One example that showed promising results for cargo delivery into \nvarious gram -negative bacteria are siderophores. [44,45] Coupling these Fe(III) -chelating \nmolecules to a cargo allows hijacking of the siderophore -mediated iron uptake pathway. This \napproach was extensively studied for antibiotic conjugates but was also shown to deliver \nlarger molecules like nucleic acid therapeutics into bacteria. [45–50] Carbohydrate conjugates \nwere used to deliver imaging probes into bacteria through the bacteria -specific maltodextrin \ntransport pathway.  [51,52] Other approaches involve the use of nanoparticles [41,53], cell -\npenetrating peptides [54–56], boron clusters[57], or disulfides.[58,59] \nAn established method to control the release of biologically active molecules inside cells \nor tissue is the introduction of photolabile protecting groups, so called photocages. [60–64] \nThese structures are covalently bound to the bioactive compound rendering them biologically \ninert and can be cleaved by light irradiation. The use of light allows temporal and spatial \ncontrol over the release of the biomolecule. One commonly used caging g roup is based on the \ncoumarin scaffold. The coumarin photocage stands out through high biocompatibility, easy \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint \n\n5 \n \nsynthesis, flexibility of structural modifications and a well -studied mechanism of \nphotocleavage.[65,66]  \nHere, we demonstrate that coumarin caged MSN can be delivered into living E. coli cells \nusing a modified cyclodextrin as additive and that they can be released on demand by \nirradiation with light.   \nResults and Discussion  \nSynthesis  of Caged MSNs  \nThe synthesis of caged MSNs followed our previously reported synthetic methods employing \nchemoselective phosphorylation with phosphoramidites and regioselective RNase T2 \nhydrolysis (Scheme 1).[67–71] The synthesis started with guanosine or 15N–labeled guanosine, \nwhich was treated with pyrophosphoryl chloride. Controlled hydrolysis leads to the 2 ′-3′ \ncyclophosphate which was regioselectively opened with RNase T2 to form pGp ( 3) or 15N–\npGp ( 4).[69,70] Unlabeled and 15N–labeled pGp was phosphorylated with bisfluorenmethyl \nphosphoramidite (5), oxidized and Fm -deprotected to form ppGpp ( 6) and 15N–ppGpp ( 7). \n15N–ppGpp was purified, while unlabeled ppGpp was used without purification. T reatment \nwith RNase T2 and purification by strong anion-exchange chromatography yielded ppGp (8) \nand 15N–ppGp (9).[69,70] \nFor photocaging, we adopted the 7-(diethylamino)-4-(but-3-yn-1-yl)coumarin (DEACBY) \nchromophore introduced by Seyfried et al.[72]. We chose to use this photocage because of its \neasy access and further because the alkyne group would enable late -stage functionalization \nof the caged MSNs by copper -catalyzed azide-alkyne cycloadditions. [73,74] \nTreatment of ppGp ( 8) and 15N–ppGp ( 9) with mixed P–amidite 10 containing the \nphotocage DEACBY  followed by oxidation with mCPBA led to the 3′-diphosphate 5′-\ntriphosphate nucleo tides with triesters  at the terminal phosphates . The triester at the 3 ′-end \nacts as a leaving group when the molecules are dissolved  in MeOH allowing fast and efficient \ncyclization to the corresponding 2 ′-3′ cyclophosphates. The fluorenmethyl (Fm) group was \nremoved with piperidine and the cyclophosphate was regioselectively opened with RNase T2 \nleading to 5 ′-DEACBY protected pppGp (11) and 15N – pppGp (12) in 58  % (97 % \nconsidering recovered starting material) and 52  % yield, respectively. Bisfluorenmethyl \nphosphoramidite reacted chemoselective ly with the monoester phosphate. Oxidation and Fm \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint \n\n6 \n \ndeprotection with 1,8-diazabicyclo[5.4.0]undec -7-en led to the caged MSNs DpppGpp (13) \nand 15N-DpppGpp (14) in 65 % and 41 % yield respectively.  The efficiency of this synthesis \nenabled the production of 13  mg 15N labeled caged pppGpp ( 14) starting from 70  mg 15N-\nlabeled ppGpp (7). \n \nScheme 1. Synthesis of caged and isotope labeled MSN derivatives.[69,70] a) P2Cl4O3, (11 eq), 0 °C, 3 h. b) NaHCO3 \n(1 M), 0 °C. c) RNase T2, pH 7.5, 37 °C, 12 h. d) 5 (3.0 eq), ETT (5.0 eq), DMF, rt, 15 min. e) mCPBA (3.0 eq), –\n20 °C, 10 min. f) DBU, 0 °C→rt, 30 min. g) RNase T2, aq. HCl, pH 5.5, 37  °C. h) 9 (2.3 – 2.4 eq), ETT (5.0 eq), \nDMF, rt, 1 h. i) MeOH, rt, 5 min j) Piperidine, DMF, rt, 5 min. k) 5 (1.5 – 1.7 eq), ETT (3.0 – 3.5 eq), DMF, rt, 15 min. \nl) mCPBA (1.5 – 2.1 eq), -20 °C, 10 min. m) Azide (1.5 eq), Na-ascorbate (5.0 eq), CuSO4 (0.1 eq),THPTA (0.5  eq), \nH2O, rt, 3 h. Abbreviations: brsm: based on recovered starting material, DBU: 1,8- diazabicyclo(5.4.0)undec-7-ene, \nETT: 5-(ethylthio)-1H-tetrazole, Fm: fluorenmethyl,  Gua: guanine, mCPBA: meta-chloroperbenzoic acid, rt: room \ntemperature. THPTA: tris(3-hydroxypropyltriazolylmethyl)amine. \nTo synthesize the caged magic spot nucleotide DppGpp, pGp was  treated with mixed amidite \n9, oxidized, cyclized in methanol  and deprotected. Regioselective ring -opening with RNase \nT2 led to DppGp  (14) in 77 % yield on a 700  mg scale. Another cycle of P-amidite coupling \nwith bisfluorenmethyl phosphoramidite,  that selectively only reacts with terminal \nphosphates,[75] oxidation and Fm deprotection with DBU led to the caged guanosine \ntetraphosphate DppGpp (15) in 69 % yield. \nThe alkyne handle on the photocage now offered the possibility to easily introduce \nstructures that could facilitate uptake into bacterial cells , while photocleavage would \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint \n\n7 \n \neventually remove them again. Various carbohydrates (glucose, galactose, mannose, maltose, \nlactose, trehalose) and a biomimetic enterobactin  analogue[76] were readily coupled to \nDppGpp via copper-catalyzed azide-alkyne cycloadditions [73,74] with yields of 24–79 % (S1–\n8). \nFor control experiments, caged adenosine triphosphate (D -ATP, S9) and caged guanosine \ndiphosphate (D -GDP, S10) were synthesized by treating commercial ADP and GMP with \nmixed amidite 9. Oxidation, and Fm deprotection, yield ed D-ATP and D-GDP. \nPhotolysis  Behavior  \nTo study the  photolysis behavior of the coumarin photocages, the caged MSNs DppGpp \n(16) and DpppGpp (13) were irradiated with a 400  nm LED in a small glass vial (25  nmol, \n50 µM, 14  mW), and the uncaging was monitored by HPLC ( Figure 2). After 2 min \nirradiation, only trace  amounts of caged MSN s remained and the corresponding free MSN s \nwere formed without detectable nucleotide byproducts  indicating a clean and efficient \nphotocleavage.  \n \nFigure 2. HPLC chromatograms (254 nm) of photolysis of DppGpp (A) and DpppGpp (B). Chromatograms were \nmeasured before and after irradiation with a 400 nm LED (25 nmol, 50 µM, 14 mW). \nUptake of MSN Probes into E. coli  \nNext, uptake into bacteria  was assessed by incubating E. coli  cell suspensions with \nsolutions of the caged nucleotides. After incubation, the cells were separated by centrifugation, \nwashed, resuspended and fluorescence of the cell suspension was measured. The intrinsic \nfluorescence of the DEACBY caging group enabled  this rapid initial study of potential \ndelivery. MSN derivatives coupled to carbohydrates and siderophore  (S1-8) did not produce \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint \n\n8 \n \nfluorescence above the autofluorescence  of E. coli  cells (Figure 3A, S2) under the \nexperimental conditions and thus further optimization was required . \nTherefore, we studied the synthetic nucleotide transporter ( SNT, 17, Figure 3A) developed \nby Kraus. [34] This commercially available compound was mixed with DppGpp ( 16) and \nDpppGpp ( 13) and then incubated with an E. coli  suspension. After washing , the cell \nsuspension exhibited a substantial increase in fluorescence , a first indication of efficient \ndelivery (Figure 3B). Flow cytometry confirmed these results ( Figure 3C), demonstrating \nlargely increased fluorescence of nearly all bacterial cells.  \n \nFigure 3. Uptake measurements of MSN in E. coli. (A) Structure of the synthetic nucleotide transporter (SNT). (B) \nCellular uptake determined by measuring fluorescence of cell suspensions. Values are  normalized to the \nautofluorescence of cells  treated with DppGpp or DpppGpp alon e. Mean ± standard deviation is shown.  (C) \nHistogram plots of flow cytometry measurements indicate MSN cage fluorescence in nearly every cell. \nConfocal Microscopy  Analysis of  Cellular Distribution  \nThe previously described experiments  cannot distinguish whether the MSN s are just bound \nto the exterior of the E. coli  membrane or if they are internalized  into the periplasm  or \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint \n\n9 \n \ncytoplasm. Thus, uptake was additionally evaluated with confocal microscopy  under \noptimized conditions using the high throughput methods described above  (Figure 4A+B). The \ncellular membranes were stained with Nile red. The intensity profiles of Nile red fluorescence \ncompared to DEACBY fluorescence show the distribution of the caged MSN s inside the \nbacterial cells ( Figure 4C+D). While Nile red fluorescence is maximal at the start and at the \nend of the intensity profiles representing the cellular membrane, DEACBY fluorescence is \nmaximal between the Nile red maxima. This clearly demonstrates that the caged MSNs enter \nthe cytoplasm of E. coli cells. Additionally, confocal fluorescence images reveal ed \nheterogeneous caged MSN uptake into different  bacterial cells. Some cells exhibit high \nintracellular fluorescence, while others show low or even  no DEACBY fluorescence, \ncontrasting with the flow cytometry measurements in Figure 3C.  \nCell viability was checked next. It was found that using 50  µl/OD600/ml incubation led to \nno reduced viability, while 200  µl/OD600/ml led to a decrease of viability to 50% (Figure S6). \n \nFigure 4. Microscopy images of E. coli treated with a mixture of DppGpp (16, B + D) or DpppGpp (13, A + C) with \nSNT (17) and the membrane stain Nile Red (red fluorescence). The fluorescence of the photolabile protecting group \nDEACBY is shown in cyan. C + D) Intensity profiles through a single cell normalized to the highest and lowest grey \nvalues. Scale bar = 2 µm in large images and 1 µm in magnified images. \nMetabolic Tracking  of MSNs  \nIn order to confirm that uncaging inside E. coli  is possible and to track the fate of the \nreleased MSNs , we applied an extraction and quantification assay using  capillary \nelectrophoresis mass spectrometr y (CE-MS).[69,77] After incubation with DpppGpp ( 13) and \nSNT (17) and successive washing, the E. coli cells were resuspended in medium the first \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint \n\n10 \n \nsample for MSN extraction was taken and the cell suspension was irradiated by a 400  nm \nLED (14 mW, 3  min) to release the free nucleotides  (Figure 5A). The culture was then \nincubated at 37  °C for 1 h and samples were collected a t predetermined time points before \nand after irradiation . The samples were  lysed, spiked with heavy isotope -labeled standards \nand extracted using weak anion exchange. Quantification was subsequently performed by CE-\nMS. \nAfter irradiation  with 400 nm light , we observed a direct and  sharp increase in pppGpp \nlevels, confirming successful photolysis  (Figure 5B). Over time , pppGpp levels decreased  \nwhile ppGpp increased, consistent with phosphatase -mediated hydrolysis. In E. coli GppA is \nthe phosphatase responsible for the conversion of pppGpp to ppGpp. In fact, ppGpp ist the \nmain alarmone in E. coli due to GppA activity. [78,79] This was confirmed using 15N-DpppGpp \n(14): we detected a corresponding increase in [ 15N]5-ppGpp as [ 15N]5-pppGpp declined \n(Figure 5C), indicating that the ppGpp originates mostly from the uncaged 15N-labeled \npppGpp and not through indirect effects of the uncaging process . Bacteria treated with caged \nGDP or bacteria that were not  irradiated with 400  nm light did not show this change in MSN \nlevels (Figure 5B). \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint \n\n11 \n \n \nFigure 5. MSNs extracted from E. coli after incubation with caged MSNs. (A) Experimental timeline of extraction \nexperiments. (B) pppGpp and ppGpp concentrations extracted from cell suspension of E. coli cells incubated with \nDpppGpp/SNT or D-GDP/SNT. Cell suspension was irradiated with 400 nm light before t = 0 min. (C) Ratio of 15N \nlabeled pppGpp and ppGpp extracted from E. coli cell suspension incubated with 15N-DpppGpp/SNT and irradiated \nwith 400 nm light. Before irradiation, no 15N-labeled nucleotides were detected. (D) pppGpp and ppGpp \nconcentrations extracted from cell suspension of E. coli  ΔgppA mutant incubated with DpppGpp/SNT or D -\nGDP/SNT. Cell suspension was irradiated with 400 nm light before t = 0 min. (E) Ratio of 15N labeled pppGpp and \nppGpp extracted from E. coli cell suspension that was incubated with 15N-DpppGpp/SNT and irradiated with 400 \nnm light. After uncaging, cells were separated by centrifugation (5 min, 5 kG) and then immediately lysed in 1 M \nformic acid. \nTo further validate this assumption, we also performed  the extraction experiment with a  \nΔgppA mutant[80] (Figure 5D). In this strain, pppGpp levels remained stable due to the absence \nof GppA , however, ppGpp still accumulated, consistent with  previous reports that pppGpp \nallosterically activates RelA, thereby enhancing ppGpp synthesis .[12,15] \nWe performed another control:  Bacteria were treated with DpppGpp ( 13) and SNT  (17) \nand [15N]5-pppGpp was spiked into the medium directly after uncaging . The conversion of \nthe heavy -isotope labeled  [15N]5–pppGpp to [ 15N]5–ppGpp resembled the conversion of  \npppGpp to ppGpp (Figure  S7), even though [ 15N]5–pppGpp should only be present in the \nmedium, while pppGpp should be located inside the bacterial cells. These results could imply \nthat periplasmic or extracellular phosphatases , such as PhoA[81] and/or phosphatases released \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint \n\n12 \n \nfrom damaged cells can also process MSN s. Furthermore, damaged cells may release MSNs \ninto the culture medium or take up 15N-labelled MSNs from the medium.  \nTo better understand which portion of  the quantified nucleotides were truly intracellular, \nwe separated the cell pellets and  the culture supernatants after photolysis  by centrifugation  \nand analyzed both fractions. We could detect MSNs in both fractions, but s urprisingly, the \nsupernatant contained more pppGpp but less ppGpp than the pellet fraction (Figure S8). This \nshows that MSNs leak into the medium, potentially through the action of the SNT . However, \nrapid intracellular metabolism could reduce detectable MSN levels inside the cells, \nexaggerating the relative abundance measured in the supernatant. When we repeated these \nexperiments with 15N-DpppGpp ( 14), we found that pppGpp -to-ppGpp conversion was \nindeed markedly faster in the pellet fraction, whereas extracellular conversion was slower \n(Figure 5E).  \nNext, we investigated whether a drug efflux system could be responsible for MSN leakage \ninto the medium.[82–84] Therefore, we compared knockout strains lacking the major drug efflux \ncomponents AcrA, AcrB and TolC with the wild type. [80] In a simple experiment , we \nincubated the cell suspensions with DpppGpp/SNT and performed four washing steps where \nwe centrifuged the cell suspension, removed the supernatant and resuspended the pellet. We \nthen analyzed the washing solution for DEACBY fluorescence to measure DpppGpp leakage. \nEven after four washing steps, significant amounts of DpppGpp could still be detected in the \nwashing solution of all the strains investigated  (Figure S9). This finding suggests that SNT -\ninduced membrane perturbation, as  opposed to active export  of internalized caged MSN , \nserve as the predominant contributors to the observed phenomenon . \nTaken together, these data suggest that the extracellular MSN pool arises largely from \nmembrane damage, which releases (p)ppGpp into the medium. At the same time, rapid \nintracellular turnover depletes the internal MSN pool, leading to lower intracellular levels \nrelative to extracellular ones.  Consequently, while CE -MS provides robust tracking of MSN \nphoto-release and metabolism, careful interpretation is required, as a portion of the detected \nsignal may not reflect true intracellular pools.  Notwithstanding,  confocal microscopy \nunambiguously showed internalization of the probes into a fraction of cells. Taken together, \nour current interpretation is that part of the probe is released inside of the cells, while another \nfraction is leaking from cells due to SNT action.  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint \n\n13 \n \nBacterial Growth Assay  \nThe bacterial growth rate is apparently regulated by MSN. [85–87] As simple read -out we \nenvisioned that photo-release of caged MSN should slow down bacterial growth . Therefore, \nthe growth of E. coli was monitored after incubation with  the D(p)ppGpp/SNT mixture with \nand without following photo -release.  \nFor wild type E. coli, we could not detect this projected difference in growth rate. As shown \nin the confocal microscopy experiments described above, uptake of the caged MSN s was \nheterogeneous. We therefore attribute the absence of a measurable population -level effect to \nthis cell-to-cell variability: while some cells internalized high amounts of D(p)ppGpp, others \nlikely contained only minimal levels, resulting in no apparent change in overall growth.  \nTo overcome this limitation , we switched to a mutant strain incapable of synthesizing \nMSNs ((p)ppGpp0) due to deletion of the relA and spoT genes . As basal levels of MSNs are \nimportant to maintain cellular component homeostasis, t his mutant has multiple disabilities \nin cell division, transcription  and translation and can have difficulties to adapt to \nenvironmental changes .[4,6] Due to the absence of MSNs in this mutant, the influence of \nartificially added MSNs on the growth rate should be pronounced , but in this case potentially \nincrease the growth rate due to compensation of above -mentioned defects.  We observed that \ntreatment with SNT alone decreases the bacterial  growth rate, likely due to its toxicity by \ndamaging the bacterial envelope. To evaluate the influence of DppGpp and DpppGpp mixed \nwith SNT on the growth rate of the E. coli (p)ppGpp0 strain, we used D-ATP (S9) mixed with \nSNT as a control . Interestingly, incubation of (p)ppGpp 0 with SNT and caged (p)ppGpp \nalready recovered the loss in growth rate caused by SNT. Caged ATP did not show this effect \n(Figure 6A). One possible conclusion is that caged MSNs, due to their versatile binding \npatterns,[88] can already interact with some of the binding partners of MSN and thus restore \ngrowth. In line with this interpretation, uncaging of (p)ppGpp did not further affect the \nrecovery of the growth rate ( Figure 6B). This interpretation is in line with previous findings \nfor caged molecules.[63,89,90]  Future studies will now address the increase of bulkiness in the \ncage through click chemistry on the alkyne handle to abrogate unwanted binding prior to \nuncaging.[91] \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint \n\n14 \n \n \nFigure 6. Growth rate of the E. coli (p)ppGpp0 mutant treated with SNT and different nucleotides. (A) Comparison \nof the different treatment conditions without light irradiation. Analyzed by Brown-Forsythe and Welch ANOVA test \nwith Dunnett′s T3 multiple comparisons test; individual variances computed for each comparison. ns: p =0.125; **: \np = 0.0054. (B) Comparison of samples irradiated or not irradiated with 400 nm light. Analyzed with two-way ANOVA \nwith Šídák multiple comparisons test, with a single pooled variance. ns: p > 0.99. Mean and standard error of mean \nof at least four separate experiments is shown. \nSummary and Conclusion  \nIn this study , we present the first  photocaged derivatives of the MSNs  pppGpp and ppGpp. \nDppGpp (16), Dpp pGpp (13), and the heavy isotope labeled 15N-DpppGpp (14) were \nefficiently synthesized in high yields using a chemoenzymatic synthesis approach . By \ncomparing different delivery strategies,  we achieve the delivery of highly negatively charged, \ncaged MSNs through the complex cell envelope of living E. coli. Several analytical \napproaches combined provide evidence for intracellular delivery into a subpopulation of cells . \nUpon 400 nm irradiation, these probes release free nucleotides, enabling tracking of pppGpp -\nto-ppGpp conversion by CE -MS. Moreover, initial growth curve studies in wild type and \n(p)ppGpp0 strains demonstrate the potential of these probes for in vivo studies after further \noptimization: While photolysis and metabolic conversion were clearly demonstrated, \nextraction experiments revealed substantial leakage of MSNs into the extracellular medium, \ncomplicating accurate quantification of intracellular levels. Growth studies with a (p)ppGpp⁰ \nstrain confirmed that caged MSNs can modulate bacterial physiology, but no light -dependent \ngrowth effect was yet observed, likely due to residual activity of the caged species.  \nFuture work should prioritize improved transporter strategies and the development of \nbulkier, biologically inert cages. Single -cell studies of cells that have received large amounts \nof caged nucleotide could help attribute heterogeneous uptake to specific cellular states . \nDespite its current limitations, this study  provides first significant strategies how to deliver \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint \n\n15 \n \nhighly charged signaling molecules into living bacteria and control cellular concentrations on \na minute time-scale through photo -uncaging.  \nAcknowledgement  \nWe thank Dr. Guizhen Liu, Dr. Mengsi Lu and Dr. Anuj Shukla for CE -MS measurements. \nFurthermore, w e thank the staff of the Life Imaging Center (LIC) of the University of  \nFreiburg, in particular I. Bierschenk, A. Neumann and R. Nitschke  for help with their \nmicroscopy resources, and the excellent  support in image recording and analysis. The \nfollowing instrument funded  by the Deutsche Forschungsgemeinschaft (DFG, German \nResearch Foundation) was used: ZEISS LSM 880 Observer / Fast Airyscan with inverted \nmicroscope (Instrument Grant Number 317784314). In addition, we thank D. Herchenbach \nand M. Follo of the Lighthouse Core Facility, funded in part by the Medical Faculty, \nUniversity of Freiburg (Project Number 2023/B3 -Fol). Hans-Georg Koch  acknowledges \nfunding by the SFB1381 (Project-ID 403222702). This project has received funding from the \nEuropean Research Council (ERC) under the European Union’s Horizon 2020 research and \ninnovation program (grant agreement no. 864246, to H.J.J.).  This study was supported by the \nDeutsche Forschungsgemeinschaft (DFG) under Germany's excellence strategy (CIBSS, \nEXC-2189, Project ID 390939984, to H. J. J ) \nAuthor Contributions  \nChristoph Popp: Synthesis, Investigation, Formal analysis, Visualization, Writing; Patrick \nMoser: Synthesis of 15N-DpppGpp (14); Xinwei Liu: Supervision and discussion of in vivo \nexperiments; Johannes Freitag: Supervision and discussion of in vivo experiments; Isabel \nPrucker: Discussion of CE-MS results; Robert Zscherp: Synthesis of S11; Xuan Wang: \nSynthesis of S13; Philipp Klahn: Supervision and discussion ; Hans-Georg Koch: Supervision \nand discussion; Gert Bange: Supervision and discussion of in vivo experiments; Henning J. \nJessen: Conceptualization, Supervision, Funding acquisition, Writing – review & editing \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint \n\n16 \n \nConflict of  Interest  \n The authors declare no conflict of interest. \nBibliography  \n[1] G. Bange, D. E. Brodersen, A. Liuzzi, W. Steinchen, “Two P or Not Two P: \nUnderstanding Regulation by the Bacterial Second Messengers (p)ppGpp” Annu Rev \nMicrobiol 2021, 75, 383–406. \n[2] O. Pacios, L. Blasco, I. Bleriot, L. Fernandez-Garcia, A. Ambroa, M. López, G. Bou, \nR. Cantón, R. Garcia-Contreras, T. K. Wood, M. Tomás, “(p)ppGpp and Its Role in \nBacterial Persistence: New Challenges” Antimicrob Agents Chemother 2020, 64, 1–14. \n[3] S. E. Irving, N. R. Choudhury, R. M. Corrigan, “The stringent response and \nphysiological roles of (pp)pGpp in bacteria” Nat Rev Microbiol 2021, 19, 256–271. \n[4] W. Steinchen, V. Zegarra, G. Bange, “(p)ppGpp: Magic Modulators of Bacterial \nPhysiology and Metabolism” Front Microbiol 2020, 11, 2072. \n[5] M. Cashel, J. Gallant, “Two Compounds implicated in the Function of the RC Gene of \nEscherichia coli” Nature 1969, 221, 838–841. \n[6] L. Fernández-Coll, M. Cashel, “Possible Roles for Basal Levels of (p)ppGpp: Growth \nEfficiency Vs. Surviving Stress” Front Microbiol 2020, 11, 592718. \n[7] S. Han, J. Min, Y. Park, W. Park, “Fine‐tuning regulation of (p)ppGpp‐driven outer \nmembrane vesicle formation in Acinetobacter baumannii” FEBS J 2025, 292, 3696–\n3717. \n[8] D. K. Fung, J. T. Barra, J. Yang, J. W. Schroeder, F. She, M. Young, D. Ying, D. M. \nStevenson, D. Amador-Noguez, J. D. Wang, “A shared alarmone–GTP switch controls \npersister formation in bacteria” Nat Microbiol 2025, 10, 1617–1629. \n[9] M. Zhu, H. Mu, X. Dai, “Integrated control of bacterial growth and stress response by \n(p)ppGpp in Escherichia coli: A seesaw fashion” iScience 2024, 27, 108818. \n[10] U. Mechold, K. Potrykus, H. Murphy, K. S. Murakami, M. Cashel, “Differential \nregulation by ppGpp versus pppGpp in Escherichia coli” Nucleic Acids Res 2013, 41, \n6175–6189. \n[11] V. Shyp, S. Tankov, A. Ermakov, P. Kudrin, B. P. English, M. Ehrenberg, T. Tenson, \nJ. Elf, V. Hauryliuk, “Positive allosteric feedback regulation of the stringent response \nenzyme RelA by its product” EMBO Rep 2012, 13, 835–839. \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint \n\n17 \n \n[12] P. Kudrin, I. Dzhygyr, K. Ishiguro, J. Beljantseva, E. Maksimova, S. R. A. Oliveira, V. \nVarik, R. Payoe, A. L. Konevega, T. Tenson, T. Suzuki, V. Hauryliuk, “The ribosomal \nA-site finger is crucial for binding and activation of the stringent factor RelA” Nucleic \nAcids Res 2018, 46, 1973–1983. \n[13] W. Steinchen, J. S. Schuhmacher, F. Altegoer, C. D. Fage, V. Srinivasan, U. Linne, M. \nA. Marahiel, G. Bange, “Catalytic mechanism and allosteric regulation of an \noligomeric (p)ppGpp synthetase by an alarmone” Proc Natl Acad Sci USA 2015, 112, \n13348–13353. \n[14] B. W. Anderson, D. K. Fung, J. D. Wang, “Regulatory Themes and Variations by the \nStress-Signaling Nucleotide Alarmones (p)ppGpp in Bacteria” Annu Rev Genet 2021, \n55, 115–133. \n[15] M. Roghanian, K. Van Nerom, H. Takada, J. Caballero-Montes, H. Tamman, P. \nKudrin, A. Talavera, I. Dzhygyr, S. Ekström, G. C. Atkinson, A. Garcia-Pino, V. \nHauryliuk, “(p)ppGpp controls stringent factors by exploiting antagonistic allosteric \ncoupling between catalytic domains” Mol Cell 2021, 81, 3310-3322.e6. \n[16] T. J. Silhavy, D. Kahne, S. Walker, “The Bacterial Cell Envelope” Cold Spring Harb \nPerspect Biol 2010, 2, a000414. \n[17] G. K. Auer, D. B. Weibel, “Bacterial Cell Mechanics” Biochemistry 2017, 56, 3710–\n3724. \n[18] V. T. Sterrenberg, D. Stalling, J. I. H. Knaack, T. K. Soh, J. B. Bosse, C. Meier, “A Tri \nPPP ro‐Nucleotide Reporter with Optimized Cell‐Permeable Dyes for Metabolic \nLabeling of Cellular and Viral DNA in Living Cells” Angew Chem Int Ed 2023, 62, \ne202308271. \n[19] T. Gollnest, T. D. de Oliveira, D. Schols, J. Balzarini, C. Meier, “Lipophilic prodrugs \nof nucleoside triphosphates as biochemical probes and potential antivirals” Nat \nCommun 2015, 6, 8716. \n[20] T. Bittner, C. Wittwer, S. Hauke, D. Wohlwend, S. Mundinger, A. K. Dutta, D. Bezold, \nT. Dürr, T. Friedrich, C. Schultz, H. J. Jessen, “Photolysis of Caged Inositol \nPyrophosphate InsP 8 Directly Modulates Intracellular Ca 2+ Oscillations and Controls \nC2AB Domain Localization” J Am Chem Soc 2020, 142, 10606–10611. \n[21] C. McGuigan, M. Derudas, B. Gonczy, K. Hinsinger, S. Kandil, F. Pertusati, M. Serpi, \nR. Snoeck, G. Andrei, J. Balzarini, T. D. McHugh, A. Maitra, E. Akorli, D. \nEvangelopoulos, S. Bhakta, “ProTides of N-(3-(5-(2′-deoxyuridine))prop-2-\nynyl)octanamide as potential anti-tubercular and anti-viral agents” Bioorg Med Chem \n2014, 22, 2816–2824. \n[22] S. V Vinogradov, A. D. Zeman, E. V Batrakova, A. V Kabanov, “Polyplex Nanogel \nformulations for drug delivery of cytotoxic nucleoside analogs.” J Control Release \n2005, 107, 143–57. \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint \n\n18 \n \n[23] I. Pavlovic, D. T. Thakor, J. R. Vargas, C. J. McKinlay, S. Hauke, P. Anstaett, R. C. \nCamuña, L. Bigler, G. Gasser, C. Schultz, P. A. Wender, H. J. Jessen, “Cellular \ndelivery and photochemical release of a caged inositol-pyrophosphate induces PH-\ndomain translocation in cellulo” Nat Commun 2016, 7, 10622. \n[24] J. Ma, J. Wehrle, D. Frank, L. Lorenzen, C. Popp, W. Driever, R. Grosse, H. J. Jessen, \n“Intracellular delivery and deep tissue penetration of nucleoside triphosphates using \nphotocleavable covalently bound dendritic polycations” Chem Sci 2024, 15, 6478–\n6487. \n[25] A. E. Fouda, M. K. H. Pflum, “A Cell‐Permeable ATP Analogue for Kinase‐Catalyzed \nBiotinylation” Angew Chem Int Ed 2015, 54, 9618–9621. \n[26] A. Maya-Mendoza, P. Olivares-Chauvet, F. Kohlmeier, D. A. Jackson, “Visualising \nchromosomal replication sites and replicons in mammalian cells” Methods 2012, 57, \n140–148. \n[27] K. Koberna, D. Staněk, J. Malínský, M. Eltsov, A. Pliss, V. Čtrnáctá, Š. Cermanová, I. \nRaška, “Nuclear organization studied with the help of a hypotonic shift: Its use permits \nhydrophilic molecules to enter into living cells” Chromosoma 1999, 108, 325–335. \n[28] S. M. Hacker, A. Buntz, A. Zumbusch, A. Marx, “Direct Monitoring of Nucleotide \nTurnover in Human Cell Extracts and Cells by Fluorogenic ATP Analogs” ACS Chem \nBiol 2015, 10, 2544–2552. \n[29] E. M. M. Manders, H. Kimura, P. R. Cook, “Direct Imaging of DNA in Living Cells \nReveals the Dynamics of Chromosome Formation” J Cell Biol 1999, 144, 813–822. \n[30] D. Zink, T. Cremer, R. Saffrich, R. Fischer, M. F. Trendelenburg, W. Ansorge, E. H. \nK. Stelzer, “Structure and dynamics of human interphase chromosome territories in \nvivo” Hum Genet 1998, 102, 241–251. \n[31] D. A. Malyshev, K. Dhami, T. Lavergne, T. Chen, N. Dai, J. M. Foster, I. R. Corrêa, F. \nE. Romesberg, “A semi-synthetic organism with an expanded genetic alphabet” Nature \n2014, 509, 385–388. \n[32] Y. Zhang, B. M. Lamb, A. W. Feldman, A. X. Zhou, T. Lavergne, L. Li, F. E. \nRomesberg, “A semisynthetic organism engineered for the stable expansion of the \ngenetic alphabet” Proc Natl Acad Sci USA 2017, 114, 1317–1322. \n[33] H. K. Lembke, A. Espinasse, M. G. Hanson, C. J. Grimme, Z. Tan, T. M. Reineke, E. \nE. Carlson, “Cationic Polymers Enable Internalization of Negatively Charged Chemical \nProbes into Bacteria” ACS Chem Biol 2023, 18, 2063–2072. \n[34] Z. Zawada, A. Tatar, P. Mocilac, M. Buděšínský, T. Kraus, “Transport of Nucleoside \nTriphosphates into Cells by Artificial Molecular Transporters” Angew Chem Int Ed \n2018, 57, 9891–9895. \n[35] J. L. Young, D. A. Dean in Adv Genet, Academic Press, 2015, pp. 49–88. \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint \n\n19 \n \n[36] S. N. Cohen, A. C. Y. Chang, L. Hsu, “Nonchromosomal Antibiotic Resistance in \nBacteria: Genetic Transformation of Escherichia coli by R-Factor DNA” Proc Natl \nAcad Sci USA 1972, 69, 2110–2114. \n[37] M. Mandel, A. Higa, “Calcium-dependent bacteriophage DNA infection” J Mol Biol \n1970, 53, 159–162. \n[38] D. Hanahan, “Studies on transformation of Escherichia coli with plasmids” J Mol Biol \n1983, 166, 557–580. \n[39] M. M. Islam, M. Odahara, T. Yoshizumi, K. Oikawa, M. Kimura, M. Su’etsugu, K. \nNumata, “Cell-Penetrating Peptide-Mediated Transformation of Large Plasmid DNA \ninto Escherichia coli” ACS Synth Biol 2019, 8, 1215–1218. \n[40] V. V. de Souza, P. A. M. Vitale, F. H. Florenzano, R. K. Salinas, I. M. Cuccovia, “A \nnovel method for DNA delivery into bacteria using cationic copolymers” Braz J Med \nBiol Res 2021, 54, e10743. \n[41] R. van der Meel, P. A. Wender, O. M. Merkel, I. Lostalé-Seijo, J. Montenegro, A. \nMiserez, Q. Laurent, H. Sleiman, P. Luciani, “Next-generation materials for nucleic \nacid delivery” Nat Rev Mater 2025, 10, 490–499. \n[42] A. Nazli, D. L. He, D. Liao, M. Z. I. Khan, C. Huang, Y. He, “Strategies and \nprogresses for enhancing targeted antibiotic delivery” Adv Drug Deliv Rev 2022, 189, \n114502. \n[43] A. V. Cheng, W. M. Wuest, “Signed, Sealed, Delivered: Conjugate and Prodrug \nStrategies as Targeted Delivery Vectors for Antibiotics” ACS Infect Dis 2019, 5, 816–\n828. \n[44] J. Kramer, Ö. Özkaya, R. Kümmerli, “Bacterial siderophores in community and host \ninteractions” Nat Rev Microbiol 2020, 18, 152–163. \n[45] P. Klahn, M. Brönstrup, “Bifunctional antimicrobial conjugates and hybrid \nantimicrobials” Nat Prod Rep 2017, 34, 832–885. \n[46] M. J. Pals, L. Wijnberg, Ç. Yildiz, W. A. Velema, “Catechol‐Siderophore Mimics \nConvey Nucleic Acid Therapeutics into Bacteria” Angew Chem Int Ed 2024, 63, \ne202402405. \n[47] R. Zscherp, J. Coetzee, J. Vornweg, J. Grunenberg, J. Herrmann, R. Müller, P. Klahn, \n“Biomimetic enterobactin analogue mediates iron-uptake and cargo transport into E. \ncoli and P. aeruginosa” Chem Sci 2021, 12, 10179–10190. \n[48] J. H. Boyce, B. Dang, B. Ary, Q. Edmondson, C. S. Craik, W. F. DeGrado, I. B. Seiple, \n“Platform to Discover Protease-Activated Antibiotics and Application to Siderophore–\nAntibiotic Conjugates” J Am Chem Soc 2020, 142, 21310–21321. \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint \n\n20 \n \n[49] M. Madaoui, O. Vidal, A. Meyer, M. Noël, J. Lacroix, J. Vasseur, A. Marra, F. \nMorvan, “Modified Galacto‐ or Fuco‐Clusters Exploiting the Siderophore Pathway to \nInhibit the LecA‐ or LecB‐Associated Virulence of Pseudomonas aeruginosa” \nChemBioChem 2020, 21, 3433–3448. \n[50] T. Zheng, J. L. Bullock, E. M. Nolan, “Siderophore-Mediated Cargo Delivery to the \nCytoplasm of Escherichia coli and Pseudomonas aeruginosa : Syntheses of \nMonofunctionalized Enterobactin Scaffolds and Evaluation of Enterobactin–Cargo \nConjugate Uptake” J Am Chem Soc 2012, 134, 18388–18400. \n[51] X. Ning, S. Lee, Z. Wang, D. Kim, B. Stubblefield, E. Gilbert, N. Murthy, \n“Maltodextrin-based imaging probes detect bacteria in vivo with high sensitivity and \nspecificity” Nat Mater 2011, 10, 602–607. \n[52] A. Zlitni, G. Gowrishankar, I. Steinberg, T. Haywood, S. Sam Gambhir, “Maltotriose-\nbased probes for fluorescence and photoacoustic imaging of bacterial infections” Nat \nCommun 2020, 11, 1250. \n[53] R. Guo, K. Li, J. Qin, S. Niu, W. Hong, “Development of polycationic micelles as an \nefficient delivery system of antibiotics for overcoming the biological barriers to reverse \nmultidrug resistance in Escherichia coli” Nanoscale 2020, 12, 11251–11266. \n[54] H.-M. Lee, J. Ren, K. M. Tran, B.-M. Jeon, W.-U. Park, H. Kim, K. E. Lee, Y. Oh, M. \nChoi, D.-S. Kim, D. Na, “Identification of efficient prokaryotic cell-penetrating \npeptides with applications in bacterial biotechnology” Commun Biol 2021, 4, 205. \n[55] G. Inoue, D. Toyohara, T. Mori, T. Muraoka, “Critical Side Chain Effects of Cell-\nPenetrating Peptides for Transporting Oligo Peptide Nucleic Acids in Bacteria” ACS \nAppl Bio Mater 2021, 4, 3462–3468. \n[56] A. Schmitt, H. Wennemers, “Amphipathic Proline-Rich Cell Penetrating Peptides for \nTargeting Mitochondria” ACS Chem Biol 2025, 20, 2298–2307. \n[57] A. Barba-Bon, G. Salluce, I. Lostalé-Seijo, K. I. Assaf, A. Hennig, J. Montenegro, W. \nM. Nau, “Boron clusters as broadband membrane carriers” Nature 2022, 603, 637–642. \n[58] Q. Laurent, R. Martinent, B. Lim, A.-T. Pham, T. Kato, J. López-Andarias, N. Sakai, S. \nMatile, “Thiol-Mediated Uptake” JACS Au 2021, 1, 710–728. \n[59] I. S. Shchelik, K. Gademann, “Thiol- and Disulfide-Containing Vancomycin \nDerivatives Against Bacterial Resistance and Biofilm Formation” ACS Med Chem Lett \n2021, 12, 1898–1904. \n[60] G. C. R. Ellis-Davies, “Caged compounds: photorelease technology for control of \ncellular chemistry and physiology” Nat Methods 2007, 4, 619–628. \n[61] Y. Li, M. Wang, F. Wang, S. Lu, X. Chen, “Recent progress in studies of photocages” \nSmart Molecules 2023, 1, e20220003. \n[62] R. Weinstain, T. Slanina, D. Kand, P. Klán, “Visible-to-NIR-Light Activated Release: \nFrom Small Molecules to Nanomaterials” Chem Rev 2020, 120, 13135–13272. \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint \n\n21 \n \n[63] G. C. R. Ellis‐Davies, “Reverse Engineering Caged Compounds: Design Principles for \ntheir Application in Biology” Angew Chem Int Ed 2023, 62, e202206083. \n[64] J. H. Kaplan, B. Forbush, J. F. Hoffman, “Rapid photolytic release of adenosine 5’-\ntriphosphate from a protected analog: utilization by the sodium:potassium pump of \nhuman red blood cell ghosts” Biochemistry 1978, 17, 1929–1935. \n[65] M. J. Hansen, W. A. Velema, M. M. Lerch, W. Szymanski, B. L. Feringa, \n“Wavelength-selective cleavage of photoprotecting groups: strategies and applications \nin dynamic systems” Chem Soc Rev 2015, 44, 3358–3377. \n[66] P. Klán, T. Šolomek, C. G. Bochet, A. Blanc, R. Givens, M. Rubina, V. Popik, A. \nKostikov, J. Wirz, “Photoremovable protecting groups in chemistry and biology: \nReaction mechanisms and efficacy” Chem Rev 2013, 113, 119–191. \n[67] T. M. Haas, D. Qiu, M. Häner, L. Angebauer, A. Ripp, J. Singh, H. Koch, C. Jessen-\nTrefzer, H. J. Jessen, “Four Phosphates at One Blow: Access to Pentaphosphorylated \nMagic Spot Nucleotides and Their Analysis by Capillary Electrophoresis” J Org Chem \n2020, 85, 14496–14506. \n[68] T. M. Haas, B. Laventie, S. Lagies, C. Harter, I. Prucker, D. Ritz, R. Saleem‐Batcha, D. \nQiu, W. Hüttel, J. Andexer, B. Kammerer, U. Jenal, H. J. Jessen, “Photoaffinity \nCapture Compounds to Profile the Magic Spot Nucleotide Interactomes**” Angew \nChem Int Ed 2022, 61, e202201731. \n[69] D. Qiu, E. Lange, T. M. Haas, I. Prucker, S. Masuda, Y. L. Wang, G. Felix, G. Schaaf, \nH. J. Jessen, “Bacterial Pathogen Infection Triggers Magic Spot Nucleotide Signaling \nin Arabidopsis thaliana Chloroplasts through Specific RelA/SpoT Homologues” J Am \nChem Soc 2023, 145, 16081–16089. \n[70] T. M. Haas, P. Ebensperger, V. B. Eisenbeis, C. Nopper, T. Dürr, N. Jork, N. Steck, C. \nJessen-Trefzer, H. J. Jessen, “Magic spot nucleotides: tunable target-specific \nchemoenzymatic synthesis” Chem Commun 2019, 55, 5339–5342. \n[71] G. S. Cremosnik, A. Hofer, H. J. Jessen, “Iterative Synthesis of Nucleoside \nOligophosphates with Phosphoramidites” Angew Chem Int Ed 2014, 53, 286–289. \n[72] P. Seyfried, L. Eiden, N. Grebenovsky, G. Mayer, A. Heckel, “Photo-Tethers for the \n(Multi-)Cyclic, Conformational Caging of Long Oligonucleotides” Angew Chem Int Ed \n2017, 56, 359–363. \n[73] V. V Rostovtsev, L. G. Green, V. V Fokin, K. B. Sharpless, “A Stepwise Huisgen \nCycloaddition Process: Copper(I)-Catalyzed Regioselective ‘Ligation’ of Azides and \nTerminal Alkynes” Angew Chem Int Ed 2002, 41, 2596–2599. \n[74] C. W. Tornøe, C. Christensen, M. Meldal, “Peptidotriazoles on Solid Phase: [1,2,3]-\nTriazoles by Regiospecific Copper(I)-Catalyzed 1,3-Dipolar Cycloadditions of \nTerminal Alkynes to Azides” J Org Chem 2002, 67, 3057–3064. \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint \n\n22 \n \n[75] H. J. Jessen, T. Dürr-Mayer, T. M. Haas, A. Ripp, C. C. Cummins, “Lost in \nCondensation: Poly-, Cyclo-, and Ultraphosphates” Acc Chem Res 2021, 54, 4036–\n4050. \n[76] R. Zscherp, J. Coetzee, J. Vornweg, J. Grunenberg, J. Herrmann, R. Müller, P. Klahn, \n“Biomimetic enterobactin analogue mediates iron-uptake and cargo transport into E. \ncoli and P. aeruginosa” Chem Sci 2021, 12, 10179–10190. \n[77] D. Qiu, M. S. Wilson, V. B. Eisenbeis, R. K. Harmel, E. Riemer, T. M. Haas, C. \nWittwer, N. Jork, C. Gu, S. B. Shears, G. Schaaf, B. Kammerer, D. Fiedler, A. Saiardi, \nH. J. Jessen, “Analysis of inositol phosphate metabolism by capillary electrophoresis \nelectrospray ionization mass spectrometry” Nat Commun 2020, 11, 6035. \n[78] A. Hara, J. Sy, “Guanosine 5’-triphosphate, 3’-diphosphate 5’-phosphohydrolase. \nPurification and substrate specificity.” J Biol Chem 1983, 258, 1678–1683. \n[79] C. R. Somerville, A. Ahmed, “Mutants of Escherichia coli defective in the degradation \nof guanosine 5’-triphosphate, 3’-diphosphate (pppGpp)” Mol Gen Genet 1979, 169, \n315–323. \n[80] T. Baba, T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, K. A. Datsenko, M. \nTomita, B. L. Wanner, H. Mori, “Construction of Escherichia coli K‐12 in‐frame, \nsingle‐gene knockout mutants: the Keio collection” Mol Syst Biol 2006, 2, DOI \n10.1038/msb4100050. \n[81] Chung Nan Chang, K. Wun-Jing, E. Y. Chen, “Nucleotide sequence of the alkaline \nphosphatase gene of Escherichia coli” Gene 1986, 44, 121–125. \n[82] D. Du, X. Wang-Kan, A. Neuberger, H. W. van Veen, K. M. Pos, L. J. V. Piddock, B. \nF. Luisi, “Multidrug efflux pumps: structure, function and regulation” Nat Rev \nMicrobiol 2018, 16, 523–539. \n[83] K. Nishino, S. Yamasaki, R. Nakashima, M. Zwama, M. Hayashi-Nishino, “Function \nand Inhibitory Mechanisms of Multidrug Efflux Pumps” Front Microbiol 2021, 12, \nDOI 10.3389/fmicb.2021.737288. \n[84] K. M. Pos, “Drug transport mechanism of the AcrB efflux pump” Biochim Biophys \nActa 2009, 1794, 782–793. \n[85] K. Potrykus, H. Murphy, N. Philippe, M. Cashel, “ppGpp is the major source of growth \nrate control in E. coli” Environ Microbiol 2011, 13, 563–575. \n[86] N. C. E. Imholz, M. J. Noga, N. J. F. van den Broek, G. Bokinsky, “Calibrating the \nBacterial Growth Rate Speedometer: A Re-evaluation of the Relationship Between \nBasal ppGpp, Growth, and RNA Synthesis in Escherichia coli” Front Microbiol 2020, \n11, 574872. \n[87] M. Zhu, X. Dai, “Growth suppression by altered (p)ppGpp levels results from non-\noptimal resource allocation in Escherichia coli” Nucleic Acids Res 2019, 47, 4684–\n4693. \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint \n\n23 \n \n[88] G. S. Kushwaha, A. Patra, N. S. Bhavesh, “Structural Analysis of (p)ppGpp Reveals Its \nVersatile Binding Pattern for Diverse Types of Target Proteins” Front Microbiol 2020, \n11, 575041. \n[89] H. Thirlwell, J. A. Sleep, M. A. Ferenczi, “Inhibition of unloaded shortening velocity \nin permeabilized muscle fibres by caged ATP compounds” J Muscle Res Cell Motil \n1995, 16, 131–137. \n[90] S. Geibel, A. Barth, S. Amslinger, A. H. Jung, C. Burzik, R. J. Clarke, R. S. Givens, K. \nFendler, “P3-[2-(4-hydroxyphenyl)-2-oxo]ethyl ATP for the Rapid Activation of the \nNa+,K+-ATPase” Biophys J 2000, 79, 1346–1357. \n[91] R. Durand-de Cuttoli, P. S. Chauhan, A. Pétriz Reyes, P. Faure, A. Mourot, G. C. R. \nEllis-Davies, “Optofluidic control of rodent learning using cloaked caged glutamate” \nProc Natl Acad Sci USA 2020, 117, 6831–6835. \n  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}