Phenoxazinone Synthase Functional Mimic Activity of Mixed-ligand Copper(II) Complexes of L-proline and Diimine Ligands Conjugated with Chitosan

Phenoxazinone Synthase Functional Mimic Activity of Mixed-ligand Copper(II) Complexes of L-proline and Diimine Ligands Conjugated with Chitosan | 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 Phenoxazinone Synthase Functional Mimic Activity of Mixed-ligand Copper(II) Complexes of L-proline and Diimine Ligands Conjugated with Chitosan Liji Muthirakalayil Abraham, Manikandan Varadhan, Kugan Mahalingam, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7918334/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 Phenoxazinone synthase (PHS) is a copper-containing bacterial enzyme that is involved in vital biological oxidation processes. Mimicking these enzymatic functions using synthetic copper complexes provides valuable insights and potential applications in green catalysis. In this work, chitosan-conjugated ligands were synthesized and anchored with Cu(II) to yield mixed-ligand complexes ( 1 and 2 ) of the general formula [Cu( L )(bpy)(H 2 O)] 2+ (1) , and [Cu( L )(phen)(H 2 O)] 2+ (2) , where L represents chitosan-conjugated-proline polymer, and diimine ligands bpy and phen represent 2,2’-bipyridine and 1,10-phenanthroline, respectively. SEM, PXRD, FTIR, and various spectroscopic methods were used to characterize the ligands and complexes. ICP-OES data revealed that complexes 1 and 2 contain 10.72% and 9.81% of copper, respectively. The oxidation of o-aminophenol (OAP), used as a model substrate, monitored via UV-Visible spectrophotometry, provides valuable kinetics and mechanistic insights into substrate interactions. Interestingly, among all the complexes, 2 exhibited the highest catalytic activity, which features phenanthroline as a co-ligand, followed by 1 and then the free ligand ( L ), highlighting the influence of the co-ligand in the oxidation of o-aminophenol to phenoxazinone as the functional mimic of phenoxazinone enzyme. Notably, the same complex 2 demonstrates 72% conversion of o-aminophenol (OAP) to 2-aminophenoxazine-3-one (APX). Kinetic analysis revealed pseudo-first-order rate constants consistent with efficient oxidase mimics. These results demonstrate the potential of Cu-chitosan hybrids as sustainable, bioinspired catalysts. Copper(II) Chitosan o-aminophenol Phenoxazinone synthase Biomimetic catalysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 1. Introduction Chitosan, a linear polysaccharide derived from chitin through deacetylation, is a highly valuable biopolymer in various research fields due to its biocompatibility, biodegradability, feasibility, affordability, green nature, and versatility in different physical forms, making it suitable for use in heterogeneous catalysis, which offers numerous active sites.(Aranaz et al. 2021 ) As a supporting material in biomimetic catalysis, chitosan serves as a solid support or immobilization surface for species that can coordinate with metal complexes and can create enzyme-like conditions.(Eric et al. 2007 ) The unique structure of chitosan, with β-(1→4)-linked polymer chain units of D-glucosamine and N-acetyl-D-glucosamine, provides free amino (-NH₂) and hydroxyl (-OH) functional groups that can be used as active reaction sites to coordinate with metal ions.(Aranaz et al. 2021 ) Its hydrophilic and porous nature enhances diffusion of the aqueous-phase substrate to the catalytically active region of the chitosan support, which is beneficial in a green catalytic setting.(Reddy et al. 2006 ) The metamorphism of native chitosan is limited by its solubility in neutral solutions, mechanical strength, and thermal stability; however, the properties of chitosan can be further enhanced by cross-linking with glutaraldehyde or mixing with other polymers or inorganic supports.(Gonçalves et al. 2024 ) The ability of chitosan to mimic biological scaffolds has placed chitosan as a significant platform for sustainable and enzyme-like catalytic development.(Wang et al. 2019 ) The early development of organocatalysis was significantly influenced by a series of key discoveries made by various research groups.(Eder et al. 1971 ; Hajos and Parrish 1974 ; Agami et al. 1985 ; Karmakar and Mukhopadhyay 2024 ; Micheli et al. 1975 ; List et al. 2004 ) These studies revealed that the amino acid L-proline is an effective catalyst for direct asymmetric Michael additions and asymmetric aldol reactions. Since then, the use of L-proline and its derivatives has become fundamental in the field of organocatalysis.(Karmakar and Mukhopadhyay 2024 ) L-proline has been utilized in a wide range of organic transformations, including the asymmetric Michael and Aldol reactions, Mannich reactions, epoxidations, Knoevenagel condensations, transaminations, asymmetric α-hydroxyaminations, and various multi-component reactions.(Karmakar and Mukhopadhyay 2024 ; List et al. 2004 ) More recently, List and Barbas demonstrated that L-proline is a highly effective catalyst in asymmetric intramolecular direct aldol reactions. The unique structure of L-proline, particularly the proximity of its amine and carboxylic acid groups, enhances its ability to chelate and participate in diverse catalytic pathways.(Karmakar and Mukhopadhyay 2024 ; List et al. 2004 ) As a result, L-proline is considered one of the most efficient and versatile organocatalysts available. In 1992, Chessi and co-workers reported the spectroscopic features of Cu(II) complexes with polymeric ligands of chitosan, together with results on the air oxidation of catecholamines, such as adrenaline, etc.(Chiessi et al. 1992 ) While in 2009, Zhang et al. synthesized a novel chitosan proline conjugated palladium catalyst for direct asymmetric aldol condensation reaction.(Zhang et al. 2009 ) In 2012, Hajipour and co-workers created a novel method for the synthesis of chitosan proline conjugate. The supported palladium (Pd) nano-catalyst demonstrated moderate efficiency in Suzuki cross-coupling reactions.(Hajipour et al. 2015 ) In 2015, Baran and colleagues studied Copper(II) and palladium(II) complexes derived from water-soluble O-carboxymethyl chitosan Schiff bases.(Baran and Menteş 2015) In 2017, Baran and co-workers introduced a novel chitosan Schiff base-stabilized Pd(II) complex, which was used as a catalyst in the microwave-assisted synthesis of biaryl compounds.(Baran 2017 ) In that year, that same team designed a pincer-type Pd(II) complex anchored on a chitosan biopolymer and explored its catalytic efficiency in Suzuki cross-coupling reactions.(Baran and Menteş 2017) Khalafi and co-workers developed a chitosan-derived magnetic ionic liquid that served as a recyclable biopolymer-supported catalyst for synthesizing 1 and 5-substituted 1H-tetrazoles from various amines and nitriles using sodium azide.(Khalafi-Nezhad and Mohammadi 2013 ) Continuing efforts in this area, Anuradha et al. (2017) synthesized two novel chitosan-supported Cu(II)-Schiff base complexes and demonstrated their catalytic utility in the formation of 5-substituted 1H-tetrazoles from oximes and sodium azide, as well as in the oxidative homo-coupling of terminal alkynes.(Anuradha et al., n.d.) In 2021, Vanessza Judit Kolcsár et al. used chitosan as a chiral ligand in Ruthenium-catalyzed asymmetric transfer hydrogenation and as an organo-catalyst in asymmetric Michelle addition reaction.(Kolcsár and Szőllősi 2021) As per our literature survey, it came to know that chitosan and its metal complexes are mainly used for catalytic reactions for organic synthesis, antimicrobial activity, as a pollutant control agent, etc. Even though they have the catalytic potential, their biomimicking enzymatic behavior is not explored much. Twenty-five years after the discovery of the enzyme, the use of transition metals and their complexes as a synthetic mimic of PHS started, even though the structure of the enzyme was not yet known.(Dey and Mukherjee 2016 ; Guo et al. 2025 ) Simándi et al. ( 1987 ) first demonstrated that cobalt(II) perchlorate could oxidize OAP to APX.(SIMANDI et al. 1987) In the year 1991, they performed a kinetics study of 2-aminophenol (OAP) oxidation with a cobalt(II) complex, and discovered that catalysis followed Michaelis-Menten-type kinetics.(Szeverényi et al. 1991 ) They also determined that there was radical intermediate involvement in the oxidation of OAP. Many other groups worked on these OAP oxidation catalysed by different transition metal complexes.(Hassanein et al. 2008 ; Panja 2014 a; 2014 b; Horváth et al. 2004; Maurya et al. 2005 ; Mukherjee et al. 2007 ; Mandal et al. 2023 ; Reja et al. 2024 ; Abo El-Kheir et al. 2024 ; Shaban et al. 2019 ) Recently in 2024, El-Khalafy group studied the catalytic behavior of chitosan-supported tetra(p-methoxyphenyl) porphyrin complexes for the heterogeneous oxidation of ortho-aminophenol (OAP) to aminophenoxazinone (APX) in the presence of bicarbonate, effectively mimicking the activity of phenoxazinone synthase. Among these, the Cu(II) complex, anchored on chitosan support, demonstrated the highest catalytic performance, achieving 87% conversion of OAP within 90 minutes under optimized conditions (El-Khalafy et al. 2024) Relying on our survey, there is only one chitosan-supported Cu(II) complex reported, which explores the phenoxazinone synthase activity of OAP to APX.(El-Khalafy et al. 2024) So, our focus is mainly on observing the biomimicking enzymatic behavior of our novel chitosan proline conjugated mixed ligand copper(II) complexes. Our research group has discovered previously that the mixed ligand copper(II) complexes containing diimines have better anticancer and biomimicking catalytic behaviors.(Karpagam et al. 2019 ; Kartikeyan et al. 2023 ) Herein, we investigated the catalytic potential of two novel Cu(II) complexes of the general formula [Cu( L )(bpy)(H 2 O)] 2+ ( 1 ), and [Cu( L )(phen)(H 2 O)] 2+ ( 2 ), where L represents chitosan-conjugated-proline polymer, and diimine ligands bpy and phen represent 2,2’-bipyridine and 1,10-phenanthroline, respectively. The synthesized complexes were characterized by FT-IR, powder XRD, solid-state absorption spectroscopy, and SEM analysis. The amide bond formation of the chitosan-L-proline polymer was confirmed by FT-IR spectroscopy. The oxidation of o-aminophenol as a model substrate, monitored via UV-Visible spectrophotometry, provides the kinetics and mechanistic insights into substrate interactions. Interestingly, phen-containing complex 2 exhibited the highest catalytic activity compared to 1 and free ligand ( L ), highlighting the importance of the copper(II) center and influence of the co-ligand phen in the oxidation of o-aminophenol to phenoxazinone as the functional mimic of phenoxazinone enzyme. Extensive kinetic calculations have been performed for catalytic studies to put forward the most probable mechanistic pathways. The spectroscopic data reveal distinct electronic transitions corresponding to oxidation processes, providing a way to examine the influence of the ligand environment on catalytic efficiency. 2. Experimental Section 2.1 Materials and Methods The chemicals utilized in this study, including Chitosan with 75% deacetylation, L-Proline, Cu(II) chloride dihydrate (CuCl 2 ⋅2H 2 O), are obtained from HI-MEDIA (India), and the substrate o-aminophenol was purchased from Merck. We procured dichloromethane from Finar, diethyl ether from SRL, ethanol and methanol from Emplura, and ultra-pure water (18.2 µΩ) was utilized for all the experiments. The solvents were dried according to standard procedures. Thin-layer chromatography was performed on Merck Pre-coated silica gel plates, and all column chromatography was conducted using Merck Silica Gel (60–120 mesh size) with analytical-grade solvent. The NMR spectra were recorded on a Bruker Advance II-400 MHz frequency for proton nuclear magnetic resonance ( 1 H NMR) and 101 MHz frequency for carbon nuclear magnetic resonance. An Agilent Cary 630FTIR operating at a resolution of 2 cm − 1 from 4000 to 400 cm − 1 was used for obtaining the infrared spectra. Powder X-ray diffraction (PXRD) studies of the ligands and complexes were performed using a PANalytical diffractometer with Cu-K α radiation (λ = 1.5406 Å) to analyze the crystalline and amorphous nature. Surface morphology of the ligands was obtained using a TESCAN VEGA3 SBH scanning electron microscope at an accelerating voltage of 15 kV. The UV-Vis absorption titrations were performed using a Shimadzu UV-2450 (Kyoto, Japan) spectrophotometer with cuvettes featuring a path length of 1 cm. The copper-metal content of the complexes was studied with an Agilent 5800 Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES). 2.2 Synthesis of ligand L and complexes 1 and 2 The ligand ( L ) (Scheme 1a) was synthesized by following the reported procedures. The corresponding complexes, [Cu( L )(bpy)(H 2 O)] 2+ ( 1 ), and [Cu( L )(phen)(H 2 O)] 2+ ( 2 ), were synthesized by reacting the corresponding ligand with copper (II) chloride dihydrate in methanol, followed by adding the diimines (bpy and phen) as illustrated in Scheme 1b. 2.2. Synthesis of Boc-L-proline L-proline (20 mmol) was dissolved in 1N NaOH (20 mL) in isopropanol (20 mL), and to this mixture, a Di-tert-butyl pyrocarbonate (BOC-anhydride) (6 mL, 26 mmol) dissolved in isopropanol (10 mL) was added drop-wise following 1N NaOH (20 mL) to the above reaction mixture. The resulting solution was allowed to stir for 2 h at room temperature. Then the reaction mixture was washed with light petroleum ether (bp 40–60 ⁰C) (20 mL), acidified to pH 3.0 with 2N H 2 SO 4, and finally extracted with chloroform (3 × 20 mL). The organic layer was dried over anhydrous Na 2 SO 4 and evaporated under reduced pressure to yield the crude product (Scheme 1a), which was then crystallized from a mixture of chloroform and petroleum ether (bp 40–60°C). (Yield = 80%). 2.3. Synthesis of Chito-proline Conjugate-Ligand (L) To synthesize the ligand L , 1g of chitosan powder was dissolved in 2% (w/v) acetic acid, and the mixture was vigorously stirred by a magnetic stirrer at room temperature until the polymer was completely dissolved. To the above solution, 1.07g of Boc-proline and 1.50 g of DCC in 10 mL of methanol were added dropwise, and the mixture was stirred for 2 h. After the reaction was completed, the product was filtered, concentrated under vacuum, and washed with 95% ethanol to remove excess DCC. The deprotection of Boc was carried out by using TFA/DCM (1:1 ratio). The final product (Scheme 1a ) was dried in a vacuum oven at 35 ⁰C for 4 h. (Yield = 86%) 2.4 Synthesis of Complex 1 0.5 g of Ligand ( L ) was dissolved in 50 mL of 2% acetic acid, and then 1 mM of the methanolic solution of CuCl 2 ⋅2H 2 O was added dropwise. This solution was then allowed to reflux at 65°C for 3 hours. Then allowed the reaction mixture to cool to room temperature. To this clear solution, a methanolic (2 mL) solution of 2,2′-bipyridine (1 mmol, 0.156 g) was added dropwise, and stirring was continued for another 1.5 hours. The blue color precipitate (Scheme 1b) formed was collected by filtration, washed with cold diethyl ether, and dried in a vacuum desiccator over anhydrous CaCl 2 . (Yield = 78%) 2.5 Synthesis of Complex 2 To synthesize complex 2 [Cu( L )(phen)(H 2 O)] 2+ , 1,10-phenanthroline was added instead of 2,2’-bipyridine, and the same procedure was followed as that for the synthesis of complex 1 (Scheme 1b). Blue color precipitate was isolated, washed with cold diethyl ether, and dried in a vacuum desiccator containing anhydrous CaCl 2 (Yield = 76%) 2.6 Phenoxazinone Synthase-like Activity of Chitosan-Proline Cu(II) Complexes The phenoxazinone synthase-like activity of ligand L and its complexes 1 and 2 was studied using o-aminophenol (OAP) as a model substrate in a methanol medium under aerobic conditions at room temperature.(El-Khalafy et al. 2024) For this study, 0.2 mg/mL solutions of complexes 1 and 2 were treated with 0.5 to 3 mM of OAP under aerobic conditions in methanol. The reaction was monitored spectrophotometrically with an increase in the absorbance band of the phenoxazinone at 425 nm at 5-minute time intervals. The catalytic activity was monitored as per the Michaelis-Menten kinetics model. All the compounds showed saturation kinetics, and K m , V max , and rate constant for dissociation of substrates (i.e., turnover number, k cat ) were calculated for all the compounds using a Lineweaver-Burk plot of 1/V vs 1/[S], using the equation, $$\:\:\:\frac{1}{V}=\frac{{K}_{M}}{{V}_{max}}+\frac{1}{\left[S\right]}+\frac{1}{{V}_{max}}$$ Where V is the reaction velocity (the reaction rate), K m is the Michaelis-Menten constant, V max is the maximum reaction velocity, and [S] is the substrate concentration.(Johnson and Goody 2011) 2.8 Catalytic conversion of OAP using complex 2 As confirmation of the above spectroscopic catalytic conversion of OAP to APX, we attempted the same process using the best catalyst, i.e., complex 2 (Scheme 2 ). Where 0.5 mmol of OAP was dissolved in 15 mL of methanol, to this solution 5 mg of complex 2 was added. The solution was kept stirring for over 6 hr. Then, the product was separated by column chromatography and confirmed by 1 H and 13 C NMR techniques ( Yield = 72%). 3. Results and Discussion The synthesized ligand and complexes were characterized using advanced analytical techniques like NMR, FT-IR, UV-Vis, scanning electron microscope, and powder X-ray analyses. The percentage of copper present in each complex is measured using ICP-OES, which showed a value of 10.72% for complex 1 and 9.81% for complex 2 . The formation of the oxidative product of the catalytic reaction is further confirmed by NMR analysis. 3.1 NMR Analysis of Chitosan-proline Conjugate (L) 1 H NMR spectra of Boc-L-proline and chito-proline conjugate (L) is showed in Figure S1 (a) and S1(b) respectively. The peaks appear at δ = 1.50–2.01 ppm (m, 2H, CH 2 ), δ = 2.40 ppm (m, 2H, CH 2 ), δ = 3.32–3.34 ppm (m, 2H, CH 2 ), and δ = 4.31–4.32 ppm (t, 1H, CH) are due to the L-proline moiety present in the molecule. Figure S1 (a) shows a singlet at δ = 2.18 ppm, corresponding to the Boc protection, and the same peak disappeared in Figure S1 (b), confirming the formation of the title compound. In the spectra, the chemical shift value of δ = 4.5 ppm representing the H 1 proton of chitosan, and the other peaks are δ = 3–4 ppm (H 2 -H 6 protons), δ = 2 ppm respect to acetamido proton (-NHCO-CH 3 ), δ = 3.25 ppm respect to -CH 2 -NH 2 protons (CH 2 ) and δ = 4.5 ppm is due to acetyl proton (C-H) of chitosan. The de-acetyl proton of the chitosan appeared at δ = 3.3–3.4 ppm. So, the above observation indicates that the deacetylated chitosan reacted with Boc L-proline and formed the desired title ligand L (chito-proline conjugate). In Figure S1 (c), the ¹³C NMR spectrum of Boc-L-proline shows distinct signals characteristic of the BOC protecting group. A very intense peak at δ = 30.15 ppm corresponds to the three methyl carbons of the BOC group, while a signal at δ = 81.50 ppm is attributed to the tertiary carbon of the BOC moiety. Additionally, the carbonyl carbon of the BOC group appears at δ = 179.96 ppm. In contrast, Figure S1 (d), representing the spectrum of chito-proline conjugate L , shows the complete disappearance of these peaks, confirming the successful removal of the BOC group from the chitosan-proline amide structure. 3.2 Scanning Electron Microscopy (SEM) Analysis The characterization of the morphology of the chitosan and chito-proline conjugate ( L ) composite was carried out using the SEM technique. The SEM images of the chitosan polymer were a bed-like folding surface before the reaction,(Castanheiro 2021 ) whereas after incorporating L-proline, the surface morphology was changed to a rod-like structure, as shown in Figs. 1 – 2 , revealing that the polymer chitosan has successfully reacted with L-proline. This also demonstrates that the biopolymer is formed with a nearly uniform distribution, with no intense particle agglomeration due to the formation of cross-linkages. 3.3 FT-IR Spectral Analysis The FTIR spectral analysis (Figure S2-S5) provides strong evidence for the successful formation of the chito-proline conjugate ligand L and its coordination with metal centers in complexes 1 and 2. The characteristic ν(O–C–O) stretching vibration, appearing consistently in the range of 1050–1100 cm⁻¹, is observed for both the free ligand and all three complexes, confirming the presence of the chitosan backbone and its retained structural integrity upon complexation. A strong and sharp ν(C–H) out-of-plane bending vibration observed in the 610–620 cm⁻¹ region further supports the conjugation of the chitosan ring with the proline moiety, characteristic of the polymeric structure of the ligand. The amide carbonyl stretching (ν(C = O)) band is observed at 1699 cm⁻¹ in the ligand L , indicating the presence of an amide linkage from the proline-chitosan conjugate. Interestingly, this band shifts to 1601 cm⁻¹ in complex 1 and 1610 cm⁻¹ in complex 2 , suggesting coordination of the carbonyl oxygen to the metal center, which weakens the C = O bond and shifts its frequency to lower wavenumbers. The N–H stretching vibrations (ν(N–H)) are found at 3268 cm⁻¹ for the free ligand, more prominently shifted to 2897 cm⁻¹ and 3081 cm⁻¹ in complexes 1 and 2 , respectively. These shifts are indicative of changes in the hydrogen bonding environment and potential coordination involving the nitrogen atom. In complexes 1 and 2 , the ν(N-H) and ν(C-H) stretching bands are merged and broadened, reflecting a complex and possibly more rigid coordination environment. Additionally, new bands appearing at 1598 cm⁻¹ for 1 and 1566 cm⁻¹ for 2 can be assigned to ν(C-N) stretching vibrations, suggesting the involvement of a nitrogen donor atom (likely from a co-ligand such as 2,2’-bipyridine or 1,10-phenanthroline) coordinating to the metal center. Finally, the appearance of low-frequency bands in the 440 − 410 cm⁻¹ region in all metal complexes corresponds to ν(Cu-N) stretching vibrations, affirming the successful coordination of the ligand/co-ligand system with the Cu(II) ion. All the data is tabulated and given in Table S1 . These data collectively validate the formation of metal-ligand complexes with chitosan-proline as the primary ligand and the co-ligands aiding in chelation. 3.4 Powder X-Ray Diffraction (PXRD) Analysis The diffractive region of chitosan (Fig. 3 a) shows the expected broad amorphous hump centered at 2θ ≈ 20°, characteristic of semi-crystalline chitosan. While peaks of L-proline (Fig. 3 b) are observed at 2θ = 20 0 , 25 0 , 30 0 , 35 0 , 40 0 . The peaks of the chitosan-proline conjugate (Fig. 3 c) are observed at 2θ = 20 0 -40 0 . The figure shows a significant shift in the diffractioun peaks and the diffraction pattern, with a broad amorphous peak, indicating that there was molecular miscibility and interaction between the components. In complex 1 , the PXRD pattern shows peaks at 2θ = 12.2°, 18.54°, 19.56°, 26°, 28°, 40°, and 50° (Fig. 4 ). The intense peak at 12.2° is particularly notable and may indicate complex formation with 2,2’-bipyridine. The broad peak typically seen around 20° in pure chitosan becomes split into sharper peaks at 18.54° and 19.56°, suggesting a transition from an amorphous to a more crystalline structure upon metal-ligand coordination, which is further confirmed by analyzing the shift or the disappearance of the sharper peak at 16°, which is seen for the pure CuCl 2 · 2H 2 O metal salt.(AbouElleef et al. 2021 ) For complex 2 , diffraction peaks are observed at 2θ = 12°, 18°, 20.54°, 35°, 40°, and 46° (Fig. 5 ). Similar to 1 , the strong peak at 12° suggests complexation, likely with 1,10-phenanthroline. The broad peak of the ligand splits into two distinct peaks at 18° and 20.54°, reinforcing the idea that the coordination process induces crystallinity. Other peaks support the crystalline nature of the complexes. The PXRD patterns of complexes 1 and 2 exhibit sharp and intense peaks, indicating a crystalline nature in contrast to the broad, diffuse pattern observed for the amorphous ligand. This suggests that upon coordination with metal ions, the molecular chains become highly ordered, forming a repeating crystalline structure throughout the material. 3.5. Solid-state UV-Vis Spectroscopy The solid-state UV-Vis spectra of the ligand L and the complexes 1–2 are shown in Fig. 6 , and the intermediate complex [CuLCl 2 (H 2 O)] in Fig. 7 . The chito-proline conjugate ligand exhibits absorption bands below 300 nm, attributable to intraligand π→π* and n→π* transitions around 210 nm and 241 nm, and displays no absorption features in the visible region.(González-Martínez et al. 2023 )(González-Martínez et al. 2023 )(González-Martínez et al. 2023 )(González-Martínez et al. 2023 )(González-Martínez et al. 2023 )(González-Martínez et al. 2023 )(González-Martínez et al.) Whereas the Cu(II) complexes 1 and 2 display intense ligand-based bands in the UV region, together with broad absorptions extending into the visible region. The bands in the range 250–320 nm may be assigned to ligand-to-metal charge transfer (LMCT) transitions from nitrogen donor atoms to the Cu(II) center. The intermediate complex, [CuLCl₂(H 2 O)], exhibited a broad band centered at 847 nm, assignable to the transition typical of a \(\:{d}^{9}\) Cu(II) center with a N, O donor environment. Coordination of the strong field diimine co-ligands to the metal center shifted the corresponding absorption maxima distinctly toward higher energy. Complex 1 showed a band at 673 nm, and complex 2 displayed a similar band at 663 nm. This blue shift (Δλ ≈ 170–180 nm) indicates a considerable increase in ligand-field strength around the copper center upon the introduction of the aromatic diimine ligands. The slightly greater shift for the phenanthroline complex (663 nm) compared to bipyridine (673 nm) correlates with its higher π-acceptor ability, increased aromaticity, and more extensive conjugation. These d-d bands are typical of Cu(II) complexes in distorted square-planar or square-pyramidal environments and reflect the influence of Jahn-Teller distortion. Appearance of charge transfer and d-d bands compared to the free ligand clearly confirms coordination of Cu(II) and supports the assignment of new coordination domains observed in PXRD. 3.6 UV-Vis Kinetic Analysis of Catalytic Oxidation of OAP The uncoordinated ligand L and complexes 1 and 2 were tested for PHS synthase activity under homogeneous conditions by dissolving 0.2 mg/mL of them and 0.5–3 mM of o-aminophenol (OAP) in methanol using UV-Vis absorption spectroscopy. Initially, oxidation of OAP to phenoxazinone chromophore was monitored with respect to the rate of increase in absorption band at λ max ~ 425 nm for 60 minutes with 5-minute intervals, which is a characteristic band of the phenoxazinone chromophore. Later, the spectra were monitored at 1-hour intervals for 6 hours (Figs. 8 b and 8 c), and the time was increased until the reaction reached saturation (Figs. 9 ). The resulting absorption spectrum is used for further analysis. Interestingly, both complexes 1 and 2 increase the absorption intensity at 425 nm (Figs. 8 b and 8 c) compared to the free ligand L (Fig. 8 a). A blank experiment without a catalyst under identical conditions does not show any significant increment in the band intensity at 425 nm. At 0.5 mM OAP, the ligand L doesn’t show any increment in the absorption band around the 415–435 region; rather, the band intensity decreases over the period of 60 minutes (Fig. 8 a). Among 1 and 2 , the phen complex 2 displays a higher intensity of absorption band at 425 nm than 1 , revealing that 2 involves more catalytic conversion than 1 . These spectral behaviours clearly reveal that both complexes are active catalysts for the aerobic oxidation of OAP to phenoxazinone chromophore. So the kinetic study of 1 and 2 was performed by the initial slope method following the rate of increase in absorption of the band at λ max ~ 425 nm. The initial first-order rate constants for the reactions were determined from the plots of log(A ∞/ A ∞− A t ) versus time (Fig. 10 ). To investigate the dependence of rate constants on substrate concentration, the catalytic reactions were conducted using six varying concentrations of the substrate while keeping the catalyst concentration constant (Fig. 11 ). The rate ( k obs ) is calculated by taking the slope of the log(A ∞/ A ∞− A t ) v/s time plot of the initial substrate concentration. At higher substrate concentrations, the initial rate method followed saturation kinetics, while at lower concentrations, first-order kinetics were observed. Hence, the Michaelis-Menten model commonly used in enzyme kinetics, along with its linearized form (Lineweaver-Burk plot), was utilized to calculate the rate of the reactions and various kinetic parameters (Fig. 12 ). From the Lineweaver-Burk plot, the V max value is obtained by taking the y-intercept value of the plot, which gives 1/ V max ; hence, V max can be calculated by taking the reciprocal of the y-intercept. Further, the slope of the Lineweaver-Burk plot gives the value corresponding to [K m /V max ]. And the K m value is computed by multiplying V max by the slope of the graph. Where the free ligand ( L ) showed no measurable activity, confirming that Cu(II) coordination is essential for catalysis. Complex 1 displayed a lower V max ( 2.17 × 10⁻³ ) and a higher K m ( 1.84 ), indicating weaker substrate binding and reduced efficiency compared to that of complex 2 , which exhibited the highest catalytic performance, with a V max of 3.13 × 10⁻³ and a relatively low K m of 1.02 , reflecting both a fast turnover rate and good substrate affinity. These results highlight the most promising phenoxazinone synthase mimic efficiency of the complex 2 , in this series, consistent with its formation of stronger absorption intensity at 425 nm in the UV-Vis spectra. Notably, the same complex 2 demonstrates 72% conversion of OAP to APX. The possible mechanism of the catalytic cycle is illustrated in Fig. 13 .(Shaban et al. 2019 ) Table 1 Comparison of the catalytic activity of ligand L and complexes 1 and 2. S/no Substrate Catalyst V max (10 − 3 ) K m 1. OAP Ligand (L) - - 2. OAP Complex 1 2.17 1.84 3. OAP Complex 2 3.13 1.02 3.7 Characterization of APX Isolated from the Reaction After the catalytic conversion of OAP to APX by the efficient catalyst 2 , the product APX was isolated through column chromatography using 1% MeOH/ CHCl 3 . The product formation was confirmed by the 1 H NMR spectrum and ESI-MS illustrated in Figures S6 and S7, respectively. The 1 H NMR spectrum of APX shows broad singlet δ = 5.143(s, 2H, NH 2 ) corresponds to protons of NH 2 attached to the APX and an peak comes around δ = 6.489(s, 1H, CH) which corresponds to proton attached next to the NH 2 group and a peak appears at δ = 7.383–7.351(dd,1H, CH) which correspond to proton attached near to the carbonyl group; and two doublet arises at δ = 7.390 (d,1H, CH) and δ = 7.557(d,1H, CH) of the aromatic ring and an δ = 7.756–7.745 (m,1H, CH), δ = 7.745–7.748 (m, 1H, CH) corresponds to the proton of aromatic hydrocarbon ring. 3.8 Conclusions The synthesis and rigorous evaluation of phenoxazinone synthase functional mimetic activity of two novel mixed ligand Cu(II) complexes containing chitosan-conjugated-proline and diimines ( 1 and 2) , of the general formula [Cu( L )(bpy)(H 2 O)] 2+ ( 1 ), and [Cu( L )(phen)(H 2 O)] 2+ ( 2 ), where L represents chitosan-conjugated-proline polymer, and diimine ligands bpy and phen represent 2,2’-bipyridine and 1,10-phenanthroline, respectively. The characterization of these synthesized materials was comprehensive, employing Fourier-transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), nuclear magnetic resonance spectroscopy, powder X-ray diffraction (XRD), and solid-state absorption spectroscopic techniques. FT-IR spectroscopy confirmed the successful synthesis of the ligand L and its complexation with Cu(II) ions and the addition of co-ligands 2,2’-bipyridine and 1,10-phenanthroline to form complexes 1 and 2 , respectively. ICP-OES data revealed that complexes 1 and 2 contain 10.72% and 9.81% of copper, respectively. Powder XRD analysis demonstrated the amorphous and crystalline properties of ligands and complexes, which tells the proper arrangement of copper ions incorporated in the Chitosan proline amide polymer, ultimately forming the active catalyst complexes 1 and 2 . This is further supported by the solid-state absorption spectroscopy, with the broad absorptions of d-d transitions of copper complexes in the 550–800 nm region, which were absent in the ligand spectra. Using Michaelis-Menten kinetics, the catalytic activity of the complexes for the conversion of OAP to APX was assessed by means of electronic absorption spectroscopy, and the results ascertained significant activity as a phenoxazinone synthase mimic, with a V max value of 0.00217 min − 1 with a K m value of 1.84 for complex 1 and V max value of 0.00313 min − 1 with a K m value of 1.02 for complex 2. This indicates that complex 2 was superior to complex 1 both in V max , which is higher, and the K m value, which is close to zero, indicating strong substrate-catalyst binding affinity. Notably, the same complex 2 demonstrates 72% conversion of OAP to APX. Further convincing evidence for the oxidation of OAP was the identification of the oxidized product APX by NMR and ESI-MS spectral analysis. Thus, when we compared with previously reported chitosan-based complexes, whose V max (1.4459 min − 1 ) and K m (0.0750 M) values are better than those of our complexes. Though our complexes are less effective than already reported porphyrin-containing Cu(II) complexes, our complexes are low-cost and easy to handle compared to those. Overall, the findings underscore the potential of the chitosan-based polymeric metal complexes to act as a robust catalyst for OAP oxidation, giving hope for expanding their utility in synthetic and industrial chemistry. This research aims to focus on the advances in the field of biomimetic catalysis and also provides a foundation for future studies exploring similar complexes for various catalytic applications. Declarations Supplementary Information The online version contains supplementary material available at Conflict of interest : The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contribution Liji Muthirakalayil Abraham: Formal analysis, Investigation, Methodology, Conceptualization, Data curation, Validation, Writing – original draft. Manikandan Varadhan: Formal analysis, Investigation, Conceptualization, Methodology. Kugan Mahalingam: Formal analysis, Investigation, Data curation. Thangaraja Chinnathangavel: Data curation, Investigation. Venugopal Rajendiran: Conceptualization, Data curation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – review & editing. Acknowledgement V. 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Schemes Schemes 1 and 2 are available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files VRLMAChemPapersSupplementaryFile.docx GA.png Graphical Abstract Schemes.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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1","display":"","copyAsset":false,"role":"figure","size":568697,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images represent the state before the conjugation of L-proline with chitosan.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7918334/v1/3e7f284aed17fcd2606f6a59.png"},{"id":94162702,"identity":"85eaf983-50b7-48a8-87e4-9e512d25cd24","added_by":"auto","created_at":"2025-10-23 05:19:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":561688,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images are represented after the conjugation of L-proline with chitosan.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7918334/v1/19d11e1776a2bc6967670beb.png"},{"id":94161943,"identity":"6660eb4c-0d4b-44cf-bd0c-5ed400c54093","added_by":"auto","created_at":"2025-10-23 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05:11:40","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":63892,"visible":true,"origin":"","legend":"\u003cp\u003eUV-Vis spectral scans showing a decrease of the APX band at 425 nm after the addition of 0.5 mM OAP to the \u003cstrong\u003e(a)\u003c/strong\u003e ligand (\u003cstrong\u003eL\u003c/strong\u003e), and an increase of the same band for \u003cstrong\u003e(b)\u003c/strong\u003e complex \u003cstrong\u003e1\u003c/strong\u003e, and \u003cstrong\u003e(c)\u003c/strong\u003e complex \u003cstrong\u003e2\u003c/strong\u003e, in methanol at 25 ⁰C. The spectra were recorded at 5-minute intervals for 1 hour.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7918334/v1/ca9181475ed623b871fe502f.png"},{"id":94162540,"identity":"ece89508-a0f6-485d-94d7-e40fe8e2febd","added_by":"auto","created_at":"2025-10-23 05:11:40","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":49979,"visible":true,"origin":"","legend":"\u003cp\u003eUV-Vis spectral scanning shows the saturation curve for all four concentrations of OAP after addition to \u003cstrong\u003e(a)\u003c/strong\u003e complex \u003cstrong\u003e1, \u003c/strong\u003eand\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Complex\u003cstrong\u003e 2 \u003c/strong\u003ein methanol at 25 \u003csup\u003eᴼ\u003c/sup\u003eC. The spectra were recorded at 5-minute intervals for 1 hour, then at 1-hour intervals for 6 hours, and at the 12\u003csup\u003eth\u003c/sup\u003e hour.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7918334/v1/c28c3d439a5648f1c10009a4.png"},{"id":94161956,"identity":"6f2d896b-eebf-4009-b646-55a474cb17a7","added_by":"auto","created_at":"2025-10-23 05:03:40","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":54846,"visible":true,"origin":"","legend":"\u003cp\u003ePlot of log(A\u003csub\u003e∞/\u003c/sub\u003eA\u003csub\u003e∞-\u003c/sub\u003eA\u003csub\u003et\u003c/sub\u003e) vs time observed for \u003cstrong\u003e(a)\u003c/strong\u003e complex \u003cstrong\u003e1 \u003c/strong\u003eand\u003cstrong\u003e (b) \u003c/strong\u003ecomplex\u003cstrong\u003e 2\u003c/strong\u003e for the reaction of 0.1 mg/mL complex with 5×10\u003csup\u003e-4 \u003c/sup\u003eM of o-aminophenol in methanol at 25 \u003csup\u003eᴼ\u003c/sup\u003eC.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-7918334/v1/0d5aea54df231de54d6b4f6b.png"},{"id":94161968,"identity":"0ec892d5-9903-4371-8a8a-abf4ab36a575","added_by":"auto","created_at":"2025-10-23 05:03:40","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":43834,"visible":true,"origin":"","legend":"\u003cp\u003eKinetic plot for the oxidation of OAP to APX by varying the substrate concentration from 0-3 mM \u003cstrong\u003e(a)\u003c/strong\u003e complex\u003cstrong\u003e 1 \u003c/strong\u003eand \u003cstrong\u003e(b)\u003c/strong\u003e complex \u003cstrong\u003e2,\u003c/strong\u003ewhere the catalyst concentration was kept as 0.1 mg/mL.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-7918334/v1/662182f6df6c20697b60975b.png"},{"id":94162705,"identity":"624148d2-b376-4f19-94b2-acf61f4d7a62","added_by":"auto","created_at":"2025-10-23 05:19:40","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":51776,"visible":true,"origin":"","legend":"\u003cp\u003eLineweaver-Burk plot for the oxidation of OAP to APX by \u003cstrong\u003e(a)\u003c/strong\u003e complex \u003cstrong\u003e1\u003c/strong\u003e, and (b) complex \u003cstrong\u003e2.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-7918334/v1/d3addc198bdc23fe50e8c8c2.png"},{"id":94162539,"identity":"ef268b91-f50e-46ef-8da9-998bf184f0a0","added_by":"auto","created_at":"2025-10-23 05:11:40","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":67531,"visible":true,"origin":"","legend":"\u003cp\u003eProposed scheme for the plausible mechanism for the catalytic oxidation of o-aminophenol by the Cu(II) complexes.\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-7918334/v1/4dda214a7ba9f8447f3f1d23.png"},{"id":95226843,"identity":"dec08f9d-7aae-4224-9e47-6b4754b22920","added_by":"auto","created_at":"2025-11-05 16:31:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2412262,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7918334/v1/7d4c3f9e-7ceb-495d-a1dc-54cf8985a9c6.pdf"},{"id":94161959,"identity":"db8208f4-18d2-4b57-be0d-bd2607f5409b","added_by":"auto","created_at":"2025-10-23 05:03:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6605399,"visible":true,"origin":"","legend":"","description":"","filename":"VRLMAChemPapersSupplementaryFile.docx","url":"https://assets-eu.researchsquare.com/files/rs-7918334/v1/54b489e6d5bc13b22ae0e579.docx"},{"id":94161944,"identity":"e450d3d8-58ac-46d9-aea3-f7307961ea67","added_by":"auto","created_at":"2025-10-23 05:03:40","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":100596,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical Abstract\u003c/p\u003e","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-7918334/v1/6d29252dc125f68f8efc18da.png"},{"id":94162532,"identity":"5c01c977-efdc-41b7-9c8d-30bcd23f1804","added_by":"auto","created_at":"2025-10-23 05:11:40","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":71802,"visible":true,"origin":"","legend":"","description":"","filename":"Schemes.docx","url":"https://assets-eu.researchsquare.com/files/rs-7918334/v1/399acb835436ee82e0e52db3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Phenoxazinone Synthase Functional Mimic Activity of Mixed-ligand Copper(II) Complexes of L-proline and Diimine Ligands Conjugated with Chitosan","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eChitosan, a linear polysaccharide derived from chitin through deacetylation, is a highly valuable biopolymer in various research fields due to its biocompatibility, biodegradability, feasibility, affordability, green nature, and versatility in different physical forms, making it suitable for use in heterogeneous catalysis, which offers numerous active sites.(Aranaz et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) As a supporting material in biomimetic catalysis, chitosan serves as a solid support or immobilization surface for species that can coordinate with metal complexes and can create enzyme-like conditions.(Eric et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) The unique structure of chitosan, with β-(1\u0026rarr;4)-linked polymer chain units of D-glucosamine and N-acetyl-D-glucosamine, provides free amino (-NH₂) and hydroxyl (-OH) functional groups that can be used as active reaction sites to coordinate with metal ions.(Aranaz et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) Its hydrophilic and porous nature enhances diffusion of the aqueous-phase substrate to the catalytically active region of the chitosan support, which is beneficial in a green catalytic setting.(Reddy et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) The metamorphism of native chitosan is limited by its solubility in neutral solutions, mechanical strength, and thermal stability; however, the properties of chitosan can be further enhanced by cross-linking with glutaraldehyde or mixing with other polymers or inorganic supports.(Gon\u0026ccedil;alves et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) The ability of chitosan to mimic biological scaffolds has placed chitosan as a significant platform for sustainable and enzyme-like catalytic development.(Wang et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eThe early development of organocatalysis was significantly influenced by a series of key discoveries made by various research groups.(Eder et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1971\u003c/span\u003e; Hajos and Parrish \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Agami et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Karmakar and Mukhopadhyay \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Micheli et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1975\u003c/span\u003e; List et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) These studies revealed that the amino acid L-proline is an effective catalyst for direct asymmetric Michael additions and asymmetric aldol reactions. Since then, the use of L-proline and its derivatives has become fundamental in the field of organocatalysis.(Karmakar and Mukhopadhyay \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) L-proline has been utilized in a wide range of organic transformations, including the asymmetric Michael and Aldol reactions, Mannich reactions, epoxidations, Knoevenagel condensations, transaminations, asymmetric α-hydroxyaminations, and various multi-component reactions.(Karmakar and Mukhopadhyay \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; List et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) More recently, List and Barbas demonstrated that L-proline is a highly effective catalyst in asymmetric intramolecular direct aldol reactions. The unique structure of L-proline, particularly the proximity of its amine and carboxylic acid groups, enhances its ability to chelate and participate in diverse catalytic pathways.(Karmakar and Mukhopadhyay \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; List et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) As a result, L-proline is considered one of the most efficient and versatile organocatalysts available.\u003c/p\u003e\u003cp\u003eIn 1992, Chessi and co-workers reported the spectroscopic features of Cu(II) complexes with polymeric ligands of chitosan, together with results on the air oxidation of catecholamines, such as adrenaline, etc.(Chiessi et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1992\u003c/span\u003e) While in 2009, Zhang et al. synthesized a novel chitosan proline conjugated palladium catalyst for direct asymmetric aldol condensation reaction.(Zhang et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) In 2012, Hajipour and co-workers created a novel method for the synthesis of chitosan proline conjugate. The supported palladium (Pd) nano-catalyst demonstrated moderate efficiency in Suzuki cross-coupling reactions.(Hajipour et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) In 2015, Baran and colleagues studied Copper(II) and palladium(II) complexes derived from water-soluble O-carboxymethyl chitosan Schiff bases.(Baran and Menteş 2015) In 2017, Baran and co-workers introduced a novel chitosan Schiff base-stabilized Pd(II) complex, which was used as a catalyst in the microwave-assisted synthesis of biaryl compounds.(Baran \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) In that year, that same team designed a pincer-type Pd(II) complex anchored on a chitosan biopolymer and explored its catalytic efficiency in Suzuki cross-coupling reactions.(Baran and Menteş 2017) Khalafi and co-workers developed a chitosan-derived magnetic ionic liquid that served as a recyclable biopolymer-supported catalyst for synthesizing 1 and 5-substituted 1H-tetrazoles from various amines and nitriles using sodium azide.(Khalafi-Nezhad and Mohammadi \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) Continuing efforts in this area, Anuradha et al. (2017) synthesized two novel chitosan-supported Cu(II)-Schiff base complexes and demonstrated their catalytic utility in the formation of 5-substituted 1H-tetrazoles from oximes and sodium azide, as well as in the oxidative homo-coupling of terminal alkynes.(Anuradha et al., n.d.) In 2021, Vanessza Judit Kolcs\u0026aacute;r et al. used chitosan as a chiral ligand in Ruthenium-catalyzed asymmetric transfer hydrogenation and as an organo-catalyst in asymmetric Michelle addition reaction.(Kolcs\u0026aacute;r and Szőllősi 2021)\u003c/p\u003e\u003cp\u003eAs per our literature survey, it came to know that chitosan and its metal complexes are mainly used for catalytic reactions for organic synthesis, antimicrobial activity, as a pollutant control agent, etc. Even though they have the catalytic potential, their biomimicking enzymatic behavior is not explored much. Twenty-five years after the discovery of the enzyme, the use of transition metals and their complexes as a synthetic mimic of PHS started, even though the structure of the enzyme was not yet known.(Dey and Mukherjee \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Guo et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) Sim\u0026aacute;ndi et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1987\u003c/span\u003e) first demonstrated that cobalt(II) perchlorate could oxidize OAP to APX.(SIMANDI et al. 1987) In the year 1991, they performed a kinetics study of 2-aminophenol (OAP) oxidation with a cobalt(II) complex, and discovered that catalysis followed Michaelis-Menten-type kinetics.(Szever\u0026eacute;nyi et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1991\u003c/span\u003e) They also determined that there was radical intermediate involvement in the oxidation of OAP. Many other groups worked on these OAP oxidation catalysed by different transition metal complexes.(Hassanein et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Panja \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003ea; \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003eb; Horv\u0026aacute;th et al. 2004; Maurya et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Mukherjee et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Mandal et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Reja et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Abo El-Kheir et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Shaban et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) Recently in 2024, El-Khalafy group studied the catalytic behavior of chitosan-supported tetra(p-methoxyphenyl) porphyrin complexes for the heterogeneous oxidation of ortho-aminophenol (OAP) to aminophenoxazinone (APX) in the presence of bicarbonate, effectively mimicking the activity of phenoxazinone synthase. Among these, the Cu(II) complex, anchored on chitosan support, demonstrated the highest catalytic performance, achieving 87% conversion of OAP within 90 minutes under optimized conditions (El-Khalafy et al. 2024)\u003c/p\u003e\u003cp\u003eRelying on our survey, there is only one chitosan-supported Cu(II) complex reported, which explores the phenoxazinone synthase activity of OAP to APX.(El-Khalafy et al. 2024) So, our focus is mainly on observing the biomimicking enzymatic behavior of our novel chitosan proline conjugated mixed ligand copper(II) complexes. Our research group has discovered previously that the mixed ligand copper(II) complexes containing diimines have better anticancer and biomimicking catalytic behaviors.(Karpagam et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kartikeyan et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) Herein, we investigated the catalytic potential of two novel Cu(II) complexes of the general formula [Cu(\u003cb\u003eL\u003c/b\u003e)(bpy)(H\u003csub\u003e2\u003c/sub\u003eO)]\u003csup\u003e2+\u003c/sup\u003e (\u003cb\u003e1\u003c/b\u003e), and [Cu(\u003cb\u003eL\u003c/b\u003e)(phen)(H\u003csub\u003e2\u003c/sub\u003eO)]\u003csup\u003e2+\u003c/sup\u003e (\u003cb\u003e2\u003c/b\u003e), where \u003cb\u003eL\u003c/b\u003e represents chitosan-conjugated-proline polymer, and diimine ligands bpy and phen represent 2,2\u0026rsquo;-bipyridine and 1,10-phenanthroline, respectively. The synthesized complexes were characterized by FT-IR, powder XRD, solid-state absorption spectroscopy, and SEM analysis. The amide bond formation of the chitosan-L-proline polymer was confirmed by FT-IR spectroscopy. The oxidation of o-aminophenol as a model substrate, monitored via UV-Visible spectrophotometry, provides the kinetics and mechanistic insights into substrate interactions. Interestingly, phen-containing complex \u003cb\u003e2\u003c/b\u003e exhibited the highest catalytic activity compared to \u003cb\u003e1\u003c/b\u003e and free ligand (\u003cb\u003eL\u003c/b\u003e), highlighting the importance of the copper(II) center and influence of the co-ligand phen in the oxidation of o-aminophenol to phenoxazinone as the functional mimic of phenoxazinone enzyme. Extensive kinetic calculations have been performed for catalytic studies to put forward the most probable mechanistic pathways. The spectroscopic data reveal distinct electronic transitions corresponding to oxidation processes, providing a way to examine the influence of the ligand environment on catalytic efficiency.\u003c/p\u003e"},{"header":"2. Experimental Section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials and Methods\u003c/h2\u003e\u003cp\u003eThe chemicals utilized in this study, including Chitosan with 75% deacetylation, L-Proline, Cu(II) chloride dihydrate (CuCl\u003csub\u003e2\u003c/sub\u003e\u0026sdot;2H\u003csub\u003e2\u003c/sub\u003eO), are obtained from HI-MEDIA (India), and the substrate o-aminophenol was purchased from Merck. We procured dichloromethane from Finar, diethyl ether from SRL, ethanol and methanol from Emplura, and ultra-pure water (18.2 \u0026micro;Ω) was utilized for all the experiments. The solvents were dried according to standard procedures. Thin-layer chromatography was performed on Merck Pre-coated silica gel plates, and all column chromatography was conducted using Merck Silica Gel (60\u0026ndash;120 mesh size) with analytical-grade solvent. The NMR spectra were recorded on a Bruker Advance II-400 MHz frequency for proton nuclear magnetic resonance (\u003csup\u003e1\u003c/sup\u003eH NMR) and 101 MHz frequency for carbon nuclear magnetic resonance. An Agilent Cary 630FTIR operating at a resolution of 2 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e from 4000 to 400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was used for obtaining the infrared spectra. Powder X-ray diffraction (PXRD) studies of the ligands and complexes were performed using a PANalytical diffractometer with Cu-K\u003csub\u003eα\u003c/sub\u003e radiation (λ\u0026thinsp;=\u0026thinsp;1.5406 \u0026Aring;) to analyze the crystalline and amorphous nature. Surface morphology of the ligands was obtained using a TESCAN VEGA3 SBH scanning electron microscope at an accelerating voltage of 15 kV. The UV-Vis absorption titrations were performed using a Shimadzu UV-2450 (Kyoto, Japan) spectrophotometer with cuvettes featuring a path length of 1 cm. The copper-metal content of the complexes was studied with an Agilent 5800 Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Synthesis of ligand L and complexes 1 and 2\u003c/h2\u003e\u003cp\u003eThe ligand (\u003cb\u003eL\u003c/b\u003e) (Scheme 1a) was synthesized by following the reported procedures. The corresponding complexes, [Cu(\u003cb\u003eL\u003c/b\u003e)(bpy)(H\u003csub\u003e2\u003c/sub\u003eO)]\u003csup\u003e2+\u003c/sup\u003e (\u003cb\u003e1\u003c/b\u003e), and [Cu(\u003cb\u003eL\u003c/b\u003e)(phen)(H\u003csub\u003e2\u003c/sub\u003eO)]\u003csup\u003e2+\u003c/sup\u003e (\u003cb\u003e2\u003c/b\u003e), were synthesized by reacting the corresponding ligand with copper (II) chloride dihydrate in methanol, followed by adding the diimines (bpy and phen) as illustrated in Scheme 1b.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Synthesis of Boc-L-proline\u003c/h2\u003e\u003cp\u003eL-proline (20 mmol) was dissolved in 1N NaOH (20 mL) in isopropanol (20 mL), and to this mixture, a Di-tert-butyl pyrocarbonate (BOC-anhydride) (6 mL, 26 mmol) dissolved in isopropanol (10 mL) was added drop-wise following 1N NaOH (20 mL) to the above reaction mixture. The resulting solution was allowed to stir for 2 h at room temperature. Then the reaction mixture was washed with light petroleum ether (bp 40\u0026ndash;60 ⁰C) (20 mL), acidified to pH 3.0 with 2N H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4,\u003c/sub\u003e and finally extracted with chloroform (3 \u0026times; 20 mL). The organic layer was dried over anhydrous Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and evaporated under reduced pressure to yield the crude product (Scheme 1a), which was then crystallized from a mixture of chloroform and petroleum ether (bp 40\u0026ndash;60\u0026deg;C). (Yield\u0026thinsp;=\u0026thinsp;80%).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Synthesis of Chito-proline Conjugate-Ligand (L)\u003c/h2\u003e\u003cp\u003eTo synthesize the ligand \u003cb\u003eL\u003c/b\u003e, 1g of chitosan powder was dissolved in 2% (w/v) acetic acid, and the mixture was vigorously stirred by a magnetic stirrer at room temperature until the polymer was completely dissolved. To the above solution, 1.07g of Boc-proline and 1.50 g of DCC in 10 mL of methanol were added dropwise, and the mixture was stirred for 2 h. After the reaction was completed, the product was filtered, concentrated under vacuum, and washed with 95% ethanol to remove excess DCC. The deprotection of Boc was carried out by using TFA/DCM (1:1 ratio). The final product (Scheme 1a\u003cb\u003e)\u003c/b\u003e was dried in a vacuum oven at 35 ⁰C for 4 h. (Yield\u0026thinsp;=\u0026thinsp;86%)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Synthesis of Complex 1\u003c/h2\u003e\u003cp\u003e0.5 g of Ligand (\u003cb\u003eL\u003c/b\u003e) was dissolved in 50 mL of 2% acetic acid, and then 1 mM of the methanolic solution of CuCl\u003csub\u003e2\u003c/sub\u003e\u0026sdot;2H\u003csub\u003e2\u003c/sub\u003eO was added dropwise. This solution was then allowed to reflux at 65\u0026deg;C for 3 hours. Then allowed the reaction mixture to cool to room temperature. To this clear solution, a methanolic (2 mL) solution of 2,2\u0026prime;-bipyridine (1 mmol, 0.156 g) was added dropwise, and stirring was continued for another 1.5 hours. The blue color precipitate (Scheme 1b) formed was collected by filtration, washed with cold diethyl ether, and dried in a vacuum desiccator over anhydrous CaCl\u003csub\u003e2\u003c/sub\u003e. (Yield\u0026thinsp;=\u0026thinsp;78%)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Synthesis of Complex 2\u003c/h2\u003e\u003cp\u003eTo synthesize complex \u003cb\u003e2\u003c/b\u003e [Cu(\u003cb\u003eL\u003c/b\u003e)(phen)(H\u003csub\u003e2\u003c/sub\u003eO)]\u003csup\u003e2+\u003c/sup\u003e, 1,10-phenanthroline was added instead of 2,2\u0026rsquo;-bipyridine, and the same procedure was followed as that for the synthesis of complex \u003cb\u003e1\u003c/b\u003e (Scheme 1b). Blue color precipitate was isolated, washed with cold diethyl ether, and dried in a vacuum desiccator containing anhydrous CaCl\u003csub\u003e2\u003c/sub\u003e (Yield\u0026thinsp;=\u0026thinsp;76%)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Phenoxazinone Synthase-like Activity of Chitosan-Proline Cu(II) Complexes\u003c/h2\u003e\u003cp\u003eThe phenoxazinone synthase-like activity of ligand \u003cb\u003eL\u003c/b\u003e and its complexes \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2\u003c/b\u003e was studied using o-aminophenol (OAP) as a model substrate in a methanol medium under aerobic conditions at room temperature.(El-Khalafy et al. 2024) For this study, 0.2 mg/mL solutions of complexes \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2\u003c/b\u003e were treated with 0.5 to 3 mM of OAP under aerobic conditions in methanol. The reaction was monitored spectrophotometrically with an increase in the absorbance band of the phenoxazinone at 425 nm at 5-minute time intervals. The catalytic activity was monitored as per the Michaelis-Menten kinetics model. All the compounds showed saturation kinetics, and \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e, and rate constant for dissociation of substrates (i.e., turnover number, \u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e) were calculated for all the compounds using a Lineweaver-Burk plot of 1/V vs 1/[S], using the equation,\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\:\\:\\frac{1}{V}=\\frac{{K}_{M}}{{V}_{max}}+\\frac{1}{\\left[S\\right]}+\\frac{1}{{V}_{max}}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWhere V is the reaction velocity (the reaction rate), \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e is the Michaelis-Menten constant, \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e is the maximum reaction velocity, and [S] is the substrate concentration.(Johnson and Goody 2011)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Catalytic conversion of OAP using complex 2\u003c/h2\u003e\u003cp\u003eAs confirmation of the above spectroscopic catalytic conversion of OAP to APX, we attempted the same process using the best catalyst, i.e., complex \u003cb\u003e2\u003c/b\u003e (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Where 0.5 mmol of OAP was dissolved in 15 mL of methanol, to this solution 5 mg of complex \u003cb\u003e2\u003c/b\u003e was added. The solution was kept stirring for over 6 hr. Then, the product was separated by column chromatography and confirmed by \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR techniques \u003cb\u003e(\u003c/b\u003eYield\u0026thinsp;=\u0026thinsp;72%).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cp\u003eThe synthesized ligand and complexes were characterized using advanced analytical techniques like NMR, FT-IR, UV-Vis, scanning electron microscope, and powder X-ray analyses. The percentage of copper present in each complex is measured using ICP-OES, which showed a value of 10.72% for complex \u003cb\u003e1\u003c/b\u003e and 9.81% for complex \u003cb\u003e2\u003c/b\u003e. The formation of the oxidative product of the catalytic reaction is further confirmed by NMR analysis.\u003c/p\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.1 NMR Analysis of Chitosan-proline Conjugate (L)\u003c/h2\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH NMR spectra of Boc-L-proline and chito-proline conjugate \u003cb\u003e(L)\u003c/b\u003e is showed in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e(a) and S1(b) respectively. The peaks appear at δ\u0026thinsp;=\u0026thinsp;1.50\u0026ndash;2.01 ppm (m, 2H, CH\u003csub\u003e2\u003c/sub\u003e), δ\u0026thinsp;=\u0026thinsp;2.40 ppm (m, 2H, CH\u003csub\u003e2\u003c/sub\u003e), δ\u0026thinsp;=\u0026thinsp;3.32\u0026ndash;3.34 ppm (m, 2H, CH\u003csub\u003e2\u003c/sub\u003e), and δ\u0026thinsp;=\u0026thinsp;4.31\u0026ndash;4.32 ppm (t, 1H, CH) are due to the L-proline moiety present in the molecule. Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e(a) shows a singlet at δ\u0026thinsp;=\u0026thinsp;2.18 ppm, corresponding to the Boc protection, and the same peak disappeared in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e(b), confirming the formation of the title compound. In the spectra, the chemical shift value of δ\u0026thinsp;=\u0026thinsp;4.5 ppm representing the H\u003csub\u003e1\u003c/sub\u003e proton of chitosan, and the other peaks are δ\u0026thinsp;=\u0026thinsp;3\u0026ndash;4 ppm (H\u003csub\u003e2\u003c/sub\u003e-H\u003csub\u003e6\u003c/sub\u003e protons), δ\u0026thinsp;=\u0026thinsp;2 ppm respect to acetamido proton (-NHCO-CH\u003csub\u003e3\u003c/sub\u003e), δ\u0026thinsp;=\u0026thinsp;3.25 ppm respect to -CH\u003csub\u003e2\u003c/sub\u003e-NH\u003csub\u003e2\u003c/sub\u003e protons (CH\u003csub\u003e2\u003c/sub\u003e) and δ\u0026thinsp;=\u0026thinsp;4.5 ppm is due to acetyl proton (C-H) of chitosan. The de-acetyl proton of the chitosan appeared at δ\u0026thinsp;=\u0026thinsp;3.3\u0026ndash;3.4 ppm. So, the above observation indicates that the deacetylated chitosan reacted with Boc L-proline and formed the desired title ligand \u003cb\u003eL\u003c/b\u003e (chito-proline conjugate).\u003c/p\u003e\u003cp\u003eIn Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e(c), the \u0026sup1;\u0026sup3;C NMR spectrum of Boc-L-proline shows distinct signals characteristic of the BOC protecting group. A very intense peak at δ\u0026thinsp;=\u0026thinsp;30.15 ppm corresponds to the three methyl carbons of the BOC group, while a signal at δ\u0026thinsp;=\u0026thinsp;81.50 ppm is attributed to the tertiary carbon of the BOC moiety. Additionally, the carbonyl carbon of the BOC group appears at δ\u0026thinsp;=\u0026thinsp;179.96 ppm. In contrast, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e(d), representing the spectrum of chito-proline conjugate \u003cb\u003eL\u003c/b\u003e, shows the complete disappearance of these peaks, confirming the successful removal of the BOC group from the chitosan-proline amide structure.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Scanning Electron Microscopy (SEM) Analysis\u003c/h2\u003e\u003cp\u003eThe characterization of the morphology of the chitosan and chito-proline conjugate (\u003cb\u003eL\u003c/b\u003e) composite was carried out using the SEM technique. The SEM images of the chitosan polymer were a bed-like folding surface before the reaction,(Castanheiro \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) whereas after incorporating L-proline, the surface morphology was changed to a rod-like structure, as shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, revealing that the polymer chitosan has successfully reacted with L-proline. This also demonstrates that the biopolymer is formed with a nearly uniform distribution, with no intense particle agglomeration due to the formation of cross-linkages.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.3 FT-IR Spectral Analysis\u003c/h2\u003e\u003cp\u003eThe FTIR spectral analysis (Figure S2-S5) provides strong evidence for the successful formation of the chito-proline conjugate ligand \u003cb\u003eL\u003c/b\u003e and its coordination with metal centers in complexes \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2.\u003c/b\u003e The characteristic ν(O\u0026ndash;C\u0026ndash;O) stretching vibration, appearing consistently in the range of 1050\u0026ndash;1100 cm⁻\u0026sup1;, is observed for both the free ligand and all three complexes, confirming the presence of the chitosan backbone and its retained structural integrity upon complexation. A strong and sharp ν(C\u0026ndash;H) out-of-plane bending vibration observed in the 610\u0026ndash;620 cm⁻\u0026sup1; region further supports the conjugation of the chitosan ring with the proline moiety, characteristic of the polymeric structure of the ligand. The amide carbonyl stretching (ν(C\u0026thinsp;=\u0026thinsp;O)) band is observed at 1699 cm⁻\u0026sup1; in the ligand \u003cb\u003eL\u003c/b\u003e, indicating the presence of an amide linkage from the proline-chitosan conjugate. Interestingly, this band shifts to 1601 cm⁻\u0026sup1; in complex \u003cb\u003e1\u003c/b\u003e and 1610 cm⁻\u0026sup1; in complex \u003cb\u003e2\u003c/b\u003e, suggesting coordination of the carbonyl oxygen to the metal center, which weakens the C\u0026thinsp;=\u0026thinsp;O bond and shifts its frequency to lower wavenumbers. The N\u0026ndash;H stretching vibrations (ν(N\u0026ndash;H)) are found at 3268 cm⁻\u0026sup1; for the free ligand, more prominently shifted to 2897 cm⁻\u0026sup1; and 3081 cm⁻\u0026sup1; in complexes \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2\u003c/b\u003e, respectively. These shifts are indicative of changes in the hydrogen bonding environment and potential coordination involving the nitrogen atom. In complexes \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2\u003c/b\u003e, the ν(N-H) and ν(C-H) stretching bands are merged and broadened, reflecting a complex and possibly more rigid coordination environment. Additionally, new bands appearing at 1598 cm⁻\u0026sup1; for \u003cb\u003e1\u003c/b\u003e and 1566 cm⁻\u0026sup1; for \u003cb\u003e2\u003c/b\u003e can be assigned to ν(C-N) stretching vibrations, suggesting the involvement of a nitrogen donor atom (likely from a co-ligand such as 2,2\u0026rsquo;-bipyridine or 1,10-phenanthroline) coordinating to the metal center. Finally, the appearance of low-frequency bands in the 440\u0026thinsp;\u0026minus;\u0026thinsp;410 cm⁻\u0026sup1; region in all metal complexes corresponds to ν(Cu-N) stretching vibrations, affirming the successful coordination of the ligand/co-ligand system with the Cu(II) ion. All the data is tabulated and given in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. These data collectively validate the formation of metal-ligand complexes with chitosan-proline as the primary ligand and the co-ligands aiding in chelation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Powder X-Ray Diffraction (PXRD) Analysis\u003c/h2\u003e\u003cp\u003eThe diffractive region of chitosan (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) shows the expected broad amorphous hump centered at 2θ\u0026thinsp;\u0026asymp;\u0026thinsp;20\u0026deg;, characteristic of semi-crystalline chitosan. While peaks of L-proline (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) are observed at 2θ\u0026thinsp;=\u0026thinsp;20\u003csup\u003e0\u003c/sup\u003e, 25\u003csup\u003e0\u003c/sup\u003e, 30\u003csup\u003e0\u003c/sup\u003e, 35\u003csup\u003e0\u003c/sup\u003e, 40\u003csup\u003e0\u003c/sup\u003e. The peaks of the chitosan-proline conjugate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec) are observed at 2θ\u0026thinsp;=\u0026thinsp;20\u003csup\u003e0\u003c/sup\u003e-40\u003csup\u003e0\u003c/sup\u003e. The figure shows a significant shift in the diffractioun peaks and the diffraction pattern, with a broad amorphous peak, indicating that there was molecular miscibility and interaction between the components. In complex \u003cb\u003e1\u003c/b\u003e, the PXRD pattern shows peaks at 2θ\u0026thinsp;=\u0026thinsp;12.2\u0026deg;, 18.54\u0026deg;, 19.56\u0026deg;, 26\u0026deg;, 28\u0026deg;, 40\u0026deg;, and 50\u0026deg; (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The intense peak at 12.2\u0026deg; is particularly notable and may indicate complex formation with 2,2\u0026rsquo;-bipyridine. The broad peak typically seen around 20\u0026deg; in pure chitosan becomes split into sharper peaks at 18.54\u0026deg; and 19.56\u0026deg;, suggesting a transition from an amorphous to a more crystalline structure upon metal-ligand coordination, which is further confirmed by analyzing the shift or the disappearance of the sharper peak at 16\u0026deg;, which is seen for the pure CuCl\u003csub\u003e2\u003c/sub\u003e\u0026middot; 2H\u003csub\u003e2\u003c/sub\u003eO metal salt.(AbouElleef et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) For complex \u003cb\u003e2\u003c/b\u003e, diffraction peaks are observed at 2θ\u0026thinsp;=\u0026thinsp;12\u0026deg;, 18\u0026deg;, 20.54\u0026deg;, 35\u0026deg;, 40\u0026deg;, and 46\u0026deg; (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Similar to \u003cb\u003e1\u003c/b\u003e, the strong peak at 12\u0026deg; suggests complexation, likely with 1,10-phenanthroline. The broad peak of the ligand splits into two distinct peaks at 18\u0026deg; and 20.54\u0026deg;, reinforcing the idea that the coordination process induces crystallinity. Other peaks support the crystalline nature of the complexes. The PXRD patterns of complexes \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2\u003c/b\u003e exhibit sharp and intense peaks, indicating a crystalline nature in contrast to the broad, diffuse pattern observed for the amorphous ligand. This suggests that upon coordination with metal ions, the molecular chains become highly ordered, forming a repeating crystalline structure throughout the material.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Solid-state UV-Vis Spectroscopy\u003c/h2\u003e\u003cp\u003eThe solid-state UV-Vis spectra of the ligand \u003cb\u003eL\u003c/b\u003e and the complexes \u003cb\u003e1\u0026ndash;2\u003c/b\u003e are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, and the intermediate complex [CuLCl\u003csub\u003e2\u003c/sub\u003e(H\u003csub\u003e2\u003c/sub\u003eO)] in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. The chito-proline conjugate ligand exhibits absorption bands below 300 nm, attributable to intraligand π\u0026rarr;π* and n\u0026rarr;π* transitions around 210 nm and 241 nm, and displays no absorption features in the visible region.(Gonz\u0026aacute;lez-Mart\u0026iacute;nez et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)(Gonz\u0026aacute;lez-Mart\u0026iacute;nez et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)(Gonz\u0026aacute;lez-Mart\u0026iacute;nez et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)(Gonz\u0026aacute;lez-Mart\u0026iacute;nez et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)(Gonz\u0026aacute;lez-Mart\u0026iacute;nez et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)(Gonz\u0026aacute;lez-Mart\u0026iacute;nez et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)(Gonz\u0026aacute;lez-Mart\u0026iacute;nez et al.) Whereas the Cu(II) complexes \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2\u003c/b\u003e display intense ligand-based bands in the UV region, together with broad absorptions extending into the visible region. The bands in the range 250\u0026ndash;320 nm may be assigned to ligand-to-metal charge transfer (LMCT) transitions from nitrogen donor atoms to the Cu(II) center. The intermediate complex, [CuLCl₂(H\u003csub\u003e2\u003c/sub\u003eO)], exhibited a broad band centered at 847 nm, assignable to the transition typical of a \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{d}^{9}\\)\u003c/span\u003e\u003c/span\u003eCu(II) center with a \u003cem\u003eN, O\u003c/em\u003e donor environment. Coordination of the strong field diimine co-ligands to the metal center shifted the corresponding absorption maxima distinctly toward higher energy. Complex \u003cb\u003e1\u003c/b\u003e showed a band at 673 nm, and complex \u003cb\u003e2\u003c/b\u003e displayed a similar band at 663 nm. This blue shift (Δλ\u0026thinsp;\u0026asymp;\u0026thinsp;170\u0026ndash;180 nm) indicates a considerable increase in ligand-field strength around the copper center upon the introduction of the aromatic diimine ligands. The slightly greater shift for the phenanthroline complex (663 nm) compared to bipyridine (673 nm) correlates with its higher π-acceptor ability, increased aromaticity, and more extensive conjugation. These d-d bands are typical of Cu(II) complexes in distorted square-planar or square-pyramidal environments and reflect the influence of Jahn-Teller distortion. Appearance of charge transfer and d-d bands compared to the free ligand clearly confirms coordination of Cu(II) and supports the assignment of new coordination domains observed in PXRD.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e3.6 UV-Vis Kinetic Analysis of Catalytic Oxidation of OAP\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eThe uncoordinated ligand \u003cb\u003eL\u003c/b\u003e and complexes 1 and \u003cb\u003e2\u003c/b\u003e were tested for PHS synthase activity under homogeneous conditions by dissolving 0.2 mg/mL of them and 0.5\u0026ndash;3 mM of o-aminophenol (OAP) in methanol using UV-Vis absorption spectroscopy. Initially, oxidation of OAP to phenoxazinone chromophore was monitored with respect to the rate of increase in absorption band at λ\u003csub\u003emax\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;425 nm for 60 minutes with 5-minute intervals, which is a characteristic band of the phenoxazinone chromophore. Later, the spectra were monitored at 1-hour intervals for 6 hours (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec), and the time was increased until the reaction reached saturation (Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The resulting absorption spectrum is used for further analysis. Interestingly, both complexes \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2\u003c/b\u003e increase the absorption intensity at 425 nm (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec) compared to the free ligand L (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). A blank experiment without a catalyst under identical conditions does not show any significant increment in the band intensity at 425 nm. At 0.5 mM OAP, the ligand \u003cb\u003eL\u003c/b\u003e doesn\u0026rsquo;t show any increment in the absorption band around the 415\u0026ndash;435 region; rather, the band intensity decreases over the period of 60 minutes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). Among \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2\u003c/b\u003e, the phen complex \u003cb\u003e2\u003c/b\u003e displays a higher intensity of absorption band at 425 nm than \u003cb\u003e1\u003c/b\u003e, revealing that \u003cb\u003e2\u003c/b\u003e involves more catalytic conversion than \u003cb\u003e1\u003c/b\u003e. These spectral behaviours clearly reveal that both complexes are active catalysts for the aerobic oxidation of OAP to phenoxazinone chromophore. So the kinetic study of \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2\u003c/b\u003e was performed by the initial slope method following the rate of increase in absorption of the band at λ\u003csub\u003emax\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;425 nm.\u003c/p\u003e\u003cp\u003eThe initial first-order rate constants for the reactions were determined from the plots of log(A\u003csub\u003e\u0026infin;/\u003c/sub\u003eA\u003csub\u003e\u0026infin;\u0026minus;\u003c/sub\u003eA\u003csub\u003et\u003c/sub\u003e) versus time (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). To investigate the dependence of rate constants on substrate concentration, the catalytic reactions were conducted using six varying concentrations of the substrate while keeping the catalyst concentration constant (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). The rate (\u003cem\u003ek\u003c/em\u003e\u003csub\u003eobs\u003c/sub\u003e) is calculated by taking the slope of the log(A\u003csub\u003e\u0026infin;/\u003c/sub\u003eA\u003csub\u003e\u0026infin;\u0026minus;\u003c/sub\u003eA\u003csub\u003et\u003c/sub\u003e) v/s time plot of the initial substrate concentration.\u003c/p\u003e\u003cp\u003eAt higher substrate concentrations, the initial rate method followed saturation kinetics, while at lower concentrations, first-order kinetics were observed. Hence, the Michaelis-Menten model commonly used in enzyme kinetics, along with its linearized form (Lineweaver-Burk plot), was utilized to calculate the rate of the reactions and various kinetic parameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFrom the Lineweaver-Burk plot, the \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e value is obtained by taking the y-intercept value of the plot, which gives 1/\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e; hence, \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e can be calculated by taking the reciprocal of the y-intercept. Further, the slope of the Lineweaver-Burk plot gives the value corresponding to \u003cem\u003e[K\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e/V\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e]. And the \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e value is computed by multiplying \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e by the slope of the graph. Where the free ligand (\u003cb\u003eL\u003c/b\u003e) showed no measurable activity, confirming that Cu(II) coordination is essential for catalysis. Complex \u003cb\u003e1\u003c/b\u003e displayed a lower \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e (\u003cb\u003e2.17 \u0026times; 10⁻\u0026sup3;\u003c/b\u003e) and a higher \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e (\u003cb\u003e1.84\u003c/b\u003e), indicating weaker substrate binding and reduced efficiency compared to that of complex \u003cb\u003e2\u003c/b\u003e, which exhibited the highest catalytic performance, with a \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e of \u003cb\u003e3.13 \u0026times; 10⁻\u0026sup3;\u003c/b\u003e and a relatively low \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e of \u003cb\u003e1.02\u003c/b\u003e, reflecting both a fast turnover rate and good substrate affinity. These results highlight the most promising phenoxazinone synthase mimic efficiency of the complex \u003cb\u003e2\u003c/b\u003e, in this series, consistent with its formation of stronger absorption intensity at 425 nm in the UV-Vis spectra. Notably, the same complex \u003cb\u003e2\u003c/b\u003e demonstrates 72% conversion of OAP to APX. The possible mechanism of the catalytic cycle is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e.(Shaban et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparison of the catalytic activity of ligand \u003cb\u003eL\u003c/b\u003e and complexes \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2.\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS/no\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSubstrate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCatalyst\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e(10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eK\u003csub\u003em\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e1.\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOAP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLigand (L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e2.\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOAP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eComplex 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.84\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e3.\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOAP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eComplex 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Characterization of APX Isolated from the Reaction\u003c/h2\u003e\u003cp\u003eAfter the catalytic conversion of OAP to APX by the efficient catalyst \u003cb\u003e2\u003c/b\u003e, the product APX was isolated through column chromatography using 1% MeOH/ CHCl\u003csub\u003e3\u003c/sub\u003e. The product formation was confirmed by the \u003csup\u003e1\u003c/sup\u003eH NMR spectrum and ESI-MS illustrated in Figures S6 and S7, respectively. The \u003csup\u003e1\u003c/sup\u003eH NMR spectrum of APX shows broad singlet δ\u0026thinsp;=\u0026thinsp;5.143(s, 2H, NH\u003csub\u003e2\u003c/sub\u003e) corresponds to protons of NH\u003csub\u003e2\u003c/sub\u003e attached to the APX and an peak comes around δ\u0026thinsp;=\u0026thinsp;6.489(s, 1H, CH) which corresponds to proton attached next to the NH\u003csub\u003e2\u003c/sub\u003e group and a peak appears at δ\u0026thinsp;=\u0026thinsp;7.383\u0026ndash;7.351(dd,1H, CH) which correspond to proton attached near to the carbonyl group; and two doublet arises at δ\u0026thinsp;=\u0026thinsp;7.390 (d,1H, CH) and δ\u0026thinsp;=\u0026thinsp;7.557(d,1H, CH) of the aromatic ring and an δ\u0026thinsp;=\u0026thinsp;7.756\u0026ndash;7.745 (m,1H, CH), δ\u0026thinsp;=\u0026thinsp;7.745\u0026ndash;7.748 (m, 1H, CH) corresponds to the proton of aromatic hydrocarbon ring.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.8 Conclusions\u003c/h2\u003e\u003cp\u003eThe synthesis and rigorous evaluation of phenoxazinone synthase functional mimetic activity of two novel mixed ligand Cu(II) complexes containing chitosan-conjugated-proline and diimines (\u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2)\u003c/b\u003e, of the general formula [Cu(\u003cb\u003eL\u003c/b\u003e)(bpy)(H\u003csub\u003e2\u003c/sub\u003eO)]\u003csup\u003e2+\u003c/sup\u003e (\u003cb\u003e1\u003c/b\u003e), and [Cu(\u003cb\u003eL\u003c/b\u003e)(phen)(H\u003csub\u003e2\u003c/sub\u003eO)]\u003csup\u003e2+\u003c/sup\u003e (\u003cb\u003e2\u003c/b\u003e), where \u003cb\u003eL\u003c/b\u003e represents chitosan-conjugated-proline polymer, and diimine ligands bpy and phen represent 2,2\u0026rsquo;-bipyridine and 1,10-phenanthroline, respectively. The characterization of these synthesized materials was comprehensive, employing Fourier-transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), nuclear magnetic resonance spectroscopy, powder X-ray diffraction (XRD), and solid-state absorption spectroscopic techniques. FT-IR spectroscopy confirmed the successful synthesis of the ligand \u003cb\u003eL\u003c/b\u003e and its complexation with Cu(II) ions and the addition of co-ligands 2,2\u0026rsquo;-bipyridine and 1,10-phenanthroline to form complexes \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2\u003c/b\u003e, respectively. ICP-OES data revealed that complexes \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2\u003c/b\u003e contain 10.72% and 9.81% of copper, respectively. Powder XRD analysis demonstrated the amorphous and crystalline properties of ligands and complexes, which tells the proper arrangement of copper ions incorporated in the Chitosan proline amide polymer, ultimately forming the active catalyst complexes \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2\u003c/b\u003e. This is further supported by the solid-state absorption spectroscopy, with the broad absorptions of d-d transitions of copper complexes in the 550\u0026ndash;800 nm region, which were absent in the ligand spectra. Using Michaelis-Menten kinetics, the catalytic activity of the complexes for the conversion of OAP to APX was assessed by means of electronic absorption spectroscopy, and the results ascertained significant activity as a phenoxazinone synthase mimic, with a \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e value of 0.00217 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with a \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e value of 1.84 for complex \u003cb\u003e1\u003c/b\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e value of 0.00313 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with a \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e value of 1.02 for complex \u003cb\u003e2.\u003c/b\u003e This indicates that complex \u003cb\u003e2\u003c/b\u003e was superior to complex \u003cb\u003e1\u003c/b\u003e both in \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e,\u003c/sub\u003e which is higher, and the \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e value, which is close to zero, indicating strong substrate-catalyst binding affinity. Notably, the same complex \u003cb\u003e2\u003c/b\u003e demonstrates 72% conversion of OAP to APX. Further convincing evidence for the oxidation of OAP was the identification of the oxidized product APX by NMR and ESI-MS spectral analysis. Thus, when we compared with previously reported chitosan-based complexes, whose \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e (1.4459 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e (0.0750 M) values are better than those of our complexes. Though our complexes are less effective than already reported porphyrin-containing Cu(II) complexes, our complexes are low-cost and easy to handle compared to those. Overall, the findings underscore the potential of the chitosan-based polymeric metal complexes to act as a robust catalyst for OAP oxidation, giving hope for expanding their utility in synthetic and industrial chemistry. This research aims to focus on the advances in the field of biomimetic catalysis and also provides a foundation for future studies exploring similar complexes for various catalytic applications.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eSupplementary Information\u003c/h2\u003e\u003cp\u003eThe online version contains supplementary material available at\u003c/p\u003e\u003ch2\u003e\u003cb\u003eConflict of interest\u003c/b\u003e:\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLiji Muthirakalayil Abraham: Formal analysis, Investigation, Methodology, Conceptualization, Data curation, Validation, Writing \u0026ndash; original draft. Manikandan Varadhan: Formal analysis, Investigation, Conceptualization, Methodology. Kugan Mahalingam: Formal analysis, Investigation, Data curation. Thangaraja Chinnathangavel: Data curation, Investigation. Venugopal Rajendiran: Conceptualization, Data curation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eV. R. acknowledges the Department of Biotechnology (DBT), New Delhi (BT/PR36476/NNT/28/1723/2020) and DST-FIST(SR/FST/CS-1/ 2021/215), New Delhi, for financial assistance. V. R also acknowledges the Ponnus Natural Products, Virudhanagar for ICP-OES analysis.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbo El-Kheir, Dina A. H., Shaban Y. Shaban, Mohamed M. Ibrahim, Abd El-Motaleb M. Ramadan, and Ahmed M. Fathy. (2024). 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Aldol Reaction Catalyzed by a Hydrophilic Catalyst in Aqueous Micelle as an Enzyme Mimic System. \u003cem\u003eChirality\u003c/em\u003e, 21 (5), 492\u0026ndash;96. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/chir.20621\u003c/span\u003e\u003cspan address=\"10.1002/chir.20621\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Schemes","content":"\u003cp\u003eSchemes 1 and 2 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"Copper(II), Chitosan, o-aminophenol, Phenoxazinone synthase, Biomimetic catalysis","lastPublishedDoi":"10.21203/rs.3.rs-7918334/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7918334/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhenoxazinone synthase (PHS) is a copper-containing bacterial enzyme that is involved in vital biological oxidation processes. Mimicking these enzymatic functions using synthetic copper complexes provides valuable insights and potential applications in green catalysis. In this work, chitosan-conjugated ligands were synthesized and anchored with Cu(II) to yield mixed-ligand complexes (\u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2\u003c/b\u003e) of the general formula [Cu(\u003cb\u003eL\u003c/b\u003e)(bpy)(H\u003csub\u003e2\u003c/sub\u003eO)]\u003csup\u003e2+\u003c/sup\u003e \u003cb\u003e(1)\u003c/b\u003e, and [Cu(\u003cb\u003eL\u003c/b\u003e)(phen)(H\u003csub\u003e2\u003c/sub\u003eO)]\u003csup\u003e2+\u003c/sup\u003e \u003cb\u003e(2)\u003c/b\u003e, where \u003cb\u003eL\u003c/b\u003e represents chitosan-conjugated-proline polymer, and diimine ligands bpy and phen represent 2,2\u0026rsquo;-bipyridine and 1,10-phenanthroline, respectively. SEM, PXRD, FTIR, and various spectroscopic methods were used to characterize the ligands and complexes. ICP-OES data revealed that complexes \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2\u003c/b\u003e contain 10.72% and 9.81% of copper, respectively. The oxidation of o-aminophenol (OAP), used as a model substrate, monitored via UV-Visible spectrophotometry, provides valuable kinetics and mechanistic insights into substrate interactions. Interestingly, among all the complexes, \u003cb\u003e2\u003c/b\u003e exhibited the highest catalytic activity, which features phenanthroline as a co-ligand, followed by \u003cb\u003e1\u003c/b\u003e and then the free ligand (\u003cb\u003eL\u003c/b\u003e), highlighting the influence of the co-ligand in the oxidation of o-aminophenol to phenoxazinone as the functional mimic of phenoxazinone enzyme. Notably, the same complex \u003cb\u003e2\u003c/b\u003e demonstrates 72% conversion of o-aminophenol (OAP) to 2-aminophenoxazine-3-one (APX). Kinetic analysis revealed pseudo-first-order rate constants consistent with efficient oxidase mimics. These results demonstrate the potential of Cu-chitosan hybrids as sustainable, bioinspired catalysts.\u003c/p\u003e","manuscriptTitle":"Phenoxazinone Synthase Functional Mimic Activity of Mixed-ligand Copper(II) Complexes of L-proline and Diimine Ligands Conjugated with Chitosan","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-23 05:03:35","doi":"10.21203/rs.3.rs-7918334/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":"1f9b52fb-cf71-4ea7-936d-cfbb7211e3fc","owner":[],"postedDate":"October 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-05T07:39:06+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-23 05:03:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7918334","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7918334","identity":"rs-7918334","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}