Nitrogen-enriched chitosan. Strategies for Amino Acid Molecule Attachment

Nitrogen-enriched chitosan. 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Strategies for Amino Acid Molecule Attachment Marta Wolczko This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4323766/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 Chitosan, a polysaccharide derived from chitin, has gained significant attention as a promising solid adsorbent due to its unique properties, such as biocompatibility, biodegradability, and good adsorption capacity. Its abundant amino and hydroxyl groups offer numerous functionalization opportunities, allowing for the development of tailored adsorbents with enhanced performance in various applications. This publication explores the potential of chitosan as a solid adsorbent and focuses specifically on strategies for attaching amino acid molecules to chitosan. Many attempts have been made to combine chitosan with an amino acid, but the best results were obtained by combining the activation of chitosan with hydroxybenzotriazole (HOBt) and simultaneous activation of the amino acid with the carbodiimide EDC. The reaction occurs in water, thus adhering to the principles of green chemistry. Substitution of every fourth chitosan sugar unit with a natural amino acid L-glycine was obtained. Other amino acids have also been successfully substituted into the chitosan sugar backbone. Furthermore, chitosan is derived from waste materials generated by the seafood industry, the utilization of materials based on it represents a way to reduce the quantity of such waste. chitosan amino acid amide bond formation nitrogen enrichment seafood waste Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction 1.1. The importance of chitosan Chitosan is a deacetylated derivative of chitin which, in turn, is the second most abundant biopolymer in Nature after cellulose.[ 1 – 3 ] Chitin is naturally found in the shellfish (prawn, shrimp, crab, etc.) and fish, can be obtained from waste of fish industries and from household garbage. It can also be extracted from bacteria and fungi by the enzymes called chitosanases. It is estimated that the annual production of chitin in the biosphere is about 10 11 tons, and the seafood industry produces 60,000–80,000 tons of waste annually.[ 4 ] Chitosan is therefore a very common raw material in Nature, and at the same time quite cheap. Moreover, its non-toxicity, biodegradability and chemical stability, make it a valuable candidate as substrate for chemical synthesis. Among its notable features is the presence of one amino group in each sugar unit of this polymer, which provide sites for chemical modifications and functionalization. These characteristics make chitosan an attractive candidate for solid adsorbent applications, where the selective and efficient removal of target molecules from complex mixtures is desired. In recent years, extensive research has been conducted to explore the potential of chitosan as a solid adsorbent in diverse fields, including environmental remediation, water purification, biomedicine, and pharmaceutical industries. Chitosan's capability to adsorb heavy metals, dyes, organic pollutants, and even biological entities like proteins and enzymes has garnered significant interest.[ 2 , 5 – 8 ] However, to further enhance its adsorption performance and expand its application range, efforts have been directed towards the development of functionalized chitosan-based adsorbents. Among various processes used, including liquid solvent sorption, membrane separation and cryogenics, the CO 2 adsorption in solids seems to be simple, low cost, and effective.[ 9 , 10 ] Chitosan, as a biopolymer rich in amine groups, has already been used in the studies of CO 2 adsorption. The highest CO 2 adsorption capacity was reported as 5 mmol/g for chitosan-based carbons (chitosan spheres) at 0 o C and at atmospheric pressure.[ 11 ] However, the preferred conditions (for industry and economic impact) consist in the adsorption at ambient temperature. In 2014, the subject of research was chitosan modified by polyethylenimine (PEI).[ 12 ] Material, in the form of an aerogel, adsorbed CO 2 according to the carbamate mechanism, with maximum adsorption capacity 2.9 mmol/g (at ambient conditions).[ 12 ] Two years later, chitosan-polybenzoxazine polymer was used as a precursor for high CO 2 adsorbing porous carbon material resulting from aerogel carbonization. Nitrogen enrichment of the carbon material allowed to achieve high adsorption capacity – 5.72 mmol/g at ambient conditions.[ 3 ] A bit later, in another research group, mesoporous chitosan − SiO 2 nanoparticles showed a maximum CO 2 adsorption capacity of 4.39 mmol/g at ambient temperature.[ 10 ] On the other hand, the adsorption of CO 2 in solid amino acids was also studied. Taurine gave the best result (3.7 mmol/g) at ambient temperature and increased pressure. Serine and valine gave results of approximately 2.5 mmol/g.[ 13 ] So far, the research on the modification of chitosan by natural amino acids has been carried out for L-glutamic acid and γ-aminobutyric acid, in order to immobilize the enzyme cellulase.[ 14 ] Another research group combined chitosan with L-glycine, L-lysine, L-isoleucine and L-glutamic acid to increase the Cu 2+ adsorption (in solution).[ 15 ] However, the use of such derivatives was to serve completely different goals. The mechanism of CO 2 capture by solid amine adsorbents is based on electrostatic, hydrogen and acid-base interactions, but also on the active attachment of a carbon dioxide molecule to the amino group according to carbamate mechanism.[ 16 ] It is very likely, that the addition of an amino acid to the chitosan structure can significantly increase the CO 2 adsorption after material carbonisation.[ 3 , 17 – 20 ] One promising approach involves the attachment of amino acid molecules to chitosan, utilizing the available amino groups as reactive sites. Amino acids, as the building blocks of proteins, exhibit unique physicochemical properties and specific binding capabilities towards various target molecules. By combining the advantages of chitosan and amino acids, it becomes possible to design tailored adsorbents with improved selectivity and affinity towards specific contaminants or biomolecules. 1.2. Strategies for amino acid molecule attachment The following part of the article focuses on the review of the methods of creating an amide bond between chitosan and natural L-amino acids. Amino acids are the building blocks of proteins, having at least one nitrogen atom, so introducing them to the chitosan structure increases the nitrogen to carbon ratio in the final material. In 2017, authors from the University of Gujarat presented a simple synthesis of a sulfur derivative of chitosan by azeotropic distillation, starting from chitosan and natural L-methionine. The amino acid and chitosan were equimolarly suspended in toluene. The reaction was carried out at 90–100°C for 24 hours in a Dean-Stark apparatus. During the reaction, the release of water was observed in the apparatus, what according to the Le Chatelier and Braun's rule, shifts the reaction equilibrium towards the formation of the product.[ 21 ] The authors present the NMR spectrum of the product, as well as the FTIR spectrum, comparing them with the spectra of chitosan, but they do not specify the efficiency of the synthesis. The reaction scheme is shown in Fig. 1 . By far, most methods of creating an amino bond from the -NH 2 group of chitosan are based on coupling reactions used to synthesize the same bond in peptides. A commonly used coupling reagent is EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), which activates the carboxyl group of an amino acid or other substrate. EDC is well known for its effectiveness in coupling two protein molecules, proteins and peptides, proteins and oligonucleotides, and proteins with other small molecules.[ 22 ] One of the most important advantages of this carbodiimide is the solubility of its by-product (urea derivative), which greatly facilitates the treatment of the mixture after the reaction. The second reagent often used together with EDC is NHS ( N -hydroxysuccinimide), which additionally stabilizes the active carboxylic acid derivative and thus increases the coupling efficiency. The group from Madrid presented a method of modifying chitosan with lactobionic acid, using a duo of EDC-NHS coupling reagents.[ 23 ] Lactobionic acid was first activated using EDC and NHS in an aqueous medium, and then it was added dropwise to chitosan previously dissolved in 1% aqueous acetic acid. The reaction was carried out for 72 hours at room temperature, then the product was purified by dialysis, obtaining a polymer with a degree of substitution from 3 to 16%, depending on the ratio of lactobionic acid to chitosan used, as shown in Fig. 2 . In 2019, a group from the Szczecin University of Technology published the results of research on optimizing the attachment of long-chain linoleic acid to the chitosan molecule.[ 24 ] The carboxyl group of the acid was activated with EDC alone or with EDC and NHS. In the first case, the esterification reaction of the hydroxyl group at C6 of chitosan was dominant, while in the reactions with the addition of NHS, the main product was the amide. The authors explain that the intermediate product – an ester of linoleic acid and NHS – selectively reacts with the amino group, resulting in the amide being the dominant product. Schematics of both synthetic paths are shown in Fig. 3 below. Due to the overlapping of key signals in the NMR spectrum, the authors do not provide quantitative information on the degree of substitution of the products, but they write that reactions with carbodiimide alone are more efficient compared to the EDC-NHS duo, and they are characterized by the opposite selectivity (O-acylation vs. N-acylation ).[ 24 ] Another research group in 2006 described research on the attachment of various carboxylic acids, including amino acids, to chitosan.[ 25 ] The authors of the study discovered that chitosan forms a water-soluble salt with hydroxybenzotriazole (HOBt). The structure of this salt was confirmed experimentally on nuclear magnetic resonance spectra. The chitosan-HOBt complex was successfully coupled to an amino acid (Boc-L-Phenylalanine) previously activated by EDC, resulting in a product with a degree of substitution of 28%. The authors emphasize that their method of modifying the chitosan amino group is characterized by high substitution efficiency and, importantly, is carried out under mild conditions (water-ethanol environment and room temperature). The reaction scheme is shown in Fig. 4 . By understanding the underlying principles and methodologies, researchers can advance the development of chitosan-based adsorbents with enhanced efficiency and selectivity, paving the way for future sustainable and effective adsorption technologies. 2. Experimental 2.1 Material and methods General Information: All starting materials and reagents were obtained from commercial sources, and were used as received unless otherwise noted. All solvents were freshly distilled before use. Chitosan (molecular weight 100-300 kDa and a degree of deacetylation of about 85% determined on the 1 H NMR spectrum), Boc-Gly-OH (>98%), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, >98%) 1-Hydroxybenzotriazole hydrate (HOBt, >99%), deuterium oxide (D 2 O, deuteration degree min. 99.9%) and CD 3 COOD (99.5%) were used for research. 1 H NMR spectra were recorded with spectrometers operating at 600 MHz in D 2 O with addition of CD 3 COOD. The infrared analysis of materials was carried out using a PerkinElmer Frontier FT-IR spectrometer. The analyses were performed in transmittance mode in a wavenumber range 4000–516 cm -1 , in ATR technique. Spectra were processed in SpectraGryph 1.2.15 . 2.2 Results The aim of the research was to obtain a "nitrogen-enriched" chitosan derivative. The initial plan was to substitute an amino acid containing two nitrogen atoms, such as L-lysine. The first choice was the method of azeotropic distillation of chitosan with L-lysine, in toluene, acc. the procedure described in section 1.2. Unfortunately, despite several repetitions of the reaction, it was not possible to obtain even traces of the product. Changing the amino acid to the smaller L-glycine also had no positive effect. Moreover, the obtained 1 H NMR spectra were almost identical to those of the authors,[21] unfortunately the only change in relation to the spectrum of pure chitosan concerned the signal at 3.34 ppm, coming from traces of methanol after washing the product (see S1 in Supplementary Material). Therefore, the azeotropic distillation method was abandoned in favor of amide bond coupling. The synthesis of L-lysine and chitosan was carried out using coupling reagents EDC and NHS, according to the procedure successfully used for lactobionic acid.[23] Unfortunately, the product was not obtained, probably due to the presence of two unprotected amino groups in the amino acid. These groups can react with the activated carboxyl group of the second molecule, condensing into oligopeptides before being introduced into the chitosan solution, thus preventing the reaction from occurring. For this reason, in the further part of the research, only amino acids with protected amino groups were used. L-Glycine with a tert -butoxycarbonyl (Boc) group was selected as the first model substrate. Two reactions were carried out according to procedures of the group from the Szczecin University of Technology.[24] In order for the N -acyl form to be the dominant product, both EDC and NHS were used in the reaction. Syntheses were carried out in 1% acetic acid at pH 4.7 and 4.0. In both cases, only traces of the product were obtained – slightly more (3%) at higher pH. The presence of the product was confirmed in the 1 H NMR spectrum, but due to the low yield, this method did not give hope for optimization. The next step was an attempt to activate chitosan by HOBt, with simultaneous activation of the amino acid by carbodiimide EDC according to procedure described above for phenylalanine.[25] This time the synthesis was successful – chitosan substituted with Boc-L-glycine in 26% was obtained. The highest degree of L-glycine substitution (26%) was obtained using a two-fold excess of coupling reagents (Table 1., No. 1). Attempting to change the solvent to MeOH, as well as eliminating and reducing the amount of water, resulted in a huge decrease in the reaction yield (Table 1, No. 2, 5, 6). We managed to reduce the amount of coupling reagents to one equivalent, which is a great success from the point of view of green chemistry and future application in industry (Table 1., No. 3). Table 1. Reaction of chitosan and Boc-L-glycine – optimization No. Chitosan (mmol) HOBt (mmol) Boc- L-Gly (mmol) EDC (mmol) Solvent DS* 1 1 2 1 2 H 2 O/EtOH (1:1) 26% 2 1 2 1 2 H 2 O/MeOH (1:1) 5% 3 1 1 1 1 H 2 O/EtOH (1:1) 21% 4 1 1 1 1 H 2 O/EtOH (1:1) 11.5%** 5 1 2 1 2 Anhydrous EtOH <0.1% 6 1 2 1 2 H 2 O/EtOH (1:16) 3% *The degree of substitution (DS) was calculated from the 1 H NMR spectrum **twice the concentration of reactants After selecting the optimal reaction conditions, substitution with other L-amino acids were carried out. Syntheses with L-phenylalanine, L-valine and L-proline protected with a Boc group have been carried out. Compared to L-glycine, worse but quite satisfactory results were obtained (Table 2.). Table 2. Reactions with another amino acids Reaction conditions: chitosan (1 mmol), HOBt (2 mmol), EDC (2 mmol), amino acid (1 mmol) H 2 O/EtOH (1:1, 16 ml); *The degree of substitution (DS) was calculated from the 1 H NMR spectrum The structures of the products were confirmed by 1 H NMR and FTIR spectra (see Fig. S2-S10 in Supplementary Material ) 3. Discussion The developed chemical modifications are the pilot studies in the synthesis of adsorbents based on chitosan – a biopolymer obtained from seafood industry waste. The aim of the research was to attach natural L-amino acids to the amino group of chitosan by an amide bond. The best result was obtained for glycine – slightly more than every fourth sugar unit in the chitosan molecule was substituted (degree of substitution: 26%). The methodology was successfully transferred to other amino acids, with slightly lower yields. By understanding the underlying principles and methodologies, researchers can advance the development of chitosan-based adsorbents with enhanced efficiency and selectivity, thereby paving the way for future sustainable and effective adsorption technologies. Furthermore, the conducted research provides invaluable contributions to the methodology of modifying the amino group in the chitosan molecule. Moreover, the use of chitosan as a substrate in the synthesis of adsorbents is not only inexpensive and environmentally friendly (due to its biodegradability) but also contributes to reducing the amount of waste from the seafood industry. Declarations Funding: Research project supported by program „Excellence initiative – research university” for the AGH University of Science and Technology”. 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Molecules. 24 , 3047 (2019). https://doi.org/10.3390/molecules24173047 Fangkangwanwong, J., Akashi, M., Kida, T., Chirachanchai, S.: Chitosan-Hydroxybenzotriazole Aqueous Solution: A Novel Water-Based System for Chitosan Functionalization. Macromol. Rapid Commun. 27 , 1039–1046 (2006). https://doi.org/10.1002/marc.200600152 Additional Declarations No competing interests reported. Supplementary Files graphicalabstract.png 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4323766","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":297274235,"identity":"29f59193-04d3-4ec7-a0b1-57dd06321b31","order_by":0,"name":"Marta Wolczko","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYHACxgMMDAkM/AwMbEAOMwg3ENQD1iLZANfCSKQWgwPEatGdkXzgwI8/aXLGx49fe8BQYZ3YIN2IX4vZjbSEgz08OcZmZ3LKDRjOpCc2yBwkoOV2jsFhBomKxG0HctIkGNsOJzZIJBKjxaAicXP/G6CWf0RrSchJ3CCRfkyCsYEYLfefAf1yIM1Y4sYbNomEY+nGbQT9cubwwQc//iTL8fenP5P4UGMt2y/dfACvFiTAYwCMHmAakCBWAwMD+wMITYKWUTAKRsEoGBkAAIvnTodHFTxkAAAAAElFTkSuQmCC","orcid":"","institution":"AGH University of Krakow","correspondingAuthor":true,"prefix":"","firstName":"Marta","middleName":"","lastName":"Wolczko","suffix":""}],"badges":[],"createdAt":"2024-04-25 11:23:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4323766/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4323766/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55707403,"identity":"e73523e2-93b7-47bd-b401-ef5302fd5543","added_by":"auto","created_at":"2024-05-02 05:16:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":45480,"visible":true,"origin":"","legend":"\u003cp\u003eReaction of chitosan with L-methionine by the method of azeotropic distillation\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4323766/v1/f292075fd87b9d85fb058ee2.png"},{"id":55707902,"identity":"4dff0f27-10d8-4564-8296-7b6faa069369","added_by":"auto","created_at":"2024-05-02 05:24:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":78029,"visible":true,"origin":"","legend":"\u003cp\u003eReaction of chitosan and lactobionic acid using EDC-NHS activating system\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4323766/v1/1f92f8b29dc1b91238b42eca.png"},{"id":55707406,"identity":"d3908c70-40f9-4667-8f38-54390442859f","added_by":"auto","created_at":"2024-05-02 05:16:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":96128,"visible":true,"origin":"","legend":"\u003cp\u003eReaction of chitosan and linoleic acid using EDS or EDC-NHS activating system\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4323766/v1/1d8d2e8ee245ed5ca950e03d.png"},{"id":55707408,"identity":"6c037d5d-a992-4d66-8835-48322613f53c","added_by":"auto","created_at":"2024-05-02 05:16:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":66390,"visible":true,"origin":"","legend":"\u003cp\u003eActivation of chitosan by HOBt and further reaction with an amino acid\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4323766/v1/8d10f5aebdd1bdb53649f56e.png"},{"id":55707404,"identity":"08771513-e7f9-4987-9032-6577034a17be","added_by":"auto","created_at":"2024-05-02 05:16:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":53380,"visible":true,"origin":"","legend":"\u003cp\u003eReaction of chitosan and Boc-L-glycine with HOBt-EDC as coupling agents\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4323766/v1/ebdb5bb5fcb5ddd381d05e88.png"},{"id":55915415,"identity":"3c5403f1-a228-401f-befd-8f2c0ca60ae3","added_by":"auto","created_at":"2024-05-06 08:54:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":799478,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4323766/v1/db62fc23-3a54-4766-b368-43fec4a51c75.pdf"},{"id":55707409,"identity":"8d86e16f-6bec-4b1f-bd4a-df05735739fa","added_by":"auto","created_at":"2024-05-02 05:16:27","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":59885,"visible":true,"origin":"","legend":"","description":"","filename":"graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-4323766/v1/bccb163cca02096be78a21a1.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Nitrogen-enriched chitosan. Strategies for Amino Acid Molecule Attachment","fulltext":[{"header":"1. Introduction","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003e1.1. The importance of chitosan\u003c/h2\u003e \u003cp\u003eChitosan is a deacetylated derivative of chitin which, in turn, is the second most abundant biopolymer in Nature after cellulose.[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] Chitin is naturally found in the shellfish (prawn, shrimp, crab, etc.) and fish, can be obtained from waste of fish industries and from household garbage. It can also be extracted from bacteria and fungi by the enzymes called chitosanases. It is estimated that the annual production of chitin in the biosphere is about 10\u003csup\u003e11\u003c/sup\u003e tons, and the seafood industry produces 60,000\u0026ndash;80,000 tons of waste annually.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] Chitosan is therefore a very common raw material in Nature, and at the same time quite cheap. Moreover, its non-toxicity, biodegradability and chemical stability, make it a valuable candidate as substrate for chemical synthesis.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAmong its notable features is the presence of one amino group in each sugar unit of this polymer, which provide sites for chemical modifications and functionalization. These characteristics make chitosan an attractive candidate for solid adsorbent applications, where the selective and efficient removal of target molecules from complex mixtures is desired.\u003c/p\u003e\u003cp\u003eIn recent years, extensive research has been conducted to explore the potential of chitosan as a solid adsorbent in diverse fields, including environmental remediation, water purification, biomedicine, and pharmaceutical industries. Chitosan's capability to adsorb heavy metals, dyes, organic pollutants, and even biological entities like proteins and enzymes has garnered significant interest.[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] However, to further enhance its adsorption performance and expand its application range, efforts have been directed towards the development of functionalized chitosan-based adsorbents.\u003c/p\u003e\u003cp\u003eAmong various processes used, including liquid solvent sorption, membrane separation and cryogenics, the CO\u003csub\u003e2\u003c/sub\u003e adsorption in solids seems to be simple, low cost, and effective.[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] Chitosan, as a biopolymer rich in amine groups, has already been used in the studies of CO\u003csub\u003e2\u003c/sub\u003e adsorption. The highest CO\u003csub\u003e2\u003c/sub\u003e adsorption capacity was reported as 5 mmol/g for chitosan-based carbons (chitosan spheres) at 0 \u003csup\u003eo\u003c/sup\u003eC and at atmospheric pressure.[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] However, the preferred conditions (for industry and economic impact) consist in the adsorption at ambient temperature. In 2014, the subject of research was chitosan modified by polyethylenimine (PEI).[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] Material, in the form of an aerogel, adsorbed CO\u003csub\u003e2\u003c/sub\u003e according to the carbamate mechanism, with maximum adsorption capacity 2.9 mmol/g (at ambient conditions).[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] Two years later, chitosan-polybenzoxazine polymer was used as a precursor for high CO\u003csub\u003e2\u003c/sub\u003e adsorbing porous carbon material resulting from aerogel carbonization. Nitrogen enrichment of the carbon material allowed to achieve high adsorption capacity \u0026ndash; 5.72 mmol/g at ambient conditions.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] A bit later, in another research group, mesoporous chitosan\u0026thinsp;\u0026minus;\u0026thinsp;SiO\u003csub\u003e2\u003c/sub\u003e nanoparticles showed a maximum CO\u003csub\u003e2\u003c/sub\u003e adsorption capacity of 4.39 mmol/g at ambient temperature.[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eOn the other hand, the adsorption of CO\u003csub\u003e2\u003c/sub\u003e in solid amino acids was also studied. Taurine gave the best result (3.7 mmol/g) at ambient temperature and increased pressure. Serine and valine gave results of approximately 2.5 mmol/g.[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] So far, the research on the modification of chitosan by natural amino acids has been carried out for L-glutamic acid and γ-aminobutyric acid, in order to immobilize the enzyme cellulase.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] Another research group combined chitosan with L-glycine, L-lysine, L-isoleucine and L-glutamic acid to increase the Cu\u003csup\u003e2+\u003c/sup\u003e adsorption (in solution).[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] However, the use of such derivatives was to serve completely different goals.\u003c/p\u003e\u003cp\u003eThe mechanism of CO\u003csub\u003e2\u003c/sub\u003e capture by solid amine adsorbents is based on electrostatic, hydrogen and acid-base interactions, but also on the active attachment of a carbon dioxide molecule to the amino group according to carbamate mechanism.[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] It is very likely, that the addition of an amino acid to the chitosan structure can significantly increase the CO\u003csub\u003e2\u003c/sub\u003e adsorption after material carbonisation.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eOne promising approach involves the attachment of amino acid molecules to chitosan, utilizing the available amino groups as reactive sites. Amino acids, as the building blocks of proteins, exhibit unique physicochemical properties and specific binding capabilities towards various target molecules. By combining the advantages of chitosan and amino acids, it becomes possible to design tailored adsorbents with improved selectivity and affinity towards specific contaminants or biomolecules.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1.2. Strategies for amino acid molecule attachment\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe following part of the article focuses on the review of the methods of creating an amide bond between chitosan and natural L-amino acids. Amino acids are the building blocks of proteins, having at least one nitrogen atom, so introducing them to the chitosan structure increases the nitrogen to carbon ratio in the final material.\u003c/p\u003e \u003cp\u003eIn 2017, authors from the University of Gujarat presented a simple synthesis of a sulfur derivative of chitosan by azeotropic distillation, starting from chitosan and natural\u003c/p\u003e \u003cp\u003eL-methionine. The amino acid and chitosan were equimolarly suspended in toluene. The reaction was carried out at 90\u0026ndash;100\u0026deg;C for 24 hours in a Dean-Stark apparatus. During the reaction, the release of water was observed in the apparatus, what according to the Le Chatelier and Braun's rule, shifts the reaction equilibrium towards the formation of the product.[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] The authors present the NMR spectrum of the product, as well as the FTIR spectrum, comparing them with the spectra of chitosan, but they do not specify the efficiency of the synthesis. The reaction scheme is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBy far, most methods of creating an amino bond from the -NH\u003csub\u003e2\u003c/sub\u003e group of chitosan are based on coupling reactions used to synthesize the same bond in peptides. A commonly used coupling reagent is EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), which activates the carboxyl group of an amino acid or other substrate. EDC is well known for its effectiveness in coupling two protein molecules, proteins and peptides, proteins and oligonucleotides, and proteins with other small molecules.[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] One of the most important advantages of this carbodiimide is the solubility of its by-product (urea derivative), which greatly facilitates the treatment of the mixture after the reaction. The second reagent often used together with EDC is NHS (\u003cem\u003eN\u003c/em\u003e-hydroxysuccinimide), which additionally stabilizes the active carboxylic acid derivative and thus increases the coupling efficiency.\u003c/p\u003e \u003cp\u003eThe group from Madrid presented a method of modifying chitosan with lactobionic acid, using a duo of EDC-NHS coupling reagents.[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] Lactobionic acid was first activated using EDC and NHS in an aqueous medium, and then it was added dropwise to chitosan previously dissolved in 1% aqueous acetic acid. The reaction was carried out for 72 hours at room temperature, then the product was purified by dialysis, obtaining a polymer with a degree of substitution from 3 to 16%, depending on the ratio of lactobionic acid to chitosan used, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn 2019, a group from the Szczecin University of Technology published the results of research on optimizing the attachment of long-chain linoleic acid to the chitosan molecule.[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] The carboxyl group of the acid was activated with EDC alone or with EDC and NHS. In the first case, the esterification reaction of the hydroxyl group at C6 of chitosan was dominant, while in the reactions with the addition of NHS, the main product was the amide. The authors explain that the intermediate product \u0026ndash; an ester of linoleic acid and NHS \u0026ndash; selectively reacts with the amino group, resulting in the amide being the dominant product. Schematics of both synthetic paths are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e below.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eDue to the overlapping of key signals in the NMR spectrum, the authors do not provide quantitative information on the degree of substitution of the products, but they write that reactions with carbodiimide alone are more efficient compared to the EDC-NHS duo, and they are characterized by the opposite selectivity (O-acylation \u003cem\u003evs.\u003c/em\u003e N-acylation ).[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eAnother research group in 2006 described research on the attachment of various carboxylic acids, including amino acids, to chitosan.[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] The authors of the study discovered that chitosan forms a water-soluble salt with hydroxybenzotriazole (HOBt). The structure of this salt was confirmed experimentally on nuclear magnetic resonance spectra. The chitosan-HOBt complex was successfully coupled to an amino acid (Boc-L-Phenylalanine) previously activated by EDC, resulting in a product with a degree of substitution of 28%. The authors emphasize that their method of modifying the chitosan amino group is characterized by high substitution efficiency and, importantly, is carried out under mild conditions (water-ethanol environment and room temperature). The reaction scheme is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBy understanding the underlying principles and methodologies, researchers can advance the development of chitosan-based adsorbents with enhanced efficiency and selectivity, paving the way for future sustainable and effective adsorption technologies.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"2. Experimental","content":"\u003cp\u003e\u003cstrong\u003e2.1 Material and methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneral Information:\u003c/strong\u003e All starting materials and reagents were obtained from commercial sources, and were used as received unless otherwise noted. All solvents were freshly distilled before use.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eChitosan (molecular weight 100-300 kDa and a degree of deacetylation of about 85% determined on the \u003csup\u003e1\u003c/sup\u003eH NMR spectrum), Boc-Gly-OH (\u0026gt;98%), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, \u0026gt;98%) 1-Hydroxybenzotriazole hydrate (HOBt, \u0026gt;99%), deuterium oxide (D\u003csub\u003e2\u003c/sub\u003eO, deuteration degree min. 99.9%) and CD\u003csub\u003e3\u003c/sub\u003eCOOD (99.5%) were used for research.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH NMR spectra were recorded with spectrometers operating at 600 MHz in D\u003csub\u003e2\u003c/sub\u003eO with addition of CD\u003csub\u003e3\u003c/sub\u003eCOOD. The infrared analysis of materials was carried out using a \u003cem\u003ePerkinElmer Frontier FT-IR\u003c/em\u003e spectrometer. The analyses were performed in transmittance mode in a wavenumber range 4000\u0026ndash;516 cm\u003csup\u003e-1\u003c/sup\u003e, in ATR technique. Spectra were processed in \u003cem\u003eSpectraGryph 1.2.15\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe aim of the research was to obtain a \u0026quot;nitrogen-enriched\u0026quot; chitosan derivative. The initial plan was to substitute an amino acid containing two nitrogen atoms, such as L-lysine. The first choice was the method of azeotropic distillation of chitosan with L-lysine, in toluene, acc. the procedure described in section 1.2. Unfortunately, despite several repetitions of the reaction, it was not possible to obtain even traces of the product. Changing the amino acid to the smaller L-glycine also had no positive effect. Moreover, the obtained \u003csup\u003e1\u003c/sup\u003eH NMR spectra were almost identical to those of the authors,[21] unfortunately the only change in relation to the spectrum of pure chitosan concerned the signal at 3.34 ppm, coming from traces of methanol after washing the product (see S1 in Supplementary Material).\u003c/p\u003e\n\u003cp\u003eTherefore, the azeotropic distillation method was abandoned in favor of amide bond coupling. The synthesis of\u0026nbsp;L-lysine and chitosan was carried out using coupling reagents EDC and NHS, according to the procedure successfully used for lactobionic acid.[23] Unfortunately, the product was not obtained, probably due to the presence of two unprotected amino groups in the amino acid. These groups can react with the activated carboxyl group of the second molecule, condensing into oligopeptides before being introduced into the chitosan solution, thus preventing the reaction from occurring. For this reason, in the further part of the research, only amino acids with protected amino groups were used.\u003c/p\u003e\n\u003cp\u003eL-Glycine with a \u003cem\u003etert\u003c/em\u003e-butoxycarbonyl (Boc) group was selected as the first model substrate. Two reactions were carried out according to procedures of the group from the Szczecin University of Technology.[24] In order for the \u003cem\u003eN\u003c/em\u003e-acyl form to be the dominant product, both EDC and NHS were used in the reaction. Syntheses were carried out in 1% acetic acid at pH 4.7 and 4.0. In both cases, only traces of the product were obtained \u0026ndash; slightly more (3%) at higher pH. The presence of the product was confirmed in the \u003csup\u003e1\u003c/sup\u003eH NMR spectrum, but due to the low yield, this method did not give hope for optimization.\u003c/p\u003e\n\u003cp\u003eThe next step was an attempt to activate chitosan by HOBt, with simultaneous activation of the amino acid by carbodiimide EDC according to procedure described above for phenylalanine.[25] This time the synthesis was successful \u0026ndash; chitosan substituted with Boc-L-glycine in 26% was obtained.\u003c/p\u003e\n\u003cp\u003eThe highest degree of L-glycine substitution (26%) was obtained using a two-fold excess of coupling reagents (Table 1., No. 1). Attempting to change the solvent to MeOH, as well as eliminating and reducing the amount of water, resulted in a huge decrease in the reaction yield (Table 1, No. 2, 5, 6). We managed to reduce the amount of coupling reagents to one equivalent, which is a great success from the point of view of green chemistry and future application in industry (Table 1., No. 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Reaction of chitosan and Boc-L-glycine \u0026ndash; optimization\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.321428571428571%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.714285714285714%\"\u003e\n \u003cp\u003e\u003cstrong\u003eChitosan (mmol)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.178571428571429%\"\u003e\n \u003cp\u003e\u003cstrong\u003eHOBt (mmol)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.892857142857142%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBoc-\u003c/strong\u003e\u003cstrong\u003eL-Gly (mmol)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.928571428571429%\"\u003e\n \u003cp\u003e\u003cstrong\u003eEDC (mmol)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.428571428571427%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSolvent\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.535714285714286%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDS*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.321428571428571%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.714285714285714%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.178571428571429%\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.892857142857142%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.928571428571429%\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.428571428571427%\"\u003e\n \u003cp\u003e\u003cstrong\u003eH\u003csub\u003e2\u003c/sub\u003eO/EtOH (1:1)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.535714285714286%\"\u003e\n \u003cp\u003e\u003cstrong\u003e26%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.321428571428571%\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.714285714285714%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.178571428571429%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.892857142857142%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.928571428571429%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.428571428571427%\"\u003e\n \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO/MeOH (1:1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.535714285714286%\"\u003e\n \u003cp\u003e5%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.321428571428571%\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.714285714285714%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.178571428571429%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.892857142857142%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.928571428571429%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.428571428571427%\"\u003e\n \u003cp\u003e\u003cstrong\u003eH\u003csub\u003e2\u003c/sub\u003eO/EtOH (1:1)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.535714285714286%\"\u003e\n \u003cp\u003e\u003cstrong\u003e21%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.321428571428571%\"\u003e\n \u003cp\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.714285714285714%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.178571428571429%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.892857142857142%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.928571428571429%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.428571428571427%\"\u003e\n \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO/EtOH (1:1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.535714285714286%\"\u003e\n \u003cp\u003e11.5%**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.321428571428571%\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.714285714285714%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.178571428571429%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.892857142857142%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.928571428571429%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.428571428571427%\"\u003e\n \u003cp\u003eAnhydrous EtOH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.535714285714286%\"\u003e\n \u003cp\u003e\u0026lt;0.1%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.321428571428571%\"\u003e\n \u003cp\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.714285714285714%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.178571428571429%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.892857142857142%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.928571428571429%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.428571428571427%\"\u003e\n \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO/EtOH (1:16)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.535714285714286%\"\u003e\n \u003cp\u003e3%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*The degree of substitution (DS) was calculated from the \u003csup\u003e1\u003c/sup\u003eH NMR spectrum\u003c/p\u003e\n\u003cp\u003e**twice the concentration of reactants\u003c/p\u003e\n\u003cp\u003eAfter selecting the optimal reaction conditions, substitution with other\u0026nbsp;L-amino acids were carried out. Syntheses with\u0026nbsp;L-phenylalanine,\u0026nbsp;L-valine and\u0026nbsp;L-proline protected with a Boc group have been carried out. Compared to\u0026nbsp;L-glycine, worse but quite satisfactory results were obtained (Table 2.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Reactions with another amino acids\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"557\" height=\"546\"\u003e\u003c/p\u003e\n\u003cp\u003eReaction conditions: chitosan (1 mmol), HOBt (2 mmol), EDC (2 mmol), amino acid (1 mmol) H\u003csub\u003e2\u003c/sub\u003eO/EtOH (1:1, 16 ml);\u003c/p\u003e\n\u003cp\u003e*The degree of substitution (DS) was calculated from the \u003csup\u003e1\u003c/sup\u003eH NMR spectrum\u003c/p\u003e\n\u003cp\u003eThe structures of the products were confirmed by \u003csup\u003e1\u003c/sup\u003eH NMR and FTIR spectra (see Fig. S2-S10 in\u0026nbsp;Supplementary Material\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eThe developed chemical modifications are the pilot studies in the synthesis of adsorbents based on chitosan \u0026ndash; a biopolymer obtained from seafood industry waste. The aim of the research was to attach natural L-amino acids to the amino group of chitosan by an amide bond. The best result was obtained for glycine \u0026ndash; slightly more than every fourth sugar unit in the chitosan molecule was substituted (degree of substitution: 26%). The methodology was successfully transferred to other amino acids, with slightly lower yields.\u003c/p\u003e \u003cp\u003eBy understanding the underlying principles and methodologies, researchers can advance the development of chitosan-based adsorbents with enhanced efficiency and selectivity, thereby paving the way for future sustainable and effective adsorption technologies. Furthermore, the conducted research provides invaluable contributions to the methodology of modifying the amino group in the chitosan molecule.\u003c/p\u003e \u003cp\u003eMoreover, the use of chitosan as a substrate in the synthesis of adsorbents is not only inexpensive and environmentally friendly (due to its biodegradability) but also contributes to reducing the amount of waste from the seafood industry.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eResearch project supported by program \u0026bdquo;Excellence initiative \u0026ndash; research university\u0026rdquo; for the AGH University of Science and Technology\u0026rdquo;.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMW - full contribution at every stage of research and preparation of the manuscript\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eResearch project supported by program \u0026bdquo;Excellence initiative \u0026ndash; research university\u0026rdquo; for the AGH University of Science and Technology\u0026rdquo;.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFirdous, K., Chakraborty, S.: A review: naturally available sources of chitosan and analysis of chitosan derivatives for its antimicrobial activity. 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Rapid Commun. \u003cb\u003e27\u003c/b\u003e, 1039\u0026ndash;1046 (2006). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/marc.200600152\u003c/span\u003e\u003cspan address=\"10.1002/marc.200600152\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"chitosan, amino acid, amide bond formation, nitrogen enrichment, seafood waste","lastPublishedDoi":"10.21203/rs.3.rs-4323766/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4323766/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChitosan, a polysaccharide derived from chitin, has gained significant attention as a promising solid adsorbent due to its unique properties, such as biocompatibility, biodegradability, and good adsorption capacity. Its abundant amino and hydroxyl groups offer numerous functionalization opportunities, allowing for the development of tailored adsorbents with enhanced performance in various applications.\u003c/p\u003e \u003cp\u003eThis publication explores the potential of chitosan as a solid adsorbent and focuses specifically on strategies for attaching amino acid molecules to chitosan. Many attempts have been made to combine chitosan with an amino acid, but the best results were obtained by combining the activation of chitosan with hydroxybenzotriazole (HOBt) and simultaneous activation of the amino acid with the carbodiimide EDC. The reaction occurs in water, thus adhering to the principles of green chemistry. Substitution of every fourth chitosan sugar unit with a natural amino acid L-glycine was obtained. Other amino acids have also been successfully substituted into the chitosan sugar backbone.\u003c/p\u003e \u003cp\u003eFurthermore, chitosan is derived from waste materials generated by the seafood industry, the utilization of materials based on it represents a way to reduce the quantity of such waste.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Nitrogen-enriched chitosan. Strategies for Amino Acid Molecule Attachment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-02 05:16:22","doi":"10.21203/rs.3.rs-4323766/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":"fcea0363-1f6c-4ef6-878a-cae834ad2f60","owner":[],"postedDate":"May 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-06T08:51:47+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-02 05:16:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4323766","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4323766","identity":"rs-4323766","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}