Click reactions in chitosan chemistry - Springer Link

Russian Chemical Bulletin, International Edition, Vol. 66, No. 5, pp. 769—781, May, 2017

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Click reactions in chitosan chemistry A. S. Kritchenkova,b and Yu. A. Skorika,c aInstitute

of Macromolecular Compounds, Russian Academy of Sciences, 31 Bol´shoi prosp. Vasilévskogo ostrova, 199004 St. Petersburg, Russian Federation. Fax: +7 (812) 328 6869. Email: [email protected] bRUDN University, 6 ul. MiklukhoMaklaya, 117198 Moscow, Russian Federation cFederal Almazov NorthWest Medical Research Center, 2 ul. Akkuratova, 197341 St. Petersburg, Russian Federation The review provides the first generalized and systematized information on the use of click reactions in chitosan chemistry for the preparation of novel polymers with attractive physicochemical and biological properties. The reactions of coppercatalyzed azide—alkyne cycloaddition and the click reactions of chitosan derivatives occurring in the absence of salts or metal complexes are discussed in detail. The data on the preclick modification of chito san (i.e., the introduction of azide function, alkyne fragment, highly dipolarophilic moieties, and thiol group into the polymer) are reviewed. Special attention is given to the application of new chitosan derivatives obtained by click modification. Key words: chitosan, click reactions, azide—alkyne cycloaddition, thiolene addition, chitosan derivatives, preclick modification.

Introduction Chitosan is one of the most abundant natural poly mers.* Chitosan itself and its modified derivatives are objects of wide research aimed at their use in various technological and biomedical applications.1—7 For these purposes, chitosan often requires chemical modification to change its structure and properties to meet the re quirements imposed by specific applications. The tradi tional approach to the chemical modification of chitosan is simple and technologically convenient and is based on the synthesis of ethers and esters. Another approach uses nucleophilic substitution reactions involving the amino group (i.e., alkylation up to amino group quaternization, formation of amide bonds during conjugation with carb oxylic acids, etc.). Other methods, such as those based on oxidation reactions, partial polymer destruction, and grafted copolymerization are used to a lesser extent. Since chitosan is a heterofunctional compound, many reac tions to occur require the introduction of protective groups followed by their removal. The introduction of protective groups is also important when working with bifunctional agents. Classical methods used for chemical modifica tion of chitosan therefore often need rather drastic condi * Strictly speaking, chitosan does not occur in nature but is a product of the chemical or enzymatic Ndeacetylation of the natural polysaccharide chitin.

tions, which are inevitably accompanied by destructive side reactions, particularly ones that change the degree of polymerization and the degree of acetylation of the amino group.8—11 One alternative approach to the classical methods of chitosan derivatization is the use of click reactions, which can be used for the preparation of new derivatives that have the desired physicochemical and biological proper ties. The term "click chemistry" was first proposed by K. B. Sharpless in 200112 to describe chemical reactions suitable for the fast and reliable preparation of chemical substances by connecting individual units. Click chemis try is not specific to any particular reaction but is con ceived as an imitation of the formation of complicated compounds in nature from module units. The click chem istry should be modular; have a broad field of application; occur with yields close to quantitative; give safe byprod ucts or proceed without formation of byproducts; be stereospecific, regio and chemiselective, and thermo dynamically favorable (–ΔG > 84 kJ mol–1) for the for mation of a single product; and possess a high economy of atoms. The use of simple reaction conditions and avail able materials and reagents is desirable, such as the ab sence of solvents or the application of harmless solvents (preferably water) and the isolation of the product by nonchromatographic methods.13,14 Click reactions in clude [3+2] cycloadditions, particularly, azide—alkyne cycloadditions (coppercatalyzed variant and the reac

Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 0769—0781, May, 2017. 10665285/17/66050769 © 2017 Springer Science+Business Media, Inc.

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Russ. Chem. Bull., Int. Ed., Vol. 66, No. 5, May, 2017

tion promoted by angular strain in the cyclic system),15,16 thiolene additions,17 Diels—Alder reactions,18 [4+1] cycloadditions between isonitriles and tetrazines,19 nucleo philic substitution in strained small rings (epoxides and aziridines),20 and additions to double carbon—carbon bonds (for example, dihydroxylation).21 Click reactions using chitosan that are currently found in the literature focus on (i) coppercatalyzed azide— alkyne dipolar cycloaddtions, (ii) metalfree* cyclo additions of azides to highly dipolarophilic systems, and (iii) metalfree thiolene additions. Since chitosan con tains no functional groups appropriate for the occurrence of the listed click reactions, so called preclick modifica tions are used to introduce these groups. These preclick modifications of chitosan include the introduction of azide functional group or alkyne fragments, highly dipo larophilic fragments, and thiol groups or double bonds. Recent reviews that discuss the click reactions involv ing chitosan are either focused on the production of new materials,22,23 or are devoted to the click chemistry of polysaccharides as a whole.24,25 In the present review, we therefore provide the first generalization and systemati zation of the currently described examples of click reac tions in the chitosan chemistry. We also discuss the approaches used for the preclick modification of the polymer and direct click transformations that lead to the formation of new chitosan derivatives. Coppercatalyzed azide—alkyne [3+2] cycloaddition Introduction of an azide group into position 6 Several strategies for preclick modification of chito san were developed to continue coppercatalyzed dipolar azide—alkyne cycloaddition. The first strategy is the in troduction of an azide function into position 6. This introduction requires a very laborconsuming route, as a rule, based on a threestep synthesis that includes the protection of the amino group of chitosan (Scheme 1, steps A and G), substitution of the hydroxy group at the С(6) atom by a good leaving group (steps B, E, and H), and substitution of the introduced function with the azide group (steps C, F, and I). This preclick modification of the polymer is followed by the click reaction with the alkyne component (see Scheme 1, steps D and J). Amino group protection is achieved by two methods: synthesis of the phthalimide derivative of chitosan (see Scheme 1, step A) or condensation with aromatic aldehy des to form Schiff bases (step G). Bromide (see Scheme 1, step B)26—31 or a tosyl group (steps E and H)29,32,33 is used, as these are good leaving groups. Bromo derivatives * Hereinafter, the reactions occurring in the absence of salt or metal complexes are implied.

Kritchenkov and Skorik

are obtained by the treatment with Nbromosuccinimide or CBr4 in the presence of triphenylphosphine, whereas tosyl derivatives are prepared by the treatment with tosyl chloride. The azide function is subsequently introduced by the nucleophilic substitution of the bromide or tosylate using sodium azide (see Scheme 1, steps C, F, and I). The Nprotective group is then removed if necessary with hydr azine (in the case of phthalimide protection) or 5% acetic acid at room temperature (in the case of protection via Schiff base formation). The "bromide" route (see Scheme 1, step B) has been applied most frequently. This method was used to obtain chitosan derivatives containing an azide group in posi tion 6.27 These derivatives were involved in the click reac tions of coppercatalyzed 1,3dipolar cycloaddition with terminal alkynes. The reaction is characterized by high regioselectivity, because of the predominant formation of 1,4disubstituted triazole heterocyclic systems (1, 2). In this and subsequent examples, copper ions were removed during polymer purification using ethylenediaminetet raacetic acid disodium salt. An alkyne with quaternized amino group (Me3N+CH2C≡CH) was conjugated30 with the chitosan derivatives with a degree of substitution of 35% according to 1Н NMR spectroscopy data. The obtained watersolu ble cationic derivative 3 forms polyplexes with DNA. Gel electrophoresis revealed a much more efficient binding of derivative 3 to DNA when compared to nonmodified chitosan and the formation of strong complexes at a nitr ogen to phosphorus molar ratio of N/P = 0.8 (the lower threshold of N/P for chitosan is 2). Derivative 3 mani fested high transfection activity in vitro in the cell lines HEK 293 and MDAMB468. The obtained26 azide derivatives of chitosan react with electrophilic alkynes RОС(О)C≡CH and due to the click reaction, the form, with high regioselectivity, 1,2,3tri azole heterocyclic systems containing the ethoxycarbon yl substituent in position 4 of the triazole ring. Subse quent treatment with hydrazine results in the transforma tion of the ester function into the carboxy group. As a result, polymers 4 bearing both basic (NH2) and acidic (COOH) groups are formed, with a degree of substitution of nearly 100%. The obtained amphoteric polymers are highly soluble in both acidic and alkaline aqueous solu tions, whereas they form nanoparticles in neutral solu tions due to the interaction between the positively (NH3+) and negatively (COO–) charged groups of the chitosan derivative. The obtained azide derivative was involved in the click reaction with the spiropyran derivative containing an alkynyl group (degree of substitution 28%).34 The intro duction of the spiran moiety into the structure of polymer 5 induces new photochromic properties. Under UV irra diation the colorless film gains an intensive color due to the transition of the spiropyran structure into the mero

Click reactions in chitosan chemistry


771

Scheme 1

CD is cyclodextrin.

cyanine form. The excited merocyanine form, being co valently bonded with the polymer template, is character ized by a long lifetime (more than 24 h). The search for new biologically active chitosan deriv atives led to the synthesis of triazole derivatives 6—11 containing substituents with hydroxy group in position 4 of the 1,2,3triazole ring35 from azide derivatives (degree of substitution 85—93%). The protective group was re moved and the amino group of chitosan was then tri methylated to form a cationic derivative. The obtained polymers exhibited high fungicidal activity against the

plant parasites Colletotrichum lagenarium and Puccinia asparagi. The copolymer of polylactide, chitosan, and polyeth ylene oxide (PEO) was synthesized using the "bromide" route.36 The azide derivative of chitosan was conjugated with the alkynyl derivative of PEO that also contained a nitrosyl fragment. The result was the synthesis of a co polymer of PEO and chitosan with a nitrosyl group (de gree of crosslinking 43%), which was then introduced into the reaction with the bromide derivative of poly lactide via the oneelectron transfer reaction (Scheme 2).

772



Scheme 2

Chitosanfuran derivative 12 was synthesized via CuI catalyzed 1,3dipolar cycloaddition.31 The acetylene de rivative of furan was used as an alkyne component, while the chitosan derivative served as an azide component (see Scheme 1). The obtained polymers had a degree of sub stitution of 10%. New hydrogels were obtained by the Diels—Alder bis(maleimide) crosslinking of the furan derivatives of chitosan. The second variant is the "tosyl" one (see Scheme 1, step Е, Х = Ts) and is based on the transformation of the hydroxy group at the С(6) atom into a good leaving tosy late. This method also provides phthalimide protection of the amino group. A comparison of the routes for the prep aration of the azide derivative of chitosan via bromide or tosyl derivative (see Scheme 1, steps B or Е, respectively) shows that the "tosyl" method is more preferable.29 This is primarily due to the better leaving ability of the tosyl group and the mild synthesis conditions. The tosyl derivative is obtained by the treatment of the phthalimide derivative of chitosan with tosyl chloride at room temperature, while the synthesis of the bromide derivative needs prolonged heating at 80—90 °С, which results in partial polymer destruction. The azide derivatives of chitosan (degree of substitution 26—28%) were used in click reactions with phenylacetylene (with the formation of polymer 2) and with terminal diacetylenes containing the triple bond at both ends of the molecule: octadi1,7yne and 1,4diethynyl benzene. In the case of terminal diacetylenes, crosslink ing of the polymer chains resulted in the formation of gels 13 and 14 characterized by pHdependent swelling. The azide derivative of chitosan obtained by the "tosyl" method was involved in cycloaddition with the alkynyl derivative of lactose.33 The formed derivatives 15 containing lactose residues in position 6 showed high affinity for lectins. Mesylate is close in structure to tosylate and is also characterized by good leaving ability. In addition, mesyl

chloride is a more reactive electrophilic agent than tosyl chloride. The mesylation of chitosan with phthalimide protection at the amino group was shown32 to occur at 10 °С (see Scheme 1, step Е, Х = SO2Me). A subsequent treatment with sodium azide leads to the chitosan deriva tive with the azide group in position 6. The obtained polymer enters the click reaction with the terminal di acetylene derivative of PEO НС≡СO(O)CHNCH2CH2 (CH2CH2O)mCH2NHC(O)OС≡СН. The crosslinking of chitosan and PEO molecules affords hydrogel of co polymer 16.32 The phthalimide group used for protection of the amino group of chitosan requires drastic conditions for its re moval (heating for 7 h at 130—140 °С), which is inevita bly accompanied by partial polymer destruction. A more efficient method was developed37 for amino group pro tection that requires no drastic conditions. The method is the treatment of chitosan in an aqueousalcohol solution with aromatic aldehydes in the presence of 2% acetic acid at 60 °С. The protective group is removed by the treat ment of the polymer with 5% acetic acid at 0 °С within several hours (see Scheme 1, step G). This protection method allowed the synthesis of the azide chitosan derivative which reacts with the alkynyl derivatives of cyclodextrins to form derivatives 17.37 The azide derivative of chitosan (degree of substitution 78%) was also involved in the click reaction with alkynes con taining a quaternized nitrogen atom (R3N+CH2C≡CH). The obtained cationic polymers 18 showed high anti microbial activity against Staphylococcus aureus and Escherichia coli.38 Introduction of an azide group through a spacer The azide function can be introduced into the poly mer template also through a spacer, which is covalently



bonded, as a rule, to the nitrogen atom of chitosan. The azide derivative of chitosan containing the 4N3С6Н4С(О) group with different degrees of substitution (from 2 to 46%) was synthesized39 by the method of activated esters (Scheme 3, route А). Further quantitative cycloaddition

between the terminal groups of the alkyne derivative of cellulose and azide functions of the chitosan derivative resulted in the first reported formation of functional co polymers 19 of the cellulose—"click"chitosan type. The thermal stability of the obtained material significantly

Scheme 3

Gr is graphene.

R = CH2OH (20), CO2Me (21),

773

(23)

774


exceeds that of the starting polysaccharides. The method of activated esters was also used in the reaction of chito san with 5azidovaleric acid.40 PEO modified by alkyne groups was added to the obtained azide derivative. An azide group was introduced into chitosan through a phthalimide spacer (see Scheme 3, route B).27 The bro mophthalimide derivative of chitosan was obtained in the first step and was transformed by the subsequent treat ment with sodium azide into the azidophthalimide deriv ative (degree of substitution 87%). The obtained polymer was used in the click reaction of dipolar quantitative cyclo addition with propargyl alcohol and with methyl propi onate to form derivatives 20 and 21, respectively. The azide group was also introduced through a 2hydr oxypropylene spacer via the reaction of 2(azidomethyl) oxirane with the amino group of chitosan (see Scheme 3, route С).41 The acetylene derivative of graphene was used as an alkyne component for the click reaction of dipolar cycloaddition. The obtained polymer 22 has good me chanical properties. The introduction of a minor amount (2%) of covalently bonded graphene into chitosan in creases the Young´s modulus for film extension by more


than 200% when compared to the film produced using nonmodified chitosan. Chitosan derivatives that have the azide function bound to the polymer through the spacer have also been used in the click reaction42 to obtain empty nanoparticles of zinc oxide loaded with taxanes. The nanoparticle surface was covered with Ncarboxymethylchitosan (see Scheme 3, route D) and then the azidecontaining fragments were introduced by the peptide chemistry methods. The alkyne derivative of folic acid was then conjugated by cycloaddi tion to these fragments to provide targeted delivery of nanoparticles of 23 to tumor cells. Other examples also demonstrate linkage of the azide group through the spacer in position 6 of monomeric chitosan units. For instance, micelles for targeted deliv ery of doxorubicine were prepared by transforming the bromosubstituted derivative of the threecomponent co polymer chitosangpoly( εcaprolacton)(gpoly(oligo ethylene glycol)methacrylate)43 into the corresponding azido derivative (Scheme 4, route А), which was then conjugated with chitosan at position 6 (degree of substi tution 10%). The obtained polymer 24 tends to selfas

Scheme 4



semble into micelles, which were loaded with doxorubi cine. Targeted delivery was provided by an additional con jugation of the alkyne derivative of folic acid to the azide groups of polymer 24 using the click reaction. Grafted copolymers of chitosan and polycaprolacton (PCL) were also obtained44 using click chemistry. In this case, the azide group was also linked to the О(6) atom of chitosan through the spacer. The ester derivative was syn thesized by the treatment of the phthalimide derivative with 2bromo2,2dimethylacetic acid bromoanhydride (see Scheme 4, route В). The nucleophilic substitution of the bromine atom with an azide group was then carried out followed by the cycloaddition of PCL containing ter minal alkyne groups to form polymers 25. The graft ratio ranged from 493 to 537 wt.%. An example of nonselective introduction of azide group through a spacer is also known.45 Chloroacetyl chloride reacts with both the primary hydroxy group at the С(6) atom and the amino group of chitosan (see Scheme 4, route С). The polymer formed was converted to an azide derivative, which was used in the click reac tion with the alkyne derivative of nicotinic acid. The formed polymer 26 (degree of substitution 30%) was high ly active against Rhizoctonia solani Kühn, Stemphylium solani Weber, and Alternaria porri. Replacement of the amino group of chitosan by an azide group Another method for introducing an azide function into a chitosan molecule is based on the conversion of the amino group into an azide one to form polymer 27. High conversion of amino to azide group can be achieved using trifluoromethanesulfonylazide (Scheme 5).24,46 Howev er, its multistep character, technical complexity, and re quirement for drastic conditions (prolonged heating at temperatures higher than 100 °С) restrict the application of this method. Three approaches to chitosan azidation at position 2 (see Scheme 5, routes А—С) were com pared47 using sodium nitrate and azide (A), trifluoro methanesulfonylazide (B), and imidazole1sulfonylazide hydrochloride (C) as azidating agents. Trifluoromethane

775

sulfonylazide and imidazole1sulfonylazide hydrochlor ide were more convenient for azidation of chitosan, since they allowed the process to proceed in one step under significantly milder conditions. Nevertheless, the maxi mum degree of azidation of chitosan obtained with these azidation reagents was only 40 and 65%, respectively. The obtained azidated chitosan derivative was conju gated by the click reaction with calixarene modified in the alkyne fragment.48 The synthesized conjugates were good sorbents for heavy metals.49 In addition, modifica tion of hydroxyapatite with propiolic acid and its use as an alkyne component in the click reaction with azidated chitosan improved the hydroxyapatite surface properties for the production of biomaterials.50 Introduction of an alkyne fragment into the chitosan molecule Chitosan derivatives can also act as alkyne compo nents in coppercatalyzed azide—alkyne cycloaddition. In this case, the preclick modification is the introduc tion of the acetylene fragment into chitosan. The necessity of protecting the amino group appears upon the selective introduction of the alkyne fragment at position O(6) in the chitosan molecule. Only phthalimide protection of chitosan amino group is described in the present context. The phthalimide chitosan derivative was transformed into the carbamate derivative with alkyne fragments by the action of 1,1ćarbonyldiimidazole and propargylamine (Scheme 6, route А). The click reactions of aliphatic or ganic azides with this derivative were quantitative and regioselective.51 After removal of the phthalimide pro tection derivatives 28—30 were obtained with degrees of substitution of 13—24%, and nanoparticles were formed from them using the ionic gelation method. Polymers 28—30 and correspondent nanoparticles were tested for antimicrobial activity against E. coli and S. aureus, and high antimicrobial activity was confirmed for the nano particles.51 Click reactions can be used for conjugation of alkynyl chitosan derivatives with biologically active molecules containing a grafted azide function. Various proteins,

Scheme 5

776



Scheme 6

R=

(28), —CH2Ph (29),

(30), —CH2(CH2CH2O)3CH2CH2NH2 (31),

including enzymes, have been immobilized on chitosan in this way.52 A similar approach was used27 to obtain de rivatives 31, which turned out to be poorly soluble in both water and organic solvents. The authors assert that owing to these properties the derivative can potentially be used for the removal of toxicants from the gastrointestinal tract. Acetylene carboxylic acids can be conjugated at posi tion О(6) of chitosan by the method of activated esters using, for example, a mixture of dicyclohexylcarbodiimide and hydroxybenzotriazole as activators. 4Oxohept6 ynoic acid was thus conjugated with chitosan (see Scheme 6, route В).53 The obtained polymer with an alkyne func tion (degree of substitution 36%) was further utilized in quantitative click reactions for cycloaddition with the PCL derivative containing the azide functional group and pyrene residue. The obtained grafted copolymers 32 self organize into nanoparticles that exhibit fluorescence with a high quantum yield due to the grafted pyrene fragment in the polymer.53 An alkyne fragment can also be introduced using the nucleophilic properties of the nitrogen atom of chitosan amino group. One approach has been based on the conju gation of acetylene carboxylic acids with chitosan by the

(32)

method of activated esters (Scheme 7). An advantage of this method is that the functional groups of the polymer do not require protection. For example, a biologically active chitosanbased polymer with an original design was synthesized using click reactions.54,55 Chitosan was conjugated with pent4ynoic acid by the method of activated esters to obtain alkyne chitosan derivatives with a degree of substitution of 10—40%. The azidated deriva tive containing the diazenium1,2diolate fragment, whose azoxy group is protected by the galactose residue, was used as an azide component for the subsequent click reaction. The introduction of polymer 33 obtained by the click reaction results in the enzymatic elimination of the galactose residue in the blood stream followed by azoxy group destruction. This leads to the release of NO, an endotheliumrelaxing factor that prevents ischemic phenomena.54,55 The same strategy was used for the conjugation of propynoic acid with chitosan amino group followed by the click reaction with the azidated derivative of cyclo dextrin.56 The obtained polymer with a degree of substi tution of 6—7% forms nanoparticles that can be used for targeted delivery of doxorubicin to tumors.



777

Scheme 7

R=

(33)

In watersoluble oligochitosans,* the terminal hemi acetal hydroxyl can undergo fairly active cyclooxo tauto merism. Oligochitosan can be condensed with aromatic amines to form Schiff bases due to the contribution of the oxo group to tautomeric equilibrium. Chitosan was condensed with 4ethynyloxyaniline followed by the re duction of the obtained Schiff base.57 Oligochitosan with triple bond in the 4ethynyloxyphenyl fragment was used as an alkyne component in the click reaction with the azidated PCL derivative.58 The obtained grafted copoly mers form micelles, which can be used for targeted de livery of antitumor drugs. Other click reactions59,60 involve the action of one chitosan derivative as an azide component and another as an alkyne component. An azide chitosan derivative (the molar fraction of the azide groups in the polymer was 6%) was obtained using the method of activated esters by the conjugation of 2bromo2methylpropionic acid with chitosan amino group followed by the substitution of the bromine atom with the azide function. An alkyne chitosan derivative was also synthesized using the method of activat ed esters by the conjugation with prop2yn1ylcarb aminic acid (degree of substitution 10%). The click reac tion between these chitosan derivatives affords microcap sules that are assumed useful for targeted drug delivery. Metalfree click reactions The most important advantage of metalfree click re actions in chitosan chemistry is that purification of the formed polymer from metal ions is not necessary. This is usually an issue when metals are present because of the high chelating ability of chitosan and its derivatives. Under the conditions of normal electron distribution (according to Sustman´s classification) in the consistent * Higher chitosan oligomers and their mixtures with the mo lecular weight from 2 to 16 kDa are considered.

pericyclic cycloaddition process, azide is an electron den sity donor and alkyne acts as an acceptor. In this case, the reaction is controlled by the interaction of the HOMO of azide and the LUMO of the alkyne.61 The difference be tween the HOMO and LUMO determines the possibility for the reaction to occur and corresponds to the activa tion barrier of cycloaddition. The introduction of elec tronwithdrawing substituents into the alkyne molecule decreases the level of LUMO, thereby bringing together the frontier molecular orbitals of the reactants and de creasing the activation energy.62 When electrophilic alkynes (e.g., dibenzylcyclooctyne) are used, azide cyclo addition is characterized by a low activation barrier and needs no catalyst. This phenomenon was exploited in a series of cases that used metalfree click reactions of the azide chitosan derivatives. The azide group was bound63—65 to glycolchitosan through the tetraethylene glycol spacer at the amino group of the polymer (5 wt.% of azide group) (Scheme 8, route А). In addition, glycolchitosan was additionally conjugated with cholic acid, which resulted in its selfassembly into nanoparticles with azide groups on the surface. The ob tained nanoparticles with azide functions were used in the metalfree click reactions with the dibenzocyclooctyne derivative containing the 1,4,7,10tetraazacyclodode cane1,4,7,10tetraacetic acid moiety, which was linked to it through a spacer containing polyethylene glycol (PEG) and lysine residue (see Scheme 8). Owing to the chelant, the nanoparticles of polymer 34 can be marked with an appropriate isotope (for example, 64Cu2+), which is con venient for evaluation of their biodistribution in vivo. The preparation of nonspherical microparticles of the chitosan copolymer with PEG was developed66 as a template with a high surface for the conjugation of nucleic acids. The chitosan/PEG microparticles were modified at the amino groups with dibenzocyclooctyne using acti vated sulfoNhydroxysuccinimide ester. Under metal free conditions, DNA with the grafted azide group was

778



Scheme 8

R = —(CH2)4N3 (a), —(CH2)3SS(CH2)3N3 (b)

conjugated to the obtained microparticles with the active alkyne function. An alternative approach has also been used involving the introduction of a highly active dipolarophile (for ex ample, oxanorbornadiene) into chitosan. The interaction of this dipolarophile with an organic azide molecule does

not require a catalyst (see Scheme 8, route В). An oxa norbornadiene chitosan derivative was obtained (degree of substitution 15%) and used in the metalfree click reac tions for conjugation of molecules with grafted azide functional groups to form polymers 35.67,68 A similar method was utilized to obtain hydrogels for tissue engi

Scheme 9



779

Scheme 10

neering by using the click reaction to crosslink the high ly dipolarophilic oxanorbornadiene chitosan derivative with the azide derivative of hyaluronic acid.69 Another example of metalfree [2+1] cycloaddition involves the use of the azide chitosan derivative obtained by the addition of N3CH2OC(O)CH2COOH to the ami no group. The obtained azide formed nitrene on heating, and the latter reacted with graphene (degree of grafting of graphene 3 wt.%) to form polymer with high strength characteristics.70 Thiolene addition is also described among metal free reactions in the click chemistry. The thiolene addi tion was carried out71 for the conjugation of chitosan and heparin, which was aimed at preparing microcapsules of interest for targeted drug delivery. A UVirradiated allyl heparin derivative (degree of substitution 80%) is added to the thiol chitosan derivative (content of thiol group 650 μmol g–1) to form the corresponding copolymer 36 (Scheme 9). A phthalimide chitosan derivative capable of undergoing thiolene addition of aromatic thiols was pre pared later. The obtained modified chitosan with 100% degree of substitution was used as an efficient initiator of polymerization reactions.72 An example of the metalfree Diels—Alder reaction has also been described.73 A chitosan derivative containing a furan fragment was used as a diene, and bismaleimide served as a dienophile (Scheme 10). Hydrogels and microspheres, which were loaded with methylene blue, were synthesized from the obtained chitosan derivat ives 37 (degree of substitution 8—23%). Good profiles of the kinetics of methylene blue release allows evalua tion of the prospects of loading these microspheres with drugs.

Conclusion Success in synthetic organic chemistry has undoubt edly favored significant progress in the field of chemical modification of chitosan and copolymerization of the re lated polymeric materials. For example, the use of such reagents as dicyclohexyl carbodiimide has made possible the application of methods of peptide chemistry for chito san conjugation with carboxylic acids, whereas the devel opment of the approach of phthalimide and benzylidene amino group protection has permitted selective transfor mations at this or other functional groups of the polymer. These changes have extended the potential of chitosan chemistry, but the diversity of derivatives of this polymer has been restricted by the limitations of the classical or ganic synthesis methods using traditional polymeranal ogous transformations consisting of the formation of chitosan ethers, esters, and amides. The concept of click chemistry has now provided chitosan chemistry with powerful synthetic methods. In particular, the azide—alkyne cycloaddition catalyzed by copper ions as the first approach in chitosan click chem istry led to the development of methods for synthesis of new chitosan derivatives. This reaction is characterized by an effective orthogonal combination of the azide and alkyne components and occurs under mild conditions with quantitative yields, although some restrictions arise re lated to toxicity and the explosive risk of azides. The metal free click reactions (cycloaddition of azides to electro philic alkynes and other highly active dipolarophilic sys tems, Diels—Alder reaction, and thiolene addition) also offer mild conditions for the preparation of chitosan de rivatives with a number of attractive physicochemical and

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biological characteristics and have the important advan tage that no purification of the obtained polymer from metal ions is necessary. Thus, the application of methods of the modern click chemistry for chitosan derivatization opens up simple and attractive ways for controlled, selec tive, and technically convenient synthesis of biocompati ble and biodegradable polymers with specified structures and properties. This work was financially supported by the Russian Foundation for Basic Research (Project No. 163460173) and the Ministry of Education and Science of the Rus sian Federation (Program for Enhancement of Competi tiveness of the RUDN University among the Word´s Leading Research and Education Centers in the 2016—2020, agreement No. 02.a03.21.0008). References 1. A. S. Berezin, E. A. Lomkova, Yu. A. Skorik, Russ. Chem. Bull., 2012, 61, 781 [Izv. Akad. Nauk, Ser. Khim., 2012, 778]. 2. M. Dash, F. Chiellini, R. M. Ottenbrite, E. Chiellini, Progr. Polymer Sci., 2011, 36, 981. 3. E. Guibal, Progr. Polymer Sci., 2005, 30, 71. 4. R. Jayakumar, M. Prabaharan, P. T. S. Kumar, S. V. Nair, H. Tamura, Biotechnol. Adv., 2011, 29, 322. 5. M. N. V. R. Kumar, R. A. A. Muzzarelli, C. Muzzarelli, H. Sashiwa, A. J. Domb, Chem. Rev., 2004, 104, 6017. 6. M. Rinaudo, Progr. Polymer Sci., 2006, 31, 603. 7. A. S. Kritchenkov, S. Andranovitš, Yu. A. Skorik, Russ. Chem. Rev., 2017, 86, 231. 8. N. M. Alves, J. F. Mano, Int. J. Biol. Macromolecules, 2008, 43, 401. 9. K. Kurita, Progr. Polymer Sci., 2001, 26, 1921. 10. V. K. Mourya, N. N. Inamdar, React. Funct. Polymers, 2008, 68, 1013. 11. H. Sashiwa, S. I. Aiba, Progr. Polymer Sci., 2004, 29, 887. 12. H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem., Int. Ed., 2001, 40, 2004. 13. M. Arseneault, C. Wafer, J.F. Morin, Molecules, 2015, 20, 9263. 14. J. Escorihuela, A. T. M. Marcelis, H. Zuilhof, Adv. Materials Interfac., 2015, 2, 1500135. 15. J. C. Jewett, E. M. Sletten, C. R. Bertozzi, J. Am. Chem. Soc., 2010, 132, 3688. 16. C. Spiteri, J. E. Moses, Angew. Chem., Int. Ed. Engl., 2010, 49, 31. 17. C. E. Hoyle, C. N. Bowman, Angew. Chem., Int. Ed. Engl., 2010, 49, 1540. 18. M. L. Blackman, M. Royzen, J. M. Fox, J. Am. Chem. Soc., 2008, 130, 13518. 19. H. Stockmann, A. A. Neves, S. Stairs, K. M. Brindle, F. J. Leeper, Org. Biomol. Chem., 2011, 9, 7303. 20. B. A. Kashemirov, J. L. F. Bala, X. Chen, F. H. Ebetino, Z. Xia, R. G. G. Russell, F. P. Coxon, A. J. Roelofs, M. J. Rogers, C. E. McKenna, Bioconjugate Chem., 2008, 19, 2308. 21. S. Tyagi, E. A. Lemke, in Laboratory Methods in Cell Biology: Imaging, Vol. 113, Methods in Cell Biology, Ed. P. M. Conn, 2012, 169.


22. M. Barbosa, C. Martins, P. Gomes, U. Porto J. Eng., 2015, 1, 22. 23. A. Uliniuc, M. Popa, T. Hamaide, M. Dobromir, Cellulose Chem. Technol., 2012, 46, 1. 24. X. Meng, K. J. Edgara, Progr. Polymer Sci., 2016, 53, 52. 25. P.H. Elchinger, P.A. Faugeras, B. Boë ns, F. Brouillette, D. Montplaisir, R. Zerrouki, R. Lucas, Polymers, 2011, 3, 1607. 26. S. Ifuku, C. Matsumoto, M. Wada, M. Morimoto, H. Saim oto, Int. J. Biol. Macromolecules, 2013, 52, 72. 27. J. R. Oliveira, M. C. L. Martins, L. Mafra, P. Gomes, Carbo hydr. Polymers, 2012, 87, 240. 28. S. Ifuku, M. Wada, M. Morimoto, H. Saimoto, Carbohydr. Polymers, 2011, 85, 653. 29. G. Zampano, M. Bertoldo, F. Ciardelli, React. Funct. Poly mers, 2010, 70, 272. 30. Y. Gao, Z. Zhang, L. Chen, W. Gu, Y. Li, Biomacromole cules, 2009, 10, 2175. 31. M. MontielHerrera, A. Gandini, F. M. Goycoolea, N. E. Jacobsen, J. Lizardi Mendoza, M. T. RecillasMota, W. M. ArgueellesMonal, Iranian Polymer J., 2015, 24, 349. 32. P. Tirino, R. Laurino, G. Maglio, M. Malinconico, G. G. dÁyala, P. Laurienzo, Carbohydr. Polymers, 2014, 112, 736. 33. K. Koshiji, Y. Nonaka, M. Iwamura, F. Dai, R. Matsuoka, T. Hasegawa, Carbohydr. Polymers, 2016, 137, 277. 34. M. Bertoldo, S. Nazzi, G. Zampano, F. Ciardelli, Carbo hydr. Polymers, 2011, 85, 401. 35. Q. Li, W. Tan, C. Zhang, G. Gu, Z. Guo, Carbohydr. Res., 2015, 418, 44. 36. K. Zhang, P. Zhuang, Z. Wang, Y. Li, Z. Jiang, Q. Hu, M. Liu, Q. Zhao, Carbohydr. Polymers, 2012, 90, 1515. 37. Y. Chen, Y. Ye, R. Li, Y. Guo, H. Tan, Fibers and Polymers, 2013, 14, 1058. 38. Y. Chen, F. Wang, D. Yun, Y. Guo, Y. Ye, Y. Wang, H. Tan, J. Appl. Polymer Sci., 2013, 129, 3185. 39. P. Peng, X. Cao, F. Peng, J. Bian, F. Xu, R. Sun, J. Polymer Sci., Part A: Polymer Chem., 2012, 50, 5201. 40. V. X. Truong, M. P. Ablett, H. T. J. Gilbert, J. Bowen, S. M. Richardson, J. A. Hoyland, A. P. Dove, Biomaterials Sci., 2014, 2, 167. 41. H. J. Ryu, S. S. Mahapatra, S. K. Yadav, J. W. Cho, Eur. Polymer J., 2013, 49, 2627. 42. N. Puvvada, S. Rajput, B. N. P. Kumar, S. Sarkar, S. Ko nar, K. R. Brunt, R. R. Rao, A. Mazumdar, S. K. Das, R. Basu, P. B. Fisher, M. Mandal, A. Pathak, Sci. Reports, 2015, 5, 11760. 43. L. Yu, M. Cao, H. Zhang, H. Xiong, Y. Jin, Y. Lu, H. Jia, D. Luo, L. Wang, H. Jiang, J. Polymer Sci., Part A: Polymer Chem., 2012, 50, 4936. 44. W. Yuan, Z. Zhao, S. Gu, J. Ren, J. Polymer Sci., Part A: Polymer Chem., 2010, 48, 3476. 45. Y. Qin, S. Liu, R. Xing, K. Li, H. Yu, P. Li, Int. J. Biol. Macromolecules, 2013, 61, 58. 46. F. Zhang, B. Bernet, W. Bonnet, O. Dangles, F. Sarabia, A. Vasella, Helv. Chim. Acta, 2008, 91, 608. 47. R. Kulbokaite, G. Ciuta, M. Netopilik, R. Makuska, React. Funct. Polymers, 2009, 69, 771. 48. M. M. Lakouraj, F. Hasanzadeh, E. N. Zare, Iranian Poly mer J., 2014, 23, 933. 49. S.Y. Zhu, H.Y. Guo, F.F. Yang, Z.S. Wang, Chinese Chem. Lett., 2015, 26, 1091.



50. J. Wei, P. GuoWang, L. Cui, H. Zhang, Y. Dai, T. Liu, Chem. Res. Chinese Universities, 2014, 30, 1063. 51. A. Sarwar, H. Katas, S. N. Samsudin, N. M. Zin, Plos One, 2015, 10, e0123084. 52. A. K. Shakya, H. Sami, A. Srivastava, A. Kumar, Progr. Polymer Sci., 2010, 35, 459. 53. W. Yuan, X. Li, S. Gu, A. Cao, J. Ren, Polymer, 2011, 52, 658. 54. Q. Zhao, J. Zhang, L. Song, Q. Ji, Y. Yao, Y. Cui, J. Shen, P. G. Wang, D. Kong, Biomaterials, 2013, 34, 8450. 55. X.W. Shi, L. Qiu, Z. Nie, L. Xiao, G. F. Payne, Y. Du, Biofabrication, 2013, 5, 041001. 56. L. Lu, X. Shao, Y. Jiao, C. Zhou, J. Appl. Polymer Sci., 2014, 131, 41034. 57. A. Guerry, J. Bernard, E. Samain, E. Fleury, S. Cottaz, S. Halila, Bioconjugate Chem., 2013, 24, 544. 58. A. Guerry, S. Cottaz, E. Fleury, J. Bernard, S. Halila, Carbo hydr. Polymers, 2014, 112, 746. 59. L. Hu, P. Zhao, H. Deng, L. Xiao, C. Qin, Y. Du, X. Shi, RSC Adv., 2014, 4, 13477. 60. J. Zhang, C. Li, Z.Y. Xue, H.W. Cheng, F.W. Huang, R.X. Zhuo, X.Z. Zhang, Acta Biomaterial., 2011, 7, 1665. 61. A. S. Kritchenkov, K. V. Luzyanin, N. A. Bokach, M. L. Kuznetsov, V. V. Gurzhiy, V. Y. Kukushkin, Organometal lics, 2013, 32, 1979. 62. Y. D. Samuilov, A. I. Konovalov, Russ. Chem. Rev., 1984, 53, 332. 63. D.E. Lee, J. H. Na, S. Lee, C. M. Kang, H. N. Kim, S. J. Han, H. Kim, Y. S. Choe, K.H. Jung, K. C. Lee, K. Choi, I. C. Kwon, S. Y. Jeong, K.H. Lee, K. Kim, Mol. Pharma ceutics, 2013, 10, 2190.

781

64. S. Lee, S.W. Kang, J. H. Ryu, J. H. Na, D.E. Lee, S. J. Han, C. M. Kang, Y. S. Choe, K. C. Lee, J. F. Leary, K. Choi, K.H. Lee, K. Kim, Bioconjugate Chem., 2014, 25, 601. 65. J.K. Rhee, O. K. Park, A. Lee, D. H. Yang, K. Park, Marine Drugs, 2014, 12, 6038. 66. S. Jung, H. Yi, Langmuir, 2012, 28, 17061. 67. J. Jirawutthiwongchai, A. Krause, G. Draeger, S. Chira chanchai, ACS Macro Lett., 2013, 2, 177. 68. J. Jirawutthiwongchai, G. Draeger, S. Chirachanchai, Macromol. Rapid Commun., 2014, 35, 1204. 69. M. Fan, Y. Ma, J. Mao, Z. Zhang, H. Tan, Acta Biomaterial., 2015, 20, 60. 70. S. K. Yadav, S. S. Mahapatra, M. K. Yadav, P. K. Dutta, RSC Adv., 2013, 3, 23631. 71. V. Calcagno, R. Vecchione, A. Sagliano, A. Carella, D. Guarnieri, V. Belli, L. Raiola, A. Roviello, P. A. Netti, Colloids Surf. B: Biointerfaces, 2016, 142, 281. 72. S. R. Valandro, A. L. Poli, T. Venancio, J. Pina, J. S. de Melo, H. D. Burrows, C. C. Schmitt, J. Photochem. Photo biol. A: Chem., 2016, 327, 15. 73. M. MontielHerrera, A. Gandini, F. M. Goycoolea, N. E. Jacobsen, J. Lizardi Mendoza, M. RecillasMota, W. M. ArgueellesMonal, Carbohydr. Polymers, 2015, 128, 220.

Received October 18, 2016; in revised form February 1, 2017