CA1183482A - Derivatives of chitins, chitosans and other polysaccharides - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Derivatives have been formed from chitins and chitosans in which the amine residues on the polyglucosamine have been modified to 0 form the groups; (a) -N=CHR or -NHCH2R or IMAGE

(b) -NHR' or (c) -NHR" or and, (d) -NH-CH2C02H or -NH-glyceryl where R is a substituent having a chelating function, R' is an aldose or ketose cr lactone residue, and R" is an organometallic aldehyde residue.
These derivatives are useful in chelating metals, in pharmaceutical formulations, in cosmetics, in chromatographic separations, in enzyme imnmobilization, as catalysts, etc. Galactomannans having selected amine-containing side chains have also been prepared by reductive amination.

Description

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FieId of the Invention .
This invention is directed to derivatives of natural polysac-charides, e.g. chitins and chitosans in which free amino groups are sub-stituted with selected groups attached through a nitrogen-carbon linkage.
Certain groups have been found to be introduceable by reductive alkyla-tion, and the carboxymethyl group has been introduced from monochloro-acetic acid. Selected amine moieties have been introduced into galacto-mannans.
Background and Prior Art Chitin is very widely distributed in the plant and, to an even greater extent, animal kingdom, where its main function is the provision of structural and skeletal support. Chitin is found most abundantly in fungi, while chitosan is obtained mainly by N-deacetylation of chitin, but also occurs in nature. The importance of chitin is emphasized by its natural abundance, an estimated 101 - 1011 tons annually, which makes it one of the most abundant organic materials on earth.
Chitin is a linear polymer o~ ~-D ~1-4) linked 2-acetamido-2-deoxy-D-glucopyranose units, of which a proportion, typically ~15%, is N-deacetylated (the fully acetylated poly~er i6 called chitan). The molecular weight of the chitin has been estimated at 1.04 x 1069 while that of chltosan ranges between 1.45-1.80 x 105. Chitin, like cellu-lose, has a ribbon-like structure. Three crystalline forms, a, ~, and y-chitin are distinguished on the basis of different cllain arrangements and the preserlce of bound water. In contrast to most other polysaccha-~5 rldes, chitln and chlto~an have basic characteristics (PKa Of chitosan~ 6~3) whlch impart them with unique propertie~ in terms of solubility, viscoaity, polyelectrolyte behaviour, membrane forming ability and metal chelatlon.
~oth these aminopoly~accharldes are insoluble in common organic 3P ~olvent~, watQr, dllute ~cld~, or cold alkalis o arly concentra~ion.
Thcre are only a few solvents or solvent systems whLch do not give rise to hydrolysis, degradation, or N-deacetylation. Chitin dissolves, for example, in 9N tlCl, or >9N H2S04 with hydrolysis of the glycosidic and amide linkages. The solvent systems for chltin which are more satis-factory include hexafl~loroisopropanol, hexafl~loroacetone sesquihydrate, .
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certain chloroalcohols, and hot concentrated solutions of neutral saltswhich are capable of high degrees of hydration, such as saturated lithium thiocyanate solution at 95~C. Chitosan is soluble in a number oE organic acids, including formic and 'acetic acid. Chitosan products have found a wide range of industrial applications and certain derivatives have been implicated in medical applications.
The outstand;ng industrial importance of polysaccharides arises from their usef~l interactions with a wide varlety of molecllles ranging ~rom food ingredients and pharmaceuticals, to inorganic particles, clay slips and oil well muds. These interactions occur mostly in aqueous media and the solubility properties of polysaccharides are therefore of great interest. Their unique soLubility properties constitute, at the samè time, one of the major problems in the development of polysaccharide chemlstry. Although these properties are known to follow certain trends, a systematlc undPrstanding of the relation of primary polysaccharide structure to physical behaviour in aqueous solution, and of polysaccha-ride-solute lnteractions, etc., remains to be esta'blished.
Natural polysaccharides can be classed into different solubil-ity groups according to their structure and conformation. Thus7 linear polysaccharides with a regular, ribbon-like structure such as cellulose and chitin, form highly ordered, often crystalline arrays which are di~ficult to dissolve due to strong cohesive forces, whereas branching usually leads to an enhancement in solubility and a reduction in the intermolecular interactions. Highly branched polysaccharides are almost ~5 always water~so]alble~ Solubillty can also be affected by a series of other Pactors such as lonic chargen, structural irregularLtles, glyco-~idic 'llnka5tes whlch preclude ri'bbon structures, Iow mo'Lecular welg'ht7 and a wide molecular weight distrLbutLon~ The possLbilLty oE modifying polysMccharldc solublli~y by chemlcal derivatlz~tioQ ls a relntLvely novel concept w`hlch ls gainin~ incrcasLnf,~ Lmportance Eor a wicle varlety o~ medLclnal ap~licntlolls, includlng polymer-medlated drug release, PrevLous workers have appllcd varlous synthetic reactlon routes to form modified products such as copolymerizatlon, orthoesters, aceto-bromosugars, hydrazones, or enzymic glycolysatlons to poly~acc:harides such as cellulose, amylose, and alginic acid. These procedures, however, ~ ~ ~39r8~2 suffer from various limitations since they require (i) specific protec-tion of the linear polysaccharide, such as ln the reaction of 1,2-ortho-- acetate sugars with 2,3-di-0-phenylcarbomoyl derivatives of amylose andcellulose; (ii~ activation of the sugar which is to form the side chain;
or (iii) reaction conditions which lead to partial or extensive polysac-charide degradation, e.g. using hydrazinehydrate. Most of the reactions are also labo~ious and low-yielding, all reasons which mitigate against routine or large-scale adaption.
The primary amine functions of chitosan have been derivatized with a number of anhydrides (S. Hirano, Y. Ohe and H. Ono, Carbohydr.
Res. 47, 315, 1976), and common aliphatic and aromatic aldehydes (S.
Hirano, N. Matsuda, O. Mlura, and H. Iwaki, Carbohydr. Res. 71, 339, 1979; and S. Elirano, N. Matsuda, O. Mitra, and T. Tanaka, Carbohydr. Res.
71, 344, 1979), invariably affording insoluble products (Schiff's base derlvative). Although certain water-soiuble ether and salt derivatives of chltosan are known, no attempts had previously been made to affect the ~olubilization of chitosan by introducing suitable hydrophilic moieties lnto the polymer.
The application of polymers as support matrlces for chelation, clinical use, catalysis, as well as for synthesis has grown rapidly since their use in peptide synthesis was demonstrated by Merrifield. One facet of this effort has been directed at incorporating metal ions or metal complexes into polymers using a ~ariety of chelating groups. Some prob-lems encountered ln many of these studles derlve from the often complex and co~tly synthesis, from lnefficient metal chelation, and ~rom the leaching into solution oP the metal complex from the polymer. The search for new and ef~icient chelatirlg polymers constitlltes, therefore, a major area of reseflrch, Numerous other reasons exist for intere~t in metal-polymer conJu~ates including the study oE metal complexes ln blological ~y~temf~, met~l-hased af~lnity chromatography, and the treatment of envi-ronmental polLIlt~nt~, In spite o~ a number oE studies o~ metal chelation by the nfltive polymers, only a few very recent attempts to improve their chelating capacity by means of chemical derivati~ation have been descrlb-ed in the llterature [M. Takahashi, K. Shinoda, T. Morl, and T. Kikyo, Japanese Patent 78 03982, ]978, (Chem. Abstr. 89, 1978, 64708g~;

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T. Sakaguchi, ~. Nakajlma and T. 7,lorikoshi, Nippon Nogei l~agaku Kai~hi, 53, 149, 1979, (Chem. Abstr. 91, 1970, 126598y)~.
United States Patent No. 3,~79,376, April 227 1975, to Vanler-berge, describes chitosan derivatives formed by acylation 7~Lth org~anic diacid anhydrides to introduce some substituent groups of the type -Nll-CO-P~-COOH. These derivatives are used as skin moisturizing agents in cosmetic compositions.
It woùld be desirable to introduce a variety of substituents so that the amino groups in the polysaccharide are attached directly to a carbon ato~n in the substituents.
Summary of the Invention .
~ mine-containing polysaccharides, in which at least a portion of thc amino groups are ~ree to react, have been reductively alkylated with various aldehyde or keto compounds to introduce substituents of the 5 following types: aromatic compounds, macrocyclic ligands, various sugars, lactones, organometallic compounds and, the carboxymethyl or glyceryl groups.
Using chitin and chitosan as examples, a method was devised whlch i8 sultable for the transformatlon of amine-containing polysaccha-rides lnto stable, branched-chain derivatives. One reaction used repre-sents a further example of the reductive amination procedure which is compatible with essentially any al~ehydo compound or compound r~adily oxldized or hydrolyzed to an alrlehyde. Under typical conditions, chito~
sans, dis50lved in a mLxture (l:]) of dilute (1%) aqueous acetic acid and methallol, was reductlvely alkylated usin~s a solutLon of the carbonyl-containLng reactant (1.1-3.2 molar equivalents per hexosamLne residue (mOl/~71CN) Q~. room temperature.
The reductLve alkylation i5 ,imply and convenLently eEected in ~,ood yield~ preferably wLth ;odLnm cyanoborohydrlde. The 7anle alkyla~lon Lll proceed ~o ~he Schlfe's baue stag,e (-N,~CllR) ln the abf;ence of re~lucln~ a~ent (cyanoborohydrLde). In the case of the carboxymethyl groups, monochloroacetic acid is utilized.
The versatile combination oE Schiff's base-formatlon and reduc-tive amlnation prov:Ldes a convenient route for attaching a wide range ofmedicinally or otherwlse important molecules to chitosan, whlch itself Ls .33~

a blodegradable material. Medicinal application, in particular, would c]early benefit from the additional options o~ selectively either solu-bilizing the polymer backbone, (an example of which will be described subsequently) or conversely, reducing its solubility by reaction of chitosan with another species.
Amlde products can be obtained from carboxylic acids or their derivatives (which can be obtained by transesterification, carbodlimide-Omediation, etc.) e.g. -~ICR. For amide products, the carboxylic acid compo~lnd to be reacted is activated with a water-soluble carbodiimide reagent (e.g. l-ethyl-3(3-dimethylaminopropyl)carbodiimide for 0.5 h) prior to addition to a solution o~ chitosan, e.g. in dilute aqueous acetlc acid.
This invention comprises as products, derivatives of amine-containlng ~olysaccharides wherein at least a portion o~ the amine groups are covalently bound through a =CII- or -C~I2- or -C- linka~e to substi-tuents selected ~rom: those having a metal chelating function includlng pyridines, aroma~ic compounds having at least one hydroxyl or carboxyl group; or macrocyclic ligands with an aldehyde or keto carbon atom;
aldose or ketose residues; organometallic residues; and a carboxymethyl or glyceryl group.
The invention includes derLvatives o~ chitin and chitosan and the substituents, including the amine N, having the structure:

(a) -N~CtlR or -~IC~I2R or -NHCR

~b) -N~IR' or -~ICR' (c) -~MR" or -~I~R"
~ nd, (cl) ~ CII2C02II or -~I-~lyceryl; respectively, where, R Ls an aromatic moiety having at least one hydroxyl or carboxyl group, or a pyridine moiety or a macrocyclic ligand, R' is an aldose or ketose or lactone residue attached via the aldehyde or keto or lactone carbon atom, and, K" ls an organometallic aldehyde resLdue attached vLa the alclehyde carhon atom.

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- The invention also includes the chitin and chitosan derivatives with substituents of the type (a), and (d) in the form of metal chelates thereof.
The invention further includes chitosan deri~atives having a mixture of substituents selected from (a), (b), (c), and (d) to give a desired balance of properties. For example, a mixture of aromatic and sugar substituents can be used to giv~ both high water solubility and high metal chelàtion capacity. A mixture of hydrophobic and hydr~philic subs~ituents can be used to achieve solubility in both aqueous and organic solvents (e.g. mixture of n-propyl and l-deoxylactit-l-yl substi-tuents).
Detailed Description and Preferred Embodiments ~ ny amine-group-containing polysaccharides can be utilized with naturally-occurring chitins and chitosans being preferred.
Chltosans with at least about 50% of their amino groups free to react are most suitable.
The aromatic substituents can be derivéd from any aromatic - aldehyde havlng at least one hydrophilic group selected from hydroxyl and carboxyl. Suitable aromatic and other chelating compounds include sali-cylaldehyde, 3-formyl-2-hydroxybenzoic acid, pyridine aldehydes and their derlvatives, 2,2-pyridil, alpha-pyridoin, and formylcinnamic acid~ and cyclodextrins, crown-ethers and cryptands.
The saccharide or sugar ~ubstituent can be derived from any aldo~e or ketose, or sllgars oxidizable or hydrolyæable to aldoses or keto~c~. The su~ar should not have steric hlndrance which would hinder reaction wlth the amine group on the chitin or chitosan. Suitable aldose~ or su~ars include glucoae, galactose~ arahinose, xylose, M-acetylglllco8amlne7 lactose, cellobiose, maltose, mellbiose, D-fructose, maltotriose, dextran, streptomycln sulfate, and glucoheptonlc acid lac-tone~ C71yceraldehyde can be utlllzed to lntroduce the glyceryl group.
The flromatlc sub~tltuents, and to some extent the (b) and (d) substltuent~7, chelate metal ions and form stable chelates ~7hich can be used as catalysts, etcO Suitable metals include iron, nickel, copper, zinc, lead, silver, mercury, palladium, uranium, chromium and platinum.
0rganometallic aldehydes can be incorporated slmilarly and the ~3~

products may also be used as catalys~s and in medical trea~ments. Suit-able organometallic aldehydes or ketones include ferrocene aldehydes, 1,1-fer~ocene dicarboxylic acid, haemin and m-toluidine chro~ium tricar-bonyl.
The carboxymethyl group can be introduced using monochloro-acetic acid as detailed in the examples.
The followlng examples are illustrative. Chitin (from crab shells) and chitosan (from shrlmp shells~ were obtained commercially and used without f~rther purification. Reactions were conducted at ambient temperature unless noted otherwise.
(L) General Procedure Chitosan (500 mg, 3 mM) was dissolved with stirring in a mi~-ture (1:1) of methanol and 1~ aqueous acetic acid (solvent A) or in the latter medium (solvent B) (30 ml). To the resulting viscous solution was added wlth vlgorous stirring a solution (10-20 ml) of the carbonyl-con-taining compound (3.3-10 mM) and sodium cyanoborohydride (20 mM). The reactlon mixture was left stir~ing at room temperature for 3-18 hr until a gel had formed. The solvent excluded by the gel was decanted, the gel was broken up, repeatedly washed with methanol (150 ml) and finally with diethyl ether (150 ml). ~le solid producta thus obtained were first air-dried for several hours, then dried in vacuo at 56C for 12-18 hr, and flnally crushed into a fine powder. In the cases where no gel was form-ed, the reaction mixture was dialyzed in dialysis bags against distilled water for perlods of 4-6 d with frequent (~15-20) changes of water to obtain, after lyophilization, mostly whlte materials.
(ll) Reac~ion~ o~ Chito~an wlth Reduclng Sugars ~x~mpLc 1 [1-deo~y-1-~alactit-1-yl] chito~an Addltion o~ galactos~ (1.20 g, 6.7 mM) led to thc formation of a stlff, glassy gel withLn 1-2 hr. The product [degree of substLtutlon (d,~.) O.9J w~ lvory coloured.
~nfll. for [(C8~l3Nos)o.l(cl2tl23 9 0-9 2 calcd. C 40.07, H 7.49, N 4.03; found C 40.09, 11 7.58, N 3.97.
When a smaller amount oE galactose (1.10 g, 6.1 1nM) was used, the resulting product had a lower d.s. (0.7).

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Anal- for ~(C6HllN4)o 3(C12H23N9)~-7 2 calcdO C 42.65, H 7.24, N 4.88; found C 42.59, H 7.20, N 4.97.
Example 2 [l-deoxy-l-glucit-l-yl~ chitosan ~ddition of glucose (0.72 g, 4 mM) in aqueous methanol produced no gel. Further addition of 0.9 ~ (5 mM) afforded a firm white gel which was washed and dried. The solid obtained (0.86 g~ appeared to be inhomo-geneous and was dialyzed for 4 days. After work-up, the product had d.s.
0.9 (0.64 g, 71%).
Anal. for [(C8Hl3NO5)0 02(C6HllN4)0.08(C12H23 9)0-9 2 10 calcd. C 43.05, H 7.22, N 4.39; found C42.80, H 7.10, N 4.60.
~xample 3 [1-deoxy-1-(2-deoxy-2-N-acetylamino) glucit-l-yl] chitosan Addition of N-acetylglucosamine (1.07 g, 4.84 mM) produced an elastic clear gel after standing overnight, which hardened and turned white after 24 hr. The product had d.s. 0.97.
A~lal. for [(C8~ll3NO5)0.o3(cl4~l25 2 9)0.97 2 calcd. C 40.20, H 7.~s4, N 6.69; found C 39.99, H 6.90, N 6.55.
Lxample ll [l-deoxy-l-cellobiit-l-yl] chitosan .
Addition oE cellobiose (3.5-6.6 mM) produced no gel after 2 d~ys. The product had d.s. 0.3.
Anal. for [(C8~113N05)o 05(C6HllN04)0 6s(~l8H33Nol4)]-o-7~ H20 calcd. C 42.47, H 7.04, N 5.11; found C 42.38, H 7.06, N 5.15.
~xample 5 ~l-deoxy~l-lactLt-l-yl] chitosan Addition of lactose (1.2 g, 3.5 mM) produced a milky solution hut no gel when the reactLon mixture was left stlrring for 10 hr. This product ~A) had d.s. 0.23. SlmLlarly, no gel was formed when the lactose to ~luco~3amlne (L/G) ratlo was increased to 1.5 (1.5 g, 4.5 mM lactose);
th~ product isolated after 30 hr had d.s. 0~12 ~B), whLle the same L/G
ratlo proclllced a whltc ~l when the reactLon ml~ture was left undlsturbed or ll~ hr ~wlth occaf3Lonal addition of reducing agent). This product (C) had d.s. 0.78 after dialysis. ~Ihen the L/G ratio was lncreased to

2.9~t (3.0 g lactose) a whlte soft gel was formed within 24 hr, which, ~4 hr, which, after nine washes with methanol (150 ml) and e~her (150 ml), produced a materlal (D) whose elemental analysis Lndicated a fulJy sub~tltllted (d.s. 0.9lf) prodllct contain~ng one equivalent of ~ ~ ~3~12 unreacted lactose per re~eating unit. Subsequent ex~ensive dialysis (5 d) of (D) produced a clear sol (E) with d.s. 0.94~
When the reaction ~as carried out in the absence ~f sodium cyanoborohydride, using an L/G ratio of 3.90, no gel formed after 28 hr and the resulting Schiff7s base product (F) had d.s. 0.1. The analyses for these lactityl derivatives are su~marized in Table 1.

Table 1 ( 8 13N05)m(C6H13N04)n(Clg~l23N014) ] x H 0 Product Formula _ N
(d.s.) mn p x calcd found calcd found calcd found _ _ _ _ ;
A(0.23) 0.07 0.7 0.23 _ 44.01 44.2 6.99 7.27 5.85 5.96 B(0.12) 0.07 0.81 0.12 0.79 41.85 41.69 7.12 7.0~ 6.44 6.60 C(0.78) 0.07 0.15 0.78 2.9 39.52 39.29 7.30 6.~ 2.98 3.00 F(0.1) 0.03 0.87 0.1 0.92 41.22 41.0] 7-13 7.01 6.63 6.71 8 13 5 0.Ol(c18~33Nl4)0 g4t 0-05(C12H330 )-1-62H 0;
calcd. C 41.72, H 7.06, N 2.63; found C 41.44, H 7.06, N 2.81.
Anal- or (D) [(C8~ll3No5)o.ol(cl8H33Nol4)o.94~ ~12H22ll] 1-9 2 ;
calcd. C ~1.80, ~l 6.86, N 1.59; ound C 41.49, H 6 96, N 1.55.

~xnmple 6 [l-deoxy-l-maltit-l-yl] chitosan Addltlon o malto~e (1.7~ g, 5.09 mM) produced a relatlvely vi~cou~ Bollltion aEter ~tandln~ of the reactlon ml~ture for 12 hr, and a ~tl~, whl~e ~el was Rormed withln 24 hr. Th~ product had d.~. 0.6.
1( B~l3No5)o.ol(c6tll~No~t)o 3g(Cl~tl33N()l~)0 Gl-l 98 ~l o;
c~ cd. C 40.38, d 7.23, N 3.96; found C 40.14, H 6.72, 9 3.67.

~ 7~ ~ ~

Example 7 [1-deoxy-1-melibiit~1~yl] chitosan Addition of melibiose (1.20 g, 3.5 mM) led to the formation of an initially (4 hr) soft gel which hardened on standing ~12 hr). The product had d.s. 0.60 8 13 05)o.ol{C6HllN04)o 3g(C18~33N 4) ] 0 59 H o calcd. C 43.12, H 6.96, N 3.81; found C 43.05, H 6.87, N 3.80.

The reactions of chitosan with various aldehyde-, keto-, lac-tone-, and non-reducing sugars, are summarized in Table 2. These reslllts as in Table 2 revealed the followLng points. The reactions of chitosan with aldehydo sugars proceed, in general, smoothly yielding products with mostly high degrees of substitution (d.s.) in almost all cases, these re-actions were accompanied by the formation of soft to very rlgid, trans-parent or mllky-whlte gels with, in the latter case, attendant synereses.
Monosaccharides produced gels at almost twice the rate of disaccharides and the d.s. of the chitosan products increased with increaslng amounts of aldehydo sugar used (with one exception for lactose).
In this series of experiments, no products were obtained for the reactions with glucosamine and galactoaamine despite the fact that very soft white gels were produced with glucosamine. This lack of prod-ucts i8 only true for low (i.e.<3 mol/GlcN) hexosamine concentrations.CouloMbic repulsion between the protonated amine groups of chitosan (pH
4.5) and those at C2 of the respective hexosamines seems to be primarily responsible for the lack of product formation at low concentrations in the latter two caMe~ since a fully substltwted chito~an derivative wa~
obtalned u~lng ~T~acetylgluco~amine. The amine function at C2 of the 3lde~chaln of thts latter sub~tituetlt provides, upon M-deacetylatLon, a convenient locus or eurther reductlve alkylatit7n reactLons which afford branched, tree-like derLvatives.
~,xample 8 Oxldations wltll GalactoMe OxidaMe [1-d~oxy-6'-aldehydo-:lactLt-l~yl] chltosan [1-deoxy-1-lactit-1-yl] chltosan fro~ Ex. 5 (103 mg, 0.13 mequiv. galactose) was dlspersed in phosphate buffer (25 mM, p~l 7, 10 m]) and formed a soft glassy gel which was purged with 2 for 1 minute.

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Table 2 - Reactions of Chitosan with Carbohydrates Conditions Product Compound (mol/GlcN)Titne Gela d.s~
added (hr) glucose 1.33 8 6 n.d.
3.00 ~ 1,5 0.9 galactose 2.03 4 1,3 0.7 2.22 4 1,3 1.0 glucosamine 1.3-1.6 8 4,5 0 2.7 ;72 4,5 0 7.ô 48 1,5 0.67 galactosamine 1.17-2.73 72 6 0 N-acetylglucosamine 1~6 12 1,3 n.d.
1.6 24 1,5 1.0 cellobiose 1.17 12 7 n.d.
2.2 36 7 0.3 2 9 456 __1,5 0 7 lactose 1 17 10 6 0 25 1~5 30 6 0.1 1.54 144 1,5 0.~
2.92-2.94 24 1,5 "2~0 maltose 1.7 12 7 n.d.
1.7 30 1,5 0.6 2~9 168 1,5 0.87 melibiose 1.11 4 1,4 n.d.
1.11 18 1,5 0.6 maltotriose 1.3 12 6 0 _ 4.2 ll~ 6 0.54 fructose 2.2-3.2 30 7 0 5.6 168 6 0.1 20.4 _ 624 ~ 0.7 ~-~lucoheptonlc lactone 3.2 72 6 0 3 6 1~ 1,5 0.77 ... _.-- . . . _ ~_ _ . . _ . . . ~ _ . .
meleæito~e* 1.05 2l~ 7 n.a.
1~05 240 2 n.a.
tre.ll~llo~e 1.3 72 6 n.a.
__ __ _ a - (1-5): gel~ formed; (1) rigLd; (2) ropy; (3) transparent; (4) very soft; (5) white; (6) gel not Eormed; (7) very viscous solution b - obtained Erom microanalysis c - not deter~ined d - see Ex. 5 * ~lelezitose does not react chemically wLth chitosan, but can be used to increase ~he ~olution visco~ity o~ chitosan~
n.a. ~ not appllcable.

~ ~3~2 Catalase (14400 units) and galactose oxidase (90 UDitS) solutions were added and a viscous, ropy materlal formed after a few hours. The poly-saccharide was diluted with water (10 ml) after 2 d and then poured into absolute ethanGl (150 ml). The precipitate was collected by centriEuga-tion (9000 rpm, 40 min) and dried, yielding 93 mg of the oxidi~ed productl-deoxy-6'-aldehydo-lactit-1-yl chitosan.
[l-deoxy-6'-aldehydo-melibiit-1-yl] chitosan [l-deoxy-l-melibiit-l-yl] chitosan from Ex. 7 (100 mg., 0.13 mequiv. galactose) was dissolved in dilute acetic acid and the p~ was raised to 4.5 by addition of aqueous NaHC03 solution yielding a gel which was treated as above. 95 mg of the oxidized product were iso-lated.
Reductive Amination of C-6 Aldehyde Chitosan Derivatives (a) The C6' aldehydo lactityl chitosan (43 mg, 0.06 mequiv~
galactose) was suspended in aqueous methanol (15 ml) to which was added a solutlon of amine spin label [32] (150 mg, 0.9 mM) and NaCNBH3 (0.1 g, 2 mM) in the sa~e solvent (5 ml). The amination was carried out for 12 hr and the product was purified by dialysis (3 d); esr double :Lntegration gave a d.s. ~0.7 (the microanalytical results could not be exactly matched with a molecular formula; found C 40.82, ~1 6.53, N 3.87); 132~ =

4-amirlo-2,2,6,6-tetramethylpiperidin-1-oxyl.
(b) The C6' aldehydo mellblityl chitosan (5S mg, 0.08 mequivO
gal~ctose) was treated as above in (a) yielding a product with d.s.~ 0.15 (from esr); found C 41.47, ~1 6.77, N 3.92.
x ~ [N~Sallcylidene] Chitosan To chito~an (500 mg, 3.0 mM), dissolved in a mixture (1:1, 25 ml) Oe methanol and 1~ aqueous acetic acld, was added dropwise and with vlgorous ~tirring, salicyLaldehyde (0.35 ml, 3.5 mM)~ The resulting yellow, inltially very viscou8 ~olutlon turned into a thick gel wlthln mln~te~ aturated aqueou~ ~olutlon of Na~lC03 was adlled (2 ml) ~o prevent acld hydroly~is of the Schlf e ~s base. ~ furtller portion of sall-cylaldehyde (0.35 m:l, 3.5 mM) was added dropwise resultlng in a further stlefening of the gel, to which was then added methanol (80 ml) and NaHC03 solutlon (1 ml). After a few minutes, the solvents were decanted and the wash was repeated twice, the p~l of the final wash being neutral. The gel was left standing in methanol (100 ml) for 4 hr, then ~3~

fil~ered on a sintered glass funnel, washed with methanol and diethyl ether (100 ml each), air-dried for several hours and finally dried in vacuo at 56C. Yield 0.75 ~, d.s. 0.97.
Anal. for [(C8~13NO5)0.02(cl3~ll5No5~o.98 2 calcd. C 56.20, 11 5.94, N 5.08; found C 56.36, H 5.84, N 5.09.
Addition of a total 4.2 mM salicylaldehyde resulted in a product with lower dos~ (0.7~.
Anal. for 1(C8~13N5)0.03(C6H11N4)0.27( 13 15 5 0.7 2 calcd. C 53.02, H 6.21, M 5.64; found C 52.84, H 5.97, N 5.67.
~xample 10 [N-(2-cresol)]-chitosan . . .
The same procedure as for Ex. 9 was employed using NaCNB~3 (0.2 g) concommitant with the addition of salicylaldehyde. Addition of the reducing agent caused the yellow colour of the product to fade and no gel was formed initially. The solution was left stirring overnight re~ulting in a soft gel. Further addition of the same quantities of ~alicylaldehyde and NaCNBH3 afforded a soft, ivory coloured gel which was dialyzed (3 d) against distilled water, and lyophilized to give a ~luEfy white material (0.44 g), d.s. 0.7.
Anal. for [(C8~113NO5)0 03(C6HllN~)0-27(C13 17 5 0-7 2 20 calcd. C 52.55, 11 6.76, N 5.59; found C 52.30, H 6.50, N 5.55.
When the reduction was carried out after the Schiff ~9 base gel had formed, the latter retained lts rigidlty and, to a large extent, its yellow colour. After the usual workup, a yellow product with d.s. 0.85 was obtaLned (0.70 g).
2S ~nal. ~or l(C8tll~N05)0 02(c6~lllN~4)o.l3(cl3~ll7No5)o-85] 2 ;
calcd. C 54.63, 11 6.66, ~ 5.32; ~ouncl C 54.42, ~1 6.34, N 5.49 R~ample 11 [N-(3-carboxy-)~al~cylldene] chLto~an A~ in ~x. 9, chitos.m (0.33 g, 2.0 mM) was conde~ed wlth 3-~ormyl~2~ydrDxy-ben~olc acLd (0.39 g, 2.35 m~) dls~olved ln l~lethanol (10 ml) tc produce a brl~ht yelLow and very rlgLd gel wLthln 3 rnln7 The produc~ wa~ yellow and odourle~s and had a d.s. 1.0 (0.53 Anal. for [(C8Hl3NOs)o.o2(cl4~llsNo7)o.98] 2 ;
calcd. C 51.24, H 5.26, N 4,31; found C 51,26, 11 5.20, N 4.17.

~ 1~34~2 Example 12 Carboxymethyl derivatives of chitosan and chitin Chitin or chitosan was suspended in DMS~ (15 ml) for 1 d prior to the treatment which was used for the preparation of both derivativesO
The polysaccharides (0.5 g) were suspended in an aqueous (65%) NaOH solu-tion (50 ml) fo 0.5 hr to produce the alkali deri~atives which werefiltered, pressed, and then added to a solution of monochloroacetic acid (2.6 g) in i-propanol (50 ml). The suspensions were stirred for 1 hr, filtered, resuspènded in water (100 ml) and the solution pH was raised (from 3.5 - 4.0) with conc. NaOH solution to neutral. The chitosan derivative formed a gel at this stage, which was lyophilized. The solid carboxymethyl chitin was filtered, pressed and dried.
Anal. for chitosan derivative d.s. 1.2 [( 12 17Nl0)0.l(c8~ll3No6)o 9]-1.02 H2O, calcd. C 40.46, H 6.25, N 5.62; found C 40.29, U 6.67, N 5.56.
Anal. Por chitin derivative d.s. 1.0 [(c~l3No6)0.l(cloHl5No7)o.9];
calcd. C 45.78, ~l 5.81, N 5.45; found C 45.89, ~1 6~86, N 5.46.
Example 13 Solubilized salicylidene chltosan (mixed substituents) To chitosan (0.50 g, 3.0 mM), dissolved in a mixture (1:1) of methanol and 1% aqueous IIOAc, was added lactose (0.30 g, 0.9 ~) in MeOII
(4 ml) and subsequently, salicylaldehyde (0.35 ml, 3.5 mM) and NaCNBH3 ~0.3 g, 4.8 t~) dissolved in water (4 ~L). The vigorously stirred mixture lost its yellow colour after a short time and produced a soEt, faintly ycllow gel. The product had an overall d.s. 1.0 with 25% sugar ~ubstltutlon.
~ na~- Eor [(C8~13N5)0 05(C13~117N5j0 71(C18~l33N014)0 21;
calcd. C 52.85~ H 6.57, N 4.42; Eound C 52.51, H 5.95, N 4.20.
~xam ~ ~ errocenyl chltosan To chitosan ~0.20 g, 1.2 mM), dissolved ln a mi~ture oE methan-ol and 1% aqucou~ ac~tlc acLd (1:1, 50 I~l) was careEully added, wlthstlrrlng, a soLutlon of Eerrocenealdehyde (0.30 g, 1.4 mM) and NaCNBH3 (0.9 g~ 14.4 mM) itl methanol (10 ml). The initially red reaction mi~ture was leEt stirring overnight yielding a ~ine brown precipitate which was Eiltered, washed (methanol), and dried. 0.38 g of the brown product (d.s. 0.l~45) was isolated.

33~

[~ 8113N5)0 02(C6MllN4)0 5~(C17~21FeN ) ];
calcd. C 48.83, H 5.83, N 5.26, Fe 9.37; found C 48.60, H 5.90, N 5.32, Fe 10.06.
Example 15 ~nhancement of Chelating Performance Chitosan was condensed with salicylaldehyde as described above to afford the Schiff's base derivative salicylidene-chitosan (1).
Reductlon of the acid-labile, base-stable azomethine function of salicylidene-chitosan (1), with sodium cyanoborohydride simultaneous with its formation produced a very soft, ivory coloured gel, wh~ch aEter dialysis and lyophilization, gave the amine (2) (d.s. 0.6) as a fluffy, off-white material. (Attempts to carry out the reduction consecutive to the formation of (1) were only partially successful as indicated by the retention of tnost oE the yellow colour and rigidity of the gel initially produced.) The analogous Schiff's base derivative (3) (d.s. 1~0) was produced in similar fashion fro~l 3-formyl-2-hydroxy benzoic acid.
The salicylidene chitosans (1), (2) and (3), like chitin (4) and chitosan (5), readily reacted wtih copper(II) acetate in either aqueou6 or methanolic (no substantial difEerences in Cu-~helation capa-city between these medla were observed) solutlon to produce coloured com-plexes (see Table 3), which could be characterlzed by esr spectroscopy.
Atomic absorption spectroscopy was used to quantitativelydetermine the amounts of Cu(II) incorporated into the derivatives. Table 3 ~hows that the copper chelation capacity of the amine (2), sampled aEter 12 hr reactton time, was enhanced by a actor of four over that of ~1), and a 50 ~nctor over that oE (3) or chitosan (5).
This lncrea~ed chelatillg capaclty o~ ~2) over (1) is in line wLth the observed stabLLlty con~tants o~ the copper(ll) complexe~ o~
related llgand systems. Furthermore, the greater porosity oP (2), a ~ ~y, water~ln~ol-lble materlaL whLch Ln contrast to it~ solid analogue (1) arld otller derlv~tlves, swelled consLderably in aqueous or alcoholic ~olutlon, is presumably also partly responsible for this observation, More inEormation was gained Erom experiments in which attempts were made to elute the copper ions Erom the complexes (1) and (2), uslng 0.1 M EDTA
solutlon at p~l 8. This proceeded successEully for the former case, whereas ~30-~0% oE the copper was retained by the latter complex, whlch ~ ~3~

Table 3 Copper Chelation Performance of some Chitin and Chitosan Derivativesa . . . ~
Copper Content Colour Cmpd Timeb mmol/gC % of parent polymer Cu(II~ complex (hr) theory , .. ....
(1) 1 0.54 23 deep yellow light green 12 0.62 26 dark green (2) 1 2.19 72 white green 12 3.03 100 dark green .
t3) 1 0.02 1 deep yellow deep yellow 12 0.06 2 deep yellow - - green yellow (6~ 1 0.42 13 whlte l-lght blue 12 2.64 80 turquoise (7) 1 0.26 7 llght yellow light green 12 0.40 11 turquoise

(5) 12 0.06 1 whlte blue (4) 12 0.18 4 yellow light blue ~8) 12 0.01 0.3 llght yellow light yellow n - ln meth~nol ~t 25C
b - contat time c - millimole per gram polyMer ~3~1~2 was, however, completely "demetallated" by treatment with aqueous acid (pH 2). These findings testify again to the greater chelating ability of (2).
It is interesting to note (see Table 3) that the amine (2)~ its analogue t1), and chitosan (5) chelated a relatively large proportion (72%, 87Z, and 86%, respectively) of their total uptake within a short period (1 hr), whereas (3) complexed a relatively smaller amount (30%) of copper~ The high chelating rate of (2) was also reflected in the al~ost instantaneous colouration (green) when this material was added to a solution of cupric or nickelous ions.
The carboxymethyl derivatives of chitin (6) (d.s. 1.0) and chitosan (7) (d.s. 1.2) formed turquoise copper(II) complexes. Both of these derivatives had a greater chelation ability that their non-deriva-tized precursors (5) and (4) - 20 times and ll greater, respectlvely, see Table 3. The carboxymethyl derlvatLves are good candidates for cGmmer-clal scale metal complexation. The N-galactosyl chitosan (8) on exposure to Cu(II) Lons did not alter its colour nor produce a detectable esr sig-nal (see Table 3). This chelation inhibiTion is presumably due to the non-porous nature of the material which appears (SEM) relatively imperv-10UB to metal ions.
Recently, we have also found that galactose-containing polymers partLcularly galactomannans such as guar gum and locust bean gum, can be derivati~ed by introducing selected amine moieties or by flrst lntroduc~
ing a prLmary amLne group and then further reactlng as for chitosan. The operatLve polymer~ ~hould have pendant galactose residues.
Galactomannan3 are assumLng an ever LncreasLng role Ln various branches o~ industry, notably ln foods, pharmaceutLcals, paper products, co~)metLcs, palnt~, drllllng, and explosive~. Guar gum and locust bean ~um are two n~' the Tnore 1mPOrtant galactomannan polysacctlarkles whLch are malnly derLved ~rom ~he seeds of legumLnous plant~ or from microblaL
source6. '~leir prLmary physiological function appears to be the reten-tion of water (by solvation), preventing the drying out of the seeds, and also their cayacity as food reserves. In addition, galactomaTtnans assume important roles in the inhibition of vLruses and ln interEeron induc-tlon.

-Both ~lar gum (MW 220,000) and locust bean gum (~W 3103000) contain a ~-D (1-4) linked mannan backbone which carries ~-D-galactosyl residues at the C-6 p~sitions and assumes a ribbon-like structure. The mannose (M) to galactose (G) ratio varies from 1.8:1 for guaran to 4:1 for locust bean gum.
The rheological properties of aqueous solutions of both gums are of particular interest for many reasons: they behave as non-Newton-ian solutions which, by themselves, form no gels unless borate or transi-tion metal ions are added. The gums are stable over a wide pH range and are compatible with many salts. The viscosity and shear stability of guar and its derivatives have made these preferred gelling agents for fracturing fluids in the oil industry.
To form the derivatives, the galactose moieties are first oxicli~ed to introduce aldehyde groups. Galactose oxidase was used to introduce an aldehyde group at C-6 of the pendant galactose units of guar g~m or locust bean gum.
The galactornannan oxidation procedures using galactose oxidase were shown to be high-yielding and specific, but may, nevertheless be not directly compatible with industrial applications in view of the costs lnvolved. Enzyme immobilization and controlled periodate oxidation can be used as two alternative, less expensive routes.

The ~ollowlng examples are lllustrative of these oxidation procedures.

~xnmple 16 - Oxldatlon Procedures on Galactomannan~
~ a) Galactose oxiclase was used to ln~roduce an aldehyde group a~ GG Oe tlle pendflnt ~alactoDc Imits o~ ~uar ~um, reductive aminatlon o whLch, u~lrl~ any prLmary or secvndary amine and sodlum cyanoborohydride afeords a polymer bearlng a substituent of choice at C6~ For example, pure guar gum (60 mg, 0.12 mM equivalents galactose) in pho~phate buffer (pH 7, 25 mM~ 15 ml) reacted with galactose oxidase (90 uni~s) in the presence of catalase (E.C. 1.11.1.6, 10500 unlts) for 24 hr, afforded a ~ ~3~

very viscous, ropy material. Omission of catalase led to an approxi-mately four-fold reduction in the yields. Reductive amination could be performed in situ by the addition of aqueous solutions of amine, e.g.
4-amino-2,2,6,6-tetramethyliperidine-1-oxyl (92 mg, 0~54 ~M) and sodium cyanoborohydride (300 mg, 4.4 ~M) over 36 hr. Alternatively the aldehyde derivativé could be isolated by ethanol precipitation, followed by cen-trifugation before reductive amination. Purlfication of the aminated product could be achieved either by dialysis (4 days) or by ethanol precipitation followed by careful washing. Although only preliminary attempts have so far been made to fully optimize the reaction conditions, the yields (based on galactose content) obtained are encouraging, typi-cally varying between 60-70% as determined by elemental microanalysis, 3C nmr, or by esr double in~egration.
Following the same procedures, locust bean gum was oxidized to the corresponding C6 aldehyde. The oxidation in this case proceeded with a greater e~iciency as ~udged by elemental analysis and esr double lntegratLon o~ the spin-labelled derivative, typical yields ranging from 70-90%.
Preferred procedure. The galactomannan (70-200 mg) was dis-~olved in phosphate buffer (25 mM, pH 7, 20 ml) by shaking for severalhours. The resulting solution was purged with oxygen for several minu~es before adding catalase (90000 units/0.1 mM galactose equivalen~s), and galactose oxLdase (l00 Imits/0.l mM galactose equivalents). The eamples were kept at 24C on a constant temperature shaker Eor 24-36 hr. The viscoslty o~ the reaction mixture increased sharply during the course of the oxLdatlon a~ordlng a ropy ~el ~or both guar and locust bean gum polys~ccharldes ~ter fl ~ew hours. '~e gel formation could be avolded by perormlng the reaction at greater dLlutlons (r~50 ml solution volume).
The aldehyde product~ were l~olated by (l? dilutLng the sampLe wLth an ~0 equal volullle o~ pho~pha~ buer prlor to etharlol precLpitation (250 ml).
The preclpLtate was then collecte~l by centri~ugation (7000 rpm, 40 min), or (ii) extensive dlalysLs and lyophilization.
(b) Periodate oxidations. The polysaccharlde (0.3 mM galac-tose equivalents) was dissolved in phosphate buffer (20 ml) as before and l-propanol (1 ml) was added followed by aqueous solutions (2 ml) of 3~82 - 20 - ~ -sodium metaperiodate (0.23 r~M/mol hexose unit for guaran and 0.17 mM/mol hexose unlt for locust bean gum). The oxidation was conducted at 5C in the dark for 15 hr after which it wa~ stopped by addition of ethylene glycol (1 ml). 11hen the oxidation was carried out in smaller solution volumes (~8 ml) the same ropy gels were obtained as in the case of galac-tose oxidase treatments. The dialdehyde products were isolated after dialysis (3 d).
I`he amine moiety is introduced into tha oxidized galactomannan by reductive amination. The amine moiety can be derived from variou~
ammonium carboxylic acid salts, aromatic and aliphatic amines including N-heterocyclic amines, hydroxyalkylamines (alkyl group from 1-6 C atoms), amino acids and peptides or biological proteins, especially enzymes.
Of the various products obtained by us by reductive amina~ion, the bovine serum albumin BSA-derivative constitutes a novel type of poly-saccharide-blolo~ical protein conjtlgate which typifies a wide range of practically useful derivatives including immobilized enzymes. Slmilarly, reactloQ of the aldehydo-galactomannans with amino acids, e.g. glycine or lysine, to yield another anionic species, illustrates the preparation of a new cla~s of glycopeptides. The hydroxyalkylamine guar derivatlves, e.g. hydroxypropylamine (d.s. ~0.8) are of interest for the-lr similarity to hydroxyethyl and hydroxypropyl derivatives of guar, prepared from the respective alkylene oxides, which have applications as efficient fracturing fluids for oil well sti~ulation. In contrast to the alkylene oxlde derivative, the hydroxyalkylamine derivative is specifically ?.5 ~ub~titlltcd at C6 of the galacto3e units whlch results in s;ubstan~ial dle~erenc¢s Ln the rheological properties. The he~erocyclic imidazole guaran prepared l~ typical Oe other medlcinal derivatLves.

6-[~1-3-amino-1-propanol] guar derivative ~eactlon o aldehyde~guar wlth 3-amino-1-propRnol (7.5 m~1/aldo-3~ hexo~e e~luLv~len~) aeEorded thi~ product. The d.s. was en~imated from13C~IImr to he ca. 0.8. MLcroanaly6ls gave a C/N ratlo of 17.33;
calcd. for d.s. 1.0; C/N 16.86).
6-(N-glycine) guarar1 derivative Reaction of aldehyde-guar with glyclne (~.0 mM/aldohexose eql1Lvalent) afforded the title product of d.s. 0.2. Analy~is for ~33 [(C6HloO5)0 64(C6Hg~jo.l6(C8H13N7)0-2] 2 calcd. C 39.35, M 6.31, N 1.43; found C 39.14, H 6.16, N 1.~7.
6-[N-(4-amino)-5-imidazolecarboxamide] guaran derivative Reaction o~ aldehyde-guar with 4-amino-5-imidazolecarboxamide HCl (4.3 mM/aldohexose equival~nt) afforded the title product of d.s.
O.05. Anal. for [(C6~l105)0 64(C6H96~0.31(c10~l15N4 5)0.05 2 calcd. C 38.94, H 6.35, N 1.48; found C 38.83, H 6.28, N 1.46.
Bovine Serum Albumin--conjugate of guaran 10To a solution of the alde~yde-guar in distilled water (119 mg, 0.71 mM, 20 ml) was added an aqueous solution of BSA (96.1 mg, 1.4 ~M) and sodium cyanoborohydride (250 mg, 4 mM, 5 ml). After stirring the reaction mixture for 2 d, the title product was isolated after dialysis.
The resulting aldehyde groups alternatively are reductively amlnated to introduce a primary amine function and provide a key catlonic lntermediate. Thi~ amination is carried out with an ammonium salt, preferably ammonlum acetate.
The reductive aminations of the C-6 aldehyde derivatives were carried out in situ after oxidation or by dissolving the isolated alde-hyde products ln aqueous solution followed by treatment with the amine(4-8 mol/galactose equivalent) and sodium cyanoborohydrlde (20-40 mol/galactose equivalent) at ambient temperature for 24-36 hrn The products were isolated by dialysis (4-6 d) and lyophilization.
The ~ollowlng examples are illu~tratlve of reductive aminatlon wlth ammonlum carboxyLate and o~ further reductive alkylation (as for chito~an) o~ the lnserted amlno groups.
17~- 6-[N-amino] guaran derlvative Reductlve amlnation Oe aldehyde-guar wlth ammonium acetate (6 mol/aldohexose unl~) ~or 3 d yLelded the tltle product having cl~s. 0.55.
30An~ or ~(c6~lloo5)o~6l~(c6~lloos~o.l6(c6lll2Nos)o~2oJ ~-56 ~l2 calcd. C l~0.48, H 6.52, N 1.56; found C 40.27, H 6~6, N 1.54.
Example 18 - 6-[N-l-deoxy-l-lactit-l-yl amine] guaran derivatlve C6-N-amino guaran (55 mg, 0.33 mM) was reductively alkylated with lactose (400 mg, 1.17 m~l) to a~ford, aEter 2 d, the title derlvative of d,~. O.b, (ba~ed on amino-gunran). An~ o-1~3~3?s~

[( 6 10 5)0.64( 6 10 6)0.16 6 12 5 0.12 18 34 15 0.08 2 calcd. C 38.94, H 6.74, N 1.41; found C 38.65, H 6.49, N 1.29.

All of the reactants operative with chitin or chitosan to introduce substituents of th~ type (a), (b), (c) and (d) can be used to de~iva-tize the prima~y amine functions in the oxidized aminated galac-tose-containing polymers.

The metal complexes involving Cu, Co~ Fe, Cr, Rh, Ru, can be used for both homogeneous and heterogeneous catalysis, pol~nerization, hydrogenation, decarbonylation, e.g. Cu(II) Schiff:s base complexes are homogeneous catalysts for the oxidative coupling of phenols. The corre-sponding Co(II) complexes are homogeneous catalysts for the oxygenation for alkyl pherlols, Co(II) Schiff's base co~plexPs are homogeneous cata-lysts for vinyl polmerlzations.
Other applications include use in chromatography, treatment of erlvlronmental pollutants~ waste water treatment, recovery oE trace metals (such as U) from sea water~ blood decontaminatiorl from radionuclides (such as plutonium), and as supports or carriers for new metalcontaining drugs.
lt 18 particularly important to note that chitosan can be con-verted into water-soluble and organic soluble derlvatives wherein the substituents are of formuLa (b), or a combination of ~b) and either (a) or (d). ~1ater solubllLty can be accompllshed at relatively low degrees of substltu~ion (d.s.) l.e., d.s. 0~ for derivatives of formula (b) nnd d.s. 0.25 for a mlxture of (b) and substLtuents (a), (c) or (d).
The chlto~an derL~atives ofer a wlde ran~e of solubllLty, gel-ling rhcological and compatibillty propertles. Thus, deriva~ivos of ~.xampLes (~), (5) and (6) form rLgld gels at concentratlon~ above 3-5~ ln aquec)u~l solutlon; derlva~lve~ of examplefi (1) and (3) geLIed ln acLdic f~oluîlollfi; thc derl-vc-ltlve oE example (7) gelled in baslc ~ol~ltions, 'rhe de~ivative~ of examples (4) and (5) exhlbit stabllity to alkaline media, while examples (4) and (7) were also compatible with 50% arlueous alcohol solutlons. Aqueous solutions of example (5) exhLbLt viscoelastic and thixotroplc behavlour and did not gel or precipitate when mLxed with ~ ~3~

calciuln chloride, chromium chloride, tin chloride, potassium chromate, boric acid, or combinations thereof. The derivative of example (4), which by itself did not gel; formed rigid gels when mixed with alginate, and very viscous so]u~ions, when mixed with guaran or locust bean gum.
The chitosan derivatives from glucoheptonic acid lactone exhibi~ unusu-ally hlgh viscosity at 1% concentrations.
Recent work has shown that metal chelating derivatives of chitosan can be obtained with substituents other than of the type (a), where R is a macrocyclic ligand with an aldehyde or keto carbon atom.
Suitable macrocyclic ligands include oxidized cyclodextrins, crown ethers, cryptands, and porphyrins.
The derivatives with macrocyclic substituents greatly extend the range of types of metals and elements which can be chelated or com plexed. Thl1s, aLkali, alkall earth, lanthanide, actlnide, halogen or noble gas elements can be incorporated into the chitosan derivatives, whereas chitosan derivatlves with substituents of the type (a) exhibit affinity towards transition metals only.
Derivatives of chitosan with macrocyclic substituents can be used for phase transfer catalysis and chromatography.
~xample 19 - [l-glucoheptonamide] chitosan To chltosan (0.50 g, 3 mmol~ in solvent (methanol ~ 1% aq.
acetic acid 1:1) was added a solution of a-glucoheptonic acid-~-lactone (2.2~ g, 10.95 mmol). After stLrring the reaction mixture for several dayr~, a soEt, whlte gel separated out. The ahove named product (d.s.
0.77) wari isolated a~ter 6 d reaction tlme, filtered and washed with ekhanol. Anal. ~or [(C~ 31105)o 03(c6HllNo4)o.2(cl3~l23Noll)o-77] 2 ;
c~lcd. C ~1.52, 11 6.~8, N ~.23; found C ~1.22, 1l 7.20, N 4.22.
L,x~m~__ 2~ - ChLtor~arl strcpromycLn lerLvatlverJ
Method A - To a r~olution of ch:Ltosan (1 mmol) Ln soLvent (1%
aq. acetic acld, 20 mL) was added a solutlon (15 mI.) of streptomycin sulfate (2.50 g, 3.~ mmol) and NaCNBH3 (1.2 g, 19 mmol). The reactlon mixture was stirred for 15 hr in the dark, and then dialyæed (~ d) to yield the derlvative (d.s. 0.07) as r;esquiacetate. Anal. for 3~3Z

8 13 5 0.03 27 49 815)0.07(C21142)0 105(C6HllN4)0 go] 3 57 H O
calcd. C 34.09, H 7.87, N 7.66 (C~N 4.45); found C 33.65, ~ 6.92, N 7.58 (C/N 4.44).
Method B - A solution of streptomycin sulfate (1.00 g, 1.36 mmol) and NaCNBH3 (0.4 g, 6.4 mmol, 15 mL) was added to an aqueous solution (pH 7) of (l-deoxylactit-l-yl) chitosan (d.s. 0.1, 0.25 g, 20 mL). The corresponding Schiff's base analogue was prepared with omission of the reducing agent to afford the yellowish sesquiacetate p~oduct (d.s.
0~08). Anal. for [(C8H13N4)0 03(C27H47NgOl5)0 og(C2H42)0 12(C6HllN04)o 89]-3.87H20 calcd. C 33.69, H 7.86, N 7.69 (C/N 4.38); found C 33.23~ ~1 6.98, N 7.66 (C/N 4.34).
Example 21 - Dextran derivative of chitosan An aqueous solution of dextran T10 (MW - 10,000) t5.00 g, ca.
0.5 mmol) and NaCNB~13 (0.4 g, 6.3 mmol, 20 mL) was added to chitosan (500 mg~ 3 mmol) in solvent (1% aq. acetic acid) containing 0.02% NaN3.
The reaction mixtura was stirred for 24 h at ambient temperature, and ~ubsequently lor 16 h at 40C~ The product was dialyzed (3 d) and iso-lated after gel chromatography. The d.s. of the dextranized product was estimated to be ca. 0.15 (found C 40.42, H 6.78, N 1.0o, C/N 37.43).
Example 22 - [2-amido-(2~6-diaminoheptanoic acid)] chitosan An aqueous solution of D,L-2,6-diaminopimelic acid (DL~2,6-dl-aminoheptanedlolc acid, mixture of LL-DD- ancl meso isomers) (1.0 g, 4.58 mmol, 40 mL) was acldifled wlth 3 drops 4N HCl before EDC (0.92 g, 5.3 mmol) wa~ add~d. A~ter ~tirring eor 0.5 h, the mixture was comblned with a ~oLutlon o~ chitosan (0.5 g, 3 mmol) in dilute (1~) aqueous acetic acid and the reactlon mixture was stirred or 24 h at amblent temperature.
The restlltin~ clear ~olutlon wa~ dlalyzed (3 d) and lyophllized, to yield th~ ~ta~ed prodllct, who~e d.~. corre~ponded approxlmately to 0.75 (C/N
3~ 3.82).
Example 23 [(1-deoxylactit-1-yl)-2-(N-cyclohexyl)pyrrolideayl] chitosan An aqueous solution containing lactose (l.00 g, 2.92 mmol) and N-cyclohexyl-2-pyrrolidone (2.00 g~ 11.96 mmol, 20 m~) wa~ added to chito~an (500 mg~ 3 mmol) in solvent (methanol - 1% aq. acetic acid 1:1).
~fter 48 h, the viscous mixture was precLpltated (methanol), exten~ively 3~

washed with 300 mL each oE methanol, acetone, ether, and finally dialyzed (2 d) to yield the product (found C 41.74, H 7.28, N 4.83).

(l-deoxylactit-l-yl)-2-(N-cocalkyl)pyrrolidenyl] chitosan The same procedure was employed, using N-cocoalkyl-2-pyrrolid-one (2.00 g, ca. 7.8 mmol) to yield this product (found C 44.33, H 7.60, N 5.15).
Example 24 - l-deoxylactit-l-yl chitin ~ Method ~ - Chitin (0.50 g) was dispersed in hexafluo~oisopro-panol (10 mL) and stirred for 12 h. After removal of undissolved material by filtration, a solution of lactose (0.50 g) and NaCNB~3 (0.15 g) in the same solvent (15 mL) was added~ After stirring the reac-tion mixture for 14 h, aqueous methanol (15 mL) was added. The resulting white precipitate was left standing Eor a further 12 h, filtered, washed wlth water (150~mL) and subsequently dialyzecl for 3 d, yielding 153 mg of an ivory-coloured, fluffy material~ d.s. 0.09. Anal. for l(C8ll13N5)0 8(c6~l11N4)0.l1(c18~33N14)0-o9] 2 ;
calcd. C 44.09, H 6~78, N 5.93; found C 44.30, H 6.77, N 5.97.

Method B - Chitin (1.00 g) was suspended in 40% aqueous sodium hydroxide (40 mL) for 30 min, then filtered, pressed, and washed succes-sivel~ with water and methanol (40 mL), subsequentl~ resuspended in a 1:1 mixture o~ 5% aqueous acetic acid and methanol (50 ~L) to which was added lactose (3.00 g) and NaCNBH3 (0.4 g). The reaction mixture was stirred ~or 2l~ h, then ~iltered and wa~hed wlth water (200 mL), methanol (150 mL), acetone (100 mL) and ether (40 mL). Anal. ~or 8 13 5)0.8(C6~ o4)o~l2(cl8~l33Nol~)o o~l-0-67 H20;
calcd. C ~4.10, ~1 6.79, N 6.01; ~ound C 43.92, ~1 6.85, N 6.18

Claims (11)

1. Derivatives of amine-containing polysaccharides wherein at least part of the amine groups are covalently linked, through a =CH- or or -CH2- linkage, to substituents selected from:
(a) those having a metal chelating function, (b) aldose or ketose or lactone residues, (c) organometallic residues, and (d) carboxymethyl or glyceryl groups.

2. Derivatives of claim 1 wherein the polysaccharides are chitin or chitosan and the substituents, including the amine N, have the structure:
(a) -N-CHR or -NHCH2R or (b) -NHR' or (c) -NHR" or and (d) -NH-CH2C02H or -NH-glyceryl;
respectively; where R is an aromatic moiety having at least one hydroxyl or carboxyl group, or a pyridine moiety, or a macrocyclic ligand, R' is an aldose or ketose or lactone residue attached via the aldehyde or keto or lactone carbon atom, and R" 1B an organometallic aldehyde residue attached via the aldehyde carbon atom.

3. The chitin or chitosan derivatives of claim 2 wherein the sub-stituents are of the type in (a) and are aromatic and the aromatic moiety its derived from at least one off salicylaldehyde, 3-formyl-2-hydroxylbenz-oic acid, pyridine aldehydes, and formylcinnamic acid.

4. The chitin or chitosan derivatives of claim 2 wherein the substituents are of formula (a) or (d), and are in the form of metal chelates.

CLAIMS (cont.)

5. The derivatives of claim 4 wherein the chelated metals are selected from silver, mercury, copper, lead, zinc, nickel, iron, pallad-ium, uranium, and platinum.

6. The chitin and chitosan derivatives of claim 2 wherein the sub, stituents have the formula (b) and R' is derived from aldoses, ketoses, and sugars oxidizable or hydrolyzable to aldoses or ketoses, selected from glucose, galactose, arabinose, xylose, glucosamine, N-acetylglucos-amine, lactose, cellobiose, maltose, melibiose, D-fructose, maltotriose, dextran, streptomycin sulfate, and glucoheptonic acid lactone.

7. The chitin and chitosan derivatives of claim 2 wherein the substituents are of formula (e) and the organometallic residues are selected from ferrocenes, haemin and m-toluidine chromium tricarbonyl.

8. The chitin and chitosan derivatives of claim 2 having a mixture of substituents selected from one or more of types (a), (b), (c) and (d) to give a desired balance of properties.

9. The chitin and chitosan derivatives of claim 6 wherein the substituents are derived from galactose and the galactosyl substituent is oxidized to form an aldehyde group and reductively aminated in situ to introduce an amine moiety and provide further branching.

10. The chitin and chitosan derivatives of claim 6 wherein the substituents are derived from glucosamine or from N-acetylglucosamine and any N-acetylglucosamine substituent is deacetylated, and the amine group reductively alkylated in situ to provide further branching.

11. The chitin and chitosan derivatives of claim 8 having a mixture of both aromatic hydrophobic and sugar type substituents.