THIOL-MODIFIED POLYMER COMPOUND, AND PREPARATION METHOD THEREFOR AND APPLICATION THEREOF
20230081482 · 2023-03-16
Inventors
Cpc classification
C08B37/003
CHEMISTRY; METALLURGY
C08L5/08
CHEMISTRY; METALLURGY
C08L5/08
CHEMISTRY; METALLURGY
C08G65/3342
CHEMISTRY; METALLURGY
C08F122/1006
CHEMISTRY; METALLURGY
C08L89/06
CHEMISTRY; METALLURGY
C08B37/0072
CHEMISTRY; METALLURGY
C08B37/0069
CHEMISTRY; METALLURGY
International classification
Abstract
A polymer compound modified by an acryloyl derivative and a polythiol compound are used for preparing a thiol-modified polymer compound by a Michael addition reaction of thiol and conjugated double bonds. In addition to achieving the structural goal of the thiol-modified polymer compound, the preparation method further has the following advantages: flexibly and effectively controlling the structure and constitution of a synthetic product, and the types and contents of a large number of compound molecular functional groups; using reagents having high biocompatibility, and effectively controlling the production cost and reducing the toxicity in the synthesis process; and obtaining, under the conditions of using safety reagents and simple reaction steps, thiol-modified biocompatible polymer compounds that can be used as extracellular matrix materials and maintain good raw material structure and biological activity, with the types and contents of functional groups adjusted according to requirements, and meeting multiple clinical application requirements.
Claims
1. A sulfhydryl-modified polymer compound, wherein a polymer compound to be modified comprises at least one of —COOH, —NH.sub.2, —OH, an acrylate group of formula a, an acrylamide group of formula b, and an acryloyl group of formula c in the structure, ##STR00020## wherein part or all of the —COOH and/or the —NH.sub.2 and/or the —OH and/or the acrylate group and/or the acrylamide group and/or the acryloyl group are modified to form a side chain with the following terminal group: ##STR00021## in the above group, * represents a linking site; R.sub.1 is selected from hydrogen, halogen, an aliphatic group, an aromatic group, and the like; R.sub.2 and R.sub.3 are the same or different and independently from each other are selected from hydrogen, halogen, an aliphatic group, an aromatic group, and the like; and R.sub.4 is a fragment of a polysulfhydryl compound.
2. The sulfhydryl-modified polymer compound according to claim 1, characterized in that, part or all of the —COOH and/or the —NH.sub.2 and/or the —OH and/or the acrylate group and/or the acrylamide group and/or the acryloyl group are modified to form at least one of the following structures: ##STR00022## in the above structures, R is selected from ##STR00023## hydrocarbylene, arylene, an amide residue, a hydrazide residue, and the like; * represents a linking site; .sub.1* represents a linking site to a left-hand group of R; .sub.2* represents a linking site to a right-hand group of R; R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are defined as above; preferably, at least one of the —COOH, the —NH.sub.2, the —OH, the acrylate group of formula a, the acrylamide group of formula b, and the acryloyl group of formula c can be directly linked to the main chain of the polymer compound, or linked to the main chain of the polymer compound via an R′ group, and the R′ group can be a heteroatom-containing group, hydrocarbylene, arylene or the following linker: ##STR00024## in the above formula, R″ is hydrocarbylene or arylene, n′ is an integer from 1 to 1000, and * represents a linking site.
3. The sulfhydryl-modified polymer compound according to claim 1, wherein the polymer compound to be modified comprises natural mucopolysaccharide polymers, proteins and/or synthetic polymers.
4. The sulfhydryl-modified polymer compound according to claim 1, wherein a sulfhydryl content of the sulfhydryl-modified polymer compound as determined by an Ellman method is 0.01-30 mmol/g.
5. The sulfhydryl-modified polymer compound according to claim 1, wherein the sulfhydryl-modified polymer compound comprises at least one of the following structures: ##STR00025## ##STR00026## ##STR00027## ##STR00028## ##STR00029## ##STR00030## ##STR00031## in the above structures, A is a fragment of the un-modified polymer compound comprising at least one of the —COOH, the —NH.sub.2, the —OH, the acrylate group of formula a, the acrylamide group of formula b and the acryloyl group of formula c in the structure; R, R′, R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are defined as above; (n2+n3)/(n1+n2+n3) represents a degree of acryloylation; n3/(n1+n2+n3) represents a degree of sulfhydrylation corresponding to the above sulfhydryl content of the sulfhydryl-modified polymer compound as determined by the Ellman method; the n1 can be 0, and if it is 0, the degree of acryloylation is not limited, and n3/(n2+n3) alone represents the degree of sulfhydrylation corresponding to the above sulfhydryl content of the sulfhydryl-modified polymer compound as determined by the Ellman method; the n2 can be 0, and if it is 0, n3/(n1+n3) represents both the degree of acryloylation and the degree of sulfhydrylation corresponding to the above sulfhydryl content of the sulfhydryl-modified polymer compound as determined by the Ellman method.
6. The sulfhydryl-modified polymer compound according to claim 1, wherein R.sub.4 is a fragment of a polysulfhydryl compound, for example, an —S—R.sub.4—SH fragment can be introduced from the following polysulfhydryl compounds including but not limited to: ##STR00032## wherein n4 is an integer from 2 to 30, such as 2, 3, 4, 5 or 6; n5 is an integer from 1 to 30, such as 1, 2, 3, 4 or 5; n6 is an integer from 1 to 30, such as 1, 2, 3, 4 or 5 etc.; 4-arm-PEG-SH represents a PEG polymer containing four sulfhydryl groups; 6-arm-PEG-SH represents a PEG polymer containing six sulfhydryl groups; 8-arm-PEG-SH represents a PEG polymer containing eight sulfhydryl groups; the PEG is an abbreviation for polyethylene glycol.
7. A preparation method for the sulfhydryl-modified polymer compound according to claim 1, comprising the following steps: 1) acryloylating the polymer compound comprising at least one of the —COOH, the —NH.sub.2 and the —OH in the structure, namely linking at least one of the —COOH, the —NH.sub.2 and the —OH comprised in the structure of the polymer compound, directly or indirectly, to the following group: ##STR00033## wherein R.sub.1, R.sub.2 and R.sub.3 are defined as above, and * represents a linking site; alternatively, directly using the polymer compound comprising at least one of the acrylate group of formula a, the acrylamide group of formula b, and the acryloyl group of formula c in the structure as a reaction starting material; 2) reacting at least one of polymer compounds obtained in step 1) with a polysulfhydryl compound HS—R.sub.4—SH to obtain the sulfhydryl-modified polymer compound, wherein R.sub.4 is defined as above.
8. The preparation method according to claim 7, comprising the following steps: 1) acryloylating the polymer compound comprising at least one of the —COOH, the —NH.sub.2 and the —OH in the structure, namely linking at least one of the —COOH, the —NH.sub.2 and the —OH comprised in the structure of the polymer compound, via an —R— group or directly, to the following group: ##STR00034## wherein R, R.sub.1, R.sub.2 and R.sub.3 are defined as above, and * represents a linking site; alternatively, directly using the polymer compound comprising at least one of the acrylate group of formula a, the acrylamide group of formula b, and the acryloyl group of formula c in the structure as a reaction starting material; 2) reacting at least one of polymer compounds obtained in step 1) with a polysulfhydryl compound HS—R.sub.4—SH to obtain the sulfhydryl-modified polymer compound, wherein R.sub.4 is defined as above.
9. The preparation method according to claim 7, wherein in step 1), the acryloylating step can be performed by reacting the polymer compound to be modified with an acrylate compound, or by reacting the polymer compound to be modified with an acryloyl chloride compound or an acrylic anhydride compound; preferably, in step 1), the acryloylating step is performed by reacting acryloyl chloride and derivatives thereof or acrylic anhydride and derivatives thereof with the polymer compound comprising at least one of —OH and —NH.sub.2; or performed by reacting glycidyl acrylate and derivatives thereof with the polymer compound comprising at least one of —COOH, —OH and —NH.sub.2; preferably, in step 1), the acryloylating step can be an unconventional reaction step, namely using a method other than the above method to synthesize a polymer compound comprising a structure of formula c; preferably, in step 2), the reaction with the polysulfhydryl compound HS—R.sub.4—SH is performed in a solvent; preferably, the solvent is water or an organic solvent, and further can be deionized water or dimethylformamide; preferably, in step 2), the reaction with the polysulfhydryl compound HS—R.sub.4—SH is performed under low to high temperature conditions; for example, the reaction temperature is 0-80° C., and further can be 10-70° C., and for example, the reaction can be performed at room temperature; preferably, in step 2), the reaction time for the reaction with the polysulfhydryl compound HS—R.sub.4—SH is 0.1-100 h; Preferably, in step 2), the pH range for the reaction with the polysulfhydryl compound HS—R.sub.4—SH is −1 to 15; for example, the reaction pH can be 6-8; and for another example, 7.
10. Use of the sulfhydryl-modified polymer compound according to claim 1, for use in the fields such as antioxidation health products, biopharmaceuticals, medical cosmetology and cosmetics.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[Sulfhydryl-modified Polymer Compound]
[0112] As described above, the present disclosure provides a sulfhydryl-modified polymer compound, wherein a polymer compound to be modified comprises at least one of —COOH, —NH.sub.2, —OH, an acrylate group of formula a, an acrylamide group of formula b, and an acryloyl group of formula c in the structure,
##STR00004##
wherein part or all of the —COOH and/or the —NH.sub.2 and/or the —OH and/or the acrylate group and/or the acrylamide group and/or the acryloyl group are modified to form a side chain with the following terminal group:
##STR00005##
in the above group, * represents a linking site;
R.sub.1 is selected from hydrogen, halogen, an aliphatic group, an aromatic group, and the like; specifically, the halogen, the aliphatic group and the aromatic group are further defined as below; preferably, R.sub.1 is selected from hydrogen, halogen, and an aliphatic group; more preferably, R.sub.1 is selected from hydrogen, halogen and C.sub.1-6 alkyl (e.g., methyl and ethyl);
R.sub.2 and R.sub.3 are the same or different and independently from each other are selected from hydrogen, halogen, an aliphatic group, an aromatic group, and the like; specifically, the halogen, the aliphatic group and the aromatic group are further defined as below;
R.sub.4 is a fragment of a polysulfhydryl compound.
[0113] In a specific embodiment, part or all of the —COOH and/or the —NH.sub.2 and/or the —OH and/or the acrylate group and/or the acrylamide group and/or the acryloyl group are modified to form at least one of the following structures:
##STR00006##
wherein in the above structures, R is selected from
##STR00007##
hydrocarbylene, arylene, an amide residue, a hydrazide residue, and the like; * represents a linking site; .sub.1* represents a linking site to a left-hand group of R; .sub.2* represents a linking site to a right-hand group of R; R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are defined as above;
wherein at least one of the —COOH, the —NH.sub.2, the —OH, the acrylate group of formula a, the acrylamide group of formula b, and the acryloyl group of formula c can be directly linked to the main chain of the polymer compound, or linked to the main chain of the polymer compound via an R′ group, and the R′ group can be a heteroatom-containing group, hydrocarbylene, arylene or the following linker:
##STR00008##
wherein in the above formula, R″ is hydrocarbylene or arylene, n′ is an integer from 1 to 1000, and * represents a linking site.
[0114] The heteroatom-containing group includes, but is not limited to an ester group, an amide residue or a hydrazide residue. Specifically, the ester group, the amide residue or the hydrazide residue are further defined as below.
[0115] The polymer compound to be modified comprises natural mucopolysaccharide polymers, such as at least one of chitosans (specifically chitosan, ethylene glycol chitosan, carboxymethyl chitosan, etc.), chondroitin sulfate, hyaluronic acid, and alginate; proteins such as gelatin, fibrin and serum proteins; and/or, synthetic polymers, such as at least one of polyvinyl alcohol, poly(meth)acrylic acid, polyhydroxyalkyl(meth)acrylate (e.g., polyhydroxyethyl(meth)acrylate), and hyperbranched polyethylene glycol.
[0116] A sulfhydryl content of the sulfhydryl-modified polymer compound as determined by the Ellman method is 0.01-30 mmol/g, for example, 0.1-10.0 mmol/g, for another example, 0.3-5.0 mmol/g, and for yet another example, 0.5-3.0 mmol/g.
[0117] The molecular weight of the sulfhydryl-modified polymer compound is substantially unchanged as compared to the molecular weight of the polymer compound before modification.
[0118] For example, the sulfhydryl-modified polymer compound of the present disclosure comprises at least one of the following structures:
##STR00009## ##STR00010## ##STR00011## ##STR00012## ##STR00013## ##STR00014##
in the above structures, A is a fragment of the un-modified polymer compound comprising at least one of the —COOH, the —NH.sub.2, the —OH, the acrylate group of formula a, the acrylamide group of formula b and the acryloyl group of formula c in the structure; R, R′, R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are defined as above, (n2+n3)/(n1+n2+n3) represents a degree of acryloylation; n3/(n1+n2+n3) represents a degree of sulfhydrylation corresponding to the above sulfhydryl content of the sulfhydryl-modified polymer compound as determined by the Ellman method; the n1 can be 0, and if it is 0, the degree of acryloylation is not limited, and n3/(n2+n3) alone represents the degree of sulfhydrylation corresponding to the above sulfhydryl content of the sulfhydryl-modified polymer compound as determined by the Ellman method; the n2 can be 0, and if it is 0, n3/(n1+n3) represents both the degree of acryloylation and the degree of sulfhydrylation corresponding to the above sulfhydryl content of the sulfhydryl-modified polymer compound as determined by the Ellman method.
[0119] Specifically, the A can be a structure shown as follows:
##STR00015##
[0120] In each of the above structures, * represents a linking site between repeating units of the main chain, ** represents a linking site between —COOH, —NH.sub.2, —OH, an acrylate group of formula a, an acrylamide group of formula b or an acryloyl group of formula c and the fragment, or a linking site between an R′ group and the fragment.
[0121] The A can also be a fragment or a repeating unit remaining in the following polymers Gelatin-A, Gelatin-MA, CTS-A, CTS-MA, PHEMA-A, PHEMA-MA, HB-PEG, PVA-A, PVA-MA, CHS-A or CHS-MA with the side chain containing the acrylamide group removed:
##STR00016##
[0122] It should be noted that Gelatin-A, Gelatin-MA, CTS-A, CTS-MA, PHEMA-A, PHEMA-MA, HB-PEG, PVA-A, PVA-MA, CHS-A and CHS-MA are abbreviations for the names of polymers having the above structures, and letters therein, when being separated, are not related to the meaning of letters appearing elsewhere in the present disclosure.
[0123] As described above, R.sub.4 is a fragment of the polysulfhydryl compound, for example, an —S—R.sub.4—SH fragment can be introduced from the following polysulfhydryl compounds including but not limited to:
##STR00017##
wherein n4 is an integer from 2 to 30, such as 2, 3, 4, 5 or 6 etc.; n5 is an integer from 1 to 30, such as 1, 2, 3, 4 or 5 etc.; n6 is an integer from 1 to 30, such as 1, 2, 3, 4 or 5 etc.;
4-arm-PEG-SH represents a PEG polymer containing four sulfhydryl groups: 6-arm-PEG-SH represents a PEG polymer containing six sulfhydryl groups; 8-arm-PEG-SH represents a PEG polymer containing eight sulfhydryl groups; the PEG is an abbreviation for polyethylene glycol.
[0124] In the present disclosure, n, n′, n1, n2, n3, n4, n5, n6, m1, m2, i, j, k1 and h are the number of repeating units in the structural formula unless otherwise specified. The range of values falls within conventional ranges known in the art.
[0125] As described above, R.sub.1 is selected from hydrogen, halogen, an aliphatic group, an aromatic group, and the like; R.sub.2 and R.sub.3 are the same or different and independently from each other are selected from hydrogen, halogen, an aliphatic group, an aromatic group, and the like.
[0126] As described above, the R may be selected from hydrocarbylene, arylene, an amide residue, a hydrazide residue, and the like.
[0127] As described above, the R′ may be selected from a heteroatom-containing group, hydrocarbylene, arylene, and the like.
[0128] As described above, the R″ may be selected from hydrocarbylene, arylene, and the like.
[0129] The halogen refers to fluorine, chlorine, bromine or iodine.
[0130] The aliphatic group is, for example, a straight-chain or branched saturated/unsaturated aliphatic group, specifically may be alkyl, alkenyl or alkynyl.
[0131] The “hydrocarbyl” used herein alone or as a suffix or prefix is, for example, a straight-chain or branched saturated/unsaturated aliphatic group, specifically may be alkyl, alkenyl or alkynyl.
[0132] The “alkyl” used herein alone or as a suffix or prefix is intended to include both branched and straight-chain saturated aliphatic hydrocarbyl groups having 1-20, preferably 1-6, carbon atoms. For example, “C.sub.1-6 alkyl” refers to a straight-chain or branched alkyl group having 1, 2, 3, 4, 5 or 6 carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl.
[0133] The “alkenyl” used herein alone or as a suffix or prefix is intended to include both branched and straight-chain aliphatic hydrocarbyl groups comprising alkenyl or alkene having 2-20, preferably 2-6, carbon atoms (or the specific number of carbon atoms if provided). For example, “CM, alkenyl” refers to an alkenyl group having 2, 3, 4, 5 or 6 carbon atoms. Examples of alkenyl include, but are not limited to, ethenyl, allyl, 1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methylbut-2-enyl, 3-methylbut-1-enyl, 1-pentenyl, 3-pentenyl, and 4-hexenyl.
[0134] The “alkynyl” used herein alone or as a suffix or prefix is intended to include both branched and straight-chain aliphatic hydrocarbyl groups comprising alkynyl or alkyne having 2-20, preferably 2-6 carbon atoms (or the specific number of carbon atoms if provided). For example, ethynyl, propynyl (e.g., 1-propynyl, 2-propynyl), 3-butynyl, pentynyl, hexynyl and 1-methylpent-2-ynyl.
[0135] The aromatic group refers to an aromatic ring structure composed of 5-20 carbon atoms. For example, the aromatic ring structure containing 5, 6, 7 and 8 carbon atoms may be a monocyclic aromatic group, e.g., phenyl; the ring structure containing 8, 9, 10, 11, 12, 13 or 14 carbon atoms may be a polycyclic aromatic group, e.g., naphthyl. The aromatic ring may be substituted at one or more ring positions with substituents such as alkyl and halogen, e.g., tolyl. The term “aryl” also includes polycyclic ring systems having two or more rings in which two or more carbons are common to two adjacent rings (the rings are “fused rings”), and at least one of the rings is aromatic and the other rings may be, for example, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl and/or heterocyclyl. Examples of polycyclic rings include, but are not limited to, 2,3-dihydro-1,4-benzodioxine and 2,3-dihydro-1-benzofuran.
[0136] The “hydrocarbylene” used herein is a group obtained by removing one hydrogen from the “hydrocarbyl”.
[0137] The “arylene” used herein is a group obtained by removing one hydrogen from the “aromatic group”.
[0138] The “alkylene” used herein is a group obtained by removing one hydrogen from the “alkyl”.
[0139] The “amide group” used herein alone or as a suffix or prefix refers to the R.sup.a—C(═O)—NH— group, wherein R.sup.a is selected from the following groups unsubstituted or optionally substituted with one or more R.sup.b: alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, and the like; R.sup.b is selected from the following groups unsubstituted or optionally substituted with one or more R.sup.b1: halogen, hydroxyl, sulfhydryl, nitro, cyano, alkyl, alkoxy, cycloalkyl, alkenyl, alkynyl, heterocyclyl, aryl, heteroaryl, amino, carboxyl, an ester group, hydrazino, acyl, sulfinyl, sulfonyl, phosphoryl, and the like; each R.sup.b1 is independently selected from halogen, hydroxy, alkyl and aryl.
[0140] The “hydrazide group” used herein alone or as a suffix or prefix refers to the R.sup.a—C(═O)—NH— group, wherein R.sup.a is defined as above.
[0141] The “amide residue” used herein is a group obtained by removing one hydrogen from the “amide group”.
[0142] The “hydrazide residue” used herein is a group obtained by removing one hydrogen from the “hydrazide group”.
[0143] The term “cycloalkyl” used herein is intended to include saturated cyclic groups having the specified number of carbon atoms. These terms may include fused or bridged polycyclic ring systems. The cycloalkyl has 3-40 carbon atoms in its ring structure. In one embodiment, the cycloalkyl has 3, 4, 5 or 6 carbon atoms in its ring structure. For example, “C.sub.3-6 cycloalkyl” refers to a group such as cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.
[0144] The term “cycloalkenyl” used herein is intended to include cyclic groups comprising at least one alkenyl group having the specified number of carbon atoms. These terms may include fused or bridged polycyclic ring systems. The cycloalkenyl has 3-40 carbon atoms in its ring structure. In one embodiment, the cycloalkenyl has 3, 4, 5 or 6 carbon atoms in its ring structure. For example, “C.sub.3-6 cycloalkenyl” refers to a group such as cyclopropenyl, cyclobutenyl, cyclopentenyl or cyclohexenyl.
[0145] The term “cycloalkynyl” used herein is intended to include cyclic groups comprising at least one alkynyl group having the specified number of carbon atoms. These terms may include fused or bridged polycyclic ring systems. The cycloalkynyl has 6-40 carbon atoms in its ring structure. In one embodiment, the cycloalkynyl has 6 carbon atoms in its ring structure. For example, “C.sub.3-6 cycloalkynyl” refers to a group such as cyclopropynyl, cyclobutynyl, cyclopentynyl or cyclohexynyl.
[0146] The “heteroaryl” used herein refers to a heteroaromatic heterocycle having at least one ring heteroatom (e.g., sulfur, oxygen, or nitrogen). The heteroaryl include monocyclic ring systems and polycyclic ring systems (e.g., having 2, 3 or 4 fused rings). Examples of heteroaryl include, but are not limited to, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrrolyl, oxazolyl, benzofuryl, benzothienyl, benzothiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl, benzothienyl, purinyl, carbazolyl, benzimidazolyl, benzoxazolyl, azabenzoxazolyl, imidazothiazolyl, benzo[1,4]dioxanyl, benzo[1,3]dioxolyl, and the like. In some embodiments, the heteroaryl has 3-40 carbon atoms, and in other embodiments, 3-20 carbon atoms. In some embodiments, the heteroaryl contains 3-14, 4-14, 3-7, or 5-6 ring atoms. In some embodiments, the heteroaryl has 1-4, 1-3, or 1-2 heteroatoms. In some embodiments, the heteroaryl has 1 heteroatom.
[0147] The term “heterocyclyl” used herein refers to a saturated, unsaturated or partially saturated monocyclic, bicyclic or tricyclic ring containing 3-20 atoms, wherein 1, 2, 3, 4 or 5 ring atoms are selected from nitrogen, sulfur, oxygen or phosphorus, which, unless otherwise stated, may be linked through carbon or nitrogen, wherein the —CH.sub.2— group is optionally replaced by —C(O)—; wherein unless otherwise stated to the contrary, the ring nitrogen atom or the ring sulfur atom is optionally oxidized to form an N-oxide or S-oxide, or the ring nitrogen atom is optionally quaternized; wherein —NH in the ring is optionally substituted with acetyl, formyl, methyl, or methanesulfonyl; and the ring is optionally substituted with one or more halogens. It should be understood that when the total number of S and O atoms in the heterocyclic group exceeds 1, these heteroatoms are not adjacent to each other. If the heterocyclyl is a bicyclic or tricyclic ring, at least one ring may optionally be a heteroaromatic or aromatic ring, provided that at least one ring is non-heteroaromatic. If the heterocyclyl is a monocyclic ring, it cannot be aromatic. Examples of heterocyclyl include, but are not limited to, piperidyl, N-acetylpiperidyl, N-methylpiperidyl, N-formylpiperazinyl, N-methanesulfonylpiperazinyl, homopiperazinyl, piperazinyl, azetidinyl, oxetanyl, morpholinyl, tetrahydroisoquinolyl, tetrahydroquinolyl, indolinyl, tetrahydropyranyl, dihydro-2H-pyranyl, tetrahydrofuranyl, tetrahydrothiopyranyl, tetrahydrothiopyran-1-oxide, tetrahydrothiopyran-1,1-dioxide, 1H-pyridin-2-one, and 2,5-dioxoimidazolidinyl.
[0148] The term “acyl” used herein refers to the R.sup.a—C(═O)— group, wherein R.sup.a is defined as above.
[0149] The term “sulfinyl” used herein refers to the R.sup.a—S(═O)— group, wherein R.sup.a is defined as above.
[0150] The term “sulfonyl” used herein refers to the R.sup.a—S(═O).sub.2— group, wherein R.sup.a is defined as above.
[0151] The term “phosphoryl” used herein refers to the R.sup.c—P(═O)(R.sup.d)— group, wherein R.sup.c and R.sup.d are the same or different and independently from each other are selected from the following groups, unsubstituted or optionally substituted with one or more R.sup.b: alkyl, cycloalkyl, alkoxy, hydroxyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, and the like; R.sup.b is defined as above.
[0152] The term “hydrazino” used herein refers to the —NHNHR.sup.a group, wherein R.sup.a is defined as above.
[0153] The term “amine group” used herein refers to the —NHR.sup.a group or the —N(R.sup.a).sub.2 group, wherein R.sup.a is defined as above.
[0154] The term “amino” used herein refers to the —NH.sub.2 group.
[0155] The term “carboxyl” used herein refers to the —COOH group.
[0156] The term “ester group” used herein refers to the R.sup.a—C(═O)—O— group or the R.sup.a—O—C(═O)— group, wherein R.sup.a is defined as above.
[0157] [Preparation Method for Sulfhydryl-Modified Polymer Compound]
[0158] As described above, the present disclosure provides a preparation method for the sulfhydryl-modified polymer compound, which comprises the following steps:
1) acryloylating the polymer compound comprising at least one of the —COOH, the —NH.sub.2 and the —OH in the structure, namely linking at least one of the —COOH, the —NH.sub.2 and the —OH comprised in the structure of the polymer compound, directly or indirectly, to the following group:
##STR00018##
wherein R.sub.1, R.sub.2 and R.sub.3 are defined as above, and * represents a linking site;
alternatively, directly using the polymer compound comprising at least one of the acrylate group of formula a, the acrylamide group of formula b, and the acryloyl group of formula c in the structure as a reaction starting material;
2) reacting at least one of polymer compounds obtained in step 1) with a polysulfhydryl compound HS—R.sub.4—SH to obtain the sulfhydryl-modified polymer compound, wherein R.sub.4 is defined as above.
[0159] In one specific embodiment of the present disclosure, the method comprises the following steps:
1) acryloylating the polymer compound comprising at least one of the —COOH, the —NH.sub.2 and the —OH in the structure, namely linking at least one of the —COOH, the —NH.sub.2 and the —OH comprised in the structure of the polymer compound, via an —R— group or directly, to the following group:
##STR00019##
wherein R, R.sub.1, R.sub.2 and R.sub.3 are defined as above, and * represents a linking site:
alternatively, directly using the polymer compound comprising at least one of the acrylate group of formula a, the acrylamide group of formula b, and the acryloyl group of formula c in the structure as a reaction starting material;
2) reacting at least one of polymer compounds obtained in step 1) with a polysulfhydryl compound HS—R.sub.4—SH to obtain the sulfhydryl-modified polymer compound, wherein R.sub.4 is defined as above.
[0160] In step 1), the acryloylating step can be performed by reacting the polymer compound to be modified with an acrylate compound, or by reacting the polymer compound to be modified with an acryloyl chloride compound or an acrylic anhydride compound.
[0161] The acrylate compound may be one or more of an alkyl acrylate compound, an aryl acrylate compound and a glycidyl acrylate polyol compound.
[0162] The polyol in the glycidyl acrylate polyol compound is, for example, a triol, specifically, glycerin, butanetriol, pentanetriol, and the like.
[0163] In step 1), the acryloylating step may be a conventional reaction step, which can be performed under existing conventional conditions. Generally, it is performed by reacting acryloyl chloride and derivatives thereof or acrylic anhydride and derivatives thereof with a polymer compound comprising at least one of —OH and —NH.sub.2. It can also be performed by reacting glycidyl acrylate and derivatives thereof with the polymer compound comprising at least one of —COOH, —OH and —NH.sub.2.
[0164] In step 1), the acryloylating step can be an unconventional reaction step, namely using a method other than the above method to synthesize a polymer compound comprising a structure of formula c.
[0165] In step 2), the reaction with the polysulfhydryl compound HS—R.sub.4—SH is performed in a solvent. The solvent is, for example, water or an organic solvent, and further can be deionized water or dimethylformamide.
[0166] In step 2), the reaction with the polysulfhydryl compound HS—R.sub.4—SH is performed under low to high temperature conditions. For example, the reaction temperature is 0-80° C., and further can be 10-70° C., and for example, the reaction can be performed at room temperature.
[0167] In step 2), the reaction time for the reaction with the polysulfhydryl compound HS—R.sub.4—SH is 0.1-100 h.
[0168] In step 2), the pH range for the reaction with the polysulfhydryl compound HS—R.sub.4—SH is −1 to 15. For example, the reaction pH can be 6-8, and for another example, 7.
[0169] In step 2), the reaction further comprises a post-treatment step.
[0170] In the post-treatment step, a dialysis method is adopted. Specifically, the solution after the reaction is filled into a dialysis bag (for example, a dialysis bag with a molecular weight cutoff of 2 kDa or more), dialyzed against a hydrochloric acid solution (for example, at pH 4) for several days (for example, 1-10 days, for another example, 5 days, and the like), optionally refreshed with water (for example, refreshed with water every day or every other day) for several times (for example, twice or more, and the like), and finally collected and dried (for example, lyophilized) to obtain a solid or viscous liquid, i.e., the sulfhydryl-modified polymer compound.
[0171] The present disclosure firstly provides a preparation method for the sulfhydryl-modified polymer compound by the Michael addition reaction of the sulfhydryl of the polysulfhydryl compound with the carbon-carbon double bond in the acryloyl group. The method has a high degree of sulfhydrylation, mild conditions for the sulfhydrylation reaction (can be performed at room temperature in an aqueous solution) and no pollution, and the prepared sulfhydryl-modified polymer compound has high purity and is particularly suitable for further use in the fields such as pharmaceuticals, cosmetology and medicine.
[Application of Sulfhydryl-Modified Polymer Compound]
[0172] As described above, the sulfhydryl-modified polymer compound of the present disclosure has a high degree of sulfhydrylation, and is suitable for any existing application field of the sulfhydryl-modified polymer compound. Specifically, it can be used in the fields such as antioxidation health care products, biopharmaceuticals, medical cosmetology and cosmetics (e.g., at least one of antioxidation cosmetics, water retention and moisture supplement cosmetics).
[0173] Taking sulfhydryl-modified hyaluronic acid as an example, it is known that hyaluronic acid (HA) is a linear, non-branched macromolecular acidic mucopolysaccharide polymer composed of repeated and alternating linkages of disaccharides (the structural units are β-(1,4)-N-acetyl-
[0174] In the present disclosure, the .sup.1H-NMR spectrum is determined by a Varian 400 MHz nuclear magnetic resonance spectrometer, with the test temperature of 25° C., the relaxation time of 1 s, and the number of scanning of 8. Specifically, 8-10 mg of the test sample is dissolved in 750 μL of deuterated water, and the obtained sample solution is determined for the .sup.1H-NMR spectrum.
Preparation Example 1. Synthesis of Acrylate-Modified Hyaluronic Acid (HA-A1)
[0175] To a 200 mL beaker were added 1 g of hyaluronic acid (Bloomage Freda, weight-average molecular weight: about 300 kDa), 50 mL of deionized water, 50 mL of dimethylformamide, 12 mL of triethylamine, and 14 mL of glycidyl acrylate. After being stirred at room temperature until uniform and transparent, the mixture was stirred for an additional 48 h. 300 mL of acetone was added, and a large amount of white precipitate was generated. The reaction solution was centrifuged, and the resulting precipitate was dissolved in 100 mL of deionized water to obtain a colorless transparent solution. The resulting solution was filled into a dialysis bag (molecular weight cutoff: 8 kDa) and dialyzed against 5 L of deionized water for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain HA-A1 (921 mg, yield 92.1%) as a white flocculent solid.
[0176] The structural formula of HA-A1 is shown in
[0177] The .sup.1H-NMR spectrum of HA-A1 is shown in
Preparation Example 2. Synthesis of Acrylate-Modified Hyaluronic Acid (HA-A2)
[0178] To a 200 mL beaker were added 1 g of hyaluronic acid (Bloomage Freda, weight-average molecular weight: about 400 kDa), 50 mL of deionized water, 50 mL of dimethylformamide, and 6.3 g of acrylic anhydride, and the mixture was dissolved with stirring. The solution was maintained at pH 8±0.5 with 1 mol/L NaOH, and stirred for an additional 24 h. 300 mL of acetone was added, and a large amount of white precipitate was generated. The reaction solution was centrifuged, and the resulting precipitate was dissolved in 100 mL of deionized water to obtain a colorless transparent solution. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of deionized water for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain HA-A2 (789 mg, yield 78.9%) as a white flocculent solid.
[0179] The structural formula of HA-A2 is shown in
[0180] The .sup.1H-NMR spectrum of HA-A2 is shown in
Preparation Example 3. Synthesis of Methacrylate-Modified Hyaluronic Acid (HA-MA1)
[0181] To a 200 mL beaker were added 1 g of hyaluronic acid (Bloomage Freda, weight-average molecular weight: about 400 kDa), 50 mL of deionized water, 50 mL of dimethylformamide (Sigma), 12 mL of triethylamine (Sigma), and 15 mL of glycidyl methacrylate. After being stirred at room temperature until uniform and transparent, the mixture was stirred for an additional 48 h. 300 mL of acetone (Sigma) was added, and a large amount of white precipitate was generated. The reaction solution was centrifuged, and the resulting precipitate was dissolved in 100 mL of deionized water to obtain a colorless solution. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of deionized water for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain HA-MA1 (859 mg, yield 85.9%) as a white flocculent solid.
[0182] The structural formula of HA-MA1 is shown in
[0183] The .sup.1H-NMR spectrum of HA-MA1 is shown in
Preparation Example 4. Synthesis of Methacrylate-Modified Hyaluronic Acid (HA-MA2)
[0184] To a 200 mL beaker were added 1 g of hyaluronic acid (Bloomage Freda, weight-average molecular weight: about 400 kDa) and 100 mL of deionized water, and the mixture was dissolved with stirring at room temperature. Further, 7.7 g of methacrylic anhydride was added and dissolved with stirring. The solution was maintained at pH 8 f 0.5 with 1 mol/L NaOH, and stirred for an additional 24 h. 200 mL of acetone (Sigma) was added, and a large amount of white precipitate was generated. The reaction solution was centrifuged, and the resulting precipitate was dissolved in 100 mL of deionized water to obtain a colorless transparent solution. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of deionized water for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain HA-MA2 (846 mg, yield 84.6%) as a white flocculent solid.
[0185] The structural formula of HA-MA2 is shown in
[0186] The .sup.1H-NMR spectrum of HA-MA2 is shown in
Preparation Example 5. Synthesis of Acrylate-Modified Chondroitin Sulfate (CHS-A)
[0187] To a 200 mL beaker were added 1.2 g of chondroitin sulfate (weight-average molecular weight: about 80 kDa), 50 mL of deionized water, 50 mL of dimethylformamide, and 5.4 g of acrylic anhydride, and the mixture was dissolved with stirring. The solution was maintained at pH 8±0.5 with 1 mol/L NaOH, and stirred for an additional 24 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 3.5 kDa) and dialyzed against 5 L of deionized water for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain CHS-A (781 mg, yield 65.1%) as a light yellow flocculent solid.
[0188] The structural formula of CHS-A is shown in
[0189] The .sup.1H-NMR spectrum of CHS-A is shown in
Preparation Example 6. Synthesis of Methacrylate-Modified Chondroitin Sulfate (CHS-MA)
[0190] To a 200 mL beaker were added 1.2 g of chondroitin sulfate (weight-average molecular weight: about 90 kDa), 50 mL of deionized water, and 50 mL of dimethylformamide, followed by 6.5 g of methacrylic anhydride, and the mixture was dissolved with stirring. The solution was maintained at pH 8 f 0.5 with 1 mol/L NaOH, and stirred for an additional 24 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 3.5 kDa) and dialyzed against 5 L of deionized water for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain CHS-MA (776 mg, yield 64.7%) as a light yellow flocculent solid.
[0191] The structural formula of CHS-MA is shown in
[0192] The .sup.1H-NMR spectrum of CHS-MA is shown in
Preparation Example 7. Synthesis of Acrylate-Modified Gelatin (Gelatin-A)
[0193] To a 200 mL beaker were added 1 g of gelatin (strength: 300 Blooms), 50 mL of deionized water, and 50 mL of dimethylformamide, followed by 10 g of acrylic anhydride, and the mixture was dissolved with stirring. The solution was maintained at pH 8 f 0.5 with 1 mol/L NaOH, and stirred for an additional 24 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of deionized water for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain Gelatin-A (781 mg, yield 78.1%) as a light yellow flocculent solid.
[0194] The condensed structural formula of Gelatin-A is shown in
[0195] The .sup.1H-NMR spectrum of Gelatin-A is shown in
Preparation Example 8. Synthesis of Methacrylate-Modified Gelatin (Gelatin-MA)
[0196] To a 200 mL beaker were added 1 g of gelatin (strength: 300 Blooms), 50 mL of deionized water, and 50 mL of dimethylformamide, followed by 10 g of methacrylic anhydride, and the mixture was dissolved with stirring. The solution was maintained at pH 8 f 0.5 with 1 mol/L NaOH, and stirred for an additional 24 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of deionized water for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain Gelatin-MA (824 mg, yield 82.4%) as a light yellow flocculent solid.
[0197] The condensed structural formula of Gelatin-MA is shown in
[0198] The .sup.1H-NMR spectrum of Gelatin-MA is shown in
Preparation Example 9. Synthesis of Acrylate-Modified Ethylene Glycol Chitosan (CTS-A)
[0199] To a 200 mL beaker were added 1 g of ethylene glycol chitosan (weight-average molecular weight: about 250 kDa), 50 mL of deionized water, 50 mL of dimethylformamide, 8 mL of triethylamine (Sigma), and 13 mL of glycidyl acrylate. After being stirred at room temperature until uniform and transparent, the mixture was stirred for an additional 48 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 3.5 kDa) and dialyzed against 5 L of deionized water for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain CTS-A (694 mg, yield 69.4%) as a light yellow flocculent solid.
[0200] The structural formula of CTS-A is shown in
[0201] The .sup.1H-NMR spectrum of CTS-A is shown in
Preparation Example 10. Synthesis of Methacrylate-Modified Ethylene Glycol Chitosan (CTS-MA)
[0202] To a 200 mL beaker were added 1 g of ethylene glycol chitosan (weight-average molecular weight: about 200 kDa), 50 mL of deionized water, 50 mL of dimethylformamide, 8 mL of triethylamine (Sigma), and 13 mL of glycidyl methacrylate. After being stirred at room temperature until uniform and transparent, the mixture was stirred for an additional 48 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 3.5 kDa) and dialyzed against 5 L of deionized water for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain CTS-MA (726 mg, yield 72.6%) as a light yellow flocculent solid.
[0203] The structural formula of CTS-MA is shown in
[0204] The .sup.1H-NMR spectrum of CTS-MA is shown in
Preparation Example 11. Synthesis of Acrylate-Modified Polyhydroxyethyl Methacrylate (PHEMA-A)
[0205] To a 200 mL beaker were added 2 g of polyhydroxyethyl methacrylate (Sigma, Mv: 20 kDa), 50 mL of deionized water, and 50 mL of dimethylformamide, followed by 16.5 g of acrylic anhydride, and the mixture was dissolved with stirring. The solution was maintained at pH 8±0.5 with 1 mol/L NaOH, and stirred for an additional 24 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 2 kDa) and dialyzed against 5 L of deionized water for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain PHEMA-A (1.42 g, yield 71.0%) as a white solid.
[0206] The structural formula of PHEMA-A is shown in
[0207] The .sup.1H-NMR spectrum of PHEMA-A is shown in
Preparation Example 12. Synthesis of Methacrylate-Modified Polyhydroxyethyl Methacrylate (PHEMA-MA)
[0208] To a 200 mL beaker were added 2 g of polyhydroxyethyl methacrylate (Sigma, Mv: 20 kDa), 50 mL of deionized water, and 50 mL of dimethylformamide, followed by 16.8 g of methacrylic anhydride, and the mixture was dissolved with stirring. The solution was maintained at pH 8 t 0.5 with 1 mol/L NaOH, and stirred for an additional 24 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 2 kDa) and dialyzed against 5 L of deionized water for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain PHEMA-MA (1.48 g, yield 74.0%) as a white solid.
[0209] The structural formula of PHEMA-MA is shown in
[0210] The .sup.1H-NMR spectrum of PHEMA-MA is shown in
Preparation Example 13. Synthesis of Acrylate-Modified Polyvinyl Alcohol (PVA-A)
[0211] To a 200 mL beaker were added 2 g of polyvinyl alcohol (Sigma, Mw: 61 kDa), 50 mL of deionized water, and 50 mL of dimethylformamide, followed by 13 g of acrylic anhydride, and the mixture was dissolved with stirring. The solution was maintained at pH 8 f 0.5 with 1 mol/L NaOH, and stirred for an additional 24 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 3.5 kDa) and dialyzed against 5 L of deionized water for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain PVA-A (1.57 g, yield 78.5%) as a white solid.
[0212] The structural formula of PVA-A is shown in
[0213] The .sup.1H-NMR spectrum of PVA-A is shown in
Preparation Example 14. Synthesis of Methacrylate-Modified Polyvinyl Alcohol (PVA-MA)
[0214] To a 200 mL beaker were added 2 g of polyvinyl alcohol (Sigma, Mw: 61 kDa), 50 mL of deionized water, and 50 mL of dimethylformamide, followed by 13.4 g of methacrylic anhydride, and the mixture was dissolved with stirring. The solution was maintained at pH 8 f 0.5 with 1 mol/L NaOH, and stirred for an additional 24 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 3.5 kDa) and dialyzed against 5 L of deionized water for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain PVA-MA (1.51 g, yield 75.5%) as a white solid.
[0215] The structural formula of PVA-MA is shown in
[0216] The .sup.1H-NMR spectrum of PVA-MA is shown in
Example 1. Synthesis of Sulfhydryl-Acrylate-Modified Hyaluronic Acid (HA-A1-SH1)
[0217] To a 200 mL beaker were added 1 g of HA-A1 prepared according to the method of Preparation Example 1, 0.3 g of dithiothreitol (VWR) and 100 mL of deionized water, and the mixture was dissolved with stirring at room temperature to obtain a transparent solution. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain HA-A1-SH1 (842 mg, yield 84.2%) as a white flocculent solid.
[0218] The reaction equation for HA-A1-SH1 is shown in
[0219] The .sup.1H-NMR spectrum of HA-A1-SH1 is shown in
Example 2. Synthesis of Sulfhydryl-Acrylate-Modified Hyaluronic Acid (HA-A2-SH1)
[0220] To a 200 mL beaker were added 1 g of HA-A2 prepared according to the method of Preparation Example 2, 0.3 g of dithiothreitol (VWR) and 100 mL of deionized water, and the mixture was dissolved with stirring at room temperature to obtain a transparent solution. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain HA-A2-SH1 (827 mg, yield 82.7%) as a white flocculent solid.
[0221] The reaction equation for HA-A2-SH1 is shown in
[0222] The .sup.1H-NMR spectrum of HA-A2-SH1 is shown in
Example 3. Synthesis of Sulfhydryl-Methacrylate-Modified Hyaluronic Acid (HA-MA1-SH1)
[0223] To a 200 mL beaker were added 1 g of HA-MA1 prepared according to the method of Preparation Example 3, 0.3 g of dithiothreitol (VWR) and 100 mL of deionized water, and the mixture was dissolved with stirring at room temperature. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain HA-MA1-SH1 (854 mg, yield 85.4%) as a white flocculent solid.
[0224] The reaction equation for HA-MA1-SH1 is shown in
[0225] The .sup.1H-NMR spectrum of HA-MA1-SH1 is shown in
Example 4. Synthesis of Sulfhydryl-Methacrylate-Modified Hyaluronic Acid (HA-MA2-SH1)
[0226] To a 200 mL beaker were added 1 g of HA-MA2 prepared according to the method of Preparation Example 4, 0.3 g of dithiothreitol (VWR) and 100 mL of deionized water, and the mixture was dissolved with stirring at room temperature. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain HA-MA2-SH1 (833 mg, yield 83.3%) as a white flocculent solid.
[0227] The reaction equation for HA-MA2-SH1 is shown in
[0228] The .sup.1H-NMR spectrum of HA-MA2-SH1 is shown in
Example 5. Synthesis of Sulfhydryl-Acrylate-Modified Chondroitin Sulfate (CHS-A-SH1)
[0229] To a 200 mL beaker were added 1 g of CHS-A prepared according to the method of Preparation Example 5, 0.25 g of dithiothreitol (VWR) and 100 mL of deionized water, and the mixture was dissolved with stirring at room temperature. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 3.5 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain CHS-A-SH1 (629 mg, yield 62.9%) as a light yellow flocculent solid.
[0230] The reaction equation for CHS-A-SH1 is shown in
[0231] The .sup.1H-NMR spectrum of CHS-A-SH1 is shown in
Example 6. Synthesis of Sulfhydryl-Methacrylate-Modified Chondroitin Sulfate (CHS-MA-SH1)
[0232] To a 200 mL beaker were added 1 g of CHS-MA prepared according to the method of Preparation Example 6, 0.25 g of dithiothreitol (VWR) and 100 mL of deionized water, and the mixture was dissolved with stirring at room temperature. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 3.5 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain CHS-MA-SH1 (642 mg, yield 64.2%) as a light yellow flocculent solid.
[0233] The reaction equation for CHS-MA-SH1 is shown in
[0234] The .sup.1H-NMR spectrum of CHS-MA-SH1 is shown in
Example 7. Synthesis of Sulfhydryl-Acrylate-Modified Gelatin (Gelatin-A-SH1)
[0235] To a 200 mL beaker were added 1 g of Gelatin-A prepared according to the method of Preparation Example 7, 0.19 g of dithiothreitol (VWR) and 100 mL of deionized water, and the mixture was dissolved with stirring at room temperature. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain Gelatin-A-SH1 (763 mg, yield 76.3%) as a light yellow flocculent solid.
[0236] The reaction equation for Gelatin-A-SH1 is shown in
[0237] The .sup.1H-NMR spectrum of Gelatin-A-SH1 is shown in
Example 8. Synthesis of Sulfhydryl-Methacrylate-Modified Gelatin (Gelatin-MA-SH1)
[0238] To a 200 mL beaker were added 1 g of Gelatin-MA prepared according to the method of Preparation Example 8, 0.19 g of dithiothreitol (VWR) and 100 mL of deionized water, and the mixture was dissolved with stirring at room temperature. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain Gelatin-MA-SH1 (787 mg, yield 78.7%) as a light yellow flocculent solid.
[0239] The reaction equation for Gelatin-MA-SH1 is shown in
[0240] The .sup.1H-NMR spectrum of Gelatin-MA-SH1 is shown in
Example 9. Synthesis of Sulfhydryl-Acrylate-Modified Ethylene Glycol Chitosan (CTS-A-SH1)
[0241] To a 200 mL beaker were added 1 g of CTS-A prepared according to the method of Preparation Example 9, 0.25 g of dithiothreitol (VWR) and 100 mL of deionized water, and the mixture was dissolved with stirring at room temperature. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 3.5 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain CTS-A-SH1 (602 mg, yield 60.2%) as a light yellow flocculent solid.
[0242] The reaction equation for CTS-A-SH1 is shown in
[0243] The .sup.1H-NMR spectrum of CTS-A-SH1 is shown in
Example 10. Synthesis of Sulfhydryl-Methacrylate-Modified Chitosan (CTS-MA-SH1)
[0244] To a 200 mL beaker were added 1 g of CTS-MA prepared according to the method of Preparation Example 10, 0.25 g of dithiothreitol (VWR) and 100 mL of deionized water, and the mixture was dissolved with stirring at room temperature. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 3.5 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain CTS-MA-SH1 (643 mg, yield 64.3%) as a white flocculent solid.
[0245] The reaction equation for CTS-MA-SH1 is shown in
[0246] The .sup.1H-NMR spectrum of CTS-MA-SH1 is shown in
Example 11. Synthesis of Sulfhydryl-Acrylate-Modified Polyhydroxyethyl Methacrylate (PHEMA-A-SH1)
[0247] To a 200 mL beaker were added 2 g of PHEMA-A prepared according to the method of Preparation Example 11, 0.42 g of dithiothreitol (VWR), 50 mL of deionized water and 50 mL of dimethylformamide, and the mixture was dissolved with stirring at room temperature. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 2 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain PHEMA-A-SH1 (1.67 g, yield 83.5%) as a white solid.
[0248] The reaction equation for PHEMA-A-SH1 is shown in
[0249] The .sup.1H-NMR spectrum of PHEMA-A-SH1 is shown in
Example 12. Synthesis of Sulfhydryl-Methacrylate-Modified Polyhydroxyethyl Methacrylate (PHEMA-MA-SH1)
[0250] To a 200 mL beaker were added 2 g of PHEMA-MA prepared according to the method of Preparation Example 12, 0.41 g of dithiothreitol (VWR), 50 mL of deionized water and 50 mL of dimethylformamide, and the mixture was dissolved with stirring at room temperature. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 2 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain PHEMA-MA-SH1 (1.62 g, yield 81%) as a white solid.
[0251] The reaction equation for PHEMA-MA-SH1 is shown in
[0252] The .sup.1H-NMR spectrum of PHEMA-MA-SH1 is shown in
Example 13. Synthesis of Sulfhydryl-Modified Hyperbranched PEG Polymer (HB-PEG-SH1)
[0253] To a 200 mL beaker were added 5 g of hyperbranched PEG (HB-PEG, Blafar Ltd., Mw: 20 kDa), 0.86 g of dithiothreitol (VWR) and 100 mL of deionized water, and the mixture was dissolved with stirring at room temperature. The resulting transparent solution was stirred for an additional 12 h.
[0254] The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 2 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain HB-PEG-SH1 (3.84 g, yield 76.8%) as a colorless viscous liquid.
[0255] The reaction equation for HB-PEG-SH1 is shown in
[0256] The .sup.1H-NMR spectrum of HB-PEG-SH1 is shown in
Example 14. Synthesis of Sulfhydryl-Acrylate-Modified Polyvinyl Alcohol (PVA-A-SH1)
[0257] To a 200 mL beaker were added 1 g of PVA-A prepared according to the method of Preparation Example 13 and 100 mL of deionized water, and the solution was heated with stirring until the PVA-A was completely dissolved. Subsequently, the solution was added with 0.47 g of dithiothreitol (VWR) and dissolved with stirring at room temperature. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain PVA-A-SH1 (737 mg, yield 73.7%) as a white solid.
[0258] The reaction equation for PVA-A-SH1 is shown in
[0259] The .sup.1H-NMR spectrum of PVA-A-SH1 is shown in
Example 15. Synthesis of Sulfhydryl-Methacrylate-Modified Polyvinyl Alcohol (PVA-MA-SH1)
[0260] To a 200 mL beaker were added 1 g of PVA-MA prepared according to the method of Preparation Example 14 and 100 mL of deionized water, and the solution was heated with stirring until the PVA-MA was completely dissolved. Subsequently, the solution was added with 0.47 g of dithiothreitol (VWR) and dissolved with stirring at room temperature. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain PVA-MA-SH1 (718 mg, yield 71.8%) as a white solid.
[0261] The reaction equation for PVA-MA-SH1 is shown in
[0262] The .sup.1H-NMR spectrum of PVA-MA-SH1 is shown in
Example 16. Synthesis of Sulfhydryl-Acrylate-Modified Hyaluronic Acid (HA-A1-SH2)
[0263] To a 200 mL beaker were added 1 g of HA-A1 prepared according to the method of Preparation Example 1, 0.42 g of 1,4-butanedithiol (Sigma) and 100 mL of deionized water, and the mixture was dissolved with stirring at room temperature to obtain a transparent solution. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain HA-A1-SH2 (852 mg, yield 85.2%) as a white flocculent solid.
[0264] The reaction equation for HA-A1-SH2 is shown in
[0265] The .sup.1H-NMR spectrum of HA-A1-SH2 is shown in
Example 17. Synthesis of Sulfhydryl-Acrylate-Modified Hyaluronic Acid (HA-A1-SH3)
[0266] To a 200 mL beaker were added 1 g of HA-A1 prepared according to the method of Preparation Example 1, 0.43 g of 2-amino-1,4-butanedithiol hydrochloride (Sigma) and 100 mL of deionized water, and the mixture was dissolved with stirring at room temperature to obtain a transparent solution. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain HA-A1-SH3 (843 mg, yield 84.3%) as a white flocculent solid.
[0267] The reaction equation for HA-A1-SH3 is shown in
[0268] The .sup.1H-NMR spectrum of HA-A1-SH3 is shown in
Example 18. Synthesis of Sulfhydryl-Acrylate-Modified Hyaluronic Acid (HA-A2-SH2)
[0269] To a 200 mL beaker were added 1 g of HA-A2 prepared according to the method of Preparation Example 2, 0.42 g of 1,4-butanedithiol (Sigma) and 100 mL of deionized water, and the mixture was dissolved with stirring at room temperature to obtain a transparent solution. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain HA-A2-SH2 (827 mg, yield 82.7%) as a white flocculent solid.
[0270] The reaction equation for HA-A2-SH2 is shown in
[0271] The .sup.1H-NMR spectrum of HA-A2-SH2 is shown in
Example 19. Synthesis of Sulfhydryl-Acrylate-Modified Hyaluronic Acid (HA-A2-SH3)
[0272] To a 200 mL beaker were added 1 g of HA-A2 prepared according to the method of Preparation Example 2, 0.43 g of 2-amino-1,4-butanedithiol hydrochloride (Sigma) and 100 mL of deionized water, and the mixture was dissolved with stirring at room temperature to obtain a transparent solution. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain HA-A2-SH3 (833 mg, yield 83.3%) as a white flocculent solid.
[0273] The reaction equation for HA-A2-SH3 is shown in
[0274] The .sup.1H-NMR spectrum of HA-A2-SH3 is shown in
Example 20. Synthesis of Sulfhydryl-Acrylate-Modified Hyaluronic Acid (HA-A2-SH4)
[0275] To a 200 mL beaker were added 1 g of HA-A2 prepared according to the method of Preparation Example 2, 0.38 g of 1,3-propanedithiol (Sigma) and 100 mL of deionized water, and the mixture was dissolved with stirring at room temperature to obtain a transparent solution. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain HA-A2-SH4 (814 mg, yield 81.4%) as a white flocculent solid.
[0276] The reaction equation for HA-A2-SH4 is shown in
[0277] The .sup.1H-NMR spectrum of HA-A2-SH4 is shown in
Example 21. Synthesis of Sulfhydryl-Acrylate-Modified Hyaluronic Acid (HA-A2-SH5)
[0278] To a 200 mL beaker were added 1 g of HA-A2 prepared according to the method of Preparation Example 2, 0.52 g of 1,3-phenyldithiophenol (Sigma) and 100 mL of deionized water, and the mixture was dissolved with stirring at room temperature to obtain a transparent solution. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain HA-A2-SH5 (836 mg, yield 83.6%) as a white flocculent solid.
[0279] The reaction equation for HA-A2-SH5 is shown in
[0280] The .sup.1H-NMR spectrum of HA-A2-SH5 is shown in
Example 22. Synthesis of Sulfhydryl-Acrylate-Modified Hyaluronic Acid (HA-A2-SH6)
[0281] To a 200 mL beaker were added 1 g of HA-A2 prepared according to the method of Preparation Example 2, 0.52 g of 1,4-phenyldithiophenol (Sigma) and 100 mL of deionized water, and the mixture was dissolved with stirring at room temperature to obtain a transparent solution. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain HA-A2-SH6 (831 mg, yield 83.1%) as a white flocculent solid.
[0282] The reaction equation for HA-A2-SH6 is shown in
[0283] The .sup.1H-NMR spectrum of HA-A2-SH6 is shown in
Example 23. Synthesis of Sulfhydryl-Acrylate-Modified Hyaluronic Acid (HA-A2-SH7)
[0284] To a 200 mL beaker were added 1 g of HA-A2 prepared according to the method of Preparation Example 2, 0.96 g of sulfhydryl polyethylene glycol (Sigma) and 100 mL of deionized water, and the mixture was dissolved with stirring at room temperature to obtain a transparent solution. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain HA-A2-SH7 (894 mg, yield 89.4%) as a white flocculent solid.
[0285] The reaction equation for HA-A2-SH7 is shown in
[0286] The .sup.1H-NMR spectrum of HA-A2-SH7 is shown in
Example 24. Synthesis of Sulfhydryl-Acrylate-Modified Hyaluronic Acid (HA-A2-SH8)
[0287] To a 200 mL beaker were added 1 g of HA-A2 prepared according to the method of Preparation Example 2, 0.74 g of trimethylolpropane-tris(3-sulfhydrylpropionate) (Sigma), 50 mL of deionized water and 50 mL of dimethylformamide, and the mixture was dissolved with stirring at room temperature to obtain a transparent solution. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain HA-A2-SH8 (785 mg, yield 78.5%) as a white flocculent solid.
[0288] The reaction equation for HA-A2-SH8 is shown in
[0289] The .sup.1H-NMR spectrum of HA-A2-SH8 is shown in
Example 25. Synthesis of Sulfhydryl-Methacrylate-Modified Hyaluronic Acid (HA-MA1-SH5)
[0290] To a 200 mL beaker were added 1 g of HA-MA1 prepared according to the method of Preparation Example 3, 0.50 g of 1,3-phenyldithiophenol (Sigma) and 100 mL of deionized water, and the mixture was dissolved with stirring at room temperature to obtain a transparent solution. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain HA-MA1-SH5 (828 mg, yield 82.8%) as a white flocculent solid.
[0291] The reaction equation for HA-MA1-SH5 is shown in
[0292] The .sup.1H-NMR spectrum of HA-MA1-SH5 is shown in
Example 26. Synthesis of Sulfhydryl-Methacrylate-Modified Hyaluronic Acid (HA-MA1-SH6)
[0293] To a 200 mL beaker were added 1 g of HA-MA1 prepared according to the method of Preparation Example 3, 0.50 g of 1,4-phenyldithiophenol (Sigma) and 100 mL of deionized water, and the mixture was dissolved with stirring at room temperature to obtain a transparent solution. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain HA-MA1-SH6 (833 mg, yield 83.3%) as a white flocculent solid.
[0294] The reaction equation for HA-MA1-SH6 is shown in
[0295] The .sup.1H-NMR spectrum of HA-MA1-SH6 is shown in
Example 27. Synthesis of Sulfhydryl-Methacrylate-Modified Hyaluronic Acid (HA-MA2-SH7)
[0296] To a 200 mL beaker were added 1 g of HA-MA2 prepared according to the method of Preparation Example 4, 0.92 g of sulfhydryl polyethylene glycol (Sigma) and 100 mL of deionized water, and the mixture was dissolved with stirring at room temperature to obtain a transparent solution. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain HA-MA2-SH7 (876 mg, yield 87.6%) as a white flocculent solid.
[0297] The reaction equation for HA-MA2-SH7 is shown in
[0298] The .sup.1H-NMR spectrum of HA-MA2-SH7 is shown in
Example 28. Synthesis of Sulfhydryl-Methacrylate 2-Modified Hyaluronic Acid (HA-MA2-SH8)
[0299] To a 200 mL beaker were added 1 g of HA-MA2 prepared according to the method of Preparation Example 4, 0.68 g of trimethylolpropane-tris(3-sulfhydrylpropionate) (Sigma), 50 mL of deionized water and 50 mL of dimethylformamide, and the mixture was dissolved with stirring at room temperature to obtain a transparent solution. The resulting transparent solution was stirred for an additional 12 h. The resulting solution was filled into a dialysis bag (Spectrumlabs, molecular weight cutoff: 8 kDa) and dialyzed against 5 L of hydrochloric acid solution at pH 4 for 5 days, with water refreshed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain HA-MA2-SH8 (825 mg, yield 82.5%) as a white flocculent solid.
[0300] The reaction equation for HA-MA2-SH8 is shown in
[0301] The .sup.1H-NMR spectrum of HA-MA2-SH8 is shown in
Example 29. Determination of Sulfhydryl Content of Sulfhydryl-Modified Polymer Compounds by Ellman Method
Preparation Process:
[0302] 1. Preparation of a test buffer: 0.1 mol/L Na.sub.2HPO.sub.4 (containing 1 mM EDTA, adjusted to pH 8.0 with concentrated hydrochloric acid).
[0303] 2. Preparation of a standard working solution: 30 mmol/L cysteine solution.
[0304] 3. Preparation of an Ellman reagent stock solution: 0.1 mol/L Ellman reagent solution.
[0305] 4. Preparation of a standard solution:
TABLE-US-00001 Molar concentration 0 0.5 1.0 1.5 2.0 of sulfhydryl mmol/L mmol/L mmol/L mmol/L mmol/L Standard 0 4 8 12 16 working solution (μL) Buffer (μL) 240 236 232 228 224 Total volume 240 240 240 240 240 (μL)
[0306] 5. Preparation of a test sample solution: an appropriate amount of the sulfhydryl-modified polymer compound sample was dissolved in the buffer to prepare 1 mg/mL solution to be tested (triplicates for each sample).
Testing Process:
[0307] 1. The cysteine standard solution was prepared in a 0.5 mL centrifuge tube according to step 4 above.
[0308] 2. In an additional 1.5 mL centrifuge tube, 50 μL of Ellman assay solution was added to 1 mL buffer to obtain an assay solution.
[0309] 3. 240 μL, of the standard solution/test sample solution were each mixed with the Ellman assay solution in step 2 of the testing process, and reacted at room temperature for 15 min.
[0310] 4. After 15 min, the absorbance at 412 nm was determined using a microplate reader.
[0311] 5. The thiol content in the product can be calculated according to the absorbance/concentration standard curve of the obtained standard solution.
[0312] The standard curve of the sulfhydryl content is shown in
Example 30. Determination of Dynamic Viscosity of Sulfhydryl-Modified Polymer Compounds
[0313] 500 mg of sulfhydryl-modified polymer compound was dissolved in 50 mL of deionized water to obtain a 1% w/v solution. The dynamic viscosity of the resulting solution was determined at 25° C. using a rotary viscometer. The results are shown in Table 1 (PHEMA-A-SH1 and PHEMA-MA-SH1 were not determined for dynamic viscosity, because they were too viscous after dissolution and appeared to be gel-like).
TABLE-US-00002 TABLE 1 Determination results of sulfhydryl content and dynamic viscosity of sulfhydryl-modified polymer compounds Ellman assay Determination results results of viscosity Sulfhydryl content Dynamic viscosity Sample (mmol/g) (mPa .Math. s) HA-A1-SH1 1.357 420 HA-A2-SH1 1.424 416 HA-MA1-SH1 1.322 387 HA-MA2-SH1 1.362 441 CHS-A-SH1 1.152 206 CHS-MA-SH2 1.245 284 Gelatin-A-SH1 0.626 317 Gelatin-MA-SH1 0.614 288 CTS-A-SH1 1.832 265 CTS-MA-SH1 1.763 292 PHEMA-A-SH1 1.736 — PHEMA-MA-SH1 1.719 — HB-PEG-SH1 0.756 76 PVA-A-SH1 1.982 64 PVA-MA-SH1 1.857 69 HA-A1-SH2 1.428 452 HA-A1-SH3 1.347 398 HA-A2-SH2 1.471 435 HA-A2-SH3 1.413 412 HA-A2-SH4 1.458 487 HA-A2-SH5 1.347 437 HA-A2-SH6 1.298 416 HA-A2-SH7 0.974 473 HA-A2-SH8 2.471 451 HA-MA1-SH5 1.242 436 HA-MA1-SH6 1.317 429 HA-MA2-SH7 0.857 481 HA-MA2-SH8 2.146 447
Example 31. Determination of Molecular Weight and Distribution of Sulfhydryl-Modified Polymer Compounds by GPC
[0314] The mobile phase of GPC was 0.05 M sodium sulfate solution, with a flow rate of 1 mL/min and a column temperature of 30° C. The curve of the standard polyethylene glycol polymer was used as the standard curve.
[0315] 5 mg of sulfhydryl-modified polymer compound was dissolved in 1 mL of 0.05 M sodium sulfate solution, filtered through a 0.22 μM filter, and determined by GPC. The results of molecular weight and distribution are shown in Table 2 (PHEMA-A-SH4 and PHEMA-MA-SH1 were not determined for molecular weight, because they were too viscous after dissolution to pass through the filter).
TABLE-US-00003 TABLE 2 Determination results of the molecular weight and molecular weight distribution of sulfhydryl-modified polymer compounds Sample Mn (Da) Mw (Da) PDI HA-A1-SH1 114599 338178 2.95 HA-A2-SH1 90222 426199 4.72 HA-MA1-SH1 74053 454233 6.13 HA-MA2-SH1 52132 427591 8.20 CHS-A-SH1 34178 84127 2.46 CHS-MA-SH2 28114 90174 3.21 Gelatin-A-SH1 14524 57898 3.99 Gelatin-MA-SH1 17204 55217 3.21 CTS-A-SH1 54127 264214 4.88 CTS-MA-SH1 47517 214285 4.51 PHEMA-A-SH1 — — — PHEMA-MA-SH1 — — — HB-PEG-SH1 8841 20412 2.31 PVA-A-SH1 79854 95874 1.20 PVA-MA-SH1 75471 98627 1.31 HA-A1-SH2 102221 454335 4.44 HA-A1-SH3 72720 429952 5.91 HA-A2-SH2 103770 373400 3.60 HA-A2-SH3 83819 411866 4.91 HA-A2-SH4 73382 417944 5.70 HA-A2-SH5 57322 416987 7.27 HA-A2-SH6 76601 337406 4.40 HA-A2-SH7 77628 419439 5.40 HA-A2-SH8 61804 375555 6.08 HA-MA1-SH5 63887 391562 6.13 HA-MA1-SH6 108308 397962 3.67 HA-MA2-SH7 69148 373393 5.40 HA-MA2-SH8 99257 337377 3.39
Example 32. Determination of Water Retention of Sulfhydryl-Modified Polymer Compounds
[0316] 50 mg of sulfhydryl-modified polymer compound was added to a 20 mL glass bottle weighed in advance, and dissolved with 5 mL of deionized water to obtain a 1% solution. The mass of the solution m.sub.0 was obtained by the mass subtraction method. The glass bottle was placed in a shaker at 37° C., and weighed at regular intervals to obtain the mass of the solution m.sub.t. The water retention of the sulfhydryl-modified polymer compounds can be calculated according to the following formula:
Water retention percentage (%)=m.sub.t/m.sub.0×100%.
[0317] The test results are listed in Table 3.
TABLE-US-00004 TABLE 3 Comparison table of water retention rate index Water retention rate (%) Time point (day) Sample 0 1 2 4 6 HA-A1-SH1 100 92.7 81.1 50.3 25.9 HA-A2-SH1 100 93.4 81.7 49.1 27.6 HA-MA1-SH1 100 91.6 79.1 46.9 25.4 HA-MA2-SH1 100 92.6 82.3 48.5 28.1 HA-A1-SH2 100 89.4 76.1 39.5 16.9 HA-A1-SH3 100 89.2 77.6 41.6 18.5 HA-A2-SH2 100 88.6 74.4 36.7 15.6 HA-A2-SH3 100 86.9 77.8 33.2 13.9 HA-A2-SH4 100 91.5 81.5 38.3 21.4 HA-A2-SH5 100 89.4 80.5 38.8 21.1 HA-A2-SH6 100 87.8 76.8 47.5 24.5 HA-A2-SH7 100 88.5 77.4 46.5 26.2 HA-A2-SH8 100 86.8 74.9 44.4 24.8 HA-MA1-SH5 100 87.7 78.3 46.5 26.6 HA-MA1-SH6 100 84.7 72.1 37.4 16.0 HA-MA2-SH7 100 84.3 73.5 39.4 18.4 HA-MA2-SH8 100 83.9 70.5 34.8 14.8
Example 33. Determination of Oxidation Resistance of Sulfhydryl-Modified Polymer Compounds by DPPH Free Radical Capture Method
[0318] An absolute ethanol solution of 25 μmol/L 1,1-diphenyl-2-trinitrophenylhydrazine (TNBS) was precisely prepared as a working solution. A certain amount of working solution was diluted with ethanol to obtain a series of standard solutions (0 μmol/L, 5 μmol/L, 10 μmol/L, 15 μmol/L, 20 μmol/L and 25 μmol/L).
[0319] The PBS solutions of sulfhydryl substituted polymer compounds were precisely prepared to obtain a series of test sample solutions at 0.1 mg/mL.
[0320] 90 μL of TNBS working solution and 10 μL of test sample solution were mixed well, and stored away from light at room temperature for 30 min. After that, the absorbance of the TNBS standard solution and the test sample mixed solution was determined at 517 nm for plotting a standard curve, and the concentration of the residual DPPH in the test sample was calculated according to the obtained standard curve. The free radical capture capacity (%) of the test sample was calculated according to the following formula:
Free radical capture capacity (%)=(1−(C.sub.sample/C.sub.DPPH))×100%.
[0321] The DPPH standard curve is shown in
[0322] The examples of the present disclosure have been described above. However, the present disclosure is not limited to the above examples. Any modification, equivalent, improvement and the like made without departing from the spirit and principle of the present disclosure shall fall within the protection scope of the present disclosure.