阅读说明：本技术 一类用于多肽合成的含硫聚乙二醇树脂 (Sulfur-containing polyethylene glycol resin for polypeptide synthesis ) 是由 向双春 高峰 李云平 于 2021-01-08 设计创作，主要内容包括：一类用于多肽合成的含硫聚乙二醇树脂。本发明通过聚乙二醇单烯丙基醚与含有胺基或羧基的巯基化合物反应得到含硫元素的聚乙二醇,同时将胺基或羧基引入了聚乙二醇,这样非常方便地将修饰聚乙二醇引入到树脂上,从而获得不同功效的含硫聚乙二醇树脂。含硫聚乙二醇树脂再引入多肽合成子,可以用于固相多肽合成,特别适用于连续流固相多肽合成。(A sulfur-containing polyethylene glycol resin for polypeptide synthesis. The invention obtains the polyethylene glycol containing sulfur element by the reaction of polyethylene glycol monoallyl ether and sulfhydryl compound containing amino or carboxyl, and introduces the amino or carboxyl into the polyethylene glycol, thus conveniently introducing the modified polyethylene glycol into the resin and obtaining the sulfur-containing polyethylene glycol resin with different effects. The sulfur-containing polyethylene glycol resin is introduced with a polypeptide synthon, can be used for solid phase polypeptide synthesis, and is particularly suitable for continuous flow solid phase polypeptide synthesis.)

1. A sulfur-containing polyethylene glycol resin is characterized in that the chemical structural general formula is as follows:

a and B are one of the following chemical structure fragments:

a isB isOr, A isB isOr the like, or, alternatively,

a isB isOr the like, or, alternatively,

a isB isOr the like, or, alternatively,

a isB isOr the like, or, alternatively,

a isB is

Wherein X is an S atom or a Sulfone (SO)₂)，

W₁A resin of the structure:

W₂a resin of the structure:

one of (1); w is matrix resin, and the matrix resin is one of polystyrene resin, polyacrylamide resin and polymethacrylate resin;

n is 5-80, q1 and q2 are respectively 1-5, m1 is 1-2, m2 is 1-2, R1 is hydrogen, methyl, carboxy methyl ester, carboxy tert-butyl ester, carboxy benzyl ester, -CH₂OCH₃、-CH₂OCH₂CH₃One of (1); r2 is one of hydrogen, methyl, ethyl and benzyl; r3 represents one of hydrogen, methyl, dimethylamino, diethylamino and dibenzylamino; r4 represents one of hydrogen, methyl, ethyl and benzyl; r5 represents one of hydrogen, methyl, ethyl and benzylSeed growing;

the K1 is one of the following structural formulas:

the K is one of the following structural formulas:

。

2. the sulfur-containing polyethylene glycol resin of claim 1, wherein the sulfur-containing polyethylene glycol resin is used for solid phase polypeptide synthesis after a linker for polypeptide synthesis is attached to the sulfur-containing polyethylene glycol resin.

3. According to claim 2, the sulfur-containing polyethylene glycol resin connected with the polypeptide synthesis linker is used for polypeptide synthesis, when the end of polyethylene glycol on the sulfur-containing polyethylene glycol resin is an amine group, the steps for polypeptide synthesis are as follows:

1) introducing a polypeptide synthesis linker containing carboxyl into the sulfur-containing polyethylene glycol resin through a coupling reagent to obtain the sulfur-containing polyethylene glycol resin connected with the polypeptide synthesis linker.

2) The polypeptide is prepared by using sulfur-containing polyethylene glycol resin connected with a polypeptide synthesis linker according to a conventional solid-phase polypeptide synthesis process.

4. According to claim 2, the sulfur-containing polyethylene glycol resin connected with the polypeptide synthesis linker is used for polypeptide synthesis, and when the end of polyethylene glycol on the sulfur-containing polyethylene glycol resin is carboxyl, the steps for polypeptide synthesis are as follows:

1) coupling the alkyl diamine with single end Boc protection to resin with coupling reagent, and eliminating Boc protecting group with trifluoroacetic acid to obtain sulfur-containing polyglycol resin with amino end on the resin.

2) Introducing a polypeptide synthesis linker containing carboxyl into the sulfur-containing polyethylene glycol resin with amino at the tail end of polyethylene glycol through a coupling reagent to obtain the sulfur-containing polyethylene glycol resin connected with a polypeptide synthesis linker.

3) The polypeptide is prepared by using sulfur-containing polyethylene glycol resin connected with a polypeptide synthesis linker according to a conventional solid-phase polypeptide synthesis process.

Technical Field

The invention belongs to the field of organic synthesis, and particularly relates to preparation and application of sulfur-containing polyethylene glycol resin.

Background

Merrifield et al reported the concept of solid phase peptide synthesis as early as 1960 s, and synthesized styrene into different types of chloromethylated polystyrene by crosslinking

[ Wu M, Yang G, Chen Z. Polymer supported peroxidization of A mil and clean oxidation of organic compounds under non-reactive and amorphous conditions read Funct Polymer, 2000,44: 97-100; hari A, Miller BL. expanding differentiation in solution vs. solid-supported reactivity for the synthesis of sulfonic acid derivatives, org Lett,1999,1: 2109-2111 ] as a carrier resin for solid phase synthesis of polypeptides (Merrifield resin, see below). Since then, polymeric solid supports have become more and more popular for use in the synthesis and purification of products.

Merrifield resin and polyethylene glycol (PEG) structure

Despite the many known advantages of crosslinked polystyrene support resins, in the solid phase synthesis of polypeptides, the polymers are mixed with polar solvents such as dimethylformamide, which are conventionally used in the synthesis of polypeptides

(DMF)[Santini R,Griffith MC,Qi M.A measure of solvent effects on swelling of resi ns for solid phase organic synthesis.Tetrahedron Lett,1998,39:8951—8954；

The compatibility of The Vaino AR, Goodin DB, Janda KD.investing resins for solid phase organic synthesis, The relative between The engineering and microbiological as probe by EPR and f cellular science J Comm Chem,2000,2: 330-336, is poor, as well as The absorption and swelling properties of The solvent. Furthermore, in heterogeneous synthesis, the poorly soluble polymer changes the traditional liquid phase synthesis into solid phase synthesis, the reaction becomes more complicated, the reaction kinetics is non-linear, and it is difficult to evaluate the completion of the reaction and the purity of the synthesized product on the support. To overcome the drawbacks of Merrifield et al resins, polyethylene glycol (PEG, represented by formula (a) below), such as TentaGel (formula (a) below) and formula (b) below, has been grafted onto a hydrophobic polystyrene core, which resins have good swelling in both non-polar and polar solvents [ Becker H, Lucas HW, Maul J, Pilai VNR, Anziger H, Mutter M.PEG-PS resin hav e benzene descriptions for colloidal-phase peptide synthesis, Macromol Chem Rapid Commun.1982,3: 217-223;

wan LS, Ke BB, Li XK, Honeycom-patterned films of polystyrene/poly (ethylene glycol), Preparation, surface aggregation and protein adsorption, Sci China Ser B-Chem, 2009,52: 969-974 ]. Currently, modified polystyrene resins have been greatly developed in research and development and commercialization.

Structures of TentaGel resin and ArgoGel resin X represents a functional group

Polyethylene glycol and the like having good swelling properties can be introduced into the resin by various methods. The first method is that polyethylene glycol forms an ether bond with the resin. TentaGel resin and ArgoGel resin. The preparation method is to form a polyethylene glycol chain by polymerizing the resin with hydroxyl and ethylene oxide under the catalysis of alkali and high pressure. The length of the polyethylene glycol On the surface of the resin obtained by the method is within 600, and the molecular weight is small, so that the method cannot meet the Synthesis of most of polypeptides [ Owen W.Gooding, Sylvee Baudart, Tracy L.Deegan, On the Development of New Poly R resin support for Solid-Phase Organic Synthesis J.Comm.Chem.1999, 1,113-122 ].

The second is the introduction of polyethylene glycol to the resin surface through a lipid group. The resin containing lipid group is easy to hydrolyze or aminolyze in the process of polypeptide synthesis, and has no practical application.

The third method is to introduce polyethylene glycol through an alkyldiamide bond. The method can be realized by the reaction of resin with amido and polyethylene glycol containing carboxyl. Introducing carboxyl group into Polyethylene Glycol with two amino groups by using alkyl dianhydride, and reacting with resin containing amino groups [ High-Load "Polyethylene Glycol-Polystyrene (PEG-PS) Graft Supports for Solid-Phase Synthesis, Kates et al biopolymers (Peptide Science), Vol.47, 365-380 (1998) ]; patent US 5,545,698] is for example a structural formula in the following formula.

Resin formed by reaction of carboxyl resin and polyethylene glycol containing amino

In the third method, the single-end carboxyl polyethylene glycol and the two-end carboxyl polyethylene glycol are easily obtained when the alkyl dianhydride reacts with the two-end amino polyethylene glycol, the two cannot be separated, complex components can be generated in subsequent synthesis, and impurity components also react with the amino group of the resin, so that the quality of the obtained polyethylene glycol resin is poor. The resin has less reports of subsequent applications except for the patent applied by the inventor and the article published in Peptide Science.

The fourth synthesis method is to introduce carboxyl to polyethylene glycol through carbamate, then introduce amine group to the other end of polyethylene glycol to obtain polyethylene glycol with amine group at one end and carboxyl group at one end, and finally couple polyethylene glycol to amino resin [ Reactive polymers,22(1994), 243-. The resin structure is as follows:

the modified polyethylene glycol corresponding to the resin is prepared by the following route:

the article mentions that several short peptides were synthesized by modifying the resin with polyethylene glycol, and no data were given for evaluating further properties of the resin. When the method is used for synthesizing the chlorinated polyethylene glycol with one end, the chlorinated polyethylene glycol with two ends and unreacted polyethylene glycol are easily obtained, so that a large amount of polyethylene glycol with carboxyl groups at two ends, polyethylene glycol with amino groups at two ends and other impurities are generated later, and different modified polyethylene glycols are grafted on the modified polyethylene glycol resin, so that the quality is poor. The urethane bond is unstable when a strong organic base such as piperidine is encountered during the synthesis of the polypeptide, and the quality of the synthesized polypeptide product is expected to be poor, so that the polyethylene glycol resin has not been commercialized so far.

The commercially available polyethylene glycol modified resins mainly include PEG-PS resins, such as TentaGel resin, PEG-based PEGA, AM PEGMatrix resin and ChemMatrix resin, and many of the polyethylene glycol modified resins reported in other documents are not used. The commercial TentaGel resin has the defects that the PEG chain is short, the length of the connected PEG is not uniform, and the quality of the synthesized polypeptide is unstable. PEG as a matrix generally has the defects of too strong swellability, poor washing, too large expansion of resin when TFA cuts polypeptide and difficult filtration.

The resin has the problems of complex production process of modified polyethylene glycol, organic alkali intolerance of the modified polyethylene glycol resin, disorder or non-uniformity of polyethylene glycol on the surface of the resin, too large swelling property, inconvenient operation and the like.

Disclosure of Invention

Aiming at the problems that the modified polyethylene glycol resin does not reach the standard or the preparation difficulty is high, the polyethylene glycol containing sulfur element is obtained by the reaction of polyethylene glycol monoallyl ether and sulfhydryl compound containing amino or carboxyl, and the amino or carboxyl is introduced into the polyethylene glycol, so that the polyethylene glycol is very conveniently introduced into the resin, and the polyethylene glycol resin with good performance is obtained. The sulfur-containing polyethylene glycol resin provided by the invention greatly reduces the synthesis cost of macromolecules such as polypeptide and nucleic acid, greatly improves the production efficiency and has wide application prospect.

A sulfur-containing polyethylene glycol resin has a chemical structural general formula as follows:

(Ⅰ)

a and B are one of the following chemical structure fragments:

a isB isOr the like, or, alternatively,

a isB isOr the like, or, alternatively,

a isB isOr the like, or, alternatively,

a isB isOr the like, or, alternatively,

a isB isOr the like, or, alternatively,

a isB is

Wherein X is an S atom or a sulfone (SO2),

W₁a resin of the structure:

W₂a resin of the structure:

one of (1);

w is matrix resin, and the matrix resin is one of polystyrene resin, polyacrylamide resin and polymethacrylate resin;

n is 5-120, preferably n is 5-80, q1 and q2 are respectively 1-5, m1 is 1-2, m is₂1-2, R1 is hydrogen, methyl, carboxy methyl ester, carboxy tert-butyl ester, carboxy benzyl ester, -CH₂OCH₃、-CH₂OCH₂CH₃One of (1); r2 is one of hydrogen, methyl, ethyl and benzyl; r3 represents one of hydrogen, methyl, dimethylamino, diethylamino and dibenzylamino; r4 represents one of hydrogen, methyl, ethyl and benzyl; r5 represents one of hydrogen, methyl, ethyl and benzyl;

the K1 is one of the following structural formulas:

the K is one of the following structural formulas:

a synthetic method of sulfur-containing polyethylene glycol resin comprises the following steps:

in the first step, sulfur-containing polyethylene glycol is synthesized. According to the difference of the structural general formula, the synthesis method of the sulfur-containing polyethylene glycol is divided into the following methods:

the synthesis method 1:

the structural general formula (I) is as follows:

and

the sulfur-containing polyethylene glycol resin is represented by the structural formula

The sulfur-containing polyethylene glycol is obtained by reacting with resin,

the specific reaction steps of the sulfur-containing polyethylene glycol when X is S are as follows:

step 1, condensing polyethylene glycol monoallyl ether 1 with a mercapto tert-butyl ester compound 2 under the catalysis of AIBN in a solvent, evaporating the solvent under reduced pressure to remove the solvent to obtain a reaction concentrated liquid, adding water, extracting the unreacted mercapto tert-butyl ester compound 2 with petroleum ether, extracting the water layer with the solvent, and evaporating the solvent under reduced pressure to obtain polyethylene glycol-R6 ester compound thioether 3.

And 2, mixing an acid-binding agent, the polyethylene glycol-R6 ester compound thioether 3 and p-toluenesulfonyl chloride in a solvent for reaction, evaporating the solvent under reduced pressure, washing with acid water for a plurality of times, directly purifying by using a silica gel column to remove unreacted p-toluenesulfonyl chloride after a dichloromethane layer is dried, and evaporating the eluent under reduced pressure to obtain the Tos ester 4 of the polyethylene glycol-R6 ester compound thioether.

Step 3, reacting Tos ester 4 of polyethylene glycol-tert-butyl ester compound thioether with sodium azide in a proper solvent, evaporating under reduced pressure to remove the solvent to obtain a thick liquid, dissolving the thick liquid with dichloromethane, washing with water to remove the sodium azide, and evaporating the dichloromethane under reduced pressure to obtain polyethylene glycol-R₆Ester compound thioether azide compound 5.

Step 4, polyethylene glycol-R₆Ester compound thioether azide compound 5 is reacted with triphenylphosphine in a suitable solvent for 2 to 12 hours. Adding water into the reaction solution, adjusting the pH value to 3-4 by using dilute acid, extracting triphenylphosphine and triphenylphosphine oxide from the obtained mixture by using ethyl acetate, basifying the water layer until the pH value is 8-9, extracting by using dichloromethane, drying, decompressing and rotary steaming to obtain amino-polyethylene glycol-R₆An ester compound thioether 6.

Step 5, amino-polyethylene glycol-R₆Removal of R from the ester Compound thioether 6 with an acid or base in a suitable solvent₆And (4) a base. Then adjusting the pH to 7 with an acid or a base to obtain H₂N-PEGn-CH₂-CH₂CH₂-S-(CH₂)q₁-(R₃)CH-COOH7。

The acid for removing tert-butyl adopts: p-toluenesulfonic acid, methanesulfonic acid, hydrogen chloride ether solution and hydrogen chloride 1, 4 dioxane solution; removal of R₆The base used in (1) is: sodium hydroxide, potassium hydroxide.

The method for synthesizing the compound 8-9 comprises the following specific steps:

step 1, amino compound and polyethylene glycol-R₆Reacting Tos ester 4 of ester compound thioether for 1 to 5 hours, evaporating the solvent under reduced pressure, adding water, acidifying until the pH value is equal to 6-7, and evaporating under reduced pressure to obtain R amino-polyethylene glycol-R₆An ester compound thioether 8.

Step 2, R₅Amino-polyethylene glycol-R₆Ester compound thioether8 removing R with acid or base in suitable solvent₆. Then adjusting the pH to 7 with an acid or a base to obtain R₅-HN-PEGn-CH₂CH₂CH₂-S-(CH₂)q₁-(R₃) CH-COOH 9. The structural formulae of the above-mentioned compounds 1 to 9 are as follows:

wherein R is₅Is one of hydrogen, methyl, ethyl, benzyl and isopropyl, R₆Is tert-butyl or benzyl. When X is Sulfone (SO)₂) When the compound 7 or 9 is oxidized by the following method, X is Sulfone (SO)₂) Is/are as follows

The sulfur-containing polyethylene glycol comprises the following specific reaction steps:

(1)R₅-HN-PEGn-CH₂CH₂CH₂-S-(CH₂)q₁-(R₃) CH-COOH was dissolved in alkaline water and BOC anhydride was added. After the reaction, the aqueous solution was extracted with petroleum ether. Adjusting the pH of a water layer to 3-4 by using dilute acid, extracting by using dichloromethane, and removing the dichloromethane by reduced pressure distillation to obtain Boc-R₅N-PEGn-CH₂CH₂CH₂-S-(CH₂)q₁-(R₃)CH-COOH10；

(2)Boc-R₅N-PEGn-CH₂CH₂CH₂-S-(CH₂)q₁-(R₃) Dissolving CH-COOH10 in ethyl acetate, adding sodium tungstate aqueous solution and hydrogen peroxide, and heating to react until the thioether is completely converted into sulfone. Distilling off ethyl acetate under reduced pressure, extracting the obtained aqueous solution with dichloromethane, washing dichloromethane with water for several times, distilling off dichloromethane under reduced pressure to obtain Boc-R₅N-PEGn-CH₂CH₂CH₂-SO₂-(CH₂)q₁-(R₃)CH-COOH11；

(3)Boc-R₅N-PEGn-CH₂CH₂CH₂-SO₂-(CH₂)q₁-(R₃) The tert-butoxycarbonyl group was removed from CH-COOH11 with an acid in an appropriate solvent. After the reaction is finished for 3-24 hours, the solvent is evaporated to dryness under reduced pressure to obtain a viscous liquid, the viscous liquid is dissolved by methanol, and then the pH value is adjusted to be equal to 7 by alkali to obtain R₅-HN-PEGn-CH₂-CH₂CH₂-SO₂-(CH₂)q₁-(R₃) CH-COOH 12. The structural formulas of compounds 10-12 are as follows:

R₅is one of hydrogen, methyl, ethyl, benzyl and isopropyl.

The synthesis method 2 comprises the following steps:

the structural general formula (I) is as follows:

and

the sulfur-containing polyethylene glycol resin is represented by the structural formula

The sulfur-containing polyethylene glycol is obtained by reacting with resin,

the specific reaction steps of the sulfur-containing polyethylene glycol when X is S are as follows:

(1) polyethylene glycol monoallyl ether 1 was condensed with a mercapto Boc amide group-containing compound 13 in a solvent under AIBN catalysis. Evaporating the solvent under reduced pressure to obtain a reaction thick liquid, adding water, extracting the unreacted Boc acylamino-containing compound 13 by using petroleum ether, extracting a water layer by using the solvent, and evaporating the solvent under reduced pressure to obtain a polyethylene glycol-thioether-Boc acylamino compound 14;

(2) dissolving the polyethylene glycol-thioether-Boc acylamino compound 14 and tert-butyl acrylate in an organic solvent, and adding alkaline water for reaction. After the reaction, water was added and stirred uniformly, and the aqueous layer was removed by layering. Drying the organic layer, and then evaporating to dryness under reduced pressure to obtain Boc-R₂-N-(R₁)CH-(CH₂)q₂-S-CH₂CH₂CH₂-PEGn-CH₂CH₂COOtBu15；

(3) Removing Boc and tert-butyl from tert-butyl propionate-polyethylene glycol-thioether-Boc amide compound 15 with acid in appropriate solvent, reacting, evaporating solvent under reduced pressure after reaction to obtain viscous liquid, dissolving with methanol, and adjusting pH to 7 with alkali to obtain R2-NH- (R)₁)CH-(CH₂)q₂-S-CH₂CH₂CH₂-PEGn-CH₂CH₂COOH16。

The synthesis of 16-1 comprises the following specific steps:

(1) dissolving the polyethylene glycol-thioether-Boc amido compound 14 and tert-butyl bromoacetate in an organic solvent, and adding sodium hydride in batches for reaction. After the reaction, the reaction mixture was slowly poured into a saturated ammonium chloride solution, and the aqueous layer was removed by separation. Drying the organic layer, and then evaporating to dryness under reduced pressure to obtain Boc-R₂-N-(R₁)CH-(CH₂)q₂-S-CH₂CH₂CH₂-PEGn-CH₂COOtBu15-1；

(2) Removing Boc and tert-butyl by acid in proper solvent for reaction, evaporating solvent under reduced pressure after reaction, dissolving viscous liquid with methanol, and adjusting pH to 7 with alkali to obtain R₂-NH-(R₁)CH-(CH₂)q₂-S-CH₂CH₂CH₂-PEGn-CH₂COOH 16-1. The specific structural formulas of the compounds 13-16 and the compounds 15-1 and 16-1 are as follows:

R₂is one of hydrogen, methyl, ethyl, benzyl and isopropyl.

II, when X is Sulfone (SO)₂) In this case, compound 15 or 15-1 is oxidized by the following method, and the protecting group is removed to give X as Sulfone (SO)₂) Is/are as follows

The specific reaction steps are as follows:

(1) dissolving tert-butyl propionate-polyethylene glycol-thioether-Boc amide compound 15 in ethyl acetate, adding sodium tungstate aqueous solution and hydrogen peroxide, and heating for reaction until thioether is completely converted into sulfone. Distilling off ethyl acetate under reduced pressure, extracting the obtained aqueous solution with dichloromethane, washing dichloromethane with water for several times, distilling off dichloromethane under reduced pressure to obtain Boc-R₂-N-(R₁)CH₂-(CH₂)q₂-SO₂-CH₂CH₂CH₂-PEGn-(CH₂)_m1COOtBu17；

(2)Boc-R₂-N-(R₁)CH₂-(CH₂)q₂-SO₂-CH₂CH₂CH₂-PEGn-(CH₂)_m1COOtBu17 Boc and t-butyl were removed with acid in a suitable solvent. After the reaction is finished for 3-24 hours, the solvent is evaporated to dryness under reduced pressure to obtain a viscous liquid, the viscous liquid is dissolved by methanol, and then the pH value is adjusted to be equal to 7 by alkali to obtain R₂-NH-(R₁)CH-(CH₂)q₂-SO₂-CH₂CH₂CH₂-PEGn-(CH₂)_m1COOH 18. The compound Boc-R₂-N-(R₁)CH₂-(CH₂)q₂-SO₂-CH₂CH₂CH₂-PEGn-(CH₂)_m1COOtBu17 and R₂-NH-(R₁)CH-(CH₂)q₂-SO₂-CH₂CH₂CH₂-PEGn-(CH₂)_m1The structural formula of COOH18 is as follows:

thirdly, the structural general formula (I) is as follows:

the sulfur-containing polyethylene glycol resin is represented by the following structural formula:

the specific synthetic steps of the sulfur-containing polyethylene glycol are as follows:

the synthesis method 3:

when X is S, according to K and m₂Different structure of value, sulfur-containing polyethylene glycol

R₂-NH-(R₁)CH₂-(CH₂)q₂-S-CH₂CH₂CH₂-PEGn-(CH₂)m₂The specific structure of PH-K is as follows:

the method comprises the following specific steps:

1. preparation of Compounds 19, 21, 23

(1) Dissolving the polyethylene glycol-thioether-Boc amido compound 14 in an anhydrous solvent, slowly adding alkali, and then adding tBu-CP₁-Tos. After the reaction, slowly pouring the reaction solution into saturated ammonium chloride solution, and extracting the obtained mixed solution with petroleum to removeThe remaining tBu-CP₁-Tos. Extracting the water layer with dichloromethane for several times, washing the dichloromethane layer with saturated sodium chloride aqueous solution for several times, drying with anhydrous sodium sulfate, and distilling under reduced pressure to remove dichloromethane to obtain tBu-CP₁-polyethylene glycol-thioether-Boc amide compound 39.

The polyethylene glycol-thioether-Boc amido compound 14, alkali and tBu-CP₁-molar ratio of Tos of 1: (3-8): (2-5);

the reaction temperature was controlled at 0 ℃ to 50 ℃.

The alkali is one of sodium hydride and potassium hydride.

(2)tBu-CP₁-polyethylene glycol-thioether-Boc amide compound 39 Boc and tert-butyl are removed with acid in a suitable solvent. And after the reaction is finished for 3-24 hours, evaporating the solvent to dryness under reduced pressure to obtain a viscous liquid, dissolving the viscous liquid with methanol, and then adjusting the pH to be equal to 7 with alkali to obtain compounds 19, 21 and 23.

The acid, tBu-CP₁The molar ratio of the polyethylene glycol-thioether-Boc amido compound 39 is (1-5): 1 the reaction solvent adopts: one or more of chloroform, dichloromethane, 1, 4 dioxane, ethyl acetate and toluene.

The reaction temperature was controlled at-10 ℃ to 30 ℃.

The acid for removing tert-butyl adopts: one or more of p-toluenesulfonic acid, methanesulfonic acid, hydrogen chloride ether solution and hydrogen chloride 1, 4 dioxane solution.

The tBu-CP₁Specific structural formula of Compound corresponding to Tos and tBu-CP₁-polyethylene glycol-thioether-Boc amide compound 39 as follows:

2. preparation of Compounds 20, 22

(1) Polyethylene glycol-thioether-Boc amide Compound 14, tBu-CP₂Dissolving triphenylphosphine in a proper solvent, slowly dropwise adding an azo catalyst, reacting for 12-48 hours, washing the reaction solution for several times by using water, drying by using anhydrous sodium sulfate, purifying by using a silica gel column to remove triphenylphosphine and triphenylphosphine oxide, and performing reduced pressure spin drying on the eluent to obtain tBu-CP₂-polyethylene glycol-thioether-Boc amide compound 40.

The polyethylene glycol-thioether-Boc amido compound 14, tBu-CP₂The molar ratio of-OH, azo catalyst and triphenylphosphine is 1: (1-3): (1-4): (1 to 4)

The reaction temperature was controlled at 10 ℃ to 70 ℃.

(2)tBu-CP₂-polyethylene glycol-thioether-Boc amido compound 40 Boc and tert-butyl are removed with acid in a suitable solvent. And after the reaction is finished for 3-24 hours, decompressing and evaporating the solvent to dryness to obtain a viscous liquid, dissolving the viscous liquid by using methanol, and then adjusting the pH value to be 7 by using alkali to obtain the compounds 20 and 22.

The acid, tBu-CP₂The molar ratio of the polyethylene glycol-thioether-Boc amido compound 40 is (1-5): 1. the reaction solvent adopts: one or more of chloroform, dichloromethane, 1, 4 dioxane, ethyl acetate and toluene.

The reaction temperature was controlled at-10 ℃ to 30 ℃.

The acid for removing tert-butyl adopts: one or more of p-toluenesulfonic acid, methanesulfonic acid, hydrogen chloride ether solution and hydrogen chloride 1, 4 dioxane solution. The tBu-CP₂The specific structure of-OH and tBu-CP 2-polyethylene glycol-thioether-Boc amide compound 40 is shown below:

when X is Sulfone (SO)₂) When, according to the structure of K and m₂The different structure of the values is such that,

R₂-NH-(R₁)CH₂-(CH₂)q-SO₂-CH₂-CH₂CH₂-PEGn-(CH₂)m₁the specific structure of PH-K is as follows:

the specific synthesis method comprises the following steps: reference compound 18 was prepared by oxidation and deprotection using compound 39 or 40 as starting material.

And step two, reacting the sulfur-containing polyethylene glycol synthesized in the step one with different types of resins to synthesize the resin modified by the sulfur-containing polyethylene glycol:

the preparation method comprises the following steps:

the method comprises the following steps:

1) and (3) reacting sulfur-containing polyethylene glycol and Fmoc-OSu in an organic water mixed solvent by using alkali as an acid-binding agent to obtain the Fmoc-sulfur-containing polyethylene glycol.

The reaction solvent is one of acetone/water, tetrahydrofuran/water, 1, 4 dioxane/water and acetonitrile/water.

The acid-binding agent is selected from one of sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, etc.

2) Fmoc-sulfur-containing polyethylene glycol corresponding to the resin, a polypeptide condensing agent, an organic base, and PS1 (aminomethyl resin or substituted aminomethyl resin) are subjected to coupling reaction in a proper solvent, and the Fmoc-sulfur-containing polyethylene glycol is introduced into the resin.

The molar ratio of the reaction feeding materials is Fmoc-sulfur-containing polyethylene glycol: polypeptide condensing agent: an organic base and an aminomethyl resin or a substituted aminomethyl resin (2-4): (2-4): (3-6): 1;

the polypeptide condensing agent includes one of common reagents such as DIC, HBTU, HATU and COMU. The organic base is one of DIEA, triethylamine and N-methylmorpholine.

The solvent used in the synthesis adopts one of DMF, NMP and dichloromethane.

3) Fmoc-sulfur-containing polyethylene glycol resin is synthesized by using a common Fmoc removal solvent to remove Fmoc protecting groups, so that the sulfur-containing polyethylene glycol modified resin is obtained.

The Fmoc removal solution is 20% piperidine solution, 1-5%/1-5% DBU/1-octyl mercaptan solution or 1-5%/19-15% DBU/piperidine solution, and the solvent adopted by the solution comprises one or more of DMF, NMP, tetrahydrofuran, dimethyl tetrahydrofuran, 1, 4 dioxane and acetonitrile.

The method 2 comprises the following steps:

reacting sulfur-containing polyethylene glycol with one of sulfonyl chloride resin, isocyanate resin or isothiocyanate resin and organic base in proper solvent at 5-80 deg.c for several hr. And after the reaction is finished, draining the reaction solution, and washing the obtained resin with the solvent for several times to obtain the target resin.

The molar ratio of the reaction feeding materials is that the sulfur-containing polyethylene glycol: organic base: sulfonyl chloride resin ═ (2-4): (3-6): 1;

the organic base is one of triethylamine, nitrogen methyl morpholine and the like.

The reaction solvent is one of dichloromethane, ethyl acetate, DMF, NMP, tetrahydrofuran, dimethyl tetrahydrofuran, etc.

The method 3 comprises the following steps:

carboxyl resin, polypeptide condensation reagent and organic alkali react in proper solvent at room temperature for half an hour to form active ester, then sulfur-containing polyethylene glycol corresponding to the resin is added to react for hours at room temperature, reaction liquid is pumped, and the obtained resin is washed by the solvent for several times to obtain the target resin. The carboxyl group of the sulfur-containing polyethylene glycol can be unprotected by the method.

The molar ratio of the reaction feeding materials is (2-4): (2-4): (3-6): 1.

the polypeptide condensing agent is one of N, N' -disuccinimidyl carbonate, TSTU and the like.

The organic base adopts one or more of DIEA, DBU, triethylamine and N-methyl morpholine.

The solvent used in the synthesis adopts one or more of DMF, NMP and dichloromethane.

The sulfone group-containing polyethylene glycol resin may also be prepared from a thioether-containing polyethylene glycol resin.

When the carboxyl end is exposed outside the resin, directly mixing the resin with sodium tungstate aqueous solution and hydrogen peroxide, stirring for several hours at room temperature, filtering the aqueous solution, washing the resin with an organic solvent, and drying in the air to obtain the sulfone-containing polyethylene glycol resin with the exposed carboxyl end.

When the amine end is exposed, the thioether-containing polyethylene glycol resin with the amine end protecting group is mixed with sodium tungstate aqueous solution and hydrogen peroxide, the mixture is stirred for a plurality of hours at room temperature, the aqueous solution is filtered, the resin is washed by an organic solvent, and then the protecting group is removed to obtain the sulfone-containing polyethylene glycol resin.

Application of sulfur-containing polyethylene glycol resin in polypeptide synthesis

According to different polypeptide synthesis strategies, corresponding polypeptide synthesis connecting agents are introduced into sulfur-containing polyethylene glycol resin, and then the preparation of the polypeptide is completed according to the conventional polypeptide synthesis process.

When the end of polyethylene glycol on the sulfur-containing polyethylene glycol resin is an amine group, the procedure for polypeptide synthesis is as follows:

1) introducing a polypeptide synthesis linker containing carboxyl into the sulfur-containing polyethylene glycol resin through a coupling reagent to obtain the sulfur-containing polyethylene glycol resin connected with the polypeptide synthesis linker.

2) The polypeptide is prepared by using sulfur-containing polyethylene glycol resin connected with a polypeptide synthesis linker according to a conventional solid-phase polypeptide synthesis process.

When the end of polyethylene glycol on the sulfur-containing polyethylene glycol resin is carboxyl, the procedure for polypeptide synthesis is as follows:

1) coupling the alkyl diamine with single end Boc protection to resin with coupling reagent, and eliminating Boc protecting group with trifluoroacetic acid to obtain sulfur-containing polyglycol resin with amino end on the resin.

2) Introducing a polypeptide synthesis linker containing carboxyl into the sulfur-containing polyethylene glycol resin with amino at the tail end of polyethylene glycol through a coupling reagent to obtain the sulfur-containing polyethylene glycol resin connected with a polypeptide synthesis linker.

3) The polypeptide is prepared by using sulfur-containing polyethylene glycol resin connected with a polypeptide synthesis linker according to a conventional solid-phase polypeptide synthesis process.

The chemical property stability, the yield of the synthesized polypeptide, the quality of the synthesized polypeptide and the operational convenience of the sulfur-containing polyethylene glycol resin are obtained by comparing with the commercial polypeptide synthetic resin.

Compared with the prior art, the sulfur-containing polyethylene glycol resin has great difference in material structure with the prior art, has some performances in polypeptide synthesis equivalent to those of the prior art, and has some performances superior to those of the prior art. Therefore, the sulfur-containing polyethylene glycol resin applied to polypeptide synthesis has great creativity.

The invention has the beneficial effects that:

the polyethylene glycol containing sulfur element is obtained by the reaction of polyethylene glycol monoallyl ether and sulfhydryl compound containing amino or carboxyl, the polyethylene glycol is very conveniently introduced onto resin through amido bond, thiourea, urea and sulfamide, the obtained polyethylene glycol resin containing thioether bond or sulfone bond has stable chemical performance, and organic alkali resistance is realized in the polypeptide synthesis process. Secondly, the sulfur-containing polyethylene glycol on the surface of the resin has uniform length and almost no heterogeneous polyethylene glycol, and the polypeptide synthesized by the resin has stable quality. Thirdly, long-chain polyethylene glycol can be introduced into the resin by the method, and the obtained polyethylene glycol resin is more suitable for synthesizing long peptide and difficult peptide. Fourthly, the PEG chain of the sulfur-containing polyethylene glycol resin is dissolved in an organic solvent during polypeptide synthesis, so that the polypeptide coupling speed is greatly improved compared with that of the cross-linked polystyrene carrier resin, and the resin does not have the problem that the polyethylene glycol matrix resin is too large in swelling and difficult to operate during polypeptide cutting.

Abbreviations and English used in the present invention have the following meanings:

drawings

FIG. 1 shows Fmoc-NH-CH in example 10₂CH₂-S-CH₂CH₂CH₂-PEG_30-40-CH₂CH₂COOH nuclear magnetic spectrum.

FIG. 2 shows Fmoc-NH-CH of example 0₂CH₂-S-CH₂CH₂CH₂-PEG_30-40-CH₂CH₂COOH mass spectrum.

FIG. 3 shows example 11NH₂-CH₂CH₂-S-CH₂CH₂CH₂-PEG_30-40-CH₂CH₂CO-AM-polystyrene resin 4 IR spectrum.

FIG. 4 shows example 11NH₂-CH₂CH₂-S-CH₂CH₂CH₂-PEG_30-40-CH₂CH₂CO-AM-polystyrene resin 4 appearance.

FIG. 5 is a high performance liquid chromatogram of the crude product of example 21 Ac-Ser-Val-Val-Val-Arg-Thr-OH.

FIG. 6 is a mass spectrum of the crude product of example 21 Ac-Ser-Val-Val-Val-Arg-Thr-OH.

FIG. 7 is a high performance liquid chromatogram of the crude thaumalutide of example 22.

FIG. 8 is a mass spectrum of crude thaumatin of example 22.

FIG. 9 is an HPLC chart of crude sertraline acetate obtained by synthesis of Alanine resin 1 in example 20.

FIG. 10 is an HPLC chart of crude sertraline acetate obtained by synthesis of Alanine resin 2 in example 20.

FIG. 11 is an HPLC chart of crude sertraline acetate from the TentaGel synthesis in example 20.

FIG. 12 is an HPLC chart of crude sertraline acetate from the Chemmatrix synthesis in example 20.

FIG. 13 is an HPLC chart of crude sertraline acetate obtained by Rink amide-PS synthesis in example 20.

Detailed Description

The invention discloses a sulfur-containing polyethylene glycol resin and a preparation method thereof, and the examples are only a part of examples, but not all examples. The embodiments are only for illustrating the technical concept and features of the present invention, and the purpose of the embodiments is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

The invention is further illustrated by the following examples.

Example 1H₂N-PEG_(60～80)-CH₂CH₂CH₂-S-CH₂CH₂-COOH (Compound 7, R)₃＝H)

1)HO-PEG_(60～80)-CH₂CH₂CH₂-S-CH₂CH₂-COOtBu Synthesis

840g (about 0.24mol) of APEG3500 is dissolved in 7L of 1, 4 dioxane, 88.9g (0.6mol) of 2-mercaptoacetic acid tert-butyl ester is added under the protection of nitrogen, the temperature is raised to 55 ℃, 9.4g (0.03mol) of AIBN is added, the temperature is stabilized to 55 ℃, the protection of nitrogen is removed, the reaction is carried out for 13h at 55 ℃, and the reaction is detected to be complete by MS.

About 4L of 1, 4-dioxane was removed by rotary evaporation under reduced pressure, 10L of water was added, and a large amount of unreacted tert-butyl 2-mercaptoacetate was extracted with petroleum ether (5L. times.3). Adjusting pH of water layer to 4 with 40% phosphoric acid (small amount of acid), extracting with dichloromethane (5L 2), washing with water, drying dichloromethane solution with anhydrous sodium sulfate, and evaporating under reduced pressure to obtain HO-PEG (60-80) -CH₂CH₂CH₂-S-CH₂CH₂-COOtBu 780g。

2)Tos-O-PEG_(8～12)-CH₂CH₂CH₂-S-CH₂CH₂-COOtBu Synthesis

595g (0.17mol) of HO-PEG are added under ice-bath conditions_(60～80)-CH₂CH₂CH₂-S-CH₂-COOtBu was dissolved in 6L of methylene chloride, and 90g (0.46mol) of p-toluenesulfonyl chloride was added. 57.4g (0.56mol) of triethylamine are added in portions. After the addition, the temperature naturally rises to room temperature, and the reaction lasts for 3 hours. The dichloromethane reaction solution was washed with water, dilute phosphoric acid and saturated brine, respectively, and the dichloromethane layer was dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure to give 623g of a brown oil.

3)N₃-PEG_(60～80)-CH₂CH₂CH₂-S-CH₂CH₂-COOtBu Synthesis

0.9kg (1.4mol) of Tos-O-PEG_(60～80)-CH₂CH₂CH₂-S-CH2-COOtBu, 160g (2.5mol) of sodium azide were mixed with 7 liters of DMF, heated to 60 ℃ and stirred overnight. To the reaction mixture was added 10L of water, and the mixture was extracted with ethyl acetate (1L. times.7), which was washed with water (1L. times.7) and then with 8L of saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure to give 0.71kg of a yellow oil.

4)NH₂-PEG_(60～80)-CH₂CH₂CH₂-S-CH₂CH₂-COOtBu Synthesis

455g (0.13mol) of N are added under ice bath₃-PEG_(60～80)-CH₂CH₂CH₂-S-CH₂CH₂COOtBu was dissolved in 6L of toluene, 85g (0.32mol) of triphenylphosphine was added in portions, and the mixture was stirred overnight with natural warming. Most of the triphenylphosphine oxide formed was removed by filtration, 5l of dilute acid water was added, the layers were separated and the aqueous phase was extracted 3 times with ethyl acetate until the triphenylphosphine was completely extracted. The aqueous phase was adjusted to pH 8 with sodium carbonate, extracted with ethyl acetate (2 l × 2), washed with water and saturated brine, dried, and then spin-dried to give 410g of a pale yellow oil.

5)NH₂-PEG_(60～80)-CH₂CH₂CH₂-S-CH₂CH₂-COOH synthesis

420g(0.12mol)NH₂-PEG_(60～80)-CH₂CH₂CH₂-S-CH₂CH₂-COOtBu is dissolved in 2.5L of dichloromethane, 2.5L of trifluoroacetic acid is added,the reaction was carried out at room temperature for 3 hours. Spin-drying the solvent under reduced pressure, adding 500ml water into the concentrate, adjusting pH to 7.0 with sodium hydroxide aqueous solution, and evaporating under reduced pressure to remove water to obtain NH₂-PEG_(60～80)-CH₂CH₂CH₂-S-CH₂-CH₂COOH 396g。

H¹NMR(400MHz，DMSO-d6)：δ1.71～1.75(m，2H)，2.34～2.36(m，2H)，2.47～2.58(m， 4H)，2.59～2.61(m，2H)，2.64～2.71(m，4H)，3.41～3.60(m，284H)，7.93(s，1H)。

Example 2 Me-HN-PEG_(8～12)-CH₂CH₂CH₂-S-CH₂CH₂COOH (Compound 9, R₃Hydrogen, R₅Armenine methyl

1)Me-NH-PEG_(8～12)-CH₂CH₂CH₂-S-CH₂CH₂-COOtBu

70g (0.1mol) of Tos-O-PEG_(8～12)-CH₂CH₂CH₂-S-CH₂CH₂-COOtBu is dissolved in 400ml tetrahydrofuran, cooled to-60 ℃, 100ml3M methylamine tetrahydrofuran solution is dripped, and after the dripping is finished, the mixture is kept at-60 ℃ and stirred for 3 hours. The solvent was evaporated to dryness under reduced pressure to give a pale yellow oil, 400ml of water was added to dissolve the oil, the aqueous phase was extracted with ethyl acetate (200 ml. times.3), acetic acid was washed once with 200ml of saturated saline, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure to give 53g of a yellow oil.

2)Me-NH-PEG_(8～12)-CH₂CH₂CH₂-S-CH₂CH₂-COOH

35g(52mmol)NH₂-PEG_(20～30)-CH₂CH₂CH₂-S-CH₂CH₂COOtBu was dissolved in a mixed solution of 150ml of methylene chloride and 150ml of trifluoroacetic acid, and reacted at room temperature for 5 hours. The solvent was spin-dried under reduced pressure, the obtained oily substance was dissolved in 150ml of water, the obtained aqueous layer was adjusted to pH 7.0 with aqueous sodium hydroxide solution, and finally water was distilled off under reduced pressure to obtain Me-NH-PEG containing sodium trifluoroacetate_(20～30)-CH₂CH₂CH₂-S-CH₂CH₂-COOH27g。

H¹NMR(400MHz，DMSO-d6)：δ1.70～1.74(m，2H)，2.33～2.35(m，2H)，2.47～2.58(m， 4H)，2.59～2.61(m，2H)，2.64～2.71(m，4H)，3.31～3.53(m，35H)，7.91(s，1H)。

Example 3

NH₂-CH₂-CH₂-S-CH₂CH₂CH₂-PEG(30～40)-CH₂CH₂COOH (compound 16, R1 ═ H, R2 ═ H)

1) Boc-NH-CH2-CH2-S-CH2CH2CH2-PEG (30-40) -OH (compound 14) synthesis

150g (about 0.1mol) of APEG1500 was dissolved in 600ml of 1, 4-dioxane, 106g (0.6mol) of Boc-cysteamine (compound 13) was added under nitrogen protection, the temperature was raised to 55 ℃, 3.3g (0.02mol) of AIBN was added, and when the temperature stabilized to 55 ℃, the nitrogen protection was removed, the reaction was reacted at 55 ℃ for 9 hours, and the reaction was detected to be complete by MS.

About 400ml of 1, 4-dioxane was removed by rotary evaporation under reduced pressure, 400ml of water was added, and petroleum ether (400 ml. times.3) was added to extract a large amount of unreacted Boc-cysteamine. Adjusting pH of water layer to 4 with 40% phosphoric acid, extracting with dichloromethane (300ml × 2), washing with water, drying dichloromethane solution with anhydrous sodium sulfate, and evaporating to dryness under reduced pressure to obtain Boc-NH-CH₂-CH₂-S-CH₂CH₂CH₂-PEG_(30～40)-OH103g。

2)Boc-NH-CH₂-CH₂-S-CH₂CH₂CH₂-PEG_(30～40)-CH₂CH₂COOtBu (Compound 15) Synthesis of 80g (ca. 50mmol) of Boc-NH-CH₂-CH₂-S-CH₂CH₂CH₂-PEG_(30～40)OH was dissolved in 400ml of methylene chloride, cooled to-5 ℃ and added with 14.8g (116mmol) of tert-butylacrylate and 1.87g (5.8mmol) of tetrabutylammonium fluoride, and 30ml of 20% aqueous NaOH solution was added to complete the reaction at 25 ℃ overnight. MS detection reaction complete, adding 400ml water, using 40% phosphoric acid to adjust aqueous phase pH3-4, layering, DCM using water (200ml x2), anhydrous sodium sulfate drying, finally decompression evaporation to dryness to obtain 73g light yellow Boc-NH-CH₂-CH₂-S-CH₂CH₂CH₂-PEG_(20～30)-CH₂CH₂COOtBu。

3)NH₂-CH₂-CH₂-S-CH₂CH₂CH₂-PEG_(30～40)-CH₂CH₂Synthesis of COOH (Compound 16)

70g(44mol)Boc-NH-CH₂-CH₂-S-CH₂CH₂CH₂-PEG_(20～30)-CH₂CH₂COOtBu was dissolved in 800ml of methylene chloride, and 17.6g (92.8mmol) of p-toluenesulfonic acid was added to the solution to react at room temperature for 3 hours. Adding 500ml of water into the reaction solution, layering, adjusting the pH of the obtained water layer to 7.0 by using sodium hydroxide water solution, and finally removing water by reduced pressure distillation to obtain NH containing p-toluenesulfonic acid sodium salt₂-CH₂-CH₂-S-CH₂CH₂CH₂-PEG_(30～40)-CH₂CH₂COOH57g。

H¹NMR(400MHz，DMSO-d6)：1.74～1.76(m，2H)，2.39～2.42(m，2H)，2.49～2.53(m， 2H)，3.13～3.14(m，2H)，3.50～3.67(m，163H)，7.82(s，2H)，10.17(s，1H)。

Example 4:

NH₂-CH₂-CH₂-SO₂-CH₂CH₂CH₂-PEG_(20～30)-CH₂COOH (Compound 18, R₁＝H，R₂＝H， m＝1)

1)Boc-NH-CH₂-CH₂-SO₂-CH₂CH₂CH₂-PEG_(20～30)-CH₂COOtBu (Compound 15-1) Synthesis of 138g (100mmol) of Boc-NH-CH₂CH₂-S-CH₂CH₂CH₂-PEG_(20～30)Reaction of-OH (Compound 14) and 40g (1mol) of 60% sodium hydride with 0.5l of anhydrous tetrahydrofuran, cooling to 0 ℃ and adding 117g (600mmol) of tert-butyl bromoacetate, reacting at 0-5 ℃After 72 hours, the reaction mixture was slowly poured into 1L of a saturated ammonium chloride solution, and the resulting mixture was extracted with petroleum ether to remove unreacted t-butyl bromoacetate (600mlx2), and then the product (300mlx3) was extracted with methylene chloride, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure to obtain 69g of a pale yellow viscous liquid.

2)NH₂-CH₂-CH₂-SO₂-CH₂CH₂CH₂-PEG_(20～30)-CH₂COOtBu

58gBoc-NH-CH₂-CH₂-S-CH₂CH₂CH₂-PEG_(20～30)-CH₂COOtBu was dissolved in 250ml of ethyl acetate, and 25ml of an aqueous solution containing 2.4g of sodium tungstate and 20ml of 27% hydrogen peroxide were added, followed by heating to react until the thioether was completely converted into sulfone. The ethyl acetate was distilled off under reduced pressure, the resulting aqueous solution was extracted with dichloromethane (100mlx2), the dichloromethane was washed with water several times, and the dichloromethane was distilled off under reduced pressure to give NH₂-CH₂-CH₂-SO₂-CH₂CH₂CH₂-PEG_(20～30)-CH₂COOtBu53g

3)NH₂-CH₂-CH₂-SO₂-CH₂CH₂CH₂-PEG_(20～30)-CH₂COOH

50g(33mmol)Boc-NH-CH₂CH₂-SO₂-CH₂CH₂CH₂-PEG_(20～30)-CH₂COOtBu was dissolved in 200ml of methylene chloride, and 12.5g (66mmol) of p-toluenesulfonic acid was added to the solution to react at room temperature for 3 hours. Adding 2L of water into the reaction solution, layering, adjusting the pH of the obtained water layer to 7.0 by using sodium hydroxide aqueous solution, and finally removing water by reduced pressure distillation to obtain 43gNH containing p-toluenesulfonic acid sodium salt₂-CH₂-CH₂-SO_2-CH₂CH₂CH₂-PEG_(20～30)-COOH

H¹NMR (400MHz, DMSO-d 6): 1.74-1.76 (m, 2H), 2.54-2.58 (m, 2H), 2.67-2.70 (m, 2H), 2.96-2.98 (m, 2H), 3.45-3.56 (m, 124H), 4.01-4.08 (m, 2H), 7.82(s, 2H). Example 5: compound 19 was synthesized (h ═ 0, n ═ 20 to 30, q ═ 1,R₁＝H，R₂＝H)

1) p-methylene benzoic acid tBu ester-PEG_(20～30)-CH₂-CH₂-CH₂-S-CH₂CH₂-NH-Boc (Compound 39) Synthesis

138g(100mmol)Boc-NH-CH₂CH₂-S-CH₂CH₂CH₂Adding PEG (20-30) -OH (compound 14) and 120g (0.5mol) of sodium hydride into 1 liter of anhydrous tetrahydrofuran, cooling to-5 ℃, dropwise adding 135.5g (0.5mol) of p-bromomethylbenzoic acid tert-butyl ester, reacting at 0-5 ℃ for 40 hours, slowly pouring a reaction solution into 2L of saturated ammonium chloride solution, extracting the obtained mixed solution with petroleum ether to remove the unreacted p-bromomethylbenzoic acid tert-butyl ester (800mlx2), extracting a product (500mlx3) with dichloromethane, drying with anhydrous sodium sulfate, and evaporating to dryness under reduced pressure to obtain 42g of light yellow viscous liquid.

2) Synthesis of Compound 19

30g (19mmol) of tBu ester-PEG of p-methylenebenzoic acid_(20～30)-CH₂CH₂CH₂-S-CH₂CH₂-NH-Boc was dissolved in 50ml of dichloromethane, 50ml of trifluoroacetic acid was added, and the reaction was carried out at room temperature for 1 hour. Evaporating the reaction solution under reduced pressure to dryness, dissolving the obtained concentrate with 60ml of water, adjusting pH to 7.0 with sodium hydroxide water solution, and evaporating under reduced pressure to remove water to obtain 26g p-methylenebenzoic acid-PEG containing trifluoroacetic acid sodium salt_(20～30)-CH₂-CH₂-CH₂-S-CH₂CH₂-NH₂(Compound 19)

H¹NMR(400MHz，DMSO-d6)：1.76～1.78(m，2H)，2.52～2.56(m，2H)，2.67～2.70(m， 2H)，2.96～2.98(m，2H)，3.46～3.62(m，124H)，4.11～4.13(s，2H)，7.47～7.49(d， 2H)，7.78～7.80(d，2H)，7.82(s，2H)，13.92(s，1H)。

Example 6: preparation of Compounds 21, 23

Compound 21(h ═ 1, n ═ 20 to 30, q ═ 1, R₁＝H，R₂＝H)

Compound 23(h ═ 1, n ═ 40 to 60, q ═ 1, R₁＝H，R₂＝H)

The synthesis method was the same as in example 5. The preparation results are as follows:

compound 21, 29g, yield 74%. H¹NMR(400MHz，CDCl₃-d6)：1.76～1.78(m，2H)， 2.52～2.59(t，2H)，2.67～2.70(t，2H)，2.96～2.98(t，2H)，3.46～3.62(m，102H)， 4.33～4.35(s，2H)，4.79～4.81(s，4H)，7.20～7.23(d，4H)，12.25(s，1H)。

Compound 23, 28.7g, yield 76%. H¹NMR(400MHz，CDCl₃-d6)：1.76～1.78(m，2H)， 2.52～2.59(t，2H)，2.67～2.70(t，2H)，2.96～2.98(t，2H)，3.46～3.62(m，102H)， 4.33～4.35(s，2H)，4.79～4.81(s，2H)，5.19～5.21(s，2H)，6.87～6.89(d，2H)， 6.98～7.02(d，2H)，7.45～7.48(d，2H)，8.09.～8.11(d，2H)，12.67(s，1H)。

Example 7: benzyl acetate-PEG_(30～45)-CH₂-CH₂-CH₂-S-CH₂CH₂-NH₂Synthesis of Compound 22 (h ═ 1, n ═ 30 to 45, q ═ 1, R₁＝H，R₂＝H)

1) Tert-butyl acetate benzyl ether-PEG_(30～45)-CH₂-CH₂-CH₂-S-CH₂CH₂-NH-Boc

158g(100mmol)Boc-NH-CH₂CH₂-S-CH₂CH₂CH₂-PEG_(30～45)-OH (Compound 40), 47.6g (200mmol) of tert-butyl acetate p-hydroxybenzyl ether (tBu-CP)₂-OH 33), 78.6g (300mmol) triphenylphosphine in dichloromethane, slowly adding 52g (300mmol) DEAD dropwise, reacting at 50 ℃ for 16 hours, washing the reaction solution with water several times, drying with anhydrous sodium sulfate, purifying with silica gel column to remove triphenylphosphine and triphenylphosphine oxide, and drying the eluate under reduced pressure to obtain 61g tert-butyl acetate benzyl ether-PEG_(30～45)-CH₂-CH₂-CH₂-S-CH₂CH₂-NH-Boc。

2)42g (26.2mmol) of tert-butyl acetate benzyl ether-PEG_(30～45)-CH₂-CH₂-CH₂-S-CH₂CH₂The solution of-NH-Boc was dissolved in 80ml of dichloromethane, and 9.9g (52.4mmol) of p-toluenesulfonic acid was added to the solution to react at room temperature for 6 hours. Evaporating the reaction solution under reduced pressure to dryness, dissolving the obtained concentrate with 60ml of water, adjusting pH to 7.0 with sodium hydroxide water solution, and evaporating under reduced pressure to remove water to obtain 24g of benzyl acetate-PEG containing p-toluenesulfonate_(30～45)-CH₂-CH₂-CH₂-S-CH₂CH₂-NH₂

H¹NMR(400MHz，DMSO-d6)：1.76～1.78(m，2H)，2.52～2.56(m，2H)，2.67～2.70(m， 2H)，2.96～2.98(m，2H)，3.46～3.62(m，124H)，4.50～4.52(s，2H)，4.70～4.72(s， 2H)，6.87～6.89(d，2H)，6.97～6.99(d，2H)，7.82(s，2H)，13.90(s，1H)。

Example 8: preparation of Compounds 20, 22

Compound 20(h ═ 1, n ═ 8 to 12, q ═ 1, R₁＝H，R₂＝H)

The synthesis method was the same as in example 6. The preparation results are as follows:

compound 20, 21.3g, yield 51.6%. H¹NMR(400MHz，CDCl₃-d6)：1.76～1.78(m，2H)， 2.52～2.59(m，6H)，2.67～2.70(m，2H)，2.96～2.98(m，2H)，3.46～3.62(m，44H)， 4.23～4.25(s，2H)，4.70～4.72(t，2H)，6.87～6.89(d，2H)，7.17～7.19(d，2H)，12.23(s， 1H)。

Example 9

H₂N-PEG_(8～12)-CH₂CH₂CH₂-S-CH₂CH[N(CH₃)₂]-COOH (Compound 7, R)₃Two methylamino)

1)HO-PEG_(8～12)-CH₂CH₂CH₂-S-CH₂CH[N(CH₃)₂]-COOBzl Synthesis

120g (about 0.24mol) of APEG500 is dissolved in 7L1, 4 dioxane, 143g (0.6mol) of N, N' -dimethyl cysteine benzyl ester is added under the protection of nitrogen, the temperature is raised to 55 ℃, 9.4g (0.3mol) of AIBN is added, the temperature is stabilized to 55 ℃, the protection of nitrogen is removed, the reaction is carried out for 17 hours at 55 ℃, and the reaction is detected to be complete by MS.

About 400ml of 1, 4-dioxane was removed by rotary evaporation under reduced pressure, 1L of water was added, and petroleum ether (0.5L. times.3) was added to extract a large amount of unreacted N, N' -dimethylcysteine benzyl ester. Adjusting pH of water layer to 4 (small amount of acid) with 40% phosphoric acid, extracting with dichloromethane (5L 2), washing with water, drying dichloromethane solution with anhydrous sodium sulfate, and evaporating under reduced pressure to obtain HO-PEG (8-12) -CH₂CH₂CH₂-S-CH₂CH[N(CH₃)₂]-COOBzl104g。

2)Tos-O-PEG_(8～12)-CH₂CH₂CH₂-S-CH₂CH[N(CH₃)₂]-COOBzl Synthesis

108g (170mmol) of HO-PEG (8-12) -CH2CH2CH2-S-CH2CH- [ N (CH3)2] -COOBzl are dissolved in 600ml of dichloromethane under ice bath condition, and then 45g (0.23mol) of p-toluenesulfonyl chloride is added. 28g (0.28mol) of triethylamine are added in portions. After the addition, the temperature naturally rises to room temperature, and the reaction lasts for 3 hours. The dichloromethane reaction solution was washed with water, dilute phosphoric acid, and saturated brine, respectively, and the dichloromethane layer was dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure to give 94g of a brown oil.

3)N₃-PEG_(8～12)-CH₂CH₂CH₂-S-CH₂CH[N(CH₃)₂]-COOBzl Synthesis

90g (0.14mol) of Tos-O-PEG_(8～12)-CH₂CH₂CH₂-S-CH₂CH[N(CH₃)₂]-COOBzl, 16g (0.25mol) sodium azide were mixed with 600ml DMF, heated to 60 ℃ and stirred overnight. To the reaction mixture was added 1L of water, and the mixture was extracted with ethyl acetate (300 ml. times.7), which was washed with water (300 ml. times.7), and then once with 700ml of saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure to give 68g of a yellow oil.

4)NH₂-PEG_(8～12)-CH₂CH₂CH₂-S-CH₂CH[N(CH₃)₂]-COOBzl Synthesis

75g (0.13mol) N are added under ice-bath conditions₃-PEG_(8～12)-CH₂CH₂CH₂-S-CH₂CH[N(CH₃)₂]-COOBzl is dissolved in 500ml of toluene, 85g (0.32mol) of triphenylphosphine are added in portions, the temperature is naturally raised after the addition, and the mixture is stirred overnight. Most of the triphenylphosphine oxide produced was removed by filtration, 1l of dilute acid water was added, the layers were separated and the aqueous phase was extracted 3 times with ethyl acetate until the triphenylphosphine oxide was completely extracted. The aqueous phase was adjusted to pH 8 with sodium carbonate, extracted with ethyl acetate (500ml × 2), washed with water and saturated brine, dried, and then spin-dried under reduced pressure to give 61g of a pale yellow oil.

5)NH₂-PEG_(8～12)-CH₂CH₂CH₂-S-CH₂CH[N(CH₃)₂]-COOH synthesis

66g(0.11mol)NH₂-PEG_(20～30)-CH₂CH₂CH₂-S-CH₂CH[N(CH₃)₂]-COOBzl was dissolved in 300ml of a methanol solution containing 12g of potassium hydroxide and reacted at room temperature for 3 hours. Adjusting the pH of the reaction solution to 7.0 by 6N hydrochloric acid, and finallyThe solvent is distilled off under reduced pressure to obtain NH₂-PEG_(8～12)-CH₂CH₂CH₂-S-CH₂CH[N(CH₃)₂]-COOH53g。

H¹NMR(400MHz，DMSO-d6)：δ2.11～2.13(m，2H)，2.32(s，6H)，2.47～2.58(m， 4H)，2.64～2.71(m，2H)，3.31～3.70(m，46H)，4.47～4.49(m，1H)，7.93(s，1H)。

Example 10

NH₂-PEG_8-12-CH₂CH₂CH₂-SO₂-CH₂CH₂-CO-AM-polystyrene resin preparation:

1)Fmoc-NH-PEG_8-12-CH₂CH₂CH₂-S-CH₂CH₂-COOH

33.8gFmoc-OSu、50gH₂N-PEG_8-12-CH₂CH₂CH₂-S-CH₂CH₂-COOH, 10.6g sodium carbonate, mixed in 200ml 1: 1 acetone/water mixed solution, and reacting at room temperature for 3 hours. 100ml of water was added, the reaction mixture was extracted with petroleum ether (100ml x3), acidified to pH 2.0 with 6N hydrochloric acid, extracted with ethyl acetate (100ml x2), and the resulting ethyl acetate solution was washed with saturated brine (100ml x2), dried over anhydrous sodium sulfate, and dried under reduced pressure to give 59g of an oil.

2)Fmoc-NH-PEG_8-12-CH₂CH₂CH₂-SO₂-CH₂CH₂-CO-AM-polystyrene resin

70g of aminomethyl-polystyrene resin with a degree of substitution of 0.67 are soaked in 800ml of dichloromethane for 10 minutes, 60g of Fmoc-NH-PEG_8-12-CH₂CH₂CH₂-S-CH₂CH₂-COOH, 25.2g DIC, 27g HOBt and 35ml triethylamine were added to the aminomethyl-polystyrene resin and stirred mechanically for 3 hours, the ninhydrin color detection showed complete blocking of the amine groups on the resin. Filtering, treeThe resin was washed with 800ml dichloromethane and repeated 3 times, the resulting resin was washed with methanol (700ml x2) and then with water ((700ml x2) the resin was mixed with 600ml water first, then 120ml of an aqueous solution containing 16.4g sodium tungstate and 160ml 27% hydrogen peroxide were added, and the mixture was heated at 50 ℃ for 3 hours to react until the thioether was completely converted to sulfone. the aqueous solution was drained, the resin was washed with water (700ml x2), methanol (700ml x2), then DCM (700ml x2), air dried, and vacuum dried to give 91g resin with a degree of substitution of 0.31.

3)NH₂-PEG_8-12-CH₂CH₂CH₂-SO₂-CH₂CH₂-CO-AM-polystyrene resin

40gFmoc-NH-PEG_8-12-CH₂CH₂CH₂-SO₂-CH₂CH₂-CO-AM-resin 1 was soaked in 500ml DMF for 10 min, solvent was drained, Fmoc20 min was removed from the swollen resin with 500ml 20% piperidine/DMF solution, reaction solution was drained, washed with DMF (500ml 2), dichloromethane (500ml 2), methanol (500ml 1), air dried, and finally dried under vacuum to give 38.2g of finished product.

Example 11

NH₂-CH₂CH₂-S-CH₂CH₂CH₂-PEG_30-40-CH₂CH₂CO-MBHA-polystyrene resin

1)Fmoc-NH-CH₂CH₂-S-CH₂CH₂CH₂-PEG_30-40-CH₂CH₂COOH

4.2gFmoc-OSu、14gH₂N-CH₂CH₂-S-CH₂CH₂CH₂-PEG_25-35-CH₂CH₂COOH, 2.0g sodium carbonate, mixed in 80ml 1: 1 tetrahydrofuran/water mixed solution, and reacted at room temperature for 3 hours. 100ml of water were added, the reaction mixture was extracted with petroleum ether (100mlx3), acidified to pH 2.0 with 6N hydrochloric acid and extracted with ethyl acetate (100 mlx)2) The obtained ethyl acetate solution was washed with saturated brine (100ml x2), dried over anhydrous sodium sulfate, and then dried by spin-drying under reduced pressure to obtain 15.5g of an oil.

The nuclear magnetism is shown in figure 1, and the mass spectrum is shown in figure 2.

2)Fmoc-NH-CH₂CH₂-S-CH₂CH₂CH₂-PEG_30-40-CH₂CH₂CO-MBHA-polystyrene resin 50g aminomethyl-polystyrene resin with a degree of substitution of 0.67 was soaked in 500ml dichloromethane for 10 min, 107g Fmoc-NH-CH₂CH₂-S-CH₂CH₂CH₂-PEG_30-40-CH₂CH₂COOH, 25.4g HBTU, 9g HOBt and 11ml DIEA were added to the aminomethyl-polystyrene resin, respectively, and mechanically stirred for 3 hours, the ninhydrin color detection showed that the amine groups on the resin were completely blocked. Filtration, resin with 500ml dichloromethane washing, repeated washing 3 times, the resin, air drying, and room temperature under reduced pressure drying, 89g resin, degree of substitution is 0.44.

3)NH₂-CH₂CH₂-S-CH₂CH₂CH₂-PEG_30-40-CH₂CH₂CO-MBHA-polystyrene resin

50gFmoc-NH-CH₂CH₂-S-CH₂CH₂CH₂-PEG_30-40-CH₂CH₂Soaking CO-MBHA-polystyrene resin in 600ml of DMF for 10 minutes, draining the solvent, removing Fmoc of the swelled resin in 600ml of 20% piperidine/DMF solution for 20 minutes, draining the reaction solution, washing with DMF (500ml of 2), washing with dichloromethane (500ml of 2), washing with methanol (500ml of 1), drying in the air, and finally drying in vacuum to obtain 47g of finished product, wherein the infrared spectrogram is shown in figure 3, and the appearance is shown in figure 4.

Example 12

HOOC-PH-PEG_8-12-CH₂CH₂CH₂-S-CH₂CH₂-NH-CO-polystyrene resin

20g of carboxyl-polystyrene resin with a degree of substitution of 0.76 were soaked for 10 minutes in 200ml of DMF, 11.6g of N, N' -disuccinimidyl carbonate and 8ml of pyridine were added, stirred for 1 hour, filtered with suction and the resin was washed 2 times with DMF. Then 200ml DMF was added and 27g HOOC-PH-PEG was added_8-12-CH₂CH₂CH₂-S-CH₂CH₂-NH₂And 11ml of triethylamine, stirred for 3 hours, filtered, and the resin was washed with 500ml of dichloromethane, and the washing was repeated 3 times, and the resulting resin was air-dried and then dried under reduced pressure at room temperature to obtain 26g of a resin having a degree of substitution of 0.23.

Example 13

HOOC-CH₂CH₂-PEG_30-40-CH₂CH₂CH₂-S-CH₂CH₂-Sulfamide-polystyrene resin

40g of sulfonyl chloride resin with a degree of substitution of 0.72 were soaked in 300ml of dichloromethane for 10 minutes, 92g of HOOC-CH₂CH₂-PEG_30-40-CH₂CH₂CH₂-S-CH₂CH₂-NH₂And 10ml of pyridine were mixed with the resin and stirred at room temperature for 4 hours. Suction filtration, resin with 200ml dichloromethane washing, repeated washing 2 times, then 1M toluene sulfonic acid dichloromethane washing 1 times, the resin, air drying, and room temperature under reduced pressure drying, 78g resin, substitution degree of 0.34.

Example 14

HOOC-CH₂CH₂-SO₂-CH₂CH₂CH₂-PEG_8-12-CH₂CH₂-Urea-polystyrene resin

18gHOOC-CH₂CH₂-S-CH₂CH₂CH₂-PEG_8-12-CH₂CH₂-NH₂25g of an isocyanate resin having a degree of substitution of 0.61 and 5.5ml of pyridine were mixed with 200ml of ethyl acetate and stirred at 60 ℃ for 4 hours.

Suction filtration, resin washing with 200ml ethyl acetate, repeated washing 3 times, and then dichloromethane washing (200ml x2), the resin obtained, methanol washing (200ml x2), then water washing ((200ml x2) resin with first and 100ml water mixing, then adding 30ml containing 4.8g sodium tungstate aqueous solution and 40ml 27% hydrogen peroxide, heating reaction at 50 ℃ for 3 hours until the thioether is completely converted into sulfone, suction drying the aqueous solution, resin washing with water (200ml x2), methanol washing (200ml x2), then DCM washing (200ml x2), drying, vacuum drying to obtain 32g resin, the degree of substitution is 0.44.

Example 15

HOOC-CH₂-PEG_8-12-CH₂CH₂CH₂-S-CH₂CH₂-Thiourea-polystyrene resin

15gHOOC-CH₂-PEG_8-12-CH₂CH₂CH₂-S-CH₂CH₂-NH₂25g of an isothiocyanate resin having a degree of substitution of 0.63 and 5ml of triethylamine were mixed with 200ml of ethyl acetate, and the mixture was stirred at 70 ℃ for 4 hours.

Suction filtration, resin with 200ml ethyl acetate washing, repeated washing 3 times, then dichloromethane washing (200ml x2), the resin, air drying, and room temperature under reduced pressure drying, 29g resin, substitution degree of 0.28.

Example 16

HOOC-CH₂CH₂-PEG_30-40-CH₂CH₂CH₂-S-CH₂CH₂-NH-CO-polystyrene resin

20g of degree of substitution 076 the carboxy-polystyrene resin was soaked in 200ml of DMF for 10 minutes, 11.6g of N, N' -disuccinimidyl carbonate and 8ml of pyridine were added, stirred for 1 hour, filtered with suction and the resin was washed 2 times with DMF. Then 200ml DMF was added followed by 48g HOOC-CH₂CH₂-PEG_30-40-CH₂CH₂CH₂-S-CH₂CH₂-NH₂And 11ml of triethylamine, stirred for 3 hours, filtered, and the resin was washed with 500ml of dichloromethane, and the washing was repeated 3 times, and the resulting resin was air-dried and then dried under reduced pressure at room temperature to obtain 38g of a resin having a degree of substitution of 0.29.

Example 17

Part of other resins

1. Coupling of carboxyl end of sulfur-containing polyethylene glycol with amino-containing resin

50g of an amino resin having a degree of substitution of 0.67mmol/g were weighed out, the procedure and the charge ratio were as in example 9, and the preparation data were as follows:

2. coupling of amine terminal of sulfur-containing polyethylene glycol with carboxyl-containing resin

20g of carboxyl resin with a degree of substitution of 0.76mmol/g were weighed out, the procedure and the charge ratios were as in example 14, and the preparation data were as follows:

3. reacting amine end of sulfur-containing polyethylene glycol with resin containing sulfonyl chloride

40g of sulfonyl chloride resin with a degree of substitution of 0.72 were weighed out, the procedure and the charge ratio were as in example 11, and the preparation data were as follows:

4. reaction of amine terminal of sulfur-containing polyethylene glycol with isocyanate-containing resin

25g of an isocyanate resin having a degree of substitution of 0.61 are weighed out, the procedure and the charge ratios are referred to in example 12, and the preparation data are as follows:

5. reaction of amine terminal of sulfur-containing polyethylene glycol with isothiocyanate-containing resin

25g of an isothiocyanate resin having a degree of substitution of 0.63 was weighed out, the procedure and the charge ratio were each as in example 13, and the preparation data were as follows:

example 18

Rink amide linker-NH-CH₂CH₂-S-CH₂CH₂CH₂-PEG_30-40-CH₂CH₂CO-AM-polystyrene resin

20g of NH having a degree of substitution of 0.42mmol/g₂-CH₂CH₂-S-CH₂CH₂CH₂-PEG_30-40-CH₂CH₂CO-AM-polystyrene resin, 6.8g (12.6mmol) of 4- [ (2, 4-dimethoxyphenyl) (Fmoc-amino) methyl]Phenoxyacetic acid, 4.8g (12.6mmol) HBTU and 5.6ml (33.6mmol) DIEA were mixed in 220ml DMF, shaken at room temperature for 3 hours, added with 2ml acetic anhydride and shaken for another 1 hour. Filtering, washing the resin with DMF(200ml x3) times, DCM washing (200ml x3), air drying, and vacuum drying at room temperature overnight, 23.9gRInk amide resin, substitution degree of 0.38 mmol/g.

Example 19

Wang-NHCH₂CH₂NHCO-CH₂CH₂-SO₂-CH₂CH₂CH₂-PEG_8-12-CH₂CH₂-Urea-polystyrene resin

1)10g of HOOC-CH with a degree of substitution of 0.46mmol/g₂CH₂-SO₂-CH₂CH₂CH₂-PEG_8-12-CH₂CH₂-Urea-polystyrene resin, 1.5g (9.2mmol) Boc-NH-CH₂CH₂-NH₂1.2g (9.2mmol) DIC and 1.2g (6.9mmol) HOBt were mixed in a 250ml single neck flask, 100ml DMF was added and shaken at room temperature for 3 hours. Filtration, resin washed with DMF (100ml x3) times, DCM (100ml x3), air dried and dried under vacuum at room temperature overnight to give 10.5g Boc-HNCH₂ CH₂-NHCO-CH₂CH₂-SO₂-CH₂CH₂CH₂-PEG_8-12-CH₂CH₂-Urea-polystyrene resin.

2) To a flask containing 10.5g Boc-HNCH₂CH₂-NHCO-CH₂CH₂-SO₂-CH₂CH₂CH₂-PEG_8-12-CH₂CH₂A flask of Urea-polystyrene resin was charged with 100ml of 50% TFA/DCM solution and shaken at room temperature for 1 hour. Filtration and washing of the resin with DCM (100 ml. times.3), air drying and further drying in vacuo at room temperature overnight gave 10.4g TFA. H₂NCH₂CH₂-NHCO-CH₂CH₂-SO₂-CH₂CH₂CH₂-PEG_8-12-CH₂CH₂-Urea-polystyrene resin.

3)10.4g TFA.H₂NCH₂CH₂-NHCO-CH₂CH₂-SO₂-CH₂CH₂CH₂-PEG₈-₁₂-CH₂CH₂-Urea-polystyreneThe ester, 1.7g (9.2mmol) of 4- (hydroxymethyl) phenoxyacetic acid, 3.5g (9.2mmol) of HBTU and 3ml (18.4mmol) of DIEA were mixed in 100ml of DMF, and after shaking at room temperature for 3 hours, 2ml of acetic anhydride was added, and further shaking was carried out for 1 hour. Filtration and washing of the resin with DMF (100 ml. times.3) and DCM (100 ml. times.3) followed by air drying and drying under vacuum overnight at room temperature gave 11.2g of Wang resin with a degree of substitution of 0.37 mmol/g.

Example 20

Performance testing of Sulfur-containing polyethylene glycol resins

Synthesizing a polypeptide: sertraline acetate, molecular weight: 3357.9

The following resins were selected for comparative testing:

sulfur-containing polyethylene glycol resin 1: rink amide-NH-PEG_40-60-CH₂CH₂CH₂-S-CH₂CH₂-CO-AM-polystyrene resin (Alanine resin 1 for short)

Sulfur-containing polyethylene glycol resin 2: rink

amide-NH-CH₂CH₂NHCO-CH₂CH₂-SO₂-CH₂CH₂CH₂-PEG_30-50-CH₂CH₂-Urea-polystyrene resin (Alanine resin for short 2)

Rink amide-aminomethyl-polystyrene carrier resin (Rink amide-PS resin for short)

Rink amide-Chemmatrix resin (Chemmatrix for short)

Rink amide-TentaGel resin (TentaGel for short)

The experimental method comprises the following steps: the polypeptide is prepared by adopting an Fmoc solid-phase polypeptide synthesis method, 2g of each resin is weighed, a polypeptide synthesis tube is selected as a reactor, and nitrogen is introduced from the bottom of the synthesis tube for stirring.

The molar ratio of reaction feeding is Fmoc amino acid, HBTU, HOBt, DIEA and resin is 3: 3: 3: 6: the 1 Fmoc removal reagent was 20% piperidine DMF solution.

DMF is used as a coupling solvent and a washing solvent, and the reaction temperature is kept between 22 and 26 ℃.

The polypeptide releasing agent is 81% TFA, 5% phenol, 5% thioanisole, 2.5% EDT, 3% water, 2% dimethylsulfide, 1.5% triisopropylsilane (w/w)

HPLC profiles of crude sertraline acetate from 5 resin syntheses are shown in FIGS. 9-13:

and (4) testing conclusion:

the Alanine resin 1 and the Alanine resin 2 both have the advantages of rapid suction filtration, easy rinsing of the resins, easy judgment of reaction endpoint detection, rapid formation of peptide bonds all the time in the synthesis of the polypeptide, high purity of crude polypeptide, and the like.

Example 21 Synthesis of Ac-Ser-Val-Val-Arg-Thr-OH

Step 1, Fmoc-Thr (tBu) -HMPA-PEG_40-50-S-Carboxyl-PS resin Fmoc removal

Structure of Fmoc-Thr (tBu) -HMPA-PEG-S-Carboxyl-PS resin

14g of Fmoc-Thr (tBu) -HMPA-PEG with a degree of substitution of 0.34mmol/g were weighed out_40-50-S-Carboxyl-PS resin is filled into the polypeptide synthesis tube.

The resin was swollen by soaking in 150ml DMF for 10 minutes. The resin was drained, 120mL of 20% piperidine/DMF solution was added, and the mixture was stirred under nitrogen for 20 minutes. The resin was drained and washed sequentially with solvent, DMF (100ml x2), DCM (100ml x2), methanol (100ml x2), DMF (100ml x 2).

Step 2, synthesizing Fmoc-Arg (pbf) -Thr (tBu) -HMPA-PEG_40-50-S-Carboxyl-PS resin

6.2g (9.52mmol) Fmoc-Arg (Pbf) -OH and 3.1mL (19mmol) DIEA were dissolved in 20mL DMF, 3.6g (9.52mmol) HBTU and 1.3g (9.52mmol) HOBt were dissolved in 20mL DMF and 2 solutions were added to the polypeptide synthesis tube containing the resin of step 1, supplemented with 60mL DMF and stirred with nitrogen for 3 hours. After the coupling is complete, the resin is washed as in step 1.

Preparation of the polypeptide Using step 1 and step 2The method completes the coupling and deprotection of 3 Fmoc-Val-OH and Fmoc-Ser (tBu) -OH according to the amino acid sequence to obtain H-Ser-Val-Val-Val-Thr (tBu) -HMPA-PEG_40-50the-S-Carboxyl-PS resin was finally blocked with acetic anhydride.

Protecting amino acid, condensing agent, organic base, and polypeptide synthetic resin (HMPA-PEG)_40-50-S-Carboxyl-PS resin) in a molar ratio of 3: 3: 6: 1; the polypeptide synthesis solvent is DMF, and the mass ratio of the polypeptide synthesis solvent to the polypeptide synthetic resin is 10-20: 1; the organic base is DIEA, and the condensing agent is HBTU/HOBt.

The polypeptide resin is processed by trifluoroacetic acid cutting solution to obtain 2.48g of crude product of Ac-Ser-Val-Val-Val-Arg-Thr-OH, the purity is 81.8 percent, and the total yield of the crude product is as follows: 81.6 percent. The crude HPLC is shown in FIG. 5 and the crude mass spectrum is shown in FIG. 6.

According to Fmoc-Thr (tBu) -HMPA-PEG_40-50And 4 kinds of sulfur-containing polyethylene glycol resin are selected under the same reaction conditions of the-S-Carboxyl-PS resin to prepare Ac-Ser-Val-Val-Val-Arg-Thr-OH. The resin structure is as follows: Fmoc-Thr (tBu) -HMPA-NHCH₂CH₂NHCO-CH₂CH₂-SO₂-CH₂CH₂CH₂-PEG_8-12-CH ₂CH₂Thiourea-PS resin (abbreviated as A resin)

2.Fmoc-Thr(tBu)-HMPA-NHCH₂CH₂NHCO-CH₂CH₂-S-CH₂CH₂CH₂-PEG_20-30-CH₂ CH₂-NH-CO-PS resin (abbreviation: B resin)

3.Fmoc-Thr(tBu)-HMPA-NHCH₂CH₂CH₂NHCO-CH₂CH₂-SO₂-CH₂CH₂CH₂-PEG_20-30-CH₂CH₂-NH-sulfoamide-PS resin (abbreviation: C resin)

4.Fmoc-Thr(tBu)-HMPA-NHCH₂CH₂CH₂-S-CH₂CH₂CH₂-PEG_20-30-CH₂CH₂CO-A M-PS resin (D resin for short)

The test data for the 4 resins are given in the following table:

name of resin	Degree of substitution (mmol/g)	Batch (g)	Purity of crude product	Total yield of crude product
					A resin	0.35	14	74.3％	82.1％
B resin	0.29	14	77.7％	75.6％
					C resin	0.29	14	82.6％	75.9％
D resin	0.31	14	83.4％	78.8％

Example 22 continuous flow Synthesis of Somalutide

The sequence of the somaglutide polypeptide:

H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala -Lys(AEEAc-AEEAc-γ-Glu-17-carboxyheptadecanoyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH

step 1, Fmoc-Gly-HMPB-PEG-CH₂CH₂-Urea-polystyrene resin Fmoc removal

Fmoc-Gly-HMPB-PEG-CH₂CH₂Structural formula of-Urea-polystyrene resin

Weighing 8g of Fmoc-Gly-HMPB-PEG-CH with the degree of substitution of 0.35mmol/g₂CH₂-Urea-polystyrene resin packing into polypeptide synthesis column 40mL of DMF was pumped into polypeptide synthesis column via circulation conduit and feed line at 40mL/min, circulated for 10 minutes to swell the resin, reagent bottles were switched, 40mL of 1% DBU/5% piperidine in DMF at 40mL/min was pumped via circulation conduit and feed lineThe material pipe is pumped into a polypeptide synthesis column, the circulation is carried out for 8 minutes, and an online monitor shows that the Fmoc is completely removed. The reagent bottle is switched to pump DMF into the polypeptide synthesis column through the circulating liquid guide pipe and the feeding pipeline at the speed of 40mL/min, and the DMF enters the waste liquid collecting bottle from the waste liquid guide pipe until the online monitor shows that the piperidine is washed clean.

Step 2:

H-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-(γ-Gl u-(OtBu))-monoButyl

Octadecanate)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly -HMPB-PEG-CH₂CH₂-Urea-polystyrene resin Synthesis

4.3g (6.6mmol) of Fmoc-Arg (Pbf) -OH is dissolved in 12mL of DMMF, 2.5(6.6mmol) of HBTU and 0.9g (6.6mmol) of HOBt is dissolved in 20mL of DMF, 2 solutions are respectively pumped into a mixer through a feeding pipeline and a feeding pipeline at the speed of 20mL/min and then enter a polypeptide synthesis column for coupling reaction, reaction liquid flows back to a reagent liquid storage bottle and then is pumped into a circulating liquid conduit and the feeding pipeline to enter a polypeptide reaction column for circulating reaction for 12 minutes. After the coupling is finished, the reagent bottle is switched to pump DMF into the polypeptide synthesis column through the circulating liquid guide pipe and the feeding pipeline at the speed of 40mL/min, and the DMF enters the waste liquid collecting bottle from the waste liquid guide pipe until the on-line monitor shows that Fmoc-Arg (Pbf) -OH is washed clean.

The rest amino acid and polypeptide fragments Fmoc-Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-OH and Fmoc-Lys (AEEA-AEEA- (gamma-Glu- (OtBu)) -monoButyl octanate) -OH fragments are coupled according to the amino acid sequence in sequence, and the molar ratio of the protected amino acid to the condensation reagent to the organic base to the polypeptide synthetic resin is 2: 2: 6: 1; the molar ratio of the polypeptide fragment, the condensation reagent, the organic base and the polypeptide synthetic resin is 2: 2: 6: 1; because the protected amino acids are different, the polypeptide synthesis solvent is DMF, and the mass ratio of the polypeptide synthesis solvent to the polypeptide synthesis resin is 10-20: 1; the organic base is DIEA, and the condensing agent is HBTU/HOBt. The flow rate of the liquid for protecting the activation of the amino acid is controlled between 30mL/min and 50 mL/min.

Fmoc removal conditions: each 40mL of 1% DBU/5% piperidine solution in DMF was controlled at a flow rate of 40mL/min, and the flow rate for washing the resin after Fmoc removal was controlled at 15 mmL/min.

And step 3:

Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(Psi(me.me)Pro)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(A EEA-AEEA-(γ-Glu-(OtBu))-monoButyl Octadecanate)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Boc)₂-Gly-Arg(Boc)₂- Gly-HMPB-CH₂CH₂-Urea-polystyrene resin (Boc-Somalipeptide-HMPB-PEG-CH)₂CH₂-Urea-polystyrene resin)

Refer to the data and operation method of step 2. 2.5g (6.6mmol) HBTU, 0.9g (6.6mmol) HOBt in 15mL DMMF, DIEA21.6mL (131.2mmol) and 82.4g (65.6mmol) Fmoc-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Ser (Psi (me. me) Pro) -OH fragment 1 in 25mL DMF, pumping the 2 solutions into the mixer through the feeding line and the feeding line at the speed of 20mL/min and 40mL/min respectively, entering the polypeptide synthesis column for coupling reaction, and circularly pumping until the coupling is finished. Fmoc removal conditions were performed with reference to step 2.

Coupling Boc-His (Boc) -Aib-Glu (OtBu) -Gly-OH to a polypeptide resin by the steps of: 2.5g (6.6mmol) of HBTU, 0.9g (6.6mmol) of HOBt are dissolved in 20mL of DMMF, Boc-His (Boc) -Aib-Glu (OtBu) -Gly-OH4.6g (6.6mmol) and DIEA2.2mL (13.1mmol) are dissolved in 25mL of DMF, 2 solutions are respectively pumped into a mixer through a feeding pipeline and a feeding pipeline at the speed of 20mL/min and 40mL/min, enter a polypeptide synthesis column for coupling reaction, are circularly pumped until the coupling is finished, and 25.9g of polypeptide resin is obtained after washing and vacuum drying.

And 4, step 4: Boc-Somalilutide-HMPB-PEG-CH₂CH₂-cutting and precipitating the Urea-polystyrene resin by using a conventional polypeptide preparation method to obtain a crude product of the somaglutide, and drying the crude product of the somaglutide in vacuum at room temperature to obtain an off-white solid of 8.3g, the purity of 89.44 percent, and the total yield of the crude product: 71.5 percent. The crude somalutide HPLC is shown in fig. 7 and the crude mass spectrum is shown in fig. 8.

Example 23 continuous flow Synthesis of Somalutide with Long-chain Sulfur-containing polyethylene glycol resin

Synthetic resin adopted

Fmoc-Gly-HMPB-NHCH₂CH₂NHCO-CH₂CH₂-PEG_60-80-CH₂CH₂-SO₂-CH₂CH₂-NH-CO-PS resin:

the synthesis method and the feeding proportion are the same as in example 19, 8g of synthetic resin is weighed, the degree of substitution is 0.22mmol/g, and finally 5.3g of crude product is obtained, the purity of the crude product is 89.37%, and the total yield of the crude product is as follows: 73.2 percent.

Experiments prove that compared with polyethylene glycol with molecular weight of about 2000 containing sulfur, the long PEG chain has less obvious effect of promoting the synthesis of long peptide.

Comparative example 1 Synthesis of Ac-Ser-Val-Val-Val-Arg-Thr-OH by Wang-resin

Synthetic resin and amount:

14g of Fmoc-Thr (tBu) -Wang-PS resin having a degree of substitution of 0.39mmol/g (Wang-PS resin parameters: degree of substitution: 0.7mmol/g, degree of crosslinking: 1%, particle size: 100-200 mesh)

Fmoc protected amino acids, condensation reagents, organic bases were as in example 21.

The stirring mode is that nitrogen is introduced from the bottom of the synthesis tube for stirring.

The molar ratio of reaction feeding is Fmoc amino acid, HBTU, HOBt, DIEA and resin is 3: 3: 3: 6: the 1 Fmoc removal reagent was 20% piperidine DMF solution.

Coupling and defmoc washes were identical to example 21, with the reaction temperature maintained at 22-26 ℃.

The polypeptide releasing agent is identical to example 21.

Treating the polypeptide resin with a trifluoroacetic acid cutting solution to obtain 1.67g of crude product of Ac-Ser-Val-Val-Val-Arg-Thr-OH, wherein the purity is 71.9 percent, and the total yield of the crude product is as follows: 60.3 percent.

The comparison experiment shows that the yield and the purity of the crude peptide of the sulfur-containing polyethylene glycol resin are superior to those of Wang-PS resin, and the thiol bond on the polyethylene glycol is indirectly proved to be stable in the strong alkaline environment of 20 percent piperidine.

Comparative example 2 Wang-resin Synthesis of Somalutide

Synthetic resin and amount:

8g of Fmoc-Gly-Wang-PS resin having a degree of substitution of 0.35mmol/g (PS resin parameters: degree of substitution: 0.7mmol/g, degree of crosslinking: 1%, particle size: 100-mesh 200.)

The preparation of the somaglutide is completed according to the conventional solid-phase polypeptide synthesis process, and the specific conditions are as follows:

fmoc protected amino acids, polypeptide fragments, condensation reagents, and organic bases were selected in accordance with example 22.

The stirring mode is that nitrogen is introduced from the bottom of the synthesis tube for stirring.

The molar ratio of reaction feeding is Fmoc amino acid, HBTU, HOBt, DIEA and resin is 3: 3: 3: 6: 1 Fmoc removal reaction was performed with 20% piperidine/DMF for 20 min.

The coupling time is based on the end point of the resin coupling detection, and is usually 2 to 4 hours.

The coupling and the Fmoc removal of the amino acid are carried out at the temperature of 22-26 ℃.

The polypeptide release agent is identical to example 22.

The polypeptide resin is processed by a cutting solution to obtain 5.9g of crude product of the Somalou peptide, the purity is 10.3 percent, and the total yield of the crude product is as follows: 51.7 percent.

The HMPB-PEG-CH can be known through comparative experiments₂CH₂The yield and purity of crude peptide of the-Urea-polystyrene resin are respectively 19.8% and 79.1% higher than those of Wang-PS resin. The long peptide not only has stable chemical properties, but also is particularly suitable for the synthesis of the long peptide, and can replace the Wang-PS resin which is popular in the market at present to prepare the long peptide.

Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the idea of the invention, also features in the above embodiments or in different embodiments may be combined, steps may be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.