JP2000290285A - Novel linker and solid-phase synthesis of glycopeptides using it - Google Patents

Abstract

(57) [Summary] [Problem] To adjust peptide C-terminal extension on a solid phase, all remain on the resin at the time of cleavage, no contamination of products, and quantification of the binding efficiency is possible. An easy linker is formed, and solid phase synthesis is performed using the linker. SOLUTION: General formula (I) (R ¹ , R ² , R ³ and R ⁴ represent an optionally substituted alkyl group, alkenyl group, cycloalkyl group,
A silyl halide compound represented by a cycloalkenyl group or a phenyl group, wherein X is a halogen atom, thereby forming a silyl linker.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel linker and a method for solid-phase synthesis of a glycopeptide using the same. More specifically, the invention of this application relates to a p-nitrophenylcarbinylsilyl halide compound which forms a novel linker having a large steric hindrance and a bulky substituent, a method for producing the same, and a method for preparing the same with a glycopeptide or a solid peptide. The present invention relates to a method used as a linker for phase synthesis.

[0002]

2. Description of the Related Art In a general solid-phase peptide synthesis method, a carboxyl group of an amino acid serving as a C-terminal of a peptide is connected to a linker fixed on a solid phase in advance by an ester bond, and then the N-terminal direction is required. A method has been used in which a number of amino acid units are sequentially linked to extend a peptide chain, and finally, an oligopeptide is cut out from a solid phase together with removal of a protecting group of an amino acid side chain. This cutting operation is generally performed under strong acidic conditions such as trifluoroacetic acid and hydrogen fluoride.

[0003] However, there is a problem in that the conventional acid-cutting operation in a general method requires careful handling since a strong acid is used, which is not practical. Thus, linkers that can be cleaved under weaker acidic conditions have also been developed. In this case, it becomes possible to take out the oligopeptide while keeping the side chain protecting group. However, it is important to carefully consider the setting of this weak acid condition. Although a resin in which various amino acids are bound to a solid phase via a residue linker for these techniques has already been marketed, the problem that the operation is not always easy has not been solved.

Under such circumstances, linkers that do not require acid conditions when cleaving the solid phase and the synthetic peptide have been developed, and one of them is to utilize the affinity between a fluorine anion and silicon. Several silicon-containing linkers that lead to the cleavage reaction are known (DGMullen, G. Barany,
J. Org. Chem. 1988, 53, 5240). However, those developed for peptide synthesis only use a silicon leaving group as a part of the substituent in the linker that connects the carboxyl group of the amino acid in an ester form.

[0005] In recent years, attempts have been made to increase the efficiency of the synthesis of sugar chains using solid-phase synthesis, or to develop combinatorial chemistry. Immobilizing the sugar residue by a silicon-oxygen bond between the hydroxyl group of the sugar residue and
It is known that glycan elongation is followed by excision with a fluorine anion (JTRandolph, KFMcClure, SJDa
nishefsky, J. Am. Chem. Soc, 1995, 117, 5712). However,
In this case, it is difficult to directly detect and estimate the number of moles of the sugar residue immobilized on the resin. Glycoprotein research is thriving, and the structure and function of glycoproteins composed of proteins and sugar chains are expected to be elucidated, and the application of glycoprotein molecules to pharmaceuticals and agricultural chemicals is expected. There is also a need to establish a technology for precise synthesis. For this purpose, a glycopeptide, which is a small fragment that should be a component of glycoprotein molecule construction, is efficiently synthesized by protecting the functional groups on its side chains such as sugar chain hydroxyl groups, and then efficiently synthesized by block condensation or the like. Techniques for assembling into large molecules by technology are considered practical.

[0006] However, the above-mentioned silyl linkers developed for the conventional peptide synthesis have the advantage that they can be cleaved out without depending on acidic conditions, but their preparation is complicated, and the cleaving of the linker is difficult at the time of cleaving. Some of them have a disadvantage that some of them are mixed into the target product as by-products. In addition, in order to use these linkers, it is usually necessary to fix the required intermediates after binding them to the solid-phase synthesis support in advance, but there has been no definitive method for verifying the immobilization.

Accordingly, an object of the present invention is to provide an excellent linker capable of overcoming these conventional problems in a solid phase synthesis reaction, and to use it as a linker for solid phase synthesis of a glycopeptide or a peptide. I have.

[0008]

Means for Solving the Problems The invention of the present application is to solve the above-mentioned problems.

[0009]

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(Wherein R ¹ , R ² , R ³ and R ⁴ are the same or different and each may have an alkyl, alkenyl, cycloalkyl, cycloalkenyl or aryl group which may have a substituent) And X represents a halogen atom). In addition, the invention of this application relates to the above compound, secondly, R ¹ and R ²
At least one of which is a methyl group,
A compound in which at least one of R ³ and R ⁴ is a phenyl group, a fourth compound in which X is a chlorine atom, and a fifth compound in which R ¹ , R ² , R ³ and R ⁴ are methyl groups To provide a compound wherein X is a chlorine atom.

Sixth, the invention of the present application relates to the general formula (I
I)

[0012]

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(Wherein R ¹ , R ² , R ³ and R ⁴ are the same or different, and each may have an alkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group or an aryl group which may have a substituent. Wherein X represents a halogen atom), wherein the phenylcarbylsilyl halide compound is hydrolyzed, nitrated, and then halogenated. A method for producing a compound is provided.

Seventh, the invention of this application relates to a method of immobilizing a sugar chain or an amino acid on a solid-phase synthesis support via a silyl linker, synthesizing a glycopeptide or peptide, and then cleaving a silicon-oxygen bond. In the method for removing a sugar or amino acid, the alcoholic hydroxyl group of the sugar or amino acid is protected by the p-nitrophenylcarbinylsilyl halide compound of the first invention, and the nitro group is reduced and then condensed with a dicarboxylic anhydride. The present invention provides a method for synthesizing a glycopeptide or an oligopeptide, wherein a dicarboxylic acid monoamide is used as a silyl linker.

Further, the invention of this application relates to the above synthesis method. Eighth, a method using succinic anhydride or glutaric anhydride as a dicarboxylic anhydride is described in ninth.
A method of cleaving a silicon-oxygen bond with a fluorine anion;
Methods for breaking silicon-oxygen bonds with 8-crown-6 or acetic acid are also provided.

That is, the invention of this application as described above is derived from the study by the inventors of a synthetic element which can be immobilized on a solid-phase synthetic support via a sugar chain, and the silyl ether is used for the synthesis. As a result of studying the structural part that becomes a linker to synthesize the glycopeptide unit containing and linking it to the solid phase through the substituent of the silyl compound, and studying the solid phase synthesis method of glycopeptides, etc., the steric hindrance is large. A novel p-nitrophenylcarbinylsilyl halide compound having a bulky substituent solves the above problems and has been completed based on the finding that it is useful as a linker for solid-phase synthesis of glycopeptides or peptides. It is a thing.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION The invention of this application has the features as described above, and the embodiments will be described below. First, as for the p-nitrophenylcarbylsilyl halide compound represented by the general formula (I) provided in the present invention, R ¹ , R ² , R
³ and R ⁴ may be an alkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, or an aryl group as a hydrocarbon group, or various kinds of these substituted hydrocarbons. Has 1 carbon atom
About 8 are suitable, and among them, methyl group, ethyl group, propyl group, isopropyl group, n-butyl group,
Those selected from lower alkyl groups such as isobutyl group and pentyl group and aryl groups such as phenyl group and tolyl group are exemplified as suitable ones.

X is a halogen atom.
From the viewpoints of synthesis and handleability, it is appropriate to use chlorine atoms. For example, specific examples of the compound of the formula (I) include (p-nitrophenyldimethylcarbyl) dimethylsilyl chloride, (p-nitrophenyldimethylcarbyl) methylethylsilyl chloride,
(P-nitrophenyldimethylcarbinyl) divinylsilyl chloride, (p-nitrophenyldimethylcarbinyl) diphenylsilyl chloride, (p-nitrophenyldimethylcarbinyl) methylphenylsilyl chloride,
(P-nitrophenyldimethylcarbinyl) methyltolylsilyl chloride, (p-nitrophenyldiethylcarbinyl) dimethylsilylchloride, (p-nitrophenylphenylmethylcarbinyl) dimethylsilyl chloride, (p-nitrophenylmethylpropylcarbinyl )
Dimethylsilyl chloride and the like.

Among them, (p-nitrophenyldimethylcarbyl) dimethylsilyl chloride, (p-nitrophenyldimethylcarbyl) methylethylsilyl chloride and (p-nitrophenyldimethylcarbyl) diethylsilyl chloride are suitable. Is exemplified. For example, the p-nitrophenylcarbylsilyl halide compound represented by the general formula (I) of the present invention as described above is a novel compound useful as a compound for forming a silyl ether type linker.

In the present invention, the p-nitrophenylcarbylsilyl halide compound is obtained from the above-mentioned phenylcarbylsilyl halide compound represented by the general formula (II) as a raw material, which is hydrolyzed, nitrated and halogenated. It is produced by reacting. This method is represented by the following general formula (III) as a reaction formula.

[0021]

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The phenylcarbylsilyl halide compound as a raw material includes (phenyldimethylcarbyl)
Dimethylsilyl chloride, (phenyldimethylcarbyl) methylethylsilyl chloride, (phenyldimethylcarbyl) divinylsilyl chloride, (phenyldimethylcarbyl) diphenylsilyl chloride, (phenyldimethylcarbyl) methylphenylsilyl chloride, (phenyldimethylcarbyl) Examples thereof include (vinyl) methyltolylsilyl chloride, (phenyldiethylcarbinyl) dimethylsilyl chloride, (diphenylmethylcarbinyl) dimethylsilyl chloride, and (phenylmethylpropylcarbinyl) dimethylsilyl chloride. Preferably, (phenyldimethylcarbyl) dimethylsilyl chloride, (phenyldimethylcarbyl) methylethylsilyl chloride and (phenyldimethylcarbyl) diethylsilyl chloride are used.

For the hydrolysis, nitration, and halogenation of the phenylcarbylsilyl halide compound as a raw material, a conventional chemical reaction method is appropriately employed. For example, in the hydrolysis, a hydrolysis method using an aqueous alkali solution is used. For example, a nitration method using ammonium nitrate and trifluoroacetic anhydride is exemplified, and a halogenation method using a oxalyl halide is exemplified for halogenation.

In the present invention, a silyl linker for solid-phase synthesis of glycopeptides or oligopeptides is provided by the p-nitrophenylcarbylsilyl halide compound represented by the general formula (I). The silyl linker is, for example,

[0025]

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(R ¹ , R ² , R ³ and R ⁴ are the same as described above, A represents a residue at the alcoholic hydroxyl group of a sugar or amino acid, and B represents a residue of a dicarboxylic acid. ). For example, as described above, the silyl linker of the present invention is obtained by reacting a p-nitrophenylcarbylsilyl halide compound represented by the above general formula (I) with an alcoholic hydroxyl group of a sugar or an amino acid to form a protected structure by a silyl-ether bond. And then reducing the nitro group to an amino group and subjecting it to a condensation reaction with a dicarboxylic anhydride to form a dicarboxylic acid monoamide.

More specifically, for example, a sugar chain or an amino acid having an organic compound hydroxyl group is used as a starting material or an intermediate as a starting material or an intermediate used in solid phase synthesis, and The corresponding silyl ether can be easily obtained by reacting nitrophenylcarbinyl halide in a solution in the presence of a deoxidizing agent. This reaction can be carried out under the conditions of ordinary silylation reaction, but it is desirable to carry out the reaction in the presence of a reaction accelerator such as sodium iodide. The obtained silyl ether is chemically stable, can be easily purified by a method such as chromatography, and the structure of the product can be easily confirmed by NMR or the like.

Next, the nitro group of the obtained silyl ether can be reduced under extremely mild conditions, for example, in the presence of zinc powder, and the corresponding aniline derivative can be obtained. This reduction reaction proceeds without being affected by the structure of the substrate, and the desired aniline derivative can be obtained. Then, when the obtained aniline derivative is reacted with a cyclic acid anhydride, for example, succinic anhydride or glutaric anhydride, a monocarboxylic acid having an amide bond (dicarboxylic acid monoamide) can be easily obtained. Of course, the acid anhydride may be of various types. In this way, the above silyl ether can be produced.

The silyl ether can form an amide bond by reacting with an amino group of an amino acid immobilized on a suitable solid phase synthesis carrier using a condensing agent.
The linker having a silyl ether type condensation obtained in the present invention binds to the solid phase using the hydroxyl group of serine or threonine, which has not been performed in the conventional solid phase peptide synthesis, and repeats the condensation to extend the peptide chain. After that, it becomes possible to specifically take out the synthetic peptide without damaging the protecting groups disposed on the N-terminal amino group, the C-terminal carboxyl group and the functional group of the side chain. Further, not only serine and threonine but also one of a large number of hydroxyl groups present in a sugar chain can be used as a binding point to the solid phase. According to the solid-phase synthesis method using the silyl linker of the present invention, both ends of the peptide main chain can be used as reaction points for peptide synthesis by selectively removing the respective protecting groups. A peptide C-terminal extension on a solid phase that has not been performed can also be performed.

The silyl linker according to the present invention has the advantages that it can be easily prepared, and that it remains on the resin at the time of cleavage, and that the separation from the target product is only a filtration operation. In addition, by binding to a commercially available amino-type resin (or amino acid-bonded resin) having a known titer,
The numerical value of the binding efficiency can be easily known by the ninhydrin method, the Fmoc quantification method, or the like.

In the present invention, no particular method is required for the quantification of the immobilization reaction. For example, the immobilization reaction can be detected very easily and with high sensitivity by a test for detecting residual amino groups using ninhydrin. And in this invention,
The solid phase synthesis reaction can be performed without any special reaction conditions, and as described above, the peptide chain can be extended to one or both of the N-terminal and the C-terminal acid terminal.

After completion of the solid phase synthesis, silicon
Although the protected glycopeptide or oligopeptide can be taken out by cleaving the oxygen bond, it can be released from the solid phase under ordinary conditions for deprotection of the silyl protecting group. When the reaction is carried out with a compound generating fluorine ions, for example, cesium fluoride, for example, in the presence or absence of acetic acid or crown ether, the cleavage can be extremely easily performed quantitatively.

[0033]

EXAMPLES Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples. (Example 1) <Production of (p-nitrophenyldimethylcarbyl) dimethylsilyl chloride> 26.57 g of (phenyldimethylcarbyl) dimethylsilyl chloride was dissolved in 75 ml of diethyl ether, and 8.4 g of potassium hydroxide was dissolved. 1: 4 (v / v) of water-methanol
100 ml of a mixed solvent was gradually added and stirred. The mixed solution was stirred at room temperature for 20 hours, separated, extracted twice with diethyl ether, washed and dried with an ether solution, and the solvent was distilled off under reduced pressure to obtain 22.89 g of (phenyldimethylcarbylvinyl) as a colorless oily substance. ) Dimethylsilanol was obtained.

5.00 g of this silanol was dissolved in 35 ml of acetonitrile, 2.60 g of ammonium nitrate was added, the suspension was adjusted, and 5.8 ml of trifluoroacetic anhydride was added little by little with stirring. After the addition,
After 50 minutes and 60 minutes, 2.0 ml each of trifluoroacetic anhydride was added, and the mixture was stirred at room temperature overnight. After adding water to the obtained reaction solution and extracting twice with ethyl acetate, the organic phase was washed and dried, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel chromatography to obtain a colorless crystalline compound.
99 g of (p-nitrophenyldimethylcarbyl) dimethylsilanol were obtained.

Next, 3.58 g of this (p-nitrophenyldimethylcarbyl) dimethylsilanol was dissolved in 50 ml of methylene chloride, and 1.50 ml of oxalyl chloride was dissolved.
Was added, 1 drop of dimethylformamide was added, and the mixture was stirred at room temperature for 1 day. The reaction solution was concentrated under reduced pressure, and the obtained crystalline residue was recrystallized from hexane to give 3.56 as colorless crystals.
g of (p-nitrophenyldimethylcarbinyl) dimethylsilyl chloride (mp. 114.5-117 ° C (hexane)) was obtained. (Example 2) The same reaction was carried out in the same manner as in Example 1 except that (phenyldimethylcarbyl) dimethylsilyl chloride was replaced with (phenyldimethylcarbyl) methylethylsilyl chloride. Dimethylcarbinyl) methylethylsilyl chloride was obtained.

Further, by performing the same method,
The following compound was obtained. (P-nitrophenyldimethylcarbinyl) divinylsilyl chloride, (p-nitrophenyldimethylcarbinyl) diphenylsilyl chloride, (p-nitrophenyldimethylcarbinyl) methylphenylsilyl chloride, (p-nitrophenyldimethylcarbinyl) methyl Tolylsilyl chloride, (p-nitrophenyldiethylcarbinyl) dimethylsilylchloride, (p-nitrophenyl-phenylmethylcarbinyl) dimethylsilylchloride, (p-nitrophenylmethylpropylcarbinyl) dimethylsilyl chloride (Example 3) <Synyl Linker Synthesis> A silyl linker was synthesized according to the following reaction formula.

[0037]

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First step: Production of silyl ether <N- (9-fluorenylmethoxycarbonyl) [O-
(4-nitrophenyldimethylcarbyl) dimethylsilyl] -L-serine allyl ester> N- (9-fluorenylmethoxycarbonyl) -L-serine allyl ester (500 mg, 1.36 mmol) and (p-nitrophenyldimethyl) Dimethylformamide (20 ml) was added to (carbinyl) dimethylsilyl chloride (420 mg, 1.63 mmol) and sodium iodide (610 mg, 4.06 mmol) and reacted at room temperature for 15 minutes. Further, N-methylmorpholine (179 μl, 1.6
After adding 2 mmol), the mixture was reacted for 20 minutes. The reaction solution was distributed between ether and saturated aqueous sodium hydrogen carbonate, and the organic layer was washed with saturated saline and dried over anhydrous sodium sulfate. The residue obtained by concentration under reduced pressure was purified by silica gel column chromatography (SiO ₂ 60 g, hexane-EtOAc ₂ : 1) to give 775 mg (97%) of the title compound as a colorless oil.
%)Obtained.

Step 2: Preparation of aniline derivative <N- (9-fluorenylmethoxycarbonyl) [O-
(4-aminophenyldimethylcarbyl) dimethylsilyl] -L-serine allyl ester> N- (9-fluorenylmethoxycarbonyl) [O- (4-nitrophenyldimethylcarbyl) dimethylsilyl] -L-serine allyl Ester (780 mg, 1.32 mmol)
Was dissolved in tetrahydrofuran (20 ml), zinc dust (5.00 g, excess) and acetic acid (1 ml) were added and reacted for 45 minutes. The reaction solution was diluted with ethyl acetate, filtered through a celite layer, washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine, and dried over anhydrous sodium sulfate. The residue obtained by concentration under reduced pressure was subjected to silica gel column chromatography (SiO ₂ 60 g, hexane-EtOAc 1: 1).
650 mg (8%) of the title compound as a colorless oil.
8%).

Third step: Preparation of amidocarboxylic acid <N- (9-fluorenylmethoxycarbonyl) ｛O-
[(4-Succinic acid monoamidophenyl) dimethylcarbyl] dimethylsilyl {-L-serine allyl ester> 9-fluorenylmethoxycarbonyl [O- (4-aminophenyldimethylcarbyl) dimethylsilyl] -L
-Serine allyl ester (650 mg, 1.16 mm
ol) and succinic anhydride (120 mg, 1.20 mmol)
l) and N-methylmorpholine (111 μl, 0.89 m
mol)) and reacted for 4 hours. Concentrate the reaction mixture to Bi
Obeads SX-3 (100 g) was spread on a column, and purified by elution with a mixed solvent of toluene-ethyl acetate (1: 1) to obtain 760 mg (99%) of the title compound. (Example 4) <Introduction into resin> The silyl linker obtained in Example 3 was introduced into a resin and immobilized by the following formula.

[0041]

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That is, N- (9-fluorenylmethoxycarbonyl) {O-[(4-amidophenylphenyl succinate) dimethylcarbyl] dimethylsilyl} -L-
Serine allyl ester (250mg, 0.38mmo
l) in N-methylmorpholine (3.0 ml)
-Benzotriazo-1-yl-1-N, N, N ', N'
-Tetramethyluronium hexafluorophosphate (HBTU: 288 mg, 0.76 mmol) and 0.5
M 1-hydroxybenzotriazole (HOBt: dimethylformamide solution, 0.76 ml, 0.38 mm
ol) and 2M diisopropylethylamine (dimethylformamide solution, 0.19 ml, 0.38 mmol
l) was added and reacted at room temperature for 1 hour and 20 minutes. Then, a glycine HMP resin (ABI, 0.95 mmol / g,
271 mg, 0.253 mmol) and stirred and mixed overnight. The obtained resin was separated by filtration and washed successively with N-methylmorpholine, 2-propanol, methylene chloride and diethyl ether to obtain 439 mg (102%: calculated from weight) of the resin. (Example 5) <Peptide solid phase synthesis> Peptide solid phase synthesis was performed following Example 4 according to the following reaction formula.

[0043]

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Removal of 9-Fluorenylmethoxycarbonyl group (Fmoc) (1) N- (9-Fluorenylmethoxycarbonyl) serine (O-linker-glycine HMP resin) allyl ester (0.59 mmol / g, 151 mg) , 0.089mm
ol) with N-methylmorpholine-piperidine (1: 1 v
/ V, 3.0 ml) and stirred and mixed for 1 hour and 40 minutes. The resulting resin was filtered off and N-methylmorpholine, 2
-Washed sequentially with propanol, methylene chloride and diethyl ether to obtain 134 mg of resin.

N- (9-fluorenylmethoxycarbonyl) [O- (2-acetamide-3,4,6-tri-O-
Introduction of acetyl-2-deoxy-α-D-galactopyranosyl] threonine (2) N- (9-fluorenylmethoxycarbonyl) [O-
(2-Acetamide-3,4,6-tri-O-acetyl-
2-Deoxy-α-D-galactopyranosyl] -L-threonine (33 mg, 0.0492 mmol) in N-methylmorpholine (1.0 ml) was added to a solution of O-benzotriazo-1-yl-1-N, N , N ', N'-tetramethyluronium hexafluorophosphate (37.3 m
g, 0.098 mmol), 1-hydroxybenzotriazole (66 mg, 0.49 mmol) and 2M diisopropylethylamine (dimethylformamide solution, 0.49 μl, 0.098 mmol) were added and reacted for 35 minutes. Then, serine (O-linker-glycine HMP resin) allyl ester (0.68 mmol / g,
66 mg, 0.0447 mmol) and stirred and mixed overnight. The resin obtained is filtered off and N-methylmorpholine,
Washed sequentially with 2-propanol, methylene chloride and diethyl ether, 88.5 mg (96%: calculated from weight)
Resin was obtained.

Removal of 9-fluorenylmethoxycarbonyl group (Fmoc) (3) N- (9-fluorenylmethoxycarbonyl) [O-
(2-Acetamide-3,4,6-tri-O-acetyl-
2-deoxy-α-D-galactopyranosyl] -L-threonyl-L-serine (O-linker-glycine HMP
Resin) allyl ester (0.47 mmol / g, 84.
3 mg, 0.0425 mmol) in N-methylmorpholine-piperidine (1: 1 v / v, 2.0 ml),
The mixture was stirred and mixed for 1 hour and 20 minutes. The resin obtained is filtered off and N
The residue was washed sequentially with -methylmorpholine, 2-propanol, methylene chloride and diethyl ether to obtain 80.4 mg of a resin.

N- (9-fluorenylmethoxycarbonyl) [O- (2-acetamide-3,4,6-tri-O-
Introduction of acetyl-2-deoxy-α-D-galactopyranosyl] threonine (4) N- (9-fluorenylmethoxycarbonyl) [O-
(2-Acetamide-3,4,6-tri-O-acetyl-
2-Deoxy-α-D-galactopyranosyl] -L-threonine (33 mg, 0.0492 mmol) in N-methylmorpholine (1.0 ml) was added to a solution of O-benzotriazo-1-yl-1-N, N , N ', N'-tetramethyluronium hexafluorophosphate (37.3 m
g, 0.098 mmol), 1-hydroxybenzotriazole (66 mg, 0.49 mmol) and 2M diisopropylethylamine (dimethylformamide solution, 0.49 μl, 0.098 mmol) were added and reacted for 35 minutes. Then, [O- (2-acetamide-3,
4,6-tri-O-acetyl-2-deoxy-α-D-
Galactopyranosyl] -L-threonyl-L-serine (O-linker-glycine HMP resin) allyl ester (0.53 mmol / g, 80.4 mg, 0.0425)
mmol) and stirred overnight. By the same treatment as in the above (2), 112 mg (102%: calculated from the weight) of the resin was obtained.

Cleavage of glycopeptide from resin (5) N- (9-fluorenylmethoxycarbonyl) [O-
(2-Acetamide-3,4,6-tri-O-acetyl-
2-deoxy-α-D-galactopyranosyl] -L-threonyl-LN- (9-fluorenylmethoxycarbonyl) [O- (2-acetamido-3,4,6-tri-O
-Acetyl-2-deoxy-α-D-galactopyranosyl] -L-threonyl-L-serine (O-linker-glycine HMP resin) allyl ester (0.39 mmol
/ G, 77.4 mg, 0.0302 mmol) in 0.2
mol / l cesium fluoride-1.0 mol / l acetic acid DM
The resultant was suspended in the F solution and stirred and mixed overnight. The obtained resin was separated by filtration, washed with N-methylmorpholine, and then sequentially washed with 2-propanol, methylene chloride and diethyl ether. The washings of 2-propanol, methylene chloride and diethyl ether were concentrated, combined with the washings of N-methylmorpholine, diluted with ethyl acetate, and partitioned with water. The aqueous layer was further extracted with ethyl acetate three times, and the combined organic layers were washed with saturated saline, dried over anhydrous sodium sulfate, and concentrated. The residue was separated by preparative thin-layer chromatography (1 mm
layer, purified by CHCl ₃ / MeOH 9: 1),
(9-fluorenylmethoxycarbonyl) [O- (2-
Acetamide-3,4,6-tri-O-acetyl-2-deoxy-α-D-galactopyranosyl] -L-threonyl-N- (9-fluorenylmethoxycarbonyl) [O
-(2-Acetamide-3,4,6-tri-O-acetyl-2-deoxy-α-D-galactopyranosyl] -L-
27.2 m of threonyl-L-serine allyl ester
g (73%).

[0049]

The (p-nitrophenylcarbyl) silyl halide compound of the present invention is a compound useful for forming a linker for solid-phase synthesis, and quantitatively binds to an aqueous alcoholic solution of a sugar or amino acid to form a silyl ether. A bond can be formed and the required compound can be immobilized on the solid phase through several further steps. Solid-phase organic synthesis using this linker is mechanically advanced, and the reaction is developed and released from the solid phase under mild conditions as a combinatorial chemistry, so that oligopeptides serving as enzyme inhibitors and peptide hormones serving as receptors can be used. Bioactive glycopeptides or peptides can be obtained.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Kazuhiko Nakamura 1-4-6-903, Miigaoka, Aoba-ku, Yokohama-shi, Kanagawa Prefecture (72) Inventor Yukinari Ito 2-23-1 Sengoku, Bunkyo-ku, Tokyo F-term (reference) 4H045 AA20 BA11 BA53 DA50 DA55 FA42 4H049 VN01 VP01 VP10 VQ16 VQ35 VQ37 VQ45 VQ57 VQ77 VR23 VR31 VR41 VS09 VS16 VS35 VS37 VS45 VS57 VS77 VT03 VT06 VT06 VT32 VT33 VT36 V42 VT40 V33 VT40 VT41

Claims (10)

[Claims] 1. A compound of the general formula (I) (Wherein R ¹ , R ² , R ³ and R ⁴ are the same or different and each may have an alkyl group,
P represents an alkenyl group, a cycloalkyl group, a cycloalkenyl group or an aryl group, and X represents a halogen atom). 2. The p-nitrophenylcarbinylsilyl halide compound according to claim 1, wherein at least ^one of R ¹ and R ² is a methyl group. ^3. The p-nitrophenylcarbinylsilyl halide compound according to claim 1, wherein at least one of R ³ and R ⁴ is a phenyl group. 4. The p-nitrophenylcarbinylsilyl halide compound according to claim 1, wherein X is a chlorine atom. 5. The p-nitrophenylcarbylsilyl halide compound according to claim ¹ , wherein R ¹ , R ² , R ³ and R ⁴ are a methyl group and X is a chlorine atom. 6. A compound of the general formula (II) (Wherein R ¹ , R ² , R ³ and R ⁴ are the same or different and each may have an alkyl group,
An alkenyl group, a cycloalkyl group, a cycloalkenyl group, or an aryl group, and X represents a halogen atom), after hydrolyzing, nitrating and then halogenating. General formula (I) (Wherein R ¹ , R ² , R ³ , R ⁴ and X are the same as those described above), and a process for producing a p-nitrophenylcarbinylsilyl halide compound represented by the formula: 7. A method for immobilizing a sugar chain or amino acid on a solid-phase synthesis support via a silyl linker, synthesizing a glycopeptide or peptide, and then cleaving a silicon-oxygen bond to extract the sugar or amino acid, Is converted to an alcoholic hydroxyl group of the general formula (I) (Wherein R ¹ , R ² , R ³ and R ⁴ are the same or different and each represents a lower alkyl group, a lower alkenyl group or an aryl group which may have a substituent, and X represents a halogen atom. The reaction is protected with a p-nitrophenylcarbylsilyl halide compound represented by the following formula), the nitro group is reduced and then condensed with a dicarboxylic anhydride to form a dicarboxylic acid monoamide, which is used as a silyl linker. Of synthesizing glycopeptides or oligopeptides. 8. The method for synthesizing a glycopeptide or oligopeptide according to claim 7, wherein succinic anhydride or glutaric anhydride is used as the dicarboxylic anhydride. 9. The method for synthesizing a glycopeptide or oligopeptide according to claim 7, wherein the silicon-oxygen bond is cleaved by a fluorine anion. 10. The method for synthesizing a glycopeptide or oligopeptide according to claim 9, wherein the silicon-oxygen bond is cleaved using 18-crown-6 or acetic acid together with cesium fluoride.