JP6198207B2 - Novel sugar donor and method for synthesizing sugar chain using the same - Google Patents

Description

  The present invention relates to a novel sugar donor and an efficient method for synthesizing sugar chains using the sugar donor.

  Protein drugs have different activities depending on the sugar chain structure on the protein. As a typical example, a technology for antibody drugs is known (Non-patent Document 1). By deleting the fucose residue at the base of the complex type sugar chain that binds to the Fc region of the antibody, ADCC activity (antibody-dependent cytotoxic activity) is enhanced and the antitumor effect is enhanced. As a result, it is attracting attention as one of the innovative technologies that lead to the reduction of medical costs by reducing the amount of antibody drugs used and the amount of expensive drugs used. However, the sugar chain structure cannot be completely controlled by the current production method. In addition, the necessity of homogenizing sugar chains has been screamed, such as the possibility that the difference in sugar chain structure of the subsequent product due to the expiration of the patent may cause unexpected side effects (Non-patent Document 2).

  Against this background, a sugar chain modification method for proteins has been developed. That is, a sugar chain derived from chicken eggs is chemically modified, and the sugar chain is introduced onto the protein by an enzyme having transglycosylation activity (endo-M). An example in which the transfer efficiency progresses quantitatively (Non-patent Document 3), and synthesis of glycoproteins having a uniform sugar chain structure has become a reality as a next-generation technology. Therefore, if a large amount of sugar chains having a uniform structure is synthesized, glycoproteins having a uniform sugar chain structure can be synthesized. Sugar chain synthesis by organic synthetic chemistry is a technology that plays a part in this. Patent Document 1 describes a method for chemically synthesizing sugar chains using a protecting group. However, since it is difficult to control the stereoselectivity in glycosylation and the synthesis process in multiple stages of sugar chain synthesis, it takes time and effort to isolate and purify. Therefore, although many sugar chain synthesis methods including the present inventors have been developed and sugar chains can be synthesized relatively easily, there has been no example of supplying biopharmaceutical-related sugar chains on a gram scale. .

LacdiNAc type complex type sugar chain 9 sugar is a skeleton very similar to complex type sugar chain that exhibits a potentiogenic effect in antibody drugs.
To date, studies on the synthesis of LacdiNAc sugar chains have been carried out, but the reports are limited. Recently, in Non-Patent Document 4, Nakahara et al. Reported the synthesis of an asparagine-linked sugar chain having a LacdiNAc structure.

  The present inventors first aimed at efficient synthesis of complex type sugar chain A having galactose at the non-reducing end by block synthesis. As a result, the stereoselectivity of the coupling reaction between the trisaccharide donor B and the trisaccharide acceptor C was low, and it was confirmed by HPLC that a mixture of α and β stereoisomers was formed (the following scheme).

  Nakahara et al. Achieved a total synthesis of complex-type glycans by changing the route to Step-wise synthesis, and applied the method to LacdiNAc synthesis (the following scheme). Nakahara et al. Constructed the β-mannoside bond using the method of Crich et al.

WO2005 / 000861

Tetsuya Shitara; YAKUGAKU ZASSHI 129 (1) 3-9 (2009) Glycotechnology in biopharmaceutical development: CMC Publishing, 2011 Tokyo Chemical Industry Co., Ltd. homepage [online], Internet <URL: http://www.tcichemicals.com/en/jp/support-download/tcimail/application/122-23.html> Hagiwara et al., J. Org. Chem. (2011) 76, 5229-5239

  In such a background, it is considered that a large amount of sugar chains can be supplied by developing an efficient chemical synthesis method of sugar chains, but such a method has not been developed.

  Accordingly, an object of the present invention is to develop an efficient chemical synthesis technique for sugar chains that enables a large supply of sugar chains. Specifically, it is an object of the present invention to develop a steric and regioselective glycosylation method while minimizing the introduction of hydroxyl protecting groups.

The inventors of the present invention have intensively studied to develop the above technique.
In the chemical synthesis of sugar chains, it is necessary to protect the sugar moiety when performing a glycosylation reaction that connects sugar to sugar. In the present invention, by optimizing the pattern of the protecting group to be introduced into the sugar residue, the glycosylation reaction proceeded sterically and regioselectively, and as a result, the hydroxyl group protection and deprotection steps were greatly improved. And successfully constructed complex sugar chains. That is, the inventors have found that an efficient chemical synthesis technique for sugar chains that meets the above-mentioned problems has been achieved, and the present invention has been completed.

  That is, the present invention is as follows.

<1> The following formula (Ia)

[Wherein R ¹ represents a pivaloyl group, R ² represents a benzylidene group together with each other, R ³ represents a hydrogen atom, or a hydroxyl group protected with a tert-butyldiphenylsilyl group or an acetyl group; Any one of ³ represents a hydrogen atom, the other represents a hydroxyl group protected with a tert-butyldiphenylsilyl group or an acetyl group, R ^4a represents an ^optionally protected hydroxyl group, and R ^5a is the same or differently protected. R ^6a is the same or different and represents a hydrogen atom or a hydroxyl protecting group, and n is an integer of 1 to 3. ] The sugar derivative represented by this.

<2> The following formula (Ib)

[Wherein R ¹ represents a pivaloyl group, R ² represents a benzylidene group together with each other, R ³ represents a hydrogen atom, or a hydroxyl group protected with a tert-butyldiphenylsilyl group or an acetyl group; Any one of ³ represents a hydrogen atom, the other represents a hydroxyl group protected with a tert-butyldiphenylsilyl group or an acetyl group, R ^4b represents a protected hydroxyl group, and R ^5b represents the same or different protected hydroxyl group or Represents a protected amino group; R ^6b is the same or different and represents a hydroxyl-protecting group; and n represents an integer of 1 to 3. In the sugar derivative represented by the following formula (II)

[Wherein, R ^5b and R ^6b are as defined above, R ⁷ represents a hydrogen atom or a hydroxyl group, one of R ⁷ represents a hydrogen atom, and the other represents a hydroxyl group. A sugar derivative represented by the following formula (III):

[Wherein, R ¹ , R ² , R ³ , R ^5b , R ^6b , R ⁷ , n are as defined above] ] The synthesis method of the sugar derivative represented by this.

<3> The following formula (Ib)

[Wherein R ¹ represents a pivaloyl group, R ² represents a benzylidene group together with each other, R ³ represents a hydrogen atom, or a hydroxyl group protected with a tert-butyldiphenylsilyl group or an acetyl group; Any one of ³ represents a hydrogen atom, the other represents a hydroxyl group protected with a tert-butyldiphenylsilyl group or an acetyl group, R ^4b represents a protected hydroxyl group, and R ^5b represents the same or different protected hydroxyl group or Represents a protected amino group; R ^6b is the same or different and represents a hydroxyl-protecting group; and n represents an integer of 1 to 3. In the sugar derivative represented by the following formula (II)

[Wherein, R ^5b and R ^6b are as defined above, R ⁷ represents a hydrogen atom or a hydroxyl group, one of R ⁷ represents a hydrogen atom, and the other represents a hydroxyl group. A sugar derivative represented by the following formula (III):

[Wherein, R ¹ , R ² , R ^5b , R ^6b , R ⁷ , n are as defined above] A sugar derivative represented by
Deprotection of the hydroxyl-protecting group represented by R ³ and sterically inversion reaction of the free hydroxyl group are carried out, and the following formula (IV)

[Wherein, R ¹ , R ² , R ^5b , R ^6b , R ⁷ , n are as defined above] ] The synthesis method of the sugar derivative represented by this.

INDUSTRIAL APPLICABILITY According to the present invention, there is provided an efficient chemical synthesis method of sugar chains and a novel sugar donor having a specific protective group pattern used in the method.
In the present invention, the construction of a complex sugar chain 9-sugar skeleton having a LacdiNAc structure has been achieved.
When a glycosylation reaction was performed on a sugar receptor trisaccharide having four free hydroxyl groups at a trisaccharide having a commonly used protective group pattern, more than 10 types of complex such as 12 sugar, 9 sugar, 6 sugar, etc. Whereas a mixture was provided, when a sugar donor trisaccharide using the protecting group pattern of the present invention was used, nine sugars were provided as a single compound. This 9-saccharide is a skeleton very similar to a complex sugar chain that exhibits a potentiogenic effect in antibody drugs.
Therefore, according to the present invention, the hydroxyl group protection and deprotection steps can be greatly shortened, and the target sugar chain can be obtained more easily than in the past.
The method of the present invention is a technique that enables supply of a uniform sugar chain structure, which is a bottleneck in biopharmaceutical production, because a wasteful deprotection process can be omitted and a simple and uniform sugar chain can be obtained.

FIG. 1 is a diagram showing the results of HPLC analysis of 9 sugar fractions obtained by reaction with sugar donor 5 and sugar acceptor 6. FIG. 2 is a diagram showing the results of HPLC analysis of 9 sugar fractions obtained by the reaction with sugar donor 50 and sugar acceptor 6. FIG. 3 is a diagram showing the results of HPLC analysis of nine sugar fractions obtained by the reaction with sugar donor 51 and sugar acceptor 6.

Abbreviations used in this specification will be described.
Ac: Acetyl group
Asn: Asparagine
Bn: Benzyl group
COZY: Correlation spectroscopy
CSA: 10-camphorsulfonic acid
CsOAc: cesium acetate
DAST: N, N-diethylaminosulfur trifluoride
^o C: degrees Celsius
DIPEA: N, N-diisopropylethylamine
DMF: N, N-dimethylformamide
DMAP: 4-dimethylaminopyridine
DBU: 1,8-diazabicyclo [5.4.0] undeca-7-ene
DTBMP: 2,6-di-tert-butyl methyl pyridine
EDA: ethylenediamine
Gal: D-galactose
GalNAc: N-acetylgalactosamine
GlcNAc: N-acetylglucosamine
HSQC: Heteronuclear single quantum correlation
Man: D-Mannose
MeOTf: methyltrifluroromethanesulfonate
MS: molecular sieves
NBS: N-bromosuccinimide
NIS: N-iodosuccinimide
Phth: Phthaloyl
Ph: Phenyl
Py: pyridine
rt: room temerature
TBAF: tetrabutylammonium fruoride
TBDMS: tert-buthyldimethylsilyl
TBDPS: tert-buthyldiphenylsilyl
Tf: (trifluoromethyl) sulfonyl
THF:
TMSN ₃ : trimethylsilylazide
TMSOTf: trimethylsilyltrifruoromethanesulfonate
TLC: Thin layer chromatography

(1) Synthetic strategy of complex type sugar chain having LacdiNAc structure The present inventors have established a synthetic strategy as shown in the following scheme for efficient synthesis of complex type sugar chain having LacdiNAc sugar chain. First, it was decided to construct a sugar chain by a block synthesis method that can efficiently construct a large sugar chain. Furthermore, the synthesis process was reduced by using a common synthetic synthon. In addition, the 9-sugar skeleton was synthesized in one step by utilizing a regioselective glycosylation reaction utilizing the difference in the reactivity of sugar hydroxyl groups at the branch, aiming to omit the protective deprotection step. On the other hand, the construction of β-mannoside bonds, which is a common problem in complex type sugar chain synthesis, was decided to use a hydroxyl inversion reaction.
Specifically, the target sugar chain was synthesized by dividing it into two blocks, a galactosylchitobiose block and a chitobiosylmannose block consisting of a common synthon, followed by regioselective glucosylation on four hydroxyl groups. A skeleton was constructed, and the target sugar chain was obtained by introducing branched β-galactose into β-mannose and the non-reducing end chitobiose structure into a LacdiNAc structure by hydroxyl group inversion reaction. There are three monosaccharide units used to construct the nine sugars: glucosamine, mannose, and galactose. After synthesizing each monosaccharide derivative, a chitobiose derivative is synthesized by condensing two residues of glucosamine. Chitobiosyl mannose is synthesized by binding a mannose derivative to chitobiose.

  The LacdiNAc structure is derived by inverting the 4-hydroxyl group of the glucosamine residue after constructing the target sugar chain. The reason why galactosamine is not used is that galactosamine is expensive and uses as much common units as possible. On the other hand, a galactosyl chitobiose block is synthesized by binding a galactose derivative to chitobiose. The reason for using a galactose residue instead of a natural mannose residue is that it is difficult to construct a β-mannose bond. On the other hand, β-galactoside bond can be easily constructed by using adjacent groups, so it can be converted to β-mannose by reversing the 2-4 position hydroxyl group after construction of the target sugar chain 9 sugar. Devised a route. Next, the position where mannoseyl chitobiose trisaccharide is reacted with the galactosylchitobiose trisaccharide having galactose residues 2,3,4,6 released, and the trisaccharide is bound only at the 3rd and 6th positions. Nine sugars are synthesized by selective glycosylation. The 6th position of galactose is a primary hydroxyl group, and the 3rd position is considered to be more reactive than other secondary hydroxyl groups due to the hydrogen bond between the hydroxyl group adjacent to the 4th position axial hydroxyl group. We thought that unnecessary protection / deprotection steps could be omitted by utilizing the difference in reactivity. Finally, the LacdiNAc structure and the β-mannose bond are converted simultaneously by the inversion reaction of the hydroxyl group to obtain the target sugar chain. The above route avoids difficult three-dimensional construction and the use of expensive raw materials, omits protective deprotection, and enables efficient sugar chain construction of the target sugar chain by block synthesis.

  Based on the above-described synthetic strategy, the present inventors have studied by various experiments, and by reacting with a sugar acceptor trisaccharide using a sugar donor trisaccharide having a specific protecting group pattern. The present inventors have found that a complex sugar chain 9-saccharide having a LacdiNAc structure is provided as a single compound. The present invention has been made based on this finding.

(2) Sugar donor TECHNICAL FIELD This invention relates to the novel sugar donor using the pattern of a specific protecting group which can be used for the above-mentioned synthesis method. Specifically, the following formula (Ia)

[Wherein R ¹ represents a pivaloyl group, R ² represents a benzylidene group together with each other, R ³ represents a hydrogen atom, or a hydroxyl group protected with a tert-butyldiphenylsilyl group or an acetyl group; Any one of ³ represents a hydrogen atom, the other represents a hydroxyl group protected with a tert-butyldiphenylsilyl group or an acetyl group, R ^4a represents an ^optionally protected hydroxyl group, and R ^5a is the same or differently protected. R ^6a is the same or different and represents a hydrogen atom or a hydroxyl protecting group, and n is an integer of 1 to 3. ] [In the following, it may be referred to as compound (Ia)].

The protecting group of the protected hydroxyl group represented by the protected the hydroxyl group which may have a protecting group and R ^4b represented by R ^4a, particularly if the active group or a hydroxyl group protecting group to be used for normal glycosylation Although it does not restrict | limit, A thioether type protective group, a trichloroacetimide type protective group, a halogen, etc. are mentioned. Here, examples of the thioether-type protecting group include a phenylthio group, a thiomethyl group, a thioethyl group, and a thiopropyl group. Examples of the trichloroacetimide type protecting group include a trichloroacetimide group. Examples of halogen include Cl, Br, I, and F.

The protecting group for the amino group which may be protected represented by R ^5a and the protecting group for the protected amino group represented by R ^5b may be any amino protecting group which is usually used in peptide synthesis. Although it does not restrict | limit in particular, A urethane type protective group, an acyl type protective group, an alkyl type protective group, a thioether type protective group, an imino type protective group, etc. are mentioned. Here, examples of the urethane-type protecting group include aralkyloxycarbonyl groups such as benzyloxycarbonyl, substituted benzyloxycarbonyl, and fluorenylmethoxycarbonyl; alkyloxycarbonyl groups represented by t-butoxycarbonyl; triphenylphosphinoethyloxycarbonyl And substituted alkyloxycarbonyl groups such as methylsulfenylethyloxycarbonyl; alkenyloxycarbonyl groups typified by allyloxycarbonyl; aryloxycarbonyl groups such as phenoxycarbonyl, and the like. Examples of the acyl protecting group include alkanoyl groups such as acetyl group and phthaloyl; arylthioacetyl groups such as o-nitrophenylthioacetyl group. Examples of the alkyl-type protecting group include aralkyl groups such as benzyl, benzhydryl, and triphenylmethyl groups. Examples of the thioether-type protecting group include aralkylthio groups such as triphenylmethylthio, dinitrophenylthio, trichlorophenylthio, and substituted phenylthio groups. Examples of the imino protecting group include a substituted benzylidene group.
The protected amino group is preferably an alkanoylamide having 2 to 6 carbon atoms, phthalimide and the like.

An ^optionally protected hydroxyl protecting group represented by R ^5a , a protected hydroxyl protecting group represented by R ^5b , and a hydroxyl protecting group represented by R ^6a and R ^6b include ether protecting groups, esters System protecting groups, acyl protecting groups and the like. Examples of the ether protecting group include a benzyl group, a p-methoxybenzyl group, an allyl group, an organic silyl group, and a trityl group. Examples of the carbamate protecting group include an allyloxycarbonyl group. Examples of the acyl protecting group include an alkanoyl group such as an acetyl group; an arylthioacetyl group such as an o-nitrophenylthioacetyl group. The acyl protecting group is preferably an alkanoyl group having 2 to 6 carbon atoms.

In the present invention, it is possible to perform a regioselective glycosylation reaction by using a specific protecting group pattern in R ¹ , R ² and R ³ of the sugar donor. The hydroxyl protecting group represented by R ^5a and R ^5b , R ^6a and R ^6b may be the same as or different from the hydroxyl protecting group represented by R ¹ , R ² or R ³ .

Compound (Ia) can be synthesized, for example, as follows.
Taking the trisaccharide donor (5) as an example, the synthesis method of compound (Ia) will be described.
The compound (45) having a benzylidene group introduced at the 4,6 position is reacted with 1.5 equivalents of pivaloyl chloride in the presence of pyridine as a starting material, thereby introducing a pivaloyl group selectively at the 3 position, and a benzylidene group at the 4,6 position. Then, a mannose derivative (7) having a pivaloyl group at the 3-position is synthesized.
A coupling reaction between the mannose derivative (7) and the chitobiose derivative (48) is performed to synthesize a novel chitobiose sylmannose donor. The trisaccharide donor (5) is synthesized by reacting the chitobiose donor (48) and the mannose acceptor (7) in dichloromethane using TMSOTf as a promoter. The reaction mixture is purified by gel filtration chromatography to give (5).

(3) Method for Synthesizing Sugar Derivative as Intermediate of Complex Type Sugar Chain Having LacdiNAc Structure Furthermore, the present invention relates to a method for synthesizing a sugar derivative as an intermediate of complex type sugar chain having LacdiNAc structure.
Specifically, the following formula (Ib)

[Wherein R ¹ represents a pivaloyl group, R ² represents a benzylidene group together with each other, R ³ represents a hydrogen atom, or a hydroxyl group protected with a tert-butyldiphenylsilyl group or an acetyl group; Any one of ³ represents a hydrogen atom, the other represents a hydroxyl group protected with a tert-butyldiphenylsilyl group or an acetyl group, R ^4b represents a protected hydroxyl group, and R ^5b represents the same or different protected hydroxyl group or Represents a protected amino group; R ^6b is the same or different and represents a hydroxyl-protecting group; and n represents an integer of 1 to 3. In the following formula (II), the sugar derivative represented by formula (II) may be referred to as the following formula (II):

[Wherein, R ^5b and R ^6b are as defined above, R ⁷ represents a hydrogen atom or a hydroxyl group, one of R ⁷ represents a hydrogen atom, and the other represents a hydroxyl group. A sugar derivative represented by the following formula (III):

[Wherein, R ¹ , R ² , R ³ , R ^5b , R ^6b , R ⁷ , n are as defined above] ] [In addition, it is related with the synthesis | combining method of the sugar derivative represented by following.

Compound (II) can be produced, for example, as follows.
Taking the trisaccharide receptor (6) as an example, the synthesis method of compound (II) will be described.
First, a compound (8) is prepared from a known compound. Coupling of the chitobiose receptor (8) and thioglycoside (9 ′) is carried out in CH ₂ Cl ₂ at −20 ° C. for 2 hours using NIS / TfOH as a promoter to obtain the trisaccharide (6 ′). Deacetylation of trisaccharide (6 ′) is performed using NaOMe / MeOH at 0 ° C. in THF to obtain galactosylchitobiose receptor (6). (Matsuo et al., J. CARBOHYDRATE CHEMISTRY, 18 (7), 841-850 (1999))

The sugar donor which is compound (Ib) is reacted with the sugar acceptor which is compound (II) to obtain compound (III).
The reaction between the compound (Ib) and the compound (II) is carried out by suspending the compound (Ib) and the compound (II) in a solvent such as dichloromethane, chloroform, ether, toluene, dichloroethane and the like, mixing them, NIS, AgOTf, etc. Of -78 ° C to 40 ° C, preferably -40
The reaction is carried out at from -20 ° C for 10 minutes to 72 hours, preferably 1 to 12 hours. The compound (III) is obtained by washing with a saturated aqueous sodium hydrogen carbonate solution and brine and drying and concentrating in a conventional manner.
After carrying out this synthesis method, if desired, the protecting group can be deprotected using a conventional deprotection reaction. It is also possible to introduce a protecting group using a normal protecting reaction. Moreover, it is also possible to introduce a substituent using a normal organic synthesis method. For example, the protected amino group represented by R ^5b can be substituted with an acetylamino group or the like. Ordinary washing, concentration, and purification can be performed. Such compounds are also encompassed within the scope of the present invention.

(4) Method for synthesizing complex sugar chain having LacdiNAc structure Furthermore, the present invention relates to a method for synthesizing complex sugar chain having LacdiNAc structure.
Specifically, the following formula (Ib)
Formula (Ib) below

[Wherein R ¹ represents a pivaloyl group, R ² represents a benzylidene group together with each other, R ³ represents a hydrogen atom, or a hydroxyl group protected with a tert-butyldiphenylsilyl group or an acetyl group; Any one of ³ represents a hydrogen atom, the other represents a hydroxyl group protected with a tert-butyldiphenylsilyl group or an acetyl group, R ^4b represents a protected hydroxyl group, and R ^5b represents the same or different protected hydroxyl group or Represents a protected amino group; R ^6b is the same or different and represents a hydroxyl-protecting group; and n represents an integer of 1 to 3. In the sugar derivative represented by the following formula (II)

[Wherein, R ^5b and R ^6b are as defined above, R ⁷ represents a hydrogen atom or a hydroxyl group, one of R ⁷ represents a hydrogen atom, and the other represents a hydroxyl group. A sugar derivative represented by the following formula (III):

[Wherein, R ¹ , R ² , R ³ , R ^5b , R ^6b , R ⁷ , n are as defined above] A sugar derivative represented by
Deprotection of the hydroxyl-protecting group represented by R ³ and sterically inversion reaction of the free hydroxyl group are carried out, and the following formula (IV)

[Wherein, R ¹ , R ² , R ^5b , R ^6b , R ⁷ , n are as defined above] ] It is related with the synthesis | combining method of saccharide derivative [it may hereafter be called compound (IV)].

Compound (III) can be obtained by reacting a sugar donor as compound (Ib) with a sugar acceptor as compound (II) in the same manner as described above.
The deprotection reaction of the protective group for a hydroxyl group represented by R ^3, in a conventional manner suitable for deprotection of the hydroxyl-protecting group represented by R ^3, can be performed.

In the stereoinversion reaction of the free hydroxyl group, the compound (III) in which R ³ is a hydroxyl group is suspended in a solvent such as dichloromethane, pyridine, chloroform, ether, toluene, dichloroethane, and an activator such as Tf ₂ O is added. The reaction is carried out at 78 ° C to 40 ° C, preferably 0 ° C to 20 ° C for 10 minutes to 72 hours, preferably 1 to 12 hours. The compound (IV) is obtained by washing with a saturated aqueous sodium hydrogen carbonate solution and brine and drying and concentrating by a conventional method.

After carrying out this synthesis method, if desired, the protecting group can be deprotected using a conventional deprotection reaction. It is also possible to introduce a protecting group using a normal protecting reaction. Moreover, it is also possible to introduce a substituent using a normal organic synthesis method. For example, the protected amino group represented by R ^5b can be substituted with an acetylamino group or the like. Ordinary washing, concentration, and purification can be performed. Such compounds are also encompassed within the scope of the present invention.

  Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited to the following examples.

The measuring method and apparatus of the physical property values described in the examples are as follows.
¹ H-NMR spectrum was measured using a JEOL-JNM-ECA300 MHZ, JEOL-JNM-ECA400 MHZ, JEOL-JNM-ECA600 MHZ nuclear magnetic resonance apparatus. Measured as an internal standard, chemical shift was indicated by δ value, and coupling constant was indicated by Hz. As shown in the figure, for the sake of convenience, in the analysis of ¹ H NMR, the sugar at the reducing end is set to a, and as it goes to the non-reducing end side, b, c, and d are written in alphabets.

Mass spectrometry was measured using a Shimadzu SHIMAZU / KRATOS matrix-assisted laser ionization time-of-flight mass spectrometer KOMPACT MALDI IV tDE.
MERCK silica gel 60 F254CE was used for thin layer chromatography (TLC).
For silica gel column chromatography, MERCK Silica Gel 60 (0.040-0.063 mm) or KANTO CHEMICAL Silica Gel 60N (spherical, neutral) 0.040-0.063 mm was used.
For gel filtration chromatography, BIO-RAD Bio-Beads (registered trademark) S-X1 (200-400 mesh) was used.

1. Regioselective glycosylation reaction using a new donor As described above, the target of the regioselective glycosylation reaction using the coupling reaction of trisaccharide donor B and trisaccharide acceptor C was the target. In addition to the compound, multiple nine sugar isomers were formed. At this time, various leaving groups and solvents were examined, but definitive conditions for obtaining a single compound were not obtained. Therefore, the present inventors paid attention to the armed-disarmed effect proposed by Freiser-Reid et al. That is, the reactivity was changed by changing the protecting group pattern of the sugar donor, and the selectivity was improved.

(Design of sugar donors with different protective group patterns)
As a position to change the pattern of the protecting group, attention was paid to a mannose residue which is considered to have the strongest influence on the reaction in the glycosylation reaction, that is, the reaction point. The following shows three types of compounds with different mannose protecting group patterns. Mannose derivative (7) protected with a benzylidene group at positions 4 and 6 and a pivaloyl group at position 3 (43), a mannose derivative with an Ac group at positions 4 and 6 (43), a benzylidene group at positions 4 and 6, and a benzyl group at position 3 A mannose derivative (44) protected with a was designed. It was expected that the acyl group-based protecting group had an electronic effect, the benzylidene group had a ring-fixing effect, and a difference in the electronic effect depending on the position of introduction of the protecting group.

(Synthesis of mannose derivatives with different protecting group patterns)
Mannose derivatives designed based on the concept described above were synthesized. The synthesis of a mannose derivative (7) having a benzylidene group at the 4th and 6th positions and a pivaloyl group at the 3rd position (7) was obtained by using 1.5 equivalents of chloride in the presence of pyridine starting from the compound (45) having a benzylidene group introduced at the 4th and 6th positions. A pivaloyl group was introduced selectively at the 3-position by reacting with pivaloyl. The yield was 81%. The structure of (7) was confirmed by confirming a peak derived from the benzylidene group at 5.3 ppm in the ¹ H NMR spectrum, a peak of 9H derived from the pivaloyl group around 1.0 ppm, and Determined from low field shift. Synthesis of the 4,6-position benzylidene group and the 3-position benzylmannose derivative (44) was performed by regioselectively relocating to (45) by stannation with dibutyltin oxide followed by reaction with TBABr and BnBr. Bn group was introduced. The yield was 96%. The structure of the 3-position benzylmannose derivative (44) was determined by observing a peak of 2H derived from the benzyl group at 4.5 ppm in the ¹ H NMR spectrum. The structure was also confirmed from the fact that the obtained proton (44) was acetylated and the proton at position 2 was shifted in a low magnetic field. The synthesis of the 4,6-position diAc mannose derivative (43) was carried out by introducing a TBDPS group by reacting with the TBDPOTf in the presence of 2,6-lutidine to the 2-position of (44). The benzylidene group was removed by hydrolysis. Thereafter, the 4- and 6-positions were converted to Ac, and the TBDPS group was removed using HF · Py. To obtain (43). From (44), the yield was 39% in 4 steps. The structure of (43) was determined by confirming a low magnetic field shift of protons corresponding to the 4th and 6th positions and a 6H peak derived from two Ac groups in the vicinity of 2.0 ppm.
As described above, three types of mannose derivatives (7), (43), and (44) with different protecting group patterns were synthesized.

(Synthesis of novel chitobiosyl mannose donor)
A novel chitobiose sylmannose donor was prepared by coupling the three previously synthesized mannose derivatives (7), (44), (43) with chitobiose derivatives (48), (25), (49). The synthesis of was studied. Trisaccharide donor (5) was synthesized by reacting chitobiose donor (48) and mannose acceptor (7) in dichloromethane using TMSOTf as a promoter. The reaction mixture was purified by gel filtration chromatography to give (5) in 23% yield. The structure of (5) was confirmed by ¹ H NMR spectrum that a peak for 9 protons derived from the pivaloyl group was observed around 1.0 ppm, a methine peak of the benzylidene group was observed around 5.0 ppm, and 1 derived from chitobiose. The structure was confirmed by observing the peak of the position and observing the target molecular ion peak with MALDI-TOF MS. On the other hand, the synthesis of the trisaccharide donor (50) protected at the 3-position with a benzyl group is performed by reacting the chitobiose donor (25) and the mannose acceptor (44) under the same conditions as described above, and after completion of the reaction, Was obtained by purification by gel filtration chromatography. The yield was 70%. The structure of (50) was observed from the ¹ H NMR spectrum by observing the methine peak of the benzylidene group at around 5.0 ppm and the peak at the 1-position proton of glucosamine derived from chitobiose. The structure was confirmed by observing the target molecular ion peak with MALDI-TOF MS. The novel trisaccharide donor (51) was synthesized using a chitobiose donor (49) and a mannose acceptor (43) having an Ac group at the 4,6 positions. The reaction mixture was purified by gel filtration chromatography and then purified by silica gel column chromatography to obtain the desired trisaccharide donor (51) in a yield of 61%. From the ¹ H NMR spectrum, the structure of (51) was observed with two peaks derived from the Ac group at around 2.0 ppm and the proton at the 1-position of glucosamine derived from chitobiose. The structure was determined from the observed molecular ion peak.

(Investigation of regioselective glycosylation using a new donor)
Using the least reactive (5) with the introduction of an electroattractive substituent, the 2, 3, 4 and 6 positions were reacted with free galactosylchitobiose (6). The reaction conditions were NIS and AgOTf at −78 ° C., similar to the conditions used so far. However, the sugar donor was not activated under these conditions, and when the reaction temperature was room temperature, the progress of the reaction was confirmed. The reaction mixture was purified by gel filtration chromatography to obtain a 9-saccharide fraction with a yield of 78%. The obtained 9-saccharide fraction was analyzed by HPLC. The result is shown in FIG. One peak was observed. As a result of analyzing the obtained compound by ¹ H NMR, it was found that the sugar donor and the sugar acceptor were 9 sugars (4) having a target structure in which α-bonded at the 3rd and 6th positions. Next, the reaction was carried out using (50) in which the 4,6 position was protected with a benzylidene group and the 3 position was protected with an electron donating Bn group. The reaction was carried out by raising the temperature from −78 ° C. to −20 ° C. using NIS and AgOTf. The resulting reaction mixture was purified by gel filtration chromatography to obtain a 9-saccharide fraction with a yield of 94%. The results of analyzing the obtained 9-saccharide fraction by HPLC are shown in FIG. Finally, a glycosylation reaction was carried out using (51) in which positions 4,6 were protected with an Ac group as an electron-withdrawing protecting group, and position 3 was protected with a Bn group as an electron-donating protecting group. The reaction was carried out by raising the temperature from −78 ° C. to −20 ° C. using NIS and AgOTf. The resulting reaction mixture was purified by gel filtration chromatography to obtain a 9-saccharide fraction in a yield of 58%. The results of analyzing the obtained 9-saccharide fraction by HPLC are shown in FIG.

(Structural analysis of 9-sugar (4))
The single NMR analysis result of (4) will be described. In order to determine the binding position of the sugar donor and sugar acceptor of Compound (4), the galactose residue at the branched portion was converted to Ac (54) (the following scheme), and NMR was measured.

First, H at the 1st position of galactose was determined by HSQC spectrum. Thereafter, H at positions 2, 3 and 4 of galactose was determined by HH COZY spectrum. Furthermore, it was confirmed by the TOCSY spectrum whether H determined by the COZY spectrum was correct. As a result, a low magnetic field shift at the 2nd and 4th positions of the galactose in the branched portion was confirmed, and it was revealed that sugar donors were bound at the 3rd and 6th positions. In addition, the ¹ J _CH of mannose and galactose was 175 Hz and 172 Hz in non-decoupling HSQC.

  Focusing on the armed-disarmed effect, we synthesized three types of trisaccharides with different mannose residue protecting group patterns of sugar donors and reacted with galactosylchitobiose. As a result, high steric and regioselectivity can be realized when sugar donor (5) and sugar acceptor (6) are used. This high selectivity is thought to be enhanced by the anomeric effect by the pivaloyl group, which is an electron-withdrawing group, and the stereoselectivity is improved. In addition, it was considered that chitobiose, the 2-position substituent, became sterically hindered and gave high regioselectivity because mannose was fixed in a chair-type conformation with a benzylidene group and the 2-position substituent stood axially.

2. Construction of LacdiNAc structure and β-mannoside bond by hydroxyl reversal reaction β-Mannoside bond is an important bond contained in the core structure of N-type sugar chain. One of the methods for constructing β-mannoside bonds is a hydroxyl inversion reaction. Matsuo et al. Introduced a mannose derivative at the 3,6-position of the β-galactosylchitobiose derivative, then converted the β-galactose residue's 2-4, 2-hydroxyl groups to Tf, and reversed the 3-D of the hydroxyl group. We have succeeded in efficiently constructing the common core pentasaccharide of the asparagine-linked sugar chain. Therefore, with reference to this method, we combined the glucosamine residue 4-position on the non-reducing end side with the 2,4-position of β-galactose residue on 9 sugars that are more sterically crowded than 5 sugars. We decided to challenge simultaneous stereoinversion of four hydroxyl groups.

(Construction of LacdiNAc structure and β-mannoside bond by simultaneous stereoinversion of hydroxyl groups)
First, the 9-saccharide derivative (4) was purified using HF · Py. To remove the TBDPS group at the 4-position of the non-reducing end side and purified by PTLC to obtain (55) in a yield of 49%. The structure of (55) was measured by ¹ H NMR. As a result, the disappearance of the peak derived from the TBDPS group observed around 1.0 ppm was confirmed, and the molecular ion peak of the target compound was confirmed by MALDI-TOF MS. Determined from. The resulting 9-sugar de-TBDPS body (55) was subjected to simultaneous steric inversion of four hydroxyl groups. First, triflation of the hydroxyl group was performed using trifluoromethanesulfonic anhydride and pyridine. After stopping the reaction of Tf ₂ O with saturated aqueous sodium hydrogen carbonate, the solvent was removed, and azeotropy with toluene was performed, followed by vacuum drying. Thereafter, the obtained residue was dissolved in toluene and reacted with cesium acetate and 18-crown-6 in the presence of ultrasonic waves. After the reaction mixture was purified by PTLC, the ¹ H NMR spectrum was measured and found to be a 3: 1 mixture. From the result of analyzing the fraction of the obtained mixture by HPLC, it was confirmed that the mixture was a 3: 1 mixture. Each peak was fractionated by preparative HPLC, and each NMR spectrum was measured. As a result, it was clarified that the compound obtained as the main product was the target product (2) in which four hydroxyl groups were inverted. The yield was 28%. The structure of (2) was determined as follows. First, the 1st position of the mannose at the branched portion was determined by HSQC spectrum. The proton correlation was traced from the determined 1st position in the HH COZY spectrum, and the 2nd, 3rd and 4th positions of mannose were determined. It was confirmed that the β-mannoside bond was constructed by inverting the hydroxyl group at the branching site because a low magnetic field shift at the 2nd and 4th positions was observed. The LacdiNAc structure was determined by confirming a peak characteristic for the galacto conformation at 5.6 ppm in the ¹ H NMR spectrum for two protons. Furthermore, the structure was confirmed by observing the target molecular ion peak by MALDI-TOF MS. The by-product had three Ac group peaks observed around 2.0 ppm, and the results of the analysis by MALDITOF MS showed that the three hydroxyl groups were inverted, and the 2-position hydroxyl group was eliminated. It was concluded that it was sugar. From this, the cause of the formation of by-products was considered. Since a desorber was obtained, it was considered that the S _N 2 inversion reaction and the E2 desorption reaction competed. Since anti-elimination takes precedence, the 5-position proton is extracted and the 4-position Tf body is desorbed.

  Using the inversion reaction of four hydroxyl groups, we succeeded in constructing a rare structure LacdiNAc and a difficult β-mannoside bond with a low yield of 28% (2 mg). We believe that the yield can be sufficiently improved if conditions for determining the end point of the reaction in Tf formation are established. As described above, the construction of the target 9 sugars has been achieved.

<Synthesis Example 1> Synthesis of Mannose Donor The known compound (7) was synthesized according to literature methods.
The benzylidene compound (44) (500 g, 1.4 mmol) was dissolved in 15 mL of pyridine. Under an argon stream, pivaloyl chloride 1 (340 μg, 2.8 mmol) was added dropwise at room temperature and stirred for 16 hours. After the reaction solution had zero ice, an aqueous sodium hydrogen carbonate solution was added to stop the reaction. The reaction mixture was dissolved in ethyl acetate, the ethyl acetate layer was washed with 1M hydrochloric acid, saturated aqueous sodium hydrogen carbonate solution and brine, the organic layer was dried over sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (Hexane: EtOAc, 4: 1) to obtain (7) (518 mg, 88%).

R _f = 0.25 (Hexane: EtOAc = 4: 1)
¹ H NMR (400MHz, CDCl ₃ ) δ: 5.616 (1H, s, PhCH), 5.59 (1H, d, H-1), 5.59 (1H, dd, J
= H-3), 4.33 (H, ddd, H-5), 4.31 (1H, d, H-6), 4.25 (1H, dd, H-6), 4.19 (1H, dd, H-2), 3.86 (1H, dd, H-4), 2.31 (1H, brs, OH), 1.24 (9H, s, (C = O (CHMALDI-TOF-MS Calcd for C ₂₄ H ₂₈ NaO ₆ S m / z [M + Na] ⁺ : 467.15 found: 467.79.

<Synthesis Example 2> Synthesis of disaccharide TBDPS body

Compound (22) (206 mg, 0.21 mmol) was converted to dichloroethane. Dissolved in (5 mL), and 2,6-lutidine (111 μL, 1.0 mmol) and TBDPSOTf (200 μL, 0.52 mmol) were added at room temperature. After stirring the reaction solution for 30 minutes, the reaction solution was ice-cooled, and an aqueous sodium hydrogen carbonate solution was added to stop the reaction. The reaction mixture was dissolved in ethyl acetate, the ethyl acetate layer was washed with 1M hydrochloric acid, saturated aqueous sodium hydrogen carbonate solution and brine, the organic layer was dried over sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (Hexane: EtOAc, 4: 1) to obtain (48a) (249 mg, 98%).

R _f = 0.50 (Toluene: EtOAc = 10: 1), R _f = 0.30 (Hexane: EtOAc = 4: 1).
¹ H NMR (400MHz, CDCl ₃ ) δ: 7.72-6.50 (38H, Ar), 5.24 (1H, d, J = 8.4Hz, H-1 ^a ), 5.12 (1H, d, J = 9.2 Hz, H- 1 ^b ), 4.76 (1H, d, H-1 ^a ), 4.74-4.31 (8H, m), 4.18 (1H, dd, J = 8.8 Hz), 4.11-3.96 (5H, m), 3.82 (1H, d, J = 12.8 Hz), 3.71 (1H, J = 9.6 Hz), 3.571-3.47 (3H, m), 3.41 (1H, dd, J = 3.6 Hz, J = 11.2 Hz), 3.31 (1H, dd, J = 2.4 Hz, J = 9.6 Hz), 0.97 ((CH ₃ ) ₃ CSiPh ₂ ).
¹³ C NMR (150 MHz, CDCl ₃ ) δ: 96.81, 85.61, 80.95, 76.42, 75.21, 74.58, 73.09, 72.96, 72.77, 72.39, 67.95, 56.66, 55.23, 27.03.
MALDI-TOF-MS: calcd for C ₇₂ H ₆₉ N ₅ O ₁₂ SiNa (M + Na) ⁺ m / z: 1246.46, found: 1247.43.

<Synthesis Example 3> Synthesis of hemiacetal (48c)

Compound (48a) (250 mg, 0.20 mmol) was dissolved in THF (3 mL), Ph ₃ P (94 mg, 0.35 mmol) and water (0.1 mL) were added, and the mixture was stirred at 40 ° C. overnight. Amberlyst was added to the reaction solution, and the mixture was stirred at 40 ° C. for 7 days. After removing the resin, the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (Hexane: EtOAc, 3: 1) to obtain (48c) (238 g, 98%).

Synthesis Example 4 Synthesis of Disaccharide Donor (48)

2,2,2-Trifluoro-N-phenylacetimidoyl Chloride (14 · L, 0.12 mmol) was added to a dichloromethane solution (1.0 mL) of compound (48c) (39 mg, 0.032 mmol), and then further added.
K ₂ CO ₃ (9 mg, 0.10 mmol) was added and stirred for 1 hour. The reaction solution was purified by silica gel column chromatography (Hexane: EtOAc, 3: 1, TEA 0.1%) to obtain (48) (40 mg, 92%).
R _f = 0.6 (Toluene: EtOAc = 3: 1), 0.27 (Hexane: EtOAc = 3: 1).

<Synthesis Example 5> Synthesis of trisaccharide donor (5)

  Dichloromethane (15 mL) was added to a mixture of Compound (48) (110 mg, 0.080 mmol), 7 (71 mg, 0.16 mmol) and AW-300, and the mixture was stirred at −78 ° C. under an argon stream. 0.024 mmol) was added and stirred for 15 min. Triethylamine was added to the reaction solution to stop the reaction. The reaction solution was diluted with ethyl acetate, filtered through celite, the ethyl acetate layer was washed with saturated aqueous sodium hydrogen carbonate solution and brine, the organic layer was dried over sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified with Bio-Beads S-X1 (Toluene: EtOAc 1: 1) to obtain compound (5) (30 mg, 23%).

R _f = 0.53 (Toluene: EtOAc = 10: 1), R _f = 0.45 (Hexane: EtOAc = 3: 1).
¹ H NMR (400MHz, CDCl ₃ ) δ: 7.79-6.47 (38H, Ar), 5.37 (1H, s, PhCH), 5.14 (1H, d, J
= 8.4 Hz, H-1 ^c ), 5.03 (1H, s, H-1 ^a ), 4.90 (1H, dd, J _1,2 = 3.2 Hz, H-3 ^c ), 4.88 (1H, d, J = 8.8 Hz, H-1 ^b ), 4.91 (1H, dd, J _3,2 = 3.6 Hz), 4.74-4.19 (8H, d, CH ₂ -Ph), 4.24 (1H, dd, J = 1.6 Hz), 4.13-3.93 (6H, m), 3.82 (1H, m), 3.54-3.46 (3H, m), 3.38-3.30 (2H, m), 3.11 (1H, dd, J = 2.4 Hz, J = 10.0 Hz) , 2.94 (1H, dd, J = 10.0 Hz), 1.14 (9H, s, (CH ₃ ) ₃ CC = O), 0.96 ((CH ₃ ) ₃ CSiPh ₂ ).
MALDI-TOF-MS: calcd for C ₉₆ H ₉₆ N ₂ O ₁₈ SiNa (M + Na) ⁺ m / z: 1647.80, found: 1648.74.

<Synthesis Example 6> Synthesis of trisaccharide donor (6)

A mixture of NIS (1.05 g, 4.67 mmol), TfOH (130 μL, 1.46 mmol) and molecular sieves (3.0 g, type 4A) in CH ₂ Cl ₂ (5 mL) was stirred at 0 ° C. for 30 minutes. Then, it cooled at -78 degreeC. To this mixture was added a solution of glycosyl donor (9 ′) (0.76 g, 2.01 mmol) and glycosyl donor (8) (1.53 g, 1.55 mmol) in CH ₂ Cl ₂ (10 mL) over 10 minutes. Added dropwise. The reaction was stirred at −20 ° C. for 2 hours. The reaction was quenched with saturated aqueous sodium bicarbonate. The reaction mixture was diluted with EtOAc and filtered through celite. The filtrate was washed with an aqueous Na ₂ S ₂ O ₅ solution and brine. The solution was dried over MgSO _{4 and} concentrated in vacuo. The residue was purified by silica gel column chromatography (Toluene: EtOAc 4: 1) to obtain compound (6 ′) (1.77 g, 86%).

R _f = 0.26 (Toluene: EtOAc = 4: 1)
[α] _D +8 ^c (c1.0, CHCl ₃ )
¹ H NMR (500MHz, CDCl ₃ ) δ: 5.29 (1H, d, J = 8.6 Hz, H-1b), 5.26 (1H, dd, J = 0.7 Hz, J = 3.4 Hz, H-4c), 5.15 ( 1H, dd, J = 0.7 Hz, J = 3.4 Hz, H-4c), 5.15 (1H, d, J
= 9.5 Hz, H-1a), 5.14 (1H, dd, J = 10.5 Hz, H-2c), 4.62 (1H, d, J = 8.1 Hz, H-1c), 2.04, 1.99, 1.962, 1.958 (3H (× 4, s, Ac)
¹³ C NMR (125 MHz, CDCl ₃ ) δ: 100.45 (C-1 c), 97.09 (C-1b), 85.62 (C-1a)
Anal.Calcd for C ₇₀ H ₆₉ O ₂₁ N ₅ : C, 63.87; H, 5.28; N 5.32
Found: C, 64.13; H, 5.31; N, 5.39

To a stirred solution of compound (6 ′) (134 mg, 0.102 mmol) in THF: MeOH (2: 3, 5 mL) was added 1M NaOMe / MeOH (100 μL) at 0 ° C. The mixture was stirred for 1 hour, neutralized with Amberlyst 15 (H ⁺ ) resin, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CHCl ₃ : MeOH 50: 1 to 20: 1) to obtain compound (6) (103 mg, 88%).
R _f = 0.33 (CHCl ₃ : MeOH = 15: 1)
[α] _D +24 ^c (c1.0, CHCl ₃ )
¹ H NMR (500MHz, CDCl ₃ ) δ: 5.32 (1H, d, J = 7.9 Hz, H-1b), 5.16 (1H, d, J = 9.5 Hz, H-1a)
¹³ C NMR (125 MHz, CDCl ₃ ) δ: 102.95 (C-1 c), 97.08 (C-1b), 85.63 (C-1a), 73.76 (C-3c), 62.67 (C-6c)
Anal.Calcd for C ₆₂ H ₆₁ O ₁₇ N ₅ : C, 64.86; H, 5.35; N 6.10
Found: C, 64.33; H, 5.36; N, 6.11

<Example 1> Synthesis of 9-sugar (4)

A dichloromethane solution of the compound (5) (24 mg, 0.015 mmol), (6) (8 mg, 0.0068 mmol) was mixed with NIS (5 mg, 0.189 mmol), AgOTf (4 mg, 0.0126 mmol) and molecular sieves 4A at -78 ° C. Added to the mixture. The reaction solution was heated to -20 ° C and stirred for 7 hours. Triethylamine was added to the reaction solution to stop the reaction, and the mixture was diluted with ethyl acetate and filtered through celite. The ethyl acetate layer was washed with a saturated aqueous sodium hydrogen carbonate solution and brine, the organic layer was dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by Bio-Beads S-X1 (Toluene: EtOAc 1: 1) to obtain 9-saccharide (4) (19.8 mg, 78%).
After gel filtration, analysis by HPLC gave a single peak.

R _f : 0.22 (Toluene: EtOAc = 5: 1)
¹ H NMR (600MHz, CDCl ₃ ) δ: 7.86-6.43 (114H, m, Ar), 5.38, 5.33 (1H, s, PhCH), 5.16-5.05 (3H, m), 4.93-3.06 (55H, m) , 2.97-2.86 (3H, m), 2.71 (1H, J = 10.2Hz), 1.14, 1.10 (9H, s, (CH ₃ ) ₃ CC = O), 0.95, 0.94 (9H, s, (CH ₃ ) ₃ CSiPh ₂ ).
MS (MALDI-TOF) calcd for C ₂₄₂ H ₂₄₁ N ₉ O ₅₇ Si ₂ Na (M + Na) ⁺ m / z: 4199.59, found: 4201.80.

<Example 2> Synthesis of 9-sugar desilylated product (55)

  Compound (4) (16 mg, 0.0038 mmol) was dissolved in pyridine (500 μL), 70% hydrogen fluoride pyridine solution (100 μL) was added at 0 ° C., and the mixture was stirred at 40 ° C. for 9 hours. Further, 70% hydrogen fluoride pyridine solution (100 μL) was added and stirred for 13 hours. The reaction solution was diluted with ethyl acetate, the ethyl acetate layer was washed with a saturated aqueous sodium hydrogen carbonate solution and brine, the organic layer was dried over sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by PTLC (Toluene: EtOAc, 2: 1) to obtain compound (55) (6.9 mg, 49%).

R _f : 0.20 (Toluene: EtOAc = 3: 1)
¹ H NMR (600MHz, CDCl ₃ ) δ: 7.87-6.70 (96H, m, Ar), 5.38, 5.33 (1H, s, PhCH), 5.26 (1H, d, J = 8.4Hz), 5.23 (1H, d , J = 7.8Hz), 5.21 (1H, d, J = 8.4Hz), 5.13 (1H, d, J = 8.4Hz), 4.97-4.65 (10H, m), 4.61-4.42 (19H, m), 4.35 -4.01 (21H, m), 3.86-3.55 (1
5H, m), 3.51-3.31 (13H, m), 3.25-3.18 (4H, m), 3.10 (1H, d, J = 9.6Hz), 2.99 (1H, dd, J = 3.0Hz), 2.93 (1H , dd, J = 4.8Hz), 2.84 (1H, d, J = 4.8Hz), 1.11, 1.07 (9H, s,
(CH ₃ ) ₃ CC = O).
MS (MALDI-TOF) calcd for r C ₂₁₀ H ₂₀₅ N ₉ O ₅₃ Na (M + Na) ⁺ m / z: 3723.35, found: 3723.35.

Example 3 Synthesis of Compound (2)

Compound (55) (17 mg, 0.0046 mmol) was dissolved in dichloromethane (2.0 mL), and pyridine (79 μL, 0.98 mmol) was added. Tf ₂ O (39 μL, 1.68 mmol) was added at 10 ° C., and the mixture was stirred at room temperature for 9 h. The reaction solution was diluted with ethyl acetate, the ethyl acetate layer was washed with a saturated aqueous sodium hydrogen carbonate solution and brine, the organic layer was dried over sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was dissolved in toluene (700 μL), CsOAc (10 mg, 0.052 mmol) and 18-crown-6 (14 mg, 0.052 mmol) were added, and the mixture was reacted for 30 hours under ultrasound. The reaction solution was diluted with ethyl acetate, the ethyl acetate layer was washed with a saturated aqueous sodium hydrogen carbonate solution and brine, the organic layer was dried over sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by HPLC to obtain compound (2) (9 mg, 51%).

R _f : 0.22 (Toluene: EtOAc = 5: 1)
¹ H NMR (600MHz, CDCl ₃ ) δ: 7.87-6.70 (96H, m, Ar), 5.59 (2H, d, J = 3.0Hz, H-4 ^f , H-4 ⁱ ), 5.31 (1H, d, H-1, J = 9.0Hz), 5.30 5.29 (1H, s, PhCH), 5.26 (1H, d, J = 9.0Hz), 5.19 (1H, d, J = 7.8Hz), 5.11 (1H, d, J = 8.4Hz), 5.08 (1H, d, J = 3.0Hz, H-2 ^c ), 4.91-4.78 (7H, m), 4.82 (1H, dd, J = 10.2Hz, H-4 ^c ), 4.74 (1H, dd, J = 3.0Hz), 4.63-3.99 (47H, m), 3.87 (1H, bs), 3.81 (1H, bd, J = 3.0Hz), 3.69-3.25 (27H, m), 3.15 ( 1H, d, J = 9.6Hz), 3.05 (1H, dd, J = 9.0Hz), 2.90 (1H, m, J = 3.0Hz), 2.77 (1H, dd, J = 10.2Hz), 2.69 (1H, dd, J = 10.2Hz), 2.02, 1.99, 1.97, 1.93 (3H, s, CH ₃ C = O), 1.05, 1.00 (9H, s, (CH ₃ ) ₃ CC = O).
MS (MALDI-TOF) calcd for r C ₂₁₈ H ₂₁₃ N ₉ O ₅₇ Na (M + Na) ⁺ m / z: 3891.39, found: 3891.20.

<Example 4> Synthesis of 9-sugar

In anhydrous CH ₂ Cl ₂ (3 mL), trisaccharide donor (30 mg, 0.021 mmol), trisaccharide acceptor (10.5 mg, 0.009 mmol), NIS (7 mg, 0.032 mmol), AgOTf (6 mg, 0.021 mmol) and dried The mixture of molecular sieves 4A was stirred at −78 ° C. under an argon stream. The resulting mixture was stirred at −20 ° C. for 7 hours, and the reaction was stopped by adding TEA. The reaction mixture was diluted with EtOAc and filtered through celite. The organic layer was washed successively with NaS ₂ O ₃ , saturated aqueous sodium hydrogen carbonate solution and brine, dried over anhydrous Na ₂ SO _{4 and} concentrated under reduced pressure. The crude product was chromatographed on Bio-Beads S-X1 with Toluene-EtOAc to give a fraction consisting of 9 sugars. The nine sugar fraction was chromatographed on PTLC (Toluene: EtOAc = 3: 1) (17 mg, 50%).

R _f : 0.25 (Toluene: EtOAc = 3: 1)
¹ H NMR (600MHz, CDCl ₃ ) δ: 7.85-6.63 (94H, m, Ar), 5.58 (1H, d, J = 3.6Hz), 5.30,
5.28 (1H, s, PhCH), 5.26-5.19 (3H, m), 5.14 (1H, d, J = 9.6Hz), 4.96, 4.79 (1H, dd,
J = 3.0Hz), 4.93 (1H, d, J = 8.4Hz), 4.88 (1H, d, J = 12.0Hz), 4.58-4.45 (13H, m),
4.37-3.87 (30H, m), 3.74-3.26 (26H, m), 3.14 (1H, d, J = 3.6Hz, J = 9.6Hz), 3.01 (1H, bd, J = 9.0Hz), 2.97 (1H , dd, J = 3.0Hz, J = 9.6Hz), 2.90 (1H, m), 2.77 (2H, m),
2.34 (1H, d, (1H, d, J = 3.6Hz), 1.99, 1.92 (3H, s, CH ₃ CO), 1.02, 0.95 (9H, s, (CH ₃ ) ₃ CSiPh ₂ ).
MS (MALDI-TOF) calcd for C ₂₄₂ H ₂₄₁ N ₉ O ₅₇ Si ₂ Na (M + Na) ⁺ m / z: 4199.59, found: 4199.88

  The present invention can be used for protein preparations, sugar chain affinity ligands, enzyme activity measurement kits, sugar chain analysis standards, and the like.

Claims (3)

The following formula (Ia)
[Wherein R ¹ represents a pivaloyl group, R ² represents a benzylidene group together with each other, R ³ represents a hydrogen atom, or a hydroxyl group protected with a tert-butyldiphenylsilyl group or an acetyl group; Any one of ³ represents a hydrogen atom, the other represents a hydroxyl group protected with a tert-butyldiphenylsilyl group or an acetyl group, R ^4a represents a thioether group, a trichloroacetimide group, or a halogen , and R ^5a is the same or different. R ^6a is the same or different and represents a hydrogen atom or a hydroxyl protecting group, and n represents an integer of 1 to 3. ] The sugar derivative represented by this. Formula (Ib) below
[Wherein R ¹ represents a pivaloyl group, R ² represents a benzylidene group together with each other, R ³ represents a hydrogen atom, or a hydroxyl group protected with a tert-butyldiphenylsilyl group or an acetyl group; Any one of ³ represents a hydrogen atom, the other represents a hydroxyl group protected with a tert-butyldiphenylsilyl group or an acetyl group, R ^4b represents a thioether group, a trichloroacetimide group, or a halogen , and R ^5b is the same or different. A protected hydroxyl group or a protected amino group, R ^6b is the same or different and represents a hydroxyl protecting group, and n represents an integer of 1 to 3. In the sugar derivative represented by the following formula (II)
[Wherein, R ^5b and R ^6b are as defined above, R ⁷ represents a hydrogen atom or a hydroxyl group, one of R ⁷ represents a hydrogen atom, and the other represents a hydroxyl group. A sugar derivative represented by the following formula (III):
[Wherein, R ¹ , R ² , R ³ , R ^5b , R ^6b , R ⁷ , n are as defined above] ] The synthesis method of the sugar derivative represented by this. Formula (Ib) below
[Wherein R ¹ represents a pivaloyl group, R ² represents a benzylidene group together with each other, R ³ represents a hydrogen atom, or a hydroxyl group protected with a tert-butyldiphenylsilyl group or an acetyl group; Any one of ³ represents a hydrogen atom, the other represents a hydroxyl group protected with a tert-butyldiphenylsilyl group or an acetyl group, R ^4b represents a thioether group, a trichloroacetimide group, or a halogen , and R ^5b is the same or different. A protected hydroxyl group or a protected amino group, R ^6b is the same or different and represents a hydroxyl protecting group, and n represents an integer of 1 to 3. In the sugar derivative represented by the following formula (II)
[Wherein, R ^5b and R ^6b are as defined above, R ⁷ represents a hydrogen atom or a hydroxyl group, one of R ⁷ represents a hydrogen atom, and the other represents a hydroxyl group. A sugar derivative represented by the following formula (III):
[Wherein, R ¹ , R ² , R ³ , R ^5b , R ^6b , R ⁷ , n are as defined above] A sugar derivative represented by
The protective group of the protected hydroxyl group represented by R ³ is deprotected, and a steric inversion reaction of a free hydroxyl group is carried out, and the following formula (IV)
[Wherein, R ¹ , R ² , R ^5b , R ^6b , R ⁷ , n are as defined above] ] The synthesis method of the sugar derivative represented by this.