JP5429734B2 - Glycosphingolipid synthesis method - Google Patents

Description

  The present invention relates to a method for chemically synthesizing glycolipids. Specifically, it relates to chemical synthesis of glycosphingolipids. The glycosphingolipid of the present invention particularly relates to ganglioside, which is a glycosphingolipid containing sialic acid.

  Being a collection of inanimate molecules, the background of our function as living organisms is the existence of biomolecules that control a vast amount of information and enable advanced organization. When looking at complex life phenomena, such as cell proliferation and its control, differentiation and expression, immunity, and pathologies caused by these abnormalities, it is always present on the cell surface and intercellular matrix It is a series of carbohydrates called complex carbohydrates. Complex carbohydrates are ubiquitously present on the surface layer of cells in a living body, and act as a structure of biological tissue or a specific functional bearer. Carbohydrates acquired a function as a molecule that brings diversity to life by being slightly separated from nucleic acid control in the homogeneity of life controlled by nucleic acids. The characteristics of carbohydrates that can form a wide variety of structures have been closely related to the evolution of organisms, responding well to the role of transmitting various information in the living body to the outside world and receiving information from the outside world. Among saccharides, ganglioside, which is a glycosphingolipid containing sialic acid, is present on the cell surface of animals, and its sialyl sugar chain is oriented outside the cell, revealing the recognition of external information and the existence of self. In addition, it is a molecular species that is deeply involved in basic life phenomena such as hormones, viruses, bacteria, cytotoxins, other receptor functions, cell-to-cell recognition, cell differentiation / proliferation, canceration, and immunity. It has been demonstrated by past studies (Non-Patent Documents 1 to 3).

  Various glycoconjugates including gangliosides are present in the raft fraction of biological membranes and have various functions in vivo. Rafts that exist continuously after the glycolipid reaches the cell membrane are thought to be formed by the interaction of the sphingolipid and cholesterol in the lipid bilayer. The molecular interactions underlying raft formation are not well understood, but there are many reports that glycoconjugates exhibit functions at the biological level by exerting a cluster effect in rafts. (Non-patent Document 4) It is an important future research subject to further clarify what functions the aggregated glycoconjugates specifically play.

  Gangliosides are extremely small in vivo, and in addition to the diversity of oligosaccharide chain structures, sialic acid and ceramide molecules, which are lipid moieties, also have diversity, and can be obtained as a pure single compound from nature. Is extremely difficult. This has denied rigorous elucidation of the structure and function of gangliosides. In order to solve this problem, the present inventors synthesize derivatives and analogs as well as natural gangliosides using organic chemistry techniques, and use the resulting various synthetic preparations as sugar chain probes. So, we have clarified the physiological functions of various sugar chains at the "molecular level" and have been aiming at the development of medicine and biology widely. Synthetic chemical methods are considered to be an indispensable field even in today's advanced genetic engineering and protein engineering in order to enable the provision of all analogs and derivatives in addition to providing pure and large-scale preparations. . In fact, in the research on sugar chains recognized by influenza virus, structure of sugar chain ligand recognized by leukocyte adhesion molecule “Selectin”, high affinity ligand of novel sialic acid recognition lectin “Siglec”, receptor glycolipid of bacterial toxin, etc. Numerous new findings have been found by using various derivatives and analogs in which the structure and bonding position of the sugar and lipid moieties, the bonding mode, and the functional group are changed (Non-patent Document 5).

As described above, there are still difficult problems in sugar chain synthesis chemistry that has synthesized various sugar chains and contributed to the development of life science. It is the introduction of lipids into sugar chains. Introduction of lipids into sugar chains is one of the most important reactions in the synthesis of gangliosides, and there are two possible synthetic strategies. One is a method of introducing a lipid into glucose or lactose at the reducing end and then extending the sugar chain in the direction of the non-reducing end. The other is a method of introducing a lipid after completing the sugar chain construction. (Formula)
(1) A method for extending sugar chains toward the non-reducing end

(2) Method of introducing lipid after completion of sugar chain construction

As an example of the first method, Hashimoto et al. Prepared 80% glucosylceramide acceptor from appropriately protected glucose and ceramide and introduced it into the disaccharide of Siaα (2 → 3) Gal. Hematoside ganglioside GM3 was synthesized in a yield (Non-patent Document 6). (Formula: Method of extending sugar chain toward non-reducing end)

In this method, ceramide and sugar chain condensation, which is considered to be the most difficult, is carried out at the monosaccharide (glucose) stage, and further, it is condensed with a sialylgalactose donor as an acceptor, thereby convergingly synthesizing GM3. Yes. For sialylgalactose, GM3 was successfully constructed in a high yield of 80% by using a 1,4-lactone body, which is considered to have good reactivity, and using phosphoramidate as the leaving group. Yes. However, in the presence of an unsaturated bond of ceramide, when deprotecting the Bn group used as a protecting group, Birch reduction must be used, and the reactivity when the sugar chain becomes more complicated No report has been made yet. In addition, since the disaccharide Siaα (2 → 3) Gal on the non-reducing end side forms a 1,4-lactone body, it is difficult to further extend the sugar chain from the Gal4 position such as GM1.

In conventional ganglioside synthesis, the second method of introducing a lipid after constructing a sugar chain has been widely used. However, there are problems with this method. The reactivity of the primary hydroxyl group of ceramide is lowered due to the influence of hydrogen bond with the 2-position amide group, and the yield is liable to decrease due to the by-product of orthoester. As a solution to this problem, two methodologies have been devised. One is an improvement on the sugar donor side, which is a method of introducing a protecting group (typically a pivaloyl group) that is sterically bulky and hardly generates an orthoester into the 2-position hydroxyl group of the reducing terminal glucose (the following formula) (Non-Patent Document 7). The other is an improvement on the lipid receptor side, which uses azidosphingosine, which is a precursor of ceramide, as a lipid (lower formula below) (Non-patent Document 8).
(Method of introducing lipid after completion of sugar chain construction (improved sugar donor side))

(Ac is an acetyl group, Me is a methyl group, MS4A is a molecular sieve, Bz is a benzyl group, Piv is a pivaloyl group, Et is an ethyl group.)
(Method of introducing lipid after completing sugar chain construction (improvement on the lipid receptor side))

(WSC is 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide, hydrochloride.)
By introducing a sterically bulky protecting group into the hydroxyl group at the 2-position of the reducing terminal glucose, the formation of orthoester is reduced, and nucleophilic attack of ceramide becomes possible from the target β-plane. The method of using azidosphingosine as a lipid is intended to reduce the steric hindrance of the two alkyl chains of ceramide, and to prevent a reduction in the nucleophilicity of the primary hydroxyl group due to the hydrogen bond between the primary hydroxyl group and the amide by using azide. is there. In terms of condensation yield, the latter method using azidosphingosine is superior, but preparation of azidosphingosine, reduction of azido group, introduction of fatty acid and complicated synthesis steps before and after that are necessary, each having advantages and disadvantages. .

  The present inventors have widely used a method (formula) using azidosphingosine, which is a ceramide precursor, for introducing ceramide when synthesizing gangliosides. In this method, the 1-position of glucose on the sugar chain reducing end side is imidated, followed by steps of i) condensing an azido sphingosine derivative, ii) selective reduction of the azido group, iii) introduction of fatty acid, and deprotection. .

  The yields at each stage of ganglio ganglioside and lacto ganglioside are listed below (table below).

  (Table 1 Yield at each stage in the conventional ceramide introduction method)

In many cases, i) condensation of azidosphingosine derivatives is in low yield. Sugar chains and ceramides have a bulky molecular structure with both extreme properties of hydrophilicity and hydrophobicity, and the reactivity is significantly lowered due to overlapping of factors that inhibit glycosylation such as steric hindrance and hydrogen bonding. In order to overcome the problem of ceramide introduction, improvement of this point is considered essential.

  Non-Patent Document 9 describes the result of a reaction for introducing a ceramide precursor called azidosphingosine. In this method, after the reaction for introducing the precursor, it is necessary to undergo reduction of the azide group and condensation with stearic acid. Non-patent document 9 achieves a certain yield, but many of the reagents used in this method are expensive and are not suitable for industrialization.

  Non-Patent Document 14 describes the synthesis of ganglioside GQ1b and related polysialogangliosides. However, the yield and selectivity are not so high and are not suitable for industrialization.

  Non-Patent Document 15 describes the synthesis of tumor-related gangliosides. However, the yield and selectivity are not so high and are not suitable for industrialization.

  Non-patent document 16 describes the synthesis of gangliosides GM1b and GD1a. However, the yield and selectivity are not so high and are not suitable for industrialization.

  Non-Patent Document 17 describes intramolecular α-glycosylation via glycosides linked by succinyl. However, yield and selectivity are not so high. However, the yield and selectivity are not so high and are not suitable for industrialization.

  Non-Patent Document 18 is a review on the synthesis of gangliosides such as GM3. However, there is no description about improvement in yield and selectivity, and there is no description about improvement for industrialization.

However, Non-Patent Documents 14 to 18 describe that glucosylceramide obtained by intramolecular condensation is used as a synthetic intermediate and is a versatile method that can also be applied to the synthesis of higher-order gangliosides. There is no suggestion.
Ando, T .; Ando, H .; Kiso, M. Trends in Glycosci. Glycotechnol. 2001, 13, 573-586. Hutley et al., Science, 2001, 291, 2337 Varki, A. Glycobiology. 1993, 3, 97-130. Yasuo Suzuki. Biochemistry 2004, 76, (3), 227-233 Hasegawa, A .; Kato, M .; Ando, T .; Ishida, H .; Kiso, M. Carbohydr. Res. 1995, 274, 155-163. Sakamoto, H .; Nakamura, S .; Tsuda, T .; Hashimoto, S. Tetrahedron Lett. 2000, 41, 7691-7695. Numata, M .; Sugimoto, M .; Koike, K .; Ogawa, T .; Carbohydr. Res. 1990, 203, 205-217. Murase, T .; Ishida, H .; Kiso, M .; Hasegawa, A. Carbohydr. Res. 1989, 188, 71-80. Hasegawa, A .; Nagahama, T .; Ohki, H .; Kiso, MJ Carbohydr. Chem. 1992, 11, 699-714. Ishida, H.-K .; Ishida, H .; Kiso, M .; Hasegawa, A. Carbohydr. Res. 1994, 260, C1-C6. Ando, H .; Ishida, H .; Kiso, M .; Hasegawa, A. Carbohydr. Res. 1997,300, 207-217. Ando, T .; Ishida, H .; Kiso, M. Carbohydr. Res. 2003, 338, 503-514. Kameyama, A .; Ishida, H .; Kiso, M .; Hasegawa, A. Cabohydr. Res. 1990,200, 269-285 Ishida, H.-K .; Ishida, H .; Kiso, M .; Hasegawa, A. Tetrahedron: Asymmetry, vol.5, No.12, 2493-2512 Kameyama, A .; Ishida, H .; Kiso, M .; Hasegawa, AJ Carbohydrate Chemistry, 1991, 10, 549-560 Hasegawa, A .; Ishida, H .; Nagahama, T .; Kiso, MAJ CarbohydrateChemistry, 1993, 12, 703-718 Ziegler, T .; Dwttmann, R .; Ariffadhillah; Zettl, UJ CarbohydrateChemistry, 1999, 18, 1079-1095 Ishida, H .; Kiso, M. Trends in Glycoscience and Glycotechnology2001, 13, 57-64

  As described above, conventionally, in the introduction of a lipid amide such as ceramide, a method using azidosphingosine, which is a precursor of ceramide, has been used. This method is said to have the best yield of introduction of lipids into the sugar chain, but still a satisfactory introduction yield has not been achieved. This method is also not an efficient method because it requires cumbersome experimental procedures and the use of harmful reagents in the preparation of azidosphingosine and reduction of the azido group.

  For these reasons, a method for introducing a lipid amide such as ceramide, which is simple and has a high yield, is expected without affecting the type or size of the sugar chain. As mentioned earlier, establishing an efficient lipid introduction method in addition to the conventional method for constructing a sugar chain structure has been reported on the importance of raft concepts and complex carbohydrates, as well as lipids themselves. It is very useful today and is expected to contribute to the further development of glycan life science. Therefore, the purpose of this study was to reconsider the lipid introduction method, protecting groups, convenience and versatility, and to develop an efficient glycolipid synthesis method. The present invention also provides:

It is also aimed to prepare gangliosides such as GM3 exemplified in (wherein Ac represents acetyl).

  The present inventors synthesized glucosylceramide in a high yield by using intramolecular condensation for introduction of ceramide into sugar (glucose), and the obtained glucosylceramide was used as an acceptor as it was as a further sugar chain. The above problem has been solved by finding that it can be used for expansion. Therefore, the present invention also provides a method for synthesizing glycolipids and a method for using the same.

  For example, the method of Non-Patent Document 9 is not suitable for industrialization because it requires a complicated method such as the use of a ceramide precursor and a high reagent is required. On the other hand, in the method of the present invention, it was possible to achieve a high yield of ceramides including not only plant type but also animal type ceramides even if they were used.

In order to achieve the above object, the present invention provides, for example, the following means.
(Item 1)
A method for producing a glycolipid, comprising:
(A) reacting a protected sugar and a lipid amide protector under conditions under which the protected sugar and the lipid amide protector bind to produce a sugar-lipid amide acceptor precursor;
(B) reacting the sugar-lipid amide acceptor precursor under conditions where an intramolecular condensation reaction in the sugar-lipid amide acceptor precursor proceeds to produce a sugar-lipid amide acceptor;
(C) reacting the sugar-lipid amide acceptor with a protected sugar chain donor under a condition in which the sugar-lipid amide acceptor and the protected sugar chain donor are linked to form a protected sugar And (d) subjecting the protected glycolipid to a deprotection reaction under conditions under which the protected sugar chain donor deprotects to produce a glycolipid. .
(Item 2)
The protected sugar is

A sugar protector selected from the group consisting of:
PRO is a protecting group independently selected from the group consisting of benzoyl (Bz), pivaloyl (Piv), MPM (p-methoxybenzyl), methoxyphenyl (MP), acetyl, benzyl, and methyl;
L is independently -SPh, -SCH ₃ , -SCH ₂ CH ₃ , -F, -OPO (OPh) ₂ (where Ph is phenyl), -OPO (N (CH ₃ ) ₂ The method according to the above item, which is a leaving group selected from the group consisting of ₂ and trichloroacetimidate.
(Item 3)
The protected lipid amide has the following formula:

Wherein R ₁ and R ₂ are independently selected from alkyl or alkenyl groups;
The _{R 3} are, tert- butyldiphenylsilyl (TBDPS), tert- butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS), trityl (Tr), is selected from the group consisting of isopropylidene ketals and methoxy acetal;
R ₄ is a protecting group selected from the group consisting of succinyl, malonyl, phthaloyl, oxalyl, carbonyl, benzoyl, acetyl and pivaloyl.
The method according to the above item.
(Item 4)
The protected lipid amide is

Selected from the group consisting of:
The sugar-lipid amide acceptor precursor is

And the sugar-lipid amide acceptor is

Where R ₁ is p-methoxybenzyl (MPM), methoxyphenyl (MP), allyl;
R ₂ is MPM, Bz, MP, allyl;
Said R ₃ is selected from the group consisting of TBDPS, TBDMS, TIPS, Tr, isopropylidene ketal and methoxybenzylidene acetal;
R ₄ and R ₅ are independently a protecting group selected from the group consisting of succinyl, malonyl, phthaloyl, oxalyl, carbonyl, benzoyl, acetyl and pivaloyl, and Ph is phenyl.
The method according to the above item.
(Item 5)
The method according to the above item, wherein the condition for binding the protected sugar and the protected lipid amide is a condition for binding an alcohol and a carboxylic acid.
(Item 6)
The step (a) includes mixing and reacting the protected sugar chain and the lipid amide in a solvent in the presence of a reagent at a predetermined reaction temperature and reaction time.
The reaction temperature is room temperature or higher;
The solvent is tetrahydrofuran _{(THF), CH 2 Cl 2} , benzene, toluene, N, is selected from the group consisting of N- dimethylformamide (DMF) and combinations thereof;
The reagent is triphenylphosphine (PPh ₃ ), azodicarboxylic acid diethyl ester (DEAD), 1-methyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (WSC), 2,4,6-trichlorobenzoyl Selected from the group consisting of chloride, triethylamine (Et ₃ N), and 4-dimethylaminopyridine (DMAP) and combinations thereof; and the reaction time is from 2 hours to 4 hours;
The method according to the above item.
(Item 7)
The method according to the above item, wherein the reagent is PPh ₃ and DEAD, the solvent is THF, and the reaction temperature is 90 ° C. or higher.
(Item 8)
In the above item, the reagent is WSC or 2,4,6-trichlorobenzoyl chloride, Et ₃ N and DMAP, the solvent is CH ₂ Cl ₂ , and the reaction temperature is 30 to 60 ° C. The method described.
(Item 9)
The method according to the above item, wherein the reagent is WSC, the solvent is CH ₂ Cl ₂ , and the reaction temperature is room temperature.
(Item 10)
The method according to the above item, wherein the reaction time is 3 hours.
(Item 11)
The solvent is tetrahydrofuran (THF);
The reagent is triphenylphosphine (PPh ₃ : 3.0 equivalent) and DEAD (3.0 equivalent), and the corresponding amount is equivalent to the protected sugar chain,
The reaction temperature is 90 ° C. and the reaction is carried out under reflux;
The method according to the above item.
(Item 12)
The solvent is CH ₂ Cl ₂ ;
The reagent is 1-methyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (WSC: 3.0 equivalents), and the corresponding amount is equivalent to the protected sugar chain,
The temperature is room temperature;
The method according to the above item.
(Item 13)
The solvent is CH ₂ Cl ₂ ;
The reagents are 2,4,6-trichlorobenzoyl chloride (1.1 equivalent), triethylamine (Et ₃ N: 1.5 equivalent) and 4-dimethylaminopyridine (DMAP: 3.0 equivalent). , The corresponding amount is equivalent to the protected sugar chain,
The temperature is room temperature;
The method according to the above item.
(Item 14)
In the above item, the step (a) includes adding a spacer precursor to the sugar and the protected lipid amide to further bind the protected lipid amide to the sugar via a spacer. The method described.
(Item 15)
The method according to the above item, wherein the spacer and the sugar or the protected lipid amide are previously bound.
(Item 16)
The method according to the above item, further comprising a step of deprotecting the hydroxyl group at the 1-position by reacting the protected lipid amide under a condition where the hydroxyl group at the 1-position of the protected lipid amide is deprotected.
(Item 17)
When the spacer precursor is succinic acid and the succinic acid binds to R ₃ of the lipid amide protector,
R ₃ is isopropylidene ketal or methoxybenzylidene acetal, and R ₄ is

(Ac) or

(Bx) and R ₅ is Ac or Bx.
(Item 18)
When the spacer precursor is succinic acid and the succinic acid binds to R ₄ of the lipid amide protector,
R ₃ is selected from the group consisting of trityl (Tr), TBDPS and TBDMS;
R ₄ is selected from the group consisting of succinyl, malonyl, oxalyl, carbonyl, glutaryl and phthaloyl, and R ₅ is acetyl or benzoyl.
The method according to the above item.
(Item 19)
When the spacer precursor is succinic acid and the succinic acid binds to R ₅ of the lipid amide protector,
R ₃ is selected from the group consisting of Tr, TBDPS and TBDMS,
R ₄ is selected from the group consisting of succinyl, malonyl, oxalyl, carbonyl, glutaryl and phthaloyl, and R ₅ is acetyl or benzoyl.
The method according to the above item.
(Item 20)
The method according to the above item, wherein the sugar residue on the reducing end side of the oligosaccharide is bound to the protected lipid amide through a spacer.
(Item 21)
The step (b) is performed in the presence of an activator for activating the intramolecular condensation reaction, and the activator includes N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH), A process according to the preceding item, selected from the group consisting of trimethylsilyl trifluoromethanesulfonic acid (TMSOTf), dimethyl (methylthio) sulfonium triflate (DMTST), N-bromosuccinimide (NBS) and combinations thereof.
(Item 22)
The step (b)
At a reaction temperature of −80 ° C. to room temperature;
In a solvent selected from the group consisting of CH ₂ Cl ₂ , diethyl ether, acetonitrile, diethyl ether, acetonitrile, propionitrile, toluene, nitromethane and combinations thereof;
From the group consisting of N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH), dimethyl (methylthio) sulfonium triflate (DMTST), molecular sieve 4 angstrom (MS4Å), molecular sieve 3 Å (MS3Å) and combinations thereof In the presence of a selected reagent; and for a reaction time of 1-48 hours,
The method according to the above item.
(Item 23)
The method according to the above item, wherein the reaction temperature is -20 to 0 ° C.
(Item 24)
The method according to the above item, wherein the solvent is dichloromethane.
(Item 25)
The method according to the above item, wherein the reagent is N-iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH).
(Item 26)
The method according to the above item, wherein the reaction time is 1 hour to 5 hours.
(Item 27)
The step (b)
Reaction time of 1.5 hours using CH ₂ Cl ₂ as a solvent, N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH), TMSOTf and molecular sieves 4 angstrom (MS4Å) as reagents, The method as described above, comprising reacting at a reaction temperature of 0 ° C.
(Item 28)
The step (b)
Using CH ₂ Cl ₂ as a solvent, N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH) and molecular sieves 4 angstrom (MS 4ＭＳ) as reagents, a reaction time of 5 hours at −40 ° C. The method according to the above item, comprising reacting.
(Item 29)
The step (b)
Using CH ₂ Cl ₂ as a solvent, N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH) and molecular sieves 4 angstrom (MS 4Å) as reagents, a reaction time of 36 hours, initially −80 A process according to the preceding item comprising reacting at a reaction temperature of 0 ° C, then -60 ° C, then -40 ° C, then 0 ° C.
(Item 30)
The step (b)
As a solvent, acetonitrile (MeCN) was used, and N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH) and molecular sieves 3 angstrom (MS3Å) were used as reagents, with a reaction time of 48 hours, initially −40 A process as described above, comprising reacting at 0 ° C and then at a reaction temperature of 0 ° C.
(Item 31)
The step (b)
As a solvent, acetonitrile (MeCN) was used, N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH) and molecular sieves 3 angstrom (MS3Å) were used as reagents, with a reaction time of 1.5 hours, −0 The method according to the above item, comprising reacting at a reaction temperature of ° C.
(Item 32)
The step (b)
Using acetonitrile (MeCN) as a solvent, N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH) and molecular sieves 3 angstrom (MS3Å) as reagents, a reaction time of 3 hours, −20 ° C. The method as described above, which comprises reacting at a reaction temperature.
(Item 33)
The step (b)
Using diethyl ether as a solvent, N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH) and molecular sieves 3 angstrom (MS3Å) as reagents using a reaction time of 25 hours, initially at 0 ° C. Next, the method according to the above item, comprising reacting at a reaction temperature of room temperature.
(Item 34)
The step (b)
Using acetonitrile (MeCN) as a solvent and dimethyl (methylthio) sulfonium triflate (DMTST) and molecular sieves 3 angstrom (MS3Å) as reagents, reacting at a reaction temperature of 0 ° C. for 1 hour reaction time. The method according to the above item, comprising:
(Item 35)
The step (b)
CH ₂ Cl ₂ as a solvent, N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH) and molecular sieves 4 angstrom (MS 4Å) as reagents, reaction time of 5 hours, reaction at 0 ° C. The method according to the above item, comprising reacting at a temperature.
(Item 36)
The step (b)
Using CH ₂ Cl ₂ as a solvent, N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH) and molecular sieves 4 angstrom (MS 4 と し て) as reagents, a reaction time of 1.5 hours, −20 The method according to the above item, comprising reacting at a reaction temperature of ° C.
(Item 37)
The step (b)
Including reaction with CH ₂ Cl ₂ as a solvent using dimethyl (methylthio) sulfonium triflate (DMTST) and molecular sieves 4 angstrom (MS4Å) as reagents for 2 hours reaction time at 0 ° C. The method described in the above item.
(Item 38)
The method according to the above item, wherein the intramolecular condensation reaction is glycosylation.
(Item 39)
The step (c)
In a donor equivalent to more than 2.5 equivalents; at a reaction temperature of −40 to 0 ° C .; in a solvent of CH ₂ Cl ₂ ; in the presence of a trimethylsilyl trifluoromethanesulfonic acid (TMSOTf) reagent, 1 Including reacting for ~ 48 hours reaction time,
The method according to the above item.
(Item 40)
The equivalent of donor to the acceptor is 2.5 equivalents;
The reaction temperature is 0 ° C .;
The solvent is CH ₂ Cl ₂ ;
The reagent is TMSOTf; and the pre-reaction time is 7 hours;
The method according to the above item.
(Item 41)
The protected sugar chain donor is

_{-SPh, -SCH 3, -SCH 2 CH} 3, -F, -OPO (OPh) 2 ( where, Ph is phenyl.) And _{-OPO (N (CH 3) 2} ) selected from the group consisting of ₂ The method according to the above item.
(Item 42)
The step (d)
In a solvent of CH ₂ Cl ₂ ;
In the presence of a reagent for trifluoroacetic acid;
At a reaction temperature of room temperature; and for a reaction time of 2 to 12 hours,
The method according to the above item.
(Item 43)
The method according to the above item, wherein the reaction time is 2 hours.
(Item 44)
(E) The method according to the above item, further comprising a step of reacting and deprotecting the product of the step (d) under conditions under which the acyl protecting group is deprotected.
(Item 45)
The step (e)
In a solvent of methanol (CH ₃ OH) or water (H ₂ O);
In the presence of sodium methoxide (NaOCH ₃ ) or KOH reagent;
Carried out at a reaction temperature of room temperature to 100 ° C .; and for a reaction time of 1 hour to 1 week,
The method according to the above item.
(Item 46)
The solvent is methanol (CH ₃ OH);
The reagent is sodium methoxide (NaOCH ₃ );
The reaction temperature is room temperature; and the reaction time is 12 hours,
The method according to the above item.
(Item 47)

Wherein the method comprises an acceptor compound

And donor compounds

(A), under the condition that the acceptor compound and the donor compound bind to each other
Where
R ¹ is Ac or H;
R ² is Ac or 2,2,2-trichloroethoxycarbonyl (Troc)

Is;
SPh is

Is;
MP is

Is;
SE is

Is;
Ac is

Is;
Bz is

And
Bn is

Is;
The method wherein Me is methyl.
(Item 48A)
The step (A) includes the following:
At a reaction temperature of −40 ° C. to room temperature;
In solvents of CH ₃ CN, CH ₂ Cl ₂ , diethyl ether, acetonitrile, propionitrile, toluene, nitromethane and combinations thereof;
In the presence of a catalyst of N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH), trimethylsilyltrifluoromethanesulfonic acid (TMSOTf) and combinations thereof; and carried out over a reaction time of 1 hour to 3 days.
The method according to the above item.
(Item 48B)
The step (A) includes the following:
At a reaction temperature of −50 ° C. to room temperature;
In solvents of CH ₃ CN, CH ₂ Cl ₂ , diethyl ether, acetonitrile, propionitrile, toluene, nitromethane and combinations thereof;
In the presence of a catalyst of N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH), trimethylsilyltrifluoromethanesulfonic acid (TMSOTf) and combinations thereof; and carried out over a reaction time of 1 hour to 3 days.
The method according to the above item.
(Item 49)
The process of any of the preceding items, wherein the catalyst is selected from the group consisting of N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH), and combinations thereof.
(Item 50)
The method as described above, wherein the solvent is CH ₃ CN, CH ₂ Cl ₂ or a mixed solution of CH ₃ CN and CH ₂ Cl ₂ .
(Item 51)
The method according to the above item, wherein the reaction temperature is −30 ° C. to 0 ° C.
(Item 52)
The method according to the above item, wherein the reaction time is 1 hour to 1 day.
(Item 53)
The step (A)
First using CH ₃ CN as a solvent, then using a mixture of CH ₃ CN and CH ₂ Cl ₂ , using N-iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH) as catalysts for 2 days The process according to the preceding item, comprising reacting at a reaction temperature of initially -30 ° C and then at a reaction temperature of room temperature.
(Item 54)
The step (A)
Using a mixed solution of CH ₃ CN and CH ₂ Cl ₂ as a solvent, using N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH) and TMSOTf as catalysts, a reaction time of 3 days, initially A process as described above, comprising reacting at -30 ° C and then at a reaction temperature of room temperature.
(Item 55)
The step (A)
Using a mixed solution of CH ₃ CN and CH ₂ Cl ₂ as a solvent and N-iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH) as catalysts, a reaction time of 2 days, initially −30 A process as described above, comprising reacting at 0 ° C and then at a reaction temperature of 0 ° C.
(Item 56)
The step (A)
Using a mixed solution of CH ₃ CN and CH ₂ Cl ₂ as a solvent, N-iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH) as a catalyst, a reaction time of 3 days, −30 ° C. Including reacting at the reaction temperature,
The method according to the above item.
(Item 57)
The step (A)
Including reaction with CH ₂ Cl ₂ as a solvent and N-iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH) as a catalyst at a reaction temperature of −30 ° C. for a reaction time of 1 day. To
The method according to the above item.
(Item 58)
The step (A)
Using a mixed solution of propionitrile and CH ₂ Cl ₂ as a solvent, N-iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH) as a catalyst, a reaction time of 6 hours, −50 ° C. Including reacting at the reaction temperature,
The method according to the above item.
(Item 59)
The following formula:

A compound represented by
(Item 60) An amino sugar having an amino group protected with trichloroethoxycarbonyl (Troc), a sugar protected with methoxyphenyl (MP), an amino sugar protected with Troc, and a sugar protected with MP A method for synthesizing oligosaccharides, which comprises a step of reacting under a condition where and bind to each other.
(Item 61) The method according to item 60, wherein the amino sugar has a leaving group L.
(Item 62) The leaving group L is —SPh, —SCH ₃ , —SCH ₂ CH ₃ , —F, —OPO (OPh) ₂ (where Ph is phenyl) and —OPO (N ( CH _{3) 2)} is selected from the group consisting of _2, the method of claim 61.
(Item 63) The Troc-protected amino sugar is the following:

And
Pro is a protecting group independently selected from the group consisting of acetyl (Ac), benzyl (Bn), benzoyl (Bz), pivaloyl (Piv), MPM (p-methoxybenzyl) and methoxyphenyl (MP). And
R ¹ is alkyl,
61. The method according to item 60.
(Item 64) The MP-protected sugar is the following:

Selected from the group consisting of
Pro is a protecting group independently selected from the group consisting of acetyl (Ac), benzyl (Bn), benzoyl (Bz), pivaloyl (Piv), MPM (p-methoxybenzyl) and methoxyphenyl (MP). Is,
61. The method according to item 60.
(Item 65) The method according to any one of Items 60 to 64, comprising a step of deprotecting the Troc group in the presence of Zn (Cu).
(Item 66) The following structure:

A compound having a structure selected from the group consisting of:
(Item 67)
The following formula:

A compound represented by the formula:
Said R ₁ and R ₂ are independently selected from alkyl and alkenyl groups;
The R ₃ is tert-butyldiphenylsilyl (tert-butyldimethylsilyl (TBDPS)), tert-butyldimethylsilyl (tert-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS), tripyryl (TIPS), and trityl (Triden). And selected from the group consisting of methoxybenzylidene acetal;
The compound, wherein R ₄ is a protecting group selected from the group consisting of succinyl, malonyl, phthaloyl, oxalyl, carbonyl, benzoyl, acetyl and pivaloyl.
(Item 68)
The following formula:

Wherein R ₁ and R ₂ are independently selected from alkyl and alkenyl groups;
Said R ₃ is selected from the group consisting of TBDPS, TBDMS, TIPS, Tr, isopropylidene ketal and methoxybenzylidene acetal;
The compound, wherein R ₄ is a protecting group selected from the group consisting of succinyl, malonyl, phthaloyl, oxalyl, carbonyl, benzoyl, acetyl and pivaloyl.

  Accordingly, these and other advantages of the invention will be apparent upon reading the following detailed description.

  According to the present invention, ceramide can be introduced into a sugar in a high yield without complicated preparation of a ceramide precursor, and can be used for further sugar chain elongation without subsequent steps. Deprotection can also be carried out simply and quickly.

  In the synthesis method of the present invention, the yield of condensing the ceramide body is remarkably superior to that of the prior art. In addition, the present invention provides a highly versatile synthesis method in which glucosylceramide obtained by intramolecular condensation is used as a synthetic intermediate, so that the synthetic intermediate can be applied to the synthesis of higher-order gangliosides. provide.

  In the present invention, in view of the importance of glycan synthesis chemistry in glycan life science, for the purpose of developing an efficient glycolipid synthesis method that has not yet been established, (1) condensation condensation between a glycan and a lipid is performed. The research was conducted with the main points of (2) stereoselectivity in lipid introduction; (3) preparation of lipid amides such as ceramide and simplification of conversion. As a result, the present invention typically provides the following remarkable effects.

  (1) Improvement of the condensation yield of sugar chains and lipids was achieved by improving the yield of glycosylation itself using intramolecular glycosylation. In the method of the present invention, in the intramolecular glycosylation and condensation with a sialylgalactose donor, it was not necessary to protect the glucose 4-position with any other group, including the chloroacetyl group. The difference in reactivity with the ceramide hydroxyl group was sufficient, and the reaction process could be greatly simplified by omitting the steps of protection and deprotection. Reducing the number of processes is an important factor for expansion to an industrial scale, that is, industrialization. However, the present invention is considered to be almost impossible by achieving such a large reduction in the number of processes. It can be said that there is a remarkable effect in that the industrial production of glycolipids such as GM3 which has been made possible has been made possible.

  In addition, by using 2.5 equivalents of a sialylgalactose donor in a glycosylceramide acceptor, the GM3 analog was successfully derived in high yield. While saving starting materials is important in industrialization, this desire could also be achieved.

  (2) The stereoselectivity in lipid introduction was completely β-selective due to the participation of adjacent groups when a Bz group was used at the 2-position of glucose. However, when the MPM group was used at the 2-position of glucose, the expected steric control of intramolecular glycosylation by succinic acid was not exerted, and a mixture of α and β was obtained. As a result of examining the reaction solvent and reaction temperature, β selectivity due to the solvent effect of acetonitrile was obtained.

  Usually, it is said that if the stereoselectivity is about 1/5 or more, there is sufficient industrial applicability, but such a highly selective method has not been known in the prior art method. Therefore, it can be said that a synthesis method having excellent stereoselectivity on an industrial scale has not been known. The present invention should have a remarkable effect in that this problem has also been solved.

  (3) Regarding the preparation and conversion of ceramide, the conversion efficiency was improved by using ceramide for glycosylation as it was. In the prior art, it was necessary to use the precursor azidosphingosine for the synthesis of glycolipids. Subsequent conversion often resulted in low yields, and the sugar chains synthesized through multiple steps were wasted. In the present invention, since ceramide can be used for glycosylation as it is, the waste of sugar chain can be omitted, and the preparation and conversion of ceramide can be simplified. In addition, the final yield is also increased.

  It should be understood that the effects achieved by the present invention are not limited to those described above, but include all those that can be understood by those skilled in the art from the matters described in this specification.

  Preferred embodiments of the present invention will be shown below, but those skilled in the art can appropriately implement the embodiments and the like from the description of the present invention and well-known and common techniques in the field, and the effects and effects of the present invention can be easily achieved. It should be recognized to understand.

  Hereinafter, embodiments of the present invention will be described in detail.

  The present invention will be described below. Throughout this specification, singular articles (eg, “a”, “an”, “the”, etc. in the English language) are understood to include the concept of the plural unless specifically stated otherwise. Should be. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. In case of conflict, the present specification, including definitions, will control.

(List of abbreviations)
In the present specification, the following abbreviations are used as necessary.
Ac: Acetyl
Ac ₂ O: acetic anhydride
BDA: benzaldehyde dimethyl acetal
BF ₃ · OEt ₂ : trifluoroborane diethyl etherate (trifluoroborane diethyl etherate)
Bn: benzyl
Bz: Benzoyl
Bz ₂ O: benzoic anhydride
CSA: (±) -camphorsulfonic acid ((±) -camphorsulfonic acid)
DBTO: dibutyltin oxide
DBU: 1,8-diazabicyclo [5.4.0] undec-7-ene (1,8-diazabiccyclo [5.4.0] undec-7-ene)
DEAD: azodicarboxylic acid diethyl ester
DMAP: 4-dimethylaminopyridine (4-dimethylaminopyridine)
DMF: N, N-dimethylformamide (N, N-dimethylformamide)
DMTST: dimethyl (methylthio) sulfonium triflate (dimethylthiol sulfonium triflate)
Et: ethyl Me: methyl MP: p-methoxyphenyl (p-methoxyphenyl)
MPM: p-methoxyphenylmethyl MS3A: molecular sieves 3Å (molecular sieves 3Å)
MS4A: molecular sieves 4 Å (molecular sieves 4 Å)
PPh ₃ : triphenylphosphine
TBAB: tetrabutylammonium bromide
TEA: triethylamine
TFA: trifluoroacetic acid
THF: tetrahydrofuran
p-TsOH: p-toluenesulfonic acid
WSC: 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (1-ethyl-3- (3-dimethylaminopropyl) -carbohydrate)
(General technology)
In carrying out the organic chemistry method in the present invention, the Laboratory Chemistry Course (The Chemical Society of Japan, Maruzen, 4th edition, 1992), Organic Chemistry Experiment Guide 1-5 Kasei Kogyo Co., Ltd. Kodansha Scientific, bioactive sugar chain research method Biochemical experiment method 42 Society publication center, precision organic synthesis-experiment manual- You can refer to experiment manuals such as Nanedo, etc. Is incorporated by reference in its entirety.

  For the synthesis of gangliosides, see Kameyama, A .; Ishida, H .; Kiso, M .; Hasegawa, A .; Cabohydr. Res. 1990, 200, 269-285; Hasegawa, A .; Nagahama, T .; Ohki, H .; Kiso, M .; J. et al. Carbohydr. Chem. 1992, 11, 699-714; Ando, H .; Ishida, H .; Kiso, M .; Hasegawa, A .; Carbohydr. Res. 1997, 300, 207-217; Ishida, H .; -K. Ishida, H .; Kiso, M .; Hasegawa, A .; Carbohydr. Res. References such as 1994, 260, C1-C6 can be referred to, and the contents thereof are incorporated by reference in their entirety.

(Definition of terms)
Listed below are definitions of terms particularly used in the present specification.

  In the present specification, the “sugar chain” refers to a compound formed by connecting one or more unit sugars (monosaccharide and / or a derivative thereof). When two or more unit sugars are connected, each unit sugar is bound by dehydration condensation by a glycosidic bond. Examples of such sugar chains include polysaccharides (glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, sialic acid, and complexes and derivatives thereof) contained in the living body. Other examples include, but are not limited to, a wide range of sugar chains that are decomposed or derived from complex biomolecules such as degraded polysaccharides, glycoproteins, proteoglycans, glycosaminoglycans, glycolipids, and the like. Therefore, in the present specification, the sugar chain can be used interchangeably with “polysaccharide”, “carbohydrate”, and “carbohydrate”. Further, unless otherwise specified, the “sugar chain” in the present specification may include both sugar chains and sugar chain-containing substances. Typically, a substance in which about 20 types of monosaccharides (glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, sialic acid, and complexes and derivatives thereof) are connected in a chain. It is attached to proteins and lipids inside and outside the cells of the body. The functions differ depending on the sequence of monosaccharides, and they are usually branched in a complex manner. The human body is expected to have several hundreds of sugar chains with various structures, and there are tens of thousands of useful structures in the human body. It is believed that there are more than types. Although it is considered to be related to higher-order functions that proteins and lipids perform in vivo, such as cell-to-cell molecular and cell recognition functions, there are many unexplained mechanisms. It is attracting attention in current life science as the third life chain after nucleic acid and protein. In particular, the function of a sugar chain as a ligand (information molecule) in cell recognition is expected, and its application to the development of highly functional materials is being studied.

  In this specification, the “sugar chain group” is a name given when a sugar chain is bonded to another group. The sugar chain group refers to a monovalent or divalent group depending on the case. For example, examples of the sugar chain group include a sialyl Lewis X group, an N-acetyllactosamine group, and an α1-6 mannobiose group.

  As used herein, “sugar” or “monosaccharide” refers to polyhydroxyaldehyde or polyhydroxyketone containing at least one hydroxyl group and at least one aldehyde group or ketone group, and constitutes the basic unit of a sugar chain. In this specification, sugar is also referred to as carbohydrate and both are used interchangeably. In this specification, when specifically mentioned, a sugar chain refers to a chain or a sequence in which one or more sugars are linked, and when referred to as a sugar or a monosaccharide, it refers to one unit constituting the sugar chain. Here, those in which n = 2, 3, 4, 5, 6, 7, 8, 9 and 10 are referred to as diose, triose, tetrose, pentose, hexose, heptose, octose, nonose and decourse, respectively. Generally, it corresponds to an aldehyde or ketone of a chain polyhydric alcohol. The former is called aldose and the latter is called ketose. In the present invention, any type can be used.

  The sugar chain that can be used in the present invention can be synthesized by a general sugar chain synthesis method. These methods include (1) chemical synthesis, (2) fermentation using genetically modified cells or microorganisms, (3) synthesis using sugar hydrolase (glycosidase), (4) glycosyltransferase And a method of synthesis using (glycosyltransferase). (WO 2002/081723, JP-A-9-31095, JP-A-11-42096, JP-A-2004-180676, Kenichi Hatanaka, Shinichiro Nishimura, Goro Ouchi and Kazuyoshi Kobayashi (1997) Glucose Science and Industry, Kodansha See Tokyo etc.).

  The nomenclature and abbreviations used to describe sugars in the present invention follow conventional nomenclature. For example, β-D-galactose

Is Gal;
N-acetyl-α-D-galactosamine

Is GalNAc;
α-D-mannose

Is Man;
β-D-glucose

Is Glc;
N-acetyl-β-D-glucosamine

Is GlcNAc;
α-L-Fucose

Is Fuc;
α-N-acetylneuraminic acid

Is Neu5Ac;
Ceramide

Is represented by Cer. It should be noted that Cer is usually classified as a lipid, but in this specification, it can also be included in the definition of a kind of sugar constituting a sugar chain. Two cyclic anomers are represented by α and β. For reasons of display, it may be expressed as a or b. Therefore, in the present specification, α and a, and β and b are used interchangeably for the anomeric notation.

  In the present specification, “galactose” refers to any isomer, but is typically β-D-galactose, and is used to refer to β-D-galactose unless otherwise specified.

  As used herein, “acetylgalactosamine” refers to any isomer, but is typically N-acetyl-α-D-galactosamine, and unless otherwise specified, N-acetyl-α-D-galactosamine. Used to refer to

  In the present specification, “mannose” refers to any isomer, typically α-D-mannose, and is used to refer to α-D-mannose unless otherwise specified.

  In the present specification, “glucose” refers to any isomer, but is typically β-D-glucose, and is used to refer to β-D-glucose unless otherwise specified.

  In the present specification, “acetylglucosamine” refers to any isomer, typically N-acetyl-β-D-glucosamine, and unless otherwise specified, N-acetyl-β-D-glucosamine. Used to refer to

  In the present specification, “fucose” refers to any isomer, but is typically α-L-fucose, and is used to refer to α-L-fucose unless otherwise specified.

  In the present specification, “sialic acid” is a general term for derivatives of neuraminic acid. N-acyl (N-acetyl or N-glycolyl) neuraminic acid and N-acyl-O-acetylneuraminic acid are naturally occurring. A generic term for amino sugars having 9 or more carbon atoms. It can be represented by an N- or O-acyl derivative of neuraminic acid.

  In the present specification, “acetylneuraminic acid” refers to any isomer, but is typically α-N-acetylneuraminic acid, and α-N-acetylneuraminic acid is referred to unless otherwise specified. Used as a pointer.

  In this specification, it is noted that the symbol, designation, abbreviation (Glc, etc.), etc. of sugars are different when they represent monosaccharides and when used in sugar chains. In the sugar chain, the unit sugar exists in a form in which hydrogen or a hydroxyl group is removed from the other side because dehydration condensation occurs between the unit sugar and another unit sugar to be bound. Therefore, when these sugar abbreviations are used to represent monosaccharides, all hydroxyl groups are present, but when used in a sugar chain, the hydroxyl groups of the sugars to which the hydroxyl groups are bound are dehydrated. It is understood that only oxygen remains after condensation.

  When a sugar is covalently bonded to albumin, the reducing end of the sugar is aminated and can be bound to other components such as albumin via its amine group, in which case the hydroxyl group at the reducing end is an amine group. Note that it has been replaced with.

  Monosaccharides are generally joined by glycosidic bonds to form disaccharides and polysaccharides. The bond orientation with respect to the plane of the ring is indicated by α and β. Specific carbon atoms that form a bond between two carbons are also described.

  As necessary, in this specification, the sugar chain is

However, the present invention is not limited to this. When this method is used, for example, the β glycoside bond between C-1 of galactose and C-4 of glucose is represented by β1,4. Thus, for example, sialyl Lewis X (SLX) is represented as Neu5Acα2,3Galβ1,4 (Fucα1,3) GlcNAc. N-acetyllactosamine (G4GN) is represented as Galβ1,4GlcNAc. α1-6 mannobiose (A6) is represented as Manα1,6Man. In the present specification, other display methods can be used as necessary.

  The branch of the sugar chain is represented by parentheses and is described by being placed immediately to the left of the unit sugar to be bound. For example,

And in parentheses are

It is written. Thus, for example, when β-glycosidic bond is formed between C-1 of galactose and C-4 of glucose, and C-3 of glucose is further α-glycoside bonded to C-1 of fucose, Galβ1,4 ( It is expressed as Fucα1,3) Glc.

  Monosaccharides are represented on the basis of numbering as low as possible to (latent) carbonyl groups. Even when an atomic group superior to a (latent) carbonyl group is introduced into a molecule according to the general standard of organic chemical nomenclature, it is usually represented by the above numbering.

Examples of the sugar chain used in the present specification include sugar chains selected from the group consisting of sialyl Lewis X, N-acetyllactosamine, α1-6 mannobiose, and combinations of two or more thereof. It is not limited to. The reason why two or more combinations can be used is not limited by theory, but each of the sugar chains has specificity for a lectin localized in the tissue or cell of the target delivery site, and is mixed. This is because it is considered that the specificity is exhibited.

  As used herein, “protected sugar” refers to a sugar protected with a protecting group. Any protecting group can be used as long as the sugar group can be protected from certain reactions. This is because the protecting group is generally used not only for protecting a hydroxyl group or amino group but also for stereocontrol of glycosyl, but in the method of the present invention, stereocontrol is achieved by the solvent effect. Thus, the role of the protecting group is to protect the sugar from certain reactions, and if it can be achieved, glycolipids can be produced. Various protecting groups can be used in consideration of the reactivity. Thus, protected sugars are, for example,

Although not limited to these, it is not limited to these. The protected sugar chains exemplified above are those using the main sugars existing in nature, and various sugar chains can be constructed by combining these. In addition, it is understood that the display of oligosaccharides in the formula is an example, and the number of sugars contained may be any number as long as it is 2 or more. In addition, the sugar may be a sugar derivative. This is because even non-natural sugar derivatives have been reported to have physiological activity.

  In one embodiment, in the protected sugar used in the present invention, an acetyl group, a benzoyl group, a benzyl group, or the like can be used as a hydroxyl protecting group, and a methyl group, benzyl can be used as a carboxyl group protection. Groups and the like can be used, but are not limited to these. As the leaving group, a phenylthio group, a fluorine group, a trichloroacetimidate group and the like can be used, but are not limited thereto.

  As used herein, “lipid amide protector” refers to the following formula:

Wherein R ₁ and R ₂ are independently selected from alkyl or alkenyl groups; R ₃ is TBDPS , TIPS, Tr, isopropylidene ketal, methoxybenzylidene acetal; the R ₄ is a protecting group which can be succinyl, malonyl, phthaloyl, oxalyl, carbonyl, benzoyl, acetyl, pivaloyl. Various protecting groups can be used in consideration of the reactivity. It is understood that these protecting groups may be any protecting group other than the exemplified protecting groups as long as they perform a protecting function. This is because in the method of the present invention, if only the primary hydroxyl group at the 1-position is glycosylated and the other hydroxyl groups can be protected, glycolipids can be produced even if other lipid amide protectors are used. Because.

  For example, as a protected lipid amide,

However, it is not limited to these. It is understood that these protecting groups R ₃ , R ₄ and R ₅ may be any protecting group other than the exemplified protecting groups as long as they perform a protecting function. This is because in the method of the present invention, if only the primary hydroxyl group at the 1-position is glycosylated and the other hydroxyl groups can be protected, glycolipids can be produced even if other lipid amide protectors are used. Because.

  In the present specification, the “conditions for bonding a protected sugar and a protected lipid amide” may be any conditions for bonding the protected sugar and a protected lipid amide, and those skilled in the art It can be implemented appropriately with reference to the textbooks listed in the technology. Exemplary conditions may be, for example, conditions where an alcohol and a carboxylic acid are combined. This is because the protected sugar has a free hydroxyl group that is not protected only at one site, and the protected lipid amide has a spacer having a carboxyl group. Although not wishing to be bound by theory, a reasonable explanation is presented that the conditions for binding the protected sugar and the protected lipid amide may be those for binding the alcohol and the carboxylic acid. .

As another illustrative condition, for example, solvents, tetrahydrofuran _{(THF), CH 2 Cl 2} , benzene, toluene, N, N-dimethylformamide (DMF), although a combination thereof, without limitation.

Reagents are triphenylphosphine (PPh ₃ ), DEAD, 1-methyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (WSC), 2,4,6-trichlorobenzoyl chloride, triethylamine (Et ₃ N) , 4-dimethylaminopyridine (DMAP), combinations thereof, and the like.

  The reaction time may be 2 hours to 4 hours, but may be a time shorter than this range or a reaction time exceeding this range. This is because if the reaction does not proceed sufficiently, the reaction time can be extended, while if the reaction proceeds efficiently, the reaction time is shortened.

  The reaction temperature is, for example, 0 ° C., 0 ° C. to room temperature (eg, 0 ° C., 5 ° C., 10 ° C., 15 ° C., 20 ° C., etc.), room temperature or higher (eg, room temperature to 90 ° C., reflux 90 ° C., 90 ° C. or higher). ), But is not limited thereto. This is because the reaction temperature can vary depending on other conditions such as the solvent.

  These reaction conditions can be appropriately selected by those skilled in the art while considering the progress of the reaction.

  As used herein, “room temperature” refers to a temperature in the range of about 15 ° C. to about 30 ° C., preferably about 20 ° C. to about 25 ° C.

  In the present specification, the “sugar-lipid amide acceptor precursor” is not a sugar-lipid amide acceptor, but is any substance that becomes a sugar-lipid amide acceptor when it is further reacted (for example, condensed). Say. When another expression method is used, it is a general term for compounds in which a sugar and a lipid amide acceptor precursor are bound. When the lipid amide is ceramide, it can also be referred to as a sugar-ceramide acceptor precursor.

  Examples of sugar-lipid amide acceptor precursors include:

The R ₁ and R ₂ may be independently an alkyl group, an alkenyl group, etc .; the R ₃ may be tert-butyldiphenylsilyl (tert-butyldiphenylsilyl (TBDPS)), tert-butyldimethylsilyl (tert-butyldimethylsilyl). (TBDMS), triisopropylsilyl (TIPS), trityl (Tr), isopropylidene ketal, methoxybenzylidene acetal; the R ₄ is succinyl, malonyl, phthaloyl, oxalyl, carbonyl, benzoyl, acetyl, pivaloyl However, the protecting group may be any protecting group other than the exemplified protecting groups as long as it performs a protecting function. It is understood that a thioglycoside is used as a leaving group in the method of the present invention, and a protecting group other than the above may be used as long as the activation condition is resistant. This is because glycolipids can also be produced using the used sugar-lipid amide acceptor precursor.

  In the present specification, “the conditions under which the intramolecular condensation reaction in the sugar-lipid amide acceptor precursor proceeds” may be any conditions under which the intramolecular condensation reaction in the sugar-lipid amide acceptor precursor proceeds. Those skilled in the art can appropriately implement the textbooks listed in the above technology.

  As exemplary conditions, the reaction temperature may be, for example, -80 ° C to room temperature (eg, -40 ° C to room temperature, -20 ° C to 0 ° C, -80 ° C to 0 ° C), but is not limited thereto. .

  In one embodiment, the reaction temperature may vary during the reaction. As an example of changing the reaction temperature, for example, the temperature may be first increased to −80 ° C., then −60 ° C., then −40 ° C., then 0 ° C. Or may be raised from 0 ° C. to room temperature. This is because the reaction temperature only needs to proceed sufficiently. Accordingly, those skilled in the art can appropriately change the reaction temperature in consideration of the reaction situation.

As the solvent, for example, CH ₂ Cl ₂ , diethyl ether ((CH ₂ CH ₃ ) ₂ O), diethyl ether, acetonitrile, diethyl ether, acetonitrile, propionitrile, toluene, nitromethane, combinations thereof and the like can be used. However, it is not limited to these. This is because any solvent may be used as long as the target reaction proceeds. A person skilled in the art can appropriately select a solvent to be used in consideration of the reaction situation.

  Examples of the reagent include N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH), dimethyl (methylthio) sulfonium triflate (DMTST), molecular sieve 4 angstrom (MS4Å), molecular sieve 3 angstrom (MS3Å) and the like. Can be used. Here, it is understood that as the reagent used, a desiccant and the like can be appropriately present in addition to the catalyst. This is because any reagent may be used as long as the target reaction proceeds. A person skilled in the art can appropriately select the reagent to be used in consideration of the reaction situation.

  The reaction time is, for example, 1 to 48 hours (for example, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, 36 hours). 48 hours) (preferably 1 hour to 5 hours), etc., but other ranges may be used. This is because if the reaction does not proceed sufficiently, the reaction time can be extended, while if the reaction proceeds efficiently, the reaction time is shortened.

  In the present specification, the “sugar-lipid amide acceptor” refers to a compound having a function of receiving a sugar chain and having a sugar-lipid structure in the structure.

  Examples of sugar-lipid amide acceptors include:

The _{R 1} and said _{R 2} are independently an alkyl group, an alkenyl group obtained; the _{R 3} is TBDPS, TBDMS, TIPS, Tr, isopropylidene ketal, methoxy an acetal obtained; the _{R 4} is , Succinyl, malonyl, phthaloyl, oxalyl, carbonyl, benzoyl, acetyl, pivaloyl. It is understood that these protecting groups may be any protecting group other than the exemplified protecting groups as long as they perform a protecting function. This is because, in the method of the present invention, thioglycoside is used as a leaving group, and a sugar-lipid amide acceptor using a protecting group other than the above can be used as long as the activation conditions are resistant. This is because glycolipids can be produced.

  In the present specification, the “protected sugar chain donor” refers to a “sugar chain donor” protected with a protecting group. It is understood that the protecting group used may be any protecting group other than the exemplified protecting groups as long as it performs a protecting function. Examples of these protected sugar chain donors include:

Although not limited to these, it is not limited to these. The protected sugar chain donor may be any sugar as long as it has a leaving group at the reducing end of the sugar chain and other protecting groups are protected. This is because, in glycosylation, only the anomeric position of the sugar residue at the reducing end of the sugar chain is eliminated by the activator, and the sugar-lipid amide acceptor causes a nucleophilic attack there. Accordingly, examples of such a leaving group include -SPh, -SCH ₃ , -SCH ₂ CH ₃ , -F, -OPO (OPh) ₂ , -OPO (N (CH ₃ ) ₂ ) ₂ (wherein , Ph is phenyl), but is not limited thereto.

  As used herein, “sugar chain donor” refers to a compound that provides a sugar chain to a sugar acceptor. Therefore, the “sugar chain donor” may be any substance capable of providing a sugar chain to a sugar acceptor, and typically consists of a sugar moiety and other moieties. In the present specification, “sugar chain donor” and “sugar donor” may be used interchangeably.

  In the present specification, “conditions for linking a sugar-lipid amide acceptor and the protected sugar chain donor” are arbitrary conditions for linking the sugar-lipid amide acceptor and the protected sugar chain donor. Any person skilled in the art can appropriately implement the textbooks listed in the above-mentioned technology.

  As an illustrative example, for example, greater than about 2.5 equivalents of donor equivalent to acceptor may be used, but is not limited thereto. This is because a person skilled in the art can appropriately change the equivalent of the donor in consideration of other conditions.

  The reaction temperature is, for example, −40 to 0 ° C. (for example, −40 ° C., −35 ° C., −30 ° C., −25 ° C., −20 ° C., −15 ° C., −10 ° C., 5 ° C., 4 ° C., 3 ° C. , 2 ° C, 1 ° C, 0 ° C, etc.), but is not limited thereto. This is because the reaction temperature only needs to proceed sufficiently. Accordingly, those skilled in the art can appropriately change the reaction temperature in consideration of the reaction situation.

Examples of the solvent include, but are not limited to, CH ₂ Cl _{2 and the} like. This is because any solvent may be used as long as the target reaction proceeds. A person skilled in the art can appropriately select a solvent to be used in consideration of the reaction situation.

  Examples of the reagent include, but are not limited to, trimethylsilyl trifluoromethanesulfonic acid (TMSOTf) and the like. This is because any reagent may be used as long as the target reaction proceeds. A person skilled in the art can appropriately select the reagent to be used in consideration of the reaction situation.

  The reaction time is, for example, 1 to 48 hours (for example, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, 36 hours). 48 hours) (preferably 1 hour to 5 hours), etc., but other ranges may be used. This is because if the reaction does not proceed sufficiently, the reaction time can be extended, while if the reaction proceeds efficiently, the reaction time is shortened.

  As used herein, “protected glycolipid” refers to a glycolipid protected with a protecting group. It is understood that the protecting group used may be any protecting group other than the exemplified protecting groups as long as it performs a protecting function. This is because, in the method of the present invention, thioglycoside is used as a leaving group, and as long as the activation conditions are resistant, sugars using protected glycolipids using other protecting groups may be used. This is because lipids can be produced.

  In the present specification, “glycolipid” is a general term for complex lipids containing carbohydrate residues. Examples of these that are classified into glycosphingolipids, glyceroglycolipids, and other glycolipids according to the type of lipid part include, but are not limited to, ganglioside GM3 and ganglioside GM4. Representative glycosphingolipids include neutral glycosphingolipids such as galactocerebroside, glucocerebroside, and globoside, and acidic glycosphingolipids such as ganglioside. Examples of glyceroglycolipids include mono and digalactosyl diacylglycerols. it can. Other types such as those containing uronic acid (uronic acid-containing glycolipid), phosphonoglycolipids containing phosphonic acid, and phosphoglycolipids containing phosphoric acid have also been found.

In the present specification, the “conditions for the alcohol and the carboxylic acid to bond” may be any conditions for the reaction between the alcohol and the carboxylic acid, and those skilled in the art can appropriately determine the conditions by referring to the textbooks listed in the above general techniques. Can be implemented. Exemplary conditions include the following.

In the present specification, the “spacer precursor” refers to a “spacer precursor” that is interposed between a sugar and a glycolipid amide protector and binds the sugar and the glycolipid amide protector.

  In the present specification, the “spacer” refers to a substance that binds a sugar and a protected glycolipid amide. For example, examples of the spacer include, but are not limited to, succinic acid, malonic acid, phthalic acid, oxalic acid, carboxylic acid, isopropylidene ketal, and methoxybenzylidene acetal. This is because, in the intramolecular reaction, the relative position between the reactive functional groups is important, but in the method of the present invention, it is sufficient to perform a condensation reaction with a molecule having a relatively large degree of freedom (for example, succinic acid). This is because good results were obtained, and crosslinking with molecules other than those described above is considered to be sufficiently effective.

  In the present specification, “the conditions under which a protected sugar chain donor is deprotected” may be any conditions under which the protected sugar chain donor deprotects, and those skilled in the art can use the textbooks listed in the above technique. It can implement suitably with reference.

  As an illustrative example, the reaction temperature can be, for example, but not limited to room temperature, preferably room temperature. This is because the reaction temperature only needs to proceed sufficiently. Accordingly, those skilled in the art can appropriately change the reaction temperature in consideration of the reaction situation.

Examples of the solvent include, but are not limited to, CH ₂ Cl _{2 and the} like. This is because any solvent may be used as long as the target reaction proceeds. A person skilled in the art can appropriately select a solvent to be used in consideration of the reaction situation.

  Examples of the reagent include, but are not limited to, trifluoroacetic acid and the like. This is because any reagent may be used as long as the target reaction proceeds. A person skilled in the art can appropriately select the reagent to be used in consideration of the reaction situation.

  The reaction time may be, for example, 2 to 12 hours (for example, 2 hours, 2.5 hours, 3 hours, 5 hours, 10 hours, 12 hours) or the like, but may be in a range other than these. This is because if the reaction does not proceed sufficiently, the reaction time can be extended, while if the reaction proceeds efficiently, the reaction time is shortened.

  In this specification, “under the condition that the 1-position hydroxyl group of the lipid amide protected body is deprotected” means that only the 1-position hydroxyl group of the lipid amide protected body is selectively deprotected and affects other protecting groups. There are no conditions. For example, when the 1-position hydroxyl group of ceramide is protected with a silyl protecting group, this condition can occur at 0 ° C. in the presence of TBAF and AcOH in a THF solvent.

  In the present specification, “acyl protecting group” refers to a protecting group having an acyl group, and examples thereof include, but are not limited to, an acetyl group, a benzoyl group, and a pivaloyl group.

  In the present specification, the “conditions for the acyl protecting group to be deprotected” may be any conditions for the acyl protecting group to be deprotected, and those skilled in the art can appropriately refer to the textbooks listed in the above-mentioned technology. Can be implemented.

  As an illustrative example, the reaction temperature is, for example, room temperature to 100 ° C. (for example, 20 ° C., 25 ° C., 30 ° C., 35 ° C., 40 ° C., 45 ° C., 50 ° C., 55 ° C., 60 ° C., 65 ° C., 70 ° C. , 75 ° C, 80 ° C, 85 ° C, 90 ° C, 95 ° C, 100 ° C), but is not limited thereto. This is because the reaction temperature only needs to proceed sufficiently. Accordingly, those skilled in the art can appropriately change the reaction temperature in consideration of the reaction situation.

Examples of the solvent include methanol (MeOH) and water (H ₂ O), but are not limited thereto. This is because any solvent may be used as long as the target reaction proceeds. A person skilled in the art can appropriately select a solvent to be used in consideration of the reaction situation.

Examples of the reagent include, but are not limited to, sodium methoxide (NaOCH ₃ ) and KOH. This is because any reagent may be used as long as the target reaction proceeds. A person skilled in the art can appropriately select the reagent to be used in consideration of the reaction situation.

  Examples of the reaction time include 1 hour to 1 week (for example, 1 hour, 2 hours, 2.5 hours, 3 hours, 5 hours, 10 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week), etc., but other ranges may be used. This is because if the reaction does not proceed sufficiently, the reaction time can be extended, while if the reaction proceeds efficiently, the reaction time is shortened.

(Description of Preferred Embodiment)
Hereinafter, preferred embodiments of the present invention will be described. The embodiments provided below are provided for a better understanding of the present invention, and the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification.

(Glycolipid production method)
In one aspect, the present invention provides a method for producing a glycolipid. In this method, (a) a protected sugar and a lipid amide protector are reacted under conditions under which the protected sugar and the lipid amide protected substance are bound, and a sugar-lipid amide acceptor precursor is reacted. (B) reacting the sugar-lipid amide acceptor precursor under a condition in which an intramolecular condensation reaction proceeds in the sugar-lipid amide acceptor precursor to generate a sugar-lipid amide acceptor. Step (c): protecting the sugar-lipid amide acceptor and the protected sugar chain donor by reacting them under conditions where the sugar-lipid amide acceptor and the protected sugar chain donor are linked. And (d) subjecting the protected glycolipid to a deprotection reaction under conditions under which the protected sugar chain donor deprotects to produce a glycolipid. .

  (A) reacting a protected sugar with a lipid amide protector under conditions under which the protected sugar and the lipid amide protector bind to produce a sugar-lipid amide acceptor precursor; It can be implemented as follows.

  Appropriate reagents such as WSC and DMAP are allowed to act on the appropriately protected sugar and the protected lipid amide in dichloromethane solvent at room temperature to bond the carboxyl group of the protected lipid amide and the hydroxyl group of the sugar. Subsequently, the protecting group at position 1 of the protected lipid amide is deprotected. It is understood that these conditions can be arbitrarily designed by those skilled in the art based on the present specification and taking into consideration well-known techniques.

  (B) The step of reacting the sugar-lipid amide acceptor precursor under a condition in which an intramolecular condensation reaction in the sugar-lipid amide acceptor precursor proceeds to produce a sugar-lipid amide acceptor is as follows. Can be implemented.

  The sugar-lipid amide acceptor precursor is subjected to an intramolecular glycosylation reaction (for example, using 2 equivalents of NIS and 0.3 equivalents of TfOH as an activator at 0 ° C. in a dichloromethane solvent). It is understood that these conditions can be arbitrarily designed by those skilled in the art based on the present specification and taking into consideration well-known techniques.

  (C) reacting the sugar-lipid amide acceptor with a protected sugar chain donor under a condition in which the sugar-lipid amide acceptor and the protected sugar chain donor are linked to form a protected sugar The step of generating lipid can be performed as follows.

The resulting sugar-lipid amide acceptor and the protected sugar chain donor are combined with an activator (eg, TMSOTf (for example, 0 ° C.) in a suitable solvent (eg, dichloromethane solvent). Using 0.04 equivalents of trimethylsilyl trifluoromethanesulfonic acid) is carried out to carry out the condensation reaction. It is understood that these conditions can be arbitrarily designed by those skilled in the art based on the present specification and taking into consideration well-known techniques.
(D) The step of producing a glycolipid by subjecting the protected glycolipid to a deprotection reaction under the condition that the protected sugar chain donor deprotects can be carried out as follows.

The resulting protected glycolipid is allowed to react with a deprotecting agent (for example, TFA (trifluoromethanesulfonic acid)) in a suitable solvent (for example, dichloromethane solvent) at room temperature, thereby protecting the group (for example, MPM group). Followed by the action of another deprotecting agent (eg NaOMe (sodium methoxide)) in a suitable solvent (eg methanol solvent) at room temperature and another protecting group (eg this In this case, the acyl protecting group) is deprotected. Finally, if there is a methyl ester possessed by sialic acid, an appropriate hydrolyzing agent (for example, water (H ₂ O)) is added and subjected to alkaline hydrolysis to deprotect the methyl ester possessed by sialic acid Lead to lipids. It is understood that these conditions can be arbitrarily designed by those skilled in the art based on the present specification and taking into consideration well-known techniques.

  In one embodiment, protected sugars that can be used by the method include, for example,

Is a sugar protector that can include, but is not limited to:

  In one embodiment, in the protected sugar used in the present invention, an acetyl group, a benzoyl group, a benzyl group, or the like can be used as a hydroxyl protecting group, and a methyl group, benzyl can be used as a carboxyl group protection. Groups and the like can be used, but are not limited to these. As the leaving group, a phenylthio group, a fluorine group, a trichloroacetimidate group and the like can be used, but are not limited thereto.

  In other embodiments, protected lipid amides that can be used in the present methods include, for example:

Wherein R ₁ and R ₂ are independently selected from alkyl or alkenyl groups;
The R ₃ can be TBDPS, TBDMS, TIPS, Tr, isopropylidene ketal, methoxybenzylidene acetal; the R ₄ is a protecting group that can be succinyl, malonyl, phthaloyl, oxalyl, carbonyl, benzoyl, acetyl, pivaloyl. is there. It will be understood that these protecting groups may be other protecting groups as long as they perform their function. This is because in the method of the present invention, if only the primary hydroxyl group at the 1-position is glycosylated and the other hydroxyl groups can be protected, glycolipids can be produced even if other lipid amide protectors are used. Because.

  In a preferred embodiment, the lipid amide protector that can be used in the present invention is:

The sugar-lipid amide acceptor precursor can be

The sugar-lipid amide acceptor is

Where R ₁ is MPM; R ₂ is MPM, Bz; and R ₃ is from the group consisting of TBDPS, TBDMS, TIPS, Tr, isopropylidene ketal and methoxybenzylidene acetal. R ₄ and R ₅ are independently protecting groups that can be succinyl, malonyl, phthaloyl, oxalyl, carbonyl, benzoyl, acetyl and pivaloyl. It will be understood that these protecting groups may be other protecting groups as long as they perform their function. This is because, in the method of the present invention, thioglycoside is used as a leaving group, and a sugar-lipid amide acceptor using a protecting group other than the above can be used as long as the activation conditions are resistant. This is because glycolipids can be produced.

  In another embodiment, in the present method, the condition for binding the protected sugar and the protected lipid amide may be, for example, the condition for binding an alcohol and a carboxylic acid. This is because the protected sugar has a free hydroxyl group that is not protected only at one site, and the protected lipid amide has a spacer having a carboxyl group. Although not wishing to be bound by theory, a reasonable explanation is presented that the conditions for binding the protected sugar and the protected lipid amide may be those for binding the alcohol and the carboxylic acid. .

In another embodiment, the step (a) includes mixing and reacting the protected sugar chain and the lipid amide in a solvent in the presence of a reagent at a predetermined reaction temperature and reaction time. To do. Here, the solvent is tetrahydrofuran _{(THF), CH 2 Cl 2} , benzene, toluene, N, N-dimethylformamide (DMF) and is obtained combinations thereof; the reagent, triphenylphosphine _(PPh 3), DEAD, 1-methyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (WSC), 2,4,6-trichlorobenzoyl chloride, triethylamine (Et ₃ N), 4-dimethylaminopyridine (DMAP) or them And the reaction time can be from 2 hours to 4 hours.

  In another embodiment, in this step (a), the reaction temperature may be room temperature or higher, preferably room temperature to 90 ° C., but these vary depending on the solvent and the like.

One exemplary embodiment may include conditions where the reagents are PPh ₃ and DEAD, the solvent is THF, and the reaction temperature is 90 ° C. or higher.

In a preferred embodiment, in step (a), the solvent is tetrahydrofuran (THF), the reagents are triphenylphosphine (PPh ₃ : 3.0 equivalents) and DEAD (3.0 equivalents), and the temperature Can be at reflux (90 ° C.).

In another preferred embodiment, in step (a), the solvent is CH ₂ Cl ₂ and the reagent is 1-methyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (WSC: 3. 0 equivalents) and 2,4,6-trichlorobenzoyl chloride (1.1 equivalents), and the temperature can be room temperature. Here, an equivalent is an equivalent with respect to the main substance (for example, protected sugar chain) of reaction.

In another preferred embodiment, in step (a), the solvent is CH ₂ Cl ₂ , and the reagent is triethylamine (Et ₃ N: 1.5 equivalents) and 4-dimethylaminopyridine (DMAP: 3. 0 equivalents), and the temperature is room temperature.

In another embodiment, the step (a) comprises adding a spacer precursor to the sugar and the lipid amide protector to bind the lipid amide protector to the sugar via a spacer. Can be included. In order to bind the protected lipid amide to the sugar via a spacer, for example, (1) in the presence of WSC, DMAP, CH ₂ Cl ₂ , room temperature; (2) in the presence of PPh ₃ , DEAD, THF , Reflux, (3) 2,4,6-trichlorobenzoyl chloride, Et ₃ N, DMAP, conditions such as room temperature in the presence of CH ₂ Cl ₂ , but are not limited thereto. This spacer and the sugar or the protected lipid amide may be bonded in advance.

  In a further embodiment, step (a) includes the step of reacting the protected lipid amide under a condition in which the 1-position hydroxyl group of the lipid amide protector is deprotected to deprotect the 1-position hydroxyl group. Can do. As a condition for deprotecting the hydroxyl group at the 1-position of the protected lipid amide, when the hydroxyl group at the 1-position of ceramide is protected with a silyl protecting group, this condition is 0 ° C. in the presence of TBAF, AcOH in a THF solvent. Can be mentioned.

In another embodiment, when the spacer precursor is succinic acid and the succinic acid is bound to R ₃ of the lipid amide protector, R ₃ can be isopropylidene ketal, methoxybenzylidene acetal, and R ₄ Is

And R ₅ can be, but is not limited to, Ac, Bx.

In another embodiment, when the spacer precursor is succinic acid and the succinic acid binds to R ₄ of the lipid amide protector, R ₃ can be Tr, TBDPS, TBDMS, and R ₄ is It can be succinyl, malonyl, oxalyl, carbonyl, glutaryl, phthaloyl and the like, and R ₅ can be, but is not limited to, acetyl, benzoyl.

In another embodiment, the spacer precursor is succinic acid, and when the succinic acid binds to R ₅ of the lipid amide protector, R ₃ can be Tr, TBDPS, TBDMS, etc., and R ₄ is , Succinyl, malonyl, oxalyl, carbonyl, glutaryl, phthaloyl, and the like, and R ₅ can be, but is not limited to, acetyl, benzoyl, and the like. This is because, in the intramolecular reaction, the relative position between the reactive functional groups is important, but in the method of the present invention, it is sufficient to perform a condensation reaction with a molecule having a relatively large degree of freedom (for example, succinic acid). This is because good results were obtained, and crosslinking with molecules other than those described above is considered to be sufficiently effective.

  In another embodiment, the sugar residue on the reducing end side of the oligosaccharide can be bound to the lipid amide protector via a spacer.

  In one embodiment, the step (b) is performed in the presence of an activator for activating the intramolecular condensation reaction, and the activator activates the SPh group of sialic acid. As long as it is N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH) TMSOTf, dimethyl (methylthio) sulfonium triflate (DMTST), N-bromosuccinimide (NBS) and combinations thereof. However, it is not limited to these. This is because if the SPh group of sialic acid can be activated, activation of the intramolecular condensation reaction can be achieved, and at the same time, the sugar-lipid amide acceptor can be purified.

In another embodiment, the step (b) is performed at a reaction temperature of −80 ° C. to room temperature at a reaction temperature of CH ₂ Cl ₂ , diethyl ether ((CH ₂ CH ₃ ) ₂ O), diethyl ether, acetonitrile, diethyl ether, In solvents such as acetonitrile, propionitrile, toluene, nitromethane and combinations thereof; N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH), dimethyl (methylthio) sulfonium triflate (DMTST), molecular sieves 4 In the presence of an angstrom (MS4Å), molecular sieves 3 Å (MS3Å) reagent (wherein it is understood that a desiccant and the like can be appropriately present in addition to the catalyst as the reagent used); ~ 48 hours reaction time Or can be done.

  In a preferred embodiment, the reaction temperature may be −20 to 0 ° C., but is not limited thereto. Although not wishing to be bound by theory, it is because the reaction proceeds favorably, and the reaction temperature of glycosylation is generally carried out at a temperature as low as possible without inhibiting the progress of the reaction. .

  In a preferred embodiment, the solvent can be dichloromethane, but is not limited thereto. While not wishing to be bound by theory, it is understood that diethyl ether, acetonitrile, propionitrile, toluene, nitromethane may also be used, although dichloromethane has shown the highest reactivity.

  In a preferred embodiment, the reagents can be, but are not limited to, N-iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH).

  In a preferred embodiment, the reaction time may be 1 hour to 5 hours, but is not limited thereto. However, it is understood that it varies depending on the reaction solvent and temperature.

In another embodiment, in the step (b), the reaction temperature is 0 ° C .; the solvent is CH ₂ Cl ₂ ; the reagent is N-iodosuccinimide (NIS), trifluoromethanesulfonic acid. (TfOH), TMSOTf, Molecular Sieves 4 Angstrom (MS4Å); the reaction time may be 1.5 hours.

In another embodiment, in the step (b), the reaction temperature is −40 ° C .; the solvent is CH ₂ Cl ₂ ; the reagent is N-iodosuccinimide (NIS), trifluoromethanesulfone. Acid (TfOH), molecular sieves 4 angstroms (MS 4Å); the reaction time may be 5 hours.

In another embodiment, in the step (b), the reaction temperature is −80 ° C. → −60 ° C. → −40 ° C. → 0 ° C .; the solvent is CH ₂ Cl ₂ ; N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH), molecular sieves 4 angstrom (MS 4Å); the reaction time can be 36 hours.

In another embodiment, in the step (b), the reaction temperature is −40 ° C. → 0 ° C .;
The solvent is acetonitrile (MeCN); the reagent is N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH), molecular sieves 3 angstrom (MS3Å); the reaction time is 48 hours obtain.

  In another embodiment, in the step (b), the reaction temperature is −0 ° C .; the solvent is acetonitrile (MeCN); and the reagent is N-iodosuccinimide (NIS), trifluoromethanesulfone. Acid (TfOH), molecular sieves 3 angstroms (MS 3Å); the reaction time may be 1.5 hours.

  In another embodiment, in the step (b), the reaction temperature is −20 ° C .; the solvent is acetonitrile (MeCN); and the reagent is N-iodosuccinimide (NIS), trifluoromethanesulfone. Acid (TfOH), Molecular Sieves 3 Angstrom (MS 3Å); the reaction time can be 3 hours.

In another embodiment, in the step (b), the reaction temperature is 0 ° C. → room temperature; the solvent is diethyl ether (Et ₂ O); and the reagent is N-iodosuccinimide (NIS). , Trifluoromethanesulfonic acid (TfOH), molecular sieves 3 angstrom (MS 3Å); the reaction time can be 25 hours.

  In another embodiment, in the step (b), the reaction temperature is 0 ° C .; the solvent is acetonitrile (MeCN); the reagent is dimethyl (methylthio) sulfonium triflate (DMTST), molecular Thieves 3 angstrom (MS3Å); the reaction time can be 1 hour.

In another embodiment, in the step (b), the reaction temperature is 0 ° C .; the solvent is CH ₂ Cl ₂ ; the reagent is N-iodosuccinimide (NIS), trifluoromethanesulfonic acid. (TfOH), Molecular Sieves 4 Angstrom (MS4Å); the reaction time may be 5 hours.

In another embodiment, in the step (b), the reaction temperature is −20 ° C .; the solvent is CH ₂ Cl ₂ ; the reagent is N-iodosuccinimide (NIS), trifluoromethanesulfone. Acid (TfOH), molecular sieves 4 angstroms (MS4Å); the reaction time may be 1.5 hours.

In another embodiment, in the step (b), the reaction temperature is 0 ° C .; the solvent is CH ₂ Cl ₂ ; the reagent is dimethyl (methylthio) sulfonium triflate (DMTST), molecular. Sieves 4 angstrom (MS 4 Å); the reaction time can be 2 hours.

  The “condition for the intramolecular condensation reaction in the sugar-lipid amide acceptor precursor to proceed” in the step (b) is not the above condition, but the intramolecular condensation reaction in the sugar-lipid amide acceptor precursor proceeds. Any conditions may be used as long as the conditions are satisfied. Because the purpose of this method is to introduce lipid sites efficiently, the conditions are not limited. Moreover, it is because a sugar-lipid amide acceptor will be synthesize | combined simultaneously if intramolecular glycosylation is achieved.

  In another embodiment, the intramolecular condensation reaction can be, but is not limited to, glycosylation. By using glycosylation, the present invention has found that lipid amides can be appropriately introduced into sugars.

In one embodiment, step (c) of the process is at a donor equivalent to more than 2.5 equivalents; at a reaction temperature of −40 to 0 ° C .; in a solvent of CH ₂ Cl ₂ ; It may include reacting for a reaction time of 1 to 48 hours in the presence of a trimethylsilyl trifluoromethanesulfonic acid (TMSOTf) reagent.

In a preferred embodiment, in step (c), the donor equivalent to the acceptor is 2.5 equivalents; the reaction temperature is 0 ° C .; the solvent is CH ₂ Cl ₂ ;
The reagent is TMSOTf; the reaction time can be 7 hours.

  In another embodiment, the protected sugar chain donor is, for example,

Although not limited to these, it is not limited to these. The protected sugar chain donor may be any sugar as long as it has a leaving group at the reducing end of the sugar chain and other protecting groups are protected. This is because, in glycosylation, only the anomeric position of the sugar residue at the reducing end of the sugar chain is eliminated by the activator, and the sugar-lipid amide acceptor causes a nucleophilic attack there. Accordingly, examples of such a leaving group include -SPh, -SCH ₃ , -SCH ₂ CH ₃ , -F, -OPO (OPh) ₂ , -OPO (N (CH ₃ ) ₂ ) ₂ (wherein , Ph is phenyl), but is not limited thereto.

In one embodiment, step (d) of the process is carried out in a solvent of CH ₂ Cl ₂ ; in the presence of a reagent of trifluoroacetic acid; at a reaction temperature of room temperature; over a reaction time of 2 to 12 hours. However, it is not limited to these. The step (d) may be under any conditions as long as the protected sugar chain donor is deprotected.

In a preferred embodiment, in step (d), the solvent is CH ₂ Cl ₂ ; the reagent is trifluoroacetic acid; the reaction temperature is room temperature; and the reaction time is 2 hours. obtain.

  In one embodiment, the step (e) of the method may further include a step of reacting and deprotecting the product of the step (d) under conditions where the acyl-based protecting group is deprotected.

In another embodiment, step (e) is carried out in a solvent of methanol (MeOH) or water (H ₂ O); in the presence of a reagent of sodium methoxide (NaOCH ₃ ) or KOH; At the reaction temperature; it can be carried out over a reaction time of 1 hour to 1 week, but is not limited to this condition. This is because in the step (e), it is only necessary to deprotect the acyl protecting group.

  In a preferred embodiment, the conditions under which step (e) is carried out are as follows: the solvent is methanol (MeOH); the reagent is sodium methoxide (NaOMe); the reaction temperature is room temperature; The reaction time can be 12 hours.

(Sugar chain synthesis method)
In one aspect, the present invention provides:

Provides a method of generating This method uses an acceptor compound

And donor compounds

(A), wherein R ¹ is Ac or H; R ² is Ac or Troc. SPh is

MP is

SE is

And Ac is

Bz is

And Bn is

Me is methyl.

In another embodiment, step (A) comprises the following: at a reaction temperature of −40 ° C. to room temperature; CH ₃ CN, CH ₂ Cl ₂ , diethyl ether, acetonitrile, propionitrile, toluene, nitromethane and their In a solvent of a mixture with the combination; in the presence of a catalyst of N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH), TMSOTf or a combination thereof; run for a reaction time of 1 hour to 3 days However, it is not limited to these.

  In a preferred embodiment, the catalyst includes, but is not limited to, N-iodosuccinimide (NIS) or trifluoromethane sulfonic acid (TfOH) and combinations thereof. The catalyst that can be used in the present method may be any catalyst as long as it dissolves well with a sialyl donor and a lactosyl acceptor and achieves steric control (α-sialylation) during condensation. . This is because the condensation reaction proceeds as long as there is an activated sialyl donor and lactosyl acceptor.

In a preferred embodiment, the solvent may be, for example, CH ₃ CN, CH ₂ Cl ₂ , a mixture of CH ₃ CN and CH ₂ Cl ₂ , but is not limited thereto. The solvent that can be used in the present method may be any solvent as long as it activates the SPh group of sialic acid. This is because if the sialyl donor is activated, the reaction proceeds rapidly due to the nucleophilicity of the free hydroxyl group of lactose.

  In a preferred embodiment, the reaction temperature may be, for example, -30 ° C to 0 ° C, but is not limited thereto. In another preferred embodiment, the reaction temperature may be −50 ° C.

  In a preferred embodiment, the reaction time can be, but is not limited to, for example, 2-3 days, about 4-8 hours, preferably about 6 hours. Glycosylation is usually within 1 hour to 1 day, but the reaction time can be arbitrarily set depending on the progress of the reaction, but is not limited thereto.

In another embodiment, the step (A) comprises:
At a reaction temperature of −30 ° C. → room temperature; using a mixture of CH ₃ CN → CH ₃ CN and CH ₂ Cl ₂ as a solvent; using N-iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH) as a catalyst Using; and reacting for a reaction time of 2 days.

In another embodiment, the step (A) comprises:
The reaction temperature is −30 ° C. → room temperature; the solvent is a mixed solution of CH ₃ CN and CH ₂ Cl ₂ ; the catalyst is N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH) ) And TMSOTf; the reaction time may be 3 days.

In another embodiment, in the step (A), the reaction temperature is −30 ° C. → 0 ° C .; the solvent is a mixed solution of CH ₃ CN and CH ₂ Cl ₂ ; -Iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH); the reaction time may be 2 days.

In another embodiment, in step (A), the reaction temperature is −30 ° C .; the solvent is a mixture of CH ₃ CN and CH ₂ Cl ₂ ; and the catalyst is N-iodosuccinimide. (NIS) and trifluoromethanesulfonic acid (TfOH); the reaction time may be 3 days.

In another embodiment, step (A), the reaction temperature, there at room temperature; the solvent, be _CH 2 Cl _2; the catalyst, N- iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH The reaction time can be 1 day.

In another embodiment, in the step (A), the reaction temperature is −50 ° C .; the solvent is a mixed solution of propionitrile and CH ₂ Cl ₂ ; and the catalyst is N-iodo. Succinimide (NIS) and trifluoromethanesulfonic acid (TfOH); the reaction time can be about 6 hours.

(Sugar chain)
In one aspect, the present invention provides a sugar chain produced by the method for synthesizing sugar chains of the present method. Here, as the method for synthesizing the sugar chain, any form described in the above (Method for synthesizing sugar chain) can be used.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range from the description of specific preferred embodiments of the present invention based on the description of the present specification and the common general technical knowledge. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

(Use)
The sugar or sugar derivative prepared according to the present invention can be used as a pharmaceutical material. The pharmaceutical material produced in this way may have the advantage of being easy to validate. If the present invention is used, it is possible to recombine pharmaceutical raw materials and pharmaceuticals and not use biological raw materials including animal-derived raw materials. When used, since it has a large cost and risk (unknown in vivo behavior, contamination, etc.), the present invention is excellent in that these can be avoided.

  When the present invention is used, it is possible to improve uniformity and stability in medicines and the like. This is due to the removal of impurities.

  When the present invention is used, safe and stable supply of pharmaceuticals and the like is possible. For example, necessary scale-up is possible, and contamination and shortage do not occur. Only this raw material can be used as an extract of animal material.

  For example, the present invention can be used in the same manner as ganglioside contained in milk. Milk mainly contains GM3 and GD3, but GM1 is also contained in a trace amount. Gangliosides contained in breast milk have the effect of inhibiting the binding of toxins such as bacteria and viruses to mucosal epithelial cells such as the oral cavity, pharynx or digestive tract of an infant (host). Accordingly, the products of the present invention may also have similar utility.

  GM3, GD3, and GM1 have an action of inhibiting adherence of toxic E. coli to intestinal epithelial cells. Therefore, the product produced by the present invention can also be expected to have the same effect. GM3 and GM1 are composed of lactosylceramide (Galβ1-4Glcβ1-1Cer) and sialic acid. However, neither lactosylceramide nor sialic acid inhibits adhesion of toxic E. coli to intestinal epithelial cells such as GM3 and GM1.

  In addition, by utilizing the present invention, it is possible to suppress the decrease in the amount of ganglioside in the brain and improve learning behavior by oral inoculation with gangliosides such as GM3 and GD3. It can also be used as an antioxidant (Japanese Patent Laid-Open Nos. 11-126418 and 11-209756).

  By utilizing the present invention, the effects of influenza virus inhibition and angiogenesis inhibition by GM3 can be achieved (JP-A-8-59684, JP-A-6-145069).

  By utilizing the present invention, GM3 glioma chemotherapeutic agents can be provided (Japanese Patent Laid-Open No. 2002-510291).

  Furthermore, by using this method, it is possible to induce differentiation of the human leukemia cell line HL60 into a monocyte / macrophage system by GM3.

  By utilizing the present invention, GM4 can provide an inhibitor of epidermal exfoliation toxin. GM4 is considered to be related to demyelination of the central nervous system, and is also attracting attention as an antiviral agent (Japanese Patent Laid-Open No. 2001-233773).

  In addition, by using the present invention, it is possible to produce a product involved in the suppression of cancer cell growth and the extension of neural cell processes.

  Recently, glycoconjugates such as glycoproteins, proteoglycans and glycolipids have attracted attention from a biochemical point of view. In addition, the physiological role of polysaccharides present in plant and bacterial cell walls has been gradually revealed. Accordingly, the construction of oligosaccharide moieties (sugar chains) has been recognized as an important issue in organic synthetic chemistry (Paulsen, H. Angew. Chem. Int. Ed. Engl. 1982, 21 , 155-). The structures of sugar chains are extremely diverse when the types, number, sequence order, stereochemistry of glycosidic bonds, and positions of sugar residues are combined. Therefore, in order to efficiently synthesize sugar chains, a means for forming glycosidic bonds in a steric and regioselective manner is required. Numerous studies have been made on the O-glycosylation reaction, which is the basic methodology at that time. However, it is hard to say that a sufficient understanding of the factors governing the stereochemistry has been reached. In addition, there is currently no universal reaction that can be applied to all types of O-glycoside bonds. However, existing glycosylation reactions have slightly different properties with respect to factors that affect stereoselectivity. Therefore, it is possible to give a wide variation in stereoselectivity by combining a leaving group and a protecting group and reaction conditions such as a solvent, an activator, and temperature. Indeed, with respect to pyranosides, it has now become possible to control the stereochemistry to a considerable extent for most O-glycosylation reactions (Wulff, G .; Rohl, G. Angew. Chem. Int. Ed. 1974, 13, 157-.).

(Stereochemical control in glycosylation reaction)
The O-glycosylation reaction is represented by the following general formula (O-glycosylation reaction). Usually, (1) is called a sugar donor (glycosyl donor) and (2) is called a sugar acceptor (glycosyl acceptor) or aglycon. Substituent X at the anomeric position of the sugar donor is called a leaving group. In order to carry out the glycosylation reaction, a catalytic or stoichiometric amount of an activator (activator or promoter) is required in order to increase the ability to remove the sugar donor. In addition, the activator may serve as a scavenger for the acid generated with the reaction.
(O-glycosylation reaction)

O-glycosides are roughly classified from the viewpoint of synthetic chemistry, and are classified into 2-hydroxy, 2-amino and 2-deoxy groups. Among these, 2-hydroxy type and 2-amino type glycosides are considered to be divided into 1,2-cis type and 1,2-trans type. Further, in the synthesis of 2-hydroxy glycosides, different methodologies are used for mannose type (manno type) and glucose type (gluco type) for 1,2-cis and 1,2-trans, respectively. The methodologies used for stereochemical control in the O-glycosylation reaction can be roughly classified into the following three. (1) Synthesis of α-glycoside by in situ anomerization, (2) Synthesis of β-glycoside by S _N 2 type inversion, and (3) Synthesis of 1,2-trans glycoside utilizing participation of adjacent groups. The following table (classification and synthesis method of O-glycoside) summarizes the basic structure of O-glycoside and the methodology used for its synthesis. In the following, the basic idea will be described taking a glycosyl halide having Br or Cl as a leaving group as an example.
(Classification and synthesis of O-glycosides)

(A. In situ anomerization method)
Most of glucosyl halides are usually present as thermodynamically stable α-form (3a). However, when the α-form (3a) is activated with a heavy metal salt, a quaternary ammonium salt or the like, an oxocarbenium ion (4a) is generated, and the β-halide (3b) is formed via a β-type ion pair. An equilibrium is established between them. Here, if alcohol (R—OH) is present in the system, (4a) is considered to give β-glycoside (5b), and (4b) gives α-glycoside (5a). Sugar donors that do not participate in adjacent groups react in this manner, and their stereochemistry is determined by the relative rates of these series of reactions. Here, when the anomeric effect is taken into consideration, it is considered that (4b) is more reactive than (4a) (K ₂ > K ₁ ). Therefore, if this equilibrium is fast enough (K ₃ >> K ₁ ), the reaction proceeds mainly via (4b) to give α-glycoside (5a). This type of reaction is useful for the synthesis of α-gluco and α-manno glycosides. As the protecting group for the hydroxyl group at the C-2 position, ether-based protecting groups such as a benzyl group and an allyl group are used in order to prevent the participation of adjacent groups. 2-Amino glycosides can be similarly α-selective by selecting the type of C-2 substituent equivalent to the amino group. It should be noted here that in order to obtain high selectivity, it is necessary to select reaction conditions that are as gentle as possible in consideration of the reactivity of the substrate. That is, if the activator is too strong for the reactivity of the substrate or the reaction temperature is too high, the reaction proceeds along the route of (3a) → (4a) → (5b), and β-form is considerably produced as a by-product. There are things to do. On the other hand, a method of preparing β-halide in advance and directly using it for the reaction is also known. This method is useful for α-glycosylation of highly reactive substrates.
(Glycosylation reaction pathway (1))

(B. Glycosylation with S _N type 2 inversion)
When a certain insoluble catalyst is used as an activator, the ion pair generated by the activation of the halide is fixed on the catalyst surface, so that the equilibrium between halide anomers is suppressed. Under such conditions, α-halide reacts with S _N 2 -like stereochemical inversion to give β-glycoside. This type of reaction is particularly useful for the synthesis of β-manno-type glycosides. In addition, when a very strong activator and a sugar donor with high detachment ability are used, the rate of substitution reaction exceeds the rate of anomerization (K ₁ > K ₃ ) to obtain a β-form as a main product. Can do.

(C. Control of stereochemistry by neighboring group participation)
When an acyl group such as an acetyl group or a benzoyl group is used as a protecting group for the hydroxyl group at the C-2 position of the sugar donor, β-gluco and α-manno type glycosides can be selectively synthesized. This phenomenon is explained by assuming the neighboring group participation of the acyl group. That is, the acyloxy group causes the adjacent group to participate in the oxocarbenium ion (7) generated from the glycosyl halide (6), and isomerizes to the more stable cyclic acyloxynium ion (8). For (8), the direction of the nucleophilic attack of the alcohol on the anomeric position is limited (path (a)), so that only a 1,2-trans coordination product (9) is obtained. This method is a very reliable reaction in terms of stereochemical control. However, the route (b) often competes, and the by-production of the orthoester (10) becomes a problem. Further, since an electron-withdrawing acyl group is present next to the anomeric position, the reactivity of the halide is lowered, and generally strong reaction conditions are required.

For 2-amino sugars, the participation of adjacent groups such as N-acyl groups and N-phthaloyl groups is also used. On the other hand, the 2-deoxy sugar as it is cannot naturally participate in adjacent groups, but attempts have been actively made to control stereochemistry by introducing substituents such as halogen, sulfur, selenium and the like at the C-2 position.
(Glycosylation reaction pathway (2))

Another problem is the control of the bonding position between sugar residues. Oligosaccharides have a large number of hydroxyl groups in the molecule, and it is necessary to react only with the desired hydroxyl group by strictly distinguishing them. As one of means for that purpose, there is a method using the difference in reactivity of hydroxyl groups. For example, since the primary hydroxyl group at the C-6 position is much more reactive than other hydroxyl groups, sugar residues can be selectively introduced. It is also often possible to perform regioselective glycosylation utilizing the fact that equatorial hydroxyl groups are less sterically hindered than axial hydroxyl groups. However, these are only special examples, and more generally, it is desirable to increase the solubility of a saccharide in an organic solvent by selectively introducing a protecting group, and to leave only the required hydroxyl group free. When actually synthesizing complex oligosaccharides, it is necessary to use various protecting groups in accordance with the characteristics, and the appropriate selection has a crucial importance for the correctness of the synthesis.

(Reaction condition setting)
It is of course desirable to carry out the glycosylation reaction under strictly anhydrous conditions. Therefore, it is necessary to remove moisture from the solvent, reagent, substrate, and reaction vessel as much as possible. In particular, silver salt is highly hygroscopic and requires attention. At present, the reaction is generally performed by injecting the solution using a syringe under an atmosphere of nitrogen or argon. It is better to carry out the reaction using silver salt while blocking light. On the other hand, most glycosylation reactions are not hindered by molecular sieves (MS) or anhydrous calcium sulfate (Drierite). Particularly in small-scale precision synthesis, the reaction is often carried out in the presence of these desiccants to reduce the technical difficulty. Commonly used solvents include halogenated hydrocarbons such as dichloromethane and 1,2-dichloroethane, aromatic hydrocarbons such as toluene and benzene, and diethyl ether, and polar solvents such as nitromethane and acetonitrile are often used. Although it is difficult to uniformly interpret the effect of the solvent in the glycosylation reaction, the reaction is generally accelerated in a polar solvent such as nitromethane. A lot of data has been accumulated on the relationship with stereoselectivity. Of particular interest is the solvent effect of acetonitrile and diethyl ether. That is, in certain reactions, the β form is obtained as the main product in acetonitrile and the α form is obtained as the main product in diethyl ether. The difference in directionality with acetonitrile does not appear to be simply due to the difference in polarity, since it is often observed that alpha selectivity is improved in ether compared to other nonpolar solvents such as dichloromethane. Although the reaction temperature is extremely wide from −70 ° C. to around 100 ° C. depending on the reactivity of the substrate, it is usually desirable to carry out the reaction at a low temperature as long as it does not interfere with the progress of the reaction. Although systematic studies have not been conducted on the effect of concentration, it is considered better to conduct a high concentration as a general intermolecular reaction. However, in some cases, good results are obtained only under high dilution (Nicolaou, K.C .; Daines, R.A .; Ogawa, Y .; Chakraboty, T. K. J. Am. Chem.). Soc. 1988, 110, 4696-)), those skilled in the art can appropriately select appropriate conditions based on the description in this specification in consideration of such information.

(Drawing a favorable production example of a new synthesis strategy for ceramide introduction)
As described above, when synthesizing a complex ganglioside, the present inventors have widely used a method for introducing a ceramide site using azidosphingosine (see below). The reason is that, as the molecular weight of the sugar chain increases, it is very difficult to introduce lipids by other methods. By using an azide, the steramide skeleton is reduced by reducing the steric hindrance of the two alkyl chains of the ceramide and further suppressing the decrease in nucleophilicity of the primary hydroxyl group caused by the hydrogen bond between the primary hydroxyl group and the amide of the ceramide Is easy to introduce.

  Method of introducing lipid after completion of sugar chain construction (improved lipid receptor side)

However, even with this prior art method, a satisfactory introduction yield has not been obtained. In addition, this method requires complicated synthesis steps before and after preparation of azidosphingosine, reduction of the azido group, introduction and condensation of fatty acids. Therefore, as a result of examining these problems, it is considered that there are the following three points for improvement. (1) Improvement of condensation yield of sugar chain and lipid (2) Stereoselectivity in lipid introduction (3) Simplification of preparation and conversion of ceramide. In the present invention, a new synthesis strategy using an intramolecular condensation reaction was devised in consideration of the above points. It is believed that properly designed intramolecular glycosylation can ameliorate all of the above problems.

  The main advantage of intramolecular glycosylation is improved reactivity, stereoselectivity and regioselectivity. Furthermore, application to solid phase synthesis and ligation synthesis is also possible. The intramolecular reaction is considered to be entropy advantageous because the two molecules become one compared to the intermolecular reaction, and as a result, the reactivity is considered to be greatly increased. By designing the compound appropriately, it can be expected that the two molecules collide more frequently at the reaction point and tilt the equilibrium state of the reaction toward the target compound. In addition, regarding the stereoselectivity and regioselectivity, both molecules are considered to be stereotyped by considering the type of spacer used (length, nature of the functional group, flexibility) and position, or the solvent and reaction temperature. As a result of various restrictions, selectivity can be improved.

  In the field of synthetic sugar chemistry, many syntheses utilizing this characteristic have been used. Intramolecular glycosylation has a long history, and various reactions have been studied so far (Jung, KH; Muller, M .; Schmidt, RR Chem. Rev. 2000, 100, 4423-4442. .). Intramolecular glycosylation can be broadly classified into three types according to spacers that bind sugar donors and sugar acceptors.

  (Classification of intramolecular glycosylation)

Leaving group-based intramolecular glycosylation is a process in which a sugar acceptor is bound to a leaving group of a sugar donor (donor) and is accepted simultaneously with the elimination of the leaving group. The body attacks the anomeric carbon of the donor.

  An acceptor atom (link of the accepting atom via a bifunctional group) bound to a bifunctional spacer is a sugar donor (typically a donor via a bifunctional spacer). Is bonded to the hydroxyl group at the 2-position, and when the leaving group is eliminated, the acceptor atom acts simultaneously or after the post-treatment to remove the spacer.

  In a spacer-mediated linkage via non-reacting center, the acceptor binds to a sugar donor by a spacer that does not participate in the reaction regardless of function or position. In general, the reaction proceeds with an unprotected hydroxyl group present in one (or more) receptors.

  One of the most prominent proposals utilizing intramolecular glycosylation is the synthesis of β-mannosides by Ito, Ogawa et al. (Ito, Y .; Ogawa, T .; J. Am. Chem. Soc. 1997, 119, 5562-.). Due to the structure of mannose, β-mannoside cannot be used in the in situ anomerization method described above or the steric control method by adjacent groups, and forms a β-glycoside that is unstable both thermodynamically and kinetically. Must. Therefore, even today, when various three-dimensional control methods are reported, it exists as one of the most difficult problems. The following shows the main glycoside binding mode and synthesis difficulty in higher animals.

  (Main glycoside binding mode and synthesis difficulty in higher animals)

Ito, Ogawa et al. Applied intramolecular glycosylation to solid phase synthesis and succeeded in the synthesis of highly stereoselective and efficient β-mannosides (Ito, Y .; Ogawa, T .; J. Am. Chem. Soc. 1997, 119, 5562-.).

  (Synthesis of β-mannoside using intramolecular glycosylation by Ito Ogawa et al.)

In the above reaction, the purification using conventional column chromatography was omitted by binding the sugar donor to the resin. In addition, β-mannosidation, which is considered difficult due to a properly designed structure, is stereoselectively performed.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range from the description of specific preferred embodiments of the present invention based on the description of the present specification and the common general technical knowledge. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  Hereinafter, although an example explains the composition of the present invention in detail, the present invention is not limited to this. The reagents used in the following were commercially available unless otherwise specified.

(Synthesis example)
In the present invention, when intramolecular glycosylation is used, which hydroxyl group of reducing saccharide (glucose) and ceramide is cross-linked, what is used as a spacer, and which method of the three types of intramolecular glycosylation We examined whether to use. First, as for which hydroxyl group of ceramide is cross-linked with sugar (glucose) at the reducing end, as a result of considering the binding yield, the 6-position hydroxyl group of highly reactive glucose and the 3-position hydroxyl group of ceramide are bonded. This is considered appropriate. Various studies are possible as to what to use as the spacer. Therefore, it is generally used in intramolecular glycosylation, is easy to handle, and in final deprotection, Zemplen condition (Plattner, JJ; Gless, RD; Rapoport, HJ Am. Chem). Soc., 1972, 94, 8613-.) Succinic acid that can be efficiently deprotected is used. For the above reasons, among the three types of intramolecular glycosylation, (Spacer-mediated linkage via reacting centers) was used.

  Two synthetic strategies are also conceivable for the introduction of ceramide using intramolecular glycosylation. The first is a method using intramolecular glycosylation after sugar chain construction (the following formula). In addition, as a second method, there is also a method in which ceramide is first introduced into glucose which is a sugar chain reducing end by intramolecular condensation via succinic acid, and then introduced into a sugar chain as a glucosylceramide acceptor. (The following formula).

  (Method using intramolecular glycosylation after glycosylation)

(Method using glucosylceramide acceptor using intramolecular glycosylation for glucose)

First, we decided to verify that ceramide can be introduced into sugars by intramolecular glycosylation. Therefore, monosaccharide glucose was used as the sugar chain. Glucose was cross-linked with ceramide and intramolecular glycosylation was performed to discuss the results of intramolecular glycosylation. Thereafter, two synthetic strategies were examined respectively.

  For the above reasons, first, appropriately protected glucose donor and ceramide acceptor are prepared, and GlcH-6 and CerH-3 are cross-linked using succinic acid. Intramolecular glycosylation is then performed. Considering the result, introduction into a sugar chain is performed using the obtained glucosylceramide acceptor. And we decided to investigate intramolecular glycosylation even in more complex sugar chains (Scheme 1).

  (Scheme 1)

(Preparation of glucose donor)
In natural gangliosides, glucose is located at the reducing end of the sugar chain, and in most gangliosides except for the gala system (glycolipid composed of β-galactoside bond between sugar chain and ceramide), Galβ (1-4) Glcβ (1- 1) It has a Cer structure.

  (Basic sugar chain structure of typical glycosphingolipid)

Based on the above structure, it is considered that the most reliable method is to introduce an acyl-based Bz group that can be expected to participate in the adjacent group into the protective group at the 2-position of the glucose donor. Moreover, since a sugar chain is introduced into the 4-position of glucose, the protecting group at the 3-position of glucose is more preferably a protecting group that does not cause a decrease in the reactivity of the 4-position hydroxyl group when the 4-position sugar chain is introduced. From the above, it is considered that the 3-position protecting group is suitably protected by an ether-based Bn group or MPM group, which has been confirmed to be useful as an electron donating protecting group. Catalytic hydrogenation is mainly used for deprotection of the Bn group, but if this method is used, the unsaturated bond of the ceramide is reduced, so it seems better to use the MPM group than the Bn group. It is. The 4th and 6th positions of glucose are formed with a free hydroxyl group at the time of crosslinking with ceramide using succinic acid, and the 6th primary hydroxyl group having the highest reactivity and the 4th secondary hydroxyl group having the lowest reactivity. By utilizing the difference in reactivity, crosslinking with succinic acid was selectively performed at the 6-position. After crosslinking with succinic acid, the hydroxyl group at the 4-position of glucose was protected with a chloroacetyl group capable of selective deprotection, leading to intramolecular glycosylation. In addition, various protecting groups can be considered for protecting the anomeric position. However, an SPh group that is relatively stable as a protecting group and that can be used as a leaving group when condensed with ceramide is used. did. The SPh group has a variety of activators and is characterized by being easily derivatizable to other leaving groups (the following formula: required glucose acceptor protection mode).

  (Required form of glucose acceptor protection)

In addition, a more efficient glucose acceptor was devised and synthesized. Specifically, the 2-position of the glucose acceptor is converted from the Bz group to an electron-donating MPM group, thereby simplifying the preparation of the glucose acceptor and, as an armed sugar, the ability to desorb the 1-position SPh group. The improvement of the reactivity in 4-position sugar-chain introduction | transduction was anticipated (the following formula | equation: The protection mode 2 of the glucose acceptor calculated | required) expected. By converting to an MPM group, the participation of the neighboring group at the 2-position is lost, but the high stereoselectivity of intramolecular glycosylation using succinic acid is utilized to control the stereoselectivity during ceramide introduction. I want to do it.

  (Required glucose acceptor protection mode 2)

(Preparation example of glucose donor (2-Bz))
First, Ac ₂ O and pyridine (pyr.) Were allowed to act on Compound 1 at 45 ° C. to obtain Compound 2 quantitatively. Thereafter, in order to protect the anomeric position of glucose with the SPh group, PhSH and BF ₃ · OEt ₂ were allowed to act at room temperature in a CH ₂ Cl ₂ solvent under an argon atmosphere, leading to compound 3 in a yield of 67%. It was. Subsequently, MeONa was allowed to act in an MeOH solvent at room temperature under an argon atmosphere to deprotect the Ac groups at the 2, 3, 4, and 6 positions to obtain Compound 4 at a yield of 93%. Subsequently, BDA and p-TsOH were allowed to act on Compound 4 in a MeCN solvent at room temperature under an argon atmosphere to obtain Compound 5 at a yield of 93%. In order to determine the structure of the product, the compound 5 was converted to Ac, and the ¹ H-NMR spectrum was confirmed. As a result, the signal δ (ppm) 3.4 (m, 1 H) of the 2-position proton of the compound 5 and the 3-position proton Signal 3.8 (m, 1H) showed a low magnetic field shift of 5.0 (t, 1H), 5.3-5.4 (t, 1H), respectively. This confirmed the introduction of benzylidene groups at positions 4 and 6.

Next, DBTO, TBAB, and MPMC1 were allowed to act on Compound 5 under reflux in a toluene solvent, leading to Compound 6 at a yield of 77%. It is said that Bu ₂ SnO forms a cyclic stannylene compound and activates cis-glycol first, and the reaction is likely to occur at the hydroxyl group of the equivalent. In compound 5, cis glycol is not present, and positions 4 and 6 are protected with a benzylidene group. Therefore, it is considered that a stanylene compound is formed at positions 2 and 3 and MPM progressed selectively at position 3. It is done. In order to determine the structure of the product, Compound 6 was converted to Ac and the ¹ H-NMR spectrum was confirmed. As a result, the signal δ (ppm) 3.5 (m, 1 H) of the 2-position proton of Compound 6 was 5.2. The magnetic field shift was as low as 5.3 (t, 1H). From this, it was confirmed that the 2-position was converted to Ac, that is, an MPM group was introduced at the 3-position.

  Subsequently, compound 7 was allowed to react with benzoyl chloride and DMAP in a pyridine (pyr.) Solvent at room temperature under an argon atmosphere to obtain compound 7 in a yield of 80%. Compound 7 was deprotected from the 4,6-position benzylidene group at 85 ° C. in an aqueous solution of AcOH at 40 ° C. to obtain Compound 8 in a yield of 82% (Scheme 2).

  (Scheme 2)

(Preparation example of glucose donor (2-MPM))
To prepare a more efficient glucose acceptor, it was decided to prepare a glucose acceptor in which the 2-position of the glucose acceptor was converted from a Bz group to an electron donating MPM group. By making the 2-position an MPM group, preparation of a glucose acceptor was simplified, and as an armed sugar, an improvement in the elimination ability of the 1-position SPh group and an improvement in reactivity in introducing a 4-position sugar chain were expected. . By converting to an MPM group, the participation of the neighboring group at the 2-position is lost, but the stereoselectivity of intramolecular glycosylation using succinic acid is utilized, and the stereoselectivity at the anomeric position in ceramide introduction is well controlled. I want to do it.

  In the preparation of the glucose donor (2-MPM), NaOMe was allowed to act catalytically on the prepared compound 7 in a MeOH / THF mixed solvent at room temperature to deprotect the Bz group at position 2, followed by DMF. NaH and MPMCl were allowed to act in a solvent at room temperature to convert the 2-position to MPM, and compound 9 was obtained in a two-step yield of 70%. Subsequently, 83% acetic acid was allowed to act on Compound 9 at 30 ° C. to prepare a glucose donor (2-MPM) in a yield of 86% (Scheme 3).

  (Scheme 3)

(Ceramide preparation example)
In the present invention, research was initially conducted using sphingosine-type ceramide widely present in mammals. However, while the usefulness of intramolecular glycosylation could not be confirmed, the unsaturation bond of sphingosine-type ceramide forced various difficulties in carrying out the research. Specifically, the catalytic hydrogenation method used for deprotecting the Bn group and the like cannot be easily used because it reduces the unsaturated bond, and in glycosylation, Activating agents that can add to unsaturated bonds cannot be used. He also suffered from a shortage of ceramide samples. Currently, several methods for preparing ceramides have been reported ((a) Berg, R.V .; Korevaar, C .; Overkleft, H .; Marel, G.V .; Boom JV. V.J. Chem. 2004, 69, 5699-5704. (B) Alexander, M .; Richard, JKT; Robert, J. W .; Norman, Lewis. Synthesis. 1994, 31-33. ) Murakami, T .; Furusawa.K .; Tetrahedron. 2002, 58, 9257-9263.) Was urgently desired to confirm the utility of intramolecular glycosylation. Therefore, first, we decided to develop a ceramide introduction method using phytosphingosine-type ceramide, which is available in large quantities, has few restrictions on protecting groups, activators, etc., and allows detailed examination of conditions.

  In addition, although the Example which mainly used the phytosphingosine type | mold ceramide was described in the present Example, as described elsewhere of this specification, it exists widely in mammals in this invention. It was revealed that glycolipids can be synthesized by intramolecular condensation even when sphingosine type ceramide is used. Therefore, it can be said that the present invention is remarkable in that it has been found that glycolipids can be produced by intramolecular condensation even in general ceramides that could not be predicted in the past, particularly sphingosine type ceramides.

  Sphingosine-type ceramide has a secondary hydroxyl group at the 3-position in addition to the primary hydroxyl group at the 1-position. Therefore, succinic acid was previously bonded to the hydroxyl group at the 3-position, and the hydroxyl group at the 1-position was used as a ceramide acceptor by protecting it with a protective group capable of selective protection / deprotection. On the other hand, phytosphingosine-type ceramide has secondary hydroxyl groups at the 3-position and 4-position in addition to the primary hydroxyl group at the 1-position. The purpose of this study is to develop a ceramide introduction method applicable to sphingosine type ceramide using phytosphingosine type ceramide. Therefore, in phytosphingosine type ceramide, a ceramide acceptor having succinic acid at position 3 is also used. We decided to prepare. Further, for the protection of ceramide 1-position, a protective group capable of primary selective protection and having selectivity with other protective groups at the time of deprotection was required. Since the glucose donor uses an MPM group that is weak against acidic conditions, a Tr group that is widely used as a protective group for primary hydroxyl groups is not suitable, and a protective group that can be protected and deprotected under basic conditions has been sought. . As protecting groups meeting this requirement, silyl-based TBDPS groups and TBDMS groups were considered. Silyl protecting groups can be selectively deprotected by nucleophilic attack of a fluorine anion, and TBDPS and TBDMS groups are widely used for selective protection of primary hydroxyl groups due to their bulk. The 4-position hydroxyl group of ceramide was easy to introduce, and an acyl-based Bz group that was simultaneously deprotected by Zemplencondition in the final deprotection was used (the following formula).

  (Examples of required ceramide protection styles)

(Ceramide preparation 1)
Using phytosphingosine as a starting material, stearic acid was introduced into the 2-position amino group. Subsequently, when CerH-1 and CerH-3 were protected using a benzylidene group, a 3,4-position benzylidene body was generated in a ratio of 1: 1 in addition to the 1,3-position benzylidene body. It became. Furthermore, in the 1,3-position benzylidene compound, when 1 equivalent of benzoic anhydride is allowed to act at room temperature in a pyridine (pyr.) Solvent and the ceramide 4-position hydroxyl group is converted to Bz, the reaction proceeds only slightly. Was confirmed. From this, it was considered that steric hindrance around the 4-position hydroxyl group of ceramide might inhibit Bzation (Scheme 4).

  (Scheme 4)

Therefore, the original preparation plan was changed to convert 1-position of phytosphingosine ceramide to TBDPS and allow 1 equivalent of succinic anhydride to act at the room temperature in a pyridine (pyr.) Solvent with a hydroxyl group at positions 3 and 4 free. For example, it is considered that succinic acid can be introduced selectively at the 3-position due to steric hindrance around the 4-position hydroxyl group. However, contrary to expectations, succinic acid was introduced preferentially at the 4-position. For this reason, a ceramide derivative having succinic acid at the 3-position of ceramide initially planned was not obtained, and a ceramide derivative having succinic acid at the 4-position was obtained (Scheme 5).

  (Scheme 5)

(Ceramide Preparation Example 2)
In the preparation 1 (Scheme 5) of the ceramide acceptor described above, problems remained in the selectivity and synthesis efficiency at the 3rd and 4th positions, but the 1st and 3rd benzylidene formation was studied and these problems were improved. Up to now, BDA and p-TsOH were allowed to act at room temperature in an acetonitrile solvent for protection using a benzylidene group at the 1- and 3-positions of ceramide. Here, paying attention to the fact that the benzylidene group forms a 6-membered ring at the 1,3-position of the ceramide and forms a 5-membered ring at the 3,4-position, the heating was carried out at 40 ° C. As a result, the benzylidene group preferentially formed a thermodynamically stable six-membered ring structure, and an improvement in the yield of protection using the 1,3-position benzylidene group was achieved. Thereafter, succinic anhydride and DMAP were reacted with Compound 15 in a pyridine (pyr.) Solvent at 40 ° C. to introduce succinic acid at the 4-position. Since compound 16 had a carboxyl group, it was very high in polarity and was extremely difficult to purify. Therefore, purification was performed after protecting the carboxyl group with the Bn group. Subsequently, the benzylidene group was deprotected using Compound 17 with an 80% aqueous acetic acid solution. Here, compounds in which succinic acid was rearranged to the 1-position and 3-position hydroxyl groups from which the benzylidene group was deprotected were confirmed. It is considered that the reaction temperature of 60 ° C. promoted the rearrangement under acidic conditions. Subsequently, TBDPSCl, TEA, and DMAP were allowed to act on Compound 18 in a dichloromethane solvent at 40 ° C. to selectively protect the hydroxyl group at the 1-position of ceramide. Subsequently, the 3-position of ceramide was protected with an Ac group, leading to compound 20. Finally, the catalytic hydrogenation method was used to lead to a ceramide acceptor (Scheme 6).

  (Scheme 6)

(Synthesis example of glucosylceramide acceptor)
(Condensation of glucose donor and ceramide)
As described above, a ceramide derivative having succinic acid at the 3-position of the ceramide originally planned was not obtained, and a ceramide derivative having succinic acid at the 4-position was obtained. However, we believe that it will be a good reference for the development of intramolecular glycosylation that forms a macrocycle with Glc and the development of new glucosylceramide acceptors, and use the obtained ceramide derivative with succinic acid at the 4-position as it is. It was.

(Condensation of glucose (2-Bz 4-CA) and ceramide)
Using the previously prepared glucose donor 8 and ceramide acceptor 14, 2,4,6-trichlorobenzoyl chloride, TEA, and DMAP were allowed to act at room temperature in a dichloromethane solvent, and glucose and ceramide were combined at a yield of 75%. Coupling via succinic acid (Scheme 7). The condensate of the 4-position hydroxyl group of glucose and ceramide, which was a concern, did not form. Thereafter, the 4-position of Glc was protected with a chloroacetyl group. Subsequently, the TBDPS group at the 1-position of ceramide was deprotected. Entry 1 was carried out under the condition that 2 equivalents of TBAF was allowed to act at room temperature in a THF solvent (Table 3). The starting material disappeared in 3 hours from the start of the reaction, but nearly 60%, the acetyl group at the 3-position of ceramide was transferred to the 1-position and the chloroacetyl group at the 4-position of Glc was eliminated. Therefore, under the condition of entry 2, 7.5 equivalents of acetic acid that buffers the basicity of TBAF was added, and 1.5 equivalents of TBAF were allowed to act. In this case, it took 18 hours to complete the reaction, but no by-product was observed, and the yield was as high as 92%. Entry3 was performed under the same conditions as entry2 with a little scale up. However, even after 18 hours, the reaction was not completed, so 0.5 equivalent of TBAF was added. When the reaction was further continued for 18 hours, a large amount of by-products in which the CA group at the Glc4 position was deprotected were produced, and the yield of the target compound was 36%. The detailed cause of the inability to obtain reproducibility here cannot be mentioned, but since 7.5 equivalents of acetic acid is used with respect to 1.5 equivalents of TBAF, the progress of the reaction is slow and the tendency is stronger with scale-up. Seems to have appeared.

  (Scheme 7)

(Deprotection of ceramide 1-TBDPS group)

(Condensation example of glucose (2-MPM 4-CA) and ceramide)
Glucose donor 10 and ceramide acceptor 14 were allowed to act on WSC and DMAP in a dichloromethane solvent at room temperature to obtain compound 24 in a two-step yield of 61%. Subsequently, chloroacetylation at position Glc4 was performed with a yield of 95%. Next, the TBDPS group at the 1-position of ceramide was deprotected (Scheme 8). In the above de-TBDPS conversion, it was found that the production of bipro tends to increase as the reaction time becomes longer. Therefore, in entry1, the reaction was carried out using 1 equivalent each of acetic acid and TBAF in THF solvent at room temperature. (Table 4). The start did not disappear after 2 hours, but the reaction was stopped and the product was confirmed. Two main products were identified. One of them was the target compound 26 with a yield of 22%. The by-product was slightly higher in polarity than the target compound and had a molecular weight of 1342. As a result of analyzing this compound by ¹ H-NMR, it almost coincided with ¹ H-NMR of the target compound, but only the methylene (—CH ₂ ) hydrogen of the CA group was different, and the magnetic field was shifted. For this reason, it was thought that a compound in which Cl in the CA group was replaced with F was generated, but the molecular weight obtained from the mass spectrum did not match, and identification was not achieved. Subsequently, in entry 2, the reaction was performed under low temperature conditions for the purpose of suppressing the rearrangement of the 3-position Ac group and the influence of the 4-position of glucose on the CA group. The reaction was carried out in THF solvent at 0 ° C. using 1 equivalent of acetic acid and 1 equivalent of TBAF. On TLC, it seemed to proceed at a relatively high yield, but a by-product in which the Ac group at the 3-position of ceramide was rearranged to the 1-position of ceramide was also obtained. This by-product is considered to increase due to the acidity of silica gel column chromatography during purification, in addition to the one produced during the reaction. By lowering the reaction temperature, the rearrangement of the 3-position Ac group was somewhat suppressed.

  (Scheme 8)

(Table 4 Deprotection of ceramide 1-TBDPS group)

(Condensation of glucose (2-Bz 4-OH) and ceramide)
Until now, the 4-position of glucose was protected with a CA group before intramolecular glycosylation, but the necessity was examined in consideration of efficiency. Due to the presence of the CA group, various side reactions occurred during de-TBDPS conversion at the 1-position of ceramide, which was a problem. Therefore, we investigated the difference in reactivity between the primary hydroxyl group at the 1-position of ceramide and the secondary hydroxyl group at the 4-position of glucose, the difference in steric environment when crosslinked with succinic acid, and the difference in reactivity between molecules and within molecules. . As a result, it is considered that the desired intramolecular glycosylation is achieved without protecting the hydroxyl group at the 4-position of glucose. Therefore, it was decided to carry out intramolecular glycosylation without protecting the 4-position of glucose (Scheme 9).

  Glucose donor 8 and ceramide acceptor 14 were reacted with WSC and DMAP in a solvent at room temperature to lead to compound 21 in a two-step yield of 68%. Subsequently, the TBDPS group at the 1-position of ceramide was deprotected. As described above, it was revealed that de-TBDPS under low temperature conditions suppressed the rearrangement of the Ac group at the 3-position of the ceramide to the 1-position, so the reaction temperature was set to 0 ° C. Compound 21 was reacted in THF solvent at 0 ° C. using 3.0 equivalents of acetic acid and 3.0 equivalents of TBAF. After 12 hours, the reaction was stopped and purification was performed using silica gel column chromatography to obtain the target compound in a yield of 75%. Under these reaction conditions, almost no compound in which the Ac group at the 3-position of ceramide was rearranged to the 1-position was confirmed.

  (Scheme 9)

(Condensation example of glucose (2-MPM 4-OH) and ceramide)
Glucose donor 10 and ceramide acceptor 14 were allowed to act on WSC and DMAP in a solvent at room temperature to obtain compound 24 in a two-step yield of 61%. Next, the TBDPS group at the 1-position of ceramide was deprotected. The reaction was performed in THF solvent at 0 ° C. using 1 equivalent of acetic acid and 1 equivalent of TBAF. On TLC, it seemed to proceed at a relatively high yield, but the yield was 58%. As a by-product, a by-product in which the Ac group at the 3-position of ceramide was rearranged to the 1-position of ceramide was also obtained. This by-product is thought to increase due to the acidity of silica gel column chromatography during purification in addition to the one produced during the reaction (Scheme 10).

  (Scheme 10)

(Intramolecular glycosylation example)
(Intramolecular glycosylation of glucose (2-Bz 4-CA) and ceramide)
Compound 23 was reacted in dichloromethane solvent at 0 ° C. using NIS and TfOH as activators. The reaction was completed in 6 hours, and the target compound 29 was obtained in a yield of 60%. . ^{By 1} H-NMR, the coupling constant of protons at the 1st and 2nd positions of glucose was 7.813, confirming that it was a β-isomer. Complete B-selective glycosylation proceeded with the Bz group at the glucose 2-position. As a result of intramolecular glycosylation once again under the same conditions for the purpose of obtaining reproducibility, the reaction was completed in 3 hours, and the yield was 69% (the following formula).

  (Intramolecular glycosylation)

(Intramolecular glycosylation example of glucose (2-MPM 4-CA) and ceramide)
Compound 26 was reacted in an dichloromethane solvent at 0 ° C. using 2 equivalents of NIS and 0.2 equivalents of TfOH. Although the compound was expected to be reactive as an armed sugar, the progress of the reaction was not confirmed. After 2 hours, 0.2 equivalent of TfOH was added and the temperature was raised to room temperature, but only a slight amount of lactol was produced.

  (Intramolecular glycosylation)

(Intramolecular glycosylation example of glucose (2-Bz 4-OH) and ceramide)
Compound 27 was reacted in dichloromethane solvent at 0 ° C. using 2.0 equivalents of NIS and 0.3 equivalents of TfOH as activators. This reaction was completed in 5 hours, and the target compound 31 was obtained in a yield of 85%. ^{From 1} H-NMR, since the coupling constant between GlcH-1 and GlcH-2 was 7.8 Hz, it was confirmed to be β-form. Subsequently, Compound 27 was reacted in an dichloromethane solvent at −20 ° C. using 3.0 equivalents of NIS and 0.3 equivalents of TfOH (entry 2). Compound 31 was obtained. ^{Since the} coupling constant between GlcH-1 and GlcH-2 was 7.8 Hz by ¹ H-NMR, it was confirmed to be β-form. Entry 3 was reacted in dichloromethane solvent at 0 ° C. using 1.5 equivalents of DMTST as an activator. The reaction was completed in 2 hours, and the target compound 31 was obtained in a yield of 75%. Got. ^{Since the} coupling constant between GlcH-1 and GlcH-2 was 7.8 Hz by ¹ H-NMR, it was confirmed to be β-form. Since the reaction proceeded even in the system using DMTST as the activator (entry3), this method is expected to be applied to sphingosine-type ceramides that have an unsaturated bond and cannot use NIS as an activator. It was possible result. In all of Entry 1 to 3, the target compound was obtained in a β-selective manner due to the participation of the adjacent group of the glucose 2-position Bz group. However, the presence of by-products was confirmed slightly in all the reactions of entries 1 to 3. This compound has a molecular weight that matches that of the target compound, but a difference appears on the ¹ H-NMR spectrum. GlcH-2 was shifted in a high magnetic field despite the presence of the Bz group, and GlcH-3 and GlcH-5 were shifted slightly in the low magnetic field as compared with the target compound. And although GlcH-4 exists in free state, GlcH-6 had a low magnetic field shift. Further, the coupling constant between GlcH-1 and GlcH-2 was 10.9 Hz. From these points, the by-product was expected to be an orthoester. It is considered that the carbonyl group of the 2-position Bz group disappeared due to the formation of the ortho ester, and the electron withdrawing property was lowered. In addition, the low magnetic field shift of GlcH-3, GlcH-5, and GlcH-6 is presumed to be due to distortion of the pyranose ring due to succinic acid crosslinking. The coupling constant between GlcH-1 and GlcH-2 at the time of ortho ester production is a little as high as 10.9 Hz, but it is considered that this is also influenced by crosslinking with succinic acid.

  (Intramolecular glycosylation)

(Intramolecular glycosylation example of glucose (2-MPM 4-OH) and ceramide)
First, Compound 28 was reacted in a dichloromethane solvent at 0 ° C. using 2.0 equivalents of NIS and 0.2 equivalents of TfOH as activators. This reaction was completed in about an hour and a half, and the target compound 32 was obtained in a yield of 55% (α / β ratio is 1/1). Subsequently, in entry 2, the reaction was carried out in a dichloromethane solvent at −40 ° C. using 2.0 equivalents of NIS and 0.2 equivalents of TfOH as the activator. The reaction was completed in 5 hours, yield 74 % (Α / β ratio is 1/1) to obtain the target compound 32. Subsequently, in entry 3, the reaction was carried out in a dichloromethane solvent at −80 ° C. using 2.0 equivalents of NIS and 0.2 equivalents of TfOH as activators. In entry 3, since the progress of the reaction was not observed, 2.0 equivalents of NIS and 0.2 equivalents of TfOH were added, and the reaction temperature was gradually increased to −60 ° C., −40 ° C., and 0 ° C. Although the reaction was carried out for 36 hours, the raw materials did not disappear, and a small amount of hemiacetal was confirmed as a by-product. Subsequently, in entry 4, when the reaction was carried out at −40 ° C. in an acetonitrile solvent using NIS 2.0 equivalents and 0.2 equivalents of TfOH as an activator, the reaction progressed slowly and the reaction temperature was increased after 35 hours. 0 ° C. The reaction was completed in 48 hours, and the target compound 32 was obtained in a yield of 28% (α / β ratio was 1 / 6.3). At this time, a large amount of lactol was confirmed as a by-product. Due to the solvent effect of acetonitrile, β selectivity was improved as compared with dichloromethane solvent, but the reaction progressed slowly because the reaction temperature of −40 ° C. was lower in acetonitrile solvent. The reaction using acetonitrile, which is a polar solvent, is usually accelerated and combined with the nitrile solvent effect is considered to increase β selectivity. However, it was considered that the original β selectivity was not exhibited because the reaction proceeded slowly. Subsequently, in entry 5, the reaction was carried out in an acetonitrile solvent at 0 ° C. using 3.0 equivalents of NIS and 0.3 equivalents of TfOH, and the reaction was completed in 1.5 hours. The target compound 32 was obtained in a yield of 81% (α / β ratio was 1 / 8.4). In this case, it is considered that the reaction proceeded quickly, and that the β-coordinated counter anion was not passed, and the solvent effect of acetonitrile was combined to obtain high β selectivity. Next, in entry 6, the reaction was performed using NIS 3.0 equivalents and 0.3 equivalents of TfOH as an activator in an acetonitrile solvent at −20 ° C., and the reaction was completed in 3 hours. The yield was 47% (α The target compound 32 was obtained at a β ratio of β / β. Compared with entry 5 at 0 ° C., the progress of the reaction was slow, and a hemiacetal was confirmed as a by-product. In Entry7, for the purpose of α selectivity, the reaction was carried out in a diethyl ether solvent at 0 ° C. using 3.0 equivalents of NIS and 0.3 equivalents of TfOH, and the reaction progressed slowly for 20 hours. The reaction temperature was later brought to room temperature. The reaction was completed in 25 hours, and the target compound 32 was obtained in a yield of 44% (α / β ratio is 1 / 1.9). When diethyl ether is used, the reaction generally proceeds slowly, and α-selective glycosylation occurs by in situ anomerization. However, no remarkable α-selectivity was observed due to inadequate crosslinking with succinic acid. In Entry8, the reaction was carried out in an acetonitrile solvent at 0 ° C. using 4.0 equivalents of DMTST as an activator. The reaction was completed in 1 hour, and the yield was 76% (the α / β ratio was 1 / The target compound 32 was obtained in 6). Since the reaction proceeded even in a system using DMTST as an activator, this method was expected to be applied to sphingosine-type ceramide having an unsaturated bond and not using NIS as an activator. .

  In intramolecular glycosylation using a glucose donor with an MPM group at the 2-position, high reactivity as an armed sugar was expected, that is, a high yield, but the reactivity was particularly conspicuous as compared to a glucose donor with a Bz group at the 2-position. No improvement was observed. Moreover, although high stereoselectivity was anticipated by bridge | crosslinking by a succinic acid, the stereoselectivity by bridge | crosslinking was hardly seen but it resulted in only obtaining the stereoselectivity by solvent effects, such as acetonitrile. Therefore, it has become clear that the present invention can be implemented by appropriately designing based on such information.

  (Intramolecular glycosylation)

(Synthesis example of GM3 analog)
(Condensation of sialylgalactose donor and glucosylceramide acceptor)
Two synthetic strategies are also conceivable for the introduction of ceramide using intramolecular glycosylation. The first is a method using intramolecular glycosylation after sugar chain construction (the above formula: a method using intramolecular glycosylation after sugar chain construction). The second method is a method in which ceramide is first introduced into glucose serving as a sugar chain reducing end by intramolecular condensation via succinic acid, and then introduced into a sugar chain as a glucosylceramide acceptor. (Formula: Method using glucosylceramide acceptor using intramolecular glycosylation on glucose). In the previous synthesis strategy, he stated that the monosaccharide glucose was first used to crosslink with ceramide, followed by intramolecular glycosylation, and based on the results, each of the two synthesis strategies will be examined. Therefore, first, it was decided to introduce into a sugar chain (Neuα (2-3) Gal) using the glucosylceramide acceptor obtained by the second method of the synthesis strategy.

2.5 equivalents of sialyl α (2-3) galactose donor 33 and glucosylceramide acceptor 32 with respect to the acceptor were reacted in a dichloromethane solvent at 0 ° C. using 0.02 equivalent of TMSOTf as an activator. Since the progress of the reaction no longer appeared, 0.02 equivalent of TMSOTf was added after 2 hours. The reaction was completed after 7 hours, and the yield was 72% (β only). It was confirmed by ¹ H-NMR that the coupling constant of GalH-1 and GalH-2 was 8.1 Hz and β-form. Sialylgalactose lactol was confirmed as a by-product. When 2.5 equivalents of sialylgalactose donor was used, the reaction was completed because there was a sufficient amount of donor for the mild reactivity of the acceptor.

1.2 equivalents of sialyl α (2-3) galactose donor 33 and glucosylceramide acceptor 32 with respect to the acceptor were reacted in a dichloromethane solvent at 0 ° C. using 0.02 equivalent of TMSOTf as an activator. After 4 hours, 0.02 equivalent of TMSOTf was added. After 6 hours, the reaction was completed and the yield was 18% (β only). The acceptor did not disappear and the donor became lactol. As a by-product, a compound in which the 2-position Bz group of sialylgalactose lactol and sialylgalactose galactose was rearranged to 1-position was confirmed. It was confirmed by ¹ H-NMR that the coupling constant of GalH-1 and GalH-2 was 8.1 Hz and β-form. When 1.2 equivalents of sialyl-lacto-donor were used, the donor became lactol before the acceptor attack occurred, and the reaction was not completed. From the above results, it is difficult to say that the glucosylceramide acceptor has high reactivity. However, in entry 1 using a sufficient amount of donor, glycosylation has been achieved at a high yield, and in entry 2, unreacted acceptor can be recovered, so it can be said that the acceptor is highly stable. Therefore, it is considered that the yield of glycosylation can be improved by converting a very unstable imidate as a donor leaving group into another stable leaving group.

  (NeuGal donor + GlcCer acceptor)

(Example of deprotection)

(Example 1. General procedure)
¹ H and ¹³ C NMR spectra were measured by Varian INOVA 400 and 500. Chemical shifts are shown in ppm (δ) relative to the signal of Me ₄ Si adjusted to δ0 ppm. MALDI-TOF MS spectra were recorded in the estimated ion format on a Bruker Autoflex using α-cyano-4-hydroxycinnamic acid (CHCA) as the matrix. Molecular sieves were obtained from Wako Chemicals Inc. And dried in a muffle furnace at 300 ° C. for 2 hours before use. The solvent as the reaction medium was dried with molecular sieves and used without purification. TLC analysis was performed on Merck TLC (silica gel 60F ₂₅₄ on glass plate). Fuji Silysia Co. Silica gel (80 mesh and 300 mesh) prepared by the method used for flash column chromatography. The amount of silica gel was usually estimated as 200 to 400 times the weight of the packed sample. The solvent system in column chromatography is described as v / v. Evaporation and concentration were performed under reduced pressure. Specific rotation was measured with a Horiba SEPA-300 high sensitivity polarimeter.

(Preparation example of glucose donor (2-Bz))
First, Ac ₂ O and pyridine (pyr.) Were allowed to act on Compound 1 at 45 ° C. to obtain Compound 2 quantitatively. Thereafter, in order to protect the anomeric position of glucose with the SPh group, PhSH and BF ₃ · OEt ₂ were allowed to act at room temperature in a CH ₂ Cl ₂ solvent under an argon atmosphere, leading to compound 3 in a yield of 67%. It was. Subsequently, MeONa was allowed to act in an MeOH solvent at room temperature under an argon atmosphere to deprotect the Ac groups at the 2, 3, 4, and 6 positions to obtain Compound 4 at a yield of 93%. Subsequently, BDA and p-TsOH were allowed to act on Compound 4 in a MeCN solvent at room temperature under an argon atmosphere to obtain Compound 5 at a yield of 93%. In order to determine the structure of the product, the compound 5 was converted to Ac, and the ¹ H-NMR spectrum was confirmed. As a result, the signal δ (ppm) 3.4 (m, 1 H) of the 2-position proton of the compound 5 and the 3-position proton Signal 3.8 (m, 1H) showed a low magnetic field shift of 5.0 (t, 1H), 5.3-5.4 (t, 1H), respectively. This confirmed the introduction of benzylidene groups at positions 4 and 6.

Next, DBTO, TBAB, and MPMCl were allowed to act on Compound 5 under reflux in a toluene solvent, leading to Compound 6 at a yield of 77%. It is said that Bu ₂ SnO forms a cyclic stannylene compound and activates cis-glycol first, and the reaction is likely to occur at the hydroxyl group of the equiurial. In compound 5, cis glycol is not present, and positions 4 and 6 are protected with a benzylidene group. Therefore, it is considered that a stanylene compound is formed at positions 2 and 3 and MPM progressed selectively at position 3. It was. In order to determine the structure of the product, Compound 6 was converted to Ac and the ¹ H-NMR spectrum was confirmed. As a result, the signal δ (ppm) 3.5 (m, 1 H) of the 2-position proton of Compound 6 was 5.2. The magnetic field shift was as low as 5.3 (t, 1H). From this, it was confirmed that the 2-position was converted to Ac, that is, an MPM group was introduced at the 3-position.

(Preparation of phenyl 2-O-benzoyl-4,6-O-benzylidene-1-deoxy-3-Op-methoxybenzyl-1-thio-β-D-glucoplanoside (7))
To a solution of compound 6 (171 mg, 0.356 mmol) in pyridine (3.56 mL) was added benzoyl chloride (62.0 μL, 0.534 mmol) and DMAP (4.35 mg, 0.036 mmol). Was added. The mixture was then stirred at room temperature for 4 hours. Complete consumption of the starting material was confirmed by TLC analysis (toluene / MeOH 50/1). Methanol was added to the reaction mixture at 0 ° C. and co-evaporated with toluene. The mixture was diluted with CHCl ₃ , washed with 2 M HCl, H ₂ O, saturated aqueous NaHCO ₃ and brine, dried (Na ₂ SO ₄ ) and concentrated. The residue was purified by column chromatography on silica gel (CHCl ₃ ) to give 7 (166 mg, 80%).

(Preparation of phenyl 2-O-benzoyl-1-deoxy-3-Op-methoxybenzyl-1-thio-β-D-glucopyranoside (8))
To a solution of compound 7 (200 mg, 0.330 mmol) in AcOH (15.0 mL) was added H ₂ O (3.00 mL). 12 hours at 40 ° C.: After (TLC monitoring toluene / EtOAc 1/1), and the mixture was diluted with CHCl _3, and washed with _H 2 O, NaHCO ₃ and brine, saturated aqueous, dried (Na ₂ SO _4), and concentrated. The residue was purified by silica gel chromatography (toluene / EtOAc 3/1) to give 8 (145 mg, 86%).

(Preparation of phenyl 4,6-O-benzylidene-1-deoxy-2,3-di-Op-methoxybenzyl-1-thio-β-D-glucopyranoside (9))
The following experiments were conducted based on the description of Hiromune Ando, Yusuke Koike, Hideharu Ishida, and Makito Kiso et al., Tetrahedron Letters 44 (2003) 6883-6886.

  For example, a solution of compound 7 in MeOH / THF = 2/1 containing MeONa was stirred at room temperature to give compound 7a.

To a solution of compound 7a (4.11 g, 8.56 mmol) in DMF (85.6 mL) was added NaH (307 mg, 12.8 mmol) and stirred at room temperature for 1 hour, then at 0 ° C. To this mixture was added MPMC1 (1.74 mL, 12.5 mmol). The mixture was stirred at room temperature for 12 hours. Complete consumption of the starting material was confirmed by TLC analysis (toluene / EtOAc 20/1). The mixture was diluted with EtOAc, washed with 2 M HCl, H ₂ O, saturated aqueous NaHCO ₃ and brine, dried (Na ₂ SO ₄ ) and concentrated. The residue was purified by silica gel chromatography (toluene / EtOAc 200/1) to give 9 (3.60 g, 70%).

(Preparation of glucose donor 10 (2-MPM))
_{The following experiment was performed using a method similar to the preparation of compound 8.} For example, 83% acetic acid was allowed to act on compound 9 at 30 ° C. to prepare glucose donor 10 (2-MPM) at a yield of 86%.

(Preparation of (2S, 3S, 4R) -1-O-tert-butyldiphenylsilyl-2-octadecanamide-octadecane (13))
Stearic acid was added to a solution of compound 11 in CH ₂ Cl ₂ in the presence of WSC. Stir at room temperature to give 12 (96%).

To a solution of compound 12 (500 mg, 0.857 mmol) and triethylamine (10.0 mL) in CH ₂ Cl ₂ (8.57 mL) was added TBDPSCl (0.260).
mL, 1.03 mmol) and DMAP (209 mg, 1.71 mmol) were added. After stirring at room temperature for 20 hours (TLC monitoring: CHCl ₃ / MeOH 50/1), MeOH was added to the mixture at 0 ° C. After concentration, the residue was purified by silica gel column chromatography (CHCl ₃ / MeOH 100/1) to obtain 13 (533 mg, 76%).

(Preparation 1 of ceramide derivative 14)
Numata, M .; Sugimoto, M .; Koike, K .; Ogawa, T .; Carbohydr. Res. 1990, 203, 205-217. _{Based on the description, the following experiment was conducted} . For example, compound 13 was allowed to react with 1 equivalent of succinic anhydride in the presence of DMAP in a pyridine solvent at room temperature, and then heated to 40 ° C. This product was treated with Ac ₂ O to obtain a ceramide derivative 14 having succinic acid at the 4-position (yield 40% in two steps).

(Preparation of Compound 16)
Numata, M .; Sugimoto, M .; Koike, K .; Ogawa, T .; Carbohydr. Res. 1990, 203, 205-217. _{Based on the description, the following experiment was conducted. For example,} Compound 12 was added with BDA and p-TsOH in acetonitrile solvent, and heated at 40 ° C. to obtain Compound 15. This compound 15 was reacted with succinic anhydride and DMAP in a pyridine solvent at 40 ° C. to obtain compound 16 in which succinic acid was introduced at the 4-position.

(Preparation of (2S, 3S, 4R) -1,3-O-benzylidene-1-deoxy-4-O-succinoylbenzyl ester-2-octadecanamide-octadecane (17))
To a solution of compound 16 (10.0 g, 13.0 mmol) in MeCN (130 mL) and DMF (130 mL) was added K ₂ CO ₃ (1.80 g, 13.0 mmol) and BnBr (1. 70 mL, 14.3 mmol) was added. The mixture was stirred at room temperature for 16 hours (TLC monitoring: toluene / EtOAc 2/1). The reaction mixture was coevaporated with toluene and extracted with CHCl ₃ . The organic phase was washed with 2 M HCl, H ₂ O, saturated aqueous NaHCO ₃ and brine, dried (Na ₂ SO ₄ ) and concentrated. Purification by column chromatography on silica gel (toluene / EtOAc 40/1) gave 17 (9.0 g, 80%).

(Preparation of (2S, 3S, 4R) -4-O-succinoylbenzyl ester-2-octadecanamide-octadecane (18))
To a solution of compound 17 (20.3 mg, 0.023 mmol) in AcOH (0.8 mL) was added H ₂ O (0.2 mL). After stirring at 60 ° C. for 12 hours (TLC monitoring: toluene / EtOAc 2/1), the mixture was diluted with CHCl ₃ , washed with H ₂ O, saturated aqueous NaHCO ₃ and brine, and dried (Na ₂ SO ₄ ) and concentrated. Purification by column chromatography on silica gel (toluene / EtOAc 2/1) gave 18 (11.0 mg, 62%).

(Preparation of (2S, 3S, 4R) -3-O-acetyl-1-O-tert-butyldiphenylsilyl-4-O-succinoylbenzyl ester-2-octadecanamide-octadecane (20))
To a solution of compound 18 (100 mg, 0.129 mmol) and triethylamine (1.0 mL) in 1,2-dichloroethane (1.3 mL) was added TBDPSCl (66.5 μL, 0.259 mmol) and DMAP. (30.6 mg, 0.250 mmol) was added. The mixture was stirred at 40 ° C. for 12 hours. After complete consumption of starting material was confirmed by TLC analysis (toluene / EtOAc 1/1), MeOH was added at 0 ° C. The reaction mixture was evaporated and pulled on a vacuum line for 30 hours. This residue was dissolved in pyridine (3.0 mL) and Ac ₂ O was added. The mixture was stirred at room temperature for 16 hours (TLC monitoring: toluene / EtOAc 2/1). The reaction mixture was coevaporated with toluene and extracted with CHCl ₃ . The organic phase was washed with 2 M HCl, H ₂ O, saturated aqueous NaHCO ₃ and brine, dried (Na ₂ SO ₄ ) and concentrated. Purification by column chromatography on silica gel (toluene / EtOAc 4/1) gave 20 (98.0 mg, 72%).

(Preparation 2 of ceramide derivative 14)
Numata, M .; Sugimoto, M .; Koike, K .; Ogawa, T .; Carbohydr. Res. 1990, 203, 205-217. Based on the description, the following experiment was conducted. For example, the ceramide acceptor 14 was obtained by using the catalytic hydrogenation method for the compound 20 at 40 ° C. using Pd (OH) ₂ in EtOH.

(Phenyl 6-O-[{(2S, 3S, 4R) -3-O-acetyl-1-O-tert-butyldiphenylsilyl-2-octadecanamido-octadecan-4-yloxy} carbonylpropanoyl] -2- Preparation of O-benzoyl-1-deoxy-3-Op-methoxybenzyl-1-thio-β-D-glucopyranoside (21) 1)
To a solution of compound 14 (204 mg, 0.212 mmol) in CH ₂ Cl ₂ (2.12 mL) was added 2,4,6-trichlorobenzoyl chloride (50.0 μL, 0.318 mmol), DMAP ( 39.0 mg, 0.318 mmol), triethylamine (44.4 μL, 0.318 mmol) and compound 8 (105 mg, 0.212 mmol) were added. The mixture was stirred at room temperature for 2 hours. Complete consumption of the starting material was confirmed by TLC analysis (toluene / EtOAc 2/1). The mixture was diluted with CHCl ₃ and washed with saturated aqueous NaHCO ₃ , H ₂ O, and brine, dried (Na ₂ SO ₄ ), and concentrated. The residue was purified by column chromatography on silica gel (EtOAc / hexane 1/6) to give 21 (229 mg, 75%) _.

(Phenyl 6-O-[{(2S, 3S, 4R) -3-O-acetyl-1-O-tert-butyldiphenylsilyl-2-octadecanamido-octadecan-4-yloxy} carbonylpropanoyl] -2- Preparation of O-benzoyl-4-O-chloroacetyl-1-deoxy-3-Op-methoxybenzyl-1-thio-β-D-glucopyranoside (22))
To a solution of compound 21 (200 mg, 0.138 mmol) in CH ₂ Cl ₂ (2.20 mL) was added triethylamine (38.7 μL, 0.277 mmol) and chloroacetic anhydride (47.4 mg, 0 .277 mmol) was added. The mixture was stirred at room temperature for 2 hours. Complete consumption of the starting material was confirmed by TLC analysis (EtOAc / hexane 1/3). To the reaction mixture was added methanol at 0 ° C. and the mixture was diluted with CHCl ₃ , washed with 2 M HCl, H ₂ O, saturated aqueous NaHCO ₃ and brine, dried (Na ₂ SO ₄ ), and concentrated. did. The residue was purified by column chromatography on silica gel (EtOAc / hexane 1/8) to give 22 (208 mg, 98%).

(Phenyl 6-O-[{(2S, 3S, 4R) -3-O-acetyl-2-octadecanamido-octadecan-4-yloxy} carbonylpropanoyl] -2-O-benzoyl-4-O-chloroacetyl Preparation of -1-deoxy-3-Op-methoxybenzyl-1-thio-β-D-glucopyranoside (23))
To a solution of compound 22 (17.0 mg, 0.011 mmol) in THF (224 μL) was added AcOH (5.00 μL, 0.084 mmol) and tetrabutylammonium fluoride (16.8 μL, 0.001 mmol). 017 mmol) was added. The mixture was stirred at room temperature for 18 hours. Complete disappearance of starting material was confirmed by TLC analysis (EtOAc / Hexane 1/1). The mixture was diluted with CHCl ₃ , washed with 2 M HCl, saturated aqueous NaHCO ₃ , dried (Na ₂ SO ₄ ), and concentrated. The residue was purified by column chromatography on silica gel (EtOAc / hexane 2/3) to give 23 (13.2 mg, 92%).

(Phenyl 6-O-[{(2S, 3S, 4R) -3-O-acetyl-1-O-tert-butyldiphenylsilyl-2-octadecanamido-octadecan-4-yloxy} carbonylpropanoyl] -1- Preparation of deoxy-2,3-di-Op-methoxybenzyl-1-thio-β-D-glucopyranoside (24))
To a solution of compound 10 (490 mg, 0.957 mmol) and compound 14 (1.11 g, 1.15 mmol) in CH ₂ Cl ₂ (9.6 mL) was added WSC (550 mg, 2.87 mmol). ) Was added. The mixture was stirred at room temperature for 6 hours. The end of the reaction was confirmed by TLC (toluene / EtOAc 3/1). The reaction mixture was diluted with CHCl ₃ , washed with H ₂ O and brine, dried (Na ₂ SO ₄ ) and concentrated. Purification by column chromatography on silica gel (EtOAc / hexane 1: 3) gave 24 (851 mg, 61%).

(Phenyl 6-O-[{(2S, 3S, 4R) -3-O-acetyl-1-O-tert-butyldiphenylsilyl-2-octadecanamido-octadecan-4-yloxy} carbonylpropanoyl] -4- Preparation of O-chloroacetyl-1-deoxy-2,3-di-Op-methoxybenzyl-1-thio-β-D-glucopyranoside (25))
To a solution of compound 24 (150 mg, 0.103 mmol) in CH ₂ Cl ₂ (1.6 mL) was added triethylamine (28.8 μL, 0.206 mmol) and chloroacetic anhydride (35.1 mg, 0 .205 mmol) was added. The mixture was stirred at room temperature for 2 hours. After complete consumption of the starting material was confirmed by TLC analysis (EtOAc / Hexane 1/3), MeOH was added at 0 ° C. The mixture was diluted with CHCl ₃ , washed with 2 M HCl, H ₂ O, saturated aqueous NaHCO ₃ and brine, dried (Na ₂ SO ₄ ) and concentrated. Purification by column chromatography on silica gel (EtOAc / hexane 2/9) gave 25 (158 mg, quantitative).

(Phenyl 6-O-[{(2S, 3S, 4R) -3-O-acetyl-2-octadecanamido-octadecan-4-yloxy} carbonylpropanoyl] -4-O-chloroacetyl-1-deoxy-2 , 3-Di-Op-methoxybenzyl-1-thio-β-D-glucopyranoside (26))
THF To a solution of compound 25 (157 mg, 0.102 mmol) in (1.0 mL), AcOH (6.4 μL, 0.102 mmol) and tetrabutylammonium fluoride (102 μL, 0.102 mmol) Was added. After stirring for 3 hours at 0 ° C. (TLC monitoring: EtOAc / hexane 1/1), the mixture was diluted with CHCl ₃ , washed with saturated aqueous NaHCO ₃ and brine, and dried (Na ₂ SO ₄ ). And concentrated. Purification by column chromatography on silica gel (EtOAc / hexane 1/4) gave 26 (76 mg, 58%).

(Phenyl 6-O-[{(2S, 3S, 4R) -3-O-acetyl-2-octadecanamido-octadecan-4-yloxy} carbonylpropanoyl] -2-O-benzoyl-1-deoxy-3- Preparation of Op-methoxybenzyl-1-thio-β-D-glucopyranoside (27))
To a solution of compound 21 (48.8 mg, 0.034 mmol) in THF (338 μL) was added AcOH (6.0 μL, 0.101 mmol) and tetrabutylammonium fluoride (102 μL, 0.102 mmol). ) Was added. After stirring at 0 ° C. for 12 hours (TLC monitoring: EtOAc / hexane 1/2), the mixture was diluted with CHCl ₃ , washed with saturated aqueous NaHCO ₃ and brine, dried (Na ₂ SO ₄ ), and Concentrated. Purification by column chromatography on silica gel (toluene / EtOAc 1.5: 1) gave 27 (30 mg, 75%).

(Phenyl 6-O-[{(2S, 3S, 4R) -3-O-acetyl-2-octadecanamido-octadecan-4-yloxy} carbonylpropanoyl] -1-deoxy-2,3-di-O- Preparation of p-methoxybenzyl-1-thio-β-D-glucopyranoside (28))
To a solution of compound 24 (157 mg, 0.108 mmol) in THF (1.1 mL) was added AcOH (6.5 μL, 0.108 mmol) and tetrabutylammonium fluoride (110 μL, 0.110 mmol). ) Was added. After stirring for 3 hours at 0 ° C. (TLC monitoring: EtOAc / hexane 1/1), the mixture was diluted with CHCl ₃ , washed with saturated aqueous NaHCO ₃ and brine, and dried (Na ₂ SO ₄ ). And concentrated. Purification by column chromatography on silica gel (toluene / EtOAc 3/2) gave 28 (76 mg, 58%).

(Phenyl 6-O-[{(2S, 3S, 4R) -3-O-acetyl-1-O-tert-butyldiphenylsilyl-2-octadecanoylamino-octadecan-4-yloxy} carbonylpropanoyl]- 2-O-benzoyl-3-Op-methoxybenzyl-1-thio-β-D-glucopyranoside (7)

To a solution of compound 6 (204 mg, 0.212 mmol) in CH ₂ Cl ₂ (2.1 mL) was added 2,4,6-trichlorobenzoyl chloride (50.0 μL, 0.318 mmol), DMAP ( 39.0 mg, 0.318 mmol), triethylamine (44.4 μL, 0.318 mmol) and compound 3 (105 mg, 0.212 mmol) were added. The mixture was stirred for 2 hours at room temperature. After confirming consumption of the starting material by TLC analysis (toluene / EtOAc 2/1), the mixture was diluted with CHCl ₃ and washed with sat. Washed with NaHCO ₃ , H ₂ O and brine, dried (Na ₂ SO ₄ ) and concentrated. The residue was subjected to column chromatography on silica gel (EtOAc / hexane 1/6) to give compound 7 (229 mg, 76%):
[Α] _D + 0.39 ° (c 1.9, CHCl ₃ ); ¹ H NMR (500 MHz, CDCl ₃ ): d 6.60-8.10 (m, 24H, 5Ph), 5.95 ( s, 1H, J2 _{, NH} = 9.5 Hz, NH), 5.35 (dd, 1H, H-3 ^Cer ), 5.19 (t, 1H, _J1,2 = _J2,3 = 9 .9 Hz, H-2 ^Glc ), 4.95 (dt, 1H, H-4 ^Cer ), 4.76 (d, 1H, _J1,2 = 9.9 Hz, H-1 ^Glc ), 4. _{66 (d, 1H, J gem} = 11.0 Hz, PhCH 2), 4.63 (d, 1H, J gem = 11.0 Hz, PhCH 2), 4.46 (dd, 1H, H-6 ' ^{Glc), 4.39 (dd, 1H} , H-6 'Glc), 4.22 (m, 1H, ^{-2 Cer), 3.72 (m,} 1H, H-4 Glc), 3.71 (s, 3H, OMe), 3.63 (m, 1H, H-3 Glc), 3.53 (m, ^{1H, H-5 Glc),} 2.60-2.66 (2d, 4H, -OCOCH 2 CH 2 COO-), 1.00-2.20 (m, 58 H, -CH 2 -), 0. 90 (t, 6H, 2-CH ₃ ); ¹³ C NMR (125 MHz, CDCl ₃ ): d 172.7, 172.2, 171.9, 170.5, 165.1, 159.3, 135. 7, 135.5, 133.2, 132.8, 132.6, 129.9, 129.8, 129.7, 128.7, 128.4, 127.9, 127.8, 127.8, 113.8, 86.3, 82.9, 73.9, 72 .1, 71.6, 69.8, 63.0, 62.3, 55.1, 49.2, 36.8, 31.9, 29.7, 29.7, 29.6, 29.6, 29.5 , 29.4, 29.4, 29.4, 27.9, 26.8, 25.7, 22.7, 20.7, 19.2, 14.1; MALDI-TOF-MS : M / z = 1464 [M + Na] ⁺ .
(Phenyl 6-O-[{(2S, 3S, 4R) -3-O-acetyl-2-octadecanoylamino-octadecan-4-yloxy} carbonylpropanoyl] -2-O-benzoyl-3-O- 4-Methoxybenzyl-1-thio-β-D-glucopyranoside (8)
THF To a solution of compound 7 (48.8 mg, 0.034 mmol) in (338 μL) was added AcOH (6.0 μL, 0.101 mmol) and tetrabutylammonium fluoride (102 μL, 0.102 mmol). . After stirring at 0 ° C. for 12 hours (TLC monitoring: EtOAc / hexane 1/2), the mixture was diluted with CHCl ₃ and washed with sat. Washed with NaHCO ₃ and brine, dried (Na ₂ SO ₄ ) and concentrated. The residue was subjected to column chromatography on silica gel (toluene / EtOAc 1.5 / 1) to give compound 8 (30 mg, 77%):
[Α] _D + 7.4 ° (c 0.6, CHCl ₃ ); ¹ H NMR (500 MHz, CDCl ₃ ): d 6.66-8.05 (m, 13H, 3 Ph), 6.23 _{(d, 1H, J 2,} NH = 9.0 Hz, NH), 5.21 (t, 1H, J 1,2 = J 2,3 = 10.0 Hz, H-2 Glc), 5.01 -5.06 (m, 2H, H-3 ^Cer , H-4 ^Cer ), 4.78 (d, 1H, J1, ₂ = 10.0 Hz, H-1 ^Glc ), 4.63 (2d _{, 2H, J gem = 11.5 Hz} , PhCH 2), 4.48 (dd, 1H, J gem = 12.0 Hz, H-6 'Glc), 4.39 (dd, 1H, J gem = 12 ^{.0 Hz, H-6 Glc)} , 4.19 (m, 1H, H-2 Cer), 3.6 -3.74 (m, 6H, H- 1 'Cer, H-3 Glc, H-4 Glc, OMe), 3.54-3.59 (m, 2H, H-1 Cer, H-5 Glc) _{, 2.60-2.75 (m, 4H, -OCOCH} 2 CH 2 COO-), 2.15 (s, 3H, OAc), 1.20-2.20 (m, 58H, -CH 2 -) , 0.89 (t, 6H, 2-CH ₃ ); ¹³ C NMR (100 MHz, CDCl ₃ ): d 173.3, 172.6, 171.9, 171.5, 165.2, 159.3 , 133.2, 132.8, 132.5, 129.8, 129.8, 129.7, 129.0, 128.7, 128.4, 128.2, 127.8, 125.3, 113 .7, 86.4, 82.9, 77.8, 74 5, 73.4, 73.2, 72.0, 69.7, 63.3, 61.4, 55.1, 49.6, 36.7, 31.9, 29.7, 29.6, 29.5, 29.3, 29.3, 29.1, 28.6, 25.6, 25.5, 22.7, 20.9, 14.1; MALDI-TOF-MS: m / z = 1226 [M + Na] ⁺ .
(2-O-benzoyl-3-O-4-methoxybenzyl-β-D-glucopyranosyl- (1 (R) 1)-(2S, 3S, 4R) -3-O-acetyl-2-octadecanoylamino -Octadecane-4,6-succinate (9)
To a solution of compound 8 (43 mg, 0.036 mmol) in CH ₂ Cl ₂ (1.2 mL) was added MS4Å (40 mg). After stirring for 1 hour, NIS (16.0 mg, 0.071 mmol) and TfOH (0.6 μL, 0.0079 mmol) were added to the suspension. The mixture was stirred for 5 hours. Completion of the reaction was confirmed by TLC (toluene / EtOAc 1/1). The reaction mixture was filtered through Celite. The combined filtrate and washings were extracted with CHCl ₃ and washed with sat. NaHCO _3, sat. Washed with Na ₂ S ₂ O ₃ and brine, dried (Na ₂ SO ₄ ) and concentrated. The residue was subjected to silica gel column chromatography (toluene / EtOAc 3: 1) to give compound 9 (33 mg, 85%):
[Α] _D + 3.7 ° (c 1.1, CHCl ₃ ); ¹ H NMR (500 MHz, CDCl ₃ ): d 6.67-8.00 (m, 9H, 2 Ph), 6.02 (D, 1H, J2 _{, NH} = 9.0 Hz, NH), 5.18 (m, 2H, _J1,2 = 7.5 Hz, H-4 ^Cer , H-2 ^Glc ), 5.10 ^{(t, 1H, H-3} Cer), 4.66 (d, 1H, J gem = 11.5 Hz, PhCH 2), 4.55 (d, 1H, J gem = 11.5 Hz, PhCH 2) _{, 4.47 (d, 1H, J} 1,2 = 7.5 Hz, H-1 Glc), 4.39 (m, 3H, J 1,2 = 5.5 Hz, H-2 Cer, H- ^6Glc , H- ^6'Glc ), 3.81 (dd, 1H, J _gem = 11.5 Hz, J _{1, 2} = 5.5 Hz, H-1 ^Cer ), 3.73 (s, 3H, OMe), 3.61-3.69 (m, 3H, H-1 ^Cer , H-3 ^Glc , H- 5 ^Glc ), 3.49 (dt, 1H, H-4 ^Glc ), 2.50-2.80 (m, 4H, —OCOCH ₂ CH ₂ COO—), 2.10 (s, 3H, OAc), 1.10-2.00 (m, 58H, —CH ₂ —), 0.84 (t, 6H, 2-CH ₃ ); ¹³ C NMR (100 MHz, CDCl ₃ ): d 172.9, 171. 4, 171.2, 170.8, 165.0, 159.3, 133.3, 129.8, 129.7, 129.6, 129.5, 128.5, 114.0, 113.9, 100.1, 81.5, 74.7, 74.3, 73.8 73.7, 72.7, 70.4, 63.8, 55.1, 47.4, 37.4, 37.1, 36.5, 33.5, 32.7, 31.9, 30. 2, 30.0, 29.7, 29.6, 29.6, 29.5, 29.5, 29.5, 29.4, 29.3, 29.3, 29.2, 27.4, 27.1 25.4, 25.0, 24.4, 22.7, 21.0, 19.7, 14.1; MALDI-TOF-MS: m / z = 1116 [M + Na] ⁺ .
(Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-β-D-galacto-2-nonuropyranosylate)-(2 → 3) -(2,4,6-tri-O-benzoyl-β-D-galactopyranosyl)-(1 → 4) -2-O-benzoyl-3-O-4-methoxybenzyl-β-D-glucopyranosyl -(1 → 1)-(2S, 3S, 4R) -3-O-acetyl-2-octadecanoylaminooctadecane-4,6-succinate (11)

To a solution of compound 9 (63 mg, 0.0578 mmol) and compound 10 (152 mg, 0.137 mmol) in CH ₂ Cl ₂ (1.1 mL), molecular sieve (AW-300) (200 mg) Was added. After stirring for 1 hour, TMSOTf (1.0 μL, 0.00548 mmol) was added to the suspension. The course of the reaction was monitored by TLC (toluene / EtOAc 1/3). After 4 hours of stirring, the reaction mixture was filtered through a pad of Celite. The filtrate and washings were combined and extracted with CHCl ₃ , sat. Washed with NaHCO ₃ and brine, dried (Na ₂ SO ₄ ) and concentrated. The residue was chromatographed on silica gel (toluene / EtOAc 1/2) to give compound 11 (83 mg, 70%):
[Α] _D + 24.4 ° (c 0.7, CHCl ₃ ); ¹ H NMR (400 MHz, CDCl ₃ ): d 7.34-8.21 (m, 20H, 4Ph), 6.43-7 .01 (2d, 4H, PMB), 5.94 (d, 1H, J2 _{, NH} = 8.2 Hz, NH ^Cer ), 5.68 (m, 1H, H-8 ^Neu ), 5.55 ( near t, 1H, J _1,2 = 8.2 Hz, H-2 ^Gal ), 5.36 (d, 1H, H-4 ^Gal ), 5.24 (dd, 1H, H-7 ^Neu ), 5 .04-5.12 (m, 3H, J _1,2 = 8.2 Hz, H-1 ^Gal H-3 ^Cer , J _1,2 = 7.8 Hz, H-2 ^Glc ), 4.91- 4.98 (m, 3H, H-4 ^Cer , NH ^Neu , H-3 ^Gal ), 4.81 ^{(M, 1H, H-4} Neu), 4.66-4.87 (2d, 2H, J gem = 11.0 Hz, PhCH 2), 4.42 (dd, 1H, J gem = 12.4 Hz , H-9 ′ ^Neu ), 4.14-4.37 (m, 7H, H-5 ^Gal , H-6 ^Gal , H-6 ′ ^Gal , H-6 ^Glc , H-6 ′ ^Glc , J _{1, 2} = 7.8 Hz, H-1 ^Glc , H-2 ^Cer ), 4.05 (dd, 1H, J _gem = 12.4 Hz, H-9 ^Neu ), 3.71-3.82 (m, 8H, H-1 ^Cer , H-1 ′ ^Cer , H-3 ^Glc , H-4 ^Glc , H-5 ^Neu , OMe), 3.61 (dd, 1H, H-6 ^Neu ), 3.57 (s , 3H, OMe), 3.50 ( t, 1H, H-5 Glc), 2.3 _{-2.67 (m, 4H, -OCOCH 2} CH 2 COO-), 2.48 (dd, 1H, H-3eq Neu), 1.64 (m, 1H, H-3ax Neu), 1.52- 2.18 (6s, 18H, OAc), 1.10-1.40 (m, 58H, —CH ₂ —), 0.87 (t, 6H, 2-CH ₃ ); ¹³ C NMR (100 MHz, CDCl ₃ ): d 172.84, 170.97, 170.90, 170.75, 170.67, 170.61, 1700.25, 170.08, 168.16, 165.73, 165.56, 165 .05, 158.82, 133.33, 133.12, 130.36, 130.15, 129.88, 129.78, 129.72, 129.63, 129.35, 128. 6, 128.53, 128.31, 113.37, 101.29, 98.78, 96.94, 79.50, 78.72, 77.66, 74.53, 74.36, 73.36, 73.12, 73.06, 71.95, 71.55, 71.41, 70.94, 69.39, 68.23, 67.61, 66.59, 63.33, 62.35, 61. 72, 54.97, 53.23, 48.82, 46.55, 37.35, 36.34, 31.93, 30.80, 30.04, 29.67, 29.62, 29.55, 29.45, 29.38, 29.20, 25.30, 25.15, 23.16, 22.70, 21.47, 20.93, 20.83, 20.73, 20.41, 14. 13; MALDI-TOF- MS: m / z = 2064 [M + Na] ^< +>.

The reaction was carried out under the conditions described in Table 2 above to crosslink Glc and Cer.

  (Preparation of Compound 29)

For example, using a method similar to that described above, compound 29 was treated at 55 ° C. in the presence of DABCO in EtOH to give compound 35 (yield 60%).

(Preparation of Compound 30)
For example, using the same method as described above, reaction was performed under the conditions described in Table 1 below to obtain Compound 30.

(2-O-benzoyl-3-Op-methoxybenzyl-β-D-glucopyranoside- (1 → 1)-(2S, 3S, 4R) -3-O-acetyl-2-octadecanamide-octadecane-4 , 6-Preparation of succinate (31))
To a solution of compound 27 (43 mg, 0.036 mmol) in CH ₂ Cl ₂ (1.2 mL) was added molecular sieve 4Å (MS4Å) (40 mg). After stirring for 1 hour, NIS (16.0 mg 0.071 mmol) and TfOH (0.6 μL, 0.0079 mmol) were added to the suspension. The mixture was stirred for 5 hours. The end of the reaction was confirmed by TLC (toluene / EtOAc 1/1). The reaction mixture was filtered through celite. The combined filtrate and washes were extracted with CHCl ₃ and washed with saturated aqueous NaHCO _3, saturated aqueous Na ₂ S ₂ O ₃ and brine, dried (Na ₂ SO ₄ ) and concentrated. Purification by column chromatography on silica gel (toluene / EtOAc 3: 1) gave 31 (33 mg, 85%).

(2,3-di-Op-methoxybenzyl-α-D-glucopyranoside- (1 → 1)-(2S, 3S, 4R) -3-O-acetyl-2-octadecanamido-octadecane-4,6 -Preparation of succinate (32))
To a solution of compound 28 (32 mg, 0.026 mmol) in MeCN (870 μL), molecular sieve 3 Å (MS3 Å) (30 mg) was added. After stirring for 1 hour, NIS (17.7 mg 0.079 mmol) and TfOH (0.7 μL, 0.0079 mmol) were added to the suspension and stirring was continued for 1.5 hours. . The end of the reaction was confirmed by TLC (toluene / EtOAc 1/1). The reaction mixture was filtered through celite. The combined filtrate and washings were extracted with CHCl ₃ , washed with saturated aqueous NaHCO _3, saturated aqueous Na ₂ S ₂ O ₃ and brine, dried (Na ₂ SO ₄ ) and concentrated. Purification by column chromatography on silica gel (toluene / EtOAc 3/1) gave 32 (2.5 mg, 9%).

(2,3-Di-Op-methoxybenzyl-β-D-glucopyranoside- (1 → 1)-(2S, 3S, 4R) -3-O-acetyl-2-octadecanamide-octadecane-4,6 -Preparation of succinate (32))
To a solution of compound 28 (32 mg, 0.026 mmol) in MeCN (870 μL) was added MS3Å (30 mg). After stirring for 1 hour, NIS (17.7 mg 0.079 mmol) and TfOH (0.7 μL, 0.0079 mmol) were added to the suspension and stirring was continued for 1.5 hours. It was. The end of the reaction was confirmed by TLC (toluene / EtOAc 1/1). The reaction mixture was filtered through celite. The combined filtrate and washes were extracted with CHCl ₃ and washed with saturated aqueous NaHCO _3, saturated aqueous Na ₂ S ₂ O ₃ and brine, dried (Na ₂ SO ₄ ) and concentrated. Purification by column chromatography on silica gel (toluene / EtOAc 3/1) gave 32 (21 mg, 72%).

(Preparation of sialyl α (2-3) galactose donor 33)
The following experiments were conducted based on the description of Hiromune Ando, Yusuke Koike, Hideharu Ishida, and Makito Kiso et al., Tetrahedron Letters 44 (2003) 6883-6886.

  For example, sialyl α (2-3) galactose donor 33 was prepared using a method similar to (Preparation of Compound 49A).

((Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonuropyranosylate- (2 → 3) -(2,4,6-tri-O-benzoyl-β-D-galactopyranosyl)-(1 → 4) -2,3-di-Op-methoxybenzyl-β-D-glucopyranoside- ( 1 → 1)-(2S, 3S, 4R) -3-O-acetyl-2-octadecanamide-octadecane-4,6-succinate (34))
To a solution of compound 32 (23.9 mg, 0.022 mmol) and compound 33 (60.0 mg, 0.054 mmol) in CH ₂ Cl ₂ (430 μL), molecular sieve (AW300) (84 mg) was added. Added. After stirring for 1 hour, TMSOTf (0.55 M solution in CH ₂ Cl ₂ , 0.2 μL, 0.001 mmol) was added to the suspension. The progress of the reaction was monitored by TLC (toluene / EtOAc 1/3). After 7 hours of stirring, the reaction mixture was filtered through a pad of Celite®. The filtrate was diluted with CHCl ₃ and the organic phase was washed with saturated aqueous NaHCO _3, saturated aqueous Na ₂ S ₂ O ₃ and brine, dried (Na ₂ SO ₄ ) and concentrated. Purification by column chromatography on silica gel (toluene / EtOAc 2/5) gave 34 (32 mg, 72%).

Molecular sieve (AW300) is added to a solution of compound 29 and a protected sugar donor (eg, compound 33) in CH ₂ Cl ₂ . After stirring, TMSOTf (in CH ₂ Cl ₂ ) is added to the suspension. The progress of the reaction is monitored by TLC. After stirring, the reaction mixture is filtered through a pad of Celite®. The filtrate is diluted and the organic phase is washed with saturated aqueous NaHCO _3, saturated aqueous Na ₂ S ₂ O ₃ and brine, dried (Na ₂ SO ₄ ) and concentrated. Purify by column chromatography on silica gel (toluene / EtOAc) to give the product.

Molecular sieve (AW300) is added to a solution of compound 30 and a protected sugar donor (eg, compound 33) in CH ₂ Cl ₂ . After stirring, TMSOTf (in CH ₂ Cl ₂ ) is added to the suspension. The progress of the reaction is monitored by TLC. After stirring, the reaction mixture is filtered through a pad of Celite®. The filtrate is diluted and the organic phase is washed with saturated aqueous NaHCO _3, saturated aqueous Na ₂ S ₂ O ₃ and brine, dried (Na ₂ SO ₄ ) and concentrated. Purify by column chromatography on silica gel (toluene / EtOAc) to give the product.

Molecular sieve (AW300) is added to a solution of compound 31 and a protected sugar donor (eg, compound 33) in CH ₂ Cl ₂ . After stirring, TMSOTf (in CH ₂ Cl ₂ ) is added to the suspension. The progress of the reaction is monitored by TLC. After stirring, the reaction mixture is filtered through a pad of Celite®. The filtrate is diluted and the organic phase is washed with saturated aqueous NaHCO _3, saturated aqueous Na ₂ S ₂ O ₃ and brine, dried (Na ₂ SO ₄ ) and concentrated. Purify by column chromatography on silica gel (toluene / EtOAc) to give the product.

(Deprotection)
(Deprotection of phytosphingosine GM3)

O- (5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonuropyranosylonic acid)-(2 → 3)-(O-β-D-galactopyranosyl) -(1 → 4) -O-β-D-glucopyranosyl- (1 → 1)-(2S, 3S, 4R) -2-octadecanamide-octadecane-1,3,4-triol (3).
To a solution of compound 1 (38 mg, 0.0184 mmol) in 1,2-dichloroethane (800 μL) was added trifluoroacetic acid (370 μL). The mixture was stirred for 7 hours at room temperature. After complete consumption of the starting material was confirmed by TLC analysis (EtOAc / Hexane 6/1), Et ₃ N was added at 0 ° C. The reaction mixture was evaporated and coevaporated with toluene. After evaporation, the pressure was reduced by a pump for 30 hours. The residue was dissolved in MeOH (1.0 mL) and NaOMe (7.2 mM MeOH solution, 100 μL, 0.00518 mmol) was added at 0 ° C. After stirring at room temperature for 26 hours (TLC monitoring: BuOH / MeOH / H ₂ O 10/1/1), H ₂ O was added to the mixture at 0 ° C. The mixture was stirred at room temperature for 10 hours (TLC monitoring: BuOH / MeOH / H ₂ O 10/1/1). The reaction mixture was neutralized with Dowex (H ⁺ ) and filtered. The filtrates were combined and the washings were concentrated. The residue was subjected to column chromatography (MeOH) with Sephadex LH-20 200 (g) to give compound 3 (22.1 mg, quantitative).

Using a method similar to that described above, for example, compound 34 was stirred in CH ₂ Cl ₂ at room temperature in the presence of trifluoroacetic acid, then with NaOMe in MeOH and H ₂ O at room temperature. Treatment yields the deprotected product.

  (Crosslinking of sphingosine ceramide with glucose (Glc))

Compound 2 (1.6 g, 2.07 mmol) was dissolved in CH ₂ Cl ₂ (19 ml) under argon, WSC (1.1 g, 5.64 mmol), DMAP (69 mg, 0.564 mmol), Compound 1 (935 mg, 1.88 mmol) was added and stirred at room temperature for 4 hours. The reaction solution was diluted with chloroform, washed with water and brine, and then purified by column chromatography (AcOEt / hexane = 1/3) to obtain Compound 3 (1.64 g).

  Compound 3 (280 mg, 0.223 mmol) was dissolved in THF (2.3 ml) under argon, and AcOH (40 μL, 0.669 mmol) and TBAF (670 μL, 0.669 mmol) were added at 0 ° C. For 1 hour. After 1 hour, the reaction temperature was raised to room temperature and stirred for 4 hours. The reaction solution was diluted with chloroform, washed with sodium bicarbonate and brine, and then purified by column chromatography (AcOEt / hexane = 1 / 1.3) to obtain Compound 4 (225 mg).

  (Sphingosine-type ceramide-succinic acid-glucose (Glc), intramolecular glycosylation)

Compound 4 (72 mg, 0.0630 mmol) was dissolved in CH ₂ Cl ₂ (13 ml) under argon, MS4A (70 mg) was added, and the mixture was stirred at room temperature for 1 hr. After 1 hour, the reaction temperature was brought to 0 ° C. and DMTST (102 mg, 0.189 mmol) was added. The mixture was stirred at 0 ° C. for 2 hours. The filtrate was diluted with chloroform, washed with sodium bicarbonate and brine, and then purified by column chromatography (CHCl ₃ / MeOH = 130/1) to obtain Compound 5 (58 mg).

  (Synthesis of GM3: (NeuGal donor + GlcCer acceptor))

Compound 6 (21 mg, 0.0189 mmol) and compound 5 (13 mg, 0.0126 mmol) were dissolved in CHCl ₃ (420 μL) under argon, AW300 (100 mg) was added, and the mixture was stirred at room temperature for 1 hour. After 1 hour, the reaction temperature was brought to 0 ° C., TMSOTf (0.3 μL, 0.00151 mmol) was added, and the mixture was stirred at 0 ° C. for 2.5 hours. After completion of the reaction, the reaction mixture was filtered through Celite, and the filtrate was diluted with chloroform, washed with sodium hydrogen carbonate and brine, and purified by column chromatography (toluene / AcOEt / MeOH = 50/20/1) to give compound 7 ( 23 mg) was obtained.

  (Deprotection of GM3)

Compound 7 (35 mg, 0.0177 mmol) was dissolved in CH ₂ Cl ₂ (700 μl) under argon, TFA (350 μl) was added at 0 ° C., and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, Et ₃ N (2 ml) was added at 0 ° C., toluene azeotropy was performed, and the residue was dried under reduced pressure for 12 hours (Compound 8). Subsequently, Compound 8 was dissolved in MeOH (1.0 ml) under argon, NaOMe (0.34 mg, 0.00177 mmol) was added at 0 ° C., and the mixture was stirred at room temperature for 17 hours. After 17 hours, H ₂ O (0.5 ml) was added and stirred for 13 hours. After completion of the reaction, neutralization with Dowex (H ⁺ ) was performed, cotton filtration was performed, and the filtrate was gel filtered with Sephadex LH-20 (MeOH) to obtain Compound 9 (19 mg).

  (Synthesis of GM2: (GM2 donor + GlcCer acceptor))

Compound 10 and compound 5 are dissolved in CHCl ₃ under argon, AW300 is added and stirred at room temperature. The reaction temperature is brought to 0 ° C., TMSOTf is added, and the mixture is stirred at 0 ° C. After completion of the reaction, the reaction mixture is filtered through Celite, and the filtrate is diluted with chloroform, washed with sodium bicarbonate and brine, and then purified by column chromatography (toluene / AcOEt / MeOH) to obtain Compound 11.

  (Deprotection of GM2)

Compound 11 is dissolved in CH ₂ Cl ₂ under argon, TFA is added at 0 ° C., and the mixture is stirred at room temperature. After completion of the reaction, Et ₃ N is added at 0 ° C., toluene azeotropy is performed, and the residue is dried under reduced pressure (Compound 12). Subsequently, Compound 12 is dissolved in MeOH under argon, NaOMe is added at 0 ° C., and the mixture is stirred at room temperature. Then add H ₂ O and stir. After completion of the reaction, neutralization with Dowex (H ⁺ ) is performed, cotton filtration is performed, and the filtrate is gel filtered with Sephadex LH-20 (MeOH) to obtain compound 13.

  (Synthesis of GM1: (GM1 donor + GlcCer acceptor)

Compound 14 and compound 5 are dissolved in CHCl ₃ under argon, AW300 is added and stirred at room temperature. The reaction temperature is brought to 0 ° C., TMSOTf is added, and the mixture is stirred at 0 ° C. After completion of the reaction, the reaction mixture is filtered through Celite, and the filtrate is diluted with chloroform, washed with sodium bicarbonate and brine, and then purified by column chromatography (toluene / AcOEt / MeOH) to obtain Compound 15.

  (Deprotection of GM1)

Compound 15 is dissolved in CH ₂ Cl ₂ under argon, TFA is added at 0 ° C., and the mixture is stirred at room temperature. After completion of the reaction, Et ₃ N is added at 0 ° C., toluene azeotropy is performed, and the residue is dried under reduced pressure (Compound 16). Subsequently, Compound 16 is dissolved in MeOH under argon, NaOMe is added at 0 ° C., and the mixture is stirred at room temperature. Then add H ₂ O and stir. After completion of the reaction, neutralization is performed with Dowex (H ⁺ ), cotton filtration is performed, and the filtrate is subjected to gel filtration with Sephadex LH-20 (MeOH) to obtain Compound 17.

  (Synthesis of GM4: (GM4 donor + GlcCer acceptor))

Compound 18 and compound 5 are dissolved in CHCl ₃ under argon, AW300 is added and stirred. Add TMSOTf and stir. After completion of the reaction, the reaction mixture is filtered through Celite, and the filtrate is diluted with chloroform, washed with sodium bicarbonate and brine, and then purified by column chromatography to obtain Compound 19.

  (Deprotection of GM4)

Compound 19 is dissolved in CH ₂ Cl ₂ under argon, TFA is added and stirred. After completion of the reaction, Et ₃ N is added, toluene azeotropy is performed, and the residue is dried under reduced pressure (Compound 20). Subsequently, compound 20 is dissolved in MeOH under argon, NaOMe is added and stirred. Add H ₂ O and stir. After completion of the reaction, neutralization is performed with Dowex (H ⁺ ), cotton filtration is performed, and the filtrate is subjected to gel filtration with Sephadex LH-20 (MeOH) to obtain Compound 21.

  (Preparation of lactose site)

A reaction was carried out under the conditions described in the above table based on the description of Hiromune Ando, Yusuke Koike, Hideharu Ishida, and Makito Kiso et al., Tetrahedron Letters 44 (2003) 6883-6886.

  (Preparation of sialic acid site)

A reaction was carried out under the conditions described in the above table based on the description of Hiromune Ando, Yusuke Koike, Hideharu Ishida, and Makito Kiso et al., Tetrahedron Letters 44 (2003) 6883-6886.

(Preparation of sialic acid site)
(Synthesis of trisaccharide)

A reaction was carried out under the conditions described in the above table based on the description of Hiromune Ando, Yusuke Koike, Hideharu Ishida, and Makito Kiso et al., Tetrahedron Letters 44 (2003) 6883-6886.

  (Change of sialic acid moiety 1)

Based on the description of Hiromune Ando, Yusuke Koike, Hideharu Ishida, and Makito Kiso et al., Tetrahedron Letters 44 (2003) 6883-6886, the sialic acid site was changed under the conditions described in the above table.

  (Change of sialic acid moiety 2)

Based on the description of Hiromune Ando, Yusuke Koike, Hideharu Ishida, and Makito Kiso et al., Tetrahedron Letters 44 (2003) 6883-6886, the sialic acid site was changed under the conditions described in the above table.

Example 2. Preparation of GM3
(Preparation of Compound 31A)

4-methoxyphenyl [methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5- (2,2,2-trichloroethoxycarbamoyl) -D-glycero-α-D-galacto- 2-nonuropyranosylate]-(2 → 3)-(2,6-O-di-benzyl-β-D-galactopyranosyl)-(1 → 4) -2,3,6 -O-tri-benzyl-β-D-glucopyranoside (31A)

Compound 25A (500 mg, 0.557 mmol: Borbas, A .; Csavas, M .; Szilagii, L .; Majer, G .; Liptak, A. J. Carbohydr. Chem. 2004, 23, 146-146. Prepared) and compound 30A (796 mg, 1.11 mmol: prepared according to Ando, H .; Koike, Y .; Ishida, H .; Kiso, M. Tetrahedron Lett. 2003, 44, 6883-6886.). It was dissolved in 10: 1 C ₂ H ₅ CN—CH ₂ Cl ₂ (8.5 ml) and stirred at room temperature for 1 hour in the presence of MS-4A (1.3 g). Thereafter, N-iodosuccinimide (501 mg, 2.22 mmol) was added, cooled to −50 ° C., trifluoromethanesulfonic acid (24 μl, 0.274 mmol) was added, and the mixture was stirred at −50 ° C. for 6 hours. After neutralization with TEA, the solid was filtered through Celite and washed with chloroform. The filtrate and washings were combined and NaHCO ₃ sat. , H ₂ O, Na ₂ S ₂ O ₃ , H ₂ O, sat. The organic layer was washed with Na ₂ SO ₄ and separated into an organic layer and a solid. The filtrate and washings were combined and concentrated under reduced pressure, and the syrup obtained was purified by silica gel chromatography (toluene: AcOEt = 6: 1) to obtain compound 31α (468 mg, 45%). Further, Compound 31β (146 mg, 14%) was obtained as a by-product.

(Preparation of Compound 33A)

4-methoxyphenyl [methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonuropyranosylate]-( 2 → 3)-(4-O-acetyl-2,6-O-di-benzyl-β-D-galactopyranosyl)-(1 → 4) -2,3,6-O-tri-benzyl- β-D-glucopyranoside (33A)
Compound 31A (50 mg, 0.0333 mmol) was dissolved in 3: 1 AcOH- (CH ₂ Cl) ₂ (1 ml) and stirred at 50 ° C. for 1.5 hours in the presence of Zn (Cu) (250 mg). did. Thereafter, Celite filtration was performed, and the solid matter was separated by filtration and washed with chloroform. The filtrate and washings were combined and H ₂ O, NaHCO ₃ sat. , H ₂ O, brine. The obtained organic layer was dried with Na ₂ SO ₄ , the organic layer and the solid were separated by filtration, and concentrated under reduced pressure with toluene, and the resulting syrup was dried under reduced pressure for 1 hour. Then, after dissolving in pyridine and cooling in an ice bath, acetic anhydride (50 μl) and 4-dimethylaminopyridine (DMAP) catalyst amount were added, the ice bath was removed, and the mixture was stirred for 2.5 hours. Thereafter, MeOH was added to stop the reaction, and the syrup obtained by concentration under reduced pressure was added to 2N HCl, H ₂ O, NaHCO ₃ sat. , H ₂ O, brine. The obtained organic layer was dried with Na ₂ SO ₄ , the organic layer and the solid were separated by filtration, the filtrate and the washing solution were combined and concentrated under reduced pressure, and the syrup obtained was subjected to silica gel chromatography (CHCl ₃ : MeOH = 60: 1). ) To give compound 33A (35 mg, 75%).

(Preparation of compound 35A)

4-methoxyphenyl [methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonuropyranosylate]-( 2 → 3)-(2,4,6-O-acetyl-β-D-galactopyranosyl)-(1 → 4) -2,3,6-O-acetyl-β-D-glucopyranoside (35A)
Compound 33A (450 mg, 0.318 mmol) was dissolved in 1: 1 EtOH-THF (5 ml), Pd (OH) ₂ -C (1.0 g) was added, and 1 at 40 ° C. under a hydrogen gas stream. Stir for hours. After completion of the reaction, the solid was filtered through celite and washed with kucloform. The filtrate and washings were combined and concentrated under reduced pressure with toluene. The obtained syrup was dried under reduced pressure for 1 hour. Thereafter, the mixture was replaced with Ar, dissolved in pyridine, cooled in an ice bath, and then added with catalytic amounts of acetic anhydride (1 ml) and 4-dimethylaminopyridine (DMAP), and the ice bath was removed and the mixture was stirred for 5 hours. After cooling in an ice bath, MeOH was added to stop the reaction, and the syrup obtained by concentration under reduced pressure was added to 2N HCl, H ₂ O, NaHCO ₃ sat. , H ₂ O, brine. The obtained organic layer was dried with Na ₂ SO ₄ , the organic layer and the solid were separated by filtration, the filtrate and the washing solution were combined and concentrated under reduced pressure, and the syrup obtained was subjected to silica gel chromatography (CHCl ₃ : MeOH = 50: 1). ) To give compound 35A (358 mg, 96%).

(Preparation of Compound 37A)

Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonuropyranosylate]-(2 → 3)- (2,4,6-O-acetyl-β-D-galactopyranosyl)-(1 → 4) -2,3,6-O-acetyl-α-D-glucopyranosyl trichloroacetimidate ( 37A)
Compound 35A (53 mg, 0.0452 mmol) was dissolved in 6: 5: 3 toluene-MeCN-H ₂ O (2 ml), cooled in an ice bath, diammonium cerium nitrate (IV) (CAN) ( 74 mg) was added. After 15 minutes, the ice bath was removed and the mixture was stirred at room temperature for 5.5 hours. After completion of the reaction, the solution was diluted with AcOEt, and H ₂ O, NaHCO ₃ sat. , H ₂ O, brine. The obtained organic layer was dried with Na ₂ SO ₄ , the organic layer and the solid were separated by filtration, the filtrate and the washing solution were combined and concentrated under reduced pressure, and the syrup obtained was subjected to silica gel chromatography (CHCl ₃ : MeOH = 25: 1). The compound 36A was obtained.

The obtained compound 36A was dissolved in CH ₂ Cl ₂ (2 ml), cooled in an ice bath, and CCl ₃ CN (452 μl, 4.516 mmol) and DBU (3.4 μl, 0.021 mmol) were added. It was. After the addition, the ice bath was removed and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, the solution was concentrated under reduced pressure, and the syrup obtained was purified by silica gel chromatography (toluene: acetone = 5: 1) to obtain Compound 37A (44 mg, 81%, 2 steps).

Preparation of (Compound 38A)

Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonuropyranosylate]-(2 → 3)- (2,4,6-O-acetyl-β-D-galactopyranosyl)-(1 → 4) -2,3,6-O-acetyl-β-D-glucopyranosyl- (1 → 1) -2 -(Tetradecyl) -hexadecanol (38A)
Compound 8A

(91 mg, 0.207 mmol) and compound 37A (50 mg, 0.0414 mmol) were dissolved in CH ₂ Cl ₂ (2.5 ml) and stirred at room temperature in the presence of AW300 (150 mg) for 1 hour. Then, it cooled at 0 degreeC, TMSOTf (1.5 microliters, 0.0082 mmol) was added, and it stirred at 0 degreeC for 10 hours. After neutralization with TEA, the solid was filtered through Celite and washed with chloroform. The filtrate and washings were combined and NaHCO ₃ sat. , H ₂ O, sat. And then washed with brine. The obtained organic layer was dried with Na ₂ SO ₄ , the organic layer and solid were separated by filtration, the filtrate and washings were combined and concentrated under reduced pressure, and the syrup obtained was subjected to silica gel chromatography (toluene: acetone = 2: 1). To give compound 38A (22 mg, 36%).

(Preparation of Compound 3A)

5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonuropyranosylate- (2 → 3) -β-D-galactopyranosyl- (1 → 4) -β -D-glucopyranosyl- (1 → 1) -2- (tetradecyl) hexadecanol (3A)
Compound 38A (22 mg, 0.0148 mmol) was suspended in MeOH (1.0 ml), NaOMe (0.040 mg, 0.00074 mmol) was added, and the mixture was stirred at room temperature for 21 hours. After confirming the deprotection of all Ac groups in the compound by MALDI-TOFMS, H ₂ O was added and stirred for 3.5 hours, and then the formation of carboxylic acid was confirmed by MALDI-TOFMS. The solution was neutralized to PH7 using Dowex (H ⁺ ), the solution and Dowex (H ⁺ ) were separated by filtration, the solution obtained by filtration was concentrated under reduced pressure, and the resulting syrup was subjected to column chromatography (Sephadex LH- 20, MeOH) to obtain Compound 3A (11 mg, 69%).

(Preparation of Compound 39A)

Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonuropyranosylate]-(2 → 3)- (2,4,6-O-acetyl-β-D-galactopyranosyl)-(1 → 4) -2,3,6-O-acetyl-β-D-glucopyranosyl- (1 → 1)-( 2S, 3R, 4E) -3,4-O-dibenzoyl-2-octadecanamido-4-octadecene-1,3-diol (39A)
Compound 6A

(55 mg, 0.0826 mmol) and compound 37A (50 mg, 0.0413 mmol) were dissolved in CH ₂ Cl ₂ (1 ml) and stirred at room temperature in the presence of AW300 (150 mg) for 1 hour. Then, it cooled to 0 degreeC, TMSOTf (1.5 microliters, 0.00818 mmol) was added, and it stirred at 0 degreeC for 22 hours. After neutralization with TEA, the solid was filtered through Celite and washed with chloroform. The filtrate and washings were combined and NaHCO ₃ sat. , H ₂ O, brine. The obtained organic layer was dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure, and the resulting syrup was purified by silica gel chromatography (toluene: acetone = 2: 1) to obtain compound 39A (13 mg, 18%).

Example 3. Preparation of GM4
(Preparation of Compound 45A)

4-methoxyphenyl (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl 3,5-dideoxy D-glycero-α-D-galacto-2-nonuropyranosylate)-(2 → 3) -4-O-acetyl-2,6-di-O-benzyl-β-D-galactopyranoside (45A)
Compound 43A in 1,2-dichloroethane (15.0 mL)

(2.0 g, 1.87 mmol: Fuse, T .; Ando, H .; Imamura, A .; Sawada, N .; Ishida, H .; Kiso, M .; Ando, T .; Li, S. C .; Li li Y.-T. Glycoconj. J. 2006, 23, 329-343) was added to acetic acid (45.0 mL) and Zn—Cu (10 0.0 g) was added. The mixture was stirred at 40 ° C. for 1.5 hours while monitoring the reaction progress by TLC (CHCl ₃ : MeOH = 15: 1). The reaction mixture was filtered through Celite. The filtrate and washings were combined and extracted with CHCl ₃ and the organic layer was washed with H ₂ O, sat. Washed with Na ₂ CO ₃ , and brine, dried over Na ₂ SO ₄ and concentrated. To a solution of the residue in pyridine (9.0 mL), acetic anhydride (614 μL) was added at 0 ° C. under an argon atmosphere. The mixture was stirred for 13 hours at ambient temperature while monitoring the reaction progress by TLC (CHCl ₃ : MeOH = 15: 1). The reaction mixture was evaporated simultaneously with toluene and extracted with CHCl ₃ . The organic phase was washed with 2M HCl, H ₂ O, sat. Washed with NaHCO ₃ and brine, dried over Na ₂ SO ₄ and concentrated. The residue was purified by column chromatography on silica gel (EtOAc: hexane = 3: 1) to give 45A (1.69 g, 92%);

(Preparation of Compound 47A)
4-Methoxyphenyl (methyl 5-acetamido-4,7,8,9-tetra O-acetyl 3,5-dideoxy D-glycero-α-D-galacto-2-nonuropyranosylate)-(2 → 3 ) -4-O-acetyl-2,6-di-O-benzoyl-β-D-galactopyranoside (47A)

To a solution of compound 45A (385 mg, 392 μmol) in EtOH (30 mL) was added palladium hydroxide [Pd (OH) ₂ ] (20 wt.% Pd on carbon) (400 mg) at ambient temperature with argon. Added under atmosphere. While the mixture was monitored by TLC (CHCl ₃ : MeOH = 15: 1), the reaction progress was vigorously stirred at ambient temperature under a hydrogen atmosphere for 4 hours. The reaction mixture was filtered through Celite and the combined filtrate and washings were concentrated. To a solution of the residue in pyridine (5.0 mL) was added benzoic anhydride (354 mg, 1.57 mmol) at 0 ° C. under an argon atmosphere. The mixture was stirred for 16 hours at ambient temperature, monitoring the progress of the reaction by TLC (CHCl ₃ : MeOH = 15: 1). The reaction mixture was evaporated simultaneously with toluene and extracted with CHCl ₃ . The organic phase was washed with 2M HCl, H ₂ O, sat. Washed with NaHCO ₃ and brine and concentrated. The residue was purified by column chromatography on silica gel (EtOAc: hexane = 3: 1) to give 47A (380 mg, 95%);

Compound 47A has the chemical formula C ₄₉ H ₅₅ NO ₂₂ , the exact mass is 1009.3216, and the molecular weight is 1009.9545. Na salt is a chemical formula _C 49 _H 55 NNaO _22, exact mass is 1032.3113, the molecular weight is 1032.9443. The K salt has the chemical formula C ₄₉ H ₅₅ NKO ₂₂ , the exact mass is 1048.2853 and the molecular weight is 1049.528.

  (Preparation of Compound 49A)

Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonuropyranosylate- (2 → 3) -4 -O-acetyl-2,6-di-O-benzoyl-β-D-galactopyranosyl trichloroacetimidate (49A)
To a solution of compound 47A (164 mg, 162 μmol) in a mixed solvent (MeCN-PhMe-H ₂ O = 3.5 mL: 2.9 mL: 1.7 mL) was added diammonium cerium (IV) nitrate (CAN). ) (445 mg, 812 μmol) was added. The progress of the reaction was stirred for 5 hours at ambient temperature while this mixture was monitored by TLC (CHCl ₃ : MeOH = 20: 1). The reaction mixture was extracted with CHCl ₃ and the organic layer was washed with H ₂ O, sat. Washed with NaHCO ₃ and brine, dried over Na ₂ SO ₄ and concentrated. The residue was purified by silica gel column chromatography (CHCl ₃ : MeOH = 65: 1) to obtain the target compound (147 mg). To a solution of the compound in CH ₂ Cl ₂ (5.0 mL) was added trichloroacetonitrile (410 μL, 407 μmol) and 1,8-diazabicyclo [5,4,0] -7-undecene (DBU) (4.9). μL, 33.0 μmol) was added. The mixture was stirred at 0 ° C. for 2 hours while monitoring the reaction progress by TLC (CHCl ₃ : MeOH = 20: 1). The reaction mixture was concentrated and the residue was purified by column chromatography on silica gel (CHCl ₃ : MeOH = 75: 1) to give 49A (132 mg, 78%);

(Preparation of Compound 50A)

Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonuropyranosylate- (2 → 3) -4 —O-acetyl-2,6-O-dibenzoyl β-D-galactopyranosyl)-(1 → 1) -2- (tetradecyl) -hexadecanol (50A)
Compound 8A

(136 mg, 0.310 mmol) and compound 49A (65 mg, 0.0621 mmol) were dissolved in CH ₂ Cl ₂ (3 ml) and stirred at room temperature for 1 hour in the presence of AW300 (200 mg). Then, it cooled to 0 degreeC, TMSOTf (2.2 microliters, 0.0124 mmol) was added, and it stirred at 0 degreeC for 20 hours. After neutralization with TEA, the solid was filtered through Celite and washed with chloroform. The filtrate and the washing solution are combined, and sat. Washed with NaHCO ₃ , H ₂ O, and brine in that order. The obtained organic layer was dried with Na ₂ SO ₄ , the organic layer and the solid were separated by filtration, the filtrate and the washing solution were combined, and concentrated under reduced pressure. Silica gel chromatography (toluene: acetone = 2: 1) was obtained. ) To obtain Compound 50A (62 mg, 76%).

(Preparation of Compound 51A)

5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonuropyranosylate- (2 → 3) -β-D-galactopyranosyl- (1 → 1) -2 -(Tetradecyl) -hexadecanol (51A)
Suspended in Compound 50A (28 mg, 0.0212 mmol) and MeOH (5.0 ml), added a catalytic amount of NaOMe, stirred for 21 hours at room temperature, heated to 55 ° C., stirred for 48 hours, and then each hydroxyl group by MALDI-TOFMS. The deprotection of the Ac group and the Bz group was confirmed. Then stirred for 3.5 hours added _{H 2} O, confirmed the formation of carboxylic acids by MALDI-TOFMS. The solution was neutralized to pH 7 using Dowex (H ⁺ ), the solution and Dowex (H ⁺ ) were separated by filtration, the solution was concentrated under reduced pressure, and the resulting syrup was subjected to column chromatography (Sephadex LH-20, MeOH). Purification gave compound 51A (15 mg, 79%).

(Preparation of Compound 52A)

Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonuropyranosylate]-(2 → 3)- (2,4,6-O-acetyl-β-D-galactopyranosyl)-(1 → 1)-(2S, 3R, 4E) -3,4-O-benzoyl-2-octadecanamide-4- Octadeca-1,3,4-triol (52A)
Compound 49A (76 mg, 0.956 mmol) and compound 7A (50 mg, 0.0478 mmol) were dissolved in CH ₂ Cl ₂ (1.5 ml) and stirred at room temperature for 1 hour in the presence of AW300 (150 mg). did. Then, it cooled to 0 degreeC, TMSOTf (1.8 microliters, mmol) was added, and it stirred at 0 degreeC for 24 hours.

After neutralization with TEA, the solid was filtered through Celite and washed with chloroform. The filtrate and the washing solution are combined, and sat. Washed with NaHCO ₃ , H ₂ O, and brine in that order. The obtained organic layer was dried with Na ₂ SO ₄ , the organic layer and solid were separated by filtration, the filtrate and washings were combined and concentrated under reduced pressure, and the syrup obtained was subjected to silica gel chromatography (toluene: acetone = 2: 1). To give compound 52A (56 mg, 70%).

(Preparation of Compound 53A)

5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonuropyranosylate- (2 → 3) -β-D-galactopyranosyl- (1 → 1)-( 2S, 3R, 4E) -3,4-O-benzoyl-2-octadecanamido-4-octadeca-1,3,4-triol (53A)
The mixture was suspended in Compound 52A (35 mg, 0.0208 mmol) and MeOH (5.0 ml), a catalytic amount of NaOMe was added, and the mixture was stirred at 55 ° C. for 60 hours. Deprotection of the Ac group and Bz group of each hydroxyl group in the compound was confirmed by MALDI-TOFMS. Thereafter, H ₂ O was added and stirred for 3.5 hours, and then the formation of carboxylic acid was confirmed by MALDI-TOFMS. The solution was neutralized to pH 7 using Dowex (H ⁺ ), the solution and Dowex (H ⁺ ) were separated by filtration, the solution was concentrated under reduced pressure, and the resulting syrup was subjected to column chromatography (Sephadex LH-20, MeOH). Purification gave compound 53 (21 mg, quant.).

As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range from the description of specific preferred embodiments of the present invention based on the description of the present specification and the common general technical knowledge. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  The application of the present invention to various sugars and aglycones, and the introduction of ceramide using intramolecular condensation after sugar chain synthesis are also conceivable, and applications in various fields such as food, pharmaceuticals, agrochemicals are expected. Is done.

Claims (42)

A method for producing a glycolipid, comprising:
(A) reacting a protected sugar and a lipid amide protector under conditions under which the protected sugar and the lipid amide protector bind to produce a sugar-lipid amide acceptor precursor;
(B) reacting the sugar-lipid amide acceptor precursor under conditions where an intramolecular condensation reaction in the sugar-lipid amide acceptor precursor proceeds to produce a sugar-lipid amide acceptor;
(C) reacting the sugar-lipid amide acceptor with a protected sugar chain donor under a condition in which the sugar-lipid amide acceptor and the protected sugar chain donor are linked to form a protected sugar Producing a lipid; and (d) subjecting the protected glycolipid to a deprotection reaction under conditions under which the protected sugar chain donor deprotects to produce a glycolipid,
The protected sugar is
A sugar protector selected from the group consisting of:
Pro is a protecting group independently selected from the group consisting of benzoyl (Bz), pivaloyl (Piv), MPM (p-methoxybenzyl), methoxyphenyl (MP), acetyl, benzyl, and methyl;
L is independently -SPh, -SCH ₃ , -SCH ₂ CH ₃ , -F, -OPO (OPh) ₂ (where Ph is phenyl), -OPO (N (CH ₃ ) ₂ A leaving group selected from the group consisting of ₂ and a trichloroacetimidate group;
The protected lipid amide is

Selected from the group consisting of:
The sugar-lipid amide acceptor precursor is

And the sugar-lipid amide acceptor is

Where R ₁ is p-methoxybenzyl (MPM), methoxyphenyl (MP), allyl;
R ₂ is MPM, Bz, MP, allyl;
The _{R 3} are, tert- butyldiphenylsilyl (TBDPS), tert- butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS), trityl (Tr), is selected from the group consisting of isopropylidene ketals and methoxy acetal;
The R ₄ and the R ₅ is independently a protecting group selected from the group consisting of base Nzoiru, acetyl and pivaloyl, Ph is phenyl,
The protected sugar chain donor is

The method of claim 1, wherein the method is selected from the group consisting of: The method according to claim 1, wherein the condition for binding the protected sugar and the protected lipid amide is a condition for binding an alcohol and a carboxylic acid. The step (a) includes mixing and reacting the protected sugar chain and the lipid amide in a solvent in the presence of a reagent at a predetermined reaction temperature and reaction time.
The reaction temperature is room temperature or higher;
The solvent is tetrahydrofuran _{(THF), CH 2 Cl 2} , benzene, toluene, N, is selected from the group consisting of N- dimethylformamide (DMF) and combinations thereof;
The reagent is triphenylphosphine (PPh ₃ ), azodicarboxylic acid diethyl ester (DEAD), 1-methyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (WSC), 2,4,6-trichlorobenzoyl Selected from the group consisting of chloride, triethylamine (Et ₃ N), and 4-dimethylaminopyridine (DMAP) and combinations thereof; and the reaction time is 2-4 hours;
The method of claim 1. The method according to claim 3, wherein the reagents are PPh ₃ and DEAD, the solvent is THF, and the reaction temperature is 90 ° C. or higher. The reagent is WSC or 2,4,6-trichlorobenzoyl chloride, Et ₃ N and DMAP, the solvent is CH ₂ Cl ₂ , and the reaction temperature is 30-60 ° C. The method described in 1. The method according to claim 3, wherein the reagent is WSC, the solvent is CH ₂ Cl ₂ , and the reaction temperature is room temperature. The method according to claim 3, wherein the reaction time is 3 hours. The solvent is tetrahydrofuran (THF);
The reagent is triphenylphosphine (PPh ₃ : 3.0 equivalent) and DEAD (3.0 equivalent), and the corresponding amount is equivalent to the protected sugar chain,
The reaction temperature is 90 ° C. and the reaction is carried out under reflux;
The method of claim 3. The solvent is CH ₂ Cl ₂ ;
The reagent is 1-methyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (WSC: 3.0 equivalents), and the corresponding amount is equivalent to the protected sugar chain,
The temperature is room temperature;
The method of claim 3. The solvent is CH ₂ Cl ₂ ;
The reagents are 2,4,6-trichlorobenzoyl chloride (1.1 equivalent), triethylamine (Et ₃ N: 1.5 equivalent) and 4-dimethylaminopyridine (DMAP: 3.0 equivalent). , The corresponding amount is equivalent to the protected sugar chain,
The temperature is room temperature;
The method of claim 3. The step (a) includes adding the spacer precursor to the sugar and the lipid amide protected body, thereby binding the lipid amide protected body to the sugar via the spacer, and the spacer precursor. The method of claim 1, wherein the body is succinic acid. The method according to claim 1, wherein the spacer and the sugar or the protected lipid amide are previously bound. The method according to claim 12, further comprising the step of reacting the protected lipid amide under a condition that the 1-position hydroxyl group of the protected lipid amide is deprotected to deprotect the 1-position hydroxyl group. When the succinic acid binds to R ₃ of the protected lipid amide,
R ₃ is isopropylidene ketal or methoxybenzylidene acetal;
R ₄ is
(Ac) or
A (Bx), and _{R 5} is Ac or Bx, The method of claim 11. The method according to claim 1, wherein the sugar residue on the reducing end side of the oligosaccharide is bound to the protected lipid amide through a spacer. The step (b) is performed in the presence of an activator for activating the intramolecular condensation reaction, and the activator includes N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH), 2. The method of claim 1 selected from the group consisting of trimethylsilyl trifluoromethane sulfonic acid (TMSOTf), dimethyl (methylthio) sulfonium triflate (DMTST), N-bromosuccinimide (NBS), and combinations thereof. The step (b)
At a reaction temperature of −80 ° C. to room temperature;
In a solvent selected from the group consisting of CH ₂ Cl ₂ , diethyl ether, acetonitrile, diethyl ether, acetonitrile, propionitrile, toluene, nitromethane and combinations thereof;
From the group consisting of N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH), dimethyl (methylthio) sulfonium triflate (DMTST), molecular sieve 4 angstrom (MS4Å), molecular sieve 3 Å (MS3Å) and combinations thereof In the presence of a selected reagent; and for a reaction time of 1 to 48 hours,
The method of claim 1. The reaction temperature is -20 to 0 ° C., The method of claim 1 7. The method of claim 17 , wherein the solvent is dichloromethane. The method according to claim 17 , wherein the reagents are N-iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH). The method according to claim 17 , wherein the reaction time is 1 hour to 5 hours. The step (b)
Reaction time of 1.5 hours using CH ₂ Cl ₂ as a solvent, N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH), TMSOTf and molecular sieves 4 angstrom (MS4Å) as reagents, The process of claim 17 comprising reacting at a reaction temperature of 0 ° C. The step (b)
Using CH ₂ Cl ₂ as a solvent, N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH) and molecular sieves 4 angstrom (MS 4ＭＳ) as reagents, a reaction time of 5 hours at −40 ° C. The method of claim 17 comprising reacting. The step (b)
Using CH ₂ Cl ₂ as a solvent, N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH) and molecular sieves 4 angstrom (MS 4Å) as reagents, a reaction time of 36 hours, initially −80 The process according to claim 17 comprising reacting at a reaction temperature of 0C, then -60C, then -40C, and then 0C. The step (b)
As a solvent, acetonitrile (MeCN) was used, and N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH) and molecular sieves 3 angstrom (MS3Å) were used as reagents, with a reaction time of 48 hours, initially −40 The process according to claim 17 , comprising reacting at 0 ° C. and then at a reaction temperature of 0 ° C. The step (b)
As a solvent, acetonitrile (MeCN) was used, N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH) and molecular sieves 3 angstrom (MS3Å) were used as reagents, with a reaction time of 1.5 hours, −0 The process according to claim 17 comprising reacting at a reaction temperature of 0C. The step (b)
Using acetonitrile (MeCN) as a solvent, N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH) and molecular sieves 3 angstrom (MS3Å) as reagents, a reaction time of 3 hours, −20 ° C. The process of claim 17 comprising reacting at the reaction temperature. The step (b)
Using diethyl ether as a solvent, N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH) and molecular sieves 3 angstrom (MS3Å) as reagents using a reaction time of 25 hours, initially at 0 ° C. The method according to claim 17 , comprising reacting at a reaction temperature of room temperature. The step (b)
Using acetonitrile (MeCN) as a solvent and dimethyl (methylthio) sulfonium triflate (DMTST) and molecular sieves 3 angstrom (MS3Å) as reagents, reacting at a reaction temperature of 0 ° C. for 1 hour reaction time. The method of claim 17 , comprising: The step (b)
CH ₂ Cl ₂ as a solvent, N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH) and molecular sieves 4 angstrom (MS 4Å) as reagents, reaction time of 5 hours, reaction at 0 ° C. The method according to claim 17 , comprising reacting at a temperature. The step (b)
Using CH ₂ Cl ₂ as a solvent, N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH) and molecular sieves 4 angstrom (MS 4 と し て) as reagents, a reaction time of 1.5 hours, −20 The process according to claim 17 comprising reacting at a reaction temperature of 0C. The step (b)
Including reaction with CH ₂ Cl ₂ as a solvent using dimethyl (methylthio) sulfonium triflate (DMTST) and molecular sieves 4 angstrom (MS4Å) as reagents for 2 hours reaction time at 0 ° C. The method according to claim 17 . The method of claim 1, wherein the intramolecular condensation reaction is glycosylation. The step (c)
In a donor equivalent to more than 2.5 equivalents; at a reaction temperature of −40 to 0 ° C .; in a solvent of CH ₂ Cl ₂ ; in the presence of a trimethylsilyl trifluoromethanesulfonic acid (TMSOTf) reagent, 1 Including reacting for ~ 48 hours reaction time,
The method of claim 1. The equivalent of donor to the acceptor is 2.5 equivalents;
The reaction temperature is 0 ° C .;
The solvent is CH ₂ Cl ₂ ;
The reagent is TMSOTf; and the pre-reaction time is 7 hours;
The method of claim 3 4. The step (d)
In a solvent of CH ₂ Cl ₂ ;
In the presence of a reagent for trifluoroacetic acid;
At a reaction temperature of room temperature; and for a reaction time of 2 to 12 hours,
The method of claim 1. The reaction time is 2 hours, The method of claim 3 6. The method according to claim 1, further comprising the step of (e) reacting and deprotecting the product of step (d) under conditions where the acyl-based protecting group is deprotected. The step (e)
In a solvent of methanol (CH ₃ OH) or water (H ₂ O);
In the presence of sodium methoxide (NaOCH ₃ ) or KOH reagent;
Carried out at a reaction temperature of room temperature to 100 ° C .; and for a reaction time of 1 hour to 1 week,
40. The method of claim 38 . The solvent is methanol (CH ₃ OH);
The reagent is sodium methoxide (NaOCH ₃ );
The reaction temperature is room temperature; and the reaction time is 12 hours,
40. The method of claim 39 . The following formula:
A compound represented by the formula:
Said R ₁ and R ₂ are independently selected from alkyl and alkenyl groups;
The compound, wherein R ₄ is a protecting group selected from the group consisting of benzoyl, acetyl and pivaloyl. The following formula:
Wherein R ₁ and R ₂ are independently selected from alkyl and alkenyl groups;
The compound, wherein R ₄ is a protecting group selected from the group consisting of benzoyl, acetyl and pivaloyl.