CN1714155A - Sugar chain asparagine derivatives, sugar chain asparagine, sugar chain, and processes for producing these - Google Patents
Sugar chain asparagine derivatives, sugar chain asparagine, sugar chain, and processes for producing these Download PDFInfo
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Abstract
An asparagine derivative of an alpha-2,3-linked sugar chain having 11 to 7 monosaccharide units; an asparagine derivative of an alpha-2,6-linked sugar chain having fluorinated 11 to 7 monosaccharide units; and a sugar chain asparagine derivative which comprises a sugar chain asparagine in which the amino-group nitrogen of the asparagine has been protected by a lipid-soluble protective group and the N-acetylglucosamine on the non-reducing end side contains at least one fucose. And production method thereof is also provided.
Description
Technical field
The present invention relates to sugar chain asparagine derivative, sugar chain asparagine and sugar chain and their manufacture method.
In addition, the invention still further relates to sugar chain asparagine derivative and the manufacture method thereof that contains Fucose.
Background technology
In recent years, as the 3rd chain biomolecule after nucleic acid (DNA), protein, the sugar chain molecule is noticeable.Human body is that all cell surfaces are all divided subcovering by sugar chain by a maxicell society of about 60,000,000 cellularities.For example, ABO formula blood group is by the difference decision of cell surface sugar chain.
Sugar chain has identification of the iuntercellular of participation and interactional function, becomes the key element that constitutes cell society.The disorder of cell society and cancer, chronic disease, infection disease, aging etc. relevant.
For example, if known cell cancerization will cause the structural changes of sugar chain.Also know vibrio cholerae or influenza virus etc. in addition by identification, in conjunction with a certain specific sugar chain, intrusion, cells infected.
Illustrating of sugar chain function, expection can drive according to the pharmaceuticals of new principle and development of food etc., and the prevent and treat that gives of disease is treated and had contribution to wait widely to use.
Sugar chain because the sequence of monose, in conjunction with the diversity of pattern and position, chain length and branch's pattern, whole higher order structure etc., so compare, be very complicated structure with nucleic acid or proteinic structure.Therefore, compare with protein with nucleic acid from the biological information of its structure, varied.Recognize the importance that sugar chain is studied though present situation is people, because its complex structure and diversity are compared with protein with nucleic acid, progress is slow.
Resemble be present in surface of cell membrane or serum etc. above-mentioned a lot of protein all in conjunction with sugar chain.The covalently bound molecule of sugar chain and protein is called sugar-protein, because sugar is different with the combination of proteins pattern, can be divided into 2 classes.One class is the amino and the sugar chain bonded l-asparagine mating type sugar chain (N-glucosides of bonding) of l-asparagine (Asn) side chain.Another kind of is the Saliva Orthana mating type sugar chain (O-glycosides of bonding) that combines sugar chain at the hydroxyl place of Serine (Ser) or Threonine (Thr).All l-asparagine mating type sugar chains have the basic framework that is made of 5 saccharide residues, because the saccharide residue kind difference of the non-reduced end of bonded sugar chain is divided into high mannose type, compound, heterozygous subclass.Saliva Orthana mating type sugar chain is divided into 4 classes again according to the difference of basic framework (core) in addition.
As mentioned above, though sugar chain is important compound, the absolute magnitude deficiency of sugar chain.Means as obtaining sugar chain have only to make sugar chain from being present in the method that biological intravital sugar-protein dissociates out.Yet it is difficult wanting in a large number sugar chain to be cut out from sugar-protein, because the sugar chain that exists a lot of structures to exactly like in the organism will be that the single sugar chain of a large amount of acquisitions is difficult to.In addition in the organism non-existent sugar chain to obtain in a large number also be difficult.
Problem of the present invention is to be provided at new sugar chain asparagine derivative and the manufacture method thereof that non-reduced end contains sialic acid more than a kind or sialic acid derivative at least.
In addition, problem of the present invention also is to be provided at new sugar chain asparagine and the manufacture method thereof that non-reduced end contains sialic acid more than a kind or sialic acid derivative at least.
In addition, problem of the present invention also is to be provided at new sugar chain and the manufacture method thereof that non-reduced end contains sialic acid more than a kind or sialic acid derivative at least.
The N-acetyl-glucosamine of a non-reduced terminal side of the sugar chain asparagine that the amino nitrogen that problem of the present invention also is to be provided at l-asparagine is protected by fat-soluble blocking group contains the new sugar chain asparagine derivative and the manufacture method thereof of an above Fucose at least.
Summary of the invention
The present invention relates to following invention.
1. the α that contains 11～7 sugar 2,3 sugar chain asparagine derivatives and the manufacture method thereof shown in the formula (1).
[in the formula, R
1And R
2Be the group of hydrogen atom, formula (2)～(5) expression, can be identical, also
Can be different.But R
1And R
2In a side must be the group of formula (2) expression.]
R, R ', R " be expressed as follows combination.
(a)R＝F、R’＝OH、R”＝OH
(b)R＝OH、R’＝F、R”＝OH
(c)R＝OH、R’＝OH、R”＝F
(d)R＝OH、R’＝OH、R”＝OH
2. the fluorine-containing α that contains 11～7 sugar 2,6 sugar chain asparagine derivatives and the manufacture method thereof shown in the formula (6).
[in the formula, R
XAnd R
YBe the group of hydrogen atom, formula (7) expression or the group of formula (3)～(5) expression.But R
XAnd R
YIn a side is arranged must be the group of formula (7) expression.]
R, R ', R " be expressed as follows combination.
(a)R＝F、R’＝OH、R”＝OH
(b)R＝OH、R’＝F、R”＝OH
(c)R＝OH、R’＝OH、R”＝F
3. the α that contains 11～7 sugar 2,3 sugar chain asparagines and the manufacture method thereof shown in the formula (8).
[in the formula, R
1And R
2As mentioned above.]
4. the fluorine-containing α that contains 11～7 sugar 2,6 sugar chain asparagines and the manufacture method thereof shown in the formula (9).
[in the formula, R
XAnd R
YAs mentioned above.]
5. the α that contains 11～7 sugar 2,3 sugar chains and the manufacture method thereof shown in the formula (10).
[in the formula, R
1And R
2As mentioned above.]
6. the fluorine-containing α that contains 11～7 sugar 2,6 sugar chains and the manufacture method thereof shown in the formula (11).
[in the formula, R
XAnd R
YAs mentioned above.]
7. (α 2,3) (α 2, the 6) sugar chain asparagine derivative that contains 11 sugar shown in the formula (22).
[in the formula, R
1Be the group of formula (2) expression, R
YIt is the group of following formula (7) expression.]
R, R ', R " be expressed as follows combination.
(a)R＝F、R’＝OH、R”＝OH
(b)R＝OH、R’＝F、R”＝OH
(c)R＝OH、R’＝OH、R”＝F
(d)R＝OH、R’＝OH、R”＝OH
8. (α 2,3) (α 2, the 6) sugar chain asparagine derivative that contains 11 sugar shown in the formula (2 3).
[in the formula, R
2Be the group of formula (2) expression, R
XIt is the group of following formula (7) expression.]
R, R ', R " be expressed as follows combination.
(a)R＝F、R’＝OH、R”＝OH
(b)R＝OH、R’＝F、R”＝OH
(c)R＝OH、R’＝OH、R”＝F
(d)R＝OH、R’＝OH、R”＝OH
The N-acetyl-glucosamine that the present invention relates to a non-reduced terminal side of the sugar chain asparagine protected by fat-soluble blocking group at the amino nitrogen of l-asparagine contains the new sugar chain asparagine derivative and the manufacture method thereof of an above Fucose at least.
Developed the manufacture method of the sugar chain asparagine derivative that can be very easy to and obtain various isolating sugar chain asparagine derivatives in large quantities compared with the past, sugar chain asparagine, sugar chain in the patent 2001-185685 that the inventor formerly applies for number (hereinafter referred to as before applying for a patent), and the new sugar chain asparagine derivative, sugar chain asparagine, the sugar chain that combine the sugar chain of any disappearance saccharide residue.
The method that should before apply for for example comprises:
(1) comprises the manufacture method from the sugar chain asparagine derivative of sugar chain asparagine of following operation: (a) fat-soluble protecting group is imported in this sugar chain asparagine that is contained in the mixture that contains sugar chain asparagine more than a kind or 2 kinds; obtain the operation of sugar chain asparagine derivative mixture, and
(b) mixture that obtains after the sugar chain asparagine derivative hydrolysis that this sugar chain asparagine derivative mixture or this sugar chain asparagine derivative mixture are contained supplies to chromatogram, and each sugar chain asparagine derivative is carried out isolating operation.
(2) manufacture method of above-mentioned (1) described sugar chain asparagine derivative, it also comprises the operation that (b ') is hydrolyzed to isolating sugar chain asparagine derivative in the operation (b) with glycosylhydrolase.
(3) manufacture method of above-mentioned (1) or (2) described sugar chain asparagine derivative, the mixture that wherein contains sugar chain asparagine more than a kind or 2 kinds are the mixtures that contains the compound of following formula (A) and/or lack the compound of 1 above saccharide residue in this compound.
(4) manufacture method of each described sugar chain asparagine derivative of above-mentioned (1)～(3), wherein fat-soluble protecting group are fluorenes methoxy carbonyl (Fmoc) bases.
(5) manufacture method of each described sugar chain asparagine derivative of above-mentioned (1)～(3), wherein operation (a) is to have this contained in the mixture of sugar chain asparagine more than a kind or 2 kinds of sialic acid residues sugar chain asparagine at non-reduced end and import the Fmoc base to containing, thereby and imports the operation that benzyl obtains the sugar chain asparagine derivative mixture to sialic acid residues.
(6) comprise the manufacture method of the sugar chain asparagine of following operation:
(a) fat-soluble protecting group is imported in contained this sugar chain asparagine of the mixture that contains the sugar chain asparagine more than a kind or 2 kinds, obtains the operation of sugar chain asparagine derivative mixture,
(b) mixture that obtains after the sugar chain asparagine derivative hydrolysis that this sugar chain asparagine derivative mixture or this sugar chain asparagine derivative mixture are contained supplies to chromatogram, and each sugar chain asparagine derivative is carried out isolating operation, and
(c) remove the protecting group of isolating sugar chain asparagine derivative in the operation (b), obtain the operation of sugar chain asparagine.
(7) manufacture method of above-mentioned (6) described sugar chain asparagine, it also comprises:
The operation that (b ') is hydrolyzed to isolating sugar chain asparagine derivative in operation (b) with glycosylhydrolase and/or
The operation that (c ') is hydrolyzed to the sugar chain asparagine that obtains in operation (c) with glycosylhydrolase.
(8) manufacture method of above-mentioned (6) or (7) described sugar chain asparagine, the mixture that wherein contains sugar chain asparagine more than a kind or 2 kinds are the mixtures that contains the compound of following formula (A) and/or lack the compound of 1 above saccharide residue in this compound.
(9) manufacture method of each described sugar chain asparagine of above-mentioned (6)～(8), wherein fat-soluble protecting group is the Fmoc base.
(10) manufacture method of each described sugar chain asparagine of above-mentioned (6)～(8), wherein operation (a) is to import the Fmoc base to containing to have in the mixture of sugar chain asparagine more than a kind or 2 kinds of sialic acid residues at non-reduced end in contained this sugar chain asparagine, thereby and imports the operation that benzyl obtains the sugar chain asparagine derivative mixture to sialic acid residues.
The detailed description of the manufacturing of relevant these sugar chain asparagine derivatives and sugar chain asparagine is owing to narrating in above-mentioned previous application, so quote this application.If but talk about the content of some previous applications; the big feature of manufacture method one of the sugar chain asparagine derivative of previous application is that this sugar chain asparagine that for example contains in the mixture of proteinic sugar chain asparagine, the sugar chain asparagine that preferably obtained by l-asparagine mating type sugar chain from natural sugar imports (combination) fat-soluble protecting group; obtaining the mixture of sugar chain asparagine derivative, is each sugar chain asparagine derivative with this mixture separation then.In this manual, so-called " sugar chain asparagine " refers to the sugar chain that combines the l-asparagine state.And so-called " l-asparagine mating type sugar chain " refers to the sugar chain group that the N-acetyl-glucosamine that is present in reduction end is connected with the acid amino of l-asparagine (Asn) in the protein and peptide by the N-glycosidic link, with the sugar chain group of Man (β 1-4) GlcNac (β 1-4) GlcNac as parent nucleus.So-called " sugar chain asparagine derivative " refers to the sugar chain asparagine that combines fat-soluble protecting group state on asparagine residue.In the structural formula of compound, " AcHN " represents acetamido.
As mentioned above, be the mixture of sugar chain that has lacked the saccharide residue of non-reduced end at random from the proteinic sugar chain of natural sugar.The inventor unexpectedly also find by to from natural sugar proteinic sugar chain, be that this sugar chain asparagine that contains in the sugar chain asparagine mixture imports fat-soluble protecting group specifically; utilize well-known stratographic method, can easily the mixture separation that has imported the sugar chain asparagine derivative of this protecting group be each sugar chain asparagine derivative.Can prepare sugar chain asparagine derivative respectively in a large number by such operation with various structures.For example, the separation between the sugar chain asparagine derivative of the similar structures of separation difficulty in the past becomes possibility, can be easily and prepare these compounds in large quantities respectively.In addition, as starting raw material, for example, remove saccharide residue, also can further synthesize various sugar chain asparagine derivatives by glycosylhydrolase is acted on successively with the sugar chain asparagine derivative that obtains.
Therefore make it become derivative by import fat-soluble protecting group to sugar chain asparagine; each sugar chain asparagine derivative be separated into possibility; think that this is because by importing fat-soluble protecting group; the fat-soluble raising of sugar chain asparagine derivative integral body; for example; significantly improve with the interaction of the anti-phase bitt that is fit to use, its result can reflect the difference of sugar chain structure, the cause that can separate each sugar chain asparagine derivative more observantly.
According to previous application,, can manually obtain various sugar chain asparagines easily and in large quantities in addition by removing the protecting group of the sugar chain asparagine derivative that obtains.
Yet sugar chain asparagine derivative, sugar chain asparagine and the sugar chain that obtains in the above-mentioned previous application all is α 2,6 combinations.
In addition, sugar chain asparagine derivative, sugar chain asparagine and the sugar chain that obtains in the above-mentioned previous application all is the sugar chain asparagine derivative that does not have in conjunction with Fucose.
And in the present invention, can obtain not have in the above-mentioned previous application α 2 of report, the sugar chain asparagine derivative of 3 combinations, sugar chain asparagine and sugar chain and α 2,6 combination respective substance, and contain the new sugar chain asparagine derivative of being of fluorine, sugar chain asparagine and sugar chain.
In addition, can obtain not having in the above-mentioned previous application sugar chain asparagine derivative of the Fucose combination of report in the present invention.
Below describe with regard to the difference of α 2,3 combinations and α 2,6 combinations.
So-called α 2,3 combinations and α 2,6 combinations are the combining form between expression sialic acid and the semi-lactosi.The former refers to sialic 2 carbon and carries out α with 3 carbon of semi-lactosi and combine, and the latter refers to sialic 2 carbon and carries out α with 6 carbon of semi-lactosi and combine.Both differences are the carbon differences in conjunction with semi-lactosi.
And this difference, for example influenza virus identification end has sialic sugar chain as acceptor.And in the influenza virus of people and bird, the specificity difference of acceptor.The former discerns sialic acid and galactose alpha 2,6 bonded sugar chains specifically, and the latter discerns sialic acid and galactose alpha 2,3 bonded sugar chains specifically.Combining form difference between known sialic acid-semi-lactosi, and sialic difference plays very big effect on the host domain of restriction influenza virus.
Relate in the present invention in the above-mentioned previous application less than new sugar chain asparagine derivative, sugar chain asparagine and the sugar chain of report and their manufacture method.
In the method for the invention; at first; will be with sialytransferase as sugar chain asparagine (9 sugar-Asn-Fmoc) sialic acid or sialic derivative are shifted of the fat-soluble protecting group protection of the usefulness of initial compounds; the sugar chain asparagine of the fat-soluble protecting group protection of the usefulness that obtains is supplied with chromatogram separate, can obtain bifunctional sialyltransferase sugar chain asparagine derivative and 2 kinds of single sialic acid sugar chain asparagine derivatives with fat-soluble protecting group protection.
Then, the bifunctional sialyltransferase sugar chain asparagine derivative that obtains and 2 kinds of single sialic acid sugar chain asparagine derivatives are separated by carrying out syrup, can obtain containing 9～7 sugar chain asparagine derivatives of sialic acid or sialic acid derivative.
In addition, (α 2 with above-mentioned 11～7 sugar chain asparagine derivatives that obtain or bifunctional sialyltransferase sugar chain asparagine, 6-11 sugar-Asn-Fmoc) as starting raw material, separate and to obtain 10～6 sugar chain asparagine derivatives by it being carried out syrup then, by shifting Fucose to 10～6 sugar chain asparagine derivatives, can obtain containing 13～7 sugar chain asparagine derivatives of Fucose with sugared transferring enzyme.
Be not particularly limited as this protecting group, for example can use the protecting group of carbonic ethers such as Fmoc base, tertiary butyloxycarbonyl (Boc) base, benzyl, allyl group, allyloxycarbonyl, ethanoyl system or acid amides system etc.Be directly used in the synthetic viewpoint of desired glycopeptide from the sugar chain asparagine derivative that will obtain, as this protecting group, preferred Fmoc base or Boc base etc., more preferably Fmoc base.The Fmoc base exists unsettled when sugar effective especially under comparison tart conditions such as sialic acid in the sugar chain.The importing of protecting group can be according to well-known method (for example with reference to Protecting groups in Organic Chemistry, John Wiley ﹠amp in addition; Sons INC., New York 1991, ISBN 0-471-62301-6) carry out.
For example, when using the Fmoc base, after in sugar chain asparagine, adding proper amount of acetone, adding 9-fluorene methyl-N-succinimdyl carbonate and sodium bicarbonate then dissolves, by under 25 ℃, carrying out the association reaction of Fmoc basal orientation asparagine residue, can import the Fmoc base to the asparagine residue of this sugar chain asparagine.
Can obtain to have imported the sugar chain asparagine derivative of fat-soluble protecting group by above operation.
As sialic acid, can use the general commercially available sialic acid or the sialic acid of chemosynthesis.
As sialic derivative, can use the general commercially available sialic acid derivative or the sialic acid derivative of chemosynthesis.Specifically, can enumerate compound after replacing with hydrogen atom or halogen atom with sialic 7,8 or 9 carbon bonded hydroxyls.As halogen atom, can enumerate fluorine, chlorine, bromine etc., preferred fluorine.
As sialytransferase, can use usually commercially available, from natural, by the sialytransferase that gene recombination is produced, can suitably select according to the kind of sialic acid that will shift or sialic acid derivative.Specifically, can enumerate enzyme as α 2,3 transferring enzymes from RatRecombinant, as the enzyme from Rat Liver of α 2,6 transferring enzymes.By using sialidase, carry out pH adjustment etc. balance is staggered in addition, sialic acid or sialic acid derivative are shifted.
Above-mentioned sugar chain asparagine derivative adopts stratographic to separate can be by suitably independent or a plurality ofly be used in combination well-known chromatogram and carry out.
For example, behind the sugar chain asparagine derivative mixture usefulness gel-filtration column chromatogram purification that obtains, use the HPLC purifying again.As operable pillar in HPLC, anti-phase bitt is suitable, for example ODS, Phenyl system, nitrile system, anionresin bitt can be utilized, specifically, the モ ノ Q post of for example Pharmacia company production, ィ ァ ト ロ PVC-ズ post that ィ ャ ト ロ Application company produces etc. can be utilized.Separation conditions etc. can suitably be adjusted with reference to well-known condition.Can obtain each desired sugar chain asparagine derivative by the sugar chain asparagine derivative mixture by above operation.
By above operation, for example when protecting group is the Fmoc base, can obtain the sugar chain asparagine derivative of formula (12), (13), (17), (18), (22), (23) with independent or form of mixtures.
Be hydrolyzed by the sugar chain asparagine derivative that above-mentioned separation is obtained then, can obtain to have the sugar chain asparagine derivative of desired sugar chain structure effectively.For example separating the sugar chain asparagine derivative stage, kind to the sugar chain asparagine derivative that contains in the mixture limits, sugar chain asparagine derivative is separated roughly, be hydrolyzed then, for example, can obtain to have the sugar chain asparagine derivative of desired sugar chain structure effectively by using glycosylhydrolase to be hydrolyzed.Hydrolysis can be carried out as described above.The viewpoint that particularly has the sugar chain asparagine derivative of desired sugar chain structure from more effective acquisition, the cut-out mode of preferred saccharide residue uses clear and definite glycosylhydrolase to be hydrolyzed.
For example, the removal of galactose residue can be dissolved in damping fluid (for example phosphoric acid buffer, acetate buffer solution, グ ッ De damping fluid etc.) by the compound with hydrolysis, according to well-known condition, the cut-out reaction of using galactohydrolase to carry out galactose residue can be finished.And no matter the compound that is hydrolyzed is the difference isolated compound, or mixture can.The galactohydrolase that uses in this reaction preferably utilizes the enzyme of commercially available well-known circumscribed-type.So long as have same activity, no matter be new isolating enzyme, still the enzyme of creating on the gene engineering can.With above-mentioned same, the reaction solution (mixture of the cut sugar chain asparagine derivative of saccharide residue) that obtains after the reaction is supplied with chromatogram then, can obtain each sugar chain asparagine derivative.For example, separate and preferably to use HPLC (ODS post, eluting solvent are the 50mM ammonium acetate aqueous solution: acetonitrile=82: 18) carry out.
The removal of N-acetyl-glucosamine residue is dissolved in damping fluid (for example phosphoric acid buffer, acetate buffer solution, グ ッ De damping fluid etc.) by the compound with hydrolysis, according to well-known condition, the cut-out reaction of using the N-acetyl-glucosamine lytic enzyme to carry out the N-acetyl-glucosamine residue can be finished.Also can use N-acetylaminohexose esterase hydrolyzed enzyme.And no matter the compound that is hydrolyzed is the difference isolated compound, or mixture can.Each enzyme that uses in this reaction preferably utilizes the enzyme of commercially available circumscribed-type.So long as have same activity, no matter be new isolating enzyme, still the enzyme of creating on the gene engineering can.With above-mentioned same, the reaction solution (mixture of the cut sugar chain asparagine derivative of saccharide residue) that obtains after the reaction is supplied with chromatogram then, can obtain each sugar chain asparagine derivative.For example, separate and preferably to use HPLC (ODS post, eluting solvent are the 50mM ammonium acetate aqueous solution: methyl alcohol=65: 35 or 50mM ammonium acetate aqueous solution: acetonitrile=82: 18) carry out.
The removal of mannose residue is dissolved in damping fluid (for example phosphate buffer solution, hac buffer, グ ッ De buffered soln etc.) by the compound with hydrolysis, according to well-known condition, the cut-out reaction of using the seminose lytic enzyme to carry out mannose residue can be finished.And no matter the compound that is hydrolyzed is the difference isolated compound, or mixture can.The seminose lytic enzyme that uses in this reaction preferably utilizes the enzyme of commercially available circumscribed-type.So long as have same activity, no matter be new isolating enzyme, still the enzyme of creating on the gene engineering can.With above-mentioned same, the reaction solution (mixture of the cut sugar chain asparagine derivative of saccharide residue) that obtains after the reaction is supplied with chromatogram then, can obtain each sugar chain asparagine derivative.For example, (ODS post, eluting solvent can have fat-soluble water-miscible organic solvent with buffered soln such as the ammonium acetate about 10～200mM and acetonitrile or ethanol or methyl alcohol or butanols or propyl alcohol etc. and suitably mix the back use to separate the preferred HPLC of use.Here as eluting solvent, be preferably the 50mM ammonium acetate aqueous solution: acetonitrile=82: 18) carry out.
After being operated each sugar chain asparagine derivative that obtains as described above; by Fucose is shifted, the amino nitrogen that can make l-asparagine is contained the new sugar chain asparagine derivative of an above Fucose at least by the N-acetyl-glucosamine of the non-reduced end side of the sugar chain asparagine of fat-soluble blocking group protection of the present invention.
Can use the general commercially available Fucose or the Fucose of chemosynthesis as Fucose.
As fucosyl transferase can use general commercially available, from natural or by the fucosyl transferase that gene recombination is produced, can suitably select according to the kind of the Fucose that is transferred.Specifically, can enumerate as making Fucose transfer to the Fucosyltransferase V (Human, Recombinant, from blood plasma, from serum, from milk, from liver) etc. of enzyme of N-acetyl-glucosamine of the non-reduced end side of sugar chain asparagine.In addition, carry out pH adjustment etc. by using the Fucose lytic enzyme, the balance that staggers also can shift Fucose.
Above-mentioned sugar chain asparagine derivative adopts stratographic to separate can be by suitably independent or a plurality ofly be used in combination well-known chromatogram and carry out.
For example, behind the sugar chain asparagine derivative mixture usefulness gel-filtration column chromatogram purification that obtains, use the HPLC purifying again.As operable pillar in HPLC, preferred anti-phase bitt, for example ODS, Phenyl system, nitrile system, anionresin bitt can be utilized, specifically, the モ ノ Q post of for example Pharmacia company production, ィ ァ ト ロ PVC-ズ post that ィ ャ ト ロ Application company produces etc. can be utilized.Separation conditions etc. can suitably be adjusted with reference to well-known condition.Can obtain each desired sugar chain asparagine derivative by the sugar chain asparagine derivative mixture by above operation.
After obtaining each sugar chain asparagine derivative by aforesaid operations, again by using various glycosylhydrolases etc. that this derivative is hydrolyzed, remove the saccharide residue of the non-reduced end of sugar chain, can obtain for example inhomogenous various sugar chain asparagine derivatives of branched structure of the end of sugar chain as each simplification compound.And use various glycosylhydrolases, by changing order or its kind of hydrolysis, can make a greater variety of sugar chain asparagine derivatives.
According to method in the past, the sugar chain asparagine derivative with ultimate sugar chain structure that obtains AG needs huger time and cost, but then do not need special device and reagent by the present invention, only be to use habitual gel-filtration column, HPLC post, at least 3 kinds of glycosylhydrolases (for example, galactohydrolase, seminose lytic enzyme, N-acetyl-glucosamine lytic enzyme) etc., with just making the sugar chain asparagine derivative that has desired sugar chain structure about 1 gram about 2 time-of-weeks.
By above operation, for example when protecting group is the Fmoc base, can obtain the sugar chain asparagine derivative of formula (14)～(16), (19)～(21) with the form of independent or mixture.
In addition, the invention provides the manufacture method that can obtain the sugar chain asparagine of various isolating sugar chain asparagines in a large number.This method is made on the manufacturing process basis of sugar chain asparagine derivative in the manufacture method according to above-mentioned sugar chain asparagine derivative, also comprises the operation of removing protecting group from the sugar chain asparagine derivative that obtains.
Removing protecting group by sugar chain asparagine derivative can carry out according to well-known method (for example, with reference to Protecting groups in Organic chemistry, John Wiley﹠amp; Sons INC., New York 1991, ISBN 0-471-62301-6).For example, when protecting group is the Fmoc base,, in the dinethylformamide (DMF), react, can remove the Fmoc base by in sugar chain asparagine derivative, adding morpholine at N.And the Boc base can be removed by making the weak acid reaction.After protecting group is removed, can suitably utilize well-known method as required, for example use the various chromatograms of gel-filtration column, ion exchange column etc., or utilize HPLC to carry out isolating method and carry out purifying, can obtain sugar chain asparagine.
By above operation, can obtain for example sugar chain asparagine of formula (8), (9) with independent or form of mixtures.
In addition, the invention provides the manufacture method that can obtain the sugar chain of various isolating sugar chains in a large number.This method is made on the manufacturing process basis of sugar chain asparagine in the manufacture method according to above-mentioned sugar chain asparagine, also comprises the operation of removing asparagine residue from the sugar chain asparagine that obtains.
Removing asparagine residue from sugar chain asparagine can carry out according to well-known method.For example, make sugar chain asparagine and anhydrous hydrazine the reaction after, remove asparagine residue by acetylize and can obtain sugar chain.In addition, sugar chain asparagine after the reflux, is removed asparagine residue by acetylize and also can be obtained sugar chain in alkaline aqueous solution.After removing asparagine residue, can for example use the various chromatograms of gel-filtration column, ion exchange column etc., or utilize the separation method of HPLC to carry out purifying suitably by well-known method as required.
By above operation, can obtain for example sugar chain of formula (10), (11) with independent or form of mixtures.
Therefore, can be low-cost by the present invention and a large amount of effectively sugar chain asparagine derivative, sugar chain asparagine and sugar chain with desired sugar chain structure (following sometimes the three is called the sugar chain class in the lump) of making.
Such sugar chain class is very useful in fields such as pharmaceuticals exploitations.For example, as the example application in the pharmaceuticals exploitations, can enumerate vaccine synthetic of cancer for example.Known cell one cancerization, the sugar chain that will occur not having in the body.Synthetic this sugar chain of known chemical gives individuality as vaccine in addition, can suppress the propagation of cancer.Therefore, if can make the sugar chain class of hope, just can carry out synthetic to the effective vaccine of treatment of cancer by the present invention.In addition, by with chemical reaction and utilize the combinations such as reaction of sugared transferring enzyme to make the sugar chain class that obtains by the present invention, also can carry out synthesizing of new vaccine again in conjunction with new saccharide residue.
Embodiment
Below enumerate reference example, embodiment describes, but the present invention is not limited to these embodiment.
Reference example 1 α 2,6-bifunctional sialyltransferase sugar chain asparagine synthetic
To be dissolved in from thick purifying SGP (sialoglycopeptide) 2.6g of ovum among ト リ ス-hydrochloric acid-calcium chloride damping fluid (TRI ZMA BASE 0.05mol/l, calcium chloride 0.01mol/l, pH7.5) 100mL.Add sodiumazide 58mg (772 μ mol) and ァ Network チ Na-ゼ-E (production of scientific research drugmaker) 526mg then, under 37 ℃, leave standstill.After 65 hours, add 263mg ァ Network チ Na-ゼ-E again, under 37 ℃, left standstill 24 hours again.After this solution lyophilize, residue with gel-filtration column chromatography (SephadexG-25,2.5 φ * 1m, launch be dissolved as water, flow velocity 1.0mL/min) purifying 2 times, is obtained 1.3g (555 μ mol) α 2,6-bifunctional sialyltransferase sugar chain asparagine.
The physical data of the bifunctional sialyltransferase sugar chain asparagine that obtains is as follows.
1H-NMR(D
2O，30℃)
δ5.13(s，1H，Man4-H-1)，5.07(d，1H，J＝9.5Hz，GlcNAcl-H-1)，4.95(s，1H，Man4-H-1)，4.77(s，1H，Man3-H-1)，4.61(d，1H，J＝7.6Hz，GlcNAc2-H-1)，4.60(d，2H，J＝7.6Hz，GlcNAc5，5-H-1)，4.44(d，2H，J＝8.0Hz，Ga16，6-H-1)，4.25(bd，1H，Man3-H-2)，4.20(bdd，1H，Man4-H-2)，4.12(bd，1H，Man4-H-2)，2.94(dd，1H，J＝4.5?H?z，17.2Hz，Asn-βCH)，2.85(dd，1H，J＝7.0Hz，17.2Hz，Asn-βCH)，2.67，2.66(dd，2H，J＝4.6Hz，12.4Hz，NeuAc7，7-H-3
eq)，2.07(s，3H，Ac)，2.06(s，6H，Ac×2)，2.02(s，6H，Ac×2)，2.01(s，3H，Ac)，1.71(dd，2H，J＝12.4Hz，12.4Hz，NeuAc7，7-H-3
ax.)
Reference example 2 compounds 1,2,3 and 4 synthetic
With the α 2 that obtains in the reference example 1,6-bifunctional sialyltransferase sugar chain asparagine (609mg, 261 μ mol) is dissolved in the 20.7mL water, adds 0.1 equivalent hydrochloric acid 13.8mL again.After 35 minutes, carry out ice-coldly in 70 ℃ of heating rapidly this solution, add saturated sodium bicarbonate aqueous solution, be transferred to pH7.After it is carried out lyophilize, (SephadexG-25,2.5 φ * 1m, expansion are dissolved as water with the gel-filtration column chromatography with residue, flow velocity 1.0mL/min) carries out purifying, obtain α 2,6-bifunctional sialyltransferase sugar chain asparagine, 2 kinds of α 2, the mixture 534mg of 6-single sialic acid sugar chain asparagine and sialic acid disappearance sugar chain asparagine.These 4 kinds of compositions do not separate respectively, directly carry out following operation.
The physical data of the sugar chain mixture that obtains is as follows.
1H-NMR(D
2O，30℃)
5.13(s，Man4-H1)，5.12(s，Man4-H1)，5.01(d，GlcNAcl-H1)，4.94(s，Man4’-H1)，4.93(s，Man4’-H1)，4.82(s，Man3-H1)，4.60(d，GlcNAc2-H1)，4.58(d，GlcNAc5，5’-H1)，4.47(dd，Gal6，6’-H1)，4.44(d，Ga16，6’-H1)，4.24(d，Man3-H2)，4.19(d，Man4’-H?2)，4.11(d，Man4-H2)，2.97(bdd，AsN-βCH)，2.72(dd，NeuAc7-H3eq，NeuAc7-H3eq)，2.64(bdd，AsN-βCH)，2.15(s×5，-Ac)，1.79(dd，NeuAc7-H3ax，NeuAc7’-H3ax)
The sugar chain mixture 429mg that obtains is dissolved in 16.3mL acetone and water 11.2mL.Add 9-fluorene methyl-N-succinimdyl carbonate (155.7mg, 461.7 μ mol) and sodium bicarbonate (80.4mg, 957 μ mol) then, under room temperature, stirred 2 hours.This solution is removed acetone by vaporizer, (SephadexG-25,2.5 φ * 1m, eluting solvent are water to remaining solution with the gel-filtration column chromatography, flow velocity 1.0mL/min) carries out purifying, obtain the mixture 309mg of compound 1, compound 2 and 3, compound 4.With this mixture by HPLC (ODS post, eluting solvent are the 50mM ammonium acetate aqueous solution: methyl alcohol=65: 35,2.0 φ * 25cm, flow velocity 3mL/min) purifying, compound 1 under wash-out after 51 minutes, compound 2 and 3 mixture under wash-out after 67 minutes, compound 4 under the wash-out after 93 minutes.After each fraction carried out lyophilize respectively,, obtain the mixture 150mg of purpose compound 2 and 3 by carrying out desalination with gel-filtration column chromatography (SephadexG-2 5,2.5 φ * 30cm, eluting solvent are water, flow velocity 1.0mL/min).
The physical data of the compound 1 that obtains is as follows.
1H-NMR(D
2O，30℃)
7.99(2H，d，Fmoc)，7.79(2H，d，Fmoc)，7.55(4H，m，Fmoc)，5.15(1H，s，Man?4-H1)，5.06(1H，d，GlcNAcl-H1)，4.95(1H，s，Man4’-H1)，4.82(1H，s，Man?3-H1)，4.69(1H，d，GlcNAc2-H1)，4.67(2H，d，GlcNAc5，5’-H1)，4.53(2H，d，Ga16，6’-H1)，4.34(1H，d，Man?3-H2)，4.27(1H，d，Man4’-H2)，4.19(1H，d，Man4-H2)，3.03(1H，bdd，AsN-βCH)，3.00(1H，bdd，AsN-βCH)，2.76(2H，dd，NeuAc7，7’-H3eq)，2.15(18H，s×6，-Ac)，1.79(2H，dd，NeuAc7，7’-H3ax)；HRMS?Calcdfor?C
103H
154N
8NaO
66[M+Na+]2581.8838，found，2581.8821
Dummy suffix notation in the above-mentioned sugar chain structure is expressed as follows:
NeuAc: sialic acid Gal:D-semi-lactosi GlcNAc:N-acetylglucosamine Man:D-seminose Asn: l-asparagine.
The physical data of compound 2 that obtains and 3 mixture is as follows.
1H-NMR(D
2O，30℃)
7.99(d，Fmoc)，7.79(d，Fmoc)，7.55(m，Fmoc)，5.14(s，Man4-H1)，5.12(s，Man4-H)，5.00(d，GLcNAc1-H1)，4.94(s，Man4’-H1)，4.93(s，Man4’-H1)，4.82(s，Man3-H1)，4.60(d，GlcNAc2-H1)，4.58(d，G1cNAc5，5’-H1)，4.46(dd，GaK6，6’-H1)，4.44(d，Ga16，6’-H1)，4.24(d，Man3-H2)，4.19(d，Man4’-H2)，4.11(d，Man4-H2)，2.97(bdd，AsN-βCH)，2.72(dd，NeuAc7-H3eq，NeuAc7-H3eq)，2.64(bdd，AsN-βCH)，2.15(s×5，-Ac)，1.79(dd，NeuAc7-H3ax，NeuAc7’-H3ax)
The physical data of the compound 4 that obtains is as follows.
1H-NMR(D
2O，30℃)
7.99(2H，d，Fmoc)，7.79(2H，d，Fmoc)，7.55(4H，m，Fmoc)，5.12(1H，s，Man4-H1)，5.06(1H，d，GlcNAc1-H1)，4.93(1H，s，Man4’-H1)，4.82(1H，s，Man3-H1)，4.69(1H，d，GlcNAc2-H1)，4.67(2H，d，GlcNAc5，5’-H1)，4.53(2H，d，Gal6，6’-H1)，4.34(1H，d，Man3-H2)，4.27(1H，d，Man4′-H2)，4.19(1H，d，Man4-H2)，3.03(1H，bdd，AsN-βCH)，3.00(1H，bdd，AsN-βCH)，2.15(12H，s×4，-Ac)；HRMS?Calcd?for?C
81H
120N
6NaO
50[M+Na+]1999.6930，found，1999.6939
Table 2 has provided the designs simplification form of above-claimed cpd 4.
Reference example 3 compounds 2,3 synthetic and separating
The mixture (5.0mg, 2.2 μ mol) of the compound 2,3 that reference example 2 is obtained is dissolved in the water of 220 μ L, adds the 22mM cesium carbonate aqueous solution of 100 μ L, adjusts to pH7.0.With this solution lyophilize.Add 430 μ L N in dried solids, dinethylformamide adds bromotoluene/N of 6.6 μ mol, dinethylformamide solution 20 μ L again.Under argon atmosphere, this solution is stirred.After 48 hours, (eluting solvent uses 1MNH by TLC
4OAc: after Virahol=1: 2) the affirmation raw material disappears, add the 4.4mL diethyl ether and make compound precipitation.Sedimentary sugar chain is filtered, with remaining sugar chain dissolving and carry out lyophilize.(YMC Packed Column D-ODS-5 S-5120A ODS No.2020178,20 * 250mm, eluting solvent are the 50mM ammonium acetate aqueous solution: acetonitrile=78: 22 by classification HPLC with the residue after the lyophilize, flow velocity 4.0mL/min) carries out purifying, the result is compound 3 under wash-out after 88 minutes, and compound 2 under wash-out after 91 minutes.Collect respectively, (コ ス モ シ-Le 75C18-OPN, 15 * 100mm, at first 50mL water is flow through by the ODS post again, 2 5% acetonitriles are flow through carry out wash-out) desalination, obtain the benzyl body 1.6mg as purpose compound 2, the benzyl body 1.8mg of compound 3.
Make the benzyl body (10 sugar, 13.6mg, 5.8mmol) of compound 2 under ice-cold condition, be dissolved in NaOHaq. (pH=12) 1.4mL.On one side by the HPLC monitoring reaction, on one side stir about 8 hours.When reaction proceeds to terminal point, reaction solution is transferred to pH=7.0 with 40mM HCl.After solution after the neutralization filtered with membrane filter, concentrate, then by HPLC (YMC-PackODS-AM, SH-343-5AM, 20 * 250mm, AN/25mM ammonium acetate buffer=20/80,7.0mL/min., wavelength: 274nm) carry out classification, purifying.After the fractionated solution concentration, again by ODS post (コ ス モ シ-Le 75C
18-OPN, Na カ ラ ィ テ ス Network company produce) carry out desalting treatment, concentrated, lyophilize obtain purpose compound 2 (6.2mg, 47.4%).The physical data of the compound that obtains is as follows.Table 1 has provided the designs simplification form of compound 2.
1H-NMR(400MHz，D
2O，30℃，HOD＝4.81)
8.00(d，2H，J＝7.2，Fmoc)，7.79(d，2H，J＝?7.2，F?m?o?c)，7.5?9(d?d，2?H，J＝7.2，Fmoc)，7.53(dd，2H，J＝7.2，Fmoc)，5.22(s，1H，Man4-H1)，5.09(d，1H，J＝9.8，GlcNAc2-H1)，5.01(s，1H，Man4’-H-1)，4.85(s，1H)，4.58-4.75(m，5H)，4.57(dd，2H，J＝8.0)，4.38-4.48(m，2?H)，4.33(s，1H)，4.28(bs，1H，Man4-H2)，4.19(bs，1H)，2.64-2.85(m，3H，Asn-βCHx2，NeuAc7-H3eq)，2.16，2.13，2.12(eachs，12H，Acx4)，1.98(s，3H，Ac)1.80(dd，1H，Ja＝12.0，Jb＝12.0，NeuAc7-H3ax).
Make the benzyl body (10 sugar, 5.0mg, 2.1mmol) of compound 3 under ice-cold condition, be dissolved in NaOHaq. (pH=12) 2.0mL.On one side by the HPLC monitoring reaction, on one side stir about 5 hours.When reaction proceeds to terminal point, reaction solution is transferred to pH=7.0 with 40mM HCl.After solution after the neutralization filtered with membrane filter, concentrate, then by HPLC (YMC-PackODS-AM, SH-343-5AM, 20 * 250mm, AN/25mM ammonium acetate buffer=20/80,7.0mL/min., wavelength: 274nm) carry out classification, purifying.After the fractionated solution concentration, again by ODS post (コ ス モ シ-Le 75C
18-OPN, Na カ ラ ィ テ ス Network company produce) carry out desalting treatment, concentrated, lyophilize obtain purpose compound 3 (2.5mg, 52.0%).The physical data of the compound that obtains is as follows.Table 1 has provided the designs simplification form of compound 3.
1H-NMR(400MHz，D
2O，30℃，HOD＝4.81)δ8.01(d，2H，J＝7.6，Fmoc)，7.80(d，2H，J＝7.6，Fmoc)，7.59(dd，2H，J＝7.6，Fmoc)，7.52(dd，2H，J＝7.6，Fmoc)，5.21(s，1H，Man4-H1)， 5.09(d，1H，J＝9.5，GlcNAc1-H1)，5.03(s，1H，Man4’-H-1)，4.58-4.71(m，5H)，4.54(t，2H，J＝7.5)，4.40-4.50(b，2H)，4.34(s，1H)，4.28(bs，1H，Man4-H2)，4.19(bs，1H)，2.70-2.85(m，2H，Asn-βCH，NeuAc7-H3eq)，2.55-2.70(m，1H，Asn-βCH)，2.16，2.15，2.13，2.11(eachs，12H，Acx4)，1.98(s，3?H，Ac)1.80(dd，1H，Ja＝12.4，Jb＝12.4，NeuAc7-H3ax).
Reference example 4 compounds 5 and 6 synthetic
Make the mixture (224mg, 97 μ mol) of compound 2 that reference example 2 obtains and 3 and the HEPES damping fluid (50mM that bovine serum albumin(BSA) 24mg is dissolved in 22mL, pH6.0) in, add beta-galactosidase enzymes (1.35U) again from Diplococcus pneumoniae.This solution after leaving standstill 15 hours under 37 ℃, is carried out lyophilize.By HPLC (ODS post, 2.0 φ * 25cm, eluting solvent are the 50mM ammonium acetate aqueous solution: acetonitrile=85: 15, flow velocity 3mL/min) purifying, compound 5 under wash-out after 129 minutes with residue, and compound 6 under wash-out after 134 minutes.Collect respectively, carry out lyophilize.Be water in initial 15 minutes by HPLC[ODS post, 2.0 φ * 25cm, eluting solvent then, after 16 minutes to 30 minutes according to water: acetonitrile (volume ratio)=10: 0 to 85: 15, after 31 minutes to 45 minutes according to water: acetonitrile=85: 15 to 80: 20 carries out gradient elution, flow velocity is 3.0mL/min], carry out desalting treatment, obtain purpose compound 5 and be 81mg, compound 6 is 75mg.
The physical data of the compound 5 that obtains is as follows.
1H-NMR(D
2O，30℃)7.99(2H，d，Fmoc)，7.79(2H，d，Fmoc)，7.55(4H，m，Fmoc)，5.15(1H，S，Man4-H1)，5.06(1H，d，GlcNAc1-H1)，4.95(1H，s，Man4’-H1)，4.82(1H，s，Man3-H1)，4.69(1H，d，GlcNAc2-H1)，4.67(2H，d，GlcNAc5，5’-H1)，4.53(1H，d，Gal6’-H1)，4.34(1H，d，Man3-H2)，4.27(1H，d，Man4’-H2)，4.19(1H，d，Man4-H2)，2.97(1H，bdd，AsN-βCH)，2.76(1H，dd，NeuAc7’-H3eq)，2.61(1H，bdd，AsN-βCH)，2.15(15H，s×5，-Ac)，1.79(1H，dd，NeuAc7’-H3ax)；HRMS?Calcd?forC
86H
127N
7NaO
53[M+Na+]2128.7356，found，2128.7363
The physical data of the compound 6 that obtains in addition is as follows.
1H-NMR(D
2O，30℃)
7.99(2H，d，Fmoc)，7.79(2H，d，Fmoc)，7.55(4H，m，Fmoc)，5.15(1H，S，Man4-H1)，5.06(1H，d，GlcNAc1-H1)，4.95(1H，s，Man4’-H1)，4.82(1H，s，Man3-H1)，4.69(1H，d，GlcNAc2-H1)，4.67(2H，d，GlcNAc5，5’-H1)，4.53(1H，d，Gal6-H1)，4.34(1H，d，Man?3-H2)，4.27(1H，d，Man4’-H2)，4.19(1H，d，Man4-H2)，2.97(1H，bdd，AsN-βCH)，2.76(1H，dd，NeuAc7-H3eq)，2.60(1H，bdd，AsN-βCH)，2.15(15H，s×5，-Ac)，1.79(1H，dd，NeuAc7-H3ax)；HRMS?Calcd?for?C
86H
125N
7Na
3O
53[M+Na+]2172.6995，found，2172.7084
Reference example 5 compounds 7 and 8 synthetic
The compound 5 that reference example 4 is obtained does not separate respectively with 6 mixture (90mg, 47.3 μ mol), direct and bovine serum albumin(BSA) 8mg is dissolved in the HEPES damping fluid (50mM of 8.1mL, pH6.0) in, add the β-glycosamine enzyme (シ グ マ ァ Le De リ ッ チ company produce, from Ren Bovis seu Bubali) of 2.88U again from Ren Bovis seu Bubali.This solution after leaving standstill 18 hours under 37 ℃, is carried out lyophilize.By HPLC (ODS post, 2.0 φ * 25cm, eluting solvent are the 50mM ammonium acetate aqueous solution: methyl alcohol=65: 35, flow velocity are 3mL/min) purifying, compound 7 under wash-out after 117 minutes with residue, and compound 8 under wash-out after 127 minutes.Collect respectively, carry out lyophilize.(ODS post, 2.0 φ * 25cm, eluting solvent were water in initial 15 minutes to continue to use HPLC, after 16 minutes to 30 minutes according to water: acetonitrile=10: 0 to 85: 15, after 31 minutes to 45 minutes according to water: acetonitrile=85: 15 to 80: 20 carries out gradient elution, flow velocity is 3mL/min] carry out desalting treatment, obtain 40mg purpose compound 7,37mg purpose compound 8.
The physical data of the compound 7 that obtains is as follows.
1H-NMR(D
2O，30℃)
8.01(2H，d，Fmoc)，7.80(2H，d，Fmoc)，7.56(4H，m，Fmoc)，5.22(1H，s，Man4-H1)，5.08(1H，d，GlcNAc1-H1)，4.94(1H，s，Man4’-H1)，4.84(1H，s，Man3-H1)，4.69(1H，d，GlcNAc2-H1)，4.67(1H，d，GlcNAc5-H1)，4.55(1H，d，Gal6-H1)，4.33(1H，dd，Man3-H2)，4.20(1H，dd，Man4-H2)，4.15(1H，dd，Man4’-H2)，2.97(1H，bdd，AsN-βCH)，2.76(2H，dd，NeuAc7，7’-H3eq)，2.62(1H，bdd，AsN-βCH)，2.15(12H，s×4，-Ac)，1.79(2H，dd，NeuAc7，7’-H3ax)；HRMS?Calcd?for?C
78H
114N
6NaO
48[M+Na+]1925.6562，found，1925.6539
The physical data of the compound 8 that obtains is as follows.
1H-NMR(D
2O，30℃)
7.99(2H，d，Fmoc)，7.79(2H，d，Fmoc)，7.55(4H，m，Fmoc)，5.15(1H，S，Man4-H1)，5.06(1H，d，GlcNAc1-H1)，4.95(1H，s，Man?4’-H1)，4.82(1H，s，Man3-H1)，4.69(1H，d，GlcNAc2-H1)，4.67(2H，d，GlcNAc5，5’-H1)，4.53(2H，d，Gal6，6’-H1)，4.34(1H，d，Man3-H2)，4.27(1H，d，Man4’-H2)，2.97(1H，bdd，AsN-βCH2)，2.76(1H，dd，NeuAc7’-H3eq)，2.61(1H，bdd，AsN-βCH2)，2.15(12?H，s×4，-Ac)，1.79(1H，dd，NeuAc7’-H3ax)；HRMS?Calcd?for?C
78H
114N
6NaO
48[M+Na+]1925.6562，found，1925.6533
Synthesizing of reference example 6 compounds 9
The HEPES damping fluid that compound 7 (30mg, 473 μ mol) that reference example 5 is obtained and bovine serum albumin(BSA) 3mg are dissolved in 6mL (50mM, pH6.0) in, add the alpha-Mannosidase of 10U again from Jack Beans.With this solution after leaving standstill 21 hours under 37 ℃, carry out lyophilize, then (oDS post, 2.0 φ * 25cm, eluting solvent were water in initial 15 minutes with HPLC, after 16 minutes to 30 minutes according to water: acetonitrile=10: 0 to 85: 15, after 31 minutes to 45 minutes according to water: acetonitrile=85: 15 to 80: 20 carries out gradient elution, flow velocity is 3mL/min) carry out purifying, obtain the purpose compound 9 of 20mg.
The physical data of the compound 9 that obtains is as follows.
1H-NMR(D
2O，30℃)
8.01(2H，d，Fmoc)，7.80(2H，d，Fmoc)，7.56(4H，m，Fmoc)，5.00(1H，d，GlcNAc1-H1)，4.95(1H，s，Man4’-H1)，4.84(1H，s，Man3-H1)，4.67(1H，d，GlcNAc2-H1)，4.56(1H，d，GlcNAc5-H1)，4.44(1H，d，Gal6-H1)，4.11(1H，dd，Man4’-H2)，4.07(1H，dd，Man?3-H2)，2.97(1H，bdd，AsN-βCH)，2.76(1H，dd，NeuAc7’-H3eq)，2.62(1H，bdd，AsN-βCH)，2.15(12H，s×4，-Ac)，1.79(2H，dd，NeuAc?7’-H3ax)；HRMS?Calcd?forC
72H
104N
6NaO
43[M+Na+]1763.6034，found，1763.6074
Synthesizing of reference example 7 compounds 10
The HEPES damping fluid that compound 8 (40mg, 630 μ mol) that reference example 5 is obtained and bovine serum albumin(BSA) 5g are dissolved in 7.8mL (50mM, pH6.0) in, add the alpha-Mannosidase of 38U again from Jack Beans.With this solution after leaving standstill 63 hours under 37 ℃, carry out lyophilize, then (ODS post, 2.0 φ * 25cm, eluting solvent were water in initial 15 minutes with HPLC, after 16 minutes to 30 minutes according to water: acetonitrile=10: 0 to 85: 15,31 minutes to 45 minutes is 85: 15 to 80: 20, carry out gradient elution, flow velocity is 3mL/min) carry out purifying, obtain the purpose compound 10 of 30mg.
The physical data of the compound 10 that obtains is as follows.
1H-NMR(D
2O，30℃)
1H-NMR(30℃)
δ7.91(d，2H，J＝7.5Hz，Fmoc)，7.71(d，2H，J＝7.5Hz，Fmoc)，7.51(dd，2H，J＝7.2Hz，Fmoc)，7.43(dd，2H，J＝7.5Hz，Fmoc)，5.12(s，1H，Man4-H-1)，4.99(d，1H，J＝9.5Hz，GlcNAcl-H-1)，4.92(s，1H，Man4’-H-1)，4.76(s，1H，Man3-H-1)，4.58(d，1H，J＝8.0Hz，GlcNAc2-H-1)，4.55(d，1H，J＝8.4Hz，GlcNAc5’-H-1)，4.47(d，1H，J＝7.8Hz，Gal6’-H-1)，4.34(t，1H，Fmoc)，4.24(bd，1H，J＝1.9Hz，Man3-H-2)，4.18(bdd，1H，J＝1.4Hz，3.3Hz，Man4-H-2)，4.11(bdd，1H，J＝1.4Hz，3.5Hz，Man4’-H-2)，2.72(bdd，1H，J＝3.0Hz，15.7Hz，AsN-βCH)，2.52(bdd，1H，J＝8.7Hz，15.7Hz，AsN-βCH)，2.06，2.05，2.04，
Synthesizing of reference example 8 compounds 11
Compound 5 (28mg, 21.3 μ mol) and bovine serum albumin(BSA) 1.0mg are dissolved in the HEPES damping fluid (50mM, pH5.0,454 μ L), and (SigmaAldrich company produces, from Viblio Cholerae, 198mU) to add neuraminidase again.This solution after leaving standstill 20 hours under 37 ℃, is finished by the reaction of HPLC analysis confirmation.Reaction solution is carried out purifying by HPLC (YMCPacked Column D-ODS-5S-5120AODS No.2020178,20 * 250mm, eluting solvent are the 50mM ammonium acetate aqueous solution: acetonitrile=80: 20, flow velocity 4.0mL/min).Again by ODS post (コ ス モ シ-Le 75C
18-OPN, 15 * 100mm, at first 50mL water is flow through, 25% acetonitrile is flow through carry out wash-out) desalination, obtain purpose compound 11 (17mg, yield 70%).The physical data of the compound 11 that obtains is as follows.
The reduced form of compound 11 structures is as shown in table 2.
1H-NMR(30℃)
δ7.91(d，2H，J＝7.5Hz，Fmoc)，7.71(d，2H，J＝7.5Hz，Fmoc)，7.51(dd，2H，J＝7.5Hz，Fmoc)，7.43(dd，2H，J＝7.5Hz，Fmoc)，5.12(s，1H，Man4-H-1)，4.99(d，1H，J＝9.5Hz，GlcNAcl-H-1)，4.92(s，1H，Man?4’-H-1)，4.76(s，1H，Man3-H-1)，4.58(d，1H，J＝8.0Hz，GlcNAc2-H-1)，4.55(d，1H，J＝8.4Hz，GlcNAc5’-H-1)，4.47(d，1H，J＝7.8Hz，Gal6’-H-1)，4.34(t，1H，Fmoc)，4.24(bd，1H，J＝1.9Hz，Man3-H-2)，4.18(bdd，1H，J＝1.4Hz，3.3Hz，Man4-H-2)，4.11(bdd，1H，J＝1.4Hz，3.5Hz，Man4’-H-2)，2.72(bdd，1H，J＝3.0Hz，15.7Hz，AsN-βCH)，2.52(bdd，1H，J＝8.7Hz，15.7Hz，AsN-βCH)，2.06，2.05，2.04，1.89(eachs，each3H，Ac)；HRMS?Calcd?for?C
75H
110N
6NaO
45[M+Na+]1837.6402，found1837.6471
Synthesizing of reference example 9 compounds 12
Compound 6 (20mg, 9.4 μ mol) and bovine serum albumin(BSA) 1.6mg are dissolved in the HEPES damping fluid (50mM, pH5.0,323 μ L), and (SigmaAldrich company produces, from Viblio Cholerae, 141mU) to add neuraminidase again.This solution after leaving standstill 18 hours under 37 ℃, is finished by the reaction of HPLC analysis confirmation.Carry out purifying by HPLC (YMC PackedColumn D-ODS-5 S-5120A ODS No.2020178,20 * 250mm, eluting solvent are the 50mM ammonium acetate aqueous solution: acetonitrile=80: 20, flow velocity 4mL/min) then.By ODS post (コ ス モ シ-Le 75C18-OPN, 15 * 100mm, at first 50mL water is flow through, 25% acetonitrile is flow through carry out wash-out) desalination, obtain purpose compound 12 (13mg, yield 76%) again.The structure of the compound that obtains by
1H-NMR and standard substance-cause and confirm.
The reduced form of compound 12 structures is as shown in table 2.
Synthesizing of reference example 10 compounds 13
Compound 7 (45mg, 24 μ mol) and bovine serum albumin(BSA) 1.7mg are dissolved in the HEPES damping fluid (50mM, pH5.0,820 μ L), and (SigmaAldrich company produces, from Viblio Cholerae, 134mU) to add neuraminidase again.This solution after leaving standstill 14 hours under 37 ℃, is finished by the reaction of HPLC analysis confirmation.Then reaction solution is carried out purifying by HPLC (YMCPacked Column D-ODS-5 S-5120A ODS No.2020178,20 * 250mm, eluting solvent are the 50mM ammonium acetate aqueous solution: acetonitrile=80: 20, flow velocity 4mL/min).By ODS post (コ ス モ シ-Le 75C18-OPN, 15 * 100mm, at first 50mL water is flow through, 25% acetonitrile is flow through carry out wash-out) desalination, obtain purpose compound 13 (28mg, yield 74%) again.The physical data of the compound that obtains is as follows.
The reduced form of compound 13 structures is as shown in table 2.
1H-NMR(30℃)
δ7.92(d，2H，J＝7.5Hz，Fmoc)，7.71(d，2H，J＝7.5Hz，Fmoc)，7.51(dd，2H，J＝7.5Hz，Fmo.c)，7.44(dd，2H，J＝7.5Hz，Fmoc)，5.10(s，1H，Man4-H-1)，4.99(d，1H，J＝9.5Hz，GlcNAc1-H-1)，4.92(s，1H，Man4’-H-1)，4.76(s，1H，Man3-H-1)，4.58(d，2H，GlcNAc2，5’-H-1)，4.47(d，1H，J＝8.0Hz，Gal6’-H-1)，4.35(t，1H，Fmoc)，4.24(bd，1H，J＝1.9Hz，Man3-H-2)，4.11(bs，1H，Man4’-H-2)，4.07(bs，1H，Man4-H-2)，2.72(bd，1H，J＝15.5Hz，AsN-βCH)，2.52(bdd，1H，J＝8.7Hz，15.5Hz，AsN-βCH)，2.06，2.04，1.89(eachs，each?3H，Ac)；HRMS?Calcd?for?C
67H
97N
5NaO
40[M+Na+1634.5608，found，1634.5564
Synthesizing of reference example 11 compounds 14
Compound 8 (47mg, 25 μ mol) and bovine serum albumin(BSA) 1.9mg are dissolved in the HEPES damping fluid (50mM, pH5.0,840 μ L), and (SigmaAldrich company produces, from Viblio Cholerae, 369mU) to add neuraminidase again.This solution after leaving standstill 37 hours under 37 ℃, is finished by the reaction of HPLC analysis confirmation.With this reaction solution lyophilize, carry out purifying by HPLC (YMC Packed Column D-ODS-5 S-5 120A ODSNo.2020178,20 * 250mm, eluting solvent are the 50mM ammonium acetate aqueous solution: acetonitrile=80: 20, flow velocity 4.0mL/min) then.By ODS post (コ ス モ シ-Le 75C18-OPN, 15 * 100mm, at first 50mL water is flow through, 25% acetonitrile is flow through carry out wash-out) desalination, obtain purpose compound 14 (26mg, yield 65%) again.The physical data of the compound that obtains is as follows.
The reduced form of compound 14 structures is as shown in table 2.
1H-NMR(30℃)
δ7.92(d，2H，J＝7.5Hz，Fmoc)，7.71(d，2H，J＝7.5Hz，Fmoc)，7.51(dd，2H，J＝7.5Hz，Fmoc)，7.43(dd，2H，J＝7.5Hz，Fmoc)，5.12(s，1H，Man4-H-1)，4.99(d，1H，J＝9.4Hz，GlcNAc1-H-1)，4.91(s，1H，Man4’-H-1)，4.77(s，1H，Man3-H-1)，4.57(bd，2H，GlcNAc2，5’-H-1)，4.46(d，1H，J＝7.5kz，Gal6’-H-1)，4.34(t，3H，Fmoc)，4.24(bs，1H，Man4’-H-2)，4.19(bs，1H，Man4-H-2)，2.72(bd，1H，J＝15.5Hz，AsN-βCH)，2.52(bdd，1H，J＝9.2Hz，15.5Hz，AsN-βCH)，2.06，2.05，1.89(each?s，each?3H，Ac)；HRMSCalcd?for?C
67H
97N
5NaO
40[M+Na+]1634.5608，found，1634.5644
Synthesizing of reference example 12 compounds 15
Compound 9 (32mg, 18.4 μ mol) and bovine serum albumin(BSA) 2.5mg are dissolved in the HEPES damping fluid (50mM, pH5.0,713 μ L), and (Sigma Aldrich company produces, from Viblio Cholerae, 134mU) to add neuraminidase again.This solution after leaving standstill 17 hours under 37 ℃, is finished by the reaction of HPLC analysis confirmation.Then reaction solution is carried out purifying by HPLC (YMCPacked Column D-ODS-5S-5120A ODS No.2020178,20 * 250mm, eluting solvent are the 50mM ammonium acetate aqueous solution: acetonitrile=80: 20, flow velocity 4mL/min).By ODS post (コ ス モ シ-Le 75C18-OPN, 15 * 100mm, at first 50mL water is flow through, 25% acetonitrile is flow through carry out wash-out) desalination, obtain purpose compound 15 (13mg, yield 52%) again.The physical data of the compound that obtains is as follows.
The reduced form of compound 15 structures is as shown in table 2.
1H-NMR(30℃)
δ7.92(d，2H，J＝7.5Hz，Fmoc)，7.71(d，2H，J＝7.5Hz，Fmoc)，7.51(dd，2H，J＝7.5Hz，Fmoc)，7.44(dd，2H，J＝7.5Hz，Fmoc)，5.00(d，1H，J＝9.9H?z，GlcNAc1-H-1)，4.92(s，1H，Man4’-H-1)，4.75(s，1H，Man3-H-1)，4.58(d，2H，J＝7.5Hz，GlcNAc2，5’-H-1)，4.47(d，1H，J＝7.8Hz，Gal6’-H-1)，4.34(t，1H，Fmoc)，4.10(bd，1H，Man3-H-2)，4.07(bs，1H，Man?4’-H-2)，2.72(bdd，1H，J＝15.5Hz，AsN-βCH)，2.52(bdd，1H，J＝9.2Hz，15.5Hz，AsN-βCH)，2.07，2.05，1.89(eachs，each?3H，Ac)；MS(Fab)，Calcd?forC
61H
88N
5O
35[M+H+]1450.5，found，1450.3
Synthesizing of reference example 13 compounds 16
Compound 10 (28mg, 16 μ mol) and bovine serum albumin(BSA) 1.7mg are dissolved in the HEPES damping fluid (50mM, pH5.0,624 μ L), and (SigmaAldrich company produces, from Viblio Cholerae, 117mU) to add neuraminidase again.This solution after leaving standstill 17 hours under 37 ℃, is finished by the reaction of HPLC analysis confirmation.Then reaction solution is carried out purifying by HPLC (YMCPacked Column D-ODS-5S-5120A ODS No.2020178,20 * 250mm, eluting solvent are the 50mM ammonium acetate aqueous solution: acetonitrile=80: 20, flow velocity 4mL/min).By ODS post (コ ス モ シ-Le 75C18-OPN, 15 * 100mm, at first 50mL water is flow through, 25% acetonitrile is flow through carry out wash-out) desalination, obtain purpose compound 16 (14.6mg, yield 68%) again.The physical data of the compound that obtains is as follows.
The reduced form of compound 16 structures is as shown in table 2.
1H-NMR(30℃)
δ7.92(d，2H，J＝7.5Hz，Fmoc)，7.71(d，2H，J＝7.5Hz，Fmoc)，7.50(dd，2H，J＝7.5Hz，Fmoc)，7.43(dd，2H，J＝7.5Hz，Fmoc)，5.12(s，1H，Man4-H-1)，4.99(d，1H，J＝9.5Hz，GlcNAc1-H-1)，4.77(s，1H，Man?3-H-1)，4.57(d，2H，J＝7.2Hz，GlcNAc2-H-1)，4.46(d，1H，J＝7.8Hz，Gal6-H-1)，4.34(t，1H，Fmoc)，4.22(bd，1H，J＝2.7Hz，Man3-H-2)，4.19(b，1H，Man4-H-2)，2.72(bdd，1H，J＝15.5Hz，AsN-βCH)，2.52(bdd，1H，J＝9.8Hz，15.5Hz，AsN-βCH)，2.05(s，6H，Ac×2)，1.89(s，3H，Ac)；MS(Fab)，Calcd?for?C
61H
88N
5O
35[M+H+]1450.5，found，1450.3
Reference example 14 (5-ethanamide-3,5,7-three deoxidations-7-fluoro-D-glyceryl-β-D-galactosyl-2-nine pyranose thuja acids (7-fluorine sialic acid)
5-acetamide-3,5,8-trideoxy-8-fluoro-D-glycero-β-D-galacto-2-nonulopyranosidonic acid's 25 is synthetic)
(1) compound 17 is synthetic
With sodium acetate (5g 69mmol) is dissolved in the diacetyl oxide (60mL), add bit by bit after the heating D-semi-lactosi (G) (10g, 55mmol).(toluene: ethyl acetate=5: 1) the affirmation reaction finishes reflux by TLC after 2 hours.After reaction soln returned to room temperature, be injected among the frozen water 300cc.Filter back collecting precipitation thing.Throw out is dissolved in ethanol (14mL) carries out recrystallization, obtain 9.0g compound 17 (yield 41%).
(2) compound 18 is synthetic
With compound 17 (4.3g, 11mmol) be dissolved in methylene dichloride (120mL) after, under argon gas stream, be cooled to-20 ℃.Then, (3.1g 12mmol), stirs after 20 minutes, and (2.3g 22mmol), returns to room temperature with temperature of reaction to add benzylalcohol to add tin tetrachloride in reaction soln.With TLC (hexane: after ethyl acetate=1: 1) the affirmation reaction finishes, reaction soln is injected in the saturated sodium bicarbonate aqueous solution, extracts with methylene dichloride.Dichloromethane layer filters, concentrating under reduced pressure behind anhydrous magnesium sulfate drying.Residue with after the moisture eliminator drying, is dissolved in the methyl alcohol (80mL) after the distillation, and (431mg 5.5mmol), stirs under the argon gas stream to add sodium methylate.With TLC (ethyl acetate: methyl alcohol: after water=10: 5: 1) the affirmation reaction finishes, neutralize, reaction is finished with Zeo-karb IR-120 (+).After resin filter removed, filtrate is carried out concentrating under reduced pressure.After the residue drying device drying, be dissolved in pyridine (44mL), reaction soln is cooled to 0 ℃.In reaction soln, add trimethyl-acetyl chloride (4.6g, 38.5mmol) after, return to room temperature, under argon gas stream, stirred 1 hour.With TLC (hexane: ethyl acetate=2: 1) confirm that reaction finishes after, be cooled to 0 ℃ after, add methyl alcohol and make reaction terminating.After reaction soln directly carried out concentrating under reduced pressure, residue is dissolved in ethyl acetate, cleans, make the ethyl acetate drying with anhydrous magnesium sulfate with the saturated common salt aqueous solution, water.After sal epsom removed by filter, filtrate is carried out concentrating under reduced pressure.Residue is by silica gel column chromatography (eluting solvent hexane: ethyl acetate=2: 1) make with extra care, obtain compound 18 (2.8g, yield 58%).
(3) compound 19 is synthetic
With compound 18 (200mg 0.455mmol) is dissolved in methylene dichloride (7.8mL) and the pyridine (1.3mL), add sym-dichloroacetic anhydride (155mg, 0.91mmol), in reacting while stirring 15 minutes under the argon gas stream, under-15 ℃.After confirming that reaction finishes, with the sym-dichloroacetic anhydride quenching, the limit is carried out azeotropic 3 times with toluene with methyl alcohol (5mL), and concentrating under reduced pressure is carried out on the limit.With ethyl acetate residue is extracted, clean with saturated aqueous common salt.Make the organic layer drying with anhydrous magnesium sulfate, concentrate after the filtration.Residue is by silica gel column chromatography (ethyl acetate: hexane=1: 4) make with extra care, obtain compound 19 (receipts amount 172mg, yield 73.5%).
1H-NMR(400MHz，CDCl
3)
δ7.37-7.29(m，5H，Ph)，5.39(dd，1H，J
1，2＝8.0Hz，J
2，3＝10.4Hz，H-2)，4.89(dd，1H，J
3，4＝3.4Hz，H-3)，4.89，4.62(2d，2H，J＝12.5Hz，OCH
2Ph)，4.53(d，1H，H-1)，4.37(dd，1H，J
6a，6b＝11.5Hz，J
6a，5＝6.0Hz，H-6a)，4.32(dd，1H，J
6b，5＝6.6Hz，H-6b)，4.00(m，1H，H-4)，3.92(s，2H，COCH
2Cl)，3.75(dd，1H，H-5)，1.23，1.19〔2s，18H，COC(CH
3)
3〕
13C-NMR(400MHz，CDCl
3)
δ178.33，177.57，165.92，(C＝O)，136.66，128.48，128.07，127.89(Ph)，99.16(C-1)，72.82(C-3)，72.35(C-5)，70.92(C-2)，70.49(OCH
2Ph)，67.29(C-4)，62.30(C-6)，40.40(OCCH
2Cl)，38.95，38.80〔COC(CH
3)
3〕，27.14，26.98〔COC(CH
3)
3〕
1H-NMR,
13C-NMR measures by the AVANCE 400 (being expressed as 400MHz) of Bruker.When solvent is attached most importance to chloroform, use trimethyl silane as internal standard.When using other heavy solvent with solvent peak as benchmark.Chemical shift uses J (Hz) to represent with δ (ppm) expression, binding constant.Silica gel column chromatography uses Merck Silicage160,70-230 order or 230-400 order, the Silica Gel 60 (Spherical) that spherical silica-gel uses Northeast chemical company to produce, use DC-Platten kieselgel 60 F254 (Artl, 05715) of E.Merk company production with (following TLC) as reaction detection.The COSMOSIL 5C that high performance liquid chromatography (HPLC) post uses Na カ ラ ィ テ ス Network company to produce
18(φ 4.6 * 150mm), the FP-210 Spectrofluorometer that spectrofluorophotometer uses JASCO company to produce for-ARPaeked Column.
(4) compound 20 is synthetic
(300mg 0.583mmol) is dissolved in the methylene dichloride (5.8mL), under the argon gas stream, under-15 ℃, adds diethylamino sulfanilamide (SN) trifluoride (diethylaminosulfatrifluoride) while stirring (DAST) with compound 19.Add DAST after 10 minutes, return to room temperature, reacted 1 hour.Confirm that with TLC raw material disappears, with methyl alcohol (3mL) with the DAST quenching after, carry out concentrating under reduced pressure.(ethyl acetate: purifying hexane=1: 6) obtains compound 20 (receipts amount 211mg, yield 70%) to residue by silica gel column chromatography.
1H-NMR(400MHz，CDCl
3)
δ7.37-7.27(m，5H，Ph)，5.31(ddd，1H，J
3，F＝14.3Hz，J
3，4＝9.69Hz，J
2，3＝9.63Hz，H-3)，5.04(dd，1H，J
1，2＝7.93Hz，H-2)，4.86(d，1H，J＝12.2Hz，OCH
2Ph)，4.60(d，1H，H-1)，4.59(d，1H，OCH
2Ph)，4.44(ddd，1H，J
4，5＝9.04Hz，J
4，F＝50.6Hz，H-4)，4.43(ddd，1H，J
6a，6b＝12.1Hz，J
6a，5＝2.41Hz，J
6a，F＝2.23Hz，H-6a)，4.24(ddd，1H，J
6b，5＝5.67Hz，J
6b，F＝1.28Hz，H-6b)，3.93(s，2H，OCOCH
2Cl)，3.75(m，1H，H-5)，1.25，1.18〔2s，18H，OCOC(CH
3)
3〕
13C-NMR(400MHz，CDCl
3)
δ177.94，117.43，165.88(C＝O)，136.34，128.55，138.23，127.92(Ph)，98.68(C-1)，87.35(d，J
4，F＝188.62Hz，C-4)，72.65(d，J
2，F＝7.96Hz，C-2)，72.05(d，J
3，F＝20.02Hz，C-3)，71.49(d，J
5，F＝23.09Hz，C-5)，70.80(OCH
2Ph)，62.12(C-6)，40.30(OCOCH
2Cl)，38.87〔OCOC(CH
3)
3〕，27.17，26.92〔OCOC(CH
3)
3〕
(5) compound 21 is synthetic
With compound 20 (625mg 1.21mmol) is dissolved in methyl alcohol (24.2mL), under argon gas stream ,-15 ℃, add while stirring sodium methylate (13.1mg, 0.6mmol).After 30 minutes, after the disappearance of TLC affirmation raw material, neutralize (pH6-7), will carry out concentrating under reduced pressure behind the resin filter with Zeo-karb IR-120 (+).Residue is with silica gel column chromatography (ethyl acetate: hexane=1: 4) carry out purifying, obtain compound 21 (receipts amount 395mg, yield 74%).
1H-NMR(400MHz，CDCl
3)
δ7.38-7.29(m，5H，Ph)，5.18(ddd，1H，J
3，F＝14.8Hz，J
3，4＝9.51Hz，J
2，3＝8.99Hz，H-3)，4.90(d，1H，J＝11.7，OCH
2Ph)，4.63(d，1H，OCH
2Ph)，4.47(ddd，1H，J
5，6a＝2.43Hz，J
6a，F＝2.2Hz，H-6a)，4.47(d，1H，J
1，2＝7.7Hz，H-1)，4.38(ddd，1H，J
4，5＝8.96Hz，J
3，4＝9.67Hz，J
4，F＝50.8Hz，H-4)，4.23(ddd，1H，J
6a，6b＝12.0Hz，J
6b，5＝6.05Hz，J
6b，F＝1.26Hz，H-6b)，3.75(m，1H，H-5)，3.54(m，1H，J
2，OH＝2.70Hz，H-2)，1.27，1.26〔2s，18H，OCOC(CH
3)
3〕
13C-NMR(400MHz，CDCl
3)
δ178.17，177.94(C＝O)，136.54，128.54，128.17，128.12(Ph)，101.31(C-1)，87.45(d，J
4，F＝187.39Hz，C-4)，74.17(d，J
3，F＝18.88Hz，C-3)，72.45(d，J
2，F＝7.56Hz，C-2)，71.45(d，J
5，F＝23.26Hz，C-5)，71.09(OCH
2Ph)，62.44(C-6)，38.90，38.85〔OCOC(CH
3)
3〕，27.14，26.99〔OCOC(CH
3)
3〕
(6) compound 22 is synthetic
(22.2 μ L, (46 μ L 0.274mmol), after 15 minutes, are dissolved with the solution of the methylene dichloride (1mL) of compound 21 in 0 ℃ of dropping to drip trifluoromethanesulfanhydride anhydride in methylene dichloride 0.274mmol) (the 370 μ L) solution to being dissolved with pyridine under 0 ℃.Confirm that with TLC raw material disappears, and dilutes reaction mixture with methylene dichloride.Clean organic layer with saturated sodium bicarbonate water, saturated aqueous common salt, water, carry out concentrating after the drying with anhydrous magnesium sulfate.With residue with the further drying of vacuum pump after, be dissolved in benzene (1mL), under argon gas stream, at room temperature add sodiumazide (13mg, 0.206mmol), (57mg 0.206mmol), makes its reaction to four ammonium chlorides under 40 ℃.With after the disappearance of TLC affirmation raw material, carry out concentrating under reduced pressure after 2 hours.With ethyl acetate residue is extracted, after cleaning with saturated aqueous common salt, water, behind anhydrous magnesium sulfate drying, concentrate.(ethyl acetate: purifying hexane=1: 4) obtains compound 22 (receipts amount 30.4mg, yield 95%) to residue by silica gel column chromatography.
1H-NMR(400MHz，CDCl
3)
δ7.39-7.32(m，5H，Ph)，4.99(ddd，1H，J
3，F＝13.18Hz，J
3，4＝9.27Hz，J
2，3＝3.87Hz，H-3)，4.93(d，1H，J＝12.07Hz，OCH
2Ph)，4.67(d，1H，J
1，2＝1.18Hz，H-1)，4.63(d，1H，OCH
2Ph)，4.51(ddd，1H，J
6a，6b＝11.95Hz，J
6a，5＝2.54Hz，J
6a，F＝2.08Hz，H-6a)，4.23(ddd，1H，J
6b，5＝6.14Hz，J
6b，F＝1.14Hz，H-6b)，4.08(m，1H，H-2)，3.64(m，1H，H-5)，1.26〔2s，18H，OCOC(CH
3)
3〕
13C-NMR(400MHz，CDCl
3)
δ178.01，177.68(C＝O)，136.06，128.63，128.31，128.14(Ph)，97.25(C-1)，85.51(d，J
4，F＝183.97，C-4)，72.01(d，J
5，F＝23.89，C-5)，71.73(d，J
3，F＝18.98，C-3)70.57(OCH
2Ph)，62.42(C-2，C-6)，39.08，38.90〔OCOC(CH
3)
3〕，27.18，26.95〔OCOC(CH
3)
3〕
(7) compound 23 is synthetic
(180mg 0.387mmol) is dissolved in methyl alcohol (8mL), and (922mg 9.67mmol) also stirs, and makes its reaction under 40 ℃ to add sodium methylate with compound 22.4.5 confirm to concentrate on 1 point with TLC after hour, after Zeo-karb IR-120 (+) neutralization, filter, concentrate.(ethyl acetate: purifying hexane=1: 1) obtains compound 23 (receipts amount 105.3mg, yield 91.6%) to residue through silica gel column chromatography.
1H-NMR(400MHz，CDCl
3)，
δ7.40-7.31(m，5H，Ph)，4.96(d，1H，J＝12.13Hz，OCH
2Ph)，4.71(d，1H，J
1，2＝1.33Hz，H-1)，4.69(d，1H，OCH
2Ph)，4.49(ddd，1H，J
4，F＝51.06Hz，J
4，5＝9.19Hz，J
3，4＝9.20Hz，H-4)，4.02(m，1H，H-2)，3.93(dddd，1H，J
6a，6b＝12.19Hz，J
6a，5＝2.31Hz，J
6a，F＝2.32Hz，J
6a，OH＝6.20Hz，H-6a)，3.89-3.77(m，2H，H-3，H-6b)，3.39(m，1H，H-5)，
13C-NMR(400MHz，CDCl
3)，
δ136.39，128.62，128.24，127.83(Ph)，98.63(C-1)，88.19(d，J
4，F＝178.91Hz，C-4)，73.95(d，J
5，F＝25.48Hz，C-5)，71.18(OCH
2Ph)，71.16(d，J
3，F＝19.69Hz，C-3)，64.48(d，J
2，F＝8.42Hz，C-2)，61.39(C-6)
(8) compound 24 is synthetic
With compound 23 (105mg 0.353mmol) is dissolved in methyl alcohol (7mL), add diacetyl oxide (333 μ L, 3.53mol) back, under argon gas stream, add 10% Pd/C of catalytic amount, carry out the hydrogen displacement after, under room temperature, stir.Confirm that with TLC raw material disappears, and concentrates behind the activated carbon filtration after 2 hours.(ethyl acetate: purifying methyl alcohol=5: 1) obtains compound 24 (receipts amount 57mg, yield 72%) to residue through silica gel column chromatography.
1H-NMR(400MHz，D
2O)，
δ5.23(dd，1H，J
1，2＝2.69Hz，J
1，F＝1.44Hz，H-1-α)，4.65(ddd，1H，J
4，F＝50.94Hz，J
3，4＝9.06Hz，J
4，5＝9.58Hz，H-4-α)，4.47(m，1H，H-2-α)，4.43(ddd，1H，J
3，F＝14.28Hz，J
2，3＝4.9Hz，H-3-α)，4.16(m，1H，H-5-α)，3.95(m，2H，H-6a-α，H-6b-α)，2.14(s，3H，NHCOCH
3-α)
13C-NMR(400MHz，D
2O)，
δ175.27(C＝O-α)，93.46(C-1-α)，88.30(d，J
4，F＝177.00Hz，C-4-α)，69.91(d，J
5，F＝24.41Hz，C-5-α)，67.60(d，J
3，F＝18.74Hz，C-3-α)，60.36(C-6)，54.12(d，J
2，F＝8.68Hz，C-2-α)，22.31(NHCOCH
3-α)
(9) compound 25 is synthetic
With compound 24 (50mg, 0.224mmol), Sodium.alpha.-ketopropionate (123mg, 1.12mmol) and bovine serum albumin(BSA) (5mg) be dissolved in sodium phosphate buffer (100mM, pH7.5 3.4mL), add acetylneuraminate aldolase (50U) then, under room temperature, begin the reaction.Make the reaction soln lyophilize after 24 hours, be dissolved in the less water, be added in the anion-exchange resin column (AG1-X8,200-400 order, formate form).After water 300mL is flow through, with the object wash-out, carry out concentrating under reduced pressure,, obtain compound 25 (receipts amount 40mg, yield 58.9%) with gel-filtration column (Sephadex G-15, water) purifying with 1M formic acid.
1H-NMR(400MHz，D
2O)，
δ4.61(dd，1H，J
7，8＝8.97Hz，J
7，F＝45.56Hz，H-7)，4.18(dd，1H，J
5，6＝10.63Hz，J
6，F＝29.86Hz，H-6)，4.15(m，1H，H-4)，4.07(m，1H，H-8)，4.02(dd，1H，J
4，5＝10.10Hz，H-5)，3.90(ddd，1H，J
9a9b＝12.18Hz，J
9a，8＝2.77Hz，J
9a，F＝2.86Hz，H-9a)，3.76(ddd，1H，J
9b，8＝5.33Hz，J
9b，F＝2.06Hz，H-9b)，2.40(dd，1H，J
3eq，3ax＝13.00，J
3eq，4＝4.88Hz，H-3eq)，2.15(s，3H，OCOCH
3)，2.00(dd，1H，J
3ax，4＝11.70Hz，H-3ax)，
13C-NMR(400MHz，D
2O)，
δ175.17，173.68(C＝O)，96.01(C-1)，89.12(d，J
7，F＝179.23Hz，C-7)，69.67(d，J
6，F＝17.41Hz，C-6)，68.31(d，J
8，F＝26.50Hz，C-8)，67.26(C-4)，62.70(C-6)，52.17(C-5)，39.19(C-3)，22.61(NHCOCH
3)，
Reference example 15 (5-ethanamide-3,5,8-three deoxidations-8-fluoro-D-glyceryl-β-D-galactosyl-2-nine pyranose thuja acids (8-fluorine sialic acid)
5-acetamide-3,5,8-trideoxy-8-fluoro-D-glycero-β-D-galacto-2-nonulopyranosidonic acid's 27 is synthetic)
Synthesize 5-ethanamide-3,5,8-three deoxidations-8-fluoro-D-glyceryl-β-D-galactosyl-2-nine pyranose thuja acids (27) according to following proposal by sialic acid (26).
The sialic NMR data of 8-fluorine have below been provided.
1H-NMR(400MHz，D
2O)，
δ4.69(dddd，1H，J
8，F＝48.7Hz，J
8，9a＝5.0Hz，J
8，9b＝3.5Hz，H-8)，4.03(ddd，1H，J
4，5＝10.0Hz，J
3ax，4＝11.1Hz，J
3eq，4＝4.7Hz，H-4)，3.95(dd，1H，J
4，5＝10.0Hz，J
5，6＝9.9Hz，H-5)，3.94(ddd，1H，J
6，7＝～0Hz，J
7，8＝6.8Hz，J
7，F＝14.0Hz，H-7)，3.88(ddd，1H，J
9a9b＝13.3Hz，J
9a，8＝3.5Hz，J
9b，F＝28.0Hz，H-9b)，3.86(dd，1H，J
5，6＝9.9Hz，J
6，7＝～0Hz，H-6)，3.72(ddd，1H，J
9a，9b＝5.33H?，J
9a，8＝5.0Hz，J
9a，F＝30.6Hz，H-9a)，2.28(dd，1H，J
3eq， 3ax＝13.00，J
3eq，4＝4.6Hz，H-3eq)，2.05(s，3H，Ac)，1.87(dd，1H，J
3ax，4＝11.1Hz，J
3eq，3ax＝13.00，H-3ax)
Reference example 16 (5-ethanamide-3,5,9-three deoxidations-9-fluoro-D-glyceryl-β-D-galactosyl-2-nine pyranose thuja acids (9-fluorine sialic acid)
5-acetamide-3,5,9-trideoxy-9-fluoro-D-glycero-β-D-galacto-2-nonulopyranosidonic acid's 28 is synthetic)
According to following proposal, by the synthetic 5-ethanamide-3,5 of sialic acid (26), 9-three deoxidations-9-fluoro-D-glyceryl-β-D-galactosyl-2-nine pyranose thuja acids (28).
Reference example 17CMP-is sialic synthetic
(a)(1)Dowex?50-X8，MeOH，(2)Ac
2O，60％HClO
4；
(b)(1)1H-Tetrazole，CH
3CN，(2)t-BuOOH，CH
3CN，(3)DBU，CH
3CN，(4)NaOMe，MeOH，H
2O
Sialic acid (0.074mmol) is dissolved in the distillating carbinol (3mL),, reacted 3 hours in adding Dowex-50W-X8 (65mg) under the argon gas stream, under the room temperature while stirring.After confirming that reaction finishes, filter the back concentrating under reduced pressure.Residue is dissolved in diacetyl oxide (200 μ L), under-20 ℃, adds diacetyl oxide while stirring: 60% perchloric acid=15: 1 solution (22 μ L), reacted 40 minutes down in 10 ℃.After confirming that reaction finishes, reaction soln is diluted with ethyl acetate, clean with saturated sodium bicarbonate water.Use the anhydrous magnesium sulfate drying organic layer, filter the back concentrating under reduced pressure, obtain containing the residue of the protected sialic acid of carboxyl (29).Residue and CMP-5 '-phosphoramidic acid ester derivative (30) (0.23mmol) are carried out azeotropic respectively 3 times with benzene, be dissolved in distillatory acetonitrile (100 μ L) respectively, mix.Under argon gas stream, in the frozen water, add while stirring the 1H-tetrazolium (17mg, 0.23mmol).Return to room temperature after 5 minutes, answered again 10 minutes.After confirming that reaction finishes, solution dilutes with ethyl acetate, cleans with saturated sodium bicarbonate water, saturated aqueous common salt.Make the organic layer drying with anhydrous magnesium sulfate, filter the back after concentrating below 30 ℃, carry out azeotropic 2 times with toluene again, remove and anhydrate.Add distilled acetonitrile (400 μ L) to residue, under argon gas stream, on one side ice-cold one side splash into the t-BuOOH toluene solution (290 μ L) of 2.5M.Return to room temperature after 5 minutes, restir 20 minutes.After confirming that reaction finishes, drip methyl-sulfide (53 μ L), stirred 10 minutes, t-BuOOH is carried out quenching.The DBU (18 μ L) that drips then stirred 20 minutes under room temperature.Confirm that reaction finishes the back, adds methyl alcohol (0.67mL), water (1.35mL), sodium methylate (360mg), in room temperature reaction 16 hours.After confirming that reaction finishes, use water extraction, clean with methylene dichloride.In below 25 ℃, water layer is evaporated to about 8mL.(gel column chromatography (eluting solvent: 20mM ammoniacal liquor, flow velocity: the 0.3mL/ minute) purifying of 1.8 φ * 90cm) obtains cmp sialic acid to this aqueous solution by using Sephadex G-15.
Reference example 18CMP-7 "-deoxidation-7 "-fluoro-is sialic synthetic
Except using compound (25) to replace the sialic acid, other are same with reference example 7, synthesize CMP-7 "-deoxidation-7 "-the fluoro-sialic acid.The NMR data have below been provided.
1H-NMR(400MHz，50mM?ND
4DCO
3?in?D
2O)，
δ8.04(d，1H，J
5，6＝7.6Hz，H-6)，6.20(d，1H，J
6，5＝7.6Hz，H-5)，6.06(d，1H，J
1’，2’＝4.5Hz，H-1’)，4.54(dd，1H，J
7”，8”＝9.5Hz，J
7”，F＝45.9Hz，H-7”)，4.42～4.20(m，7H，H-2’，H-3’，H-4’，H-5’a，H-5’b，H-6”，H-8”)，4.16(ddd，1H，J
4”，3”eq＝4.7Hz，J
4”，3”ax＝11.3Hz，J
4，5＝10.3Hz，H-4”)，4.03(dd，1H，J
5”，4”＝J
5”，6”＝10.3Hz，H-5”)，3.91(ddd，1H，J
9”a，9”b＝12.2Hz，J
9”a，8”＝2.8Hz，J
9”a，F＝2.8Hz，H-9”a)，3.75(ddd，1H，J
9”a，9”b＝12.2Hz，J
9”b，8”＝5.4Hz，J
9”b，F＝2.1Hz，H-9”b)，2.61(dd，1H，J
3”eq，4”＝4.7Hz，J
gem＝13.3Hz，H-3”eq)，2.14(s，3H，Ac)，1.76(ddd，1H，J
3”ax，4”＝11.5Hz，J
gem＝13.3Hz，J
3”ax，P＝5.6Hz，H-3”ax)，
Reference example 19CMP-8 "-deoxidation-8 "-fluoro-is sialic synthetic
Except using compound (27) to replace the sialic acid, other are same with reference example 7, synthesize CMP-8 "-deoxidation-8 "-the fluoro-sialic acid.The NMR data have below been provided.
1H-NMR(400MHz，50mM?ND
4DCO
3?in?D
2O)，
δ8.08(d，1H，J
5，6＝7.6Hz，H-6)，6.20(d，1H，J
6，5＝7.6Hz，H-5)，6.09(d，1H，J
1’，2’＝4.1Hz，H-1’)，4.90(m，1H，H-8”)，4.42(dd，1H，J
3’，2’＝J
?3’，4’＝4.9Hz，H-3’)，4.39(dd，1H，J
2’，1’＝4.1Hz，J，
2’，3’＝4.9Hz，H-2’)，4.31-4.28(m，3H，H-4’，H-5’a，H-5’b)，4.15(ddd，1H，J?
4”，3”eq＝4.4Hz，J
4”，3”ax＝11.5Hz，J
4，5＝10，5Hz，H-4”)，4.10-3.90(m，5H，H-5”，H-6”，H-7”，H-9”a，H-9”b)，2.60(dd，1H，J
3”eq，4”＝4.4Hz，J
gem＝13.1Hz，H-3”eq)，2.13(s，3H，Ac)，1.77(ddd，1H，J
3”ax，4”＝11.5Hz，J
gem＝13.1Hz，J
3”ax，P＝4.5Hz，H-3”ax)，
Reference example 20CMP-9 "-deoxidation-9 "-fluoro-is sialic synthetic
Except using compound (28) to replace the sialic acid, other are same with reference example 7, synthesize CMP-9 "-deoxidation-9 "-the fluoro-sialic acid.
The amino nitrogen of embodiment 1 l-asparagine is by the amino nitrogen of bifunctional sialyltransferase base α 2,3 sugar chain asparagines (C1-1) of Fmoc base protection and l-asparagine synthesizing by 2 kinds of single sialic acid base α 2,3 sugar chain asparagines (C1-2 and C1-3) of Fmoc base protection
Use sialytransferase that cmp sialic acid is transferred to the amino nitrogen of the l-asparagine that reference example 3 obtains by the asialoglycoprotein base sugar chain asparagine of Fmoc base protection.
As α 2,3 transferring enzymes of sialytransferase use from commercially available Rat, Recombinant.
Asialoglycoprotein base 9 sugar (20mg, 10.1 μ mol) that reference example 3 is obtained be dissolved in 50mM cacodylic acid damping fluid (pH=6.0,5mL) after, add bovine serum albumin(BSA) (BSA, 5mg).(5 μ L 125unit), and carry out all-change to add cmp sialic acid (26mg, 40.4 μ mol), alkaline phosphatase then.Add α 2 at last, 3-sialytransferase (CALBIOCHEM company produces, 100 μ L) left standstill under 37 ℃ 48 hours.By the HPLC monitoring reaction, reduce to the purpose amount at raw material and make reaction terminating constantly, reaction solution filters with membrane filter.Filtrate is concentrated, after solution amount is reduced, by HPLC fractionation column (YMC-PackR﹠amp; D ODS, D-ODS-5-A, 20 * 250mm, AN/25mM ammonium acetate buffer=18/82,7.5mL/min., wavelength: 274nm) carry out purifying, after 25 minutes, bifunctional sialyltransferase base 11 sugar compounds (C1-1) elute, each single sialic acid base 10 sugar compounds (C1-2) and (C1-3) eluted respectively after 30 minutes, after 34 minutes.After collecting respectively, carry out desalting treatment, carry out lyophilize then, obtain compound 1,2,3 each 0.7mg (2.7%), 1.9mg (8.3%), 3.5mg (15.3%) respectively.The NMR data of each compound are as follows.
Compound (C1-1)
1H?NMR(400MHz，D
2O，30℃，HOD＝4.81)
δ7.90(d，2H，Fmoc)，7.69(d，2H，Fmoc)，7.49(dd，2H，Fmoc)，7.42(dd，2H，Fmoc)，5.10(s，1H，Man4-H1)，4.97(d，1H，GlcNAc1-H1)，4.91(s，1H，Man4’-H-1)，4.50-4.60(m，4H)，4.34(1H，Fmoc)，4.24(bs，1H，Man3-H2)，4.18(bs，1H，Man?4-H2)，4.10(m，2H)，2.74(m，3H，Asn-βCH，NeuAc7，7’-H3eq)，2.40-2.60(m，1H，Asn-βCH)，2.05，2.03，2.02(eachs，Ac)，1.77(dd，2H，NeuAc7，7’-H3ax).
Compound (C1-2)
1H?NMR(400MHz，D
2O，30℃，HOD＝4.81)
δ7.90(d，2H，Fmoc)，7.69(d，2H，Fmoc)，7.49(dd，2H，Fmoc)，7.42(dd，2H，Fmoc)，5.10(s，1H，Man4-H1)，4.97(d，1H，GlcNAc1-H1)，4.90(s，1H，Man?4’-H-1)，4.47-4.60(m)，4.43(d，1H)，4.32(1H，Fmoc)，4.22(bs，2H)，4.17(bs，1H，Man4-H2)，4.06-4.13(m，2H)，2.72(m，2H，Asn-βCH，NeuAc7-H3eq)，2.50-2.60(m，1H，Asn-βCH)，2.05，2.03，2.01(eachs，Ac)，1.77(dd，1H，NeuAc7-H3ax).
Compound (C1-3)
1H?NMR(400MHz，D
2O，30℃，HOD＝4.81)
δ7.90(d，2H，Fmoc)，7.69(d，2H，Fmoc)，7.49(dd，2H，Fmoc)，7.42(dd，2H，Fmoc)，5.10(s，1H，Man4-H1)，4.97(d，1H，GlcNAc1-H1)，4.90(s，1H，Man4’-H-1)，4.50-4.60(m)，4.45(d，1H)，4.33(1H，Fmoc)，4.22(m，2H)，4.17(bs，1H，Man4-H2)，4.09(m，2H)，2.74(m，2H，Asn-βCH，NeuAc7-H3eq)，2.45-2.60(m，1H，Asn-βCH)，2.05，2.03，2.02，2.00(eachs，Ac)，1.77(dd，1H，NeuAc7-H3ax)
Embodiment 2
HEPES damping fluid (the 50mM that compound (C1-2) (2mg, 0.88 μ mol) that embodiment 1 is obtained and bovine serum albumin(BSA) 1mg are dissolved in 100 μ L, pH5.0) in, (biochemical industrial produces, from Jack Beans to add beta-galactosidase enzymes again, 5 μ L, 100mU).This solution after leaving standstill 15 hours under 37 ℃, is filtered with membrane filter.With filtrate by HPLC (ODS post, 2.0 φ * 25cm, eluting solvent are the 50mM ammonium acetate aqueous solution: acetonitrile=82: 18, flow velocity are 7.5mL/min) purifying after, solvent is concentrated, carry out lyophilize then.Residue is dissolved in the 200 μ L water, by ODS-column chromatography (コ ス モ シ-Le 75C
18-OPN, initial water clean, and carry out wash-out with 25% acetonitrile solution then) carry out desalting treatment, obtain purpose compound (C2) 0.5 μ g.The NMR data are as follows.
1H?NMR(400MHz，D
2O，30℃，HOD＝4.81)
δ7.90(d，2H，Fmoc)，7.69(d，2H，Fmoc)，7.49(dd，2H，Fmoc)，7.42(dd，2H，Fmoc)，5.10(s，1H，Man4-H1)，4.98(d，1H，GlcNAc1-H1)，4.90(s，1H，Man4’-H-1)，4.50-4.60(m)，4.33(1H，Fmoc)，4.22(m，2H)，4.17(bs，1H，Man4-H2)，4.10(m，2H)，2.74(m，2H，Asn-βCH，NeuAc7-H3eq)，2.45-2.60(m，1H，Asn-βCH)，2.05，2.03，2.01(each?s，Ac)，1.78(dd，1H，NeuAc7-H3ax)
Embodiment 3
Compound (C2) (1.8mg, 0.86 μ mol) and bovine serum albumin(BSA) 1mg-that embodiment 2 is obtained work the HEPES damping fluid (50mM that is dissolved in 90 μ L; pH5.0) in; add 4 μ L (250mU) N-ethanoyl-β-glycosamine enzymes (SigmaAldrich company produces, from Jack Beans) again.This solution after leaving standstill 24 hours under 37 ℃, is filtered with membrane filter.To filter night by HPLC (ODS post, 2.0 φ * 25cm, eluting solvent are the 50mM ammonium acetate aqueous solution: acetonitrile=82: 18, flow velocity are 7.5mL/min) purifying after, solvent is concentrated, carry out lyophilize then.Residue is dissolved in the 200 μ L water, by ODS-column chromatography (コ ス モ シ-Le 75C
18-OPN, initial water clean, and carry out wash-out with 25% acetonitrile solution then) carry out desalting treatment, obtain purpose compound (C3) 0.9 μ g.
1H?NMR(400MHz，D
2O，30℃，HOD＝4.81)
δ8.01(d，2H，J＝7.6，Fmoc)，7.80(d，2H，J＝7.6，Fmoc)，7.60(dd，2H，J＝7.6，Fmoc)，7.53(dd，2H，J＝7.6，Fmoc)，5.21(s，1H，Man4-H1)，5.09(d，1H，J＝8.8，GlcNAc1-H1)，5.00(s，1H，Man4’-H-1)，4.87(s，1H)，4.60-4.78(m，5H)，4.40-4.50(bm，2H)，4.34(s，1H)，4.28(bs，1H，Man4-H2)，4.20(dd，1H，Ja＝3.0，Jb＝9.9)，2.80-2.95(m，2H，Asn-βCH，NeuAc7-H3eq)，2.65-2.75(m，1H，Asn-βCH)，2.16，2.14，2.12(eachs，Acx3)，1.98(s，3H，Ac)，1.89(dd，1H，Ja＝12.1，Jb＝11.9，NeuAc7-H3ax).
Embodiment 4
Compound (C3) (0.8mg, 0.42 μ mol) and bovine serum albumin(BSA) 1mg-that embodiment 3 is obtained work the HEPES damping fluid (50mM that is dissolved in 50 μ L, pH5.0) in, add α-seminase (SigmaAldrich company produces, from Jack Beans) 30 μ L (2.9U) again.This solution after leaving standstill 63 hours under 37 ℃, is filtered with membrane filter.To filter night by HPLC (ODS post, 2.0 φ * 25cm, eluting solvent are the 50mM ammonium acetate aqueous solution: acetonitrile=80: 20, flow velocity are 7.5mL/min) purifying after, solvent is concentrated, carry out lyophilize then.Residue is dissolved in the 200 μ L water, by ODS-column chromatography (コ ス モ シ-Le 75C
18-OPN, initial water clean, and carry out wash-out with 25% acetonitrile solution then) carry out desalting treatment, obtain purpose compound (C4) 0.6 μ g.
1H?NMR(400MHz，D
2O，30oC，HOD＝4.81)
δ8.00(d，2H，J＝7.2，Fmoc)，7.79(d，2H，J＝7.2，Fmoc)，7.59(dd，2H，J＝7.2，Fmoc)，7.52(dd，2H，J＝7.2，Fmoc)，5.21(s，1H，Man4-H1)，5.09(d，1H，J＝10.0，GlcNAc1-H1)，4.60-4.75(m，)，4.40-4.50(m，2H)，4.32(bd，1H，J＝2.3)，4.28(bs，1H)，4.22(bdd，1H，Ja＝9.7，Jb＝2.8，Man4-H2)，2.80-2.95(m，2H，Asn-βCH，NeuAc7-H3eq)，2.60-2.75(m，1H，Asn-βCH)，2.14，2.14，2.12(eachs，Acx3)，1.98(s，3H，Ac)，1.88(dd，1H，Ja＝12.1，Jb＝12.0，NeuAc7-H3ax).
Embodiment 5
HEPES damping fluid (the 50mM that compound (C1-3) (1mg, 0.44 μ mol) that embodiment 1 is obtained and bovine serum albumin(BSA) 1mg are dissolved in 50 μ L, pH5.0) in, (biochemical industrial produces, from Jack Beans to add beta-galactosidase enzymes again, 5 μ L, 100mU).This solution after leaving standstill 15 hours under 37 ℃, is filtered with membrane filter.To filter night by HPLC (ODS post, 2.0 φ * 25cm, eluting solvent are the 50mM ammonium acetate aqueous solution: acetonitrile=82: 18, flow velocity are 7.5mL/min) purifying after, solvent is concentrated, carry out lyophilize then.Residue is dissolved in the 200 μ L water, by ODS-column chromatography (コ ス モ シ-Le 75C
18-opn, initial water clean, and carry out wash-out with 25% acetonitrile solution then) carry out desalting treatment, obtain purpose compound (C5) 0.3 μ g.
1H?NMR(400MHz，D
2O，30℃，HOD＝4.81)
δ8.01(d，2H，J＝7.2，Fmoc)，7.81(d，2H，J＝7.2，Fmoc)，7.60(dd，2H，J＝7.2，Fmoc)，7.53(dd，2H，J＝7.2，Fmoc)，5.21(s，1H，Man4-H1)，5.09(d，1H，J＝9.6，GlcNAc1-H1)，5.02(s，1H，Man4’-H-1)，4.55-4.70(m)，4.44(1H，Fmoc)，4.30-4.38(bm，2H)，4.28(bd，1H，Man4-H2)，4.17-4.25(m，2H)，2.78-2.95(m，2H，Asn-βCH，NeuAc7-H3eq)，2.55-2.70(m，1H，Asn-βCH)，2.16，2.15，2.14，2.12(eachs，12H，Acx4)，1.98(s，3H，Ac)，1.89(dd，1H，Ja＝12.2，Jb＝12.0，NeuAc7-H3ax).
Embodiment 6
Compound (C5) (1.0mg, 0.48 μ mol) and bovine serum albumin(BSA) 1mg-that embodiment 5 is obtained work the HEPES damping fluid (50mM that is dissolved in 50 μ L; pH5.0) in; add 4 μ L (250mU) N-ethanoyl-β-glycosamine enzymes (SigmaAldrich company produces, from Jack Beans) again.This solution after leaving standstill 22 hours under 37 ℃, is filtered with membrane filter.To filter night by HPLC (ODS post, 2.0 φ * 25cm, eluting solvent are the 50mM ammonium acetate aqueous solution: acetonitrile=82: 18, flow velocity are 7.5mL/min) purifying after, solvent is concentrated, carry out lyophilize then.Residue is dissolved in the 200 μ L water, by ODS-column chromatography (コ ス モ シ-Le 75C
18-opn, initial water clean, and carry out wash-out with 25% acetonitrile solution then) carry out desalting treatment, obtain purpose compound (C6) 0.6 μ g.
1H?NMR(400MHz，D
2O，30℃，HOD＝4.81)
δ8.01(d，2H，J＝7.6，Fmoc)，7.80(d，2H，J＝7.6，Fmoc)，7.60(dd，2H，J＝7.6，Fmoc)，7.53(dd，2H，J＝7.6，Fmoc)，5.19(s，1H，Man4-H1)，5.09(d，1H，J＝9.2，GlcNAc1-H1)，5.02(s，1H，Man4’-H-1)，4.85(s，1H)4.58-4.75(m，5H)，4.38-4.48(m，2H，Fmoc)，4.40(bd，J＝2.4，1H)，4.18-4.25(m，2H)，4.15(m，1H)，2.80-2.95(m，2H，Asn-βCH，NeuAc7-H3eq)，2.65-2.75(m，1H，Asn-βCH)，2.16，2.13，2.12(eachs，9H，Acx3)，1.98(s，3H，Ac)，1.89(dd，1H，Ja＝12.2，Jb＝12.0，NeuAc7-H3ax).
Embodiment 7
HEPES damping fluid (the 50mM that compound (C6) (1.0mg, 0.53 μ mol) that embodiment 6 is obtained and bovine serum albumin(BSA) 1mg are dissolved in 50 μ L, pH5.0) in, add α-seminase (SigmaAldrich company produces, from Jack Beans) 10 μ L (0.9U) again.This solution after leaving standstill 20 hours under 37 ℃, is filtered with membrane filter.To filter night by HPLC (ODS post, 2.0 φ * 25cm, eluting solvent are the 50mM ammonium acetate aqueous solution: acetonitrile=80: 20, flow velocity are 7.5mL/min) purifying after, solvent is concentrated, carry out lyophilize then.Residue is dissolved in the 200 μ L water, by ODS-column chromatography (コ ス モ シ-Le 75C
18-opn, initial water clean, and carry out wash-out with 25% acetonitrile solution then) carry out desalting treatment, obtain purpose compound (C7) 0.5 μ g.
1H?NMR(400MHz，D
2O，30℃，HOD＝4.81)
δ8.01(d，2H，J＝7.6，Fmoc)，7.81(d，2H，J＝7.6，Fmoc)，7.60(dd，2H，J＝7.2，Fmoc)，7.53(dd，2H，J＝7.6，Fmoc)，5.D9(d，1H，J＝9.2，GlcNAc1-H1)，5.01(s，1H，Man4’-H-1)，4.84(s，1H)，4.55-4.70(m，5H)，4.44(t，1H，J＝6.0，Fmoc)，4.30-4.38(bs，1H)，4.15-4.25(m，2H)，4.17(s，1?H)，2.80-2.95(m，2H，Asn-βCH，NeuAc7-H3eq)，2.55-2.70(m，1H，Asn-βCH)，2.16，2.13，2.12(eachs，Acx3)，1.98(s，3H，Ac)1.89(dd，1H，Ja＝12.2，Jb＝12.3，NeuAc7-H3ax).
Embodiment 7A
The amino nitrogen of l-asparagine is synthetic by bifunctional sialyltransferase base (α 2,6) (α 2, the 3) sugar chain asparagine of Fmoc base protection
Use sialytransferase that cmp sialic acid is transferred to the amino nitrogen of the l-asparagine that reference example 3 obtains by the asialoglycoprotein sugar chain asparagine (compound 2) of Fmoc base protection.
Use from commercially available Rat α 2,3 transferring enzymes of Recombinant as sialytransferase.
After the compound 2 (1.7mg, 0.75 μ mol) that reference example 3 is obtained is dissolved in 50mM cacodylic acid damping fluid (pH=5.0,85 μ L), add bovine serum albumin(BSA) (BSA, 1mg).Add cmp sialic acid (4.8mg, 7.5 μ mol) then, (1 μ L 75unit), and carries out homogenization to alkaline phosphatase.Add α 2 at last, 3-sialytransferase (CALBIOCHEM company produces, 75 μ L, 34mU) left standstill under 37 ℃ 3.5 hours.By the HPLC monitoring reaction, the moment in that raw material disappears, make reaction terminating, reaction solution filters with membrane filter.Filtrate is concentrated, after solution amount is reduced, by HPLC fractionation column (YMC-Pack R﹠amp; D ODS, D-ODS-5-A, 20 * 250mm, AN/25mM ammonium acetate buffer=18/82,7.5mL/min., wavelength: 274nm) carry out purifying, after 25 minutes, compound (C7A) is eluted.After collecting respectively, carry out desalting treatment, carry out lyophilize then, can obtain compound (C7A) 1.3mg (67.8%).The NMR data of compound are as follows.
1H?NMR(400MHz，D
2O，30℃，HOD＝4.81)
δ8.00(d，2H，J＝7.2，Fmoc)，7.79(d，2H，J＝7.2，Fmoc)，7.60(dd，2H，J＝7.2，Fmoc)，7.52(dd，2H，J＝7.2，Fmoc)，5.21(s，1H，Man4-H1)，5.09(d，1H，J＝8.8，GlcNAc1-H1)，5.03(s，1H，Man4’-H-1)，4.86(s，1H)，4.58-4.72(m，5H)，4.54(d，1H，J＝8.0)，4.38-4.48(m，2H)4.34(bs，1H)，4.28(bs，1H)，4.15-4.25(m，2H)，2.80-2.86(dd，1H，Ja＝4.4，Jb＝12.4，NeuAc7-H3eq)，2.73-2.83(m，dd，3H，Ja＝4.4，Jb＝12.4，Asn-βCH，NeuAc7-H3eq)，2.60-2.72(m，1H，Asn-βCH)，2.16，2.15，2.14，2.12(each?s，Ac?x5)，1.98(s，3H，Ac)，1.89(dd，1H，Ja＝12.4，Jb＝12.0，NeuAc7-H3ax)，1.81(dd，1H，Ja＝12.4，Jb＝12.0，NeuAc7-H3ax).
Embodiment 7B
The amino nitrogen of l-asparagine is synthetic by bifunctional sialyltransferase base (α 2,3) (α 2, the 6) sugar chain asparagine of Fmoc base protection
Use sialytransferase that cmp sialic acid is transferred to the amino nitrogen of the l-asparagine that reference example 3 obtains by the asialoglycoprotein sugar chain asparagine (compound 3) of Fmoc base protection.
Use from commercially available Rat α 2,3 transferring enzymes of Recombinant as sialytransferase.
After the compound 3 (1.2mg, 0.53 μ mol) that reference example 3 is obtained is dissolved in 50mM cacodylic acid damping fluid (pH=5.0,60 μ L), add bovine serum albumin(BSA) (BSA, 1mg).Add cmp sialic acid (3.4mg, 5.3 μ mol) then, (1 μ L 75unit), and carries out homogenization to alkaline phosphatase.Add α 2 at last, (CALBIOCHEM company produces the 3-sialytransferase, and 52.9 μ L 24mU), left standstill under 37 ℃ 3 hours.By the HPLC monitoring reaction, the moment in that raw material all consumes, make reaction terminating, reaction solution filters with membrane filter.Filtrate is concentrated, after solution amount is reduced, by HPLC fractionation column (YMC-Pack R﹠amp; DODS, D-ODS-5-A, 20 * 250mm, AN/25mM ammonium acetate buffer=18/82,7.5mL/min., wavelength: 274nm) carry out purifying, after 23 minutes, compound (C7B) is eluted.After collecting respectively, carry out desalting treatment, carry out lyophilize then, can obtain compound (C7B) 1.1mg (81.2%).The NMR data of each compound are as follows.
1H?NMR(400MHz，D
2O，30℃，HOD＝4.81)
δ8.00(d，2H，J＝7.6，Fmoc)，7.79(d，2H，J＝7.6，Fmoc)，7.59(dd，2H，J＝7.6，Fmoc)，7.51(dd，2H，J＝7.6，Fmoc)，5.21(s，1H，Man4-H1)，5.08(d，1H，J＝10.0，GlcNAc1-H1)，5.00(s，1H，Man4’-H-1)，4.84(s，1H)，4.60-4.72(m，5H)，4.52(d，1H，J＝7.6)，4.35-4.45(m，2H)，4.33(bs，1H)，4.27(bs，1H)，4.15-4.25(m，2H)，2.80-2.86(dd，1H，Ja＝4.8，Jb＝12.4，NeuAc7-H3eq)，2.73-2.83(bs，dd，3H，Ja＝4.8，Jb＝12.4，Asn-βCH，NeuAc7-H3eq)，2.60-2.72(m，1H，Asn-βCH)，2.15，2.12，2.10(each?s，Ac?x5)，1.97(s，3H，Ac)，1.88(dd，1H，Ja＝12.4，Jb＝12.4，NeuAc7-H3ax)，1.80(dd，1H，Ja＝12.4，Jb＝12.4，NeuAc7-H3ax).
The amino nitrogen of embodiment 8 l-asparagines is by 7 of the protection of Fmoc base "-deoxidation-7 "-fluoro-saliva acidic group α 2; the amino nitrogen of 3-sugar chain asparagine (C8-1) and l-asparagine is by 2 kinds of lists 7 of Fmoc base protection "-deoxidation-71 "-fluoro-saliva acidic group α 2,3-sugar chain asparagine (C8-2 and C8-3) synthetic
Except the CMP-7 that uses reference example 8 to obtain "-deoxidation-7 "-the fluoro-sialic acid; other is operated similarly to Example 1; the amino nitrogen that obtains l-asparagine as follows is by 7 of the protection of Fmoc base "-deoxidation-7 "-fluoro-saliva acidic group α 2; the amino nitrogen of 3-sugar chain asparagine and l-asparagine is by 2 kinds of lists 7 of Fmoc base protection "-deoxidation-7 "-fluoro-saliva acidic group α 2,3 sugar chain asparagines.
Embodiment 9
Except the compound (C8-2) that uses embodiment 8 to obtain replaced the compound (C1-2) of embodiment 2, other was operated similarly to Example 2, obtains purpose compound (C9).
Embodiment 10
Except the compound (C9) that uses embodiment 9 to obtain replaced the compound (C2) of embodiment 3, other was operated similarly to Example 3, obtains purpose compound (C10).
Embodiment 11
Except the compound (C10) that uses embodiment 10 to obtain replaced the compound (C3) of embodiment 4, other was operated similarly to Example 4, obtains purpose compound (C11).
Embodiment 12
Except the compound (C8-3) that uses embodiment 8 to obtain replaced the compound (C1-3) of embodiment 5, other was operated similarly to Example 5, obtains purpose compound (C12).
Embodiment 13
Except the compound (C12) that uses embodiment 12 to obtain replaced the compound (C5) of embodiment 6, other was operated similarly to Example 6, obtains purpose compound (C13).
Embodiment 14
Except the compound (C13) that uses embodiment 13 to obtain replaced the compound (C6) of embodiment 7, other was operated similarly to Example 7, obtains purpose compound (C14).
The amino nitrogen of embodiment 15 l-asparagines is by 8 of the protection of Fmoc base "-deoxidation-8 "-fluoro-saliva acidic group α 2; the amino nitrogen of 3 sugar chain asparagines (C15-1) and l-asparagine is by 2 kinds of lists 8 of Fmoc base protection "-deoxidation-8 "-fluoro-saliva acidic group α 2,3 sugar chain asparagines (C15-2 and C15-3) synthetic
Except the CMP-8 that uses reference example 9 to obtain "-deoxidation-8 "-the fluoro-sialic acid; other is operated similarly to Example 1; the amino nitrogen that obtains l-asparagine as follows is by 8 of the protection of Fmoc base "-deoxidation-8 "-fluoro-saliva acidic group α 2; the amino nitrogen of 3 sugar chain asparagines and l-asparagine is by 2 kinds of lists 8 of Fmoc base protection "-deoxidation-8 "-fluoro-saliva acidic group α 2,3 sugar chain asparagines.
This sugar chain asparagine is equivalent to the R of formula (1)
1=R
2=formula (2), R=OH, R '=F, R "=OH.
This sugar chain asparagine is equivalent to the R of formula (1)
1=formula (3), R
2=formula (2), R=OH, R '=F, R "=OH.
This sugar chain asparagine is equivalent to the R of formula (1)
1=formula (2), R=OH, R '=F, R "=OH, R2=formula (3).
Embodiment 16 (galactohydrolase of embodiment 15)
Except the compound (C15-2) that uses embodiment 15 to obtain replaced the compound (C1-2) of embodiment 2, other was operated similarly to Example 2, obtains purpose compound (C16).
This sugar chain asparagine is equivalent to the R of formula (1)
1=formula (4), R
2=formula (2), R=OH, R '=F, R "=OH.
Embodiment 17 (the N-acetyl-glucosamine lytic enzyme of embodiment 16)
Except the compound (C16) that uses embodiment 16 to obtain replaced the compound (C2) of embodiment 3, other was operated similarly to Example 3, obtains purpose compound (C17).
This sugar chain asparagine is equivalent to the R of formula (1)
1=formula (5), R
2=formula (2), R=OH, R '=F, R "=OH.
Embodiment 18 (the seminose lytic enzyme of embodiment 17)
Except the compound (C17) that uses embodiment 17 to obtain replaced the compound (C3) of embodiment 4, other was operated similarly to Example 4, obtains purpose compound (C18).
This sugar chain asparagine is equivalent to the R of formula (1)
1=H, R
2=formula (2), R=OH, R '=F, R "=OH.
Embodiment 19 (galactohydrolase of embodiment 15)
Except the compound (C15-3) that uses embodiment 15 to obtain replaced the compound (C1-3) of embodiment 5, other was operated similarly to Example 5, obtains purpose compound (C19).
This sugar chain asparagine is equivalent to the R of formula (1)
1=formula (2), R=OH, R '=F, R "=OH, R
2=formula (4).
Embodiment 20 (the N-acetyl-glucosamine lytic enzyme of embodiment 19)
Except the compound (C19) that uses embodiment 19 to obtain replaced the compound (C5) of embodiment 6, other was operated similarly to Example 6, obtains purpose compound (C20).
This sugar chain asparagine is equivalent to the R of formula (1)
1=formula (2), R=OH, R '=F, R "=OH, R
2=formula (5).
Embodiment 21 (the seminose lytic enzyme of embodiment 20)
Except the compound (C20) that uses embodiment 20 to obtain replaced the compound (C6) of embodiment 7, other was operated similarly to Example 7, obtains purpose compound (C21).
This sugar chain asparagine is equivalent to the R of formula (1)
1=formula (2), R=OH, R '=F, R "=OH, R
2=H.
The amino nitrogen of embodiment 22 l-asparagines is by 9 of the protection of Fmoc base "-deoxidation-9 "-fluoro-saliva acidic group α 2; the amino nitrogen of 3 sugar chain asparagines (C22-1) and l-asparagine is by 2 kinds of lists 9 of Fmoc base protection "-deoxidation-9 "-fluoro-saliva acidic group α 2,3 sugar chain asparagines (C22-2 and C22-3) synthetic
Except the CMP-9 that uses reference example 10 to obtain "-deoxidation-9 "-the fluoro-sialic acid; other is operated similarly to Example 1; the amino nitrogen that obtains above-mentioned l-asparagine is by 9 of the protection of Fmoc base "-deoxidation-9 "-fluoro-saliva acidic group α 2; the amino nitrogen of 3 sugar chain asparagines and l-asparagine is by 2 kinds of lists 9 of Fmoc base protection "-deoxidation-9 "-fluoro-saliva acidic group α 2,3 sugar chain asparagines.
(C22-1) be equivalent to the R of formula (1)
1=R
2=formula (2), R=OH, R '=OH, R "=sugar chain asparagine of F.
(C22-2) be equivalent to the R of formula (1)
1=formula (3), R
2=formula (2), R=OH, R '=OH, R "=sugar chain asparagine of F.
(C22-3) be equivalent to the R of formula (1)
1=formula (2), R=OH, R '=F, R "=OH, R
2The sugar chain asparagine of=formula (3).
Embodiment 23 (galactohydrolase of embodiment 22)
Except the compound (C22-2) that uses embodiment 22 to obtain replaced the compound (C1-2) of embodiment 2, other was operated similarly to Example 2, obtains purpose compound (C23).
(C23) be equivalent to the R of formula (1)
1=formula (4), R
2=formula (2), R=OH, R '=OH, R "=sugar chain asparagine of F.
Embodiment 24 (the N-acetyl-glucosamine lytic enzyme of embodiment 23)
Except the compound (C23) that uses embodiment 23 to obtain replaced the compound (C2) of embodiment 3, other was operated similarly to Example 3, obtains purpose compound (C24).
(C24) be equivalent to the R of formula (1)
1=formula (5), R
2=formula (2), R=OH, R '=OH, R "=sugar chain asparagine of F.
Embodiment 25 (the seminose lytic enzyme of embodiment 24)
Except the compound (C24) that uses embodiment 24 to obtain replaced the compound (C3) of embodiment 4, other was operated similarly to Example 4, obtains purpose compound (C25).
(C25) be equivalent to the R of formula (1)
1=H, R
2=formula (2), R=OH, R '=OH, R "=sugar chain asparagine of F.
Embodiment 26 (galactohydrolase of embodiment 22)
Except the compound (C22-3) that uses embodiment 22 to obtain replaced the compound (C1-3) of embodiment 5, other was operated similarly to Example 5, obtains purpose compound (C26).
(C26) be equivalent to the R of formula (1)
1=formula (2), R=OH, R '=OH, R "=F, R
2The sugar chain asparagine of=formula (4).
Embodiment 27 (the N-acetyl-glucosamine lytic enzyme of embodiment 26)
Except the compound (C26) that uses embodiment 26 to obtain replaced the compound (C5) of embodiment 6, other was operated similarly to Example 6, obtains purpose compound (C27).
(C27) be equivalent to the R of formula (1)
1=formula (2), R=OH, R '=OH, R "=F, R
2The sugar chain asparagine of=formula (5).
Embodiment 28 (the seminose lytic enzyme of embodiment 27)
Except the compound (C27) that uses embodiment 27 to obtain replaced the compound (C6) of embodiment 7, other was operated similarly to Example 7, obtains purpose compound (C28).
(C28) be equivalent to the R of formula (1)
1=formula (2), R=OH, R '=OH, R "=F, R
2The sugar chain asparagine of=H.
The amino nitrogen of embodiment 29 l-asparagines is by 7 of Fmoc base protection "-deoxidation-7 "-amino nitrogen of fluoro-saliva acidic group (2-6) sugar chain asparagine (C29-1) and l-asparagine is by 2 kinds of lists 7 of Fmoc base protection "-deoxidation-7 "-fluoro-saliva acidic group (2-6) sugar chain asparagine (C29-2 and C29-3) synthetic
Except the CMP-7 that uses reference example 7 to obtain "-deoxidation-7 "-the fluoro-sialic acid; use is as α 2; the commercially available enzyme from Rat Liver of 6 transferring enzymes is as sialytransferase; the pH of cacodylic acid damping fluid is transferred to beyond 6.0; other is operated similarly to Example 1, and the amino nitrogen that obtains l-asparagine as follows is by 7 of the protection of Fmoc base "-deoxidation-7 "-amino nitrogen of fluoro-saliva acidic group (2-6) sugar chain asparagine and l-asparagine is by 2 kinds of lists 7 of Fmoc base protection "-deoxidation-7 "-fluoro-saliva acidic group (2-6) sugar chain asparagine.(C29-1)～(C29-3) chemical formula is as follows.
Embodiment 30 (galactohydrolase of embodiment 29)
Except the compound (C29-2) that uses embodiment 29 to obtain replaced the compound (C1-2) of embodiment 2, other was operated similarly to Example 2, obtains purpose compound (C30).(C30) chemical formula is as follows.
Embodiment 31 (the N-acetyl-glucosamine lytic enzyme of embodiment 30)
Except the compound (C30) that uses embodiment 30 to obtain replaced the compound (C2) of embodiment 3, other was operated similarly to Example 3, obtains purpose compound (C31).(C31) chemical formula is as follows.
Embodiment 32 (the seminose lytic enzyme of embodiment 31)
Except the compound (C31) that uses embodiment 31 to obtain replaced the compound (C3) of embodiment 4, other was operated similarly to Example 4, obtains purpose compound (C32).(C32) chemical formula is as follows.
Embodiment 33 (galactohydrolase of embodiment 29)
Except the compound (C29-3) that uses embodiment 29 to obtain replaced the compound (C1-3) of embodiment 5, other was operated similarly to Example 5, obtains purpose compound (C33).(C33) chemical formula is as follows.
Embodiment 34 (the N-acetyl-glucosamine lytic enzyme of embodiment 33)
Except the compound (C33) that uses embodiment 33 to obtain replaced the compound (C5) of embodiment 6, other was operated similarly to Example 6, obtains purpose compound (C34).(C34) chemical formula is as follows.
Embodiment 35 (the seminose lytic enzyme of embodiment 34)
Except the compound (C34) that uses embodiment 34 to obtain replaced the compound (C6) of embodiment 7, other was operated similarly to Example 7, obtains purpose compound (C35).(C35) chemical formula is as follows.
Embodiment 36～49
Below same operation synthesized sugar chain asparagine derivative shown below.
In addition,, below provided 8F α 2, the NMR data of 6-11 sugar-Asn-Fmoc (C36-1) as typical example.
1H?NMR(400MHz，D
2O，30℃，HOD＝4.81)
δ?8.01(d，2H，J＝7.4，Fmoc)，7.80(d，2H，J＝7.4，Fmoc)，7.59(dd，2H，J＝7.4，Fmoc)，7.52(bdd，2H，J＝7.4，Fmoc)，5.22(s，1H，Man4-H1)，5.08(d，1H，J＝9.4，GlcNAc1-H1)，5.05(s，1H，Man4’-H-1)，4.85-4.95(m，1H)，4.55-4.75(m)，4.53(d，1H，J＝7.9)，4.43(m，1H)，4.35(bs，2H，Man3-H2)，4.28(bs，1H，Man4-H2)，4.10-4.25(m，2H)，2.75-2.85(m，1H，Asn-βCH)，2.63-2.70(dd，2H，Ja＝3.9，Jb＝12.0，NeuAc7，7’-H3eq)，2.55-2.65(m，1H，Asn-βCH)，2.16，2.11，2.08(eachs，15H，Acx5)，1.84(s，3H，Ac)，1.74(dd，1H，Ja＝12.3，Jb＝12.2，NeuAc7-H3ax).
Embodiment 50 (deprotection of the Fmoc base of sugar chain asparagine derivative)
In all sugar chain asparagine derivatives, all carry out the deprotection of Fmoc base in the following order.At first, add the N of 240 μ L for per 1 μ mol sugar chain asparagine Fmoc body, the morpholine of dinethylformamide, 160 μ L makes its reaction under room temperature, argon atmosphere.(eluting solvent uses the 1M ammonium acetate: after confirming that Virahol=8: 5) reaction finishes, cool off in frozen water by TLC.Add the diethyl ether that is equivalent to 10 times of amounts of reaction soln then, stir after 15 minutes, the throw out of separating out is filtered.Make the residue that obtains be dissolved in water, under 35 ℃, evaporate.Add 3mL toluene again, carry out above-mentioned evaporation operation repeatedly 3 times.Residue is passed through reversed-phase column chromatography (コ ス モ シ-Le 75C
18-OPN, 15 * 100mm, eluting solvent are water) carry out purifying, obtain corresponding sugar chain asparagine.
Embodiment 51 (removing of the asparagine residue of sugar chain asparagine)
After making the sugar chain asparagine and anhydrous hydrazine reaction that embodiment 50 obtains, by acetylize, remove asparagine residue after, obtain corresponding sugar chain.
Embodiment 52～69
Make each Fmoc-sugar chain asparagine 2nmol that makes among reference example 2,3,8～13, the embodiment 1～7 be dissolved in the Tris-HCl damping fluid of about 10mL.In this solution, add GDP-Fucose 200nmol, (Human, Recombinant) 0.5mU left standstill under 37 ℃ about 2 hours fucosyltransferase V, made its reaction.After reaction solution usefulness ultrapure water 20mL dilution, by capillary electrophoresis (fused silica capillary, 50mm i.d., 60cm, buffer; 100mM Tris-borate, pH=8.3,100mM Heptane sulfonate, impressed voltage 27kV, 25 ℃ of temperature 214mm) are separated, and obtain each object.
Raw material and object thereof among each embodiment have below been provided.
Table 1
Embodiment 52
Raw material
(compound 2)
Object
Embodiment 53
Raw material
(compound 3)
Object
Table 2
Embodiment 54
Raw material
(compound 4)
Object
Embodiment 55
Raw material
(compound 11)
Object
Embodiment 56
Raw material
(compound 12)
Object
Embodiment 57
Raw material
(compound 13)
Object
Embodiment 58
Raw material
(compound 14)
Object
Embodiment 59
Raw material
(compound 15)
Object
Embodiment 60
Raw material
(compound 16)
Object
Table 3
Embodiment 61
Raw material
(compound 17)
Object
Embodiment 62
Raw material
(compound 18)
Object
Table 4
Embodiment 63
Raw material
(compound 19)
Object
Embodiment 64
Raw material
(compound 20)
Object
Embodiment 65
Raw material
(compound 21)
Object
Embodiment 66
Raw material
(compound 22)
Object
Embodiment 67
Raw material
(compound 23)
Object
Embodiment 68
Raw material
(compound 24)
Object
Embodiment 69
Raw material
(compound 25)
Object
Claims (27)
1. the α that contains 11～7 sugar 2,3 sugar chain asparagine derivatives shown in the following formula (1),
In the formula, R
1And R
2Be the group of hydrogen atom, formula (2)～(5) expression, can be identical, also can be different, but R
1And R
2In a side must be the group of formula (2) expression,
R, R ', R " be expressed as follows combination.
(a)R＝F、 R’＝OH，?R”＝OH
(b)R＝OH、?R’＝F， R”＝OH
(c)R＝OH、?R’＝OH，?R”＝F
(d)R＝OH、?R’＝OH，?R”＝OH
2. the fluorine-containing α that contains 11～7 sugar 2,6 sugar chain asparagine derivatives shown in the following formula (6),
In the formula, R
XAnd R
YBe the group of hydrogen atom, formula (7) expression or the group of formula (3)～(5) expression, but R
XAnd R
YIn a side must be the group of formula (7) expression,
R, R ', R " be expressed as follows combination.
(a)R＝F、 R’＝OH，?R”＝OH
(b)R＝OH、?R’＝F， R”＝OH
(c)R＝OH、?R’＝OH，?R”＝F
3. the α that contains 11～7 sugar 2,3 sugar chain asparagines shown in the following formula (8),
In the formula, R
1And R
2As mentioned above.
4. the fluorine-containing α that contains 11～7 sugar 2,6 sugar chain asparagines shown in the following formula (9),
In the formula, R
XAnd R
YAs mentioned above.
5. the α that contains 11～7 sugar 2,3 sugar chains shown in the following formula (10),
In the formula, R
1And R
2As mentioned above.
6. the fluorine-containing α that contains 11～7 sugar 2,6 sugar chains shown in the following formula (11),
In the formula, R
XAnd R
YAs mentioned above.
7. the α that contains 11 sugar 2 shown in the following formula (12); the manufacture method of 3 bifunctional sialyltransferase base sugar chain asparagine derivatives; it is characterized in that: sialic acid or sialic derivative are transferred on the sugar chain asparagine of protecting with fat-soluble protecting group with sialytransferase; the sugar chain asparagine of the fat-soluble protecting group protection of the usefulness that obtains is supplied with chromatogram to be separated
In the formula, R
1And R
2It all is the group of formula (2) expression.
8. the α that contains 10 sugar 2 of following formula (13) expression; the manufacture method of 3 single sialic acid base sugar chain asparagine derivatives; it is characterized in that: sialic acid or sialic derivative are transferred on the sugar chain asparagine of protecting with fat-soluble protecting group with sialytransferase; the sugar chain asparagine of the fat-soluble protecting group protection of the usefulness that obtains is supplied with chromatogram to be separated
In the formula, R
1, R
2A side be the group of formula (2) expression, the opposing party is the group of formula (3) expression.
9. the manufacture method of the α that contains 9 sugar 2, the 3 single sialic acid base sugar chain asparagine derivatives of following formula (14) expression is characterized in that: is hydrolyzed with the single sialic acid base sugar chain asparagine derivative of galactohydrolase to formula (13) expression,
In the formula, R
1, R
2A side be the group of formula (2) expression, the opposing party is the group of formula (4) expression.
10. the manufacture method of the α that contains 8 sugar 2, the 3 single sialic acid base sugar chain asparagine derivatives of following formula (15) expression, it is characterized in that: the single sialic acid base sugar chain asparagine derivative of formula (14) being represented with the N-acetyl-glucosamine lytic enzyme is hydrolyzed,
In the formula, R
1, R
2A side be the group of formula (2) expression, the opposing party is the group of formula (5) expression.
11. the manufacture method of the α that contains 7 sugar 2, the 3 single sialic acid base sugar chain asparagine derivatives of following formula (16) expression is characterized in that: be hydrolyzed with the single sialic acid base sugar chain asparagine derivative of seminose lytic enzyme to formula (15) expression,
In the formula, R
1, R
2A side be the group of formula (2) expression, the opposing party is a hydrogen atom.
12. the α that contains 11 sugar 2 of following formula (17) expression; the manufacture method of 6 bifunctional sialyltransferase base sugar chain asparagine derivatives; it is characterized in that: sialic acid or sialic derivative are transferred on the sugar chain asparagine of protecting with fat-soluble protecting group with sialytransferase; the sugar chain asparagine of the fat-soluble protecting group protection of the usefulness that obtains is supplied with chromatogram to be separated
In the formula, R
XAnd R
YIt all is the group of formula (7) expression.
13. the α that contains 10 sugar 2 of following formula (18) expression; the manufacture method of 6 single sialic acid base sugar chain asparagine derivatives; it is characterized in that: sialic acid or sialic derivative are transferred on the sugar chain asparagine of protecting with fat-soluble protecting group with sialytransferase; the sugar chain asparagine of the fat-soluble protecting group protection of the usefulness that obtains is supplied with chromatogram to be separated
In the formula, R
XAnd R
YA side be the group of formula (7) expression, the opposing party is the group of formula (3) expression.
14. the manufacture method of the α that contains 9 sugar 2, the 6 single sialic acid base sugar chain asparagine derivatives of following formula (19) expression is characterized in that: be hydrolyzed with the single sialic acid base sugar chain asparagine derivative of galactohydrolase to formula (18) expression,
In the formula, R
XAnd R
YA side be the group of formula (7) expression, the opposing party is the group of formula (4) expression.
15. the manufacture method of the α that contains 8 sugar 2, the 6 single sialic acid base sugar chain asparagine derivatives of following formula (20) expression is characterized in that: be hydrolyzed with the single sialic acid base sugar chain asparagine derivative of N-acetyl-glucosamine lytic enzyme to formula (19) expression,
In the formula, R
XAnd R
YA side be the group of formula (7) expression, the opposing party is the group of formula (5) expression.
16. the manufacture method of the α that contains 7 sugar 2, the 6 single sialic acid base sugar chain asparagine derivatives of following formula (21) expression is characterized in that: be hydrolyzed with the single sialic acid base sugar chain asparagine derivative of seminose lytic enzyme to formula (20) expression,
In the formula, R
XAnd R
YA side be the group of formula (7) expression, the opposing party is a hydrogen atom.
17. the manufacture method of the α that contains 11～7 sugar 2,3 sugar chain asparagines of formula (8) expression is characterized in that: the protecting group of removing the α that contains 11～7 sugar 2,3 sugar chain asparagine derivatives of formula (1) expression.
18. the manufacture method of the α that contains 11～7 sugar 2,6 sugar chain asparagines of formula (9) expression is characterized in that: the protecting group of removing the α that contains 11～7 sugar 2,6 sugar chain asparagine derivatives of formula (6) expression.
19. the manufacture method of the α that contains 11～7 sugar 2,3 sugar chains of formula (10) expression is characterized in that: the asparagine residue of removing the α that contains 11～7 sugar 2,3 sugar chain asparagines of formula (8) expression.
20. the manufacture method of the α that contains 11～7 sugar 2,6 sugar chains of formula (11) expression is characterized in that: the asparagine residue of removing the α that contains 11～7 sugar 2,6 sugar chain asparagines of formula (9) expression.
21. (α 2,3) (α 2, the 6) sugar chain asparagine derivative that contains 11 sugar of following formula (22) expression,
In the formula, R
1Be the group of formula (2) expression, R
YBe the group of following formula (7) expression,
R, R ', R " be expressed as follows combination.
(a)R＝F、 R’＝OH，?R”＝OH
(b)R＝OH、?R’＝F，?R”＝OH
(c)R＝OH、?R’＝OH，?R”＝F
(d)R＝OH、?R’＝OH、?R”＝OH
22. (α 2,3) (α 2, the 6) sugar chain asparagine derivative that contains 11 sugar of following formula (23) expression,
In the formula, R
2Be the group of formula (2) expression, R
XBe the group of following formula (7) expression,
R, R ', R " be expressed as follows combination.
(a)R＝F、 R’＝OH，?R”＝OH
(b)R＝OH、?R’＝F， R”＝OH
(c)R＝OH、?R’＝OH，?R”＝F
(d)R＝OH、?R’＝OH、?R”＝OH
23. sugar chain asparagine derivative, it contains the Fucose more than at least 1 on the N-acetyl-glucosamine of amino nitrogen with the non-reduced end side of the sugar chain asparagine of fat-soluble protecting group protection of l-asparagine.
24. the described sugar chain asparagine derivative that contains Fucose of claim 23, wherein the amino nitrogen of l-asparagine is the α that contains 11～7 sugar 2,3 sugar chain asparagine derivatives shown in the formula (1) with the sugar chain asparagine of fat-soluble protecting group protection.
25. the described sugar chain asparagine derivative that contains Fucose of claim 23; wherein the amino nitrogen of l-asparagine is the fluorine-containing α that contains 11～7 sugar 2,6 sugar chain asparagine derivatives shown in the formula (6) with the sugar chain asparagine of fat-soluble protecting group protection.
26. the described sugar chain asparagine derivative that contains Fucose of claim 23, wherein the amino nitrogen of l-asparagine is the α that contains 11～6 sugar 2,6 sugar chain asparagine derivatives shown in the following formula (6) with the sugar chain asparagine of fat-soluble protecting group protection,
In the formula, R
XAnd R
YBe the group of hydrogen atom, following formula (7) expression, or the group of above-mentioned formula (3)～(5) expression, but R
XAnd R
YIn a side must be the group of formula (7) or formula (3) expression,
Wherein, R=OH, R '=OH, R "=OH.
27. the N-acetyl-glucosamine of the non-reduced end side of the sugar chain asparagine that the amino nitrogen of l-asparagine is protected by fat-soluble protecting group contains the manufacture method of the sugar chain asparagine derivative of an above Fucose at least; it is characterized in that: make on the sugar chain asparagine that amino nitrogen that Fucose transfers to l-asparagine protected by fat-soluble blocking group with fucosyl transferase, the sugar chain asparagine of being protected by fat-soluble protecting group that obtains is supplied with chromatogram separate.
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| JP2002373213 | 2002-12-24 | ||
| JP373213/2002 | 2002-12-24 | ||
| JP202708/2003 | 2003-07-28 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104020216A (en) * | 2014-06-19 | 2014-09-03 | 江南大学 | Method for relatively quantitatively analyzing carbohydrate chain in two-end labeling manner |
| CN107522779A (en) * | 2011-09-04 | 2017-12-29 | 株式会社糖锁工学研究所 | The polypeptide of additional sugar chain and the medical composition containing the polypeptide |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH089989A (en) * | 1994-07-04 | 1996-01-16 | Meiji Milk Prod Co Ltd | Production of beta-mannosyloligosaccharide |
| JP3124199B2 (en) * | 1994-12-28 | 2001-01-15 | 日本たばこ産業株式会社 | Method for producing sugar containing sialic acid |
| JP4346713B2 (en) * | 1998-12-07 | 2009-10-21 | 生化学工業株式会社 | Method for producing oligosaccharide containing sialic acid |
| JP2002045196A (en) * | 2000-08-02 | 2002-02-12 | Toyobo Co Ltd | Method for carrying out enzymatic synthesis of hybrid sugar chain |
| CN100343264C (en) * | 2001-06-19 | 2007-10-17 | 大塚化学株式会社 | Process for producing sugar chain asparagine derivative |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107522779A (en) * | 2011-09-04 | 2017-12-29 | 株式会社糖锁工学研究所 | The polypeptide of additional sugar chain and the medical composition containing the polypeptide |
| CN107522779B (en) * | 2011-09-04 | 2021-10-26 | 株式会社糖锁工学研究所 | Polypeptide having sugar chain attached thereto and pharmaceutical composition containing the same |
| CN104020216A (en) * | 2014-06-19 | 2014-09-03 | 江南大学 | Method for relatively quantitatively analyzing carbohydrate chain in two-end labeling manner |
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