EP0736101A1 - Methode enzymatique pour la synthese d'acide amine ou de peptide o-glycosyle ou de derives de ces derniers - Google Patents

Methode enzymatique pour la synthese d'acide amine ou de peptide o-glycosyle ou de derives de ces derniers

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Publication number
EP0736101A1
EP0736101A1 EP95903439A EP95903439A EP0736101A1 EP 0736101 A1 EP0736101 A1 EP 0736101A1 EP 95903439 A EP95903439 A EP 95903439A EP 95903439 A EP95903439 A EP 95903439A EP 0736101 A1 EP0736101 A1 EP 0736101A1
Authority
EP
European Patent Office
Prior art keywords
peptide
amino acid
group
derivatives
synthesis
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP95903439A
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German (de)
English (en)
Inventor
Kurt Nilsson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bioflexin AB
Original Assignee
Bioflexin AB
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Publication date
Application filed by Bioflexin AB filed Critical Bioflexin AB
Publication of EP0736101A1 publication Critical patent/EP0736101A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/005—Glycopeptides, glycoproteins

Definitions

  • the present invention describes a new method to produce O- glycosylated amino acids, O-glycosylated peptides or derivatives of these.
  • the present invention relates to products produced by the above method as well as uses of the resulting products.
  • Glycoconjugates contain oligosaccharide chains with up to twenty monosaccharide units and several sequences have been shown to have biological activity e.g. in the binding to different cells, pathogens, toxins, antibodies or other proteins on cell surfaces, in cancer metastasis, in inflammation processes (for example selectin-carbohydrate interactions) , as a modifier of the activity, stability and biological activity of proteins, and as immunogenic substances which have potential for vaccination against different diseases.
  • An extensive literature has been developed during the last few years in this field and there are several review articles on this type of biology, glycobiology, e.g. in Annual Review of Biochemistry and in Current Opinion in Structural Biology (see for example volume 3, 1993) incorporated herein by reference.
  • glycoconjugate the glycoproteins
  • the glycoproteins contain carbohydrate-peptide sequences in which the carbohydrate unit is bound to the peptide or protein chain via mainly three different types of linkages, the O-glycosidic linkage represented by GalNAc ⁇ -OSer and GalNAc ⁇ -OThr, that is the linkage between N- acetyl-D-galactosamine and the hydroxyl group on a L-serine or a L-threonine residue in the peptide or protein chain (several such linkages can be found in a peptide or protein chain depending on the number of serine or threonine units in the molecule) , the N-glycosidic linkage between N-acetyl-D- glucosamine and the amide function in asparagine, GlcNAc/3-N-Asn, and the O-glycosidic linkage between galactose or xylose and different hydroxyl group containing amino
  • glycosidic N-linkage e.g. a galactosyl-serine or a galactosyl- threonine linkage (Parekh, R.B., (1991), Curr. Opin. Struct. Biol. 1: 750-754).
  • Glycosidases have been utilized in a few cases. Thus, it was found that glycosylated products were obtained under equilibrium conditions employing monosaccharide and non-modified serine or threonine in high concentrations (Johansson et al 1991) . However, in this type of reaction, the N-glycosylation of the reducing end of the monosaccharide is a severe chemical side-reaction. Interestingly, enzymatic glycosylation of serine or threonine was not observed under kinetic (transglycosylation) reaction conditions, i.e. employing disaccharides or activated glycosides as glycosyl donors.
  • R represents H in an equilibrium reaction, or represents an organic group, i.e. a saccharide unit or an aromatic or aliphatic group in a kinetic reaction.
  • Simple monosaccharides have been used as donor, Sugar-OR, in equilibrium synthesis, whereas various O-glycosides thereof including lactose, have been used in transglycosylation reactions.
  • the enzyme is chosen with regard to the sugar substrate and linkage to be prepared (with ⁇ -N-acetyl- galactosaminidase one obtains GalNAc ⁇ -Ser/Thr, as is known in the art) .
  • glycosylated amino acid or peptides or derivatives thereof is produced by the reaction of (a) a simple sugar in pyranose or furanose form as glycosyl donor, for example N- acetyl-glucosamine (GlcNAc) , N-acetyl-galactosamine (GalNAc) , mannose (Man) , xylose (Xyl) , galactose (Gal) , fucose (Fuc) , or specific oligosaccharide sequences which contain one or more of any of these monosaccharides, and (b) hydroxyl group containing amino acid or peptide derivative as acceptor which contains at least one hydroxyl group and which has been modified in its (N-) terminal ⁇ -amino function but which has a non-modified C- terminal carboxyl group, employing an exo- or endoglycosidase as catalyst (enzyme)
  • glycopeptide fragments as well as derivatives thereof the type X-GalNAc ⁇ -OR, X-GalNAc/3-OR, XGalcx-OR, XGal3-OR, XMan ⁇ -OR, XMan ⁇ - OR, X-Glc ⁇ -OR, X-Glc/3-OR, X-Xyl ⁇ -OR, X-Xyl ⁇ -OR, X-GlcNAc ⁇ -OR, X- GlcNAc/3-OR, X-Fucct-OR, wherein X is a glycosidically bound carbohydrate-group (by using endoglycosidase) , or, when an exoglycosidase is used, X is not present (i.e.
  • X-GalNAc ⁇ -OR etc is the same as GalNAc ⁇ -OR, etc.).
  • the acceptor, HOR consists of a hydroxyl group containing amino acid or peptide which has been modified in at least the terminal N-group, but which has a free (non-modified) carboxyl group.
  • acceptor substances are R'-serine, R'-threonine, R'-Ser-Ala-OH, R'-Ser-Val- OH and so on, where R 1 is a protection group on the amino function of the amino acid or peptide such as an acetyl (Ac-) , t-butyloxycarbonyl (Boc-) or Fmoc-group.
  • R 1 is a protection group on the amino group, or in the case when the acceptor is a peptide R 1 represents an amino protection group or an amino acid- or peptide group which is protected in the ⁇ -N-terminal group with an amino protection group, and where R 2 consists of an -OH group, or represents an amino acid or peptide group (bound via a C(0)-NH linkage) which has a non-modified carboxyl group in the carboxyl terminal unit.
  • the amino protection group is described above and below.
  • the donor is used in high concentration (preferably higher than IM when buffered water is used as the only solvent) or in excess over the acceptor, since the donor (simple monosaccharides with exoglycosidases) is abundant and have high solubility in water.
  • relatively high concentrations of enzyme e.g., 1-50 U/ml where 1 U is defined as the hydrolysis of 1 ⁇ mole of glycoside substrate per minute
  • temperature room temperature or higher, e.g. 25°— 65°C
  • yields may be improved if lower temperatures are used, such as when organic solvents are used.
  • the glycosidase can be used preferably in a relative pure form to obtain a stereospecific synthesis.
  • N-protection groups which may be used according to the invention may be mentioned alkoxy and aliphatic protection groups e.g. benzyloxycarbonyl-, allyloxycarbonyl-, t-butyloxycarbonyl- (Boc-) , formyl-, acetyl-, fenacetyl-, 4-metoxybenzyloxycarbonyl- (Moz-) , 9-fluorenyl methyloxycarbonyl (Fmoc-) , etc. (see for example Houben-Weyl, Bd 15/1, Griffine 3/79, 14 and especially Synthetic Peptides, G.A. Grant, Editor, W.H.
  • peptide derivate is acceptor in the reaction according to the invention, the size is preferably di- to penta peptide but even bigger peptide fragments can be glycosylated according to the invention.
  • glycosylation can be achieved also of hydrophobic amino acid or peptide derivatives because of the high concentration of donor and the equilibrium type of reaction facilitates glycosylation of acceptors which are inefficient acceptors in kinetic reactions (which require an efficient acceptor and a relatively high concentration of acceptor to minimize the hydrolytic side reaction) .
  • acceptors used in the method according to the invention have a free carboxyl group which facilitate their dissolution in solvents with high water content and thus no or low amounts of cosolvents are required.
  • the reactions according to the invention may also be carried out in mixed solvents, i.e.
  • the enzyme may be used in immobilized, cross-linked or in soluble form.
  • the enzyme may be used adsorbed to a solid phase such as e.g. glass or polystyrene. Examples are the use of enzyme adsorbed to e.g. celite or XAD R resins. The latter may be especially useful in the cases when organic solvents (acetone, acetonitrile, tetrahydrofurane) are used as cosolvents (in high concentrations e.g. > 70%) or in two-phase systems.
  • the product can be isolated by for example (a) slight acidification of the reaction mixture (pH 4-5; neutralizing the charge on the carboxyl group) followed by (b) precipitation or extraction of unreacted acceptor with a suitable organic solvent, followed by for example (c) column chromatography (e.g. on a Sephedex R , silica, reversed phase or ion-exchange column) or another extraction step with a less hydrophobic solvent to remove the product from the reaction mixture (the unmodified sugar will stay in the water phase) , or followed by evaporation of the water phase and extraction with for example methanol eventually followed by column chromatography of the methanol phase.
  • column chromatography e.g. on a Sephedex R , silica, reversed phase or ion-exchange column
  • Mannose was dissolved in water (>40% w/w) together with Boc-L-serine (in this example 0.6 M) ⁇ -Mannosidase was added with pH corrected to 6.5.
  • the reaction was carried out at 40° Celsius and after a suitable reaction time, depending on the amount of enzyme activity added and reaction temperature, the product (N-t-Boc)-L-serine ⁇ -mannoside (compound III in the Figure below) , was isolated by (a) extraction of the water phase to remove the excess of the acceptor followed by column chromatography of the water-phase.
  • the donor can be used in extremely high concentrations (up to ca 85% w/w or higher) and at higher temperatures than mentioned above.
  • the acceptor can also be used in high concentrations, often higher than 0.5 M, because the non-modified deprotonated carboxyl group increases the water solubility of the acceptor.
  • a typical equilibrium controlled synthesis of Man ⁇ -Ser(N- Boc) (compound III in the figure below) was carried out by dissolving Boc-L-serine (compound I in the figure below) (700 mg) in buffer (0.05 M) , the pH was adjusted to 5.0, the liquid volume adjusted to 1.6 ml followed by the addition of mannose (2.5 g) . After the addition of ⁇ -mannosidase (20 U) the reaction was carried out at 25°C for five days.
  • Gal ⁇ -Ser(N-Boc) A typical equilibrium controlled synthesis of (N-t- Boc) -L-serine ⁇ -galactoside (compound V below) Gal ⁇ -Ser(N-Boc) was carried out at pH 5 in a total liquid volume of 2 ml following the above procedure for dissolving Boc-L-serine (900 mg) and galactose (2.1 g) . After the addition of ⁇ - galactosidase (100 U) , the reaction was carried out at 35°C for ten days. Termination of the reaction and isolation of product was performed as above.
  • N- X N- XY-
  • X is an N ⁇ - amino protection group, such as for example Boc-, or Fmoc- or another ⁇ -N-protection group which has been exemplified above
  • Y can be-serine or threonine or a peptide residue containing any of those amino acids according to the formula above
  • the above mentioned types of donors and enzymes can be used in a corresponding manner to produce the corresponding glycosylated derivative of the respective type of acceptor.
  • Amino acid or peptide derivatives of the D-configuration or which contain a mixture of amino acid residues of the D-and the L-configurations may be used as acceptors according to the invention.
  • Glycosidases are abundant in all living organisms and the source of enzyme do not limit the scope of the invention. Examples of suitable sources are different microorganisms such as bacteria and yeast (e.g. Aspergillus niger and Asperg ⁇ llus oryzae) , plants and different animal tissues.
  • the enzyme can be used in more or less isolated form, if a stereospecific reaction is required the enzyme should be free of any contaminating glycosidase which may lead to formation of the non-desired anomeric configuration.
  • the enzyme may be used in soluble or immobilized form (to allow reuse if desired) as bound covalently or adsorbed to a solid support (e.g. a glass, silica, polystyrene, another plastic, a polysaccharide (e.g. cellulose or agarose) ) or enriched in a water phase in a two-phase system.
  • a solid support e.g. a glass, silica, polystyrene, another plastic, a polysaccharide (e.g. cellulose or agarose)
  • the product can be used directly, or after the removal of the N-protection groups, e.g. by chemical or enzymatic methods such as treatment with lipase or protease, or after the binding to other molecules via e.g. the amino group or the carboxyl group, e.g. attachment to polymers, plastics, metal surfaces, including gold surfaces via e.g.
  • a mercaptopropionic acid spacer ELISA-plates, proteins, lipids (the carboxyl or amino group can be used for covalent binding) in biological studies or can be used in a continued chemical or enzymatic synthesis according to the invention.
  • the product can for example also be used for synthesis of disaccharide amino acid or peptide derivatives.
  • glycosylated derivatives as mentioned above and (b) use such a glycosylated derivative as acceptor for synthesis with glycosyltransferase or glycosidase of for example the following type of structures (the (N-X)Y structure has been defined above; this group may have been modified as described above before the synthesis described below) :
  • Gal / Sl-3GlcNAc / S-(N-X)Y or Gal/3l-3GalNAc ⁇ -(N-X)Y by using ⁇ - D-galactosidase (for instance from ox testes) as catalyst, nitrophenyl ⁇ -D-galactoside or lactose as donor and GlcNAc/?-(N- X)Y or GalNAc ⁇ -(N-X)Y as acceptor, respectively,
  • ⁇ - D-galactosidase for instance from ox testes
  • nitrophenyl ⁇ -D-galactoside or lactose as donor
  • GlcNAc/?-(N- X)Y or GalNAc ⁇ -(N-X)Y as acceptor
  • Gal/51-4GlcNAcjS-(N-X)Y by using a suitable -galactosidase as catalyst, nitrophenyl ⁇ -D-galactoside or lactose as donor and GlcNAc/3-(N-X)Y as acceptor,
  • Man ⁇ l-2Man ⁇ -(N-X)Y by using ⁇ -D-mannosidase as catalyst, nitrophenyl ⁇ -D-mannopyranoside as donor and Man ⁇ -(N-X)Y as acceptor.
  • oligosaccharide sequences can be produced with other glycosidases (e.g. by the use of glycosidases mentioned above) and/or other acceptors.
  • Glycosylation with mannose as the glycosyl donor is attractive for the synthesis of O-mannosylated serine or threonine derivatives due to the abundant donor substrate and the stereospecific reaction.
  • the formation of product was more rapid at the higher temperatures (in the range 25°-50°C) .
  • the yield of product increased with the reaction temperature in the range 25°-50°C.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
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  • Health & Medical Sciences (AREA)
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  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)

Abstract

L'invention concerne un procédé pour la synthèse d'acide aminé ou de peptide O-glycosylé ou de dérivés de ces derniers. Ce procédé est caractérisé par l'utilisation d'un donneur de glycosyle qui est un monosaccharide se présentant sous forme de pyranose ou de furanose ou qui est un oligosaccharide, et par l'utilisation d'un accepteur qui est un groupe hydroxyle contenant un dérivé d'acide aminé ou de peptide ayant été modifié dans son groupe N-terminal α-aminé et dans lequel le groupe carboxyle C-terminal n'est pas modifié. Ce procédé utilise un exo- ou un endoglycosidase (EC 3.2) comme catalyseur dans une réaction d'équilibre et la substance produite est isolée du mélange de réaction ou est utilisée directement ou après isolement partiel ou complet dans une synthèse continue effectuée par des méthodes chimiques ou enzymatiques.
EP95903439A 1993-12-24 1994-12-27 Methode enzymatique pour la synthese d'acide amine ou de peptide o-glycosyle ou de derives de ces derniers Withdrawn EP0736101A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
SE9304316 1993-12-24
SE9304316A SE9304316D0 (sv) 1993-12-24 1993-12-24 Aminosyra-konjugat
PCT/IB1994/000444 WO1995018232A1 (fr) 1993-12-24 1994-12-27 Methode enzymatique pour la synthese d'acide amine ou de peptide o-glycosyle ou de derives de ces derniers

Publications (1)

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EP0736101A1 true EP0736101A1 (fr) 1996-10-09

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EP (1) EP0736101A1 (fr)
AU (1) AU1249095A (fr)
SE (1) SE9304316D0 (fr)
WO (1) WO1995018232A1 (fr)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69518246T2 (de) * 1994-09-06 2000-12-21 Ab Lund Bioflexin Aminosäuren konjugaten
US6284494B1 (en) 1995-12-12 2001-09-04 The University Of British Columbia Methods and compositions for synthesis of oligosaccharides using mutant glycosidase enzymes
US5716812A (en) * 1995-12-12 1998-02-10 The University Of British Columbia Methods and compositions for synthesis of oligosaccharides, and the products formed thereby
DE19802748A1 (de) * 1998-01-26 1999-07-29 Chemtec Leuna Ges Fuer Chemie Verfahren zur enzymatischen Synthese von Glycoaminosäurederivaten
DE602005019038D1 (de) 2004-05-04 2010-03-11 Novo Nordisk Healthcare Ag O-verknüpfte glykoformen von faktor vii und verfahren zu deren herstellung
CA2868395C (fr) 2012-04-04 2017-08-08 Pepsico, Inc. Formation d'une proteine conjuguee par filage electrostatique

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SE451849B (sv) * 1985-12-11 1987-11-02 Svenska Sockerfabriks Ab Sett att syntetisera glykosidiska bindningar samt anvendning av pa detta sett erhallna produkter
DE4013077A1 (de) * 1990-04-25 1991-10-31 Hoechst Ag Verfahren zur glycosidasekatalysierten synthese von glycokonjugaten
SE9102292L (sv) * 1991-08-06 1993-02-07 Kurt G I Nilsson Enzymatisk metod
EP0551107A2 (fr) * 1992-01-09 1993-07-14 Hoechst Aktiengesellschaft Méthode pour réaliser une transglycosilation catalysée par bêta-galactosidase en utilisant des donneurs de glycosyl non-physiologiques

Non-Patent Citations (1)

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Title
See references of WO9518232A1 *

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WO1995018232A1 (fr) 1995-07-06
SE9304316D0 (sv) 1993-12-24
AU1249095A (en) 1995-07-17

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