WO2012171966A1 - Procédé pour l'hydrolyse régiosélective de monosaccharides - Google Patents

Procédé pour l'hydrolyse régiosélective de monosaccharides Download PDF

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Publication number
WO2012171966A1
WO2012171966A1 PCT/EP2012/061222 EP2012061222W WO2012171966A1 WO 2012171966 A1 WO2012171966 A1 WO 2012171966A1 EP 2012061222 W EP2012061222 W EP 2012061222W WO 2012171966 A1 WO2012171966 A1 WO 2012171966A1
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WO
WIPO (PCT)
Prior art keywords
process according
group
acetyl
monosaccharide
tri
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Ceased
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PCT/EP2012/061222
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English (en)
Inventor
Marco Terreni
Teodora Bavaro
Alassal Qais ABU
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Universita degli Studi di Pavia
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Universita degli Studi di Pavia
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Publication of WO2012171966A1 publication Critical patent/WO2012171966A1/fr
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Classifications

    • 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
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/14—Acyclic radicals, not substituted by cyclic structures attached to a sulfur, selenium or tellurium atom of a saccharide radical
    • 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
    • C12P19/02—Monosaccharides
    • 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
    • C12P19/12—Disaccharides
    • 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
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides

Definitions

  • the present invention relates to a method for the regioselective hydrolysis of monosaccharides having a thiocyanomethyl (-SCH 2 CN) group in the anomeric position.
  • the first step consists of the direct acetylation and bromuration in the anomeric position (C-l) of -D-mannopyranoside .
  • the substitution of the halogen with thiourea and the subsequent reaction with chloroacetonitrile allows the corresponding cyanomethyl 2 , 3 , 4 , 6-tetra-O-acetyl- 1-thio- -D-mannopyranoside to be obtained.
  • IME-thiomannopyranoside synthesis is also reported in WO 2010/087612 according to the above method.
  • the monosaccharide thus activated is conjugated to the human serum albumin in an aqueous media at room temperature for 1.5 hours.
  • the same author describes the application of the glycoconjugate obtained in the radiopharmaceutical field .
  • lipase and other hydrolases have previously been used in the hydrolysis of monosaccharides completely protected with acetylic groups or bearing an alkyl group in anomeric position.
  • the regioselectivity of these enzymes is highly influenced by the type of substituent present in the anomeric position.
  • hydrolases in particular the lipase from Candida rugosa, are able to selectively hydrolyse the protective acyl group in only one position of monosaccharides bearing a thiocyanomethyl group in anomeric position.
  • a first object of the present invention is therefore represented by a process for the selective hydrolysis of one acetyl group in a fully acethylated monosaccharide bearing a thiocyanomethyl group in anomeric position.
  • said monosaccharide is mannose.
  • the present invention describes the products cyanomethyl-6-O- ( 2 , 3 , 4 , 6-tetra-O-acetyl- -D-mannopyranosil ) -2,3, 4-tri-O-acetyl- -D- thiomannopyranoside, cyanomethyl ( -D-2 ' , 3 ' , 5 ' -tri-O- acetyl-arabinofuranosyl ) - ( 1 ⁇ 6 ) -2 , 3 , 4-tri-O-acetyl- - D- thiomannopyranoside and cyanomethyl ( -D- 2' ',3' ',5' '-tri-0-acetyl-arabinofuranosyl)-( 1 ⁇ 6 ) - ( - D-2 ' , 3 ' , 4 ' -tri-O-acetyl-mannopyranosyl ) - ( 1 ⁇ 6
  • Figure 1 shows the preparation of cyanomethyl 6-0- (2,3,4, 6-tetra-O-acetyl- -D- mannopyranosil ) -2,3, 4-tri-O-acetyl- -D- thiomannopyranoside according to the present invention
  • FIGS 2, 3 and 4 show particular embodiments of the invention .
  • a first object of the present invention is represented by a process for the regioselective hydrolysis of one protecting group in a monosaccharide fully protected with acyl groups and bearing a thiocyanomethyl group (-SCH 2 CN) in anomeric position .
  • said residue is mannose.
  • reaction of hydrolysis may therefore be represented as follows:
  • R is a protective group, thus showing that the acyl protecting group in position C6 has been hydrolysed.
  • acyl group is preferably represented by the acetyl group.
  • hydrolysis is performed using suitable enzymes, in particular hydrolase enzymes such as, for example, lipase and esterase.
  • hydrolase enzymes such as, for example, lipase and esterase.
  • the lipase is obtained from Candida rugosa (CRL) .
  • a CRL Sigma-Aldrich srl, Milan, Italy
  • a specific activity equal to 2.3 U/mg (116 U/g of support for the immobilised enzyme) .
  • the enzymes are previously immobilised onto a suitable solid carrier.
  • the lipases can be immobilised onto octyl-agarose or on decaoctyl Sepabeads .
  • supports such as octyl-agarose gel (Octyl Sepharose ® CL-4B) or polymethacrylic-based and butyl (Sepabeads FP-BU) or decaoctyl (Sepabeads EC-OD/S) type resins, completely derivatized with hydrophobic groups, (respectively butyl and decaoctyl chains) may be used.
  • the immobilisation may take place on a macroporous matrix of silica or silicates, on a matrix formed of acrylic type adsorbent resins, even cross-linked (Amberlite ® XAD-8 or Lewatit ® E 2001/85), by an amphiphilic substrate containing lipophilic chains, on a matrix of styrene and divinylbenzene optionally containing epoxy groups (Lewatit ® R 259 K or R 260 K or Diaion ® HP-40), on a polyacrylic resin containing epoxy groups (FP 4000) or, optionally, on a polymethacrylic resin containing epoxy groups (Sepabeads ® FP-EP or Eupergit ® C) suitably derivatized with hydrophobic groups.
  • this is performed in an aqueous environment, optionally buffered to a pH of between 3 to 6 and preferably 4 to 5.
  • Buffers which can be usefully used include the TRIS buffer and the phosphate buffer (solution of KH 2 PO 4 10-100 mM, preferably 50 mM) or other commonly used buffers.
  • an organic co-solvent is possible, preferably chosen from among acetonitrile or acetone.
  • the co-solvent may be added to the reaction mixture and, preferably, it is between about 10-30% (v/v the reaction mixture) .
  • the temperature is preferably kept constant in the range of about 0- 25°C.
  • OAg octyl-agarose (Octyl Sepharose CL-4B)
  • mannose obtained from the regioselective hydrolysis reaction described above is conjugated with a suitably protected saccharide residue which is activated in the anomeric position, so as to obtain di- or polysaccharide units .
  • the conjugation may be performed with a unit of mannopyranose bearing a protective group in position 2,3,4 and 6; in this way oligomannans may be obtained.
  • hydroxyl groups can be suitably protected with acyl groups, such as acetyl groups .
  • the activator group is preferably chosen from the outgoing groups commonly used in the glycosylation reactions, such as groups active in anomeric position of the sugar donor, such as halogen groups or preferably the trichloroacetamide group.
  • the conjugation reaction is performed according to methods known to persons skilled in the art and is followed by appropriate isolation and purification steps of the di- or polysaccharide obtained .
  • the present invention allows to obtain the products: cyanomethyl 6-0- ( 2 , 3 , 4 , 6-tetra-O- acetyl- -D-mannopyranosil ) -2,3, 4-tri-O-acetyl- -D- thiomannopyranoside
  • the first product is prepared starting from cyanomethyl-2 , 3 , 4-tri-O-acetyl- -D- thiomannopyranoside, obtained by the selective hydrolysis of cyanomethyl 2 , 3 , 4-tri-O-acetyl- -D- thiomannopyranoside, subsequently conjugated with 2,3,4, 6-Tetra-O-acetyl- -D-mannopyranose
  • the di- or polysaccharide obtained according to the present invention protected and functionalised in the anomeric position with the thiocyanomethyl group, has to be deprotected by the protective groups and activated in position CI.
  • said operation is performed in a single step with NaOMe/NaOH so as to obtain the IME- thioglycoside ready for conjugation to the protein.
  • the coupling with the protein is then performed in phosphate buffer 20 mM at pH 9 and a ratio of protein: thioglycoside of 1:1.
  • the reaction is kept under magnetic stirring at room temperature.
  • the glycoprotein is subsequently purified by molecular exclusion chromatography and can be analysed using the MALDI technique.
  • the solvent was removed in a vacuum and the residue was purified by means of a silica gel chromatographic column using a mixture (hexane- ethyl acetate 50:50) as eluent, thus supplying the cyanomethyl 6-0- (2,3,4, 6-tetra-O-acetyl- -D- mannopyranosil ) -2,3, 4-tri-O-acetyl- -D- thiomannopyranoside with a yield of 70%.
  • the pH during hydrolysis was controlled using a Metrohm automatic pH-Stat 718 Stat Tritino (Herisau, Switzerland) .
  • the HPLC analysis was conducted using a HPLC Merck Hitachi L-7100 (E.Merck, Darmstadt, Germany) provided with detector UV L-7400 and an injection valve with 20 pL loop.
  • a Gemini RP Cig 250 x 4.6 mm, 5 pm; Phenomenex, Castel Maggiore (BO), Italy
  • the residue was purified by means of a silica gel chromatographic column using a mixture of hexane-ethyl acetate 40:60 as the eluent thus obtaining the cyanomethyl-2 , 3 , 4- tri-O-acetyl- -D-thiomannopyranoside and cyanomethyl 3 , 4 , 6-tri-O-acetyl- -D-thiomannopyranoside with an overall yield of 30%.
  • OAg octylagarose (Octyl Sepharose CL-4B)

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

Abstract

La présente invention concerne un procédé pour la préparation de disaccharides et de polysaccharides au moyen de l'hydrolyse régiosélective d'unités de monosaccharide dans la position primaire.
PCT/EP2012/061222 2011-06-13 2012-06-13 Procédé pour l'hydrolyse régiosélective de monosaccharides Ceased WO2012171966A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IT001064A ITMI20111064A1 (it) 2011-06-13 2011-06-13 Procedimento di idrolisi regioselettiva di monosaccaridi
ITMI2011A001064 2011-06-13

Publications (1)

Publication Number Publication Date
WO2012171966A1 true WO2012171966A1 (fr) 2012-12-20

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IT (1) ITMI20111064A1 (fr)
WO (1) WO2012171966A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070253942A1 (en) 2001-04-03 2007-11-01 Isis Innovation Limited Lectin-directed prodrug delivery system
US20080069802A1 (en) 2004-07-23 2008-03-20 Davis Benjamin G Modified Virus
WO2010087612A1 (fr) 2009-01-28 2010-08-05 Snu R&Db Foundation Complexe à agent(s) chélateur(s) bifonctionnel(s) et sérumalbumine humaine mannosylée

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070253942A1 (en) 2001-04-03 2007-11-01 Isis Innovation Limited Lectin-directed prodrug delivery system
US20080069802A1 (en) 2004-07-23 2008-03-20 Davis Benjamin G Modified Virus
WO2010087612A1 (fr) 2009-01-28 2010-08-05 Snu R&Db Foundation Complexe à agent(s) chélateur(s) bifonctionnel(s) et sérumalbumine humaine mannosylée

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
CORDOVA ARMANDO ET AL: "Esterification of methyl glycoside mixtures by lipase catalysis", 1997, BIOTECHNOLOGY LETTERS, VOL. 19, NR. 1, PAGE(S) 15-18, ISSN: 0141-5492, XP002669927 *
DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1980, STOWELL C P ET AL: "PREPARATION OF SOME NEW NEO GLYCO PROTEINS BY AMIDINATION OF BOVINE SERUM ALBUMIN USING 2 IMINO-2-METHOXYETHYL 1 THIO GLYCOSIDES", Database accession no. PREV198171028603 *
DATABASE CAPLUS [online] CHEMICAL ABSTRACTS SERVICE, COLUMBUS, OHIO, US; 1982, LEE, R.T.: "Binding site of the rabbit liver lectin specific for galactose/N-acetylgalactosamine", XP002683659, Database accession no. 1982:102251 *
DATABASE CAPLUS [online] CHEMICAL ABSTRACTS SERVICE, COLUMBUS, OHIO, US; 1982, STOWELL C. P. ET AL.: "Preparation of neoglycoproteins using 2-imino-2-methoxyethyl-1-thioglycosides", XP002683660, Database accession no. 1982:541265 *
EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY, vol. 45, 2010, pages 2713 - 2718
LEE R T: "BINDING SITE OF THE RABBIT LIVER LECTIN SPECIFIC FOR GALACTOSE OR N ACETYL GALACTOSAMINE", 1982, BIOCHEMISTRY, VOL. 21, NR. 5, PAGE(S) 1045-1050, ISSN: 0006-2960 *
Y. C. LEE, BIOCHEMISTRY, vol. 15, 1976, pages 3956 - 3962
Y. C. LEE, LEE BIOCHEMISTRY, vol. 15, 1976, pages 3956 - 3962

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