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WO1990004596A1 - Isolation d'oligosaccharides - Google Patents

Isolation d'oligosaccharides Download PDF

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Publication number
WO1990004596A1
WO1990004596A1 PCT/GB1989/001294 GB8901294W WO9004596A1 WO 1990004596 A1 WO1990004596 A1 WO 1990004596A1 GB 8901294 W GB8901294 W GB 8901294W WO 9004596 A1 WO9004596 A1 WO 9004596A1
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oligosaccharide
groups
oligosaccharides
column
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PCT/GB1989/001294
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Elizabeth Fay Hounsell
Mark Sebastian Stoll
Ten Feizi
Alexander Macnair Lawson
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3I Research Exploitation Limited
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3204—Inorganic carriers, supports or substrates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C07H1/08—Separation; Purification from natural products
    • 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
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • 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
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C07H15/10—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical containing unsaturated carbon-to-carbon bonds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/552—Glass or silica
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8836—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving saccharides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose

Definitions

  • This invention is concerned with the
  • glycosylation The majority of proteins are post-translationally modified by glycosylation.
  • the degree of glycosylation and the structural pattern of the oligosaccharide chains is dependent on the origin and status of the cell. For example differences in glycosylation pattern are noted for
  • the oligosaccharide chains of glycoproteins have specific sequences recognized by antibodies and mammalian cell lectins. These sequences can be present on both O- and N-linked cores.
  • a short form of 0-linked chain also exists (mono - to tetrasaccharide) which may direct an antigenic response to the surrounding amino acid sequence or be part of an oligosaccharide-protein combined antigenic epitope.
  • protein-linked monosaccharide e.g. at the core region N- acetylgalactosamine different core region sequences are present and the relatively large chains (found e.g. in mucins) are very heterogeneous.
  • microscale quantities e.g. ⁇ g or less
  • the released oligosaccharide groups may be coupled with a lipid molecule post separation from glycoprotein on the PBA column and, more preferably, a glyceride or
  • neoglycolipid The combination of the oligosaccharide and the lipid is referred to as a "neoglycolipid”.
  • a neoglycolipid may comprise a long chain fatty acid moiety of from 5 to 50, preferably 5 to 30 and more preferably 10 to 25, carbon atom length, an example being stearylamine.
  • Simple amino hydrocarbons may also be
  • the amino group is necessary for chemical bonding to the oligosaccharide to form the conjugate.
  • Neoglycolipids may be formed by reacting an oligosaccharide having at least one aldehyde group with an amine having one or more hydrophobic groups.
  • Neoglycolipids of particular interest have the general formula: o
  • n, and m have a value within tne range 5 to 50, preferably 5 to 30, and desirably 10 to 25.
  • the carbohydrate group R 1 may be derived from a monosaccharide, oligosaccharide, or even a polysaccharide which contains an aldehyde group.
  • a carbohydrate group R 1 which does not in itself contain the necessary aldehyde group, may be subjected to oxidation, for example using periodate oxidation.
  • lipids may be derived from phosphatidyl ethanolamine dipalmitoyl 9PPEADP) and its analogues,
  • L-alpha-phosphatidyl-L-serine L-alpha-phosphatidyl- ethanolamine (PPEA) dilauroyl, PPEA dimyristoyl, PPEA distearoyl, PPEA beta-oleoyl-gamma-palmitoyl.
  • PBA phenyl boronic acid
  • oligosaccharide derivatives formed by reductive amination. Reduction amination has been used for the preparation of derivatives for sensitive detection on HPLC (Hase et al., 1984) for increased ionization on MS analysis (Wang et al., 1984) and for antigenic analysis as neoglycolipids (Tang et al., 1986; Tang and Feizi, 1987).
  • the PBA column also has potential applications in other fields where reduced oligosaccharides are obtained from glycoproteins (e.g. by treatment with mild base/borohydride degradation or by hydrazinolysis and reduction, where a simple reduction step can be carried out (e.g. analysis of galatose levels in plasma; Henderson and Fales, 1980) or where reduced oligosaccharides are used as in vivo tracers (e.g. mannitol; Laker et al., 1982).
  • a simple reduction step can be carried out (e.g. analysis of galatose levels in plasma; Henderson and Fales, 1980) or where reduced oligosaccharides are used as in vivo tracers (e.g. mannitol; Laker et al., 1982).
  • Phenylboronic acid is an example, when bound to an inert matrix, of an immobilised support comprising the reactive dihydroxyboryl group, which we have found can exclusively bind the planar vicinal diol groups of
  • the support can be in the form of a silica-bound
  • the present invention therefore provides (1) a method for the sequential isolation and preferably
  • identification of 0-linked and/or N-linked oligosaccharide chains released from a glycoprotein or peptide sample and further provides (2) an analytical method for determining oligosaccharide structure in such a sample, (3) a kit-of- parts suitable for carrying into effect method (1) and/or (2), and (4) apparatus comprising means capable of carrying into effect one or more of said methods.
  • a method of separating specific oligosaccharide chains from a glycoprotein or peptide sample containing O- and/or N-linked oligosaccharide chains which comprises:-
  • phenylboronic acid (PBA) column such that O- linked chains are adsorbed thereon, optionally retaining the effluent for further treatment, and
  • step (d) subjecting the effluent obtained in step (b) to a process of hydrazinolysis to release N-linked chains, followed by reduction
  • step (e) passing the treated sample from step (d) through the same or different phenylboronic acid (PBA) column such that N-linked chains are adsorbed thereon, and
  • a fluorescent or radioactive label is introduced on ⁇ -elimination or on reduction.
  • Preferred agents include, for example, a tritium-labelled borohydride or fluorescent cyanoacetamide.
  • the sample is applied to the support, e.g. the PBA column, in alkaline pH and the support is subsequently eluted under acidic pH.
  • the support e.g. the PBA column
  • the invention also provides an analytical method for determining oligosaccharide structure in a glycoprotein or peptide sample containing O- and/or N- linked oligosaccharide chains which comprises a separation method as defined in the first aspect (preferably including the use of labelled reducing agents in steps (a) and/or d) followed by:-
  • a mild oxidising agent such as periodate to cleave the terminal (protein- proximal) monosaccharide chain
  • step (i) subjecting the product, formed in step (h) to an analytical technique capable of
  • the invention provides an analytical kit for carrying out the methods of the first and/or second aspects of the invention which comprises:-
  • the present invention embraces a method of separating 0-linked and/or N-linked glycosylation and facilitating their subsequent identification by combining the selective oxidative cleavage of the C4-C5 bond of the protein-proximal monosaccharide, together with the high resolving power of t.l.c.
  • the mild oxidising conditions selected are most preferably highly specific, i.e. selective for the alditol moiety, without oxidising other parts of the oligosaccharide groups. This can be achieved by using periodate at
  • the mild periodate oxidation step has surprisingly been found to specifically cleave the C4-C5 bond of the terminal monosaccharide unit e.g. N-acetylgalactosaminitol disubstituted at C3 and C6, or monosubstituted at C3 or C6.
  • the terminal monosaccharide unit e.g. N-acetylgalactosaminitol disubstituted at C3 and C6, or monosubstituted at C3 or C6.
  • oligosaccharides branched at the core N-acetylgalactosaminitol each give two derivatives.
  • Two lipid-linked fragments thus obtained from each oligosaccharide alditol are well resolved on thin layer chromatography and can be sensitively analysed by liquid secondary ion mass spectrometry to assign the sequence and branching patterns of oligosaccharides linked at C6 and C3 to the N-acetylgalactosaminitol.
  • Such neoglycolipids therefore confer considerable advantage for biological and immunological assessment.
  • oligosaccharide profiling is given by way of example only.
  • oligosaccharide alditols isolated from the PBA column can be treated by mild periodate oxidation and the resslting aldehydes specifically formed at the "protein linkage" sugar coupled by reductive amination to lipid by a known procedure or any amine containing molecule (e.g. those giving
  • oligosaccharides prior to the synthesis of neoglycolipids for antigenic and functional studies of oligosaccharides is most desirable.
  • the present method ensures that antigenic or receptor specificity is only assigned to oligosaccharides. All of the
  • oligosaccharides which bind to PBA will be oxidised by periodate which is a necessary step in the production of neoglycolipids. This is because both PBA and periodate require access to planar vicinal hydroxyl groups.
  • the PBA column can potentially be used to isolate reducing mono- and oligosaccharides from body fluids. Only the reducing sugars present in such mixtures would be labelled by reduction or reductive amination and
  • Aluminium backed t.l.c. plates the derivatives can be analysed by direct liquid secondary ion mass spectrometry. This method permits elucidation of the sequence, linkage positions and core region branching patterns of O-linked chains of glycoproteins on less than 5 nmole oligosaccharide and is therefore a powerful tool for their microscale profiling.
  • N-acetylgalactosaminitol at the C4-C5 bond described here accounts for two observations in the earlier study [4]: (a) the successful derivatisation of the oligosaccharide alditol Nl in situ after TLC and (b) the preservation of reactivity of the products with the monoclonal antibody, anti-I Ma [4].
  • the two lipid derivatives thus obtained are obtained:
  • the former would contain the epitope for this antibody which is known to
  • glucitols substituted at C3 or at C4 have differing susceptibilities to fission of C5-C6 and C1-C2 sites.
  • oligosaccharides with reduced end N-acetylgalactosaminitol have been observed to exhibit exclusive fission of the C4-C5 bond of branched oligosaccharides and that the C5-C6 bond which is available in linear oligosaccharides with 3-substitution is not a site of primary oxidation. With the oligosaccharides tested we have found no mass spectrometric evidence for degradation of the oligosaccharides outside the core N- acetylgalactosaminitol.
  • neoglycolipids enabled structural assignments to be made. Thus essential information was obtained for the
  • FIGURE 1 shows a chromatographic standard for
  • FIGURE 2 a mixture of reduced and non-reduced glucose oligomers derived from dextran by hydrolysis fractionated on a PBA column under condition A.
  • the figure shows TLC (solvent 1) with orcinol detection of aliquots of the eluted fractions.
  • G1-G7 oligosaccharides with 1-7 glucose units and the
  • FIGURE 3 a mixture of reduced (GM 1 -ol) and non-reduced octasaccharide GM 1 fractionated on a PBA column under condition A.
  • the figure shows TLC (solvent II) with orcinol detection of aliquots of the eluted fractions 1-10.
  • FIGURE 4 the triasaccharide alditol GlcNAc ⁇ 1-6[Gal ⁇ 1- 3]GalNAc-ol passed through a PBA column under condition A.
  • the figure shows TLC (solvent II) with orcinol detection of
  • FIGURE 5 a mixture of 20 common amino acids and reduced dextran hydrolysate on a PBA column under condition B.
  • the figure shows TLC (solvent)) of aliquots of the eluted fractions 1-9.
  • FIGURE 6 TLC (solvent II) of aliquots of the eluted fractions (1-9) from a PBA column run under condition B.
  • (a) A mixture of GA, Asn, Gly-Ser and fetuin detected with ninhydrin.
  • (b) A mixture of GM 1 -ol, GM 1 and NGGA detected with orcinol.
  • FIGURE 7 a mixture of reduced (LacNAc-ol) and unreduced (Lac-NAc) N-acetyllactosmine fractionated on a PBA column under condition B.
  • the figure shows TLC (solvent II) with orcinol detection of aliquots of the eluted fractions 1- 10.
  • FIGURE 8 a mixture of reduced (2-3ol and 2-6ol) and unreduced (2-3 and 2-6) NeuAc ⁇ 2-3(6 )Gal ⁇ 1-4Glc fractionated on a ABA column under condition B.
  • the figure shows TLC (solvent II) with orcinol detection of aliquots of the eluted fractions 1-10.
  • FIGURE 9 the reaction mixture obtained in the reductive amination of lactose with PPEADP and soium
  • FIGURE 10 proposed mechanism of action of the PBA column, (a) Activation of matrix bound PBA to give
  • FIGURE 11 TLC of 12 oligosaccharide alditol fractions derived from human meconium glycopeptides. Each lane contained approximately lnmol oligosaccharide. Large arrow indicates origin. Staining was with orcinol. The continuous band
  • FIGURE 12 TLC of the neoglycolipids derived from the di-, tri-, and tetrasaccharide alditols R1, O1 and N1. Each lane contained 1-2 nmol of carbohydrate. Panel A, orcinol staining; panel B, primulin staining; arrow indicates origin; L, excess lipid. The numbered hands at positions 1,2 and 8 were analysed by LSIMS; and
  • FIGURE 13 shows primulin stained chromatograph of the neoglycolipids obtained from the oligosaccharide fractions K5(3), K2(3), K3(4), K6(2), K6(3), K4(2), K4(3) and K3(2) in panel A, and from standard oligosacchardies designated 03 and 07, (panel B). Each lane contained 1-2nmol of oligosaccharide. Bands at positions 1-13 are indicated; arrow indicates origin. Flow chart for isolation of O- and N-linked chains
  • Glycoprotein (having both O- and N-linked chains)
  • dipalmitoyl(PPEADP), the dipeptide Gly-Ser (GS), amino acids and dextran T200 were from Sigma Chemical Co. Ltd [Poole, Dorset, UK].
  • a mixture of glucose oligomers was obtained from dextran T200 by hydrolysis in 01M H 2 SO 4 for 16h at 100oC.
  • NeuAcc(2-3Gal ⁇ 1-4GlcNAc ⁇ 1-Asn (NGGA) from the urine of a patient with aspartyijlucosaminuria were kindly supplied by Drs S. G. Strecker and J.C. Michalski [U.S.T.L. Flanders Artois,
  • GlcNAc-Asn was chemically synthesized according to the method of Spinola and Jeanloz (1970).
  • the trisaccharide GlcNAc ⁇ 1-6[Gal ⁇ 1-3]GalNAc-ol was obtained from meconium glycopeptides by base-borohydride degradation (Hounsell et al., 1985).
  • Hexose containing material was detected by spraying with orcinol reagent (2% orcinol w/v in ethanol containing 5% sulphuric acid) and heating at 105oC for 5 min.
  • Material containing amino acids was detected by spraying with ninhydrin reagent (0.3% w/v ninhydrin and 3% v/v acetic acid in butan-1- ol) and heating at 105oC for 5 min.
  • PPEADP and PPEADP derivatives were chromatographed in solvent system chloroform/methanol/ water, 60:35:8 v/v (Solvent III) and detected by spraying with primulin reagent (1 mg primulin/100 ml acetone/water, 4:1 v/v) and visualizing under long wave UV.
  • the PBA column was activated by consecutive washing with 2 x 1 mL of methanol, 1 mL of 0.1 M HCl, 2 x lmL water and 4 x 1 mL of 0.2 M NH 4 OH. Samples were then applied to the column under one of three conditions (A-C described below).
  • Condition A the column was washed with 2 x 1 mL water and the sample applied in 200 ⁇ L water (fraction 1) followed by elution with 3 x 200 ⁇ L water (fractions 2-4), 5 x 200 ⁇ L 0.1 M acetic acid (fractions 5-9) and 1 mL 0.1 M HC1 (fraction 10).
  • Condition B the sample was applied in 200 ⁇ L 0.2 M NH 4 OH (fraction 1) without a prior water wash of the column and elution carried out with 2 x 100 ⁇ L 0.2 M NH 4 OH (fractions 2 and 3), 2 x 200 ⁇ L water (fractions 4 and 5) and 4 x 0.1 M acetic acid (fractions 6-10).
  • Condition C the column was washed with 2 x 1 mL water, 2 x 1mL methanol and 2 x 1mL chloroform/methanol (1:1 v/v).
  • the products of a reductive amination reaction between lactose and PPEADP (details to be reported elsewhere) where then applied in 350 ⁇ L chloroform/methanol (1:1 v/v) (fraction 1) and elution carried out with chloroform/methanol (1:1 v/v) (5 x 200 ⁇ L;
  • Figure 2 shows that for oligosaccharides of increasing molecular size those having glucose at the reducing end are not retained by the PBA column in mild alkali (eluted in fractions 2 and 3) whereas the corresponding alditols are retained and specifically eluted with decreasing pH (in fractions 6 and 7).
  • the same behaviour (Fig. 3) was shown by the glycoprotein derived octasaccharides GM 1 which could be also separated from its alditol, the latter being retained by the PBA column.
  • glycoproteins (Hounsell et al., 1985), was also retained by the column and eluted in fractions 5-9 (Fig. 4).
  • the column therefore is generally applicable to the separation of oligosaccharides from their alditols.
  • oligosaccharides could also be separated from proteinaceous material, first a mixture of the 20 common amino acids was passed down the column under the identical conditions of Figs. 2 to ⁇ (condition A). Some retention of the more basic amino acids (Arg, Lys, hydroxyLys and His) was observed but this could be obviated by applying the samples in 0.2M NH 4 OH (having omitted the water wash after the 0.2 M NH 4 OH column activation step;
  • condition B there was some retention of non- reduced oligosaccharides, e.g. GM 1 (Fig. 6b) and LacNAc (Fig. 7). Therefore, although all the alditols tested could be separated from non-sugar components under condition B, condition A is required to completely separate some oligosaccharides from their alditols.
  • Condition B was better suited than condition A to separation of sialyllactose from sialyllactitol.
  • condition B the non-reduced isomers eluted in fractions 1-3 and the alditols in fractions 3-8, giving greater than 90%
  • sialylated alditols from non-reduced siayylated oligosaccharides, non-carbohydrate material and glycopeptides.
  • a less specific and weaker mechanism of binding to PBA appears to be by cation exchange which would explain the observed
  • HP-TLC plates (aluminium-backed 5 ⁇ m silica, Merck) were from BDH, Poole, Dorset, UK.
  • C 18 Bond Elut columns (100mg in 1ml) were from Jones Chromatography, Hengoed, Glamorgan, UK.
  • Sodium cyanoborohydride, PPEADP, primulin, orcinol, lactose and imidazole were from Sigma Chem. Co. Ltd., Poole, Dorset, UK.
  • Chloroform, ethanol, methanol, sodium periodate, butan-2,3-diol, nitrobenzyl alcohol, tetramethylurea and triethanolamine were Analytical Reagent grade.
  • Imidazole buffer was 40mM adjusted to pH 6.5 with hydrochloric acid.
  • Primulin reagent was 0.001% in acetone/water, 4:1 by vol.
  • Orcinol reagent was 0.2%. (wt/vol) in ethanol containing water (5.6%, by vol) and concentrated sulphuric acid (11.1%, by vol).
  • the oligosaccharides investigated were di- to hexasaccharide alditols released from human meconium
  • Gal ⁇ 1-3GlcNAc ⁇ 1-3Gal and Gal ⁇ 1-4GlcNAc ⁇ 1-3Gal (designated 03 and 07, respectively) were donated.
  • carbohydrate-PPEADP conjugates was applied as a 5mm band and developed in solvent system chloroform/methanol/water (130:5:9, by vol).
  • the lipids were visualised by spraying with primulin reagent and viewing under long-wave UV and carbohydrate (hexose) was visualised by spraying with orcinol reagent and heating at 105o for 5 min.
  • neoglycolipid bands were cut out as strips (5x1.5mm) on the aluminium backed silica gel and placed on a standard stainless steel LSIMS target probe.
  • Oligosaccharide 01 gave rise to two neoglycolipid bands (Fig. 12) consistent with its known structure (Table 1). The first at position 1 was identical to that derived from
  • Oligosaccharide fraction Nl gave rise to two major neoglycolipid bands (Fig. 12), in accord with the structure proposed (Table 1), and several minor bands which were not investigated further.
  • the faster migrating band at position 1 had a spectrum identifying it as Gal ⁇ 1-3-OX (Table 2).
  • the other major band at position 8 stained with both orcinol and primulin and gave a molecular ion [M-H] m/z 1099.7 and fragment ions m/z 937 and 734 arising from the loss of Gal and Gal- GlcNAc, respectively, consistent with the derivative Gal ⁇ 1- 4GlcNAc ⁇ 1-6-OY.
  • oligosaccharides shows that these components are difficult to resolve. However they are mostly separated as neoglycolipid derivatives (Fig. 13 panel A), thus allowing analysis of the individual components by LSIMS. These are discussed below in the order shown in the chromatogram except for the previously
  • Gal-GlcNAc-6-OY identified above and in the presence of Ga1-3-OX at position 1 would be derived from the minor contaminant oligosaccharide Nl, previously detected (Table 1).
  • GlcNAc-3-OX sequence remains an alternative possibility in the absence of sequence ions).
  • the relative amounts of the -6-OY and -3-OX sequences detected in this band suggest the presence of a previously undetected pentasaccharide with the structure:
  • the minor neoglycolipid bands at position 1 and 12 having [M-H] 997.6 and 1464.8 are consistent with the presence of oligosaccharides related to Nl and to the major component of fraction K4(3) as detected previously (Table 1).
  • a third minor band at position 13 with [M-H]- m/z 1727.9 may arise from the linear sequence 13a (Table 2) in the absence of GC-MS data for the branched Gal residue in this fraction [3].
  • Fraction K6(2) gave one prominent neoglycolipid band at position 11 (Fig.13) with [M-H] m/z 1565.9 and fragment ions m/z 1403, 1200, 1038 and 835 consistent with the sequence
  • Gal ⁇ 1-3GlcNAc ⁇ 1-3Gal ⁇ 1-4GlcNAc-3-OX (Table 2) which would arise from the previously identified major oligosaccharide in this fraction (Table 1).
  • a minor band at position 12 had a negative ion spectrum identical to that from band 11 and was deduced to arise from the minor oligosaccharide isomer, position 12
  • Fraction K6(3) gave two major neoglycolipid bands (Fig. 13 Panel A) of which the slower migrating band at position 9 gave a LSIMS spectrum identical to that derived from fraction K2(3) (Table 2) consistent with the structure proposed (Table 1) for the 6-linked branch of the major oligosaccharide alditol in this fraction.
  • the faster migrating band at position 3 was shown by LSIMS (Table 2) to correspond to derivative (Fuc ⁇ 1- 2Gal ⁇ 1-3-OX) from t e proposed 3-linked branch giving a molecular ion [M-H] m/z
  • Fraction K2(4) gave rise to two neoglycolipid bands: one migrated at position 8 and gabe a negative ion spectrum (Table 2) consistent with the sequence Gal ⁇ 1-4GlcNAc ⁇ 1-6-OY that is predicted from the 6-linked branch of the major
  • Fraction K4(2) gave two prominent neoglycolipid bands migrating at positions 4 and 9. The latter band gave a negative ion spectrum identical to the derivative from fraction K2(3) (Table 2) which is the expected derivative from the 6-linked branch of the major oligosaccharide characterised previously in this fraction (Table 1). The band at position 4 gave a spectrum (Table 2) having a molecular ion [M-H]- m/z 1200.5 and
  • Fraction K4(3) was shown previously to contain several oligosaccharides of which only the major component was
  • Gal-GlcNAc and Gal-GlcNAc-Gal consistent with the sequence 12b given in Table 2:
  • LSIMS analysis of the prominent slower migrating band at position 13 enabled a second oligosaccharide sequence to be identified in this fraction:
  • oligosaccharides was given by the characteristic satellite peaks at -18 and +28 daltons from the fragment peak.
  • Additional oligosaccharides suggested to be present from MS analysis of free oligosaccharides could be identified by LSIMS analysis of the minor neoglycolipid bands migrating at positions 4, 8, 9 and 10 (Table 2). These were hexasaccharides related to the major components in fractions K2(4), K3(4) and K4(2), deduced from the band pairs at positions 8a and 9b, 8a and 10a and at positions 4 and 9a, respectively. Derivatives from a fourth minor oligosaccharide related to K2(3) suggested by MS analysis of the free oligosaccharides, would be contained within neoglycolipid bands at positions 1 and 9a.
  • This fraction consisted mainly of pentasaccharides as deduced by LSIMS analysis of the native fraction which gave only one molecular ion species [M-H] m/z 952.
  • M-H molecular ion species
  • the fraction was too heterogeneous to be characterised by the previous MS and NMR analyses except for the following: HI proton signals at 4.852 and 4.853 ppm (at 285K) indicated the presence of non- reducing terminal GlcNAccL residues and major H2 and H5 signals from GalNAcol were within 0.006 ppm upfield of those for
  • oligosaccharide K2(3) indicating that the major oligosaccharides were branched with GlcNAc at C6 and Gal at C3 of the core region GalNAcol.
  • the neoglycolipids from fraction K3(2) resolved into 6 discrete bands at positions 1, 2, 5, 8, 9 and 10 and a broad band at position 12.
  • the bands at positions 1 and 2 were identified (Table 2) from their LSIMS spectra as the derivatives Gal ⁇ 1-3-OX and GlcNAc ⁇ 1-6-OY.
  • the weak band at position 5 gave a LSIMS spectrum consistent with HexNAc-Hex-3-OX from the [M-H] ion m/z 1200.7 and fragment ions m/z 997 and 835 given by loss of HexNAc and HexNAc-Hex respectively (Table 2).
  • the slower migration of this band compared to that at position 4
  • positions 9 and 10 each give a spectrum consistent with the presence of two sequences; the first GlcNAc-Gal-GlcNAc-6-OY having [M-H] m/z 1302.9, with fragment ions m/z 1099 and 937 corresponding to loss of GlcNAc and GlcNAc-Gal respectively, and the second Gal-GlcNAc-Gal-3-OX having [M-H] m/z 1362.8 and fragment ions 1200 and 997 corresponding to loss of Gal and Gal-GlcNAc respectively (Table 2).
  • the band at position 12 gave a spectrum (Fig.
  • HexNAc- HexNAc-Hex-3-OX having [M-H] m/z 1565.9 and fragment ions 1362, 1200, 997 and 835 resulting from loss of HexNAc, HexNAc-Hex, HexNAc-Hex-HexNAc and HexNAc-Hex-HexNAc-Hex, respectively, and a minor component with the sequence Fuc-Hex-HexNAC-Hex-3-OX ( [M- H]- m/z 1508.9).

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Abstract

Procédé de séparation de groupes d'oligosaccharides à liaison -O et/ou à liaison -N d'un milieu comprenant une glycoprotéine, un protéoglycan ou un glycopeptide auxquels lesdits groupes sont liés, dans lequel lesdits groupes sont libérés desdits glycoprotéine, protéoglycan ou glycopeptide dans ledit milieu et successivement mis en contact, dans leur forme alditol, avec un support portant un groupe dihydroxyboryle réactif afin de sélectivement lier lesdits groupes d'oligosaccharides alditol en l'absence de liaison d'autres groupes audit support. Ledit procédé consiste ensuite à libérer du support les groupes liés. On utilise de préférence une colonne d'acide phénylboronique (APB) pour la liaison et la libération sélectives. Les fragments d'oligosaccharides sont ensuite accouplés après clivage C4-C5 par oxydation sélective, à des molécules de lipides afin d'aider à la détection, à l'identification ainsi que dans d'autres procédés analytiques mettant en ÷uvre la séparation chromatographique et des techniques spectrographiques de masse.
PCT/GB1989/001294 1988-10-28 1989-10-30 Isolation d'oligosaccharides WO1990004596A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994021656A3 (fr) * 1993-03-12 1994-11-10 Genzyme Corp Nouveaux conjugues phospholipide-saccharide
WO1998045700A3 (fr) * 1997-04-09 1999-03-11 Joachim W Engels Procede de sequençage de biopolymeres par spectrometrie de masse
WO2008006373A1 (fr) * 2006-07-12 2008-01-17 Merck Patent Gmbh Détection en phase solide de monosaccharides terminaux clivés de substrats glycosylés
WO2014040066A1 (fr) * 2012-09-10 2014-03-13 The Johns Hopkins University Analyse, en phase solide, de glycanes et de glycopeptides et puce microfluidique pour l'extraction et l'analyse glycomiques, et ses procédés d'utilisation
WO2014074756A1 (fr) * 2012-11-09 2014-05-15 Children's Medical Center Corporation Procédés et réactifs de glycoprotéomique
US9625468B1 (en) * 2015-11-19 2017-04-18 Michael A. Madson Method of isolating and analyzing oligosaccharides in glycoproteins

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4269605A (en) * 1978-06-28 1981-05-26 Amicon Corporation Method and kit for separation of glycoproteins
US4406792A (en) * 1981-11-16 1983-09-27 Glad Magnus J Separation agent
WO1987002777A1 (fr) * 1985-10-24 1987-05-07 Research Corporation Limited Reactif biochimique
EP0227240A1 (fr) * 1985-10-16 1987-07-01 Farmos-Yhtyma Oy Procédé pour un essai immunologique d'une analyte macromoléculaire

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4269605A (en) * 1978-06-28 1981-05-26 Amicon Corporation Method and kit for separation of glycoproteins
US4406792A (en) * 1981-11-16 1983-09-27 Glad Magnus J Separation agent
EP0227240A1 (fr) * 1985-10-16 1987-07-01 Farmos-Yhtyma Oy Procédé pour un essai immunologique d'une analyte macromoléculaire
WO1987002777A1 (fr) * 1985-10-24 1987-05-07 Research Corporation Limited Reactif biochimique

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Biomedical Chromatography, Vol. 2, No. 6, 1988 M.S. Stoll et al: "Selective Purification of Reduced Oligosaccharides Using a Phenylboronic Acid Bond Elut Column: Potential Application in HPLC, Mass Spectometry, Reductive Amination Procedures and Antigenic/Serum Analystis. ", and the whole article. *
Chemical Abstracts, volume 104, no. 13, 31 March 1986, (Columbus, Ohio, US), P.W. Tang et al : "Novel approach to the study of the antigenicities and receptor functions of carbohydrate chains of glycoproteins. ", see page 542, abstract 107512m, & Biochem. Biophys. Res. Commun. 1985, 132( 2), 474- 480 *
Chemistry and Industry, Vol.,30 May 1959 (London) M.J. Clancy et al: "Selective periodate oxidation of reducing-end groups in oligosaccharides. ", see page673 - page 675. *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994021656A3 (fr) * 1993-03-12 1994-11-10 Genzyme Corp Nouveaux conjugues phospholipide-saccharide
WO1998045700A3 (fr) * 1997-04-09 1999-03-11 Joachim W Engels Procede de sequençage de biopolymeres par spectrometrie de masse
WO2008006373A1 (fr) * 2006-07-12 2008-01-17 Merck Patent Gmbh Détection en phase solide de monosaccharides terminaux clivés de substrats glycosylés
JP2009542233A (ja) * 2006-07-12 2009-12-03 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツング グリコシル化された基質から切断される末端単糖の固相検出
US8445288B2 (en) 2006-07-12 2013-05-21 Merck Patent Gmbh Solid-phase detection of terminal monosaccharides cleaved from glycosylated substrates
WO2014040066A1 (fr) * 2012-09-10 2014-03-13 The Johns Hopkins University Analyse, en phase solide, de glycanes et de glycopeptides et puce microfluidique pour l'extraction et l'analyse glycomiques, et ses procédés d'utilisation
WO2014074756A1 (fr) * 2012-11-09 2014-05-15 Children's Medical Center Corporation Procédés et réactifs de glycoprotéomique
US10745737B2 (en) 2012-11-09 2020-08-18 Children's Medical Center Corporation Methods and reagents for glycoproteomics
US9625468B1 (en) * 2015-11-19 2017-04-18 Michael A. Madson Method of isolating and analyzing oligosaccharides in glycoproteins

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