OLIGOSACCHARIDE AMINO ALDITOLS AND ASSAY METHOD

Abstract: Disclosed is a method of detecting the presence of a compound of interest (such as an enzyme), or the spatial distribution within a sample of a compound of interest. The method comprises the step of impregnating or coating a suitable base material with a marker substance capable of reacting or interacting with the compound of interest, the marker substance being labelled in a detectable manner (for example fluorescently, radioactively or being coloured) and having a substantially different affinity for the base material than the product of the reaction or interaction thereof with the compound of interest. The method further comprises the steps of bringing the impregnated base material into contact with the sample under test, to permit the labelled marker substance to react or interact with any target compound present in the sample, removing either unreacted labelled marker substance or labelled reacted product with a suitable solvent, and detecting the presence of labelled marker substance remaining on said base material, or detecting the liberation of labelled reacted product from said base material, to give an indication of the presence or absence of the compound of interest. The marker substance may be an oligosaccharide, oligosaccharidyl-1-amino-1-deoxy-alditols (OADs) being particularly preferred. A method of manufacturing OADs is also disclosed.

Description: OLIGOSACCHARIDE AMINO ALDITOLS AND ASSAY METHOD. The invention relates to methods for locating and measuring the activities of compounds such as enzymes, e.g. glycosylhydrolases (enzymes that catalyse the hydrolysis of glycosidic linkages) , transglycosylaseε (enzymes that catalyse transglycosylation) , and lyases (enzymes that catalyse the eliminative cleavage of glycosidic linkages) . Living organisms possess a wide variety of glycosylhydrolase and transglycosylase enzymes, which play numerous diverse roles in the lives of the organisms by acting on carbohydrate substrates including xyloglucan, other hemicelluloses, cellulose, starch, glycogen, chitin and pectic polysaccharides. Laboratory methods for detecting and measuring theεe enzymes usually employ aqueous solutions of appropriate substrates placed in vessels (such as test tubes) , which are monitored for enzyme-catalysed changes e.g. by colorimetric, radiochemical, chromatographic or viscometric methods . However, such methods tend to be time-consuming and do not readily lend themselves to the screening of numerous samples of the enzymes. In addition, the methods do not readily provide spatial information on the distribution or localisation of the enzymes within a specimen such as a gel electrophoretogram or part of an organism. In one aspect, the present invention provides a method of detecting the presence of a compound of interest, or the spatial distribution within a sample of a compound of interest, the method comprising the step of impregnating or coating a suitable base material with a marker substance capable of reacting or interacting with the compound of interest, the marker substance being labelled in a detectable manner (for example fluorescently, radioactively or being coloured) and having a substantially different affinity for the baεe material than the product of the reaction or interaction with the compound of interest, the method further comprising the steps of bringing the impregnated base material into contact with the sample under test to permit the labelled marker substance to react or interact with any target compound present in the sample, removing either unreacted labelled marker substance or labelled reacted product with a suitable solvent, and detecting the presence of labelled marker substance remaining on said base material, or detecting the liberation of labelled reacted product from said base material, to give an indication of the presence or absence of the compound of interest . The invention exploits the ability of some carbohydrates but not others to bind to the base material in the chosen solvent . Preferably, the compound of interest is an enzyme, and the marker substance is a substrate for the enzyme. Said substrate for the enzyme may be a protein, a peptide, a polysaccharide or an oligosaccharide. In a particularly preferred method, the substrate is an oligosaccharidyl-1- amino-l-deoxy-alditol These compounds have been developed by the applicants and are believed to be new in themselves. They are referred to where appropriate in this specification as "OADs", which term is to be taken to include 0_-glycosyl -1-amino- 1 -deoxy-alditols , 0_- oligoglycosyl-1-amino-*1-deoxy-alditols, O-glycosyl-6-amino- 6-deoxy-aldonic acids, 0-oligoglycosyl-6-amino-6-deoxy- aldonic acids and/or their lactones, and related products of reductive amination. It may in some cases be an advantage that the reducing carbohydrate not only possesses one reducing site but possesses two or more reducing sites randomly placed in said reducing carbohydrate. These additional reducing sites may be obtained with an oxidation agent such as sodium periodate. The invention also provides a method of manufacturing OADs involving the reaction of a reducing carbohydrate with an ammonium salt and a reducing agent. Preferably, the ammonium salt is ammonium hydrogencarbonate and the reducing agent is sodium cyanoborohydride . Further preferred features of the invention are described in the subsidiary claims . As stated above the method of the invention may be used for detecting enzyme activity and/or for detecting the spatial distribution within a sample of an enzyme. In particular (but not exclusively) the method of the invention may be used for the following enzymes : transglycosylases such as dextransucrase, amylosucrase, inulosucrase, glycogen phosphorylase, starch phosphorylase, xylan synthase, glucan synthase, pectin synthase , glycogen synthase, starch synthase, callose synthase, chitin synthase,* glycosylhydrolases (endoacting) such as xylanase, endoglucanase, chitinase, mannanase, polygalacturonase, β- amylase, arabinanase, galactanase, rhamnogalacturonase; lyases such as pectin/pectate lyase, alginate lyase; glycosylhydrolases (exoacting) such as α-amylase, amyloglucosidase , -L-arabinofuranosidase , α-D- galactosidase, /3-D-galactosidase, β-D-glucosidase, α-L- fucosidase, β-D-mannoεidase, α-D-mannosidase, exo- polygalacturonase, N-acetyl-3-D~glucosaminidase, cκ-D- xylosidase . A specific embodiment of the invention will now be described by way of example. The enzyme to be tested for in this example is xyloglucan endotransglycosylase (XET) [ref 1] . 1. Preparation of labelled oligosaccharide The reducing oligosaccharide 4-0- [4-0- [4-0- [6-0-α-D- Xylopyranosy1 - β -D- glueopyranosy1 ] -6-0 . - ( 2 - 0_- β - D- galactopyranosyl) -α-D-xylopyranosyl-3-D-glucopyranosyl] -6-0- ( 2 -0_- β - D- galactopyranosy1 ) - α-D-xy1opyranosy1 - β -D- glucopyranoεyl] -D-glucose ("XLLG", uεing the abbreviated nomenclature of ref 2) (1 gram) is dissolved in 25 ml of a saturated aqueous solution of ammonium hydrogencarbonate containing 1 gram of sodium cyanoborohydride (NaCNBH ) and incubated in the dark at 25°C for 7 days to permit reductive amination. The ammonium hydrogencarbonate is then removed by drying, and the (ninhydrin-reactive) aminated derivative of XLLG is purified e.g. by gel-permeation chromatography or cation-exchange chromatography. The product is believed to be an oligosaccharidyl-1-amino-1-deoxyalditol, i.e. a derivative of XLLG in which the reducing terminal D-glucose moiety has been replaced by 1-amino-1-deoxy-D-glucitol . The oligosaccharidyl-1-amino-1-deoxyalditol (50mg) is dissolved in 3 ml of 3% borax (di-sodium tetraborate; pH = 9.0-9.5) and a freshly-prepared solution of lOmg lissamine rhodamine sulphonyl chloride [purchased from Molecular Probes Inc., USA] in 0.75 ml of dry dimethylformamide (DMF) is added gradually, with stirring, and the mixture is incubated in the dark overnight. A further 0.75 ml of DMF containing 10 mg lissa.mine rhodamine sulphonyl chloride is added and the mixture incubated for a further 8 h. The bright pink oligosaccharidyl-1-amino-1-deoxyalditol- lissamine-rhodamine conjugate (referred to as XLLGol-SR) is purified by gel-permeation chromatography followed by reversed-phase chromatography on a C 1B -silica gel column. After washing of the latter column with water, a methanol gradient is applied and the XLLGol-SR elutes in about 50% methanol . 2. Preparation of xyloglucan-impregnated paper Whatman No. 1 filter paper is moistened with a 1% aqueous solution of xyloglucan and dried. The XLLGol-SR preparation is diluted into enough 75% aqueous acetone to give an absorbance at 580 nm (A sβ0 ) of 0.2; the xyloglucan- coated sheet of Whatman No. 1 paper is then dipped through this solution and re-dried,* the product is referred to as "XET-paper" . Suitably sized pieces (e.g. 72 x 108 mm) of the XET-paper may then be glued with a non-aqueous adheεive onto a non- bsorbent medium such as a sheet of transparent acetate. 3. Conduct of assay (a) Dot-blot test for XET activity in aqueous solutions (i) The source (e.g. the stem of a growing plant) to be tested for XET activity is homogenised in a suitable buffer such as 250 mM succinate, pH 5.5, containing 10 mM calcium chloride and 10 mM dithiothreitol, and the homogenate is centrifuged to give a clear supernatant. Other samples that might be tested for XET activity include fractions eluted from a chromatography column, samples of spent culture media from plant cell suspenεion culture, or samples of XET that have been treated with subεtances whose effect on XET activity are to be investigated. (ii) A spot of each enzyme solution is pipetted on to a marked position in a piece of XET-paper. If the spots are 4 μl, the spacing between the samples can conveniently be 9 mm (centre-to-centre, i.e. aε in a standard 96-well test plate format) . (iii) The XET paper is then quickly (before the spots have dried) clamped between two sheets of plastic (e.g. acetate sheets, as used on overhead projectors) and incubated e.g. at 20°C for 1 hour. (iv) The incubated XET-paper and its plastic backing is then placed (paper-side down) in a diεh containing about 150 ml of a solvent [e.g. freshly prepared ethanol/formic acid/water (1:1:1 by volume) ] that will remove from the paper the unreacted XLLGol-SR but not any XLLGol-SR that haε become incorporated into the xyloglucan owing to XET- catalysed transglycosylation. The paper now readily detaches from the plastic backing. (v) The paper is then rinsed in running water for 5 minutes, then in approximately 100ml of acetone for 5 minutes, and then dried thoroughly. If deεired, drying can be expedited by a 5-minute treatment in an oven at 80°C. (vi) The paper is then examined under a short- wavelength ultraviolet lamp (e.g. emitting at 254 nm; suitable eye- and skin-protection should be worn) . Active XET is indicated by a pink (orange-fluorescing) spot, which can be quantified, e.g. by use of a scanning spectrofluorimeter. The assay method is useful for testing for the presence of substanceε that promote or inhibit XET activity. (b) Activity printing, e.g. to localise XET activity within plant tissue or an zymograms The XET-paper is buffered, e.g. by spraying with 500 mM succinate, pH 5.5, containing 10 mM calcium chloride and 10 mM dithiothreitol . The plant tisεue is sliced to produce an exposed surface; for mapping XET activity over the surface area of a leaf, it may be beneficial to press the leaf between the test paper and a rough surface (e.g. sandpaper) or to press the leaf on the test paper under a very cold (eg -80°C) , heavy flat metal place (which iε subsequently allowed o reach room temperature) to promote tissue disruption. The exposed tissue surface, or a gel electrophoretogram, o:r other surface in which XET activity is to be mapped, is then pressed on to the damp paper for about 1 minute so that the enzyme is transferred on to the paper. The enzyme source is then removed from the XET- paper, and steps (iii) to (vi) , above, are performed. The above example illustrates the invention as applied to a transglycosylase enzyme, XET. The method is also applicable to the assay of glycosylhydrolase enzymes. For example, Whatman No. 1 paper that has been impregnated with XLLGol-SR (and no xyloglucan) can be used as a dot blot to test for the activity of the glycosylhydrolases (a- xylosidaεe, 3-glucosidaεe and /8-galactosidase) that together catalyse the exo-hydrolysiε of XLLGol . The assay is conducted in a manner similar to that described in 3 (a) , above, except that: in step (iv) a solvent is selected (e.g. 90% ethanol) that removes from the paper the hydrolysis products of XLLGol-SR below a certain εize but not XLLGol-SR itself; and step (v) involves no further solvents, but only drying. Activity of the relevant glycosylhydrolase enzymes thus yields non-fluorescent spots on a fluorescing paper background. The method deεcribed above for preparing XLLGol-SR is readily adaptable to form a generally valuable means of preparing fluorscently (or otherwise) labelled reducing oligosaccharides or polysaccharideε . Such labelled εaccharides (e.g. oligo-SRs) can be used: - in diverse enzymes assays for endo- and exo- glycosylhydrolases and transglycosylases, including those involved in starch metabolism,* in detecting, separating and characterising oligosaccharides of interest by HPLC or TLC in combination with the use of pure glycosylhydrolaεeε, the oligo-SRs acting aε authentic markers; as a probe which is suitable for application, with or without added unlabelled xyloglucan, to plant tissue in order to map the distribution of XET activity histochemically, by uεe of fluoreεcence microεcopy. O-Glycoεyl-1-amino-1-deoxy-alditols and/or 0- oligoglycosyl-1-amino-1-deoxy-alditols can be prepared by the method (reductive amination) described, starting from any reducing oligosaccharide. Analogous products are obtained from oligosaccharides in which the reducing terminus is a modified sugar,* for example, in the case of an oligosaccharide whose reducing terminus is a hexuronic acid moiety, the reaction product is an 0-oligoglycosyl-6-amino- 6-deoxy-aldonic acid or its lactone. Any of these products of the reductive amination reaction can be detected via their amino group, e.g. by staining with ninhydrin, and can themselveε be utiliεed for diverεe purposes including for the chromatographic and electrophoretic analysiε of oligoεaccharides,* aε modulatorε of enzyme action. References [1] Fry SC, Smith RC, Renwick KF, Martin DJ, Hodge SK, Matthewε KJ (1992) : Xyloglucan endotransglycoεylaεe, a new wall-loosening enzyme activity from plants. Biochemical Journal 282: 821-828. [2] Fry SC, York WS, Alberεheim P, Darvill A, Hayaεhi T, Joεeleau J-P, Kato Y, Lorences EP, Maclachlan GA, McNeil M, Mort AJ, Reid JSG, Seitz HU, Selvendran RR, Voragen AGJ, White AR (1993) : An unambiguous nomenclature for xyloglucan- derived oligosaccharides. Physiologia Plan tarum 89: 1-3

Claims: CLAIMS 1. A method of detecting the presence of a compound of interest, or the spatial distribution within a εample of a compound of interest, the method comprising the εtep of impregnating or coating a suitable base material with a marker substance capable of reacting or interacting with the compound of interest, the marker substance being labelled in a detectable manner (for example fluorscently, radioactively or being coloured) and having a substantially different affinity for the base material than the product of the reaction or interaction thereof with the compound of interest, the method further comprising the steps of bringing the impregnated base material into contact with the sample under test, to permit the labelled marker subεtance to react or interact with any target compound preεent in the sample, removing either unreacted labelled marker substance or labelled reacted product with a εuitable solvent, and detecting the presence of labelled marker substance remaining on said base material, or detecting the liberation of labelled reacted product from said base material, to give an indication of the presence or absence of the compound of interest . 2. A method according to claim 1, wherein the compound of interest is an enzyme and the marker substance is a substrate for the enzyme. 3. A method according to claim 2, wherein the enzyme iε a glycosylhydrolase, a tranεglycosylase, or a lyase. 4. A method according to claim 3, wherein the enzyme is one which acts upon xyloglucan, other hemicelluloses, εtarch, glycogen, chitin or pectic polysaccharides. 5. A method according to any of claims 1 to 4, wherein the marker substance is an oligosaccharidyl-1-amino- 1-deoxy-alditol (OAD) , as defined. 6. A method according to any preceding claim, wherein the marker substance is labelled with liεsamine rhodamine sylphonyl chloride. 7. A method according to claim 5 or claim 6, suitable for (a) conducting dot-blot assays of aqueous solutions (i) for the activity of α-xylosidase, /3-glucoεidaεe and β - galactosidase or (ii) for the presence of subεtanceε that increase or decrease the activity of α-xylosidase, β- glucosidase or jβ-galactosidase, or (b) conducting tissue- printing to εtudy the diεtribution of α-xyloεidaεe, β- glucosidase or 0-galactosidase activity within plant tiεsue, or (c) conducting blots of gel eletrophoretograms to study the distribution of oz-xylosidase, /3-glucosidaεe or β- galactosidase activity on zymograms, wherein the substrate is an oligosaccharide (such as XLLG) derived from xyloglucan. 8. A method according to claim 2 , or any claim appendant thereto, wherein the base material is further impregnated with an unlabelled co-substrate for said enzyme. 9. A method according to claim 8, suitable for (a) conducting dot-blot assays of aqueous solutions (i) for the activity of xyloglucan endotranεglycosylase (XET) or (ii) for the presence of substanceε that promote or inhibit XET activity, or (b) conducting tissue-printing to study the distribution of XET activity within plant tissue, or (c) conducting blots of gel eletrophoretograms to study the diεtribution of XET activity on zymograms, wherein the εubstrate is an oligosaccharide (such as XLLG) derived from xyloglucan and the base material is further impregnated with unlabelled xyloglucan. 10. Anoligosaccharidyl-1-amino-1-deoxy-alditol (OAD) , as defined. 11. A method of manufacturing an OAD according to claim 10, comprising the reaction of a reducing carbohydrate with an ammonium salt and a reducing agent. 12. A method according to claim 11, wherein the ammonium salt is ammonium hydrogencarbonate. 13. A method according to claim 11 or claim 12, wherein the reducing agent is sodium cyanoborohydride. 14. A method of labelling an OAD according to claim 10, comprising reacting the OAD with lissamine rhodamine sulphonyl chloride . 15. A method according to any of claims 1 to 9, wherein the base material is filter paper, a nitrocellulose sheet, or an electrophoresis gel (such as polyacrylamide, agarose or starch) . 16. A method according to claim 15, as dependent from any of claims 1 to 9, wherein the base substance is a suitable gel (e.g. polyacylamide) , the method involving the electrophoretic separation of proteins and the m situ identification of those proteins that are glycosylhydrolases and transglycosylaseε capable of acting on the εubstrates impregnated, in order to produce a zymogram. 17. The use of a labelled conjugate formed between lissamine rhodamine sulphonyl chloride and an OAD according to claim 10 and derived from XLLG, as a probe suitable for application, with or without added unlabelled xyloglucan, to plant tissue in order to map the distribution of XET activity histochemically. 18. The use of a ninhydrin-reactive OAD according to claim 10 as an authentic standard (marker) in the analysis of oligosaccharides, e.g. by chromatography (including HPLC, and ca ion-exchange chromatography) or by electrophoresis (including capillary electrophoresis or high-voltage paper electrophoresis) . 19. The use of a labelled conjugates of an OAD according to claim 10 as an authentic standard (marker) in the analysis of oligosaccharides, e.g. by chromatography (including HPLC, thin-layer chromatography or paper chromatography) or by electrophoresis . 20. An assay kit, for example for XET, comprising a base material impregnated with an OAD according to claim 10.

Inventors: FRY STEPHEN CHARLES, FRY, STEPHEN, CHARLES
Applicants: FRY STEPHEN CHARLES, NOVONORDISK AS, FRY, STEPHEN, CHARLES, NOVO NORDISK A/S
Application year: 1996
Main classification: C12Q1
IPC codes: C07H15, C12Q1