WO1992019974A1

WO1992019974A1 - Sequencing of oligosaccharides

Info

Publication number: WO1992019974A1
Application number: PCT/GB1992/000830
Authority: WO
Inventors: Rajesh Bhikhu Parekh
Original assignee: Oxford Glycosystems Limited
Priority date: 1991-05-07
Filing date: 1992-05-07
Publication date: 1992-11-12
Also published as: JPH06507242A; EP0586419A1

Abstract

The present invention relates to the analysis of oligosaccharides and more particularly to the form of analysis known as sequencing of oligosaccharides. The invention provides, inter alia, a process suitable for the sequencing of an oligosaccharide wherein an oligosaccharide entity is immobilised on a support material. The oligosaccharide entity may be, for example, an oligosaccharide, a product of an oligosaccharide or a species having an oligosaccharide portion. The support material may be, for example, a solid support material such as 1,1' carbonyldiimidazole activated agarose.

Description

Sequencing of Oligosaccharides

The present invention relates to the analysis of oligosaccharides and more particularly to the form of analysis known as sequencing of oligosaccharides.

According to one aspect of the present invention there is provided a process suitable for use in the sequencing of an oligosaccharide wherein an oligosaccharide entity is immobilised on a support material.

According to another aspect of the present invention there is provided a process suitable for use in the sequencing of an oligosaccharide which process includes the step of immobilising an oligosaccharide entity on a support material.

According to a further aspect of the present invention there is provided apparatus suitable for use in the sequencing of an oligosaccharide which apparatus includes an immobilised oligosaccharide entity.

According to yet a further aspect of the present invention, there is provided apparatus suitable for use in the sequencing of an oligosaccharide which apparatus includes a support material upon which an oligosaccharide entity may be immobilised.

The oligosaccharide entity may be, for example, an oligosaccharide, or a product of an oligosaccharide, or a species having an oligosaccharide portion. A product of an oligosaccharide may be, for example, a product produced by previously applying a sequencing agent to an oligosaccharide; by way of example, the product may itself be an oligosaccharide. Thus, it is to be understood that, by way of example, an oligosaccharide as such may be immobilised and subjected to sequencing in accordance with the present invention; by way of further example, as an alternative, a product of an oligosaccharide may be subjected to sequencing in accordance with the present invention. (It will be appreciated that the product may itself be an oligosaccharide.)

Alternatively, by way of example, an oligosaccharide provided as an oligosaccharide portion of a species having an oligosaccharide portion (e.g. an oligosaccharide linked to a conjugate) may be subjected to sequencing in accordance with the. present invention.

Glycoproteins and glycolipids are examples of species (having a portion comprising an oligosaccharide) which may be immobilised and subjected to sequencing in accordance with the present invention such that oligosaccharide is subjected to sequencing.

Thus, by way of further example, an oligosaccharide may, if desired, be immobilised and subjected to sequencing in accordance with the present invention whilst still attached to a conjugate thereof (e.g. a peptide chain) provided that the conjugate does not interfere in the sequencing to any unacceptable degree.

From the foregoing disclosure it will be appreciated that, by way of example, an oligosaccharide to be subjected to sequencing may be provided in any suitable form and in any suitable manner.

Also, from the foregoing disclosure it will be appreciated that in accordance with the present invention sequencing of an oligosaccharide may include, for example, applying a sequencing agent to an oligosaccharide, or a product of an oligosaccharide, or a species having an oligosaccharide portion.

An example of a species having an oligosaccharide portion is a species comprising an oligosaccharide attached to a conjugate.

An oligosaccharide entity may be immobilised on a support material by any suitable means.

Thus, where for example, the oligosaccharide entity is an oligosaccharide, or a product thereof, immobilisation may be effected, for example, by means of chemical attachment (e.g. covalent linkage) via a reducing terminus of an oligosaccharide.

An oligosaccharide entity comprising an oligosaccharide, or a product thereof, may be immobilised in accordance with the present invention for example by direct covalent linkage with a support material, or by direct non-covalent (e.g. hydrophilic) linkage with a support material.

Alternatively, by way of example, an oligosaccharide entity comprising a species having an oligosaccharide portion may be immobilised on a support material before being subjected to sequencing.

By way of example, where it is desired to immobilise an oligosaccharide, or a product thereof, whilst still attached to a conjugate the conjugate may be linked (e.g. by covalent linkage or non-covalent (e.g. hydrophilic) linkage) to a support material such that the oligosaccharide, or product thereof, is indirectly linked to the support material via the conjugate. By way of example, where an oligosaccharide entity is an oligosaccharide, or a product of an oligosaccharide, immobilisation of the oligosaccharide, or the product of an oligosaccharide, on a support material may be effected by means of the following procedure: unreduced oligosaccharide + 2-amino pyridine/ NaBH-_jCN→oligo-pyridylamino derivative→conjugation.

By way of further example, where an oligosaccharide entity is an oligosaccharide, or a product of an oligosaccharide, the oligosaccharide, or the product of the oligosaccharide, may be immobilised on a support material by means of the following procedure: unreduced oligosaccharide + dansyl hydrazine/TFA -> oligo-dansyl hydrazine derivative, oligo-dansyl hydrazine derivative + NaBH₄/H₂0 → conjugation.

It is to be understood that attachment of an oligosaccharide, via a reducing terminus, to a support material, is preferably independent of the oligosaccharide structure; the attachment may not, for example, require the reducing terminus monosaccharide to be retained in a ring-closed configuration.

Any suitable support material may be used in accordance with the present invention.

An example of a support material for use in accordance with the present invention is a solid support material comprising 1,1'carbonyldiimidazole-activated agarose.

Oligosaccharides form a class of chemical compounds which are each made up of a number of monosaccharide units linked together by glycosidic bonds. Important sources of naturally occurring oligosaccharides are glycoproteins in which saccharides are found linked to a peptide chain either by an N-glycosidic bond or by an 0- glycosidic bond; these oligosaccharides may vary from a few monosaccharide units to highly branched structures containing many (e.g. over 30) monosaccharide units.

The "sequencing" of an oligosaccharide involves deducing certain information concerning the structure of the oligosaccharide such as (i) the type of each monosaccharide unit in the oligosaccharide, (ii) the order in which the monosaccharide units are arranged in the oligosaccharide, (iii) the position of linkages between each of the monosaccharide units (e.g. 1-3, 1-4, etc.), and hence any branching pattern and/or (iv) the orientation of linkage between each of the monosaccharide units (i.e. whether a linkage is an α linkage or a β linkage) .

Where it is desired to obtain as much information as possible regarding the structure of an oligosaccharide then "sequencing" of the oligosaccharide may be carried out to obtain as much information as possible in relation to features (i) to (iv) inclusively immediately hereinbefore disclosed.

An agent which assists in obtaining information in relation to some or all of features (i) to (iv) inclusively on being applied to an oligosaccharide entity may be regarded as a "sequencing agent". By way of example, a sequencing agent may be a physical agent or a chemical agent. Examples of physical sequencing agents are proton n. .r., carbon-13 n. .r. and mass spectrometry for molecular weight determinations. Also, by way of example, a sequencing agent may be capable of causing cleavage of a chemical bond or capable of causing formation of a chemical bond.

Where, for example, a sequencing agent is a chemical reagent (which may be, for example, a chemical reagent or a biochemical reagent) the sequencing agent may be regarded as a sequencing reagent. Examples of sequencing reagents are enzymes (such as exoglycosidases and endoglycosidases) and chemical reagents (e.g. a periodate) capable of effecting chemical cleavage of an oligosaccharide and/or a chemical modification of an oligosaccharide which assists in obtaining information regarding the structure of the oligosaccharide as hereinbefore disclosed.

Examples of enzymes which may be used as sequencing reagents are given in Table 1.

In Table 1 there is presented a list of enzymes commonly used for cleaving monosaccharides from N-linked oligosaccharides and the rules showing which monosaccharides are cleaved by each of these enzymes and from which part of an oligosaccharide structure cleavage can be expected.

A sequencing agent which is capable of bringing about a cleaving of a particular linkage or linkages in an oligosaccharide may be an agent capable of effecting a specific transformation on the oligosaccharide.

It will be appreciated that a sequencing agent may be chosen such that the reaction products obtained when it is applied to the oligosaccharide entity (e.g. contacted with the oligosaccharide entity in the case of a^' chemical or biochemical reagent) will reveal the presence or absence of a particular structural sub-unit TABLE 1

TABLE 1 (continued)

(e.g. a monosaccharide unit) in the oligosaccharide entity.

Thus, in one embodiment of the present invention there is provided a process for the sequencing of an oligosaccharide which process includes applying a sequencing agent to an oligosaccharide entity and analysing for a component of the oligosaccharide entity, which component has been released from the oligosaccharide entity by means of the sequencing agent.

It will be appreciated that in accordance with the immediately foregoing embodiment of the present invention it is not the oligosaccharide entity which is analysed after application of the sequencing agent to obtain information regarding the structure of the oligosaccharide entity. Rather, in accordance with the immediately foregoing embodiment of the present invention, it is a component which is released or cleaved from an oligosaccharide entity that is analysed for in order to facilitate "sequencing".

Thus, for example, when analysis is carried out, the detection of a component comprising a particular monosaccharide in the products of a cleaving reaction may be used to confirm the presence of a particular linkage and monosaccharide in the original oligosaccharide structure of the oligosaccharide entity.

A set of possible structures for an oligosaccharide (i.e. a set of "candidate" structures) may be prepared in any suitable way.

For example, a set of "candidate" structures may be drawn up from literature surveys. By way of further example, a set of "candidate" structures may be prepared by considering possible permutations of putting together monosaccharide units.

Alternatively, by way of further example, a concept of structures and sub-structures may be used (as discussed further hereafter) to prepare a set of candidate structures for an unknown oligosaccharide.

It will also be appreciated that, for example, by sequentially applying different sequencing agents to an oligosaccharide entity and analysing the products obtained by use of each sequencing agent it is possible to eliminate certain structures from consideration (i.e. eliminate certain structures from a postulated set of possible "candidate" structures for the oligosaccharide entity) and to confirm the presence of a certain structure or structures thereby enabling information regarding the structure of the oligosaccharide entity to be deduced.

Thus, for example, by sequentially applying different sequencing agents to a given oligosaccharide or to a product thereof (being a product produced by previously applying a sequencing agent to the oligosaccharide) or to a species having an oligosaccharide portion, and analysing the products obtained by use of each sequencing agent it is possible to eliminate certain structures from consideration (i.e. eliminate certain structures from a postulated set of possible "candidate" structures for the oligosaccharide) and to confirm the presence of a certain structure or certain structures thereby enabling information regarding the structure of the oligosaccharide (which may be, for example, an oligosaccharide as such or an oligosaccharide portion of a species having an oligosaccharide portion) to be deduced. The presence and linkage of a particular monosaccharide at an end of an oligosaccharide structure of an oligosaccharide entity may be determined, for example, by the ability of a given sequencing agent (e.g. a biochemical reagent such as an enzyme (e.g. an exoglycosidase) ) to cause cleavage of that linkage; thus, if cleavage occurs, then detection of the particular monosaccharide in the products of the cleaving reaction will confirm the presence of that linkage in the original oligosaccharide structure.

Thus, by sequentially using a plurality of different sequencing agents having known specific linkage cleaving capabilities it is possible to deduce increasing amounts of information regarding the structure of the oligosaccharide entity under analysis. It is to be understood that where no single sequencing agent can be found which can distinguish between candidate oligosaccharide structures then consideration may be given to possible combinations of two, three or more agents to be applied one after the other; at each stage of consideration an agent which has no effect on any of a set of candidate structures may be eliminated. Consideration may also be given to using a combination of two or more agents simultaneously as this may lead to a reduction in the time required to carry out a sequencing analysis.

Thus, an iterative process may be used whereby a cycle of analysis, application of a sequencing agent (or a combination of sequencing agents) and subsequent analysis is repeated until as much information as possible has been obtained regarding the structure of an oligosaccharide entity with the agents available or the sample of oligosaccharide entity is exhausted.

From the foregoing it will be appreciated that in certain circumstances a particular sequencing agent may be such that it does not react with the oligosaccharide entity to give products thereby permitting the fact that it did not so react to allow deductions to be made regarding the structure of the oligosaccharide entity.

The effectiveness of the sequencing of an oligosaccharide entity may be seen as depending upon the choice of sequencing agent to be applied at various stages in sequencing and the accuracy of interpretation of the results of applying a given sequencing agent to an oligosaccharide entity.

To a large degree, a good choice of sequencing agent depends upon the skill of an experienced operator who has already made some intelligent guesses about the type of oligosaccharide structure being investigated.

A poor choice of sequencing agent may result in little or no additional information being revealed by a particular application of a sequencing agent and thus lead to a waste of time and materials. Also there is present the danger that prejudices of an operator will mask ambiguities in the interpretation of results; for example, an operator may assign a single structure which is consistent with experimental results, whereas in reality there may be more than one structure consistent with the same experimental results.

A further difficulty may arise in defining the point in sequencing at which no further information can be revealed by the use of available sequencing agents.

For oligosaccharide entities (e.g. oligosaccharides) obtained from glycoproteins the sequencing thereof may be assisted by a knowledge of the biosynthetic pathways involved in building up oligosaccharide structures. Thus, for example, for N-linked oligosaccharides it is known that there is a characteristic core structure and that additional monosaccharides may only add on in certain well defined orders and branching patterns.

This knowledge may be used to develop for oligosaccharides a concept of structures and sub¬ structures; this is discussed further hereinafter with reference to Figures 1, 2, and 3 of the accompanying drawings.

For example, if an oligosaccharide entity to be subjected to sequencing in accordance with the present invention is an oligosaccharide which has been obtained from a mixture of oligosaccharides released from a glycoprotein by the enzyme peptide-N-glycosidase F, it may be assumed that the oligosaccharide is an N-glycan and that the structure thereof is likely to be a structure similar to those of Figure l. Figure 2 or Figure 3 of the accompanying drawings or a sub-structure generated from the structures similar to those of Figures 1, 2 and 3 of the accompanying drawings.

In one embodiment of the present invention there is provided a process for the sequencing of an oligosaccharide which process includes applying a sequencing agent, or a combination of sequencing agents, to an oligosaccharide entity.

In another embodiment of the present invention there is provided a process for the sequencing of an oligosaccharide entity which process includes applying a sequencing agent to an oligosaccharide entity, immobilised on a support material, and analysing the products obtained by applying the sequencing agent to the oligosaccharide entity. In a further embodiment of the present invention there is provided a process for the sequencing of an oligosaccharide which process includes applying a sequencing agent to an oligosaccharide entity, immobilised on a support material, analysing the products obtained by applying the sequencing agent to the oligosaccharide entity, and applying a further sequencing agent to an oligosaccharide entity, or a product thereof.

By way of example a sequencing agent, or a combination of sequencing agents, may be applied to an oligosaccharide entity in accordance with the present invention in any suitable manner. For example, a sequencing agent, or a combination of sequencing agents, may be applied to an oligosaccharide entity attached to a support material in a suitable reaction vessel.

The sequencing agent or a combination of sequencing agents may be introduced in a suitable solvent to a means for contacting together a sequencing agent and an oligosaccharide entity.

It will be appreciated that any suitable method or process of sequencing an oligosaccharide entity may be applied to the sequencing of an oligosaccharide entity immobilised on a support material in accordance with the present invention.

Thus, for example, a means for selecting a sequencing agent to be applied to an oligosaccharide entity may be used.

Such a means for selecting a sequencing agent may be, for example, a unit capable of making logical choices (e.g. a logic unit) . Also, the unit may be such that it is capable of interpreting results generated by an analysing means and capable of selecting a sequencing agent (or a combination of sequencing agents) to be applied to an oligosaccharide entity. Thus, for example, the unit may be such as to be capable of requesting the application of a sequencing agent (or a combination of sequencing agents) to an oligosaccharide entity, the application of another sequencing agent (or combination of sequencing agents) to an oligosaccharide entity being a product of an oligosaccharide entity or the application of another or a further sequencing agent (or combination of sequencing agents) to an oligosaccharide entity, or to an oligosaccharide entity being a product of an oligosaccharide entity.

An immobilised oligosaccharide entity, or any product thereof produced by application of a sequencing agent and retained on the support material, may be readily separated from released products, such as species ( with new reducing termini) generated by the application of a sequencing agent or agents. Thus, for example, the product of the oligosaccharide entity may be retained on the support material and species with new reducing termini may be removed by suitable washing.

It will be appreciated that when sequencing of an oligosaccharide structure of an oligosaccharide is conducted in free solution, analysis of an effect of a sequencing agent upon an oligosaccharidestructure of an oligosaccharide entity, will generally involve the loss of some oligosaccharide entity. Thus, in taking a sample of reaction mixture (produced by application of a sequencing agent to an oligosaccharide entity) for analysis, some of the oligosaccharide entity, may be removed along with any other species present in the reaction mixture. By use of immobilisation in accordance with the present invention, oligosaccharide entity (e.g. an oligosaccharide, or product thereof, or species having an oligosaccharide portion) , may be retained on a support material and thus not removed when a sample of reaction mixture is taken for analysis.

The use of immobilisation in accordance with the present invention may also permit more accurate determination of species produced by application of a sequencing agent to an oligosaccharide entity. Thus, for example, in a sample of reaction mixture (produced by application of a sequencing agent to an oligosaccharide entity) taken for analysis there may be substantially no oligosaccharide entity (e.g. oligosaccharide, or product thereof, or species having an oligosaccharide portion) , to interfere with analysis since the oligosaccharide entity is retained on the support material.

The use of immobilisation in accordance with the present invention permits, if desired, the removal of one sequencing agent before application of a second sequencing agent; this offers the advantage of avoiding unwanted application of the first sequencing agent to a product of an oligosaccharide entity generated by application of the second sequencing agent.

A process in accordance with the present invention may also include the step of carrying out a preliminary analysis of an oligosaccharide entity of unknown structure prior to application of a sequencing agent.

It will be appreciated that a preliminary compositional analysis may enable the number of candidate oligosaccharide structures, that need to be considered during subsequent structural analysis, to be reduced; thus, such a preliminary structural analysis may enable the number of sequencing agent applications to be reduced.

The analysis of the oligosaccharide entity (e.g. a preliminary analysis or any subsequent analysis) may be effected in any suitable way. Thus, for example, the type and number of each monosaccharide in the oligosaccharide entity may be analysed (i.e. a compositional analysis may be effected) by any suitable method. For example, complete degradation of an oligosaccharide structure of an oligosaccharide entity into its monosaccharide components may be effected by treatment with suitable reagents (e.g.. a mixture of digesting reagents such as exoglycosidases) and the resulting reaction mixture analysed using a suitable monosaccharide detection method such as those hereinafter disclosed. Alternatively, by way of further example, an oligosaccharide entity may be subjected to methanolysis, N-acetylation (if required) and silylation and the resulting substances subjected to gas chromatography/mass spectrometry.

It will be appreciated that, by way of further example, information regarding an oligosaccharide structure of an oligosaccharide entity may also be obtained by observing its retention time on a chromatographic column; by way of example the chromatographic column may be such that the retention time of an oligosaccharide entity is expressed in glucose units.

Monosaccharide detection may be effected in any suitable manner, examples of which are the following HPLC-based methods: (i) use of an SP 1010 reverse phase column, (ii) HPAE with PAD using a Dionex instrument and (iii) capillary electrophoresis. In one embodiment of the present invention there is provided apparatus suitable for use in the sequencing of an oligosaccharide entity which apparatus includes an immobilised oligosaccharide entity and analysing means for analysing for a component of the oligosaccharide entity, which component has been released from the oligosaccharide by means of a sequencing agent.

An analysing means for use in accordance with the present invention may include a detector capable of measuring the types and relative amounts of monosaccharides present in an oligosaccharide and/or monosaccharides produced by applying a sequencing agent to an oligosaccharide entity, (e.g. by bringing together a sequencing agent and an oligosaccharide entity) . By way of example, the monosaccharides may be measured as monosaccharides or as derivatised products thereof.

The present invention will now be further described by way of example only with reference to the accompanying drawings and with reference to the Examples.

In the accompanying drawings: Figure 1 shows a structure for N-linked oligo¬ saccharides of high mannose types (Man 9) ;

Figure 2 shows a structure for N-linked oligosaccharides of hybrid types (Hy 2) ;

Figure 3 shows a structure for N-linked oligosaccharides of ulti-antennary types (Hex 2);

Figure 4 shows a total ion current chromatogram of TMS- methyl glycosides of standard monosaccharides; Figures 5 to 23 show Gas Chromatography-Mass Spectra for TMS-methyl glycosides of standard monosaccharides. The relevant monosaccharide is indicated in the top right-hand corner of each Figure; it will be appreciated that M/Z in the Figures indicates mass/charge ratio;

Figure 24 shows a structure of an oligosaccharide entity to which reference is made in Example 1;

Figure 25 shows a total ion current chromatogram of TMS- methyl glycosides obtained as disclosed in relation to Example 1(a);

Figures 26 to 29 show Gas Chromatography-Mass Spectra for TMS-methyl glycosides obtained as disclosed in relation to Example 1(a);

Figure 30 shows a total ion current chromatogram of TMS- methyl glycosides obtained as disclosed in relation to Example 1(b);

Figures 31 to 33 show Gas Chromatography-Mass Spectra for TMS-methyl glycosides obtained as disclosed in relation to Example 1(b);

Figure 34 shows a total ion current chromatogram of TMS- methyl glycosides obtained as disclosed in relation to Example 1(c) ;

Figures 35 to 37 show Gas Chromatography-Mass Spectra of TMS-methyl glycosides obtained as disclosed in relation to Example 1(c);

Figure 38 shows a structure of an oligosaccharide entity to which reference is made in Example 2; Figure 39 shows a structure of an oligosaccharide entity to which reference is made in Example 3; and

Figure 40 shows a structure of an oligosaccharide entity to which reference is made in Example 4.

In Figures 1, 2 and 3 of the accompanying drawings and elsewhere in this Specification the abbreviations Man, Fuc, Gal and Glcnac mean, respectively, D-mannose, L-fucose, D-galactose and N-acetyl-D- glucosamine.

Referring now to Figure 1 of the accompanying drawings there is shown a structure for N-linked oligosaccharides of high mannose types.

It will be appreciated that the structure shows a number of mannose and N-acetyl glucosamine monosaccharide units, linked by a variety of linkages; it will also be appreciated that the N-acetyl glucosamine unit to the extreme right of the Figure may be identified as the reducing terminus of the structure. The concept of structures and sub-structures was hereinbefore disclosed and it may now be stated that it may be assumed that an oligosaccharide, for which a particular structure is relevant, is either the structure itself or is a member of a set of sub¬ structures of the structure, which sub-structures may be generated by performing a specific transformation on the structure. This leads to a possibility that successive sequencing of an oligosaccharide structure of an oligosaccharide entity will eliminate more and more candidate structures from a set of structures until no further information can be obtained.

Thus, an unknown oligosaccharide structure of an oligosaccharide entity may be identified as one of the structures remaining. In the case of the structure shown in Figure 1 (and in Figures 2 and 3) the transformations used to generate sub-structures are successive deletions of terminal monosaccharides in all possible ways; this forms all unique sub-structures having the same root as the structure where the combination of the monosaccharides existing in the sub-structure follows that of the structure.

Referring now to Figure 2 of the accompanying drawings there is shown a structure for N-linked oligosaccharides of hybrid types (Hy 2) .

The disclosure regarding structure and sub- structures hereinbefore given in relation to Figure 1 applies mutatis mutandis in relation to Figure 2.

Referring now to Figure 3 of the accompanying drawings there is shown a structure for N-linked oligosaccharides of multi-antennary types (Hex 2) .

The disclosure regarding structure and sub¬ structures hereinbefore given in relation to Figure 1 applies mutatis mutandis in relation to Figure 3.

Example 1

In this Example an oligosaccharide entity comprising an oligosaccharide of the structure given in Figure 24 of the accompanying drawings was used to demonstrate use of the present invention.

The oligosaccharide was confirmed as having a purity of > 95% by 500 MH₃ Η-NMR (1-dimensional) and high performance anion-exchange chromatography. The oligosaccharide was immobilised by being conjugated to a support material comprising, 1,1' carbonyl diimidazole-activated agarose using reductive amination as follows:

1 mg of the oligosaccharide was heated with 80 μl of a reagent prepared by dissolving 100 mg 2-amino pyridine in 65 μl of concentrated hydrochloric acid at 90°C for 12 minutes. Subsequently, 8 μl of a dimethyl sulphoxide solution of sodium cyanoborohydride at concentration 1.66 gm/ml was added and the resulting mixture heated at 90°C for a further 90 minutes. After cooling, the mixture was diluted with 0.5 ml n- butanol:ethanol (4:1) then applied to a column of cellulose (4 ml) and eluted ith 20 ml n- butanol:ethanol:water [4:4:1], followed by methanol (3 ml) then water (5 ml) . The methanol and water fractions were combined and concentrated by rotary-evaporation to 0.2 ml. A slurry of diimidazolecarbonyl activated agarose in acetone (0.5 ml) was added and the resulting mixture stirred at room temperature for 24 hours.

The combination of oligosaccharide conjugated to the support material (which combination will be referred to as "conjugated material" in this Example) was separated from reaction mixture and any unconjugated substances by rinsing in 0.1M sodium chloride followed by centrifugation (1000 g for 1 minute) , the liquid supernatant being discarded; the rinsing, centrifugation and discarding of liquid was repeated five times.

The immobilised oligosaccharide was subjected to sequencing by incubating conjugated material with exoglycosidases and identifying and quantifying any released monosaccharides as follows: A preliminary analysis of the oligosaccharide was carried out and the following monosaccharide units were identified: Man (3 units) , Gal (2 units) and Glcnac (4 units) .

The results of the preliminary analysis were used, in conjunction with a means for selecting a sequencing agent to be applied to an oligosaccharide entity (said means including a logic unit) , to identify candidate structures and to select a sequencing agent to be applied to the conjugated material.

Thus:

(a) to conjugated material in a plastic tube was added 100 μl of a solution consisting of 0.1M sodium citrate/phosphate, pH 3.5 containing 1.0 unit of purified β-D-galactosidase enzyme (obtained from jack bean) to form a mixture. The tube was capped and the mixture incubated at 37°C for 6 hours. Conjugated material was separated by rinsing in 0.1M sodium chloride followed by centrifugation (1000 g for 1 minute) . Liquid supernatant was separated from conjugated material and collected. This was repeated and all liquid supernatant was pooled and desalted by passage through an ion- exchange column consisting of 0.5 ml Dowex AG50X 12(H⁺) resin below 0.5 ml Dowex AG3X 4A(OH') resin. (Both resins were purchased from Bio RAD.) Eluent from the column was collected, rotary-evaporated to dryness and converted to the 1-0-methyl trimethylsilyl glycoside exactly according to the standard procedure of Chaplin (Analytical Biochemistry 123 p.336 (1982)). The resulting l-O- methyl trimethylsilyl glycoside was quantitated by GC-MS and identified by reference to known standard compounds based on retention time during GC and mass spectrum. The total ion current chromatogram of standard monosaccharides is shown in Figure 4 of the accompanying drawings and mass spectra of standard monosaccharides are shown in Figures 5 to 23 of the accompanying drawings. The total ion current chromatogram and mass spectra for the liquid supernatant recovered after incubating the conjugated material with the jS-D-galactosidase are shown respectively in Figure 24 and 26 to 29 of the accompanying drawings. From this information it can be concluded that the action of the β-O- galactosidase led to the separation from the conjugated material of 511 nano oles of galactose and of no other monosaccharide;

(b) the information obtained in (a) above was used in conjunction with a means for selecting a sequencing agent to be applied to an oligosaccharide entity (said means including a logic unit) , to select a further sequencing agent to be applied to the conjugated material obtained after treatment as disclosed in (a) above.

Thus to conjugated material (as obtained after treatment as disclosed in (a) above) in a plastic tube was added 100 μl of a solution consisting of 0.1M sodium cacodylate, pH 6.0 containing 48 microunits of purified jS-N-acetyl-D-hexosaminidase enzyme (obtained from Streptococcus pneumoniae) to form a mixture. The tube was capped and the mixture incubated at 37°C for 6 hours. Conjugated material was separated by rinsing in 0.1M sodium chloride followed by centrifugation (1000 g for 1 minute) . Liquid supernatant was separated from conjugated material and collected. This was repeated and all liquid supernatant was pooled and desalted by passage through an ion-exchange column consisting of 0.5 ml Dowex AG50X 12(H⁺) resin below 0.5 ml Dowex AG3X 4A(0H^") resin. (Both resins were purchased from Bio RAD.) Eluent from the column was collected, rotary-evaporated to dryness and converted to the l-0-methyl trimethylsilyl glycoside exactly according to the standard procedure of Chaplin (Analytical Biochemistry 123 p.336 (1982)). The resulting 1-O-methyl trimethylsilyl glycoside was quantitated by GC-MS and identified by reference to known standard compounds based on retention time during GC and mass spectrum. The total ion current chromatogram of standard monosaccharides is shown in Figure 4 of the accompanying drawings and mass spectra of standard monosaccharides are shown in Figures 5 to 23 of the accompanying drawings. The total ion current chromatogram and mass spectra for the liquid supernatant recovered after incubating the conjugated material with the β-N-acetyl-D- hexosaminidase are shown respectively in Figure 30 and Figures 31 to 33 of the accompanying drawings. From this information it can be concluded that the action of the β-N-acetyl-D-hexosaminidase led to the separation from the conjugated material of 487 nanomoles of N-acetylglucosamine and of no other monosaccharide;

(c) the information obtained in (b) above was used in conjunction with a means for selecting a sequencing agent to be applied to an oligosaccharide entity (said means including a logic unit) , to select a further sequencing agent to be applied to the conjugated material obtained after treatment as disclosed in (b) above.

Thus, to conjugated material (as obtained after treatment as disclosed in (b) above) in a plastic tube was added 100 μl of a solution consisting of 0.1M sodium acetate/0.01M zinc acetate, pH 5.0 containing 6 units of the purified α-D-mannosidase enzyme (obtained from jack bean) to form a mixture. The tube was capped and the mixture incubated at 37°c for 6 hours. Conjugated material was separated by rinsing in 0.1M sodium chloride followed by centrifugation (1000 g for 1 minute) . The liquid supernatant was separated from conjugated material and collected. This was repeated and all liquid supernatant was pooled and desalted by passage through an ion-exchange column consisting of 0.5 ml Dowex AG50X 12(H⁺) resin below 0.5 ml Dowex AG3X 4A(OH") resin._. (Both resins were purchased from Bio RAD.) Eluent from the column was collected, rotary-evaporated to dryness and converted to the 1-O-methyl trimethylsilyl glycoside exactly according to the standard procedure of Chaplin (Analytical Biochemistry 123 p.336 (1982)). The resulting l-O-methyl trimethylsilyl glycoside was quantitated by GC-MS and identified by reference to known standard compounds based on retention time during GC and mass spectrum. The total ion current chromatogram of standard monosaccharides is shown in Figure 4 of the accompanying drawings and mass spectra of standard monosaccharides are shown in Figures 5 to 23 of the accompanying drawings. The total ion current chromatogram and mass spectra for the liquid supernatant recovered after incubating the conjugated material with the α-D-mannosidase are shown respectively in Figure 34 and Figures 35 to 37 of the accompanying drawings. From this information it can be concluded that the action of the α-D-mannosidase led to the separation from the conjugated material of 527 nanomoles of mannose and of no other monosaccharide; (d) the information obtained in (c) above was used, in conjunction with a means for selecting a sequencing agent to be applied to an oligosaccharide entity (said means including a logic unit) , to select a further sequencing agent to be applied to the conjugated material obtained after treatment as disclosed in (c) above.

Thus, to conjugated material (as obtained after treatment as disclosed in (c) above) in a plastic tube was added 100 μl of a solution consisting of 0.1M sodium acetate, pH 4.0 containing 0.3 units of the purified 3-D-mannosidase enzyme (obtained from Helix pomatia) to form a mixture. The tube was capped and the mixture incubated at 37°C for 6 hours. Conjugated material was separated by rinsing in 0.1M sodium chloride followed by centrifugation (1000 g for 1 minute) . The liquid supernatant was separated from conjugated material and collected. This was repeated and all liquid supernatant was pooled and desalted by passage through an ion-exchange column consisting of 0.5 ml Dowex AG50X 12(H⁺) resin below 0.5 ml Dowex AG3X 4A(0H') resin. (Both resins were purchased from Bio RAD.) Eluent from the column was collected, rotary-evaporated to dryness and converted to the 1-O-methyl trimethyl-silyl glycoside exactly according to the standard procedures of Chaplin (Analytical Biochemistry 123 p.336 (1982)). The resulting 1-O-methyl trimethylsilyl glycoside was quantitated by GC-MS and identified by reference to known standard compounds based on retention time during GC and mass spectrum. The total ion current chromatogram of standard monosaccharides is shown in Figure 4 of the accompanying drawings and mass spectra of standard monosaccharide are shown in Figures 5 to 23 of the accompanying drawings. The total ion current chromatogram and mass spectra for the liquid supernatant recovered after incubating the conjugated material with the 3-D-mannosidase were essentially the same as those shown, respectively, in Figure 34 and Figures 35 to 37 of the accompanying drawings. From this information it can be concluded that the action of the β-O- mannosidase led to the separation from the conjugated material of 271 nanomoles of mannose and of no other monosaccharide;

(e) the information obtained in (d) above was used, in conjunction with a means for selecting a sequencing agent to be applied to an oligosaccharide entity (said means including a logic unit) to select a further sequencing agent to be applied to the conjugated materials obtained after treatment as disclosed in (d) above.

Thus, to conjugated material (as obtained after treatment as disclosed in (d) above) in a plastic tube was added 100 μl of a solution consisting of 0.1M sodium citrate/phosphate, pH 4.5 containing 2.5 units of the purified /S-N-acetyl-D- hexosaminidase (obtained from jack bean) to form a mixture. The tube was capped and the mixture incubated at 37°C for 6 hours. Conjugated material was separated by rinsing in 0.1M sodium chloride followed by centrifugation (1000 g for 1 minute) . The liquid supernatant was separated from conjugated material and collected. This was repeated and all liquid supernatant was pooled and desalted by passage through an ion-exchange column consisting of 0.5 ml Dowex AG50X 12(H⁺) resin below 0.5 ml Dowex AG3X 4A(OH") resin. (Both resins were purchased from Bio RAD.) Eluent from the column was collected, rotary-evaporated to dryness and converted to the 1-O-methyl trimethylsilyl glycoside exactly according to the standard procedure of Chaplin (Analytical Biochemistry 123 p.336 (1982)). The resulting 1-O-methyl trimethylsilyl glycoside was quantitated by GC-MS and identified by reference to known standard compounds based on retention time during GC and mass spectrum. The total ion current chromatogram of standard monosaccharides is shown in Figure 4 of the accompanying drawings and mass spectra of standard monosaccharides are shown in Figures 5 to 23 of the accompanying drawings. The total ion current chromatogram and mass spectra for the liquid supernatant recovered after incubating the conjugated material with 3-N-acetyl-D- hexosaminidase were essentially the same as those shown, respectively, in Figure 30 and Figures 31 to 33 of the accompanying drawings. From this information it can be concluded that the action of the S-N-acetyl-D-hexosaminidase led to the separation from the conjugated material of 267 nanomoles of N-acetylglucosamine and of no other monosaccharide.

From the information obtained as above disclosed in this Example and the well known specificities of the exoglycosidases employed, it is clear that monosaccharides were released from the conjugated material in the order and ratio stated below:

D-galactose jSl → 4 2 residues

N-acetyl-D-glucosamine j3l 2 residues

Mannose αl → 6,3 2 residues

Mannose j8l → 4 1 residue N-acetyl-D-glucosamine βl 1 residue The monosaccharide attaching the starting oligosaccharide to the resin is not susceptible to an exoglycosidase enzyme, since it is not attached by an 0- glycosidic linkage. This terminal N-acetyl glucosamine (Glcnac) is therefore understood to exist. From this information, the sequence of the initial oligosaccharide can clearly only be that as shown in Figure 24 of the accompanying drawings.

This sequence is consistent with NMR studies of a solution form of the oligosaccharide.

Example 2

In this Example an oligosaccharide entity comprising an oligosaccharide of the structure given in Figure 38 of the accompanying drawings was used to demonstrate use of the present invention.

A preliminary analysis of the oligosaccharide was carried out and the following monosaccharide units were identified: Gal (2 units) , Glcnac (5 units) , Man (3 units) , Fuc (1 unit) .

The oligosaccharide (0.6 mg) was attached to a support material as disclosed in relation to Example 1 to give a conjugated material.

The conjugated material was subjected to successive treatments with various sequencing agents (in this Example exoglycosidase enzymes) , the choice of each successive sequencing agent being based upon the results of analysis and use of a means for selecting a sequencing agent to be applied to an oligosaccharide entity (said means including a logic unit) ; the procedures used in this Example were substantially similar to those disclosed in relation to Example 1.

The order in which the various sequencing agents (exoglycosidases in this Example) were applied and the results are set out in Table 2.

The order and ratio of monosaccharides release was as follows: D-galactose βl→4 2 residues

N-acetyl-D-hexosamine 3l->2 (Man αl->3) 1 residue

N-acetyl-D-hexosamine jSl-+2 (Man αl→6) 1 residue

N-acetyl-D-hexosamine /3l->4 (Man βl→4 ) 1 residue

D-mannose αl→6,3 2 residues D-mannose 3l→4 1 residue

L-fucose αl→6 1 residue

N-acetyl-D-hexosamine jSl→4 1 residue

From this information the sequence of the initial oligosaccharide can clearly only be that as shown in Figure 38 of the accompanying drawings.

TABLE 2

Example 3

In this Example an oligosaccharide entity comprising an oligosaccharide of the structure given in Figure 39 of the accompanying drawings was used to demonstrate use of the present invention.

A preliminary analysis of the oligosaccharide was carried out and the following monosaccharide units were identified: Man (3 units) , Glcnac (3 units) .

The oligosaccharide (0.3 mg) was attached to a support material as disclosed in relation to Example 1 to give a conjugated material.

The order in which the various sequencing agents (exoglycosidases in this Example) were applied and the results are set out in Table 3. TABLE 3

The order and ratio of monosaccharides release was as follows:

D-mannose αl→3 1 residue N-acetyl-D-glucosamine /31-+2 1 residue D-mannose αl->6 1 residue D-mannose |3l→4 1 residue N-acetyl-D-glucosamine βl→-% 1 residue

From this information the sequence of the initial oligosaccharide can clearly only be that as shown in Figure 39 of the accompanying drawings.

Example 4

In this Example an oligosaccharide entity comprising an oligosaccharide of the structure given in Figure 40 of the accompanying drawings was used to demonstrate the use of the present invention.

A preliminary analysis of the oligosaccharide was carried out and the following monosaccharide units were identified: NANA (sialic acid) (2 units) , Gal (2 units) , Glcnac (4 units) , Man (3 units) .

The oligosaccharide (0.4 mg) was attached to a support material as disclosed in relation to Example 1 to give a conjugated material.

The order in which the various sequencing agents (exoglycosidases in this Example) were applied and the results are set out in Table 4.

The order and ratio of monosaccharides released was as follows: D-NANA α2→6 2 residues

D-galactose jSl→4 2 residues

N-acetyl-D-glucosamine j8l→2 2 residues

Mannose αl-+6,3 2 residues

Mannose |8l→4 1 residue N-acetyl-D-glucosamine /31-+4 1 residue

From this information the sequence of the initial oligosaccharide can clearly only be that as shown in Figure 40 of the accompanying drawings.

TABLE 4

Claims

1. A process suitable for use in the sequencing of an oligosaccharide wherein an oligosaccharide entity is immobilised on a support material.

2. A process as claimed in Claim 1 wherein an oligosaccharide entity is immobilised on a support material and analysis is carried out for a component of the oligosaccharide entity which component has been released from the oligosaccharide entity, by means of a sequencing agent.

3. A process as claimed in Claim 1 or Claim 2 wherein the oligosaccharide entity is an oligosaccharide, or a product of an oligosaccharide, or a species having an oligosaccharide portion.

4. A process as claimed in any one of Claims 1 to 3 wherein the process includes the step of immobilising an oligosaccharide entity on a support material.

5. A process as claimed in any one of the preceding Claims wherein the process includes applying a sequencing agent, or a combination of sequencing agents, to an oligosaccharide entity immobilised on a support material.

6. A process as claimed in any one of the preceding Claims wherein the process includes applying a sequencing agent to an oligosaccharide entity immobilised on a support material and analysing the products obtained by applying the sequencing agent to the oligosaccharide entity.

7. A process as claimed in any one of the preceding Claims wherein the process includes applying a sequencing agent to an oligosaccharide entity immobilised on a support material, analysing the products obtained by applying the sequencing agent to the oligosaccharide entity, and applying a further sequencing agent to the oligosaccharide entity, or a product thereof.

8. A process as claimed in any one of the preceding Claims wherein an iterative process is used whereby a cycle of analysis, application of a sequencing agent, or a combination of sequencing agents, and subsequent analysis is repeated.

9. A process as claimed in any one of Claims 1 to 8 wherein an oligosaccharide as such is immobilised and subjected to sequencing.

10. A process as claimed in any one of Claims 1 to 7 wherein an oligosaccharide is immobilised and subjected to sequencing whilst still attached to a conjugate.

11. A process as claimed in any one of Claims 1 to 7 wherein the oligosaccharide entity is a species having an oligosaccharide portion said species being a glycoprotein or a glycolipid.

12. A process as claimed in any one of the preceding Claims wherein an oligosaccharide entity is immobilised on a support material by means of chemical attachment.

13. A process as claimed in any one of the preceding Claims wherein an oligosaccharide entity is an oligosaccharide, or a product thereof, and the oligosaccharide, or product thereof, is immobilised by a direct covalent linkage, or by a direct non-covalent linkage, with a support material.

14. A process as claimed in any one of Claims 10 to 12 wherein an oligosaccharide, or a product thereof, is indirectly linked to a support material via a conjugate.

15. A process as claimed in any one of the preceding Claims wherein the support material is a solid support material comprising 1,1' carbonyldiimadazole activated agarose.

16. A process as claimed in any one of the preceding

Claims wherein the sequencing agent is a physical agent or a chemical agent.

17. A process as claimed in Claim 16 wherein the physical agent is proton n.m.r., carbon-13 n.m.r. or mass spectroscopy.

18. A process as claimed in any one of Claims 1 to 16 wherein the sequencing agent is capable of causing cleavage of a chemical bond or capable of causing formation of a chemical bond.

19. A process as claimed in any one of Claims 1 to 16 or Claim 18 wherein the sequencing agent is a chemical reagent.

20. A process as claimed in Claim 19 wherein the sequencing agent is a periodate.

21. A process as claimed in Claim 19 wherein the sequencing agent is an enzyme.

22. A process as claimed in Claim 21 wherein the enzyme is an exoglycosidase or an endoglycosidase.

23. A process as claimed in Claim 22 wherein the enzyme is achatina fulica beta mannosidase, a.saitoi alpha mannosidase, jack bean alpha mannosidase, bovine testis beta galactosidase, jack bean beta galactosidase, c.lampas beta xylosidase, s.pneu beta N-acetyl hexosaminidase, jack bean beta n-acetyl hexosaminidase, bovine epididymis alpha fucosidase, c.lampas alpha fucosidase, coffee bean alpha galactosidase, or almond alpha fucosidase.

24. A process as claimed in any one of Claims 16, 18, 20 or 21 to 23 wherein the sequencing agent is a sequencing agent capable of effecting a specific transformation on an oligosaccharide.

25. Apparatus suitable for use in the sequencing of an oligosaccharide entity which apparatus includes an immobilised oligosaccharide entity.

26. Apparatus as claimed in Claim 25 wherein the apparatus includes an immobilised oligosaccharide entity, and analysing means for analysing for a component of the oligosaccharide entity, which component has been released from the oligosaccharide entity, by means of a sequencing agent.

27. Apparatus suitable for use in the sequencing of an oligosaccharide which apparatus includes a support material upon which an oligosaccharide entity may be immobilised.