Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1993-03-12
2001-01-30
Geist, Gary (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S004100, C536S055300, C536S120000, C530S395000, C530S397000, C436S071000
Reexamination Certificate
active
06180779
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to a method for the release of O-glycans (i.e. O-linked type oligosaccharides) from glycoconjugates and to isolation of such glycans.
Examples of glycoconjugates are glycoproteins, glycohormones and glycolipids.
An “O-linked type” oligosaccharide is an oligosaccharide which is covalently bonded to a conjugate by an O-glycosidic linkage. An example of such linkages as typically found on glycoproteins is shown in
FIG. 1
of the accompanying Drawings. For the purposes of this invention, release refers to an event or process Leading to cleavage of the covalent O-glycosidic bond attaching an ‘O-linked-type’ oligosaccharide to a conjugate, and isolation refers to an event or process leading to physical separation of released oligosaccharides from conjugate or conjugate-derived materials.
The release and subsequent isolation of an “O-linked type” oligosaccharide or ‘O-linked type’ oligosaccharides (hereinafter referred to respectively as an “O-glycan” or “O-glycans”) from a glycoconjugate are important for several reasons. Principal among these are the following: Firstly, a detailed structural characterization of a glycoconjugate requires structural analysis of any associated O-glycans. Since several such O-glycans may be attached to a single conjugate, and since structural analysis of O-glycans is most accurately performed on single, purified glycans, such an analysis requires prior release of O-glycans from the conjugate and subsequent isolation of the O-glycans from the conjugate, and then purification of glycans from one another. Secondly, O-glycans are increasingly recognized as important biological molecules in their own right. A study of the biological properties of O-glycans is, preferentially undertaken using O-glycans free of the conjugate.
The criteria that ought to be simultaneously satisfied for any successful method for the release and isolation of an O-glycan from a glyconjugate are as follows:
(i) The method of release should preferably cleave the O-glycosidic linkage in a manner independent of the nature of both the oligosaccharide component and the conjugate component.
(ii) The method of release should preferably achieve cleavage without permanent (i.e. not easily reversible) chemical damage to the cleaved glycans.
(iii) The method of isolation should preferably separate an O-glycan from a conjugate in a manner independent of the nature of both the oligosaccharide component and the conjugate component, as in (i), above.
(iv) The method of isolation should preferably achieve isolation without causing chemical damage to the cleaved glycans.
In addition, it is preferable if the method also achieves recovery in high yield (e.g. ≧85%) of O-glycans irrespective of the amount of starting glycoconjugate.
Previously, two different methods have been used for the release of O-glycans. These are briefly summarized below and assessed with respect to the above criteria.
A. Enzymatic Methods—For example, the use of proteolytic enzymes such as Pronase to obtain glycopeptides from glycoproteins, and/or the use of an enzyme such as O-glycanase™ (E.C. number 3.2.1.97) to cleave some O-glycans from the glycoconjugate. Such methods are generally unsatisfactory for several reasons, but principally because of the selectivity of O-glycan release. Enzymes are, by their very nature specific, and release only certain O-glycans, and then in a manner influenced by the attached conjugate. That is, enzymatic methods as currently practiced and understood, do not satisfy criterion (i), above.
B. Chemical Methods—Two chemical methods have been practiced for the release of O-glycans. These are:
(i) The use of alkaline solutions. The O-glycosidic linkage attaching an O-glycan to a conjugate is alkali labile, and alkaline solutions can therefore be employed for release of an O-glycan. For example, incubation with 50 mM NaOH at 45° C. for 16 hours has been successfully employed. An established and accepted disadvantage of this method is the alkali-lability of most O-glycans. Most: O-glycans that are attached to a peptide are so attached through the structures shown in
FIG. 2
of the accompanying Drawings. Upon cleavage (see
FIG. 2
) by alkali, the reducing terminal monosaccharide is further degraded by the alkali. Such degradation can be prevented by the reduction of the reducing terminal monosaccharide residue by performing the alkali-induced release in the presence of a vast excess of reducing agent. Typically 1M NaBH
4
is used. This reduction involves conversion of released O-glycan to the alditol form (FIG.
2
). Irrespective, therefore, of the presence or absence of excess reducing agent, the cleaved O-glycan will be recovered in a permanently chemically altered form (i.e. not as the native compound). This method does not therefore satisfy criterion (ii), above.
(ii) The use of anhydrous hydrazine. A few reports in the scientific literature concerning O-glycans suggest that O-glycans may be released from a peptide conjugate after incubating an anhydrous glycoprotein at high temperature (typically ≧100° C.) for several hours (typically ≧10 hours) with anhydrous hydrazine. Each such report also indicates that any O-glycans so released are subject to extensive chemical degradation and alteration of the released O-glycans by the hydrazine. Indeed, the general scientific opinion indicates that:- ‘The behavior towards hydrazine of O-glycosidically linked glycans has not yet been elucidated’. However some results show that they are profoundly degraded until an alkali-stable linkage stops the ‘peeling’ reaction. (J. Montreuil et al., in ‘Carbohydrate Analysis—A Practical Approach’—Ed. M. F. Chaplin and J. F. Kennedy—IRL Press, 1986). In the method of Rademacher and Dwek (European Patent Application No. 0 215 766A2) conditions are defined in which anhydrous hyzadrine is used to release glycans from glycoproteins, but these conditions are discussed only for N-glycans. Further these conditions are not suitable for O-glycans. In general therefore, the use of hydrazine is not widely considered by scientists to release intact O-glycans.
In summary, none of the above mentioned methods as currently practiced and understood is suitable for the release of intact, unaltered, O-glycans.
BRIEF DESCRIPTION OF THE INVENTION
In accordance with one aspect of the present invention there is provided a method for releasing an O-glycan from a glycoconjugate which includes subjecting a glycoconjugate to the influence of a hydrazine reagent, said glycoconjugate being substantially salt-free and substantially anhydrous and said hydrazine reagent being substantially anhydrous, and controlling conditions under which the glycoconjugate is subjected to the influence of the hydrazine reagent so as to cause release of O-glycan from the glycoconjugate in such a way as to allow subsequent recovery of O-glycan in substantially unreduced and intact form.
In one embodiment the present invention may be used to achieve a high yield of O-glycan.
In accordance with the present invention a high yield may be defined as ≧85%, but may be less than this as long as the yield is useful in practice.
The glycoconjungate optionally may be subjected to the influence of the hydrazine reagent and subjected to an energy input. Thus, for example, glycoconjugate and the hydrazine reagent may be brought together and the resulting reaction mixture heated by any suitable means. Other forms of energy input may be employed if desired (e.g. microwave radiation or infra-red radiation).
The hydrazine reagent may be hydrazine itself or any suitable derivative or compound thereof (e.g. related salt) capable of bringing about a desired cleavage of an O-glycosidic bond linking an O-glycan to a conjugate. The hydrazine reagent may be used, for example, in liquid form or in vapor form.
By way of example, the present invention provides a method for releasing unreduced and intact O-glycans from glycoconjugates, which comprises heating a substantially salt-free and essentially anhydrous glycoconjugate with pri
Bruce James
Merry Anthony Hugh
Parekh Rajesh Bhikhu
Geist Gary
Nixon & Vanderhye P.C.
Oxford GlycoSystems Limited
White Everett
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