Method of producing derivatives of lactosamine

Details Method of producing derivatives of lactosamine Method of producing derivatives of lactosamine

1996-06-28

2003-11-25

Zeman, Mary K. (Department: 1631)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S205000, C435S206000, C435S207000, C435S209000, C435S224000

Reexamination Certificate

active

06653109

ABSTRACT:

INTRODUCTION AND BACKGROUND
The present invention describes a new method for the production of certain carbohydrate containing compounds related to glycoconjugates; namely, lactosamine derivatives and substances derived therefrom. In a further aspect the present invention relates to products produced by the above method as well as uses of the resulting products.
Glycoconjugates contain saccharide chains with from one up to twenty monosaccharide units and in which certain sequences have been shown to have biological activity, for example in the binding of different cells, pathogens, toxins, as well as antibodies or other proteins to cell surfaces, in cancer metastasis, in inflammatory processes, for instance selectin-carbohydrate interactions in the binding of white blood cells to the blood vessel wall, as a modifier of the biological activity and stability of glycoproteins, as immunogenic substances, which have potential in the vaccination against different diseases (See for instance Annual Review of Biochemistry, vol. 58 (1989), pages 309-350, and Current Opinion in Structural Biology, for example review articles in vol. 3 (1993) and references therein).
Structures containing the sequence Gal&bgr;1-4GlcNAc, called N-acetyl-lactosamine below, are especially of importance and are found for instance in glycoconjugate oligosaccharides of the lactosamine type. The structure is found in blood group structures, for instance Lewis-x (e.g. Gal&bgr;1-4(Fuc&agr;1-3)GlcNAc), sialylated Lewis-x and 3′-sulfated Lewis-x, and is of importance in e.g. selectin-carbohydrate interactions (as reviewed by J. B. Lowe, in Molecular Glycobiology, pages 163-205, Fukuda and Hindsgaul, Eds., IRL Press at Oxford University Press, Oxford, 1994; see also Curr. Opin. Struct. Biol. vol. 3 (1993)).
It is of interest to be able to produce derivatives of lactosamine in large quantities for biological/clinical studies/tests, for example for inhibition of the selectin-carbohydrate interaction in vivo to inhibit/modify cell-mediated inflammatory processes (for instance in acute septic shock, ARDS, reperfusion injuries, rheumatoid arthritis, virus-induced pneumonia, psoriasis and the like).
Chemical methods known heretofore to produce N-acetyl-lactosamine and derivatives thereof have demanded multi-step synthesis and are often expensive and labor intensive. Enzymatic methods used before the present method were mainly based on the use of galactosyltransferase (EC 2.4), a cofactor dependent enzyme which requires UDP-galactose as a glycosyl donor (e.g. Wong et al., J. Org. Chem. (1982), pages 5416-5418). This type of enzyme also has disadvantages as high acceptor selectivity and exhibits low efficiency with unnatural acceptors, for instance derivatives of glucosamine (e.g. as reviewed by Khan and Hindsgaul in Molecular Glycobiology, pages 206-229, Fukuda and Hindsgaul, Eds., IRL Press at Oxford University Press, Oxford, 1994). For a general review of enzymatic methods, see K. G. I. Nilsson, Trends in Biotechnology, 1988, p. 256-264 (the nomenclature used in that review article and in this application follow the same IUPAC-rules). Glycosidases have been used to produce N-acetyl-lactosamine and N-acetyl-allolactosamine from galactosides and N-acetyl-glucosamine (Sakai et al., J. Carbohyd. Chem, 11: 553-565, 1992).
Earlier methods with glycosidases (EC 3.2) for production of derivatives of N-acetyl-lactosamine gave generally low yields because of low or wrong regioselectivity. Thus, for example, &agr;-galactosidase from
E. coli
or from ox-testes give solely Gal&bgr;1-6GlcNPht, (K. G. I. Nilsson, unpublished result; Pht symbolizes a phthalimido group which generally is used as a temporary protection group on the amino group of glucosamine) when lactose is used as the glycosyl donor and GlcNPht is used as the acceptor.
SUMMARY OF THE INVENTION
The present invention describes a method which with unexpectedly high specificity gives the &bgr;1-4 linkage in the synthesis of different lactosamine derivatives, using abundant donor substances such as lactose and other low cost galactosyl donor substances. In one embodiment of the invention, the method is carried out by using the yeast
Bullera singularis
as a catalyst (classified as
Bullera singularis
according to Yeasts, second edition by Barnett et al., Cambridge University Press, 1990).
In a second embodiment, the process of the invention is carried out by using enzymes (which belongs to the group of glycosidases, EC Group 3.2), preferably in a crude, partially isolated or isolated form, especially &bgr;-galactosidase from
Bullera singularis
but also other &bgr;-galactosidase e.g. recombinant, of the same structure or of a similar structure (e.g., containing similar active site structure) as the one from
Bullera singularis.
DETAILED DESCRIPTION OF THE INVENTION
According to the more detailed aspects of the present invention, the process for producing lactosamine derivatives can be carried out as an equilibrium (reversed hydrolysis) reaction or preferably as a kinetic (transglycosylation) reaction. As is known in the art, the principles of an equilibrium reaction and a kinetic reaction are well understood (e.g. see K. G. I. Nilsson, Trends in Biotechnol. (1988), pages 256-264).
In the case where the reaction is carried out as a transglycosylation reaction, the glycosyl donor is a glycoside, e.g. of D-galactose (Gal) modified in the C-1 position (anomeric position) but it can also be an oligosaccharide, such as lactose (Gal&bgr;1-4Glc or aglycoside thereof, e.g.:
Gal&bgr;OR+GlcNR″—R′″→Gal&bgr;1-4GlcNR″—R′″+ROH
R can be a glycosidically linked organic group, for example sugar (e.g. C
n
H
2n
O
n
or C
n
H
2n−2
O
n−1
such as glucose), lower alkyl group (e.g. -Me, -Et) or an aromatic group (e.g. phenyl (-Ph), umberriferyl or m-, o-, or p-nitrophenyl group), preferably R is Glc (glucose) or nitrophenyl. Other glycosides (e.g. F-, N- or S-glycosides) may be selected.
It is known in the art that glycosidases allow some modification of the glycon part (i.e., the galactosyl part in the present invention) of the glycosyl donor. Therefore, in addition to Gal&bgr;OR, donors where the galactosyl part have been partially modified in a way still allowing the transglycosylation reaction to occur, resulting in the &bgr;1-4 linkage between the glycon part of the glycosyl donor and the glucosamine derivative, can be selected by the person skilled in the art for use with the method according to the present invention. Examples of such modifications of the glycon are modifications where at least one of the hydroxyl groups have inverted configuration (e.g. inversion in position 4 means that Gal is substituted for by Glc, that is Glc&bgr;OR, i.e. a &bgr;-glucoside, is used as donor), or where one of the hydroxyl groups of Gal has been modified or substituted for by an inorganic (e.g. -F, -H) or an organic group, e.g. a lower alkyl (e.g. methyl), allyl or an acetyl group. The selection of such a donor in the method according to the invention thus gives a &bgr;1-4 linked product in which the galactosyl part is correspondingly modified. Products of the type R′-Gal&bgr;1-4GlcNR″—R′″ may thus be prepared where R′-Gal relates to a modified glycon of the glycosyl donor. In the case of a transglycosylation reaction, the glycosyl donor in the scheme shown below, R′-Gal-R, is a &bgr;-glycoside:
R′-Gal-R+GlcNR″—R′″→R′-Gal&bgr;1-4GlcNR″—R′″+RH
In the transglycosylation reaction,
Gal&bgr;OR+GlcNR″—R′″→Gal&bgr;1-4GlcNR″—R′″+ROH
the reaction rate is higher than in the equilibrium reaction since the glycoside or disaccharide is more reactive than the non-activated sugar D-galactose used as donor in the equilibrium reaction. An enzyme of less purity, even a non-purified enzyme, can be used in the reaction since the enzymes are substrate/linkage specific and contaminating enzymes (e.g., &agr;-

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