Production of fucosylated carbohydrates by enzymatic...

Details Production of fucosylated carbohydrates by enzymatic... Production of fucosylated carbohydrates by enzymatic...

1992-10-14

2001-11-20

Lankford, Jr., Leon B. (Department: 1651)

Chemistry: molecular biology and microbiology

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

Preparing compound containing saccharide radical

C435S084000

Reexamination Certificate

active

06319695

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention provides for improved methods of enzymatic production of carbohydrates especially fucosylated carbohydrates. The invention provides for improved synthesis of glycosyl 1- or 2-phosphates using both chemical and enzymatic means. These phosphorylated glycosides are then used to produce sugar nucleotides which are in turn used as donor sugars for glycosylation of acceptor carbohydrates. Especially preferred herein is the use of the disclosed methods for fucosylation.
2. Summary of the Invention
This invention provides for a method of producing a fucosylated carbohydrate in a single reaction mixture comprising the steps of: using a fucosyltransferase to form an O-glycosidic bond between a nucleoside 5′-diphospho-fucose and an available hydroxyl group of a carbohydrate acceptor molecule to yield a fucosylated carbohydrate and a nucleoside 5′-diphosphate; and recycling in situ the nucleoside 5′-diphosphate with fucose to form the corresponding nucleoside 5′-diphospho-fucose. Preferred methods of this invention include the use of guanine as a base for the nucleoside, the use of catalytic amounts of nucleosides, the use of N-acetylglucosamine, galactose, N-acetylgalactosamine or N-acetyllactosamine as the carbohydrate acceptor molecule, and the use of a sialylated carbohydrate acceptor molecule.
This invention further contemplates the above method for producing fucosylated sialylated carbohydrate molecule through enzymatic formation of glycosidic linkages in a single reaction mixture comprising: forming a first glycosidic linkage between an diphosphonucleoside-activated glycosyl donor such as UDP-Gal and an available hydroxyl group of a carbohydrate acceptor molecule such as GlcNAc using a first glycosyltransferase such as &bgr;1,4-galactosyltransferase in preparing Gal&bgr;1,4GlcNAc; forming a second glycosidic linkage between a monophosphonucleoside-activated sialyl donor such as CMP-NeuAc and an available hydroxyl group of the sugar acceptor molecule such as the 3-position hydroxyl of the Gal of Gal&bgr;1,4GlcNAc using a sialyltransferase such as &agr;2,3sialyltransferase; forming a third glycosidic linkage between a diphosphonucleoside-activated fucosyl donor such as GDP-Fuc and an available hydroxyl group of the sugar acceptor molecule such as the 3-position hydroxyl of the GlcNAc of Gal&bgr;1,4GlcNAc using a fucosyltransferase such as &agr;1,3/4fucosyltransferase wherein at least one of steps (a) (b) or (c) further comprise the in situ formation of the phosphonucleotide-activated glycosyl donor from a catalytic amount of the corresponding monophosphate and diphosphate nucleoside. Especially preferred are methods of this invention wherein the fucosylated sialylated carbohydrate moiety product is a sialylated Lewis ligand such as sialyl Le
x
(SLe
x
) or sialyl Le
a
(SLe
a
) and wherein the fucose is transferred from a fucosyl donor to a hydroxyl group of a N-acetylglucosamine or galactose residue of the carbohydrate acceptor molecule.
This method embraces multiple glycosyltransferases catalyzing reactions in a single reaction mixture and preferred are those methods where one glycoslytransferase is a sialyltransferase selected from the group consisting of: &agr;2,3 sialyltransferase, an &agr;2,4 sialyltransferase an &agr;2,6 sialyltransferase and &agr;2,8 sialyltransferase. The invention contemplates the fucosylation of an oligosaccharide and preferred are those fucosyltransferases selected from the group consisting of: a &agr;1,2 fucosyltransferase, &agr;1,3/4 fucosyltransferase, &agr;1,3 fucosyltransferase, &agr;1,6 fucosyltransferase and &agr;1,4 fucosyltransferase. Especially preferred fucosyltransferases include &bgr;-galactosidase &agr;1,2 fucosyltransferase, N-acetylglucosamine &agr;1,3 fucosyltransferase, N-acetylglucosamine &agr;1,4 fucosyltransferase and N-acetyl-glucosamine &agr;1,6 fucosyltransferase.
The carbohydrate acceptor molecules are virtually unlimited because the glycosyltransferases are not selective beyond the adjacent sugar positions. Thus they may be any carbohydrate substituted molecule wherein the carbohydrate is a Gal&bgr;1,4GlcNAc molecule or an analog thereof, or terminates in a Gal&bgr;1,4GlcNAc-X moiety and where X is an organic molecule. Additional carbohydrate acceptor molecules that are substrates for a fucoylase include analogs of Gal&bgr;1,4GlcNAc and Gal&bgr;1,4GlcNAc-X. Exemplary of such molecules as lactose, NeuAc&agr;1,6Gal&bgr;1,4GlcNAc, Gal&bgr;1,3GlcNAc, Gal&bgr;1,4Glucal (lactal), NeuAc&agr;2,3Gal&bgr;1,4Glucal, the 2-halo-substituted reaction products of the above glucals, Gal&bgr;1,4(5-thio)Glc, Gal&bgr;1,4GlcNAc&bgr;-O-allyl and the like. It is to be understood that the carbohydrate acceptor molecule must contain an available hydroxyl group on the saccharide to which the donated fucosyl or other sugar group is linked, and the hydroxyl that must be present is determined by the glocsyltransferase enzyme that is utilized in the reaction.
The method contemplated herein further comprises regeneration of catalytic amounts of nucleotides used to form nucleoside sugars. A preferred bases for the nucleotides are either cytidine, guanine, or uridine. Monosaccharide donors are activated nucleotide sugars such as cytidine 5′-monophospho-N-acetylneuraminic acid, guanidine 5′-diphospho-fucose and uridine 5′-diphospho-galactose.
In addition to the above methods, this invention also contemplates in vitro reaction systems. Such systems refer to an inert or nonreactive container or compartment housing the reagents used to conduct the above described reactions. More specifically, these reaction systems have at a minimum a fucosyltransferase and a nucleoside diphosphofucose forming enzyme. These reaction systems can further comprise guanosine diphosphofucose pyrophosphorylase as the GDP-fucose-forming enzyme, a kinase such as pyruvate kinase or fructose-1,6-diphosphate kinase, acetyl kinase or fucose kinase. Other reagents can include a NADPH regeneration system or guanosine diphosphate mannose and guanosine diphospho mannose pyrophosphorylase. If a NADPH regeneration system is present it can include a catalytic amount of NADP, isopropanol in about 1 percent to about 10 percent, preferably about 2 percent to 4 percent w/v of the reaction system, and an alcohol dehydrogenase.
A number of chemical methods for synthesizing oligosaccharides are also disclosed herein. One method includes the production of a glycosyl 1- or 2-phosphate by reacting a blocked glycosyl ring having a hydroxyl at the anomeric position (1- or 2-position) with a trivalent phosphitylation reagent to yield a blocked glycosyl 1- or 2-phosphite-substituted ring. The blocked phosphite is oxidized to form a corresponding phosphate that is utilized in an enzymatic reaction. The glycosyl ring can include a galactosyl, glucosyl, fucosyl, N-acetylglucosyl and mannosyl as well as other saccharides. The preferred trivalent phosphitylating reagents are dibenzyl N,N-dialkylphosphoroamidite such as dibenzyl N,N-diethylphosphoroamidite. Such dialkyls are lower alkyls of 1-5 carbons inclusive and they can be the same or different. This method further utilizes blocking reagents such as acetyl or benzyl. The glycosyl ring is optionally from the group consisting of D- or L-aldoses having four, five or six carbons or from the group consisting of D- or L-ketoses having four, five or six carbons, as well as saccharides having up to nine carbons in the saccharide chain.
This invention further contemplates novel intermediates for the production of glycosyl 1- or 2-phosphates. A preferred intermediate is a blocked phosphityl monosaccharide of the formula I:
wherein R
1
is aryl or lower alkyl;
X is independently oxygen or nitrogen;
R
2
in independently an acyl, benzyl, silyl or alkyl blocking group;
R
3
is independently —CH
3
, —OR
2
, —CH
2
OR
2
, —CH(OR
2
)—CH(OR
2
), or —CH(OR
2
)—CH(OR
2
)—CH(OR
2
);
R
4
is hydrogen (H), carboxyl or C
1
-C
5
or benzyl carboxylate ester; and
n is 1 or 2.
In

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