Oligosaccharide enzyme substrates and inhibitors: methods...

Details Oligosaccharide enzyme substrates and inhibitors: methods... Oligosaccharide enzyme substrates and inhibitors: methods...

1998-05-05

2001-01-02

Peselev, Elli (Department: 1623)

Chemistry: molecular biology and microbiology

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

Preparing compound containing saccharide radical

C435S074000, C435S085000, C435S100000, C435S101000, C435S183000

Utility Patent

active

06168934

ABSTRACT:

TECHNICAL FIELD
The present invention relates to oligosaccharide compounds, and more particularly to di-, tri- and tetrasaccharides that are substrates or inhibitors of glycosyltransferase and glycosidase enzymes, their manufacture and use.
BACKGROUND ART
The stereocontrolled synthesis of oligosaccharides based on sophisticated protection/deprotection, activation and coupling strategies has been well established. See, e.g., Danishefsky et al.
J. Am. Chem. Soc.,
111:6656 (1989); Okamoto et al.,
Tetrahedron,
46:5835 (1990); and Ito et al.,
Tetrahedron
46:89 (1990). A useful alternative to the chemical synthesis is enzymatic oligosaccharide synthesis based on glycosyltransferase or glycosidase enzymes. Toone et al.,
Tetrahedron,
45:5.365 (1989). One advantage of such enzymatic synthesis is the lack of extensive protection and deprotection steps. A disadvantage of such enzymatic synthesis is the apparent limitation of product formation that results from the specificity of glycosyltransferase and glycosidase enzymes.
Glycosyltransferases are highly specific enzymes that catalyze the transfer of activated donor monosaccharides to acceptor saccharides. That transfer results in the elongation or synthesis of an oligo- or polysaccharide.
A number of glycosyltransferase types have been described including sialyltransferases, fucosyltransferases, galactosyltransferases, N-acetylgalactosaminyltransferases, N-acetylglucos-aminyltransferases and the like. Beyer, et al.,
Adv. Enzymol.,
52:23 (1981). The designation of those enzymes indicates the nature of the donor substrate. Thus, for example, a sialyltransferase transfers a sialic acid moiety to an acceptor molecule.
Within each of the general enzyme types set forth above, specific transferase enzymes are additionally designated by the type of glycosidic linkage formed. For example, a &bgr;1,4-galactosyltransferase transfers a galactosyl moiety to an acceptor molecule, forming a &bgr;1,4-glycosidic linkage with such acceptor.
Further, glycosyltransferases are characterized by the acceptor molecule to which the donor glycosyl compound is transferred. A &bgr;1,4-galactosyltransferase from bovine milk (GalT, EC 2.4.1.22) is known to accept N-acetylglucosamine (GlcNAc) and its glycosides (p is better than a-glycoside) as acceptor substrates. See.e.g., Schanbacher, et al.,
J. Biol. Chem.,
245:5057 (1970); Berliner, et al.,
Mol. Cell. Biochem.,
62:37 (1984); Nunez, et al.,
Biochemistrv,
19:495 (1980); Beyer, et al.,
Adv. Enzymol.,
52:23 (1981); Barker, et al.,
J. Biol. Chem.,
247:7135 (1972); and Babad, et al.,
J. Biol. Chem.,
241:2672 (1966). Glucose and its &agr;- and &agr;-glucosides are also acceptable; however, lactalbumin is required for &agr;-glucosides. Beyer, et al., supra.
Taken together with the donor and linkage specificity set forth above, such acceptor specificity is used to define unique products of glycosyltransferase activity.
Oligosaccharides are considered to have a reducing end and a non-reducing end, whether or not the saccharide at the reducing end is in fact a reducing sugar. In accordance with accepted nomenclature, oligosaccharides are depicted herein with the non-reducing end on the left and the reducing end on the right.
All oligosaccharides described herein are, thus, described with the name or abbreviation for the non-reducing saccharide (i.e., Gal), followed by the configuration of the glycosidic bond (&agr; or &bgr;), the ring position of the non-reducing saccharide involved in the bond (1 or 2), the ring position of the reducing saccharide involved in the bond (2, 3, 4, 6 or 8), and then the name or abbreviation of the reducing saccharide (i.e., GlcNAc).
It is often extremely difficult to make synthetic saccharides that can be used to study naturally occurring synthetic routes by inhibiting the synthetic reactions. The lack of such synthetic inhibitors hampers attempts to investigate the effects of metabolic changes on carbohydrate production and turnover.
It is also often difficult to prepare novel, non-naturally occurring oligo- and polysaccharides that are useful as carriers or solubilizing agents for drugs and, which because of their non-natural structures, are resistant to degradation in vivo.
There is, therefore, a pressing need for oligosaccharide compounds and efficient methods of making the same that serve as substrates or inhibitors of transferase and glycosidase enzymes.
BRIEF SUMMARY OF THE INVENTION
The present invention provides novel oligosaccharides that are substrates for some glycosyltransferases and that inhibit other glycosyltransferase and glycosidase enzymes as well as a method for making such oligosaccharides. Those oligosaccharides are also useful as building blocks in the synthesis of other oligosaccharides such as sialyl Le
x
and its analogs.
In one aspect, the present invention contemplates an oligosaccharide that corresponds to structural Formula I:
wherein X is O, S, SO, SO
2
or NR
16
, wherein R
16
is hydrogen, C
1
-C
12
acyl, C
1
-C
12
alkyl, C
1
-C
4
alkoxycarbonyl or >NR
16
is a C
1
-C
12
alkyl N-oxide;
R
1
is absent, hydrogen, hydroxyl, C
1
-C
4
acyl, C
1
-C
4
alkoxycarbonyloxy, a saturated or unsaturated alkoxide or alkoxy alkoxide containing up to five carbon atoms or a glycosidially linked saccharide;
R
1
′ is hydrogen or R
1
and R
1
′ together form an oxo group;
R
2
is absent, hydrogen, hydroxyl, halide, C
1
-C
5
alkoxy or NR
17
R
18
where R
17
is hydrogen or C
1
-C
4
alkyl and R
18
is hydrogen, C
1
-C
4
alkyl, C
1
-C
4
acyl, or C
1
-C
4
alkoxycarbonyl, or NR
17
R
18
together form a cyclic imido group containing 4-8 carbon atoms;
R
3
and R
4
are independently hydrogen, C
1
-C
4
alkyl, hydroxyl, thiophenyl, C
1
-C
3
alkylthio, a saturated or unsaturated alkoxide or alkoxy alkoxide containing up to five carbon atoms, a glycosidically linked glucosyl, N-acetylglucosaminyl, galactosyl,
N-acetylgalactosaminyl, fucosyl, mannosyl, rhamnosyl, sialyl group or a disaccharide thereof, or R
3
and R
4
together form an oxo group, with the proviso that at least one of R
3
and R
4
is hydrogen except when (i) R
3
and R
4
together form an oxo group, (ii) R
2
and R
3
are absent with their bonds forming ethylenic unsaturation or (iii) X is NR
16
;
R
5
is absent, hydrogen, hydroxyl, methyl, C
1
-C
4
acyl or C
1
-C
4
alkoxycarbonyloxy;
R
6
is absent, hydroxymethyl, methyl, trihydroxyropyl, methylene C
1
-C
4
acyloxy or benzyloxy;
R
7
is hydrogen or carboxyl;
R
8
is hydrogen, hydroxyl or acetamido;
R
9
is hydroxymethyl, methyl, trihydroxypropyl, methylene C
1
-C
4
acyloxy or benzyloxy, and 3-acetoxy-1,2-dihydroxypropyl, 3-lactyloxy-1,2-dihydroxypropyl, 3-azido-1,2-dihydroxypropyl, and 3-fluoro-1,2-dihydroxypropyl when R
8
is hydrogen and R
11
is N-acetylamino;
R
10
is absent, hydroxyl or acetamido;
R
11
is absent, hydroxyl or acetamido;
R
12
is hydroxyl or acetamido;
R
13
is hydroxymethyl or trihydroxypropyl, and 3-acetoxy-l,2-dihydroxypropyl, 3-lactyloxy-1,2-dihydroxypropyl, 3-azido-l,2-dihydroxypropyl, and 3-fluoro-1,2-dihydroxypropyl when R
15
is hydrogen and R
12
is N-acetylamino;
R
14
is hydrogen or carboxyl;
R
15
is hydrogen, hydroxyl or acetamido; and
m is zero or one such that when m is zero, ring C is absent and when m is one, ring C is present;
with the provisos (a) that one of substituents R
1
, R
2
and R
5
or a hydroxyl group of R
6
is absent from ring B and ring B is joined to ring A through a glycosidic bond to the ring B carbon of the absent substituent, and that a numbered substituent or hydroxyl is only absent when ring A is joined to ring B at the position of that substituent or hydroxyl except as enumerated herein; (b) that when m is one, one of substituents R
10
and R
11
or a hydroxyl group of R
9
is absent from ring A and ring C is joined to ring A through a glycosidic bond to the ring A carbon of the absent substituent or hydroxyl, and that numbered substituent or hydroxyl is only absent when ring C is joined to ring A at the position of that substituent or hydroxyl, or a second of

Affiliated with

Also associated with

Rating

Oligosaccharide enzyme substrates and inhibitors: methods... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Oligosaccharide enzyme substrates and inhibitors: methods..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Oligosaccharide enzyme substrates and inhibitors: methods... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2440994