Enzymatic synthesis of L-fucose and L-fucose analogs

Details Enzymatic synthesis of L-fucose and L-fucose analogs Enzymatic synthesis of L-fucose and L-fucose analogs

1995-10-24

2004-03-30

Prats, Francisco (Department: 1651)

Chemistry: molecular biology and microbiology

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

Preparing compound containing saccharide radical

C435S094000, C435S072000

Reexamination Certificate

active

06713287

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to methods for synthesizing L-fucose and L-fucose analogs. More particularly, the invention is directed to enzymatic methods for synthesizing L-fucose and L-fucose analogs and to the L-fucose analogs synthesized thereby.
BACKGROUND
L-fucose 1 is a naturally occurring sugar widely found in Nature. It is found in many bacterial and plant glycosides and polysaccharides (Lindberg et. al.,
MTP Int. Rev. Ser. One, Carbohydr
. 1973, 7, 319; Jann, X et. al. In I. W. Shutherland (Ed.),
Surface Carbohydrates of the Prokaryotic Cell
, Academic Press, New York, 1977, pp 247-287; Aspinall, G. O. et. al.
MTP Int. Rev. Sci., Ser. One, Carbobydr
. 1973, 7, 285). L-fucose is 25 sometimes found sulfated (Percival, E et. al.
Methods Carbohydr. Chem
. 1962, 1, 195; Larsen, B et. al.,
Acta Chem. Scand
. 1966, 20, 219; Chandrasekaran, E. V. et. al.
Biochemistry
1995, 34, 2925). It has also been found in oligosaccharides of human milk (Kobata, A. In M. I. Horowitz, W. Pigman (Eds.),
The Glycoconjugates
, Vol. 1, Academic Press, New York, 1977, pp 423-440). In addition, L-fucose is found in many glycolipids (Hakomori, S.-I.
Prog. Biochem. Pharmacol
. 1975, 10, 167; Mckibbin, J. M.
J. Lipid Res
. 1978, 19, 131) and glycoproteins including several families of blood-group antigens (Loyd, K. O.
MTP Int. Rev. Sci., Ser. Two, Carbohydr
. 1976, 7, 251; Kornfeld et. al.
Annu. Rev. Biochem
. 1976, 45, 217). It is found in cell-surface oligosaccharides, such as the tetrasaccharide sialyl Lewis x (Le
x
) on neutrophils, as part of selectin ligands involved in cell adhesion (Phillips, M. et. al.
Science
1990, 250, 1130; Waltz, G. et. al.,
Science
1990, 250, 1132; Lowe, J et. al.
Cell
1990, 63, 475) and cancer metastasis processes (Paulson, J. C.
In The Receptors
; Conn, M., Ed.; Academic Press: New York, 1985; Vol. 2, pp 131-219; Paulson, J. C.
In Adhesion: its role in inflammatory disease
; Harlan, J.; Liu, D., Eds.; W. H. Freeman: New York, 1992; Chapter 2, p 19; Springer, T. et. al.
Nature
1991, 349, 196; Lasky, L.
Science
1992, 258, 964; Rice, G. et. al.
Science
1989, 246, 1303; Bevilacqua, M P et. al.
Proc. Natl. Acad. Sci. U.S.A
. 1987, 84, 9238.
Given the importance of sialyl Le
x
and derivatives as potential therapeutic agents for the treatment of inflammatory diseases, the development of an efficient synthesis of 1 has been a subject of interest in glycotechnology (Wong et. al.
J. Am. Chem. Soc
. 1992, 114, 9283; Wong, et. al.
J. Am. Chem. Soc
. 1993, 115, 7549; Wong, et. al.
J. Am. Chem. Soc
. 1995, 117, 66; Mulligan, M. S. et. al.
Nature
1993, 364, 149; Flowers, H. M.
Adv. Carbohydr. Chem. Biochem
. 1981, 39, 279).
Organic synthesis may be employed to produce L-fucose 1. Tanimura discloses an organic synthesis of L-fucose 1 in 9 steps with a 1% overall yield, starting from L-arabinose (Tanimura, A. et. al.
Eisei Shikenjo Hokoku
1959, 77, 123
; Chem. Abstr
. 1961, 55, 12306). An improved organic synthesis of L-fucose 1 is disclosed by Dejter-Juszyzynski using D-galactose as a starting material and providing a 15% overall yield (4.88 mmol) in 4 steps (Dejter-Juszynski, M. et. al.
Carbohydr. Res
. 1973, 28, 144). A synthesis for the preparation of L-fucose from D-glucose has produced 19.3% of L-fucose and afforded 1.69 mmol of L-fucose in 5 steps (Chiba, T.,
Chem. Pharm. Bull
. 1979, 27, 2838). L-fucose has also been synthesized using D-mannose in 54.8% yield and 6 steps (Gesson, J. et. al.
Tetrahedron Lett
. 1992, 33, 3637) or using methyl &agr;-D-mannopyranoside in 24% overall yield affording 0.26 mmol of L-fucose in 8 steps (Wong, C.-C.
Carbohydr. Res
. 1981, 95, 131). In general, the known organic syntheses of L-fucose 1 employ multichemical transformations and result in low yields.
There is no known method for synthesizing L-fucose 1 enzymatically.
Commercially, L-fucose 1 is obtained from natural sources. The preferred natural source is Fucoidan, i.e. a substance extracted from kelp (Schweiger, R. G. (To Kelco Co.), U. S. Pat. No. 3,240,775 Mar. 15, 1966, Appl. Jul. 30, 1962
; Chem. Abstr
. 1966, 65, 2342).
The synthesis of L-fuculose-1-phosphate 4 has been previously carried out from dihydroxyacetone phosphate 2 and L-lactaldehyde 3 via recombinant L-fuculose aldolase-catalyzed aldolic condensation. In addition, the synthesis of L-fuculose-1-phosphate 4 has been performed via L-fucose isomerase-catalyzed isomerization of L-fucose 1, coupled with L-rhamnulose kinase (Fessner et. al.
Tetrahedron: Asymmetry
1993, 4, 1183; Fessner et. al.
Angew. Chem. Int. Ed. Engl
. 1991, 30, 555; Fessner et. al.
Tetrahedron Lett
. 1992, 33, 5231; Wong, C.-H. et. al.
J. Org. Chem
. 1991, 56, 6280). D/L lactaldehyde dimethylacetal 6 has been synthesized and reported by Wong et. al.
J. Am. Chem. Soc
. 1986, 108, 7812.
L-fuculose 5 has been prepared from L-fucose 1 using a cell free extract of an
E. coli
mutant strain in the presence of a borate buffer (Green, M.; Cohen, S. S.
J. Biol. Chem
. 1956, 219, 557) via bacterial oxidation of L-fucitol by Aerobacter suboxidans (Williams, D. T.; Jones, J. K. N.
Can. J. Chem
. 1967, 45, 741) and from 4 via enzyme-catalyzed phosphate hydrolysis (Fessner et. al.
Tetrahedron: Asymmetry
1993, 4, 1183; Wong, C.-H. et. al.
J. Org. Chem
. 1991, 56, 6280).
L-fuculose-1-phosphate aldolase is prepared from recombinant
E.Coli
cells using the methodology as described in Wong, C.-H. et. al.
J. Am. Chem. Soc
. 1994, 116, 6191 and Wong, C.-H.
Bioorg. Med. Chem
. 1995, 3, 945
. E.coli
which express recombinant aldolase may be obtained from American Type Culture Collection (ATCC number 86984).
E.coli
which express recombinant isomerase may be obtained from American Type Culture Collection (ATCC number 87024). Acid phosphatase is commercially available and can be purchased from Sigma chemical company.
What is needed is a simple method for enzymatically synthesizing L-fucose 1 and L-fucose analogs for providing good yields at low cost.
SUMMARY
One aspect of the invention is directed to a method for enzymatically synthesizing L-fucose and L-fucose analogs. The method for enzymatically synthesizing L-fucose includes three steps, viz. providing L-fuculose-1-phosphate, enzymatically converting the L-fuculose-1-phosphate to L-fuculose; and then enzymatically converting the L-fuculose to L-fucose.
In a preferred mode, the L-fuculose-1-phosphate is obtained by means of an aldol addition reaction between dihydroxyacetone phosphate and DL-lactaldehyde catalyzed by aldolase or more particularly by L-fucolose-1-phosphate aldolase. The DL-lactaldehyde may be obtained by conversion from DL-lactaldehyde dimethylacetal. In one mode of the invention, the L-fuculose-1-phosphate is purified after its production by the aldol addition reaction and prior to its conversion to L-fuculose. In an alternative mode of the invention, the L-fuculose-1-phosphate is not purified after the aldol addition reaction, i.e., it is employed without purification in a dephosphorylation reaction which converts the L-fuculose-1-phosphate to L-fuculose. In this later instance, the aldol condensation reaction and the dephosphorylation reaction may be performed in a single reaction vessel.
In a preferred mode the dephosphorylation of L-fuculose-1-phosphate to form L-fuculose is catalyzed by acid phosphatase (E.C. 3.1.3.2). The isomerization of the L-fuculose to form L-fucose may then be catalyzed by L-fucose isomerase. In one mode of the invention, the L-fuculose is purified prior to its isomerization to L-fucose. In an alternative mode of the invention, the L-fuculose is not purified, i.e., it is employed without purification in the isomerization reaction which converts the L-fuculose to L-fucose. In this later instance, the dephosphorylation reaction and the isomerization reactions may be performed in a single reaction vessel.
A second aspect of the invention is directed to a method for enzymatically synthesizing L-fucose analogs represented by the following structure:
wherein R is a substituent selected from the group consisting of H,

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