Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-01-14
2002-03-12
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S105000, C435S072000, C435S137000, C536S018700, C536S055300, C536S124000
Reexamination Certificate
active
06355453
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the enzymatic synthesis of fluorinated sugars (including fluorinated amino or amido sugars) which have a cyclic structure and a side chain and hence have a nucleus of more than 6 carbon atoms. An aspect of this invention relates to the synthesis of fluorinated sialic acids or fluorinated nonulosaminic acids, a family of amino sugars containing at least 8, more typically at least 9, carbon atoms.
Another aspect of this invention relates to the enzymatic rid synthesis of 3-fluoroneuraminic acid (5-acetamido-3, 5-didesoxy-3-fluoro-D-glycero-D-galacto-nonulopyranosonic acid) and other 3,5-didesoxy-3-fluorononulopyranosonic acid derivatives and their use.
DESCRIPTION OF THE PRIOR ART
The sialic acids are generally cyclic pentoses (furanoses) or cyclic hexoses (pyranoses) with a side chain (typically three carbon atoms long); accordingly, the sugar nucleus of these compounds has 8 or 9 carbon atoms, and this nucleus can be substituted with an amino group which can be in turn be substituted with acyl groups and the like. The most important embodiments of this class of compounds can be considered to be derivatives of amino sugars such as neuraminic acid, C
9
H
17
NO
8
. At least five sialic acids occur in nature; they are widely distributed throughout the animal kingdom (including some bacteria as well as more complex organisms such as mammals) and appear to be regular components of glycoproteins and glycolipids (where they typically occupy terminal positions). The most important of the sialic acids is generally considered to be the N-acetyl derivative of neuraminic acid (“Neu5Ac”), i.e. 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulopyranosonic acid.
Preparations of sialic acids having 8- as well as 9-carbon nuclei are described in the scientific literature. For an example of a synthesis involving a furanose such as D-arabinose, see U. Kragl et al,
J. Chem. Soc. Perkin. Trans
. 1:119-124 (1994).
Because Neu5Ac and similar sugars occur so widely in higher animals and are so intimately involved in the physiology of mammals, they can be used as diagnostic and investigative tools, provided that they are suitably labeled with an isotope which can be measured or detected by various imaging techniques, radioactivity measurements, and other non-invasive procedures.
Fluorine is one of the most important labeling elements. The stable isotope,
19
F, provides opportunities for in vivo investigation with
19
F-NMR spectroscopy and has advantages over
1
H-NMR spectroscopy used in MR-tomography. In the body, fluorine occurs naturally only in teeth and bones. Therefore, it is possible to observe the kinetics of the biodistribution of fluorine-labeled compounds.
Fluorine has several isotopes in addition to
19
F, all of which are unstable, but only one of these has practical significance: the isotope,
18
F, which is radioactive and has the longest half-life of the unstable isotopes (the other unstable isotopes have half-lives lasting less than 3 minutes). The
1
8F isotope has a half-life of 110 minutes and is very useful in biological studies and in medicine, but a half-life of less than 2 hours does impose some limits on its utility. Examples of the uses of
18
F include non-invasive measurement of pharmacokinetic phenomena and the localization of tumors with
18
F-labeled 2-fluorodesoxyglucose (e.g. by positron emission tomography).
The short half-life of the
18
F isotope can impose severe requirements upon methods for synthesizing the
18
F-labeled compound. The yield of labeled compound should be high, and, even more important, the synthesis must be very rapid.
In any method in which an F-labeled compound is used, it is generally necessary that its physiological properties (e.g. its properties as a substrate for an enzyme) be similar to the endogenous, non-fluorinated compound it is supposed to mimic. The fluorine atom has the advantage of being fairly small in its covalent radius and hence does not differ too markedly from hydrogen in terms of steric hindrance. A fluorine substituent does differ from other substituents in terms of charge density, due to its high electronegativity and electronic density. But generally speaking, the advantages of fluorine as a labeling substituent far outweigh its disadvantages.
A number of F-labeled derivatives of Neu5Ac are known. For example, the 3-fluorine derivative (“Neu5Ac3F”), which has the systemic name 5-acetamido-3,5-didesoxy-3-fluoro-D-glycero-D-galacto-nonulopyranosonic acid, can be prepared by the aldol condensation of N-acetylmannosamine (“ManNAc”) and fluoropyruvate or &bgr;-fluoropyruvic acid (F—CH
2
—CO—COOH, systemic name 1-fluoro-2-oxopropanoic acid). The yield, however, is moderate (1.5%) and the purification is labor intensive. A recent paper discloses an electrophilic selective fluoridation which provided yields up to 80% and diastereomer selectivity of 75%. But the need for a faster and more stereospecific synthesis still exists.
The biosynthesis of Neu5Ac is enzyme-catalyzed and would appear to provide a model for a quick synthesis. In the biosynthesis, ManNAc is reacted with the pyruvate (pyruvic acid, CH
3
—CO—COOH), and the enzyme catalyst is N-acetylneuraminic acid aldolase. N-acetylneuraminic acid aldolase EC 4.1.3.3 can be found in animal tissue and some bacteria. This enzyme has also been produced by biotechnology methods involving common microorganisms such as
E. coli
. The natural sialic acid Neu5Ac has been made successfully by enzymatic synthesis, but the preparation of Neu5Ac derivatives (particularly Neu5Ac3F) is more problematic.
The enzymatic synthesis of Neu5Ac3F has been investigated. For example, the amounts of substrates (the sugar component, ManNAc, and the pyruvate, &bgr;-fluoropyruvate) have been varied considerably. The ManNAc would be expected to react with the &bgr;-fluoropyruvate (“F-pyr”) to form Neu5Ac F-substituted at the 3-position (Neu5Ac3F), but under the conditions chosen in the If literature (50 mM of both substrates, 0.1 U*/ml and N-acetylneuraminic acid aldolase [“Neu5Ac-aldolase”] from
E. coli
in water, buffered at a pH of 7.7, maintained at 37° C. and incubated for 24 hours), no conversion to Neu5Ac3F was observed. (The expression U*, a convenient enzyme activity unit employed in presenting data hereafter, was devised by the enzyme supplier, Toyobo, and is the enzyme activity with respect to the standard reaction of “Pyr” (pyruvate)+ManNAc to obtain Neu5Ac. The expression U [without the asterisk], also used hereafter, is the enzyme activity with respect to the reaction of F-Pyr+ManNAc to Neu5Ac3F.)
Accordingly, further investigation of the enzymatic synthesis of these fluorinated sugars (particularly fluorinated amino sugars such as Neu5Ac3F) is needed.
SUMMARY OF THE INVENTION
It has now been discovered that the manipulation of enzyme synthesis conditions can provide a high-yield and preferably rapid preparation of fluorinated ≧8-carbon sugars from the appropriate substrates using an aldolase enzyme. First and foremost, the enzyme concentration must be increased drastically in comparison to the concentration reported in the literature. This drastic increase in enzyme concentration does not create any serious economic drawback. The enzyme obtained through biotechnology methods, e.g. from
E. Coli
, is relatively inexpensive. The enzyme Neu5Ac-aldolase from
C. perfringens
is also commercially available and can be used in this invention with results similar to the enzyme from
E. coli
. Moreover, the enzyme exhibits remarkable stability and can be recovered and reused in subsequent cycles or batches.
To insure that the synthesis will be fast enough to permit the use of
18
F-labeled compounds, the sugar component is preferably present, in molar terms, in a large excess by comparison to the fluorinated component. The fluorinated component concentration should be sufficient also, however, and it is preferred, in the synthesis of
18
F-labeled compounds, to introduce a significant amount of
19
F-containing subs
Beliczey Juliane
Coenen Heinz Hubert
Hamacher Kurt
Kragl Udo
Liese Andreas
Connolly Bove & Lodge & Hutz LLP
Forschungszentrum Julich GmbH
Prats Francisco
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