Coenzymes useful for the synthesis of L-carnitine

Details Coenzymes useful for the synthesis of L-carnitine Coenzymes useful for the synthesis of L-carnitine

2001-04-11

2002-01-08

Weber, Jon P. (Department: 1651)

Chemistry: molecular biology and microbiology

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

Preparing oxygen-containing organic compound

C435S157000, C435S128000, C435S195000

Reexamination Certificate

active

06337197

ABSTRACT:

The invention described herein relates to enzymes useful for the synthesis of L-carnitine, particularly a compound of coenzyme A, and more particularly gamma-butyrobetainyl-coenzyme A and crotonobetainyl-coenzyme A, to procedures for their preparation and to their use for the production of L(−)-carnitine from crotonobetaine and D(−)-carnitine.
To date we have no knowledge of isolated enzymes which are useful for the synthesis of L-carnitine, and in particular neither gamma-butyrobetainyl-coenzyme A nor crotonobetainyl-coenzyme A are known substances. When the abilities of these compounds to be used in the L(−)-carnitine production process are verified, the known technique and the knowledge relating to L(−)-carnitine production processes must be taken into consideration Numerous chemical and biochemical or biotechnological processes are known for obtaining L(−)-carnitine. Most of the chemical synthesis processes yield D,L-carnitine as the result, by reaction of the racemic mixture with optically active separation acids, for example with optical isomers of tartaric acid, camphoric acid or camphorsulphonic acid, and via subsequent fractionated crystallization it is possible to obtain the L-carnitine enantiomer (for example, patents granted DD 23 217; DD 93 347; and published application DE 2997 672). All the synthesis processes to date present the disadvantage that D(+)-carnitine results as a discard product and must be disposed of and that only a maximum of 50% of the synthesized product is obtained as L(−)-carnitine. The therapeutic use of D,L-carnitine is non-substitutable, in that D(+)-carnitine is not substitutable, inasmuch as D(+)-carnitine is not only ineffective as regards the oxidation of fatty acids, but is also more competitive as a substance inhibiting the various transport systems and specific enzymes of L(−)-carnitine (Life Sciences 28[191]2931-2938). For this reason, processes have been developed in recent years for stereospecific synthesis from initial achiral stages (for example, Tetrahedron, [1992], Vol. 48, 319-324).
Alternatives to the chemical synthesis of L(−)-carnitine are microbiological or enzymatic processes. In this way it proves possible to exploit the inverse reaction of L(−)-carnitine dehydrogenase (EC 1.1.1.108) to produce L(−)-carnitine from 3-dihydrocarnitine (U.S. Pat. No. 4,221,869). Being an NADH-dependent enzyme, the preparation of reduction equivalents must be guaranteed. 3-Dihydrocarnitine, moreover, is very unstable. Various Entero-bacteriaceae strains are capable of transforming L(−)-carnitine into gamma-butyrobetaine via crotonobetaine in anaerobic conditions (patents granted DD 221 905, JP 6167 494, JP 61 234 794, JP 61 234 788). The metabolisation of L(−)-carnitine to crotonobetaine is reversible and is catalyzed by a stereospecific enzyme, L(−)-carnitine dehydratase (patents granted DD 281 735, DD 281,919). In this way, crotonobetaine can be used as the achiral end compound for the synthesis of L(−)-carnitine. A number of Proteus strains can also form L(−)-carnitine from crotonobetaine in aerobic conditions (U.S. Pat. No. 5,300,430). For the enzymatic synthesis of L(−)-carnitine from the discard product D(+)-carnitine, a racemate has been described (patent granted DD 300 181). A third possibility consists in obtaining L(−)-carnitine from gamma-butyrobetaine via gamma-butyrobetaine hydroxylase (EC 1.14.11.1) (publication DE 3123975). In patents EP 158 194, EP 195 944 and JP 61 199 793 processes are described based on the production of L(−)-carnitine from crotonobetaine or gamma-butyrobetaine by cultivating selected micro-organisms on a supplementary source of C, for example glycinebetaine, in the presence of crotonobetaine or gamma-butyrobetaine.
L(−)-carnitine dehydratase (EC 4.2.1.89) catalyses the reversible transformation of crotonobetaine to L(−)-carnitine only in the presence of a low-molecular-weight factor F isolated from
Escherichia coli
and as yet unidentified (patent granted DD 281 735). From the immobilization of an L(−)-carnitine dehydratase isolated from the Enterobactenaceae family a process has been developed for the synthesis of L(−)-carnitine without the formation of subproducts (DD 281 910). The aforesaid low-molecular-weight factor F is equally indispensable for the racemization of D(+)- to L(−)-carnitine. The reduction of crotonobetaine to gamma-butyrobetaine also occurs only in the presence of this factor.
L(−)-carnitine (3-hydroxy-4-trimethylaminobutyrate) is a ubiquitous, naturally occurring compound. It is of fundamental importance as a carrier of acyl groups for the transport of long-chain fatty acids across the internal mitochondrial membrane. As a result of its central role in the metabolism of superior organisms, L(−)-carnitine is used in the therapy and prophylaxis of patients with various heart diseases as well as in the treatment of patients on dialysis (see: Pathology 17, [1985], 116-169). L(−)-carnitine supplementation is indispensable in the parenteral nutrition of neonates in the postnatal period and also in adults for longer periods (Gürtler and Löster, Carnitin [1996], 21-30). L-(−)-carnitine is a dietary supplement of growing importance.
The microbiological processes used for the synthesis of L(−)-carnitine present the disadvantage that the micro-organisms, owing to their very limited number, may be poorly separated from their culture medium and new nutrient media must be made available continually for the cultivation. The result is that a substantial effort must be made to regenerate the evaporated L(−)-carnitine-containing culture fluid. In the synthesis of L(−)-carnitine from crotonobetaine in microbiological systems there is the possibility of transforming crotonobetaine to gamma-butyrobetaine via crotonobetaine reductase as a reaction competing with the synthesis reaction.
Of by no means secondary importance is the problem of using micro-organisms for the production of substances for pharmaceutical use. At times, the micro-organisms used come from pathogenic strains, or are highly engineered and contain extraneous genes, which is an aspect to which the Regulatory Authorities devote a great deal of attention.
The process for the enzymatic synthesis of L(−)-carnitine from crotonobetaine presents the disadvantage that a solution obtained from
E. coli
and devoid of proteins, through immobilized L(−)-carnitine dehydratase, must contain not only crotonobetaine, but also an unidentified factor F, which is essential for activation of the enzyme. Influences disturbing factor F produced by components of the protein-free solution cannot be excluded. The enzymatic synthesis of L(−)-carnitine can be optimized only to a limited extent, particularly in view of the fact that the amount of factor F used cannot be exactly quantified. The same factor F is of fundamental importance for the racemization of D(+)- to L(−)-carnitine described in
E. coli
. Factor F cannot be replaced by known coenzymes or cofactors of enzymes (Jung et al., Biochim. Biophys. Acta 1003 [1989] 270-276).
The cause of the disadvantages mentioned consists in our poor knowledge of the structure of factor F and of its role in activating the apoenzyme of L(−)-carnitine dehydratase. No other compounds are known which could stimulate activation of the apoenzyme of L(−)-carnitine dehydratase in the same way.
The purpose of the invention described herein is to make the stereospecific synthesis of L(−)-carnitine from crotonobetaine in an a cellular medium enzymatically possible as well as the racemization of the discard product D(+)-carnitine to L(−)-carnitine. These enzymatically catalyzed reactions represent an alternative to the pure chemical synthesis of L(−)-carnitine or to the use of microbiological processes to obtain L(&

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