Simultaneous enzymatic synthesis of (S)-3-fluoroalanine and (R)-3-fluorolactic acid

Article abstract of DOI:10.1016/S0957-4166(00)00096-3

A coupled enzymatic system for the simultaneous synthesis of (S)-3-fluoroalanine (1a) and (R)-3-fluorolactic acid (3) with L-alanine dehydrogenase (L-AlaDH) from Bacillus subtilis and rabbit muscle L-lactate dehydrogenase (L-LDH) using rac-1 and NAD⁺ is described. Analysis of isolated products of the laboratory preparative scale process revealed 1a in 60% yield and 88% ee and 3 in 80% yield and over 99% ee. The compounds 1a and 3 represent chiral building blocks for the synthesis of several products with pharmacological activity. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0957-4166(00)00096-3

Tetrahedron: Asymmetry 11 (2000) 1465±1468
Simultaneous enzymatic synthesis of (S)-3-¯uoroalanine and
(R)-3-¯uorolactic acid
L. P. B. Goncalves, O. A. C. Antunes,* G. F. Pinto and E. G. Oestreicher
Â
Instituto de Quõmica, UFRJ, Cidade Universitaria CT Bloco A, Rio de Janeiro, RJ 21945-970, Brazil
Â
Received 10 February 2000; accepted 2 March 2000
Abstract
A coupled enzymatic system for the simultaneous synthesis of (S)-3-¯uoroalanine (1a) and (R)-3-¯uoro-
lactic acid (3) with l-alanine dehydrogenase (l-AlaDH) from Bacillus subtilis and rabbit muscle l-lactate
dehydrogenase (l-LDH) using rac-1 and NAD+ is described. Analysis of isolated products of the labora-
tory preparative scale process revealed 1a in 60% yield and 88% ee and 3 in 80% yield and over 99% ee.
The compounds 1a and 3 represent chiral building blocks for the synthesis of several products with phar-
macological activity. # 2000 Elsevier Science Ltd. All rights reserved.
Fluorinated a-hydroxyacids and a-aminoacids are considered to be highly versatile chiral
building blocks in asymmetric synthesis for the production of several compounds of pharmaco-
logical interest.1
(S)-3-Fluoroalanine or 3-¯uoro-d-alanine (1a) is recognized as an antibiotic via irreversible
inactivation of bacterial alanine racemase.2 It also acts as an inhibitor of serine palmitoyl-
transferase3 and it is a potential precursor for the synthesis of ¯uoroamino compounds.1
(R)-3-Fluorolactic acid (3), on the other hand, represents an important three carbon, chiral
building block, 1,2,3-trisubstituted for the synthesis, for instance, of b-adrenergic blocking agents
of the aryloxypropanolamine group such as (S)-propranolol and (S)-moprolol. It is also an
intermediate for the synthesis of other products of pharmaceutical interest such as (S)-hydroxy-
pyrrolidine-2-one which in its open form [(S)-4-amino-2-hydroxybutanoic acid] is considered to
be one of the most potent inhibitors known of the neurotransmitter GABA, also showing anti-
cancer activity. Compound 3 is also a potential intermediate for the production of carnitin and
GABOB.4
A useful methodology for the synthesis of chiral building blocks is represented by the utilization
of enzymes as catalysts as widely reported in the literature.4,5 The use of dehydrogenases in
organic synthesis is limited by the fact that the coenzymes (NAD+ or NADP+) are very expensive
* Corresponding author. E-mail: octavio@iq.ufrj.br
0957-4166/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(00)00096-3

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reagents to be used in stoichiometric concentrations. Overwhelmingly, the use of catalytic
amounts of coenzymes, that can be achieved with a convenient method for recycling the coenzyme,
is an important step for the economical viability of the process.4
(R)-3-Fluoroalanine (1b) was continuously produced by Ohshima et al. in the reductive sense
with l-AlaDH from Bacillus sphaericus from 2 and ammonium formate in a proper reactor.6 In
that system, the recycle of the coenzyme was made possible by oxidation of the formate to produce
CO2, catalyzed by yeast formate dehydrogenase.
Our group has recently described the enzymatic production of 3.4 We envisaged an enzymatic
system capable of simultaneously producing 1a via resolution of rac-1 by oxidation of 1b, and 3
by reduction of 2. The overall reaction system devised is shown in Scheme 1.
Scheme 1.
In the proposed system, l-AlaDH and l-LDH were designed to be used in the presence of
catalytic concentrations of NAD+ with rac-1 as starting material. Therefore, l-AlaDH will cat-
alyse the oxidative deamination of the l-enantiomer of rac-1 in the presence of NAD+, thus
producing NADH, NH+4 and 2. This latter compound will then be enantioselectively reduced to 3,
in the presence of NADH, by the action of l-LDH, so allowing the desirable recycling of NADH
to NAD+.
This process presents the advantage that the product of the principal reaction is the appro-
priate feedstock of the recycling reaction. In addition, as soon as 2 is formed, it is reduced,
establishing then a steady state condition that will avoid inhibition of l-AlaDH by either the
main reaction product (2) or by NADH. Thus, the overall system can only be vulnerable to
inhibition by ammonium and/or by the product of the recycling reaction (3).
To test the performance of the proposed enzymatic system, preliminary experiments were
conducted with rac-alanine, in which the l-enantiomer is the natural substrate for l-AlaDH. The
results obtained showed that l-alanine was converted into lactic acid salt in 98% yield, therefore
con®rming the validity of the proposed model under experimental conditions.
Based on these results, we turned our attention to the ¯uorinated feedstock (rac-1), which was
obtained from 2, via reductive amination with satisfactory yield.7,8
The assays were carried out with rac-1 on analytical scale by using the coupled enzymatic system.
After 90 h of reaction, over 90% of 1b was consumed. Once the eciency of the coupled system on
analytical scale was proved, we undertook this reaction on a preparative scale (100 mg) in order

L. P. B. GoncËalves et al. / Tetrahedron: Asymmetry 11 (2000) 1465±1468
1467
to isolate and characterize the reaction products. After 24 h, about 93% of 1b was converted into
3, via reduction of the intermediate 2 by action of l-LDH.
(S)-3-Fluoroalanine (1a) was isolated with 60% yield and 88% ee.9 The results of IR, 1H NMR
and elemental analysis (C,H,N) of 1a agreed with the literature.10 In this system, 3 was obtained
in 80% yield and over 99% ee, which is consistent with our previously published results.4
In conclusion, in the main reaction of the coupled system, rac-1 was submitted to an enzymatic
resolution, thus leaving unreacted 1a in good yield and high ee. As far as we know, this is the ®rst
example in which l-AlaDH was used in a preparative scale reaction in the oxidative sense, which
is thermodynamically unfavorable for the isolated reaction. The driving force for the whole system
was provided by the recycle system, which also a€orded another important chiral building block,
3.
Acknowledgements
Financial support from CNPq, FAPERJ, FUJB, FINEP (PADCT, FNDCT) and PRONEX is
acknowledged. A CNPq fellowship award to LPBG is also acknowledged. We also thank the
`Nucleo de Pesquisas de Produtos Naturais-UFRJ' for running NMR analyses.
References
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6. Ohshima, T.; Wandrey, C.; Conrad, D. Biotechnol. Bioeng. 1989, 34, 394.
7. rac-Fluoroalanine was prepared by reductive amination of sodium 3-¯uoropyruvate (3.49 mmols) with NaBH4 (20
mmols) in the presence of NH4OH (68 mmols).8
8. Dolling, U. H.; Douglas, A. W.; Grabowski, E. J. J.; Schoenewaldt, E. F.; Sohar, P.; Sletzinger, M. J. Org. Chem.
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9. (a) l-AlaDH from Bacillus subtilis (EC 1.4.1.1, suspension in 2.4 M (NH4)2SO4, pH 7.0), l-LDH from rabbit
muscle (EC 1.1.1.27, solution in 50% glycerol containing 10 mM potassium phosphate bu€er, pH 7.5), l-GluDH
from bovine liver type III (lyophilized), NAD+ (grade III-C), NADH (grade III), bovine serum albumin (fraction V),

1468
L. P. B. GoncËalves et al. / Tetrahedron: Asymmetry 11 (2000) 1465±1468
sodium pyruvate, sodium ¯uoropyruvate and d,l-alanine were obtained from Sigma. a-Ketoglutarate and NaBH4
were purchased from Aldrich. All other chemicals used were of analytical grade and obtained from Merck. (b)
Reaction mixture contained in a total volume of 50 mL: 0.05 M phosphate bu€er (pH 7.9), 20 mM rac-
¯uoroalanine, 373 UI of l-AlaDH, 4250 UI of l-LDH and 0.05% (w/v) BSA. (c) Reaction mixture was
maintained under magnetic stirring and the temperature was kept at 25ꢀC by circulating water with a
thermocirculating bath. The reaction was started by adding 0.25 mM NAD+. The extent of the reaction was
measured by removing at di€erent times aliquots (10±50 mL) of the medium that were immediately diluted with 0.5
mL of 0.05 M phosphate bu€er (pH 7.9) and heated at 100ꢀC for 5 min in order to inactivate the enzymes. (d) The
concentration of ammonium present in these samples was determined by `end point' assays in the presence of 0.4
mM NADH, 0.5 mM of a-ketoglutarate and 1 mg of l-GluDH in a total volume of 2 mL at 25ꢀC. The decrease of
NADH absorption was followed at 340 nm before and after the addition of the enzyme in silica cuvettes with a 1
cm light path using a Beckman DU 70 recording spectrophotometer until consumption of the substrate. The
concentration of ammonium was calculated from the di€erence of NADH absorption (before and after the
addition of l-GluDH) by using a molar absorption coecient of NADH of 6220 M^1 cm^1. (e) The commercial
suspension of l-AlaDH was desalinized by ultra®ltration with Centricon-10 concentrators (AMICON), through
®ve consecutives centrifugations at 5ꢀC for 1 h and 2000 G. The original volume was made up with phosphate
bu€er (10 mM, pH 7.2). (f) Preparative work up was conducted by thermal treatment of reaction medium (100ꢀC, 15
min), HCl (3 M) addition until pH 2, active charcoal addition, stirring during 15 min, ®ltration and ionic exchange
chromatography (40 mL of wet Dowex 50 W-X8 H+) eluted successively with water (for isolation of 3) and 1 M
NH4OH (for isolation of 1a). An overall yield of 80% and over 99% ee were obtained for 3 and 60% yield and
88% ee were obtained for 1a [1H NMR spectrum (D2O): ꢀ 4.02 (ddd) JH,F=29.5 Hz, JH,H=4.5 and 3.1 Hz; ꢀ 4.81
(ddd) JH,F=47.4 Hz, JH,H=4.5 and 3.0 Hz. IR (KBr) 3428, 3061, 2987, 2923, 1615, 1356 and 1022 cm^1]. The
determination of ees for 1a and 3 were carried out by chiral high performance liquid chromatography, which was
performed by using a chiral column (ET 250/8/4 Nucleosil1 Chiral-1); eluent: 1 mM copper sulfate, pH 4.6; ¯ow
rate: 1 mL/min; temperature 23ꢀC; UV detection at 235 nm. Compound 1a: ‰ꢁŠD25=^8.7 (c=0.8, 1 M HCl), and 3
(as methyl ester):4 ‰ꢁŠD25=^4 (c=1, EtOH).
10. Kollonitsch, J.; Barash, L. J. Am Chem. Soc. 1976, 98, 5581.