Stereoinversion of β- and γ-substituted α-amino acids using a chemo-enzymatic oxidation-reduction procedure

Article abstract of DOI:10.1039/b309787k

Both D- and L-β- and γ-substituted α-amino acids can be interconverted to their respective L- and D- diastereoisomers by treatment with an enantioselective amino acid oxidase and a chemical reducing agent.

Full text of DOI:10.1039/b309787k

Stereoinversion of b- and g-substituted a-amino acids using a
chemo-enzymatic oxidation–reduction procedure
Alexis Enright,a Francois-Rene Alexandre,a Geoffrey Roff,a Ian G. Fotheringham,a Michael J.
Dawsonb and Nicholas J. Turner*a
a School of Chemistry, The University of Edinburgh, King’s Buildings, West Mains Road, Edinburgh, UK
EH9 3JJ. E-mail: n.j.turner@ed.ac.uk
b GlaxoSmithKline R&D, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, UK
SG1 2NY
Received (in Cambridge, UK) 14th August 2003, Accepted 8th September 2003
First published as an Advance Article on the web 23rd September 2003
Both
D
- and
L
-b- and g-substituted a-amino acids can be
range of different amino acid oxidases and chemical reducing
agents.
interconverted to their respective
L
- and - diastereoisomers
D
by treatment with an enantioselective amino acid oxidase
and a chemical reducing agent.
Initially it was important to establish that b-substituted a-
amino acids were indeed substrates for the various
L
- and -
D
amino acid oxidases. Thus (2RS,3RS)-2-amino-3-methylhex-
anoic acid 2 and (2RS,3RS)-b-methyl-phenylalanine 3 were
prepared in racemic form, and as a mixture of diastereomers (ca.
40% d.e.), from the corresponding racemic aldehydes via the
Strecker reaction. Each substrate was subjected to stereoinver-
Enantiomerically pure b-substituted a-amino acids are valuable
building blocks for the synthesis of peptidomimetics and
enzyme inhibitors.1 The incorporation of a b-substituent into an
a-amino acid (e.g. b-methylphenylalanine) generally leads to
restricted conformational flexibility in peptides offering the
prospect of developing high affinity ligands for receptors.
Methods for the preparation of b-substituted a-amino acids
include resolution,2 asymmetric catalysis,3 and also asymmetric
synthesis employing chiral auxiliaries.4 A particularly versatile
approach involves the catalytic asymmetric hydrogenation of
b,b-disubstituted dehydroamino acids using chiral phosphine
ligands.5 Although such substrates tend to undergo hydro-
genation slowly, Burk et al. have developed appropriate ligands
(e.g. Me-DuPHOS) that result in high enantioselectivities.6
However even these ligands tend to be more selective and
reactive towards (Z)-configured b-substituted enamides com-
pared to the (E)-isomers resulting in preferential access to only
two of the four possible diastereoisomers. In addition, a number
of natural b-substituted a-amino acids are more readily
sion using -amino acid oxidase from Trigonopsis variabilis
D
and either NaBH4 or NaCNBH3 as the reducing agent (Scheme
2).13 The progress of the reactions was monitored by chiral
HPLC allowing the concentration of all four stereoisomers to be
assessed during the reaction. For both substrates, rapid
consumption of the pair of (2R)-diastereoisomers was observed,
albeit at slightly different rates, with concomitant production of
the (2S)-isomers (Table 1). For substrate 2, the reaction was
found to be complete ( > 99% e.e. for each diastereomer) after
18 h using NaCNBH3 and only 3.5 h with the more reactive
NaBH4. At the end of the reaction a 1 : 1 mixture of the (2S,3S)-
and (2S,3R)-isomers was observed (NB the final ratio of
diastereoisomers is independent of the initial diastereomeric
composition generated via the Strecker reaction and always
approaches 1 : 1). In contrast to 2, (2RS,3RS)-b-methyl-
phenylalanine 3 was found to react more slowly although again
the use of NaBH4 enabled complete conversion after 6 h. The
reduced rate with this substrate may be a consequence of partial
inhibition of the reaction by some 3-methylphenylpyruvic acid
produced via hydrolysis of the intermediate imine.14
available in one diastereoisomeric form than the other (cf.
L
-
isoleucine vs. -allo-isoleucine). With this in mind we sought to
D
develop methodology for interconverting diastereoisomers of
amino acids bearing two stereogenic centres.
Recently we have developed a general and practical method
for the stereoinversion of a-amino acids7,8 and amines.9,10 The
overall process relies upon the combined action of an enantiose-
lective oxidase and a non-selective chemical reducing agent
(e.g. Pd/C–HCO2NH4; amine : borane; sodium borohydride/
Having established the basic reaction conditions we next
tested the generality of the stereoinversion procedure by
examining two different amino acid oxidases and an alternative
cyanoborohydride). For example, the use of an
oxidase ( -AAO) enables conversion of a racemic mixture of a-
amino acids to the optically pure
-enantiomer (Scheme 1).8
L
-amino acid
L
D
Provided that the amino acid oxidase is highly enantioselective
then only 7 oxidation/reduction cycles are required to achieve
an e.e. of > 99% with yields ranging between 80–99%.11,12
Extension of this approach to a-amino acids containing one
or more additional stereogenic centres would provide a method
for interconverting diastereoisomers rather than enantiomers.
Herein we demonstrate the success of this approach using a
Scheme 2 Stereoinversion of b-substituted a-amino acids.
Table 1 Stereoinversion of 2 and 3 using T. variabilis D-AAO and NaBH4/
NaCNBH3
D
-AAO
Yield
(%)
e.e.
(%)
Substrate (U)
[H] (equiv.)
Time/h
2
2
2
3
3
3
3
0.68
0.68
0.68
0.68
1.70
3.40
0.68
NaCNBH3 (25)
NaBH4 (40)
NaBH4 (40)
NaCNBH3 (25)
NaCNBH3 (40)
NaCNBH3 (60)
NaBH4 (40)
18
3
3.5
2
22
22
6
63
94
99
69
68
86
71
> 99
> 99
> 99
33a
48 a
50 a
> 99
Scheme 1 Deracemisation of an a-amino acid
D/L-1 using a combination of
an -amino acid oxidase ( -AAO) and reducing agent.
a denotes e.e. of one of the diastereomers indicating incomplete reaction.
L
L
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CHEM. COMMUN., 2003, 2636–2637
This journal is © The Royal Society of Chemistry 2003

reducing agent (NH3 : BH3) which has previously been shown
to be effective.8 In this case we chose to use enantiomerically
pure (2R,3R)-3 and (2S,3R)-3 as substrates and successfully
conversion to (2RS,4S)-6 (66% yield; > 99% e.e. and d.e.). The
-AAO catalysed oxidation of (2R,4S)-6 proceeded rapidly
suggesting that the presence of a g-methyl group has relatively
little effect on the reaction rate compared to simple a-amino
acids.
D
interconverted the pair of diastereoisomers using
D
-AAO from
porcine kidney and -AAO from snake venom (Scheme 3).
L
In addition to NH3 : BH3, catalytic transfer hydrogenation
(Pd/C–HCO2NH4) is also compatible with the oxidase reac-
tions.8 Both reagents were examined for their ability to convert
In conclusion we have developed a novel procedure for
interconverting b- and g-substituted a-amino acid diastereo-
isomers using amino acid oxidases in combination with a range
of chemical reducing agents. The procedure appears highly
versatile in that so far we have examined four different AAOs
and four different reducing agents and in all cases the reactions
proceeded in good to high yield and excellent stereoselectiv-
ities.
L
-isoleucine 4 to
D
-allo-isoleucine 5 in the presence of -AAO
L
from Proteus myxofaciens (Scheme 4). This enzyme has been
over-expressed in Escherichia coli15 and can be used either as a
whole cell biocatalyst or partially disrupted cells. The use of
NH3 : BH3 (40 equiv.) with disrupted cells yielded
D-allo-
isoleucine 5 in 87% yield and 99% d.e. whereas by comparison
Pd/C–HCO2NH4 with intact cells gave 5 in 61% yield and 96%
d.e. The use of snake venom -AAO with NH3 : BH3 resulted in
We are grateful to the BBSRC and GlaxoSmithKline (A.E.)/
DowPharma (G.R.) for CASE awards and to Great Lakes for a
postdoctoral fellowship (F-R.A.). We also acknowledge finan-
cial support from the Wellcome Trust.
L
a modest improvement (89% yield; > 99% d.e.).
Finally to further explore the range of substrates amenable to
stereoinversion, we examined g-substituted a-amino acids.
(2RS,4S)-2-Amino-4-methylhexanoic acid 6 was prepared by
alkylation of imine 716 using (S)-(+)-1-bromo-2-methylbutane 8
(e.e. > 98%) followed by hydrolysis (conc. HCl) to give
(2RS,4S)-6 in 65% yield and 88% d.e (Scheme 5). Treatment of
Notes and references
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2 R. M. Williams, Synthesis of Optically Active a-Amino Acids, Pergamon
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6 with T. variabilis -AAO and NaCNBH3 resulted in rapid
D
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6 M. J. Burk, M. F. Gross and J. P. Martinez, J. Am. Chem. Soc., 1995,
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Scheme 3 Interconversion of (2R,3R)- and (2S,3R)-b-methylphenylalanine
3.
10 R. Carr, M. Alexeeva, A. Enright, T. S. C. Eve, M. J. Dawson and N. J.
Turner, Angew. Chem., Int. Ed., 2003, 42, in press.
11 For a recent review on the kinetics and stereochemistry of deracemisa-
tion reactions see: U. T. Strauss, U. Felfer and K. Faber, Tetrahedron
Asymmetry, 1999, 10, 107.
12 The final e.e. of the product from a deracemisation reaction is
Scheme 4 Conversion of L-isoleucine 4 to D-allo-isoleucine 5.
determined by the ratio of rate constants k1/k2 for oxidation of the
D and
L
-enantiomers since the latter are in equilibrium via the imino acid.
Hence the e.e. is identical to that achieved by a standard kinetic
resolution process. The e.e. is also given by (E 2 1)/(E + 1) as for
dynamic kinetic resolutions: H. Stecher and K. Faber, Synthesis, 1997,
1.
13 For initial reports on the use of NaBH4 in the deracemisation of a-amino
acids see: E. W. Hafner and D. Wellner, Proc. Natl. Acad. Sci. USA,
1971, 68, 987; J. W. Huh, K. Yokoigawa, N. Esaki and K. Soda, J.
Ferment. Bioeng., 1992, 74, 189; J. W. Huh, K. Yokoigawa, N. Esaki
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Enzym., 2001, 11, 795.
16 M. J. O’Donnell, J. M. Boniece and S. E. Earp, Tetrahedron Lett., 1978,
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Scheme 5 Synthesis of (2S,4S)-4-methyl-2-aminohexanoic acid 6.
CHEM. COMMUN., 2003, 2636–2637
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