Chemoenzymatic synthesis of 4-amino-2-hydroxy acids: A comparison of mutant and wild-type oxidoreductases

Article abstract of DOI:10.1021/jo980821a

We describe a new chemoenzymatic synthesis of enantiopure 4-amino-2-hydroxy acids using two biotransformations in a single-pot process in aqueous medium. These compounds are valuable as γ-turn mimics for investigations into the secondary structure of peptides. The enzyme substrates are a series of carbobenzyloxy (CBZ)-protected 4-amino-2-keto esters, prepared efficiently from the L-amino acids, alanine, leucine, phenylalanine, and valine. First, the α-amino acids were converted to the corresponding β-amino acids in a simple five-step procedure. A further one-carbon homologation via ozonolysis of the corresponding β-keto cyanophosphoranes gave the required α-keto esters in good yield. The enzyme catalyzed hydrolyses of all the α-keto esters to the corresponding α-keto acids proceeded smoothly with the lipase from Candida rugosa. Using the same reaction pot, it was found that wild-type lactate dehydrogenases from either Bacillus stearothermophilus CBS-LDH) or Staphylococcus epidermidis (SE-LDH) could be used to specifically reduce the ketone of the alanine-derived α-keto acid 2, giving the (S)- and CR)-2-hydroxy acids, respectively, in good yields. However, the more bulky α-keto acids 3, 4, and 5 (derived from valine, leucine, and phenylalanine) were not substrates for these enzymes. In contrast, the genetically engineered H205Q mutant of D-hydroxyisocaproate dehydrogenase proved to be an ideal catalyst for the reduction of all the α-keto acids 2-5, giving excellent yields of the CBZ-protected (2R,4S)-4-amino2-hydroxy acids as single diastereomers. This genetically engineered oxidoreductase has great potential value in synthesis due to its broad substrate specificity and high catalytic activity. For example, reduction of 1 mmol of N-protected (S)-4-amino-2-oxopentanoic acid 2 took just 4 h with the H205Q mutant giving, after esterification, the CR)-2-alcohol 25 in 85% yield, whereas with SE-LDH the reaction required 4 days to give a 67% yield of 25.

Full text of DOI:10.1021/jo980821a

7764
J . Org. Chem. 1998, 63, 7764-7769
Ch em oen zym a tic Syn th esis of 4-Am in o-2-h yd r oxy Acid s:
A Com p a r ison of Mu ta n t a n d Wild -Typ e Oxid or ed u cta ses
Andrew Sutherland and Christine L. Willis*
School of Chemistry, University of Bristol, Cantock’s Close, Bristol BS8 1TS, U.K.
Received May 1, 1998
We describe a new chemoenzymatic synthesis of enantiopure 4-amino-2-hydroxy acids using two
biotransformations in a single-pot process in aqueous medium. These compounds are valuable as
γ-turn mimics for investigations into the secondary structure of peptides. The enzyme substrates
are a series of carbobenzyloxy (CBZ)-protected 4-amino-2-keto esters, prepared efficiently from the
L-amino acids, alanine, leucine, phenylalanine, and valine. First, the R-amino acids were converted
to the corresponding â-amino acids in a simple five-step procedure. A further one-carbon
homologation via ozonolysis of the corresponding â-keto cyanophosphoranes gave the required R-keto
esters in good yield. The enzyme catalyzed hydrolyses of all the R-keto esters to the corresponding
R-keto acids proceeded smoothly with the lipase from Candida rugosa. Using the same reaction
pot, it was found that wild-type lactate dehydrogenases from either Bacillus stearothermophilus
(BS-LDH) or Staphylococcus epidermidis (SE-LDH) could be used to specifically reduce the ketone
of the alanine-derived R-keto acid 2, giving the (S)- and (R)-2-hydroxy acids, respectively, in good
yields. However, the more bulky R-keto acids 3, 4, and 5 (derived from valine, leucine, and
phenylalanine) were not substrates for these enzymes. In contrast, the genetically engineered
H205Q mutant of D-hydroxyisocaproate dehydrogenase proved to be an ideal catalyst for the
reduction of all the R-keto acids 2-5, giving excellent yields of the CBZ-protected (2R,4S)-4-amino-
2-hydroxy acids as single diastereomers. This genetically engineered oxidoreductase has great
potential value in synthesis due to its broad substrate specificity and high catalytic activity. For
example, reduction of 1 mmol of N-protected (S)-4-amino-2-oxopentanoic acid 2 took just 4 h with
the H205Q mutant giving, after esterification, the (R)-2-alcohol 25 in 85% yield, whereas with
SE-LDH the reaction required 4 days to give a 67% yield of 25.
Sch em e 1
In tr od u ction
2-Hydroxy acids are valuable building blocks for use
in the synthesis of complex molecules and may be
prepared in good yields and with excellent enantioselec-
tivities by the lactate dehydrogenase-catalyzed reduction
of the corresponding R-keto acids. These reactions are
simple to perform on a multigram scale.1 L- and D-lactate
dehydrogenases have been obtained from various mam-
malian and bacterial sources and exhibit reasonably
broad substrate specificities. For example, LDHs from
Bacillus stearothermophilus (BS-LDH) and Staphylococ-
cus epidermidis (SE-LDH) have been used to prepare a
series of (S)- or (R)-2-hydroxy acids, respectively, which
in the main possess either a saturated hydrocarbon side
chain or an aromatic ring (Scheme 1).2,3 The utility of
the resultant 2-hydroxy acids as intermediates in syn-
thesis is limited to manipulation of the alcohol and/or
the carboxylic acid. Clearly, the utility of these products
would be increased by the presence of further functional-
ity in the side chain. Examples of the use of LDHs for
the synthesis of functionalized 2-hydroxy acids include
the reduction of 2,4-dioxo acids,4 3-chloropyruvate,5
substituted aromatic rings,6 and unsaturated 2-keto
acids.7 However, in some cases, the reduction was so
slow that it is not a viable route for the production of
sufficient quantities of the enantioenriched alcohols for
use in synthesis. One way to overcome this problem is
to genetically modify the enzymes to give enhanced
turnover rates and broader substrate specificities.8
Holbrook and co-workers have genetically altered sites
in both the mobile polypeptide chain and other residues
around the active site of BS-LDH, giving the MVS/GG
mutant with an active site that is more hydrophobic and
flexible.9 Casy et al. have shown that reduction of
4-methyl-2-oxopent-3-enoic acid using wild-type BS-LDH
proceeded very slowly, whereas reduction with this
(6) Tsantrizos, Y. S.; Lunetta, J . F.; Boyd, M.; Fader, L. D.; Wilson,
M.-C. J . Org Chem. 1997, 62, 5451.
(1) Roberts, S. M. Preparative Biotransformations; J . Wiley and
Sons: Chichester, 1993.
(2) Bur, D.; Luyten, M. A.; Wynn, H.; Provencher, L. R.; J ones, J .
B.; Gold, M.; Friesen, J . D.; Clarke, A. R.; Holbrook, J . J . Can. J . Chem.
1989, 67, 1065. Kim, M.-J .; Whitesides, G. M. J . Am. Chem. Soc. 1988,
110, 2959.
(7) (a) Casy, G.; Lee, T. V.; Lovell, H. Tetrahedron Lett. 1992, 33,
817; (b) MacRitchie, J .; Silcock, A.; Willis, C. L. Tetrahedron: Asym-
metry 1997, 8, 3895.
(8) Luyten, M. A.; Bur, D.; Wynn, H.; Parris, W.; Gold, M.; Friesen,
J . D.; J ones, J . B. J . Am. Chem. Soc. 1989, 111, 6800. Gerlt, J . A. Chem.
Rev. 1987, 87, 1079.
(3) Kim, M.-J .; Kim, J . Y. J . Chem. Soc., Chem. Commun. 1991, 326.
(4) Casy, G. Tetrahedron Lett. 1992, 33, 8159.
(5) Hirschbein, B. L.; Whitesides, G. M. J . Am. Chem. Soc. 1982,
104, 4458.
(9) Wilks, H. M.; Halsall, D. J .; Atkinson, T.; Chia, W. N.; Clarke,
A. R.; Holbrook, J . J . Biochemistry 1990, 29, 8587.
(10) Casy, G.; Lee, T. V.; Lovell, H.; Nichols, B. J .; Sessions, R. B.;
Holbrook, J . J . J . Chem. Soc., Chem. Commun. 1992, 924.
10.1021/jo980821a CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/09/1998

Comparison of Mutant and Wild-Type Oxidoreductases
J . Org. Chem., Vol. 63, No. 22, 1998 7765
Sch em e 2a
genetically altered MVS/GG mutant proceeded signifi-
cantly faster, giving the corresponding (S)-2-hydroxy acid
in 91% yield and 99% ee.10
Recently, it has been reported that the D-2-hydroxy-
4-methylvalerate dehydrogenase (also known as D-hy-
droxyisocaproate dehydrogenase11) from Lactobacillus
delbrueckii bulgaricus (LB-hicDH) has more favorable
kinetic parameters than D-LDHs for certain substrates.12
Holbrook and co-workers13 have used genetic engineering
to prepare a rational series of mutants of this enzyme
and so analyzed the important residues involved in its
active site. They found a much enhanced rate for the
reduction of 2-oxo-4-phenylbutanoic acid with the H205Q
mutant (exchange of the His-205 for Gln) of LB-hicDH
than with LDHs.
We now describe our investigations on the comparison
of this H205Q mutant dehydrogenase with wild-type
LDHs for the synthesis of R-hydroxy acids with nitrogen-
containing functionality in the side chain. These prod-
ucts are of particular value as γ-turn mimics to investi-
gate the secondary structure of peptides as well as
building blocks for use in synthesis.
Resu lts a n d Discu ssion
We have shown that carbobenzyloxy (CBZ)-protected
4-amino-2-oxobutanoic acid 1 is reduced by the com-
mercially available wild-type BS-LDH and SE-LDH,
giving the corresponding (S)- and (R)-2-hydroxy acids,
respectively, in good yields and >99% ee.14 However, the
reaction was very slow with SE-LDH and, on a 1 mmol
scale, took 7 days to reach completion. Hence, we
reasoned that the analogous CBZ-protected 4-substituted
4-amino-2-oxobutanoic acids 2-5 would be interesting
a
Reagents: (a) BOC2O, 2 M NaOH; (b) EtOCOCl, Et3N then
NaBH4; (c) TsCl, Et3N, DMAP; (d) NaCN, DMF; (e) NaCN, DMF;
(e) 6 M HCl; (f) BnOCOCl, 2 M NaOH; (g) Ph3PCHCN, DMAP;
(h) O3, MeOH.
the conformational features of a γ-turn by forming
intramolecular hydrogen bonds between the carbonyl
group of one residue and the hydroxyl group of the
4-amino-2-hydroxy acid. This appears to be the only
reported synthesis of these compounds.
The required R-keto esters 19-22 were prepared from
the L-amino acids as shown in Scheme 2. There are many
examples of the conversion of an R-amino acid to the
homologous â-amino acid, including the Arndt-Eistert
reaction17 and the stereoselective ring opening of aziri-
dine 2-carboxylates.18 We favored an approach involving
nucleophilic displacement of a tosylate by cyanide19
followed by hydrolysis. van den Broek et al.20 have
reported that such tosylates are unstable, and so the
tosylates 7-10 were immediately treated with sodium
cyanide in DMF to give the required nitriles 11-14 in
63-84% yields. Under acidic conditions, the nitrile was
hydrolyzed and the BOC group removed. The resultant
â-amino acids were reacted directly with benzyl chloro-
formate to give the acids 15-18 in 50-70% yields over
the two steps. An efficient method for the preparation
of R-keto esters from carboxylic acids via ozonolysis of
â-ketocyanophosphoranes was described by Wasserman
compounds for our substrate specificity studies with the
wild-type and mutant oxidoreductases. Furthermore, the
resultant R-hydroxy acids would be of value to examine
the γ-turn associated with the secondary structure of
proteins.15 Reetz and co-workers16 have prepared a series
of allyl-substituted 4-amino-2-hydroxy acids 6 via a
Wittig rearrangement of 4-amino(allyloxy)acetates (with
typical diastereoselectivities of 80%) and shown that
when incorporated into peptides these compounds display
(11) Bernard, N.; J ohnsen, K.; Ferain, T.; Garmyn, D.; Hols, P.;
Holbrook, J . J .; Delcour, J . Eur. J . Biochem. 1994, 224, 439.
(12) Alvarez, J . A.; Gelpi, J . L.; J ohnsen, K.; Bernard, N.; Delcour,
J .; Clarke, A. R.; Holbrook, J . J .; Cortes, A. Eur. J . Biochem. 1997,
244, 203.
(13) Bernard, N.; J ohnsen, K.; Gelpi, J . L.; Alvarez, J . A.; Ferain,
T.; Garmyn, D.; Hols, P.; Cortes, A.; Clarke, A. R.; Holbrook, J . J .;
Delcour, J . Eur. J . Biochem. 1997, 244, 213.
(14) Bentley, J . M.; Wadsworth, H. J .; Willis, C. L. J . Chem. Soc.,
Chem. Commun. 1995, 231.
(15) Callahan, J . F.; Newlander, K. A.; Burgess, J . L.; Eggleston,
D. S.; Nichols, A.; Wong, A.; Huffman, W. F. Tetrahedron 1993, 49,
3479. Milner-White, E. J . J . Mol. Biol. 1990, 216, 385.
(16) Reetz, M. T.; Griebenow, N.; Goddard, R. J . Chem. Soc., Chem.
Commun. 1995, 1605.
(17) Plucinska, K.; Liberek, B. Tetrahedron 1987, 43, 3509.
(18) Baldwin, J . E.; Adlington, R. M.; O’Neil, I. A.; Schofield, C.;
Spivey, A. C.; Sweeney, J . B. J . Chem. Soc., Chem. Commun. 1989,
1852.
(19) Kaseda, T.; Kikuchi, T.; Kibayashi, C. Tetrahedron Lett. 1989,
30, 4539.
(20) van den Broek, L. A. G. M.; Lazaro, E.; Zylicz, Z.; Fennis, P. J .;
Missler, F. A. N.; Leleieveld, P.; Garzotto, M.; Wagener, D. J . T.;
Ballesta, J . P. G.; Ottenheijm, H. C. J . J . Med. Chem. 1989, 32, 2002.
(21) Wasserman, H. H.; Ho, W.-B. J . Org. Chem. 1994, 59, 4364.

7766 J . Org. Chem., Vol. 63, No. 22, 1998
Sutherland and Willis
Sch em e 3
and Ho,21 and we used this method to prepare the
required R-keto esters. Activation of the acids 15-18
with EDCI/DMAP and coupling with (cyanomethylene)-
triphenylphosphorane gave the analogous â-keto cyano-
phosphoranes, which were ozonolyzed in MeOH/CH2Cl2
to give the required R-keto esters 19-22 in 58-65%
yields over the two steps.
For the hydrolysis of the N-protected 4-amino-2-keto
esters 19-22, lipase from Candida rugosa was used in
order to allow mild saponification of the ester at a pH
close to 7.0 under aqueous conditions. An advantage of
using this method is that the conditions required for the
hydrolysis are compatible with the dehydrogenase-
catalyzed reduction step, thus allowing the two reactions
to be carried out in a one-pot procedure. The oxidoreduc-
tases require a stoichiometric amount of the expensive
cofactor NADH; however, by use of the formate-formate
dehydrogenase (FDH) protocol developed by Shaked and
Whitesides,22 the NADH may be recycled in situ, enabling
the economic synthesis of multigram quantities of R-hy-
droxy acids. For ease of handling and purification, the
R-hydroxy acids isolated from the enzyme-catalyzed
reactions were directly converted to the corresponding
R-hydroxy esters using trimethylsilyldiazomethane
(Scheme 3).
diastereomers in 78-90% yield over the three steps from
the corresponding 2-keto esters 19-22.
The genetically engineered H205Q mutant D-dehydro-
genase clearly has great potential value in organic
synthesis. This present study indicates that it has a
much broader substrate specificity than wild-type LDHs.
This may be attributed to the presence of four extra
residues in the mobile loop closing the active site, thus
allowing accommodation of larger substrates.12 In ad-
dition, the remarkable catalytic activity should enable
the efficient synthesis of large quantities of (R)-2-hydroxy
acids. It has been proposed that the protonated H205
residue forms an ionic interaction with the negative
pyrophosphate backbone of NADH allowing tight binding
of the cofactor in the active site.13 Loss of this interaction
in the H205Q mutant allows weaker binding of the
coenzyme, thus allowing faster release of NAD+ and
therefore resulting in more rapid overall reduction of the
R-keto acid. Indeed, our investigations have revealed
that on a 1 mmol scale reduction of the CBZ derivative
of (S)-4-amino-2-oxopentanoic acid 2 with SE-LDH re-
quired 4 days, giving the (R)-2-hydroxy ester 25 in 67%
yield over the three steps from the corresponding keto
ester 19, whereas with the H205Q mutant the reduction
was complete in 4 h, giving 25 in 85% yield.
In conclusion, we have shown that a combination of
two biotransformations (a hydrolysis and specific reduc-
tion) in a single-pot process enables the clean and
efficient synthesis of novel R-hydroxy acids in excellent
yields. The products are to be used as γ-turn mimics to
explore the secondary structure of peptides. The broad
substrate specificity and enhanced turnover rates of the
H205Q mutant of LB-hicDH gives this enzyme exciting
potential as an efficient catalyst for the large-scale
production of novel R-hydroxy acids for use in organic
synthesis.
The 4-amino-2-oxo esters 19-22 were all accepted as
substrates by lipase. However, only the alanine-derived
R-keto acid 2 was then reduced by BS-LDH and SE-LDH,
giving after esterification the (S)- and (R)-2-hydroxy
esters 24 and 25, respectively. In the cases of the
substrates derived from valine, leucine, and phenylalaine
20-22 with more bulky substituents at C-4, only the
corresponding R-keto acids 3-5 were isolated from the
enzyme-catalyzed reactions. In contrast, the mutant
H205Q dehydrogenase proved to an excellent catalyst for
the reduction of all of the R-keto acids giving the
corresponding (R)-2-hydroxy esters 25-28 as single
Exp er im en ta l Section
Gen er a l Meth od s.7b The enzymes used were purchased
and stored as follows: Lipase from C. rugosa (Sigma Protein)
(22) Shaked, Z.; Whitesides, G. M. J . Am. Chem. Soc. 1980, 102,
7104.

Comparison of Mutant and Wild-Type Oxidoreductases
J . Org. Chem., Vol. 63, No. 22, 1998 7767
(1 000 000 eU) was dissolved in Tris buffer (100 mL, 5 mM)
and stored at 4 °C; L-lactate dehydrogenase from Bacillus
stearothermophilus (BS-LDH) (Genzyme) and D-lactate dehy-
drogenase from Staphylococcus epidermidis (SE-LDH) (Sigma)
were both stored at -20 °C. D-Hydroxyisocaproate dehydro-
genase from Lactobacillus delbrueckii bulgaricus (LB-hicDH)
(a gift from Professor J . J . Holbrook, Department of Biochem-
istry, University of Bristol); formate dehydrogenase from
Candida boidinii (FDH) (Boerhinger), and â-nicotinamide
adenine dinucleotide hydride (NADH) (Genzyme) were all
stored at 4 °C.
Gen er a l P r oced u r e for th e Syn th esis of N-(ter t-Bu -
toxyca r bon yl)-L-a m in o Acid s. The amino acid (0.1 mol) was
dissolved in 2 M sodium hydroxide (100 mL) and cooled to 0
°C. Di-tert-butyl dicarbonate (1.2 equiv) was slowly added.
After 0.5 h, the reaction mixture was warmed to room
temperature and allowed to stir for a further 2 h. The reaction
mixture was then acidified to pH 2 using concentrated
hydrochloric acid and extracted with ethyl acetate (3 × 50 mL).
The resulting organic layer was then dried (MgSO4) and
concentrated in vacuo. The resulting white solid was then
recrystallized using the appropriate solvent.
(0.1 mol) was dissolved in dichloromethane (100 mL) and
cooled to 0 °C under a nitrogen atmosphere. Triethylamine
(1.1 equiv), DMAP (0.2 g), and p-toluenesulfonyl chloride (1.3
equiv) were all added. The reaction mixture was warmed to
room temperature and allowed to stir overnight under a
nitrogen atmosphere. The reaction mixture was then acidified
to pH 2 with 2 M hydrochloric acid. The two layers were
separated, and the aqueous layer was washed with dichlo-
romethane (2 × 50 mL). The combined organic layers were
then dried (MgSO4) and concentrated in vacuo.
A solution of the crude tosylate (0.1 mol) in DMF (40 mL)
was slowly added to a solution of sodium cyanide (3 equiv) in
DMF (50 mL). The reaction mixture was allowed to stir at
room temperature for 24 h under a nitrogen atmosphere. The
reaction mixture was then diluted with water (70 mL) and
extracted with ethyl acetate (3 × 60 mL). The organic layer
was then washed with water (3 × 50 mL), dried (MgSO4), and
concentrated in vacuo. The resulting white solid was then
purified by recrystallization from the appropriate solvent.
(S)-3-[(ter t-Bu toxyca r bon yl)a m in o]bu ta n en itr ile (11):
84% over two steps; mp 68-70 °C (from dichloromethane and
hexane) (lit.26 mp 69-71 °C (from dichloromethane and
hexane)); [R]25 -84.3 (c 1.0, CHCl3) (lit.26 [R]25 -87.0 (c 1.0,
N-(ter t-Bu toxyca r bon yl)-L-alanine: 92% yield; mp 82-84
D
D
°C (from ethyl acetate) (lit.23 mp 82-83 °C); [R]22 -19.6 (c
CHCl3)).
D
1.6, CH2Cl2) (lit.23 [R]23 -19.2 (c 2.0, CH2Cl2)).
(R)-3-[(ter t-Bu toxycar bon yl)am in o]-4-m eth ylpen tan en -
itr ile (12): 63% yield over two steps; mp 80-82 °C (from
dichloromethane and hexane) (lit.26 mp 82-84 °C (from
dichloromethane and hexane)); [R]25D -83.4 (c 0.3, CHCl3) (lit.26
D
N-(ter t-Bu toxyca r bon yl)-L-valine: 94% yield; mp 74-76
°C (from ethyl acetate) (lit.24 mp 73-79 °C); [R]23 -7.1 (c 1.2,
D
AcOH) (lit.24 [R]25 -6.4 (c 1.0, AcOH)).
D
[R]25 -81.5 (c 0.65, CHCl3)).
D
N-(ter t-Bu toxyca r bon yl)-L-leucine: 89% yield; mp 81-83
°C (from ethyl acetate and light petroleum) (lit.24 mp 82-83
°C); [R]23D -23.6 (c 0.9, AcOH) (lit.24 [R]23D -24.5 (c 2.0, AcOH)).
N-(ter t-Bu toxyca r bon yl)-L-phenylalanine: 81% yield; mp
75-78 °C (from ethyl acetate and light petroleum) (lit.25 mp
76-79 °C (from ethyl acetate and light petroleum)); [R]25D -6.2
(S)-3-[(ter t-Bu toxyca r bon yl)a m in o]-5-m eth ylh exa n en -
itr ile (13): 70% over two steps; mp 75-77 °C (from dichlo-
romethane and hexane) (lit.26 mp 75-77 °C (from dichlo-
romethane and hexane)); [R]24D -86.2 (c 1.4, CHCl3) (lit.26 [R]25
-83.2 (c 0.82, CHCl3)).
D
(c 1.0, AcOH) (lit.25 [R]25 -3.91 (c 0.8, AcOH)).
(S)-3-[(ter t-Bu toxyca r bon yl)a m in o]-4-p h en ylbu ta n en i-
tr ile (14): 63% yield over two steps; mp 118-120 °C (from
dichloromethane and hexane) (lit.26 mp 119-120 °C (from
dichloromethane and hexane)); [R]25D -19.6 (c 0.3, CHCl3) (lit.26
D
Gen er a l P r oced u r e for th e Red u ction of N-(ter t-Bu -
toxyca r bon yl)-L-a m in o Acid s Usin g Eth yl Ch lor ofor m a te
a n d Sod iu m Bor oh yd r id e. The protected amino acid (0.1
[R]25 -18.7 (c 0.48, CHCl3)).
D
mol) was dissolved in THF (100 mL) under
a nitrogen
Gen er a l P r oced u r e for t h e P r ep a r a t ion of CBZ-
P r otected â-Am in o Acid s 15-18. The amino nitrile (0.1
mol) was dissolved in 6 M hydrochloric acid and heated under
reflux for 12 h. The reaction mixture was then cooled, and 2
M sodium hydroxide was added until a pH of 7 was achieved.
The solution was concentrated in vacuo. The crude â-amino
acid was then dissolved in 2 M sodium hydroxide (100 mL)
and cooled to 0 °C. Benzyl chloroformate (1.2 equiv) was
slowly added. After 0.5 h, the reaction mixture was warmed
to room temperature and allowed to stir for a further 2 h. The
reaction mixture was then acidified to pH 2 using concentrated
hydrochloric acid and extracted with ethyl acetate (3 × 50 mL).
The resulting organic layer was then dried (MgSO4) and
concentrated in vacuo. The resulting white solid was then
recrystallized using the appropriate solvent.
atmosphere and cooled to 0 °C. Triethylamine (1.1 equiv) and
ethyl chloroformate (1.1 equiv) were then added to form a
white precipitate. After 0.25 h, the solution was filtered. The
filtrate was added dropwise to a solution of sodium borohydride
(1.5 equiv) in water (40 mL) at 0 °C. After 0.5 h, the reaction
mixture was warmed to room temperature and allowed to stir
for a further 2 h. The reaction mixture was then acidified to
pH 2 using 2 M hydrochloric acid and extracted with ethyl
acetate (3 × 50 mL). The organic layer was then washed with
a saturated solution of sodium hydrogen carbonate, dried
(MgSO4), and concentrated in vacuo.
(S)-2-[(ter t-Bu toxyca r bon yl)a m in o]p r op a n -1-ol: 69%
yield; mp 57-58 °C (from ethyl acetate and light petroleum)
(lit.26 59-61 °C (from ethyl acetate and light petroleum)); [R]24
D
-6.9 (c 0.3, CHCl3) (lit.26 [R]25 -8.9 (c 1.01, CHCl3)).
D
(S)-3-[(Ben zyloxyca r bon yl)a m in o]bu ta n oic a cid (15):
70% yield over two steps; mp 104-106 °C (from diethyl ether
and light petroleum) (lit.27 mp 105-107 °C (from diethyl ether
and light petroleum)); [R]24 -24.1 (c 0.4, CHCl3) (lit.27 [R]20
(S)-2-[(ter t-Bu toxyca r bon yl)a m in o]-3-m eth ylbu ta n -1-
ol: oil; 90% yield; [R]24D -17.9 (c 1.0, CHCl3) (lit.26 [R]25D -17.0
(c 1.7, CHCl3)).
(S)-2-[(ter t-Bu toxyca r bon yl)a m in o]-4-m eth ylp en ta n -1-
ol: oil; 70% yield; [R]24D -26.8 (c 0.8, CHCl3) (lit.26 [R]25D -22.0
(c 1.66, CHCl3)).
D
D
-26.0 (c 1.0, CHCl3)).
(R)-3-[(Ben zyloxyca r b on yl)a m in o]-4-m et h ylp en t a n o-
ic a cid (16): oil; 50% yield over two steps; [R]23 -33.6 (c 0.2,
D
(S)-2-[(ter t-Bu toxyca r bon yl)a m in o]-3-p h en ylp r op a n -1-
ol: 75% yield; mp 93-95 °C (from ethyl acetate and light
petroleum) (lit.26 mp 95-97 °C (from ethyl acetate and light
petroleum)); [R]23 -27.9 (c 0.2, CHCl3) (lit.26 [R]25 -27.7 (c
CHCl3); νmax(film)/cm-1 3327 (NH), 1707 (br, 2 × CO); δH (270
MHz) 0.94 (3H, d, J ) 7Hz, 5-H3), 0.95 (3H, d, J ) 7Hz, 4-CH3),
1.87 (1H, m, 4-H), 2.89 (1H, dd, J ) 15, 9Hz, 2-HH), 3.07 (1H,
dd, J ) 15, 4Hz, 2-HH), 4.00 (1H, m, 3-H), 5.01 (1H, d, J )
12Hz, PhCHH), 5.03 (1H, d, J ) 12Hz, PhCHH), 7.26-7.36
(5H, m, Ph); Found (CI) [MH]+ 266.1390, C14H19NO4 requires
[MH]+ 266.1392; m/z (CI) 266 ([MH]+, 6), 91 (100).
D
D
1.11, CHCl3)).
Gen er a l P r oced u r e for th e Syn th esis of th e Am in o
Nitr iles 11-14 via th e Tosyla tes 7-10. The amino alcohol
(S)-3-[(Ben zyloxyca r b on yl)a m in o]-5-m et h ylh exa n oic
a cid (17): 60% yield over two steps; mp 76-78 °C (from ethyl
(23) Buckley, T. F., III; Rapoport, H. J . Am. Chem. Soc. 1981, 103,
6157.
(24) Colombo, R. Chem. Lett. 1980, 1119.
(25) Kita, Y.; Haruta, J .-I.; Yasuda, H.; Fukunaga, K.; Shirouchi,
Y.; Tamura, Y. J . Org. Chem. 1982, 47, 2697.
(26) Caputo, R.; Cassano, E.; Longobardo, L.; Palumbo, G. Tetra-
hedron 1995, 51, 12337.
(27) Drey, C. N. C.; Mtetwa, E. J . Chem. Soc., Perkin Trans. 1 1982,
1587.
(28) El Marini, A.; Roumestant, M. L.; Viallefont, P.; Razafindram-
boa, D.; Bonato, M.; Follet, M. Synthesis 1992, 1104.

7768 J . Org. Chem., Vol. 63, No. 22, 1998
Sutherland and Willis
acetate and light petroleum) (lit.28 mp 80 °C); [R]23 -31.6 (c
3.02 (2H, m, 3-H2), 3.83 (3H, s, OMe), 4.33 (1H, m, 4-H), 5.03
(2H, s, PhCH2O), 5.07 (1H, d, J ) 9Hz, NH), 7.13-7.38 (10H,
2 × Ph); δC (75.5 MHz) 40.2 and 43.1 (3-C and 5-C), 48.9 (4-
C), 53.1 (OMe), 66.7 (PhCH2O), 126.9, 128.0, 128.1, 128.5,
128.7, 129.2, 136.2, 137.0 (aromatics), 155.6, (1-C), 160.8
(PhCH2OCO), 192.1 (2-C); m/z (CI) 356 ([MH]+, 56), 91 (100).
Anal. Calcd for C20H21NO5: C, 67.6; H, 5.9; N, 3.9. Found C,
67.2; H, 5.9; N, 3.9.
D
1.3, CHCl3) (lit.28 [R]20 -29.0 (c 2.5, CHCl3)).
D
(S)-3-[(Ben zyloxyca r b on yl)a m in o]-4-p h en ylb u t a n oic
a cid (18): 69% yield over two steps; mp 116-118 °C (from
ethyl acetate and light petroleum) (lit.17 mp 118-119 °C); [R]22
D
-37.2 (c 1.0, AcOH) (lit.17 [R]20 -38.0 (c 1.0, AcOH)).
D
Gen er a l P r oced u r e for th e Syn th esis of 2-Keto Ester s
19-22 via th e 3-Keto Cya n op h osp h or a n es. The (cyano-
methyl)triphenylphosphorane chloride was prepared according
to the method of Trippett and Walker.29 The salt (1.8 equiv)
was dissolved in water (50 mL) and dichloromethane (50 mL).
Sodium hydroxide (6 equiv) in water (15 mL) was slowly added.
After 0.3 h, the two layers were separated. The dichlo-
romethane layer was dried (MgSO4) and added to a solution
of the carboxylic acid (0.1 mol), DMAP (0.2 g), and EDCI (1.8
equiv) in dichloromethane (100 mL) at 0 °C under a nitrogen
atmosphere. The reaction mixture was then warmed to room
temperature and allowed to stir for 16 h. Water (100 mL) was
added, and the two layers were separated. The organic layer
was washed with a saturated solution of sodium hydrogen
carbonate (2 × 50 mL), dried (MgSO4), and concentrated in
vacuo. The crude â-keto cyanophosphorane was then dissolved
in dichloromethane (100 mL) and methanol (100 mL) and
cooled to -78 °C. Ozone was then bubbled through the
solution until the reaction mixture had turned blue. Oxygen
was then passed through the solution to remove the excess
ozone. The solution was warmed to room temperature and
concentrated in vacuo. The crude product was purified by
recrystallization from the appropriate solvent or by column
chromatography, eluting with an increasing ratio of ethyl
acetate in light petroleum.
Gen er a l P r oced u r e for th e Syn th esis of N-P r otected
4-Am in o-2-h yd r oxy Ester s. The 2-keto ester (1 mmol) was
dissolved in 5 mM tris buffer (40 mL). C. rugosa lipase (10 000
eU) was added and the pH adjusted to 7.5 by the addition of
1.0 M hydrochloric acid. The pH was maintained at a value
between 7.0 and 7.5 by the addition of 0.1 M sodium hydroxide
until the pH had stopped changing or until 1 equiv of base
had been added. The solution was deoxygenated by bubbling
a stream of nitrogen through for 1 h. DTT (20 µL) was then
added followed by the dehydrogenase enzyme (10 mg if BS-
LDH, 6 mg if SE-LDH, and 1 mL if LB-hicDH), FDH (10 mg),
sodium formate (1 g, 15 equiv), and NADH (10 mg). The
reaction was allowed to stir under a nitrogen atmosphere with
the pH being kept constant at approximately 6.1 by the
addition of 1.0 M hydrochloric acid. After the pH had stopped
changing or 1 equiv of acid was added, the reaction mixture
was acidified to pH 2 by the addition of 2 M hydrochloric acid
and extracted with ethyl acetate (3 × 40 mL). The organic
layer was dried (MgSO4) and concentrated in vacuo. The
resulting 2-hydroxy acid was dissolved in methanol (10 mL)
and toluene (10 mL). Trimethylsilyldiazomethane (2.0 M) in
hexane (0.6 mL, 1.1 equiv) was slowly added, turning the
solution a bright yellow color. The solution was stirred for
0.5 h and then concentrated in vacuo. The resulting residue
was purified by recrystallization from the appropriate solvent
or by column chromatography, eluting with an increasing ratio
of ethyl acetate in light petroleum.
Meth yl (S)-4-[(Ben zyloxyca r bon yl)a m in o]-2-oxop en -
ta n oa te (19): oil; 65% over two steps; [R]23 -15.8 (c 0.5,
D
CHCl3); νmax(film)/cm-1 3460 (NH), 1732 (br, 3 x CO); δH (400
MHz) 1.26 (3H, d, J ) 8Hz, 5-H3), 3.03 (2H, m, 3-H2), 3.86
(3H, s, OMe), 4.20 (1H, m, 4-H), 5.06 (1H, br m, NH), 5.08
(2H, s, PhCH2) and 7.31-7.38 (5H, m, Ph); δC (75.5 MHz) 20.6
(5-C), 43.7 (3-C), 45.8 (4-C), 53.1 (OMe), 66.8 (PhCH2), 128.1,
128.2, 128.5, 136.3 (aromatics), 155.5 (1-C), 161 (PhCH2OCO),
193.0 (2-C); found (CI) [MH]+, 280.1176, C14H17NO5 requires
[MH]+ 280.1185; m/z (CI) 280 ([MH]+, 7), 91 (100).
Met h yl (2R,4S)-4-[(Ben zyloxyca r b on yl)a m in o]-2-h y-
d r oxyp en ta n oa te (25). The lipase hydrolysis was complete
after 4 days, and the LB-hicDH reduction was complete after
4 h. Recrystallization from ethyl acetate and light petroleum
gave 25 as a white crystalline solid (85% yield): mp 72-73
°C (from ethyl acetate and light petroleum); [R]22 +16.7 (c
D
2.0, CHCl3); νmax(Nujol mull)/cm-1 3343 (OH, NH), 1691 (br, 2
× CO); δH (270 MHz) 1.24 (3H, d, J ) 7Hz, 5-H3), 1.81 (2H, m,
3-H2), 3.77 (3H, s, OMe), 4.02 (1H, m, 4-H), 4.30 (1H, dd, J )
10, 4Hz, 2-H), 5.03 (1H, d, J ) 7Hz, NH), 5.09 (2H, s, PhCH2),
7.26-7.37 (5H, m, Ph); m/z (CI) 282 ([MH]+, 50), 264 ([MH -
H2O]+, 29), 91 (100). Anal. Calcd for C14H19NO5: C, 59.8; H,
6.8; N, 5.0. Found: C, 59.8; H, 7.0; N, 5.2.
Meth yl (R)-4-[(Ben zyloxyca r bon yl)a m in o]-5-m eth yl-2-
oxoh exa n oa te (20): oil; 58% over two steps; [R]23 -31.7 (c
D
0.3, CHCl3); νmax(film)/cm-1 3388 (NH), 1730 (br, 3 × CO); δH
(270 MHz) 0.94 (6H, d, J ) 7Hz, 5-CH3 and 6-H3), 1.89 (1H,
oct, J ) 7Hz, 5-H), 2.94 (1H, dd, J ) 16, 8Hz, 3-HH), 3.06
(1H, dd, J ) 16, 4Hz, 3-HH), 3.86 (3H, s, OMe), 3.94 (1H, m,
4-H), 4.91 (1H, br d, J ) 9Hz, NH), 5.05 (2H, s, PhCH2), 7.27-
7.40 (5H, m, Ph); δC (75.5 MHz) 14.2 (6-C), 19.1 (5-CH3), 32.0
(5-C), 43.0 (3-C), 53.1 (OMe), 61.0 (4-C), 78.0 (PhCH2), 128.2,
128.5, 135.8 (aromatics), 155.0 (1-C), 161.0 (PhCH2OCO), 192.0
(2-C); found (CI) [MH]+ 308.1499, C16H21NO5 requires [MH]+
308.1498; m/z (CI) 308 ([MH]+, 0.1), 91 (100).
The above reaction was repeated using SE-LDH, and the
reduction was complete after 4 days. Recrystallization from
ethyl acetate and light petroleum gave 25 as a white crystal-
line solid (67% yield).
Met h yl (2S,4S)-4-[(Ben zyloxyca r b on yl)a m in o]-2-h y-
d r oxyp en ta n oa te (24). The lipase hydrolysis was complete
after 4 days, and the BS-LDH reduction was complete after 3
days. Recrystallization from ethyl acetate and light petroleum
gave 24 as a white crystalline solid (86% yield): mp 48-50
Meth yl (S)-4-[(ben zyloxyca r bon yl)a m in o]-6-m eth yl-2-
oxoh ep ta n oa te (21): oil; 60% over two steps; [R]22 -29.0 (c
D
0.3, CHCl3); νmax(film)/cm-1 3336 (NH), 1732 (br, 3 × CO); δH
(400 MHz) 0.93 (3H, d, J ) 6Hz, 7-H3), 0.94 (3H, d, J ) 6Hz,
6-CH3), 1.26-1.68 (3H, m, 5-H2 and 6-H), 2.96 (1H, dd, J )
17, 7Hz, 3-HH), 3.09 (1H, dd, J ) 17, 5Hz, 3-HH), 3.86 (3H, s,
OMe), 4.15 (1H, m, 4-H), 4.89 (1H, br d, J ) 8Hz, NH), 5.06
(2H, s, PhCH2), 7.29-7.38 (5H, m, Ph); δC (75.5 MHz) 22.0
(7-C), 22.8 (6-CH3), 25.0 (6-C), 43.7 (5-C), 44.9 (3-C), 53.1
(OMe), 60.0 (4-C), 66.8 (PhCH2), 128.1, 128.2, 128.5, 136.0
(aromatics), 156.0 (1-C), 161.0 (PhCH2OCO), 193.0 (2-C); found
(CI) [MH]+ 322.1661, C17H23NO5 requires [MH]+ 322.1654; m/z
(CI) 322 ([MH]+, 1), 91 (100).
°C (from ethyl acetate and light petroleum); [R]22 -11.4 (c
D
2.0, CHCl3); νmax(Nujol mull)/cm-1 3335 (OH, NH), 1702 (br, 2
× CO); δH (270 MHz) 1.21 (3H, d, J ) 7Hz, 5-H3), 1.94 (2H, m,
3-H2), 3.72 (3H, s, OMe), 4.02 (1H, m, 4-H), 4.27 (1H, t, J )
6Hz, 2-H), 4.81 (1H, br m, NH), 5.09 (2H, s, PhCH2), 7.27-
7.37 (5H, m, Ph); m/z (CI) 282 ([MH]+, 2), 264 ([MH - H2O]+,
2), 91 (100). Anal. Calcd for C14H19NO5: C, 59.8; H, 6.8; N,
5.0. Found: C, 59.4; H, 7.1; N, 5.1.
Met h yl (2R,4R)-4-[(Ben zyloxyca r b on yl)a m in o]-2-h y-
d r oxy-5-m eth ylh exa n oa te (26). The lipase hydrolysis was
complete after 4 days, and the LB-hicDH reduction was
complete after 5 h. Purification by flash column chromatog-
raphy, eluting with 46% ethyl acetate in light petroleum, gave
Meth yl (S)-4-[(ben zyloxycar bon yl)am in o]-2-oxo-5-ph en -
ylp en ta n oa te (22): 62% yield over two steps; mp 107-109
°C (from ethyl acetate and light petroleum); [R]22 -15.1 (c
D
0.3, CHCl3); νmax(Nujol mull)/cm-1 3312 (NH), 1750 (ester, CO),
1732 (ketone, CO), 1693 (amide, CO); δH (270 MHz) 2.86 (1H,
dd, J ) 14, 7Hz, 5-HH), 2.95 (1H, dd, J ) 14, 7Hz, 5-HH),
26 as a viscous oil (90% yield): [R]22D +9.0 (c 2.0, CHCl3); νmax
-
(film)/cm-1 2246 (OH, NH), 1694 (br, 2 × CO); δH (270 MHz)
0.92 (3H, d, J ) 7Hz, 6-H3), 0.94 (3H, d, J ) 7Hz, 5-CH3), 1.76
(3H, m, 3-H2 and 5-H), 3.73 (1H, m, 4-H), 3.77 (3H, s, OMe),
4.25 (1H, dd, J ) 8Hz, 6, 2-H), 4.83 (1H, d, J ) 9Hz, NH),
(29) Trippett, S.; Walker, D. M. J . Chem. Soc. 1959, 3874.

Comparison of Mutant and Wild-Type Oxidoreductases
J . Org. Chem., Vol. 63, No. 22, 1998 7769
5.10 (2H, s, PhCH2), 7.26-7.37 (5H, m, Ph); found (CI) [MH]+,
310.1661, C16H23NO5 requires [MH]+ 310.1654; m/z (CI) 310
([MH]+, 8), 292 ([MH - H2O]+, 11), 91 (100).
(4) as a viscous oil (0.31 g, 100%): [R]22 -25.1 (c 0.5, CHCl3);
D
ν
max(film)/cm-1 3329 (OH, NH), 1694 (br, 3 × CO); δH (400
MHz) 0.95 (6H, d, J ) 6Hz, 6-CH3 and 7-H3), 1.28-1.71 (3H,
m, 5-H2 and 6-H), 2.96 (1H, dd, J ) 17, 7Hz, 3-HH), 3.05 (1H,
dd, J ) 17, 5Hz, 3-HH), 4.18 (1H, m, 4-H), 5.05 (2H, s, PhCH2),
7.26-7.41 (5H, m, Ph); found (CI) [MH]+ 308.1480, C16H21NO5
requires [MH]+ 308.1498; m/z (CI) 308 ([MH]+, 1), 91 (100).
Met h yl (2R,4S)-4-[(Ben zyloxyca r b on yl)a m in o]-2-h y-
d r oxy-5-p h en ylp en ta n oa te (28). Lipase hydrolysis was
complete after 4 days, and the LB-hicDH reduction was
complete after 5 h. Purification by recrystallization from ethyl
acetate and light petroleum gave 28 as white crystals (0.30 g,
85%): mp 89-91 °C (from ethyl acetate and light petroleum);
Tr ea tm en t of Meth yl (R)-4-[(Ben zyloxyca r bon yl)a m i-
n o]-5-m eth yl-2-oxoh exa n oa te (20) w ith Lip a se a n d BS-
LDH. The reactions were carried out according to the above
general procedure using methyl (R)-4-[(benzyloxycarbonyl)-
amino]-5-methyl-2-oxohexanoate (20) (0.31 g, 1 mmol). The
lipase hydrolysis was complete after 4 days. No pH change
was noted after addition of BS-LDH, NADH, sodium formate,
and FDH. After 2 weeks, the reaction mixture was worked
up to give (R)-4-[(benzyloxycarbonyl)amino]-5-methyl-2-oxo-
hexanoic acid (3) as a viscous oil (69% yield): [R]26 -16.3 (c
D
1.0, CHCl3); νmax(film)/cm-1 3335 (OH, NH), 1732 (br, 3 × CO);
δH (400 MHz) 0.93 (3H, d, J ) 7Hz, 6-H3), 0.95 (3H, d, J )
7Hz, 5-CH3), 1.86 (1H, m, 5-H), 2.87 (1H, dd, J ) 15, 9Hz,
3-HH), 3.07 (1H, dd, J ) 15, 4Hz, 3-HH), 3.99 (1H, m, 4-H),
5.01 (1H, d, J ) 12Hz, PhCHH), 5.05 (1H, d, J ) 12Hz,
PhCHH), 7.26-7.36 (5H, m, Ph); m/z (CI) 279 ([MH]+ - CH3,
2), 91 (100).
[R]23 +2.3 (c 0.2, CHCl3); νmax(Nujol mull)/cm-1 3491 (OH),
D
1732 (CO, ester), 1694 (CO, amide); δH (270 MHz) 1.80 (2H,
m, 3-H2), 2.81 (1H, dd, J ) 13, 7Hz, 5-HH), 2.92 (1H, dd, J )
13, 7Hz, 5-HH), 3.74 (3H, s, OMe), 4.17 (1H, m, 4-H), 4.29 (1H,
dd, J ) 10, 4Hz, 2-H) 4.97 (1H, d, J ) 9Hz, NH), 5.07 (2H, s,
PhCH2O), 7.15-7.39 (10H, m, 2 × Ph); m/z (CI) 358 ([MH]+,
1), 91 (100). Anal. Calcd for C20H23NO5: C, 67.2; H, 6.4; N,
3.9. found: C, 67.3; H, 6.5; N, 4.1.
Met h yl (2R,4S)-4-[(Ben zyloxyca r b on yl)a m in o]-2-h y-
d r oxy-6-m eth ylh ep ta n oa te (27). The lipase hydrolysis was
complete after 4 days, and the LB-hicDH reduction was
complete after 4 h. Recrystallization from ethyl acetate and
light petroleum gave 27 as a white crystalline solid (78%
yield): mp 72-73 °C (from ethyl acetate and light petroleum);
Tr eatm en t of Meth yl (S)-4-[(Ben zyloxycar bon yl)am in o]-
2-oxo-5-p h en ylp en ta n oa te (22) w ith Lip a se a n d BS-LDH.
The reactions were carried out according to the above general
procedure using methyl (S)-4-[(benzyloxycarbonyl)amino]-2-
oxo-5-phenylpentanoate (22) (0.35 g, 1 mmol). Lipase hydroly-
sis was complete after 5 days. No pH change was noted after
the addition of BS-LDH, NADH, sodium formate, and FDH.
After 2 weeks, the reaction mixture was workedup to give (S)-
4-[(benzyloxycarbonyl)amino]-2-oxo-5-phenylpentanoic acid (5)
[R]22 -1.0 (c 2.0, CHCl3); νmax(Nujol mull)/cm-1 3353 (OH,
D
NH), 1682 (br, 2 × CO); δH (270 MHz) 0.92 (6H, d, J ) 6Hz,
6-CH3 and 7-H3), 1.24-1.71 (3H, m, 5-H2 and 6-H), 1.78 (2H,
m, 3-H2), 3.78 (3H, s, OMe), 3.99 (1H, m, 4-H), 4.28 (1H, t, J
) 7Hz, 2-H), 4.85 (1H, d, J ) 9Hz, NH), 5.10 (2H, s, PhCH2),
7.29-7.40 (5H, m, Ph); m/z (CI) 324 ([MH]+, 1), and 91 (100).
Anal. Calcd for C17H25NO5: C, 63.2; H, 7.7; N, 4.3. Found:
C, 63.0; H, 7.7; N, 4.1.
as a white solid (0.28 g, 83%): [R]23D -17.4 (c 2.0, CHCl3); νmax
-
(Nujol mull)/cm-1 3394 (OH, NH), 1717 (br, 3 × CO); δH (400
MHz) 2.87 (2H, m, 5-H2), 3.01 (2H, m, 3-H2), 4.36 (1H, m, 4-H),
5.03 (2H, s, PhCH2O), 5.13 (1H, d, J ) 8Hz, NH), 7.11-7.48
(10H, m, 2 × Ph); found (CI) [MH]+ 342.1339, C19H19NO5
requires [MH]+ 342.1341; m/z (CI) 342 ([MH]+, 26), 91 (100).
Tr eatm en t of Meth yl (S)-4-[(Ben zyloxycar bon yl)am in o]-
6-m eth yl-2-oxop en ta n oa te (21) w ith Lip a se a n d BS-LDH.
The reactions were carried out according to the above general
procedure using methyl (S)-4-[(benzyloxycarbonyl)amino]-6-
methyl-2-oxoheptanoate (21) (0.32 g, 1 mmol). The lipase
hydrolysis was complete after 4 days. No pH change was noted
after addition of BS-LDH, NADH, sodium formate, and FDH.
After 2 weeks, the reaction mixture was worked up to give
(S)-4-[(benzyloxycarbonyl)amino]-6-methyl-2-oxoheptanoic acid
Ack n ow led gm en t. We thank the University of
Bristol for a Scholarship to A.S., and we are most
grateful to Professor J . J . Holbrook for the gift of the
LB-hicDH.
J O980821A