Hydrolase-catalyzed stereoselective preparation of protected α,α-dialkyl-α-hydroxycarboxylic acids

Article abstract of DOI:10.1016/j.tetasy.2008.07.005

The title compounds were prepared by a Passerini multi-component reaction and subjected to enzymatic kinetic resolution using 40 enzymes known to be active towards esters of tertiary alcohols. This identified a protease (P1), a thermophilic esterase (PestE) and an esterase of metagenome origin (esterase 8) as the most active and enantioselective biocatalysts. After further optimization of reaction conditions, two compounds were prepared in preparative scale using esterase 8 yielding (+)-1-(tert-butylamino)-2-methyl-1-oxobutyl-2-yl acetate with 93%ee and (-)-1-(tert-butylamino)-2-trifluoro-methyl-1-oxobutyl-2-yl acetate with 47%ee.

Full text of DOI:10.1016/j.tetasy.2008.07.005

Tetrahedron: Asymmetry 19 (2008) 1839–1843
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Tetrahedron: Asymmetry
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Hydrolase-catalyzed stereoselective preparation of protected
a,a-dialkyl-a-hydroxycarboxylic acids
Robert Kourist a, Giang-Son Nguyen a, Dirk Strübing a, Dominique Böttcher a, Klaus Liebeton b,
Christian Naumer b, Jürgen Eck b, Uwe T. Bornscheuer a,
*
a Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Strasse 4, D-17487 Greifswald, Germany
b B.R.A.I.N. AG, Darmstädter Strasse 34–36, D-64673 Zwingenberg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The title compounds were prepared by a Passerini multi-component reaction and subjected to enzymatic
kinetic resolution using 40 enzymes known to be active towards esters of tertiary alcohols. This identified
a protease (P1), a thermophilic esterase (PestE) and an esterase of metagenome origin (esterase 8) as the
most active and enantioselective biocatalysts. After further optimization of reaction conditions, two
compounds were prepared in preparative scale using esterase 8 yielding (+)-1-(tert-butylamino)-2-
methyl-1-oxobutyl-2-yl acetate with 93%ee and (À)-1-(tert-butylamino)-2-trifluoro-methyl-1-oxobu-
tyl-2-yl acetate with 47%ee.
Received 11 June 2008
Accepted 2 July 2008
Available online 28 July 2008
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
enzymatic synthesis of enantiopure a,a-disubstituted carboxylic
acids has so far been mainly attempted by enantioselective hydro-
lysis of carboxylic acid esters,14 while the resolution of the tertiary
hydroxyesters has received much less attention. To overcome ste-
ric hindrance, Recuero et al. used a linker between the stereogenic
centre and the reaction centre, that allowed them to prepare key
precursors of (S)-oxybutynin with high enantioselectivity using li-
pase from Pseudomonas cepacia or from Candida antarctica B.15
Ohta et al. studied the kinetic resolution of benzyloxy-protected
esters of 1 and several other derivatives. Whilst the enantioselec-
tivities for several substrates varied up to E = 52, (S)-1 was pre-
pared with an enantioselectivity of only E = 10.16
We attempted to resolve this class of compounds by resolving
the tertiary alcohol and therefore protecting the acid with an
amide group. Even though the reaction mechanisms of enzymatic
hydrolysis of amides and esters are highly similar, promiscuous
amidase activity of esterases, if present, is usually very low,17,18
which has already been successfully applied to the selective enzy-
matic deprotection of tert-butyl esters in the presence of other pro-
tected functional groups.19
The synthesis of enantiomerically pure tertiary alcohols is an
important, but also challenging task in organic synthesis.1,2 Never-
theless, in recent years a number of enzymatic processes have
emerged, including the hydrolase-catalyzed kinetic resolution of
tertiary structures such as cyanohydrins,3 epoxides4 and carboxyl
esters.5 The identification of esterases and lipases acting on tertiary
alcohols was greatly facilitated by the discovery of an amino acid
motif, the so-called GGG(A)X-motif, consisting of three glycines
(or two glycines and an alanine) in the active site,6,7 which was
identified in a computer-based study of structure–function rela-
tionships of a large number of hydrolases8 and later related to
the ability to convert esters of tertiary alcohols.9 The application
of the GGG(A)X-motif in a guided screening led to the identifica-
tion of several highly enantioselective esterases in databases5,6
and metagenome-derived enzyme libraries.10
Enantiomerically pure
a,a-dialkyl-a-hydroxycarboxylic acids
are important building blocks in organic synthesis. For instance,
(S)-2-hydroxy-2-methylbutyric acid 1 is present in the natural
product clerodendrin-A11 and has been applied to the synthesis
of a cyclooxygenase inhibitor.12 Only very recently has a biocata-
lytic route using oxynitrilases that affords 1 with high enantio-
meric purity (ee >99%) been reported.13 It is, however, hampered
by the required use of HCN as a substrate. Enzyme-catalyzed reso-
lution would represent an environmentally more beneficial pro-
cess. The enzymatic conversion is difficult because the crowded
chiral centre significantly lowers the reaction rates. Hence the
Preparation of the protected esters can be easily accomplished
in one step by the Passerini-reaction, a multi-component reaction
(MCR) widely applied in combinatorial chemistry.20 Enzymatic ki-
netic resolution and subsequent deprotection would directly give
the desired
a,a-dialkyl-a-hydroxycarboxylic acid. MCRs have al-
ready been efficiently combined with biocatalytic methods, both
for the synthesis of enantiopure precursors for the Ugi MCR21
and for the synthesis of racemic secondary alcohols by Passerini-
MCR and subsequent enantioselective enzymatic hydrolysis.22
Herein we report the synthesis of protected
hydroxycarboxylic acid esters by the Passerini-reaction and the
a,a-dialkyl-a-
* Corresponding author. Tel.: +49 3834 86 4367; fax: +49 3834 86 80066.
E-mail address: uwe.bornscheuer@uni-greifswald.de (U.T. Bornscheuer).
0957-4166/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2008.07.005

1840
R. Kourist et al. / Tetrahedron: Asymmetry 19 (2008) 1839–1843
O
O
R3
R1
OH
R2
R1
HO
R2
O
R1
O
R2
Passerini
reaction
O
R1
O
R2
O
H
H
Hydrolase
Buffer
H
N
N
N
+
R3
R3
O
O
NC
1-5a
rac-
1-5a
1-5b
1
2
3
1a R1=Et, R2=CF3, R3=Me
2a R1=Et, R2=Me, R3=Me
3a R1=Ph, R2=CF3, R3=Me
R =Et, R =CF3, R =n-Pr
4a
5a R1=Et, R2=CF3, R3=Phenyl
Scheme 1. Synthesis of protected a,a-dialkyl-a-hydroxycarboxylic acid esters by Passerini-MCR and subsequent enzymatic kinetic resolution.
subsequent enantioselective hydrolysis and deprotection in a sim-
ple two-step route (Scheme 1) to afford these building blocks.
Table 1
Enantioselectivity of metagenome-derived GGG(A)X-hydrolases in the hydrolysis of
acetates of arylaliphatic tertiary alcohols 6a and 7a
cb (%)
Ec (–)
a
a
2. Results and discussion
Entry
Enzyme
eeS (%)
eeP (%)
6a
6a
6a
6a
6a
7a
7a
7a
Esterase 56
Esterase 63
Esterase 8
M48cD5
Esterase 5
Esterase 56
Esterase 8
M48cD5
27
4
60
40
0
39
62
2
43
4
44
37
0
91
63
8
40
52
58
52
37
56
51
25
3
1
4
3
1
35
8
1
2.1. Synthesis of protected
acids
a,a-dialkyl-a-hydroxy-carboxylic
As reported in the literature,20 the Passerini-reaction proceeded
considerably faster in aqueous media than in an organic solvent. In
aqueous solution, however, a certain amount of hydrolysis of the
substrate to the alcohol was observed, which made purification
by column chromatography necessary. Alternatively, the reactions
were performed in organic solvent and here the products could be
directly isolated with excellent purity although longer reaction
times were required.
a
As determined by GC analysis.
b
c
Conversion calculated as c = eeS/(eeS + eeP).
Calculated according to Chen et al.25
O
2.2. Identification of metagenome-derived hydrolases acting on
tertiary alcohols
O
R
Activity towards tertiary alcohols has been found in several a,b-
fold hydrolases bearing the GGG(A)X-motif in the active site9 or
with high structural similarity to peptidases.23,24 We have already
reported on the screening of 35 esterases and lipases from metage-
nome origin, previously identified by a high-throughput screening
for activity in the hydrolysis of several esters of tertiary alcohols
including 6a.10
6a
7a
R=CH3
R=CF3
Scheme 2. Esters of tertiary alcohols used in the initial screening of metagenome-
In a similar approach, we investigated 58 enzymes from another
metagenomic library initially for principle hydrolytic activity
against the acetates of tertiary alcohols 6a and 7a, as these com-
pounds were readily available in our laboratory. In this screening
14 enzymes were identified as active and five enzymes having
the highest activity were used further for biocatalysis experiments.
Three enzymes, esterases 8, 56 and M48cD5, had low to moderate
enantioselectivity in the hydrolysis of the arylaliphatic tertiary es-
ters 6a and 7a (Table 1, Scheme 2).
derived hydrolases.
enzymes, esterase 8, esterase PestE and Chirazyme P1 showed
activity and thus were investigated further.
Chirazyme P1 displayed low enantioselectivity towards sub-
strates 1a and 2a. The thermophilic esterase PestE showed activity
towards 1a at temperatures above 50 °C but at this temperature
low chemical hydrolysis was observed, which led to a decrease
in the enantiomeric purity of the product. Interestingly, the enan-
tiopreference of PestE was inverse to that of esterase 8 but the
enantioselectivity (E = 10) was too low for preparative purposes
and the overall activity was unsatisfactory. Surprisingly, PestE
had almost no activity towards analogue 2a (E = 3) and was inac-
tive towards the bulky compound 3a.
Esterase 8 showed a promising E-value (E = 19) in the kinetic
resolution of 1a at 30 °C. Optimization of the reaction conditions
including the addition of water-miscible co-solvents [tert-butanol,
dimethyl sulfoxide (DMSO), dimethyl formamide (DMF)] and a de-
crease of the temperature slightly increased the enantioselectivity
to E = 22 for 1a and E = 42 for 2a at 4 °C and in the presence of 5%
DMF (v/v). The kinetic resolution of 2a was performed under opti-
2.3. Identification of enzymes with activity towards amide-
protected a,a-dialkyl-a-hydroxycarboxylic acid acetates
For the kinetic resolution of the Passerini-MCR reaction prod-
ucts, 40 carboxyl hydrolases were screened for activity towards
1a by small-scale biocatalysis experiments, including the metage-
nome-derived enzymes, commercially available hydrolases such as
Candida rugosa lipase, pig liver esterase, Candida antarctica lipase A
and Chirazyme P1. Furthermore, recombinantly expressed esterase
BS2 from Bacillus subtilis5,6 and esterase PestE from the thermo-
philic organism Pyrobaculum calidifontis were used.26,27 Only three

R. Kourist et al. / Tetrahedron: Asymmetry 19 (2008) 1839–1843
1841
Table 2
Results of kinetic resolution of 1a and 2a after 4 h reaction time
d
e
Entry
Enzyme
Ua
40b
T (°C)
Co-solvent
eeS (%)
eeP (%)
c (%)
Eg (–)
1a
2a
1a
2a
1a
1a
2a
PestE
PestE
P1
P1
Esterase 8
Esterase 8
Esterase 8
50
50
37
37
30
4
10% DMSO
10% DMSO
10% DMSO
10% DMSO
5% DMF
n.d.
n.d.
n.d.
n.d.
80 (À)
54 (+)
49 (+)
38 (À)
73 (+)
87 (+)
85 (À)
11f
2f
10
3
4
100b
856b
856b
230b
680c
400c
40f
24f
54f
36g
53g
2
n.d.
19
22
42
5% DMF
5% DMF
47 (À)
4
93 (+)
a
Determined using p-nitrophenyl acetate.
0.055 mmol substrate.
0.55 mmol substrate.
Determined after hydrolysis to 2b.
Determined by GC analysis.
b
c
d
e
f
Determined by NMR spectroscopy.
g
Calculated according to Chen et al.25
mized conditions on a 120-mg scale and yielded the remaining
substrate (+)-2a (38%, 93%ee) and the product (À)-2b (46%,
85%ee) with good yields and enantiomeric purities (Table 2). The
attempt to vary the chain length and structure of the acyl moiety
was not successful, as 4a and 5a were not converted by esterase
8. The bulky 3a was not converted by any of the enzymes.
and synthesis, analytical-scale biocatalysis and chiral analytics of
6a and 7a were performed as described.7 All metagenomic ester-
ases were produced by B.R.A.I.N. AG and used as glycerol-stabilized
crude cell extracts or lyophylizate.28
4.2. General procedure for the synthesis of protected racemic
a,a-dialkyl-
a-hydroxycarboxylic acids
3. Conclusion
These were prepared by adapting a previously reported proce-
dure:20 (a) in organic solvent: to a stirred solution of 3.0 mmol iso-
cyanide in dichloromethane (20 mL), ketone (3.3 mmol, 1.1 equiv)
and acid (3.3 mmol, 1.1 equiv) were added. After 7 d, the organic
phase was washed three times with distilled water. (b) In aqueous
solution: to a stirred solution of 3.0 mmol isocyanide in an aqueous
LiCl-solution (1 M, 20 mL), ketone (3.3 mmol, 1.1 equiv) and acid
(3.3 mmol, 1.1 equiv) was added. The product was extracted three
times with dichloromethane. In both cases, after drying over
Na2SO4, the solvent and the remaining reactants were removed
by distillation under reduced pressure.
It has been shown that a structure-guided search for hydrolases
acting on tertiary alcohols can provide enzymes active towards
difficult compounds such as the bulky amide-protected
a,a-dial-
kyl- -hydroxycarboxylic acids. Two esterases bearing the
a
GGG(A)X-motif in their active sites, and a protease were active
and enantioselective in the hydrolysis of the tertiary alcohol ace-
tates and one enzyme, esterase 8, displayed good enantioselectiv-
ity. This underlines the importance of the GGG(A)X-motif for the
identification of enzymes for the conversion of tertiary alcohols.
In connection with the easy preparation of the substrates by the
Passerini-reaction, hydrolase-catalyzed kinetic resolution provides
a fast, straight-forward route for the synthesis of enantiomerically
4.2.1. (R,S)-2-(tert-Butylcarbamoyl)-1,1,1-trifluorobut-2-yl
acetate, 1a
enriched protected a,a-dialkyl-a-hydroxycarboxylic acids.
Compound 1a was obtained after 7 d in dichloromethane as a
white powder (1.5 g, 5.6 mmol, 84%, mp 52 °C). 1H NMR: d = 0.92
(t, J = 7.48, 3H, CH3), 1.4 (s, 9H (CH3)3), 2.2 (s, 3H, CH3), 2.5 (m,
2H, CH2), 5.8 (s, 1H, N). 13C NMR: d = 7.5, 22.4, 21.2, 28.3, 51.9,
83.9 (q, J = 27.9), 123.3, (q, J = 285.9), 163.1, 167.3. MS (EI): 269
(M+), 254, 226, 212, 194, 171, 154, 97, 43. Anal. Calcd for
4. Experimental
4.1. General
All chemicals were purchased from Fluka (Buchs, Switzerland),
Sigma (Steinheim, Germany) and Merck (Darmstadt, Germany),
unless stated otherwise. NMR spectroscopy experiments were per-
formed on an ARX300 (300.13 MHz for 1H and 75.5 MHz for 13C,
Bruker, Karlsruhe, Germany), using the d scale (ppm) for chemical
shifts; 13C-spectra were edited using DEPT techniques. Mass spec-
tra were recorded on a QP2010 GC–MS device (electron impact,
70 eV, Shimadzu, Japan). High-resolution MS was determined by
direct injection on
Germany) with nitrogen as nebulizer gas and drygas. Chiral GC-
analyses were performed by using a Heptakis-(2,3-di-O-acetyl-6-
O-t-butyldimethylsilyl)-b-cyclodextrin-column
Düren, Germany) on a GC-14A gas chromatograph (Shimadzu,
Tokyo, Japan). Chiral HPLC-analyses were performed by using the
column OD-H (Daicel Chemical Industries Ltd., Osaka, Japan) with
n-hexane and 2-propanol as solvent on a Hitachi Elite LaChrom de-
vice (Hitachi High Technologies America Inc., San Jose, USA). The
sign of the optical rotation was detemined by using a chiral
HPLC-detector (IBZ-Messtechnik, Hannover, Germany). Chirazyme
P1 was obtained from Roche Diagnostics GmbH (Mannheim, Ger-
many). Production of esterase Pest E from Pyrobaculus calidifontis26
C
11H18F3NO3 (269.26): C, 49.07; H, 6.74; N, 5.20. Found: C, 49.18;
H, 6.68; N, 5.04. HRMS: 292.111[M + Na+].
4.2.2. 1-(tert-Butylamino)-2-methyl-1-oxobut-2-yl acetate, 2a
Compound 2a was obtained after 4 h in aqueous LiCl-solution
and by column chromatography (n-pentane/ethyl acetate) as white
powder (775 mg, 3.6 mmol, 31%, mp 46 °C). 1H NMR: d = 0.8 (t,
J = 7.45, 3H, CH3), 1.7 (s, 3H, CH3), 1.4 (s, 9H, C(CH3)3), 2.0 (m,
2H, CH2), 2.1 (s, 3H, CH3), 6.0 (s, 1H, NH). 13C NMR: d = 8.0, 22.0,
22.1, 28.7, 29.7, 51.0, 85.5, 168.7, 171.3. MS (EI): 215 (M+), 187,
172, 155, 116, 100. Anal. Calcd for C11H21NO3 (215.15): C, 61.37;
H, 9.83; N, 22.29. Found: C, 60.55; H, 9.98; N, 6.33. HRMS:
238.139 [M+Na+].
a micrOTOF device (Bruker, Karlsruhe,
(Machey-Nagel,
4.2.3. (R,S)-3-(tert-Butylamino)-1,1,1-trifluoro-3-oxo-2-
phenylpropan-2-yl acetate, 3a
Compound 3a was obtained after 4 h in aqueous LiCl-solution
and by column chromatography (n-pentane/ethyl acetate) as white
powder (464.7 mg, 1.5 mmol, 48%, mp 88 °C). In dichloromethane,
3a was obtained after 7 d with higher yield (666 mg, 2.1 mmol,

1842
R. Kourist et al. / Tetrahedron: Asymmetry 19 (2008) 1839–1843
70%). 1H NMR: d = 1.4 (s, 9H, C(CH3)3), 2.3 (s, 3H, CH3), 5.9 (s, 1H,
NH), 7.4 (m, 3H, H-Ar), 7.5 (m, 2H, H-Ar).13C NMR: d = 21.4, 28.3,
52.3, 83.6 (q, J = 28.3), 122.7 (q, J = 285.5), 127.3, 128.4, 129.6,
131.3, 162.8, 167.9. MS (EI): 318 (M+), 274, 260, 242, 219, 156,
77, 57, 43. Anal. Calcd for C15H18F3NO3: C, 56.78; H, 5.72; N,
4.41. Found: C, 57.07; H, 55.74; N, 4.33. HRMS: 298.101
[MÀCH3COOH+Na+].
(s, 1H, OH), 6.2 (s, 1H, NH), 7.4 (m, 3H, H-Ar), 7.7 (m, 2H, H-Ar).
13C NMR: d = 28.4, 52.9, 77.8 (q, J = 28.5), 123.9 (q, J = 286.1),
128.8, 128.9, 129.6, 134.8, 166.0. MS (EI): 232 (MÀ43), 176, 156,
105, 84, 77, 57. HRMS: 298.121 [M+Na+].
4.4. High-throughput screening
The test kit for the determination of released acetic acid was
from R-Biopharm GmbH (Darmstadt, Germany) and applied
according to the manufacturer’s protocol as described previously.29
4.2.4. (R,S)-2-(tert-Butylcarbamoyl)-1,1,1-trifluorobut-2-yl
butyrate, 4a
Compound 4a was obtained after 7 d in dichloromethane as a
white powder (1064 mg, 3.6 mmol, 92%, mp 72 °C). 1H NMR:
d = 0.9 (t, J = 7.52, 3H, CH3), 1.0 (t, 3H, CH3), 1.4 (s, 9H, (CH3)3),
1.7 (m, 2H, CH2), 2.5 (t, 2H, CH2), 2.6 (m, 2H, CH2), 5.8 (s. 1H,
NH). 13C NMR: d = 7.5, 22.4, 13.5, 18.3, 28.3, 36.6, 51.9, 83.8, (q,
J = 27.8), 123.4 (q, J = 285.6), 163.2, 170.0. MS (EI): 297 (M+), 279,
254, 242, 172, 100, 71, 57. Anal. Calcd for C13H22F3NO3: C, 52.52;
H, 7.46; N, 4.71; O, 16.14. Found: C, 52.52; H, 7.46; N, 4.71. HRMS:
320.144 [M+Na+].
Enzyme solution (20
of racemic 6a were added to a mixture (150
l
L) and substrate solution (20
lL; 7.5 mg/mL)
lL) of the test kit com-
ponents. The increase of NADH was monitored at 340 nm by using
the Fluostar Galaxy or Fluostar Optima (BMG, Offenburg, Ger-
many). Mixtures of test kit components with buffer or cell lysate
from induced Escherichia coli, that contained pUC18 without an
esterase gene, served as controls.
4.5. General procedure for esterase-catalyzed small-scale
resolutions
4.2.5. (R,S)-2-(tert-Butylcarbamoyl)-1,1,1-trifluorobut-2-yl
benzoate, 5a
To a stirred solution of the acetate (25 mM) in a phosphate buf-
fer (100 mM, pH 7.5) and the appropriate amount of co-solvent,
esterase solution was added to a total volume of 2 mL. The reaction
mixture was stirred in a thermoshaker (Eppendorf, Hamburg, Ger-
many) at the appropriate temperature. Samples were taken after
1 h, 4 h and 24 h. The reaction mixture was extracted twice with
Compound 5a was obtained after 2 d in dichloromethane and
column chromatography (n-pentane/ethyl acetate) as colourless
oil (845 mg, 2.8 mmol, 93%, mp 102 °C). 1H NMR: d = 0.9 (t,
J = 7.45, 3H, CH3), 2.5 (m, 2H, CH2), 5.9 (s, 1H, NH), 7.3 (m, 2H, H-
Ar), 7.6 (m, 1H, H-Ar), 8.1 (m, 1H, H-Ar). 13C-NMR: d = 7.6, 22.6,
28.3, 52.0, 84.1 (q, J = 27.9), 123.6 (q, J = 283.2), 129.3, 129.8,
130.1, 134.0, 163.2, 163.3. MS (EI): 332 (M+), 316, 259, 232, 105,
77, 57. Anal. Calcd for C15H17F3NO3: C, 58.00; H, 6.08; N, 4.23.
Found: C, 58.45; H, 6.03; N, 4.215. HRMS: 354.129 [M + Na+].
400 lL dichloromethane, the combined organic layers were dried
over anhydrous sodium sulfate and the organic solvent was re-
moved under nitrogen. For the determination of the enantiomeric
purity of the remaining substrate a it was hydrolyzed to the corre-
sponding alcohol b. Enantioselectivity and conversion were calcu-
lated according to Chen et al.25
4.3. General procedure for the synthesis of racemic N-tert-
butyl-2-hydroxyamides (serving as standards)
4.6. Esterase-catalyzed preparative-scale resolution of 2a
General procedure for the preparation of hydroxyamides: 2 mmol
of tertiary ester of protected a,a-dialkyl-a-hydroxycarboxylic acid
To a solution of 2a (120 mg, 0.55 mmol) in phosphate buffer
(100 mM, pH 7.5) and 5% (v/v) dimethyl formamide, esterase solu-
tion was added to a total volume of 40 mL. The solution was stirred
for 4 h at 4 °C, and the product was extracted three times with
methyl tert-butyl ether. After drying over Na2SO4 the solvent was
removed by distillation under reduced pressure, and the product
was purified by column chromatography (n-pentane/ethyl
acetate).
were dissolved in an aqueous solution of NaOH (0.1 N, 50 mL) with
DMSO (10%, v/v) as co-solvent and stirred for 1 h. The product was
extracted three times with dichloromethane. After drying over
Na2SO4, the solvent was removed by distillation under reduced
pressure.
4.3.1. (R,S)-N-tert-Butyl-2-hydroxy-2-(trifluoromethyl)-
butanamide, 1b
Compound 1b was obtained after as yellowish powder (69 mg,
0.3 mmol, 83%). 1H NMR: d = 0.92 (t, J = 7.41, 3H, CH3), 1.4 (s, 9H,
(CH3)3), 1.9 (m, 2H, CH2), 4.5 (s, 1H, OH), 6.1 (s,1H, NH). 13C
NMR: d = 6.33, 25.61, 28.35, 52.18, 77.15 (t, J = 28.0), 126.2 (t,
J = 285.4), 166.0. MS (EI): 212 (M+À15). HRMS: 228.119 [M+H+].
GC analysis: 5.3 min (+), 8.0 min (À) at a column temperature of
75 °C.
4.6.1. (À)-2-(tert-Butylcarbamoyl)-1,1,1-trifluorobut-2-yl
acetate, 1a
Compound (À)-1a was obtained after column chromatography
(n-pentane/ethyl acetate) as a white powder (30 mg, 0.11 mmol,
30%). Enantiomeric excess was determined after hydrolysis to 1b
as 47%ee.
4.3.2. (R,S)-N-tert-Butyl-2-hydroxy-2-methyl butanamide, 2b
Compound 2b was obtained after as white powder 72 mg,
0.4 mmol, 90%, mp 71 °C. 1H NMR: d = 0.9 (t, J = 7.44, 3H, CH3),
1.4 (s, 3H, CH3), 1.3 (s, 9H, (CH3)3), 1.7 (m, 2H, CH2), 2.4 (2H,
CH2), 2.9 (s, 1H, OH), 6.5 (s, 1H, NH). 13C NMR: d = 7.8, 26.6, 33.4,
27.7, 50.7, 75.9, 174.8. MS (EI): 144 (M+À29), 130, 101, 73, 58,
41. HRMS: 174.150 [M+H+]. GC analysis: 41.2 min (À), 44.0 min
(+) at a column temperature of 90 °C.
4.6.2. (+)-N-tert-Butyl-2-hydroxy-2-(trifluoromethyl)-
butanamide, 1b
Compound (+)-1b was obtained after column chromatography
(n-pentane/ethyl acetate) as a white powder (25 mg, 0.12 mmol,
32%, 87%ee).
4.6.3. (+)-1-(tert-Butylamino)-2-methyl-1-oxobutyl-2-yl
acetate, 2a
Compound (+)-2a was obtained after column chromatography
(n-pentane/ethyl acetate) as a white powder (46 mg, 0.22 mmol,
38%). Enantiomeric excess was determined after hydrolysis to 2b
as 93%ee.
4.3.3. (R,S)-N-tert-Butyl-3,3,3-trifluoro-2-hydroxy-2-
phenylpropanamide, 3b
Compound 3b was obtained after as white powder (61 mg,
0.26 mmol, 67%, mp 115 °C). 1H NMR: d = 1.31 (s, 9H, (CH3)3), 5.3

R. Kourist et al. / Tetrahedron: Asymmetry 19 (2008) 1839–1843
1843
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G.S. is grateful to the DAAD (Grant: A/07/95194) and the Viet-
namese ministry of Education and Training (Grant: 3413/QD-
BGDDT-VP) for financial support. The authors thank H. Atomi (Kyo-
to University, Kyoto, Japan) for providing the gene encoding PestE,
Dr. Thomas Jira (Institute of Pharmacy, University of Greifswald)
for useful discussions about chiral analysis and Dr. Michael Lalk
(Institute of Pharmacy, University of Greifswald) for support with
mass spectrometry.
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