Application of a modified Mosher's method for the determination of enantiomeric ratio and absolute configuration at C-3 of chiral 1,3-dihydroxy ketones

Article abstract of DOI:10.1016/j.tetasy.2009.07.023

The enantiomeric ratio and absolute configuration of products of the transketolase reaction are typically determined by comparison of the specific rotation or derivatisation and HPLC or GC. A Mosher's ester method has been developed via ester formation at

Full text of DOI:10.1016/j.tetasy.2009.07.023

Tetrahedron: Asymmetry 20 (2009) 1828–1831
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Application of a modified Mosher’s method for the determination
of enantiomeric ratio and absolute configuration at C-3
of chiral 1,3-dihydroxy ketones
*
James L. Galman, Helen C. Hailes
Department of Chemistry, University College London, 20 Gordon Street, London WC1H 0AJ, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The enantiomeric ratio and absolute configuration of products of the transketolase reaction are typically
determined by comparison of the specific rotation or derivatisation and HPLC or GC. A Mosher’s ester
method has been developed via ester formation at the primary alcohol C-1 which can be used to deter-
mine the stereoselectivity of the reaction, as well as the absolute configuration of the product at C-3.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 17 June 2009
Accepted 16 July 2009
Available online 21 August 2009
1. Introduction
Several assays have been described which detect a,
a0-dihy-
droxy ketones in biocatalytic conversions including spectrophoto-
metric and colorimetric methods.11a,15 However, chiral assays that
both establish the enantiomeric purity and determine the absolute
stereochemistry of these structural motifs are most important. For
TK-generated products this has predominantly been carried out by
comparison of the specific rotation, and enantiomeric purities have
frequently not been determined. Recently, an acetate derivatisa-
tion and chiral HPLC and GC method has been reported, together
with Ender’s SAMP methodology to confirm absolute stereochem-
istries.12 The use of chiral auxiliaries and a six-step procedure to
determine absolute stereochemistries however is time consuming,
and although suitable for a range of compounds, the reaction fails
to accommodate derivatives when R is Ar. A more convenient and
Several methods are available for determining the absolute ste-
reochemistries of compounds including comparison of specific
rotation data to reported values, chiral HPLC and GC with correla-
tion to known or synthesised compounds,1 X-ray crystallography,
and NMR spectroscopy via the use of lanthanide shift reagents2
or chiral derivatising agents.3,4 The latter approach, in particular
the application of the Mosher’s method using 2-methoxy-2-(tri-
fluoromethyl) phenylacetic acid (MTPA) has been extensively
used:4–6 it should be noted that it has been reliably utilised for
determining the absolute configuration of secondary alcohols and
primary amines.5,6 For example, chiral secondary alcohols have
typically been coupled in two separate, analogous experiments
using each enantiomer of the Mosher’s acid. The chemical shift
differences of the ester protons at R1 and R2 (Fig. 1) were then
calculated (
D
d = dS-MTPA ꢀ dR-MTPA) and assigned. Protons at R1 in
H
O
H
O
the (R)-MTPA diastereoisomer were observed upfield relative to
the (S)-MTPA ester due to the diamagnetic effect of the aromatic
ring.4
(S)
(R)
CF3
CF3
R2
R2
O
O
R1
R1
OMe
Ph
Ph
MeO
a,
a0-Dihydroxy ketone functionalities are found in several bio-
(S)-diastereoisomer
(R)-diastereoisomer
logically important compounds, and can be used as synthons in
further structural elaboration to a range of compounds including
ketosugars and aminodiols.7–10 The enzyme transketolase (TK)
(EC 2.2.1.1) has been shown to catalyse in vitro the condensation
of hydroxypyruvate (Li-HPA, 1) with a range of aldehydes to stere-
Figure 1. Two Mosher conformers showing the (S)-MTPA and (R)-MTPA
derivatives.
oselectively generate a,
a0-dihydroxy ketones 2 (Scheme 1); a bio-
mimetic route to these compounds has also been reported.8–13 In
addition, asymmetric routes to such chiral dihydroxyacetones have
been established using Ender’s chiral auxiliary methodology.14
O
O
TK
R
H
OH
R
OH
LiO2C
Mg2+, ThDP
+
O
OH
2
1
* Corresponding author. Tel.: +44 (0) 20 7679 4654; fax: +44 (0) 20 7679 7463.
E-mail address: h.c.hailes@ucl.ac.uk (H.C. Hailes).
Scheme 1. TK-catalysed reaction with cofactors Mg2+ and thiamine diphosphate
(ThDP) to generate
a0-dihydroxy ketones.
a
,
0957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.07.023

J. L. Galman, H. C. Hailes / Tetrahedron: Asymmetry 20 (2009) 1828–1831
1829
rapid approach would be the application of the modified Mosher’s
method for determining both the enantiomeric purity and absolute
stereochemistry at the C-3 carbinol stereocentre. To date, a modi-
fied Mosher’s method has been applied to 1,3-diol motifs via deri-
vatisation as di-MTPA esters which was successfully used with
acyclic syn-diols and cyclic diols, however irregularities in the
Dd
of acyclic anti-1,3-diols made this method not applicable in all
cases.16 Interestingly, the modified Mosher’s method has been
used to determine the absolute configuration of primary alcohols
with chiral methyl groups at C-2: chemical shifts of the oxymeth-
ylene protons in each MTPA ester (attached at C-1) were noted, and
a larger chemical shift difference was observed in the (2S,20S)- and
(2R,20R)-isomers compared to the (2S,20R)- and (2R,20S)-isomers.17
This was attributed to restricted rotation along the C2–C3 bond.
Due to the presence of the ketone at C-2 in 2, and the conforma-
tional restriction this may confer, application of the modified
Figure 2. 1H NMR signals for diastereotopic protons Ha and Hb in the (R)-MTPA
ester 4a (spectrum A) and (S)-MTPA ester 5a (spectrum B).
As with the work of Kobayashi et al. on chiral primary alcohols
with methyl groups at C-2, the oxymethylene proton separation
0
0
0
0
varied and
D
d2HR;3 R and
D
d2HS;3 S
>
D
d2HR;3 S and
D
d2HS;3 R. The ee could
be determined to within 5% of the value from GC analysis.
a, -
a0
Mosher’s method, via coupling at C-1, to
a,
a0-dihydroxy ketones
Dihydroxyketones with reported absolute configurations were
was investigated to establish whether it could be used to deter-
then used to investigate the validity of the method.
mine the absolute configuration and ee at C-3.
First, commercially available L-erythrulose 2b, prepared from 1
and glycolaldehyde 3b using TK, was converted into the corre-
sponding (R)- and (S)-MTPA esters using MTPA chloride. The 1H
2. Results and discussion
NMR analysis revealed that Ha and Hb in the (R)-MTPA ester 4b
0
(major isomer (2R,30S)) were at dH 5.11 and 5.19 ppm (
D
d2HR;3 S is
The absolute stereochemistry and enantiomeric purity of (3S)-
1,3-dihydroxypentan-2-one 2a have been established previously,12
and so were used in the initial studies for the attachment of MTPA
at the primary hydroxyl position. The diastereotopic protons Ha
and Hb in 2a are normally observed at 4.59 and 4.70 ppm, and
may be shifted in each MTPA ester as described previously for C-
2 methyl primary alcohol systems.17 Compound 2a was prepared
using wild-type (WT)-TK.12 Coupling of 2a to either (R)- or (S)-
MTPA was performed under standard conditions (Scheme 2), and
after purification to remove the remaining starting materials, urea
side products and di-MTPA esters which were formed in low yields,
the MTPA esters 4a and 5a were analysed by 1H NMR spectroscopy
in CDCl3. The 1H NMR signals for diastereotopic protons Ha and Hb
0.08) and negligible signals for the minor isomer were observed.
For the (S)-MTPA ester 5b, the major isomer (2S,30S) signals for
0
Ha and Hb were observed at dH 5.05 and 5.23 ppm (
D
d2HS;3 S is
0.18) while no minor isomer was detected. Integration of the sig-
nals for Ha and Hb as before indicated -erythrulose to 0be of >95%
ee. Again for the oxymethylene protons > D
d2HS;3 S dH2R;3 S enabling,
L
0
D
by formation of both MTPA esters, confirmation of absolute stereo-
chemistry as well as the enantiomeric purity.
2-Methoxy ethanal 3c has been described as an aldehyde accep-
tor in the TK reaction with spinach TK, where an ee of 60% was ob-
served (S-major isomer).18 This transformation was performed
using WT Escherichia coli TK to validate Mosher0s method for ste-
reochemical determination. Compound 2c was formed in 30% yield
and coupling to the MTPA chlorides gave 4c and 5c with Ha and Hb
for the major isomers at 5.13 and 5.17 ppm (2R,30S) and 5.02 and
in the (R)-MTPA ester 4a for the major isomer (2R,30S) were at dH
0
5.05 ppm (
and 5.17 ppm (
D
d2HR;3 S 0) and for the minor isomer were at dH 4.93
0
0
0
d2HR;3 R 0.24) (Fig. 2, spectrum A). For the (S)-MTPA
5.29 ppm (2S,30S), respectively. Again
D
d2HS;3 S (0.27) >
D
d2HR;3 S
D
ester 5a the major isomer (2S,30S0) signals for Ha and Hb were at dH
(0.04) (Table 1), confirming the application of the method, and
integration of Ha and Hb for both esters 4c and 5c as before gave
an average value for the ee of 57%, which was comparable to that
reported for spinach-TK.18 The synthesis of the 1,3-dihydroxy-1-
phenylpropan-2-one 2d has been described previously and the
MTPA method was also explored with this aromatic analogue to
4.93 ppm and 5.17 ppm (
D
d2HS;3 S 0.24) and the minor isomer
0
(2S,30R) signals were at dH 5.05 ppm (
D
d2HS;3 R is 0) (Fig. 2, spectrum
B). Averaging of the two ee values calculated from the integration
of HaHb signals in spectra A and B gave an ee of 55% which was
comparable to that of 58% determined by GC methods.12
O
O
TK
R
H
OH
2a R = CH2CH3
2b R = CH2OH
2c R = CH2OMe
R
OH
3'
LiO2C
+
Mg2+, ThDP
O
Ha Hb
HO
1
3a R = CH2CH3
3b R = CH2OH
3c R = CH2OMe
or Et3N
DCC, DMAP
(R)- or (S)-MTPA
(S)- or (R)-MTPACl
R = Ph
ref. 13
O
O
O
MeO Ph
Ph OMe
2
Ph
OH
2
R
3'
O
R
O
3'
CF3
(R)
CF3
2d
(S)
or
HO
Ha Hb
Ha Hb
HO
O
Et3N
(S)-MTPACl
HO
O
4a R = CH2CH3
4b R = CH2OH
4c R = CH2OMe
5a R = CH2CH3
5b R = CH2OH
5c R = CH2OMe
O
MeO Ph
Ph
O
2
3'
CF3
4d
Ha Hb
HO
O
Scheme 2. TK-catalysed reactions using 3 to generate ketones 2 and the formation of MTPA esters 4 and 5.

1830
J. L. Galman, H. C. Hailes / Tetrahedron: Asymmetry 20 (2009) 1828–1831
4.2. (2R,30S)-3,3,3-Trifluoro-2-methoxy-2-phenyl propionic acid
Table 1
Chemical shifts for MTPA esters and absolute stereochemistries
30-hydroxy-20-oxo-pentyl ester 4a
R
MTPA ester
D
dH HaHb (major isomer)
eea of 2
55% 2a
Lit. ee
The reaction was carried out under anhydrous conditions. To a
stirred solution of 2a (0.030 g, 0.25 mmol) in CH2Cl2 (5 mL) were
added DCC (0.057 g, 0.28 mmol) and DMAP (0.012 g, 0.02 mmol)
in CH2Cl2 (10 mL) at 0 °C and the reaction mixture was stirred for
2 min. (S)-MTPA (0.030 g, 0.08 mmol) was added and the reaction
mixture was stirred for 30 min, after which further (S)-MTPA
(0.020 g, 0.08 mmol) was added and the mixture was stirred for
48 h at rt. The solvent was removed in vacuo, and the crude prod-
uct was dry loaded onto silica gel and purified using flash chroma-
tography (EtOAc/hexane, 3:2) to afford 5a as a colourless oil
(0.035 g, 59%), (2R,30S)-55% ee (from the integrations of Ha and
Hb 1H NMR signals from 4a and 5a). Rf = 0.45 (EtOAc/hexane,
CH2CH3
CH2CH3
4a (R)
5a (S)
0 (2R,30S)
0.24 (2S,30S)
58% (S)12
CH2OH
CH2OH
4b (R)
5b (S)
0.08 (2R,30S)
0.18 (2S,30S)
>95% 2b
57% 2c
>98% (S)12
60% (S)18
CH2OMe
CH2OMe
4c (R)
5c (S)
0.04 (2R,30S)
0.27 (2S,30S)
a
Determined from the integration of 1H NMR signals for Ha and Hb from both the
(R)- and (S)-MTPA esters.
establish whether the separation of Ha and Hb was similar to that
for the aliphatic series.13,19 Compound 2d was prepared using the
biomimetic TK reaction described previously13 and was converted
to the mono-MTPA diastereoisomeric esters 4d using (S)-MTPA
chloride. Protons Ha and Hb were observed as before and by anal-
ogy to the assignment for the aliphatic series,
3:2); ½a 2D0
ꢁ
¼ þ13:3 (c 0.15, CHCl3); mmax (KBr)/cmꢀ1 3429br s,
2925s, 1712s; 1H NMR (500 MHz; CDCl3) d 1.00 (3H, m, CH3),
1.65 (1H, m, CHHCH3) 1.89 (1H, m, CHHCH3), 2.86 (1H, d, J 6.0,
OH), 3.64 (3H, s, OMe), 4.29 (1H, m, CHOH), 4.93 (0.26H, d, J
17.0, CHHO (2R,30R)), 5.05 (1.48H, s, CH2O (2R,30S)), 5.17 (0.26H,
d, J 17.0, CHHO (2R,30R)), 7.54 (3H, m, Ph), 7.61 (2H, m, Ph); 13C
NMR (125 MHz; CDCl3) d 8.8 (CH3), 27.2 (CH2CH3), 55.8 (OCH3),
67.2, 76.2, 123.2 (q, JCF 283, CF3), 127.6, 128.6, 129.9, 131.8,
166.3 (C@O ester), 204.2 (C@O, ketone); 19F NMR (282 MHz; CDCl3)
d ꢀ72.2; m/z (FTMS) [M+NH4] calcd for C15H21F3O5N, 352.1366;
found 352.1371.
0
D
d2HR;3 R was 0.31
0
while
D
d2HS;3 R was 0.05, highlighting the potential application of
this approach with aromatic analogues.
3. Conclusion
The stereoselectivity of E. coli WT-TK using 2-methoxy ethanal
as a substrate was comparable to that observed with spinach TK,
and was consistent with previous reports indicating a preference
for formation of the (S)-isomer in the biotransformation. In addi-
tion, by using (R)- and (S)-MTPA we have demonstrated that the
4.3. (2S,30S)-3,3,3-Trifluoro-2-methoxy-2-phenyl propionic acid
30-hydroxy-20-oxo-pentyl ester 5a
ees of a,
a0-dihydroxyketones can routinely be determined, to with-
in 5% of values determined using GC or HPLC method, as well as the
The same procedure was used as that described above for 4a
using (S)-MTPA to give 5a as a colourless oil (0.030 g, 57%)
(2S,30S)-55% ee (from integrations of Ha and Hb 1H NMR signals
absolute stereochemistries. The method can be used with a range
of aliphatic
a,
a0-dihydroxyketones 2 with lipophilic and polar R
groups. Determination of the absolute stereochemistries is more
convenient than the multistep chiral auxiliary procedures de-
scribed previously, and is also applicable to a wider range of com-
pounds including aromatic systems (R is Ar). This is important for
from 4a and 5a). Rf = 0.45 (EtOAc/hexane, 3:2); ½a D20
¼ ꢀ37:0
ꢁ
(c 0.1, CHCl3); 1H NMR (300 MHz; CDCl3) d 1.00 (3H, t, J 7.4, CH3),
1.69 (1H, m, CHHCH3) 1.89 (1H, m, CHHCH3), 2.87 (1H, d, J 5.3,
OH), 3.64 (3H, s, OMe), 4.29 (1H, m, CHOH), 4.93 (0.82H, d, J
17.0, CHHO (2S,30S)), 5.05 (0.36H, s, CH2O (2S,30R)), 5.17 (0.82H,
d, J 17.0, CHHO (2S,30S)), 7.54 (3H, m, Ph), 7.61 (2H, m, Ph); 13C
NMR (75 MHz; CDCl3) d 8.7 and 8.8 (CH3), 27.2, 55.8 (OCH3),
67.2, 76.2, 123.2 (q, JCF 283, CF3), 127.6, 128.5, 128.7, 129.9,
166.2 (C@O ester), 204.2 (C@O, ketone); 19F NMR (282 MHz; CDCl3)
d ꢀ72.2.
future work using chemical or biocatalytic routes to
dihydroxyketones.
a, -
a0
4. Experimental
4.1. General information
4.4. (2R,30S)-3,3,3-Trifluoro-2-methoxy-2-phenyl propionic acid
Unless otherwise noted, solvents and reagents were of reagent
grade from commercial suppliers (Sigma–Aldrich) and were used
without further purification. Dry CH2Cl2 was obtained using anhy-
drous alumina columns.20 All moisture-sensitive reactions were
performed under a nitrogen or argon atmosphere using oven-dried
glassware. Reactions were monitored by TLC on Kieselgel 60 F254
plates with detection by UV, potassium permanganate and phos-
phomolybdic acid stains. Flash column chromatography was carried
30,40-dihydroxy-20-oxo-butyl ester 4b
The reaction was carried out under anhydrous conditions. To a
stirred solution of 2b (0.030 g, 0.25 mmol) in CH2Cl2 (5 mL) were
added triethylamine (34
lL, 0.25 mmol) and (S)-MTPA chloride
(34 L, 0.18 mmol) in CH2Cl2 (2 mL) and the reaction mixture
l
was stirred for 12 h at rt. The product was dry loaded onto silica
gel and purified using flash chromatography (EtOAc) to afford 4b
as a colourless oil (0.054 g, 63%), (2R,30S) >95% ee (from integra-
tions of Ha and Hb 1H NMR signals of 4b and 5b). Rf = 0.45 (EtOAc);
out using silica gel (particle size 40–63 l
m). 1H NMR and 13C NMR
spectra were recorded at the field indicated using Bruker
AMX300 MHz and Avance 500 and 600 MHz machines. Coupling
constants are measured in hertz (Hz) and unless otherwise specified,
NMR spectra were recorded at 298 K. Mass spectra were recorded on
a LTQ Orbitrap XL. Infrared spectra were recorded on a Shamadzu
FTIR-8700 infrared spectrophotometer. Optical rotations were re-
corded on an Optical Activity Limited PolAAR2000 polarimeter at
589 nm, quoted in deg cm2 gꢀ1 and concn (c) in g/100 mL.
½
a 2D0
ꢁ
¼ þ10:2 (c 0.4, CHCl3); mmax (KBr)/cmꢀ1 3429br s, 2925s,
1712s; 1H NMR (300 MHz; CDCl3) d 3.64 (3H, s, OMe), 3.89 (1H,
dd, J 10.6 and 3.8, CHHOH), 3.95 (1H, dd, J 10.6 and 3.8, CHHOH),
4.35 (1H, dd, J 3.8 and 3.8, CHOH), 5.11 (1H, d, J 17.0, CHHO
(2R,30S)), 5.19 (1H, d, J 17.0, CHHO (2R,30S)), 7.43 (3H, m, Ph),
7.62 (2H, m, Ph), no (2R,30R isomer detected); 13C NMR (75 MHz;
CDCl3) d 55.9 (OCH3), 63.5, 68.0, 76.4, 84.6 (q, JCF 28, CCF3), 123.1
(q, JCF 288, CF3), 127.5, 128.6, 129.9, 131.7, 166.5 (C@O ester),
203.3 (C@O, ketone); 19F NMR (282 MHz; CDCl3) d ꢀ72.2; m/z
(FTMS) [M+NH4] calcd for C14H19F3O6N, 354.1159; found 354.1162.
Lithium hydroxypyruvate was synthesised as described
previously.21 (3S)-1,3-Dihydroxypentan-2-one 2a (58% ee) was
prepared as described previously using WT E. coli TK,12 and
rulose 2b was commercially available.
L-eryth-

J. L. Galman, H. C. Hailes / Tetrahedron: Asymmetry 20 (2009) 1828–1831
1831
4.5. (2S,30S)-3,3,3-Trifluoro-2-methoxy-2-phenyl propionic acid
nals of 4c and 5c). Rf = 0.45 (EtOAc/hexane, 1:1); ½a D20
ꢁ
¼ ꢀ10:6 (c
30,40-dihydroxy-20-oxo-butyl ester 5b
0.25, CHCl3); 1H NMR (500 MHz; CDCl3) d 3.38 (3H, s, OCH3), 3.61
(3H, s, OCH3) 3.57–3.70 (2H, m, CH2), 4.36 (1H, m, CHOH), 5.02
(0.74H, d, J 17.5, CHHO (2S,30S)), 5.13 (0.81H, d, J 17.5, CHHO
(2S,30R)), 5.17 (0.38H, d, J 17.5, CHHO (2S,30R)), 5.29 (0.81H, d, J
17.5, CHHO (2S,30S)), 7.42 (3H, m, Ph), 7.62 (2H, m, Ph); 13C NMR
(150 MHz; CDCl3) d 55.8 (OCH3), 59.5 (OCH3), 68.1, 72.8, 75.0,
84.5 (q, JCF 27, CCF3), 123.1 (q, JCF 285, CF3), 127.5, 128.5, 129.8,
131.5, 166.1 (C@O ester), 202.9 (C@O, ketone); 19F NMR
(282 MHz; CDCl3) d ꢀ72.2.
The same procedure was used as that described above for 4b
using (R)-MTPA chloride to give 5b as a colourless oil (0.044 g,
51%) (2S,30S) >95% ee (from integrations of Ha and Hb 1H NMR sig-
nals of 4b and 5b). Rf = 0.45 (EtOAc); ½a D20
ꢁ
¼ ꢀ14:4 (c 0.5, CHCl3), 1H
NMR (500 MHz; CDCl3) d 3.63 (3H, s, OMe), 3.92 (2H, m, CH2OH),
4.35 (1H, dd, J 4.0 and 3.8, CHOH), 5.05 (1H, d, J 17.0, CHHO
(2S,30S)), 5.23 (1H, d, J 17.0, CHHO (2S,30S)), 7.43 (3H, m, Ph), 7.62
(2H, m, Ph), no (2S,30R isomer detected); 13C NMR (125 MHz;
CDCl3) d 55.8 (OCH3), 63.5, 67.9, 76.3, 84.6 (q, JCF 28, CCF3), 123.1
(q, JCF 288, CF3), 127.5, 128.5, 129.9, 131.7, 166.4 (C@O ester),
203.1 (C@O, ketone); 19F NMR (282 MHz; CDCl3) d ꢀ72.2.
4.9. (2R,30RS)-3,3,3-Trifluoro-2-methoxy-2-phenyl propionic
acid 30-hydroxy-20-oxo-30-phenylpropyl ester 4d
The same procedure was used as that described above for 4c
using (S)-MTPA chloride and 2d.13 The product was purified using
flash silica chromatography (EtOAc/hexane, 1:4) to give 4d as a col-
ourless oil (0.014 g, 61%). 1H NMR (500 MHz; CDCl3) d 3.62 (3H, s,
OCH3), 4.71 (0.25H, d, J 16.9, CHHO (2R,30R)), 4.86 (0.25H, d, J 16.9,
CHHO (2R,30S)), 4.91 (0.25H, d, J 16.9, CHHO (2R,30S)), 5.02 (0.25H,
d, J 16.9, CHHO (2R,30R)), 5.25 (1H, m, CHOH), 7.34–7.54 (6H, m,
Ph), 7.70 (2H, m, Ph), 8.01 (2H, J 8.6, Ph).
4.6. 1, 3-Dihydroxy-4-methoxy-butan-2-one 2c
ThDP (22 mg, 48
lmol) and MgCl2ꢂ6H2O (39 mg, 180
lmol)
were dissolved in H2O (10 mL) and the pH was adjusted to 7 with
0.1 M NaOH. To this stirred solution, at 25 °C, was added WT-TK
clarified lysate (2 mL)11c,12 and the mixture was stirred for
20 min. In another flask, 1 (110 mg, 1 mmol) and 3c (74 mg,
1 mmol) were dissolved in H2O (8 mL) and the pH was adjusted
to 7 with 0.1 M NaOH. Following the 20-min enzyme/cofactor
pre-incubation, the 1/3c mixture was added to the enzyme solu-
tion and the mixture was stirred at 25 °C for 24 h. During this time,
the pH was maintained at 7.0 by addition of 1 M HCl using a pH
stat (Stat Titrino, Metrohm). Silica was added and the reaction mix-
ture was concentrated to dryness before dry loading onto a flash
silica gel column. Following column purification (EtOAc/CH3OH,
Acknowledgements
We thank the EPSRC for a DTA studentship to J.L.G, and Abil
Aliev for helpful discussions. We thank the EPSRC National Mass
Spectrometry Service Centre, Swansea University, for the provision
of high resolution MS data.
80:20), 2c was isolated as an oil (40 mg, 30%). ½a D20
¼ þ2:0 (c 2.0,
ꢁ
CHCl3), lit.18
½
a 2J 5
ꢁ
¼ þ3:0 (c 0.017, MeOH); mmax (KBr)/cmꢀ1
References
3415br s, 2923s, 1727s; 1H NMR (300 MHz; CDCl3) d 3.36 (3H, s,
OCH3), 3.61 (1H, dd, J 9.9 and 4.4, CHHOMe), 3.70 (1H, dd, J 9.9
and 4.4, CHHOMe), 4.38 (1H, dd, J 4.4 and 4.4, CHOH), 4.43 (1H,
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d,
J 19.7, CHHOH), 4.53 (1H, d, J
19.7, CHHOH); 13C NMR
(75 MHz; CDCl3) d 59.5 (OCH3), 66.7 (CH2), 73.4 (CH2), 74.8 (CHOH)
210.8 (C@O); m/z (FTMS) [M+NH4] calcd for C5H14O4N, 152.0917;
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4.7. (2R,30S)-3,3,3-Trifluoro-2-methoxy-2-phenyl propionic acid
30-hydroxy-40-methoxy-20-oxo-butyl ester 4c
The reaction was carried out under anhydrous conditions. To a
stirred solution of 2c (0.010 g, 0.07 mmol) in CH2Cl2 (3 mL) were
added triethylamine (34
lL, 0.25 mmol) and (S)-MTPA chloride
(20 L, 0.11 mmol) in CH2Cl2 (2 mL) and the reaction mixture
l
was stirred for 12 h at rt. The product was dry loaded onto silica
gel and purified using flash chromatography (EtOAc/hexane, 1:1)
to afford 4c as a colourless oil (0.015 g, 61%), (2R,30S) 57% ee (from
integrations of Ha and Hb 1H NMR signals of 4c and 5c). Rf = 0.40
(EtOAc/hexane,1:1); ½a D20
¼ þ23:2 (c 0.25, CHCl3); mmax (KBr)/
ꢁ
cmꢀ1 3415br s, 2923s, 1727s; 1H NMR (300 MHz; CDCl3) d 3.38
(3H, s, OCH3), 3.61 (3H, s, OCH3) 3.57–3.70 (2H, m, CH2), 4.36
(1H, m, CHOH), 5.02 (0.16H, d, J 17.5, CHHO (2R,30R)), 5.13
(0.84H, d, J 17.5, CHHO (2R,30S)), 5.17 (0.81H, d, J 17.5, CHHO
(2R,30S)), 5.29 (0.16H, d, J 17.5, CHHO (2R,30R)), 7.42 (3H, m, Ph),
7.62 (2H, m, Ph); m/z (FTMS) [M+NH4] calcd for C15H21F3O6N,
368.1315; found 368.1318.
4.8. (2S,30S)-3,3,3-Trifluoro-2-methoxy-2-phenyl propionic acid
30-hydroxy-40-methoxy-20-oxo-butyl ester 5c
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20. Pangborn, A. B.; Giardello, M. A.; Grubbs, R. H.; Rosen, R. K.; Timmers, F. J.
Organometallics 1996, 15, 1518–1520.
The same procedure was used as that described above for 4c
using (R)-MTPA chloride to give 5c as a colourless oil (0.010 g,
40%) (2S,30S) 57% ee (from integrations of Ha and Hb 1H NMR sig-
21. Dickens, F. Biochem. Prep. 1962, 9, 86–91.