C. Moberg, K. Hult et al.
(
0.08 mm), MOPS (4 mm, pH 7.2) and acetonitrile (0.7%). The absorb-
ance was recorded at 405 nm with a Cary Bio spectrophotometer in
.5 mL semi-micro UV cuvettes from Plastibrand. Analyses were carried
acetonitrile (0.8%). Analysis was carried out with a volume of 300 mL of
this solution in each well. Measurement of absorbance was carried out in
1
cycles. Initially a starting level of absorbance was recorded for 27 mi-
À1
out with 1.2 mL of the solution in each cuvette. The absorbance was mea-
sured continuously during the different reaction steps. A starting value of
nutes. Addition of 3 mL of HLADH solution (10 mgmL
Na
, 100 mm
x
H
3ÀxPO
4
, pH 7.0) was performed automatically from pump 1. The ab-
the absorbance was recorded over 10 minutes to verify a stable level of
sorbance was measured for 46 minutes to ensure depletion of unreacted
À1
absorbance. Afterwards, CALB solution (10 mgmL
,
12 mL, 5 mm
aldehyde. After completion of the first reaction step, pump 1 was washed
À1
MOPS, pH 7.2) was added. The reaction time for stabilization of the ab-
with buffer and filled with CALB solution (10 mgmL
Na
(10 mgmL , 100 mm Na
, 100 mm
sorbance level, corresponding to depletion of the S enantiomer, was
x
H
3ÀxPO
4
À1
,
pH 7.2), whilst pump 2 was filled with PLE solution
, pH 7.0). Absorbance was recorded for
À1
3
0 minutes. For the second reaction step, PLE solution (10 mgmL
,
x 4
H3ÀxPO
1
2 mL, 5 mm MOPS, pH 7.2) was added and the reaction time for stabili-
8 minutes (two cycles) to compensate for any differences between meas-
urements. Addition of CALB solution (3 mL) was then performed from
pump 1. The hydrolysis of the S enantiomer was monitored for 40 mi-
nutes, after which PLE solution (3 mL) was added from pump 2. The hy-
drolysis of remaining R enantiomer was observed after 44 minutes of re-
action time. The last cycle of absorbance measurements after each reac-
tion step was used for calculations of ee and conversion. The samples
were run in quadruplicate. The mean value from 16 control samples,
identical to the reaction mixture but without substance 1a, was used for
subtraction of any background decrease in absorbance.
zation of the absorbance level, corresponding to depletion of the remain-
ing R enantiomer, was 30 minutes. A control sample, not containing the
reaction mixture, was used for subtraction of any background decrease in
absorbance.
Enzymatic determination of enantiomeric excess and conversion by use
of NADH: Compounds 1b–1r were tested by conventional spectropho-
tometry. For each of the substances 1b–1i, 1l–1m and 1o, a racemic
sample and two samples exhibiting high ee values, one of the R and one
of the S enantiomer, were tested. Racemic samples were tested for the
substances 1k, 1n and 1p–1r. The measurements were performed at
3
3
40 nm in 1.5 mL semi-micro UV cuvettes from Plastibrand in a Cary
00 Bio spectrophotometer. Two different dilutions were made for each
sample, one giving a total change in absorbance of 0.4–0.6, and one
giving a total absorbance change of 0.2–0.3. Typically, these were 600-
fold and 1200-fold dilutions of the samples from the reaction mixture,
Acknowledgements
which also contained NADH (0.17 mm), Na
and acetonitrile (0.8%). Analysis was carried out with a volume of
.2 mL of the solution in each cuvette. The absorbance was measured
x
H
3ÀxPO
4
(83m m, pH 7.0)
This work was supported by the Swedish Foundation for Strategic Re-
search. We are grateful to Prof. S. J. Haswell for the generous loan of a
microreactor.
1
continuously during the different reaction steps. A starting value of the
absorbance was recorded over 10 minutes to verify a stable level of ab-
À1
sorbance. Afterwards, HLADH solution (10 mgmL , 12 mL, 100 mm
[
1] a) K. D. Shimizu, M. L. Snapper, A. H. Hoveyda, Chem. Eur. J.
998, 4, 1885–1889; b) B. Jandeleit, D. J. Schaefer, T. S. Powers,
H. W. Turner, W. H. Weinberg, Angew. Chem. 1999, 111, 2648–
689; Angew. Chem. Int. Ed. 1999, 38, 2494–2532; c) S. Dahmen, S.
Bräll, Synthesis 2001, 1431–1449; d) M. T. Reetz, Angew. Chem.
001, 113, 292–320; Angew. Chem. Int. Ed. 2001, 40, 284–310;
e) M. T. Reetz, Angew. Chem. 2002, 114, 1391–1394; Angew. Chem.
Int. Ed. 2002, 41, 1335–1338; f) J. P. Stambuli, J. F. Hartwig, Curr.
Opin. Chem. Biol. 2003, 7, 420–426.
x 4
Na H3ÀxPO , pH 7.0) was added to the sample solution. The reaction
time necessary to obtain a stable absorbance level, corresponding to de-
pletion of unconverted aldehyde, was typically 20 minutes. In the follow-
1
2
À1
ing reaction step, CALB solution (10 mgmL
Na
,
12 mL, 100 mm
x
H
3ÀxPO
4
, pH 7.2) was added. The reaction time for stabilization of
the absorbance level, corresponding to depletion of the S enantiomer,
was typically 45 minutes. For the last reaction step, PLE solution
2
À1
(
10 mgmL , 12 mL, 100 mm Na
x
H
3ÀxPO
4
, pH 7.0) was added and the re-
action time for stabilization of the absorbance level, corresponding to de-
pletion of the remaining R enantiomer, was typically 45 minutes. Varia-
tions in reaction times were less than 10 minutes, depending on the activi-
ty for the enzymes towards the different substrates. A control sample,
without the reaction mixture, was used for subtraction of any background
decrease in absorbance.
[
[
2] a) C. Gennari, U. Oiarulli, Chem. Rev. 2003, 103, 3071–3100.
3] a) E. M. Vogl, H. Grçger, M. Shibasaki, Angew. Chem. 1999, 111,
1
672–1680; Angew. Chem. Int. Ed. 1999, 38, 1570–1577.
4] a) M. T. Reetz, T. Sell, A. Meiswinkel, G. Mehler, Angew. Chem.
003, 115, 814–817; Angew. Chem. Int. Ed. 2003, 42, 790–793; b) K.
Ding, H. Du, Y. Yuan, J. Long, Chem. Eur. J. 2004, 10, 2872–2884.
[
2
General procedure for microreactor-based reactions: A T-shaped micro-
reactor design with three reservoirs, two inlets (A and B) and one outlet
[5] a) P. Watts, QSAR Comb. Sci. 2005, 24, 701–711; b) K. Geyer,
J. D. C. CodØe, P. H. Seeberger, Chem. Eur. J. 2006, 12, 8434–8442.
[6] C. Jçnsson, S. Lundgren, S. J. Haswell, C. Moberg, Tetrahedron
2004, 60, 10515–10520.
[7] a) T. J. Edkins, D. R. Bobbitt, Anal. Chem. 2001, 73, 488A–496A;
b) M. Tsukamoto, H. B. Kagan, Adv. Synth. Catal. 2002, 344, 453–
(
C), with approximate channel dimensions of 10050 mm and outer di-
[
10,20]
mensions of 202025 mm, was used.
and S2, were prepared:
Two standard solutions, S1
S1: Salen-Ti
2 (Figure 4, 60 mg, 0.049 mmol, 5 mol%), Lewis base
4
63.
(
10 mol%) and benzaldehyde (100 mL, 0.98 mmol) were dissolved in dry
[
8] a) M. T. Reetz, A. Zonta, K. Schimossek, K. Liebeton, K.-E. Jaeger,
Angew. Chem. Int. 1997, 109, 2961–2963; Angew. Chem. Int. Ed.
Engl. 1997, 36, 2830–2832; b) P. Abato, C. T. Seto, J. Am. Chem.
Soc. 2001, 123, 9206–9207; c) F. Taran, C. Gauchet, B. Mohar, S.
Meunier, A. Valleix, P. Y. Renard, C. CrØminon, J. Grassi, A.
Wagner, C. Mioskowski, Angew. Chem. 2002, 114, 132–135; Angew.
Chem. Int. Ed. 2002, 41, 124–127; d) M. B. Onaran, C. T. Seto, J.
Org. Chem. 2003, 68, 8136–8141; e) Li, L. Bꢁtikofer, B. Witholt,
Angew. Chem. 2004, 116, 1730–1734; Angew. Chem. Int. Ed. 2004,
dichloromethane (1 mL) and the solution was cooled to 08C.
S2: Pyruvonitrile (140 mL, 1.98 mmol) was dissolved in dry dichlorome-
thane (1 mL) and the solution was cooled to 08C.
The channels of the microreactor were primed with dry dichloromethane
prior to the addition of standard solutions S1 and S2 (50 mL of each) to
À1
reservoirs A and B. A pressure generating a flow of 1 mLmin was ap-
plied. Reactions were performed at room temperature over a 20 or
4
0 min period. The microreactor was washed with dry dichloromethane
prior to each experiment to remove any residue from the system.
4
3, 1698–1702; f) S. Dey, K. R. Karukurichi, W. Shen, D. B. Berko-
High-throughput screening: The screening was performed on a Greiner
witz, J. Am. Chem. Soc. 2005, 127, 8610–8611; g) C. M. Sprout, C. T.
Seto, Org. Lett. 2005, 7, 5099–5102.
bio-one PS microplate (96 well microtitre plate) in
a FLUOstar
OPTIMA plate reader fitted with two separate injection pumps (pumps 1
and 2). Absorbance was recorded at 340 nm. The following solution was
prepared individually for each of the 20 samples: 600-fold dilution of re-
[9] a) M. T. Reetz, K. M. Kꢁhling, A. D. Deege, H. Hinrichs, D. Belder,
Angew. Chem. 2000, 112, 4049–4052; Angew. Chem. Int. Ed. 2000,
39, 3891–3893; b) M. Ludwig, F. Kohler, D. Belder, Electrophoresis
2003, 24, 3233–3238.
x 4
action mixture in NADH (0.17 mm), Na H3ÀxPO (83m m, pH 7.0) and
4340
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 4334 – 4341