Double-cuvette ISES: In situ estimation of enantioselectivity and relative rate for catalyst screening

Article abstract of DOI:10.1021/ja052010b

Described is a new method for the screening of an array of catalysts, in situ, to estimate enantioselectivity and relative rates. We term this approach "double-cuvette ISES (in situ enzymatic screening)". The Co(III)-salen mediated hydrolytic kinetic resolution (HKR) of (±)-propylene oxide is used as a model reaction to demonstrate proof of principle. In two parallel cuvettes, a lower CHCl₃-based organic layer is loaded with the epoxide and the chiral salen catalyst. Aqueous reporting layers, containing distinct "reporting enzymes" and their nicotinamide cofactors, are layered above the organic layers. The 1,2-propanediol enantiomers formed by the chiral catalyst diffuse into the aqueous layer and are oxidized there by the reporting enzymes at rates dependent upon the diol concentration, the R:S ratio of the diol, and the enantioselectivity of the reporting enzymes. A focused chiral salen library was constructed from seven chiral 1,2-diamines, derived from amino acid, terpenoid, and carbohydrates skeletons, and seven salicylaldehyde derivatives. Double-cuvette ISES identified a couple of interesting combinatorial hits in this salen array, wherein either the sense or magnitude of enantioselection for a given chiral diamine depends significantly upon the choice of "salicylaldehyde" partner. A comparison of predicted ee's and relative rates using this new screening tool with those independently measured is provided. Copyright

Full text of DOI:10.1021/ja052010b

Published on Web 05/26/2005
Double-Cuvette ISES: In Situ Estimation of Enantioselectivity and Relative
Rate for Catalyst Screening
Sangeeta Dey, Kannan R. Karukurichi, Weijun Shen, and David B. Berkowitz*
Department of Chemistry, UniVersity of Nebraska, Lincoln, Nebraska 68588-0304
Received March 29, 2005; E-mail: dbb@unlserve.unl.edu
Scheme 1. Double-Cuvette ISES
Combinatorial methods have made an important impact on
catalyst discovery in recent years.1 Notable examples include the
discovery of catalysts for asymmetric acylation2a and Stetter-type
chemistry,2b,c Pd(0)-2d and Cu(I)-mediated2e allylic alkylations, Ag-
based carbene insertion,2f FeCl2-mediated epoxidation,2g and early
transition-metal-based additions to imines.2h,i These successes have
spurred interest in catalyst screening.3 Screens for active lead
catalysts, based upon IR thermography,4a,b fluorescence,4c-f and dye
formation4g/bleaching4h have been reported. Particularly valuable
screens also provide information on enantioselectiVity.5 To predict
kinetic resolution efficiency in situ, several elegant parallel enan-
tiomer competition assays are available.4a,6 In situ screens for
organic catalysts that detect product handedness and thereby apply
to enantioselective catalysis (achiral educts) or actual resolutions
(racemic educts) are much rarer. One such method has recently
been reported by Morken and employs an isotopically chiral 13C
NMR probe substrate.7
Table 1. Focused 7 × 7 Chiral Salen Array for
Co(III)-Salen-Mediated HKR of rac-Propylene Oxidea
We have described the use of enzymes to monitor relative rates
in real time for allylic substitution catalysts.8,9 In its first iteration,
ISES (in situ enzymatic screening) was run in a bilayer, with an
aqueous enzymatic layer reporting on the turnover of an allylic
ethyl carbonate substrate in an organic layer. An asymmetric, Ni-
(0)-mediated allylic amination was uncovered in the process.8a,b
Herein, we disclose a second iteration of the ISES technique, in
which the reporting enzymes provide information on both relative
rate and enantioselectivity. This method differs significantly from
the first version of ISES: (i) One uses the reporting enzymes to
observe the reaction product directly (a chiral 1,2-diol here) rather
than a byproduct of catalyst turnover (e.g., EtOH previously); this
allows one to take full advantage of the chirality in the enzymatic
“sensor”. (ii) Because one wishes to glean information on enantio-
selectivity, as well as relative rates, two reporting enzymes are
employed, in parallel cuvettes. Two ISES reporting rates are needed
to distinguish the situation in which catalyst A has the same rate
as catalyst B but greater R:S selectivity from the one in which the
two catalysts display similar enantioselectivities, with catalyst A
possessing the greater rate. We term this approach “double-cuvette”
ISES (Scheme 1).
To demonstrate proof of principle, we chose the hydrolytic kinetic
resolution (HKR) of (()-propylene oxide, a reaction known to be
catalyzed efficiently by chiral Co(III)-salen complexes from the
pioneering work of Jacobsen.11 To provide an information-rich data
set requires that the two reporting enzymes display different-ideally
opposite-enantiomeric preferences. Screening revealed that alcohol
dehydrogenase from horse liver (HLADH) and from Thermoa-
naerobium brockii (TBADH) fulfill this criterion. The former
enzyme prefers (S)-1,2-propanediol,12 whereas the latter favors the
(R)-antipode.13
a Each box provides HKR data for the Co(III)-salen acetate derived
from the indicated salen. Presented are the % ee of the 1,2-propanediol
product [“+” ) (S) and “-” ) (R)] as predicted by double-cuvette ISES
(indigo) and as observed by chiral HPLC (black). Where available, observed
catalyst S values10 are also provided (enclosed boxes). The cuvette
experiments are run in a bilayer of pH 8.6 buffer over 7.2 M epoxide in
CHCl3, containing 0.25 mol % catalyst, for 15-35 min. “Inherent” catalyst
ee’s are judged by running the HKR in neat propylene oxide, containing
0.55 equiv of H2O, also at 0.25 mol % catalyst. §These catalysts gave ISES
†
signals < 20 mAbs min-1 over 35 min. This catalyst was tested at 0.05
mol %, as it was especially fast. #The catalysts derived from 2g and 4e
displayed ISES rates of 14.9 and 18.1 mAbs min-1, respectively, in the
HLADH cuvette, over 35 min. ¶Difficulty was encountered in synthesizing
appreciable quantities of these salens. *The 3,5-dinitrobenzoate counterion
was employed for these Co(III) catalysts.
terpenoid, amino acid, and carbohydrate skeletons) with sterically
and electronically diverse “salicylaldehydes”.
In the experiment, each Co(III)-salen catalyst (at 0.25 mol %) is
placed in a lower organic layer (CHCl3 and expoxide, 300 µL total
volume) in each of two parallel cuvettes. Aqueous reporting layers
containing TBADH/NADP+ (cuvette 1) and HLADH/NAD+
A focused 7 × 7 “salen” array was designed (Table 1) so as to
explore the interplay of novel chiral diamine scaffolds (from
9
8610
J. AM. CHEM. SOC. 2005, 127, 8610-8611
10.1021/ja052010b CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Acknowledgment. The authors thank the NSF (CHE-0317083),
the Nebraska Research Initiative, and the Alfred P. Sloan Founda-
tion (fellowship to D.B.B.) for support.
Supporting Information Available: Experimental methods and
synthetic characterization data. This material is available free of charge
via the Internet at http://pubs.acs.org.
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Figure 1. Predicted (double-cuvette ISES) vs observed enantioselectivities
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Figure 2. Predicted (ISES) vs observed (NMR) relative rates (normalized
to 4b) for the Co(III)-salen-mediated HKR of (()-propylene oxide.
(cuvette 2), respectively, are then added. The increase in absorbance
at 340 nm can be followed for several such sets in parallel by UV-
vis spectrophotometry. The kinetic data are then used to make the
double-cuvette ISES predictions. A comparison of the predicted
and measured ee’s is presented in Figure 1. Of 49 new salens
targeted, 42 were obtained, and 25 gave active Co(III)-catalysts.
Above a 65% ee threshold in either direction, ISES found 9 of 11
true hits (18% false negatives) and succeeded in 9 of its 12
predictions (25% false positives). Note that the measured ee’s are
for the HKR under the neat conditions typically used in Jacobsen’s
studies,11 whereas the predicted ee’s are for the bilayer used in
double-cuvette ISES. Figure 2 compares the relative rates predicted
1
by ISES with those observed in a bilayer by H NMR. Observed
relative rates agree with ISES predictions to within 25% in 9 of 10
cases measured, with the Co(III)-5d catalyst displaying slightly
greater variance.
Interesting “combinatorial hits” include the finding that 2-hy-
droxy-1-naphthaldehyde (c) yields catalysts with very low activity,
whereas 1-hydroxy-2-naphthaldehyde (d) is the best partner for
â-pinene-derived diamine 1. Furthermore, a remarkable inversion
of stereoselectivity is observed for salens emanating from the new
â-D-fructopyranose-based diamine 7, upon going from the 3,5-di-
tert-butylsalicylaldehyde partner to the sterically less encumbered
3,5-diiodo congener.
More work is needed to define the scope and limitations of
double-cuvette ISES. Candidate reactions must tolerate some water,
if run under biphasic conditions, but may be run under inert
atmosphere.8 The ISES method has the advantage that the reactant
under study need not be altered by installing a chromophore.
However, appropriate reporting enzymes (e.g., dehydrogenases)
must be available that recognize the reaction product. In addition
to other kinetic resolutions related to the HKR, it should be possible
to apply double-cuvette ISES to reactions in which chirality is
installed de novo in achiral substrates. Transformations such as
carbonyl additions, ketone reductions, and alkene oxidations, for
example, would appear to be good target reaction types with which
to explore this approach.
(12) Matos, J. R.; Smith, M. B.; Wong, C. H. Bioorg. Chem. 1985, 13, 121-30.
(13) HLADH displays an S:R relative velocity that increases from 2.6 to 3.9,
in going from 100 to 4 mM fixed diol concentrations, whereas TBADH
displays an R:S relative velocity that ranges from 8.4 to 10.7 between 80
and 3 mM. Consistent with this, for HLADH, [(Vmax/Km)S ÷ (Vmax/Km)R]
) 3.6 and, for TBADH, [(Vmax/Km)R ÷ (Vmax/Km)S] ) 11. Relative
velocities do not vary greatly with concentration as the antipodal diol
Km’s for each enzyme are in the range of 20 ( 5 mM. We thus set an
approximate (enantio)selectivity factor for each enzyme across the
concentration range of the ISES experiment. Expressions were then derived
for the enantioselectivity (i.e., [(R)-diol]/[(S)-diol]) and for relative rate
(i.e., total diol), assuming that observed ISES rates (∆Abs/time) could be
approximated as the {R-Units} × {[(R)-diol] + ([(S)-diol]/Sel)} (see SI
for details).
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