High-throughput measurement of the enantiomeric excess of chiral alcohols by using two enzymes

DOI: 10.1002/anie.200353055

Source and publish data:

Angewandte Chemie - International Edition p. 1698 - 1702 (2004)

Update date:2022-08-30

Topics:

Authors:

Li, Zhi Li, Zhi

Buetikofer, Lukas

Witholt, Bernard

Read Full Text PDF DownLoad Join now for total 90,000,000 free articles

Article abstract of DOI:10.1002/anie.200353055

Rapid ee determination: Enantioselective alcohol dehydrogenases A and B were used to oxidize chiral alcohols in a sensitive, accurate, high-throughput method (see scheme). The reaction rates were determined by monitoring the formation of NAD(P)H by UV spectroscopy. The ee value was calculated from the reaction rates and the kinetic constants of the enzymes.

Full text of DOI:10.1002/anie.200353055

Communications

Chiral Chemistry

followed by UV spectroscopy^[11a]or infrared thermogra-

phy.^[11b]In the former method, known as EMDee (enzymatic

method for determining enantiomeric excess), an enantio-

specific alcohol dehydrogenase is used to oxidize one

enantiomer of an alcohol at a known concentration, the

oxidation rate is determined by following the formation of

NAD(P)H (NAD, nicotinamide-adenine dinucleotide), and

the ee value of the alcohol is calculated by referring to a

standard curve of rate versus the ee value established at the

same alcohol concentration. This method is high-throughput

and sensitive and has been successfully used to determine the

ee value of the alcohol generated from a chemical catalysis in

which 100% conversion is achieved. However, the general

application of such a method in high-throughput screening of

enantioselective catalysts is rather limited since conversion is

often below 100%, which means that the product concen-

tration must be determined by another high-throughput

method before analysis of the ee value. Herein, we describe

a method for determining the ee value based on the new

concept of using two enantioselective enzymes to modify the

product. This method allows the determination of the ee value

of the product independent of concentration. In contrast to

the concept of Schoofs and Horeau, which applies to pseudo-

enantiomeric mixtures of reagents in one reaction, our

analysis of the ee value involves two separate enzymatic

reactions. Our method allows sensitive, accurate, high-

throughput measurement of the enantiomeric excess of a

chiral alcohol. In addition, the alcohol concentration can be

estimated within the same process.

Chiral alcohols 4 were used as model compounds to

demonstrate the principle of our approach. These alcohols

represent an important class of intermediates for the synthesis

of pharmaceuticals and fine chemicals^[1]and can be prepared

by various enantioselective catalytic transformations, such as

biohydroxylation of hydrocarbons 1, chemical or enzymatic

reduction of ketones 2, or enzymatic hydrolysis of esters 3 (or

the reverse reaction, namely formation of an ester;

Scheme 1). To analyze the ee value of 4, enantioselective

NAD(P)⁺-dependent alcohol dehydrogenases A and B were

each used to oxidize alcohol 4. The reaction rates for

enzyme A (v^a) and enzyme B (v^b) were determined by

following the formation of NAD(P)H through its absorption

at 340 nm. The enantiomers of chiral alcohols are generally

competing substrates for enantioselective alcohol dehydro-

genases so the reaction rates v^aand v^bcan be expressed by

Equations (1) and (2):^[12]

High-Throughput Measurement of the

Enantiomeric Excess of Chiral Alcohols by

Using Two Enzymes**

Zhi Li,* Lukas Bütikofer, and Bernard Witholt

Dedicated to Professor Karl Schlögl

on the occasion of his 80th birthday

The development of enantioselective catalytic processes has

attracted increasing attention as a result of the importance of

enantiomerically pure organic compounds in pharmaceutical,

agricultural, and fine chemical syntheses.^[1]Attempts to

discover new enantioselective catalysts have recently focused

on the screening of libraries of chemical catalysts^[2]created by

combinatorial synthesis, and enzymes and enzyme mutants^[3,4]

generated by molecular biotechnology techniques such as the

error-prone polymerase chain reaction and gene shuffling.^[5]

Since the catalyst libraries are huge, analysis of the catalyst

enantioselectivity is the bottleneck in such approaches. Many

methods have been developed for high-throughput enantio-

selectivity analysis.^[6]The enantioselectivity factor E for

kinetic resolution can be estimated quickly by measuring

separately the reaction rates of the R and S enantiomers of

the substrate.^[7]This technique can also be applied to the

reversible asymmetric reaction of a prochiral substrate by

examining separately the reverse reactions of the R and S

products.^[7d]On the other hand, high-throughput analysis of

the product enantiomeric excess (ee) can be used for the

evaluation of the catalyst enantioselectivity in asymmetric

transformations. The product ee can be determined by GC-

GC on a column with a chiral stationary phase,^[8a]HPLC with

CD/UV,^[8b,c]or by using optical rotation/refractive index (OR/

RIU) detection^[8c], chirally modified capillary electrophore-

sis,^[8d]electrospray ionization tandem MS,^[8e]colour indicators

based on doped liquid crystals,^[8f]or competitive enzyme

immunoassays.^[8g]The concept developed by Schoofs and

Horeau^[9a]allows the ee value of the product to be established

by exploiting kinetic resolution effects and using mass-tagged

or fluorescence-tagged pseudo-enantiomeric mixtures of

acylating agents, with mass spectrometry (MS)^[9b,c]or fluo-

rescence^[9d]detection. Elegant approaches for determination

of the ee values also include the use of isotopically labeled

pseudo-enantiomeric or pseudo-meso substrates and MS^[10a,b]

or NMR detection.^[10c,d]When the product concentration is

known, the ee value of the product can be determined by

using an enzyme to catalyze a further reaction that can be

V_m^a_ax;SK^a_m;R½S þ V_m^a_ax;RK_m^a_;S½R

v^a

v^b

ð1Þ

ð2Þ

K^a_m;R½S þ K^a_m;S½R þ K_m^a_;SK^a_m;R

V_m^b_ax;SK^b_m;R½S þ V_m^b_ax;RK_m^b_;S½R

K^b_m;R½S þ K^b_m;S½R þ K_m^b_;SK^b_m;R

[*] Dr. Z. Li, L. Bütikofer, Prof. Dr. B. Witholt

Institute of Biotechnology

ETH-Hönggerberg, HPT C71

8093 Zürich (Switzerland)

Fax: (+41)1-633-1051

V_maxis the maximum reaction velocity, K_mis the Michaelis

constant, a and b refer to the two enzymes, and S and R refer

to the alcohol enantiomers. Equations (1) and (2) lead to

Equations (3) and (4):

E-mail: zhi@biotech.biol.ethz.ch

[**] Financial support from the ETH research office is gratefully

acknowledged.

1698

ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

DOI: 10.1002/anie.200353055

Angew. Chem. Int. Ed. 2004, 43, 1698 –1702

Angewandte

Chemie

the alcohol dehydrogenases from Lacto-

bacillus kefir (LKADH),^[13a]Thermo-

anaerobium brockii (TBADH, a thermo-

stable enzyme),^[13b]and Rhodococcus

erythropolis

(READH).^[13c]

These

enzymes are relatively stable and their

stock solutions, when kept at 48C, can be

used for analysis for at least 10 h without

loss of activity. LKADH and TBADH

were used for the oxidation of 4a,

whereas LKADH and READH were

used for the oxidation of the alcohols

4b and 4c. Bioconversions were per-

Scheme 1. Synthetic routes to alcohols 4 and catalysis of the oxidation of these alcohols with

alcohol dehydrogenases A and B.

formed with a 200-mL solution containing

buffer, substrate, NAD(P)⁺, and enzyme

in a deep well microtiter plate and the

UVabsorption at 340 nm of the produced

NAD(P)H was measured for 5 min. The kinetic data for the

enzyme-catalyzed oxidation of (S)-4 and (R)-4 were estab-

lished for each enzyme in separate experiments and are

summarized in Table 1.

The measurement of the ee value of 4a is a typical

example of an analysis using two enantioselective but not

enantiospecific enzymes. In total, 54samples with 37 different

ee values and 22 different concentrations (0.4–3.5 mm) were

analyzed in a microtiter plate by treatment with LKADH and

TBADH, respectively. The results are shown in Figure 1, in

which each point represents the average of two independent

measurements. Figure 1a is a plot of the measured versus

actual ee values. In every case (from ꢀ100% to 100% ee) but

one (0% ee), the measured value falls within 9% ee of the

true value. The analysis of the ee values is independent of

sample concentration; nearly the same ee values were

measured for samples with identical actual ee values but

different concentrations.

V^a_max;Rꢀv^a

V^b_max;Rꢀv^b

ꢀ

K^a_m;Rv^a

K^b_m;Rv^b

½S ¼

½R ¼

ð3Þ

ð4Þ

ðV^a_max;R^ꢀv^aÞðV^b_max;S^ꢀv^bÞ ðV^b_max;R^ꢀv^bÞðV_m^a_ax;S^ꢀv^aÞ

ꢀ

K^a_m;R^v^aK^b_m;Sv^b

K^b_m;R^v^bK^a_m;Sv^a

V^b_max;Sꢀv^b

K^b_m;Sv^b

V^a_max;Sꢀv^a

ꢀ

K^a_m;Sv^a

ðV_m^a_ax;Rꢀv^aÞðV_m^b_ax;Sꢀv^bÞ ðV^b_max;Rꢀv^bÞðV_m^a_ax;Sꢀv^aÞ

ꢀ

K^a_m;R^v^aK^b_m;Sv^b

K^b_m;R^v^bK^a_m;Sv^a

The ee value can thus be calculated by using Equation (5):

V^b_max;Sꢀv^bV_m^a_ax;Sꢀv^a

K^b_m;Sv^b

V^b_max;Sꢀv^bV_m^a_ax;Sꢀv^a

K^b_m;Sv^b

V^a_max;Rꢀv^a

V^b_max;Rꢀv^b

ꢀ

K^a_m;Sv^a

K^a_m;Rv^a

K^b_m;Rv^b

½Rꢀ½S

ee ¼

ð5Þ

V^a_max;Rꢀv^a

V^b_max;Rꢀv^b

K^b_m;Rv^b

½R þ ½S

ꢀ

K^a_m;Sv^a

K^a_m;Rv^a

If two enantiospecific alcohol dehydrogenases are used,

Equation (5) can be simplified. Suppose enzyme A specifi-

cally oxidizes (R)-4 and enzyme B specifically oxidizes (S)-4.

In this case, V^a_max;S= 0, K^a_m;S= K^a_I, V_m^b_ax;R= 0, K^b_m;R= K_I^b, where

K_Iis the inhibition constant (see the Experimental Section).

Therefore,

The concentrations (C = [R] + [S]) of alcohol 4a were also

calculated from the results of the same experiments by using

Equations (3) and (4). As shown in Figure 1b, the measured

concentrations corresponded well to the actual concentra-

V^b_max;Sꢀv^b

V^a_max;Rꢀv^a

Table 1: Kinetic data for the oxidation of enantiopure secondary alcohols 4a–c catalyzed by

alcohol dehydrogenases LKADH, TBADH, and READH.

ꢀ

K_I^a

K^a_m;Rv^a

K^b_m;Sv^b

K_I^b

½Rꢀ½S

ee ¼

ð6Þ

V^b_max;Sꢀv^b

K^b_m;Sv^b

V^a_max;Rꢀv^a

½R þ ½S

Substrate Enzyme^[a]

Enzyme conc.

[mgmL^ꢀ1[UmL^{ꢀ1 [b]}

NADP⁺NAD⁺

K_m

K_IV_max

K_I^a

K^a_m;Rv^a

K_I^b

]

[mm]

[mm] [mm] [mmmin^ꢀ1

]

The kinetic constants V^a_max;RV^a, K_m^a_;RK^a

m;S

(R)-4a

(S)-4a

(R)-4a

(S)-4a

(R)-4b

(S)-4b

(R)-4b

(S)-4b

(R)-4c

(S)-4c

(R)-4c

(S)-4c

LKADH

TBADH

LKADH

READH

LKADH

READH

0.10

0.015

0.10

1.0

365

663

900

1250

2600

–

5.4

max;S

V_m^b_ax;R_,V_m^b_ax;S, K^b_m;R_,K_m^b_;S_,K_I^a, and K_I^bcan be quickly

established by separate oxidation of (R)-4, (S)-4,

and mixtures of (R)- and (S)-4 in known ratios

with enzymes A and B, respectively. We used an

enzyme preparation with a constant concentra-

tion to establish the kinetic constants and analyze

unknown samples. The ee values could then be

calculated by applying Equation (5) or (6). The

use of V_maxis advantageous because this approach

does not require accurate determination of the

enzyme concentration in each experiment.

3070

400

0.036

2.0

–

792

136

–

326

0.050

1.0

360

2630

0.036

2.0

[a] The enzymatic assays were carried out in tris(hydroxymethyl)aminomethane buffer (50 mm,

pH 7.5) at 258C for LKADH and in potassium phosphate buffer (50 mm, pH 8.5) at 408C and

258C for TBADH and READH, respectively. [b] The specific activity U refers to the reduction of p-

chloroacetophenone.

To illustrate this method, three commercially

available enzymes were used for the oxidations:

Angew. Chem. Int. Ed. 2004, 43, 1698 –1702

www.angewandte.org

ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

1699

Communications

(0.2–5.0 mm). Figure 2 shows plots of the measured ee values

versus the actual values for 4b and 4c. For 4b, each point

represents the average of three independent measurements

and demonstrates the accuracy of the method: 60 of the 64

measured values show an error of less than 5% ee of the true

Figure 1. a) Plot of the actual ee values of 4a versus those measured

by using two enantioselective alcohol dehydrogenases, LKADH and

TBADH. b) Plot of the actual concentrations (c) of 4a versus those

measured by using two enantioselective alcohol dehydrogenases,

LKADH and TBADH.

Figure 2. Plots of the actual ee values of 4b (a) and 4c (b) versus the

ee values measured by using two enantiospecific alcohol dehydrogen-

ases, LKADH and READH.

tions from 0.4to 2.0 m m, but large errors were observed at

higher concentrations. The measured oxidation rate may not

be very accurate when the concentration of 4a is much higher

than the value of K_m. According to Equations (3)–(5), small

errors in v^aand v^bcreate much bigger errors in the

concentration than in the ee value. Nevertheless, such con-

centration measurements could still be useful for qualitative

estimates of the extent of conversion for catalyst screening. In

the case of a high product concentration, the sample could be

diluted several fold for analysis.

As a representative example of the use of two enantio-

specific alcohol dehydrogenases for analysis of the ee value,

an experiment was carried out with LKADH and READH.

We tested 64samples of 4b with 35 different ee values and 29

different concentrations (0.4–10 mm), as well as 83 samples of

4c with 51 different ee values and 33 different concentrations

value, and the other four values have errors of 5–7% ee. For

4c, only one measurement was made at each ee value. Of the

83 measured ee values, 79 are within 10% ee of the true value,

and four results have an error of 10–12% ee.

Our method for the fast determination of the enantio-

meric excess of secondary alcohols by using two alcohol

dehydrogenases has several distinctive features: 1) The ee can

be determined with satisfactory accuracy, independent of the

concentration. 2) The enzymes do not have to be specific to,

or highly active towards the alcohol. A large number of

alcohol dehydrogenases with broad substrate ranges are now

available and it is thus easy to find appropriate alcohol

1700

ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.angewandte.org

Angew. Chem. Int. Ed. 2004, 43, 1698 –1702

Angewandte

Chemie

kinetic data, to calculate the ee values from Equation (5) or (6). The

measured ee values were plotted against the actual values.

dehydrogenases for analysis of the ee value of a given alcohol.

3) The analysis method is very sensitive. The technique can be

used to determine the ee value of a sample with a concen-

tration as low as 200 mm and is therefore particularly useful for

biocatalyst screening, where product concentration is often

low. 4) The analysis is performed with UV spectroscopy; no

special instrument is required. 5) Experiments are performed

in a 96-well microtiter plate, which allows analysis of the

ee values of 48 samples within 5 min. Given a preparation

time of 5 min, about 288 samples can be analyzed in an hour,

which is a high enough throughput for most practical

applications. 6) The method can be extended to measure the

ee values of other types of compounds. Two enzymes of

another type may be applied, coupled with detection by UV

spectroscopy, MS, or even HPLC or GC for short analysis

times. 7) The method also provides the possibility to measure

concentration. For high concentrations, samples may be

diluted and then analyzed, which makes the method useful

for screening catalyst activities.

Received: October 10, 2003

Revised: January 8, 2004[Z53055]

Keywords: alcohols · asymmetric catalysis · dehydrogenation ·

enantioselectivity · high-throughput screening

[1] a) Chiralityin Industr:y The Commercial Manufacture and

Applications of OpticallyActive Compounds

(Eds.: A. N.

Collins, G. N. Sheldrake, J. Crosby), Wiley, Chichester, 1992;

b) Chiralityin IndustryII: Developments in the Commercial

Manufacture and Applications of OpticallyActive Compounds

(Eds.: A. N. Collins, G. N. Sheldrake, J. Crosby), Wiley, Chi-

chester, 1997; c) Chiralityin Agrochemicals (Eds.: N. Kurihara, J.

Miyamoto), Wiley, Chichester, 1998; d) Chiralityin Liquid

Crystals (Ed.: H.-S. Kitzerow), Springer, New York, 2001.

[2] For a review, see: a) A. Berkessel, Curr. Opin. Chem. Biol. 2003,

7, 409 – 419; b) J. P. Stambuli, J. F. Hartwig, Curr. Opin. Chem.

Biol. 2003, 7, 420 – 426; c) A. Hagemeyer, B. Jandeleit, Y. Liu,

D. M. Poojary, H. W. Turner, A. F. Volpe, Jr., W. H. Weinberg,

Appl. Catal. A 2001, 221, 23 – 43; d) K. W. Kuntz, M. L. Snapper,

A. H. Hoveyda, Curr. Opin. Chem. Biol. 1999, 3, 313 – 319.

[3] For a review, see: a) M. T. Reetz, K.-E. Jaeger, Chem. Eur. J.

2000, 6, 407 – 412; b) K.-E. Jaeger, M. T. Reetz, Curr. Opin.

Chem. Biol. 2000, 4, 68 – 73; c) M. T. Reetz, Tetrahedron 2002,

58, 6595 – 6602.

Further investigations will focus on the application of this

method to the discovery of enantioselective biological and/or

chemical catalysts. The scope and possible limitations of the

method will also be explored.

[4] For examples, see: a) M. T. Reetz, A. Zonta, K. Schimossek, K.

Liebeton, K.-E. Jaeger, Angew. Chem. 1997, 109, 2961 – 2963;

Angew. Chem. Int. Ed. Engl. 1997, 36, 2830 – 2832; b) M. T.

Reetz, S. Wilensek, D. Zha, K.-E. Jaeger, Angew. Chem. 2001,

113, 3701 – 3703; Angew. Chem. Int. Ed. 2001, 40, 3589 – 3591;

c) D. Zha, S. Wilensek, M. Hermes, K.-E. Jaeger, M. T. Reetz,

Chem. Commun. 2001, 2664– 2665; d) E. Henke, T. Bornscheu-

er, Biol. Chem. 1999, 380, 1029 – 1033; e) O. May, P. T. Nguyen,

F. H. Arnold, Nat. Biotechnol. 2000, 18, 317 – 320.

[5] a) E. T. Farinas, T. Bulter, F. H. Arnold, Curr. Opin. Biotechnol.

2001, 12, 545 – 551; b) W. P. C. Stemmer, Proc. Natl. Acad. Sci.

USA 1994, 91, 10747 – 10751; c) W. P. C. Stemmer, Nature 1994,

370, 389 – 391.

[6] For a review, see: a) M. T. Reetz, Angew. Chem. 2001, 113, 292 –

320; Angew. Chem. Int. Ed. 2001, 40, 284– 310; b) M. T. Reetz,

Angew. Chem. 2002, 114, 1391 – 1394; Angew. Chem. Int. Ed.

2002, 41, 1335 – 1338; c) D. Wahler, J.-L. Reymond, Curr. Opin.

Biotechnol. 2001, 12, 535 – 544; d) J. F. Traverse, M. L. Snapper,

Drug DiscoveryToday 2002, 7, 1002 – 1012; e) M. G. Finn,

Chirality 2002, 14, 534– 540.

[7] a) M. T. Reetz, A. Zonta, K. Schimossek, K. Liebeton, K.-E.

Jaeger, Angew. Chem. 1997, 109, 2961 – 2963; Angew. Chem. Int.

Ed. Engl. 1997, 36, 2830 – 2832; b) M. T. Reetz, M. H. Becker,

K. M. Kuehling, A. Holzwarth, Angew. Chem. 1998, 110, 2792 –

2795; Angew. Chem. Int. Ed. 1998, 37, 2647 – 2649; c) L. E. Janes,

R. J. Kazlauskas, J. Org. Chem. 1997, 62, 4560 – 4561; d) G. Klein,

J.-L. Reymond, Helv. Chim. Acta 1999, 82, 400 – 407; e) F.

Badalassi, D. Wahler, G. Klein, P. Crotti, J.-L. Reymond, Angew.

Chem. 2000, 112, 4233 – 4236; Angew. Chem. Int. Ed. 2000, 39,

4067 – 4070.

Experimental Section

Enzymes: LKADH was obtained from Fluka as a lyophilized powder

with an activity of 0.4Umg ^ꢀ1for the reduction of acetophenone.

TBADH was purchased from Aldrich as a lyophilized powder with an

activity of 4.7 Umg^ꢀ1for the oxidation of 2-propanol. READH was

obtained from Juelich Fine Chemicals as a solution with an activity of

144 UmL^ꢀ1for the reduction of p-chloroacetophenone. The enzyme

stock solutions were freshly prepared each day, kept in aqueous

buffer at 48C, and used for measurement of the kinetic data and for

the analysis of the ee value.

General procedure for measurement of the ee value: A mixture

of buffer, alcohol 4, NAD(P)⁺, and enzyme with a total volume of

200 mL was pipetted into individual deep wells in a microtiter plate

and the absorption of the produced NAD(P)H at 340 nm was

measured for 5 minutes at 25 or 408C on a Spectra Max Plus

photometer with an optical density resolution of 0.001. Data points

were recorded every 12 s, which gave 26 points for every measure-

ment. The absorption data were used to calculate the NAD(P)H

concentration from a previously established standard curve. The

initial reaction velocity was calculated by linear regression as the

slope of a plot of [NAD(P)H] versus time.

The kinetic constants were quickly obtained by oxidizing (R)-4,

(S)-4, and mixtures of the enantiomers at various concentrations by

treatment with a single enzyme on a microtiter plate. The average

velocities calculated from three measurements and the alcohol

concentrations were used as input for the program Enzfit, which

was used to calculate the values of K_mand V_max. To determine the

inhibition constant K_I, Lineweaver–Burk (L-B) plots of v^ꢀ1versus

[S]^ꢀ1were generated for various inhibitor concentrations. A graph of

the slopes of the L–B plots versus the inhibitor concentration was

created. The value of K_Iwas determined by dividing the intercept by

the slope of this plot.

[8] a) M. T. Reetz, K. M. Kꢀhling, S. Wilensek, H. Husmann, U. W.

Hꢁusig, M, Hermes, Catal. Today 2001, 67, 389 – 396; b) K.

Ding, A. Ishii, K. Mikami, Angew. Chem. 1999, 111, 519 – 523;

Angew. Chem. Int. Ed. 1999, 38, 497 – 501; c) K. Mikami, R.

Angelaud, K. Ding, A. Ishii, A. Tanaka, N. Sawada, K. Kudo, M.

Senda, Chem. Eur. J. 2001, 7, 730 – 737; d) M. T. Reetz, K. M.

Kuehling, A. Deege, H. Hinrichs, D. Belder, Angew. Chem. 2000,

112, 4049 – 4052; Angew. Chem. Int. Ed. 2000, 39, 3891 – 3893;

To measure the ee value, samples containing the two enantiomers

in various ratios and at a range of concentrations were prepared in

microtiter plates and separately oxidized with each of two enzymes.

The oxidation velocities were measured and used, together with

Angew. Chem. Int. Ed. 2004, 43, 1698 –1702

www.angewandte.org

ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

1701

Communications

e) W. A. Tao, R. G. Cooks, Angew. Chem. 2001, 113, 779 – 782;

Angew. Chem. Int. Ed. 2001, 40, 757 – 760; f) R. A. van Delden,

B. L. Feringa, Angew. Chem. 2001, 113, 3298 – 3300; Angew.

Chem. Int. Ed. 2001, 40, 3198 – 3200; g) F. Taran, C. Gauchet, B.

Mohar, S. Meunier, A. Valleix, P. Y. Renard, C. Creminon, J.

Grassi, A. Wagner, C. Mioskowski, Angew. Chem. 2002, 114,

132 – 135; Angew. Chem. Int. Ed. 2002, 41, 124– 127.

[9] a) A. Schoofs, A. Horeau, Tetrahedron Lett. 1977, 18, 3259 –

3262; b) J. Guo, J. Wu, G. Siuzdak, M. G. Finn, Angew. Chem.

1999, 111, 1868 – 1871; Angew. Chem. Int. Ed. 1999, 38, 1755 –

1758; c) S. Yao, J.-C. Meng, G. Siuzdak, M. G. Finn, J. Org.

Chem. 2003, 68, 2540 – 2546; d) G. A. Korbel, G. Lalic, M. D.

Shair, J. Am. Chem. Soc. 2001, 123, 361 – 362.

[10] a) M. T. Reetz, M. H. Becker, H. W. Klein, D. Stockigt, Angew.

Chem. 1999, 111, 1872 – 1875; Angew. Chem. Int. Ed. 1999, 38,

1758 – 1761; b) W. Schrader, A. Eipper, D. J. Pugh, M. T. Reetz,

Can. J. Chem. 2002, 80, 626 – 632; c) M. T. Reetz, A. Eipper, P.

Tielmann, R. Mynott, Adv. Synth. Catal. 2002, 344, 1008 – 1016;

d) M. A. Evans, J. P. Morken, J. Am. Chem. Soc. 2002, 124, 9020 –

9021.

[11] a) P. Abato, C. T. Seto, J. Am. Chem. Soc. 2001, 123, 9206 – 9207;

b) N. Millot, P. Borman, M. S. Anson, I. B. Campbell, S. J. F.

Macdonald, M. Mahmoudian, Org. Process Res. Dev. 2002, 6,

463 – 470.

[12] H. Bisswanger, Enzyme Kinetics: Principles and Methods, Wiley-

VCH, Weinheim, 2002, pp. 106 – 108.

[13] a) C. W. Bradshaw, W. Hummel, C. H. Wong, J. Org. Chem.

1992, 57, 1532 – 1536. b) E. Keinan, K. K. Seth, R. J. Lamed,

Ann. N. Y. Acad. Sci. 1987, 501, 130 – 150; c) J. Peters, T. Minuth,

M. R. Kula, Tetrahedron: Asymmetry 1993, 4, 1683 – 1692.

1702

ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.angewandte.org

Angew. Chem. Int. Ed. 2004, 43, 1698 –1702

Products guided by the article

Product name:2-Hexanone

Cas No:591-78-6

R&D Labs maybe for 591-78-6

Suzhou Chenke Biotech Co., Ltd.

Contact:+86-18662408853

Address:NO.150, Renai Road, Suzhou Industrial Park
ZHEJIANG JIANYE CHEMICAL CO.,LTD.

Contact:86-571-64149273,64149234

Address:No. 48, Fuxi Road, Meicheng Town
Zhenjiang Haitong Chemical Industry Co., Ltd.

Contact:+86 (511) 8448-0369

Address:Baoyan Town, Dantu District, Zhenjiang City, Jiangsu, China
Xi'an HO-SHINE Bio-Technology Co., Ltd

Contact:+86 (29) 8248-5435/18292020086

Address:No.6 Shiyuan Road Xian City Shaanxi Province, 710048 China
Suzhou Ryan Pharmachem Technology Co.,Ltd

Contact:+86-0512-68780025

Address:B-301,No.2 Taishan Road,Suzhou New District,Jiangsu,P.R. China

Relevant to this article

STEROIDAL SAPONINS FROM SMILAX RIPARIA AND S. CHINA

Doi:10.1016/0031-9422(92)83295-A
(1992)
Synthesis and bioevaluation of 3-(substitutedphenyl)-1-(4-hydroxyphenyl)-2-propen-1-ones and their carbamate derivatives against root - knot nematode (meloidogyne javanica)

Doi:10.13005/ojc/300346
(2014)
Regulating the Solvation Sheath of Li Ions by Using Hydrogen Bonds for Highly Stable Lithium–Metal Anodes

Doi:10.1002/anie.202101976
(2021)
Kinetics and mechanism of the reaction of substituted O-benzoylbenzamidoximes with sodium methoxide in methanol

Doi:10.1002/(SICI)1099-1395(199908)12:8<626::AID-POC173>3.0.CO;2-W
(1999)
Structure-based switch of regioselectivity in the flavin-dependent tryptophan 6-halogenase Thal

Doi:10.1074/jbc.RA118.005393
(2019)
Ionic conductivity in solid solutions of PbF₂ and YF₃

Doi:10.1016/S0025-5408(01)00634-1
(2001)

Article Doi

High-throughput measurement of the enantiomeric excess of chiral alcohols by using two enzymes

DOI: 10.1002/anie.200353055

Source and publish data:

Authors:

Article abstract of DOI:10.1002/anie.200353055

Full text of DOI:10.1002/anie.200353055

Products guided by the article

R&D Labs maybe for 591-78-6

Relevant to this article

Hot Product