Stereoselective Reduction of α-Fluoro-β-keto Esters by NADH and NADPH-Dependent Ketoreductases

Article abstract of DOI:10.1002/ejoc.201900644

Racemic α-fluoro-β-keto esters were stereoselectively transformed to α-fluoro-β-hydroxy esters through dynamic reductive kinetic resolution (DYRKR) using commercially available NAD(P)H-dependent ketoreductases. Aromatic, alkenyl, and alkyl substrates were

Full text of DOI:10.1002/ejoc.201900644

A Journal of
Accepted Article
Title: Stereoselective Reduction of α-Fluoro-β-ketoesters by NADH and
NADPH-dependent Ketoreductases
Authors: Thomas Green, Anil Damarancha, Matthew Vanagel, Brandon
Showalter, Sandra Kolberg, and Alexander Thompson
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201900644
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10.1002/ejoc.201900644
European Journal of Organic Chemistry
Stereoselective Reduction of α-Fluoro-β-ketoesters by NADH and
NADPH-dependent Ketoreductases
Thomas K. Green*, Anil Damarancha, Matthew Vanagel, Brandon Showalter, Sandra Kolberg,
and Alexander Thompson
Abstract: Racemic α-Fluoro-β-ketoesters were stereoselectively transformed to α-fluoro-β-hy-
droxyesters through dynamic reductive kinetic resolution (DYRKR) using commercially-available
NAD(P)H-dependent ketoreductases. Aromatic, alkenyl and alkyl substrates were all reduced in
high optical purities and yields. For most substrates, either anti or syn diastereomers could be
produced with high enantiomeric excess, depending on the enzyme employed. The enzyme reac-
tions with ethyl -fluoroacetoacetate were conveniently monitored in real time by in situ 19F NMR
spectroscopy. These commercially-available enzymes provide convenient access to stereoisomers
of α-fluoro-β-hydroxyesters from easily accessible racemic substrates.
Introduction
The introduction of the C-F bond into a compound can dramatically alter its physical and
chemical properties[1] including its solubility, pKa, and molecular conformation. Fluorine can also
have a dramatic effect on the bioactive properties of pharmaceutical drugs, including potency,
membrane permeability, enzyme binding, and metabolic stability.[2–7] Indeed, some of the best
performing drugs on the market contain one or more fluorine atoms incorporated into their struc-
ture.[1,8] Due to the high, and often beneficial, impact of fluorine incorporation into drugs, the de-
velopment of new methodologies for the synthesis of fluorine-containing compounds remains an
active area of research. Given that most drugs possess one or more chiral centers, it comes as no
surprise that recent efforts have focused on the stereoselective incorporation of fluorine. This paper
focuses on the stereoselective synthesis of α-fluoro-β-hydroxyesters, with two chiral centers, using
a set of commercially-available ketoreductases (KRED) from Codexis, Inc.
A number of chemical methods have been developed for the stereoselective synthesis of α-
fluoro-β-hydroxyesters including radical reduction of α-bromo-α-fluoro-β-hydroxyesters,[9] using
either trimethyl aluminum (syn) or tris(trimethylsilyl)silane (anti) as reductant. Using similar con-
ditions, the α-allyl-α-fluoro-β-hydroxyesters could be produced with high diastereomeric excess.
[10] β-Fluoro-α-hydroxyesters have been synthesized by enantioselective Pd-catalyzed β-fluorina-
tion of the α-ketoester followed by diastereoselective chemical reduction of the keto function.[11]
Anti-α-fluoro-β-hydroxy ketones were synthesized with high stereoselectivity by L-prolinol-cata-
lyzed aldol condensation.[12] Chiral fluorinated hydroxyketones, derived from p- and m-trifluoro-
acetyl substituted acetophenones, have also been synthesized in high yield and enantiomeric ex-
cess (>98%) using commercially available KREDs.[13] Racemic ethyl-α-fluoroacetoacetate was
hydrogenated over a Cinchona-modified Pt catalyst to favor (2S,2R)-α-fluoro-β-hydroxyester in
82% enantiomeric excess and a 99/1 anti/syn ratio.[14] The aldolase-catalyzed reaction of fluoro
pyruvate with aromatic and heteroaromatic aldehydes leads to syn α-fluoro β-hydroxy carboxylic
acids with high diastereo- and enantioselectivity.[15]
[a] Department of Chemistry & Biochemistry, Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, AK 99775
Phone: (+1)-907-474-1559
Email: tkgreen@alaska.edu
URL: https://www.uaf.edu/chem/faculty/tgreen/
1
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10.1002/ejoc.201900644
European Journal of Organic Chemistry
There are numerous reports of KRED-catalyzed enantioselective reductions of β-ketoesters to
β-hydroxyesters possessing one chiral center.[16–21] KREDs have also been employed in the syn-
thesis of α-alkylated- and α-chloro-β-hydroxyesters possessing two chiral centers (four stereoiso-
mers).[22,23] These products form by reduction of the prochiral ketone with epimierzation [24] at the
α-carbon, termed dynamic reductive kinetic resolution (DYRKR)[25], to often yield a single stere-
oisomer.[26] The stereoselective reduction of the ethyl α-fluoroacetoacetate on a large scale using
a dehydrogenase has been accomplished, with 85% diastereomeric and >98% enantiomeric ex-
cess.[27] In this paper, we sought to broaden the scope of α-fluorinated-β-ketoesters as substrates
that might be candidates for DYRKR. We employ 24 isolated NAD(P)H dependent ketoreductases
from Codexis to reduce alkyl-, alkenyl- and aryl-substituted α-fluorinated-β-ketoesters. In many
instances, a single stereoisomer of four possible α-fluorinated-β-hydroxyesters can be synthesized
in high yield and high diastereo- and enantioselectivity.
Results and Discussion
The enzymatic reduction -fluoro--ketoesters is shown in Scheme 1. Ethyl -fluoroaceto-
acetate 1a was obtained commercially as a racemic mixture. Substrates 1b – 1i were synthesized
by Reformatsky reaction of the corresponding aldehyde with ethyl bromofluoroacetate.[28,29] fol-
lowed by oxidation with Dess-Martin periodinane to the corresponding ketone (Supporting Infor-
mation).[30] The Reformatsky product was a mixture of all four stereoisomers shown in Scheme 1,
which was used as a standard for stereochemical analysis by NMR spectroscopy.
R: a, methyl; b, pentyl; c, heptyl; d, 1-heptenyl; e, 1-undecenyl;
f, phenyl; g, p-methoxyphenyl; h, p-trifluoromethylphenyl; i, styrenyl
Scheme 1. Enzymatic reduction of -fluoro--ketoesters.
2
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European Journal of Organic Chemistry
Initially, the substrate (+/-)-ethyl-α-fluoroacetoacetate 1a (R=CH3) was subjected to KRED-
catalyzed reduction for 24 hours at 37 C using a set of 24 commercially-available ketoreductases
obtained from Codexis, Inc. using conditions previously employed (Supporting Information).[16]
Typically, 25, 50, 100 or 200 μmol of substrate was reacted in water/methanol with either isopro-
pyl alcohol or glucose/glucose dehydrogenase for co-factor recycling. All KREDs utilize NADPH
except for two enzymes, which utilize NADH. Crude products were analyzed by quantitative 19F
NMR spectroscopy to assess diastereomeric ratio (anti:syn ratio) and percent conversion, which
were typically 86-97% isolated yields. Downfield 19F NMR signals correspond to the anti config-
uration (2S,3S and 2R,3R) according to the literature.[28,31] The anti and syn configuration exhibit
characteristic three-bond 19F-1H couplings of 17.8 and 22.1 Hz, respectively, which aids in identi-
fication. The anti and syn signals in the 1H NMR spectra were identified by the three-bond 1H-1H
coupling of 4.0 Hz and 3.3 Hz, respectively.[31]
KRED products were subjected directly without purification to in-NMR-tube Mosher ester
derivatization[32,33] and analyzed by 19F NMR spectroscopy to establish carbinol stereochemistry
(C-3) and relative amounts of each stereoisomer (Supporting Information). The percent anti or
19
syn diastereomer determined from separate integration of the F resonances of the KRED and
MPTA products agreed to within a standard deviation of ±1.1% (23 product analyses), indicative
of complete and nonselective MPTA derivatization. The anti/syn ratios in Tables 1 and 2 were
determined by integration of the MPTA ester 19F resonances.
Preliminary screening results with all 24 enzymes with 1a as substrate revealed that conver-
sions were typically >99%, with no substrate detected by NMR spectroscopy after product work-
up. Most KREDs show anti selectivity. Seven KREDs resulted in at least 90% anti configuration
in high yield, as shown in Table I. Five KREDs produced the 2S,3S enantiomer as the predominant
anti configuration (≥ 88%). Two KREDs produced the 2R,3R enantiomer as the predominant ste-
reoisomer (88%). Six enzymes show syn selectivity, although less impressively at 53-73% relative
to anti (Supporting Information).
3
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European Journal of Organic Chemistry
Table 1. Enzyme-Catalyzed Stereoselective Reduction of (+/-)-Ethyl-α-fluoroacetoacetate (1a).[a],[b]
Entry KRED
Anti/Syn[c] 2R,3R 2S,3S
2S,3R
nd
1
2R,3S
12
14
nd
nd
nd
2
1
2
119
130
88/12
85/15
97/3
nd[d]
1
88
84
41
nd
9
3
NADH-110
P1-A04
P1-H10
P2-C11
P2-G03
P2-H07
P3-B03
P3-G09
P3-H12
56
83
88
32
6
3
4
83/17
97/3
17
3
5
6
94/6
62
92
nd
95
92
93
4
7
98/2
1
1
8
88/12
95/5
88
nd
nd
1
12
nd
nd
nd
nd
5
9
10
11
92/8
8
94/6
6
[a] Conditions: Entries 1-3; β-ketoester (50 µmol), methanol (100 μL), KRED Mix N
(114.8 mg in 2.0 mL H2O), ketoreductase (2 mg), 37 ± 1 °C, 24 h: Entries 4-11; Same
conditions except 2-propanol (800 µL) added and KRED Mix P (58.2 mg).
[b] Conversion to product >99%, except for P3-B03 (95%). Conversion determined from
integration of 19F resonances of products upon work-up. In situ 19F NMR analysis of P1-
H10, P2-C11, and P3-G09 reactions revealed formation of 8-20% of acid from ester hy-
drolysis, which was not recovered upon work-up.
[c] Percentages of stereoisomers determined by integration of the 19F NMR resonances of
the corresponding MPTA esters.
[d] Not detected.
The reactions of 1a were also screened by in situ 19F NMR spectroscopy for some enzymes to
establish conversion products and rates. The reactions were carried out in both H2O and D2O. In
each case, the substrate was mixed with co-factor recycling mix supplied by Codexis dissolved in
H2O or D2O, methanol and/or isopropyl alcohol. Under these conditions, the ketoester 1a was in
equilibrium with the hydrate (~40%), as shown in Figure 1. Then enzyme was added, and the
reaction monitored at 37C by 19F NMR spectroscopy. In D2O, prior to adding enzyme, deuterium
exchange takes place rapidly at the α-carbon (C-2), with exchange up to 98% deuterium incorpo-
ration in less than one hour. For all reactants and products, the 19F chemical shifts of the α-deuter-
ated form appeared upfield of the α-protiated form, as shown in Figure 1, with a change in multi-
19
plicity consistent with two-bond F-D coupling. KRED DYRKR reactions in D2O may offer
convenient access to diastero- and enantiomerically pure [2-d]-2-fluoro-3-hydroxyacids and re-
lated derivatives. For example, stereochemically pure [2-d]-2-methyl-3-hydroxyacids have
proved valuable in mechanistic studies of epimerization and reduction by ketoreductases,[24] but a
lengthy six-step process was required for their synthesis.
4
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10.1002/ejoc.201900644
European Journal of Organic Chemistry
Figure 1. In situ monitoring of 1a with KRED P03-G09 by 19F NMR spectroscopy at 37C; (A) Over-
view of reaction; (B) KRED P3-09 in D2O; (C) P2-09 in H2O. Reaction times were <30 min.
Final product distributions were 2S,3S (92%) and 2R,3S (8%). Protiated forms of reactants
appear at ~0.6 ppm higher frequency (left) of the deuterated forms. Stereochemistry was
determined by 19F NMR Mosher ester analysis.
For some enzymes, the conversion rate of 1a was monitored in H2O by 19F NMR spectroscopy.
Using P1-H10, P2-C11, and P3-G09, conversion to 2a was complete in 1-2 hours. For P1-H10 and
5
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European Journal of Organic Chemistry
P3-G09, disappearance of 1a was linear up to ~80-90% at 17.2 mM substrate/mg KRED, but con-
version using P2-C11 was linear only to ~20-30% at this concentration, as shown in Figure 2.
Reactions were also monitored at higher concentrations of 1a for P2-C11 (68.8 and 138 mM) using
1 mg of enzyme (Figure 3). Initial rates of disappearance of 1a increased with substrate concen-
tration, as shown in Figure 3, indicating that substrate concentrations were below saturation levels
19
for P2-C11. The in situ F NMR technique offers potential of establishing Michaelis-Menten
constants for these reactions. KRED NADH-110 quantitatively converts 1a to anti 2a (anti:syn
97:3, Table I) in less than 10 min, which was too rapid to follow by in situ 19F NMR spectroscopy.
For the in situ reactions, we observed that 8 -20% of substrate is hydrolyzed to carboxylic acid
as identified by the19F doublet at 184 ppm (J=51.3 Hz).[34] Hydrolysis of the ester competes with
ketoreduction by the enzyme and will lower yield. Hydrolysis occurs both in the absence and
presence of enzyme; substrate 1a was quantitative hydrolyzed within 5 h in the absence of enzyme
using the Mix P system (Supporting Information). More rapid reduction (e.g. NADH-110) mini-
mizes hydrolysis product. The carboxylic acid is, however, not recovered upon workup, presum-
ably due to its high water solubility.
Figure 2. Conversion of 1a with KRED P2-C11 monitored by in situ 19F NMR spectroscopy at 37 ºC.
Conditions: 100 µmol in 400 μL isopropanol and 50 μL methanol, KRED Mix P (29.1 mg in
1.0 mL H2O) followed by 1.0 mg ketoreductase. Substrate concentration was 68.8 mM. ●
1a, 1a-H2O; ■ anti 2a; ▲ syn 2a; × acid of 1a.
6
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10.1002/ejoc.201900644
European Journal of Organic Chemistry
Figure 3. Disappearance of 1a as a function of substrate concentration using KRED P2-C11. Condi-
tions: See Figure 2. Concentrations: ■ 200 µmol, 138 mM; ● 100 µmol, 68.8 mM; ▲ 25
µmol, 17.2 mM. Initial slopes from 0-27 min: ■ -1.56; ● -1.18; ▲ -0.284 mM/min. Insert
plot is rate of disappearance of 1a (slope) against initial [1a].
Initial KRED reductions of substrates with larger R groups were carried out and analyzed in a
similar manner using KREDs NADH-110 and 130, since these KRED resulted in rapid conver-
19
sions with either high anti or syn selectivity. In situ F NMR studies also indicated that these
substrates were more resistant to ester hydrolysis than 1a. For reactions, conversions were >99%
by 19F NMR spectroscopy. For all substrates, the 19F NMR resonances of the anti diastereomers
1
were more downfield. Additionally, the anti three-bond H-19F coupling constants were smaller
(14-20 Hz) than the corresponding syn coupling constants (22-24 Hz), consistent with the litera-
ture. [28,31]
The results in Table 2 indicate that NADH-110 selects for anti configuration for all substrates,
with 2S,3S isomer strongly favored in high enantiomeric ratio, especially for the aromatic sub-
strates. KRED 130 showed syn selectivity for all substrates except 1a, favoring the formation of
the 2S,3R configuration in all cases. For the major diastereomer, high enantiomeric ratio was
achieved especially for the aromatic substrates (1f-1h). Substrate 1g was scaled (200 mg) using
the same reactions conditions to obtain the same result in 77% yield. Substrate 1g was also exam-
ined by in situ 19F NMR with both KRED NADH-110 and 130. Using NADH-110, 1g undergoes
rapid, quantitative reduction to >99% 2S,3S-2g in less than 10 min. Using KRED 130, 1g is
reduced quantitatively to 90% 2S,3R in less than 30 min. In contrast, certain KREDs resulted in
poor yield, for example, 1g and P1-H10 gave only about ~10% 2g (19F NMR yields). Our studies
also reveal that certain Codexis enzymes favor the syn 2R,3S isomer (P2-C02 and P1-B02, Table
2), demonstrating that 3 of 4 possible stereoisomers can be synthesized with high diastereomeric
and enantiomeric ratio.
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European Journal of Organic Chemistry
Table 2. Enzyme-Catalyzed Stereoselective Reduction of Racemic α-fluoro--ketoesters.[a],[b]
NADH-110
Anti/Syn [c]
2R,3R 2S,3S
2S,3R
3
2R,3S
nd
1a
1b
97/3
56
41
96
99/1
4
nd
1
0
nd
1c
86/14
78/22
93/7
86
3
11
1d
77
nd
22
1e
nd
nd
nd
nd
nd
2R,3R
1
93
nd
7
1f
99/1
>99
>99
>99
>99
2S,3S
84
nd
nd
1g
99/1
nd
nd
1h
99/1
nd
nd
1i
99/1
nd
nd
130
1a
Anti/Syn
85/15
17/83
13/87
14/86
8/92
2S,3R
1
2R,3S
14
1c
3
14
83
84
79
92
90
89
77
2S,3R
2
nd
1d
6
7
3
1e
5
9
7
1f
8
nd
nd
1g
10/90
11/89
23
10
11
77
2R,3R
4
nd
nd
1h
nd
nd
1i
16
nd
P2-C02
1d
Anti/Syn
17/83
12/88
Anti/Syn
9/91
2S,3S
13
2R,3S
81
1e
2
10
7
81
P1-B02
1d
2R,3R
5
2S,3S
4
2S,3R
5
2R,3S
86
1g
1/99
nd
nd
18
82
[a] Conditions: NADH-110 and 130; β-ketoester (50 µmol), methanol (100 μL), KRED
Mix N (114.8 mg in 2.0 mL H2O), ketoreductase (2 mg), 37 ± 1 °C, 24 h: P2-C02 and P1-
B02; Same conditions except 2-propanol (800 µL) added and KRED Mix P (58.2 mg).
[b] Conversion to product >99%. Conversion determined from integration of 19F reso-
nances of products upon work-up.
[c] Percentages of stereoisomers determined by integration of the 19F NMR resonances of
the corresponding MPTA esters.
8
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European Journal of Organic Chemistry
Conclusions
Of the substrates employed in this study, except for 1a, we conclude that KRED NADH-110 se-
lects for the 2S,3S configuration with high enantiomeric and diastereomeric ratio. For at least two
substrates, 1a and 1g, reduction is rapid and quantitative, with reaction complete in less than 10
min. KRED 130 can be used to select for the 2S,3R configuration, and KREDs P1-B02 and P2-
C02 can be used to select for the 2R,2S configuration. The presence of the 19F nucleus allows for
convenient in situ 19F NMR monitoring of the enzyme reaction, as well as convenient analysis of
Mosher ester products for stereochemical assignments and quantification. These α-fluoro-β-hy-
droxyesters may serve as useful intermediates in the synthesis of 2-fluorinated D-erythro sphingo-
sine analogues by α-amination reactions.[35] Such intermediates may also prove useful toward syn-
thesis of α-fluoro-α-amino acids.[36,37]
Experimental Section
Synthesis of racemic 2-fluoro-3-hydroxyesters: Racemic 2b-2i were synthesized by Refor-
matsky reactions using zinc dust, ethyl bromofluoroacetate and the corresponding aldehyde as de-
scribed in the literature.[28,29] See Supporting Information for experimental procedure, yield data
and NMR characterization, including 1H, 13C, and 19F NMR spectra.
Synthesis of 2-fluoro-3-ketoesters: Ketoesters 1b and 1c were synthesized by alkylation of ethyl-
2-fluoro-acetoacetate 1a according to the literature.[38] Ketoesters 1d-1i were synthesized by Dess-
Martin periodinane oxidation of racemic 2d-2i according to the literature.[30] See Supporting
Information for experimental procedure, yield data and NMR characterization, including 1H, 13C,
and 19F NMR spectra
KRED Screening Reactions: A procedure similar to previously described [16] was used. NADH
system (Entries 1-3, Table I): Into a solution of β-ketoester (50 µmol) in 100 μL methanol, was
added KRED Mix N (114.8 mg in 2.0 mL deionized H2O) and 2.0 mg ketoreductase. The mixture
was stirred at 37 ± 1 °C. After 24 h, the reaction was extracted by EtOAc (2 x 1 mL). The combined
organic extract was dried over anhydrous Na2SO4 and was subjected to 1H and 19F NMR spectros-
copy.
NADPH system (Entries 4-11, Table I): Into a solution of β-ketoester (50 µmol) in 800 μL iso-
propanol and 100 μL methanol, was added KRED Mix P (58.2 mg in 2.0 mL deionized H2O) and
2.0 mg ketoreductase. The mixture was stirred at 37 ± 1 °C. After 24 h, the reaction was extracted
by EtOAc (2 x 1 mL). The combined organic extract was dried over anhydrous Na2SO4 and was
subjected to 1H and 19F NMR spectroscopy.
1
1H and 19F NMR spectra for crude KRED products were obtained in CDCl3 at 300 MHz. H NMR
data for H-2 and 19F NMR data were both diagnostic for anti and syn configuration. 2a: Ethyl-2-
fluoro-3-hydroxybutanoate (2a).[14] 1H NMR (300 MHz, CDCl3) anti δ 4.83 (dd, J = 48.5, 4.0 Hz,
1H, H-2), syn δ 4.75 (dd, J = 48.3, 3.3 Hz, 1H, H-2); 19F NMR (282 MHz, CDCl3) anti δ -201.05
(dd, J = 48.5, 17.8 Hz), syn δ -206.6 (dd, J = 48.3, 22.1 Hz).
KRED Scale-up reaction (100x): Ethyl (2S,3S)-2-fluoro-3-(4-methoxyphenyl)propanoate
(2g):[39] The reaction was scaled by a factor of 100 compared to the screening reactions. Into a
solution of 1g (200 mg, 0.823 mmol) in 1.67 mL methanol, was added KRED Mix N (1.91 g in
9
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10.1002/ejoc.201900644
European Journal of Organic Chemistry
33.3 mL deionized H2O) and 33.3 mg ketoreductase. The mixture was stirred at 37 ± 1 °C. After
24 h, the reaction was extracted by EtOAc (2 x 10 mL). The combined organic extract was dried
over anhydrous Na2SO4 and was subjected to 1H, 13C, and 19F NMR spectroscopy; 156 mg, 77%,
1
>99% 2S,2S. H NMR (300 MHz, CDCl3);[39] anti δ 7.29 (m, 2H), 6.86 (m, 2H), 5.03 (dd, J =
16.4, 4.9 Hz, 1H, H-3), 5.01 (dd, J = 49.5, 4.9 Hz, 1H, H-2), 4.17 (q, J = 7.1 Hz, 2H), 1.19 (t, J =
7.1 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 167.9 (d, J = 23.1 Hz), 159.7, 129.8 (d, J = 2.6 Hz), ,
128.2 (d, J = 1.4 Hz), 113.8, 91.1 (J = 190.5 Hz), 73.2 (d, J = 21.9 Hz), 61.8, 55.2, 14.0; 19F NMR
(282 MHz, CDCl3) anti δ -198.4 (dd, J = 49.8, 14.9 Hz).
Mosher ester synthesis and analysis. An in-tube derivatization similar to that previously de-
scribed was used to synthesize MPTA (α-methoxy-α-trifluoromethylphenylacetic acid) esters of
the product alcohols.[33] The NMR tube contained approximately 50 μmol of KRED-reduced prod-
uct in 0.6 mL of CDCl3. Half of the NMR tube volume (0.3 mL) was mixed with a CDCl3 solution
(0.3 mL) containing either R-MPTA or S-MPTA (0.0176 g, 75 μmol, 3 eq), N,N'-dicyclohexylcar-
bodiimide (DCC) (0.0154 g, 75 μmol, 3 eq), and 4-dimethylaminopyridine (DMAP) (0.00915 g,
75 μmol, 3 eq). A small quantify of 3 Å molecular sieves was added (0.5 cm in height). The NMR
tube was then allowed to react overnight at 37C. A solid precipitate sometimes formed on top,
and usually redissolved upon mixing. The NMR tube contents was analyzed directly by 19F NMR
spectroscopy to assess stereoisomeric ratios. A minimum of 2000 transients was collected to min-
imize S/N ratio. A typical spectrum yielded the following chemical shift and coupling infor-
mation. 19F NMR spectrum of S-MPTA esters of 2d (R = heptenyl). 19F NMR (282 MHz, CDCl3)
2S,3S, δ -199.0 (dd, J = 48.6, 21.2 Hz); 2R,3R, -201.1 (dd, J = 48.8, 21.9 Hz); 2R,3S -201.4 (dd, J
= 47.4, 22.0 Hz); 2S,3R, -203.3 (dd, J = 47.2, 23.3 Hz).
Monitoring reaction by in situ 19F NMR Spectroscopy. Substrate 1a (25, 50, 100 or 200 µmol)
was mixed with 400 μL isopropanol and 50 μL methanol, KRED Mix P (29.1 mg in 1.0 mL H2O)
followed by 1.0 mg KRED P1-H10, P2-C11, or P3-GO9. A capillary insert containing D2O was
placed in the NMR tube for locking and shimming purposes. A sequence of spectra was obtained
in an arrayed experiment using the available software, with 64 transients per spectrum, collected
every 10 minutes for 120 minutes. Spectra were processed, phased and baseline corrected prior to
integration. Absolute areas of each integral were summed and used to calculate relative amounts
of reactant and products.
Keywords: enantioselectivity, enzymes, ketoreductases, α-fluoro-β-hydroxyesters, α-fluoro-β-
ketoesters
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European Journal of Organic Chemistry
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