A Hammett Study of Clostridium acetobutylicum Alcohol Dehydrogenase (CaADH): An Enzyme with Remarkable Substrate Promiscuity and Utility for Organic Synthesis

Article abstract of DOI:10.1055/s-0039-1691576

Described is a physical organic study of the reduction of three sets of carbonyl compounds by the NADPH-dependent enzyme Clostridium acetobutylicum alcohol dehydrogenase (CaADH). Previous studies in our group have shown this enzyme to display broad substrate promiscuity, yet remarkable stereochemical fidelity, in the reduction of carbonyl compounds, including α-, β- and γ-keto esters (d -stereochemistry), as well as α,α-difluorinated-β-keto phosphonate esters (l -stereochemistry). To better mechanistically characterize this promising dehydrogenase enzyme, we report here the results of a Hammett linear free-energy relationship (LFER) study across three distinct classes of carbonyl substrates; namely aryl aldehydes, aryl β-keto esters and aryl trifluoromethyl ketones. Rates are measured by monitoring the decrease in NADPH fluorescence at 460 nm with time across a range of substrate concentrations for each member of each carbonyl compound class. The resulting v ₀ versus [S] data are subjected to least-squares hyperbolic fitting to the Michaelis-Menton equation. Hammett plots of log(V _max) versus σ _X yield the following Hammett parameters: (i) for p -substituted aldehydes, ρ = 0.99 ± 0.10, ρ = 0.40 ± 0.09; two domains observed, (ii) for p -substituted β-keto esters ρ = 1.02 ± 0.31, and (iii) for p -substituted aryl trifluoromethyl ketones ρ = -0.97 ± 0.12. The positive sign of ρ indicated for the first two compound classes suggests that the hydride transfer from the nicotinamide cofactor is at least partially rate-limiting, whereas the negative sign of ρ for the aryl trifluoromethyl ketone class suggests that dehydration of the ketone hydrate may be rate-limiting for this compound class. Consistent with this notion, examination of the ¹³ C NMR spectra for the set of p -substituted aryl trifluo romethyl ketones in 2percent aqueous DMSO reveals significant formation of the hydrate (gem -diol) for this compound family, with compounds bearing the more electron-withdrawing groups showing greater degrees of hydration. This work also presents the first examples of the CaADH-mediated reduction of aryl trifluoromethyl ketones, and chiral HPLC analysis indicates that the parent compound α,α,α-trifluoroacetophenone is enzymatically reduced in 99percent ee and 95percent yield, providing the (S)-stereoisomer, suggesting yet another compound class for which this enzyme displays high enantioselectivity.

Full text of DOI:10.1055/s-0039-1691576

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© Georg Thieme Verlag Stuttgart · New York
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G. P. Kudalkar et al.
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A Hammett Study of Clostridium acetobutylicum Alcohol Dehydro-
genase (CaADH): An Enzyme with Remarkable Substrate Promis-
cuity and Utility for Organic Synthesis
Linear free-energy relationships with Clostridium acetobutylicum ADH
Gaurav P. Kudalkar
Aldehyde Carbonyls
OH
Virendra K. Tiwari
O
Joshua D. Lee
H
X
X
NADP+ + H+
David B. Berkowitz*
NADPH
b-Keto Ester Carbonyls
OH
O
Department of Chemistry, University of Nebraska, Lincoln, NE
O
O
68588, USA
dberkowitz1@unl.edu
OEt
OEt
X
X
Published as part of the Cluster Biocatalysis
Trifluoromethyl Ketones
OH
CF3
O
CF3
X
X
Received: 11.12.2019
Accepted after revision: 08.01.2020
Published online: 16.01.2019
Biocatalysis is emerging as a key element in the synthe-
sis of both chiral building blocks and advanced synthetic in-
termediates, particularly in process chemistry laboratories.1
This includes the use of individual enzymes, enzyme cas-
cades2 and in indeed, most recently, entire retrosynthetic
pathways3 designed around evolved enzymes,4 not to men-
tion whole cell processes.5 Such biocatalytic approaches
play a pivotal role in the catalysis of a large number of in-
dustrially relevant reactions such as the production of bio-
fuels by fermentation of glucose, the synthesis of polymer
monomers by engineered enzymatic pathways and the spe-
cialized application of enzymes for key steps in pharmaceu-
tical process chemistry. Landmark achievements in the lat-
ter area include (i) the remarkable engineering of a trans-
aminase to efficiently set a key stereocenter in the
synthesis of sitagliptin by the collaborative Codexis/Merck
team,6 and (ii) the spectacular biocatalytic total synthesis [9
enzymes (5 evolved)/2 pots] of islatravir by a team from the
same two companies.7
Transaminases (pyridoxal phosphate) utilized for the
sitagliptin case, and the dehydrogenases/ketoreductases
(nicotinamide adenine dinucleotide(s)) discussed here, re-
quire active molecular cofactors stoichiometric with en-
zyme for catalytic turnover. Thus cofactor regeneration be-
comes a key component of such applications of enzymes in
synthesis or process chemistry, with isopropylamine serv-
ing as a terminal reductant in the engineered transaminase
case noted and, for example, isopropyl alcohol, formate,
glucose, phosphite or ethanol serving as the terminal re-
ductant for alcohol dehydrogenases, depending on the sys-
tem, recycling enzyme(s) chosen.8 With dehydrogenases,
there have also been creative examples of either formal
substrate disproportionation9 approaches or ‘self-sufficient
hydride transfer’10 processes that allow for internal recy-
cling of the cofactor. The focus of this article will be upon
DOI: 10.1055/s-0039-1691576; Art ID: st-2010-r0669-c
Abstract Described is a physical organic study of the reduction of
three sets of carbonyl compounds by the NADPH-dependent enzyme
Clostridium acetobutylicum alcohol dehydrogenase (CaADH). Previous
studies in our group have shown this enzyme to display broad substrate
promiscuity, yet remarkable stereochemical fidelity, in the reduction of
carbonyl compounds, including -, - and -keto esters (D-stereochem-
istry), as well as ,-difluorinated--keto phosphonate esters (L-stereo-
chemistry). To better mechanistically characterize this promising dehy-
drogenase enzyme, we report here the results of a Hammett linear
free-energy relationship (LFER) study across three distinct classes of
carbonyl substrates; namely aryl aldehydes, aryl -keto esters and aryl
trifluoromethyl ketones. Rates are measured by monitoring the de-
crease in NADPH fluorescence at 460 nm with time across a range of
substrate concentrations for each member of each carbonyl compound
class. The resulting v0 versus [S] data are subjected to least-squares hy-
perbolic fitting to the Michaelis–Menton equation. Hammett plots of
log(Vmax) versus X yield the following Hammett parameters: (i) for p-
substituted aldehydes,  = 0.99 ± 0.10,  = 0.40 ± 0.09; two domains
observed, (ii) for p-substituted -keto esters  = 1.02 ± 0.31, and (iii) for
p-substituted aryl trifluoromethyl ketones  = –0.97 ± 0.12. The posi-
tive sign of  indicated for the first two compound classes suggests that
the hydride transfer from the nicotinamide cofactor is at least partially
rate-limiting, whereas the negative sign of  for the aryl trifluoromethyl
ketone class suggests that dehydration of the ketone hydrate may be
rate-limiting for this compound class. Consistent with this notion, ex-
amination of the 13C NMR spectra for the set of p-substituted aryl trifluo-
romethyl ketones in 2% aqueous DMSO reveals significant formation
of the hydrate (gem-diol) for this compound family, with compounds
bearing the more electron-withdrawing groups showing greater de-
grees of hydration. This work also presents the first examples of the
CaADH-mediated reduction of aryl trifluoromethyl ketones, and chiral
HPLC analysis indicates that the parent compound ,,-trifluoroace-
tophenone is enzymatically reduced in 99% ee and 95% yield, providing
the (S)-stereoisomer, suggesting yet another compound class for which
this enzyme displays high enantioselectivity.
Key words linear free-energy relationships, Hammett study, Clostridi-
um acetobutylicum ADH, stereochemical fidelity, enzyme promiscuity,
trifluoromethyl ketones
© 2020. Thieme. All rights reserved. Synlett 2020, 31, A–K
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G. P. Kudalkar et al.
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alcohol dehydrogenase (ADH) enzymology, specifically the
physical organic characterization of a ‘privileged’ ADH en-
zyme that we have uncovered in our laboratory.
The Berkowitz group has a longstanding interest in the
creative use of enzymes in synthesis, including the use of li-
pases in unnatural amino acid synthesis17 and in lignan nat-
ural product total synthesis.18 Alcohol dehydrogenase and
alcohol oxidase enzymes have become the key workhorse
enzymes in our efforts to develop enzyme-based screens
for the discovery of new organic/organometallic reaction
manifolds,19 new catalytic combinations20 and new types of
chiral ligands.21 In this effort, these ‘reporting enzymes’
serve as analytical tools to report back to the experimental-
ist, in real time, on the relative rates, and where possible,
enantioselectivity of the reactions being screened. There-
fore, we label this approach in situ enzymatic screening (IS-
ES).22 Important for this effort is the identification and
characterization of new reporting enzymes and this moti-
vation was a big driver for the exploration of the CaADH en-
zyme that is the subject of the studies described herein.
CaADH: Substrate Promiscuity/Stereochemical Fidelity
Our group expressed, purified and characterized the
CaADH enzyme in 2011; the Clostridium acetobutylicum
species to which it is native is unusual in that it naturally
In hybrid biocatalytic ventures, dehydrogenases have
become powerful tools to set the absolute and relative ste-
reochemistry for value-added targets of use in medicinal
chemistry or chemical biology.11 Indeed, in the Merck pro-
cess group ‘isolated (ADH) enzymes have clearly supplanted
whole cell bioreductions and, in most instances, chemocat-
alytic ketone reductions.’12 Our group has utilized a SsADH-
10, a dehydrogenase from an archaeal hyperthermophile, to
access a wide range of (S)-profen scaffolds13 from racemic
precursors via dynamic reductive kinetic resolution
(DYRKR)14 at elevated temperature. The Brenna group has
used a set of ADH enzymes to dial in each of the four possi-
ble stereoisomers in a 4-methyl-3-heptanol insect phero-
mone system.15 Others have developed one-pot enzyme
cascade reactions in which ADH enzymes play key roles to
access stereochemically homogeneous, functionalized
building blocks for synthesis and chemical biology.2d,16
CaADH Applications in Asymmetric Synthesis
HN
O
H
N
O
OH
a-Keto Ester
OH
H2N
N
H
N
N
H
L
OEt
O
Ph
NADP+ + H+
O
O
NADPH
N
CO2H
OEt
O
H
O
Ph
Platelet Fibrinogen Receptor antagonist (antithrombotic)
(90%, 90% ee)
O
CF3
GDH
Fluoxetine
b-Keto Ester
OH
O
D-gluconolactone
D-glucose
O
O
O
Ph
OEt
N
H
L
Ph
OEt
(93%, 99% ee)
Me
g-Keto Ester
OH
O
O
OEt
O
Ph
OEt
O
O
O
HN
Cl
Ph
O
O
O
(84%, 99% ee)
N
H
O
OMe
Me
β-Keto-a,a-difluorophosphonates
Cryptophycin A
OAr
OAr
OH
O
P
O
O
P
Thiono–Claisen
Rearrangement
S
O
O
P
OR'
OR'
OR'
OR'
S
O
O
δ
α
R
OR'
OR'
β
*
F
R
*
δ
β
R
F
α P
F
γ
OR'
OR'
F
*
F
F
R
γ
(86–91%, 93–99% ee)
F
F
SH
δ
O
P
SH
O
P
β
β
γ
δ
γ
O
α
α
Asp-Ile
O
O
O
O
*
O
*
versus
F
H3N
F
O
O
Me
platform for new potential
Zn-aminopeptidase (APA) inhibitors
known Zn-APA inhibitor (Roques)
Figure 1 Use of CaADH for the enantioselective reduction of a broad range of carbonyl compounds to provide access to building blocks for the synthe-
sis of important target molecules in medicinal chemistry and chemical biology
© 2020. Thieme. All rights reserved. Synlett 2020, 31, A–K

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undergoes a solventogenic phase in its life cycle in which
the microorganism produces acetone, butanol and ethanol
(ABE phase).23 For this reason, Clostridial enzymes and met-
abolic pathways have been of interest to bioengineers seek-
ing to build viable biobutanol refinery technology.24 From
our point of view, this seemed a promising microbial source
to pan for organic-solvent-compatible ADHs. Indeed, the
CaADH enzyme is well expressed heterologously in E. coli
and catalyzes the enantioselective reduction of -, - and -
keto esters into the corresponding alcohols with good yield
and excellent enantioselectivity, giving the D-enantiomers
as key value-added building blocks for pharmaceutical and
chemical biology applications (Figure 1).25
In more recent work, CaADH was found to enantioselec-
tively reduce another distinct class of substrates, namely -
keto-,-difluoroalkyl phosphonates of real significance
because ,-difluorinated phosphonates are phosphatase-
inert phosphate surrogates of considerable value in chemi-
cal biology and medicinal chemistry.26 This enzymatic reac-
tion proceeds with the opposite sense of facial selectivity,
as compared to -keto-carboxylate ester reduction, giving
rise to the corresponding L--hydroxy-,-difluorophos-
phonates. Molecular modeling suggests that CaADH exhib-
its considerable active-site plasticity to accommodate these
structurally distinct substrate classes.27 Conversion of the
enzymatic products into -pentafluorophenyl allylic thion-
ocarbonates leads to an exceptionally facial [3,3]-sigmato-
tropic rearrangement in which the CaADH-imprinted -hy-
droxy stereocenter is parlayed into a -thio stereocenter in
the product. This sequence provides for a hybrid biocatalyt-
ic/sigmatropic rearrangement route that is valuable for the
synthesis of potential new Zn-aminopeptidase A inhibitors,
as is illustrated in Figure 1. Given the utility of CaADH in
asymmetric synthesis, and its ability to reduce varied class-
es of carbonyl compounds, we set out to examine the elec-
tronics of CaADH enzymology more quantitatively, using
physical organic chemical tools.
Experimentally determined LFERs have been a staple in
mechanistic organic chemistry for years, and have found re-
newed application in reaction and catalyst optimization
with, for example, the use of sterimol parameters.30 The use
of such tools to study enzyme mechanism was first pro-
posed and explored several decades ago,31 but is not with-
out challenges. Indeed, Sinnott, Greig and others have not-
ed that the quantitative analysis of the free-energy relation-
ships for enzyme-catalyzed reactions presents some
inherent challenges.32 There are several criteria that en-
zyme active site environments may not strictly obey that
could lead to deviations from LFER behavior in a Hammett
study. On the one hand, aromatic substrates must be well
tolerated for such an enzymatic LFER study to be possible.
Beyond this, if steric and/or electronic variations in the aro-
matic substitutents lead to significant diffferences in bind-
ing orientation or affinity (perhaps leading to a change in
the rate-limiting step) or simply lead to a change in mecha-
nism due to inherent chemical differences in reactivity
with the enzyme, one expects to see deviations from linear-
ity.
Illuminating Examples of Hammett LFERs in Enzy-
mology
Despite these challenges, Hammett-type LFER tools
have been utilized to help elucidate active-site reaction
pathways in enzymology.33 For example, in a classic study,
Kanerva and Klibanov examined how the workhorse hydro-
lase Carlsberg subtilisin performed in organic solvents.33n
They were able to measure the enzymatic acylation rates of
a series of substituted phenyl acetates in THF, acetone, ace-
tonitrile, tert-amyl alcohol and butyl ether.
Because the enzymatic acylation step was being stud-
ied, the authors were able to substitute hexanol for water as
the nucleophile in these organic solvents. These results
demonstrated remarkably similar values for the Hammett
reaction constant,  = 0.83–0.93 across this panel of sol-
vents, similar to the value of 0.87 measured in water,33n sug-
gesting that the acylation transition state for subtilisin does
not significantly change upon immersion in organic sol-
vents. These mechanistic fingerprinting results obtained by
Hammett LFERs agree nicely with subsequent crystallo-
graphic studies showing that the enzyme retains key wa-
ters of hydration, even upon immersion in organic solvents,
presumably preserving key active site structural, dipolar
and H-bonding elements.34
The Hammett LFER tool has also been used to study my-
eloperoxidase-mediated sulfoxidation of aryl sulfides [por-
phyrin-Fe(IV)=O active site species], suggesting the inter-
mediacy of a sulfenium radical cation intermediate.33j
Davidson and co-workers used Hammett studies to glean
evidence for a carbanionic intermediate in the methyl-
amine-dehydrogenase-mediated oxidation [tryptophan
tryptophyl-o-quinone (TTQ) cofactor] of a series of p-sub-
stituted benzylamines.33m Recently, the Guo group per-
formed a Hammett study of a promiscuous C–C bond-form-
Hammett Analysis: A Physical Organic Tool in Enzy-
mology
Experimental probes for linear free-energy relation-
ships (LFERs) are established tools of physical organic
chemistry based upon transition-state theory that can, in
principle, also provide insight into enzyme catalysis. The
Hammett LFER tool, in particular, examines how the rate or
equilibrium constant of a reaction under study responds to
substituent effects, provided that the reaction supports an
aromatic substrate or educt platform.28 Of course, there is
the further assumption that, across a broad range of aro-
matic substituents, the series of compounds under study all
react via the same rate-determining elementary steps. If
this holds, then one expects an LFER between the logarithm
of the rate constants for that series of reactions, plotted as a
function of substituent and the logarithm of the associated
equilibrium constants for benzoic acid dissociation, for the
same substituents (i.e., the X values).29
© 2020. Thieme. All rights reserved. Synlett 2020, 31, A–K

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G. P. Kudalkar et al.
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ing reaction (Henry reaction) catalyzed by an acyl peptide
releasing enzyme from the archaeal thermophile Sulfolobus
tokodaii (ST0779).33b The Guo team studied the condensa-
tion of nitromethane with a series of substituted benzalde-
hydes and obtained a  value of 1.33, indicative of nitronate
anion condensation with the aldehyde in the rate-deter-
mining step. For o-, m- and p-NO2-substituted aldehydes, as
well as the p-CN-benzaldehyde coupling partners, high (S)-
enantioselectivity and good catalytic efficiency were seen.
In another case, Bugg and co-workers obtained a Hammett
 value of –0.71 for the retro-Claisenase-type enzyme,
BpHD.33h That electron-rich substituents so significantly fa-
vor this reaction suggests that the C–C bond cleavage pro-
ceeds out of a gem-diol intermediate leading to an oxocar-
benium ion species in the rate-determining step.
be an excellent candidate to explore the possibility of mea-
suring  values for more than one carbonyl class in the
same active site (Scheme 1).
LFERs in the CaADH Active Site?
Aldehyde Carbonyl Class
O
OH
NADP+ + H+
NADPH
H
X
X
β-Keto Ester Carbonyl Class
OH
O
O
O
OEt
OEt
X
X
Trifluoromethyl Ketone Carbonyl Class
OH
CF3
Hammett LFERs with Other NAD(P)H-Dependent De-
hydrogenases
O
CF3
There have been several Hammett LFER studies with
ADH enzymes as summarized in Table 1. To our knowledge,
these have all examined ADH behavior across a panel of
substituted benzaldehyde substrates in the active sites of
Oryctolagus cuniculus glyceraldehyde DH,31c Solanum tu-
berosum aromatic alcohol dehydrogenase,35 Candida tenuis
xylose reductase36 and Saccharomyces cerevisiae ethanol
dehydrogenase.37 All the observed  values were in the 1.2–
1.7 range, apart for the latter enzyme in which a  value of
>2 was observed by Klinman.37 These results are consistent
with hydride transfer from the nicotinamide cofactor to the
benzaldehyde carbonyl center being at least partially rate-
limiting. The elevated  value in the case of the yeast en-
zyme has been attributed to the fact that this ADH is a zinc-
metalloenzyme, with the expectation that Zn2+-mediated
polarization of the substrate carbonyl leads to a greater par-
tial positive charge at the benzylic center in the benzalde-
hyde educt and therefore a greater change in charge density
along the hydride transfer reaction coordinate.
X
X
Scheme 1 Hammett study of CaADH-catalyzed carbonyl reduction
LFERs as a function of compound class
RESULTS: CaADH-Mediated Reduction of p-Substitut-
ed Benzaldehydes
In line with this objective, we experimentally measured
the rate of NADPH-mediated reduction of eleven p-substi-
tuted benzaldehydes as substrates for the CaADH enzyme.
These assays were run in a plate-reading fluorimeter with
enzyme expressed and purified, in house, as described ear-
lier.25,27 Each well was irradiated at 340 nm (NADPH max),
with fluorescence emission being monitored at 460 nm.
Each aldehyde was monitored for reduction rate across a
range of a half-dozen concentrations, with each initial ve-
locity measurement being run at least in duplicate. The vo
versus [S] data were worked up by performing a least-
squares hyperbolic fit to the Michaelis–Menton equation.
The fastest rates were observed for the reduction of p-nitro-
benzaldehyde and the slowest for p-methoxybenzaldehyde.
A Hammett plot of log kcat versus X showed pseudo-bipha-
sic behavior as can be seen in Figure 2a. That is, the fastest
substrates appear to bifurcate, with those containing the
most polarizable (‘soft’) electron-withdrawing substituents
Given the aforementioned utility of the C. acetobutyli-
cum alcohol dehydrogenase (CaADH) in asymmetric syn-
thesis, we set out to examine the electronic dependence of
its carbonyl reduction chemistry using the Hammett LFER
tool. Moreover, given the substrate promiscuity that we
have observed with CaADH (Figure 1), this ADH seemed to
Table 1 Prior Hammett Studies of Alcohol Dehydrogenase Enzymes: All Performed with Substituted Benzaldehydes
Entry
1
Enzyme
/+

Vmax/(Vmax/Km)
Vmax
Ref.
37
ethanol (alcohol) dehydrogenase
(Saccharomyces cerevisiae)
+
2.17
2
3
4
glyceraldehyde 3-phosphate DH
(Oryctolagus cuniculus)



1.27, 1.24
0.42, 1.38
1.4–1.7
Vmax, Vmax/Km
Vmax (m-), Vmax (p-)
both
31c
aromatic alcohol dehydrogenase
(Solanum tuberosum)
35
36
xylose reductase
(Candida tenuis)
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G. P. Kudalkar et al.
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(p-I, p-Br, p-SCF3) tracking along a nice linear free-energy
relationship with those bearing electron-donating groups.
These substrates provide for an LFER corresponding to
 = 0.99 ± 0.13 (Figure 2a, red trace). However, another set
of four fast substrates bearing less polarizable (‘hard’) elec-
tron-withdrawing substituents with significant dipoles (p-
F, p-Cl, p-OCF3, p-CF3) project out a second LFER with a sig-
nificantly less positive slope, but a very good correlation
with  = 0.40 ± 0.06 (Figure 2a, blue trace). The best fit ap-
pears to occur with bifurcation of the data at the OMe sub-
stituent.
that these two sets of substituents interact differently in
the enzyme active site, with the more dipolar substituents
engaging in favorable dipole–dipole or charge–dipole inter-
actions with active-site pocket residue(s) that lead to a
slower dissociation rate for this substrate subclass. In such a
case, the 0.99  value associated with the faster substrate
subclass would reflect a kinetic profile in which NADPH-
mediated hydride delivery is largely, if not fully, rate-limit-
ing. The 0.40  value associated with the subclass of sub-
strates bearing dipolar EWGs would then be indicative of
partially rate-limiting hydride transfer that is punctuated
by partially rate-limiting product dissociation. We are cur-
rently working to obtain an X-ray crystallographic structure
of the title enzyme; such structural biological data would
help us to evaluate these postulates, in the longer term. In
the meantime, we have used homology model building and
molecular docking to examine possible substrate binding
modes in light of the discussion above (vide infra).
We are aware of only one other report of a biphasic
Hammett plot for alcohol dehydrogenase based chemistry.
As in another example of ADH LFER, Towers and co-workers
examined the Solanum tuberosum ADH-mediated reduction
of m- and p-substituted benzaldehydes.35 The Hammett
plot of log(Vmax) versus X reported in their work appears,
in the first analysis, to show concave upward biphasic ki-
netic behavior with two  values of ~0.4 for the more elec-
tron-donating groups and of ~1.4 for the more electron-
withdrawing groups. This upward concavity would suggest
a change in mechanism.32a However, as the authors note,
the lower value of  generally correlates with the kinetic
behavior of the m-substituted substrates, whereas the larg-
er  generally correlates with the p-substituted substrates.
The authors argue for a difference in binding mode for the
m- versus p-substituted substrates, perhaps suggesting the
p-substitution allows for better positioning of the aldehyde
carbonyl with respect to typical Bronsted acid proton-do-
nating residues in the active site. This could give rise to a
larger + in the ground state and the carbonyl carbon and a
larger accumulation in negative charge with NADH-mediat-
ed hydride transfer for the p-configured substrates.
A classic case of a concave-upward biphasic Hammett
plot that more clearly translates into a likely change in
mechanism as a function of substituent can be found in the
pioneering work on lysozyme by Tsai and co-workers.38
These workers studied the enzymatic hydrolysis of a set of
p-substituted aryl -di-N-acetylchitobiosides. The biphasic
Hammett plot obtained gave a reaction constant  value of
–2.96 for electron-donating substituents (X ≤ 0) and a 
value of +0.55 for electron-withdrawing substituents (X ≥
0). These data and supporting evidence suggest that for
electron-donating substituents an oxocarbenium ion mech-
anism is followed, whereas for electron-withdrawing sub-
stituents a direct displacement ensues, whereby an active-
site carboxylate residue directly displaces an anomeric p-
substituted phenolate leaving group in the rate-determin-
Figure 2 Correlations between the logarithm of turnover number of
CaADH-catalyzed reduction of (a) aldehydes, and (b) -keto esters
This Hammett plot is essentially a biphasic, concave-
downward LFER plot and this pattern of kinetic behavior
normally indicates a change in the rate-limiting step.32a
That is to say, in moving from substrates with more polariz-
able EWG substituents to those with harder, less polariz-
able substituents, there may be a change in the rate-limit-
ing kinetic profile. One possibility, for example, would be
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G. P. Kudalkar et al.
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ing step. Several other instances of biphasic kinetic behav-
ior for enzymatic Hammett LFER plots have been reported
through the years.32a,39
CaADH-Mediated Reduction of p-Substituted -Keto
Esters
kinetics by monitoring the decrease in absorbance at 340
nm with NADPH consumption ( = 6220 M–1 cm–1). As can
be seen from Figure 3a, we observed a negative sign for the
reaction constant  = –0.97 ± 0.12 for this class of sub-
strates. This is the first enzymatic Hammett LFER study
with aryl trifluoromethyl ketones of which we aware.
Next, we examined electronic substituent effects on the
CaADH-mediated reduction of a second carbonyl class,
namely a set of p-substituted aryl -keto esters. The
Hammett plot of log kcat versus X shows a linear correlation
with  = 1.02 ± 0.31 (Figure 2b). The positive value of 
shows that in this substrate class electron-withdrawing
substituents also speed the enzymatic reduction catalyzed
by CaADH, and this result also suggests an increase in nega-
tive charge at the carbonyl center in the rate-limiting tran-
sition state, implying that for this less electronegative car-
bonyl class, hydride delivery from NADPH is largely rate-de-
termining. This is the first example of which we are aware
of the examination of this substrate class in an ADH active
site (see Table 1 for reported  values seen for aldehyde sub-
strates across a range of ADH active sites).
CaADH-Mediated Reduction of p-Substituted Aryl
Trifluoromethyl Ketones
Given the notable substrate promiscuity exhibited by
CaADH, we next set out to examine yet another substrate
class in this active site, namely aryl trifluoromethyl ketones
bearing a range of p-substituents. Pleasingly, we observed
that CaADH also readily accepts this new substrate class
and preliminary indications are that these reductions also
proceed with high facial selectivity. Namely, for the parent
compound, phenyl trifluoromethyl ketone (X = H), chiral
HPLC analysis indicates that the CaADH-mediated reduc-
tion proceeds with very high enantioselectivity for the
product -trifluoromethyl benzyl alcohol.
This result is significant as such organofluorine com-
pounds have value for pharmaceutical, agrochemical, and
materials science applications.40 Earlier, several groups had
reported such enzymatic reductions of aryl trifluoromethyl
ketones under yeast whole cell conditions41 and with puri-
fied ADH enzymes,11b–d,42 giving the enantioenriched (R)- or
(S)--trifluoromethyl benzyl alcohols, depending on the
choice of enzyme. The CaADH-catalyzed reduction of phe-
nyl trifluoromethyl ketone observed here is among the
most efficient (95% yield) and the most enantioselective yet
seen (99% ee). The absolute stereochemistry of the product
-trifluoromethyl benzyl alcohol was established as (S)
based upon retention time on chiral HPLC for the reported
compound (see the Supporting Information).
These promising results encouraged us to synthesize a
complete set of aryl trifluoromethyl ketones and to perform
a Hammett study across this new CaADH carbonyl substrate
class. In contrast to the kinetic studies with the aryl alde-
hyde and -keto ester substrate classes for which it was
found advantageous to measure NADPH-fluorescence ow-
ing to significant substrate absorbance, for the aryl trifluo-
romethyl ketone substrate class, we were able to follow the
Figure 3 (a) Relation between the logarithm of turnover number of
CaADH-catalyzed reduction of ,,-trifluoroacetophenone vs X (b)
Data replotted from Stewart and Van Dyke45 for the log(dehydration
+
equilibrium constant) for trifluoroacetophenone hydrates vs X
The negative  value seen in these studies stands in con-
trast to the  value of 3.14 observed by Teo and Stewart for
sodium borohydride mediated ketone reduction of aryl tri-
fluoromethyl ketones in an organic medium.43a Another
non-enzymatic Hammett LFER study examines the electro-
chemical reduction of aryl trifluoromethyl ketones in aceto-
nitrile with an organic-soluble electrolyte (NBu4ClO4), for
which Yang and co-workers43b reported  = 0.52–0.61. Per-
haps more germane to our studies, Ohno and co-workers
have examined model Hantzsch ester reductions of aryl tri-
fluoromethyl ketones in anhydrous acetonitrile and report
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G. P. Kudalkar et al.
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 values ranging from 1.3–3, depending upon whether a di-
valent cation is present.44 The net positive  values ob-
served in these model studies are consistent with either
rate-limiting single-electron transfer (in the electrochemi-
cal reduction, in particular) or hydride transfer (Hantzsch
ester studies) to the electrophilic carbonyl center in the aryl
trifluoromethyl ketone substrates.
However, our experimental data for the CaADH-mediat-
ed reduction chemistry suggest that a net positive charge
builds up in the rate-limiting transition state and in turn,
this suggested to us that perhaps the true substrates are ac-
tually the trifluoromethyl ketone hydrates, under the con-
ditions of these enzymatic experiments. Indeed, the
Van Dyke group has systematically studied the effect of the
substituents on the hydration/dehydration equilibrium
constant for aryl trifluoromethyl ketones. In Figure 3b, we
have replotted their equilibrium Hammett data; on the or-
dinate is the logarithm of the equilibrium constant for de-
hydration of the ketone hydrate, log(Kd), plotted versus +,
on the abscissa. This gives a nice equilibrium linear free-en-
ergy relationship with  = –1.62 indicating that electron-
donating groups on the aryl trifluoromethyl ketones favor
dehydration.45
NMR spectra acquired. The results are displayed in Figure 4,
in stack plot form, from most electron-withdrawing to most
electron-donating p-substituent, front to back. The results
clearly show that in our hands, as well, the carbonyl hydra-
tion equilibrium for this class of aryl trifluoromethyl ke-
tones shows a strong dependence upon the p-substituent,
with electron-withdrawing substituents favoring hydra-
tion. Thus, the p-Cl-,,-trifluoroacetophenone substrate
is almost completely hydrated under the conditions of this
NMR experiment (2 vol% H2O) whereas the p-MeO-substi-
tuted counterpart exists largely as the free ketone. Accord-
ingly, it is expected that under the conditions of our en-
zyme kinetic studies [10% (v/v) DMSO in aqueous buffer],
our aryl trifluoromethyl ketone substrates exist largely in
hydrated form.
All that said, it is expected that NADPH reduction in the
CaADH active site will require dehydration of the geminal-
diol (ketone hydrate) such that hydride transfer from the
bound nicotinamide cofactor can ensue. It is therefore pos-
sible that dehydration of the gem-diol or carbonyl hydrate is
largely rate-determining for this class of substrates. This
would be consistent with both the negative -value seen in
the Hammett plot (Figure 3a) and the propensity for these
aryl trifluoromethyl ketones to exist in hydrated form (Fig-
ure 4). So, the mechanism for enzymatic reduction may
first involve dehydration of the gem-diol in the CaADH ac-
tive site, followed by NAPDH-mediated hydride transfer.
To examine this further, we conducted a 13C NMR exper-
iment to examine the tendency of our own set of aryl trifluo-
romethyl ketones to hydrate in solution. The substrates
were each dissolved in 2% (v/v) aqueous DMSO-d6 and the
Figure 4 Hydration of aryl trifluoromethyl ketones as a function of the p-substituent in 2% aqueous DMSO-d6 as monitored by 13C NMR (175 MHz). The
results are displayed from electron-withdrawing group to electron-donating group (front to back). One sees the quartet diagnostic of the hydrate at
~95 ppm fade out and the corresponding quartet for the free ketone at ~180–185 ppm fade in, front to back
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Such a mechanism is reminiscent of the proposed mecha-
nism for lysozyme-catalyzed hydrolysis of p-substituted
aryl -di-N-acetylchitobiosides discusssed above.38
generated structure as judged by inspection of substrate
positioning, particularly with regard to hydride transfer
from the NADPH cofactor to the substrate carbonyl.
There, the negative value of  = –2.96 for electron-do-
nating substituents was interpreted as evidence that en-
zyme-mediated acetal cleavage is rate-limiting, presumably
yielding an oxocarbenium-ion-like intermediate, prior to
addition of water. Here, a very similar acetal-like substrate
would undergo partially rate-limiting cleavage to an oxo-
carbenium-like species (protonated carbonyl) in the active
site, followed by highly favorable hydride transfer. Below we
discuss this and the mechanisms for the other two sub-
strate classes in the context of molecular modeling studies.
Molecular Modeling
In order to examine how substrates of all three carbonyl
classes studied here bind to the CaADH active site, molecu-
lar modeling was undertaken. Given that no three dimen-
sional structure of CaADH has yet been published, we con-
structed a homology model using Clustal W,46 and relaxed
this structure (GROMACS47) as described previously.27
Docking was carried out using Autodock Vina48 (Yasara
package49). Carbonyl substrates were prepared and mini-
mized in Spartan and exported as protein databand (pdb)
files. Dockings were performed so as to generate a total of
25 bound poses for each ligand studied. Poses were evaluated
based upon both Autodock-estimated binding energies for
each pose and the reasonableness of the docking algorithm-
The resulting models of the ternary CaADH-NADPH-car-
bonyl complexes for each substrate class are presented in
Figure 5. Notice the important substrate binding and hydro-
gen bond donor roles played by the S140 and Y153 residues
in all docked structures. These residues are part and parcel
of the canonical carbonyl binding pocket in the family of
short-chain dehydrogenases of which CaADH is a mem-
ber.50 Docking suggests that these residues may also inter-
act with the hydroxy groups in the gem-diols derived from
the aryl trifluoromethyl ketone substrates and may facili-
tate their dehydration (see Figure 5d) as described above.
The arene rings of the aryl -keto ester substrates (Figure
5b) and of the aryl trifluoromethyl ketone substrates (Fig-
ure 5c) are postulated to enjoy favorable edge-to-face -
interactions with F192 and F241, respectively. This results
in proper positioning of these carbonyl substrates so as to
enantioselectively give the D--hydroxy ester and (S)--tri-
fluoromethyl benzyl alcohol products, as observed.
In summary, we report here what to our knowledge is
the first example of a Hammett LFER study of more than
one carbonyl substrate class in a single dehydrogenase ac-
tive site. The dehydrogenase under study, CaADH from Clos-
tridium acetobutylicum, is an enzyme that has shown enor-
mous potential in asymmetric synthesis, acting to enantio-
selectively reduce substrates of the -keto ester, -keto
ester, -keto ester, -keto-,-difluoroalkyl phosphonate
Figure 5 Molecular modeling of NADPH cofactor in the CaADH active site (homology model) with the following substrates: (a) p-nitrobenzaldehyde,
(b) ethyl benzoyl acetate (-keto ester), (c) phenyl trifluoromethyl ketone, and (d) the hydrate (gem-diol) form of phenyl trifluoromethyl ketone
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I
G. P. Kudalkar et al.
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and aryl trifluoromethyl ketone substrate classes, the latter
example having been first demonstrated in this work. The
Hammett  values obtained for the aryl aldehyde (Figure
2a:  = 0.99 ± 0.10 and  = 0.40 ± 0.09) and aryl -keto ester
(Figure 2b:  = 1.02 ± 0.31 ) substrate classes are, in these
cases, consistent with at least partially rate-limiting hy-
dride transfer from NADPH to the substrate carbonyl. It
should be noted that rate-limiting single-electron transfer
cannot be ruled out solely on the LFER data that we present.
This would lead to a transient substrate ketyl radical anion
and a nicotinamide radical cation. Though such mecha-
nisms are not generally considered likely for NAD(P)H-me-
diated dehydrogenase chemistry, the recent work of Hys-
ter51 shows that under appropriate conditions (e.g., photo-
initiation, haloketone substrates), nicotinamide enzymes
can undergo one-electron chemistry.
For our studies described here with CaADH, the pseudo-
biphasic, concave-downward nature of the Hammett plot
observed for the p-substituted benzaldehyde substrate class
(Figure 2a) suggests that there may be a change in the rate-
determining step in moving from substrates bearing ‘softer’
highly polarizable substituents (I, SCF3, Br) to those bearing
‘harder’ substituents with a significant dipole (F, Cl, OCF3,
CF3, NO2). One interpretation of this bifurcating kinetic pat-
tern would be that for the former class, hydride transfer is
rate-limiting, whereas for the latter substrate class, product
dissociation becomes partially rate-limiting, owing to spe-
cific interactions of these dipolar substituents with the en-
zyme, giving rise to the lower value of  seen for this sub-
strate subclass. Consistent with this notion, molecular
docking of the p-nitrobenzaldehyde substrate with the
CaADH homology model shows the possibility for signifi-
cant ion–dipole and dipole–dipole interactions between
the polar p-NO2 group and the ammonium group of K207
and the hydroxy group of Y211, respectively (Figure 5a).
The negative  value observed for the aryl trifluoro-
methyl ketone (,,,trifluoroacetophenone) substrate class
together with model NMR studies suggests that these sub-
strates are largely hydrated and that substrate dehydration,
favored for substrates bearing electron-donating groups,
may be rate-limiting for this interesting substrate class. Im-
portantly, the efficient and highly enantioselective manner
in which CaADH processes this new substrate class [95%
yield, 99% ee, favoring the (S)-enantiomer of -trifluoro-
methyl benzyl alcohol)52 augers well for future applications
of the CaADH enzyme in asymmetric synthesis and process
chemistry.
RR-06307) and the NSF (Grant Nos. CHE-0091975 and MRI-0079750)
for NMR instrumentation and the NIH, National Center for Research
Resources (Grant No. RR016544) for facilities.
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Supporting information for this article is available online at
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References and Notes
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Funding Information
This work was supported by the National Science Foundation (NSF),
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(52) Enzymatic Reduction/Preparation of (S)-2,2,2-Trifluoro-1-
phenylethanol; Typical Procedure
To a solution of NADPH (21 mg, 0.0287 mmol, 0.01 equiv), D-
glucose (3.09 g, 17.2 mmol, 6 equiv), CaADH (50 units) and
NADP+-dependent glucose dehydrogenase (2% w/w) in KPO4
buffer (100 mM, pH 8.0) was added trifluoromethylphenyl
ketone (500 mg, 2.87 mmol) in DMSO so that the final DMSO
concentration was 10% (v/v). The reaction mixture was allowed
to stir at rt for 8–10 h and the reaction progress was monitored
by TLC. The product was extracted with EtOAc and dried over
Na2SO4. Following vacuum filtration and concentration, the
crude residue was purified by flash column chromatography on
silica gel (EtOAc/hexane, 1:4) to give the title alcohol as a color-
less oil (484 mg, 96%) in homogeneous form. The ee was deter-
mined to be 99.6% (S) by HPLC with a chiral stationary phase
(see the Supporting Information).
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1H NMR (400 MHz, CDCl3):  = 7.55–7.47 (m, 2 H), 7.47–7.41 (m,
3 H), 5.11–4.97 (m, 1 H), 2.66 (d, J = 4.5 Hz, 1 H). 13C NMR (100
MHz, CDCl3):  = 180.88 (q, J = 35 Hz), 135.85, 130.47 (q, J = 2.8
Hz), 128.91, 107.01 (q, J = 294 Hz). 19F NMR (376 MHz, CDCl3):
 = –78.36 (d, J = 6.7 Hz).
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© 2020. Thieme. All rights reserved. Synlett 2020, 31, A–K