Bioorganometallic chemistry: Co-factor regeneration, enzyme recognition of biomimetic 1,4-NADH analogs, and organic synthesis; tandem catalyzed regioselective formation of N-substituted-1,4-dihydronicotinamide derivatives with [Cp*Rh(bpy)H]+, coupled to chiral S-alcohol formation with HLADH, and engineered cytochrome P450s, for selective C-H oxidation reactions

Article abstract of DOI:10.1016/j.jorganchem.2017.02.013

Two novel tandem catalysis approaches for the chiral synthesis of S-alcohols from reduction of their prochiral ketones with Horse Liver Alcohol Dehydrogenase (HLADH), and selective C-H oxidation reactions with protein engineered Cytochrome P450s, are presented. We utilized a co-factor regeneration procedure with three biomimetic NAD⁺ models that do not contain the pyrophosphate, nor the adenosine group, and either/or a ribose, N-1-benzylnicotinamide triflate, 1, N-4-methoxybenzylnicotinamide triflate, 2, and β-nicotinamide-5′-ribose methyl phosphate, 3, in conjunction with in situ formed [Cp*Rh(bpy)H]⁺ from [Cp*Rh(bpy)(H₂O)]²⁺ (Cp*?=?η⁵-C₅Me₅, bpy?=?2,2'-bipyridyl) and the hydride source, sodium formate, to regioselectively provide their 1,4-NADH analogs, N-benzyl-1,4-dihydronicotinamide, 4, N-4-methoxybenzyl-1,4-dihydronicotinamide, 5, and 1,4-dihydronicotinamide-5′-ribose methylphosphate, 6. Surprisingly, the 1,4-NADH biomimics, 4 and 6, were recognized, in the second tandem catalysis approach, by the natural 1,4-NADH dependent enzyme, HLADH, for catalyzed, highly enantioselective conversions of prochiral ketones to chiral S-alcohols. For example, with phenethylmethyl ketone and benzylmethyl ketone, the corresponding chiral alcohols were formed in >93% ee (S-enantiomer). Thus, 1,4-NADH biomimetic model recognition by HLADH does not significantly depend on the presence of the ribose, pyrophosphate, or adenosine groups to provide chiral products. We will also propose a plausible active site (HLADH)Zn-H intermediate, generated via a hydride transfer from bound 4/6 to Zn, for the enzymatic reduction of prochiral aryl/alkyl ketones to their chiral aryl/alkyl S-alcohols. Furthermore, the use of protein engineered cytochrome P450 enzymes provided improved molecular recognition of the above mentioned 1,4-NADH biomimetic co-factors, 4 and 5, for selective C-H oxidation reactions. For example, 1,4-NADH dependent mutants of natural 1,4-NAD(P)H dependent P450 BM-3 and 1,4-NADH dependent P450 CAM, with biomimetic co-factors 4 and 5, provided selective oxidation of p-nitrophenoxydecanoic acid to ω-oxydecanocarboxylic acid and p-nitrophenol, via C-H hydroxylation and β-hydrogen elimination, while oxidation of camphor provided hydroxycamphor, respectively. We will discuss the various parameters that effect molecular recognition of the biomimics, including protein engineering of both P450 BM-3 and P450 CAM enzymes, while determining the effect of the co-factor regeneration procedure on HLADH and P450 enzyme activity. These important observations have created new paradigms for the synthesis of organic compounds of interest, with the economically more favorable biomimics of NAD⁺, 1,4-NADH, and 1,4-NAD(P)H as co-factors, in tandem with the use of [Cp*Rh(bpy)(H)]⁺ as a regioselective catalytic reagent for co-factor regeneration.

Full text of DOI:10.1016/j.jorganchem.2017.02.013

Accepted Manuscript
Bioorganometallic chemistry: Co-factor regeneration, enzyme recognition
of biomimetic 1,4-NADH analogs, and organic synthesis; tandem catalyzed
regioselective formation of N-substituted-1,4-dihydronicotinamide derivatives with
+
[Cp*Rh(bpy)H] , coupled to chiral S-alcohol formation with HLADH, and engineered
cytochrome P450s, for selective C-H oxidation reactions
H. Christine Lo, Jessica D. Ryan, John B. Kerr, Douglas S. Clark, Richard H. Fish
PII:
S0022-328X(17)30083-9
10.1016/j.jorganchem.2017.02.013
JOM 19802
DOI:
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 25 November 2016
Revised Date: 6 February 2017
Accepted Date: 10 February 2017
Please cite this article as: H.C. Lo, J.D. Ryan, J.B. Kerr, D.S. Clark, R.H. Fish, Bioorganometallic
chemistry: Co-factor regeneration, enzyme recognition of biomimetic 1,4-NADH analogs, and organic
synthesis; tandem catalyzed regioselective formation of N-substituted-1,4-dihydronicotinamide
+
derivatives with [Cp*Rh(bpy)H] , coupled to chiral S-alcohol formation with HLADH, and engineered
cytochrome P450s, for selective C-H oxidation reactions, Journal of Organometallic Chemistry (2017),
doi: 10.1016/j.jorganchem.2017.02.013.
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ACCEPTED MANUSCRIPT
Bioorganometallic Chemistry: Co-Factor Regeneration, Enzyme Recognition of
Biomimetic 1,4-NADH Analogs, and Organic Synthesis; Tandem Catalyzed
Regioselective Formation of N-Substituted-1,4-Dihydronicotinamide Derivatives
+
with [Cp*Rh(bpy)H] , Coupled to Chiral S-Alcohol Formation with HLADH, and
Engineered Cytochrome P450s, for Selective C-H Oxidation Reactions
1
2
1
2
,1
H. Christine Lo, Jessica D. Ryan, John B. Kerr, Douglas S. Clark, Richard H. Fish*
1
Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720
Department of Chemical and Biomolecular Engineering, University of California,
2
Berkeley, CA 94720
Abstract: Two novel tandem catalysis approaches for the chiral synthesis of S-alcohols
from reduction of their prochiral ketones with Horse Liver Alcohol Dehydrogenase
(HLADH), and selective C-H oxidation reactions with protein engineered Cytochrome
P450s, are presented. We utilized a co-factor regeneration procedure with three
+
biomimetic NAD models that do not contain the pyrophosphate, nor the adenosine
group, and either/or a ribose, N-1-benzylnicotinamide triflate, 1,
N-4-methoxybenzylnicotinamide triflate, 2, and β-nicotinamide-5'-ribose methyl
+
phosphate, 3, in conjunction with in situ formed [Cp*Rh(bpy)H] from
2
+
5
[
Cp*Rh(bpy)(H O)] (Cp* = η -C Me , bpy = 2,2'-bipyridyl) and the hydride source,
2 5 5
sodium formate, to regioselectively provide their 1,4-NADH analogs, N-benzyl-1,4-
dihydronicotinamide, 4, N-4-methoxybenzyl-1,4-dihydronicotinamide, 5, and 1,4-
dihydronicotinamide-5'-ribose methylphosphate, 6. Surprisingly, the 1,4-NADH
biomimics, 4 and 6, were recognized, in the second tandem catalysis approach, by the
natural 1,4-NADH dependent enzyme, HLADH, for catalyzed, highly enantioselective
conversions of prochiral ketones to chiral S-alcohols. For example, with phenethylmethyl
ketone and benzylmethyl ketone, the corresponding chiral alcohols were formed in
>
93 % ee (S-enantiomer). Thus, 1,4-NADH biomimetic model recognition by HLADH
does not significantly depend on the presence of the ribose, pyrophosphate, or adenosine
groups to provide chiral products. We will also propose a plausible active site
(HLADH)Zn-H intermediate, generated via a hydride transfer from bound 4/6 to Zn, for
the enzymatic reduction of prochiral aryl/alkyl ketones to their chiral aryl/alkyl
S-alcohols. Furthermore, the use of protein engineered cytochrome P450 enzymes
provided improved molecular recognition of the above mentioned 1,4-NADH biomimetic
co-factors, 4 and 5, for selective C-H oxidation reactions. For example, 1,4-NADH
dependent mutants of natural 1,4-NAD(P)H dependent P450 BM-3 and 1,4-NADH
dependent P450 CAM, with biomimetic co-factors 4 and 5, provided selective oxidation
of p-nitrophenoxydecanoic acid to ω-oxydecanocarboxylic acid and p-nitrophenol, via
C-H hydroxylation and ß-hydrogen elimination, while oxidation of camphor provided
hydroxycamphor, respectively. We will discuss the various parameters that effect
molecular recognition of the biomimics, including protein engineering of both P450
BM-3 and P450 CAM enzymes, while determining the effect of the co-factor
regeneration procedure on HLADH and P450 enzyme activity. These important
observations have created new paradigms for the synthesis of organic compounds of
+
interest, with the economically more favorable biomimics of NAD , 1,4-NADH, and
1

ACCEPTED MANUSCRIPT
+
1
,4-NAD(P)H as co-factors, in tandem with the use of [Cp*Rh(bpy)(H)] as a
regioselective catalytic reagent for co-factor regeneration.
Introduction
The biocatalysis discipline has focused on the generation of important chiral and
selectively oxidized organic compounds, by the use of numerous enzymes, usually in the
presence of critical natural co-factors [1a-j]. Thus, biocatalytic, enzymatic reactions have
the ability to transform precursor molecules to high chirality, or selective oxidation
products, usually in one step, that may be useful as pharmaceuticals and specialty
chemicals[2]. More importantly, the economics for the utilization of natural co-factors
+
such as nicotinamide adenine dinucleotide (NAD ), and the reduced form, 1,4-
dihydronicotinamide adenine dinucleotide (1,4-NADH), might ultimately drive this
nascent biocatalysis field, while a new paradigm concerning this critical parameter may
be necessary for the future successful use of an array of enzymes in the production of
such chiral or selective oxidation compounds [1a-j].
Furthermore, practical methods for the regeneration of the co-enzyme,
1
,4-NADH, have continued to be of significance in the applications of biocatalysis.
In this vain, a variety of transition metal hydrides have been evaluated as catalysts for the
+
+
regioselectivereduction of NAD and NAD models to the corresponding 1,4-NADH
derivatives, in order to attempt to develop faster rates and a more economical
regeneration process [3b]. In one of the most pertinent examples, Steckhan and co-
workers have described the use of the in situ generated catalytic hydride species,
+
5
[
Cp*Rh(bpy)(H)] (Cp* = η -C Me , bpy = 2,2'-bipyridyl), for the regiospecific
5 5
reduction of natural NAD+ to 1,4-NADH,[3a-l], and then demonstrated the co-factor
regeneration process in enzymatic, chiral reduction reactions with prochiral ketones [4,5].
More importantly, we previously reported on the source of this unusually high
+
regioselectivity and other mechanistic aspects with a biomimetic model NAD
compound, N-benzylnicotinamide triflate, 1, (Figure 1) in 1:1 H
Cp*Rh(bpy)(H O)](OTf) as the catalyst precursor, and sodium formate as the hydride
source, to exclusively provide the kinetic product, N-benzyl-1,4-dihydronicotinamide, 4
6, 7a]. Basically, we found that the high regioselectivity for the 1,4-NADH biomimic
2
O/THF using
[
2
2
[
+
was due to the binding of the carbonyl of the 3-amide group of the NAD model to the
Cp*Rh metal ion center that provided a constricted six-member ring transition state for
concerted regiospecific hydride transfer to C4[6,7a]. This allowed us to categorize
+
[
Cp*Rh(bpy)(H)] as a potential biomimetic hydride, via its innate ability to bind to
substrates for regiospecific hydride transfer. In addition, we also utilized an aqueous
+
NAD model, β-nicotinamide-5'-ribose methyl phosphate, 3, and demonstrated its similar
+
regioselective reduction with in situ formed [Cp*Rh(bpy)(H)] to the corresponding 1,4-
dihydronicotinamide-5'-ribose methyl phosphate, 6, at pH 6.5[6,7a]. It is important to
+
+
note that NAD model, 3, has some structural similarities to NAD itself, with particular
emphasis on the mono-ribose phosphate moiety, but with no pyrophosphate nor
+
adenosine substituents, while also recognizing that the structure of the NAD biomimic, 3,
has a simple N-benzyl group in place of the ribose, pyrophosphate, and adenosine groups
(Scheme 1).
2

ACCEPTED MANUSCRIPT
+
Figure 1: Biomimetic models of NAD , compounds 1-3, and 1,4-NADH,
+
+
compounds 4-6. Natural NAD , NAD(P) , and 1,4-NADH, are also shown.
3

ACCEPTED MANUSCRIPT
+
Scheme 1. Proposed Catalytic Cycle for the Regioselective Reduction of NAD
Biomimic, 1, with in situ Formed [Cp*Rh(bpy)H] [6,7a].
+
The forward reaction shown in Scheme 1 was found to be totally reversible with
the use of deuterium labeling experiments, as shown in Scheme 1a. Thus, by utilizing
protio 4 in 1:1 D O/THF over a prolonged period of > 24h, in the presence of
2
[
Cp*Rh(bpy)(D)](OTf) and [Cp*Rh(bpy)(D O)](OTf) , we observed the biomimetic
2 2
1
,4-NADH mono and dideutero compounds, 4-d and 4-d , while also observing the
2
+
1
deuterated biomimetic NAD analogue, 1-d, via H NMR analysis [7a]. The latter
product, 1-d, we speculate, emanates from the reaction of [Cp*Rh(bpy)(D O)](OTf)
2
2
with monodeutero, 4-d, to provide [Cp*Rh(bpy)(H)](OTf) and 1-d [7a]. The deuteride
exchange of [Cp*Rh(bpy)(H)](OTf) to [Cp*Rh(bpy)(D)](OTf) is a well-known exchange
4

ACCEPTED MANUSCRIPT
reaction. More importantly, a control experiment with protio 1 or 4 clearly showed, via
1
2 2
H NMR analysis, that in the absence of [Cp*Rh(bpy)(D O)](OTf) , no deuterium
exchange was evident over ~ 24h. The reversibility of biomimetic 1,4-NADH, 4, to
+
NAD biomimic, 1, with [Cp*Rh(bpy)(D O)](OTf) , shown in Scheme 1a, was utilized
2
2
by Sadler et al. to show unequivocally the formation of [CpIr(L)H complexes, via
1
+
H NMR analysis, from the conversion of natural 1,4-NADH to NAD [7b].
Scheme 1a: Plausible mechanism for observing the deuterium labeling, forward and
reverse reactions; i.e., the role of [Cp*Rh(bpy)(D)](OTf) and [Cp*Rh(bpy)(D O)](OTf) .
2
2
Therefore, in the first of our two preliminary communications on the use of
biomimetic 1,4-NADH co-factors, 4-6, which did not contain the pyrophosphate, nor the
adenosine group, while also stripping the sugar moiety entirely in two examples, the
natural 1,4-NADH dependent enzyme, Horse Liver Alcohol Dehydrogenase (HLADH),
still recognized biomimics, 4 and 6, with the common feature of a N-substituted
nicotinamide nucleus, to provide S-chiral alcohols from prochiral ketones, in comparison
to a very similar result as the natural 1,4-NADH co-factor [8]. We also observed that the
reverse reaction predominately occurred with the S-enantiomer of a chiral alcohol, and
oxidized co-factor, 1, that provided the prochiral ketone [8]. We then extended these
biomimetic 1,4-NADH co-factors, 4 and 5, to Cytochrome P450 enzymes, P450 BM-3
5

ACCEPTED MANUSCRIPT
and P450 CAM, for selective C-H oxidation reactions, including the use of site directed
mutagenesis to enhance the molecular recognition process of the biomimetic
co-factors with the P450 enzymes [9].
These important discoveries led our group, and other workers in the biocatalysis
+
discipline, to also extend these results to several more enzymes that were NAD /1,4-
+
NADH/NAD(P) /1,4-NAD(P)H dependent. For example, in a further extension, of this
discovery, we also developed this concept for reactions with the HbpA enzyme that
converts phenols to catechols[10]. In this latter study, we found that the biomimetic
1
,4-NADH co-factor, 4, was able to reduce FAD ~ 40X faster than natural 1,4-NADH,
+
and interestingly, faster than [Cp*Rh(bpy)(H)] , the co-factor regeneration catalyst; i.e.,
the co-factor regeneration cycle did not interfere with the reduction of FAD by
biomimetic cofactor, 4. This was a significant discovery that was also recently
implemented by other workers with biomimetic co-factor, 4, in conjunction with utilizing
Styrene Monooxygenase for epoxidation of substituted styrene derivatives [11].
Similarly, natural 1,4 NAD(P)H and FMN dependent co-factors for enolate reductases
were also shown to be able to substitute biomimetic co-factor, 4, for 1,4-NAD(P)H, in the
2
conversion of FMN to FMNH [12]. Thus, in this full paper of our previous two
communications [8,9], we present our extensive studies that clearly demonstrates that
+
simplified, biomimetic NAD , and 1,4 NADH co-factors are recognized by HLADH and
mutant P450 BM-3 and P450 CAM enzymes for the synthesis of chiral alcohols and
selective C-H hydroxylation compounds, respectively.
Results and Discussion
Tandem Catalysis: Co-factor Regeneration of 1, 4-NADH Biomimics, 4 and 6, with
+
[
Cp*Rh(bpy)(H)] , Followed by, Chiral Synthesis of Aryl and Alkyl S-Alcohols
from Prochiral Ketones, Utilizing the Enzyme, HLADH.
We previously determined the initial rates, r
i
, turnover rate, kcat, and turnover
-1
frequencies, TOF (h ), of the regioselective reduction of 3 in H O at pH 6.5, with in situ
2
+
generated [Cp*Rh(bpy)(H)] , to its corresponding 1,4-dihydro analog, 6, and found,
+
surprisingly, that it was very comparable to natural NAD itself, in H O at pH 6.5 [7].
2
-1
-8
For example, the biomimetic co-factor, 3, at 303K: r
i
was 9.70 x 10 M s , kcat was
-
1
-1 -1
-1
3
.68 x 10 M s , with a TOF of 25 h , while the comparable numbers for natural
+
-8
-1
-1
-1 -1
-1
NAD , respectively, were: 7.57 x 10 M s , 3.06 x 10 M s , and 21 h .
Therefore, we decided, in a serendipitous manner, to attempt to use both 1 and 3 as
+
biomimics of NAD in chiral reduction reactions, in conjunction with the above
mentioned co-factor regeneration method, with the added stipulation that the enzyme we
were to utilize, 1,4-NADH dependent HLADH, would hopefully recognize the simplified
reduced forms, 4 and 6, for ultimate hydride transfer to substrate prochiral ketones, to
provide chiral alcohols [8]. Thus, in several preliminary communications, we provided
the answer to this postulate, and the theme of the present paper, of utilizing these 1,4-
NADH biomimics, is to detail all the important parameters that affect molecular
recognition in reduction and selective oxidation reactions [8,9].
6

ACCEPTED MANUSCRIPT
+
Furthermore, X-ray structures of the NAD /1,4-NADH binding site in the
HLADH enzyme showed that adenosine stabilized this interaction by being in a
hydrophobic area of the enzyme, while the two faces of the 1,4-dihydronicotinamide
group were found in a hydrophilic and a hydrophobic site, with the two different C4
hydrogens now being in a chiral environment; only one specific hydrogen is transferred
to the enzyme site for reduction of prochiral ketones [13]. Therefore, the pertinent
questions we previously asked ourselves, but are still valid today, was: what is the role of
each substituent on the 1,4-dihydronicotinamide nucleus, including ribose,
pyrophosphate, and adenosine groups, and will enzymatic molecular recognition prevail
to provide chiral reduction or oxidation products with the most important structural
feature still present in our biomimetic 1,4-NADH models; namely, the
1
,4-dihydronicotinamide nucleus?
The tandem co-factor regeneration and chiral synthesis experiments with
+
+
substrates 7-11 (Chart 1) and NAD biomimics, 1 and 3, with natural NAD itself as a
comparison, provided quite dramatic results in that; for example, the aryl substituted,
2 2 3
prochiral ketone, PhCH CH C(O)CH , 7, in the presence of HLADH, was reduced to
give
the corresponding alcohol, PhCH CH CH(OH)CH , with >93 % ee (S-enantiomer), at
2
2
3
o
-1
+
3
0 C, and a turnover frequency (TOF) of ~30 d ; both NAD models, 1 and 3, gave
+
similar chiral synthesis results compared to natural NAD itself (Table 1). Clearly, all
that was apparently necessary for HLADH chiral recognition of the 1, 4-NADH
biomimics, 4 and 6, was the 1,4-dihydronicotinamde moiety, while the 1-benzyl and
ribose-5'-methyl phosphate role in binding in the hydrophobic pocket of HLADH
surprisingly did not effect the chiral transfer of hydride to the prochiral ketone substrate.
7

ACCEPTED MANUSCRIPT
Furthermore, a review of the biomimetic co-factor's role in biocatalysis studies
was recently published[14]; however, these authors raised the question of whether the
preliminary results we published in our initial paper on utilizing 1 and 3, as biomimetic
+
NAD co-factors with the enzyme HLADH [8], were invalid, due to the possible
+
presence of the natural co-factor, NAD , without stating any proof. Therefore, we will
demonstrate, with definitive control experiments, that our results, as published, were
correct. Thus, several important control experiments were accomplished to clearly show
+
that if we eliminated the biomimetic NAD cofactors, 1 and 3, in the presence of the
+
[
Cp*Rh(bpy)(H)] , the HLADH enzyme, and for example, the prochiral ketone substrates,
2
-pentanone, 10, or phenethylmethyl ketone, 7, then no chiral alcohols were produced;
+
i.e., the HLADH enzyme had trace (< 5%) or no natural NAD contamination, that would
mask our findings, under our tandem catalysis reaction conditions! We particularly paid
attention to this latter thought, the essence of this allegation, that the HLADH enzyme
+
had incorporated traces of natural NAD that would be converted to natural 1,4-NADH,
and mask our results with the biomimetic co-factors; to reiterate, no chiral alcohol
products were formed from the prochiral ketones, 2-pentanone, 10, and
+
phenethylmethyl ketone, 7, in the absence of biomimetic NAD co-factors, 1 and 3, with
8

ACCEPTED MANUSCRIPT
0
% ee for racemic 2-pentanol/2-phenethylmethyl alcohol, which emanated from the
+
non-enzymatic, direct reaction of [Cp*Rh(bpy)(H)] with 2-pentanone, 10, or
phenethylmethyl ketone, 7; a reaction, we have studied extensively (Scheme 2)[15].
+
Scheme 2: Proposed catalytic cycle for the direct reaction of [Cp*Rh(bpy)(H)] with
2
-pentanone, 10, and HLADH, to form racemic 2-pentanol, and in the absence of the
+
NAD biomimetic co-factors, 1 or 3[15].
More importantly, a structure-reactivity study (Chart 1) of a variety of prochiral
ketone substrates (Table 1), further showed that benzylmethyl ketone, PhCH C(O)CH ,
2
3
8
, also provided the corresponding chiral alcohol, PhCH CH(OH)CH (>99 % ee,
2
3
-1
S-enatiomer, TOF, 18 d ), while benzophenone, PhC(O)CH , 9, was extremely slow
3
-1
(TOF, 2 d ) in providing the chiral product, PhCH(OH)CH , after 24 h, but the final
3
result was still >93 % ee for the S-enantiomer. Therefore, as the number of methylene
groups is reduced (2 to 0) between the phenyl and carbonyl groups, the turnover
frequency of chiral reduction product was progressively slower, showing that a specific
non-covalent π-π interaction may be prevalent in the phenyl ketone binding site, in
proximity to the (HLADH)Zn active site.
9

ACCEPTED MANUSCRIPT
The alkyl ketone, 2-pentanone, 10, was also studied, and provided an 85% ee for
+
the predominate S-alcohol, 2-pentanol, which was similar to natural NAD itself, and
may demonstrate that the binding site for this type of alkyl ketone is not as rigid as the
phenyl ketone binding site to engender higher chirality to the alcohol product.
Interestingly, we have also shown that the microscopic reverse reaction; i.e., S-2-pentanol
+
to 2-pentanone, 10, occurs with NAD biomimics, 1 and 3, and HLADH, while the
R-enantiomer was found to be extremely slow to form 10; the relative rate ratio of the
S- and R-2-pentanols, in the first 24 hr, was ~ S/R = 4:1 (Scheme 3).
HLADH
Enzyme
O
O
NH
2
NH
2
O
P
+
+
N
-
N
-O
OTf
O
O
H
or
!
CH O
3
HO
OH
S-CH CH CH CCH
3
3
2
2
substrate OH
CH CH CH CCH
3
3
2
2
H
O
H O
H
H O
O
NH2
NH
2
O
P
2-pentanone
N
-
O
N
O
or
CH O
3
HO
OH
co-factor biomimics
Scheme 3: Microscopic Reverse of Chiral S-2-Pentanol (> 93% ee) to
-Pentanone with Co-factors, 1 and 3, and HLADH.
2
Alternatively, the bulky, aliphatic ketone, norcamphor, 11, which is actually a
commercial mixture of two enantiomeric ketones, reacted not only at different reduction
rates, but also provided a different diastereomeric mixture of predominantly endo-chiral
alcohols. As shown in Table 2, one enantiomer of norcamphor, 11, gave an
approximately equal mixture of the exo and endo alcohols, while the other gave
exclusively the endo-alcohol with no exo-alcohol detected. Clearly, the endo-alcohol
product must be a consequence of the putative chiral (HLADH)Zn-H (vide infra)
attacking the C=O group from predominately the side with the bridge position carbon
atom, to provide the endo-alcohol diastereomer (Table 2).
10

ACCEPTED MANUSCRIPT
The tandem catalysis experiments demonstrated that the regeneration regime with
+
in situ formed [Cp*Rh(bpy)(H)] , was found to be kinetically independent with the
transfer of hydride from 1,4-NADH biomimetic co-factors, 4 and 6, to (HLADH)Zn, and
from the putative chiral Zn-H (vide infra) to the prochiral ketone, since our control
experiment detected no alcohol products in the presence of the prochiral ketone substrate,
+
the biomimetic co-factors, 1 or 3, and the in situ formed [Cp*Rh(bpy)(H)] [15].
+
Therefore, it seems definitive that biomimetic NAD co-factors, 1 and 3, preferentially
bind to the Cp*Rh moiety, in the presence of the prochiral ketone, and was preferentially
reduced to 4 and 6 (Scheme 4). We also observed that there was no significant effect on
the enzyme activity of HLADH, when we maximized the concentration of
+
[
Cp*Rh(bpy)(H
2
O)](OTf)
2
for the formation of [Cp*Rh(bpy)(H)] , and the reduction of
the co-factors.
11

ACCEPTED MANUSCRIPT
Scheme 4: Plausible tandem catalytic reduction cycle for the synthesis of
1
,4-NADH biomimics, 4 and 6, followed by a catalytic cycle for chiral alcohol
synthesis from prochiral ketone substrates, with the enzyme, HLADH.
2
+
The role of the Zn metal ion center of HLADH has still not been fully resolved,
in our opinion, with regard to binding the 1,4-NADH co-factor, and its role in the hydride
2
+
transfer to prochiral, Zn bound ketones to form S-chiral alcohols[1k,l]. To reiterate, we
+
recently reported on the binding regime of the amide carbonyl of NAD biomimics, 1 and
+
3
, to [Cp*Rh(bpy)H] that allowed concerted, regiospecific hydride transfer to C4, via a 6
membered ring transition state, to provide 1,4-NADH biomimics, 4 and 6 (Scheme
2
+
1
)[6,7]. Therefore, we propose a similar binding regime of 6 to the Zn metal ion center
of HLADH, which essentially is the microscopic reverse of the latter process (Scheme
)[16]. This potentially provides a low energy pathway to a putative, chiral Zn-H
5
intermediate, emanating from one specific hydrogen on the1,4-dihydronicotinamide
nucleus, that can then transfer this hydride, via a process that also might bind the phenyl
2
+
+
ketone substrate to the Zn metal ion center, now devoid of the oxidized NAD model, 3,
to a chiral re face of the carbonyl group of the substrate, phenethylmethyl ketone, 7, to
give the S-enantiomer of the chiral alcohol. The role of the imidazole ligand might
possibly entail opening a coordination site, and to also increase electron density at the
2
+
Zn metal ion center; for example, the chiral Zn-H reaction with prochiral ketones.
The recent LADH active site modeling result of Parkin et al [17] demonstrated
that a similar biomimetic (Tp)Zn-O-Et intermediate complex could transfer a hydride to
+
reduce a NAD model, the p-NO
2
-C
OH to the TpZn moiety; i. e., TpZnOCH
and form in this process, MeCHO; this is similar to the microscopic reverse of the
6
H
5
CHO substrate, and in this process, bind the
corresponding p-NO
2
-C
6
H
5
CH
2
2
C
6
H
5
-NO -p,
2
reduction reaction, which is shown in Scheme 3. Moreover, Kimura et al. [18] have also
intimated a biomimetic (HLADH)Zn-Oalkyl complex to form a putative Zn-H
+
intermediate that could further reduce NAD biomimic 1 to 3; however, these latter
12

ACCEPTED MANUSCRIPT
workers did not suggest a putative Zn-H as the intermediate in the reduction of the aryl
+
aldehyde or NAD model, but their results, in our opinion, synergize our presented
hypothesis on the intermediacy of a putative chiral Zn-H complex (Scheme 5).
Interestingly, it has also been previously shown by Mimoun et al. [19a] that chiral Zn-H
complexes were able to reduce prochiral ketones to chiral alcohols, and in that process,
defined a similar mechanism; i.e., Zn-H formation, ketone binding to a five coordinate
2
+
Zn site, followed by hydride transfer to provide the Zn-aryl/alkoxide complex, that is
conceivably displaced by another 1,4-NADH biomimic, such as 4/6, or by a proton, as
shown in Scheme 5. Furthermore, Riant et al. [19b] also more recently developed a
chiral Zn-H complex for reduction of prochiral ketones to chiral alcohols, with
polymethylhydrosiloxane, as the hydride transfer agent.
Scheme 5: Plausible active site model for (HLADH)Zn with 1,4-NADH biomimic, 6, as
hydride source for the putative chiral Zn-H, and phenethylmethyl ketone, 7, as substrate.
+
Therefore, we have clearly shown, for the first time, that biomimetic NAD
models, 1 and 3, structurally modified to retain the nicotinamide nucleus and either one
similar substituent, the ribose-5’-phosphate group, or none, the 1-benzyl group, compared
+
to natural NAD , can be converted to their 1,4-dihydronicotinamide analogs using in situ
+
generated [Cp*Rh(bpy)H] , and indeed, these 1,4-NADH biomimics are recognized by
HLADH for chiral synthesis of aryl/alkyl substituted alcohols (Scheme 4). We have
13

ACCEPTED MANUSCRIPT
further verified this molecular recognition process between the HLADH and the
+
NAD /1,4-NADH biomimics, 3 and 6, and compared these results with the natural
co-factors, by conducting stoichiometric reduction and oxidation reactions with prochiral
ketone, 10, and the product, S-2-pentanol, which clearly provided similar results (see the
experimental section). Another critical molecular recognition parameter for the chiral
reduction, we believe, is the 1,4-dihydronicotinamide amide carbonyl binding to the
2
+
HLADH-Zn metal ion center (Scheme 5). This allows specific, chiral transfer of one of
2
+
+
the C4 hydrogens as hydride to Zn and, in turn, generates the NAD biomimic, 3, which
+
is recycled to 6, via the co-factor regeneration process with [Cp*Rh(bpy)H] .
The microscopic reverse oxidation reaction with biomimic 3/natural NAD , HLADH, and
+
S-2-pentanol, also constitutes, a molecular recognition process for the nicotinamide
amide carbonyl group for hydride transfer to form the 1,4-NADH biomimic 6/natural
1
,4-NADH, and 2-pentanone (Scheme 3).
Cytochrome P-450 Enzymes: Site Directed Mutagenesis for Molecular Recognition
of 1,4-NADH Biomimics, and Selective C-H Oxidation Reactions.
Cytochrome P450s are heme enzymes that can selectively oxidize a broad range
of compounds, often at unreactive C-H bonds, with high regio- and
enantioselectivity[1h-j]. Two bacterial P450s, P450 BM-3 and P450 CAM, have been
extensively studied, and have served as credible model P450 enzymes. They are water
soluble, easily expressed in a bacterial host, such as E. coli, and were found to be
relatively stable. However, these P450 enzymes have many barriers for their commercial
utilization, including the economics of the required cofactor, 1,4-NAD(P)H[20].
Furthermore, the search for alternative cofactors and/or regeneration regimes has
been a continuing topic of significant interest, in the general area of biocatalysis. Several
alternative electron sources have been evaluated for their ability to support P450 catalysis,
including sodium dithionite[21], ascorbic acid[22], and electrochemical techniques[23,
2
4]; however, the substrate conversion and initial rates for such systems were found to be
significantly lower compared to those using natural 1,4-NAD(P)H[25]. Important
characteristics of biomimetic co-factors include being inexpensive to synthesize, should
not inhibit the P450 catalyst, and should support 1,4-NADH dependent oxidoreductases.
As we reported, Lo and Fish showed that 4 and 6 (generated in situ with
+
[
Cp*Rh(bpy)H] ) can be used by horse liver alcohol dehydrogenase (HLADH) to
catalyze the reduction of prochiral ketones to chiral alcohols[8], while Fish et al. reported
that 4 can supply the electrons necessary for phenol oxidation by 2-hydroxybiphenyl 3-
monooxygenase, HbpA[10]. Since 4, or other 1,4-NADH biomimics, had not been
evaluated with a Cytochrome P450 enzyme[9], thus, our objectives were to evaluate the
ability of 4 and N-4-methoxybenzyl-1,4-dihydronicotinamide, 5 (Figure 1), to support
selective C-H oxidation reactions catalyzed by Cytochrome P450s, and to determine
whether the activity of P450s towards the 1,4-NADH biomimetic cofactors, 4 and 5,
could be enhanced by protein engineering.
In a previous study directed toward improving P450 BM-3 (Figure 2) for
industrial oxidation reactions, Maurer et al. created the mutants W1064A, W1064S, and
R966D/W1064S, which enabled the substitution of 1,4-NAD(P)H with 1,4-NADH, thus
M
reducing the overall biocatalysis process economics[26]. The K and kcat values for the
14

ACCEPTED MANUSCRIPT
1
,4-NAD(P)H-dependent reduction of Cytochrome C by the mutant P450s were
compared, and the double mutant, R966D/W1064S, had the greatest catalytic efficiency
k /K ) for reactions with 1,4-NADH. This double mutant also had a lower preference
(
cat
M
for 1,4-NAD(P)H over 1,4-NADH, than the wild-type enzyme[26]; therefore, we utilized
the same P450 BM-3 mutant, and then assayed its ability to reduce Cytochrome C with
1
,4-NADH, and biomimics, 4 and 5 (Table 3).
Figure 2: Structure of P450 BM-3
Table 3. K and k values for Cytochrome C reduction by wild-type and double mutant
M
cat
P450 BM-3 using 1,4-NADH biomimics, 4 and 5, as the co-factors, as well as natural
1
,4-NADH, for comparison [9].
[
a]
[a]
[a]
P450
BM-3
K
M
k
cat
kcat /K
M
Cofactor
-1
-1
-1
[µM]
[min ]
[min µM ]
1
,4-
8
10
346
0.43
Wild
Type
NADH
4
5
No reaction
No reaction
--
--
--
--
1
,4-
10.5
4260
405
R966D/
W1064S
NADH
4
5
29.5
96.0
1220
2500
41.3
26.0
[a] The reported values are the averages of three measurements for
which the standard deviations were less than 10%
15

ACCEPTED MANUSCRIPT
More importantly, the R966D/W1064S double mutant was capable of using both
4
and 5 to reduce Cytochrome C, while the wild-type enzyme had no activity with either
biomimic. Furthermore, the catalytic efficiencies for the double mutant using both 4 and
were much greater than that of the wild-type enzyme with 1,4-NADH.
5
Moreover, p-Nitrophenoxydecanoic acid (p-NCA) has been shown to be an important
model substrate for P450 BM-3, which upon selective hydroxylation of the alpha C-H
bond to the ether oxygen, forms
ω-oxydecanocarboxylic acid and p-nitrophenol, via a
β-hydrogen elimination reaction (Scheme 6)[27]. The wild-type enzyme did not
hydroxylate p-NCA, using either 4 or 5, as evidenced by the lack of Uv-vis detection of
p-nitrophenol after 1 h. However, both 1,4-NADH biomimics, 4 and 5, supported p-NCA
hydroxylation by the R966D/W1064S double mutant. Interestingly, the double mutant
achieved the same conversion for p-NCA oxidation (>95%) using 1,4-NAD(P)H, 1,4-
NADH, or 4, as did the wild-type enzyme using its preferred cofactor, 1,4-NAD(P)H.
However, reactions with the double mutant and 5, a 1,4-NADH biomimetic co-factor
with an electron donating p-methoxy group for potential enhanced molecular recognition
at the co-factor binding site, provided only a 42% conversion based on the initial
concentration of p-NCA.
The initial rates of p-NCA hydroxylation with wild-type and R966D/W1064S
P450 BM-3 are shown in Table 4. The reactions with the wild-type enzyme and 1,4-
NAD(P)H had the highest initial rate; however, the initial rate of the double mutant
reaction with 4 was only 1.9X slower. The initial rate with 5 was 1.6X slower, in
comparison to 4, indicating that the electron donating p-methoxy group may not be
available for critical non-covalent interactions at the co-factor binding site, as we had
envisioned. These results clearly demonstrate that protein engineering can be beneficial
for generating a mutant of P450 BM-3 that has good activity, with the 1,4-NADH
biomimics 4 and 5, while the 1,4-NAD(P)H dependent wild-type enzyme was inactive.
Scheme 6: Reaction Pathway for the Selective Oxidation of p-Nitrophenoxydecanoic
16

ACCEPTED MANUSCRIPT
Acid (p-NCA) with Double Mutant P450 BM-3, using the 1,4-NADH Biomimetic
Co-factor, 4, as the Reductant.
Table 4. Initial rates of p-NCA hydroxylation by
wild-type and double mutant P450 BM-3
using 1,4-NAD(P)H, 1,4-NADH, 4, and 5, as the
co-factors.
[
a]
[a]
WT [nmol/s
BM-3]
·
mg R966D/W1064S
Cofactor
,4-
[nmol/s mg BM-3]
·
1
43.6
30.4
34.5
NAD(P)H
,4-
NADH
1
1
.6
4
5
no reaction
no reaction
23.3
14.7
[
a]
Reactions contained 60
µ
M p-NCA, 200
µ
M
co-factor, and 0.13
µM wild-type or 0.15 µM
R966D/W1064S, P450 BM-3 mutant.
The reported values are averages of three
measurements for which the standard deviations were
less than 10%.
_
___________________________________________
Even though P-450 BM-3 was considered a model system for reductase proteins
that have both an FAD and FMN ligand, such as human P-450 reductase[28], the
reductase of P-450 CAM was a more reliable model for other oxygenase-coupled 1,4-
NADH-dependent ferredoxin reductases (ONFRs), which was necessary for electron
transport to a number of hydroxylases, including alkane, benzene, and diphenyl
hydroxylases[29]. Moreover, wild-type (WT) P-450 BM-3, was very different than WT
P-450 CAM, which was able to catalyze substrate oxidation, utilizing camphor, with both
1
,4-NADH biomimics, 4 and 5 (Table 5). In contrast to the results for P-450 BM-3, the
initial rate of WT P-450 CAM was nearly the same with both 4 and 5, but these initial
rates were much less than the rate with the natural cofactor, 1,4-NADH.
Furthermore, the P450 CAM requirements included three proteins for catalytic
activity; i.e., putidaredoxin reductase (PdR), putidaredoxin (PdX), and cytochrome-m
(cyt-m). Previous studies have shown that the initial rate of WT P450 CAM varied with
changing the PdX and cyt-m concentrations, when 1,4-NADH was the co-factor[31,32].
However, when either 4 or 5 was used, the rate of camphor oxidation was not influenced
by the concentration of either PdX (range: 0.5-7.5
In contrast, the initial rate did moderately increase 1.3X, when the concentration of PdR
was increased from 0.75 to 2.8 M, suggesting that the reduction of the FAD ligand
within PdR was rate limiting, when using 4 or 5, and that the rate could be further
µM) nor cyt-m (range: 0.5 to 1.5µM).
µ
17

ACCEPTED MANUSCRIPT
improved by using a mutant of PdR that was more active towards the 1,4-NADH
biomimics; this e-transfer pathway is shown in Scheme 7.
_
__________________________________
Table 5. Initial rates of P450 CAM
mediated camphor oxidation to
hydroxycamphor supported by 4, 5, or
,4-NADH.
1
Initial Rate
[nmol/s·mg
cyt-m]
Cofactor Reductase
WT
0.72
1.80
0.92
0.82
1.93
1.11
130
78
[
[
a]
a]
4
5
E300A
E300L
WT
E300A
E300L
WT
1
,4- [
b]
E300A
E300L
NADH
125
[
a] Reactions contained 5 mM of either 4
or 5, 1.0 mM camphor, 1.5 M cyt-m,
.5 M PdX, and 2.8 M PdR. [b]
Reactions contained stoichiometric
amount of 1,4-NADH and 0.5 M cyt-m,
.5 M PdX, and 0.25 M PdR. The
µ
7
µ
µ
µ
2
µ
µ
reported values are averages of three
measurements for which the standard
deviations were less than 10%.
18

ACCEPTED MANUSCRIPT
S
2
e-
transfer
Fe2+
PdXr Fe3+
Fe3+
Fe2+
NADH
H+
O
FAD
+
S
S
e-
Fe4+
R-OH
R-H
NAD+
Fe2+ PdXoxFe3+
FADH2
+ O2
S
-H O
2
e- flow
Putidaredoxin Reductase
Putidareredoxin
Cytochrome M
Iron-Sulfur Heme, 45 kD
FeS Protein, 11 kD
FAD -Flavo Protein, 45. 5 kD
Each Protein Cloned and Expressed in E. Coli
Scheme 7: P450 CAM Electron Pathway with PdR, PdX, and Cytochrome M, the
needed Components, to Convert Camphor to Hydroxycamphor.
Moreover, there was no tryptophan residue available, corresponding to the W1064
of P450 BM-3, in the 1,4-NADH binding pocket of PdR, for mutation. Alternatively, the
corresponding residue had a tyrosine (Y159), which has a different conformation relative
to the FAD, in contrast to W1064 of P450 BM-3[32]. Therefore, we chose to mutate
E300, which was found to participate in non-covalent H-bonding with the ribose ring
nearest to the nicotinamide portion of 1,4-NADH[32]. Two single point mutations,
E300A and E300L, were inserted, and then these mutants were bioassayed for camphor
oxidation with both 4 and 5 (Table 5). To our total surprise, both mutations improved
the initial rate of camphor oxidation using the 1,4-NADH biomimics, with E300A
affording a greater increase than E300L, for both 4 and 5 (Scheme 8). For example, with
4
, the E300A/E300L ratio, provided an initial rate increase of 2X, as well as, 2.5X the
WT P450 CAM, while with 5, the E300A/E300L was 1.7X, and with WT P450 CAM the
ratio was 2.4X. Interestingly, the WT P450 CAM with natural 1,4-NADH had an initial
rate increase of 1.7X over E300A, but equal to the rate of E300L.
19

ACCEPTED MANUSCRIPT
Scheme 8: Mutation of P450 CAM, Glu 300 exchanged by Ala (A) or Leu (L).
+
Biomimetic NAD Counter Anion Effects on the Rates and Total Turnover
Numbers (TTNs) of the Selective Oxidation Reactions with the P450
Enzymes.
+
-
In order to understand if the biomimetic NAD counter anion, X , had any effect
-
on the rate of regeneration of 4 from 1(X , Figure 3) in the P450 BM-3 and CAM
selective C-H oxidation reactions, we evaluated three different anions. Therefore, we
used the same non-enzymatic regeneration strategy, employed using the HLADH
enzyme, with the P450 reactions, which included 5mM 1(Cl), 1(Br), and 1(OTf), with
[
Cp*Rh(bpy)(H
source for cofactor regeneration; for example, Scheme 4, as it pertains to Scheme
[6,7a]. As shown in Table 6, the initial rates of substrate oxidation depended on the
2 2
O)](OTf) as the catalyst precursor, and sodium formate as the hydride
7
anion associated with the oxidized biomimetic analogs 1(Cl), 1(Br), and 1(OTf).
In general, reactions containing triflate anions, 1(OTf)), were faster than those with
chloride, 1(Cl), or bromide anions, 1(Br). These results suggested that counter anions,
-
-
Cl and Br , possibly hinder binding of the co-factors to the Cp*Rh metal ion center.
Moreover, the initial rates of substrate oxidation by both P450 enzymes were nearly the
same, intimating that the rate of the biomimetic co-factor reduction, with 1 and 2, were
-
rate limiting, while the TTN values showed clearly that the weak binding anion, OTf ,
-
-
provided higher numbers, and further suggested some inhibition by Cl and Br counter
anions could be prevalent.
20

ACCEPTED MANUSCRIPT
+
-
Figure 3: Anion effect on the regeneration of 4 with biomimetic co-factor 1 (X ), where
-
X = Cl, Br, or OTf.
Table 6. Initial rates and TTNs of camphor oxidation by P450 CAM, and
p-NCA oxidation by R966D/W1064S P450 BM-3 with co-factor
regeneration of 4 with 1(Cl), 1(Br), or 1(OTf), containing different
counter ions, in tandem with [Cp*Rh(bpy)(H
2 2
O)](OTf) and sodium formate.
_
____________________________________________________
[
a]
Initial Rate
[a]
P450
Cofactor
[pmol/s mg
P450]
TTNs
1
(Cl)
118
90
33
24
P450CAM
1(Br)
1
(OTf)
150
125
83
38
1
(Cl)
264
180
312
R966D/W1064
P450 BM-3
1
(Br)
1(OTf)
144
[
a]
The reported values are the averages of three measurements
for which the standard deviations were less than 10%.
21

ACCEPTED MANUSCRIPT
2 2
Effect of the Concentration of [Cp*Rh(bpy)(H O)](OTf) on P450 Enzyme
Stability and Efficiency, in Co-factor Regeneration and Selective C-H
Oxidation Reactions.
We wanted to understand the effect of the concentration of
[
Cp*Rh(bpy)(H O)](OTf) during co-factor regeneration and P450 enzyme C-H
2
2
oxidation reactions. When assayed, after incubation with 50
µM
[
Cp*Rh(bpy)(H O)](OTf) for 1 h, P450 CAM retained only 10% of its oxidation
2
2
activity, while P450 BM-3 was relatively stable in the presence of the organorhodium
precatalyst complex, and maintained 70% of its oxidation activity, under the same
conditions. It might be possible that the organorhodium precatalyst effected the substrate
binding domain of P450 CAM more than P450 BM-3, at high concentrations of
+
[
Cp*Rh(bpy)(H
2
O)](OTf)
2
, and in the absence of the biomimetic NAD co-factors, 1 or
2
, while the structural differences between the active sites of P450 CAM and P450 BM-3
might also possibly account for the observed inhibition reactions. Table 6 also shows the
total turnover numbers (TTNs), defined as the concentration of product formed divided
by the enzyme concentration. Despite the similarity in initial rates, the TTNs were much
larger for P450 BM-3 than for P450 CAM, apparently due to the greater stability and
reactivity of P450 BM-3 in comparison to P450 CAM, under the reaction conditions.
+
We also measured the rate of NAD reduction by in situ generated
+
[
Cp*Rh(bpy)(H)] , after incubation for 1 h with P450 BM-3, P450 CAM, or bovine
+
serum albumin. None of these proteins inhibited the activity of [Cp*Rh(bpy)H] .
Thus, the lysine and cysteine residues did not appear to inhibit the reduction of natural
co-factor, NAD , by promiscuous reactivity of precatalyst, [Cp*Rh(bpy)(H O)](OTf) ,
+
2
2
with peripheral NH or SH groups, as suggested by Fish et al., for the HbpA enzyme[10].
2
The critical difference between the two scenarios for Cytochrome P-450 enzyme stability
+
and efficiency was the absence or presence of the natural or biomimetic NAD co-factors,
+
which we found to react preferentially with the in situ formed [Cp*Rh(bpy)H] complex,
to provide the biomimetic 1,4-NADH co-factors, and thus, have no effect on P450
enzyme C-H oxidation reactivity, nor co-factor regeneration. Therefore, it appears
imperative that tandem biocatalysis reactions must include all components,
2 2
[Cp*Rh(bpy)(H O)](OTf) , sodium formate, co-factors, substrate, and enzyme, to
mitigate any potential deliterious effects of the precatalyst, [Cp*Rh(bpy)(H O)](OTf) ,
2
2
on
enzyme reactivity.
Conclusions
+
We have clearly demonstrated that NAD and 1,4-NADH biomimetic co-factors,
1
and 3, and 4 and 6, respectively, are recognized by the natural 1,4-NADH dependent
enzyme, HLADH, and this led to the tandem catalysis regime (Scheme 3) of co-factor
+
regeneration, with [Cp*Rh(bpy)(H)] as the catalytic species, and chiral synthesis
utilizing prochiral ketones (Chart 1) being reduced to chiral S-alcohols (Table 1).
We found, surprisingly, that the turnover numbers and the high enantioselectivity for the
S-alcohols, with the biomimics, were similar to those found for natural 1,4-NADH. More
importantly, a critical control experiment provided unequivocal evidence that when we
22

ACCEPTED MANUSCRIPT
+
eliminated the biomimetic NAD co-factors, 1 and 3, in this tandem catalysis experiment,
no chiral S-alcohols were detected by GC-MS, only racemic alcohols. Thus, the
significance of our results is that simple N-substituted-nicotinamide triflate salts, and
their 1,4-dihydronicotinamide analogs, could be readily substituted for the more
+
economically expensive natural NAD and 1,4-NADH co-factors in HLADH, and many
more important enzymes [10-12,14,33].
Therefore, the favorable economic benefits of this discovery are obvious for
biocatalytic applications of industrial importance, and interestingly, that some structural
+
features of natural NAD /1,4-NADH need not be present to afford enzyme recognition
and chiral organic compound synthesis, as well as, selective C-H oxidation reactions; i.e.,
ribose, pyrophosphate, or adenosine groups. We especially highlighted 1,4-NADH
biomimetic co-factor, 4, for being more stable under conditions that might cause natural
1
,4-NADH to be hydrolytically compromised[34].
Furthermore, the 1,4-NADH dependent WT P450 CAM oxidation reactions were
supported by biomimetic 1,4-NADH co-factors, 4 and 5, while the 1,4-NAD(P)H
dependent WT P450 BM-3 was not active with these 1,4-NADH biomimetic analogs.
However, we have clearly shown that mutations of a key residue in the reductase (PdR)
for P450 CAM afforded only a moderate increase in initial rate, while mutations in the
P450 BM-3 reductase domain enabled the use of the 1,4-NADH analogs, 4 and 5, at rates
comparable to those of the natural co-factor, 1,4-NAD(P)H. Presumably, other P450 and
oxidoreductase mutants with comparable, or even greater activity than the wild-type
enzymes with the biomimics, can be generated via protein engineering[1h]. We also
found that P450 BM-3 was stable in the presence of pre-incubated
[
Cp*Rh(bpy)(H O)](OTf) , in the absence of the natural or biomimetic co-factors, while
2 2
non-enzymatic co-factor regeneration appears to be promising for select enzymatic
reactions under optimized buffer conditions.
We hope the biocatalysis discipline in general, along with biocatalysis industrial
+
groups, utilize these biomimetic NAD /1,4-NADH co-factors for their economical
advantage, in the future, as epitomized by 1,4-NADH biomimic, 4, which could be used
stoichiometrically for many 1,4-NADH dependent enzyme reactions. In addition, mutant
P450 enzymes, such as P450 BM-3, whose natural co-factor is 1,4-NAD(P)H, but by
judicial protein engineering, the double mutant P450 BM-3 enzyme was found to be
1
(
,4-NADH dependent, via the crucial molecular recognition of biomimics, 4 and 5
Tables 4 and 6).
Finally, we have clearly demonstrated that organometallic chemistry is fully
compatible with biocatalysis, in that [Cp*Rh(bpy)(H)](OTf) controlled the biocatalytic
+
reaction, by selectively binding to the biomimetic NAD co-factors, and not the substrate;
2
+
for example, 2-pentanone, to provide chiral S-2-pentanol. We also believe that the Zn
2
+
metal ion binds to the biomimetic 1,4-NADH co-factor for hydride transfer to Zn ,
2
+
followed by chiral Zn -H transfers to the substrate prochiral ketone (Scheme 5). With
the P450 enzymes, it was the role of the generated biomimetic 1,4-NADH co-factor, from
+
the reduction of the biomimetic NAD co-factor, with [Cp*Rh(bpy)(H)](OTf), to deliver
-
+
-
H /2 electrons/H to the FAD component to form FADH
2
, on a e pathway to reduce the
3
+
2+
Cytochrome P450 Fe center to Fe , and the subsequent binding of O , and formation of
the ferryl ion, Fe =O, for hydroxylation reactions (Scheme 7). We envision this
2
4
+
bioorganometallic chemistry-biocatalysis connection to play a broader role in many
23

ACCEPTED MANUSCRIPT
future biological reactions of importance.
Experimental Section
Apparatus.
The pH values were obtained with an Orion 601A pH meter equipped with an
Orion semimicro combination pH electrode. The GC chromatogram analyses were
obtained on a Hewlett-Packard instrument (HP 5890) equipped with a modified chiral
β-cyclodextrin capillary column (Supelco, β-DEX-225, 30m, i.d. 0.32mm, df 0.25µm).
The products were identified by comparing the retention times with those of authentic
chiral samples in the GC chromatograms, and were confirmed by GC/MS analyses on a
Hewlett-Packard instrument (HP 5971 GC/MS with HP 5890 Series II GC) equipped
with a J&W, DB-Wax column (30m, i.d. 0.25mm, df 0.25µm).
Materials.
Lyophilized powder of horse liver alcohol dehydrogenase (HLADH, >95% by
weight, 0.43 unit/mg) was purchased from Sigma and was used as received.
+
β
-Nicotinamide adenine dinucleotide (NAD , sodium salt, >95%), and
β-1,4-NADH
(disodium salt, >95%) were purchased from Sigma, and were used without further
purification. Water (HPLC grade) was purchased from Aldrich. Cyclohexanol (HPLC &
UV grade, >99%) was purchased from Alfa Aesar and used as received. Optically pure
S-2-pentanol (98%, 99% ee), R-2-pentanol (98%, 99% ee), and racemic 2-pentanol (98%)
were purchased from Aldrich and were de-oxygenated prior to use. Prochiral ketone
substrates were purified under N atmosphere according to published methods, prior to
2
use[35]. All other corresponding optically pure alcohol products were purchased from
Aldrich, and were used as authentic samples in GC chromatogram analysis.
R-α-methylbenzyl isocyanate (>99%, >99.5% ee) was purchased from Aldrich, and was
used as received, in derivatization of 2-pentanol for GC chromatogram analysis.
Compounds 1-3, and 4-6 and [Cp*Rh(bpy)(H O)](OTf) were prepared according to
2
2
literature methods[6,7].
General GC-Analysis Process.
According to the substrate reactivity, a suitable amount of cyclohexanol in 1 mL
of phosphate buffer (100 mM, pH 7.02) was used as an internal standard solution in all
the GC chromatogram analysis. The progress of the reaction was determined as follows:
during the reaction course, each reaction aliquot (1 µL) was mixed well with the
cyclohexanol internal standard solution in a vial prior to injecting a proper amount of the
mixture for GC chromatogram analysis, and the measured relative areas in the
chromatograms were then calibrated with their corresponding response factors, with
respect to cyclohexanol (internal standard). The yields of the reaction were directly
24

ACCEPTED MANUSCRIPT
calculated according to the calibrated areas of prochiral ketone substrates, and of the
combined alcohol products, in the GC chromatograms. Unless otherwise stated, the ee
values of the products were determined directly by measuring the alcohol enantiomer’s
relative areas in the obtained chromatograms (Conditions for GC analysis of chiral
o
alcohols from prochiral ketones 7-10, isothermal at 110 or 115 C, while with racemic
o
ketone 11, for the exo- and endo-norbornanol diasteromers, the initial temp was 74 C to
o
o
final temp, 90 C, at 10 C /min). In the case of 2-pentanone, the ee of the S-2-pentanol
was determined by derivatization with optically pure R-α-methylbenzyl isocyanate, after
the reaction was quenched (24h)[6].
General Procedure for the Catalytic, Enantioselective Reduction of Prochiral
Ketones with Precatalyst [Cp*Rh(bpy)(H O)](OTf) , Sodium Formate, and NAD
Biomimics, 1 or 3, in the Presence of the HLADH Enzyme.
+
2
2
The reported reaction conditions, such as the reactant ratio, reaction temperature,
and the pre-catalyst amount, were obtained after numerous trial tests; however, the
reaction conditions were not optimized. For reaction temperatures tested, from RT to
o
o
3
9 C, while 30 C exhibited the highest ee (85%~99%) for the chiral alcohol products. A
+
general reaction procedure is given as follows, by using NAD biomimic, 1, and substrate,
PhCH CH COCH , 7, as an example: [Cp*Rh(bpy)(H O)](OTf) (1.90 mg, 2.61 x 10
mmol), sodium formate (17.80 mg, 261.69 x 10 mmol), NAD biomimic, 1, (4.10 mg,
1.32 x 10 mmol), and HLADH (23.80 mg, 10.23 units) were placed in a 10 mL
-3
2
2
3
2
2
-3
+
-3
1
Schlenk flask capped with a septum, and Schlenk techniques were carefully followed to
deoxygenate the solid mixture. Potassium phosphate buffer (5 mL, 100 mM, pH = 7.02,
de-O ) was added to give a clear solution,followed by the addition of the ketone substrate
2
-3
(
12.5 µL, 83.41 x 10 mmol, de-O ) via syringe, respectively. Under a gentle N pressure,
2
2
the septum was removed, and the reaction flask was capped with a glass-stopper sealed
with a small amount of grease and a metal clip. The stopcock of the reaction flask to N
2
gas was then shut, and the reaction flask was immediately shaken in a water-bath shaker
o
at 30 C, with the water level above the reaction mixture solution. The reaction was
monitored by means of GC for 24h (yield, 90%; 93% ee,). Small amounts of a
precipitate were observed in all reactions tested after 24h. The SDS-PAGE
analysis indicated the observed precipitate to be mainly the HLADH enzyme.
Biomimetic Reduction of Norcamphor (racemic).
+
The procedure as described above in the NAD biomimic case, 1, was followed;
however, due to the sublimation of norcamphor in vacuo, the addition of norcamphor was
conducted under a positive argon pressure, followed by the addition of phosphate buffer
via syringe.
Biomimetic Reduction of 2-Pentanone.
The procedure described in the General Procedure example was followed;
however, the enantiomeric excess of the products was determined by derivatization of the
o
alcohols with an optically pure (-)S-1-phenylethyl isocyanate in toluene at 140 C,
25

ACCEPTED MANUSCRIPT
followed by separation with a
After 24 h, the reaction mixture was extracted with CH
detectable alcohols in the mother liquor. The combined extracts were dried with Na SO ,
β-cyclodextrin column. The workup is detailed as follows:
2
Cl , until there was no GC-
2
2
4
followed by distilling off the solvent, along with any residual water, which could be
removed azeotropically with CH Cl , in an oil-bath under argon. The resulting residue
2
2
was brought into a glove box, and 1.5 mL of toluene (CaH dried) was used to make a
2
solution, which was then transferred to a Schlenk tube. (-)S-1-phenylethyl isocyanate (in
excess) was then added to the above-stated solution, and the Schlenk tube was sealed and
o
brought out of the glove box. The reaction flask was put in a 140 C oil-bath for 12~20 h.
The diastereomeric purity of the formed diastereomers was determined by their relative
areas obtained in the chromatogram, and was confirmed by authentic compounds and
GC-MS. The activities were virtually the same between the three co-factors, 1, 3, and
+
NAD , in the first "24 to ~48h". For a prolonged reaction time, the reactivity for natural
+
NAD was better.
+
The Consequences of an NAD Contamination of the HLADH Enzyme, on the
Formation of Chiral S-2-Pentanol.
Since the purchased HLADH enzyme contained sodium phosphate, and a very
+
small amount of NAD , per the description by Sigma (combined impurities, <5% by
weight), a series of control experiments were performed to verify the possible influence
+
of the stated NAD impurity in HLADH, used in our biomimetic reduction reactions for
the synthesis of chiral alcohols.
Control Experiment (1): Using 2-pentanone, 10, and PhCH CH C(O)CH , 7, as the
2
2
3
ketone substrates, and under the same conditions as described in the General Procedure,
+
but in the absence of the NAD biomimic, 3, only racemic alcohols, without a measurable
-1
ee in the products, could be detected in 24h, and both at RT (0% ee; TOF, 10 d ), and at
o
-1
3
0 C (0% ee; TOF, 14 d ), respectively. The reaction rates were found to be
comparable to the direct reduction of ketone substrates by precatalyst
Cp*Rh(bpy)(H O)](OTf) with sodium formate, under similar conditions. For example,
[
2
2
-1
a yield of 33% (TOF, 11 d ) was obtained in the case of 2-pentanone, 10, for racemic, 2-
pentanol, after 24h at room temperature[15].
Control Experiment (2): Under the same conditions as described in the Control
Experiment (1), but using twice the amounts of the ketone substrates, at room
temperature, provided only racemic 2-pentanol, without a measurable % ee, detected after
2
4h. The yield was not determined. As a result of these control experiments, there was
+
no NAD in the HLADH used in our biomimetic reactions, and could be excluded as the
responsible source for the obtained enantioselectivity in our biomimetic studies. That is,
the recognition of the formed 1,4-NADH biomimics, 4 and 6, by HLADH, was clearly
shown under the given conditions in our biomimetic system.
26

ACCEPTED MANUSCRIPT
+
Biomimetic Oxidation of S-2-Pentanol Using Natural NAD as the Co-factor with
HLADH.
+
-3
NAD (83.58 x 10 mmol) and HLADH (10 units) were placed in a 10 mL
Schlenk flask, and Schlenk techniques were followed to deoxygenate the solid mixture.
Under positive argon pressure, 5 mL of potassium phosphate buffer (100 mM, pH = 7.04,
-3
de-O ) and S-2-pentanol (83.58 x 10 mmol) were added via syringe, respectively. The
2
reaction flask was immediately capped securely with a glass-stopper and shaken with a
shaker at room temperature. The progress of the reaction was monitored by GC, and the
products were identified by comparing the retention time with that of authentic samples.
Under similar conditions, the oxidation of R-2-pentanol was tested as well, and its
reaction rate was found to be much slower relative to that of S-2-pentanol. The relative
rates of S- and R-2-pentanols in the first 24 hr were ~ 4:1. Additionally, the reactions
were found to reach an equilibrium after ~ 60 h (in the case of S-2-pentanol), where both
2
-pentanol and 2-pentanone were present in the reaction mixture in a ratio of ~ 40 to 60.
Biomimetic Oxidation of S-2-Pentanol Using NAD+ Biomimetic Cofactor, 3, with
HLADH.
+
The same procedure described in the NAD example was followed; however, the
+
+
NAD was replaced with the water soluble NAD biomimic, 3. Both racemic 2-pentanol
+
and S-2-pentanol were tested, and similar results were obtained as stated in the NAD
case, except that the reaction rates became slower after 24 h.
Molecular Recognition Experiments: Stoichiometric Reactions were Conducted to
+
Further Confirm the Molecular Recognition of NAD /1,4-NADH Biomimics by
HLADH.
The purpose of these experiments was to insure that the HLADH enzyme can
+
recognize our NAD /1,4-NADH biomimics, 3/6, providing good yields and high
+
enantioselectivities, as does the natural NAD /1,4-NADH co-factors, under the same
conditions.
Stoichiometric Reduction of 2-Pentanone with 1,4-NADH Biomimic, 6, and HLADH
at Room Temperature.
-3
1
,4-NADH biomimic, 6 (21.80 mg, 82.77 x 10 mmol), and HLADH (23.80 mg,
1
0.23 units) were placed in a 10 mL Schlenk flask capped with a septum, and Schlenk
techniques were carefully followed to deoxygenate the solid mixture. Potassium
phosphate buffer (5 mL, 100 mM, pH = 7.02, de-O ) was added to give a clear solution,
2
-3
followed by the addition of 2-pentanone (8.8
respectively. Under a gentle N pressure, the septum was removed, and the reaction flask
was capped with a glass-stopper sealed with a small amount of grease and a metal clip.
The stopcock of the reaction flask to N gas was then shut, and the reaction flask was
µL, 82.66 x10 mmol) via syringes
2
2
27

ACCEPTED MANUSCRIPT
immediately shaken in a water-bath shaker at room temperature with the water level
above the reaction mixture solution. The reaction was monitored by means of GC and
was quenched after 24h. After the workup and derivatization of the reaction mixture as
stated in General GC-Analysis Process, the ee was found to be 25% (yield, 35%);
o
however, if the reaction temperature was raised to 30 C, we found a dramatic
o
increase in 85% ee, and yield, 41%, See Table 1 for all 30 C results with prochiral
ketones 7-10.
Stoichiometric Reduction of 2-Pentanone, 10, with 1,4-NADH and HLADH at Room
Temperature[8].
The same procedure described with 1,4-NADH biomimic, 6, was followed with
1
1
,4-NADH (after 24h:, 52% ee; yield, 42%). Although both the biomimic, 6, and natural
,4-NADH provided comparable reactivities toward the HLADH enzyme, at room
temperature (35% vs 42%), the enantioselectivity of S-2-pentanol with 1,4-NADH, was
much higher than with biomimic 6, under the given conditions. The results observed in
+
the stoichiometric oxidation reactions clearly illustrate that not only can NAD /1,4-
NADH biomimics 3/6 be recognized by HLADH in effectively carrying out the
transformation between 2-pentanol and 2-pentanone, but the S-alcohol equally displays
+
much higher reactivities toward HLADH with NAD biomimic 3 than the R-alcohol, as
+
was found with NAD in the case of oxidation reactions. Therefore, the coordination
+
between HLADH and the NAD biomimics, 1 and 3, have been further substantiated.
+
Stoichiometric Oxidation of S-2-Pentanol with NAD Biomimic, 3, and HLADH at
Room Temperature [6].
Biomimic 3 (29.30 mg, 82.93 x 10-3 mmol) and HLADH (23.80 mg, 10.23 units)
were placed in a 10 mL Schlenk flask capped with a septum, and Schlenk techniques
were carefully followed to deoxygenate the solid mixture. Potassium phosphate buffer (5
mL, 100 mM, pH = 7.02, de-O
addition of S-2-pentanol (9.0
gentle N pressure, the septum was removed, and the reaction flask was capped with a
2
) was added to give a clear solution, followed by the
-3
µ
L, 82.90 x 10 mmol) via syringes, respectively. Under a
2
glass-stopper sealed with a small amount of grease and a metal clip. The stopcock of the
reaction flask to N gas was then shut, and the reaction flask was immediately shaken in a
2
water-bath shaker at room temperature with the water level above the reaction mixture
solution. The reaction was monitored by means of GC for 72h. The product was
identified by comparing the retention time with that of 2-pentanone (after 24h: yield,
5
3%). Under the same conditions, stoichiometric oxidation of R-2-pentanol
was tested as well, and its reaction rate was found to be much slower compared to that of
S-2-pentanol. The reaction rates of S- and R-2-pentanols, in the first 24h, were found to
be~ 4:1. Additionally, the reactions appeared to reach an equilibrium after ~ 60h (in the
case of S-2-pentanol), where both 2-pentanol and 2-pentanone were present in the
reaction mixture in a ratio of ~ 40 to 60. While the reactivities of S- and R-2-pentanols
toward HLADH with biomimic 3 were tested separately, racemic 2-pentanol was also
examined under the same conditions. Identical results were obtained no matter S- and R-
2
-pentanols were co-present or separately in the reaction mixture.
28

ACCEPTED MANUSCRIPT
+
Stoichiometric Oxidation of S-2-Pentanol with NAD and HLADH at Room
Temperature[6].
The same procedure described for the biomimetic model 3 was followed with
NAD . Based on GC chromatogram analyses, both biomimic 3 and NAD exhibited
+
+
almost identical reactivities toward HLADH under the same conditions (after 24h: yield,
5
6%), except the reactions with biomimic 3 became much slower after 24h, and that the
enzyme containing precipitate in the case of 3 was slightly more significant compared to
+
cases observed with NAD . Stoichiometric oxidation with biomimic, 1, was not tested,
due its limited solubility in phosphate buffers, under the given conditions.
Synthesis of N-4-Methoxybenzylnicotinamide Bromide[7a].
To a 100 ml roundbottom flask was added 3g (0.025 mmol) of nicotinamide and
5
g (0.025mmol) of 4-methoxybenzyl bromide dissolved in 40 ml of dry THF. The salt
precipitated on mixing and was filtered and washed with diethyl ether and dried under
1
vacuum to provide 7g (88 % yield). H NMR (D O): d 9.30 (s, 1H, H2, Py); 9.15, (d, J =
2
5
8
.0, 1H, H6, Py); 8.86 (d, J = 8.0, 1H, H4, Py); 8.15 (t, J = 8.0, 1H, H5, Py); 7.46 (d, J =
.0, 2H, Ph H3); 7.03 (d, J = 8.0, 2H, Ph H2); 5.81 (s, 2H, Ph-CH ), 3.81 (s, Ph-OCH ).
2
3
Synthesis of N-4-Methoxy-1,4-dihydronicotinamide[7a].
To 2g (0.0062 mmol) of N-4-methoxybenzylnicotinamide bromide was added 2.6
g (24.4 mmol) of sodium carbonate and 4.3 g (24.4 mmol) of sodium dithionite dissolved
in 30 ml of deoxygenated water, and under Ar gas. The reaction mixture turned cloudy
and a yellow oil appears on the walls of the 100 ml flask. The work-up consisted of
washing the chilled in ice reaction mixture 3X with water, and drying on a high vacuum
line to provide 1.48 g (~100%) of the yellow glass product, N-4-methoxybenzyl-1,4-
1
dihydronicotinamide. H NMR (CDCl ): d 7.17-7.15 (m, 4H, H2, H3, Ph); 6.87, (d, J =
3
1
4
3
.6, 1H, H2, dihydropy); 5.72 (qd, J = 1.6, 8.0, 1H, H6, dihydropy); 5.17 (bs, 2H, NH2);
.73 (td, J =3, 8, 1H, H5, dihydropy); 4.22 (s, 2H, Ph-CH ), 3.81 (s, Ph-OCH );
.15 ( dd, J = 1.6, 3.5, 2H, H4 dihydropy). Due to the hydroscopic and light sensitivity
2
3
nature of the 1,4-NADH biomimics, we were not able to obtain reliable elemental or
1
mass spectral data, but the H NMR data was unequivocal for the structure assigned.
29