Coenzyme analogs: Excellent substitutes (not poor imitations) for electrochemical regeneration

Article abstract of DOI:10.1039/c1cc14313a

For the first time, employment of nicotinamide coenzyme NAD analogs has overcome the limitations of NAD in electrochemical regeneration. It has been shown that NAD analogs, APAD and PAAD, were electrochemically reduced more efficiently than original NAD and that the stability of their reduced products was also much higher than NADH.

Full text of DOI:10.1039/c1cc14313a

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2011, 47, 12538–12540
www.rsc.org/chemcomm
COMMUNICATION
Coenzyme analogs: excellent substitutes (not poor imitations) for
electrochemical regenerationw
a
b
b
a
Hye Jung Lee, Sahng Ha Lee, Chan Beum Park* and Keehoon Won*
Received 18th July 2011, Accepted 27th September 2011
DOI: 10.1039/c1cc14313a
For the first time, employment of nicotinamide coenzyme NAD
analogs has overcome the limitations of NAD in electrochemical
regeneration. It has been shown that NAD analogs, APAD and
PAAD, were electrochemically reduced more efficiently than
original NAD and that the stability of their reduced products
was also much higher than NADH.
enzymatic method. We recently introduced metallic nano-
particles to significantly enhance the electrochemical reduction
8
rate of NAD. Another major limitation of NAD(P)H for
industrial application is its low stability in aqueous solution. It
is well known that NADH decomposes under neutral and
9
acidic conditions at ambient temperatures.
This work, for the first time, employed NAD analogs to
improve the efficiency of electrochemical cofactor regeneration
Enzymes have long been successfully employed as biocatalysts
in organic synthesis because they possess high specificity and
1
catalytic activity even under mild conditions. Among six
(
Scheme 1). Analogs of pyridine nucleotide coenzymes have
different optical and chemical properties, even reduction
potentials, from those of NAD, and thus have been applied
classes of enzymes, oxidoreductases are promising because
they can catalyze complex reactions involving oxygen
insertion, hydrogen extraction, electron transfer, proton
1
0
to various biochemical researches. While the low reduction
potential of NAD makes it reluctant to electrochemical
reduction, its analog can be reduced more efficiently if the
analog’s potential is more positive. However, little attention
has been paid to the coenzyme analogs as substitutes for NAD
in electrochemical regeneration. We tested four commercially
available NAD analogs with the 3-substituted pyridine
ring: thionicotinamide adenine dinucleotide (thio-NAD),
2
abstraction, hydride transfer, and so on. However, the
applications of redox enzymes are limited, mainly because of
their requirement for nicotinamide coenzymes (nicotinamide
adenine dinucleotide [NAD(H)] and its respective phosphate
[
NADP(H)]), which are too expensive to be used in stoichio-
metric quantities. Therefore, efficient regeneration of the
coenzymes is essential for successful application of redox
enzymes to chemical processes. Compared with their oxidized
counterparts, the reduced forms of nicotinamide coenzymes,
NAD(P)H, are used for more diverse, useful redox reactions
3
-acetylpyridine adenine dinucleotide (APAD), 3-pyridine-
aldehyde adenine dinucleotide (PAAD), and nicotinic acid
adenine dinucleotide (NAAD). Their spectral and electro-
1
1
chemical properties are shown in Table S1, ESIw.
3
such as reductions, hydroxylations, and epoxidation.
Enzymatic, electrochemical, and photochemical methods have
3–5
been developed for the regeneration of nicotinamide coenzymes.
In particular, electrochemical coenzyme regeneration is often
favoured owing to no requirement of impurities such as a
second enzyme, co-substrate, a photosensitizer, and sacrificial
3
,5
electron donors, when compared with the other methods.
Furthermore, the use of organometallic mediators such as
2
+
[
Cp*Rh(bpy)H
2
O]
(Mox) solved the problem of isomer
,7
6
and dimer formation. However, the rate of electrochemical
cofactor regeneration is still unsatisfactory compared to the
a
Department of Chemical and Biochemical Engineering,
Dongguk University-Seoul, 26 Pil-dong 3-ga, Jung-gu,
Seoul 100-715, Republic of Korea. E-mail: keehoon@dongguk.edu;
Fax: +82 2 2268 8729; Tel: +82 2 2260 8922
b
Department of Materials Science and Engineering, Korea Advanced
Institute of Science and Technology, Science Road 335, Yuseong-gu,
Daejeon 305-701, Republic of Korea. E-mail: parkcb@kaist.ac.kr;
Fax: +82 42 350 3310; Tel: +82 42 350 3340
w Electronic supplementary information (ESI) available. Spectral and
electrochemical properties of the NAD analogs. See DOI: 10.1039/
c1cc14313a
Scheme 1 Schematic illustration of electrochemical reduction of
2+
O]
NAD analogs mediated by [Cp*Rh(bpy)H
2
.
1
2538 Chem. Commun., 2011, 47, 12538–12540
This journal is c The Royal Society of Chemistry 2011

View Article Online
3
,7
1
,4-NADH (selectivity 4 99%). However, there has been
no report on the regioselectivity of Mred toward NAD analogs
such as APAD and PAAD. To determine the amount of
enzymatically active coenzymes, the electrochemically reduced
NAD and its analogs were enzymatically oxidized with 20 mM
a-ketoglutarate and 50 mM ammonium sulfate using
L-glutamate dehydrogenase (L-GDH) in a phosphate buffer
(
0.1 M, pH 7). The absorbance decrease at lmax of each
coenzyme is caused by oxidation of only enzymatically active
reduced coenzymes. If the ratio of active reduced coenzymes to
total electrochemically reduced coenzymes (yield of active
coenzyme) is 100%, the absorbance at lmax will eventually
be decreased to 0 by the enzymatic reaction. As expected, the
NAD electrochemically reduced by Mred (i.e., NADH) was
found to be nearly 100% active. It was revealed that the
electrochemically reduced APAD and PAAD (i.e., APADH
and PAADH) showed about 80% yield of active coenzyme,
slightly lower than that of NADH (Fig. 1). For reference, we
also performed the reduction of NAD, APAD, and PAAD
with formate using formate dehydrogenase, which is most
widely used for enzymatic NADH regeneration, and then
measured the yields of active coenzyme in the same manner.
We observed that the absorbance at lmax decreased to 0 in all
cases. The yields of active APADH and PAADH are accep-
table considering that their production rate and conversion
were far greater than those of NADH. Moreover, their yields
might be improved by changing metals and/or ligands of the
Fig. 1 Electrochemical reduction of 1 mM NAD and its analogs in
the presence of the rhodium complex mediator (Mox) and yield of their
enzymatically active reduced products.
The indirect electrochemical reduction of NAD and its
analogs was performed in a three-electrode cell immersed in
1
2
a temperature-controlled water bath. As shown in Fig. 1,
NAD as well as its analogs were successfully reduced—except
for thio-NAD and NAAD. In particular, APAD and PAAD
showed a two- to three-fold increased conversion compared
with NAD. This happened because they have more positive
reduction potentials (Table S1, ESIw). The equilibrium is much
more favourable towards the reduction reaction for APAD
1
5
organometallic mediator.
Fig. 1 indicates that the concentration of the produced
NADH reached 0.3 mM and then decreased with time,
whereas the PAADH concentration increased continuously.
This is due to the decomposition of NADH, which is unstable
in an aqueous solution. NADH decomposes via general
acid-catalyzed protonation at C-5 of the nicotinamide ring,
followed by nucleophilic attack on C-6 of the resulting
(
À258 mV) and PAAD (À262 mV) than for NAD (À320 mV).
In fact, the equilibrium constants for enzymatic reduction of
these analogs turned out to be approximately 200 times higher
9
,16,17
imine.
NADH’s acid-catalyzed addition reaction is
1
3
than that of NAD. The small reduction of thio-NAD cannot
be explained by the redox potential because it also has more
positive redox potential (À285 mV) than NAD. The reduced
verified by rapid loss of the UV-absorbing chromophore at
1
7
340 nm of the dihydro-nicotinamide ring. In addition, even
in neutral solutions in which hydrogen ion concentration is
low, some buffer ions, particularly phosphate ions, interact
with NADH through complexation, resulting in the accelera-
+
rhodium complex [Cp*Rh(bpy)H] (Mred) generated from Mox
might have no specificity for thio-NAD. However, it was
reported that 1-benzylthionicotinamide in which benzene is
substituted for adenosine 5-diphosphoribose (ADPR) of thio-
NAD exhibited a higher reaction rate and turnover frequency
than 1-benzylnicotinamide in regioselective reduction with
1
8–20
tion of NADH decomposition.
In this respect, we attempted to investigate stability of the
reduced analogs and to contrast them with that of NADH. We
incubated NADH, APADH, and PAADH in a phosphate
buffer (0.1 M, pH 7) at four different temperatures of 4 1C,
25 1C, 37 1C, and 60 1C for 48 h, during which the concentra-
tions of the enzymatically active coenzymes were measured at
predetermined intervals. The natural logarithm of the relative
concentrations was plotted against the incubation time.
Apparently the decrease in the relative concentration followed
first-order kinetics. At the same temperature, the relative
concentration of NADH decreased with time more sharply
compared to that of APADH and PAADH, as shown in
Fig. 2(a)–(c). For example, at 37 1C—an optimum tempera-
ture for most enzymatic reactions—the relative concentration
of NADH was observed to be only 5% after 24 h, whereas
those of APADH and PAADH were as high as 60% and 80%,
respectively. We also evaluated the thermal decomposition
1
,
4
M
red
indicating that the rhodium mediator can also display
activity on thio-NAD because the adenosine residue is not
directly involved in coordination of the Rh-central atom to
6
,14
the carbonyl-O-atom of NAD.
Therefore, for the present, it
is unclear why thio-NAD was not reduced electrochemically.
Similarly, the reason is not obvious for lack of electrochemical
reduction for NAAD—for which redox potential and affinity
for Mred are not reported—and is a question for future studies.
We further investigated whether the electrochemically
reduced forms of NAD, APAD, and PAAD were active as a
coenzyme for oxidoreductases, which is essential for coenzyme
regeneration to be used in an enzymatic reaction. Mred is
known to function as a regioselective hydride transfer agent
on NAD, yielding only the enzymatically active form of
This journal is c The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 12538–12540 12539

View Article Online
J. H. Ryu and Y. J. Yoo, Biotechnol. Bioprocess Eng., 2010,
1
5, 371; (d) A. Salic, A. Tusek, Z. Kurtanjek and B. Zelic,
Biotechnol. Bioprocess Eng., 2011, 16, 495; (e) H. G. Kim,
G. H. Han and S. W. Kim, Biotechnol. Bioprocess Eng., 2010,
1
5, 476.
3
4
F. Hollmann and A. Schmid, Biocatal. Biotransform., 2004, 22, 63.
(a) S. H. Lee, J. Ryu, D. H. Nam and C. B. Park, Chem. Commun.,
2
011, 47, 4643; (b) S. H. Lee, D. H. Nam and C. B. Park, Adv.
Synth. Catal., 2009, 351, 2589; (c) S. H. Lee, D. H. Nam,
J. H. Kim, J.-O. Baeg and C. B. Park, ChemBioChem, 2009,
10, 1621; (d) D. H. Nam, S. H. Lee and C. B. Park, Small, 2010,
6, 922; (e) E. Siu, K. Won and C. B. Park, Biotechnol. Prog., 2007,
23, 293.
5
6
(a) W. A. van der Donk and H. Zhao, Curr. Opin. Biotechnol.,
2
2
003, 14, 421; (b) W. Liu and P. Wang, Biotechnol. Adv., 2007,
5, 369.
(a) R. Ruppert, S. Herrmann and E. Steckhan, Tetrahedron Lett.,
987, 28, 6583; (b) F. Hollmann, A. Schmid and E. Steckhan,
Angew. Chem., Int. Ed., 2001, 40, 169.
1
7
8
F. Hollmann, B. Witholt and A. Schmid, J. Mol. Catal. B: Enzym.,
2
(a) H.-K. Song, S. H. Lee, K. Won, J. H. Park, J. K. Kim, H. Lee,
S.-J. Moon, D. K. Kim and C. B. Park, Angew. Chem., Int. Ed.,
2008, 47, 1749; (b) S. H. Lee, K. Won, H.-K. Song and C. B. Park,
Small, 2009, 5, 2162.
003, 19, 167.
Fig. 2 Decrease in relative concentrations of enzymatically active (a)
NADH, (b) APADH, and (c) PAADH over time at different
temperatures and (d) their deactivation energy. NADH was purchased
from Sigma (USA), and the others were obtained from the electro-
chemical reduction experiment in Fig. 1.
9
P. L. Hentall, N. Flowers and T. D. H. Bugg, Chem. Commun.,
001, 2098.
2
0 (a) B. M. Anderson, in The Pyridine Nucleotide Coenzymes, ed.
1
J. Everse, B. Anderson and K.-S. You, Academic Press, New York,
982, pp. 91–133; (b) C. Woenckhaus and R. Jeck, in Pyridine
1
d
constant (k ) from the slopes shown in Fig. 2(a)–(c) and found
Nucleotide: Coenzymes Part A, ed. D. Dolphin, R. Poulson and
O. Avramovic, John Wiley & Sons, New York, 1987, pp. 449–568;
(c) B. Ferrier, Anal. Biochem., 1990, 186, 229.
that the k of each coenzyme decreased as the substituent at
d
the 3 position acts as an electron sink, which is in agreement
1
6,18
11 (a) P. M. Weiss, S. R. Gavva, B. G. Harris, J. L. Urbauer,
W. W. Cleland and P. F. Cook, Biochemistry, 1991, 30, 5755;
with the literature.
This is attributed to the acceleration of
the acid-catalyzed addition reaction of NADH by electron
(
b) M. Lamborg, F. E. Stolzenbach and N. O. Kaplan, J. Biol.
1
9
donating or basicity-increasing substituents. An Arrhenius
plot of ln k versus 1/T allowed us to calculate the deactivation
energy (E ). As shown in Fig. 2(d), the E values reveal
Chem., 1958, 231, 685; (c) A. Chiba, T. Eguchi, T. Oshima and
K. Kakinuma, Biosci., Biotechnol., Biochem., 1999, 63, 1647.
2 The electrochemical reduction reaction was carried out in a
phosphate buffer (0.1 M, pH 7) containing 0.5 mM Mox and
d
1
d
d
that APADH and PAADH have much higher stabilities
than NADH.
1
mM NAD or its analogs with stirring at 25 1C for 24 h while
a constant potential of À0.8 V (vs. Ag/AgCl) was applied by a
potentiostat (Gamry G750, USA). A conventional three-electrode
configuration was used: the working, counter, and reference
electrodes were 10 mm  10 mm  0.025 mm gold foil (width Â
height  thickness), a platinum wire, and Ag/AgCl (saturated
KCl), respectively. The reaction was monitored by measuring the
absorbance at the maximum wavelength (lmax) of each coenzyme
from which the concentration of the reduced NAD and its analogs
was estimated.
In conclusion, we have introduced NAD analogs to electro-
chemical regeneration to overcome NAD’s limitations, such as
low reduction conversion and poor stability. It was found that
APAD and PAAD were electrochemically reduced more
efficiently while they showed the yield of the enzymatically
active reduced coenzyme comparable to original NAD. More-
over, at ambient temperatures, the stability of APADH and
PAADH was found to be much higher than that of NADH.
This work has demonstrated that NAD analogs can be
excellent coenzymes to be reduced via an electrochemical
regeneration method and to be applied to redox enzymatic
reactions. The present work has encouraged us to introduce
these coenzyme analogs for photochemical regeneration, a
study currently underway.
13 (a) B. M. Anderson and N. O. Kaplan, in Pyridine Nucleotide:
Coenzymes Part A, ed. D. Dolphin, R. Poulson and O. Avramovic,
John Wiley & Sons, New York, 1987, pp. 569–611; (b) N. O.
Kaplan, M. M. Ciotti and F. E. Stolzenbach, J. Biol. Chem., 1956,
221, 833.
14 (a) H. C. Lo, O. Buriez, J. B. Kerr and R. H. Fish, Angew. Chem.,
Int. Ed., 1999, 38, 1429; (b) H. C. Lo, C. Leiva, O. Buriez,
J. B. Kerr, M. M. Olmstead and R. H. Fish, Inorg. Chem., 2001,
4
15 (a) E. Steckhan, S. Herrmann, R. Ruppert, E. Dietz, M. Frede and
0, 6705.
E. Spika, Organometallics, 1991, 10, 1568; (b) J. Canivet,
ka, Eur. J. Inorg. Chem., 2007, 4736.
This research was supported by the Converging Research
Center Program (2011-K000845) and the Original Technology
Research Program for Brain Science (2011-0030168) through
the National Research Foundation of Korea (NRF) funded by
the Ministry of Education, Science and Technology.
ˇ
ss-Fink and P. Ste
G. Su
¨
ˇ
pnic
ˇ
1
6 A. G. Anderson Jr. and G. Berkelhammer, J. Am. Chem. Soc.,
958, 80, 992.
1
17 N. J. Oppenheimer, in Pyridine Nucleotide Coenzymes: Part A,
ed. D. Dolphin, R. Poulson and O. Avramovic, John Wiley &
Sons, New York, 1987, pp. 323–365.
1
8 S. G. A. Alivisatos, F. Ungar and G. Abraham, Nature, 1964,
03, 973.
Notes and references
2
1
(a) T. Hudlicky and J. W. Reed, Chem. Soc. Rev., 2009, 38, 3117;
b) K. Faber, Biotransformations in Organic Chemistry, Springer,
Berlin, 5th edn, 2004.
(a) F. Xu, Ind. Biotechnol., 2005, 1, 38; (b) V. B. Urlacher and
R. D. Schmid, Curr. Opin. Chem. Biol., 2006, 10, 156; (c) K. Min,
19 S. L. Johnson and P. T. Tuazon, Biochemistry, 1977, 16, 1175.
20 (a) C.-H. Wong and G. M. Whitesides, J. Am. Chem. Soc., 1981,
103, 4890; (b) L. Rover Jr., J. C. B. Fernandes, G. O. Neto,
L. T. Kubota, E. Katekawa and S. H. P. Serrano, Anal. Biochem.,
(
2
1
998, 260, 50.
1
2540 Chem. Commun., 2011, 47, 12538–12540
This journal is c The Royal Society of Chemistry 2011