Synthesis and Biochemical Evaluation of Nicotinamide Derivatives as NADH Analogue Coenzymes in Ene Reductase

Article abstract of DOI:10.1002/cbic.201800661

Nicotinamide and pyridine-containing conjugates have attracted a lot of attention in research as they have found use in a wide range of applications including as redox flow batteries and calcium channel blockers, in biocatalysis, and in metabolism. The interesting redox character of the compounds’ pyridine/dihydropyridine system allows them to possess very similar characteristics to the natural chiral redox agents NAD⁺/NADH, even mimicking their functions. There has been considerable interest in designing and synthesizing NAD⁺/NADH mimetics with similar redox properties. In this research, three nicotinamide conjugates were designed, synthesized, and characterized. Molecular structures obtained through X-ray crystallography were obtained for two of the conjugates, thereby providing more detail on the bonding and structure of the compounds. The compounds were then further evaluated for biochemical properties, and it was found that one of the conjugates possessed similar functions and characteristics to the natural NADH. This compound was evaluated in the active enzyme, enoate reductase; like NADH, it was shown to help reduce the C=C double bond of three substrates and even outperformed the natural coenzyme. Kinetic data are reported.

Full text of DOI:10.1002/cbic.201800661

Accepted Article
Title: Synthesis and Biochemical Evaluation of Nicotinamide
Derivatives as NADH analogues in Ene Reductase
Authors: Natashya Falcone, Zhe She, Jebreil Syed, Alan Lough, and
Heinz-Bernhard Kraatz
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10.1002/cbic.201800661
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Synthesis and Biochemical Evaluation of Nicotinamide
Derivatives as NADH analogues in Ene Reductase
Natashya Falcone, [a, b] Zhe She, [b] Jebreil Syed, [b] Alan Lough, [c] and Heinz-Bernhard Kraatz*[a, b, c]
Abstract: Nicotinamide and pyridine-containing conjugates have
attracted a lot of attention in research as they have found use in a
wide range of applications including as redox flow batteries, calcium
channel blockers, and in biocatalysis, and metabolism. The interesting
redox character of the compounds’ pyridine/dihydropyridine system
allows them to possess very similar, if not able to mimic, the functions
and characteristics of the natural chiral redox agents NAD+/NADH.
Considerable interest has been given in designing and synthesizing
NAD+/NADH mimics with similar redox properties. In this research,
three nicotinamide conjugates were designed, synthesized and
characterized. Molecular structures obtained through X-ray
crystallography were obtained for two conjugates providing more
detail into the bonding and structure of the compounds. The
compounds were then further evaluated for biochemical properties
and it was found that one of the conjugates possessed similar
functions and characteristics to the natural NADH compound.
Compound 4 was evaluated in the active enzyme, Enoate Reductase
and compared to NADH, it was shown to be successful in reducing
the C=C double bond of three substrates and outperformed the
natural coenzyme, kinetic data has been reported.
critical sense that it is a catalytic hydride donor similar to NADH
rather than a stoichiometric hydride reagent.[9] This is due to the
fact that the pyridines catalytic behaviour lies in the chemistry of
the 1,2-dihydropyridine/pyridine redox couple driven by
a
dearomatization-aromatization process. The biologically
important redox couple provides a reversible formation of a C-H
bond centered on the nicotinamide ring representing an efficient
system for numerous biological hydrogen-transfer reactions.[9]
Considerable interest has been given in creating these
nicotinamide conjugates that are able to mimic the activity of the
NAD+/NADH redox couple[10,11] and mimic the activity from the
viewpoints of the electrochemical behavior. Many groups have
studied the electrochemical behaviour and regeneration of
NAD+/NADH.[12–15] Sanford et al. designed and synthesized
various pyridine containing compounds and used its redox
properties in the application of redox flow batteries.[16] They
reported the evolutionary design of a series of pyridine-based
anolyte materials that exhibit up to two reversible redox couples
at low potentials in the presence of Li-ion supporting electrolytes.
The
Colbran
group
reported
the
synthesis
of
pyridine/dihydropyridine conjugates for bio-inspired reduction and
multi-electron reduction,[17] and demonstrated that a combination
of an organic hydride pyridinium compound and a metal center
can complex and lead to an efficient catalyst for transfer
hydrogenation. In addition, Musgrave et al. reported the reduction
of CO2 to methanol catalyzed by a biomimetic organo-hydride
produced from pyridine, where the 1,2-dihydropyridine acts as a
recyclable organo-hydride that reduced CO2 to methanol via three
hydride and proton transfer steps.[18] Dihydropyridine compounds
have also been heavily reported to act as calcium channel
blockers and are explored for the development of pain
therapeutics.[4,20] Given the potential application in biocatalysis, it
is not surprising that the synthesis of these pyridine conjugates
Introduction
Nicotinamide and pyridine-containing conjugates have attracted a
lot of attention in the past years due to their variety of applications
in different fields, from playing a key role in metabolism, to being
an electron carrier in many biological pathways, and for their roles
in medicinal scaffolds and as redox flow batteries.[1–6] The most
common and biologically significant nicotinamide conjugate is
NAD+/NADH, nicotinamide adenine dinucleotide, which is a
cofactor involved in over 300 biochemical reactions and biological
processes including redox reactions, cellular metabolism, and
cellular respiration.[7,8] Nicotinamide conjugates containing the
pyridine/dihyropyridine system, which NAD+ possesses, are the
closest analogues to the natural cofactor found in nature. It has
been reported that dihydropyridine compounds are unique in the
have attracted the attention of numerous researchers for years.[21–
27]
Not only has considerable interest been given in creating
model compounds that mimic the activity of the NAD+/NADH
redox couple, but mimics containing the nicotinamide moiety
have been synthesized and tested in active enzymes as
replacements to the natural cofactors. Lowe et al. developed an
artificial coenzyme, CL4, which retained the nicotinamide portion
and replaced the phosphate and adenine moieties with other
functional groups. They showed its activity in the active enzyme
horse liver alcohol dehydrogenase (HLADH).[28] A couple years
later Fish et al. demonstrated that a simpler nicotinamide
coenzyme mimic, also retaining the nicotinamide portion and
substituting the rest with a simple benzyl group, displayed activity
in HLADH.[29,30] This group continued to report on these mimics
being able to catalyze the reduction of aldehyde compounds to
chiral alcohol products and for other hydrogenation
applications.[30–33] Through many studies it was demonstrated
that the nicotinamide moiety of NAD+ is all that is necessary for
enzyme recognition and redox function.[29,34,35] This concept was
[a]
N. Falcone, Prof. H-B. Kraatz
Department of Chemical Engineering and Applied Chemistry
University of Toronto
200 College Street, Toronto, Canada, M5S 3E5
E-mail: bernie.kraatz@utoronto.ca
[b]
[c]
N. Falcone, Dr. Z. She, J. Syed, Prof. H-B. Kraatz
Department of Physical and Environmental Sciences
University of Toronto Scarborough
1065 Military Trail, Scarborough, Canada, M1C 1A4
Dr. A. Lough, Prof. H-B. Kraatz
Department of Chemistry
University of Toronto
80 St. George Street, Toronto, Canada, M5S 3H6
Supporting information for this article is given via a link at the end of
the document.
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reductive half reaction, a hydride is transferred from NADH (or a mimic of).
NADH oxidation can be followed spectroscopically by monitoring decrease of
the characteristic absorption band at 340-360 nm. In the oxidative half reaction,
the hydride is then transferred from the bound-flavin, oxidizing the flavin, to the
alkene substrate for C=C reduction.
later further applied by Clark et al. in cytochrome P450 enzymes
where they engineered heme-containing enzymes for improved
activity towards the same biomimetic cofactor.[36] In addition, Paul
and Hollman designed and evaluated a range of NADH biomimics
in enoate reductase (ER) enzymes.[34,37] Although biomimics of
NAD(H) have been developed and have found applications within
chemo-enzymatic synthesis, to date, only a few examples have
been published including reports on kinetic data for
reductases.[34,37,38] In addition, many of the reported NADH mimics
mentioned above are similar in structure and other functional
groups conjugated to the pyridine ring have not been explored for
further alterations.
Herein, we report the synthesis and characterization of
three nicotinamide-containing conjugates. These were
characterized, including two of the compounds, which were
characterized by single X-ray crystallography, allowing the
determination of intra- and intermolecular interactions.
Specifically, these NAD-mimics were designed with future
modifications in mind that may allow further alterations and
conjugations. From the X-ray structures we observe the
engagement of intermolecular bonding between neighboring
nicotinamide moieties through the carbonyl and amide nitrogen
Compound 4 displayed biological activity with ER allowing us to
drive the enzymatic transformation of three enoate substrates.
Here, we report a class 2 Ene reductase (see SI) that accepts a
nicotinamide coenzyme biomimetic with
a side by side
comparison of kinetics to the natural coenzyme, NADH. We
provide details of this study including the design and synthesis of
the compounds, the biochemical evaluation of them, and detailed
enzyme kinetic analysis.
Results and Discussion
Three pyridine-containing conjugates were synthesized using
nicotinamide and nicotinic acid as the starting materials.
Conjugate 1, a phenylalanine derivative, was synthesized by
peptide coupling, followed by pyridine-modification using benzyl
bromide. Conjugates 2 and 3, used nicotinamide as the starting
material and were synthesized by a simple one-step reflux
reaction with 5-bromo valeric acid and 4-bromomethyl benzoic
acid, respectively. It was reported that the nicotinamide moiety is
all that is necessary for enzyme recognition and for the
involvement in the oxidation/reduction process, while the adenine
moiety infers catabolic from anabolic reactions which is irrelevant
in catalytic applications. All three compounds retained the
nicotinamide moiety and modifications at the pyridine nitrogen
and amide nitrogen were explored. These compounds were
chosen with further modifications in mind including modifications
for surface studies, specifically, for compounds 2 and 3 by adding
the additional carboxylic acid functional group to the reported
benzyl group. The three nicotinamide-containing compounds and
corresponding dihydropyridines are reported in Table 1 and
characterization can be found in the SI.
functional groups with hydrogen bonding distances in
a
comparable range to the natural cofactor. Through
electrochemical evaluation of the compound we found that the
three conjugates also all exhibit a characteristic NADH reduction
peak of compounds at -1.1-(-1.6) V.[12,13] We then further explored
the reduction of the compounds into their corresponding
dihydropyridine derivatives. This was followed by enzymatic
transformations using the dihydropyridine derivatives as hydride
transfer agents involving the enzyme ER, a yeast recombinant
made in E. coli (EC 1.3.1.16), as represented in Scheme 1. ERs
are a class of redox enzymes that are capable of catalyzing the
asymmetric reduction of activated C=C bonds forming
stereogenic centers.[39–41] The ability to catalyze reductions and
the wide acceptance of different substrates are driving the
exploitation of ERs towards novel applications in redox
biocatalysis and their implementation in industrial processes.[42a]
The ER catalytic reaction resembles an asymmetric Michael-type
addition of a chiral hydride to an enone. The catalytic cycle can
be divided into two separated half reactions. In the first, a hydride
is transferred from NADH to the enzyme-bound flavin.[34] After the
release of the oxidized NAD+, the hydride is then transferred from
the bound flavin to the activated alkene substrate (Scheme 1).
Table 1. Table showing the molecular structure of compounds 1 - 5 and their
corresponding dihydropyridine.
Compound
Corresponding dihydropyridine
O
O
R
O
N
O
N
R
N
N
O
O
H
H
O
O
O
N+
N
N
R
NH
(1)
N
(5)
FMNox
Reductive
Oxidative
half reaction
O
half reaction
NH2
R
N
H
N
R
N+
O
O
N+
R
NH
N
H
O
O
OH
FMNred
Scheme 1. Scheme showing the catalytic cycle of ER using nicotinamide
(2)
N/A
compounds as the coenzyme and 2-methyl-2-pentenal as the substrate. In the
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corresponding product 4 and 5, while no discernable reduction
products involving 2 were obtained. (See Table 1) This is
surprising since all three conjugates are reducible
electrochemically (vide infra). At this point, we can only speculate
that the aliphatic substituent on the pyridine nitrogen interferes
with the chemical reduction using dithionite. Potential differences
in the electronic interactions between the benzoic acid versus
valeric acid may play a role, since reductions using dithionite can
be dependent on electron withdrawing groups.[42b,c,d] Both
O
O
NH2
NH2
N
N+
(3)
(4)
OH
OH
O
O
Figure 1. Molecular structure obtained by single crystal X-ray crystallography for A) Compound 1 and B) Compound 2. c) Hydrogen bonding interactions in
compound 2 involving the O4, O1, N2, and Br1 atoms between adjacent molecules, d[N2—O1] = 2.870(3) Å, d[N2-Br1] = 3.356(2) Å, and d[O4—Br1] = 3.1811(19)
Å, resulting in the formation of a one-dimensional chain exhibiting a zig-zag pattern.
Reduction of compounds 1-3 was explored using sodium
dithionite as the reducing agent to form the corresponding 1,2-
dihydropyridine. We are able to reduce conjugates 1 and 3 to the
compounds show a strong absorption λmax = 360 nm which is
characteristic of dihydropyridines, and which is also present for
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NADH at 340 nm.[43] The UV spectra of compounds 1, 3, 4 and 5,
can be seen in Figure S22.
remains planar.[48] A more detailed look of the intermolecular
patterns between many molecules of compound 2 can be seen in
Figure S33. Next, the ability of the conjugates to be used as
NADH mimics that enable biochemical hydride transfer reactions
was probed.
Electrochemical reduction of the nicotinamide conjugates 1 - 3
was also explored by cyclic voltammetry (CV).[12,13] All CV
experiments were carried out in a 1 mM solution of the
compounds in 100 mM phosphate buffer (pH 7.05). A glassy
carbon working and counter electrode was used with reference to
a Ag/AgCl reference electrode. At a scan rate of 100 mV/s, the
cyclic voltammograms of the compounds exhibited a reduction
signal at -1.1 - -1.6V shown in Figure S21, which is characteristic
of reduction of the nicotinamide moiety.[12,13,44,45a]
Biochemical Evaluation of Nicotinamide Derivatives
The nicotinamide conjugates were biochemically evaluated in the
enzyme ER, (EC 1.3.1.16) to explore the ability of the compounds
to act as a coenzyme mimic of NADH. ER catalyzed reactions in
the absence of NADH analogs, served as negative controls and
thus it was decided to investigate these reactions further
employing our synthetic nicotinamide derivatives as a NADH
analogue. In addition, research into NADH-dependent biocatalytic
alkene reduction catalyzed by these enzymes have been
described in respect to their applications in industrial
processes.[52] ER also exhibits cofactor promiscuity and was
reported to accept a wide range of substrates which emphasizes
the catalytic versatility and the expansion of ER use.[52] Therefore,
the NADH analogues were then evaluated in ER where clear
controls were run prior and precise results were drawn. Three
substrates were chosen in this study due to their wide use but lack
of kinetic data reported in ER. Due to solubility issues of
compound 5, it was not possible to carry out an accurate
measurement of enzyme activity. In addition, based on the
bulkiness of the amino acid substituent on the amide nitrogen, a
proper fit in the active site may not be possible similar to the
Hantzsch ester previously tested. However, compound 4 acts as
a coenzyme mimic in the active enzyme and enzyme kinetics
were performed for the enzymatic Ene reductions (see Table 2).
These reductions were conveniently followed by GC-MS analysis,
allowing the identification of the reduced product. This data is
presented in Figures S26-28 (compound 4) and S32-34 (NADH),
and the retention times for the substrates and products are
reported in Table S6. In addition, the enzymatic transformations
were followed spectroscopically by monitoring the characteristic
absorbance of the dihydropyridine derivative 4 at λmax = 360 nm.
A decrease at 360 nm is the result of substrate conversion.
Scheme 2 shows the reduction of the C=C bond in the substrate
2-methyl-2-pentenal in ER using compound 4 as the coenzyme.
Firstly, we were able to calculate the conversion of compound 4
into compound 3 using the calculated molar extinction coefficient,
reported in Table S5, and the absorbance values at beginning of
Stability of the mimics were also assessed (see Figure S44-45)
as it has been reported the stability of dihydropyridines varies
under different conditions.[45b] Compound 4 has been assessed in
3 different temperatures, 20, 30, and 40 oC, at 5 different pH each.
From this study we have concluded that compound 4, similar to
other dihydropyridine compounds, are stable in all temperatures
in slightly basic to basic conditions. In acidic, weakly acidic, and
neutral conditions, the compound had a fast rate of dissociation.
Therefore, the studies were performed and reported at pH 7.5 or
greater in order to ensure the compound was stable through all
tests while still maintaining a physiological pH for the enzyme to
functionally operate at.
Crystallographic Studies
Single crystals suitable for X-ray crystallography analysis were
obtained for compounds 1 and 2. Compound 1 crystallized from
dimethyl sulfoxide in the trigonal space group P31, while crystals
from compound 2 were obtained by slow evaporation from
chloroform and diethyl ether and crystallized in the triclinic space
group P-1. Details of the crystallographic analyses are provided
in Tables S1 - S4. The molecular structures for compounds 1 and
2 are shown in Figure 1a and b. Compound 1 exhibits a single
hydrogen bonding interaction between the amide nitrogen and the
bromide anion (d[N1-Br1] = 3.524(5) Å). Hydrogen bonding
involving amide NH and Br anions were also reported for a similar
nicotinamide
conjugate,
N-(3-(aden-9-yl)propyl)-3-
carbamoylpyridinium bromide, with a distance of 3.397(7) Å.[46] In
contrast, compound 2 engages in three intermolecular hydrogen
bonding interactions resulting in the formation of
a
supramolecular assembly involving the amide and carbonyl
groups of adjacent molecules, and a linkage through a bromide
anion to neighboring molecules with distances of d[N1—O1] =
2.870(3) Å, d[N2-Br1] = 3.356(2) Å, and d[O4—Br1] = 3.1811(19)
Å. Similar to the molecular structure of nicotinamide,[47] N-benzyl
nicotinamide, [48] a very commonly reported NAD+ mimic, and the
natural NAD+ itself,[49–51] the nicotinamide moiety of compound 2
engages in intermolecular bonding between neighboring
nicotinamide moieties through the carbonyl and amide nitrogen
functional groups. See figure 1c. The hydrogen bonding distances
between the amide and carbonyl groups for the nicotinamide
compounds stated above are reported to be in the range of 2.87-
2.99 Å, which compound 2 also falls into (2.870(3) Å). In addition,
compound 2 crystalized in the triclinic space group similar to the
crystal structure of NAD+ and as expected the pyridine group
Ene Reductase
O
O
6
7
O
H H
O
NH2
NH2
N+
N
OH
OH
O
O
4
3
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the reaction and after 6 h. The highest conversion of compound 4
to compound 3 was found with the substrate 2-methyl-2-pentenal
(6), with a conversion of 93%. The conversion when using the
other two substrates with ER was not nearly as high with values
of 31% for carvone (8) and 25% with the ketoisophorone (10).
absorbance corresponding to substrate conversion was
monitored. Next the kinetics of the enzymatic transformation was
explored with compound 4 and the three substrates.
Figure 2: A) UV/vis spectra showing the decrease in absorbance at 360 nm
with compound 4 as the coenzyme in 50 mM MOPS buffer + 5 mM CaCl2 pH
7.5 containing 0.002umol of ER with 0.67 mM 2-methyl-2-pentenal as the
substrate at 30 oC. B) UV/vis spectra showing the decrease in absorbance at
360 nm with NADH as the coenzyme in 50 mM MOPS buffer + 5 mM CaCl2 pH
7.5 containing 0.002umol of ER with 0.67 mM 2-methyl-2-pentenal as the
substrate at 30 oC. C) Absorbance recorded at T=21600 s (black), T= 14400 s
(green), T= 9000 s (purple), T= 4500 s (red), T= 60s (blue). B) Reaction profile
of the enzymatic C=C reduction using compound 4 as the coenzyme and 2-
methyl-2-pentenal as the substrate over 6 hours. D) Absorbance recorded at
T=21600 s (black), T= 14400 s (green), T= 9000 s (purple), T= 4500 s (red), T=
60s (blue). B) Reaction profile of the enzymatic C=C reduction using NADH as
the coenzyme and 2-methyl-2-pentenal as the substrate over 6 hours.
Scheme 2: Scheme showing the enzymatic transformation in Ene Reductase
of compound 6 to compound 7 using compound 4 as the coenzyme.
Secondly, we were able to confirm that compound 4 was capable
of functionally substituting the natural coenzyme NADH in the
enzymatic reaction in ER. Like NAD+/NADH, compounds 3 and 4
exhibit absorbance’s at 280 nm for the oxidized compounds and
the additional peak at 340-360 nm for the reduced compounds.
We were able to monitor the enzymatic reaction by observing the
decrease in absorbance at 360 nm further proving that compound
4 can substitute NADH in the reductase enzyme. Figure 2 shows
the decrease in the
absorbance at 360 nm for compound 4 (a) and for NADH (b) over
a 6-hour time period at times 60 s, 4500 s, 9000 s, 14400 s, and
21600 s. Figure 2c and d shows the reaction profile of the
enzymatic reaction with compound 4 and NADH over time, and
clearly shows the decrease in absorbance from the start of the
reaction to the end of the 6-hour time course. Through both GC-
Kinetic Evaluation of Compound 4 as a NADH Mimic
Michaelis-Menten kinetics were evaluated where the enzyme and
coenzyme mimic concentrations remained constant while the
substrate concentrations were varied. Experiments were
performed with five different concentrations, 0.16, 0.33, 0.67, 1.67,
and 3.33 mM for each of the three substrates, 2-methyl-2-
pentenal (4), carvone (6), and ketoisophorone (8). Each reaction
was monitored over 6 hours and readings were taken in 5 minute
intervals. This was repeated with the natural NADH compound for
a side by side comparison. All raw kinetic data is provided in the
Supplemental Information section in Figures S23-34. From this,
Michaelis-Menten curves and Lineweaver-Burk plots were
constructed for each substrate (Figure S33-43) in order to
determine the Km and Vmax values. See Table 2. In addition, the
catalytic efficiencies (Km/kcat) were calculated and reported in
Table 2 using the Vmax values.
From the side by side comparison of our compound 4 and NADH,
we now provide a second example of a coenzyme mimic
outperforming the natural NADH coenzyme. In addition, we
provide kinetic data for a Class II type ER and show its
Table 2: Table listing the substrate and products used in ER with compound 4 and NADH as the coenzyme and the reported experimental Michaelis-
Menten constant, Km, (mM), Vmax values mU) and turnover number, kcat, (mM/min) found for each substrate.
Substrate
Product
Km
Vmax (compound
4)
kcat/Km
(compound 4)
Km (NADH)
Vmax (NADH)
0.8 ± 0.05
kcat/Km
(NADH)
(compound 4)
0.07 ± 0.016
15 ± 0.04
111.36
0.03 ± 0.0094
11.62
O
O
(6)
(8)
(7)
0.22 ± 0.067
4 ± 0.6
7.97
0.36 ± 0.0503
0.48 ± 0.299
0.9 ± 0.04
0.6 ± 0.04
1.22
O
O
(9)
0.26 ± 0.046
3 ± 0.07
5.32
0.61
O
O
O
O
(10)
(11)
MS and UV studies it is concluded that all three products of the
enzymatic reaction were detected and the decrease in
acceptance to a nicotinamide biomimetic and to a range of
substrates. As shown in Figure 2, in over 6 hours, compound 4
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General Considerations
shows a decrease of the peak at 360 nm to an absorbance of
almost 0. While NADH also shows a decrease, it is not nearly as
fast as the turnover of compound 4 over the 6 hour period. When
comparing the catalytic constants, it further proves that compound
4 outperformed NADH as a higher Vmax and catalytic efficiency
was reported for each substrate (Table 2). For both compound 4
and NADH, a preference for substrate 6 is found in the enzyme
over the other two substrates 8 and 10 as a lower Km value was
reported and a higher conversion. The Km for substrate 6 was
0.077 mM and the Vmax was 0.0147 µmol/min while for substrates
8 and 10, which has industrial relevance due to the importance of
the product, levodione 11 in carotenoids synthesis,[40] were 0.175
mM and 0.295 mM with a Vmax value of 0.0033 and 0.0028
µmol/min, respectively. These values are in a similar range of
other reported substrates used in ER transformations.[53][54] From
this data, it can be concluded that conjugate 4 is an effective
NADH mimic in the three reactions examined here allowing the
transformation of substrates 6, 8, and 10 into their corresponding
products.
Nicotinamide, nicotinic acid, n-hydroxysuccinamide, pyridine, sodium
dithionite, 2-methyl-2-pentenal, carvone, ketoisophorone, DCC, 1,2-
dimethoxyethane, 4-bromomethyl benzoic acid, 5-bromo valeric acid, and
Ene Reductase were all purchased from Sigma-Aldrich. Boc-Phe-OMe
was purchased through Advanced ChemTech. All reagents were used
without further modifications unless specified. Milli-Q water was used
throughout this study for all purposes including electrochemistry, sample
solutions and rinsing.
Synthesis of Compound 1
Compound 1 was prepared by synthesizing a nicotinic acid ester. N-
hydroxysuccinamide (35 mmol) and DCC (35 mmol) were added into a
solution of nicotinic acid (35 mmol) dissolved in 70 mL of pyridine. The
solution was stirred 20 hours then filtered, re-dissolved in chloroform and
washed 3 times with water. The crude product was then recrystallized with
ethanol to give white needles.
The ester was then dissolved in 1,2-dimethoxyethane and cooled in an ice
bath. A sodium bicarbonate solution containing 16 mmol of the amino acid
phenylalanine was added to the ester solution drop wise over half an hour.
o
The solution was left to stir for 2 hours at 0 C then at room temperature
Conclusions
for 14 hours. The solution was then filtered and acidified with 10% citric
acid to a pH of 4 and then washed with ethyl acetate. The organic layer
was dried with sodium sulfate and evaporated under pressure. The oil
product was purified by silica column chromatography using
dichloromethane and methanol as eluents.
Due to the expanding interest in exploring nicotinamide
derivatives as mimics for biochemical transformations involving
NADH and NAD+, three novel conjugates were synthesized and
their chemical properties, including their redox behavior were
examined as potential mimics for the natural redox agents. It was
possible to reduce compounds 1 and 3 into their corresponding
dihydropyridines, 4 and 5. The reduced compounds were shown
to exhibit properties of typical dihydropyridine systems including
displaying the characteristic absorbance at 360 nm. Compound 4
was exploited in biochemical transformations involving the
enzyme Ene reductase, which allowed the enzymatic reduction of
three unsaturated substrates into their corresponding products,
and was proved to act as a NADH mimic. In addition, compound
4 outperformed the natural coenzyme having a higher catalytic
efficiency and Vmax value providing the second example of a
nicotinamide biomimetic that outperforms the natural NADH in ER.
Kinetic data is reported for the biotransformation with three
substrates 6, 8, and 10 and each were shown to have comparable,
if not better, catalytic values to the natural NADH. Both NADH and
compound 4 performed best with substrate 6, having the lowest
Km value of the three substrates and a low value compared to
other similar substrates reported in ER in the literature.[34,53,54]
Here, we also provide kinetic data for substrates that have been
used in ER but lack the kinetic values, as well as kinetics for a
class 2 ER which is not heavily reported compared to classes 1
and 3. Conjugate 4 is able to take on the role of a NADH mimic,
possessing comparable kinetics in ER catalyzed biochemical
transformations, suggesting that there is potential of 4 to be use
useful in other enzymatic reactions that use NADH as the
coenzyme.
7 mmol of the nicotinoyl-L-phenylalanine compound was dissolved in 50
mL of 1,4-doixane/ 12.5 mL methanol solution. 15 mmol of benzyl bromide
was added to this solution and was refluxed for 5 hours in an oil bath. The
product was filtered and washed with hexanes to obtain the final pure
product.
1H NMR (500 MHz, chloroform-d) 10.53 (s, 1H, pyr-1), 9.73 (d, 1H, amide
NH), 8.91 (d, 1H, pyr-5), 8.84 (d, 1H, pyr-3), 7.97 (m, 2H, pyr-4), 7.62 (dd
2H, aromatic C-H), 7.49 (m, 5H, aromatic C-H), 7.26 (t, 2H, aromatic C-H),
7.18 (t, 1H, aromatic C-H), 6.11 (s, 2H, CH2), 4.94 (m, 1H, CHα), 3.75 (s,
3H, CH3), 3.54(dd, 1H, CH2β), 3.44 (dd, 1H, CH2β). 13C NMR (DMSO-d6):
δ 171.76 (1C of COOMe), δ 161.81 (1C of CONH), δ 145.24 (3C, pyr-
1,3,5), δ 137.45 (2C, pyr-2 & aromatic C), δ 133.98 (2C, aromatic C and
pyr-4), δ 129.34 (10C, aromatic C), δ 64,10 (1C, CH2), δ 55.08 (1C, α C),
δ 52.70 (1C, CH3), δ 36.76 (2C, β C), Expected MS: m/z 375.1703 [M +],
Experimental MS: m/z 375.1704 C23H23N2O3+ Yield: 76%
Synthesis of Compound 2
15 mmol of nicotinamide was dissolved in a 50 mL 1,4-dioxane/25 mL
methanol mixture. 7 mmol of 5-bromo valeric acid was added and the
solution was refluxed for 6 hours. The solution was filtered and the solid
was washed with dichloromethane and hexanes to obtain the pure product.
1H NMR (500 MHz, DMSO-d6) 12.14 (s, 1H, COOH), 9.51 (m, 1H, pyr-1),
9.22 (d, 1H, pyr-5), 8.94 (d, 1H, pyr-3), 8.57(s, 1H, NH2), 8.28 (dd, 1H, pyr-
4), 8.19 (s, 1H, NH2), 4.67(t, 2H, CH2), 2.29 (t, 2H, CH2), 1.97 (m, 2H, CH2),
1.51 (p, 2H, CH2), 13C NMR (DMSO-d6): δ 174.51(1C of COOH, δ 163.28
(1C of CONH2), δ 146.81 (1C, pyr-3), δ 145.28 (1C, pyr-1), δ 143.65 (1C,
pyr-5), δ 134.35 (1C, pyr-2), δ 128.33(1C, pyr-4), δ 61.28 (1C, CH2), δ
33.29 (1C, CH2), δ 30.54 (1C, CH2), δ 21.33 (1C, CH2) Experimental MS:
m/z 223.1077 [M +], Expected MS: m/z 223.1074[M +], C11H15N2O3+ Yield:
67%
Experimental Section
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Synthesis of Compound 3
GC-MS Studies
15 mmol of nicotinamide was dissolved in a 50 mL 1,4-dioxane/25 mL
methanol mixture. 7 mmol of 4-bromomethyl benzoic acid was added and
the solution was refluxed for 6 hours. The solution was filtered and the
solid was washed with dichloromethane and hexanes to obtain the pure
product.
GC-MS studies were carried out on an Agilent DC-624 (30m x 0.32 (1.0
µm df)). The enzymatic reactions were set-up in a temperature-controlled
water bath. The buffer (MOPS, 50 mM, pH 7.5 with 5 mM CaCl2), substrate
(2-methyl-2-pentenal, carvone, and ketoisophorone), coenzyme mimic (4)
and enzyme (ER) were added in this order. 250 uL were taken at different
time intervals over 6 hours and extracted with ethyl acetate (600 uL) and
centrifuged for 2 minutes. The ethyl acetate layer was then analyzed by
GC-MS.
1H NMR (500 MHz, DMSO-d6) 13.17 (s, 1H, COOH), 9.62 (s, 1H, pyr-1),
9.28 (d, 1H, pyr-5), 8.98 (d, 1H, pyr-3), 8.59 (s, 1H, NH2), 8.31 (dd, 2H,
pyr-4), 8.19 (s, 1H, NH2), 8.00 (d, 2H, aromatic C-H), 7.63 (d, 2H, aromatic
C-H), 6.00 (s, 2H, CH2), 13C NMR (DMSO-d6): δ 167.20 (1C of COOH), δ
163.16 (1C of CONH2), δ 147.05 (1C, pyr-5), δ 138.93 (1C, aromatic C), δ
134.68 (2C, pyr-2), δ 132.08 (1C, pyr-4), δ 130.47 (2C, aromatic C), δ
129.48 (2C, aromatic C), δ 128.83 (1C, aromatic C), δ 63.51 (1C, CH2),
Expected MS: m/z 257.0921 [M +], Experimental MS: m/z 257.0918
C14H13N2O+ 78%
Nuclear Magnetic Resonance (NMR) Studies
Compounds 1-3 were characterized using NMR spectroscopy (Bruker
spectrometer 500 MHz). Compound 1 and 5 was dissolved in chloroform-
d while compound 2, 3 and 4, was dissolved in dimethoxy-sulfoxide-d6 all
at a concentration of 2 mM. Spectra are attached in the Supplemental
Information section.
Synthesis of Compound 4
Electrochemical Studies
6 mmol of sodium bicarbonate was added to 20 mL of Mili-Q water. Once
dissolved 1 mmol of compound 3 was added and dissolved. Under N2 and
in the dark, 4 mmol of sodium dithionite was added slowly to the mixture.
The reaction was stirred vigorously for 3 hours at room temperature. The
pure compound was precipitated out using concentrated HCl.
The electrochemical experiments were carried out at room temperature
using a CHI660B electrochemical workstation (CH Instruments Inc). All
electrochemical experiments used a glassy carbon working electrode,
glassy carbon as a counter electrode, and Ag/AgCl (saturated with 3M KCl)
reference electrode. The glassy carbon electrodes were polished with 0.05
µM Al2O3, then sonicated in Milli-Q water, ethanol, and Milli-Q water again
for 10 minutes each to fully remove any absorbed Al2O3. The electrodes
were thoroughly rinsed with Milli-Q water after each sonication. Both
electrodes were electrochemically cleaned using a 1 mM KOH solution. 1
mM solutions of compounds 1-3 were prepared in 1 mM phosphate buffer
pH 7.05 and were used for all electrochemical experiments. Cyclic
voltammetry (CV) was performed at a 100 mV/s scan rate.
1H NMR (500 MHz, DMSO-d6) 13.16 (s, 1H, COOH), 7.92 (d, 2H, aromatic
CH), 7.36 (d, 2H, aromatic CH), 6.99 (s, 1H, pyr-1), 6.58 (s, 2H, NH2), 5.94
(d 1H, pyr-5), 4.63 (dt 1H, pyr-4), 4.39 (s, 2H, CH2), 2.97 (d, 2H, pyr-3),
13C NMR (DMSO-d6): δ 169.44 (1C of COOH), δ 167.69 (1C of CONH2),
δ 143.72 (3C, pyr-1), δ 138.26 (1C, aromatic C), δ 130.07 (2C, aromatic
C), δ 128.77 (1C, pyr-5), δ 127.83 (1C, aromatic C) δ 127.60 (1C, aromatic
C), δ 102.44 (1C, pyr-2), δ 101.14 (2C, pyr-4), δ 55.97 (2C, CH2) δ 22.76
(1C, pyr-3) Expected for MS: m/z 259.1004 [M +H+], Experimental MS:
m/z 259.1069 C14H14N2O3 Yield: 57%
X-ray Crystallography Studies
Synthesis of Compound 5
Crystallographic data were collected on a Bruker Kappa APEX-DUO
diffractometer using a Copper ImuS tube with multilayer optics. The data
were processed using APEX2 and SAINT. Absorption corrections were
carried out using SADABS. The structure was solved with SHELXT and
refined using SHELXL-2014 for full-matrix least-squares refinement that
was based on F2. All H atoms were included in calculated positions and
allowed to refine in riding-motion approximation with U ∼ iso ∼ tied to the
carrier atom
6 mmol of sodium bicarbonate was added to 20 mL of Mili-Q water. Once
dissolved 1 mmol of compound 1 was added along with 15 mL of
dichloromethane until fully dissolved. Under N2 and in the dark, 4 mmol of
sodium dithionite was added slowly to the mixture. The reaction was stirred
vigorously for 3 hours at room temperature. The compound was collected
in the organic layer and was purified through silica column chromatography.
1H NMR (500 MHz, chloroform-d) 8.68 (s, 1H, amide NH), 7.14-7.48 (m,
10H, aromatic CH), 5.73 (dd, 1H, pyr-1), 4.99 (dq, 1H, pyr-5), 4.49 (d, 1H,
CHα), 4.41 (dd, 1H, pyr-4), 4.30 (q, 2H, CH2), 3.73 (s, 3H, CH3), 3.34 (d,
2H, pyr-2), 3.15 (m, 2H, CH2β). 13C NMR (chloroform-d6): δ 172.77 (1C
of COOMe), δ 167.47 (1C of CONH), δ 141.39 (1C, pyr-1), δ 137.48 (1C,
aromatic C), δ 136.24 (1C, pyr-5), δ 128.24 (10C, aromatic C), δ 99.77 (1C,
pyr-2), δ 98.66 (1C, pyr4), δ 84.52 (1C, CH2), δ 57.81 (1C, CHα), δ 54.95
(1C, CH3), δ 52.83 (1C, CH2β), δ 38.27 (1C, pyr-2). Expected MS: m/z
377.1787 [M +H+], Experimental MS: m/z 377.1864 C23H24N2O3 Yield:
37%
Acknowledgements
We gratefully acknowledge funding from the Natural Science and
Engineering Research Council of Canada (NSERC) and the
University of Toronto. We also thank our TRACES manager Tony
Adamo for his help.
Keywords: nicotinamide• redox • NADH mimics • enzyme
kinetics • biocatalysis
UV/vis studies of the Enzymatic Transformations with compound 4
In a quartz cuvette, 100 ug/mL of Ene Reductase, 0.1 mM of compound 4,
3 mL of MOPS buffer, and varying concentrations of each substrate from
0.16 mM- 3.33 mM was added. The cell was kept at 30 oC. The decrease
of the absorbance at 360 nm which is characteristic of the dihydropyridine
was monitored with an 8453 w Peltier UV/vis spectrophotometer.
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In
the
present
study
three
Natashya Falcone, Zhe She, Jebriel
Syed, Alan Lough, Heinz-Bernhard
Kraatz*
nicotinamide-containing conjugates
are synthesized, and biochemically
evaluated in ER. Two molecular
structures were obtained by X-ray
crystallography of compounds 1 and
2. Compound 4 was biochemically
evaluated and it was found that it can
functionally substitute and outperform
the natural coenzyme NADH with
comparable kinetic values for three
substrates.
1 – 8
Synthesis, and Biochemical
Evaluation of Nicotinamide
Derivatives as NADH Analogues in
Ene Reductase
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