Synthesis and electrochemical behavior of triazole-containing nicotinamide adenine dinucleotide analogs

Article abstract of DOI:10.1139/V09-145

The coupling of 2′,3′-di-O-acetyl nicotinamide mononucleotide with 3-butyn-1-ol in the presence of 2,4,6- triisopropylbenzenesulfonyl chloride quantitatively afforded a terminal alkyne-containing intermediate. Furthermore, copper(I)-mediated Huisgen [3 + 2] cycloaddition with a series of azido compounds in a two-phase solvent system gave eight triazole-containing nicotinamide adenine dinucleotide analogs with yields over 88%. The cyclic voltammetric behaviors of these novel analogs were investigated with a glassy carbon electrode, and structural features of these analogs on their electrochemical properties were briefly discussed.

Full text of DOI:10.1139/V09-145

35
Synthesis and electrochemical behavior of
triazole-containing nicotinamide adenine
dinucleotide analogs
Wujun Liu, Shuhua Hou, and Zongbao Kent Zhao
Abstract: The coupling of 2’,3’-di-O-acetyl nicotinamide mononucleotide with 3-butyn-1-ol in the presence of 2,4,6-
triisopropylbenzenesulfonyl chloride quantitatively afforded a terminal alkyne-containing intermediate. Furthermore,
copper(I)-mediated Huisgen [3 + 2] cycloaddition with a series of azido compounds in a two-phase solvent system gave
eight triazole-containing nicotinamide adenine dinucleotide analogs with yields over 88%. The cyclic voltammetric be-
haviors of these novel analogs were investigated with a glassy carbon electrode, and structural features of these analogs
on their electrochemical properties were briefly discussed.
Key words: nicotinamide adenine dinucleotide, analog, click chemistry, cyclic voltammetry.
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Resume : Le couplage du mononucleotide du 2’,3’-di-O-acetylnicotinamide avec le but-3-yn-1-ol, en presence de chlorure
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de 2,4,6-triisopropylbenzenesulfonyle conduit a la formation pratiquement quantitative d’un intermediaire comportant un
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alcyne terminal. Une cycloaddition ulterieure [3 + 2] de Huisgen, catalysee par le cuivre(I), avec une serie de composes
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azide, dans un systeme liquide a deux phases, conduit a la formation, avec des rendements globaux superieurs a 88 %,
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d’analogues dinucleotides d’adenine nicotinamide contenant un triazole en position 8. On a etudie les comportements en
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voltamperometrie cyclique de ces nouveaux analogues a l’aide d’une electrode de carbone vitreux et on discute brievement
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des caracteristiques structurales de ces analogues sur leurs proprietes electrochimiques.
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Mots-cles : dinucleotide de l’adenine nicotinamide, analogue, chimie par clic, voltamperometrie cyclique.
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[Traduit par la Redaction]
study showed that a simple phosphodiester-type NAD+ ana-
log could be recognized by horse liver alcohol dehydrogen-
ase, and that the system could be applied for reduction of
prochiral ketones in moderate yields.10,11 Thus, it is essential
to have the NMN part in NAD+ analogs so that their pros-
pects as a redox chemistry mediator can be further explored.
In these reactions, a synthetic cofactor was regenerated with
valuable rhodium complexes, but low-cost electrochemical
or photo driven regeneration of the NAD+ cofactor led to a
complex reaction.12,13 One strategy to overcome these defi-
ciencies is to modify the structure of the cofactor to improve
electrode reaction activity.
We were recently interested in the preparation of NAD+
analogs in which one phosphorus atom is removed from the
original skeleton to meliorate electrode reaction activity of
the natural coenzyme. In this work, we wish to report our
efforts on the construction of 1,2,3-triazole-containing
NAD+ analogs (Fig. 1) and their electrochemical properties.
We developed an efficient synthetic strategy for synthesis of
these analogs based on CuSO4/sodium ascorbate-mediated
Huisgen’s 1,3-dipolar cycloaddition, or click reaction.14
Introduction
Nicotinamide adenine dinucleotide (NAD+) and its re-
duced form, NADH, are involved in numerous biological
systems, especially as cofactors for enzymes mediating oxi-
doreductive reactions.1 In those oxidoreductive reactions,
NAD(H) transfers hydrogen and electron shuttles, along
with chemical potential change. The chemical potential of
NAD+ or its derivatives play critical roles in calcium ho-
meostasis,2 cross-membrane transportation,3 oxidative stress
resistance,4,5 and reactive oxygen species clearance.6
Although electrochemical studies have been done on a few
model compounds,7 and on applying a novel method8 or ma-
terials9 for modification of the surface of the electrode to
improve the sensitivity of electrode response to NAD(H),
there are few reports on testing the electrocatalytic reduction
of synthetic NAD+ analogs.
NAD+ is a coupled product of the nicotinamide mononu-
cleotide (NMN) part and the adenosine monophosphate
(AMP) moiety (Fig. 1). The NMN part bestows its oxidore-
ductive property, whereas the AMP moiety is largely in-
volved in binding in the biological environment. Early
Received 10 July 2009. Accepted 14 September 2009. Published on the NRC Research Press Web site at canjchem.nrc.ca on
16 December 2009.
W. Liu and S. Hou. Dalian Institute of Chemical Physics, CAS, Dalian 116023, P. R. China; Graduate School of the Chinese Academy
of Sciences, Beijing 100039, P. R. China.
Z.K. Zhao.1 Dalian Institute of Chemical Physics, CAS, Dalian 116023, P. R. China; Dalian National Laboratory of Clean Energy,
Dalian 116023, P. R. China.
1Corresponding author (e-mail: zhaozb@dicp.ac.cn).
Can. J. Chem. 88: 35–41 (2010)
doi:10.1139/V09-145
Published by NRC Research Press

36
Can. J. Chem. Vol. 88, 2010
Scheme 1. Synthesis of triazole-containing NAD+ analogs. Reagents and conditions: (i) Ac2O/Py (1:1), 0–5 8C, 24 h, 98%; (ii) 3-butyn-1-ol
(3 equiv.), TIPS-Cl (3 equiv.), DMF/Py (1:1), RT, 3 h, 99%; (iii) (a) R–N3, CuSO4/sodium ascorbate, H2O/DCM (1:1), RT, 0.5–6 h, 90%–
98%, (b) NH4HCO3 (1 mol/L), in H2O/MeOH (1:10), 98%. Data in the parentheses indicate the total isolated yield of each compound on
Ac2NMN.
Compared to NAD+, these analogs showed relatively low re-
ductive peak potentials (Ep) and a distinct improvement of
the electrode reaction rate constant (Ks).
Fig. 1. Structures of NAD+ and its triazole-containing analogs.
Results and discussion
Synthesis of triazole-containing NAD+ analogs
Our in-house preparation of NMN was realized via selec-
tive hydrolysis of NAD+ using ZrCl4 as catalyst.15 To syn-
thesize the triazole-containing NAD+ analogs, we originally
tried to couple NMN with the corresponding alkynyl alcohol
followed by click reaction with azides (Fig. 1). Because
NMN is liable to form an inactive inner salt and has notori-
ously low solubility in most organic solvents (i.e., DMF (di-
methylformamide), pyridine, MeCN), it was difficult to find
an appropriate solvent system to realize the coupling reac-
tion. Moreover, C–N bond breakage of the NMN molecule
was substantial in many experiments. We then treated NMN
with a mixture of Ac2O and pyridine at 0–5 8C, leading to
the acetylated product of NMN, 2’,3’-di-O-acetyl nicotina-
mide mononucleotide (Ac2NMN). It turned out that
Ac2NMN had much better solubility and higher activity.
Thus, the coupling of Ac2NMN with excess 3-butyn-1-ol in
DMF/Py (1:1, v/v) in the presence of 2,4,6-triisopropylben-
zenesulfonyl chloride (TIPS-Cl) at room temperature for 3 h
afforded the key intermediate of 2’,3’-di-O-acetyl b-nicotina-
mide ribose-5’-3-butynyl phosphate, 3, in a 99% yield based
on 31P NMR analysis (Scheme 1).
carried out cycloaddition experiments in the presence of
copper sulfate and sodium ascorbate according to the strat-
egy described by Sharpless and co-workers.17 Upon testing
a couple of solvent systems, i.e., water mixed with DMSO,
DMF, DCM, THF, MeCN, or t-BuOH, we found that there
was no significant difference whether the organic solvent
was soluble or insoluble in water. This was likely because
compound 3 and the coupled products had excellent solubil-
ity in water. To facilitate an easier recycling of azides, our
synthesis was carried out in a two-phased system made of
DCM and H2O. Using this strategy, we were successful in
coupling azides a–h with 3 at room temperature in over
90% yields within 6 h. For most reactions, 5 mol% Cu(I)
catalyst was enough to convert 3 within 2 h. The bulky
azides f and h required 4 h for better yields. Because azide
g had a poor solubility, up to 6 h was necessary to achieve
To convert compound 3 into triazole-containing NAD+
analogs, we prepared eight azides (Scheme 1). These com-
pounds were selected to mimic the adenine moiety of the
NAD+ structure, mainly depending on their accessibility and
aromatic ring structures. Azides a–c were obtained from the
corresponding halides via a nucleophilic substitution using
sodium azide, whereas azides d–h were made via acylation
of the corresponding amines with 5-chloropentanoyl chloride
followed by a nucleophilic substitution reaction using so-
dium azide.16
With intermediate 3 and azide compounds in hand, we
Published by NRC Research Press

Liu et al.
37
Table 1. Electrochemical dynamic constants of tria-
zole-containing NAD+ analogs (4a–4h) and related
compounds.
excellent conversion. We also tried the combination CuSO4/
Cu to mediate the coupling reaction. However, the products
seemed liable to coordinate with copper, leading to a tedious
purification process and low isolated yields.
It was interesting to note that those triazole-containing in-
termediates could be easily deacetylated by 1 mol/L
NH4HCO3 solution in H2O/MeOH (1:10, v/v). Thus, crude
compound 4 was achieved during the reverse phase silica
gel chromatography purification.
Compound
4a
4b
4c
4d
Ep (V)
–1.157
–1.145
–1.148
–1.154
–1.159
–1.142
–1.145
–1.141
–1.269
–1.183
–1.211
Ks (s–1)
0.24
0.23
0.23
0.23
0.20
0.23
0.23
0.24
0.11
0.12
0.12
a
0.39
0.38
0.36
0.35
0.34
0.36
0.36
0.40
0.22
0.28
0.21
4e
4f
4g
4h
Because NAD+ analogs are amphiphilic molecules con-
taining both positive and negative charges, purification usu-
ally presents a major barrier to achieve an appreciable
quantity of target compounds in the lab. Thus, successful
procedures varied from case to case for the isolation of ana-
logs 4a–4h. Chromatography with various supporting mate-
rials, including activated charcoal, silica gel, octyl-
functionalized silica gel, ion exchange resins, and Bio-Gel
P-2 resin has been applied in different combinations to ful-
fill valid purification. Compound 4a was purified with octyl-
NAD+
NMN
NAR
Note: All data were the average value of three replicates.
Fig. 2. Overlay of the continuous reductive curves of NAD+ and 4h
under the same conditions.
–
functionalized silica gel and 201 ꢀ 2 HCO2 form of the
anion resin followed by Bio-Gel P-2 resin. For compounds
4b, 4d, and 4e, chromatography on octyl-functionalized
silica gel and anion resin, at different elution conditions,
gave the best results. For compounds 4c and 4f–4h, chroma-
tography on octyl-functionalized silica gel and Bio-Gel P-2
resin gave our target molecules.
Electrochemical behavior of triazole-containing NAD+
analogs
With novel analogs in hand, we investigated their electro-
chemical performance on a glassy carbon (GC) electrode in
aqueous solution. Oxido-reductive behaviors of these tria-
zole-containing NAD+ analogs were estimated by cyclic vol-
tammetry. The redox curves were also a two electron
mechanism according to the Angulo et. al method.18 Upon a
reversal scan of the NAD+ analogs, both the reduction peak
and reoxidation peak were observed, albeit the latter was
much weaker than the former. The corresponding reoxida-
tion peaks were stronger than that of NAD+ under the same
conditions. Thus, the overall processes are not reversible.
The reductive potential (Ep) of the triazole-containing
analog (–1.141 V) was much lower than that of natural
NAD+ (–1.269 V; Table 1). Upon four cyclic reversal scans,
marginal decreases in the reductive peak currents of NAD+
analogs were observed, and the decrease range of 4h was
much smaller than that of NAD+ under the same conditions
(Fig. 2). This indicated that triazole-containing analogs had
relatively weak absorption on GC than natural NAD+.
The cathodic peak currents (ip) of triazole-containing ana-
logs increased curvilinearly with the square root of the scan
rate (v), an upper warp curve in the range of 20–200 mV s–1.
All the upper data resulted in the reduction of triazole-con-
taining NAD+, which was an absorption-controlled irreversi-
ble process under our conditions.
potential, logarithm of the scan rate (v), gas constant, abso-
lute temperature, Faraday constant, the transfer electron
number (n = 2 for NAD+ or its derivatives), electrode reac-
tion rate constant, and electron transfer coefficient, respec-
tively. The value of E8 of all compounds (vs the standard
calomel electrode (SCE)) was obtained from the intercept
of the plot of Ep vs. v (data not shown). The electrode reac-
tion rate constant (Ks) and the electron transfer coefficient
(a) were calculated from the slope and intercept of Ep vs.
ln v according to the above equation (Fig. 3a). Accordingly,
all triazole-containing NAD+ analogs and related compounds
were tested and results were listed in Table 1.
It was obvious that analogs with bulky aromatic rings and
4b have relatively low reductive peak potentials. Compound
4a and 4h have the largest Ks and a. Compound 4e shows
the lowest Ks and a in tris(hydroxymethyl)aminomethane
(Tris)/NaCl buffer, which may be due to the presence of ar-
omatic fluorine atoms in the molecule. To investigate the
electrochemical property in more detail, we carried out addi-
tional experiments on 4h.
According to Laviron theory,19 absorption-controlled irre-
versible process potential (Ep) is determined based on the
following equation:
To make a comparison, we also obtained the correlative
constants of NAD+, NMN, and nicotinamide ribose (NAR)
under identical conditions. It was clear that NAD+ and
NAR had similar reduction peak potentials (Ep) and NMN
had a more positive Ep value. However, all NAD+ analogs
had even more positive Ep values. Thus, substitution of
½1ꢁ
Ep ¼ Eꢂ ꢃ ðRT=anFÞlnðanF=RTKsÞ ꢃ ðRT=anFÞln v
where E8, ln v, R, T, F, n, Ks, and a, represent the standard
Published by NRC Research Press

38
Can. J. Chem. Vol. 88, 2010
Fig. 3. (a) Linear plot of Ep vs. ln v of NAD+ analog 4h (r = 0.9986). (b) Linear plot of Ep vs. pH of the NAD+ analog 4h.
AMP moiety of the NAD+ skeleton with a triazole-contain-
ing structure could increase the Ep of the corresponding
product.
These analogs were more liable to reduce than NAD+ on a
GC electrode in Tris/NaCl buffer. Triazole analogs showed
a stable redoxide property at different pHs. The reductive
peak potential of 4h increased linearly with the solution pH.
The triazole-containing analogs could be broadly applied in
cofactor regeneration or biosensor. We are now exploring
other special biological functions with these compounds in
a wide variety of chemical and biological areas, and results
will be reported in due course.
Ks and a of 4h were more than twofold larger than that of
other compounds, especially to natural NAD+. That indi-
cated that the reduction of NAD+ analog 4h on a GC elec-
trode was faster than natural NAD+ and related compounds.
Thus, modification of pyrophosphate made NAD+ reduce at
a relatively lower potential and the triazole ring in the ana-
log molecule made it reduce faster than natural NAD+. The
triazole ring increased the charge transfer efficiency, which
is similar to Zhou et al.’s20 results. Thus, we concluded that
triazole-containing NAD+ analogs were more liable to be re-
duced than other compounds on a GC electrode.
Experimental section
General
All reagents were analytical grade, obtained from com-
mercial suppliers (ABCR, ACROS, or Sigma-Aldrich), and
used without further purification. NMR spectra were meas-
ured with a Bruker DRX-400 spectrometer (400.3 MHz for
1H NMR, 100.6 MHz for 13C NMR, 160.1 MHz for 31P
NMR, and 376 MHz for 19F NMR) at 298 K. High resolu-
tion mass spectroscopy was obtained on a LC/Q-TOF-MS
and operated with an electrospray source in positive ion
mode. F254 thin-layer and silica gel (400 mesh) were pur-
chased from Yantai Jiangyou Silica Co., Ltd., China. Octyl-
functionalized silica gel was purchased from Sigma-Aldrich.
Ion exchange resin (100*200 mesh) was purchased from
the Chemical Plant of Nankai University, Tianjin, China.
Bio-Gel P-2 resin (45 mm) was obtained from Bio-Rad Lab-
oratories, Inc. All reactions were carried out under a nitro-
gen atmosphere. UV–vis spectra were obtained at room
temperature on a JASCO V-530 UV–vis spectrophotometer.
The electrochemical measurements were performed with a
CHI 600C electrochemical analyzer (CH Instruments, USA)
connected to a personal computer. All experiments were
performed using a conventional three-electrode system with
a glassy carbon (GC) disc (CHI 104, 3 mm diameter) work-
ing electrode, a saturated calomel reference electrode (CHI
150), and a platinum wire as the counter electrode (CHI
115).
To investigate the reoxide stability of 4h at different pHs,
the peak reductive potential of the NAD+ analog was meas-
ured in 1 mol/L phosphate buffer containing 10–3 mol/L 4h
at different pHs (pH = 4–9). It was found that the Ep in-
creased linearly with pH and that reduction of 4h would be
much easier at a higher pH (Fig. 3b). It should be noted that
4h was stable at these selected pHs and could be recovered
by reverse-phase column chromatography in a 96% yield.
However, no such correlations were observed for NAD+,
NMN, or NAR at identical conditions (data not shown).
Based on the pKa of nicotinamide and triazole, the amino
group of nicotinamide and nitrogen atom of the triazole ring
was protonated below pH 6. Thus, the NAD+ analogs ex-
isted as an inner salt in solution at a wide range of pHs.
The triazole ring had an interaction with H+ and promoted
conductance of the charge at a relatively high concentration
of H+, so a higher Ep was needed to reduce analogs. When
the pH was higher than 7, interaction between the triazole
ring and proton on the NAD+ analog gradually disappeared.
The repulsion of the triazole ring with the hydroxide anion
made analog 4h reduce at a relatively lower potential.
Conclusion
An efficient strategy to prepare novel triazole-containing
NAD+ analogs was developed. These compounds had a tria-
zole ring and phosphatediester linkage as mimics of the ad-
enosine heterocyclic ring and pyrophosphate, respectively.
Preparation of Ac2NMN (2)
To a mixture of pyridine (7.5 mL) and Ac2O (7.5 mL)
was added NMN (275 mg, 0.82 mmol) in H2O (160 mL)
Published by NRC Research Press

Liu et al.
39
at –5 8C. The suspension was kept at 0 8C until all solids
dissolved. The solvents were removed and the residue was
dissolved in a mixture of H2O (2 mL) and pyridine (2 mL).
The yellow solution was stirred at room temperature for 2 h
and the solvents were evaporated at reduced pressure. The
residue was lyophilized to give Ac2NMN as an amorphous
solid, which was used directly in the next reaction without
additional purification.21
1H), 8.84 (d, J = 8.12 Hz, 1H), 9.09 (d, J = 6.28 Hz, 1H),
9.28 (s, 1H). 13C NMR (100 MHz, D2O, ppm) d: 168.05,
162.82, 148.47, 147.04, 144.86, 142.22, 136.41, 136.28,
130.95, 126.58, 120.34, 102.28, 89.59, 89.51, 80.13, 73.30,
67.21, 66.76, 52.03, 36.21, 28.83, 28.75, 22.56. 31P NMR
(160 MHz, D2O, pp,) d: 0.067. HR-MS (ESI) calcd. for
C19H26N5O8P [M + H]+: 484.1597; found: 484.1588.
Compound 4b
–
Preparation of alkynyl ester (3)
Compound 4b was purified by anion resin (HCO2 form)
Ac2NMN (344 mg, 0.82 mmol) and 3-butyn-1-ol (3 equiv.
to Ac2NMN) were dissolved in Py/DMF (1:1, v/v) under a
nitrogen atmosphere. TIPS-Cl (3 equiv. to Ac2NMN) was
added and the mixture was stirred at room temperature for
3 h in a 99% yield based on 31P NMR. The solvent was re-
moved, and the residue was added to 10 mL of H2O and ex-
tracted with DCM (15 mL ꢀ 3). The aqueous solution was
concentrated, purified via ion exchange column chromatog-
column chromatography eluted with water to give 4b as a
colorless solid syrup in a 94% yield. H NMR (400 MHz,
1
D2O, ppm) d: 2.84 (t, J = 5.92 Hz, 2H), 3.52 (ddd, J =
1.84, 4.64, 11.88 Hz, 1H), 3.70 (ddd, J = 2.24, 4.40, 12 Hz,
1H), 3.91 (dd, J = 5.92, 11.84 Hz, 2H), 4.10 (dd, J = 2.48,
4.76 Hz, 1H), 4.15 (t, J = 2.16 Hz, 1H), 4.25 (t, J = 5.16 Hz,
1H), 5.35 (s, 2H), 5.93 (d, J = 5.48 Hz, 1H), 7.2 (m, 5H),
7.76 (s, 1H), 7.98 (dd, J = 6.48, 7.76 Hz, 1H), 8.73 (d, J =
8.12 Hz, 1H), 8.94 (d, J = 7.88 Hz, 1H), 9.16 (s, 1H). 13C
NMR (100 MHz, D2O, ppm) d: 168.0, 148.42, 147.53,
144.70, 142.22, 142.22, 137.50, 136.43, 131.64, 131.26,
130.95, 130.72, 126.67, 102.29, 89.52, 80.15, 73.33, 67.20,
66.71, 56.33, 28.82. 31P NMR (160 MHz, D2O, ppm) d:
0.01. HR-MS (ESI) calcd. for C22H27N5O8P [M + H]+:
520.1597; found: 520.1617.
–
raphy on anion resin (201 ꢀ 2, HCO2 form) eluted with
H2O. Fractions were concentrated and lyophilized to give
crude 3, and it was used in the next reaction without further
purification.
General method for the preparation of azide compounds
(a–h)
Azides (a–c) in Scheme 1 were synthesized via a nucleo-
philic substitution using sodium azide, whereas azides d–h
were made via acylation of the corresponding amines with
5-chloropentanoyl chloride followed by a nucleophilic sub-
stitution reaction using sodium azide.16
Compound 4c
Compound 4c was purified by size-exclusion chromatog-
raphy Bio-Gel1 P-2 Gel polyacrylamide gel, eluted with
25 mmol/L NH4HCO3 water solution, to give 4c as a color-
1
less solid syrup in a 92% yield. H NMR (400 MHz, D2O,
General method for the preparation of NAD+ analogs
(4a–4h)
ppm) d: 2.91 (t, 2H), 3.61 (t, 2H), 3.94 (dd, J = 10.64,
5.16 Hz, 1H), 3.99 (dd, J = 2.76 Hz, 1H), 4.02 (t, 2H), 4.07
(t, 1H), 5.51 (d, J = 2.76 Hz, 1H), 5.56 (d, J = 4.36 Hz, 1H),
7.31 (d, J = 8.6 Hz, 1H), 7.35 (dd, J = 6.16, 3.0 Hz, 2H),
7.47 (t, J = 7.52 Hz, 1H), 7.62 (m, 4H), 7.90 (s, 1H), 8.39
(d, J = 6.16 Hz, 1H), 8.44 (d, J = 8.04 Hz, 1H), 8.61 (s,
1H). 13C NMR (100 MHz, D2O, ppm) d: 167.32, 147.92,
147.49, 143.43, 141.31, 135.46, 134.98, 134.84, 131.22,
130.33, 130.14, 130.02, 129.94, 129.43, 128.38, 126.41,
101.91, 89.46, 79.93, 73.32, 67.04, 66.83, 56.36, 28.83. 31P
NMR (160 MHz, D2O, ppm) d: 0.14. HR-MS (ESI) calcd.
for C26H28N5O8P [M + H]+: 570.1754; found: 570.1735.
A solution of compound 3 in H2O and azide in DCM (or
other solvent) was mixed. To the mixture was added an ap-
propriate amount of CuSO4/sodium ascorbate (5 mol%) in
one portion. After the reaction was stirred at room tempera-
ture for 0.5–6 h, the organic layer was removed, and the
aqueous phase was purified by octyl-functionalized silica
gel eluted with water and 1 mol/L NH4HCO3 solution in
CH3OH/H2O (1:10, v/v) to give the crude product. The prod-
–
uct was further purified via anion resin (201 ꢀ 2, HCO2
form) or size-exclusion chromatography Bio-Gel1 P-2 Gel
polyacrylamide gel. The corresponding fractions were
pooled and freeze dried to give the triazole-containing ana-
logs.
Compound 4d
–
Compound 4d was purified by anion resin (HCO2 form)
column chromatography eluted with water to give 4d as a
colorless solid syrup in a 95% yield. H NMR (400 MHz,
Compound 4a
1
–
Compound 4a was purified by anion resin (HCO2 form)
D2O, ppm) d: 1.35 (m, 2H), 1.68 (m, 2H), 2.15 (t, 2H), 2.77
(t, 2H), 3.70 (ddd, J = 2.08, 5.08, 11.96 Hz, 1H), 3.78–3.86
(m, 3H), 4.13–4.18 (m, 3H), 4.26 (t, 1H), 5.87 (d, J = 5.24
Hz, 1H), 6.92 (m, 1H), 7.10 (d, 5H), 7.64 (s, 1H), 7.94 (t, J
= 6.6 Hz, 1H), 8.67 (d, J = 8.12 Hz, 1H), 8.89 (d, J = 6.24
Hz, 1H), 9.08 (s, 1H). 13C NMR (100 MHz, D2O, ppm) d:
177.13, 173.46, 168.11, 167.85, 162.90, 148.26, 147.17,
144.61, 142.04, 139.20, 136.22, 131.55, 130.84, 127.82,
126.42, 124.07, 102.17, 89.34, 80.01, 73.10, 67.21, 66.68,
52.33, 37.96, 31.24, 28.70, 24.51. 31P NMR (160 MHz,
D2O, ppm) d: 0.01. HR-MS (ESI) calcd. for C26H33N6O9P
[M + H]+: 605.2125; found: 605.2103.
column chromatography eluted with H2O and the fraction
was concentrated, followed by size-exclusion chromatogra-
phy Bio-Gel1 P-2 Gel polyacrylamide gel, eluted with a
25 mmol/L NH4HCO3 water solution, to give 4a as a color-
1
less syrupy solid in a 96% yield. H NMR (400 MHz, D2O,
ppm) d: 2.46 (q, 2H), 2.85 (t, J = 6.16 Hz, 2H), 3.83 (ddd, J
= 11.96, 5.00, 2.2 Hz, 1H), 3.94 (q, 2H), 3.96 (ddd, J =
11.96, 4.56, 2.4 Hz, 1H), 4.22 (dd, J = 4.96, 2.72 Hz, 1H),
4.29 (t, J = 6.68 Hz, 2H), 4.34 (t, J = 5.2 Hz, 1H), 4.41 (t, J
= 2.44 Hz, 1H), 4.82 (dd, J = 8.84, 1.48 Hz, 1H), 4.87 (d, J
= 1.52 Hz, 1H), 5.62 (ddd, J = 23.96, 17.28, 10.44 Hz, 1H),
6.06 (d, J = 5.44 Hz, 1H), 7.72 (s, 1H), 8.12 (t, J = 6.48 Hz,
Published by NRC Research Press

40
Can. J. Chem. Vol. 88, 2010
Compound 4e
Compound 4h
–
Compound 4e was purified by anion resin (HCO2 form)
column chromatography, eluted with 5% HCO2NH4 solu-
tion, followed by octyl-functionalized silica gel eluted with
water, to give 4e as a colorless solid syrup in a 95% yield.
1H NMR (400 MHz, D2O, ppm) d: 1.48 (m, 2H), 1.80 (m,
2H), 2.31 (t, 2H), 2.86 (t, 2H), 3.80 (dd, J = 3.72, 10.6 Hz,
1H), 3.91–3.96 (m, 3H), 4.13–4.18 (m, 3H), 4.20 (s, 1H),
4.26–4.32 (m, 3H), 4.38 (s, 1H), 6.02 (d, J = 5.24 Hz, 1H),
6.84–6.91 (m, 2H), 7.28 (dd, J = 2.72, 8.76 Hz, 1H), 7.31 (s,
1H), 8.09 (t, J = 6.72 Hz, 1H), 8.81 (d, J = 8.12 Hz, 1H),
9.05 (d, J = 6.28 Hz, 1H), 9.24 (s, 1H). 13C NMR
(100 MHz, D2O, ppm) d: 178.13, 168.01, 148.45, 144.82,
142.19, 136.40, 130.94, 130.05, 126.52, 114.00, 113.78,
107.08, 106.83, 106.58, 102.30, 89.56, 80.13, 73.30, 67.26,
66.81, 52.38, 37.43, 31.26, 28.78, 24.60. 31P NMR
(160 MHz, D2O, ppm) d: 0.08. 19F NMR (376 MHz, D2O,
ppm) d: –111.8, –118.9. HR-MS (ESI) calcd. for
C26H31F2N6O9P [M + H]+: 641.1936; found: 641.1960.
Compound 4h was purified by size-exclusion chromatog-
raphy Bio-Gel1 P-2 Gel polyacrylamide gel, eluted with
25 mmol/L NH4HCO3 water solution, to give 4h as a color-
1
less solid syrup in a 91% yield. H NMR (400 MHz, D2O,
ppm) d: 1.45 (m, 2H), 1.62 (m, 2H), 2.17 (t, J = 7 Hz, 2H),
2.86 (t, J = 6 Hz, 2H), 3.75 (dd, J = 11.4, 4.15 Hz, 1H),
3.92–4.11 (m, 3H), 4.14 (t, J = 6.8 Hz, 2H), 4.18 (t, J =
2.4 Hz, 1H), 4.24 (t, J = 5.4 Hz, 1H), 4.35 (s, 1H), 5.91 (d,
J = 5.2 Hz, 1H), 7.28–7.39 (m, 5H), 7.5 (s, 3H), 7.68 (d, J =
7.8 Hz, 2H), 7.75 (t, J = 8.2 Hz, 2H), 7.91 (t, J = 6.8 Hz,
2H), 8.63 (d, J = 8.08 Hz, 1H), 8.92 (d, J = 6.16 Hz, 1H),
9.08 (s, 1H). 13C NMR (100 MHz, D2O, ppm) d: 176.29,
167.64, 148.39, 146.84, 144.82, 143.86, 142.1, 136.33,
130.95, 129.60, 126.01, 102.35, 89.66, 80.16, 73.22, 67.16,
66.76, 59.23, 52.04, 37.19, 31.47, 28.94, 24.83. 31P NMR
(160 MHz, D2O, ppm) d: 0.15. HR-MS (ESI) calcd. for
C33H39N6O9P [M + H]+: 695.2594; found: 695.2615.
Electrochemical experiment
All data were recorded on a CHI 600C electrochemical
analyzer, and further processed using the software Origin
8.0 when necessary.22 The working concentration of analytes
was 2 ꢀ 10–3 mol/L. Solutions of either 0.01 mol/L Tris/
NaCl (0.005 mol/L) or phosphate (1 mol/L) buffer were
used as supporting electrolytes. The pH was adjusted with
NaOH or H3PO4. Stock solutions of all compounds were
stored at 4 8C to avoid decomposition. Solutions were soni-
cated and purged with purified nitrogen and the temperature
was kept at 25 0.2 8C.
To use this electrode it was necessary to activate its sur-
face. For this purpose, several methods were tried, namely,
polishing with alumina powder, sonication, activation with
sulfochromic mixture, etc. The treatment selected for the
electrode, made before each measurement, was the follow-
ing: washing with distilled water, putting the electrode into
a sulfochromic mixture for 30 s, washing with distilled
water, polishing with 0.3 mm alumina powder and 0.05 mm
alumina powder, sonication for 2 min in distilled water,
washing with distilled water, and drying with soft paper.
Under such conditions measurements were highly reproduci-
ble.
Compound 4f
Compound 4f was purified by size-exclusion chromatog-
raphy Bio-Gel1 P-2 Gel polyacrylamide gel, eluted with
25 mmol/L NH4HCO3 water solution, to give 4f as a color-
1
less solid syrup in a 90% yield. H NMR (400 MHz, D2O)
d: 1.36 (m, 2H), 1.54 (m, 2H), 2.10 (t, 2H), 2.82 (t, 2H),
3.80 (ddd, J = 11.96, 5.16, 1.92 Hz, 1 H), 3.94 (m, 3H),
4.05 (t, 2H), 4.17 (dd, J = 4.52, 2.64 Hz, 1H), 4.22 (t, 1H),
4.34 (s, 1H), 4.52 (s, 2H), 5.88 (d, J = 5.32 Hz, 1H), 7.32–
7.17 (m, 4H), 7.44 (s, 1H), 7.64 (d, J = 2.44 Hz, 1H), 7.66
(dd, J = 3.6 Hz, 1H), 7.87 (t, J = 7.64 Hz, 1H), 8.58 (d, J =
8.08 Hz, 1H), 8.87 (d, J = 6.24 Hz, 1H), 9.04 (s, 1H). 13C
NMR (100 MHz, D2O) d: 178.03, 167.54, 162.80, 148.02,
146.99, 144.49, 141.87, 135.85, 135.46, 132.88, 131.16,
130.64, 129.01, 128.66, 128.40, 128.12, 126.13, 125.48,
102.21, 89.37, 80.05, 73.11, 67.21, 66.74, 52.18, 43.43,
37.40, 31.21, 28.81, 24.82. 31P NMR (160 MHz, D2O, ppm)
d: 0.11. HR-MS (ESI) calcd. for C31H37O9N6P [M + H]+:
669.2438; found: 669.2444.
Compound 4g
Compound 4g was purified by size-exclusion chromatog-
raphy Bio-Gel1 P-2 Gel polyacrylamide gel, eluted with
25 mmol/L NH4HCO3 water solution, to give 4g as a color-
To maintain the activity of NAD+, all buffer pHs must be
in the range of 5–7. Tris/NaCl (pH 7) was a better choice
than the same pH phosphate for a sensitive GC electrode re-
sponse. In NaCl (0.01 mol/L) / Tris (0.005 mol/L) buffer at
pH 7, peak currents was invariable. The potential range used
for NAD+ analog detection was –0.4 to –1.4 V, which was
the most suitable (small peak current changes). The scan
rate was 0.1 V/s.
Cyclic voltammogram for determination of dynamic con-
stants was performed with a glassy carbon electrode vs. SCE
in supporting electrolyte (0.01 mol/L NaCl / 0.005 mol/L
Tris, pH 7). The response to 2 ꢀ 10–3 mol/L NAD+ analogs
and corresponding compounds were measured in 10 mL of
supporting electrolyte at 298 0.2 8C under a nitrogen at-
mosphere.
1
less solid syrup in an 88% yield. H NMR (400 MHz, D2O,
ppm) d: 1.46 (m, 2H), 1.70 (m, 2H), 2.18 (t, J = 6.96 Hz,
2H), 2.80 (t, J = 6.62 Hz, 2H), 3.77 (dd, J = 11.36,
4.04 Hz, 1H), 3.88–3.93 (m, 3H), 4.17–4.22 (m, 5H), 4.27
(t, J = 5.16 Hz, 1H), 4.32 (s, 1H), 5.90 (d, J = 5.48 Hz,
1H), 7.16 (d, J = 7.96 Hz, 2H), 7.21 (d, J = 7.12 Hz, 1H),
7.27 (t, J = 7.24 Hz, 2H), 7.41 (m, 5H), 7.64 (s, 1H), 8.01 (t,
J = 6.88 Hz, 1H), 8.72 (d, J = 8.08, 1H), 8.98 (d, J =
6.2 Hz, 1H), 9.16 (s, 1H). 13C NMR (100 MHz, D2O, ppm)
d: 177.21, 167.63, 148.39, 144.79, 142.09, 141.24, 140.09,
136.32, 131.16, 130.97, 130.33, 129.61, 129.02, 128.81,
102.36, 89.63, 80.17, 73.23, 67.15, 66.76, 52.15, 44.87,
37.43, 31.51, 28.98, 24.88. 31P NMR (160 MHz, D2O, ppm)
d: 0.01. HR-MS (ESI) calcd. for C33H39N6O9P [M + H]+:
695.2594; found: 695.2569.
The cyclic voltammogram for pH dependence of 4h was
performed in 1 mol/L phosphate buffer at different pHs
(pH 4–9) containing 2 ꢀ 10–3 mol/L 4h.
Published by NRC Research Press

Liu et al.
41
(11) Lo, H. C.; Fish, R. H. Angew. Chem. Int. Ed. 2002, 41 (3),
Acknowledgement
478.
doi:10.1002/1521-3773(20020201)41:3<478::AID-
We are grateful for the support of the National Natural
Science Foundation of China (No. 20472084).
ANIE478>3.0.CO;2-K.
(12) Song, H. K.; Lee, S. H.; Won, K.; Park, J. H.; Kim, J. K.;
Lee, H.; Moon, S. J.; Kim, D. K.; Park, C. B. Angew.
Chem. Int. Ed. 2008, 47 (9), 1749. doi:10.1002/anie.
200703632.
(13) Lee, S. H.; Nam, D. H.; Kim, J. H.; Baeg, J. O.; Park, C. B.
ChemBioChem 2009, 10 (10), 1621. doi:10.1002/cbic.
200900156. PMID:19551795.
(14) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem.
Int. Ed. 2001, 40 (11), 2004. doi:10.1002/1521-
3773(20010601)40:11<2004::AID-ANIE2004>3.0.CO;2-5.
(15) Liu, R. H.; Visscher, J. Nucleosides Nucleotides 1994, 13
References
(1) Haigis, M. C.; Mostoslavsky, R.; Haigis, K. M.; Fahie, K.;
Christodoulou, D. C.; Murphy, A. J.; Valenzuela, D. M.;
Yancopoulos, G. D.; Karow, M.; Blander, G.; Wolberger,
C.; Prolla, T. A.; Weindruch, R.; Alt, F. W.; Guarente, L.
Cell 2006, 126 (5), 941. doi:10.1016/j.cell.2006.06.057.
PMID:16959573.
(2) Liu, Q.; Kriksunov, I. A.; Graeff, R.; Lee, H. C.; Hao, Q. J.
Biol. Chem. 2007, 282 (8), 5853. doi:10.1074/jbc.
M609093200. PMID:17182614.
(5),
1215.
doi:10.1080/15257779408011891.
PMID:
11539878.
(3) Alano, C. C.; Ying, W. H.; Swanson, R. A. J. Biol. Chem.
2004, 279 (18), 18895. doi:10.1074/jbc.M313329200.
PMID:14960594.
(4) Olek, R. A.; Ziolkowski, W.; Kaczor, J. J.; Greci, L.; Popini-
gis, J.; Antosiewicz, J. J. Biochem. Mol. Biol. 2004, 37 (4),
416. PMID:15469728.
(5) Waypa, G. B.; Schumacker, P. T. J. Appl. Physiol. 2005, 98
(1), 404. doi:10.1152/japplphysiol.00722.2004. PMID:
15591310.
(16) (a) Theocharis, A. B.; Alexandrou, N. E.; Terzis, A. J. Het-
erocycl. Chem. 1990, 27 (6), 1741. doi:10.1002/jhet.
5570270643.; (b) van der Peet, P.; Gannon, C. T.; Walker,
I.; Dinev, Z.; Angelin, M.; Tam, S.; Ralton, J. E.; McCon-
ville, M. J.; Williams, S. J. ChemBioChem 2006, 7 (9),
1384. doi:10.1002/cbic.200600159. PMID:16841351.; (c)
Gunter, M. J.; Farquhar, S. M.; Mullen, K. M. N. J. Chem.
2004, 28 (12), 1443. doi:10.1039/b411583j.
(17) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K.
B. Angew. Chem. Int. Ed. 2002, 41 (14), 2596. doi:10.1002/
1521-3773(20020715)41:14<2596::AID-ANIE2596>3.0.
CO;2-4.
(6) (a) Barker, C. D.; Reda, T.; Hirst, J. Biochemistry (Mosc.)
2007, 46 (11), 3454. doi:10.1021/bi061988y.; (b) Zhang, Z.;
Blake, D. R.; Stevens, C. R.; Kanczler, J. M.; Winyard, P.
G.; Symons, M. C.; Benboubetra, M.; Harrison, R. Free Ra-
(18) Angulo, M.; Ruiz Montoya, M.; Marin Galvin, R.; Rodri-
guez Mellado, J. Electroanalysis 1997, 9 (4), 345. doi:10.
1002/elan.1140090417.
(19) Laviron, E. J. Electroanal. Chem. 1974, 52 (3), 355. doi:10.
1016/S0022-0728(74)80448-1.
(20) Zhou, Z.; Li, S. W.; Zhang, Y. L.; Liu, M. L.; Li, W. J. Am.
Chem. Soc. 2005, 127 (31), 10824. doi:10.1021/ja052280u.
PMID:16076176.
(21) Graham, S. M.; Macaya, D. J.; Sengupta, R. N.; Turner, K.
B. Org. Lett. 2004, 6 (2), 233. doi:10.1021/ol036152r.
PMID:14723536.
dic.
Res.
1998,
28
(2),
151.
doi:10.3109/
10715769809065801. PMID:9645392.
(7) Damian, A.; Omanovic, S. J. Mol. Catal. Chem. 2006, 253
(1–2), 222. doi:10.1016/j.molcata.2006.03.020.
(8) Xu, X. H.; Chen, J. H.; Li, W.; Nie, Z.; Yao, S. Z. Electro-
chem. Commun. 2008, 10 (10), 1459. doi:10.1016/j.elecom.
2008.07.033.
(9) Salimi, A.; Izadi, M.; Hallaj, R.; Soltanian, S.; Hadadzadeh,
H. J. Solid State Electrochem. 2009, 13 (3), 485. doi:10.
1007/s10008-008-0583-6.
(10) Lo, H. C.; Leiva, C.; Buriez, O.; Kerr, J. B.; Olmstead, M.
M.; Fish, R. H. Inorg. Chem. 2001, 40 (26), 6705. doi:10.
1021/ic010562z. PMID:11735482.
(22) OriginLab Corporation. Origin 8.0; MicroCal, Inc.: North-
ampton, MA, USA, 2007.
Published by NRC Research Press