Synthesis of phosphodiester-type nicotinamide adenine dinucleotide analogs

Article abstract of DOI:10.1016/j.tet.2009.08.007

Fourteen phosphodiester-type β-nicotinamide adenine dinucleotide (NAD⁺) analogs were prepared starting from nicotinamide. The phosphodiester linkage was effectively assembled in 69-93% yields via condensation reaction between 2′,3′-di-O-acetyl nicotinamide mononucleotide and alcohols in the presence of 2,4,6-triisopropylbenzenesulfonyl chloride. The analog β-nicotinamide ribose-5-(2-phenylethyl) phosphate showed beneficial effects on cell growth of model microorganisms.

Full text of DOI:10.1016/j.tet.2009.08.007

Tetrahedron 65 (2009) 8378–8383
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of phosphodiester-type nicotinamide adenine dinucleotide analogs
,
,
,
,c,
Wujun Liu a b, Siguo Wu a b, Shuhua Hou a b, Zongbao (Kent) Zhao a
*
a Dalian Institute of Chemical Physics, CAS, Dalian 116023, PR China
b Graduate School of the Chinese Academy of Sciences, Beijing 100039, PR China
c Dalian National Laboratory of Clean Energy, Dalian 116023, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 June 2009
Received in revised form 31 July 2009
Accepted 6 August 2009
Available online 9 August 2009
Fourteen phosphodiester-type
starting from nicotinamide. The phosphodiester linkage was effectively assembled in 69–93% yields via
condensation reaction between 20,30-di-O-acetyl nicotinamide mononucleotide and alcohols in the
presence of 2,4,6-triisopropylbenzenesulfonyl chloride. The analog
ethyl) phosphate showed beneficial effects on cell growth of model microorganisms.
Ó 2009 Elsevier Ltd. All rights reserved.
b
-nicotinamide adenine dinucleotide (NADþ) analogs were prepared
b-nicotinamide ribose-5-(2-phenyl-
Keywords:
Nicotinamide adenine dinucleotide
Coupling reaction
Phosphodiesters
Bioorganic chemistry
1. Introduction
induce T cell death.8 NADþ uptake by mammalian cells has also
been firmly recognized.9 These findings have greatly enriched our
understanding on the essential functions of pyridine nucleotides.10
When NADþ functions as a coenzyme of dehydrogenase, the
adenosine pocket and the NAR binding site are specially isolated.11
This appeals to engineering of the adenosine binding pocket of
dehydrogenase12 to match elaborated NADþ analogs in an approach
as found in kinases research.13 In such scenario, NADþ analogs are
of great potential to be developed for elucidation of complicated
problems at the interface of chemistry and biology.
Nicotinamide adenine dinucleotide (NADþ, Fig. 1) and its re-
duced form, NADH, serve as cofactors of many oxidoreductive en-
zymes.1 They are also functional molecules in many aspects of life. It
has been established that NADþ plays critical roles in calcium ho-
meostasis,2 cell proliferation,3 aging,4 apoptosis,5 covalent protein
modification,6 gene expression, and regulation of numerous NADþ-
dependent non-oxidoreductive enzymes.7 Furthermore, recent
studies showed that NADþ could act as an immune modulator or
Although modifications on adenosine, ribose, PPi, and nicotin-
amide moiety of the NADþ structure have been achieved, those with
substantial changes of the NADþ skeleton are limited.14 In particular,
preparation of diversified phosphodiester-type NADþ analogs
(Fig. 1) remains challenging in terms of efficient coupling strategy
and product purification. The simplest phosphodiester-type NADþ
O
OH
OH
NH2
O
P
OH
O
P
OH
O
N
N
O
O
O
N
N
O
N
NH2Adenosine
PPi
NAR
OHOH
analog, b
-nicotinamide ribose-50-methyl phosphate, was prepared
NAD+
using the N,N0-dicyclohexylcarbodiimide (DCC)/4-dimethylamino-
pyridine (DMAP) system.15 The reduced form of the analog acted as
a biomimic cofactor binding to horse liver alcohol dehydrogenase
and facilitated reduction of prochiral ketones in moderate yields.16
Although the results were stimulating, no other phosphodiester-
type NADþ analogs have been documented so far. Because a methyl
group could barely provoke tight interactions with protein, more
complex structures are intriguing to mimic the adenosine moiety.
Here, we designed novel phosphodiester-type NADþ analogs (Fig.1),
of which the adenine moiety, the ribose moiety, and the PPi linkage
was replaced with other organic constituents, ether or alkane
chains17 and monophosphate functionality, respectively.
O
X
R
NH2
O
P
OH
O
O
N
O
X=O or CH2
OHOH
NAD+ analogs
Figure 1. Structural function of NADþ and its phosphodiester-type analogs.
* Corresponding author. Tel./fax: þ86 411 8437 9211.
E-mail address: zhaozb@dicp.ac.cn (Z.(Kent) Zhao).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.08.007

W. Liu et al. / Tetrahedron 65 (2009) 8378–8383
8379
We now report our efforts on preparation of these NADþ ana-
2.2. Synthesis
logs. We also present preliminary results of microbial cell growth
and activity assay of dehydrogenases in the presence of a typical
asymmetrical phosphodiester NADþ analog.
In the literatures, 4 has been prepared by selective hydrolysis21
of NADþ or chemical synthesis.22 To facilitate in-house preparation
of various NADþ analogs, we prepared 4 starting from commercially
available nicotinamide according to a modified procedure and
obtained a total isolated yield of 63%.22c,23 Briefly, nicotinamide was
treated with 1,1,1,3,3,3-hexamethyldisilazane (HMDS) and TMSCl,
then reacted with 1,2,3,5-tetra-O-acetylribofuranose (2) in the
presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) to
form 20,30,50-tri-O-acetyl nicotinamide riboside via a glycosylation
mechanism. The intermediate was deacetylated by methanolic
ammonia to give nicotinamide ribose (NAR, 3), which was phos-
phorylated with OPCl3 to afford 4. With NMN in hand, our attention
turned to assemble the phosphodiester linkage en route to the
NADþ analogs.
2. Results and discussion
2.1. Synthetic plan
Construction of phosphatediesters including positive charged
constituents remains challenge, although many strategies have
been developed to assemble phosphodiester via either P(III)18 or
P(V)19 reagents in nucleic acid chemistry.20 P(III) strategy required
more steps such as oxidation, protection, and deprotection. This
resulted in tedious purification work and breakdown of the
anomeric C–N bond in NADþ analog assembly. Thus, we focused on
P(V) chemistry. Based on the retrosynthetic analysis, we planned
our synthesis work by one of the two routes (Scheme 1). In-
termolecular condensation reaction between nicotinamide mono-
nucleotide (NMN, 4) and alcohols was designed as the key step in
Route I. However, it was difficult to find an activation system to
realize the coupling reaction, because NMN is liable to form an
inner salt and has notorious solubility in most organic solvents.
Moreover, breakdown of 4 via scission of the C–N bond was sub-
stantial in many experiments. According to Route II, we successfully
prepared 6 in dichloromethane. However, attempts were in vain to
assemble the C–N bond via glycosylation reaction with nicotin-
amide or its N-protected derivatives in the presence of Lewis acids.
Therefore, these NADþ analogs were prepared in a way similar to
Route I with NMN derivatives (vide infra).
The DCC/DMAP strategy was first applied to couple 4 with 2-
phenylethanol (c), a conceptive adenosine analog, in various sol-
vents.15 It was found that long reaction time (>3 d) and large excess
DCC were required to achieve good conversion, yet low yield and
complicated byproducts were observed based on 31P NMR analysis.
In the reaction, significant amount of 4 dimerized to form
the symmetric pyrophosphate product. Reactions involved the
hydroxyl groups at 20- and 30-positions of 4 also occurred. Conse-
quently, it was difficult to recover
b
-nicotinamide ribose-50-(2-
phenylethyl) phosphate 9c.
We then prepared 20,30-di-O-acetyl nicotinamide mono-
nucleotide (Ac2NMN, 7) in around 62% overall yield started from
nicotinamide (Scheme 2).24 It was found that Ac2NMN had rea-
sonable solubility and high reactivity in solvents such as DMF,
O
O
O
NH2
O
P
O
P
NH2
NH2
-OTf
O
O
O
R
N
HO
O
HO
O
N
N
NH2
OAc
O-
O
O
O-
O
Route I
N
OHOH
OHOH
4
OH OH
3
NAD+ analogs
Route II
1
O
P
O
P
OH
R
O
O
HO
AcO
OAc
OAc
O
O
O
OH
OAcOAc
OAcOAc
OAcOAc
2
6
5
Scheme 1. Retrosynthetic analysis for phosphodiester-type NADþ analogs.
O
O
O
O
HO P
O-
O
NH2
ii
NH2
iii
NH2
O
HO
R
O
O P O
N
N
N
i
-OTf
NH2
O-
O
O
O
N
1OR1
OHOH
3
OR
AcO OAc
7
8: R1=Ac
9: R1=H
1
iv
ROH:
OH
OH
OH
a (85%)
O
OH
O
OH
H3C
H3C
O
d (86%)
b (89%)
c (93%)
e (85%)
O
O
O
F
OH
O
OH
O
OH
O
OH
O
O
OH
OMe
OMe
f (89%)
g (88%)
h (87%)
i (85%)
j (74%)
O
Cl
O
Cl
O
O
N
O
OH
O
F
O
OH
OH
OH
H
l (70%)
m (69%)
n (76%)
k (70%)
Scheme 2. Synthesis of phosphodiester-type NADþ analogs. Reagents and conditions: (i) (a) TMSCl, HMDS, 120 ꢀC, 8 h; (b) 2, 1,2-dichloroethane, TMSOTf, 45 ꢀC, 2 h; (c) NH3/CH3OH,
ꢁ5 ꢀC, 20–48 h; (ii) (a) POCl3 (4 equiv), PO(OMe)3, ꢁ5 to 0 ꢀC, 12 h, 63% for four steps; (b) Ac2O/pyridine (1:1), 0–5 ꢀC, 24 h; (iii) (a) ROH (a–n), coupling reagent, DMF/pyridine (1:1),
25 ꢀC, 6 h; (iv) 1 M NH3/CH3OH, ꢁ5 ꢀC, 4 h, 96–98%. Data in the parentheses indicated the coupling yield.

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W. Liu et al. / Tetrahedron 65 (2009) 8378–8383
DMSO, and pyridine, which were routinely used in coupling re-
actions. Moreover, blocking the free hydroxyl groups on the ribose
ring also eliminated side reactions and improved the stability of the
precursor.
When Ac2NMN and 2-phenylethanol was treated with 2,4,6-
triisopropylbenzenesulfonyl chloride (TIPS-Cl), 31P NMR analysis
indicated that the singlet at 3.7 ppm of the starting material dis-
appeared within 6 h and signals at 0.3 and ꢁ11.2 ppm, corre-
sponding to the product and the pyrophosphate byproduct,
respectively, evolved. A typical yield was about 93% (Table 1, entry
8) that was comparable to nucleotide or glycosyl phosphate diester
synthesis using P(III) agents.18 Seth et al. improved the yield of
phosphodiester in nucleotide synthesis using 1H-tetrazole and
TIPS-Cl.26 However, dimerization of Ac2NMN increased noticeably
if 1H-tetrazole was applied.
The alcohols (a–n) used in NADþ analogs preparation were ei-
ther commercially available (a–c) or obtained via Williamson ether
synthesis (d–n) using the corresponding phenol and the halide
(Scheme 2).25
To attain these analogs, we evaluated commercially available
condensation agents found in the preparations of DNA or RNA
fragments (Fig. 2). Table 1 summarizes the results of the coupling
reaction between Ac2NMN (7) and 2-phenylethanol (c) with
various condensation reagents using an anhydrous DMF/pyridine
solvent system at room temperature. Conventional carbodiimides
derivatives including DCC, N,N0-diisopropylcarbodiimide (DIC)
and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydro-
chloride (EDCI) showed unsatisfactory results (Table 1, entries
1–3). Although about 100% conversion for 7 was achieved with
these mediators, the product 8c was less than 10% based on 31P
NMR analysis. Difficulties also occurred to recover 8c in the
presence of excess byproducts and the coupling reagent
derivatives. Acyl chlorides, commonly used for assembling of H-
phosphonate linkage in nucleic acid chemistry, such as pivaloyl
chloride (Piv-Cl) and 1-adamantanecarbonyl chloride (AC-Cl)
were estimated.20 Both Piv-Cl and AC-Cl facilitated the coupling
reaction, yet the reaction was rather slow. It took over 10 d for
Piv-Cl or AC-Cl to reach 31P NMR yields of 51% and 34%, re-
spectively (Table 1, entries 4 and 5). Moreover, large excess
2-phenylethanol and the acyl chlorides were required. Chloro-
phosphates were also tested, including diphenyl phosphoro-
chloridate (DPP-Cl) and bis(2-oxo-3-oxazolidinyl) phosphinic
chloride (BOP-Cl), which are known to prompt asymmetrical
pyrophosphate synthesis. Near quantitative conversion of 7 was
achieved within 24 h in the presence of DPP-Cl or BOP-Cl (Table
1, entries 6 and 7).
Another agent with a similar structure to that of TIPS-Cl, 1-(2,4,6-
triisopropylbenzenesulfonyl)-3-nitro-1H-1,2,4-triazole (TIPS-N) was
estimated, however, the NMR yield was only 24% after 20 h (Table 1,
entry 9).
As demonstrated herein, TIPS-Cl was the favored agent to pre-
pare 8c. When it was applied for the coupling reaction with other
alcohols, NADþ analogs 8a–8n were obtained in isolated yields
ranged from 69% to 93% (Scheme 2). It was interesting to note that
analogs 8j–8n, resulted from alcohols with an electron withdraw-
ing or bulky group, were obtained in slightly lower yields. We
reasoned that nucleophilic attack on the phosphorus atom of
phosphate–sulfonate anhydride intermediate was less effective due
to stronger repulsion forces between these alcohol substrates and
the TIPS group.
2.3. Preliminary bioactivity assays
Preliminary tests were performed using the analog 9c for its
effects on cell growth of model microorganisms. Compared to the
control samples, Escherichia coli DH5
evisiae ATCC 26108 cells grew faster in the presence of 100
a
and Saccharomyces cer-
M 9c
m
(Fig. 3). The maximal growth increments for E. coli and S. cer-
evisiae cells reached 16% and 52%, respectively. It is known that
NADþ or NAR can have beneficial effects on the culture of
mammalian cells, because these compounds can change the
metabolism of pyridine nucleotide cofactors.9 The analog 9c
promoted microbial cell growth might also suggest a similar
mechanism.
HCl
N
N
C N
DIC
N
C N
N
C N
DCC
EDCI
COCl
O
P
O
COCl
A
24
21
18
15
12
9
O
OPh
P
PhO
N
N
O
Cl
Cl
O
O
Piv-Cl
AC-Cl
DPP-Cl
BOP-Cl
O
S N
O
N
SO2Cl
6
3
N
NO2
0
0
2
4
Time (h)
6
8
TIPS-N
TIPS-Cl
Figure 2. Structures of condensation agents used in this work.
80
70
60
50
40
30
20
10
0
B
Table 1
Results and conditions with different condensation reagentsa
Entry
Reagents
Loading (equiv)
Time (h)
NMR yield (%)
1
2
3
4
5
6
7
8
9
DCC
DIC
EDC
Piv-Cl
AC-Cl
DPP-Cl
BOP-Cl
TIPS-Cl
TIPS-N
3
3
3
10
10
3
3
3
3
36
76
78
240
240
24
20
6
20
10
10
8
51
34
39
33
93
24
0
6
15
17
Time (h)
Figure 3. Growth improvements of (A) E. coli DH5
the presence of 100
M NADþ analog 9c. Cell growth was indicated using OD600 data
and normalized with the control cultures.
a and (B) S. cerevisiae ATCC 26108 in
a
Conditions: Ac2NMN and 3 equiv of 2-phenylethanol were employed in the
presence of a condensation agent in DMF/pyridine (1:1) at 25 ꢀC.
m

W. Liu et al. / Tetrahedron 65 (2009) 8378–8383
8381
Enzymatic activity of alcohol dehydrogenase (ADH) from S.
cerevisiae was also tested in the presence of analog 9c. Compared to
the control sample, there was no discernible reaction rate change
even the concentration of 9c was fivefold more than that of NADþ
(Fig. 4). Similar results were also observed in the case of lactate
dehydrogenase (LDH) from Lactobacillus leichmanii (data not
shown). These results indicated that the synthetic analogs were
unlikely recognized by wild-type NADþ-dependent oxidoreduc-
tases. Extensive screening work is ongoing to find mutated en-
zymes from designed libraries.12
Ac2NMN 7 was prepared starting from nicotinamide in five steps
using a modified procedure of the literatures.22c,23
4.3. General procedure for preparation of NADD
analogs (9a–9n)
All NADþ analogs were synthesized following the procedure
described for 9c. All purifications were performed at 25 ꢀC except
size exclusion chromatography (4 ꢀC).
4.3.1.
b
-Nicotinamide ribose-5-(2-phenylethyl) phosphate (9c).
L,
Ac2NMN 7 (263 mg, 0.63 mmol) and 2-phenylethanol c (200
m
NAD+ + 9c + ADH
0.95
1.67 mmol) were dissolved in pyridine/DMF (1:1, 25 mL). TIPS-Cl
(604 mg, 2 mmol) was added and the suspension was stirred at
room temperature for 6 h. After removing the solvents, the residue
was taken by H2O (15 mL), extracted with DCM (3ꢂ15 mL). The
supernatant was purified by column chromatography on octyl-
functionalized silica gel eluted with CH3OH/H2O (30:1) to give the
crude intermediate 8c (306 mg, 0.58 mmol). This was treated with
1 M NH3/MeOH (0.2 mL) at ꢁ5 ꢀC for 4 h. The solvent was evapo-
rated and the residue was purified by column chromatography on
anion resin (201ꢂ2, HCOꢁ2 form, H2O). The corresponding fraction
was pooled and lyophilized to give syrupy solid 9c (251 mg, 91%).
NAD+ + ADH
0.90
0.85
0.80
0.75
0.70
0.65
0.60
1H NMR (400 MHz, D2O)
d (ppm) 3.65 (m, 3H), 3.94 (m, 3H), 4.11 (t,
J¼2.2 Hz, 1H), 4.35 (s, 1H), 6.01 (d, J¼5.4 Hz, 1H), 6.68 (m, 3H), 7.15
0
20
40
60
80
(m, 2H), 8.06 (t, J¼7.6 Hz, 1H), 8.80 (d, J¼8.0 Hz, 1H), 9.06 (d,
J¼6.0 Hz, 1H), 9.22 (s, 1H). 13C NMR (100 MHz, D2O)
d (ppm) 62.3,
Figure 4. Time course of ADH activity in the presence of 9c.
65.3, 66.7, 69.2, 73.4, 80.1, 89.6, 89.7, 102.2, 128.9, 130.8, 131.0, 131.6,
136.2, 141.2, 141.9, 144.7, 148.4, 167.0. 31P NMR (160 MHz, D2O)
3. Conclusions
d
(ppm) 0.2. HRMS (ESI) calcd for C19H23N2O8P [MþH]þ 439.1270;
found: 439.1284.
We have developed an effective and general strategy to prepare
phosphodiester-type NADþ analogs starting from nicotinamide
through coupling of Ac2NMN with alcohols in the presence of TIPS-
Cl. Preliminary study indicated that one representative analog 9c
promoted microbial cell growth. We are now exploring the bi-
ological function with these compounds in a wide variety of chem-
ical biologyareas, and detailed results will be reported in due course.
4.3.2.
b
-Nicotinamide ribose-5-(1-butyl) phosphate (9a). Compound
9a (syrupy solid, 203 mg, 82%) was obtained from 7 (263 mg,
0.63 mmol) as described for the synthesis of 9c, followed by size
exclusion chromatography (polyacrylamide gel, 25 mM NH4HCO3).
1H NMR (400 MHz, D2O)
d
(ppm) 0.73 (t, J¼7.2 Hz, 2H), 1.19 (m, 2H),
1.44 (m, 2H), 3.73 (q, 2H), 4.01 (d, J¼5.3 Hz, 1H), 4.18 (d, J¼10.3 Hz,
1H), 4.3 (s, 1H), 4.40 (t, J¼5.1 Hz, 1H), 4.49 (s, 1H), 6.08 (d, J¼4.3 Hz,
1H), 8.15 (t, J¼6.9 Hz,1H), 8.86 (d, J¼7.9 Hz,1H), 9.14 (d, J¼6.0 Hz,1H),
4. Experimental
4.1. General
9.34 (s, 1H). 13C NMR (100 MHz, D2O)
d (ppm) 15.4, 20.8, 34.4, 66.8,
68.7, 73.5, 80.2, 89.7, 102.3, 130.9, 136.4, 142.2, 144.9, 148.4, 168.2. 31P
NMR (160 MHz, D2O) d (ppm) 0.5. HRMS (ESI) calcd for C15H23N2O8P
All reagents wereanalytical grade and obtained fromcommercial
suppliers (ABCR, ACROS or Sigma). NMR spectra were measured
with a Bruker DRX-400 spectrometer (400.3 MHz for 1H,100.6 MHz
for 13C, 160.1 MHz for 31P, and 376.4 for 19F) at 298 K. HRMS was
obtainedona Q-TOF-MSandoperatedwith anelectrospraysource in
positive ion mode. Optical density at 600 nm (OD600) was recorded
on JASCOV-530UV–vis spectrophotometer. F254 thin-layerand silica
gel (400 mesh) were purchased from Yantai Jiangyou Silica Co., Ltd.,
China. Octyl-functionalized silica gel was purchased from Sigma. Ion
exchange resin (100–200 mesh) was purchased from the Chemical
[MþH]þ 391.1270; found: 391.1277.
4.3.3.
b-Nicotinamide ribose-5-[2-(1-adamantane)ethyl] phosphate
(9b). Compound 9b (syrupy solid, 271 mg, 87%) was obtained from
7 (263 mg, 0.63 mmol) as described for the synthesis of 9c, fol-
lowed by size exclusion chromatography (polyacrylamide gel,
25 mM NH4HCO3). 1H NMR (400 MHz, D2O)
d (ppm) 1.20 (t,
J¼7.4 Hz, 2H), 1.28 (d, J¼2.0 Hz, 6H), 1.39–1.51 (m, 6H), 1.69 (s, 3H),
3.74 (q, 2H), 4.01 (ddd, J¼1.9, 4.9, 12.0 Hz, 1H), 4.17 (ddd, J¼2.2, 4.0,
12.1 Hz, 1H), 4.28 (s, 1H), 4.43 (s, 1H), 4.48 (s, 1H), 6.12 (d, J¼5.0 Hz,
1H), 8.21 (t, J¼7.2 Hz, 1H), 8.92 (d, J¼7.9 Hz, 1H), 9.19 (d, J¼5.8 Hz,
Plant of Nankai University, Tianjin, China. Bio-Gel P2 resin (45
mm)
was obtained from Bio-Rad Laboratories, Inc. E. coli DH5 and S.
a
1H), 9.37 (s, 1H). 13C NMR (100 MHz, D2O)
d (ppm) 30.8, 33.7, 39.1,
cerevisiae ATCC 26108 were purchased from Beijing Ding Guo Bio-
tech. Co., Ltd and Invitrogen Co., Ltd, respectively. ADH from S. cer-
evisiae (CAS No. 9031-72-5) and LDH from L. leichmanii (CAS No.
9028-36-8) were purchased from Sigma. All organic reactions were
carried out under a nitrogen atmosphere.
44.5, 46.5, 65.0, 66.8, 73.3, 80.2, 89.7, 102.5, 131.1, 136.5, 142.3, 145.2,
148.7, 167.8. 31P NMR (160 MHz, D2O)
d (ppm) 0.6. HRMS (ESI) calcd
for C23H33N2O8P [MþH]þ 497.2053; found: 497.2061.
4.3.4.
b-Nicotinamide ribose-5-[5-(m-tolyloxy)pentyl] phosphate
(9d). Compound 9d (syrupy solid, 266 mg, 83%) was obtained from
4.2. Procedure for preparation of intermediate compounds
7 (263 mg, 0.63 mmol) as described for the synthesis of 9c. 1H NMR
(400 MHz, D2O) d (ppm) 1.34 (m, 2H), 1.56 (m, 4H), 2.10 (s, 3H), 3.77
Adenosine analogs d–n in Scheme 2 were synthesized via Wil-
liamson ether synthesis using corresponding phenol and the halide
according to known procedure (yield 80–92%).25
(m, 4H), 4.00 (ddd, J¼2.5, 4.8, 10.4 Hz, 1H), 4.14 (ddd, J¼2.0, 4.0,
12.0 Hz, 1H), 4.27 (m, 1H), 4.32 (t, J¼4.8 Hz,1H), 4.44 (s, 1H), 5.99 (d,
J¼5.1 Hz, 1H), 6.53 (t, J¼7.2 Hz, 2H), 6.65 (d, J¼7.5 Hz, 1H), 7.01 (t,

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W. Liu et al. / Tetrahedron 65 (2009) 8378–8383
J¼7.8 Hz, 1H), 8.07 (t, J¼6.4 Hz, 1H), 8.73 (d, J¼8.1 Hz, 1H), 9.07 (d,
from 7 (263 mg, 0.63 mmol) as similar procedure described for the
synthesis of 9c (anion resin, aqueous 5% HCO2NH4), followed by
column chromatography on octyl-functionalized silica gel with
J¼6.2 Hz, 1H), 9.20 (s, 1H). 13C NMR (100 MHz, D2O)
d (ppm) 22.9,
24.3, 30.4, 31.9, 66.8, 68.7, 70.5, 73.1, 80.1, 89.6, 102.3, 114.0, 117.7,
124.5, 130.9, 132.1, 136.2, 142.2, 142.7, 144.7, 148.3, 160.5, 167.7. 31P
CH3OH/H2O (30:1) as eluent. 1H NMR (400 MHz, D2O)
d (ppm) 1.37
NMR (160 MHz, D2O)
d
(ppm) 0.6. HRMS (ESI) calcd for
(q, 2H), 1.54 (m, 2H), 1.63 (m, 2H), 3.66 (s, 3H), 3.76 (q, 2H), 3.84 (t,
J¼6.2 Hz, 2H), 4.01 (dd, J¼4.5, 12.2 Hz, 1H), 4.16 (dd, J¼4.1, 12.0 Hz,
1H), 4.27 (m,1H), 4.34 (t, J¼5.0 Hz,1H), 4.45 (s,1H), 5.99 (d, J¼5.2 Hz,
1H), 6.81 (m, 4H), 8.08 (t, J¼7.3 Hz, 1H), 8.72 (d, J¼8.0 Hz, 1H), 9.06
C23H31N2O9P [MþH]þ 511.1845; found: 511.1839.
4.3.5.
b-Nicotinamide ribose-5-{2-[2-(m-tolyloxy)ethoxy]ethyl} phos-
phate (9e). Compound 9e (syrupy solid, 267 mg, 83%) was obtained
(d, J¼6.2 Hz, 1H), 9.22 (s, 1H). 13C NMR (100 MHz, D2O)
d (ppm) 24.2,
from 7 (263 mg, 0.63 mmol) as described for the synthesis of 9c. 1H
30.3, 31.9, 57.9, 66.8, 68.7, 71.2, 73.1, 80.1, 89.6, 114.5, 115.7, 123.9,
NMR (400 MHz, D2O)
d
(ppm) 2.04 (s, 3H), 3.59 (t, J¼4.2 Hz, 2H), 3.67
124.0, 130.6, 130.9, 136.2, 142.2, 148.3, 149.7, 150.7, 162.7, 167.8. 31P
(t, J¼7.2 Hz, 2H), 3.88 (m, 4H), 3.97 (ddd, J¼1.9, 4.8, 12.1 Hz, 1H), 4.15
(ddd, J¼1.8, 3.4, 12.1 Hz, 1H), 4.21 (m, 1H), 4.25 (t, J¼5.1 Hz, 1H), 4.34
(s, 1H), 5.86 (d, J¼5.2 Hz, 1H), 6.43 (m, 2H), 6.57 (d, J¼7.5 Hz, 1H),
6.93 (t, J¼8.3 Hz, 1H), 7.99 (t, J¼7.5 Hz, 1H), 8.63 (d, J¼8.1 Hz, 1H),
NMR (160 MHz, D2O) d (ppm) 0.6. HRMS (ESI) calcd for
C23H31N2O10P [MþH]þ 527.1795; found: 527.1802.
4.3.10.
b-Nicotinamide ribose-5-{2-[2-(3-fluorophenoxy)ethoxy]ethyl}
9.00 (d, J¼6.2 Hz, 1H), 9.08 (s, 1H). 13C NMR (100 MHz, D2O)
d
(ppm)
phosphate (9j). Compound 9j (syrupy solid, 234 mg, 72%) was
obtained from 7 (263 mg, 0.63 mmol) as similar procedure described
for the synthesis of 9c (anion resin, aqueous 5% HCO2H). 1H NMR
66.8, 67.6, 69.4, 71.6, 72.7, 73.0, 80.1, 89.5, 102.2, 117.5, 124.6, 129.2,
130.9, 132.0, 136.0, 141.9, 142.6, 144.6, 148.1, 160.1, 162.8, 167.6. 31P
NMR (160 MHz, D2O)
d
(ppm) 0.3. HRMS (ESI) calcd for
(400 MHz, D2O)
d
(ppm) 3.65 (s, 2H), 3.75 (t, J¼3.8 Hz, 2H), 3.91 (m,
C22H29N2O10P [MþH]þ 513.1638; found: 513.1632.
2H), 3.99 (m, 3H), 4.17 (dd, J¼3.9, 11.9 Hz, 1H), 4.26 (m, 1H), 4.33 (m,
1H), 4.40 (m, 1H), 5.97 (d, J¼5.2 Hz, 1H), 6.56 (m, 3H), 7.12 (m, 1H),
8.10 (t, J¼8.1 Hz, 1H), 8.73 (d, J¼8.7 Hz, 1H), 9.08 (d, J¼6.2 Hz, 1H),
4.3.6.
b-Nicotinamide ribose-5-{2-[2-(2-phenoxy)ethoxy]ethyl} phos-
phate (9f). Compound 9f (syrupy solid, 242 mg, 86%) was obtained
9.21 (s, 1H). 13C NMR (100 MHz, D2O)
d (ppm) 66.9, 67.6, 69.9, 71.5,
from 7 (263 mg, 0.63 mmol) as described for the synthesis of 9c. 1H
72.7, 72.8, 73.2, 80.2, 89.5, 89.6, 102.3, 104.4, 104.7, 110.3, 110.6, 112.9,
NMR (400 MHz, D2O)
d
(ppm) 3.64 (t, J¼4.3 Hz, 2H), 3.74 (t, J¼4.2 Hz,
131.0, 133.1, 133.2, 136.2, 142.1, 144.8, 148.3, 161.6, 167.8. 31P NMR
2H), 3.91 (m, 2H), 3.99 (m, 3H), 4.15 (dd, J¼2.5, 5.5 Hz, 2H), 4.25 (d,
J¼5.3 Hz,1H),4.29(d, J¼2.6 Hz,1H), 4.38(s,1H),5.93(d, J¼5.4 Hz,1H),
6.73 (d, J¼8.2 Hz, 2H), 6.82 (t, J¼7.4 Hz,1H), 7.13 (t, J¼7.7 Hz,1H), 8.05
(t, J¼6.7 Hz, 1H), 8.69 (d, J¼8.1 Hz, 1H), 9.06 (d, J¼6.2 Hz, 1H), 9.15 (s,
(160 MHz, D2O) d d (ppm)
(ppm) 0.3. 19F NMR (376 MHz, D2O)
ꢁ111.5. HRMS (ESI) calcd for C21H26FN2O10P [MþH]þ 517.1406;
found: 517.1397.
1H).13C NMR (100 MHz, D2O)
d
(ppm) 66.9, 67.6, 69.6, 71.7, 72.7, 73.2,
4.3.11. b-Nicotinamide ribose-5-[5-(3-fluorophenoxy)pentyl] phos-
80.2, 89.6, 102.3, 117.0, 124.0, 129.4, 131.0, 132.3, 136.2, 142.1, 144.8,
phate (9k). Compound 9k (syrupy solid, 217 mg, 67%) was obtained
from 7 (263 mg, 0.63 mmol) as similar procedure described for the
synthesis of 9c (anion resin, aqueous 5% HCO2H). 1H NMR
148.3, 160.2, 167.9. 31P NMR (160 MHz, D2O)
d (ppm) 0.3. HRMS (ESI)
calcd for C21H27N2O10P [MþH]þ 449.1482; found: 449.1493.
(400 MHz, D2O) d (ppm) 1.37 (q, 2H), 1.57 (m, 2H), 1.64 (m, 2H), 3.78
4.3.7.
b
-Nicotinamide ribose-5-(5-phenoxypentyl) phosphate
(q, 2H), 3.87 (t, J¼6.2 Hz, 2H), 4.05 (dd, J¼4.5, 11.6 Hz, 1H), 4.20 (dd,
J¼4.3, 12.0 Hz, 1H), 4.31 (m, 1H), 4.38 (t, J¼5.0 Hz, 1H), 4.48 (s, 1H),
6.05 (d, J¼5.3 Hz, 1H), 6.60 (m, 3H), 7.15 (m, 1H), 8.14 (t, J¼7.2 Hz,
1H), 8.78 (d, J¼8.0 Hz, 1H), 9.12 (d, J¼6.1 Hz, 1H), 9.28 (s, 1H). 13C
(9g). Compound 9g (syrupy solid, 265 mg, 85%) was obtained
from 7 (263 mg, 0.63 mmol) as described for the synthesis of
9c. 1H NMR (400 MHz, D2O)
d (ppm) 1.30 (m, 2H), 1.48 (m, 2H),
1.54 (m, 2H), 3.71 (q, 2H), 3.75 (t, J¼6.4 Hz, 2H), 3.95 (ddd,
J¼2.2, 4.9, 12.0 Hz, 1H), 4.10 (ddd, J¼2.4, 4.2, 12.0 Hz, 1H), 4.22
(m, 1H), 4.28 (t, J¼5.0 Hz, 1H), 4.39 (s, 1H), 5.96 (d, J¼5.2 Hz,
1H), 6.68 (d, J¼8.1 Hz, 2H), 6.77 (t, J¼7.4 Hz, 1H), 7.09 (t,
J¼8.4 Hz, 2H), 8.02 (t, J¼7.8 Hz, 1H), 8.69 (d, J¼8.1 Hz, 1H), 9.02
NMR (100 MHz, D2O) d (ppm) 66.8, 68.8, 71.0, 73.2, 80.2, 89.6, 89.7,
102.4, 104.5, 104.7, 110.1, 110.3, 113.1, 131.0, 133.1, 133.2, 136.3, 142.2,
144.8, 148.4, 162.4, 167.5. 31P NMR (160 MHz, D2O) (ppm) 0.6. 19
d
F
NMR (376 MHz, D2O)
d
(ppm) ꢁ111.7. HRMS (ESI) calcd for
C22H28FN2O9P [MþH]þ 515.1595; found: 515.1592.
(d, J¼6.3 Hz, 1H), 9.16 (s, 1H). 13C NMR (100 MHz, D2O)
d (ppm)
24.2, 30.4, 31.8, 31.9, 66.8, 68.7, 70.5, 73.0, 80.1, 89.4, 102.3,
117.0, 123.7, 130.9, 132.2, 136.2, 142.1, 144.7, 148.3, 160.4, 162.7,
4.3.12. b-Nicotinamide ribose-5-{2-[2-(2-chlorophenoxy)ethoxy]ethyl}
phosphate (9l). Compound 9l (syrupy solid, 231 mg, 69%) was
obtained from 7 (263 mg, 0.63 mmol) as similar procedure described
for the synthesis of 9c (anion resin, aqueous 5% HCO2H). 1H NMR
167.8. 31P NMR (160 MHz, D2O)
d (ppm) 0.5. HRMS (ESI) calcd
for C22H29N2O9P [MþH]þ 497.1689; found: 497.1680.
(400 MHz, D2O)
d
(ppm) 3.61 (t, J¼4.1 Hz, 2H), 3.70 (t, J¼4.2 Hz, 2H),
4.3.8.
b-Nicotinamide ribose-5-{2-[2-(2-methoxyphenoxy)ethoxy]ethyl}
3.86 (m, 2H), 3.99 (m, 3H), 4.13 (ddd, J¼2.4, 3.9, 11.9 Hz, 2H), 4.18 (m,
1H), 4.23 (t, J¼5.1 Hz, 1H), 4.32 (t, J¼2.4 Hz, 1H), 5.87 (d, J¼5.2 Hz,
1H), 6.70 (t, J¼7.6 Hz, 2H), 6.79 (d, J¼8.3 Hz,1H), 7.03 (m, 2H), 8.00 (t,
J¼6.4 Hz, 1H), 8.63 (d, J¼8.1 Hz, 1H), 8.99 (d, J¼6.3 Hz, 1H), 9.10 (s,
phosphate (9h). Compound 9h (syrupy solid, 297 mg, 86%) was
obtained from 7 (263 mg, 0.63 mmol) as similar procedure described
for the synthesis of 9c (anion resin, aqueous 5% HCO2NH4), followed by
column chromatography on octyl-functionalized silica gel with
1H). 13C NMR (100 MHz, D2O)
d (ppm) 66.8, 67.7, 67.8, 71.0, 71.5, 72.8,
CH3OH/H2O (30:1) as eluent. 1H NMR (400 MHz, D2O)
d
(ppm) 3.61 (s,
73.0, 80.1, 89.3, 89.4, 102.2, 116.6, 124.0, 124.6, 130.8, 130.9, 132.6,
3H), 3.64 (t, J¼2.7 Hz, 2H), 3.75 (t, J¼4.2 Hz, 2H), 3.87–4.08 (m, 5H),
4.20 (ddd, J¼2.1, 4.5, 12.2 Hz, 1H), 4.24 (dd, J¼2.3, 4.7 Hz, 1H), 4.26 (t,
J¼5.1 Hz, 1H), 4.38 (s, 1H), 5.88 (d, J¼5.3 Hz, 1H), 6.77–6.91 (m, 4H),
8.03 (t, J¼7.7 Hz, 1H), 8.68 (d, J¼8.0 Hz, 1H), 9.02 (d, J¼6.2 Hz, 1H), 9.14
136.0, 142.0, 144.7, 148.2, 155.7, 162.7, 167.6. 31P NMR (160 MHz, D2O)
d
(ppm) 0.2. HRMS (ESI) calcd for C21H26ClN2O10P [MþH]þ 533.1092;
found: 533.1092.
(s, 1H). 13C NMR (100 MHz, D2O)
d
(ppm) 57.8, 66.9, 67.7, 70.2, 71.6,
4.3.13. b-Nicotinamide ribose-5-[5-(2-chlorophenoxy)pentyl] phos-
72.7, 73.2, 80.1, 89.5, 102.2, 114.4, 115.5, 123.8, 124.2, 130.9, 136.1, 141.8,
phate (9m). Compound 9m (syrupy solid, 224 mg, 68%) was
obtained from 7 (263 mg, 0.63 mmol) as similar procedure de-
scribed for the synthesis of 9c (anion resin, aqueous 5% HCO2H). 1H
144.7, 148.1, 149.3, 150.4, 162.8. 31P NMR (160 MHz, D2O)
d (ppm) 0.3.
HRMS (ESI) calcd for C22H29N2O11P [MþH]þ 551.1407; found: 551.1402.
NMR (400 MHz, D2O) d (ppm) 1.42 (q, 2H),1.56 (m, 2H),1.66 (m, 2H),
4.3.9.
b
-Nicotinamide ribose-5-[5-(2-methoxyphenoxy)pentyl] phos-
3.80 (q, 2H), 3.93 (t, J¼5.6 Hz, 2H), 4.00 (ddd, J¼2.3, 4.6,12.1 Hz,1H),
phate (9i). Compound 9i (syrupy solid, 277 mg, 82%) was obtained
4.16 (ddd, J¼2.0, 4.1, 12.0 Hz 1H), 4.29 (m, 1H), 4.36 (m, 1H), 4.46 (m,

W. Liu et al. / Tetrahedron 65 (2009) 8378–8383
8383
1H), 6.03 (d, J¼5.1 Hz, 1H), 6.81 (t, J¼7.7 Hz, 1H), 6.92 (d, J¼8.3 Hz,
Supplementary data
1H), 7.13 (t, J¼7.7 Hz, 1H), 7.2 (d, J¼9.9 Hz, 1H), 8.11 (t, J¼7.7 Hz, 1H),
8.73 (d, J¼8.1 Hz, 1H), 9.11 (d, J¼6.2 Hz, 1H), 9.27 (s, 1H). 13C NMR
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2009.08.007.
(100 MHz, D2O)
d (ppm) 24.3, 30.3, 31.9, 66.8, 68.8, 71.8, 73.1, 80.2,
89.5, 102.4, 116.9, 124.5, 130.8, 131.0, 132.6, 136.2, 142.3, 144.8, 148.3,
156.0, 167.7. 31P NMR (160 MHz, D2O)
d (ppm) 0.6. HRMS (ESI) calcd
References and notes
For C22H28ClN2O9P [MþH]þ 533.1119; found: 533.1111.
1. Stryer, L. Biochemistry; W.H. Freeman: New York, NY, 1995.
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4.3.14.
b-Nicotinamide ribose-5-{2-[2-(N-benzoyl-3-aminophenoxy)-
ethoxy]ethyl} phosphate (9n). Compound 9n (syrupy solid, 287 mg,
74%) was obtained from 7 (263 mg, 0.63 mmol) as similar procedure
described for the synthesis of 9c (octyl-functionalized silica gel,
CH3OH/H2O, 30:1), followed by size exclusion chromatography
(polyacrylamide gel, 25 mM NH4HCO3). 1H NMR (400 MHz, D2O)
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d
(ppm) 3.69 (m, 2H), 3.79 (m, 2H), 4.02 (m, 4H), 4.08 (m, 1H), 4.17
(d, J¼11.9 Hz 1H), 4.30 (m, 2H), 4.39 (s, 1H), 5.89 (d, J¼5.2 Hz, 1H),
6.64 (d, J¼8.3 Hz, 1H), 6.94 (d, J¼8.0 Hz, 2H), 6.99 (s, 1H), 7.18 (t,
J¼8.1 Hz, 2H), 7.43 (t, J¼7.5 Hz, 2H), 7.53 (d, J¼7.4 Hz, 1H), 7.72
(d, J¼8.0 Hz, 2H), 8.06 (t, J¼7.2 Hz, 1H), 8.70 (d, J¼8.0 Hz, 1H), 9.06
(d, J¼6.1 Hz, 1H), 9.13 (s, 1H). 13C NMR (100 MHz, D2O)
d (ppm) 66.9,
67.7, 69.7, 71.6, 72.8, 73.2, 80.1, 89.6, 102.3, 110.4, 113.7, 117.0, 129.8,
130.9, 131.3, 132.6, 134.9, 136.0, 136.2, 140.9, 142.1, 144.6, 148.2,
160.7, 171.5. 31P NMR (160 MHz, D2O)
d (ppm) 0.2. HRMS (ESI) calcd
for C28H32N3O11P [MþH]þ 618.1853; found: 618.1857.
9. (a) Billington, R. A.; Travelli, C.; Ercolano, E.; Galli, U.; Roman, C. B.; Grolla, A. A.;
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4.4. Preliminary tests of the effects of 9c on microbial
cell growth
The detailed procedure for cell culture in the presence of NADþ
analog 9c was as follows: 55 mL sterilized Luria–Bertani medium
(peptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, pH 7.2) supple-
mented with 100
culture of E. coli DH5
m
M analog 9c was inoculated with 8-h-old pre-
cells (500
L), and cultured at 37 ꢀC,
a
m
200 rpm. The control sample was run in the absence of analog 9c.
Aliquots were withdrawn every 2 h and OD600 was recorded on
a UV–vis spectrophotometer.
For yeast S. cerevisiae ATCC 26108, 50 mL sterilized YEPD me-
dium (glucose 20 g/L, yeast extract 10 g/L, peptone 20 g/L, pH 6.2)
supplemented with 100
old preculture (250
L), and cultured at 30 ꢀC, 200 rpm. The control
sample was run in the absence of analog 9c. Aliquots were with-
mM analog 9c was inoculated with 18-h-
m
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drawn every 5 h and OD600 was recorded on
spectrophotometer.
a UV–vis
´
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4.5. Activity assay of ADH in the presence of analog 9c
To a solution of ethanol (10 mM), NADþ (5 mM), and analog 9c
(25 mM) in Tris–HCl buffer (50 mM, pH 8.8) was added ADH
(0.002 U). The mixture was vortexed and transferred into a colori-
metric cuvette. Time course measurement was recorded for 90 s at
340 nm on a UV–vis spectrophotometer at 25 ꢀC. Activity of LDH
was similarly tested.
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Acknowledgements
We are grateful for the support of the National Natural Science
Foundation of China (No. 20472084).
26. Seth, A. K.; Jay, E. Nucleic Acids Res. 1980, 8, 5445.