Synthesis and calcium mobilization activity of cADPR analogues which integrate nucleobase, northern and southern ribose modifications

Article abstract of DOI:10.3390/molecules17044343

Novel cADPR mimics, which integrate nucleobase, northern and southern ribose modifications were synthesized. The key steps of the synthesis were a Cu(I)-catalyzed Hueisgen [3+2] cycloaddition and a microwave-assisted intramolecular pyrophosphorylation. Preliminary biological investigations showed that these cADPR mimics are membrane-permeating agonists of the calcium signaling pathway. The introduction of chlorine or fluorine at the 2'-position of the southern riboses led to a decrease of activity. The existence of a hydrophobic group on the 3'-OH of the southern riboses does not obviously alter the agonistic activity.

Full text of DOI:10.3390/molecules17044343

Molecules 2012, 17, 4343-4356; doi:10.3390/molecules17044343
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Calcium Mobilization Activity of cADPR
Analogues Which Integrate Nucleobase, Northern and
Southern Ribose Modifications
Yue Zhou 1, Peilin Yu 1, Hongwei Jin 1, Zhenjun Yang 1, Jianbo Yue 2, Liangren Zhang 1,*
and Lihe Zhang 1
1
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences,
Peking University, Beijing 100191, China
2
Department of Physiology, University of Hong Kong, Hong Kong, China
* Author to whom correspondence should be addressed; E-Mail: liangren@bjmu.edu.cn;
Tel: +86-10-8280-2567, Fax: +86-10-8280-5063.
Received: 24 February 2012; in revised form: 31 March 2012 / Accepted: 5 April 2012 /
Published: 10 April 2012
Abstract: Novel cADPR mimics, which integrate nucleobase, northern and southern
ribose modifications were synthesized. The key steps of the synthesis were a
Cu(I)-catalyzed Hüisgen [3+2] cycloaddition and a microwave-assisted intramolecular
pyrophosphorylation. Preliminary biological investigations showed that these cADPR
mimics are membrane-permeating agonists of the calcium signaling pathway. The
introduction of chlorine or fluorine at the 2'-position of the southern riboses led to a
decrease of activity. The existence of a hydrophobic group on the 3'-OH of the southern
riboses does not obviously alter the agonistic activity.
Keywords: cADPR analogue; nucleotide; synthesis; calcium mobilization
1. Introduction
Cyclic adenosine disphosphate ribose (cADPR, 1, Figure 1) is a universal Ca2+ mobilizing
secondary messenger first identified in the sea urchin egg system [1]. Since its discovery, numerous
cell systems that utilize the cADPR/ryanodine receptor (RyR) Ca2+ signaling system to control
Ca2+-dependent cellular responses, such as fertilization, secretion, contraction, proliferation and so on have
been described [2,3]. A large number of proteins are involved in specifically shaping Ca2+ signals,

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however, it is still unclear exactly how cADPR elicits calcium release, which includes whether specific
cADPR binding proteins exist. Since the discovery of cADPR, a number of structurally diverse
cADPR analogues have been synthesized and used to elucidate the molecular mechanism of calcium
signaling [4–6]. The syntheses could be divided into chemo-enzymatic or chemical synthesis [7,8].
The structures include modifications of the pyrophosphate [9–12], purine [13–16] and southern and
northern riboses [17–20] of cADPR. These analogues can agonize or antagonize cADPR/RyR calcium
signal pathway.
Figure 1. Structures of cADPR analogues.
cADPR analogues with modifications on the northern and southern riboses are the most
investigated. Shuto et al. found that the effect of modification of the northern ribose on calcium
signaling depended on the cell system [21,22]. Our previous studies showed that the northern ribose of
cADPR tolerated structural modifications to some extent. The agonistic activity is mostly maintained
even if the northern ribose is replaced by ether or alkane linkages, such as in cIDPRE (2), and this
modification makes analogues cell-permeant [18]. Potter et al. investigated the importance of the
2'-OH on the calcium signaling behavior of 8-substituted cADPR derivatives. They found that the
2'-OH group does not affect the Ca2+-mobilizing ability of cADPR itself, but that it is an important
motif for the antagonistic activities of 8-substituted cADPR analogues [23]. The C2'-endo/syn
conformation is crucial for agonistic or antagonistic activity in sea urchin egg homogenates [24]. The
findings implied the coordinating effect of nucleobase and riboses on the activity of cADPR analogues.
The nucleobase of cADPR was simplified in our previous study; we have found that triazole-based
cADPR analogues 3,4 are cell-permeating mild agonists of the cADPR/RyR calcium pathway [25]. To
elucidate the structure-activity relationships of cADPR analgues in more detail, and provide probes for
investigation of the molecular mechanism of cADPR regulated calcium pathways, we have designed
and synthesized novel cADPR analogues which integrate three types of modifications of the
nucleobase, northern and southern riboses (compounds 5). In this study, the nucleobase is replaced by
a simplified triazole moiety, the northern ribose is replaced by an ether linkage and the southern ribose
is replaced by 2'-deoxy or 2'-deoxy-2'-haloribofuranoses, respectively.

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2. Results and Discussion
2.1. Chemistry
The synthesis could be generalized to three steps, i.e., the synthesis of phosphorylated northern and
southern moieties, the assembly of the triazole moiety and intramolecular cyclization (Scheme 1). The
phosphorylation was performed before the coupling of the northern and southern moieties.
Microwave-assisted intramolecular cyclization was used to form the pyrophosphates of the
cADPR analogues.
Scheme 1. Retrosynthesis of cADPR analogues.
For the syntheses of the southern moieties, various strategies were adopted to construct
1-azido-2-modified sugars 16, which are summarized in Scheme 2.
Scheme 2. Synthesis of 1-azido-2-modified sugars.
N3
N3
a
c
b
O
O
O
O
HO
AcO
AcO
HO
OH
OAc
93%
83%
92%
HO
AcO
AcO
HO
7
8
9
6
N3
N3
e
f
d
O
O
O
O
HO
BzO
BzO
BzO
OBz
Br
75%
two steps
95%
HO
R
BzO
R
BzO
R
BzO
R
10
12
13 a: R = F
11
b: R = Cl
N3
N3
N3
f
g
O
O
O
h
BzO
BzO
HO
9 or 13
83%
two steps
75%
HO
14
R
TBDMSO
R
TBDMSO
R
15
a: R = H
b: R = Cl
c: R = F
16
Reagents and conditions: (a) Ac2O, pyridine, 1,4-dioxane, reflux, 3 h; (b) SnCl4, TMSN3 TMSOTf,
CH2Cl2, ice-bath; (c) K2CO3, CH3OH, rt, 12 h; (d) 33% HBr, AcOH, CH2Cl2, rt, 3 h; (e) NaN3,
DMF, 40 C, 10 h; (f) NH3/CH3OH, rt, 10–12 h; (g) BzCl, pyridine, CH2Cl2, ice-bath;
(h) TBDMSCl, imidazole, DMF, 60–65 C; (f) NH3/CH3OH, rt, 2–3 h.

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1--D-Azido-2-deoxyribose (9) was prepared starting from 2-deoxyribose based on Greenberg’s
method [26]. The azidation of triacetylribose 7 gave a pair of anomers. The α:β isomer ratio was 1:8
and they could be separated by silica gel column chromatography. The fluroarabinose 10a was used
directly from commercial material [27]. The chloroarabinose 10b was synthesized by the triflation of
commercial available 1,3,5-tri-O-benzoyl-α-D-ribofuranose, followed by treatment with LiCl in
N-methylpyrrolidinone (NMP) [28]. Treatment of 10 with HBr/CH3COOH in dichloromethane
followed by the addition of sodium azide in DMF at 30 C for 12 h gave 12 in a yield of 75%.
Considering the instability of the anomeric bromide, intermediate 11 was used without purification.
The 3-OH groups of 9 and 13 were protected by tert-butyldimethyl silyl (TBDMS) groups by reaction
with TBDMSCl after their 5-OH groups were selectively protected as benzoyl groups, thus giving 15.
After treatment with NH3/CH3OH, compound 16 was obtained.
Cu(I)-catalyzed Hüisgen [3+2] cycloaddition between terminal alkyne and azide groups was
adopted to build the 1,2,3-triazole unit (Scheme 3). The terminal alkyne building block 17 which
carried a S,S-diphenylphosphate group was prepared by the reported procedure [29]. Several classical
Cu(I) catalysis systems were used to catalyze the Hüisgen [3+2] cycloaddition reaction of 16 and 17.
Under optimal conditions, compound 18 was obtained in a yield of 85%. Compound 18 was
phosphorylated by using POCl3/DIPEA in CH3CN, followed with purification by HPLC eluting with
0.05M triethylammonium bicarbonate (TEAB, pH = 7.5). It was found that the S,S-diphenylphosphate
group was sensitive to alkaline conditions and was decomposed to S-phenylphosphate. Compound 20
was obtained as a triethylammonium salt in a yield of 54% for two steps.
Scheme 3. Synthesis of compound 5.
O
O
O
PhS P O
SPh
O
PhS P O
SPh
O
O
16
+
N
N
N
N
N
H
N
H
N
R
N
R
a
b
O
O
O
18a: 84%
18b: 85%
18c: 83%
(PhS)2P O
N
O
O
O
H
HO
HO P O
OH
17
OTBDMS
OTBDMS
19
18
O
O
O
O
O
O
O
O
N
N
N
N
N
N
H
N
N
N
N
PhS P O
OH
N
N
R
H
H
O
O
c
e
d
O P OH
O
O P OH
O
O
O
O
54%
two steps
21a: 87%
21b: 82%
21c: 81%
5a: 36%
5b: 31%
5c: 29%
O
O
R
HO P O
OH
R
P
P
O
OH
O
OH
21
OH
OTBDMS
OTBDMS
20
a: R = H
b: R = Cl
c: R = F
5
Reagents and conditions: (a) CuI, DIPEA, CH3CN; (b) POCl3, DIPEA, CH3CN; (c) 1M TEAB;
(d) I2, pyridine; (e) TBAF, THF, Ac2O.
Microwave-assisted organic chemistry has been recently developed for the efficient synthesis of
functional compounds in many areas [30,31]. In most previous works, intermolecular reactions for the
formation of pyrophosphates of cADPR analogues were performed in super-dilute solutions [8]. Under

Molecules 2012, 17
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these super-dilute solution conditions, the reaction was usually completed by using a syringe-pump
over 20 h. In addition, the reaction was very sensitive to traces of water. This strictly dry environment
and the long reaction time in a super-dilute solution made the experimental work-up tedious, and thus,
the large-scale preparation of cADPR analogues was difficult. Under microwave-assisted conditions,
the overall efficiency of intramolecular pyrophosphorylation has been greatly improved [29]. We
applied this technology to compound 20 to prepare 21. Optimization of the reaction conditions was
done by changing temperature and reaction time. The reaction of 20 with I2 in pyridine (75 C/15 min)
gave cyclic compound 21 as its triethylammonium salts in a yield of 81%–87%. Finally, the removal
of the TBDMS group of 21 was carried out in a solution of 1M TBAF/THF at room temperature for
1 h to obtain the target compound 5. Compound 5 was identified by 1H- and 31P-NMR, and HRMS.
2.2. Pharmacology
The Ca2+-mobilizing ability of the newly synthesized cADPR analogues 5a–c were evaluated in
Jurket-T cells. For studying the effect of the 3'-OH on activity, the 3'-O-TBDMS substituted precursors
21a–c were also studied. The results are shown in Figure 2.
Figure 2. Ca2+ mobilization activities of compounds 21 and 5 in intact Jurkat T cells.
F/F0 Compounds 21 and 5
Conditions: The response to the application of the cIDPRE served as a positive control while
addition of buffer was used as blank. Jurkat T-cells were loaded with Fura-2AM and subjected to
Ca2+ imaging. Changes in mean F/F0 after the application of compounds or buffer control were
averaged for at least three independent experiments for each concentration. (A) The peak values of
F/F0 were analyzed as the maximum within 200 s after application. (B) Time course of Ca2+
mobilization by addition of compounds 21a–c and 5a–c. The time point’s addition of 750 μM
positive control, compound 21a–c and 5a–c are indicated by arrows.

Molecules 2012, 17
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In general, the synthesized cADPR analogues are membrane-permeating agonists of the calcium
signaling pathway. All compounds showed typical biphasic Ca2+ mobilizing kinetics with an initial
immediate Ca2+ peak and a subsequent plateau phase. The induction of the immediate peak was
strongest for compound 5a at 750 M, which was almost as active as the positive control cIDPRE,
while for the other 2'-deoxy cADPR analogues a less pronounced initial Ca2+ peak was observed. The
result indicates that the introduction of chlorine (5b) or fluorine (5c) to the 2'-deoxy position will lead
to a decrease of activity.
In comparison to cIDPRE, however, 2'-deoxy cADPR analogues 21a–c and 5a–c sustained longer
Ca2+ mobilization (Figure 2B), which implied their different molecular mechanism. 2'-Deoxy-cADPR
was found to be inactive in Jurkat T cells [32]. Potter’s finding implied that the 2'-OH group is an
important motif for the antagonistic activities of 8-substituted cADPR analogues and 2'-OH deletion
may also result in an increase in the agonistic effects of the 8-substituted cADPR analogues [23,24].
This study shows that the agonistic activity is maintained in the case of disappearance of the 2'-OH,
which emphasizes the importance of the structure of the nucleobase on the effect of the 2'-OH on
calcium mobilization.
The existence of a large hydrophobic tert-butyldimethylsilyl (TBDMS) group on the 3'-OH of the
southern ribose (compounds 21a–c) lowers activity, but most of the agonistic properties are still
maintained. Potter et al. reported that the 3'-hydroxyl group is essential for Ca2+ releasing activity of
cADPR in sea urchin eggs [23]. In our case, TBDMS attached compound 21 did not much alter the
agonistic activity of compound 5. This result may come from the existence of a hydrophobic group on
the 3'-OH that makes compound more membrane permeant and partially compensates the negative effect.
The conformations of the modified cADPR analogues were investigated by molecular modeling.
Their optimized conformations were superimposed with cADPR (Figure 3). The result clearly shows
that 5a–c and cADPR have very similar conformations. All riboses are in 2'-endo/3'-exo conformations
and the whole backbones are in similar alignments. The consistence of conformations might result in
the agonistic activities of cADPR analogues 5a–c, even if the structures were simplified both in the
southern ribose and nucleobase regions. For clearly understanding the structure-activity relationship,
further detailed chemistry and/phamarcology investigations are needed.
Figure 3. Superimposition of minimized conformations of cADPR and compounds 5a (a),
5b (b), 5c (c) and 5a–c (d).
(a)
(b)
(c)
(d)

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3. Experimental
3.1. Chemistry
3.1.1. General
1
HR-ESI-MS and ESI-MS were performed with a Bruker BIFLEX III instrument. H-NMR and
13C-NMR were recorded with a JEOL AL300 or a Bruker AVANCE III 400; CDCl3, D2O were used as
solvents. Chemical shifts are reported in parts per million downfield from TMS (1H and 13C).
31P-NMR spectra (121.5 MHz) were recorded at room temperature by use of a JEOL AL300
spectrometer. Orthophosphoric acid (85%) was used as external standard. 19F-NMR spectra (470 MHz)
19
were recorded on a Varian VXR-500 spectrometer. Chemical shifts of F-NMR are reported in ppm
with reference to CF3COOH as external standard. Compounds were purified on an Alltech preparative
C18 reversed-phase columns (2.2 × 25 cm) with a Gilson HPLC using MeCN/TEAB (pH 7.5) buffer
system as eluent. Analytical TLC was performed using commercial glass plates coated to a thickness
of 0.25 mm with Kieselgel 60 GF254 silica and visualized under UV light. Flash chromatography
was performed using Qingdao (230–400 mesh) silica under a slight positive pressure of air.
Microwave-assisted reactions were performed using a Biotage unit (400 w, 2.45 GHz). Solvents and
regents for anhydrous reactions were dried prior to use by conventional methods.
3.1.2. General Procedure for the Synthesis of 18a–c
To a solution of compound 17 (185 mg, 0.44 mmol) and compound 16a–c (0.44 mmol) in CH3CN
(15 mL) was added CuI (10 mg, 0.05 mmol) and DIPEA (78 μL, 0.5 mmol), and the mixture was
stirred at room temperature for 3 h. The mixture was evaporated in vacuo and the residue was
partitioned between EtOAc and H2O. The aqueous phase was extracted again with EtOAc, then the
organic layers were combined and dried (Na2SO4), filtered and concentrated in vacuo. The residue was
purified by silica gel column chromatography (DCM-MeOH = 40:1–30:1) to afford a yellow oil.
N-(2-(2-O-Bis(phenylthio)phosphorylethoxy)ethyl)-1H-1,2,3-triazole-4-carboxamid-1-yl-2'-deoxy-3'-
tert-butyldimethylsilyl-1'-β-D-ribofuranoside (18a): Yield: 84%. ESI-TOF+: 695.2 [(M+H)+]. 1H-NMR
(400 MHz, CDCl3): δ 8.50 (s, 1H, H-5), 7.54–7.37 (m, 10H, Ar-H), 7.42 (t, 1H, –NH–) 6.44 (dd, 1H,
J = 8 Hz, H-1'), 4.49–4.47 (m, 1H, H-5'a), 4.25–4.23 (m, 1H, H-5'b), 4.34–4.30 (m, 2H, H-1"),
3.67–3.64 (m, 2H, H-3', H-4'), 3.62–3.56 (m, 4H, H-2", H-3"), 2.77–2.73 (m, 1H, H-2'a), 2.45 (dd, 1H,
J2'a,2'b = 16 Hz H-2'b), 0.82 (s, 9H, 3×CH3), 0.06 (s, 6H, 2×–Si(CH3)–). 13C-NMR (75 MHz, CDCl3): δ
160.3, 143.2, 135.7, 129.4, 126.2, 90.1, 89.5, 76.7, 70.1, 66.8, 62.0, 42.5, 39.1, 25.6, 17.9, −5.0.
31P-NMR (D2O, decoupled with 1H): δ 50.91 (s).
N-(2-(2-O-Bis(phenylthio)phosphorylethoxy)ethyl)-1H-1,2,3-triazole-4-carboxamid-1-yl-2'-chloro-3'-
1
tert-butyldimethylsilyl-1'-β-D-arabinoside (18b): Yield: 85%. ESI-TOF+: 729.2 [(M+H)+]. H-NMR
(400 MHz, CDCl3): δ 8.34 (s, 1H, H-5), 7.54–7.24 (m, 10H, Ar-H), 6.33 (dd, 1H, J = 8 Hz, H-1'), 4.73
(dd, 1H, J5'a,5'b = 12Hz, H-5'a), 4.50 (dd, 1H, J5'a,5'b = 12 Hz, H-5'b), 4.36–4.30 (m, 2H, H-4', H-3'),
4.41–3.90 (m, 2H, H-1"), 3.85–3.80 (m, 1H, H-2'), 3.68–3.61 (m, 2H, H-2"), 3.59–3.58 (m, 2H, H-4",
13
H-3"), 0.89 (s, 9H, 3×CH3), 0.13 (s, 6H, 2×–Si(CH3)–). C-NMR (75 MHz, CDCl3): δ 160.2, 142.5,

Molecules 2012, 17
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135.3, 129.5, 126.3, 88.5, 77.3, 69.7, 66.8, 60.6, 38.6, 25.0, 17.8, −4.5. 31P-NMR (D2O, decoupled
with 1H): δ 51.27 (s).
N-(2-(2-O-Bis(phenylthio)phosphorylethoxy)ethyl)-1H-1,2,3-triazole-4-carboxamid-1-yl-2'-fluoro-3'-
1
tert-butyldimethylsilyl-1'-β-D-arabinoside (18c): Yield: 83%. ESI-TOF+: 713.2 [(M+H)+]. H-NMR
(400 MHz, CDCl3): δ 8.40 (s, 1H, H-5), 7.50–7.30 (m, 10H, Ar-H), 6.44 (dd, 1H, J = 8 Hz, H-1'),
5.08–4.93 (m, 1H, H-5'a) 4.65–4.59 (m, 1H, H-5'b), 4.30–4.28 (m, 2H, H-2', H-4'), 4.01–4.00 (m, 1H,
H-3'), 3.78–3.54 (m, 8H, H-1'', H-4'', H-2'', H-3''), 0.86 (s, 9H, 3×CH3), 0.08 (s, 6H, 2×–Si(CH3)–).
13C-NMR (75 MHz, CDCl3): δ 160.0, 143.1, 135.3, 129.4, 126.3, 96.2, 66.6, 60.7, 53.5, 38.7, 25.5,
17.8, 5.2. 31P-NMR (D2O, decoupled with 1H): δ 51.09 (s).
3.1.3. General Procedure for the Synthesis of 20a–c
Compound 18a–c (0.10 mmol) was dissolved in anhydrous CH3CN (5 mL). DIPEA (0.42 mmol)
and POCl3 (0.35 mmol) were added successively to the solution at −20 °C. The mixture was stirred at
0 °C for 16 h, and then TEAB (5 mL, 1 M, pH 7.5) were added at 0 °C and the stirring was continued
for 1 h at room temperature. After evaporation under reduced pressure, the residue was partitioned
between H2O and CHCl3, and the aqueous layer was washed with CHCl3 and evaporated
in vacuo. The residue was dissolved in TEAB buffer (5 mL, 0.05 M, pH 7.5), and applied to a C18
reversed-phase column (2.2 × 25 cm). The column was eluted using a linear gradient of 0–80%
CH3CN in TEAB buffer (0.05 M, pH 7.5) to give 19a–c as its triethylammonium salt. The residue was
dissolved in 1 M TEAB (5 mL) and stirred at room temperature for 12 h, and then evaporated in vacuo.
The residue was partitioned between H2O and CHCl3, and the aqueous layer was washed with CHCl3.
Evaporated in vacuo, dissolved in TEAB buffer (1 mL, 0.05 M, pH 7.5), then applied to a C18
reversed-phase column (2.2 cm × 25 cm) eluted by a linear gradient of 0–80% CH3CN in TEAB buffer
(0.05 M, pH 7.5) to give 20a–c as triethylammonium salts.
N-(2-(2-O-Phenylthiophosphorylethoxy)ethyl)-1H-1,2,3-triazole-4-carboxamid-1-yl-2'-deoxy-3'-tert-
butyldimethylsilyl-5'-phosphoryl-1'-β-D-ribofuranoside (20a): Yield: 54%. HRMS (ESI-TOF−) Calcd
1
for C24H40N4O11P2SSi: [(M−H)+] 681.1586; Found: 681.1557. H-NMR (400 MHz, D2O): δ 8.47 (s,
1H, H-5), 7.47–7.17 (m, 5H, Ar-H) 6.42 (d, 1H, J = 8 Hz, H-1'), 4.48 (t, 1H, J5'a,5'b = 4Hz, H-5'a), 4.41
(t, 1H, J5'a,5'b = 4Hz, H-5'b), 3.98–3.96 (m, 2H, H-4', H-3'), 3.57–3.44 (m, 4H, H-2'',H-3''), 3.43–3.40
(m, 2H, H-4''), 3.07 (q, –NCH2–), 2.70–2.68 (m, 1H, H-2'a), 2.34 (d, 1H, H-2'b), 1.02 (t, –CH3–), 0.58
31
1
(s, 9H, 3×CH3), 0.07 (s, 6H, 2×–Si(CH3)–). P-NMR (D2O, decoupled with H): δ 15.01 (br, s), 1.70
(br, s).
N-(2-(2-O-Phenylthiophosphorylethoxy)ethyl)-1H-1,2,3-triazole-4-carboxamid-1-yl-2'-chloro-3'-tert-
butyldimethylsilyl-5'-phosphoryl-1'-β-D-arabinoside (20b): Yield: 52%. HRMS (ESI-TOF−) Calcd for
C24H39ClN4O11P2SSi: [(M−H)+] 715.1196; Found: 715.1186. 1H-NMR (400 MHz, D2O): δ 8.55 (s, 1H,
H-5), 7.36–7.06 (m, 5H, Ar-H), 6.47–6.45 (m, 1H, H-1'), 4.48–4.46 (m, 2H, H-5'a, H-5'b), 4.59–4.03
(m, 2H, H-4', H-3'), 4.41–3.90 (m, 2H, H-1''), 3.96–3.95 (m, 1H, H-2'), 3.53–3.49 (m, 4H, H-2'', H-3''),
3.39–3.37 (m, 2H, H-4''), 3.02 (q, –NCH2–), 1.02 (t, –CH3–), 0.67 (s, 9H, 3×CH3), 0.1 (s, 6H).
31P-NMR (D2O, decoupled with 1H): δ 18.24 (br, s), 1.16 (br, s).

Molecules 2012, 17
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N-(2-(2-O-Phenylthiophosphorylethoxy)ethyl)-1H-1,2,3-triazole-4-carboxamid-1-yl-2'-fluoro-3'-tert-
butyldimethylsilyl-5'-phosphoryl-1'-β-D-arabinoside (20c): Yield: 50%. HRMS (ESI-TOF−) Calcd for
C24H39FN4O11P2SSi: [(M−H)+] 699.1492; Found: 699.1511. 1H-NMR (400 MHz, D2O): δ 8.46 (s, 1H,
H-5), 7.36–7.09 (m, 5H, Ar-H), 6.50–6.46 (m, 1H, H-1'), 5.30–5.08 (m, 1H, H-4'), 4.11–4.10 (m, 1H,
H-5'a), 3.98–3.96 (m, 1H, H-5'b), 3.94–3.91 (m, 3H, H-2', H-1''), 3.57–3.52 (m, 4H, H-2'',H-3''),
3.42–3.39 (m, 2H, H-4''), 3.02 (q, –NCH2–), 1.09 (t, –CH3–), 0.74 (s, 9H, 3×CH3), 0.01 (s, 6H,
2×–Si(CH3)–). 31P-NMR (D2O, decoupled with 1H): δ 18.28 (br, s), 1.30 (br, s).
3.1.4. General Procedure for the Synthesis of 21a–c
The mixture of compound 20a–c (12.4 mmol) and iodine (73 mg, 0.29 mmol) in pyridine (8 mL)
was stirred at 75 C, assisted by microwave irradiation, for 15 min. Then the pyridine was evaporated,
and the residue was partitioned between CHCl3 and H2O. The aqueous layer was evaporated and the
residue was dissolved in 0.05 M TEAB buffer (5.0 mL), and applied to a C18 reversed-phase column
(2.2 × 25 cm). The column was eluted using a linear gradient of 0–80% CH3CN in TEAB buffer
(0.05 M, pH 7.5) to give 21a–c as triethylammonium salts.
N-(2-(2-O-Phosphorylethoxy)ethyl)-1H-1,2,3-triazole-4-carboxamid-1-yl-2'-deoxy-3'-tert-butyldimethylsilyl-
5'-phosphoryl-1'-β-D-ribofuranoside 2,5'-cyclicpyrophosphate (21a): Yield: 87%. HRMS (ESI-TOF−)
Calcd for C18H34N4O11P2Si: [(M−H)+] 571.1395; Found: 571.1380. 1H-NMR (400 MHz, D2O): δ 8.77
(s, 1H, H-5), 6.39–6.36 (m, 1H, H-1'), 4.47–4.46 (m, 1H, H-5'a), 4.42–4.41 (m, 1H, H-5'b), 4.35–4.34
(m, 2H, H-4', H-3'), 3.96–3.95 (m, 4H, H-2'', H-3''), 3.64–3.59 (m, 2H, H-4''), 3.04 (q, –NCH2–),
2.57–2.52 (m, 1H, H-2'a), 2.32–2.27 (m,1H, H-2'b), 1.12 (t, –CH3–), 0.52 (s, 9H, 3×CH3), 0.12 (s, 6H).
31P-NMR (D2O, decoupled with 1H): δ −11.69 (br, s), −12.01 (br, s).
N-(2-(2-O-Phosphorylethoxy)ethyl)-1H-1,2,3-triazole-4-carboxamid-1-yl-2'-chloro-3'-tert-butyldimethylsilyl-
5'-phosphoryl-1'-β-D-arabinoside 2,5'-cyclicpyrophosphate (21b): Yield: 82%. HRMS (ESI-TOF−)
1
Calcd for C18H33ClN4O11P2Si: [(M−H)+] 605.1006; Found: 605.1002. H-NMR (400 MHz, D2O): δ
9.13 (s, 1H, H-5), 6.60 (d, 1H, J = 8 Hz, H-1'), 4.76 (d, 1H, J5'a,5'b = 8Hz, H-5'a), 4.55–4.53 (d, 1H,
J5'a,5'b = 8 Hz, H-5'b), 4.26–4.21 (m, 1H, H-4'), 4.14–4.13 (2H, m, H-1''), 4.05–4.01 (1H, m, H-3'),
3.91–3.88 (1H, m, H-2'), 3.72–3.64 (m, 4H, H-2'',H-3''), 3.58–3.35 (m, 2H, H-4''), 3.02 (q, –NCH2–),
1.16 (t, –CH3–), 0.79 (s, 9H, 3×CH3), 0.09 (s, 6H, 2×–Si(CH3)–). 31P-NMR (D2O, decoupled with 1H):
δ −12.93 (br, s), −14.43 (br, s).
N-(2-(2-O-Phosphorylethoxy)ethyl)-1H-1,2,3-triazole-4-carboxamid-1-yl-2'-fluoro-3'-tert-butyldimethylsilyl-
5'-phosphoryl-1'-β-D-arabinoside 2,5'-cyclicpyrophosphate (21c): Yield: 81%. HRMS (ESI-TOF−)
1
Calcd for C18H33FN4O11P2Si: [(M−H)+] 589.1302; Found: 589.1325. H-NMR (400 MHz, D2O): δ
9.09 (s, 1H, H-5), 6.63 (dd, 1H, J = 8 Hz, H-1'), 5.36–5.21 (m, 1H, H-4'), 4.20–4.16 (m, 1H, H'-5a),
4.16–4.14 (m, 1H, H-5'b), 4.10–3.91 (m, 3H, H-2', H-1''), 3.68–3.52 (m, 4H, H-2'', H-3''), 3.54–3.34
(m, 2H, H-4''), 3.01 (q, –NCH2–), 1.13 (t, –CH3–), 0.79 (s, 9H, 3×CH3), 0.09 (s, 6H, 2×–Si(CH3)–).
31P-NMR (D2O, decoupled with 1H): δ −12.90 (br, s), −14.22 (br, s).

Molecules 2012, 17
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3.1.5. General Procedure for the Synthesis of 5a–c
Compound 21a–c (33.6 μmol) was dissolved in 1M TBAF/THF (0.5 mL) and the solution was
stirred for 2 h, and then was evaporated under reduced pressure. The residue was dissolved in 0.05 M
TEAB buffer (2.0 mL), which was loaded to C18 reversed-phase column (2.2 × 25 cm). The column
was eluted using a linear gradient of 0–80% CH3CN in TEAB buffer (0.05 M, pH 7.5) to afford 5a–c
as triethylammonium salts.
N-(2-(2-O-Phosphorylethoxy)ethyl)-1H-1,2,3-triazole-4-carboxamid-1-yl-2'-deoxy-5'-phosphoryl-1'-β-
D-ribofuranoside 2,5'-cyclicpyrophosphate (5a): Yield: 36%. HRMS (ESI-TOF−) Calcd for
C12H20N4O11P2: [(M+H)+] 459.1051; Found: 459.1040. 1H-NMR (400 MHz, D2O): δ 8.47 (s, 1H, H-5),
6.48–6.42 (m, 1H, H-1'), 4.48–4.47 (m, 1H, H'-5a), 4.37–4.30 (m, 1H, H-5'b), 3.99–3.97 (m, 2H, H-1''),
3.87–3.86 (m, 1H, H-3'), 3.61–3.55 (m, 4H, H-3'', H-4''), 3.46–3.43 (m, 2H, H-2''), 3.09 (q, –NCH2–),
2.89–2.77 (m, 1H, H-2'a), 2.43–2.40 (m, 1H, H-2'b), 1.17 (t, –CH3–). 31P-NMR (D2O, decoupled with
1H), δ −9.77 (br, s), −10.05 (br, s).
N-(2-(2-O-Phosphorylethoxy)ethyl)-1H-1,2,3-triazole-4-carboxamid-1-yl-2'-chloro-5'-phosphoryl-1'-β-
D-arabinoside 2,5'-cyclicpyrophosphate (5b): Yield: 31%. HRMS (ESI-TOF−) Calcd for
1
C12H19ClN4O11P2: [(M−H)+] 491.0141; Found: 491.0142. H-NMR (400 MHz, D2O): δ 9.17 (s, 1H,
H-5), 6.60 (d, 1H, J = 8 Hz, H-1'), 4.77–4.73 (m, 2H, H-5'a, H-5'b), 4.26–4.21 (m, 1H, H-4'),
4.24–4.09 (2H, m, H-1''), 4.00–3.98 (1H, m, H-3'), 3.69–3.66 (1H, m, H-2'), 3.50–3.40 (m, 4H, H-2'',
H-3''), 3.10–3.04 (m, 2H, H-4''). 3.04 (q, –NCH2–), 1.15 (t, –CH3–). 31P-NMR (D2O, decoupled with 1H):
δ −7.58 (br, s), −9.00 (br, s).
N-(2-(2-O-Phosphorylethoxy)ethyl)-1H-1,2,3-triazole-4-carboxamid-1-yl-2'-fluoro-5'-phosphoryl-1'-β-
D-arabinoside 2,5'-cyclicpyrophosphate (5c): Yield: 29%. HRMS (ESI-TOF−) Calcd for
1
C12H19FN4O11P2: [(M−H)+] 475.0437; Found: 475.0449. H-NMR (400 MHz, D2O): δ 9.14 (s, 1H,
H-5), 6.64 (m, 1H, H-1'), 5.46–5.43 (m, 1H, H-5'a), 5.33–5.32 (m, 1H, H-5'b), 4.20–4.15 (m, 2H,
H-1''), 4.02–3.99 (m, 2H, H-2', H-4'), 3.69–3.67 (m, 2H, H-2''), 3.52–3.41 (m, 4H, H-3'', H-4''), 3.06 (q,
–NCH2–), 2.96–2.94 (m, 1H, H-3'), 1.13 (t, –CH3–). 31P-NMR (D2O, decoupled with 1H): δ −9.57 (br,
s), −10.53 (br, s). 19F-NMR (D2O, CF3COOH), δ-202.13–202.32 (m).
3.2. Biological Studies
Cell Culture. The human Jurkat T-lymphocyte cell line was obtained from the Hongkong University
Physiology Department Professor Lee Hon Cheung’s lab Culture Collection. It was cultured in RPMI
Medium 1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS),
100 units/mL penicillin, and 2% Hepes Buffer (1 M, pH 7.4) in a humidified (5% CO2) atmosphere
at 37 °C.
Calcium agonistic activity measurement. The cells (3 × 105 cells/well) were plated in 24-well plate
with poly L-lysine (100 μg/mL) and incubated in no FBS and penicillin medium at 37 °C overnight for
adherence. Before measurement, cells were incubated with Fura-2 AM (2 μM) in HBSS for 20 min in
dark at 37 °C. Then cells were washed with calcium measurement buffer HBSS twice and added

Molecules 2012, 17
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200 μL HBSS for measurement. Changes in Fura-2 fluorescence were measured using an Olympus
CellR Live-cell confocal imaging system, operating in ratio mode (alternating excitation at 340 and
380 nm). Each compound was dissolved at tested concentrations in 50 μL HBSS and added into the
cells at a time point 40 s after the start of each measurement. Data (F: ratio between emission at 340
and 380 nm) were recorded for the next 500 s. Changes in mean F/F0 (F0: Ratio between emission at
340 and 380 nm at the start point of measurement) after the application of compounds were averaged
for at least three independent experiments for each concentration. The peak values of F/F0 were
analyzed as the maximum within 200 s after compound application, shown in the ordinate of Figure 2.
Response to the application of cIDPRE was served as a positive control while addition of buffer was
used as blank.
3.3. Calculations
The conformations of studied cADPR analogues were optimized with density functional theory
(DFT) quantum chemical method by using Gaussian 09 program package [33]. Equilibrium geometries
of all molecules were fully optimized at the B3LYP/6–31G(d) level of theory [34]. Vibrational frequencies,
calculated at the same levels, were used to determine the nature of the stationary points and to give the
thermodynamic date and zero-point vibrational energies. Then SYBYL-X 1.1 software was used to align
the calculated structures of three cADPR analogs with crystal structure of cADPR, respectively.
4. Conclusions
In summary, novel nucleobase-modified and sugar-modified cADPR mimics 5 were synthesized, in
which halogen atoms were introduced into the southern ribose for the first time. The synthesis
employed a Cu(I)-catalyzed Hüisgen [3+2] cycloaddition for the building of the triazole moiety and
microwave-assisted reactions were used for the pyrophosphate bond formation. Biological evaluation
reveals that these new kinds of cADPR analogues are membrane permeant agonists of the calcium
signaling pathway. The introduction of chlorine or fluorine into the 2'-position of the southern ribose
led to a decrease of activity. The existence of a hydrophobic group on the 3'-OH of the southern ribose
does not alter much the agonistic activity. The result of this study is helpful for the understanding of
structure-activity relationships of cADPR analogues and to help design new probes to investigate the
cADPR-mediated calcium signaling pathway.
Acknowledgements
This study was supported by the National Natural Sciences Foundation of China (Grant No.
90713005, 20910094) and the Ministry of Education of China (Grant No. 200800010078).
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Sample Availability: Samples of the compounds 5a–c are available from the authors.
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