A novel method for the synthesis of sulfur substituted-cyclopyrophosphate of cADPR analogs

Article abstract of DOI:10.1016/j.cclet.2014.07.010

A facile and efficient protocol for the synthesis of sulfur substituted-cyclopyrophosphate of cIDPRE (PS1-cIDPRE) was developed. The key step was the cyclization process which was completed by the sulfur substituted cyclization precursor 1b via the one-pot phosphoramidite strategy.

Full text of DOI:10.1016/j.cclet.2014.07.010

Chinese Chemical Letters 25 (2014) 1583–1585
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
A novel method for the synthesis of sulfur substituted-
cyclopyrophosphate of cADPR analogs
*
Ren-Min Wu, Na Qi, Yu-Wen Jia, Zhu Guan, Liang-Ren Zhang, Li-He Zhang, Zhen-Jun Yang
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
A R T I C L E I N F O
A B S T R A C T
Article history:
A facile and efficient protocol for the synthesis of sulfur substituted-cyclopyrophosphate of cIDPRE
(PS1-cIDPRE) was developed. The key step was the cyclization process which was completed by the sulfur
substituted cyclization precursor 1b via the one-pot phosphoramidite strategy.
ß 2014 Zhen-Jun Yang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights
reserved.
Received 3 June 2014
Received in revised form 10 July 2014
Accepted 14 July 2014
Available online 23 July 2014
Keywords:
cADPR
cIDPRE
Phosphoramidite strategy
Cyclopyrophosphate
1. Introduction
On the other hand, Slama and his coworkers discovered that the
substituted methylenebisphosphonate of the pyrophosphate
Cyclic adenosine diphosphoribose (cADPR, Fig. 1) was discov-
ered as a Ca2+ mobilizing second messenger by Lee [1]. Since its
discovery, many cell systems have been proved to utilize the
cADPR/ryanodine receptor (RyR) Ca2+ signaling system to control
Ca2+-dependent cellular responses, such as fertilization, secretion,
contraction, proliferation and many more [2,3]. In order to
elucidate the specific molecular mechanism with which cADPR
intervenes calcium releasing, numerous analogs of cADPR with
improved stability and permeability have been obtained and used
as molecular probes for the mechanistic studies. These derivatives
include the modification of southern and northern riboses [4–6],
purine [7–10], and pyrophosphate [11,12] of cADPR. They are
agonists or antagonists of cADPR/RyR Ca2+ signaling pathway.
As far as we know, there were only a few analogs modified on
the pyrophosphate moiety of cADPR have been synthesized and all
of them were shown to be agonists (Fig. 1) [6,13,10]. The ‘‘caged’’
cADPR-2-nitrophenyl ethyl ester of cADPR was reported as a
membrane permeant by Lee et al. Specifically, this compound is
biologically inactive, but it can be uncaged and activated by UV
induced photolysis. Sih and colleagues synthesized a cADPR analog
consist of a triphosphate bridge (cATPR). The cATPR was proved to
be considerably more potent than cADPR in inducing Ca2+ release.
moiety in cADPR synthesized the cADPR and 3-deaza-cADPR
analogs (cADPR[CH2] and 3-deaza-cADPR[CH2], Fig. 1) which were
15- and 5-fold less positive than cADPR in sea urchin egg
homogenates.
The N1-ethoxy-methyl-substituted cyclic inosine diphosphor-
ibose (cIDPRE, Fig. 1) was proven to be a membrane-permeable and
stable cADPR agonist in Jurkat T cells in our previous works [14].
This novel cADPR analog can be applied as an important tool to
study the cADPR-mediated Ca2+ signaling system in intact cells.
Recently, our laboratory developed a new concise method, the
one-pot phosphoramidite strategy, which was characterized by
simple operation, time-saving and profited phosphate modifica-
tion. And the cIDPRE as well as sulfur- and selenium-substituted
pyrophosphate cIDPRE analogs (PS1-cIDPRE, PS1e-cIDPRE, PS2-cIDPRE
and PS2e-cIDPRE, Fig. 1) were obtained by application of this method
[11]. Interestingly, the PS1-cIDPRE-1 and PS1-cIDPRE-2 have shown
the opposite biological activities with regard to Ca2+ signaling. It
meant these two membrane permeant molecules were promising
probe tool molecules to reveal the specific molecular mechanism
of the cADPR/RyR Ca2+ signaling pathway.
2. Experimental
In the two routes, the key step, the cyclization, was completed
by the same method- the one-pot phosphoramidite strategy. So the
procedure is similar. The general procedure of the one-pot
*
Corresponding author.
E-mail address: yangzj@bjmu.edu.cn (Z.-J. Yang).
http://dx.doi.org/10.1016/j.cclet.2014.07.010
1001-8417/ß 2014 Zhen-Jun Yang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

1584
R.-M. Wu et al. / Chinese Chemical Letters 25 (2014) 1583–1585
Fig. 1. Structures of pyrophosphate modified cADPR analogs.
phosphoramidite strategy was as follows, took the synthesis of
buffer (pH 7.0) to yield 3b-1, 3b-2 in the ratio 1: 1.5. Ultimately, the
PS1-cIDPRE-1 and PS1-cIDPRE-2 were obtained after deprotection of
propylidene.
compound 2b as an example: the 2-cyanoethoxy-N,N,N0,N0-
tetraisopropyl-phosphoramidite (4, 41
mL, 0.13 mmol) was added
to a solution of 1b (50 mg, 86 mol) and 1H-tetrazole (19 mg,
m
0.27 mmol) in CH3CN (30 mL) under argon (Scheme 1). The
reaction was continued for 0.5 h at room temperature. Then tert-
BuOOH (0.4 mmol) was added and the reaction was continued in
one pot to give the compounds 2b in 61% yield for oxidation. After
deprotection, a mixture of two diastereoisomers 3b was obtained,
which can be separated by HPLC on a C18 column (Agela Venusil
XBP preparative C18 reversed-phase column, 250 mm ꢀ 22 mm,
10 mm) eluting by the buffer system: CH3CN/0.05 mol/L TEAA
3. Results and discussion
As showed in the Scheme 1, although the cyclization
precursor 1a and 1b were different, the products were both
PS1-cIDPRE-1 and PS1-cIDPRE-2. This is an interesting experimen-
tal result, because the sulfhydryl group was more affinitive with
phosphorus than hydroxyl did [15–17]. However, there was only
cyclic-pyrophosphate of oxygen bridge products 2b instead of
Scheme 1. Synthesis of the PS1-cIDPRE-1 and PS1-cIDPRE-2 by two different routes. Reagents and conditions: (i) (a) 2-cyanoethoxy-N,N,N0,N0-tetraisopropyl -phosphoramidite
(4), 1H-tetrazole, CH3CN, r.t.; (b) S, CS2, r.t., overnight; (ii) 1 mol/L TEAB; (iii) 60% HCOOH; (iv) (a) 4, 1H-tetrazole, CH3CN, r.t.; (b) tert-BuOOH, r.t., 30 min. *Compound 1a and
1b was prepared using a modified procedure according to the Supporting information.
Scheme 2. One puzzle generated from the experimental results.

R.-M. Wu et al. / Chinese Chemical Letters 25 (2014) 1583–1585
1585
the expected sulfur bridge compounds 2b0 (Scheme 2). We were
Appendix A. Supplementary data
assured this conclusion by the following objective evidences:
There were only four cyclized compounds after the cyclization of
1b via the one-pot phosphoramidite strategy according to the
graphs of separation of HPLC and molecular ion peaks of MS
(details in the supporting information). If the reaction reacted in
path B or in path A and path B, there would be only two or six
cyclized products. This indicated that path A was the only route
(Scheme 2). After deprotection of acrylonitrile, the northern
chiral phosphorus of compounds 2b or 2b0 was racemized. Only
two diastereoisomers, 3b-1 and 3b-2, were obtained (Scheme 1).
If there were sulfur-bridge compounds 2b0, it would produce
only one symmetrical compound. Moreover, the 31P NMR of
compounds 3b-1 and 3b-2 were both around 43 and ꢁ11 ppm.
These signal peaks were the same as the 31P NMR of 3a, including
3a-1 and 3a-2, which were synthesized by previous route in our
lab from cyclization precursor 1a (details in the Supporting
information).
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.cclet.2014.07.010.
References
[1] H.C. Lee, Physiological functions of cyclic ADP-ribose and NAADP as calcium
messengers, Annu. Rev. Pharmacol. Toxicol. 41 (2001) 317–345.
[2] A.H. Guse, Biochemistry, biology, and pharmacology of cyclic adenosine dipho-
sphoribose (cADPR), Curr. Med. Chem. 11 (2004) 847–855.
[3] L. Xu, T.F. Walseth, J.T. Slama, Cyclic ADP-ribose analogues containing the methy-
lenebisphosphonate linkage: effect of pyrophosphate modifications on Ca2+
release activity, J. Med. Chem. 48 (2005) 4177–4181.
[4] X. Huang, M. Dong, J. Liu, et al., Concise syntheses of trifluoromethylated cyclic
and acyclic analogues of cADPR, Molecules 15 (2010) 8689–8701.
[5] B.V.L. Potter, T.F. Walseth, Medicinal chemistry and pharmacology of cyclic ADP-
ribose, Curr. Mol. Med. 4 (2004) 303–311.
[6] J. Xu, Z.J. Yang, W. Dammermann, et al., Synthesis and agonist activity of cyclic
ADP-ribose analogues with substitution of the northern ribose by ether or alkane
chains, J. Med. Chem. 49 (2006) 5501–5512.
[7] A.H. Guse, Second messenger function and the structure–activity relationship of
cyclic adenosine diphosphoribose (cADPR), FEBS. J. 272 (2005) 4590–4597.
[8] S. Shuto, M. Fukuoka, A. Manikowsky, et al., Total synthesis of cyclic ADP-
carbocyclic-ribose, a stable mimic of Ca2+-mobilizing second messenger cyclic
ADP-ribose1, J. Am. Chem. Soc. 123 (2001) 8750–8759.
In summary, the experimental results showed only oxygen-
bridge products 2b produced from cyclization precursor 1b via the
one-pot phosphoramidite strategy (Scheme 2) [11].
[9] M. Dong, T. Kirchberger, X.C. Huang, et al., Trifluoromethylated cyclic-ADP-ribose
mimic: synthesis of 8-trifluoro-methyl-N1-[(500-O-phosphorylethoxy)methyl]-
50-O-phosphorylinosine-50,500-cyclic-pyrophosphate (8-CF3-cIDPRE) and its calci-
um release activity in T cells, Org. Biomol. Chem. 8 (2010) 4705–4715.
[10] F.J. Zhang, S. Yamada, Q.M. Gu, C.J. Sih, Synthesis and characterization of cyclic
ATP-ribose: a potent mediator of calcium release, Bioorg. Med. Chem. Lett. 6
(1996) 1203–1208.
[11] N. Qi, K. Jung, M. Wang, et al., A novel membrane-permeant cADPR antagonist
modified in the pyrophosphate bridge, Chem. Commun. 47 (2011) 9462–9464.
[12] X. Gu, Z.J. Yang, L.R. Zhang, et al., Synthesis and biological evaluation of novel
membrane-permeant cyclic ADP-ribose mimics: N1-[(500-O-phosphorylethoxy)
methyl]-50-O-phosphoryl-inosine 50,500-cyclic-pyrophosphate (cIDPRE) and 8-
substituted derivatives, J. Med. Chem. 47 (2004) 5674–5682.
4. Conclusion
In summary, a novel and concise strategy was developed to
synthesize sulfur substituted-cyclopyrophosphate of cIDPRE
(PS1-cIDPRE). Especially, this strategy has shortened the synthetic
route from 10 steps to 7 steps compared with the previous route
to synthesize the promising probe molecules PS1-cIDPRE-1 and
PS1-cIDPRE-2. Moreover, the experimental results generated an
intriguing question why there was no sulfur-bridge compounds
obtained or detected. There may be hiding vital clues in those
results for the study of mechanism of the cyclization of the
one-pot phosphoramidite strategy. The work of the mechanism
is on-going.
[13] R. Aarhus, K. Gee, H.C. Lee, Caged cyclic ADP-ribose synthesis and use, J. Biol.
Chem. 270 (1995) 7745–7749.
[14] A.H. Guse, X.F. Gu, L.R. Zhang, et al., A minimal structural analogue of cyclic ADP-
ribose synthesis and calcium release activity in mammalian cells, J. Biol. Chem.
280 (2005) 15952–15959.
[15] J. Wang, J. Zhou, G.P. Donaldson, et al., Conservative change to the phosphate moiety
of cyclic diguanylic monophosphate remarkably affects its polymorphism and ability
to bind DGC, PDE, and PilZ proteins, J Am. Chem. Soc. 133 (2011) 9320–9330.
[16] E. Krawczyk, J. Mikolajczak, A. Skowronska, J. Michalski, Reaction of tricoordinate
phosphorus compounds with organophosphorus pseudohalogens: desulfuriza-
tion and deoxygenation of oxophosphoranesulfenyl chlorides, scope and mecha-
nism, J. Org. Chem. 57 (1992) 4963–4970.
Acknowledgment
We are grateful for scientific research funding from the National
Natural Science Foundation of China (Nos. 21332010, 21172010,
21002004).
[17] L.X. Na, X.L. Sun, M. Wang, et al., Atropisomerism of diastereomer diribonucleo-
side phosphotriester, Chin. Chem. Lett. 24 (2013) 13–16.