Chinese Chemical Letters  2014, Vol.25 Issue (12):1583-1585   PDF    
A novel method for the synthesis of sulfur substitutedcyclopyrophosphate 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
Abstract: 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.
Key words: cADPR     cIDPRE     Phosphoramidite strategy     Cyclopyrophosphate    
1. Introduction

Cyclic adenosine diphosphoribose (cADPR,Fig. 1) was discovered 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, 5, 6], purine [7, 8, 9, 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. On the other hand,Slama and his coworkers discovered that the substituted methylenebisphosphonate of the pyrophosphate 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.

Fig. 1.Structures of pyrophosphate modified cADPR analogs.

The N1-ethoxy-methyl-substituted cyclic inosine diphosphoribose (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 modification. And the cIDPRE as well as sulfur- and selenium-substituted pyrophosphate cIDPRE analogs (PS1-cIDPRE,PSe1-cIDPRE,PS2 -cIDPRE and PSe2-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 phosphoramidite strategy was as follows,took the synthesis of compound 2b as an example: the 2-cyanoethoxy-N,N,N',N'- tetraisopropyl-phosphoramidite (4,41 μL,0.13 mmol) was added to a solution of 1b (50 mg,86 μmol) and 1H-tetrazole (19 mg, 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 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.

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,N',N'-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.
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 experimental result,because the sulfhydryl group was more affinitive with phosphorus than hydroxyl did [15, 16, 17]. However,there was only cyclic-pyrophosphate of oxygen bridge products 2b instead of the expected sulfur bridge compounds 2b' (Scheme 2). We were assured this conclusion by the following objective evidences: Therewere 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 2b' was racemized. Only two diastereoisomers,3b-1 and 3b-2,were obtained (Scheme 1). If there were sulfur-bridge compounds 2b',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 peakswere the same as the 31P NMRof 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).

Scheme 2.One puzzle generated from the experimental results.

In summary,the experimental results showed only oxygenbridge products 2b produced from cyclization precursor 1b via the one-pot phosphoramidite strategy (Scheme 2) [11]. 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.


Weare grateful for scientific research funding from the National Natural Science Foundation of China (Nos. 21332010,21172010, 21002004).

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version,at

[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 diphosphoribose (cADPR), Curr. Med. Chem. 11 (2004) 847-855.
[3] L. Xu, T.F. Walseth, J.T. Slama, Cyclic ADP-ribose analogues containing the methylenebisphosphonate 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 ADPribose, 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 ADPcarbocyclic-ribose, a stable mimic of Ca2+-mobilizing second messenger cyclic ADP-ribose1, J. Am. Chem. Soc. 123 (2001) 8750-8759.
[9] M. Dong, T. Kirchberger, X.C. Huang, et al., Trifluoromethylated cyclic-ADP-ribose mimic: synthesis of 8-trifluoro-methyl-N1-[(5''-O-phosphorylethoxy)methyl]-50-O-phosphorylinosine-50,500-cyclic-pyrophosphate (8-CF3-cIDPRE) and its calcium 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-[(5''-O-phosphorylethoxy) methyl]-5'-O-phosphoryl-inosine 5',5''-cyclic-pyrophosphate (cIDPRE) and 8-substituted derivatives, J. Med. Chem. 47 (2004) 5674-5682.
[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 ADPribose 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 phosphatemoiety of cyclic diguanylicmonophosphate remarkably affects its polymorphismand 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: desulfurization and deoxygenation of oxophosphoranesulfenyl chlorides, scope and mechanism, J. Org. Chem. 57 (1992) 4963-4970.
[17] L.X. Na, X.L. Sun, M. Wang, et al., Atropisomerism of diastereomer diribonucleoside phosphotriester, Chin. Chem. Lett. 24 (2013) 13-16.