Article pubs.acs.org/jmc Aberrant Cyclization Affords a C-6 Modified Cyclic Adenosine 5′-Diphosphoribose Analogue with Biological Activity in Jurkat T Cells Christelle Moreau,† Tanja Kirchberger,‡ Bo Zhang,† and Barry V. L. Potter*,† † ‡ Mark P. Thomas, Karin Weber, Andreas H. Guse, ‡ †Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath, BA2 7AY, United Kingdom ‡Calcium Signalling Group, Department of Biochemistry and Signal Transduction, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany *S Supporting Information ABSTRACT: Two nicotinamide adenine dinucleotide (NAD+) analogues modified at the 6 position of the purine ring were synthesized, and their substrate properties toward Aplysia californica ADP-ribosyl cyclase were investigated. 6-N-Methyl NAD+ (6-N-methyl nicotinamide adenosine 5′-dinucleotide 10) hydrolyzes to give the linear 6-N-methyl ADPR (adenosine 5′-diphosphoribose, 11), whereas 6-thio NHD+ (nicotinamide 6-mercaptopurine 5′-dinucleotide, 17) generates a cyclic dinucleotide. Surprisingly, NMR correlation spectra confirm this compound to be the N1 cyclic product 6-thio N1-cIDPR (6-thio cyclic inosine 5′-diphosphoribose, 3), although the corresponding 6-oxo analogue is well-known to cyclize at N7. In Jurkat T cells, unlike the parent cyclic inosine 5′-diphosphoribose N1-cIDPR 2, 6-thio N1-cIDPR antagonizes both cADPR- and N1- cIDPR-induced Ca2+ release but possesses weak agonist activity at higher concentration. 3 is thus identified as the first C-6 modified cADPR (cyclic adenosine 5′-diphosphoribose) analogue antagonist; it represents the first example of a fluorescent N1- cyclized cADPR analogue and is a new pharmacological tool for intervention in the cADPR pathway of cellular signaling. ■ INTRODUCTION Cyclic adenosine 5′-diphosphoribose (cADPR, 1, Figure 1), discovered by Lee et al. in 1987,1 is one of the principal second Figure 1. C-6 modified cADPR analogues and numbering system. messenger molecules that mobilize intracellular Ca2+ in a different way to the well-established D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3),2 by gating the ryanodine receptor.3,4 The cADPR/ Ca2+ signaling system is active in diverse mammalian cellular systems such as cardiac muscle, acinar cells, and plant cells.5 cADPR is a metabolite of nicotinamide adenine dinucleotide (NAD+) and is produced enzymatically by ADP-ribosyl cyclases (ADPRC). Its structure was fully characterized by Lee et al.6 as a cyclic 18-membered dinucleotide featuring two glycosidic bonds and a pyrophosphate linkage. Several excellent reviews dealing with the chemistry of cADPR and the cADPR/Ca2+ signaling system have appeared in recent years.3,5,7−11 cADPR is readily hydrolyzable at the N1 glycosidic bond linkage to give ADPR in both neutral aqueous solution and under physiological conditions,12,13 thus rendering chemical synthesis of analogues challenging. The main choice has been between total chemical synthesis14 and a chemoenzymatic ap- proach15,16 developed earlier and modeled on the biosynthesis of cADPR from NAD+. By combination of both approaches, a large number of cADPR analogues have been synthesized over the years and their Ca2+ release activities examined in several systems, such as sea urchin egg homogenate (SUH) and T cells inter alia. Modification at the 8-position with 8-amino and 8-bromo groups in particular has been shown to convert cADPR from an agonist to an antagonist,16−19 although at high concen- trations this does not apparently hold.20 However, 8-substituted cyclic adenosine 5′-diphosphocarbocyclic ribose (8-X cADPcR) analogues developed by Shuto21 were reported to be agonists in sea urchin egg homogenates, indicating that 8-substitution alone may not be responsible for antagonist activity. 3′-O-Methyl cADPR was also found to have antagonist properties, at least in SUH.13 The hydrolysis-resistant 7-deaza 8-bromo cADPR has found several biological applications22,23 as a cell permeant competitive antagonist.17,24,25 Analogues with structural mod- ifications of the “northern” ribose12 showed agonist activities in T cells, including those with more radical changes.26,27 Received: May 12, 2011 Published: January 16, 2012 © 2012 American Chemical Society 1478 dx.doi.org/10.1021/jm201127y | J. Med. Chem. 2012, 55, 1478−1489