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Salvadenosine, a 5′-Deoxy-5′-(methylthio) Nucleoside from the Bahamian Tunicate Didemnum sp.

Salvadenosine, (1) a rare 5′-deoxy-5′-(methylthio) nucleoside, was isolated from the deep-water Bahaman tunicate Didemnum sp. The structure was solved by integrated analysis of MS and 1D and 2D NMR data. We revise the structure of the known natural product, hamiguanosinol, which is a constitutional isomer of 1, to 5 by interpretation of the spectroscopic data and comparison with synthesized nucleosides.


■ INTRODUCTION
Modified nucleosides are relatively rare from marine organisms, but they have a long history. Early pioneering work 1 by Bergmann on Caribbean sponges of the genera Tethya and Cryptotethya resulted in the isolation of spongosine, spongothymidine, 2 and spongouridine 2b,c (the first arabino-nucleosides) and others. Predating the modern era of marine natural products chemistry, these seminal discoveries were the inspiration 3 for development of the clinically important antitumor drugs Ara A 4 and Ara C. 5 In our investigations of antifungal and antitumor compounds using "nanomole-scale" techniques, 6 we examined several extracts of rare tunicates that displayed antifungal activity against a panel of Candida spp. and Cryptococcus spp. Here, we report salvadenosine (1), an uncommon 5′-deoxy-5′-(methylthio) nucleoside from an encrusting deep-water tunicate Didemnum sp. (Figure 1). In addition, we revise the structure of the known compound, hamiguanosinol (2), reported by Proksch and co-workers from the Pacific sponge Hamigera hamigera. 7 Salvadenosine (1) joins the family of rare marine-derived nucleosides that include Bergmann's arabino-nucleosides from Cryptotethya sp. 2 and the antiproliferative trachycladine A (3) from the sponge Trachycladus laevispirulifer. 8

■ RESULTS
The n-BuOH soluble partition of the methanol extract of Didemnum sp. was separated by reversed-phase HPLC to give 1 in addition to tryptamine and the known natural product 6bromotryptamine. 9 The molecular formula of 1, C 11 H 15 (Table 1). COSY correlations confirmed the 5′-deoxy-5′-(methylthio)ribose moiety. Cross peaks arising from a modified ribose corresponded to the following contiguous spin system: anomeric proton H-1′ (δ H 5.87, d, J Distinct differences were apparent between the 1 H and 13 C chemical shifts of 1 and those published for hamiguanosinol (2, Table 2), particularly the sp 2 13 C NMR signals. An HSQC correlation from the sole downfield nonexchangeable aromatic signal (δ H 8.00, δ C 150.8, 1 J CH = 203.4 Hz) revealed a 13 C chemical shift that was deshielded (Δδ 15.8 ppm) compared to that of the H-8 of 2 (δ H 7.90, δ C 135.0). Another notable difference was observed for the most shielded sp 2 13 C signal of 1 (δ C 103.6, s) and 2 (δ C 118.0, s, C-8; Δδ 14.4 ppm). HMBC correlations of 1 were observed from the anomeric proton H-1′ signal, and sp 2 13 C signals were also inconsistent with guanine; for example, no correlation was observed between the downfield 1 H NMR signal (δ H 8.08, s, 1H) and the anomeric carbon, C-1′, a correlation common to guanosine nucleosides. 10 The HSQC (DMSO-d 6 ) showed the aforementioned downfield proton was attached to a carbon with a 13 C signal (δ C 151.7, s) more compatible with an imidazolone CO group than the imidazole C-8 chemical shift of 2 (δ C 135.0, s). A better match for the NMR data of 1 was obtained by replacement of guanine with 8-oxoadenine, a nucleobase isomeric with the former. The downfield 1 H NMR signal (δ H 8.08, s, 1H) was assigned to H-2, and all inconsistencies were resolved. For example, the expected long-range correlation between C-1′ and H-8 for a guanine ring systembut missing in 2is replaced by 2,4 J correlations of H-2 to C-4 and C-5 in an 8-oxoadenine, respectively. The 13 C chemical shifts (DMSO-d 6 , Table 2) of the quaternary ring junction carbons C-4 and C-5 (δ C 146.7, s; 103.6, s) of 1 are more polarized (Δδ [C-4 − C-5] = 43.1 ppm) than the corresponding signals of 2 (Δδ = 35 ppm), guanosine, or adenosine but closer in magnitude than the 8-oxoadenosines, aplidiamine, erinacean, and caissarone. 11 Additionally, we measured the heteronuclear coupling constants of the downfield sp 2 1 H NMR singlets in 1 and several purine nucleosides (Table 3), revealing a better match between 8-oxoadenosine and the natural product (1: H-2, 1 J CH = 203.4 Hz; 8oxoadenosine: H-2 1 J CH = 201.5 Hz; guanosine: H-8 1 J CH = 213.5 Hz).
The natural products salvadenosine (1) and hamiguansinol are clearly not identical, but isomeric ( Figure 1). Naturally, the   question of validity of the structure of 2 arises. The nucleobase in hamiguanosinol (incorrectly named "6-hydroxyguanine") 7 was assigned as the enol tautomer (cf. enol form of guanosine 4a, Figure 1) based on a 13 C shift for C-6 of δ C 157.2 "instead of a ca.170 ppm for a keto amide f unction"; 7 however, we find this less convincing for three reasons. Keto groups in purines (guanine, inosine, etc.) are not electronically equivalent to simple amides and often exhibit relatively high field chemical shifts in the range observed for 3; the C-6 13 C chemical shift reported for hamiguanosinol is not incompatible with the 6keto tautomer 4b. Numerous studies have shown the keto form of guanosine (4b) and deoxyguanosine are the naturally stable tautomers in protic solvents: in fact, Watson−Crick pairing of G−C in DNA is crucially dependent upon it. Given that electronic differences between guanosine and 2 are insignificant, there are no compelling reasons to expect that 2 would be "locked" in the enol form and unable to spontaneously tautomerize to the keto form in protic solvent. Lastly, compound 5 has been synthesized by van Tilburg and coworkers 12 who characterized it as the keto tautomer. We repeated the synthesis of 5 (Scheme 1) from guanosine (4b) and showed the 13 C chemical shifts of the product were essentially the same as those reported for natural hamiguanosinol (Supporting Information, Table S2). 13 Therefore, the structure of hamiguanosinol is the keto tautomer 5, 14 not the enol 2: synthetic 5 and hamiguanosinol are identical. In order to verify the structure of salvadenosine (1), the natural product was synthesized by extension of the sequence of reactions used to prepare 5. N,O-Protected adenosine 15 was subjected to bromination (Scheme 2, saturated Br 2 −H 2 O, pH 4 NaOAc buffer), but only the cyclized product 7 was formed presumably through intramolecular attack by the 5′-OH group after bromination at C-8. 16 The same result was observed under aprotic conditions (NBS, DMF; 5,5-dimethyl-1,3-dibromohydantoin, DBH, DMF); 17 consequently, we turned to a "protecting-group free" strategy (Scheme 2). Adenosine (8) was converted to 8-bromoadenosine (9) (saturated Br 2 −H 2 O, pH 4 buffer) followed by an efficient conversion to the 8oxoadenosine (10) under Chatgilialoglus conditions. 18 Compound 10 was converted to primary chloride 11 with improved yield (SOCl 2 , DMPU, 70%); the latter, in turn, was subjected to nucleophilic substitution with sodium thiomethoxide to yield 1 in four steps and 17% yield from adenosine. Since the sample of natural 1 was purified as the formate salt, synthetic 1 was also converted to the formate salt for comparison. The 1 H and 13 C NMR spectra (Supporting Information, Table S3) as well as UV, IR, and CD data of the two samples 1·HCO 2 H matched in every way. Co-injection of natural 1 with synthetic 1 by HPLC resulted in a single peak (see the Supporting Information, Figure S1). Therefore, salvadenosine is assigned the structure 1 with high confidence.

■ DISCUSSION
The nature of the 5′-(methylthio) group in 1 and 5 deserves some comment. One plausible origin of 1 is from S-adenosyl methionine (SAM). The two CH 2 groups and one CH 3 group bonded to the S in SAM are electrophilic in nature. Common biological methylation involves S N 2 nucleophilic substitution of the electrophilic Me-S bond through attack by C-, N-, O-, Scentered nucleophiles, generating S-adenosylhomocysteine: the latter is cycled back to adenosine and homocysteine. In the biosynthesis of both the chlorinated antitumor drug salinosporamide A 19 and rare fluorinated natural products, substantial biochemical and structural evidence supports participation of the 5′-CH 2 −S bond of SAM in S N 2 substitutions by halide ions (Figure 2). For example, Streptomyces cattleya produces 5′fluoroadenosine through nucleophilic attack at the 5′-CH 2 of SAM by F − (path a), catalyzed by the enzyme fluorinase; 20 the former in turn is catabolized to fluoroacetate. A homologous "chlorinase" catalyzes displacement at 5′-CH 2 of SAM by Cl − (path a) in salinosporamide A biosynthesis. 19 Phylogenetic and biochemical studies 21 have shown a bifurcation of this biosynthetic motif. The gene duf62, represented in Nature in about 100 bacterial and archael genomes, expresses a protein, DUF62, that has high structural homology to the halogenases. DUF62, a "protein of unknown function," lacks halogenase activity, but carries out hydrolysis of SAM by nucleophilic attack at the 5′-CH 2 group of adenosine group by HO − (path b) to liberate L-methionine and adenosine. Attack at the 5″-CH 2 of SAM is similarly rare, with the best-characterized example occurring in the biosynthesis of nocardicin. 22 Transfer of the 3-amino-3-carboxypropyl group from SAM has also been proposed in the biosynthesis of modified bases for bacterial and yeast tRNAPhe, 23 the natural product discadenine, 24 nicotianamine (a precursor of plant siderophores), 25 and homoserinebased betaine lipids. 26 In addition, transfer of the 3-aminopropyl group from decarboxylated SAM is involved in polyamine biosynthesis. 27 Common to these biological reactions is the release of 5′-deoxy-5′-(methylthio)adenosine.
We speculate that 1 may arise through displacement of 5′deoxy-5′-(methylthio)adenosine from SAM (path b), possibly via a LuxI-type mechanism with release of homoseryl lactone. 28,29 The product may then be converted to 1 by electrophilic or free-radical attack at C-8 with reactive oxygen species (ROS), a reaction known from purine metabolism and the biology of DNA damage. 30 The anomaly is 5; no guanosine analogue of SAM has been demonstrated yet, but 5 has been detected in human urine as a byproduct of nucleotide catabolism. 14 In conclusion, we confirmed the structure of a new nucleoside salvadenosine (1, 5′-deoxy-5′-(methylthio)-8-oxoadenosine) from the tunicate Didemnum sp. through integrated analysis of spectroscopic data and total synthesis. Re-evaluation of the published structure of hamiguanosinol and its synthesis from guanosine requires revision of the structure of hamiguanosinol 7 from the enol tautomer 2 to the keto form 5.

■ EXPERIMENTAL SECTION
General Experimental Procedures. Inverse detected 2D NMR spectra were measured on a 500 MHz spectrometer equipped with a 5 mm 1 H{ 13 C} 5 mm probe or a 600 MHz NMR spectrometer with a 1.7 mm 1 H{ 13 C} microcryoprobe. 13 C NMR spectra were collected on a 125 MHz spectrometer equipped with a 5 mm 13 C{ 1 H} cryoprobe. NMR spectra were referenced to residual solvent signals, CD 3 OD (δ H 3.31, δ C 49.00), (CD 3 ) 2 SO (δ H 2.50, δ C 39.52). High-resolution mass data were obtained with an ESI-TOF system. Low-resolution MS measurements were made on a UHPLC coupled to an MSD singlequadrupole detector. Automated medium-pressure chromatography was carried out using a 30 g C 18 cartridges under specified gradient elution conditions. Optical rotations were measured at the D-double emission line of Na. FTIR spectra were collected on a ZnSe plate. CD spectra were measured in quartz cells (1 or 2 mm path length). DMPU and triethylamine were distilled from CaH 2 under N 2 . Other solvents were dried by passage through activate alumina or molecular sieves under Ar. Reactions were performed under N 2 .
Salvadenosine (1) 6-Bromotryptamine: colorless powder; 1 H, 13 C, and HRMS data were consistent with previously published data. 9 Tryptamine: colorless powder; MS and 1 H NMR spectra were identical to those of an authentic sample.
■ ASSOCIATED CONTENT * S Supporting Information 1 H, 13 C, and 2D NMR spectra of 1 and 7 as well as 1 H NMR spectra of 6 and 11. This material is available free of charge via the Internet at http://pubs.acs.org.