In the course of our systematic screening for antiviral, antifungal, antibacterial, and cytotoxic compounds from cyanobacteria (blue-green algae), we found that a MeOH extract of Anabaena affinis strain VS-1 showed strong cytotoxicity to L1210 murine leukemia cells, and we assign here structures to two compounds, 1 and 2, responsible for the cytotoxicity of the organism. This is the first report of the isolation and characterization of pyrrolo[3,2-d]pyrimidine derivatives as biosynthetic products. A. affinis strain VS-1 was isolated from a cyanobacterial water-bloom collected from Star Lake, Norwich, VT and cultivated in 2-8 mineral medium according to the conditions reported by Carmichael. The lyophilized cells were extracted with MeOH-H2O (4:1), and the aqueous residue obtained after evaporation of the MeOH was passed through a CHP-2OP column. The column was rinsed with H2O, and the active components were eluted with 15% EtOH-H2O and evaporated. Two active components, 1 (0.25% of dried cell weight) and 2 (0.028%), were isolated by bioassay-guided separation of the residue using HPLC with an ODS column. Compound 1, [α]2'D +21.9° (c 0.051, H2O), showed a molecular ion peak at m/z 429.1627 (C17H24N4O9, M+ H, Δ -0.5 mDa) in the high-resolution (HR) FAB mass spectrum obtained with dithiothreitol/dithioerythritol (magic bullet) as matrix. The 1H NMR spectrum of 1 contained two aromatic proton signals and 13 one-proton signals ascribable to a pentose and a hexose. Six heteroatom-substituted aromatic 13C signals were detected in the 13C NMR spectrum of 1, together with 11 signals due to the sugar units. These data and the UV spectrum of 1 [λmax (H2O) 287 (sh), 276, 268, and 229 nm; (0.01 N HCl) 272 and 235 nm] suggested that 1 is a nucleoside with two sugar units. Collisionally induced tandem FABMS (FABMS/CID/MS) of 1 showed three major fragment ion peaks at m/z 177, 163, and 147, together with a strong fragment ion peak at m/z 267 generated by the loss of the hexose unit, but a prominent peak due to (base + H2)+ was not detected, suggesting a C-nucleoside. This was confirmed by the chemical shifts of the anomeric center (δH, 4.87; δC, 78.1), which were observed at relatively high fields in the 1H and 13C NMR spectra of 1. Subtraction of the sum of the two sugar units from the molecular formula of 1 gave C6H5N4 (133 Da) as the base unit. One-bond 1H-13C coupling constants of 13C signals at δ 128.0 (1JC,H = 190 Hz) and 149.9 (1JC,H = 207 Hz) were characteristic for carbons attached to, respectively, one and two nitrogen atoms. The 1H signal (δ, 7.72) for the hydrogen attached to the former carbon (δ, 128.0) showed long-range coupling (J = 1.0 Hz) to an anomeric proton (δ, 4.89, H-1'). These data suggested that the base unit is either pyrrolo[2,3-d]pyrimidine (i.e., 7-deazaadenine) or pyrrolo[3,2-d]pyrimidine (i.e., 9-deazaadenine). The 13C signals due to the aromatic carbons of 1 resemble those reported for 9-deazaadenine derivatives rather than 7-deazaadenine derivatives, although no pyrrolo[3,2-d]pyrimidine derivative has been reported from natural sources. UV spectra of 1, especially the shifts of absorption maxima in acidic solution, were also more like those of 9-deazaadenine than those of tubercidin (7-deazaadenosine). Accordingly, the base unit in 1 is most likely 9-deazaadenine. The 13C signals assigned to the hexose unit of 1 closely resembled those of methyl α-D-glucopyranoside, suggesting that 1 is the α-D-glucopyranoside of 9-deazaadenosine. 5'-α-D-Glucopyranosides of tubercidin and toyocamycin have been isolated from cyanobacteria. 1H and 13C NMR data for the sugar units of 1 were very similar to those for these compounds, except for the signals due to the C-1' position. Moreover, enzymatic deglycosidation of 1 with α-D-glucosidase gave D-glucose and 2, which was isolated as the minor component (11% of 1) from the same cyanobacterium. Compound 2, [α]BD -28.4° (c 0.016, H2O), showed a molecular ion peak at m/z 267.1090 (C11H15N4O4, M + H, Δ +0.3 mDa) by HRFABMS. FABMS/CID/MS of 2 gave the same fragment ion peaks at m/z 177, 163, and 147 observed for 1. The 1H NMR spectrum of 2 showed the signals ascribable to a ribose unit and two aromatic proton signals. From the results above, the structure of 2 can be assigned as 9-deazaadenosine, which has been synthesized by Lim and Klein as a cytotoxic C-nucleoside isostere of adenosine. The direct comparison of 2 with a synthetic sample of 9-deazaadenosine by HPLC, TLC, and UV spectra confirmed that 2 was identical to synthetic 9-deazaadenosine. 1H NMR data for natural 2 hydrochloride were also identical with those for synthetic 2 hydrochloride. Consequently, the structure of 1 was assigned as the 9-deazaadenosine 5'-α-D-glucopyranoside. Compounds 1 and 2 are pyrrolo[3,2-d]pyrimidine derivatives which have not been reported previously as biosynthetic products, i.e., from natural sources. Their biosynthesis will be of considerable interest. The IC50 of 1 and 2 vs L1210 murine leukemia cells were 0.01 and 0.002 μg/mL, respectively. These compounds also showed lethal toxicity to the aquatic invertebrate Ceriodaphnia dubia; the LC50 for acute (48 h) and chronic (7 day) toxicities were, respectively, 0.5 and 0.3 μg/mL for 1 and 0.3 and 0.1 μg/mL for 2.