Polyketide origin of 3-alkylpyridines in the marine mollusc Haminoea orbignyana Adele Cutignano, Guido Cimino, Antonella Giordano, Giuliana dÕIppolito and Angelo Fontana * Istituto di Chimica Biomolecolare (ICB), CNR, Via Campi Flegrei 34, 80078 Pozzuoli, Napoli, Italy Received 23 December 2003; revised 26 January 2004; accepted 27 January 2004 Abstract—The paper reports the biosynthesis of the main alkylpyridine alkaloids, haminol-1 (1) and -2 (2), in the Mediterranean mollusc Haminoea orbignyana. Experiments were carried out by in vivo incorporation of [1,2- 13 C 2 acetate]. Data give full account for a polyketide origin of haminols in the Mediterranean molluscs, showing the biosynthesis of these 3-alkylpyridine alkaloids by elongation with acetate of a starter unit of nicotinic acid. Ó 2004 Elsevier Ltd. All rights reserved. 3-Alkylpyridine alkaloids are a family of marine metabolites showing an outstanding variety of chemical structures formally derived by single or multiple units of a basic 3-alkylpyridine motif. 1;2 Mediterranean of the genus Haminoea (Gastropoda: Cephalaspidea) show the simplest members of this family of compounds and, therefore, represent ideal systems to investigate the biogenesis of this class of natural products. 3 Recently, we have first described the in vivo biosynthesis of the main alkylpyridine compounds, haminol-1 (1) and -2 (2), in the Mediterranean Haminoea orbignyana. 4 Clas- sical feeding experiments with isotopically labelled pre- cursor proved the origin of the 3-alkylpyridine motif from nicotinic acid and acetate units. However, these experiments did not allow us to fully elucidate the bio- chemical pathway leading to haminols since at least three different routes could be in agreement with experimental results. 4 Herein, we report a further experiment with H. orbignyana and give a complete interpretation of the biogenesis of 3-alkylpyridine alk- aloids in this mollusc. The animals (65 specimens) were maintained in aquar- ium and injected twice (day 1 and 4) over a period of 1 week with [1,2- 13 C 2 ]-acetate (4.6 mg/specimen in 40 lL of distilled water). 5 Subsequent extraction and isolation of the ether-soluble fractions 4 gave 1.0 mg of haminol-2 (2, 0.016 mg/specimen). 13 C NMR spectrum 6 of this product showed evident differences with that previously reported in the literature (Fig. 1). 4 The changes were particularly severe for the signals of the pyridine ring, thus suggesting the protonation of the heterocyclic moiety as probable origin of the observed spectroscopic variation. In fact, in agreement with the literature of protonated pyridine ring, 7 C-2 0 and C-6 0 (145.2 and 145.1 ppm) were significantly shifted towards high-field regions (literature 4 values 149.9 and 147.1 ppm), and whereas C-4 0 (140.5 ppm) experienced a low-field shift (literature 4 values 135.9 ppm). The bulky effect of these changes was an evident overlapping of the signals in the region of the sp 2 carbons (from 125 to 145 ppm) and a consequent difficulty to discriminate the double bond carbons of the alkyl chain. However, although the sig- nals of the entire spectrum suffered slight variation within respect to the values reported in the literature, 4 the effect was rather marginal on the other carbons and did not preclude the analysis of the 13 C– 13 C coupling constants and the identification of the biogenetically related C 2 -units. In fact, besides the evident correlation (J c–c ¼ 59:6 Hz) between the methyl (21.3 ppm) and the carbonyl signals of the acetyl residue (170.4 ppm), very clear was the presence of the coupled doublets flanking the signals of C2 (70.7 ppm, J c–c ¼ 38:4 Hz) and C3 (39.2 ppm, J c–c ¼ 38:4 Hz) (Fig. 2). On the other hand, N OR 1 R=H 2 R = Ac 3 4 11 12 2' 4' 5' 6' 2 1 * Corresponding author. Tel.: +39-081-8675096; fax: +39-081-80417- 70; e-mail: afontana@icmib.na.cnr.it 0040-4039/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tetlet.2004.01.138 Tetrahedron Letters 45 (2004) 2627–2629 Tetrahedron Letters