A multi-factored complex of structurally-unique macrolides was isolated from culture broths of a new species of Saccharopolyspora. The core structure consists of a 5,6,5-cis-anti-trans-tricyclic ring system fused to a 12-membered macrocyclic lactone, which is further substituted by an amino- and a neutral sugar. Fermentation broths of soil microorganisms have provided a continuous source of novel secondary metabolites possessing useful biological activities. Within this wide array of natural products, the macrolide class has been exceptionally diverse in both structure and biological activity.1, 2 Screening of culture broths for mosquito larvicidal activity has recently led to the discovery of a series of structurally-unique macrolides, designated as the A83543 complex. Since such activity is not found within the class of traditional macrolide antibiotics, these compounds represent a novel addition to the list of diverse macrolide structures warranting synthetic and biosynthetic studies. The multi-factored A83543 complex was produced in fermentation broths of a new species of Saccharopolyspora (S. spinosa). 3 The active components were extracted from the culture biomass with methanol, and the resultant solution was concentrated and extracted with ether. The ether-soluble material was purified by sequential reverse phase HPLC (Lobar RP-8, then Dynamax C-18), eluting with methanolacetonitrile-water mixtures. The major factor of the complex was A83543A, whose empirical formula was established by high resolution mass spectrometry as C41H65NO10 (MW 731). A83543D, the second most abundant factor, differed from A83543A by addition of the elements CH2 (C42H67NO10, MW 745). Using MS/MS techniques, both compounds were shown to contain forosamine, an aminosugar also present in the macrolide antibiotic spiramycin. 4 Minor factors A83543B and A83543C differed from factor A by loss of the elements CH2 and C2H4, respectively, with empirical formulas of C40H63NO10 (MW 717) for A83543B and C39H61NO10 (MW 703) for A83543C. NMR data from 1H homonuclear decoupling, 13C DEPT, 2D one-bond heteronuclear correlation, and 2D long range heteronuclear correlation (FULCOUP) experiments were then combined with mass spectral fragmentation data to provide the gross structure of A83543A (Table 1, Figure 1). Relative stereochemistry at C-3, 4, 7, 11, and 12 was determined from coupling constants and difference NOE experiments. Crystallization of A83543A from ethanol-water provided white crystals which were successfully analyzed in a single crystal x-ray diffraction study to confirm the gross structure of the molecule and to establish the relative stereochemistry at C-9, 16, 17, and 21, as depicted in Figure 1. Once the structure of A83543A had been established, the structures of the other factors were readily determined from comparative mass spectrometric and NMR spectroscopic studies. 5 Critical data for A83543B and A83543C were the intensity (or absence) of their respective N-methyl resonance and the chemical shifts for the aminosugar in their 13C spectra. 6 A83543D was shown to be 6-methyl-A83543A by comparison of 13C spectra, homonuclear decoupling of H-4, downfield shift of C-7, and slight upfield shift of C-8 (see Table 1). The absolute stereochemistry of A83543A was determined by comparing samples of forosamine which had been independently produced from A83543A and spiramycin by acidic hydrolysis. 7 The two samples of forosamine were identical in all respects, including sign and degree of optical rotation, thus establishing that A83543A contained D-(+)-forosamine, the same as found in spiramycin. 8 Combined with the X-ray crystal data, the absolute stereochemistry of A83543A was thereby established as depicted in Figure 1. The most closely related structures previously reported in the literature are those of ikarugamycin and capsimycin.9,10 However, these latter compounds are derived from a mixed polyketide-amino acid pathway, whereas the aglycone of A83543 is completely polyketide in origin and two saccharides are attached. Although A83543 and ikarugamycin both contain 5,6,5-cis-anti-trans-tricyclic ring systems fused to a macrocyclic ring, their tricyclic systems have opposite absolute stereochemistries, suggesting that they arise by different biogenetic pathways. Furthermore, A83543 contains a simple lactone rather than a tetramic acid imbedded within a lactam as its macrocyclic ring system, and the ring substitution patterns are substantially different, including the presence of both an amino- and a neutral saccharide in A83543. In contrast to conventional macrolides such as erythromycin and tylosin, 11 the A83543 factors were devoid of antibacterial activity. In further contrast to the macrolide antibiotics, which do not possess insecticidal activity, the A83543 factors possessed potent mosquito larvicidal activity. The major factor, A83543A, exhibited a minimum inhibitory concentration (MIC) of 0.016 μg/ml against first instar mosquito larvae. Thus, the A83543 factors are structurally- and biologically-unique macrolides.