Pamamycin-607 (MW 607) was isolated from the producing strain of Streptomyces alboniger IFO 12738 as an aerial mycelium-inducing substance. It is a sixteen-membered macrodiolide with a dimethylamino group-bearing side chain1>2). We earlier isolated 14 homologues of pamamycin having different substituents at RX~R5and ranging in MWfrom 593 to 649, and confirmed that a change from CH3to CH2CH3at R3 or R4 caused loss of aerial mycelium-inducing activity3). We also reported that desdimethylaminopamamycin-607 prepared by Hofmann degradation and subsequent catalytic hydrogenation entirely lost this activity4). We therefore investigated the structure-activity relationship of the side chain part and continued our search for pamamycin-related compounds in cultured S. alboniger. We here report the isolation and structures of de-7Vmethylpamamycin-593A and B (Fig. 1). A crude parnamycin fraction3) showed a new Dragendorff reaction-positive spot at Rf 0.30 along with spots of pamamycins (Rf 0.66) on TLC analysis (EtOAc-w-hexane-diisopropylamine, 3 : 7 : 0.5). This crude fraction (40 mg obtained from 14 liters of cultured material) was chromatographed in a silica gel column which first was flushed with the solvent system EtOAc- /2-hexane-(z-Pr)2NH (2: 8 :0.5) to wash out the pamamycins then with the same solvent system (5:5:0.5) to elute the new compounds. The fraction containing the new compounds was chromatographed by ODS-HPLC (0.2% AcONH4 in 90% aq. MeOH) then by NH2-HPLC (rc-pentane-MeOH-rc-BuNH2, 100:0.5 :0.5), giving two new compounds (2.55 and 2.38mg). These compounds seemed to be pamamycin-related ones, because EI-MS analysis showed an M+ion at m\z 593 and an M+-43 ion atm\z 550, similar to the pattern of pamamycin-593. There was, however, a characteristic peak at m/z 86 rather than at m/z 100 the peak for pamamycins. Because the fragment ion of the pamamycins with m/z 100 is derived from a-cleavage of the dimethylamino group (fragment ion x in Table 1), these new compounds are thought to be de-7V-methyl derivatives (Rx=CH3, Ry=H, Rz=rc-C3H7 in Table 1) or derivatives with the dimethylamino group and the shorter alkyl side chain (Rx=Ry=CH3, Rz=C2H5) of pamamycin. To confirm this, the incorporation of methioninemethyl-d3 into pamamycins and the new compounds present in cultures ofS. albonigerà" (1 mg/ml medium) were examined. Pamamycin-607 obtained from the feeding experiment had isotope peaks of 3 and 6 units larger mass at the M+ion as well as fragment ions x and y (Table 1). This resulted from the replacement of CH3 at Rx and Ry with CD3, confirmation that the two methyl groups on the dimethylamino group of pamamycin are derived from methionine. The two new compounds obtained from the feeding experiment also had isotope peaks of 3 units larger mass at the M+ion and fragment ions x and y, but no peaks of6 units larger mass (such as m/z 599, 92 or 556) were detected. These findings confirm that the two new compounds are de-TV-methyl derivatives of pamamycin but not derivatives with the shorter alkyl side chain. We named them de-7Vmethylpamamycin-593A and B (Fig. 1). The alkyl substituents RX ~ R5 of de-jV-methylpamamycin-593A and B were identified by GC-MS analysis of the respective diol products obtained by LiAlH4 degradation of each compound2'3). The respective small diol fragments from de-Af-methylpamamycin-593A and B were identical to those obtained from pamamycin-607 and 621B by GC-MS analysis. The structures of the large diol fragments were determined by comparing their fragment ions in the MS spectra with those of large fragments obtained from known pamamycins (Table 2). De-Af-methylpamamycin-593A has the same alkyl substituent pattern as pamamycin-607 (Fig. 1). De-Af-methylpamamycin-593A and B showed aerial mycelium-inducing activity comparable to that of pamamycin-607. Their growth-inhibitory activity, however, was only half that of pamamycin. These findings support the idea3), based on the structure-activity relationship of Rt~R5 in pamamycins, that aerial mycelium-inducing and growth-inhibitory activities are produced by different mechanisms. Wenow are investigating ways to improve the yield of the de-7V-methyl derivatives using methylation inhibitors to search for dide-7V-methyl derivatives. Preparation of various compounds by chemical modification of these pamamycin derivatives will help to clarify the structure-activity relationship of the side chain and the mechanism of aerial mycelium-induction by pamamycin.