The chemical structure of tautomycin (C41H66O13)was determined by chemical degradation, spectroscopic analysis and 2D INADEQUATE of tautomycin labeled with [l,2-13C]acetate. Tautomycin exists in methanol-buffer solution (1% diethylamine-formic acid, pH7.3) as an equilibrium mixture of a 2,3-dialkylmaleic anhydride and its dicarboxylic acid in a ratio of approximately 5 : 4.In the course of screening of soil microorganisms for new antibiotics for agricultural use, a strain of Streptomyces spiroverticillatus was found to produce a new antibiotic, tautomycin (1), which has high activity against Sclerotinia sclerotiorum. Later, the antibiotic was found to induce the morphological change (bleb formation) of human leukemia cells K562, which is correlated with protein phosphorylation. The biological activity of tautomycin has been described1'2*. This paper presents detailed experimental results and discussion of the structure elucidation of tautomycin. The purified tautomycin gave two peaks on the HPLCcolumn1*. Each peak was collected and analyzed by HPLCin the same condition. Immediately after the separation, each fraction showed virtually a single peak. However, a counter peak appeared and gradually increased. Equilibrium was reached after standing over 12 hours at room temperature. The ratio of the mixture was approximately 5 : 4. HR positive FAB-MSof the pseudo molecular ion (M+Na)+ from tautomycin was recorded as m/z 789.4380, and corresponds to a formula of C41H66O13Na. FD-MS(m/z 767, (M+H)+) and the total number of carbons detected by 13C NMRspectrum confirmed the molecular formula. IR absorption bands at 1825 and 1755cm"1, and UV maximumat 250nm in CH3CNsuggested the presence of a 2,3-dialkylmaleic anhydride structure3*. IR absorption bands at 1730 and 1700cm" 1 suggested the presence of ester and ketone. XHNMRspectrum of tautomycin in DMSO-J6showed three deuterium exchangeable hydroxy protons (5.84, 4.66 and 4.46ppm). After a week in a NMRtube in DMSO-d6, two carboxyl protons at 12.5 ppm appeared. This suggests that the acid anhydride had been hydrolyzed by a trace amount of water in DMSO~d6. The quaternary carbon at 95.4ppm in 13C NMRspectrum suggests the presence of a six memberedspiroketal moiety4). Homonuclear proton spin decoupling experiment in connection with^-^C COSY, WHCOSYand heteronuclear multiple bond correlation (HMBC) spectra of 1 revealed the partial structures (A), (B), (C), (D) and (E) as shown in Fig. 1. Treatment of 1 at pH9 (20% Cs2CO3, MeOH)resulted in the hydrolysis of an ester bond, dehydration between C-21 and C-22 and gave the anhydrodeacyltautomycin 2 and an acid 3 as shown in Fig. 2. The fully-decoupled (COM) and INEPT 13C NMRspectrum and^-^C COSYspectrum of 2 indicated the presence of8xCH3, 8xCH2, 6xCH, 5xCHO, 2xCH=, 2xC=O, 1xOCO and 1xOCH3. SI-MS (m/z 567 (M+H)+) and the NMRdata of 2 indicated the molecular formula of C33H58O7accounting for three double bonds and two rings. The coupling constant (/= 16Hz) between 21-H at 6.36ppm and 22-H at