Structure of cyclothiazomycin, a novel renin inhibitor isolated from cultured broth of Streptomyces sp. NRO516, was determined to be a unique polythiazole-containing peptide related to thiostrepton and noshiheptide. Its structure including stereochemistry was established based on NOESY experiments. In the preceding paper, we reported the five structural fragments of cyclothiazomycin (1), a very unique polythiazole-containing peptide antibiotic. As summarized in Fig. 1, these fragments (A to E) consist of each one mole of glycine, L-threonine, L-proline, L-aspartic acid, four moles of L-cysteine, heterocyclic derivatives containing thiazoline and thiazole residues. The remaining problems to be solved were to prove the connectivities of five unexplained bonds A to E (one of them being ascribed to a free carboxylic acid) and to define the absolute stereochemistries at two chiral centers of the unusual amino acid analogs (Q-7 in fragment D and Pyr-2 in fragment E). A NOESY experiment in CD3OH-H2O (9:1) (observed NOEs are illustrated by arrows in Fig. 1) enabled us to connect these five fragments. Fragments A and D were combined each other through Q-7NH, which showed NOEs to protons of the proline moiety in fragment A. Similarly, fragments C and D were linked through the Asn-1NH based on its NOEs to protons on the substituted pyridine moiety. The bonds between fragments C and E, and fragments A and E were also determined based on the NOEs from the amide protons as shown by the arrows in Fig. 1. Since the presence of a ketone in 1 was excluded by the 13C-NMR spectral data (no signal below 180 ppm), terminal E was assigned to a free carboxylic acid. The information about the connectivity of fragment B to other fragments was obtained from the structures of the degradation products 2 and 3 prepared by hydrolysis of 1. Since 2 was reasonably assumed to be derived from fragment B, the terminal C should be a masked carboxylic acid function. Similarly the structure of 3 suggested that the terminal A in fragment D was also a carboxylic acid equivalent. These evidences required that fragment D was connected to fragment B through a bond between terminal A and B, and consequently fragment B was linked to fragment E through a bond between C and D. Therefore, the structure of 1 was established to be a unique polythiazole-containing bicyclic peptide as shown in Fig. 4. In order to determine the absolute configurations of two chiral centers (Pyr-2 and Q-7), use was made of conformational analysis mainly based on NOESY experimental data (see Fig. 2). At first, the temperature dependency of the 1H chemical shifts over the range 30°C to 60°C were measured. Most of non-labile protons of 1 were slightly temperature dependent except for relatively large temperature dependency observed with Pro-4CH2 (ca. 5.0 ppb/°C downfield), Pro-3CH (ca. 5.0 ppb/°C downfield), Q-7H (ca. 5.0 ppb/°C upfield), Deala-2Hc (ca. 5.6 ppb/°C downfield), Deala-3Ht (ca. 3.3 ppb/°C downfield) and Thz(1)-SH (ca. 5.0 ppb/°C downfield). No significant change in nature of the proton spin couplings suggested that the conformational change of 1 could be neglected over this temperature range. The chemical shift changes of the amide resonances with temperature are known to be related to their ability to participate in intramolecular hydrogen-bonding. Generally, temperature gradients exceeding 4 ppb/°C are considered to be an evidence for an external NH orientation, while values smaller than 2 ppb/°C are indicative of intramolecular hydrogen bonding. The amide proton of Gly was almost unaffected by changing temperature (0.0 ppb/°C). Taking into consideration of the NOE data, this proton was deduced to be hydrogen-bonded to CO of But(2) (But(2)-1) as part of a β-loop (Fig. 2). Similarly, one of the amide protons of the Asn side chain (Asn3NH2) was also unaffected by the change of temperature (1.0 ppb/°C) suggesting the formation of hydrogen-bonding with the nearby carbonyl group (Q-9). All other amide proton shifts exhibited slopes explaining their external orientation. Four amide protons (Asn-NH, Q-NH, Gly-NH and Thr-NH) were coupled with their neighboring α-proton; other seven amides being observed as broad singlets in both the solvents (CD3OH-H2O and DMF-d7). The observed coupling constants (3JHN-CH; 6.0 Hz, d, for Asn, 5.0 Hz, d, for Q, 4.0 Hz, t, for Gly and 9.0 Hz, d, for Thr) were depended on their possible dihedral angles and gave information about the backbone conformation. The peptide ring, cycle-(Q-Asn-Cys(2)-But(2)-Pyr-Thr-But(1)-Pro), was concluded to have relatively rigid conformation by taking into consideration of the presence of a β-loop [hydrogen bonding between Gly-NH and But(2)-CO] and NOE contacts, and by assuming normal configurations for the amide bonds except for the cis But(1)-Pro amide bond (Fig. 3). The NOEs used to deduce the above peptide conformation were, i) NOE from Asn-1NH to Gly-2H and Q-7H, ii) NOE between Asn-1H and Q-7H, iii) NOE from Gly-2H to Q-7H, iv) NOE between Gly-2NH and But(1)-4CH3, and v) NOE from But(2)-3H to Pyr-3CH3 and Cys(4)-3H. The configurations at Q-7 and Pyr-2 were established based on this rigid conformation of the peptide backbone. The following three additional NOEs could only be accounted for by assuming the R configuration at Q-7; i) although NOE was observed between Gly-2H and Q-7H, no NOE was observed between Gly-2H and Q-8CH3, ii) Asn-1NH showed strong NOE to Q-7H with weak NOE to Q-8CH3, and iii) weak NOE existed between Q-8CH3 and But(2)-4CH3. Similarly the S configuration at Pyr-2 was established from the following results; i) Thr-4CH3 showed strong NOE to Pyr-3CH3 but not to Cys(4)-3H, and ii) strong NOEs existed from But(2)-3H to Cys(4)-2H and Cys(4)-3H. Based on the results described above, the stereostructure of cyclothiazomycin was established as shown in Fig. 4. It is a bicyclic peptide and structurally related to thiostrepton and noshiheptide. Since cyclothiazomycin exhibited inhibitory activity against renin, it may be interesting to investigate the renin inhibitory activity of other polythiazole-containing peptides.