The all-light-atom X-ray crystal-structure analysis of cyclosporin A (l), a cyclic undecapeptide containing seven N-methylated amino acids, reveals a conformation very similar to that of the previously analysed iodo derivative which is characterised by a twisted P-pleated sheet involving the residues Me-Val-1 1, MeBmt-1, Abu-2, Sar-3, MeLeu-4, Val-5, MeLeu-6, and Ala-7. The@-bend at Sar-MeLeu-4 is of type II', and the loop of the residual amino acids involves a cis-peptide bond between MeLeu-9 and MeLeu-10. The NH proton of D-Ala-8 closes a $-bend with a H-bond to the MeLeu-6 CO group. The crystal was grown from acetone. A closely similar backbone conformation in apolar solvents such as CDCl, or C6D6 has been derived from the interpretation of the NMR spectral parameters (homo-and heteronuclear NOE effects, coupling constants, chemical-shift values of the C-, H-, and N-atoms). A minor variation in the backbone conformation between crystal and solution is observed in the region of o-Ala-8, where in solution a 3-center H-bond is established between the NH of D-Ala-8 und the carbonyl 0-atoms of both MeLeu-6 ($-turn) and o-Ala-8 (C5-bend). A recently proposed technique to identify intramolecular H-bond via heteronuclear NOE from NH proton to carbonyl C-atoms is critically analysed. The main difference between crystal and solution conformations lies in the orientation of the side chains of the unusual amino acid MeBmt kl = f60" in solution, -168" in the crystal) and of MeLeu-10 k1 = -60" in solution, +60' in the crystal). The differences in crystal and solution are caused by the break ofthe intermolecular H-bond of the OH group of MeBmt on dissolution of the crystal. The bifurcated H-bond ofo-Ala-8 twists the backbone in this region. Molecular modeling demonstrates that this is the origin of the change in the side chain conformation of MeLeu-10. The intramolecular flexibility in the crystal indicated by the thermal parameters obtained from the X-ray refinement, and in solution by an analysis of spin-lattice relaxation times in the NMR experiments, indicate a fairly rigid backbone and fixed conformations for all the side chains except for that of Abu-2 and the distal atoms of MeBmt.

Cyclosporin A, a Peptide Metabolite from Tvichoderrna polysporum (LINK ex PERS.) Rifai, with a remarkable immunosuppressive activity. -Summary. Two antifungal metaboliies, cyclosporins A and C, were isolated from a strain of Trichoderma polysporum (LINK ex PERS.) Rzfai. Cyclosporin A, the main component, has the molecular formula C,jzHlllNnOlz as deduced by NMR. and mass spectra. Hydrolytic cleavage of cyclosporin A furnished 11 amino acids as fragments, among them an artefact of a new Cg-amino acid. The amino acid sequence was determined by Edman degradation of iso-cyclosporin A, a basic rearrangement product formed from cyclosporin A by N , O-acyl migration. In an independent investigation an X-ray analysis of the iodo derivative 10 was performed (see the following paper). On the basis of the chemical, spectroscopic and crystallographic evidencc the complete structure of cyclosporin A was elucidated as the neutral cyclic oligopeptide 1. Cyclosporin A and C show remarkable immunosuppressive and antiphlogistic activities in several pharmacological models.Aus Kulturen eines neuen Stammes der Gattung Trichoderma (fungi imperfect;) wurde ein Gemisch von Metaboliten isoliert, die durch antifungische Aktivitaten auffielen. Der neue Pilzstamm, der aus einer bei Hardanger, Norwegen, gesammelten Bodenprobe erhalten wurde, liess sich aufgrund seiner wesentlichen morphologischen Merkmale und des Wachstums auf verschiedenen Nahrboden als Trichoderma polysporum (LINK ex PERS.) Rifai bestimmenl) 2). Zur Isolierung der aktiven Stoffwechselprodukte wurden Kulturbriihen des Pilzes mit n-Butylacetat ausgeriihrt und der Extrakt durch Verteilen zwischen 90proz. Methanol und Petrolather entfettet. Chromatographie des rohen Gernisches an Kieselgel fiihrte zu einem angereicherten Wirkstoffkomplex. Weitere Chromatographien an Sephadex LH-20 und an neutralem Aluminiumoxid lieferten schliesslich zwei einheitliche Komponenten, die als Cyclosporin A und C bezekhnet wurden3). Die zwei Metaboliten scheinen strukturell nahe verwandt zu sein ; sie lassen sich mittels Dunnschichtchromatographie auf ((Polygram Sil G D gut voneinander unterscheiden. Zur Reinheitspriifung ist auch die Hochdruckfliissigkeits-Chromatographie (4Reversed-phase a-Methodik) geeignet.

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The 1H‐, 13C‐, and 15N‐NMR spectra of the immunosuppressive cyclic undecapeptide cyclosporin A (1) have been analyzed at 300 MHz in CDCl3, C6D6, and mixtures of these solvents. A combination of different homo‐ and heteronuclear two‐dimensional NMR techniques enable complete assignment of all H‐, C‐ and 4 N‐signals. Recognition of the proton spin systems has been achieved via 1H,1H–COSY and double‐quantum‐1H‐NMR spectroscopy. NOESY spectra yield some sequence assignments, but two techniques using coupling across amide bonds have been applied to get independent assignments of all amino acids in the sequence: (i) An 1H,1H‐COSY spectrum optimized for small coupling constants enables the detection of long‐range couplings from N‐methyl groups to both α‐protons attached to that amide bond. (ii) An 1H, 13C‐COSY spectrum optimized for C,H‐long‐range couplings (J = 5 to 10 Hz) to the eleven CO groups again yields coupling to both α‐protons attached to that amide bond. Additionally these two experiments yield the assignment of N‐methyl protons and carbonyl C‐atoms. Normal and relayed 1H,13C‐COSY in both solvents have been applied to assign all C‐atoms via their directly attached and remote protons. An 1H,13C‐COLOC spectrum at 500 MHz in CDCl3, which uses H,C‐long‐range couplings confirms the assignment of all proton spin systems as well as the C‐signals of each individual amino acid. Ambiguities in the assignment of the C(δ)'s of MeLeu have thus been removed. An 1H,15N‐COSY spectrum enables the assignment of the 4 NH N‐atoms.