L-a-Aminobutyric acid

Cyclosporin A contains II amino acids, joined in a cyclic strncture by peptide bonds. The structure is also stabilized by intramolecular hydrogen bonds. Only two of the amino acids, i.e. alanine and valine, are typical of proteins. The compound contains several A-methylated amino acid residues, together with the even less common L-a-aminobutyric acid and an Ai-methylated butenylmethylthreonine. There is one o-amino acid, i.e. o-alanine, and the assembly of the polypeptide chain is known to start from this residue. Many of the other natural cyclosporin structures differ only with respect to a single amino acid (the a-aminobutyric acid residue) or the number of amino acids that have the extra Ai-methyl group. 

The second major metabolite from T. inflation is structurally closely related to cyclosporin A, as can be deduced by elemental analysis, mass spectrum (m/z 1217), IR and NMR spectra. Furthermore, the presence of the double bond and OH group of the unusual MeBmt was established. Sulphonic acids in methanol or dioxane effected the typical rearrangement reaction by N, O-acyl migration to the iso-compound (13). Hydrolysis furnished the same amino acids as cyclosporin A with the exception of L-a-aminobutyric acid, which is replaced in cyclosporin C (12) by L-threonine. The amino-acid sequence could be deduced by conversion of cyclosporin C into cyclosporin A via the corresponding tosylate (14) and iodo derivatives (15) [7]. Position 2 for L-threonine as well as the assumed twisted -pleated sheet conformation of the molecule were confirmed by 13C-NMR spectra. 

A non-ribosomal biosynthetic pathway is clearly indicated for cyclosporin A, considering the uncommon structural elements MeBmt, L-a-aminobutyric acid and D-alanine as well as the plethora of isolated congeners [20,21]. Non-ribosomal biosynthesis directed by multienzyme thiotemplates have been reported for other small peptides of microbial origin, for example, gramicidin S [22] and enniatin [23]. Experimental data for cyclosporin A were obtained by feeding appropriate labelled precursors to cultures of T. inflation strains. The distribution profile of the labelled atoms in cyclosporin A was determined by 3H- or 13C-NMR spectroscopy. In preliminary trials with several tritium and carbon-14 labelled precursors, [met/y>/-3H]methionine proved to be the most suitable marker for the biosynthetic preparation of radiolabelled cyclosporin A for pharmacokinetic and metabolic studies [24],... 

By contrast, substitution of L-a-aminobutyric acid in position 2 by other amino acids is quite well tolerated, as proven by the high potency of cyclosporins C (LThr2) and G (LNva2). However, shortened or branched side-chains of the substituting amino acid, as in cyclosporin B (iAla2) and D (LVal2), may diminish the immunosuppressive efficacy. The marked decrease in activity of the synthetic [Ser2]cyclosporin A can be explained by a partially disturbed interaction with the cyclosporin receptor. 

Figure 2 Order and organization of enniatin synthetase and cyclosporin synthetase as deduced from gene sequence and biochemical characterization. Symbols in the adenylateforming modules (black boxes) indicate the corresponding activated amino acids. M stands for A -methyltransferase domain. Condensation domains are represented by white boxes. (A) Top Structure of enniatin synthetase. EA represents the D-Hiv-activating module EB represents the L-valine-activating module D-Ehv is D-2-hydroxyisovaleric acid. Bottom Structural features of the wild-type A -methyltransferase domain M of esynl. The black boxes indicate conserved motifs which can be found within methyltransferases and A -methyltransferase domains of peptide synthetases (see also Fig. 3). The numbers indicate the amino acid position in the sequence of Esyn. (B) Structure of cyclosporin synthetase. Abu = L-a-aminobutyric acid Bmt = (4A)-4-[(E)-2-butenyl]-4-methyl-L-threonine.

Cyclosporins, produced by the filamentous fungus Tolypocladium niveum and by numerous strains of Fusaria and Neocosmospora, are a class of cyclic undecapep-tides which are composed of hydrophobic aliphatic amino acids [73-75], They exhibit antiinflammatory, immunosuppressive, antifungal, and antiparasitic properties [74], The main metabolite, cyclosporin A, is in clinical use worldwide under the trade name SANDIMMUN to prevent allograft rejection [77,78], Besides cyclosporin A, there are 24 naturally occuring cyclosporins which have substitutions of amino acids in positions 1, 2, 4, 5, 7, and 11 and/or contain unmethylated peptide bonds in positions 1,4,6,9,10, or 11 [79-82], Cyclosporin A contains three nonproteinogenic amino acids D-alanine in position 8, L-a-aminobutyric acid in position 2, and, in position 1, the unusual amino acid 4(R)-4-[(E)-2-butenyl]-4-methyl-L-threonine (Bmt) (Fig. 8). All three amino acids have to be synthesized by a pathway independent of the primary metabolism. In addition, several peptide bonds of the cyclosporin molecule are A -mcthylated similar to the depsipeptides enniatin, beauvericin and PF1022A-related peptides. 

Figure 8 Structure of cyclosporin A. Abu = L-a-aminobutyric acid Bmt = (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine Sar = sarcosine (A-methylglycine).

Dimethyldithiocarbamates are metabolised by fungi and bacteria to y-N,N-dimethylthio-(carbamoylthio)-L-a-aminobutyric acid (56) (Kaslander, 1966). 

L-a-aminobutyric acid, D-a-aminobutyiic acid, L-a,y-diamino-butyric acid... 

The occurrence of numerous secondary metabolites and the unusual structural elements l-a-aminobutyric acid, D-alanine, and the C9-amino acid (2S,3/(,4/(,6 )-2-amino-3-hydroxy-4-methyl-6-octenoic acid [(4/ )-4-(( )-2-butenyl)-4-methyl-L-threonine=Bmt] indicate that this peptide is not produced ribosomally but rather post-translationally by a multi-enzyme complex. Additionally offered foreign amino acids are incorporated, e.g., further cyclosporins are obtained in directed biosyntheses by replacement of the a-aminobutyric acid in position 2 by D- or L-allylglycine, of d-alanine in position 8 by D-setine or 3-fluoroalanine, and of butenylmethylthreonine in position 1 by L-/S-cyclohexylalanine... 

The cyclic 11-peptide contains mainly normal amino acids, one D-alanine and one L-a-aminobutyric acid, but, strikingly, not less than seven iV-methyl groups and a quite uncommon amino acid, a C9-compound (4R)-4[(F)-2-butenyl]-4-methyl-N-methyl-L-threonine. 

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