No abstract

The conformational properties of the atrial natriuretic peptide atriopeptin III were investigated by Fourier-transform infrared spectroscopy. Infrared spectra in the amide I region were analyzed quantitatively using deconvolution and band-fitting procedures. According to this analysis, in aqueous solution the monomeric peptide has a random structure. Binding to bilayer vesicles of dimyristoyl phosphatidylglycerol results in drastic conformational changes. The lipid-complexed atriopeptin III adopts a highly ordered structure of predominantly fl-sheets. A transition to a similar, but not identical, f-structure occurs upon self-association of the peptide. The results of model experiments suggest that the binding of this atrial peptide to the target cell membrane is associated with the induction of fl-sheet structure and that it is this latter conformation that is predominant in the active form of the hormone. Ser-Ser-Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-GlyAla-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe.Arg-Tyr The molecular dynamic basis for the physiological action of the atrial peptides and, in particular, the conformational properties of the peptides are, however, largely unknown. In the present communication we have used infrared spectroscopy to establish the conformation of atriopeptin III (molar ratio 10:1) in buffer, followed by rapid dispersion on a Vortex mixer. Alternatively, samples were prepared by rapidly mixing a freshly prepared peptide solution (2 mM) with a suspension of sonicated lipid vesicles. The infrared spectra of samples prepared by the two procedures were identical. Infrared spectra were recorded at 30'C with a Digilab FTS-15 instrument using a high-sensitivity mercury cadmium telluride detector. For each spectrum, 512 interferograms were added and Fourier-transformed to give a resolution of 2 cm-1. The spectra in the 1500 to 1800 cm-' region were corrected for the weak underlying 2H20 absorption. To eliminate spectral contributions of atmospheric water vapor, the instrument was continuously purged with dry nitrogen. Overlapping infrared bands were resolved using Fourier self-deconvolution procedures (4,5). Curve fitting was performed as described by Fraser and Suzuki (6). Fig. 1A shows an infrared spectrum, between 1500 and 1800 cm-1, of a freshly prepared 4 mM atriopeptin III solution in 2H20 buffer (recorded within 10 min after sample preparation). The conformation-sensitive amide I mode between 1600 and 1700 cm-' is dominated by a broad, featureless band with a maximum at 41645 cm-'. Whereas the freshly prepared solution of atriopeptin III was optically clear, prolonged incubation of the sample (at 30'C) resulted in an increase in turbidity, suggesting a time-dependent aggregation of the monomeric peptide molecules. This process was accompanied by drastic changes in the infrared spectrum. Upon aggregation of atriopeptin III, the maximum of the amide I band was shifted by =20 cm-1 to lower frequency (Fig. 1B). The process of self-association was dependent on the peptide concentration. ...

A structure (the disulfide form of cyclo-D-cysteinyl-L-valyl-D-cysteinyl-D-leucyl-L-isoleucyl), previously proposed for malformin A, was reexamined. On the basis of chemical degradations, a different structure (the disulfide form of cyclo-D-cysteinyl-D-cysteinyl-L-valyl-Dleucyl-L-isoleucyl) was established. Accordingly, a compound with this structure was synthesized and was found to be identical with malformin A. The synthetic product causes curvatures on corn roots; maximum effect was seen at a concentration of 0.1 Lg/ml, the optimal concentration for malformin A.Malformin, a metabolic product of Aspergillus niger, induces curvatures on bean plants and on corn roots. It was discovered (1-3) and isolated by Roy W. Curtis, who also demonstrated that malformin consists of a family of cyclic peptides. The best-studied member of this family, malformin A (4), is composed of two half-cystine residues, and one each of leucine, valine, and isoleucine, while other malformins contain alloisoleucine (5) or valine (6) instead of isoleucine. The configuration of the amino acid residues and their sequence was determined by Marumo and Curtis (5). Synthesis of a compound with the cyclic pentapeptide structure (Fig. 1, compound I) proposed for malformin A was reported by Isono and Curtis (7) in 1964; however, the synthetic preparation showed no malformin-like activity.Malformin was called to our attention by Prof. David Perlman of the University of Wisconsin, who pointed out that in addition to causing distortions on plants, malformins also have antibiotic activity (8) and inhibit mitosis as well (9); therefore, they are compounds of general biochemical interest. At the time when our engagement in research on malformin was under consideration, a series of papers (10-14) appeared from the laboratory of Alfons Sch6berl, describing the synthesis of compound I. The synthetic cyclopentapeptide could I I t D-Cys-Val-D-Cys-D-Leu-ile

11) Antonini, E.; Brunori, M.; Wyman, J. Biochemistry 1965, 4, 545-551. (12) Berzinis, A. Unpublished work. (13) (a) Collman, J. P.; Brauman, J. I.; Halbert, T. R.; Suslick, K. S. Proc. Natl. Acad. Sci. U S A . 1976, 73, 3333-3337. (b) Collman, J. P.; Brauman. J. I.; Doxsee, K. M.: Halbert, T. R.; Suslick, K. S. Abstract:The synthesis, characterization, and application of a poly(N-acrylylpyrrolidine) resin as an improved insoluble support for the preparation of peptides is described. The three-dimensional polymer was obtained by reverse-phase suspension copolymerization of the water-soluble monomers N-acrylylpyrrolidine, N,N'-bis(acryly1)-I ,2-diaminoethane, and N-acrylyl-I ,6-diaminohexane hydrochloride to yield a resin in beads of predetermined size. This method of polymer preparation appears to be generally applicable for water-soluble monomers. The particular resin studied contained 4.4% cross-linking, 0.7 mmol of primary amine per g of polymer and was prepared with a monomer-to-solvent dilution ratio of 1:4. The resin has favorable swelling properties in solvents covering a broad range of polarities (CHlClr, alcohols, AcOH, DMF, and H20), which allow the adaptation of most of the techniques developed for the Merrifield method of solid-phase peptide synthesis as well as the use of procedures not previously compatible with polystyrene. The feasibility of amino acid and peptide attachment and detachment has been demonstrated with three types of polymer-to-peptide bridging groups: ( I ) a new S-carbamoyl group for the side-chain attachment of cysteine was used for a high yield bidirectional synthesis of deaminooxytocin, a potent biologically active analogue of oxytocin; (2) the dinitrophenylene group for the side-chain attachment of histidine was used for the bidirectional synthesis of thyrotropin-releasing hormone; ( 3 ) the benzyl ester group for COOH-terminal attachment and, moreover, for the side-chain attachment of glutamic acid. N-Benzyloxycarbonylprolylglutamyl-05-[4-(oxymethyl)phenylacetamidopoly(N-acrylylpyrrolidine)]glycinamide was assembled bidirectionally to demonstrate the feasibility of preparing both glutamic acid and glutamine-containing peptides from the same polymer-attached peptide intermediate; saponification and ammonolysis were the respective methods of choice for the liberation of the Z-Pro-Glu-Gly-NHz and 2-Pro-Gln-Gly-NHz model peptides. Reaction conditions used for detachment are nonacidic and different from those normally used for removal of amino group protection. Coupling reactions on the resin have been demonstrated in such diverse solvents as CH2C12, DMF, and aqueous media.

DG-051B is a first-in-class small molecule inhibitor of leukotriene A4 hydrolase (LTA4H), currently in Phase II clinical development for the prevention of heart attack. Process optimization led from a linear seven-step synthetic procedure to a convergent four-step manufacturing sequence that has been used to manufacture at 100-kg scale. The entire process can be telescoped due to high conversion reactions, low impurity levels, efficient separations, and a very effective final purification. Two key aspects of the process are: (a) bypassing the isolation of a reactive electrophile by using its aqueous-washed reaction mixture directly into a coupling reaction with a phenoxide nucleophile and (b) modulating the properties of the final product solutions for optimal extraction, purification, and crystallization.