Studies of peptide antibiotics. XLIV. Syntheses and biological activities of gramicidin S analogs containing delta-hydroxy-L-norvaline or glycine

The antibiotic gramicidin S (GS) has the structure of cyclo (-L-Val1-L-Orn2-L-Leu3-D-Phe4-L-Pro5-L-Val1'-L-Orn2'-L-Leu3'-D-Phe4'-L-Pro5'-) and is basic in character. Five GS analogs including [Gly1,1']-GS and the neutral [L-Hnv2,2']-GS (Hnv represents delta-hydroxynorvaline) were synthesized by the solid-phase method to evaluate the role of L-Val1,1' and L-Orn2,2' residues in GS. The hybrid analogs [( Gly1]-GS and [L-Hnv2]-GS) and [D-Tyr4,4']-GS showed high antibacterial activities, whereas [Gly1,1']-GS and [L-Hnv2,2']-GS possessed no activity. Inhibitory effects by these analogs for the adsorption of 14C-labeled GS on cells of bacteria sensitive to GS were determined. The structure-activity relationship of GS is discussed on the basis of the results on these GS analogs.     

Studies of peptide antibiotics. XLVI. Syntheses of gramicidin S analogs containing D-alpha,beta-diaminopropionic acid or alpha,beta-dehydroalanine

A gramicidin S (GS) analog ([D-Dpr4,4'] GS) containing D-alpha,beta-diaminopropionic acid (D-Dpr) in place of D-Phe at 4,4' positions was derived from [L-Orn(delta-formyl)2,2', D-Dpr(beta-Z)4,4']GS, which was synthesized by conventional method in solution. An analog [delta Ala4,4']GS was synthesized from [L-Orn(delta-Boc)2,2', D-Dpr4,4']GS through Hofmann degradation of the D-Dpr residues. Antimicrobial activities of these analogs were tested; [D-Dpr(beta-Z)4,4']GS and [delta Ala4,4']GS showed high antimicrobial activities against Gram-positive bacteria. [D-Dpr4,4']-GS showed an appreciable activity against Gram-negative bacteria such as Escherichia coli. Four semigramicidin S (semiGS) analogs such as [delta Ala4]semiGS were synthesized; these had no antimicrobial activity. Analogs containing delta Ala residues were hydrogenated, and the formation of L-Ala or D-Ala residues was determined. The delta Ala residues in [delta Ala4,4'] GS were reduced to DL-Ala, and delta Ala in [delta Ala4]semiGS mostly to L-Ala. The relationships of the antimicrobial activity, CD curves and asymmetric hydrogenation to the structure were discussed.     

Cyclic peptides. VI. Asymmetric hydrogenation of dehydroalanine or dehydroaminobutanoic acid residue in cyclodipeptides.

Several cyclo(-L-aminoacyl-deltaAla-) (aminoacyl = Ala, Val, Leu, Phe, Pro and Lys (epsilon-Ac)) were prepared by tosylation and successive detosylation of cyclo(-L-aminoacyl-L-Ser-), which were synthesized via the Nitecki and Fischer methods. Hydrogenation of the double bond of dehydroalanine residues in cyclodipeptides was carried out using Pd black in methanol at 1-atm pressure and room temperature. The degree of asymmetric hydrogenation was assessed by determining the amounts of L- and D-alanine by a modified Manning and Moore procedure. When L-valine was used as a chiral source, L-alanine residue with chiral induction of 98.4% was derived from cyclo(-L-Val-deltaAla-). L-Amino acids other than L-proline also were effective in inducing remarkable asymmetric hydrogenation. Hydrogenation of alpha,beta-dehydro-alpha-aminobutanoic acid residues in cyclodipeptides produced L-alpha-aminobutanoic acid residues with effective chiral induction to the same extent as observed with dehydroalanine residues. Optically pure l-alanine was prepared from cyclo(-L-Lys(epsilon-Ac)-deltaAla-) via asymmetric hydrogenation. A mechanism of chiral induction is discussed.     

Comparison of conformation and antimicrobial activity of synthetic analogs of gramicidin S: Stereochemical consideration of the role of D-phenylalanine in the antibiotic.

In order to study the role of D -amino acid residues in keeping the stable β-sheet conformation and in the antimicrobial activity of gramicidin S (GS), the four analogs of GS containing D -Ala, L -Ala, Gly, and Aib (α-aminoisobutyric acid) in place of D -Phe were synthesized. D -Ala-and Gly-containing analogs showed antimicrobial activity, while those containing L -Ala and Aib showed no activity. Conformation of these analogs and their derivatives were studied by comparison of ORD and CD spectra and by slective methylation method. It is concluded that the biologically active analogs have β-sheet conformation while inactive analogs have a much different conformation from that of GS. This indicates that D -Ala-Pro and Gly-Pro sequences favor taking a β-bend form but L -Ala-Pro and Aib-Pro sequences do not because the presence of L -side methyl group on the α-carbon atom of L Ala and Aib residues destabilizes the β-bend form. This would explain why the inactive analogs which take a different conformation from that of the active ones result in the loss of activity.     

Syntheses of two trimannose analogs each containing C-mannosyl or 5-thio-C-mannosyl residue: their affinities to concanavalin A

C-Mannosyl residue-containing trimannose ManC alpha(1,6)[Man alpha(1,3)Man] (2) and 5-thio-C mannosyl residue-containing trimannose 5SManC alpha(1,6)[Man alpha(1,3)Man] (3) were synthesized via a glycosyl radical addition to enone derivative of mannose (6). Dissociation constants for the binding of these trisaccharides to concanavalin A (ConA) were determined by a fluorescence anisotropy inhibition assay: Kd = 198 and 31 microM, respectively. The unexpectedly large Kd value for the compound 2 compared with the compound 3 and the natural trimannose 1 demonstrates a characteristic of C-glycoside.     

Studies of peptide antibiotics. XLI. Syntheses of 6-L-valine-tyrocidine A and 7-L-ornithine-tyrocidine A

Tyrocidine A (TA) is an antibiotic cyclic decapeptide with the sequence of cyclo (-L-Val1-L-Orn2-L-Leu3-D-Phe4-L-Pro5-L-Phe6-D-Phe7-L-Asn8-L-Gln9-L-Tyr10-). Gramicidin S (GS) regarded as a homolog of TA is also a cyclic decapeptide with the sequence of cyclo (-L-Val1-L-Orn2-L-Leu3-D-Phe4-L-Pro5-L-Val5-L-Orn7-L-Leu8-D-Phe9-L-Pro10-). GS shows higher antibacterial activity, whereas TA exhibits inhibitory activity on the biosynthesis of RNA. Two analogs of TA, [L-Val6]-TA (12a) and [L-Orn7]-TA (12b), were synthesized by the conventional method in order to study the interrelationships between the two related antibiotics TA and GS. Antibacterial activities of 12a and TA are nearly the same, but the activity of 12b is significantly lower. The optical rotatory dispersion spectra of 12a, 12b, and TA showed a trough at 233 nm region; the troughs of 12a and TA are nearly the same in depth, but the trough of 12b is shallower. Relationships between structure and activity of 12a and 12b compared with TA and GS were discussed.     

Gramicidin S analogs with a D-Ala, Gly, or L-Ala residue in place of the D-Phe residue: molecular conformations and interactions with phospholipid membrane

The proton nmr and CD spectra of gramicidin S (GS) cyclic-(Val^1,1′-Orn^2,2′-Leu^3,3′-D-Phe^4,4′-Pro^5,5′)₂ and of GS analogs—namely, [D-Ala^4,4′]-GS, [Gly^4,4′]-GS, and [L-Ala^4,4′]-GS—were analyzed. The molecular conformation of [D-Ala^4,4′]-GS is similar to that of GS, with the trans form about the D-Ala-Pro peptide bond. The molecular conformation of [Gly^4,4′]-GS depends on the solvent composition of dimethylsulfoxide-d₆/trifluoroethanol (DMSO)-d₆/TFE and DMSO-d₆/H₂O as well as the solute concentration. In DMSO-d₆ solution, [Gly^4,4′]-GS forms the GS-type conformation of the monomer at lower concentration. At higher concentration, the GS-type conformer is converted to the other one that forms molecular aggregates. The cis form about the X-Pro peptide bonds is found for [Gly^4,4′]-GS and [L-Ala^4,4′]-GS in DMSO-d₆ and for [L-Ala^4,4′]-GS in TFE solution. The large temperature dependences of α-proton chemical shifts of [L-Ala^4,4′]-GS in DMSO-d₆ solution indicate that the conformer equilibrium changes with temperature. The GS-type conformation is not formed in [L-Ala^4,4′]-GS. The two active peptide analogs, [D-Ala^4,4′]-GS and [Gly^4,4′]-GS, interact with the phospholipid membrane, taking the GS-type conformation. By contrast, an inactive analog, [L-Ala^4,4′]-GS, does not interact with phospholipid membrane. The activities of GS analogs are found to correlate to the formation of the GS-type conformation upon binding with phospholipid membrane.     

DNA-supercoiling is affected in vitro by the peptide antibiotics tyrocidine and gramicidin

Tyrocidine, a peptide antibiotic produced by Bacillus brevis (ATCC 8185), relaxes superhelical DNA in a biphasic manner and induces 'packaging' of the DNA at higher concentrations. This was concluded from studies using the sensitive 4,5',8-trimethylpsoralen photobinding technique [Sinden, R. R., Carlson, J. O. & Pettijohn, D.-E. (1980) Cell 21, 773-783]. Relaxed DNA is not affected by tyrocidine whereas linearized molecules become packaged. The linear gramicidin synthesized by the same strain reverses the tyrocidine-induced relaxation as well as the packaging, an observation which might be of biological relevance.     

Syntheses and biological activities of sandostatin analogs containing stereochemical changes in positions 6 or 8

In a continuation of our research efforts on the design and synthesis of novel peptidomimetic structures, we have synthesized a series of sandostatin amide analogs in which stereoisomers of threonine and beta-hydroxyvaline(beta-Hyv) are employed. The analogs D-Phe1-c[Cys2-Phe3-D-Trp4-Lys5-Xaa6-Cys 7]-Xbb8-NH2 (Xaa = allo-Thr, D-allo-Thr, D-beta-Hyv, beta-Hyv, D-Thr, and Xbb = Thr or Xaa = Thr and Xbb = allo-Thr, D-allo-Thr, beta-Hyv, D-Thr) explore the effects on biological activity of stereochemical modifications and beta-methylation at positions 6 or 8. By these modifications, we examine the role of the two residues in binding to somatostatin receptors. We describe the synthesis and biological activity of these analogs. In combination with the results of the conformational analysis, this study provides new insights into the structural requirements for the binding affinity of somatostatin amide analogs to somatostatin receptors [Mattern et al., Conformational analyses of sandostatin analogs containing stereochemical changes in positions 6 or 8].     

Organization of the biosynthesis genes for the peptide antibiotic gramicidin S.

A recombinant bacteriophage containing the intact Bacillus brevis gene for gramicidin S synthetase 1, grsA, and the 5' end of the gramicidin S synthetase 2 gene, grsB, was identified by screening an EMBL3 library with anti-GrsA antibodies. This clone, EMBL315, has a 14-kilobase (kb) insert that hybridizes to the previously isolated 3.9-kb fragment of the grsB gene, which encodes the 155-kilodalton ornithine-activating domain of gramicidin S synthetase 2. Deletion and subcloning experiments with the 14-kb insert located the grsA structural gene and its putative promoter on a 4.5-kb PvuII fragment which encoded the full-length 120-kilodalton protein in Escherichia coli. In addition, hybridization analysis revealed that the 5' end of the grsB gene is located approximately 3 kb from the grsA structural gene. Furthermore, these studies indicated that grsA and grsB are transcribed in opposite orientations.     

Transmembrane channel activity of gramicidin A analogs: effects of modification and deletion of the amino-terminal residue.

The relationship between the nature of the side-chain at position 1 and the activity of the gramicidin A channel was investigated. The N-formyl group was removed, one round of an Edman degradation was carried out, and a new N-formyl residue was inserted in place of N-formyl-l-valine. The conductance properties of semisynthetic gramicidins containing N-formyl derivatives of d-valine, desvaline, glycine, cysteine, S-methyl-l-cysteine, S-carboxamidomethyl-l-cysteine, S-(spin-labeled)-l-cysteine, p-iodo-l-phenylalanine, and p-fluoro-l-phenylalanine were investigated. We found that the nature of the side-chain at position 1 markedly affects channel activity. A highly hydrophobic l-residue is required.     

Exploring relationships between mimic configuration, peptide conformation and biological activity in indolizidin-2-one amino acid analogs of gramicidin S.

Indolizidin-2-one amino acids (I2aas, 6S- and 6R-1) possessing 6S- and 6R-ring-fusion stereochemistry were introduced into the antimicrobial peptide gramicidin S (GS) to explore the relationships between configuration, peptide conformation and biological activity. Solution-phase and solid-phase techniques were used to synthesize three analogs with I2aa residues in place of the d-Phe-Pro residues at the turn regions of GS: [(6S)-I2aa4-5,4'-5']GS (2), [Lys2,2',(6S)-I2aa4-5,4'-5']GS (3) and [(6R)-I2aa4-5,4'-5']GS (4). Although conformational analysis of [I2aa4-5,4'-5']GS analogs 2-4 indicated that both ring-fusion stereoisomers of I2aa gave peptides with CD and NMR spectral data characteristic of GS, the (6S)-I2aa analogs 2 and 3 exhibited more intense CD curve shapes, as well as greater numbers of nonsequential NOE between opposing Val and Leu residues, relative to the (6R)-I2aa analog 4, suggesting a greater propensity for the (6S)-diastereomer to adopt the beta-turn/antiparallel beta-pleated sheet conformation. In measurements of antibacterial and antifungal activity, the (6S)-I2aa analog 2 exhibited significantly better potency than the (6R)-I2aa diastereomer 4. Relative to GS, [(6S)-I2aa4-5,4'-5']GS (2) exhibited usually 1/2 to 1/4 antimicrobial activity as well as 1/4 hemolytic activity. In certain cases, antimicrobial and hemolytic activities of GS were shown to be dissociated through modification at the peptide turn regions with the (6S)-I2aa diastereomer. The synthesis and evaluation of GS analogs 2-4 has furnished new insight into the importance of ring-fusion stereochemistry for turn mimicry by indolizidin-2-one amino acids as well as novel antimicrobial peptides.     

A method for the facile solid-phase synthesis of gramicidin S and its analogs

Summary A simple method is described for the facile synthesis of gramicidin S and six other analogs, using standard solidphase synthetic technology and a single solution-phase cyclization step. The peptides were purified to homogeneity and characterized by plasma desorption time-of-flight mass spectrometry and NMR spectroscopy. Complete ¹H NMR assignments for all seven peptides (in aqueous solution) are presented. Unlike previous approaches, the presented method is simple, automatable, rapid (less than three days), high-yielding, requires no side-chain protection during cyclization, and appears to be generally applicable to the preparation of a variety of related head-to-tail cyclic peptides.Abbreviations Boc t-butyloxycarbonyl - BOP benzotriazoyl N-oxytris(dimethylamino)phosphonium hexafluorophosphate - Bzl benzyl - DCC N,N-dicyclohexylcarbodiimide - DCM dichloromethane - DIEA N,N-diisopropylethylamine - DMF N,N-dimethylformamide - DQF-COSY double-quantum-filtered correlation spectroscopy - DSS 2,2-dimethyl-2-silapentane-5-sulfonate, sodium salt - EDAC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide - HBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate - HOBt 1-hydroxybenzotriazole - 4-MeBzl 4-methylbenzyl - NHS N-hydroxysuccinimide - NOESY nuclear Overhauser effect spectroscopy - PAM phenylacetamidomethyl (resin) - RP-HPLC reversed-phase high-performance liquid chromatography - TFA trifluoroacetic acid - TOCSY total correlation spectroscopy - Tos tosyl - Troc 2,2,2-trichloroethylcarbamate.     

The effects of ring-size analogs of the antimicrobial peptide gramicidin S on phospholipid bilayer model membranes and on the growth of Acholeplasma laidlawii B.

We have examined the effects of three ring-size analogs of the cyclic beta-sheet antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior and permeability of phospholipid model membranes and on the growth of the cell wall-less Gram-positive bacteria Acholeplasma laidlawii B. These three analogs have ring sizes of 10 (GS10), 12 (GS12) or 14 (GS14) amino acids, respectively. Our high-sensitivity differential scanning calorimetric studies indicate that all three of these GS analogs perturb the gel/liquid-crystalline phase transition of zwitterionic phosphatidylcholine (PtdCho) vesicles to a greater extent than of zwitterionic phosphatidylethanolamine (PtdEtn) or of anionic phosphatidylglycerol (PtdGro) vesicles, in contrast to GS itself, which interacts more strongly with PtdGro than with PtdCho and PtdEtn bilayers. However, the relative potency of the perturbation of phospholipid phase behavior varies markedly between the three peptides, generally decreasing in the order GS14 > GS10 > GS12. Similarly, these three GS ring-size analogs also differ considerably in their ability to cause fluorescence dye leakage from phospholipid vesicles, with the potency of permeabilization also generally decreasing in the order GS14 > GS10 > GS12. Finally, these GS ring-size analogs also differentially inhibit the growth of A. laidlawii with growth inhibition also decreasing in the order GS14 > GS10 > GS12. These results indicate that the relative potencies of GS and its ring-size analogs in perturbing the organization and increasing the permeability of phospholipid bilayer model membranes, and of inhibiting the growth of A. laidlawii B cells, are at least qualitatively correlated, and provide further support for the hypothesis that the primary target of these antimicrobial peptides is the lipid bilayer of the bacterial membrane. The very high antimicrobial activity of GS14 against the cell wall-less bacteria A. laidlawii as compared to various conventional bacteria confirms our earlier suggestion that the avid binding of this peptide to the bacterial cell wall is primarily responsible for its reduced antimicrobial activity against such organisms. The relative magnitude of the effects of GS itself, and of the three ring-size GS analogs, on phospholipid bilayer organization and cell growth correlate relatively well with the effective hydrophobicities and amphiphilicities of these peptides but less well with their relative charge density, intrinsic hydrophobicities or conformational flexibilities. Nevertheless, all of these parameters, as well as others, may influence the antimicrobial potency and hemolytic activity of GS analogs.     

Induction of sporulation in Bacillus brevis. 2. Dependence on the presence of the peptide antibiotics tyrocidine and linear gramicidin

This paper presents evidence that the two peptide antibiotics tyrocidine and linear gramicidin, produced by Bacillus brevis ATCC 8185, are required for the induction of sporulation in the producer organism. When tyrocidine synthesis was specifically blocked with 2-amino-3-hydroxy-3-phenylpropanoic acid [Mach, B., Reich, E., and Tatum, E. L. (1963) Proc. Natl Acad. Sci. USA, 50, 175-181], sporulation and gramicidin synthesis were inhibited, but both processes could be restored by the addition of tyrocidine. Certain other amino acids such as L-tyrosine inhibited both sporulation and peptide antibiotic synthesis in nitrogen-limited cultures. When either tyrocidine or linear gramicidin was added together with L-tyrosine, neither sporulation nor peptide antibiotic synthesis was restored. On the other hand, the addition of both tyrocidine and linear gramicidin effectively reversed the inhibition of sporulation by L-tyrosine. These experiments demonstrate that sporulation of B. brevis depends on either the endogenous synthesis or the addition of both tyrocidine and linear gramicidin. The fact that endogenous as well as exogenous peptides could effect sporulation argues against the involvement of artifacts, such as the depletion of intracellular nucleotide pools caused by the surfactant properties of added peptide antibiotics.     

Cystamine-enkephalin dimer. Syntheses and biological activities of enkephalin analogs containing cystamine and cysteamine

A cystamine-enkephalin dimer, containing two molecules of [D-Ala2, Leu5] enkephalin cross-linked at the COOH-terminal leucine residue with cystamine, (NH2-CH2-CH2-S-)2, has been synthesized in order to examine directly the dimerization effect of an enkephalin molecule on the opiate receptor interactions. In a comparison of potencies against [3H]-[D-Ala2,D-Leu5] enkephalin (3H-DADLE) and [3H]-[D-Ala2,MePhe4,Gly-ol5] enkephalin (3H-DAGO) as delta and mu tracers, respectively, enkephalin dimer showed a very high affinity, especially for the delta opiate receptors. Dimer was almost threefold more potent than DADLE, which is one of the most utilized delta ligand to date. When the binding affinity of cystamine-dimer was compared with that of its reduced thiol-monomer, namely [D-Ala2,Leu5,cysteamine6] enkephalin, the increment in affinity was four to fivefold for both delta and mu receptors. The results strongly indicate that the dimeric enkephalin is more potent presumably due to the simultaneous interaction with the two binding sites of the opiate receptors.     

Preparation and properties of novel gramicidin S analogs possessing a tri-, tetra- or pentamethylene bridge between ornithine side chains.

Gramicidin S (GS) analogs in which the Ndelta atoms of the two Orn side chains are linked by an oligomethylene bridge [-(CH2)n-; n=3-5] were prepared via the bis(p-nitrobenzenesulfonyl) derivative [Orn(NBS)2,2']GS. For comparison the nonbridged secondary amino group-containing analog [Orn(Me)2,2']GS was also prepared. 1H NMR and CD spectral analysis indicated that these analogs adopt the same beta-sheet conformation as GS. The antimicrobial activities of these analogs were very similar, but were slightly dependent on the bridge chain length, the trimethylene-bridged analog being the most potent.