Convenient preparation of [Orn(Tfa)2]- and [Orn(Boc)2, Orn(Tfa)2′]gramicidin S,versatile unsymmetrically protected derivatives of gramicidin S

Abstract: Treatment of gramicidin S (GS) with trifluoroacetic anhydride afforded a derivative in which only one of the two Orn side chains was trifluoroacetylated in 72% yield, furnishing the first efficient method for the preparation of a monoprotected derivative of GS. The mono(Tfa) derivative [Orn(Tfa)²]GS was treated with di-tert-butyl dicarbonate to yield dually protected derivative [Orn(Boc)²,Orn(Tfa)^2′]GS from which another monoprotected derivative [Orn(Boc)²]GS was prepared in high yield. These unsymmetrically protected GS derivatives are versatile starting materials for the preparation of various other GS derivatives. As an example of application of the unsymmetrically protected derivatives, a dimeric GS derivative was prepared via a singly p-nitrobenzenesulfonyl(NBS)-activated derivative [Orn(Boc)²,Orn(NBS)^2′]GS.     

Proline residue‐modified polycationic analogs of gramicidin S with high antibacterial activity against both Gram‐positive and Gram‐negative bacteria and low hemolytic activity*

Abstract: Novel polycationic analogs of the cyclic decapeptide antibiotic, gramicidin S, possessing NH₂, d /l ‐Phe‐NH or l ‐Lys‐NH groups at the 4α‐ or 4β‐positions of the l ‐Pro residues, were synthesized. While l ‐Pro(4α/β‐NH₂)‐containing analogs exhibited much weaker antibacterial activity, the d /l ‐Phe and l ‐Lys‐substituted analogs exhibited higher antibacterial activity against Gram‐negative bacteria than the parent gramicidin S. All of these additional amino group‐containing analogs showed substantially reduced toxicity against human blood cells.     

Synthesis and NMR conformational analysis of a β-turn mimic incorporated into gramicidin S

The solution structure of a gramicidin S (GS) analog containing a β-turn mimic[BTD^4-5, Lys^2,2′]GS has been compared to that of native GS. The linear [BTD^4-5, Lys^2,2′]GS was synthesized by solid phase methodology and the cyclized peptide was analyzed by NMR. In the peptide portion of [BTD^4-5, Lys^2,2′]GS, the intramolecular hydrogen bonding pattern, inter-residue NOEs, including a transannular Hα-Hα NOE, and J^Nα coupling constants all describe a solution structure which is equivalent to that of native GS. These data confirm that the BTD group is a competent Type II' β-turn mimic since it does not disrupt the native conformation of GS. It also supports the use of GS as a conformational model in which to test β-turn mimics.     

Effects of single d‐amino acid substitutions on disruption of β‐sheet structure and hydrophobicity in cyclic 14‐residue antimicrobial peptide analogs related to gramicidin S

Abstract: Gramicidin S (GS) is a 10‐residue cyclic β‐sheet peptide with lytic activity against the membranes of both microbial and human cells, i.e. it possesses little to no biologic specificity for either cell type. Structure–activity studies of de novo‐designed 14‐residue cyclic peptides based on GS have previously shown that higher specificity against microbial membranes, i.e. a high therapeutic index (TI), can be achieved by the replacement of a single l ‐amino acid with its corresponding d ‐enantiomer [Kondejewski, L.H. et al. (1999) J. Biol. Chem. 274 , 13181]. The diastereomer with a d ‐Lys substituted at position 4 caused the greatest improvement in specificity vs. other l to d substitutions within the cyclic 14‐residue peptide GS14, through a combination of decreased peptide amphipathicity and disrupted β‐sheet structure in aqueous conditions [McInnes, C. et al. (2000) J. Biol. Chem. 275 , 14287]. Based on this information, we have created a series of peptide diastereomers substituted only at position 4 by a d ‐ or l ‐amino acid (Leu, Phe, Tyr, Asn, Lys, and achiral Gly). The amino acids chosen in this study represent a range of hydrophobicities/hydrophilicities as a subset of the 20 naturally occurring amino acids. While the d ‐ and l ‐substitutions of Leu, Phe, and Tyr all resulted in strong hemolytic activity, the substitutions of hydrophilic d ‐amino acids d ‐Lys and d ‐Asn in GS14 at position 4 resulted in weaker hemolytic activity than in the l ‐diastereomers, which demonstrated strong hemolysis. All of the l ‐substitutions also resulted in poor antimicrobial activity and an extremely low TI, while the antimicrobial activity of the d ‐substituted peptides tended to improve based on the hydrophilicity of the residue. d ‐Lys was the most polar and most efficacious substitution, resulting in the highest TI. Interestingly, the hydrophobic d ‐amino acid substitutions had superior antimicrobial activity vs. the l ‐enantiomers although substitution of a hydrophobic d ‐amino acid increases the nonpolar face hydrophobicity. These results further support the role of hydrophobicity of the nonpolar face as a major influence on microbial specificity, but also highlights the importance of a disrupted β‐sheet structure on antimicrobial activity.     

Studies of peptide antibiotics. XLVI. Syntheses of gramicidin S analogs containing D-α, β-diaminopropionic acid or α,β-dehydroalanine*

A gramicidin S (GS) analog ([d -Dpr^4,4] GS) containing d -α,β-diaminopropionic acid (D-Dpr) in place of D-Phe at 4,4′ positions was derived from [l -Orn(δ-formyl)^2,2, d -Dpr(β-Z)^4,4′]GS, which was synthesized by conventional method in solution. An analog [ΔAla^4,4′]GS was synthesized from [l -Orn(δ-Boc)^2,2′, d -Dpr^4,4′]GS through Hofmann degradation of the d -Dpr residues. Antimicrobial activities of these analogs were tested; [d -Dpr(β-Z)^4,4′]GS and [ΔAla^4,4′]GS showed high antimicrobial activities against Gram-positive bacteria. [d -Dpr^4,4′]-GS showed an appreciable activity against Gram-negative bacteria such as Escherichia coli. Four semigramicidin S (semiGS) analogs such as [ΔAla⁴]semiGS were synthesized; these had no antimicrobial activity. Analogs containing ΔAla residues were hydrogenated, and the formation of l -Ala or d -Ala residues was determined. The ΔAla residues in [ΔAla^4,4′] GS were reduced to dl -Ala, and ΔAla in [ΔAla⁴]semiGS mostly to l -Ala. The relationships of the antimicrobial activity, CD curves and asymmetric hydrogenation to the structure were discussed.     

Studies of peptide antibiotics. XLIV. Syntheses and biological activities of gramicidin S analogs containing δ-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 [Gly^1,1′]-GS and the neutral [l -Hnv^2,2′]-GS (Hnv represents δ-hydroxynorvaline) were synthesized by the solid-phase method to evaluate the role of l -Val^1,1′ and l -Orn^2,2′ residues in GS. The hybrid analogs ([Gly¹]-GS and [l -Hnv²]-GS) and [d -Tyr^4,4′]-GS showed high antibacterial activities, whereas [Gly^1,1′]-GS and [l -Hnv^2,2′]-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.     

Conformation of a water-soluble β-sheet model peptide. A circular dichroism and Fourier-transform infrared spectroscopie study of double D-amino acid replacements

Among peptide secondary structures β-sheet domains have been much less intensively studied than α-helical conformations, mainly because of the lack of well characterized model peptides. In the present paper the secondary structure of a water-soluble de novo peptide consisting of 26 amino acids (DPKGDPKGVTVTVTVTVTGKGDPKPD-NH₂) and the corresponding double D-amino acid replacement set have been studied by circular dichroism and Fourier-transform infrared spectroscopy. The model peptide was found to be unstructured in aqueous solution at peptide concentrations < 10^?3 mol/L but to adopt a predominantly β-sheet structure in the presence of 15 mM sodium dodecyl sulfate or at apolar/water interfaces. Although the peptide is composed of amino acids with low helical propensity, it formed a single-stranded helical structure in aqueous trifluoroethanol. The D-amino acid replacement set was synthesized in order to study the conformational stability of the model peptide selectively in distinct regions. The data show that both the α-helix present in 50% trifluoroethanol as well as the β-sheet domain formed in the presence of sodium dodecyl sulfate or at apolar/water interfaces, are located in the region between Val⁹ and Thr¹⁸. Pairwise substitution of adjacent amino acids by their corresponding D-amino acids provides a pronounced β-sheet disturbance. These findings demonstrate that double D-amino acid replacements may be used to locate β-sheet domains in peptides.     

New patterns of hydrogen bonded interactions between polypeptide chains

Crystals of glycylglycylglycine (C₆H₁₁N₃O₄), grown from an aqueous methanol solution, are triclinic, space group P1, with the unit cell dimensions (at 22 ± 3°) a= 11.656(3), b= 14.817(3), c= 4.823(2) Å, α= 88.45(3), β= 95.96(3), γ= 105.42(3)°, Z = 4 (with two molecules in the asymmetric unit) with a density of D_obs= 1.58g·cm^-3 and D_calc= 1.572g·cm^-3. The crystal structure was solved by a combination of multisolution and trial and error methods and refined with full-matrix least-squares method to a final R value of 0.036 for the observed 3021 reflections (I ≥ 2s?). The conformation of the two molecules I and II in the asymmetric unit is very similar (except around the N-terminal end); they have the fully extended trans-planar conformation, and have ω values ranging from 2 to 4°. The peptide chain repeating distances (C¹_α - C³_α) are 7.27 Å and 7.18 Å in the two molecules as compared with the value of 6.68 Å for extended β-sheets with β-carbons. There are four different interactions between these two molecules characterized by different hydrogen bonding. Molecule I is hydrogen bonded to a neighboring molecule I using four hydrogen bonds. Molecule II is hydrogen bonded to another II, using bifurcated interactions involving the peptide nitrogen. Molecule I is hydrogen bonded to two different molecules II forming distinctly different hydrogen bonding patterns from the two mentioned above. The molecules are packed in rows, in a head-to-tail fashion (C-terminal opposite N-terminal) and are held together in sheets by hydrogen bonds between carbonyl and amide groups, corresponding to the very familiar anti-parallel pleated sheet arrangement for polypeptides. The hydrogen bonds involving the amino nitrogens as donors are significantly longer and presumably weaker compared to those involving the NH⁺₃ group. The C=O distances show variations that correlated with hydrogen bonding. The N-H … O angle varies from 152 to 174° and the bent N-H … O hydrogen bonds show bifurcated interactions.     

Observation of a mixed antiparallel and parallel β-sheet motif in the crystal structure of Boc-Ala-Ile-Aib-OMe

A diastereomeric mixture of the tripeptide Boc-Ala-Ile-Aib-OMe crystallized in the space group Pl from CH₃OH/H₂O. The unit cell parameters are a= 10.593(2) Å, b= 14.377(3) Å, c= 17.872(4) Å, α= 104.41(2)°, β= 90.55(2)°, γ= 106.91(2)°, V= 2512.4 ų, Z=4. X-Ray crystallographic studies shows the presence of four molecules in the asymmetric unit consisting of two pairs of diastereomeric peptides, Boc-l -Ala-l -Ile-Aib-OMe and Boc-l -Ala-d -Ile-Aib-OMe. The four molecules in the asymmetric unit form a rarely found mixed antiparallel and parallel β-sheet hydrogen bond motif. The Ala and (l ,d )-Ile residues in all the four molecules adopt the extended conformations, while the φ, ψ values of the Aib residues are in the right-handed helical region. In one of the molecules the Ile sidechain adopts the unusual gauche conformation about the C^β-C^γ bond. © Munksgaard 1996.     

Simulations of conformers of tuftsin and a cyclic tuftsin analog

The conformational properties of the configurational isomers of tuftsin, a linear tetrapeptide with the sequence Thr-Lys-Pro-Arg, were investigated with six 1 ns molecular dynamics simulations in explicit water and in a 1.0 M NaCl solution. The average conformation of the cis isomer is a type VI β-turn. Our results indicate that water-peptide hydrogen bonding, in addition to intramolecular hydrogen bonds, stabilizes the cis conformer. The trans isomer is neither a β- nor a γ-turn. Results are compared with parallel studies on a cyclic analog of tuftsin, cyclo(Thr-Lys-Pro-Arg-Gly). The addition of salt does not influence the backbone conformation of the peptide. Differences between the structures are confined to the side-chain orientations of the Lys and Arg residues. © Munksgaard 1995.     

Solid phase synthesis of human growth hormone-releasing factor analogs containing a bicyclic β-turn dipeptide

Three analogs derived from the N-terminal 29-residue fragment of human growth hormone-releasing factor (hGRF) which contained a bicyclic β-turn dipeptide (BTD) at 7-8,8-9, and 9-10 positions were synthesized by solid phase methodology to ascertain if the β-turns are important for the biological activity of hGRF and also to show the applicability of the BTD unit to solid phase synthesis. All three analogs were obtained in good yield and purity indicating that the BTD unit can be used in the usual condition of solid phase synthesis. The capacity of these analogs to release growth hormone (GH) was tested in an in vitro bioassay using rat anterior pituitary cells. All three BTD-containing analogs showed the same maximal GH secretion with parallel dose-response curves to that of hGRF(1-29)NH₂, except their relative potencies were very low.     

Design,synthesis, and cytotoxic evaluation of novel scopoletin derivatives

A series of scopoletin derivatives were designed and synthesized by introducing α‐aminoacetamide, acrylamide and β‐aminopropamide, respectively, to 3‐position of scopoletin, and their chemical structures were confirmed by ESI‐MS, IR, ¹H NMR, and ¹³C NMR spectra. All target compounds were evaluated in vitro against four human cancer cell lines (MDA‐MB‐231, MCF‐7, HepG2, and A549) by MTT method. Cytotoxic assay showed that compounds 7a , 7b , 7e , 7f , 8a , and 8e exhibited more potent cytotoxicities compared to scopoletin. Besides, we have further evaluated the growth inhibitory activities of these selected compounds against normal tissue cell lines HFL‐1. Although compound 8a showed the strongest antiproliferative activity in vitro, it exhibited strong cytotoxicity on normal cells HFL‐1, which limited its further study. Compound 7a and 7b exhibited higher antiproliferative activity against MDA‐MB‐231 and HepG2 cells and weak cytotoxicity on HFL‐1, which suggested that 7a and 7b might be ideal anticancer candidates. The SARs showed that the introduction of the acrylamide and its analogues β‐aminopropamide could significantly improve activity, while the α‐aminoacetamide failed to enhance potency obviously. Therefore, the mechanism of compound 7a and 7b is worthy of further research and the structure of compound 8a should be further optimized.     

Vibrational analysis of peptides,polypeptides and proteins

Normal mode calculations have been carried out on three low-energy structures of gramicidin S obtained from conformational energy calculations. When the results on the amide modes are compared with observed bands in the infrared and Raman spectra of crystalline gramicidin S and its N-deuterated derivative, one of the structures is clearly disfavored. Of the other two, one is slightly favored, and it corresponds to the lowest-energy structure obtained from the energy calculations. Spectra from solutions in DMSO and CH₃ OH suggest that the molecular conformation is essentially retained in these solvents.     

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

Abstract: 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)‐I²aa^{4?5,4′?5′}]GS ( 2 ), [Lys^2,2′,(6S)‐I²aa^{4?5,4′?5′}]GS ( 3 ) and [(6R)‐I²aa^{4?5,4′?5′}]GS ( 4 ). Although conformational analysis of [I²aa^{4?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 β‐turn/antiparallel β‐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)‐I²aa^{4?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.     

Crystal structures of a D-residue containing tetrapeptides 1. tert-Boc-D-Valyl-alanyl-leucyl-alanyl methoxide,butanol solvate

The crystal structure of a heterochiral peptide, viz. Boc-D-Val-Ala-Leu-Ala-OMe, with a D-residue in the beginning of the sequence has been determined (a= 9.464(5), b= 35.615(5), c= 9.703(2) Å, space group P2₁2₁2, Z= 4, R= 0.09). The peptide is in the extended β-conformation and the packing is stabilised by four N—H. O hydrogen bonds in an antiparallel β-sheet arrangement. The solvent molecule is disordered and does not have any specific interactions with the peptide.     

Design,synthesis, and evaluation of novel Akt1 inhibitors based on an indole scaffold

A new series of potential Akt1 inhibitors with indole scaffold were designed and synthesized. The antiproliferative activity against PC‐3 cell line and enzyme inhibitory activity against Akt1 were evaluated. Among them, some compounds showed much more potent antiproliferative activity and stronger Akt1 inhibitory activity compared to the positive control of GSK690693. In particular, compound 19b exhibited the most potent inhibitory activity against Akt1 with inhibition rate of 70.3% at a concentration of 10 nm . Furthermore, compound 19b could dose dependently reduce the phosphorylation of the downstream GSK3β protein in the PC‐3 cell line and displayed fivefold higher antiproliferative activity against PC‐3 cell line with IC₅₀ value of 3.1 ± 0.1 μm than positive control (15.5 ± 0.4 μm ). Herein, compound 19b may serve as a promising lead for further optimization and development of novel Akt1 inhibitors based on an indole scaffold.     

The crystal structure of Boc-Pro-Val-Gly-NH2, with a remark on the conformation of the valyl residues in peptides

The crystal structure of Boc-Pro-Val-Gly-NH₂ has been determined: monoclinic; P2₁; a = 9.331 (3) Å, b = 9.532 (4), c = 23.080 (9), β= 91.33 (3)R, Z = 4; R = 0.053 for 3400 reflections with ˙Fo˙,>α(Fo). There are two independent but very similar molecules in the crystal. The peptide main chains are in an extended form, and packed in two kinds of antiparallel β sheets, the (φ, Φ) angles of the central Val residues are (-156°, 146°) and (-139°, 155°), and the mean length of the N- H . 0 hydrogen bonds in the sheets is 2.965 Å. A detailed study of the conformations of the Val residues in oligopeptide crystals shows that the preferred conformation of Val in peptides is: the (φ, Φ) angles close to those of the antiparallel β sheet, and C^γ1 and C^γ2, against N with respect to the C^α– C^β bond, at either (trans, gauche) or (-gauche, gauche). The mean π(NC^αC') angle of such Val residues is 107.9(9)°. A twisting in the β sheets is also discussed.     

Synthesis and properties of equine β-melanotropin analogs with substitution in residue position 1

Five analogs of equine β-melanotropin have been synthesized by the solid phase method. The NH₂-terminal aspartic acid was substituted with amino acids (Gly, Trp, Ile, Lys and Nα-acetyl-Asp) differing widely in physicochemical properties. On the basis of their lipolytic potencies it was concluded that this position plays a negligible role in this activity.     

Anti‐bronchoconstrictor activities and side effect potential of inhaled formoterol and (S,R)‐epiformoterol in the rhesus monkey

In a recent patent, the (S,R) isomer of epiformoterol was claimed to be a bronchodilator with reduced adverse effects compared to racemic formoterol. We initiated the present study to seek direct evidence for the claim. Anaesthetised, spontaneously breathing rhesus monkeys were set up for measuring airways resistance. Blood pressure and heart rate were measured concomitantly to gauge systemic exposure and the potential for side effects. Formoterol, 1.2µg/kg, administered by aerosol, induced rapidly developing, sustained inhibition of the bronchoconstrictor responses to aerosolised methacholine accompanied by tachycardia. (S,R)‐epiformoterol, 63µg/kg, induced anti‐bronconstrictor effects and an associated tachycardia, which were superimposable with those induced by racemic formoterol at 1.2µg/kg. Thus, (S,R)‐epiformoterol is a long‐acting anti‐bronchoconstrictor agent in the rhesus monkey and causes an associated tachycardia. This profile is not qualitatively different from that of racemic formoterol but does differ from that of salmeterol, which, in this model, shows lower efficacy and more pronounced tachycardia for an equivalent degree of anti‐bronchoconstrictor activity. Thus, the claim that (S,R)‐epiformoterol shows bronchodilator activity with reduced adverse effect potential compared to racemic formoterol is not supported by the present data. Drug Dev. Res. 57:1–5, 2002. © 2002 Wiley‐Liss, Inc.     

Synthesis,biology, NMR and conformation studies of the topographically constrained δ‐opioid selective peptide analogs of [β‐iPrPhe3]deltorphin I

Abstract: Replacement of Phe³ in the endogenous δ‐opioid selective peptide deltorphin I with four optically pure stereoisomers of the topographically constrained, highly hydrophobic novel amino acid β‐isopropylphenylalanine (β‐iPrPhe) produced four pharmacologically different deltorphin I peptidomimetics. Radiolabeled ligand‐binding assays and in vitro biological evaluation indicate that the stereoconfiguration of the iPrPhe residue plays a crucial role in determining the binding affinity, bioactivity and selectivity of [β‐iPrPhe³]deltorphin I analogs: a (2S,3R) configuration of the iPrPhe³ residue in [β‐iPrPhe³]deltorphin I provided the most desirable biological properties with binding affinity (IC₅₀ = 2 n m ), bioassay potency (IC₅₀ = 1.23 n m in MVD assay) and exceptional selectivity for the δ‐opioid receptor over the µ‐opioid receptor (30 000). Further conformational studies based on two‐dimensional NMR and computer‐assisted molecular modeling suggested a model for the possible bioactive conformation in which the Tyr¹ and (2S,3R)‐β‐iPrPhe³ residues adopt trans side‐chain conformations, and the linear peptide backbone favors a distorted β‐turn conformation.