VI. Synthesis,conformation, and ion-binding of two bicyclic nonapeptides *

Two related homodetic bicyclic nonapeptides (cyclo Glu-X-Pro-Gly-Lys-X-Pro-Gly)-cyclo (lγ→ 5?), X = Ala(BCP2), X = Leu(BCP3) have been synthesized using conventional solution phase methods involving mixed anhydride coupling reactions starting with appropriately protected naturally occurring amino acids. The conformation and ion binding properties of BCP2 have been studied by nuclear magnetic resonance and circular dichroism techniques. The results of these studies have been compared to those of BCP3. The presence of Ala caused both Ala-Pro bonds to be trans in free BCP2. This characteristic imparted subtle differences to the ion-binding properties of BCP2 as compared to free BCP3 which has one cis Leu-Pro bond and one trans Leu-Pro bond.     

Synthesis and backbone conformations of cyclic hexapeptides cyclo-(Xxx-Pro-D-Gln)2

The solution syntheses of cyclo-(Xxx-Pro-D-Gln)₂, where Xxx=Gly, Ala, Leu, Phe and Val are described. Several routes were examined, the most successful involving the intermediate Z-Xxx-Pro-D-Gln-O-tBu and proceeding to cyclization of H-Xxx-Pro-D-Gln-Xxx-Pro-D-Gln-OH using diphenylphosphoryl azide. The N-H regions of the proton magnetic resonance spectra of aqueous solutions of these peptides were examined, and in the Xxx=Leu and Val peptides an unsymmetrical backbone, presumably with one cis Xxx-Pro peptide bond, was found to be important. Previous reports of cyclo-(Xxx-Pro-D-Yyy)₂ peptides have shown only C₂-symmetric forms.     

17O and 14 N n.m.r. studies of the Co (II) interaction with cyclo(Ala*-Ala) in aqueous solution

The complexation of cyclo(Ala*-Ala) with the cobaltous ions in aqueous solution was investigated by ¹⁷O and ¹⁴N n.m.r. spectroscopy. The ¹⁷O and ¹⁴N transverse relaxation time (T_2p) and chemical shift (Δω_a) of cyclo(Ala*-Ala) were measured as a function of the temperature at pH = 7.03 ± 0.02, and pH = 6.45 ± 0.02, and as a function of pH at room temperature. No effects of pH on the transverse relaxation time and chemical shift were observed. Complementary ¹⁷O studies of the solvent water molecules were also carried out. The hyperfine coupling constant and the entropy and enthalpy of activation for the exchange of cyclo(Ala*-Ala) and water molecules between the coordinated and noncoordinated states were determined by least-square fit of theoretical equation for the chemical shift Δω_a to experimental data. The hyperfine coupling constant of the peptide bound oxygen was determined to be (– 1.6 ± 0.1) ± 10⁵ Hz and the entropy and enthalpy (32.0 ± 3.0) kJ/mol and (– 12.0 ± 1.0)e.u, respectively. Information obtained from ¹⁷O n.m.r. study allows some inferences concerning the probable coordination sphere of the cobaltous ion. There are three types of complexes: Co(H₂O)²⁺₆, CoL ± 5H₂O and CoL₂ ± 4H₂O, with relative concentrations 19.9%, 2.9%, and 77.2%, respectively. The ¹⁷O n.m.r. line broadening is due to the presence of a minor species, CoL ± 5H₂O, in rapid exchange and not due to the major species, CoL₂ ± 4H₂O, in solution, which has a large ±t_m. ¹⁴N n.m.r. transverse relaxation rates are unaffected by the cobaltous ion, indicating either that the cobaltous ions do not bind to the nitrogen peptide bond or that the rate of exchange is very slow and does not affect the free peptide.     

Comparative conformational studies on cyclic hexapeptides corresponding to message sequence His-Phe-Arg-Trp of α-Melanotropin by NMR

Solution conformation of cyclo(Gly¹-His²-Phe³-Arg⁴-Trp⁵-Gly⁶) and its d -Phe analog corresponding to the message sequence [Gly-α-MSH_5-10] of α-MSH has been studied by 1D and 2D proton magnetic resonance spectroscopy in dimethyl sulfoxide (DMSO)-d₆ solution and in a DMSO-d₆/H₂O cryoprotective mixture. The NMR data for both the analogs in solution at 300 K cannot be interpreted based on a single ordered conformation, as evidenced by the broadening of only -NH resonances as well as the temperature coefficients of the amide protons. An analysis of the nuclear Overhauser effect (NOE) cross-peaks in conjunction with temperature coefficient data indicates an equilibrium of multiple conformers with a substantial population of particular conformational states at least in the d -analog. The molecular dynamics simulations without and with NOE constraints also reveal numerous low-energy conformers with two γ-turns, a γ-turn and a β-turn, two β-turns, etc. for both the analogs. The observed NMR spectra can be rationalized by a dynamic equilibrium of conformers characterized by a γ-bend at Gly⁶, two γ-bends at Phe³ and Gly⁶ and a conformer with a single β-turn and a γ-bend for the l -Phe analog. On the other hand, a conformation with two fused β-turns around the two tetrads His²-d -Phe³-Arg⁴-Trp⁵ and Trp⁵-Gly⁶-Gly¹-His² dominates the equilibrium mixture for the d -Phe analog. For the d -Phe analog, the experimentally observed average conformation is corroborated by molecular dynamics simulations as well as by studies in cryoprotective solvent.     

Influence of zinc (II) binding on the structure of bovine α-lactalbumin

The effect of Zn(II) binding on the structure of bovine α-lactalbumin (LA) was investigated. α-Lactalbumin, a regulatory subunit of lactose synthase, binds Ca(II) and Zn(II) at different sites in a mutually non-exclusive manner. The structures of the metal-depleted form of LA (apo-LA) and Ca(II)-bound LA (holo-LA) have been well characterized. Here, the effect of Zn(II) binding on the structure of holo-LA has been investigated by comparison with the structure of holo-LA and apo-LA using CD and NMR spectroscopy. The CD spectrum of Zn(II)-holo-LA was similar to that of holo-LA, but the intensity of the negative peak in near-UV region was decreased. Zn(II) binding to holo-LA produced only small changes in NMR chemical shifts, but the integral volumes of the cross-peaks of NOESY signals in cluster II, which is in the vicinity of Zn(II) binding site, were affected. Zn(II) binding induces a local structural change on the holo-LA, but it does not induce a large backbone conformational change, © Munksgaard 1996.     

Conformations of cyclo(L-alanyl-L-alanyl-ε-aminocaproyl) and of cyclo(L-alany1-D-alanyl-ε-aminocaproyl); cyclized dipeptide models for specific types of β-bends

Conformational energy calculations indicate that the peptide backbones of the low-energy conformations of the cyclized dipeptide derivatives cyclo (L-alanyl-L-alanyl-ε-aminocaproyl) and cyclo (L-alanyl-D-alanyl-ε-aminocaproyl) are constrained to form β-bends of types I + III and II, respectively. Thus, the two compounds can serve as models for the spectroscopic properties of β-bends of these types. The coupling constants obtained from ¹H n.m.r. spectra in DMSO-d₆ are consistent with the dihedral angles of the computed lowest-energy conformations. Differences in ¹³C chemical shifts between the two compounds can be correlated with differences in shielding by C=O groups in bends of various types. ¹H and ¹³C chemical shifts suggest association of cyclo (L-Ala-L-Ala-Aca) but not of cyclo (L-Ala-D-Ala-Aca) in dimethylsulfoxide. The different tendencies to associate can be explained in terms of the difference in conformation. The circular dichroism spectra of the two compounds are quite different. In methanol, trifluoroethanol and water, the L-Ala-L-Ala derivative has a positive extremum near 190 nm and two negative extrema near 206 and 220 nm, whereas the L-Ala-D-Ala derivative has a positive extremum at about 203 nm and negative extrema at about 187 and 229 nm. The spectra can be used to estimate the contribution of various bend types in a related series of compounds. A normal mode analysis of the vibrations of the computed low-energy conformations was compared with solid state infrared and Raman spectra, in order to determine the predominant conformations. The bend types determined by this comparison fully agree with the predictions of the theoretical computations for both derivatives.     

Conformational investigation of α,β-dehydropeptides VII*. Conformation of Ac-Pro-ΔAla-NHCH3 and Ac-Pro-(E)-ΔAbu-NHCH3: comparison with (Z)-substituted α,β-dehydropeptides†

The crystal structure and solution conformation of Ac-Pro-ΔAla-NHCH₃ and the solution conformation of Ac-Pro-(E)-ΔAbu-NHCH₃ were investigated by X-ray diffraction method and NMR, FTIR and CD spectroscopies. Ac-Pro-ΔAla-NHCH, adopts an extended-coil conformation in the crystalline state, with all-trans peptide bonds and the ΔAla residue being in a C₅ form, φ₁=– 71.4(4), ψ₁=– 16.8(4), φ₂=– 178.4(3) and ψ₂= 172.4(3)°. In inert solvents the peptide also assumes the C₅ conformation, but a γ-turn on the Pro residue cannot be ruled out. In these solvents Ac-Pro-(E)- ΔAbu-NHCH₃ accommodates a βII-turn, but a minor conformer with a nearly planar disposition of the CO—NH and C=C bonds (φ₂～0°) is also present. Previous spectroscopic studies of the (Z)-substituted dehydropeptides Ac-Pro-(Z)- ΔAbu-NHCH, and Ac-Pro-ΔVal-NHCH₃ reveal that both peptides prefer a βII-turn in solution. Comparison of conformations in the family of four Ac-Pro-ΔXaa-NHCH₃ peptides let us formulate the following order of their tendency to adopt a β-turn in solution: (Z)- ΔAbu > (E)- δAbu > ΔVal; ΔAla does not. None of the folded structures formed by the four compounds is stable in strongly solvating media. © Munksgaard 1996.     

Three‐dimensional structure of the α‐MSH‐derived candidacidal peptide [Ac‐CKPV]2

Abstract: Previous research has shown that the immunomodulatory peptide α‐melanocyte‐stimulating hormone (α‐MSH) and its carboxy‐terminal tripeptide KPV (Lys‐Pro‐Val α‐MSH_11?13) have antimicrobial influences. By inserting a Cys‐Cys linker between two units of KPV, we designed the dimer [Ac‐CKPV]₂ that showed excellent candidacidal effects in pilot tests and was the subject of further investigations. [Ac‐CKPV]₂ was active against azole‐resistant Candida spp. Therefore, the molecule appeared a promising candidate for therapy of fungal infections and was the subject of a structural study. ¹H‐NMR and restrained mechanic and dynamic calculations suggest that the peptide adopts an extended backbone structure with a β‐turn‐like structure. These results open a pathway to development of additional novel compounds that have candidacidal effects potentially useful against clinical infections.     

Conformational investigation of αβ-dehydropeptides

Solution conformations of three series of model peptides, homochiral Ac-Pro-L-Xaa-NHCH₃ and heterochiral Ac-Pro-D-Xaa-NHcH₃ (Xaa = Val, Phe, Leu, Abu. Ah) as well as αβ-unsaturated Ac-Pro-ΔXaa-NHCH₃ [Δ Xaa =ΔVal, (Z)-ΔPhe, (Z)-ΔLeu, (Z)-ΔAbu] were investigated in CDCl₃ and CH₂Cl₂ by ¹H-, ¹³C-NMR, and FTIR spectroscopy. NH stretching absorption spectra, solvent shifts Δδ for NH (Xaa) and NHCH₃ on going from CDCl₃ to (CD₃)₂SO, diagnostic interresidue proton NOEs, and trans-cis isomer ratios were examined. These studies performed showed the essential difference in conformational propensities between homochiral peptides (L-Xaa) on the one hand and heterochiral (D-Xaa) and αβ-dehydropeptides (ΔXaa) on the other. Former compounds are conformationally flexible with an inverse γ-bend, a β-turn, and open forms in an equilibrium depending on the nature of the Xaa side chain. Conformational preferences of heterochiral and αβ-dehydropeptides are very similar, with the type-II β-turn as the dominating structure. There is no apparent correlation between conformational properties and the nature of the Xaa side chain within the two groups. The β-turn formation propensity seems to be somewhat greater in αβ-unsaturated than in heterochiral peptides, but an estimation of β-folded conformers is risky.     

Improved synthesis and resolution of β-(3-pyridyl)-DL-α-alanine

β-Benzamido-α-(3-pyridyl)-DL-α-alanine hydrochloride was synthesized from 3-pyridinecarboxyaldehyde via the azlactone which was hydrolyzed to the acrylic acid before hydrogenation. The methyl ester was effectively resolved with subtilisin. The optical purity of the D-isomer was established, since the D-isomer was used in synthesis of antagonists of the luteinizing hormone releasing hormone.     

Solution structure of a biologically active cyclic LDV peptide analogue containing a type II′β-turn mimetic

The solution structure of cyclo-[Gly-Leu-Asp-Val-BTD] (BTD=β-turn dipeptide) has been determined by two-dimensional ¹H-NMR (nuclear magnetic resonance) spectroscopy and systematic conformational searching combined with molecular dynamics studies. The structure contains two hydrogen bonds between the Gly and Val residues, and a type I β-turn with Leu and Asp at the (i+ 1) and (i+ 2) positions of the turn. The cyclic compound shows activity in a scintillation proximity assay (SPA) for the inhibition of the interaction between the integrin α₄β₁ and vascular cell adhesion molecule-1 (VCAM-1). The structure-activity relationship of the LDV sequence is discussed. © Munksgaard 1996.     

Synthesis and properties of human β-endorphin-(1–9) and its analogs

Four analogs of human β-endorphin (β_h-EP) were synthesized by the solid-phase method: β_h-EP-(1–9) (I), [D-Ala²]-β_h-EP-(1–9) (II), [Gln⁸]-β_h-EP-(1–9) (III), and [D-Ala², Gln⁸]-β_h-EP-(1–9) (IV). Measurement in a radioreceptor binding assay with use of tritiated β_h-EP as primary ligand gave relative potencies as follows: Met-enkephalin, 100; I, 76; II, 100; III, 200; IV, 200. Two new amino acid derivatives were prepared and used for synthesis of the analogs: N^α-t-butyloxycarbonyl-O-(cyclopentyl) -tyrosine and N^α-t-butyloxycarbonyl-γ-(cyclopentyl)-glutamic acid.     

β-ENDORPHIN: SYNTHESIS AND RADIORECEPTOR BINDING ACTIVITY OF βh-ENDORPHIN-(1–27) AND ITS ANALOGS

β_h-Endorphin-(1–27) (I), [Ac-Tyr¹]-β_h-endorphin-(1–27) (II), [Gln⁸]-β_h-endor-phin-(1–27) (III), and [Ac-Tyr¹, Gln⁸]-β_h-endorphin-(1–27) (IV) were synthe sized by the solid-phase method. The binding potency of I-IV to rat brain membrane preparations was measured by radioreceptor binding assay. The relative potencies were: β_h-endorphin, 100; I, 30; II, 0.04; III, 90; IV, 0.07.     

Peptides from chiral Cα,α-disubstituted glycines Synthesis and characterization,conformational energy computations and solution conformational analysis of Cα-methyl,Cα-isopropylglycine [(αMe)Val] derivatives and model peptides*

Conformational energy computations on Ac-l -(αMe)Val-NHMe indicate that turns and right-handed helical structures are particularly stable conformations for this chiral Cα-methyl, Cα-alkylglycyl residue. We have synthesized and characterized a variety of l -(αMe)Val derivatives and peptides (to the pentamer level). The results of the solution conformational analysis, performed using infrared absorption, ¹H nuclear magnetic resonance, and circular dichroism, are in general agreement with those obtained from the theoretical investigation, in the sense that the l -(αMe)Val residue turns out to be a strong β-turn and right-handed helix former. A comparison is also made with the conclusions extracted from published work on peptides rich in other Cα-methyl, Cα-alkylglycyl residues.     

β-Turn induction by C60-based fulleroproline: synthesis and conformational characterization of Fpr/Pro small peptides

We have recently described the preparation of Fpr (C₆₀-based fulleroproline). In this paper the synthesis and a conformational characterization of heterochiral di-and tripeptides containing this new α-amino acid are reported. A folded structure, induced by the-l -Fpr-d -Ala-sequence in chloroform solution and detected by Fourier transform infrared absorption and H nuclear magnetic resonance, has been compared with the known propensity of the cognate-l -Pro-d -Ala-sequence to adopt a βII-turn conformation, which has also been confirmed in this work. The βII-turn structure is retained in the crystal state by the Pro-peptides, as shown by the X-ray diffraction structures of Ibu-l -Pro-d -Ala-NHtBu and Z-l -Pro-d -Ala-l -Ala-OtBu. © Munksgaard 1997.     

NMR solution structure of the receptor binding domain of Pseudomonas aeruginosa pilin strain P1. Identification of a β-turn

The solution structure of the peptide antigen from the receptor binding domain of Pseudomonas aeruginosa strain P1 has been determined using two-dimensional ¹H NMR techniques. Ensembles of solution conformations for the trans form of this 23-residue disulfide bridged peptide have been generated using a simulated annealing procedure in conjunction with distance and torsion angle restraints derived from NMR data. Comparison of the NMR-derived solution structures of the P1 peptide with those previously determined for the 17-residue PAK, PAO and KB7 strain peptides [Mclnnes, C., et al. (1993) Biochemistry 32 , 13432–13440; Campbell, A.P., et al. (1995) Biochemistry 34 , 16255–16268] reveals the common structural motif of a β-turn, which may be the necessary structural requirement for recognition of a common cell surface receptor and a common cross-reactive antibody to which all four strains bind. The importance of this conserved β-turn in the PAK, PAO, KB7 and P1 peptides is discussed with regard to the design of a synthetic peptide vaccine effective against multiple strains of Pseudomonas aeruginosa infections.     

[7-Methyltryptophan 9] -β-corticotropin-(1–24): a hyperactive Synacthen analogue

[7-Methyltryptophan 9] -β-corticotropin-(1–24) has been synthesised. In an isolated adrenal cell bioassay, it had 2.7 times the steroidogenic activity of β-corticotropin-(1–24) (Synacthen).     

Preparation and preliminary biological evaluation of 177Lu‐labeled GluDTPA‐cyclo(RGDfK) for integrin ανβ3 receptor‐positive tumor targeting

Integrin α_νβ₃ is a receptor and is highly expressed on activated and proliferating endothelial cells during the growth and metastasis of solid tumors but not on resting endothelial cells and normal organs. Because RGD peptide binds to integrin α_νβ₃ receptor, a variety of radiolabeled RGD peptides have been evaluated for non‐invasive imaging of integrin α_νβ₃‐positive tumors. In an attempt to develop RGD‐based radiopharmaceuticals, a novel GluDTPA‐cyclo arginine‐glycine‐aspartic acid‐d ‐phenylalanine‐lysine (GluDTPA‐cycloRGDfK) was simply synthesized and radiolabeled with ¹⁷⁷Lu. Also, tumor targeting and retention of the radiolabeled complex were evaluated in U87MG glioma‐bearing mice. The ¹⁷⁷Lu‐labeled GluDTPA‐cyclo(RGDfK) was formulated with a high radiolabeling yield (>98%) under mild condition, and the radiochemical purity was sustained in both saline and serum for over 4 days at 37°C. The radiolabeled compounds were rapidly cleared from the blood pool and non‐target tissue. Tumor‐to‐blood ratio was 12.09 at 2 h post injection and increased to 134.67 at 24 h, while tumor to liver ratio was 2.01 at 24 h similar to that of 2 h. Though it is inappropriate for targeted therapy due to its low uptake in tumor (~ 1 %ID/g), the acceptable results on radiochemistry and biodistribution propose to take a further assessment for non‐invasive imaging and detection of integrin α_νβ₃‐positive tumors by applying diagnostic radionuclides. Copyright © 2011 John Wiley & Sons, Ltd.     

Synthesis and properties of β-endorphin analogs containing the dynorphin-(1–13) sequence

Two analogs of β_h-endorphin containing dynorphin-(5 - 13) or dynorphin-(6 - 13) sequence have been synthesized by the solid-phase method. Biological activities of the analogs have also been investigated. It was found that the opiate activity is about three times more than β_h-endorphin in the guinea pig ileum assay. Both analgesic potency and opiate receptor-binding activity of the analogs are lower when compared with β_h-endorphin. However, the analogs have interesting behavioral effects in mice.     

Antimicrobial and cancer cell growth inhibitory activities of 3β-acetoxy-17β-( -prolyl)amino-5α-androstane in vitro

The in vitro activity of the steroidal amide 3β-acetoxy-17β-( -prolyl)amino-5α-androstane against 179 Gram-positive clinical isolates was examined. The minimum bactericidal concentration (MBC)/MIC ratios were ≤2 for 73% of methicillin-resistant Staphylococcus aureus, 59% of vancomycin-resistant Enterococcus spp. and 88% of penicillin-resistant Streptococcus pneumoniae. The androstane derivative was bactericidal for a variety of other Gram-positive genera, including Nocardia, Corynebacterium and Listeria. Variation in MICs is pH 6–8 media was slight. The frequency of occurrence of bacterial spontaneous mutations to resistance ranged from 10⁻⁶ to 10⁻⁹. Kill curve analysis confirmed the bactericidal nature of the steroidal amide, and demonstrated that killing was time dependent but not concentration dependent for all organisms. The ability of 3β-acetoxy-17β-( -prolyl)amino-5α-androstane to inhibit human cancer cell growth was also evaluated. The concentration required to inhibit 50% of cell growth (GI₅₀) was <2.5 mg/l for all cell lines examined. In single-dose murine toxicity evaluations, the androstane derivative was non-toxic at doses up to 400 mg/kg.