The evaluation of type I and type II β-turn mixtures

Circular dichroism (CD) and¹ H-{¹H}NOE spectra were obtained for Piv-Pro-Ser-NHCH₃(1),[Piv-(CH₃)₃-C-CO], Boc-Pro-Ser-NHCH₃ (2) and Boc-Val-Ser-NHCH₃ (3), to determine the solution conformation of these p-turn models. In the crystal, 1 and 3 adopt an ideal type I β-turn, while 2 is characterized by a semifolded backbone geometry incorporating a cis Boc-Pro tert-amide bond. The predominance of a β-turn conformation in solution was suggested for models 1-3 on the basis of ¹H-{¹H}NOE data. In a nonpolar solvent the prevailing trans rotamer form (>80%) of 2 has a β-turn conformation according to heteronuclear NOE measurement. Positive ¹H-{¹H} NOEs were detected between the H^α(Pro)/NH(Ser), Hα(Ser)/NH(Ser) and NH(NHCH₃3)/HN(Ser) protons in the trans Boc-Pro rotamer form of 2 at -20° in CDCl₃. Similar positive homonuclear NOE enhancements were also observed on the appropriate proton signals in other models, such as Boc-Val-Ser-NHCH₃ (3). Boc-Val-D-Ser-NHCH₃ (4) and Boc-Pro-D-Ser-NHCH₃ (5), in various solvents. The ¹H- {¹H)NOE experiments carried out in CD₃CN clearly showed that besides the type I (or III) β-turn structure, one of the main conformations of models 1-5 is close to the type II β-turn backbone geometry in a nonpolar solvent. Unexpectedly, the conformational mixture of models 1-3 were characterized by class C (helix-like) CD spectra, although class C spectra are generally only correlated with the type I β-turn conformation. These acyclic models are the first carefully investigated examples of -L-L- triamide systems, containing a significant amount of a type II β-turn, as well as the type I p-turn and, however, yielding a class C circular dichroism spectra. The CD spectra recorded for 3 and 4 in acetonitrile were ‘calibrated’ using the ¹H-{¹H}NOE data. Such a “calibration”, as well as the semi-quantitative CD and NMR comprehensive analyses, demonstrated that class C, class B, as well as class C’ CD spectra may be obtained from the linear combination of the same two-component spectra, with different conformational weights. Therefore, it is suggested that the extraction of the conformational components of such models, simply on the basis of their CD spectra, must be made with caution.     

Crystal structure analysis of a β-turn mimic in hydrazino peptides

The crystal structures of four hydrazino peptides (Piv-Pro-h(N^α-Bzl)Gly-NHiPr 1 , Piv-Pro-hAla-NHiPr 2 , Moc-hPro-NHiPr 3 , and Boc-hPro-Gly-N(OH)Me 4 ) deriving from the hydrazino analogues of glycine (hGly), l -alanine (hAla) or l -proline (hPro) have been solved. They reveal a common folded structure of the α-hydrazino acid residue characterized by a bifurcated hydrogen bond closing an eight-membered cycle. This folded structure is topologically similar to the βII′-turn in peptides, and the CO-NH-N hydrazide link can be considered as a good turn-inducer in peptide analogues.     

Studies on β-turn of peptides

The effect of changing 1st and 4th amino acid residues on β-turn preference of tetrapeptide sequences was studied by use of CD spectra of the chromophoric derivatives, which have Dnp- and pNA-groups as the amino and carboxyl substituents, respectively. The effect was examined with the tetrapeptides having such sequences at the 2nd and 3rd positions as -L-Pro-L-Asn-, -L-Pro-Gly-, -L-Pro-D-Ala-, -L-Ala-D-Leu-, -L-Ala-L-Pro-, and -D-Ala-L-Pro-. The β-turn preferences estimated from the CD intensities of the bands due to exciton interaction were found to depend largely on the configurations of the 1st and 4th amino acid residues. When 1st and 2nd (or 3rd and 4th) residues had the same configuration, decreased intensity of the CD band was observed even if the internal sequence had high β-turn preference. Terminal Gly residues were favorable for the β-turn conformation in many of the tetrapeptide sequences examined.     

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.     

Stabilization of β-turn conformations in enkephalins

Stereochemical constraints have been introduced into the enkephalin backbone by substituting α-aminoisobutyryl (Aib) residues at positions 2 and 3, instead of Gly. ¹H n.m.r. studies of Tyr-Aib-Gly-Phe-Met-NH₂, Tyr-Aib-Aib-Phe-Met-NH₂ and Tyr-Gly-Aib-Phe-Met-NH₂ demonstrate the occurrence of folded, intramolecularly hydrogen bonded structures in organic solvents. Similar conformations are also favoured in the corresponding t-butyloxycarbonyl protected tetrapeptides, which lack the Tyr residue. A β-turn centred at positions 2 and 3 is proposed for the Aib²-Gly³analog. In the Gly²-Aib³analog, the β-turn has Aib³-Phe⁴as the corner residues. The Aib²-Aib³analog adopts a consecutive β-turn or 3₁₀ helical conformation. High in vivo biological activity is observed for the Aib²and Aib²-Aib³analogs, while the Aib³peptide is significantly less active.     

Evidence for a β-turn in an azadipeptide sequence

Crystalline Bu^tCO-Pro-AzaAla-NHPrⁱ, prepared by the active ester procedure, has been subjected to single-crystal X-ray diffraction analysis which evidences a type II β-turn.     

Crystal structure of tert.-butyloxycarbonyl-L-prolyl-D-alanyl-D-alanyl-N-methylamide Dimeric β-sheet formation

The crystal structure of the tripeptide t-Boc-L-Pro-D-Ala-D-Ala-NHCH₃, monohydrate, (C₁₇H₃₀N₄O₅·H₂O, molecular weight = 404.44) has been determined by single crystal X-ray diffraction. The crystals are mono-clinic, space group P2₁, a = 9.2585(4), b = 9.3541(5). c = 12.4529(4) Å, β= 96.449(3)°, Z = 2. The peptide units are in the trans and the tBoc-Pro bond in the cis orientation. The first and third peptide units show significant deviations from planarity (Δω=5.2° and Δω=3.7°, respectively). The backbone torsion angles are: φ¹, = -60°, ψ_1/= 143.3°, ω₁= -174.8°, φ₂= 148.4°, ψ₂= -143.1°, ω₂= -179.7°, φ₃= 151.4°, ψ₃= -151.9°, ω₃= -176.3°. The pyrrolidine ring of the proline residue adopts the C₂— C^γ conformation. The molecular packing gives rise to an antiparallel β-sheet structure formed of dimeric repeating units of the peptide. The surface of the dimeric β-sheet is hydrophobic. Water molecules are found systematically at the edges of the sheets interacting with the urethane oxygen and terminal amino groups. Surface catalysis of an L-Ala to D-Ala epimerization process by water molecules adsorbed on to an incipient β-sheet is suggested as a mechanism whereby crystals of the title peptide were obtained from a solution of tBoc-Pro-D-Ala-Ala-NHCH₃.     

Crystal structures of a nonapeptide helix containing α, α-di-n-butylglycine (Dbg), Boc-G1y-Dbg-Ala-Val-Ala-Leu-Aib-Val-Leu-OMe

Two crystal structures of a nonapeptide (anhydrous and hydrated) containing the amino acid residue α,α-di-n-butylglycyl, reveal a mixed 3₁₀-α-helical conformation. Residues 1-7 adopt φ, ψ values in the helical region, with Val(8) being appreciably distorted. The Dbg residue has φ, ψ values of -40, -37° and -46, -407° in the two crystals with the two butyl side chains mostly extended in each. Peptide molecules in the crystals pack into helical columns. The crystal parameters are: C₅₀ H₉₁ N₉ O₁₂, space group P2₁, with a= 9.789(1)Å;, b= 20.240(2) Å. c= 15.998(3) Å. β= 103.97(1): Z= 2, R=10.3% for 1945 data observed < 3σ(F) and C₅₀H₉₁N₉O₁₂· 3H₂O, space group P2₁ with a= 9.747(3)Å, b= 21.002(8) Å, c= 15.885(6) Å, β= 102.22(3). Z= 2. R=13.6% for 2535 data observed < 3σ(F) The observation of a helical conformation at Dbg suggests that the higher homologs in the α,α-dialkylated glycine series also have a tendency to stabilize peptide helices. © Munksgaard 1996.     

Conformational investigation of α,β-dehydropeptides Part VI. Molecular and crystal structure of benzyloxycarbonylglycyl-(Z)-dehydrophenylalanine

The structure of a peptide containing C-terminal dehydrophenylalanine, Z-Gly-(Z)-δPhe (C₁₉H₁₈N₂O₅, MW = 354) was determined from single-crystal X-ray diffraction data. Needle-shaped crystals were grown from a 1:1 mixture of methanol-acetone in the monoclinic space group P2₁ with a= 14.717(4), b= 4.941(2), c= 12.073(4) Å, β= 103.72(4)?; V= 852.86(8) ų, Z= 2 and D_c= 1.32 g cm ^?3. The structure was solved by direct methods using SHELXS-86 and refined to a final R-index of 0.032 for 1714 observed reflections. The peptide adopts a conformation folded at the glycine residue, and principal torsion angles are ω₀= 167.6(2)?, pHGR;₁= -71.8(3)?, ψ₁= -31.6(4)?, ω_l= - 165.7(3)?, pHGR;₂= 65.6(4)?, ψ1/2 = -174.4(3)? and ψ2/2 = 5.2(4)?. Two intermolecular hydrogen bonds, N₁—H…O_o and O₂—H…O′₁, join the folded molecules into columns and link columns to each other, respectively. FTIR spectroscopy shows the presence of three hydrogen bonds. This third one has been interpreted as an intramolecular hydrogen bond of the N₂—H…N₁ type. © Munksgaard 1994.     

Conformational study of a peptide epitope shows large preferences for β-turn conformations

The conformational preferences of the 7-residue peptide Glu-Val-Val-Pro-His-Lys-Lys was investigated using a global search algorithm, namely the Electrostatically Driven Monte Carlo (EDMC) method, and the ECEPP/2 potential energy function. This particular sequence corresponds to the N-terminal portion of a 19-residue peptide antigen whose three dimensional structure, when complexed to a cognate antibody, was reported recently. As a result of this study a series of low-energy conformations were identified showing a common folding pattern with residues Val-3, Pro-4, His-5 and Lys-6 forming a β turn. A comparison of the computed conformations with the one determined by X-ray crystallography in the antibody-antigen complex reveals marked similarities. In most of the cases rms deviations smaller than 1.1 Å were found for the backbone atoms of the four residues forming the turn. These results suggest that the recognition process is accomplished in this case through the interaction of the antibody with relatively stable conformers of the antigenic peptide.     

Pharmacological profiles of β-funaltrexamine (β-FNA) and β-chlornaltrexamine (β-CNA) on the mouse vas deferens preparation

The profiles of action of β-funaltrexamine (β-FNA) and β-chlornaltrexamine (β-CNA) have been assessed in the mouse vas deferens preparation. β-FNA, but not β-CNA, demonstrated a reversible agonist action that appeared to be mediated via κ-receptor interaction. β-CNA produced an irreversible antagonism of μ-, κ- and δ-mediated agonist actions, whereas β-FNA irreversibly antagonized μ-mediated agonist effects only. This selective action of β-FNA could also be seen following administration in vivo. β-CNA and particularly β-FNA should prove valuable in the elucidation of multiple opioid receptors.     

Synthesis and radiolabelling of Re(CO)3‐β‐elemene derivatives as potential therapeutic radiopharmaceuticals

β‐Elemene, (1S, 2S, 4R)‐(?)‐(1‐methy‐1‐vinyl‐2,4‐diisopropenyl cyclohexane) is an anticancer agent from the Traditional Chinese Herb Medicinal. Three novel Re(CO)₃‐β‐elemene derivatives including their radioactive conjugates containing N,N,N tridentate ligands and tricarbonyl rhenium (complex 12, 13, 14) were synthesized. Their structures were characterized by infrared (IR), ¹H‐NMR and HRMS. Good radioactive yield (above 90%) and radioactive chemical purity with Re‐188 (above 95%) were obtained for all of the three derivatives (complex 15, 16, 17). The antiproliferative activity of non‐radioactive β‐elemene‐Re(CO)₃ derivatives on Lewis lung cancer cells and HeLa cell lines were evaluated by WST‐1 methods. The result shows substantial decrease in IC₅₀ values compared with the parent compound β‐elemene. The synthesis and radiosynthesis of β‐elemene tricarbonyl rhenium conjugates provide the possibility to find a new kind of potential radiopharmaceuticals on β‐elemene. Copyright © 2009 John Wiley & Sons, Ltd.     

Secondary structure of a dynamic type I’β‐hairpin peptide

Abstract: A spontaneously folding β‐hairpin peptide (Lys‐Lys‐Tyr‐Thr‐Val‐Ser‐Ile‐Asn‐Gly‐Lys‐Lys‐Ile‐Thr‐Val‐Ser‐Ile) and related cyclic (cyclo‐Gly‐Lys‐Tyr‐Ile‐Asn‐Gly‐Lys‐Ile‐Ile‐Asn) and linear (Ser‐Ile‐Asn‐Gly‐Lys) controls were studied to determine the effects of various factors on secondary structure. Secondary structure was evaluated using circular dichroism (CD) and 1D and 2D ¹H nuclear magnetic resonance (NMR). The effects of chemical modifications in the peptide and various solution conditions were investigated to determine their impact on peptide structure. The β‐hairpin peptide displayed a CD minimum at 216 nm and a TOCSY i + 1 ? i + 2 and i + 2 ?i + 3 interaction, confirming the expected structure. Using NMR α‐proton (H) chemical shifts, the extents of folding of the β‐hairpin and linear control were estimated to be 51 and 25% of the cyclic control (pH 4, 37 °C), which was taken to be maximally folded. Substitution of iso‐aspartic acid for Asn reduced the secondary structure dramatically; substitution of aspartic acid for Asn also disrupted the structure. This result suggests that deamidation in unconstrained β‐turns may have adverse effects on secondary structure. N‐terminal acetylation and extreme pH conditions also reduced structure, while the addition of methanol increased structure.     

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.     

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.     

Radiosynthesis of [123I]N‐(3‐iodoprop‐(2E)‐enyl)‐2α‐(imino‐methyl)‐3β‐(3′,4′‐dichlorophenyl) nortropane as a potential SPET tracer for dopamine transporter

The radiosynthesis of a novel tropane derivative [¹²³I]KUC‐25019, [[¹²³I];N‐(3‐iodoprop‐(2E)‐enyl)‐2α‐(imino‐methyl)‐3β‐(3′,4′‐dichlorophenyl)nortropane], a potential inhibitor of the dopamine transporter is reported. The synthetic routes include the preparation of standard reference, the stannyl precursor and the ¹²³I‐labeling synthesis. The no‐carrier‐added ¹²³I‐labeling has about 20% yield, the specific activity of [¹²³I]KUC‐25019 is > 107 GBq/µmol and the radiochemical purity of [¹²³I] KUC‐25019 is >95%. Copyright © 2002 John Wiley & Sons, Ltd.     

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.     

Antinociceptive Synergism of MD‐354 and Clonidine. Part II. The α2‐Adrenoceptor Component

Abstract: Previously, we reported that antinociceptive synergism of a 5‐HT₃/α₂‐adrenoceptor ligand MD‐354 (m‐chlorophenylguanidine) and clonidine combination occurs, in part, through a 5‐HT₃ receptor antagonist mechanism. In the present investigation, a possible role for α₂‐adrenoceptors was examined. Mechanistic studies using yohimbine (a subtype non‐selective α₂‐adrenoceptor antagonist), BRL 44408 (a preferential α_2A‐adrenoceptor antagonist) and imiloxan (a preferential α_2B/C‐adrenoceptor antagonist) on the antinociceptive actions of a MD‐354/clonidine combination were conducted. Subcutaneous pre‐treatment with all three antagonists inhibited the antinociceptive synergism of MD‐354 and clonidine in the mouse tail‐flick assay in a dose‐dependent manner (AD₅₀ = 0.33, 2.1, and 0.17 mg/kg, respectively). Enhancement of clonidine antinociception by MD‐354 did not potentiate clonidine’s locomotor suppressant activity in a mouse locomotor assay. When [ethyl‐³H]RS‐79948‐197 was used as radioligand, MD‐354 displayed almost equal affinity to α_2A‐ and α_2B‐adrenoceptors (K_i = 110 and 220 nM) and showed lower affinity at α_2C‐adrenoceptors (K_i = 4,700 nM). MD‐354 had no subtype‐selectivity for the α₂‐adrenoceptor subtypes as an antagonist in functional [³⁵S]GTPγS binding assays. MD‐354 was a weak partial agonist at α_2A‐adrenoceptors. Overall, in addition to the 5‐HT₃ receptor component, the present investigation found MD‐354 to be a weak partial α_2A‐adrenoceptor agonist that enhances clonidine’s thermal antinociceptive actions through an α₂‐adrenoceptor‐mediated mechanism without augmenting sedation.     

Fast and efficient peptide bond formation using bis‐[α,α‐bis(trifluoromethyl)‐benzyloxy]diphenylsulfur. Part I

Abstract: This study towards the development of sulfurane‐based coupling agents shows that bis‐[α,α‐bis(trifluoromethyl)‐benzyloxy]diphenylsulfur (BTBDS) can facilitate rapid amide bond formation between N^α‐urethane‐protected l ‐amino acids and l ‐phenylalanine ethyl ester in the absence of an external base. The corresponding dipeptide esters were obtained in excellent yields and with no detectable racemization, as judged by analysis of the formed dipeptides by chiral‐phase HPLC. In addition, BTBDS‐mediated condensation of benzoyl‐l ‐phenylalanine with l ‐phenylalanine ethyl ester was also investigated. The results indicate that sulfuranes can be useful for application in racemization‐sensitive systems, such as segment condensation.