Conformational comparison of µ‐selective endomorphin‐2 with its C‐terminal free acid in DMSO solution,by 1H NMR spectroscopy and molecular modeling calculation

Abstract: In order to make clear the structural role of the C‐terminal amide group of endomorphin‐2 (EM2, H‐Tyr‐Pro‐Phe‐Phe‐NH₂), an endogenous µ‐receptor ligand, in the biological function, the solution conformations of endomorphin‐2 and its C‐terminal free acid (EM2OH, H‐Tyr‐Pro‐Phe‐Phe‐OH), studied using two‐dimensional ¹H NMR measurements and molecular modeling calculations, were compared. Both peptides were in equilibrium between the cis and trans isomers around the Tyr‐Pro ω bond in a population ratio of ≈ 1 : 2. The lack of significant temperature and concentration dependence of NH protons suggested that the NMR spectra reflected the conformational features of the respective molecules themselves. Fifty possible 3D structures for the each isomer were generated by the dynamical simulated annealing method under the proton?proton distance constraints derived from the ROE cross‐peaks. These energy‐minimized conformers, which were all in the φ torsion angles estimated from J_NHCαH coupling constants within ± 30°, were then classified in groups one or two according to the folding backbone structures. All trans and cis EM2 conformers adopt an open conformation in which their extended backbone structures are twisted at the Pro²–Phe³ moiety. In contrast, the trans and cis conformers of EM2OH show conformational variation between the ‘bow’‐shaped extended and folded backbone structures, although the cis conformers of its zwitterionic form are refined into the folded structure of the close disposition of C‐ and N‐terminal groups. These results indicate clearly that the substitution of carboxyl group for C‐terminal amide group makes the peptide flexible. The conformational requirement for µ‐receptor activation has been discussed based on the active form proposed for endomorphin‐1 and by comparing conformational features of EM2 and EM2OH.     

Antibacterial,antitumor and hemolytic activities of α‐helical antibiotic peptide,P18 and its analogs

Abstract: The α‐helical antibiotic peptide (P18: KWKLFKKIPKFLHLAKKF‐NH₂) designed from the cecropin A(1–8)–magainin 2 (1?12) hybrid displayed strong bactericidal and tumoricidal activity without inducing hemolysis. The effect of the Pro⁹ residue at central position of P18 on cell selectivity was investigated by Pro⁹ → Leu or Pro⁹ → Ser substitution. Either substitution markedly reduced the antibacterial activity of P18 and increased hemolysis, although it did not significantly affect cytotoxicity against human transformed tumor and normal fibroblast cells. These results suggest that a proline kink in α‐helical antibiotic peptide P18 serves as a hinge region to facilitate ion channel formation on bacterial cell membranes and thus plays an important role in providing high selectivity against bacterial cells. Furthermore, to investigate the structure?antibiotic activity relationships of P18, a series of N‐ or C‐terminal deletion and substitution analogs of P18 were synthesized. The C‐terminal region of P18 was related to its antibiotic activity and α‐helical conformation on lipid membranes rather than N‐terminal one. Higher α‐helicity of the peptides was involved in the hemolytic and antitumor activity rather than antibacterial activity. Except for [L⁹]‐P18 and [S⁹]‐P18, all the designed peptides containing a Pro residue showed potent antibacterial activity, although they did not induce a cytolytic effect against human erythrocyte and normal fibroblast cells at the concentration required to kill bacteria. In particular, P18 and some analogs (N‐1, N‐2, N‐3, N‐3L and N‐4L) with potent bactericidal and tumoricidal activity and little or no normal cell toxicity may serve as an attractive candidate for the development of novel anti‐infective or antitumor agents.     

Conformational properties of hybrid peptides containing α‐ and ω‐amino acids*

Abstract: This review briefly surveys the conformational properties of guest ω‐amino acid residues when incorporated into host α‐peptide sequences. The results presented focus primarily on the use of β‐ and γ‐residues in αω sequences. The insertion of additional methylene groups into peptide backbones enhances the range of accessible conformations, introducing additional torsional variables. A nomenclature system, which permits ready comparisons between α‐peptides and hybrid sequences, is defined. Crystal structure determination of hybrid peptides, which adopt helical and β‐hairpin conformations permits the characterization of backbone conformational parameters for β‐ and γ‐residues inserted into regular α‐polypeptide structures. Substituted β‐ and γ‐residues are more limited in the range of accessible conformation than their unsubstituted counterparts. The achiral β,β‐disubstituted γ‐amino acid, gabapentin, is an example of a stereochemically constrained residue in which the torsion angles about the C^β–C^γ (θ₁) and C^α–C^β (θ₂) bonds are restricted to the gauche conformation. Hybrid sequences permit the design of novel hydrogen bonded rings in peptide structures.     

Bioactive N‐terminal undecapeptides derived from parathyroid hormone: the role of α‐helicity*

Abstract: The N‐terminal 1–34 segment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it can reproduce all biological responses in bone characteristic of the native intact PTH. Recent studies have demonstrated that N‐terminal fragments presenting the principal activating domain such as PTH(1–11) and PTH(1–14) with helicity‐enhancing substitutions yield potent analogues with PTH(1–34)‐like activity. To further investigate the role of α‐helicity on biological potency, we designed and synthesized by solid‐phase methodology the following hPTH(1–11) analogues substituted at positions 1 and/or 3 by the sterically hindered and helix‐promoting C^α‐tetrasubstituted α‐amino acids α‐amino isobutyric acid (Aib), 1‐aminocyclopentane‐1‐carboxylic acid (Ac₅c) and 1‐aminocyclohexane‐1‐carboxylic acid (Ac₆c): Ac₅c‐V‐Aib‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( I ); Aib‐V‐Ac₅c‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( II ); Ac₆c‐V‐Aib‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( III ); Aib‐V‐Ac₆c‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( IV ); Aib‐V‐Aib‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( V ); S‐V‐Aib‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( VI ), S‐V‐Ac₅c‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( VII ); Ac₅c‐V‐S‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( VIII ); Ac₆c‐V‐S‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( IX ); Ac₅c‐V‐Ac₅c‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( X ); Ac₆c‐V‐Ac₆c‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( XI ). All analogues were biologically evaluated and conformationally characterized in 2,2,2‐trifluoroethanol (TFE) solution by circular dichroism (CD). Analogues I – V , which cover the full range of biological activity observed in the present study, were further conformationally characterized in detail by nuclear magnetic resonance (NMR) and computer simulations studies. The results of ligand‐stimulated cAMP accumulation experiments indicated that analogues I and II are active, analogues III , VI and VII are very weakly active and analogues IV , V , VIII–XI are inactive. The most potent analogue, I exhibits biological activity 3500‐fold higher than that of the native PTH(1–11) and only 15‐fold weaker than that of the native sequence hPTH(1–34). Remarkably, the two most potent analogues, I and II , and the very weakly active analogues, VI and VII , exhibit similar helix contents. These results indicate that the presence of a stable N‐terminal helical sequence is an important but not sufficient condition for biological activity.     

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.     

Prediction of γ‐turns from amino acid sequences

Abstract: We predicted γ‐turns from amino acid sequences using the first‐order Markov chain theory and enlarged representative data sets corresponding to protein chains selected from the Protein Data Bank (PDB). The following data sets were used for training and deriving the probability values: (1) an initial data set containing 315 protein chains comprising 904 γ‐turns and (2) a later data set in order to include new entries in the PDB, containing 434 protein chains and comprising 1053 γ‐turns. By excluding 93 protein chains that were common to these two training data sets, we generated two mutually exclusive data sets containing 222 and 341 protein chains for testing our predictions. Applying amino acid probability values derived from training data sets on to testing data sets yielded overall prediction accuracies in the range 54–57%. We recommend the use of probability values derived from the data set comprising 315 protein chains that represents more γ‐turns and also provides better predictions.     

Potent Opioid Peptide Agonists Containing 4′‐[N‐((4′‐phenyl)‐phenethyl)carboxamido]phenylalanine (Bcp) in Place of Tyr

Analogues of the opioid peptides H‐Tyr‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ (non‐selective), H‐Tyr‐d ‐Arg‐Phe‐Lys‐NH₂ (μ‐selective) and dynorphin A(1‐11)‐NH₂ (κ‐selective) containing 4′‐[N‐((4′‐phenyl)‐phenethyl)carboxamido]phenylanine (Bcp) in place of Tyr¹ were synthesized. All three Bcp¹‐opioid peptides retained high μ opioid receptor binding affinity, but showed very significant differences in the opioid receptor selectivity profiles as compared with the corresponding Tyr¹‐containing parent peptides. The cyclic peptide H‐Bcp‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ turned out to be an extraordinarily potent, μ‐selective opioid agonist, whereas the Bcp¹‐analogue of dynorphin A(1‐11)‐NH₂ displayed partial agonism at the μ receptor. The obtained results suggest that the large biphenylethyl substituent contained in these compounds may engage in a hydrophobic interaction with a receptor subsite and thereby may play a role in the ligand’s ability to induce a specific receptor conformation or to bind to a distinct receptor conformation in a situation of conformational receptor heterogeneity.     

Synthesis of [1,3, NH2‐15N3] (5′S)‐8,5′‐cyclo‐2′‐deoxyguanosine

To facilitate NMR studies and low‐level detection in biological samples by mass spectrometry, [1,3, NH₂‐¹⁵N₃] (5′S)‐8,5′‐cyclo‐2′‐deoxyguanosine was synthesized from imidazole‐4,5‐dicarboxylic acid in 21 steps. The three ¹⁵N isotopes were introduced during the chemo‐enzymatic preparation of [1,3, NH₂‐¹⁵N₃]‐2′‐deoxyguanosine using an established procedure. The ¹⁵N‐labeled 2′‐deoxyguanosine was converted to a 5′‐phenylthio derivative, which allowed the 8‐5′ covalent bond formation via photochemical homolytic cleavage of the C–SPh bond. SeO₂ oxidation of C‐5′ followed by sodium borohydride reduction and deprotection gave the desired product in good yield. The isotopic purity of the [1,3, NH₂‐¹⁵N₃] (5′S)‐8,5′‐cyclo‐2′‐deoxyguanosine was in excess of 99.94 atom% based on liquid chromatography–mass spectrometry measurements. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

Synthesis,Biological Activity,and NMR‐Based Structural Studies of Deltorphin I Analogs Modified in Message Domain with a New α,α‐Disubstituted Glycines

This article describes new deltorphin I analogs in which phenylalanine residues were replaced by the corresponding (R) or (S)‐α‐benzyl‐β‐azidoalanine, α‐benzyl‐β‐(1‐pyrrolidinyl)alanine, α‐benzyl‐β‐(1‐piperidinyl)alanine, and α‐benzyl‐β‐(4‐morpholinyl)‐alanine residues. The potency and selectivity of the new analogs were evaluated by a competitive receptor binding assay in the rat brain using [³H]DAMGO (a μ ligand) and [³H]DELT (a δ ligand). The affinity of analogs containing (R) or (S)‐α‐benzyl‐β‐azidoalanine in position 3 to δ‐receptors strongly depended on the chirality of the α,α‐disubstituted residue. The conformational behavior of peptides modified with (R) or (S)‐α‐benzyl‐β‐(1‐piperidinyl)Ala, which displays the opposite selectivity, was analyzed by ¹H and ¹³C NMR. The μ‐selective Tyr‐d ‐Ala‐(R)‐α‐benzyl‐β‐(1‐piperidinyl)Ala‐Asp‐Val‐Val‐Gly‐NH₂ lacks the helical conformation observed in the δ‐selective Tyr‐d ‐Ala‐(S)‐α‐benzyl‐β‐(1‐piperidinyl)Ala‐Asp‐Val‐Val‐Gly‐NH₂. Our results support the proposal that differences between δ‐ and μ‐selective opioid peptides are attributable to the presence or absence of a spatial overlap between the N‐terminal message domain and the C‐terminal address domain.     

Identification of five pyrrolidinyl substituted cathinones and the collision‐induced dissociation of electrospray‐generated pyrrolidinyl substituted cathinones

This article reports on the analytical properties of five pyrrolidinyl substituted cathinones: α ‐pyrrolidinononaphenone (α ‐PNP, 1 ), 4‐chloro‐α ‐pyrrolidinopropiophenone (4‐Cl‐α ‐PPP, 2 ), 4‐chloro‐α ‐pyrrolidinovalerophenone (4‐Cl‐α ‐PVP, 3 ), 5‐dihydrobenzofuranpyrovalerone (5‐DBFPV, 4 ), and 2‐(pyrrolidin‐1‐yl)‐1‐(5,6,7,8‐tetrahydronaphthalen‐2‐yl)hexan‐1‐one (β ‐THNPH, 5 ). These identifications were based on liquid chromatography–quadrupole time‐of‐flight‐mass spectrometry (LC–QTOF–MS), gas chromatography–mass spectrometry (GC–MS) and nuclear magnetic resonance spectroscopy (NMR). To our knowledge, no analytical data about α ‐PNP, 4‐Cl‐α ‐PPP, 4‐Cl‐α ‐PVP, and β ‐THNPH have appeared until now, making this the first report on these compounds. Moreover, in order to study the collision‐induced dissociation (CID) characteristic fragmentation routes of pyrrolidinyl substituted cathinones, a total number of 13 pyrrolidinyl substituted cathinones were selected and discussed. The major fragmentation pathways under CID mode are produced, leading to the formation of characteristic ions. Product ions of [M‐C₄H₉N]⁺ and C_nH_2nN⁺ indicate the presence of pyrrolidinyl substitution. Characteristic fragments are also produced via the cleavages of the CH–N(CH₂)₄ bond and the CO‐CHN bond. Copyright © 2016 John Wiley & Sons, Ltd.     

Incorporation of vinylogous scaffolds in the C‐terminal tripeptide of substance P

Abstract: Glycine‐9 and leucine‐10 of substance P (SP) are critical for (NK)‐1 receptor recognition and agonist activity. Proψ(Z)‐CH=CH(CH₃)‐CONH)Leu (or Met) and Proψ((E)‐CH=CH(CH₃)‐CONH)Leu (or Met) have been introduced in the sequence of SP, in order to restrict the conformational flexibility of the C‐terminal tripeptide, Gly‐Leu‐Met‐NH₂, of SP. Proψ((Z)‐CH=C(CH₂CH(CH₃)₂)‐CONH)Met‐NH₂, with an isobutyl substituent to mimic the Leu side‐chain, was also incorporated in place of the C‐terminal tripeptide. The substituted‐SP analogs were tested for their affinity to human NK‐1 receptor specific binding sites (NK‐1M and NK‐1m) and their potency to stimulate adenylate cyclase and phospholipase C in Chinese Hamster ovary (CHO) cells transfected with the human NK‐1 receptor. The most potent SP analogs [Pro⁹ψ((Z)CH=C(CH₃)CONH)Leu¹⁰]SP and [Pro⁹ψ ((E)CH=C(CH₃)CONH)Leu¹⁰]SP, are about 100‐fold less potent than SP on both binding sites and second messenger pathways. These vinylogous (Z)‐ or (E)‐CH=C(CH₃)‐ or (Z)‐CH=C(CH₂CH(CH₃)₂) moieties hamper the correct positioning of the C‐terminal tripeptide of SP within both the NK‐1M‐ and NK‐1m‐specific binding sites. The origin of these lower potencies is related either to an incorrect peptidic backbone conformation and/or an unfavorable receptor interaction of the methyl or isobutyl group.     

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.     

Comparative biological activities of potent active-site analogues of α-melanotropin. Effect of tyrosine substitution at position-4

We have prepared several α-melanotropin (α-MSH) analogues with tyrosine substituted for methionine at the 4-position and determined their melanotropic activities on the frog (Rana pipiens), lizard (Anolis carolinensis) and S-91 (Cloudman) mouse melanoma adenylate cyclase bioassays. The potencies of Ac-[Tyr⁴]-α-MSH_4–10-NH₂ and Ac-[Tyr⁴]-α-MSH_4–11-NH₂ were compared with α-MSH and with their corresponding methionine and norleucine substituted analogues. The Tyr-4 analogues were found to be less active than the Nle-4 analogues on both the frog and lizard assays. Ac-[Tyr⁴]-α-MSH_4–10-NH₂ was found to be less active than Ac-[Tyr⁴]-α-MSH_4–11-NH₂ on the lizard bioassay, but more active than the longer fragment on the frog skin assay. Ac-[Tyr⁴]-α-MSH_4–10-NH₂ exhibited extremely prolonged biological activity on frog skin, but not on lizard skin, while the melanotropic activity of Ac-[Tyr⁴]-α-MSH_4–11-NH₂ was rapidly reversed on both assay systems. The increased potency of Ac-[Tyr⁴]-α-MSH_4–10-NH₂ over Ac-[Tyr⁴]-α-MSH_4–11-NH₂ on frog melanocytes may be related to the fact that the shorter 4–10 analogue exhibits prolonged biological activity. Interestingly, it was found that both Tyr-4 analogues were partial agonists on the mouse melanoma adenylate cyclase bioassay, and stimulated the enzyme to only about 50% of the maximal activity of α-MSH. We reported previously that replacement of L-Phe-7 by its D-enantiomer in [Nle⁴]-α-MSH and its Nle-4 containing analogues resulted in peptides with increased potency and in some instances prolonged activity. Similarly, incorporation of D-Phe-7 into Tyr-4 containing melanotropin fragments produced analogues Ac-[Tyr⁴, D-Phe⁷]-αMSH_4–10-NH₂ and Ac-[Tyr⁴, D-Phe⁷]-α-MSH_4–11-NH₂, which also exhibited greatly increased biological activity in all three assay systems. Both of these analogues were also found to have prolonged activity in the frog skin bioassay but little or no prolonged activity in the lizard skin bioassay. These two analogues turned out to be full agonists in the mouse melanoma adenylate cyclase bioassay and were equipotent to α-MSH. These results demonstrate that substitution of tyrosine for methionine at position-4 dramatically affects the potency and prolonged activity of these melanotropin analogues and the melanotropic activities observed as a result of such substitutions are themselves affected by concomitant substitutions at the 7(Phe) and 11 (Lys) positions of the analogues.     

Structures of neuropeptide γ from goldfish and mammalian neuropeptide γ, as determined by 1H NMR spectroscopy

Abstract: Neuropeptide γ belongs to tachykinin families which have a common C‐terminal amino acid sequence (Phe‐X‐Leu‐Met‐NH₂) and which induce various biological responses including salivation, hypotension, and contraction of gastrointestinal, respiratory, and urinary smooth muscle. In the present study, we present the solution structures of neuropeptide γ (NPγ) from gold fish (G‐NPγ) and mammalian NPγ (M‐NPγ), as determined by nuclear magnetic resonance (NMR) spectroscopy in 50% trifluoroethanol (TFE)/water (1 : 1, v/v) solution and 200 mm sodium dodecyl sulfate (SDS) micelles. In aqueous TFE solution, G‐NPγ has a α‐helical conformation in the region of His¹²–Met²¹ and a short helix in the N‐terminal region, and has a β‐turn from Arg⁹ to Arg¹¹ in between. In aqueous TFE solution, M‐NPγ also has α‐helical conformations both in the C‐terminal region and the N‐terminal region and a β‐turn from His⁹ to Arg¹¹ in between. In SDS micelle, the structure of G‐NPγ contains a stable α‐helix from His¹² to Met²¹ and a β‐turn from Arg⁹ to Arg¹¹, while M‐NPγ has a short helix from Ser¹⁶ to Met²¹. The region from His¹² to Met²¹ corresponds to the amino acid sequence of neurokinin A. Neuropeptide γ may act as a precursor of neurokinin A and the post‐translational processing of this peptide involves the enzymatic attack of the basic β‐turn region from residue 9 to residue 11 in the middle. From our relaxation study, it could be suggested that in fish system G‐NPγ induces the biological actions corresponding to those of substance P in mammalian system. The structures of G‐NPγ and M‐NPγ contain α‐helical structures at the C‐terminus and this helix seems to promote the affinity for NK₁ and/or NK₂ receptor.     

Synthesis of an M + 7 stable isotope of γ‐cyhalothrin

γ‐Cyhalothrin is a single isomer, synthetic pyrethroid insecticide. This material was originally developed and marketed by Pytech Chemicals, a joint venture between Dow AgroSciences and Cheminova A/S. Cheminova A/S now wholly owns Pytech Chemical. As a part of registration studies, there was a need for a stable isotope of γ‐cyhalothrin to serve as an internal standard. This paper will discuss the 11‐step synthesis that was used to prepare an M + 7 stable isotope of γ‐cyhalothrin by utilizing triethyl phosphonoacetate‐d₂ and acetone‐d₆ to incorporate deuterium into the molecule. In the end, the diastereomers were separated by preparative reverse‐phase HPLC to give γ‐cyhalothrin‐d₇ in an overall yield of 6%. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthesis of all‐trans‐[10′‐3H]‐8′‐apo‐β‐carotenoic acid

The enzyme, 15,15′‐β‐carotene dioxygenase (BCDOX), facilitates the oxidation of β‐carotene to yield retinal. This is a remarkable process in which one of 11 double bonds in β‐carotene is selectively oxidized. To further probe the mechanistic aspects of BCDOX, the synthesis of all‐trans‐[10′‐³H]‐8′‐apo‐β‐carotenoic acid is reported. This compound will be used as a photoaffinity labeling reagent to probe the β‐carotene binding pocket within BCDOX. The synthesis outlines a simple and efficient route for the incorporation of tritium at the 10′ olefinic carbon of 8′‐apo‐β‐carotenoic acid. Copyright © 2002 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.     

Biological and conformational study of β‐substituted prolines in MT‐II template: steric effects leading to human MC5 receptor selectivity*

Abstract: To investigate the molecular basis for the interaction of the χ‐constrained conformation of melanotropin peptide with the human melanocortin receptors, a series of β‐substituted proline analogs were synthesized and incorporated into the Ac‐Nle‐c[Asp‐His‐d ‐Phe‐Arg‐Trp‐Lys]‐NH₂ (MT‐II) template at the His⁶ and d ‐Phe⁷ positions. It was found that the binding affinities generally diminished as the steric bulk of the p‐substituents of the 3‐phenylproline residues increased. From (2S, 3R)‐3‐phenyl‐Pro⁶ to (2S, 3R)‐3‐(p‐methoxyphenyl)‐Pro⁶ analogs the binding affinity decreased 23‐fold at the human melanocortin‐3 receptor (hMC3R), 17‐fold at the hMC4R, and eight‐fold at the hMC5R, but selectivity for the hMC5R increased. In addition, the substitution of the d ‐Phe⁷ residue with a (2R, 3S)‐3‐phenyl‐Pro resulted in greatly reduced binding affinity (10³–10⁵) at these melanocortin receptors. Macromodel's Large Scale Low Mode (LLMOD) with OPLS‐AA force field simulations revealed that both MT‐II and SHU‐9119 share a similar backbone conformation and topography with the exception of the orientation of the side chains of d ‐Phe⁷/d ‐Nal (2′)⁷ in χ space. Introduction of the dihedrally constrained phenylproline analogs into the His⁶ position (analogs 2 – 6 ) caused topographical changes that might be responsible for the lower binding affinities. Our findings indicate that hMC3 and hMC4 receptors are more sensitive to steric effects and conformational constraints than the hMC5 receptor. This is the first example for melanocortin receptor selectivity where the propensity of steric interactions in χ space of β‐modified Pro⁶ analogs of MT‐II has been shown to play a critical role for binding as well as bioefficacy of melanotropins at hMC3 and hMC4 receptors, but not at the hMC5 receptor.