Conformational analysis of endomorphin‐1 by molecular dynamics methods

Abstract: Endomorphin‐1 (EM1, H‐Tyr‐Pro‐Trp‐Phe‐NH₂) is a highly potent and selective agonist for the μ‐opioid receptor. A conformational analysis of this tetrapeptide was carried out by simulated annealing and molecular dynamics methods. EM1 was modeled in the neutral (NH₂‐) and cationic (NH‐) forms of the N‐terminal amino group. The results of NMR measurements were utilized to perform simulations with restrained cis and trans Tyr¹‐Pro² peptide bonds. Preferred conformational regions in the Φ₂–Ψ₂, Φ₃–Ψ₃ and Φ₄–Ψ₄ Ramachandran plots were identified. The g(+), g(?) and trans rotamer populations of the side‐chains of the Tyr¹, Trp³ and Phe⁴ residues were determined in χ₁ space. The distances between the N‐terminal N atom and the other backbone N and O atoms, and the distances between the centers of the aromatic side‐chain rings and the Pro² ring were measured. The preferred secondary structures were determined as different types of β‐turns and γ‐turns. In the conformers of trans‐EM1, an inverse γ‐turn can be formed in the N‐terminal region, but in the conformers of cis‐EM1 the N‐terminal inverse γ‐turn is absent. Regular and inverse γ‐turns were observed in the C‐terminal region in both isomers. These β‐ and γ‐turns were stabilized by intramolecular H‐bonds and bifurcated H‐bonds.     

Synthesis and evaluation of potential affinity labels derived from endomorphin‐2

Abstract: In an attempt to identify potential peptide‐based affinity labels for opioid receptors, endomorphin‐2 (Tyr‐Pro‐Phe‐PheNH₂), a potent and selective endogenous ligand for µ‐opioid receptors, was chosen as the parent peptide for modification. The tetrapeptide analogs were prepared using standard Fmoc‐solid phase peptide synthesis in conjunction with incorporation of Fmoc‐Phe(p‐NHAlloc) and modification of the p‐amino group. The electrophilic groups isothiocyanate and bromoacetamide were introduced into the para position on either Phe³ or Phe⁴; the corresponding free amine‐containing peptides were also prepared for comparison. The peptides bearing an affinity label group and their free amine analogs were evaluated in a radioligand‐binding assay using Chinese hamster ovary (CHO) cells expressing µ‐ and δ‐opioid receptors. Modification on Phe⁴ was better tolerated than on Phe³ for µ‐receptor binding. Among the analogs tested, [Phe(p‐NH₂)⁴]endomorphin‐2 showed the highest affinity (IC₅₀ = 37 nm ) for µ‐receptors. The Phe(p‐NHCOCH₂Br)⁴ analog displayed the highest µ‐receptor affinity (IC₅₀ = 158 nm ) among the peptides containing an affinity label group. Most of the compounds exhibited negligible binding affinity for δ‐receptors, similar to the parent peptide.     

Novel endomorphin‐2 analogs with μ‐opioid receptor antagonist activity

Abstract: A series of position 4‐substituted endomorphin‐2 (Tyr‐Pro‐Phe‐Phe‐NH₂) analogs containing 3‐(1‐naphthyl)‐alanine (1‐Nal) or 3‐(2‐naphthyl)‐alanine (2‐Nal) in l ‐ or d ‐configuration, was synthesized. The opioid activity profiles of these peptides were determined in the μ‐opioid receptor representative binding assay and in the Guinea‐Pig Ileum assay/Mouse Vas Deferens assay (GPI/MVD) bioassays in vitro, as well as in the mouse hot‐plate test of analgesia in vivo. In the binding assay the affinity of all new analogs for the μ‐opioid receptor was reduced compared with endomorphin‐2. The two most potent analogs were [d ‐1‐Nal⁴]‐ and [d ‐2‐Nal⁴]endomorphin‐2, with IC₅₀ values 14 ± 1.25 and 19 ± 2.1 nm , respectively, compared with 1.9 ± 0.21 nm for endomorphin‐2. In the GPI assay these analogs were found to be weak antagonists and they were inactive in the MVD assay. The in vitro GPI assay results were in agreement with those obtained in the in vivo hot‐plate test. Antinociception induced by endomorphin‐2 was reversed by concomitant intracerebroventricula (i.c.v.) administration of [d ‐1‐Nal⁴]‐ and [d ‐2‐Nal⁴]‐endomorphin‐2, indicating that these analogs were μ‐opioid antagonists. Their antagonist activity was compared with that of naloxone. At a dose 5 μg per animal naloxone almost completely inhibited antinociceptive action of endomorphin‐2, while [d ‐1‐Nal⁴]endomorphin‐2 in about 46%.     

Binding of endomorphin‐2 to μ‐opioid receptors in experimental mouse mammary adenocarcinoma

Abstract: Endomorphin‐2 (Tyr‐Pro‐Phe‐Phe‐NH₂) binds with high affinity and selectivity to the μ‐opioid receptor. In the present study, [¹²⁵I]endomorphin‐2 has been used to characterize μ‐opioid‐binding sites on transplantable mouse mammary adenocarcinoma cells. Cold saturation experiments performed with [¹²⁵I]endomorphin‐2 (1 nm ) show biphasic binding curves in Scatchard coordinates. One component represents high affinity and low capacity (K_d = 18.79 ± 1.13 nm , B_max = 635 ± 24 fmol/mg protein) and the other shows low affinity and higher capacity (K_d = 7.67 ± 0.81 μm , B_max = 157 ± 13 pmol/mg protein) binding sites. The rank order of agonists competing for the [¹²⁵I]endomorphin‐2 binding site was [d ‐1‐Nal³]morphiceptin > endomorphin‐2 ? [d ‐Phe³]morphiceptin > morphiceptin > [d ‐1‐Nal³]endomorphin‐2, indicating binding of these peptides to μ‐opioid receptors. The uptake of ¹³¹I‐labeled peptides administered intraperitoneally to tumor‐bearing mice was also investigated. The highest accumulation in the tumor was observed for [d ‐1‐Nal³]morphiceptin, which reached the value of 8.19 ± 1.14% dose/g tissue.     

Synthesis of different types of dipeptide building units containing N‐ or C‐terminal arginine for the assembly of backbone cyclic peptides*

Abstract: Different types of dipeptide building units containing N‐ or C‐terminal arginine were prepared for synthesis of the backbone cyclic analogues of the peptide hormone bradykinin (BK: Arg‐Pro‐Pro‐Gly‐Phe‐Ser‐Pro‐Phe‐Arg). For cyclization in the N‐terminal sequence N‐carboxyalkyl and N‐aminoalkyl functionalized dipeptide building units were synthesized. In order to avoid lactam formation during the condensation of the N‐terminal arginine to the N‐alkylated amino acids at position 2, the guanidino function has to be deprotected. The best results were obtained by coupling Z‐Arg(Z)₂‐OH with TFFH/collidine in DCM. Another dipeptide building unit with an acylated reduced peptide bond containing C‐terminal arginine was prepared to synthesize BK‐analogues with backbone cyclization in theC‐terminus. To achieve complete condensation to the resin and to avoid side reactions during activation of the arginine residue, this dipeptide unit was formed on a hydroxycrotonic acid linker. HYCRAM^? technology was applied using the Boc‐Arg(Alloc)₂‐OH derivative and the Fmoc group to protect the aminoalkyl function. The reduced peptide bond was prepared by reductive alkylation of the arginine derivative with the Boc‐protected amino aldehyde, derived from Boc‐Phe‐OH. The best results for condensation of the branching chain to the reduced peptide bond were obtained using mixed anhydrides. Both types of dipeptide building units can be used in solid‐phase synthesis in the same manner as amino acid derivatives.     

Synthesis and biological evaluation of constrained analogues of the opioid peptide H‐Tyr‐d‐Ala‐Phe‐Gly‐NH2 using the 4‐amino‐2‐benzazepin‐3‐one scaffold

Abstract: The synthesis of conformationally restricted dipeptidic moieties 4‐amino‐1,2,4,5‐tetrahydro‐2‐benzazepin‐3‐one (Aba)‐Gly ([(4S)‐amino‐3‐oxo‐1,2,4,5‐tetrahydro‐1H‐2‐benzazepin‐2‐yl]‐acetic acid) and 8‐hydroxy‐4‐amino‐1,2,4,5‐tetrahydro‐2‐benzazepin‐3‐one (Hba)‐d ‐Ala ([(4S)‐amino‐8‐hydroxy‐3‐oxo‐1,2,4,5‐tetrahydro‐benzo[c]azepin‐2‐yl]‐propionic acid) was based on a synthetic strategy that uses an oxazolidinone as an N‐acyliminium precursor. Introducing these Aba scaffolds into the N‐terminal tetrapeptide of dermorphin (H‐Tyr‐d ‐Ala‐Phe‐Gly‐Tyr‐Pro‐Ser‐NH₂)‐induced remarkable shifts in affinity and selectivity towards the opioid μ‐ and δ‐receptors. This paper provides the synthesis and biological in vitro and in vivo evaluation of constricted analogues of the N‐terminal tetrapeptide H‐Tyr‐d ‐Ala‐Phe‐Gly‐NH₂, which is the minimal subunit of dermorphin needed for dermorphin‐like opiate activity.     

Tritium labelling and degradation studies of Dmt1‐endomorphin 2

Two tritiated derivatives of Dmt¹‐endomorphin 2 (Dmt¹‐EM2) were prepared by the catalytic tritiodehalogenation of 3′,5′‐diiodo‐Dmt¹‐EM2 and the saturation of ^3,4ΔPro²‐Dmt¹‐EM2, resulting in [3′,5′‐³H₂]Dmt¹‐EM2 and [³H₂]Pro²‐Dmt¹‐EM2 with specific activities of 2.88 TBq/mmol (77.8 Ci/mmol) and 1.95 TBq/mmol (52.8 Ci/mmol), respectively. 3′,5′‐Diiodo‐Dmt¹‐EM2 was synthesized by the chloramine T method from Dmt¹‐EM2. ^3,4ΔPro²‐Dmt¹‐EM2 was synthesized by using the Merrifield solid‐phase method. The distributions of the tritium in the labelled peptides were investigated by reversed‐phase high‐performance liquid chromatography after acidic hydrolysis. The stability of Dmt¹‐EM2 in a rat brain membrane homogenate was also determined. Copyright © 2007 John Wiley & Sons, Ltd.     

Cyclization of several linear penta‐ and heptapeptides with different metal ions studied by CD spectroscopy*

Abstract: A cyclic pentapeptide c(Tyr‐Leu‐Ala‐Gly‐Pro) ( I ), which was isolated and identified from Pseudostellaria heterophylla medicinal herbs, and two cyclic heptapeptides, c(Gly‐Tyr‐Gly‐Gly‐Pro‐Phe‐Pro) ( II ) and c(Gly‐Ile‐Pro‐Tyr‐Ile‐Ala‐Ala) ( III ), which were isolated and identified from Stellaria yunnanensis Franch (M), were synthesized by using 3‐(diethoxyphosphoryloxy)‐1,2,3‐benzotriazin‐4(3 H)‐one (DEPBT) as a coupling reagent in solution, and mediated by different metal ions, from their linear peptide precursors H‐Tyr‐Leu‐Ala‐Gly‐Pro‐OH ( I‐1 ) and H‐Ala‐Gly‐Pro‐Tyr‐Leu‐OH ( I‐2 ), H‐Gly‐Tyr‐Gly‐Gly‐Pro‐Phe‐Pro‐OH ( II‐1 ) and H‐Gly‐Ile‐Pro‐Tyr‐Ile‐Ala‐Ala‐OH ( III‐1 ), respectively. The results show that alkali metal ions can improve the cyclization yields and/or the cyclization rates of linear peptide precursors, such as Na⁺ ion is favorable for the cyclization of linear pentapeptides and Cs⁺ ion is favorable for the cyclization of linear heptapeptides, while some bivalent and trivalent metal ions, such as Mg²⁺, Ca²⁺, Zn²⁺, Fe²⁺, Ni²⁺ and Cr³⁺ reduced/inhibited both the cyclization yields and the cyclization rates of the linear peptide precursors. The circular dichroism spectra of I‐1 , II‐1 and III‐1 with different metal ions were studied to elucidate the changes in their secondary structures. It is shown that Cs⁺ can induce and stabilize the type I β‐turn conformation in the linear heptapeptide II‐1 and the type II β‐turn conformation in the linear heptapeptide III‐1 .     

Insect neuropeptide antagonist. Part II. Synthesis and biological activity of backbone cyclic and precyclic PBAN antagonists

Abstract: A new approach for the design and synthesis of pheromone biosynthesis activating neuropeptide (PBAN) agonists and antagonists using the backbone cyclization and cycloscan concepts is described. Two backbone cyclic (BBC) libraries were synthesized: library I (Ser library) was based on the active C‐terminal hexapeptide sequence Tyr‐Phe‐Ser‐Pro‐Arg‐Leu‐NH₂ of PBAN1‐33NH₂; whereas library II (d ‐Phe library) was based on the sequence of the PBAN lead linear antagonist Arg‐Tyr‐Phe‐d ‐Phe‐Pro‐Arg‐Leu‐NH₂. In both libraries the Pro residue was replaced by the BBC building unit N^α‐(ω‐aminoalkyl) Gly having various lengths of alkyl chain. The peptides of the two libraries were tested for agonistic and antagonistic activity. Four precyclic peptides based on two of the BBC antagonists were also synthesized; their activity revealed that a negative charge at the N‐terminus of the peptide abolished antagonistic activity. We also describe the use of the reagent SiCl₃I for selective deprotection of the Boc group from the building unit prior to on‐resin amino‐end to backbone‐nitrogen (AE‐BN) cyclization, during solid‐phase synthesis with Fmoc chemistry.     

Characteristic Structural Features of Indolicidin: Effects of the cis‐trans Isomerism on its Conformation

Indolicidin is an antimicrobial peptide showing a broad spectrum of antibacterial and antifungal activities, and according to the cis‐trans isomerism of three Xaa‐Pro peptide bonds, eight different stereoisomers could be distinguished for this peptide. As the cis‐trans isomerism about the Xaa‐Pro peptide bonds was not considered in previous studies, the structural features of distinct stereoisomeric forms were not characterized in detail, so far. In this theoretical study, the influences of cis‐trans isomerism on the conformation of indolicidin were investigated, as well as the typical structural properties of each stereoisomer were determined, focusing on the secondary structures and intramolecular interactions. Based on the results derived from the molecular dynamics simulations, it could be concluded that the eight different stereoisomeric forms of indolicidin adopted characteristic conformational features. Nevertheless, the appearance of various turn structures and intramolecular interactions proved to be dependent on the cis or trans nature of Xaa‐Pro peptide bonds, indicating the relevant role of Pro amino acids in determining the three‐dimensional structure of this peptide.     

Cyclic Opioid Peptide Agonists and Antagonists Obtained Via Ring‐Closing Metathesis

The opioid peptide H‐Tyr‐c[D‐Cys‐Phe‐Phe‐Cys]NH₂ cyclized via a methylene dithiother is a potent and selective μ opioid agonist (Przydial M.J. et al., J Peptide Res, 66, 2005, 255). Dicarba analogues of this peptide with Tyr, 2′,6′‐dimethyltyrosine (Dmt), 3‐[2,6‐dimethyl‐4‐hydroxyphenyl)propanoic acid (Dhp) or (2S)‐2‐methyl‐3‐(2,6‐dimethyl‐4‐hydroxyphenyl)propanoic acid [(2S)‐Mdp] in the 1‐position were prepared. The peptides were synthesized on solid‐phase by substituting d ‐allylglycine and (2S)‐2‐amino‐5‐hexenoic acid in position 2 and 5, respectively, followed by ring‐closing metathesis. Mixtures of cis and trans isomers of the resulting olefinic peptides were obtained, and catalytic hydrogenation yielded the saturated –CH₂–CH₂– bridged peptides. All six Tyr¹‐ and Dmt¹‐dicarba analogues retained high μ and δ opioid agonist potency and showed only slight or no preference for μ over δ receptors. As expected, the six Dhp¹‐ and (2S)‐Mdp¹‐dicarba analogues turned out to be μ opioid antagonists but, surprisingly, displayed a range of different efficacies (agonism, partial agonism or antagonism) at the δ receptor. The obtained results indicate that the μ versus δ receptor selectivity and the efficacy at the δ receptor of these cyclic peptides depend on distinct conformational characteristics of the 15‐membered peptide ring structure, which may affect the spatial positioning of the exocyclic residue and of the Phe³ and Phe⁴ side chains.     

C‐Terminal 18F‐fluoroethylamidation exemplified on [Gly‐OH9] oxytocin

The no‐carrier‐added (n.c.a.) ¹⁸F‐fluoroethylamidation of the acid function of the protected nonapeptide Boc–Cys–Tyr(tBu)–Ile–Gln(Mtt)–Asn(Mtt)–Cys–Pro–Leu–Gly–OH forming the labelled peptide hormone derivative [Gly‐(2‐[¹⁸F]fluoroethyl)NH⁹]‐oxytocin is described. The labelling conditions were elaborated using a protected tripeptide, identical to the C‐terminal sequence of oxytocin. The prosthetic group n.c.a. 2‐[¹⁸F]fluoroethylamine was synthesised via cryptate mediated n.c.a. ¹⁸F‐fluorination of N‐Boc‐2‐(p‐toluenesulfonyloxy)ethylamine in DMSO (RCY: ca. 60%) and subsequent deprotection with a radiochemical yield of 46±5%. [¹⁸F]Fluoroethylamine was reacted with Z–Pro–Leu–Gly–OH in presence of the coupling reagent TBTU or with activated esters of the model‐tripeptide. The activated ester method as well as the condensation in presence of TBTU yielded ?90% of the ¹⁸F‐fluoroethyl‐amidated tripeptide. TBTU‐mediated condensation of n.c.a. 2‐[¹⁸F]fluoro‐ethylamine with the C‐terminal free acid group of protected oxytocin gave the radiochemical yield of about 75%. Deprotection under acidic conditions led to the formation of [Gly–(2‐[¹⁸F]fluoroethyl)NH⁹]oxytocin within 75 min with a radiochemical yield of about 30% as measured by analytical HPLC. Copyright © 2002 John Wiley & Sons, Ltd.     

Conformational study on trans- and cis-N-acetyl-N′-methylamides of Pro-Xaa dipeptides

Conformational free energy calculations using an empirical potential ( ECEPP /2) and the hydration shell model were carried out on the N-acetyl-N′-methylamides of Pro-Xaa dipeptides (Xaa = Ala, Leu, Val, Gly, Cys, Met, Phe, Tyr, Asn, Asp, and Ser) with trans and cis peptide bonds preceding proline residue in the unhydrated and hydrated states. As compared with the results obtained by using the earlier version of ECEPP, the values of β-bend probabilities are about doubled. The average calculated population of cis-dipeptide is about 4%, which is close to the abundance obtained from the analysis of X-ray crystal structures of proteins. The β-bends are the most dominant structures of cis-dipeptides. Type I, usually having intramolecular hydrogen bonds, contributes greatly to the β-bend conformations of trans- and cis-dipeptides. However, type I β-bends of cis-dipeptides do not have any hydrogen bonds. By including the hydration, the β-bend probabilities for trans- and cis-dipeptides decreased, indicating that the interactions of water molecules with a backbone or side-chain may force the dipeptides to be more distorted or extended. In particular, type II is found to be a dominant β-bend conformation of trans- and cis-Pro-Gly dipeptides in both the unhydrated and hydrated states. In general, the calculated propensities for Pro-Xaa dipeptides to adopt β-bend conformations are reasonably consistent with available experimental data. From comparing conformations of Pro and Xaa residues in the dipeptides and single residues, we found that inter-residue interactions and hydration are of importance in determining the conformational properties of the Pro-Xaa dipeptide.     

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.     

Temperature effects on the stereospecificity of nucleophilic fluorination: formation of trans‐[18F]4‐fluoro‐l‐proline during the synthesis of cis‐[18F]4‐fluoro‐l‐proline

Fluorine‐18 labeled (2S,4S)‐4‐fluoro‐l ‐proline (cis‐[¹⁸F]4‐FPro) has been reported to be a potential positron emission tomography tracer to study abnormal collagen synthesis occurring in pulmonary fibrosis, osteosarcomas, mammary and colon carcinomas. In this paper, we report the stereospecific radiofluorination of (2S,4R)‐N‐tert‐butoxycarbonyl‐4‐(p‐toluenesulfonyloxy) proline methyl ester (at 110°C) to produce diastereomerically pure cis‐[¹⁸F]4‐FPro in 38% radiochemical yield at the end of a 90‐min synthesis. Investigation of the effect of temperature on the stereospecificity of nucleophilic fluorination showed that diasteriomerically pure cis‐[¹⁸F]4‐FPro or trans‐[¹⁸F]4‐FPro was produced at lower temperatures (85°C–110°C) during the fluorination of (2S,4R) or (2S,4S) precursors, respectively. However, at higher temperatures (130°C–145°C), fluorination of (2S,4R) precursor produced a mixture of cis‐[¹⁸F]4‐FPro and trans‐[¹⁸F]4‐FPro diastereomers with cis‐[¹⁸F]4‐FPro as the predominant isomer. Hydrolysis of the purified fluorinated intermediate was carried out either in one step, using 2 m triflic acid at 145°C for 10 min, or in two steps where the intermediate was heated in 1 m HCl at 110°C for 10 min followed by stirring at room temperature in 1 N NaOH for 5 min. The aqueous hydrolysis mixture was loaded onto an anion exchange column (acetate form for one‐step hydrolysis) or an ion retardation column (two‐step hydrolysis) followed by a C₁₈ Sep‐Pak® (Waters Corporation, Milford, MA, USA). Pure cis‐[¹⁸F]4‐FPro was then eluted with sterile water. We also report that epimerization of cis‐[¹⁸F]4‐FPro occurs during the two‐step hydrolysis (H⁺ followed by OH^?) of the intermediate, resulting in 5 ± 3% trans‐[¹⁸F]4‐FPro, whereas the one‐step acid hydrolysis yielded pure cis‐[¹⁸F]4‐FPro in the final product. Copyright © 2011 John Wiley & Sons, Ltd.     

Synthesis of dermorphin‐(1–4) derivatives catalyzed by proteases in organic solvents*

Abstract: In order to extend the use of proteases to organic synthesis and seek the rules of enzymatic reactions in organic media, we focused on unnatural substrates for proteases to form amide bonds. In this paper, the study of unnatural substrates containing d ‐amino acid residue, which act as acyl acceptors as well as acyl donors for proteases in organic media, is reported. Dermorphin is a heptapeptide (H‐Tyr‐d ‐Ala‐Phe‐Gly‐Tyr‐Pro‐Ser‐NH₂) with potent analgesic activity. The N‐terminal tetrapeptide is the minimum sequence that retains dermorphin activity, and is selected as the model compound in our study. Two dermorphin‐(1–4) derivatives, Boc‐Tyr‐d ‐Ala‐Phe‐Gly‐N₂H₂Ph and Boc‐Tyr‐d ‐Ala‐Phe‐Gly‐NH₂, which contained a d ‐amino acid residue, were synthesized by proteases in organic media for the first time. The synthesis of these two dermorphin‐(1–4) derivatives could be catalyzed by subtilisin with Boc‐Tyr‐d ‐Ala‐OCH₂CF₃ as an acyl donor substrate in AcOEt. The synthesis of dermorphin‐(1–2) derivative Boc‐Tyr‐d ‐Ala‐N₂H₂Ph was catalyzed by α‐chymotrypsin in different organic solvents and d ‐Ala‐N₂H₂Ph was used as an acyl acceptor substrate. Factors influencing the above enzymatic reactions were systematically studied.     

X-Ray structure of Tyr-D-Tic-Phe-Phe-NH2 (D-TIPP-NH2), a highly potent μ-receptor selective opioid agonist. Comparisons with proposed model structures

Tyr-D-Tic-Phe-Phe-NH₂ (D-TIPP), a linear tetrapeptide containing the conformationally restricted Tic residue (tetrahydroisoquinoline-3-carboxylic acid), is an opioid agonist which exhibits high affinity and selectivity for the μ-receptor. Its conformational features have been studied using a combination of solid-state (X-ray) and modeling (molecular mechanics and Monte Carlo simulations) methods. The results of the X-ray study showed two distinct conformers for D-TIPP, with the main differences lying in the orientation of the Tyr side-chain and the presence of both D-Tic(+) and D-Tic(—) conformations for the D-Tic residue. The peptide backbone is folded and stablized by the formation of one intramolecular hydrogen bond. The modeling results also indicated a folded backbone for the peptide and both cis and trans conformers for the D-Tic residue are found in the lowest-energy structures. Comparison of the X-ray and modeling results shows many similarities especially around the D-Tic residue. © Munksgaard 1997.     

Chemical synthesis and receptor binding of catfish somatostatin: a disulfide‐bridged β‐d‐Galp‐(1→3)‐α‐d‐GalpNAc O‐glycopeptide*

Abstract: The glycopeptide hormone catfish somatostatin (somatostatin‐22) has the amino acid sequence H‐Asp‐Asn‐Thr‐Val‐Thr‐Ser‐Lys‐Pro‐Leu‐Asn‐Cys‐Met‐Asn‐Tyr‐Phe‐Trp‐Lys‐Ser‐Arg‐Thr‐Ala‐Cys‐OH; it includes a cyclic disulfide connecting the two Cys residues, and the major naturally occurring glycoform contains d ‐GalNAc and d ‐Gal O‐glycosidically linked to Thr⁵. The linear sequence was assembled smoothly starting with an Fmoc‐Cys(Trt)‐PAC‐PEG‐PS support, using stepwise Fmoc solid‐phase chemistry. In addition to the nonglycosylated peptide, two glycosylated forms of somatostatin‐22 were accessed by incorporating as building blocks, respectively, N^α‐Fmoc‐Thr(Ac₃‐α‐D‐GalNAc)‐OH and N^α‐Fmoc‐Thr(Ac₄‐β‐D‐Gal‐(1→3)‐Ac₂‐α‐D‐GalNAc)‐OH. Acidolytic deprotection/cleavage of these peptidyl‐resins with trifluoroacetic acid/scavenger cocktails gave the corresponding acetyl‐protected glycopeptides with free sulfhydryl functions. Deacetylation, by methanolysis in the presence of catalytic sodium methoxide, was followed by mild oxidation at pH 7, mediated by N^α‐dithiasuccinoyl (Dts)‐glycine, to provide the desired monomeric cyclic disulfides. The purified peptides were tested for binding affinities to a panel of cloned human somatostatin receptor subtypes; in several cases, presence of the disaccharide moiety resulted in 2‐fold tighter binding.     

Design,Synthesis and Pharmacological Characterization of Endomorphin Analogues with Non‐Cyclic Amino Acid Residues in Position 2

Abstract: A series of endomorphin‐1 (EM‐1) and endomorphin‐2 (EM‐2) analogues, containing non‐cyclic amino acids (Ala, d ‐Ala, β‐Ala, NMeAla, d ‐NMeAla or Sar) instead of Pro in position 2 was synthesized, where NMeAla = N‐methylalanine and Sar = N‐methylglycine, sarcosine. The opioid activity profiles of these peptides were determined in μ and δ opioid receptor (MOR and DOR)‐representative binding assays and bioassays in vitro, as well as in the mouse hot‐plate test in vivo. Finally, the degradation rates of all analogues in the presence of either rat brain homogenate or selected proteolytic enzymes were determined. Analogues of EM‐2 were generally more potent than the respective analogues of EM‐1. EM‐2 analogues with d ‐Ala or d ‐NMeAla were about twofold more potent than the parent peptide and were least prone to degradation by brain homogenate, dipeptydyl peptidase IV and aminopeptidase M. In the in vivo test, [d ‐Ala²]EM‐2 and [d ‐NMeAla²]EM‐2 showed much higher analgesic potency than EM‐2 which confirmed the usefulness of structural modifications in obtaining new leads for pain‐relief therapeutics.     

The detection of a synthetic Interleukin‐1 receptor antagonist peptide in a seized product from a racing stable

A synthetic Interleukin‐1 receptor antagonist peptide with the sequence Acetyl‐Phe‐Glu‐Trp‐Thr‐Pro‐Gly‐Tyr‐Trp‐Gln‐Pro‐Tyr‐Ala‐Leu‐Pro‐Leu‐OH has been identified in a vial seized during a stable inspection. The use of peptide‐based Interleukin‐1 receptor antagonists as anti‐inflammatory agents has not been previously reported, making this peptide the first in a new class of sports doping peptides. The peptide has been characterized by high‐resolution mass spectrometry and a detection method developed based on solid‐phase extraction and liquid chromatography ‐ triple quadrupole mass spectrometry. Using in vitro and in vivo models to study the properties of the peptide after administration, the peptide was shown to be highly unstable in plasma and was not detected in urine after administration in a rat. The poor stability of the peptide makes detection challenging but also suggests that it has limited effectiveness as an anti‐inflammatory drug. Copyright © 2015 John Wiley & Sons, Ltd.