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.     

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.     

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.     

Cyclic enkephalin analogs containing various para‐substituted phenylalanine derivatives in place of Tyr1 are potent opioid agonists*

Abstract: The cyclic enkephalin analog H‐Tyr‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ is a highly potent opioid agonist with IC₅₀s of 35 pm and 19 pm in the guinea‐pig ileum (GPI) and mouse vas deferens (MVD) assays, respectively. The Phe¹‐analog of this peptide showed 370‐fold and 6790‐fold lower agonist potency in the GPI and MVD assays, respectively, indicating the importance of the Tyr¹ hydroxyl‐group in the interaction with μ and δ opioid receptors. In the present study, the effect of various substituents (‐NH₂, ‐NO₂, ‐CN, ‐CH₃, ‐COOH, ‐COCH₃, ‐CONH₂) introduced in the para‐position of the Phe¹‐residue of H‐Phe‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ on the in vitro opioid activity profile was examined. Most analogs showed enhanced μ and δ agonist potencies in the two bioassays, except for the Phe(pCOOH)¹‐analog, which was weakly active, probably as a consequence of the negative charge. The most potent compounds were the Phe(pCOH₃)¹‐ and the Phe(pCONH₂)¹‐analogs. The latter compound showed subnanomolar μ and δ agonist potencies and represents the most potent enkephalin analog lacking the Tyr¹ hydroxyl‐group reported to date. Taken together, these results indicate that various substituents introduced in the para‐position of Phe¹ enhance opioid activity via hydrogen bonding or hydrophobic interactions with the receptor. Comparison with existing structure‐activity relationship on phenolic hydroxyl replacements in morphinans indicates that these nonpeptide opiates and some of the cyclic enkephalin analogs described here may have different modes of binding to the receptor.     

Structure–bioactivity of C‐terminal pentapeptide of osteogenic growth peptide [OGP(10–14)]

The amino acid sequence of osteogenic growth peptide (OGP) consists of 14 residues identical to the C‐terminal tail of histone H4. Native and synthetic OGP are mitogenic to osteoblastic and fibroblastic cells and enhance osteogenesis and hematopoiesis in vivo. The C‐terminal truncated pentapeptide of OGP, H‐Tyr‐Gly‐Phe‐Gly‐Gly‐OH [OGP(10–14)], is a naturally occurring osteoblastic mitogen, equipotent to OGP. The present study assesses the role of individual amino acid residues and side chains in the OGP(10–14) mitogenic activity which showed a very high correlation between osteoblastic and fibroblastic cell cultures. Truncation of either Tyr¹⁰ or its replacement by Ala or d ‐Ala resulted in substantial, but not complete, loss of activity. Nevertheless, only a small loss of activity was observed following removal of the Tyr¹⁰ amino group. No further loss occurred consequent to the monoiodination of desaminoTyr¹⁰ on meta‐position. However, a marked decrease in proliferative activity followed removal of the Tyr¹⁰ phenolic or the Phe¹² aromatic group. Loss of activity of a similar magnitude also occurred subsequent to replacing Gly¹¹ with l ‐ or d ‐Ala. Approximately 50% loss of mitogenic activity occurred subsequent to truncation of Gly¹⁴ or blocking the C‐terminal group as the methyl ester. All other modifications of the C‐terminus and l ‐ or d ‐Ala substitution of Gly¹³ resulted in 70–97% decrease in activity. Collectively, these data suggest that the integrity of the pharmacophores presented by Tyr and Phe side chains, as well as the Gly residues at the C‐terminus, are important for optimal bioactivity of OGP(10–14).     

Synthesis of Met‐enkephalin by solution‐phase peptide synthesis methodology utilizing para‐toluene sulfonic acid as N‐terminal masking of l‐methionine amino acid

The Met‐enkephalin, Tyr‐Gly‐Gly‐Phe‐Met, was synthesized by the solution‐phase synthesis (SPS) methodology employing ‐OBzl group as carboxyls' protection, while the t‐Boc groups were employed for the N‐terminal α‐amines' protection for the majority of the amino acids of the pentapeptide sequence. The l ‐methionine (l ‐Met) amino acid was used as PTSA.Met‐OBzl obtained from the simultaneous protection of the α‐amino, and carboxyl group with para‐toluene sulfonic acid (PTSA) and as‐OBzl ester, respectively in a C‐terminal start of the 2 + 2 + 1 fragments condensation convergent synthetic approach. The protection strategy provided a short, single‐step, simultaneous, orthogonal, nearly quantitative, robust, and stable process to carry through the protected l ‐methionine and l ‐phenylalanine coupling without any structural deformities during coupling and workups. The structurally confirmed final pentapeptide product was feasibly obtained in good yields through the current approach.     

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 .     

Synthesis of tritium labeled isotopomers of L‐tyrosine

The synthesis of four selectively labeled isotopomers of L ‐tyrosine, (L ‐Tyr), using chemical and enzymatic methods is reported. Four tritium labeled isotopomers of L ‐phenylalanine (L ‐Phe) – [2‐³H]‐, [2′,6′‐³H₂]‐, [3R‐³H]‐ and [3S‐³H]‐ have been synthesized using a combination of chemical and enzymatic methods. The labeled isotopomers of L ‐Phe have been converted into [2 ‐³H]‐, [2′,6′‐³H₂]‐, [3R‐³H]‐, and [3S‐³H]‐L ‐Tyr by using the enzyme L ‐phenyl‐alanine 4′‐monooxygenase. Copyright © 2002 John Wiley & Sons, Ltd.     

New chromogenic dipeptide substrate for continuous assay of the d‐alanyl‐d‐alanine dipeptidase VanX required for high‐level vancomycin resistance

Abstract: A direct continuous UV–Vis spectrophotometric assay has been developed for VanX, a d ‐alanyl‐d ‐alanine aminodipeptidase necessary for vancomycin resistance. This method is based on the hydrolysis of the alternative substrate d ‐alanyl‐α‐(R)‐phenylthio‐glycine d ‐Ala‐d ‐Gly(S‐Ph)‐OH (H‐d Ala‐d Psg‐OH, 5a ). Spontaneous decomposition of the released phenylthioglycine generates thiophenol, which is quantified using Ellman's reagent. The dipeptide behaved as an excellent substrate of VanX, exhibiting Michaelis–Menten kinetics with a k_cat of 76 ± 5/s and a K_M of 0.83 ± 0.08 mm (k_cat = 46 ± 3/s and K_M = 0.11 ± 0.01 mm for d ‐Ala‐d ‐Ala). Determination of the kinetic parameters of the previously reported mechanism‐based inhibitor d ‐Ala‐d ‐Gly(SΦp‐CHF₂)‐OH (H‐d ‐Ala‐d Pfg‐OH, 5c ) [Araoz, R., Anhalt, E., René, L., Badet‐Denisot, M.‐A., Courvalin, P. & Badet, B. (2000) Biochemistry 39, 15971–15979] using the substrate reported in the present study yielded values of K_irr of 22 ± 1 μm and k_inact of 9.3 ± 0.4/min in good agreement with values previously obtained in our laboratory (K_irr = 30 ± 1 mm ; k_inact = 7.3 ± 0.3/min). In addition, inhibition by the competing substrate d ‐Ala‐d ‐Ala resulted in determination of a K_i = 70 ± 6 μm close to the previously reported K_M value. These results demonstrate that the present assay is a convenient, rapid and sensitive tool in the search for VanX inhibitors.     

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.     

Transport characteristics of peptides and peptidomimetics: I. N‐methylated peptides as substrates for the oligopeptide transporter and P‐glycoprotein in the intestinal mucosa

Abstract: Peptides and peptidomimetics often exhibit poor oral bioavailability due to their metabolic instability and low permeation across the intestinal mucosa. N‐Methylation has been used successfully in peptide‐based drug design in an attempt to improve the metabolic stability of a peptide‐based lead compound. However, the effect of N‐methylation on the absorption of peptides through the intestinal mucosa is not well understood, particularly when transporters, i.e. the oligopeptide transporter (OPT) and P‐glycoprotein (P‐gp), modulate the passive diffusion of these types of molecules. To examine this, terminally free and terminally modified (N‐acetylated and C‐amidated) analogs of H‐Ala‐Phe‐Ala‐OH with N‐methyl groups on either the Ala‐Phe or Phe‐Ala peptide bond were synthesized. Transport studies using Caco‐2 cell monolayers, an in vitro model of the intestinal mucosa, showed that N‐methylation of the Ala‐Phe peptide bond of H‐Ala‐Phe‐Ala‐OH stabilized the molecule to protease degradation, and the resulting analog exhibited significant substrate activity for OPT. However, N‐methylation of the Phe‐Ala peptide bond of H‐Ala‐Phe‐Ala‐OH did not stabilize the molecule to protease degradation, and the substrate activity of the resulting molecule for OPT could not be determined. Interestingly, N‐methylation of the Phe‐Ala peptide bond of the terminally modified tripeptide Ac‐Ala‐Phe‐Ala‐NH₂ decreased the substrate activity of the molecule for the efflux transporter P‐gp. In contrast, N‐methylation of the Ala‐Phe peptide bond of the terminally modified tripeptide Ac‐Ala‐Phe‐Ala‐NH₂ increased the substrate activity of the molecule for P‐gp.     

Synthesis and activity of the melanocortin Xaa‐d‐Phe‐Arg‐Trp‐NH2 tetrapeptides with amide bond modifications

Abstract: The melanocortin receptor (MCR) pathway has been identified as participating in several physiologically important pathways including pigmentation, energy homeostasis, inflammation, obesity, hypertension, and sexual function. All the endogenous MCR agonists contain a core His‐Phe‐Arg‐Trp sequence identified as important for receptor molecular recognition and stimulation. Several structure–activity studies using the Ac‐His‐d ‐Phe‐Arg‐Trp‐NH₂ tetrapeptide template have been performed in the context of modifying N‐terminal ‘capping’ groups and amino acid constituents. Herein, we report the synthesis and pharmacologic characterization of modified Xaa‐d ‐Phe‐Arg‐Trp‐NH₂ (Xaa = His or Phe) melanocortin tetrapeptides (N‐site selective methylation, permethylation, or amide bond reduction) at the mouse MC1, MC3, MC4 and MC5 receptors. The modified peptides generated in this study resulted in equipotent or reduced MCR potency when compared with control ligands. The reduced amide bond analog of the Phe‐d ‐Phe‐Arg‐Trp‐NH₂ peptide converted its agonist activity into an antagonistic at the central mMC3 and mMC4 receptors involved in the regulation of energy homeostasis, while retaining full agonist activity at the peripheral MC1 and MC5 receptors.     

Role of azaamino acid residue in β‐turn formation and stability in designed peptide

Abstract: The structural perturbation induced by C^αH→N^α exchange in azaamino acid‐containing peptides was predicted by ab initio calculation of the 6‐31G* and 3‐21G* levels. The global energy‐minimum conformations for model compounds, For‐azaXaa‐NH₂ (Xaa = Gly, Ala, Leu) appeared to be the β‐turn motif with a dihedral angle of φ = ± 90°, ψ = 0°. This suggests that incorporation of the azaXaa residue into the i + 2 position of designed peptides could stabilize the β‐turn structure. The model azaLeu‐containing peptide, Boc‐Phe‐azaLeu‐Ala‐OMe, which is predicted to adopt a β‐turn conformation was designed and synthesized in order to experimentally elucidate the role of the azaamino acid residue. Its structural preference in organic solvents was investigated using ¹H NMR, molecular modelling and IR spectroscopy. The temperature coefficients of amide protons, the characteristic NOE patterns, the restrained molecular dynamics simulation and IR spectroscopy defined the dihedral angles [ (φ_i+1, ψ_i+1) (φ_i+2, ψ_i+2)] of the Phe‐azaLeu fragment in the model peptide, Boc‐Phe‐azaLeu‐Ala‐OMe, as [(?59^°, 127^°) (107^°, ?4^°)]. This solution conformation supports a βII‐turn structural preference in azaLeu‐containing peptides as predicted by the quantum chemical calculation. Therefore, intercalation of the azaamino acid residue into the i + 2 position in synthetic peptides is expected to provide a stable β‐turn formation, and this could be utilized in the design of new peptidomimetics adopting a β‐turn scaffold.     

Roles of residues 3 and 4 in cyclic tetrapeptide ligand recognition by the κ‐opioid receptor

Abstract: A series of cyclic, disulfide‐ or dithioether‐containing tetrapeptides based on previously reported potent μ‐ and δ‐selective analogs has been explored with the aim of improving their poor affinity to the κ‐opioid receptor. Specifically targeted were modifications of tetrapeptide residues 3 and 4, as they presumably interact with residues from transmembrane helices 6 and 7 and extracellular loop 3 that differ among the three receptors. Accordingly, tetrapeptides were synthesized with Phe³ replaced by aliphatic (Gly, Ala, Aib, Cha), basic (Lys, Arg, homo‐Arg), or aromatic sides chains (Trp, Tyr, p‐NH₂Phe), and with d ‐Pen⁴ replaced by d ‐Cys⁴, and binding affinities to stably expressed μ‐, δ‐, and κ‐receptors were determined. In general, the resulting analogs failed to exhibit appreciable affinity for the κ‐receptor, with the exception of the tetrapeptide Tyr‐c[d ‐Cys‐Phe‐d ‐Cys]‐NH₂, cyclized via a disulfide bond, which demonstrated high binding affinity toward all opioid receptors (Ki^μ = 1.26 nm , Ki^δ = 16.1 nm , Ki^κ = 38.7 nm ). Modeling of the κ‐receptor/ligand complex in the active state reveals that the receptor‐binding pocket for residues 3 and 4 of the tetrapeptide ligands is smaller than that in the μ‐receptor and requires, for optimal fit, that the tripeptide cycle of the ligand assume a higher energy conformation. The magnitude of this energy penalty depends on the nature of the fourth residue of the peptide (d ‐Pen or d ‐Cys) and correlates well with the observed κ‐receptor binding affinity.     

Characterization of d-boroAla as a novel broad-spectrum antibacterial agent targeting d-Ala-d-Ala ligase

d ‐boroAla was previously characterized as an inhibitor of bacterial alanine racemase and d ‐Ala‐d ‐Ala ligase enzymes (Biochemistry, 28, 1989, 3541). In this study, d ‐boroAla was identified and characterized as an antibacterial agent. d ‐boroAla has activity against both Gram‐positive and Gram‐negative organisms, with minimal inhibitory concentrations down to 8 μg / mL. A structure–function study on the alkyl side chain (NH₂‐CH R ‐B(OR’)₂) revealed that d ‐boroAla is the most effective agent in a series including boroGly, d ‐boroHomoAla, and d ‐boroVal. l ‐boroAla was much less active, and N‐acetylation completely abolished activity. An LC‐MS / MS assay was used to demonstrate that d ‐boroAla exerts its antibacterial activity by inhibition of d ‐Ala‐d ‐Ala ligase. d ‐boroAla is bactericidal at 1× minimal inhibitory concentration against Staphylococcus aureus and Bacillus subtilis, which each encode one copy of d ‐Ala‐d ‐Ala ligase, and at 4× minimal inhibitory concentration against Escherichia coli and Salmonella enterica serovar Typhimurium, which each encode two copies of d ‐Ala‐d ‐Ala ligase. d ‐boroAla demonstrated a frequency of resistance of 8 × 10⁻⁸ at 4× minimal inhibitory concentration in S. aureus. These results demonstrate that d ‐boroAla has promising antibacterial activity and could serve as the lead agent in a new class of d ‐Ala‐d ‐Ala ligase targeted antibacterial agents. This study also demonstrates d ‐boroAla as a possible probe for d ‐Ala‐d ‐Ala ligase function.     

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.     

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

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

Novel Side‐Chain Glucosylated and Adamantylated [Asp2/Glu2]Enkephalin Analogs: Synthesis and In Vitro Growth Inhibition of Human Tumor Cells

A series of new backbone‐modified Leu‐ and Met‐enkephalin analogs ( 13 – 20 a and b ) were synthesized. Backbone manipulations involved the replacement of the Gly² residue in Tyr‐Gly‐Gly‐Phe‐Leu/Met with side‐chain glucosylated or adamantylated D /L ‐aspartic or ‐glutamic acids. The in vitro antiproliferative activity of these compounds was evaluated for several cell lines and the results were compared with the effect of Met‐enkephalin, the native opioid growth factor. The tested compounds modestly inhibited the growth of the tumor cells (20–50% inhibition at millimolar concentrations). Among the tested compounds, Tyr‐D ‐Glu(AdNH)‐Gly‐Phe‐Met ( 20b ) showed significant antiproliferative activity, somewhat more pronounced on MCF‐7 (breast carcinoma) and MOLT‐4 (lymphoblastic leukemia) cells.     

The chemical syntheses and bioactivities of novel peptide‐based endothelin antagonists

Abstract: Endothelin antagonists, novel tripeptides containing a series of unnatural amino acids, were synthesized and characterized. A linear peptide BQ‐485, perhydroazepin‐1‐yl‐l ‐leucyl (1)‐d ‐tryptophanyl (2)‐d ‐tryptophan (3), was selected as the parent compound. The introduction of d ‐Phe derivatives into these peptidic ET antagonists resulted in potent activity against the contraction of rat aortic smooth muscles induced by ET‐1 (10 nm ) which activated the ET receptors. Among these compounds, 15 tripeptides had high enough antagonistic activity at the level of 10^?7 mol/L (IC₅₀). The activity of three compounds was 10^?6 mol/L (IC₅₀). These HIM‐CO‐Leu‐d ‐Trp‐d ‐Phe(‐R)‐OH compounds as ET_A antagonists may provide a tool for the development of therapeutic agents in the treatment of putative ET‐1‐related disorders.     

Dermorphin interaction with rat brain opioid receptors: involvement of hydrophobic sites in the binding domain

Dermorphin structural analogues were utilized to determine the nature of opioid receptor subsite specificity, affinity, and selectivity in rat brain membranes. The data suggest that these parameters are influenced by the amino acid composition and sequence and the known solution conformation of dermorphin, in addition to the conformation of the membrane receptor. Two hydrophobic components of dermorphin are required for optimal binding. One component encompasses the stacked phenol groups in Tyr1 and Tyr5; the second involves the phenyl group of Phe3. Evidence to support this proposal includes the following results: (a) removal of aromaticity, as occurs in [des-Tyr5]- and [Gly5]dermorphin, drastically reduced binding to both mu and delta sites; (b) inversion of the Phe3-Gly4 sequence in dermorphin to the Gly3-Phe4 in enkephalin enhanced binding to delta receptor sites, yet the peptide remained mu-selective; (c) substitution of Pro4 for Gly4 disrupted the solution conformation of dermorphin and decreased affinities at both receptor subsites, substantiating the requirement for the Phe3-Gly4-Tyr5 sequence in dermorphin to interact with mu sites; and (d) modification of the serine residue, as occurs in [Ser(Bzl7)] dermorphin and [Ser-NHNHZ7]dermorphin, enhanced interaction with delta opioid receptors; the apparent delta affinity increased over 50-fold with [Ser(Bzl7)]dermorphin, although it retained a weak mu-selectivity. However, both [Ser(Bzl7)]- and [Ser-NHNHZ7]dermorphin exhibited high affinity for mu receptor sites. Furthermore, the D-configuration about the alpha-carbon of residue 2 and the alpha-amine function and hydroxyl group on Tyr1 are essential for receptor binding. We conclude that mu-opioid receptors contain distinct regions that accomodate the stacked phenol groups of Tyr1 and Tyr5 residues and the phenyl group of Phe3.