Isolation and characterization of insulin from the Brockmann body of Dissostichus mawsoni,an Antarctic teleost fish

Abstract: The Brockmann body of fish synthesizes and secretes insulin. The Brockmann body of Antarctic fish has been described anatomically and shown to contain insulin immunoreactive sites, however, the primary structure of an Antarctic fish insulin has yet to be reported. Insulin was isolated from the Brockmann bodies of the Antarctic perciform teleost, Dissostichus mawsoni. The peptide was purified to homogeneity by gel filtration and reversed‐phase HPLC. Insulin‐containing fractions were identified by radioimmunoassay using antisera raised against porcine insulin. Electrospray ionization‐mass spectrometry determined the mass of the isolated product to be 5725.27 a.m.u. The amino acid composition and primary structure were determined for the pyridylethylated A‐ and B‐ chains. The amino acid sequences of the A chain and B chain were H‐Gly‐Ile‐Val‐Glu‐Gln‐Cys‐Cys‐His‐Gln‐Pro¹⁰‐Cys‐Asn‐Ile‐Phe‐Asp‐Leu‐Gln‐Asn‐Tyr‐Cys²⁰‐Asn‐OH and H‐Ala‐Pro‐Gly‐Pro‐Gln‐His‐Leu‐Cys‐Gly‐Ser¹⁰‐His‐Leu‐Val‐Asp‐Ala‐Leu‐Tyr‐Leu‐Val‐Cys²⁰‐Gly‐Glu‐Arg‐Gly‐Phe‐Phe‐Tyr‐Asn‐Pro‐Lys³⁰‐OH, respectively. The primary structure of insulin from Antarctic fish is compared with known structures of insulin from other vertebrates.     

Synthetic protein design: construction of a four‐stranded β‐sheet structure and evaluation of its integrity in methanol–water systems

Abstract: The characterization of a four‐stranded β‐sheet structure in a designed 26‐residue peptide Beta‐4 is described. The sequence of Beta‐4 (Arg‐Gly‐Thr‐Ile‐Lys‐^Dpro‐Gly‐Ile‐Thr‐Phe‐Ala‐^DPro‐Ala‐Thr‐Val‐Leu‐Phe‐Ala‐Val‐^DPro‐Gly‐Lys‐Thr‐Leu‐Tyr‐Arg) was chosen such that three strategically positioned ^DPro‐Xxx segments nucleate type II′β‐turns, which facilitate hairpin extension. A four‐stranded β‐sheet structure is determined in methanol from 500 MHz ¹H NMR data using a total of 100 observed NOEs, 11 dihedral restraints obtained from vicinal J_CαH‐NH values and 10 hydrogen bonding constraints obtained from H/D exchange data. The observed NOEs provide strong evidence for a stable four‐stranded sheet and a nonpolar cluster involving Ile⁸, Phe¹⁰, Val¹⁵ and Phe¹⁷. Circular dichroism studies in water–methanol mixtures provide evidence for melting of the β‐sheet structure at high water concentrations. NMR analysis establishes that the four‐stranded sheet in Beta‐4 is appreciably populated in 50% (v/v) aqueous methanol. In water, the peptide structure is disorganized, although the three β‐turn nuclei appear to be maintained.     

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.     

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.     

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.     

A series of simple detection systems for genetic variants of flavin-containing monooxygenase 3 (FMO3) with impaired function in Japanese subjects

Increasing numbers of single-nucleotide substitutions of the human flavin-containing monooxygenase 3 (FMO3) gene are being recorded in mega-databases. Phenotype–gene analyses revealed impaired FMO3 variants associated with the metabolic disorder trimethylaminuria. Here, a series of reliable FMO3 genotyping confirmation methods was assembled and developed for 45 impaired FMO3 variants, mainly found in Japanese populations, using singleplex or duplex polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) methods and singleplex, duplex, or tetraplex allele-specific PCR methods. Nine PCR-RFLP procedures with single restriction enzymes and fourteen duplex PCR-RFLP procedures (for p.Trp41Ter and p.Thr329Ala, p.Met66Val and p.Leu163Pro, p.Pro70Leu and p.Glu308Gly, p.Asn114Ser and p.Ser195Leu, p.Glu158Lys and p.Ile441Thr, p.Cys197Ter and p.Trp388Ter, p.Arg205Cys and p.Val257Met, p.Arg205His and p.Cys397Ser, p.Met211ArgfsTer10 and p.Arg492Trp, p.Arg223Gln and p.Leu473Pro, p.Met260Val and p.Thr488Ala, p.Tyr269His and p.Ala311Pro, p.Ser310Leu and p.Gly376Glu, and p.Gln470Ter and p.Arg500Ter) were newly established along with eight singleplex (for p.Pro153GlnfsTer14, p.Gly191Cys, p.Pro248Thr, p.Ile486Met, and p.Pro496Ser, among others), one duplex (p.Ile199Ser and p.Asp286Tyr), and one tetraplex (p.Ile7Thr, p.Val58Ile, p.Thr201Lys, and p.Gly421Val) allele-specific PCR systems. This series of systems should facilitate the easy detection in a clinical setting of FMO3 variants in Japanese subjects susceptible to low drug clearances or drug reactions possibly caused by impaired FMO3 function.     

Synthesis and effect of shortened oostatic decapeptide (TMOF) analogs with isosteric structures on reproduction of Neobellieria bullata

Abstract: Oligopeptides 2a ? 2d derived from the oostatic decapeptide (TMOF) sequence, H‐Tyr‐Asp‐Pro‐Ala‐Pro‐Pro‐Pro‐Pro‐Pro‐Pro‐OH ( 1a ) and containing isosteric structures were synthesized and assayed to determine their effect during reproduction in the flesh fly Neobellieria bullata. The N‐terminal linear tetra‐ and pentapeptides 2a , 2b containing the Pro‐ψ[CH₂O]Ala isosteric linkage affect egg development in 80–90% of ovarioles resulting in some resorbed egg chambers, abnormal yolk deposition, the formation of large eggs with irregular yolk granules and proliferation of follicular epithelium. In comparison with their nonisosteric precursors 1b , 1c they exhibit even more accelerated oostatic activity. However, peptides 2c , 2d containing a Pro‐ψ[CH₂S]Ala isosteric linkage are less active.     

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.     

Solvolysis and aminolysis on peptidyl‐Kaiser oxime resin assisted by Ca2+and Eu3+: a mild procedure to prepare α‐methyl and ‐ethyl esters of protected peptides

Abstract: Ca²⁺ and Eu³⁺ were able to assist solvolysis on peptidyl‐Kaiser oxime resins generating α‐methyl and ‐ethyl esters of protected peptides. The methanolysis assistance was at least twice as effective as that of acetic acid, the common catalyst used in aminolysis of the ester oxime linkage. No molar excess of Ca²⁺ or Eu³⁺ was needed to enhance this reaction efficiency. Ca²⁺ also assisted aminolysis on peptidyl‐Kaiser oxime resins. Solvolysis and aminolysis rates depended on the nature of theC‐terminal residue attached to the resin and on the alcohol used. Both reactions were selective to the ester oxime linkage since no significant amount of secondary products, resulting from rearrangements or simultaneous transesterification of the β‐benzyl or cyclohexyl esters, was detected in the reaction media. The α‐methyl and ‐ethyl esters of Ac‐Ala‐Gly‐X [where, X = Gly, Ala, Phe or Lys (2‐Cl‐Z)] and of Ac‐Ile‐Ser (Bzl)‐Asp(OZ) (where, Z = Bzl or cHex) were essentially the only products formed in the solvolyses performed. Ac‐Ile‐Ser(Bzl)‐Asp(OcHex)Arg(HCl)‐OMe and Ac‐Ile‐Ser(Bzl)‐Asp(OcHex)Arg (HCl)‐OEt were the major products formed in the aminolysis reactions. In the presence of the metal ions, the resin‐cleavage yields were > 50%. In their absence, they were < 15%.     

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.     

Context‐dependent conformation of diethylglycine residues in peptides

Abstract: Diethylglycine (Deg) residues incorporated into peptides can stabilize fully extended (C₅) or helical conformations. The conformations of three tetrapeptides Boc‐Xxx‐Deg‐Xxx‐Deg‐OMe (Xxx = Gly, GD4; Leu, LD4 and Pro, PD4) have been investigated by NMR. In the Gly and Leu peptides, NOE data suggest that the local conformations at the Deg residues are fully extended. Low temperature coefficients for the Deg(2) and Deg(4) NH groups are consistent with their inaccessibility to solvent, in a C₅ conformation. NMR evidence supports a folded β‐turn conformation involving Deg(2)‐Gly(3), stabilized by a 4 → 1 intramolecular hydrogen bond between Pro(1) CO and Deg(4) NH in the proline containing peptide (PD4). The crystal structure of GD4 reveals a hydrated multiple turn conformation with Gly(1)–Deg(2) adopting a distorted type II/II′ conformation, while the Deg(2)–Pro(3) segment adopts a type III/III′ structure. A lone water molecule is inserted into the potential 4 → 1 hydrogen bond of the Gly(1)–Deg(2) β‐turn.     

Evaluation of geranylazide and farnesylazide diphosphate for incorporation of prenylazides into a CAAX box‐containing peptide using protein farnesyltransferase*

Abstract: Protein farnesyltransferase (PFTase) catalyzes the attachment of a geranylazide (C10) or farnesylazide (C15) moiety from the corresponding prenyldiphosphates to a model peptide substrate, N‐dansyl‐Gly‐Cys‐Val‐Ile‐Ala‐OH. The rates of incorporation for these two substrate analogs are comparable and approximately twofold lower than that using the natural substrate farnesyl diphosphate (FPP). Reaction of N‐dansyl‐Gly‐Cys(S‐farnesylazide)‐Val‐Ile‐Ala‐OH with 2‐diphenylphosphanylbenzoic acid methyl ester then gives a stable alkoxy‐imidate linked product. This result suggests future generations whereby azide groups introduced using this enzymatic approach are functionalized using a broad range of azide‐reactive reagents. Thus, chemistry has been developed that could be used to achieve highly specific peptide and protein modification. The farnesylazide analog may be useful in certain biological studies, whereas the geranylazide group may be more useful for general protein modification and immobilization.     

Bisabolangelone, a gastric H+/K+-ATPase inhibitor: homology modeling and docking study

The homology model of human gastric H⁺/K⁺-ATPase has been produced based on the template provided by pig gastric H⁺/K⁺-ATPase (PDB code: 3IXZ). After molecular mechanics optimization, induced-fit docking simulation between gastric H⁺/K⁺-ATPase and bisabolangelone that has significantly inhibition activity of H⁺/K⁺-ATPase was performed. The results of ligand docking showed that the binding pocket involves the amino acid residues Asp101, Asp102, Tyr105, Leu106, Val296, Phe297, Met299, Ala300, Tyr764, Tyr767, Leu774, Gly777, Cys778, Ile779, Gln889, Tyr893, and Ile952. The hydrogen bonds are formed between bisabolangelone and the amino acid residues Cys778, Gln889, and Tyr893.     

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).     

Development of highly potent and ective dynorphin A analogues as new medicines

Abstract: Dynorphin A (Dyn A), a 17 amino acid peptide H‐Tyr‐Gly‐Gly‐Phe‐Leu‐Arg‐Arg‐Ile‐Arg‐Pro‐Lys‐Leu‐Lys‐Trp‐Asp‐Asn‐Gln‐OH, is a potent opioid peptide which interacts preferentially with κ‐opioid receptors. Research in the development of selective and potent opioid peptide ligands for the κ‐receptor is important in mediating analgesia. Several cyclic disulphide bridge‐containing peptide analogues of Dyn A, which were conformationally constrained in the putative message or address segment of the opioid ligand, were designed, synthesized and assayed. To further investigate the conformational and topographical requirements for the residues in positions 5 and 11 of these analogues, a systematic series of Dyn A_1?11‐NH₂ cyclic analogues incorporating the sulphydryl‐containing amino acids l ‐ and d ‐Cys and l ‐ and d ‐Pen in positions 5 and 11 were synthesized and assayed. Cyclic lactam peptide analogues were also synthesized and assayed. Several of these cyclic analogues, retained the same affinity and selectivity (vs. the μ‐ and δ‐receptors) as the parent Dyn A_1?11‐NH₂ peptide in the guinea‐pig brain (GPB), but exhibited a much lower activity in the guinea‐pig ileum (GPI), thus leading to centrally vs. peripherally selective peptides. Studies of the structure–activity relationship of Dyn A peptide provide new insights into the importance of each amino acid residue (and their configurations) in Dyn A analogues for high potency and good selectivity at κ‐opioid receptors. We report herein the progress towards the development of Dyn A peptide ligands, which can act as agonists or antagonists at cell surface receptors that modulate cell function and animal behaviour using various approaches to rational peptide ligand‐based drug design.     

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.     

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.     

Structure–Function Relationship of Val/Arg‐rich Peptides: Effects of Net Charge and Pro on Activity

Our previous study reported Val/Arg‐rich peptides, and the relationship was linear between hydrophobicity and antimicrobial potency within a certain range. Here, we further develop a new series of analogs to investigate the effect of net charge and Pro residue on activity. Replacement of Gly with Ala or Pro led to the decrease in antimicrobial activity. The substitution of Gly with Ala retained its hemolytic activity, while the substitution with Pro significantly decreased the toxicity, suggesting positive effect of Pro on hemolytic activity. The increase in net charge from +4 to +6 significantly improved antimicrobial activity and decreased the hemolysis. However, antibacterial and hemolytic activities were not affected by increasing the net charge from +6 to +8, indicating a moderate net positive charge. The peptides produced larger blue shifts in PE/PG than in PC/cholesterol, suggesting a stronger affinity with negatively charged membrane over zwitterionic membrane. Lowering the net charge or insert of Pro led to the lack of α‐helical structure in SDS micelles, which may be correlated with weakened antimicrobial potency. This study indicated that Val/Arg‐rich peptides should have moderate net charge and Pro may play a role in reducing the toxicity against red blood cells.     

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.     

Structure–activity relationships of arodyn,a novel acetylated kappa opioid receptor antagonist*,†

Abstract: We previously reported that the novel dynorphin A (Dyn A, Tyr‐Gly‐Gly‐Phe‐Leu‐Arg‐Arg‐Ile‐Arg‐Pro‐Lys‐Leu‐Lys‐Trp‐Asp‐Asn‐Gln) analog arodyn (Ac[Phe^1,2,3,Arg⁴,d ‐Ala⁸]Dyn A‐(1–11)NH₂, Bennett, M.A., Murray, T.F. & Aldrich, J.V. (2002) J. Med. Chem. vol. 45, pp. 5617–5619) is a κ opioid receptor‐selective peptide [K_i(κ) = 10 nm , K_i ratio (κ/μ/δ) = 1/174/583] which exhibits antagonist activity at κ opioid receptors. In this study, a series of arodyn analogs was prepared and evaluated to explore the structure–activity relationships (SAR) of this peptide; this included an alanine scan of the entire arodyn sequence, sequential isomeric d ‐amino acid substitution in the N‐terminal ‘message’ sequence, NMePhe substitution individually in positions 1–3, and modifications in position 1. The results for the Ala‐substituted derivatives indicated that Arg⁶ and Arg⁷ are the most important residues for arodyn's nanomolar binding affinity for κ opioid receptors. Ala substitution of the other basic residues (Arg⁴, Arg⁹ and Lys¹¹) resulted in lower decreases in affinity for κ opioid receptors (three‐ to fivefold compared with arodyn). Of particular interest, while [Ala¹⁰]arodyn exhibits similar κ opioid receptor binding as arodyn, it displays higher κ vs. μ opioid receptor selectivity [K_i ratio (κ/μ) = 1/350] than arodyn because of a twofold loss in affinity at μ opioid receptors. Surprisingly, the Tyr¹ analog exhibits a sevenfold decrease in κ opioid receptor affinity, indicating that arodyn displays significantly different SAR than Dyn A; [Tyr¹]arodyn also unexpectedly exhibits inverse agonist activity in the adenylyl cyclase assay using Chinese hamster ovary cells stably expressing κ opioid receptors. Substitution of NMePhe in position 1 gave [NMePhe¹]arodyn which exhibits high affinity [K_i(κ) = 4.56 nm ] and exceptional selectivity for κ opioid receptors [K_i ratio (κ/μ/δ) = 1/1100/>2170]. This peptide exhibits antagonistic activity in the adenylyl cyclase assay, reversing the agonism of 10 nm Dyn A‐(1–13)NH₂. Thus [NMePhe¹]arodyn is a highly κ opioid receptor‐selective antagonist that could be a useful pharmacological tool to study κ opioid receptor‐mediated activities.