The biological consequences of replacing hydrophobic amino acids in deltorphin I with amphiphilic α‐hydroxymethylamino acids

Abstract: New analogues of deltorphin I (DT I), in which the Phe residue in position 3, and the Val residue in position 5 or 6 are replaced with respective amphiphilic α‐hydroxymethylamino acid residues (HmAA), were synthesized and tested for receptor affinity and selectivity to μ and δ opioid receptors. The analogue with (R)‐HmPhe at position 3 lost receptor selectivity, as a result of a partial decrease of affinity to δ and a significant increase of affinity to μ receptors. In contrast, an analogue with (S)‐HmPhe in the same position, was very potent and more specific to δ receptors than parent DT I. The analogue with (R)‐HmVal at position 5 expressed higher δ affinity and selectivity than parent DT I. The analogue with other possible isomer (S)‐HmVal was less selective for δ opioid receptors, as a result of decreasing affinity to δ and increasing affinity to μ receptors. The analogues with (R)‐ or (S)‐HmVal in position 6 expressed equally low receptor affinity and selectivity. The data obtained support a previously proposed model of active conformation of deltorphins.     

An affinity label for δ‐opioid receptors derived from [d‐Ala2]deltorphin I*

Abstract: A series of potential affinity label derivatives of the amphibian opioid peptide [d ‐Ala²]deltorphin I were prepared by incorporation at the para position of Phe³ (in the ‘message’ sequence) or Phe⁵ (in the ‘address’ sequence) of an electrophilic group (i.e. isothiocyanate or bromoacetamide). The introduction of the electrophile was accomplished by incorporating Fmoc‐Phe(p‐NHAlloc) into the peptide, followed later in the synthesis by selective deprotection of the Alloc group and modification of the resulting amine. While para substitution decreased the δ‐opioid receptor affinity, selected analogs retained nanomolar affinity for δ receptors. [d ‐Ala²,Phe(p‐NCS)³]deltorphin I exhibited moderate affinity (IC₅₀ = 83 nm ) and high selectivity for δ receptors, while the corresponding amine and bromoacetamide derivatives showed pronounced decreases in δ‐receptor affinity (80‐ and >1200‐fold, respectively, compared with [d ‐Ala²]deltorphin I). In the ‘address’ sequence, the Phe(p‐NH₂)⁵ derivative showed the highest δ‐receptor affinity (IC₅₀ = 32 nm ), while the Phe(p‐NHCOCH₂Br)⁵ and Phe(p‐NCS)⁵ peptides displayed four‐ and tenfold lower δ‐receptor affinities, respectively. [d ‐Ala²,Phe(p‐NCS)³]deltorphin I exhibited wash‐resistant inhibition of [³H][d ‐Pen²,D‐Pen⁵]enkephalin (DPDPE) binding to δ receptors at a concentration of 80 nm . [d ‐Ala², Phe(p‐NCS)³]deltorphin I represents the first affinity label derivative of one of the potent and selective amphibian opioid peptides, and the first electrophilic affinity label derivative of an agonist containing the reactive functionality in the ‘message’ sequence of the peptide.     

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.     

The Biological Consequences of Replacing d‐Ala in Biphalin with Amphiphilic α‐Alkylserines

Biphalin, a synthetic opioid peptide with a broad affinity for all opioid receptors (δ, μ, and κ) and high antinociceptive activity, has been under extensive study as a potential analgesic drug. This study presents the synthesis and biological properties of four new analogues of biphalin containing amphiphilic α‐alkylserines in position 2 and 2′. The incorporation of bulky α,α‐disubstituted amino acids in the peptide chain using standard peptide chemistry is often unsuccessful. We synthesized depsipeptides, and then, the desired peptides were obtained by internal O,N‐migration of the acyl residue from the hydroxyl to the amino group under mild basic conditions. The potency and selectivity of the new analogues were evaluated by a competitive receptor‐binding assay in the rat brain using [³H]DAMGO (a μ ligand) and [³H]DELT (a δ ligand). Their binding affinity is strongly dependent on the chirality of α‐alkylserine, as analogues containing (R)‐α‐alkylserines displayed higher μ receptor affinity and selectivity than those incorporating the (S)‐isomers.     

Dermorphin‐based potential affinity labels for µ‐opioid receptors*

Abstract: Dermorphin and [Lys⁷]dermorphin, selective µ‐opioid receptor ligands originating from amphibian skin, have been modified with various electrophiles in either the ‘message’ or ‘address’ sequences as potential peptide‐based affinity labels for µ‐receptors. Introduction of the electrophilic isothiocyanate and bromoacetamide groups on the para position of Phe³ and Phe⁵ was accomplished by incorporating Fmoc‐Phe(p‐NHAlloc) into the peptide followed by selective deprotection and modification. The corresponding amine‐containing peptides were also prepared. The pure peptides were evaluated in radioligand binding experiments using Chinese hamster ovary (CHO) cells expressing µ‐ and δ‐opioid receptors. In dermorphin, introduction of the electrophilic groups in the ‘message’ domain lowered the binding affinity by > 1000‐fold; only [Phe(p‐NH₂)³]dermorphin retained nanomolar affinity for µ‐receptors. Modifications in the ‘address’ region of both dermorphin and [Lys⁷]dermorphin were relatively well tolerated. In particular, [Phe(p‐NH₂)⁵,Lys⁷]dermorphin showed similar affinity to dermorphin, with almost 2‐fold higher selectivity for µ‐receptors. [Phe(p‐NHCOCH₂Br)⁵]‐ and [Phe(p‐NHCOCH₂Br)⁵,Lys⁷]dermorphin exhibited relatively high affinity (IC₅₀ = 27.7 and 15.1 nm , respectively) for µ‐receptors. However, neither of these peptides inhibited [³H]DAMGO binding in a wash‐resistant manner.     

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.     

Molecular modeling of salsolinol,a full Gi protein agonist of the μ‐opioid receptor,within the receptor binding site

(R/S)‐Salsolinol is a full agonist of the μ‐opioid receptor (μOR) G_i protein pathway via its (S)‐enantiomer and is functionally selective as it does not promote β‐arrestin recruitment. Compared to (S)‐salsolinol, the (R)‐enantiomer is a less potent agonist of the G_i protein pathway. We have now studied the interactions of the salsolinol enantiomers docked in the binding pocket of the μOR to determine the molecular interactions that promote enantiomeric specificity and functional selectivity of (R/S)‐salsolinol. Molecular dynamics simulations showed that (S)‐salsolinol interacted with 8 of the 11 residues of the μOR binding site, enough to stabilize the molecule. (R)‐Salsolinol showed higher mobility with fewer prevalent bonds. Hence, the methyl group bound to the (S)‐stereogenic center promoted more favorable interactions in the μOR binding site than in the (R)‐orientation. Because (S)‐salsolinol is a small molecule (179.2 Da), it did not interact with residues implicated in the binding of larger morphinan agonists that are located toward the extracellular portion of the binding pocket: W318^7.35, I322^7.39, and Y326^7.43. Our results suggest that contact with residues which (S)‐salsolinol interacts with are enough to elicit G_i protein activation, and possibly define a minimum set required by μOR ligands to promote activation of the G_i protein pathway.     

Probing opioid receptor–ligand interactions by employment of indolizidin‐9‐one amino acid as a constrained Gly2‐Gly3 surrogate in a leucine‐enkephalin mimic

Abstract: The relationship between the conformation and biological activity of Leu‐enkephalin was studied using (2S,6R,8S)‐9‐oxo‐8‐N‐(Boc)amino‐1‐azabicyclo[4.3.0]nonane‐2‐carboxylic acid [(2S,6R,8S)‐ 1 , I⁹AA] as a constrained Gly²‐Gly³ dipeptide surrogate. [I⁹AA]^2,3‐Leu‐enkephalin 12 was assembled using solid‐phase peptide synthesis on Merrifield resin with TBTU as the coupling reagent. The in vitro assays indicated that [I⁹AA]^2,3‐Leu‐enkephalin 12 exhibited affinities for the µ‐ and δ‐opioid receptors that were three orders of magnitude lower than that of Leu‐enkephalin, as well as partial agonist character for both receptors. In in vivo assays for spinal analgesia, the indolizidinone analog 12 showed significantly enhanced duration of action, indicating an increased metabolic stability. Conformational analysis was performed using NMR and CD spectroscopy. The amide temperature coefficients and ³J_NH‐CαH coupling constants for 12 could not support a hydrogen‐bonded β‐turn structure; however, its CD spectrum indicated a turn conformation. Incorporation of indolizidinone amino acid 1 into Leu‐enkephalin thus provided additional support for the importance of a turn conformation for the biological activity of the native peptide.     

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.     

Entrapping unusual folding characteristics of the β‐Ala residues in a model peptide: Boc‐β‐Ala‐Aib‐β‐Ala‐NHCH3

Abstract: Crystal structure analysis of a model peptide: Boc‐β‐Ala‐Aib‐β‐Ala‐NHCH₃ (β‐Ala: 3‐amino propionic acid; Aib: α‐aminoisobutyric acid) revealed distinct conformational preferences for folded [φ≈136°, µ ≈ ?62°, ψ ≈100°] and semifolded [φ ≈ 83°, µ ≈ ?177°, ψ ≈ ?117°] structures of the N‐ and C‐terminus β‐Ala residues, respectively. The overall folded conformation is stabilized by unusual N_i···H‐N_i+1 and nonconventional C–H···O intramolecular hydrogen bonding interactions.     

Synthesis and biological properties of β-MePhe3 analogues of deltorphin I and dermenkephalin: influence of biased X1 of Phe3 residues on peptide recognition for δ-opioid receptors

Using the method of conformational constraint, we have designed and synthesized analogues of deltorphin I and dermenkephalin containing each of the four stereoisomers (2S, 3S: 2S, 3R; 2R, 3S; 2R, 3R) of the unusual amino acid ß-methylphenylalanine in position three. The potency and selectivity of these analogues were evaluated by radioreceptor binding assays in the rat brain using [³H]CTOP (δ-ligand) and [³H][p-CβPhe⁴]DPDPE (β-ligand), and by bioassay using the mouse vas deferens (β-receptor assay) and guinea pig ileum (β-receptor assay) assays. The substitution of a ß-MePhe for Phe³ in deltorphin I and dermenkephalin has a large and variable effect on the bioactivities of the synthesized analogues. The synthesized analogues are somewhat less potent than the native peptides. Both [(2S, 3R)-ß-MePhe³]deltorphin and [(2S, 3R)-ß-MePhe³]dermenkephalin are more selective, however, and interact essentially specifically with the receptor in the binding assays and bioassays. The bioassay data in vitro of the synthesized analogues of deltorphin I and dermenkephalin follow the same general trends as the receptor binding data. These results demonstrate that topographical modifications of the side-chain conformation of critical structural moieties in a ligand can significantly modulate both the potency and receptor selectivity for ligands that have multiple sites of biological activity, and they illustrate that this approach has general application to peptide and peptidomimetic ligand design.     

Structural analysis of fertilinβ cyclic peptide mimics that are ligands for α6β1 integrin

Abstract: The NMR structural analysis of two fertilinβ mimics cyclo(EC²DC¹)YNH_2, 1 , and cyclo(D²EC²D¹C¹)YNH_2, 2 is described. Both of these mimics are moderate inhibitors of sperm?egg binding with IC₅₀ values of 500 µm in a mouse in vitro fertilization assay. For peptide 1 , the optimized conformations that best match the NMR data have a pseudo‐type II′β‐turn with the linker and Glu at the i+1 and i+2 positions, respectively. The EC²D¹C¹ sequence is in a nonclassical (type IV) β‐turn. For peptide 2 , the conformation that best matches the NMR data has two turns: a pseudo‐type II′β‐turn in the D²EC²D¹ sequence followed by a nonclassical β‐turn in the EC²D¹C¹ sequence. The Cβ?Cβ distance between E and D¹ in peptide 1 is 9.1 Å, in peptide 2 , it is 7.7 Å. Thus, one possibility for the high IC₅₀ values of these cyclic peptides is that the acidic residues are not constrained to a sufficiently tight turn, and thus much entropy must still be lost upon binding to the α₆β₁ integrin. This explains why the cyclic peptides are the same as linear peptides at inhibiting sperm?egg binding.     

Novel 19F‐MRS β‐galactosidase reporter molecules incorporated nitrogen mustard analogues

In this study, we propose a novel molecular platform‐integrated fluorinated antitumor nitrogen mustards for ¹⁹F‐MRS assay of β‐galactosidase (β‐gal) activity. Following this idea, we have designed, synthesized, and characterized 2‐fluoro‐4‐[bis(2′‐chloroethyl)amino]phenyl β‐D‐galactopyranoside 5 , 2‐fluoro‐4‐{bis[2′‐O‐(β‐D‐galactopyranosyl)ethyl]amino}phenyl β‐D‐galactopyranoside 8 , 2‐fluoro‐4‐{bis[[1″‐(β‐D‐galactopyranosyl)‐1″, 2″, 3″‐triazol‐4″‐yl]methyl] amino}phenyl β‐D‐galactopyranoside 14 and 2‐fluoro‐4‐{bis[[1″‐(β‐D‐glucopyranosyl)‐1″, 2″, 3″‐triazol‐4″‐yl]methyl]amino}phenyl β‐D‐galactopyranoside 15 through glycosylation and click reaction strategies, and their structures were confirmed by NMR and HRMS or elemental analysis data. Among them, 2‐fluoro‐4‐[bis(2′‐chloroethyl)amino]phenyl β‐D‐galacto‐pyranoside 5 was found very sensitive to β‐gal (E801A) in PBS at 37°C with big Δδ_F response. Here, we demonstrated the feasibility of this platform for assessing β‐gal activity in solution, and in vitro with lacZ‐transfected human MCF7 breast and PC3 prostate tumor cells, by the characterization of β‐gal‐responsive ¹⁹F‐chemical shift changes Δδ_F and hydrolytic kinetics.     

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.     

Some Hydrazones of 2‐Aroylamino‐3‐methylbutanohydrazide: Synthesis,Molecular Modeling Studies,and Identification as Stereoselective Inhibitors of HIV‐1

In accordance with our antiviral drug development attempt, acylhydrazone derivatives bearing amino acid side chains were synthesized for the evaluation of their antiviral activity against various types of viruses. Among these compounds, 8 _S , 11 _S , and 12 _S showed anti‐HIV‐1 activity with a 50% inhibitory concentration (IC₅₀) = 123.8 µM (selectivity index, SI > 3), IC₅₀ = 12.1 µM (SI > 29), IC₅₀ = 17.4 µM (SI > 19), respectively. Enantiomers 8 _R , 11 _R , and 12 _R were inactive against the HIV‐1 strain III_B. Hydrazones 8 _S , 11 _S , and 12 _S which were active against HIV‐1 wild type showed no inhibition against a double mutant NNRTI‐resistant strain (K103N;Y181C). Molecular docking calculations of R‐ and S‐enantiomers of 8 , 11 , and 12 were performed using the hydrazone‐bound novel site of HIV‐1 RT.     

Identify old drugs as selective bacterial β‐GUS inhibitors by structural‐based virtual screening and bio‐evaluations

Irinotecan (CPT‐11) is a cytotoxic drug that has wide applicability and usage in cancer treatment. Despite its success, patients suffer dose‐dependent diarrhea, limiting the drug's efficacy. No effective therapy is available for this unmet medical need. The bacterial β‐glucuronidase (β‐GUS) plays pivotal role in CPT‐11‐induced diarrhea (CID) via activating the non‐toxic SN‐38G to toxic SN‐38 inside intestine. By using structural‐based virtual screening, three old drugs (N‐Desmethylclozapine, Aspartame, and Gemifloxacin) were firstly identified as selective bacterial β‐GUS inhibitors. The IC₅₀ values of the compounds in the enzyme‐based and cell‐based assays range from 0.0389 to 3.6040 and 0.0105 to 5.3730 μM, respectively. The compounds also showed good selectivity against mammalian β‐GUS and no significant cytotoxicity in bacteria. Molecular docking and molecular dynamics simulations were performed to further investigate the binding modes of compounds with bacterial β‐GUS. Binding free energy decomposition revealed that the compounds formed strong interactions with E413 in catalytic trail from primary monomer and F365′ on the bacterial loop from the other monomer of bacterial β‐GUS, explaining the selectivity against mammalian β‐GUS. The old drugs identified here may be used as bacterial β‐GUS inhibitors for CID or other bacterial β‐GUS‐related disorders.     

Influence of hydrophobic interactions on the conformational adaptability of the β‐Ala residue

Abstract: The chemical synthesis and X‐ray crystal structure analysis of a model peptide incorporating a conformationally flexible β‐Ala residue: Boc‐β‐Ala‐Pda, 1 (C₂₃H₄₆N₂O₃: molecular weight = 398.62) have been described. The peptide crystallized in the crystal system triclinic with space group P2₁: a = 5.116(3) Å, b = 5.6770(10) Å, c = 21.744(5) Å; α = 87.45°, β = 86.87°, γ = 90.0°; Z = 1. An attractive feature of the crystal molecular structure of 1 is the induction of a reasonably extended backbone conformation of the β‐Ala moiety, i.e. the torsion angles φ ≈ ?115°, µ ≈ 173° and ψ ≈ 122°, correspond to skew^?, trans and skew⁺ conformation, respectively, by an unbranched hydrophobic alkyl chain, Pda, which prefers an all‐anti orientation (θ₁ ≈ ?153°, θ₂ ≈ … θ₁₄ ≈ ±178°). The observation is remarkable because, systematic conformational investigations of short linear β‐Ala peptides of the type Boc‐β‐Ala‐Xaa‐OCH₃ (Xaa = Aib or Acc⁶) have shown that the chemical and stereochemical characters of the neighboring moieties may be critical in dictating the overall folded and/or unfolded conformational features of the β‐Ala residue. The overall conformation of 1 is typical of a ‘bar’. It appears convincing that, in addition to a number of hydrophobic contacts between the parallel arranged molecules, an array of conventional N‐H…O=C intermolecular H‐bonding interactions stabilize the crystal molecular structure. Moreover, the resulting 14‐membered pseudo‐ring motif, generated by the amide–amide interactions between the adjacent molecules, is completely devoid of nonconventional C?H…O interaction. The potentials of the conformational adaptation of the β‐Ala residue, to influence and stabilize different structural characteristics have been highlighted.     

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%.     

Exploring relationships between mimic configuration,peptide conformation and biological activity in indolizidin‐2‐one amino acid analogs of gramicidin S*

Abstract: Indolizidin‐2‐one amino acids (I²aas, 6S‐ and 6R‐ 1 ) possessing 6S‐ and 6R‐ring‐fusion stereochemistry were introduced into the antimicrobial peptide gramicidin S (GS) to explore the relationships between configuration, peptide conformation and biological activity. Solution‐phase and solid‐phase techniques were used to synthesize three analogs with I²aa residues in place of the d ‐Phe‐Pro residues at the turn regions of GS: [(6S)‐I²aa^{4?5,4′?5′}]GS ( 2 ), [Lys^2,2′,(6S)‐I²aa^{4?5,4′?5′}]GS ( 3 ) and [(6R)‐I²aa^{4?5,4′?5′}]GS ( 4 ). Although conformational analysis of [I²aa^{4?5,4′?5′}]GS analogs 2?4 indicated that both ring‐fusion stereoisomers of I²aa gave peptides with CD and NMR spectral data characteristic of GS, the (6S)‐I²aa analogs 2 and 3 exhibited more intense CD curve shapes, as well as greater numbers of nonsequential NOE between opposing Val and Leu residues, relative to the (6R)‐I²aa analog 4 , suggesting a greater propensity for the (6S)‐diastereomer to adopt the β‐turn/antiparallel β‐pleated sheet conformation. In measurements of antibacterial and antifungal activity, the (6S)‐I²aa analog 2 exhibited significantly better potency than the (6R)‐I²aa diastereomer 4 . Relative to GS, [(6S)‐I²aa^{4?5,4′?5′}]GS ( 2 ) exhibited usually 1/2 to 1/4 antimicrobial activity as well as 1/4 hemolytic activity. In certain cases, antimicrobial and hemolytic activities of GS were shown to be dissociated through modification at the peptide turn regions with the (6S)‐I²aa diastereomer. The synthesis and evaluation of GS analogs 2?4 has furnished new insight into the importance of ring‐fusion stereochemistry for turn mimicry by indolizidin‐2‐one amino acids as well as novel antimicrobial peptides.