Tryptophan‐containing peptide helices: interactions involving the indole side chain*

Abstract: Two designed peptide sequences containing Trp residues at positions i and i + 5 (Boc‐Leu‐Trp‐Val‐Ala‐Aib‐Leu‐Trp‐Val‐OMe, 1 ) as well as i and i + 6 (Boc‐Leu‐Trp‐Val‐Aib‐Ala‐Aib‐Leu‐Trp‐Val‐OMe, 2 ) containing one and two centrally positioned Aib residues, respectively, for helix nucleation, have been shown to form stable helices in chloroform solutions. Structures derived from nuclear magnetic resonance (NMR) data reveal six and seven intramolecularly hydrogen‐bonded NH groups in peptides 1 and 2 , respectively. The helical conformation of octapeptide 1 has also been established in the solid state by X‐ray diffraction. The crystal structure reveals an interesting packing motif in which helical columns are stabilized by side chain–backbone hydrogen bonding involving the indole N?1H of Trp(2) as donor, and an acceptor C=O group from Leu(6) of a neighboring molecule. Helical columns also associate laterally, and strong interactions are observed between the Trp(2) and Trp(7) residues on neighboring molecules. The edge‐to‐face aromatic interactions between the indoles suggest a potential C‐H…π interaction involving the Cζ3H of Trp(2). Concentration dependence of NMR chemical shifts provides evidence for peptide association in solution involving the Trp(2) N?1H protons, presumably in a manner similar to that observed in the crystal.     

Synthesis,crystal structure and molecular conformation of Nα‐Boc‐l‐leucyl‐(Z)‐β‐(3‐pyridyl)‐α,β‐ dehydroalanyl‐l‐leucine methyl ester*

Abstract: A protected tridehydropeptide containing (Z)‐β‐(3‐pyridyl)‐α,β‐dehydroalanine (Δ^Z3Pal) residue, Boc‐Leu‐Δ^Z3Pal‐Leu‐OMe ( 1 ), was synthesized via Erlenmeyer azlactone method. X‐ray crystallographic analysis revealed that the peptide 1 adopts an extended conformation, which is similar to that of a Δ^ZPhe analog, Boc‐Leu‐Δ^ZPhe‐Leu‐OMe ( 2 ).     

Role of N‐t‐Boc group in helix initiation in a novel tetrapeptide

Abstract: Protecting groups in N‐ and C‐terminal positions play a decisive role in the conformational preference of smaller peptides. Conformational analysis of tetrapeptide derivatives containing Ala, Ile and Gly residues was performed. Peptide 1 , Boc‐Ala‐Ile‐Ile‐Gly‐OMe (Boc: tert‐butyloxycarbonyl) has a predominantly helical turn conformation in all the alcoholic solvents studied, whereas in the solid state it has a β‐sheet conformation. In contrast, peptide 2 , Ac‐Ala‐Ile‐Ile‐Gly‐OMe (Ac: acetyl) has a random coil conformation in solution. The FTIR spectrum of peptide 1 shows a lower frequency of urethane carbonyl, indicating involvement of the carbonyl group in hydrogen bonding in the helical turn.     

Inhibition of β‐amyloid toxicity by short peptides containing N‐methyl amino acids

Abstract: Single N‐methyl amino acid‐containing peptides related to the central hydrophobic region β_16–20 (Lys‐Leu‐Val‐Phe‐Phe) of the β‐amyloid protein are able to reduce the cytotoxicity of natural β_1–42 in PC12 cell cultures. N‐methyl phenylalanine analogs yield statistically significant increments in cell viability (Student's t‐test < 0.01%) and are nontoxic in the same assay. These promising results indicate that these peptide molecules could be a starting point for the development of potential therapeutic compounds for the treatment of Alzheimer's disease.     

New Alloferon Analogues: Synthesis and Antiviral Properties

We have extended our study on structure/activity relationship studies of insect peptide alloferon (H‐His‐Gly‐Val‐Ser‐Gly‐His‐Gly‐Gln‐His‐Gly‐Val‐His‐Gly‐OH) by evaluating the antiviral effects of new alloferon analogues. We synthesized 18 alloferon analogues: 12 peptides with sequences shortened from N‐ or C‐terminus and 6 N‐terminally modified analogues H‐X¹‐Gly‐Val‐Ser‐Gly‐His‐Gly‐Gln‐His‐Gly‐Val‐His‐Gly‐OH, where X¹ = Phe ( 13 ), Tyr ( 14 ), Trp ( 15 ), Phg ( 16 ), Phe(p‐Cl) ( 17 ), and Phe(p‐OMe) ( 18 ). We found that most of the evaluated peptides inhibit the replication of Human Herpesviruses or Coxsackievirus B2 in Vero, HEp‐2 and LLC‐MK₂ cells. Our results indicate that the compound [3‐13]‐alloferon ( 1 ) exhibits the strongest antiviral activity (IC₅₀ = 38 μm ) among the analyzed compound. Moreover, no cytotoxic activity against the investigated cell lines was observed for all studied peptides at concentration 165 μm or higher.     

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.     

Structure and property of self‐assemble valinyl bolaform amides having different chirality

Abstract: Bolaform amides were designed from N,N'‐bis(carboethoxy‐l ‐valinyl)‐diaminoethane ( 1 ) by linking t‐butyloxycarbonyl‐valine through ethylenediamine (EDA) to enable spectroscopic and X‐ray diffraction analyses. N,N'‐Bis(Boc‐l ‐valinyl)‐diaminoethane ( 2 ) and N,N'‐bis(Boc‐d ‐valinyl)‐diaminoethane ( 3 ) were composed of l ‐Val and d ‐Val, respectively. N‐(Boc‐l ‐valinyl)‐N'‐(Boc‐d ‐valinyl)‐diaminoethane ( 4 ) was composed of both l ‐Val and d ‐Val, and was achiral (meso‐peptide). Peptide 5 was a 1 : 1 mixture of 2 and 3 , and was also achiral (racemate). These peptides mediated gelation of corn oil at a concentration of approximately 1%. Within crystals, the peptides formed β‐sheet ribbons, but differences were observed in hydrogen‐bonding patterns and side‐chain arrangements. These differences were also deduced from temperature dependence of amide protons. Force‐field calculations based on the crystal structures indicated that association of β‐sheet ribbons had energy benefits, and it was assumed that molecular aggregation progressed spontaneously. These structural studies indicated the chirality of amino acids affected for the properties of bolaform amides.     

Anticancer activity of VmCT1 analogs against MCF‐7 cells

Antimicrobial peptides are considered promising drug candidates due to their broad range of activity. VmCT1 (Phe–Leu–Gly–Ala–Leu–Trp–Asn–Val–Ala–Lys–Ser–Val–Phe–NH₂) is an α‐helical antimicrobial peptide that was obtained from the Vaejovis mexicanus smithi scorpion venom. Some of its analogs showed to be as antimicrobial as the wild type, and they were designed for understanding the influence of physiochemical parameters on antimicrobial and hemolytic activity. Some cationic antimicrobial peptides exhibit anticancer activity so VmCT1 analogs were tested to verify the anticancer activity of this family of peptides. The analogs were synthesized, purified, characterized, and the conformational studies were performed. The anticancer activity was assessed against MCF‐7 mammary cancer cells. The results indicated that [Glu]⁷‐VmCT1‐NH₂, [Lys]³‐VmCT1‐NH₂, and [Lys]⁷‐VmCT1‐NH₂ analogs presented moderated helical tendency (0.23–0.61) and tendency of anticancer activity at 25 μmol/L in 24 hr of experiment; and [Trp]⁹‐VmCT1‐NH₂ analog that presented low helical tendency and moderated anticancer activity at 50 μmol/L. These results demonstrated that single substitutions on VmCT1 led to different physicochemical features and could assist on the understanding of anticancer activity of this peptide family.     

A combined extended and helical backbone for Boc‐(Ala‐Leu‐Ac7c‐)2‐OMe*

Abstract: The structure of the peptide Boc‐Ala‐Leu‐Ac₇c‐Ala‐Leu‐Ac₇c‐OMe (Ac₇c,1‐aminocycloheptane‐1‐carboxylic acid) is described in crystals. The presence of two Ac₇c residues was expected to stabilize a 3₁₀‐helical fold. Contrary to expectation the structural analysis revealed an unfolded amino terminus, with Ala(1) adopting an extended β‐conformation (φ = ?93°,ψ = 112°). Residues 2–5 form a 3₁₀‐helix, stabilized by three successive intramolecular hydrogen bonds. Notably, two NH groups Ala(1) and Ac₇c(3) do not form any hydrogen bonds in the crystal. Peptide assembly appears to be dominated by packing of the cycloheptane rings that stack against one another within the molecule and also throughout the crystal in columns.     

Conformation of the transmembrane domains in peripheral myelin protein 22. Part 1. Solution‐phase synthesis and circular dichroism study of protected 17‐residue partial peptides in the first putative transmembrane domain*

Abstract: Charcot‐Marie‐Tooth disease (CMT) is the most commonly inherited peripheral neuropathy. DNA duplication and point mutation of the gene encoding peripheral myelin protein 22 (PMP22) have been found in CMT type 1A dominants. To investigate the influence of the point mutation of PMP22 on the secondary structure, protected partial peptides in the putative first transmembrane domain, wild type Boc‐IVLH(Bom)VAVLVLLFVSTIV‐OMe ( 1 ) and its Pro¹⁶ mutant Boc‐IVLH(Bom)VAVPVLLFVSTIV‐OMe ( 2 ) were synthesized. Circular dichorism (CD)‐spectral analysis suggested that peptide 1 adopts a stable α‐helical conformation in membrane‐mimetic solvent,1‐BuOH/1,1,1,3,3,3‐hexafluoro‐2‐propanol (HFIP) system. On the contrary, the mutant 2 favors β‐sheet conformation in the same solvent system. Interestingly, α‐helix to β‐sheet transition of 2 was observed at higher contents of 1‐BuOH than 70%.     

Pseudopeptides containing the 2‐hydrazonoacyl fragment: analogs of the chemotactic agent HCO‐Met‐Leu‐Phe‐OMe

Abstract: In order to further examine the properties of pseudopeptides containing the 2‐hydrazonoacyl fragment, two new series of analogs of the prototypical chemotactic N‐formyl‐tripeptide HCO‐Met‐Leu‐Phe‐OMe were designed and synthesized. The first group contains the new fragment as the N‐terminal residue and is represented by the N‐aryl derivatives p‐Cl‐C₆H₄‐NH‐N=C(R)‐CO‐Leu‐Phe‐OMe ( 2 and 3 ) and by the corresponding N‐aroyl analogs p‐CH₃‐C₆H₄‐CO‐NH‐N=C(R)‐CO‐Leu‐Phe‐OMe ( 4 ). The second group contains the new fragment in place of the central Leu residue and is represented by compounds HCO‐Xaa‐NH‐N=C(R)‐CO‐Phe‐OMe ( 7a and 7b ) where Xaa is Nle and Met, respectively. The conformational and biochemical properties of the new products were examined.     

Chromogenic and fluorogenic glycosylated and acetylglycosylated peptides as substrates for serine,thiol and aspartyl proteases

We synthesized short chromogenic peptidyl–Arg–p-nitroanilides containing either (Galβ)Ser or (Glcα,β)Tyr at P₂ or P₃ sites as well as O-acetylated sugar moieties and studied their hydrolysis by bovine trypsin, papain, human tissue kallikrein and rat tonin. For comparison, the susceptibility to these enzymes of Acetyl–X–Arg–pNa and Acetyl–X–Phe–Arg–pNa series, in which X was Ala, Phe, Gln and Asn were examined. We also synthesized internally quenched fluorescent peptides with the amino acid sequence Phe⁸–His–Leu–Val–Ile–His–Asn¹⁴ of human angiotensinogen, in which [GlcNAcβ]Asn was introduced before Phe⁸ and/or after His¹³ and ortho-aminobenzoic acid (Abz) and N-[2-, 4-dinitrophenyl]–ethylenediamine (EDDnp) were attached at N- and C-terminal ends as a donor/receptor fluorescent pair. These peptides were examined as substrates for human renin, human cathepsin D and porcine pepsin. The chromogenic substrates with hydrophilic sugar moiety increased their susceptibility to trypsin, tissue kallikrein and rat tonin. For papain, the effect of sugar depends on its position in the substrate, namely, at P₃ it is unfavorable, in contrast to the P₂ position that resulted in increasing affinity, as demonstrated by the higher inhibitory activity of Ac–(Galβ)Ser–Arg–pNa in comparison to Ac–Ser–Arg–pNa, and by the hydrolysis of Ac–(Glcα,β)Tyr–Arg–pNa. On the other hand, the acetylation of sugar hydroxyl groups improved hydrolysis of the susceptible peptides to all enzymes, except tonin. The P^′₄ glycosylated peptide [Abz–F–H–L–V–I–H–(GlcNAcβ)N–E–EDDnp], that corresponds to one of the natural glycosylation sites of angiotensinogen, was shown to be the only glycosylated substrate susceptible to human renin, and was hydrolysed with lower K_m and higher k_cat values than the same peptide without the sugar moiety. Human cathepsin D and porcine pepsin are more tolerant to substrate glycosylation, hydrolysing both the P^′₄ and P₄ glycosylated substrates.     

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.     

Isopeptide bonds in chemotactic tripeptides. Synthesis and activity of lysine‐containing fMLF analogs

Abstract: In order to explore the properties of chemotactic N‐formylpeptides containing isopeptide bonds within their backbones, a group of lysine‐containing analogs of the prototypical chemotactic tripeptide N‐formylmethionyl‐leucyl‐phenylalanine (fMLF) was synthesized. The new analogs were designed by adding to the HCO‐Met or Boc‐Met residue a dipeptide fragment made up of Lys and Phe residues joined through Lys N^α or N^ε bonds, in all possible combinations. Thus, the following six pairs of tripeptides were synthesized and examined for their bioactivity: RCO‐Met‐Lys(Z)‐Phe‐OMe ( 2a , b ), RCO‐Met‐Lys(Z‐Phe)‐OMe ( 3a , b ), Z‐Lys(RCO‐Met)‐Phe‐OMe ( 4a , b ), Z‐Phe‐Lys(RCO‐Met)‐OMe ( 5a , b ), RCO‐Met‐Phe‐Lys(Z)‐OMe ( 6a , b ) and Z‐Lys(RCO‐Met‐Phe)‐OMe ( 7a , b ), with R=OC(CH₃)₃ and R=H for compounds a and b , respectively. All the new models were characterized fully and their activity (chemotaxis, superoxide anion production and lysozyme release) on human neutrophils determined as agonists (compounds b ) and antagonists (compounds a ). All N‐formyl derivatives 2b ? 7b are less potent than fMLF‐OMe as chemoattractants, but compound 7b exhibits selective activity as superoxide anion producer. Derivatives 2a ? 7a do not show antagonistic activity towards fMLF induced chemotaxis and O₂^? production, however, all these compounds except 4a antagonize lysozyme release by 60%.     

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.     

Synthesis and backbone conformations of cyclic hexapeptides cyclo-(Xxx-Pro-D-Gln)2

The solution syntheses of cyclo-(Xxx-Pro-D-Gln)₂, where Xxx=Gly, Ala, Leu, Phe and Val are described. Several routes were examined, the most successful involving the intermediate Z-Xxx-Pro-D-Gln-O-tBu and proceeding to cyclization of H-Xxx-Pro-D-Gln-Xxx-Pro-D-Gln-OH using diphenylphosphoryl azide. The N-H regions of the proton magnetic resonance spectra of aqueous solutions of these peptides were examined, and in the Xxx=Leu and Val peptides an unsymmetrical backbone, presumably with one cis Xxx-Pro peptide bond, was found to be important. Previous reports of cyclo-(Xxx-Pro-D-Yyy)₂ peptides have shown only C₂-symmetric forms.     

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.     

Enhanced cleavage of diaminopimelate-containing isopeptides by leucine aminopeptidase and matrix metalloproteinases in tumors: application to bioadhesive peptides

We prepared (2S,6S)-Z-Dpm(Z)(OMe) ( 4 ) by protease-mediated hydrolysis of (R,R/S,S)-Z-Dpm(Z)(OMe)-OMe ( 3 ), converted it to (2S,6S)-Dpm(Z)(OMe) ( 6 ) via PCl₅ to an NCA intermediate and hydrolysis, protected the amino group with Boc to give (2S,6S)-Boc-Dpm(Z)(OMe) ( 7 ), which upon ammonolysis of the Me ester afforded (2S,6S)-Boc-Dpm(Z)(NH₂) ( 8 ). Hydrogenolysis of 8 and protection with Fmoc gave (2S,6S)-Boc-Dpm(Fmoc)(NH₂) ( 10 ). Using 10 and SPPS, we prepared three Dpm-containing peptides and their corresponding Lys peptides. Enzymatic studies with mLAP and cLAP showed that the Leu moiety in Ac-Gly-(2S,6S)-Dpm(Leu)(NH₂)-Ala ( 14 ) was hydrolyzed 68-fold and> 1000-fold more rapidly, respectively, than that in Ac-Gly-Lys(Leu)-Ala ( 12 ). The enhanced rate of Leu formation from 14 compared to 12 was also observed with homogenates of mouse C3 sarcomas. This homogenate also hydrolyzed Ac-Gly-(2S,6S)-Dpm(Ac-Gly-Pro-Gln-Gly-Leu)(NH₂)-Ala ( 16 ) to Ac-Gly-(2S,6S)-Dpm(NH₂)-Ala ( 13 ), Leu and Ac-Gly-Pro-Gln-Gly ( 17 ). This implies the side chain is cleaved first by endopeptidases, such as matrix metalloproteinases (MMPs), and then the remaining Leu is cleaved by LAP-like exopeptidases. The rate of liberation of 17 from 16 and the corresponding Lys isopeptide, Ac-Gly-Lys(Ac-Gly-Pro-Gln-Gly-Leu)-Ala ( 15 ), was not significantly different. The rate of formation of 13 was faster from 16 than Ac-Gly-Lys-Ala ( 11 ) was from 15 . Thus, the entire isopeptide side chain can be removed by the cooperative action of LAP-like and MMP-like peptidases present in tumor tissue, which occurs faster in the Dpm peptide 16 than in the Lys peptide 15 . The rate of formation of 13 from 16 by lung, liver, and intestine homogenates (from the same C3 tumor-bearing mice) was comparable to or higher than from the tumor homogenates, but the rate by blood was only 4% the value of the tumor homogenates. Analogs of a bioadhesive fragment from the laminin α1 chain were prepared by replacing the essential Lys with Dpm(NH₂) ( 20 ) and Dpm(Leu)(NH₂) ( 21 ). Both Dpm-containing peptides were active, although considerably weaker than the corresponding Lys peptides 18 and 19 , in a cell attachment assay with human fibrosarcoma HT-1080 cells.     

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.     

Conformation of dehydropeptides

Six model dipeptide methyl amides containing dehydroaminobutyric acid (Δ Abu) of the type Boc-X-δ^ZAbu-NHCH₃ and Box-X-δ^EAbu-NHCH₃, X = Ala, Val, Phe (Boc =tert-butoxycarbonyl), have been synthesized and their solution conformations explored using 300 MHz ¹H NMR and IR spectroscopy. Studies based on delineation of intramolecularly hydrogen bonded NH groups in CDCl₃ and (CD₃)₂SO revealed that none of the NH groups is appreciably solvent shielded. Difference NOE (Nuclear Overhauser Effect) studies have also failed to detect the presence of any discernible turn structure in these peptides. These studies indicate that the conformational preferences of peptides containing, α, β-dehydroaminobutyric acid are different from those of Δ^ZPhe and Δ^ZLeu. It appears that steric interactions due to the β-substituent in the dehydroamino acid moiety play an important role. Unlike Δ^ZPhe and Δ^ZLeu, which have relatively large β-substituents, phenyl and isopropyl, respectively, and stabilize a β-turn, the β-methyl group of Δ^ZAbu or Δ^EAbu is readily accommodated in extended conformation. Clearly, the size of β-substituent in dehydroamino acid crucially influences the conformational preferences. Thus, it may be possible to use different dehydroamino acids to introduce variable but definite constraints in synthetic peptides.