Protease‐catalyzed fragment condensation via substrate mimetic strategy: a useful combination of solid‐phase peptide synthesis with enzymatic methods

Abstract: The concept of substrate mimetic strategy represents a new powerful method in the field of enzymatic peptide synthesis. This strategy takes advantage of the shift in thesite‐specific amino acid moiety from the acyl residue to the ester‐leaving group of the carboxyl component enabling acylation of the enzyme by nonspecific acyl residues. As a result, peptide bond formation occurs independently of the primary specificity of proteases. Moreover, because of the coupling of nonspecific acyl residues, the newly formed peptide bond is not subject to secondary hydrolysis achieving irreversible peptide synthesis. Here, we report the combination of solid‐phase peptide synthesis with substrate mimetic‐mediated enzymatic peptide fragment condensations. First, the utility of the oxime resin strategy for the synthesis of peptide fragments in the form of substrate mimetics esterified as 4‐guanidinophenyl‐, phenyl‐ and mercaptopropionic acid esters was investigated. The study was completed by using the resulting N^α‐protected peptide esters as acyl donors in trypsin‐, α‐chymotrypsin‐ and V8 protease‐catalyzed fragment condensations.     

Solvent effects on coupling yields during rapid solid‐phase synthesis of CGRP(8‐37) employing in situ neutralization*

Abstract: The success of solid‐phase peptide synthesis is often dependent upon solvation of the resin and the growing resin‐bound peptide chain. We investigated the relationship between solvent properties and solvation of the resin and peptide‐resin in order to obtain satisfactory coupling yields for the rapid solid‐phase peptide synthesis, using butyloxycarbonyl‐(Boc)‐amino acid derivatives, of human‐α‐calcitonin gene‐related peptide(8‐37) (CGRP(8‐37)). Solvation of (p‐methylbenzhydrylamine)copoly(styrene–1% divinylbenzene (DVB) (resin) and resin covalently bound to the fully protected amino acid sequence of CGRP(8‐37) (peptide–resin) was correlated to solvent Hildebrand solubility (δ) and hydrogen‐bonding (δ_h) parameters. Contour solvation plots of δ_h vs. δ revealed maximum solvation regions of resin and peptide–resin. Maximum resin solvation occurred with N‐methylpyrrolidinone (NMP), NMP : dimethylsulfoxide (DMSO) (8 : 2) and DMSO. Inefficient solvation of the peptide–resin occurred with these solvents and resulted in poor syntheses with average coupling yields of 78.1, 88.9 and 91.8%, respectively. Superior peptide–resin solvation was obtained using dimethylacetamide (DMA) and dimethylformamide (DMF), resulting in significantly higher average coupling yields of 98.0 and 99.5%, respectively. Thus, the region of maximum peptide–resin solvation shifts to solvents with higher δ_h values. DMF provided the most effective peptide–resin solvation and was the only solvent from which CGRP(8‐37) was obtained as a single major product in the crude cleaved material.     

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.     

Improving the Specificity of the Prostate‐Specific Antigen Substrate Glutaryl‐Hyp‐Ala‐Ser‐Chg‐Gln as a Promoiety

To develop PSA peptide substrates with improved specificity and plasma stability from the known substrate sequence glutaryl‐Hyp‐Ala‐Ser‐Chg‐Gln, systematic replacements of the N‐terminal segment with D‐retro‐inverso‐peptides were performed with the incorporation of 7‐amino‐4‐methylcoumarin (7‐AMC) after Gln for convenient fluorometric determination and ranking of the PSA substrate activity. The D‐retro‐inverso‐peptide conjugates with P2‐P5 D‐amino acid substitutions were moderate but poorer PSA substrates as compared to the original peptide, suggesting that inversion of the amide bonds and/or incorporation of the additional atom as in the urea linker adversely affected PSA binding. However, P5 substitution of Hyp with Ser showed significant improvements in PSA cleavage rate; the resulting AMC conjugate, glutaryl‐Ser‐Ala‐Ser‐Chg‐Gln‐AMC ( 11 ), exhibited the fastest PSA cleavage rate of 351 pmol/min/100 nmol PSA. In addition, GABA←mGly‐Ala‐Ser‐Chg‐Gln‐AMC (conjugate 6 ) was the second best PSA substrate and released 7‐AMC at a rate of 225 pmol/min/100 nmol PSA as compared to 171 pmol/min/100 nmol PSA for the control conjugate glutaryl‐Hyp‐Ala‐Ser‐Chg‐Gln‐AMC. Incubations of selected AMC conjugates with mouse and human plasma revealed that GABA←D‐Ser‐ψ[NH‐CO‐NH]‐Ala‐Ser‐Chg‐Gln‐AMC ( 5 ) and GABA←mGly‐Ala‐Ser‐Chg‐Gln‐AMC ( 6 ) were most stable to non‐PSA‐mediated proteolysis. Our results suggest that the PSA specificity of glutaryl‐Hyp‐Ala‐Ser‐Chg‐Gln is improved with Ser and mGly substitutions of Hyp at the P5.     

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.     

In vitro characterization of an in situ microdialysis sampling assay for elastase activity detection

A microdialysis sampling method has been developed to detect the in vitro presence of a proteolytic enzyme, porcine elastase, external to a microdialysis probe. Elastase converts the substrate, succinyl(Ala)(3)-p-nitroanilide (suc(Ala)(3)-p-NA), to p-nitroaniline (p-NA). The substrate, suc(Ala)(3)-p-NA, was locally delivered through the microdialysis probe to external solutions containing different elastase activities (0.025-0.5 units/mL). The product, p-NA, was recovered back into the probe. Dialysates containing both suc(Ala)(3)-p-NA and p-NA were quantified using HPLC-UV. Different microdialysis suc(Ala)(3)-p-NA extraction efficiencies (EE) were observed among different elastase-containing solutions (buffer and 0.3% agar solutions). The p-NA concentrations recovered back into the microdialysis probe correlated with the elastase activity external to the microdialysis probe. The greatest fraction of p-NA recovered as compared to substrate lost occurred with the highest flow rate used (5.0 microL/min). However, the highest concentrations of p-NA recovered occurred at the lowest flow rates. This method may allow for microdialysis sampling to be used as a means to study localized enzyme activity.     

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

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

Optimized aminolysis conditions for cleavage of N‐protected hydrophobic peptides from solid‐phase resins

Abstract: Solid‐phase synthesis and aminolysis cleavage conditions were optimized to obtain N‐ and C‐terminally protected hydrophobic peptides with both high quality and yield. Uncharged ‘WALP’ peptides, consisting of a central (Leu‐Ala)_n repeating unit (where n = 5, 10.5 or 11.5) flanked on both sides by Trp ‘anchors’, and gramicidin A (gA) were synthesized using 9‐fluorenylmethoxycarbonyl chemistry from either Wang or Merrifield resins. For WALP peptides, the N‐terminal amino acid was capped by coupling N‐acetyl‐ or N‐formyl‐Ala or ‐Gly to the peptide/resin or by formylation of the completed peptide/resin with para‐nitrophenylformate (p‐NPF). N‐Terminal acetyl‐ or formyl‐Ala racemized when coupled as an HOBt‐ester to the resin‐bound peptide, but not when the peptide was formylated with p‐NPF. Racemization was avoided at the last step by completing the peptide with acetyl‐ or formyl‐Gly. For both WALP peptides and gA, cleavage conditions using ethanolamine or ethylenediamine were optimized as functions of solvent, time, temperature and resin type. For WALP peptides, maximum yields of highly pure peptide were obtained by cleavage with 20% ethanolamine or ethylenediamine in 80% dichloromethane for 48 h at 24°C. N‐Acetyl‐protected WALP peptides consistently gave higher yields than those protected with N‐formyl. For gA, cleavage with 20% ethanolamine or ethylenediamine in 80% dimethylformamide for 48 h at 24°C gave excellent results. For both WALP peptides and gA, decreasing the cleavage time to 4 h and increasing the temperature to 40–55°C resulted in significantly lower yields. The inclusion of hexafluoroisopropanol in the cleavage solvent mixture did not improve yields for either gA or WALP peptides.     

Synthetic peptide derived from α‐fetoprotein inhibits growth of human breast cancer: investigation of the pharmacophore and synthesis optimization

Abstract: A synthetic peptide that inhibits the growth of estrogen receptor positive (ER+) human breast cancers, growing as xenografts in mice, has been reported. The cyclic 9‐mer peptide, cyclo[EMTOVNOGQ], is derived from α‐fetoprotein (AFP), a safe, naturally occurring human protein produced during pregnancy, which itself has anti‐estrogenic and anti‐breast cancer activity. To determine the pharmacophore of the peptide, a series of analogs was prepared using solid‐phase peptide synthesis. Analogs were screened in a 1‐day bioassay, which assessed their ability to inhibit the estrogen‐stimulated growth of uterus in immature mice. Deletion of glutamic acid, Glu1, abolished activity of the peptide, but glutamine (Gln) or asparagine (Asn) could be substituted for Glu1 without loss of activity. Methionine (Met2) was replaced with lysine (Lys) or tyrosine (Tyr) with retention of activity. Substitution of Lys for Met2 in the cyclic molecule resulted in a compound with activity comparable with the Met2‐containing cyclic molecule, but with a greater than twofold increase in purity and corresponding increase in yield. This Lys analog demonstrated anti‐breast cancer activity equivalent to that of the original Met‐containing peptide. Therefore, Met2 is not essential for biologic activity and substitution of Lys is synthetically advantageous. Threonine (Thr3) is a nonessential site, and can be substituted with serine (Ser), valine (Val), or alanine (Ala) without significant loss of activity. Hydroxyproline (Hyp), substituted in place of the naturally occurring prolines (Pro4, Pro7), allowed retention of activity and increased stability of the peptide during storage. Replacement of the first Pro (Pro4) with Ser maintains the activity of the peptide, but substitution of Ser for the second Pro (Pro7) abolishes the activity of the peptide. This suggests that the imino acid at residue 7 is important for conformation of the peptide, and the backbone atoms are part of the pharmacophore, but Pro4 is not essential. Valine (Val5) can be substituted only with branched‐chain amino acids (isoleucine, leucine or Thr); replacement by d ‐valine or Ala resulted in loss of biologic activity. Thus, for this site, the bulky branched side chain is essential. Asparagine (Asn6) is essential for activity. Substitution with Gln or aspartic acid (Asp), resulted in reduction of biologic activity. Removal of glycine (Gly8) resulted in a loss of activity but nonconservative substitutions can be made at this site without a loss of activity indicating that it is not part of the pharmacophore. Cyclization of the peptide is facilitated by addition of Gln9, but this residue does not occur in AFP nor is it necessary for activity. Gln9 can be replaced with Asn, resulting in a molecule with similar activity. These data indicate that the pharmacophore of the peptide includes side chains of Val5 and Asn6 and backbone atoms contributed by Thr3, Val5, Asn6, Hyp7 and Gly8. Met2 and Gln9 can be modified or replaced. Glu1 can be replaced with charged amino acids, and is not likely to be part of the binding site of the peptide. The results of this study provide information that will be helpful in the rational modification of cyclo[EMTOVNOGQ] to yield peptide analogs and peptidomimetics with advantages in synthesis, pharmacologic properties, and biologic activity.     

Synthesis of peptide sequences related to thrombospondin: factors affecting aspartimide by‐product formation

Abstract: Aspartimide formation is one of the most common secondary reactions on solid phase peptide synthesis. In the present work, we describe the optimization of the synthesis of two thrombospondin fragments containing an Asp‐Gly sequence that show a strong tendency to form cyclic aspartimide derivatives in an unusual high percentage. Several different strategies were applied changing type of resin, Fmoc‐deprotection reagents, coupling additives, resin cleavage cocktails and the use of Hmb‐Gly derivative to minimize the extension of this byproduct. Best results were obtained with cross‐linked ethoxylate acrylate (CLEAR^®‐cross‐linked ethoxylate Acrylate Resin)‐type resin and pip/dimethylformamide deprotection. Besides, as in biological assays the aspartimide containing sequence resulted to be more active than the linear one, the optimization of its synthesis was also carried out.     

Syntheses,characterization and application of cross‐linked polystyrene−ethyleneglycol acrylate resin (CLPSER) as a novel polymer support for polypeptide syntheses

Abstract: Cross‐linked polystyrene?ethyleneglycol acrylate resin (CLPSER) was developed for the solid‐phase synthesis of peptide by introducing a cross‐linker, O,O′‐bis(2‐acrylamidopropyl)polyethylene glycol₁₉₀₀ (Acr₂PEG), into polystyrene. The cross‐linker was prepared by treating acryloyl chloride with O,O′‐bis(2‐aminopropyl) polyethylene glycol₁₉₀₀[(NH₂)₂PEG] in the presence of diisopropylethylamine. The copolymer was prepared either by bulk or inverse suspension copolymerization of Acr₂PEG₁₉₀₀ and styrene using sorbitan monolaurate as the suspension stabilizer, and a mixture of ammonium peroxodisulfate and benzoyl peroxide as the radical initiators. The resin was characterized using gel‐phase ¹³C NMR, infrared (KBr) spectroscopic techniques and the morphological features of the resin were investigated using scanning electron microscopy photographs. CLPSER showed excellent swelling in a broad range of solvents and was found to be chemically inert to various reagents and solvents used in solid‐phase peptide synthesis. To demonstrate the usefulness of the new resin in polypeptide synthesis, the support was derivatized with an ‘internal reference’ amino acid (norleucine) and a handle 4‐(4‐hydroxymethyl‐3‐methoxy)butyric acid. The new resin was compared with commercial supports such as Merrifield and Sheppard resins by synthesizing an acyl carrier protein (65?74) fragment under the same experimental conditions. HPLC profiles revealed the high efficiency of the newly developed support. Resin capability in peptide synthesis was further demonstrated by the solid phase synthesis of a 25‐residue peptide from the E2/NS1 region hepatitis C viral polyprotein.     

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.     

Facile solid‐phase synthesis of C‐terminal peptide aldehydes and hydroxamates from a common Backbone Amide‐Linked (BAL) intermediate*†

Abstract: C‐Terminal peptide aldehydes and hydroxamates comprise two separate classes of effective inhibitors of a number of serine, aspartate, cysteine, and metalloproteases. Presented here is a method for preparation of both classes of peptide derivatives from the same resin‐bound Weinreb amide precursor. Thus, 5‐[(2 or 4)‐formyl‐3,5‐dimethoxyphenoxy]butyramido‐polyethylene glycol‐polystyrene (BAL‐PEG‐PS) was treated with methoxylamine hydrochloride in the presence of sodium cyanoborohydride to provide a resin‐bound methoxylamine, which was efficiently acylated by different Fmoc‐amino acids upon bromo‐tris‐pyrrolidone‐phosphonium hexafluorophosphate (PyBrOP) activation. Solid‐phase chain elongation gave backbone amide‐linked (BAL) peptide Weinreb amides, which were cleaved either by trifluoroacetic acid (TFA) in the presence of scavengers to provide the corresponding peptide hydroxamates, or by lithium aluminum hydride in tetrahydrofuran (THF) to provide the corresponding C‐terminal peptide aldehydes. With several model sequences, peptide hydroxamates were obtained in crude yields of 68–83% and initial purities of at least 85%, whereas peptide aldehydes were obtained in crude yields of 16–53% and initial purities in the range of 30–40%. Under the LiAlH₄ cleavage conditions used, those model peptides containing t‐Bu‐protected aspartate residues underwent partial side chain reduction to the corresponding homoserine‐containing peptides. Similar results were obtained when working with high‐load aminomethyl‐polystyrene, suggesting that this chemistry will be generally applicable to a range of supporting materials.     

Tri(propylene glycol) glycerolate diacrylate cross‐linked polystyrene: a new resin support for solid‐phase peptide synthesis

Abstract: A highly flexible, mechanically and chemically stable copolymer, tri(propylene glycol) glycerolate diacrylate cross‐linked polystyrene (PS‐TRPGGDA), was synthesized by the suspension polymerization and employed as a solid support for peptide synthesis. The beaded polymer support containing secondary hydroxyl functional groups in the cross‐linker was used as the growth site for peptide synthesis. The procedure is unique and cost‐effective in that it avoids the initial functionalization steps required for most of the styrene‐based polymer supports. The resin was characterized by ¹³C‐CP‐MAS NMR spectroscopy and the morphologic features of the resin were investigated using scanning electron microscopy. Swelling studies conducted on the new support revealed that the PS‐TRPGGDA resin undergoes more effective swelling and solvation than PS‐DVB resin in all solvents used in peptide synthesis. The efficiency of the new support was demonstrated by synthesizing a ‘difficult’ sequence Ala‐Arg‐(Ala)₆‐Lys and comparing it with commercially available Merrifield and Sheppard resins. The synthetic efficiency was further demonstrated by the synthesis of a 24‐residue NR 2A peptide substrate of calcium/calmodulin‐binding peptide. The high yield and purity of the peptide synthesized on the novel support indicates the positive role of the flexible and hydrophilic cross‐linking agent in the solid support.     

111In‐ and 203Pb‐labeled cyclic arginine‐glycine‐aspartic acid peptide conjugate as an αvβ3 integrin‐binding radiotracer

Methodology for site‐specific modification and chelate conjugation of a cyclic arginine‐glycine‐aspartic acid (cRGD) peptide for the preparation of a radiotracer molecular imaging agent suitable for detecting α_vβ₃ integrin is described. The method involves functionalizing the peptide with an aldehyde moiety and conjugation to a 1,4,7,10‐tetraazacyclododecane‐N,N′,N″,N?‐tetraacetic acid derivative that possesses an aldehyde reactive aminooxy group. The binding assay of the ¹¹¹In‐labeled peptide conjugate with α_vβ₃ integrin showed 60% bound when four equivalents of the integrin was added, a reasonable binding affinity for a monovalent modified RGD peptide.     

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 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.     

Cis/trans conformational equilibrium across the N‐methylphenylalanine2‐ N‐methylphenylalanine3 peptide bond of arginine vasopressin analogs

Abstract: Two cyclic analogs of vasopressin, ‐Pro‐Arg‐Gly‐NH₂ ( 1 ) and ‐Pro‐Arg‐Gly‐NH₂ ( 2 ) were synthesized by the solid phase method. Their structure was determined in aqueous solution by two‐dimensional NMR techniques and simulated annealing algorithm from an extended template in X‐PLOR. The total chemical shift correlation spectroscopy and rotating‐frame Overhauser enhancement spectroscopy of the peptides displayed four distinct sets of residual proton resonances. This suggests that both analogs adopt four families of conformations in H₂O/D₂O (9 : 1) (one major and three minor species). In further analysis only signals of major species (M) and of one minor species (m₁) were considered. The major species of both peptides include a trans peptide bond between the first and second residue, and a cis form between the second and third residue. In the minor species, all peptide bonds were found to exist in trans geometry.     

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.     

Effect of viscosity on the deamidation rate of a model Asn‐hexapeptide

Abstract: The effect of viscosity on the deamidation rate of a model Asn‐containing hexapeptide (l ‐Val‐l ‐Tyr‐Pro‐l ‐Asn‐Gly‐l ‐Ala) was assessed in aqueous solution and in solids containing varying amounts of poly(vinyl pyrrolidone) (PVP) and water. Stability studies were conducted at 0.1 mg/mL peptide and 0–50% PVP (w/w) in aqueous solution, and at 5% (w/w) peptide and different relative humidities (31.6, 53.1, 74.4 and 96%) in the solid state. The parent peptide and its deamidation products were analysed by reverse‐phase high‐performance liquid chromatography. Deamidation rates decreased with increasing solvent viscosity in a manner described by a semi‐empirical mathematical model developed to describe this relationship. The results suggest that the motion of the Asn side‐chain along the reaction coordinate is a function of the macroscopic solvent viscosity. However, the apparent energy barrier for the diffusive movement of the side‐chain appears to be less than the energy barrier for that associated with macroscopic viscosity. The dependence of the deamidation rate on viscosity in both viscous solution and hydrated solids further demonstrates the importance of mobility in peptide deamidation.