Synthesis and biological activities of new side chain and backbone cyclic bradykinin analogues

Abstract: A series of conformationally constrained cyclic analogues of the peptide hormone bradykinin (BK, Arg‐Pro‐Pro‐Gly‐Phe‐Ser‐Pro‐Phe‐Arg) was synthesized to check different turned structures proposed for the bioactive conformation of BK agonists and antagonists. Cycles differing in the size and direction of the lactam bridge were performed at the C‐ and N‐terminal sequences of the molecule. Glutamic acid and lysine were introduced into the native BK sequence at different positions for cyclization through their side chains. Backbone cyclic analogues were synthesized by incorporation of N‐carboxy alkylated and N‐amino alkylated amino acids into the peptide chain. Although the coupling of Fmoc‐glycine to the N‐alkylated phenylalanine derivatives was effected with DIC/HOAt in SPPS, the dipeptide building units with more bulky amino acids were pre‐built in solution. For backbone cyclization at the C‐terminus an alternative building unit with an acylated reduced peptide bond was preformed in solution. Both types of building units were handled in the SPPS in the same manner as amino acids. The agonistic and antagonistic activities of the cyclic BK analogues were determined in rat uterus (RUT) and guinea‐pig ileum (GPI) assays. Additionally, the potentiation of the BK‐induced effects was examined. Among the series of cyclic BK agonists only compound 3 with backbone cyclization between positions 2 and 5 shows a significant agonistic activity on RUT. To study the influence of intramolecular ring closure we used an antagonistic analogue with weak activity, [d ‐Phe⁷]‐BK. Side chain as well as backbone cyclization in the N‐terminus of [d ‐Phe⁷]‐BK resulted in analogues with moderate antagonistic activity on RUT. Also, compound 18 in which a lactam bridge between positions 6 and 9 was achieved via an acylated reduced peptide bond has moderate antagonistic activity on RUT. These results support the hypothesis of turn structures in both parts of the molecule as a requirement for BK antagonism. Certain active and inactive agonists and antagonists are able to potentiate the bradykinin‐induced contraction of guinea‐pig ileum.     

Insect neuropeptide antagonist. Part II. Synthesis and biological activity of backbone cyclic and precyclic PBAN antagonists

Abstract: A new approach for the design and synthesis of pheromone biosynthesis activating neuropeptide (PBAN) agonists and antagonists using the backbone cyclization and cycloscan concepts is described. Two backbone cyclic (BBC) libraries were synthesized: library I (Ser library) was based on the active C‐terminal hexapeptide sequence Tyr‐Phe‐Ser‐Pro‐Arg‐Leu‐NH₂ of PBAN1‐33NH₂; whereas library II (d ‐Phe library) was based on the sequence of the PBAN lead linear antagonist Arg‐Tyr‐Phe‐d ‐Phe‐Pro‐Arg‐Leu‐NH₂. In both libraries the Pro residue was replaced by the BBC building unit N^α‐(ω‐aminoalkyl) Gly having various lengths of alkyl chain. The peptides of the two libraries were tested for agonistic and antagonistic activity. Four precyclic peptides based on two of the BBC antagonists were also synthesized; their activity revealed that a negative charge at the N‐terminus of the peptide abolished antagonistic activity. We also describe the use of the reagent SiCl₃I for selective deprotection of the Boc group from the building unit prior to on‐resin amino‐end to backbone‐nitrogen (AE‐BN) cyclization, during solid‐phase synthesis with Fmoc chemistry.     

Potent bradykinin antagonists containing N‐benzylglycine or N‐benzyl‐l‐alanine in position 8

Abstract: Two new analogues of a previously designed bradykinin (BK) antagonist, d ‐Arg‐Arg‐Pro‐Hyp‐Gly‐Thi‐Ser‐d ‐Phe‐Thi‐Arg, substituted in position 8 by N‐benzylglycine and N‐benzyl‐l ‐alanine were designed, synthesized and bioassayed. The results show an impressive enhancement of B₂ antagonistic potencies of both peptides in comparison with the model. In two further analogues these modifications were combined with acylation of the N‐terminus with 1‐adamantanacarboxylic acid. Acylated analogues exhibited higher antagonistic potency in comparison with the parent compounds, however, the range of effect was not as high as in previously described cases. The activity of analogues was assessed by their ability to inhibit vasodepressor response to exogenous BK (rat blood pressure test). Our results may be of value in the design of more potent BK antagonists.     

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.     

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.     

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.     

Conformational comparison of µ‐selective endomorphin‐2 with its C‐terminal free acid in DMSO solution,by 1H NMR spectroscopy and molecular modeling calculation

Abstract: In order to make clear the structural role of the C‐terminal amide group of endomorphin‐2 (EM2, H‐Tyr‐Pro‐Phe‐Phe‐NH₂), an endogenous µ‐receptor ligand, in the biological function, the solution conformations of endomorphin‐2 and its C‐terminal free acid (EM2OH, H‐Tyr‐Pro‐Phe‐Phe‐OH), studied using two‐dimensional ¹H NMR measurements and molecular modeling calculations, were compared. Both peptides were in equilibrium between the cis and trans isomers around the Tyr‐Pro ω bond in a population ratio of ≈ 1 : 2. The lack of significant temperature and concentration dependence of NH protons suggested that the NMR spectra reflected the conformational features of the respective molecules themselves. Fifty possible 3D structures for the each isomer were generated by the dynamical simulated annealing method under the proton?proton distance constraints derived from the ROE cross‐peaks. These energy‐minimized conformers, which were all in the φ torsion angles estimated from J_NHCαH coupling constants within ± 30°, were then classified in groups one or two according to the folding backbone structures. All trans and cis EM2 conformers adopt an open conformation in which their extended backbone structures are twisted at the Pro²–Phe³ moiety. In contrast, the trans and cis conformers of EM2OH show conformational variation between the ‘bow’‐shaped extended and folded backbone structures, although the cis conformers of its zwitterionic form are refined into the folded structure of the close disposition of C‐ and N‐terminal groups. These results indicate clearly that the substitution of carboxyl group for C‐terminal amide group makes the peptide flexible. The conformational requirement for µ‐receptor activation has been discussed based on the active form proposed for endomorphin‐1 and by comparing conformational features of EM2 and EM2OH.     

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.     

Facile synthesis of orthogonally protected amino acid building blocks for combinatorial N‐backbone cyclic peptide chemistry

Abstract: Protected N^α‐(aminoallyloxycarbonyl) and N^α‐(carboxyallyl) derivatives of all natural amino acids (except proline), and their chiral inverters, were synthesized using facile and efficient methods and were then used in the synthesis of N^α‐backbone cyclic peptides. Synthetic pathways for the preparation of the amino acid building units included alkylation, reductive amination and Michael addition using alkylhalides, aldehydes and α,β‐unsaturated carbonyl compounds, and the corresponding amino acids. The resulting amino acid prounits were then subjected to Fmoc protection affording optically pure amino acid building units. The appropriate synthetic pathway for each amino acid was chosen according to the nature of the side‐chain, resulting in fully orthogonal trifunctional building units for the solid‐phase peptide synthesis of small cyclic analogs of peptide loops (SCAPLs^?). N^α‐amino groups of building units were protected by Fmoc, functional side‐chains were protected by t‐Bu/Boc/Trt and N‐alkylamino or N‐alkylcarboxyl were protected by Alloc or Allyl, respectively. This facile method allows easy production of a large variety of amino acid building units in a short time, and is successfully employed in combinatorial chemistry as well as in large‐scale solid‐phase peptide synthesis. These building units have significant advantage in the synthesis of peptido‐related drugs.     

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.     

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.     

Preparation of protected peptidyl thioester intermediates for native chemical ligation by Nα‐9‐fluorenylmethoxycarbonyl (Fmoc) chemistry: considerations of side‐chain and backbone anchoring strategies,and compatible protection for N‐terminal cysteine*,†

Abstract: Native chemical ligation has proven to be a powerful method for the synthesis of small proteins and the semisynthesis of larger ones. The essential synthetic intermediates, which are C‐terminal peptide thioesters, cannot survive the repetitive piperidine deprotection steps of N^α‐9‐fluorenylmethoxycarbonyl (Fmoc) chemistry. Therefore, peptide scientists who prefer to not use N^α‐t‐butyloxycarbonyl (Boc) chemistry need to adopt more esoteric strategies and tactics in order to integrate ligation approaches with Fmoc chemistry. In the present work, side‐chain and backbone anchoring strategies have been used to prepare the required suitably (partially) protected and/or activated peptide intermediates spanning the length of bovine pancreatic trypsin inhibitor (BPTI). Three separate strategies for managing the critical N‐terminal cysteine residue have been developed: (i) incorporation of N^α‐9‐fluorenylmethoxycarbonyl‐S‐(N‐methyl‐N‐phenylcarbamoyl)sulfenylcysteine [Fmoc‐Cys(Snm)‐OH], allowing creation of an otherwise fully protected resin‐bound intermediate with N‐terminal free Cys; (ii) incorporation of N^α‐9‐fluorenylmethoxycarbonyl‐S‐triphenylmethylcysteine [Fmoc‐Cys(Trt)‐OH], generating a stable Fmoc‐Cys(H)‐peptide upon acidolytic cleavage; and (iii) incorporation of N^α‐t‐butyloxycarbonyl‐S‐fluorenylmethylcysteine [Boc‐Cys(Fm)‐OH], generating a stable H‐Cys(Fm)‐peptide upon cleavage. In separate stages of these strategies, thioesters are established at the C‐termini by selective deprotection and coupling steps carried out while peptides remain bound to the supports. Pilot native chemical ligations were pursued directly on‐resin, as well as in solution after cleavage/purification.     

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.     

Aromatic interactions in tryptophan‐containing peptides: crystal structures of model tryptophan peptides and phenylalanine analogs*

Abstract: The crystal structures of the peptides, Boc‐Leu‐Trp‐Val‐OMe ( 1) , Ac‐Leu‐Trp‐Val‐OMe ( 2a and 2b), Boc‐Leu‐Phe‐Val‐OMe ( 3 ), Ac‐Leu‐Phe‐Val‐OMe ( 4 ), and Boc‐Ala‐Aib‐Leu‐Trp‐Val‐OMe ( 5 ) have been determined by X‐ray diffraction in order to explore the nature of interactions between aromatic rings, specifically the indole side chain of Trp residues. Peptide 1 adopts a type I β‐turn conformation stabilized by an intramolecular 4→1 hydrogen bond. Molecules of 1 pack into helical columns stabilized by two intermolecular hydrogen bonds, Leu(1)NH…O(2)Trp(2) and IndoleNH…O(1)Leu(1). The superhelical columns further pack into the tetragonal space group P4₃ by means of a continuous network of indole–indole interactions. Peptide 2 crystallizes in two polymorphic forms, P2₁ ( 2a ) and P2₁2₁2₁ ( 2b ). In both forms, the peptide backbone is extended, with antiparallel β‐sheet association being observed in crystals. Extended strand conformations and antiparallel β‐sheet formation are also observed in the Phe‐containing analogs, Boc‐Leu‐Phe‐Val‐OMe ( 3 ) and Ac‐Leu‐Phe‐Val‐OMe ( 4 ). Peptide 5 forms a short stretch of 3₁₀‐helix. Analysis of aromatic–aromatic and aromatic–amide interactions in the structures of peptides, 1 , 2a , 2b are reported along with the examples of 14 Trp‐containing peptides from the Cambridge Crystallographic Database. The results suggest that there is no dramatic preference for a preferred orientation of two proximal indole rings. In Trp‐containing peptides specific orientations of the indole ring, with respect to the preceding and succeeding peptide units, appear to be preferred in β‐turns and extended structures.     

A convenient incorporation of conformationally constrained 5,5‐dimethylproline into the ribonuclease A 89–124 sequence by condensation of synthetic peptide fragments

Abstract: The presence of l ‐5,5‐dimethylproline (dmP) within an amino acid sequence results in the formation of an X‐dmP peptide bond predominantly locked in a cis conformation. However, the common use of this unnatural amino acid has been hampered by the difficulty of the economical incorporation of the dmP residue into longer peptide segments due to the steric hindrance imposed by the dimethyl moieties. Here, we describe synthesis of the C‐terminal 36‐residue peptide, corresponding to the 89–124 sequence of bovine pancreatic ribonuclease A (RNase A), in which dmP is incorporated as a substitute for Pro⁹³. The peptide was assembled by condensation of protected 5‐ and 31‐residue peptide fragments, which were synthesized by solid‐phase peptide methodology using fluorenylmethyloxycarbonyl chemistry. We focused on optimizing the synthesis of the Fmoc‐Ser(^tBu)‐Ser(^tBu)‐Lys(Boc)‐Tyr(^tBu)‐dmP‐OH pentapeptide (residues 89–93) with efficient acylation of the sterically hindered dmP residue. In a comparative study, the reagent O‐(7‐azabenzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate was found to be superior to bromo‐tris‐pyrrolidino‐phosphonium hexafluorophosphate and tetramethylfluoroformamidinium hexafluorophosphate for the synthesis of the ‐Tyr(^tBu)‐dmP‐ peptide bond in solution as well as on a resin.     

Synthesis of N-carboxyalkyl and N-aminoalkyl functionalized dipeptide building units for the assembly of backbone cyclic peptides

Abstract: To improve the assembly of backbone cyclic peptides, N-functionalized dipeptide building units were synthesized. The corresponding N-aminoalkyl or N-carboxyalkyl amino acids were formed by alkylation or reductive alkylation of amino acid benzyl or tert-butyl esters. In the case of N-aminoalkyl amino acid derivatives the aldehydes for reductive alkylation were obtained from N,O-dimethyl hydroxamates of N-protected amino acids by reduction with LiAlH₄. N-carboxymethyl amino acids were synthesized by alkylation using bromoacetic acid ester and the N-carboxyethyl amino acids via reductive alkylation using aldehydes derived from formyl Meldrums acid. Removal of the carboxy protecting group leads to free N-alkyl amino acids of very low solubility in organic solvents, allowing efficient purification by extraction of the crude product. These N-alkyl amino acids were converted to their tetramethylsilane-esters by silylation with N,O-bis-(trimethylsilyl)acetamide and could thus be used for the coupling with Fmoc-protected amino acid chlorides or fluorides. To avoid racemization the tert-butyl esters of N-alkyl amino acids were coupled with the Fmoc-amino acid halides in the presence of the weak base collidine. Both theN-aminoalkyl and N-carboxyalkyl functionalized dipeptide building units could be obtained in good yield and purity. For peptide assembly on the solid support, the allyl type protection of the branching moiety turned out to be most suitable. The Fmoc-protected N-functionalized dipeptide units can be used like any amino acid derivative under the standard conditions for Fmoc-solid phase synthesis.     

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

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

Facile synthesis of Nα‐protected‐l‐α,γ‐diaminobutyric acids mediated by polymer‐supported hypervalent iodine reagent in water

Abstract: Hofmann rearrangement of N^α‐Boc‐l ‐Gln‐OH mediated by a polymer‐supported hypervalent iodine reagent poly[(4‐diacetoxyiodo)styrene] (PSDIB) in water afforded N^α‐Boc‐l ‐α,γ‐diaminobutyric acid (Boc‐Dab‐OH, 1 ) in 87% yield. N^α‐Z‐derivative (Z‐Dab‐OH, 2 ) was prepared with PSDIB in 83% yield. Since the reaction of N^α‐Fmoc‐Gln‐OH by this procedure did not proceed because of the insolubility of Fmoc‐Gln‐OH in aqueous media, we synthesized Fmoc‐Dab(Boc)‐OH ( 5 ) from 2 in 54% yield. Polymyxin B heptapeptide (PMBH) which contains four Dab residues was successfully synthesized in a solution‐phase synthesis.     

From peptide libraries to optimized nonpeptide ligands in the search for S‐farnesyltransferase inhibitors

Abstract: A complete 331 776‐member library of tetrapeptides made of 24 amino acid building blocks was synthesized robotically on solid phase and subjected to a deconvolution based on the inhibitory potency of the sublibraries in a HPLC assay of the S‐farnesyltransferase activity in vitro. One of the non‐natural peptide and noncysteine‐containing leads Nip‐Trp‐Phe‐His (Nip = p‐nitrophenyl‐l ‐alanine) was optimized chemically to give a proteolytically stable pseudopeptide with a 200‐fold potency compared with the original lead. The final compound was converted to the C‐terminal ethyl ester: p‐F‐C₆H₄‐CO(CH₂)₂‐CO‐Bta‐d ‐Pheψ[CH2NH]His‐OEt (Bta = benzothienyl‐l ‐alanine) and shown to behave as a prodrug which was hydrolyzed back to the C‐terminal acid following cell penetration. The method confirmed that several structurally original leads can be discovered in large libraries when deconvolution relies upon a highly specific assay and that these leads can be optimized by chemical modification to impart the final compound the desired pharmacological and pharmacokinetic properties.     

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%.