Synthesis of different types of dipeptide building units containing N- or C-terminal arginine for the assembly of backbone cyclic peptides.

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 the C-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.     

Synthesis of differentially protected N-acylated reduced pseudodipeptides as building units for backbone cyclic peptides.

Backbone cyclization has become an important method for generating or stabilizing the bioactive conformation of peptides without affecting the amino acid side-chains. Up to now, backbone cyclic peptides were mostly synthesized with bridges between N-amino- and N-carboxy-functionalized peptide bonds. To study the influence of a more flexible backbone on the biological activity, we have developed a new type of backbone cyclization which is achieved via the N-functionalized moieties of acylated reduced peptide bonds. As described in our previous publications, the formation of N-functionalized dipeptide units facilitates the peptide assembly compared with the incorporation of N-alkyl amino acids. Besides the racemization-free synthesis of Fmoc-protected pseudodipeptide esters with reduced peptide bonds, the new type of backbone modification allows the use of a great variety of omega-amino- and alpha,omega-dicarboxylic acids differing in chain length and chemical properties. Best results for the coupling of the omega-amino- and alpha,omega-dicarboxylic acids to the reduced peptide bond were obtained by the formation of mixed anhydrides with alkyl chloroformates. Whereas the protecting group combination of Z/OBzl in the dipeptide unit and Boc/OtBu for the N-functionalized moiety leads to the formation of 2-ketopiperazine during hydrogenation, the combination of Fmoc/OtBu and Alloc/OAll is very suitable for the synthesis of backbone cyclic peptides on solid support.     

Novel method for the synthesis of urea backbone cyclic peptides using new Alloc‐protected glycine building units

Cyclization of bioactive peptides, utilizing functional groups serving as natural pharmacophors, is often accompanied with loss of activity. The backbone cyclization approach was developed to overcome this limitation and enhance pharmacological properties. Backbone cyclic peptides are prepared by the incorporation of special building units, capable of forming amide, disulfide and coordinative bonds. Urea bridge is often used for the preparation of cyclic peptides by connecting two amine functionalized side chains. Here we present urea backbone cyclization as an additional method for the preparation of backbone cyclic peptide libraries. A straightforward method for the synthesis of crystalline Fmoc‐N^α [ω‐amino(Alloc)‐alkyl] glycine building units is presented. A set of urea backbone cyclic Glycogen Synthase Kinase 3 analogs was prepared and assessed for protein kinase B inhibition as anticancer leads. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of novel protected Nalpha(omega-thioalkyl) amino acid building units and their incorporation in backbone cyclic disulfide and thioetheric bridged peptides.

General methods for the preparation of protected Nalpha(omega-thioalkyl) amino acids building units for backbone cyclization using reductive alkylation and on-resin preparation are described. The synthesis of non-Gly Fmoc-protected S-functionalized N-alkylated amino acids is based on the reaction of readily prepared protected omega-thio aldehyde with the appropriate amino acid. Preparation of Fmoc-protected S-functionalized N-alkylated Gly building units was carried out using two methods: reaction of glyoxylic acid with Acm-thioalkylamine and an on-resin reaction of bromoacetyl resin with Trt-thioalkylamines. Three model peptides were prepared using these building units. The GlyS2 building unit was incorporated into a backbone cyclic analog of somatostatin that contains a disulfide bridge. Formation of the disulfide bridge was performed by on-resin oxidation using 12 or Tl(CF3COO-)3. Both methods resulted in the desired product in a high degree of purity in the crude. The AspS3 building unit was also successfully incorporated into a model peptide. In addition, the in situ generation of sulfur containing Gly building units was demonstrated on a Substance P backbone cyclic analog containing a thioether bridge.     

Novel Gly building units for backbone cyclization: synthesis and incorporation into model peptides

We report the preparation of novel building units for backbone cyclization that have the general formula Fmoc-N[CH(R)CO₂Al]Gly-OH. These building units were prepared by the reductive alkylation method using allyl esters of several amino acids as starting material and hence, respectively, contain the side chain of these amino acids. These N-alkylated Gly building units were incorporated in model backbone cyclic peptides. The resulting crude backbone cyclic peptides were obtained in high degree of purity according to HPLC and mass spectrometric analyses.     

Studies on cyclic peptides. II. Synthesis of cyclic peptides containing sarcosine.

Cyclic peptides containing sarcosine, cyclo-(Pro-Sar-Gly)₂, cyclo-(Sar-Sar-Gly)₂, cyclo-(Sar₄), and cyclo-(Sar₆) have been synthesized by the cyclization of the p-nitrophenyl ester of linear peptides. The tert-butoxycarbonyl group was used as the N^α-protecting group, which was removed by acid. Benzyl ester was used to protect the C-terminal. tert-butoxycarbonylpeptide was obtained by the stepwise elongation of the peptide bond by the carbodiimide method. Deblocking and cyclization of the linear peptides gave the cyclic peptides.     

Conformational properties of tripeptides having cyclic dipeptide backbone and their catalytic properties for enantiomer-selective hydrolysis

Conformation in aqueous solution at pH 6.95 of tripeptides having cyclic dipeptide backbones, cyclo[l-Glu(l-Leu-OBzl)-l-His] and cyclo[l-Glu(l-Leu-OH)-l-His], was investigated by u.v., c.d. and n.m.r. spectroscopy and by the lanthanide probe method. In the major conformation of cyclo[l-Glu(l-Leu-OBzl)-l-His], the cyclic dipeptide backbone takes a flagpole-boat conformation in which the sidechain of the l-His residue is nearly parallel with the backbone plane and the sidechain of the l-Glu residue protrudes outside the backbone plane. In the major conformation of cyclo[l-Glu(l-Leu-OH)-l-His], the cyclic dipeptide backbone takes a flagpole-boat conformation in which the sidechains of the l-His and l-Glu residues are accommodated in the same side of the backbone plane so that the imidazolyl sidechain of l-His residue is twisted slightly. Tripeptides were not found to change the conformation when metal salts or ammonium salts such as Cl^?H₃N^?(CH₂)₁₁ COOEt, Gly-OEt-HCl, dl-Val-OEt-HCl and l-Leu-OEt-HCl were added, but a significant conformation change occurred upon adding d-Leu-OEt·HCl. If the same situation holds with the addition of α-amino acid p-nitrophenyl ester hydrochlorides, the previously reported enantiomer-selective catalysis by the tripeptides which hydrolysed d-Leu-OPh(NO₂·HCl faster than l-Leu-OPh(NO₂)·HCl can be explained; that is, the tripeptides change the conformation only when d-Leu-OPh(NO₂)·HCl is bound and consequently the intramolecular reaction is facilitated. This phenomenon may be compared with that of ‘induced fit’ in enzyme catalysis.     

A safety catch linker for Fmoc-based assembly of constrained cyclic peptides.

A new safety-catch linker for Fmoc solid-phase peptide synthesis of cyclic peptides is reported. The linear precursors were assembled on a tert-butyl protected catechol derivative using optimized conditions for Fmoc-removal. After activation of the linker using TFA, neutralization of the N-terminal amine induced cyclization with concomitant cleavage from the resin yielding the cyclic peptides in DMF solution. Several constrained cyclic peptides were synthesized in excellent yields and purities.     

Hydration of peptides. I. Calculation of accessible surface areas for several conformations of a cyclic dipeptide

The concept of “static accessibility” to water has been used to determine the accessible surface area of a cyclic dipeptide: c(l-Thr-l-His). Different calculated and experimental conformations of this model molecule have been examined, which allows us to analyse the variations of accessibility of the hydration sites localized on the peptide backbone and on the polar side chains. The maximum solvation criterion involves a large destabilization of conformations governed by intramolecular interactions. The variations of the amphiphilic character with the conformations are relatively small. Nevertheless, the experimental conformation seems to reflect such a behaviour, especially in the crystal, in which the amphiphilic character is compatible with intermolecular interactions. The accessibility studies must be regarded only as a preliminary step to a more quantitative analysis of peptide hydration.     

Synthesis of cyclic tryptathionine peptides

The helicity of the tryptathionine moiety of the phallotoxins has been recognized by comparison with cyclic tryptathionine tripeptides. In order to investigate the influence of the configuration of the component amino acids on the conformation of the cyclic peptides, six analogue thioether tripeptides containing L- and D-alanine and L- and D-cysteine, respectively, have been synthesized. The CD spectra of the peptides are very similar to each other, showing mirror images of the CD of phalloidin and, therefore, negative helicity. The spectra of the D-cysteine containing compounds differ from the L-cysteine containing compounds by their weakly positive ellipticity values around 270 nm. The cyclization reaction of Boc-Hpi-D-Ala-D-Cys(STrt)OCH3, along with the cyclic tripeptide, afforded a cyclic hexapeptide by dimerization. The CD spectrum of the dimer is very similar to that of phalloidin, thus pointing to a positive helicity of its two tryptathionine moieties. The dimeric thioether peptide forms a rather strong complex with Cu2+ ions.     

Columnar assembly of cyclic beta-amino acid functionalized with pyranose rings

A novel cyclic trimer and tetramer of protected beta-glycamino acids were synthesized and investigated on conformation and assembly formation. A characteristic point of these cyclic beta-glycamino acids is their better solubility than other cyclic beta-amino acids due to the pyranose rings. Thus, the assembling process of the cyclic molecules could be examined by CD or NMR spectroscopy. FT-IR and NMR measurements and geometry optimization revealed a highly symmetric and planar conformation for each cyclic beta-peptide with all-trans amide groups. The amide groups in the cyclic peptides took a vertical orientation against the cyclic skeleton to be suitably arranged for intermolecular hydrogen bonds, which should promote formation of molecular assembly in a columnar structure through molecular stacking. These cyclic beta-peptides were successfully crystallized to yield rod-shaped molecular assemblies in nanometer sizes. Evidence for the columnar structure in the crystals was obtained by electron diffraction analysis, which showed a layer spacing of ca. 4.8 A. Interestingly, the molecular assembly of the cyclic trimer showed a high aspect ratio, width less than 40 nm, and length more than 2 mum, suggesting stable molecular stacking in the column.     

Synthesis of fluorine-containing bioisosteres corresponding to phosphoamino acids and dipeptide units

It has been shown that fluorinated analogues of naturally occurring biological active compounds including amino acids often exhibit unique physiological activity. Among wide varieties of fluorine-containing amino acids, nonhydrolyzable phosphoamino acids possessing a substituent of the difluoromethylene (CF(2)) unit for the phosphoryl ester oxygen are of value in the medicinal and biological fields. We have engaged in the synthesis of these classes of nonhydrolyzable phosphoamino acids corresponding to pTyr 3, pSer 4, and pThr 5 with their incorporation into peptides using newly developed deprotecting procedures. In this article, stereoselective synthesis of the CF(2)-substituted pThr mimetics and development of a two-step deprotecting methodology for the nonhydrolyzable analogues are reviewed. In the course of the above synthetic study, we found that gamma,gamma-difluoro-alpha,beta-enoates were reduced to gamma-fluoro-beta,gamma-enoates by organocopper reagents and then applied to the synthesis of (Z)-fluoroalkene dipeptide isosteres, which have served as potential dipeptide mimetics having structural as well as electrostatic similarity to the parent peptide bonds. Furthermore, mechanistic investigation of the organocopper-mediated reduction led us to development of a SmI(2)-mediated approach toward the synthesis of the fluoroalkene isosteres.     

Hydration of peptides. II. Determination of the preferred sites of interactions of a cyclic dipeptide with water

As a first step in the determination of the hydration scheme of small peptides, the hydration sites of the cyclic dipeptide c(l-Thr-l-His) have been determined by two empirical potential treatments. In the first approach the energy is calculated by using the “Caillet-Claverie's” potentials, including electrostatic, dispersion-repulsion and polarization contributions. In the second approach (EMPWI method), the energy is calculated by simplified treatment, taking into account the electrostatic interactions of a suitable charge distribution and the dispersion-repulsion contributions. In this study, only the crystalline conformation of the cyclic dipeptide is considered. The hydration sites determined can be classified in three groups: (a) bridging sites, in which water interacts with both side chains, (b) bridging sites in which water interacts with one side chain and the DKP ring, (c) individual sites in which water interacts only with one polar group. The agreement between the results obtained by the two calculations is sufficiently satisfactory. This allows us to use EMPWI potential for calculations of more complex systems.     

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

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.     

Synthesis of biologically active cyclic peptides

1. The extent of racemization and the coupling yield in peptide synthesis were studied under high dilution conditions. The azide method yielded the best results. 2. Five linear penta-peptide precursors related to gramicidin S were subjected to cyclization in order to study how the difference in the sequence influences the yield and the ratio of cyclic dimer to monomer. The azide with the sequence of -L -Pro-L -Val-L -Orn(Z)-L -Leu-D -Phe- afforded diZ-gramicidin S in a high yield of 63%. 3. Alternaria mali toxin III, a cyclotetradepsipeptide phytotoxin, was synthesized. The activated linear tetradepsipeptide containing a D -Dap(Z) (N³-Z-D -2,3-diaminopropionic acid) residue at the N-terminus afforded the cyclic precursor (53%). The Dap residue in the precursor was converted into a ΔAla residue by Hofmann degradation to give the desired product.     

Synthesis of cyclic α-MSH peptides

Summary Cyclic lactam analogs of α-melanocyte stimulating hormone (α-MSH) have been shown to be potent agonists in the frog skin bioassay [Al-Obeidi, F. et al., J. Med. Chem., 32 (1989) 2555], demonstrating melanocortin-1 (MC1) receptor activity. We synthesized cyclic α-MSH(1–13) and α-MSH(4–10) lactam analogs. The peptides were synthesized using Fmoc chemistry. We improved the cyclization procedure: side chains of Asp⁵ and Lys¹⁰ from the deprotected peptide were coupled in DMF to form a cyclic lactam, using an excess of PyBOP reagent and DIEA as a base. The cyclization reaction was completed within 1 h and was almost quantitative. We also synthesized an α-MSH analog cyclized via a disulphide bridge. The peptides were tested for their selectivity for the rat MC4 receptor. Cyclization and substitutions at position 7 dramatically influenced the selectivity for the rMC4 receptor.     

Synthesis and alkylation of aza‐glycinyl dipeptide building blocks

Aza‐glycinyl dipeptides are useful building blocks for the synthesis of a diverse array of azapeptides. The construction of the aza‐glycine residue is however challenging, because of the potential for side reactions, such as those leading to formation of oxadiazalone, hydantoin and symmetric urea by‐products. Employing N,N′‐disuccinimidyl carbonate to activate benzophenone hydrazone, we have developed a more efficient approach for the synthesis of aza‐glycinyl dipeptides. Alkylation of the semicarbazone of the resulting protected aza‐glycinyl dipeptides using tetraethylammonium hydroxide and propargyl bromide provided an efficient entry into the aza‐propargylglycinyl peptide building blocks, which have served previously in various reactions including Sonogashira cross‐couplings, dipolar cycloadditions and intramolecular exo‐dig cycloadditions to furnish a variety of azapeptide building blocks. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Stereochemical studies on cyclic peptides. IX. Conformational studies on cyclic tetrapeptides containing alternating cis and trans peptide units

Conformational analyses of cyclic tetrapeptides consisting of alternating cis and trans peptide units have been made using contact criteria and energy calculations. This study has been restricted to those structures having a symmetry element in the backbone ring, such as a twofold axis (d) or a center of inversion (i). There are five main results. (1) There are two distinct types of conformations, which are stereochemically favorable corresponding to each of twofold and inversion-symmetrical structures, designated as d₁, d₂ (for twofold symmetrical) and i₁, i₂ (for inversion-symmetrical). Among these, the i₁ type has the lowest energy when glycyl residues occur at all four α-carbon atoms. (2) With the glycyl residue at all four α-carbon atoms, methyl substitution at the cis peptide nitrogen atoms is possible in all the four types, whereas the substitution at trans peptide nitrogen atoms is possible only for the i₁ type. Thus only in the i₁ type can all the nitrogen atoms be methylated simultaneously. The conformation of the molecule in the crystal structure of cyclotetrasarcosyl belongs to the i₁ type. (3) When alanyl residues occur at all four α-carbon atoms, the possible symmetrical type is dependent on the enantiomorphic form and the actual sequence of the alanyl residues. (4) The methyl substitution at peptide nitrogen atoms for cyclic tetrapeptides having alanyl residues causes more stereochemical restriction in the allowed conformations than with glycyl residues. (5) The prolyl residue can be incorporated favorably at the cis-trans junction of both d and i types of structures. The results of the present study are compared with the data on cyclic tetrapeptides available from the crystal structure and nmr studies. The results show an overall agreement both regarding the type of symmetry and the conformational parameters.