Synthesis of sequential oligopeptides and polypeptide having the sequence of L-alanyl-L-leucylglycine

A series of sequential oligopeptides having simple nonpolar side chains, Nps-(L -Ala-L -Leu-Gly)_n- OEt has been prepared by a stepwise fragment-condensation method using Nps-L -alanyl-L -leucylglycine N-hydroxysuccinimide ester, which was prepared by the Nps-N-carboxy α-amino-acid-anhydride method. The success of the synthesis of the peptide having a high-molecular weight, such as octadecapeptide, results from the highest solubility of the tripeptide unit, L -alanyl-L -leucylglycine. The sequential polypeptide having the same tripeptide sequence was also prepared by polycondensation of the tripeptide N-hydroxy-succinimide ester.     

Synthesis of oligopeptides consisting of L-leucyl-L-leucyl-L-ananyl and L-methionyl-L-methionyl-L-leucyl repeating units with an improved preparation of Nps-amino acids

Two series of peptides with hydrophobic side chains, Nps-(L -Leu-L -Leu-L -Ala)_n-OEt and Nps-(L -Met-L -Met-L Leu)_n-OEt (n = 1–6), were synthesized by the fragment condensation method using dicyclohexylcarbodiimide in the presence of N-hydroxysuccinimide. The tripeptide fragments were prepared stepwise by dicyclohexylcarbodiimide-mediated reaction of Nps-amino acids, which were synthesized by an improved rapid procedure.     

Convergent solid‐phase and solution approaches in the synthesis of the cysteine‐rich Mdm2 RING finger domain

The RING finger domain of the Mdm2, located at the C‐terminus of the protein, is necessary for regulation of p53, a tumor suppressor protein. The 48‐residues long Mdm2 peptide is an important target for studying its interaction with small anticancer drug candidates. For the chemical synthesis of the Mdm2 RING finger domain, the fragment condensation on solid‐phase and the fragment condensation in solution were studied. The latter method was performed using either protected or free peptides at the C‐terminus as the amino component. Best results were achieved using solution condensation where the N‐component was applied with the C‐terminal carboxyl group left unprotected. The developed method is well suited for large‐scale synthesis of Mdm2 RING finger domain, combining the advantages of both solid‐phase and solution synthesis. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and intramolecular reactions of Tyr-Gly and Tyr-Gly-Gly related 6-O-glucopyranose esters

6-O-(L-Tyrosylglycyl)- and 6-O-(L-tyrosylglycylglycyl)-D-glucopyranose were synthesized by condensation of the pentachlorophenyl esters of the respective di- and tripeptide with fully unprotected D-glucose. The intramolecular reactivity of the sugar conjugates was studied in pyridine-acetic acid and in dry methanol, at various temperatures and for various incubation times. The composition of the incubation mixtures was monitored by a reversed-phase HPLC method that permits simultaneous analysis of the disappearance of the starting material and the appearance of rearrangement and degradation products. To determine the influence of esterification of the peptide carboxy group on its amino group reactivity, parallel experiments were done in which free peptides were, under identical reaction conditions, incubated with D-glucose (molar ratios 1:1 and 1:5). Depending on the starting compound, different types of Amadori products (cyclic and bicyclic form), methyl ester of peptides, and Tyr-Gly-diketopiperazine were obtained.     

Synthesis and conformational characterization in the solid state of peptides consisting of L-phenylalanyl-L-phenylalanylglycyl and L-phenylalanyl-L-leucylglycyl residues

Two series of peptides containing L -phenylalanine, Nps-(L -Phe-L -Phe-Gly)_n-OEt (n = 1–6) and Nps-(L -Phe-L -Leu-Gly)_n-OEt (n = 1–7), were prepared by the fragment-condensation method using the tripeptide N-hydroxysuccinimide esters. Conformational characterization of these peptides in the solid state was performed by ir spectroscopy and x-ray powder diffraction measurement. The peptides Nps-(L -Phe-L -Phe-Gly)_n-OEt take the β-structure, but the pentadecapeptide and higher peptides of Nps-(L -Phe-L -Leu-Gly)_n-OEt form the α-helix, although the lower homologs take the β-structure.     

The Use of Crown Ethers in Peptide Chemistry – V. Solid-phase Synthesis of Peptides by the Fragment Condensation Approach using Crown Ethers as Non-covalent Protecting Groups

We have previously described the conditions by which peptide synthesis by the solid-phase fragment condensation approach can be carried out using crown ethers as non-covalent protection for the N^α -amino group. Here we demonstrate that the procedure can be extended to large, partially protected peptide fragments possessing free Lys and/or Arg residues. The first step was to ensure that complex formation on the side chain of amino acids was not detrimental to the methodology and exhibited the same solubility and coupling properties as N^α -complexed peptides. Thus, a model hexapeptide was synthesized using Fmoc chemistry containing Lys and Arg residues, which, when complexed with 18-Crown-6, was readily soluble in DCM and coupled quantitatively to a resin-bound tetrapeptide. Two tripeptides were then prepared, one containing a free Ser residue, the other free Tyr, to examine the possible occurrence of side reactions. After coupling using standard conditions only the former tripeptide exhibited the formation of the O-acylation by-product (5%). Another model hexapeptide containing Lys, Tyr, Ser and Asp protected with a TFA-stable adamantyl group was complexed with 18-Crown-6 and coupled to the resin-bound tetrapeptide with near quantative yield. Extending the length of the peptide to 21 and 40 residues, which represent sequences Gly⁵² to Leu⁷² (21-mer) and Pro³³ to Leu⁷² (40-mer) from Rattus norvegicus chaperonin 10 protein, respectively, resulted in partially protected fragments that were readily soluble in water, thus enabling purification by RP-HPLC. Complexation with 18-Crown-6 gave two highly soluble products that coupled to resin-board tetramer with 68% and 50% coupling efficiencies for the 21-mer and 40-mer, respectively. Treatment with 1% DIEA solutions followed by acidolytic cleavage and purification of the major product confirmed that the correct product had been formed, when analysed by amino acid analysis and ESI-MS. These results served to extend the methodology of non-covalent protection of large partially protected peptide fragments for the stepwise fragment condensation of polypeptides.     

Sequential oligopeptides. Synthesis and characterization of the oligopeptides and a polypeptide with the repeating sequence L-norvalyl-glycyl-L-proline

The synthesis and characterization of a series of oligopeptides (from the tripeptide to the octadecapeptide) with the repeating sequence L -norvalyl-glycyl-L -proline and a polytripeptide with this sequence are reported. The oligomers were synthesized step by step using the mixed anhydride method. All the products were chemically and optically pure. The polymer was prepared by the active ester method, using the p-nitrophenyl ester as the polymerizable tripeptide derivative. Good yield of relatively high average molecular-weight polymer was obtained. In the accompanying paper conformational investigations, both in solution and in the solid state, on the oligomers and the polymer are described.     

Synthesis of 2-acetamido-1-N[N-(tert-butoxycarbonyl)-l-aspart-1-oyl-(l-phenylalanyl-l-serine methyl ester)-4-oyl]-2-deoxy-β-d-glucopyranosylamine and analogs

2-Acetamido-1-N-[N-(tert-butoxycarbonyl)-l-aspart-1-oyl-(l-phenylalanyl-l-serine methyl ester)-4-oyl]-2-deoxy-β-d-glucopyranosylamine and analogs containing d-glucopyranosyl, 4-O-β-d-glucopyranosyl-d-glucopyranosyl, l-Phe-l-Ala, and d-Phe-l-Ser were synthesized by condensation of glycosylamines having free hydroxyl groups with tripeptide esters activated with N-hydroxysuccinimide.     

Incorporation of N‐amidino‐pyroglutamic acid into peptides using intramolecular cyclization of α‐guanidinoglutaric acid

N‐terminal modification of peptides by unnatural amino acids significantly affects their enzymatic stability, conformational properties and biological activity. Application of N‐amidino‐amino acids, positively charged under physiological conditions, can change peptide conformation and its affinity to the corresponding receptor. In this article, we describe synthesis of short peptides, containing a new building block—N‐amidino‐pyroglutamic acid. Although direct guanidinylation of pyroglutamic acid and oxidation of N‐amidino‐proline using RuO₄ did not produce positive results, N‐amidino‐Glp‐Phe‐OH was synthesized on Wang polymer by cyclization of α‐guanidinoglutaric acid residue. In the course of synthesis, it was found that literature procedure of selective Boc deprotection using TMSOTf/TEA reagent is accompanied by concomitant side reaction of triethylamine alkylation by polymer linker fragment. It should be mentioned that independently from cyclization time and coupling agent (DIC or HCTU), the lactam formation was incomplete. Separation of the cyclic product from the linear precursor was achieved by HPLC in ammonium formate buffer at pH 6. HPLC analysis showed N‐amidino‐Glp‐Phe‐OH stability at acidic and physiological pH and fast ring opening in water solution at pH 9. The suggested method of N‐amidino‐Glp residue formation can be applied in the case of short peptide chains, whereas synthesis of longer ones will require fragment condensation approach. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Coformational aspects of polypeptide structure XL. The synthesis of poly((S)-thiazolidine-4-carboxylic acid)

The synthesis of poly[(S)-thiazolidine-4-carboxylic acid] is described. The polymer is obtained by the polymerization of the N-carboxyanhydride of (S)-thiazolidine-4-carboxylic acid in pyridine or nitrobenzene using triethylamine as an initiator. The amino acid is prepared by the condensation of cysteine and formaldehyde. N-Acetyl-(S)-thiazolidine-4-carboxylic acid methyl ester is also prepared as a model compound by standard acetylation and esterification reactions.     

Peptide synthesis using o-nitrophenylsulfenyl N-carboxy alpha-amino acid anhydrides. Sequential oligopeptides having alternate gamma-methyl L-glutamyl and L-phenylalanyl residues.

A series of sequential oligopeptides having the sequence alternating γ-methyl L -glutamyl and L -phenylalanyl residues have been successfully prepared by a rapid method involving the reaction of o-nitrophenylsulfenyl N-carboxy α-amino acid anhydrides with amino acid and peptide esters. The sequential oligopeptides, which are interesting from a conformational aspect, were obtained in optical pure forms above 74% yields. This result demonstrates that the o-nitrophenylsulfenyl N-carboxy α-amino acid anhydride method is especially useful for easy synthesis of protected oligopeptides with o-nitrophenylsulfenyl group.     

The Solution Synthesis of Antisense Oligonucleotide‐Peptide Conjugates Directly Linked via Phosphoramide Bond by Using a Fragment Coupling Approach

To improve antisense oligonucleotide penetration inside cells, conjugates of oligonucleotides and cell‐penetrating peptides, covalently linked through a phosphoramide bond, were prepared by a fragment coupling approach in the liquid phase. Two methods were used for this synthesis, i.e., phosphorylation of a peptide amino group by an oligonucleotide terminal phosphate 1‐hydroxybenzotriazole ester in aqueous media or condensation of phosphate and amino groups in presence of triphenylphosphine, 2,2′‐dithiopyridine and 4‐dimethylaminopyridine in organic media. Several oligonucleotides, including a 18‐mer antisense oligodeoxyribonucleotide complementary to an internal coding region of the reporter gene of the green fluorescent protein (GFP) were prepared. Peptides derived from the third helix of the homeodomain of Antennapedia, the influenza envelope hemagglutinin subunit as well as melittin and polymyxin B were used for the conjugates' synthesis. The peptides with various amino acid composition were chosen to confirm that these coupling methods are of a general use.     

Enzymatic Peptide Synthesis in Frozen Aqueous Solution: Use of Nα-unprotected Peptide Esters as Acyl Donors

The ability of the endopeptidase α-chymotrypsin (EC 3.4.21.1) to catalyse the reaction of various N^α- unprotected di- and tripeptide ester derivatives with H-Leu-NH₂, and with a series of C-terminal free di- and tripeptides at −15° C in frozen aqueous solution was investigated. The enzyme is able to synthesize N- and C-terminal unprotected penta- and hexapeptides in up to 92% yield, depending on the amino component used, in a single-step segment-condensation reaction. Freezing the reaction mixture resulted in significantly increased peptide yields compared with the reaction at room temperature. The enzyme shows a modified nucleophilic specificity in frozen solution compared with room temperature. Nucleophilic amino components with positively charged amino acids in P₂^′ -position are accepted. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and utilization of 13C and 15N backbone-labeled proline: NMR study of synthesized oxytocin with backbone-labeled C-terminal tripeptide amide

Summary. The ¹³C and ¹⁵N backbone-labeled proline was prepared using Oppolzer’s method based on application of a sultam as chiral auxiliary. This isotopomer was used in the synthesis of the ¹³C, ¹⁵N backbone-labeled C-terminal tripeptide amide fragment of neurohypophyseal hormone oxytocin. Finally, this tripeptide amide was coupled by segment condensation with N-Boc- or N-Fmoc-tocinoic acid, followed by N-deprotection with TFA or piperidine. The labeled oxytocin exhibited biological activity identical with that of natural oxytocin. A detailed ¹H, ¹³C and ¹⁵N NMR study confirmed the assigned oxytocin conformation containing a β-turn in the cyclic part of the molecule, stabilized by H-bond(s) that can be perturbed by the C-terminal tripeptide amide moiety as indicated by comparison of NMR data for both the tocine ring in oxytocin and tocinoic acid.     

Intermolecular association of tetra-(γ-benzyl-L-glutamate)s in ethylene dichloride solutions

Light scattering from ethylene dichloride solutions of tetra-(γ-benzyl-L -glutamate)s has been measured and their association in solution is examined. One of the peptides is monodisperse o-nitrophenylthio-tetra-(γ-benzyl-L -glutamate) ethylamide prepared by a stepwise condensation method, and the other is low-molecular-weight poly(γ-benzyl-L -glutamate) prepared by the N-carboxyanhydride method with n-hexilamine initiation at [A]/[I] = 4 and factionated by dosslution in formic acid. Concentration-dependent association of both peptides occurs noncooperatively, without giving critical micelle concentrations. The aggregate size is small: about 23 for the former tetrapeptide and about 7 for the latter polypeptide. While angular dissymmetry is close to unity, light scattering shows anomalous angular dependence, the intensity being symmetrically low with respect to the scattering angle of 90°. The observed angular dependence is interpreted in terms of the effect of optical anisotropy of peptide units. Formation of the anisotropic phase in concentrated solutions of these peptides is also examined briefy.     

Synthesis of sequential polypeptides containing lysine and tyrosine

Sequential polypeptides with the repeating units L -tyrosyl-L -lysyl, L -tyrosyl-(L -lysyl)₂, and L -tyrosyl-(L -lysyl)₃ have been synthesized by solution polymerization of the N-hydroxy-succinimide esters of the corresponding di-, tri-, and tetrapeptides. The monomers for the polytripeptide and polytetrapeptide were prepared by fragment condensation, using the mixed carbonic anhydride coupoling method. Moderately high molecular weight polypeptides were obtained.     

Sequential polypeptides by matrix-controlled thermal polymerization. Synthesis and racemization studies

A new method for the synthesis of sequential polypeptides is described. The approach consists of the thermal condensation in vacuo of the trifluoroacetate salts of tripeptide pentachlorophenyl esters deposited on a micron-size celite matrix. The synthesis and polymerization of the trifluoroacetate salts of L -valy-L -valyl-L -alanine pentachlorophenyl ester, glycyl-L -valyl-L -alanine pentachlorophenyl ester, and L -valyl-L -alanylglycine pentachlorophenyl ester are described. High-molecular-weight polymers are obtained rapidly in high yield by this method. Comparison of the extents of racemization in the last two polymers constitutes a general approach for assessing racemization during the synthesis of sequential polypeptides since both yield the same polymer, but only the C-terminal alanine may recemize in the former case.     

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.     

N-Methylated Analogs of hIAPP Fragments 18–22, 23–27, 33–37 Inhibit Aggregation of the Amyloidogenic Core of the Hormone

Amylin (hIAPP) aggregation leads to the formation of insoluble deposits and is one of the factors in the development of type II diabetes. The aim of this research was to find N-methylated analogs of the aggregating amylin fragments 18–22, 23–27, and 33–37, which would not themselves be susceptible to aggregation and would inhibit the aggregation of the amyloidogenic cores of the hormone. None of the analogs of fragment 18–22 containing one or two N-methylated amino acid residues showed any tendency to aggregate. Only the peptide H−F(N−Me)GA(N−Me) IL−OH ( 6 ) derived from the 23–27 hIAPP hot spot did not form fibrous structures. All analogs of the 33–37 amylin fragment were characterized by the ability to form aggregates, despite the presence of N-methylated amino acids in their structures. N-Methylated peptides 1 – 5 demonstrated inhibitory properties against the aggregation of fragment 18–22. Aggregation of the amyloidogenic core of 23–27 was significantly inhibited by N-methylated peptides 1 – 3 derived from the (18–22) H−HSSNN−OH fragment and by the H−F(N-Me)GA(N−Me)IL−OH ( 6 ) fragment derived from the 23–27 amylin hot spot. Fragment (33–37) H−GSNTY−NH₂ was found to be inhibited in the presence of N-methylated peptides 1 – 3 derived from the 18–22 fragment and by the double methylated peptide H−F(N−Me)GA(N−Me)IL−OH ( 6 ). Research on the possibility of using N-methylated analogs of amyloidogenic amylin cores as inhibitors of hormone aggregation is ongoing, with a focus on finding the minimum concentration of N-methylated peptides capable of inhibiting the aggregation of hIAPP hot spots.     

Thiocarbamate‐linked peptides by chemoselective peptide ligation

Peptide chemical ligation chemistries, which allow the chemoselective coupling of unprotected peptide fragments, are useful tools for synthesizing native polypeptides or unnatural peptide‐based macromolecules. We show here that the phenylthiocarbonyl group can be easily introduced into peptides on α or ε amino groups using phenylthiochloroformate and standard solid‐phase method. It reacts chemoselectively with cysteinyl peptides to give an alkylthiocarbamate bond. S,N‐shift of the alkylaminocarbonyl group from the Cys side chain to the α‐amino group did not occur. The method was used for linking two peptide chains through their N‐termini, for the synthesis of a cyclic peptide or for the synthesis of di‐ or tetravalent multiple antigenic peptides (MAPs). Thiocarbamate ligation is thus complementary to thioether, thioester or disulfide ligation methods. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.