Synthesis by conventional methods of human growth hormone peptide fragments. I.

The synthesis of two protected peptides which correspond to positions 139-146 and 147-156 of the HGH primary structure is described. These peptides prepared by the stepwise procedure, are: Boc-Phe-Lys(Z)-Gln-Thr(Bzl)-Ser(Bzl)-Lys(Z)-Phe-OMe and Boc-Asp(OBzl)-Asn-Ser(Bzl)-His(Dnp)-Asn(OBzl)-Asp(OBzl)-Ala-Leu-OBzl. All protected intermediates were isolated and characterized for homogeneity.     

Biological activity in human neutrophils of di-tripeptides, analogues of the chemotactic fMLP

for-Met-Ser(for-Met-Leu-Phe)-Phe-OMe 1 and for-Met-Lys(for-Met-Leu-Phe)-Phe-OMe 2 were synthesized in order to investigate biological activities on human neutrophils of crosslinked di-tripeptides. Our results seem to highlight that the tested di-tripeptides (i) do not step up chemotaxis, (ii) can elicit superoxide anion production which is dependent on the nature of the residue at position 2, chosen in the tripeptide that is crosslinked to the fMLP-OMe.     

Conformational effects of chiral alpha,alpha-dialkyl amino acids. I. C-terminal tetrapeptides of emerimicin containing alpha-ethylalanine

The syntheses and the crystal structures of the C-terminal tetrapeptide fragments of emerimicin IV and III, Boc-R-EtA-Hyp(Bzl)-Ala-Phol and Boc-R-EtA-Hyp(Bzl)-MeA-Phol, containing the chiral alpha,alpha-dialkyl amino acid, R-alpha-ethylalanine (R-EtA) are reported. The two peptides are isomorphous and assume a 3(10)-helical conformation in the crystal. A comparison of the crystal data on alpha,alpha-dialkyl amino acids indicates that alkyl substituents larger than a methyl group do not preclude peptides containing these amino acids from assuming the conformations associated with minima which have been well characterized for alpha-methylalanine.     

Synthesis and Immunostimulating Activity of Cysteine-Containing Derivatives of Glycyrrhizic Acid

New cysteine-containing derivatives of glycyrrhizic acid were synthesized by its coupling with Cys(Bzl) esters or the Cys(Bzl)-Val-OBu ^t dipeptide by the active ester method (DCC/HOSu) or by Woodward's reagent K. The derivatives with Cys(Bzl) and Cys(Bzl)-Val residues attached to the carbohydrate part of the molecule stimulated the primary immune response and the reaction of delayed-Type hypersensitivity in mice at a dose of 2 mg/kg.     

Cyclic peptides. XXVI. Synthesis of AM-toxin II analogs by cyclization through ester bond formation

In order to explore the route for the preparation of cyclodepsipeptide by cyclization through an ester bond formation, two analogs of AM-toxin II, cyclotetradepsipeptide, were synthesized. As a preliminary experiment, synthesis of [L-Phe3, L-Ser(Bzl)4]-AM-toxin II, containing L-Phe and L-Ser(Bzl) in place of L-App (2-amino-5-phenyl-pentanoic acid) and delta Ala (alpha, beta-dehydroalanine), respectively, was attempted. Cyclization of H-L-Hmb-L-Phe-L-Ser(Bzl)-L-Ala-OH in CH2Cl2 at 10 mM concentration using water-soluble carbodiimide (EDC) and 4-dimethylaminopyridine (DMAP) successfully gave a cyclic monomer in 16% yield. Cyclization of H-L-Hmb-L-App-L-Ser(Bzl)-L-Ala-OH under the same conditions also afforded a cyclic monomer, [L-Ser(Bzl)4]AM-toxin II, in 19% yield. Analytical parameters of these cyclic monomers obtained were identical to those of the authentic samples obtained by cyclization through a peptide bond formation.     

Thermolysin as a catalyst in enzymatic synthesis of asparagine-containing peptides II

Thermolysin was used as a catalyst to obtain the following protected di- and tripeptide esters: Z-Asn-Leu-OEt, Z-Asn-Phe-OEt, Moz-Asn-Leu-Gly-OEt, Boc-Asn-Leu-Gly-OEt, Z-Asn-Leu-Gly-OEt, Moz-Asn-Leu-Gly-OBzl, Moz-Asn-Leu-Gly-OtBu, Moz-Gln-Leu-Gly-OEt, Moz-Asn-Ile-Gly-OEt, and Moz-Asn-Leu-Ala-OEt. These compounds were obtained in pure form and the yields exceeded 50%, except for Moz-Asn-Leu-Gly-OtBu and Boc-Asn-Leu-Gly-OEt. H-Cys(Bzl)-OtBu and H-Cys(Bzl)-Pro-Leu-Gly-NH2 were both inadequate as amino components for obtaining Moz-Asn-Cys(Bzl)-OtBu, Z-Asn-Cys(Bzl)-OtBu and Moz-Asn-Cys(Bzl)-Pro-Leu-Gly-NH2 in the thermolysin-catalyzed reactions. In the attempted synthesis of the protected pentapeptide amide, this protease cleaved the Pro-Leu bond of the amino component H-Cys(Bzl)-Pro-Leu-Gly-NH2 and catalyzed the coupling between the resulting dipeptide amide and Moz-Asn-OH, thus yielding Moz-Asn-Leu-Gly-NH2 as the main product.     

Relationship between long-term resistance to Trypanosoma cruzi and latent infection,examined by antibody production and polymerase chain reaction in mice

Trypanosoma cruzi infections persist for the lifetime of humans and laboratory animals as either latent or pathogenic parasitism. Mice inoculated with a nonpathogenic, attenuated strain (TCC) display resistance against virulent challenge, with a strong control of parasitemia and protection against tissue lesions for more than 12 mo. Three main approaches were used to test whether protection by TCC inocula is based on a latent infection or on a "sterile" immunological memory: curative Benznidazole (Bzl) treatment, serological reactions, and detection of infection by polymerase chain reaction (PCR). If resistance is maintained in the absence of infection, it should not be reduced by Bzl treatment and TCC-inoculated animals should not maintain long-term serological or PCR reactivity. The Bzl treatment after TCC inoculations did not reduce, after periods of up to 420 days, TCC-induced resistance to challenge. But TCC inocula given during Bzl treatment conferred short-term, but not long-term. protection. Maintenance of high antibody levels and protection were better in the virulent Tulahuen (TUL) strain than in the attenuated TCC strain infections, and trypomastigote inocula of either strain were better inducers of antibodies and resistance than epimastigotes. PCR detection of T. cruzi DNA was positive in almost all TUL strain-inoculated animals and negative in immunocompetent animals inoculated with TCC epimastigotes, although high numbers of TCC trypomastigotes produced persistent PCR signals of infection in newborn BALB mice. Thus, 2 polar models were developed, where latent infection by TCC was either demonstrated or excluded. In both, resistance to virulent challenge was maintained during long periods. But late declination of antibody titers (>200 days) and resistance to challenge (>350 days) was observed in animals displaying clearance of all signals of infection.     

Investigations on peptide synthesis with polymeric supports: synthesis and characterization of the deamido-calcitonin-M-(19-32)-tetradecapeptide by use of the soluble support polyethyleneglycol and coupling of the products to the insoluble polystyryl-benzhydrylamine resin (author's transl)]

The synthesis of the deamido-calcitonin-M-(19-32)-tetradecapeptide Boc-Phe-His-Thr(Bzl)-Phe-Pro-Gln-Thr(Bzl)-Ala-Ile-Gly-Val-Gly-Ala-Pro-OH (I) by the use of the soluble polymeric support polyethyleneglycol is described. For product characterization the following compounds were prepared:H-Phe-His-Thr(Bzl)-Phe-Pro-Gln-Thr(Bzl)-Ala-Ile-Gly-Val-Gly-Ala-Pro-OH and H-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Ala-Pro-OH. The analyses of these products by ion exchange chromatography indicates that the correct tetradecapeptide sequence was synthesized to more than 90%. Despite high yields in the coupling reactions, crude I was inhomogenous due to partial cleavage to O-benzyl ether bonds. In order to use I for the total synthesis of calcitonin M, it was coupled to the insoluble polystyryl-benzhydrylamine resin. 0.08 mmol of peptide was coupled per g of resin. Treatment of this peptide resin with hydrogen fluoride yielded H-Phe-His-Thr-Phe-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2.     

Identification and Suppression of β-Elimination Byproducts Arising from the Use of Fmoc-Ser(PO3Bzl,H)-OH in Peptide Synthesis

The formation of 3-(1-piperidinyl)alanyl-containing peptides via phosphoryl β-elimination was identified from the application of Fmoc-Ser(PO₃Bzl,H)-OH in peptide synthesis as shown by RP-HPLC, ES-MS and ³¹P-NMR analysis. An N ^α -deprotection study using the model substrates, Fmoc-Xxx(PO₃Bzl,H)-Val-Glu(O^tBu)-Resin (Xxx = Ser, Thr or Tyr) demonstrated that piperidine-mediated phosphoryl β-elimination occurred in the N-terminal Ser(PO₃Bzl,H) residue at a ratio of 7% to the target phosphopeptide, and that this side reaction did not occur in the corresponding Thr(PO₃Bzl,H)- or Tyr(PO₃Bzl,H)- residues. The generation of 3-(1-piperidinyl)alanyl-peptides was also shown to be enhanced by the use of microwave radiation during Fmoc deprotection. An examination of alternative bases for the minimization of byproduct formation showed that cyclohexylamine, morpholine, piperazine and DBU gave complete suppression of β-elimination, with a 50% cyclohexylamine/DCM (v/v) deprotection protocol providing the crude peptide of highest purity. Piperidine-induced β-elimination was found only to occur in Ser(PO₃Bzl,H) residues that were in the N-terminal position, since the addition of the next residue in the sequence rendered the phosphoseryl residue stable to multiple piperidine treatments. The application of the alternative N ^α -deprotection protocol using 50% cyclohexylamine/DCM (v/v) is therefore recommended for deprotection of the Fmoc group from the Fmoc-Ser(PO₃Bzl,H) residue, with particular benefit anticipated for the synthesis of multiphosphoseryl peptides.     

Synthesis and immunostimulating activity of cysteine-containing glycopeptide derivatives of glycyrrhizic acid

New cysteine-containing derivatives of glycyrrhizic acid were synthesized by its coupling with Cys(Bzl) esters or the Cys(Bzl)-Val-OBu(t) dipeptide by the active ester method (DCC/HOSu) or by Woodward's reagent K. The derivatives with Cys(Bzl) and Cys(Bzl)-Val residues attached to the carbohydrate part of the molecule stimulated the primary immune response and the reaction of delayed-type hypersensitivity in mice at a dose of 2 mg/kg. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.     

Synthesis,conformation and biological activity of centrally modified pseudopeptidic analogues of For-Met-Leu-Phe-OMe

Summary. For-Met-βAlaψ[CSNH]-Phe-OMe (3), For-Met-βAlaψ[CH₂NH]-Phe-OMe (5), For-Met-NH-pC₆H₄-SO₂-Phe-OMe (8a), For-Met-NH-mC₆H₄-SO₂-Phe-OMe (8b) and the corresponding N-Boc precursors (2, 4, 7a, b) have been synthesized and their activity towards human neutrophils has been evaluated in comparison with that shown by the reference tripeptide For-Met-Leu-Phe-OMe (fMLF-OMe). Chemotaxis, lysozyme release and superoxide anion production have been measured. ¹H NMR titration experiments and IR spectra have been discussed in order to ascertain the preferred solution conformation adopted by the tripeptide 3 with particular reference to the presence of a folded conformation centred at the centrally positioned thionated β-residue.     

Synthesis of peptides by fragment condensation on a solid support. II. A scheme for preparation of 4,8-disubstituted vasopressins evaluated on 8-arginine-vasopressin.

A convenient scheme for the synthesis of 4,8-disubstituted vasopressins has been designed and its usefulness evaluated in the preparation of one of the parent hormones, 8-arginine-vasopressin. The main feature of the scheme involves preparation of two protected tripeptide fragments, Z-Cys(Bzl)-Tyr(Bzl)-Phe and Boc-Asn(Mbh)-Cys(Bzl)-Pro which are incorporated in a synthesis on a solid support. Both tripeptides were prepared conventionally with the carboxyl groups protected as benzyl esters. The benzyl-ester groups were removed by transesterification with 2-dimethylaminoethanol and subsequent hydrolysis. To avoid racemization in the coupling step with the fragment containing a C-terminal phenylalanine, N-hydroxysuccinimide was added. After removal of the peptide from the resin, deprotection, oxidation and desalting, final purification was effected by ion-exchange chromatography. Apart from the main product, which exhibited full pressor activity, only small amounts of impurities could be isolated.     

Synthesis and characterization of Tyr(Bzl)9,11,13,15 and Tyr9,11,13,15 gramicidin A

Tyr(Bzl) and Tyr gramicidin A were prepared by the solid phase method using a 4-(oxymethyl)-Pam resin and Bpoc as alpha-amino-protecting group. The benzylated analog [Gr.T(Bzl)] was purified by chromatography on silica gel and then on LH60 Sephadex. Removal of benzyl groups was carried out by hydrogenolysis and the debenzylated derivative (Gr.T) was purified in the same way. Both gramicidins were checked and characterized by t.l.c., HPLC, circular dichroism, 1H n.m.r. and single channel measurements. CD spectra were found to be different for Gr.T(Bzl) and Gr.T and strongly dependent upon the solvent and the concentration. Single channel conductance of Gr. T is slightly lower than that of Gr.A (A Gr.T approximately equal to 0.7 A Gr.T).     

Solvolysis and aminolysis on peptidyl-Kaiser oxime resin assisted by Ca2+ and Eu3+: a mild procedure to prepare alpha-methyl and -ethyl esters of protected peptides.

Ca2+ and Eu3+ were able to assist solvolysis on peptidyl-Kaiser oxime resins generating alpha-methyl and -ethyl esters of protected peptides. The methanolysis assistance was at least twice as effective as that of acetic acid, the common catalyst used in aminolysis of the ester oxime linkage. No molar excess of Ca2+ or Eu3+ was needed to enhance this reaction efficiency. Ca2+ also assisted aminolysis on peptidyl-Kaiser oxime resins. Solvolysis and aminolysis rates depended on the nature of the C-terminal residue attached to the resin and on the alcohol used. Both reactions were selective to the ester oxime linkage since no significant amount of secondary products, resulting from rearrangements or simultaneous transesterification of the beta-benzyl or cyclohexyl esters, was detected in the reaction media. The alpha-methyl and -ethyl esters of Ac-Ala-Gly-X [where, X = Gly, Ala, Phe or Lys (2-Cl-Z)] and of Ac-Ile-Ser (Bzl)-Asp(OZ) (where, Z = Bzl or cHex) were essentially the only products formed in the solvolyses performed. Ac-Ile-Ser(Bzl)-Asp(OcHex)Arg(HCl)-OMe and Ac-Ile-Ser(Bzl)-Asp(OcHex)Arg (HCl)-OEt were the major products formed in the aminolysis reactions. In the presence of the metal ions, the resin-cleavage yields were > 50%. In their absence, they were < 15%.     

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

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 alpha or N epsilon 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 O(2)(-) production, however, all these compounds except 4a antagonize lysozyme release by 60%.     

Synthesis of linear,cyclic and polypeptides having the sequence -Asp-βAla-Gly-Ser-βAla-Gly-His-βAla-Gly- as catalysts in hydrolytic reactions

Boc-Asp-βAla-Gly-Ser-βAla-Gly-His-βAla-Gly-OEt, Boc-Asp-βAla-Gly-Ser-βAla-Gly-Ser-βAla-Gly-His-βAla-Gly-OH, cyclo(Asp-βAla-Gly-Ser-βAla-Gly-His-βAla-Gly) and poly(Asp-βAla-Gly-Ser-βAla-Gly-His-βAla-Gly) were synthesized as catalysts in hydrolytic reactions. Asp and Ser residues were protected with a benzyl group, but the His residue was not protected during the synthesis. The protected linear-nonapeptides were prepared by synthesis and subsequent fragment condensation of three kinds of tripeptide which have the sequences-Asp(OBzl)-βAla-Gly-,-Ser(Bzl)-βAla-Gly- and -His-βAla-Gly-, respectively. The protected cyclic-nonapeptide was obtained by cyclization of H-Asp(OBzl)-βAla-Gly-Ser(Bzl)-βAla-Gly-His-βAla-Gly-OH with DCC-HOBr and the subsequent purification by silica gel column chromatography. The protected poly-nonapeptide was prepared by polymerization of H-Asp(OBzl)-βAla-Gly-Ser(Bzl)-βAla-Gly-His-βAla-Gly-OH with DPPA. Deprotection was performed with catalytic hydrogenation for the protected linear- and cyclic-nonapeptides, and with methanesulphonic acid for the protected polynonapeptide, respectively, to give final products. Catalytic actions of the linear-, cyclic- and polypeptides for hydrolysis of p-nitrophenyl acetate are also reported briefly.     

Synthesis of oxytocin using iodine for oxidative cyclization and silica gel adsorption chromatography for purification.

Oxytocin (OT) was synthesized employing the solid phase method. Resins made of copolymers of polystyrene-1%-crosslinked with divinylbenzene gave better yields (73-95%) of Z-Cys(Bzl)-Tyr(Bzl)-Ile-Gln-Asn-Cys(Bzl)-Pro-Leu-Gly-NH2 (I) than 2%-crosslinked resins (10--56%). Reduction of I with Na-liq.NH3 and oxidation with I2-MeOH at -40 degrees minimized dimer and polymer formation, and resulted in good yields (49--54%) of OT. The large volumes of MeOH required when several grams of I are reduced and then oxidized were rapidly evaporated in vacuo, and the residue was desalted by dissolving the peptide in a small volume of glacial acetic acid and filtering to remove the salt. OT was purified by adsorption chromatography on a silica gel column with combinations of MeOH-CHCl3 of graded polarity. Oxytocin elutes with 33% MeOH-CHCl3. After two purification steps by adsorption chromatography, the resulting OT was found to be homogeneous. The hormone was characterized chemically and found to be active biologically.     

Comparative Study of Chemical Approaches to the Solid-Phase Synthesis of a Tumor-Seeking α-MSH Analogue

The synthesis of a cyclic melanocortin analogue (H-pz-βAla-Nle-cyclo[Asp-His-DPhe-Arg-Trp-Lys]-NH₂), where the Boc-protected derivative of a metal-chelating pyrazolyl ligand (pz) was inserted as N-terminal residue, was addressed by several different Fmoc/tBu and Boc/Bzl solid-phase strategies. On-resin cyclization was achieved immediately following incorporation of Asp, by condensation of the Asp side chain carboxyl with the Lys side chain primary amine after selective and simultaneous removal of side chain protecting groups. The success of the synthesis was highly dependent on the chemical strategy employed, with Boc/Bzl chemistry giving the best results. On the light of our findings, Fmoc/tBu strategies are not advantageous for the solid-phase synthesis of this particular type of lactam-bridged peptides. Last, but not least, the target peptide was recently found to have promising tumor-seeking properties (J Biol Inorg Chem 13:449–459, 2008).     

Degradation of d-Biotin by Microorganisms

For the classical solution synthesis of human epidermal growth factor (h-EGF), five protected peptide derivatives, Boc-Leu-Asp(OcHex)-Lys(Cl-Z)-Tyr(Br-Z)-Ala-OH (5), Boc-Val-Cys(MeBzl)-Met-Tyr(Br-Z)-Ile-Glu(OcHex)-Ala-OH (12), Boc-Tyr(Br-Z)-Cys(MeBzl)-Leu-His-Asp(OcHex)-Gly-OH (18), Boc-Cys(MeBzl)-Pro-Leu-Ser(Bzl)-His-Asp(OcHex)-Gly-O H (23) and Boc-Asn-Ser(Bzl)-Asp(OcHex)-Ser(Bzl)-Glu(OcHex)-OH (28) were synthesized to build up the sequence corresponding to 1–30.     

Synthesis of phosphopeptides by the Multipinast method: Evaluation of coupling methods for the incorporation of Fmoc- Tyr(PO3Bzl,H)-OH, Fmoc- Ser(PO3Bzl,H)-OH and Fmoc- Thr(PO3Bzl,H)-OH

astMultipin is a trademark of Chiron Technologies Pty. Ltd., Clayton, Victoria, Australia.The efficiency of various coupling methods for the incorporation of the three monobenzyl phosphorodiester-protected derivatives, Fmoc- Tyr(PO3Bzl,H)-OH, Fmoc-Ser(PO3Bzl,H)-OH and Fmoc-Thr(PO3Bzl,H)-OH, was examined through the test synthesis of Ala-Ser-Gln-Gly-Xxx(PO3H2)-Leu- Glu-Asp-Pro-Ala-NH2 (Xxx = Tyr, Ser, Thr) using the Multipin method of multiple peptide synthesis. The coupling methods examined were (1) PyBrop/DIEA; (2) BOP/HOBt/NMM; (3) BOP/HOBt/DIEA; (4) HBTU/HOBt/DIEA; (5) HATU/HOAt/DIEA; (6) HATU/DIEA; (7) DIC/HOBt; (8) DIC/HOBt/DIEA; (9) DIC/HOAt; (10) DIC/HOAt/DIEA. While all four DIC-based coupling procedures resulted in incomplete incorporation, both the HBTU/HOBt/DIEA and HATU/HOAt/DIEA coupling procedures provided most efficient incorporation of the three Fmoc- Xxx(PO3Bzl,H)-OH derivatives. In the subsequent synthesis of the -helical Tyr(P)-peptide, Glu-Thr-Gly-The-Lys- Ala-Glu-Leu-Leu-Ala-Lys-Tyr(PO3H2)-Glu-Ala-Thr- His-Lys-NH2, analysis of the crude peptide by electrospray MS confirmed that several residue deletions had occurred but that complete incorporation of the Tyr(P)-residue had been accomplished using HBTU/HOBt/DIEA coupling of Fmoc- Tyr(PO3Bzl,H)-OH.