Synthesis,conformation, and biological activity of the carbohydrate-free vespulakinin 1

Synthesis of the carbohydrate-free heptadecapeptide corresponding to the amino acid sequence of vespulakinin 1 was achieved by the continuous flow solid phase procedure on 4-hydroxymethyl-phenoxyacetyl-norleucyl derivatized Kieselguhr-supported polydimethylacrylamide resin, as well as by a combination of solid phase and solution syntheses. Preformed Fmoc-amino acid symmetrical anhydrides (Boc derivative for the N-terminal residue) were used for amine acylation in the continuous flow method. Serine and threonine were side chain protected as tert.-butyl ethers and the 4-methoxy-2, 3, 6,-trimethyl-benzenesulfonyl group was used for masking the guanidino function of arginine residues. After cleavage from the resin the final peptide was purified by ion exchange chromatography and characterized by amino acid analysis, high voltage electrophoresis, and RP-HPLC analysis. Alternatively, the protected N-terminal octapeptide, Fmoc-Thr(Bu^t)-Ala-Thr(Bu^t)-Thr(Bu^t)-Arg(Mtr)-Arg-(Mtr)-Arg(Mtr)-Gly-OH was prepared on 4-hydroxymethyl-3-methoxyphenoxyacetyl-norleucyl derivatized Kieselguhr-supported polydimethylacrylamide resin and the C-terminal nonapeptide H-Arg(NO₂)-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-(NO₂)-OBzl was synthesized in solution through the fragment condensation method. The two fragments were coupled by the DCC-HOBt procedure and the resulting heptadecapeptide was deblocked and purified. The conformational features of the synthesized peptides are reported. Preliminary pharmacological experiments indicated that carbohydrate-free vespulakinin 1 is more potent than bradykinin in lowering rat blood pressure.     

Synthesis and characterization of human α‐defensins 4‐6

Abstract: Human α‐defensins are small, Cys‐rich, cationic proteins expressed predominantly in neutrophils and intestinal epithelia. They play important roles in innate and adaptive immunity against infection. Progress in studying these molecules can be accelerated by access to large quantities of high‐quality materials, which have been obtained mainly from natural sources. Here, we report total synthesis of human α‐defensins 4, 5, and 6, also known as HNP4, HD5, and HD6, using the optimized N,N‐diisopropylethylamine (DIEA) in situ neutralization/2‐(1 H‐benzotriazolyl)‐1,1,3,3‐tetramethyluroniumhexafluorophosphate (HBTU) activation protocol for solid‐phase Boc chemistry. Oxidative folding/disulfide formation was achieved directly using crude peptides, resulting in an overall synthetic yield of 10–16% with high purity. Antimicrobial activity assays were performed with Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, using colony‐counting methods, and the results demonstrated differential activity against these strains. Our report describes a highly efficient synthetic approach that enables thorough structural and functional studies of these three important immunologic molecules.     

Synthesis,Preferred Conformation,and Membrane Activity of Medium-Length Peptaibiotics: Tylopeptin B

The solid-phase synthesis and full chemical characterization of the medium-length (14-amino acid residues) peptaibol with antibiotic properties of tylopeptin B, originally extracted from the fruiting body of the mushroom Tylopilus neofelleus, are described. These data are accompanied by the results on the solution-phase synthesis via the segment condensation approach of a selected, side-chain protected, analog. A solution conformational analysis, performed by the combined use of FTIR absorption, circular dichroism, and 2D-NMR (the latter technique coupled to molecular dynamics calculations), favors the conclusion that the 3D-structure of tylopeptin B is largely helical with a preference for the α- or the 3₁₀-helix type depending upon the nature of the solvent. Helix topology and (partial) amphiphilic character are responsible for the observed membrane-modifying properties of this peptaibiotic.     

From pro defensins to defensins: synthesis and characterization of human neutrophil pro α‐defensin‐1 and its mature domain

Abstract: Human neutrophil α‐defensins (HNPs) are small, cationic, Cys‐rich antimicrobial proteins that play important roles in innate immunity against infectious microbes such as bacteria, fungi and enveloped viruses. Synthesized as inactive precursors in vivo (pre‐proHNPs), HNPs are activated through proteolytic removal of the inhibitory pro‐peptide required for subcellular sorting and correct folding. We seek to understand the molecular basis for the recognition between the 45‐residue pro‐peptide and the C‐terminal functional domain. Here we described, total chemical synthesis of the 75‐residue human neutrophil pro α‐defensin‐1 (proHNP1) via native chemical ligation. After oxidative folding, proHNP1 is cleaved by cyanogen bromide at the Met⁴⁵–Ala⁴⁶ peptide bond to release the mature form. The native disulfide connectivity in HNP1, i.e. Cys¹–Cys⁶, Cys²–Cys⁴ and Cys³–Cys⁵, is verified by mass mapping of peptide fragments generated by proteolytic digestion and Edman degradation. Fluorescence spectroscopy studies and antimicrobial activity assays further support that synthetic proHNP1 and HNP1 are correctly folded. While largely unstructured in aqueous solution, the pro‐peptide binds to HNP1 intermolecularly with an apparent K_d value of 6.2 μm at pH 7.4, confirming the mode of intramolecular inactivation of human α‐defensin precursors.     

兔防御素NP2酵母表达液抗菌活性的研究

兔防御素NP2是防御素家族成员之一,从兔子的多形核嗜中性细胞中分离出来,对革兰阴性细菌、阳性细菌、分枝杆菌、真菌、某些恶性肿瘤细胞和被膜病毒如艾滋病病毒有较强的毒杀作用,对正常功能的细胞几乎无毒害作用[1].在免疫调控中可促进抗原递呈细胞、单核巨嗜细胞的靶趋向性,增加机体的免疫能力,加快伤口的愈合[2].为深入研究和开发防御素,我们将鉴定正确的酵母穿梭质粒pGAPZ αA-NP2转入Pichia pastoris SMD1168酵母表达;选择对传统抗生素耐药性较强的细菌为研究对象,通过抗菌活性试验,初步确定酵母表达兔防御素的抗菌活性.     

Synthesis and characterization of the colistin peptide polymyxin E1 and related antimicrobial peptides

Abstract: Two strategies were developed to synthesize the acylated cyclic peptides know as polymyxins. Synthesis of polymyxin E1 and several analogs enabled us to evaluate the minimum inhibitory concentration of individual compounds against Gram‐negative bacteria. In this study we also report the first identification of two component peptides in the complex polymyxin fermentation product colistin, a Thr2Ser isoform and an acyl group isomer. Both of these peptides, as well as a known component peptide, Leu7Ile, were similar to polymyxin E1 in potency, suggesting that conservative mutations in the colistin family are functionally inconsequential. In contrast, the acyclic analogs of all of these peptides were inactive, indicating that the characteristic lariat structure of the polymyxins is necessary for antimicrobial activity.     

Synthesis,biological activity,conformational analysis by NMR and molecular modeling of N-formyl-L-Met-L-Pro-L-Phe-OMe,a praline analogue of the chemotactic peptide N-formyl-L-Met-L-Leu-L-Phe-OH

The tripeptide N-formyl-Met-Pro-Phe-OMe (f-MPF-OMe), an analogue of the signal peptide N-formyl-Met-Leu-Phe-OH (f-MLF-OH), was synthesized and its chemotactic activity evaluated; it showed no activity in either superoxide production or calcium mobility with human neutrophils. However, the corresponding acid f-MPF-OH retained about 25% activity in the production of superoxide. The conformation of the f-MPF-OMe analogue was evaluated by NMR spectroscopy and molecular simulation and shown to predominate in a γ-turn with a hydrogen bond between Met CO and Phe NH. Since this analogue is not chemotactic, it is suggested that for recognition the receptor prefers a peptide with a flexible backbone, favoring an extended conformation in the binding site.     

Synthesis and biologic activity of conformationally constrained analogs of L‐363,301

Abstract: We report the synthesis, biological activity and conformational analysis of analogs of the cyclic hexapeptide L‐363,301, c[Pro⁶‐Phe⁷‐d ‐Trp⁸‐Lys⁹‐Thr¹⁰‐Phe¹¹] (numbering as in the native hormone somatostatin‐14). The d ‐Trp in position 8 was replaced with (2R,3S)‐ and (2R,3R)‐β‐MeTrp respectively, with an added methyl group in the beta position of Trp. The objective of our study was to determine the potency and selectivity generated by the added constraint in the beta position of the d ‐Trp upon binding to human somatostatin receptors hsst1‐5. We synthesized the building blocks enantioselectively and incorporated them into the peptides by SPPS. Competition binding assays revealed that both compounds 2 and 3 were selective for hsst2 over hsst5. The (2R,3S) analog 2 was approximately 30 times more potent at hsst2 than the (2R,3R) analog 3 . Interestingly, the (2R,3R) compound showed no binding affinity at hsst5.     

靶向TRPV1受体的蜈蚣毒素环肽cRhTx的合成及活性研究

摘 要：目的 合成首尾环化的蜈蚣毒素环肽（cRhTx）,评价它对TRPV1配体门控离子通道的影响。方法 采用Fmoc-固液相联用多肽合成技术和反相高效液相色谱-质谱联用技术制备野生型蜈蚣毒素（RhTx）与cRhTx,利用圆二色谱法分析两者的结构特点,并进一步采用电生理学法检测两者对TRPV1受体激活的影响。结果 首次通过引入GGAAGG柔性连接片段合成了结构新颖的cRhTx,cRhTx和RhTx具有较高的结构相似性,且cRhTx对TRPV1受体的激动作用与RhTx相似。结论 引入GGAAGG柔性连接片段,对野生型RhTx进行环化,可以保持对TRPV1的激动作用,获得的cRhTx具有重要应用潜力。     

Synthesis,purification and characterization of thymosin α11, a new thymic peptide

A synthesis of thymosin α₁₁, a new 35 amino acid thymic peptide structurally related to thymosin α₁ (J. Caldarella, G.J. Goodall, A.M. Felix, E.P. Heimer, S.B. Salvin & B.L. Horecker (1983) Proc. Natl. Acad. Sci US 80, 7424–7427), is reported. The synthesis was accomplished by the solid phase method employing the 4-(aminoacyloxymethyl) phenylacetamidomethyl(Pam)-copoly(styrene-1% divinylbenzene) resin. The synthetic material was purified by preparative high pressure liquid chromatography and was found to be equivalent to the natural compound in its chemical properties.     

Methylation in positions 1 and 7 of angiotensin II A structure-activity relationship study

Six analogues of angiotensin II (Ang) were synthesized with modifications in positions 1 and 7. The study was undertaken in order to learn more about the influence of alkylations in positions 1 and 7 and their interdependence. Previous studies have shown that x,x-dimethylation of Gly (aminoisobutyric acid, Aib) in position 1 produces quite potent analogues, as does N-methylation of Gly (sarcosine). Combination of both C^x- and N^x-methylations to N-Me-Aib¹, however, did not produce an affinity increase. Decyclisation of the Pro⁷-residue produced moderately active analogues with position 7 N-methylation and inactive analogues if the N-alkylation was suppressed. In order to investigate a possible stereochemical interdependence of positions 1 and 7, a group of peptides with combinations of position 1 and 7 alkylations were investigated. The following analogues were prepared: [Sar¹, Aib⁷]Ang, [Sar¹,Aib⁷,Leu⁸]Ang, [Aib^1,7]Ang, [Aib^1,7, Leu⁸]Ang, [N-Me-Aib¹,Aib⁷]Ang, [N-Me-Aib¹,Aib⁷,Leu⁸]Ang. They were synthesized by classical solid phase synthesis using the BOC-TFA-HF scheme. The biological properties of these peptides were assessed on the rabbit aorta preparation and their binding potencies were measured on bovine adrenal membranes. Both on agonistic and antagonistic [Leu⁸]Ang analogues single Aib substitutions in position 1 or 7 induced affinity reduction in both bioassays. Simultaneous Aib modifications in positions 1 and 7 induced more important affinity loss in a synergic manner in both bioassays and as well for agonists and antagonists. The N-Me-Aib¹ modifications induce similar affinity loss with or without concomitant Aib⁷ modification. Agonistic (Phe⁸) analogues containing Aib in position 7 all have reduced intrinsic activity, indicating for the first time an influence of this position on the activation mechanism of the Ang receptor of the Type AT₁. © Munksgaard 1994.     

Synthesis and properties of an all-D model ribonuclease S-peptide

In order to examine the effect of a defined enantiomeric sequence on protein structure, the all-d model ribonuclease S-peptide, H-Ala-Glu-Ala₄-Lys-Phe-Ala-Arg-Ala-His-Met-Ala₂-OH, has been synthesized by the solid phase method. The all-L peptide has been synthesized previously and shown to possess 36% of ribonuclease S activity when added to ribonuclease S-protein (Komoriya, A. & Chaiken, I.M. (1982) J. Biol. Chem. 257, 2599–2604). The synthetic d -peptide was purified by gel filtration and semipreparative reverse phase HPLC. Amino acid composition of the synthetic peptide was in agreement with theory and gas chromatographic analysis showed that no significant racemization had occurred during synthesis. Circular dichroism (CD) studies of the d -peptide showed a peak of positive ellipticity in the 220–230 nm region, whereas a negative ellipticity peak for the l -peptide was observed. The effects of temperature and trifluoroethanol on the far-ultraviolet CD spectra of d - and l -peptides were similar but of opposite sign, confirming the expectation that the d -peptide has the propensity to form an α-helical structure which is enantiomeric with respect to that formed by the l -peptide. In the presence of S-protein, the l -peptide showed hydrolytic activity against the substrate cytidine-2′:3′-monophosphate, whereas the d -peptide was inactive. Addition of the d -peptide to mixtures of l -peptide and S-protein did not lead to inhibition of enzymatic activity. These results indicate lack of binding of d -peptide to S-protein to produce either an active or inactive species.     

环肽类化合物的合成与生物活性研究进展

对近年来在环肽化合物的化学合成和生物活性等方面的研究进展及该类化合物潜在的临床应用前景作一综述。环肽类化合物是一类结构新颖、生物活性广泛、作用机制独特的化合物,在天然产物中占据十分重要的地位,环肽的研究正在受到人们越来越多的关注。     

Synthesis and biological studies of dermorphin and its analogs substituted at positions 5 and 7

Dermorphin and seven of its analogs substituted at positions 5 and/or 7, have been synthesized by the solid phase method employing mainly 9-fluorenylmethyloxycarbonylamino acid trichlorophenyl esters in presence of 1-hydroxybenzotriazole, the solid support being the Merrifield resin. Among the analogs synthesized, the most interesting is [Tyr⁷]dermorphin. It is one of the most potent dermorphin analogs reported so far. Compared to the natural peptide, it is about two times more potent in the GPI (in vitro) and nearly 1.4 times more potent in its analgesic activity in mice by the hot plate test (in vivo). Further, its antidiarrhoeal activity in mice (in vivo) is comparable to that of dermorphin. On the other hand, [Thr⁷]dermorphin is almost as potent as dermorphin.     

Synthesis of [1, 6-cyclo(Acetyl-1-L-glutamic acid, 2-D-phenylalanine, 3-D-tryptophan, 6-D-lysine)] luteinizing hormone-releasing hormone on poly-N-acrylylpyrrolidine resin

The protected peptide, Ac-Glu(OBu^t)-D-Phe-D-Trp-Ser(Bu^t)-Tyr(Bu^t)-D-Lys(Z)-Leu-Arg(Tos)-Pro-Gly-NH₂ was synthesized in a stepwise manner on a resin of poly-N-acrylylpyrrolidine using both acid cleavable N^α-tert.-butyloxy-carbonyl and base cleavable N^α-fluorenylmethyloxycarbonyl protecting groups. After cleavage by ammonolysis in methanol, the tert.-butyl and benzyloxy-carbonyl side-chain protecting groups were cleaved with CF₃-CO₂H-thioanisole and the 1–6 amide ring formed by cyclization with diphenylphosphorylazide, after which the remaining tosyl protecting group was cleaved in HF-anisole. [1,6-Cyclo (Ac-Glu¹, D-Phe², D-Trp³, D-Lys⁶] LH-RH exhibited less than 10% of the antiovulatory potency of [D<Glu¹, D-Phe², D-Trp^3,6] LH-RH, a potent linear antagonist.     

Backbone—side-chain interactions in serine Synthesis,crystal structure and solution conformation of a linear model peptide N-Boc-l-Ser-l-Phe-OCH3

The peptide Boc-Ser-Phe-OCH₃ was synthesised by a solution-phase method using the usual workup procedure. The peptide was crystallized from a 70:30 (v/v) methanol-water mixture. The crystals are monoclinic, space group P2₁ with a= 5.128(2), b=17.873(2), c=11.386(2) Å, and β=98.03(3)°. The structure was determined by direct methods and refined by a structure factor least-squares procedure. The final R-value for 1499 observed reflections was 0.041. The structure contains one peptide and one solvent water molecule. The peptide adopts a β-strand-like conformation with φ₁=- 100.3(5), ψ_l= 99.9(5), φ₂= - 122.2(5), ψ^T₂= -172.5(6)°. The Ser side-chain assumes an extended conformation with χ¹₁= - 177.0(4)°. The O^γH group of serine acts as a proton donor in an intramolecular weak hydrogen bond with (Ser) O′₁; [O^γ₁;-H^γ₁?O′₁= 3.253(6) Å]. The Phe side-chain adopts a staggered conformation with χ¹₂= -70.9(6), χ²_2,1= 88.4(7)°, χ^2,2₂= -89.2(6)°. The water molecule generates a loop through two hydrogen bonds with O^γ₁ [OW?O^γ₁= 2.893(5) Å] and O′₂ [OW-O′₂= 2.962(7) Å] atoms. The unit-translated peptide molecules along the α-axis are held by hydrogen bonds: N₁-H₁?O₂ (x-1, y, z) = 2.954(4) Å and N₂-H₂?O′₁ (x+1, y, z) = 2.897(6) Å in a manner similar to those observed in parallel β-pleated sheet structures. There is an additional interaction involving O^γ₁ and the water molecule [OW?O^γ₁ (x= 1, y, z) = 2.789(4) Å]. The strong NOE peak of C_i(H)?N_i+1 (H) and a simultaneous weak NOE peak of N_i(H)?N_i+l (H) in the ROESY spectra of two-dimensional NMR in dimethyl sulfoxide indicate a β-strand-like conformation for the peptide in solution. © Munksgaard 1996.     

Synthesis and properties of human γ-lipotropin

The synthesis of human γ-lipotropin by the solid-phase method is described. The synthetic product was characterized by R_f in partition chromatography on Sephadex G-50, paper electrophoresis, polyacrylamide gel electrophoresis, thin-layer chromatography, HPLC, end group determination, peptide mapping of a tryptic digest, and amino acid analyses of acid and enzymic digests. The synthetic material is identical to natural human γ-lipotropin when assayed against natural human β-lipotropin for lipolytic activity.     

Synthesis and properties of equine β-melanotropin analogs with substitution in residue position 1

Five analogs of equine β-melanotropin have been synthesized by the solid phase method. The NH₂-terminal aspartic acid was substituted with amino acids (Gly, Trp, Ile, Lys and Nα-acetyl-Asp) differing widely in physicochemical properties. On the basis of their lipolytic potencies it was concluded that this position plays a negligible role in this activity.     

Synthesis and opioid activity of partial retro-inverso analogs of dermorphin

We studied the effect of partial retro-inverso modification of selected peptide bonds of dermorphin (H-Tyr-d -Ala-Phe-Gly-Tyr-Pro-Ser-NH₂. The modifications concern two consecutive peptide bonds (Phe³-Cly⁴-Tyr⁵, I) or a single one (Gly⁴-Tyr⁵-, II or Phe³-Gly⁴, III). All pseudoheptapeptides showed low opioid activity in the in vitro and in vivo tests. Compound III has a biological potency comparable to that of morphine but only 2–5% of original dermorphin when tested in guinea pig ileum preparation and in mice tail-flick assay after intra-cerebro or subcutaneous administration.     

Synthesis and properties of equine β-melanotropin and its naturally occurring des-Asp analog

The syntheses of equine β-melanotropin and newly isolated des-Asp¹-β-melanotropin are described. The syntheses have been accomplished by the solid-phase method. The lipolytic potency of des-Asp¹-β_e-MSH is about 5 times higher than that of β_e-MSH.