Antibacterial peptides designed as analogs or hybrids of cecropins and melittin

Eight new analogs of cecropin A, two new analogs of melittin and 30 hybrid peptides containing sequences from cecropins and melittin have been synthesized. The lengths of the peptides have varied from 37 residues (the length of cecropin A) to 18 residues. The peptides have been assayed for lysis of sheep red blood cells and for antibacterial activity against two Gram negative and three Gram positive bacteria. The best analogs of cecropin A maintained the anti-Escherichia coli activity of the parental peptide, and were not lytic for red blood cells. Melittin and its replacement analogs were all lytic for red blood cells, but an analog with transposed segments was not. Several of the hybrid peptides were found to be both non-hemolytic and highly active against all test bacteria. The data were used to define the structural requirements for antibacterial activity.     

Chemical synthesis of a designed β-protein through the flow-polyamide method

A designed, 61-residue long, metal-binding protein was synthesized through the flow-polyamide method. This protein, named Minibody (Mini-antibody), contains a β-sheet scaffold and two regions corresponding to immunoglobulin hypervariable loops (H1 and H2), onto which a metal binding site was engineered. The protein is extremely hydrophobic, with a 70%β structure, Accordingly, it was anticipated, and actually found, to be a “difficult sequence” for all its length. Comparison of the standard, “minimum redundancy” protocol (single coupling, low excess of activated species) with a different protocol (double and triple coupling plus capping) showed that the two produced approximately the same amount of full-length product (3.7% after extensive purification). Capping was effective in blocking the unreacted amino groups, converting most failure sequences into truncated ones, a possible aid to implement affinity-type chromatographic protocols. Purification was complicated by the very low solubility of the molecule, coupled to a high tendency to aggregate even in concentrated chaotropic media (8 M urea). Nevertheless, a multi-dimensional purification scheme produced a highly homogeneous product, with the expected IonSpray mass spectrum. The Minibody produced by recDNA methods showed identical chromatographic, spectroscopic and biochemical properties to those of the synthetic product.     

Disulfide linkage to polyacrylic resin for automated Fmoc peptide synthesis. Immunochemical applications of peptide resins and mercaptoamide peptides

The use of disulfide bonds for peptide–resin linkage in solid-phase peptide synthesis was investigated using polyacrylic polymers (Expansin^TM) and automated Fmoc methodology. The disulfide moiety was bound to the support either by coupling a protected bifunctional handle or by an original stepwise procedure. Among the three different disulfide handles that were investigated, only the aminoethyldithio-2-isobutyric acid (AEDI) handle was stable enough to achieve peptide synthesis. A series of peptides of up to 10–20 amino acids were prepared in this manner, in good yield and purity. Rapid and quantitative peptide release was obtained by reduction with equimolecular amounts of dithiothreitol at pH 9 or tris(2-carboxymethyl) phosphine at pH 4.5. This allowed direct and rapid coupling of the released cysteamide peptides to an activated protein carrier and the use of free or resin-bound forms of the antigen in immunoassays.     

Solid-phase synthesis of a range of O-phosphorylated peptides by post-assembly phosphitylation and oxidation

A completely general method for the O-phosphorylation of peptides of any given composition using solid-phase methodology is described. Peptides were assembled using Fmoc amino acid active esters, with base used for Fmoc deprotection. Unprotected amino acid side chain hydroxyl groups were phosphitylated and oxidised at the end of the assembly using bis (benzyloxy)(diisopropylamino)phosphine and tert.-butylhydroperoxide respectively. TFA was used for final deprotection of the amino acid side chains and for simultaneous cleavage from the resin. The synthesis of O-phosphopeptides of up to 15 residues in length is described.     

Anti-writhing activity of some peptides related to neurotensin and tuftsin

Several small peptides related to neurotensin (NT) and tuftsin were synthesized and tested for analgesic activity against acetic acid induced writhing in mice. None of the peptides approached the activity shown by NT or NT hexapeptide. Tuftsin itself was found to be weakly active. An isosteric dipeptide related to a cobra venom peptide was found to have considerable anti-writhing activity at a high intracerebroventricular dose.     

Chemical synthesis and characterization of the epidermal growth factor-like module of human complement protease Clr

Clr is one of the two serine proteases of Cl, the first component of complement, in which it is associated in a calcium-dependent manner to the homologous serine protease Cls. This interaction is mediated by the N-terminal region of Clr, which comprises a single epidermal growth factor (EGF)-like module containing the consensus sequence required for calcium binding, surrounded by two CUB modules. With a view to determine the structure of the EGF-like module of Clr and evaluate its contribution to calcium binding, this module [Clr(123–175)] was synthesized by automated solid-phase methodology using the Boc strategy. A first synthesis using the Boc-His(Z) derivative gave very low yield, due to partial deprotection of His residues leading to chain termination by acetylation, and to insertion of glycine residues. This could be circumvented by using the Boc-His(DNP) derivative and by condensation of appropriate glycine-containing segments. The synthetic peptide was efficiently folded under redox conditions to the species with three correct disulfide bridges, as determined by mass spectrometry and N-terminal sequence analyses of thermolytic fragments. The homogeneity of the synthetic peptide was assessed by reversed-phase HPLC and electrospray mass spectrometry. One-dimensional ¹H NMR spectroscopic analysis provided evidence that the EGF-like module had a well defined structure, and was able to bind calcium with an apparent K_d of 10 mM. This value, comparable to that found for the isolated EGF-like modules of coagulation factors IX and X, is much higher than that measured for native Clr. As already proposed for factors IX and X, it is suggested that neighbouring module(s), most probably the N-terminal CUB module, contribute(s) to the calcium binding site. © Munksgaard 1997.     

芋螺毒素B179的化学合成及其氧化折叠的研究

目的合成新型芋螺毒素B179,并对其氧化折叠进行研究。方法采用Fmoc固相合成法合成芋螺毒素多肽B179,研究了不同的氧化缓冲液、谷胱甘肽浓度、还原型与氧化型谷胱甘肽的比例、以及温度对氧化折叠的影响。结果采用Fmoc固相合成法可以成功合成线性肽,其氧化折叠产物中主要为含一对二硫键的产物,但还出现了其他副产物,可能是含有分子间二硫键的折叠产物;在高浓度还原型谷胱甘肽环境下,折叠副产物更易形成;随着氧化反应温度升高,折叠反应速率加快。结论固相合成法可以有效合成芋螺毒素B179,且不同因素对其二硫键的形成有着较大的影响,本研究结果可为新型芋螺毒素的合成及其氧化折叠提供借鉴。     

Use of the 3-nitro-2-pyridine sulfenyl protecting group to introduce Nε-branching at lysine during solid-phase peptide synthesis I. Application to the synthesis of a peptide template containing two addressable sites

TASPs (template-assembled synthetic peptides) are generated by the covalent attachment of linear peptides to a common peptide backbone, thus generating larger synthetic peptides/proteins with prefolded structure. In this work we present a strategy for the synthesis of a heterotemplate-assembled synthetic peptide containing two addressable sites. This orthogonal protection strategy would allow the selective introduction of different peptide chains via the ε-amino functions of template lysines being protected by either fluorenylmethoxycarbonyl (Fmoc) or 3-nitro-2-pyridine sulfenyl (Npys) groups. The N^α-Boc-N^ε-Npys-l -lysine required for solid-phase peptide synthesis (SPPS) is not readily available at a reasonable cost. To facilitate the more widespread use of this reagent we have compared the two published procedures for synthesizing this protected amino acid and evaluated the suitability of the products for SPPS. Two resin-bound peptides, a tripeptide Ac-G-K-Npys)-G-resin and an octapeptide template Ac-P₁-K₂-K₃-L₄-K_s-K₆-P₇-G₈-resin, were synthesized by SPPS. The ε-amino functions of lysines K₂ & K₆ and K₃ & K₅ of the octapeptide were protected by Fmoc and Npys groups, respectively. Secondly, these peptides were used to evaluate various reagents and reaction conditions for the deprotection of the ε-amino function of lysines bearing the N_ε-Npys protecting group. A procedure for the optimized selective and quantitative deprotection of the Npys group from the ε-amino function of lysine in a resin-bound peptide using 2-mercaptopyridine-N-oxide is described. © Munksgaard 1995.     

Solid phase synthesis and characterization of two canine gut gastrin-releasing peptides

Two canine gastrin-releasing peptides originally isolated from gut tissue extracts have been synthesized by solid phase methodology and purified by preparative reverse phase high performance liquid chromatography (RP-HPLC). The synthetic gastrin-releasing peptides GRP1-27 and GRP 5-27 were characterized with regard to homogeneity and composition using nine different RP-HPLC systems, mass spectroscopy, amino acid analysis, Edman degradation, methionine oxidation, and peptide mapping with tryptic, Staph. aureus V8 protease and cyanogen bromide cleavage (the latter two systems performed only with GRP 1-27). Although a scarcity of the natural products prevented quantitative biological comparison of the synthetic and natural peptides, they were found to elute identically on RP-HPLC co-chromatography and similar dose dependent biological potencies were observed in canine antral muscle tissue contraction experiments. Indeed, all the peptides containing the bombesin-like car-boxyl terminal decapeptide sequence studied to date have similar biological activities.     

Epitopic characterization of the human wild-type and mutant ras proteins using membrane-bound peptides

Overlapping octapeptides encompassing the entire sequences of the human oncogene products Ha-ras, K-ras and N-ras protein were synthesized as spots on polypropylene membrane sheets. The binding of anti-row protein monoclonal antibodies (mAbs) to the membrane-bound peptides was assessed using an enzyme-linked immunosorbent assay. Epitopes of 10 of 18 mAbs to the human ras proteins were mapped and identified by this procedure. The epitopes of nine of the mAbs are within residues 28-39 in the constant domain common to the three ras proteins, whereas the epitope of the tenth (mAb 21) spans residues 136-144 in Ha-ras. The minimal lengths of epitopes of all ten of the mAbs were further precisely mapped using peptides of varying length, and the tolerance for mAb binding of mutated epitopes was determined by systematically replacing each residue in the epitope with each of the 20 common amino acids. The results show that most of these mAbs have essentially the same binding specificity, namely for the sequence YDPT (residues 32–35) or for slightly longer sequences containing these residues. This site is in the switch 1 region (residues 32-38) in the ras effector loop, indicating that some of the same residues important for the interaction of ras with other proteins (GTPase-activating protein, neurofibromin or raf) are highly antigenic. In addition, we investigated epitopes and specificity of five mAbs against the activated human ras proteins by the same procedure. The information gained from this study should be useful both for study of the complicated functions of ras proteins and for clinical detection of ras oncogenes in human tumor cells.     

Synthesis and conformational studies of peptides encompassing the carboxy-terminal helix of thermolysin

The 21-residue fragment Tyr-Gly-Ser-Thr-Ser-Gln-Glu-Val-Ala-Ser-Val-Lys-Gln-Ala-Phe-Asp-Ala-Val-Gly-Val-Lys, corresponding to sequence 296–316 of thermolysin and thus encompassing the COOH-termi-nal helical segment 301–312 of the native protein, was synthesized by solid-phase methods and purified to homogeneity by reverse-phase high performance liquid chromatography. The peptide 296–316 was then cleaved with trypsin at Lys³⁰⁷ and Staphylococcus aureus V8 protease at Glu³⁰², producing the additional fragments 296–307, 308–316, 296–302, and 303–316. All these peptides, when dissolved in aqueous solution at neutral pH, are essentially structureless, as determined by circular dichroism (CD) measurements in the far-ultraviolet region. On the other hand, fragment 296–316, as well as some of its proteolytic fragments, acquires significant helical conformation when dissolved in aqueous trifluoroethanol or ethanol. In general, the peptides mostly encompassing the helical segment 301–312 in the native thermolysin show helical conformation in aqueous alcohol. In particular, quantitative analysis of CD data indicated that fragment 296–316 attains in 90% aqueous trifluoroethanol the same percentage (～58%) of helical secondary structure of the corresponding chain segment in native thermolysin. These results indicate that peptide 296–316 and its subfragments are unable to fold into a stable native-like structure in aqueous solution, in agreement with predicted location and stabilities of isolated subdomains of the COOH-terminal domain of thermolysin based on buried surface area calculations of the molecule     

Efficient Fmoc/solid-phase peptide synthesis of O-phosphotyrosyl-containing peptides and their use as phosphatase substrates

A general synthetic method for the efficient preparation of Tyr(P) -containing peptides is described by the use of Fmoc-Tyr(PO₃¹Bu₂) -OH in Fmoc/solid-phase synthesis followed by simultaneous cleavage of the peptide from the resin and peptide deprotection by acidolytic treatment. The applicability of this approach is demonstrated by the synthesis of H-Ser-Ser-Ser-Tyr(P) -Tyr(P) -OH.TFA and the synthesis of the phosphorylated forms of the two physiological peptides, angiotensin II and neurotensin 8–13. In addition, the three phosphorylated peptides were used as substrates in the study of the local specificity determinants of T-cell protein tyrosine phosphatase. In a competition assay using ³²P-radiolabeled [Tyr(P)]⁴-angiotensin II, both un-labeled synthetic [Tyr(P)]⁴-angiotensin II and Ser-Ser-Ser-Tyr(P) -Tyr(P) reduced the release of ³²P and indicated that they efficiently competed as substrates for the phosphatase. Conversely, [Tyr(P)]⁴-neurotensin 8–13 was ineffective as a competitive substrate and indicated that this particular Tyr(P) -containing peptide sequence was not recognized by the enzyme. The marked difference in the recognition of Asp-Arg-Val-Tyr(P) -Ile-His-Pro-Phe and Arg-Arg-Pro-Tyr(P) -Ile-Leu is consistent with the presence of an acidic residue in the -3 position relative to the Tyr(P) residue.     

Chemical synthesis and purification of proteins: a methodology

Classical stepwise solid-phase peptide synthesis (SPPS) has been used successfully for the synthesis of proteins up to 150 residues in length, although usually with poor yields and homogeneity. The major limitation has been the inability to separate chromatographically similar deletion and truncated impurities from the target sequence. We have developed a highly effective protocol for stepwise SPPS and‘one-step’purification of small proteins. To demonstrate the effectiveness of the methodology we synthesised the 101 residue chaperonin 10 protein from Rattus norvegicus (Rat Cpnl0) using three different chemical protocols. Highly homogeneous Rat Cpnl0 was obtained using an optimised synthetic strategy and one-step purification procedure (method C), involving (i) HBTU/HOBt activation, (ii) N-(2-chlorobenzyloxy-carbonyloxy)succinimide as capping agent and (iii) the incorporation of a reversible Fmoc-based chromatographic probe, derivatised with a lipophilic group for fast one-step RP purification, to give an overall yield of 9.6%. Analysis by ESI-MS indicated that the product was virtually free of deletion impurities, while RP-HPLC under four different conditions and CZE indicated that the protein was 100 and 84% pure, respectively. The spontaneous folding of Rat Cpnl0 into its biologically active form was found to correlate well with the degree of purity as assessed by chromatography, ESI-MS and sequencing, since 29 (A), 55 (B) and 81% (C) of correctly folded heptameric structure was obtained. The degree of homogeneity was also reflected in the ability of purified Rat Cpnl0 to facilitate the refolding of yeast enolase. © Munksgaard 1996.     

Studies on gliadin related peptides. I. Synthesis,purification and 1H n.m.r. characterization of the pentapeptide H-Tyr-(Gln)3-Pro-OH

The pentapeptide H-Tyr-(Gln)₃-Pro-OH has been recently postulated to be the basic repetitive unit of a sequential polypeptide contained in wheat bread α-gliadins, which are believed to be toxic factors in coeliac disease, gluten-dependent enteropathy. Solid-phase synthesis, purification and ¹H n.m.r. characterization in water solution of this peptide are described.     

Issues related to targeted delivery of proteins and peptides

While modern genomic and proteomic technology enables rapid screening of novel proteins and peptides as potential drug candidates, design of delivery systems for these biologics remains challenging especially to achieve site-specific pharmacological actions. This article discusses the issues associated with targeted delivery of protein and peptide drugs at physiochemical, physiological, and intracellular levels with a special focus on cancer therapy.     

Chemical synthesis of phosphorylated peptides of the carboxy-terminal domain of human p53 by a segment condensation method

A segment condensation method was developed for the chemical synthesis of large (>90 amino acid) phosphopeptides and was used to produce phosphorylated and non-phosphorylated derivatives of the C-terminal tetramerization and regulatory domains of human p53 (residues 303-393). Efficient condensation synthesis of the 91 residue p53 domain was achieved in two steps. The non-phosphorylated N-terminal segment p53(303-334) (1) and its derivative phosphorylated at serine 315 (1P315), and the non-phosphorylated middle segment p53(335-360) (2), were synthesized as partially protected peptide thioesters in the solid phase using Boc chemistry. The C-terminal segment p53(361-393) (3) and its derivative phosphorylated at serine 392 (3P392) were synthesized as partially protected peptides in the solid phase using Fmoc chemistry. Phosphoamino acid was incorporated into the N-terminal segment (1P315) at the residue corresponding to p53 serine 315 as Boc-Ser(PO₃(Bzl)₂)-OH during synthesis. Serine 392 in the C-terminal segment was selectively phosphorylated after synthesis by phosphitylation followed by oxidation. A derivative phosphorylated at serine 378 was synthesized in a one-step condensation of the unphosphorylated N-terminal segment (1) and the phosphorylated long C-terminal segment p53(335-393) (2-3P378). Yields of the ligated peptides after removal of the protecting groups and HPLC purification averaged 60% for the first condensation and 35% for the second condensation. All five p53 peptides exhibited monomer-tetramer association as determined by analytical ultracentrifu-gation. Circular dichroism spectroscopy revealed that phosphorylation at Ser315 increased the α-helical content, which was abolished when Ser392 also was phosphorylated, suggesting an interaction between N-terminal and C-terminal residues of the C-terminal domain of p53. © Munksgaard 1996.     

Photochemical conjugation of peptides to carrier proteins using 1,2,3-thiadiazole-4-carboxylic acid Immunoreactivity of free C-terminal epitope with specific antibodies

A new heterobifunctional cross-linking reagent, 1,2,3-thiadiazole-4-carboxylic acid, for the photochemical conjugation of peptides to proteins is described. The title compound can be coupled directly to a protected peptide resin during solid-phase peptide synthesis (SPPS) using standard coupling procedures. The probe is stable to TFA deprotection/cleavage mixtures containing ethanedithiol commonly used in Fmoc-SPPS. Furthermore, tritium may easily be introduced into the thiadiazole ring by base-catalyzed hydrogen-exchange. Upon irradiation at 245-300 nm, parent 1,2,3-thiadiazole rapidly eliminates N₂, generating very reactive thioketene which reacts with amines to give a thioamide in high yield, even when the photolysis is carried out in hydroxylic solvents. In order to investigate the potential of the title compound as a heterobifunctional cross-linking reagent a model study with angiotensin II (AII) was conducted. The photoreactive peptide N^α-4-carbonyl-1,2,3-thiadiazole-AII (TDA-AII) was synthesized by Fmoc-SPPS and conjugated to bovine serum albumin (BSA) by photolysis at 245 and 300 nm. By use of a capture competition ELISA, the C-terminal Pro-Phe epitope of photoconjugated AII with the sequence DRVYIHPF was shown to bind specifically to antiAII antibodies (anti-AII abs), although antibodies against both the C- and N-terminal epitopes were present in the assay. A dipeptide His-Leu carboxy-extension form of AII, angiotensin I (AI), only bound to anti-AII abs at 100-200 times higher concentrations, showing that the C-terminal epitope was blocked by the dipeptide. © Munksgaard 1996.     

A systematic approach to the solid-phase synthesis of linear and cyclic pseudopeptide libraries containing ψ[CH2NH] amide bond surrogates

A systematic approach has been adopted for the synthesis and characterization of a series of linear and cyclic pseudopeptide mixtures containing the ψ[CH₂NH] amide replacement. The parent structures were based on biologically relevant compounds including an enkephalin analog, H-Tyr-d -Ala-Gly-Phe-Leu-OH, and an Arg-Gly-Asp peptide sequence. The linear mixtures containing 4 and 64 pseudopeptide components with 1, 2 or 3 amide bond surrogates were synthesized using Boc-SPPS. The amount of desired linear pseudopeptides in the mixtures ranged from 67 to 90% as determined by integration of HPLC peak areas. Comparative studies indicated: (i) racemization is not a problem in the synthesis of pseudopeptide mixtures containing the ψ[CH₂NH] surrogate; and (ii) protection of the ψ[CH₂NH] surrogate with a benzyloxycarbonyl group during the synthesis is beneficial. Cyclic mixtures containing 4 and 256 cyclic pseudopeptide components with a single amide bond surrogate were synthesized using a resin-bound cyclization approach featuring side-chain attachment of Boc-Asp-OFm to the solid support. Cyclization kinetic studies revealed that the newly developed HATU coupling reagent provided a fast cyclization rate for a pseudopeptide mixture and that the position of the reduced peptide bond within a peptide mixture had only a small effect on the cyclization rates of the mixture. Pseudopeptide libraries permit the more efficient bioassay of complex structures and can also be used to reveal more rapidly trends in physicochemical variables. For example, we observed that the expected increase in hydrophilicity with ψ[CH₂NH] substitutions during RP-HPLC analysis did not continue with several such replacements. © Munksgaard 1997.     

Solid-phase synthesis,conformational analysis,and biological activity of AVRp elicitor peptides of the fungal tomato pathogen Cladosporium falvum

The race-specific peptide elicitor AVR9 of the fungal pathogen Cladosporium fulvum specifically induces a hypersensitive response in tomato genotypes carrying the complementary resistance gene Cf-9. The total chemical syntheses of this 28-residue AVR9 peptide containing three disulfide bonds, and of three mutant peptides [R8K]AVR9, [F10A]AVR9 and [F21A]AVR9, have been accomplished. The syntheses were carried out using a stepwise solid-phase approach based on tBoc chemistry. The disulfide bridges were formed by air oxidation. The correctness of the chemical structure of all folded synthetic peptides was confirmed by combined NMR and MS analyses. The biological activity and a number of physicochemical properties of folded synthetic AVR9 are identical to those of native fungal 28-residue AVR9. The overall conformations of the folded synthetic mutant peptides were comparable to that of synthetic wild-type AVR9 as demonstrated by NMR spectroscopy. Mutant [R8K]AVR9 showed a threefold higher, and mutant [F10A]AVR9 a threefold lower necrosis-inducing activity when compared to synthetic wild-type AVR9. However, mutant [F21A]AVR9 showed hardly any necrosis-inducing activity. Affinity for polyclonal antibodies raised against native fungal AVR9 is positively correlated with the necrosis-inducing activity of the synthetic AVR9 peptides ([R8K]AVR9 > wild-type AVR9 > [F10A]AVR9 > [F21A]AVR9).     

Solid-phase synthesis and conformational studies of helper T cell immunogenic peptides that carry carbohydrate haptens linked to serine

N ^α-Fmoc serine and its corresponding pentafluorophenyl ester were glycosylated with the 1,2-trans peracetates of the disaccharides galabiose and cellobiose. Complete stereoselectivity and 52-75% yields were obtained under boron trifluoride etherate promotion. Lower yields and loss of stereoselectivity were obtained when thioglycosides. trichloroacetimidates or glycosyl bromides were employed as glycosyl donors. The glycosylated building blocks were used in solid-phase synthesis of derivatives of a helper T cell immunogenic peptide consisting of amino acids 52-61 from hen-egg lysozyme. ¹H-NMR spectroscopy in DMSO-d₆ showed that the peptide moiety of the glycopeptides assumed random conformations which were not influenced by glycosylation at different positions.