The synthesis and spectroscopic analysis of the neurotoxic prion peptide 106-126: Comparative use of manual Boc and Fmoc chemistry

Summary A peptide corresponding to residues 106–126 of the human prion protein (PrP) possesses the neurotoxic and amyloidogenic properties of the infectious form of the parental protein. This peptide is now identified as a ‘difficult sequence’ and synthesis using conventional manual Fmoc chemistry was unsuccessful with acylation terminating at a central core of hydrophobic amino acids. The use of tetramethylfluoroformamidinium hexafluorophosphate and 1-methyl-2-pyrrolidone as anti-aggregatory agents in the coupling steps improved the synthesis but still resulted in an incomplete peptide. The incorporation ofN-(2-hydroxy-4-methoxybenzyl)protection at glycine residues 119 and 124 enabled synthesis of the full length peptide in low yield. Synthesis using Boc chemistry within situ neutralisation gave the full length peptide in high yield.     

A New Approach for the Efficient Synthesis of Oligodeoxyribonucleotides Containing the Mutagenic DNA Modification 7,8-Dihydro-8-oxo-2′-deoxyguanosine at Predefined Positions

Abstract

A combination of H-phoshonate and phosphoramidite chemistry has been applied for the automated solid-phase synthesis of oligodeoxyribonucleotides containing 7, 8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) residues at predefined positions. The unmodified part of the oligomers has been synthesized by using protected standard phosphoramidites, for the incorporation of 8-oxodG the synthon 2-N-acetyl-5′-0-(4,4′-dimethoxytrityl)-7,8-dihydro-2′-deoxyguanosin-8-one-3′-H-phosphonate, prepared in a five step synthesis via 8-bromo-2′-deoxyguanosine, has been used. This approach combines the advantages of both DNA synthesis strategies in that a high yield of full length oligomers is obtained and unreacted, protected 8-oxodG monomers can be recycled, respectively.     

Influenza A virus protein PB1-F2: synthesis and characterization of the biologically active full length protein and related peptides.

Recently the discovery of a novel 87 amino acid influenza A virus (IAV) protein, named PB1-F2, has been reported that originates from an alternative reading frame in the PB1 polymerase gene and is encoded in most of the known human IAV isolates. Using optimized protocols, full length biologically active sPB1-F2 and a number of fragments have been synthesized by following either the standard elongation SPPS method or by native chemical ligation of unprotected N- and C-terminal peptide fragments at the histidine and cysteine residues located in position 41 and 42 of the native sequence, respectively. The ligation procedure afforded the most efficient synthesis of sPB1-F2 and facilitated the generation of various mutants of sPB1-F2 from pre-synthesized peptide fragments. During the synthesis of sPB1-F2, the formation of succinimide and subsequent conversion to the piperidine derivative at the aspartic acid residue in position 23 was observed. This reaction was forestalled by applying specific modifications to the SPPS protocol. The chain-elongation SPPS protocol is optimal for producing small peptides of sPB1-F2, their derivatives and precursors for a subsequent ligation protocol, while the full length protein, mutants and labelled derivatives are more conveniently and efficiently synthesized by SPPS protocols that include native chemical ligation. The molecular identity of sPB1-F2 was confirmed by peptide mapping, mass spectrometry, N-terminal sequencing, (1)H NMR spectroscopy and Western blot analysis. The latter analysis afforded direct evidence of the inherent tendency of sPB1-F2 to undergo oligomerization, a phenomenon observed both for full length sPB1-F2 and fragments thereof, as well as for its full length viral counterpart. Our synthesis protocols open the field for multiple biological and structural studies on sPB1-F2 that, similar to the molecule expressed in an IAV context, induces apoptosis and interacts with membranes in vitro and in vivo, as shown in previous studies.     

Site-specific incorporation of fluorotyrosines into the R2 subunit of E. coli ribonucleotide reductase by expressed protein ligation

Expressed protein ligation (EPL) allows semisynthesis of a target protein with site-specific incorporation of probes or unnatural amino acids at its N or C termini. Here, we describe the protocol that our lab has developed for incorporating fluorotyrosines (F(n)Ys) at residue 356 of the small subunit of Escherichia coli ribonucleotide reductase using EPL. In this procedure, the majority of the protein (residues 1-353 out of 375) is fused to an intein domain and prepared by recombinant expression, yielding the protein in a thioester-activated, truncated form. The remainder of the protein, a 22-mer peptide, is prepared by solid-phase peptide synthesis and contains the F(n)Y at the desired position. Ligation of the 22-mer peptide to the thioester-activated R2 and subsequent purification yield full-length R2 with the F(n)Y at residue 356. The procedure to generate 100 mg quantities of Y356F(n)Y-R2 takes 3-4 months.     

Solid-phase synthesis of reactive peptide crosslinker by selective deprotection

An effective and simple strategy for preparing peptide crosslinkers is described. An MMP-13 degradable peptide QPQGLAK-NH(2) was prepared on the solid-phase using Fmoc chemistry. The peptide crosslinker was synthesized on-bead by the coupling reaction between acrylic acid and the amine groups of glutamine and lysine residues. The synthetic procedure employed the acid-labile Fmoc-Lys (Mtt)-OH and base-labile Fmoc-AA-OH derivatives. Selective deprotection, of -Mtt and -Fmoc groups on-bead, freed the amine end-groups on glutamine and lysine residues for coupling reaction with acrylic acid while maintaining the peptide attached to the resin. Subsequent cleavage from the resin yielded a peptide crosslinker with two unsaturated acrylate end-groups with high yield and purity. This method can be generally employed for the synthesis of a wide range of peptides with one or more reactive groups for grafting in the fabrication of biomimetic scaffolds in tissue engineering applications.     

Synthesis of full length PB1-F2 influenza A virus proteins from 'Spanish flu' and 'bird flu'.

The proapoptotic influenza A virus PB1-F2 protein contributes to viral pathogenicity and is present in most human and avian isolates. Previous synthetic protocols have been improved to provide a synthetic full length H1N1 type PB1-F2 protein that is encoded by the 'Spanish flu' isolate and an equivalent protein from an avian host that is representative of a highly pathogenic H5N1 'bird flu' isolate, termed SF2 and BF2, respectively. Full length SF2, different mutants of BF2 and a number of fragments of these peptides have been synthesized by either the standard solid-phase peptide synthesis method or by native chemical ligation of unprotected N- and C-terminal peptide fragments. For SF2 chemical ligation made use of the histidine and the cysteine residues located in positions 41 and 42 of the native sequence, respectively, to afford a highly efficient synthesis of SF2 compared to the standard SPPS elongation method. By-product formation at the aspartic acid residue in position 23 was prevented by specific modifications of the SPPS protocol. As the native sequence of BF2 does not contain a cysteine residue two different mutants of BF2 (Y42C) and BF2 (S47C) with appropriate cysteine exchanges were produced. In addition to the full length molecules, fragments of the native sequences were synthesized for comparison of their physical characteristics with those from the H1N1 human isolate A/Puerto Rico/8/34 (H1N1). All peptides were analyzed by mass spectrometry, (1)H NMR spectroscopy, and SDS-PAGE. The protocols allow the synthesis of significant amounts of PB1-F2 and its related peptides. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Identification of Conus amadis disulfide isomerase: minimum sequence length of peptide fragments necessary for protein annotation

Protein disulfide isomerase (PDI) has been identified in a protein extract from the venom duct of the marine snail C. amadis. In-gel tryptic digestion of a thick protein band at approximately 55 kDa yields a mixture of peptides. Analysis of tryptic fragments by MALDI-MS/MS and LC-ESI-MS/MS methods permits sequence assignment. Three tryptic fragments yield two nine residue sequences (FVQDFLDGK and EPQLGDRVR ) and an eleven residue sequence (DQESTGALAFK ). Database analysis using peptides and were consistent with the sequence of PDI and peptide appears to be derived from a co-migrating protein. In identifying proteins based on the characterization of short peptide sequences the question arises about the reliability of identification using peptide fragments. Here we have also demonstrated the minimum length of peptide fragment necessary for unambiguous protein identification using fragments obtained from the experimentally derived sequences. Sequences of length > or =7 residues provide unambiguous identification in conjunction with protein molecular mass as a filter. The length of sequence necessary for unambiguous protein identification is also established using randomly chosen tryptic fragments from a standard dataset of proteins. The results are of significance in the identification of proteins from organisms with unsequenced genomes.     

Hyperpolarization-activated cation current Ih of dentate gyrus granule cells is upregulated in human and rat temporal lobe epilepsy

Phosphorylation of α-synuclein at Ser-129 is of crucial relevance to Parkinson's disease and related synucleinopathies. Here we provide biochemical evidence that PLK2 and to a lesser extent PLK3 are superior over CK2, as catalysts of Ser-129 phosphorylation both in full length α-synuclein and in a peptide reproducing the C-terminal segment of the protein. By using substituted peptides we also show that the sequence surrounding Ser-129 is optimally shaped for undergoing phosphorylation by PLK2, with special reference to the two acidic residues at positions n-3 (Glu-126) and n+2 (Glu-131) whose replacement with alanine abrogates phosphorylation.     

景东湍蛙抗菌肽jindongenin-1d的分析和活性测定

新型抗菌肽研究有助于解决细菌对抗生素的耐药性问题。本研究用SMART技术构建了景东湍蛙Amolops jingdongensis皮肤的全长cDNA文库。通过单克隆和测序获得一个抗菌肽cDNA序列,序列比对结果表明其属于jindongenin-1家族,命名为jindongenin-1d。其cDNA序列全长321bp,编码含66个氨基酸残基的多肽。该多肽包括1个信号肽和1个前肽序列。成熟jindongenin-1d多肽包含24个氨基酸残基,理论分子量为2 709.38,等电点为9.24。对人工合成的jindongenin-1d蛋白进行了抗菌和溶血活性分析,结果表明jindongenin-1d对所选的革兰氏阴性菌、革兰氏阳性菌和真菌均有显著抑制作用,同时有弱溶血活性。本研究结果有助于进一步了解两栖动物皮肤分泌物活性物质的多态性和新型抗感染药物的设计。     

Radiolabeling of RGD peptide and preliminary biological evaluation in mice bearing U87MG tumors

2-[(18)F]Fluoroethyl azide ([(18)F]FEA) and terminal alkynyl modified propioloyl RGDfK were selected in this study. [(18)F]FEA was prepared by nucleophilic radiofluorination of 2-azidoethyl 4-toluenesulfonate with radiochemical yield of 71 ± 4% (n = 5, decay-corrected). We assessed the various conditions of the CuAAC reaction between [(18)F]FEA and propioloyl RGDfK, which included peptide concentration, reaction time, temperature and catalyst dosage. The (18)F-labeled-RGD peptide ([(18)F]F-RGDfK) could be obtained in 60 min by a two-step radiochemical synthesis route, with total radiochemical yield of 60 ± 2% (n = 3, decay-corrected) through click chemistry. [(18)F]F-RGDfK showed high stability in phosphate buffered saline and new-born calf serum. Micro-PET imaging at 1 h post injection of [(18)F]F-RGDfK showed medium concentration of radioactivity in tumors while much decreased concentration in tumors in the blocking group. These results showed that [(18)F]F-RGDfK obtained by click chemistry maintained the affinity and specificity of the RGDfK peptide to integrin α(v)β(3). This study provided useful information for peptide radiofluorination by using click chemistry.     

Peptide sequences for sucrose splitting and glucan binding within Streptococcus sobrinus glucosyltransferase (water-insoluble glucan synthetase).

The gene encoding glucosyltransferase responsible for water-insoluble glucan synthesis (GTF-I) of Streptococcus sobrinus (formerly Streptococcus mutans 6715) was cloned, expressed, and sequenced. A gene bank from S. sobrinus 6715 DNA was constructed in vector pUC18 and screened with anti-GTF-I antibody to detect clones producing GTF-I peptide. Five immunopositive clones were isolated, all of which produced peptides that bound alpha-1,6 glucan. GTF-I activity was found in only two large peptides: one stretching over the full length of the GTF-I peptide and composed of about 1,600 amino acid residues (AB1 clone) and the other lacking about 80 N-terminal residues and about 260 C-terminal residues (AB2 clone). A deletion study of the AB2 clone indicated that specific glucan binding, which is essential for water-insoluble glucan synthesis, was lost prior to sucrase activity with an increase in deletion from the 3' end of the GTF-I gene. These results suggest that the GTF-I peptide consists of three segments: that for sucrose splitting (approximately 1,100 residues), that for glucan binding (approximately 240 residues), and that of unknown function (approximately 260 residues), in order from the N terminus. The primary structure of the GTF-I peptide, deduced by DNA sequencing of the AB1 clone, was found to be very similar to that of the homologous protein from another strain of S. sobrinus.     

Synthesis and properties of the red chromophore of the green-to-red photoconvertible fluorescent protein Kaede and its analogs

Green fluorescent protein (GFP) and homologous proteins possess a unique pathway of chromophore formation based on autocatalytic modification of their own amino acid residues. Green-to-red photoconvertible fluorescent protein Kaede carries His-Tyr-Gly chromophore-forming triad. Here, we describe synthesis of Kaede red chromophore (2-[(1E)-2-(5-imidazolyl)ethenyl]-4-(p-hydroxybenzylidene)-5-imidazolone) and its analogs that can be potentially formed by natural amino acid residues. Chromophores corresponding to the following tripeptides were obtained: His-Tyr-Gly, Trp-Tyr-Gly, Phe-Trp-Gly, Tyr-Trp-Gly, Asn-Tyr-Gly, Phe-Tyr-Gly, and Tyr-Tyr-Gly. In basic conditions they fluoresced red with relatively high quantum yield (up to 0.017 for Trp-derived compounds). The most red-shifted absorption peak at 595 nm was found for the chromophore Trp-Tyr-Gly in basic DMSO. Surprisingly, in basic DMF non-aromatic Asn-derived chromophore Asn-Tyr-Gly demonstrated the most red-shifted emission maximum at 642 nm. Thus, Asn residue may be a promising substituent, which can potentially diversify posttranslational chemistry in GFP-like proteins.     

Synthesis of a membrane protein with two transmembrane regions.

A membrane protein with two transmembrane domains was synthesized by means of the thioester method. The F1F0 ATP synthase subunit c (Sub.c), which consists of 79 amino acid residues (MW 8257), was chosen as a target. For synthetic purposes, two building blocks, Boc-[Lys34(Boc)]-Sub.c(1-38)-SCH2CH2CO-Ala and Sub.c(39-79), were synthesized via solid-phase methods using Boc chemistry. RP-HPLC purification conditions for the transmembrane peptide were examined. As a result, a combination of a mixture of formic acid, 1-propanol and water with a phenyl column was found to be useful for separating the transmembrane peptide. The purified building blocks were condensed in DMSO in the presence of silver chloride, 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt), N,N-diisopropylethylamine to give the product, Sub.c, after removal of Boc groups (yield 16%). The yield of the condensation reaction could be improved to 23% by raising the reaction temperature to 50 degrees C, and to 26% when a mixture of chloroform and methanol was used as a solvent.     

Transient production and secretion of human transforming growth factor TGF-beta 2

Transient transfection of simian COS cells with a recombinant plasmid encoding the human transforming growth factor TGF-beta 2 precursor protein results in the production of a latent, biologically inactive protein. Upon acidification, recombinant TGF-beta 2 exhibits full biological activity, including inhibition of mink lung epithelial cell growth, stimulation of anchorage-independent growth of murine embryonic fibroblasts, and competition for TGF-beta receptor binding. Further analysis of conditioned media with antiserum to either a pro- [amino acid (aa) residues 1-220] or mature [aa 297-414] peptide of the TGF-beta 2 precursor suggests that TGF-beta 2, similar to TGF-beta 1 production in Chinese hamster ovary cells [Gentry et al., Mol. Cell. Biol. 7 (1987) 3418-3427], is initially synthesized as a larger precursor protein which is proteolytically cleaved to yield the mature 112-aa transforming growth factor.     

A convenient incorporation of conformationally constrained 5,5-dimethylproline into the ribonuclease A 89-124 sequence by condensation of synthetic peptide fragments.

The presence of l-5,5-dimethylproline (dmP) within an amino acid sequence results in the formation of an X-dmP peptide bond predominantly locked in a cis conformation. However, the common use of this unnatural amino acid has been hampered by the difficulty of the economical incorporation of the dmP residue into longer peptide segments due to the steric hindrance imposed by the dimethyl moieties. Here, we describe synthesis of the C-terminal 36-residue peptide, corresponding to the 89-124 sequence of bovine pancreatic ribonuclease A (RNase A), in which dmP is incorporated as a substitute for Pro93. The peptide was assembled by condensation of protected 5- and 31-residue peptide fragments, which were synthesized by solid-phase peptide methodology using fluorenylmethyloxycarbonyl chemistry. We focused on optimizing the synthesis of the Fmoc-Ser(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-dmP-OH pentapeptide (residues 89-93) with efficient acylation of the sterically hindered dmP residue. In a comparative study, the reagent O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate was found to be superior to bromo-tris-pyrrolidino-phosphonium hexafluorophosphate and tetramethylfluoroformamidinium hexafluorophosphate for the synthesis of the -Tyr(tBu)-dmP- peptide bond in solution as well as on a resin.     

Novel prodrug approach to photodynamic therapy: Fmoc solid-phase synthesis of a cell permeable peptide incorporating 5-aminolaevulinic acid

The first example of the synthesis of a peptide incorporating 5-aminolaevulinic acid (5-ALA) using standard Fmoc solid-phase chemistry is reported. The synthesised peptide contains residues 52-58 of the cell-permeable peptide Penetratin and represents a prototype for the enhanced topical delivery of 5-ALA using such oligopeptide vectors. Effective intracellular conversion of the peptide to the endogenous photosensitiser, protoporphyrin IX, is observed in PAM212 cells, thus demonstrating the potential of this approach for the development of novel peptide prodrugs for use in photodynamic therapy.     

A mutant of human insulin-like growth factor II (IGF II) with the processing sites of proinsulin. Expression and binding studies of processed IGF II.

A mutant of human insulin-like growth factor II (IGF II) was constructed by site-directed mutagenesis: the nucleotides coding for Ser33 and Ser39 were changed to yield Arg and Lys, respectively, thus creating two pairs of basic residues, Arg-Arg and Lys-Arg, as flanking sequences of the remaining C domain. [Arg33, Lys39]IGF II was expressed in NIH-3T3 cells as a processed two-chain peptide with a deletion of amino acid residues 37-40 and crosslinked by three disulfide bonds. This des(37-40)[Arg33]IGF II showed 3.6-fold and 7.4-fold reduced affinities to the type 1 and type 2 IGF receptor overexpressing cells, respectively, whereas the thymidine incorporation potency was the same as that of wild-type IGF II. We speculate that the discrepancy between the reduced binding to the type 1 IGF receptor and the full thymidine incorporation potency is due to the 6.1-fold reduced affinity of the expressed mutant to the co-expressed IGF binding protein 3 (IGFBP-3). The results suggest that des(37-40)[Arg33]IGF II assumes a conformation very similar to IGF II, and that the entire length of the C domain is not essential for biological activity.     

Expressed protein ligation. Method and applications.

The introduction of noncanonical amino acids and biophysical probes into peptides and proteins, and total or segmental isotopic labelling has the potential to greatly aid the determination of protein structure, function and protein-protein interactions. To obtain a peptide as large as possible by solid-phase peptide synthesis, native chemical ligation was introduced to enable synthesis of proteins of up to 120 amino acids in length. After the discovery of inteins, with their self-splicing properties and their application in protein synthesis, the semisynthetic methodology, expressed protein ligation, was developed to circumvent size limitation problems. Today, diverse expression vectors are available that allow the production of N- and C-terminal fragments that are needed for ligation to produce large amounts and high purity protein(s) (protein alpha-thioesters and peptides or proteins with N-terminal Cys). Unfortunately, expressed protein ligation is still limited mainly by the requirement of a Cys residue. Of course, additional Cys residues can be introduced into the sequence by site directed mutagenesis or synthesis, however, those mutations may disturb protein structure and function. Recently, alternative ligation approaches have been developed that do not require Cys residues. Accordingly, it is theoretically possible to obtain each modified protein using ligation strategies.     

alpha-Chymotrypsin as the catalyst for peptide synthesis.

alpha-Chymotrypsin (EC 3.4.21.1)-catalysed syntheses of peptides were performed with various N-acylated amino acid or peptide esters as donors, and amino acid derivatives, peptides or their derivatives as acceptors. Under optimal conditions the synthesis was almost quantitative. As acceptor nucleophiles, free amino acids or the ester derivatives were inadequate, but amino acid amides or hydrazides, di- or tri-peptides, or the amides, hydrazides and esters of the peptides were useful. The nucleophile specificity for synthesis was markedly similar to the leaving-group specificity in hydrolysis; hydrophobic or bulky amino acid residues were most effecient at both P1' and P2' positions [notation of Schechter & Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162], but L-proline as well as D-amino acid residues were the worst choices. The synthesis was further dependent on the solubility of the products synthesized; a higher yield of products was expected with lower solubility. As donor esters, good substrates were all useful. Accordingly, fragment condensation was possible by using N-acylated peptide esters and various peptides. The present study suggested that alpha-chymotrypsin may become a useful tool for peptide synthesis.     

Understanding interactions of gastric inhibitory polypeptide (GIP) with its G-protein coupled receptor through NMR and molecular modeling.

Gastric inhibitory polypeptide (GIP, or glucose-dependent insulinotropic polypeptide) is a 42-amino acid incretin hormone moderating glucose-induced insulin secretion. Antidiabetic therapy based on GIP holds great promise because of the fact that its insulinotropic action is highly dependent on the level of glucose, overcoming the sideeffects of hypoglycemia associated with the current therapy of Type 2 diabetes. The truncated peptide, GIP(1-30)NH2, has the same activity as the full length native peptide. We have studied the structure of GIP(1-30)NH2 and built a model of its G-protein coupled receptor (GPCR). The structure of GIP(1-30)NH2 in DMSO-d6 and H2O has been studied using 2D NMR (total correlation spectroscopy (TOCSY), nuclear overhauser effect spectroscopy (NOESY), double quantum filtered-COSY (DQF-COSY), 13C-heteronuclear single quantum correlation (HSQC) experiments, and its conformation built by MD simulations with the NMR data as constraints. The peptide in DMSO-d6 exhibits an alpha-helix between residues Ile12 and Lys30 with a discontinuity at residues Gln19 and Gln20. In H2O, the alpha-helix starts at Ile7, breaks off at Gln19, and then continues right through to Lys30. GIP(1-30)NH2 has all the structural features of peptides belonging to family B1 GPCRs, which are characterized by a coil at the N-terminal and a long C-terminal alpha-helix with or without a break. A model of the seven transmembrane (TM) helices of the GIP receptor (GIPR) has been built on the principles of comparative protein modeling, using the crystal structure of bovine rhodopsin as a template. The N-terminal domain of GIPR has been constructed from the NMR structure of the N-terminal of corticoptropin releasing factor receptor (CRFR), a family B1 GCPR. The intra and extra cellular loops and the C-terminal have been modeled from fragments retrieved from the PDB. On the basis of the experimental data available for some members of family B1 GPCRs, four pairs of constraints between GIP(1-30)NH2 and its receptor were used in the FTDOCK program, to build the complete model of the GIP(1-30)NH2:GIPR complex. The model can rationalize the various experimental observations including the potency of the truncated GIP peptide. This work is the first complete model at the atomic level of GIP(1-30)NH2 and of the complex with its GPCR.