New methods have been developed for the synthesis of disulfide-bridged homo and hetero peptide dimers (both parallel and antiparallel), as exemplified in the oxytocin and deamino-oxytocin families. The linear sequences were assembled by 9-fluorenyl-methyloxycarbonyl (Fmoc) solid-phase synthesis techniques, with orthogonal protection for the two beta-thiols by appropriate combinations of S-[(N'-methyl-N'-phenylcarbamoyl)sulfernyl] (Snm), S-acetamido-methyl (Acm) and S-2, 4, 6-trimethoxybenzyl (Tmob) groups. Two octapeptide-resins gave rise to four different nonapeptide amides, which were brought together in defined ways to provide access to four of the six possible dimeric products. For each dimer, the first disulfide bond was formed in solution by a directed method, and then the second disulfide bond was formed, without purification of the intermediates, by iodine oxidation. Final isolated yields of the desired pure dimers were in the 20% to 40% range. Biological activities ranged from 0.2% to 6% that of oxytocin, depending on the assay, and were in some cases considerably protracted. These data are consistent with the hypothesis that dimers revert slowly to monomers under the testing conditions.
Human statherin, at low molecular weight (M 5380 Da. 43 amino acid residues) acidic tyrosine-rich phosphoprotein secreted mainly by salivary glands, has been synthesized successfully for the first time following standard solid-phase Fmoc chemistry. Synthesis of this phosphoprotein was accomplished using preformed phosphoserin building blocks. The phosphorylated protein thus synthesized was analyzed and compared with the native molecule and was found to have identical characteristics in its entirety, is evidenced by various analytical methods including mass spectral analysis. Analysis of both the synthetic and native statherin by circular dichroism spectroscopy showed an increase in helicity upon the addition of an organic cosolvent, trifluoroethanol (50%, vol/vol), indicating the presence of potentially amphipathic helical regions. Circular dichroism studies and hydrophobic moment calculations on this synthetic phosphoprotein revealed that the molecule adopts an amphipathic helical conformation at the N-terminus connected to a long poly-L-proline type II segment, which, in turn, is linked to an extended beta-strand. In correlation with previous studies. It appears that the strong binding affinity of statherin for hydroxyapatite can be attributed primarily to the N-terminal sequence, which prefers to adopted helical conformation and provides both electrostatic and hydrogen bonding interactions, thereby inhibiting its mineralization. Production of this highly homogenous synthetic statherin by chemical means may circumvent the prevailing obstacles encountered in conducting its tertiary structural investigations under various physiological conditions.
The binding specificity of a monoclonal antibody (MAb 12) known to recognize the surface antigen of hepatitis B virus (HBsAg) was studied using a positional scanning synthetic combinatorial library. A hexapeptide library totaling more than 30 x 10(6) sequences, made up of 120 mixtures having a single position defined with individual amino acids and the remaining five positions composed of mixtures of amino acids, was screened by competitive enzyme-linked immunosorbent assay (ELISA). This led to the identification of Ac-STTSMM-NH2 (IC50 = 170 nM), which specifically inhibited the interaction between HBsAg and MAb 12. One of the most active individual mixtures from the library was Ac-XXXPXX-NH2; however, none of the individual peptides synthesized containing proline at the fourth position showed significant activity (IC50 > 100,000 nM). To identify the individual peptide(s) responsible for the activity of Ac-XXXPXX-NH2, a bidirectional iterative synthesis and selection process was carried out. A completely different but active peptide sequence was identified (Ac-SVGPPH-NH2, IC50 = 165 nM). The two different hexapeptide sequences were prepared as linear homo- and heterodimer peptides in an attempt to improve the antigenicity. Of the four different sequences prepared, one heterodimer (Ac-STTSMMGGGSVGPPH-NH2) was found by ELISA to have a 10-fold improvement in activity over the two individual hexapeptides, and was equal to the inhibitory activity of the protein antigen. The equilibrium affinity constant of the MAb 12 toward this heterodimer sequence was 50-fold higher than the protein antigen when measured in a biosensor system. Since motifs from these two hexapeptides, namely, -STTS- and -GP-, were located in the primary sequence of the protein (residues 114-120, subtype ad), overlapping hexapeptides of this region were synthesized and assayed. The most active hexapeptide, namely, Ac-TTSTGP-NH2 (IC50 = 2.3 microM), was 10-fold less active than either hexapeptide found from the library. Extending the specific motif sequences to eleven residues resulted in an analog (Ac-STTSTGPSRTC-NH2) having an equilibrium affinity constant similar to the heterodimer. The combined use of positional scanning libraries and the iterative synthesis and selection process in this study illustrates the power of these methods for the identification of novel peptides that inhibit anti-protein antibodies. These methods can be directly applied to the development of improved immunodiagnostics.
The sites of the disulfide bonds in a napin protein isolated from Brassica napus have been identified by proteolytic cleavage and subsequent peptide mapping by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Napins consist of two polypeptide chains containing two and six cysteine residues, respectively, that are held together by disulfide bonds. Upon initial cleavage of native napin by Endo-Lys-C, a disulfide-linked core complex of four peptides was obtained. This core peptide was isolated by reversed-phase HPLC and further digested by thermolysin, and the resulting fragments were identified by MALDI-MS. In a separate set of experiments, intact napin was subjected to proteolysis by thermolysin, and an isolated disulfide-linked peptide of interest was subdigested again using thermolysin. The combined data resulting from these experiments allowed the assignment of the disulfide linkages in a relatively abundant napin isoform, BngNAP1, apart from an ambiguity concerning the adjacent cysteines at positions 14' and 15' of the long chain. Two intermolecular disulfide bonds link Cys10 (short chain) with Cys25' (long chain) and Cys23 with Cys14' (or Cys15'), respectively. The long chain of napin contains two intramolecular disulfide bonds connecting Cys27' with Cys80' and Cys14' (or Cys15') with Cys72'.
The molecular and crystal structures of four peptides containing one L-Trp guest residue in Aib (alpha-aminoisobutyric acid or C-alpha-methyl alanine) host oligopeptide chains have been determined by X-ray diffraction. The peptides are Z-Aib-L-Trp-Aib-OMe, Z-(Aib)(2)-L-Trp-Aib-OMe, Z-(Aib)(3)-L-Trp-Aib-OtBu and Boc-(Aib)(3)-L-Trp-Aib-OMe. Right-handed beta-turns and incipient and fully developed 3(10)-helices are formed in the crystal state by the tri-, tetra- and pentapeptides, respectively. The Trp residue is easily accommodated in these folded structures. The average geometry and preferred conformation for the Trp indolyl side chain are also discussed.
A variety of thiol-containing compounds, in combination with m-cresol, were tested as scavengers in hydrogen fluoride (HF) cocktails that are used to deprotect haloacetylated peptidyl resins. Our results indicate that brome and chloroacetyl moieties on a synthetic peptide remain intact following HF treatment when the HF cocktail contains m-cresol along with either thiophenol, m-thiocresol or 1,2-ethanedithiol. The free thiols prevent the formation of a number of impurities in the preparation of bromo- and chloroacetylated peptides that contain amino acids that could be oxidized in a nonreducing HF environment. Ethvimethylsulfide, however, could not be used with bromoacetylated peptides, but it could be used with chloroacetylated peptides.
We have designed and constructed a multiple automated robotic synthesizer, the MARS. Its novel timing procedure for handling multiple synthetic tasks eliminates unnecessary respite time by keeping the robotic arm in continuous operation. Polypropylene syringes equipped at the bottom with polypropylene frits serve as physically independent reaction vessels. All operations are performed by the robotic arm, which is equipped with a specially designed gripper to hold a syringe and to aspirate and dispense liquid. Typically, the MARS synthesizes concurrently 5 to 15 peptides of different length, and once one peptide is finished it automatically starts the synthesis of the next peptide in the queue, assuring a continuous flow of peptides.
We have developed a functional assay to verify the delivery into the cytoplasm of a 14-amino acid hydrophilic peptide, modified by the incorporation of a palmitoyl-lysine residue into the N- or C-terminal end. This assay is based on the use of a pseudo-substrate sequence for the protein kinase C (PKC) isoenzymes of alpha and beta for the quantification of PKC-mediated tumor necrosis factor (TNF) secretion after T-cell activation by phorbol ester and anti-CD3 MAb. This cellular assay is simple, and it allows for a rapid and comparative study of several peptides. The lipidic analogues of the pseudo-substrate peptide were able to inhibit TNF secretion in intact-activated Jurkat cells, with an EC50 in the 40-60 microM range, whereas the unmodified peptide was not active. Two control lipopeptides were also inactive, demonstrating that the palmitoyl-lysine group had no effect by itself. This study confirms that the modification of a relatively long peptide by the insertion of a palmitoyl-lysine into the N- or C-terminal end is sufficient to allow entry into intact cells.
We recently described the local destabilizing effect of systematic double D-amino acid replacements for characterization of amphipathic helices in peptides. The objective of this study was to determine the destabilizing effect of two adjacent D-amino acids incorporated into the center of a single-stranded amphipathic alpha-helix by hydrogen exchange and guanidine hydrochloride denaturation studies in trifluoroethanol (TFE)/water. Data from guanidine hydrochloride titration experiments in the presence of 30% TFE suggest that double D-amino acid replacements at the center of the helix destabilize the secondary structure by 4.5 kJ/mol. While the exchange rate for one backbone proton was found to vary by a factor of 10 at the replacement position, the remaining backbone protons are not markedly influenced by double D-amino acid replacement. These results confirm the hypothesis that the energy of -4.5 kJ/mol per residue is a major contribution to the stability of helical peptides in water and in solvent mixtures of TFE/water.
Intercellular junctions present a formidable challenge for the paracellular delivery of drugs. Cadherins are calcium-dependent cell-cell adhesion molecules, which are responsible for the formation and regulation of these junctions. Anti-E-cadherin monoclonal antibody can bind to E-cadherin (uvomorulin) and inhibit cell-cell adhesion through the inhibition of cadherin-cadherin interactions. The objective of this study was to utilize this monoclonal anti-E-cadherin antibody to map the extracellular domains of E- and N-cadherin. This was accomplished by using two different enzyme-linked immunosorbent assays (ELISAs), a regular indirect ELISA and an immobilized-peptide ELISA. Two peptides from each extracellular domain were recognized by this anti-E-cadherin antibody. By mapping the extracellular domains of cadherins, peptides that have discrete roles in cell-cell adhesion can be identified. This will aid in the design of synthetic peptides that can modulate intercellular junctions to improve drug delivery.
The c-Jun transcription factor contains a transactivation domain that belongs to the "acidic" type of transcription activator. To determine the secondary structure of the Jun activation domain, we synthesized a peptide corresponding to amino acids 61 to 98 of c-Jun. Jun N-terminal kinases are able to phosphorylate Ser-63 and Ser-73 in vivo, which dramatically increases the transactivation potential of Jun. As this phosphorylation event may influence the secondary structure, we synthesized a second peptide containing two phosphoserine groups instead of serine in positions 63 and 73. Secondary structure predictions did not show potential for the peptides to adopt any stable, dominating conformation. Both peptides were purified and analyzed by circular dichroism spectroscopy. The peptides appeared to be flexible and essentially unstructured in aqueous solution. At acidic pH, we observed a decrease in the negative ellipticity at 202 nm, suggesting that some ordered structure might be present under these conditions. alpha-Helical conformation, as a dominating secondary structure, was induced in the presence of trifluoroethanol, and there was no significant difference between the unphosphorylated and phosphorylated peptides.
Three different peptides, A13, A14x2 and A20, related to the Arc repressor from Salmonella phage P22, were synthesized. They all contained the 13 N-terminal residues of Arc known to form a beta-sheet interacting with the operator OArc. In the case of A20, the tripeptide Lys-Trp-Lys was added in the C-terminal position because of its propensity to increase affinity to DNA. The interaction of the three peptides with OArc and with other related (OMnt from the same phage) and unrelated oligonucleotides was followed using circular dichroism, filter binding assays and DNaseI protection experiments. While Kd = 10(-9) M for the protein, 8.7 x 10(-5) M and 7.7 x 10(-6) M Kd values were obtained for A14x2 and A20 interacting with OArc.
The original trimethylsilyl bromide (TMSBr) method of peptide resin deprotection and cleavage has been modified for convenience and applicability to larger peptides. Equal amounts of a 66-residue test peptide resin were cleaved by the standard hydrogen fluoride (HF) procedure, the original TMSBr method and the modified TMSBr method. The peptide profile from the original TMSBr cleavage procedure showed multiple products and a lower overall yield. In contrast, the modified TMSBr procedure gave high yields of crude products comparable in purity to those obtained by HF cleavage.
Plant defensins are a class of cysteine-rich peptides of which several members have been shown to be potent inhibitors of fungal growth. A series of overlapping 15-mer peptides based on the amino acid sequence of the radish antifungal protein Rs-AFP2 have been synthesized. Peptides 6, 7, 8 and 9, comprising the region from cysteine 27 to cysteine 47 of Rs-AFP2 showed substantial antifungal activity against several fungal species (minimal inhibitory concentrations of 30-60 micrograms/mL), but no activity towards bacteria (except peptide 6 at 100 micrograms/mL). The active peptides were shown to be sensitive to the presence of cations in the medium and to the composition and pH of the medium. When present at a subinhibitory concentration (20 micrograms/mL), peptides 1, 7, 8 and 10 potentiated the activity of Rs-AFP2 from 2.3-fold to 2.8-fold. By mapping the characteristics of the active peptide on the structure of Rs-AFP2 as determined by nuclear magnetic resonance, the active region of the antifungal protein appears to involve beta-strands 2 and 3 in combination with the loop connecting those strands. A cyclized synthetic mimic of the loop, cysteine 36 to cysteine 45, was shown to have antifungal activity. Substitution of tyrosine 38 by alanine in the cyclic peptide substantially reduced the antifungal activity, indicating the importance of this residue for the activity of Rs-AFP2 as demonstrated carrier by mutational analysis.
We have synthesized a hydrophilic crosslinked aminoalkyl polydimethylacrylamide-beaded support upon which peptides have been assembled using standard Fmoc chemistry in automated batch-wise equipment. The resin was prepared by the free radical-initiated co-polymerization of N,N-dimethylacryl-amide, N,N'-bisacrylyl-1,3-diaminopropane and a functional monomer N-methacrylyl-1,3-diaminopropane hydrochlorid. After coupling of N-alpha-tert-butyloxycarbonyl-glycine (Boc-glycine), amino acid analyses gave resin loading capacities of 0.66 mmol/g. The resulting polymer was highly swollen by polar solvents including aqueous buffers and had an exclusion limit of 50 kDa for soluble proteins. This resin was found to be an excellent support for peptide synthesis using Fmoc chemistry. Typical purities of crude peptides were 80%-95%, including sequences that failed on conventional polystyrene resins.
The formylpeptides formyl-L-methionyl-L-leucyl-L-N-methylphenylalanine methyl ester 1, formyl-L-methionyl-L-leucyl-L-2-oxy-3-phenylpropionic acid methyl ester 2 and formyl-L-methionyl-L-leucyl-L-2-aminoxy-3-phenylpropionic acid methyl ester 3 were synthesized to investigate the role of the amide bond at position 3 in biological activity in human neutrophiles. Our results indicate that this amide bond is required for optimal chemotactic activity, but not for superoxide anion production.
Multiple cyclic antigen peptides (McAPs) are dendrimers that have branched, multiple closed-chain architectures. We describe an approach for their stepwise, solid-phase synthesis that permits a self-assembly of cyclization reactions of a McAP with four copies of cyclic peptides in solution after their cleavage from the resin with all protecting groups removed. The conceptual framework of our approach is the development of a method favoring intrachain cyclization based on ring-chain tautomerism between an N-terminal Cys and an aldehyde attached to the side chain of Lys to form a loop linked by a thiazolidine ring. The McAP precursor contains an amino Cys(St-Bu) and an internal Lys(Ser). A trialklyphosphine is used to deblock Cys(St-Bu) on the amino terminus and to effect the concomitant thiazolidine formation with the glyoxyl moiety obtained from an oxidative conversion of the Ser on the Lys side chain. Two McAPs, each containing cyclic peptides of 17 and 24 amino acid residues, have been prepared. To evaluate intrachain cyclization yields, a cleavage site as Asp-Pro incorporated at the COOH terminus of each monomeric loop and subsequently released after completion of the cyclization by treatment with formic acid at an elevated temperature. Reversed-phase high-performance liquid chromatography analyses of the liberated cyclic peptide monomer with synthetic standards support the theory that intrachain cyclization is the predominant cyclization pathway and validate the usefulness of this ring-chain tautomerization concept in the self-assembly of cyclic peptides on a branched peptide dendrimer.
Phase clones with affinity for polystyrene/polyurethane magnetic particles were isolated from a 10-men peptide display library. Sequence analysis revealed that 40 out of 80 clones contained the consensus WXXWXXXW. Some of the selected phages showed high surface activity and adsorbed to plastic surfaces even in the presence of blocking agents or surfactants. Covalent attachment of a synthetic peptide (KG), carrying one of the selected sequences to alkaline phosphatase (AP) or bovine serum albumin (BSA) enhanced binding of AP to a wide range of materials and improved the ability of BSA to prevent binding of antibodies and phages to polystyrene. Interestingly, the WXXW/XXXW motif occurs in the beta- and gamma-chains of the natural "adhesive" protein fibrinogen, and a synthetic peptide carrying the gamma-chain 369-376 sequence turned out to have essentially the same binding properties as the KG peptide. Furthermore, adsorption in different types of polystyrene was similar for AP carrying either the KG or gamma-chain peptide intact fibrinogen and plasmin-generated fragment D1. The latter fragment contains two copies of the WXXWXXXW motif but lacks the alpha-chain: protuberances previously implicated in fibrinogen adsorption. Thus, our study may have revealed a hitherto unknown structural determinant for fibrinogen's adsorptivity, located in the 13-kDa C terminal region of the gamma-chain.
An 18-residue-long fragment of vasoactive intestinal polypeptide [VIP(11-28)-NH2] that is known to be analgesic was synthesized by solid-phase t-Boc methodology on a 4-methylbenzhydrylamine resin. Circular dichroism spectroscopy gave evidence that the peptid acquires about 60% helical structure in 50/50 methanol/phosphate buffer, pH 6.0, and 65% (+/-5%) helicity in 80/20 methanol/phosphate buffer pH 7.0, A 2.0 mM solution of VIP (11-28) NH2 in 80% methanol, 20% phosphate buffer pH 7.0 was subjected to 2-dimensional nuclear magnetic resonance (NMR) studies The NMR results suggested formation of an extended helical structure extending from residue 11 to 27 essentially the same region found to be helical in a VIP(1-28)-NH2 and log. This finding suggests that the sequence required for analgesia assumes a helical structure at the receptor.
