Synthesis of human pancreatic growth hormone-releasing factor and two omission analogs by segment-coupling method in aqueous solution

The human growth hormone-releasing factor (GRF) peptides [GlyS¹⁵]-GRF-(1–15) (IV), trifluoroacetyl-GRF-(20–44) (VI), trifluoroacetyl-GRF-(18–44) (VIII), and trifluoroacetyl-GRF-(16–44) (X) were synthesized by the solid-phase method. Each of the peptides was reacted with citraconic anhydride and the trifluoroacetyl group was removed by reaction with 10% hydrazine in water. The citraconylated GRF-(1–15) peptide was coupled to the (20–44), (18–44) or (16–44) peptides by reaction with silver nitrate/N-hydroxysuccinimide to give GRF-(1–15)-(20–44) (XII), GRF-(1–15)-(18–44) (XIII), or GRF-(1–44), respectively. GRF-(1–44) was shown to stimulate the release of rat growth hormone from rat pituitary cells with an ED₅₀= 8.8 times 10^-11 M. Peptides XII and XIII were inactive, either as agonists or as antagonists of the action of GRF-(1–44).     

Total synthesis of S-carbamoylmethyl bovine apocytochrome c by segment coupling

Three peptide segments corresponding to the complete sequence of the 104 amino acid protein bovine apocytochrome c were synthesized by the solid-phase method. The peptides Ac-[Cys(Cam)^14,17, GlyS²³]-apocytochrome c-(1–23) (I), CF₃CO-[GlyS⁶⁰]-apocytochrome c-(24–60) (II), and CF₃CO-apocytochrome c-(61–104) (III) were purified by chromatography on CM-cellulose, partition chromatography and/or HPLC. Each of the peptides was reacted with citraconic anhydride to block all of the lysine side chains, and the 61–104 peptide was treated with 10% hydrazine to remove the trifluoroacetyl group, to give the corresponding peptides Ia, IIa, and IIIa. Peptides IIa and IIIa were coupled together by reaction with silver nitrate/N-hydroxysuccinimide to give the 24–104 sequence. After removal of the trifluoroacetyl group from the amino terminus, peptide Ia was also coupled. Treatment of the peptide mixture with aqueous acetic acid removed the citraconyl groups, and purification by chromatography on CM-cellulose and HPLC gave a 0.6% yield of [Cys-(Cam)^14,17]-apocytochrome c. The synthetic product was shown to be identical to a sample derived from native bovine cytochrome c by paper or gel electrophoresis, HPLC and by chymotryptic or tryptic map.     

Simultaneous multiple synthesis and selective conjugation of cyclized peptides derived from a surface loop of a meningococcal class 1 outer membrane protein

Starting from the α-(2,4-diniethoxybcnzyl) ester of N-(9-fluorenylniethoxycarbonyl)aspartic acid [Fmoc-Asp-ODmb], side-chain-protected resin-bound Fmoc-peptides containing an N^c-l-(4,4-dimethyl-2,6-dioxocyclohexylidenc)ethyl lysY1 [Lys(Dde)] residue were prepared. The C-terminal dimethoxybenzyl esters of aspartic acid were removed with 1% trifluoroacetic acid and 10% anisole in dichloromethane, followed by Fmoc-cleavage in the usual manner. The resin-bound peptides were then cyclized using 1-benzotriazolyloxy-tris-[N-pyrrolidino]phosphonium hexafluorophosphate (PyBOP) in the presence of N-methylmorpholine. The (dimethyldioxocyclohexylidene)ethyl groups of lysine were removed with 1% hydrazine hydrate in N,N-dimethylacetarnide, and the liberated side-chain amino functions were modified by reaction with pentafluorophenyl S-acetylinercaptoacetate (SAMA-OPfp). Finally, the peptides were side-chain deprotected, with exception of the Lys(SAMA) residue. and cleaved from the solid support with trifluoroacetic acid/anisole/ water, 95/2.5/2.5. Cyclic peptides comprising 7–14 amino acid residues were obtained employing this procedure. As a model conjugation. cyclo[Thr-Asn-Asn-Asn-Leu-Lys(SAMA)-Thr-Lys-Asp] was coupled with bromoacetamide. The same peptide was also coupled with a bromoacetylpeptide to give a well defined peptide1 peptide conjugate. All peptides were conjugated to bromoacetylated tetanus toxoid for immunization purposes.     

Optimized aminolysis conditions for cleavage of N‐protected hydrophobic peptides from solid‐phase resins

Abstract: Solid‐phase synthesis and aminolysis cleavage conditions were optimized to obtain N‐ and C‐terminally protected hydrophobic peptides with both high quality and yield. Uncharged ‘WALP’ peptides, consisting of a central (Leu‐Ala)_n repeating unit (where n = 5, 10.5 or 11.5) flanked on both sides by Trp ‘anchors’, and gramicidin A (gA) were synthesized using 9‐fluorenylmethoxycarbonyl chemistry from either Wang or Merrifield resins. For WALP peptides, the N‐terminal amino acid was capped by coupling N‐acetyl‐ or N‐formyl‐Ala or ‐Gly to the peptide/resin or by formylation of the completed peptide/resin with para‐nitrophenylformate (p‐NPF). N‐Terminal acetyl‐ or formyl‐Ala racemized when coupled as an HOBt‐ester to the resin‐bound peptide, but not when the peptide was formylated with p‐NPF. Racemization was avoided at the last step by completing the peptide with acetyl‐ or formyl‐Gly. For both WALP peptides and gA, cleavage conditions using ethanolamine or ethylenediamine were optimized as functions of solvent, time, temperature and resin type. For WALP peptides, maximum yields of highly pure peptide were obtained by cleavage with 20% ethanolamine or ethylenediamine in 80% dichloromethane for 48 h at 24°C. N‐Acetyl‐protected WALP peptides consistently gave higher yields than those protected with N‐formyl. For gA, cleavage with 20% ethanolamine or ethylenediamine in 80% dimethylformamide for 48 h at 24°C gave excellent results. For both WALP peptides and gA, decreasing the cleavage time to 4 h and increasing the temperature to 40–55°C resulted in significantly lower yields. The inclusion of hexafluoroisopropanol in the cleavage solvent mixture did not improve yields for either gA or WALP peptides.     

Solid-phase synthesis of (tyrosyl-alanyl-glutamyl)n,by segment condensation

(Tyr-Ala-Glu)_n, n= 1–9, were synthesized by segment condensation using the Fmoc/tert-butyl protection strategy and solid-phase techniques. The C-terminal residue was coupled to the resin and the peptides were built out by adding Fmoc-Glu(O-r-Bu)-Tyr(t-Bu)-Ala-OH units. When the desired lengths were reached the peptides were capped with Fmoc-Tyr(t-Bu)-Ala-OH units. Fmoc-Tyr(t-Bu)-Ala-OH and Fmoc-Glu(O-t-Bu)-Tyr(t-Bu)-Ala-OH were synthesized in aqueous solution by the successive addition of N-hydroxysuccinimide esters of Fmoc-Tyr(t-Bu) and Fnioc-Glu(0-t-Bu) to the growing chain. Neither sequential amino acid addition or segment condensation techniques were successful on polystyrene supports. However, the segment condensations were highly successful on kieselguhr-supported polydimethylacrylamide based resins. (Tyr-Ala-Glu)_n, n= 1–9, were tested as inhibitors of the protein [yosine kinase, pp60C^c-src. Inhibition, as measured by IC₅₀ values, increased with increasing size of the peptide.     

Secondary structure analysis of synthetic peptides of the extracellular domain of the human follicle‐stimulating hormone receptor

Abstract: Follicle‐stimulating hormone receptor (FSHR) is a glycoprotein hormone receptor and possesses a large extracellular domain (ECD) instrumental in hormone binding. The ECD is characterized by the presence of leucine‐rich repeat (LRR) structures made up of α‐helices flanked by β‐strands. Our previous studies with the synthetic peptides corresponding to the potentially surface‐oriented regions of the ECD had led to the identification of some of these regions in either FSH‐binding or FSH‐induced cAMP production or both. This study was undertaken with an aim to correlate the findings made in vitro with the secondary structures of the respective peptides. Accordingly, all peptides were screened for their secondary structures in different biochemical environments. This study correlates the observed α‐helical signature with the previously demonstrated activity in signal generation for peptides 15–31 and 216–235 hFSHR, while FSH binding is correlated with the maintenance of β‐sheet structure in peptides 285–300 and 297–310 hFSHR as observed in vitro.     

Amino acid sequence of UP1, an hnRNP-derived single-stranded nucleic acid binding protein from calf thymus

The UP1 single-stranded nucleic acid binding protein from calf thymus (Herrick, G. & Alberts, B.M. (1976) J. Biol. Chem. 251, 2124–2132) has recently been shown to be a proteolytic fragment derived from the A₁ heterogeneous nuclear ribonucleoprotein (hnRNP)(Pandolfo et al. (1985) Nucleic Acids Res. 13, 6577–6590). The NH₂-terminus of the 22 162 dalton UP1 protein appears to be blocked, which suggests that UP1 represents the NH₂-terminal two thirds of this 32 000 dalton hnRNP protein. The complete amino acid sequence for UP1 was derived from automated sequencing of peptides that were purified by HPLC from digests with trypsin, chymotrypsin, Staphylococcus aureus protease, endoproteinase Lys-C, and cyanogen bromide. Trichloroacetic acid precipitation followed by enzymatic digestion in 2 M urea proved to be the best approach for generating UP1 peptides. By carboxymethylating after, rather than before, digestion it was possible to avoid problems associated with the insolubility of the carboxymethylated UP1. All of the resulting peptides in amounts varying from 2 to 15 nmol were coupled to aminopolystyrene prior to solid-phase sequencing. Using these methods, no difficulties were encountered in assigning glutamic acid residues or in completely sequencing peptides that contained up to 25–30 residues. The relative ease with which the UP1 protein was sequenced, requiring only about a year to complete, and the comparatively modest amount of protein required, less than 5 mg, attests to the usefulness of water soluble carbodiimide coupling and solid-phase sequencing for determining the primary structures of proteins. In addition to serving as a basis for determining structural relationships among various mammalian single-stranded nucleic acid binding proteins, the amino acid sequence of UP1 reveals that the A₁ hnRNP protein contains a region of internal sequence homology that apparently corresponds to two independent nucleic acid binding sites.     

Chromogenic and fluorogenic glycosylated and acetylglycosylated peptides as substrates for serine,thiol and aspartyl proteases

We synthesized short chromogenic peptidyl–Arg–p-nitroanilides containing either (Galβ)Ser or (Glcα,β)Tyr at P₂ or P₃ sites as well as O-acetylated sugar moieties and studied their hydrolysis by bovine trypsin, papain, human tissue kallikrein and rat tonin. For comparison, the susceptibility to these enzymes of Acetyl–X–Arg–pNa and Acetyl–X–Phe–Arg–pNa series, in which X was Ala, Phe, Gln and Asn were examined. We also synthesized internally quenched fluorescent peptides with the amino acid sequence Phe⁸–His–Leu–Val–Ile–His–Asn¹⁴ of human angiotensinogen, in which [GlcNAcβ]Asn was introduced before Phe⁸ and/or after His¹³ and ortho-aminobenzoic acid (Abz) and N-[2-, 4-dinitrophenyl]–ethylenediamine (EDDnp) were attached at N- and C-terminal ends as a donor/receptor fluorescent pair. These peptides were examined as substrates for human renin, human cathepsin D and porcine pepsin. The chromogenic substrates with hydrophilic sugar moiety increased their susceptibility to trypsin, tissue kallikrein and rat tonin. For papain, the effect of sugar depends on its position in the substrate, namely, at P₃ it is unfavorable, in contrast to the P₂ position that resulted in increasing affinity, as demonstrated by the higher inhibitory activity of Ac–(Galβ)Ser–Arg–pNa in comparison to Ac–Ser–Arg–pNa, and by the hydrolysis of Ac–(Glcα,β)Tyr–Arg–pNa. On the other hand, the acetylation of sugar hydroxyl groups improved hydrolysis of the susceptible peptides to all enzymes, except tonin. The P^′₄ glycosylated peptide [Abz–F–H–L–V–I–H–(GlcNAcβ)N–E–EDDnp], that corresponds to one of the natural glycosylation sites of angiotensinogen, was shown to be the only glycosylated substrate susceptible to human renin, and was hydrolysed with lower K_m and higher k_cat values than the same peptide without the sugar moiety. Human cathepsin D and porcine pepsin are more tolerant to substrate glycosylation, hydrolysing both the P^′₄ and P₄ glycosylated substrates.     

POSSIBLE MECHANISMS INVOLVED IN THE NATRIURETIC RESPONSE TO ATRIAL NATRIURETIC FACTOR (ANF) AND PROANF 31–67 IN THE RAT

1. The present study was conducted to compare the mechanisms involved in the natriuretic response to atrial natriuretic factor (ANF) and pro ANF 31–67. The peptides were infused intravenously into anaesthetized rats at 10 pmol/min for 40 min. 2. Only ANF produced a significant decrease in arterial pressure; the maximum decrease was 11 mmHg (P<0.05). 3. Both peptides produced significant increases in sodium excretion (P<0.05) but only ANF increased the cyclic GMP (cGMP) excretion rate (P<0.01) and neither peptide had a significant effect on plasma renin activity or glomerular filtration rate (GFR). Pro ANF 31–67 did not increase the plasma levels of ANF. 4. These results demonstrate that both ANF and proANF 31–67 have natriuretic effects via a tubular mechanism and suggest that the natriuretic effects of ANF are mediated by cGMP while the effects of pro ANF 31–67 are mediated by a different mechanism, not involving changes in cGMP excretion, changes in GFR or a reduction in renin secretion.     

INSULIN-POTENTIATING ACTION OF HUMAN GROWTH HORMONE:

Four peptides from the N-terminal region of human growth hormone have been synthesized by the solid-phase method: hGH(6–13), hGH(7–13), hGH(8–13) and hGH(9–13). Although these peptides contain the sensitive -Asp-Asn-sequence, apparently homogeneous products were obtained by synthesis on polystyrene resin, cleavage by hydrogen fluoride and purification by ion-exchange chromatography. The insulin-potentiating activity of these peptides is reported. The data indicates that extension of hGH(9–13) at its N-terminus is required for in vitro activity.     

SECRETION AND RENAL EFFECTS OF ANF PROHORMONE PEPTIDES

1. Atrial natriuretic factor (ANF) and pro ANF peptides appear to be secreted simultaneously from the atria in response to atrial stretch. 2. The major peptide forms secreted from rat atria appear to be ANF (pro ANF 99–126) as the primary C-terminal peptide and pro ANF 1–30 as the primary N-terminal peptide, as opposed to 1–67 or 1–98. 3. The plasma concentrations of ANF and pro ANF 1–30 are increased by acute stimulation with blood volume expansion and the plasma levels of ANF and N-terminal ANF prohormone peptides are chronically elevated by high salt diet. 4. Pro ANF 31–67 produces a natriuresis which is not dependent on an increase in renal cGMP excretion, decreases in plasma renin activity (PRA) or elevations in plasma ANF concentration.     

Liposomal angiogenic peptides for ischemic limb perfusion: comparative study between different administration methods

Background: We investigated the therapeutic effectiveness of PEGylated liposomes loaded with angiogenic peptides for treating hindlimb ischemia.

Methods: Rats received a femoral artery occlusion. Red blood cells collected from the animals were labeled with technetium-99m. Limb perfusion gamma imaging was performed. PEGylated liposomes loaded with angiogenic peptides were administered intra-arterially. Technetium-99m red blood cell imaging was repeated 1 week later. The animals were sacrificed the next day. The expression of angiogenic proteins was studied. Later, changes in limb perfusion after intra-arterial infusion versus intra-muscular injection were also compared to determine the therapeutic effectiveness of different administration methods.

Results: Femoral artery occlusion dramatically reduced ischemic limb perfusion (by an average of 69%, compared to contralateral limb). This was not different among groups (p?>?0.05). Liposomes loaded with angiogenic peptides significantly improved ischemic limb perfusion, compared to controls (210% of baseline, versus 100% of baseline in control; p?<?0.05 versus controls). The enhanced ischemic limb perfusion was accompanied by an increased expression of CD 31 (an average of 1.6-fold increase of controls; p?<?0.05). The liposomes or peptides treatment alone did not affect ischemic perfusion (liposomes alone: 100% of baseline; peptides alone: 120% of baseline; p?>?0.05 versus controls, respectively) or the angiogenic response (1.1-fold of controls in liposomes alone; 1.0-fold of controls in peptides alone; p?>?0.05 versus controls, respectively). Intra-muscular injection induced similar liposomal treatment effects on ischemic limb perfusion (230% of baseline) as those by intra-arterial infusion (210% of baseline; p?<?0.05 versus intra-muscular).

Conclusions: PEGylated liposomes loaded with angiogenic peptides improved ischemic limb perfusion and promoted angiogenic responses. Liposomal angiogenic treatment via intra-arterial infusion resulted in an equally effective therapeutic efficacy compared to that of intra-muscular injection. These results show the therapeutic potential of our liposomal strategy for treating peripheral limb ischemia.     

STUDIES ON TRYPSIN INHIBITORS

The general strategy for the synthesis, by conventional procedures, of the entire sequence of porcine pancreatic secretory trypsin inhibitor II (Kazal) is discussed. The synthesis of two protected peptides corresponding to positions 2–10 and 1–10 of the proposed primary structure of the inhibitor is described. The heptapeptide free base threonyl-S-acetamidomethylcysteinylthreonylseryl-γ-tert-butylglutamylvalylserine tert-butyloxycarbonylhydrazide (sequence 4–10) was acylated, by the azide procedure, with either the dipeptide benzyloxycarbonyl-γ-tert-butylglutamylalanine hydrazide (sequence 2–3) or the tripeptide Nω-benzyloxycarbonyl-Nω-nitroarginylglutamylalanine hydrazide (sequence 1–3). The stereochemical homogeneity of the resulting peptides, benzyloxycarbonyl-γ-tert-butylglutamylalanylthreonyl-S-acetamidomethyl-cysteinylthreonylseryl-γ-tert-butylglutamylvalylserine tert-butyloxycarbonylhydrazide and Nω-benzyloxycarbonyl-Nω-nitroarginylglutamylalanylthreonyl-S-acetamido-methylcysteinylthreonylseryl-γ-tert-butylglutamylvalylserine tert-butyloxycarbonylhydrazide, was assessed, after partial deprotection with liquid hydrogen fluoride, by digestion with aminopeptidase M followed by quantitative amino acid analysis.     

Efficient induction of cytotoxic T lymphocytes specific for severe acute respiratory syndrome (SARS)-associated coronavirus by immunization with surface-linked liposomal peptides derived from a non-structural polyprotein 1a

Spike and nucleocapsid are structural proteins of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) and major targets for cytotoxic T lymphocytes (CTLs). In contrast, non-structural proteins encoded by two-thirds of viral genome are poorly characterized for cell-mediated immunity. We previously demonstrated that nucleocapsid-derived peptides chemically coupled to the surface of liposomes effectively elicited SARS-CoV-specific CTLs in mice. Here, we attempted to identify HLA-A*0201-restricted CTL epitopes derived from a non-structural polyprotein 1a (pp1a) of SARS-CoV, and investigated whether liposomal peptides derived from pp1a were effective for CTL induction. Out of 30 peptides predicted on computational algorithms, nine peptides could significantly induce interferon gamma (IFN-γ)-producing CD8⁺ T cells in mice. These peptides were coupled to the surface of liposomes, and inoculated into mice. Six liposomal peptides effectively induced IFN-γ-producing CD8⁺ T cells and seven liposomal peptides including the six peptides primed CTLs showing in vivo killing activities. Further, CTLs induced by the seven liposomal peptides lysed an HLA-A*0201 positive cell line expressing naturally processed, pp1a-derived peptides. Of note, one of the liposomal peptides induced high numbers of long-lasting memory CTLs. These data suggest that surface-linked liposomal peptides derived from pp1a might offer an efficient CTL-based vaccine against SARS.     

Alignment-based design and synthesis of new antimicrobial Aurein-derived peptides with improved activity against Gram-negative bacteria and evaluation of their toxicity on human cells

Considering the worldwide increasing prevalence of resistance to traditional antibiotics, it is necessary to find new antibiotics to deal with this issue. Recently, antimicrobial peptides (AMPs) have been proposed as new antimicrobial agents. Aureins are a family of AMPs that are isolated from Green and Golden Bell Frogs. These peptides have a favorable antibacterial activity against Gram-positive bacteria. We designed two peptides derived from natural Aurein enjoying alignment-based design method. After synthesis of the peptides, their secondary structure was checked by circular dichroism. Consequently, the antibacterial effects of these peptides were investigated by determining the minimum inhibitory concentration (MIC) and bactericidal concentration. Eventually, the toxicity of these peptides was determined by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay on normal human skin cells (Hu02 cell line). Natural Aurein1.2 was used as a natural control to compare the properties in all stages. The results indicated that these new peptides had medium-upward antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis (MIC of 8–64 μg/mL) and weak bactericidal activity against Staphylococcus aureus (MIC of 128–256 μg/mL). Also, MTT assays results showed that AureinN2 is less toxic than AureinN1 and Aurein1.2. Toxicity of AureinN2 for Hu02 cell lines was between 20 and 40% at the concentration of 8–500 μg/mL. In this study, we were able to improve antimicrobial activity of two synthetic derivatives of the Aurein family against Gram-negative bacteria by using machine-learning algorithm and other in silico methods.     

A solid‐phase synthetic strategy for the preparation of peptide‐based affinity labels: synthesis of dynorphin A analogs

Abstract: Solid‐phase synthetic methodology was developed for the preparation of peptide‐based affinity labels. The initial peptides synthesized were dynorphin A (Dyn A) analogs [Phe(p‐X)⁴,d ‐Pro¹⁰]Dyn A(1–11)NH₂ containing isothiocyanate (X = –N=C=S) and bromoacetamide (X = –NHCOCH₂Br) groups. The peptides were assembled on solid supports using Fmoc‐protected amino acids, and the side chain amine to be functionalized, Phe(p‐NH₂), was protected by the Alloc (allyloxycarbonyl) group. Following removal of the Alloc group by palladium(0), the reactive isothiocyanate and bromoacetamide functionalities were successfully introduced while the peptides were still attached to the resin. Synthesis of these peptides was carried out on polystyrene (PS) and polyethylene glycol–polystyrene (PEG–PS) resins containing the PAL [peptide amide linker, 5‐(4‐Fmoc‐aminomethyl‐3,5‐dimethoxyphenoxy)valeric acid] linker. Both the rate of Alloc deprotection and the purity of the crude affinity‐labeled peptides obtained were found to be dependent on the resin used for peptide assembly.     

Influence of reaction conditions on syntheses of sweetener precursors catalyzed by thermolysin in tert-amyl alcohol

Abstract: The activity of enzymes to form a peptide bond in organic solvent was greatly influenced by observed pH and water content. The precursors of two sweeteners, P-Asp-Xaa-OR (P = Z or For, Xaa-OR = Phe-OMe or Ala-OcHex), were synthesized by enzyme, and the reaction conditions were studied systematically. Z-Asp-OH was coupled with H-Phe-OMe or H-Ala-OcHex by thermolysin in tert-amyl alcohol. The best coupling results were obtained when the optimized observed pH was 8 or 9, and the water content was about 6% (V/V). The protecting group Z is better than For under the reaction conditions and H-Phe-OMe is a better nucleophile than H-Ala-OcHex. The expected optically pure peptides were obtained when the racemic amino acids were used as amino components in the starting materials. The physical constants of P-Asp-Xaa-OR synthesized by thermolysin are identical with those of peptides synthesized by chemical method.     

Synthetic peptide vaccines: palmitoylation of peptide antigens by a thioester bond increases immunogenicity

Synthetic peptides have frequently been used to immunize animals. However, peptides less than about 20 to 30 amino acids long are poor immunogens. In general, to increase its immunogenicity, the presentation of the peptide should be improved, and molecular weight needs to be increased. Many attempts have been made to couple peptide immunogens to different carrier proteins [e.g. keyhole limpet haemocyanin (KLH) or ovalbumin]. This leads to very complex structures, however. We used a controlled conjugation of a peptide to a single long-chain fatty acid like palmitic acid by a thioester or an amide bond. It was found that these S-palmitoylated peptides were much more immunogenic than N-palmitoylated peptides and at least similar to KLH-conjugated peptides with respect to appearance and magnitude of induced antibodies (canine parvovirus) or immunocastration effect (gonadotropin-releasing hormone). For chemical synthesis of thioesters, we established conditions for solution and solid-phase synthesis. In both phases, Cys(SBu^t) could only be deprotected efficiently using phosphines, and S-acylation was accomplished using standard coupling at pH 5. We speculate that, in vivo, the presence of an appropriate fatty acid chain, chemically linked through a labile thioester bond, greatly enhances immunogenicity, because it represents a favourable substrate for cleavage by cellular thioesterases in cells of the immune system.     

Study on the synthesis and characterization of peptides containing phosphorylated tyrosine

Phosphorylation and dephosphorylation are key events in receptor-mediated and post-receptor-mediated signal transduction. Synthetic phosphopeptides have been shown to have dramatic agonist or antagonist effects in several of these signaling pathways. For its 1997 study, the Association of Biomolecular Resource Facilities (ABRF) Peptide Synthesis Research Group assessed the ability of member laboratories to synthesize phosphotyrosine peptides. Participating laboratories were requested to synthesize and submit the following crude peptide, H–Glu–Asp–Tyr–Glu–Tyr(PO₃H₂)–Thr–Ala–Arg–Phe–NH₂, for evaluation by amino acid analysis, sequence analysis, RP-HPLC, MALDI-TOF and ESI mass spectrometry. Prior to analysis of submitted peptides from ABRF members, the Peptide Synthesis Research Group synthesized and characterized the nonphosphorylated form of the peptide, the doubly phosphorylated form and the peptides singly phosphorylated on either the first or the second tyrosine. These peptide standards were separated easily by HPLC and capillary electrophoresis and the phosphotyrosine was detected readily by Edman degradation sequence analysis. No differences were seen by amino acid analysis and the expected masses were observed by mass spectrometry. The two singly phosphorylated peptides were easily distinguished by MALDI-PSD. Analysis of the peptides submitted from member facilities revealed that all but four of the 33 samples contained the correct product as determined by HPLC and mass spectrometry. HPLC analysis indicated that 20 of the 33 submitted samples contained greater than 75% correct product, five contained less than 50% correct product and four did not contain any correct product. By ESI/MS, an additional singly charged ion at m/z 535.5 was detected in five of the 33 submitted samples; this ion was subsequently shown to represent Ac-TARF-NH₂. No correlation was found to exist between coupling time and percentage correct product; however, a correlation may exist between a greater percentage of correct product and the use of non-protected phosphotyrosine.     

Helix-enhancing propensity of fluoro and alkyl alcohols: influence of pH,temperature and cosolvent concentration on the helical conformation of peptides

We have analyzed the effects of trifluoroethanol (TFE) and three other alcohols(1-propanol, 2-propanol and hexafluoro-2-propanol) on S-peptide (residues 1–20) of ribonuclease A, an analog of S-peptide (QHM → AAA, Sa-peptide) and TC-peptide (residues 295–316) of thermolysin to assess the helix-enhancing propensity of fluoro and alkyl alcohols under different environmental conditions of cosolvent concentration, pH and temperature by circular dichroism (CD). The dependence of cosolvent concentration on helix-induction showed a plateauing effect in all cases. 1-Propanol and 2-propanol were as effective as TFE in all the three peptides. Hexafluoro-2-propanol (HFIP) was a better helix enhancer in all cases however, the relative effectiveness varied with the peptide sequence. The alcohol transitions were analyzed assuming a two-state transition. The free energy decreased linearly in the cosolvent concentration range of 0–5 m for all the three peptides. The m-value (constant of proportionality) varied between peptides but was similar for any given peptide for TFE, 1-propanol or 2-propanol. The m-values of HFIP for all three peptides was much higher compared to other cosolvents. The isothermal cosolvent helix-induction curves for the three peptides exhibited similar features of shape and character for 1-propanol, 2-propanol and TFE. The additivity of cosolvent-induced helix formation was observed for different blends of alkyl and/or fluoro cosolvents. The pH-dependence of helix formation was observed in both TFE and 1-propanol solutions for S-peptide and TC-peptide, respectively, while in Sa-peptide, which was designed to perturb the pH-effect, helix formation was unaffected. The overall results provide some insight into the mechanism of cosolvent-mediated helix-enhancement in protein segments and are likely to facilitate optimization of conditions for cosolvent usage in chemistry and biology.