Peptide Synthesis Techniques Research Articles

Evaluation of the trifluoromethanosulfonic acid/trifluoroacetic acid/thioanisole cleavage procedure for application in solid-phase peptide synthesis.

As an extension of our investigation of peptidyl-resin linkage stability towards different cleavage procedures used in the solid-phase peptide synthesis (SPPS) technique, the present paper evaluated the trifluoromethanesulfonic acid (TFMSA)/trifluoroacetic acid (TFA)/thioanisole method, varying the type of resin (benzhydrylamine-resin, BHAR; methylbenzhydrylamine-resin, MBHAR and 4-(oxymethyl)-phenylacetamidomethyl-resin, PAMR) and peptide resin-bound residue (Gly and Phe). The vasoactive angiotensin II (AII, DRVYIHPF) and its [Gly8]-AII analogue linked to those resins used routinely in tert-butyloxycarbonyl (Boc)-SPPS chemistry were submitted comparatively to a time course study towards TFMSA/TFA cleavage. At 0 degrees C, [Gly8]-AII was completely removed from all resins in less than 6 h, but the hydrophobic Phe8 moiety-containing AII sequence was only partially cleaved (not more than 15%) from BHAR or MBHAR in this period. At 25 degrees C, [Gly8]-AII cleavage time decreased to less than 2 h irrespective of the solid support, and quantitative removal of AII from PAMR and MBHAR occurred in less than 3 h. However, about 10-15 h seemed to be necessary for cleavage of AII from BHAR, and in this extended cleavage reaction a significant increase in peptide degradation rate was observed. Regardless of the cleavage temperature used, the decreasing order of acid stability measured for resins was BHAR>MBHAR>PAMR. Collectively, these findings demonstrated the feasibility of applying TFMSA/TFA solution as a substitute for anhydrous HF at the cleavage step in Boc-SPPS methodology. Care should be taken however, as the cleavage efficacy depends on multiple factors including the resin, peptide sequence, the time and temperature of reaction.

The use of/ -amino acids in the design of protease and peptidase inhibitors

The use of β-amino acids as peptidomimetics has emerged in recent years with significant potential in a number of applications. The incorporation of β-amino acids has been successful in creating peptidomimetics that not only have potent biological activity, but are also resistant to proteolysis. This article reviews the recent applications of β-amino acids in the design of protease and peptidase inhibitors. Given their structural diversity, together with the ease of synthesis and incorporation into peptide sequences using standard solid-phase peptide synthesis techniques, β-amino acids have the potential to form a new platform technology for peptidomimetic design and synthesis.

Molecular and cellular actions of a structural domain of human growth hormone (AOD9401) on lipid metabolism in Zucker fatty rats.

A lipolytic domain (AOD9401) of human growth hormone (hGH) which resides in the carboxyl terminus of the molecule and contains the amino acid residues 177-191, has been synthesized using solid-phase peptide synthesis techniques. AOD9401 stimulated hormone-sensitive lipase and inhibited acetyl coenzyme A carboxylase (acetyl CoA carboxylase) in isolated rat adipose tissues, in a similar manner to the actions of the intact hGH molecule. The synthetic lipolytic domain mimicked the effect of the intact growth hormone on diacylglycerol release in adipocytes. Chronic treatment of obese Zucker rats with AOD9401 for 20 days reduced the body weight gain of the animals, and the average cell size of the adipocytes of the treated animals decreased from 110 to 80 microm in diameter. Unlike hGH, synthetic AOD9401 did not induce insulin resistance or glucose intolerance in the laboratory animals after chronic treatment. The results suggest that AOD9401 has the potential to be developed into a therapeutic agent for the control of obesity.

An Unnatural Amino Acid that Mimics a Tripeptide β-Strand and Forms β-Sheetlike Hydrogen-Bonded Dimers

Unnatural amino acid 2 (5-HO2CCONH-2-MeO-C6H3-CONHNH2) duplicates the hydrogen-bonding functionality of one edge of a tripeptide β-strand. It is composed of hydrazine, 5-amino-2-methoxybenzoic acid, and oxalic acid groups and is designated by the three-letter abbreviation “Hao” to reflect these three components. The 2,7-di-tert-butylfluorenylmethyloxycarbonyl (Fmoc*)- and tert-butyloxycarbonyl (Boc)-protected derivatives of Hao are prepared efficiently and in high yield by the condensation of suitably protected derivatives of hydrazine, 5-amino-2-methoxybenzoic acid, and oxalic acid. Fmoc*-Hao and Boc-Hao behave like typical Fmoc- and Boc-protected amino acids and can be incorporated into peptides by standard solid- and solution-phase peptide synthesis techniques using carbodiimide coupling agents. Hao-containing peptide 9 (i-PrCO-Phe-Hao-Val-NHBu) forms a β-sheetlike hydrogen-bonded dimer in CDCl3 and CD3OD−CDCl3 solutions. Peptides containing Hao and natural amino acids display hydrogen-bonding surfaces...

Mutational analysis of the IgE-binding epitopes of P34/Gly m Bd 30K

Background: Peanuts and soybeans are 2 foods that have been shown to be responsible for many atopic disorders. Because of their nutritional benefit, soybean proteins are now being used increasingly in a number of food products. Previous studies have documented multiple allergens in soybean extracts, including glycinin, β-conglycinin, and the P34/Gly m Bd 30K protein. Objective: Our overall goal was to identify soybean-specific allergens to begin to understand molecular and immunochemical characteristics of legume proteins. The specific aim of the current investigation was to identify the essential amino acid residues necessary for IgE binding in the 5 distinct immunodominant epitopes of P34/Gly m Bd 30K. Methods: Serum IgE from 6 clinically sensitive soybean-allergic individuals was used to identify P34/Gly m Bd 30K in the native and single amino acid substituted peptides with use of the SPOTS peptide synthesis technique to determine critical amino acids required for IgE binding. Results: The intensity of IgE binding and epitope recognition by serum IgE from the individuals varied substantially. With use of serum from 6 clinically soybean-sensitive individuals, 2 of the 5 immunodominant epitopes could be mutagenized to non-IgE binding peptides. Conclusions: Single-site amino acid substitution of the 5 immunodominant epitopes of Gly m Bd 30K with alanine revealed that IgE binding could be reduced or eliminated in epitopes 6 and 16 in the serum obtained from 6 soybean-sensitive patients. (J Allergy Clin Immunol 2000;105:378-84.)

One Step Synthesis of Inverted Aspartame Type Sweetener, Ac-Phe-Lys, Using Chemically Modified Chymotrypsin.

To search for techniques of simplified peptide synthesis, benzyloxycarbonyl chymotrypsin was prepared by a water-soluble acylating reagent and used to make Ac-Phe-Lys, an artificial peptide sweetener, which was selected as a target compound. As a result of using chemically modified chymotrypsin, Lys can be coupled directly with Ac-Phe and Ac-Phe-Lys made virtually in one step. Moreover, the total yield from preparation and purification steps for Ac-Phe-Lys was 13%. The value corresponds to that of the chemical synthesis method. On the contrary, enzymatic synthesis using native chymotrypsin cannot reach the level of the new method. It is expected that the method is more effective for simplified peptide synthesis as compared with other methods, especially on a large scale.

Linear Heptapeptides Containing DNA-Intercalators. Synthesis and Interaction with DNA

Five peptides of general structure X-Ser-Pro-Thr-Ser-Pro-Ser-Y (X = Tyr, quinoxaline- 2-carbonyl, acridine-9-carbonyl, Y = Tyr, (quinoxalin-2-yl)amino) were prepared using standard solid-phase peptide synthesis technique. Their interaction with DNA (calf thymus DNA and plasmids pUC9, p∆NS and pGEMEX) was studied using UV and CD spectroscopy, sedimentation analysis and agarose gel electrophoresis after treatment with topoisomerase I. In contrast to earlier findings (Suzuki M.: Nature 1990, 344, 562) intercalation into DNA structure has not been proved for any compound studied.

A reversible affinity tag for the purification of N-glycolyl capped peptides

A reversible biotin affinity tag, 2-[ N-(Biotin)-6-aminocaproate]-acetate ( 1), has been generated for the efficient purification of peptides requiring N-terminal derivatization. This methodology is compatible with solid phase peptide synthesis techniques.

A monoclonal antibody against the X protein of hepatitis B virus: fine mapping of its epitope and application in a quantitative ELISA of the X protein in sera of hepatitis B patients.

A HBx-specific mouse monoclonal antibody was developed and its epitope mapped to a hydrophilic segment 94HKRTLGL100 using the multipin peptide synthesis technique. A sensitive ELISA with a threshold of 5 to 10 ng was developed to identify the HBx-positive hepatitis B cases and measure the levels of HBx in sera. The same patient sera were also analyzed for the presence of anti-HBx using the purified recombinant antigen. HBx was present in 23% of the cases (15/65) whereas only 14% of the cases (9/65) were positive for anti-HBx. The mean value of HBx in acute hepatitis sera was higher (522 ng/ml) than in cirrhosis cases (48 ng/ml). PCR amplification of the S gene showed that all 15 HBx-positive cases were also positive for the viral DNA.

The WW Domain of Neural Protein FE65 Interacts with Proline-rich Motifs in Mena, the Mammalian Homolog of DrosophilaEnabled

The neural protein FE65 contains two types of protein-protein interaction modules: one WW binding domain and two phosphotyrosine binding domains. The carboxyl-terminal phosphotyrosine binding domain of FE65 interacts in vivo with the beta-amyloid precursor protein, which is implicated in Alzheimer disease. To understand the function of this adapter protein, we identified binding partners for the FE65 WW domain. Proline-rich sequences sharing a proline-proline-leucine-proline core motif were recovered by screening expression libraries for ligands of the FE65 WW domain. Five proteins of molecular masses 60, 75, 80, 140, and 200 kDa could be purified from mouse brain lysates by affinity to the FE65 WW domain. We identified two of these five proteins as the 80- and 140-kDa isoforms encoded by Mena, the mammalian homolog of the Drosophila Enabled gene. Using the SPOTs technique of peptide synthesis, we identified the sequences in Mena that interact with the FE65 WW domain and found that they contain the signature proline-proline-leucine-proline motif. Finally, we demonstrated that Mena binds to FE65 in vivo by coimmunoprecipitation assay from COS cell extracts. The specificity of the Mena-FE65 WW domain association was confirmed by competition assays. Further characterization of the FE65-Mena complex may identify a physiological role for these proteins in beta-amyloid precursor protein biogenesis and may help in understanding the mechanism of molecular changes that underlie Alzheimer disease.

Unnatural peptide synthesis automated

Researchers from academia and industry have developed a solid-phase peptide synthesis technique that makes it possible to incorporate unnatural amino acids into peptides in a totally automated manner. The technique could be useful for making combinatorial libraries of structurally diverse peptides containing both natural and unnatural amino acids. Until now, unnatural amino acids had to be prepared separately before they could be used as building blocks in the automated synthesis of modified peptides. The new technique—called unnatural peptide synthesis (UPS)—is faster and more convenient in that side-chain-modified peptide derivatives can now be formed on the fly in an automated peptide synthesizer. UPS was developed in a collaborative effort between chemistry professor Martin J. O'Donnell and postdoctoral fellow Changyou Zhou of Indiana University-Purdue University at Indianapolis, and chemist William L. Scott of Eli Lilly & Co., Indianapolis [ J. Am. Chem. Soc. , 118 , 6070 (1996)]. The technique add...

Comparative Total Syntheses of Turkey Ovomucoid Third Domain by Both Stepwise Solid Phase Peptide Synthesis and Native Chemical Ligation

Turkey ovomucoid third domain, OMTKY3, is a potent protein inhibitor of most serine proteinases that prefer a neutral residue at the P1 position. It has been a target of increasingly intense scrutiny by site-directed mutagenesis in an attempt to elucidate the sequence-to-reactivity algorithm of the inhibitor. Here we report the total chemical synthesis of (6−56)OMTKY3 using both the stepwise solid phase peptide synthesis (SPPS) technique and the native chemical ligation approach. After refolding and affinity purification, the resultant products were characterized by electrospray ionization mass spectrometry, analytical ion exchange chromatography, measurement of association equilibrium constants with six different serine proteinases, and thermal denaturation studies. The two synthetic proteins were found to be functionally as well as structurally identical to their recombinant counterpart. Moreover, the native chemical ligation and stepwise SPPS techniques gave rise to comparable yields and similar product quality. The native chemical ligation strategy used here presents highly efficient synthetic access to novel analogs of OMTKY3 that can be used for understanding the molecular basis of enzyme−inhibitor recognition.

Pseudo-Prolines as a Solubilizing, Structure-Disrupting Protection Technique in Peptide Synthesis

Serine-, threonine-, and cysteine-derived cyclic building blocks (pseudo-prolines, ΨPro) serve as reversible protecting groups for Ser, Thr, and Cys and prove to be versatile tools for overcoming some intrinsic problems in the field of peptide chemistry. The presence of ΨPro within a peptide sequence results in the disruption of β-sheet structures considered as a source of intermolecular aggregation during chain elongation, thus increasing solvation and coupling kinetics in peptide assembly. Due to their easy synthetic access and variability in the chemical stability by modifications introduced in the C-2 position of the oxazolidine/thiazolidine ring system, this protection technique is adaptable to all common strategies in peptide synthesis. We describe new types of ΨPro building blocks suitable for standard Fmoc/tBu-based solid phase peptide synthesis, convergent strategies, and chemoselective ligation techniques as well as their use as a structure-disrupting, solubilizing protection technique for the example of peptides generally considered as “difficult sequences”.

L-O-(2-malonyl)tyrosine: a new phosphotyrosyl mimetic for the preparation of Src homology 2 domain inhibitory peptides.

Inhibition of Src homology 2 (SH2) domain-binding interactions affords one potential means of modulating protein-tyrosine kinase-dependent signaling. Small phosphotyrosyl (pTyr)-containing peptides are able to bind to SH2 domains and compete with larger pTyr peptides or native pTyr-containing protein ligands. Such pTyr-containing peptides are limited in their utility as SH2 domain inhibitors in vivo due to their hydrolytic lability to protein-tyrosine phosphatases (PTPs) and the poor cellular penetration of the ionized phosphate moiety. An important aspect of SH2 domain inhibitor design is the creation of pTyr mimetics which are stable to PTPs and have reasonable bioavailability. To date, most PTP-resistant pTyr mimetics which bind to SH2 domains are phosphonates such as (phosphonomethyl)phenylalanine (Pmp, 2), [(monofluorophosphono)methyl]phenylalanine (FPmp, 3) or [(difluorophosphono)methyl]-phenylalanine (F2Pmp, 4). Herein we report the incorporation of a new non-phosphorus-containing pTyr mimetic, L-O-(2-malonyl)tyrosine (L-OMT, 5), into SH2 domain inhibitory peptides using the protected analogue L-N alpha-Fmoc-O'-(O",O"-di-tert-butyl-2-malonyl)tyrosine (6) and solid-phase peptide synthesis techniques. Five OMT-containing peptides were prepared against the following SH2 domains: the PI-3 kinase C-terminal p85 SH2 domain (Ac-D-(L-OMT)-V-P-M-L-amide, 10, IC50 = 14.2 microM), the Src SH2 domain (Ac-Q-(L-OMT)-E-E-I-P-amide, 11, IC50 = 25 microM, and Ac-Q-(L-OMT)-(L-OMT)-E-I-P-amide, 14, IC50 = 23 microM), the Grb2 SH2 domain (Ac-N-(L-OMT)-V-N-I-E-amide, 12, IC50 = 120 microM), and the N-terminal SH-PTP2 SH2 domain (Ac-L-N-(L-OMT)-I-D-L-D-L-V-amide, 13, IC50 = 22.0 microM). These results show that peptides 10, 11, 13, and 14 have reasonable affinity for their respective SH2 domains, with the IC50 value for the SH-PTP2 SH2 domain-directed peptide 13 being equivalent to that previously observed for the corresponding F2Pmp-containing peptide. OMT may afford a new structural starting point for the development of novel and useful SH2 domain inhibitors.

A new class of compounds, peptide derivatives of adenosine 5′-carboxylic acid, includes inhibitors of ATP receptor-mediatedresponses

A new type of ligand for the study of P 2-purinergic receptor subtypes was synthesized by combining and modifying conventional nucleoside chemistry with Fmoc solid phase peptide synthesis techniques. The tri- and tetra-aspartic acid derivatives of adenosine-5′-carboxylic acid (AdoCAsp 3 and AdoCAsp 4) were found to act as weak agonists at P 2-purinergic receptors, (activated by ATP and UTP respectively) present on C6 glioma cells. AdoCAsp 4 induced inositol 1,4,5-trisphosphate formation in the C6 cells with an EC 50 of 73 μM. In addition, AdoCAsp 4 was found to inhibit (IC 50 ≈80 nM) ATP-induced cytosolic [Ca 2+] transients in these glioma cells. The glycine derivative, AdoCGly, increased evoked release of noradrenaline from mouse vas deferens slices, probably due to the blockade of presynaptic P 2-autoreceptors. The possibility that aspartic, glutamic or γ-carboxyglutamic residues may be used to replace phosphate groups on an ATP receptor ligand, opens up new ways in ligand design.

Location of an epitope defined by an enhancing monoclonal antibody to growth hormone: some structural details and biological implications.

We have previously shown that a murine monoclonal antibody (MAb OA15) prepared against ovine GH (oGH) can enhance the somatogenic activity of bovine GH (bGH), as determined by increased incorporation of 35SO4(2-) into costal cartilage of hypopituitary Snell dwarf mice in vivo We have now established that MAb OA15 can also enhance the biological activity of oGH and porcine GH (pGH) in vivo. Using multiple pin peptide synthesis techniques a set of overlapping immobilized octamers representing the entire bGH sequence were synthesized and tested for their ability to bind MAb OA15 using an enzyme-linked immunosorbent assay. The pattern of binding showed that OA15 defined a functionally continuous epitope comprising residues 91-102. This region includes the C-terminal end of helix 2 plus a portion of the adjacent loop linking helices 2 and 3. Polyclonal antisera to a synthetic peptide representing this epitope mimicked the ability of MAb OA15 to enhance oGH, bGH, and pGH. Window size analysis showed that the heptapeptide 94-100 (SRVFTNS) represents the minimum unit to retain full recognition of MAb OA15. The fact that pGH, bGH, and oGH have identical sequences in this region also explains the ability of OA15 to both cross-react with and enhance the biological activity of each of these GHs. Replacement net analysis (where each residue in the heptamer is substituted with each of the 19 naturally occurring L-amino acids) demonstrated that residues R95 and T98 are critical for antibody binding and also indicated that the substitution of V96 with I, as found in rat GH, would permit the observed binding of OA15 to this hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Systematic study of substance P analogs

The multipin peptide synthesis technique, a method for simultaneous multiple peptide synthesis, was developed for large-scale screening of oligopeptides [Geysen et al. (1984) Proc. Natl. Acad. Sci. USA, 81, 3998-4002]. A modification of the technique allows the peptides assembled on polyethylene pins to be cleaved in their native amide form and reconstituted into physiologically compatible solutions. In this study, the suitability of these peptides for quantitative receptor binding assay was evaluated. Substance P and 18 analogs, including a set of N-terminal truncated substance P and a set of naturally occurring substance P analogs, were synthesized by the multipin methods. An average yield of 20 +/- 3 nmol of peptide per pin was obtained. The purity of the peptides was estimated to be ca. 90%. The binding activities of these peptides were determined in a competition assay against 125I-BHSP binding to a rat brain synaptosome preparation. The rank order of the affinities of these peptides depicted a typical pharmacological profile of central NK1 receptor. The IC50 values obtained were also in good agreement with data reported by other groups using similar experimental conditions, except that bulk synthesized peptides were used. This study demonstrates that the peptides synthesized with the multipin technique are suitable for quantitative receptor studies, particularly for a high-volume screening of bioactive peptides.

Application of the multipin peptide synthesis technique for peptide receptor binding studies: Substance P as A model system

The multipin peptide synthesis technique allows thousands of discrete peptides to be synthesised simultaneously. To explore the application of this technique in large scale screening of bioactive peptides, substance P and analogues were synthesised and their receptor binding affinity determined. The results are consistent with published data, suggesting that this approach is applicable to large scale, quantitative receptor studies.

Circular dichroism, luminescence, and electronic absorption of copper binding sites in metallothionein and its chemically synthesized .alpha. and .beta. domains

The stoichiometry and molecular architecture of Cu-thionein is unknown. Thus, the oligonuclear copper-binding sites of chemically synthesized α and β fragments employing continuous flow solid phase peptide synthesis technique have been studied and compared with those of the apo-MT, intact Cd 5 Zn 2 -MT and its substituted form Cd 7 -MT.

Investigation of the solid- and solution-phase binding reactivities of continuous epitopes recognized by polyclonal guinea-pig anti-recombinant bovine growth hormone antisera.

We have used the technique of multiple pin peptide synthesis to identify three major continuous epitopes in the recombinant bovine (rb) GH molecule. We have synthesized these peptides, residues 24-40, 139-152 and 179-189, as N-terminally acetylated, C-terminal amides and confirmed their reactivity in a standard solid-phase ELISA. Subsequently, for epitope 139-152, we have synthesized a peptide affinity column and used this to isolate antibodies with this epitope specificity from whole antiserum. In addition, we demonstrate that under native conditions in a liquid phase RIA, these antibodies will precipitate [125I]rbGH. Further, peptide 139-152 itself also cross-reacts in an rbGH RIA inhibiting binding by up to 20%. Our data suggest that during the immune response to rbGH in guinea-pigs a substantial part of the B-cell response is directed to the 139-152 region and that this part of the protein is a native epitope.