Biomedical subjects

R M Valerio

Publications and source records attributed to R M Valerio.

10 recordsLinked to original sources

PubMed1996

Multipin solid-phase synthesis of acyl 2, 3-diaminopropionic acid oligomers.

A modular approach for the synthesis of sets of diverse organic molecules is described. N-alpha-Fmoc-N-beta-Alloc-D-2,3-diaminopropionic acid (Fmoc-D-Dpr(Alloc)-OH) was prepared in four steps from Boc-D-asparagine and used as a scaffold for attachment of sidechains. Using the Multipin approach, a number of model acyl trimers were rapidly prepared by sequential coupling of Fmoc-D-Dpr(Alloc)-OH and acylation of the beta-amino group with a range of activated carboxylic acids.

beta-Alanine↗

PubMed1994

Multiple peptide synthesis on acid-labile handle derivatized polyethylene supports.

Using the multipin peptide synthesis approach, a range of peptides with native amide and carboxylate C-termini were generated using an acid-labile approach. Polyethylene crowns grafted with hydroxyethylmethacrylate (HEMA) polymer were functionalized with either 4-hydroxymethylphenoxyacetic acid for the generation of peptide-carboxylate or p-[(R,S)-alpha-[1-(9H-fluoren-9-yl)methoxyformamido]-2,4-dim ethoxy- benzyl]phenoxyacetic acid for peptide-amide. A range of known peptide hormone sequences and other peptides with native C-termini were assembled by sequential incorporation of N alpha-Fmoc protected amino acids. Peptides were sidechain deprotected and cleaved from crowns with TFA/scavengers within 2 mL centrifuge tubes, and isolated by a series of ether/petrol wash and centrifugation steps. In this way it was possible to avoid a cleavage and isolation botteneck, allowing rapid processing of large numbers of peptides.

Amino Acid Sequence↗

PubMed1993

The synthesis and use of pp60src-related peptides and phosphopeptides as substrates for enzymatic phosphorylation studies.

A series of peptides and phosphopeptides corresponding to the auto-phosphorylation site of pp60src, -Asn-Glu-Tyr416-Thr-Ala-, were prepared by either Boc/solution or Fmoc/solid phase peptide synthesis and used as substrates to study their enzymatic phosphorylation by various casein kinases. The Tyr(P)-containing peptide, Asn-Glu-Tyr(P)-Thr-Ala, was prepared by the use of Fmoc-Tyr(PO3Bzl2)-OH in Fmoc/solid phase peptide synthesis followed by acidolytic treatment of the peptide-resin with 5% anisole/CF3CO2H. Both Asn-Glu-Tyr-Thr-Ala and Asn-Glu-Ser(P)-Thr-Ala were prepared by the Boc/solution phase peptide synthesis and employed hydrogenolytic deprotection of the protected peptides. Enzymatic phosphorylation studies established that (A) the Tyr residue acted as an unusual positive determinant for directing phosphorylation to the Thr-residue, (B) the rate of Thr-phosphorylation was markedly facilitated by a change from the Tyr-residue to the Tyr(P)-residue, and (C) a Ser(P)-residue was as effective as the Tyr(P)-residue in facilitating Thr-phosphorylation. A subsequent structure-function study using Asn-Glu-Phe-Thr-Ala, Asn-Glu-Tyr(Me)-Thr-Ala (prepared by Fmoc/solid phase peptide synthesis) and Asn-Glu-Cha-Thr-Ala (prepared by hydrogenation of Asn-Glu-Tyr-Thr-Ala) established that the rate of Thr-phosphorylation was influenced by the extent of hydrophobic-hydrophobic interactions by the aralkyl side-chain group (either aromatic or aliphatic) of the 416-residue with casein kinase-2; the rate of Thr-phosphorylation being decreased by the introduction of methyl or hydroxyl groups at the 4-position of the aromatic group (i.e. Tyr(Me) and Tyr respectively) but enhanced by the introduction of the hydrophilic phosphate group (i.e. as Tyr(P)).

Amino Acid Sequence↗

PubMed1993

Multipin peptide synthesis at the micromole scale using 2-hydroxyethyl methacrylate grafted polyethylene supports.

The multipin peptide synthesis procedure has been adapted to allow the synthesis of peptides at micromole loadings. The original solid pin support was replaced with a detachable crown-shaped polyethylene support with an increased surface area. In addition, the polyethylene crowns were radiation-grafted with 2-hydroxyethyl methacrylate monomer instead of acrylic acid to yield hydroxy functionalized supports with a larger concentration of polymer and hence a larger peptide capacity. Fmoc-beta-Alanine was directly esterified to the HEMA hydroxy groups with subsequent addition of a diketopiperazine-forming handle for peptide attachment. Peptides varying in length from 10 to 25 residues were assembled at a number of loadings from 1.0 to 2.2 mumol. Purity of peptides at all loadings was equal to, and in some instances superior to, that achieved on conventional solid-phase supports.

Amino Acid Sequence↗

PubMed1991

Synthesis of peptide analogues using the multipin peptide synthesis method.

Modification of the multipin peptide synthesis method which allows the simultaneous synthesis of large numbers of different peptide analogues is described. Peptides were assembled on polyethylene pins derivatized with a 4-(beta-alanyloxymethyl)benzoate (beta-Ala-HMB) handle. For comparative purposes, peptides were also assembled on the diketopiperazine-forming handle N epsilon-(beta-alanyl)lysylprolyloxylactate. In model studies it was demonstrated that beta-Ala-HMB-linked peptides were cleaved from polyethylene pins with dilute sodium hydroxide or 4% methylamine/water to yield analogues with beta-Ala-free acid (beta-Ala-CO2H) and beta-Ala-methylamide (beta-Ala-CONHCH3), respectively. To assess the suitability of this approach for T-cell determinant analysis, analogues of a known T-cell determinant were synthesized with the various C-terminal endings. Peptides were characterized by amino acid analysis and fast atom bombardment-mass spectrometry. HPLC of the crude cleaved peptides indicated that 22 of the 24 peptides were greater than 95% pure. These crude peptide solutions were nontoxic in sensitive cell culture assays without further purification. All three cleavage procedures gave comparable activities in T-cell proliferation assays. These results demonstrate the potential of the multipin peptide synthesis method for the production of large numbers of different peptide analogues.

Amino Acid Sequence↗

PubMed1990

Phosphorylation of src-phosphopeptides by casein kinases-1 and -2: favourable effect of phosphotyrosine.

The synthetic phosphotyrosyl tridecapeptide H-Arg-Leu-Ile-Glu-Asp-Asn-Glu-Tyr(P)-Thr-Ala-Arg-Gln-Gly-OH, reproducing a major phosphoacceptor site of protein tyrosine kinases of the src-family, can be phosphorylated at Thr-9 by both casein kinases -1 and -2. Its shorter derivative H-Asn-Glu-Tyr(P)-Thr-Ala-OH is not affected by casein kinase-1 while representing a substrate as good as the tridecapeptide for casein kinase-2. The unphosphorylated analogue H-Asn-Glu-Tyr-Thr-Ala-OH, however, is a much poorer substrate, and no significant phosphorylation could be observed of its O-methyl ether derivative H-Asn-Glu-Tyr(Me)-Thr-Ala-OMe. These data on one side corroborate the concept that casein kinase-1 recognizes residues located on the C-terminal edge of acidic stretches, providing, on the other, the evidence that phosphotyrosyl side chains can act as specificity determinants for casein kinase-2.

Amino Acid Sequence↗

PubMedDate not supplied

Multiple synthesis by the multipin method as a methodological tool.

The multipin method of peptide synthesis is demonstrated as a potent methodological tool, where large numbers of comparative studies can be performed concurrently. Two studies are presented. In each study, the test peptides were simultaneously synthesized, and the products examined by high throughput ion spray mass spectrometry and reverse-phase HPLC. In the first study, comprising 24 experiments, peptides 1 (AELFSTHYLAFKEDYSQ-NH2) and 2 (LKDFRVYFREGRDQLWKGPG-NH2) were prepared using Fmoc-Axx/BOP/HOBt/NMM [100 : 100 : 100 : 150 mM) and Fmoc-AXX/HATU/HOAt/NMM (100 : 100 : 100 : 150 nM) with 60, 90 and 120 min coupling times. The two reagent combinations were found to give comparable results. The second study compared the N-terminal coupling of Fmoc-Asn-OH, Fmoc-Asn(Mbh)-OH, Fmoc-Asn(Mtt)-OH, Fmoc-Asn(Tmob)-OH and Fmoc-Asn(Trt)-OH in the synthesis of seven test peptides: 3, NVQAAIDYIG-cyclo(KP): 4. NTVQAAIDYIG-cyclo(KP): 5. NRVYVHPFNL: 6. NRVYVHPFHL: 7. NEAYVHDAPVRSLN: 8. NQLVVPSEGLYLIYSQVLFK; 9, NPNANPNANPNA. A total of 33 experiments were performed. Peptides 3 and 4 were selected to highlight the effect of steric bulk of each Asn derivative on coupling efficiency. Reagent efficiency, as measured by target peptide purity, was as follows: Fmoc-Asn(Tmob)-OH > Fmoc-Asn-OH > Fmoc-Asn(Mtt)-OH = Fmoc-Asn(Trt)-OH > Fmoc-Asn(Mbh)-OH.

Amino Acid Sequence↗

PubMedDate not supplied

Systematic screening for bioactive peptides.

Using simultaneous multiple peptide synthesis by the multipin approach, the feasibility of systematic large-scale pharmacological screening of peptide ligands was investigated. The method involves the assembly of small quantities of peptides (ca. 50 nmol) on plastic pins derivatized with an ester linker based on glycolate and 4-(hydroxymethyl)benzoate. These esters are stable under peptide synthesis and side-chain deprotection conditions but cleave under relatively mild basic conditions to generate peptides having C-terminal acid, amide and methylamide. A two-step approach to side-chain deprotection and cleavage from the solid support allows potentially toxic reagents to be removed (washed) from the peptide prior to cleavage. Consequently, the resulting peptide solutions can be used in bioassays with minimal processing. A series of angiotensin II and substance P analogs were synthesized and evaluated in an in vivo rat model and in vitro radioreceptor assay, respectively. Structure-activity studies on analogs of these bioactive peptides are well documented. The data obtained were consistent with that reported in the literature.

Amino Acid Sequence↗

PubMedDate not supplied

Effects of end groups on the stimulatory capacity of minimal length T cell determinant peptides.

We studied the effect of end groups on the capacity of two closely related peptides to stimulate a human T cell clone. Using multipin peptide synthesis technology, we generated solution phase peptides with six combinations of end groups for each sequence. The end modifications examined were amino-terminal acetylation, carboxy-terminal methylamidation or the addition of a dipeptide containing a diketopiperazine ring. The response to the less stimulatory of the two peptide sequences was significantly increased by acetylation of the amino-terminus, a finding which was consistent at different peptide doses. Amino-terminal acetylation was found to be more significant in affecting responses than any of the carboxy-terminal modifications tested. The use of peptides with a diketopiperazine ring structure at the carboxy-terminus did not interfere with presentation and recognition of peptides and may enhance the effectiveness of peptides for T cell epitope scanning.

Amino Acid Sequence↗

PubMedDate not supplied

Larger scale multipin peptide synthesis.

The multipin peptide synthesis approach originated as an immunological tool for epitope mapping. However, continuing evolution of the basic technology has allowed synthesis at scales up to 10 mumol per pin. At this loading, the methodology can no longer be considered just a screening tool. The overall synthesis efficiency of this approach was assessed by the synthesis of 2913 different peptides having little or no sequence homology and ranging up to a 46-mer in length. High performance liquid chromatography analysis of the crude peptides indicates overall quality of synthesis was high. The method is suitable for multi-milligram synthesis of peptides without sacrificing any of the inherent advantages of the 96-well format.

Amino Acid Sequence↗