Search PubMed⌕ Search

Biomedical subjects

J C Spetzler

Publications and source records attributed to J C Spetzler.

9 recordsLinked to original sources
PubMed1999

Preparation and application of O-amino-serine, Ams, a new building block in chemoselective ligation chemistry.

The non-codable amino acid O-amino-serine, Ams, has been prepared in both L- and D-forms as the orthogonally protected derivative, Fmoc-Ams(Boc)-OH (1 and 2). This new amino acid derivative is useful for chemoselective ligations. Under acidic conditions and in the presence of all other common amino acid functionalities, the oxyamine function selectively forms oxime linkages with aldehydes. The Ams residue has been incorporated into both ends of the peptide sequence Asp-Leu-Trp-Gln-Lys using standard SPPS. The deprotected peptide has been used for chemical ligation to afford a peptide dimer as well as a glycopeptide. Ams racemization was found to be negligible, as monitored by HPLC separation of Ams dipeptide diastereomers.

Amino Acid Sequence↗
PubMed1998

Kinetic analysis of the mechanism and specificity of protein-disulfide isomerase using fluorescence-quenched peptides.

Protein-disulfide isomerase (PDI) is an abundant folding catalyst in the endoplasmic reticulum of eukaryotic cells. PDI introduces disulfide bonds into newly synthesized proteins and catalyzes disulfide bond isomerizations. We have synthesized a library of disulfide-linked fluorescence-quenched peptides, individually linked to resin beads, for two purposes: 1) to probe PDI specificity, and 2) to identify simple, sensitive peptide substrates of PDI. Using this library, beads that became rapidly fluorescent by reduction by human PDI were selected. Amino acid sequencing of the bead-linked peptides revealed substantial similarities. Several of the peptides were synthesized in solution, and a quantitative characterization of pre-steady state kinetics was carried out. Interestingly, a greater than 10-fold difference in affinity toward PDI was seen for various substrates of identical length. As opposed to conventional PDI assays involving larger polypeptides, the starting material for this assay is homogenous. It is furthermore simple and highly sensitive (requires less than 0.5 microgram of PDI/assay) and thus opens the possibility for quantitative determination of PDI activity and specificity.

Amino Acid Sequence↗
PubMed1998

Preparation of fluorescence quenched libraries containing interchain disulphide bonds for studies of protein disulphide isomerases.

Protein disulphide isomerase is an enzyme that catalyses disulphide redox reactions in proteins. In this paper, fluorogenic and interchain disulphide bond containing peptide libraries and suitable substrates, useful in the study of protein disulphide isomerase, are described. In order to establish the chemistry required for the generation of a split-synthesis library, two substrates containing an interchain disulphide bond, a fluorescent probe and a quencher were synthesized. The library consists of a Cys residue flanked by randomized amino acid residues at both sides and the fluorescent Abz group at the amino terminal. All the 20 natural amino acids except Cys were employed. The library was linked to PEGA-beads via methionine so that the peptides could be selectively removed from the resin by cleavage with CNBr. A disulphide bridge was formed between the bead-linked library and a peptide containing the quenching chromophore (Tyr(NO2)) and Cys(pNpys) activated for reaction with a second thiol. The formation and cleavage of the interchain disulphide bonds in the library were monitored under a fluorescence microscope. Substrates to investigate the properties of protein disulphide isomerase in solution were also synthesized.

Cystine↗
PubMed1995

Unprotected peptides as building blocks for branched peptides and peptide dendrimers.

We describe two new site-specific ligation methods for preparing branched peptide dendrimers such as multiple antigen peptide (MAP). Both methods are based on the general approach of exploiting the specific reaction between a weak base and an aldehyde under acidic conditions so that unprotected peptides can be used as building blocks. A weak base such as benzoyl hydrazine or 1,2-amino thiol of cysteine was attached to the N-terminal of an unprotected peptide as nucleophile to react with the alkyl aldehyde on the core matrix of MAP to form a stable hydrazone linkage or a five-membered thiazolidine ring, respectively. Two synthetic peptides rich in basic amino acids such as lysine and arginine were used as models in the ligation reactions in solution to give peptide dendrimers containing four or eight copies of peptide immunogens. The resulting macromolecules with the MW ranging from 5 to 16 kDa were unambiguously characterized by laser-desorption mass spectrometry. Furthermore, we also optimized the conditions of these ligation reactions using elevated temperature and a water-miscible organic co-solvent to give a combination of rate enhancement about 10 fold. These optimizations allowed the ligation reactions to be completed in < 4 h instead of 2-3 days. Our ligation approach also has the advantages of flexibility so that peptides can be attached through the amino or carboxyl terminus to the core matrix. The phenyl hydrazone linkage and the five-membered ring were found to be stable at physiological pH suitable for immunization. Thus our results provide two practical and useful methods for the synthesis of macromolecular peptide dendrimers for vaccines, artificial proteins, and enzymes.

Aldehydes↗
PubMed1995

Chemoselective approaches to the preparation of peptide dendrimers and branched artificial proteins using unprotected peptides as building blocks.

Peptide dendrimers such as Multiple Antigen Peptides (MAPs) are artificial proteins with branched architectures. They hold promise in biochemical and biomedical applications such as synthetic vaccines, serodiagnostics and intracellular delivery of peptides. We have shown that a new design of MAPs containing lipidated built-in adjuvant can be delivered by oral administration to elicit systemic and mucosal immunoglobulins as well as cytotoxic T-lymphocytes. For synthetic vaccines, it is desirable to obtain highly homogeneous preparations. To provide the precision and chemical unambiguity of this class of artificial proteins, we have devised several chemoselective approaches by thiol and carbonyl chemistries to facilitate their synthesis using unprotected peptide segments as building blocks and ligating them to the core matrix. This paper describes the methods of preparation, comparative studies of stability, and presents results of the preparation of antigens containing preformed multiple disulfides.

Aldehydes↗
PubMed1994

A novel strategy for the synthesis of the cysteine-rich protective antigen of the malaria merozoite surface protein (MSP-1). Knowledge-based strategy for disulfide formation.

The most promising antigen for a protective malaria vaccine is a cysteine-rich domain at the carboxyl terminus of the merozoite surface protein (MSP-1). Passive transfer of anti-MSP-1 antibody or immunization of MSP-1 against infection challenge confers protection in primate and rodent models. The antigen belongs to the three-disulfide epidermal growth factor (EGF) family based on the alignment of the six cysteines. In the K1 strain there are, however, only four cysteines corresponding to the four carboxyl cysteines of EGF. Furthermore, disulfide pairing would produce a non-EGF pattern. Because this cysteine-rich antigen is conformation-dependent, and reduction of the disulfide bonds abolishes antigenicity, we used a synthetic analog to investigate the probable disulfide pairing of this antigen. This paper describes the synthesis, folding and disulfide pairings of two 50-residue cysteine-rich peptides. One contains two disulfides (VK-50) derived from the native sequence of MSP-1 of the Thailand K1 strain (aa 1629-1679). The other contains an EGF-like, three-disulfide [Cys-9,14]VK-50 peptide. Both peptides were synthesized by a solid-phase method using Fmoc-chemistry. The crude peptide of VK-50 was folded, and the disulfide was oxidized by the DMSO method to obtain a structure with an expected disulfide pairing of 3-4, and 5-6. The specific pairing pattern of 1-3, 2-4 and 5-6 in [Cys 9,14]VK-50 corresponding to EGF in [Cys 9,14]VK-50 was obtained using a 'knowledge-based' (KB) strategy for their formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMedDate not supplied

Synthetic hFSH peptide constructs in the evaluation of previous studies on the hFSH receptor interaction.

The human follicle-stimulating hormone (hFSH) belongs to a family of glycoprotein hormones which contains two non-identical subunits. This paper describes the design and synthesis of a series of synthetic hFSH constructs as putative ligands for the receptor. The design of these constructs is based on the crystal structure of hCG and molecular modelling using the program package Insight II/Discover. The designed constructs contain peptides ranging from 7 to 48 amino acid residues, disulphide bridges and glycan residues. All the synthetic peptides were synthesized by the stepwise solid-phase method using Fmoc chemistry. Two of the synthetic peptides contain the glycosylated amino acid. Asn(GlcNAc-GlcNAc) and both were prepared using fully protected glycosylated building blocks in the solid-phase peptide synthesis. The disulphide bridges were formed from acetamidomethyl-protected glycopeptides and peptides by a direct deprotection/oxidation method using thallium(III) trifluoroacetate. Mass spectroscopy and amino acid analysis were used for characterization of the synthetic hFSH glycopeptides and peptides. The synthetic hFSH constructs were tested for binding activity on FSH receptor assays but none showed improved binding properties compared with the naturally occurring hormone. It was finally demonstrated that non-related peptides showed non-specific binding at the same level as reported for specific peptides.

Amino Acid Sequence↗
PubMedDate not supplied

Self-assembly of cyclic peptides on a dendrimer: multiple cyclic antigen peptides.

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.

Amino Acid Sequence↗