Gary R. Matsueda

Urethane protected derivatives of 1-guanylpyrazole for the mild and efficient preparation of guanidines

Abstract Bis-urethane protected derivatives of 1-guanylpyrazole were prepared and found to readily react with relatively amines at room temperature to produce bis-protected guanidines in good yields. Simultaneous removal of both protecting groups from these products efficiently produced monosubstituted guanadines.

Preparation of peptide-protein immunogens using N-succinimidyl bromoacetate as a heterobifunctional crosslinking reagent.

Synthetic peptides derived from human fibrin were unidirectionally conjugated to three carrier proteins (bovine serum albumin, bovine alpha-lactalbumin, and keyhole limpet hemocyanin) by a method that employs N-succinimidyl bromoacetate. This heterobifunctional crosslinking reagent was prepared with a 79% yield in gram quantities from inexpensive starting materials. With this reagent, carrier proteins were first bromoacetylated, then reacted with the thiol groups of cysteine-containing peptides. The extent of peptide conjugation was assessed by amino acid analysis after acid hydrolysis, which liberated 1 mol of S-carboxymethylcysteine for each mole of thioether linkage between peptide and protein. The results of several conjugation experiments indicated that the efficiency of peptide incorporation ranged between 22 and 37% based on the recovery of S-carboxymethylcysteine relative to lysine. When the conjugates were used as immunogens, the S-carboxymethyl linkage was not antigenic in comparison with the S-maleimidobenzoyl linkage, even though their antipeptide immunoreactivities were similar.

Inhibition of clot-bound alpha 2-antiplasmin enhances in vivo thrombolysis.

Recent experiments in vitro have shown that inhibition of human alpha 2-antiplasmin by a monoclonal antibody (MAb RWR) markedly enhances clot lysis by plasminogen activators. To extend these studies in vivo, we tested whether inhibition of clot or fibrin-bound alpha 2-antiplasmin by MAb RWR could enhance the lysis of a human clot by tissue-type plasminogen activator (t-PA) in a rabbit jugular vein thrombosis model. Compared with a saline placebo or a control antibody, MAb RWR significantly increased thrombolysis by endogenous plasminogen activator in rabbits to which no t-PA was administered (p less than 0.05). In rabbits that received t-PA, the combination of MAb RWR and t-PA caused significantly greater thrombolysis than equivalent doses of t-PA alone (p less than 0.05). However, compared with equipotent doses of t-PA alone, the combination of MAb RWR and t-PA did not increase the nonspecific consumption of fibrinogen. These experiments suggest that the combination of an alpha 2-antiplasmin inhibitor and a plasminogen activator could be a more potent thrombolytic strategy.

Characterization of adhesion of "resting" and stimulated platelets to fibrinogen and its fragments.

Abstract Adhesion of resting and stimulated platelets to immobilized fibrinogen (Fg) was characterized using various forms of Fg, receptor peptide mimics, and antibodies to glycoprotein (GP) IIb/IIIa and Fg. Resting platelets adhered to Fg, but to less than half the extent of the same platelets stimulated with epinephrine/ADP. The adhesion of resting and stimulated platelets to Fg was inhibited by a receptor peptide mimic (G13, a peptide corresponding to residues 300–312 of GPIIb), anti-GPIIb/IIIa antibodies, and a monoclonal antibody (4A5) against the carboxyl terminus of the γ chain of Fg. The results presented here demonstrate that the γ chain RGD platelet recognition sites are not required to mediate the adhesion of either stimulated or resting platelets to immobilized Fg. Although stimulated platelets can adhere extensively to monomeric Fg containing one functional γ chain, resting platelets require bivalent Fg containing two functional γ chains to mediate irreversible adhesion to Fg.

Increased specificity in human cardiacmyosin radioimmunoassay utilizing two monoclonal antibodies in a double sandwich assay

In some instances, even the increased resolution that may be afforded in immunoassays by the use of monoclonal antibodies fails to effect resolution among molecules that share many epitopes. An immunoradiometric assay that simultaneously measured two different epitopes on the same molecule was devised to overcome this difficulty in the differentiation between cardiac- and skeletal-myosin light chains. Three monoclonal antibodies were examined that were 100% (1C5), 25% (2B9) and 17% (4F10) cross reactive, respectively, between the two antigens. One antibody of the pair to be studied was immobilized to cyanogen bromide-activated Sepharose 4B while the other was iodinated with 125I using the lactoperoxidase method. The antigen was mixed with the immobilized antibody, the labeled antibody was added and the precipitate then washed and counted in a gamma counter. When both antibodies of the pair to be studied (immobilized and labeled) were the same (2B9), no radioactivity above background was bound to the precipitate, indicating that the second antibody could not bind to an already occupied epitope. When two different antibodies were employed, the specificity of the assay increased over that of a single antibody. The cross reactivity of a pair approximated the product of the cross reactivities of the individual antibodies. Thus, 1C5 and 2B9 were 25% cross reactive together, 1C5 and 4F10 17% cross reactive, and 2B9 and 4F10 4.3% cross reactive.

The use of an internal reference amino acid for the evaluation of reactions in solid-phase peptide synthesis

Abstract Disubstituted (hydroxymethylphenylacetamidomethyl-; N -acetyl-α-aminobutyramidomethyl-) copoly(styrene-1% divinylbenzene) in bead form has been prepared for use in solidphase peptide synthesis. As expected, this resin retained resistance to acidolysis described by Mitchell et al. (J. Amer. Chem. Soc. 98 , 7357–7362, 1976). An additional feature of this resin is an internal reference amino acid which could be quantified following propionic acid: HCl hydrolysis for 4 h. Aminobutyric acid (Abu) was found to be stable during peptide synthesis, in particular, treatment with TFA:CH 2 Cl 2 (1:1) for 90 h at 25°C and HF:anisole (9:1) for 60 min at 0°C. The ratios of an amino acid to Abu could be used to evaluate the attachment of an amino acid to the resin, the incorporation of a subsequent amino acid, and finally, eleavage of the peptide from the resin. It is noteworthy that the weight of resin in a peptidyl resin sample could be calculated by using Abu recovery values, if the initial substitution of Abu on the resin was known. This resin which contained Abu, has been used for the extended synthesis of a heavy chain variable region from a homogeneous rabbit antibody.

An Efficient Method for The Preparation of w, w′-bis-Urethane Protected Arginine Derivatives

Abstract Fmoc-Argw, w′(Boc)2-OH and Boc-Argw, w′(Cbz)2-OH were prepared in two steps: Guanylation of Cu(II)-ornithine complex at Nδ with N, N′bis-urethane protected derivatives of 1-guanylpyrazole, followed by Nα-protection using either 9-fluorenylmethyl succinimidylcarbonate or di-t-butyl-dicarbonate in the presence of EDTA. The overall yields of these products (2 steps) were 65% and 73%.

Induction of fibrin-specific antibodies by immunization with synthetic peptides that correspond to amino termini of thrombin cleavage sites

Fibrin-specific antibodies have been produced in rabbits which were immunized with synthetic peptides. Specificity against human fibrin monomer was achieved because the synthetic peptide haptens were derived from sites unique to fibrin as compared with fibrinogen. Two undecapeptides were chemically synthesized according to the amino acid sequence of the amino termini of human fibrin alpha- and beta-chains which are revealed by thrombin cleavage. Rabbits immunized with either an alpha- or beta-chain peptide conjugate produced anti-peptide sera which reacted with fibrin monomer. Following immunoadsorption of the rabbit sera with human fibrinogen-Sepharose, fibrin-specific antibodies were detectable by solid-phase radioimmunoassay that did not react with fibrinogen. Antisera elicited by clotted human fibrin contained antibodies that reacted with fibrin and fibrinogen when treated in a similar manner.

Selectivity of angiotensin II antisera

A significant problem in the immunoassay of angiotensin II is the cross-reactivity of most available antisera with the peptides metabolic products, (des-Asp1)-angiotensin II and (des-Asp1.Arg2)-angiotensin II. In order to attempt to generate antisera of greater selectivity, a variety of conjugates between angiotensin II or derivative peptides and carrier proteins were examined as immunogens with the aim of generating antisera that would selectively identify the amino terminal region of the peptide. Selectivity for the amino terminus was achieved by either (1) immunization with N-acetylated angiotensin II-amide which had been coupled to rabbit serum albumin by its carboxy terminus, or (2) immunization with angiotensin-(1-7)-heptapeptide which was randomly coupled to thyroglobulin. The antisera produced with the N-acetylated immunogen cross-reacted with the unacetylated ligand (Asn1-Val5)-angiotensin, but did not recognize the human hormone (Asp1,Ile5)-angiotensin. Carboxy-terminal coupling of angiotensin without N-acetylation did not induce selectivity for the amino terminus, nor did a conjugate which was linked to the carrier protein via a diazo bond to His6 of the peptide. These findings may be explained by the fact that N-acetylated angiotensin II resists degradation by amino peptidases and thus retains its structure in the immunogen and by the fact that the (1-7)-heptapeptide has lost the immunodominant carboxy-terminal epitope, thus emphasizing the desired amino terminal determinant.

N,N-Diisopropyl-bis[2-(trimethylsilyl)ethyl]phosphoramidite. An attractive phosphitylating agent compatible with the Fmoc/t-butyl strategy for the synthesis of phosphotyrosine containing peptides

Abstract A new phosphitylating agent, compatible with the Fmoc/t-butyl strategy for the production of phosphotyrosine containing peptides was synthesized. Our results demonstrated that high yields and high purities of phosphotyrosine peptides can be obtained using this phosphitylating agent.