Harri G. Ramjit

Large-scale purification and characterization of recombinant tick anticoagulant peptide

Recombinant tick anticoagulant peptide (r-TAP), a potent and specific inhibitor of blood coagulation factor Xa, was purified to greater than 99% homogeneity at the multi-gram scale. Genetically engineered yeast secreted 200-250 mg/l of the heterologous protein into the medium. Cells were separated from broth by diafiltration and purification was done by two chromatographic steps, both conducive to operation on a large scale. Analysis of the purified protein by several methods indicated that it was greater than 99% homogeneous and no incompletely processed or truncated proteins were detected. Physico-chemical characterization data of r-TAP show that it exists as a monomer in solution and no evidence of post-translational modification was observed. The purified protein was fully active in inhibiting human coagulation factor Xa.

Utilization of the chiral synthon, methyl 3-O-benzyl-2,4,6-trideoxy-6-iodo-α-D-erythro-hexopyranoside in the synthesis of a potent HMG-CoA reductase inhibitor

Abstract Compound 1 , a potent synthetic HMG-CoA reductase inhibitor, has been synthesized from the chiral synthon, methyl 3-O-benzyl-2,4,6-trideoxy-6-iodo-α-D- erythro -hexopyranoside ( 2 ), utilizing a novel palladium-mediated procedure for benzyl ether cleavage.

Trends in Kinetic Behavior during Ester-Ester Exchange Reactions in Polyesters by Mass Spectrometry. I

Abstract The kinetics of the ester-ester exchange reaction between polyesters from adipic acid and various linear and branched glycols were investigated by mass spectometry using the dimer analysis method (DAM). Rate constants, activation energies, and frequency factors are given for reactions studied in the temperature range of 572–585 K. Correlation of glycol methylene ratios with activation energies and frequency factors shows an alternating trend in kinetic behavior. Reaction systems containing even numbers of methylene groups in the glycol moiety of the reactants exhibited slower reaction rates than systems with odd numbers of methylene groups, while branched reaction systems followed very similar trends when the influence of pendant groups is ignored.

HIV-1 protease inhibitory activity of L-694,746, a novel metabolite of L-689,502

L-689,502 is a potent inhibitor of HIV-1 protease activity in vitro. Microbial biotransformations of L-689,502 by cultures belonging to the genus Streptomyces sp. were performed. Extracts of culture broths were examined for the production of metabolites of L-689,502 that could inhibit HIV-1 protease activity. One culture, MA 6804 (Streptomyces lavendulae, ATCC 55095), produced L-694,746 that, while being structurally related to L-689,502, is a novel metabolite and a potent inhibitor of HIV-1 protease.

The Influence of Stereochemical Structure on the Kinetics and Mechanism of Ester-Ester Exchange Reactions by Mass Spectrometry. II

Abstract Exter-ester exchange reactions in polyesters, formulated from adipic acid and various linear and branched glycols, were studied by mass spectrometry employing the Dimer Analysis Method (DAM). Activation energies (E) and frequency factors (A) were obtained for the reaction systems at temperatures of 572–585 K. The kinetic behavior of these reactions was influenced by the stereochemical structures of carbonyl oxygens along the polymer chain backbone. Syndiotactic/syndiotactic systems reacted slower than syndiotactic/heterotactic and heterotactic/heterotactic systems, respectively. Correlation of frequency factors (A) and stereochemical structures showed an alternating trend in kinetic behavior. An “associative reaction mechanism” was postulated since it satisfied the energetic and geometric requirements necessary for the simultaneous making and breaking of alkoxide bonds, and also gives a good fit to the kinetic expression derived for an opposing bimolecular reaction.

Sensitive gas—liquid chromatographic procedure for urinary Nτ-methylimidazole acetic acid, an index of histamine turnover

A simple and specific gas-liquid chromatographic procedure, compatible with both nitrogen-phosphorous and electron-capture detection, and employing conventional packed columns, has been devised for urinary 1-methylimidazole-4-acetic acid (N tau-MeImAA), an index of whole-body histamine turnover. N tau-MeImAA is isolated by ion-exchange on Dowex 1 (CH3COO-), esterified by reaction with chloroethanol-boron trichloride and, depending upon detection employed, chromatographed on base-deactivated SP-2401 or SP-2250. [3H]N tau-MeImAA serves as internal recovery standard.

Kinetics of Disulphide–Disulphide Interchange Reactions in Polysulphide Polymers by Mass Spectrometry. I

A novel analytical method using Electron Ionization Mass Spectrometry (EI/MS) was developed for studying the kinetics of disulphide–disulphide interchange reactions in polysulphide polymer blends. During the disulphide–disulphide interchange process, the concentration or reactants and products were measured from the observed peak heights of the respective dimer units. Reactions were studied in melt blends, over a temperature range of 207–220°C. An activation energy of 79.80 KJmol−1 and pre-exponential factor log A=2.54 was observed. The dominant relaxation process for the intermolecular disulphide–disulphide interchange reaction in the polymer blend, without the influence of solvents and other reagents, is assumed to follow an associative mechanism. The model polysulphide polymers A (PS1) and B (PS2), used in this study, were formulated from bis (2-chloroethyl) ether and bis (2-chloroethoxy) methane, respectively.

Mechanism of Disulfide–Disulfide Interchange in Polysulfide Polymer Melt Blend by Electron Ionization Mass Spectrometry. II

Abstract The degree of randomization, q, of structural units in melt blends of the polysulfide homopolymers A(PS1) and B(PS2), wherein the disulfide equivalents D A/D B = 1, were studied by electron ionization mass spectrometry. Over the temperature range of 207–219°C, the relaxation process, due to the dominant disulfide–disulfide interchange reactions, is postulated to follow an associative reaction mechanism. These intermolecular disulfide–disulfide interactions promote a transient enhancement of the sulfur rank in the activated complex resulting in formation of the randomized co‐polymer AB. The mass spectrometric experimental design enabled measurement of concentrations of reactants A(PS1) and B(PS2), as well as the randomized copolymer AB, by monitoring the abundance of dimer units a2, b2, and ab, respectively as a function of time. The degree of randomization, q, was observed in the absence of catalysts or solvents, notwithstanding the solvent/solute and solute/solvent characteristics of the polymer melt blend. The mechanism of this randomization process, was rationalized on the basis of the properties of sulfur, aided by the observation of macrocyclic monomeric and dimeric units during the retro‐polymerization reactions under the EI/MS conditions employed. The model polysulfide polymers A(PS1) and B(P52), used in this study were synthesized from bis(2‐chloroethyl)ether and bis(2‐chloro ethoxy)methane, respectively.

Design and Synthesis of a Selective PSA Cleavable Peptide-Doxorubicin Prodrug Which Targets PSA Positive Tumor Cells

Prostate cancer is the second leading cause of cancer mortality in males. It is estimated that 37,000 men died of prostate carcinoma in the United States in 1999 [1]. While cancer that is confined to the prostate can be treated with surgery or radiation, the prognosis for metastatic disease is poor.