Mercuric Reagent Research Articles

Palladium-Catalyzed Synthesis of (E)-5-(3-Aminoallyl)-Uridine-5'-O-Triphosphates.

This unit describes a simple, reliable, and efficient chemical method for the synthesis of 5-(3-aminoallyl)-2'-deoxyuridine-5'-O-triphosphate (AA-dUTP) and 5-(3-aminoallyl)-uridine-5'-O-triphosphate (AA-UTP), starting from the corresponding nucleoside triphosphate. The presented strategy involves regioselective iodination of nucleoside triphosphate using N-iodosuccinimide followed by the palladium-catalyzed Heck coupling with allylamine to provide the corresponding (E)-5-aminoallyl-uridine-5'-O-triphosphate in good yields. It is noteworthy that the protocol not only provides a high-purity product but also eliminates the use of toxic mercuric reagents. © 2017 by John Wiley & Sons, Inc.

Expression inEscherichia coli, Refolding, and Purification of the Recombinant Mature Form of Human Matrix Metalloproteinase 7 (MMP-7)

In the latent pro-form of matrix metalloproteinase 7 (MMP-7), the cysteine residue in the pro-peptide binds the active-site zinc ion. Hence, recombinant active MMP-7 was prepared from pro-MMP-7 by modification of this cysteine residue with a mercuric reagent. In this study, mature MMP-7 was expressed in Escherichia coli as inclusion bodies, solubilized, and refolded with 1 M L-arginine. The purified product was indistinguishable from the one prepared from pro-MMP-7 as assessed by hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH(2).

Characterization of ATPase activity of a hepatitis C virus NS3 helicase domain, and analysis involving mercuric reagents.

The C-terminal two-thirds of nonstructural protein 3 (NS3) of hepatitis C virus (HCV) exhibits RNA-dependent NTPase/helicase activity. This enzyme is considered to be involved in viral replication and is expected to be one of the target molecules of anti-HCV drugs. In a search for NTPase inhibitors specific to HCV, we expressed and purified the truncated NS3 NTPase/helicase domain. Here, we report the characterization of its RNA-dependent ATPase activity. This enzyme preferred Mg(2+) and the optimal pH was 7.0. We further investigated the effects of heavy metal ions on the ATPase activity. The mercuric ion inhibited it significantly, the 50% inhibitory concentration being 49 nM. The fact that the inhibitory profile was competitive and that this inhibition was blocked in the presence of a large excess of cysteine or dithiothreitol, suggested that a cysteine residue in the DECH box was the main target site of mercury.

Refolding and recovery of recombinant human matrix metalloproteinase 7 (matrilysin) from inclusion bodies expressed by Escherichia coli.

The recombinant prepro-form of human matrix metalloproteinase 7 (matrilysin or MMP-7) was overexpressed in Escherichia coli as insoluble inclusion bodies. The recombinant protein was refolded by 100-fold dilution after solubilization with 6 M guanidine HCl. The refolding was monitored by the recovery of matrilysin activity. The addition of either 1.0 M arginine or 0.1% Brij-35 promoted remarkably the refolding. The refolding was dependent on pH and temperature, with lower temperature (<10 degrees C) and pH 6-8 preferable. Glutathione had no effect on refolding, and it was excluded from the refolding conditions. Starting with inclusion bodies (2.0 g, wet) containing 360 mg protein, 29.5 mg of pro-matrilysin (30 kDa) was obtained after refolding with 1.0% Brij-35 at pH 7.5 and 4 degrees C for 12 h. Pro-matrilysin (24.0 mg) was purified to homogeneity by cation-exchange HPLC with a 15-fold increase in purity and an activity yield of 81.3%. Pro-matrilysin was converted entirely to matrilysin (19.0 kDa; 15.2 mg) by activation with a mercuric reagent. The activity (k(cat)/K(m)) of matrilysin was 1.7 x 10(5) M(-1) x s(-1).

Communication: An Improved Synthesis of Methyl 2,3-Anhydro-α and β-D-Lyxofuranosides

Methyl 2,3-anhydro-α (6) and β (7)-D-lyxofuranosides are important intermediates in the synthesis of C-3 substituted derivatives of D-arabinose which show biological activity as a tumor inhibitor.1,2 Some syntheses of 6 and 7 are reported but they are either expensive or give poor yields 3-4 and generally the authors refer to Baker and coll.5 who synthesized both α and β epoxides from D-xylose in five steps; yields were 28% and 22% respectively. This synthesis is very well described but reaction times and workups are long and several intermediates are distilled with difficulty under reduced pressure. Unger and coll.6, using Baker's method, improved the yield of compound 6 and Martin and coll.7 described a three steps synthesis of 6 but the final purification is very difficult and the use of mercuric reagents is not consistent with biological activity; furthermore these two publications concern only α anomer 6.

A New Spectrophotometric Method for the Determination of Micro Amounts of Sulfite

Abstract A new spectrophotometric method for the determination of a minute quantity of sulfite was studied. When a definite amount of aqueous solution of mercuric nitrate mixed with potassium bromide and an ethanol solution of diphenylcarbazone are added to the sample solution kept at pH 7.0, sulfite reacts with mercuric reagent and the excess of the mercuric reagent forms the reddish-violet complex with diphenylcarbazone which may be easily extracted by benzene. Sulfite is determined by measuring the absorbancy of the benzene phase which decreases upon an increase in the concentration of sulfite, at the wavelength of 562 mμ, The method which is applicable to the determination of both sulfite and sulfide has also been established, because sulfide as well as sulfite reacts with mercuric reagent. By this proposed method, sulfite (0.1—2.5 ppm) and sulfide (0.01—0.5 ppm) could be determined at the same time rapidly and accurately. The composition of the complex formed between mercury(II) and sulfite was also studied.