Solid-phase synthesis of arginine-containing peptides and fluorogenic substrates using a side-chain anchoring approach

Attachment of an amino acid to a solid support by its side chain is sometimes necessary to take advantage of an alpha-carboxylic group available for diverse modifications, including the incorporation of a fluorophore for the preparation of fluorogenic substrates. In contrast to most other amino acids, anchoring the guanidinium group of an arginine to a resin requires the use of a supplementary linker. To avoid the usually multistep synthesis of such a linker as well as its difficult attachment to the guanidine group, we developed a simple method where the guanidine group is built on a Rink amide resin. Our strategy followed the steps of guanidine formation: (i) addition of an isothiocyanate derivative of ornithine to the amino group of a solid support, yielding Nomega-linked thiocitrulline; (ii) S-methylation of thiourea; (iii) guanidinylation using ammonium acetate. Cleavage of the resin generated the arginine-containing compound, the amine group of the resin becoming part of the guanidine. We have demonstrated the usefulness of this method by the synthesis of a series of fluorogenic substrates for trypsin-like serine proteases, which were obtained in high yield and purity. Then, our strategy also allowed generation from the same precursor differentially substituted arginine derivatives, including Nomega-methyl- and Nomega-ethylarginines. The ability to prepare such analogues together with the intermediates thiocitrulline and S-methylisothiocitrulline from a unique precursor while the alpha-amine and carboxylic groups remain available for modification also makes this method a powerful tool for combinatorial solid-phase synthesis of NO synthase inhibitors.     

Evaluation of the interelemental effect in X-ray fluorescence analysis by the total addition method

Summary An algorithm for quantifying interelemental effects in X-ray fluorescence techniques is developed. By applying an addition process, the ratio between the mass absorption coefficients of the analyte and the unknown sample ( _i ^* / _s ^* ) is calculated to correct the fluorescence intensity of the element to be determined and linearize the I-c calibration plot. This coefficient can be calculated graphically and numerically. The method is applied to the determination of tin in lead alloys with good results over wide concentration ranges.Notation used Q Constant of proportionality in Eq. (4) X-ray fluorescence intensity of the I _i ^o standard - I _i ^s unknown sample - I _i ^ns dilute unknown sample - I _i ^ms unknown sample after addition of i analyte Corrected fluorescence intensity of the I _i ^cs unknown sample - I _i ^msc unknown sample after addition of i analyte Relationship of fluorescence intensity between R_i sample and standard - R _i dilute sample and standard Factor of f_m addition - f_x addition equivalent to the mass fraction of the i analyte in the unknown sample Mass absorption coefficient of _i ^* analyte - _s ^* unknown sample - _ms ^* unknown sample after addition Mass fraction of c _i ^s analyte - c _i ^ms unknown sample after addition     

Determination of inorganic cations in brine solutions by ion chromatography

The method for analysis of inorganic cations in brine solutions was developed. Ion chromatography is a well-established and accepted technique in the determination of a variety of inorganic ions. However, there are significant complications when ion chromatography is used to determine trace concentrations of inorganic ions in brine matrices. The brine solution in our study was made to simulate the solution from the Waste Isolation Pilot Plant. Instrumental parameters such as eluent composition, flow-rates, and sample loop volumes were investigated to arrive at the optimum condition for the determination of the cations with minimal dilution. Separation was carried out in a Dionex CG12A/CS12A with 8.25 mM H2SO4 as eluent at 1.2 ml/min flow-rate. Our results indicated that ion chromatography is an accurate and a good alternative method for the analysis of cations in brine solution.     

The reactivity of 2-vinylindoles with dimethyl acetylenedicarboxylate

The overall rates of reaction of 1-substltuted-1- (1-methyl-2-indolyl)ethenes with dimethyl acetylenedicarboxylate are considerably lower than those of the corresponding 2-vinyl- pyrroles. Steric interaction between the N-methyl group on the indole ring and the 1-substituent on the ethenyl group prevents the diene system adopting a coplanar $\text{cisoid}$ configuration and, thereby, inhibits the _π4 + _π2 cycloaddition reaction of the system with dimethyl acetylenedicarboxylate. Under such conditions, the indolylethene preferentially undergoes a Michael addition reaction at the 3-position. The _π4 + _π2 cycloaddition reaction is promoted at elevated temperatures. No evidence was found for a Cope rearrangement of the Michael adducts to give the dihydrocarbazole.     

Asymmetric Induction in the Synthesis of 3,4,5-Trisubstituted Ferrocenyl-4,5-dihydropyrazoles

The asymmetric induction in the synthesis of 3,4,5-trisubstituted 4,5-dihydropyrazoles with ferrocenyl substituents, starting from the E and Z isomers of ,-unsaturated ketones was studied. A high diastereoselectivity was revealed at the 1,2 chiral center chiral center induction, which is independent of the configuration of the starting chalcones.     

Separation of proteins with a molecular mass difference of 2 kDa utilizing preparative double-inverted gradient polyacrylamide gel electrophoresis under nonreducing conditions: application to the isolation of 24 kDa human growth hormone

A method for separating proteins with a molecular mass difference of 2 kDa using SDS-PAGE under nonreducing conditions is presented. A sample mixture containing several human growth hormone (hGH) isoforms was initially separated on a weak anion-exchange column. Fractions rich in 24 kDa hGH as determined by analytical SDS-PAGE were pooled and further separated by cation-exchange chromatography. The fractions pooled from the cation-exchange chromatography contained two hGH isoforms with a 2 kDa molecular mass difference according to SDS-PAGE analysis, 22 and 24 kDa hGH. The 22 and 24 kDa hGH were separated using continuous-elution preparative double-inverted gradient PAGE (PDG-PAGE) under nonreducing conditions. The preparative electrophoresis gel was composed of three stacked tubular polyacrylamide matrices, a 4% stacking gel, a 13-18% linear gradient gel, and a 15-10% linear inverted gradient gel. Fractions containing purified 24 kDa hGH were pooled and Western blot analysis displayed immunoreactivity to antihGH antibodies. PDG-PAGE provides researchers with an electrophoretic technique to preparatively purify proteins under nonreducing conditions with molecular mass differences of 2 kDa.     

Determination of promethazine hydrochloride with bromophenol blue by a turbidimetric method and flow injection analysis

A flow injection analysis procedure for the turbidimetric determination of promethazine is proposed. The sample solution is injected directly into the carrier reagent stream, which is composed of 1.16 × 10⁻³M bromophenol blue at pH 1.20. The calibration graph is linear over the range 25–197 ppm of promethazine. The influence of some foreign substances was also investigated. The method is applied to promethazine determination in a pharmaceutical formulation.     

Base promoted transformation on thiadiazolopyridinium chlorides

1,2,4-Thiadiazolo[2,3-a]pyridinium chlorides undergo a very facile base promoted transformation to give bispyridilimino-1,2,4-thiadiazolidines. The unequivocal structural assignment of these last compounds was achieved by spectroscopic ¹H, 13C and ¹⁵N two dimensional methods.