Peptide compounds analysis

The development of modem methods, suitable for the analysis of ampholytes in biological fluids, provided means for isolating from urine some chemically better defined fractions containing peptide compounds. The methods used did not, however, exclude the existence of some other forms of combined amino acids in the fractions studied. 

However, 0.5 g p-thiocresol and 0.5 mL m-cresol were used as scavengers as recommended in the literature for chloroacetylated peptide compounds (33). The crude peptide was purified by RP-HPLC and, after lyophilization, was characterized by analytical HPLC, mass spectrometry, and amino acid analysis. The pure product was obtained at 65% yield. 

The dried, partially protected crude peptide is dissolved in DMF-pyridine (4 1 v/v) and treated with DMF-SO3 (125-fold molar excess) in the presence of 1,2-ethanedithiol (100-fold molar excess). After about 15 h at room temperature, the solution is applied to a size exclusion chromatography column and eluted with DMF. The target compound is collected and the solvent evaporated to dryness. After lyophilization, the crude compound is treated with a 90% TFA-based reagent at 4°C. The reaction time of this step is optimized by monitoring the acidolytic treatment of a small aliquot of the O-sulfated peptide and analysis of the synthetic products by analytical HPLC and mass spectrometry. 

In general, the first step in virtual screening is the filtering by the application of Lipinski s Rule of Five [20]. Lipinski s work was based on the results of profiling the calculated physical property data in a set of 2245 compounds chosen from the World Drug Index. Polymers, peptides, quaternary ammonium, and phosphates were removed from this data set. Statistical analysis of this data set showed that approximately 90% of the remaining compounds had ... 

The amino add analysis of all peptide chains on the resins indicated a ratio of Pro Val 6.6 6.0 (calcd. 6 6). The peptides were then cleaved from the resin with 30% HBr in acetic acid and chromatogra phed on sephadex LH-20 in 0.001 M HCl. 335 mg dodecapeptide was isolated. Hydrolysis followed by quantitative amino acid analysis gave a ratio of Pro Val - 6.0 5.6 (calcd. 6 6). Cycll2ation in DMF with Woodward s reagent K (see scheme below) yielded after purification 138 mg of needles of the desired cyc-lododecapeptide with one equiv of acetic add. The compound yielded a yellow adduct with potassium picrate, and here an analytically more acceptable ratio Pro Val of 1.03 1.00 (calcd. 1 1) was found. The mass spectrum contained a molecular ion peak. No other spectral measurements (lack of ORD, NMR) have been reported. For a thirty-six step synthesis in which each step may cause side-reaaions the characterization of the final product should, of course, be more elaborate. 

For mixture.s the picture is different. Unless the mixture is to be examined by MS/MS methods, usually it will be necessary to separate it into its individual components. This separation is most often done by gas or liquid chromatography. In the latter, small quantities of emerging mixture components dissolved in elution solvent would be laborious to deal with if each component had to be first isolated by evaporation of solvent before its introduction into the mass spectrometer. In such circumstances, the direct introduction, removal of solvent, and ionization provided by electrospray is a boon and puts LC/MS on a level with GC/MS for mixture analysis. Further, GC is normally concerned with volatile, relatively low-molecular-weight compounds and is of little or no use for the many polar, water soluble, high-molecular-mass substances such as the peptides, proteins, carbohydrates, nucleotides, and similar substances found in biological systems. LC/MS with an electrospray interface is frequently used in biochemical research and medical analysis. 

Many individual compound reports contain infrared spectral information, but there is only one in which detailed analysis appears. The 3-hydroxytriazolopyri-dine 125 used as a catalyst for peptide coupling (Section IV.J) has been studied in the solid and in solution, in association with a crystallographic study, and shown to exist as a dimer in solution (99MI1). 

The APCl ionization regime is much more harsh that ESI and this precludes its use for the study of large biomolecules, with the mass limit for APCl being generally considered as below 2000 Da. Having said this, as will be shown later, the technique may still be used for the analysis of many thermally labile compounds without their decomposition, and small peptides have been studied. 

Hoveyda and coworkers [142] developed the Cu-catalyzed allylic substitutions of phosphonate derivatives with pyridinyl peptide structures as efficient ligands. The structure of the ligands was chosen through synthesis, and analysis of libraries. Optimized compounds were used as ligands for the... 

Silica-based restricted access materials (RAM) have been developed for cleanup in bioanalysis, first for low molecular weight compounds in biofluids (Rbeida et al., 2005) and subsequently for biopolymers such as peptides (Wagner et al., 2002). A classification of different types of RAM has been given by Boos and Rudolphi (1997). Novel RAMs with strong cation-exchange functionality have been synthesized and implemented in the sample cleanup of biofluids. Racaityte et al. (2000) have shown that this type of RAM is highly suitable for the online extraction and analysis of... 

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