Isolation by HPLC

The bioluminescence spectrum of P. stipticus and the fluorescence and chemiluminescence spectra of PM are shown in Fig. 9.7. The fluorescence emission maximum of PM-2 (525 nm) is very close to the bioluminescence emission maximum (530 nm), but the chemiluminescence emission maximum in the presence of a cationic surfactant CTAB (480 nm) differs significantly. However, upon replacing the CTAB with the zwitter-ionic surfactant SB3-12 (3-dodecyldimethylammonio-propanesulfonate), the chemiluminescence spectrum splits into two peaks, 493 nm and 530 nm, of which the latter peak coincides with the emission maximum of the bioluminescence. When PM-1 is heated at 90°C for 3 hr in water containing 10% methanol, about 50% of PM-1 is converted to a new compound that can be isolated by HPLC the chemiluminescence spectrum of this compound in the presence of SB3-12 (curve 5, Fig. 9.7) is practically identical with the bioluminescence spectrum. 

Properties of luciferin precursors. About one dozen of the luciferin precursors of M. citricolor isolated by HPLC had a strong tendency of isomerization, as mentioned above. Their molecular weights could not be established by mass spectrometry, which is probably due to isomerization, although they appear to be in a range of 300-600. The precursors showed an absorption peak at about 369 nm in methanol and aqueous acetonitrile (Fig. 9.13). According to an NMR study, all precursors probably contain the following common partial structure (personal communication from Dr. H. Nakamura, 1998). 

Comparative Evaluation of Related Compounds. Structural studies are incomplete, but NMR and gas chromatographic - mass spectroscopic analysis of a component (fraction 7) isolated by HPLC indicated biphenyl characteristics (30). In an attempt to evaluate the cytolytic effect of compounds with similar structure, 4-hydroxybiphenol and 2-hydroxybiphenol were tested for lytic effects on P. brevis. Bioassays of 2- and 4-hydroxybiphenol indicated that both compounds were cytolytic to P. brevis (31). 

The reactions were cleaner than those with cis,cis-1,5-cyclooctadiene, and purification of the intermediate bis-mercurated peroxide was unnecessary. Indeed, it was a simple matter to isolate 52 (28 %) and 53 (38 %) from crude products obtained using a sample of 1,4-cyclooctadiene containing 30% of cis,cis-1,5-cyclooctadiene, which is readily accessible via hydrobromination-dehydrobromination of the 1,5-diene. Two of the diastereoisomers of 53 were separately isolated by HPLC and the most... 

Englert, G. and Vecci, M. 1980. Trans/cis isomerization of astaxanthin diacetate/isolation by HPLC and identification by 1H-NMR spectroscopy of three mono-civ- and six di-civ-isomers. Helv. Chim. Acta 63 1711-1717. 

Continued work by the same group 123) has led to the first isolation of vinblastine (1) from a multiple shoot culture of C. roseus. The most productive line, MSC-B-1, consisted of two distinctly different tissues, multiple shoots and unorganized tissue, and was maintained growing and productive for 30 months. Vinblastine (1) was isolated by HPLC, and the content was estimated to be 15 jjig/g dry weight. Production of this alkaloid was greater than that in the callus culture but less than that observed for the parent plant, even though the levels of catharanthine (4) and vindoline (3) were about the same. 

The FOX assay applied to a skatole oxidation product isolated by HPLC gave a positive result, supporting the contention that it is skatolyl hydroperoxide (40) . Mixtures of 183 and the eight diastereoisomeric hydroperoxides 184 and 185 derived from thymidine (42), as shown in equation 64, can be separated and detected by RP-HPLC with UVD at 229 nm. Each isomer is determined by applying the FOX assay using a capillary reactor heated at 60 °C to provide sufficient time for total oxidation of the Fe(ll) ions, followed by UVD at 596 mn . A commercial kit based on the FOX assay for hydroperoxide determination in plasma, serum and tissue homogenizates appears in Table 2. 

The peroxidase-catalyzed oxidation of ABTS (103) by H2O2 can also be performed by hydroperoxides, yielding a green-colored free radical. This reaction, catalyzed by HRP-C, was applied to the identification of an oxidation product of skatole isolated by HPLC, as a hydroperoxide (40). This assignment was confirmed by MS analysis (Section V.C.3) . 

Hi. Lysine. Gamma radiolysis of aerated aqueous solution of lysine (94) has been shown, as inferred from iodometric measurements, to give rise to hydroperoxides in a similar yield to that observed for valine and leucine. However, attempts to isolate by HPLC the peroxidic derivatives using the post-column derivatization chemiluminescence detection approach were unsuccessful. This was assumed to be due to the instability of the lysine hydroperoxides under the conditions of HPLC analysis. Indirect evidence for the OH-mediated formation of hydroperoxides was provided by the isolation of four hydroxylated derivatives of lysine as 9-fluoromethyl chloroformate (FMOC) derivatives . Interestingly, NaBILj reduction of the irradiated lysine solutions before FMOC derivatization is accompanied by a notable increase in the yields of hydroxylysine isomers. Among the latter oxidized compounds, 3-hydroxy lysine was characterized by extensive H NMR and ESI-MS measurements whereas one diastereomer of 4-hydroxylysine and the two isomeric forms of 5-hydroxylysine were identified by comparison of their HPLC features as FMOC derivatives with those of authentic samples prepared by chemical synthesis. A reasonable mechanism for the formation of the four different hydroxylysines and, therefore, of related hydroperoxides 98-100, involves initial OH-mediated hydrogen abstraction followed by O2 addition to the carbon-centered radicals 95-97 thus formed and subsequent reduction of the resulting peroxyl radicals (equation 55). 

To 1 volume of the hydrazone mixture there was added, with mixing, 2 volumes of 0.6 M NaBH3CN in 0.1 M NaOAc/MeCN (1 1) the apparent pH was previously adjusted (glass electrode) to 4.6 with glacial AcOH. The apparent pH was readjusted to an apparent value of 4.6 with glacial AcOH, the soln incubated for 4d at rt in the dark, and the reduced hydrazone finally isolated by HPLC. The same conditions could be used for both the ligation steps shown in Scheme 2. 

A soln of peptide 33 in degassed argon-sat. 50 mM NH4OAc buffer (pH 5.5) at 4 mM concentration was added dropwise to a 10 mM soln of 1 equiv of heterodimer 32 in 50 mM NH4OAc (pH 5.5) under exclusion of air oxygen. The reaction was monitored spectroscopically at 430 nm and by HPLC. After 2-3 h the solvent was removed and the product isolated by HPLC yield 35% the product was characterized by ESI-MS, HPLC, and amino add analysis. 

Synthesis of Z-Tyr-Gly-NH2 in Aqueous-DMF Solvent Media. Using this modified enzyme, we carried out the synAesis of "Z-Tyr-Gly-NH2", which has never been formed in 100% aqueous system (14), and compart with native chymotrypsin on the effect of organic solvent. To a solution of Z-Tyr-OH (315mg) in Tris buffer (pH 6.7,0.5ml), which contained N,N-dimethylformamide (DW) (0-100%), was added a solution of H-Gly-NH2 HC1 (1 Img) and native or modified chymotrypsin (2mg) in the same Tris buffer. The mixture was incubated at 20°C for 24 hours and heated at lOO C for IS minutes. The products were isolated by HPLC (ODS column, 278nm, 50% acetonitrile). Native chymotrypsin inactivated when concentration of DMF was 50%, while chemically modified chymotrypsin kept its activity even up to 80% (Table IV). 

The TASP molecule (10 mg, 1.35 pmol, 1.05 equiv) was dissolved in 250 mM NaOAc buffer (pH 4, 6.5 mL) containing 10% TFE and added dropwise to a vigorously stirred soln (6.2 mL) of template (1.37 mg, 1.28 pmol) in NaOAc buffer (250 mM, pH 4). The condensation reaction was complete after 9h as monitored by HPLC. T4-(4a14)-T4was isolated by HPLC purification (gradient 45-95% MeCN in 30min) yield 5.1 mg (43%). 

The blue color was disappeared by irradiation with visible (X > 500 nm) light. In the dark, however, the blue color remained stable and at room temperature never reverted to the colorless form. In toluene, the colored isomer was found to be stable even at 100 °C. The stable, colored isomer was isolated by HPLC and its molecular structure was analyzed by NMR and X-ray crystallography. Both indicated that the blue colored isomer was the closed-ring form. Therefore, the photochromism of the diarylethene derivative was ascribed to the following photocydization and cycloreversion reactions. 

The absorbance of la remained almost constant even after 850 cycles, while the absorbance of 3a gradually declined. At the same time, a photostable, violet product with an absorption maximum at 547 nm was formed. The photostable by-product could be isolated by HPLC, and was found by elemental analysis and molecular mass determination to be isomeric with compound 3a. Its molecular structure was determined by X-ray crystallographic analysis to be a six-membered condensed ring structure 9, as shown in Figure 3. The by-product was produced from the closed-ring form more efficiently by UV irradiation. 

The heating of 4-o-tolyl-l,3-dithia-2,5-diazolium hexafluoroarsenate 154 with a small excess of o-tolylcyanide 155 in liquid S02 leads to 1,2,4-dithiazolium hexafluoroarsenate 6 in low yield (Equation 24) <1996AXC2148>. The decomposition of the known pesticide, 3,5-dirncthyltctrahydro-2//-1,3,5-thiadiazine-2-thione 156 (DTTT), in water and in salt solutions was studied in detail. 4-Methyl-5-methylimino-l,2,4-dithiazolidine-3-thione 157 and 2,4-dimethyl-l,2,4-thiadiazolidine-3,5-dithione 158 were isolated by HPLC and identified among the noncyclic decomposition products (Equation 25) <1996MI503>. 

The 96-nucleotide primer can also be isolated by HPLC on RPC-5 at neutral pH (Eshaghpour and Crothers, 1978, Sanger et al., 1980). For a discussion on the application of this technique to nucleic acid fractionation see Wells et al. (1980). 

Second, the enzyme is covalently bound to the affinity label in conditions chosen according to the enzyme in question and the chemical nature of the analogue, so as to decrease nonspecific labeling. Third, the labeled enzyme is subjected to proteolysis and the radioactive peptide(s) are isolated by HPLC. The labeled peptide(s) are then sequenced, providing information about the domains of the enzyme involved in the interaction with the substrate (or with the modulators). 

Butyl-4(5)-[125I]iodo-5(4)-hydroxymethylene imidazole 349 has been prepared330 by treatment of 350 with chloramine T in the presence of [x 2 5I]sodium iodide (equation 149). The product 349 isolated by HPLC is obtained in chemical and radiochemical yields of 70% at specific activity equal to 2200 Ci mmol -1. 

The reaction mixture contained glycine, coenzyme A, buffer, a-ketoglutarate, and bone marrow lysate. After incubation for 1 hour, the reaction was terminated by addition of 10% TCA. The samples were chilled and clarified by centrifugation, and the pyrrole formed. After processing, the pyrrole was isolated by HPLC and its radioactivity was quantitated. 

The product from Step 3 (0.425 g), Cgo-fullerene (98 mg), CBr4 (45 mg), and DBU (23 mg) were dissolved in 100 ml toluene and stirred 22 hours at ambient temperature. The mixture was purified by chromatography on silica gel first using toluene to remove unreacted C o-fullerence, and using toluene/EtOAc, 1 1, and the product isolated by HPLC in 21% yield. 

These structural isomers of M Cs2 have been separated and isolated by HPLC (Section 3). The minor isomer of La Cs2, i e., La Cs2 (II), was isolated by Yamamoto et al. (1994b). Similar to the major isomer. 

The two isomers were isolated by HPLC from the products and the X-ray crystal structure of each isomer is shown in Scheme 3, while a schematic representation of each compound is illustrated in Figure 5 [171]. 

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