Molecular fluorescence excitation-emission matrices relevant to tissue spectroscopy

In vivo and ex vivo studies of fluorescence from endogenous and exogenous molecules in tissues and cells are common for applications such as detection or characterization of early disease. A systematic determination of the excitation-emission matrices (EEM) of known and putative endogenous fluorophores and a number of exogenous fluorescent photodynamic therapy drugs has been performed in solution. The excitation wavelength range was 250-520 nm, with fluorescence emission spectra collected in the range 260-750 nm. In addition, EEM of intact normal and adenomatous human colon tissues are presented as an example of the relationship to the EEM of constituent fluorophores and illustrating the effects of tissue chromophore absorption. As a means to make this large quantity of spectral data generally available, an interactive database has been developed. This currently includes EEM and also absorption spectra of 35 different endogenous and exogenous fluorophores and chromophores and six photosensitizing agents. It is intended to maintain and extend this database in the public domain, accessible through the Photochemistry and Photobiology website (http://www.aspjournal. com/).     

Simplified micro-method for the quantitative analysis of putrescine, spermidine and spermine in urine

A simplified micro-method for the quantitative analysis of urinary polyamines is described. After acid hydrolysis of urine, the polyamines are converted to fluorescent 1-dimethylaminonaphthalene-5-sulfonyl (Dns; dansyl) derivatives and separated by means of thin-layer chromatography. Dns-NH2, which has been reported to interfere with the determination of putrescine, is well separated from di-Dns-putrescine. Putrescine, spermidine and spermine are quantitated by in situ scanning of their fluorescent spots on the chromatogram. The present method is both sensitive and reproducible. It eliminates a number of time-consuming steps and thus reduces preparative losses. Yet an adequate chromatographic resolution is obtained. Representative polyamine analyses of urine from normal volunteers and from cancer patients are reported. Elevated levels occur in the urines of pregnant women and of patients with various types of cancer.     

Control of lysine reactivity in four-helix bundle proteins by site-selective pKa depression: expanding the versatility of proteins by postsynthetic functionalization

Five 42-residue polypeptides have been designed to fold into hairpin helix-loop-helix motifs that dimerize to form four-helix bundles, and to serve as protein scaffolds for the elucidation at the molecular level of the principles that control and fine-tune lysine and ornithine reactivities in a protein context. Site-selective control of Lys and Orn reactivity provides a mechanism for addressing directly individual residues and is a prerequisite for the site-selective functionalization of folded proteins. Several lysine and one ornithine residues were introduced on the surface and in the hydrophobic core of the folded motif. The reactivity of each residue was determined by measuring the degree of acylation of the trypsin cleaved fragments by HPLC and mass spectrometry. The most reactive residues were Orn34 and Lys19, both of which were located in d positions in the heptad repeat, and therefore in hydrophobic environments. Upon reaction of the helix-loop-helix dimer KA-I with one equivalent of mono-p-nitrophenyl fumarate, Orn34 was acylated approximately three times more efficiently than Lys19, whereas Lys10 (b position), Lys15 (g position), and Lys33 (c position) remained unmodified. In the sequence KA-I-A(15) Lys15 was replaced by an alanine residue and the selectivity of Orn34 over Lys19 increased to approximately a factor of six, probably because Lys15 had the capacity to reduce the pK(a) value of Lys19 and 85 % of site-selectively monoacylated product was obtained. The pH dependence of the acylation reaction was determined and showed that the pK(a) of the reactive residues were 9.3, more than a pK(a) unit below the magnitude of the corresponding residue in a solvent exposed position. Introducing Lys and Orn residues into a or d positions of the heptad repeat therefore serves as a mechanism of depressing their pK(a) to increase their reactivity site selectively. Extensive NMR and CD spectroscopic analyses showed that the sequences fold according to prediction.     

Efficient sample pre-concentration of bupivacaine from human plasma by solid-phase extraction on molecularly imprinted polymers

The ability to use imprinted polymers for solid-phase extraction is demonstrated in a model pre-concentration of bupivacaine from human plasma samples prior to gas chromatography. Imprinting of the structural analogue pentycaine yielded a sorbent which efficiently extracted analyte and internal standard, while possible interference on analyte quantification from leakage of remaining template molecules was eliminated. Human plasma samples were diluted with citrate buffer pH 5, and applied onto solid phase extraction columns containing 15 mg of imprinted sorbent. Wash steps with 20% methanol in water followed by acetonitrile preceded elution with 2% triethylamine in acetonitrile. A direct comparison with conventional sample pre-treatment methods showed the high selectivity of the imprinted sorbent resulted in distinctly cleaner chromatographic traces than were obtained both after liquid-liquid extraction and C18-based solid-phase extraction.     

Quantitative analysis of conformational exchange contributions to 1H-15N multiple-quantum relaxation using field-dependent measurements. Time scale and structural characterization of exchange in a calmodulin C-terminal domain mutant

Multiple-quantum spin relaxation is a sensitive probe for correlated conformational exchange dynamics on microsecond to millisecond time scales in biomolecules. We measured differential 1H-15N multiple-quantum relaxation rates for the backbone amide groups of the E140Q mutant of the C-terminal domain of calmodulin at three static magnetic field strengths. The differential multiple-quantum relaxation rates range between -88.7 and 92.7 s(-1), and the mean and standard deviation are 7.0 +/- 24 s(-1), at a static magnetic field strength of 14.1 T. Together with values of the 1H and 15N chemical shift anisotropies (CSA) determined separately, the field-dependent data enable separation of the different contributions from dipolar-dipolar, CSA-CSA, and conformational exchange cross-correlated relaxation mechanisms to the differential multiple-quantum relaxation rates. The procedure yields precise quantitative information on the dominant conformational exchange contributions observed in this protein. The field-dependent differences between double- and zero-quantum relaxation rates directly benchmark the rates of conformational exchange, showing that these are fast on the chemical shift time scale for the large majority of residues in the protein. Further analysis of the differential 1H-15N multiple-quantum relaxation rates using previously determined exchange rate constants and populations, obtained from 15N off-resonance rotating-frame relaxation data, enables extraction of the product of the chemical shift differences between the resonance frequencies of the 1H and 15N spins in the exchanging conformations, deltasigma(H)deltasigma(N). Thus, information on the 1H chemical shift differences is obtained, while circumventing complications associated with direct measurements of conformational exchange effects on 1H single-quantum coherences in nondeuterated proteins. The method significantly increases the information content available for structural interpretation of the conformational exchange process, partly because deltasigma(H)deltasigma(N) is a signed quantity, and partly because two chemical shifts are probed simultaneously. The present results support the hypothesis that the exchange in the calcium-loaded state of the E140Q mutant involves conformations similar to those of the wild-type apo (closed) and calcium-loaded (open) states.     

A new synthetic approach for imidazo[1,2-a]imidazoles and pyrrolo[1,2-a]imidazoles

Substituted pyrrolo[1,2-a]imidazoles and imidazo[1,2-a]imidazoles are prepared in a simple one pot reaction sequence from esters of heterocyclic or aromatic α-amino acids. The reaction involves condensation with acetoine followed by cyclization with either malonodinitrile, ethyl cyanoacetate or cyanamide.     

Heterocycles from amino acids. A novel synthetic approach for imidazo[1,5-a]pyridines and imidazo[1,5-a]quinolines

From the corresponding heterocyclic amino acids 2 and 9a the heterocyclic systems imidazo[1,5-a]pyridine ( 3 ) and imidazo[1,5-a]quinoline ( 10 ) are easily accessible. From compound 7 the tricyclic system 11 was prepared and from compound 17a a pyridyl-1,2,4-triazinone ( 18 ) could be obtained.     

Towards molecular-imprint based SPE of local anaesthetics

Summary Solidago canadensis L., Canadian goldenrod (Asteraceae) has been used in European phytotheraphy for centuries as a component of urological and antiphlogistical remedies. High-performance liquid chromatography (HPLC) coupled with diode-array detection (DAD) and online mass spectrometry (MS) has been used for the separation and quantification of phenolics (chlorogenic acid, caffeic acid, kaempferol-3-O-α-L-rutinoside (nicotiflorin), quercetin-3-O-β-D-rutinoside (rutin), quercetin-3-O-β-D-galactoside (hyperoside), quercetin-3-O-β-D-glucoside (isoquercitrin), quercetin-3-O-β-D-rhamnoside (quercitrin), kaempferol-3-O-α-L-rhamnoside (afzelin) and quercetin from Solidaginis herba. Extracts have been obtained using different technologies. Three aqueous and three alcoholic extracts were studied separately. Reversedphase high-performance liquid chromatography separation of polyphenols on octadecyl sorbent Hypersil was performed, using acetonitrile: acetic acid 2.5 v/v % as eluent in gradient elution. Our results confirm previous reports concerning the presence of several flavonoids. Quantification of the main quercetin glycosides in pharmaceuticals is also reported. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

Copolymeric (lR-trans)-N,N'-1,2-cyclohexylene-bisbenzamide oligodimethylsiloxane chiral stationary phase for gas chromatography

A copolymeric stationary phase, consisting of a chiral selective part, i.e. (1R-trans)-N,N'-1,2-cyclohexylenebisbenzamide, and an efficient siloxane oligomeric part, was successfully applied to open tubular column GC analysis. The efficiency and the chiral selectivity of this stationary phase were studied in detail, and high capacity and efficiency at elevated GC temperatures were especially noted. Several drugs and other enantiomeric pairs were separated. The shown examples demonstrate a broad application range for this type of chiral stationary phase in GC analysis.     

POLARIZED FLUORESCENCE OF 5-METHYLCYTOSINE SPECIES IN SOLUTION AT ROOM TEMPERATURE

Fluorescence yields (π_f,'s) and polarizations ( P ) are measured for aqueous 5-methylcytosine (˜ 0.1 m M ) at 20°C as a function of pH over the range 2–14. Both properties change abruptly and in parallel at pH's corresponding to the known pK_a values. Polarizations were also obtained for the 5-methylcytosine cation, neutral and anion species in ethylene glycol water glass at ˜180K. The weak fluorescence of the neutral and cation species at 20°C was polarized almost as highly as at low temperature. When the fluorescence lifetimes are assumed to be correctly given by the product of calculated radiative lifetimes and quantum yields, the polarizations are found to be consistent with rotational diffusion rates ˜4 times faster than predicted from Stokes-Einstein models for the neutral and anion species. The cation seemed to rotate about two times more slowly than the neutral and anion species. It was also shown that the properties of the three species are such that a plot of 1/P vs apparent π_f in the pH range 2–11 is fortuitously linear.