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Fluorescent monofunctionalized beta-cyclodextrins bearing a copper(II) binding side arm and a dansyl group (CD-NH-AA-CH(2)CH(2)NH-DNS) were designed as enantioselective sensors for unmodified alpha-amino acids. The side arm was derived from amino acid synthons (AA = L- and D-phenylalanine (1 and 2), L- and D-phenylglycine (3 and 4), L-proline (5), and L-cyclohexylglycine (6)) and was chosen in order to contain an amide, an amine, and a sulphonamide group. Enantioselectivity was evaluated by addition of copper(II) complexes of D- or L-valine and D- or L-proline. Chiral discrimination in the fluorescence response was observed in all cases, due to a ligand exchange process. The best conditions for these experiments were found to be the use of an excess (10:1) of the copper complex. The cyclodextrin 4 containing a D-phenylglycine unit was found to be poorly enantioselective, as found for 2, suggesting that the best design can be obtained by using L-amino acids. All L-amino acid containing cyclodextrins showed good enantioselectivities, some of which were higher than those already reported for 1. Other analytes related to amino acids were studied using cyclodextrins 1 and 3. Enantiomers of alpha,alpha-disubstituted amino acids, N-methylamino acids, and amino acid amides were found to be discriminated, while beta-phenylalanine and other molecules bearing a poor anchoring group at the alpha-carbon gave poor enantioselectivity. On the basis of the present data a model for the recognition process, based on the formation of ternary diastereomeric complexes, is proposed.

Selectively modified 6,6'-dideoxy-6,6'-L-diamino-beta-cyclodextrins (AB, AC, AD) were successfully used as chiral selectors for the enantiomeric separation of hydroxy acids and carboxylic acids (in particular, phenoxyalkanoic acid herbicides) in capillary electrophoresis (CE). Chiral separations were obtained at a low selector concentration (1 mM) with good enantioselectivity and resolution factors. Separations were optimized as a function of pH. The different position of the charged groups on the upper rim greatly influenced the separation, accounting for electrostatic interactions between the protonated amino groups of the cyclodextrins (CDs) and the carboxylate of the selectands. The best enantiomeric separation of hydroxy acids was obtained with the AC regioisomer, whereas carboxylic acids were well resolved only by the AB regioisomer. A recognition model is proposed, based on two-dimensional nuclear magnetic resonance (2-D NMR) experiments, in which the orientation of the guest in the inclusion complex is determined by the electrostatic interactions between the selectand and the CD upper rim.

Peptide nucleic acids (PNAs) are oligonucleotide mimics containing a pseudopeptide chain, which are able to bind complementary DNA tracts with high affinity and selectivity. Two mixed-sequence PNA undecamers (1 and 2) were synthesized and their double-stranded adducts with the complementary oligonucleotides (3 and 4) were revealed by the appearance of the corresponding peak in anion-exchange HPLC. A DEAE column was used and elution was performed with aqueous Tris buffer (pH 8) and an ionic strength gradient (0-0.5 M NaCl). The same effect was not observed with non-complementary oligonucleotides. The stability of the PNA-DNA adducts under the conditions used in the chromatographic system was studied as a function of temperature. Furthermore, in competition experiments double-stranded oligonucleotides were challenged by a PNA complementary to one strand: the formation of the PNA-DNA hybrid and the displacement of the non-complementary strand were observed with high specificity. The results suggest a possible use of ion-exchange HPLC for studying PNA-DNA interactions, and indicate the efficiency of PNA probes in the chromatographic analysis of DNA.

The enantiomeric separation of alpha-hydroxy acids and carboxylic acids was successfully performed by using 6-deoxy-6-N-histamino-beta-cyclodextrin (CD-hm), a monosubstituted positively charged beta-cyclodextrin (beta-CD) bearing a histamine moiety linked to the C6 of a glucose unit in the upper CD rim via the amino group. Good results were obtained at a low selector concentration (1 mM). The number of positive charges on the upper rim may be modulated as a function of pH, because of the different pKa of the amino and the imidazolyl groups, and was found to affect both the enantioselectivity and resolution factors. With the analogous 6-deoxy-[4-(2-aminoethyl)imidazolyl]-beta-cyclodextrin (CD-mh) bearing the histamine moiety linked to the C6 via the imidazolyl group, very poor results were obtained, showing that the proximity of the positive charge to the cavity plays an important role in the enantiomeric recognition. The complexation mode was studied by electrospray ionization-mass spectrometry (ESI-MS) and two-dimensional nuclear magnetic resonance (2-D NMR) ROESY experiments: the recognition model is consistent with an inclusion complexation of the aromatic ring of the analyte within the CD cavity coupled to electrostatic interactions between the carboxylate and the protonated amino group of the cyclodextrin.

A modified beta-cyclodextrin bearing a 2-aminomethylpyridine binding site for copper(II) (6-deoxy-6-[N-(2-methylamino)pyridine)]-beta-cyclodextrin, CDampy) was synthesized by C6-monofunctionalization. The acid-base properties of the new ligand in aqueous solution were investigated by potentiometry and calorimetry, and its conformations as a function of pH were studied by NMR and circular dichroism (c.d.). The formation of binary copper(II) complexes was studied by potentiometry, EPR, and c.d.. The copper(II) complex was used as chiral selector for the HPLC enantiomeric separation of underivatized aromatic amino acids. Enantioselectivity in the overall stability constants of the ternary complexes with D- or L-Trp was detected by potentiometry, whereas the complexes of the Ala enantiomers did not show and difference in stability. These results were consistent with a preferred cis coordination of the amino group of the ligand and of the amino acid in the ternary complexes ("cis effect"), which leads to the inclusion of the aromatic side chain of D-Trp, but not of that of L-Trp. In Trp-containing ternary complexes, the two enantiomers showed differences in the fluorescence lifetime distribution, consistent with only one conformer of D-Trp and two conformers of L-Trp, and the latter were found to be more accessible to fluorescence quenching by acrylamide and KI.

Indole ( 1 ) and 3- methylbut - 2 - enyl bromide ( 2 ) were reacted in aqueous solution over a wide range of pH, in absence of Lewis catalysts, leading to 3-(3'-methylbut-2'-enyl) indole ( 3 ), 2,3-di-(3'-methylbut-2-enyl) indole ( 4 ) and to 2 - (3 - indolyl) - 3,3 - di - (3' - methylbut - 2' - enyl) - 2,3 - dihydroindote ( 6 ) in acidic buffer, whereas compound 6 was not formed at basic pH. Both the reactivity and the selectivity of the reaction appear to be influenced by the acidity of the medium. The reaction extended to biologically significant 3-substituted indoles gave a series of new products. 3-Substituted indoles bearing a basic group at their β-position ( 7 , 11 , 12 and 13 ) in acetic buffer (pH = 3) gave mainly cyclization to dihydrofurano or dihydropyrrolo [2.3-b] indoles ( 15 , 16 , 20 , 21 and 23 ), whereas those with the basic group at the γ-position ( 9 , 10 ) gave the 2 - (3 - methylbut - 2 - enyl) indole derivatives ( 18 , 19 ) only.

Cultures of Aspergillus candidus produce, in addition to chlorflavonin, a closely related metabolite that has been isolated and characterized as the dechloro analogue. Radioactivity from [14C]dechlorochlorflavonin was incorporated irreversibly into chlorflavonin. Cultures supplemented with D-[U-14C]glucose, L-[methyl-14C]methionine, L-[U-14C]phenylalanine, or L-[β-14C]-phenylalanine yielded the 14C-labeled metabolites; chemical degradation of chlorflavonin labeled by the last named precursor showed that the isotope had been incorporated specifically into position 2 of the flavonoid carbon skeleton. We conclude that chlorflavonin is synthesized de novo by A. candidus and is, therefore, the first proven fungal flavonoid.

Isolation and structural determination of a novel isoprenylated dehydrotryptophyl metabolite fromAspergillus amstelodami, neoechinulin D, is reported.

Two 11mer peptide nucleic acid (PNA) beacons were synthesized and tested for the detection of full-matched or single mismatched DNA. Fluorescent measurements carried out in solution showed only partial discrimination of the mismatched sequence, while using anion-exchange HPLC, in combination with fluorimetric detection, allowed DNA analysis to be performed with high sensitivity and extremely high sequence selectivity. Up to > 90 : 1 signal discrimination in the presence of one single mismatched base was observed. The analysis was tested on both short and long DNA oligomers. Detection of DNA obtained from PCR amplification was also performed allowing the selective detection of the target sequence in complex mixtures. Label free detection of the DNA with high sequence selectivity is therefore possible using the present approach.