Hana Dvořáková

Enzymatic synthesis of oligo-D-galactofuranosides and L-arabinofuranosides: from molecular dynamics to immunological assays

D-Galactofuranosyl-containing conjugates are ubiquitous in many pathogenic microorganisms, but completely absent from mammals. As they may constitute interesting pharmacophores, recent works have been dedicated to their preparation. Besides well-reported chemical procedures, enzymatic approaches are still limited, mainly due to the lack of the corresponding biocatalysts. Based on the similarity between chemical structures, the arabinofuranosyl hydrolase Araf51 from Clostridium thermocellum was expected to recognize both the L-Araf motif and its D-Galf analogue. Molecular dynamics and STD-NMR were firstly used to confirm this hypothesis and increase our knowledge of the active site. Interestingly, this arabinofuranosidase was not only able to hydrolyze galactosyl derivatives, but was also really efficient in catalyzing oligomerisations using p-nitrophenyl furanosides as donors. The structures of the products obtained were determined using mass spectrometry and NMR. Amongst them, all the possible regioisomers of di-arabino and -galactofuranosides were synthesized, and the ratio of each regioisomer was easily tuned with respect to the reaction time. Especially, the galactofuranobioside displaying the biologically relevant sequence beta-D-Galf-(1,6)-beta-D-Galf was enzymatically prepared for the first time. All fractions going from di- to penta-arabino- and galactofuranosides were tested for their ability in eliciting the production of TNF-alpha. Interesting immunological properties were observed with arabinofuranosides as short as three sugar residues.

Dimercuration of Calix[4]arenes: Novel Substitution Pattern in Calixarene Chemistry

A mercuration reaction of tetrapropoxycalix[4]arene immobilized in the cone conformation gave a mixture of two dimercurated products (meta,meta and meta,para) in approximately a 1:1 ratio. Both regioisomers represent inherently chiral compounds, which makes them very attractive for design of novel receptors. As demonstrated by Pd-catalyzed arylation, the different reactivity of HgCl functions in the meta,para-disubstituted isomer opens the door for regioselective introductions of two different functional groups to achieve a substitution pattern so far unknown in calixarene chemistry.

meta-Bridged calix[4]arenes: a straightforward synthesis via organomercurial chemistry

The regioselective mercuration of tetraalkylated calix[4]arenes with Hg(OCOCF3)2 leads to the formation of meta-substituted products which enabled Pd-catalysed intramolecular bridging within the calixarene skeleton. Bridged derivatives represent a completely novel substitution pattern in calixarene chemistry with extremely distorted cavities and possible applications in supramolecular chemistry.

Synthesis of hapten and conjugates of coumestrol and development of immunoassay

3-O-Carboxymethylcoumestrol was prepared as the hapten for immunoassay by a partial alkylation of coumestrol with ethyl chloroacetate in acetone alkalized with potassium carbonate. 3-O-Ethoxycarbonylmethylcoumestrol was separated by column chromatography and finally was hydrolyzed with formic acid. 1H and 13C NMR data (APT, COSY, HMQC, and HMBC) revealed that the reaction was regioselective, as 3-O-ethoxycarboxymethylcoumestrol was the only monosubstituted derivative. The hapten was then conjugated to bovine serum albumin and used for immunization of rabbits. A radioimmunoassay (RIA) system was established based on the polyclonal antiserum and a 125I-labeled hapten-tyrosine methyl ester conjugate as the radioligand. Parameters of the RIA: sensitivity: 12 pg per tube, 50% intercept: 140 pg per tube, working range: 20-4000 pg per tube. The cross-reactivity of a panel isoflavonoid and lignan phytoestrogens was either negligible (e.g. formononetin 0.07%; biochanin A 0.06%) or not detectable at all. The major immunoreactive peak in HPLC fractions from an alfalfa extract had the same retention time as coumestrol standard and represented 94.8% of the signal. The remaining 5.2% of immunoreactivity was distributed between five minor peaks. We conclude that after the validation for particular matrices, the method will be a useful tool for analysis of coumestrol, especially in low volume and low concentration samples.

Use of residual dipolar couplings in conformational analysis of meta-disubstituted calix[4]arenes

Dimercuration of tetrapropoxy calix[4]arene followed by a reaction with isoamyl nitrite afforded dinitroso regioisomers with unique substitution patterns. The unusual conformational behaviour of these inherently chiral systems was revealed by the combination of dynamic NMR and residual dipolar coupling (RDC) techniques.

Regioselective Halogenation of Thiacalix[4]arenes in the Cone and 1,3-Alternate Conformations

Monohalogenation of thiacalix[4]arene in the cone conformation gave either the meta- or para-substituted isomers depending on the halogen and reaction conditions used. Surprisingly, the same reaction with the 1,3-alternate conformer led only to the meta isomer. This is the first example of such a conformation-dependent regioselectivity in calixarene/thiacalixarene chemistry. As the halogen-substituted calixarenes are useful synthetic intermediates, this provided the unique opportunity to functionalize the basic skeleton at two different positions.

Anion receptors based on ureidocalix[4]arenes immobilised in the partial cone conformation

A novel method enabling the synthesis of receptors based on the calix[4]arenes immobilized in the partial cone conformation is reported. The application of a protection/deprotection strategy using nosyl groups enables the regioselective introduction of functional groups (NO2 and NH2) into the upper rim of calix[4]arenes, leading finally to the substitution pattern so far inaccessible to calixarene chemistry. The introduction of two ureido functions at the para positions of the partial cone conformer yields a novel type of receptor which can bind anions even in a highly competitive solvent (DMSO). This revealed that the partial cone conformation, so far rather ignored in supramolecular chemistry, can be also very useful in design of novel anion receptors.

Palladium-catalysed Claisen rearrangement of 6-allyloxypurines

Abstract6-Allyloxypurines readily undergo palladium-catalysed Claisen rearrangement under mild conditions affording N1-substituted hypoxanthines. In contrast with the previously reported protocol, the Claisen rearrangement can be performed using Pd(PPh3)4 or Pd(dba)2/dppf in dry THF at 60°C. The reaction can accommodate variously substituted allyl fragments to position N1 of the hypoxanthine skeleton with high yields. Retention of the double bond configuration during rearrangement was observed.