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Hydroxytyrosol 1 is a natural polyphenolic antioxidant present in leafs and fruits of olive tree and in small amount in virgin olive oil. It presents high radical scavenging capacity so protecting cells against oxidative stress. Furthermore, it possesses antiatherogenic, anti-inflammatory and anti-platelet aggregation properties, reducing risk for CVD disease. However, 1 is highly hydrophilic and this has hampered its use as nutraceutical or antioxidant additive in oils or fats. Fatty acids hydroxytyrosyl esters has overcome these limitations. Continuing the research on new derivatives of this natural antioxidant, in order to test the effect on the antioxidant activity of the presence of a double hydroxytyrosyl moiety in a single molecule, we have developed the synthesis of a series of dihydroxytyrosyl esters of dicarboxilic acids. To obtain regioselectively the diesters 3 from 1 the catechol hydroxyls in 1 were previously protected as methyl orthoformate 2 and easily deprotected after esterification. Dihydroxytyrosyl esters 3 constitute a new class of antioxidants and were analyzed for their antioxidant activity. Both chemical (ABTS assay) and biological (DCF assay) experiments were carried out. In vitro studies evidenced in all the derivatives 3 a strong and lipophilicity-dependent enhancement of antioxidant capacity (TEAC) in comparison with that of the already known monohydroxytyrosyl esters of carboxylic fatty acids. In cell environment, MTT experiments demonstrated that these compounds do not inhibit cell proliferation, while either with L6 or HTP-1 cells, high antioxidant activities were measured, although not as strong as in in vitro measurements.

Hydroxytyrosol 1 (R=H) is a natural polyphenolic antioxidant that protects cells against oxidative stress.1 It also reduces risks connected with aging pathogenesis,2 cancer proliferation3 and HIV infection.4 In spite of these remarkable properties, its amphiphilic character makes difficult its solubilization in lipid environment and hampers its use as nutraceutical or antioxidant additive. Hydroxytyrosyl esters (1, R= CO(CH2)nCH3) overcome these limitations.5, 6 In order to test the effect on the antioxidant activity of both the alcohol chain lenght and the position of the catechol function, we have developed the synthesis of the hydroxytyrosol analogues 2 and 3 (R= H) and their esters of fatty acids (R= CO(CH2)nCH3, n=0-16) using a recently published procedure based on a chemo and regioselective aromatic hydroxylation of the corresponding phenol derivatives by using 2-iodoxybenzoic acid (IBX).5 The antioxidant activity of all compounds was evaluated by both chemical (ABTS assay) and biological (DCF assay) experiments in order to establish possible structure/antioxidant activity relationship. Experiments have evidenced interesting correlation between in vitro antioxidant capacity and chemical structure, while in cell environment the antioxidant activity follows the peculiar trend already established with hydroxytyrosyl esters.6

The lithium salts of 4-methoxy-derivatives of pyrimidinic bases undergo chemoselective N1-methylation or ethylation when treated with the appropriate alkylsulphate in dry dioxane. Under the same conditions, 4-unprotected pyrimidinic bases afford mixtures of N1-, O2- and N1,N3-alkylation products. This is explained in terms of HSAB principle.

The lithium salts of 4-methoxy-derivatives of pyrimidinic bases undergo chemoselective N1-methylation or ethylation when treated with the appropriate alkylsulphate in dry dioxane. Under the same conditions, 4-unprotected pyrimidinic bases afford mixtures of N1-, O2- and N1,N3-alkylation products. This is explained in terms of HSAB principle.

The M1 strain, able to grow on beta-myrcene as the sole carbon and energy source, was isolated by an enrichment culture and identified as a Pseudomonas sp. One beta-myrcene-negative mutant, called N22, obtained by transposon mutagenesis, accumulated (E)-2-methyl-6-methylen-2,7-octadien-1-ol (or myrcen-8-ol) as a unique beta-myrcene biotransformation product. This compound was identified by gas chromatography-mass spectrometry. We cloned and sequenced the DNA regions flanking the transposon and used these fragments to identify the M1 genomic library clones containing the wild-type copy of the interrupted gene. One of the selected cosmids, containing a 22-kb genomic insert, was able to complement the N22 mutant for growth on beta-myrcene. A 5,370-bp-long sequence spanning the region interrupted by the transposon in the mutant was determined. We identified four open reading frames, named myrA, myrB, myrC, and myrD, which can potentially code for an aldehyde dehydrogenase, an alcohol dehydrogenase, an acyl-coenzyme A (CoA) synthetase, and an enoyl-CoA hydratase, respectively. myrA, myrB, and myrC are likely organized in an operon, since they are separated by only 19 and 36 nucleotides (nt), respectively, and no promoter-like sequences have been found in these regions. The myrD gene starts 224 nt upstream of myrA and is divergently transcribed. The myrB sequence was found to be completely identical to the one flanking the transposon in the mutant. Therefore, we could ascertain that the transposon had been inserted inside the myrB gene, in complete agreement with the accumulation of (E)-2-methyl-6-methylen-2,7-octadien-1-ol by the mutant. Based on sequence and biotransformation data, we propose a pathway for beta-myrcene catabolism in Pseudomonas sp. strain M1.