An attractive hypothesis in enzyme evolution relies on the promiscuous activities that already exist in enzymes, which can be used as pipelines for the selection of new catalytic activities. [1] These new activities are usually low, but can serve as starting points in Darwinian evolution. In metalloenzymes, the diversity of promiscuous activity is increased by the variety of metallic ions that can be incorporated in the active site and can catalyse a wide range of chemical transformations. As an example, the iron-containing rubredoxin oxidase and zinc-b-lactamase are two phylogenetically related enzymes that catalyse oxidation and hydrolysis reactions with unrelated mechanisms. [2] This principle could be applicable in the field of directed evolution as a way to discover primitive artificial metalloenzymes that might catalyse challenging asymmetric transformations. In this work, we replaced the zinc ion of natural carbonic anhydrase with manganese and showed that the resulting product can catalyse the enantioselective epoxidation of styrene.Protein modification with cofactors or metal-containing complexes is an approach that has been widely used for the generation of new catalytic activities, [3] but few examples in the field of asymmetric catalysis have yet appeared.[4] The very first was reported in 1978 by Wilson and Whitesides, on the incorporation of a biotinylated achiral rhodium diphosphine complex in avidin for the hydrogenation of dehydroamino acids; [5] this concept has recently been improved through combination of structural variants of the biotinylated complex with several wild-type and mutant proteins.[6] A similar noncovalent approach was used for the enantioselective oxidation of sulfides after incorporation of a vanadate ion into a hydrolase A C H T U N G T R E N N U N G homologous to vanadium-dependent peroxidases [7] or of an A C H T U N G T R E N N U N G achiral chromium salophen complex into apomyoglobin.[8] Covalent grafting of achiral organometallic complexes has also been used as a complementary approach, with attachment of a copper phenanthroline complex to the adipocyte lipid-binding protein affording enantioselective hydrolysis of esters and amides.[9] More recently, the dual anchoring of an achiral manganese salen complex in apomyoglobin for asymmetric sulfoxidation has been reported. [10] In all these approaches the metallic ion is part of a larger achiral complex or ion and the chiral environment is provided by the protein.Our approach consists of incorporating only the catalytic metal ion in a protein featuring a suitable site for coordination, taking advantage of an attractive recently reported procedure for the manganese-catalysed epoxidation of alkenes.[11] This method uses hydrogen peroxide in hydrogen carbonate buffer, and the authors postulated the formation of peroxymonocarbonate-HCO 4 À -as the actual oxygen-transfer reagent. We selected carbonic anhydrase as a suitable candidate for hosting the manganese ion in its active site because the active species of the epoxidation...

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We hypothesized that over-expression of estrogen receptor (ER) in hormone-sensitive breast cancer could be harnessed synergistically with the tumor-migrating effect of porphyrins to selectively deliver estrogen-porphyrin conjugates into breast tumor cells, and preferentially kill the tumor cells upon exposure to red light. In the present work we synthesized four (4) conjugates of C17-alpha-alkynylestradiol and chlorin e6-dimethyl ester with varying tether lengths, and showed that all these conjugates specifically bound to recombinant ER alpha. In a cellular uptake assay with ER-positive MCF-7 and ER-negative MDA-MB 231 human breast cancer cell-lines, we observed that one such conjugate (E17-POR, XIV) was selectively taken up in a dose-dependent and saturable manner by MCF-7 cells, but not by MDA-MB 231 cells. Furthermore, MCF-7 cells, but not MDA-MB 231 cells, were selectively and efficiently killed by exposure to red light after incubation with E17-POR. Therefore, the combination approach, including drug and process modalities has the potential to be applied clinically for hormone-sensitive cancers in organs where ER is significantly expressed. This could potentially be carried out either as monotherapy involving a photo-induced selective destruction of tumor cells and/or adjuvant therapy in post-surgical treatment for the destruction of residual cancer cells in tissues surrounding the tumor.

Two efficient synthetic routes to 1alpha,25-dihydroxy-16-ene-vitamin D(3) (4a) and their C-20 analogues (3 and 4) have been developed. Key features common to both routes A and B are the introduction of side chains functionalized at C20 (17, 21, 19, and 25). In route A the CD side chain fragments 5 and 6 are prepared by S(N)2' syn displacement of allylic carbamates 8 and 9 (X = OCONHPh) by Li(2)Cu(3)R(5). The triene unit is then constructed by assembling the latter fragments with the A-ring fragment using the Wittig-Horner method (average yield of vitamin D analogue 35%, 11-13 steps from ketone 11). In route B, the S(N)2' syn displacement of the carbamate moiety by Li(2)Cu(3)R(5) is carried out on intermediates 12 and 13, both of which bear the vitamin D triene unit (average yield of vitamin D analogue 27%, 13-15 steps from ketone 11). The latter route is particularly attractive as an approach to diverse C-20 vitamin D analogues for biological screening.

The synthesis of four new analogues of calcitriol (1alpha, 25-(OH)(2)-D(3)) possessing aromatic and conjugated double bond units at the side chain are described. The triene system is introduced using the Lythgoe-Hoffmann La Roche convergent Wittig-Horner approach. The key steps in the preparation of the requisite upper fragments are the introduction of the side chain with the E-conjugated aromatic system and its photochemical conversion to the Z counterpart.