Highly Efficient Access to Both Geometric Isomers of Silyl Enol Ethers: Sequential 1,2-Brook/Wittig Reactions.

Novel sequential 1,2-Brook/Wittig reactions were developed for the preparation of silyl enol ethers. This method enables highly selective preparation of both geometric isomers of glyoxylate silyl enol ethers, using aldehydes (E-selective) and tosylimines (Z-selective) as a Wittig electrophile. The salt-free conditions of this reaction system are likely to be advantageous for switching the selectivity. The optimal reaction conditions and generality of the reaction were investigated, and plausible explanations for the observed selectivity were also discussed.

A novel method for determining peroxisomal fatty acid ß-oxidation.

The purpose of this study is to establish an assay method to screen for chemical compounds that stimulate peroxisomal fatty acid ß-oxidation activity in X-linked adrenoleukodystropy (X-ALD) fibroblasts. In this investigation, we used 12-(1-pyrene)dodecanoic acid (pyrene-C12:0), a fluorescent fatty acid analog, as a substrate for fatty acid ß-oxidation. When human skin fibroblasts were incubated with pyrene-C12:0, ß-oxidation products such as pyrene-C10:0 and pyrene-C8:0 were generated time-dependently. These ß-oxidation products were scarcely detected in the fibroblasts from patients with Zellweger syndrome, a peroxisomal biogenesis disorder. In contrast, in fibroblasts with mitochondrial carnitine-acylcarnitine translocase deficiency, the ß-oxidation products were detected at a level similar to control fibroblasts. These results indicate that the ß-oxidation of pyrene-C12:0 takes place in peroxisomes, but not mitochondria, so pyrene-C12:0 is useful for measuring peroxisomal fatty acid ß-oxidation activity. In X-ALD fibroblasts, the ß-oxidation activity for pyrene-C12:0 was approximately 40 % of control fibroblasts, which is consistent with previous results using [1-(14)C]lignoceric acid as the substrate. The present study provides a convenient procedure for screening chemical compounds that stimulate the peroxisomal fatty acid ß-oxidation in X-ALD fibroblasts.

Synthesis of Denosomin-Vitamin D3 Hybrids and Evaluation of Their Anti-Alzheimer's Disease Activities.

As an extension of previously conducted studies on developing an anti-Alzheimer's disease agent, denosomin (1-deoxy-24-norsominone, an artificial inducer of neurite elongation), derivatives were designed and synthesized based on the hypothesis that our denosomin would exhibit axonal extension activity via a 1,25D(3)-membrane-associated, rapid response steroid-binding protein (1,25D(3)-MARRS) pathway. The biological assay revealed that the hybridization of characteristic δ-lactone in denosomin and the triene moiety in VD(3) was effective to enhance the nerve re-extension activity in amyloid ß (Aß)-damaged neurons.

Synthesis and biological evaluation of N-(2-fluorophenyl)-2ß-deoxyfuconojirimycin acetamide as a potent inhibitor for α-l-fucosidases.

In this study we revealed that the addition of an N-phenylacetamide substituent to the C-1 position of 1-deoxyfuconojirimycin (DFJ) can lead to highly potent inhibitors of α-l-fucosidases. A structure-activity relationship study showed that a fluoro group on the phenyl ring greatly increased its potency and selectivity. In contrast the addition of two or three fluoro groups decreased their inhibition potency. Consequently, N-(2-fluorophenyl)-2ß-DFJ acetamide (18j) was found to display very potent and selective inhibition of bovine kidney, rat epididymis, and human lysosome α-l-fucosidases, with IC50 value of 0.012, 0.044, and 0.0079µM respectively. It is noteworthy that our designed N-phenyl-2ß-DFJ acetamide derivative exhibited about 18-fold stronger effects on human lysosomal α-l-fucosidase than original DFJ and it occupied the active-site of this enzyme. We therefore expect that this compound may find applications in new therapeutic trials against genetic deficiency disorders.

Chemical cell-surface receptor engineering using affinity-guided, multivalent organocatalysts.

Catalysts hold promise as tools for chemical protein modification. However, the application of catalysts or catalyst-mediated reactions to proteins has only recently begun to be addressed, mainly in in vitro systems. By radically improving the affinity-guided DMAP (4-dimethylaminopyridine) (AGD) catalysts that we previously reported (Koshi, Y.; Nakata, E.; Miyagawa, M.; Tsukiji, S.; Ogawa, T.; Hamachi, I. J. Am. Chem. Soc. 2008, 130, 245.), here we have developed a new organocatalyst-based approach that allows specific chemical acylation of a receptor protein on the surface of live cells. The catalysts consist of a set of 'multivalent' DMAP groups (the acyl transfer catalyst) fused to a ligand specific to the target protein. It was clearly demonstrated by in vitro experiments that the catalyst multivalency enables remarkable enhancement of protein acylation efficiency in the labeling of three different proteins: congerin II, a Src homology 2 (SH2) domain, and FKBP12. Using a multivalent AGD catalyst and optimized acyl donors containing a chosen probe, we successfully achieved selective chemical labeling of bradykinin B(2) receptor (B(2)R), a G-protein coupled receptor, on the live cell-surface. Furthermore, the present tool allowed us to construct a membrane protein (B(2)R)-based fluorescent biosensor, the fluorescence of which is enhanced (tuned on) in response to the antagonist ligand binding. The biosensor should be applicable to rapid and quantitative screening and assay of potent drug candidates in the cellular context. The design concept of the affinity-guided, multivalent catalysts should facilitate further development of diverse catalyst-based protein modification tools, providing new opportunities for organic chemistry in biological research.