A mild and efficient ligand-free Suzuki-Miyaura coupling reaction catalyzed by heterogeneous Pd/C was developed. Aryl bromides and triflates undergo the cross-coupling with aryl boronic acids in excellent yields without the presence of any additives in aqueous media at room temperature. Aryl vinyl boronic acids are also applicable to this coupling reaction and provide the trans-stilbene derivatives in high yields. The application of wet-type Pd/C to the coupling reaction was achieved without any loss of activity under aerobic conditions, and the reuse of Pd/C is feasible for a fifth run without significant loss of activity. Inductively coupled plasma (ICP) mass-spectrometric analysis of the filtrate from the reaction mixture of 4-bromonitrobenzene with phenylboronic acid demonstrated that the palladium metal hardly leached into the solution within the limits of the detector (<1 ppm), thus suggesting that the present Suzuki-Miyaura reaction proceeded by heterogeneous catalysis.

Small luminescent molecular probes based on the iridium(III) complex BTP, (btp)2Ir(acac) (btp = benzothienylpyridine, acac = acetylacetone) have been developed for sensing intracellular and in vivo O2. These compounds are BTPSA (containing an anionic carboxyl group), BTPNH2 (containing a cationic amino group), and BTPDM1 (containing a cationic dimethylamino group); all substituents are incorporated into the ancillary acetylacetonato ligand of BTP. Introduction of the cationic dimethylamino group resulted in an almost 20-fold increase in cellular uptake efficiency of BTPDM1 by HeLa cells compared with BTP. The phosphorescence intensity of BTPDM1 internalized in living cells provided a visual representation of the O2 gradient produced by placing a coverslip over cultured monolayer cells. The intracellular O2 levels (pO2) inside and outside the edge of the coverslip could be evaluated by measuring the phosphorescence lifetime of BTPDM1. Furthermore, intravenous administration of 25 nmol BTPDM1 to tumor-bearing mice allowed the tumor region to be visualized by BTPDM1 phosphorescence. The lifetime of BTPDM1 phosphorescence from tumor regions was much longer than that from extratumor regions, thereby demonstrating tumor hypoxia (pO2 = 6.1 mmHg for tumor and 50 mmHg for extratumor epidermal tissue). Tissue distribution studies showed that 2 h after injection of BTPDM1 into a mouse, the highest distribution was in liver and kidney, while after 24 h, BTPDM1 was excreted in the feces. These results demonstrate that BTPDM1 can be used as a small molecular probe for measuring intracellular O2 levels in both cultured cells and specific tissues and organs.

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Aquaporin-1 (AQP1), a water channel protein, has been shown to play an important role in tumor growth and angiogenesis in mouse endothelial cells. We recently reported that the expression of AQP1 mRNA was induced in cultured human retinal vascular endothelial cells (HRVECs) under hypoxia. In the present study, HRVECs were cultured under normoxia or hypoxia (1% O(2)) to elucidate the mechanism of hypoxic induction of AQP1. AQP1 mRNA expression was increased 1.7 ± 0.24-fold under hypoxia compared with that under normoxia (p < 0.01). This increase was almost completely blocked by the transcriptional inhibitor actinomycin D (p < 0.01). The degradation of AQP1 mRNA showed no difference under normoxia or hypoxia. These data suggest that the hypoxia-induced expression of AQP1 results from RNA transcription. The sequence located from -1338 to -1334 bp is identical to the consensus sequence of the hypoxia-inducible factor 1 (HIF-1) binding site. The promoter activities of the two constructs including this putative HIF-1 binding site showed 2.0 ± 0.67-fold increase and 2.9 ± 1.9-fold increase under hypoxia when compared with those under normoxia. However, both deletion and mutation of the HIF-1 binding site abrogated this effect. These data suggest that this sequence mediates the transcriptional activation of AQP1 by hypoxia. The chromatin immunoprecipitation assay showed that HIF-1α bound to the putative HIF-1 binding site. In conclusion, hypoxia-induced expression of AQP1 requires transcriptional activation, and the HIF-1 binding site of the 5'-promoter is necessary for transcriptional activation in HRVECs.

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