We report that azides capable of copper-chelation undergo much faster “Click chemistry” (copper-accelerated azide-alkyne cycloaddition, or CuAAC) than nonchelating azides under a variety of biocompatible conditions. This kinetic enhancement allowed us to perform site-specific protein labeling on the surface of living cells with only 10–40 µM CuI/II and much higher signal than could be obtained using the best previously-reported live-cell compatible CuAAC labeling conditions. Detection sensitivity was also increased for CuAAC detection of alkyne-modified proteins and RNA labeled by metabolic feeding.

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Growth inhibition and oxidized guanine lesion formation were studied in a number of base excision repair (BER) deficient Escherichia coli (E. coli) following chromate exposure. The only BER deficient bacterial strain that demonstrated significant growth inhibition by chromate, in comparison to its matched wild-type cell line, was the Nei deficient (TK3D11). HPLC coupled with electrospray ionization mass spectrometry showed that the Nei deficient E. coli accumulated the further oxidized guanine lesion, spiroiminodihydantoin (Sp), in genomic DNA at levels that were approximately 20-fold greater than its wild-type counterpart. However, no accumulation of the putative intermediate of Sp, 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG), was observed in the Nei deficient strain. A MutM-/MutY- double deletion mutant that was deficient in BER enzymes for the recognition and repair of 8-oxodG demonstrated no sensitivity toward chromate nor was there an associated increase in Sp accumulation over that of its wild type. However, the MutM-/MutY- double deletion mutant did show approximately 20-fold accumulation of 8-oxodG upon chromate exposure over that of the wild type and the Nei deficient E. coli. These data demonstrate that the Nei BER enzyme is critical for the recognition and repair of the Sp lesion in bacterial cell lines and demonstrates the protective effect of a specific BER enzyme on DNA lesions formed by chromate. To our knowledge, these are the first studies to show the formation and biological significance of the Sp lesion in a cellular system. This study has significant mechanistic and toxicological implications for how chromate may serve as an initiator of carcinogenesis and suggests a role for specific repair enzymes that may ameliorate the carcinogenic potential of chromate.

The copper-catalyzed azide-alkyne cycloaddition, or CuAAC, has been used extensively for the conjugation, immobilization, and purification of biomolecules. [1] Despite excellent reaction kinetics, high specificity, and bioorthogonality, CuAAC has been used to a far lesser extent in the cellular context because of toxicity caused by the Cu Imediated generation of reactive oxygen species (ROS) from O 2 . [2] One way to address this problem is to remove the Cu I requirement, by using alkynes activated by ring strain. [3][4][5] However, even the fastest of the strained cyclooctynes [6] react with azides more than tenfold slower than terminal alkynes in the presence of Cu I (k obs % 1m À1 s À1 for (aza)dibenzocyclo octyne [6] compared to k obs % 10-100 m À1 s À1 per 10-100 mm Cu I / Cu II for CuAAC [7] ). A second approach to improve cell compatibility is to use water-soluble ligands such as tris-(hydroxypropyltriazolylmethyl)amine (THPTA), [8] bis[(tertbutyltriazoyl)methyl]-[(2-carboxymethyltriazoyl)methyl]amine (BTTAA), [9] or bis(l-histidine) [10] for Cu I . These ligands both accelerate the cycloaddition reaction and act as sacrificial reductants, helping to protect cells and biomolecules from ROS. [8] Here we explore a third approach to improve the cell compatibility and performance of CuAAC. In general, decreasing the copper concentration lowers the toxicity of CuAAC to cells, but this is accompanied by a large decrease in reaction kinetics. [9] We reasoned that it might be possible to compensate for this decrease by using an azide reaction partner that contains an internal copper-chelating moiety (Figure 1 A), which would raise the effective copper concentration at the reaction site. This concept has been explored for azide-alkyne reactions in organic solvents, with Cu II rather than Cu I species, and at very high copper (10 mm) and reactant (200-400 mm) concentrations, [11,12] but never before under conditions relevant to biomolecular labeling. The goal of our study was to examine the effect of substrate chelation assistance on CuAAC kinetics and biocompatibility.The rate-determining step of CuAAC is postulated to be the formation of the metallacycle from the Cu I acetylide and the organic azide. [15] We decided to test whether an organic azide containing an internal Cu I ligand could accelerate formation of the metallacycle and hence the overall rate of the CuAAC reaction. We prepared two azides with proximal pyridine nitrogen atoms to chelate the Cu I ion (picolyl azides 2 and 4), as well as their nonchelating carbocyclic analogues, 1 and 3 ( Figure 2).CuAAC reaction timecourses were measured using 7ethynylcoumarin, a fluorogenic alkyne whose quantum yield (QY) increases from 1 % to 25 % upon reaction with azides [4] (Figure 2 A). Assays were first performed with 10 mm CuSO 4 in the absence of Cu I ligands. Reaction timecourses are shown in Figure S1 (see Supporting Information) and values for percent conversion into product after 10 and 30 minutes are given in Figure 2 B. Whereas the conventional azides 1 a...

7,8-dihydro-8-oxoguanine (8-oxoG) is thought to be a major lesion formed in DNA by oxidative attack at the nucleobase guanine. Recent studies have shown that 8-oxoG has a lower reduction potential than the parent guanine and is a hot spot for further oxidation. Spiroiminodihydantoin (Sp) has been identified as one of these further oxidation products. Chromium(VI) is a human carcinogen that, when reduced by a cellular reductant such as ascorbate, can oxidize DNA. In this study, duplex DNA was reacted with Cr(VI) and ascorbate to identify and quantify the base lesions formed. Guanine bases were observed to be preferentially oxidized with 5′ guanines within purine repeats showing enhanced oxidation. Trapping of the guanine lesions by the base excision repair enzymes hOGG1 and mNEIL2 showed nearly exclusive trapping by mNEIL2, suggesting that 8-oxoG was not the major lesion but rather a lesion recognized by mNEIL2 such as Sp. Formation of the Sp lesion in the Cr(VI)/Asc oxidation reaction with DNA was confirmed by LC-ESI-MS detection. HPLC-ECD was used to identify and quantify any 8-oxoG arising from Cr(VI)/Asc oxidation of DNA. Concentrations of Cr(VI) (3.1-50 μM) with a corresponding 1:10 ratio of Asc oxidized between 0.3% and 1.5% of all guanines within the duplex DNA strand to Sp. 8-oxoG was also identified but with the highest Cr (VI) concentration converting ~0.1% of all guanines to 8-oxoG. These results show that Sp was present in concentrations ~20 times greater than that of 8-oxoG in this system. The results indicate that 8-oxoG, while present, was not the major product of Cr(VI)/Asc oxidation of DNA and that Sp predominates under these conditions. These results further imply that Sp may be the lesion that accounts for the carcinogenicity of this metal in cellular systems.