We report an advanced chemoenzymatic strategy for the direct fluorescence detection, proteomic analysis, and cellular imaging of O-GlcNAc-modified proteins. O-GlcNAc residues are selectively labeled with fluorescent or biotin tags using an engineered galactosyltransferase enzyme and [3+2] azide-alkyne cycloaddition chemistry. We demonstrate that this approach can be used for direct ingel detection and mass spectrometric identification of O-GlcNAc proteins, identifying 146 novel glycoproteins from the mammalian brain. Furthermore, we show that the method can be exploited to quantify dynamic changes in cellular O-GlcNAc levels and to image O-GlcNAc-glycosylated proteins within cells. As such, this strategy enables studies of O-GlcNAc glycosylation that were previously inaccessible and provides a new tool for uncovering the physiological functions of OGlcNAc.Understanding posttranslational modifications to proteins is critical for elucidating the functional roles of proteins within the dynamic environment of cells. O-Linked β-Nacetylglucosamine (O-GlcNAc) glycosylation has emerged as important for the regulation of diverse cellular processes, including transcription, cell division, and glucose homeostasis. 1 While new chemical tools have provided rapid, sensitive methods for detecting the modification and enabled better control over the activity of O-GlcNAc enzymes, 1a,2 significant challenges remain with regard to elucidating the functions of O-GlcNAc in cells. For instance, a robust method for the direct fluorescence detection of O-GlcNAc proteins in gels would permit monitoring of changes in glycosylation levels in response to cellular stimuli and greatly extend the reach of existing technologies. Furthermore, new tools for imaging OGlcNAc-glycosylated proteins would enable the expression and dynamics of the modification to be monitored in cells and tissues. Here, we report an advanced chemoenzymatic labeling strategy that addresses these important needs. HHMI Author Manuscript HHMI Author Manuscript HHMI Author ManuscriptPrevious studies have shown that an engineered β-1,4-galactosyltransferase enzyme (Y289L GalT) efficiently transfers a ketogalactose moiety from an unnatural UDP substrate selectively onto O-GlcNAc-modified proteins. 2a However, treatment of cell lysates with an aminooxy fluorescein derivative resulted in some nonspecific labeling of proteins. We therefore investigated whether Y289L GalT would accept the UDP-azidogalactose substrate 1 (UDPGalNAz), which would allow for labeling of O-GlcNAc proteins using [3+2] azide-alkyne cycloaddition chemistry ( Figure 1A). 3 In addition to providing alternative dyes to potentially reduce nonspecific interactions, this Cu(I)-catalyzed cycloaddition reaction would have the advantage of being performed more rapidly and at physiological pH.We tested the approach using α-crystallin, a known O-GlcNAc-modified protein with a low extent (~10%) of glycosylation. α-Crystallin was treated with 1 and Y289L GalT, followed by reaction with CuSO 4 , sodium ascor...

Enantioenriched pyranones are important intermediates in the synthesis of natural products and the generation of compound libraries. A one-pot method for their synthesis is outlined. Catalytic asymmetric alkylation of 2-furfurals in the presence of catalytic (−)-MIB generates enantioenriched furyl zinc alkoxides. Addition of water/THF followed by NBS results in formation of pyranones with ee's >90% and yields between 46-77%.Advances in organic synthesis enable chemists to prepare most natural product targets. Even with state of the art methods, however, syntheses often require many synthetic manipulations and purifications, resulting in low overall yields and generation of large amounts of chemical waste. To address these issues, increasing synthetic efficiency and reducing E-factors (which is defined as the ratio of the mass of waste produced to the mass of product), are becoming more important in designing synthetic routes. 1 One approach to streamline organic synthesis is through tandem and sequential reactions that accomplish multiple steps in a single flask and minimize isolations, purifications, and solvent use.Herein, we present a one-pot method to prepare enantioenriched pyranones from 2-furfurals. Pyranones are valuable building blocks that have been extensively used in natural product 2, 3,4,5 and diversity oriented syntheses. 6 6-Hydroxy-(2H)-pyran-3-ones are typically prepared by the oxidative rearrangement of furyl alcohols in the Achmatowicz reaction. 7 Key intermediates in the Achmatowicz reaction are illustrated in Scheme 1. An example of the Achmatowicz reaction in the synthesis of (−)-8a-epi-swainsonine 2 by O'Doherty is shown in Scheme 2.The most popular approach to enantioenriched pyranones has relied on kinetic resolution (KR) of racemic furyl alcohols, 8 as illustrated in Fürstner's elegant synthesis of ipomoeassian E (Scheme 3). 9 Upon treatment of the racemic furyl alcohol under KR conditions with the Sharpless-Katsuki catalyst and TBHP, the matched enantiomer underwent the Achmatowicz reaction to generate the optically active pyranone. The unreacted enantiomerically enriched furyl alcohol was then isolated in 47% yield (>99% ee) and subjected to a second Achmatowicz reaction. 9Correspondence to: Patrick J. Walsh. Supporting Information Available Procedures and full characterization are available (PDF). This material is available free of charge via the Internet at http://pubs.acs.org. In a KR, the product ee decreases with rising conversion while the starting material ee increases. 11 Thus, unless the KR selectivity factor (=k fast /k slow ) is very large, it is always the enantioenriched starting material that is carried forward. NIH Public AccessOur approach to enantioenriched pyranones is based on our enantioselective carbonyl addition/ epoxidation (Scheme 4). 12 These one-pot transformations generate epoxy alcohols with three contiguous stereogenic centers with high enantio-and diastereoselectivity. In applying this approach to the synthesis of enantioenriched pyranones, we fo...

Pyran derivatives R 0340One-Pot Catalytic Asymmetric Synthesis of Pyranones. -The target products are prepared from a variety of furfural derivatives with enantioselectivities >90% e.e. The procedure involves initial asymmetric alkylation of the substrates to generate zinc furyl alkoxide intermediates. Subsequent oxidation with NBS promotes the Achmatowicz reaction to furnish the pyranone products. -(CHENG, K.; KELLY, A. R.; KOHN, R. A.; DWECK, J. F.; WALSH*, P.