Figure 3 of this Letter contains an inadvertently duplicated panel: the PBS 30 panel is identical to the aGalCer panel (top right). The corrected panels are shown here. Our results and conclusions are unaffected by this oversight. CORRIGENDUM

Glycolipid presentation by CD1 proteins has emerged as an important aspect of antigen recognition, and presentation of alpha-glycosylceramides by CD1d to natural killer T cells has become a central focus in understanding how glycolipid presentation can influence immune responses. An alpha-galactosylceramide containing relatively long lipid chains has been the subject of intense study because, when presented by CD1d to natural killer T cells, it stimulates the release of both proinflammatory and immunomodulatory cytokines. Using an efficient synthesis of alpha-galactosylceramides, we have prepared a series of glycolipids in which the lipid chain lengths have been incrementally varied. The responses of natural killer T cells to these glycolipids have been determined, and we have found that truncation of the phytosphingosine lipid chain increases the relative amounts of immunomodulatory cytokines released. In similar fashion, the length of the acyl chain in alpha-galactosylceramides influences cytokine release profiles.

CD1d presentation of alpha-galactosyl ceramides to natural killer T cells has been a focal point of the study of regulatory T cells. KRN7000, an alpha-galactosyl ceramide originally generated from structure activity studies of antitumor properties of marine sponge glycolipids, is currently the most commonly used agonist ligand and is used to stain NKT cells. However, this glycolipid suffers from poor solubility and availability. We have developed an alpha-galactosyl ceramide with improved solubility over KRN7000 that effectively stains NKT cells, both mouse and human, and stimulates cytokine release at low concentrations.

Chemoenzymatic synthesis of SAM analogs This study highlights a broadly applicable platform for the facile syntheses of SAM analogs that is directly compatible with downstream SAM utilizing enzymes. The ability to couple SAM synthesis and utilization in a single vessel circumvents issues associated with rapid SAM analog decomposition and thereby opens the door to the further interrogation of a wide range of SAM utilizing enzymes. As a proof of concept for the feasibility of natural product ‘alkylrandomization’, the coupled strategy was used to generate a small set of indolocarbazole analogs in conjunction with the rebeccamycin O-methyltransferase RebM.

Alpha-galactosylceramides are potent stimulators of human T cells. Stimulation occurs through binding of the glycolipids by CD1d, presentation to T cells, and formation of a CD1d-glycolipid-T cell receptor complex. To facilitate the elucidation of the structural features of glycolipids necessary for T cell stimulation, alpha-galactosylceramides have been prepared with small molecules appended at the C6 position of the sugar. The appended molecules do not significantly influence the abilities of the glycolipids to stimulate T cells. [reaction: see text]

A sweet library: Two variants (wild‐type (WT) and a triple mutant) of glycosyltransferase (GT) OleD have been shown to catalyze glycosylation of over 70 substrates, formation of O‐, S‐ and N‐glycosidic bonds, and iterative glycosylation (see scheme). Identified substrates include nucleophiles not previously known to act in GT reactions and span numerous natural product and therapeutic drug classes.

A comprehensive two-phase "hot spot" saturation mutagenesis strategy for the rapid evolution of glycosyltransferase (GT) specificity for nonnatural acceptors is described. Specifically, the application of a high-throughput screen (based on the fluorescent acceptor umbelliferone) was used to identify key amino acid hot spots that contribute to GT proficiency and/or promiscuity. Saturation mutagenesis of the corresponding hot spots facilitated the utilization of a lower-throughput screen to provide OleD prodigy capable of efficiently glycosylating the nonnatural acceptor novobiocic acid with an array of unique sugars. Incredibly, even in the absence of a high-throughput screen for novobiocic acid glycosylation, this approach rapidly led to improvements in the desired catalytic activity of several hundred-fold.

Summary The enediyne antibiotic calicheamicin (CLM) γ1I is a prominent antitumor agent that is targeted to DNA by a novel aryltetrasaccharide comprised of an aromatic unit and four unusual carbohydrates. Herein we report the heterologous expression and the biochemical characterization of the two ‘internal’ glycosyltransferases CalG3 and CalG2 and the first structural elucidation of an enediyne glycosyltransferase (CalG3). In conjunction with the previous characterization of the ‘external’ CLM GTs CalG1 and CalG4, this study completes the functional assignment of all four CLM GTs, extends the utility of enediyne GT-catalyzed reaction reversibility, and presents conclusive evidence of a sequential glycosylation pathway in CLM biosynthesis. This work also reveals the common GT-B structural fold can now be extended to include enediyne GTs.