Fidelity and Promiscuity of a Mycobacterial Glycosyltransferase

Members of the genus <i>Mycobacterium</i> cause devastating human diseases, including tuberculosis. <i>Mycobacterium tuberculosis</i> can resist some antibiotics because of its durable and impermeable cell envelope. This barrier is assembled from saccharide building blocks not found in mammals, including galacto­furanose (Gal<i>f</i>). Within the cell envelope, Gal<i>f</i> residues are linked together to afford an essential poly­saccharide, termed the galactan. The formation of this polymer is catalyzed by the glycosyl­transferase GlfT2, a processive carbohydrate polymerase, which generates a sequence-specific poly­saccharide with alternating regio­isomeric β(1–5) and β(1–6) Gal<i>f</i> linkages. GlfT2 exhibits high fidelity in linkage formation, as it will terminate polymerization rather than deviate from its linkage pattern. These findings suggest that GlfT2 would prefer an acceptor with a canonical alternating β(1–5) and β(1–6) Gal<i>f</i> sequence. To test this hypothesis, we devised a synthetic route to assemble oligo­saccharides with natural and non-natural sequences. GlfT2 could elongate each of these acceptors, even those with non-natural linkage patterns. These data indicate that the glycosyl­transferase is surprisingly promiscuous in its substrate preferences. However, GlfT2 did favor some substrates: it preferentially acted on those in which the lipid-bearing Gal<i>f</i> residue was connected to the sequence by a β(1–6) glycosidic linkage. The finding that the relative positioning of the lipid and the non-reducing end of the acceptor influences substrate selectivity is consistent with a role for the lipid in acceptor binding. The data also suggest that the fidelity of GlfT2 for generating an alternating β(1–5) and β(1–6) pattern of Gal<i>f</i> residues arises not from preferential substrate binding but during processive elongation. These observations suggest that inhibiting the action of GlfT2 will afford changes in cell wall structure

Enantioselective Synthesis of SM-130686 Based on the Development of Asymmetric Cu(I)F Catalysis To Access 2-Oxindoles Containing a Tetrasubstituted Carbon

Enantioselective Synthesis of SM-130686 Based on the Development of Asymmetric Cu(I)F Catalysis To Access 2-Oxindoles Containing a Tetrasubstituted Carbo

Live-Cell Protein Modification by Boronate-Assisted Hydroxamic Acid Catalysis

Selective methods for introducing protein post-translational modifications (PTMs) within living cells have proven valuable for interrogating their biological function. In contrast to enzymatic methods, abiotic catalysis should offer access to diverse and new-to-nature PTMs. Herein, we report the boronate-assisted hydroxamic acid (BAHA) catalyst system, which comprises a protein ligand, a hydroxamic acid Lewis base, and a diol moiety. In concert with a boronic acid-bearing acyl donor, our catalyst leverages a local molarity effect to promote acyl transfer to a target lysine residue. Our catalyst system employs micromolar reagent concentrations and affords minimal off-target protein reactivity. Critically, BAHA is resistant to glutathione, a metabolite which has hampered many efforts toward abiotic chemistry within living cells. To showcase this methodology, we installed a variety of acyl groups in E. coli dihydrofolate reductase expressed within human cells. Our results further establish the well-known boronic acid–diol complexation as a bona fide bio-orthogonal reaction with applications in chemical biology and in-cell catalysis

Total Synthesis of (±)-Garsubellin A

The first total synthesis of garsubellin A, a neurotrophic compound with potent choline acetyltransferase-inducing activity, is described. Keys for success were (1) stereoselective intermolecular aldol reaction at the C-4 position with acetaldehyde, (2) stereoelective Claisen rearrangement to introduce an allyl group to the most sterically crowded position at C-6, (3) ring-closing metathesis to construct the B-ring, and (4) Wacker-type oxidative C-ring formation. This synthetic route can be extended to an asymmetric synthesis of garsubellin A using the Koga catalytic enantioselective alkylation, which produced enantioenriched α-prenyl cyclohexenone with excellent enantioselectivity (95% ee)

Total Synthesis of (±)-Garsubellin A

The first total synthesis of garsubellin A, a neurotrophic compound with potent choline acetyltransferase-inducing activity, is described. Keys for success were (1) stereoselective intermolecular aldol reaction at the C-4 position with acetaldehyde, (2) stereoelective Claisen rearrangement to introduce an allyl group to the most sterically crowded position at C-6, (3) ring-closing metathesis to construct the B-ring, and (4) Wacker-type oxidative C-ring formation. This synthetic route can be extended to an asymmetric synthesis of garsubellin A using the Koga catalytic enantioselective alkylation, which produced enantioenriched α-prenyl cyclohexenone with excellent enantioselectivity (95% ee)

Two Approaches toward the Formal Total Synthesis of Oseltamivir Phosphate (Tamiflu): Catalytic Enantioselective Three-Component Reaction Strategy and l‑Glutamic Acid Strategy

Two independent formal total syntheses of oseltamivir phosphate were successfully achieved: the first utilized a copper-catalyzed asymmetric three-component reaction strategy, and the second utilized l-glutamic acid γ-ester as a chiral source to install the correct stereochemistry. Both strategies used Dieckmann condensation to construct a six-membered ring core, after which manipulation of the functional groups and protecting groups accessed Corey’s intermediate for the synthesis of oseltamivir phosphate. While the first synthesis was accomplished via four purification steps in 25.7% overall yield, albeit with moderate optical purity (76% ee), the second strategy achieved the synthesis via six purification steps in 19.8% overall yield with perfect enantiocontrol

Nuclear envelope expansion is critical for proper chromosomal segregation during a closed mitosis

Here, we screened a 10,371 library of diverse molecules using a drug-sensitive fission yeast strain to identify compounds which cause defects in chromosome segregation during mitosis. We identified a phosphorium-ylide-based compound Cutin-1 which inhibits nuclear envelope expansion and nuclear elongation during the closed mitosis of fission yeast, and showed that its target is the β-subunit of fatty acid synthase. A point mutation in the dehydratase domain of Fas1 conferred in vivo and in vitro resistance to Cutin-1. Time-lapse photomicrography showed that the bulk of the chromosomes were only transiently separated during mitosis, and nucleoli separation was defective. Subsequently sister chromatids re-associated leading to chromosomal mis-segregation. These segregation defects were reduced when the nuclear volume was increased and were increased when the nuclear volume was reduced. We propose that there needs to be sufficient nuclear volume to allow the nuclear elongation necessary during a closed mitosis to take place for proper chromosome segregation, and that inhibition of fatty acid synthase compromises nuclear elongation and leads to defects in chromosomal segregation

Designer Adaptor Proteins for Functional Conversion of Peptides to Small-Molecule Ligands toward In-Cell Catalytic Protein Modification

Peptides are privileged ligands for diverse biomacromolecules, including proteins; however, their utility is often limited due to low membrane permeability and in-cell instability. Here, we report peptide ligand-inserted eDHFR (PLIED) fusion protein as a universal adaptor for targeting proteins of interest (POI) with cell-permeable and stable synthetic functional small molecules (SFSM). PLIED binds to POI through the peptide moiety, properly orienting its eDHFR moiety, which then recruits trimethoprim (TMP)-conjugated SFSM to POI. Using a lysine-acylating BAHA catalyst as SFSM, we demonstrate that POI (MDM2 and chromatin histone) are post-translationally and synthetically acetylated at specific lysine residues. The residue-selectivity is predictable in an atomic resolution from molecular dynamics simulations of the POI/PLIED/TMP-BAHA (MTX was used as a TMP model) ternary complex. This designer adaptor approach universally enables functional conversion of impermeable peptide ligands to permeable small-molecule ligands, thus expanding the in-cell toolbox of chemical biology

Designer Adaptor Proteins for Functional Conversion of Peptides to Small-Molecule Ligands toward In-Cell Catalytic Protein Modification

Peptides are privileged ligands for diverse biomacromolecules, including proteins; however, their utility is often limited due to low membrane permeability and in-cell instability. Here, we report peptide ligand-inserted eDHFR (PLIED) fusion protein as a universal adaptor for targeting proteins of interest (POI) with cell-permeable and stable synthetic functional small molecules (SFSM). PLIED binds to POI through the peptide moiety, properly orienting its eDHFR moiety, which then recruits trimethoprim (TMP)-conjugated SFSM to POI. Using a lysine-acylating BAHA catalyst as SFSM, we demonstrate that POI (MDM2 and chromatin histone) are post-translationally and synthetically acetylated at specific lysine residues. The residue-selectivity is predictable in an atomic resolution from molecular dynamics simulations of the POI/PLIED/TMP-BAHA (MTX was used as a TMP model) ternary complex. This designer adaptor approach universally enables functional conversion of impermeable peptide ligands to permeable small-molecule ligands, thus expanding the in-cell toolbox of chemical biology