TGM2-mediated histone transglutamination is dictated by steric accessibility.

Recent studies have identified serotonylation of glutamine-5 on histone H3 (H3Q5ser) as a novel posttranslational modification (PTM) associated with active transcription. While H3Q5ser is known to be installed by tissue transglutaminase 2 (TGM2), the substrate characteristics affecting deposition of the mark, at the level of both chromatin and individual nucleosomes, remain poorly understood. Here, we show that histone serotonylation is excluded from constitutive heterochromatic regions in mammalian cells. Biochemical studies reveal that the formation of higher-order chromatin structures associated with heterochromatin impose a steric barrier that is refractory to TGM2-mediated histone monoaminylation. A series of structure-activity relationship studies, including the use of DNA-barcoded nucleosome libraries, shows that steric hindrance also steers TGM2 activity at the nucleosome level, restricting monoaminylation to accessible sites within histone tails. Collectively, our data indicate that the activity of TGM2 on chromatin is dictated by substrate accessibility rather than by primary sequence determinants or by the existence of preexisting PTMs, as is the case for many other histone-modifying enzymes.

Photocatalytic Activation of Aryl(trifluoromethyl) Diazos to Carbenes for High-Resolution Protein Labeling with Red Light.

State-of-the-art methods in photoproximity labeling center on the targeted generation and capture of short-lived reactive intermediates to provide a snapshot of local protein environments. Diazirines are the current gold standard for high-resolution proximity labeling, generating short-lived aryl(trifluoromethyl) carbenes. Here, we present a method to access aryl(trifluoromethyl) carbenes from a stable diazo source via tissue-penetrable, deep red to near-infrared light (600-800 nm). The operative mechanism of this activation involves Dexter energy transfer from photoexcited osmium(II) photocatalysts to the diazo, thus revealing an aryl(trifluoromethyl) carbene. The labeling preferences of the diazo probe with amino acids are studied, showing high reactivity toward heteroatom-H bonds. Upon the synthesis of a biotinylated diazo probe, labeling studies are conducted on native proteins as well as proteins conjugated to the Os photocatalyst. Finally, we demonstrate that the conjugation of a protein inhibitor to the photocatalyst also enables selective protein labeling in the presence of spectator proteins and achieves specific labeling of a membrane protein on the surface of mammalian cells via a two-antibody photocatalytic system.

A chemically engineered, stable oligomer mimic of amyloid ß42 containing an oxime switch for fibril formation.

Toxic aggregation of monomeric amyloid ß (Aß) into oligomers followed by the formation of fibrils is a causative process in the pathogenesis of Alzheimer's disease. The mechanism for furnishing the toxicity of Aß aggregates is elusive, however, mainly due to the transient, unstable properties of the oligomer states. Oligomer mimics stabilized by chemical protein engineering are potentially useful tools for elucidating the pathogenicity of Aß aggregates. Here we report a stable Aß oligomer mimic that is transformed into fibrils by a chemical stimulus, i.e., an oxime exchange reaction. A derivative of Aß42[Met35(O)], compound 2, containing an oxime tether between residues 23 and 28 (a salt-bridge surrogate between Asp23 and Lys28 of the Aß42 oligomer), rapidly and homogeneously formed stable, relatively large oligomers with preserved amyloid-like properties, such as the propensity to form ß-sheets and toxicity. Chemical cleavage of the tether via an oxime exchange reaction induced transformation of the oligomers into the fibril state. These results demonstrate that the oxime bond formation/cleavage can switch the aggregation state of the mimic by functionally surrogating the salt-bridge of Aß42. This novel system temporally dissects the dynamic process of Aß aggregation, and thus might offer a unique molecular tool for exploring the properties of Aß oligomers and fibrils.

Site-Selective Peptide/Protein Cleavage.

Site-selective peptide/protein degradation through chemical cleavage methods is an important modification of biologically relevant macromolecules which complements enzymatic hydrolysis. In this review, recent progress in chemical, site-selective peptide bond cleavage is overviewed, with an emphasis on postulated mechanisms and their implications on reactivity, selectivity, and substrate scope.

Directing activator-assisted regio- and oxidation state-selective aerobic oxidation of secondary C(sp(3))-H bonds in aliphatic alcohols.

The regioselective conversion of an unactivated C(sp(3))-H bond of a methylene carbon (CH2) into a C-O single bond is an attractive reaction in organic synthesis. Herein, we present a strategy for a regio- and oxidation state-selective aerobic C-H oxidation based on an N-hydroxyamide-derived directing activator (DA), which is attached to a hydroxy group in alcohol substrates. The DA reacts with NOx species generated in situ from NaNO2, a Brønsted acid, and aerobic oxygen, and effectively generates an amidoxyl radical from the N-hydroxy moiety of the DA. Then, the amidoxyl radical promotes site-selective intramolecular C-H abstraction from methylenes with γ- (or δ-) selectivity. The thus-generated methylene radicals are trapped by molecular oxygen and NO. This process results in the predominant formation of nitrate esters as products, which suppresses undesired overoxidation. The products can be easily converted into alcohols after hydrogenolysis.

Copper-catalyzed intramolecular C(sp³)-H and C(sp²)-H amidation by oxidative cyclization.

The first copper-catalyzed intramolecular C(sp(3))-H and C(sp(2))-H oxidative amidation has been developed. Using a Cu(OAc)2 catalyst and an Ag2CO3 oxidant in dichloroethane solvent, C(sp(3))-H amidation proceeded at a terminal methyl group, as well as at the internal benzylic position of an alkyl chain. This reaction has a broad substrate scope, and various ß-lactams were obtained in excellent yield, even on gram scale. Use of CuCl2 and Ag2CO3 under an O2 atmosphere in dimethyl sulfoxide, however, leads to 2-indolinone selectively by C(sp(2))-H amidation. Kinetic isotope effect (KIE) studies indicated that C-H bond activation is the rate-determining step. The 5-methoxyquinolyl directing group could be removed by oxidation.

Hydroxy Group Directed Catalytic Hydrosilylation of Amides.

Chemo- and site-selective hydrosilylation of α- or ß-hydroxy amides using organocatalyst B(C6F5)3 and commercially available hydrosilanes is described. This transformation is operative under mild conditions and tolerates a wide range of functional groups. The reaction was applied for selective reduction of a specific amide group of the therapeutically important cyclic peptide cyclosporin A, demonstrating the potential usefulness of this catalytic method in late-stage structural transformations of drug lead molecules.

Scandium(iii) triflate-promoted serine/threonine-selective peptide bond cleavage.

The site-selective cleavage of peptide bonds is an important chemical modification that is useful not only for the structural determination of peptides, but also as an artificial modulator of peptide/protein function and properties. Here we report site-selective hydrolysis of peptide bonds at the Ser and Thr positions with a high conversion yield. This chemical cleavage relies on Sc(iii)-promoted N,O-acyl rearrangement and subsequent hydrolysis. The method is applicable to a broad scope of polypeptides with various functional groups, including a post-translationally modified peptide that is unsuitable for enzymatic hydrolysis. The system was further extended to site-selective cleavage of a native protein, Aß1-42, which is closely related to the onset of Alzheimer's disease.

Chemo- and regioselective oxygenation of C(sp3)-H bonds in aliphatic alcohols using a covalently bound directing activator and atmospheric oxygen.

Chemically reactive directing groups (directing activators) represent a promising strategy for mild and regioselective C(sp3)-H functionalization. The use of a radical N-oxyl directing activator promoted the aerobic oxygenation of benzylic, propargylic, tertiary, and unactivated acyclic methylene C(sp3)-H bonds in aliphatic alcohols with γ- (or δ-) selectivity under mild conditions (room temperature to 50 °C). The reaction was unaffected by the presence of various oxidation-sensitive functional groups, which proved to be problematic in previously reported studies on the oxidation of C(sp3)-H bonds. Structural modifications on the directing activator altered the regioselectivity, and thus provided an ultra-remote aerobic C(sp3)-H oxygenation. The observed reactivity and regioselectivity could be rationalized in terms of the intramolecular conformational accessibility of the N-oxyl radical and the electronic characteristics of C(sp3)-H bonds.