Bioconjugation of Au25 Nanocluster to Monoclonal Antibody at Tryptophan.

We report the first bioconjugation of Au25 nanocluster to a monoclonal antibody at scarcely exposed tryptophan (Trp) residues toward the development of high-resolution probes for cryogenic electron microscopy (cryo-EM) and tomography (cryo-ET). To achieve this, we improved the Trp-selective bioconjugation using hydroxylamine (ABNOH) reagents instead of previously developed N-oxyl radicals (ABNO). This new protocol allowed for the application of Trp-selective bioconjugation to acid-sensitive proteins such as antibodies. We found that a two-step procedure utilizing first Trp-selective bioconjugation for the introduction of azide groups to the protein and then strain-promoted azide-alkyne cycloaddition (SPAAC) to attach a bicyclononyne (BCN)-presenting redox-sensitive Au25 nanocluster was essential for a scalable procedure. Covalent labeling of the antibody with gold nanoclusters was confirmed by various analytical methods, including cryo-EM analysis of the Au25 nanocluster conjugates.

Pharmacological intervention of cholesterol sulfate-mediated T cell exclusion promotes antitumor immunity.

Effective cancer immunotherapy requires physical contact of T cells with cancer cells. However, tumors often constitute special microenvironments that exclude T cells and resist immunotherapy. Cholesterol sulfate (CS) is a product of sulfotransferase SULT2B1b and acts as an endogenous inhibitor of DOCK2, a Rac activator essential for migration and activation of lymphocytes. We have recently shown that cancer-derived CS prevents tumor infiltration by effector T cells. Therefore, SULT2B1b may be a therapeutic target to dampen CS-mediated immune evasion. Here, we identified 3ß-hydroxy-5-cholenoic acid (3ß-OH-5-Chln) as a cell-active inhibitor of SULT2B1b. 3ß-OH-5-Chln inhibited the cholesterol sulfotransferase activity of SULT2B1b in vitro and suppressed CS production from cancer cells expressing SULT2B1b. In vivo administration of 3ß-OH-5-Chln locally reduced CS level in murine CS-producing tumors and increased infiltration of CD8+ T cells. When combined with immune checkpoint blockade or antigen-specific T cell transfer, 3ß-OH-5-Chln suppressed the growth of CS-producing tumors. These results demonstrate that pharmacological inhibition of SULT2B1b can promote antitumor immunity through suppressing CS-mediated T cell exclusion.

Targeted inhibition of EPAS1-driven IL-31 production by a small-molecule compound.

BACKGROUND: IL-31 is a major pruritogen associated with atopic dermatitis (AD). Although a specific antibody for IL-31 receptor has been shown to alleviate pruritus in patients with AD, therapeutic approaches to inhibition of IL-31 production remain unexploited. IL-31 production by TH cells critically depends on the transcription factor EPAS1, which mediates IL31 promoter activation in collaboration with SP1. OBJECTIVE: We aimed at developing small-molecule inhibitors that selectively block IL-31 production by TH cells. METHODS: We generated the reporter cell line that inducibly expressed EPAS1 in the presence of doxycycline to mediate Il31 promoter activation, and we screened 9600 chemical compounds. The selected compounds were further examined by using TH cells from a spontaneous mouse model of AD and TH cells from patients with AD. RESULTS: We have identified 4-(2-(4-isopropylbenzylidene)hydrazineyl)benzoic acid (IPHBA) as an inhibitor of IL31 induction. Although IPHBA did not affect nonspecific T-cell proliferation, IPHBA inhibited antigen-induced IL-31 production by TH cells from both an AD mouse model and patients with AD without affecting other cytokine production and hypoxic responses. In line with this, itch responses induced by adoptive transfer of IL-31-producing TH cells were attenuated when mice were orally treated with IPHBA. Mechanistically, IPHBA inhibited the association between EPAS1 and SP1, resulting in defective recruitment of both transcription factors to the specific sites of the IL31 promoter. We also determined the structure-activity relationship of IPHBA by synthesizing and analyzing 201 analogous compounds. CONCLUSION: IPHBA could be a potential drug leading to inhibition of EPAS1-driven IL-31 production.

Protein Modification at Tyrosine with Iminoxyl Radicals.

Post-translational modifications (PTMs) of proteins are a biological mechanism for reversibly controlling protein function. Synthetic protein modifications (SPMs) at specific canonical amino acids can mimic PTMs. However, reversible SPMs at hydrophobic amino acid residues in proteins are especially limited. Here, we report a tyrosine (Tyr)-selective SPM utilizing persistent iminoxyl radicals, which are readily generated from sterically hindered oximes via single-electron oxidation. The reactivity of iminoxyl radicals with Tyr was dependent on the steric and electronic demands of oximes; isopropyl methyl piperidinium oxime 1f formed stable adducts, whereas the reaction of tert-butyl methyl piperidinium oxime 1o was reversible. The difference in reversibility between 1f and 1o, differentiated only by one methyl group, is due to the stability of iminoxyl radicals, which is partly dictated by the bond dissociation energy of oxime O-H groups. The Tyr-selective modifications with 1f and 1o proceeded under physiologically relevant, mild conditions. Specifically, the stable Tyr-modification with 1f introduced functional small molecules, including an azobenzene photoswitch, to proteins. Moreover, masking critical Tyr residues by SPM with 1o, and subsequent deconjugation triggered by the treatment with a thiol, enabled on-demand control of protein functions. We applied this reversible Tyr modification with 1o to alter an enzymatic activity and the binding affinity of a monoclonal antibody with an antigen upon modification/deconjugation. The on-demand ON/OFF switch of protein functions through Tyr-selective and reversible covalent-bond formation will provide unique opportunities in biological research and therapeutics.

DOCK1 inhibition suppresses cancer cell invasion and macropinocytosis induced by self-activating Rac1P29S mutation.

Rac1 is a member of the Rho family of small GTPases that regulates cytoskeletal reorganization, membrane polarization, cell migration and proliferation. Recently, a self-activating mutation of Rac1, Rac1P29S, has been identified as a recurrent somatic mutation frequently found in sun-exposed melanomas, which possesses increased inherent GDP/GTP exchange activity and cell transforming ability. However, the role of cellular Rac1-interacting proteins in the transforming potential of Rac1P29S remains unclear. We found that the catalytic domain of DOCK1, a Rac-specific guanine nucleotide exchange factor (GEF) implicated in malignancy of a variety of cancers, can greatly accelerate the GDP/GTP exchange of Rac1P29S. Enforced expression of Rac1P29S induced matrix invasion and macropinocytosis in wild-type (WT) mouse embryonic fibroblasts (MEFs), but not in DOCK1-deficient MEFs. Consistently, a selective inhibitor of DOCK1 that blocks its GEF function suppressed the invasion and macropinocytosis in WT MEFs expressing Rac1P29S. Human melanoma IGR-1 and breast cancer MDA-MB-157 cells harbor Rac1P29S mutation and express DOCK1 endogenously. Genetic inactivation and pharmacological inhibition of DOCK1 suppressed their invasion and macropinocytosis. Taken together, these results indicate that DOCK1 is a critical regulator of the malignant phenotypes induced by Rac1P29S, and suggest that targeting DOCK1 might be an effective approach to treat cancers associated with Rac1P29S mutation.

C(sp3 )-H Cyanation Promoted by Visible-Light Photoredox/Phosphate Hybrid Catalysis.

Inspired by the reaction mechanism of photo-induced DNA cleavage in nature, a C(sp3 )-H cyanation reaction promoted by visible-light photoredox/phosphate hybrid catalysis was developed. Phosphate radicals, generated by one-electron photooxidation of phosphate salt, functioned as a hydrogen-atom-transfer catalyst to produce nucleophilic carbon radicals from C(sp3 )-H bonds with a high bond-dissociation energy. The resulting carbon radicals were trapped by a cyano radical source (TsCN) to produce the C-H cyanation products. Due to the high functional-group tolerance and versatility of the cyano group, the reaction will be useful for realizing streamlined building block syntheses and late-stage functionalization of drug-like molecules.

Development of Highly Chemoselective Oxidative Transformations by Designing Organoradicals.

To conduct organic synthesis in the field of pharmaceutical science, methodologies that can easily and quickly supply compounds with high drug-likeness are highly desirable. Based on the original catalyst design concept "Radical-Conjugated Redox Catalysis (RCRC)" established during my research, various C(sp3)-H functionalizations and protein modifications have been developed, taking advantage of the high reactivity and chemoselectivity of the single-electron transfer process. This review focuses on the eight-year research efforts by my collaborators and me, from conception to results.

Transition Metal-Free Tryptophan-Selective Bioconjugation of Proteins.

Chemical modifications of native proteins can facilitate production of supernatural protein functions that are not easily accessible by complementary methods relying on genetic manipulations. However, accomplishing precise control over selectivity while maintaining structural integrity and homogeneity still represents a formidable challenge. Herein, we report a transition metal-free method for tryptophan-selective bioconjugation of proteins that is based on an organoradical and operates under ambient conditions. This method exhibits low levels of cross-reactivity and leaves higher-order structures of the protein and various functional groups therein unaffected. The strategy to target less abundant amino acids contributes to the formation of structurally homogeneous conjugates, which may even be suitable for protein crystallography. The absence of toxic metals and biochemically incompatible conditions allows a rapid functional modulation of native proteins such as antibodies and pathogenic aggregative proteins, and this method may thus easily find therapeutic applications.

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.

Enhanced Structural Variety of Nonplanar N-Oxyl Radical Catalysts and Their Application to the Aerobic Oxidation of Benzylic C-H Bonds.

The design and synthesis of structurally variable, nonplanar N-oxyl radical catalysts and their application to the aerobic oxidation, etherification, and acetoamidation of benzylic C-H bonds are described. The catalytic oxidation of C-H bonds represents a powerful tool to synthesize oxygenated functional molecules from simple hydrocarbons in a straightforward way. Electron-deficient N-oxyl radical catalysts, such as phthalimidoyl N-oxyl (PINO) radical, generated from N-hydroxyphthalimide (1), have attracted much attention because of their applications in the oxidation of C-H bonds with high bond dissociation energy (BDE). However, a few sites in 1 are available for structural modifications and improvements of the catalytic performance. By replacing one carbonyl group in 1 with a trifluoromethyl (CF3)-substituted sp(3)-carbon, we generated an additional tunable site and a nonplanar backbone, while retaining the desirable electron-withdrawing properties and increasing the lipophilicity with respect to 1. We synthesized a variety of N-hydroxy precatalysts containing such a CF3 moiety, and investigated their utility in the aerobic oxidation of benzylic C-H bonds. Precatalysts with electron-withdrawing substituents, such as trifluoroethoxy and the acetophenone moieties, afforded higher yields than a corresponding methoxy-substituted analogue. The introduction of substituents at the aromatic ring was also effective, as evident from the performance of 7-CF3 and 4,5,6,7-tetrafluoro precatalysts. Especially the combination of trifluoroethoxy- and 4,5,6,7-tetrafluoro substitution afforded a superior performance. These catalyst systems exhibited high functional group tolerance during the aerobic oxidation of C-H bonds, and benzylic etherification and Ritter-type reactions could be carried out at room temperature when a selected precatalyst and N-bromosuccinimide (NBS) were used.

Serine-selective aerobic cleavage of peptides and a protein using a water-soluble copper-organoradical conjugate.

The site-specific cleavage of peptide bonds is an important chemical modification of biologically relevant macromolecules. The reaction is not only used for routine structural determination of peptides, but is also a potential artificial modulator of protein function. Realizing the substrate scope beyond the conventional chemical or enzymatic cleavage of peptide bonds is, however, a formidable challenge. Here we report a serine-selective peptide-cleavage protocol that proceeds at room temperature and near neutral pH value, through mild aerobic oxidation promoted by a water-soluble copper-organoradical conjugate. The method is applicable to the site-selective cleavage of polypeptides that possess various functional groups. Peptides comprising D-amino acids or sensitive disulfide pairs are competent substrates. The system is extendable to the site-selective cleavage of a native protein, ubiquitin, which comprises more than 70 amino acid residues.

Manganese-catalyzed aerobic dehydrogenative cyclization toward ring-fused indole skeletons.

We describe the first example of manganese(III)-catalyzed aerobic dehydrogenative cyclization producing ring-fused indole skeletons. This catalytic system converts from two C-H bonds of indole and malonate to a C-C bond and produces water as the sole side product. This operationally easy method was extended to an intermolecular cross-dehydrogenative coupling of indole and α-substituted malonate with complete C2-selectivity.

Practical N-to-C peptide synthesis with minimal protecting groups.

Accessible drug modalities have continued to increase in number in recent years. Peptides play a central role as pharmaceuticals and biomaterials in these new drug modalities. Although traditional peptide synthesis using chain-elongation from C- to N-terminus is reliable, it produces large quantities of chemical waste derived from protecting groups and condensation reagents, which place a heavy burden on the environment. Here we report an alternative N-to-C elongation strategy utilizing catalytic peptide thioacid formation and oxidative peptide bond formation with main chain-unprotected amino acids under aerobic conditions. This method is applicable to both iterative peptide couplings and convergent fragment couplings without requiring elaborate condensation reagents and protecting group manipulations. A recyclable N-hydroxy pyridone additive effectively suppresses epimerization at the elongating chain. We demonstrate the practicality of this method by showcasing a straightforward synthesis of the nonapeptide DSIP. This method further opens the door to clean and atom-efficient peptide synthesis.

A Germanium Catalyst Accelerates the Photoredox α-C(sp3)-H Alkylation of Primary Amines.

Site-selective C(sp3)-H functionalizations using photoredox catalysis (PC) and hydrogen atom transfer (HAT) catalysis have received increasing attention. Here, we report a Ph2GeCl2 cocatalyst that greatly improves the yield of α-C(sp3)-H alkylation of primary amines catalyzed by a PC-HAT hybrid system. The α-position of the amino group selectively reacted even when weaker C-H bonds existed in the substrates. This finding may help the design of a novel site-selective hybrid catalysis.

Crystalline covalent organic frameworks with hydrazone linkages.

Condensation of 2,5-diethoxyterephthalohydrazide with 1,3,5-triformylbenzene or 1,3,5-tris(4-formylphenyl)benzene yields two new covalent organic frameworks, COF-42 and COF-43, in which the organic building units are linked through hydrazone bonds to form extended two-dimensional porous frameworks. Both materials are highly crystalline, display excellent chemical and thermal stability, and are permanently porous. These new COFs expand the scope of possibilities for this emerging class of porous materials.

Catalytic migratory oxidative coupling of nitrones through an outer-sphere C(sp3)-H activation process.

Outer-sphere redox catalysis is key to efficient C-H activation, which has attracted increased interest in organic chemistry. In this account, we describe a Cu(I) -catalyzed oxidative coupling between nitrones and various ethers or amines as an example. Predictable site-selective C-C bond formation was achieved through activation of the C-H bonds in each coupling partner and the migration of a C-N double bond. Mechanistic studies strongly suggested that the reaction proceeded via an oxonium/iminium cation species as the key intermediate. The mechanistic information allows for future extension of outer-sphere redox catalysis.

A metal-organic framework with covalently bound organometallic complexes.

A procedure for making covalently linked organometallic complexes within the pores of metal-organic frameworks (MOFs) has been described. An N-heterocyclic carbene precursor containing link L0 was prepared and then constructed into a MOF-5-type structure (IRMOF-76). Attempts to produce covalently bound organometallic complexes in IRMOF-76 were unsuccessful. An alternative way of linking the first metalated link, L1, into the desired metalated MOF structure, IRMOF-77, was successful. IRMOF-76 and -77 were characterized by single-crystal X-ray studies. Demonstration of permanent porosity and successful substitution of the pyridine coligand in IRMOF-77 are also described.

Identification of modular chiral bisphosphines effective for Cu(i)-catalyzed asymmetric allylation and propargylation of ketones.

New modular chiral phosphines effective for two distinct Cu(I)-catalyzed asymmetric tetrasubstituted-carbon-forming reactions, namely, allylation and propargylation of ketones, were identified. The optimized phosphine 8 was readily synthesized on a gram scale in high yield via three facile transformations (O-alkylation, bisaminal formation, and phosphination) from commercially available materials. In both reactions, excellent enantioselectivity (up to 98% ee) was produced from a range of substrates, including aromatic and aliphatic ketones, using 0.1-5 mol % catalyst loading. Specifically, catalytic enantioselective propargylation was the first example, affording synthetically useful chiral building blocks that have not been easily accessed to date. In addition to the enantioselectivity, the high catalytic activity of the CuOAc-8 complex is noteworthy. Preliminary studies to elucidate the structure-catalyst activity relationship suggested that the high catalytic activity of the Cu-8 complex is due to the extraordinarily wide bite angle ( angleP-Cu-P = 137.8 degrees ), leading to the stabilization of the active monomeric catalytically active species. Furthermore, mechanistically intriguing nonconventional hydrogen bonds existed between the acetate ligand of Cu and the bisaminal hydrogen atoms, stabilizing the distorted tetrahedral coordination state of the Cu atom.

Induction of ADCC by a folic acid-mAb conjugate prepared by tryptophan-selective reaction toward folate-receptor-positive cancer cells.

We developed conjugates between monoclonal antibody (mAb) and folic acid (FA) by using a tryptophan (Trp)-selective reaction, which yields relatively homogenous products compared to conventional methods. The obtained mAb-FA conjugates showed significant cellular cytotoxicity toward folate receptor-expressing cancer cells, demonstrating that the conjugates retained the Fc region's original function.

Asymmetric reductive Mannich reaction to ketimines catalyzed by a Cu(I) complex.

A highly diastereoselective reductive Mannich coupling of ketimines and alpha,beta-unsaturated esters was developed using CuOAc-PPh(3) or CuOAc-MePPh(2) complex as a catalyst (5 mol %) and pinacolborane as a reducing reagent. The reaction was easily conducted at room temperature, and the substrate generality was broad. This platform methodology was extended to the first catalytic asymmetric reductive Mannich reaction of ketimines using CuOAc-DIFLUORPHOS as the catalyst (10 mol %). Switching the reducing reagent from pinacolborane to (EtO)(3)SiH was key to inducing the high enantioselectivity (82-93% ee). High diastereoselectivity was also maintained (3:1 approximately 30:1). Thus, products containing contiguous tetra- and trisubstituted carbons were catalytically synthesized with high stereoselectivities. Products were converted to alpha,beta,beta-trisubstituted (beta(2,3,3)) amino acid derivatives without any racemization and epimerization through simple treatment under acidic conditions. This method is the first entry of the catalytic asymmetric synthesis of beta(2,3,3)-amino acid derivatives, which constitute important chiral building blocks of biologically significant molecules.