Catalytic Approaches to Chemo- and Site-Selective Transformation of Carbohydrates.

Carbohydrates are a fundamental unit playing pivotal roles in all the biological processes. It is thus essential to develop methods for synthesizing, functionalizing, and manipulating carbohydrates for further understanding of their functions and the creation of sugar-based functional materials. It is, however, not trivial to develop such methods, since carbohydrates are densely decorated with polar and similarly reactive hydroxy groups in a stereodefined manner. New approaches to chemo- and site-selective transformations of carbohydrates are, therefore, of great significance for revolutionizing sugar chemistry to enable easier access to sugars of interest. This review begins with a brief overview of the innate reactivity of hydroxy groups of carbohydrates. It is followed by discussions about catalytic approaches to enhance, override, or be orthogonal to the innate reactivity for the transformation of carbohydrates. This review avoids making a list of chemo- and site-selective reactions, but rather focuses on summarizing the concept behind each reported transformation. The literature references were sorted into sections based on the underlying ideas of the catalytic approaches, which we hope will help readers have a better sense of the current state of chemistry and develop innovative ideas for the field.

In vivo-stable bis-iminobiotin for targeted radionuclide delivery with the mutant streptavidin.

Targeted delivery of radionuclides to tumors is significant in theranostics applications for precision medicine. Pre-targeting, in which a tumor-targeting vehicle and a radionuclide-loaded effector small molecule are administered separately, holds promise since it can reduce unnecessary internal radiation exposure of healthy cells and can minimize radiation decay. The success of the pre-targeting delivery requires an in vivo-stable tumor-targeting vehicle selectively binding to tumor antigens and an in vivo-stable small molecule effector selectively binding to the vehicle accumulated on the tumor. We previously reported a drug delivery system composed of a low-immunogenic streptavidin with weakened affinity to endogenous biotin and a bis-iminobiotin with high affinity to the engineered streptavidin. It was, however, unknown whether the bis-iminobiotin is stable in vivo when administered alone for the pre-targeting applications. Here we report a new in vivo-stable bis-iminobiotin derivative. The keys to success were the identification of the degradation site of the original bis-iminobiotin treated with mouse plasma and the structural modification of the degradation site. We disclosed the successful pre-targeting delivery of astatine-211 (211At), α-particle emitter, to the CEACAM5-positive tumor in xenograft mouse models.

Live-cell epigenome manipulation by synthetic histone acetylation catalyst system.

Chemical modifications of histones, such as lysine acetylation and ubiquitination, play pivotal roles in epigenetic regulation of gene expression. Methods to alter the epigenome thus hold promise as tools for elucidating epigenetic mechanisms and as therapeutics. However, an entirely chemical method to introduce histone modifications in living cells without genetic manipulation is unprecedented. Here, we developed a chemical catalyst, PEG-LANA-DSSMe 11, that binds with nucleosome's acidic patch and promotes regioselective, synthetic histone acetylation at H2BK120 in living cells. The size of polyethylene glycol in the catalyst was a critical determinant for its in-cell metabolic stability, binding affinity to histones, and high activity. The synthetic acetylation promoted by 11 without genetic manipulation competed with and suppressed physiological H2B ubiquitination, a mark regulating chromatin functions, such as transcription and DNA damage response. Thus, the chemical catalyst will be a useful tool to manipulate epigenome for unraveling epigenetic mechanisms in living cells.

Pathological complete remission of relapsed tumor by photo-activating antibody-mimetic drug conjugate treatment.

Antibody-mimetic drug conjugate is a novel noncovalent conjugate consisting of an antibody-mimetic recognizing a target molecule on the cancer cell surface and low-molecular-weight payloads that kill the cancer cells. In this study, the efficacy of a photo-activating antibody-mimetic drug conjugate targeting HER2-expressing tumors was evaluated in mice, by using the affibody that recognize HER2 (ZHER2:342 ) as a target molecule and an axially substituted silicon phthalocyanine (a novel potent photo-activating compound) as a payload. The first treatment with the photo-activating antibody-mimetic drug conjugates reduced the size of all HER2-expressing KPL-4 xenograft tumors macroscopically. However, during the observation period, relapsed tumors gradually appeared in approximately 50% of the animals. To evaluate the efficacy of repeated antibody-mimetic drug conjugate treatment, animals with relapsed tumors were treated again with the same regimen. After the second observation period, the mouse tissues were examined histopathologically. Unexpectedly, all relapsed tumors were eradicated, and all animals were diagnosed with pathological complete remission. After the second treatment, skin wounds healed rapidly, and no significant side effects were observed in other organs, except for occasional microscopic granulomatous tissues beneath the serosa of the liver in a few mice. Repeated treatments seemed to be well tolerated. These results indicate the promising efficacy of the repeated photo-activating antibody-mimetic drug conjugate treatment against HER2-expressing tumors.

Antibody mimetic drug conjugate manufactured by high-yield Escherichia coli expression and non-covalent binding system.

Antibody-drug conjugates (ADCs) are a major therapeutic tool for the treatment of advanced cancer. Malignant cells in advanced cancer often display multiple genetic mutations and become resistant to monotherapy. Therefore, a therapeutic regimen that simultaneously targets multiple molecules with multiple payloads is desirable. However, the development of ADCs is hampered by issues in biopharmaceutical manufacturing and the complexity of the conjugation process of low-molecular-weight payloads to biologicals. Here, we report antibody mimetic-drug conjugates (AMDCs) developed by exploiting the non-covalent binding property of payloads based on high-affinity binding of mutated streptavidin and modified iminobiotin. Miniprotein antibodies were fused to a low immunogenic streptavidin variant, which was then expressed in Escherichia coli inclusion bodies, solubilized, and refolded into functional tetramers. The AMDC developed against human epidermal growth factor receptor 2 (HER2) effectively killed cultured cancer cells using bis-iminobiotin conjugated to photo-activating silicon phthalocyanine. The HER2-targeting AMDC was also effective in vivo against a mouse KPL-4 xenograft model. This AMDC platform provides rapid, stable, and high-yield therapeutics against multiple targets.

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.

A Stable and Cleavable O-Linked Spacer for Drug Delivery Systems.

Anti-cancer chemotherapy with good efficacy and fewer side effects is highly desirable. A drug delivery system comprising a cancer-targeting module and a cytotoxic agent connected with a cleavable linker is promising for reducing side effects. The development of a cleavable linker satisfying the requirements of both stability and cleavability, however, is difficult, especially when a carbonate moiety is used for conjugating the linker to a hydroxy group in a drug of interest. We herein report a new stable linker comprising carbamate and ester spacers, which can be introduced on a hydroxy group of a drug. This linker is more stable in aqueous neutral buffer than a corresponding carbonate-type linker, and releases a payload anti-cancer drug, SN-38, through a two-step sequence upon cathepsin B treatment. This linker may have potential use in other drug delivery systems to lower side effects by selectively transporting cytotoxic drugs to tumor cells.

Cupid and Psyche system for the diagnosis and treatment of advanced cancer.

In advanced cancer patients, malignant cells invade and disseminate within normal cells and develop resistance to therapy with additional genetic mutations, which makes radical cure very difficult. Precision medicine against advanced cancer is hampered by the lack of systems aimed at multiple target molecules within multiple loci. Here, we report the development of a versatile diagnostic and therapeutic system for advanced cancer, named the Cupid and Psyche system. Based on the strong non-covalent interaction of streptavidin and biotin, a low immunogenic mutated streptavidin, Cupid, and a modified artificial biotin, Psyche, have been designed. Cupid can be fused with various single-chain variable fragment antibodies and forms tetramer to recognize cancer cells precisely. Psyche can be conjugated to a wide range of diagnostic and therapeutic agents against malignant cells. The Cupid and Psyche system can be used in pre-targeting therapy as well as photo-immunotherapy effectively in animal models supporting the concept of a system for precision medicine for multiple targets within multiple loci.

Nuclear envelope expansion is crucial 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 ß-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.

Kinetic analyses and structure-activity relationship studies of synthetic lysine acetylation catalysts.

Lysine acylation of proteins is a crucial chemical reaction, both as a post-translational modification and as a method for bioconjugation. We previously developed a chemical catalyst, DSH, which activates a chemically stable thioester including acyl-CoA, allowing the site-selective lysine acylation of histones under physiological conditions. However, a more active catalyst is required for efficient lysine acylation in more complex biological milieu, such as in living cells, but there are no rational guidelines for developing efficient lysine acylation catalysts for use under physiological conditions as opposed to in organic solvents. We, herein, conducted a kinetic analysis of the ability of DSH and several derivatives to mediate lysine acetylation to better understand the structural elements essential for high acetylation activity under physiological conditions. Interestingly, the obtained trend in reactivity was different from that observed in organic solvents, suggesting that a different principle is necessary for designing chemical catalysts specifically for use under physiological conditions compared to catalysts for use in organic solvents. Based on the obtained information, we identified a new catalyst scaffold with high activity and structural flexibility for further modification to improve this catalyst system.

Synthetic Posttranslational Modifications: Chemical Catalyst-Driven Regioselective Histone Acylation of Native Chromatin.

Posttranslational modifications (PTMs) of histones play an important role in the complex regulatory mechanisms governing gene transcription, and their dysregulation can cause diseases such as cancer. The lack of methods for site-selectively modifying native chromatin, however, limits our understanding of the functional roles of a specific histone PTM, not as a single mark, but in the intertwined PTM network. Here, we report a synthetic catalyst DMAP-SH (DSH), which activates chemically stable thioesters (including acetyl-CoA) under physiological conditions and transfers various acyl groups to the proximate amino groups. Our data suggest that DSH, conjugated with a nucleosome ligand, such as pyrrole-imidazole-polyamide and LANA (latency-associated nuclear antigen)-peptide, promotes both natural (including acetylation, butyrylation, malonylation, and ubiquitination) and non-natural (azido- and phosphoryl labeling) PTMs on histones in recombinant nucleosomes and/or in native chromatin, at lysine residues close to the DSH moiety. To investigate the validity of our method, we used LANA-DSH to promote histone H2B lysine-120 (K120) acylation, the function of which is largely unknown. H2BK120 acetylation and malonylation modulated higher-order chromatin structures by reducing internucleosomal interactions, and this modulation was further enhanced by histone tail acetylation. This approach, therefore, may have versatile applications for dissecting the regulatory mechanisms underlying chromatin function.

Fidelity and Promiscuity of a Mycobacterial Glycosyltransferase.

Members of the genus Mycobacterium cause devastating human diseases, including tuberculosis. Mycobacterium tuberculosis 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 (Galf). Within the cell envelope, Galf 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 ß(1-5) and ß(1-6) Galf 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 ß(1-5) and ß(1-6) Galf 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 Galf residue was connected to the sequence by a ß(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 ß(1-5) and ß(1-6) pattern of Galf 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.

Supramolecular Ligands for Histone Tails by Employing a Multivalent Display of Trisulfonated Calix[4]arenes.

Post-translational modification of histone tails plays critical roles in gene regulation. Thus, molecules recognizing histone tails and controlling their epigenetic modification are desirable as biochemical tools to elucidate regulatory mechanisms. There are, however, only a few synthetic ligands that bind to histone tails with substantial affinity. We report CA2 and CA3, which exhibited sub-micromolar affinity to histone tails (especially tails with a trimethylated lysine). Multivalent display of trisulfonated calix[4]arene was important for strong binding. CA2 was applicable not only to synthetic tail peptides but also to endogenous histone proteins, and was successfully used to pull-down endogenous histones from nuclear extract. These findings indicate the utility of these supramolecular ligands as biochemical tools for studying chromatin regulator protein and as a targeting motif in ligand-directed catalysis to control epigenetic modifications.

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.

A chemical catalyst enabling histone acylation with endogenous acyl-CoA.

Life emerges from a network of biomolecules and chemical reactions catalyzed by enzymes. As enzyme abnormalities are often connected to various diseases, a chemical catalyst promoting physiologically important intracellular reactions in place of malfunctional endogenous enzymes would have great utility in understanding and treating diseases. However, research into such small-molecule chemical enzyme surrogates remains limited, due to difficulties in developing a reactive catalyst capable of activating inert cellular metabolites present at low concentrations. Herein, we report a small-molecule catalyst, mBnA, as a surrogate for a histone acetyltransferase. A hydroxamic acid moiety of suitable electronic characteristics at the catalytic site, paired with a thiol-thioester exchange process, enables mBnA to activate endogenous acyl-CoAs present in low concentrations and promote histone lysine acylations in living cells without the addition of exogenous acyl donors. An enzyme surrogate utilizing cellular metabolites will be a unique tool for elucidation of and synthetic intervention in the chemistry of life and disease.

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.

Hydroxamic Acid-Piperidine Conjugate is an Activated Catalyst for Lysine Acetylation under Physiological Conditions.

Lysine acylation of proteins is an essential chemical reaction for posttranslational modification and as a means of protein modification in various applications. N,N-Dimethyl-4-aminopyridine (DMAP) derivatives are widely-used catalysts for lysine acylation of proteins; however, the DMAP moiety mostly exists in a protonated, and thus deactivated, form under physiological conditions due to its basicity. An alternative catalytic motif furnishing higher acylation activity would further broaden the possible applications of chemical lysine acylation. We herein report that the hydroxamic acid-piperidine conjugate Ph-HXA is a more active catalytic motif for lysine acetylation than DMAP under physiological conditions. In contrast to DMAP, the hydroxamic acid moiety is mostly deprotonated under aqueous neutral pH, resulting in a higher concentration of the activated form. The Ph-HXA catalyst is also more tolerant of deactivation by a high concentration of glutathione than DMAP. Therefore, Ph-HXA might be a suitable catalytic motif for target protein-selective and site-selective acetylation in cells.

Synthetic hyperacetylation of nucleosomal histones.

We report combinations of a DMAP-based catalyst and phenyl acetate with optimal electron density as a new chemical system for high-yield, selective synthetic acetylation of histone lysine residues. The utility of this chemical system as a unique biologic tool is demonstrated by applying it to Xenopus laevis sperm chromatin.

Sulfanylmethyldimethylaminopyridine as a Useful Thiol Additive for Ligation Chemistry in Peptide/Protein Synthesis.

Sulfanylmethyl-installed dimethylaminopyridine, 2-sulfanylmethyl-4-dimethylaminopyridine (2), has an acidic thiol group comparable to that in aryl thiols due to the formation of a zwitterion consisting of a thiolate anion and a pyridinium cation. It can be used as an additive for native chemical ligation. The alkyl thiol in 2 allows it to be used for the one-pot native chemical ligation-desulfurization protocol in peptide synthesis. The utility of 2 in the synthesis of cyclic peptides is demonstrated.

Enantioselective synthesis of SM-130686 based on the development of asymmetric Cu(I)F catalysis to access 2-oxindoles containing a tetrasubstituted carbon.

Two different catalytic enantioselective approaches to 3-aryl- and 3-alkenyl-3-hydroxy-2-oxindoles have been developed. First, enantioselective arylation and alkenylation reactions of isatins using aryltrimethoxysilanes and alkenyltrimethoxysilanes as nucleophiles can be catalyzed by a complex of CuF with structurally tuned Taniaphos (6) in the presence of a catalytic amount of ZnF(2). Despite the wide substrate scope, this intermolecular reaction was not applicable to a catalytic enantioselective synthesis of SM-130686 (1), a highly potent, orally active growth hormone secretagogue containing a sterically congested chiral tetrasubstituted carbon. Therefore, we developed an intramolecular catalytic enantioselective arylation of alpha-keto amides, taking advantage of the robustness of arylboronate reagents under multiple synthetic conversions and silica gel column chromatography purification. A complex of CuF with Ph-BPE (12) catalyzed the enantioselective arylation of alpha-keto amide 19, affording product 20 in 85% ee. The addition of ZnF(2) to this intramolecular reaction was not necessary. The first enantioselective synthesis of SM-130686 was achieved using this catalytic methodology. Because 2-oxyindoles are a versatile motif for biologically active compounds, the two types of Cu-catalyzed asymmetric reactions developed here will be useful for the synthesis of other natural products and pharmaceutical leads.