Evaluation of acute toxicity of cancer-targeting albumin-based artificial metalloenzymes.

Recently, the development of abiotic metal-mediated drug delivery has been significant growth in the fields of anticancer approach and biomedical application. However, the intrinsic toxicity of abiotic metal catalysts makes in vivo use difficult. Our group developed a system of cancer-targeting albumin-based artificial metalloenyzmes (ArMs) capable of performing localized drug synthesis and selective tagging therapy in vivo for cancer therapy. The toxicity of the system at higher concentrations was investigated in vitro and in vivo in the study to demonstrate its safety for potential application in clinical trials. In cell-based experiments, the study revealed that the cytotoxicity of metal catalysts anchored within the binding cavity of the cancer-targeting ArMs could be significantly reduced compared to free-in-solution metal catalysts. Moreover, the in vivo data demonstrated that the cancer-targeting ArMs did not cause considerable damage in organs or change in the hematological parameters in a single-dose (160 mg/Kg) toxicity study in rats. Therefore, the system is safe, highlighting that it could be used in clinical trials for cancer treatment.

Anticancer Approach Inspired by the Hepatotoxic Mechanism of Pyrrolizidine Alkaloids with Glycosylated Artificial Metalloenzymes.

Metabolic oxidation of pyrrolizidine alkaloids (PAs) from herbal and dietary supplements by cytochrome P450 produces dehydro-PAs (DHPs), which leads to toxicities. A highly reactive cation species generated from the active pyrrole ring of DHPs readily reacts with various cellular components, causing hepatotoxicity and cytotoxicity. Inspired by PA-induced hepatic damage, we developed a therapeutic approach based on a cyclization precursor that can be transformed into a synthetic DHP under physiological conditions through gold-catalyzed 5-endo-dig cyclization using a gold-based artificial metalloenzyme (ArM) instead of through metabolic oxidation by cytochrome P450. In cell-based assays, the synthesis of the DHP by a cancer-targeting glycosylated gold-based ArM substantially suppressed cell growth of the targeted cancer cells without causing cytotoxicity to untargeted cells, highlighting the potential of the strategy to be used therapeutically in vivo.

In vivo organic synthesis by metal catalysts.

The metal-catalyzed reactions have given various chemical modifications that could not be achieved through basic organic chemistry reactions. In the past decade, many metal-mediated catalytic systems have carried out different transformations in cellulo, such as decaging of fluorophores, drug release, and protein conjugation. However, translating abiotic metal catalysts for organic synthesis in vivo, including bacteria, zebrafish, or mice, could encounter numerous challenges regarding their biocompatibility, stability, and reactivity in the complicated biological environment. In this review, we categorize and summarize the relevant advances in this research field by emphasizing the system's framework, the design of each transformation, and the mode of action. These studies disclose the massive potential of the emerging field and the significant applications in synthetic biology.

Prodrug Activation by Gold Artificial Metalloenzyme-Catalyzed Synthesis of Phenanthridinium Derivatives via Hydroamination.

An emerging approach in the field of targeted drug delivery is the establishment of abiotic metal-triggered prodrug mechanisms that can control the release of bioactive drugs. Currently, the design of prodrugs that use abiotic metals as a trigger relies heavily on uncaging strategies. Here, we introduce a strategy based on the gold-catalyzed activation of a phenanthridinium-based prodrug via hydroamination under physiological conditions. To make the prodrug strategy biocompatible, a gold artificial metalloenzyme (ArM) based on human serum albumin, rather than the free gold metal complex, was used as a trigger for prodrug activation. The albumin-based gold ArM protected the catalytic activity of the bound gold metal even in the presence of up to 1 mM glutathione in vitro. The drug synthesized via the gold ArM exerted a therapeutic effect in cell-based assays, highlighting the potential usefulness of the gold ArM in anticancer applications.

Fluorescence "Turn-on" Lectin Sensors Fabricated by Ligand-Assisted Labeling Probes for Detecting Protein-Glycoprotein Interactions.

Elucidation of protein-protein interactions (PPIs) is often very challenging and yields complex and unclear results. Lectin-glycoprotein interactions are especially difficult to study due to the noncovalent nature of the interactions and inherently low binding affinities of proteins to glycan ligands on glycoproteins. Here, we report a "ligand-directed labeling probe (LLP)"-based approach to fabricate protein probes for elucidating protein-glycoprotein interactions. LLP was designed with dual photoactivatable groups for the introduction of an alkyne handle proximal to the carbohydrate-binding pocket of lectins, Ricinus communis agglutinin 120 (RCA120) and recombinant human Siglec-2-Fc. In proof-of-principle studies, alkynylated lectins were conjugated with a photoreactive diazirine cross-linker and an environment-sensitive fluorophore, respectively, by the bioorthogonal click reaction. The modified RCA120 or Siglec-2-Fc was used for detecting the interaction with the target glycoprotein in the solution or endogenously expressed glycoproteins on live HeLa cells. We anticipate that the fabrication of these protein probes will accelerate the discovery of novel PPIs.

Recent advances in self-adjuvanting glycoconjugate vaccines.

Compared to traditional vaccines that are formulated into mixtures of an adjuvant and an antigen, a self-adjuvanting vaccine consists of an antigen that is covalently conjugated to a well-defined adjuvant. In self-adjuvanting vaccines, innate immune receptor ligands are usually used as adjuvants. Innate immune receptor ligands effectively trigger acquired immunity through the activation of innate immunity to enhance host immune responses to antigens. When a self-adjuvanting vaccine is used, immune cells simultaneously uptake the antigen and the adjuvant because they are covalently linked. Consequently, the adjuvant can specifically induce immune responses against the conjugated antigen. Importantly, self-adjuvanting vaccines do not require co-administration of additional adjuvants or immobilization to carrier proteins, which enables avoidance of the use of highly toxic adjuvants or the induction of undesired immune responses. Given these excellent properties, self-adjuvanting vaccines are expected to serve as candidates for the next generation of vaccines. Herein, we review vaccine adjuvants, with a focus on the adjuvants used in self-adjuvanting vaccines, and then overview recent advances made with self-adjuvanting conjugate vaccines.

Enantioselective synthesis of cyclic and linear diamines by imine cycloadditions.

Imine is one of the most versatile functional groups in chemistry and biochemistry fields. Although many biochemical reactions involve imine formation, the inherently unstable property of N-alkyl-α,ß-unsaturated imines still hindered their utilization in organic synthesis. In this article, we described that the N-alkyl-α,ß-unsaturated imines, which prepared from alkylamines and acrolein, could smoothly react through [4 + 4] cycloaddition to give eight-membered diazacyclooctane derivatives in excellent yields. Under a similar condition, in the presence of formaldehyde, the [4 + 2] and [4 + 2 + 2] cycloadditions could lead to the formation of six-membered hexahydropyrimidine or eight-membered triazacyclooctanes, depending on the substituent of aldehydes. Moreover, an easy functional group manipulation of the cyclic products obtained from these cycloadditions can provide variously substituted chiral linear diamines. We can utilize these novel reactivities to reveal the unknown and essential properties of many biological processes that involve N-alkyl-unsaturated imines.

Immunological Evaluation of Co-Assembling a Lipidated Peptide Antigen and Lipophilic Adjuvants: Self-Adjuvanting Anti-Breast-Cancer Vaccine Candidates.

Co-assembling vaccines composed of a lipidated HER2-derived antigenic CH401 peptide and either a lipophilic adjuvant, Pam3 CSK4 , α-GalCer, or lipid A 506, were evaluated as breast cancer vaccine candidates. This vaccine design was aimed to inherit both antigen multivalency and antigen-specific immunostimulation properties, observed in reported self-adjuvanting vaccine candidates, by using self-assembly and adjuvant-conjugated antigens. Under vaccination concentrations, respective lipophilic adjuvants underwent co-assembly with lipidated CH401, which boosted the anti-CH401 IgG and IgM production. In particular, α-GalCer was responsible for the most significant immune activation. Therefore, the newly developed vaccine design enabled the optimization of adjuvants against the antigenic CH401 peptide in a simple preparatory manner. Overall, the co-assembling vaccine design opens the door for efficient and practical self-adjuvanting vaccine development.

Development of α-Gal-Antibody Conjugates to Increase Immune Response by Recruiting Natural Antibodies.

Cancer treatment with antibodies (Abs) is one of the most successful therapeutic strategies for obtaining high selectivity. In this study, α-gal-Ab conjugates were developed that dramatically increased cellular cytotoxicity by recruiting natural Abs through the interaction between α-gal and anti-gal Abs. The potency of the α-gal-Ab conjugates depended on the amount of α-gal conjugated to the antibody: the larger the amount of α-gal introduced, the higher the level of cytotoxicity observed. The conjugation of antibodies with an α-gal dendrimer allowed the introduction of large amounts of α-gal to the Ab, without loss of affinity for the target cell. The method described here will enable the re-development of Abs to improve their potency.

Syntheses and Immunological Evaluation of Self-Adjuvanting Clustered N-Acetyl and N-Propionyl Sialyl-Tn Combined with a T-helper Cell Epitope as Antitumor Vaccine Candidates.

Sialyl-Tn (STn) is a tumor-associated carbohydrate antigen (TACA) rarely observed on healthy tissues. We synthesized two fully synthetic N-acetyl and N-propionyl STn trimer (triSTn) vaccines possessing a T-helper epitope and a TLR2 agonist, since the clustered STn antigens are highly expressed on many cancer cells. Immunization of both vaccines in mice induced the anti-triSTn IgG antibodies, which recognized triSTn-expressing cell lines PANC-1 and HepG2. The N-propionyl triSTn vaccine induced the triSTn-specific IgGs, while IgGs induced by the N-acetyl triSTn vaccine were less specific. These results illustrated that N-propionyl triSTn is a valuable unnatural TACA for anticancer vaccines.

A photo-cleavable biotin affinity tag for the facile release of a photo-crosslinked carbohydrate-binding protein.

The use of photo-crosslinking glycoprobes represents a powerful strategy for the covalent capture of labile protein complexes and allows detailed characterization of carbohydrate-mediated interactions. The selective release of target proteins from solid support is a key step in functional proteomics. We envisaged that light activation can be exploited for releasing labeled protein in a dual photo-affinity probe-based strategy. To investigate this possibility, we designed a trifunctional, galactose-based, multivalent glycoprobe for affinity labeling of carbohydrate-binding proteins. The resulting covalent protein-probe adduct is attached to a photo-cleavable biotin affinity tag; the biotin moiety enables specific presentation of the conjugate on streptavidin-coated beads, and the photolabile linker allows the release of the labeled proteins. This dual probe promotes both the labeling and the facile cleavage of the target protein complexes from the solid surfaces and the remainder of the cell lysate in a completely unaltered form, thus eliminating many of the common pitfalls associated with traditional affinity-based purification methods.

Anticancer approach by targeted activation of a global inhibitor of sialyltransferases with acrolein.

Cells are covered with a thick layer of sugar molecules known as glycans. Abnormal glycosylation is a hallmark of cancer, and hypersialylation increases tumor metastasis by promoting immune evasion and inducing tumor cell invasion and migration. Inhibiting sialylation is thus a potential anticancer treatment strategy. However, targeting sialic acids is difficult because of the lack of selective delivery tools. Here, we present a prodrug strategy for selectively releasing the global inhibitor of sialylation peracetylated 3Fax-Neu5Ac (PFN) in cancer cells using the reaction between phenyl azide and endogenous acrolein, which is overproduced in most cancer cells. The prodrug significantly suppressed tumor growth in mice as effectively as PFN without causing kidney dysfunction, which is associated with PFN. The use of sialylated glycans as immune checkpoints is gaining increasing attention, and the proposed method for precisely targeting aberrant sialylation provides a novel avenue for expanding current cancer treatments.

Stepwise orthogonal click chemistry toward fabrication of paclitaxel/galactose functionalized fluorescent nanoparticles for HepG2 cell targeting and delivery.

In this report, we used stepwise orthogonal click chemistry (SOCC) involving strain-promoted azide-alkyne cycloaddition (SPAAC) and microwave-assisted Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) to assemble an anticancer drug (paclitaxel, PTX) and a targeting ligand (trivalent galactoside, TGal) on a fluorescent silicon oxide nanoparticle (NP) by using dialkyne linker 8 as a bridge. The fluorescent NH2@Cy3SiO2NP was fabricated using a competition method to incorporate Cy3 without loss of the original surface amine density on the NPs. The concept of SOCC was first investigated in a solution-phase model study that showed quantitative reaction yield. In the fabrication of TGal-PTX@Cy3SiO2NP, the expensive compound azido-functionalized PTX 12 used in SPAAC can be easily recovered due to the absence of other reagents in the reaction mixture. High loading of the sugar ligand on the NP surface serves a targeting function and also overcomes the low water solubility of PTX. Confocal fluorescence microscopy and cytotoxicity assay showed that TGal-PTX@Cy3SiO2NP was taken up by HepG2 cells and was affected by the microtubule skeleton in these cells and inhibited the proliferation of these cells in a dose-dependent manner. The presence of a fluorescent probe, a targeting ligand, and an anticancer drug on the multifunctional TGal-PTX@Cy3SiO2NP allows for real-time imaging, specific cancer-cell targeting, and the cell-killing effect which is better than free PTX.

Synthesis and evaluation of a photoactive probe with a multivalent carbohydrate for capturing carbohydrate-lectin interactions.

Lectins are ubiquitous carbohydrate-binding proteins of nonimmune origin that are characterized by their specific recognition of defined monosaccharide or oligosaccharide structures. However, the use of carbohydrates to study lectin has been restricted by the weak binding affinity and noncovalent character of the interaction between carbohydrates and lectin. In this report, we designed and synthesized a multifunctional photoaffinity reagent composed of a trialkyne chain, a masked latent amine group, and a photoreactive 3-trifluoromethyl-3-phenyl-diazirine group in high overall yield. Two well-defined chemistries, Huisgen-Sharpless click chemistry and amide bond coupling, were the key steps for installing the multivalent character and tag in our designed photoaffinity probe. The photolabeling results demonstrated that the designed probe selectively labeled the target lectin, RCA120 ( Ricinus communis Agglutinin), in an E. coli lysate and an asialoglycoprotein receptor (ASGP-R) on intact HepG2 cell membranes. Moreover, the probe also enabled the detection of weak protein-protein interactions between RCA120 and ovalbumin (OVA).

Synthesis of acylguanidine zanamivir derivatives as neuraminidase inhibitors and the evaluation of their bio-activities.

A series of acylguanidine-modified zanamivir analogs were synthesized and their inhibitory activities against the NAs of avian influenza viruses (H1N1 and H3N2) were evaluated. In particular, zanamivir derivative , with a hydrophobic naphthalene substituent, exhibits the best inhibitory activity against group-1 NA with an IC50 of 20 nM.

Catalytic olefin metathesis in blood.

The direct synthesis of drugs in vivo enables drugs to treat diseases without causing side effects in healthy tissues. Transition-metal reactions have been widely explored for uncaging and synthesizing bioactive drugs in biological environments because of their remarkable reactivity. Nonetheless, it is difficult to develop a promising method to achieve in vivo drug synthesis because blood cells and metabolites deactivate transition-metal catalysts. We report that a robust albumin-based artificial metalloenzyme (ArM) with a low loading (1-5 mol%) can promote Ru-based olefin metathesis to synthesize molecular scaffolds and an antitumor drug in blood. The ArM retained its activity after soaking in blood for 24 h and provided the first example of catalytic olefin cross metathesis in blood. Furthermore, the cyclic-Arg-Gly-Asp (cRGD) peptide-functionalized ArM at lower dosages could still efficiently perform in vivo drug synthesis to inhibit the growth of implanted tumors in mice. Such a system can potentially construct therapeutic drugs in vivo for therapies without side effects.

Stereoselective synthesis of S-linked α(2→8) and α(2→8)/α(2→9) hexasialic acids.

A new approach for the synthesis of S-linked α(2→8) and alternating α(2→8)/α(2→9) oligosialic acids by S-alkylation has been developed, using chemo- and stereoselective alkylation of a C2-thiolated sialoside donor (nucleophile) with either a C8- or C9-iodide-activated sialoside acceptor (electrophile). An efficient intramolecular acetyl group migration from the C7 to C9-position of the sialoside under mild basic conditions was used to generate the C8-iodide, the key sialyl acceptor (electrophile). Using this strategy, the syntheses of S-linked α(2→8) and α(2→8)/α(2→9) hexasialic acids were achieved.

Site-specific immobilization of enzymes on magnetic nanoparticles and their use in organic synthesis.

Magnetic nanoparticles (MNPs) are attractive materials that serve as a support for enzyme immobilization and facilitate separations by applying an external magnetic field; this could facilitate the recycling of enzymes and broaden their applications in organic synthesis. Herein, we report the methods for the immobilization of water-soluble and membrane-bound enzymes, and the activity difference between free and immobilized enzymes is discussed. Sialyltransferase (PmST1, from Pasteurella multocida ) and cytidine monophosphate (CMP)-sialic acid synthetase (CSS, from Neisseria meningitides ) were chosen as water-soluble enzymes and expressed using an intein expression system. The enzymes were site-specifically and covalently immobilized on PEGylated-N-terminal cysteine MNPs through native chemical ligation (NCL). Increasing the length of the PEG linker between the enzyme and the MNP surface increased the activity of the immobilized enzymes relative to the free parent enzymes. In addition, the use of a fluorescent acceptor tag for PmST1 affected enzyme kinetics. In contrast, sialyltransferase from Neisseria gonorrheae (NgST, a membrane-bound enzyme) was modified with a biotin-labeled cysteine at the C-terminus using NCL, and the enzyme was then assembled on streptavidin-functionalized MNPs. Using a streptavidin-biotin interaction, it was possible to immobilize NgST on a solid support under mild ligation conditions, which prevented the enzyme from high-temperature decomposition and provided an approximately 2-fold increase in activity compared to other immobilization methods on MNPs. Finally, the ganglioside GM3-derivative (sialyl-lactose derivative) was synthesized in a one-pot system by combining the use of immobilized PmST1 and CSS. The enzymes retained 50% activity after being reused ten times. Furthermore, the results obtained using the one-pot two-immobilized-enzyme system demonstrated that it can be applied to large-scale reactions with acceptable yields and purity. These features make enzyme-immobilized MNPs applicable to organic synthesis.

Precise immunological evaluation rationalizes the design of a self-adjuvanting vaccine composed of glycan antigen, TLR1/2 ligand, and T-helper cell epitope.

Sialyl-Tn (STn), overexpressed on various tumors, has been investigated for its application in anti-cancer vaccine therapy. However, Theratope, an STn-based vaccine, failed in the phase III clinical trial due to poor immunogenicity and epitope suppression by the foreign carrier protein. We therefore developed a self-adjuvanting STn based-vaccine, a conjugate of clustered STn (triSTn) antigen, TLR1/2 ligand (Pam3CSK4), and T-helper (Th) cell epitope, and found that this three-component self-adjuvanting vaccine effectively resulted in the production of anti-triSTn IgG antibodies. We herein analyzed immune responses induced by this self-adjuvanting vaccine in detail. We newly synthesized two-component vaccines, i.e., Pam3CSK4- or Th epitope-conjugated triSTn, as references to evaluate the immune-stimulating functions of Pam3CSK4 and Th epitope. Immunological evaluation of the synthesized vaccine candidates revealed that Pam3CSK4 was essential for antibody production, indicating that the uptake of triSTn antigen by antigen-presenting cells (APCs) was promoted by the recognition of Pam3CSK4 by TLR1/2. The function of the Th epitope was also confirmed. Th cell activation was important for boosting antibody production and IgG subclass switching. Furthermore, flow cytometric analyses of immune cells, including T cells, B cells, dendritic cells, and other monocytes, were first employed in the evaluation of self-adjuvanting vaccines and revealed that the three-component vaccine was able to induce antigen-specific immune responses for efficient antibody production without excessive inflammatory responses. Importantly, the co-administration of Freund's adjuvants was suggested to cause excessive myeloid cell accumulation and decreased plasma cell differentiation. These results demonstrate that vaccines can be designed to achieve the desired immune responses via the bottom-up construction of each immune element.

Epoc group: transformable protecting group with gold(iii)-catalyzed fluorene formation.

This study presents the novel concept of a transformable protecting group, which changes its properties through structural transformation. Based on this concept, we developed a 2-(2-ethynylphenyl)-2-(5-methylfuran-2-yl)-ethoxycarbonyl (Epoc) group. The Epoc group was transformed into an Fmoc-like structure with gold(iii)-catalyzed fluorene formation and was removable under Fmoc-like mild basic conditions post-transformation even though it was originally stable under strongly basic conditions. As an application for organic synthesis, the Epoc group provides the novel orthogonality of gold(iii)-labile protecting groups in solid-phase peptide synthesis. In addition, the high turnover number of fluorene formation in aqueous media is suggestive of the applicability of the Epoc group to biological systems.