Umpolung Phosphorylation of Tyrosine via 1,2-Phospha-Brook Rearrangement.

Phosphorylated tyrosine is a fundamental building block of bioactive peptides and proteins. However, the chemoselective phosphorylation of tyrosine over other nucleophilic amino acid residues in unprotected peptides remains a significant challenge. Here we report an umpolung strategy that converts the C-terminal tyrosine into an electrophilic spirolactone cyclohexadienone motif through hypervalent iodine oxidation, followed by a 1,2-phospha-Brook rearrangement using phosphite diesters as nucleophilic phosphoryl donors. This reaction proceeds chemoselectively at the tyrosine phenol and is applicable to a wide range of peptide substrates containing various nucleophilic amino acid residues, including serine and threonine.

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.

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.

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.

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.

Stereoconvergent 1,4-Addition Reaction of 5H-Oxazol-4-ones with E,Z Isomeric Mixture of Alkylidene ß-Ketoesters Catalyzed by Chiral Guanidines.

A 1,4-addition reaction of 5H-oxazol-4-ones to alkylidene ß-ketoesters, which was catalyzed by using chiral guanidines via a dynamic kinetic resolution process that involved geometric isomerization of the alkylidene ß-ketoesters, was developed. This method allowed using E,Z isomeric mixture of various acyclic alkylidene ß-ketoesters to obtain the products stereoselectively. The relation between the geometry and the stereoselectivity of products was investigated, and the derivatization of the products to the corresponding α-acyl-γ-lactone was performed.

Organocatalytic 1,4-Addition Reaction of 2-Formyl(thio)esters to Vinylketones: An Efficient Access to Acyclic Chiral Building Blocks with a Quaternary Carbon Stereocenter.

2-Formyl(thio)esters were utilized as pronucleophiles to obtain less-accessible acyclic chiral building blocks bearing versatile functional groups on a quaternary carbon atom for enantioselective 1,4-addition to vinylketones. To achieve high enantioselectivity in the present 1,4-addition reaction, thiourea-tertiary amines containing a bulky chiral backbone were developed as catalysts, and several derivatizations of the products were performed to demonstrate the synthetic utility of the products.