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Amphipathic peptides composed of cationic amino acids and hydrophobic amino acids have cell-penetrating ability and are often used as a delivery tool for membrane-impermeable compounds. Small interfering RNA (siRNAs) are one of the delivery targets for such cell-penetrating peptides (CPPs). Cationic CPPs can associate with anionic siRNAs by electrostatic interactions resulting in the formation of nano-sized complexes, which can deliver siRNAs intracellularly. CPPs containing unnatural amino acids offer promising tools to siRNA delivery. However, the detailed structure–activity relationship in siRNA delivery has been rarely studied. In the current study, we designed peptides containing dipropylglycine (Dpg) and explored the cellular uptake and cytotoxicity of peptide/siRNA complexes. The amphipathic structure of the peptides played a key role in complexation with siRNAs and intracellular siRNA delivery. In the amphipathic peptides, cellular uptake of siRNA increased with increasing peptide length, but cytotoxicity was reduced. A peptide containing four Dpg exhibited an effective gene-silencing effect with small amounts of peptides without cytotoxicity in medium containing serum. These findings will be helpful for the design of novel CPPs for siRNA delivery., Chemical & pharmaceutical bulletin, 71(3), pp. 250-256; 2023

SAR studies of PROTACs that target H-PGDS, focusing on the E3 ligase ligand and the H-PGDS ligand, are described.

Targeted protein degradation by proteolysis-targeting chimera (PROTAC) is one of the exciting modalities for drug discovery and biological discovery. It is important to select an appropriate linker, an E3 ligase ligand, and a target protein ligand in the development; however, it is necessary to synthesize a large number of PROTACs through trial and error. Herein, using a docking simulation of the ternary complex of a hematopoietic prostaglandin D synthase (H-PGDS) degrader, H-PGDS, and cereblon, we have succeeded in developing PROTAC(H-PGDS)-7 (6), which showed potent and selective degradation activity (DC50 = 17.3 pM) and potent suppression of prostaglandin D2 production in KU812 cells. Additionally, in a Duchenne muscular dystrophy model using mdx mice with cardiac hypertrophy, compound 6 showed better inhibition of inflammatory cytokines than a potent H-PGDS inhibitor TFC-007. Thus, our results demonstrated that in silico simulation would be useful for the rational development of PROTACs.

Understanding the characteristics of crystal polymorphism of active pharmaceutical ingredients and analyzing them with high sensitivity is important for quality of drug products, appropriate characterization strategies, and appropriate screening and selection processes. However, there are few methods to measure intra- and intermolecular correlations in crystals other than X-ray crystallography, for which it is sometimes difficult to obtain suitable single crystals. Recently, solid-state NMR has been recognized as a straightforward method for measuring molecular correlations. In this study, we selected ranitidine hydrochloride, which is known to exist in two forms, 1 and 2, as the model drug and investigated each form using solid-state NMR. In conducting the analysis, rotating the sample tube, which had a 1-mm inner diameter, increased the solid-state NMR resolution at 70 kHz. The

Proteolysis targeting chimeras (PROTACs) have emerged as a powerful technology for the degradation of disease-related proteins by the hijacking of the endogenous ubiquitin-proteasome system. A multitude of bifunctional PROTACs have been developed using small-molecule ligands; one ligand binds to the target protein of interest and one ligand binds to an E3 ligase. The characteristics of those PROTACs vary, including their reversible or irreversible covalent binding to the target protein, their binding to orthosteric and allosteric sites, their agonist or antagonist activity, and their use of multiple ligands. In addition, oligopeptides and nucleotides have recently been used as alternative targeting ligands. The properties of PROTACs, such as selectivity, delivery and sensitivity to drug resistance, can be improved through the use of a variety of targeting ligand modalities. This minireview introduces the mechanisms and behavior of small-molecule based PROTACs as well as targeted proteolysis techniques using peptides and nucleic acids as targeting ligands.

N-Nitrosodimethylamine (NDMA) is a probable human carcinogen. This study investigated the root cause of the presence of NDMA in ranitidine hydrochloride. Forced thermal degradation studies of ranitidine hydrochloride and its inherent impurities (Imps. A, B, C, D, E, F, G, H, I, J, and K) listed in the European and United States Pharmacopeias revealed that in addition to ranitidine, Imps. A, C, D, E, H, and I produce NDMA at different rates in a solid or an oily liquid state. The rate of NDMA formation from amorphous Imps. A, C, and E was 100 times higher than that from crystalline ranitidine hydrochloride under forced degradation at 110 °C for 1 h. Surprisingly, crystalline Imp. H, bearing neither the N,N-dialkyl-2-nitroethene-1,1-diamine moiety nor a dimethylamino group, also generated NDMA in the solid state, while Imp. I, as an oily liquid, favorably produced NDMA at moderate temperatures (e.g., 50 °C). Therefore, strict control of the aforementioned specific impurities in ranitidine hydrochloride during manufacturing and storage allows appropriate control of NDMA in ranitidine and its pharmaceutical products. Understanding the pathways of the stability related NDMA formation enables improved control of the pharmaceuticals to mitigate this risk.

Optimizing linker design is important for ensuring efficient degradation activity of proteolysis-targeting chimeras (PROTACs). Therefore, developing a straightforward synthetic approach that combines the protein-of-interest ligand (POI ligand) and the ligand for E3 ubiquitin ligase (E3 ligand) in various binding styles through a linker is essential for rapid PROTAC syntheses. Herein, a solid-phase approach for convenient PROTAC synthesis is presented. We designed azide intermediates with different linker lengths to which the E3 ligand, pomalidomide, is attached and performed facile PROTACs synthesis by forming triazole, amide, and urea bonds from the intermediates.

A chiral cyclic α,α-disubstituted α-amino acid, (S)-(-)-cucurbitine, which has a pyrrolidine ring with a chiral center at the α-position, was synthesized, and its homopeptides were prepared. (S)-(-)-Cucurbitine homopeptides with a Boc-protecting group formed helical structures with slight control of the helical screw sense to the left-handed form. The state of the pyrrolidine ring in (S)-(-)-cucurbitine was important for the control of the helical structures and helical screw sense of its homopeptides.

[Image: see text] Although hematopoietic prostaglandin D synthase (H-PGDS) is an attractive target for treatment of a variety of diseases, including allergic diseases and Duchenne muscular dystrophy, no H-PGDS inhibitors have yet been approved for treatment of these diseases. Therefore, the development of novel agents having other modes of action to modulate the activity of H-PGDS is required. In this study, a chimeric small molecule that degrades H-PGDS via the ubiquitin-proteasome system, PROTAC(H-PGDS)-1, was developed. PROTAC(H-PGDS)-1 is composed of two ligands, TFC-007 (that binds to H-PGDS) and pomalidomide (that binds to cereblon). PROTAC(H-PGDS)-1 showed potent activity in the degradation of H-PGDS protein via the ubiquitin-proteasome system and in the suppression of prostaglandin D(2) (PGD(2)) production. Notably, PROTAC(H-PGDS)-1 showed sustained suppression of PGD(2) production after the drug removal, whereas PGD(2) production recovered following removal of TFC-007. Thus, the H-PGDS degrader—PROTAC(H-PGDS)-1—is expected to be useful in biological research and clinical therapies.

Antimicrobial peptides (AMPs) are expected to be good candidate molecules for novel antimicrobial therapies. Most AMPs exert their antimicrobial activity through disruption of microbial membranes due to their amphipathic properties. Recently, the helical peptide 'Stripe' was reported by our group, a rationally designed amphipathic AMP focused on distribution of natural cationic and hydrophobic amino acid residues. In this study, a set of Stripe-based AMP foldamers was designed, synthesized and investigated that contain α,α-disubstituted amino acids or side-chain stapling to stabilize their helical structures. Our results showed that a peptide containing 2-aminoisobutyric acid (Aib) residues exhibited potent antimicrobial activity against both Gram-positive S.aureus (MIC value: 3.125 μM) and Gram-negative bacteria (including a multidrug-resistant strain, MDRP, MIC value: 1.56 μM), without significant hemolytic activity (>100 μM). Electrophysiological measurements revealed that this peptide formed stable pores in a 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)/1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) bilayer but not in a dioleoylphosphocholine (DOPC) bilayer. The introduction of Aib residues into Stripe could be a promising way to increase the antimicrobial activity of AMP foldamers, and the peptide could represent a promising novel therapeutic candidate to treat multidrug-resistant bacterial infection.

The purpose of this study was to elucidate the effect of high-temperature storage on the stability of ranitidine, specifically with respect to the potential formation of N-nitrosodimethylamine (NDMA), which is classified as a probable human carcinogen. Commercially available ranitidine reagent powders and formulations were stored under various conditions, and subjected to LC-MS/MS analysis. When ranitidine tablets from two different brands (designated as tablet A and tablet B) were stored under accelerated condition (40 °C with 75% relative humidity), following the drug stability guidelines issued by the International Conference on Harmonisation (ICH-Q1A), for up to 8 weeks, the amount of NDMA in them substantially increased from 0.19 to 116 ppm and from 2.89 to 18 ppm, respectively. The formation of NDMA that exceeded the acceptable daily intake limit (0.32 ppm) at the temperature used under accelerated storage conditions clearly highlights the risk of NDMA formation in ranitidine formulations when extrapolated to storage under ambient conditions. A forced-degradation study under the stress condition (60 °C for 1 week) strongly suggested that environmental factors such as moisture and oxygen are involved in the formation of NDMA in ranitidine formulations. Storage of ranitidine tablets and reagent powders at the high temperatures also increased the amount of nitrite, which is considered one of the factors influencing NDMA formation. These data indicate the necessity of controlling/monitoring stability-related factors, in addition to controlling impurities during the manufacturing process, in order to mitigate nitrosamine-related health risks of certain pharmaceuticals.

The side effects of kwao keur dietary supplements (obtained from the tuberous root of Pueraria mirifica) have recently been reported by the Ministry of Health, Labour and Welfare, Japan. To control the quality of kwao keur products, its ingredients need to be maintained by characteristic marker compounds, such as miroestrol, deoxymiroestrol, and kwakhurin (KWA). In this study, we described the facile synthesis of KWA, a marker compound of P. mirifica. Our revised synthetic method produced KWA with shorter steps and higher yield than the reported method. Furthermore, the absolute purity of KWA was determined by quantitative NMR analysis for standardization as a reagent, and its purity was 92.62 ± 0.12%.

Cell-penetrating peptides (CPPs) have gained much attention as carriers of hydrophilic molecules, such as drugs, peptides, and nucleic acids, into cells. CPPs are mainly composed of cationic amino acid residues, which play an important role in their intracellular uptake via interactions with acidic groups on cell surfaces. In addition, the secondary structures of CPPs also affect their cell-membrane permeability. Based on this knowledge, a variety of cell-penetrating foldamers (oligomers that form organized secondary structures) have been developed to date. In this account, we describe recent attempts to develop cell-penetrating foldamers containing various building blocks, and their application as DDS carriers.

Manipulation of protein stability using small molecules has a great potential for both basic research and clinical therapy. Based on our protein knockdown technology, we developed chimeric degrader molecules SNIPER(ER)s that target the estrogen receptor alpha (ERα) for degradation via the ubiquitin-proteasome system. This chapter describes the design and synthesis of SNIPER(ER) compounds and methods for the evaluation of their activity in cellular systems and in a tumor xenograft model.

Arginine (Arg)-rich peptides can penetrate the cell membrane and deliver nucleic acid-based therapeutics into cells. In this study, a helical template designed with a repeating sequence composed of two l-leucines (l-Leu) and a 2-aminoisobutyric acid (Aib) (l-Leu-l-Leu-Aib) was conjugated to nona-arginine on either the C- or N- terminus, designated as Block 1 and Block 2. Each terminal modification induced helical structure formation and improved the physicochemical properties of peptide/plasmid DNA (pDNA) complexes, resulting in efficient intracellular pDNA delivery. The introduction of a helical template may be effective for the endosomal escape of pDNA and pDNA release from complexes in cells. These results emphasized the potency of a helical template for the development of novel cell-penetrating peptides for pDNA delivery.

Purpose Antibody-drug conjugates (ADCs), which are monoclonal antibodies (mAbs) conjugated with highly toxic payloads, achieve high tumor killing efficacy due to the specific delivery of payloads in accordance with mAbs’ function. On the other hand, the conjugation of payloads often increases the hydrophobicity of mAbs, resulting in reduced stability and increased aggregation. It is considered that mAb aggregates have potential risk for activating Fcγ receptors (FcγRs) on immune cells, and are internalized into cells via FcγRs. Based on the mechanism of action of ADCs, the internalization of ADCs into target-negative cells may cause the off-target toxicity. However, the impacts of aggregation on the safety of ADCs including off-target cytotoxicity have been unclear. In this study, we investigated the cytotoxicity of ADC aggregates in target-negative cells. Methods The ADC aggregates were generated by stirring stress or thermal stress. The off-target cytotoxicity of ADC aggregates was evaluated in several target-negative cell lines, and FcγR-activation properties of ADC aggregates were characterized using a reporter cell assay. Results Aggregation of ADCs enhanced the off-target cytotoxicity in several target-negative cell lines compared with non-stressed ADCs. Notably, ADC aggregates with FcγR-activation properties showed dramatically enhanced cytotoxicity in FcγR-expressing cells. The FcγR-mediated off-target cytotoxicity of ADC aggregates was reduced by using a FcγR-blocking antibody or Fc-engineering for silencing Fc-mediated effector functions. Conclusions These results indicated that FcγRs play an important role for internalization of ADC aggregates into non-target cells, and the aggregation of ADCs increases the potential risk for off-target toxicity.

N-Nitrosodimethylamine (NDMA) is a probable human carcinogen. This study investigated the root cause of the presence of NDMA in ranitidine hydrochloride. Forced thermal degradation studies of ranitidine hydrochloride and its inherent impurities (Imps. A, B, C, D, E, F, G, H, I, J, and K) listed in the European and United States Pharmacopeias revealed that in addition to ranitidine, Imps. A, C, D, E, H, and I produce NDMA at different rates in a solid or an oily liquid state. The rate of NDMA formation from amorphous Imps. A, C, and E was 100 times higher than that from crystalline ranitidine hydrochloride under forced degradation at 110 °C for 1 h. Surprisingly, crystalline Imp. H, bearing neither the N,N-dialkyl-2-nitroethene-1,1-diamine moiety nor a dimethylamino group, also generated NDMA in the solid state, while Imp. I, as an oily liquid, favorably produced NDMA at moderate temperatures (e.g., 50 °C). Therefore, strict control of the aforementioned specific impurities in ranitidine hydrochloride during manufacturing and storage allows appropriate control of NDMA in ranitidine and its pharmaceutical products. Understanding the pathways of the stability related NDMA formation enables improved control of the pharmaceuticals to mitigate this risk.

Inducing degradation of undruggable target proteins by the use of chimeric small molecules, represented by proteolysis-targeting chimeras, is a promising strategy for drug development. We developed a series of chimeric molecules, termed "specific and nongenetic inhibitor of apoptosis protein (IAP)-dependent protein erasers" (SNIPERs) that recruit IAP ubiquitin ligases to induce degradation of target proteins. SNIPERs also induce degradation of some IAPs, including cIAP1 and XIAP, which are antiapoptotic proteins that are overexpressed in many cancers. Such protein degraders have unique properties that could be especially useful in cancer therapy. This chapter describes (1) the design and synthesis of SNIPER compounds, (2) the methods used for the detection of target protein degradation and ubiquitylation, and (3) the protocol to evaluate the antitumor activity of SNIPER.

Hematopoietic prostaglandin D synthase (H-PGDS) is an attractive target for the development of therapeutic agents for Duchenne muscular dystrophy (DMD) and other H-PGDS-related diseases. We have recently developed the H-PGDS degrader PROTAC(H-PGDS)-1, which is a chimeric molecule in which TFC-007 (that binds to H-PGDS) and pomalidomide (that binds to cereblon [CRBN]) were conjugated to the PEG5 linker. Herein, using a docking simulation of the ternary complex of the H-PGDS degrader, H-PGDS, and CRBN, we have succeeded in developing PROTAC(H-PGDS)-7, a new H-PGDS degrader that does not contain a linker. PROTAC(H-PGDS)-7 showed potent and selective degradation activity (DC50 = 17.3 pM), and potent suppression of prostaglandin D2 (PGD2) production in KU812 cells. Additionally, in a DMD model using mdxmice with cardiac hypertrophy, PROTAC(H-PGDS)-7 showed better inhibition of inflammatory cytokines than TFC-007. PROTAC(H-PGDS)-7 is expected to be a promising candidate for the treatment of DMD and other H-PGDS-related diseases.

A chiral cyclic α,α-disubstituted α-amino acid, (

Inducing degradation of undruggable target proteins by the use of chimeric small molecules, represented by proteolysis-targeting chimeras, is a promising strategy for drug development. We developed a series of chimeric molecules, termed "specific and nongenetic inhibitor of apoptosis protein (IAP)-dependent protein erasers" (SNIPERs) that recruit IAP ubiquitin ligases to induce degradation of target proteins. SNIPERs also induce degradation of some IAPs, including cIAP1 and XIAP, which are antiapoptotic proteins that are overexpressed in many cancers. Such protein degraders have unique properties that could be especially useful in cancer therapy. This chapter describes (1) the design and synthesis of SNIPER compounds, (2) the methods used for the detection of target protein degradation and ubiquitylation, and (3) the protocol to evaluate the antitumor activity of SNIPER.

Magainin 2 (Mag2), which was isolated from the skin of the African clawed frog, is a representative antimicrobial peptide (AMP) that exerts antimicrobial activity via microbial membrane disruption. It has been reported that the helicity and amphipathicity of Mag2 play important roles in its antimicrobial activity. We investigated and recently reported that 17 amino acid residues of Mag2 are required for its antimicrobial activity, and accordingly developed antimicrobial foldamers containing &alpha, &alpha, disubstituted amino acid residues. In this study, we further designed and synthesized a set of Mag2 derivatives bearing the hydrocarbon stapling side chain for helix stabilization. The preferred secondary structures, antimicrobial activities, and cell-membrane disruption activities of the synthesized peptides were evaluated. Our analyses revealed that hydrocarbon stapling strongly stabilized the helical structure of the peptides and enhanced their antimicrobial activity. Moreover, peptide 2 stapling between the first and fifth position from the N-terminus showed higher antimicrobial activity than that of Mag2 against both gram-positive and gram-negative bacteria without exerting significant hemolytic activity. To investigate the modes of action of tested peptides 2 and 8 in antimicrobial and hemolytic activity, electrophysiological measurements were performed.

Antimicrobial peptides (AMPs) are potential novel therapeutic drugs against microbial infections. Most AMPs function by disrupting microbial membranes because of their amphipathic properties and ordered secondary structures. In this minireview, we describe recent efforts to develop helical AMP foldamers containing non-proteinogenic amino acids, such as α,α-disubstituted α-amino acids, β-amino acids, γ-amino acids, side-chain stapling and N-alkyl glycines.

Although hematopoietic prostaglandin D synthase (H-PGDS) is an attractive target for treatment of a variety of diseases, including allergic diseases and Duchenne muscular dystrophy, no H-PGDS inhibitors have yet been approved for treatment of these diseases. Therefore, the development of novel agents having other mode of actions to modulate the activity of H-PGDS is required. In this study, a chimeric small molecule that degrades H-PGDS via the ubiquitin-proteasome system, PROTAC(H-PGDS)-1, was developed. PROTAC(H-PGDS)-1 is composed of two ligands, TFC-007 (that binds to H-PGDS) and pomalidomide (that binds to cereblon). PROTAC(H-PGDS)-1 showed potent activity in the degradation of H-PGDS protein via the ubiquitin-proteasome system and in the suppression of prostaglandin D2 (PGD2) production. Notably, PROTAC(H-PGDS)-1 was slightly more effective in the suppression of PGD2 production than the known inhibitor, TFC-007. Thus, the H-PGDS degrader—PROTAC(H-PGDS)-1—is expected to be useful in biological research and clinical therapies.

TGR5, a G-protein-coupled receptor (GPCR), plays an important role in several physiological functions. TGR5 activation through bile acids induces an increase in energy expenditure. Therefore, synthetic TGR5 ligands could be useful for the treatment of obesity or dyslipidemia. In this study, we designed and synthesized a set of TGR5 ligands with a 5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalene (TMN) skeleton, and evaluated their TGR5 agonistic activity. We also investigated the selectivity of the synthesized compounds for TGR5 relative to the farnesoid X receptor (FXR) and retinoic acid receptor (RAR). Our results show that compound 4 b [N-(2-chlorophenyl)-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenecarboxamide] exhibited potent TGR5 agonist activity with an IC50 value of 8.4 nM without significant cytotoxicity. In addition, compound 4 b showed only slight agonistic activity toward FXR and RAR at 1 μM treatment. These data indicate that compound 4 b is a selective TGR5 agonist, and could be a promising therapeutic agent for dyslipidemia.

The side effects of kwao keur dietary supplements (obtained from the tuberous root of Pueraria mirifica) have recently been reported by the Ministry of Health, Labour and Welfare, Japan. To control the quality of kwao keur products, its ingredients need to be maintained by characteristic marker compounds, such as miroestrol, deoxymiroestrol, and kwakhurin (KWA). In this study, we described the facile synthesis of KWA, a marker compound of P. mirifica. Our revised synthetic method produced KWA with shorter steps and higher yield than the reported method. Furthermore, the absolute purity of KWA was determined by quantitative NMR analysis for standardization as a reagent, and its purity was 92.62 ± 0.12%.

Peptide-based inducers of estrogen receptor (ER) α and androgen receptor (AR) degradations via the ubiquitin-proteasome system (UPS) were developed. The designated inducers were composed of two biologically active scaffolds: the helical peptide PERM3, which is an LXXLL-like mimic of the coactivator SRC-1, and various small molecules (MV1, LCL161, VH032, and POM) that bind to E3 ligases (IAPs, VHL, and cereblon, respectively), to induce ubiquitylation of nuclear receptors that bind to SRC-1. All of the synthesized chimeric E3 ligand-containing molecules induced the UPS-mediated degradation of ERα and AR. The PERM3 peptide was applicable for the development of the ERα and AR degraders using these E3 ligands.

Photopharmacology has attracted attention as an approach for the development of novel therapeutics because it allows regulation of the bioactivity of compounds based on their conformational change by photo-irradiation. Previously, we have reported several types of selective estrogen receptor (ER) modulators based on diphenylmethane skeleton. To develop novel photopharmacological reagents, we designed and synthesized a set of ER ligands based on azobenzene skeleton, which can switch its conformation following UV irradiation. Our results showed that after UV irradiation, the Z-form of the synthesized compound 9 interacted with ERα, with a KD value of 2.5 µM, whereas the E-form of compound 9 did not bind ability to ERα at 10 µM.

Norgestomet is a synthetic progesterone derivative applied in veterinary medicine to control estrus and ovulation in cattle. Norgestomet has been widely used in the livestock industry to promote the synchronization of estrus in cattle and increase pregnancy rates. However, highly reproducible synthetic methods for Norgestomet have been rarely reported. Here, we described a method for the synthesis of Norgestomet and performed quantitative NMR analysis to determine the purity of the products. Moreover, the agonistic activity of the synthesized compounds against progesterone receptors (PRs) was evaluated using an alkaline phosphatase assay. We synthesized Norgestomet with 97.9% purity that exhibited agonistic activity against PR with EC50 values of 4.5 nM. We also synthesized the 17β-isomer of Norgestomet with 92.7% purity that did not exhibit any PR agonistic activity. The proposed synthetic route of Norgestomet can facilitate the assessment of residual Norgestomet in foods.

Peptide-based target protein degradation inducers called PROTACs/SNIPERs have low cell penetrability and poor intracellular stability as drawbacks. These shortcomings can be overcome by easily modifying these peptides by conjugation with cell penetrating peptides and side-chain stapling. In this study, we succeeded in developing the stapled peptide stPERML-R7, which is based on the estrogen receptor alpha (ERα)-binding peptide PERML and composed of natural amino acids. stPERML-R7, which includes a hepta-arginine motif and a hydrocarbon stapling moiety, showed increased α-helicity and similar binding affinity toward ERα when compared with those of the parent peptide PERML. Furthermore, we used stPERML-R7 to develop a peptide-based degrader LCL-stPERML-R7 targeting ERα by conjugating stPERML-R7 with a small molecule LCL161 (LCL) that recruits the E3 ligase IAPs to induce proteasomal degradation via ubiquitylation. The chimeric peptide LCL-stPERML-R7 induced sustained degradation of ERα and potently inhibited ERα-mediated transcription more effectively than the unstapled chimera LCL-PERML-R7. These results suggest that a stapled structure is effective in maintaining the intracellular activity of peptide-based degraders.

Canthin-5,6-dione is a common structure of the fluorescent natural products amarastelline A and nigakinone. Here, we synthesized various derivatives, and studied their fluorescence properties. The effects of substituent groups at the N 3 -, 4- and 10-positions were clarified. N 3 -Substitution influenced the solvent-dependent fluorescent change by modulating tautomerization between canthin-5,6-dione and the 5-hydroxy-6-one form. 10-Substitution also influenced the fluorescence, especially in aqueous solution, in combination with N 3 -substitution. Among the synthesized compounds, 5 showed an ‘OFF-ON-OFF’-type fluorescence change with increase of pH, and therefore served as a novel fluorescent sensor for a specific range of pH. Our findings suggest that canthin-5,6-dione should be useful as a fluorophore moiety for fluorescent labeling of biomolecules and for development of fluorescent probes and sensors.