Second-Generation Chiral Amino Acid Derivatives Afford High Stereoselectivity and Stability in Aqueous RNA Acylation.

Activated acyl species have proven versatile in the esterification of 2'-OH groups in RNA, enabling structure mapping, caging, profiling, and labeling of the biopolymer. Nearly all reagents developed for this reaction have been achiral; however, a recent study reported that simple chiral amino acid acylimidazole derivatives could yield diastereoselective reactions at RNA 2'-OH in water, enabling up to 4:1 selectivity in screening. Here, we investigated the effect of steric bulk on the stereoselectivity of RNA reaction and on the stability of adducts with a library of 36 chiral acylimidazole scaffolds with increasing steric demand. The results document the highest stereoselectivity yet achieved in RNA acylation reactions, with as high as >99:1 diastereoselectivity at >70% conversion. Also notably, the bulky adducts were found to have markedly improved stability on RNA.

Aqueous Activation of RNA 2'-OH for Conjugation with Amines and Thiols.

Strategies for covalent modification of RNA are important for enabling biological studies of the biopolymer and for enhancing properties of therapeutic RNAs. While a number of electrophiles have been observed to react with RNA, few methods exist for reaction with nucleophiles. Here, we describe new reagents that enable efficient conjugation of amines and other nucleophiles to unmodified RNA postsynthetically via transient activation of 2'-OH groups. Reaction of single-stranded RNA in aqueous solution with phenolic imidazolecarbamates at room temperature results in stoichiometric and superstoichiometric yields of imidazolecarbonyl group adducts, and control experiments with DNA confirm the site of reaction in RNA as 2'-OH. Subsequent incubation of imidazolecarbonyl-activated RNAs with primary or selected secondary amines results in rapid, high-yield conversion to carbamate conjugates. The activation and subsequent nucleophile reaction can be carried out either stepwise or in a one-pot reaction. Thiols and phenol species react to yield (thio)carbonate adducts, and amino acid sidechains also react, suggesting possible future utility for protein conjugates and analysis of protein-RNA interactions. The activation method is found to be selective to unpaired regions of RNA, and can be directed to a specific location in a strand by use of a loop-inducing helper DNA. The results establish novel and efficient reagents and methods for modifying RNA postsynthetically with nucleophiles.

Efficient post-synthesis incorporation and conjugation of reactive ketones in RNA via 2'-acylation.

Despite the broad utility of ketones in bioconjugation, few methods exist to introduce them into RNA. Here we develop highly reactive 2'-OH acylating reagents containing strained-ring ketones, and employ them as versatile labeling handles for RNA.

Stereoselective RNA reaction with chiral 2'-OH acylating agents.

The reactivity of RNA 2'-OH groups with acylating agents has recently been investigated for high-yield conjugation of RNA strands. To date, only achiral molecules have been studied for this reaction, despite the complex chiral structure of RNA. Here we prepare a set of chiral acylimidazoles and study their stereoselectivity in RNA reactions. Reactions performed with unfolded and folded RNAs reveal that positional selectivity and reactivity vary widely with local RNA macro-chirality. We further document remarkable effects of chirality on reagent reactivity, identifying an asymmetric reagent with 1000-fold greater reactivity than prior achiral reagents. In addition, we identify a chiral compound with higher RNA structural selectivity than any previously reported RNA-mapping species. Further, azide-containing homologs of a chiral dimethylalanine reagent were synthesized and applied to local RNA labeling, displaying 92% yield and 16 : 1 diastereoselectivity. The results establish that reagent stereochemistry and chiral RNA structure are critical elements of small molecule-RNA reactions, and demonstrate new chemical strategies for selective RNA modification and probing.

Monodemethylated Metabolites of Orally Administered Nobiletin: Identification and Quantitation in Rat Plasma and Tissues.

Although nobiletin (Nob) is a promising functional food component in view of its multifaceted physiological activity, the metabolism of this flavonoid remains underexplored. Herein, we examine the pharmacokinetics and tissue distribution of orally ingested Nob in rats, focusing on the six monodemethylnobiletin (MDNob) isomers as the main Nob metabolites. Two of these metabolites, namely, 6-MDNob and 8-MDNob, are chemically prepared for the first time, and a method for the simultaneous determination of all six MDNobs is developed. The obtained results demonstrate the production of 8-MDNob as a novel Nob metabolite and confirm the previously reported generation of 6-MDNob and 7-MDNob as oral metabolites of Nob in vivo. Finally, a quantitative relationship is established between the amount of metabolically generated MDNobs and that of administered Nob. Thus, this work paves the way for the broad applications and safe usage of Nob.

Sulfonylation of RNA 2'-OH groups.

The nucleophilic reactivity of RNA 2'-OH groups in water has proven broadly useful in probing, labeling, and conjugating RNA. To date, reactions selective to ribose 2'-OH have been limited to bond formation with short-lived carbonyl electrophiles. Here we report that many activated small-molecule sulfonyl species can exhibit extended lifetimes in water and retain 2'-OH reactivity. The data establish favorable aqueous solubility for selected reagents and successful RNA-selective reactions at stoichiometric and superstoichiometric yields, particularly for aryl sulfonyltriazole species. We report that the latter are considerably more stable than most prior carbon electrophiles in aqueous environments and tolerate silica chromatography. Furthermore, an azide-substituted sulfonyltriazole reagent is developed to introduce labels into RNA via click chemistry. In addition to high-yield reactions, we find that RNA sulfonylation can also be performed under conditions that give trace yields necessary for structure mapping. Like acylation, the reaction occurs with selectivity for unpaired nucleotides over those in the duplex structure, and a sulfonate adduct causes reverse transcriptase stops, suggesting potential use in RNA structure analysis. Probing of rRNA is demonstrated in human cells, indicating possible cell permeability. The sulfonyl reagent class enables a new level of control, selectivity, versatility, and ease of preparation for RNA applications.

4″-Sulfation Is the Major Metabolic Pathway of Epigallocatechin-3-gallate in Humans: Characterization of Metabolites, Enzymatic Analysis, and Pharmacokinetic Profiling.

Epigallocatechin-3-gallate (EGCG), a major green tea polyphenol, has beneficial effects on human health. This study aimed to elucidate the detailed EGCG sulfation process to better understand its phase II metabolism, a process required to maximize its health benefits. Results show that kinetic activity of sulfation in the human liver and intestinal cytosol is 2-fold and 60- to 300-fold higher than that of methylation and glucuronidation, respectively, suggesting sulfation as the key metabolic pathway. Moreover, SULT1A1 and SULT1A3 are responsible for sulfation in the liver and intestine, respectively. Additionally, our human ingestion study revealed that the concentration of EGCG-4″-sulfate in human plasma (Cmax: 177.9 nmol·L-1, AUC: 715.2 nmol·h·L-1) is equivalent to free EGCG (Cmax: 233.5 nmol·L-1, AUC: 664.1 nmol·h·L-1), suggesting that EGCG-4″-sulfate is the key metabolite. These findings indicate that sulfation is a crucial factor for improving EGCG bioavailability, while also advancing the understanding of the bioactivity and toxicity of EGCG.

Image-based screen capturing misfolding status of Niemann-Pick type C1 identifies potential candidates for chaperone drugs.

Niemann-Pick disease type C is a rare, fatal neurodegenerative disorder characterized by massive intracellular accumulation of cholesterol. In most cases, loss-of-function mutations in the NPC1 gene that encodes lysosomal cholesterol transporter NPC1 are responsible for the disease, and more than half of the mutations are considered to interfere with the biogenesis or folding of the protein. We previously identified a series of oxysterol derivatives and phenanthridine-6-one derivatives as pharmacological chaperones, i.e., small molecules that can rescue folding-defective phenotypes of mutated NPC1, opening up an avenue to develop chaperone therapy for Niemann-Pick disease type C. Here, we present an improved image-based screen for NPC1 chaperones and we describe its application for drug-repurposing screening. We identified some azole antifungals, including itraconazole and posaconazole, and a kinase inhibitor, lapatinib, as probable pharmacological chaperones. A photo-crosslinking study confirmed direct binding of itraconazole to a representative folding-defective mutant protein, NPC1-I1061T. Competitive photo-crosslinking experiments suggested that oxysterol-based chaperones and itraconazole share the same or adjacent binding site(s), and the sensitivity of the crosslinking to P691S mutation in the sterol-sensing domain supports the hypothesis that their binding sites are located near this domain. Although the azoles were less effective in reducing cholesterol accumulation than the oxysterol-derived chaperones or an HDAC inhibitor, LBH-589, our findings should offer new starting points for medicinal chemistry efforts to develop better pharmacological chaperones for NPC1.

Switching subtype-selectivity: Fragment replacement strategy affords novel class of peroxisome proliferator-activated receptor α/δ (PPARα/δ) dual agonists.

Peroxisome proliferator-activated receptors (PPARs) are important drug targets for treatment of dyslipidemia, type 2 diabetes, cardiovascular disease, nonalcoholic fatty liver disease and nonalcoholic steatohepatitis, and great efforts have been made to develop novel PPAR ligands. However, most existing PPAR ligands contain a carboxylic acid (CA) or thiazolidinedione (TZD) structure (acidic head group) that is essential for activity. We recently discovered non-CA/TZD class PPARα/δ partial agonists, which contain an acetamide moiety and adjacent methyl group, linked to a 1,2,4-oxadiazole ring ("fragment a"). We hypothesized that the acetamide structure might interact with the CA/TZD-binding pocket. To test this idea, we firstly replaced fragment a in one of our compounds with the α-alkoxy-CA structure often found in PPAR agonists. Secondly, we replaced the α-alkoxy-CA head group of several reported PPAR agonists with our acetamide-based fragment a. The agonistic activities of the synthesized hybrid compounds toward PPARs (PPARα, PPARγ and PPARδ) were evaluated by means of cell-based reporter gene assays. All the hybrid molecules showed PPAR-agonistic activities, but replacement of the α-alkoxy-CA head group altered the maximum efficacy and the subtype-specificity. The acetamide-based hybrid molecules showed partial agonism toward PPARα and PPARδ, whereas the α-alkoxy-CA-based molecules were generally selective for PPARα and PPARγ, with relatively high activation efficacies. Thus, the fragment replacement strategy appears promising for the development of novel acetamide-based PPARα/δ dual agonists.

Structure-activity relationship studies of non-carboxylic acid peroxisome proliferator-activated receptor α/δ (PPARα/δ) dual agonists.

The peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors that contribute to the regulation of lipid, glucose and cholesterol homeostases. They are considered as therapeutic targets for metabolic diseases such as dyslipidemia and type 2 diabetes mellitus. Various PPAR agonists have been developed, but most of them contain a carboxylic acid (CA) or thiazolidinedione (TZD) moiety, which is essential for the activity. However, we recently discovered non-CA/non-TZD class PPARα/δ dual agonists having an acetamide structure. Here, we describe structure-activity relationship (SAR) studies of these novel acetamide-based PPARα/δ dual agonists. The SAR studies revealed that the acetamide functionality and adjacent methyl group contribute greatly to the agonistic activity. Compound (S)-10 was the most potent PPARα/δ dual agonist among the compounds synthesized (PPARα EC50=17nM, PPARδ EC50=23nM).

Catalytic asymmetric total synthesis of (+)-artalbic acid.

The first total synthesis of (+)-artalbic acid has been accomplished using asymmetric allylation of an acetoacetate derivative with a phase-transfer catalyst. This synthetic work was completed in 12 steps from isopropyl acetoacetate with high stereocontrol. In addition, the absolute configuration of naturally occurring artalbic acid was determined to be 7S, 9S, and 10S.