An adenine base editor variant expands context compatibility.

Adenine base editors (ABEs) are precise gene-editing agents that convert A:T pairs into G:C through a deoxyinosine intermediate. Existing ABEs function most effectively when the target A is in a TA context. Here we evolve the Escherichia coli transfer RNA-specific adenosine deaminase (TadA) to generate TadA8r, which extends potent deoxyadenosine deamination to RA (R = A or G) and is faster in processing GA than TadA8.20 and TadA8e, the two most active TadA variants reported so far. ABE8r, comprising TadA8r and a Streptococcus pyogenes Cas9 nickase, expands the editing window at the protospacer adjacent motif-distal end and outperforms ABE7.10, ABE8.20 and ABE8e in correcting disease-associated G:C-to-A:T transitions in the human genome, with a controlled off-target profile. We show ABE8r-mediated editing of clinically relevant sites that are poorly accessed by existing editors, including sites in PCSK9, whose disruption reduces low-density lipoprotein cholesterol, and ABCA4-p.Gly1961Glu, the most frequent mutation in Stargardt disease.

Transcriptome-wide profiling and quantification of N6-methyladenosine by enzyme-assisted adenosine deamination.

N6-methyladenosine (m6A), the most abundant internal messenger RNA modification in higher eukaryotes, serves myriad roles in regulating cellular processes. Functional dissection of m6A is, however, hampered in part by the lack of high-resolution and quantitative detection methods. Here we present evolved TadA-assisted N6-methyladenosine sequencing (eTAM-seq), an enzyme-assisted sequencing technology that detects and quantifies m6A by global adenosine deamination. With eTAM-seq, we analyze the transcriptome-wide distribution of m6A in HeLa and mouse embryonic stem cells. The enzymatic deamination route employed by eTAM-seq preserves RNA integrity, facilitating m6A detection from limited input samples. In addition to transcriptome-wide m6A profiling, we demonstrate site-specific, deep-sequencing-free m6A quantification with as few as ten cells, an input demand orders of magnitude lower than existing quantitative profiling methods. We envision that eTAM-seq will enable researchers to not only survey the m6A landscape at unprecedented resolution, but also detect m6A at user-specified loci with a simple workflow.

Controllable catalytic difluorocarbene transfer enables access to diversified fluoroalkylated arenes.

Difluorocarbene has important applications in pharmaceuticals, agrochemicals and materials, but all these applications proceed using just a few types of reaction by taking advantage of its intrinsic electrophilicity. Here, we report a palladium-catalysed strategy that confers the formed palladium difluorocarbene (Pd=CF2) species with both nucleophilicity and electrophilicity by switching the valence state of the palladium centre (Pd(0) and Pd(II), respectively). Controllable catalytic difluorocarbene transfer occurs between readily available arylboronic acids and the difluorocarbene precursor diethyl bromodifluoromethylphosphonate (BrCF2PO(OEt)2). From just this simple fluorine source, difluorocarbene transfer enables access to four types of product: difluoromethylated and tetrafluoroethylated arenes and their corresponding fluoroalkylated ketones. The transfer can also be applied to the modification of pharmaceuticals and agrochemicals as well as the one-pot diversified synthesis of fluorinated compounds. Mechanistic and computational studies consistently reveal that competition between nucleophilic and electrophilic palladium difluorocarbene ([Pd]=CF2) is the key factor controlling the catalytic difluorocarbene transfer.

Transition-Metal (Cu, Pd, Ni)-Catalyzed Difluoroalkylation via Cross-Coupling with Difluoroalkyl Halides.

Difluoroalkylated compounds play a remarkably important role in life and materials sciences because of the unique characteristics of the difluoromethylene (CF2) group. In particular, precise introduction of a CF2 group at the benzylic position can dramatically improve the biological properties of the corresponding molecules. As a consequence, difluoroalkylation of aromatic compounds has become a powerful strategy in modulating the bioactivities of organic molecules. However, efficient strategies to selectively synthesize difluoroalkylated arenes had been very limited before 2012. Traditional synthetic methods in this regard suffer from either harsh reaction conditions or narrow substrate scope, significantly restricting their widespread applications, particularly for late-stage difluoroalkylation of bioactive molecules. To overcome these limitations, a straightforward route to access these valuable difluoroalkylated skeletons is the direct introduction of the difluoroalkylated group (CF2R) onto aromatic rings through transition-metal-catalyzed cross-coupling. However, because of the instability of some difluoroalkylated metal species, which are prone to protonation, dimerization, and/or generation of other unknown byproducts, it is difficult to selectively control the catalytic cycle to suppress these side reactions. In this context, we proposed the use of low-cost and widely available difluoroalkyl halides as fluoroalkyl sources for transition-metal-catalyzed difluoroalkylation reactions via cross-coupling. In this Account, we summarize our major efforts on copper-, palladium-, and nickel-catalyzed difluoroalkylations of aromatics with low-cost and widely available difluoroalkyl halides as fluoroalkyl sources. Four modes of catalytic difluoroalkylation reactions, including nucleophilic difluoroalkylation, electrophilic difluoroalkylation, radical difluoroalkylation, and metal-difluorocarbene coupling (MeDiC), have been demonstrated through careful modulation of the catalytic systems. Among these reactions, the MeDiC reaction represents a new mode of fluoroalkylation. These processes enable difluoroalkylation of a variety of aryl halides and arylboron reagents under mild reaction conditions. A wide range of difluoroalkyl halides, including activated difluoroalkyl halides (Cl/BrCF2R, R = π system), unactivated difluoroalkyl halides (BrCF2R, R = alkyl, H), and especially the inert and inexpensive industrial chemical chlorodifluoromethane (ClCF2H), are applicable to these reactions, providing straightforward and facile routes to a diverse range of difluoroalkylated (hetero)arenes. These difluoroalkyl halide-based strategies can also be applied to prepare difluoroalkylated alkenes, alkynes, and alkanes and feature impressive advantages over conventional methods for the synthesis of difluoroalkylated compounds in terms of synthetic efficiency, functional group tolerance, and structural diversity. In particular, the late-stage difluoroalkylation of bioactive molecules through these processes offers good opportunities for the synthesis and development of new medicinal agents without the need for multistep de novo syntheses.

Bulky Diamine Ligand Promotes Cross-Coupling of Difluoroalkyl Bromides by Iron Catalysis.

Although iron-catalyzed cross-coupling of Grignard reagents with alkyl halides has been well established, the adoption of the reaction for fluoroalkylations has not been reported because traditional catalytic systems often lead to defluorination reactions. Described herein is the investigation of an iron-catalyzed cross-coupling between arylmagnesium bromides and difluoroalkyl bromides with modified N,N,N',N'-tetramethyl-ethane-1,2-diamine (TMEDA) as a ligand. The use of this bulky diamine, in which a butylene is substituted at one carbon atom of the ethylene backbone in TMEDA, enables the iron-catalyzed difluoroalkylation under mild reaction conditions with a wide range of difluoroalkyl bromides, including vulnerable bromodifluoromethane, thus providing a general and cost-efficient route for applications in medicinal chemistry.

Copper-Catalyzed Highly Stereoselective Trifluoromethylation and Difluoroalkylation of Secondary Propargyl Sulfonates.

It is challenging to stereoselectively introduce a trifluoromethyl group (CF3 ) into organic molecules. To date, only limited strategies involving direct asymmetric trifluoromethylation have been reported. Herein, we describe a new strategy for direct asymmetric trifluoromethylation through the copper-catalyzed stereospecific trifluoromethylation of optically active secondary propargyl sulfonates. The reaction enables propargylic trifluoromethylation with high regioselectivity and stereoselectivity. The reaction could also be extended to stereospecific propargylic difluoroalkylation. Transformations of the resulting enantiomerically enriched fluoroalkylated alkynes led to a variety of chiral fluoroalkylated compounds, thus providing a useful protocol for applications in the synthesis of fluorinated complexes.

Nickel-Catalyzed Difluoroalkylation of (Hetero)Arylborons with Unactivated 1-Bromo-1,1-difluoroalkanes.

A nickel-catalyzed cross-coupling between (hetero)arylborons and unactivated 1-bromo-1,1-difluoroalkanes has been developed. The use of two ligands (a bidentate bipyridine-based ligand, 4,4'-ditBu-bpy, and a monodentate pyridine-based ligand, DMAP) offers a highly efficient nickel-based catalytic system to prepare difluoroalkylated arenes which have important applications in medicinal chemistry.

Facile Access to Fluoromethylated Arenes by Nickel-Catalyzed Cross-Coupling between Arylboronic Acids and Fluoromethyl Bromide.

The nickel-catalyzed fluoromethylation of arylboronic acids was achieved with the industrial raw material fluoromethyl bromide (CH2 FBr) as the coupling partner. The reaction proceeded under mild reaction conditions with high efficiency; it features the use of a low-cost nickel catalyst, synthetic simplicity, and excellent functional-group compatibility, and provides facile access to fluoromethylated biologically relevant molecules. Preliminary mechanistic studies showed that a single-electron-transfer (SET) pathway is involved in the catalytic cycle.

Heteroaryldifluoromethylation of organoborons catalyzed by palladium: facile access to aryl(heteroaryl)difluoromethanes.

A first example of Pd-catalyzed heteroaryldifluoromethylation of organoborons with bromodifluoromethylated heteroarenes has been described. The use of phosphine ligand PAd2(n-Bu)·HI is critical for the reaction efficiency. With use of this ligand, a wide range of aryl(heteroaryl)difluoromethanes were obtained with high efficiency. The notable features of this reaction are its broad substrate scope and excellent functional group compatibility, thus providing a facile protocol for application in drug discovery and development.

Nickel-catalyzed cross-coupling of functionalized difluoromethyl bromides and chlorides with aryl boronic acids: a general method for difluoroalkylated arenes.

Transition-metal-catalyzed difluoroalkylation of aromatics remains challenging despite the importance of difluoroalkylated arenes in medicinal chemistry. Herein, the first successful example of nickel-catalyzed difluoroalkylation of aryl boronic acids is described. The reaction allows access to a variety of functionalized difluoromethyl bromides and chlorides, and paves the way to highly cost-efficient synthesis of a wide range of difluoroalkylated arenes. The notable features of this protocol are its high generality, excellent functional-group compatibility, low-cost nickel-catalyst, and practicality for gram-scale production, thus providing a facile method for applications in drug discovery and development.

Direct olefination of fluorinated benzothiadiazoles: a new entry to optoelectronic materials.

Fluorinated olefin-containing benzothiadiazoles have important applications in optoelectronic materials. Herein, we reported the direct olefination of fluorinated benzothiadiazoles, as catalyzed by palladium. The reaction proceeds under mild reaction conditions and shows high functional-group compatibility. A preliminary study of the properties of the resulting symmetrical and unsymmetrical olefin-containing fluorinated benzothiadiazoles in red-light-emitting dyes has also been conducted.

Palladium-catalyzed difluoroalkylation of aryl boronic acids: a new method for the synthesis of aryldifluoromethylated phosphonates and carboxylic acid derivatives.

The palladium-catalyzed difluoroalkylation of aryl boronic acids with bromodifluoromethylphosphonate, bromodifluoroacetate, and further derivatives has been developed. This method provides a facile and useful access to a series of functionalized difluoromethylated arenes (ArCF2 PO(OEt)2 , ArCF2 CO2 Et, and ArCF2 CONR(1) R(2) ) that have important applications in drug discovery and development. Preliminary mechanistic studies reveal that a single electron transfer (SET) pathway may be involved in the catalytic cycle.

Distribution of Chinese medicine syndrome patterns and its laws in patients with polycystic ovarian syndrome / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the distribution laws of the Chinese medicine syndrome patterns and its correlated symptoms in patients with polycystic ovarian syndrome (PCOS), and the possible correlation between Chinese medicine syndrome patterns and PCOS associated parameters, thus to provide a guidance for selecting proper indices in curative effectiveness assessment.</p><p><b>METHODS</b>Using clinical epidemiological methods and mathematical statistics, the Chinese medicine syndrome patterns were studied in 228 PCOS patients. The distribution features of Chinese medicine syndrome patterns were summarized.</p><p><b>RESULTS</b>Shen-deficiency blood-stasis syndrome was the most frequently seen in PCOS patients, followed by Pi-deficiency phlegm-dampness syndrome, Pi-Shen yang-deficiency syndrome, and Shen-yin deficiency syndrome. Positive correlation existed between serum levels of follicle stimulating hormone (FSH) and Pi-Shen yang-deficiency syndrome. Positive correlation existed between fasting blood sugar (FBS), waist to hip ratio (WHR), body mass index (BMI), fasting insulin (FIN), and Hirsutism score and Pi-deficiency phlegm-dampness syndrome. Positive correlation existed between serum levels of prolactin (PRL), estradiol (E2 ) and qi stagnancy and blood stasis syndrome. Positive correlation existed between serum progesterone (PRG) level, FSH, FIN, BMI, acne score and Gan stagnancy and blood heat syndrome. Positive correlation existed between luteinizing hormone (LH) and Shen-deficiency blood-stasis syndrome. Besides, LH/FSH >3 was possibly more frequently seen in Pi-deficiency phlegm-dampness syndrome and Pi-Shen yang-deficiency syndrome. Family heritability could be seen in each syndrome patterns. Among them, female heritability was more often seen in Shen-deficiency blood-stasis syndrome, while male heritability was more often seen in Shen-yin deficiency syndrome.</p><p><b>CONCLUSIONS</b>Shen-deficiency blood-stasis syndrome, Pi-deficiency phlegm-dampness syndrome, Pi-Shen yang-deficiency syndrome, and Shen-yin deficiency syndrome were most frequently seen in PCOS patients. The sex hormones (including 6 items), FBS, FIN, WHR and BMI, etc. were correlated with each Chinese medicine syndrome pattern to various extents, which could be taken as reference in Chinese medicine syndrome differentiation.</p>