Review on NMR as a tool to analyse natural products extract directly: Molecular structure elucidation and biological activity analysis.

INTRODUCTION: Natural products, the small organic molecules produced by plants, microbes and invertebrates, often present in the form of a mixture, this leads to the structural characterisation of natural extracts often requiring time-consuming multistep purification procedures. Nuclear magnetic resonance (NMR) technology is traditionally utilised as a tool for the structural elucidation of pure compounds. Contemporarily, an up-to-date trend in the application of NMR in natural product research is shifting to the direct NMR analysis of crude mixtures, to obtain molecular structure and biological activity information without performing cumbersome separation. OBJECTIVE: To review works of literature on the evolution, principle and progress of NMR technologies for analysing mixtures, we focus on the successful application of NMR technologies in direct analysis of natural product extracts. METHODOLOGY: Based on our research experience, academic tracking and extensive literature search, which involved, but not limited to, the use of various databases, like Web of Knowledge and PubMed. The keywords used, in various combinations, to retrieve recent literature on the successful application of NMR technologies to sheer natural product extracts, and excluded artificially natural product mixture and biofluid. RESULTS: NMR technologies for direct natural extracts analysis, including two-dimensional J-resolved spectroscopy (2D-JRES), pure shift NMR, diffusion-ordered NMR spectroscopy (DOSY), statistical correlation spectroscopy (STOCSY), concentration-ordered NMR spectroscopy (CORDY), saturation transfer difference (STD) and water-ligand observed via gradient spectroscopy (WaterLOGSY) were illustrated. CONCLUSIONS: By these methods, molecular structure and biological activity information will be directly obtained from NMR analysis of natural products extract, aiming to save experimental time and expenses.

Genome-wide association analysis of resistance to frogeye leaf spot China race 7 in soybean based on high-throughput sequencing.

KEY MESSAGE: FLS is a disease that causes severe yield reduction in soybean. In this study, four genes (Glyma.16G176800, Glyma.16G177300, Glyma.16G177400 and Glyma.16G182300) were tentatively confirmed to play an important role in the resistance of soybean to FLS race 7. Frogeye leaf spot (FLS) causes severe yield loss in soybean and has been found in several countries worldwide. Therefore, it is necessary to select and utilize FLS-resistant varieties for the management of FLS. In the present study, 335 representative soybean materials were assessed for partial resistance to FLS race 7. Quantitative trait nucleotide (QTN) and FLS race 7 candidate genes were identified using genome-wide association analysis (GWAS) based on a site-specific amplified fragment sequencing (SLAF-seq) approach. A total of 23,156 single-nucleotide polymorphisms (SNPs) were used to evaluate the level of linkage disequilibrium with a minor allele frequency ≥ 5 and deletion data < 3%. These SNPs covered about 947.01 MBP, nearly 86.09% of the entire soybean genome. In addition, a compressed mixed linear model was utilized to identify association signals for partial resistance to FLS race 7. A total of 15 QTNs associated with resistance were found to be novel for FLS race 7 resistance. A total of 217 candidate genes located in the 200-kb genomic region of these peak SNPs were identified. Based on gene association analysis, qRT-PCR, haplotype analysis and virus-induced gene silencing (VIGS) systems were used to further verify candidate genes Glyma.16G176800, Glyma.16G177300, Glyma.16G177400 and Glyma.16G182300. This indicates that these four candidate genes may participate in FLS race 7 resistance responses.

Detoxification mechanism of vinegar-processed Kansui revealed by systematic phytochemical analysis using ultrahigh-performance liquid chromatography diode array detection tandem mass spectrometry, ultrahigh-performance liquid chromatography high-resolution mass spectrometry and in silico drug target identification.

RATIONALE: The dried roots of Euphorbia kansui L., known as Kansui, are used to treat ascites and edema in traditional Chinese medicine. However, the toxicity of this herb has seriously restricted its clinical application. A unique vinegar-processing method has been used to reduce its toxicity since the time of ancient China. However, the detoxification mechanism underlying such vinegar processing has not been fully revealed. To find the answer, the process-induced changes in components should be carefully investigated. METHODS: We performed a systematic analysis of chemical components in raw and vinegar-processed Kansui using ultrahigh-performance liquid chromatography (UHPLC) diode array detection tandem mass spectrometry and UHPLC high-resolution mass spectrometry. Thirty-one chemical components in raw and vinegar-processed Kansui were found, the chemical structures of 28 components among them were proposed and the process-induced changes in components were then investigated. RESULTS: A comprehensive conclusion about the process-induced chemical change was drawn. It was found that jatrophane-type diterpenoids decreased markedly after vinegar processing, while ingenane-type diterpenoids were retained during vinegar processing. In silico drug target identification gave hints that jatrophane-type diterpenoids, which decreased markedly during vinegar processing, may have more intense toxicity involving cholinesterase and mitogen-activated protein kinases, while ingenane-type diterpenoids, which were retained during vinegar processing, may have a more intense therapeutic effect involving carbonic anhydrase. CONCLUSIONS: The possible detoxification mechanism of vinegar-processed Kansui is presented. The research has significance for the therapeutic/toxic chemical basis of Kansui. Also, it has significance for drug discovery from terpenoids within the herb.

Identification of glutathione transferase gene associated with partial resistance to Sclerotinia stem rot of soybean using genome-wide association and linkage mapping.

KEY MESSAGE: Association and linkage mapping techniques were used to identify and verify single nucleotide polymorphisms (SNPs) associated with Sclerotinia sclerotiorum resistance. A novel resistant gene, GmGST , was cloned and shown to be involved in soybean resistance to SSR. Sclerotinia stem rot (SSR), caused by the fungus Sclerotinia sclerotiorum, is one of the most devastating diseases in soybean (Glycine max (Linn.) Merr.) However, the genetic architecture underlying soybean resistance to SSR is poorly understood, despite several mapping and gene mining studies. In the present study, the identification of quantitative trait loci (QTLs) involved in the resistance to S. sclerotiorum was conducted in two segregating populations: an association population that consisted of 261 diverse soybean germplasms, and the MH population, derived from a cross between a partially resistant cultivar (Maple arrow) and a susceptible cultivar (Hefeng25). Three and five genomic regions affecting resistance were detected by genome-wide association study to control the lesion length of stems (LLS) and the death rate of seedling (DRS), respectively. Four QTLs were detected to underlie LLS, and one QTL controlled DRS after SSR infection. A major locus on chromosome (Chr.) 13 (qDRS13-1), which affected both DRS and LLS, was detected in both the natural population and the MH population. GmGST, encoding a glutathione S-transferase, was cloned as a candidate gene in qDRS13-1. GmGST was upregulated by the induction of the partially resistant cultivar Maple arrow. Transgenic experiments showed that the overexpression of GmGST in soybean increased resistance to S. sclerotiorum and the content of soluble pigment in stems of soybean. The results increase our understanding of the genetic architecture of soybean resistance to SSR and provide a framework for the future marker-assisted breeding of resistant soybean cultivars.

NMR-based screening for natural product subfraction to precisely identify ligand of target protein.

INTRODUCTION: The inherent diversity of natural product extracts has not only made their value for biological activity attractive, but has also presented significant challenges for separation and detection techniques to enable rapid characterisation of the biologically active component present in the complicated mixture. OBJECTIVE: Nuclear magnetic resonance (NMR) is routinely valued for its ability to shed light on molecular structure, when NMR is used as a promising tool in drug screening, it can detect and quantify molecular interactions, and at the same time provide detailed structural information with atomic level resolution. Here, we introduced an accurate and efficient strategy for discovering ligands from natural product extracts, by taking the advantage of NMR-based drug screening. METHODOLOGY: The characteristic pre-purified subfraction libraries were brought into screening, and combinatorial ligand-observed NMR interaction detection experiments were performed, once hits were found from one subfraction, the repository of the subfraction would be subject to separation and preparation, and the structure of the hits would be easily identified. RESULTS: Screening procedure of Radix Polygoni Multiflori water extract against human serum albumin (HSA) was used as an example, to discuss and verify the detailed methodology. Furthermore, human fatty acid binding protein 4 (FABP4 ) was presented as an example target protein, to illustrate the utility of this method for discovering biologically active component. CONCLUSIONS: It is proved that suitable subfraction library and well-combined ligand-detected NMR experiments will make the screening streamline more accurate and efficient. NMR is a promising tool to integrate natural product extracts into modern drug screening.

Genome-wide association and transcriptional studies reveal novel genes for unsaturated fatty acid synthesis in a panel of soybean accessions.

BACKGROUND: The nutritional value of soybean oil is largely influenced by the proportions of unsaturated fatty acids (FAs), including oleic acid (OA, 18:1), linoleic acid (LLA, 18:2), and linolenic acid (LNA, 18:3). Genome-wide association (GWAS) studies along with gene expression studies in soybean [Glycine max (L.) Merr.] were leveraged to dissect the genetics of unsaturated FAs. RESULTS: A association panel of 194 diverse soybean accessions were phenotyped in 2013, 2014 and 2015 to identify Single Nucleotide Polymorphisms (SNPs) associated with OA, LLA, and LNA content, and determine putative candidate genes responsible for regulating unsaturated FAs composition. 149 SNPs that represented 73 genomic regions were found to be associated with the unsaturated FA contents in soybean seeds according to the results of GWAS. Twelve novel genes were predicted to be involved in unsaturated FA synthesis in soybean. The relationship between expression pattern of the candidate genes and the accumulation of unsaturated FAs revealed that multiple genes might be involved in unsaturated FAs regulation simultaneously but work in very different ways: Glyma.07G046200 and Glyma.20G245500 promote the OA accumulation in soybean seed in all the tested accessions; Glyma.13G68600 and Glyma.16G200200 promote the OA accumulation only in high OA germplasms; Glyma.07G151300 promotes OA accumulation in higher OA germplasms and suppresses that in lower OA germplasms; Glyma.16G003500 has the effect of increasing LLA accumulation in higher LA germplasms; Glyma.07G254500 suppresses the accumulation of LNA in lower OA germplasms; Glyma.14G194300 might be involved in the accumulation of LNA content in lower LNA germplasms. CONCLUSIONS: The beneficial alleles and candidate genes identified might be valuable for improving marker-assisted breeding efficiency and exploring the molecular mechanisms underlying unsaturated fatty acid of soybean.

Structure characterization and optical properties investigation of the four main components of the classical phenazinium dye Safranin O.

Safranin O is an important and classical phenazinium dye; since the 19th century, it has been extensively used in the academic field as a spectroscopic probe and indicator. Surprisingly, we found that this long-used reagent is without exception a mixture. In this study, the four main components in a Safranin O sample were prepared, and their chemical structures were elucidated for the first time. Optical property investigations showed that the components had somewhat different absorbance properties and markedly different fluorescence properties, and their structure-optical activity relationships were also discussed. It could be inferred that the variation of each component in the content would unavoidably result in inconsistent optical data when using this Safranin O reagent as a spectroscopic probe or indicator. Considering the accurate transfer of measurement results between laboratories, high-purity Safranin O is in urgent demand in the academic field.

Investigation of serum amino acids involved in gallic acid detoxification of formaldehyde by liquid chromatography/tandem mass spectrometry and neutral loss scan.

RATIONALE: Gallic acid is one of the most common polyphenols in natural products and human diet. The consumption of gallic acid reduces the incidence of cardiovascular diseases, chronic metabolic disorders and cancers. Most previous publications focused on the antioxidative or prooxidative properties of gallic acid. In the present work, gallic acid as a trapping agent of blood formaldehyde was investigated by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and neutral loss scan. METHODS: Serum samples incubated with gallic acid were subjected to LC/MS/MS analysis using an LTQ XL ion trap mass spectrometer. The adduct ions of gallic acid-formaldehyde-amino acids were explored by investigation of their fragmentation patterns and neutral loss scan experiments. RESULTS: A series of Mannich adducts (namely, gallic acid-formaldehyde-alanine, gallic acid-formaldehyde-proline, gallic acid-formaldehyde-leucine or gallic acid-formaldehyde-isoleucine and gallic acid-formaldehyde-phenylalanine) were identified as metabolites by neutral loss scan experiments. CONCLUSIONS: This work demonstrated that serum amino acids are involved in gallic acid detoxification of formaldehyde. Because excessive formaldehyde in blood is implicated in a variety of disease pathologies, detoxification of formaldehyde, especially endogenous formaldehyde, may be another health beneficial effect of gallic acid. It also suggested that more attention should be paid to Mannich-type metabolites of polyphenol-formaldehyde-amino acids in research into the pharmacokinetics and bioavailability of polyphenols.

Effects of First High-Dose Atorvastatin Loading in Patients With ST-Segment Elevation Myocardial Infarction Undergoing Percutaneous Coronary Intervention.

To determine the effects of 80-mg atorvastatin administration for the first time in patients with acute ST segment elevation myocardial infarction (STEMI) before emergency percutaneous coronary intervention (PCI). A total of 118 patients with STEMI who underwent emergency PCI were enrolled in this study. The patients were divided into 80-mg group (n = 59) and 40-mg group (n = 59), according to the loading dose of atorvastatin firstly before operation. The occurrence of no-reflows and changes of HbA1c were observed preoperatively and postoperatively on second and fifth days. All patients were followed up for 1 year with major adverse cardiac events (MACE) recorded. The incidence of no-reflow in 80-mg group was obviously lower than in 40-mg group (13.56% vs. 25.42%) (χ = 4.374, P = 4.374). The preoperative HbA1c levels exhibited no significant difference between 80-mg group and 40-mg group (P > 0.05). The postoperative HbA1c levels in 2 groups showed a trend of gradual decline, which were lower in 80-mg group than in 40-mg group for second day, fifth day, first month, sixth month, and 12th month (all P < 0.05). The postoperative incidence of MACE in 80-mg group was significantly lower than in 40-mg group for sixth and 12th months (both P < 0.05). The incidence of MACE in patients with reflow in 80-mg and 40-mg groups was significantly higher than in patients with no-reflow who were in 80-mg and 40-mg groups for postoperative 12th month (both P < 0.05). The first loading high dose of atorvastatin can significantly prevent occurrence of postoperative no-reflow in patients with STEMI after PCI, reduce HbA1c levels and the incidence of MACE. Clinical randomized controlled trial with larger sample size is required to confirm this finding.

Quantitative 31P-NMR spectroscopy for the determination of fosfomycin and impurity A in pharmaceutical products of fosfomycin sodium or calcium.

A quantitative 31P-NMR method for the determination of fosfomycin and impurity A in pharmaceutical products of fosfomycin sodium or calcium has been developed. In this method, coaxial inserts containing trimethyl phosphate are used as external standard. The method is convenient and robust, and gives both high accuracy and precision. It is shown that an accurate determination is possible using different probes and coaxial inserts.

Advancing Solid Oxide Fuel Cell Performance: Enhanced Electrochemical Properties of Pr1-xCaxBaFe2O5+δ Nanofiber Cathodes via Ca Doping.

The double perovskite oxide PrBaFe2O5+δ has great potential as a cathode material for solid oxide fuel cells (SOFCs). However, the electrochemical characteristics of Fe-based double perovskites are relatively inferior. To improve its electrochemical performance, Ca is investigated to partially replace Pr, forming Pr1-xCaxBaFe2O5+δ (PCBFx, x = 0.0-0.3) by an electrospinning technique. The PCBFx nanofibers exhibited a crystalline structure characterized by orthorhombic symmetry and space group P4/mmm. Furthermore, these PCBFx nanofibers displayed exceptional chemical compatibility with the Sm0.2Ce0.8O1.95 (SDC) electrolyte when sintered at a temperature of 900 °C for 5 h. The X-ray photoelectron spectroscopy (XPS) analysis reveals a progressive increase in the Fe4+ concentration as the Ca doping level rises. The polarization resistances (Rp) of the PCBF00, PCBF01, PCBF02, and PCBF03 nanofiber cathodes were 0.103, 0.079, 0.056, and 0.048 Ω cm2 at 750 °C. In the meantime, doping Ca increases the peak power density of the single cell by 46%, from 762.80 (PCBF00) to 1114.85 (PCBF03) mW cm-2 at 750 °C. The results demonstrate that PCBF03 double perovskite nanofibers exhibit great potential as cathode materials for SOFCs.

Building a Charge Transfer Bridge between g-C3N4 and Perovskite with Molecular Engineering to Achieve Efficient Perovskite Solar Cells.

Effective defect passivation and efficient charge transfer within polycrystalline perovskite grains and corresponding boundaries are necessary to achieve highly efficient perovskite solar cells (PSCs). Herein, focusing on the boundary location of g-C3N4 during the crystallization modulation on perovskite, molecular engineering of 4-carboxyl-3-fluorophenylboronic acid (BF) on g-C3N4 was designed to obtain a novel additive named BFCN. With the help of the strong bonding ability of BF with both g-C3N4 and perovskite and favorable intramolecular charge transfer within BFCN, not only has the crystal quality of perovskite films been improved due to the effective defects passivation, but the charge transfer has also been greatly accelerated due to the formation of additional charge transfer channels on the grain boundaries. As a result, the champion BFCN-based PSCs achieve the highest photoelectric conversion efficiency (PCE) of 23.71% with good stability.

A dihydrochalcone and several homoisoflavonoids from Polygonatum odoratum are activators of adenosine monophosphate-activated protein kinase.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a major cellular energy sensor and master regulator of metabolic homeostasis; thus, AMPK plays a central role in studies on diabetes and related metabolic diseases. From the rhizomes of Polygonatum odoratum (Mill.) Druce, six homoisoflavonoids (1-6) and one dihydrochalcone (7) were isolated, and the structures of polygonatones A-D (4-7) were elucidated by various spectroscopic analyses. Compounds 1-7 were evaluated for their effect on AMPK activation. The amount of active phosphorylated AMPK and acetyl-CoA carboxylase in rat liver epithelial IAR-20 cells increased when the cells were incubated with the aforementioned compounds. Specifically, (3R)-5,7-dihydroxyl-6-methyl-8-methoxyl-3-(4'-hydroxylbenzyl)-chroman-4-one (1), (3R)-5,7-dihydroxyl-6,8-dimethyl-3-(4'-hydroxylbenzyl)-chroman-4-one (2), (3R)-5,7-dihydroxyl-6-methyl-3-(4'-hydroxylbenzyl)-chroman-4-one (3), and polygonatone D (7) exhibited significant activation effects.

Correction: Regioselective O-alkylation of 2-pyridones by TfOH-catalyzed carbenoid insertion.

Correction for 'Regioselective O-alkylation of 2-pyridones by TfOH-catalyzed carbenoid insertion' by Zhewei Yan et al., Chem. Commun., 2023, 59, 106-109, https://doi.org/10.1039/d2cc05676c.

Identification of QTL, QTL-by-environment interactions, and their candidate genes for resistance HG Type 0 and HG Type 1.2.3.5.7 in soybean using 3VmrMLM.

Introduction: Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is an important disease affecting soybean yield in the world. Potential SCN-related QTLs and QTL-by-environment interactions (QEIs) have been used in SCN-resistant breeding. Methods: In this study, a compressed variance component mixed model, 3VmrMLM, in genome-wide association studies was used to detect QTLs and QEIs for resistance to SCN HG Type 0 and HG Type 1.2.3.5.7 in 156 different soybean cultivars materials. Results and discussion: The results showed that 53 QTLs were detected in single environment analysis; 36 QTLs and 9 QEIs were detected in multi-environment analysis. Based on the statistical screening of the obtained QTLs, we obtained 10 novel QTLs and one QEI which were different from the previous studies. Based on previous studies, we identified 101 known genes around the significant/suggested QTLs and QEIs. Furthermore, used the transcriptome data of SCN-resistant (Dongnong L-10) and SCN-susceptible (Suinong 14) cultivars, 10 candidate genes related to SCN resistance were identified and verified by Quantitative real time polymerase chain reaction (qRT-PCR) analysis. Haplotype difference analysis showed that Glyma.03G005600 was associated with SCN HG Type 0 and HG Type 1.2.3.5.7 resistance and had a haplotype beneficial to multi-SCN-race resistance. These results provide a new idea for accelerating SCN disease resistance breeding.

Quality control and product differentiation of LMWHs marketed in China using 1H NMR spectroscopy and chemometric tools.

Low molecular weight heparins (LMWHs) are heterogeneous mixtures of glycosaminoglycan chains composed of mixture of different lengths and substitution patterns. Structural characterization and quality control of LMWHs have always been challenging. The Chinese drug regulatory authorities have been committed to improve the supervision standards of LMWHs to better regulate the quality and safety of LMWHs in current Chinese market. In the present paper, 80 batches of three types LMWHs (dalteparin, enoxaparin and naldroparin) marketed in China from different manufacturers were studied by 1H NMR experiments and chemometric analysis. The method can be used not only to monitor impurities and contaminants, but also to check the batch-to-batch consistency of each manufacture. Moreover, for the biosimilar LMWHs from different manufactures, they can be differentiated and clustered according to their slightly different structural compositions originated from production process. By using this method, the quality and safety of LMWHs marketed in China were initially assessed.

CircRNA ACAP2 induces myocardial apoptosis after myocardial infarction by sponging miR-29.

BACKGROUND: The aim of this study was to investigate the effect of circular RNA circ ACAP2 on apoptotic phenotype of rat cardiomyocytes and its molecular mechanism. METHODS: Left anterior descending ligation was used to establish a rat myocardial infarction (MI) model, and real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of circ ACAP2 in apoptotic rat cardiomyocytes. The stability of circ ACAP2 was tested by actinomycin D and RNase R. H9C2 cells were infected by recombinant circ ACAP2 adenovirus (rAd-circ ACAP2), and the expression of apoptotic related genes Bax and BCL-2 in H9C2 was detected. Double luciferase reporter gene experiment, RNA antisense purification experiment and RNA Pull-Down experiments verified the binding effect of circ ACAP2 and miR-29. RESULTS: RT-qPCR results showed that the expression of circ ACAP2 was increased in cardiomyocytes of MI rats. Actinomycin D and RNase R experiment confirmed that circ ACAP2 had better stability and resistance to RNase R degradation than its host gene ACAP2 mRNA. Overexpression of circ ACAP2 promotes apoptosis. The double luciferase reporter gene assay, RNA antisense purification assay and RNA Pulldown assay consistently confirmed the specific binding effect between circ ACAP2 and miR-29, and circ ACAP2 promoted the apoptotic phenotype in rats. CONCLUSIONS: The expression of circ ACAP2 increased in cardiomyocytes after MI and promoted apoptosis by binding to miR-29.

Weighted gene co-expression network analysis identifies genes related to HG Type 0 resistance and verification of hub gene GmHg1.

Introduction: The soybean cyst nematode (SCN) is a major disease in soybean production thatseriously affects soybean yield. At present, there are no studies on weighted geneco-expression network analysis (WGCNA) related to SCN resistance. Methods: Here, transcriptome data from 36 soybean roots under SCN HG Type 0 (race 3) stresswere used in WGCNA to identify significant modules. Results and Discussion: A total of 10,000 differentially expressed genes and 21 modules were identified, of which the module most related to SCN was turquoise. In addition, the hub gene GmHg1 with high connectivity was selected, and its function was verified. GmHg1 encodes serine/threonine protein kinase (PK), and the expression of GmHg1 in SCN-resistant cultivars ('Dongnong L-204') and SCN-susceptible cultivars ('Heinong 37') increased significantly after HG Type 0 stress. Soybean plants transformed with GmHg1-OX had significantly increased SCN resistance. In contrast, the GmHg1-RNAi transgenic soybean plants significantly reduced SCN resistance. In transgenic materials, the expression patterns of 11 genes with the same expression trend as the GmHg1 gene in the 'turquoise module' were analyzed. Analysis showed that 11genes were co-expressed with GmHg1, which may be involved in the process of soybean resistance to SCN. Our work provides a new direction for studying the Molecular mechanism of soybean resistance to SCN.

Regioselective O-alkylation of 2-pyridones by TfOH-catalyzed carbenoid insertion.

Selective alkylation of 2-pyridone could solve a challenge in chemistry and streamline the synthesis of important molecules. Here we report the regioselective O-alkylation of 2-pyridones by TfOH-catalyzed carbenoid insertion. In the catalytic system, alkylation of 2-pyridone was achieved with unprecedented regioselectiviy (>99 : 1). This protocol is characterized by mild reaction conditions, metal-free, and simplicity. Moreover, this method provides the desired products in good yield and demonstrates a broad substrate scope in this transformation.

TfOH-catalyzed regioselective N2-alkylation of indazoles with diazo compounds.

Selective alkylation of indazoles is still a highly challenging topic in chemistry and the synthesis of important molecules. Herein, a novel highly selective N2-alkylation of indazoles with diazo compounds is described in the presence of TfOH. Unlike the traditional metal- and base-catalysed version, this protocol highlights the regioselectivity of alkylation of indazoles and a metal-free catalysis system, affording N2-alkylated products in good to excellent yields with high regioselectivity (N2/N1 up to 100/0) and excellent functional group tolerance. Furthermore, a gram scale synthesis was conducted successfully to give rise to the corresponding products. Mechanistic studies through control experiments provide plausible mechanistic proposals.