s2fieldsofstudy: Chemistry Biology Medicine

text:
Anaphylactic Rare Saponins Separated from Panax notoginseng Saponin and a Proteomic Approach to Their Anaphylactic Mechanism

In recent years, many traditional Chinese medicine injections based on Panax notoginseng saponin (PNS) have been reported to cause anaphylaxis. Previous studies on the anaphylactic saponins of PNS and their mechanism are inadequate. In this study, potential anaphylactic saponins were obtained by the separation of PNS and preparation of each individual component through comprehensive techniques, such as liquid chromatography, preparative chromatography, HPLC, NMR, and MS. The anaphylactic abilities of these saponins were tested using RBL-2H3 cells via a β-hexosaminidase release rate test. The results for the mechanism of anaphylaxis were obtained by a proteomic analysis using RBL-2H3 cells. The results indicate that, among all the saponins prepared, gypenoside LXXV and notoginsenoside T5 showed strong anaphylactic abilities and notoginsenoside ST-4 and ginsenoside Rk3 showed weak anaphylactic abilities. These 4 saponins can induce anaphylaxis via direct stimulation of effector cells. The gene oncology enrichment analysis results showed that, among these saponins, only gypenoside LXXV was related to organelles of the endoplasmic reticulum and Golgi apparatus and biological processes in response to organic cyclic compounds. Four proteins in RBL-2H3 cells with the accession numbers A0A0G2JWQ0, D3ZL85, D4A5G8, and Q8K3F0 were identified as crucial proteins in the anaphylactic process. This research will help traditional Chinese medicine injection manufacturers strengthen their quality control and ensure the safety of anaphylactic saponins.

ability, such as ginsenoside F2 [13] and Compound K [13]. e mechanism underlying the anaphylactoid reactions is distinct from that of IgE-mediated allergies, although they both have the same symptoms.
e complement system plays a key role in the process of anaphylaxis induced by many agents (e.g., radiocontrast media, liposomes, and TCM injections) [7]. Xu et al. [5,10] studied the anaphylactoid reaction induced by Xuesaitong injection and discovered that >10 kDa molecules could activate classical complement pathways through direct stimulation to cause anaphylaxis; Tween-80 can activate the complement system through classical and alternative pathways; and tannic acid can induce anaphylactoid reactions through coactivation of the complement system, the kallikrein-kinin system, and coagulation. Another mechanism is direct stimulation, which occurs through either direct G protein activation or opioid receptors [7]. For example, some TCM injections can also directly induce β-hexosaminidase and histamine release through mast cell degranulation [7].
Panax notoginseng saponin (PNS), a common material for TCM injections administered to cure cardiovascular diseases, is produced from Panax notoginseng (Burk.) Chen [5]. ADR reports indicated that the main TCM injections based on PNS as the crude material could induce more than 12% of the total TCM injection ADRs; moreover, anaphylactoid reactions mainly occurred within 30 min after the first administration [3,4]. However, previous studies on PNS, especially the crude PNS extract used in TCM injections, are insufficient.
To determine the specific anaphylactic constituents in PNS (from the crude extract of the root of Panax notoginseng (Burk.) F.H. Chen) and elucidate the mechanism, we prepared potential anaphylactic saponins via liquid chromatography, HPLC, and preparative chromatography and identified these saponins via ESI-MS and NMR. We further analyzed the cytotoxicity and β-hexosaminidase release rate (β-HexRR) of these saponins in vitro using RBL-2H3 cells. A proteomic analysis was performed to identify the possible mechanism underlying the anaphylactoid reactions.
is study will help manufacturers of TCM injections based on PNS ensure that those anaphylactic saponins are within safe levels and control the quality of TCM injections to avoid clinical anaphylactoid reactions.
All reagents were analytically or chromatographically pure.

Cell
Culture. RBL-2H3 cells were maintained in RPMI 1640 media with 10% FBS and 1% penicillin-streptomycin solution (100x) at 37°C and 5% CO 2 . Single-cell suspensions were plated at a density of 5-8 × 10 5 cells/mm 2 on a culture dish (D � 100 mm). When the cells reached over 80% confluence, they were harvested and passaged at a ratio of 1 : 3.

Anaphylactic Properties of the Subfractions.
Five milliliters of each subfraction was concentrated and freezedried in a Heidolph Rotary Evaporator (Heizbad Hei-VAP, Heidolph Instruments GmbH & CO. KG, Germany) and Biocool Freeze Dryer (Biocool FD-1C-50, Boyikang, Beijing), respectively. e obtained powders were weighed and added to the RPMI 1640 culture media with 1% DMSO, and the final concentration of each solution was 200 μg/mL.
Exponentially growing RBL-2H3 cells in 6-well dishes were washed with PBS 3 times. Two milliliters of the tested supernatant was added to the corresponding wells, including the negative control group (RPMI 1640 culture media with 1% DMSO) and the positive control group (C 48/80; 20 μg/mL). Each group was made in triplicate. e 6well dishes were incubated in an incubator (37°C, 5% CO 2 ) for 30 min before the supernatants were discarded. e 2 Evidence-Based Complementary and Alternative Medicine dishes were placed under an inverted microscope (Nikon Eclipse TS100-F, Japan) for observation. e groups with high degranulation rates were considered to have the potential constituents of the anaphylactoid reaction.

Preparation and
Identification of the Potential Saponins of the Anaphylactoid Reaction. Isocratic elution and preparative separation of Fr. 3, Fr. 5, and Fr. 7-8 were conducted using preparative chromatography with mobile phases of 68% methanol/water, 74% methanol/water, and 80% methanol/water, respectively. e eluents were subsequently concentrated and freeze-dried into powders before analysis by ESI-MS and NMR for molecular weight and 13 C-NMR information.

IC 50 Test.
Each saponin was dissolved in RPMI 1640 media into solution or dispersed suspension. Exponentially growing RBL-2H3 cells in a 96-well dish were washed with PBS before they were incubated with the corresponding saponins. e control group only contained the culture medium and RBL-2H3 cells, while the blank group only contained the culture medium. Cells were incubated for 24 h, and the supernatants were discarded. After washing with PBS 3 times, 100 μL culture medium was added to each well, and then MTS reagents were added for an incubation of approximately 30 min. e dish was subsequently placed on a microplate reader to record the absorbance.

β-Hexosaminidase Release Rate.
Exponentially growing RBL-2H3 cells in 24-well dishes were washed with RPMI 1640 media 3 times. en, 100 μL of RPMI 1640 medium was added to each well before incubation for 10 min. Another 100 μL of each tested constituent at different concentrations was added to the corresponding wells. Incubations of 15 min, 30 min, 1 h, and 2 h were conducted before an ice bath for 10 min to end the reaction. Fifty microliters of the supernatant in each well together with the substrate (β-D-hexosamine; 4 mM) were added to a 96-well dish before incubation for 1 h at 37°C. en, 150 μL of stop solution (glycine buffer; pH 10.7; 200 mM) was added to each well to stop the reaction. e absorbance (absorbance of supernatant, AoS) at 405 nm was measured for all wells. e rest of the supernatant in the 24-well dishes was removed, and 200 μL of 0.5% Triton X-100 was added to each well to lyse the cells. e lysate of each well was centrifuged at 3000 rpm for 1 min, and 50 μL of the supernatant in each well was transferred to a 96-well dish. β-D-Hexosamine (4 mM) as the substrate was added to each well at the same time. e 96-well dish was incubated at 37°C for 1 h before 150 μL of stop solution was added to each well. e absorbance (absorbance of lysate, AoL) at 405 nm was measured for all wells. β-HexRR can be calculated using the following equation: (1)

Proteomic Analysis. RBL-2H3 cells of different groups
were collected into the corresponding microtubes and centrifuged at 4000 rpm for 4 min before the supernatant was discarded. e cells were washed with PBS and centrifuged once again, and the supernatant was discarded. RIPA lysis buffer and protease inhibitor were added before treatment in an ice bath for 30 min. e cells were then lysed via ultrasonication. Protein concentrations were measured using the bicinchoninic acid (BCA) method.
Urea (8 M) and DTT (final concentration 10 mM) were added to the microtubes containing the cell lysate at volume ratios of 1 : 1 and 1 : 2, respectively. e mixture was incubated at 37°C for 4 h before IAA was added (IAA : DTT � 5 : 1, mol/mol). e mixture was further incubated in the dark for 1 h at room temperature. NH 4 HCO 3 (50 mM) was added to the mixture to dilute urea to below 1 M. Sequencing grade modified trypsin was added (trypsin: protein � 1 : 50, w/w). e mixture was incubated in a water bath at 37°C for 10-16 h. e mixture was desalted before the peptide concentration was measured via a NanoDrop ( ermo Fisher, Waltham, MA, USA). LC-MS/MS analysis was performed on a Q-Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer ( ermo Fisher Scientific) coupled online to a nanoflow LC system (EASY-nLC 1000, ermo Fisher Scientific). Peptides were delivered onto a 2 cm self-packed trap column (100 μm inner diameter, 3 μm resin, ReproSil-Pur C18-AQ, Dr Maisch GmbH) in solvent A (0.1% formic acid FA in HPLC grade water). After loading and washing, the peptides were transferred to a 12 cm column (150 μm inner diameter, 1.9 μm resin, ReproSil-Pur C18-AQ, Dr Maisch GmbH) and separated over 78 min nonlinear gradients from 6% to 95% solvent B (0.1% FA in ACN) at a flow rate of 600 nL/min. e exact elution gradients were as follows: 0-16 min, 6%-10% B; 16-51 min, 10%-24% B; 51-71 min, 24%-34% B; 71-72 min, 34%-95% B; 72-78 min, 95% B. e peptides were ionized using a 2.0 kV spray voltage and a capillary temperature of 320°C. e data acquisition was performed in the OT-IT mode. Full MS scans (300 to 1,400 m/z) were performed at a resolution of 120,000, a maximum injection time of 80 ms, and an AGC target value of 3e6. Tandem mass spectra were generated for up to 20 precursors by HCD with a normalized collision energy of 27%. e dynamic exclusion was set to 12 s. e MS2 spectra were read out in the Orbitrap at a resolution of 15,000 with an AGC target value of 5e4 and a maximum injection time of 19 ms.
e parameters and settings were as follows: precursor mass tolerance, 15 ppm; fragment mass tolerance, 20 mmu; enzyme name, trypsin; max missed cleavage sites, 2; static modification, carbamidomethyl; dynamic modifications, acetyl and oxidation. e confidence level was set to 95%.

Evidence-Based Complementary and Alternative Medicine
Differentially expressed proteins were determined by the ratio of protein expression of the adjacent groups. e expression of upregulated proteins in the high dose group/ medium dose group, medium dose group/low dose group, and low dose group/control group was all above 1.2; downregulated proteins in the high dose group/medium dose group, medium dose group/low dose group, and low dose group/control group were all below 1/1.2. Cluster heatmaps were drawn based on the upregulated proteins and downregulated proteins. Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were based on the upregulated proteins, downregulated proteins, and the DAVID (Database for Annotation, Visualization and Integrated Discovery) tool.

Anaphylactic Properties of the Subfractions.
Photographs of RBL-2H3 cells incubated with each subfraction are presented in Figure 1. e cells in the positive control groups were strongly degranulated and showed blurred cell membranes and obvious granules. In contrast, few cells in the negative control groups showed degranulation. Fr. 3, Fr. 5, and Fr. 7-8 were shown to induce degranulation of RBL-2H3 cells (>50% degranulated cells) while Fr. 1-2, Fr. 4, Fr. 6, Fr. 9-10, and Fr. 11 showed no anaphylactic properties.

Structural Identification of the Potential Saponins of the
Anaphylactoid Reaction. Compounds 1, 2, and 3 were obtained from the eluents of Fr. 3. Compounds 2, 3, and 4 were obtained from the eluents of Fr. 5. Compounds 5, 6, and 7 were obtained from the eluents of Fr. 7-8. All compounds were white amorphous powders that showed a red color on thin-layer plates when reacting with 10% ethanol sulfate solution. e 13 C-NMR spectra are shown in Figure S1, and the data are listed in Supplementary Materials. e data for compounds 1 and 4 were in accordance with the data in [14], and they were identified as notoginsenoside T5 (noto-T5) and notoginsenoside T4 (noto-T3), respectively. e data for compounds 2 and 3 were in accordance with the data in [15]; therefore, they were identified as ginsenoside Rk3 (Rk3) and ginsenoside Rh4 (Rh4), respectively. e data for compounds 5, 6, and 7 were in accordance with the data in [16][17][18], respectively; therefore, they were identified as ginsenoside Rg3 (Rg3), gypenoside LXXV (gyp-LXXV), and notoginsenoside ST-4 (noto-ST-4), respectively. Notably, all of the above saponins are rare saponins in PNS.

Basic Physical and Chemical Information of Each Saponin.
Saponins on a microgram scale were weighed and mixed with 1 mL of RPMI 1640 media with 1% DMSO at room temperature. e chemical structure, chemical formula, molecular weight, and solubility of each saponin are shown in Table 1.
Noto-T3 was not involved in the following studies because it presented flocculent precipitate and aggregation in the solution and was hard to be solved or shattered into small pieces, which was not suitable for the follow-up experiments.

IC 50 Test.
To prepare an appropriate concentration gradient of each saponin, an IC 50 test was performed based on RBL-2H3 cells using the MTS assay. e average cell viability of each group is shown in Table S1.
e IC 50 value of each saponin was obtained by linear interpolation. According to the IC 50 value of each saponin, the concentrations of the low, medium, and high dose groups were set geometrically, following the rules that the dose of high dose groups was close to but lower than the IC 50 value ( Table 2). According to the experiments, C 48/80 could induce abnormal absorbance in the IC 50 test. erefore, the low, medium, and high dose groups of C 48/80 were set based on [12].

β-Hexosaminidase Release Rate.
e β-hexosaminidase release rate is the gold standard to quantitatively evaluate the level of anaphylactoid reactions [7,10,19]. According to clinical reports, anaphylactoid reactions induced by TCM injections containing PNS mainly occur within 30 min after administration, and few ADR cases still occur within 30 min to 2 h after administration [20]. erefore, 4 periods of time (15 min, 30 min, 1 h, and 2 h) were set to investigate the β-HexRR of the control group, C 48/80 group, and 6 saponin groups (Figures 2(a)-2(d)).
e results indicated that, compared with the control and C 48/80 groups, Rg3 had no anaphylactic ability, gyp-LXXV and noto-T5 had strong anaphylactic abilities, and noto-ST-4 and Rk3 had weak anaphylactic abilities (Figure 2(e)). For Rh4, the results cannot support the conclusion that it possesses anaphylactic ability. Surprisingly, its high dose group with an incubation time of 1 h even induced a significantly lower β-HexRR.
ese results also demonstrated that gyp-LXXV, noto-T5, noto-ST-4, and Rk3 can directly activate β-hexosaminidase release and degranulation from RBL-2H3 cells, which suggested that these saponins can induce anaphylaxis via direct stimulation of effector cells.

Proteomic Analysis.
To investigate the mechanism of the anaphylactoid reaction induced by the potential anaphylactic saponins in PNS, differentially expressed proteins of each saponin group with an incubation time of 30 min were studied. According to the filter conditions, proteins were screened and classified into upregulated proteins and downregulated proteins (Table 3). Based on the upregulated and downregulated proteins, cluster heatmaps were drawn ( Figure 3); GO and KEGG enrichment analyses were also carried out via the DAVID tool. GO enrichment analyses of the biological process (BP) (p < 0.005), cellular component (CC) (p < 0.005), and molecular function (MF) (p < 0.005) categories are displayed in Figure 4. e KEGG enrichment pathway analysis results are shown in Figure S2.   Evidence-Based Complementary and Alternative Medicine     Table 2. Each group was repeated 3 times, and the data are expressed as the mean ± SEM (n � 3). * p < 0.05 and * * p < 0.01, compared with the corresponding control group (t-test). e β-HexRR of the high dose group of Rh4 with an incubation time of 1 h was significantly lower than that of the control group, while all the other significant differences were significantly higher. Table 3: Number of upregulated and downregulated proteins and total identified proteins of each saponin group with an incubation time of 30 min.

Discussion
e β-HexRR results were in accordance with a previous study showing that Rg3 could inhibit mast cell-mediated allergies by blocking degranulation [21].
Further analysis demonstrated that there were 4 proteins in common in the differentially expressed proteins of the strong anaphylactic groups (gyp-LXXV groups and noto-T5 groups), namely, A0A0G2JWQ0, D3ZL85, D4A5G8, and Q8K3F0, all of which were downregulated proteins and not included in the differentially expressed proteins of the other 4 groups (Figure 5(b)). is result indicated that these 4 proteins had a higher possibility of playing a key role in the anaphylactoid reactions of RBL-2H3 cells.
For gyp-LXXV, KEGG enrichment pathway analysis results were not obtained after data filtration. For the noto-T5 and other 4 groups, the KEGG enrichment pathway analysis result was not consistent with RBL-2H3 cells and anaphylactoid reactions ( Figure S1). e results indicate that the molecular mechanism of the anaphylactoid reactions induced by gyp-LXXV and noto-T5 requires further in-depth study.
Although gyp-LXXV and noto-T5 were identified as strong anaphylactic saponins and noto-ST-4 and Rk3 were identified as weak anaphylactic saponins, these findings do not necessarily mean that every TCM injection based on PNS will induce anaphylaxis. In fact, incomplete clinical reports indicated that anaphylactoid reactions induced by several main TCM injections based on PNS had an overall incidence rate of less than 1% and varied according to the batch [20,22], which also indicates that anaphylactic saponins are most likely to be rare saponins and that the occurrence of anaphylaxis depends on the total content of these rare saponins. Other related factors include the dripping speed, age, sex, and body constitution [3,4]. Here we only discuss the potential anaphylactic abilities of these saponins and offer a guide for TCM injection manufacturers.

Conclusions
In conclusion, among the 7 saponins identified, gyp-LXXV and noto-T5 had strong anaphylactic abilities, noto-ST-4 and Rk3 had weak anaphylactic abilities, and Rg3 had no anaphylactic ability. Anaphylactic saponins can induce anaphylaxis via direct stimulation of effector cells. e results of proteomic studies indicated that the anaphylactoid reaction of gyp-LXXV was related to the biological processes in the response to organic cyclic compounds and the cellular components of the endoplasmic reticulum and Golgi apparatus. Four proteins were most likely to play key roles in the anaphylactoid reactions of RBL-2H3 cells, and their accession numbers were A0A0G2JWQ0, D3ZL85, D4A5G8, and Q8K3F0. To avoid clinical anaphylactoid reactions, manufacturers should strictly control the ingredients of TCM injections based on PNS and ensure that gyp-LXXV, noto-T5, noto-ST-4, and Rk3 are within safe levels.

Data Availability
All data used to support the findings of this study are available from the corresponding author upon request.


s2fieldsofstudy:
Chemistry
Agricultural And Food Sciences

text:
Centrifugation-Assisted Solid-Phase Extraction Coupled with UPLC-MS/MS for the Determination of Mycotoxins in ARECAE Semen and Its Processed Products

Mycotoxins can occur naturally in a variety of agriculture products, including cereals, feeds, and Chinese herbal medicines (TCMs), via pre- and post-harvest contamination and are regulated worldwide. However, risk mitigation by monitoring for multiple mycotoxins remains a challenge using existing methods due to their complex matrices. A multi-toxin method for 22 mycotoxins (aflatoxin B1, B2, G1, G2, M1, M2; ochratoxin A, B, C; Fumonisin B1, B2, B3; 15-acetyldeoxynivalenol, 3-acetyldeoxynivalenol, diace-toxyscirpenol, HT-2, T-2, deepoxy-deoxynivalenol, deoxynivalenol, neosolaniol, zearalenone, and sterigmatocystin) using centrifugation-assisted solid-phase extraction (SPE) clean-up prior to ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis for Arecae Semen and its processed products was developed and validated. Several experimental parameters affecting the extraction and clean-up efficiency were systematically optimized. The results indicated good linearity in the range of 0.1–1000 μg/kg (r2 > 0.99), low limits of detection (ranging from 0.04 μg/kg to 1.5 μg/kg), acceptable precisions, and satisfactory recoveries for the selected mycotoxins. The validated method was then applied to investigate mycotoxin contamination levels in Areca catechu and its processed products. The mycotoxins frequently contaminating Areca catechu were aflatoxins (AFs), and the average contamination level and number of co-occurring mycotoxins in the Arecae Semen slices (Binlangpian) were higher than those in commercially whole Arecae Semen and Arecae Semen Tostum (Jiaobinlang). Sterigmatocystin was detected in 5 out of 30 Arecae Semen slices. None of the investigated mycotoxins were detected in Arecae pericarpium (Dafupi). The results demonstrated that centrifugation-assisted SPE coupled with UHPLC-MS/MS can be a useful tool for the analysis of multiple mycotoxins in Areca catechu and its processed products.

Introduction
Mycotoxins are the secondary metabolites of fungal origin produced by various genera (Aspergillus, Fusarium, Penicillium, etc.) and have been a global concern for their ability to Mycotoxins are the secondary metabolites of fungal origin produced by various genera (Aspergillus, Fusarium, Penicillium, etc.) and have been a global concern for their ability to induce acute to chronic toxicity in humans and animals due to their teratogenic, mutagenic, carcinogenic, immunosuppressive, and endocrine-disrupting effects [1]. Up to now, more than 400 mycotoxins have been identified with various chemical structures. Mycotoxin contamination can occur in different matrices, such as foods, cereals, feeds, and traditional Chinese medicines (TCMs), as well as related products [2][3][4]. In these matrices, several mycotoxin groups are frequently found, including aflatoxins, ochratoxin, fumonisins, zearralenone, T-2/HT-2, and deoxynivalenol. Despite efforts to control fungal infection, extensive mycotoxin contamination has been reported in foods and TCMs.
Areca catechu Linn, an evergreen areca tree in the palm family, is widely distributed and cultivated in several tropical southern and southeastern Asian countries such as China, India, Bangladesh, Indonesia, Myanmar, Thailand, Malaysia, Vietnam, and the Philippines [5]. The main producing areas in China are Hainan, Guangdong, Guangxi, Yunnan, and Taiwan provinces. Arecanut palm is a popular crop in the south and southeast of Asia for both social and economic reasons. Arecae Semen is an addictive substance that is widely used as a mouth freshener and to assist digestion. As a traditional herb medicine, Arecae Semen has been used to kill parasites and promote digestion, with over 100 prescriptions containing Arecae semen Semen and its processed products [6]. The most common clinically used forms of Areca catechu are Arecae Semen, Arecae Semen Tostum, and Arecae pericarpium. Arecae Semen (Binglang in Chinese, BL) is the dried ripe seed of Areca catechu. Slices of Arecae Semen are stir-baked until a charred yellow color appears to obtain Arecae Semen Tostum (Jiaobinglang in Chinese, JBL). Arecae pericarpium (Dafupi in Chinese, DFP) is the dried pericarp of Areca catechu ( Figure 1). Arecae Semen is susceptible to contamination by pathogenic fungi during various stages of harvesting, storage, and transportation. Furthermore, Arecanut palm is frequently cultivated in countries with high temperature and humidity climates [7]. Because of these conditions, as well as post-harvest environmental factors, fungi are capable of producing mycotoxins in areca products and medicines. The safety of the areca nut has gradually captured attention due to its extensive medicinal and edible value. Several LC-MS/MS (or fluorescence detection, FLD) methods have been reported for the analysis of mycotoxins in Areca catechu. However, none so far have focused on the occurrence and detection of mycotoxin residues in Areca catechu-processed products [7][8][9]. Hongmei Liu et al. reported the development of a UFLC-MS/MS based on a one-step extraction without any further clean-up for the quantitative analysis of 11 mycotoxins in Several LC-MS/MS (or fluorescence detection, FLD) methods have been reported for the analysis of mycotoxins in Areca catechu. However, none so far have focused on the occurrence and detection of mycotoxin residues in Areca catechu-processed products [7][8][9]. Hongmei Liu et al. reported the development of a UFLC-MS/MS based on a one-step extraction without any further clean-up for the quantitative analysis of 11 mycotoxins in Areca catechu [8], whereas Muhammad Asif Asghar et al. described an HPLC-FLD method for the simultaneous analysis of aflatoxins (AFs, AFB 1 , AFB 2 , AFG 1 , AFG 2 ) in betel nuts, and compared the level of contamination amongst Asian countries. Samples were extracted with a mixture of ACN/H 2 O, followed by immunoaffinity column clean-up [7]. Hung-Yu Lin et al. developed an HPLC-MS/MS to determine nine mycotoxins in the betel  [9]. Despite satisfactory recovery and repeatability, these methods included only a limited number of mycotoxins, involved significant matrix effects, or required complex pretreatments that are time-consuming and can result in inaccurate results. Low levels of mycotoxins and complex matrices of Arecae Semen and its processed products require an efficient clean-up method to improve the sensitivity and selectivity of the analytical method. Solid-phase extraction is one of the most commonly used methods to remove matrix interferences. Traditionally, during the wash, eluent solvent passes through the SPE column by gravity or negative pressure. However, this tedious procedure increases the analysis time or requires vacuum pump equipment, leading to a reduction in the number of samples that can be processed at one time. Hence, it is necessary to develop a rapid and sensitive approach to detect the presence of multi-mycotoxins in Arecae Semen and its processed products.

Optimization of the UPLC-MS/MS System
Careful optimization of the UPLC-MS/MS parameters was achieved in order to obtain suitable separation and better sensitivity for target mycotoxins. An initial investigation was carried out to optimize the MS parameters for mycotoxin detection. A standard solution of 1.0 µg/mL of each mycotoxin was injected directly into the MS using flow injection, and a full scan was executed in the first quadrupole in electrospray ionization (ESI) positive and negative modes. The results showed that 22 target mycotoxins had better responses in the positive ion mode. For most mycotoxins (AFs, OTS, FBs, etc.), there is a high abundance of the [M + H] + peak in positive ESI mode since they contain methyl or carbonyl groups. The [M + NH 4 ] + ions among the precursor of T-2 and HT-2 showed the highest ion abundance. The high DAS ion abundance was in the [M + Na] + ion form. The signal acquisition information of each mycotoxin in the optimized multiple reaction monitoring (MRM) mode, including characteristic ion pairs, fragmentor voltage, and collision energies, are listed in Table 1. For each mycotoxin, two mass transitions with the highest abundances were selected. One quantifier ion and one qualifier transition were monitored for each mycotoxin, whereas only one transition was chosen for the internal standard (IS). Finally, the MRM experiments were segmented according to the separation time of target mycotoxins with the goal of increasing the sensitivity of the analysis since the MS scans only these ions during that specific time interval. The higher specificity and sensitivity obtained when employing MRM segmentation, and multiple transitions are expected to result in better quantification [10]. Because of the diverse structure and physio-chemical properties of mycotoxins, it is important to optimize the chromatography column and the composition of the mobile phase. Based on previous experience, a Waters Acquity BEH C 18 column (100 mm × 2.1 mm, 1.7 µm, Waters) was selected as the separation column for the current application. The mobile phase was further evaluated, including both organic (MeOH and ACN) and aqueous phases with different buffer solutions (acetic acid (AA), formic acid (FA), ammonium formate, and ammonium acetate). For all mycotoxins, a better response was observed when using the MeOH/H 2 O 2 as a mobile phase compared to ACN/H 2 O 2 . A similar report pointed out that this trend might be attributed to the protic nature of MeOH, which enhances the response of [M + H] + in ESI + mode [11]. An acidic mobile phase could improve the signal intensities for some mycotoxins (FBs, OTA) due to the presence of carboxylic groups in the molecular structure [12]. Moreover, the acidification of the mobile phase with ammonium acetate could promote T-2 and HT-2 to form ammonium adducts [M + NH 4 ] + as precursor ions. Additionally, it could avoid the formation of unwanted adducts (e.g., [M + Na] + ) [13]. When the mobile phase was fixed as 2 mM ammonium acetate and MeOH (containing 0.1% FA), all mycotoxins were separated with resolved symmetrical peaks and at high abundances. Other parameters, including column temperature, flow rate, and injection volume, were also optimized. The MRM chromatograms of the target mycotoxins under the optimized UPLC-MS/MS conditions are shown in Figure 2.

Optimization of Sample Preparation
For the detection of multiple mycotoxins, the method of extraction and clean-up is a crucial step prior to UPLC-MS/MS analysis. An excellent extraction capability could be considered the most important factor in the selection of the extraction solvent. MeOH and acetone were not considered due to the insolubility of the mycotoxins in MeOH and the unselective solvent power of acetone [14]. ACN was chosen because it has been shown to extract the broadest range of organic compounds without the co-extraction of large amounts of lipophilic material [15]. However, when ACN was used as the extraction solvent, the extraction effect of mycotoxins sensitive to the polarity range was not ideal. Adding acid to the extraction solution can improve the extraction rate of some acid toxins (such as FBs, OTA, and T-2) and AFs [16]. Therefore, the extraction recoveries of mycotoxins by ACN (80% and 84% aqueous ACN) with different concentrations of FA were evaluated by spiking blank samples with target mycotoxins. Figure 3 shows that the recovery of most of the mycotoxins extracted with 84% aqueous ACN was better than using 80% aqueous ACN. In addition, when FA was added to the extraction solvent, it resulted in increased recovery of some target mycotoxins (OTA, OTB, OTC, DON, DAS, HT-2, T-2, and NEO). It is important to note that when the concentration of FA in the extraction solvent is above 0.3%, there will be reduced recovery of some toxins, such as AFs, OTA, OTB, OTC, DOM-1, DON, 15-AcDON, 3-AcDON, etc. Thus, ACN with 0.2% FA was selected for further experiments. Next, the effects of the volume of extraction agent (10,15, and 20 mL) and extraction time (5, 10, and 15 min) were evaluated. When the volume of the extraction solvent was above 15 mL, there was no significant increase in the recovery of the mycotoxins. As for the extraction time, it can be highlighted that the recovery of the mycotoxins increased from 5 to 10 min and then decreased or kept constant. Using the proposed extraction method with these conditions achieved satisfactory extraction efficiency with recoveries ranging from 67.1 to 111.2%.

Optimization of Sample Preparation
For the detection of multiple mycotoxins, the method of extraction and clean-up is a crucial step prior to UPLC-MS/MS analysis. An excellent extraction capability could be considered the most important factor in the selection of the extraction solvent. MeOH and acetone were not considered due to the insolubility of the mycotoxins in MeOH and the unselective solvent power of acetone [14]. ACN was chosen because it has been shown to extract the broadest range of organic compounds without the co-extraction of large amounts of lipophilic material [15]. However, when ACN was used as the extraction solvent, the extraction effect of mycotoxins sensitive to the polarity range was not ideal. Adding acid to the extraction solution can improve the extraction rate of some acid toxins (such as FBs, OTA, and T-2) and AFs [16]. Therefore, the extraction recoveries of mycotoxins by ACN (80% and 84% aqueous ACN) with different concentrations of FA were evaluated by spiking blank samples with target mycotoxins. Figure 3 shows that the recovery of most of the mycotoxins extracted with 84% aqueous ACN was better than using 80% aqueous ACN. In addition, when FA was added to the extraction solvent, it resulted in increased recovery of some target mycotoxins (OTA, OTB, OTC, DON, DAS, HT-2, T-2, and NEO). It is important to note that when the concentration of FA in the extraction solvent is above 0.3%, there will be reduced recovery of some toxins, such as AFs, OTA, OTB, OTC, DOM-1, DON, 15-AcDON, 3-AcDON, etc. Thus, ACN with 0.2% FA was selected for further experiments. Next, the effects of the volume of extraction agent (10, 15, and 20 mL) and extraction time (5, 10, and 15 min) were evaluated. When the volume of the extraction solvent was above 15 mL, there was no significant increase in the recovery of the mycotoxins. As for the extraction time, it can be highlighted that the recovery of the mycotoxins increased from 5 to 10 min and then decreased or kept constant. Using the proposed extraction method with these conditions achieved satisfactory extraction efficiency with recoveries ranging from 67.1 to 111.2%.
Matrix effects caused by the co-extraction of compounds from the matrix are a known limitation of LC-MS/MS analysis. An efficient clean-up procedure following extraction can serve to decrease matrix effects. In recent years, multi-functional purification columns have been popular in LC-MS/MS preprocessing for the simple and fast removal of impurities [17]. In our preliminary research, SPE (MycoSpin TM 400) was more effective and convenient compared with the other clean-up pretreatments. Thus, the centrifugation-assisted SPE method was selected as the clean-up method in this study. In what follows, two parameters that affected the extraction recoveries, pH and load volume of sample extraction solvent, were carefully optimized. BLS was used as a blank matrix, and the test sample was prepared by spiking in a standard mixture (5.0 µg/kg for AFB 1 , AFB 2 , AFG 1 , AFG 2 , AFM 1 , and AFM 2 ; 25.0 µg/kg for OTA, OTB, and OTC; 50.0 µg/kg for DON, DOM-1, HT-2, T-2, ST, ZEN, 15-Ac DON, 3-Ac DON NEO, and DAS; and 125.0 µg/kg for FB 1 , FB 2 , and FB 3 ) into the blank. The purification effect was investigated using the recovery rate as the index. An evaluation of the pH adjustment with AA, prior to clean-up, was performed. In order to be convenient for sample testing, the concentration of AA (%, v/v) was used for optimization instead of the pH value. The recoveries of FB 1 , FB 2 , and FB 3 were 0 when using the sample extraction solvent (pH = 4.66) without adding AA. Alongside with FBs, the recoveries of OTA, OTB, AFM 2 , AFG 2 , etc., were below 40%. Figure 4 shows the effect of AA concentration on the recovery of mycotoxins after clean-up. It can be observed that the recoveries of FBs, OTA OTB, OTC, AFM 2 , and AFG 2 increased significantly when the concentration of AA ranged from 0.5% (pH = 4.6) to 15% (pH = 3.15). In particular, increasing the AA concentration gradually increased the recoveries of NEO, 15-AcDON, 3-AcDON, DAS, T-2, and ST, approaching 100%, although a concentration of AA above 15% (20%, pH = 2.8; 25%, pH = 2.48) did not yield a significant increase in the recoveries of most mycotoxins. However, there was a significant decrease in the recoveries of DON, DOM-1, 15-AcDON, 3-AcDON, DAS, ZEN, and ST. Additionally, the influence of AA concentration on the purification efficiency was investigated by comparing the color of the purified extract. Increasing the concentration of AA caused the color of the purified extract to become dark ( Figure S1), which indicated that the concentration of acid affected the binding ability to adsorb and the co-extractive components in the matrix. It was therefore decided to maintain the concentration of AA at 15% in order to obtain maximum recovery. Hence, the pH value of the loading solution was a key factor affecting the accuracy of the SPE method. For WBL, JBL, and DFP, the trend for color change in the purified solution with different acid concentrations was similar to that of BLS. In addition, after adding different concentrations of AA, the pH values of the loading solvents from different samples (WBL, JBL, and DFP) were not significantly different from that of BLS (within ±0.25). Under the optimized conditions, the recoveries of most selected mycotoxins in WBL, JBL, and DFP ranged from 70% to 120% (Table S1), which indicated that the developed clean-up method is suitable for these herbs.  similar to that of BLS. In addition, after adding different concentrations of AA, the pH values of the loading solvents from different samples (WBL, JBL, and DFP) were not significantly different from that of BLS (within ±0.25). Under the optimized conditions, the recoveries of most selected mycotoxins in WBL, JBL, and DFP ranged from 70% to 120% (Table S1), which indicated that the developed clean-up method is suitable for these herbs. As we know, high concentrations of acid in the mobile phase and sample extraction solvent would inhibit MS response for all analytes. To investigate the effectiveness of AA on the target mycotoxins in the present study, a mycotoxin-free BLS sample was used. The sample preparation method was the same as detailed above. After SPE purification, one sample was evaporated to dryness under a gentle nitrogen steam and reconstituted with an ACN/H2O2/FA (84/15.8/0.2, v/v/v) solution (Sa), and the other sample was not evaporated with nitrogen (Sb). The MS signal responses of mycotoxins in both solvents were compared using the following equation: IA (%) = (1 − Sa/Sb) × 100. As shown in Figure 5, the addition of AA (15%) had different inhibitory effects on the MS signal response of selected mycotoxins. The inhibition rate was in the range of 16.76% (DAS) ~ 45.19% (ST). Therefore, after the sample was purified by SPE, the acid was removed with nitrogen from the eluent. As we know, high concentrations of acid in the mobile phase and sample extraction solvent would inhibit MS response for all analytes. To investigate the effectiveness of AA on the target mycotoxins in the present study, a mycotoxin-free BLS sample was used. The sample preparation method was the same as detailed above. After SPE purification, one sample was evaporated to dryness under a gentle nitrogen steam and reconstituted with an ACN/H 2 O 2 /FA (84/15.8/0.2, v/v/v) solution (Sa), and the other sample was not evaporated with nitrogen (Sb). The MS signal responses of mycotoxins in both solvents were compared using the following equation: IA (%) = (1 − Sa/Sb) × 100. As shown in Figure 5, the addition of AA (15%) had different inhibitory effects on the MS signal response of selected mycotoxins. The inhibition rate was in the range of 16.76% (DAS)~45.19% (ST). Therefore, after the sample was purified by SPE, the acid was removed with nitrogen from the eluent.
Next, different load volumes (300, 450, 600, 750, and 900 µL) of extract were evaluated to estimate the capacity of saturation of the solid phase. The recovery rates shown in Figure S2 indicated that acceptable recovers were obtained with a load volume of 750 µL. Therefore, this load volume was chosen as the optimum volume. Finally, the rotation speed and time of centrifugation were evaluated and shown to have no significant effect on the recovery of the target mycotoxins. ated to estimate the capacity of saturation of the solid phase. The recovery rates shown in Figure S2 indicated that acceptable recovers were obtained with a load volume of 750 μL. Therefore, this load volume was chosen as the optimum volume. Finally, the rotation speed and time of centrifugation were evaluated and shown to have no significant effect on the recovery of the target mycotoxins.

Matrix Effect
In mycotoxin analysis, the matrix effect (ME) is often observed when ESI is used as the ionization technique in LC-MS/MS. The ion intensity of target analytes is suppressed or enhanced by co-extractive compounds coming from the matrix [18,19]. ME affects the reproducibility and accuracy of the proposed LC-MS/MS method, which is considered one of the major drawbacks. In our team's previous research, we observed that BLS has a significant ME on some mycotoxins [8]. To evaluate the possible occurrence of ME, the MS/MS peak area of the mycotoxins spiked in the extraction solvent was compared to those spiked in mycotoxin-free sample extraction solvent after SPE clean-up at the same concentration level. In general, ME is considered to be tolerable with a value in the range between 80~120% [20]. Values outside of this range are considered to have strong signal suppression or enhanced effect. As shown in Table 2, the ME of WBL was acceptable, with values for most toxins in the range of 80~120%, except for DON (78.30%), NEO (128.25%), 15-AcDON (78.98%), AFG2 (77.91%), AFG1 (71.68%), DAS (124.47%), and FB1 (109.10%). Although the ME values of these seven toxins were outside the acceptable range, they were very close to the critical value. Similar ME values were found in BLP and JBL. However, for DFP, six mycotoxins (DOM-1, AFG2, AFG1, FB1, FB2, and FB2) showed a strong suppressive effect in the range of 56.50~79.97%. Apparently, ME still remained high for a small number of toxins (especially in DFP), although SPE clean-up had been applied to remove co-extracted components. The use of stable isotopically labeled internal standards is one of the commonly employed strategies to compensate for the bias caused by ME. Therefore, the quantification of multi-mycotoxins in Arecae Semen and its processed products was carried out using the internal standard curve method, even if strong ME was only noticed in DFP.

Matrix Effect
In mycotoxin analysis, the matrix effect (ME) is often observed when ESI is used as the ionization technique in LC-MS/MS. The ion intensity of target analytes is suppressed or enhanced by co-extractive compounds coming from the matrix [18,19]. ME affects the reproducibility and accuracy of the proposed LC-MS/MS method, which is considered one of the major drawbacks. In our team's previous research, we observed that BLS has a significant ME on some mycotoxins [8]. To evaluate the possible occurrence of ME, the MS/MS peak area of the mycotoxins spiked in the extraction solvent was compared to those spiked in mycotoxin-free sample extraction solvent after SPE clean-up at the same concentration level. In general, ME is considered to be tolerable with a value in the range between 80~120% [20]. Values outside of this range are considered to have strong signal suppression or enhanced effect. As shown in Table 2, the ME of WBL was acceptable, with values for most toxins in the range of 80~120%, except for DON (78.30%), NEO (128.25%), 15-AcDON (78.98%), AFG 2 (77.91%), AFG 1 (71.68%), DAS (124.47%), and FB 1 (109.10%). Although the ME values of these seven toxins were outside the acceptable range, they were very close to the critical value. Similar ME values were found in BLP and JBL. However, for DFP, six mycotoxins (DOM-1, AFG 2 , AFG 1 , FB 1 , FB 2 , and FB 2 ) showed a strong suppressive effect in the range of 56.50~79.97%. Apparently, ME still remained high for a small number of toxins (especially in DFP), although SPE clean-up had been applied to remove co-extracted components. The use of stable isotopically labeled internal standards is one of the commonly employed strategies to compensate for the bias caused by ME. Therefore, the quantification of multi-mycotoxins in Arecae Semen and its processed products was carried out using the internal standard curve method, even if strong ME was only noticed in DFP.

Method Validation
Due to its selectivity, this analytical method was chosen to evaluate obvious sources of co-extractive compounds at definitive retention times in blank samples. The retention times of the target mycotoxins in the chromatograms of the blank samples showed high levels of repeatability without interfering peaks, indicating that this approach has high selectivity. The comprehensive characteristic performance parameters are listed in Table 3. Linear calibration curves were obtained over the range of 0.1-1000 µg/kg for the target mycotoxins with linear regression coefficients (r 2 ) exceeding 0.99. The limits of detection (LOD) and limits of quantification (LOQ) values were in the range of 0.04-1.5 µg/kg and 0.1-5.0 µg/kg, respectively. The precisions of intra-and inter-day were lower than 8%. The average recoveries of mycotoxins at three different concentration levels ranged from 70% to 120%, with the exception of DON and DOM-1, and the relative standard deviation (RSD) values were mostly <15.0% (Table 4). These validation data indicated that the developed method was appropriate for the simultaneous determination of 22 mycotoxins in BL with satisfactory performance according to the European Commission regulations [21].

Application
The proposed SPE-UPLC-ESI-MS/MS method was applied to determine the target mycotoxins in BL and its processed products purchased from local markets. Each sample was prepared in triplicate for extraction, clean-up, and LC-MS/MS analysis under the optimized conditions. The results of the mycotoxin contamination in the WBL, BLS, and JBL samples are presented in Table 5. The MRM chromatogram of the detected mycotoxins in the different positive samples is shown in Figures S3-S6. None of the DFP collected was contaminated with the selected mycotoxins. AFs were detected in the WBL, BLS, and JBL samples. This result was similar to that found in a previous study [8], in which large amounts of AFs were detected in BLS with an incidence of 16.7%. In addition, ST was detected in 5 BLP samples. be related to the contaminated fungal species. In aflatoxigenic species, in which ST is converted into AFB 1 , the accumulation of ST is rarely seen. Although, some species (A. versicolor, A. nidulans, etc.) are unable to convert ST into AFB 1 , probably due to the lack of genes encoding the specific methyltransferase required for this conversion [26]. For this reason, substrates colonized by these fungi can contain high amounts of ST. Therefore, it can be speculated that the species contaminated with areca nut may be Aspergillus flavus or A. parasiticus. In these fungi, ST does not accumulate in large amounts since it is consumed to produce AFB 1 .
A previous report found that the fungal counts of A. flavus in WBL, BLS, and JBL were significantly different (BLS > JBL > WBL) [27]. This information indicated that these results are closely related to the consequence of the present study. Additionally, the components and processing method may affect fungal growth. The areca nut is rich in polysaccharides, fatty acids, and lipids, which may be beneficial for fungal growth and the production of toxins. Alternatively, DFP contains a large amount of fiber, and triterpenes in the DFP possess significant antifungal activity [28]. Furthermore, the processing itself may destroy aflatoxins or may cause chemical changes that render boiled and baked nuts less favorable substrates for fungal growth. Consequently, to minimize the risk of mycotoxin contamination in Areca catechu and its processed products, attention could be paid to specific factors, such as suitable culture management, appropriate drying, processing procedures, and storage conditions.

Conclusions
In this study, a centrifugation-assisted SPE coupled with UHPLC-MS/MS was developed and validated for the fast and sensitive detection of multi-mycotoxins in Areca catechu and its processed products. This method could be applied for the qualitative and quantitative analysis of the 22 selected mycotoxins in herbs, providing quality and safety to the herb industry and consumers. However, it should be emphasized that the pH value of the extract prior to SPE purification may be adjusted according to the tested TCMs. Mycotoxin analysis showed that Areca catechu is susceptible to AF contamination caused by inadequate harvesting and storage conditions. Therefore, continuous monitoring for multiple mycotoxins by the proposed method is important for confirmation of current contamination and further assurance of the safe consumption of Areca catechu and its related products. Considering the toxicity of ST and its involvement in the biosynthesis of AFB 1 , setting a limit for ST in TCMs is essential to ensure their safety.

UPLC-MS/MS Conditions
A Waters Acquity UPLC-tandem quadrupole (TQD) mass spectrometer (Waters, Milford, MA, USA) with ESI was used for detection. The target mycotoxins were separated on an Acquity UPLC BEH C 18 (1.7 µm, 2.1 mm × 100 mm) column with the column temperature set at 40 • C. The mobile phase was comprised of methanol containing 0.1% FA as eluent A and 2 mM ammonium formate in water as eluent B. The gradient elution system was as follows: 0 min: 25% A; 1 min: 25% A; 4 min: 45% A; 6 min: 60% A; 14 min: 95% A; 19 min: 95% A; 20 min: 25% A. The flow rate was set at 0.3 mL min −1 , and the injection volume was 2 µL. The system was re-equilibrated with 25% eluent A for 5 min prior to the next injection. The detection and qualitative analysis were operated in positive electrospray ionization mode with MRM scanning mode. Nitrogen was used as the cone and desolvation gas, and argon was used as the collision gas. The MS conditions were as follows: capillary voltage, 3.5 kV; desolvation temperature, 350 • C; source temperature, 150 • C; desolvation gas, 650 L h −1 ; cone nitrogen gas, 30 L h −1 . The data were acquired using the software program MassLynx 4.1. The optimal collision energies and cone voltages for each transition of the target mycotoxins are summarized in Table 1. Data acquisition was performed under time-segmented conditions based on the chromatographic separation of the target mycotoxins to maximize detection sensitivity. A segment at 0-6 min was detected for DON, DOM-1, NEO, 3-AcDON, 15-AcDON, and AFM 2 ; a segment at 4.5-7.5 min was detected for AFG 2 , AFM 1 , AFG 1 , AFB 2 , AFB 1 , and DAS; and a segment at 7.5-12 min was detected for HT-2, OTB, FB 1 , T-2, OTA, FB 3 , ZEN, ST, FB 2 , and OTC.

Samples
A total of 75 samples of Arecae Semen and its processed products (including 15 WBL, 30 BLS, 15 JBL, and 15 DFP) were analyzed for multi-mycotoxins content. All samples were obtained from pharmacies or markets in the provinces of Hainan, Guangxi, Guangdong, and Yunnan in China P. R. and identified by the authors. Samples were homogenized using a laboratory mill and sieved through a 0.85 mm mesh filter before they were finally stored at −20 • C until the time of the experimental analysis.

Sample Preparation
Homogenized powder from each sample was accurately weighed (2.00 g) and placed in a 50 mL centrifuge tube with 15 mL of ACN/H 2 O 2 /FA (84/15.8/0.2, v/v/v) solution, followed by vortex mixing for 1 min and ultrasonic extraction at room temperature for 10 min. The solution was cooled to room temperature and adjusted to the original weight with the ACN/H 2 O 2 /FA (84/15.8/0.2, v/v/v) solution and then centrifuged at 4000 rpm for 10 min. Subsequently, 150 µL of AA was added to 850 µL of the supernatant and mixed by vortex for 30 s. Then, 750 µL of the mixture was transferred into the MycoSpin TM 400 column, which was capped, vortexed for one minute, and turned upside down before breaking the bottom tip off and placing the column in the centrifuge tube for centrifugation at 2000 rpm for 2 min. Last, 500 µL of the eluting solvent was transferred into 1.5 mL dark vials, and aliquots were evaporated to dryness under a gentle nitrogen steam at 40 • C, reconstituted to 500 µL with the extraction solvent, and filtered through a 0.22 µm PTFE syringe filter for UPLC-MS/MS analysis.

Method Validation
Method validation was carried out based on the regulation guidelines of the European Commission [21] in terms of the determination of selectively, linearity, LOD, LOQ, precision (intra and inter-day variability), and accuracy. Linearity was evaluated through the coefficient of determination (r 2 ) of the calibration curves using the internal standard method. The LOD and LOQ were determined as the concentration of the mycotoxin above a signal-to-noise ratio (S/N) of 3 and 10 times, respectively. Intra-day precision was assessed by carrying out six replicates of the working solution in one day and calculating the RSD of the six values. The inter-day precision was determined by measuring the working solutions on three successive days. To evaluate the stability of the analytes in the blank sample extracts, spiked samples were extracted, and mycotoxin concentrations were determined at 0, 2, 4, 8, 12, 16, and 24 h under the optimized UPLC-MS/MS conditions. The results were expressed as RSD for peak areas of the target mycotoxins. Accuracy was determined by spiking a mixed standard solution into the blank sample at three different levels. The spiked samples were extracted and analyzed by UPLC-MS/MS as described above. The recovery was evaluated using the following formula: Recovery (%) = (C 2 /C 1 ) × 100, where C 1 is the fortification concentration of the mycotoxin and C 2 is the measured concentration of the mycotoxin standard in the blank sample.

Matrix Effect Evaluation
The ME was evaluated by comparing the peak responses of the mycotoxin standards in pure solvent to those in the spiked extract at three levels. The ME was calculated using the following formula: ME (%) = (A 2 /A 1 ) × 100, where A 1 is the average peak area of the mycotoxin standard in the blank solvent (ACN) at a specific concentration, and A 2 is the average peak area of the mycotoxin standard in the blank extract sample at the same concentration. With an acceptable compliance interval between 80% and 120%, the ME can be ignored. Otherwise, when the values were below 80%, it indicated a substantial ion suppression effect, and when the values were above 120%, it indicated a substantial ion enhancement effect [29].


s2fieldsofstudy: Chemistry Biology

text:
Cobalt-catalyzed branched selective hydroallylation of terminal alkynes

Here, we reported a cobalt-hydride-catalyzed Markovnikov-type hydroallylation of terminal alkynes with allylic electrophile to access valuable and branched skipped dienes (1,4-dienes) with good regioselectivity. This operationally simple protocol exhibits excellent functional group tolerance and exceptional substrate scope. The reactions could be carried out in gram-scale with TON (turn over number) up to 1160, and the products could be easily derivatized. The preliminary mechanism of electrophilic allylation of α-selective cobalt alkenyl intermediate was proposed based on deuterium labeling experiment and kinetic studies.

Here, we reported a cobalt-hydride-catalyzed Markovnikov-type hydroallylation of terminal alkynes with allylic electrophile to access valuable and branched skipped dienes (1,4-dienes) with good regioselectivity. This operationally simple protocol exhibits excellent functional group tolerance and exceptional substrate scope. The reactions could be carried out in gram-scale with TON (turn over number) up to 1160, and the products could be easily derivatized. The preliminary mechanism of electrophilic allylation of α-selective cobalt alkenyl intermediate was proposed based on deuterium labeling experiment and kinetic studies.
In this work, we report cobalt-hydride-catalyzed branched selective hydroallylation of terminal alkynes with allylic bromides as electrophiles to access branched terminal skipped dienes with good regioselectivity and excellent functional group tolerance (Fig. 1d).

Reaction optimization
We performed the study by using 4-ethynylanisole 1a as a model substrate, 2-methylallylbromide 2a as an allylic electrophile, and PMHS as a hydride source ( Table 1). The Co(OAc) 2 was used as a catalyst, N-(2-(4,5-dihydrooxazol-2-yl)phenyl)quinoline-2-carboxamide (L1) and LiOtBu were used as the ligand and base, respectively. The reaction was performed in a solution of tetrahydrofuran (THF) at 50°C for 24 h to afford electrophilic hydroallylation product in 42% yield with 83/17 b/l (entry 1). A significant increase in the regioselectivity was observed when a larger gem-dimethyl group was used on the oxazoline moiety, which gave rise to skipped diene in 70% yield with >95/5 rr (ratio of regioselectivity; entries 1-3). However, when the size of the substituent was further increased, the regioselectivity slightly decreased (entries 4-6) which implied that the steric hindrance of the substituents on the oxazoline moiety might affect the yield and regioselectivity. Changing the steric effect on pyridine moiety, the selectivity of the reaction decreased slightly (entries 7-8). Using various hydrosilane, such as PhSiH 3 , (EtO) 3 SiH, and Ph 2 MeSiH led to poor yield and selectivity (entries 9-11). Using Ni(OAc) 2 instead of Co(OAc) 2 , a poor yield of hydroallylation was observed (entry 12). Additionally, Cu(OAc) 2 could not promote this transformation. However, the Sonogashira coupling reaction of allyl bromides with terminal alkynes could be promoted under mild conditions (entry 13) (We should thank one of the reviewers for the suggestion of using nickel or copper catalyst to performing the control experiments.). Using CoBr 2 instead of Co(OAc) 2 , this transformation could process smoothly (entry 14). The model reaction could be completed in 20 min (entry 15). The (L3-H)•CoBr complex reported in our previous studies 46 could also be used as an efficient catalyst (entry 16). The standard conditions were identified as 1.0 mmol of terminal alkyne, 0.50 mmol of allylic electrophile, 5 mol% of (L3-H) •CoBr, 0.75 mmol of LiOtBu, and 0.75 mmol of PMHS in a solution of THF (1 mL) at 50°C.

Substrate scope
Compared to other olefins, 2,4-disubstituted skipped dienes are difficult to obtain through classic Wittig reaction, due to the ketoenol tautomerism of 1,3-diketone. Thus, with the optimized conditions in hand, we mainly examined the substrate scope of 2,4disubstituted skipped dienes (Fig. 2). However, due to the presence of Lewis acid metal catalysts and corresponding bases, the inevitable side reaction of terminal alkynes self-polymerization and dehalogenation of allylic bromides would reduce the yield of allylation products. Respectively, 3b and 3c could be obtained on gram scale. The volatile dienes (3d, 3af) could also be obtained via distillation on gram-scale. Various allylic bromide (2d-2i) could be tolerated in this transformation to deliver skipped diene with moderate yield and excellent regioselectivity. Z−1,2-disubstituted bromide (2j) was also investigated. Interestingly, S N 2′-type skipped alkene (3j) was obtained as a major product in this transformation. The significant regioselectivitity differences between 3 f and 3j might owe to the different E/Z stereoconfigurations and steric hindrance of allyl bromides between 2 f and 2j. Besides allylic bromides, different allylic electrophilic reagents such as allyl iodide and allyl phosphate, could also be tolerated in this transformation with slightly decreased yield and regioselectivity (3b, 3d). Benzyl electrophiles, which exhibit similar properties with allylic reagent, could also be transformed smoothly on gram-scale with excellent regioselectivity 47,48 . The 1,4bis(bromomethyl)benzene (2 l) could also react with terminal alkynes smoothly to obtained the 1,1-disubstituted alkenes with excellent regioselectivity. For broader synthetic interests, a variety of functional groups on phenyl rings were investigated. Methyl, trifluoromethyl, fluorine, bromine, chlorine, protected alcohol, and ester could be well tolerated to afford the skipped dienes (3m-3v) in moderate yields with good to excellent regioselectivity (55-76% yield, 92/8 ->95/5 b/l). The alkynes containing heterocycles, such as pyridine 1w and thiophene 1x, could also be tolerated to deliver branched terminal skipped dienes in 48-50% yield. Conjugated enyne (1 y), silyl alkyne (1z), and cyclopropyl acetylene (1aa) could undergo this hydroallylation reaction smoothly. Simple terminal alkynes (1ab-1ae) were also amenable to this transformation to deliver the corresponding product in 55-67% yields with 91/9 to >95/5 rr. Additionally, terminal alkynes contained in bioactive molecules were investigated. Naproxen, menthol, and geraniol derivative (1ag-1ai) could be employed to deliver corresponding products in 45-69% yield.

Mechanistic studies
To elucidate the C(sp 2 )-C(sp 3 ) bond forming process in this transformation, control experiments were conducted. 3(E)-deuterated allylic bromide 10 was prepared to distinguish substitution at the 1-and 3-positions of the electrophile. This reaction was performed to give the  deuterated skipped dienes 11 in 42% yield with 0.17 D at terminal carbon and 0.69 D at sp 3 carbon (Fig. 4a). This result indicated that substitution occurred through the S N 2′-like process accompanied with the partial S N 2 process via attack of the postulated cobalt species at the 1,3position of the allylic bromides. To elucidate the hydrometallation process, the hydroallylation of deuterium labeling phenylacetylene 12 was performed in 30 min to give the deuterated skipped alkene in 59% NMR yield with 0.40 D at C(sp 2 ) 1(E)-position and 0.40 D at C(sp 2 ) 1(Z)-position (Fig. 4b). Due to the presence of strong bases, deuterium atoms might loss during this process. This result combined with our previous mechanistic studies indicated that the E/Z ratio of deuterated product might owe to the relatively fast Crabtree-Ojima-type isomerization 42,46 . The reaction could undergo smoothly in the presence of 1,1-diphenylethylene or butylated hydroxytoluene (BHT), which might rule out the radical reaction pathway (Fig. 4c). The hydroallylation product (3 g, h, j, k) showed that the configuration of    the allylic bromides would affect the S N 2 and S N 2′ selectivity of this transformation. Additionally, the π-allyl pathway could not be exclusively ruled out. Quantitative kinetic studies were also performed to determine the roles of alkyne, allylic bromide, hydrosilane, and (L3-H)•CoBr complex. Kinetic studies on alkyne showed that with a zero-order rate dependence on alkyne, however, as the concentrations of alkyne increased, the initial rates (k in ) of the reaction decreased (Fig. 5a). This result demonstrated excessive alkyne might be a ligand to coordinate with cobalt catalyst leading to the reduction of the rate of reaction. Measurements of the initial rates (k in ) of the reaction with different concentrations of allylic bromide and (L3-H)•CoBr complex showed a corresponding rise in the rates of the reactions. Plots of k in versus the concentrations of allylic bromide and (L3-H)•CoBr complex (Fig. 5b, c) gave two linear curves (slope = 1.19 × 10 −4 Ms −1 ; 6.79 × 10 −3 Ms −1 ), which suggested a first-order rate dependence on allylic bromide and (L3-H) •CoBr complex. Similar kinetic studies on PMHS showed no change in k in within a certain concentrations range (Fig. 5d), indicating a zeroorder rate dependence on hydrosilane. These quantitative kinetic studies suggests that the nucleophilic substitution of cobalt(II) alkenyl intermediate with allylic bromide could be the turnover-limiting step.
Based on the experimental studies and previously reported literatures 35,42,46,[57][58][59] , a possible mechanism is shown in Fig. 6. The cobalt hydride species C was obtained from the reaction of active intermediate (L3-H)•CoBr with LiOtBu and hydrosilane. The alkyne coordination with species C followed by the insertion of terminal alkyne into the cobalt hydride bond delivering majorly α-selective cobalt-alkenyl intermediate E. The quick isomerization balance between E and cobalt carbene zwitterion F led to the E/Z ratio variation based on the deuterium labeling experiment. The following S N 2′ and S N 2-like process of E with allylic electrophile generates species to deliver the corresponding skipped dienes. The hydrosilane and alkoxide might be likely responsible for the observed regioselectivity increase during the catalysis process.

Discussion
In summary, we reported an efficient cobalt-hydride catalyzed branched selective electrophilic hydroallylation of terminal alkynes with allylic electrophile to access terminal skipped dienes with good regioselectivity and functional group tolerance under mild conditions. The reaction could be carried out on gram scale, and the TON was up to 1160. The primary mechanism of electrophilic allylation of α-selective cobalt alkenyl intermediate was proposed based on deuterium labeling experiment and kinetic studies. Various metal-hydridecatalyzed regioselective hydrofunctionalization of terminal alkynes will be further explored in our laboratory.

Materials
For NMR spectra of compounds in this manuscript, see Supplementary  General procedure for hydroallylation of terminal alkynes A 25 mL Schlenk flask equipped with a magnetic stirrer and a flanging rubber plug was dried with flame under vacuum. When cooled to ambient temperature, it was vacuumed and flushed with N 2 . This degassed procedure was repeated for three times. Then (L3-H)•CoBr (0.025 mmol, 5 mol%), THF (1.0 mL, 0.5 M), PMHS (0.75 mmol, 1.5 equiv.), terminal alkynes (1.0 mmol, 2 equiv.), allylic bromides (0.5 mmol, 1.0 equiv.), and LiOtBu (0.75 mmol, 1.5 equiv.) were added sequentially. The reaction was run at 50°C for 30 min to 4 h. Then the resulting solution was quenched with 10 mL of PE and filtered through a pad of silica gel, washed with PE/ EtOAc (5/1) (3 × 20 mL). The combined filtrate was concentrated under vacuum and the ratio of b/l was monitored by 1 H NMR analysis. The mixture was purified by flash column chromatography to give the corresponding product. Article https://doi.org/10.1038/s41467-022-32291-3


Catalytic asymmetric synthesis of carbocyclic C-nucleosides

Access to carbocyclic C-nucleosides (CC-Ns) is currently restricted. The few methods available to make CC-Ns suffer from long syntheses and poor modularity, hindering the examination of potentially important chemical space. Here we report an approach to CC-Ns which uses an asymmetric Suzuki-Miyaura type reaction as the key C-C bond forming step. After coupling the densely functionalized racemic bicyclic allyl chloride and heterocyclic boronic acids, the trisubstituted cyclopentenyl core is elaborated to RNA analogues via a hydroborylation-homologation-oxidation sequence. We demonstrate that the approach can be used to produce a variety of enantiomerically enriched CC-Ns, including a carbocyclic derivative of Showdomycin.

N ucleosides and their analogs are widely studied antiviral and anticancer agents [1][2][3] . Carbocyclic nucleoside analogs show a broad spectrum of antiviral activity and are known to exhibit enhanced flexibility, lipophilicity and metabolic stability [4][5][6] . For instance, Entecavir is clinically used to treat hepatitis B; 7,8 Aristeromycin and Abacavir are active against HIV 9,10 . Additionally, nucleoside analog Galidesivir is a potent inhibitor of Ebola and Zika viruses 11,12 . There has been much recent interest in using nucleoside in the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2); Remdesivir 13,14 and Molnupiravir 15,16 demonstrate activity against SARS-COV-2 and have been approved for high-risk patients with severe symptoms (Fig. 1).
The synthesis and bioactivities of N-nucleosides, C-nucleosides, and carbocyclic N-nucleosides ( Fig. 2A) are extensively studied [17][18][19][20][21] . Nucleophilic addition to carbohydrate-derived oxocarbeniums is widely used to access N-and C-nucleosides 20 . Strategies for the synthesis of carbocyclic nucleosides revolve around construction of the carbocycle and the mode of nucleobase addition 18 , for example Pd-catalyzed allylic amination followed by late-stage construction of the nucleobase allows access to carbocyclic N-nucleoside derivatives 19 .
Carbocyclic C-nucleosides (CC-Ns) are rare, and this is almost certainly due to difficulties in their synthesis ( Fig. 2A) [22][23][24][25] . Few methods for the synthesis of CC-Ns are known, and these methods tend to be long and non-modular. For instance, the synthesis of a potential Alzheimer's disease drug by Merck is reported in 17 steps from D-Ribose (Fig. 2B) 23 . Here, we report a cross-coupling approach to CC-Ns which uses an asymmetric Suzuki-Miyaura type reaction as the key C-C bond forming step followed by hydroborylation-homologation-oxidation strategy to access the RNA analogs.

Strategy
Using a cross-coupling strategy to solve the CC-N synthesis problem may be ideal as it could provide a general and modular route to access many derivatives. With the development of a catalytic asymmetric cross-coupling strategy in mind, we envisaged that coupling heteroaryl boronic acids 2 with a suitably functionalized halide such as racemic 1 could provide enantioenriched 3. Allyl halide 1 is racemic, but is pseudo-symmetrical about the allyl halide unit, which would allow a successful desymmetrizing reaction to set the absolute and relative stereochemistry of the three contiguous stereocenters on the cyclopentene core while simultaneously forming the key C-C bond. These cyclopentenes may be suitable precursors to CC-Ns (Fig. 2C) if modification of 3 by alkene functionalization could produce RNA analogs.
While we felt that relatively simple boronic acids (Fig. 3A) may be suitable for the asymmetric cross-coupling strategy to CC-Ns, the use of these coupling partners would give relatively simple (although almost unknown) products.
Accessing more typical nucleobases would require using functionalized heterocyclic boronic acids (Fig. 3B) that are significantly more complex than have been used in comparable asymmetric transformations. At the outset of this project it was not at all clear from the literature if appropriately complex boronic acid derivatives would undergo SCM reactions, what impact they would have on enantioselectivity over using benzenederived boronic acids, or if they would even be stable 32,33 . The incompatibility of complex heterocycles with transition-metal catalyzed reactions (particularly asymmetric transformations) remains a major challenge for a number of reasons, including catalyst poisoning, undesired reactivity patterns, and protodemetallation (or deborylation) [34][35][36][37][38][39] . The use of complex boronic acids early in the sequence would also require that the installed nucleobase units were compatible with the chemistry used to convert the cyclopentene core 3 to CC-Ns.
Another strategy to potentially access CC-Ns involves using relatively less complex boronic acids (Fig. 3C) which are designed so that late-stage modifications would reveal or allow construction of heterobase units. While this approach involves more steps than Strategy B, it has significant advantages in terms of how easy it would be to perform each step in the sequence. This would likely make Strategy C more suitable for the production of libraries of related compounds, as having to heavily optimize each individual reaction for each target molecule would be unwelcome. Despite this approach's relative length, it would still compare favorably to known approaches to CC-Ns involving long, nonmodular sequences starting from the chiral pool (c.f. Fig. 2B).

Results and discussion
We first targeted relatively simple CC-Ns derived from the addition of six-membered rings; benzenes, pyridines and pyrimidines. Previous reports of asymmetric SMC conditions, extensively explored variables such as solvent and ligand 28 , and using these conditions addition of PhB(OH) 2 to racemic bicycle 1 gave 3a as a single diastereoisomer in 91% yield with very high enantioselectivity (95% ee, Table 1, Entry 1). The absolute configuration was assigned by analogy to previous work 28 . The efficiency and selectivity of this reaction, combined with previous observations that a wide variety of benzene-derived nucleophiles are generally well tolerated in related reactions, encouraged us not to dwell on simple all carbon nucleophiles and to instead explore nitrogen containing six-membered rings.
When addition of pyridines and pyrimidines was attempted no desired product was observed (Table 1, entries 2-4). We chose to next look at addition of 2-halo-pyridyl boronic acids, which have two advantages over simple pyridines, (i) that the presence of the halogen at the 2-position moderates the Lewis-basicity of the heterocycle, and (ii) that the halogen can either be used as a handle in further functionalization reactions or simply removed to reveal the parent pyridine 32,34-39 . Addition of 2-chloro-5pyridyl boronic acid 2e to allyl chloride 1 provided~60% conversion to the desired product, and 3b was isolated in 50% yield and 95% ee ( Table 1, Entry 5). The reaction could be improved by adding water as co-solvent which led to the full consumption of starting material and 3b could be isolated as a single diastereoisomer in 70% yield and 94% ee (   Reaction scope A variety of 2-halo-pyridyl boronic acid isomers were then used to give products 3b-g as shown in Fig. 4. The reaction was conducted at 50°C for 2-halo-pyridyl-4-boronic acids, and 3d and 3e were isolated in 85% yield and 90% ee, and 72% yield and 93% ee respectively. 2-chloro-pyridyl-6-boronic acid provided 3 f in 80% yield and 96% ee. Ortho-substitution of these pyridines such as 2-chloro-pyridyl-3-boronic acid, 2,6difluoropyridyl-3-boronic acid and 2,6-difluoropyridyl-3boronic acid gave very poor results, but we did find we could use 2-fluoro-pyridyl-3-boronic acid to obtain 3 g in 32% yield and 85% ee. The use of 5-membered O-and N-containing heterocyclic nucleophiles was then explored, with 2-and 3-furan boronic acids providing 3 h (80%, 97% ee) and 3 l (81%, 97% ee). Pyrrole derived boronic acids similarly undergo highly efficient reactions to give highly enantioenriched (>94% ee) products, but 3j and 3k are a bit sensitive to decomposition and were isolated in 55% and 65% yield, respectively.
Encouraged by these results, we decided to explore more heavily functionalized 5-and 6-membered heterocycles. In the few examples we examined, di-halo-substituted pyridines performed poorly in reactions at 60°C, we are unsure why but by simply doing the reaction at room temperature 3l and 3m were found to give a single diastereoisomer of product in >80% yield, and reasonable levels of enantioselectivity (80% ee).
A 2-chloro-aminopyridine derived boronic acid, which features both hydrogen-bond donating and accepting moieties, was also used and gave 3n in 79% yield (80% ee). Similarly, we were able to add 2-cyano pyrrol-5-boronic acid to give 3o (48%, 94% ee). We note that this 2-cyano pyrrole moiety is important because it is used in the late-stage construction of the pyrrolo-triazine nucleobase found in Remdesivir, and nucleobase which is otherwise difficult to make (Fig. 4) 40 .
Boronic acids featuring more than one heteroatom are a further step away from benzene rings to more elaborate cross coupling partners that are unexplored in asymmetric catalysis and have less well understood reactivity patterns in the context of asymmetric cross-coupling reactions; 2-chloro-pyrimidine-5boronic acid was successfully employed and 3p was isolated in 70% yield (94% ee).
The ability to run reactions on scale is tremendously important to any multi-step synthetic sequence and so we investigated the synthesis of 3h and 3k on a 5 mmol scale (~1 g). We found these could repeatedly make these products with consistent yields and enantioselectivities, and so these reactionswith challenging nucleophilesshould allow them to be used in the synthesis of complex products such as CC-Ns.

Synthesis of carbocyclic C-nucleosides
In order to produce carbocyclic ribose analogs from 3 a 5'-hydroxymethyl group must be added to the 4' carbon in the ring (ribose numbering). From the carbocyclic alkene in 3 both the regiochemistry (attachment at the 4' position) and stereochemistry (cis or trans relative to the nucleobase) must be controlled with the cis or β-stereochemistry desired as it mimics the stereochemistry most often found in natural nucleosides. After considerable exploratory work, including examining metal catalyzed direct carbonylation and carboxylation, hydrozirconation followed by trapping and photochemical approaches, we were able to identify a useful 3-step hydroborylation-homologationoxidation sequence in order to add the hydroxymethyl group with complete stereo-and regio-chemical control in roughly 50% yield (Fig. 5) however it does involve the use of fairly reactive species and so it would not be expected to be compatible with all functional groups.
The homologation first uses Wilkinson's catalyst (2.5 mol%) with pinacol borane to give boronic ester 4 as a single isomer. The presence of cesium carbonate is crucial for the suppression of competitive and undesired alkene reduction. Matteson homologation using n-BuLi and CH 2 ICl was then used to convert the secondary boronic ester 4 to primary ester 5, and subsequent hydrogen peroxide oxidation furnished alcohol 6 as a key CC-N precursor (Fig. 5). The route successfully furnished phenyl (6a), fluoropyridines (6c, 6e, 6l), furan (6h) and pyrrole (6j and 6k) derivatives in high yields. However, most chloropyridines (except 6b) and the cyano-pyrrole decomposed during attempted hydroborylation. The hydroborylation-homologation-oxidation sequence was successfully performed on 4 mmol scales to give 6h and 6k.
By performing a final acetonide deprotection under standard acidic conditions (Fig. 6), a series of phenyl, pyridine and furan derived CC-Ns (7a-7c, 7e, 7h, and 7k) was isolated in high yields. Encouraged by the success in obtaining a collection of CC-Ns bearing simple nucleobase moieties we became interested in the problem of how to make more elaborate CC-Ns. We started preparing some complex heterocyclic boronic acids and exploring their synthesis and compatibility with the SMC reaction, (for example see SI; See supplemantry data for (a) Attepmts toward complex boronic acids. (b) Detailed synthetic schemes and procedures) Overall, we found that a wide selection of what we imagined to be suitably complex boronic acids (and corresponding esters) were difficult to prepare and not very stable.
In the absence of a specific target molecule that would provide the motivation to overcome stability problems we decided to focus our efforts on nucleobase 9, featured in Remdesivir (Fig. 1). These pyrrolo-triazines contain 4 nitrogen atoms, and were additionally attractive because boronic ester 9d had been reported and seemed easy to prepare 25 . Our first attempts at making suitable boronic acid derivatives were based on the above success with 2-halo-substitution, and we chose to examine the synthesis of 9a and 9b. While a halogen-lithium exchange approach was unsuccessful, presumably due to incompatibility with nBuLi, we were able to make boronic esters using Ir-catalyzed C-H borylation [41][42][43] . However, borylation to give 9a-c was complicated because mixture of regioisomers were obtained which proved difficult to separate. Eventually, we found that 9d and 9e could be prepared as single isomers and these were examined in the asymmetric cross-coupling reaction. Repeated attempts to add 9d gave poor conversion, however when using 9e (Fig. 7) we obtained 3q in 40% yield and 93% ee when doubling the normal catalyst loading.
The pyrrolo-triazine carbocycle 3q underwent hydroborylation to 4q, but attempts to homologate this boronic ester to add the hydroxymethyl group were unsuccessful and gave decomposition products, almost certainly due to incompatibility of Matteson homologation using n-BuLi and CH 2 ICl with the rich array of nitrogen atoms in 4q. We were able to access carbocyclic triol 10 however by oxidation followed by trifloroacetic acid mediated global deprotection to give the TFA salt in 43% yield over 3 steps (Fig. 7).
Overall, the synthesis of complex boronic acids featuring nucleobases was not only difficult, but they were more often than not either inherently unstable or incompatible with the relatively mild conditions of the asymmetric SMC reaction. Most of the complex boronic acids led to protodeborylation and it was difficult to see any obvious correlation between heterocycle structure and boronic acid stability/reactivity.
As the approach (Fig. 3: Strategy 2) of adding highly complex boronic acids proved to be difficult and would likely require extensive optimization in cases where it was viable, and the addition of the 5' hydroxyl methyl group also appeared challenging in the presence of complex heterocycles we decided to pursue the alternative approach (Fig. 3: Strategy 3) of adding relatively simple boronic acids which could later be transformed into more elaborate units.
We envisaged that the oxidative cleavage of furan could provide carboxylic acid moiety primed for the construction of complex heterocycles. Alcohol 6h was first TBS-protected and then oxidative cleavage of furan moiety was accomplished by the combination of ruthenium chloride (10 mol%) and sodium periodate. The resulting carboxylic acid 12 was isolated in 65% yield over 2 steps (Fig. 8). As a simple proof of concept of this approach we chose to make the benzoimidazole derived carbocyclic C-nucleoside 13 which was accomplished via in situ generation of the corresponding acid chloride, and addition of 1,2diaminobenzene to form the amide. Acetic acid mediated cyclization and global deprotection gave CC-N 11 in 52% isolated yield from 12 (Fig. 8). We note that carboxylic acids can readily be transformed into a whole range of structures and so it seems very likely that intermediate 12 44 could also be used to access many other complex CC-Ns.  Showdomycin is a C-nucleoside with antiviral, antibacterial and antitumor properties 22,45,46 . We chose to produce a carbocyclic analog of Showdomycin in order to showcase our approach to enantiomerically enriched CC-N derivatives of natural products with important biological activity (Fig. 9). After hydroborylation-homologation-oxidation sequence of 3k (94% ee) to 6k (Fig. 5, 62% yield over three steps), the alcohol was subjected to TBS protection followed by pyrrole Boc-deprotection to give 14 (65% yield over 2 steps). Pyridinium chlorochromate (PCC) oxidation of the pyrrole group followed by deprotection with trifluoroacetic acid gave carbocyclic Showdomycin analog 16 in 53% isolated yield over 2 steps (Fig. 9).

Conclusions
We present new synthetic strategies for the synthesis of carbocyclic C-nucleosides. The approaches use a key asymmetric Suzuki-Miyaura-type coupling reaction followed by late-stage addition of the hydroxymethyl group to give ribose analogs. Using relatively simple boronic acids nucleophiles Strategy A provides a route to simple CC-N's featuring benzene derivatives or heterocycles featuring a single heteroatom in the 'nucleobase' moiety. We identified two strategies (B and C, Fig. 3) which could produce more complex CC-Ns.
While strategy B would provide direct access to complex CC-N's by using complex boronic acid derivatives, these nucleophiles  are difficult to prepare, and if they are compatible with the sequence then it seems very likely that optimization of many of the individual steps would be required to prepare each substrate (Fig. 7).
We have demonstrated that strategy C is capable of producing CC-Ns featuring more complex nucleobases including the carbocyclic analog of Showdomycin, a biologically active natural product. Strategy C involves the addition of relatively less complex boronic acids followed by late-stage modification, and therefore requires more steps than strategy B but we would recommend this approach to anyone seeking to produce more complex CC-N's in the future (although there may of course be shorter alternative routes that can be devised to access a particular target); carboxylic acid 12 can obviously be converted into a wide range of structures and there are many relatively simple heterocycles available which would be expected to be compatible with our sequence and could very likely be converted into complex CC-N targets.

Methods
General procedure for enantioselective Suzuki-Miyaura coupling. [Rh(COD) OH] 2 (4.6 mg, 0.010 mmol, 2.5 mol%) and (S)-Segphos (14.6 mg, 0.026 mmol, 6.0 mol%) were added to a 7 mL dram vial, sealed with a rubber septum under an argon atmosphere, dissolved in THF (0.80 mL) and stirred at 60°C. After 30 min, a solution (or suspension) of boronic acid (0.80 mmol, 2.0 equiv) and allylic chloride (62 µL, 0.40 mmol, 1.0 equiv) in THF (0.8 mL) and H 2 O (0.2 mL) was added via syringe and the flask was rinsed with THF (0.4 mL). Lastly, CsOH (50 wt% aq. solution, 70 µL, 0.40 mmol, 1.00 equiv) was added and the resulting mixture was then stirred at indicted temperature for the period of time indicated. The mixture was then cooled to room temperature and diluted with Et 2 O (2 mL) before passing through a plug of SiO 2 . The plug was washed with an additional 10 mL of Et 2 O and the solvents were removed in vacuo. Purification by flash chromatography afforded the desired product 3.
General procedure for synthesis of 7a-l. [Rh(PPh 3 ) 3 Cl] (23.1 mg, 0.025 mmol, 2.5 mol%) and Cs 2 CO 3 (325.8 mg, 1.0 mmol, 1 equiv) were added to a flame dried 25 mL round bottom flask, sealed with a rubber septum under an argon atmosphere, dissolved in THF (0.5 mL) and stirred at room temperature. After 5 min, a solution (or suspension) of 3 (1 mmol, 1.0 equiv) in THF (3 mL) was added via syringe and the flask was rinsed with THF (0.5 mL). Pinacolborane (0.29 mL, 2 mmol, 2.0 equiv) was added dropwise to the reaction mixture at 50°C and stirred for 16 h at 50°C. The mixture was then cooled to room temperature and diluted with Et 2 O (5 mL) before passing through a plug of silica. The plug was washed with an additional 10 mL of Et 2 O and the solvents were removed in vacuo. The crude product boronic acid pinacol ester 4 was charged to the next step without further purification.
In a flamed dried flask under nitrogen, n-butyllithium (2.5 M in hexanes, 0.75 mL, 1.88 mmol, 2.5 equiv) was added dropwise to a solution of boronic acid pinacol ester 4 obtained above (0.75 mmol, 1.0 equiv) and iodochloromethane (165 µL, 2.25 mmol, 3 equiv) in THF (3.5 mL) at −78°C. The reaction mixture was slowly allowed to warm to ambient temperature over 12 h. The mixture was diluted with Et 2 O (5 mL) and the organic layer was washed with an aq. sat. solution of NH 4 Cl (2 × 3 mL) and dried over Na 2 SO 4 , filtered, and the solvent was removed in vacuo. The crude product boronic acid pinacol ester 5 was charged to the next step.
30%wt. H 2 O 2 (1 mL) was added to a solution of boronic acid pinacol ester 5 obtained above (0.75 mmol, 1.0 equiv) and 2 N aq. NaOH (1.5 ml) in THF (4 mL) at 0°C. The reaction mixture was slowly allowed to warm to ambient temperature. After 3 h, the mixture was diluted with Et 2 O (7 mL). The organic layer was separated and the aqueous layer was extracted with Et 2 O (2 × 5 mL). The combined organic layers were dried over anhydrous Na 2 SO 4 , filtered, and the solvent was removed in vacuo. Purification by flash chromatography afforded alcohol 6.
A solution of alcohol 6 (0.2 mmol) in AcOH (0.5 mL) and H 2 O (0.5 mL) was stirred at 50°C for 16 h. The reaction mixture was concentrated under reduced pressure and purification by flash chromatography afforded compound 7.
Data availability