Biocatalytic Regioselective O-acylation of Sesquiterpene Lactones from Chicory: A Pathway to Novel Ester Derivatives.

We report the first biocatalytic modification of sesquiterpene lactones (STLs) found in the chicory plants, specifically lactucin (Lc), 11ß,13-dihydrolactucin (DHLc), lactucopicrin (Lp), and 11ß,13-dihydrolactucopicrin (DHLp). The selective O-acylation of their primary alcohol group was carried out by the lipase B from Candida antarctica (CAL-B) using various aliphatic vinyl esters as acyl donors. Perillyl alcohol, a simpler monoterpenoid, served as a model to set up the desired O-acetylation reaction by comparing the use of acetic acid and vinyl acetate as acyl donors. Similar conditions were then applied to DHLc, where five novel ester chains were selectively introduced onto the primary alcohol group, with conversions going from >99 % (acetate and propionate) to 69 % (octanoate). The synthesis of the corresponding O-acetyl esters of Lc, Lp, and DHLp was also successfully achieved with near-quantitative conversion. Molecular docking simulations were then performed to elucidate the preferred enzyme-substrate binding modes in the acylation reactions with STLs, as well as to understand their interactions with crucial amino acid residues at the active site. Our methodology enables the selective O-acylation of the primary alcohol group in four different STLs, offering possibilities for synthesizing novel derivatives with significant potential applications in pharmaceuticals or as biocontrol agents.

Metabolic transitions regulate global protein fatty acylation.

Intermediary metabolites and flux through various pathways have emerged as key determinants of post-translational modifications. Independently, dynamic fluctuations in their concentrations are known to drive cellular energetics in a bi-directional manner. Notably, intracellular fatty acid pools that drastically change during fed and fasted states act as precursors for both ATP production and fatty acylation of proteins. Protein fatty acylation is well regarded for its role in regulating structure and functions of diverse proteins; however, the effect of intracellular concentrations of fatty acids on protein modification is less understood. In this regard, we unequivocally demonstrate that metabolic contexts, viz. fed and fasted states, dictate the extent of global fatty acylation. Moreover, we show that presence or absence of glucose that influences cellular and mitochondrial uptake/utilization of fatty acids and affects palmitoylation and oleoylation, which is consistent with their intracellular abundance in fed and fasted states. Employing complementary approaches including click-chemistry, lipidomics, and imaging, we show the top-down control of cellular metabolic state. Importantly, our results establish the crucial role of mitochondria and retrograde signaling components like SIRT4, AMPK, and mTOR in orchestrating protein fatty acylation at a whole cell level. Specifically, pharmacogenetic perturbations that alter either mitochondrial functions and/or retrograde signaling affect protein fatty acylation. Besides illustrating the cross-talk between carbohydrate and lipid metabolism in mediating bulk post-translational modification, our findings also highlight the involvement of mitochondrial energetics.

Enzymatic Acylation of Flavonoids from Bamboo Leaves: Improved Lipophilicity and Antioxidant Activity for Oil-Based Foods.

The goal of this study was to expand the applications of bamboo leaf flavonoids (BLFs) by improving their lipophilicity through enzymatic acylation with vinyl cinnamate. Characterization of the acylated BLFs using Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, high-resolution electrospray ionization mass spectrometry, electrospray ionization with tandem mass spectrometry, and 1H nuclear magnetic resonance spectroscopy indicated that acylation occurred at the C6-OH position of glucoside moieties. The highest degree of acylation (18.61%) was obtained by reacting BLFs with vinyl cinnamate (1:5, w/w) at 60 °C for 48 h. Acylation significantly improved the lipophilicity of BLFs and their capacity to inhibit lipid peroxidation, as evidenced by the reduced production of lipid hydroperoxides and malondialdehyde in rapeseed oil and rapeseed oil-in-water emulsions during storage at 37 °C for 15 days. The study findings provide important data that will enable the use of BLFs in lipid or lipophilic matrices, such as oil-based foods.

Unique acyl chains mark phosphoinositides for recycling.

Two recent complementary studies show that, after phospholipase C cleavage, the characteristic acyl chain composition of phosphoinositide-derived diacylglycerol funnels them back into the PI cycle.

Metabolic stimulation-elicited transcriptional responses and biosynthesis of acylated triterpenoids precursors in the medicinal plant Helicteres angustifolia.

BACKGROUND: Helicteres angustifolia has long been used in Chinese traditional medicine. It has multiple pharmacological benefits, including anti-inflammatory, anti-viral and anti-tumor effects. Its main active chemicals include betulinic acid, oleanolic acid, helicteric acid, helicterilic acid, and other triterpenoid saponins. It is worth noting that some acylated triterpenoids, such as helicteric acid and helicterilic acid, are characteristic components of Helicteres and are relatively rare among other plants. However, reliance on natural plants as the only sources of these is not enough to meet the market requirement. Therefore, the engineering of its metabolic pathway is of high research value for enhancing the production of secondary metabolites. Unfortunately, there are few studies on the biosynthetic pathways of triterpenoids in H. angustifolia, hindering its further investigation. RESULTS: Here, the RNAs of different groups treated by metabolic stimulation were sequenced with an Illumina high-throughput sequencing platform, resulting in 121 gigabases of data. A total of 424,824 unigenes were obtained after the trimming and assembly of the raw data, and 22,430 unigenes were determined to be differentially expressed. In addition, three oxidosqualene cyclases (OSCs) and four Cytochrome P450 (CYP450s) were screened, of which one OSC (HaOSC1) and one CYP450 (HaCYPi3) achieved functional verification, suggesting that they could catalyze the production of lupeol and oleanolic acid, respectively. CONCLUSION: In general, the transcriptomic data of H. angustifolia was first reported and analyzed to study functional genes. Three OSCs, four CYP450s and three acyltransferases were screened out as candidate genes to perform further functional verification, which demonstrated that HaOSC1 and HaCYPi3 encode for lupeol synthase and ß-amyrin oxidase, which produce corresponding products of lupeol and oleanolic acid, respectively. Their successful identification revealed pivotal steps in the biosynthesis of acylated triterpenoids precursors, which laid a foundation for further study on acylated triterpenoids. Overall, these results shed light on the regulation of acylated triterpenoids biosynthesis.

Preparation of functionalized pectin through acylation with alkyl gallates: Experiments coupled with density functional theory.

The covalent grafting of alkyl gallates onto pectin using a lipase-catalyzed reaction in a tetrahydrofuran/aqueous medium process acylated pectin molecules with excellent antioxidant and antibacterial properties. The alkyl gallates including methyl, ethyl, and propyl gallates were enzymatically grafted onto pectin molecule, in order to study the effect of alkyl gallates on the functional modification of pectin. The grafting mechanism was analyzed by ultraviolet-visible spectrum (UV-Vis), Fourier transform infrared spectrum (FTIR), proton nuclear magnetic resonance (1HNMR), and density functional theory (DFT). Results suggested that lipase grafted 4-OH of alkyl gallate onto pectin by catalyzing esterification in organic/aqueous solution, and the grafting rate was affected by the length of alkyl chain of the gallates molecule. In vitro experiments, the acylated pectins exhibited stronger antioxidant activity in the DPPH test and ß-carotene bleaching test and were found to have obvious antimicrobial performance against Escherichia coli and Staphylococcus aureus.

Characterization of Glucuronosyl-diacyl/monoacylglycerols and Discovery of Their Acylated Derivatives in Tomato Lipid Extracts by Reversed-Phase Liquid Chromatography with Electrospray Ionization and Tandem Mass Spectrometry.

Glucuronic acid containing diacylglycerols (3-(O-α-d-glucuronopyranosyl)-1,2-diacyl-sn-glycerols, GlcA-DAG) are glycolipids of plant membranes especially formed under phosphate-depletion conditions. An analytical approach for the structural characterization of GlcA-DAG in red ripe tomato (Solanum lycopersicum L.) extracts, based on reversed-phase liquid chromatography (RPLC) coupled with electrospray ionization (ESI) and tandem mass spectrometry (MS/MS) using a linear ion trap, is described in this paper. At least 14 GlcA-DAG (R1/R2) species, including four regioisomers, containing three predominant fatty acyl chains C16:0, C18:2, and C18:3, were identified for the first time. Moreover, 29 GlcA-DAG acylated on the glucuronosyl ring (acyl-R3 GlcA-DAG) were discovered, alongside 15 acylated lyso-forms, i.e., acylated 3-(O-α-d-glucuronosyl)monoacylglycerols, abbreviated as acyl-R3 GlcA-MAG (R1/0) or (0/R2). Although many of these acylated lyso-forms were isomeric with GlcA-DAG (i.e., acyl chains with equivalent sum composition), they were successfully separated by reversed-phase liquid chromatography (RPLC) using a solid-core C18 column packed with 2.6 µm particle size. Tandem MS (and eventually MS3) data obtained from sodium adducts ([M + Na]+) and deprotonated molecules ([M - H]-) were fundamental to detect diagnostic product ions related to the glucuronosyl ring and then determine the identity of all investigated glycolipids, especially to recognize the acyl chain linked to the ring. A classification of GlcA-MAG, GlcA-DAG, and acylated GlcA-DAG and GlcA-MAG was generated by an in house-built database. The discovery of acylated derivatives emphasized the already surprising heterogeneity of glucuronic acid-containing mono- and diacylglycerols in tomato plants, stimulating interesting questions on the role played by these glycolipids.

Recent Research Progress in Taxol Biosynthetic Pathway and Acylation Reactions Mediated by Taxus Acyltransferases.

Taxol is one of the most effective anticancer drugs in the world that is widely used in the treatments of breast, lung and ovarian cancer. The elucidation of the taxol biosynthetic pathway is the key to solve the problem of taxol supply. So far, the taxol biosynthetic pathway has been reported to require an estimated 20 steps of enzymatic reactions, and sixteen enzymes involved in the taxol pathway have been well characterized, including a novel taxane-10ß-hydroxylase (T10ßOH) and a newly putative ß-phenylalanyl-CoA ligase (PCL). Moreover, the source and formation of the taxane core and the details of the downstream synthetic pathway have been basically depicted, while the modification of the core taxane skeleton has not been fully reported, mainly concerning the developments from diol intermediates to 2-debenzoyltaxane. The acylation reaction mediated by specialized Taxus BAHD family acyltransferases (ACTs) is recognized as one of the most important steps in the modification of core taxane skeleton that contribute to the increase of taxol yield. Recently, the influence of acylation on the functional and structural diversity of taxanes has also been continuously revealed. This review summarizes the latest research advances of the taxol biosynthetic pathway and systematically discusses the acylation reactions supported by Taxus ACTs. The underlying mechanism could improve the understanding of taxol biosynthesis, and provide a theoretical basis for the mass production of taxol.

Sublethal heat stress-induced O-GlcNAcylation coordinates the Warburg effect to promote hepatocellular carcinoma recurrence and metastasis after thermal ablation.

The malignant transformation of residual hepatocellular carcinoma (HCC) cells after thermal ablation is considered as the main factor promoting postoperative HCC progression, which greatly limits the improvement of long-term survival, and at present there is no effective targeted therapeutic strategies. The Warburg effect is a metabolic feature correlated highly with malignant transformation (e.g. epithelial-to-mesenchymal transition [EMT]). Here, we showed that sublethal heat stress triggered a stronger Warburg effect of HCC cells, which contributed to the thermotolerance and invasion of HCC cells. Sublethal heat stress-induced O-GlcNAcylation was involved in this process. Such enhanced Warburg effect in HCC cells may be eliminated through O-GlcNAcylation inhibition, resulting in impaired thermotolerance and EMT, and thereby preventing tumor recurrence and metastasis of HCC-bearing mice after insufficient thermal ablation. Finally, we present evidence that sublethal heat stress-induced O-GlcNAcylation regulates the Warburg effect in HCC cells by promoting hypoxia-inducible factor 1α (HIF-1α) stability. In conclusion, the present study suggests that O-GlcNAcylation coordinates the Warburg effect to promote HCC progression after thermal ablation, which may serve as a novel potential target for controlling postoperative HCC recurrence and metastasis.

Preparation of acylated blueberry anthocyanins through an enzymatic method in an aqueous/organic phase: effects on their colour stability and pH-response characteristics.

To explore the potential of anthocyanins in pH-colour responsive intelligent packaging and improve the stability of the pigments, 3,4,5-trimethoxybenzoic acid and gallic acid were grafted onto blueberry anthocyanins via enzyme-catalysed grafting. The structural analysis based on UV-vis and IR spectroscopy showed that the two acids were successfully grafted onto the blueberry anthocyanins. The acylation degrees of the 3,4,5-trimethoxybenzoic acid-acylated anthocyanin (Tr-An) and gallic acid-acylated anthocyanin (Ga-An) were 6.38% and 6.51%, respectively. The results from the DPPH radical scavenging assay and ferric reducing antioxidant power assay implied that the antioxidant capacity of Tr-An was worse than that of natural anthocyanin (Na-An), but the antioxidant capacity of Ga-An was stronger than that of Na-An. The grafting of the two acids enhanced the stability of the blueberry anthocyanins and had little effect on the pH-colour response characteristics of the blueberry pigments.

Acylated Anthocyanins from Red Cabbage and Purple Sweet Potato Can Bind Metal Ions and Produce Stable Blue Colors.

Red cabbage (RC) and purple sweet potato (PSP) are naturally rich in acylated cyanidin glycosides that can bind metal ions and develop intramolecular π-stacking interactions between the cyanidin chromophore and the phenolic acyl residues. In this work, a large set of RC and PSP anthocyanins was investigated for its coloring properties in the presence of iron and aluminum ions. Although relatively modest, the structural differences between RC and PSP anthocyanins, i.e., the acylation site at the external glucose of the sophorosyl moiety (C2-OH for RC vs. C6-OH for PSP) and the presence of coordinating acyl groups (caffeoyl) in PSP anthocyanins only, made a large difference in the color expressed by their metal complexes. For instance, the Al3+-induced bathochromic shifts for RC anthocyanins reached ca. 50 nm at pH 6 and pH 7, vs. at best ca. 20 nm for PSP anthocyanins. With Fe2+ (quickly oxidized to Fe3+ in the complexes), the bathochromic shifts for RC anthocyanins were higher, i.e., up to ca. 90 nm at pH 7 and 110 nm at pH 5.7. A kinetic analysis at different metal/ligand molar ratios combined with an investigation by high-resolution mass spectrometry suggested the formation of metal-anthocyanin complexes of 1:1, 1:2, and 1:3 stoichiometries. Contrary to predictions based on steric hindrance, acylation by noncoordinating acyl residues favored metal binding and resulted in complexes having much higher molar absorption coefficients. Moreover, the competition between metal binding and water addition to the free ligands (leading to colorless forms) was less severe, although very dependent on the acylation site(s). Overall, anthocyanins from purple sweet potato, and even more from red cabbage, have a strong potential for development as food colorants expressing red to blue hues depending on pH and metal ion.

Ascorbic acid-induced degradation of liposome-encapsulated acylated and non-acylated anthocyanins of black carrot extract.

BACKGROUND: In the presence of ascorbic acid, the degradation of acylated (sinapic, ferulic and p-coumaric acid derivatives of cyanidin-3-xylosylglucosylgalactoside) and non-acylated anthocyanins of black carrot extract (BCE) encapsulated in liposomes was studied. BCEs (0.2% and 0.4% w/w) were encapsulated in liposomes using different lecithin concentrations (1%, 2% and 4% w/w). RESULTS: The liposomes were prepared with particle diameters of less than 50 nm and zeta potentials of about -21.3 mV for extract-containing liposomes and -27.7 mV for control liposomes. The encapsulation efficiency determined using high-performance liquid chromatography (HPLC) showed that increasing lecithin levels increased the efficiency to 59% at the same extract concentration. The concentrations of total anthocyanins and individual anthocyanins were determined for ascorbic acid (0.1% w/w)-degraded extract and liposomes (containing 0.2% w/w extract). Anthocyanin quantification of both liposomal and extract samples was performed by HPLC using cyanidin-3-O-glucoside chloride as standard. Five anthocyanins in the extract and encapsulated liposomes were quantified during 24 h (0-24 h): cyanidin-3-xylosylglucosylgalactoside 1.0-0.51 and 0.82-0.58 mg g-1 , cyanidin-3-xylosylgalactoside 2.5-1.1 and 2.2-1.7 mg g-1 , cyanidin-3-xylosyl(sinapoylglucosyl)galactoside 0.51-0.14 and 0.35-0.28 mg g-1 , cyanidin-3-xylosyl(feruloylglucosyl)galactoside 1.37-0.41 and 1.06-0.98 mg g-1 , and cyanidin-3-xylosyl(coumaroylglucosyl)galactoside 0.28-0.08 mg g-1 for extract and 0.27-0.26 mg g-1 for liposomes, respectively. CONCLUSIONS: This study demonstrates the potential beneficial effect of liposomal encapsulation on individual, particularly acylated, anthocyanins after addition of ascorbic acid during a storage time of 24 h.

Pectin modified with phenolic acids: Evaluation of their emulsification properties, antioxidation activities, and antibacterial activities.

Three phenolic acids including p-hydroxybenzoic acid (PHBA), 3,4-dihydroxybenzoic acid, (DHBA), and gallic acid (GA) were grafted onto native pectin (Na-Pe) through enzymatic method. Ultraviolet-visible spectrometry, Fourier transform infrared spectroscopy, and 1H NMR analyses were used to explore the reaction mechanism. Results indicated that the p-hydroxyl of the phenolic acids reacted with the methoxycarbonyl of pectin through transesterification, and a covalent connection was formed. The phenolic acid contents of PHBA modified pectin (Ph-Pe), DHBA modified pectin (Dh-Pe), and GA modified pectin (Ga-Pe) were 20.18%, 18.87%, and 20.32%, respectively. After acylation with phenolic acids, the 1,1-diphenyl-2-picryl hydrazine clearance of pectin changed from 7.68% (Na-Pe) to 6.88% (Ph-Pe), 40.80% (Dh-Pe), and 90.30% (Ga-Pe), whereas its inhibition ratio of pectin increased from 3.11% (Na-Pe) to 35.02% (Ph-Pe), 66.36% (Dh-Pe), and 77.89% (Ga-Pe). Moreover, compared with Na-Pe, modified pectins exhibited better emulsification properties and stronger antibacterial activities against both Escherichia coli and Staphylococcus aureus.

Different Reactivity of Raw Starch from Diverse Potato Genotypes.

Potato starch is one of the most important renewable sources for industrial manufacturing of organic compounds. Currently, it is produced from mixed potato varieties that often are harvested from different fields. Meanwhile, tuber starches of various potato breeds differ in their crystallinity, granule morphology, and other physical and chemical parameters. We studied the reactions of raw potato starches of different origins to chemical and biochemical reactions typically used for industrial starch modification. The results clearly demonstrate that there is a significant difference in the reactivity of the starches of different potato genotypes. While the main products of the transformations are the same, their preparative yields differ significantly. Thus, tuber starch of certain potato varieties may be more suitable for specific industrial purposes. Starch reactivity may potentially be a phenotypical trait for potato breeding to obtain potato starches for various industrial applications.

Matrix-assisted laser desorption/ionization mass spectrometry-guided visualization analysis of intestinal absorption of acylated anthocyanins in Sprague-Dawley rats.

It is unknown whether intestinal absorption of acylated anthocyanins occurs in their intact or metabolized form. In this study, with the aid of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging, intestinal absorption of acylated anthocyanins was visually investigated. Anthocyanin extracts from purple carrots were orally administered to Sprague-Dawley rats. Acylated cyanidins were absorbed into portal and circulating blood systems in their intact form, and aglycon; cyanidin 3-O-(6-O-feruloyl-ß-d-glucopyranosyl)-(1 â†’ 6)-[ß-d-xylopyranosyl-(1 â†’ 2)]-ß-d-galactopyranoside (Cy3XFGG), and showed a high absorption of 39.3 ± 0.1 pmol/mL-plasma at 60 min after administration. MALDI-MS imaging analysis of the rat jejunum membranes showed that an organic anion transporting polypeptide (OATP) transporter was involved in Cy3XFGG transport, while deacylated anthocyanins were incorporated through both the glucose transporter 2 and OATP routes. In conclusion, acylated anthocyanin, Cy3XFGG, can be absorbed in its intact form through intestinal OATP.

Disaccharide anthocyanin delphinidin 3-O-sambubioside from Hibiscus sabdariffa L.: Candida antarctica lipase B-catalyzed fatty acid acylation and study of its color properties.

Enzymatic lipophilization is an important process to extend the use of anthocyanins in lipidic media. In this work delphinidin 3-O-sambubioside (Dp3sam) isolated from Hibiscus sabdariffa L. flower was esterified with octanoic acid using Candida antarctica lipase B. The physical-chemical properties of the new lipophilic pigment were studied by UV-vis spectroscopy. Dp3sam with chloride, acetate and formate as counter ions were employed to study the lipophilization reaction. The hydrolysis of the reagent was avoided with a formate counter ion and the expected product was achieved with a noteworthy change of solubility. 1D and 2D NMR characterization of Dp3sam-C8 confirmed that the lipophilization took place at the primary alcohol of the glucoside moiety. Overall, the Dp3sam-C8 ester presents a stabilization of the quinoidal base (blue color) at neutral or moderate alkaline pH, which foresees a potential use of this pigment as a broad kind of industries on lipo-soluble formulations.

Characterization of teaghrelin-like compounds from tea cultivars.

Teaghrelins, identified originally in Chin-shin oolong tea, are unique acylated flavonoid tetraglycosides and proposed to be potential oral analogues of ghrelin. In the present study, two new teaghrelin-like compounds were characterized from tea cultivars (TTES No. 12), and their chemical structures were established by the spectroscopic and spectrometric analysis. However, due to the different location of rhamnose, these two teaghrelin-like compounds may not show significant ghrelin receptor affinity.[Figure: see text].

Preparation of acylated pectin with gallic acid through enzymatic method and their emulsifying properties, antioxidation activities and antibacterial activities.

In this study, native pectin (Na-Pe) was acylated with gallic acid through enzymatic method. UV-Vis, Fourier transform infrared spectroscopy and proton NMR analyses demonstrated that the phenolic hydroxyl group on gallic acid attacked the carbomethoxy of Na-Pe and replaced the methoxy group to form a new ester group under catalysis. The galloyl content of acylated pectin prepared via 24-h reaction (Ac1-Pe) was 16.8%, while that prepared via 48-h reaction (Ac2-Pe) reached 20.7%. The emulsifying properties, antioxidation activities and antibacterial activities of acylated pectin was significantly improved compared with those of Na-Pe. The emulsion activity and emulsion stability of the pectin emulsion improved from 1.08% and 56.13% (Na-Pe) to 1.57% and 88.27% (Ac1-Pe) and 1.71% and 93.3% (Ac2-Pe), respectively. The DPPH clearance of the pectin improved from 2.68% (Na-Pe) to 68.92% (Ac1-Pe) and 76.98% (Ac2-Pe) and the inhibition ratio in the ß-carotene bleaching assay of the pectin increased from 3.15% (Na-Pe) to 73.02% (Ac1-Pe) and 78.96% (Ac2-Pe). The inhibition rate of the pectin against Escherichia coli and Staphylococcus aureus also improved from 2.93% and 8.92% (Na-Pe) to 26.95% and 42.18% (Ac1-Pe) and 31.56% and 47.87% (Ac2-Pe), respectively.

Pennelliisides A-C, 2,3,4-Trisubstituted Acyl Glucoses Isolated from Solanum pennellii.

Solanum species accumulate a variety of secondary metabolites in their trichomes, and it is well known that acyl sugars are specialized metabolites secreted by the trichomes. However, very few reports provide detailed information on the chemical structure of polyacylated glucose derivatives, due to the α and ß isomerization that can occur at the C-1 position. In this study, a strategy was established to isolate polyacylated glucose derivatives. According to the developed strategy, hydroxy groups were derivatized to a benzyloxy group using TriBOT. After isolation of the compounds in pure form and deprotection of the benzyloxy group, the chemical structures of pennelliisides A-C were determined as 2,3,4-O-triisobutyryl-d-glucose, 3-O-(8-methylnonanoyl)-2,4-O-diisobutyryl-d-glucose, and 3-O-decanoyl-2,4-O-diisobutyryl-d-glucose, respectively. Structural elucidation was performed using spectroscopic techniques, including 1D and 2D NMR, FD-MS, and GC-MS. It was also found that the fatty acid moiety contributes to the allelopathic properties of the isolated compounds.

Targeted characterization of acylated compounds from Scrophulariae Radix using liquid chromatography coupled with Orbitrap mass spectrometry and diagnostic product ion-based data analysis.

Acylated compounds are often present in herbal medicines. In this study, a diagnostic product ion-based strategy was established to comprehensively characterize acylated compounds in Scrophulariae Radix. After untargeted data acquisition using ultra-high performance liquid chromatography coupled with Orbitrap mass spectrometry, the data were processed by three-stage diagnostic product ions. First, diagnostic product ions corresponding to the acyl groups (cinnamoyl, p-coumaroyl, feruloyl, and caffeoyl) were used to search 90 compounds. Second, these compounds were divided into three categories using diagnostic product ions for phenylethanoid glycosides, iridoid glycosides, and phenylpropanoids, respectively. Last, the linkage position of the acyl group to iridoid glycosides was discriminated via the third-stage diagnostic product ions. As a result, 90 acylated compounds were characterized, and 37 of them were reported from Scrophulariae Radix for the first time.