Regioselective Hydrolysis Research Articles

Detection of seco-macrotetrolides caused by regiospecific hydrolysis in Indonesian Streptomyces strains isolated at the karst area in Sulawesi Island

Among a total of 30 endophytic Streptomyces strains collected at the karst area in Sulawesi Island, Indonesia, four strains showed antimicrobial activity together with distinct purple-colored spot(s) on TLC after anisaldehyde staining and baking. Four endophytic strains AA004, AA010, AA034, and AA035 accumulated ionophore macrotetrolides (cyclic tetraesters) including nonactin, monactin, dinactin, trinactin, and tetranactin. Occasionally, strains AA034 and AA035 accumulated considerable amount of seco-macrotetrolides (ring-opened, linear macrotetrolides) including seco-dinactin, seco-monactin, and seco-trinactin, together with macrotetrolides as above mentioned. Time-course increase of seco-macrotetrolides indicates the subsequent hydrolysis of macrotetrolides. Seco-macrotetrolides in strain AA034 were separated from macrotetrolides and other metabolites through Sephadex LH20 and silica gel chromatographies, although seco-macrotetrolides contain inseparable five analogs. We here investigated the hydrolyzed positions in seco-macrotetrolides. ESI-CID-MS/MS analysis of seco-dinactin (m/z 805 as [M+Na]+) and its derivatives, methyl ester (m/z 819 as [M+Na]+) and O-acetyl methyl ester (m/z 861 as [M+Na]+), revealed that seco-dinactin obtained in strains AA034/AA035 is a single regioisomer with a cleavage at an ester bond between the alcohol of (−)-homononactate and the carboxylate of (+)-nonactate, suggesting the regioselective hydrolysis on the occurrence of seco-dinactin. Further ESI-CID-MS/MS analysis revealed that seco-monactin (m/z 791 as [M+Na]+) is also single regioisomer with the ester-bond cleavage between the alcohol of (−)-homononactate and the carboxylate of (+)-nonactate. In the case of seco-trinactin (m/z 819 as [M+Na]+), additional regioisomer with the ester-bond cleavage between the alcohol of (−)-homononactate and the carboxylate of (+)-homononactate was detected, together with the regioisomer with the ester-bond cleavage between the alcohol of (−)-homononactate and the carboxylate of (+)-nonactate. A mixture of seco-macrotetrolides exhibited no antimicrobial activity, indicating the importance of macrocyclic structure for antimicrobial activity.

Dinuclear platinum(II) complexes with 1,5-nphe bridging ligand: Spectroscopic and molecular docking study of the interactions with N-acetylated L-methionylglycine and human serum albumin

The hydrolysis of N-acetylated L-methionylglycine dipeptide (Ac-L-Met-Gly) in the presence of dinuclear platinum(II)-aqua complexes with general formula [{Pt(L)(H2O)}2(μ-1,5-nphe)]4+ (1,5-nphe is 1,5-naphthyridine; L is bidentately coordinated ethylenediamine (1w), (±)-1,2-propylenediamine (2w) and 1,3-propylenediamine (3w)) is described in detail. The hydrolytic activity of these complexes was monitored by 1H NMR spectroscopy, which confirmed the regioselective cleavage of the Met-Gly-amide bond in this peptide. Quantum-chemical calculations revealed the primary reaction path with coordination of dipeptide via the terminal amide nitrogen of methionine followed by its coordination for the sulphur atom, in which the decomposition of the resulted platinum(II)-dipeptide complex and the coordination of its glycine residue precedes the hydrolytic cleavage of the Met-Gly amide bond. In the secondary reaction path, in which Ac-L-Met-Gly is coordinated via the methionine sulphur atom, the decomposition of the starting [{Pt(L)(H2O)}2(μ-1,5-nphe)]4+ complex is followed by the regioselective hydrolysis of the investigated dipeptide. The binding affinity of the chloride derivatives of the corresponding dinuclear platinum(II)-aqua complexes, [{Pt(L)Cl}2(μ-1,5-nphe)]2+ (1–3) towards the human serum albumin (HSA), a transport protein, was investigated using electronic absorption and fluorescence emission measurements, and results indicated the static interactions between these complexes and HSA. A docking study revealed a unique binding site on HSA, based on the electrostatic and the hydrogen bonding, which does not have a methionine residue nearby, therefore does not exist danger of HSA hydrolysis by these complexes.

A concise synthesis of l-gulose and its C-6 derivatives

A convenient route for the preparation of l-gulose and its C-6 derivatives starting from commercially available 2,3:5,6-diisopropylidene-d-mannofuranose via C-5 epimerization as the key step was developed. 1-O-Benzylation followed by regioselective hydrolysis of the 5,6-isopropylidene group furnished benzyl 2,3-isopropylidene-α-d-mannofuranoside, which was subjected upon regioselective one-pot 6-O-benzoylation and 5-O-mesylation, providing the corresponding 5-OMs-6-OBz derivative in excellent selectivity. Treatment of this mesylate compound with potassium t-butoxide to remove the benzoyl group followed by intramolecular SN2 inversion led to benzyl 5,6-anhydro-2,3-isopropylidene-β-l-gulofuranoside, which could undergo not only nucleophilic substitutions to open the epoxide ring to give various C-6 derivatives, but also acidic hydrolysis to yield 1,6-anhydro-β-l-gulopyranose for further transformation into l-gulopyranosyl pentaacetate.

Engineering of reaction specificity, enantioselectivity, and catalytic activity of nitrilase for highly efficient synthesis of pregabalin precursor.

Simultaneous evolution of multiple enzyme properties remains challenging in protein engineering. A chimeric nitrilase (BaNITM0 ) with high activity towards isobutylsuccinonitrile (IBSN) was previously constructed for biosynthesis of pregabalin precursor (S)-3-cyano-5-methylhexanoic acid ((S)-CMHA). However, BaNITM0 also catalyzed the hydration of IBSN to produce by-product (S)-3-cyano-5-methylhexanoic amide. To obtain industrial nitrilase with vintage performance, we carried out engineering of BaNITM0 for simultaneous evolution of reaction specificity, enantioselectivity, and catalytic activity. The best variant V82L/M127I/C237S (BaNITM2 ) displayed higher enantioselectivity (E = 515), increased enzyme activity (5.4-fold) and reduced amide formation (from 15.8% to 1.9%) compared with BaNITM0 . Structure analysis and molecular dynamics simulations indicated that mutation M127I and C237S restricted the movement of E66 in the catalytic triad, resulting in decreased amide formation. Mutation V82L was incorporated to induce the reconstruction of the substrate binding region in the enzyme catalytic pocket, engendering the improvement of stereoselectivity. Enantio- and regio-selective hydrolysis of 150 g/L IBSN using 1.5 g/L Escherichia coli cells harboring BaNITM2 as biocatalyst afforded (S)-CMHA with >99.0% ee and 45.9% conversion, which highlighted the robustness of BaNITM2 for efficient manufacturing of pregabalin.

Regioselective Bond-Forming and Hydrolysis Reactions of Doubly Charged Vanadium Oxide Anions in the Gas Phase

The gas-phase reactivity of vanadium-containing dianions, NaV3O92− and its hydrated form H2NaV3O102−, were probed towards sulphur dioxide at room temperature by ion-molecule reaction (IMR) experiments in the collision cell of an ion trap mass spectrometer. The sequential addition of two SO2 molecules to the NaV3O92− dianion leads to the breakage of the stable V3O9 backbone, resulting in a charge separation process with the formation of new V-O and S-O bonds. On the contrary, the H2NaV3O102− hydroxide species reacts with SO2, promoting regioselective hydrolysis and bond-forming processes, the latter similar to that observed for the NaV3O92− reactant anion. Kinetic analysis shows that these reactions are fast and efficient with rate constants of the 10−9 (±30) cm3 s−1 molecule−1 order of magnitude.

Highly regioselective hydrolysis of the glycosidic bonds in ginsenosides catalyzed by snailase

Minor ginsenosides display higher physiological activities than those of major ginsenosides, but their low abundance limits their clinical application. In this study, snailase was employed to transform protopanaxadiol-type (PPD) ginsenosides, protopanaxatriol-type (PPT) ginsenosides, and Ro to minor ginsenosides. The results showed that snailase hydrolyzed the saccharide chains at the C-3 position of Rb1, Rc, Rb2 Rg3 and Ro and the saccharide chains at the C-28 position of Ro efficiently. The end glucosyl unit of the saccharide chain at the C-6 position of Rf was hydrolyzed entirely. However, the rhamnosyl of Re was intact. The end glucosyl unit of the ginsenoside Rb1 was hydrolyzed entirely, but both arabinopyranosyl of Rb2 and arabinofuranosyl of Rc were not hydrolyzed. So snailase is selective for glycoside bond locations and saccharide types of ginsenoside, and it can be used to produce Rg3, CK, Rh1 and so on.

Enzyme-Assisted Synthesis of High-Purity, Chain-Deuterated 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.

1-Palmitoyl-d31-2-oleoyl-d32-sn-glycero-3-phosphocholine (POPC-d63) with the palmitoyl and oleoyl chains deuterium-labeled was produced in three steps from 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine, deuterated palmitic acid, and deuterated oleic anhydride. Esterification at the sn-2 position was achieved under standard chemical conditions, using DMAP to catalyze the reaction between the 2-lysolipid and oleic anhydride-d64. Complete regioselective sn-1 acyl substitution was achieved in two steps using operationally simple, enzyme-catalyzed regioselective hydrolysis and esterification to substitute the sn-1 chain for a perdeuterated analogue. This method provides chain-deuterated POPC with high chemical purity (>96%) and complete regiopurity, useful for a variety of experimental techniques. This chemoenzymatic semisynthetic approach is a general, modular method of producing highly pure, mixed-acyl phospholipids, where the advantages of both chemical synthesis (efficiency, high yields) and biocatalytic synthesis (specificity, nontoxicity) are realized.

Hydrolysis of the Amide Bond in L-Methionine- and L-Histidine-Containing Dipeptides in the Presence of Dinuclear Palladium(II) Complexes with Benzodiazines Bridging Ligands

1H NMR spectroscopy was applied to study the catalytic activity of dinuclear Pd(II)-aqua complexes with different benzodiazine bridging ligands, [{Pd(en)(H2O)}2(μ-qx)]4+ (Pd1), [{Pd(en)(H2O)}2(μ-qz)]4+ (Pd2) and [{Pd(en)(H2O)}2(μ-phtz)]4+ (Pd3) (qx, qz and phtz denote quinoxaline, quinazoline and phthalazine, respectively), in the hydrolytic cleavage of the amide bond in N-acetylated L-methionylglycine (Ac–L–Met–Gly) and L-histidylglycine (Ac–L–His–Gly) dipeptides. All reactions were investigated with an equimolar amount of the reactants at pH = 2.0–2.5 in D2O and at 37 °C. The obtained data for the catalytic activity of Pd1–Pd3 complexes are compared with those previously reported for [{Pt(en)(H2O)}2(μ-L)]4+ (L denotes benzodiazine: qx, qz and phtz), [{Pd(en)(H2O)}2(μ-L)]4+ and [{Pt(en)(H2O)}2(μ-L)]4+ (L denotes diazine: pyrazine and pyridazine) complexes. It was found that catalytic activity of these complexes in peptide cleavage is strongly related to the position of the nitrogen atoms in the benzodiazine or diazine bridging ligand. The investigated dinuclear Pd(II) and Pt(II) complexes show catalytic activity in the selective hydrolysis of the Met–Gly amide bond of Ac–L–Met–Gly dipeptide. Moreover, all the above mentioned Pd(II) complexes were also able to catalyze the regioselective hydrolysis of the His–Gly amide bond of Ac–L–His–Gly dipeptide. However, in the reaction with Ac–L–His–Gly, only Pt(II) aqua complexes containing bridging ligands with two nitrogen atoms in the para-position (quinoxaline and pyrazine) were able to cleave this dipeptide.

De Novo Biosynthesis of (S)- and (R)-Phenylethanediol in Yeast via Artificial Enzyme Cascades.

Due to oil depletion and global climate change, sustainable manufacturing of fine chemicals from renewable feedstocks has gained increasing attention in the scientific community. In the present study, we attempted to engineer Saccharomyces cerevisiae towardde novo synthesis of (S)- or (R)-phenylethanediol, an important pharmaceutical intermediate. More specifically, the biocatalytic cascades contain the following: l-phenylalanine undergoes deamination/decarboxylation to styrene by using phenylalanine ammonia lyase (PAL) and ferulic acid decarboxylase (FDC), followed by S-selective epoxidation of styrene to give (S)-styrene oxide with styrene monooxygenase (SMO); regioselective hydrolysis of (S)-styrene oxide with epoxide hydrolase from Sphingomonas HXN-200 (SpEH) or from potato (StEH) gives rise to (S)- or (R)-phenylethanediol. In this work, we found that the artificial enzyme cascades could be functionally expressed in the heterologous host of S.cerevisiae. Small-scale shake flask studies revealed that the engineered yeast cell factories produced approximately 100-120 mg/L of (S)- or (R)-phenylethanediol after 96 h cultivation. To the best of our knowledge, this is the first attempt to explore an artificial route with styrene as an intermediate for producing phenylethanediol in S.cerevisiae. We envision that our engineering strategy will open a new research field for synthesizing other vicinal diol derived chemicals in yeast.

Lipase-Catalyzed Regioselective Ester Hydrolysis as a Key Step in an Alternative Synthesis of a Buprenorphine Pro-Drug

This paper describes the development of an alternative route toward a hemiadipic acid pro-drug of buprenorphine. Buprenorphine was acylated with adipic acid monoethyl ester. A regioselective ester hydrolysis using C. antarctica lipase B cleaved the sterically less-hindered alkyl ester in the presence of the more labile phenolic ester. In this manner the pro-drug could be isolated in good yield and high purity.

Enhanced catalytic stability and reusability of nitrilase encapsulated in ethyleneamine-mediated biosilica for regioselective hydrolysis of 1-cyanocycloalkaneacetonitrile.

Nitrilase-catalyzed regioselective hydrolysis of 1-cyanocyclohexaneacetonitrile (1-CHAN) is a green and efficient approach for the preparation of 1-cyanocyclohexaneacetic acid (1-CHAA), a key precursor for the synthesis of gabapentin. Here, a mesoporous biosilica particles prepared by the ethyleneamine-mediated silicification have been used as carrier for the encapsulation of nitrilase from Acidovorax facilis (NitA). The silica-encapsulated NitA (NitA@silica) with triethylenetetramine as an initiator showed the highest immobilization efficiency (98.3%) and specific activity (672.6 U/g). Both free and encapsulated NitA were optimally active at 40 °C and pH 7.0, however, the encapsulated enzyme exhibited wider optimum temperature range, and enhanced thermal stability compared with the free enzyme. The kinetic parameters Km and Vmax for free and encapsulated NitA were calculated to be 141 mM and 9.97 mM min-1, and 280 mM and 9.02 mM min-1, respectively. The encapsulated NitA showed good reusability and retained about 94.2% of its initial activity even after 16 cycles of reaction. Also, the storage experiments revealed high activity maintenance of encapsulated NitA after 17-day storage at 4 °C. A preparative scale regioselective hydrolysis of 1-CHAN to 1-CHAA with encapsulated NitA as biocatalyst was carried out in a 2 L stirred bioreactor. The concentration of 1-CHAA reached 152 g/L after 8 h reaction and the conversion was 90.9%. These results showed that the encapsulation of NitA in ethyleneamine-mediated biosilica is an efficient and simple way for preparation of stable nitrilase and have a great potential for application in enzymatic production of carboxylic acids.

Flow-based biocatalysis: Application to peracetylated arabinofuranosyl-1,5-arabinofuranose synthesis

Abstract The lipase-catalyzed regioselective hydrolysis of peracetylated arabinose was performed in a packed bed flow reactor (PBR). In particular, the hydrolysis of the α anomer of peracetylated arabinose catalyzed by Novozym® 435 resulted in the monodeprotection of C5 in only 5 min and 91% yield. By using the immobilized Pseudomonas stutzeri lipase, the regioselective hydrolysis of the β anomer was also accomplished affording the C1 deacetylated derivative in 30 min with good yields. The high local concentration of the immobilized biocatalyst in the PBR allow for a significant reduction of the reaction time; moreover, repeated re-use, and easy downstream processing increase the efficiency and the productivity of the process, if compared to the classical batch procedure. In fact, under optimized conditions, the specific reaction rate of the biocatalyzed flow reaction showed an increase of more than 300 times compared to the batch one. The obtained building blocks were prepared in gram scale and then used for the synthesis of peracetylated arabinofuranosyl-1,5-arabinofuranose.

Development and Optimization of a New Chemoenzymatic Approach for the Synthesis of Peracetylated Lactosamine (Intermediate for the Synthesis of Pharmacologically Active Compounds) Monitored by RP- HPLC Method.

Purpose: To describe a chemoenzymatic approach joining an enzymatic regioselective hydrolysis of peracetylated N-acetyl-α-D-glucosamine (A) with a mild controlled acyl relocation which resulted 2-acetamido-2 deoxy-1,3,6-tri-O-acetyl-α-D-glucopyranose (1B). Methods: Immobilization of lipase on decaoctyl (DSEOD) and octyl-agarose (OSCL) was carried out as reported by the work of Bastida et al. The newly developed RP-HPLC method for examining the enzymatic hydrolysis was carried out isocratically utilizing a HPLC system. Results: The new approach resulted the target compound (B) in 95% yield after purification utilizing flash column chromatography. Candida rugosa-lipase immobilized ondecaoctyl-sepabeads was the best catalyst in terms of activity and region-selectivity in the hydrolysis of substrate (A), delivering the deacetylation at C6 position (98% general yield). Also, a reversed-phase high-performance liquid-chromatographic (RP-HPLC) method for controlling the region-selective hydrolysis of peracetylated N-acetyl-α-D-glucosamine (A) with a mild monitored acyl movement which led to 2-acetamido-2-deoxy-1,3,6-tri-O-acetyl-α-D-glucopyranose (1B) has additionally been developed. The developed RP-HPLC method was utilized as fingerprints to follow the hydrolysis of substrate (A) and to determine its purity and additionally yield. Furthermore, the acquired compound (B) was further purified by flash chromatography. Compound (B) was further characterized utilizing 1HNMR and mass spectrometry. Conclusion: An efficient chemoenzymatic procedure to optimize the preparation of peracetylated lactosamine B containing acetyl ester as extraordinary protecting group is presented. Compound B is a significant intermediate for the synthesis of pharmacologically active compound (e.g. complex oligosaccharides for biochemical, biophysical, or biological examinations). Besides, reaction monitoring utilizing HPLC proposes more exact information than spectroscopic methods.

Enzymatic tailoring of oleuropein from Olea europaea leaves and product identification by HRMS/MS spectrometry

Oleuropein, a bioactive compound found in all parts of olive tree, especially in leaves and branches, presents numerous health promoting properties that increase research and market interest the last few years. In addition, oleuropein degradation products, such as hydroxytyrosol, elenolic acid, and the aglycones also exhibit biological activities with different properties compared to the starting compound. Under this view, a commercial lipase preparation Lipolase 100L and a thermophilic β-glucosidase from Myceliophthora thermophila were used for the regioselective hydrolysis of oleuropein towards the production of the corresponding biologically active compounds. The enzymatic degradation products of oleuropein, such as hydroxytyrosol, elenolic acid and its glucoside, and oleuropein aglycones were identified by LC-HRMS/MS and NMR spectroscopy. The latter, was found as a mix of diastereomers of the monoaldehydic form of oleuropein aglycone, identified as (5S, 8R, 9S)-, (5S, 8S, 9S)- and (5S, 8R, 9R). The high substrate specificity exhibited by both lipase and β-glucosidase allows the successful tailoring of oleuropein towards the production of different biologically active compounds with significant potential in the cosmeceutical and food industry.

Short and Efficient Chemoenzymatic Syntheses of non‐Natural (−)‐Muscarine and (+)‐allo‐Muscarine from Cyano‐Sugar Precursors Catalyzed by Immobilized Burkholderia cepacia Lipase

AbstractEnantiopure (2R)‐configured non‐natural (−)‐muscarine and (+)‐allo‐muscarine were efficiently synthesized by a chemoenzymatic approach from an easily accessible cyano‐sugar available on a large‐scale. The key enzymatic hydrolysis step was accomplished by immobilized Burkholderia cepacia lipase (also known as Pseudomonas cepacia lipase, PSL‐IM). The PSL‐IM‐mediated regioselective hydrolysis of the 5‐O‐toluoyl ester group in α‐ and β‐cyano‐sugars, furnished 3‐O‐toluoyl‐α‐cyano‐sugar and 3‐O‐toluoyl‐β‐cyano‐sugar, respectively, with total selectivity and in excellent yields. To demonstrate the industrial utility of this method, 3‐O‐toluoyl‐β‐cyano‐sugar was synthesized on a 10 g scale using PSL‐IM and the catalyst was reused. A key feature of the synthesis route described herein is the simultaneous hydrogenolysis of a tosyl group, reduction of a nitrile and deprotection of a toluoyl group using LiAlH4 in a one‐pot procedure. This study offers a green protocol for synthesis of two muscarine derivatives in high yields employing a concise four‐step strategy.magnified image

Immobilization of Neutral Protease from Bacillus subtilis for Regioselective Hydrolysis of Acetylated Nucleosides: Application to Capecitabine Synthesis.

This paper describes the immobilization of the neutral protease from Bacillus subtilis and its application in the regioselective hydrolysis of acetylated nucleosides, including building blocks useful for the preparation of anticancer products. Regarding the immobilization study, different results have been obtained depending on the immobilization procedure. Epoxy hydrophobic carriers gave a poorly stable derivative that released almost 50% of the immobilized protein under the required reaction conditions. On the contrary, covalent immobilization on a differently activated hydrophilic carrier (agarose) resulted in very stable enzyme derivatives. In an attempt to explain the obtained enzyme immobilization results, the hypothetical localization of lysines on the enzyme surface was predicted in a 3D structure model of B. subtilis protease N built in silico by using the structure of Staphylococcus aureus metalloproteinase as the template. The immobilized enzyme shown a high regioselectivity in the hydrolysis of different peracetylated nucleosides. A stable enzyme derivative was obtained and successfully used in the development of efficient preparative bioprocesses for the hydrolysis of acetylated nucleosides, giving new intermediates for the synthesis of capecitabine in high yield.

ChemInform Abstract: Regioselective Hydrolysis and Transesterification of Dimethyl 3‐Benzamidophthalates Assisted by a Neighboring Amide Group.

An efficient, highly regioselective hydrolysis and transesterification of dimethyl 3-benzamidophthalates into the corresponding carboxylic acid monoesters and mixed esters (including tert-butyl esters) under basic conditions is presented. The selectivity is governed by the neighboring 3-benzamido moiety’s participation and by the nature of the solvent. In alcohols the reaction occurred exclusively at the ortho-position to the benzamido functionality, in pyridine or acetonitrile at both ester groups. An insight into the mechanistic pathway was obtained from a 1H NMR study in perdeuteromethanol.

Regioselective Functionalization of 3-Hydroxy-pyridine Carboxylates by Neighboring Group Assistance.

Regioselective hydrolysis, transesterification, and aminolysis of unactivated, highly substituted pyridine esters were realized under mild conditions by employing neighboring group assisted catalysis. Excellent yields were achieved without active removal of the alcohol byproduct. Regioselective aminolysis had a considerable substrate scope ([hetero]aryl, alkyl and amino acid). A mechanism involving assistance by the deprotonated phenolic OH-group is suggested for hydrolysis and transesterification.

Regioselective Hydrolysis and Transesterification of Dimethyl 3-Benzamidophthalates Assisted by a Neighboring Amide Group.

An efficient, highly regioselective hydrolysis and transesterification of dimethyl 3-benzamidophthalates into the corresponding carboxylic acid monoesters and mixed esters (including tert-butyl esters) under basic conditions is presented. The selectivity is governed by the neighboring 3-benzamido moiety's participation and by the nature of the solvent. In alcohols the reaction occurred exclusively at the ortho-position to the benzamido functionality, in pyridine or acetonitrile at both ester groups. An insight into the mechanistic pathway was obtained from a (1)H NMR study in perdeuteromethanol.

Understanding the regioselective hydrolysis of ginkgolideB under physiological environment based on generation, detection, identification, and semi-quantification of the hydrolyzed products.

A liquid chromatography-mass spectrometry (LC-MS) method coupled with specialized sample-preparation strategies was developed to investigate the hydrolysis of ginkgolideB (GB) in physiological environments in comparison with that of ginkgolide A (GA). The rapid hydrolysis processeswere captured by thedirect injectionof samples prepared in the volatile buffers. The LC-MS behavior of the hydrolyzed products, including three monocarboxylates and three dicarboxylates, was acquired. The monocarboxylates were identified by fragmentation analysis, and the dicarboxylates were accordingly tentatively identified by reaction sequences. The base-catalyzed hydrolysis of GB and GA was characterized at 4°C within pH7.0-10.7. The regioselective reactions on the lactone-C and lactone-F were revealed by thermodynamic studies at pH6.8 and 7.4. It was revealed that the 1-hydroxyl group on the skeleton of GB blocks the reactivity of the lactone-E. On the basis of these results, a distinctive hydrolysis phenomenon of GB was confirmed in plasma of humans, rats, and dogs as a rapid degradation of the trilactonealongwith the onlyproduction of the lactone-F-hydrolyzed product. This phenomenon is also closely associated with the 1-hydroxyl group, because it was not observed in GA. More interestingly, the underlying mechanism was revealed not to be associated with any typical enzyme-catalyzed process, but to be potentially involved with a selective reaction of the intact or broken lactone-C moiety with endogenous small-moleculereactants in plasma. This in-depth knowledge of the hydrolysis of GB versus GA not only facilitated understanding of their pharmacological mechanisms but also provided potential routes to study the structure-activity relationships of ginkgolides. Graphical Abstract Regioselective hydrolysis of ginkgolide B in pH 7.4 buffers and plasma.