Investigations of taxoids by electrospray and atmospheric pressure chemical ionization with tandem mass spectrometry

The fragmentation behavior of taxoids was studied using electrospray (ESI) and atmospheric pressure chemical ionization (APCI) sources with multi-stage tandem mass spectrometry. In the positive ion mode taxoids gave prominent [M+Na]+ and [M+K]+ ions with the ESI source, and [M+NH4]+ or [M+H]+ ions with the APCI source. The MS/MS fragmentations of ions produced by APCI and ESI sources were very similar. For both sources, the presence of cinnamoyl or benzoyl groups could be characterized by initial losses of 148 or 122 u, respectively, from molecular adduct ions. However, the elimination of cinnamic acid was relatively difficult for the molecular adduct ions formed by APCI, and was comparable in importance to the loss of acetic acid. The other fragments involved losses of CH2CO, CO, and H2O. The 5/7/6 type taxoids underwent characteristic losses of 58 or 118 u from ions produced by both APCI and ESI sources. The fragmentation behavior was remarkably influenced by substitution locations. The elimination of the C-10 benzoyl group was usually the first fragmentation step, while that of the C-2 benzoyl group was relatively difficult. The acetoxyl group at C-7 was more active than those at C-2, C-9, and C-10, which in turn were more active than that at C-4. These fragmentation rules could facilitate the rapid screening and structural characterization of taxoids in plant extracts by high-performance liquid chromatography/mass spectrometry (HPLC/MS).     

A facile approach for the construction of the oxetane ring from 5α-acyloxy-Δ~(4(20)) - taxoids

An oxetane ring can be constructed from 5α-acyloxy-Δ4(20)-taxoids. Hie facile intramolecular acyl migration from 5- to 20-position under slightly basic conditions enabled the construction of the oxetane ring in a convenient short cut, whereas the acyl migration from 2- to 20-position left the 2-hydroxyl accessible to a later benzoylation. An unexpected five-mem-bered 4-O, 20- O sulfite ring was formed in the attempted construction of the oxetane ring with 5α-triflate as a leaving group. After the construction of the oxetane ring, treatment with strong base LiHMDS and acetyl chloride gave the expected 4-O-acetate while treatment with acetic anhydride and DMAP gave a 4-O-acetoacetate.     

A selective intramolecular transacylation of taxoids accompanying with the oxetane ring opening

A selective intramolecular transacylation from C-4 to C-5 of taxoids, which occurred simultaneously with the oxetane D-ring opening and was promoted by TiCl4, was presented. The optimal condition was found to be treatment of substrates in double dose of dichloromethane (per mg of substrate dissolved in 2?mL DCM) with 1?eq of TiCl4 at 25°C for 10?min.     

Dual parallel electrospray ionization and atmospheric pressure chemical ionization mass spectrometry (MS), MS/MS and MS/MS/MS for the analysis of triacylglycerols and triacylglycerol oxidation products

Two mass spectrometers, in parallel, were employed simultaneously for analysis of triacylglycerols in canola oil, for analysis of triolein oxidation products, and for analysis of triacylglycerol positional isomers separated using reversed-phase high-performance liquid chromatography. A triple quadrupole mass spectrometer was interfaced via an atmospheric pressure chemical ionization (APCI) interface to two reversed-phase liquid chromatographic columns in series. An ion trap mass spectrometer was coupled to the same two columns using an electrospray ionization (ESI) interface, with ammonium formate added as electrolyte. Electrospray ionization mass spectrometry (ESI-MS) under these conditions produced abundant ammonium adduct ions from triacylglycerols, which were then fragmented to produce MS/MS spectra and then fragmented further to produce MS/MS/MS spectra. ESI-MS/MS of the ammoniated adduct ions gave product ion mass spectra which were similar to mass spectra obtained by APCI-MS. ESI-MS/MS produced diacylglycerol fragment ions, and additional fragmentation (MS/MS/MS) produced [RCO](+) (acylium) ions, [RCOO+58](+) ions, and other related ions which allowed assignment of individual acyl chain identities. APCI-MS of triacylglycerol oxidation products produced spectra like those reported previously using APCI-MS. APCI-MS/MS produced ions related to individual fatty acid chains. ESI-MS of triacylglycerol oxidation products produced abundant ammonium adduct ions, even for those molecules which previously produced little or no intact molecular ions under APCI-MS conditions. Fragmentation (MS/MS) of the [M+NH(4)](+) ions produced results similar to those obtained by APCI-MS. Further fragmentation (MS/MS/MS) of the diacylglycerol fragments of oxidation products provided information on the oxidized individual fatty acyl chains. ESI-MS and APCI-MS were found to be complementary techniques, which together contributed to a better understanding of the identities of the products formed by oxidation of triacylglycerols.     

Study on fragmentation behavior of 5/7/6-type taxoids by tandem mass spectrometry

The mass spectrometric behaviors of seven compounds, namely four 4beta(20),5-oxetane 5/7/6-type taxoids (i.e. taxayuntin A, taxayuntin B, taxayuntin and taxayuntin C) and three 4(20)-methylene 5/7/6-type taxoids (i.e. brevifoliol, taxchinin A and 7-acetyl-10-deacetyl-7-debenzoylbrevifoliol) have been investigated by the positive ion FAB-MS/MS technique. The fragmentation has been correlated with the types and positions of substituents of these compounds. It has been found that with the OH group at the C-10 position, taxayuntin A, taxayuntin B and 7-acetyl-10-deacetyl-7-debenzoylbrevifoliol are dominated by the loss of H2O, while with the BzO group at the C-10 position, taxayuntin, taxayuntin C, brevifoliol and taxchinin A preferentially eliminate the BzO group. In addition, C-2 is an active site, and neutral loss from the C-2 position readily occurs. The four 4beta(20),5-oxetane 5/7/6-type taxoids produce the terminal product ion with a stable conjugated system at m/z 311, while the 4(20)-methylene 5/7/6-type taxoids brevifoliol and 7-acetyl-10-deacetyl-7-debenzoylbrevifoliol produce this ion at m/z 237, and taxchinin A at m/z 253. Interestingly, characteristic fragment ions involving the loss of a 118 u group were observed for taxayuntin, and a possible fragmentation mechanism is given. The major fragmentation pathways and mechanisms of ion formation for the compounds are proposed on the basis of CID spectra and accurate mass measurements. The results of this paper will be helpful for structural analysis of analogs.     

Four new epoxy taxanes from needles of Taxus cuspidata (Taxaceae)

Four new epoxy taxoids were isolated from the needles of Taxus cuspidata. Their structures were established as 2a,9a-diacetoxy-5a-cinnamoyloxy-11,12-epoxy-10ss-hydroxytax-4(20)-en-13-one (1), 2a,10ss-diacetoxy-5a-cinnamoyloxy-11,12-epoxy-9a-hydroxytax-4(20)-en-13-one (2), 2a,9a-diacetoxy-11,12-epoxy-10ss,20-dihydroxytax-4-en-13-one (3) and 2a,10ss-diacetoxy-11,12-epoxy-9a,20-dihydroxytax-4-en-13-one (4) on the basis of spectral analysis including 1H-NMR, 13C-NMR, 1H-1H-COSY, HSQC, HMBC and HRFABMS. Compounds 3 and 4 are the first example of 11,12-epoxy taxoids with C-4 double bond found in T. cuspidata.     

Organometallic dithiolene complexes of the group 8-10 metals: reactivities, structures and electrochemical behavior

Research progress in the organometallic dithiolene complexes such as [Cp(or Cp*)M(dithiolene)] (M = Co, Rh, Ir, Ni), [(C(6)R(6))Ru(dithiolene)] and [(C(4)R(4))Pt(dithiolene)] complexes during the past decade is described and the reactivities, structures and electrochemical behavior are summarized in this paper. The five-membered metalladithiolene ring (MS(2)C(2)) undergoes addition reactions to the M[double bond, length as m-dash]S bond to form 18-electron adducts by an imido, alkylidene, alkene or norbornene group and also undergoes dimerizations on the basis of the unsaturation in the ring. The aromaticity of the ring causes substitution reactions on the dithiolene carbon by a C-centered radical, S-centered radical or succinimide group when the ring has a C-H bond. Furthermore a dithiolene-dithiolene homo-coupling reaction by an acid or dithiolene-aryl cross-coupling occurs based on the aromaticity in the ring. Dissociations of the 18-electron adducts are observed by those thermolyses, photolyses, electrochemical redox reactions and other chemical reactions with tertiary phosphorus compounds. One representative example of them is the imido adduct dissociation with PR(3) under heating toward the intramolecular imido migration to a Cp ligand. Since all products are rearomatized by those adduct dissociations, it is concluded that the 'coexistence of aromaticity and unsaturation' in the metallacycle mediates the diverse chemical reactions.     

Direct analysis in real time mass spectrometry (DART‐MS) of highly non‐polar low molecular weight polyisobutylenes

Low molecular weight polyisobutylenes (PIB) with chlorine, olefin and succinic acid end‐groups were studied using direct analysis in real time mass spectrometry (DART‐MS). To facilitate the adduct ion formation under DART conditions, NH₄Cl as an auxiliary reagent was deposited onto the PIB surface. It was found that chlorinated adduct ions of olefin and chlorine telechelic PIBs, i.e. [M + Cl]^? up to m/z 1100, and the deprotonated polyisobutylene succinic acid [M? H]^? were formed as observed in the negative ion mode. In the positive ion mode formation of [M + NH₄]⁺, adduct ions were detected. In the tandem mass (MS/MS) spectra of [M + Cl]^?, product ions were absent, suggesting a simple dissociation of the precursor [M + Cl]^? into a Cl^? ion and a neutral M without fragmentation of the PIB backbones. However, structurally important product ions were produced from the corresponding [M + NH₄]⁺ ions, allowing us to obtain valuable information on the arm‐length distributions of the PIBs containing aromatic initiator moiety. In addition, a model was developed to interpret the oligomer distributions and the number average molecular weights observed in DART‐MS for PIBs and other polymers of low molecular weight. Copyright © 2015 John Wiley & Sons, Ltd.     

Data-directed scan sequence for the general assignment of C-glycosylflavone O-glycosides in plant extracts by liquid chromatography-ion trap mass spectrometry

An ion trap LC-MS/MS method is described for the analysis of C-glycosylflavone O-glycosides in crude methanolic extracts of plants. The method employs survey scans with and without the application of up-front collision induced dissociation (CID) to generate diagnostic ions for data-directed MS/MS. The spectra acquired allow assignment of the C-linked sugar to either the C-6 or C-8 position of the aglycone and provide data on the molecular mass of the compound, the number and type of O-linked sugars and the molecular mass of the flavone aglycone. These data for the majority of C-glycosylflavone O-glycosides in an extract are obtained automatically in one LC-MS/MS analysis without manual pre-programming. Key to the assignment of the C-6 or C-8 site of C-glycosylation is the generation, by up-front CID, of the (0,1)X+ product ion formed by internal cleavage of the C-linked sugar. MS/MS of this ion is found to have diagnostic value in addition to the (0,2)X+ product ion described by other authors. Ion trap MS/MS spectra of [M+H]+ of the 6,8-di-C-glycosylflavones schaftoside and isoschaftoside show an additional and previously unreported diagnostic product ion that is useful in determining the type of sugar at the C-6 position. The product ion spectra of protonated kaempferol 3-O-glucosylrhamnosides show similarities to the spectra of C-glycosylflavone O-glycosides; this is a potential source of confusion if the analysis of such glycosides is limited solely to MS/MS of [M+H]+.     

Synthesis and in vitro evaluation of taxol oxetane ring D precursors

A series of potential taxoid substrates was prepared in radiolabeled form to probe in vitro for the oxirane formation step and subsequent ring expansion step to the oxetane (ring D) presumably involved in the biosynthesis of the anticancer agent Taxol. None of the taxoid test substrates underwent transformation in cell-free systems from Taxus suggesting that these surrogates bore substitution patterns inappropriate for recognition or catalysis by the target enzymes, or that taxoid oxiranes and oxetanes arise by independent biosynthetic pathways.     

Synthesis of extremely simplified compounds possessing the key pharmacophore units of taxol, phenylisoserine and oxetane moieties.

Straight chain compounds having a phenylisoserine unit and an oxetane ring at the alpha- and omega- position, respectively as extremely simplified analogues of taxol were prepared. None of these compounds showed promising tubulin inhibitory activity.     

Elucidation of the double-bond position of long-chain unsaturated fatty acids by multiple-stage linear ion-trap mass spectrometry with electrospray ionization

Linear ion-trap (LIT) MS2 mass spectrometric approach toward locating the position of double bond(s) of unsaturated long-chain fatty acids and toward discerning among isomeric unsaturated fatty acids as dilithiated adduct ([M-H+2Li]+) ions are described in this report. Upon resonance excitation in a LIT instrument, charge-remote fragmentation that involves beta-cleavage with gamma-H shift (McLafferty rearrangement) is the predominant fragmentation pathway seen for the [M-H+2Li]+ ions of monoenoic long-chain fatty acids. The fragmentation process results in a dilithiated product ion of terminally unsaturated fatty acid, which undergoes consecutive McLafferty rearrangement to eliminate a propylene residue, and gives rise to another dilithiated adduct ion of terminally unsaturated fatty acid. In addition to the above-cited fragmentation process, the [M-H+2Li]+ ions of homoconjugated dienoic long-chain fatty acids also undergo alpha-cleavage(s) with shift of the allylic hydrogen situated between the homoconjugated double bonds to the unsaturated site. These fragmentation pathways lead to two types of CC bond cleavages that are allylic (alpha-cleavage) or vinylic, respectively, to the proximal CC double bond, resulting in two distinct sets of ion series, in which each ion series is separated by a CH2CHCH (40 Da) residue. These latter fragmentations are the predominant processes seen for the polyunsaturated long-chain fatty acids. The spectrum feature dependent on the position of unsaturated double bond(s) affords unambiguous assignment of the position of double bond(s) of long-chain unsaturated fatty acids.     

Multi-stage mass spectrometric analysis of saponins in glycyrrhiza radix

The fragmentation pathways of six triterpenoid saponins from Glycyrrhiza radix were investigated using LC-MS/MS. Depending on the structure and the substitution pattern, different molecular adduct ions, [M+NH4]+ or [M+H]+, were observed in the positive ESI spectra. In the positive MSn spectra from the molecular adduct ions, characteristic product ions corresponding to the loss of dehydrated glucuronic acid or glucuronic acid were detected and they indicated the type of substitution and structural modification. Fragment ions originating from the sapogenin moiety in the positive mass spectra were predominantly provided by saponins having an 11-oxo-12-ene structure. On the other hand, the saponins gave fragment ions corresponding to the sugar moiety in the negative mass spectra. These results indicate the specific property of saponins that have the 11-oxo-12-ene structure to localize positive or negative charge in the mass spectrometric ionization and fragmentation process. Information obtained from the present study can be utilized for structural elucidation of triterpenoid saponins in the Glycyrrhiza radix by LC-MS.     

Exclusive formation of alpha-methyleneoxetanes in ketene-alkene cycloadditions. Evidence for intervention of both an alpha-methyleneoxetane and the subsequent 1,4-zwitterion

This paper describes a new mechanistic feature for the Staudinger ketene-alkene cycloaddition reactions to give cyclobutanones. Low-temperature NMR (13C, 19F, and 1H) monitoring of a reaction between bis(trifluoromethyl)ketene (1) and ethyl vinyl ether (2) has shown that the Staudinger reaction proceeds to form initially and exclusively an alpha-methyleneoxetane (3) by [2 + 2](C=O) cycloaddition across the ketene C=O bond. The initial intermediate 3 undergoes ring cleavage to produce a 1,4-zwitterion (4), which is converted to the final [2 + 2](C=C)-type product, cyclobutanone (5). The key intermediate 3 has been isolated in its pure form and was found to be converted to the final products 5 on warming, via the 1,4-zwitterion 4. The alpha-methyleneoxetane 3 is so reactive that it reacts with methanol rapidly even at -80 degrees C via solvolysis to afford an adduct 7. The ion 4 derived from the pure isolated oxetane 3 was intercepted with acetone by a 1,4-dipolar cycloaddition to give a 1,3-dioxane 8. An open-chain alpha,beta-enone (6) has been also obtained from 3. We conclude that the (1 + 2) reaction proceeds in a new three-step mechanism; formation of an alpha-methyleneoxetane 3, a [2 + 2]-type cycloadduct across the C=O bond of ketene, followed by ring cleavage to give the zwitterion 4 and by recombination to form the final product, cyclobutanone 5. The zwitterion 4 is not equilibrating with reactants 1 and 2 but comes from the alpha-methyleneoxetane 3. Exclusive formation of another oxetane 12 has been observed in a reaction between diphenylketene (9) and methyl isopropenyl ether (11). The selectivity of initial formation of cyclobutanone or oxetane has been generalized with aid of frontier-orbital theory and ab initio calculations.     

Analysis of triterpenoids in <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> using liquid chromatography coupled with electrospray ionization mass spectrometry

Triterpenoids extracted from Ganoderma lucidum (Leyss. ex Fr.) Karst were separated and characterized using optimized reversed-phase liquid chromatography with diode array detection and electrospray ion trap tandem mass spectrometry (HPLC-DAD-ESI-MS(n)). They could be classified into five types depending on the fragmentation behavior. All triterpenoids gave [M - H](-) and [2M - H](-) ions by electrospray ionization monitored in the negative ion mode; in addition, compounds of types III and IV gave prominent [M - H - H(2)O](-) ions and the unsaturated bond at C-20, 22 would reduce the abundance of [M - H - H(2)O](-) ion. The key fragmentation information was cleavage at C- and D-rings despite the predominant losses of H(2)O and CO(2). Compounds with hydroxyls at C-7 and C-15 would produce a list of b, b - 1, b - 2, and b - 16 ions attributed to cleavage of D-ring; if the second alcohol at C-15 were oxidized to ketone, the prominent cleavage would occur at C-ring and produce a group of ions of a; if C-7 were oxidized to ketone, transference of two hydrogen atoms would occur during the cleavage of rings and a list of ions about a + 2 and/or b + 2 would appear instead. The above fragmentations and regularities in fragmentation pathways were reported for the first time, and were implemented for the analysis of triterpenoids in G. lucidum. The chloroform extract was separated on a Zorbax SB-C(18) column, eluting with an acetonitrile-0.2% acetic acid gradient. A total of 32 triterpenoids, including six new ones, were identified or tentatively characterized based on the tandem mass spectra of the HPLC peaks.     

Use of the tubulin bound paclitaxel conformation for structure-based rational drug design

A new computational docking protocol has been developed and used in combination with conformational information inferred from REDOR-NMR experiments on microtubule bound 2-(p-fluorobenzoyl)paclitaxel to delineate a unique tubulin binding structure of paclitaxel. A conformationally constrained macrocyclic taxoid bearing a linker between the C-14 and C-3'N positions has been designed and synthesized to enforce this "REDOR-taxol" conformation. The novel taxoid SB-T-2053 inhibits the growth of MCF-7 and LCC-6 human breast cancer cells (wild-type and drug resistant) on the same order of magnitude as paclitaxel. Moreover, SB-T-2053 induces in vitro tubulin polymerization at least as well as paclitaxel, which directly validates our drug design process. These results open a new avenue for drug design of next generation taxoids and other microtubule-stabilizing agents based on the refined structural information of drug-tubulin complexes, in accordance with typical enzyme-inhibitor medicinal chemistry precepts.     

Formation of a Pentacoordinate 1,2-Oxastannetanide by Novel Base-Induced Rearrangement of a Bis(β-Hydroxyalkyl)Stannane

Treatment of a bis(β-hydroxyalkyl)diphenylstannane with potassium hydride in THF in the presence of 18-crown-6 gave the corresponding pentacoordinate t-alkoxy-l,2-oxastannetanide as stable crystals instead of a hexacoordinate stannate with two oxetane rings. This compound is considered to be formed by novel tin-1,3-migration reaction from carbon to oxygen reaction involving the formation of an oxetane ring and the subsequent tin-carbon bond cleavage.     

Acetonitrile covalent adduct chemical ionization tandem mass spectrometry of non-methylene-interrupted pentaene fatty acid methyl esters

Acetonitrile covalent adduct chemical ionization tandem mass spectrometry (CACIMS/MS) has shown to be an efficient method for the identification of double‐bond position in homoallylic, conjugated and several polyene non‐methylene‐interrupted (NMI) fatty acid methyl esters. However, it has not been thoroughly evaluated for NMI highly unsaturated fatty acids (HUFA) with more than four double bonds. Docosahexaenoic acid (DHA)‐rich single cell oil (DHASCO^®; Martek Biosciences, Corp.) was partially hydrogenated (partially hydrogenated DHASCO; PHDO) producing ten novel 22:5 and 22:6 HUFA isomers. In single‐stage MS, the ratio of [M+54]⁺/[M+54‐32]⁺ for the 22:5 and 22:6 isomers indicated the presence of homoallylic or partially conjugated double‐bond systems. The CACIMS/MS spectra revealed six 22:5 isomers with diagnostic ions corresponding to the homoallylic 22:5n‐6 and 22:5n‐3 isomers, and four distinct NMI 22:5 isomers. Diagnostic ions for four 22:6 isomers were identical to the native DHA illustrating that CACIMS/MS is sensitive to double‐bond position but not geometry. Three gas chromatography (GC) peaks for partially conjugated 22:6 isomers were also detected and clearly distinguishable from homoallylic 22:6 isomers, but their CACIMS/MS spectra did not yield prominent ions indicative of double‐bond position, possibly due to co‐elution. Overall, CACIMS/MS was effective for determining double‐bond position in NMI 22:5 isomers. Further investigations are warranted to evaluate and determine fragmentation patterns for partially conjugated and NMI 22:6 HUFA. Copyright © 2011 John Wiley & Sons, Ltd.     

The oxetane ring in taxol

Numerous structure-activity studies combining synthesis and bioassay have been performed for the anti-cancer drug Taxol. The four-membered D-ring, an oxetane, is one of four structural features regarded to be essential for biological activity. This proposition is examined by application of a Taxol-epothilone minireceptor, K(i) estimation for microtubule binding and docking of Taxol analogues into a model of the Taxol-tubulin complex. In this way, we evaluate the two characteristics considered responsible for oxetane function: (1) rigidification of the tetracyclic Taxol core to provide an appropriate framework for presenting the C-2, C-4, C-13 side chains to the microtubule protein and (2) service as a hydrogen-bond acceptor. An energy decomposition analysis for a series of Taxol analogues demonstrates that the oxetane ring clearly operates by both mechanisms. However, a broader analysis of four-membered ring containing compounds, C- and D-seco derivatives, and structures with no oxetane equivalent underscores that the four-membered ring is not necessary for Taxol analogue bioactivity. Other functional groups and ligand-protein binding characteristics are fully capable of delivering Taxol biobehavior as effectively as the oxetane D-ring. This insight may contribute to the design and development of novel anticancer drugs.     

Synthesis and biological evaluation of novel A-seco-taxoids derived from 1-deoxybaccatin VI

Three novel nor-seco-taxoids 13, 15, 23 in which the A rings are cleaved but the B, C, and D rings are retained were prepared from 1-deoxybaccatin VI via its nor-dioxo derivative and their structures were confirmed by ¹H NMR, ¹³C NMR and high resolution MS. Oxidative introduction of C-1 hydroxyl to 1-deoxybaccatin VI with oxidising agent KBrO₃ and catalyst RuCl₃ led to the dioxo derivative 6 and its structure is determined by X-ray crystallographic analysis. A-seco taxoids 13, 15, 23 with a C-13 ester linkage were tested for cytotoxic activity and all compounds showed no measurable cytotoxic activity against HCT-116 cell line. However, 1-deoxy-9a-dihydrotaxane analogue 4 semi-synthesised from 1-deoxybaccatin VI is 10-fold less cytotoxic than paclitaxel, indicating the indispensible nature of the A ring double bond for the bioactivity of paclitaxel.