Retro Diels-Alder Fragmentation Research Articles

Rationalising the retro-Diels-Alder fragmentation pattern of viscutins using electrospray interface-tandem mass spectrometry coupled to theoretical modelling.

Although mass spectrometry (MS) is a powerful tool in structural elucidation of unknown flavonoids based on their unique fragmentation patterns, proposing the correct fragmentation mechanism is still a challenge from tandem mass spectrometry data only. In recent years, computational tools such as molecular networking and MS2LDA have played a major role in the identification of structurally related compounds through an in-depth survey of their fragmentation patterns. Therefore, in this study, three viscutin molecules in Viscum combreticola Engl. crude extracts were characterised using ultra-high-performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry and MS2LDA, a computational tool. Ion-trap mass spectrometry and density functional theoretical modelling were used as confirmatory tools to rationalise the unique fragmentation patterns observed for these molecules. Here, MS2LDA revealed the presence of a unique Mass2Motif in all the three viscutin molecules at m/z 137, which was confirmed to be a 1,3 A- RDA (retro-Diels-Alder) fragmentation product using liquid chromatography-ion-trap mass spectrometry and density functional theoretical modelling. Moreover, MS2LDA proved to be useful in differentiating this spectral feature that was specific to viscutin molecules in the presence of other isobaric ions at m/z 137 occurring in compounds in other molecular families. Therefore, the results of the current study showed that computational tools such as MS2LDA are essential in uncovering some gas-phase fragmentation reactions of molecules in MS and that theoretical modelling is a powerful tool in rationalising these reactions in metabolite identification.

The Mechanism of a Retro-Diels-Alder Fragmentation of Luteolin: Theoretical Studies Supported by Electrospray Ionization Tandem Mass Spectrometry Results.

The mechanisms of retro-Diels–Alder fragmentation of luteolin are studied theoretically using the Density Functional Theory method (B3LYP hybrid functional) together with the 6-311++G(d,p) basis set and supported by electrospray ionization tandem mass spectrometry (ESI-MS) results. The reaction paths leading to the formation of 1,3A− and 1,3B− fragment ions observed as the main spectral features in the ESI-MS spectrum are described and discussed, including the structures of the transition states and intermediate products. The heights of the activation energy barriers which have to be overcome along the reaction paths corresponding to 1,3-retrocyclization cleavage of the ionized luteolin are predicted to span the 69–94 kcal/mol range (depending on the initial isomeric structure) for the concerted retrocyclization mechanism and the 60–89 kcal/mol (first barrier) and 24–52 kcal/mol (second barrier) barriers for the stepwise mechanism (also depending on the initial isomeric structure). It is also demonstrated that the final fragmentation products (1,3A− and 1,3B−) are in fact represented by various isomeric systems which are not experimentally distinguishable. In addition, the absence of the spectral feature corresponding to the [M-B]− fragment ion formed by the rupture of the C-C bond connecting luteolin’s B and C rings (which does not occur during the ESI-MS experiment) is explained by much larger energy barriers predicted for such a process.

Hydroxyl Group Stabilization for Increased Yields of Low-Molecular-Weight Products in the Copyrolysis of Cellulose and Thermoplastics

Biomass is a promising renewable and sustainable resource to produce energy and value-added chemicals. Fast pyrolysis is one of the simplest thermochemical methods to convert biomass into high yields of liquid products that can be upgraded into drop-in fuels or platform chemicals; however, its diverse product distributions and low product selectivity incur significant cost due to subsequent upgrading and separation operations. In this work, a strategy to promote yields of low-molecular-weight products (LMWPs) from cellulose pyrolysis via hydroxyl group stabilization using molten polymers (MPs) is presented. Three types of thermoplastics, high-density polyethylene (HDPE), polyethylene glycol (PEG), and polystyrene (PS), were copyrolyzed with cellulose to investigate the possible hydroxyl group stabilization effects caused by the ether and aromatic moieties in MPs during cellulose pyrolysis. A custom-made batch pyrolysis reactor was employed for the copyrolysis experiments. Our results showed that the combined yields of levoglucosan (LG) and LMWPs significantly increased in the presence of MPs due to the physical inhibition of anhydrosugar oligomer evaporation. The product distributions were varied dependent on the MPs used. In particular, both ether groups in PEG and aromatic groups in PS were found to stabilize the cellulosic hydroxyl groups during glycosidic bond cleavage, inhibiting the formation of LG. Aromatic moieties in MPs were observed to create a stronger inhibition effect on the glycosidic bond cleavage than ether moieties. Our experiments also suggest that both ether and aromatic groups in MPs stabilize the hydroxyl groups during dehydration, leading to increased yields of products from retro-Diels–Alder fragmentation. Ether moieties were found to be more effective at inhibiting dehydration than aromatic moieties. Yields of the HDPE-derived products increased during copyrolysis, suggesting a possible catalytic effect in HDPE pyrolysis caused by the LMWPs produced from carbohydrate pyrolysis. Yields of the PEG-derived products increased only in the presence of cellulose and glucose, whereas yields of the PS-derived products were unaffected in all copyrolysis experiments. A possible reaction mechanism accounting for the hydroxyl group stabilization effects is proposed based on our experimental findings.

Tandem high-resolution electrospray ionization mass spectrometry of fluorinated thevinols and 18,19-dihydrothevinols

Fragmentation of fluorinated 18,19-dihydrothevinols and thevinols under electrospray ionization with collisionally activated dissociation accompanied by the elimination of an amine fragment from the cycle and the subsequent loss of H2O and CH3OH have been studied. For thevinol derivatives with a double bond at C18=C19, the [M + H]+ ions undergo the retro-Diels–Alder fragmentation, the abundance of which depends on substituents at the nitrogen and C20 atoms. The retro-Diels–Alder fragmentation of the [M + H]+ ions of thevinols bearing–CH3,–CH2CH=CH2, and $$ - C{H_2} - \triangleleft $$ groups at the nitrogen atom occurs due to the formation of a protonated diene molecule, whereas the elimination of a neutral diene in thevinols with hydrogen at the nitrogen atom has been observed.

Gas-Phase Retro-Diels-Alder Reactions of Cyclohexene, 1-Methylcyclohexene, and 4-Methylcyclohexene following Photoexcitation at 193 nm: A Velocity-Map Imaging Study.

We present the results of a velocity-map imaging study into the retro-Diels-Alder (RDA) reactions of cyclohexene, 1-methylcyclohexene, and 4-methylcyclohexene following photoexcitation at 193 nm. Universal detection of all neutral fragments via vacuum-ultraviolet photoionization at 118 nm allows imaging of both RDA fragments in all cases. Fragment kinetic energy distributions reveal contributions from both dissociative ionization and retro-Diels-Alder reaction of the respective parent molecules, yielding reaction products with a high degree of internal excitation. Together with the observed isotropic product angular distributions, this is consistent with a mechanism in which the RDA reaction occurs from high vibrational levels of the electronic ground state following internal conversion from a higher-lying state initially populated in the photoexcitation process, as predicted by frontier molecular orbital theory. Velocity-map images and total translational energy distributions for the RDA products of 1-methylcyclohexene and 4-methylcyclohexene are very similar to those for unsubstituted cyclohexene, indicating that methyl substitution either adjacent to or far from the double bond has little effect on the dynamics of the RDA process.

UPLC–ESI-Q-TOF-MSE identification of urinary metabolites of the emerging sport nutrition supplement methoxyisoflavone in human subjects

Methoxyisoflavone (5-methyl-7-methoxyisoflavone) is a synthetic isoflavone used by bodybuilders for its ergogenic properties. A recent study demonstrated that methoxyisoflavone metabolites can induce false-positive results in urinary immunoassay screening tests for cannabinoids, and only one metabolite has been identified. To improve the knowledge on the metabolic pathways of methoxyisoflavone, ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC–Q-TOF) was applied. Urine samples were obtained from methoxyisoflavone regular users. After enzymatic hydrolysis and liquid–liquid extraction, the samples were analyzed by UPLC–Q-TOF fitted with an electrospray ionization source (ESI) operating under positive ion mode. Mass data were acquired with the MSE method. Five metabolites were identified. Those were divided into two metabolic pathways, depending on whether the B ring hydroxylation was preceded or not by the O-demethylation of the methoxy group. The MSE mass spectra of methoxyisoflavone and its metabolites are specific of isoflavones structures and revealed 1,3 retro Diels-Alder fragmentation and double CO loss. Losses of small neutral molecules CO and H2O, and radical CH3, typical of flavonoids, were also observed. This study illustrates the capacity of the sensitive UPLC–Q-TOF analytical system, combined with the MSE method of collection of fragmentation data, to rapidly elucidate the unknown xenobiotics metabolism.

Identification of regioisomers of methylated kaempferol and quercetin by ultra high performance liquid chromatography quadrupole time-of-flight (UHPLC–QTOF) tandem mass spectrometry combined with diagnostic fragmentation pattern analysis

The O-methylation of active flavonoids can enhance their antiallergic, anticancerous, and cardioprotective effects depending on the methylation position. Thus, it is biologically and pharmacologically important to differentiate methylated flavonoid regioisomers. In this study, we examined the regioisomers of methylated kaempferol and quercetin using ultra high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry. The methyl groups on the flavonoids can generally be cleaved as methyl radicals in a position-independent manner. We found that methyl groups can be cleaved as methane. If there are protons adjacent the methoxy on the flavonol rings, intra-molecule proton transfer can occur via collision-induced dissociation, and one molecule of methane can then be eliminated. The remaining charged fragment ([M+H−CH4]+) reflects the adjacent structure and is specific to the methoxy position. Furthermore, the retro Diels–Alder (RDA) fragmentation of methylated flavonols can generate fragments with the methoxy at the original methylated ring. Combining the position-specific [M+H−CH4]+ fragment with the RDA fragments provides a diagnostic pattern for rapidly identifying methylated regioisomeric flavonols. Along with their retention behaviour, we have successfully identified ten regioisomers of methylated kaempferol and quercetin, which include six compounds previously reported in plants and shown to be biologically active. The developed approach is sensitive, rapid, reliable, and requires few standard compounds. It is highly efficient for characterising the specificity of novel flavonoid O-methyltransferases and can help direct enzymatic or chemical syntheses during the early stages of drug discovery. This method also has potential for use in identifying other methylated isomeric flavonoids.

Degradable Conjugates from Oxanorbornadiene Reagents

Oxanorbornadienedicarboxylate (OND) reagents were explored for purposes of binding and releasing drugs from serum albumins as representative macromolecular carriers. Being highly reactive Michael acceptors, ONDs form adducts with thiols and amines, which then undergo retro-Diels-Alder fragmentation. A study of more than 30 model adducts revealed a number of modifications that can be used to influence adduct stability. For the most reactive OND linkers, the labeling of the single available bovine serum albumin (BSA) cysteine residue was complete within minutes at a mid-micromolar concentration of reactants. While a selectivity of greater than 1000-fold for thiol over amine was observed with model amino acids, the labeling of protein amines with ONDs is fast enough to be practical, as demonstrated by the reaction with thiol-depleted BSA. The OND-amine adducts were found to be up to 15 times more stable than OND-thiol adducts, and to be sensitive to acid by virtue of a stereochemically dependent acceleration of cycloreversion. The release rate of fluorescent cargo from serum albumins was tuned by selecting the coupling partners: the available half-lives ranged from 40 min to 7 days at 37 °C. Such versatility of release profiles from protein carriers, controlled by the nature of the OND linkage, is a useful addition to the drug delivery toolbox.

Identification of prenylated pterocarpans and other isoflavonoids in Rhizopus spp. elicited soya bean seedlings by electrospray ionisation mass spectrometry

Phytoalexins from soya are mainly characterised as prenylated pterocarpans, the glyceollins. Extracts of non-soaked and soaked soya beans, as well as that of soya seedlings, grown in the presence of Rhizopus microsporus var. oryzae, were screened for the presence of prenylated flavonoids with a liquid chromatography/mass spectrometry (LC/MS)-based screening method. The glyceollins I-III and glyceollidins I-II, belonging to the isoflavonoid subclass of the pterocarpans, were tentatively assigned. The formation of these prenylated pterocarpans was accompanied by that of other prenylated isoflavonoids of the subclasses of the isoflavones and the coumestans. It was estimated that approx. 40% of the total isoflavonoid content in Rhizopus-challenged soya bean seedlings were prenylated pterocarpans, whereas 7% comprised prenylated isoflavones and prenylated coumestans. The site of prenylation (A-ring or B-ring) of the prenylated isoflavones was tentatively annotated using positive-ion mode MS by comparing the (1,3) A(+) retro-Diels-Alder (RDA) fragments of prenylated and non-prenylated isoflavones. Furthermore, the fragmentation pathways of the five pterocarpans in negative-ion (NI) mode were proposed, which involved the cleavage of the C-ring and/or D-ring. The absence of the ring-closed prenyl (pyran or furan) gave exclusively -H(2) O(x,y) RDA fragments, whereas its presence gave predominantly the common RDA fragments.

A novel biomimetic synthesis of ( S)-(−)-zearalenone: via macrocyclization and transannular aromatization

On heating, a hydroxy-keto-dioxinone underwent retro-Diels–Alder fragmentation and the resultant α,γ-diketo-ketene was efficiently trapped intramolecularly by a secondary alcohol to provide a macrocyclic triketo-lactone. Following ketal hydrolysis, transannular aromatization gave the resorcylate natural product, ( S)-(−)-zearalenone.

Reaction of 2,5-bis-trifluoromethyl-1,3,4-oxadiazole with 7-oxanorbornenes revisited: Experimental and quantum-chemical study of reaction stereoselectivity

The stereochemical outcome of reaction of 2,5-bis-trifluoromethyl-1,3,4-oxadiazole with 7-oxanorbornenes under various conditions was investigated. For the first time, microwave irradiation in carrying this type of reactions was used, resulting in comparable yields in significantly shorter reaction times. Regardless on substrate, in all reactions mixtures of the two isomeric O3-[3]polynorbornanes, bent and linear were obtained, with slight preference for bent structure. In some cases, retro Diels–Alder fragmentation was observed resulting in formation of isobenzofuran species. Reaction mechanism was also studied computationally (RHF/6-31G* method), and the origin of stereoselectivity explained by repulsive lone pair interactions between oxygen bridges in the transition state of the 1,3-dipolar addition.

Flavonoid–matrix cluster ions in MALDI mass spectrometry

The behaviour of 2,5-dihydroxybenzoic acid (2,5-DHB) matrix under matrix-assisted laser desorption/ionisation (MALDI) conditions was investigated, and the formation of 2,5-DHB cluster ions, mainly dehydrated 2,5-DHB ions, is reported. Interestingly, in the mass spectra of this compound, besides dimers and trimers, protonated tetramers, pentamers, hexamers and heptamers were also found with significant abundance.The MALDI behaviour of four flavonoids, quercetin, myricetin, luteolin and kaempferol, using 2,5-DHB as matrix, was also investigated. The mass spectra of the flavonoids studied revealed a number of flavonoid-2,5-DHB cluster ions (mainly with the dehydrated 2,5-DHB). The number of clusters formed is dependent on the structure of the analyte. For luteolin and kaempferol, in particular, evidence was found for the formation of cluster ions involving retro Diels Alder fragments and intact flavonoids molecules, as well as the corresponding protonated retro Diels Alder fragments with dehydrated DHB molecules. All ion compositions were attributed taking into account high accuracy mass measurements and tandem mass spectrometry experiments.

X-ray Structural Analysis for the Prediction on the Nature of the Retro Diels - Alder Pathway: Concerted or Stepwise. Structural Studies on Nitrosobenzene Cycloadducts

Crystal structures of nitrosobenzene cycloadducts 5–7 reveal structural effects consistent with the early stages of the retro Diels–Alder fragmentation. There is a clear differentiation between the structure parameters of cycloadduct 5, which reacts by a concerted synchronous pathway and that of cycloadduct 6, which must react by a two-step pathway. Based on these data, cycloadduct 7 is predicted to react by a highly asynchronous or two-step pathway.

Isomerization of delta-9-THC to delta-8-THC when tested as trifluoroacetyl-, pentafluoropropionyl-, or heptafluorobutyryl- derivatives

For GC-MS analysis of delta-9-tetrahydrocannabinol (delta-9-THC), perfluoroacid anhydrides in combination with perfluoroalcohols are commonly used for derivatization. This reagent mixture is preferred because it allows simultaneous derivatization of delta-9-THC and its acid metabolite, 11-nor-delta-9-THC-9-carboxylic acid present in biological samples. When delta-9-THC was derivatized by trifluoroacetic anhydride/hexafluoroisopropanol (TFAA/HFIPOH) and analyzed by GC-MS using full scan mode (50-550 amu), two peaks (P1 and P2) with an identical molecular mass of 410 amu were observed. On the basis of the total ion chromatogram (TIC), P1 with a shorter retention time (RT) was the major peak (TIC 84%). To identify the peaks, delta-8-THC was also tested under the same conditions. The RT and spectra of the major peak (TIC 95%) were identical with that of P1 for delta-9-THC. A minor peak (5%) present also correlated well with the latter peak (P2) for the delta-9-THC derivative. The fragmentation pathway of P1 was primarily demethylation followed by retro Diels-Alder fragmentation (M - 15-68, base peak 100%) indicating P1 as a delta-8-THC-trifluoroacetyl compound. This indicated that delta-9-THC isomerized to delta-8-THC during derivatization with TFAA/HFIPOH. Similar results were also observed when delta-9-THC was derivatized with pentafluoropropionic anhydride/pentafluoropropanol or heptafluorobutyric anhydride/heptafluorobutanol. No isomerization was observed when chloroform was used in derivatization with TFAA. In this reaction, the peaks of delta-8-THC-TFA and delta-9-THC-TFA had retention times and mass spectra matching with P1 and P2, respectively. Because of isomerization, perfluoroacid anhydrides/perfluoroalcohols are not suitable derivatizing agents for analysis of delta-9-THC; whereas the TFAA in chloroform is suitable for the analysis.

Does the electron ionization induced fragmentation of partly saturated stereoisomeric pyrrolo‐ and isoindoloquinazolinones show stereospecificity?

The electron ionization mass spectrometric behavior of pyrroloquinazolinones (1-6) and isoindoloquinazolinones (7-14) was studied. These compounds were further classified as partly saturated pyrroloquinazolinones (1-3), benzologues (7-11), methylene-bridged derivatives (4-6, 12, 13) and a bisacyl compound (14). The mass spectra of the pyrrolo- and isoindoloquinazolinones did not exhibit stereospecific retro-Diels-Alder (RDA) fragmentations. The cyclohexane-fused compounds 7 (cis annelated) and 8 (trans annelated) did display some other ions differing in their abundances that could be used to differentiate this pair of stereoisomers. Also the cyclohexene-fused compounds 2, 3, 9 and 10 exhibited somewhat different ion abundances pairwise that could be utilized for isomeric differentiation. Earlier hypothesis of pyrrolo ring cleavage via the loss of C(3)H(5)O(.) was strengthened by the fragmentation of compounds 1-4. RDA(+/-H) fragmentation is more favorable than the formation of [M-R](+) ions (R=H, C(6)H(4)CH(3), or C(6)H(4)Cl) when an unsaturated bicyclic group is present but both RDA fragmentation and [M-R](+) formation occur for cyclohexene-fused compounds, possibly because of the lower ring strain than with norbornene-fused compounds. The [M-H](+) ion was abundant for compounds 7 and 8 as was [M-Ar](+) for 1-4 and 11. Although the compounds studied might participate in amide-imidol tautomerism, no indication of such tautomerism was detected.

Apicin, a New Flavonoid from Artemisia apiacea.

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Apicin, A New Flavonoid from Artemisia apiacea

Artemisia species spreads widely in nature and are genus of the family Compositae consisting of more than 350 species. A. apiacea is distributed at wasteland and river beaches of Korea, Japan and China, and has been used as traditional medicine to treat eczema and jaundice. Investigations on the compounds from A. apiacea have revealed the presence of campesterol, stigmasterol, β-sitosterol, 7methoxycoumarin, 7,8-dimethoxycoumarin and 7,8-methylenedioxycoumarin, scopoletin, protocatechualdehyde and ethyl and methyl caffeates, daphnetin, 7-hydroxy-8-methoxycoumarin and 7-isopentenyloxy-8-methoxycoumarin, volatile constituents like α-pinene and artemisia ketone and artemicapin C, apigenin, daucosterol, cacticin, hyperin, α-amyrin, β-amyrin, β-sitosterol and 5,6,7-trimethoxycoumarin. During the course of our continued studies on the compounds from A. apiacea, a new flavonoid (1) was isolated and identified. Compound 1 is described here for the first time as a naturally occurring compound. Compound 1 was obtained as yellow crystals from MeOH. It responded positively to the Shinoda test. In the EI-MS of 1, the molecular ion peak showed at m/z 360 corresponding to the molecular formula C18H16O8. The characteristic fragment ion peaks at m/z 153 showed the retro Diels-Alder fragmentation of flavonoids. The IR spectrum of 1 showed absorption bands for hydroxyl at 3383 cm−1, α,β-unsaturated C=O at 1612 cm−1 and C-O at 1015 cm−1. In the H-NMR spectrum of 1, the typical flavonoid signals were observed. The singlets of aromatic 5-OH at δ 13.03 and three -OMe signals at δ 3.91, 3.80 and 3.71 were observed. The singlets of H-8, -2', -4', and -6' were observed at δ 6.95, 7.09, 6.55 and 7.44, respectively. Its C-NMR spectrum of 1 showed C=O at δ 183.1 and three -OMe at δ 57.3, 57.6 and 60.9. In the homonuclear COSY spectrum, the correlation of proton signals is not indicated. The assignments of the Hand CNMR signals derived hetero nuclear direct and long-range correlations on 1 are listed in Table 1. Accordingly, compound 1 was assigned as a new flavonoid and named apicin (3,5,3'-trihydroxy-6,7,5'-trimethoxyflavone).

Structural analysis of selected characteristic flavones by electrospray tandem mass spectrometry.

Seven structure analogical flavonoid aglycones have been analyzed using electrospray ionization tandem mass spectrometry (ESI-MSn) in the negative-ion mode. The spectra obtained ESI-MSn allowed us to propose plausible schemes for their fragmentation mechanism. By analyzing the product ions spectra of deprotonated molecule ions [M-H](-), some neutral diagnostic losses and specific retro Diels-Alder fragments were obtained. By using all of these characteristic fragment ions we can specially differentiate the flavone isomer.

Endogenous alkaloids in man: XXXVIII. “Chiral” and “achiral” determination of the neurotoxin TaClo (1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline) from blood and urine samples by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry

An improved sensitive assay for the determination of the dopaminergic and serotonergic neurotoxin 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo) is presented, based upon on-line coupling of high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (HPLC–ESI-MS–MS). Applying synthetic [D 4]TaClo as a fourfold deuterated internal standard, TaClo was detected and reliably quantified as a trace constituent of blood samples (0.5 up to 70 ng g −1 of clot) obtained from six patients orally treated with the hypnotic chloral hydrate. Unambiguous identification of this tricyclic “endogenous alkaloid” was achieved by selected reaction monitoring (SRM) experiments. The molecular ion peaks of TaClo, m/ z 289 (for [ 35Cl 3]TaClo) and m/ z 291 (for its [ 37Cl 35Cl 2]isotopomer), were both monitored to undergo a retro-Diels–Alder fragmentation by loss of a CH 2NH portion (−29 u) as typical of a tetrahydropyrido ring system of tetrahydro-β-carbolines. Detection of the resulting fragments, m/ z 260 and m/ z 262, with the expected statistical chlorine isotopic intensities of 100:96 confirmed the identity of the TaClo molecule. In addition, an enantiomer-specific device was developed for TaClo, by employing a chiral reversed-phase HPLC column in combination with circular dichroism (CD) spectroscopy and MS–MS analysis (LC–CD and LC–MS–MS coupling). In a human clot sample, both TaClo enantiomers were found in equimolar concentration (i.e., as a racemate) corroborating a spontaneous, non-enzymatic formation of TaClo from biogenic tryptamine and therapeutically administered chloral. In urine samples of TaClo-treated rats, by contrast, the ( S)-antipode was found to predominate, hinting at an enantiomer-differentiating metabolism of the compound.

Mass-spectrometric differentiation of di exo- and di endo-fused isomers of norbornane/ene-condensed 2-thiouracil and 1,3-thiazino[3,2- a]pyrimidine derivatives: stereoselectivity of retro-diels-alder fragmentations under EI and CI conditions

The stereoisomers of the title compounds produce nearly identical electron ionization (EI) mass spectra, which are dominated in the case of the norbornene-condensed derivatives by retro-Diels-Alder (RDA) fragmentation of the hydrocarbon ring. The RDA fragmentation mainly occurs with H transfer and gives rise to [M–C 5H 5] +. For the norbornane-condensed derivatives, the main fragmentation routes include the formation of [M–C 5H 7] + (protonated thiouracil) and [M–C 7H 9] + (only from thiazinopyrimidines). The latter species are formed via RDA decomposition of the pyrimidone subunit of the heterocyclic system, a process previously observed for cyclohexane-condensed analogs of these compounds. Only minor differences could be detected between the EI spectra of the di exo and di endo isomers. Under chemical ionization (CI) conditions, the norbornane-condensed compounds produced no significant fragment peaks with either isobutane or methane as reagent gas. In contrast, the isobutane and methane CI spectra of the norbornene-condensed compounds exhibited prominent peaks of [MH–C 5H 6] + and [(M+C xH y)–C 5H 6] + originating from moderately stereoselective RDA fragmentations. The relative abundances of the RDA ions obtained from the respective stereoisomers with the same reagent gas were consistently different over a range of experimental conditions. The non-occurrence of RDA fragmentation of the thiazinopyrimidine ring under CI conditions suggested that its energy of activation is higher than that for either of the norbornene-ring RDA fragmentations (with or without H transfer) observed under EI and CI conditions.