Leveraging ToF-SIMS imaging to investigate tenofovir disoproxil fumarate degradation at excipient interfaces in oral compressed tablets.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging has been used to study the hydrolysis of tenofovir disoproxil fumarate (TDF) to tenofovir monosoproxil (TM) within an oral compressed tablet. The ToF-SIMS images displayed a heterogenous distribution of the matrix components. Evaluation of the TM distribution revealed that it was primarily co-localized with areas of higher excipient concentration pointing toward excipient driven degradation. To support these observations, a compatibility study of TDF with each tablet component was performed via liquid chromatography. The ToF-SIMS imaging and compatibility study indicated that the excipient, Avicel® PH-102, was the primary driver of TM formation in the tablet. The hydrolysis degradation mechanism within the tablet is further rationalized through discussion of chemical and physical properties of the matrix components. The sum of this work demonstrates a new analytical workflow for probing and understanding matrix driven degradation in oral compressed tablets utilizing ToF-SIMS imaging.

Differentiation of the Aziridine Functionality from Related Functional Groups in Protonated Analytes by Using Selective Ion-Molecule Reactions Followed by Collision-Activated Dissociation in a Linear Quadrupole Ion Trap Mass Spectrometer.

Aziridines are commonly used as reagents for the synthesis of drug substances although they are potentially mutagenic and genotoxic. Therefore, their unambiguous detection is critically important. Unfortunately, tandem mass spectrometry (MS2) based on collision-activated dissociation (CAD), a powerful method used for the identification of many unknown compounds in complex mixtures, does not provide diagnostic fragmentation patterns for ionized aziridines. Therefore, a different mass spectrometry approach based on MS3 experiments is presented here for the identification of the aziridine functionalities. This approach is based on selective gas-phase ion-molecule reactions of protonated analytes with tris(dimethylamino)borane (TDMAB) followed by diagnostic CAD reactions in a modified linear quadrupole ion trap (LQIT) mass spectrometer. TDMAB reacts with protonated aziridines by forming adduct ions that have lost a dimethylamine (DMA) molecule ([M + H + TDMAB - HN(CH3)2]+). CAD on these product ions generated diagnostic fragment ions with m/z-values 25- and 43-units lower than those of the ion-molecule reaction product ions. None of the ion-molecule reaction product ions formed from other, structurally related, protonated analytes produced related fragment ions. Quantum chemical calculations were employed to explore the mechanisms of the observed reactions.

A Diagnostic Nitrosamine Detection Approach for Pharmaceuticals by Using Tandem Mass Spectrometry Based on Diagnostic Gas-Phase Ion-Molecule Reactions.

N-Nitrosamines are strictly regulated in pharmaceutical products due to their carcinogenic nature. Therefore, the ability to rapidly and reliably identify the N-nitroso functionality is urgently needed. Unfortunately, not all ionized N-nitroso compounds produce diagnostic fragment ions and hence tandem mass spectrometry based on collision-activated dissociation (CAD) cannot be used to consistently identify the N-nitroso functionality. Therefore, a more reliable method was developed based on diagnostic functional-group selective ion-molecule reactions in a linear quadrupole ion trap mass spectrometer. 2-Methoxypropene (MOP) was identified as a reagent that reacts with protonated N-nitrosamines in a diagnostic manner by forming an adduct followed by the elimination of 2-propenol (CH3C(OH)═CH2]). From 18 protonated N-nitrosamine model compounds studied, 15 formed the diagnostic product ion. The lack of the diagnostic reaction for three of the N-nitrosamine model compounds was rationalized based on the presence of a pyridine ring that gets preferentially protonated instead of the N-nitroso functionality. These N-nitrosamines can be identified by subjecting a stable adduct formed upon ion-molecule reactions with MOP to CAD. Further, the ability to use ion-molecule reactions followed by CAD to differentiate protonated O-nitroso compounds with a pyridine ring from analogous N-nitrosamines was demonstrated This methodology is considered to be robust for the identification of the N-nitroso functionality in unknown analytes. Lastly, HPLC/MS2 experiments were performed to determine the detection limit for five FDA regulated N-nitrosamines.

Differentiation of Protonated Sulfonate Esters from Isomeric Sulfite Esters and Sulfones by Gas-Phase Ion-Molecule Reactions Followed by Diagnostic Collision-Activated Dissociation in Tandem Mass Spectrometry Experiments.

Sulfonate esters, a class of potentially mutagenic drug impurities, are strictly regulated in pharmaceuticals. On the other hand, sulfite esters and sulfones, analogs of sulfonate esters, have limited safety concerns. However, previously developed analytical methods for sulfonate ester identification cannot be used to differentiate sulfonate esters from the isomeric sulfite esters and sulfones. A tandem mass spectrometric method is introduced here for the differentiation of these compounds. Diisopropoxymethylborane (DIMB) reacts with protonated sulfonate esters, sulfite esters, and sulfones (and many other compounds) in the gas phase to form the product ion [M + H + DIMB - CH3CH(OH)CH3]+. Upon collision-activated dissociation (CAD), these product ions generate diagnostic fragment ions that enable the differentiation of sulfonate esters, sulfite esters, and sulfones from each other. For example, SO2 elimination enabled the unambiguous identification of sulfite esters. On the other hand, elimination of CH3B═O followed by elimination of (CH3)2C═O was only observed for sulfonate esters. Neither type of diagnostic fragment ions was detected for the products of sulfones. However, the product ions formed for sulfones with an additional hydroxyl substituent underwent the elimination of another CH3CH(OH)CH3 molecule, which enabled their identification. Finally, ion-molecule reactions of DIMB with various other functionalities were also examined. Some of them yielded the product ions [M + H + DIMB - CH3CH(OH)CH3]+ but none of these product ions underwent the diagnostic CAD reactions discussed above. Quantum chemical calculations were employed to explore the mechanisms of the reactions. The limits of detection for the diagnostic ion-molecule reaction product ions in high-performance liquid chromatography (HPLC)/mass spectrometry (MS2) experiments were found to range from 0.075 to 1.25 nmol.

Fast Determination of the Lignin Monomer Compositions of Genetic Variants of Poplar via Fast Pyrolysis/Atmospheric Pressure Chemical Ionization Mass Spectrometry.

The proportional content of the phenylpropanoid monomeric units (4-hydroxyphenyl (H), guaiacyl (G), and syringyl (S)) in lignin is of paramount importance in germ plasm screening and for evaluating the results of plant breeding and genetic engineering. This content is usually determined using a tedious and slow (2 days/sample) method involving derivatization followed by reductive cleavage (DFRC) combined with GC/MS or NMR analysis. We report here a fast mass spectrometric method for the determination of the monomer content. This method is based on the fast pyrolysis of a lignin sample inside the ion source area of a linear quadrupole ion trap mass spectrometer. The evaporated pyrolysis products are promptly deprotonated via negative-ion mode atmospheric pressure chemical ionization ((-)APCI) and analyzed by the mass spectrometer to determine the monomer content. The results obtained for the wild-type and six genetic variants of poplar were consistent with those obtained by the DFRC method. However, the mass spectrometry method requires only a small amount of sample (50 µg) and the use of only small amounts of three benign chemicals, methanol, water, and ammonium hydroxide, as opposed to DFRC that requires substantially larger amounts of sample (10 mg or more) and large amounts of several hazardous chemicals. Furthermore, the mass spectrometry method is substantially faster (3 min/sample), more precise, and the data interpretation is more straightforward as only nine ions measured by the mass spectrometer are considered.

Characterization of ionized lignin model compounds with α-O-4 linkages by positive- and negative-ion mode electrospray ionization tandem mass spectrometry based on collision-activated dissociation.

RATIONALE: The biggest obstacle in the rational conversion of biomass into aromatic chemicals is the identification of unknown compounds in lignin degradation mixtures that are highly complex. As opposed to lignin degradation products with ß-O-4 linkages, very little is known about the mass spectrometric analysis of lignin degradation products with α-O-4 linkages. METHODS: Lignin model compounds with an α-O-4 and another linkage, as well as lignin model compounds with only ß-O-4 linkages, were ionized by attachment of lithium or sodium cations under positive-ion mode electrospray ionization (ESI) or by deprotonation in negative-ion mode ESI in a linear quadrupole ion trap mass spectrometer. The ions were subjected to collision-activated dissociation in multiple-stage tandem mass spectrometry experiments to characterize their fragmentation patterns. RESULTS: All studied compounds formed abundant sodium and lithium cation adducts in positive-ion mode ESI with no fragmentation. Model compounds with ß-O-4 linkages displayed stable [M - H]- ions in negative-ion mode ESI whereas compounds with α-O-4 linkages only showed fragment ions. CAD of the lithiated α-O-4 compounds provided more structural information than CAD of sodiated compounds. However, both sodiated and lithiated compounds with α-O-4 linkages showed losses of monomer units at the MS2 stage, which is useful for sequencing of lignins with this type of linkage. CONCLUSIONS: An ionization and sequencing method has been developed for lignin model compounds with α-O-4 linkages that spontaneously fragment upon ionization via (-)ESI.

Associations of the MTHFR rs1801133 polymorphism with gastric cancer risk in the Chinese Han population.

In recent years, increasing evidence has implicated the importance of mutations in the MTHFR gene in the risk of gastric cancer risk. A single nucleotide polymorphism (SNP) in the MTHFR gene (rs1801133) may serve a critical role in gastric cancer. A hospital-based case-controlled study was performed to assess the risk of the rs1801133 polymorphism on gastric cancer. A total of 307 patients with gastric cancer and 560 patients in the control group were recruited. Genomic DNA was extracted from peripheral blood and genotyped for rs1801133 using the ligase detection reaction. The relationship between rs1801133 and gastric cancer risk was evaluated by unconditional logistical regression analysis. The rs1801133-TT genotype was associated with a borderline significantly decreased risk of gastric cancer [(TT vs. CC, adjusted odds ratio (OR)=0.54, 95% confidence intervals (CI)=0.35-0.83; P=0.006; CT vs. CC, adjusted OR=0.59, 95% CI=0.44-0.79, P<0.001; and TT/CT vs. CC, adjusted OR=0.61, 95% CI=0.44-0.83, P=0.001), and further analysis showed the relationship was evident amongst older patients and patients who never drank alcohol. The C>T mutation at rs1801133 of the MTHFR gene was associated with a decreased risk of gastric cancer in older individuals and those who never drink.

Studies of the Fragmentation Mechanisms of Deprotonated Lignin Model Compounds in Tandem Mass Spectrometry.

Unlabeled and deuterium-labeled dimeric lignin model compounds with ß-O-4 linkages were evaporated and ionized using negative ion mode electrospray ionization, transferred into a linear quadrupole ion trap, isolated, and subjected to collision-activated dissociation (CAD; MS2 experiments). The elemental compositions of the fragment ions were determined by using a high-resolution Orbitrap mass analyzer, and their structures were examined using further CAD experiments (MSn experiments wherein n = 2-5). Data analysis was facilitated by determining the fragmentation pathways for several deprotonated model compounds. The structures of the key fragment ions of several pathways were determined by comparison of the CAD mass spectra measured for undeuterated and deuterated analogues and for deprotonated authentic compounds. Some of the proposed reaction mechanisms were tested by examining additional deprotonated synthetic model compounds. Quantum chemical calculations were used to delineate the most likely reaction pathways and reaction mechanisms. This work provides basic information needed for the design of tandem mass spectrometry-based CAD sequencing strategies for mixtures of lignin degradation products.

Development of an automated and High throughput UHPLC/MS based workflow for cleaning verification of potent compounds in the pharmaceutical manufacturing environment.

Cleaning verification (CV) is a critical step in the pharmaceutical manufacturing process to eliminate or reduce unacceptable contamination of a product as a result of insufficiently cleaned equipment surfaces. The main concern is cross contamination with active pharmaceutical ingredients (APIs) from previous runs that may impact patient safety. Current conventional approaches involve rather tedious sample preparation and analytical methods with relative lengthy analysis time. Potent APIs possessing low acceptable daily intake (ADI) values require analytical methods for CV with very low detection limits to confirm that these APIs are below their acceptance limits prior to the next manufacturing process. In this work, a novel end to end CV workflow was developed, which includes the automated sample and calibration solution preparation as well as high throughput analysis by ultra-high-performance liquid chromatography (UHPLC) coupled with single quadrupole mass spectrometry in multiple injection chromatography and selected ion monitoring mode (MIC-MS-SIM). The method was validated using ten model compounds. Acceptable specificity, linearity (R2 > 0.997) and single digit ng/mL LOQ and LOD were achieved for all model compounds. This approach was also successfully applied to the analysis of 22 internal CV samples from an internal program.

Development of a highly efficient decontamination approach for ceftolozane in the pharmaceutical manufacturing environment.

The ß-lactam core is a key structure responsible for inducing both IgE-mediated acute-onset hypersensitivity and T-cell-mediated delayed-onset hypersensitivity with penicillins in humans. There is essentially no clinically significant immunologic cross-reactivity noted between the ß-lactam cores of penicillins and cephalosporins based on challenge studies in humans. The side-chains appear to be more important in inducing IgE-mediated acute-onset hypersensitivity and T-cell delayed-onset hypersensitivity with cephalosporins in humans. Despite these clinical findings, the U. S. Food and Drug Administration (FDA) still requires the level of ß-lactam-related antibiotic residues to be controlled at very low levels in manufacturing facilities. Ceftolozane is Merck & Co., Inc., Kenilworth, NJ, USA's (MSD's) 5th generation broad spectrum cephalosporin antibiotic against gram-negative bacteria. In searching for the optimal decontamination method of ceftolozane, most methods were found to be very slow in opening the ß-lactam ring in ceftolozane. Moreover, most of the previously reported decontamination methods applied analytical methods that only monitored the disappearance of the parent molecule as the endpoint of degradation. In this way, many of the ß-lactam-containing degradation products could be overlooked. In order to develop an efficient decontamination solution for ceftolozane, a sensitive ultra high performance liquid chromatography-high resolution-electrospray ionization-tandem mass spectrometry (UHPLC-HRMS/MS) method was first developed to ensure the detection of the ß-lactam ring in all degradation products. Through online UHPLC-UV-HRMS monitoring, 2.5 N KOH in 50% aqueous MeOH or 50% aqueous EtOH was identified as the best condition to fully degrade the ß-lactam ring in ceftolozane. This decontamination could be done within 15 min, even at 100 mg/mL concentration, and thus enable a quick turnaround time for equipment cleaning in the ß-lactam manufacturing facility. This method was also successfully applied to 12 other commercially available ß-lactam antibiotics.

[Application of metabonomics examination in differential diagnosis of non-small cell lung cancer (NSCLC)].

Objective To explore the preliminary application of metabonomics in the qualitative diagnosis of non-small cell lung cancer (NSCLC). Methods According to the pathological type, 201 patients with NSCLC were divided into squamous cell carcinoma (SCC) group (n=71) and adenocarcinoma (AC) group (n=130). Immunohistochemistry was used to detect the expression of ki67, cytokeratin 5/6 (CK5/6) and CK7. Ultra-high performance liquid chromatography-time-of-flight mass spectrometry (UPLC-Q/TOF-MS) was used to detect serum metabolomics. Results SCC group showed typical SCC structure; ki67 proliferation index was 21.9%; CK5/6 expression was positive; CK7 expression was negative or weakly positive. The typical adenoid structure was found in the AC group; the proliferation index of ki67 was 17.6%; CK7 was positive; and CK5/6 was negative or weakly positive. UPLC-Q/TOF-MS screened 28 different metabolites, of which 6 were the most significant ones: L-leucine, carnitine, C16 sphinganine, 13, 16, 19-docosatrienoic acie (DA), LysoPE (18:2/0:0), PC (20:4/P-16:0). These metabolites had good diagnostic value, among which L-Leucine had the highest specificity and LysoPE had the highest sensitivity. Conclusion Metabolomic analysis of lung SCC and AC provides a new index for the differential diagnosis of NSCLC.

Differentiation of Deprotonated Acyl-, N-, and O-Glucuronide Drug Metabolites by Using Tandem Mass Spectrometry Based on Gas-Phase Ion-Molecule Reactions Followed by Collision-Activated Dissociation.

Glucuronidation, a common phase II biotransformation reaction, is one of the major in vitro and in vivo metabolism pathways of xenobiotics. In this process, glucuronic acid is conjugated to a drug or a drug metabolite via a carboxylic acid, a hydroxy, or an amino group to form acyl-, O-, and/or N-glucuronide metabolites, respectively. This process is traditionally thought to be a detoxification pathway. However, some acyl-glucuronides react with biomolecules in vivo, which may result in immune-mediated idiosyncratic drug toxicity (IDT). In order to avoid this, one may attempt in early drug discovery to modify the lead compounds in such a manner that they then have a lower probability of forming reactive acyl-glucuronide metabolites. Because most drugs or drug candidates bear multiple functionalities, e.g., hydroxy, amino, and carboxylic acid groups, glucuronidation can occur at any of those. However, differentiation of isomeric acyl-, N-, and O-glucuronide derivatives of drugs is challenging. In this study, gas-phase ion-molecule reactions between deprotonated glucuronide metabolites and BF3 followed by collision-activated dissociation (CAD) in a linear quadrupole ion trap mass spectrometer were demonstrated to enable the differentiation of acyl-, N-, and O-glucuronides. Only deprotonated N-glucuronides and deprotonated, migrated acyl-glucuronides form the two diagnostic product ions: a BF3 adduct that has lost two HF molecules, [M - H + BF3 - 2HF]-, and an adduct formed with two BF3 molecules that has lost three HF molecules, [M - H + 2BF3 - 3HF]-. These product ions were not observed for deprotonated O-glucuronides and unmigrated, deprotonated acyl-glucuronides. Upon CAD of the [M - H + 2BF3 - 3HF]- product ion, a diagnostic fragment ion is formed via the loss of 2-fluoro-1,3,2-dioxaborale (MW of 88 Da) only in the case of deprotonated, migrated acyl-glucuronides. Therefore, this method can be used to unambiguously differentiate acyl-, N-, and O-glucuronides. Further, coupling this methodology with HPLC enables the differentiation of unmigrated 1-ß-acyl-glucuronides from the isomeric acyl-glucuronides formed upon acyl migration. Quantum chemical calculations at the M06-2X/6-311++G(d,p) level of theory were employed to probe the mechanisms of the reactions of interest.

Mechanistic insight into the oxazoline decomposition of DFC-M, a synthetic intermediate of florfenicol.

2-(dichloromethyl)-5[4-(methylsulfonyl)-phenyl]-4-(fluoromethyl)-oxazoline (DFC-M, 1) is a key oxazoline-containing intermediate in commercial process for the synthesis of Florfenicol (3), a marketed broad spectrum veterinary antibiotic. DFC-M was not stable in solution due to the presence of oxazoline moiety, which provided further hindrance for analytical sample preparation and HPLC analysis. Hence, the mechanistic study on the in-solution degradation of DFC-M was carried out via online and offline UPLC-HR-ESI-MS as well as in-situ NMR, and the degradation pathways were proposed. This mechanistic information, together with the follow-up solution stability study, provided crucial information regarding the solution handling and mobile phase selection for DFC-M analysis during commercial processing.

Structural elucidation of a dimeric impurity in the process development of ceftolozane using LC/HRMS and 2D-NMR.

Ceftolozane is MSD's 5th generation cephalosporin antibiotic with broad-spectrum gram-negative activity. While refining the synthetic route to the drug substance, a dimeric impurity was observed in the final active pharmaceutical ingredient (API) of ceftolozane. Through impurity enrichment and preparative isolation, the structure of the impurity was subsequently established through LC/HRMS, HRMSMS, H/D exchange and 2D-NMR studies. A kinetic study of the impurity formation was conducted to determine the best conditions to control it in the final chemical process.

Polar Effects Control the Gas-Phase Reactivity of para-Benzyne Analogs.

We report herein a gas-phase reactivity study on a para-benzyne cation and its three cyano-substituted, isomeric derivatives performed using a dual-linear quadrupole ion trap mass spectrometer. All four biradicals were found to undergo primary and secondary radical reactions analogous to those observed for the related monoradicals, indicating the presence of two reactive radical sites. The reactivity of all biradicals is substantially lower than that of the related monoradicals, as expected based on the singlet ground states of the biradicals. The cyano-substituted biradicals show substantially greater reactivity than the analogous unsubstituted biradical. The greater reactivity is rationalized by the substantially greater (calculated) electron affinity of the radical sites of the cyano-substituted biradicals, which results in stabilization of their transition states through polar effects. This finding is in contrast to the long-standing thinking that the magnitude of the singlet-triplet splitting controls the reactivity of para-benzynes.

Differentiating Isomeric Deprotonated Glucuronide Drug Metabolites via Ion/Molecule Reactions in Tandem Mass Spectrometry.

Isomeric O- and N-glucuronides are common drug metabolites produced in phase II of drug metabolism. Distinguishing these isomers by using common analytical techniques has proven challenging. A tandem mass spectrometric method based on gas-phase ion/molecule reactions of deprotonated glucuronide drug metabolites with trichlorosilane (HSiCl3) in a linear quadrupole ion trap mass spectrometer is reported here to readily enable differentiation of the O- and N-isomers. The major product ion observed upon reactions of HSiCl3 with deprotonated N-glucuronides is a diagnostic HSiCl3 adduct that has lost two HCl molecules ([M - H + HSiCl3 - 2HCl]-). This product ion was not observed for deprotonated O-glucuronides. Reaction mechanisms were explored with quantum chemical calculations at the M06-2X/6-311++G(d,p) level of theory.

Mapping the dark space of chemical reactions with extended nanomole synthesis and MALDI-TOF MS.

Understanding the practical limitations of chemical reactions is critically important for efficiently planning the synthesis of compounds in pharmaceutical, agrochemical, and specialty chemical research and development. However, literature reports of the scope of new reactions are often cursory and biased toward successful results, severely limiting the ability to predict reaction outcomes for untested substrates. We herein illustrate strategies for carrying out large-scale surveys of chemical reactivity by using a material-sparing nanomole-scale automated synthesis platform with greatly expanded synthetic scope combined with ultrahigh-throughput matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS).

Identification of ortho-Substituted Benzoic Acid/Ester Derivatives via the Gas-Phase Neighboring Group Participation Effect in (+)-ESI High Resolution Mass Spectrometry.

Benzoic acid/ester/amide derivatives are common moieties in pharmaceutical compounds and present a challenge in positional isomer identification by traditional tandem mass spectrometric analysis. A method is presented for exploiting the gas-phase neighboring group participation (NGP) effect to differentiate ortho-substituted benzoic acid/ester derivatives with high resolution mass spectrometry (HRMS1). Significant water/alcohol loss (>30% abundance in MS1 spectra) was observed for ortho-substituted nucleophilic groups; these fragment peaks are not observable for the corresponding para and meta-substituted analogs. Experiments were also extended to the analysis of two intermediates in the synthesis of suvorexant (Belsomra) with additional analysis conducted with nuclear magnetic resonance (NMR), density functional theory (DFT), and ion mobility spectrometry-mass spectrometry (IMS-MS) studies. Significant water/alcohol loss was also observed for 1-substituted 1, 2, 3-triazoles but not for the isomeric 2-substituted 1, 2, 3-triazole analogs. IMS-MS, NMR, and DFT studies were conducted to show that the preferred orientation of the 2-substituted triazole rotamer was away from the electrophilic center of the reaction, whereas the 1-subtituted triazole was oriented in close proximity to the center. Abundance of NGP product was determined to be a product of three factors: (1) proton affinity of the nucleophilic group; (2) steric impact of the nucleophile; and (3) proximity of the nucleophile to carboxylic acid/ester functional groups. Graphical Abstract ᅟ.

Rapid Characterization of Insulin Modifications and Sequence Variations by Proteinase K Digestion and UHPLC-ESI-MS.

Discovery of novel insulin analogs as therapeutics has remained an active area of research. Compared with native human insulin, insulin analog molecules normally incorporate either covalent modifications or amino acid sequence variations. From the drug discovery and development perspective, methods for efficient and detailed characterization of these primary structural changes are very important. In this report, we demonstrate that proteinase K digestion coupled with UPLC-ESI-MS analysis provides a simple and rapid approach to characterize the modifications and sequence variations of insulin molecules. A commercially available proteinase K digestion kit was used to process recombinant human insulin (RHI), insulin glargine, and fluorescein isothiocynate-labeled recombinant human insulin (FITC-RHI) samples. The LC-MS data clearly showed that RHI and insulin glargine samples can be differentiated, and the FITC modifications in all three amine sites of the RHI molecule are well characterized. The end-to-end experiment and data interpretation was achieved within 60 min. This approach is fast and simple, and can be easily implemented in early drug discovery laboratories to facilitate research on more advanced insulin therapeutics. Graphical Abstract ᅟ.

Substituent Effects on the Reactivity of the 2,4,6-Tridehydropyridinium Cation, an Aromatic σ,σ,σ-Triradical.

2,4,6-Tridehydropyridinium cation (7) undergoes three consecutive atom or atom group abstractions from reagent molecules in the gas phase. By placing a π-electron-donating hydroxyl group between two radical sites, their reactivity can be quenched by enhancing their through-space coupling via a favorable resonance structure. Indeed, 3-hydroxy-2,4,6-tridehydropyridinium cation (8) abstracts only one atom or group of atoms from reagents. On the other hand, an electron-withdrawing cyano group between two of the radical sites (9) destabilizes the analogous resonance structure and diminishes through-space coupling between the radical sites, resulting in abstraction of three atoms, just like 7. However, the cyano-substituent also increases acidity to the point that 9 reacts pre-dominantly via proton transfer instead of undergoing radical reactions. Therefore, acidic triradicals may undergo nonradical, barrierless proton transfer reactions faster than radical reactions, which are usually accompanied by barriers. Examination of the analogous cyano-substituted mono-and biradicals revealed behavior similar to that of the corresponding unsubstituted species, with the exception of substantially greater reactivities due to their greater (calculated) vertical electron affinities. Finally, the 3-cyano-2,6-didehydropyridinium cation with a singlet ground state (S-T splitting: -11.9 kcal mol-1) was found to react exclusively from the lowest-energy triplet state by fast proton transfer reactions.