JMS Letters Received: 18 April 2014
Revised: 10 October 2014
Accepted: 22 October 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3534
The competing radical eliminations in the tandem mass spectrometry of the OHdeprotonated benzyl vanillate Additional supporting information may be found in the online version of this article at the publisher’s web site.
Dear Sir,
432
Combination of electrospray ionization mass spectrometry (ESI-MS) with collision-induced dissociation (CID) technique has emerged as a particularly useful analytical tool for the structural elucidation of various compounds.[1–4] As a rule, the ions generated by ESI-MS are mainly even-electron (EE) ions with closed-shell electronic structures, which undergo the low energy CID to commonly afford the EE ions.[5–7] However, several exceptional cases have been documented in literatures over the years that dissociation of an EE ion affords an odd-electron (OE) ion in the CID-MS process, which has attracted the interest of many mass spectrometrists.[7–17] Generation of radical ions can be attributed to resonance stabilization of the product ions,[13] or the dynamically favorable process via an intramolecular electronic excitation of a transient structure, rather than a simple homolytic cleavage.[12,14,15] Katritzky et al. proposed a concept of the radical merostabilization, in which an electron-accepting group and an electron-donating group can significantly stabilize a radical.[17] Many phenolic plant metabolite compounds, such as ferulic acid and vanillic acid, exhibit antioxidative properties.[18,19] In our previous work, methyl radical elimination has been found to occur efficiently in dissociation of deprotonated ferulic acid, while it is inaccessible to lose ethyl radical from the deprotonated ethyl ferulate.[12] As part of a continuing effort to study the radical loss of the EE ions, a series of benzyl vanillate derivatives and benzyl isovanillate (Scheme 1) were designed for an ESI CID-MS investigation. The results demonstrated that two interesting competing eliminations of the benzyl and the methyl radicals are obtained in dissociation of the deprotonated benzyl vanillate. Methanol HPLC grade was purchased from Sigma-Aldrich (St. Louis, MO, USA). Vanillic acid (VA) and isovanillic acid (IVA) (purity 99.0%) were obtained from Sigma-Aldrich (Milwaukee, WI, USA). Benzyl vanillate derivatives (compounds 1–7), benzyl isovanillate (compound 8) and benzyl-d7 vanillate (compound 9) were synthesized by reaction of VA with the corresponding benzyl bromide, IVA with benzyl bromide and VA with benzyl-d7 bromide, respectively (Scheme 1).[20] All the compounds were purified after synthesis, and their structures were confirmed by NMR and high resolution mass spectrometry. The samples were analyzed on an LCQ advantage IT-MS equipped with an ESI interface in the negative ion mode (ThermoFisher, San Jose, CA). Every diluted solution (1 μg mL 1 in methanol) was infused into the source chamber at a flow rate of 5 μL min 1. The ESI source conditions were as follows: the
J. Mass Spectrom. 2015, 50, 432–436
ion-spray voltage, 4.5 kV, the nebulizing gas (N2), 25 arbitrary units (a.u.), the drying gas, 5 a.u. and the capillary temperature, 250 °C. The CID MS spectra of the deprotonated molecules were obtained by activation of the precursor ions at the normalized collision energy of 32%.† High-resolution mass spectrometry experiments were performed on a micrOTOF (time-of-flight) mass spectrometer (Bruker, Billerica, MA, USA), equipped with an ESI interface. Theoretical calculations were performed using the Gaussian 03 program.[21] The geometries of the target species were optimized using the density functional theory (DFT) method at the uB3LYP/6-31+G(d,p) level. The optimized structures were identified as a true minimum in energy by the absence of imaginary frequencies. Vibrational frequencies of all the key species were calculated at the same level of theory. Hard data on geometries of all structures involved are available as supplementary data. The energies discussed here are the sum of electronic and thermal free energy. The CID spectrum recorded from the m/z 257 ion for deprotonated benzyl vanillate ([1 H] ) is depicted in [Fig. 1(a)], and the CID-MS results for compound 1 are summarized in Table 1. The most abundant fragment peak at m/z 242 is assigned as 2-oxido-5-benzoxyl (oxo) methyl-phenoxyl, which results from the methyl radical elimination from [1 H] .[12] The product ion spectrum recorded from the m/z 242 ion shows an intense peak at m/z 151 via losing a benzyl radical [Fig. S1(a)]. The other dominant peak at m/z 166 is also proposed to be a radical anion, 2-methoxyl-4-carboxylate-phenoxyl, formed by the benzyl radical elimination from [1 H] via the homolytic cleavage of the ester C―O bond. The m/z 166 ion subsequently undergoes the CO2 elimination to afford the peak at m/z 122 [Fig. S1(b)]. The minor peak at m/z 213 is proposed to be generated through expulsion of CO2 from [1 H] . The elemental compositions of these proposed fragment ions
* Correspondence to: Zuguang Li, College of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310014, China. E-mail: lzg@zjut. edu.cn * Correspondence to: Kezhi Jiang, Key Lab of Organosilicon Chemistry and Material Technology, Hangzhou Normal University, Hangzhou, 31121, China. E-mail: [email protected] †
Note that the software of Finnigan ion trap applies a ‘normalized collision energy’ (see Finnigan Product Support Bulletin 104: http://www.thermo.com/ eThermo/CMA/PDFs/Articles/articlesFile_21418.pdf) by which mass of the target ion is automatically considered in the calculation of the excitation amplitude in the CID experiments.
Copyright © 2015 John Wiley & Sons, Ltd.
JMS Letters
Scheme 1. Structures of benzyl vanillate derivatives (1–7), benzyl isovanillate (8) and benzyl-d7 vanillate (9).
have been confirmed by high-resolution mass spectrometry experiments (Table S1 and Fig. S2). Thus, two predominant reactions involving the competing eliminations of methyl and benzyl radicals have been obtained in fragmentation of [1 H] (Scheme 2). In addition, results from tandem mass spectrometric experiment conducted with deuterium-labeled compound 9 has also consolidated the proposed fragmentation pathways [Fig. 1(c)]. As can be seen, the m/z value of the ion 1-b (bearing a benzyl group in the structure) is observed to shift to 249 from 242 in the CID spectrum of deprotonated compound 9 (Scheme 1), while there is no mass shift observed for the ion 1-a, suggesting the elimination of benzyl-d7 radical. Compounds 2–7 bearing different substituents on the paraposition of the benzyl ring (Scheme 1) have been designed for the tandem MS analysis to investigate the electronic effect on the competing radical eliminations. All of these compounds exhibit similar fragmentation patterns, whereas change of the substituent significantly affects the relative abundances of the radical anions (Table 1 and Fig. S3). On the whole, the presence of the electronwithdrawing groups effectively promotes the direct decomposition to lose the substituted benzyl radical (R-C6H4CH•2, Path-a), while that of the electron-donating group inhibits such fragmentation pathway. As shown in Fig. 2, the logarithmic values of the relative peak intensity ratios of the two competing product ions, ln[Intensity(C8H6O4 •)/Intensity(R-C14H9O4 •)], are in line with the Hammett substituent constants, σp .[22] To probe the effect of the substitution pattern on the competing radical eliminations, fragmentation of the deprotonated benzyl isovanillate [8 H] [Fig. 1(b)] has been investigated and compared with that of [1 H] . Interestingly,
Table 1. The collision-induced dissociation mass spectra data of the deprotonated benzyl vanillate derivatives (1–7) and benzyl isovanillate (8) at the normalized collision energy of 32% Compounds Substituent (R-)
[M H] m/z (%)
Path-a • C8H6O4 m/z (%)
1 2 3 4 5
257 (31.6) 287 (28.1) 271 (28.7) 275 (21.3) 291 (20.9) 293 (9.20) 335 (7.5) 337 (9.2) 302 (0.7) 257 (3.7)
166 (96.9) 166 (88.5) 166 (90.9) 166 (91.2) 166 (100) 166 (100) 166 (100) 166 (100) 166 (25.1) 166 (7.2)
6 7 8
H OCH3 CH3 F 35 Cl 37 Cl 79 Br 81 Br NO2 –
Path-b • R-C14H9O4 m/z (%) 242 (100) 272 (100) 256 (100) 260 (100) 276 (68.2) 278 (53.8) 320 (74.1) 322 (74.0) 287 (5.4) 242 (100)
the relative intensity of the m/z 257 from [8 H] is much lower than that from [1 H] (3.7% in [8 H] vs 31.6% in [1 H] ) under the same collisional energy, indicating a more fragile structure of [8 H] . The ESI-generated [8 H] undergoes dissociation to generate a single dominant peak at m/z 242 and several minor peaks at m/z 166, 151 and 122. The peak at m/z 166 (2-methoxyl-5-carboxylate-phenoxyl) resulting from the benzyl elimination from [8 H] is distinctly less abundant than the corresponding peak from [1 H] (7.2% in [8 H] vs 96.9% in [1 H] ), indicating that benzyl elimination from [8 H] is significantly less favorable than
433
Figure 1. Collision-induced dissociation mass spectra of [1 H] (a), [8 H] (b) and [9 H] (c).
J. Mass Spectrom. 2015, 50, 432–436
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JMS Letters
Scheme 2. Proposed reaction pathways for the fragmentation of [1 H] and [8 H] .
•
•
Figure 2. Plot of ln[Intensity (C8H6O4 )/Intensity (R-C14H9O4 )] versus the Hammett substituent constants σp in the CID-MS spectra of the deprotonated benzyl vanillate derivatives.
434
that from [1 H] . A comparison of the breakdown curves of [1 H] and [8 H] consolidates the above CID-MS results (Fig. S4). Thereby, the two isomers can be easily differentiated solely by tandem MS based on the significant differences of the relative intensity of the major fragment peaks. The mechanistic investigation of the above differences will be further performed in the succeeding section. Further scrutiny of the competing radical eliminations and the optimized structures of the key species was conducted by DFT theoretical calculations at the uB3LYP/6-31+G(d,p) level (Fig. S5). The schematic potential energy diagrams for the fragmentation pathways of [1 H] and [8 H] are depicted in Fig. 3. It should be noteworthy that the energy barrier for the homolytic cleavage is estimated by frequency analysis of the transient structure with the electronic excitation energy of almost zero in the potential energy surface scans of the breaking bond of
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Figure 3. The schematic potential energy diagrams for the fragmentation of [1 H] and [8 H] .
the precursor ion.[12] For the product ion 1-a (m/z 166), there are many resonance forms in structure (Scheme S1), of which the most predominant one is 1-a-h by analyzing the calculated results in Table 2, albeit direct fragmentation of [1 H] leads to 4-oxido-3-methoxyl-benzoyloxyl (1-a-a). Similarly, dissociation of [8 H] via homolytic cleavage of the ester C―O bond leads to 3-oxido-4-methoxyl-benzoyloxyl (8-a-a, m/z 166), but it subsequently undergoes electron transition to afford the ground state 8-a-h (Table 2 and Scheme S2). The spontaneous electron transition has been consolidated by analysis of the molecular orbits of 8-a, in which the HOMO, the LUMO and the (LUMO+1) orbits are mainly located at the carboxylic group (8-a-h), the phenyl group and the (phenyl and phenoxyl) moiety (8-a-a), respectively (Fig. S6). The negative charge in [1 H] can be delocalized over the phenyl and the carboxyl groups, while that in [8 H] is not
Copyright © 2015 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2015, 50, 432–436
JMS Letters Table 2. The Breneman charge distribution and the spin density distribution in the two radical fragment ions at m/z 166 from [1 H] and [8 H] Atom
Breneman charge Spin density Breneman charge Spin density C1 C2 C3 C4 C5 C6 C7 O8 O9 O10 O11 C12
0.257 0.284 0.016 0.110 0.242 0.479 0.850 0.775 0.600 0.795 0.383 0.260
0.289 0.200 0.419 0.114 0.227 0.040 0.041 0.056 0.324 0.030 0.047 0.003
0.506 0.301 0.034 0.010 0.306 0.262 0.863 0.809 0.356 0.790 0.590 0.137
0.005 0.179 0.129 0.377 0.189 0.342 0.003 0.021 0.069 0.010 0.353 0.002
Acknowledgements The authors gratefully acknowledge financial support from the National Science Foundation of China (21205025), the Department of Education of Zhejiang Province (Pd2013016), the Analysis and Detection Foundation of Science and Technology Department of Zhejiang Province (2013C37055) and the Key Innovation Team of Science and Technology in Zhejiang Province (2010R50018). Computer time was made available on the SGI Aktix 450 sever at Computational Center for Molecular Design of Organosilicon Compounds, Hangzhou Normal University. Yours, Xiaoping Zhang, a,b Peixi Zhu,c Huarong Zhang,b Zuguang Li,a* Kezhi Jiang,b* and Maw-Rong Leed a
College of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310014, China b
Key Laboratory of Organosilicon Chemistry and Material Technology, Hangzhou Normal University, Hangzhou 311121, China
c
Zhejiang Institute for Food and Drug Control, Hangzhou 310004, China d
Department of Chemistry, National Chung-Hsing University, Taichung 40227, Taiwan
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References [1] K. W. Cheng, C. C. Wong, M. Wang, Q. Y. He, F. Chen. Identification and characterization of molecular targets of natural products by mass spectrometry. Mass Spectrom. Rev. 2010, 29, 126. [2] A. S. Fang, X. Miao, P. W. Tidswell, M. H. Towle, W. K. Goetzinger, J. N. Kyranos. Mass spectrometry analysis of new chemical entities for pharmaceutical discovery. Mass Spectrom. Rev. 2008, 27, 20. [3] G. Y. He, X. Y. Xu, D. M. Fang, S. W. Luo, L. X. Wang, G. L. Zhang, Z. J. Wu. + Unexpected [M-H] ions in cyclopenta[b]indoles detection by electrospray ionization mass spectrometry. J. Mass Spectrom. 2013, 48, 1266. [4] E. Lattová, H. Perreault. The usefulness of hydrazine derivatives for mass spectrometric analysis of carbohydrates. Mass Spectrom. Rev. 2013, 32, 366. [5] A. B. Attygalle, J. Ruzicka, D. Varughese, J. B. Bialecki, S. Jafri. Low-energy collision-induced fragmentation of negative ions derived from ortho-, meta-, and para-hydroxyphenyl carbaldehydes, ketones, and related compounds. J. Mass Spectrom. 2007, 42, 1207. [6] K. Levsen, H. M. Schiebel, J. K. Terlouw, K. J. Jobst, M. Elend, A. Preiss, H. Thiele, A. Ingendoh. Even-electron ions: a systematic study of the neutral species lost in the dissociation of quasi-molecular ions. J. Mass Spectrom. 2007, 42, 1024. [7] A. B. Attygalle, J. B. Bialecki, U. Nishshanka, C. S. Weisbecker, J. Ruzicka. Loss of benzene to generate an enolate anion by a site-specific doublehydrogen transfer during CID fragmentation of o-alkyl ethers of orthohydroxybenzoic acids. J. Mass Spectrom. 2008, 43, 1224. [8] M. Chahma, X. Li, J. P. Phillips, P. Schwartz, L. E. Brammer, Y. Wang, J. M. Tanko. Activation/driving force relationships for cyclopropylcarbinyl→homoallyl-type rearrangements of radical anions. J. Phys. Chem. A 2005, 109, 3372. [9] J. Tanko, J. P. Phillips. Rearrangements of radical ions: what it means to be both a radical and an ion. J. Am. Chem. Soc. 1999, 121, 6078. [10] K. B. Herath, C. S. Weisbecker, S. B. Singh, A. B. Attygalle. Circumambulatory movement of negative charge ("ring walk") during gas-phase dissociation of 2,3,4-trimethoxybenzoate anion. J. Org. Chem. 2014, DOI: 10.1021/jo500234w.
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dispersed to the carboxyl group (Table S2). Thus, [8 H] lies 41.7 kJ/mol above [1 H] in free energy, while their product ions at m/z 242 via the methyl radical loss are the same radical anion with the different resonance forms (Scheme 2), indicating that [8 H] is thermodynamically more favorable to undergo methyl elimination than [1 H] . As shown Fig. 3, the energy barrier for methyl elimination (path-b, 195.4 kJ mol 1) is slightly less that that of benzyl elimination (path-a, 217.0 kJ mol 1) in dissociation of [1 H] , whereas the reaction pathway of path-b is thermodynamically more favorable than that of path-a by 14.3 kJ mol 1. Thus, both occur efficiently in the fragmentation of [1 H] . In contrast, methyl elimination of [8 H] has the estimated energy barrier of 172.2 kJ mol 1, which is significantly more feasible than the benzyl elimination of [8 H] . The above calculation results are in a good agreement with the CID-MS data. In this study, an interesting benzyl radical elimination, competing with methyl radical elimination, was obtained and proved in gas-phase dissociation of the deprotonated benzyl vanillate (1). These two competing radical elimination pathways are significantly affected by the substitution pattern (8) and the electronic effect of different substituent groups (2–7). Elimination of benzyl radical from [1 H] was significantly more favorable than that from [8 H] , indicating that the two isomers can be differentiated solely by tandem MS. In addition, the presence of the electron-withdrawing groups (R) on the para-position of the benzyl ring effectively promotes the direct decomposition to lose the substituted benzyl radical (R-C6H4CH•2), while that of the electron-donating group inhibits such fragmentation pathway. Last, these observations are important to the MS-based isomeric differentiation and the proper interpretation of the mass spectra of related compounds. Further works in this field will be carried out in our laboratory.
JMS Letters [11] Y. Liu, J. He, R. Zhang, J. Shi, Z. Abliz. Study of the characteristic fragmentation behavior of hydroquinone glycosides by electrospray ionization tandem mass spectrometry with optimization of collision energy. J. Mass Spectrom. 2009, 44, 1182. [12] X. Zhang, F. Li, H. Lv, Y. Wu, G. Bian, K. Jiang. On the origin of the methyl radical loss from deprotonated ferulic and isoferulic acids: electronic excitation of a transient structure. J. Am. Soc. Mass Spectrom. 2013, 24, 941. [13] Y. Cai, Z. Mo, N. S. Rannulu, B. Guan, S. Kannupal, B. C. Gibb, R. B. Cole. Characterization of an exception to the ’even-electron rule’ upon low-energy collision induced decomposition in negative ion electrospray tandem mass spectrometry. J. Mass Spectrom. 2010, 45, 235. [14] M. Butler, P. A. Mañez, G. M. Cabrera. An experimental and computational study on the dissociation behavior of hydroxypyridine N-oxides in atmospheric pressure ionization mass spectrometry. J. Mass Spectrom. 2010, 45, 536. [15] C. Denekamp, E. Tenetov, Y. Horev. Homolytic cleavages in pyridinium ions, an excited state process. J. Am. Soc. Mass Spectrom. 2003, 14, 790. [16] R. Vessecchi, Z. Naal, J. N. Lopes, S. E. Galembeck, N. P. Lopes. Generation of naphthoquinone radical anions by electrospray ionization: solution, gas-phase, and computational chemistry studies. J. Phys. Chem. A 2011, 115, 5453. [17] A. R. Katritzky, P. A. Shipkova, M. Qi, D. A. Nichols, R. D. Burton, C. H. Watson, J. R. Eyler, T. Tamm, M. Karelson, M. C. Zerner. Study of radical merostabilization by electrospray FTICR/MS. J. Am. Chem. Soc. 1996, 118, 11905. [18] H. Palafox-Carlos, J. Gil-Chávez, R. R. Sotelo-Mundo, J. Namiesnik, S. Gorinstein, G. A. González-Aguilar. Antioxidant interactions between major phenolic compounds found in ‘Ataulfo’ mango pulp: chlorogenic, gallic, protocatechuic and vanillic acids. Molecules 2012, 17, 12657.
[19] M. Alzweiri, Y. AI-Hiari. Evaluation of vanillic acid as inhibitor of carbonic anhydrase isozyme III by using a modified HummelDreyer method: approach for drug discovery. Biomed. Chromatogr. 2013, 27, 1157. [20] Q. Zhang, K. S. Raheem, N. P. Botting, A. M. Slawin, C. D. Kay, D. O’Hagan. Flavonoid metabolism: the synthesis of phenolic glucuronides and sulfates as candidate metabolites for bioactivity studies of dietary flavonoids. Tetrahedron 2012, 68, 4194. [21] M. Frisch,G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, V. G. Zakrzewski, J. A. Montgomery, Jr., R. E. Stratmann, J. C. Burant, S. Dapprich, J. M. Millam, A. D. Daniels, K. N. Kudin, M. C. Strain, O. Farkas, J. Tomasi, V. Barone, M. Cossi, R. Cammi, B. Mennucci, C. Pomelli, C. Adamo, S. Clifford, J. Ochterski, G. A. Petersson, P. Y. Ayala, Q. Cui, K. Morokuma, D. K. Malick, A. D. Rabuck, K. Raghavachari, J. B. Foresman, J. Cioslowski, J. V. Ortiz, B. B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R. Gomperts, R. L. Martin, D. J. Fox, T. Keith, M. A. Al-Laham, C. Y. Peng, A. Nanayakkara, C. Gonzalez, M. Challacombe, P. M. W. Gill, B. Johnson, W. Chen, M. W. Wong, J. L. Andres, C. Gonzalez, M. E. Head-Gordon, S. Replogle, J. A. Pople. Gaussian 03, Revision B.03, Gaussian, Inc.: Wallingford, CT, 2004. [22] C. Hansch, A. Leo, R. Taft. A survey of Hammett substituent constants and resonance and field parameters. Chem. Rev. 1991, 91, 165.
Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web site.
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Copyright © 2015 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2015, 50, 432–436
Revised: 10 October 2014
Accepted: 22 October 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3534
The competing radical eliminations in the tandem mass spectrometry of the OHdeprotonated benzyl vanillate Additional supporting information may be found in the online version of this article at the publisher’s web site.
Dear Sir,
432
Combination of electrospray ionization mass spectrometry (ESI-MS) with collision-induced dissociation (CID) technique has emerged as a particularly useful analytical tool for the structural elucidation of various compounds.[1–4] As a rule, the ions generated by ESI-MS are mainly even-electron (EE) ions with closed-shell electronic structures, which undergo the low energy CID to commonly afford the EE ions.[5–7] However, several exceptional cases have been documented in literatures over the years that dissociation of an EE ion affords an odd-electron (OE) ion in the CID-MS process, which has attracted the interest of many mass spectrometrists.[7–17] Generation of radical ions can be attributed to resonance stabilization of the product ions,[13] or the dynamically favorable process via an intramolecular electronic excitation of a transient structure, rather than a simple homolytic cleavage.[12,14,15] Katritzky et al. proposed a concept of the radical merostabilization, in which an electron-accepting group and an electron-donating group can significantly stabilize a radical.[17] Many phenolic plant metabolite compounds, such as ferulic acid and vanillic acid, exhibit antioxidative properties.[18,19] In our previous work, methyl radical elimination has been found to occur efficiently in dissociation of deprotonated ferulic acid, while it is inaccessible to lose ethyl radical from the deprotonated ethyl ferulate.[12] As part of a continuing effort to study the radical loss of the EE ions, a series of benzyl vanillate derivatives and benzyl isovanillate (Scheme 1) were designed for an ESI CID-MS investigation. The results demonstrated that two interesting competing eliminations of the benzyl and the methyl radicals are obtained in dissociation of the deprotonated benzyl vanillate. Methanol HPLC grade was purchased from Sigma-Aldrich (St. Louis, MO, USA). Vanillic acid (VA) and isovanillic acid (IVA) (purity 99.0%) were obtained from Sigma-Aldrich (Milwaukee, WI, USA). Benzyl vanillate derivatives (compounds 1–7), benzyl isovanillate (compound 8) and benzyl-d7 vanillate (compound 9) were synthesized by reaction of VA with the corresponding benzyl bromide, IVA with benzyl bromide and VA with benzyl-d7 bromide, respectively (Scheme 1).[20] All the compounds were purified after synthesis, and their structures were confirmed by NMR and high resolution mass spectrometry. The samples were analyzed on an LCQ advantage IT-MS equipped with an ESI interface in the negative ion mode (ThermoFisher, San Jose, CA). Every diluted solution (1 μg mL 1 in methanol) was infused into the source chamber at a flow rate of 5 μL min 1. The ESI source conditions were as follows: the
J. Mass Spectrom. 2015, 50, 432–436
ion-spray voltage, 4.5 kV, the nebulizing gas (N2), 25 arbitrary units (a.u.), the drying gas, 5 a.u. and the capillary temperature, 250 °C. The CID MS spectra of the deprotonated molecules were obtained by activation of the precursor ions at the normalized collision energy of 32%.† High-resolution mass spectrometry experiments were performed on a micrOTOF (time-of-flight) mass spectrometer (Bruker, Billerica, MA, USA), equipped with an ESI interface. Theoretical calculations were performed using the Gaussian 03 program.[21] The geometries of the target species were optimized using the density functional theory (DFT) method at the uB3LYP/6-31+G(d,p) level. The optimized structures were identified as a true minimum in energy by the absence of imaginary frequencies. Vibrational frequencies of all the key species were calculated at the same level of theory. Hard data on geometries of all structures involved are available as supplementary data. The energies discussed here are the sum of electronic and thermal free energy. The CID spectrum recorded from the m/z 257 ion for deprotonated benzyl vanillate ([1 H] ) is depicted in [Fig. 1(a)], and the CID-MS results for compound 1 are summarized in Table 1. The most abundant fragment peak at m/z 242 is assigned as 2-oxido-5-benzoxyl (oxo) methyl-phenoxyl, which results from the methyl radical elimination from [1 H] .[12] The product ion spectrum recorded from the m/z 242 ion shows an intense peak at m/z 151 via losing a benzyl radical [Fig. S1(a)]. The other dominant peak at m/z 166 is also proposed to be a radical anion, 2-methoxyl-4-carboxylate-phenoxyl, formed by the benzyl radical elimination from [1 H] via the homolytic cleavage of the ester C―O bond. The m/z 166 ion subsequently undergoes the CO2 elimination to afford the peak at m/z 122 [Fig. S1(b)]. The minor peak at m/z 213 is proposed to be generated through expulsion of CO2 from [1 H] . The elemental compositions of these proposed fragment ions
* Correspondence to: Zuguang Li, College of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310014, China. E-mail: lzg@zjut. edu.cn * Correspondence to: Kezhi Jiang, Key Lab of Organosilicon Chemistry and Material Technology, Hangzhou Normal University, Hangzhou, 31121, China. E-mail: [email protected] †
Note that the software of Finnigan ion trap applies a ‘normalized collision energy’ (see Finnigan Product Support Bulletin 104: http://www.thermo.com/ eThermo/CMA/PDFs/Articles/articlesFile_21418.pdf) by which mass of the target ion is automatically considered in the calculation of the excitation amplitude in the CID experiments.
Copyright © 2015 John Wiley & Sons, Ltd.
JMS Letters
Scheme 1. Structures of benzyl vanillate derivatives (1–7), benzyl isovanillate (8) and benzyl-d7 vanillate (9).
have been confirmed by high-resolution mass spectrometry experiments (Table S1 and Fig. S2). Thus, two predominant reactions involving the competing eliminations of methyl and benzyl radicals have been obtained in fragmentation of [1 H] (Scheme 2). In addition, results from tandem mass spectrometric experiment conducted with deuterium-labeled compound 9 has also consolidated the proposed fragmentation pathways [Fig. 1(c)]. As can be seen, the m/z value of the ion 1-b (bearing a benzyl group in the structure) is observed to shift to 249 from 242 in the CID spectrum of deprotonated compound 9 (Scheme 1), while there is no mass shift observed for the ion 1-a, suggesting the elimination of benzyl-d7 radical. Compounds 2–7 bearing different substituents on the paraposition of the benzyl ring (Scheme 1) have been designed for the tandem MS analysis to investigate the electronic effect on the competing radical eliminations. All of these compounds exhibit similar fragmentation patterns, whereas change of the substituent significantly affects the relative abundances of the radical anions (Table 1 and Fig. S3). On the whole, the presence of the electronwithdrawing groups effectively promotes the direct decomposition to lose the substituted benzyl radical (R-C6H4CH•2, Path-a), while that of the electron-donating group inhibits such fragmentation pathway. As shown in Fig. 2, the logarithmic values of the relative peak intensity ratios of the two competing product ions, ln[Intensity(C8H6O4 •)/Intensity(R-C14H9O4 •)], are in line with the Hammett substituent constants, σp .[22] To probe the effect of the substitution pattern on the competing radical eliminations, fragmentation of the deprotonated benzyl isovanillate [8 H] [Fig. 1(b)] has been investigated and compared with that of [1 H] . Interestingly,
Table 1. The collision-induced dissociation mass spectra data of the deprotonated benzyl vanillate derivatives (1–7) and benzyl isovanillate (8) at the normalized collision energy of 32% Compounds Substituent (R-)
[M H] m/z (%)
Path-a • C8H6O4 m/z (%)
1 2 3 4 5
257 (31.6) 287 (28.1) 271 (28.7) 275 (21.3) 291 (20.9) 293 (9.20) 335 (7.5) 337 (9.2) 302 (0.7) 257 (3.7)
166 (96.9) 166 (88.5) 166 (90.9) 166 (91.2) 166 (100) 166 (100) 166 (100) 166 (100) 166 (25.1) 166 (7.2)
6 7 8
H OCH3 CH3 F 35 Cl 37 Cl 79 Br 81 Br NO2 –
Path-b • R-C14H9O4 m/z (%) 242 (100) 272 (100) 256 (100) 260 (100) 276 (68.2) 278 (53.8) 320 (74.1) 322 (74.0) 287 (5.4) 242 (100)
the relative intensity of the m/z 257 from [8 H] is much lower than that from [1 H] (3.7% in [8 H] vs 31.6% in [1 H] ) under the same collisional energy, indicating a more fragile structure of [8 H] . The ESI-generated [8 H] undergoes dissociation to generate a single dominant peak at m/z 242 and several minor peaks at m/z 166, 151 and 122. The peak at m/z 166 (2-methoxyl-5-carboxylate-phenoxyl) resulting from the benzyl elimination from [8 H] is distinctly less abundant than the corresponding peak from [1 H] (7.2% in [8 H] vs 96.9% in [1 H] ), indicating that benzyl elimination from [8 H] is significantly less favorable than
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Figure 1. Collision-induced dissociation mass spectra of [1 H] (a), [8 H] (b) and [9 H] (c).
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JMS Letters
Scheme 2. Proposed reaction pathways for the fragmentation of [1 H] and [8 H] .
•
•
Figure 2. Plot of ln[Intensity (C8H6O4 )/Intensity (R-C14H9O4 )] versus the Hammett substituent constants σp in the CID-MS spectra of the deprotonated benzyl vanillate derivatives.
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that from [1 H] . A comparison of the breakdown curves of [1 H] and [8 H] consolidates the above CID-MS results (Fig. S4). Thereby, the two isomers can be easily differentiated solely by tandem MS based on the significant differences of the relative intensity of the major fragment peaks. The mechanistic investigation of the above differences will be further performed in the succeeding section. Further scrutiny of the competing radical eliminations and the optimized structures of the key species was conducted by DFT theoretical calculations at the uB3LYP/6-31+G(d,p) level (Fig. S5). The schematic potential energy diagrams for the fragmentation pathways of [1 H] and [8 H] are depicted in Fig. 3. It should be noteworthy that the energy barrier for the homolytic cleavage is estimated by frequency analysis of the transient structure with the electronic excitation energy of almost zero in the potential energy surface scans of the breaking bond of
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Figure 3. The schematic potential energy diagrams for the fragmentation of [1 H] and [8 H] .
the precursor ion.[12] For the product ion 1-a (m/z 166), there are many resonance forms in structure (Scheme S1), of which the most predominant one is 1-a-h by analyzing the calculated results in Table 2, albeit direct fragmentation of [1 H] leads to 4-oxido-3-methoxyl-benzoyloxyl (1-a-a). Similarly, dissociation of [8 H] via homolytic cleavage of the ester C―O bond leads to 3-oxido-4-methoxyl-benzoyloxyl (8-a-a, m/z 166), but it subsequently undergoes electron transition to afford the ground state 8-a-h (Table 2 and Scheme S2). The spontaneous electron transition has been consolidated by analysis of the molecular orbits of 8-a, in which the HOMO, the LUMO and the (LUMO+1) orbits are mainly located at the carboxylic group (8-a-h), the phenyl group and the (phenyl and phenoxyl) moiety (8-a-a), respectively (Fig. S6). The negative charge in [1 H] can be delocalized over the phenyl and the carboxyl groups, while that in [8 H] is not
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JMS Letters Table 2. The Breneman charge distribution and the spin density distribution in the two radical fragment ions at m/z 166 from [1 H] and [8 H] Atom
Breneman charge Spin density Breneman charge Spin density C1 C2 C3 C4 C5 C6 C7 O8 O9 O10 O11 C12
0.257 0.284 0.016 0.110 0.242 0.479 0.850 0.775 0.600 0.795 0.383 0.260
0.289 0.200 0.419 0.114 0.227 0.040 0.041 0.056 0.324 0.030 0.047 0.003
0.506 0.301 0.034 0.010 0.306 0.262 0.863 0.809 0.356 0.790 0.590 0.137
0.005 0.179 0.129 0.377 0.189 0.342 0.003 0.021 0.069 0.010 0.353 0.002
Acknowledgements The authors gratefully acknowledge financial support from the National Science Foundation of China (21205025), the Department of Education of Zhejiang Province (Pd2013016), the Analysis and Detection Foundation of Science and Technology Department of Zhejiang Province (2013C37055) and the Key Innovation Team of Science and Technology in Zhejiang Province (2010R50018). Computer time was made available on the SGI Aktix 450 sever at Computational Center for Molecular Design of Organosilicon Compounds, Hangzhou Normal University. Yours, Xiaoping Zhang, a,b Peixi Zhu,c Huarong Zhang,b Zuguang Li,a* Kezhi Jiang,b* and Maw-Rong Leed a
College of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310014, China b
Key Laboratory of Organosilicon Chemistry and Material Technology, Hangzhou Normal University, Hangzhou 311121, China
c
Zhejiang Institute for Food and Drug Control, Hangzhou 310004, China d
Department of Chemistry, National Chung-Hsing University, Taichung 40227, Taiwan
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dispersed to the carboxyl group (Table S2). Thus, [8 H] lies 41.7 kJ/mol above [1 H] in free energy, while their product ions at m/z 242 via the methyl radical loss are the same radical anion with the different resonance forms (Scheme 2), indicating that [8 H] is thermodynamically more favorable to undergo methyl elimination than [1 H] . As shown Fig. 3, the energy barrier for methyl elimination (path-b, 195.4 kJ mol 1) is slightly less that that of benzyl elimination (path-a, 217.0 kJ mol 1) in dissociation of [1 H] , whereas the reaction pathway of path-b is thermodynamically more favorable than that of path-a by 14.3 kJ mol 1. Thus, both occur efficiently in the fragmentation of [1 H] . In contrast, methyl elimination of [8 H] has the estimated energy barrier of 172.2 kJ mol 1, which is significantly more feasible than the benzyl elimination of [8 H] . The above calculation results are in a good agreement with the CID-MS data. In this study, an interesting benzyl radical elimination, competing with methyl radical elimination, was obtained and proved in gas-phase dissociation of the deprotonated benzyl vanillate (1). These two competing radical elimination pathways are significantly affected by the substitution pattern (8) and the electronic effect of different substituent groups (2–7). Elimination of benzyl radical from [1 H] was significantly more favorable than that from [8 H] , indicating that the two isomers can be differentiated solely by tandem MS. In addition, the presence of the electron-withdrawing groups (R) on the para-position of the benzyl ring effectively promotes the direct decomposition to lose the substituted benzyl radical (R-C6H4CH•2), while that of the electron-donating group inhibits such fragmentation pathway. Last, these observations are important to the MS-based isomeric differentiation and the proper interpretation of the mass spectra of related compounds. Further works in this field will be carried out in our laboratory.
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