IonâMolecule Reactions for Mass Spectrometric Identification of

Anal. Chem. 2004, 76, 964-976

Ion-Molecule Reactions for Mass Spectrometric Identification of Functional Groups in Protonated Oxygen-Containing Monofunctional Compounds Michael A. Watkins,† Jason M. Price,†,‡ Brian E. Winger,§ and Hilkka I. Kentta 1 maa*,†

Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, and Eli Lilly and Company, Indianapolis, Indiana 46285

Protonated oxygen-containing monofunctional compounds react with selected methoxyborane reagents by proton transfer followed by nucleophilic substitution of methanol at the boron atom in a Fourier transform ion cyclotron resonance mass spectrometer. The derivatized oxygen functionality can be identified by H/D exchange, collisionactivated dissociation, or both. This information on the identity of the functionalities in the analyte, in conjunction with molecular formula information obtained from exact mass measurements on either the protonated or derivatized analyte, facilitates structure elucidation of unknown organic compounds in a mass spectrometer. In the past two decades, mass spectrometry (MS) has contributed greatly to the area of mixture analysis. This is primarily due to the advent of “soft” ionization techniques, such as electrospray ionization (ESI), which lead to little or no fragmentation.1 ESI provides a convenient way to volatilize and ionize condensed-phase mixture components by the addition or removal of a proton. These features have led to the widespread employment of ESI-MS for the analysis of complex mixtures in both academic and industrial laboratories.2 Mixture analyses often require the identification of each individual mixture component. Employing techniques such as exact mass measurements and collision-activated dissociation (CAD), the elemental composition and some connectivity information, respectively, can be obtained by using mass spectrometry. This information is sometimes sufficient for the elucidation of the structures of the mixture components. However, often the identity of the functional groups in mixture components still remains elusive. The employment of spectroscopic techniques, such as NMR, FT-IR, and X-ray crystallography, can provide elemental connectivity information and complete the structure elucidation process. These methods, however, require the isolation of each mixture component prior to analysis. Yet some samples contain mixture components that are present in very low abundances. * To whom correspondence should be addressed. E-mail: [email protected]. † Purdue University. ‡ Present address: Procter & Gamble, Cincinnati, OH, 45201. § Eli Lilly and Co. (1) Cole, R. B. Electrospray Ionization Mass Spectrometry; John Wiley & Sons: New York, 1997. (2) Fenn, J. B., Presented at the 2002 Nobel Prize Ceremony, Stockholm, Sweden, October 9, 2002.

964 Analytical Chemistry, Vol. 76, No. 4, February 15, 2004

In these cases, rather large quantities of sample are required in order to isolate enough of each component for analysis due to the low sensitivity of these spectroscopic methods.3 Obtaining samples in this quantity may prove to be extremely expensive, if not impossible. Furthermore, processing such large sample quantities may be slow, tedious, or impractical. Hence, it is desirable to obtain more detailed structural information, especially information concerning the functionalities present, by rapid direct mass spectrometric analysis of mixtures introduced and ionized by ESI. In contrast to the above spectroscopic techniques, mass spectrometry is a very sensitive technique and typically requires less than 1 µg of material for analysis.3 The usefulness of ion-molecule reactions for functional group identification in mass spectrometry has attracted interest for a long time. There are many documented cases of ions that react preferentially with specific functionalities in neutral analyte molecules.4 In fact, the bulk of the literature focuses on the identification of functionalities in neutral molecules by using selective ionic reagents. Very few reports have appeared wherein neutral reagents are being used to identify the functionalities present in a protonated analyte.5,6 Herein we introduce a generally useful approach for obtaining functional group information for protonated monofunctional oxygen-containing compounds in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. EXPERIMENTAL SECTION Instrumentation. All experiments were performed using an Extrel model FTMS 2001 dual-cell FT-ICR mass spectrometer equipped with a 3-T superconducting magnet, a Finnigan Odyssey data station, and inlets for introduction of solid, liquid, and gaseous (3) Winger, B. E.; Kemp, C. A. J. Am. Pharm. Rev. 2001, 4, 55-63. (4) For examples of documented cases of ions reacting preferentially with specific functionalities in neutral compounds, see: (a) Donovan, T.; Brodbelt, J. Biol. Mass Spectrom. 1992, 21, 254-258. (b) Eichmann, E. S.; Brodbelt, J. S. Org. Mass Spectrom. 1993, 28, 1608-1615. (c) Eichmann, E. S.; Brodbelt, J. S. J. Am. Soc. Mass Spectrom. 1993, 4, 97-105. (d) Alvarez, E. J.; Brodbelt, J. S. J. Mass Spectrom. 1995, 30, 625-631. (e) Ramos, L. E.; Cardoso, A. M.; Correia, A. J. F.; Nibbering, N. M. M. Int. J. Mass Spectrom. 2000, 203, 101-110. (f) Moraes, L. A. B.; Gozzo, F. C.; Eberlin, M. N.; Vainiotalo, P. J. Org. Chem. 1997, 62, 5096-5103. (g) Reid, G. E.; Tichy, S. E.; Perez, J.; O’Hair, R. A.; Simpson, R. J.; Kentta¨maa, H. I. J. Am. Chem. Soc. 2001, 123, 1184-1192. (5) Kentta¨maa, H. I.; Cooks, R. G. J. Am. Chem. Soc. 1989, 111, 4122-4123. (6) Kentta¨maa, H. I.; Pachuta, R. R.; Rothwell, A. P.; Cooks, R. G. J. Am. Chem. Soc. 1989, 111, 1654-1665. 10.1021/ac034946d CCC: $27.50

© 2004 American Chemical Society Published on Web 01/10/2004

reagents. One of the key features of this instrument is that it contains a differentially pumped dual cell.7 The two cells share a common trap plate that can be temporarily held at 0 V, allowing ions to be transferred from one cell into the other through a 2-mm hole in the center of this plate. The efficiency of this transfer event is often increased by the use of quadrupolar axialization (QA) for axial and radial ion cloud compression prior to transfer.8 Furthermore, QA is inherently mass-selective and hence isolates the desired ion population from unwanted ions prior to transfer. The dual cell allows for one cell region to be used for ion generation while the other is used for ion-molecule reactions and detection. A series of protonated monofunctional alcohols, ketones, aldehydes, esters, ethers, carboxylic acids, amides, and amines were studied. All reagents used were purchased from SigmaAldrich and used as received. Since the FT-ICR mass spectrometer used for these experiments is not equipped with an ESI source, the ions were generated in one of the cells by self-protonation chemical ionization (CI), or “self-CI”. This was accomplished by allowing the molecular ion and its ionic fragments, produced upon electron ionization (25 eV electron energy; 500 ms) of the analyte, to react (∼3 s) with the analyte (3 × 10-8 Torr nominal pressure). The ions were transferred into the other cell where they were allowed to cool (0.5 s) by IR photon emission9 and then react with selected neutral reagents present at static pressures of ∼3.5 × 10-8 Torr (nominal pressure). The structures of the reaction products were probed by subsequent isolation via a series of stored-waveform inverse Fourier transform10 (SWIFT) excitation pulses followed by additional ion-molecule reactions or CAD. The CAD experiments performed herein utilized on-resonance excitation of the ion (both 10B and 11B isotopes were individually analyzed) for ∼300 µs in the presence of an inert target gas (∼10-5 Torr of argon). All spectra were subjected to background subtraction. The background spectra were generated by SWIFT ejection of the ion of interest prior to reaction time or CAD. Kinetics. Reactions studied under the conditions described above inherently follow pseudo-first-order kinetics. The secondorder reaction rate constants (kreaction) of some of the reactions studied were extracted from the semilogarithmic plot of the relative abundance of the reactant ion over time. The theoretical collision rate constants (kcollision) were calculated using a parametrized trajectory theory.11 Reaction efficiencies (the fraction of collisions leading to reaction; a reaction with an efficiency of 1 proceeds at collision rate) are given as kreaction/kcollision. The reagent pressures used to obtain the second-order reaction rate constants (kreaction) were measured using ion gauges. These values were corrected for the sensitivity of the ion gauge toward each neutral reagent12 and its distance from the center of the ICR cell13 by measuring the rates of highly exothermic electron-transfer reactions between the argon radical cation and the reagent of interest. (7) Littlejohn, D. P.; Ghaderi, S. U.S. Patent 4,581,533, 1986. (8) Schweikhard, L.; Guan, S.; Marshall, A. G. Int. J. Mass Spectrom. Ion Processes 1992, 120, 71-83. (9) Dunbar, R. C. Mass Spectrom. Rev. 1992, 11, 309-339. (10) Marshall, A. G.; Wang, T. C. L.; Chen, L.; Ricca, T. L. ACS Symp. Ser. 1987, No. 359, 21-33. (11) Su, T.; Chesnavich, W. J. J. Chem. Phys. 1982, 76, 5183-5185. (12) Bartmess, J. E.; Georgiadis, R. M. Vacuum 1983, 33, 149-153. (13) For examples, see (and references therein): (a) Leeck, D. T.; Stirk, K. M.; Zeller, L. C.; Kiminkinen, L. K. M.; Castro, L. M.; Vainiotalo, P.; Kentta¨maa, H. I. J. Am. Chem. Soc. 1994, 116, 3028-3038. (b) Tho¨lmann, D.; Gru ¨ tzmacher, H.-F. J. Am. Chem. Soc. 1991, 113, 3281-3287.

This reaction is assumed to proceed at collision rate; therefore, its efficiency is adjusted to 1 (or 100%). Computational Studies. All theoretical energies reported in this work were calculated with the Gaussian 98 suite of programs.14 Geometry optimizations and vibrational frequency calculations of stationary points on the potential energy surface (PES) of the reaction of protonated ethanol with diethylmethoxyborane were performed using density functional theory at the BLYP/631+G(d,p) level of theory. Since the proposed substitution reaction mechanism involves proton transfer and nucleophilic addition into an empty boron p-orbital, polarization and diffuse functions were added to the 6-31G split valence basis set. The proton affinity (PA) of diethylmethoxyborane was calculated at the BLYP/6-311G(d,p) level of theory by using protonated methanol15 as the Brønsted acid in an isodesmic reaction scheme. The synchronous transitguided quasi-Newton (STQN) method16 was employed at the BLYP/6-31G(d) level of theory to locate the transition-state structures for the amide and amine PESs. All stationary points were verified by vibrational frequency analysis to possess the correct number of imaginary frequencies: 0 for minimums, 1 for transition states. All theoretical energies are presented at 0 K and include zero-point vibrational energy corrections. RESULTS AND DISCUSSION Neutral Reagent. The desired characteristics of the neutral reagent used for functional group identification include the following: (1) it reacts universally and rapidly with functional groups that have some common feature (i.e., contain oxygen) and derivatizes only these groups, while screening out others (e.g., amino), and (2) it modifies the functional groups (e.g., hydroxyl, carbonyl, etc.) in such a way that they are distinguishable via further mass spectrometric experiments. The functional groups to be identified are part of a protonated molecule when ESI is used in positive ion mode to introduce and ionize the molecules. Most ion-molecule reactions are controlled by the charged site of a molecule,17,18 and by far the most common type of an ion-molecule reaction observed for protonated molecules is an acid/base reaction.19,20 In fact, most compounds either do not react with protonated molecules or react only by abstraction of a proton. To take advantage of this selectivity, controlled by the acid/base properties of the protonated analyte and the neutral (14) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A., Jr.; Stratmann, R. E.; Buran, J. C.; Dapprich, S.; Millam, J. M.; Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.; Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz, J. V.; Baboul, A. G.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Andres, J. L.; Gonzalez, C.; Head-Gordon, M.; Replogle, E. S.; Pople, J. A. Gaussian 98, Revision A.9; Gaussian: Pittsburgh, PA, 1998. (15) Hunter, E. P.; Lias, S. G. Proton Affinity Evaluation. In NIST Chemistry WebBook, NIST Standard Reference Database Number 69; Linstrom, P. J., Mallard, W. G., Eds.; National Institute of Standards and Technology: Gaithersburg, MD, 20899, March 2003; http://webbook.nist.gov. (16) Peng, C.; Schlegel, H. B. Isr. J. Chem. 1994, 33, 449-454. (17) Nibbering, N. M. M. Acc. Chem. Res. 1990, 23, 279-285. (18) Gronert, S. Chem. Rev. 2001, 101, 329-360. (19) Munson, M. S. B.; Field, F. H. J. Am. Chem. Soc. 1966, 88, 2621-2630. (20) Munson, M. S. B.; Franklin, J. L.; Field, F. H. J. Phys. Chem. 1964, 68, 3098-3107.

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Scheme 1

reagent, deprotonation was chosen to be the first step of the analytical reaction. The next step of the reaction was designed to be a fast substitution of the protonated moiety of the reagent by the analyte, while both species are still in the solvation complex. Therefore, this reaction leads to derivatization of the unknown functional group. The first attempt at employing this type of a reaction scheme for the structural elucidation of analytes with oxygen-containing functionalities involved using ethyl vinyl ether as the neutral reagent. The basic vinyl group was to be used to deprotonate the molecule of interest. This would make the R-carbon of the ethyl group electrophilic and available as a center for a substitution reaction, with acetaldehyde as the leaving group.6 However, this SN2 reaction is slow relative to proton transfer, and only the protontransfer reaction product was observed (Scheme 1). A more electrophilic and less sterically crowded center than an sp3-carbon is needed to promote rapid substitution. Boranes contain a boron atom with an empty p-orbital and hence are often rapidly attacked by nucleophiles. Therefore, a commercially available borane, diethylmethoxyborane, was chosen for the initial experiments. This compound possesses a methoxy oxygen to serve as the basic site and an electron-deficient boron center susceptible to nucleophilic attack. The methoxy group was chosen over other possible functionalities, such as amino, because it is basic enough to deprotonate protonated oxygen-containing compounds within a gas-phase collision complex but not so basic that separated protontransfer products would be formed in large abundance. Subsequent examination of this reagent proved its ability to react by proton abstraction followed by nucleophilic substitution (see Figure 1 for an example; see Scheme 2 for a proposed mechanism). In addition, theoretical studies showed that proton transfer to diethylmethoxyborane is essentially a barrierless process for protonated analytes having proton affinities less than that of diethylmethoxyborane (PAcalc ) 191.4 kcal/mol; BLYP/ 6-311G(d,p)). This makes the nucleophilic addition the first barriered reaction on the potential energy surface (PES). This is evident in the PES calculated (BLYP/6-31+G(d,p)) for the re966


action of protonated ethanol (PA ) 185.6 kcal/mol15) with diethylmethoxyborane (Figure 2). Furthermore, these calculations suggest that the proposed mechanism (Scheme 2) corresponds to a spontaneous reaction. After a barrierless proton transfer, nucleophilic addition of ethanol into the empty borane p-orbital occurs via a transition state that is predicted to lie 14.1 kcal/mol below the energy of the separated reactants. This transition state has a lower energy than the separated reactants due to ion-molecule solvation.21 The result of this addition is the formation of a triply charged (dritterionic) intermediate (net charge is +1). Despite the fact that this intermediate is somewhat unusual, it is predicted to be favorable, lying 20.5 kcal/mol below the separated reactants. In fact, the existence of energetically favorable dritterionic intermediates formed by nucleophilic addition22 and the formation of gas-phase borane ion salt-bridge structures have been reported previously.23 The second transition state, leading to methanol loss, is predicted to lie 16.7 kcal/mol below the energy of the separated reactants. The formation of the separated reaction products, protonated diethylethoxyborane and methanol, from the reaction of protonated ethanol with diethylmethoxyborane, is estimated to be exothermic by 11.6 kcal/ mol. Borane Derivatization Reactions. A series of monofunctional alcohols, ketones, aldehydes, esters, ethers, carboxylic acids, and amides were chosen as the oxygen-containing monofunctional analytes for this study. A few amines were also examined to test the specificity of our method for oxygen-containing compounds. All analytes were protonated using self-chemical ionization (vide supra). The protonated analytes were isolated, transferred into the analyzer cell of the dual cell instrument, and allowed to react with diethylmethoxyborane present at a static pressure (∼3.5 × 10-8 Torr nominal pressure). Diethylmethoxyborane was found to react with most of the protonated analytes by deprotonation (21) Brauman, J. I. Mass Spectrom. 1995, 30, 1649-1651. (22) Nelson, E. D.; Artau, A.; Price, J. M.; Tichy, S. E.; Jing, L.; Kentta¨maa, H. I. J. Phys. Chem. A 2001, 105, 10155-10168. (23) Gronert, S.; Huang, R. J. Am. Chem. Soc. 2001, 123, 8606-8607.

Figure 1. Reaction of protonated acetone (m/z 59) with diethylmethoxyborane. This reaction results in a product that corresponds to addition accompanied by methanol loss (m/z 126, 127). The boron-containing species are easily identified in the mass spectrum due to the unique boron isotope ratio (25% 10B relative to 11B).

Scheme 2

accompanied by substitution of methanol (Figure 1; Scheme 2). In contrast, the methoxy group of the borane is not basic enough to deprotonate some of the most basic nitrogen-containing functionalities studied here (see Figure 3 for an example). The absence of the substitution product for these molecules provides a prescreening for these functionalities. Since the reaction products of the other compounds contain boron, they are easily identified in the mass spectrum due to the unique boron isotope ratio (25% 10B relative to 11B). Furthermore, the reaction products

are simply a derivatized form of the original unknown compound (net addition of diethylborenium ion to the functional group), thus allowing for further structure probing by CAD or additional ionmolecule reactions. Reaction Kinetics. In order for functional group identification by ion-molecule reactions to be practical (e.g., for analysis of HPLC effluent), the reactions must be fast. Nearly all of the protonated oxygen-containing molecules studied were found to react very rapidly with diethylmethoxyborane. For example, Analytical Chemistry, Vol. 76, No. 4, February 15, 2004

967

Figure 2. Calculated potential energy surface for the reaction of protonated ethanol with diethylmethoxyborane (BLYP/6-31+G(d,p) + ZPVE).

Figure 3. Basicity of diethylmethoxyborane. Diethylmethoxyborane is not basic enough to deprotonate amines, tertiary amides, or most secondary amides. Therefore, no derivatization product was observed for these compounds.

protonated 1-pentanol reacts with diethylmethoxyborane with an efficiency in excess of 98%, protonated acetone at near 70%, and protonated methyl acetate at 88%. The relatively high proton affinities of amides compared to that of diethylmethoxyborane drastically influence their reaction rates. For example, protonated acetamide (PA ) 206.4 kcal/mol15) was found to react with diethylmethoxyborane by nucleophilic substitution at an efficiency of only 18%. The only secondary amide found to display reactivity toward diethylmethoxyborane is the simplest secondary amide, N-methylacetamide (PA ) 212.4 kcal/mol15). Reactions of this amide proceeded with very low efficiency (0.4%). None of 968


the tertiary amides examined were found to react with diethylmethoxyborane. Protonated Analytes. Alcohols. Primary, secondary, and tertiary alcohols, including alcohols with various chain lengths and an unsaturated alcohol, were studied (2-propanol, tert-butyl alcohol, 1-pentanol, 2-butanol, and allyl alcohol; Table 1). All of these alcohols, except for one, react to form substitution products by a mechanism consistent with the one proposed in Scheme 2. In addition to nucleophilic substitution reactivity, each of the alcohols studied react via proton transfer (see Table 1). The observed reactions proceed very rapidly to completion. The one

Table 1. Reactions of Various Protonated Alcohols with Diethylmethoxyborane protonated reagent (m/z of [M+H]+) ethanol (47) 2-propanol (61) tert-butanol (75) 1-pentanol (89) 2-butanol (75) allyl alcohol (59)

observed product ions,a m/z (%)

reaction

115 (56) 101 (44) 129 (75) 101 (25) 119 (54) 101 (46) 157 (76) 101 (24) 143 (75) 101 (25) 127 (81) 101 (19)

nucleophilic substitution (adduct - methanol) proton transfer to diethylmethoxyborane nucleophilic substitution (adduct - methanol) proton transfer to diethylmethoxyborane salt-bridge (adduct - isobutene) proton transfer to diethylmethoxyborane nucleophilic substitution (adduct - methanol) proton transfer to diethylmethoxyborane substitution (adduct - methanol) proton transfer to diethylmethoxyborane nucleophilic substitution (adduct - methanol) proton transfer to diethylmethoxyborane

a Only primary products containing the most abundant isotope (11B) are listed (all products observed also contain a abundance of 25% relative to that of the 11B isotope).

10B

isotope present in an

Scheme 3

reaction that deviates from the proposed mechanism is that of protonated tert-butyl alcohol. Rather than forming an adduct that has lost methanol, as all other alcohols, protonated tert-butyl alcohol reacts by adduct formation followed by isobutene loss (m/z 118, 119). The deviation in the reactivity of tert-butyl alcohol from that observed for the other alcohols is likely due to steric crowding. The mechanism shown in Scheme 3 explains the observed reaction by proposing the formation of a dritterionic, salt-bridge intermediate and product. Probing the structure of the reaction product (m/z 118, 119) by CAD revealed loss of water (formation of ions of m/z 100 and 101), thus providing additional support to the proposed ion structure (Scheme 3). Ethers. Diethyl ether, butyl methyl ether, tetrahydrofuran, and tetrahydropyran were examined (Table 2). The reaction of each of these ethers is consistent with the mechanism shown in Scheme 2. Ketones. Cyclic and acyclic ketones, including ketones of differing chain lengths (acetone, cyclohexanone, 4-heptanone, and

3-nonanone; Table 3), were investigated to determine their reactivity toward diethylmethoxyborane. The variation in the constitution of the ketones does not affect their reactivity. All the ketones studied react rapidly by a mechanism consistent with that shown in Scheme 2. Esters. Several esters of varying constitution were tested (methyl acetate, ethyl butyrate, methyl butyrate, methyl isobutyrate, and methyl propanoate; Table 4). The reactivity of all these esters was predictable based on the proposed mechanism shown in Scheme 2. Aldehydes. Aldehydes possessing aliphatic carbon chains of various lengths and branching, as well as an aromatic aldehyde, were allowed to react with diethylmethoxyborane (acetaldehyde, butanal, hexanal, 2-methylbutyraldehyde, and benzaldehyde; Table 3). All these aldehydes react to form products consistent with the proposed mechanism shown in Scheme 2. However, it should be noted that protonated acetaldehyde and butanal (due to their low proton affinity) react largely by proton transfer to diethylmethoxyborane. Analytical Chemistry, Vol. 76, No. 4, February 15, 2004

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Table 2. Reactions of Various Protonated Ethers with Diethylmethoxyborane protonated reagent (m/z of [M+H]+)


reaction

diethyl ether (75) butylmethyl ether (89) tetrahydrofuran (73) tetrahydropyran (87)

143 (100) 157 (100) 141 (100) 155 (100)

nucleophilic substitution (adduct - methanol) nucleophilic substitution (adduct - methanol) nucleophilic substitution (adduct - methanol) nucleophilic substitution (adduct - methanol)

a Only primary products and the most abundant isotope (11B) are listed (all products observed also contain a 10B isotope present in an abundance of 25% relative to that of the 11B isotope).

Table 3. Reactions of Various Protonated Aldehydes and Ketones with Diethylmethoxyborane protonated reagent (m/z of [M+H]+) acetaldehyde (45) butanal (73) hexanal (101) 2-methylbutyraldehyde (86) benzaldehyde (107) acetone (59) cyclohexanone (99) 4-heptanone (115) 3-nonanone (143)


reaction

113 (5) 101 (95) 141 (68) 101 (32) 169 (100) 155 (90) 101 (10) 175 (100) 115 (93) 101 (7) 167 (100) 183 (100) 211 (100)

nucleophilic substitution (adduct - methanol) proton transfer to diethylmethoxyborane nucleophilic substitution (adduct - methanol) proton transfer to diethylmethoxyborane nucleophilic substitution (adduct - methanol) nucleophilic substitution (adduct - methanol) proton transfer to diethylmethoxyborane nucleophilic substitution (adduct - methanol) nucleophilic substitution (adduct - methanol) proton transfer to diethylmethoxyborane nucleophilic substitution (adduct - methanol) nucleophilic substitution (adduct - methanol) nucleophilic substitution (adduct - methanol)


Table 4. Reactions of Various Protonated Esters with Diethylmethoxyborane protonated reagent (m/z of [M+H]+)


reaction

methyl acetate (75) ethyl butyrate (117) methyl butyrate (103) methyl isobutyrate (103) methyl propanoate (89)

143 (100) 185 (100) 171 (100) 171 (100) 157 (100)

nucleophilic substitution (adduct - methanol) nucleophilic substitution (adduct - methanol) nucleophilic substitution (adduct - methanol) nucleophilic substitution (adduct - methanol) nucleophilic substitution (adduct - methanol)


Carboxylic Acids. A group of carboxylic acids were selected for examination, namely, butanoic acid, hexanoic acid, heptanoic acid, and benzoic acid (Table 5). All carboxylic acids studied react by a mechanism consistent with that shown in Scheme 2, forming the expected primary reaction product (nucleophilic addition and loss of methanol). However, the carboxylic acids differ from all the other monofunctional compounds in that the primary reaction products formed with diethylmethoxyborane possess an acidic hydrogen that is capable of being transferred to another diethylmethoxyborane molecule. Accordingly, the primary reaction product undergoes a second substitution reaction with another diethylmethoxyborane molecule (Figure 4). Therefore, carboxylic acids undergo two substitutions. A proposed mechanism for the complete reaction of the carboxylic acids, both primary and secondary, is shown in Scheme 4. Amides. Primary, secondary, and tertiary amides were studied, namely, acetamide, N-methylacetamide, N,N-dimethylacetamide, 970 Analytical Chemistry, Vol. 76, No. 4, February 15, 2004

N,N-diethylacetamide, N-methylpropanamide, and N-ethylacetamide (Table 6). Although each of these species possesses the amido functionality, their reactivity toward diethylmethoxyborane is varied. The reactivity of the amides is greatly affected by their proton affinity. It was found computationally, using the STQN method for transition structure searching at the BLYP/6-31G(d) level of theory, that if the analyte has a proton affinity higher than that of diethylmethoxyborane, the proton transfer and nucleophilic addition events occur in a single step and together make up the first barrier encountered in the PES for the reaction. Therefore, as the proton affinity of the analyte increases, so does the energy required to surmount the first transition state on the PES for its reaction with diethylmethoxyborane. This is evident when considering the experimental observations of acetamide (PA ) 206.4 kcal/mol), N-methylacetamide (PA ) 212.4 kcal/mol), and N,Ndimethylacetamide (PA ) 217.0 kcal/mol) (Table 6).15 Nucleophilic substitution reactions were observed for acetamide and

Table 5. Reactions of Various Protonated Carboxylic Acids with Diethylmethoxyborane protonated reagent (m/z of [M+H]+) butanoic acid (89) hexanoic acid (117) heptanoic acid (131) benzoic acid (123)

observed product ions, m/z (%)

reaction

157 (92) 101 (8) 225 (100) 185 (100) 253 (100) 199 (100) 267 (100) 191 (100) 259 (100)

nucleophilic substitution (adduct - methanol)a proton transfer to diethylmethoxyboranea nucleophilic substitution (adduct - methanol)b nucleophilic substitution (adduct - methanol)a nucleophilic substitution (adduct - methanol)b nucleophilic substitution (adduct - methanol)a nucleophilic substitution (adduct - methanol)b nucleophilic substitution (adduct - methanol)a nucleophilic substitution (adduct - methanol)b

a Primary products. The most abundant isotope (11B) is listed (all primary products observed also contain a 10B isotope present in an abundance of 25% relative to that of the 11B isotope). b Secondary products (possess two boron atoms). The most abundant isotope (11B) is listed (all secondary products observed also contain a 10B isotope present in an abundance of 50% relative to that of the 11B isotope).

Figure 4. Consecutive reactions with the borane. The primary reaction product (m/z 156, 157) of butanoic acid can transfer its acidic hydrogen as a proton to another diethylmethoxyborane molecule, which allows a second substitution reaction to occur to form the ions of m/z 224 and 225. Note: the isotope ratio for the secondary product is 50% 10B relative to 11B, indicating that two boron atoms are incorporated in the secondary product.

N-methylacetamide, but the reaction efficiency of N-methylacetamide is substantially lower than that of acetamide (vide supra). No reaction was observed for N,N-dimethylacetamide. These observations are readily rationalized by the partial PES calculated at the BLYP/6-31G(d) level of theory for these reactions (Figure 5). Computations predict the proton transfer/nucleophilic addition barrier for the reaction of protonated acetamide, N-methylacetamide, and N,N-dimethylacetamide with diethylmethoxyborane, to be -8.7, -2.6, and +0.1 kcal/mol, respectively, relative to the energy of the separated reactants. As the energy of this transition state approaches the total energy of the system, the sum of states, that is, the number of ways to pass through the transition state, decreases. This explains the observed reduction in the reaction rate of N-methylacetamide relative to that of acetamide. No reactivity was observed for N,N-dimethylacetamide, which is explained by the fact that its proton affinity is higher than that of

the amides that do react with diethylmethoxyborane. This increase in proton affinity leads to a higher-energy proton transfer/ nucleophilic addition transition state, which requires more energy to surmount than is available to the system. Amines. Several protonated amines were studied in order to demonstrate the selectivity of diethylmethoxyborane toward oxygen-containing functionalities. The reagents selected for analysis are dimethylamine, diethylamine, aniline, tert-butylamine, cyclohexylamine, isopropylamine, and triethylamine (Table 6). None of these compounds react with diethylmethoxyborane. The lack of reactivity of amines was examined by calculating a portion of the PES at the BLYP/6-31G(d) level of theory for the reaction of protonated dimethylamine with diethylmethoxyborane (Figure 6). Computations predict that the first transition state is 20.7 kcal/ mol above the total energy of the system. Theory also predicts that this transition state involves both the deprotonation of the Analytical Chemistry, Vol. 76, No. 4, February 15, 2004

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Scheme 4

Table 6. Reactions of Various Protonated Amides and Amines with Diethylmethoxyborane protonated reagent (m/z of [M+H]+)


reaction

acetamide (60) N-methylacetamide (74) N,N-dimethylacetamide (88) N,N-diethylacetamide (116) N-methylpropanamide (88) N-ethylacetamide (88) dimethylamine (46) diethylamine (74) aniline (94) tert-butylamine (100) isopropylamine (60) triethylamine (102)

128 (100) 142 (100) n/ab n/a n/a n/a n/a n/a n/a n/a n/a n/a

nucleophilic substitution (adduct - methanol) nucleophilic substitution (adduct - methanol) no reaction observed no reaction observed no reaction observed no reaction observed no reaction observed no reaction observed no reaction observed no reaction observed no reaction observed no reaction observed

a Only primary products and the most abundant isotope (11B) are listed (all products observed also contain a 10B isotope present in an abundance of 25% relative to that of the 11B isotope). b n/a, not applicable.

amine and the nucleophilic addition of the amine to the boron center. The high proton affinity of dimethylamine (222.2 kcal/ mol15), relative to that of diethylmethoxyborane (191.4 kcal/mol; calculated), is most likely the reason that the required energy to surmount this transition state is greater than that of the system. Since none of the amines studied react with diethylmethoxyborane, their proton transfer/nucleophilic addition transition states are expected to lie above the energy of the separated reactants. 972 Analytical Chemistry, Vol. 76, No. 4, February 15, 2004

H/D Exchange Reactions. Since virtually all of the oxygencontaining monofunctional compounds react with diethylmethoxyborane by the same substitution pathway, an additional step is necessary in order to differentiate between the functionalities. The reaction products of alcohols, carboxylic acids, and primary amides have acidic hydrogens. These acidic hydrogens may be exchanged for deuteriums when exposed to a proper H/D exchange reagent, such as perdeuterated methanol. Indeed, when allowed to react

Figure 5. Partial PES (calculated at the BLYP/6-31G(d) + ZPVE level of theory) for the reaction of protonated (a) N,N-dimethylacetamide, (b) N-methylacetamide, and (c) acetamide with diethylmethoxyborane. As the proton affinity of the analyte increases, so does the height of the proton transfer/nucleophilic addition barrier. Overcoming the reaction barrier calculated for N,N-dimethylacetamide requires more energy than is available to the system and the reaction is not feasible.

Figure 6. Partial PES (calculated at the BLYP/6-31G(d) + ZPVE level of theory) for the reaction of protonated dimethylamine with diethylmethoxyborane. Because of the relatively high proton affinity of amines compared to diethylmethoxyborane, the barrier to proton transfer/ nucleophilic substitution is greater than the total energy of the system.

with perdeuterated methanol, these reaction products undergo one H/D exchange, whereas the reaction products of ketones, ethers, aldehydes, and esters do not (see Figure 7 for an example). Probing the reaction products by H/D exchange thus allows for distinction of derivatives that possess acidic hydrogens from those that do not. Furthermore, primary amide derivatives

can be positively identified based on the fact that these ions exchange two hydrogens for deuteriums rather than just one, as is the case for alcohol and carboxylic acid derivatives. Carboxylic acids are readily distinguished from alcohols and primary amides based on their double substitution product (vide supra) (Figure 4; Scheme 4). Therefore, the combination of derivatization of a Analytical Chemistry, Vol. 76, No. 4, February 15, 2004

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Figure 7. H/D exchange. (a) The derivatization product (m/z 127) arising from the reaction of protonated acetone with diethylmethoxyborane is shown not to undergo H/D exchange with perdeuterated methanol. (b) The derivatization product (m/z 129) arising from the reaction of protonated 2-propanol with diethylmethoxyborane is shown to undergo one H/D exchange with perdeuterated methanol.

protonated molecule with diethylmethoxyborane, along with H/D exchange reactions with perdeuterated methanol, allows a distinction between a protonated carboxylic acid, alcohol, and primary amide. CAD Reactions. Ketones, esters, ethers, and aldehydes do not react by H/D exchange and, therefore, remain unidentified when the above method is used. However, our previous work has shown that reactions of dimethoxyborenium ion with ketones, esters, ethers, and aldehydes are very exothermic and result in fragmentation that is characteristic to the functional group in the analyte.24-28 The same type of fragmentation can be expected for (24) Thoen, K. K.; Tutko, D.; Ranatunga, T. D.; Kentta¨maa, H. I. J. Am. Soc. Mass Spectrom. 1996, 7, 1138-1143. (25) Ranatunga, T. D.; Kentta¨maa, H. I. Inorg. Chem. 1995, 34, 18-27. (26) Leeck, D. T.; Ranatunga, T. D.; Smith, R. L.; Partanen, T.; Vainiotalo, P.; Kentta¨maa, H. I. Int. J. Mass Spectrom. Ion Processes 1995, 141, 229-240.

974 Analytical Chemistry, Vol. 76, No. 4, February 15, 2004

CAD of the derivatized analyte ions examined here. However, the ethyl groups bound to the boron in the derivatized analyte ions tend to cleave readily as ethylenes under CAD conditions, complicating the CAD spectra of these derivatives. To make the CAD spectra easier to interpret, a slight modification was made to the borane reagent to reduce the number of fragments. Replacement of the two ethyl groups of diethylmethoxyborane with a butylene chain yields a new reagent, 1-methoxyborolane. This reagent reacts with the protonated analytes to produce substitution reaction products that are analogous to those with diethylmethoxyborane. In addition, these reaction products do not yield significant fragments due to dissociation of their alkyl chain (27) Ranatunga, T. D.; Kentta¨maa, H. I. J. Am. Chem. Soc. 1992, 114, 86008604. (28) Ranatunga, T. D.; Kennady, J. M.; Kentta¨maa, H. I. J. Am. Chem. Soc. 1997, 119, 5200-5207.

Figure 8. (a) CAD of the derivatization product (m/z 181) formed from the reaction between protonated 4-heptanone and 1-methoxyborolane. Note: ions of m/z 71 and 83 are not boron-containing fragments (based on 10B CAD data, which are not shown). (b) CAD of the derivatization product (m/z 197) formed from the reaction between protonated dibutyl ether and 1-methoxyborolane.

Table 7. Identification (Dark Boxes) of the Functionality Present in Protonated Monofunctional Alcohols, Ketones, Ethers, Carboxylic Acids, and Primary Amides by Using Borane Derivatization Followed by H/D Exchange or CADa

a NN refers to events that are not necessary for identification. Aldehydes and esters are expected to be distinguishable by borane derivatization and CAD based on cited literature (see refs 25 and 29).

upon CAD. Protonated ketones and ethers derivatized by the reaction of 1-methoxyborolane were indeed found to undergo structurally diagnostic fragmentations upon CAD (Figure 8). Based upon previous work,24,28 CAD of ester and aldehyde derivatization products is also expected to produce characteristic fragmentation. This method therefore appears to provide differentiation between protonated monofunctional ketones, esters, ethers, and aldehydes.

CONCLUSIONS We have shown that protonated monofunctional oxygencontaining compounds react in a mass spectrometer with selected methoxyborane reagents by rapid substitution reactions that yield a derivatized version of the original neutral oxygen-containing compound. In addition, we have demonstrated the ability to distinguish between and identify protonated monofunctional alcohols, ketones, ethers, carboxylic acids, and primary amides Analytical Chemistry, Vol. 76, No. 4, February 15, 2004

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(identification of aldehydes and esters is also expected) by applying this borane derivatization reaction followed by H/D exchange or CAD (Table 7). After being extended to include other common functionalities, this analytical method has great potential for being able to provide complete structure elucidation for unknown mixture components in a mass spectrometer when used in conjunction with molecular formula information obtained from exact mass measurements. Its likely applications include analysis of mixtures of unknown drug degradation products and metabolites for drug development and discovery. ACKNOWLEDGMENT The authors gratefully acknowledge Eli Lilly Corp. for financial support of this work. The authors thank Dr. Steven W.

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Baertschi of Eli Lilly for useful discussions and guidance during this project, and Dr. P. V. Ramachandran and Mr. Subash C. Jonnalagadda of the H. C. Brown Center for Borane Research for the synthesis of the 1-methoxyborolane reagent. Dr. Christopher J. Petzold is acknowledged for providing insights into many of the complex problems that were encountered during this project.

Received for review August 12, 2003. Accepted November 25, 2003. AC034946D

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