Anal. Chem. 1999, 71, 1574-1578
Positive-Ion Analysis of Boropeptides by Chemical Ionization and Liquid Secondary Ionization Mass Spectrometry Michael J. Haas,*,† Karl F. Blom,† and Carl H. Schwarz III‡
The DuPont Pharmaceutical Company, DuPont Experimental Station, P.O. Box 80500, Wilmington, Delaware 19880-0500, and ThermoQuest Corporation, 255 Old New Brunswick Road, Piscataway, New Jersey 08854
Techniques for the characterization of two boronic acid peptides as their cyclic boronate ester derivatives by positive-ion ammonia chemical ionization (CI) and positive-ion liquid secondary ionization (LSIMS) are described and results presented. These techniques avoid the complications introduced by the thermally induced processes that boronic acids may undergo when the mass spectrometric characterizations of free boronic acids are attempted. Derivatizations for CI analysis were accomplished via a simple benchtop method using several polyfunctional nucleophilic derivatizing agents (ethylene glycol, glycerol, et al.), while derivatizations for LSIMS analysis were accomplished via both benchtop and previously established in situ methods using the same derivatizing agents. Certain previously held misconceptions about the LSIMS mass spectrometry of boronic acids are examined. The preparation of cyclic boronate esters has long been a useful technique for the characterization of polyfunctional nucleophilic compounds by gas chromatography (GC) and gas chromatography/electron ionization mass spectrometry (GC/EIMS). This method was first applied to 1,2-diols and simple sugars in order to facilitate the characterization of these compounds by GC1 (Figure 1). The technique has since been applied to the GC and GC/MS characterization of many polyfunctional nucleophilic compounds.2-8 Cyclic esters, typically n-butylboronates or benzeneboronates, have been preferred as derivatives over dialkyl esters, since the dialkyl esters are more hydrolytically unstable.9 † The DuPont Pharmaceutical Co. Formerly: The DuPont Merck Pharmaceutical Co. ‡ ThermoQuest Corp. (1) Kuivila, H. G.; Keough, A. H.; Soboczenski, E. J. J. Org Chem. 1954, 19, 780-783. (2) Brooks, C. J. W.; Watson, J. Gas Chromatography 1968, Proc. 7th Int. Symp., Copenhagen, 1968; p 129. (3) Anthony, G. M.; Brooks, C. J. W.; Maclean, I.; and Sangster, I. Proc. 5th Int. Symp. Advances in Chromatography, Las Vegas, 1969; p 146. (4) Brooks, C. J. W.; Harvey, D. J. Biochem. J. 1969, 114, 15P. (5) Brooks, C. J. W.; Maclean, I. J. Chromatogr. Sci. 1971, 9, 18-24. (6) Wood, P. J.; Siddiqui, I. R. Carbohydr. Res. 1974, 36, 247-256. (7) Oliw, E. H. J. Chromatography Biomed. Appl. 1983, 275, 245-259. (8) Giachetti, C.; Zanolo, G.; Assandri, A.; Poletti, P. Biomed. Environ. Mass Spectrom. 1989, 18, 592-597. (9) Singhawangcha, S.; Hu, L.-E. C.; Poole, C. F.; Zlatkis, A. J. High. Resolut. Chromatogr. Chromatogr. Commun. 1978, 1, 304-306.
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Figure 1. Reaction of a free boronic acid with a diol to produce a cyclic boronate ester, where R1, R2, and R3 are alkyl groups and/or other nonreactive functionalities.
Figure 2. Thermally induced cyclization reaction of free boronic acid to produce a boroxine.
Free boronic acids are known to undergo conversion to boroxines via a dehydration/trimerization reaction in the injection port of a gas chromatograph at elevated temperatures9 (Figure 2). While this reaction has been induced deliberately for the study of the electron ionization (EI) mass spectra of boroxines,10 it can interfere with the GC characterization of free boronic acids. It has been demonstrated that boroxine formation is prevented by the derivatization of a free boronic acid to an ester in a benchtop reaction with pinacol9 and in an on-line reaction (in the GC injector port) with 1,3-propanediol,11 according to Figure 1. Difficulties are also encountered in the characterization of free boronic acids by mass spectrometry when techniques other than gas chromatography are used to introduce the sample into the ion source. The introduction of a free boronic acid via a heatable direct insertion probe (DIP) can induce the conversion of the acid to a boroxine, just as in high-temperature GC injectors. A simple analytical-scale derivatization method, in which the free boronic acid is esterified with an excess of 1,3-propanediol, has been shown to eliminate boroxine formation in the DIP/EI characterization of boronic acids.12 In the present work, it was observed that under direct insertion probe/chemical ionization (DCI) free boronic acids may undergo mono and bisdehydrations (via thermal- and/or ionic-decomposition pathways), a complication which has not been reported previously. (10) Brooks, C. J. W.; Harvey, D. J.; and Middleditch, B. S. Org. Mass Spectrom. 1970, 3, 231-235. (11) Rose, M. E.; Longstaff, C.; Dean, P. D. G. J. Chromatogr. 1982, 249, 174179. (12) Longstaff, C.; Rose, M. E. Org. Mass Spectrom. 1982, 17, 508-518. 10.1021/ac9810112 CCC: $18.00
© 1999 American Chemical Society Published on Web 03/12/1999
More recently, several chlorinated cis dihydroxycyclohexadienes, catechols, and other bifunctional compounds were characterized as boronate esters, both by positive-ion EI and electroncapture chemical ionization (ECCI) in methane.13-14 No positive CI studies of boronic acids or boronate esters have been reported. The derivatization technique was extended to negative-ion liquid secondary ion mass spectrometry (LSIMS) where it was presumed that mixtures of boronic acids and certain trifunctional nucleophilic compounds (e.g., glycerol) reacted in situ to produce negatively charged boronate complexes which were subsequently sputtered from the liquid surface and mass analyzed.15 This approach has been used to study a number of polyfunctional nucleophilic compounds as their corresponding boronate esters.15-19 In situ derivatization was also used to characterize 20-hydroxyecdysteroids as their phenylboronate esters by positive-ion LSIMS,20-21 which seems to belie the mechanism of direct boronate complex ion formation (“pre-ionization”) postulated by Rose et al.15 Subsequently, there have been broader negative-ion LSIMS studies of boron-containing compounds.22-26 The presumption that pre-ionization of a boronic acid to a boronate complex anion occurs in situ may account for the preferential use of negative LSIMS mode in conjunction with the derivatization technique. However, this assumption has apparently precluded the investigation of the positive-ion LSIMS of boronic acids and esters; no positive-ion LSIMS studies of boronic acids have been reported. In this report we address the positive-ion mass spectral characterization of two representative boronic acid peptides using the derivatization method in conjunction with the DCI and LSIMS techniques. Under positive DCI conditions, we found that the established derivatization method eliminates the problems of boroxine formation and dehydration; we present results for the two boronic acid peptides using four derivatizing agents: ethylene glycol, 1,3-propanediol, 1,4-butanediol, and glycerol. As an alternate to DCI analysis, we present results for positive-ion LSIMS studies in which boronate esters of the two boronic acid peptides have been formed both in situ (using the derivatizing agent as the liquid matrix) and by benchtop derivatization. These studies indicate that in LSIMS mode the boronate complex ions are formed by a (13) Kirsch, N. H.; Stan, H.-J. J. Chromatogr., A 1994, 684, 277-287. (14) Simpson, J. T.; Markey, S. P.; Pu, Y. M.; Ziffer, H. Proc. 41st ASMS Conf. Mass Spectrometry and Allied Topics, 1993; p 338a. (15) Rose, M. E.; Longstaff, C.; Dean, P. D. G. Biomed. Mass Spectrom. 1983, 10, 512-527. (16) Yang, H.-J.; Chen, Y.-Z. J. Carbohydr. Chem. 1993, 12, 39-48. (17) Yang, H.; Chen, Y.; Zhao, F.; Li, H.; Zhai, J.; Chen, N. Chem. Res. Chin. Univ. 1992, 8, 231-238. (18) New, A. P.; Haskins, N. J.; Games, D. E. Rapid Commun. Mass Spectrom. 1993, 7, 1099-1107. (19) Yan, L.; Fang, Y. Chin. J. Chem. 1993, 11, 53-58. (20) Pı´s, J.; Hykl, J.; Vaisar, T.; Harmatha, J. J. Chromatogr., A 1994, 658, 7782. (21) Vaisar, T.; Pı´s, J. Rapid Commun. Mass Spectrom. 1993, 7, 46-52. (22) Okamoto, Y.; Takei, Y.; Rose, M. E. Int. J. Mass Spectrom. Ion Processes 1989, 87, 225-235. (23) Rose, M. E.; Webster, M. J. Org. Mass Spectrom. 1989, 24, 567-572. (24) Rose, M. E.; Wycherley, D.; Preece, S. Org. Mass Spectrom. 1992, 27, 876882. (25) Aubagnac, J.-L.; Claramunt, R.-M.; Lopez, C.; and Elguero, J. Rapid Commun. Mass Spectrom. 1991, 5, 113-116. (26) Aubagnac, J.-L.; Man, M. W.-C.; Elguero, J. Rapid Commun. Mass Spectrom. 1991, 5, 469-471.
Figure 3. Structure of the free base of Ac-(D)Phe-Pro-boroArgOH‚HCl, designated herein as “boroarginine peptide”.
Figure 4. Structure of the free base of [N-(C(O)(CH2)2Ph)-N-CH3]Gly-boroLys-OH‚HCl, designated herein as “borolysine peptide”.
two-step process: (1) formation of the neutral boronate ester and (2) ionization of the ester by LSIMS. EXPERIMENTAL SECTION Materials. All of the compounds used in this work were obtained from Aldrich Chemical Co. except for the following two boronic acid peptides, both synthesized within the Research & Development Division of The DuPont Pharmaceutical Co.: Ac(D)Phe-Pro-boroArg-OH‚HCl (Figure 3), designated herein as “boroarginine peptide”27 and [N-(-C(O)(CH2)2Ph)-N-CH3]Gly boroLys-OH‚HCl (Figure 4), designated herein as “borolysine peptide”.28 The syntheses of these compounds have been described elsewhere.29-31 All materials were used as received, without further characterization or purification. Preparation of Free Boronic Acid Solutions. Stock solutions of phenylboric acid (1.0 M), boroarginine peptide (0.1 M), and borolysine peptide (0.1 M) in 9:1 acetonitrile/water were prepared. These samples were diluted with acetonitrile to 0.01 M for direct (underivatized) analysis by DCI and were derivatized to boronate esters as described below for DCI and LSIMS analysis. Derivatization of Boronic Acids to Boronate Esters. Stock solutions (1.0 M) in acetonitrile were made of the following derivatizing agents: ethylene glycol, 1,3-propanediol, 1,4-butanediol, and glycerol. For DCI analysis, boronate esters of phenylboric acid, boroarginine peptide, and borolysine peptide were prepared on the (27) The systematic (CAS) designation for this compound is: (R)-N-acetyl-Dphenylalanyl-N-{4-[(aminoiminomethyl)amino]-1-boronobutyl}-L-prolinamide monohydrochloride. Molecular formula of the free base is C22H33N6O5B, and the monoisotopic molecular weight of the free base is 460.26. (28) The systematic (CAS) designation for this compound is: N-{2-[(5-amino1-boronopentyl)amino]-2-oxoethyl}-N-methyl-R-oxo-N-(phenylmethyl)-benzenepropanaminium chloride. Molecular formula of the free base is C17H28N3O4B, and the monoisotopic molecular weight of the free base is 349.11 Da. (29) Kettner, C.; Mersinger, L.; Knabb, R. J. Biol. Chem. 1990, 265, 18289. (30) Kettner, C. A.; Shenvi, A. B. U.S. Patent 5, 187, 157, 1993. (31) Galemmo, R. A., Jr.; Abelman, M. M.; Amparo, E. C.; Fevig, J. M.; Knabb, R. M.; Miller, W. H.; Pacofsky, G. J.; and Weber, P. C. WO 95/09634, PCT/ US94/11280, 1995 (see pages 24, 162 and 173).
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µg scale. A 1-µL aliquot of boronic acid stock solution was dissolved in a 3-fold molar excess of the derivatizing-agent stock solution. The reaction mixture was diluted with acetonitrile to a total volume of 200 µL. The reaction was allowed to proceed for about 5 min at ambient temperature before an aliquot of the mixture was drawn and analyzed by low-resolution ammonia DCI (see below). For LSIMS analysis, the boronate esters were prepared by benchtop derivatization, as described above, and directly on the stage of the LSIMS probe (see below). Ammonia Direct Chemical Ionization (NH3-DCI). Chemical ionization spectra were obtained on a Finnigan MAT 8230 mass spectrometer. Approximately 0.5 µL of analyte was placed on the DCI probe filament and the solvent allowed to evaporate. The probe was inserted into the ion source (125 °C) to which 0.5 Torr of NH3 had been admitted. The probe filament current was ramped at the rate of 20-40 mA/s to vaporize the sample and, if the derivatization reaction had been carried out, to achieve some separation by distillation of the excess derivatizing agent from the boronate ester. The spectra were acquired by magnet scan in centroid mode over the mass range of 95-1150 Da, at a rate of 0.5 s/decade and a mass resolution of 1000. Ions resulting from the excess derivatization agent were partially or fully removed from each spectrum by background subtraction. Liquid Secondary Ionization (LSIMS). LSIMS spectra were obtained on a VG-70 VSE mass spectrometer equipped with a Csion gun. Derivatizations of boroarginine and borolysine peptides were carried out in two ways: (1) Benchtop Derivatization: 2 µL of the 0.1 M boronic acid stock solution was dissolved in 6 µL of 1.0 M derivatizing-agent stock solution. The reaction was allowed to proceed for five minutes before a 3-µL aliquot of the reaction mixture was dissolved in 5 µL of a 3-nitrobenzyl alcohol (NBA) matrix on the stage of the LSIMS probe. (2) In Situ Derivatization: the derivatizing agent was the LSIMS matrix. 1 µL of 0.1 M boronic acid stock solution was dissolved in 5 µL of pure derivatizing agent (i.e. not stock solution) on the stage of the LSIMS probe. The in situ reaction was allowed to proceed for 5 min before LSIMS analysis. The probe was inserted into the ion source (ambient temperature), where the matrix-sample mixture was bombarded by a beam of fast Cs ions (22 keV, 5 µA) to vaporize and ionize the analyte mixture. The spectra were acquired by magnet scan in multichannel analysis (MCA) mode over the mass range of 1001100 Da, at a rate of 15 s/scan and a mass resolution of 1200. Ions due solely to the matrix were selectively removed from each spectrum by the computer software. RESULTS DCI-NH3 Characterization of Boronic Acid Peptides as Cyclic Boronate Ester Derivatives. The DCI-NH3 mass spectrum of underivatized phenylboric acid is shown in Figure 5. The spectrum exhibits only molecular-ion species for the corresponding boroxine; no molecular ions of the free phenylboric acid are observed. The benchtop derivatization of phenylboric acid to various boronate esters, followed by DCI-NH3 analysis, demonstrates the utility of the derivatization method in characterizing boronic acids. The formula of any neutral cyclic boronate ester, M′, is given by 1576 Analytical Chemistry, Vol. 71, No. 8, April 15, 1999
Figure 5. The DCI-NH3 mass spectrum of phenylboric acid (underivatized), which exhibits ions of the corresponding triphenylboroxine instead of the free boronic acid. Table 1. DCI-NH3 Spectra of Derivatization Reaction Mixtures of Phenylboric Acid and Several Polyfunctional Derivatizing Agents derivatizing agent
m/za (% rel abundance)
ethylene glycol 1,3-propanediol 1,4-butanediol glycerol
149 (100), 166 (53.9) 162 (32.8), 180 (100) 108 (100), 177 (15.2), 194 (42.2) 110 (15.6), 161 (34.0), 179 (46.9), 196 (100)
a Ions with intensities of
