Chiral Analysis Using the Kinetic Method with Optimized Fixed

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Anal. Chem. 2003, 75, 678-684

Chiral Analysis Using the Kinetic Method with Optimized Fixed Ligands: Applications to Some Antibiotics Lianming Wu and R. Graham Cooks*

Department of Chemistry, Purdue University, West Lafayette, Indiana 47907

A new version of the kinetic method for chiral analysis, which employs a fixed (nondissociating) ligand as well as the usual analyte and chiral reference ligands, is introduced to simplify the kinetics of this experiment. Singly charged clusters containing the divalent transition metal ion MnII, a peptide which serves as a fixed ligand, an amino acid chiral reference, and the analyte 4-benzyl-2oxazolidinone were generated by electrospray ionization (ESI). The cluster ion of interest was mass-selected, and the kinetics of its competitive unimolecular dissociations was investigated in an ion trap mass spectrometer. The chiral selectivity (Rfixed chiral), the ratio of the two fragment ion abundances when the cluster contains one pure enantiomer of the analyte expressed relative to that for the other enantiomer, varies with increasing size of the fixed peptide ligands. The metal-ligand and the ligand-ligand interactions that produce chiral discrimination are optimized in the tetrapeptide fixed ligand Gly-Gly-Ala-Gly, as shown by data for 15 fixed ligands. The difference in the free energies of activation for the two competitive reactions is estimated to be ∼7 kJ/mol for this particular fixed ligand. The sensitive nature of the methodology and the linear relationship between the logarithm of the fragment ion abundance ratio and the optical purity (intrinsic to the kinetic method) allows mixtures to be analyzed for as little as 1% enantiomeric excess (ee), by simply recording the ratios of fragment ion abundances in a tandem mass spectrum. These features are demonstrated in the case of the pharmacologically important 4-benzyl-2-oxazolidinones and in the case of penicillamine. The invention of novel strategies for the synthesis and analysis of enantiomerically pure compounds continues to grow in importance.1 Chirally pure compounds are required not only by the fine chemicals industry (pharmaceuticals and agrochemicals),2 but are also of interest in polymer, electronics, and other applications.3 Oxazolidinones have become popular as chiral auxiliaries because they allow very high stereoselectivities to be achieved.4 A range * Corresponding author. Tel: (765) 494-5265. Fax: (765) 494-9421. E-mail: [email protected]. (1) Stinson, S. C. Chem. Eng. News 2000, 78, 55-79. (2) Stinson, S. C. Chem. Eng. News 1999, 77, 101-120. (3) Feringa, B. L.; van Delden, R. A.; Koumura, N.; Geertsema, E. M. Chem. Rev. 2000, 100, 1789-1816. (4) Ager, D. J.; Prakash, I.; Schaad, D. R. Aldrichim. Acta 1997, 30, 3-12.

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of reaction types, such as Wittig reactions5 and Michael additions,6 are amenable to stereocontrol using oxazolidinones. In addition, oxazolidinones represent a new class of totally synthetic antibiotics that are active against a wide range of multidrug-resistant pathogens, with such promising features as potent antibacterial activity against all important Gram-positive bacteria, oral bioavailability, a novel mechanism of action, and no observable resistance.7 Recent approval of the first drug of this class, linezolid, (Zyvox),8,9 has sparked further interest in the development of novel antibiotics using oxazolidinone as the template. Quantification of enantiomeric forms of drugs and their metabolites has become a requirement in both drug discovery and clinical pharmacy.10 Therefore, the enantiomeric determination of chiral oxazolidinones is of fundamental importance as well as being of practical significance. In recent years, mass spectrometry has emerged as a powerful tool for direct gas-phase chiral analysis11-16 and differentiation of isomers.17 Among other methods, that based on the kinetics of competitive dissociations18-21 of transition metal-bound complexes,16 allows quantitative analysis of various R-amino acids,16 R-hydroxy acids,22 dipeptides,23,24 β-blockers,25 and the antiviral nucleoside 2′-fluoro-5-methyl-β-arabinofuranosyluracil.26 Mass(5) Reddy, G. V.; Rao, G. V.; Iyengar, D. S. 1999, 40, 775-776. (6) Cai, C.; Soloshonok, V. A.; Hruby, V. J. J. Org. Chem. 2001, 66, 13391350. (7) Bouza, E.; Munoz, P. Clin. Microbiol. Infect. 2001, 7, 75-82. (8) Marchese, A.; Schito, G. C. Clin. Microbiol. Infect. 2001, 7, 66-74. (9) Lucio, V.-C.; Alejandra, G.-F.; W. G., E.-F.; Oliverio, W. Antimicrob. Agents Chemother. 2001, 45, 3629-3630. (10) Bakhtiar, R.; Tse, F. L. S. Rapid Commun. Mass Spectrom. 2000, 14, 11281135. (11) Filippi, A.; Giardini, A.; Piccirillo, S.; Speranza, M. Int. J. Mass Spectrom. 2000, 198, 137-163. (12) Smith, G.; Leary, J. A. J. Am. Chem. Soc. 1998, 120, 13046-13056. (13) Sawada, M.; Takai, Y.; Yamada, H.; Hirayama, S.; Kaneda, T.; Tanaka, T.; Kamada, K.; Mizooku, T.; Takeuchi, S.; Ueno, K.; Hirose, K.; Tobe, Y.; Naemura, K. J. Am. Chem. Soc. 1995, 117, 7726-7736. (14) Guo, J.; Wu, J.; Siuzdak, G.; Finn, M. G. Angew. Chem., Int. Ed. 1999, 38, 1755-1758. (15) Grigorean, G.; Lebrilla, C. B. Anal. Chem. 2001, 73, 1692-1698. (16) Tao, W. A.; Zhang, D.; Nikolaev, E. N.; Cooks, R. G. J. Am. Chem. Soc. 2000, 122, 10598-10609. (17) Voinov, V. G.; Claeys, M. Int. J. Mass Spectrom. 2001, 205, 57-64. (18) Cooks, R. G.; Kruger, T. L. J. Am. Chem. Soc. 1977, 99, 1279-1281. (19) Cooks, R. G.; Patrick, J. S.; Kotiaho, T.; McLuckey, S. A. Mass Spectrom. Rev. 1994, 13, 287-339. (20) Cooks, R. G.; Wong, P. S. H. Acc. Chem. Res. 1998, 31, 379-386. (21) Cheng, X. H.; Wu, Z. C.; Fenselau, C. J. Am. Chem. Soc. 1993, 115, 48444848. (22) Wu, L.; Tao, W. A.; Cooks, R. G. Anal. Bioanal. Chem. 2002, 373, 618627. (23) Tao, W. A.; Cooks, R. G. Angew. Chem., Int. Ed. 2001, 40, 757-760. 10.1021/ac0260948 CCC: $25.00

© 2003 American Chemical Society Published on Web 12/21/2002

Figure 1. ESI mass spectrum of a sample containing (R)-4-benzyl-2-oxazolidinone (100 µM), the amino acid valine (100 µM), GGAG (100 µM), and manganese(II) chloride (25 µM) in a 1:1 methanol/water solution. Major cluster ions are assigned, the analyte (An) being (R)-4-benzyl2-oxazolidinone. Note the peaks marked by asterisks have doubly charged components; the rest are singly charged cluster ions, as confirmed using high-resolution zoom scans shown by the insert figures (high-resolution zoom scan spectra of peaks at m/z ) 608 and 382).

selected trimeric cluster ions, [MII(An)(ref*)2 - H]+, where MII designates the transition metal ion, An designates the analyte, and ref* designates the chiral reference, undergo collision-induced dissociation to yield two dimeric clusters (eq 1) with a branching ratio dictated by the stereochemistry of the analyte An.

The relative branching ratio R (eq 2) for the two competitive dissociation channels is defined as

R ) [MII(An)(ref*) - H]+/[MII(ref*)2 - H]+

(2)

RR and RS are the relative branching ratios for the pure enantiomeric R and S forms of the analyte An. Rchiral (a measure of chiral selectivity) is given by the ratio of RR and RS (eq 3). Note that although it is unusual to use R and S nomenclature for amino acids, we have done this here so that a consistent set of abbreviations can be used in this paper. (24) Tao, W. A.; Wu, L.; Cooks, R. G. J. Am. Soc. Mass Spectrom. 2001, 12, 490-496. (25) Tao, W. A.; Gozzo, F. C.; Cooks, R. G. Anal. Chem. 2001, 73, 1692-1698. (26) Tao, W. A.; Wu, L.; Cooks, R. G. J. Med. Chem. 2001, 44, 3541-3544.

Rchiral )

RR [MII(AnR)(ref*) - H]+/[MII(ref*)2 - H]+ ) RS [MII(An )(ref*) - H]+/[MII(ref*) - H]+ S 2 (3)

The difference in energy required to generate the diastereomeric forms of the fragment ions [MII(An)(ref*) - H]+, due to the two configurations (R or S-form) of the analyte An, is reflected in the degree of chiral distinction (Rchiral). The more different the Rchiral value from unity, the higher the degree of chiral recognition. Chiral discrimination can be increased by optimizing the choice of the central metal and ligands.16 In most previous studies,16,22,25,26 two molecules of the same reference ligand were used in the activated trimeric clusters (eq 1). This procedure limits flexibility in optimizing the interactions that allow chiral discrimination. It also results in the creation and sampling of a variety of isomeric forms of the dissociating trimeric complex, since the loss of the proton can occur from more than one molecule that comprises the complex. This complicates fundamental studies of the energetics and kinetics. In this study, a novel variant of the kinetic method is introduced and shown to improve chiral recognition. The key innovation is to select one of the reference ligands so that it is not lost in the collision-induced dissociation (CID) step. This ligand can be optimized to maximize chiral distinction, which is based on the relative extents of loss the other two ligands. This procedure has the further advantages that the deprotonation site can be confined to the fixed ligand, avoiding the problem referred to the above and producing complexes with unambiguous structures. Analytical Chemistry, Vol. 75, No. 3, February 1, 2003

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Figure 2. MS/MS product ion spectra of [(MnII + Lfixed - H)(ref*)(An)]+ (Lfixed ) GGAG, ref* ) V, An ) (R),(S)-4-benzyl-2-oxazolidinone, m/z ) 608) using a CID activation level 7.5%, corresponding to ∼188 mV zero-to-peak AC excitation amplitude.

Figure 4. MS/MS product ion spectra of [(MnII + Lfixed - H)(ref*)(An)]+ (Lfixed ) GGAG, ref* ) V, An ) (R),(S)-penicillamine, m/z ) 580) using a CID activation level 8.0%, corresponding to ∼200 mV zero-to-peak AC excitation amplitude.

Figure 3. Fixed ligand effects on chiral recognition of 4-benzyl-2oxazolidinone using MnII as the central metal ion, the amino acid V as the reference ligand, and the fixed ligand shown.

Figure 5. Collision energy effects on chiral distinction by the dissociation of the cluster ion [(MnII + Lfixed - H)(ref*)(An)]+ (Lfixed ) GGAG, ref* ) V, An ) (R),(S)-4-benzyl-2-oxazolidinone, m/z ) 608).

EXPERIMENTAL SECTION All experiments were performed using a commercial LCQ ion trap mass spectrometer (ThermoFinnigan, San Jose, CA), equipped with an ESI source and operated in the positive ion mode under the following conditions: spray voltage, 5.00 kV; capillary voltage, 20 V; heated capillary temperature, 150 °C; tube lens offset voltage, 20 V; and sheath gas (N2) flow rate, 30 units (roughly 0.45 L/min). For the ion trap mass analyzer, the automatic gain control (AGC) settings were 5 × 107 counts for a full-scan mass spectrum and 2 × 107 counts for a full product ion mass spectrum with a maximum ion injection time of 200 ms. In the full-scan MS/MS mode, the parent ion of interest was isolated by using multiple waveforms to remove undesired ions through broadband excitation. The isolated ions were then subjected to a supplementary AC potential to resonantly excite them and so cause CID. The Mathieu qz values chosen for resonance excitation and the resonance ejection mass scan were 0.25 and 0.83, respectively. The excitation time used 680 Analytical Chemistry, Vol. 75, No. 3, February 1, 2003

was 30 ms. The excitation amplitude could be varied from 0 to 100% relative collision energy, corresponding to 0-2.5 V zero-topeak resonant excitation potential; the value was optimized in each experiment but was kept constant for the measurement of the R and S enantiomers. Spectra shown represent the average of ∼50 scans, where each scan is an average of five individual microscans. Mass/charge ratios (m/z) are reported using the Thomson unit (1 Th ) 1 atomic mass unit per unit positive charge).27 The error bars at each point in the plots represent the standard deviations for triplicate measurements at 95% confidence level. The amino acids in almost all naturally occurring peptides are in the S enantiomeric form (see above for comment on nomenclature). They are indicated here using the standard one-letter abbreviations. Gas-phase transition metal ion complexes were generated simply by electrospraying 50/50 water/methanol solu(27) Cooks, R. G.; Rockwood, A. L. Rapid Commun. Mass Spectrom. 1991, 5, 93.

Scheme 1. Collision-Induced Dissociation (CID) of a Metal-Bound Trimeric Complex Ion Occurs via Two Channels, Loss of the Reference Ligand and the Analyte, but the Strongly Chelated Fixed Ligand Is Not Lost

Figure 6. Calibration curve for chiral analysis of (R),(S)-4-benzyl2-oxazolidinone using MnII as the central core ion, V as the reference, and GGAG as the fixed ligand. fixed Table 1. Improved Chiral Selectivity (R chiral ) and Increased Differences in Free Energies of Activation When Replacing One Reference Ligand with a Fixed Ligand

ligands, and manganese chloride, were purchased from Sigma Chemical Co. (St. Louis, MO) and were used without further purification. Methanol (HPLC grade) was obtained from Fisher Co. (Pittsburgh, PA).

a The CID collision energy is 9.0%, corresponding to ∼225 mV AC zero-to-peak excitation amplitude. b The standard deviation (SD) is based on triplicate measurements on separate occasions (95% confidence level). c Effective temperature Teff is assumed to be 850 K on the basis of a study of the dissociation of proton-bound and lithiumbound clusters in an ion trap mass spectrometer.38-41 d The standard deviation (SD) was calculated on the basis of the propagation of fixed e variance from the error associated with Rchiral . These cases employ the usual trimeric clusters [MII(An)(ref*)2 - H]+, not a fixed ligand.

RESULTS AND DISCUSSION Formation and Dissociation of Complex Ions Chiral recognition and quantitative analysis using the kinetic method is commonly achieved by the formation of singly charged trimeric ions of the type [MII(An)(ref*)2 - H]+ followed by CID.16,26 The method employs a divalent transition metal ion (MII) as the central core ion, so deprotonation of one of the ligands is needed to complete the coordination shell. Oxazolidinones (abbreviated as An, for analyte) do not easily undergo deprotonation, as indicated by the difficulty met in attempting to form transition-metal-bound homocluster ions. The reference ligands (ref*) are therefore chosen to allow deprotonation as well as to give measurable ion abundance ratios for the two fragmentation channels.26 Among 19 chiral R-amino acids, only a few of them, including A (alanine), V (valine), L (leucine), and I (isoleucine), are eligible as reference ligands on the basis of these criteria. However, chiral recognition was not successful using these references (Table 1), in part because of their small size and lack of additional functional groups.16 The key to success was to replace one of the two reference ligands with an easily deprotonated compound having high metal affinity (thereafter called the fixed ligand, Lfixed). Fragmentation still occurs via two channels, loss of the reference ligand and the analyte, but the strongly chelated fixed ligand is not lost, as illustrated in Scheme 1. Under these conditions, eq 1 takes the form of eq 4; and accordingly, eq 2 and eq 3 become eq 5 and eq 6, respectively

tions containing a mixture of 4-benzyl-2-oxazolidinone, a peptidefixed ligand, and a chiral reference ligand at a concentration of 100 µM each, with the metal salt present at 25 µM concentration. Pentapeptides GGAGG and GGGAG were synthesized at the Purdue Cancer Center (Purdue University, West Lafayette, IN 47907). The identity and purity of the synthetic pentapeptides was confirmed by high-performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS). Tandem mass spectrometry (MS/MS) was used to further confirm the structures of the synthetic pentapeptides. The other peptides, along with (R)-(+)-4-benzyl-2-oxazolidinone and (S)-(-)-4-benzyl2-oxazolidinone (each >99% purity), chiral amino acid reference

Here, Rfixed and Rfixed are the branching ratios for the R S analytes of AnR and AnS when the fixed ligand is used. Note that the equations are written showing the fixed ligand as the only deprotonation site, which is the ideal situation, even if not always achieved in practice. As a result, chiral differentiation resulting

ref*

fixed ligand (Lfixed)

fixed Rchiral ( SDb

∆(∆Gfixed)c ( SDd (kJ/mol)

A

Ae GA GGA GGAG GGAGG

0.943 ( 0.033 1.12 ( 0.04 1.30 ( 0.03 2.89 ( 0.04 1.12 ( 0.07

-0.415 ( 0.247 0.801 ( 0.252 1.85 ( 0.16 7.50 ( 0.10 0.801 ( 0.442

V

Ve GA GGA GGAG GGAGG

0.959 ( 0.031 1.13 ( 0.04 1.32 ( 0.03 3.01 ( 0.03 1.15 ( 0.07

-0.296 ( 0.228 0.864 ( 0.250 1.96 ( 0.16 7.79 ( 0.07 0.988 ( 0.430

L

Le GA GGA GGAG GGAGG

0.949 ( 0.042 1.14 ( 0.04 1.31 ( 0.05 3.12 ( 0.05 1.13 ( 0.08

-0.370 ( 0.238 0.926 ( 0.250 1.91 ( 0.27 8.04 ( 0.113 0.864 ( 0.500

I

Ie GA GGA GGAG GGAGG

0.946 ( 0.040 1.13 ( 0.05 1.36 ( 0.05 3.35 ( 0.05 1.15 ( 0.08

-0.392 ( 0.261 0.864 ( 0.313 2.17 ( 0.26 8.54 ( 0.11 0.988 ( 0.492

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from both the metal-ligand interaction and the ligand-ligand interaction can be finely optimized by changing the properties of the fixed ligand. The facile deprotonation of peptides makes it easy to form the desired singly charged cluster ions by electrospraying a solution containing a transition metal ion, such as MnII, and the analyte 4-benzyl-2-oxazolidinone, as well as a peptide (fixed ligand) and an amino acid (reference ligand). This system also has the convenience of allowing easy changes in the size of the fixed ligand without changes in the chemistry. Figure 1 shows formation of abundant cluster ions in the ESI-MS spectrum in a typical case, including protonated, sodiated, and especially transition-metalbound clusters, all the coordination numbers of the ligands responsible being represented by prominent ions. The most interesting classes of ions are singly charged transition-metalbound trimers formed by deprotonation of one of the constituent ligands. In a higher resolution zoom scan (Figure 1, insert), no half mass between 608 and 609 is observed, confirming the lack of interference by doubly charged cluster ions with the signal due to the desired singly charged trimeric complex ion [MnII + GGAG - H)(V)(4-benzyl-2-oxazolidinone)]+ (m/z ) 608). All of the other singly charged cluster ion assignments labeled in Figure 1 were confirmed by using high-resolution zoom scans. Several peaks marked with asterisks in Figure 1, including *309, *382, *440, *470, and *558, do have doubly charged components, as shown by peaks at half mass between two isotopic peaks. Figure 1 (insert) shows the zoom scan of the peak at m/z 382, a typical case. Moreover, although the peak at m/z of 382 is prominent in the ESI mass spectrum (Figure 1), its intensity decreases significantly when it is subjected to mild collisional activation. The fragile characteristics of these doubly charged ions resulting in part from internal columbic repulsion make them less efficiently isolated. Gas-phase basicities of oligopeptides have been measured using the kinetic method28-30 and have been observed to increase with increasing peptide length.31 Similarly, peptides often have higher metal ion affinities than amino acids, especially when bonding to the transition metal ions, where their special coordination properties allow them to bind strongly at multiple bonding sites to form metallopeptides.32 Therefore, by selecting peptides as fixed ligands, only the chiral amino acid reference ligand and the analyte oxazolidinone will be lost upon collisional activation. That this expectation is achieved in practice is evident from the dissociation behavior of the singly charged cluster ion [(MnII + GGAG - H)(V)(4-benzyl-2-oxazolidinone)]+ (Figure 2). This has the important advantage that there is only one isomeric form of the trimeric ion, that in which the fixed ligand is the chelating ligand. In previous experiments, there has been concern that there might be differential sampling or formation of isomeric complexes, because either the reference or the analyte could be the deprotonated and chelating ligand.16,33 In the fixed-ligand experiments, (28) Wu, Z. C.; Fenselau, C. Tetrahedron 1993, 49, 9197-9206. (29) Kaltashov, I. A.; Fenselau, C. C. Int. J. Mass Spectom. Ion Processes 1995, 146, 339-347. (30) Kaltashov, I. A.; Fabris, D.; Fenselau, C. C. J. Phys. Chem. 1995, 99, 1004610051. (31) Harrison, A. G. Mass Spectrom. Rev. 1997, 16, 201-217. (32) Huang, X.; Pieczko, M. E.; Long, E. C. Biochem. 1999, 38, 2160-2166. (33) Tao, W. A.; Zhang, D.; Wang, F.; Thomas, P.; Cooks, R. G. Anal. Chem. 1999, 71, 4427-4429.

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the branching ratio for these two fragmentations of the trimeric complex ion depends more strongly on the stereochemistry of 4-benzyl-2-oxazolidinone when the same reference and suitable fixed ligands are employed. At a relative CID activation energy of 7.5%, when An is the pure (R)-4-benzyl-2-oxazolidinone, the is 0.398 (Figure 2a), whereas Rfixed is 0.105 branching ratio Rfixed R S for the pure (S)-4-benzyl-2-oxazolidinone (Figure 2b). The chiral fixed selectivity, Rchiral , is therefore 3.79 in this case. Improved Chiral Differentiation with the Optimized Fixed Ligand It is believed that chiral recognition results from both enthalpic contributions (e.g., metal-ligand bonding and electrostatic interactions) and entropic contributions (e.g., steric interactions).34 Both contributions are relatively small when using amino acids as references for 4-benzyl-2-oxazolidinone, and so no obvious chiral differentiation is observed. Keeping the analyte and the reference the same and changing the size of the fixed ligand but not its functionality produces an increasing degree of chiral differentiation. Hence, the simplest chiral amino acid, alanine, was chosen as the base group in a series of fixed ligands and glycinealanine peptides of various sequences, and increasing sizes, including GA/AG, GGA/GAG/AGG, GGAG/GGGA/AGGG, and GGAGG/GGGAG, were examined. Increasing the size of the fixed ligand forces the analyte and the reference ligand closer together, resulting in stronger metal-ligand and ligand-ligand interactions, and accordingly, this is expected to lead to improved chiral discrimination. The effect of the size of the fixed ligand on chiral selectivity, fixed Rchiral , is shown graphically in Figure 3. The fixed ligands GGGA, AGGG, and especially GGAG display dramatically enhanced chiral selectivity compared to the other fixed ligands, including the tri- and pentapeptides. As shown in Table 1, very similar trends were also observed when the amino acids valine, leucine, and isoleucine (V, L, and I) were used as the reference compounds. In particular, the maximum of chiral selectivity is observed using the tetrapeptides. The results can be interpreted in terms of a trade-off between the enthalpic and entropic contributions. If the fixed ligand is relatively small, chiral recognition mainly results from the enthalpic contribution. On the other hand, if the fixed ligand is too large, it results in weaker metalligand interactions, and chiral differentiation mainly comes from the entropy contribution. Only when the fixed ligand is a suitable intermediate size do the overall contributions from both enthalpic and entropic terms maximize, as is the case when using GGAG as the fixed ligand. The effect of changes in the position of the residue A in the peptides was also investigated. It was found that there is no significant difference in chiral distinction when the amino acid A is at any position of the dipeptides and tripeptides. Similarly, when using the pentapeptides GGAGG and GGGAG as the fixed ligands, minor differences in chiral recognition were observed. Furthermore, when the amino acid A is at the N terminus of the tetrapeptide, the fixed-ligand effect decreases, and this decrease becomes larger where the amino acid A is at the C terminus. Because these peptides represent the simplest chiral peptides, further experiments were performed by replacing the amino acids A with G to probe whether A plays a role in chiral discrimination. The results show that the chiral selectivity decreases dramatically when amino acids A are replaced by G (34) Schneider, H.-J. Angew. Chem., Int. Ed. Engl. 1991, 30, 1417-1436.

(i.e., using GGG, GGGG, and GGGGG as the fixed ligands). To investigate this point further, the nonchiral peptide GG(β-A)G (smallest backbone change away from the chiral peptide GGAG) was also examined as the fixed ligand, and the results (Figure 3) show that the chiral selectivity is almost completely removed. When the amino acid A is at one terminus of the tetrapeptide (AGGG or GGGA), it becomes either binding group, and hence, its chiral effect decreases. When the amino acid A is in the middle of the tetrapeptide as in GGAG, the desired coordination geometry with MnII is produced, and a large steric effect involving A leads to improved chiral distinction.35 The effects just ascribed to the positions of the chiral amino acid A are supported by molecular mechanics modeling of the GGAG system (see Supporting Information). However, using the tripeptide GAG as the fixed ligand, the chiral effects become much smaller (Figure 3). Hence, the chiral effects maximize only when the MnII-bound peptide has both a suitable size and geometry. As expected from these ligands, none of the nonchiral peptides (GGG, GGGG, GGGGG, and GG(β-A)G) when used as the fixed ligand allows significant chiral recognition. Note that each transition metal ion has its specific coordination properties with a peptide, and it is predicted that changing either MnII or GGAG will alter the optimized coordination geometry. On the other hand, it can also be predicted that the use of (MnII + GGAG) for chiral recognition of other kinds of compounds will also give enhanced chiral selectivity, and this prediction was confirmed by the case of chiral analysis of the sulfur fixed containing antibiotic penicillamine (Figure 4). In this case, Rchiral is 4.88, an even greater value than that for 4-benzyl-2-oxazolidinone under the same conditions. Chiral Recognition from Free Energy of Activation A linear relationship (eq 7) is expected on the basis of the kinetic method18 between the difference in the free energy ∆(∆Gfixed) requirements of the two competitive reactions and the natural logarithm of the fixed ) chiral selectivity (ln Rchiral

ln Rfixed chiral )

∆(∆Gfixed) RTeff

(7)

Here, R is the gas law constant and Teff is the effective temperature (the average temperature of the activated complex ions for the two competitive reactions36), and ∆(∆Gfixed) is defined as the difference in free energies between reactions 8 and 9, the reverse barriers of which are considered negligible or equal.

[(MII + Lfixed - H)(An)(ref*)]+ f [(MII + Lfixed - H)(An)]+ + ref* (8) [(MII + Lfixed - H)(An)(ref*)]+ f [(MII + Lfixed - H)(ref*)]+ + An (9)

∆(∆Gfixed)

When the analyte consists of a pure R or S enantiomer, becomes ∆(∆Gfixed)R or ∆(∆Gfixed)S, respectively, and eq 7 takes the forms of eq 10 and eq 11. (35) Gyurcsik, B.; Vosekalna, I.; Larsen, E. J. Inorg. Biochem. 2001, 85, 89-98. (36) Laskin, J.; Futrell, J. H. J. Phys. Chem. A 2000, 104, 8829-8837.

ln RR )

∆(∆Gfixed)R RTeff

(10)

ln RS )

∆(∆Gfixed)S RTeff

(11)

For an enantiomeric mixture with an enantiomeric excess of the R enantiomer given by ee, one can write

∆(∆Gfixed) ) ∆(∆Gfixed)R

1 + ee 1 - ee + ∆(∆Gfixed)S ) 2 2

[∆(∆Gfixed)R + ∆(∆Gfixed)S] + 2 [∆(∆Gfixed)R - ∆(∆Gfixed)S] ee (12) 2

Therefore, the relationship between R and ee can be expressed by combining eqs 6, 10, 11, and 12 to obtain eq 13.

ln Rfixed )

[

] [

]

fixed ln(Rfixed ) ln (Rfixed R ) + ln (RS chiral) + ee (13) 2 2

Equation 13 predicts a linear relationship between ln Rfixed and ee using the kinetic method. The predicted behavior is analogous to the observed linear relationships in the nonfixed ligand case of R-amino acids16,33,37 R-hydroxy acids,22 and various chiral drugs,25,26 as well as both chiral23 and isomeric24 dipeptides. It is worth pointing out that eq 13 gives physical meaning to the calibration curve of ln Rfixed versus ee. The slope is equal to onehalf of the natural logarithm of the chiral selectivity, and the intercept is an average of the natural logarithm of branching ratios when the analyte is the pure R or S isomer. It is clear that the larger the chiral selectivity, the larger the contribution to the measured ratio Rfixed made by a given change in ee and the higher the accuracy that will be obtained. Because the energy difference, ∆(∆Gfixed), dictates chiral recognition, the novel method shown here for improvement of chiral selectivity is of fundamental and practical importance in understanding the underlying driving forces behind chiral differentiation. The energy parameter in eq 7, the effective temperature, is assumed to be 850 K on the basis of studies of the dissociation of proton-bound and alkali-metal-bound amino acid and peptide clusters.38-41 Using this temperature, the value ∆(∆Gfixed) can be estimated as summarized in Table 1. In the favorable case when using the amino acids A, V, L, and I as the reference ligands and GGAG as the fixed ligand, an energy difference of more than ∼7 kJ/mol for two competitive dissociations was observed. (37) Zhang, D.; Tao, W. A.; Cooks, R. G. Int. J. Mass Spectrom. 2001, 204, 159169. (38) Vekey, K.; Gomory, A. Rapid Commun. Mass Spectrom. 1996, 10, 14851496. (39) Vekey, K.; Czira, G. Anal. Chem. 1997, 69, 1700-1705. (40) Cerda, B. A.; Hoyau, S.; Ohanessian, G.; Wesdemiotis, C. J. Am. Chem. Soc. 1998, 120, 2437-2448. (41) Nold, M. J.; Cerda, B. A.; Wesdemiotis, C. J. Am. Soc. Mass Spectrom. 1999, 10, 1-8.

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Table 2. Enantiomeric Measurement (ee) on (R)-4-Benzyl-2-oxazolidinonea enantiomeric excess (ee) of (R)-4-benzyl-2-oxazolidinone sample

actual (%)

experimental (%)

1 99 97.9 2 66 66.7 3 32 31.3 4 1 1.92 overall av rel error (%)

99.8 65.8 33.2 0.462

99.3 67.6 32.8 0.511

av (%)

rel error (%)

99.0 66.7 32.4 0.964

0.0 1.0 1.3 3.6 1.5

a Using MnII as the central metal ion, GGAG as the fixed ligand, and V as the reference ligand.

Construction of Calibration Curves Using Different Collision Energies. Figure 5 illustrates how chiral differentiation varies with CID collision energy when using GGAG as the fixed ligand. The chiral recognition decreases with increasing collision energy and then becomes constant. This behavior is consistent with the expectation that the higher the collision energy, the higher the effective temperature of the activated cluster ion.42 At high CID energy, the difference in dissociation energetics is overwhelmed by the increasing internal energy of the excited trimeric complex ion. The occurrence of multiple collisions in an ion trap mass spectrometer makes the specific activation energy supplied difficult to characterize. For most reproducible measurefixed ments of chiral selectivity, the region where Rchiral is constant should be used, even though some loss in chiral discrimination is experienced. There is actually an offsetting increase in the probability of dissociation at higher collision energy. Based in Figure 5, the relative CID collision energy of 9.5% was chosen for constructing the calibration curve, and a correlation coefficient (r2) of 0.9995 was obtained (Figure 6). The highly linear relationship exhibited by the calibration curve and predicted by eq 13 as discussed in the previous section allows very low ee to be determined. Unknown solutions were prepared to test the constructed calibration curve, especially for the extreme cases of very low and very high ee values. Each sample was measured three times on separate occasions to improve precision, and the results that are tabulated in Table 2 show that samples with 1% ee could be determined with an average error of ∼1.5%. This novel approach to chiral analysis offers improved accuracy over the standard kinetic method procedure. It is clearly useful for pharmaceutical applications in which enantiomeric contamination is usually of concern at very low ee values.

formed by ESI-MS and have been used previously for chiral recognition and quantification. New to this study is the use of a fixed ligand with properties that are intended to prevent it from being lost upon dissociation of the complex ion. In comparison with the dissociation of trimeric complex ions with two identical reference ligands, the metal-ligand and ligand-ligand interactions in the dissociation of trimeric complexes containing one fixed ligand are easier to optimize, and hence, improved chiral recognition is more readily obtained.16 Another advantage of this approach for chiral analysis is that it simplifies the dissociation kinetics in that only the reference ligand or the analyte can be lost. In an application that uses peptides as fixed ligands, it is shown that the amino acid A in the middle of the tetrapeptide GGAG can most effectively transfer its chiral effect to the glycine containing MnII-chelating rings, resulting in a large improvement in chiral selectivity for the antibiotics, including oxazolidinone and penicillamine. This interpretation is supported by molecular mechanics modeling. In future work, other fixed ligands, including oligonucleotides, enzymes, ion channel receptors, and R,ω-diamines or their metalcoordinated complexes, will be selected together with deprotonated chiral reference compounds, such as carboxylic acids. Further investigation of the change of chirality of the fixed ligands and the references is ongoing. This version of the kinetic method provides the basis for controlled chiral discrimination in the gas phase while yielding a linear relationship between the natural logarithm of two fragment ion abundance ratios and the enantiomeric composition of antibiotics, such as 4-benzyl-2-oxazolidinone and penicillamine. SUPPORTING INFORMATION AVAILABLE Molecular mechanics modeling showing improved chiral selectivity for analysis of (R),(S)-4-benzyl-2-oxazolidinone using the fixed ligand GGAG is available as Supporting Information. This material is available free of charge via the Internet at http:// pubs.acs.org. ACKNOWLEDGMENT This work was supported by the U.S. Department of Energy, Office of Basic Energy Sciences, and by the National Science Foundation (CHE 97-32670). Fellowship support from Merck & Co. is gratefully acknowledged.

CONCLUSIONS Cluster ions containing a divalent transition metal ion, a fixed peptide ligand, a chiral reference ligand, and an analyte are readily

Received for review August 30, 2002. Accepted November 15, 2002.

(42) Vekey, K. J. Mass Spectrom. 1996, 31, 445-463.

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684 Analytical Chemistry, Vol. 75, No. 3, February 1, 2003