Anal. Chem. 1997, 69, 1700-1705
Distinction of Amino Acid Enantiomers Based on the Basicity of Their Dimers Ka´roly Ve´key* and Ga´bor Czira
Central Research Institute for Chemistry, H-1025 Budapest, Pusztaszeri u´ t 59-67, Hungary
Mixtures of several amino acid pairs, in all four chiral combinations, were studied. The protonated trimers (A2BH+) fragment, forming ABH+ and A2H+ dimers. Abundance ratios of these fragments were measured in the mass-analyzed ion kinetic energy spectra of the trimers. These were found to depend on the stereochemistry (homo- or heterochiral form) of the ABH+ dimer. The results were evaluated using the kinetic method, and the chiral discrimination was related to a difference in gasphase basicity (GB) between the homo- and the heterochiral dimers. Four amino acid pairs (proline-tryptophan, phenylalanine-alanine, phenylalanine-proline, and phenylalanine-valine) were studied. Chiral discriminations were observed in all cases, relating to 0.4-4 kJ/ mol differences in GB. The technique described here can generally be used to study enantiomers by mass spectrometry and is capable of reliably distinguishing energy differences as small as 0.2 kJ/mol in cluster ions. A challenging area of mass spectrometry is the study of isomers,1 and in particular optically active (chiral) compounds. If a molecule has one chiral center, it may exist in two alternative forms, which are called enantiomers. These are mirror images of each other and are designated typically as R or S, D or L, or + or -. Isolated, or in a nonchiral environment, enantiomers have identical chemical characteristics, but they display different behavior toward other chiral compounds. Most biomolecules are optically active, so chiral interactions have received much attention. Such processes can also be studied by mass spectrometry, which has the advantage of isolating the interacting molecules in the gas phase, so that intrinsic properties of the chiral effect can be studied. A recent example is the study of proton transfer reactions of cytochrome c, which reacts more favorably with (R)than with (S)-sec-butylamine.2 Chiral reactions can also be utilized to distinguish enantiomers. In isolation, these have identical properties and so, by definition, have identical mass spectra. If two enantiomers react with another chiral compound, however, they may be distinguished, e.g., by studying the reaction product or the reaction rate by mass spectrometry. The first such example was observed 20 years ago:3 in electron impact ionization diisopropyl tartrate molecules associate, forming proton-bound dimers. There are four possible (1) Splitter, J. S., Turecek, F., Eds. Applications of mass spectrometry to organic stereochemistry; VCH Publishers: New York, 1994. (2) Camara, E. J. O.; Green, M. K.; Penn, S. G.; Lebrilla, C. B. Presented at the 44th ASMS Conference on Mass Spectrometry and Allied Topics, Portland, OR, May 12-16, 1996. (3) Fales, H. M.; Wright, G. J. J. Am. Chem. Soc. 1977, 99, 2339.
1700 Analytical Chemistry, Vol. 69, No. 9, May 1, 1997
forms (R,R; S,S; R,S; and S,R). Among these, R,R and S,S are enantiomers, having identical chemical and physical properties; likewise, so are R,S and S,R. The “homochiral” dimers (R,R or S,S) are not mirror images of the “heterochiral” ones (R,S or S,R), but they are diastereomers. It was found that, in the mass spectra, the ion intensities of the homochiral tartrate dimers were higher than those of the heterochiral ones, indicating higher stability of the former. Since this initial study, several more recent examples of enantiomer distinction by mass spectrometry have been published (refs 1-11 and references therein). Chemical ionization is an often-applied technique in this field (refs 1 and 9 and references therein). The largest differences observed were related to tartrates, which have been studied in detail.3,10 Crown etheramine complexes often show a relatively large degree of chiral recognition.4-8 Amino acids form another class of compounds of biochemical interest, where chiral effects were studied using mass spectrometry.11 In spite of these efforts, only limited success is achieved in the study of chirality by mass spectrometry, mainly because differences in the spectra of enantiomers, if any, are usually very small. Studies on chiral molecules indicate that, to observe differences in the spectra, two chiral reactants should form of a polydentate complex. The fragmentation patterns of homo- and heterochiral dimers are usually identical. The spectral differences observed were, nearly without exception, related to a difference in the stability between the (protonated or cationized) homo- and heterochiral dimer. Energy differences up to 4 kJ/mol can be expected in favorable cases. To study such small energy differences, special experimental techniques and/or methods for data evaluation are highly desirable. Such developments were discussed in relation to chiral crown ether-amine complexes: the use of stability constants measured from ion abundances in the spectra4 were found to be more advantageous than the conventional approach of using relative peak intensities;6,7 recently the idea of using two internal standards for the study of bimolecular complex formation (one for each reactant) has been suggested.5 (4) Po´csfalvi, G.; Lipta´k, M.; Huszthy, P.; Bradshaw, J. S.; Izatt, R. M.; Ve´key, K. Anal. Chem. 1996, 68, 792. (5) Dobo´, A.; Lipta´k, M.; Huszthy, P.; Ve´key, K. Eur. Mass Spectrom., submitted for publication. (6) Sawada, M.; Shizuma, M.; Takai, Y.; Yamada, H.; Kaneda, T.; Hanafusa, T. J. Am. Chem. Soc. 1992, 114, 4405. (7) Sawada, M.; Okumura, Y.; Yamada, H.; Takai, Y.; Takahashi, S.; Kaneds, T.; Hirose K.; Misumi, S. Org. Mass Spectrom. 1993, 28, 1525. (8) Chu, I.-H.; Dearden, D. V.; Bradshaw, J. S.; Huszthy, P.; Izatt, R. M. J. Am. Chem. Soc. 1993, 115, 4318. (9) Sellier N. M.; Bouillet, C. T; Douay, D. L.; Tabet, J.-C. E. Rapid Commun. Mass Spectrom. 1994, 8, 891. (10) Winkler, J.; Krause, H. J. Chromatogr. A 1994, 666, 549. (11) Shen, W.; Patrick J. S.; Cooks, R. G., unpublished results. S0003-2700(96)00931-6 CCC: $14.00
© 1997 American Chemical Society
A frequently used technique for determination of thermochemical values is the “kinetic method” developed by Cooks and co-workers.12-15 The central idea of this method is the observation of two competitive reactions of an ion. Typically a proton-bound dimer is selected which fragments, forming monomers, either of which can be protonated. Very small changes in the relative critical energy of the two reaction channels, which correlate very closely with product stabilities, cause large changes in the respective rate constants, which are directly proportional to fragment ion abundances, provided there are equal detection efficiencies for ions of different masses. This means that very small differences in product ion stability result in large changes in relative fragment ion abundances. Energy differences smaller than 1 kJ/mol cause easily detectable changes in the relative abundances. Derivation of gas-phase basicities using the kinetic method is based on the following equations:12-15
A-H+-B f AH+ + B
(kA)
f BH+ + A
(kB)
(1)
ln(kA/kB) ) ln([AH+]/[BH+])
(2)
) {GB(A) - GB(B)}/RTeff In the equations above, A and B are the two monomers; GB(A) and GB(B) are their gas-phase basicities; kA and kB are the rate constants of formation of AH+ and BH+; square brackets ([AH+] and [BH+]) indicate abundances (peak areas) of the fragment ions; Teff is the “effective” temperature (related to the internal energy) of the dissociating dimer. Using a series of reference bases (B), the basicity of A can be determined. There are requirements for the structural similarity of A and B which, if properly observed, allow determination of proton affinities as well.12-15 Application of the kinetic method to the distinction of enantiomers is likely to have the advantage of detecting stability differences, which are too small to measure using other techniques. The stability of a proton-bound dimer (ABH+) formed from chiral compounds A and B can be characterized by the dissociation of a proton-bound trimer (ABRH+), formed from A, B, and a reference compound, R. Besides other fragmentation channels, this protonated trimer dissociates into a protonated dimer, ABH+, and a protonated reference compound, RH+, in competitive processes. The abundance ratio [ABH+]/[RH+] will characterize the basicity of AB compared to that of R. If there is a difference in the stability of ABH+ of homochiral and heterochiral stereochemistry, this will result in different [ABH+homo]/[RH+] and [ABH+hetero]/[RH+] abundance ratios. From the point of view of analytical chemistry, this abundance ratio can characterize whether the ABH+ dimer is of homo- or of heterochiral configuration. If one of the reactants is of known configuration (used as a “chiral reference” compound), the chirality of the other can be determined. From the [ABH+]/[RH+] abundance ratios, ther(12) Cooks, R. G.; Kruger, T. L. J. Am. Chem. Soc. 1979, 99, 1279. (13) McLuckey, S. A.; Cameron, D.; Cooks, R. G. J. Am. Chem. Soc. 1981, 103, 1313. (14) Majumdar, T. K.; Clairet, F.; Tabet, J. C.; Cooks, R. G. J. Am. Chem. Soc. 1992, 114, 2897. (15) Patrick, J. S.; Kotiaho, T.; McLuckey, S. A.; Cooks, R. G. Mass Spectrom. Rev. 1994, 13, 287.
modynamic properties of ABH+ dimers can also be derived. While this approach is conceptionally straightforward, it presents practical difficulties: (1) ABRH+ clusters in a ternary mixture have lower abundance than A2BH+ clusters in binary mixtures. (2) More importantly, protonated trimers preferentially form protonated dimers in their CID-MIKE fragmentation (and even more so in metastable MIKE experiments), the protonated monomer fragments have usually low abundance (if observable at all). This means that measurement of the [ABH+]/[RH+] fragment abundance ratio will not be precise, if possible at all. Information on the relative stability of a proton-bound homochiral or heterochiral ABH+ dimer can also be obtained from the fragmentation of proton-bound A2BH+-type trimers. This approach is discussed below in detail. In this approach, the [ABH+]/[A2H+] fragment ion abundance ratio is used, which is easy to measure (typically both peaks are abundant fragments). Such an extension of the kinetic method was described recently for the determination of molecular pair gas-phase basicities (MPGBs).16 The A2BH+type trimer has four reaction channels (two of them degenerate), and it was shown that the various reaction channels are competitive. Taking the case of the dissociation of an A2BH+ protonbound trimer, eq 3 indicates the alternative reaction channels, while others like eqs 4 and 5 can be used for MPGB determination.
A2BH+ f A2H+ + B
(kA2)
f BH+ + A2
(kB) (3)
f ABH + A
(kAB)
f AH+ + AB
(kA)
+
ln(kA2/kB) ) ln([A2H+]/[BH+])
(4)
) {GB(A2) - GB(B)}/RTeff ln({kAB/2}/kA2) ) ln({[ABH+]/2}/[A2H+])
(5)
) {GB(AB) - GB(A2)}/RTeff The equations required have been derived and discussed in detail before, together with examples of MPGB values of some amino acids. Using eq 4, one has to consider that the monomer may be formed by a sequential process, like A2BH+ f ABH+ + A f BH+ + 2A. This, however, would require a larger activation energy than the direct cleavage shown in eq 3 by the amount of dissociation energy of A2 to 2A, and so would be less abundant, probably insignificant, compared to BH+ formation by direct cleavage. In the case of amino acids, the basicity of B2 is larger than that of B by ∼15 kJ/mol;16 were the dissociation energy of A2 only half this amount (i.e., 7.5 kJ/mol), sequential processes would introduce only 0.16 kJ/mol error in the MPGB value (were the dissociation energy of A2 zero, the error would be still less than 0.7 kJ/mol). Reproducibility of MPGB measurements was better than ∼0.5 kJ/mol, and the internal consistency for glycine, alanine, and valine dimers was ∼0.8 kJ/mol.16 Using eq 5, the difference in basicity between dimers A2 and AB can be determined. In the present paper, only this equation (16) Ve´key, K.; Czira, G. Rapid Commun. Mass Spectrom. 1995, 9, 783.
Analytical Chemistry, Vol. 69, No. 9, May 1, 1997
1701
will be used and will be related to chiral recognition. Division by 2 in eq 5 reflects the number of alternative reaction channels leading to the same product. Sequential processes will have an even smaller impact on the use of eq 5 than on MPGB values: less than 0.01 kJ/mol using the numerical example above. Chiral compounds in general, and amino acid enantiomers in particular, can be distinguished by a number of techniques, most importantly by their optical rotation (circular dichroism or optical rotatory dispersion spectroscopies17,18) by gas or liquid chromatography using a chiral stationary phase.19 In the present paper, we attempt to use mass spectrometric techniques for the same purpose, not to compete with existing methods of amino acid enantiomer analysis but to show that such subtle differences can be studied by mass spectrometry. Further development may, in the future, offer advantages in practical analysis as well. The present approach involves the use of the kinetic method to distinguish homochiral and heterochiral amino acid dimers. This is compared to other mass spectrometric techniques that which are often used to characterize isomers. Emphasis is placed on the measurement of the chiral effect, but by this approach the difference in the MPGB of homo- vs heterochiral amino acid dimers is also possible. EXPERIMENTAL SECTION Experiments were run on a reverse geometry VG-ZAB-SEQ instrument. Saturated solutions of D- and L-amino acid samples in water (containing 1% HCl) were prepared. One-microliter aliquots of two optically pure amino acid solutions were added to 1 µL of glycerol matrix on the probe. Spectra were taken using FAB ionization (Cs+ bombardment at 30 kV energy, also called liquid secondary ion mass spectrometry, LSIMS). Fragmentation of cluster ions was studied by the mass-analyzed ion kinetic energy (MIKE) technique. Metastable fragmentations were studied introducing no collision gas into the instrument. Fragmentation by collision-induced decomposition (CID) was also studied (8 kV accelerating voltage, Ar collision gas). Main beam transmission was 80%, where mainly single collisions occur. To separate the metastable contribution from the CID process, the collision cell was floated at 0.5 kV, when the CID and metastable peaks were separated. In the subsequent analysis, only the CID fragments were used. MIKE spectra were recorded using “raw” (noncentroided) data, where peak areas were measured. This provides much better accuracy, especially for peaks of small abundance, than the use of centroided data. To obtain a good signal-to-noise ratio, spectra were measured for 10 min, and data in the period between 2 and 8 min (starting from sample insertion) were summed. Amino acid pairs were studied in all four chiral combinations, and the measurements were repeated at least three times. Measurements on a given amino acid pair were made within one day. RESULTS AND DISCUSSION Mixtures of two optically pure amino acids, tryptophan (Trp) and proline (Pro), were studied by FAB ionization mass spectrometry. A typical spectrum is depicted in Figure 1, showing protonated molecular ions (TrpH+, ProH+), protonated dimers (17) Eliel, E. L.; Wilen, S. H.; Mander, N. L. Stereochemistry of Organic Compounds; Wiley-Interscience: New York, 1994; Chapter 13. (18) Nakanishi, K., Perova, N., Woody, R. V., Eds. Circular Dichroism: Principles and Applications; VCH Publishers: New York, 1994. (19) Pirkle, W. H.; Pochapsky, T. C. Chem. Rev. 1989, 89, 327.
1702 Analytical Chemistry, Vol. 69, No. 9, May 1, 1997
Figure 1. FAB mass spectrum of a mixture of tryptophan and proline. Major cluster ions are indicated.
(Trp2H+, TrpProH+, Pro2H+), and protonated trimers (Trp3H+, Trp2ProH+, TrpPro2H+, Pro3H+). Other peaks also appear in the spectrum due to fragments, matrix (glycerol, G) peaks, matrixcontaining clusters (e.g., ProGH+), and, with low abundance, protonated amino acid tetramers. The mass spectra were studied using optically pure amino acids in all four combinations: Dtryptophan mixed with D-proline, L-tryptophan mixed with Dproline, L-tryptophan mixed with L-proline, and D-tryptophan mixed with L-proline. These mixtures will be abbreviated as (D,D), (L,D), (L,L), and (D,L), respectively. Peak abundances in the single-stage mass spectra showed no measurable differences among optical isomer pairs. The relative abundances of protonated dimers and trimers were checked with particular care. Variations in the abundance ratio of, e.g., [TrpProH+]/[Trp2H+] were less than 5%, suggesting random errors rather than isomeric differences. These results indicate that the difference in stability between the homoand the heterochiral TrpProH+ dimers, if any, is not sufficient to cause an observable change in the abundance of the various TrpProH+ ions in the FAB spectra. Regarding terminology, D-TrpH+-D-Pro and L-TrpH-L-Pro+ are defined as homochiral dimers, while D-TrpH-L-Pro+ and L-TrpH-D-Pro+ are defined as heterochiral dimers, and these will be abbreviated as D,D; L,L; D,L; and L,D, respectively. Fragmentation of the TrpProH+ dimer was also studied using metastable and CID mass-analyzed ion kinetic energy (MIKE) spectra. The main fragment ion was TrpH+, but ProH+ was also observed, corresponding to the typical behavior of a proton-bound dimer. The peak abundance ratio [TrpH+]/[ProH+] reflects the relative gas-phase basicity of the two amino acids (which is often approximated by the relative proton affinity); the stereochemistry (homo- or heterochiral dimer) did not have any effect on this ratio. (The proton affinities of tryptophan and proline are 944 and 922 kJ/mol, respectively.20) Other (low-abundance) fragments present in the CID-MIKE spectrum also did not show stereoisomeric differences. The fragmentation rate of the protonated dimer should depend on its stability: The more stable stereoisomer (i.e., the homo- or the heterochiral dimer) should have a lower fragmentation rate, decreasing the abundance of the fragment ion relative to the main beam (the ion ratio [TrpH+]/[TrpProH+]) in the metastable or CID-MIKE spectrum. These ratios, however, did not show any stereoisomeric difference. (20) Lias, S. G.; Liebman, J. F.; Levin, R. D. J. Chem. Phys. Ref. Data 1984, 13, 695.
Table 1. Fragment Ion Abundance Ratios [ABH+]/ [A2H+] As Observed in the Metastable or CID MIKE Spectra of A2BH+ a
metastable
A
B
enantiomer form
Trp
Pro
D,D
[ABH+]/ [A2H+] (av)
SD of average
0.241
0.001
0.239 0.303 0.289 1.528 1.529 1.656 1.643 2.936 3.049 3.429 3.461 0.291 0.291 0.315 0.313 15.221 15.098 9.346 9.901 0.786 0.776 0.744 0.749
0.004 0.002 0.001 0.003 0.003 0.010 0.008 0.017 0.030 0.027 0.030 0.002 0.002 0.004 0.002 0.310 0.447 0.412 0.350 0.003 0.004 0.003 0.002
L, L D,L L, D
CID
Trp
Pro
D,D L, L D,L L, D
CID
Pro
Trp
D,D L, L
Figure 2. Relevant part of the CID-MIKE spectrum of the protonated trimer cluster, Trp2ProH+. The metastable (“META”) and CID components of the fragment ions (TrpProH+ and Trp2H+) are separated due to the voltage on the collision cell. Peaks outside the range shown (e.g., the protonated monomers) have much lower intensity (5% or less).
D,L L, D
CID
Phe
Ala
D,D L, L D,L L, D
CID
Phe
Pro
D,D L, L
The “conventional” approaches of optical isomer distinction having failed, we have used the idea of the kinetic method, as described in the introduction. The CID-MIKE fragmentation of an A2BH+-type cluster ion, the proton-bound Trp2ProH+ trimer, was studied. The two main fragments were TrpProH+ and Trp2H+ (Figure 2). All combinations of amino acid enantiomers were used. The resulting Trp2H+ fragment is always a homochiral dimer (D,D or L,L) and its peak is used as a reference peak. TrpProH+, on the other hand, could be either be a homo- or a heterochiral dimer. If the heterochiral TrpProH+ would be more stable than the homochiral one, the [TrpProH+]/[Trp2H+] abundance ratio would be higher for the heterochiral dimer. This, indeed, was the case: the ratios were 1.649 for the heterochiral and 1.528 for the homochiral protonated dimers (Tables 1 and 2). The chiral discrimination, ∆Rchiral, can be defined by the following equation:
∆Rchiral(AB) )
D,L L, D
CID
Val
D,D L, L D,L L, D
SD of ∆Rchiral
1.233
0.007
1.079
0.004
1.151
0.008
1.079
0.008
0.645
0.015
0.956
0.004
a Metastable and CID components were separated by floating the collision cell to 0.5 kV. Standard deviations and the chiral discrimination (∆Rchiral) are also shown.
Table 2. Fragment Ion Abundance Ratios [TrpProH+]/ [Trp2H+] As Observed in the CID MIKE Spectra of Trp2ProH+ enantiomer form D,D L,L D,L L,D
[ABH+hetero]/[A2H+]
Phe
∆Rchiral
[ABH+]/[A 1.533 1.523 1.660 1.632
1.528 1.537 1.623 1.639
+ 2H ]
1.535 1.525 1.676 1.669
1.517 1.529 1.663 1.633
SD
[ABH+]/ [A2H+] (av)
SD of average
0.007 0.005 0.020 0.015
1.528 1.529 1.655 1.643
0.003 0.003 0.010 0.008
) [ABH+homo]/[A2H+]
{[ABH+]/[A2H+]}DD + {[ABH+]/[A2H+]}LL {[ABH ]/[A2H ]}DL + {[ABH ]/[A2H ]}LD +
+
+
(6)
+
The ∆Rchiral value in the present case is 1.079, with an accuracy of ∼0.004 (Table 1). All possible care was taken to assure that the results quoted here are due to “real” differences of chiral compounds and not to occasional “random” errors, impurities or badly designed experiments. Standard solutions of the four compounds (D- and L-tryptophan, D- and L-proline) were made, and 1 µL aliquots were mixed with 1 µL of matrix on the probe (even though sample preparation and concentration should not affect the MIKE spectra of a given trimer). The spectra were studied for 10 min, and the average between 2 and 8 min was used to determine peak areas for TrpProH+ and Trp2H+, a procedure similar to that used in the case of some crown ethers.4,5 Spectra of mixtures D,D; D,L; L,L; and L,D were studied in this sequence, and then this procedure was repeated four times. The detailed results are shown in Table 2, where a statistical evaluation is also presented.
The two homochiral dimers D,D and L,L are enantiomers, and, therefore, should have identical behavior, among others an identical [TrpProH+]/[Trp2H+] abundance ratio in the CID-MIKE spectrum. Tables 1 and 2 shows that this is, indeed, the case; the measured peak ratios for D,D and L,L (on average 1.528 and 1.529) are well within experimental error (standard deviation of this average is 0.003 in both cases). The heterochiral dimers D,L and L,D show likewise identical fragment ion ratios: 1.656 ( 0.010 and 1.643 ( 0.008, respectively. The identical behavior of the two homochiral and the two heterochiral ProTrpH+ dimers is a necessary prerequisite and a very strong indication that the observed behavior reflects, indeed, a stereochemical difference between the homo- and the heterochiral dimers. The behavior of the Trp2ProH+ cluster ion was also studied by its unimolecular fragmentation (metastable MIKE spectrum). The fragment ion abundance ratio [TrpProH+]/[Trp2H+] is much lower in this case, reflecting the lower internal energy of the parent ion and indicating that Trp2 has a higher basicity than TrpPro. The resulting TrpProH+ fragment has a higher abundance in the case of the heterochiral than in the homochiral dimer, similar to Analytical Chemistry, Vol. 69, No. 9, May 1, 1997
1703
Table 3. Relative Gas-Phase Basicities (∆GBchiral) of Hetero- and Homochiral Protonated Amino Acid Dimers, in kJ/mol Units, Calculated from Data in Table 1 Using Eq 7 dimer
∆GBchiral a
comment
TrpProH+
0.6 1.1 1.0 0.6 -3.3 -0.4
from Trp2ProH+; CID from Pro2TrpH+; CID from Trp2ProH+; metastable from Phe2AlaH+; CID from Phe2ProH+; CID from Phe2ValH+; CID
PheAlaH+ PheProH+ PheValH+ a
Figure 3. Relevant part of the CID-MIKE spectrum of the protonated trimer cluster, Pro2TrpH+. The metastable (“META”) and CID components of the fragment ions (TrpProH+ and Pro2H+) are separated due to the voltage on the collision cell. Peaks outside the range shown (e.g., the protonated monomers) have much lower intensity (5% or less).
the observation in CID-MIKE. The relative abundances for the two homochiral and also for the two heterochiral dimers are identical within experimental error (Table 1). The relative stability of the homo- and the heterochiral TrpProH+ dimers was checked using fragmentation of a different cluster ion as well. The protonated trimer cluster, Pro2TrpH+, yields TrpProH+ and Pro2H+ fragment ions in CID-MIKES (Figure 3). The spectra were studied for all four combinations of amino acid enantiomers, and the abundance of the homo- or heterochiral TrpProH+ was compared to that of the always homochiral Pro2H+ ([TrpProH+]/[Pro2H+]). The result (Table 1) confirms that the relative abundance of the heterochiral dimer is higher than that of the homochiral dimer. The ratio of fragment ion abundances for the two homochiral (D,D or L,L) dimers is identical within experimental error; the same is true for the two heterochiral (D,L or L,D) dimers. The relative ion abundances discussed above indicate that the heterochiral TrpPro dimer has a higher basicity (TrpProH+ is more stable) than the homochiral dimer. The results discussed above can be evaluated by the recent modification of the kinetic method.16 The gas-phase basicity of TrpPro relative to that of Trp2 can be determined on the basis of eq 5, provided the effective temperature (Teff) is known. In the present case, the effective temperature is estimated to be 970 K in CID-MIKE spectra, taken from the previous study on MPGB of various amino acid dimers,16 which was done using the same experimental conditions. Using the results from CID experiments listed in Table 1, the gas-phase basicity (at ∼970 K) of the homochiral TrpPro dimer can be calculated from eq 5 to be 2.2 kJ/mol lower than that of Trp2 and 3.3 kJ/mol higher than that of Pro2. Combining these values, the gas-phase basicity of Trp2 is 5.5 kJ/mol higher than that of Pro2. Errors in the effective temperature would change these values only slightly, by ∼(0.5 kJ/mol. The results are in accord with earlier data16 that (a) mixed dimers have a gas-phase basicity close to the average of the “pure” dimers and (b) differences between the MPGB values of amino acids are lower than differences between GBs. Basicities are (effective) temperature-dependent values, but this dependence is not studied or discussed in the present paper. 1704
Analytical Chemistry, Vol. 69, No. 9, May 1, 1997
Positive values indicate higher basicity for the heterochiral dimer.
From eqs 5 and 6, the relative gas-phase basicity of homo- and heterochiral dimers can be derived:
∆GBchiral(AB) ) GB(ABhetero) - GB(ABhomo) (7) ) RTeff ln{∆Rchiral(AB)} A positive ∆GBchiral value indicates that the heterochiral dimer is more basic (suggesting that its protonated form is more stable) than the homochiral one. In the case of the TrpPro dimer, ∆GBchiral was calculated using eq 7, ion abundance ratios listed in Table 1, and 970 K effective temperature. ∆GBchiral(ProTrp) calculated from the fragmentation of Trp2ProH+ is 0.6 kJ/mol. This indicates that the heterochiral TrpProH+ is more stable than the homochiral one. The relative basicity calculated using the fragmentation of Pro2TrpH+ is of the same sign but larger (1.1 kJ/mol, Table 3). The metastable fragmentation of Trp2ProH+ can also be used to determine ∆GBchiral, but the effective temperature in this case is lower; estimated to be 600 K.21 ∆GBchiral(TrpPro) determined this way is 1.0 kJ/mol. The standard deviation among the three different ∆GBchiral values is 0.2 kJ/mol, somewhat larger than random errors in the calculations (∼(0.1 kJ/mol). This suggests that systematic errors (probably estimation of the effective temperature) are likely to have some effect on the calculation of ∆GBchiral. The overall accuracy of the ∆GBchiral(TrpPro) values, taking into account systematic errors, is estimated to be better than (0.3 kJ/ mol. Other chirally pure amino acid dimers (PheAla, PheVal, and PhePro) were also studied by this technique; the results are listed in Tables 1 and 3. As for the tryptophan-proline mixture, all four chiral combinations were studied in each case. The D,D and L,L mixtures on the one hand, and D,L and L,D mixtures on the other hand, always gave the same results. Peak abundances were measured in CID-MIKE spectra. The spectra were reproduced several times, and the reproducibility was similar to that described above. In the case of the phenylalanine-alanine mixture, fragmentation of the Phe2AlaH+ cluster was studied. The abundance of PheAlaH+ was compared to that of Phe2H+; their ratio was higher in the case of the heterochiral PheAlaH+ dimer, similar to the case of ProTrpH+. The calculated ∆GBchiral value is 0.6 kJ/mol. In the case of the phenylalanine-proline mixture, fragmentation of the Phe2ProH+ cluster was studied. In this case, the (21) Ve´key, K.; Czira, G., unpublished results.
abundance of PheProH+ was higher in the case of the homochiral than in the heterochiral PheProH+ dimer, in contrast to the case of TrpProH+. The calculated ∆GBchiral value is a high negative value, -3.3 kJ/mol. This indicates that, in this case, the homochiral dimer is the more stable stereochemical form. This is the case also for the PheValH+ dimer, but with a small ∆GBchiral ) -0.4 value. The corresponding difference of relative peak abundances between the stereoisomers is quite small in the case of PheValH+ (4%, see Tables 1 and 3), though still easily measurable (random errors are an order of magnitude lower, ∼0.4%). Data in Table 1 give the reproducibility (standard deviation) of ∆Rchiral values; Table 2 shows that the distribution of individual data is close to statistical (normal distribution). The reproducibility of ∆Rchiral shown in Table 1 also suggests that random errors are smaller in those cases when ∆Rchiral is closer to unity. The average standard deviation of the three ∆Rchiral values closest to unity is 0.005. This can be converted to a standard deviation of 0.05 kJ/mol in the ∆GBchiral value. This suggests that a difference as small as 0.1-0.2 kJ/mol between the basicities of homochiral and heterochiral dimers can be detected by the present approach. CONCLUSIONS Protonated amino acid trimer clusters fragment, forming protonated dimers. The relative rate of the two competitive reactions (A2BH+ f ABH+ and A2BH+ f A2H+) is measured by the fragment ion abundance ratio [ABH+]/[A2H+]. If optically pure amino acids are mixed together, A2H+ will always be of homochiral stereochemistry, so A2BH+ f A2H+ can be used as a reference process. ABH+, on the other hand, can be of either homochiral or heterochiral stereochemistry. The [ABH+homo]/ [A2H+] and [ABH+hetero]/[A2H+] ratios were found to be characteristically different. The values obtained for the two homochiral (D,D and L,L) clusters were identical, and so were those of the two heterochiral (D,L and L,D) dimers. The alternative AB2H+ f ABH+ and AB2H+ f B2H+ processes were also studied, and these yielded analogous results. The success of the present method (all amino acid pairs studied so far showed a chiral difference) is related to the fact
that reaction rates depend very strongly on small changes in the activation energy, and the peak ratios studied are directly proportional to reaction rates. The observed ion abundance ratios can be evaluated in terms of gas-phase basicity of amino acid dimers using the “kinetic” method.12-16 Differences as small as 0.2 kJ/mol between the GBs of homochiral and heterochiral dimers can be reliably measured. It should be noted that measurement of chiral discrimination (dependence of [ABH+]/ [A2H+] ratio on stereochemistry) does not depend on approximations of the kinetic method; it is only used to convert chiral discrimination into an MPGB difference. The present method can have important analytical applications as well, as it shows that mass spectrometric techniques are suitable for studying chirality. Applying the kinetic method was suitable to detect such small differences in the interaction of two chiral compounds, which showed identical behavior in typical mass spectra. Chiral discrimination of the present method seems high enough to be generally useful for various compound classes. The absolute chirality of a given analyte (an amino acid in the present case) can be determined by mixing it with a “reagent” of known configuration (here another amino acid). Peak ratios in the CID-MIKE spectra, as used above, could be used to determine if the analyte-reagent dimer is of homo- or heterochiral configuration. The main limitation is that trimer clusters have to be produced, which requires fairly high sample concentration. Using other techniques for ion production and analysis (e.g., electrospray or Fourier transform mass spectrometry) may alleviate this limitation. The technique described here is also suitable for studies on host-guest chemistry and molecular recognition. The main advantages are that spectral differences can be converted to energetical data and that the technique is suitable to detect differences in ion stability as small as 0.1-0.2 kJ/mol. Received for review September 12, 1996. November 12, 1996.X
Accepted
AC960931M X
Abstract published in Advance ACS Abstracts, March 1, 1997.
Analytical Chemistry, Vol. 69, No. 9, May 1, 1997
1705
