Chiral Melamine Derivatives: Design, Synthesis, and Application to

A novel class of chiral melamine derivatives has been designed and synthesized. The ability of these compounds to perform chiral recognition toward 19...
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Anal. Chem. 2005, 77, 5302-5310

Chiral Melamine Derivatives: Design, Synthesis, and Application to Mass Spectrometry-Based Chiral Analysis Qin Liu, Shuzhen Zhang, Bidong Wu, Jifen Guo, Jianwei Xie,* Mingsong Gu, Yimin Zhao, Liuhong Yun, and Keliang Liu

Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing 100850, P. R. China

A novel class of chiral melamine derivatives has been designed and synthesized. The ability of these compounds to perform chiral recognition toward 19 natural chiral r-amino acids has been investigated by electrospray ionization tandem mass spectrometry for the first time. The enantioselectivities of these new chiral selectors are encouraging. To elucidate some mechanism and regularity in the chiral recognition process using chiral melamine derivatives as chiral selectors, the effect of different noncovalent interactions caused by various chiral or achiral moieties in melamine derivatives on the chiral recognition in the gas phase has been studied at the same time. The result shows that electrostatic, hydrogen bond, π-π stacking, and steric interaction between selector and analyte play important roles in the association and enantioselective recognition of amino acids with the chiral melamine derivatives as chiral selectors. Enantiodiscrimination for analytes with different structures and properties could be improved by modifying substituents in melamine derivatives on purpose. Chirality is of increasing importance in many fields such as pharmaceutics, chemical industry, and agriculture. More and more single enantiomers have been researched and developed. As a result, demand for chiral recognition and separation of chiral compounds has increased in the past few decades. Enantiomers have identical physical and chemical properties expect for optical rotation. Because of these similarities, chiral discrimination of enantiomers is a very challenging task. The most often used methods in chiral analysis are circular dichroism, polarimetry, nuclear magnetic resonance (NMR), chromatography, and capillary electrophoesis. An attractive alternative method is mass spectrometry, due to its many advantages such as the speed of analysis, high sensitivity, molecular specificity, and small amount of sample. The key procedure for chiral analysis using MS technique is to add a chiral selector in the compound-matrix system to generate diastereomers. Because the diastereomers formed from the analyte (chiral compound) and the chiral selector have unique reactivity and stability, the resulting mass spectrum contains different relative abundances that reflect these differences and provides a method for chiral analysis.1 * To whom correspondence should be addressed. E-mail: [email protected]. Fax: (8610)68211656.

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Chemical ionization2-5 and fast atom bombardment6-13 were the main ionization modes used in the early development of chiral mass spectrometry, whereas electrospray ionization mass spectrometry (ESI-MS),14-20 especially electrospray ionization tandem mass spectrometry (ESI-MS/MS),21-38 has recently emerged as (1) Sawada, M. Mass Spectrom. Rev. 1997, 16, 73-90. (2) Hua, S.; Chen, Y.; Jiang, L.; Xue, S. Org. Mass Spectrum. 1986, 21, 7-10. (3) Chen, Y. Z.; Li, H.; Yang, H. J.; Hua, S. M.; Li, H. Q.; Zhao, F. Z.; Chen, N. Y. Org. Mass Spectrom. 1988, 23, 821-824. (4) Tabet, J. C. Tetrahedron 1987, 43, 3413-3420. (5) Yang, H.; Chen, Y. Org. Mass Spectrom. 1992, 27, 736-740. (6) Chen, Y.; Wu, Y. Chin. J. Anal. Chem. 1998, 26, 737-741. (7) Sawada, M.; Okumura, Y.; Shizuma, M.; Takai, Y.; Hidaka, Y.; Yamada, H.; Tanaka, T.; Kaneda, T.; Hirose, K.; Misumi, S.; Takahashi, S. J. Am. Chem. Soc. 1993, 115, 7381-7388. (8) Sawada, M.; Shizuma, M.; Takai, Y.; Yamada, H.; Kaneda, T.; Hanafusa, T. J. Am. Chem. Soc. 1992, 114, 4405-4406. (9) Ve´key, K.; Czira, G. Anal. Chem. 1997, 69, 1700-1705. (10) Smith, G.; Leary, J. A. J. Am. Chem. Soc. 1996, 118, 3293-3294. (11) Sawada, M.; Takai, Y.; Yamada, H.; Nishida, J.; Kaneda, T.; Arakawa, R.; Okamoto, M.; Hirose, K.; Tanaka, T.; Naemura, K. J. Chem. Soc., Perkin Trans. 2 1998, 701-710. (12) Sawada, M.; Yamaoka, H.; Takai, Y.; Kawai, Y.; Yamada, H.; Azuma, T.; Fujioka, T.; Tanaka, T. Int. J. Mass Spectrom. 1999, 193, 123-130. (13) Shizuma, M.; Imamura, H.; Takai, Y.; Yamada, H.; Takeda, T.; Takahashi, S.; Sawada, M. Int. J. Mass Spectrom. 2001, 210/211, 585-590. (14) Chu, I. H.; Dearden, D. V.; Bradshaw, J. S.; Huszthy, P.; Izatt, R. M. J. Am. Chem. Soc. 1993, 115, 4318-4320. (15) Dearden, D. V.; Dejsupa, C.; Liang, Y. J.; Bradshaw, J. S.; Izatt, R. M. J. Am. Chem. Soc. 1997, 119, 353-359. (16) Ramirez, J.; He, F.; Lebrilla, C. B. J. Am. Chem. Soc. 1998, 120, 73877388. (17) Grigorean, G.; Ramirez, J.; Ahn, S. H.; Lebrilla, C. B. Anal. Chem. 2000, 72, 4275-4281. (18) Ramirez, J.; Ahn, S.; Grigorean, G.; Lebrilla, C. B. J. Am. Chem. Soc. 2000, 122, 6884-6890. (19) Grigorean, G.; Lebrilla, C. B. Anal. Chem. 2001, 73, 1684-1691. (20) Cheng, Y.; Hercules, D. M. J. Mass. Spectrom. 2001, 36, 834-836. (21) Tao, W. A.; Cooks, R. G. Anal. Chem. 2003, 75, 25A-31A. (22) Tao, W. A.; Zhang, D.; Wang, F.; Thomas, P. D.; Cooks, R. G. Anal. Chem. 1999, 71, 4427-4429. (23) Tao, W. A.; Zhang, D.; Nikolaev, E. N.; Cooks, R. G. J. Am. Chem. Soc. 2000, 122, 10598-10609. (24) Tao, W. A.; Gozzo, F. C.; Cooks, R. G. Anal. Chem. 2001, 73, 1692-1698. (25) Wu, L.; Tao, W. A.; Cooks, R. G. Anal. Bioanal. Chem. 2002, 373, 618627. (26) Zhang, D.; Tao, W. A.; Cooks, R. G. Int. J. Mass Spectrom. 2001, 204, 159169. (27) Tao, W. A.; Wu, L.; Cooks, R. G. J. Med. Chem. 2001, 44, 3541-3544. (28) Chen, J.; Zhu, C. J.; Chen, Y.; Zhao, Y. F. Rapid Commun. Mass Spectrom. 2002, 16, 1251-1253. (29) Wu, L.; Lemr, K.; Aggerholm, T.; Cooks, R. G. J. Am. Soc. Mass Spectrom. 2003, 14, 152-160. (30) Angusti, D. V.; Carazza , F.; Augusti, R.; Tao, W. A.; Cooks, R. G. Anal. Chem. 2002, 74, 3458-3462. 10.1021/ac050318f CCC: $30.25

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a powerful technique for chiral recognition. ESI is a very gentle ionization technique that can maintain weakly bound complexes; thus, noncovalent diastereomers can be characterized by mass spectrometry. Tandem mass spectrometry has been used to discriminate chiral compounds by comparing differences in fragmentation patterns via collision-induced dissociation (CID) of disatereomers that are formed from an analyte and a chiral selector or by the kinetic method. Compared to single-stage mass spectra, tandem mass spectra are more stable and insensitive to ionization efficiencies, ion-transfer coefficients, impurities, and changes in concentration. Very small energy differences between diastereomeric complexes can be easily detected by MS/MS.9 No matter which method is used, the chiral selector is the most important part of the chiral analysis system. Thus, great effort has been devoted to searching and constructing new and versatile chiral selectors. Some well-known selectors used in chiral mass spectrometry are as follows: (1) Chiral host molecules11,12,16-20 like cyolodextrins and chiral crown ethers. When they interact with a racemic guest, diastereomeric complexes having different thermodynamic stability are formed. If one enantiomer of the analyte is isotopically labeled, the corresponding mixture of diastereomeric complexes can be mass resolved.11,12 Chiral recognition based on gas-phase guest exchange reactions has also been reported.14-20 Diastereomeric complex ions are mass-selected and allowed to exchange the chiral guest in a reaction with a neutral reagent. Chiral distinction is achieved because the exchange rate varies with the chirality of the analyte incorporated into the adduct ion. (2) Metal ion complex.22-36 Metal complexes with chiral coanalytes are used to produce diastereomeric complexes that are probed by CID. Chiral discriminations are performed by monitoring the relative abundances of the dissociation products that varied with the chirality of the analyte. According to the kinetic method used by Cooks and coworkers,22-27,29-31 an aqueous methanol solution containing an analyte (chiral compound, A), a chiral reference compound (chiral amino acids, ref*), and a transition metal ion (M) is introduced into the ESI source and singly charged trimeric cluster ions [M(ref*)2(A) - H]+ are generated. CID of [M(ref*)2(A) - H]+ cluster ions typically produce dimeric complexes [M(ref*)(A) H]+ and [M(ref*)2 - H]+ by competitive loss of the natural reference compound, ref*, and the analyte A, respectively. Due to the two configurations of the analyte A, the differences in energy required to generate the diastereomeric forms of the fragment ions [M(ref*)(A) - H]+ result in differences in their abundance, measured relative to the abundance of [M(ref*)2 - H]+. In this method, chiral references are required to have structures similar to that of the analytes, to yield both product ions [M(ref*)(A) H]+ and [M(ref*)2 - H]+ in the process of fragmentation. So far (31) Wu, L.; Meurer, E. C.; Cooks, R. G. Anal. Chem. 2004, 76, 663-671. (32) Paladini, A.; Calcagni, C.; Palma T. D.; Speranza M.; Lagana, A.; Fago, A.; Filippi, A.; Satta M.; Guidoni, A. G. Chirality 2001, 13, 707-711. (33) Hofmeister, G.; Leary, J. A. Org. Mass Spectrom. 1991, 26, 811-812. (34) Fago, G.; Filippi, A.; Giardini, A.; Lagana`, A.; Paladini, A.; Speranza M. Angew. Chem., Int. Ed. 2001, 40, 4051-4054. (35) Desaire, H.; Leary, J. A. Anal. Chem. 1999, 71, 1997-2002. (36) Arakwa, R.; Kobayashi, M.; Ama, T. J. Am. Soc. Mass Spectrom. 2000, 11, 804-808. (37) Yao, Z. P.; Wan, T. S. M.; Kwong, K. P.; Che, C. T. Anal. Chem. 2000, 72, 5383-5393. (38) Yao, Z. P.; Wan, T. S. M.; Kwong, K. P.; Che, C. T. Anal. Chem. 2000, 72, 5394-5401.

this method has been successfully applied to chiral discrimination of amino acids,23,26 hydroxy acids,25 chiral peptides,28,29 and sugars.30 (3) Small chiral molecules.1-5,37,38 These selectors can interact with chiral analyte by noncovalent forces such as hydrogen bonds and van der Waals forces to generate diastereomeric complexes. Yao37,38 et al. reported the chiral analysis of 19 common chiral amino acids using modified amino acids as chiral selectors. CID of protonated trimers formed between the chiral selector and the amino acid to give protonated dimers was studied. Chiral distinction was indicated by different dissociation efficiency (the intensity ratio of the product to the precursor ion) between the homochiral and heterochiral trimers. However, limited selectivity and limited application range of recognition of the chiral selectors are still main problems that should be solved in chiral MS analyses. The search for novel either broadly or dedicatedly applicable chiral selectors is always an open field attracting the creativity and efforts of the researchers. An interesting way of achieving chiral selectors, having satisfactory enantiodiscriminating capabilities covering a wide range of chiral analyte, is to have an achiral polyfunctional unity available to link several different chiral moieties.40 Melamine is a promising compound for these purposes owning to its special structure. On one hand, melamine has both proton acceptors and proton donors in its molecule, which make it very easy to form a hydrogen bond with other molecules. The hydrogen bond-directed assemblies of melamine and cyanurate derivatives are one of the famous and widely studied systems in supermolecular chemistry. On the other hand, introducing and modifying the functional groups in the N-substituents could provide multiple interactions with other compounds. According to the Dalgliesh’s three-point model,39 enantiomer recognition is achieved as a result of three simultaneous attractive interactions (donor-acceptor interactions such as hydrogen bonding, π-stacking, and dipoledipole interactions) between the selector and one of enantiomers separated. At least one of these interactions must be stereochemically dependent. Chiral melamine derivatives (melamines functionalized with chiral moieties, M) are thus quite attractive for their potential application to chiral discrimination as chiral selectors. However, very few chiral melamine derivatives used as chiral selectors have been reported so far. Barretta40 et al. had investigated the enantiodiscriminating ability of 2-hexylamino-4,6bis-L-valyl-L-valine isopropyl ester-1,3,5-triazine as modified 2,4,6trichloro-1,3,5-triazine toward the N-3,5-dinitrobenzoyl deriviatives of 1-phenylethylamine or valine methyl ester by 1H NMR spectroscopy, but the result was not satisfactory. Now our interest is focused on using chiral melamine derivatives as chiral selectors while a MS-based method is employed. The most outstanding advantage of developing this kind of chiral selector is that selectors could be designed and synthesized according to the structure of the selectant molecule. Special recognition sites can be introduced to the chiral selectors easily in order to improve the enantiodiscriminating capabilities. The goal of this study is to try to elucidate some mechanism and regularity in the chiral recognition process using chiral melamine derivatives as chiral selectors by chiral mass spectrometry. To better understand the role played by the structural (39) Dalgliesh, C. E. J. Chem. Soc. 1952, 3940-3942. (40) Barretta, G. U.; Iuliano, A.; Menicagli, R.; Peluso, P.; Pieroni, E.; Salvadori, P. Chirality 1997, 9, 113-121.

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Chart 1

features of the melamine derivatives on the enantioselective properties and to identify more effective chiral selectors, we synthesized a series of new chiral melamine derivatives (Chart 1) starting from cyanuric chloride (2,4,6-trichloro-1,3,5-triazine), in which amine synthons with different structures and properties were used. Cyanuric chloride is a rather low cost and easily available material. Its trifunctionality and reactivity toward nuclephiles, such as different amines, make it very attractive for the synthesis of various melamine derivatives.41-43 To evaluate the chiral recognition ability of the chiral melamine derivatives, we have attempted to detect diastereomers formed from the chiral melamine derivatives and amino acids by mass spectrometry. The 19 natural chiral R-amino acids (A) have been selected as “probe” selections since they are important compounds of chemical and biological systems, and their chiral recognition attracts considerable interest. Moreover, amino acids have unique structures, which can afford multiple noncovalent interactions and provide essential conditions for chiral recognition. The side chains of 19 natural chiral amino acids vary a lot from each other, making it convenient for us to systematically study the impact of various noncovalent interactions on chiral recognition in the gas phase. By modifying groups in N-substituents of melamine derivatives, the effect of different noncovalent interactions, such as hydrogen bonding, π-π interactions, ionic forces, and steric effects, on the chiral discrimination ability of these modified melamines for amino acids were investigated using ESI-MS/MS. Since the interfering effect of the solvent was eliminated in the gas phase, some chiral (41) Kurteva,V. B.; Afonso, C. A. M. Green Chem. 2004, 6, 183-187. (42) Bielejewska, A. G.; Marjo, C. E.; Prins, L. J.; Thnmerman, P.; Jong, F. D.; Reinhoudt, D. N. J. Am. Chem. Soc. 2001, 123, 7518-7533. (43) Mathias, J. P.; Seto, C. T.; Simanek, E. E.; Whitesides, G. M. J. Am. Chem. Soc. 1994, 116, 1725-1736.

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recognition mechanism and regularity of chiral melamine derivatives as chiral selectors were discussed. EXPERIMENTAL SECTION Chemicals. All D- and L-amino acids were purchased from Sigma. Cyanuric chloride and all chiral or achiral amines used to synthesize various chiral melamine derivatives were purchased from Arcos. All were used without further purification. Chiral amino acid methyl esters used were provided by the laboratory of Prof. Keliang Liu. Anhydrous THF was distilled from Na/ benzophenone. Synthesis. Chiral melamine derivatives were synthesized by the selective nucleophilic substitution to cyanuric chloride (2,4,6trichloro-1,3,5-triazine) using different amines in THF in the presence of N,N-diisopropylethylamine.42 All the compounds were identified by 1H NMR, polarimetry, and high-resolution mass spectrometry. The detailed synthetic procedures can be obtained from the Supporting Information. Mass Spectrometry. Mass spectrometry was performed by using a commercial API3000 triple-quadrpole (QQQ) mass spectrometer (ABI), equipped with an ESI source and operated in the positive ion mode under the following conditions: spray voltage, 4 kV; capillary temperature, 50 °C; curtain gas, 9 units. The sample was infused via a syringe pump at a flow rate of 10 µL/min. In the full scan QQQ-MS2 mode, the parent ion of interest which isolated in the first quadrupole Q, was excited in the second quadrupole Q on which only a radio frequency voltage is placed by collision with N2 (collision gas 1 unit, collision energy range 5-8 V) and then analyzed in the third quadrupole Q. The collision energy was optimized in each experiment but kept constant for all the four chiral combinations of the chiral selector and amino acid: RS, SR, RR, and SS. Spectra were measured for 8 min, and

data in the period between 2 and 8 min were averaged. The measurements for each chiral combination were repeated three times. For the measurement of chiral recognition ratio, 2 mM solutions of L- or L-amino acids were prepared in water. The 2 mM solutions of chiral melamine derivatives were prepared in methanol. The solution of an amino acid and a chiral selector were mixed in 1:1 ratio and then diluted to 0.2 mM by 50% methanol prior to mass spectrometric analysis. RESULTS AND DISCUSSION Design of the Chiral Melamine Derivatives. Though 19 natural chiral amino acids have roughly similar structures since they all possess an amino group and a carboxyl group simultaneously, they differ widely in their side-chain structure. According to side-chain structure, the amino acids can be divided into different types, i.e., aliphatic, aromatic, unsaturated, and amino acids with a reactive hydrogen-containing side chain. The interactions that different amino acids are likely to form are different. To gain insight into the role played by the different noncovalent interactions in the recognition, we designed and synthesized 10 enantiomer pairs of chiral melamine derivatives with various N-substituents (1-10, Chart 1). Our current study only involves simple synthetic, because the simpler the selector, the easier it is to single out clearly the specific interactions that promote the chiral discrimination toward particular selections. The substituents are uncomplicated: amino (1-8) to investigate mainly the effect of hydrogen bond on chiral discrimination; dibutylamino (1-4, 9, 10) to investigate mainly the effect of static electrostatic interactions and steric hindrance; 4-tert-butylanilino (5, 6) to investigate mainly the effect of steric hindrance and π-π stacking interactions; different chiral amino, such as 1-phenylethylamino (1, 5, 7, 8, 10), 1-(1-naphthyl)ethylamino (2, 6, 8), 1-methyl-3phenylpropylamino (3), and 2-butylamino (4), to investigate mainly the effect of chiral group size, rigidity, and structure on enantiomer discrimination; single (1-6, 10) or double (7-9) chiral moieties to investigate mainly the effect of the number of chiral groups. In addition, arrangement and realignment of these substituents are helpful to better understand the mechanism of chiral recognition. Association of Chiral Melamine Derivatives and Amino Acids. As we know, the recognition mechanism involves the aggregation of the chiral selector and the enantiomeric pair R/S into two diastereomeric complexes held together by a different combination of noncovalent intermolecular interactions and, therefore, endowed with different stability and reactivity.44 In other words, association of a chiral selector and a chiral selection is the basis of the chiral recognition. Thus, we studied the association of the chiral melamine derivatives and amino acids in ESI-MS spectra first. Typical ESI mass spectra of chiral melamine derivatives/amino acids mixtures are depicted in Figure 1. The mass spectra were relatively simple, and some valuable information on the combination process of the melamine derivatives and the amino acids were obtained. Except protonated molecular ions MH+ and protonated dimer M2H+, most amino acids such as acidic and neutral amino acids usually bond with melamine derivatives (44) Filippi, A.; Giardini, A.; Piccirillo, S.; Speranza, A. Int. J. Mass Spectrom. 2000, 198, 137-163.

Figure 1. ESI spectra of mixtures of amino acids (0.2 mM) and chiral melamine derivatives (0.2 mM): (a) 8a + D-Phe; (b) 9a + D-Phe; (c) 1b + D-Lys. ESI conditions: ion spray voltage 4 kV, nebulizer gas 2 units, curtain gas 9 units, and temperature 50 °C.

to form trimer ions M2AH+(Figure 1a and b), whereas basic amino acids (Arg, His, Lys) bond with the chiral selectors to form dimer ions MAH+ (Figure 1c). Increasing the concentration of the solution resulted in similar spectra. Protonated amino acids AnH+ were not observed as products ions in all cases, probably because the proton affinity of amino acids is much lower than that of the melamine derivatives. Nearly all synthesized chiral melamine derivatives except 10 could combine with amino acids with aromatic side chains (His, Phe, Trp, Tyr) or amino acids possessing unsaturated groups in side chains (Arg, Asn, Asp, Gln, Glu), suggesting the existence of the π-π interactions. When amino acid side chains had neither aromatic group nor unsaturated group, only chiral melamine derivatives bearing both dibutylamino and amino (1-4) could bind with them. It might be a reasonable explanation that the basicity of the dibutylamino improved the salt-forming or static electrostatic interaction with the carboxyl group of the amino acids, and the amino increased hydrogen bonding between the molecules. Among all chiral melamine derivatives we synthesized, Analytical Chemistry, Vol. 77, No. 16, August 15, 2005

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Figure 2. ESI spectra of all four complexes of Phe and 1: (a) 1a + D-Phe; (b) 1a + L-Phe; (c) 1b + D-Phe; (d) 1b + L-Phe. ESI conditions: ion spray voltage 4 kV, nebulizer gas 2 units, curtain gas 9 units, and temperature 50 °C.

1-4 showed the greatest ability to combine with 19 amino acids according to the abundance of the protonated complexes in MS spectra, and whether there were aromatic rings in chiral moieties did not have any influence on the combination or the intensity of the combination, which indicates that the chiral moieties are not the leading factor in the association processes. Chiral amino acid methyl esters, such as LeuOCH3, PheOCH3, and TrpOCH3, were also employed as analytes in the experiment, but combinations between the melamine derivatives and these amino acid methyl esters were not observed by ESI-MS. This suggests again that salt-forming interaction plays an important role in the association of the melamine derivatives and the amino acids and is one of the driving forces for combination. As for 10 characterized by ESI-MS, neither the combination with the amino acids nor the aggregation of itself was observed by ESI-MS, probably because too large a steric hindrance of the two dibutylamino groups blocked the close of the molecules. Chiral Recognition of Amino Acids with Chiral Melamine Derivatives. When we studied the association of the melamine derivatives and the amino acids by ESI-MS, we found that some obvious isomeric differences in combination, especially combination between the melamine derivatives with aromatic chiral moieties and the amino acids with aromatic side chain, could be observed directly from the peak abundances in the single-stage 5306 Analytical Chemistry, Vol. 77, No. 16, August 15, 2005

mass spectra. A typical example of these, as observed in the ESIMS spectra of all four complexes of an amino acid (Phe) and a chiral selector (1), is depicted in Figure 2, showing that the combination between 1a (R configuration of 1) and L-Phe was apparently stronger than that between 1a and D-Phe, whereas the combination between 1b (L configuration of 1) and D-Phe was apparently stronger than that between 1b and L-Phe. This suggests that association of the chiral melamine derivatives and the amino acids is related to the chirality, and the isomeric differences indeed exist. However, we did not make a thorough study of this phenomenon, considering that single-stage mass spectra are sensitive to many external factors, such as ionization efficiencies, ion-transfer coefficients, concentration, and impurities, which might result in poor reproducibility and indistinction of subtle differences. Further investigation of the chiral discrimination was carried out by ESI-MS/MS. As mentioned above, Cooks,21-27,30,31 Yao,37,38 and others9 recently exploited a mass spectrometric methodology for gasphase chiral discrimination, which is based on the CID of the trimeric adducts X2YH+, where X is the chiral selector and Y is the chiral analyte. We applied this technique to enantiodiscriminate the chiral amino acids by CID of their protonated trimer M2AH+ with chiral melamine derivatives. For Arg, His, and Lys, CID of protonated dimer MAH+ were studied.

Figure 3. CID spectra of protonated trimers from (a) 1a + D-Asp and (b) 1a + D-Asn. Collision gas 1 unit and collision energy 5.

The dissociation processes of a protonated trimer depend on the structure of the precursor ion and the proton affinity of the product ions.9 In this study, since the structure and properties of the amino acids as well as the melamine derivatives are quite different from each other, the dissociation paths of the trimer ions formed are different, resulting in the different CID spectra of M2AH+: (1) In the majority of cases, precursor ion M2AH+ and product ions M2H+ and MH+ were observed in CID spectra. Product ion MAH+ was not observed by varying CID conditions. A typical spectrum is shown in Figure 3a. (2) In the minority of cases, precursor ion M2AH+ and product ions M2H+, MH+, and MAH+ were observed in CID spectra. A typical spectrum is shown in Figure 3b. As for Arg, His, and Lys, which bound with the chiral selectors to form dimer ions MAH+, only precursor ion MAH+ and product ions MH+ were observed in CID spectra. Chiral discrimination can be measured by the difference of the dissociation efficiency (the intensity ratios of the protonated dimer to the protonated trimer or the protonated monomer to the protonated dimer) between the heterochiral and the homochiral cases. For each complex ion, all four possible forms, i.e., RS and SR (heterochiral) and RR and SS (homochiral), were studied. As to R-amino acids, the D configuration is equal to the R configuration, and the L configuration is equal to the S configuration. The chiral discrimination ratio Rchiral can be defined by eq 1, where n ) 1 or 2. The equation is similar to that used by Yao37 and Ve´key.9

Rchiral )

([MnH+]/[MnAH+])heterochiral ([MnH+]/[MnAH+])homochiral

)

([MnH+]/[MnAH+])RS + ([MnH+]/[MnAH+])SR ([MnH+]/[MnAH+])RR + ([MnH+]/[MnAH+])SS

(1)

We use the intensity ratios of the product ion MnH+ to the precursor ion MnAH+ to calculate Rchiral since the MnH+ could be observed in all CID spectra. Using a unified calculation method to measure chiral discrimination makes the experiment data of different complexes more comparable. When Rchiral < 1, the dissociation is more favorable in the homochiral case, whereas when Rchiral > 1, the dissociation is more favorable in the heteochiral case. For Rchiral ) 1, there is no observable chiral discrimination. The further the Rchiral value is from unity, the higher the degree of chiral recognition observed. Figure 4 shows the CID spectra of all four chiral combinations of the protonated trimers (1)2(Asn)H+. The Rchiral values for 19

natural chiral amino acids with the chiral melamine derivatives as chiral selectors are summarized in Table 1. According to the RSD data in Table 1, which are all no more than 4.0%, at the 95% confidence level, Rchiral values fall within the confidence interval of 1.00 ( 0.10 (i.e., 1.00 ( 4.30 × 4.0%/30.5)45 would mean a lack of chiral discrimination. In other words, an Rchiral value larger than 1.1 or smaller than 0.9 indicates a significant chiral discrimination. As shown in Table 1, 1 and 2 have the most extensive chiral recognition ability among all synthesized chiral melamine derivatives. Dibutylamino and amino existing simultaneously in their structure allows them to form complexes easily with amino acids. Even though their chiral moieties bear the benzene ring and naphthalene ring, respectively, 1 and 2 displayed similar chiral selectivity. In both cases, superior chiral recognition was achieved when the amino acid had an aromatic side chain (Phe, Trp, Tyr) or an unsaturated side chain (Arg, Asn, Glu), suggesting that π-π interactions may play a role in the setereospecificity. A subtle chiral selectivity of His was observed when 1 and 2 were employed as the chiral selectors. This is probably because the chiral selectivity of His was measured based on the CID spectra of the protonted dimers (1)(His)H+ and (2)(His)H+, while the chiral recognition of other aromatic amino acids was measured based on the CID sprectra of protonated trimers. More aggregation in the complexion might increase steric hindrance and enhance discrimination, so protonated trimers usually show larger chiral discriminations than protonated dimers.37 Data in Table 1 show that 1 and 2 give low chiral selectivity for two acidic amino acids, Asp and Glu. This may be related to the carboxyl group presenting in both amino acid side chains. The additional carboxyl group in Asp and Glu, as compared to other amino acids, would greatly increase salt-forming interaction for the complexes involving melamine derivatives, but such increased interaction might not be helpful to chiral discrimination, because the effect of other interactions, including interactions on chiral moieties, might be reduced correspondently. So it would be better for chiral recognition if the multiple interactions formed between a chiral selector and a chiral analyte are comparatively balanced. It’s interesting to note that four D- and L-aliphatic amino acids (Ala, Leu, Ile, Val), which are usually hard to resolve by other methods, were chirally differentiated when using 1 or 2 as selector. The results were even better than that of those amino (45) Manahan, S. E. Quantitative Chemical Analysis; Brooks/Cole Publishing Co.: Monterey, CA, 1986; pp 71-72.

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Figure 4. Chiral discrimination of Asn with 1 as observed in the CID spectra of protonated trimers: (a) 1a + D-Asn (b) 1a + L-Asn (c) 1b + D-Asn, and (d) 1b + L-Asn. MS/MS conditions: collision gas 1 unit and collision energy 6. Table 1. RChirala Values According to Eq 1 and Obtained from the CID Spectra of the Protonated Trimers of 19 Amino Acids with Chiral Melamine Derivatives 1-10 1 aa

Rchiral

Argb Asn Gln Asp Glu Phe Trp Tyr Hisb Lysb Met Cys Ser Thr Ala Leu Ile Val Pro

0.51 0.52 0.65 0.96 0.97 0.64 0.73 0.52 0.85 0.75 0.83 0.88 1.13 1.14 0.78 0.61 0.70 0.73 1.40

a

2

3

5c

4

6c

7c

8c

9c

10c

RSD RSD RSD RSD RSD RSD RSD RSD RSD RSD (%) Rchiral (%) Rchiral (%) Rchiral (%) Rchiral (%) Rchiral (%) Rchiral (%) Rchiral (%) Rchiral (%) Rchiral (%) 2.1 1.9 3.8 1.5 1.8 2.0 2.8 2.3 2.2 2.9 3.5 2.0 2.7 2.9 3.7 2.1 3.1 3.4 1.4

0.46 0.51 0.58 0.95 0.88 0.63 0.70 0.67 0.88 0.79 0.86 0.91 1.11 1.27 0.81 0.72 0.74 0.81 1.22

3.4st 2.2 2.0 1.5 1.6 3.9 1.9 3.0 2.2 3.4 3.9 3.1 2.1 1.1 1.7 3.6 2.7 2.7 1.1

0.63 0.65 0.69 1.07 0.80 0.84 0.74 1.05 1.06 0.88 0.91 1.05 1.22 1.25 1.07 1.11 1.21 1.02 0.86

2.1 1.8 3.3 1.3 2.9 1.5 2.6 2.1 2.9 2.0 2.5 1.8 2.6 1.9 3.6 2.1 2.7 1.8 2.0

0.99 0.98 1.04 1.01 0.99 0.88 0.91 1.09 1.13 1.09 1.02 1.06 1.03 1.00 0.97 0.93 0.90 0.95 0.97

1.2 1.8 2.1 2.0 0.9 1.3 2.5 1.9 2.7 2.1 1.9 1.2 2.0 2.3 3.3 2.6 3.9 4.0 2.4

0.89 0.77 0.85 1.23 1.29 1.54 1.76 1.63 0.61 / / / / / / / / / /

3.3 2.3 3.2 1.8 1.5 1.5 2.2 1.3 3.8 / / / / / / / / / /

0.84 0.80 0.80 1.28 1.20 1.75 1.81 1.66 0.72 / / / / / / / / / /

3.3 2.6 2.9 1.6 0.9 1.3 1.0 0.8 2.6 / / / / / / / / / /

0.66 0.59 0.77 1.07 0.93 1.34 1.41 1.46 0.89 / / / / / / / / / /

2.6 2.7 3.3 2.5 2.0 2.1 0.9 1.0 2.6 / / / / / / / / / /

0.54 0.58 0.71 0.99 0.94 1.23 1.29 1.33 0.82 / / / / / / / / / /

2.8 2.6 1.8 2.1 3.1 2.5 1.4 1.7 3.2 / / / / / / / / / /

1.71 1.53 1.56 0.86 1.03 0.77 0.88 1.13 0.90 / / / / / / / / / /

1.0 0.8 0.9 2.4 1.3 2.6 2.2 2.8 1.9 / / / / / / / / / /

/ / / / / / / / / / / / / / / / / / /

/ / / / / / / / / / / / / / / / / / /

n ) 3. b The CID spectra of protonated dimers were measured. c The complex ions were not observed.

acids with reactive hydrogen-containing side chains (Ser, Thr, Cys, Lys), showing that some interactions in stereostructure of the trimer clusters are hard to predict. The amino acids with larger side chains (Leu, Ile) generally showed a little better chiral discrimination than those processing smaller side chains (Ala, Val). In most conditions, the heterochiral complex was more stable than the homochiral one with 1 or 2 as chiral selector, except in 5308 Analytical Chemistry, Vol. 77, No. 16, August 15, 2005

cases where Ser, Thr, or Pro was used as the analyte. The reason is not well understood, which also means that the mechanism involved in chiral recognition is extremely complicated. Similar to 1 and 2, melamine derivative 3 also possesses dibutylamino, amino, and aromatic chiral moieties. But the chiral selectivity decreased dramatically for most amino acids with 3 as chiral selector compared to 1 and 2. Apparently the presence of an extra CH2-CH2 group in the aromatic chiral moiety of 3, as

compared to 1, increases the flexibility of the chiral group, inferring a rigid chiral moiety is more favorable to chiral selectivity, which is consistent with most reports. To investigate the role played by the aromatic chiral moieties of the chiral selector, we synthesized 4 which differs from 1, 2, and 3 in that it does not possess an aromatic group in the chiral moiety. The results showed that no distinct chiral selectivity was observed for all amino acids with 4 as chiral selector, although 4 was not apparently different from 1, 2, and 3 in the number of groups and the ability to form a hydrogen bond, indicating that π-π interactions played an important role in the chiral recognition of amino acids. It also suggests that chiral moieties are the leading factor for chiral discrimination. As mentioned above, chiral melamine derivatives 5 and 6 can only combine with amino acids with aromatic (Phe, Tyr, Trp, His) or unsaturated (Arg, Asn, Gln, Asp, Glu) side chains. However, they exhibited enantioselectivity for most of these amino acids, especially the amino acids possessing an aromatic group, inferring that π-π interactions between the selector and the amino acid are the principal attractive interactions. 5 and 6 were the only melamine derivatives that exhibited good chiral recognition for both Asp and Glu. This is probably because the initial dibutylaminos in 1 and 2 are replaced by 4′-tert-butylamilinos. On one hand, the basicity of 4′-tert-butylamilino is much weaker than that of dibutylamino, since the N-H group and the conjugated benzene ring are linked directly, which decreases the electron density of the nitrogen atom dramatically and weakens the ability of the nitrogen atom to accept proton consequently. In fact, 5 and 6 have the weakest basicity among all 10 melamine derivatives according to their structure. Therefore, the strength of salt-forming interaction between them and two acidic amino acids Asp and Glu would not be too strong. On the other hand, 4′-tert-butylamilino might increase π-π interactions and steric hindrance. Thus it can be seen that modifying substituents in melamine derivatives on purpose could enhance the enantiodiscrimination for analytes with different structures and properties. Both 7 and 8 contain two chiral centers, and they can only bind amino acids with an aromatic side chain or unsaturated side chain too. However, the presence of the second chiral center does not apparently increase their chiral selectivity, suggesting that the more chiral centers introduced into the chiral selector does not mean the better the chiral selectivity. 7 and 8 showed the same trend of chiral discrimination, just like 5 and 6, proving again that a chiral moiety bearing benzene ring or naphthalene ring would not make much difference to chiral selectivity. Changing the amino in 7 to dibutylamine resulted in 9. Although the basicity of 9 is stronger than that of 7, its ability to bind amino acids is not improved, inferring that the NH2 group that can be involved in a hydrogen bond is important in the process of combination. Similar to 7, melamine derivative 9 also possesses two aromatic chiral moieties, but it showed better chiral selectivity for the amino acids with an unsaturated side chain (Arg, Asm, Glu) than for aromatic amino acids (Phe, Tyr, Trp, His), which indicates that, besides π-π interactions, some other factors also affect chiral recognition. In addition, the homochiral complexes formed by 9 and amino acids Arg, Asn, or Glu were more stable than the corresponding heterochiral complexes, just contrary to the case using other melamine derivatives as chiral

selectors, inferring that the amino has a significant effect on combination and recognition. For the melamine derivatives without dibutylamino, such as 5, 6, 7, and 8, the homochiral complexes formed with aromatic amino acids except for His were more stable than the corresponding heterchiral complexes, which were different from the other melamine derivatives, showing that the enantiodiscrimination for different amino acids could be strongly influenced by modifying substituents in melamine derivatives. From what has been discussed above, we have a preliminary understanding of the fundamental mechanism as well as some regularity underlying the chiral recognition process for amino acids with chiral melamine derivatives as chiral selectors in the gas phase: (1) The chiral selector and the analyte are pulled together by hydrogen bond, salt-forming interaction, and π-π interaction, which are the main driving forces for association. Then, some other interactions, especially the interactions on chiral moieties, such as π-π stacking, steric interaction, etc., will help to achieve chiral recognition. (2) Electrostatic interaction, hydrogen bond, π-π stacking, and steric interaction between selector and analyte may play important roles in the association and enantioselective recognition of amino acids. The phenomenon that different melamine derivatives appear to selectively combine and recognize different types of amino acids suggests us designing selector according to the structure properties of the analyte and introducing some groups, which can provide adequate interactions for the association with the analyte, into the melamine derivatives. Then proper chiral groups are brought in to improve chiral selectivity. (3) Mutiple interactions formed between chiral selectors and analytes should be comparatively balanced. Too strong interactions outside the chiral moieties might decrease the chiral recognition ability. (4) Introducing more chiral centers into the chiral selector does not mean the chiral selectivity will be improved. A rigid chiral moiety is helpful to improve chiral discrimination. A chiral moiety-bearing benzene ring or naphthalene ring would not make much difference to chiral selectivity. (5) Enantiodiscrimination for analytes with different structures and properties could be improved by modifying substituents in melamine derivatives on purpose. CONCLUSION In this work, we synthesized a series of new chiral melamine derivatives using a simple route starting from cyanuric chloride. The ability of these compounds to perform chiral recognition toward 19 natural chiral R-amino acids has been investigated by ESI-MS/MS. Melamine derivatives have unique structures and properties that make it easy for them to possess various groups that can provide multiple interactions for chiral recognition. Moreover, they can produce polymer clusters with amino acids in ESI mass spectrometry easily. So they are very suitable for employment as chiral selectors for MS analysis. The enantioselectivities obtained are encouraging. Something meriting special attention is that 1 and 2 could provide excellent chiral discriminations for 17 of the 19 natural amino acids, and by modifying substituents in melamine derivatives, the chiral discrimination for all 19 amino acids could be achieved and selectively enhanced, which shows the great potentialities of the chiral melamine derivatives acting as a new class of chiral selectors in MS analysis. Analytical Chemistry, Vol. 77, No. 16, August 15, 2005

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The effect of different noncovalent interactions caused by various chiral or achiral moieties on the chiral recognition in the gas phase has also been studied at the same time to elucidate some mechanism and regularity in the chiral recognition process. The result shows that electrostatic interaction, hydrogen bond, π-π stacking, and steric interaction between selector and analyte play important roles in the association and enantioselective recognition of amino acids with the chiral melamine derivatives as chiral selectors. Syntheses of the chiral melamine derivatives mentioned above are all based on some simple substituents, so only limited noncovalent interactions were involved in recognition. We are currently carrying out further modifications of the selective binding sites to achieve more specific interactions with amino

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acids or some other chiral analytes, hence, to improve chiral recognition. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (20275045, 203900508). SUPPORTING INFORMATION AVAILABLE Detailed synthetic procedures. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review February 21, 2005. Accepted June 7, 2005. AC050318F