MS for Determination of the

Aug 15, 1997 - Discovery and Characterization of NK13650s, Naturally Occurring p300-Selective Histone Acetyltransferase Inhibitors. Shigehiro Tohyama ...
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Anal. Chem. 1997, 69, 3346-3352

A Nonempirical Method Using LC/MS for Determination of the Absolute Configuration of Constituent Amino Acids in a Peptide: Elucidation of Limitations of Marfey’s Method and of Its Separation Mechanism Kiyonaga Fujii,† Yoshitomo Ikai,‡ Tsuyoshi Mayumi,† Hisao Oka,‡ Makoto Suzuki,† and Ken-ichi Harada*,†

Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468, Japan, and Aichi Prefectural Institute of Public Health, Tsuji-machi, Kita, Nagoya 462, Japan

As the first step in establishing our proposed method, the advanced Marfey’s method, which is planned for the simultaneous determination of the absolute configuration of amino acids in a peptide, we applied Marfey’s method to commercially available amino acids, and the separation behavior was examined in detail. Although good resolution of the diastereomeric pair of an individual amino acid was obtained for all amino acids tested and the applicability of the method was confirmed, the (1-fluoro-2,4dinitrophenyl)-5-L-alaninamide (FDAA) derivative of the L-amino acid was not always eluted prior to its corresponding D-amino acid derivative. Because this proposed method relies on the elution order of a derivatized amino acid with FDAA to determine its absolute configuration, its separation mechanism was carefully investigated using UV and NMR spectral techniques. The results suggested that the resulting conformations of the L- and D-amino acid derivatives are stable and that the resolution between the L- and D-amino acid derivatives is due to the difference in their hydrophobicity, which is derived from the cis- or trans-type arrangement of two more hydrophobic substituents at both r-carbons of an amino acid and L-alanine amide, so that the FDAA derivative of the cis (Z)-type arrangement interacts more strongly with ODS silica gel and has a longer retention time than that of the trans (E)type arrangement. Therefore, the L-amino acid derivative is usually eluted from the column before its corresponding D-amino acid derivative in Marfey’s method. According to this separation mechanism, the elution order of a desired amino acid can be elucidated from the average retention time of the L- and D-amino acid derivatives, and the DL-serine and -asparagine derivatives are critical for Marfey’s method. We have studied the structural determination of naturally occurring peptides produced by microorganisms1 and cyanobac* Corresponding author. Telephone: 81-52-832-1781 ext. 334. Fax: 81-52-8348780. E-mail: [email protected]. † Meijo University. ‡ Aichi Prefectural Institute of Public Health. (1) Ikai, Y.; Oka, H.; Hyakawa, J.; Matsumoto, M.; Saito, M.; Harada, K.-I.; Mayumi, T.; Suzuki, M. J. Antibiot. 1995, 48, 233-242.

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teria,2,3 and they frequently have unusual amino acids together with protein amino acids.4 Although the determination of the absolute configuration of constituent amino acids in peptides is one of the most essential processes for structural characterization of naturally occurring peptides, we occasionally encounter a problem in such a case of unusual amino acids.3 In addition, because the amount of hydrolysate from a peptide is very small in most cases, the determination of the absolute configuration becomes more and more difficult. Usually, although enzymatic or circular dichroism (CD) methods have been applied for the determination of the absolute configuration of amino acids, these direct methods, which recognize an enantiomeric relationship, require not only a comparatively larger amount of sample but also a longer time for analysis, because it is essential to isolate each constituent amino acid before the analysis. For these reasons, they have been considered minor methods for the structural characterization of amino acids. On the other hand, chromatographic methods such as GC and HPLC have been widely used instead of these direct methods, and the absolute configuration of an amino acid is determined by comparison with the retention time of a standard amino acid under a diasteromeric circumstance after appropriate derivatiztion. These chromatographic methods have the advantage that analysis using small amounts of a sample is achievable and is not time-consuming, and it is particularly possible to simultaneously analyze more than 10 amino acids. However, even chromatographic methods always require a standard amino acid, and they are inapplicable to a peptide containing unusual amino acids. The purpose of this study is to establish a nonempirical method for the determination of the absolute configuration of constituent amino acids in peptides. Our proposed method consists of a chromatography technique for the separation of amino acids into each enantiomer, the detection of an amino acid by mass spectrometry, and a procedure for obtaining the corresponding enantioner from either the L- or D-amino acid. In this paper, we choose Marfey’s method as the chromatography technique and (2) Harada, K.-I.; Mayumi, T.; Shimada, T.; Suzuki, M.; Kondo, F.; Watanabe, M. F. Tetrahedron Lett. 1993, 34, 6091-6094. (3) Harada, K.-I.; Fujii, K.; Shimada, T.; Suzuki, M.; Sano, H.; Adachi, K.; Carmichael, W. W. Tetrahedron Lett. 1995, 36, 1511-1514. (4) Fusetani, N.; Matsunaga, S. Chem. Res. 1993, 93, 1793-1806. S0003-2700(97)00179-0 CCC: $14.00

© 1997 American Chemical Society

describe the limitation of the method and the rationale of the proposed separation mechanism. EXPERIMENTAL SECTION Chemicals. Derivatizing reagents were synthesized from amino acid amide hydrochlorides (Kokusan Chemicals, Tokyo, Japan) and 1,5-difluoro-2,4-dinitrobenzene (Nacalai Tesque, Kyoto, Japan). Amino acids and amino compounds were purchased from Sigma (St. Louis, MO), Wako (Osaka, Japan), Tokyo Kasei Kogyo (Tokyo, Japan), or Nacalai Tesque. Reagent-grade acetonitrile from Nacalai Tesque was distilled and filtered for HPLC, and water was purified using a purification system (Barnstead, Dubuque, IA). Diazomethane was prepared in our laboratory to obtain amino acid methyl ester derivatives. Methanol-d4 for NMR experiments was purchased from Aldrich (Steinheim, Germany). All other chemicals and solvents were of analytical grade. Syntheses of Derivatizing Reagents. (1-Fluoro-2,4-dinitrophenyl-5)-L-alaninamide (FDAA) was prepared according to Marfey,5 and the analogs (1-fluoro-2,4-dinitrophenyl-5)-L-valinamide (FDVA), -L-phenylalaninamide (FDPA), -L-isoleucinamide (FDIA), -L-leucinamide (FDLA), and -D-alaninamide (D-FDAA) were also synthesized according to his procedure,5 in which the L-alaninamide was replaced by each amino acid amide. These derivatizing reagents synthesized were characterized by melting point and mass spectra, which were taken on a JMS-AX505W (JEOL, Tokyo, Japan) mass spectrometer equipped with an FAB ion source (matrix, mnitrobenzyl alchohol). FDAA: mp 225-227 °C (lit.5 mp 224226 °C), MS m/z 273 [M + H]+. FDVA: mp 177-179 °C (lit.6 mp 174 °C), MS m/z 301 [M + H]+. FDPA: mp 191-192 °C (lit.6 mp 191 °C), MS m/z 349 [M + H]+. FDIA: mp 205-207 °C, MS m/z 315 [M + H]+. FDLA: mp 171-173 °C, MS m/z 315 [M + H]+. D-FDAA: mp 225-227 °C, MS m/z 273 [M + H]+.

Sample Preparation. To 50 µL of a 50 mM aqueous solution of amino acid or amino compound were added 20 µL of 1 M sodium bicarbonate and then 100 µL of 1% FDAA in acetone. The solution was vortexed and incubated at 37 °C for 60 min. Reactions were quenched by addition of 20 µL of 1 N HCl. Samples were diluted with 810 µL of acetonitrile, and 1 µL of this solution was analyzed by HPLC. FDAA derivatives of amino acid methyl esters were synthesized by reaction of the amino acid derivative with diazomethane. Chromatography. HPLC was performed using a TOSOH (Tokyo, Japan) dual-pump delivery system composed of two Model CCPS pumps, a Model SD-8022 degasser, a Model MX-8010 mixer, a Model CO-8020 column oven, a Model UV-802O UV-visible detector, a Model PX-8020 system controller, a Model 991J photodiode array detector from Waters (Milford, MA) and a Model C-R6A integrator from Shimadzu (Kyoto, Japan). Separations were carried out on a Cosmosil 5C18-AR (150 mm × 4.6 mm i.d., Nacalai

Tesque) column, heated at 40 °C. For gradient elution in HPLC, mobile phase A was prepared from 0.1 M ammonium acetate in aqueous solution, which was adjusted to pH 3-7 by addition of trifluoroacetic acid, and mobile phase B was acetonitrile. In all cases (Tables 1, 2 and 3), linear gradients started with 15% B and finished with 45% B in 45 min, and mobile phase A was adjusted to pH 3. The system was allowed to equilibrate for 10 min at 15% B prior to the next analysis. The flow rate was 1 mL/min, with UV detection at an absorbance of 340 nm and 250-500 nm by photodiode array detection. Preparation of FDAA Derivatives of L- and D-Valine for NMR Experiments. The FDAA derivatives of L- and D-valine were prepared as described above and were purified by preparative TLC under the following conditions: plate, silica gel (Kieselgel 60F254, 20 × 20 cm2, layer thickness 1 mm, Merck, Darmstadt, Germany); mobile phase, chloroform/methanol/water (65/35/5, lower phase). The 1H-NMR and the NOE difference spectra of the FDAA derivatives of L- and D-valine in methanol-d4 were recorded on a JNM-GX400 (JEOL) spectrometer. 1H chemical shifts are referenced to the residual methanol-d4 signal (δ 3.30): FDAA derivative of L-valine, δ 9.20 (s, 1H, H-3 on the benzene), 5.80 (s, 1H, H-6 on the benzene); of alaninamide, δ 4.32 (q, 1H, J ) 6.7 Hz, H-2), 1.60 (d, 3H, J ) 6.7 Hz, H-3); of valine, δ 3.80 (d, 1H, J ) 5.6 Hz, H-2), 2.30 (m, 1H, H-3), 1.12 (d, 3H, J ) 6.7 Hz, H-4), 1.16 (d, 3H, J ) 6.7 Hz, H-4′); FDAA derivative of D-valine, δ 9.21 (s, 1H, H-3 on the benzene), 5.84 (s, 1H, H-6 on the benzene); of alaninamide, δ 4.34 (q, 1H, J ) 6.7 Hz, H-2), 1.58 (d, 3H, J ) 6.7 Hz, H-3), valine δ 3.83 (d, 1H, J ) 5.6 Hz, H-2), 2.33 (m, 1H, H-3), 1.14 (d, 3H, J ) 6.7 Hz, H-4), 1.18 (d, 3H, J ) 7.1 Hz, H-4′). RESULTS AND DISCUSSION A Nonempirical Method for Determination of the Absolute Configuration of Constituent Amino Acids in Peptides. In 1984, Marfey proposed a method for determination of the absolute configuration of amino acids using HPLC.5 This method is based on the principle that D- and L-amino acids can be separated into each enantiomer by HPLC after derivatization with a chiral reagent. (1-Fluoro-2,4-dinitrophenyl-5)-L-alaninamide (FDAA) was applied as a chiral derivatization reagent and has been referred to as Marfey’s reagent. The resulting amino acid derivatives are separated by the usual reversed-phase HPLC and are detected by UV at 340 nm. The L-amino acid derivative is usually eluted from the column before its corresponding D-amino acid derivative. This method can simultaneously identify amino acids with the correct absolute configuration under gradient elution conditions and is highly sensitive. For this reason, Marfey’s method has been widely used for structural characterization of peptide compounds, confirmation of racemization in peptide synthesis, and detection of small, quantitative D-amino acids. However, it is difficult to apply this chromatographic method to a peptide containing unusual amino acids without having their standard samples. We considered that it would be effective to combine Marfey’s method with an appropriate mass spectrometry for our purpose. On the basis of this consideration, we proposed a nonempirical method using LC/MS for the determination of the absolute configuration of constituent amino acids in a peptide as shown in (5) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591-596 (6) Bru ¨ ckner, H.; Keller-Hoehl, C. Chromatographia 1990, 30, 621-629.

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Figure 1. Total procedure for the method using LC/MS for determination of the absolute configuration of constituent amino acids in a peptide.

Figure 1, and we named it the “advanced Marfey’s method”.7 A mixture of constituent amino acids obtained from hydrolysis of a peptide is divided into two portions (samples 1 and 2) in this method. Sample 1 is directly analyzed by LC/MS after derivatization with FDAA, in which protein amino acids are identified on the basis of retention times and mass spectra of their derivatives. If unusual amino acids are detected, their planar structures are elucidated by mass spectra of their derivatives. Sample 2 is mainly used for determination of the absolute configuration of unusual amino acids. Because naturally occurring peptides are usually composed of amino acids with either D- or L-configuration, each constituent amino acid gives one peak on the chromatogram, whose absolute configuration cannot be determined in this step. Therefore, in order to obtain its enantiomer, amino acids in sample 2 have to be racemized under appropriate conditions. However, the resulting chromatogram after the racemization followed by derivatization with FDAA becomes complicated, and it is difficult to find a pair of each epimer. In our method, mass chromatography is applied to this process, and two peaks of each epimeric pair can be selected on the mass chromatogram monitored at the m/z value of the molecular ion species of the desired amino acid derivative. Subsequently, comparison between the retention time of the original peak in sample 1 and that of the newly formed peak in sample 2 can determine the absolute configuration based on the elution order in Marfey’s method. The following two problems had to be resolved to establish this combination method: (1) elucidation of limitations of Marfey’s method and of its separation mechanism and (2) optimization of various conditions to effectively combine Marfey’s method with mass spectrometry. Because this proposed method relies on the elution order of an amino acid derivatized with FDAA to determine its absolute configuration, it is very important to clarify why an enantiomeric mixture is separated into each enantiomer after the derivatization. In the present paper, Marfey’s method is applied to commercially available amino acids to confirm its applicability, and the separation behavior was examined as the first step. In the following step, a (7) Harada, K.-I.; Fujii, K.; Mayumi, T.; Hibino, Y.; Suzuki, M.; Ikai, Y.; Oka, H. Tetrahedron Lett. 1995, 36, 1515-1518.

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separation mechanism was elucidated on the basis of the experimental results. Finally, we would like to describe the limitations of the method and the rationale of the proposed separation mechanism. In addition, problem 2 will be discussed in detail in a future paper. HPLC Separation of Derivatized Amino Acids with FDAA. Derivatization of an amino acid with FDAA produces a diastereomer with a highly sensitive and specific UV label (e ) 3 × 104 at 340 nm) on the R-amino group.5 Because FDAA reacts mainly with uncharged amino groups, phenolic hydroxyl groups, and sulfhydryl groups, amino acids including N-methyl amino acids (which are occasionally contained in naturally occurring peptides2-4) are easily derivatized. In the original report, Marfey applied his method to only five amino acids (alanine, aspartic acid, glutamic acid, methionine, and phenylalanine), and each diastereomer of these five derivatized amino acids could be separated on a conventional reversed-phase column without the addition of chiral modifiers to the mobile phase.5 Afterward, several research groups applied his method to other amino acids, and they showed that every amino acid tested had the same elution order, in that the L-amino acid derivative was eluted from the column before its corresponding D-amino acid derivative.8-12 To our knowledge, no exception has been reported so far. Although a few researchers discussed the relationship between the separation behavior and the separation mechanism for this method, the discussions were not always clear and were not certified. To confirm the applicability of this method, we first applied Marfey’s method to commercially available amino acids, which were classified into four types (neutral, hydroxyl and acidic, basic, and N-methyl amino acids), and the separation behavior was carefully examined under common conditions. Marfey originally used triethylammonium phosphate buffer (pH 3) as the mobile phase.5 To effectively combine Marfey’s method with mass spectrometry, the nonvolatile buffer had to be replaced by volatile ammonium acetate or trifluoroacetic acid, which have usually been used for LC/MS analysis. The data in Table 1 were acquired by revered-phase HPLC with UV detection at 340 nm using ammonium acetate buffer (pH 3) as the mobile phase. When necessary, the pH was adjusted to pH 4-7. We confirmed that trifluoroacetic acid buffer also gave almost the same separation behavior as the ammonium acetate buffer (pH 3). The resolution of the diastereomeric pair of individual amino acid was evaluated by the difference (∆t) between the retention time of the D-diastereomer (tRD) and that of the L-diastereomers (tRL). When the ∆t value was above (1.0, the diastereomeric pair was completely separated into each diastereomer on the base line. Excellent resolution was obtained for neutral amino acids, and all L-diastereomers were eluted prior to the D-diastereomers without exception. The resolution became better with an increase in the length of their alkyl side chains, and the retention time also became longer, except for the imino acid, proline. As expected, tyrosine gave bis-derivatives in which FDAA is introduced into both the amino and phenolic hydroxyl groups. (8) Kochhar, S.; Christen, P. Anal. Biochem. 1989, 178, 17-21. (9) Szo´ka´n, G.; Mezo ¨, G.; Hudecz, F. Chromatogrphia 1988, 444, 115-122. (10) Szo´ka´n, G.; Mezo ¨, G.; Hudecz, F.; Majier, Z.; Scho ¨n, I.; Nye´ki, O.; Szirtes, T.; Do ¨lling, R. J. Liq. Chromatogr. 1989, 12, 2855-2875. (11) Goodlett, D. R.; Abuaf, P. A.; Savage, P. A.; Kowalski, K. A.; Mukherjee, T. K.; Tolan, J. W.; Corkem, N.; Goldstein, G.; Crowther, J. B. J. Chromatogr. A 1995, 707, 233-244. (12) Adamson, J. G.; Hoang, T.; Crivici, A.; Lajoie, G. A. Anal. Biochem. 1992, 202, 210-214.

Table 1. Analysis of FDAA Derivatives of Protein and Non-Protein Amino Acids by HPLC

elution order

tRLa (min)

tRDb (min)

alanine 2-amino-n-butyric acid norvaline norleucine valine leucine isoleucine methionine phenylalanine tyrosine (di) proline

LfD LfD LfD LfD LfD LfD LfD LfD LfD LfD LfD

14.8 17.9 24.0 29.9 23.2 29.6 28.7 20.2 27.7 37.8 15.7

19.1 23.8 30.4 36.5 29.7 35.9 35.3 26.4 33.2 42.6 18.2

4.3 5.9 6.4 6.6 6.5 6.3 6.6 6.2 5.5 4.8 2.5

serine homoserine O-methylserine threonine allo-threonine glutamine asparagine glutamic acid aspartic acid β-hydroxyaspartic acidd β-hydroxyaspartic acide

LfD LfD LfD LfD LfD LfD LfD LfD LfD DfL DfL

9.2 9.0 12.3 10.4 10.4 8.8 6.7 10.8 8.2 3.8 6.9

9.9 10.5 17.2 14.8 12.3 9.6 7.3 13.1 9.8 3.7 6.1

0.7 1.5 4.9 4.4 1.9 0.8 0.6 2.3 1.6 -0.1 -0.8

lysine (mono-R) lysine (di) ornitine (mono-R) ornitine (di) histidine (mono-R) histidine (di) arginine citruline

LfD LfD DfL DfL DfL LfD LfD DfL

10.6 31.4 9.4 28.5 6.7 21.9 9.3 10.7

10.8 34.5 8.5 26.0 6.0 25.3 9.6 10.0

0.2 3.1 -0.9 -2.5 -0.7 3.4 0.3 -0.7

N-methylalanine N-methylphenylalanine N-methylvaline N-methylleucine N-methylaspartic acid

f LfD LfD LfD DfL

18.4 30.3 28.8 32.8 11.4

18.4 30.9 32.4 35.4 9.8

0.0 0.6 3.6 2.6 -1.6

amino acid

∆tc (min)

a The retention times of L-amino acid derivatives. b The retention times of D-amino acid derivatives. c ∆t ) tRD - tRL. d β-threo-Hydroxyaspartic acid. e β-erythro-Hydroxyaspartic acid. f The diastereomers were not resolved.

The resolution of acidic and hydroxyl amino acids was generally poor in comparison with that of neutral amino acids. In connection with this behavior, their retention times were relatively short. The resolution of aspragine and glutamine containing a carboxyl amide group on the side chain was much poorer than that of aspartic acid and glutamic acid containing a carboxyl group. Among amino acids with a hydroxyl group, serine showed the worst resolution power. Although the resolution of homoserine became slightly better, one of the structural isomers, O-methylserine, showed a resolution similar to that of alanine. In addition, both diastereomeric pairs of β-hydroxyaspartic acid showed the opposite elution order (D f L), and their retention times were extremely short. In the case of basic amino acids, lysine and ornitine gave three derivatives, mono-R- mono-ω-, and bis-derivatives, and histidine

gave two derivatives, mono-R- and bis-derivatives, but arginine gave only the mono-R-derivative. While their mono-R- and bisderivatives can be resolved, their mono-ω-derivatives were not resolved, indicating that derivatization at the R-amino group is essential for the resolution. However, the mono-R-derivatives of basic amino acids showed the usual elution order (L f D) or the opposite elution order (D f L), and their elution order depended on the pH of the mobile phase used. Although the bis-derivatives of lysine and histidine showed the usual elution order, those of ornitine showed only the opposite elution order, in spite of the long retention times. Commercially available N-methyl amino acids alanine, phenylalanine, valine, leucine, and aspartic acid gave mono-derivatives in the same way. Although their retention times became longer than those of the parent amino acids, the resolution power of the N-methyl amino acids decreased in comparison with that of their parent amino acids. Particularly, N-methylalanine was not resolved, and N-methylaspartic acid was eluted in the reverse order. Throughout these experiments, it was found that the FDAA derivative of the L-amino acid was not always eluted prior to its corresponding D-amino acid derivative. Therefore, Marfey’s method cannot be applied to an unusual amino acid without the clarification of its reasonable separation mechanism. In addition, these experimental results also indicated that the following two points are critical for the elution order: hydrophobicity of an amino acid and conformation of the FDAA derivatives during separation. Separation Mechanism of Marfey’s Method. The conformation of the FDAA derivative of an amino acid during separation was discussed independently by Marfey5 and Bru¨ckner et al.6,13 They suggested that the resolution between the L- and D-amino acid derivatives is based on the different conformations, including intramolecular hydrogen bonding, between the carboxyl group of an amino acid and the carbonyl oxygen of the L-alaninamide of FDAA.5,6,13 However, their discussions were not satisfactory for the resolution, and no definitive conclusion has been proposed so far. For the elucidation of a reasonable separation mechanism for Marfey’s method, UV and NMR spectral approaches were suitable, because these instrumentations are susceptible to conformation. To obtain UV spectra of the FDAA derivatives of amino acids, they were measured using photodiode array detection. A typical UV spectrum is shown for the derivative of L-valine in Figure 2c, together with the UV spectra of the derivative of N-methyl-L-valine (Figure 2a) and the derivatized reagent, FDAA (Figure 2b), and is characterized by absorption maxima at 340 and 414 nm, which are derived from the bridge chromophore between the nitro groups of the dinitrobenzene and the amino groups of the amino acid and L-alaminamide.14 Furthermore, the UV spectrum of the D-valine derivative is consistent with that of its corresponding L-derivative. The derivatives of all amino acids tested had quite similar UV spectra, shown in Figure 2c, except for proline and N-methyl amino acids. Particularly, the bis-derivatives of D- and L-ornitine with the opposite elution order gave the characteristic UV spectra whose shorter absorption maxima at 340 nm were shifted to 320 nm. These results suggested that the stable conformation, including intramolecular hydrogen bonding, be(13) Bru ¨ ckner, H.; Gah, C. J. Chromatogr. 1991, 555, 81-95. (14) Marfey, P. S.; Nowak, H.; Uziel, M; Yphantis, D. A. J. Biol. Chem. 1965, 240, 3264-3269.

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Figure 2. UV spectra of the derivatizing reagent, FDAA (b) and FDAA derivatives of N-methyl-L-valine (a) and L-valine (c), which were measured by HPLC using photodiode array detection.

Figure 3. Plausible conformations of the L- and D-valine derivatives during separation by Marfey’s method, in which each substituent except for the amino groups of valine and L-alaninamide was oriented perpendicularly to the planar molecule of the dinitrobenzene. These are derived from an NOE experiment, in which strong NOEs are observed among the R-protons of the valine and L-alaninamide of FDAA and H-6 of the benzene ring of the derivatives of D- and L-valine and UV spectral data.

tween the two nitro groups in the benzene ring and both amino groups of the amino acid and L-alaninamide, as shown in Figure 3, is formed as a planar molecule of a three-ring system, like anthracene. The NMR spectra of L- and D-valine derivatized with FDAA were measured in methanol-d4. An NOE experiment with the L-derivative showed that strong NOEs were observed among the R-protons of the valine and L-alaninamide of FDAA and the H-6 of the benzene ring, as shown in Figure 3, whereas no NOE was observed among H-6 and the protons of the isopropyl group in valine and the methyl group in L-alaninamide. In addition, it was also found that the derivative of D-valine showed almost the same 3350 Analytical Chemistry, Vol. 69, No. 16, August 15, 1997

NMR spectral behavior as that of the L-derivative under common conditions. These results indicated that both R-protons were spatially situated near H-6 of the benzene ring in both the L- and D-amino acid derivatives. Throughout these results and UV spectral experiments, it was suggested that the resulting conformations of the L- and D-valine derivatives shown in Figure 3, in which each substituent except for the amino groups of valine and L-alaninamide was oriented perpendicularly to the planar molecule of the dinitrobenzene, were stable and predominant in solution, and it was found that the Land D-amino acid derivatives had a common conformation. Williams et al. reported a solid conformation of the derivatized 2-methyl-3-aminopentanoic acid with FDAA by X-ray analysis;15 their results definitely supported our proposed conformation shown in Figure 3. Therefore, the FDAA derivative of D-valine had the cis (Z)-type arrangement of two more hydrophobic substituents of valine and L-alaninamide, the isopropyl group and methyl group, to the plane of the dinitrobenzene, whereas the FDAA derivative of L-valine had the opposite arrangement (trans (E)-type). Based on these results and the elution behavior of FDAA derivatives of the amino acids tested, the resolution between the L- and D-amino acid derivatives was found to be due to the difference in their hydrophobicity, which is derived from the cisor trans-type arrangement of two more hydrophobic substituents at both R-carbons of an amino acid and L-alaninamide, so that the FDAA derivatives of the cis-type arrangement interact more strongly with ODS silica gel and have a longer retention time than that of the trans-type arrangement. In most cases of amino acids, because the substituent of the side chain of an amino acid is more hydrophobic than the carboxyl group, the FDAA derivatives of the D-amino acid have the cis-type arrangement. Therefore, the L-amino acid derivative is usually eluted from the column before its corresponding D-amino acid derivative in Marfey’s method. Confirmation of the Proposed Mechanism. According to our proposed mechanism for Marfey’s method, the retention times of derivatized amino acids with FDAA were dependent on the hydrophobicity of amino acids, and the elution order and the resolution between the L- and D-amino acid derivatives were due to the difference in hydrophobicity between the R-carboxyl group and the side chain of an amino acid. Namely, the FDAA derivative of an amino acid, which has a larger difference in hydrophobicity between the R-carboxyl group and the side chain, has a longer retention time and a better resolution. Indeed, the comparison with retention times of derivatized amino acids with FDAA to the Bull and Breese values for intact amino acids,16 which indicated the hydrophobicity of amino acids, showed a very good correlation. In our proposed mechanism, intramolecular hydrogen bonding including the R-carboxyl group of an amino acid was not considered at all. However, Bru¨ckner et al. concluded that such hydrogen bonding was essential for the resolution.6,13 Therefore, in order to confirm whether or not the presence of the R-carboxyl group is critical for the resolution, the separation behavior of FDAA derivatives of amino acid methyl esters and amino compounds without the R-carboxyl group, such as 1-phenylethylamine, alaninol, and valinol, was carefully examined. (15) Williams, D. E.; Burgayne, D. L.; Rettig, S. J.; Andersen, R. J. J. Nat. Prod. 1993, 56, 545-551. (16) Bull, H. B.; Breese, K. Arch. Biochem. Biophys. 1974, 161, 665-670.

Figure 4. Relationship between the retention times and the elution order of DL-amino acid derivatives. ∆t (Y axis) is the difference between the retention time of the D-amino acid derivative and that of the L-amino acid derivative. Table 2. Analysis of FDAA Derivatives of Amino Acid Methyl Ester and Amino Compounds by HPLC

elution order

tRLa (min)

tRDb (min)

Amino Acid Methyl Esters alanine d 24.8 2-amino-n-butyric acid L f D 31.9 norvaline LfD 37.8 norleucine LfD 43.6 serine DfL 17.0 alaninol valinol 1-phenylethylamine a

Amino Compound LfD 13.6 LfD 22.0 DfL 43.2

∆tc (min)

24.8 33.0 39.9 46.0 13.5

0.0 1.1 1.9 2.4 -3.5

18.2 29.6 39.2

4.6 7.6 -4.0

The retention times of L-derivatives. b The retention times of ∆t ) tRD - tRL. d The diastereomers were not resolved.

D-derivatives. c

The FDAA derivatives of the amino acid methyl esters and amino compounds tested can be resolved as shown in Table 2; their UV spectra were identical to those of the usual amino acids. While the retention times of the methyl esters became longer than those of the parent amino acids, the resolution power of the amino acid methyl ester decreased in comparison with that of the parent amino acid. Particularly, the FDAA derivative of alanine methyl ester was not resolved, and the serine methyl ester derivatives showed the opposite elution order. The retention times and resolution power of the amino compound derivatives were almost the same as those of the parent amino acids. These results indicated that the R-carboxyl group of an amino acid was not always essential for the resolution. On the other hand, the separation behavior can be easily explained using our proposed mechanism without consideration of intramolecular hydrogen bonding. As was shown previously, acidic and hydroxyl amino acids and amino acid methyl esters showed poorer resolution compared with that of neutral amino acids. In particular, β-hydroxyaspartic acids and serine methyl ester gave the

Table 3. Analysis of Derivatized Alanine with Several Derivatizing Reagents by HPLC derivatizing reagent (tR, mina)

elution order

tRLb (min)

tRDc (min)

∆td (min)

FDAA (17.4) D-FDAA (17.4) FDPA (34.9) FDVA (26.4) FDIA (32.7) FDLA (33.0)

LfD DfL LfD LfD LfD LfD

14.8 19.1 27.1 19.7 25.1 26.5

19.1 14.8 32.8 26.4 31.9 33.7

4.3 4.3 5.7 6.7 6.8 7.2

a The retention times of derivatizing reagents. b The retention times of L-alanine derivatives. c The retention times of D-alanine derivatives. d ∆t ) t RD - tRL.

opposite elution order (D f L). These data confirmed further that an amino acid FDAA derivative with a trans-type arrangement concerning two more hydrophobic substituents at both R-carbons of an amino acid and L-alaninamide moieties is usually eluted from a reversed-phase column before its corresponding amino acid derivative with the cis-type arrangement in Marfey’s method. This conclusion was also supported by the following experiments: the separation behavior of a derivatized amino acid with (1-fluoro-2,4-dinitrophenyl-5)-amino acid amide, which replaces L-alaninamide with L-valinamide, L-phenylalaninamide, L-isoleucinamide, L-leucinamide, and D-alaninamide, was examined. The separation behavior of the derivatized alanine with these reagents is shown in Table 3. As expected, the retention time also became longer, and the resolution became better with the increase of their length of alkyl side chains in the amino acid amides. The derivatized amino acid with the reagent of D-alaninamide showed the completely opposite elution order. Application Guideline for Marfey’s Method. To elucidate the elution order for DL-amino acid derivatives, a reasonable separation mechanism for Marfey’s method was proposed as mentioned above. The elution order of a desired amino acid can be elucidated from the comparison of hydrophobicity between the R-carboxyl group and the side chain of the amino acid on the basis of our proposed mechanism. However, it is difficult to evaluate definitely the hydrophobicity of the two functional groups. We considered that the retention time of the amino acid derivatives is usable for the elution order, because the R-carboxyl group is a common functional group in amino acids, and the hydrophobicity of FDAA derivatives of an amino acid is dependent on that of the Analytical Chemistry, Vol. 69, No. 16, August 15, 1997

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side chain of the derivatized amino acid. Therefore, the relationship between the retention time and the elution order of DL-amino acid derivatives was investigated using Table 1 and is shown in Figure 4. As expected, the average retention time of L- and D-amino acid derivatives, which had the usual elution order (L f D), was longer than that of derivatized amino acids containing a side chain which had the same hydrophobicity as the R-carboxyl group, such as serine and asparagine. On the other hand, the amino acid derivatives which had the opposite elution order (D f L) showed a shorter average retention time. Consequently, the elution order of a desired amino acid can be elucidated from the average retention time of the L- and D-amino acid derivatives. The DL-serine and -asparagine derivatives were regarded as critical samples, and their average retention times were situated at the turning points for the elution order. However, the bis-derivatives of ornitin showed the very curious separation behavior that the elution order was opposite, in spite of their relatively long average retention time (27.3 min), as shown in Figure 4. Study on this separation behavior is now in progress. CONCLUSIONS As the first step in establishing our proposed method, the “advanced Marfey’s method”, we applied Marfey’s method to commercially available amino acids, and the separation behavior was examined in detail. Although good resolution of the diastereomeric pair of an individual amino acid was obtained for all amino acids tested and its applicability was confirmed, the FDAA derivative of the L-amino acid was not always eluted prior to its

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corresponding D-amino acid derivative. Because this proposed method relies on the elution order of a derivatized amino acid with FDAA to determine its absolute configuration, the separation mechanism was carefully investigated using UV and NMR spectral techniques. It was suggested that the resulting conformations of the L- and D-amino acid derivatives shown in Figure 3 are stable and that the resolution between the L- and D-amino acid derivatives is due to the difference in their hydrophobicity, which is derived from the cis- or trans-type arrangement of two more hydrophobic substituents at both R-carbons of an amino acid and L-alaninamide, so that the FDAA derivative of the cis (Z)-type arrangement interacts more strongly with ODS silica gel and has a longer retention time than that of the trans (E)-type arrangement. Therefore, the L-amino acid derivative is usually eluted from the column before its corresponding D-amino acid derivative in Marfey’s method. According to this separation mechanism for Marfey’s method, the elution order of a desired amino acid can be elucidated from the average retention time of L- and D-amino acid derivatives, and the DL-serine and -asparagine derivatives are critical for Marfey’s method (Figure 4). The problem involved in the combination of Marfey’s method with mass spectrometry will be discussed in a future paper. Received for review February 12, 1997. Accepted June 6, 1997.X AC9701795 X

Abstract published in Advance ACS Abstracts, August 15, 1997.