Anal. Chem. 1997, 69, 4463-4468
Complete Enantiomeric Separation of Phenylthiocarbamoylated Amino Acids on a Tandem Column of Reversed and Chiral Stationary Phases Takayuki Iida, Hirokazu Matsunaga, Takeshi Fukushima, Tomofumi Santa, Hiroshi Homma, and Kazuhiro Imai*
Faculty of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan
The enantiomeric separation of phenylthiocarbamoyl derivatives of amino acids (PTC-AAs) was studied on a series of reversed phase HPLC columns coupled to the chiral phase HPLC columns. First, the five chiral phases (native, 0.2, 3.3, 7.5 and 16.9 phenylcarbamoylated/βcyclodextrins, Ph/CD) were newly prepared by modification of β-cyclodextrin with phenyl isocyanate and were examined for the enantiomeric separation of PTC-AAs. Among them, the 3.3Ph/CD phase gave the best enantiomeric separation (r g 1.04). However, the separation of the individual PTC-AAs was not sufficient. Next, these separations were investigated on various reversed phase HPLC columns, and octyl silica was selected in terms of the suitability of the mobile phase adopted for the enantiomeric separation mentioned above. The effects of the column temperature, the ion-pairing reagent, and the final content of methanol were also studied on the tandem column of octyl silica and the 3.3Ph/CD phase. Under the best conditions (100 mM ammonium acetate (pH 6.5) containing 1 mM butanesulfonate with 0-40% methanol as the mobile phase), all the individual PTC-AAs were well separated within 150 min. The applicability of the method was demonstrated by the sequence/configuration analysis of a peptide containing a D-amino acid ([D-Thr2]leucine enkephalin-Thr). Since the discovery of dermorphin,1 which has a D-Phe residue, many biologically active peptides containing D-amino acids have been found in snails2 and spiders.3 Recent findings of β-amyloid protein in the brain of patients with Alzheimer’s disease also clarified the occurrence of the protein containing D-Asp.4 Thus, it has become increasingly important to determine the D/Lconfiguration as well as the sequence of amino acid residue in the peptides. However, the commonly adopted procedure has (1) Montecucchi, P. C.; Castinglione, R. D.; Piani, S.; Gozzini, L.; Espamer, V. Int. J. Pept. Protein Res. 1981, 17, 275-283. (2) Kamatani, Y.; Minakata, H.; Kenny, P. T. M.; Iwashita, T.; Watanabe, K.; Funase, K.; Sun, X. P.; Yongsiri, A.; Kim, K. H.; Novales-Li, P.; Novales, E. T.; Kanapi, C. G.; Takeuchi, H.; Nomoto, K. Biochem. Biophys. Res. Commun. 1989, 160, 1015-1020. (3) Heck, S. D.; Siok, C. J.; Krapcho, K. J.; Kelbaugh, P. R.; Thadeio, P. F.; Welch, M. J.; Williams, R. D.; Ganong, A. H.; Kelly, M. E.; Lanzetti, A. J.; Gray, W. R.; Phillips, D.; Parks, T. N.; Jackson, H.; Ahlijanian, M. K.; Saccomano, N. A.; Volkmann, R. A. Science 1994, 266, 1065-1068. (4) Roher, A. E.; Lowenson, J. D.; Clarke, S.; Wolkow, C.; Wang, R.; Cotter, R. J.; Reardon, I. M.; Zurcher-Neely, H. A.; Heinrikson, R. L.; Ball, M. J.; Greenberg, B. D. J. Biol. Chem. 1993, 268, 3072-3083. S0003-2700(97)00236-9 CCC: $14.00
© 1997 American Chemical Society
been the Edman sequencing of amino acids, followed by configuration determination with the chemically synthesized peptide as the reference. In order to overcome this time-consuming procedure for confirmation, we recently reported a new Edman procedure for sequence/configuration analysis of peptides containing D-amino acids using the fluorescent Edman reagent, 7-[(N,N-dimethylamino)sulfonyl]-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS).5-7 After the coupling reaction of DBD-NCS with a peptide, DBDthiazolinone (DBD-TZ) amino acid generated by the cyclization/ cleavage reaction was separated enantiomerically on a β-cyclodextrin (β-CD) stationary phase and detected sensitively with the fluorescence detector. However, the DBD-TZ amino acids were racemized to some extent during the cyclization/cleavage reaction with trifluoroacetic acid (TFA). We have carefully investigated the mechanism of the racemization and reported that the racemization was caused by the replacement of a hydrogen atom located at the R-carbon position to the acid hydrogen of TFA. Therefore, we adopted boron trifluoride, an aprotic Lewis acid, and the derivatized DBD-TZ amino acids were scarcely racemized. We have also applied this procedure to sequence/configuration analysis with phenyl isothiocyanate (PITC), which is widely used as an Edman reagent. However, the generated anilinothiazolinone (ATZ) amino acids were unstable and had to be hydrolyzed to the corresponding phenylthiocarbamoyl amino acids (PTC-AAs), which were then separated on the β-CD stationary phase. There have been various reports which studied the enantiomeric separation methods of amino acid derivatives on the β-CD stationary phase. Recently, Rizzi et al. reported the enantiomeric separation of PTC-AAs using native β-CD for HPLC.8 However, they did not achieve the separation of all PTC-AAs. On the other hand, the modification of the hydroxyl group of β-CD brought about efficient chiral recognition of amino acid derivatives; for example, epichorohydrin β-CD,9 1-(1-naphthylethyl)carbamoylated (5) Imai, K.; Uzu, S.; Nakashima, K.; Akiyama, S. Biomed. Chromatogr. 1993, 7, 56-57. (6) Matsunaga, H.; Iida, T.; Fukushima, T.; Santa, T.; Homma, H.; Imai, K. Biomed. Chromatogr. 1996, 10, 95-96. (7) Matsunaga, H.; Santa, T.; Iida, T.; Fukushima, T.; Homma, H.; Imai, K. Anal. Chem. 1996, 68, 2850-2856. (8) Rizzi, A. M.; Cladrowa-Runge, S.; Jonsson, H.; Osla, S. J. Chromatogr. 1995, 710, 287-295. (9) Thuaud, N.; Sebille, B.; Deratani, A.; Lelievre, G. J. Chromatogr. 1991, 555, 53-64.
Analytical Chemistry, Vol. 69, No. 21, November 1, 1997 4463
Figure 1. Structure of β-CD bonded chiral stationary phases. Native CD and phenylcarbamoylated CD mean n ) 0 and 0 < n e 20, respectively.
β-CD,10,11 and phenylcarbamoylated β-CD12 for the enantiomeric separation of dansyl amino acids,9-12 dinitrobenzoyl amino acids10,11 and dinitropyridyl amino acids,10,11 respectively. In this paper, we prepare five types of phenylcarbamoylated β-CD phase HPLC columns and investigate the enantiomeric separation of PTC-AAs. The complete enantiomeric separation of the individual PTC-AAs is achieved on a tandem column of a reversed and a chiral stationary phase. The method is demonstrated to be applicable to the configuration/sequence analysis of a peptide ([D-Thr2]leucine enkephalin-Thr). EXPERIMENTAL SECTION Materials and Apparatus. Amino acids and [D-Thr2]leucine enkephalin-Thr (Tyr-D-Thr-Gly-Phe-Leu-Thr) were purchased from Sigma Chemical Co. (St. Louis, MO). Pyridine, boron trifluoride etherate, and ion-pairing reagents (ethane-, propane-, butane-, pentane-, and hexanesulfonic acid sodium salts) were purchased from Tokyo Chemical Industry (Tokyo, Japan). PITC, TFA, and methanesulfonic acid were purchased from Wako Pure Chemicals (Osaka, Japan). Water was purified on a Milli-Q system (Millipore, Bedford, MA). Methanol used for the mobile phase was of HPLC grade. All chemicals were of analytical reagent grade. Boron trifluoride etherate was distilled under reduced pressure and kept in an ampule at 4 °C until use. The HPLC system consisted of an L-7100 intelligent pump (Hitachi, Tokyo, Japan), a UV-8000 detector (Tosoh, Tokyo, Japan), and a D-2500 integrator (Hitachi). The column temperature was controlled at 10-40 °C with an LHB-200 water bath (Advantec, Tokyo, Japan) or an SF-1710 water bath (Atto, Tokyo, Japan). Stationary Phases and Columns. Five types of chiral phases were prepared from modified β-CD and silica gel support, 5 µm, by Shinwa Chemical Industries Ltd. (Kyoto, Japan). Figure 1 shows the structure of β-CD stationary phases. The degree of phenylcarbamoylation on β-CD was controlled by changing the quantity of the modified reagent, phenyl isocyanate, in a way similar to that previously reported.13 A part of each modified β-CD stationary phase was dried under reduced pressure on phosphorus pentoxide for a day and subjected to elemental analysis for carbon. The surface concentration of β-CD and the degrees of phenylcarbamoylation were calculated as previously reported.14,15 Since the surface area of the silica support is 300 m2/g, the surface concentration of native β-CD was 0.4 µmol/m2. The degrees of (10) Berthod, A.; Chang, S. C.; Armstrong, D. W. Anal. Chem. 1992, 64, 395404. (11) Lee, S. H.; Berthod, A.; Armstrong, D. W. J. Chromatogr. 1992, 603, 8393. (12) Fujimura, K.; Suzuki, S.; Hayashi, K.; Masuda, S. Anal. Chem. 1990, 62, 2198-2205. (13) Nakamura, K.; Fujima, H.; Kitagawa, H.; Wada, H.; Makino, K. J. Chromatogr. 1995, 694, 111-118. (14) Berendsen, G. E.; Galan, L. D. J. Liq. Chromatogr. 1978, 1, 561-586. (15) Armstrong, D. W.; Stalcup, A. M.; Hilton, M. L.; Duncan, J. D.; Faulkner, J. R.; Chang, S. C. Anal. Chem. 1990, 62, 1610-1615.
4464 Analytical Chemistry, Vol. 69, No. 21, November 1, 1997
phenylcarbamoylation for 1 mol of β-CD (Ph/CD) were 0.2, 3.3, 7.5, and 16.9. The preparation of the modified β-CD stationary phase was reproducible when the preparation was repeated several times. They were packed into 150 mm × 6.0 mm i.d. steel columns. Three types of alkyl-bonded silica stationary phases (methyl, phenyl, and octyl) were also used. They were Pack-TMS (250 mm × 4.6 mm i.d., 5 µm, YMC Co. Ltd, Kyoto, Japan), Pack-Ph (250 mm × 4.6 mm i.d., 5 µm, YMC), and Octyl-80Ts (150 mm × 4.6 mm i.d., 5 µm, Tosoh), respectively. HPLC Conditions. For isocratic elution, the mobile phases of 100 mM ammonium acetate (pH 6.5)/methanol (100/0-80/ 20, v/v) were studied. The flow rate was 0.7 or 1.0 mL/min, and the UV detection was done at 254 nm. For gradient elution, mobile phase A of 100 mM ammonium acetate (pH 6.5) and mobile phase B of 100 mM ammonium acetate (pH 6.5)/methanol (50/50, v/v) were used. Both mobile phases contained an alkanesulfonate ion-pairing reagent. The flow rate and the UV detection were the same as those for isocratic elution. The programs of the gradient elution and the other conditions are described in the legends of the figures and tables. Preparation of PTC-AAs. Amino acids were dissolved in 50% pyridine. Ten microliters of the solution (10 mM) was mixed with 10 µL of 50% pyridine, 5 µL of PITC, and 20 µL of ethanol. The mixture was heated at 50 °C for 30 min. After drying by a centrifugal evaporator (SPE-200, Shimadzu, Kyoto, Japan) at 50 °C for 5 min, 10 µL of water was added to the residue. The excess reagent and byproducts were removed by washing three times with 100 µL of n-heptane/ethyl acetate (7/1, v/v). Eighty microliters of water was added to the aqueous phase, and then the mixture was devided into 10 equal parts and dried by the centrifugal evaporator at 50 °C for 15 min. To the residue was added 100 µL of 5% acetonitrile, and an aliquot (20 µL) of the mixture was subjected to HPLC analysis. Sequencing of [D-Thr2]Leucine Enkephalin-Thr. [D-Thr2]Leucine enkephalin-Thr was dissolved in 50% pyridine. Ten microliters of the solution (1 mM) was mixed with 10 µL of 50% pyridine, 5 µL of PITC, and 20 µL of ethanol. The coupling and washing procedure were the same as described in Preparation of PTC-AAs. To the residue of PTC-peptide was added 30 µL of 8 mM boron trifluoride etherate in acetonitrile, and the mixture heated at 50 °C for 5 min to liberate ATZ amino acid.7 After drying under a stream of nitrogen gas, 20 µL of water was added to the residue. The solution was extracted twice with 100 µL of n-heptane/ethyl acetate (1/5, v/v). The aqueous phase was dried by centrifugal evaporator at 50 °C for 10 min and subjected to the next cycle. The combined organic phase was dried under a stream of nitrogen gas and hydrolyzed in 50 µL of TFA/water (1/1000, v/v) at room temperature for 30 min. To the resulting solution was added 50 µL of mobile phase A, and an aliquot (20 µL) of the mixture was subjected to HPLC analysis. RESULTS AND DISCUSSION Enantiomeric Separation of PTC-AAs on Phenylcarbamoylated β-CD Phases. To evaluate the enantiomeric separation, we chose PTC-Glu, -Ala, -His, -Ser, and -Phe, which represent acidic, neutral, basic, hydroxyl, and aromatic amino acids, respectively. Among the five different types of β-CD phase, the 3.3Ph/ CD gave the best results (Table 1). PTC-Ser enantiomers were only separated on the 3.3Ph/CD (separation factor R ) 1.05), and the other PTC-AAs enantiomers were also successfully separated
Table 1. Enantiomeric Separation of PTC-Amino Acids on the Five Types of β-Cyclodextrin Chiral Stationary Phase Columnsa Glu k1′ R
chiral phase native-CD 0.2Ph/CD 3.3Ph/CD 7.5Ph/CD 16.9Ph/CD
0.91 0.53 1.04 0.35 0.37
1.19 1.15 1.18 1.23 1.00
Ser k 1′ R 1.38 0.86 1.51 0.79 1.03
1.00 1.00 1.05 1.00 1.00
Ala
His k1′ R
k1′
R
1.56 0.93 1.65 0.90 1.59
1.00 1.00 1.07 1.12 1.00
2.02 1.38 2.31 1.87 2.86
1.07 1.07 1.11 1.16 1.00
Phe k1′ R 5.84 4.06 7.20 3.44 8.81
1.26 1.29 1.34 1.39 1.05
a k ′ means the capacity factor of PTC-D-amino acids. Mobile phase: 1 100 mM ammonium acetate (pH 6.5)/methanol ) 80/20 (v/v). Flow rate: 1.0 mL/min. Temperature: ambient.
Table 2. Capacity Factors (k′) and Separation Factors (r) Obtained for PTC-Amino Acids on the 3.3Ph/CD Columna PTCAAs Ala Arg Asn Asp Gln Glu Glyb His Ile Leu
k′ D-form
L-form
2.40 3.74 2.20 1.81 2.18 1.57
2.54 4.25 2.31 1.88 2.49 1.88 2.25 3.56 5.13 5.75
3.19 4.04c 4.44
R 1.06 1.14 1.05 1.04 1.14 1.20 1.12 1.27 1.30
k′
PTCAAs
D-form
L-form
R
Lys Met Phe Pro Ser Thr Trp Tyr Val
11.26 3.85 13.61 2.23 2.28 1.99 13.72 6.34 3.12
12.59 4.45 18.46 3.44 2.40 2.23 18.92 6.70 3.74
1.12 1.16 1.36 1.54 1.05 1.12 1.38 1.06 1.20
Figure 2. Effect of ammonium acetate concentration in the mobile phase on the enantiomeric separation of PTC amino acids. Mobile phase: 20-500 mM ammonium acetate (pH 6.5)/methanol ) 90/10 (v/v). The flow rate and the temperature are the same as in Table 1.
a Mobile phase: 100 mM ammonium acetate (pH 6.5)/methanol ) 90/10 (v/v). The flow rate and the temperature are the same as in Table 1. b Gly has no chirality. c This is D-allo-Ile, which is a stereoisomer of D-Ile at the β-carbon position.
(R ) 1.07-1.34). These results suggest that a suitable modification of hydroxyl groups of β-CD is available for the enantiomeric separation of PTC-AAs because of penetration into or interaction with the cavity of β-CD. Therefore, we selected the 3.3Ph/CD as the chiral stationary phase for the further experiments. The enantiomers of 18 derivatives of PTC-AAs and PTC-Gly were also well separated on the 3.3Ph/CD (Table 2). For all the PTC-AAs, the 3.3Ph/CD retained the L-form more than the D-form. In particular, the size and/or the hydrophobicity of the side chain of the amino acids influenced the retention of PTC-AAs on the 3.3Ph/CD. PTC-AAs, which have small side chains, gave small values of capacity factor (k′): Asp(L-form; k′ ) 1.88), Asn (2.31), Ser (2.40), and Ala (2.54). PTC-AAs, which have aromatic side chains, gave large k′ values: Tyr (6.70), Phe (18.46), and Trp (18.92). Since, in the case of Lys, the two amino groups of both R position and position were derivatized to PTC-Lys to afford a high hydrophobicity, a large k′ value (12.59) was obtained. These results indicate that the 3.3Ph/CD affords a kind of reversed phase mode separation in the enantiomeric separation of PTC-AAs, similar to the mode observed for the completely phenylcarbamoylated CD phase used for dansyl amino acids.12 The enantiomeric separation was also affected by the concentration of ammonium acetate in the mobile phase (Figure 2). When the concentration of ammonium acetate was changed in the range from 20 to 500 mM, the required concentration for the enantiomeric separation of PTC-Ser and PTC-Glu was found to be greater than or equal to 50 mM. With further increment in the concentration, the less separation between the individual PTC-AAs, such
Figure 3. Separation of PTC-Ser, -Gly, -Asn, and -Gln on the reversed phase silica columns and on the tandem column with the octyl silica and the 3.3Ph/CD. Mobile phase: 100 mM ammonium acetate (pH 6.5). Flow rate: 0.7 mL/min. Temperature: (a) 30 °C, (b) Octyl-80Ts at 30 °C, and 3.3PhCD at 20 °C.
as PTC-Ser and -Glu, was observed. Thus, we selected 100 mM as the optimum concentration of ammonium acetate in the mobile phase. However, the individual separation of all PTC-AAs was not successful on the 3.3Ph/CD, in spite of the modification of the mobile phases, i.e., the use of acetonitrile instead of methanol, etc. Especially, PTC-Ser, -Gly, -Asn, and -Gln were eluted at similar elution times from 15 to 20 min, even when ammonium acetate alone was adopted as the mobile phase. Tandem Coupling of the Reversed Phase and the 3.3Ph/ CD Stationary Phase Colunms. To separate PTC-Ser, -Gly, -Asn, and -Gln individually, the reversed phase separation was considered for preseparation prior to the 3.3Ph/CD. The three types of reversed phase (methyl, phenyl, and octyl silica) columns were investigated using 100 mM ammonium acetate alone as the Analytical Chemistry, Vol. 69, No. 21, November 1, 1997
4465
Figure 4. Effect of types of ion-pairing reagent in the mobile phase on the capacity factors of PTC amino acids. Open and solid symbols mean PTC amino acids of D-form and L-form, respectively. Carbon number “0” represents the absence of ion-pairing reagent in the mobile phase. Column and temperature: Octyl-80Ts (at 30 °C) + 3.3Ph/CD (at 20 °C). Mobile phase: A, 100 mM ammonium acetate (pH 6.5) containing 1 mM ion-pairing reagent; B, 100 mM ammonium acetate (pH 6.5)/methanol ) 50/50 (v/v) containing 1 mM ion-pairing reagent. Gradient: isocratic elution until 65 min (B, 0%), linear gradient elution from 65 to 115 min (B, 0-20%), then from 115 to 140 min (B, 20-60%), isocratic elution from 140 to 240 min (B, 60%). Flow rate: 0.7 mL/min.
mobile phase (Figure 3a), since these amino acid derivatives are hydrophilic and not so much retained on the columns. We selected the octyl silica, because it gave an adequate separation of the PTC-AAs with a lower column pressure than the methyl and the phenyl silica. Before the coupling of the two columns, the effect of the column temperature was examined, since the k′ values of PTCAAs were greatly dependent on the temperature. The low temperature gave large k′ values on both the stationary phases. Besides, the elution order of PTC-D-Asn and -Gly was reversed with the temperature change on the 3.3Ph/CD: k′ ) 6.8 and 6.7 for PTC-D-Asn and -Gly at 10 °C vs k′ ) 4.5 and 4.6 at 25 °C, respectively. Considering the above results, the temperatures of both the stationary phases should be controlled separately to give a suitable separation of PTC-AAs. Thus, the temperatures for the 4466 Analytical Chemistry, Vol. 69, No. 21, November 1, 1997
Figure 5. Effect of butanesulfonate concentration in the mobile phase on the capacity factors of PTC amino acids. Open and solid symbols mean PTC amino acids of D-form and L-form, respectively. The mobile phases A and B contain 0.2-5 mM sodium butanesulfonate. The other conditions are the same as in Figure 4.
octyl silica and the 3.3Ph/CD on a tandem column were selected at 30 and 20 °C, respectively (Figure 3b). The enantiomeric separations of PTC-Ser and -Asn were sufficient for identification, although baseline separation was not achieved because of the diffusion of the solutes on the octyl silica. Effect of Ion-Pairing Reagent in the Mobile Phase. When the mobile phase containing methanol was run on a tandem coupling of the octyl silica and the 3.3Ph/CD, each pair of PTCAla and -His, and of PTC-Pro and -Arg, was eluted very close. We thought that the basic amino acids could be retained more and separated from PTC-Ala or -Pro if an ion-pairing reagent was added to the mobile phase to increase the hydrophobicity of PTC-His or -Arg. Thus, the effects of several sodium alkanesulfonates on the k′ values of those pairs as well as other PTC-AAs were studied. Contrary to our expectation, the k′ value of PTC-Arg was steady with the addition of any alkanesulfonate to the mobile phase, and the k′ value of PTC-His decreased with the increase in the carbon number of the alkanesulfonate, whereas PTC-Ala and -Pro behaved similarly to PTC-His (Figure 4a). When we used a single column of the octyl silica, similar changes of the k′ values of PTC-Arg, -His, -Ala, and -Pro were also observed (data not shown).
Figure 6. Separation of a mixture of 37 PTC amino acids. Amount of each enantiomer was 1 nmol, and Gly was 2 nmol. The elution order of enantiomers was D-form and then L-form. Ile was a mixture of D-allo-Ile and L-Ile. Column and temperature: Octyl-80Ts (at 30 °C) + 3.3Ph/ CD (at 20 °C). Mobile phase: A, 100 mM ammonium acetate (pH 6.5) containing 1 mM sodium butanesulfonate; B, 100 mM ammonium acetate (pH 6.5)/methanol ) 50/50 (v/v) containing 1 mM sodium butanesulfonate. Gradient: isocratic elution until 50 min (B, 0%), linear gradient elution from 50 to 80 min (B, 0-20%), then from 80 to 110 min (B, 20-80%), isocratic elution from 110 to 150 min (B, 80%). Flow rate: 0.7 mL/min.
Therefore, these results might suggest that PTC-AAs were repelled by the negative charges of the long-chain alkanesulfonates (the carbon number g 4) which were adsorbed more on the octyl silica than the short-chain alkanesulfonates.16-18 Only PTC-Arg was retained to the same degree without the addition of the alkanesulfonates, probably because of the balance of ion-pairing and the repulsion. No great change on the retention was observed on a single column of the 3.3Ph/CD with the addition of alkanesulfonates (data not shown). According to Figure 4a, the best separations between PTC-His and -Ala, and PTC-Arg and -Pro, was observed by the addition of butanesulfonate. We thus selected butanesulfonate as the ion-pairing reagent. Next, the concentration of butanesulfonate was examined. The increase in the concentration did not induce any changes in the k′ value of PTC-Arg, while decreases in the k′ values of PTC-His, -Ala, and -Pro were observed (Figure 5a), as in the case of increasing the carbon number of the alkanesulfonate. The retention behavior of other PTC-AAs was the same as those of PTC-Ala and -Pro (Figure 5b). Considering these results, we selected 1 mM as the optimum concentration. Complete Separation of Individual PTC-AAs. To separate PTC-Leu, -Ile, -Trp, -Lys, and -Phe, which eluted later because of their high hydrophobicity, the final methanol content in the mobile phase was investigated under gradient elution condition from 35% to 50%. D-Allo-Ile (different configuration at β-carbon from D-Ile) and L-Ile were separated well with 40% methanol, with which D,LLys eluted between D- and L-Trp. Figure 6 shows a chromatogram of separation of all the PTC-AAs on a tandem column of the octyl silica and the 3.3Ph/CD. A mixture of 37 PTC-AAs was separated within 150 min. Their elution times were constant (Table 3). The reproducibility of the elution times was satisfactory, owing to the precise regulation of the column temperature of the octyl silica (16) Melin, A. T.; Ljungcrantz, M. J. Chromatogr. 1979, 185, 225-239. (17) Fekete, J.; Castilho, P. D.; Kraak, J. C. J. Chromatogr. 1981, 204, 319-327. (18) Gennaro, M. C. Adv. Chromatogr. 1995, 35, 344-381.
Table 3. Variation of the Elution Times of PTC-Amino Acids (n ) 6)a elution time (min)
elution time (min)
PTC-AAs
mean
SD
PTC-AAs
mean
SD
D-Asp
31.8 33.3 74.4 77.1 88.3 89.9
0.29 0.44 0.26 0.24 0.16 0.15
D-Arg
103.7 105.2 117.2 117.8 135.7 138.4
0.12 0.12 0.04 0.04 0.11 0.12
L-Asp D-Gln L-Gln D-Thr L-Thr a
L-Arg D-Val L-Val D-Phe L-Phe
The HPLC conditions are the same as in Figure 6.
and the 3.3Ph/CD. Detection limits of PTC-Asp and -Val were 20 pmol (highest) and 5 pmol (lowest), respectively. Amino Acid Sequence and D/L-Configuration Determination of a Peptide. In order to demonstrate the applicability of the enantiomeric separation of PTC-AAs in practice, Edman sequence analysis of a peptide containing the D-amino acid, [D-Thr2]leucine enkephalin-Thr (Tyr-D-Thr-Gly-Phe-Leu-Thr), was carried out. This procedure is different from the conventional procedure in that ATZ amino acid obtained at the cyclization/ cleavage step is transformed to PTC-AAs by hydrolysis instead of conversion to phenylthiohydantoin (PTH) amino acids. Thus, PTC-AAs obtained by the procedure were subjected to the proposed HPLC separation. As shown in Figure 7, five residues of N-terminal amino acids of the peptide were identified. In addition, the D/L-configuration of Tyr (1), Thr (2), Phe (4), and Leu (5) were identified as L-, D-, L-, and L-, respectively, although the PTC-AAs were partially racemized. This result reveals that the individually enantiomeric separation of PTC-AAs is extraordinarily useful for the simultaneous determination of the amino acid sequence and D/Lconfiguration of a peptide containing D-amino acid. Analytical Chemistry, Vol. 69, No. 21, November 1, 1997
4467
racemization ratios () 100 × D/(D + L)) of Tyr (1), Phe (4), and Leu (5) were 19%, 22%, and 35%, respectively, even with boron trifluoride during the cyclization/cleavage reaction.7 The racemization can be reduced in the future by optimizing the hydrolysis condition; for example, if we adopt the hydrolysis reagent of hydrochloric acid instead of 0.1% TFA, we should observe less than 10% racemization (data not shown). In the present experiment, C-terminal amino acid residue (Thr) was not detected. The reason is probably that the coupling and cyclization/cleavage reactions of the C-terminal residue do not result in the formation of ATZ amino acid. There are very few reports on the sequence/configuration analysis by HPLC,19-22 and most procedures involved the diastereomeric separation of amino acid derivatives using a chiral reagent. However, these procedures have some problems: Asn and Gln could not be detected,19,20 and the derivatives of acidic amino acids could not be adequately separated.21,22 The procedure described in this report resolved those problems. The application of the present method to the automated sequencing system is expected to contribute to the study of a peptide containing D-amino acid, such as β-amyloid protein. In conclusion, we described for the first time a complete enantiomeric separation of 37 PTC-AAs (18 amino acids and Gly) within 150 min on the tandem column of the octyl silica and the 3.3Ph/CD stationary phases. The separation method was demonstrated to be useful for the simultaneous determination of the amino acid sequence and D/L-configuration of a peptide containing D-amino acid.
Figure 7. Sequence and D/L-configuration determination of [D-Thr2]leucine enkephalin-Thr. The HPLC conditions are the same as in Figure 6.
It should be noted that a racemization of amino acids was observed under the procedure, except for D-Thr (2). The (19) Scaloni, A.; Simmaco, M.; Bossa, F. Anal. Biochem. 1991, 197, 305-310. (20) Scaloni, A.; Simmaco, M.; Bossa, F. Amino Acids 1995, 8, 305-313. (21) Toyo’oka, T.; Liu, Y. M. J. Chromatogr. 1995, 689, 23-30. (22) Toyo’oka, T.; Suzuki, T.; Watanabe, T.; Liu, Y. M. Anal. Sci. 1996, 12, 779782.
4468 Analytical Chemistry, Vol. 69, No. 21, November 1, 1997
ACKNOWLEDGMENT We gratefully acknowledge Dr. C. K. Lim for the revision and comments on the manuscript. We thank Shinwa Chemical Industries Ltd. for preparing the chiral stationary phases of the modified β-cyclodextrin and packing the HPLC columns. We also thank Tosoh and YMC Co. Ltd. for the gift of reversed phase HPLC columns. Received for review March 4, 1997. Accepted July 31, 1997.X AC970236U X
Abstract published in Advance ACS Abstracts, October 1, 1997.
