l -configuration of peptides using a fluorogenic Edman reagent, 7-N,N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate

Analytica Chimica Acta 415 (2000) 57–66

Detection of 7-N,N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) carbamoyl amino acids generated by post-column desulfuration in the simultaneous determination of the sequence and d/l-configuration of peptides using a fluorogenic Edman reagent, 7-N,N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate Akira Toriba, Yong Huang, Tomofumi Santa, Kazuhiro Imai∗ Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Received 27 October 1999; received in revised form 7 March 2000; accepted 7 March 2000

Abstract A modified Edman sequencing method using 7-N,N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS) for amino acid sequence and d/l-configuration determination has been developed. DBD-thiazolinone (TZ)-amino acids generated by Edman sequencing method were hydrolyzed and then oxidized to DBD-carbamoyl (CA)-amino acids, instead of the conversion reaction to thiohydantoin (TH)-amino acid in the conventional Edman method. The oxidative desulfuration step operated manually with formation of DBD-CA-amino acids was difficult to control because of the production of by-products by the side-chain oxidation of amino acid and inapplicability to the conventional automated protein sequencer. Therefore, post-column desulfuration method in which the reaction was carried out after the separation of analyte on a column was designed. DBD-thiocarbamoyl (TC)-amino acids generated by the modified sequencing method were separated on the reversed phase or Pirkle type chiral stationary phase column and then oxidized to DBD-CA-amino acids with hydrogen peroxide as a post-column reagent and detected fluorimetrically. Using the method, the determination of the amino acid sequence and d/l-configuration of a d-amino acid containing peptide was achieved and gave us the recognition to the utility of the method. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Amino acid sequence; d-amino acid containing peptide; Fluorogenic Edman reagent; Chiral separation; Post-column desulfuration

1. Introduction Although proteins and peptides in living organisms have generally been believed to consist of only l-amino acids, several peptides containing d-amino acid have been found in eukaryote [1–5] and mammals [6–8]. The presence of d-amino acid residue ∗

Corresponding author. Fax: +81-3-5841-4885.

in these peptides affected their biological activity [1,2] and on the other hand, was related to diseases with aging such as Alzheimer’s disease and cataract [6–8]. Therefore, the determination method of amino acid sequence and d/l-configuration of peptide was desirable. The modified Edman sequencing methods have been applied to the sequence and d/l-configuration determination [9–11]. They were carried out by

0003-2670/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 0 ) 0 0 8 4 8 - 5

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the detection of the liberated amino acid derivatives from N-terminal of peptides with conventional Edman method using phenylisothiocyanate (PITC) [9] or diastereomeric derivatives generated by using 4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,Ndimethylaminosulfonyl)-2,1,3-benzoxadiazole as a chiral Edman reagent [10] and 1-fluoro-2,4-dinitro-5-lalanine as a chiral reagent in a subtractive Edman method [11]. A disadvantage common to these methods was that the amino acid derivatives liberated from the N-terminal of peptides had a tendency to racemize to a considerable extent [12,13]. We reported the suppression of the racemization by use of an aprotic acid, boron trifluoride (BF3 ), instead of TFA, as a cleavage/cyclization reaction catalyst in an amino acid sequencing method for peptides [14]. Then, BF3 was used in an amino acid sequencing method using PITC to retain the d/l-configuration of amino acid residues [15] and further applied to the convenient vapor phase sequencer [16]. Usually only a small amount of peptides and proteins purified for the sequence and d/l-configuration determination are available, therefore, the improvement of detection sensitivity of the liberated amino acid derivatives in the sequencing analysis is required. Various fluorescence Edman reagents have been reported to enhance the detection sensitivity of thiohydantoin (TH)-amino acids [10,17–20]. However, these reagents are not yet utilized as compared with PITC because of the lower coupling yield derived from the bulky fluorophores of these reagents [21] and the interference of the detection of the generated TH-amino acids by the strong fluorescence of the reagents themselves and degradation products. To overcome this disadvantage, we have reported a new Edman procedure using the fluorescent Edman reagents, 7-N,N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS), 7-phenylsulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate and 7-methylsulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate, in which the thiazolinone (TZ)-amino acids generated by cleavage/cyclization reaction were detected fluorimetrically [22,23]. These reagents itself did not fluoresce and thus the reagents itself and the degradation products did not interfere with the detection of the TZ-amino acids. Furthermore, we modified the procedure mentioned above to detect more stable fluorescent derivatives; DBD-TZ amino acids

were hydrolyzed to DBD-thiocarbamoyl (TC)-amino acids and oxidized to corresponding DBD-carbamoyl (CA)-amino acids, and detected fluorimetrically [24]. However, the latter method suffered from two disadvantages. First, several peaks were observed on the chromatogram obtained from the amino acid residues such as Tyr, Met and Pro having side-chains which were sensitive to oxidation and their side reactions were difficult to control. Second, since amino acid sequence analysis is currently accomplished on an automated sequencer, the methods applicable to the convenient vapor phase sequencer are much more desirable for the simultaneous determination of the sequence and d/l-configuration of amino acids in peptides. However, this method in which DBD-TC-amino acids were converted to the corresponding CA-amino acids was difficult to apply to the automated sequencer, since a further reaction step of the oxidation to the DBD-CA-amino acids was added to the conventional Edman procedure. In this study, we modified the procedure. DBDTC-amino acids generated by the Edman sequencing procedure were separated on the reversed phase or chiral stationary phase column, and then converted to DBD-CA-amino acids by adding hydrogen peroxide to the mobile phase and detected fluorimetrically (Fig. 1). Using this post-column desulfuration method, the by-products oxidized in their side chain disappeared in the chromatogram and we could easily identify the sequence and d/l-configuration. Finally, we established a sensitive and simultaneous determination method for the sequence and d/l-configuration of peptides, which is applicable to the automated sequencer.

2. Experimental 2.1. Materials and apparatus DBD-NCS was synthesized as described previously [25]. Amino acids, l-aspartic acid ␤-methyl ester (AspMe), l-glutamic acid ␥-methyl ester (GluMe), S-␤-(4-pyridylethyl)-l-cysteine (PEC) and [d-Ala2 ]-Deltorphin II were purchased from Sigma (St. Louis, MO, USA). Hydrogen chloride-methanol reagent 5 (HCl-MeOH), pyridine and boron trifluoride diethyl etherate complex (BF3 –Et2 O) were purchased

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Fig. 1. Reaction scheme for the Edman sequencing procedure.

from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 4-Vinylpyridine, trifluoroacetic acid (TFA) and hydrogen peroxide (H2 O2 ) were obtained from Wako Pure Chemicals (Tokyo, Japan). 1,4-Dithiothreitol, citric acid monohydrate and HPLC-grade acetonitrile and methanol were purchased from Kanto Chemical (Tokyo, Japan). Water was purified using a Milli-Q system (Millipore, Bedford, MA, USA). All other reagents were of analytical or guaranteed reagent grade. HPLC was carried out using two intelligent pumps, L-7100 and L-6000 (Hitachi, Tokyo, Japan), an

L-4000H UV detector (Hitachi), an L-7480 fluorescence detector (Hitachi), a D-7500 integrator (Hitachi) and water bath TC-100 (JASCO, Tokyo, Japan). Mass spectra were measured on an M-1200H mass spectrometer (Hitachi) with an atmospheric pressure chemical ionization system (APCI-MS) or an electrospray ionization system (ESI-MS). 2.2. HPLC conditions Flow diagram of the HPLC system is shown in Fig. 2. DBD-TC-amino acids were separated

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Fig. 2. Flow diagram of HPLC system for the determination of DBD-TC-amino acids with post-column desulfuration. Mobile phase (MP); hydrogen peroxide reagent (HP); HPLC pumps (P1 and P2); injector (I); column (C); mixing device (MD); reaction coil (RC); water bath (WB); fluorescence detector (D).

using two columns in tandem, i.e. a phenyl group bonded porous silica gel column (YMC-Pack Ph, 250 mm×4.6 mm i.d., 5 ␮m; YMC Co., Ltd., Kyoto, Japan) and an ODS column (TSK gel ODS-80Ts, 250 mm×4.6 mm i.d., 5 ␮m; Tosoh, Tokyo, Japan) for the identification of individual amino acid on sequencing analysis. The following conditions were used: mobile phase A, acetonitrile/methanol/water (24/18/58, v/v/v) containing 0.1% TFA and mobile phase B, acetonitrile/methanol/water (50/20/30, v/v/v) containing 0.1% TFA; gradient, isocratic elution until 38 min (B, 0%), linear gradient from 38.1 to 70 min (B, 50–100%). Flow rate was kept at 0.7 ml/min. For chiral separation of DBD-TC-amino acids, Pirkle type chiral stationary phase (SUMICHIRAL OA-3100, 250 mm×4.6 mm i.d., 5 ␮m; Sumika Chemical Analysis Service, Osaka, Japan) was used. Isocratic elution was employed using the following two conditions: methanol containing 0.025 mM citric acid for basic amino acids (Arg, Lys, His) and methanol containing 2.5 mM citric acid for the others at a flow rate of 0.7 ml/min. The post-column hydrogen peroxide (H2 O2 ) solution was acetonitrile/water (70/30) containing 2.65 M H2 O2 at a flow rate of 0.5 ml/min. For optimization of H2 O2 concentration, the post-column H2 O2 solution composed of various concentration of H2 O2 was prepared by dilution with acetonitrile. The temperature of the reaction coil (0.5 mm i.d.×10 m) was kept at 62◦ C. Fluorescence detection was performed at 524 nm with excitation at 384 nm. For identification of DBD-TC-amino acids, the following HPLC system was also used: mobile phase A and B containing 10 mM formic acid were 30/70 (v/v) and 70/30 (v/v) of acetonitrile/water, respectively. The analytical column was ODS-80Ts at a flow rate of

1.0 ml/min using gradient elution from 0 to 25 min (mobile phase B composition; 0–100 %), and the UV detection was carried out at 385 nm. 2.3. Preparation of standard DBD-TC-amino acids Amino acids were dissolved in pyridine/water (1/1, v/v) (0.1 mM). A 100 nmol of DBD-NCS was dissolved in 20 ␮l of the solution. The mixture was vortex mixed and heated at 50◦ C for 20 min. After the coupling reaction, the mixture was evaporated to dryness using a centrifugal evaporator (SPE-200, Shimadzu, Kyoto, Japan) at 50◦ C for 15 min, and the resulting residue was dissolved in the HPLC mobile phase. The absorption peak corresponding to DBD-TC-amino acids were collected by HPLC and dried in a centrifugal evaporator at 50◦ C. After the residue was dissolved in acetonitrile, the solution was subjected to LC-MS and these DBD-TC-amino acids were identified with ESI-MS for Arg, Gln, His, and Thr derivatives or APCI-MS for other amino acid derivatives by detecting the expected molecular mass: Ala, m/z 374 ([M+H]+ ); Arg, m/z 459 ([M+H]+ ); Asn, m/z 417 ([M+H]+ ); Gln, m/z 431 ([M+H]+ ); Gly, m/z 358 ([M−H]− ); His, m/z 439 ([M]+ ); Ile, m/z 416 ([M+H]+ ); Leu, m/z 416 ([M+H]+ ); Lys, m/z 431 ([M+H]+ ); Met, m/z 434 ([M+H]+ ); Phe, m/z 450 ([M+H]+ ); Pro, m/z 400 ([M+H]+ ); Ser, m/z 390 ([M+H]+ ); Thr, m/z 404 ([M+H]+ ); Trp, m/z 489 ([M+H]+ ); Tyr, m/z 466 ([M+H]+ ); Val, m/z 402 ([M+H]+ ). dl-PEC was prepared by the reaction of dl-Cys with 4-vinylpyridine [26]. dl-Cys was dissolved in pyridine/water (1/1, v/v) (0.1 mM). To a solution of 20 ␮l, 5 ␮l of 4-vinylpyridine was added, and the

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mixture was vortex mixed and heated at 50◦ C for 2 h. The excess reagent and by-products were removed by washing two times with 200 ␮l of n-heptane/ethyl acetate (1/1, v/v). The aqueous phase was dried in a centrifugal evaporator at 50◦ C. The resulting residue (dl-PEC) was subjected to the reaction with DBD-NCS as described earlier (APCI-MS: m/z 511[M+H]+ ). DBD-TC derivatives of dl-AspMe and dl-GluMe were prepared from dipeptide according to the sequencing procedure similar to that shown below. DBD-TC-amino acids liberated from N-terminal l-AspMe and l-GluMe residues in the each dipeptides were completely racemized using TFA, instead of BF3 , as cyclization/cleavage reagent, and hence the racemic DBD-TC-AspMe and GluMe were obtained (APCI-MS: AspMe, m/z 432 [M+H]+ ; GluMe, m/z 446 [M+H]+ ).

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in acetonitrile was added to the residue. The mixture was heated at 50◦ C for 10 min and dried under a stream of nitrogen. After the cleavage/cyclization reaction, 20 ␮l of water was added to the residue. The solution was extracted twice with 100 ␮l of ethyl acetate. The aqueous phase was dried in a centrifugal evaporator and subjected to the next cycle. The combined organic phase was dried under a stream of nitrogen. The resulting residue was dissolved in 20 ␮l of acetonitrile and added with 80 ␮l of 0.1 M aqueous HCl containing 2 ␮M dithiothreitol in which a steam of nitrogen was bubbled before use. The mixture was heated at 50◦ C for 5 min for hydrolysis and diluted 10 times with acetonitrile and 5 ␮l of the solution was subjected to the HPLC. 3. Results and discussion

2.4. Optimization of oxidation reaction time DBD-TC-Leu prepared as described earlier was dissolved in acetonitrile/water (1/1, v/v) containing 0.1% TFA (0.1 mM). A 7 ␮l of the solution was added to 5 ␮l of acetonitrile/water (70/30) containing 2.65 M H2 O2 and the mixture was vortex mixed and heated at 70◦ C, and withdrawn after appropriate time intervals. After the reaction, a 10 ␮l of the mixture was subjected to HPLC and the analytes were monitored simultaneously by fluorescence and UV detectors. 2.5. Liquid phase sequencing of [d-Ala2 ]-Deltorphin II with DBD-NCS The peptide was dissolved in water (0.1 mM) and 20 ␮l of the solution was dried in a centrifugal evaporator at 50◦ C. The resulting residue was dissolved in 50 ␮l of HCl-MeOH solution and heated at 50◦ C for 5 h. After the methyl esterification, the solution was dried under a stream of nitrogen. The esterified peptide was dissolved in 20 ␮l of pyridine/water (1/1, v/v) containing 5 mM DBD-NCS. The mixture was vortex mixed and heated at 50◦ C for 25 min. After the coupling reaction, the excess reagent and by-products were removed by washing three times with 200 ␮l of n-heptane/ethyl acetate (1/4, v/v). The aqueous phase was evaporated to dryness using a centrifugal evaporator at 50◦ C for 15 min, and 30 ␮l of 1% BF3 –Et2 O

3.1. Optimization of oxidation reaction time In our previous paper, we reported the oxidative desulfuration of DBD-TC-amino acids to DBD-CA-amino acids with NaNO2 under acidic condition [24]. However, this system was inapplicable to post-column desulfuration, since when the mobile phase for the separation on reversed phase column of DBD-TC-amino acids was mixed with the solution containing NaNO2 to oxidize DBD-TC-amino acids, the organic phase and aqueous phase in the mixed solution were separated by salting-out effect. Therefore, H2 O2 [27] was adopted in the post-column desulfuration of DBD-TC-amino acids. First, we optimized the reaction time of the desulfuration of DBD-TC-Leu to DBD-CA-Leu by a batch method. An aliquot of the reaction mixture of DBD-TC-Leu and H2 O2 after appropriate intervals was subjected to RP-HPLC and the absorbance of DBD-TC-Leu and the fluorescence intensity of DBD-CA-Leu were monitored. As shown in Fig. 3, DBD-TC-Leu completely disappeared and the fluorescence of DBD-CA-Leu showed maximal intensity after 2 min. Based on this result, the length of the reaction coil between the mixing device and the fluorescence detector of 10 m was adopted and the reaction time calculated based on the sum of the dead volume of the reaction coil was 1.64 min. We selected the length of the reaction coil mentioned earlier, although the reaction time was shorter than

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Fig. 3. Time course for the desulfuration of DBD-TC-Leu to DBD-CA-Leu in a batch method. Absorbance of DBD-TC-Leu (䊉) and fluorescence intensity of DBD-CA-Leu (䊊).

optimum 2 min because of their broad peak shape observed with the length of the reaction coil more than 10 m (data not shown). 3.2. Optimization of H2 O2 concentration and reaction temperature The concentration of H2 O2 and reaction temperature were examined by using a mixture of DBD-TC-Arg, Asn, Thr, Pro and Leu. The maximal fluorescence intensities of DBD-TC-amino acids were observed with 2.65 M H2 O2 (Fig. 4A). While the optimum reaction temperature was 70◦ C (Fig. 4B), we selected 62◦ C as reaction temperature, considering the boiling point of methanol used as mobile phase for the chiral separation. 3.3. Separation of DBD-TC-amino acids on reversed phase column The fluorescence intensity of DBD-CA-amino acids increased with the increase in the organic solvent content in the HPLC mobile phase and was high under acidic condition [24]. Therefore, two analytical columns, i.e. an ODS column and a phenyl bonded silica gel column, were used in tandem to retain DBD-TC-amino acids on the columns with the mobile phase of relatively high acetonitrile content. As shown in Fig. 5, all DBD-TC-amino acids were separated within 70 min with the mobile phase con-

Fig. 4. Effects of concentration of H2 O2 (A) and reaction temperature (B) on the fluorescence intensities of DBD-CA-amino acids. Symbols show the following amino acids: Leu (䊊); Thr (䊏); Asn (4); and Arg (䉱).

taining 0.1% TFA, and DBD-CA-amino acids generated by post-column desulfuration of DBD-TC-amino acids were detected fluorimetrically. DBD-TC-Asp and -Glu were separated as methyl esters of these DBD-TC-amino acids, since these methyl esters were more stable than DBD-TC-Asp and -Glu. DBD-TC-Cys was separated as S-␤-(4-pyridylethyl) cysteine derivative, since sequence analysis of peptides containing cysteine is usually carried out after derivatization with S-pyridylethylation [28,29]. The detection limits for DBD-CA-amino acids were 90 to 350 fmol (S/N=3). These results were comparable

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Fig. 5. Separation of DBD-TC-amino acids on the reversed phase HPLC. The amounts of each DBD-TC-amino acids were 200 pmol (Trp), 100 pmol (Cysa , Pro, Tyr), 80 pmol (Gly, His, Ser, Thr), 50 pmol (Asn, Aspb , Gln, Glub , Leu, Met, Val) and 40 pmol (Ala, Arg, Ile, Lys, Phe). a DBD-TC-Cys indicates S-␤-(4-pyridylethyl) cysteine derivative. b DBD-TC-Asp and -Glu indicate those of methyl ester. HPLC conditions were the same as those described in the Section 2.

to the system in which the desulfuration step was manually operated [24]. Further, the fluorescence intensities observed in the post-column desulfuration of DBD-TC-amino acids were consistent with those of DBD-CA-amino acids [24]. The differences of the fluorescence intensities among the derivatives might be dependent on the effect of the side-chains of amino acids. 3.4. Enantiomeric separation of DBD-TC-amino acids on chiral stationary phase (CSP) In the previous study, we reported that commercially available Pirkle type CSPs (SUMICHIRAL OA series) exhibited superior enantiomeric separation for amino acids derivatized with the benzofurazan fluorogenic reagent, 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), NBD-amino acids, with mobile phase of methanol containing citric acid [30,31]. We also reported that the carboxylic acid group in the analyte such as NBD-amino acids contributed to the enantiomeric separation on Pirkle type CSPs [30]. Therefore, we selected Pirkle type CSP for the chiral separation of DBD-TC-amino acids resulting from the sequencing of the peptide. We selected the CSP

of a 3,5-dinitrophenylamino carbonyl-l-valine covalently bonded to 3-aminopropylsilanized silica gel support (OA-3100), since larger separation factors α were observed for DBD-TC-amino acids than those on other OA columns such as OA-2500 and -4700 in our preliminary study (data not shown). The capacity factors k0 and the α values for DBD-TC-amino acids on OA-3100 are summarized in Table 1. The enantiomers of 19 DBD-TC-amino acids were separated with two types of mobile phase containing different concentration of citric acid though the separation of individual DBD-TC-amino acid enantiomers on OA-3100 was not successful. The fluorescence detection for DBD-CA-amino acids was more sensitive than that on reversed phase column, since DBD-CA-amino acids gave intense fluorescence in methanol compared with aqueous solution such as the mobile phase for the reversed phase column [24]. 3.5. Amino acid sequence and d/l-configuration determination of [d-Ala2 ]-Deltorphin II A manual sequencing procedure established as described earlier was applied to the amino acid sequence and d/l-configuration determination of

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Table 1 Capacity factors (k0 ) and separation factors (α) for DBD-TC-amino acids on chiral stationary phase Amino acids

k10

k20

Ile Val Leu Cysc Thr Tyr Ala Phe Trp Met Ser Glud Gln Asn Aspd Gly Pro Arg Lys His

d 1.49b d 1.55 d 1.69 d 2.02 d 2.05 d 2.28 d 2.51 d 2.56 d 2.75 d 2.78 d 2.81 d 3.12 d 3.27 d 3.29 d 3.90 4.08 l 8.31 d 2.19 d 2.40 d 6.23

l l l l l l l l l l l l l l l d l l l

α

Mobile phasea

1.59 1.75 1.88 2.34 2.93 2.45 3.00 2.81 2.93 3.10 3.79 3.42 3.87 4.02 4.50

1.07 1.13 1.11 1.16 1.43 1.07 1.20 1.10 1.07 1.12 1.35 1.10 1.18 1.22 1.15

8.62 2.57 2.84 8.09

1.04 1.17 1.18 1.30

A A A A A A A A A A A A A A A A A B B B

a Mobile phases were methanol containing 2.5 mM (A) or 0.025 mM (B) citric acid. b This is d-allo-Ile, which is of d-Ile at the ␤-carbon position. c DBD-TC-Cys indicates S-␤-(4-pyridylethyl) cysteine derivative. d DBD-TC-Asp and -Glu indicate those of methyl ester.

d-amino acid containing peptide, [d-Ala2 ]-Deltorphin II, which was a neuroactive peptide analogous to the dermorphin family [2] (Fig. 6). BF3 was used as cleavage/cyclization reagent, instead of TFA, to suppress the racemization as reported previously [14]. The liberated DBD-TZ-amino acids were further hydrolyzed to DBD-TC-amino acids as described previously. Since the individual DBD-TC-amino acid enantiomers have not been completely separated on the CSP-HPLC, the amino acid sequence and d/l-configuration were identified under the two HPLC conditions. An aliquot of the resulting DBD-TC-amino acids was first subjected to the RP-HPLC system for amino acid sequencing and the other part of the sample was subjected to CSP-HPLC system to separate the enantiomers. The DBD-TC-amino acids were converted to DBD-CA-amino acids by post-column desulfuration and detected fluorimetrically. The amino acid sequence was identified as Tyr-Ala-Phe-Glu-Val from

the chromatograms obtained on RP-HPLC system (Fig. 6A). The Ala residue (cycle 2) of the peptide was easily identified as d-form from the chromatogram obtained on CSP-HPLC system (Fig. 6B). The Glu residue (cycle 3) was detected as more stable methyl ester of the DBD-TC derivative. The sequence in the cycle 6 was identified as Val, whereas the identification of d/l-configuration of the cycle was difficult due to the co-elution of DBD-TC-Val and by-products with the mobile phase (data not shown). For the d/l-configuration determination, the partial racemization was observed: cycles 1 (racemization ratio 16.3%), 2 (11.6%), 3 (19.0%), 4 (18.5%) and 5 (24.8%). The racemization was thought to occur at the hydrolysis step because no or little racemization occur at the cleavage/cyclization step with BF3 [14], and the racemization at the hydrolysis step was observed in the sequence determination using PITC [15]. The variation of the racemization ratio for each amino acid observed in this method (Fig. 6B) was similar to that previously observed in the hydrolysis step in the sequence determination using PITC [16]. The method developed in this study was easily applicable to the identification of d/l-configuration of the residue completely converted to d-form in peptides such as dermorphin family [2], however, the further suppression of the racemization in sequencing procedure might be required to identify the racemization ratio of the partially racemized residue such as d-Asp in ␤-amyloid peptide[6]. The repetitive yield of the sequencing with TFA as cyclization/cleavage reagent for 2 nmol of ␤-casomorphin-7 (Tyr-Pro-Phe-Pro-Gly-Pro-Ile) was 84% as calculated by the fluorescence intensity of Pro2 and Pro6 , while the yield of the sequencing with BF3 was 65%. The reason for the lower repetitive yield with BF3 has not been cleared. The peak of DBD-CA-amino acids generated by the spontaneous desulfuration of a certain amount (ca. 15%) of DBD-TC-amino acids was observed as the peak distinguished from DBD-CA-amino acids derived from DBD-TC-amino acids. This spontaneous desulfuration was completely suppressed by using the hydrolysis solvent containing dithiothreitol and the removal of oxygen in the solvent by bubbling nitrogen through the solvent. Ilse et al. also reported that the PTC-amino acids were sensitive to oxidation, even to dissolved oxygen [32].

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Fig. 6. Amino acid sequence and d/l-configuration determination of [d-Ala2 ]-Deltorphin II (2 nmol), Tyr-d-Ala-Phe-Glu-Val-Val-Gly-amide. (A) Sequence determination on the reversed phase HPLC system; (B) d/l-configuration determination on the chiral stationary phase HPLC system. HPLC conditions were the same as those described in the Section 2.

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In comparison with the method in which the oxidative desulfuration step is manually operated, the advantages of the post-column desulfuration method are as follows. The sequence and d/l-configuration of a peptide were more easily identified, since the DBD-TC-amino acids derived from all amino acid residues were detected as a single peak without the decrease in the detection sensitivity compared with the manual desulfuration method. The application of the method to the convenient vapor phase sequencer can be easily achieved only by carrying out the hydrolysis step, instead of conversion step of TZ derivatives to TH derivatives, in the conversion flask of the automated sequencer, since the manual desulfuration step was omitted. In conclusion, we developed an amino acid sequence and d/l-configuration determination procedure easily applicable to the automated sequencer using the fluorescence Edman reagent, DBD-NCS, in which DBD-TC-amino acids generated by Edman sequencing procedure were separated on the reversed phase column or the Pirkle type CSP and then converted to DBD-CA-amino acids. This method will simplify the application of DBD-NCS to commercially available automated sequencer and give us the sensitive and simultaneous determination method for amino acid sequence and d/l-configuration determination of peptides and proteins.

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Acknowledgements We thank Dr. Chang-Kee Lim, MRC Toxicology Unit, University of Leicester, for his valuable suggestions and discussion. References [1] P.C. Montecucchi, R.D. Castiglione, S. Piani, L. Gozzini, V. Erspamer, Int. J. Pept. Protein Res. 17 (1981) 275. [2] V. Erspamer, P. Melchiorri, G. Falconieri-Erspamer, L. Negri, R. Corsi, C. Severini, D. Barra, M. Simmaco, G. Kreil, Proc. Natl. Acad. Sci. USA 86 (1989) 5188. [3] Y. Kamatani, H. Minakata, P.T.M. Kenny, T. Iwashita, K. Watanabe, K. Funase, X.P. Sun, A. Yongsiri, K.H. Kim, P. Novales-Li, E.T. Novales, C.G. Kanapi, H. Takeuchi, K. Nomoto, Biochem. Biophys. Res. Commun. 160 (1989) 1015. [4] N. Ohta, I. Kubota, T. Takao, Y. Shimonishi, Y. YasudaKamatani, H. Minakata, K. Nomoto, Y. Muneoka, M.

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