Applicability of a Modified Edman Procedure for Measurement of

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Applicability of a Modified Edman Procedure for Measurement of Protein Adducts: Mechanisms of Formation and Degradation of Phenylthiohydantoins Per Rydberg,† Bjo¨rn Lu¨ning,‡ Carl Axel Wachtmeister,† Lars Eriksson,§ and Margareta To¨rnqvist*,† Department of Environmental Chemistry, Department of Organic Chemistry, and Department of Structural Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden Received December 6, 2000

Adducts to N-terminal valine residues in hemoglobin (Hb) are used for monitoring in vivo doses of electrophiles and are quantitated by means of a modified Edman procedure, the “Nalkyl Edman procedure”. In the reaction with pentafluorophenyl isothiocyanate, N-alkylated valines cyclize and detach from the protein as pentafluorophenylthiohydantoins (PFPTHs) much more efficiently than do unsubstituted N-terminal valine residues. The mechanisms of this reaction, and of possible degradation reactions, have been studied with model compounds using phenyl- and pentafluorophenyl isothiocyanate. The rapid cyclization to N-alkylvaline-PTHs occurs as a consequence of the influence of substituents on ring formation. This facilitated cyclization favors a direct attack by the thiocarbamoyl nitrogen atom on valine-C-1, and is also observed to occur slowly at unsubstituted N-terminal valines. Such cyclization is favored in protic solvents. Under alkaline conditions and in the presence of air, hydrolytic and oxidative processes give rise to degradation products. The PTH derivatives of N-alkylvaline are less apt to undergo such reactions than are the corresponding derivatives of unsubstituted valine. We conclude that the presence of an N-substituent exerts a greater influence on the cyclization process than the structure of the amino acid or of the Edman reagent. For adducts of different structures, the method has broad applicability, for which the limits, however, are not yet explored. The knowledge from the studies is valid not only for the N-alkyl Edman procedure, but also, to some extent, for the classical Edman degradation reaction. The oxidative side reaction gave rise to the invention of a novel synthesis route for insertion of nucleophiles at carbon-5 in thiohydantoins. The present investigation provides a basis for the N-alkyl Edman procedure, facilitating new toxicological applications.

Introduction It has been demonstrated earlier that in vivo electrophilic compounds can be monitored by measuring the products (adducts) of their reaction with proteins, in particular hemoglobin (Hb) (1-5). Important nucleophilic sites in Hb which are reactive under physiological conditions are the imidazole nitrogen atoms in histidine residues, sulfur atoms in cysteine and methionine residues, oxygen atoms in carboxyl groups and in hydroxyl groups in tyrosine and serine residues, and the R-nitrogen atoms in the N-terminal valine residue of all four chains of human Hb (5, 6). In attempts to develop a simple and sensitive procedure for analysis of adducts to N-terminal valine residues in Hb, a number of methods were tested, including the Edman degradation procedure used for protein sequencing (7). It was observed that the N-terminal valine

N-alkylated with a radioactively labeled 2-hydroxyethyl moiety from ethylene oxide was released spontaneously as a phenylthiohydantoin (PTH)1 under the conditions (pH >7) employed for the coupling reaction between phenyl isothiocyanate (PITC) and protein (8). This PTH could be separated by extraction from unmodified Nterminal valine residues, as well as from the rest of the protein. This observation led to the development of the so-called “N-alkyl Edman” procedure for mass spectrometric (MS) quantitation of Hb adducts (9). To attain the required sensitivity and reproducibility, considerable development was necessary (9-11). Because of its usefulness, this method has been applied in a number of laboratories for research purposes, dose monitoring, and hygienic surveillance (12-17). The method has functioned well despite the incomplete knowledge concerning the mechanism of formation and

* Correspondence should be addressed to this author at the Department of Environmental Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden. Fax: +46-8-152561, e-mail: [email protected]. † Department of Environmental Chemistry. ‡ Department of Organic Chemistry. § Department of Structural Chemistry.

1 For abbreviations of the compounds studied, see Figure 2. Other abbreviations: ATZ, anilinothiazolinone; BCPS, bis-(4-chlorophenyl)sulfone; DDQ, 2,3-dichloro-5,6-dicyano-p-benzoquinone; 2,4-DNF, 2,4dinitrophenylhydrazine; EtOAc, ethyl acetate; MeOH, methanol; MITC, methyl isothiocyanate; PITC, phenyl isothiocyanate; PTH, phenylthiohydantoin; PFPITC, pentafluorophenyl isothiocyanate; PFPTH, pentafluorophenylthiohydantoin.

10.1021/tx000247+ CCC: $22.00 © 2002 American Chemical Society Published on Web 04/15/2002

Formation and Degradation of Phenylthiohydantoins

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Figure 1. The N-alkyl Edman reaction.

detachment of the valine derivative. Modifications subsequently introduced, e.g., alterations in the conditions employed for isolation and further derivatization of the detached valine derivative (18, 19), have not involved changes in the basic reaction parameters. A more detailed understanding of the underlying mechanism is required, above all for studies on previously unknown adducts (how widely applicable is this procedure?) as well as for its further development. Due to its sensitivity and the possibility of identifying adducts by mass spectrometric techniques, this method can also be utilized for the identification and quantitation of background carcinogens (20, 21), an area in which investigation of Hb adducts often has advantages over studying DNA adducts. Problems with artifacts in connection with studies of adducts from background carcinogens are minimized in the case of the N-alkyl Edman procedure, since modified valine cannot be incorporated during synthesis of the globin protein (22). The identification of genuine valine adducts in Hb is also facilitated, since the PFPTH derivatives contain this residue from the globin. A brief description of the N-alkyl Edman procedure is presented in Figure 1. A sample of the globin (isolated from red blood cells by acid precipitation) is dissolved in formamide, and pentafluorophenyl isothiocyanate (PFPITC) is then added, together with a small amount of aqueous 1 M NaOH in order to obtain a near-neutral solution. The mixture is maintained at room temperature overnight, after which the temperature is raised to 45 °C for a couple of hours (23). The pentafluorophenylthiohydantoin (PFPTH) derivatives of the terminal Nalkylvaline residues are released in high yield by this procedure and subsequently isolated by extraction. The main goal of the present study was to elucidate why the N-alkyl Edman procedure selectively leads to detachment of N-substituted valine residues (as PTHs/ PFPTHs) from the globin, whereas unmodified valine requires acidification to effectuate such detachment. This difference has also been observed at the amino acid level: N-substituted valines, e.g., N-alkyl-, -hydroxyalkyl-, and -phenylvalines, give rise directly to PTHs/ PFPTHs, whereas valine does not give rise to these cyclic products after the initial carbamoylation (24). It was thus assumed that the major explanation for the selectivity observed could not be the reagent (PITC or PFPITC) or the structure of the substituent or the leaving group (the protein residue), but that the determining factor is probably the presence of an N-substituent. Therefore, the present study has focused primarily on comparative studies of N-methylvaline and valine as models for N-substituted and unsubstituted valine residues, respectively, in Hb. Findings with these amino acids were also confirmed by limited studies on peptides. On the basis of our findings, a mechanism, described previously, is proposed to explain the general enhance-

ment of the rate of the cyclization resulting from Nsubstitution. During these studies, it was observed that prolonged reaction times could lead to partial degradation of the products formed, i.e., the N-alkylvaline-PTHs/PFPTHs and Val-PTH/PFPTH. The conditions leading to such degradation were investigated and the degradation products characterized. Relative degradation rates were determined under alkaline conditions, in the presence and absence of molecular oxygen. Since the studies were performed primarily on free amino acids (and their derivatives), which exhibit higher pKa values than the corresponding N-terminal residues, reaction media with a higher apparent recorded pH than formamide were used (10, 13). This paper deals with the chemistry involved in the N-alkyl Edman procedure. The increased knowledge regarding both formation and degradation of the products, the thiohydantoins, is required for application to a broadened range of adducts, including previously unknown ones.

Experimental Procedures Chemicals. Structures of compounds 1-131 and abbreviations of compounds 14-24 are given in Figure 2. 2,3-Dichloro5,6-dicyano-p-benzoquinone (DDQ, >98%) was obtained from Merck; 2,4-dinitrophenylhydrazine (2,4-DNF, ∼99%) and phenyl isothiocyanate (PITC, purum) were obtained from Fluka. Bis(4-chlorophenyl)-sulfone (BCPS,1 >99%) was obtained from Sigma-Aldrich. L-Valine (Val, 19), L-valinamide (ValNH2, 20), N-methyl-D,L-valine (MeVal, 21), sodium cyanoborohydride (NaBH3CN, >90%), and 5-isopropyl-3-phenyl-2-thiohydantoin (Val-PTH, 5) were obtained from Sigma. N-Methylvalylleucylanilide (MeValLeu-NHφ, >99%, 22) and valylleucylserine [ValLeuSer (H-Val-Leu-Ser-OH), 95%, 23) were obtained from Bachem (Bubendorf, Switzerland). 18O2 (96%) was obtained from ICON (Marion, NY). (2H3)Acetonitrile (99.8% 2H), (2H)chloroform (99.8% 2H), deuterium chloride (99.5% 2H, 20% in 2H2O), deuterium oxide (99.9% 2H, 2H2O), (2H8)2-propanol (99% 2H), and (2H4)methanol (99.8% 2H) were obtained from CIL (Andover, MA). 5-Isopropyl-1-methyl-3-phenyl-2-thiohydantoin (MeValPTH, 6), 5-isopropyl-3-pentafluorophenyl-2-thiohydantoin (ValPFPTH, 12), and 5-isopropyl-1-methyl-3-pentafluorophenyl-2thiohydantoin (MeVal-PFPTH, 13) were synthesized as described earlier (24). Phenylthiourea (14) and N-methyl-N′-phenylthiourea (15) were synthesized by reacting PITC with an excess of aqueous concentrated ammonia and methylamine, respectively, according to (25). All other chemicals and solvents were of analytical grade. Instrumentation, Methods for Analysis, and Characterization. 1H and 13C NMR spectra were recorded on a JEOL GSX 270 instrument at 270 MHz. All solvents used were fully deuterated; TMS was added as internal standard in chloroform, acetonitrile, and methanol. The alkaline hydrolysis experiments were performed in 0.5 M Na2HCO3 in 2H2O/(2H8)2-propanol [1:1 (v/v)] or in 0.5 M Na2CO3 in 2H2O/(2H8)2-propanol [1:1 (v/v)] at 45 or 30 °C using sodium 3-(trimethylsilyl)propanesulfonate as

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Figure 2. The phenylthiocarbamoyls (PTCs), phenylthiohydantoins (PTHs), and other chemicals studied. internal standard. The conditions for each experiment are described below. Methods and instrumentation for GC-MS [GLC-(EI, NCI, and PCI)-MS] were as described earlier (24). The LC-MS/thermospray (TS) system comprised a Finnigan TSQ 700 triple-stage quadrupole mass spectrometer from Finnigan (San Jose, CA), connected to a Finnigan Mat TSP2 vaporization unit. The LC system components were a Waters 600 multisolvent delivery system connected to a Supelcosil LC-18 column (26 × 250 mm), a Waters 486 MS tunable absorbance detector (λ ) 254 nm, 2H2 lamp), and a Waters 600 MS controller unit. Other parameters for the LC-MS system were as follows: evaporation temperature, 112 °C; ionization chamber temperature, 230 °C; flow rate ) 1 mL/min; eluent A ) 20% aqueous acetonitrile, 0.05 M NH4HCO3 buffered to pH 5 with acetic acid; eluent B ) acetonitrile, gradient ) 0-100% B in 15 min, then 100% B; loop, 20 µL; scan range, 110-350 m/z, 530 ms/scan. The positive-ion mode (PI) was employed. The LC-MS/electrospray (ESP) system comprised a Waters 2690 separation module interfaced with a Quatro-II triple-stage quadrupole mass spectrometer from Micromass. The MS was fitted with an atmospheric pressure ionization (API) source, which was used in the electrospray mode. Mobile phase: H2O/ methanol (MeOH) (1:1), isocratic flow at 10 µL/min. The ionsource temperature was 120 °C, the capillary voltage was 3.5 kV, and the cone voltage varied between 25 and 140 V. Nitrogen was used as drying gas at a flow rate of 250 L/h. Both the PI mode and the negative-ion mode (NI) were used. Purification of 24 was performed by HPLC, on a Shimadzu LC-4A connected with a Kromasil LC-18 column (250 × 10 mm) and a Shimadzu SPD-2AS detector (λ ) 214 nm, D2 lamp). Flow rate ) 2.5 mL/min, loop 0.7 mL eluted with 2% aqueous acetonitrile buffered with 0.02% TFA. The crystallographic investigations of 8 were carried out on a Siemens STOE/AED4 diffractometer equipped with a graphite monochromator. Thin plates were obtained by recrystallization from MeOH: space group P-1(2), a ) 6.87(1) Å, b ) 11.82(1) Å, c ) 9.057(3) Å, R ) 85.62(5)°, β•• ) 86.12(6)°, and γ ) 76.05(6)°. The thin single crystals yielded a total of 1148 reflections with 547 significant reflections with I g 2σ(I). The structure was solved by direct methods, and the final model with a total of

152 parameters was refined with full matrix least-squares methods. The wR2(all data) ) 0.2162 and R1(observed) ) 0.0876. These high R values are to a great extent dependent on the large number of weak reflections. Crystallographic data (excluding structure factors) for structure 8 in this paper have been deposited at the Cambridge Crystallographic Data Center as supplementary publication no. CCDC 137560. Copies of these data can be obtained, free of charge, on application to CCDC, 12 Union Rd., Cambridge CB2 1EZ,U.K.(fax: +441223336033ore-mail: [email protected]). Kinetic measurements under anaerobic alkaline conditions gave rate constants at 45 °C ((0.1 °C) under pseudo-first-order conditions in the thermostated cell compartment of a Hitachi U-3210 spectrophotometer. The rate of the hydrolysis reaction of 5 and 6 was calculated from the change of absorbance at 270 nm. Degradation studies under aerobic alkaline conditions gave more than one product, and kinetic measurements were therefore performed by HPLC, on a Shimadzu LC-4A as described above: Supelcosil LC-18 column (26 × 250 mm), detection (λ ) 254 nm), flow rate ) 0.7 mL/min, loop 100 µL (normally 10-50 µg/compound injected); eluents: A ) 20% aqueous acetonitrile, buffered at pH 6.9 with 0.02 M phosphate; B ) acetonitrile, gradient ) 0-100% B in 35 min, 100% B in 15 min. The degradation rates of 5, 6, 12, and 13 were obtained under pseudo-first-order conditions by comparing the absorbances of the compounds with the absorbances of the respective reference compound. TLC was performed using silica gel 60 f-254 plates (SiO2, Merck); spots were developed with UV, ninhydrin, and/or potassium permanganate. Melting points were determined on a Bu¨chi 535 instrument. Mikrokemi AB, Uppsala, Sweden, performed elementary analyses. Measurements of pH were carried out on an Orion EA 920 pH-meter equipped with a Ross 8130 glass electrode. Starting Materials: Synthesis of Sodium N-Phenylthiocarbamoyl-L-valinate (PTC-Val-Na, 1), N-Phenylthiocarbamoyl-L-valine (PTC-Val, 2), and N-Phenylthiocarbamoyl-L-valine Methyl Ester (PTC-Val-OMe, 3). Compound 1 was synthesized according to Edman (26). Synthesis of 2 and 3: To an ice-cold, stirred solution of 1 (100 mg, 0.36

Formation and Degradation of Phenylthiohydantoins mmol) in EtOAc (10 mL) was added 1 M aqueous NaHSO4 (5 mL). The organic phase was dried (Na2SO4) and divided into two equal parts. One aliquot was used to obtain NMR spectra of the acid 2, and the other aliquot was methylated with an etheral solution of CH2N2 to yield 3 for NMR analysis. No further characterizations were performed. 1: 1H NMR (in acetonitrile-2H3, 25 °C): δ 0.88, 0.95 [dd, 6H, J ) 7.6 Hz, CH3(γ,γ′)], 2.23 [m, 1H, J ) 4.7, 7.6 Hz, CH(β)], 4.70 [dd, 1H, J ) 4.7, 7.6 Hz, CH(R)], 7.01-7.62 (m, 5H, ar.), 8.27 [d, 1H, J ) 7.5, NH(Val)], 10.42 (br, 1H, N′H). 13C NMR (in acetonitrile2H , 25 °C): δ 19.50, 19.75 [CH (γ,γ′)], 32.48 [CH(β)], 65.45 [CH3 3 (R)], 118.20, 124.64, 129.14, 141.40 (ar.), 178.22 (CO), 181.75 (CS). 2: 1H NMR (in chloroform-2H, 25 °C): δ 0.92, 0.95 (dd, 6H, 6.7 Hz, CH3(γ,γ′)], 2.32 [m, 1H, 4.5, 6.7 Hz CH(β)], 5.14 [d+d, 1H, J ) 4.5, 8.7 Hz CH(R)], 6.52 [d, 1H, J ) 8.7 Hz, NH(Val)], 7.25-7.46 (m, 5H, ar.), 8.76 (s, 1H, N′H), 9.80 (br, 1H, COOH). 13C NMR (in chloroform-2H, 25 °C): δ 18.13, 18.65 [CH3(γ,γ′)], 31.02 [CH(β)], 62.38 [CH(R)], 127.33, 129.14, 130.18, 135.79 (ar.), 176.02 (CO), 180.38 (CS). 3: 1H NMR (in chloroform2H, 25 °C): δ 0.90, 0.93 (d+d, 3+3H, J ) 7.0 Hz, CH (γ,γ′)], 3 2.26 [m, 1H, J ) 4.9, 7.0 Hz, CH(β)], (s, 3H, CH3-O), 5.12 [d+d, 1H, J ) 4.9, 8.5 Hz, CH(R)], 6.63 [d, 1H, J ) 8.5 Hz, NH(Val)], 7.28-7.48 (m, 5H, ar.), 8.35 (s, 1H, NH). 13C NMR (chloroform2H, 25 °C): δ 18.33, 18.56 [CH (γ,γ′)], 31.25 [CH(β)], 52.20 3 (OCH3), 62.66 [CH(R)], 124.82, 127,26, 130.12, 135.91 (ar.), 176.34 (CO), 180.59 (CS). Synthesis of N-Methylvalylleucylserine (MeValLeuSer, 24). ValLeuSer (23, 101 mg, 0.302 mmol) was dissolved in water (8 mL), and 36% aqueous formaldehyde (22 µL, 0.29 mmol) was added with stirring at room temperature. After 15 min, NaBH3CN (21 mg, 0.30 mmol, calcd on 90% purity) was added, and 18 h later, the reduction was stopped by acidification to pH ∼2 with TFA (CAUTION!).2 Purification of the crude product was performed with semipreparative HPLC as described above. Four peaks were obtained and fractions collected, retention time [compound (yield)]: 15 min [23 (32.9 mg, 34%) obtained as a white solid], 16 min [unidentified product, partially coeluted with 23 (6.9 mg)], 20 min [24 (36.1 mg, 36%) obtained as a white solid], 26 min [N-(dimethyl)-ValLeuSer (36.2 mg, 35%) obtained as a white solid]. Analysis by LC-MS(ESI) m/z [(fragment, ion), rel int.]: compound 23: 318 [(M+H)+, 5%], 213 [(M-105)+, (MSer)+ 3%], 102 [(M-216, M-LeuSer)+, 14%], 72 [(M-246, H2NCHCHMe2)+, 9%)]. Compound 24: 332 [(M+1+), 37%], 227 [(M-105, M-Ser)+, 6%], 86 [(M-246, MeNCHCHMe2)+, 78%]. N-(Dimethyl)-ValLeuSer: 346 [(M+1)+, 11%], 100 [(M-246, Me2NCHCHMe2)+, 34%]. The identity of compound 24 was also confirmed by performing the N-alkyl Edman procedure with PITC as reagent (24). The product formed, MeVal-PTH [characterized by TLC (SiO2, Tol/EtOAc 2:1) and by GC-MS (PCI)], was pure (Val-PTH could not be detected).

Cyclization to Thiohydantoins Acid-Catalyzed Cyclization of PTC-Val (2) to Val-PTH (5). Concentrated aqueous 2HCl (50 µL, 600 µmol) was added to a solution of 1 (5.0 mg) in 1 mL of (2H4)methanol containing sodium 3-(trimethylsilyl)propanesulfonate as internal standard. After removal of precipitated NaCl, four IH NMR spectra of the solution were recorded; the first, immediately at 25 °C, the second and third, 10 and 20 min later, respectively. Finally, the temperature was raised to 55 °C, and the last spectrum was recorded 15 min later. The first spectrum is consistent with that of the free acid 2. The last spectrum is identical to that of 5 (24). No intermediate anilinothiazolinone (ATZ) could be detected during the progress of the cyclization reaction. Cyclization of N-Phenylthiocarbamoyl-L-valine Methyl Ester (PTC-Val-OMe, 3) at pH ∼8. Compound 3 (20 mg) was suspended in 0.5 M aqueous NaHCO3/1-propanol [2:1 (v/v), 5 mL]. After a few minutes at room temperature, the white 2 CAUTION! Acidification must be performed in a well-ventilated hood since HCN is released.

Chem. Res. Toxicol., Vol. 15, No. 4, 2002 573 precipitate formed was dissolved in EtOAc (2 × 5 mL). Evaporation gave a crystalline solid identified as 5 by 1H NMR and l3C NMR; the data obtained were in agreement with published spectra (24). Comparative Study of the Yields of 5, 6, 12, and 13 Obtained upon Reacting MeVal (21), MeValLeu-NHφ (22), MeValLeuSer (24), and ValLeuSer (23) with PITC and PFPITC in Aqueous 0.5 M NaHCO3/1-Propanol [2:1 (v/v)]. (A) Preparation of Stock Solutions. Stock solution A: to a solution of 23 (110 mg, 347 µmol) in water (2.2 mL) was added BCPS (440 µg, internal standard) dissolved in 1-propanol (1.0 mL). Stock solution S-24: compound 24 (4.3 mg, 13 µmol) was dissolved in water (0.86 mL, giving 5.0 mg/mL, 15 mM). Stock solution S-22: compound 22 (4.8 mg, 15 µmol) was dissolved in 1-propanol (0.96 mL, giving 5 mg/mL, 16 mM). Stock solution S-21: compound 21 (6.3 mg, 48 µmol) was dissolved in water (4.3 mL, giving 1.5 mg/mL, 11 mM). (B) Preparation of Samples, General Procedure. To six glass vials (2 mL) each containing 1.25 mL of 0.5 M aqueous NaHCO3/1-propanol [2:1 (v/v)] was added stock solution A (150 µL containing 5.0 mg, 16 µmol of 23, and 19 µg of BCPS) together with 100 µL of stock solution S-24 (double sample) and S-24 diluted 10 and 100 times with water (2+2 samples). This dilution series gave molar ratios between 24 and 23 of approximately 1/10, 1/100, and 1/1000 (see Table 1), respectively. The procedure described above was also applied for preparation of dilution series from stocks S-21 and S-22 (totally 6+6 samples, S-22 was diluted with 1-propanol). Two control samples were prepared from stock solution A and PITC or PFPITC. (C) Derivatization. The samples were preheated (46 °C) and divided into two equal groups; one group was reacted with PITC (20 µL, 17 µmol/sample) and the other with PFPITC (25 µL, 17 µmol/sample), all reactions being performed with stirring. After 70 min, the reactions were stopped by fast-chilling to 0 °C (ice bath), followed by extraction with toluene (2 × 1 mL). The samples were dried with Na2SO4, analyzed by GC-MS (PCI, as described above), and quantitated on the basis of standards [containing 5, 6, 12, and 13 at concentrations of 100, 20, 4, 0.8, and 0.16 µg/mL and BCPS (19 µg/mL in all standards)]. Synthesis and Cyclization of Potassium N-Methyl-N(phenylthiocarbamoyl)-D,L-valinate (PTC-MeVal-K, 4) at pH >7. A solution of MeVal (7.8 mg, 59 µmol) in 0.1 M aqueous KOH (590 µL) was evaporated to dryness, and the residue was dissolved in 2H2O (1 mL), evaporated to dryness, and suspended in acetonitrile-2H3 (1 mL). The suspension was again evaporated and suspended in acetonitrile-2H3 (1.5 mL). PITC (7 µL) was added and the suspension stirred on a vortex mixer (5 min) and filtered. An aliquot (0.7 mL) was directly used to obtain the 1H NMR spectrum of 4 at 37 °C (TMS as internal standard). 2H2O (0.2 mL) was added, and after another 10 min, the spectrum obtained was identical with that of MeVal-PTH (6) described earlier (24). Analysis of 4: 1H NMR (acetonitrile-2H3, 37 °C) δ 0.87, 0.95 [d+d, 3+3H, J ) 6.6 Hz, CH3(γ,γ′)], 2.43 [m, 1H, J ) 6.6, Hz, CH(β)], 3.37 (s, 3H, N-CH3), 3.55 [br 1H, CH(R)], 7.59 (1H, N′H), 7.00-7.58 (m, 5H, ar.).

Degradation and Oxidation of PTHs Alkaline Anaerobic Hydrolysis of Val-PTH (5) and MeVal-PTH (6). Samples were prepared by dissolving compounds 5 or 6 in 1-propanol (1 mg/mL), and these solutions were freshly used. Buffers used were A Sørensen Glycin:NaOH [0.100 M + 0.100 M, 1:1 (v/v); measured at pH 11.2 at 22 °C, giving pH 10.6 at 45 °C according to Clark (27)] and B Tris-Cl [0.100 M; measured at pH ) 8.3 at 22 °C, giving pH 7.7 at 45 °C according to Clark (27)]. Hydrolysis was started by adding aliquots (30 µL) of the sample solutions to the preheated buffers (3 mL, 45 °C, giving 10 µg/mL 5 and 6 in buffer solutions A and B containing 1% propanol) in sealed spectrometer cuvettes under argon. The half-times of the hydrolysis reactions were obtained from measurements of the UV absorption at 270 nm. Alkaline Aerobic Hydrolysis of Val-PTH (5) and MeValPTH (6) and LC-MS of Hydrolysis Products. Compound 5

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(5.0 mg) in 5 mL of 0.5 M aqueous NaHCO3/1-propanol [2:1 (v/ v)] was kept at room temperature with p-nitroanisole as internal reference. After 4 days, an aliquot (300 µL) was neutralized with 1 M HCl (50 µL), then diluted in eluent toluene/EtOAc [1.55 mL, 1:1 (v/v)], and analyzed by LC-MS. Analysis by LC-MS(TS) [(fragment) m/z (rel int.)]: phenylthiourea (14) eluted at 8.50 min [(M+H+) 153 (100), (M-33)+ 119 (26)]; PTC-Val (2) eluted at 11.02 min [M+ missing, (M-16)+ 236 (20), (M-93)+ 159 (100), (M-134)+ 118 (19)]; Val-PTH (5) eluted at 14.40 min [(M+H)+ 235 (100)]; p-nitroanisole eluted at 15.28 min; ValPTH-R-OPr (7) eluted at 17.51 min [(M+H)+ 293 (100), (M59)+ 233 (53), (M-168)+ 124 (20), (M-177)+ 115 (53)] (see below for more detailed analysis of 7). The retention times for 2, 5, and 14 were in agreement with reference compounds analyzed under identical conditions. MeVal-PTH (6, 5.0 mg) was hydrolyzed as above in 0.5 M Na2CO3/1-propanol [2:1 (v/v), 48 °C, 4 days]. An aliquot (300 µL) was analyzed as above. Analysis by LC-MS(TS) [(fragment) m/z (rel int.)]: N-methyl-N′-phenylurea (18) eluted at 9.43 min [151 (100)]; N-methyl-N′-phenylthiourea (15) eluted at 11.28 min [(M+H)+ 167 (100)]; p-nitroanisole eluted at 15.22 min. Alkaline Aerobic Hydrolysis and Relative Rates of Hydrolysis of Phenylthiohydantoins 5 and 6 and Pentafluorophenylthiohydantoins 12 and 13. Preparation of samples (general method): compound 5 (2 mg) and p-nitroanisole (5 mg) were dissolved in 1-propanol (1.0 mL) in a Pyrex tube (8 mL) with a screw cap. The tube was heated on a thermostat-controlled water bath at 45 °C and the hydrolysis started by the addition of a freshly prepared, preheated solution of 0.5 M aqueous NaHCO3/1-propanol [2:1 (v/v), 2.0 mL] and vigorous shaking. The following samples and references were used: Compound 6 (2.5 mg) with biphenyl (0.5 mg), 12 (2.5 mg) with biphenyl (0.5 mg), 13 (2.5 mg) with p-nitroanisole (5 mg). Analysis: Aliquots (100 µL) of the hydrolysis samples were dissolved in 20% aqueous acetonitrile, buffered to pH 6.9 with 0.02 M phosphate, and injected onto a Shimadzu HPLC. The half-lives (t1/2) were calculated from the pseudo-first-order rate constants (k), inferred from log concentration (log A) vs time plots. 1H NMR Studies of Alkaline Aerobic Hydrolysis of 5 and 6. General method: Val-PTH (5, 3 mg) was dissolved in 2-propanol-2H8 (350 µL); the hydrolysis was initiated by addition of either 0.5 M Na2CO3 in 2H2O (350 µL) or 0.5 M Na2HCO3 in 2H O (350 µL; 0.5 M Na2HCO in 2H O was prepared by addition 2 3 2 of 1 equiv of 2HCl in 2H2O to a solution of Na2CO3). The samples were then hydrolyzed in NMR tubes in a water bath at 45 °C. Samples of MeVal-PTH (6) were prepared as described above. The hydrolysis of 5 and 6 was studied under the following conditions: p2H ∼9, 30 °C (t ) 20 min and t ) 30 min); p2H ∼9, 45 °C (t ) 30 min and t ∼18 h); p2H 11, 45 °C (t ) 18 h and t ) 60 h). Preparative Alkaline Aerobic Hydrolysis of 5. A solution of 5 (0.98 g, 4.2 mmol) in 0.5 M aqueous NaHCO3/1-propanol [250 mL, 1:1 (v/v)] was stirred for 50 h at 47 °C. The solution was evaporated to dryness, and the residue was extracted with acetone (3 × 50 mL). Removal of acetone yielded a crystalline solid (618 mg) that was chromatographed on a silica column (40 × 220 mm) with heptane/EtOAc [1:1 (v/v)] as eluent. Compound 7 (38 mg) eluted first, followed by 5 (160 mg) and 14 (yield not determined). Compound 7 was further purified from a highly lipophilic impurity (revealed by HPLC) by extracting with heptane (15 mL) and repeated crystallizations from heptane/CHCl3 [6:1 (v/v)] to give pure 7 (18 mg, 60 µmol, 1.4%) as white crystals, mp 185.8-186.6 °C (100% purity on HPLC). Analysis: C15H19N2O2S: C 61.25%, H 6.55%, N 9.2%. Found: C 61.83, H 6.57, N 9.61. 1H NMR (chloroform-2H, 25 °C): δ 0.95 (t, 3H, J ) 7.4 Hz, CH2-CH3), 1.02, 1.10 [d+d, 3+3H, J ) 6.8, 7.0 Hz, CH3(γ,γ′)], 1.65 (m, 2H, J ) 7.0 Hz, -CH2CH3), 2.35 [m, 1H, J ) 6.9 Hz, CH(β)], 3.40, 3.55 (2 × m, 1+1H, J ) 6.9, 7.1 Hz, OCH2), 7.25-7.53 (m, 5H, ar.). 13C NMR (chloroform2H, 25 °C): δ 10.54 (CH CH ), 15.54, 16.05 [CH (γ,γ′)], 22.78 2 3 3 (CH2CH3) 34.81 [CH(β)], 66.16 (OCH2), 92.94 [CH(R)], 128.21,

Rydberg et al. 129.23, 129.45, 132.29 (ar.), 171.34 (CO), 183.66 (CS). UV (ethanol, r.t.) λmax ) 273.1 nm, 273.1 ) 14 500. Alkaline Oxidation of 5 by Oxygen Gas in MeOH. To a solution of 5 (50 mg, 0.21 mmol) in dry MeOH (10 mL) was added 0.5 mL of 0.5 M NaOMe (0.25 mmol) in MeOH. Oxidation was induced by a gentle stream of oxygen bubbles through the solution. The progress of the reaction was monitored by TLC [SiO2, toluene/EtOAc 1:1 (v/v)] and by iodometric titration of aliquots (10 × 0.3 mL) (with 10 mM thiosulfate). After 8 h, the remaining solution (7 mL) was diluted with diethyl ether (20 mL) and extracted with H2O (3 × 15 mL). Evaporation of the etheral phase gave a colorless syrup, which was then chromatographed on SiO2 [20 × 130 mm, heptane/EtOAc 1:1 (v/v)]. After fractionation and evaporation, the solid residue was recrystallized in CHCl3/heptane (1:6, v/v) to give 8 as white needles (31 mg, 0.12 mmol, calculated yield 80%). Analysis: 1H NMR (chloroform-2H, 25 °C): δ 1.02, 1.10 [d+d, 3+3H, J ) 7.0, CH3(γ,γ′)], 2.37 [q, 1H, J ) 7.0 Hz, CH(β)], 3.36 (s, 3H, OCH3), 7.25-7.53 (m, 5H, ar.). 13C NMR (chloroform-2H, 25 °C): δ 15.46, 16.14 [CH3(γ,γ′)], 34.60 [CH(β)], 52.08 (OCH3), 93.43 [CH(R)], 128.18, 129.24, 129.46, 132.29 (ar.), 170.96 (CO), 183.83 (CS). GC-MS(PCI): m/z 265 [(M+1)+, 100], 293 [(M+29)+, 38], 305 [(M+41)+, 13]. GC-MS(NCI): m/z 264 (M-, 9), 247 [(M-17)-, 4], 232 [(M-32)-, 100]; mp 152-153.5 °C. The structure of compound 8 was determined by X-ray diffraction. Alkaline Oxidation of 6 by Oxygen Gas in MeOH. Compound 6 (52 mg, 0.21 mmol) was oxidized by a slow stream of oxygen as described for 5 above. After 8 h, the remaining solution (70%) was diluted with H2O (14 mL) and extracted with diethyl ether (3 × 15 mL). The combined etheral phases were dried with Na2SO4 and further purified by preparative TLC [SiO2, toluene/EtOAc 1:1 (v/v)] to yield MeVal-PTH-R-OMe (11, 11 mg, 0.039 mmol, calculated yield 27%) and MeVal-PTH-ROH (10, 10 mg, 0.038 mmol, calculated yield 26%), both as colorless syrups. A small amount N-methyl-N′-phenylthiourea (15) was also obtained (1 mg, 0.006 mmol, calculated yield 4%). Analysis of 10: 1H NMR (chloroform-2H, 25 °C): δ 1.00, 1.20 [d+d, 3+3H, J ) 7.0, CH3(γ,γ′)], 2.37 [q, 1H, J ) 7.0 Hz, CH(β)], 3.28 (s, 3H, NCH3), 3.47 [s, 1H, OH(R)], 7.24-7.52 (m, 5H, ar.). 13C NMR (chloroform-2H, 25 °C): δ 15.25, 15.95 [CH (γ,γ′)], 3 29.25, 29.68 [CH(β), splitted 2.3:1 ratio], 34.49 (NCH3), 89.65 [CH(R)], 128.32, 129.10, 129.27, 132.72 (ar.), 172.07 (CO), 181.69 (CS). Analysis of 11: 1H NMR (chloroform-2H, 25 °C): δ 0.98, 1.18 [d+d, 3+3H, J ) 7.0, CH3(γ,γ′)], 2.36 [q, 1H, J ) 7.0 Hz, CH(β)], 3.24, 3.27 (s+s, 3H+3H, NCH3, OCH3), 7.2-7.6 (m, 5H, ar.). 13C NMR (chloroform-2H, 25 °C): δ 15.17, 15.95 [CH3(γ,γ′)], 28.95, 29.68 [CH(β), splitted 1.2:1 ratio], 34.49 (NCH3), 52.46 (OCH3) 95.03 [CH(R)], 128.35, 129.18, 129.47, 131.10 (ar.), 169.83 (CO), 182.50 (CS). Oxidation of 5 by DDQ. DDQ (98.0 mg, 4.31 mmol) was added to a solution of 5 (101 mg, 0.431 mol) and triethylamine (300 µL) in H2O/dioxane [10 mL, 1:1 (v/v)]. The solvent was evaporated after 2 h, and the solid residue was fractionated on a silica column [toluene/EtOAc 2:1 (v/v), TLC Rf ) 0.61] to give 9 (41 mg, 0.16 mmol, 37%) as a colorless syrup. Analysis: MS (EI, direct inlet): m/z 260 [M+, 24], 259 [(M-1)+, 98], 250 [(M10)+, 13], 217 [(M-43)+, 100], 216 [(M-44)+, 47], 135 [(M-125)+, 36]. GC-MS(EI): m/z 258 [(M-2)+, 8], 232 [(M-28)+, 100]. GCMS(PCI): m/z 260 [M+, 100], 288 [(M+28)+, 30], 300 [(M+40)+, 10]. GC-MS(NCI): m/z 258 [(M-2)-, 23], 232 [(M-28)-, 100].1H NMR (chloroform-2H, 25 °C): δ 1.14, 1.31 [d+d, 3+3H, J ) 7.0 Hz, CH3(γ,γ′)], 2.58 [sep, 1H, J ) 7.0 Hz, CH(β)], 7.24-7.65 (m, 5H, ar.). 13C NMR (chloroform-2H, 25 °C): δ 15,64, 16.99 [CH3(γ,γ′)], 36.45 [CH(β)], 64.04 [CH(β)], 113.28 [CN], 128.01, 129.50, 130.01, 131.66 (ar.), 166.44 (CO), 182.90 (CS). Compound 9 was also obtained as the main product of oxidation of 5 in acetonitrile with oxygen in the presence of KCN and triethylamine. It is therefore evident that DDQ acted not only as an oxidant but also as a source of cyanide ion. Oxygen Gas (18O2) Oxidation of 5 and 6 in MeOH with an Equimolar Amount of NaOMe. Val-PTH (10.3 mg, 44 µmol) and MeVal-PTH (10.9 mg, 44 µmol) were dissolved in dry



Table 1. Relative Yields of Edman Derivatives from N-Methylated Valine as the Free Amino Acid or as the N-Terminus of a Tripeptide

compound

added amount (nmol/sample)

MeValLeuSer MeValLeuSer MeValLeuSer MeValLeu-NHφ MeValLeu-NHφ MeValLeu-NHφ MeVal MeVal MeVal control (ValLeuSer)

1510 151 15.1 1560 156 15.6 1140 114 11.4 15800

[ValLeuSer]/ [compound] molar ratio 10.5 105 1050 10.1 101 1010 13.9 139 1390

relative yields (%) of MeVal-PTH (6)

MeVal-PFPTH (13)

Val-PTH (5)

Val-PFPTH (6)

ratio of yields of MeVal-PTH/Val-PTH or (MeVal-PFPTH/Val-PFPTH)

96 96 64 95 105 111 120 124 112 0.21a (33 nmol)

57 56 91 101 89 94 69 74 128 0.17b (27 nmol)

0.46 0.40 0.41 0.40 0.41 0.41 0.40 0.41 0.40 0.37

0.052 0.045 0.037 0.062 0.043 0.044 0.062 0.043 0.041 0.046

210 (1100) 230 (1200) 160 (2600) 240 (2500) 260 (2100) 300 (2100) 300 (1100) 300 (2200) 280 (3100) 270 (1700)

a The background formation of 6 was estimated to be 33 nmol, corresponding to 0.21% (based on the added amount of 23, assuming 100% yield). b The background formation of 13 was somewhat lower, i.e., 0.17%.

MeOH (2 mL) in two septum-equipped tubes (6 mL). The solutions were bubbled with argon and evacuated. A portion of 18O (4 mL, 160 µmol/tube) was added to the tubes, and then 2 0.5 M NaOMe in MeOH (0.1 mL, 50 µmol) was added. The tubes were shaken vigorously and then kept at ambient temperature for 18 h. Purifications were performed by preparative TLC as described above for compound 6. Compound 5 gave only one product (8) without incorporation of 18O, while compound 6 gave three products (10, 11, and 15). Analysis on LC-MS showed that only compound 10 had incorporated 18O (50%). Analysis of 10: LC-MS[ES, NI, 30 V (cone voltage)]: m/z 265 [18O, (M-1)-, 7], 263 [16O, (M-1)-, 11], 192 [18O, (M-74)-, 13], 190 [16O, (M74)-, 18], 130 [18O, (M-136)-, 71], 128 [16O, (M-136)-, 100]. LC-MS[EI, pos. mode, 30 V (cone voltage)]: m/z 267 [18O, (M+1)+, 39], 265 [16O, (M+1-), 42]. Analysis of 11: GC-MS(PCI): m/z 319 [(M+41)+, 8], 307 [(M+29)+, 37], 279 [(M+1)+, 100], 247 [(M-31)+, 64], 235 [(M-43)+, 11]. GC-MS(NCI): m/z 278 (M-, 1), 246 [(M-32)-, 100].

Results Cyclization to PTH and PFPTHs. To assess the influence of N-methylation of globin N-termini on the rate of thiohydantoin formation, amino acids and peptides were employed as models for normal and N-methylated proteins, derivatized under the originally used conditions developed for the N-alkyl Edman procedure. The yields of MeVal-PTH (6) and MeVal-PFPTH (13), formed from MeVal (21), MeValLeu-NHφ (22), and MeValLeuSer (24) upon reaction with PITC and PFPITC, were compared with the yields of Val-PTH (5) and Val-PFPTH (12) formed from ValLeuSer (23) under the same conditions. To determine the yields of analytes (5, 6, 12, and 13), duplicate dilution series for each N-methylated model compound (21, 22, and 24) were mixed with fixed amounts of 23 and an internal standard (see Table 1). The samples in one of these dilution series were reacted with PITC and the other with PFPITC in mildly alkaline aqueous 1-propanol at 47 °C. After these reactions, the samples were purified by extraction and quantitated employing GC-MS(PCI), utilizing the internal standard and reference compounds. Unfortunately, the commercial tripeptide (23) was found to contain an impurity corresponding to N-methylvaline in peptide or as amino acid resulting in unrealistically high yields of 6 and 13 in the most dilute samples. The presence of this impurity (estimated to be ∼0.21 mol %; probably formed from 24) has been corrected for in Table 1. In the evaluation of the yields of measured products, some problems were encountered: the samples containing the highest concentrations of 21, 22, and 24 (ap-

proximately 1500 nmol) gave values that were out of the linear range of the calibration curve, whereas the values for samples containing the lowest concentrations (around 15 nmol) were high because of impure 23. A byproduct from PFPITC which partly coeluted with BCPS, and gave rise to common, interfering ions, disturbed measurement of compounds 12 and 13. Despite these uncertainties (estimated to amount to a maximum error of approximately 30%), the data in Table 1 permit conclusions to be drawn concerning the influence of N-alkylation on the relative yields of the different thiohydantoins and concerning the differences between the phenyl and pentafluorophenyl reagents. There was a pronounced selectivity in the yields of 6 and 13 obtained from the N-methylated model compounds as compared to the low yields of 5 and 12 formed from 23. The ratio of the yields of 6/5 was approximately 250, while the corresponding ratio of 13/12 was even higher, around 2000. This selectivity was shown to be independent of whether the N-substituted valine was present as the free amino acid or as part of a valyl peptide. However, the used reagent, PFPITC, could explain the 10-fold lower yield of Val-PFPTH compared to Val-PTH, leading to an approximately 10 times higher ratio of 13/12 than of 6/5. The inherent effect of a substituent on the R-nitrogen atom on cyclization is also reflected in the equilibrium between the open (PTC) and ring-closed (PTH) forms of the amino acid in protic solvents. While the PTH of N-methylvaline (6) was formed rapidly without discernible intermediates, ValPTH was formed only slowly in an equilibrium that favors the open valinate form (PTC-Val anion) in alkaline solution and the cyclized PTH at low pH (see Figure 3). The free acid form of N-(phenylthiocarbamoyl)valine (PTC-Val, 2, see Figure 2) was sufficiently stable to permit methylation by diazomethane to produce the methyl ester (3, see Figure 2). This methyl ester (3) cyclized to Val-PTH (5) within minutes at room temperature in aqueous 1-propanol at pH 8. Attempts to prepare the corresponding N-methyl-N-(phenylthiocarbamoyl)valine (cf. 4) resulted in the PTH derivative (6). However, this compound could be prepared by reacting the potassium salt of N-methylvaline with PITC in an aprotic solvent (dry acetonitrile). The potassium N-methyl-N(phenylthiocarbamoyl)-D,L-valinate (PTC-MeVal-K, 4) formed was stable under aprotic conditions, although addition of a catalytic amount of water or alcohol induced rapid cyclization to MeVal-PTH (6).

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Figure 3. Formation of Val-PTH (5) and N-(R)-Val-PTH (e.g., 6).

Figure 4. Proposed mechanisms for alkaline hydrolysis and oxidation of Val-PTH (5) and MeVal-PTH (6).

Degradation of PTHs and PFPTHs (Alkaline Hydrolysis and Oxidation). When Val-PTH (5) was digested in aqueous 1% 1-propanol at pH 8 (buffer A) or pH 11 (buffer B) in the absence of air, PTC-Val anion (1) was the only product observed, with t1/2 values of 320 and 5.0 min, respectively (see alkaline hydrolysis in Figure 4). The PTC-Val anion (1) obtained was found to be very stable toward further hydrolysis to valine and aniline [t1/2 ∼ 8000 min (5.5 days) at pH 11] (as monitored by TLC and ninhydrin) and could be reconverted to Val-PTH (5) by acidification. Under the same conditions, MeVal-PTH (6) disappeared more slowly than 5 [t1/2 ∼ 6000 min (4.2 days) at pH 8 and 70.5 min at pH 11] with concomitant formation of N-methylvalinanilide (17, λmax ) 238 nm; see Figure 4). The end-products of alkaline anaerobic hydrolysis of 5 and 6 reflected the equilibria between the open PTCs (1 and 4) and the PTHs (5 and 6). As expected, compound 17 could not be reconverted to MeVal-PTH. If the alkaline solution of 5 in aqueous 1-propanol was exposed to air (see alkaline oxidative hydrolysis in Figure 4), appreciable amounts of phenylthiourea (14) and a

nonpolar component were present in addition to the anion of PTC-Val (1) (7, cf. below).Under similar conditions, MeVal-PTH (6) was slowly converted to N-methyl-N′phenylthiourea (15). N-Methyl-N′-phenylurea (18) was also obtained, through the oxidation of 15 by the hydrogen peroxide formed in the reaction (28). Upon determining the relative rates of hydrolysis under aerobic conditions (pH 8, 45 °C), the following t1/2 values and retention times in the HPLC system [t1/2 (min); retention time (min)] were obtained: 5 [460; 25], 6 [4500; 29], 12 [330; 34], and 13 [3900; 36]. As shown (Figure 4), the hydrophobic compound 7 (with a retention time of 32 min) was formed in connection with the hydrolysis of 5, and a corresponding Val-PFPTH compound was assumed to be formed from 12 (with a retention time 43 min; but the identity of this product was not verified). On the other hand, hydrolysis of 6 and 13 under these conditions did not result in any hydrophobic products. The NMR spectrum of 7 showed the presence of a propoxy group, the location of which could not be assigned. An oxidation product analogous to 7 could be


Figure 5. An ORTEP plot of molecule 8.

prepared in nearly quantitative yield by bubbling oxygen for a few hours at room temperature through a solution of Val-PTH (5) in dry MeOH containing an equimolar amount of sodium methoxide. This crystalline product was demonstrated by X-ray crystallography to be 5-isopropyl-5-methoxy-3-phenylthiohydantoin (Val-PTH-ROMe, 8 in Figure 5). Formation of peroxide was also detected during the reaction, whereas when oxygen was bubbled through the alkalized solvent alone, the peroxide test was negative. When MeVal-PTH (6) was oxidized in dry MeOH as described above, three products were obtained, viz., 11, 15, and, surprisingly, 10. Upon addition of water, 10 was converted to 15. When 18O2 was employed for the oxidation of 5 and 6 in MeOH/sodium methoxide, 18O was incorporated only into 10, in 50% yield. Production of urea derivatives requires the formation of 2-oxoisovaleric acid (16), which was indeed identified by reaction with 2,4-DNF to give the 2,4-dinitrophenylhydrazone [(characterized by LC-MS/NI: (M-1)-, m/z ) 295 (100% int.)]. When Val-PTH or Me-Val-PTH was digested for as long as 60 h in aqueous (2H8)2-propanol containing 0.25 M sodium deuterium carbonate or disodium carbonate, respectively, hydrolysis could be monitored by observing selected 1H resonances. Immediately after the addition of sodium deuterium carbonate, the β-hydrogen signal (2.30 ppm in 5 and 2.55 ppm in 6) was transformed from a multiplet into a well-defined septet, indicating removal of the R-proton. An additional weak septet indicating the presence of 5a and 6a (Figure 4) appeared rapidly at 3.05 ppm during the hydrolysis of 5 and at 3.65 ppm during the hydrolysis of 6. Both of these septets disappeared as the hydrolysis approached completion.

Discussion In the present paper, the driving forces involved in the N-alkyl Edman reaction were examined. An increased understanding of this reaction can be attained by considering the present experimental data with previously published mechanistic studies on the classical Edman degradation (7, 26) and Barrett’s modifications (29) as well as Davies and Mohammed’s investigations on racemization (30) and Blagoeva and Kirby’s work on hydantoins (31-33). The stability of the derivatives formed and side-reactions such as hydrolysis and oxidation will also be discussed. The Classical Edman Degradation Reaction. The classical Edman degradation approach to peptide sequencing (7, 26) consists of the following three reaction steps: Step 1: The coupling of a protein or peptide with PITC, in an aqueous buffer at pH >7. Step 2: A cleavage


step performed in anhydrous strong acid, with concomitant cyclization. The only atom acting as a nucleophile under such acidic conditions is the sulfur of the phenylthiocarbamoyl group, resulting in the release of a thermodynamically unfavorable anilinothiazolinone (ATZ1)(see Val-ATZ in Figure 6). Step 3: The ATZ is converted to the more stable phenylthiohydantoin (PTH) in aqueous acid. These reactions have been thoroughly studied by several groups (7, 30, 34-36) and their chemistry and applications reviewed in detail by Allan (37). Barett’s Modification of the Edman Degradation Reaction. In the modified procedure of Barrett (29), the coupling of PITC to the peptide is accomplished in the same manner as in the original method. After removal of excess PITC, the temperature is raised to 75 °C, which leads to cleavage of the first peptide bond with direct formation of the phenylthiohydantoin (PTH). This cleavage is strongly accelerated by the addition of an aqueous triethylammonium acetate buffer with a pH around 7. This buffer acts at the same time as a base and as a proton donor, both of which are necessary for bond cleavage. In this case, the anilino nitrogen of the thiocarbamoyl peptide must act as the nucleophile, in a manner similar to our proposal for the N-alkyl Edman reaction (cf. Figure 6, path b; see below). This route would also explain the phenomenon of “preview” in the classical Edman degradation reaction, well-known, but scarcely mentioned in the literature (38, 39). A strong tendency to cyclize at pH 8 is also exhibited by PTC-Val methyl ester (3; see Figure 6), in which the negative charge of the carboxyl group has been eliminated. This rapid cyclization of 3 implies that the anilino nitrogen atom actually functions as the nucleophile, giving rise directly to the PTH (path b in Figure 6). An alternative attack by sulfur would lead to a short-lived thiazolinone intermediate that would hydrolyze to yield the valinate anion (1, path a in Figure 6). This route is not very probable, because of the state of the equilibrium between the open form (1) and Val-PTH (5) under the mildly alkaline conditions of this reaction. The fact that 3 gives rise immediately to 5 in high yield implies that attack by the nitrogen atom is the predominant reaction mechanism in the case of carbamoylated N-substituted as well as unsubstituted valines and valyl peptides. The difference in the cyclization rates of N-substituted and normal valine derivatives is best explained by the gemdialkyl effect (discussed below), not by a change in the reaction mechanism. Cyclization to Hydantoins. Irrespective of the substituent present [an alkyl group from methyl to octyl, isopropyl, 2-hydroxyalkyls, a phenyl group, etc. (10, 24, 40)], N-substitution enhances cyclization to the N-substituted valine-PTH/PFPTH under the conditions employed in the N-alkyl Edman procedure. This effect is observed both with valyl peptides and with free amino acids (see Table 1). It is reasonable to assume that the rapid release of N-alkylated PTH under the mild conditions of the N-alkyl Edman procedure reflects acceleration of a reaction route which is normally minor. Kirby and Blagoeva found that alkyl-substituted hydantoic acids undergo cyclization as much as 105 times more rapidly (31) than do their unalkylated analogues (32). These investigators ascribe this acceleration to the Thorpe-Ingold effect (41), i.e., release of steric strain during ring formation.

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Figure 6. Formation of Val-PTH (5) directly from PTC-Val-OMe (3).

Kirby has also investigated the conditions under which the anilino nitrogen atom can act as a nucleophile to attack a negatively charged carboxylate anion, albeit with rather weak electrophilic character. The possible mechanisms underlying this reaction, in particular the sequence of proton transfer, are discussed by Blagoeva and Kirby in some detail (31). Finally, Blagoeva et al. consider stereo-electronic factors involved in the cyclization step. These studies by Blagoeva and co-workers differ from our considerations in two essential respects: (a) these other investigations deal with the formation and stability of hydantoins, not of thiohydantoins; and (b) they do not involve protein derivatives. With respect to (a), it is evident that a thiocarbamoyl nitrogen atom should be at least as good a nucleophile as the corresponding carbamoyl nitrogen, since sulfur is less electronegative than oxygen. Concerning (b), the effects of alkyl substituents on hydantoins and thiohydantoins on cyclization rates would in general not be expected to be dependent on the nature of the leaving groups. An alternative explanation put forward by Bruice and Pandit (42) is the “Reactive Rotamer” effect. This hypothesis states that alkylation should produce a decrease in the concentration of unfavorable rotamers, thereby increasing the rate of cyclization. Modern computational methods have shown, however, that the concentration of a reactive rotamer is a less important rate-determining factor than its enthalpy. The results collected and interpreted by Parill and Dolata (43) have been referred to as the “facilitated transition” hypothesis. The rate of the cyclization reaction can also be enhanced by using MITC1 instead of PITC. This can be attributed to the higher nucleophilicity of the attacking thiocarbamoyl nitrogen atom in the N-(methyl)thiocarbamoyl peptide in comparison to the corresponding phenyl compound. The facile cyclization then gives rise to a new free N-terminal residue susceptible to attack by MITC. It was observed that MITC partially degraded an unalkylated tripeptide (Val-Leu-Ser) during coupling, whereas PITC formed stable carbamoyl products under the same conditions.3 The cyclization of N-alkyl-N-(phenylthiocarbamoyl)valine, even when still attached to the rest of the protein, thus seems to be induced by a stereo-electronic effect favoring a reaction path that is also present in the normal Edman reaction procedure as well. In this reaction, the

small, but not negligible, nucleophilic character of the anilino nitrogen atom becomes decisive, leading directly to the product. In agreement with this proposed mechanism for cyclization, it has been observed that in connection with the application of the N-alkyl Edman procedure to globin, the formation of Val-PFPTH can be observed at a low rate [approximately 0.1% of the unsubstituted N-terminal valines are detached as PFPTH (9)]; at the same time, high yields (>50%) of PFPTHs are obtained from N-substituted valines (with an abundance of 1 in 106-108 termini) (8, 9). Irrespective of which amino acid forms the N-terminal, N-alkylation is expected to enhance the rate of cyclization to the corresponding N-alkyl-PTH derivatives after initial carbamoylation. This has been demonstrated to occur in the case of N-terminal N-methylglycine (44), as well as several other N-alkylated N-termini in our present study.4 In this context, it can be mentioned that isocyanates also form stable carbamoyl adducts to N-terminal valine in Hb. These adducts are cyclized and detached as hydantoins after acidification of the reaction mixture in a procedure similar to the classical Edman degradation (45), a procedure which has been employed in studies of exposure to isocyanates or their precursors (46-49). Solvent Effects. Both the polar protein and the more unpolar PITC/PFPITC Edman reagent must dissolve in the solvent utilized for the coupling reaction. The solvent systems that have been tested in this respect are mixtures of water and organic solvents such as 1-propanol, pyridine, THF, and dioxane (10). In the case of the modified Edman reaction, formamide is now used both in order to decrease products from side-reactions and because formamide is a good solvent for globin under the reaction conditions employed. Moreover, good yields can be obtained with formamide as the solvent (10). If the solvent is changed from a protic, polar solvent (e.g., propanol/water) to an aprotic solvent (e.g., acetonitrile, THF, or Me2SO), the carbamoylation step should be accelerated because the nitrogen atom is solvated to a lesser degree and, thus, more nucleophilic. However, proteins such as globin generally precipitate in aprotic solvents. On the other hand, the cyclization reaction itself 3 4

Rydberg, P., et al., unpublished findings. To¨rnqvist, M., unpublished findings.


will also be affected by the choice of solvent, since polar, protic solvents such as water have been reported to facilitate cyclization by providing more extensive solvation of the anion of the carboxyl group in the transition state (32). In the present studies with free amino acids, we observed this effect with PTC-MeVal anion (4). In the aqueous solvent system, cyclization of this compound to MeVal-PTH (6) was too rapid to be followed by chromatographic methods (24), whereas in dry acetonitrile 4 was stable until water or ethanol was added. Degradation and Oxidation of PTHs. Since weakly alkaline aqueous alcoholic media have been employed for the N-alkyl Edman reaction, particularly for preparation of standards from N-alkylvalines (9), it was of interest to determine the stability of PTHs in such media. Furthermore, in develomental work it has been shown that high pH during preparation and purification of PFPTHs leads to decreased yields.4 To study the stability of PTH derivatives, they were maintained at pH ∼8 or higher for prolonged periods of time. Both in the absence and in the presence of air, the relative rate of disappearance of N-methylvaline-PTH (MeVal-PTH, 6) was roughly 10-20 times lower than that of the corresponding derivative of unsubstituted valine (5), in agreement with the high rates of cyclization of the corresponding N-alkyl-N(phenylthiocarbamoyl)valinates. The pentafluorophenyl derivatives (12, 13) were only slightly less stable than the corresponding phenyl compounds (5, 6). The products formed during hydrolysis of 5 and 6 included the thiourea derivatives 14 and 15, respectively, formation of which must involve an oxidation step. In the case of degradation of Val-PTH (5), an additional nonpolar component containing an alkoxy group was also formed. When degradation of 5 was performed in aqueous MeOH, this alkoxy compound was identified by X-ray diffraction as 5-isopropyl-5-methoxy-3-phenyl-2-thiohydantoin (8). This compound could be obtained in almost quantitative yield by performing the oxidation with molecular oxygen in nonaqueous basic solution (dry MeOH). Various nucleophiles, e.g., cyanide ion, can be incorporated into the 5-position. When N-methylvalinePTH (6) was oxidized under nonaqueous basic conditions, an analogous 5-methoxy compound (11) was formed simultaneously with a hydroxy compound (10) which could be converted to the thiourea derivative (15) by water. Even under these nonaqueous conditions, a substantial amount of the thiourea derivative (15) was obtained. We propose a mechanism for the oxidation of Val-PTH (5) and MeVal-PTH (6), shown in Figure 4, which involves the addition of oxygen to the carbanion moiety R to the carbonyl group (50). After a proton shift, the hydrogen peroxide anion is eliminated to form a resonancestabilized imine or immonium ion (5a and 6a) that finally accepts a nucleophile. The CH signal in the 1H NMR spectrum of the isopropyl group in Val-PTH (5) was temporarily shifted from 2.3 to 3.1 ppm during the course of the reaction, indicating formation of an intermediate imine. Analogous to this, a weak CH signal was temporarily recorded at 3.6 ppm during the oxidation of MeValPTH (6), indicating a shift from its normal location (2.4 ppm). Incorporation of 18O into 10 can be explained on the basis of the formation of a charged intermediate (6a in Figure 4) caused by the N-methyl group of 6. Retarded loss of a hydrogen peroxide anion from the ring would


favor a cleavage of the oxygen-oxygen bond. An alternative explanation might be incorporation of the 18OHarising from homolytic cleavage of the hydrogen peroxide anion. This proposed mechanism, initiated by the formation of an R-carbanion, is supported by the finding that the R-proton in both Val-PTH and MeVal-PTH exchanges with the solvent in slightly alkaline medium. According to Davies and Mohammed (30), such exchange is accompanied by complete racemization of the R-carbon center. To determine whether other oxidants can replace oxygen, DDQ1 was tested. In the presence of this compound under nonaqueous conditions, the cyano compound (9) was formed from 5. This formation of 9 indicates that the cyanide ion produced by degradation of DDQ can execute a nucleophilic attack on the imine (5a, Figure 4) formed during the oxidation. This was verified by bubbling oxygen through a solution of Val-PTH (5) in acetonitrile in the presence of potassium cyanide and triethylamine. Under these conditions, 9 was obtained in high yield. The much more rapid degradation of Val-PTH than of N-alkyl-Val-PTH under the conditions employed for the N-alkyl Edman reaction examined here is in favor of the N-alkyl compounds, although prolonged reaction times, in general, should be avoided. Our results indicate that, to obtain optimal results, the reaction should preferably be performed under anaerobic conditions.

Conclusions The occurrence and levels of several types of electrophilic compounds/intermediates in vivo have been examined by the N-alkyl Edman procedure, for specific detachment and analysis of adducts to N-terminal valines in Hb. In the present study, insight into the mechanism of the key step, i.e., the favored detachment, through cyclization, to Edman derivatives (PTHs and PFPTHs) of N-alkylvalines, and also mechanisms involved in degradation of the desired products were studied. On the basis of our study, combined with results from other studies, some general conclusions could be drawn: • The general enhancement of the rate of cyclization/ cleavage observed for N-substituted valines in comparison to unsubstituted valine is related to the gem-dialkyl effect. As a result of this effect, the rate of cyclization/ cleavage depends mainly on the presence or absence of an N-substituent, less on the nature of the N-substituent, the amino acid, and the isothiocyanate reagent employed. However, the rate of the cyclization/cleavage reaction for unsubstituted valyl peptides is in the order MITC > PITC > PFPITC. This phenomenon probably reflects the higher nucleophilicity of the methylthiocarbamoyl nitrogen atom in comparison to the same atom in reagents containing electron-withdrawing phenyl and pentafluorophenyl groups. • A predicted limitation of the N-alkyl Edman procedure is that coupling/cyclization is prevented by blocking the terminal nitrogen atom from being thiocarbamoylated, e.g., when this nitrogen atom is tertiary or when it is substituted with acyl groups (known blocking agents). • Protic, polar solvents (e.g., H2O, alcohols, and formamide) facilitate cyclization, while aprotic solvents reduce the rate of this reaction.

580


• Under the conditions employed here, the Edman derivatives formed are racemic. • A high pH and the presence of oxygen, especially for longer periods of time, lead to degradation of the Edman derivatives both of the model N-alkylvaline used in these studies, i.e., N-methylvaline, and of unsubstituted valine. However, the Edman derivatives of N-methylvaline were found to be degraded 10-20-fold more slowly than the corresponding derivative of unsubstituted valine. With respect to various physicochemical and chemical factors that affect the yield of analytes and their analytical properties, the protocol for the N-alkyl Edman procedure (9, 23) results in high yields of derivatives formed from low molecular weight adducts with a low level of undesirable products, e.g., little background formation of unalkylated valine hydantoins. In practice, when the N-alkyl Edman procedure is used for the identification of adducts, the possible limitations with regard to applicability (see above) as well as the stability and potential degradation of adducts must be considered. In real-life applications, particularly when the levels of the adducts being examined are low, it becomes important to avoid the formation of byproducts, e.g., due to oxidation or hydrolysis. From these points of view, the enhanced stability of N-alkylvaline-PTH compared to Val-PTH is advantageous and no doubt contributes to the usefulness of the N-alkyl Edman procedure. The conclusions drawn from the mechanistic studies presented in this paper provide a basis for further development of the N-alkyl Edman procedure and its applications to new areas of toxicology.

Acknowledgment. We owe thanks to Dr. Roland Stenutz for valuable discussions and for performing the NMR measurements; to Lena Liedgren for NMR measurements; to Anna Malmva¨rn, B. Sc., for skillful laboratory work; to Prof. Lars Ehrenberg for valuable discussions; and to Dr. Hans Helleberg, Ioannis Athanasiadis, Johanna Minten, B. Sc., and Dr. Thomas Alsberg for their skillful mass-spectrometric analyses. This work was supported financially by the Stockholm University Center for Research on Natural Resources and the Environment, the Swedish Natural Science Research Council, the Swedish Environmental Protection Agency, The Foundation for Strategic Environmental Research, and the Swedish Council for Working Life and Social Research.

References (1) Ehrenberg, L., Hiesche, K. D., Osterman-Golkar, S., and Wennberg, I. (1974) Evaluation of genetic risks of alkylating agents: Tissue doses in the mouse from air contaminated with ethylene oxide. Mutat. Res. 24, 83-103. (2) Osterman-Golkar, S., Ehrenberg, L., Segerba¨ck, D., and Ha¨llstro¨m, I. (1976) Evaluation of genetic risks of alkylating agents. II. Haemoglobin as a dose monitor. Mutat. Res. 34, 1-10. (3) Neumann, H. G., Baur, H., and Wirsing, R. (1980) Dose-response relationship in the primary lesion of strong electrophilic carcinogens. Arch. Toxicol. Suppl. 3, 69-77. (4) Skipper, P. L., and Tannenbaum, S. R. (1990) Protein adducts in the molecular dosimetry of chemical carcinogens. Carcinogenesis 11, 507-518. (5) To¨rnqvist, M., Fred, C., Haglund, J., Helleberg, H., Paulsson, B., and Rydberg, P. (2002) Protein adducts: quantitative and qualitative aspects of formation, analysis and application. J. Chromatogr. (in press). (6) Osterman-Golkar, S., and Ehrenberg, L. (1980) Alkylation of macromolecules for detecting mutagenic agents. Teratog., Carcinog., Mutagen. 1, 105-127.

Rydberg et al. (7) Edman, P., and Henschen, A. (1975) Sequence determination. In Protein Sequence Determination (Needleman, S. B., Ed.) pp 232279, Springer-Verlag, Berlin. (8) Jensen, S., To¨rnqvist, M., and Ehrenberg, L. (1984) Hemoglobin as a dose monitor of alkylating agents: Determination of alkylation products of N-terminal valine. In Individual Susceptibility to Genotoxic Agents in the Human Population (de Serres, F. J., and Pero, R. W., Eds.) pp 315-320, Plenum Press, New York. (9) To¨rnqvist, M., Mowrer, J., Jensen, S., and Ehrenberg, L. (1986) Monitoring of environmental cancer initiators through hemoglobin adducts by a modified Edman method. Anal. Biochem. 154, 255266. (10) To¨rnqvist, M. (1989) Monitoring and cancer risk assessment of carcinogens, particularly alkenes in urban air. Thesis, Department of Radiobiology, Stockholm University, Stockholm. (11) To¨rnqvist, M. (1996) Monitoring of human exposure to xenobiotics; Identification and quantification of cancer initiators in vivo. In Mass Spectrometry in Biomolecular Sciences (Caprioli, R. M., Malorni, A., and Sindona, G., Eds.) pp 381-395, Kluwer Academic Publishers, Dordrecht. (12) Tates, A. D., Grummt, T., To¨rnqvist, M., Farmer, P. B., van Dam, F. J., van Mossel, H., Schoemaker, H. M., Osterman-Golkar, S., Uebel, C., Tang, Y. S., Zwinderman, A. H., Natarajan, A. T., and Ehrenberg, L. (1991) Biological and chemical monitoring of occupational exposure to ethylene oxide. Mutat. Res. 250, 483497. (13) To¨rnqvist, M., Magnusson, A. L., Farmer, P. B., Tang, Y. S., Jeffery, A. M., Wazneh, L., Beulink, G. D. T., van der Waal, H., and van Sittert, N. J. (1992) Ring test for low-levels of N-(2hydroxyethyl)valine in human hemoglobin. Anal. Biochem. 203, 357-360. (14) Bergmark, E., Calleman, C. J., He, F., and Costa, L. G. (1993) Hemoglobin adducts in humans occupationally exposed to acrylamide. Toxicol. Appl. Pharmacol. 120, 45-54. (15) Fidder, A., Noort, D., de Jong, A. L., Trap, H. C., de Jong, L. P., and Benschop, H. P. (1996) Monitoring of in vitro and in vivo exposure to sulfur mustard by GC/MS determination of the N-terminal valine adduct in Hb after a modified Edman degradation. Chem. Res. Toxicol. 9, 788-792. (16) Thier, R., Lewalter, J., Kempkes, M., Selinski, S., Bruning, T., and Bolt, H. M. (1999) Haemoglobin adducts of acrylonitrile and ethylene oxide in acrylonitrile workers, dependent on polymorphisms of the glutathione transferases GSTT1 and GSTM1. Occup. Environ. Med. 56, 197-202. (17) Begemann, P., Sram, R. J., and Neumann, H. G. (2001) Hemoglobin adducts of epoxybutene in workers occupationally exposed to 1,3-butadiene. Arch. Toxicol. 74, 680-687. (18) Passingham, B. J., Farmer, P. B., Bailey, E., Brooks, A. G. F., and Yates, D. W. (1988) 2-Hydroxyethylation of haemoglobin in man. In Methods for Detecting DNA Damaging Agents in Humans: Applications in Cancer Epidemiology and Prevention (Bartsch, H., Hemminki, K., and O’Neill, I. K., Eds.) pp 279285, IARC, Lyon, France. (19) Hindsø Landin, H., Osterman-Golkar, S., Zorcec, V., and To¨rnqvist, M. (1996) Biomonitoring of epichlorohydrin by hemoglobin adducts. Anal. Biochem. 240, 1-6. (20) To¨rnqvist, M., and Kautiainen, A. (1993) Adducted proteins for identification of endogenous electrophiles. Environ. Health Perspect. 99, 39-44. (21) Tareke, E., Rydberg, P., Karlsson, P., Eriksson, S., and To¨rnqvist, M. (2000) Acrylamide: A cooking carcinogen? Chem. Res. Toxicol. 13, 517-522. (22) Kautiainen, A., Osterman-Golkar, S., and Ehrenberg, L. (1986) Misincorporation of alkylated amino acids into hemoglobinsa possible source of background alkylations. Acta Chem. Scand. B 40, 453-456. (23) To¨rnqvist, M. (1994) Epoxide adducts to N-terminal valine of hemoglobin. Methods Enzymol. 237, 650-657. (24) Rydberg, P., Lu¨ning, B., Wachtmeister, C. A., and To¨rnqvist, M. (1993) Synthesis and characterization of N-substituted valines and their phenyl-thiohydantoins and pentafluorophenyl-thiohydantoins. Acta Chem. Scand. 47, 813-817. (25) Weith, W. (1875) CHBEAM Chem. Ber. 8, 1524. (26) Edman, P. (1950) Methods for determination of the amino acid sequence in peptides. Acta Chem. Scand. 4, 283-298. (27) Clark, W. M. (1923) in The determination of hydrogen ions, Williams & Wilkins, Baltimore. (28) Hepner, B., and Frenkenberg, H. (1932) J. Prakt. Chem. 134, 249-256. (29) Barrett, G. C., and Penglis, A. A. E. (1985) Edman stepwise degradation of polypeptides: A new strategy employing mild basic cleavage conditions. Tetrahedron Lett. 26, 4375-4378.

Formation and Degradation of Phenylthiohydantoins (30) Davies, J. S., and Mohammed, K. A. (1984) Chiral analysis of the reaction stages in the Edman method for sequencing peptides. J. Chem. Soc., Perkin Trans. 2, 1723-1727. (31) Blagoeva, I. B., Pojarlieff, I. G., and Kirby, A. J. (1984) Intramolecular nucleophilic attack on carboxylate by ureide anion. General acid-base catalysis of the alkaline cyclisation of 2,2,3,3tetramethylhydantoic acid. J. Chem. Soc., Perkin Trans. 2, 745751. (32) Blagoeva, I. B., Pojarlieff, I. G., and Taschev, D. T. (1989) Intramolecular nucleophilic attack by urea nitrogen. Reactivityselectivity relationships for the general acid-base-catalyzed cyclisations of ureido acids and esters. J. Chem. Soc., Perkin Trans. 2, 347-353. (33) Blagoeva, I. B., Kirby, A. J., Koedjikov, A. H., and Pojarlieff, I. G. (1999) Multiple changes in rate-determining step in the acidand base-catalyzed cyclizations of ethyl N-(p-nitrophenyl)hydantoates caused by methyl substitution. Can. J. Chem. 77, 849859. (34) Bethell, D., Metcalfe, G. E., and Sheppard, R. C. (1965) Kinetics and mechanism of the Edman reaction. Chem. Commun. 10, 189190. (35) Tarr, G. E. (1977) Improved manual sequencing methods. Methods Enzymol. 67, 335-357. (36) Toriba, A., Adzuma, K., Santa, T., and Imai, K. (2000) Development of an amino acid sequence and D/L configuration determination method of peptide with a new fluorescence Edman reagent, 7-methylthio-4-(2,1,3-benzoxadiazolyl) isothiocyanate. Anal. Chem. 72, 732-739. (37) Allan, G. (1981) Determination of peptide sequences by the Edman degradation. In Sequencing of Proteins and Peptides (Work, T. S., and Burdon, R. H., Eds.) pp 174-228, Elsevier Science Publishers B.V., Amsterdam. (38) Blomba¨ck, B., Blomba¨ck, M., Hessel, B., and Iwanaga, S. (1967) Structure of N-terminal fragments of fibrinogen and specificity of thrombin. Nature 215, 1445-1448. (39) Sundby, F. (1972) The amino acid sequence of human glucagon. FEBS Lett. 21, 315-319. (40) Eide, I., Hagemann, R., Zahlsen, K., Tareke, E., To¨rnqvist, M., Kumar, R., Vodicka, P., and Hemminki, K. (1995) Uptake, distribution, and formation of hemoglobin and DNA adducts after inhalation of C2-C8 1-alkenes (olefins) in the rat. Carcinogenesis 16, 1603-1609.

Chem. Res. Toxicol., Vol. 15, No. 4, 2002 581 (41) Beesley, R. M., Ingold, C. K., and Thorpe, J. F. (1915) The formation and stability of spiro-compounds. Part I. Spirocompounds from cyclohexane. J. Chem. Soc. 107, 10801107. (42) Bruice, T. C., and Pandit, U. K. (1960) The effect of geminal substitution ring size and rotamer distribution on the intramolecular nucleophilic catalysis of the hydrolysis of monophenyl esters of dibasic acids and the solvolysis of the intermediate anhydrides. J. Am. Chem. Soc. 82, 5858-5865. (43) Parill, A. L., and Dolata, D. P. (1996) The “facilitated transition” hypothesis as an explanation for the gem-dialkyl effect. J. Mol. Struct. (THEOCHEM) 370, 187-202. (44) Chang, J. Y. (1978) A novel Edman-type degradation: Direct formation of the thiohydantoin ring in alkaline solution by reaction of Edman-type reagent with N-monomethyl amino acids. FEBS Lett. 91, 63-68. (45) Abderhalden, E., and Brockmann, H. (1930) The determination of the constitution of proteines and polypeptides. Biochem. Z. 225, 386-408. (46) Sriramachari, S., and Chandra, H. (1997) The lessons of Bhopal (toxic) MIC gas disaster scope for expanding global biomonitoring and environmental specimen banking. Chemosphere 34, 22372250. (47) Angerer, J., Goen, T., Kra¨mer, A., and Ka¨fferlein, H. U. (1998) N-methylolcarbamoyl adducts at the N-terminal valine of globin in workers exposed to N,N-dimethylformamide. Arch. Toxicol. 72, 309-313. (48) Mra´z, J., and Bouskova´, S. (1999) 2,4-Toluenediisocyanate and hexamethylenediisocyanate adducts with blood proteins: assessment of reactivity of amino acid residues in vitro. Chem.-Biol. Interact. 117, 173-186. (49) Sabbioni, G., Hartley, R., Henschler, D., Ho¨llrigl-Rosta, A., Koeber, R., and Schneider, S. (2000) Isocyanate-specific hemoglobin adduct in rats exposed to 4,4′-methylenediphenyl diisocyanate. Chem. Res. Toxicol. 13, 82-89. (50) Gersmann, H. R., and Bickel, A. F. (1971) Autoxidation of ketones and esters in basic solution. J. Chem. Soc. (B) 11, 2230-2237.

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Applicability of a Modified Edman Procedure for Measurement of

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