Anal. Chem. 1982, 5 4 , 213-216
213
Comparison of the Alkylation of Nicotinamide and 4-(p -Nitrobenzyl)pyridine for the Determination of Aliphatic Epoxides Hans J. C. F. Nells,' Subhash C. Alry, and J. E. Sinshelmer" College of Pharmacy, The Unlversiv of Michigan, Ann Arbor, Michigan 48 109
The alkylatlon of nlcotlnamlde by a serles of ten propylene oxldes wHh subsequent formation of a chromophore In a new procedure Is compared to the conventlonal 4-(p -nitrobenzyl)pyrldlne (NBP) test. Both reactlons exhlblted slmllar rates of alkylatlon, competlng rates of solvolysls and correlatlon of the extent of alkylatlon to the Taft u* values of substltuent groups. The nlcotlnamlde procedure has the advantage that the lnltlal alkylatlon can be run under more physlologlcal condltlons and that there Is an Increase In the stablllty of the flnal chromophore.
Alkylating agents are generally associated with deleterious biological effects. For example, nucleic acid alkylation is well documented and is an important consideration in chemical mutagenesis and carcinogenesis ( I ) . A standard in vitro procedure for comparing alkylating agents of bionucleophiles is based upon their reaction with 4-(p-nitrobenzyl)pyridine (NBP) and subsequent spectrophotometric measurement of the color generated in alkaline medium (2-7). We recently developed a fluorometric procedure for the determination of epoxides based upon their alkylation of nicotinamide (8). It is the purpose of this paper to describe an extention of that procedure which permits a spectrophotometric determination of the rate of alkylation of nicotinamide and to compare the new procedure to the standard NI3P method. Our continued interest in the structure-mutagenicity relationship of aliphatic epoxides (9) as well as the reports of Hemminki and co-workers ( I 0 , I I ) as to correlation of the mutagenicity of aliphatic epoxides to NBP reactivity led us to a reinvestigation of the alkylation reaction on an extended series of propylene oxide derivatives. This same series of ten epoxides also serves as the basis of our comparison of nicotinamide and NBP alkylation. EXPERIMENTAL SECTION Reagents. Nicotinamide was obtained from Calbiochem (Los Angeles, CA) while 4-(p-nitrobenzyl)pyridine,propylene oxide, epifluorohydrin,epibromohydrin, epichlorohydrin,glycidol, and 3,3,3-trichloropropyleneoxide were obtained from the Aldrich Chemical Co. (Milwaukee,WI). All epoxides except epifluorohydrin were redistilled under reduced pressure (6mm) before use. Epibromohydrin was further purified by passing it through a silica column with dichloromethane. Epiiodohydrin (1,2-epoxy-3-iodopropane) was prepared from epibromohydrin (12). 1,2-Epoxy-3-nitratopropane, 1,2-epoxy-3-thiocyanopropane, and 1,2-epoxy-3-nitropropanewere synthesized according to literature procedures (12-14). Reactions involved nucleophilic displacement of either the bromo or iodo group in epibromohydrin or epiiodohydrin by the appropriate anion, i.e., nitrate, thiocyanate, or nitrite. Final products were purified by distillation under vacuum. Semipreparative HPLC employing a silica column (Partisil-M9, Whatman, Clifton, NJ) was also used for the iodo
Present address: Laboratoria voor Medische Biochemie en Klinische Analyse, R.U.G., De Pintelaan 135,B-9000 Gent, Belgium. 0003-2700/82/0354-0213$01.25/0
and thiocyano compounds with mixtures of dichloromethane and pentane (7:3) and (82) as the eluent, respectively. Purity of the samples was checked in two TLC systems on silica GF plates (Uniplate,Analtech, Newark, DE) with dichloromethane or ethyl acetate. Spots were visualized under short-wavelengthUV light and by spraying with NBP (6). Estimated purity of all samples was >99%. Alkylation of Nicotinamide by Aliphatic Epoxides. In our nicotinamide alkylation approach, 700 pL of a 0.1 M phosphate buffer, pH 7.4, containing 7.1% of ethanol, 200 pL of a nicotinamide solution (250 fimol/mL in the same buffer), and 100 fiL of an ethandic epoxide solution (10 pmol/mL) were combined in a test tube. After incubation at 60 "C, tubes were immersed in dry ice-acetone to stop the reaction. For color development, 0.5 mL of acetone, 0.2 mL of 6 M aqueous potassium hydroxide, and, after 5 min, 2 mL of water were added. The absorbance of the solution was read 11 min after the addition of the water at 358 nm. Synthesis of N-Alkylnicotinamides. Three alkylated nicotinamideswere prepared as reference compounds by using excem epoxide in a modification of a procedure described for ethylene oxide by Windmueller et al. (15). (i) Reaction with Propylene Oxide. Nicotinamide (40 mmol) was dissolved in 40 mL of water and 160 mmol of the epoxide in 25 mL of ethanol was added. The pH of the reaction was kept between 6 and 10 by the addition of 1.0 M HC1 while the reaction mixture after 15 min at room temperature was gradually heated to 80 OC. The generationof base became slow after a totalreaction time of about 1h. Then the solution was decolorized with charcoal and concentrated under reduced pressure to yield a viscous residue. A quantity of 80 mL of ethanol-acetone (53) was added and the3mixture kept at 4 "C for 12 h. The white precipitate was filtered and recrystallized twice from aqueous ethanol to yield 22% of the final product. The reaction and final product were monitored for unreacted nicotinamide by TLC on silica GF (AnalTech,Newark, DE) with propanol:lO% ammonia (955 v/v). The recrystallized product showed no indication of nicotinamide but had an intense quenching spot under UV light at the origin for the product. This spot showed a greenish fluorescence after spraying with a 15% (v/v) solution of acetophenone in ethanol which contained potassium hydroxide (1mol/L). Subsequent spraying with 50% formic acid and gentle heating turned the fluorescence intensely blue. These reactions are highly specific for alkylated nicotinamides(8,16,17). IR (KBr) 3280,3140,2320, 1690,1400, and 1320 cm-'; 'H NMR (DzO360 MHz) 6 1.34 (d, 3 H, J = 6.4 Hz), 4.30 (m,1 H), 4.52 (d, d, 1 H, J = 13.4, J = 8.8 Hz), 4.86 (d, d, 1 H, J = 13.4, J = 2.9 Hz), 8.22-9.29 (m, 4 H). (ii) Reaction with Epichlorohydrin. The reaction conditions and TLC monitoring were similar to those reported above with the exception that a 5 to 1 mmol ratio of epichlorohydrin to nicotinamide was employed. Yield, 27%; IR (KBr) 3130,2360, 1685,1400,and 1300 cm-l; 'H NMR (DzO,360 MHz) 6 3.81 (m, 2 H), 4.45 (m, 1 H), 4.76 (d, d, 1 H, J = 13.4, J = 8.8Hz), 503 (d, d, 1 H, J = 13.4, J = 2.9 Hz), 8.22-9.29 (m, 4 H). (iii) Reaction with Trichloropropylene Oxide. The millimole ratio of trichloropropyleneoxide to nicotinamide was 2 to 1. Yield 51%; IR (KBr) 3100,2320,1700,1400, and 1300 cm-'; 'H NMR (CD30D/D20,360 MHz) 6 4.78 (d, d, 1 H, J = 9.5, J = 2.5 Hz), 4.92 (d, d, 1 H, J = 13.4, J = 9.8 Hz),5.48 (d, d, 1 H, J = 13.4, J = 2.4 Hz),8.27-9.43 (m, 4 H). Alkylation of NBP by Aliphatic Epoxides. The conditions used for NBP alkylation were based on the existing procedures 0 1982 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 2, FEBRUARY 1982
Table I. Linearity Data qnd Molar Absorptivities for N-Alkylnicotinamides
a
compound N-alkylnicotinamide from propylene oxide N-alkylnicotinamide from epichlorohydrin N-alkylnicotinamide from 3,3,3-trichloropropyleneoxide N' -methylnicotinamide At 358 nm.
1.0-
k'-
concn range, pmol/mL 0.03125-0.500 0.03125-0.500 0.03125-0.625
eq of the curve 1.844~t 0.0008 1.847~ - 0.0004 1.856~ - 0.006
corr coeff 0.9999 0.9999 0.9997
molar abs a (n = 6) 1849 * 13 1846 * 18 1854 * 48
0.06250-0.500
1.665~t 0.0004
0.9995
1698 t 47
0
TIMEhln)
TIME(mln)
Alkylation of NBP by aliphatic epoxides at 60 'C: (P) propylene oxide: (N) 1,2-epoxy-3-nitratopropane; (S) 1,2-epoxy-3thiocyanopropane; (C) epichlorohydrin; (T) 3,3,3-trlchloropropylene oxide, 3/4 of the actual curve. (5-7). One milliliter of a 2% ( W / V ~ solution of NBP in ethylene glycol was mixed with 0.7 mL of a 0.2 M Tris-HC1 buffer (pH 7.4),containing 27% ethanol. The epoxide (0.2pmol) was added in absolute ethanol (100pL) and the mixture incubated at 60 "C. After the appropriate incubation time, tubes were placed in dry ice-acetone and finally 2 mL of a triethylamine solution (50% v/v in acetone) was added with the absorbance read at 560 nm after exactly 1 min. Alternatively, the milder conditions of Hemminki (10) were duplicated with the following amounts of reagents: 1.7 mL of a NBP solution in ethylene glycol (3.33% w/v); 1.2 mL of a 0.1 M Tris-HC1 buffer (pH 7.4) containing 27.5% acetone; 100 pL of an acetone solution of 0.858 Fmol of epoxide.
RESULTS Nicotinamide is readily alkylated by aliphatic epoxides in buffered aqueous solutions, containing small amounts of water-miscible organic solvents (8, 15). After reaction with acetone in a basic medium, N-alkylnicotinamides are converted to cyclic products which in addition to being fluorescent (8, 18) display absorbance maxima around 360 nm (18). Linearity of the reaction was checked for the three synthesized alkylated nicotinamides as well as with N-methylnicotinamide (Sigma, St. Louis, MO) and the corresponding molar absorptivities were calculated. Table I summarizes the results. Beer's law was valid up to an absorbance value of 1. In addition, molar absorptivities of the three synthetic compounds were virtually identical and differed only about 8% from that of N-methylnicotinamide. The resulting color was
Flgure 2. Alkylation of NBP by three aliphatic epoxides at 32 'C: (P) propylene oxMe; (C) epichlorohydrin; (T) 3,3,3-trlchloropropyieneoxide, '1, of the actual curve.
reasonably stable over a sufficient time interval. That is, within the first 8 min following addition of the water, absorbance increased by rates up to 15%,but for the next 8 min, this increase had leveled off to less than 6%. Measurements were always performed after a fixed time. Initial rate studies, performed by removing samples with increasing incubation times prior to development of their chromophores, clearly indicated sharp breaks in their absorbance vs. time curves for the more reactive epoxides after 20-40 min. As this was in contrast to the linear results of Hemminki and Falck (10) but in agreement with an extensive kinetic study (3) of NBP alkylation by nitrogen mustards and ethylene imines, it was decided to reinvestigate the NBP-epoxide reaction with a series of 3-substituted propylene oxide derivatives. Figure 1shows the curves of absorbance of the final color vs. incubation time obtained for five representative compounds. For epoxides with low activity, e.g., propylene oxide, linearity extended over a 2-h period. In contrast, derivatives with medium to high activity showed a sharp break in their curves after 30-70 min. Epifluorohydrin, epibromohydrin, and epiiodohydrin yielded similar curves in the same region as the thiocyano and nitrato epoxides of Figure 1with breaks after 60,60, and 50 min, respectively. The nitro compound gave zero absorbance over the whole time course of the reaction. Linearity could be extended by performing the reaction at lower temperature. To this end, we employed the milder conditions, described by Hemminki et al. (10). Results for three epoxides are illustrated in Figure 2. While there is linearity for the propylene oxide, a slight break in the epichlorhydrin curve may occur around t = 60 min. Moreover,
ANALYTICAL CHEMISTRY, VOL. 54, NO. 2, FEBRUARY 1982
215
Table 11. Comparison of Alkylating Activities in the NBP Test at 60 "C alkylating activity std RSD, % dev compound A 560 1,2-epoxy-3-nitropropane
0.000
propylene oxide
0.114 0.232 epifluorohydrin 0.242 1,2-epoxy-3-iodopropane 0.260 1,2-epoxy-3-thiocyanopropane0.260 epibromohy drin 0.265 epichlorohydrin 0.368 1,2-epoxy-3-nitratopropane
0.014 0.014 0.002 0.01 2
12.0 6.0 3.2 4.3 0.9 4.4
0.018
5.0
0.008 0.011
Table 111. Comparison of Alkylating Activities in the NBP Test at 32 "C alkylating activity std RSD, compound A,,, dev % 0.070 0.071 0.208 3,3,3-trichloropropylene 2.271 oxide
propylene oxide glycidol epichlorohydrin
0.005 0.007 0.012 0.077
7.1 10.0 5.7 3.4
the trichloropropyleneoxide curve deviates significantly from linearity. Final comparisons between different epoxides were made on the basis of a one-point measurement, i.e., after a fixed incubation time, in the linear portions of the curves. The absorbances obtained for six samples of each epoxide incubated for 30 min and analyzed as described in the literature (5-7) are presented in Table 11. Trichloropropylene oxide had to be excluded from the above series, because its high activity results in early deviation from linearity. To overcome this drawback, this compound, and the three other epoxides used in common in the present study and that of Hemminki et al. (10) were run under the 32 "C temperature of these authors for 20 min. Results of this experiment are summarized in Table 111. Similar conditions were also used to assess the alkylating activity of 1,a-epoxy3-nitropropanein that a comparison was made with propylene oxide by incubatingboth compounds at 32 "C but for 2 h. The nitro derivative yielded an absorbance of 0.061 f 0.009 (n = 6) under those circumstances while the value for propylene oxide was 0.295 f 0.009 ( n = 6). Rate studies were conducted with nicotinamide as the nucleophile (Figure 3). Breaks in the curves for epichlorohydrin and epibromohydrin are clearly defined whereas propylene oxide yields a straight line over the whole time interval studied. The shape of the trichloropropylene oxide curve is quite comparable to the one obtained in the NBP test, a plateau being reached after about 40 min. Comparison between different epoxides was again made on the basis of replicate determinations at a fixed incubation time, ie., 20 min (Table IV). The poorly active 1,2-epoxy-3-nitropropane was compared to propylene oxide in a separate run. Incubation time was extended to 80 min to increase the sensitivity. The nitro derivative (Amnm = 0.084 f 0.003, n = 6) displayed about 40% of the activity of the propylene oxide (Amnm = 0.200 f 0.006, n = 6). This was in close agreement with the results obtained with a fluorometric approach. Both compounds (0.55 Mmol) were incubated at 37 "C for 2 h, and the extent of alkylation was estimated by our previously described fluorometric procedure (8). The fluorescence obtained for the nitro derivative
b 20
40
80
TIME(mln)
Figure 3. Aikylatlon of nicotinamide by aliphatic epoxides: (P) propylene oxide; (8)epibromohydrin; (C) epichlorohydrin; (T) 3,3,3-triof the actual curve. chloropropylene oxide; ~
~~
Table IV. Comparison of Alkylating Activities in the Nicotinamide Test std dev compound 4 5 8 0.070 0.097 0.137 0.143 1,2-epoxy-3-thiocyanopropane 0.1 50 1,2-epoxy-3-nitratopropane 0.161 0.169 1,2-epoxy-3-iodopropane epichlorohydrin 0.171 3,3,3-trichloropropyleneoxide 1.239
glycidol propylene oxide epifluorohydrin epibromohydrin
RSD, %
0.003
3.6
0.011
11.1
0.004 0.002 0.002
3.2 1.6
0.008 0.008 0.001
0.027
1.7
4.7 4.7 0.9 0.6
(24% T ) again approximated 40% of the corresponding propylene oxide value (60% T). Even under milder conditions, the same principles are still operative (Figure 2). For the epichlorohydrin, linearity over the whole range can still be assumed, although the curve can be drawn in different ways. The possibility of a slight break around 60 min has to be considered in this respect. In contrast, a very pronounced deviation from linearity can hardly be denied in the case of the trichloropropylene oxide and indeed the original curve for the NBP-trichloropropylene oxide reaction reported in the literature (10) could also have been described with a change in slope after 30-60 min.
DISCUSSION While the NBP test is well established for the qualitative detection of aliphatic epoxides both in solution and on TLC (6, 7), special care should be taken when it is to be applied for quantitative purposes. Our data confirm that the kinetic principles, as outlined by Bardos et al. (3) for other alkylating agents, are valid for these compounds as well. It can be demonstrated depending upon reaction conditions and compounds that the reaction is only linear over a limited time period and that a sharp break occurs in the absorbance vs. time curves, even for moderately active derivatives. This phenomenon has been explained by Bardos et al. (3) in terms
218
ANALYTICAL CHEMISTRY, VOL. 54, NO. 2, FEBRUARY 1982
Table V. Summary of Relative Alkylation Results re1 alkylation
compound propylene oxide 1,2-epoxy-3nitratopropane epifluorohy drin 1,2-epoxy-3iodopropane 1,2-epoxy-3thiocyanopropane epi bromohy drin epichlorohydrin glycidol 3,3,3-trichloropropylene oxide 1,2-epoxy-3nitropropane
NBP 1.00
nicotinamide
2.04
1.00 1.66
2.12 2.28
1.41 1.74
2.28
1.55
2.32 3.28
1.47 1.76
1.01
32.4 0.21
0.72
12.8
Taft values a u*
0.00 1.10
0.85
1.00 1.05 0.555 2.65
0.42
a Taft u* values are from ref 1 9 on the basis of substituents on ethylene oxide,
of two competing S312 reactions, Le., alkylation and hydrolysis. Deviation from linearity occurs at a point where a considerable amount of epoxide has been consumed in either of the two reactions, resulting in a sharp decrease of its initial concentration (3). In the case of trichloropropylene oxide (Figure l), which is chemically the most active compound of the series, a plateau was reached after 60 min, indicating completion of the reactions. As a consequence, quantitative comparisons between different derivatives can only be made in the initial linear portions of the curves. However, the need to interrupt the reaction at this point for color development may account for the poor reproducibility of the NBP test. In our hands, day-to-day variations as high as 15-20% in the slopes of the curves were observed. Therefore, accurate comparison of curves could only be obtained if all determinations were carried out under the same conditions at the same time. Since this would be inconvenient and time-consuming, measurements a t a fixed incubation time, in the linear part of the curves, offer a valuable alternative. This allows handling of increased numbers of samples with less involved standardiazation of incubation times. The reaction with nicotinamide obviously proceeds along the same kinetic principles as the NBP reaction, as evidenced from the breaks occurring in the curves. Deviation from linearity tended to show up earlier, probably because of the lower concentration of the nucleophile. The molar ratio between nicotinamide and epoxide is only 50, while it is at 465 in the case of the NBP standard procedure at 60 OC. This new method for screening alkylating activities has some distinct advantages over the NBP procedure. The spectrophotometric approach described in this paper lends itself very well for the quantitative comparison of alkylating activities, since it was demonstrated that the molar absorptivities of different N-alkylnicotinamides were nearly equal. This indicates that a major change in the nature of the alkyl chain does not affect the overall spectrophotometric characteristics of alkylated nicotinamides to an appreciable extent. Thus, differences in absorbances, as obtained for a series of epoxides, will reflect variations in alkylation rate rather than differences in molar absortivities.
Table V is a summary of the results for all ten epoxides relative to that for propylene oxide set as 1.00. The results with nicotinamide as the nucleophile show a good correlation (r = 0.9987) in a linear least-squares regression analysis compared to those for the NBP procedure. A comparison of the effect of polarity of the substituent group on the epoxide moiety as indicated by the available Taft u* values (19) was also made for each procedure. There was reasonable correlation in a least-squares analysis to Q* values shown in Table V with no appreciable difference between the two systems (r = 0.9058 for the nicotinamide system and r = 0.9051 with NBP as the nucleophile). It is unexpected that the nitropropylene oxide with its strong electron withdrtwing substituent was a weaker alkylating agent than propylene oxide in both procedures. However, this compound has been reported to undergo rapid isomerization to a y-nitroallyl alcohol (14). The nicotinamide procedure approaches physiological conditions more closely than does the NBP reaction, which proceeds in an essentially organic medium. This could be important in comparison to bioalkylation for those in vitro procedures in which solvolysis can be a significant factor. It is still possible in the nicotinamide method to decrease the alcohol content in the reaction medium, which has a favorable effect on the alkylation rate. However, a standard concentration of 15% of ethanol in buffer was chosen, to avoid possible insolubility of the epoxides in the aqueous buffer. Another decided advantage of the nicotinamide method lies in the enhanced stability of the final chromophore. While color in the NBP test fades rapidly, only a slight increase of absorbance with time is observed in the nicotinamide procedure. LITERATURE CITED (1) Slnger, B. "Progress in Nuclelc AcM Research and Molecular Biology"; Cohen, W. E., Ed.; Academlc Press: New York, 1975;Vol. 15, pp 219-285. (2) Epstein, J.; Rosenthal. R. W.; Ess, R. J. Anal. Chem. 1955, 27, 1435. (3) Bardos, T. J.; Datta-Gupta, N.; Hebborn, P.; Triggle, D. J. J. Med. Chem. 1885, 8 , 167. (4) Von Preussman, R.; Schneider, H.; Epple, F. Armelm.-Forsch. 198% 18, 1059. (5) Swalsland, A. J.; Grover, P. L.; Sims, P. Blochem. fharmacol. 1973, 22,1547. (6) Hammock, L. 0.;Hammock, B. D.; Casida, J. E. Bull. Envlron. Contam. Toxicol. 1974, 12, 759.
(7) Agarwal, S. C.; Van Duuren, B. L.; Kneip, T. J. Bull. Envlron. Contam. roxicoi. 1979, 23,825. (8) Nelis, H. J. C. F.; Slnsheimer, J. E. Anal. Blochem. 1981, 715, 151. (9) Wade, D. R.; Airy, S. C.; Slnshelmer, J. E. Mufat. Res. 1978, 5, 217. (IO) Hemminki, K.; Falck, K. Toxicol. Lett. 1979, 4 , 103. (11) Hemmlnkl, K.; Falck, K.; Valnio, H. Arch. Toxicol. 1980, 46, 277. (12) Nef, J. U. Justus Lleb/gs Ann. Chem. 1904, 335,204. (13) Engie, W. D. J. Am. Chem. SOC. 1898, 20, 668. (14) Sokovlshlna, 1. F.; Perekalin, V. V.; Lerner. 0. M.; Andreeva. L. M. Zh. 0rg. Khlm. 1965, 1 , 636. (15) Wlndmueller, H. G.;Ackerman. C. J.; Bakerman, H.; Mickelsen, 0.J. Blol. Chem. 1958, 234, 889. (16) Clark, B. R.; Halpern, R. M.; Smith, R. A. Anal. Blochem. 1975, 68, 54. (17) Nakamura, H.; Tamura, 2. Anal. Chem. 1978, 50, 2047. (18) Huff, J. W. J. Blol. Chem. 1847, 167, 151. (19) Taft, R. W. "Steric Effects in Organic Chemlstry"; Newman, M.,Ed.; Wiley: New York, 1957;p 619.
RECEIVED for review September 11, 1981. Accepted October 21, l%l. This paper was presented in part at the 182nd National Meeting of the American Chemical Society as part of paper No. 39, New York, NY, Aug 24, 1981. H.J.C.F.N. is indebted to the Belgian Fund for Medical Scientific Research (F.G.W.O.) and A. P. De Leenheer, Gent, Belgium, for the opportunity to take part in this investigation.
