[CONTRIBUTION FROM THE LABORATORIES OF THEROCKEFELLER INSTITUTE FOR MEDICALRESEARCH]
CHEMICAL REACTIONS OF T H E NITROGEN MUSTARD GASES.’ V. T H E REACTIONS O F T H E NITROGEN MUSTARD GASES TT~ITH PROTEIN CONSTITUENTS JOSEPH S. FRUTON,’ W I L L I A M H. S T E I N ,
AND
M A X BERGMANN’
Received March 99,1046
It is generally believed that many of the physiological effects of vesicant agents are a consequence of reactions of the vesicant with tissue proteins, particularly enzyme proteins (1-18). The nitrogen mustard gases have been shown to inhibit, and hence probably to react with, numerous enzymes (10-18). It follows, therefore, that an understanding of the physiological action of the nitrogen mustards requires a detailed study of the reactions of theseagents with proteins, amino acids, and peptides. Aside from their significance as protein constituents, amino acids and peptides also occur as such in blood and other body fluids and tissues. In addition to the reactive a-amino and acarboxyl. groups common t o most amino acids, certain of the amino acids possess characteristic side chains, many of which exist in an uncombined state in proteins. The reactions of these groups with the nitrogen mustards are, therefore,worthy of study. The reaction of methyl-bis(8-chloroethyl)arninelethyl-bis(/3-chlorocthyl)arninel and tris@-chtoroethy1)amine with the amino groups of amino acids and peptides. The data given in Table I show the extent of the decrease in amino nitrogen when amino acids or peptides are treated with the nitrogen mustards a t weakly alkaline pH values. The reactions were carried out at room temperature, and the disappearance of amino nitrogen was followed by means of the Van Slyke nitrous acid method. I n the case of methyl-bis(chloroethy1)amine (MBA) and ethyl-bis(8-chloroethy1)amine (EBA) the reaction mixtures were shaken for 4 hours to obtain homogeneous solutions and were then allowed to stand for 16 hours. Since under the experimental conditions employed, tris(8-chloroethy1)annine (TBA) does not pass into solution as rapidly as d:, MBA and EBA, the reaction mixtures containing TBA were shaken during the entire 20-hour period. It will be noted that in the experiments with MBA and EBA, 4 milliequivalents of amino groups were employed per millimole of nitrogen mustard ; whereas, in the experiments with TBA 6 milliequivalents of amino groups were employed. In all experiments, therefore, 2 equivalents of amino groups were employed for each chloroethyl group. From the data in Table I it is evident that extensive reaction occurs when the a-amino groups of amino acids are treated with any one of the nitrogen
* This work was done in whole under Contract No. OEMsr-313 between The Rockefeller Institute for Medical Research and the Office of Scientific Research and Development, which assumes no responsibility for the accuracy of the statements contained herein. The experiments were performed during the period June 1942-January 1944. Present address, Yale University, New Haven, Connecticut. * Died, November 7,1944. f
559
660
FRUTON, STEIN, AND BERQMANM
mustards a t pE 8. The results presented in Table I indicate that the a-amino groups of all the amino acids tested, with the exception of histidine, react at pH 8 to about the same extent with MBA. This also seems to be the case for ERA and TEA. The fact that the a-amino group of histidine reacts with any given citrogen mustard to a much lesser extent than do the a-amino groups of the other amino acids is of particular interest. As is shown later in this communication and in Paper VI of this series (19), this abnormal behavior is due to a rapid reaction of the nitrogen mustards with the imidazole group of histidine. Thus, the greater part of the available nitrogen mustard is removed from the reaction before the sloxer action of the vesicant on the a-amino group of histidine can occur. The data in Table X indicate that the ,%amino group of ,!?-alanine reacts with MBA and EBA to a slightly smaller extect than do the amino groups of the a-amino acids. This difference docs not appear to hold in the case of TBA. It will be noted from Table I that the amino group of 2-alanine does not react at pH 8 to a greater extent with any given nitrogen mustard than do the amino groups of the more complex amino acids serine, threonine, glutamic acid, arginine, and tyrosine (as amide).4 It must be concluded, therefore, that under these conditions the aliphatic hydroxyl group of serine and threonine, the y-carboxyl group of glutamic acid, the guanido group of arginizie, and the phenolic hydroxyl group of tyrosine do not interfere appreciably with the reaction of the nitrogen mustards with a-amino groups. It will be noted that in the case of the peptides investigated, the extent of the reaction with a given nitrogen mustard is considerably greater than in the case of amino acids such as alanine. On the other hand, the e-amino group of benzoyl-1-lysineamide does not react with MBA to the same degree as do the a-amino groups. It is of interest that analogous findings were obtained in the study of the reaction of mustard gas with the amino groups of amino acids and peptides (20). The amino acids phenylalanine, tryptophane, and methionine are sparingly soluble a t pII 8. They, therefore, were employed as sodium salts at pH 9.5. It mill be noted that a t pH 9.5 the a-amino group of alanine reacts with a given nitrogen mustard to a greater extent than a t pH 8. The amino groups of phenylalanine and tryptophane react a t pH 9.5 to about the same extent as does that of alanine. This indicates that the indole nucleus of tryptophane does not react appreciably with the nitrogen mustards. The amino group of methionine was found to react with TBA at pH 9.5 to less than half the extent observed for the amino groups of alanine, phenylalanine and tryptophane. 71'ith MBA and EBA, a decreased reactivity of the amino group of methionine was also observed but to a very much lesser degree. It will be shown in another paper (21) that mustard gas itself reacts readily with the --,%HI group of methionine to form a sulfonium salt. The simplest interpretation of the findings with the nitrogen mustards is that these agents 4 The amino acid tyrosine was not studied as such because of its law solubility at pH 8. In its place, the acetate of I-tyrosineamide was employed.
NITROQEN MUSTARD GAS.
561
V
also have a tendency to combine with the -SCHI group of methionine, presumably with the formation of a sulfonium salt. In the case of TBA this tendency is quite marked, although not so great as observed with mustard gas itself. For MBA and EBA, the tendency is so small as to be questionable. The discussion thus far has been concerned with the relative reactivity of a given nitrogen mustard towards various protein constituents. It is also possible, TABLE I REACTION O F METHYL-BIS(~-CHLOROETHYL)AMINE (?VIBA), EFHYL-BIS(B-CHLOR0ETHYL)AMINE (EBA), AND TRIS(~-CHLOROETHYL)AMINE (TBA) WITH THE AMINO G R ~ U P S OF AhiINo ACIDS A N D PEPTIDES Concentration of reactants per cc.: 0.134 mM of MBA or EBA; 0.127 mM of TBA. (The nitrogen mustards were employed as hydrochlorides, and one eqiivalent of NaOH was added t o liberate the free base.) 0.531 m.equiv. of "2-N (in the case of MBA or EBA); 0.762 m. equiv. of NHP-N (in the case of TBA). 0.531 m.V of NaHCOs for MBA or EB.4; 0.526 m M of NaHC03 for TBA. Temperature 25"; reection period, 20 hours (4 hours shaking, 16 hours standing for MBA and EBA; 20 hours shaking or TBA).
-
DECREASE IN
"2-N
SUBSTANCE
Glycine. . . . . . . . . . . . . . . . . . I-Alanine. . . . . . . . . . . . . . . . . . 2-Serine . . . . . . . . . . . . . . . . . . dl-Threonine . . . . . . . . . . Z-Glutamic acid. . . . . . . . . . . I-Lysine. . . . . . . . . . . . . . . . . . . l-Alanine. . . . . . . . . . . . . . . . . . 2-Phenyldanine~ dl-Methioninea . . . . . . . . . . . . 6-Alanine . . . . . . . . . . . . . . . . . 2-Tyrosineamide acetate. . . Benzoyl-!-lysineamide.. . . . Glycylglycine . . . . . . . . . . . . . 2-IJeucylglycine. l-Leucylglycylglycine. . . . . .
DTiCBEASRrN OF AGENT
PER CC.
-
MBA.
EBA,
TBA
Y.EQUIV.
Y.EQUIV.
M.EQU;V.
8 8 8 8 8 8 8 8 9.5 9.5 9.5 9.5 8 8 8 8 8 8
0.096 * 100 .loo .117 .loo .OS3 .112 .015 .147 .167 .117 .161 * 067 .133 .021 .147 .161 .148
0.114 .117 .150 .142 .136 .123 .135 .037 .156 .153 .125 .156 ,080
0.15 .145 * 19 .18 .16 .18 .16 .07 .19 .22 .09 .25 .18
.188 .190 .172
.23
1
MBA,
EBA,
TBA.
U.EQUIV.
M.EQUIV.
K.P.QUIV.
0.72 .75 .75 .87 .75 .62 .84
0.86 0.88 1.13 1.06 1.02 0.92 1.01 0.28 1.17 1.15 0.94 1.17 0.60
1.2 1.15 1.5 1.4 1.3 1.4 1.3 0.6 1.5 1.7 0.7 1.9 1.4
1.41 1.43 1.30
1.8
.ll 1.1 1.25 0.87 1.2 0.50 1.0 0.10 1.1 1.2 1.1
These sparingly soluble amino acids were dissolved as the sodium salts, and the same amount cif bicarbonate was added as in the other experiments.
on the basis of the data given in Table I, to obtain a rough comparison of the resctivit,y of the three nitrogen mustards towards the compounds listed. Since TEA contains three chloroethyl groups and MBA and EBA each contain but two chloroethyl groups, the comparison should be made on the basis of the milliequivnlents of amino nitrogen which have reacted per chloroethyl group. The data for such a comparison are given in Table 11. Before commenting upon the data presented in Table 11, it should be em-
562
FRUTOK, STEIN, AKD BERG?dANK
phasized that any conclusions which can be drawn from the data may be valid only for the experimental conditions employed, and probably have no general kinetic applicability. This reservation is necessary, because at the beginning of the experiments homogeneous solutions were not obtained, and the three nitrogen mustards passed into solution at different rates, TBA being by far the slowest. Hence, the concentration of reactive ethylenimonium groups in solution was not known and was not the same for each agent. With the above reservations it appears from Table I1 that the chloroethyl groups of all the nitrogen mustards have about the same reactivity towards the amino groups of most of the compounds listed. The order of reactivity appears to be EBA > TBA > MBA, although the differences among the three TABLE I1 COMPARISON OF THE REACTIVITIES OF TRIS(,%CHLOROETHYL)AMINE (TBA), METHYL-BIS(&CHLOROETHYL)AMINE (MBA), AND ETHYL-BIS(~-CHLOROETHYL)AMINE (EBA) WITH THE AMINOGROUPS OF AMINO ACIDSAND PEPTIDES The data in this table are calculated from those given in Table I. DECBEASE I N BUBSIAAHCE
-~ Glycine. .................... 1-Alanine . . . . . . . . . . . . . . . . . . . 1-Serine. .................... dl-Threonine. . . . . . . . . . . . . . . . LGlutamic acid.. . . . . . . . . . . . 1-Lysine. . . . . . . . . . . . . . . . . . . . t-Arginine . . . . . . . . . . . . . . . . . . Z-Histidine. . . . . . . . . . . . . . . . . . &Alanine.. . . . . . . . . . . . . . . . . . 1-Alanine . . . . . . . . . . . . . . . . . . . 2-Phenylalanine. . . . . . . . . . . . . 1-Tryptophane. . . . . . . . . . . . . . dl-Methionine . . . . . . . . . . . . . . Glycylglycine. . . . . . . . . . . . . . .
"2-N
PEK M.EQUIV. OF CHLOBOEIHYL CROUPS
PH
8 8 8 8 8 8 8 8 8 9.5 9.5 9.5 9.5 8
TBA,
MXQUIV.
0.40 .38 .50 .47 .43 .43 .47 .20 .47 .50 .57
.63 .23 .60
MBA, M.EQUIV.
0.36 .38 .38 .42 .38 .42 .31 .06 .25 .55 .62 .60 .44 .55
EBA,
M.EQUIV.
0.43 .44 .56 .53 .51 .50 .46 .14 .30 .58 .57 .58 .47 .70
agents are not very marked.6 TBA seems to have a greater reactivity toward the &amino group of &alanine and toward the sulfide group of methionine than do MBA or EBA. MBA, on the other hand, seems to have the highest reactivity toward the imidazole group of histidine, followed by EBA and TBA [see also Paper VI of this series (19)]. 6 It should be pointed out that, while the concentrations of TBA, MBA, and EBA are identical in these experiments, the concentrations of chloroethyl groups are not. The TBA reaction mixture contains half again as many chloroethyl groups per cc. as do the EBA or MBA reaction mixtures. In order to circumvent this difficulty, the experiment with alanine and TBA a t pH 8 was repeated, with the concentration of the reactants reduced t o two-thirds of its former value. In this case, the decrease in m.equiv. of amino-nitrogen per m.equiv. of chloroethyl group fell from 0.38 t o 0.33. Once again i t appears that the reactivities of TBA, MBA and EBA towards the amino group are quite similar with MBA appearing, on this basis, to be slightly more reactive than TBA.
N T R O G E N MUSTARD QAS.
563
V
The reaction of MBA with the amino groups of proteins. When aqueous solutions of crystalline egg albumin or of gelatin are treated at slightly alkaline pH values with MBA, a significant decrease in the amino nitrogen content of the protein solution is observed (Table 111). In this behavior MBA differs from mustard gas, since it has been found that the latter does not appear to combine t o an appreciable extent with the amino groups of egg albumin or gelatin (22). The data in Table 111 indicate that about one-fourth of the free amino groups of egg albumin and about' one-third of the free amino groups of gelatin have disappeared during the reaction. The mechanism of the reaction of MBA with amino groups. In view of the complex reactions that occur when the nitrogen mustards react with water TABLE I11 REACTIONOF
METHYL-BIS(@-CHLOROETHYL)AMINE (MBA) WITH AMINOGROUPSOF PROTEINS
Time of reaction, 24 hours a t 25'; pH 7.5 (bicarbonate). MB.4 was employed as the hydrochloride and one equivalent of NaOH added to liberate the base. PPOTEIN
'
PROTEIN PER CC.
MBA PEP cc.
NTIz-K~ PEE CC.
DECPBASE IN NHz-N PEP CC.
.Egg albumin Gelatin
a
mg.
mM
mg.
mg.
114
0 0.08 0 0.08
0 12.5 0 12.5
0.92 0.68 0.71 0.50
DECREASE IN
"1-N
PER
m M MBA
mg.
aY
0.24
0.017
0.21
0.21
0.015
0.19
mM
30 minutes shaking in the Van Slyke amino nitrogen apparatus.
(9), it seemed desirable to determine whether the reaction with amino groups involved direct reaction with the nitrogen mustard itself or with one or more of the transformation products of the nitrogen mustard. To this end, a study was made of the reaction of MBA with alanine in bicarbonate solution. The following analytical procedures were applied : (a) disappearance of amino nitroger,; (b) formation of chloride ions; (c) formation of hydrogen ions as measured by the consumption of bicarbonate; and (d) disappearance of the ethylenimonium ion as measured approximately by the reaction with thiosulfate in 10 minutes (23). The data in Table I V shorn the following: (a) During the first 20 minutes, when all of the MBA is transformed into the l-methyl-l-(8-chloroethy1)ethylenimoniumion, only about 0.02 m.equiv. of NH2-N disappear per mM of MBA. Thus, the direct reaction of MBA itself with amino groups is negligibly small. After 24 hours, when the decrease in amino groups has stopped, only 0.75 m.equiv. of "2-N per mM of MBA have disappeared. The maximum possible value is 2 m.equiv. of NH2-N per m M of MBA.
564
FRUTON, GTEIN, AND BERGMANN
Separate experiments with 1-glutamic acid and glycylglycine have shown that in these cases also MBA is convcrted into the ethylenimonium ion before appreciable disappearance of amino nitrogen is observed. (b) The initial rate of chloride liberation is much the same in the presence of alanine as in bicarbonate alone, but during the period when most of the amino nitrogen disappears, the chloride liberation is more rapid. In the presence of alanine the extent of the chloride liberation is also more complete (93%). (c) The liberation of H+ is more rapid during the period when most of the amino nitrogen disappears. However, the H+ liberated after 24 hours corresponds to only 47% of the amount to be expected if the two chlorine atoms had reacted directly with amino groups or with water. As in the absence of alanine, the H+ liberated corresponds to one-half of the C1- formed. TABLE I V
REACTIONS OP METHYL-BIS(&CHLOROETHYL)AMINE (MBA) IN THE PRESENCE AND ABSENCE OF ALANINE Concentration of reactants per cc.: 0.127 m M of MBAeHCl (one equivalent of NaOH added to liberate the base); 0.536 miM of l-alanine 1.526mM of NaHCOa.
NHt-N REACIION YTx’lTXZ
TIME
min.
20 40 60 360 1440
Bicarbonate plus I-alanine
20 40 60 360 1440
SULFAIS USED IN 0 MIN. PEP
XBEPAICO PER m Y
m.cguiv.
m.cpir.
m.cquia.
m.cquic.
1.10 1.21 1.31 1.53 1.69
0.17 .36 .43 .70 .a7
0.93 .85 .88 .83 .82
1.06 1.04 0.97 .58 .24
1.07 1.25 1.34 1.73 1.85
.21 .37
.86
.58
.76 .83 .92
.91 .79 .64 .17 .03
MBA
Bicarbonate alone
THIO-
CL-
DISAPPEARED PEP m Y
0.02 .14 * 24 .72 .75
MBA
MBA
-- mM MBA
.90 .93
.88
n.equie.
(d) As in the experiment without alanine, the increase in C1- after the first 20 minutes is accompanied by an approximately equivalent increase in H+. (e) The rate of disappearance of the ethylenimonium form is definitely more rapid in the presence of alanine than in bicarbonate alone. After 24 hours, the quantity of imonium ion is negligibly small. The above observations may perhaps best be summarized on the basis of the following reactions (cf. Figure 1): In bicarbonate solution, Reaction A, which occurs within the first 20 minutes, is the formation of the first ethylenimonium ring. This is shown by the liberation of more than half of the expected C1-, very little H+, and the rapid consumption of 1 equivalent of added thiosulfate. The imonium compound then undergoes several different reactions. The occurrence of Reactions B, C, and D in bicarbonate solution has been demonstrated in Paper I of this series (23).
NITROGEN MUSTARD GAS.
565
V
CHz CHIC1
/ CHaN \
CHxCHxC1
I
(A) (water)
C1- CHI CHI C1
+/
CH, N-
B
E
(water)
(alanine)
1I
1
/
CHI
I
CHICHzC1
F (water)
C (water)
G (water)
D (water)
CHa
C1- CHICHZ
+/
I
HOOCCHNHCHa CHiN CHa
Fra. 1
/ \
C1-
CH:
\+ I N CHI CHxNHCHCOOH / \
CHI CHI
CHa
566
FRUTON, STEIN, AND BERGMANN
In the presence of alanine, Reaction A takes place also. About 75% of the imonium compound then reacts with the amino group of alanine as shown by the gradual disappearance of amino groups (Reaction E). This reaction probably involves the ethylenimonium group. At the end of the reaction, 93% of the theoretical C1- and 47% of the maximum possible H+ have been liberated; consequently, the final product of the reaction of MBA with alanine must contain quaternary nitrogen. Since the final 10-minute thiosulfate titer is low, this quaternary nitrogen cannot be ethylenimonium nitrogen. It may be assumed, therefore, that whatever monomeric reaction product is formed dimerizes rapidly according to Reactions F and G. In the presence of alanine, only about 25% of the first ethylenimonium compound follows the sequenceof reactions postulated for the MBA-bicarbonate system. The isotation of a reaction product of MBA with phenylalanine. It was shown in Table I that the amino group of phenylalanine reacts at pH 9.5 with MBA. disUnder the experimental conditions employed, 1.25 m.equiv. of "2-N appeared per mM of MBA. This would indicate that at least a portion of the MBA had reacted in such a manner that 1 molecule of MBA (or its ethylenimonium form) had combined with 2 molecules of phenylalanine to give a product of the following structure: CH2 CaH6
I
/ \
CH2 CHzNHCHCOOH
CH, N
CH2 CHzNHCHCOOH
I
CH, CaH5 This substance has been isolated from the reaction mixture. As previously stated, it was necessary to perform the reaction of MBA with phenylalanine at pH 9.5 because of the slight solubility of the amino acid at pE-1 7.5-8. The relatively high yield and the structure of the product is of interest, since in the reaction of MBA with alanine at pH 8, the analytical data suggested that the principal product was formed from 2 molecules of MBA and 2 molecules of alanine and was, probably, a derivative of the dichloro cyclic dimer of MBA. The reaction of M B A with the imidazole group of histidine. As shown earlier, the imidazole group of histidine interferes markedly with the reaction of MBA with a-amino groups at pH 8. Thus, in the case of histidine, the extent of the disappearance of amino nitrogen was 0.11 m.eqEiv. of "2-N per mM of MBA, reacted per mM while with other amino acids about 0.75 m.equiv. of "2-N of MBA. In order to eliminate the complicating effect of the a-amino group of histidine, acetylhistidine was prepared and the influence of this compound on the reaction between MBA and alanine was studied. MBA (0.32 mlcf) was treated with 1.28 mM of l-alanine in the presence of 1.28 mlcf of acetyl-dl-histidine and 1.0 m31' of NaHC03. After 24 hours, 0.048 m.equiv. of amino nitrogen had disappeared (0.15 m.equiv. of KH2-N per
NITROGEN MUSTARD GAS.
V
567
mill of MBA). In a control experiment, in which acetylhistidine had been omitted, the decrease in amino nitrogen was 0.230 mM (0.72 m.equiv. of "2-N per mM of MBA). This result shows that the imidazole group of acetylhistidine reacts with MBA more readily than does the a-amino group of alanine. The reaction of M B A , EBA, and T B A with carboxyl groups. The reaction of a nitrogen mustard with carboxyl groups might be expected to lead to the formation of esters, which upon treatment with alkali, would be saponified with the disappearance of titratable alkali. Accordingly, MBA and EBA were treated with sod5um acetate or sodium hippurate for 18 to 24 hours in bicarbonate solution, and the amount of saponifiable esters was determined. Bicarbonate controls were necessary, since even in the absence of a carboxylic acid, products were formed from MBA and EBA which decomposed in alkaline solution nith the liberation of acid. In these experiments, no evidence of ester formation was obtained. These results are not surprising, in view of the later findings of Cohen and Van Artsdalen (24). Kinetic studies by these investigators showed that the first ethylenimonium compound derived from MBA reacts with propionate in aqueous solution at pH 7.4. The resulting ester of methyldiethanolamine was found to be unstable, however, saponifying at p H 7.4 with such rapidity that little or no ester could be detected in the reaction mixture after 24 hours. With TBA, unequivocal evidence for the formation of esters was obtained. In the presence of sodium acetate, sodium hippurate, sodium acetyldehydrophenylalanine (I), or sodium acetyldehydrophenylalanyldehydrophenylalanine (111), approximately 25% of the chloroethyl groups of TBA were found to have reacted to form esters. In the experiments with acetate and hippurate the products of the reaction were not isolated. The products of the reaction with acetyldehydrophenylalanine and acetyldehydrophenylalanyldehydrophenylalanine have been isolated, however, and found to be triacyl derivatives of triethanolamine (Compounds I1 and IV). C BHS
C6Hs
CH
CH
I II
I
I
CHI CONHC CO OH
STBA
(1) C&s
(CH, CONH C COO CHs CH,)s N (11) C6H6
I I CH II II CHaCONHCCONHCCOOH CH
(111) CsHs
CsHs
+TBA
568
FRETON, STEIN, AND BERGMANX DISCCSSION
The experiments outlined in this paper show that the nitrogen mustards are capable of reacting in vitro with the functional groups of a number ofprotein constituents. Since many of these functional groups are normally prcsent in intact protein molecules, it seems likely that the nitrogen mustards can react with proteins in vivo. Among the functional groups which are found free in many proteins and which might serve as possible points of attack for the nitrogen mustards, may be mentioned the €-amino groups of lysine residues or any free terminal amino groups of the peptide chains, the imidazole group of histidine residues, the sulfide group of methionine residues, the 8- or y-carboxyl groups of aspartic or glutamic acid residues, and any free terminal carboxyl groups of the peptide chains. To this list must be added sulfhydryl groups, as shown by HeHerman et al. (25). The studies on the mechanism of the reaction between MBA and the amino group of alanine, furnish strong evidence for the view now generally held that the nitrogen mustards must first cyclize to the water-soluble ethylenimonium form prior to undergoing alkylation reactions. EXPERIMENTAL
The reaction of the nitrogen mustards with amino groups of amino acids and peptides. The procedure employed (except for benzoyl-l-lysine amide) involved adding 2 cc. of a n aqueous solution containing 1.6 m M of the nitrogen mustard hydrochloride to 10 cc. of a solution containing 6.4 m.equiv. of amino nitrogen (9.6 m M in the experiments in which TBA was employed), 6.4 m M of NaHCO,, and 1.6 cc. of N NaOH. The reaction mixture was shaken for 4 hours and the solution wa8 ieft at room temperature for 16 hours longer. In the experiments employing TBA, shaking tlas continued for 20 hours. One cc. was removed, diluted determinations by the Van Slyke nitrous t o 10 cc. and 1-cc. aliquot8 were used for “2-N acid method. I n the case of the reaction with lysine and benzoyllysine amide the shaking time was 30 minutes; with the other compounds it was 5 minutes. In the case of benzoyllysine amide, the reaction Kas carried out on a micro scale. A reaction mixture (1.2 cc.) containing 0.21 m M of MBA.HCI, 0.32 mM of benzoyllysine amide HC1,0.8 m M of NaHCO1, and 0.2 mM of NaOH was shaken for 4 hours. After standing for 16 hours longer, 0.2-cc. samples were used for NH2-N determinations. T h e mechanism of the reaction c j M B A with amino groups. The reaction mixtures were made up to the concentrations indicated in Table IV in the manner described above. The C1- and H liberation and thiosulfate consumption were estimated in the manner previously described (23). The isolation o j a reaction product of MBA with phenylalanine. T o 4.23 g. of l-phenylalanine (25.6 m M ) dissolved in 26 cc. of N XaOH were added 1.6 g. of NaHC03 and 6.4 cc. of N NaOH followed by the addition of 8 cc. of an aqueous solution of MBA-HCl (6.4 7 n M ) . The mixture was shaken fcr 24 hoursat 25’. The gelatinous precipitate which separated out n7as filtered and washed with cold water. The product after drying i n vacuo over Pt0s meighed 850 mg. (31%). The substancc was sparingly soluble in water, alcohol, or organic solvents. It was soluble in dilute acid and could be precipitated by neutralization with alltcli. TCe material retained traces of ash tenaciously, and even after several reprecipitations 0.2% of ash was still present. Anal. Calc’d for C2JH31N104.tHzO:C, 65.4; H , 7.7; N, 9.9. Found: C, 65.4; 13, 7.8; N, 9.9. [a]: +29.8” (2% in N HC1) The reaction of the nitrogen mustards with carboxyt groups. The procedure employed in +
KITROCEX WUSTARD GAS.
V
569
the experiments with MBA and EBA was essentially the same, and, therefore, will be given in detail only for the latter. The carboxylic acid (12 mM, employed as the sodium salt) was treated with 4 mM of EBA-HCl in the presence of 4 m M of NaOH and 12 m M of NaHC03. After 24 hours a t 25O, the solution was acidified and evacuated to remove C 0 2 . Absolute ethanol6 (250 cc.) was added and after neutralization to phenolphthalein, 3.0 cc. of 1.06 N NaOH were introduced into the solution. After 30 minutes, the excess alkali was back-titrated t o phenolphthalein nith 0.1 AT HC1. A control experiment (without the carboxylic acid; was also performed and the plkali consumption found in the presence of the carboxylic acid was corrected accordingly. It was found that, in the presence of hippuric acid, the alkali consumption exceeded the control value by 2.1 cc. of 0.1 N NaOH. This indicates that only about 2.6% of the chloroethyl groups of EBA had rcccted to form saponifiable esters of hippuric acid. Similarly, in the presence of acetic mid, i t was found that only about 1.9% of the chloroethyl groups of MBA had reacted to form saponifiable esters. The results with hIBA were essentially simila r . The experiments with TBA were performed in the following manner: To 2.67 m M of TBA.HL'L mas added a solution containing 12 m M of the Na salt of the acid, 12 mM of NaHCOa, cnd 2.7 cc. of AT NaOH. The mixture (total volume 25 cc.) was shaken for 48 hours ct 25". I n each case a precipitzte appeared during the reaction. HCl was added and the C02was removed in vacuo. Absolute alcohol (100 cc.) was added to give a clear solution end N &OH was added to neutrality (phenolphthalein). Five cc. of N NaOH was then added and the solution was left a t room temperature for 30 minutes. Back-titration nith 0.1 N IICl gave the amount of ester so,ponified. The titration in the experiment with compound I11 was made difficult by the yellow color of the solution. Isolaizon (if triacgl dcrivatmes of iriethanolamine. Compound I I . This substance sepnrated P S :t solid nhen 2.67 mM of TBAaHCI was shaken with a solution containing 12 mM of the iSa maltof (I), 12 m M of XaHCO3, and 2.7 cc. of N NaOH. It was recrystallized by carefu! addition of water to Sn alcoholic solution of the substance; m.p. 179-180". Anal. Calc'd for C3&4&40p. C. 65.9; H, 6.0; N , 7.9. Found: C, 66.0; H , 6.1; N, 7.0. Compound IV. When 2.67 mM of TBA.HC1 was shaken with a solution containing 12 mM of the K'a, salt of I11 and 12 mM of NaHCOo, there appeared a white precipitate mixed with a yellow oil. A creamy white solid was obtained by repeated recrystallization fromaqueous alcohol. The air-dried material melted at 120-130". Anal. CLlc'd for C G ~ H , $ ~ O I ~ . ~C, H ~67.0; O : H I 5.7; N,5.3. Found: C, 66.6; H , 5.7; N,8.5.
The stuthors wish to acknowledge with thanks the helpful cooperation of Miss Rosalind E. Joseph, who assisted in the conduct of these experiments, and of Mr. Stephen M. Nagy, who performrd the microanalyses reported in this paper. NEW YORK,N. Y. REFERENCES
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WOOD (1044).a
6 Previous experiments have shown that HCl may be satisfactorily titrated in the pres. ence oi methyldiethanolamine, ethyldiethanolamine, or triethanolamine in 60-C1070 nlcoholUnpublished data obtained in the United States. 0
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* Unpublished data obtained in Great Britain.
