3686
BERNARD J. JAKDORF
T701. T S
nil. of methylene chloride was heated under reflux for 105 minutes. The resultant yellow solution was concentrated a t 40' under reduced pressure; 50 ml. of benzene was added to flush out the thionyl chloride. The residue was taken up in methylene chloride and extracted with a 5 % sodium bicarbonate solution. After concentration, the tan resinous product was dissolved in 10 ml. of benzene; after 1 hr. a t room temperature, 0.335 g. (23%) of a crystalline substance, tn.p. 212-214" dec., was obtained. An analytical sample, obtained by recrystallization from ethanol, had m.p. 214213' dec. Assignment of structurc X (2,2-dirnethyl-3carbomethoxy-.5-keto-6-benzylsulfonamido - 2,3,1,5- tetrahydro-l,4-thiazepine) was made from its ultraviolet (Fig. 2 ) m d infrared (curve E, Fig. 1 ) absorption spectra. .-lnaZ. Calcd. for Cc6H?S>O,S?: C , 40.98; H , ,5.24; S , 7.29. Found: C, 49.98; H , ,).06; X, 7.61.
From a cyclization run involving 0.250 g. (0.57 tnmole I of V I I a and 5.0 ml. of thionyl chloride in 15 ml. of methylene chloride, in which the crude reaction products were oxidized with potassium permanganate in 80% acetic acid, there was isolated 0.120 g. (51%) of the sulfone I X a , m.p. 212-213" dec. Recrystallization from acetone-water gave colorless needles, m.p. 214-215" dec. Anal. Calcd. for C16HZON207S2. C, 46.14; H, 4.85; X, 6.72. Found: C , 46.18; H , 4.89; S , 6.63. B. $-Isomer.--A sample of 0.75 g. (0.0017 mole) o f VI13 was treated with 13 ml. of thionyl chloride in 50 nil. of methylene chloride, as described above for the a-isomer. After the preliminary purification of the crude reaction products by extraction with 5% sodium bicarbonate solution, 0.30 g. (46%) of VIIIp, m.p. 128-130', was obtained directi>- by crystallization from benzene. A4n analytical sample was obtained by recrystallization from acetone The material in the benzene mother liquors from S was passed through a column containing 6.0 g. of Brockman ether-petroleum ether, m.p. 128.5-130". The mixed m.p. with VIIIa (m.p. 130-131.5') was depressed (114-124'). Grade I11 ethyl acetate neutralized alumina with benzene Anal. Calcd. for C 1 6 H a o ~ 2 0 6cS, ~49.98; : H , 5.21; N, as eluent. Crystallization of the material in the first 25 7.29. Found: C, 50.12; H , 5.22; S, 7.41. ml. of eluate from benzene-ether-petroleum ether afforded 0.625 g. (43%) of crude YIIIa, m.p. 128-131'. RecrysNone of the isomeric substance S appeared to have k e n tallization from acetone-petroleum ether gave a n analytical formed in this reaction. sample of this P-lactam,12m.p. 130-131.5'. The sulfone 1x8 was prepared bl- oxidation of the crude .11zel. Calcd. for Cl6Hnoh-rOjSa: c, 49.08; H , d.2-f; S, reaction products obtained from 0.16 g. (0.36 Inmole) of 1.116 and 5 ml. of thionyl chloride in 15 i d . of methylene 7.29. Found: C, 50.10; H , 5.30; S,7.33. chloride. The yield of crude sulfone was 86 mg. (59%), m.p. 138-169". -4n analytical sample, prepared by re(12) A D D E D Ih' P R O O F . - A preliminary i l l vi00 assay of V I I I a in male crystallization from acetone-water, had n1.p. 159.5-160'. albino mice against D . pneiimoniae type I1 using potassium penicillin A n d . Calcd. for C16H20S?OiS2. c, 46.14; H , 4.85; G as a standard indicated a potency of approximately 7 unita/mg. S,6.72. Found: C, 46.11; H , 4.91; S , 6.,59. This assay was carried out under the supervision of Dr J. k i n , Bristd Laboratories, Syracuse, New York. CAMBRIDGE 39, MASSACHUSETTS
[COXTRIBUTIOS FROM
THE
ENZYME CHEMISTRY BRANCH, CHEMICAL WARFARE LABORATORIES]
Chemical Reactions of Nerve Gases in Neutral Solution. I. Reactions with Hydroxylamine BY BERNARD J. JANDORF RECEIVEDFEBRUARY 14,1956
-
Hydroxylamine and certain of its N-substituted derivatives react with organophosphorus anticholinesterases (DFP, GI3 (R)(R'O)stoichiometrically at room temperature and pH 7 . 5 . The over-all reaction is ( R ) ( R ' O ) P : ( O ) X 4- ~ ~ H z O H P( : 0) OH H X 4- SZ S H : $ 2 H t 0 , and is accompanied by loss of anticholinesterase activity. Evidence for several steps in this over-all reaction is presented. Hydroxylamine in 2000-fold excess does not prevent inhibition of cholinesterase by GB and does not reactivate GB-inhibited cholinesterase. An adaptation of the h>-droxamic acid reaction to the colorimetric determination of micromole amounts of SH20H is described.
+
+
+
Organophosphorus anticholinesterases 'nerve gases") react with susceptible enzymes in a stoichiometric, irreversible fashion. The reaction consists of several steps, the last of which represents an alkylphosphorylation of a serine moiety. X search has been in progress in this laboratory for compounds which can react rapidly with nerve gases under physiological conditions of pH and temperature with the aim of finding substances which may successfully compete in the animal body with cholinesterase (ChE) for the inhibitor. I n the first attempt to find such a competitor, naturally occurring amino acids were screened for reactivity with DFP (diisopropyl phosphorofluoridate) and GB (Sarin, isopropylmethyl phosphonofluoridate) under physiological conditions. These experiments were completely unsuccessful, and simi(I
(1) This paper describes experiments which have been carried o u t during 1950-1961 and formed t h e basis of a classified report issued a t t h a t time. (2) B. J. Jandorf, H. 0. Michel, N. K. Schaffer, R. Egan and W. H. Summerson, F a r a d a y SOC.Disc., in press (1956).
lar negative results have been reported by The search was therefore widened to include other representative chemical structures, not necessarily known to exist in protein molecules but potentially able to react with nerve gases. There exists in many respects a similarity between the reactions of susceptible esterases with their substrates and with nerve gases (leading to acylation and phosphorylation of the enzymes, respectively). With this in mind, one of the first substances to be tested was hydroxylamine which was known to react rapidly with acyl anhydrides and esters,6 including a~etylcholine,~ with formation of the corresponding acylhydroxamic acid. A%sa working hypothesis it was assumed that an (3) E. F. Jansen, 11.-D. F. Xutting, K . Jang and A . I;. Balls, J . B i d . Chem., 186, 209 (1950). (4) T. Wagner-Jauregg, J. J. O'Neill and W, H. Summerson, T H I S JOURNAL, 73, 5201 (1961). ( 5 ) W. K. Berry, K. P. Fellowes, P. J. Fraser, J. P. Rutland and A . Todrick. Biochem. J . , 59, 1 (1955). (6) F. Lipmann and L. C. Tuttle, J . B i d . Chem., 159, 21 (1845). ( 7 ) S . Hestrin. ibid , 180, 219 (1949).
Aug. 5, 1956
CHEIIICAL
REACTIONS OF
XERVE
analogous reaction might take place between nerve gases (which contain an ester linkage) and hydroxylamine. The results to be presented show that, while hydroxylamine does react with nerve gases a t room temperature and neutral pH, the mechanism of this reaction is somewhat different from that with acyl esters and may be represented by the equation
iYz
+ NHz + H X + 2H20
(1)
The detailed study of this reaction forms the gist of this report. Results Mechanism of Reaction between NHzOH and GB.-The reaction of limiting amounts of GB with hydroxylamine, followed manometrically in bicarbonate buffer (see example under Experimental) leads to the liberation of two moles of gas per mole of GB (Fig. 1).* The half-time ( t o 5 ) of the reaction is 11-12 minutes a t all concentrations of GB employed, indicating that with this large excess of S H 2 0 H the rate-determining reaction is pseudo first order. This half-time is to be contrasted with an extrapolated t 0 . 5 of 7.5 hours for spontaneous hydrolysis of GB (indicated in Fig. 1 by a dotted curve) in this particular experiment. (Other runs which were extended over a considerably longer period of time yielded a t0.5 for spontaneous hydrolysis of 9-10 hours under these conditions.)
GASESLVITH
3687
f~YDROXTLXRIINE
in the liberation of one mole of gas per mole of added GB. This gas is absorbed by neither acid nor alkali, and has been presumptively identified as nitrogen. The tO.5 of its evolution is the same as that found in bicarbonate. Determination of the $H of the vessel contents after completion of the reaction indicates the simultaneous production of acid. These results therefore indicate that the action of NH2OH on GB is not that of a catalyst of spontaneous hydrolysis (which would also result in the production of two moles of gas in bicarbonate per mole of GB, both of which would however be CO,) since, if the latter were true, there should have occurred only acid production without gas evolution in phosphate buffer. TABLE I GASEVOLUTION IN THE REACTION BETWEES GB AND ExCESS (100 MOLES) HYDROXYLAMISE IS 0.025 M PHOSPHATE BUFFER,p H 7.4 AT 25" G B added (pmoles) 0 4 Absorber Gas evolved (pl.) 49 137 hTetgas evolved due t o reaction between G B and NH20H (pmoles) 3.93 Gas evolved per mole G B (moles) 0.98 1 0 . 5 (min.) 13 Final p H of reaction mixture
0
6
hvaOH 32 178
7.21
0
6 HCI 47.5 199
6.50
6.76
1.08 12 7.00
1.13 15 6.98
7.22
Further evidence against the catalytic nature of NHpOH in this reaction is provided by the data in Fig. 2 which show a rapid disappearance of NH20H
0
0
IO
20
30
40
50
60
m i n u t e s of incubation.
20
40
60 80 100 120 minutes o f incubation.
140
160
180
Fig. 1.-Reaction of GB with excess hydroxylamine in bicarbonateeC02 buffer, p H 7.54, t 25'. Figures on curves represent micromoles of reactants in 2.2 ml. Arrows =
Substitution of phosphate for bicarbonate buffer in an atmosphere of nitrogen (Table I) results (8) D a t a in Fig. 1 have been corrected for a small amount of gas production in a control containing 100 pmoles NHzOH b u t n o GB. This blank, amounting to about 30 p l . of gas over t h e 180-minute period, probably represents a slow decomposition of NHzOH in neutral solution.
Fig. 2.- Disappearance of hydroxylamine in reaction with GB. S H 2 0 H ( 5 X lO-3iM) with (curve a) and without (curve b) 0.1 LIT GB was incubated in 0.1 M phosphate, pH 7.2 a t room temperature. Aliquots were removed a t intervals and tested for remaining NH20H by the colorimetric procedure (see Experimental).
in the presence of excess GB. A balance experiment was therefore run in which, in addition to the measurement of gas evolution due to decomposition of GB and of NHzOH disappearance, production of ammonia was measured also, since it was suspected that the net production of one mole of acid in reality constituted the liberation of 2 moles of acid with
3688
Yol. 7s
BERN.LRD J. J.\XDORF
TABLEI1 XI-I?OH,A N D r;ORMATIOS O F SHn, IK TIIE REACTIOS H E T \ \ ' E E T S I I ? o I I .\XI1 Reaction medium: 0.025 Jf phoqphnte, PH 7.4. t = 25'. Gas phase: nitrogen. After 90 miuutesa ZIInO? was tip[)cd in, the remaining S H ? O H was determined manometrically, the vessel contents were filtered and used for SH3 cietertriinations. All figures = hnioles of compound per vessel. 30 30 30 3il Reaction N H 2 0 H: 30 mixture )(GB: 0 S 0 2 4 nISAPPEARAKCE OF
/
(1)
Gas produced in 90 min. S e t gas produced = GB reacted
Reaction between S H 2 0 H and GB 0.42 1.88 3.04 1.46 2.62 (2)
K!O produced by M n O ? KH?OH disappeared
9 H ? O H disappearance 12 64 10.80 8.16 3.68 8.96
(3) Ammonia formation 1.77 3.62 1.85
KH3 present Ket SH3 produced
4.31 3.89
5.45 5.03
7.24 10,SO
5.98 13.32
5 28 3 51
6.17 4 40
4.06 2.29
(4) Balance sheet 2 01 -1v 2 84 2 78 2 25 3 42 S H ? O H disappeared per pmole GB reacted 0 88 Av 0 98 1.26 0 88 0 91 S H 3 formed per pmole GB reacted The reaction was stopped deliberately before being complete to minimize spontaneous deconipositioii of SIIYOI I
simultaneous liberation of one mole of base. The results of such a balance study are shown in Table 11. Though the results are not as precise as might be desired (probably due in part to the fact that addition of MnOndid not stop the reaction between GB and NHzOH instantaneously) they nevertheless indicate the disappearance of 3 moles of NH20H and the production of 1 mole of NH3 per mole of GB destroyed. When a limiting amount of N H 2 0 H is allowed to react with excess of GB there is a net evolution of one mole of gas per mole of GB in bicarbonate, and no gas production in phosphate buffer (Table III).9 TABLE I11 REACTION BETWEEN EXCESS GB ASD LIMITINGAMOUKTS OF KHsOH (For experimental details, see text) NHzOII added (pmoles) GB added (pmoles)
0 100
2
1 100
100
4 100
c, 100
(1) I n 0.026 ~ l fNaHC03-CO? buffer, pH 7.4 Gas evolved a t zero time (extrap.) (1.11.) S e t zero time {(HI.) evolution (pmoles) Gas evolved per mole NHzOH (moles)
(2)
44
68 2-1 1.07
84 40 1.79
1.07
0.90
135 91
177
4.06
133 5.93
1.02
0.99
In 0.025 ;M phosphate buffer, p H 7.4
Gas evolved a t zero time (extrap.) (1.11.) S e t zero time evolution (1.11.)
12
16
14
14
4
2
2
The above results, taken together, lead to the formulation of the reaction between NH20H and GB as a multiple step reaction 0
I/ CH3-P-F I
0
il + SH20H +CHB-P-ONH~ I
+ H F (2)
OCH( CHI)?
OCH(CH8)z
1
(9) In this experiment the rates of gas production after the initial rapid evolution, which were high in all cases due t o the superimposed spontaneous hydrolysis of excess GB. were extrapolated back t o time of mixing of the reactants, and the amount ai gas production due t o the presence of NHnOH was thus determined.
0
I
I + 2SH30H +CH?-P-OH
+ Y: + S H i + " 2 0 (3)
OCH(CH3)z
which, in more general terms, yields equation 1 as the summation. The reason for representation of the intermediary (I) as pictured above, rather than as the hydroxamic acid CH,-P(: 0) [OCH(CH3)2]NHOH will be presented below. The results of Table I11 indicate, as might be expected, that in the presence of a limiting amount of NHzOH only reaction ( 2 ) occurs which results in the production of one mole of acid but no other gas. In the complete reaction, which takes place with excess NH20H, the rate-determining step appears to be the first (equation 2) since the values of t o 5 for acid production and nitrogen liberation are the same. $Then the initial course of reaction between a fixed amount of GB (3 pmoles) and a varying stoichiometric excess of N H 2 0 H (10, 25, 50 pmoles) in bicarbonate buffer is plotted as log [ a / ( n - x)] (where a = maximal gas evolution = 6 pmoles and .w = gas evolution a t time t ) os. time, straight lines are obtained (Fig. 3a) indicating that for each concentration of NHzOH the reaction shows the characteristics of a first-order reaction, a conclusion already previously drawn from other evidence. A plot of t o 6 against the molar ratio of GB :NHzOH also yields a straight line (Fig. 3b), which leads to the bimolecular rate constant 1 K z = t(NH20H) In
&- = 2.45 1. mole-' min
Effect of $H.-No systematic study of this aspect was carried out. Table IV presents first-order constants, obtained as described in a previous section, a t various values of pH, for the reaction between 4 pmoles of GB and 100 pmoles of NH20H. From these values second-order constants have been calculated, on the basis of total initial hydroxylamine concentration (Ct = 100 pmoles in 2.2 nil. = 4.54 X lo-' AI), as well as on the basis of undissoII-) ciated hydroxylamine Cu = K'Ct '(S'
+
Aug. 5 , 1956
cHEiWICAL R E A C T I O N S O F N E R V E
20
IO
30
40
minutes of incubation.
50
GASESWITH HYDROXYLAMINE
60
Fig. 3 -A, reaction between 3 pmoles of GB and 10,25and 50 pmoles of NHzOH in bicarbonate-Con buffer, pH 7.54, t 25'. For method of plotting see text B. plot of tu 6 against ratio of reactants (data derived from Fig. 3 4 .
where K' is the apparent dissociation constant of the reaction NfH30H NH20H f H+. Titration of 4.5 x lop2AMNH20H.HC1, containing 0.025 ,If NaC1, yielded a pK' = 6.03 for this reaction. While the data are not sufficiently numerous for an unequivocal conclusion to be drawn, they nevertheless indicate that NHzOH is the species involved in the reaction with GB. TABLE IV REACTIVITY BETWEEN G B (4 MMOLES) A N D SHzOH (100 ~ Y O L E S ) The pH of bicarbonate-C02 mixtures was calculated, that for other buffers determined by glass electrode; t = 25 . EFFECTO F pH
OX
Buffer
Gas phase Nz COz
K-H phthalate, 0.05 M NaHCOa, 0.025 M Na-K phosphate, 0.05 M hTaHCOs, 0.025 d l NaIICOa, 0.10 .If
?.-I
5% COz, 95% Nz 5 % COz,
pH 4.62 6.14
3G89
Second-order First-order const. based on constant NHzOH (min.3) Total Undissc. No reaction 0.0273 0.60 1.07
6.9.5 7,54
,0339 .Of307
1.17 1.34
1.31
8.04
,0606
1.33
1.31
1.37
95y0 Na
Effect of Oxygen.-A few experiments were run in bicarbonate buffer with 5y0 COZ in oxygen (instead of nitrogen), and in phosphate buffer with air as the gas phase. I n both cases there was a rather high rate of evolution of gas (not further identified) in vessels containing N H 2 0 H only. When GB was also present under these conditions the net gas evolution (;.e.] the increase over that due to NHrOH alone) appeared to occur a t about the same rate and to the same extent as under anaerobic conditions. Reactivity of Hydroxylamine with DFP and Tabun.-Hydroxylamine reacts with D F P in the same way as with GB, but its reactivity is much lower (Fig. 4), the t o . 5 for this compound being approximately 90 minutes. Comparison with the rate of spontaneous hydrolysis of D F P a t equal concentration (dotted curve) shows that a reaction does occur, and the total gas evolution approaches t h e expected 2 moles per mole of DFP.
I .
1
0.5
.
I
.
I
.
:.'.'.'.'.'/G--J
2 3 4 hours of incubation.
10
1.5
5
6
7
23
Fig. 4.-Reaction of DFP ( 4 pmoles) and Tabun (4 pmoles) with hydroxylamine (100 pmoles) in bicarbonate-Con buffer. For other experimental details and controls see text.
Tabun (ethyl N,N-dimethylphosphoramidocyanidate) presents a slightly different behavior. I t was expected that, if Tabun reacts with NH20H by a mechanism analogous to that of GB, one mole of gas (nitrogen) should be liberated in bicarbonate (or in phosphate) buffer but there should be no net acid production since HCN is too weak an acid to liberate COS from bicarbonate buffer. The results of Fig. 4 show for this compound a gas evolution which approaches one mole per mole of Tabun added, with t o . 6 of approximately 30 minutes for this phase of the reaction. This evolution is followed by a gas absorption: a t the end of 23 hours the net gas production amounts to less than 0.4 moles per mole of Tabun. A possible explanation for this phenomenon may be a slow reaction of NH2OH with the N-P linkage of Tabun, resulting in liberation of dimethylamine which is a strong enough base to cause C 0 2 absorption from the gas phase. That this explanation may be the correct one is shown by the fact that Tabun, previously hydrolyzed with alkali a t the P-CN bond and neutralized, exhibits only gas absorption when treated with NHzOH under these conditions. Alkali-hydrolyzed GB shows neither gas production nor absorp tion on subsequent treatment with NH20H. So inorganic phosphate is liberated when Tabun or D F P is treated with hydroxylamine for 24 hours. Reactivity of GB with Derivatives of Hydroxylamine.-The reactivity of some substituted hydroxylamines against GB was compared on an equimolar basis with that of the parent compound (Table V). The results show that substitution in the hydroxyl group of hydroxylamine greatly lowers or abolishes reactivity. Monobenzylation of the amino group lowers the reactivity t o approximately 50% that of NH20N; the c1at:i con-
Vol. 78
HERNARD J. J . ~ N D O K I ;
3690
TABLE 1. REACTIVITY OF HYDROXYLAMINE DERIVATIVES (100 MOLES) WITH G B (4 MOLES) (Bicarbonate-CO? buffer, pH 7 . 5 ; t = 25') Cumpound
Name
Hydroxylamine .Iminooxyacetic acid
Structure
Source
Kemarks
li.6
(1
C
0
C
b
S-Benz!.lli?.tlrox ylamirie
KH1OH SH20CH2COOH SHzOCH?COOC?Hi SHZOCH~C~H;, CcHbCHzSHOH
13
h
.\ypros. 200 25
S,SDibenzylhydroxylamirie
(CaHjCH2)jSOH
b
C
S-H!.droxyl-or-pyridone
m S 0 .
U
35
Liberates 1 mole of acid ixi bicarbonate, no gas in phosphate Very insoluble
Alcetoxime (CHa)jC=SOH C Locally synthesized. 'I Obtained through the Chemical and Biological Coordination Center. liberation equal to, or less than, that due to spontaneous hydrolysis of GB.
cerning gas evolution with this compound might indicate a reaction proceeding only through the first step of decomposition (equation 2). Results with the dibenzylated compound are equivocal since the material was present in suspension rather than in solution, and its true concentration in the reaction mixture was probably much lower than indicated. S-Hydroxy-a-pyridone may be regarded as a substituted hydroxamic acid (RCOS(R')OH) and its reactivity, albeit smaller than that of XH20H, confirms the necessity of a free OH group in the N H 2 0 H molecule. If' The System NHzOH :GB :Cholinesterase.-In connection with the general purpose of these investigations, as outlined in the Introduction, information was desired on the following points: ( 1 ) does the reaction between NHzOH and GB result i n products which no longer show anticholinesterase activity? (2) will addition of XHzOH to cholinesterase (ChE) result in their competition for GB, with a resultant lowered anticholinesTABLE 1.1 THESYSTEM S H 2 0 H:GB :CHOLISESTERASE Procedure.-The niisture, s h o i ~ - n1)eIow (total vol. = 2.0 mi.) were incuhated for 2 Iiours a t 20' (Part 1) or 30 min. a t 38' (Parts 2 , 3 ) . After ;i I :A()(! dilution (for Part 1 only) the second ac]diti,,ll , and the mixtures were incubated further ( 3 0 tnin., 38" [vater was added Trhere necessary to equalize tlii. VIIIUII . At the end of the second incubation aliquots were tested 11. ChE activity. aCc ht iEv ,
Part
Title
I
'rest of reaction
2
:3
First inciil>atic>n
NILOH :10(i
"
GB ( 5 )
Second incubation ChE ChE C'hE
(70
of
control)
"'; y8
o
Test of ti
