3258
CHRISTINEZIOUDROU AND GASTON L. SCHMIR
added t o a suspension of phenanthrenequinone (10.4 g., 50 mmoles) in dry benzene (100 ml.), under nitrogen with stirring. A clear, red-brown solution was observed after 45 rnin. a t 20' Within 2 hr., the solution was nearly colorless. Removal of the benzene in vacuo left a yellow oil which soon became a nearly colorless crystalline mass (16 g . ) . One recrystallization from hexane gave t h e colorless phenanthrenequinone-trimethyl phosphite adduct (VII), 111.p. 71-73', in 90cL yield (15 g.). The analytical sample had m . p . 74-75', Other trialkyl phosphite adducts were prepared in c a . 95ci.L yield by this method. The reaction with aliphatic a-diketones, like biacetyl, is particularly exothermic and must be moderated by external cooling. ( b ) In the Absence of Solvents. Procedure B.-Trimethyl phosphite (6.82 g., 50 mnioles) was mixed with solid benzil (10.5 g., 50 mmoles) under nitrogen. An exothermic reaction ensued, and a colorless oil resulted within 30 rnin. The oil was dissolved in hexane (30 ml.) and t h e solution was cooled a t 0". Colorless crystals of the benzil-trimethyl phosphite adduct (X), m.p. 47-49' separated within 8-10 hr.; the yield was nearly quantitative. Biacetyl (74 9.) was added dropwise t o trirnethyl phosphite (135 9.) under nitrogen, with stirring and external cooling. The
[CONTRIDCTIOX FROM
THE
VOl. 85
mixture was then kept a t 60" for 15 min. and submitted t o fractional distillation. The colorless biacetyl-trimethyl phosphite adduct (XIII) was collected a t 45-17' ( 0 . 5 mrn.); the yield was quantitative. The liquid adduct becomes slightly yellom- on standing, or in contact with air, even if the latter is dry, since biacetyl is formed by oxidation. Triphenyl Phosphite Adducts.-A suspension of phenanthrenequinone (2.08 g., 10 mmoles) in triphenyl phosphite (12.4 g . , 40 mmoles) was kept 16 hr. &t l l O o , under nitrogen with stirring. The pale yellow solution was cooled, treated with hexane, and filtered. The colorless insoluble phenanthrenequinone -triphenyl phosphite adduct ( I X ) 14.5 9 . ) had m . p . 143-145'. Onerecrystallization from benzene-hexane gave material of m . p . 145-147'.
Acknowledgment.-\.\.'e are very grateful to Dr. J . Lancaster of the American Cyanamid Co. (Stamford, Conn.) for the P3I n.m.r. spectra; to Dr. Lancaster and Prof. E. Eliel of the Univ. of Natre Dame for their cooperation in H' n.m.r. spectrometry; to Dr. D. C. Eelson of the Applied Physics Corp. (Monrovia, Calif.) for the Raman spectrum.
DEPARTMEST OF BIOCHEMISTRY, \-ALE UNIVERSITY SCHOOL O F MEDICINE, NEW HAVEN11, Cosn-
The Participation of the Amide Group in the Solvolysis of Phosphoric Acid Esters. I. Phosphotriesters in Alkaline Media BY CHRTSTINE ZIOUDROUA N D GASTONL. SCHMIR RECEIVED M A Y9. 1963 Phosphotriesters of type I containing a neighboring amide group have been synthesized Exposure of such triesters to dilute ethoxide or t-butoyide solution at room temperature results in the rapid formation of cyclic structures and expulsion of a phosphodiester fragment Detailed kinetic data and spectrophotonietric evidence support the hypothesis that t h r rate-limiting step of the reaction consists of intramolecular nucleophilic attack of the amide anion on the alkyl carbon, resulting in carbon-oxygen cleavage The ability of the phosphodiester anion t o act as a leaving group is compared to that of other anions Quantitative measure of the efficiency of the intramolecular process is provided by comparison to the solvoll-tic behavior of ethyl diphenyl phosphate.
Introduction Intramolecular nucleophilic reactions of derivatives of carboxylic acids have received close scrutiny in the past decade because of their possible relevance to the mechanisms of certain enzymatic events.' I t is less well known t h a t the solvolytic behavior of esters of phosphoric acid is also profoundly influenced by neighboring nucleophilic functions. While there has been sporadic description of phenomena best explained on the basis of intramolecular processes, detailed mechanistic studies have been few and little kinetic information is available. For example, the ease of hydrolysis of ocarboxyphenyl dihydrogen phosphate (salicyl phosphate) has been ascribed2 to participation of the o-carboxylate ion. The rapid acid-catalyzed isomerization of phosphomonoesters of 1,%diols ( e . g . , a-glycerophosphate) seems to proceed via the formation of an intermediate cyclic diester. Numerous investigation^^^,^ have established that the rate and direction of hydrolysis of phosphodiesters is markedly affected by the presence of a vicinal hydroxyl group, whose intervention in the hydrolytic process is responsible, for instance, for the alkaline lability of ribonucleic acids5 (1) (a) M .I,. Bender, C h e m . R e v . , 60, 53 (1960), [b) T C. Bruice, Brookhaven S y m p o s i a i n Biology, NO. 16, 52 (1902). ( 2 ) ( a ) J . D . Chanley, E . M . Gindler, and H Sohotka, J A m . C h e m . SOC, 74, 4317 (1Y52); ( h ) F. R. Atherton, Chem. Soc. (London) Spec. Publ. S o . 8 , 1957, p. 77. (3) ( a ) MLC. Bailly, C o m p f . r e n d . , 106, 1902 ( 1 Y 3 8 ) ; 206, 443 (1939), ( h ) L) hl. Brown and A. R. 'Todd, J . Chem SOC.,41 (1952). (4) ( a ) 0 . Bailly a n d J. G a u d Bull. soc. c h i m . France, 2, 354 (1Y35); Kates. I. J . Biol. C h e m . , 1 7 6 , 7Y (1948); 166, 61.5 (1950); (b) E Baer a n d & (c) D M ,Brown a n d .4.R . Todd, J . Chem. S o c . , .5'2 (1952); 2040 (19.53); i d ) 11 M. Brown, D . I Slagrath, a n d A . R . T o d d , zbid., 2708 ( 1 9 5 2 ) ; (e) D. M Brown a n d H . SI.Higson, i b i d . , 2034 ( 1 9 5 7 ) ; ( f ) L> A I Brown. G. E. Hall, and H Sf.Higson, rbid , 1360 (1958) (9) D . M . Brown, G . E. Hall, and R. Letters. i b i d . , 3547 (1959). ( 5 ) D. M Brown a n d 4. R! T o d d , in "The Nucleic Acids," Vol I, E. ~
Alkaline treatment of phosphotriesters derived from ethanolamine6 or ethylene glycol' affords products whose nature suggests the involvement of the neighboring amino or hydroxyl function in the solvolytic reaction. A recent report has demonstrated that the fast rate of hydrolysis of dimethyl phosphoacetoin8 in weakly basic solution is a consequence of the presence of an adjacent keto group. I t appeared of interest to examine the effects of another nucleophilic entity upon the solvolysis of phosphoric acid esters. To this end, substances incorporating an amide grouping appropriately situated vis-a-vis a pkospkotriester function were studied in alkaline media. The intramolecular interaction of the ionized amide group with the phosphotriester moiety is documented in this communication. Results In preliminary experiments, phosphotriesters of general structure I were exposed to dilute sodium ethoxide or potassium t-butoxide a t room temperature. Such treatment resulted in rapid, extensive, and irreversible spectral changes in the ultraviolet region (Fig. 1). Repetition of this procedure on a preparative scale (see Table VI, Experimental) resulted in the isolation of cyclic products I1 (in yields of 70-959&) and of phosphodiesters (yields of 30-95%). The A*-oxazoline IIa, formed from I a , Ib, and Id, was shown to be identiChargaff and J. N . Davidson, E d , Academic Press, I n c , New Y o r k , S . Y , 195.5, p . 409 ( 6 ) ( a ) D . S I , Brown a n d G 0. Oshorne, J . Chem. SOL.,2590 ( 1 9 5 7 ) . (b) G. J D u r a n t , J . H . Turnbull, and W. Wilson, Chem. I n d . i L o n d o n ) . 157
(19,58). (7) (a) 0. Bailly and J . G a u d , Bull. soc. c h t m F r a n c e , 3, 1390 (1936), (b) D. M .Brown a n d N . K . H a m e r , J . C h e m Soc., 400 (1900). (8) F. Ramirez, B. Hansen, and S . B . Desai, J. A m . C k e m . Sor , 04, 4588 (1962).
PHOSPHOTRIESTERS IN ALKALINEMEDIA
Oct. 20, 1963
c
3259 I
I
I
J
1.0
log [KOBu-t],
Fig. 1.-Spectral changes in triester I b on treatment with ethoxide solution: A, I b a t 1.3 X M in ethanol; B, after exposure t o 0.2 M sodium ethoxide for 20 min. a t 30".
cal in spectral properties and m.p. with the compound prepared by condensation of methyl p-nitrobenzimidate and ethanolamine. Similarly, the dihydrooxazine IIb (from IC) was identified by comparison with a specimen obtained both by the iminoester method and by cyclization of N-(3-hydroxypropyl)-p-nitrobenzamidewith 0 0
I1
NOz ~ ~ N H ( C H & O IP O R I
Fig. 2.-Dependence of rate of cyclization a t 28" of IC on t-butoxide concentration; triester a t M ; 0, rate measured by increase of absorbance at 310 m p ; 0 , rate measured by decrease of absorbance a t 340 mp. Solid curve is calculated according t o eq. 5, with assumed values of k, = 1 min.-I, K = 100
.-'E.
E
22
ORa
Ia, R1 = RZ = C&, n = 2 b , K I = RZ = CsHjCHz, n = 2 Rz = CgHj, n = 3 C, RI CsHj, n = 2 d, RI RP = 2-p-nitrobenzamidoethyl -3 IIa, n = 2 b,n = 3
thionyl chloride. The phosphodiesters were isolated either as the monocyclohexylammonium salt (in the case of diphenyl hydrogen phosphate) or as the free acid. To substantiate the structural assignment of 2p-nitrobenzamidoethyl phenyl hydrogen phosphate (obtained from I d ) , it was also prepared by phosphorylation of N-(2-hydroxyethyl)-p-nitrobenzamidewith one equivalent of phenyl phosphorodichloridate followed by hydrolysis. I t could be concluded that alkaline treatment of triesters I a t spectrophotometric concentrations (cu. lop4M) yields products identical with those isolated since, for example, the ultraviolet spectrum of an equimolar mixture of oxazoline TIa and dibenzyl hydrogen phosphate is the same as that shown by curve R of Fig. 1. During the cyclization of Ia, however, free phenol was formed in the amount of 3-10y0 (as measured colorimetrically) although, from spectral data, oxazoline formation appeared quantitative. The significant spectral difference between each triester and its cyclization products was utilized in a detailed study of the kinetics of cyclization in t-butoxide and ethoxide solutions. Pseudo-first-order rate constants were obtained in the presence of excess potassium t-butoxide from the increase in absorbance at 310 mp or the decrease in absorbance at 340 mp (see Discus-
-2
log [N,b,,,].
0
Fig. 3.-Dependence of rate of cyclization a t 30" of Ia on X. Solid curve ethoxide concentration; triester a t 2.5 X is calculated according t o eq. 5 with assumed values of kl = 1.13 min.-I, K = 18.4 M-'
sion). The results of these and similar experiments in the presence of excess ethoxide are given in Tables I and I1 and Fig. 2 and 3. TABLEI RATESOF CYCLIZATIOS OF TRIESTERS IN BUTOXIDE SOLUTION^ Compd.b
[KOBu-11 X 108, M
A ? mr
k , min. - 1
0.66 340 9.9 0.66 340 11 4 1 3 340 19.3 3.2 340 21 . o 12.3 340 22.6 Ib 0.66 310 0.72 0.66 340 0.66 3.2 310 1 27 3 2 340 1 20 28.2 340 1.56 Id 0.66 340 4 6 1 3 340 i 6 12.3 340 i.6 5 I n t-butyl alcohol a t 28'. At 1-2 X M. Wave length of measurement; rate determined either by increase of absorbance at 310 rnp or decrease of aosorbance a t 340 mp, Ia
CHRISTINEZIOUDROUPL~~
3260
GASTON L. SCHMIR
VOl. 85
Discussion Kinetic as well as preparative experiments indicate that alkaline treatment of phosphotriesters I , possessing a neighboring amide function, results in a cyclization reaction accompanied by the expulsion of a phosphodiester fragment. Two conclusions emerge clearly from inspection of the kinetic data presented in Table I and Fig. 2 : (a) the cyclization reaction a t 28' occurs with exceptional facility in the presence of dilute t-butoxide (e.g., t l / , in 0.003 Af t-butoxide varies from 2.9 min. for IC to 2 sec. for I a ) ; (b) the pseudo-first-order rate constant for cyclization increases with, but is not proportional to, base concentration. These kinetic observations suggest t h a t the transformation consists of a two-step process (Fig. 4 ) : (a) ionization of the amide group; (b) intramolecular, rate-determining, nucleophilic displacement on carbon by the oxygen anion. The mgchanism of Fig. 4 is described by eq. 1 and 2 , where (AH) and (a-) represent neutral amide and Figure 4. amide anion, respectively, (RO-) is t-butoxide or ethoxide ion, and (P) stands for the reaction products. Cyclization, under basic conditions, of amides of type I11 to A2-oxazolines has previously been o b ~ e r v e d . ~ The rate of formation of products or the rate of triester K For purposes of comparison to the phosphotriesters of AH
[NaOCzHsl X l o 2 , 'V
OF
(1)
ki
TABLE I1 RATE O F CYCLIZATIOS
+ RO- Jr X A-+P
(2)
TRIESTERS 1S ETHOXIDE SOLUTIOSa.b (3)
[ X ~ O C I HX ~ ] 102,
k , min. -
M
1
Compound Ib' 1 4 0.013 2.7 026 5.4 045 10.9 082 16.3 118 21 8 149 21.8 ,155
k , min. - 1
Compound Idc 0.13 0.021 ,26 042 52 . OS3 1.31 167 2.66 ,318 5.32 ,512
Rates measured by increase in absorbance a t 310 ethanol a t 30". Ib a t 2.6 X M; Id a t 1.5 X
rnp,
*
disappearance is given by eq. 3 and 4, which are related by the expressions A t = AH
K
In
M.
=
+
A [A-]/[AH][KO-]
Assumption of the mechanism of eq. 1 and 2 for the cyclization process leads to the predictions that the observed pseudo-first-order rate constant k will vary with base concentration according to eq. 3 and t h a t a plot of k us. log [RO-] will exhibit a sigmoid shape. At
TABLE 111 RATESOF CYCLIZATION OF IIIa Compd
IIIa
IIIb
a
[Pia0 C2Hal x 102, M h
0 13 26 52 1 31 2 62 5 24 10 5 21 0 0 11 36 70
ASD
k min
IIIb -1
0 007 016 031 072 125 215 368 539 2 0 3 9 7 9
IN
ALKOXIDE SOLUTIOV [KOBu 1 1 X 103 M C
0 7 3 2 3 2
k min
-1
s 9 29 2 27 2
M In ethanol ' I I I a a t 2 1-2 4 X 1 0 F M , I I I b a t 2 X a t 3 0 " , rate measured by Increase in absorbance a t 310 rnp rate measured by decrease in absorbI n t-but! I alcohol a t 28' ance a t 340 mF
this study, the rates of oxazoline formation from I I I a and I I I b in ethoxide or t-butoxide solution were also determined (Table 111) 0
IIIa, K = -KO?, 4; = -Cl b , R = - S O 2 , X = p-toluenesulfonyloxy 19) (a) H VJ. Heine, .I, A m C h e f i t . SOL., 78, 3708 (1956); (b) F. L R. E . Glick. and S.Winstein, E x p e r i e n t i a , 13, 183 (1957).
Scott,
high base concentration, the experimental rate constant k reaches the limiting value kl. The equilibrium constant K for amide ionization is evaluated simply from the inflection point of the sigmoid curve. The data of Fig. 2 indicate close agreement of the experimental values of k for the cyclization of IC with those calculated from eq. 5, employing values of kl = l min -' and K = 100 J P 1 Additional support for the existence of the aniide anion in these experiments may be derived from the following spectrophotometric observations' (a) the extrapolated zero time absorbance of the triester solution a t 310 mp was noted to increase with increasing base concentration; (b) the progress of the cyclization reaction could be followed by the decrease in absorbance at 340 mp, although the triesters exhibit lower light absorption than the products a t this wave length In the absence of added base. As a t 310 mp, zero time absorbances a t 340 mp increased with base concentration Presumably, both the instantaneous appearance of strong light absorption a t 340 mp and the increase in zero time absorbance a t 310 mp are due to the presence of the anionic amide species I t should be noted that a t t-butoxide concentrations greater than ca. 0.01 M, it was no longer possible to follow accurately oxazoline (or oxazine) formation by the increase in absorbance a t 310 mp, since sufficient amide anion was gener,ited so that its absorbance was similar to that of the reaction products. In these cases, the reaction rate was deter-
PHOSPHOTRIESTERS IN ALKALINE MEDIA
Oct. 20, 1963
mined solely from the decrease a t 340 mp. In several cases (Table I and Fig. a),rate constants were calculated from measurements both at 310 and 340 mp and found to be in reasonable agreement. The proposed mechanism is also supported by the rate measurements carried out in sodium ethoxide solution (Table I1 and Fig. 3). For Ia, the values of kl = 1.13 min.-' and K = 18.4 were chosen to calculate the curve of Fig. 3 according to eq. 5. The rate data obtained for triesters I b and Id led to the evaluation of kl and K for these substances also. In these instances, measurements were carried out over a limited range of alkali concentration, and the derived values of the constants kl and K must be considered less reliable. The values of kl and K in ethoxide solution calculated from eq. 5 are summarized in Table IV. TABLE IT CALCULATED COXSTASTS FOR CYCLIZATION IN ETHOXIDE SOLUTIOS~ Compd.
kl,
min.-J
K ,M - 1 18.4 2 25
k i K , .W-'min.-J
Ia 1.13 20.8 Ib 0.44 0.99 Id 1.23 13.1 16.1 IIIa 0 94 6 1 5 7 IIIb 1100 " Sodium ethoxide in ethanol at 30".
-Relative ki
1.2 0.4i 1 31 1. o
rateskiK
3.65 0.17 2.82 1. o 193
The observation that 0.03-0.10 mole of phenol is formed per mole of triester I a during the course of the cyclization process in ethoxide solution leads to a modification of the mechanism of Fig. 4, which accounts also for the fact that conversion of I a to oxazoline I I a is nevertheless quantitative. According t o the annexed scheme, l o Ia may undergo transformation to triester Ie (I, n = 2, R1 = C6H6, R 2 = C2&) via a transesterifireaction which results in the release of phenoxide ion. Similarly, triester Ie may be further converted to triester If (I, n = 2, R1 = R z = CzH6). The three triesters (Ia, Ie, and If) may be expected to yield oxazoline IIa, although possibly a t different rates. As is discussed below, the pathway involving If need not be considered further. Whether the rate of forma0
la
k
+I I a
!! + (C&O)2POH 0
ki CiHaO-
+ Ie -+ k3
C&O-
kr CgHsO-
C6HsO-
IIa
+ ( C Z H ~ O ) ( C B H11~ O ) P O H 0
I1 + If + IIa + (C2H50)2POH ks
tion of oxazoline I I a will approximate pseudo-firstorder kinetics with respect to Ia will be largely determined by the ratio k ! k n . If k >> k 2 , deviation from the first-order rate law will be negligible. When k and k2 are of similar magnitude, the ratio k j k 3 becomes the significant factor, since the closer this ratio is to unity, the smaller will be the deviation from first-order kinetics. To obtain an estimate of the magnitude of the transesterification rate constants k2 and kq, the rates of solvolysis in ethoxide solution of the model triester ethyl diphenyl phosphate were measured. l 2 Equation 6 (10) T h e terms k , k2, k3, e t c . , in t h e scheme represent pseudo-first-order r a t e constants tor t h e transformations shown, a t a fixed ethoxide ion concentration. (11) ( a ) H . D. Orloff,C . J. Worrel, a n d F. X . Markley, J . A m . Chem. Soc., 80, 727 (15.58); ( b ) H A . C. Montgomery, J. H . Turnbull, a n d W . Wilson, J C h e w . Soc., 1003 (1950). ( 1 2 ) R a t e s were determined spectrophotometrically a t 30° b y appearance of phenoxide ion a t 290 mG; see Table V I I , Experimental.
3261
formulates the two consecutive transesterification reactions of this substance. In the range of ethoxide concentrations of 0.015-0.18 hf, k6 varied from 0.010.13 min.-', and k j from 0.0006-0.009 min.-l. From these values and the rate data for I a (Fig. 3), i t is seen that k / k 6 varies from 24-7 and k / k , from 400-95 in the same alkali range. A comparison of the rates of cyclization of I a and Id (Table 11)suggests that ka is not much
P
0
I1
ks
(C6HsO)*POC*Hs-+ CiHaO-
I!
+
C ~ H ~ O P ( O C ~ H SCsH5O)~ ki
.1
CsHsO-
+
( C ~ H ~ , O ) J P = O CsHsO-
(6)
smaller than k . Assuming that kq = k i , it follows that k >> kq and that consequently k3 >> k?. I t may be concluded then that the species If is formed to a negligible extent. If it is assumed that the release of the first mole of phenol from ethyl diphenyl phosphate occurs a t about the same rate as from Ia (ie.,k6 = k z ) , then k,lk2 is of the order of 25-10, and the pathway Ia +.Ie + I I a is a t best a minor one. Oxazoline formation from Ia was found to obey first-order kinetics to a t least BOY0 of completion. In view of the estimated relative magnitudes of k , kz, and ka, the rate constant derived from the initial rates of oxazoline appearance is approximately equal to k . In addition, the amount of free phenol (310%) found a t the end of the reactions is consistent with the k / k 2 ratio estimated above. For a given triester, the observed rate constant k approaches the limiting value k1 a t high alkali concentration. At low alkali concentration ([RO-]
