827
THEANTIEXOTICPSICOFVR.~KINE
Feb. 20, 1960
ORGANIC AND BIOLOGICAL CHEMISTRY [CONTRIBUTION FROM
THE RESEARCH
LABORATORIES OF THEUPJOHNCO.]
Kinetics of the Hydrolytic Degradation of a Nucleoside, the Antibiotic Psicofuranine BY EDWARD R. GARRETT RECEIVED JULY 27, 1959 The nucleoside, the antibiotic psicofuranine, ~-amiiio-9-D-psicofurnnosylpurine,is degraded by hydrogen arid hydroxyl ions. The products are adenine and the sugar psieose with the former catalyst and posslbly with the latter. The rates are linear functions of the concentrations uf tlie neutral [PI, protonated [PH+] and anionic [P-1, nucleoside and can be expressed: d[P],,,,l/dt = /k?[H*] + k3[OH-]][Pi, -+ k,[H-][PH+] 4- kr[OII-] [P-]. The uncharged psicofuranine is hydrolyzed with bimolecular rate constants ca, 2 . 5 times faster than the charged species due to the repulsion of the catalytic species, H + or OH-, by the positively or negatively charged psicofuranine ions, respectively. Estimates of the p K , of the acid function in the sugar portion of the nuc1e:)siclehave been made froin the studies of the variations of the bimolecular r a t e constant for hydrolyses with pH CQ. 12.
TABLE I The new antibiotic, psicofurnnine (I),I 6-amino9-D-psicofuranosylpurine, is 2 unrqce ni-cleoside COXDITIOX: A N D OESER\-ED FIRST-ORDER RATECONSTAPXS ( h I N SEC.-') AT COSSTAXT pH FOR THE -4CID 11YDROLYSIS with antitumor and antibacterial pro; crties3 Determination of the kinetics of degradation o!' O F PSICOFURa4hTNE A?' 1 hlG./blL. Ohsd., Buffer comjxsition, .If I, possibly by hydrolysis to adenine (11) 3 r d the su10517" Run OC. pH [HCI] gar psicose (III), is of pharniaceutical iniyortance 1.51 1 30.' 2 . 0 3 0 . 0 1 for the estimation of stability in aqueous iorniula" 2
2 3
Zf,:?
5' ; 2 5'. 2 50.2
1 59 1.36 1 1.;
9
50.2
(i.9i
10 11 12 13
60.6 60.6 60 6
1.137 1.60 1 82
4 5
c. 7 8
tions, to contribute to process design for maximum yield and to determine the least degrading conditions for bioassays6 The determination of the $H profile of the rates of hydrolysis of a nucleoside a t several temperatures is also of fundamental interest since, to the author's knowledge, a complete study of hydrolytic degradation in this class of compounds has not been reported.
Experimental T h e characterization of the psicofuranine used in these studies has been reported recently.1'2a Sufficient psicofurnnine, generally 2.5 mg., previously dried a t 60' under high vacuum for 48 hours, was weighed into a tared 25-1111. volumetric flask,and made up to volume with the aqueous solvent of the appropriate hj-drochloric acid, acetic acid-sodium acetate, or sodium hydroxide concentration. Tables I arid I1 list tlie final conipositions. The solvent previously had been equilibrated in the appropriate constant temperature bath. The resultant solutions were placed in the constant tcmperature bath and assayed at recorded time intervals. (1) T. E. Eble, H. Hoeksema, G. A . Bo:-ack :&I G. M . Snvage Antib. and Cheino., 9, 419 (195gj. (2) (a) W. Schroeder and H. Hoekserna, Tms J O I J R N A L , 81, 1767 (1959). (b) This structure also ha: been proposed for angustrnycin C by Hsu Yuntsen, J . Aiiiibiotics ( J a g a n ) , 11A,2-14 (1938). (3) J. J. Vavra, A. Dietz, B. W. Churchill, P. Siminoff and H. J. Koepsell, Antib. and Chemo., 9 , 427 (19%) (4) C. Lewis, H . R. Reames and L. E. Rhulnnd, i b i d . , 9, 421 (1959). (5) J. S. Evans and J. E. Gray, ibid., 9, 675 (1959). ( 6 ) E. R . Garrett, J. Ani Phnvm. Assoc., Sci Ed., 48, 169 (1959).
1 .58
3 . 2 30.2 3'>2 5')5
1.26 l,l(i ,
60.6 1.24
4.65 1C.8
.(;2$5 ,ot5
--
16.8 19.P 01 .s 130 2(!5
./$t*>
.01 0:?5 i;Ti i!7\7 1')
262
59.6 139 293 424
.IJ~
.02 .03 .06 [CHa-
[CHICoOII.
14 15
16 17 18 19 20 21
22
coo-]
3.33 0.075 51.2 3 . 6 1 ,1756 50.7 4.04 ,1234 5 2 . 1 5.06 ,0326 52.1 5.68 ,0094 40.4 5.76 ,0100 .0100 50.0 5 . 7 5 60.2 5.75 ,0100 69.5 5.75 .0100 5'1.2
0.0122 ,0333 ,0837 ,0953 ,0900
,0900 ,0900 ,0900
1.64" l.lld 0.520e ,0731' ,0146' ,00439
,0126 .0481 ,151
KCII
23 50.5 4 . n ~ .4oo 0 ,100 . 60gh 24 5 0 . 5 4 . 0 1 ,2667 0.334 ,0666 .60Sh 25 50.5 4 . 0 2 ,2000 0.0500 ,050 a Rate constants calculated on the basis of equation 8 are in agreement with the experimental listed. Typical calculated 105kvnluesareb23.6, 1.53, 1.11,n0.519, f 0.0623, 0 0.0153 and 0.541. Thc analyses involved the reading of spectrophntometric absorharice in a Beckman moclcl B a t 630 m p of the acidtlereloped colored diphenylamine reaction product subsequent t o alkaline sodium borohydride reduction of interfering sugars. The procedure was that of Forist' which has been applied t o the chemical determination of psicofuranine in blood plasma and serum.8 Prior to the assay a 1-ml. aliquot TTas pipetted into a 5ml. volumetric flask containing sufficient S a O H t o neutral, ize the aliquot of the acid-catalyzed hydrolysate so as t o halt hydrolj-sis prior to the alkaline borohydride reduction of the liberated psicose. This solution was then brought up to (7) A . A. Forist, Anal. Chem., 31,1767 (1959). (8) A. A. Forist, S. Theal and H. Hoeksema, A n f i b . and Chcmo.,9, 685 (1959).
828
EDWARD R. GARRETT d [ P ] ~ / d l= ~ H + [ H + ] [ P ]=T ~ [ P ] T
TABLE I1 CONDITIONS A N D OBSERVED FIRST-ORDER RATECONSTANTS (k IN S E C . - ~ )FOR THE ALKALINEDEGRADATION OF PSICOFURANIXE AT 1 MG./ML. Run
26 27 28 29 30 31 32 33 34 35 36
OC.
[NaOH]
1701.
Calculated 0110
105~
40.4 0.100 12.31 0.0649 40.4 ,200 12.53 ,0919 411.8 ,300 12.89 ,150 40.5 ,400 13.00 .193 50.2 .loo 12.17 ,221 50.2 .200 12.45 306 50.2 ,300 12.62 .449 50.2 .400 12 73 .613 60.4 .025 11.35 ,361. 60.4 ,050 11.63 ,591 60.4 .075 11.80 . 671 37 60.4 .lo0 11.92 .734 38 59.9 .200 12.21 1.14 39 59.9 .300 12.37 1.35 40 59.9 .400 12.48 1.77 41 69 5 .010 10 74 0.311 42 69.5 .02o 11.03 0.556 43 69.4 .050 11.39 1.13 44 69.4 .ion 11.68 1 59 45 69.5 .200 11.97 2.72 46 69.5 .300 12.13 3.49 47 69.5 .300 12.13 3.02 48 69.5 ,400 12 25 3.51 49 69.5 ,400 12.25 4.41 Calculated from pH = p K , - pOH; where pK,'s are 13.535 a t 40", 13.262 a t SO", 13.017 a t 60" and 12.778 a t 70" from ref. 11; and pOH = - logf [ NaOH] , [ NaOH] is the experimental, and f the mean activity coefficient for NaOH a t the appropriate temperature from ref. 12.
Calculations and Results The hydrolysis of psicofuranine is subject to specific hydrogen ion and hydroxyl ion catalysis and a t constant pH, or constant acid and alkali concentration, is excellently first order. Figure 1 presents typical apparent first-order plots for the acidcatalyzed hydrolysis of psicofuranine as measured by less absorbance of the developed color a t 630 mp. Similarly, typical apparent first-order plots for the alkaline hydrolysis are given in Fig. 2. The upper part of Fig. 2 shows that in alkaline medium the color decreases to a reasonably consistent asymptote of ca.. 0.100 for 1 mg./ml. of psicofuranine initially. This asymptote is accounted for in the first-order plots given in the lower part of Fig. 2. Hydrogen Ion-catalyzed Hydrolysis.-Variation of acetic acid-sodium acetate concentration a t constant p H and ionic strength (see runs 23, 24, and 25 in Table I and curve C in Fig. 1) did not significantly affect the apparent first-order rate constant and thus did not implicate general acid-base catalysis. Variation of initial psicofuranine concentrations a t 0.42, 0.61 and 1.00 mg./ml. in hydrolysis studies a t 40" in 0.100 M HC1 also did not significantly affect the apparent first-order rate constant. Variation in pH, however, had a significant effect on first-order rate constants as is shown by the varying slopes of the plots in Fig. 1 so that the rate dependence may be postulated as
(1)
where k H t is the catalytic rate constant for specific hydrogen ion catalysis, IP]T is the total psicofuranine concentration and k is the apparent first-order rate constant a t constant pH. The conditions and observed first-order rate constants for the hydrolysis are given in Table I except for the 40" hydrolysis data in HCl solutions which have been included in another p ~ b l i c a t i o n . ~ The rate constants were determined from the slopes of plots such as are shown in Fig. 1
I
volume with water and the assay procedure applied. There was no need to neutralize the solutions subjected to alkaline hydrolysis.
82
log
[PIT
=
-
[k/2.303]t
+ lOg[p]o
(2)
where [PI0 is the initial concentration of psicofuranine. In actuality, the absorbance of the color developed a t 630 mp by the analytical method was used in the determination of the rate constants. The plots of the observed first-order rate constants, k in sec.-l, against [H+] for the various temperatures are given in Fig. 3 and the resultant slopes are listed in Table 111. These slopes as per equation 1 are the values in l./mole/sec. for the specific hydrogen ion catalytic rate constant , k', for the hydrolysis of protonated psicofuranine. These plots do not go through the origin as the psicofuranine of pKa 3.9 actually consumes some of the hydrogen ion concentration in its salt formation so that the molarity of psicofuranine (0.037 izI a t 1mg./ml.) must be subtracted from the apparent HC1 molarity to obtain the true HC1 concentration. This correction should have no effect on the slope of such plots. TABLE I11 CATALYTIC RATECONSTANTS, k, A N D k2, IN L./MoLE/SEC. FOR THE ACID HYDROLYSIS O F PROTONATED A S D NONPROTONATED PSICOFURANINE T ,O C .
10'ki'"
log kib
10kz'C
log k r d
30 2.43 -2.72 ... .... 40 8.58 -2.CI.l 0.247 -1.62 50 27.7 -1.56 0.708 -1.16 TS.9 -1.16 2.70 -0.58 60 70 ... .... 8.5 +0 01 a Determined from the slope of the observed pseudo firstorder rate constant, k in sec.-l, against the molarity of HC1. 6 Determined from the intercept of the line formed by log k 21s.pH with a slope of unity and based on PH values less than 2.5. Calculated from k in set.-' a t PH 5.75 divided Estimated from the intercept of the by [H'] = 10-5.'5. line formed by log k us. p H with a slope of unity and based on pH values greater than 4.5.
An alternative estimate of k H t involving protonated psicofuranines, i.e., kl,may be determined by the plot of log k against p H assuming a slope of unity log k = log kl
- fiH
(3)
The intercepts for such plots from the studies a t the several temperatures are derived from the plots of the acid branch in Fig. 4 and are given in Table 111. Arrhenius plots according to the classical relation of the specific rate constant, k or kl', are given in log kl -(AH,/2.303R)(l/T) log C (4) Fig. 5 as plots of log kl vs. 1/T, where R is 1.987 cal. deg.-l mole-' and T is the absolute temperature.
+
(9) E. R . Garrett and L J. Hanka, submitted for publication to J . A m , Phorm. Assoc., Sci. E d .
THEANTIBIOTICPSICOFURANINE
Feb. 20, 19GO
400
-0 80
829
1
ID\\ 0
40
80
120 160 TIME IN HOURS.
200
Fig. 1.-Typical pseudo first-order plots for the acidic degradation of psicofuranine as followed by absorbance of developed color (50"). The curves, PH and run number are: A, 5.68, 18: B, 5.06, 17; D, 3.61, 15. For curve C a t PH 4.02, the acetate ion concentration and run numbers are 0 , 0.100M, 23; A, 0.067M, 24; O.O5OM, 25.
.,
0
002
004
0.06
008
b".
010
Fig. 3.-Plots of pseudo first-order rate constants, k in sec. -1, for the acidic hydrolysis of psicofuranine as a function of the apparent molarity of HCl, [ H c l ] ~ .
TIME IN HOURS.
Fig. 4.--pH profiles of the logarithm of the apparent firstorder rate constant, k in sec-1, for the hydrolytic degradation of psicofuranine.
TIME IN HOURS.
Fig. 2.-Typical plots of the pseudo first-order alkaline degradation of psicofuranine as followed by absorbance of developed color (GO"). The curves, R'aOH concentration and run number are: A, 0.02Ei M , 34; B, 0.075 M,36; C, 0.400 M,40.
The sloDe of this Dlot is -5.056 so that the heat of activat;on, AFIa, 'is 23.1 kcal./mole and log C is 14.060.
The most complete log k vs. p H profile for the acid branch was obtained a t 50" for 0-6 p H and is plotted in Fig. 4. The slope of this 50' plot is not constant; i t shows an enhancement in log k in the 3 . 5 4 . 5 p H region. However, a ready explanation can be found for this phenomenon. Actually, the rate of total psicofuranine [PIT concentration change of equation 1 is the sum of the rates of change of the protonated, d [PH+]/dt, and non-protonated, d [P]/dt, forms so that
+
d [ P ] ~ / d t= d ( P 1 [PH+l)/dt = -kr[H+][PH+] ki[H+][P] = - ~ H + [ H + ] ( [ P H + ] [PI]
-
The ionization equilibrium of P H + zP
+
+H+
(5)
(6)
Vol. 82
EDWARD I
