DETERMINATION OF

STREPTOMYCIN

OF IONIZATION BY THE

CONSTANTS

INDICATOR

METHOD

P . S. N y s , E . M. S a v i t s k a y a , a n d T . S. K o l y g i n a

UDC 615.332(Streptomycinum)0.11.4:541~

The m e a s u r e m e n t of ionization constants of polybasic compounds is complex when the s e p a r a t e groups have s i m i l a r constants. Additional difficulties a r i s e in the determination of ionization constants lying at high pH values due to the i n a c c u r a c y of determining pH in the basic region. S t r e p t o m y c i n , C21tts?N?Oi2 , is an antibiotic having t h r e e titratable amino groups. Two of them a r e strongly basic guanidine groups, titratable in parallel in the pH interval of 10.0-12o0 (Fig. 1) and one is a weakly basic methylamino group (pKa ~ 8). In aqueous solution s t r e p t o m y c i n is able to exist in four f o r m s (BH]+, BH~+, BH +, B), the equilibrium between which is d e s c r i b e d by the corresponding ionization constants (we r e g a r d ionization of s t r e p t o m y c i n as ionization of a t r i b a s i c acid). [BH~+]aH+ . K1-

[BH +] all+

'

[B] all+ '

IBH+I'

(1)

w h e r e BH] +, BH~+, BH +, and B are t r i - , di-, and monovalent cations, and the uncharged f o r m o f streptomycin, r e spe ctively. The equilibrium BH] + ~ BH~+ (ionization of the methylamino group) d e s c r i b e d by K 1 can be examined independently, since titration of the methylamino group ends before titration of the guanidine groups begins (see Fig. 1)o Consequently, the usual methods used for calculation of dissociation constants of monobasic acids can be used to d e t e r m i n e K t. The sloping c o u r s e of the curve in the region of titration of the guanidine groups of streptomycin, and absence of a bend corresponding to titration of one group indicate that a parallel,independent o r c o n c u r r e n t ionization of guanidine groups o c c u r s in this region. As a r e s u l t of this an experimental determination of individual f o r m s of s t r e p t o m y c i n is not possible. Only ~he total n u m b e r of protons cIeaved upon ionization of I mole of BH~+ (r) can be d e t e r m i n e d experimentally. In the case of ionization of the guanidine groups of s t r e p t o m y c i n r changes f r o m 0 to 2. It is e a s y to show that r is associated with K2 and K s of s t r e p t o m y c i n in the following way:

(2)

KaaH + -]- 2K~K8

r = a~+ ~-/(2aH+ -~/(2Ka"

We did not consider the possibility of parallel ionization of the guanidine groups in deriving Eqs. (1) and (2). P r o c e s s e s which can o c c u r upon parallel ionization of the guanidine groups can be p r e s e n t e d s c h e m a t i c a l l y in the following way: ~.a BH z

BH +

~c B

Consequently, a dibasic acid undergoes two parallel ionization p r o c e s s e s having microconstants k a and kb, forming monovalent cations BH+ and HB +, r e s p e c t i v e l y . The l a t t e r , in turn, undergoes ionization All-Union S c i e n t i f i c - R e s e a r c h Institute of Antiobiotics, Moscow. T r a n s l a t e d f r o m KhimikoF a r m a t s e v t i c h e s k i i Zhurnal, Vol. 5, No. 9, pp. 58-62, September, 1971. Original a r t i c l e submitted June 22, 1970. 9 Consultants Bureau, a division of Plenum Publishing Corporation, 227 West 17th Street, New York, N. Y. 10011. All rights reserved. This article cannot be reproduced for any purpose whatsoever without permission of the publisher. A copy of this article is available from the publisher for $15.00.

576

with m i c r o c o n s t a n t s k c and kd, as a r e s u l t of which the uncharged m d e c u l e is f o r m e d . The given s y s t e m can be d e s c r i b e d by equations of m i c r o c o n s t a n t s :

pH 12

8

[BH+] an+

#

[HB+] all+

; kb=

; ko--I

[B] all+

'+l

;

[B] all+

I">----7

(3)

4 2 I

o

t

-

I

3 mg-eq/mmole 1

2

It is e a s y to show that for such a s y s t e m k a *k c = k b "k d.

I

4

F i g . 1. P o t e n t i o m e t r i c titration curve of s t r e p t o m y c i n . Explained in the text.

o, az a ,

k2 =

{ [ B N + ] + [ H ~ + l} all+_ = ks + kb;

z8.,~2

Fig. 2. Calculation of K 1 of s t r e p tomycin.

(4)

BH~+

ka=

8,6L,,,f

oa oa a4 az

Macroconstants of s t r e p t o m y c i n K2 and K 3 a r e a s s o c i a t e d with m i c r o c e n s t a n t s in the following way:

[B] an+ kc'kd - _ _ [BH+I+[HB+] kc-~-kd

(5)

If the dibasic acid undergoes two identical and independent ionization p r o c e s s e s , then both titratable groups a r e identical in their affinity for H + ions and m i c r o c o n s t a n t s in Eq. (3) will be the s a m e , i.e., k a = k b = k c = k d = k. In this case the experimentally determined constants K 2 and K a are naturally not equal to one another(Ka= 4K~; (5a), which r e s u l t s f r o m the following e x p r e s s i o n s : K2 = 2k and K 3 = k / 2 (5b). If we now substituted into Eq. (2) value of K 2 and K 3 f r o m Eqs. 5b we obtain an equation of the f o r m : r.aH+ r k = 2--r or p H = p k @ l g 2 _ r" (6)

In the case of c o n c u r r e n t parallel ionization both titratable groups have a differentaffinity with r e s p e c t to H+ ions, the m i c r o c o n s t a n t s in Eq. (3) will not be equal, and k in Eq. (6) will be variable: however, the curve d e s c r i b e d by Eq. (6) in the coordinates p K - r p e r m i t s the determination c~ both m i c r o c o n s t a n t s of ionization of a dibasic acid f r o m s e g m e n t s intercepted on the ordinate axes. We c a r r i e d out the potentiometric titration of solutions of s t r e p t o m y c i n base in solutions of potassium chloride of various concentrations. The s t r e p t o m y c i n base was obtained by passing a solution of s t r e p t o m y c i n sulfate of 98% purity through the OH f o r m of Dowex 1• 4 anion-exchange r e s i n . The potentiometric titration curve of a 0.01 N solution of s t r e p t o m y c i n base in the p r e s e n c e of a 0.1 N solution of potassium chloride with a 0.1 N solution of h y d r o c h l o r i c acid is p r e s e n t e d in Fig. 1. Ionization constant of the metl=ylamino group was determined f r o m potentiometric titration data (pH interval of 7.5-9.0). Experimental data t r e a t e d by the l i n e a r equation pK i = pH-log [BH~+]/[BH~+],are p r e s e n t e d in Fig. 2. The Ki of s t r e p t o m y c i n is equal to 3.5 9 10 -9 m m o l e / m l . Ionization constants of the guanidine groups can be calculated with equation (6) for a s e r i e s of values of all+ and r . To calculate r f r o m experimental data we used the following relation: 2c-- (b F Coil-) r , (7) C where c is the concentration of s t r e p t o m y c i n base taken for titration (mmoles/ml); b is the concentration of added acid r e c a l c u l a t e d to total volume of solution (mg-eq/ml); COH- is the concentration of hydroxyl ions ( m g - e q / m l ) . Since the guanidine groups a r e t i t r a t e d in the strongly basic region (pH 10o0-12.0) the concentration of free hydroxyl ions is c o m p a r a b l e to and, in certain c a s e s , exceeds the concentration of added acid. A c c u r a c y of d e t e r m i n i n g r is to a l a r g e degree d e t e r m i n e d by the a c c u r a c y of determining the h y d r o x y - i o n concentration, during which it is n e c e s s a r y to know namely their concentration, and not activity, the value of which can be calculated f r o m p o t e n t i o m e t r i c titration data. The impossibility of jointly using

577

Ex' 15~ 24

H/.6 H/.5_ I 'I'4 '

",..'

o,'~~.~ o.'z o.', t d, z9 Fig. 3. Calculation of K2, u and EA - of the indicator,

Fige 4. Connectionbetween PxOH and EX.

Fig. 5. Calculation of K 2 and

K 3 of s t r e p t o m y c i n .

the values of activities and concentrations for calculating r [see Eq~ (7)] and also the low a c c u r a c y of d e t e r m i n i n g pH in r e s e a r c h with glass e l e c t r o d e s in the b a s i c r e g i o n make it impossible to use potentiom e t r i c titration data to calculate ionization constants of the guanidine groups. Under such conditions the s p e c t r o p h o t o m e t r i c method is convenient. However, since s t r e p t o m y c i n itself does not a b s o r b light, the indicator s p e c t r o p h o t o m e t r i c method was used to m e a s u r e ionization constants of its guanidine groups; it consisted of the following. An indicator having a pK a value lying in the pH region of i n t e r e s t to us was added to the investigated solution in such an amount that it did not have any significant effect on the a c i d - b a s e equilibrium of s t r e p t o m y c i n . We chose the a l i z a r i n yellow indicator which is a dibasic acid. In aqueous solution it can exist in the f o r m s A 2-, A-, A 0, the divalent and monovalent anion and uncharged molecule. The equilibrium between f o r m s is described by the f i r s t and second ionization constants of the indicator: J [A e ] E x --gAo K1, u = a n + [AO---]- = a l l + EA e . EX ,

(8a)

[A2-] E x - - E A~ K,, ~ = ~n+ ~ = an+ e~__ex

(8b)

w h e r e EA 0, E A - , and EA2- a r e m o l e c u l a r extinctions of the uncharged molecule and m o n o - and divalent anions; E X is the m o l a r extinction of the investigated solution containing a mixture of f o r m s . The pH region of 5.0-9.0 is the region of existence of the uncharged molecule and monovalent anion and the pH region of 10.0-13.0 of i n t e r e s t to us is the region of existence of ionic f o r m s . We c a r r i e d out the s p e c t r o p h o t o m e t r i c determination of ionization constants of a l i z a r i n yellow, p r e l i m i n a r i l y r e c r y s t a l l i z e d two t i m e s . F o r this purpose s p e c t r a w e r e taken of the divalent anion in 2.0 and 2.5 N solutions of p o t a s s i u m hydroxide and of the uncharged indicator molecule in 0.1 N and 2.5 N solutions of h y d r o c h l o r i c acid, o b s e r v a n c e of B e e r ' s law was confirmed for these f o r m s at the absorption = 564 m a x i m u m of the divalent anion (X = 564 nm), and values of molar extinctions of the divalent anion i=X x~lm, A2- nm,J and the uncharged molecule (E x = 564 n m ) w e r e calculated. The m o l a r extinction of A 2- is equal to 21,3000 im, A and of A ~ is 210. Spectra of the indicator w e r e taken at pH 8.0 and 9.0 in the region of existence of the monoanion; they w e r e found to be different and consequently it was not possible to obtain the individual f o r m of the monovalent ion and to calculate its m o l a r extinction. To d e t e r m i n e ionization constants of the indicator 3.1 9 10 -5 M solutions of the indicator w e r e p r e p a r e d in a 0.1 N solution of p o t a s s i u m chloride and w e r e neutralized to various d e g r e e s with a 0.1 N solution of potassium hydroxide. The optical density was m e a s u r e d at the absorption maximum of the divalent anion for all samples, the m o l a r extinction was c a l culated for t h e investigated solution (Ex) containing a mixture of ionic f o r m s of the indicator (pH region of 10.1-14.4) or a mixture of the monoanion and the neutral molecule (pH region of 8.0-4.8), the pH of the solution was m e a s u r e d potentiometrically, and the concentration of OH- ions was calculated f r o m the r a t i o :

COH- =

Amount of added p o t a s s i u m hydroxide (mg-eq) Volume of solution (ml) --"

Equations n o r m a l l y used in the s p e c t r o p h o t o m e t r i c method could not be used to calculate ionization c o n stants of the indicator, since the m o l a r extinction of the monoanion is not known. T h e r e f o r e , to t r e a t the experimental data,we used equation (ab) in the following:

578

E 2 ---Ex.

~'x = EAe + ~"~,u aH+

(9)

We found the value of the m o l a r extinction of the m o n o rE x = 564 nm = 7400) f r o m the segment intercepted on the anion ~ A-, l m ZO

0

S

7

8

o

10

I1

[Z

pH

Fig. 6. Equilibrium c u r v e s of the four f o r m s of s t r e p t o m y c i n in the pH region of 6~

ordinate axis by the Iine d e s c r i b e d by Eq. (9) (Fig. 3). Tangent of the angle of inclination is equal to K2,u of the indicator (pK2,u = 12.1 • 0~ and the value of Ki,u calculated with Eq. (Sa) is equal to 1.8 x 10 -7 (pKi, u = 6.75). The dependence of optical density of indicator solutions on concentration of hydroxyl ions (Fig. 4) can be d e s c r i b e d by the e m p i r i c a l equation of the f o r m : p c O H = 3 , 2 2 - - 2 . 0 X 10 -~ ( E x - - 7500),

which p e r m i t s calculation of the concentration of hydroxyl ions in the s y s t e m by measuring E X of the indicator in the given s y s t e m . To d e t e r m i n e ionization constants of the guanidine groups of s t r e p t o m y c i n by the indicator method, a titration c u r v e of s t r e p t o m y c i n was taken in the following way. A s e r i e s of 0.01 N solutions of s t r e p t o m y c i n base was p r e p a r e d in a 0.1 N solution of potassium chloride, each of which contained indicator in a concentration of 3 9 10 -5 m o l e / l i t e r o Solutions of s t r e p t o m y c i n base w e r e neutralized a c c u r a t e I y to various d e g r e e s with 0.1 N h y d r o c h l o r i c acid. The optical density of the absorption maximum of the dianion was m e a s u r e d in each of the solutions and EX was calculated, and then used to calculate att+ with Eq. (Sb) and cOH- with Eq. (10); r was found with Eq. (7). Data of titration of s t r e p t o m y c i n obtained in this way (see Fig. 1, c i r c l e s designate t i t r a t i o n of guanidine groups by the indicator method) w e r e t r e a t e d with Eq. (6) in the coordinates pH-log r / ( 2 - r ) (Fig. 5). The l i n e a r dependence between pH and log r / 2 - r p r e s e n t e d in Fig. 5 indicates that the guanidine groups of s t r e p t o m y c i n a r e t i t r a t e d in parallel and independently. The segment intercepted on the ordinate axis by the line d e s c r i b e d by Eq. (6) is equal to the m i c r o c o n s t a n t of the guanidine groups of s t r e p t o m y c i n (pk = 11.81 • 0.05). The pK 2 and pK S of s t r e p t o m y c i n calculated f r o m Eq. (5b) a r e e q u a l to 11.5] + 0.05 and 12.11 9 0.05, r e s p e c t i v e l y . Concentrations of individual f o r m s of s t r e p t o m y c i n in a 0.1 N solution of potassium chloride in the pH r e g i o n of 6.0-14.0 (Fig. 6) w e r e calculated f r o m the known ionization constants on a digital computer.

579