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Interaction of metal ions with disubstituted purines
J. inorg, nucl. Chem., 1973, Vol. 35, pp. 1285-1292.
Pergamon Press.
Prinl~l in Great Britain
INTERACTION OF METAL IONS DISUBSTITUTED PURINES
WITH
M. M. T A Q U I K H A N Department of Chemistry, Post-Graduate Centre, Osmania University, Warangal-1, India and C. R. K R I S H N A M O O R T H Y Department of Chemistry, Indian Institute of Technology, Madras-36, India
(Received 15 February 1972) A b s t r a c t - T h e interaction of Cu(II), Ni(II), Zn(II), Co(II), Mn(II), Mg(II) and Ca(II) ions with disubstituted purines as 2 : 6 diaminopurine, 2-amino 6-mercaptopurine and 2-mercapto 6-aminopurine and 8-azaadenine has been investigated by a potentiometric method. The stability constants of the complexes containing a l : I ratio of the ligand to the metal ions have been reported at 45 ° and 0.10 M KNOa ionic strength. INTRODUCTION
It~ A PREVIOUS communication[l] we have reported the interaction of monosubstituted purines with bivalent metal ions. The present investigation has been extended to the metal chelates of disubstituted purines as 2:6 diaminopurine, 2-amino 6-mercaptopurine (6-thioguanine) and 2-mercapto 6-aminopurine and azapurines as 8-azaadenine. Apart from naturally occurring purines, synthetic purines as 6-thioguanine and 2-mercapto-6-aminopurine have been found to be potential antitumour agents and 8-azaadenine is a good antimetabolite, Physiochemical work on the metal complexes of the above mentioned substituted purines in aqueous solutions is being reported for the first time. The investigation cannot be extended to 2-amino-6-hydroxy purine (guanine) and 2:6-dihydroxypurine (xanthine) because of the insolubility of these ligands in aqueous solutions. EXPERIMENTAL The experimental method consisted of the potentiometric titration of each ligand with standard sodium hydroxide solution in the absence and presence of the metal ion being investigated. The ionic strength of the solution was maintained approximately constant in the course of the titration by the use of a medium containing 0.10 M KNOa and relatively low concentrations of ligand and metal ion. Presaturated nitrogen was passed through the solution throughout the course of the titration and the temperature was maintained at 45°---0.1 °. A Beckman model G pH meter with glass and calomel electrode was used to determine the hydrogen ion concentration. The electrode system was calibrated by direct titration with acetic acid and the observed pH meter reading was compared with actual hydrogen ion concentration as calculated from the data tabulated by Harned and Owen[2]. The pH region below 3-5 and above 10.5 were calibrated by direct measurement of the hydrogen ion concentration in hydrochloric acid and sodium hydroxide solutions respectively. The equilibrium constants reported in this investigation correspond to the reference state of infinitely dilute reacting species in a medium of 0.10 M (KNOs), thus all equilibrium constants determined are very close to the thermodynamic constants. 1. M. M. Taqni Khan and C. R. Krishnamoorthy, J. inorg, nucl. Chem. 33, 1417 (1971). 2. H. S. Harned and B. B. Owen, The Physical Chemistry of Electrolytic Solution, pp. 485-578. Reinhold, New York (1950). 1285
1286
M . M . T A Q U I K H A N and C. R. K R I S H N A M O O R T H Y
Reagents Chromatographically pure sample of the ligand prepared by Mann-Schwarz research laboratory was employed in this work. Fresh solid ligand was weighed out for every titration to ensure no loss by hydrolysis or photochemical decomposition. The metal salt solutions were standardised by titration with disodium salt of E D T A as described by Schwarzenbach [3]. Carbonate free sodium hydroxide was prepared by the method of Schwarzenbach and Biedermann[4] and was standardised by titration with pure potassium acid phthalate. Calculations The acid dissociation constants of the substituted purines were calculated by a direct algebraic method. The dissociation constants of the triprotonated iigands as 2-amino-6-mercaptopurine and 2-mercapto-6-aminopurine are related to the usual equilibrium expression:
H~L . r~ H2L- . HL 2- .
K jo
K~
, [H2L-]+[H +] " [HL2-] + [H +] • [L3-]+ H+].
By suitable combination of the material balance equations, the following expressions were derived to calculate the dissociation constants: [H +] (aTL + [H +] -- [OH-])
Ka
TL-- (aTL + [H ÷] -- [OH+])
[H +] ( (a -- 1) TL + [H +] -- [OH-])
K2a
K3a
TL -- ( (a -- 1 ) TL + [H +] -- [OH-])
(I)
(2)
[H+]((a - 2)TL + [H +] -- [OH-]) =
TL -- ((a -- 2)TL + [H +] - [OH-])
(3)
where TL represents the total concentration of the ligand species and a represents the number of moles of base added per mole of ligand. In case of 2:6-diaminopurine and 8-azaadenine the dissociation equilibria may be expressed as: Ka
H2L.
" [HL-] + [H +]
HL- . K,, , [L2-] + [H+]. The stability constants of the various 1 : 1 complexes formed by the above diprotonated and tnprotonated ligands with the bivalent metal ions were calculated by setting up suitable material balance equations by the use of the method developed by Richard et al. [5].
RESULTS
2:6-Diaminopurine The potentiometric titration of 2:6 diaminopurine shown in Fig. 1(L) indicates a steep inflection at a = 1 followed by a buffer region at higher pH. The PKa and pK2a values calculated in the lower and upper buffer regions were 5"1 0 _ 0.1 and 9-80_ 0.1 respectively. As in the case of adenine [ 1] the first dissociation of 2 : 6diaminopurine probably involves the dissociation of a proton from the basic centre at N(1) of the pyrimidine ring and the second dissociation is due to the dissociation of a proton at the 9-position of the imidazole ring. 3. G. Schwarzenbach, Complexometric Titration, pp. 77, 82. Interscience, New York (1957). 4. G. Schwarzenbach and R. Biedermann, Heir. Chim. A cta 31,337 (1948). 5. C. F. Richard, R. L. Gustafson and A. E. Marteil, J. A m . chem. Soc. 81, 1033 (1959).
1287
Interaction of metal ions with disubstituted purines
I•H2
H~_ zC,~ ~N 'N£~Z0\,C H
l•H2 ~N
/(3..
pro-5,
H
PKba=9.8
~C~ ~N
: O70) H
H2N/C~'J"~N--H
lO-
8 --
g I 6 /./f
4
2
I
I~
2.0
Q
Fig. 1. Potentiometric titration of 2:6 diaminopurine with Cu(II) and Ca(I1) in a 1:1 ratio of the ligand to metal ion at 45°,/~ = 0.10 M (KNO3) L = free ligand, A = Cu(II) and B = Ca(ll). a = moles of base added per mole of ligand.
Interaction o f metal ions with 2 : 6-diaminopurine Potentiometric titration curves of 2 : 6 diaminopurine in the presence of Cu(II), and C a ( I I ) in 1 : 1 ratio are presented in Fig. 1. (A and B) respectively. Similar titration curves were obtained for other metal ions being investigated. In the case of transition metal ions the titration could not be completed because of the separation of a solid phase before the inflection point was reached. In such cases the formation of a 1 : 1 normal complex (K~L) was assumed and the constants were calculated in the region of the titration curve between a = 0.3-0-8 well ahead of the precipitation point and presented in T a b l e 1. In the case of Mg(II) and C a ( I I ) inflection was obtained at a = 1. In such cases protonated and normal c o m p l e x e s have been assumed to be formed in the lower and u p p e r buffer regions respectively and the corresponding constants K~mL and K ~ calculated and presented in T a b l e 1. T h e stability of the I : 1 normal complexes of 2 : 6-diaminopurine decreases in the order Cu(lI) > N i ( l I ) > Z n ( I I ) > C o ( I I ) > M n ( I I ) > C a ( l I ) > Mg(lI).
6- Thioguanine T h e potentiometric titration of 6-thioguanine Fig. 2(L) shows the stepwise
1288
M.M. TAQUI KHAN and C. R. KRISHNAMOORTHY
Table 1. Equilibrium constants associated with the interaction of disubstituted purines and 8-azaadenine with bivalent metal ions [t = 45°/.t = 0.10 M (KNO3)] 2 : 6 Diaminopurine
2-Amino 6-mercaptopurine
2-Mercapto 6-amino purine
8-Azaadenine
pKa = 5"1-0"1 pK2a = 9"8 ±0"1
PKa = 3"2-----0"1 PKza = 7"9±0"1 PKaa = 9"8±0"1
PKa = 3"8---0"1 pK2a = 7'7±0'1 PKoa = 9"6±0"1
PKa = 2"4---0"1 PKza = 5"8±0"1
Metal
log KML
log KM~
log KM~L
log KML
Cu(II) Ni(II) Zn(II) Co(II) Mn(II)
9-0 8-1 7.8 7"6 7.5
3.4 3.3 3.2 3.1 3.0
3"6 3-5 3.4 3.2 3.1
5"0 4.4 4.1 4.0 4"0
log K ~IL Mg(II) Ca(II)
2"5 2.8
2"8 2.9
MH2L log KMttL 2"9 2.7
3"3 3.0
MHaL log KMH L 2"9 2"8
3"0 2.9
3"9 3.8
dissociation c o r r e s p o n d i n g to the separate neutralisation reactions as:
H2NJC~X'I~' ' ' ' ~ N - H
H,NJ
H, N~C~k"~"J""~'N/--H 9g'bo
oS
A s in the case o f o t h e r purines the first dissociation o f the ligand is due to the r e m o v a l o f a p r o t o n f r o m N(1) o f the pyrimidine ring. T h e s e c o n d dissociation c o r r e s p o n d s to the r e m o v a l o f a p r o t o n f r o m the - S H g r o u p at position 6 o f the pyrimidine ring w h i c h is m o r e acidic [6] than the p r o t o n associated with nitrogen at the position 9 o f the imidazole ring as seen in the case o f 6 - m e r c a p t o p u r i n e . T h e third dissociation is therefore due to the r e m o v a l o f the p r o t o n at position 9 o f the imidazole ring. Interaction o f m e t a l ion with 6-thioguanine P o t e n t i o m e t r i c titration c u r v e s o f 6-thioguanine in the p r e s e n c e o f N i ( I I ) and M g ( I I ) in a 1 : 1 ratio is p r e s e n t e d in Fig. 2 (A and B respectively). In the case o f N i ( I I ) , Z n ( I I ) , C o ( I I ) and M n ( I I ) the titration c o u l d not be c o m p l e t e d b e c a u s e o f the separation o f a solid p h a s e before the inflection point was reached. 6. G. B. Elion, E. Burgi and G. H. Hitchings,J. Am. chem. Soc. 74, 411 (1952).
Interaction of metal ions with disubstituted purines
1289
¥8,
j/A
2 m
0
I
I0
I
20
I
30
O
Fig. 2. Potentiometric titration of 2-amino 6-mercaptopurine with Ni(II) and Mg(II) in a 1: 1 ratio of the ligand to metal ion at 45°,/~ = 0.10 M (KNO~) L = free ligand, A = Ni(II) and B = Mg(II). a = moles of bases adder per mole ofligand. Cu(II) gave an immediate precipitate when the metal ion and ligand were mixed. In the case of Mg(II) and Ca(II) the formation of a 1 : 1 diprotonated complex (KM~L) and a monoprotonated complex (KI~,L) in the lower and upper buffer regions were assumed and the constants thus calculated are reported in Table 1. In the case of Ni(II), Zn(II), Co(II) and Mn(II), the formation of a diprotonated complex MH~L was assumed in the buffer region a = 0.3-0.7 well ahead o f the precipitation point and the constants calculated are presented in Table 1. F o r Cu(lI), the stability constant of the diprotonated 1:1 complex Cu MH2L has been extrapolated from a plot of the sum of the first and second ionization potential of the metal ions versus log K M T h e stability of the diprotonated complex of MH2L" 6-thioguanine decreases in the order Cu(ll) > Ni(II) > Zn(II) > C o ( l l ) > Mn(II) > Mg(II) > Ca(II). Further the stability of the monoprotonated complexes of Mg(II) and Ca(II) are higher than the corresponding diprotonated complexes.
2-mercapto-6-aminopurine The potentiometric titration curve of 2-mercapto 6-aminopurine Fig. 3(L) indicates stepwise dissociation corresponding to separate neutralisation reactions. H e r e again, as in the case of the isomeric compound, 6-thioguanine the first dissociation o f the ligand is due to the removal of a proton from N(1) o f the pyrimidine ring. T h e second dissociation corresponds to the removal o f the proton from the S - H group and the third dissociation is due to the removal of a proton from the position 9 of the imidazole ring.
1290
M.M. TAQUI KHAN and C. R. KRISHNAMOORTHY
l•H2 PK2affiT.7
PKa=3"8
HS...'~NJ.~'L--"~__H
I-IS
O
I•IH2 e
IC
E
~ o
T 4
2 --
I
1.0
I
2'O
I
3'0
Fig. 3. Potentiometric titration of 2-mercapto 6-aminopurine with Ni(ll) and Mg(II) in a 1:1 ratio of the ligand to metal ion at 45°,/z-- 0.10 M (KNO3) L = free ligand, A = Ni(II) and B = Mg(ll). a = moles of base adder per mole ofligand.
Interaction of metal ions with 2-mercapto 6-aminopurine Potentiometric titration curves of 2-mercapto 6-aminopurine in the presence of Ni(II) and Mg(II) in a 1 : 1 ratio is presented in Fig. 3 (A and B, respectively). Because of the separation of a solid phase before the inflection point was reached the titration could not be completed in the case of Ni(II), Zn(II), C o ( I I ) and Mn(II). In such cases the formation of a diprotonated complex was assumed and the constants were calculated in a buffer region a = 0.3--0.7 well ahead of the precipitation point and presented in Table 1. In the case of Cu(lI), an immediate
Interaction of metal ions with disubstituted purines
1291
precipitation occured when the metal ion and ligand were mixed. The stability of 1 : 1 diprotonated complex of Cu(II) was however determined by the same extrapolation method as in the case of 6-thioguanine. For Mg(II) and Ca(lI) inflections at a = 1 and a = 2 were observed, corresponding to the formation of a diprotonated complex (K~H2L)and monoprotonated complex (K~L), respectively. The stability constants for the formation of MHzL and MHL have been calculated in the respective buffer regions and reported in Table 1. The stability of the diprotonated complexes decreases in the order Cu(ll) > Ni(II) > Zn(ll) > Co(ll) > Mn(ll) > Mg(II) > Ca(lI). As in the case of the isomeric 6-thioguanine the stability of the 1 : 1 monoprotonated complexes of Ca(I1) and Mg(II) were higher than those of the corresponding diprotonated complexes.
8-,4zaadenine The potentiometric titration curve of 8-azaadenine shown in Fig. 4(L) indicates a stepwise dissociation corresponding to separate neutralisation reactions: l~H2
I~IH2
NH2
As in the case of adenine the first dissociation may correspond to the removal of a proton from the basic centre N2(1) of the pyrimidine ring and the second dis-
~o-
.~
8 g,6 -?
4~ / A
0
I
J
i,O
2.0
I
3.0
o
Fig. 4. Potentiometric titration of 8-azaadenine with Cu(II), Ni(II) and Ca(II) in a 1 : 1 ratio of the ligand to metal ion at 45 °,/~ -- 0.10 M (KNO3). L = free ligand, A = Cu(II), B = Ni(II) and C = Ca(II). a = moles of base added per mole ofligand.
1292
M.M. TAQUI KHAN and C. R. KRISHNAMOORTHY
sociation probably involves the removal of the proton from the position 9 of the imidazole ring.
Interaction of metal ion with 8-azaadenine Potentiometric titration curves of 8-azaadenine in the presence of Cu(II), Ni(II) and Ca(II) in a 1 : 1 ratio are presented in Fig. 4 (A, B and C, respectively). In the case of Cu(II), Ni(II), Zn(II), Co(II) and Mn(II) the titration could not be completed because of the separation of a solid phase before the inflection point was reached. In these cases the formation of a normal 1 : 1 complex was assumed and the constants were calculated in the region of the titration curve a = 0.3--0.7 well ahead of the precipitation point. In the case of Mg(II), and Ca(II) though an inflection was observed at a = 1, the formation of a monoprotonated complex in the lower buffer region however, could not be confirmed by mathematical treatment of the buffer region which shows the complete dissociation of a proton. The normal 1:1 complexes were accordingly calculated in the second buffer region and presented in Table 1. DISCUSSION
It is of interest to compare the effect of substitution in the purine ring on the compounds 6-aminopurine, 2:6-diaminopurine and 8-azaadenine. The substitution of another amino group at the two position of the purine ring in the adenine increases considerably the basicity of the ligand as evident from their pK values. Irrespective of the binding sites, the presence of two -NH2 groups in 2,6-diaminopurine make the donor nitrogen atoms of the - N H s and ~ N groups better donors than adenine. The complexes of 2 : 6 diaminopurine with bivalent metal ions are therefore much more stable than the complexes of adenine. Substitution of a -~ CH by .~N group in adenine makes 8-azaadenine considerably less basic than adenine (pKa and pK2a values for 8-azaadenine are 2.4 and 5.8 respectively). The basicity of the purine ring is not much affected by the interchange of groups in the 2 and 6 positions. 2-mercapto-6-aminopurine and 2-amino-6mercapto purine show almost the same basicity as indicated, by a sum of their dissociation constants (pK~ + pKz~ + pKa~, of 20"9 and 21.2, respectively) and the stability of their complexes with divalent metal ions.
Pergamon Press.
Prinl~l in Great Britain
INTERACTION OF METAL IONS DISUBSTITUTED PURINES
WITH
M. M. T A Q U I K H A N Department of Chemistry, Post-Graduate Centre, Osmania University, Warangal-1, India and C. R. K R I S H N A M O O R T H Y Department of Chemistry, Indian Institute of Technology, Madras-36, India
(Received 15 February 1972) A b s t r a c t - T h e interaction of Cu(II), Ni(II), Zn(II), Co(II), Mn(II), Mg(II) and Ca(II) ions with disubstituted purines as 2 : 6 diaminopurine, 2-amino 6-mercaptopurine and 2-mercapto 6-aminopurine and 8-azaadenine has been investigated by a potentiometric method. The stability constants of the complexes containing a l : I ratio of the ligand to the metal ions have been reported at 45 ° and 0.10 M KNOa ionic strength. INTRODUCTION
It~ A PREVIOUS communication[l] we have reported the interaction of monosubstituted purines with bivalent metal ions. The present investigation has been extended to the metal chelates of disubstituted purines as 2:6 diaminopurine, 2-amino 6-mercaptopurine (6-thioguanine) and 2-mercapto 6-aminopurine and azapurines as 8-azaadenine. Apart from naturally occurring purines, synthetic purines as 6-thioguanine and 2-mercapto-6-aminopurine have been found to be potential antitumour agents and 8-azaadenine is a good antimetabolite, Physiochemical work on the metal complexes of the above mentioned substituted purines in aqueous solutions is being reported for the first time. The investigation cannot be extended to 2-amino-6-hydroxy purine (guanine) and 2:6-dihydroxypurine (xanthine) because of the insolubility of these ligands in aqueous solutions. EXPERIMENTAL The experimental method consisted of the potentiometric titration of each ligand with standard sodium hydroxide solution in the absence and presence of the metal ion being investigated. The ionic strength of the solution was maintained approximately constant in the course of the titration by the use of a medium containing 0.10 M KNOa and relatively low concentrations of ligand and metal ion. Presaturated nitrogen was passed through the solution throughout the course of the titration and the temperature was maintained at 45°---0.1 °. A Beckman model G pH meter with glass and calomel electrode was used to determine the hydrogen ion concentration. The electrode system was calibrated by direct titration with acetic acid and the observed pH meter reading was compared with actual hydrogen ion concentration as calculated from the data tabulated by Harned and Owen[2]. The pH region below 3-5 and above 10.5 were calibrated by direct measurement of the hydrogen ion concentration in hydrochloric acid and sodium hydroxide solutions respectively. The equilibrium constants reported in this investigation correspond to the reference state of infinitely dilute reacting species in a medium of 0.10 M (KNOs), thus all equilibrium constants determined are very close to the thermodynamic constants. 1. M. M. Taqni Khan and C. R. Krishnamoorthy, J. inorg, nucl. Chem. 33, 1417 (1971). 2. H. S. Harned and B. B. Owen, The Physical Chemistry of Electrolytic Solution, pp. 485-578. Reinhold, New York (1950). 1285
1286
M . M . T A Q U I K H A N and C. R. K R I S H N A M O O R T H Y
Reagents Chromatographically pure sample of the ligand prepared by Mann-Schwarz research laboratory was employed in this work. Fresh solid ligand was weighed out for every titration to ensure no loss by hydrolysis or photochemical decomposition. The metal salt solutions were standardised by titration with disodium salt of E D T A as described by Schwarzenbach [3]. Carbonate free sodium hydroxide was prepared by the method of Schwarzenbach and Biedermann[4] and was standardised by titration with pure potassium acid phthalate. Calculations The acid dissociation constants of the substituted purines were calculated by a direct algebraic method. The dissociation constants of the triprotonated iigands as 2-amino-6-mercaptopurine and 2-mercapto-6-aminopurine are related to the usual equilibrium expression:
H~L . r~ H2L- . HL 2- .
K jo
K~
, [H2L-]+[H +] " [HL2-] + [H +] • [L3-]+ H+].
By suitable combination of the material balance equations, the following expressions were derived to calculate the dissociation constants: [H +] (aTL + [H +] -- [OH-])
Ka
TL-- (aTL + [H ÷] -- [OH+])
[H +] ( (a -- 1) TL + [H +] -- [OH-])
K2a
K3a
TL -- ( (a -- 1 ) TL + [H +] -- [OH-])
(I)
(2)
[H+]((a - 2)TL + [H +] -- [OH-]) =
TL -- ((a -- 2)TL + [H +] - [OH-])
(3)
where TL represents the total concentration of the ligand species and a represents the number of moles of base added per mole of ligand. In case of 2:6-diaminopurine and 8-azaadenine the dissociation equilibria may be expressed as: Ka
H2L.
" [HL-] + [H +]
HL- . K,, , [L2-] + [H+]. The stability constants of the various 1 : 1 complexes formed by the above diprotonated and tnprotonated ligands with the bivalent metal ions were calculated by setting up suitable material balance equations by the use of the method developed by Richard et al. [5].
RESULTS
2:6-Diaminopurine The potentiometric titration of 2:6 diaminopurine shown in Fig. 1(L) indicates a steep inflection at a = 1 followed by a buffer region at higher pH. The PKa and pK2a values calculated in the lower and upper buffer regions were 5"1 0 _ 0.1 and 9-80_ 0.1 respectively. As in the case of adenine [ 1] the first dissociation of 2 : 6diaminopurine probably involves the dissociation of a proton from the basic centre at N(1) of the pyrimidine ring and the second dissociation is due to the dissociation of a proton at the 9-position of the imidazole ring. 3. G. Schwarzenbach, Complexometric Titration, pp. 77, 82. Interscience, New York (1957). 4. G. Schwarzenbach and R. Biedermann, Heir. Chim. A cta 31,337 (1948). 5. C. F. Richard, R. L. Gustafson and A. E. Marteil, J. A m . chem. Soc. 81, 1033 (1959).
1287
Interaction of metal ions with disubstituted purines
I•H2
H~_ zC,~ ~N 'N£~Z0\,C H
l•H2 ~N
/(3..
pro-5,
H
PKba=9.8
~C~ ~N
: O70) H
H2N/C~'J"~N--H
lO-
8 --
g I 6 /./f
4
2
I
I~
2.0
Q
Fig. 1. Potentiometric titration of 2:6 diaminopurine with Cu(II) and Ca(I1) in a 1:1 ratio of the ligand to metal ion at 45°,/~ = 0.10 M (KNO3) L = free ligand, A = Cu(II) and B = Ca(ll). a = moles of base added per mole of ligand.
Interaction o f metal ions with 2 : 6-diaminopurine Potentiometric titration curves of 2 : 6 diaminopurine in the presence of Cu(II), and C a ( I I ) in 1 : 1 ratio are presented in Fig. 1. (A and B) respectively. Similar titration curves were obtained for other metal ions being investigated. In the case of transition metal ions the titration could not be completed because of the separation of a solid phase before the inflection point was reached. In such cases the formation of a 1 : 1 normal complex (K~L) was assumed and the constants were calculated in the region of the titration curve between a = 0.3-0-8 well ahead of the precipitation point and presented in T a b l e 1. In the case of Mg(II) and C a ( I I ) inflection was obtained at a = 1. In such cases protonated and normal c o m p l e x e s have been assumed to be formed in the lower and u p p e r buffer regions respectively and the corresponding constants K~mL and K ~ calculated and presented in T a b l e 1. T h e stability of the I : 1 normal complexes of 2 : 6-diaminopurine decreases in the order Cu(lI) > N i ( l I ) > Z n ( I I ) > C o ( I I ) > M n ( I I ) > C a ( l I ) > Mg(lI).
6- Thioguanine T h e potentiometric titration of 6-thioguanine Fig. 2(L) shows the stepwise
1288
M.M. TAQUI KHAN and C. R. KRISHNAMOORTHY
Table 1. Equilibrium constants associated with the interaction of disubstituted purines and 8-azaadenine with bivalent metal ions [t = 45°/.t = 0.10 M (KNO3)] 2 : 6 Diaminopurine
2-Amino 6-mercaptopurine
2-Mercapto 6-amino purine
8-Azaadenine
pKa = 5"1-0"1 pK2a = 9"8 ±0"1
PKa = 3"2-----0"1 PKza = 7"9±0"1 PKaa = 9"8±0"1
PKa = 3"8---0"1 pK2a = 7'7±0'1 PKoa = 9"6±0"1
PKa = 2"4---0"1 PKza = 5"8±0"1
Metal
log KML
log KM~
log KM~L
log KML
Cu(II) Ni(II) Zn(II) Co(II) Mn(II)
9-0 8-1 7.8 7"6 7.5
3.4 3.3 3.2 3.1 3.0
3"6 3-5 3.4 3.2 3.1
5"0 4.4 4.1 4.0 4"0
log K ~IL Mg(II) Ca(II)
2"5 2.8
2"8 2.9
MH2L log KMttL 2"9 2.7
3"3 3.0
MHaL log KMH L 2"9 2"8
3"0 2.9
3"9 3.8
dissociation c o r r e s p o n d i n g to the separate neutralisation reactions as:
H2NJC~X'I~' ' ' ' ~ N - H
H,NJ
H, N~C~k"~"J""~'N/--H 9g'bo
oS
A s in the case o f o t h e r purines the first dissociation o f the ligand is due to the r e m o v a l o f a p r o t o n f r o m N(1) o f the pyrimidine ring. T h e s e c o n d dissociation c o r r e s p o n d s to the r e m o v a l o f a p r o t o n f r o m the - S H g r o u p at position 6 o f the pyrimidine ring w h i c h is m o r e acidic [6] than the p r o t o n associated with nitrogen at the position 9 o f the imidazole ring as seen in the case o f 6 - m e r c a p t o p u r i n e . T h e third dissociation is therefore due to the r e m o v a l o f the p r o t o n at position 9 o f the imidazole ring. Interaction o f m e t a l ion with 6-thioguanine P o t e n t i o m e t r i c titration c u r v e s o f 6-thioguanine in the p r e s e n c e o f N i ( I I ) and M g ( I I ) in a 1 : 1 ratio is p r e s e n t e d in Fig. 2 (A and B respectively). In the case o f N i ( I I ) , Z n ( I I ) , C o ( I I ) and M n ( I I ) the titration c o u l d not be c o m p l e t e d b e c a u s e o f the separation o f a solid p h a s e before the inflection point was reached. 6. G. B. Elion, E. Burgi and G. H. Hitchings,J. Am. chem. Soc. 74, 411 (1952).
Interaction of metal ions with disubstituted purines
1289
¥8,
j/A
2 m
0
I
I0
I
20
I
30
O
Fig. 2. Potentiometric titration of 2-amino 6-mercaptopurine with Ni(II) and Mg(II) in a 1: 1 ratio of the ligand to metal ion at 45°,/~ = 0.10 M (KNO~) L = free ligand, A = Ni(II) and B = Mg(II). a = moles of bases adder per mole ofligand. Cu(II) gave an immediate precipitate when the metal ion and ligand were mixed. In the case of Mg(II) and Ca(II) the formation of a 1 : 1 diprotonated complex (KM~L) and a monoprotonated complex (KI~,L) in the lower and upper buffer regions were assumed and the constants thus calculated are reported in Table 1. In the case of Ni(II), Zn(II), Co(II) and Mn(II), the formation of a diprotonated complex MH~L was assumed in the buffer region a = 0.3-0.7 well ahead o f the precipitation point and the constants calculated are presented in Table 1. F o r Cu(lI), the stability constant of the diprotonated 1:1 complex Cu MH2L has been extrapolated from a plot of the sum of the first and second ionization potential of the metal ions versus log K M T h e stability of the diprotonated complex of MH2L" 6-thioguanine decreases in the order Cu(ll) > Ni(II) > Zn(II) > C o ( l l ) > Mn(II) > Mg(II) > Ca(II). Further the stability of the monoprotonated complexes of Mg(II) and Ca(II) are higher than the corresponding diprotonated complexes.
2-mercapto-6-aminopurine The potentiometric titration curve of 2-mercapto 6-aminopurine Fig. 3(L) indicates stepwise dissociation corresponding to separate neutralisation reactions. H e r e again, as in the case of the isomeric compound, 6-thioguanine the first dissociation o f the ligand is due to the removal of a proton from N(1) o f the pyrimidine ring. T h e second dissociation corresponds to the removal o f the proton from the S - H group and the third dissociation is due to the removal of a proton from the position 9 of the imidazole ring.
1290
M.M. TAQUI KHAN and C. R. KRISHNAMOORTHY
l•H2 PK2affiT.7
PKa=3"8
HS...'~NJ.~'L--"~__H
I-IS
O
I•IH2 e
IC
E
~ o
T 4
2 --
I
1.0
I
2'O
I
3'0
Fig. 3. Potentiometric titration of 2-mercapto 6-aminopurine with Ni(ll) and Mg(II) in a 1:1 ratio of the ligand to metal ion at 45°,/z-- 0.10 M (KNO3) L = free ligand, A = Ni(II) and B = Mg(ll). a = moles of base adder per mole ofligand.
Interaction of metal ions with 2-mercapto 6-aminopurine Potentiometric titration curves of 2-mercapto 6-aminopurine in the presence of Ni(II) and Mg(II) in a 1 : 1 ratio is presented in Fig. 3 (A and B, respectively). Because of the separation of a solid phase before the inflection point was reached the titration could not be completed in the case of Ni(II), Zn(II), C o ( I I ) and Mn(II). In such cases the formation of a diprotonated complex was assumed and the constants were calculated in a buffer region a = 0.3--0.7 well ahead of the precipitation point and presented in Table 1. In the case of Cu(lI), an immediate
Interaction of metal ions with disubstituted purines
1291
precipitation occured when the metal ion and ligand were mixed. The stability of 1 : 1 diprotonated complex of Cu(II) was however determined by the same extrapolation method as in the case of 6-thioguanine. For Mg(II) and Ca(lI) inflections at a = 1 and a = 2 were observed, corresponding to the formation of a diprotonated complex (K~H2L)and monoprotonated complex (K~L), respectively. The stability constants for the formation of MHzL and MHL have been calculated in the respective buffer regions and reported in Table 1. The stability of the diprotonated complexes decreases in the order Cu(ll) > Ni(II) > Zn(ll) > Co(ll) > Mn(ll) > Mg(II) > Ca(lI). As in the case of the isomeric 6-thioguanine the stability of the 1 : 1 monoprotonated complexes of Ca(I1) and Mg(II) were higher than those of the corresponding diprotonated complexes.
8-,4zaadenine The potentiometric titration curve of 8-azaadenine shown in Fig. 4(L) indicates a stepwise dissociation corresponding to separate neutralisation reactions: l~H2
I~IH2
NH2
As in the case of adenine the first dissociation may correspond to the removal of a proton from the basic centre N2(1) of the pyrimidine ring and the second dis-
~o-
.~
8 g,6 -?
4~ / A
0
I
J
i,O
2.0
I
3.0
o
Fig. 4. Potentiometric titration of 8-azaadenine with Cu(II), Ni(II) and Ca(II) in a 1 : 1 ratio of the ligand to metal ion at 45 °,/~ -- 0.10 M (KNO3). L = free ligand, A = Cu(II), B = Ni(II) and C = Ca(II). a = moles of base added per mole ofligand.
1292
M.M. TAQUI KHAN and C. R. KRISHNAMOORTHY
sociation probably involves the removal of the proton from the position 9 of the imidazole ring.
Interaction of metal ion with 8-azaadenine Potentiometric titration curves of 8-azaadenine in the presence of Cu(II), Ni(II) and Ca(II) in a 1 : 1 ratio are presented in Fig. 4 (A, B and C, respectively). In the case of Cu(II), Ni(II), Zn(II), Co(II) and Mn(II) the titration could not be completed because of the separation of a solid phase before the inflection point was reached. In these cases the formation of a normal 1 : 1 complex was assumed and the constants were calculated in the region of the titration curve a = 0.3--0.7 well ahead of the precipitation point. In the case of Mg(II), and Ca(II) though an inflection was observed at a = 1, the formation of a monoprotonated complex in the lower buffer region however, could not be confirmed by mathematical treatment of the buffer region which shows the complete dissociation of a proton. The normal 1:1 complexes were accordingly calculated in the second buffer region and presented in Table 1. DISCUSSION
It is of interest to compare the effect of substitution in the purine ring on the compounds 6-aminopurine, 2:6-diaminopurine and 8-azaadenine. The substitution of another amino group at the two position of the purine ring in the adenine increases considerably the basicity of the ligand as evident from their pK values. Irrespective of the binding sites, the presence of two -NH2 groups in 2,6-diaminopurine make the donor nitrogen atoms of the - N H s and ~ N groups better donors than adenine. The complexes of 2 : 6 diaminopurine with bivalent metal ions are therefore much more stable than the complexes of adenine. Substitution of a -~ CH by .~N group in adenine makes 8-azaadenine considerably less basic than adenine (pKa and pK2a values for 8-azaadenine are 2.4 and 5.8 respectively). The basicity of the purine ring is not much affected by the interchange of groups in the 2 and 6 positions. 2-mercapto-6-aminopurine and 2-amino-6mercapto purine show almost the same basicity as indicated, by a sum of their dissociation constants (pK~ + pKz~ + pKa~, of 20"9 and 21.2, respectively) and the stability of their complexes with divalent metal ions.