Studies on the chelation of cytosine, cytidine and cytidine phosphate with U(VI), Th(IV), Ce(III) and La(III)

Bioelectrochemistty and Bioenergetics, 12 (1984) 475-484 A section of J. Elecrroanal. Chem., and constituting Vol. 173 (1984) Elsevier Sequoia .%A., Lausanne - Printed in The Netherlands

415

706-STUDIES ON THE CHELATION OF CYTOSINE, CYTIDINE AND CYTIDINE PHOSPHATE WITH U(VI), Th(IV), Ce(II1) AND La(II1)

Y.M. TEMERK

*, M.M. KAMAL,

Chemistn_ Department, (Manuscript

M.E. AHMED

and MI.

ABD-EL-HAMID

Faculty of Science, Ass&t Unioersit_v, Assiut (Egypt)

received March

17th 1984)

SUMMARY The complexes formed between cytosine, cytidine and cytidine phosphate and U(W), Th(IV), Ce(II1) and La(II1) have been investigated using spectrophotometric and conductometric methods. The apparent formation constants of these complexes have been calculated. The mode of bonding of the ligand in the solid chelates was studied by I.R. spectrophotometry which showed that chelate formation takes place through C(Z)=0 and C=N(3) groups in the case of cytosine and cytidine. The I.R. spectra of cytidine phosphate complexes indicated a tridentate ligand which coordinated via the former two groups and phosphate groups to the central metal ion.

INTRODUCTION

Metal ions are known to bind with nucleic acids and thereby alter their conformation and biological function [l]. In this context various studies in the past [2-121 have shown that alkali and alkaline earth metal ions interact only with the phosphate moiety while transitional metal ions interact with both the phosphate and bases of nucleic acids. The metal ion base interaction depends on the nature of both metal and bases, a certain site of coordination is preferred. This base-specific interaction has, therefore, been exploited recently for a sequencing of DNA by electron microscopy, when DNA is labelled with heavy metal ions like Au and Pt [13,14]. This together with the recent finding that certain transitional metal complexes have been found to be potentially useful in cancer chemotherapy [15,16], created a renewed interest in the study of the interaction of heavy metal ion with DNA or its components. The present investigation is devoted to study the ability of cytosine, cytidine and cytidine phosphate to form stable complexes with U(VI), Th(IV), Ce(II1) and La(II1). The composition and stability constant of complexes have been determined spectrophotometrically and conductometrically.

l

To whom correspondence

0302-4598/84/$03.00

should be addressed.

0 1984 Elsevier Sequoia

S.A.

complexes

complexes

H,O

complexes

Ce(C9H,,N,0,,P,)2CI,.2 La(C,H,,N,O,,P,),CI,.2

UO,(C,H,,N301,P,),CI,

Y-CLIP

I-I,0 H,O

H,O

H,O

Ce(C,H,,N,0,P)2 Cl,.2 H,O La(C,H,jN,0,P),Cl,.2 H,O

T~(C,H,JN,O,P)*(NO~)~.~

5’-CMP

La(C9H,,N&),C1,.2

T~(CBH,,N,O,),(NO,)~.~

U~~G&,N~OS)~C~,

Cvfidine complexes

Th(C,H,NsO),(NO3),.2 H,O Ce(C,H,N,0)&1,.2 H,O

U0,C,H,N,0),C12

Qrosine

Compound

_

9.29 (9.22) 11.62 (11.45) 11.62 (11.32)

White White White

9.41 (9.07) 9.92 (9.61) 9.92 (9.57)

14.05 (14.22)

White

White White White

13.05 (13.41) 10.77 (10.42)

8.52 (8.07) 6.50 (6.11) 9.51 (9.08)

1.14 (1.53) 1.20 (1.44) 1.20(1.51)

1.56 (1.38) 1.95 (2.13) 1.95 (2.21)

2.23 (2.03)

1.58 (1.83) 1.71 (1.39)

0.89 (1.02) 1.23 (1.58) 1.80 (1.44)

H

Calculated. % (Found, %) C

Pale yellow Greenish yellow

Pale yellow Greenish yellow White

Colour

Analyticat data and colour of cytosine, cytidine and cytidine phosphate solid complexes

TABLE 1

11.46(11.51) 11.47 (11.60)

8.43 (8.63) 4.52 (4.69) 4.53 (4.74)

6.19 (6.00) 9.80 (9.62) 9.80 (9.96)

13.85 (13.76)

5.46 (5.39)

3.66 (3.81) 3.86 (3.49) 3.87 (3.62)

8.57 (8.69) _

12.81 (12.68) 21.10 (19.85)

Cl

5.08 (5.36) 9.76 (9.64)

7.46 (7.83) 13.27 (12.93) 8.32 (8.00)

N

477

EXPERIMENTAL

Preparation of solutions of ligand Cytosine, cytidine-5’-monophosphate (5’~CMP) and cytidine-5’-diphosphate (5’-CDP) were obtained from Serva, Heidelberg, F.R.G. The solutions containing different concentrations of the investigated ligands were prepared by dissolving a known amount of the chemically pure product into a definite volume of absolute ethanol. The content of ligand in the sample solution was determined spectrophotometrically. Preparation of solutions of metal salt The metal salt solutions of UO,‘+, Th4+, Ce3+ and La3’ were prepared by dissolving the appropriate amount of the Analar metal salt in absolute ethanol. The metal contents of solutions were determined by conventional methods [17]. Isolation of metal complexes (i) Cytosine and cytidine complexes. A hot ethanolic ligand solution (0.2 mole) is mixed with a hot ethanolic solution (0.1 mole) of UO$l, or Th(NO,), .5 H,O or CeCl, .7 H,O or LaCl, * 6 H,O. The mixture is refluxed for 6 h on a steam bath and then concentrated to a small volume and cooled to room temperature when pale greenish yellow for Th4’ complexes and white precipitates for other complexes are separated. The solid is filtered off, washed several times with cold ethanol and dried in a desiccator over P,Os. (ii) Cytidine phosphate complexes. 0.1 mole of the ethanolic ligand solution is refluxed with 0.05 mole of the metal salt solution for about two hours on a steam bath. The mixture is left at room temperature for 24 h, whereby a heavy white solid is precipitated. This is filtered, washed with ethanol and dried over P,Os. The solid complexes were analysed for their carbon, hydrogen, nitrogen and chlorine contents. Results of chemical analysis are given in Table 1. Working procedure Conductometric titrations were carried conductometric cell of the dipping type. recorded in a Cary 912 spectrophotometer spectra of the chelates were recorded on a KBr disc. RESULTS

out using a Pye conductance bridge and a The U.V. spectra of the solutions were using 1 cm matched silica cells. The I.R. Perkin-Elmer 599-B specrophotometer as

AND DISCUSSION

Spectrophotometric

measurements

The electronic absorption spectra of complexes formed between cytosine, cytidine and cytidine phosphate and UO;+, Th4+, Ce3+ and La3+ are recorded over the wavelength range 250-395 nm in pure ethanol (Fig. 1). As a blank, an alcoholic

478

6 0.6 -

?? C -III

0.4 -; -S 0.2 -

275

300

250

325

275

Ahm) 250 275

300

300

325

350

325

Fig. 1. (A) Spectra of Th(IV)-cytosine complexes. (cytosine]=10-4 M; [Th4’ ] = (1) 2X10m5 M. (2) 3XlO-5 M, (3) 4~10-~ M, (4) 5x10-’ M, (5) 6~10~~ M, (6) 8X1O-5 M and (7) lX10m4 M. (B) Spectra of Th(IV)-cytidine complexes. [cytidine]=10m4 M: [Th4+]= (1) 2~10-~ M, (2) 4x1O-5 M, (3) 5x10~~ M, (4) 6~10~~ M. (5) 7~10-~ M and (6) 1X10m4 M. (C) Spectra of Th(IV)-5'-CDP complexes. [5’-CDP]=10-4 M. [Th4’]=(1) 4X10m5 M. (2) 4.5X10-’ M. (3) 5X10e5 M. (4) 5.5~10-~ M, (5)6x10m5 M, (6) 6.5~10.~ M. (7) 7X1O-5 M and(g) 8X10m5 M.

solution containing the same concentration of the ligand as that in test solution was used in order to compensate the absorption due to the reagent and hence its effect on the absorption curve. The absorption band of the investigated ligands showed a red shift on complexation owing to the enhanced charge transfer through the cytosine moiety. The obvious red shift of the band in comparison with that characterising the free ligand verifies the complex formation with the metal ions. This leads to the appearance of characteristic’ absorption band which may be attributed to the chelate formation. The stoichiometry of the different complexes formed in alcoholic media was determined by Job’s continuous variation [18,19], molar ratio [20], straight line [21], slope ratio [22] and limiting logarithmic [23] methods. The variation of absorbance uersus mole fraction of the metal ions (Fig. 2) as well as the absobance uersus ligand/metal molar ratio plots (Fig. 3) denotes the formation of two types of

419 (a)

(b)

0.4 0.6 9 0.4 - 0.3 -2 d ;I”

o~2rl--/ Mole Fraction

0

0

..

0.6

0.6

0.4

0.2

OF metal

0.4

0.2

0.f3(DJ

0.0

Fig. 2. Stoichiometry of cytosine-UOi+ continuous variation method.

complexes for the metal (ligand : metal). Conductometric

(a) and

ions investigated

(a)

cytidine-La’+

(b) complexes

with stoichiometric

ratios

by

application

of

1 : 1 and 2 : 1

measurements

Solution of 10e4 mole of metal ions were titrated with lop3 mole solution of cytosine, cytidine and cytidine phosphate as shown in Fig. 4. The conductance uersus molar ratio curves are characterized by breaks denoting the formation of 1: 1 and 2 : 1 types of complexes with metal ions investigated. The results are in agreement with those of spectrophotometric measurements. In the case of all metals (91 (a!

0.5

/

0.5 - g

/

/

/

--

/A-

b

a :::E_k

0 0 0

[L / M”+] I 1 3.0 2.0

I 1.0 I 1.0

2.0

Fig. 3. Molar ratio plots: cytosine-Ce’+

(a) l(b) 3.0 (a) and S-CMP-ni”

(b) complexes.

480

162 -

152-

180 -151

142 -

t32-

160 -13

122 -

[L/ M”+] 121

I

0.5

&

1.0

Fig. 4. Conductometric titration cytidine-UOf+ (d) complexes.

1

I

I

1.5

2.0

2.5

curves for: cytidine-Th4+

(a), cytidine-Ce3+

(b), cytidine-La3’

(c) and

studied the titration curves show a slight increase in conductance as the concentration of the ligand increases. The slight increase of conductance is explained by the fact that metal ions form a coordination bond with the anions, that would exhibit an apparent covalent nature. When the reaction between the ligand and the metal ions takes place, the anion of the salt are preferentially expelled from the coordination sphere. Thus, the electrostatic attraction between two oppositely charged particles is decreased, and the anion can migrate relatively free to carry the electrical current and consequently the conductance of the medium increases. The results of conductometric titration indicated that the -OH group of ribose or ribose phosphate does not participate in the chelate formation. If the ligand would exhibit participation of the -OH group, the formation of complexes should have been accompanied by the liberation of the hydrogen ion which has high value of the ionic molar conductivity (A, = 349.82 Q-’ cm2 mole-l). This is in accordance with the result of I.R. spectroscopy of the complexes under investigation.

481

Determination

of the apparent stability constant

The apparent stability constants (p) of 1: 1 complexes formed were determined from the spectral data using molar ratio [20], continuous variation [l&19] and straight line [21] methods. The mean values of In p as well as the values of -AG* are given in Table 2. The values of -AG* have been determined using the relation AG* = - RT In p. The results obtained indicate that the stability of complexes depends on the nature of both the ligand used and the metal ions. The values of stability constants for the same metal ion with different ligands are in agreement with the following: 5’-CDP > 5’-CMP > cytidine

> cytosine

Obviously the increasing number of hydrophilic phosphate groups in the cytidine molecules increases the stability of chelates. This is connected with the participation of the phosphate group of the ligand in the coordination to the central metal ion. The values of stability constants for the same ligand with different metal ions are in agreement with the following: U(V1) > Th(IV)

> Ce(III)

> La(II1)

TABLE 2

values of In B and - AC* for cytosine, cytidine, 5’-CMP and 5’-CDP complexes (1: 1) Metal ion

In P

- AG* kJ mol-’

6.93 6.75 5.57 5.22

39.77 38.75 31.97 29.96

6.59 6.28 5.92 5.73

37.83 36.05 33.98 32.89

uwu -NV) Ce(II1) La(II1)

6.95 7.66 6.76 6.34

39.89 43.96 38.80 36.39

5’-CDP complexesU(V1) TWIV) Ce(II1) La(II1)

7.97 7.99 7.81 6.45

45.75 45.86 44.83 37.02

Qtosine

complexes

uw Th(Iv) Ce(III) La(II1) Cvtidine complexes WI) J-WV) Cc(II1) La(II1) 5’- CMP complexes

482

This order is supported by the fact that the possibility for U(V1) and Th(IV) to form complexes rather than the others can be presumably ascribed to the fact that actinide elements are much more prone to complex formation than lanthanides [24]. Moreover, the high positive charge on Th(IV) makes it susceptible for complex formation. I.R. spectra of solid chelates The I.R. spectra of complexes of cytosine and cytidine as compared to those of the ligands (Table 3) indicate that the bands due to the N(1) out of plane deformation, in-plane deformation and N(l)-H stretching vibration at 840, 1570 and 3170 cm-’ are still observed at the same positions. The presence of these bands in the I.R. spectra of cytosine complexes showed that the N(1) site is excluded from a direct interaction with metal ion. The existence of vNH, bands at 1275 and 1240 cm-’ in the I.R. spectra of tie chelates of cytosine and cytidine reveals also that the -NH, group is not involved in complex formation. However I.R. spectra of the complexes of cytosine and cytidine indicate that the bands due to C(2)=0 at 1605 cm-’ and C=N(3) at 1530 cm-’ shift to lower frequency. This indicates that the bonding of cytosine and cytidine to metal ions takes place through the C(2)=0 and the nitrogen atom of the C=N(3) group. The mode of bonding of metal ions with cytosine and cytidine can be represented as follows:

R

TABLE

3

Infrared

data for cytidine

complexes

Free ligand

Th4+ complex

Ce’+ complex

La)+ complex

uo:+ complex

Band assignment

1660 1270 945 720

1670 1280 945 720

1660 1275 945 720

1660 1270 945 720

1670 1285 945 720

“NH, deformation symmetric and asymmetric in plane and out of plane respectively

1120 1080 1045 980

1125 1080 1045 980

1120 1085 1045 980

1120 1085 1045 980

1120 1085 1050 990

Yribose ring

3460

masked

masked

masked

3465

YOH group of ribose moiety

1605 1550

1630 1530

1560 1520

1560 1520

1570 1535

YC(2)=0 YC=N(3)

483 TABLE 4 Infrared data for 5-CMP complexes Free ligand

Th4 + complex

Ce’+ complex

La” complex

uo; + complex

Band assignment

1660 1275 720

1660 1285 720

1660 1280 720

1660 1280 720

1660 1285 masked

vNH, deformation

1610 1525

1590 1510

1585 1505

1585 1505

1580 1510

vC(2)=0 vC=N(3)

1080 980 815

1105 995 805

1115 990 800

1110 985 805

1105 995 805

VP-O YPO:- group vc-O-P

where R = H (cytosine) and R = ribose moiety (cytidine). With respect to metal cytidine chelates no evidence for ribose sugar coordination is sheen as the degenerate ribose stretching frequencies at 1120, 1080, 1045 and 980 cm-’ are virtually unchanged in intensity or position upon complexation. On the otherhand the bands due to the -OH groups of ribose moiety at 3460, 1380 and 1220 cm-’ are still observed and indicated that OH group is not involved in the chelate formation. For the cytidine phosphate the 1100 and 980 cm-’ bands are shifted for all the complexes which seem to indicate coordination with phosphate (Table 4). The shifting of the phosphoric ester band indicates a strengthening C-O-P bond. On the other hand the I.R. spectra of 5’-CMP and 5’-CDP complexes as compared with that of the ligand reveal also that the bands due to C(2)=0 and C=N(3) vibration at 1610 and 1525 cm-’ respectively are shifted to lower frequency. The foregoing result indicates that the cytidine phosphate are coordinated to the central metal ion through the C(2)=0, C=N(3) and phosphate groups. The structure of cytidine phosphate metal complexes can be represented as follows: t

CH2-o-Lot

N’-‘2

I

NH2

II

o--

q_o_HhC

0-

-’

G HO

OH

484

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