Potentiometric and 1H NMR study of the interaction of hypoxanthine and inosine with H+, Cu(II), and Zn(II)

Potentiometric and ‘H NMR Study of the Interaction of Hypoxanthine and Inosine with H+, Cu(II), and Zn(I1) R. Tauler, J. F. Cid, and E. Casassas Departament de Quhica Anal&u,

Universitat de Barcelona, Barcelona, Spain

ABSTRACT The interaction of H+ , Zn(II), and Cu(I1) ions with hypoxanthine (HYPX) and inosine (INOS) in aqueous solution is studied by means of potentiometry and ‘H NMR spectroscopy. A new microvolumetric procedure for the ‘H NMR determinations, which includes an in situ calibration, is used. For the evaluation of the spectra of the pure species present in solution and of their stability constants a PC version of the program NMROPT was written. Protonation and formation constants of the 1: 1 complexes with Cu(I1) and Zn(I1) are given both for hypoxantine and inosine. On the basis of the ‘H NMR titration curves and the chemical shifts, protonation binding sites are identified. For all systems studied, metal-chelate formation involving O-C6 and N7 atoms of the ligands is postulated.

INTRODUCTION Proton and metal ion interactions with nucleic acids and their constituents is a subject of major interest in current research because of their biological and clinical implications [l]. Although some compilations about the stability constants and affinities to metal and hydrogen ions of these molecules have been published [2, 31 controversial results are usually found [4-81. The present work is a part of a wider study concerning the interpretation of metal ions and proton interactions with nucleic acids in aqueous solution under biological conditions. Special emphasis is given to the elucidation of the number and stoichiometry of the formed species and to the evaluation of their relative stabilities. Recent studies with cis-(Pt(NHs)Clz) reported that a chelate is formed between the strands of DNA and Pt(I1) which involves the N7 atoms of vicinal purine bases [9- 111. However, a complete understanding of the in vivo systems is not yet reached and therefore a clear picture of the coordination sites involved in those complexations is still required. A number of studies deals with the protonation and complex formation

Address reprint requests to: Dr. E. Casassas, Departament Barcelona, Av. Diagonal 647, 08028 Barcelona, Spain.

de Quimica

Analitica,

Journal of Inorganic Biochemistry, 39, 211-285 (1990) @ 1990 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, NY, NY 10010

Universitat

de

217 0162-0134/90/$3.50

278

R. Tauler, J. F. Cid, and E. Casassas

of nucleotides, specially 5’-nucleotides [12-151, but the presence of at least one phosphate group (mononucleotides) as an active competitor of bases for binding to metal ions make them less suitable for extrapolation to real systems (nucleic acids). On the other hand, purine and pyrimidine bases in nucleosides act similarly as in polynucleotides because their N9 atoms are involved in the glycoside bonds and phosphate groups are missing (phosphate groups in polynucleotides have a reduced metal ion binding ability due to the ribose or deoxyribose sterification). Therefore unambiguously identification of purine or pyrimidine binding sites can bc achieved by comparing base and nucieoside proton and metal-ion interactions /?I] Protonation of hypoxanthine and inosine is usually assigned to N7 atom (see Scheme [.Z. 161 or ‘H NMK [17/ data, but the possibility of i ) based on potentiometric IS not excluded, although Gmultaneous protonation nn other sites, i.e.. N3 atom, there is a general opinion that this only happens to a minor extent Assignment of the first deprotonation of hypoxanthine to NI was made previously [16, 18. r9] but the stability constants reported differ considerably, due tc the variety of conditions 7 191 for the second dcprotonation of used. The values of the CcJnStants reported [-, hypoxanthine and inosine, usually assigned to N9 atom and to one hydrclxy! group of the ribose moiety. respectively, show even 23higher Jiscrepanq. Metal complexation sites of hypoxanthine and inosinc have been srudied zither by potcntiometry [ 161 or ’ H NMR diamagnetic shiii techniques [ 121. From these studies. the weakly basic h’7 atom was identified as the main complexatmn site, thus resolving the dichotomy N 147 for purinc derivatives suggested by i;ome authors [ 181. However, a recent multi-spectroscopic study on the Cu(IIl-innsinc iystem [20] and :t previous study on the Cti( II‘i-hypoxanthinc syhlcm 181 point W; the i,ver~rmplificaFic~li I il d~h dn approach hecaii~i: hinuclcar 01 polynuciear compi~~x~~aulitcli kxwld exist. especially at weakly acidic and basic pH, arc not considered, Rciner and Weiss [Xl reported the binding according to N7-Cu-O(C’h) for the C‘u(II i-h> pouanthinc >y~cm. On the other hand. reliabic stability constants for the\e complexes are still lackrng. either because the pH-dependence of Cu(IIj and Zn(II) binding was not considered. or because biological conditions were not used in their determination. in the prrsent SCHEME

1.

R -= M in Hvpoxanthine C!

R = H in Hypoxanthine R = Ribose in lnosine

(HYPXi. R : Ribose in Inosintx tlN(SS)

HYPOXANTHINE,

INOSINE

WITH H+, Cu(II),

Zn(I1)

279

research the stability constants are reported for the protonation, and Cu(I1) ion and Zn(I1) ion complexation with hypoxanthine (HYPX) and inosine (INOS) under biological conditions (aqueous solution at 37’C and 0.15 M ionic strength). The protonation binding sites for both compounds are unambiguously identified and the analysis of potentiometric and ‘H NMR data for all the studied systems suggests metal chelate formation involving N7 and O-C6 atoms of the ligands. The influence of the N9substitution on both the stability constants and the chemical shifts changes is also discussed. EXPERIMENTAL Materials Zn(NOs)z, 4H20, and Cu(N03)~. 3H20 were Merck (analytical grade). Inosine and hypoxanthine were Fluke (puriss. grade). All aqueous solutions were prepared in COz-free deionized water at a constant ionic strength (0.15 M KN03), and their pH was adjusted as required with diluted solutions of HNOs (Merck Suprapur) or KOH (Carlo Erba). Stock solutions for the metal ions were titrimetrically standardized; for the ligands the concentration was determined from the weighted amounts. D20 99.8% was obtained from Aldrich Co. The pD in the ‘H NMR experiments was adjusted with concentrated NaOD (Na dissolved in D20) and DC1 99.8% (Stohler Isotop Chemicals). Trimethylammonium chloride (TMA) and tert-butyl alcohol (tBuOH) from Carlo Erba were used as internal standards in ‘H NMR determinations. Apparatus For pH and pD measurements an ORION pH meter 701 A and a combined Ross pH electrode (semimicro ORION 81-03) were used. Potentiometric titrations were controlled automatically with an HP-3421A data acquisition unit connected to an HP98168 computer. Readings were taken from the recorder output of the pH meter. ‘H NMR spectra were recorded on a Hitachi Perkin Elmer R-24B 60 MHz spectrometer at 32°C and on a Varian-XL 200 MHz spectrometer’ at 25”C, both with a sweep width of 10 ppm. Potentiometric and ‘H NMR titrations were conducted in thermostatized cell (37°C) compartments using cells of different volumes (100 ml and 7 ml respectively). Calculations For the evaluation of the protonation and complex formation constants from the potentiometric data, the SUPERQUAD [21] program was used. Calculations were done at an IBM 3090 computer. For the simultaneous evaluation of the formation constants and of the individual spectra of the species present in solution from the ‘H NMR data, a PC version of the Fortran program NMROPT [22] was implemented.2 ‘H NMR Determinations Protonation of hypoxanthine (HYPX) and inosine (INOS) and their complexation with Zn(I1) were studied with a microvolumetric procedure adapted from the ‘H NMR method described previously [23]. 1 Both apparatus belong to the “Servei d’Espectrosc6pia thanks are given. ’ Available upon request from R. T.

RMN” of the University of Barcelona, to whom

280

R. Tauler, J. F. Cid, and E. Casassas

TABLE 1. Experimental

Conditions

Number of Recorded

L&and h

Metal h pD Range

Internal Reference

0.0048

I .‘?-2.53

TMA

0.0050 0.0048

x.44-1 1.44 ? 26-10 19

Cone

SpeCtrd

of the ‘H NMR Determinations”

COIK.

H + HYPX = (HYPX) H’ 7 H+(HYPX)

H I=HYPX”

5 9 H+(HYPX)

H._z=(HYPX)

5

H lr 0.0040

Zn + HYPX = Zn (HYPX) H 6 6

t-BuOH &BuOH

_

1I 20-12.91

t-BuOH

0.0043 0.0050

2.97-5.13 2.66.-6.03

t-BuOH t-BuOH

0.48- I .31 0.52-2.96

TMA TMA

, + H’ 0.0043 0.0051

H + INOS = (INOS) H -

8

0.051

_.

8

0.048

-.

H + (INOS) H ~, = INOSd 8 Zn+INOS=Zn 9 6

0.046

7.3%9.41

I-BuOH

3.28-6.97 3 12-6.88

TMA t-BuOH

(INOS) H_] +H’ 0.028 0.029

0.0057 0.0130

a Conditions: 37°C and 0.15 M KNOl in aqueous solution. All the spectra for the hypoxanthme determinations were recorded on a 200 MHz spectrometer at 25°C. The spectra for the promnation and deprotonation of inosine were recorded on a 60 MHz spectrometer at 32 “r b Initial ligand and metal concentrations in mol. dtn ‘. ’ Protonation equilibrium for hypoxanthine and inosme. ’ First deprotonation equilibrium lhr hypoxanthine and inosine. ’ Second deprotonation equilibrmm for hypoxanthine. For inosine only potentiometric determinations were carried out because this deprotimation step is associated with one of the hydrnxyl woups in the riboae tnoiety

’ I : I complex formation equilibrmm for Zn(II)-hypoxanthine

and for Zn(II)-mosine

systems.

According to this procedure, small volumes ( 1-2 ~1) of the titrand ( DCl) are added from a 50 ,ul syringe into the background 0.15 M KNOJ solution, contained in a 7.0 ml cell thermostatized at the experimental temperature (37’C). thus allowing the calibration of the electrode in terms of pD to be made (in situ calibration according to the Gran method [241). After the addition of a known amount of the ligand- and of the metal ion in case of a complex formation study--and of the spectroscopic internal standard (TMA in the 0.5-6.0 pD range, t-RuOH in the 6.0-13.0 pD range), the pD is adjusted lo successive different values with concentrated NaOD or DCl, so that dilution effects can be neglected, and, at predetermined titration points. 300 Fl-volumes of solution are transferred to ‘H NMR tubes and the spectra are recorded. In Table 1, the experimental initial conditions of the ‘H NMR determinations made are shown. For measurements conducted at pD above 1, “junction potentials

HYPOXANTHINE, INOSINE WITH H+, Cu(II), Zn(I1)

281

were studied but no significant values were found [25]. The chemical shifts of the protons H(8) and H(2) were measured as a function of pD with and without the presence of Zn(I1) salt. Because of the fast proton exchange between coordinated and free species, to which Zn(I1) and H interactions are subjected, each observed peak is an average signal for each proton in the mixture of species [26]. Assignment of signals was made according to previous studies [ 17, 181. For this study, solutions were taken only where no precipitation or hydrolisis occurred.

Potentiometric

Determinations

Potentiometric determinations of Hz0 and D20 autoprotolysis constants were carried out at 37°C and 0.15 M KNOs. Gran plots [24] were used for the calibration of the cells and linear regression was applied to obtain the mean values of the autoprotolysis constants. For the study of protonation of hypoxanthine, which occurs in the l-3 pH range, four titration curves (429 experimental points) were obtained, using ligand concentrations of about 0.0047 M-0.0049 M. For the study of the two deprotonation steps occuring at pH > 8, four titration curves (161 experimental points) were recorded. Ligand concentrations varied from 0.0036 M-0.0059 M. The behavior of inosine was studied in the same way: three titration curves were performed (118 experimental points) with ligand concentrations from 0.018 M-0.021 M for the protonation step occuring at pH near 1 and seven titration curves (363 experimental points) with ligand concentrations from 0.0027 M-0.022 M for the two deprotonation steps at basic pH, the second one (at pH > 11) being associated with the hydroxyl groups of the ribose moiety. For the study of Cu(I1) complex formation with hypoxanthine and inosine, five (360 experimental points) and seven (355 experimental points) titration curves were obtained, respectively. Concentrations of hypoxanthine and inosine taken were in the range from 0.0031 M-0.0048 M and from 0.0038 M-0.04 M respectively; whereas Cu(I1) concentrations varied from 0.0006 M-0.004 M (hypoxanthine experiments) and from 0.0019 M-0.0038 M (inosine experiments). For the study of Zn(I1) complexation with hypoxanthine, three (248 experimental points) titrations were conducted, using ligand concentrations in the range 0.0045 M-0.0048 M and metal ion concentrations between 0.0012 M and 0.0042 M. For the study of the complexation of this metal ion with inosine three titration (with 3 13 experimental points) were made, with ligand and metal ion concentrations in the ranges 0.006-0.0024 M and 0.0054-0.0057 M, respectively. RESULTS

AND DISCUSSION

Calculated ‘H NMR chemical shifts for all the pure species detected are given in Table 2. From these calculated chemical shifts and from the magnitude of their relative changes, the most probable binding sites (see also Table 2) can be deduced on the assumption that the observed shifts should be larger for the protons nearer to these sites. Protonation of hypoxanthine and inosine gives a large downfield shift of H8. In contrast, H2 is shifted very little, thus showing the proton has a clear preference towards the N7 atom of the imidazole ring. The different magnitudes of these chemical shifts are clearly shown in Figure 1 by the ‘H NMR titration curves for HS and H2 (calculated as in ref. 26) obtained in the protonation of inosine. Furthermore, the presence of the ribose (sugar) has an effect on the protonation behavior of the N7

282

R. Tauler, J. F’. Cid, and E. Casassas

TABLE 2. Chemical Shifts~’ and Most Probable Binding Sites!‘ INOSINE i: F-l?

HYPOXANTHINE Species

I’ Chemical program

d H8

d HZ?

shifts of the pure specw

calculated

Site

d HX

from the analysis of the measured shifts with the N,L1ROP7

(22). The given values are related to TMA

(in ppm) with cstirndtcd

” Most probable binding

Gtes where a protonationideprotonatiorl

of hypoxanthine,

corresponds

which

hinded to N9 is removed). Deprotonation

See

’ ’ and

to the equilibrium

associated with one nf the hqdroxyl

the NMROPT

program)

occurs (c-8.

(HYPX)

Results and Discussion

” Because of the bmali change> m the chemical

Site

H

errors of 0 01 ppm

in the srccrnddrprotonation

: i H

for the 7niiIi

:tiYPX:

H

:, the proton

~ompieuation

groups oi- the rih<>.\c moiety

shrlis of both H8 and H:.

nc~r~i~;~blc 1 txluc~(calculated

urth

were c)btamed.

FIGURE 1. ’ H NMR titration curve for the protonation of inosine. Symbols refer to exper-imcntal chemical shifts (in ppm related to TMA) obtained at different pD values; continuous lines refer to the calculated values using the NMROPT 1221 program ____________-_-__-_.-..-.--. .---.-.-11----“-----7 T-ES-j EEd ;;f E4 i 33-Y s 2 A

yH\,t,

HYPOXANTHINE,

TABLE 3. Protonation

INOSINE

and Cu(I1) and Zn(I1) Complex Formation

Equilibria

HYPOXANTHINE Potent. ‘H NMR

L+H=LH LH_,+H=L LH_2+H=LH_i

2.13(2) 8.51(2) 11.40(2) 6.00(6) 4.37(3)

L+Cu=CuLH_i+H L+Zn=ZnLH_,+H

WITH H+, Cu(II),

Zn(I1)

283

Constantsa INOSINE

Potent.

‘H NMR

2.0(l) 8.8(4) 11.8(3)

0.97(2) 8.50(2) 11.65(l)

1.1(l) 8.3(5) _b

_c _d

4.27(2) 2.77(2)

_c _d

a Values of the logarithm of the stability constants for the protonation and 1: 1 complex formation equilibria obtained from the SUPERQUAD [2 l] treatment of the potentiometric data, and from the NMROPT PC version (this work) treatment of the ‘H NMR data. Conditions: 37°C and 0.15 M KNOs in aqueous solution. Values in ( ) are the standard deviations in the last figure of the logarithm of the constants, for the potentio-metric data, and are the deviations of this last figure which cause a double value of the error-square function, for the ‘H NMR data. b Deprotonation associated with the hydroxyl groups of the ribose moiety (not studied by the ’ H NMR method). ’ Cu(II) is paramagnetic and therefore can not be studied by the I H NMR chemical shift method. d See explanation in d Table 2.

atom; this effect, although small, is significant: the log of the protonation constant of inosine is lower in one unit of log K than that of hypoxanthine (see Table 3). A similar effect is also observed for adenine and adenosine but no differences are seen when adenine and 9-methyladenine are compared [26]. The first deprotonation process of hypoxanthine causes comparable H8 and H2 upfield shifts and a crossing of signals. For inosine, a crossing of H8 and H2 signals is also observed upon deprotonation, but in contrast to hypoxanthine, an upfield shift of only 0.02 ppm occurs for H2, while the more distant (referred to Nl site) H8 undergoes 0.26 ppm upfield shift. This behavior agrees with the fact, pointed out by other authors [18], that more profound electronic rearrangements should occur upon binding to the Nl atom than to the N7 atom, although there is not yet a satisfactory explanation from the chemical shift comparison criterion. An H8 upfield shift larger than the one obtained upon Nl deprotonation is observed for the second deprotonation process of hypoxanthine, while the H2 upfield shift is similar in magnitude. According to these results, in the uncharged molecule of hypoxanthine, the H2 atom is more affected by both deprotonation processes (Nl and N9) than by protonation (N7). However, H8 displays a different pattern because of the larger change in chemical shift which occurs upon protonation. On the other hand, the H2 atom of inosine seems to be nearly unaltered either upon protonation or deprotonation of the molecule. Values of the protonation constants (Table 3) of hypoxanthine and inosine obtained from the analysis of ‘H NMR spectra agree well with the values evaluated from potentiometric data, proving the reliability of the NMR method for pKa determination. Moreover, in many instances the information provided by the proposed ‘H NMR method will be very useful for the study of the molecular structure of the species in solution. The well established potentiometric method will give higher confidence for the calculation of precise values of stability constants.

284

R. Tauler, J. F. Cid, and E. Casassas

From the ‘H NMR analysis of solutions which contain Zn(I1) and hypoxanthine, the H8 and H2 chemical shifts are calculated for the proposed ZnLH __I (L = neutral hypoxanthine) species. In this case, the NMROFT program does not allow the simultaneous evaluation of the formation constants for the complex species because these species show small chemical shifts and are present at low concentration levels (lower than 20’%). Therefore, the program was run using formation constants which were kept unchanged at their potentiometric values during the refinement. The downfield shifts observed for the Zn(I1) complexes are a little smaller than those obtained for the protonated species as it has been observed in previous studies [22+ 231, proving the deshielding effect of Zn(I1) compared to I-I’ . The analysis of the potentiometric data for both the Cu(II) and Zn(I1) complex formation with both hypoxanthine and inosine shows that the prevailing complex species in the pH range 2-5 is the 1: 1 complex species. Other complexes are detected at higher pH but no identification was intended because of the simultaneous precipitation of the solutions. In the case of inosine, at higher pH the solutions which contain Cu(I1) ion redissolve, and become viscous probably due to gel formation of polynuclear species via hydroxyde bridges from ribose. This fact was also observed by other authors [20] and is planned for further study. The considerable higher values of the formation constants for the Cu(I1) and the Zn(I1) complexes with hypoxanthine and with inosine (see Table 3) compared with the ones for the complexes with adenine and adenosine or 9-methyladenine [26) cannot be explained if only monodentate coordination is considered as proposed by other authors 112, 201. Additionally, the observed proton loss associated with complex formation confirms the hypothesis of chelate complex formation. For inosine complexes this proton loss can only take place via the Nl atom (acid pH). and therefore a chelate involving N7 and O-C6 atoms of both must be proposed for both ligands. The literature data are extremely controversial about this fact [ 8, 201, but according to our potentiometric and ‘H NMR results, hypoxanthine chelates are however stronger than inosine chelates, as is seen from the comparison of their formation constants (see Ta-ble 3). These results suggest a contribution of the free N9 atom of hypoxanthine as an additional binding site in a similar way to that observed previously for adenine 1261. The overall effect would be made up of a composite of C6-O-G-N7 chelation and two N3,N9-2Cu-N9,N3 bridges between two molecules of hypoxanthme; probably the later contribution is weaker (220 complex) and of course undistinguishable by potentiometric menas. Further study using spectroscopic techniques is planned, This research was supported by the Ministerio de Educacidn y C’iencirrof’ Spain {CICYT program No. PB87-0061)

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HYPOXANTHINE, INOSINE WITH H+, Cu(II), Zn(I1)

285

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