o~~kloH~H - NISCAIRnopr.niscair.res.in/bitstream/123456789/39889/1/IJCA 34A... · 2017. 1. 31. · 48 INDIAN J CHEM. SEe. A, JANUARY 1995 Table 1- EMF values at different temperatures

Indian Journal of ChemistryVol. 34A,January 1995, pp. 47-51

Deprotonation energetics of purine anduric acid in water from emf measurements

at different temperatures

Sonali Ganguly & Kiron K Kundu"

Physical Chemistry Laboratories, Jadavpur University,Calcutta 700 032

Received 15 March 1994; revised and accepted 5 August 1994

The first step and second step deprotonation con-stants of purine and uric acid in water have been de-termined from emf measurements of cells comprisingH2 and Ag-AgI electrodes at five equidistant tempera-tures ranging from 15°-35°C. The pK values are fittedin the temperature equation pK = AT- 1 +B +cr byleast squares method and the related standard free en-ergies (aG'), entropies (as) and enthalpies (air) ofthe deprotonation processes in water have also beenevaluated using the values of the coefficients A, B andC of the respective acids.

In continuation of our earlier work' on deproton-ation energetics of uridine 5' -monophosphate andguanosine 5' -monophosphate we are reporting thedeprotonation constants and the related energeticsof purine (Pur) and its 2,6,8-trihydroxy derivativeuric acid (Uri) at zero ionic strength and in therange of 15°-35°C at 5 degree intervals as deter-mined by emf method using Hamed Ehler-typegalvanic cells without liquid junction comprisingPt, H2 (g, 1 atm) and Ag-AgI electrodes. TheHamed Ehler-type cells used for the determina-tion of pK, and pK2 and related energetics are(A)-(D)For HA = Pur; H2A + = protonated Pur andA- = conjugate base of PurPt, H2 (g, 1 atm)1H2A + 1- (ml), HA (mz),KI(m3)/AgI-Ag ... (A)Pt, H2(g, 1 atm)/HA (m,), NaA (m2),

KI(m3)/AgI-Ag ... (B)For H2A = Uri, HA - = conjugate base of Uri andN- - dianion of UriPt,H2(g, 1 atm)1H2A(ml),NaHA(m2),KI (m3)/ AgI-Ag ... (C)

Pt, H2(g, 1 atm)INAHA (m1 ). Na2A (m2)'KI (m3)/AgI-Ag ... (D)

6~~

2~~~~8H

(a) Purin. (Pur)

H~H

o~~kloH H

(b) Uric acid (Uri)

Fig. I-Structures of Pur and Uri

ExperimentalPurine (grade P-1655) and uric acid (grade

U-2625) (both from Sigma Chemical) were usedas such after drying in a vacuum desiccator. Thepurity of all the chemicals were checked by UVspectroscopy/ and were found to lie within 98%-99%. Sodium hydroxide (GR), potassium iodide(GR) and hydroiodic acid (GR) were obtainedfrom E. Mer-ck.

Cell solutions at different ionic strengths wereprepareo by mixing appropriate weighed amountsof biochemicals, NaOH and KI solutions ofknown molality and triply distilled CO2 free waterin well stoppered Jena bottles. Appropriateamounts of standard NaOH solutions were usedfor the stepwise neutralization of the acids so asto get the desired concentrations of the corre-sponding conjugate bases. For the determinationof pKl for Pur, HI acid was used instead of alkalisolution. The method of HI distillation was simi-lar to that as described elsewhere".

General experimental procedure including thepreparation of Ag-AgI and fIz(Pt) electrodes weresimilar to those described 3• The equilibrium wasattained in 3-4 hours. The constancy of emf read-ings to about ± 0.1 mV for one hour was takenas the criterion of equilibrium, The readings werefirst taken at 15°C and then successively at highertemperatures of 5°C intervals. The readings of25°C when back checked after 35°C agreed with-in ±0.1 mY.

Results and discussionThe measured emf's of cells corrected to

PHz = 1 atm gave the value of E. The emf data (E)of cells (A)-(D) at different temperatures andcorresponding molalities of KI, HzA+ 1-, HA forcell (A), KI, HA. NaA for cell (B), KI, H2A, Na-

48 INDIAN J CHEM. SEe. A, JANUARY 1995

Table 1- EMF values at different temperatures for different cell solutions in pure water for first and second step deprotona-tion constants of purine and uric acid

,., ml x 10J m2 x 103 m3 x 103 emf (V)(mol kg-I) 15 20 25 30 35"C

Pur:pKI -(pK.)H,A'; ml -mH,A" m2 -mHA, m3- ml-

.0100 4.8 6.6 10.0 .1357 .13825 .1403 .1430 .1446

.0231 9.9 26.0 23.1 .1237 .12575 .1276 .12925 .1308

.0322 19.9 16.3 32.2 .0916 .0930 .0946 .0963 .0968

.0439 9.9 26.0 43.9 .1087 .1114 .1124 .11375 .1151

.0655 19.8 16.6 65.5 .07575 .07715 .0783 .07975 .0802

.0786 19.7 16.5 78.6 .07175 .0725 .0738 .0751 .0758

.1064 20.2 16.1 106.4 .0640 .0652 .06625 .06725 .06795

Pur: pK2 - (pK.)HA; m 1- mHA,m2 - mA-, m3" ml-

.0110 14.3 5.1 5.9 .4812 .4846 .4876 .4908 .4933

.0200 12.8. 14.8 5.2 .5135 .5174 .5210 .5245 .5275

.0301 16.1 20.3 10.6 .49845 .5020 .5050 .5080 .5105

.0427 16.3 19.7 23.0 .4786 .4816 .4844 .4875 .4900

.0543 16.1 19.7 34.6 .4684 .4710 .4738 .4762 .47885

.0701 16.3 19.6 50.5 .45845 .4610 .4636 .4658 .4684

.0878 16.4 19.5 68.3 .45095 .4536 .4558 .4580 .4598

.1004 16.1 19.8 80.6 .44785 .4504 .4525 .4548 .4571

Uri:pKI-(pK.)H,~ ml - mH,Atmz- mHA-,m3- ml-

.0090 1.1 1.0 8.0 .3031 .3068 .3099 .3131 .3158

.0212 l.U 1.1 20.2 .2825 .2858 .2886 .2914 .2938

.0308 1.0 0.9 29.9 .2729 .2760 .2783 .2813 .2835

.0422 0.9 0.9 41.3 .2653 .2683 .2702 .2730 .2751

.0539 1.2 1.1 52.8 .2574 .2599 .2617 .2646 .2666

.0664 1.1 1.3 65.1 .2588 .2610 .2628 .2658 .2678

.0750 1.0 1.2 73.8 .2560 .2586 .2603 .2629 .2648

.0842 0.9 0.9 83.3 .2485 .2509 .2525 .2550 .2571

Uri: pK2 - (pK.lHK; ml - mHA-,mz - m,;-, m3 - ml-

.0110 1.0 1.2 6.4 .5870 .5911 .5946 .5980 .6027

.0207 2.2 2.4 11.3 .5726 .5715 .5810 .5851 .5900

.0334 2.5 2.1 24.6 .5455 .5502 .5542 5583 .5630

.0448 3.6 3.8 29.8 .5430 .5480 .5532 .5576 .5624

.0563 3.8 3.2 42.9 .5372 .5415 .5465 .5520 .5568

.0661 5.6 5.4 44.3 .5367 .5412 .5440 .5483 .5520

.0780 5.2 5.1 57.5 .5298 .5338 .5370 .5406 .5450

.0881 5.1 5.1 67.1. .5260 .5302 .5351 .5391 .5410

HA for cell (C) and KJ, NaHA, Na2A for cell (D)for each of the cell solutions are given in Table 1.

Similar to our previous paper', the pKI andpK2 of Pur and Uri were evaluated by using thefunctions pK1' and pK{ defined by Eqs 1-4

pKI (Pur) = -log(mwmHA/mH,A+)

-log (Yw YHA/YH,A+)

pKl'(Pur) = -log[mil+(m2 + mil+ )/(ml - mil+)]=pK1(Pur) + f(,u) ... (1)

where-log(mil.)=(E - EO)/k + log(ml-)- 2Sf(!J.dof2

... (1')

In the above equations m and Y denotes re-spectively the molality and molal activity coeffi-cients of the species involved. The standard stateis so chosen that at infinite dilution in a given sol-vent Yi of any species i is equal to unity,k = 2.3026 RT/F, !J. (ionic strength) = m1_= m3and Sf (Debye Huckel constant) = 1.824 x 106

(EwTt312, mil+ denotes the apparent molality ofhydrogen due to deprotonation of the acid andhydrolysis of conjugate base of the acid, EO thestandard electrode potential of Ag-AgI electrodesin pure water, do = density of pure water, R = uni-versal gas constant, T, absolute temperature, F,Faraday, Ew, the dielectric constant of water andf(!J.) stands for a function of !J. which is usuallylinear.

pK2(Pur)=(E- EO)/k+ 10g(mI-mHAlmK)

+ log( Yd'HA/YA-)

pK'2(Pur)=(E- EO)!k+ log [m3(m, +mow)/

(m2-mow)]=pk2(Pur)+f(!J.) (2)

where log mow = log K; + PwH (2')

PwH= -log awYow = -logawi'I-= (E - EO)!Ie + log 1nJ-

!J.= m2 + m3; moH- stands for the molality of hy-droxyl ion due to hydrolysis; Kw, the ionic productof water, PwH = Bates acidity function.

NOTES

pKI (Uri)=(E - EO)/k+ 10g(lnJ-mH,~mHK)

+ log (YI-YH,~rHA-)

pK1'(Uri)=(E- EO)/k+ log(m3m/m2) .=pK1(Uri)+f(!J.) ... (3)

where !J.= m2 + m3

pK2(Uri)=(E - EO)/k+ 10g(lnJ-mHA-/mA'-)

+ log( YI-YHA-/YA'-)

pKz(Uri) = (E- EO)/k+ log [m3(m, + mow)/

(m2 - mow)] + 2Sf(!J.dS12

= pK2(Uri)+ f(!J.) ... (4)

where mow is given by Eq (2') and!J.= m1 + 3m2 + m3

The involved activity coefficients in water areobtained by the Bronsted form of Debye-Huckelequation -log Yi= SrZf(!J.do)':!+ bi!J., whereZj= charge of the species involved and b, is a con-stant for the species i depending upon the natureof the solvent and temperature. As indicated inour previous paper", due corrections for mil+and·mow have been made for pKI and pK2 ofPur and U ri respectively. Since in all the casespK' values were found to be linear with respectto !J., the pK values at different temperatureswere obtained from. linear fit of the typepK' = pK + b!J. and their values along with thestandard deviations are listed in Table 2. For eachof the acids the pK values at different tempera-tures were fitted into Harned-Robinson type ex-pression'

pK=AT-1 + B + cr ... (5)

by the method of least squares. The thermody-namic parameters I:l.Go, I:l.S and I:l.Ir accompa-nying the deprotonation of the acids were evaluat-ed using the same relations as described in ourprevious paper'. The values of I:l.CO, I:l.S andI:l.Ir so obtained and the values of the constantsA, B and C are presented in Table 3. The maxi-mum uncertainties in I:l.GO, I:l.S and I:l.H" are± 0.01 kJ mol", ± 1 JK -1 mol- 1 and ± 0.3 kJ

Table 2- Values of pK with standard deviations of purine and uric acid in pure water at different temperature

W ~ ~ ~ 35~

pK, 2.551±.002 2.528±.001 2.501±.001 2.471±.001 2.439±.001pK2 9.248± .001 9.141± .0025 9.040± .001 8.932± .002 8.830± .002pK, 5.863±.003 5.810±.002 5.760±.003 5.710±.002 5.660±.002

pK2 10.910± .004 10.804± .001 10.690± .001 10.601± .003 10.522 ± .003

Compd

Pur

Uri

50 INDIAN J CHEM. SEe. A, JANUARY 1995

Table 3- Values of A, B, C and relevant energetics ll.G',ll.S' and ll.1r (molal scale) for purine and uric acid at 25"C

Compd A(K) B C(K-') ll.G'(kJ.mol-') ll.S'(JK-1 mol:") ll.1r(kJmol-l)

1st step dissociation

-1550 14.575 -0.02306 14.275 -15.8 9.60(± 10) (±.08) (±.00002)

Pur 2nd step dissociation1196 7.24 -0.00744 51.56 -53.7 35.60

(± 12) (±.06) (±.OOO06)

1st step dissociation0 8.74 -0.01000 32.87 -53.2 49.9

(±O) (±.05) (±.00006)Uri 2nd step dissociation

6077 -24.18 0.04872 61.23 -93.3 33.4(±5) (±.06) (±.00009)

D.t,

~6~~

~~~8H

Pur(HAl

SCHEME 1

.1 pKZ L

~OH ~ .J::.J;~~t·..,~lOH

HN NH ~7~'--... I N.10 -- HO~~ ~,,.lOt:f ~ Cc . Hf:(~ ~ ...'I pK

UrllHZA ) ~ ~ ...d- N~N

O~N I ~).Qi~ oV~.Conjugal. ba~ of lki IHA1 Dlanion of Uri rA2-J

SCHEME 2

mol-1 respectively.The sites of proton ionization from Pur and Uri

are discussed below and the equilibria involved inthe deprotonation processes are shown inSchemes 1 and 2.

The fairly low magnitude of entropy change ac-companying the first step ionization of Pur indi-cates that the ionization process is a charge trans-fer process (CfP) involving a BH+ type acid andthe deprotonation sites (with pKa = 2.50) to beeither N 1H + or N3H + group.

On the other hand, negative magnitude of en~tropy of ionization of second step dissociation ofPur suggests the ionization process involved ascharge separation process (CSP) involving a HA-type acid. In purine hydrogen was found to be lo-cated at N(7) and N(9)4. However, from chargedensity distribution' it appears that deprotonationfrom neutral Pur occurs from N(9) andpKa = 9.04 is assigned to it. Notably, pKa = 2.50for protonated Pur and pKa = 9.04 for netural Pursuggest that it is a weaker acid than pyrimidine

(pKa = 1.3) and a much stronger acid than imida-zole (pKa = 14.5). These properties are consistentwith pyrimidine ring withdrawing electrons fromthe imidazole ring of purine.

Similar to neutral Pur, the first step dissociationof Uri with a negative magnitude of entropychange is also a esp. By analogy with hypoxan-thine (Hyp) and xanthine (Xan) and from chargedensity distribution it appears that in aqueous so-lution the lactam-lactim tautomerism exists andthe deprotonation sites in Uri (with pKa = 5.96)are either 2 or 6 hydroxy group. Interestinglyenough, on comparing pK\ of 2,6,8-trihydroxypu-rine (Uri) with 6-hydroxypurine (Hyp) and 2,6-di-hydroxypurine (Xan) it is found that acidity in-creases in the order Hyp < Xan < U ri. This mightbe due to the electron withdrawing inductive ef-fect of the hydroxyl groups which tend to destabi-lize the negative charge on the oxygen of the 2 or6 hydroxyl group which deprotonates.

The l!.S values for the second step ionizationof Uri, Hyp and Xan are similar indicating thatdeprotonation occurs from HA - type acid and- N9H on the ring is the favourable ionizationsite. Moreover, the magnitude of pK2 of Uri, Xanand Hyp indicates an increase in pKa in the orderUri <Xan <Hyp which is again a result of gradedincrease in + I effect of hydroxy groups in the or-der Hyp < Xan < Uri. However, in unsubstitutedpurine N9H site is .more acidic than the same inits mono-, di- and tri-hydroxy derivatives. This isdue to the fact that deprotonation from N9H siteof hydroxy derivatives of purine occur from theirconjugate bases and negative charge on oxygen at2 or 6 position in nibits to some extent the pro-ton ionization from N9H group and thereby re-sults in a decrease in acidity.

NOTES 51

The highly negative magnitude of entropychange accompanying the second step deprotona-tion of uric acid and that l!.~.Uri is more negativethan l!.s;..Uri conform to what is expected fromthe nature of the equilibria involved", In the caseof Uri, the dissociation processes involved

K, K,

are H2A ?H+ +HA- and HA- ? H+ + A2- andthe corresponding entropy changes are given byEqs (6 and 7).

l!.S~(Uri) = (S~l,o' - S~,o)+(S~- - S~,o) (6)

l!.S'2(Uri) = (S~,o' - S~,o) + (S~.v-- S~-) (7)

(S~ 0+ - S~0) is negative as H30+ carrying aJ 2 • •

charge must be solvated resultmg m a more or-derliness and there is a decrease of entropy ofH3q+ relative to w~ter i.e. S~,o' < S'H,O'similar-ly, S'HA-< S'H,Aand SOA'-< S~-. Since entropy ofhydration of any ion is directly _proportional tothe square of ionic charge, (S~,- - S~ -)< (S~--S'H,A) i.e. AS'A'-HA-<l!.S~--H,A' In otherwords l!.~,Uri < l!.s;.,Uri'

References1 Ganguly S & Kundu K K, Indian J Chem, 33 (1994) 1099,i Windholz M, The merck index (Merck 'and Co. Rahway,

NJ.) 1983, 10th edn.3 Kundu K K, Jana D & Das M N, Electrochim Acta, 18

(1973) 75.4 Ts'o POP, iasic principles in nucleic acid chemistry

Soc B, (1972) 2034; (b) Talukder H, Rudra S P & Kundu5 Pullman B & Pullman A, Quantum biochemistry (Intersci-

ence, New York) 1963.6 (a) Kundu K K, Chattopadhyay P K & Das M N, J Chem

Soc B. (1972) 2034; (b) Talukder H,Rudra S P & KunduK K, Indian J Chern, 21A'(1988) 764.