Deprotonation Energetics of Disodium Uridine 5'-Monophosphate and Disodium Guanosine 5'-Monophosphate in Water from Emf Measurem

Indian Journal of Chemistry Vol. 33A, December 1994, pp. 1099-110 3

Deprotonation energetics of disodium uridine 5' -monophosphate and disodium H~5

guanosine 5'-monophosphate in water 9H, s' ~Nt:)6 from emf measurements HO-P-O-C~O . , o 4' H, H 1 Sonali Ganguly & Kiron K Kundu* H 'H OH HO Physical Chemistry Laboratories, Jadavpur University, S'UridlnE' monophosph att' (SUMP) Calcutta 700 032 [I-V,A] Received 25 February 1994; revised and accepted o 5 August 1994 7 HN 6 N The second step and third step deprotonation con- ,H9H~~ ~ 4 ~)e stants of 5'-uridine monophosphate and 5'-guanosine HO-P-o-gco monophosphate have been obtained in water from o 4' H, H r emf measurements of Hamed Ehler-type cells com- HH prising H2 and Ag-AgI electrodes at different tempera- OH H tures. The pK values have been fitted in the tempera- S'Guanoslne rnonapbosphcte ( S'GMP) ture equation pK = AT- I + B + cr by least squares [H4A) method and the related thermodynamic quantities viz., Fig. l=-Structures of 5'-UMP and S'-GMP tJ.Go, TtJ.So and tJ./f" have been obtained using the va- lues of the coefficients A, B and C of the respective mononucleotides. far been made on the enthalpy and entropy of ionization of second step deprotonation of 5'- GMP. We report here our findings on deprotona- A nucleotide is the phosphate ester of a sugar in tion constants (K I & K 2) and related true ther- glycosyl combination with a base molecule de- modynamic quantities of the disodium salts of 5'- rived from purine and pyrimidine '. Since the bio- UMP and 5'-GMP as determined by measuring logically most interesting pH range is 6-10, study the emf's of the Harned Ehler-type galvanic cells of second and third step ionization energetics of without liquid junction comprising Pt, H2 (g, nucleotides where deprotonation occurs in the 1 atm) and Ag-AgI electrodes and buffers of dif- above pH range is highly useful in identifying dif- ferent ionic strengths at five equidistant tempera- ferent deprotonation sites involved in various bio- tures ranging between 15°-35°C. chemical reactions. In a previous paper we have The Harned EWer-type eells used for the deter- reported the deprotonation constant data and as- mination of various pKa's and related energetics sociated energetics of 5' -adenosine monophosph- are (A) and (B) ate (5' -AMP) and other adenine derivatives+ In

this note, we present the same for 5' -uridine For H~A= 5'-UMP/5'-GMP; H3A- = conjugate monophosphate (5'-UMP) and 5'-guanosine mon- base of 5'-UMP/5'-GMP; H2N- =dianion of ophosphate (5'-GMP) [vide Fig 1] which are two 5'-UMP/5'-GMP and HN- = trianion of 5'-UMP/ of the four main ribosyl nucleotides present in 5'-GMP RNA. Pt, H2(g, 1 atm)/NaH,A(ml), Na2H2A(m2), Literature survey reveals that some deprotona- KJ(mJ)/AgI-Ag (A) tion constant data are available:', but these data were evaluated based on either half-neutralization Pt, H2(g, 1 atm)lNa:H2A(mj), Na3HA(m2), point determination of pH titration curves using KJ(mJ)/AgI-Ag ... (B) glass (H +) electrode and aqueous saturated cal- omel electrode SCE (W) forming cells with liquid Experimental junction or spectrophotometric method at differ- Disodium salt of 5'-uridine monophosphate ent ionic strengths or under a variety of tempera- (grade U 6375) and disodium salt of 5'-guanosine tures. Evidently these data are of doubtfulpreci- monophosphate (grade G 8377)(both Sigma sion and reliability. Moreover, no studies have so Chemical) were used without further pretreatment 1100 INDIAN J CHEM, SEe. A, DECEMBER 1994

Table I-emf values at different temperatures for different cell solutions in pure water for second and third step deprotonation constants at 5'-UMP and 5'-GMP

ml x 10.1 1Il~ X 10.1 m,>: 10.1 emf(V)

(mol kg-I) 15 20 25 30 35°C

5'-UMP,pKz = (pK,lH,A-; ml = mH.,A-,m, = mH,A'-' m, = ml-

.O1l2 1.1 2.2 3.5 .3827 .3909 .3998 .4077 .4162 .0202 2.5 3.2 !U .3489 .3566 .3649 .3722 .3804 .0301 2.1 2.2 21.4 .3181 .3254 .3329 .3399 .3473 .0390 4.5 5.0 19.5 .3210 .3284 .3359 .3430 .3504 .0468 4.6 4.R 27.8 .3096 .3167 .3241 .3309 .33R1 .0600 3.6 4.R 42.0 .3041 .3111 .3183 .3251 .3322 .0741 5.7 0.0 50.4 .2924 .2992 .3062 .312R .3196 .OR39 7.2 R.6 50.9 .2946 .3017 .30R6 .3150 .3219

5'-UMP, pK3 = (pK.)H,A' ; m, = mH,A>;m, = mH,v'; m., = m,

.0120 1.I l.l 2.1 .5520 .5568 .5611 .5667 .5741 .0220 2.4 2.3 1.0 .5671 .5725 .5770 .5829 .5905 .0323 3.1 3.3 3.2 .5397 .5442 .5486 .5539 .5610 .0441 4.5 4.5 3.6 .5334 .53R1 .5421 .5473 .5543 .0592 5.3 5.1 12.7 .4999 .5039 .5073 .5119 .5183 .0698 5.6 5.9 17.6 .4934 .4973 .5006 .5050 .5113 .0809 6.1 6.1 26.0 .4812 .4850 .4880 .4922 .4983 .0923 6.3 6.8 32.6 .4770 .4806 .4836 .4877 .4937

5'-GMP,pK2 = (pK.)H,A-; m, = mH,K; m2 = mH,A'-; m, = ml-

.0129 2.4 2.1 4.2 .3580 .3666 .3749 .3833 .3914 .0238 5.1 4.8' 4.3 .3569 .3655 .3737 .3821 .3901 .0331 5.6 5.8 10.1 .3368 .3450 .3528 3590 .3685 .0442 7.1 7.0 16.1 .3227 .3306 .3382 .3459 .3533 .0558 8.5 8.4 22.1 .3134 .3212 .3288 .3361 .3433 .0656 9.3 9.6 27.5 .3082 .3159 .3231 .3306 .3377 .0710 9.5 9.6 32.7 .3030 .3103 .3178 .3252 .3322 .0813 10.1 10.4 40.0 .2974 .3049 .3120 .3192 .3262

5'-GMP; pK3 = (pK.)H,A'·; m, =mH,A'-; m2 = mHA'-;mj = m..

.0091 1.1 0.9 0.4 .5941 .5988 .6026 .6077 .6121 .0218 2.3 2.1 2.3 .5507 .5546 .5583 .5620 .5656 .5524 .0332 3.1 3.3 4.1 .5385 .5421 .5450 .5490 .0450 4.2 4.3 6.6 .5246 .5279 .5305 .5343 .5375 .0570 5.4 5.7 6.9 .5230 .5263 .5289 .5326 .5357 .0683 6.2 6.0 13.7 .5028 .5058 .5080 .5113 .5141 .5204 .0782 7.6 7.6 9.8 -5115 .5146 .5169 .5233 .0881 8.2 8.5 12.5 .5056 .5086 .5108 .5142 .5170 NOTES 1101

after drying in a vacuum desiccator kept at 5°- (e;T) - 3/2, e; the dielectric constant of water, 10°C. These biochemicals assayed 99% by UV do= density of water and f(.u) denotes a function spectroscopy". Water used was triply distilled and of.u which is usually linear. CO free. KI (GR, E. Merck) was used after dry- 2 pK (5'-UMP,5'-GMP) ing at 150°C and "then in a vacuum desiccator for 3 12 h. Cell solutions of different ionic strengths = (E - EO)Ik+ log(ml- mHzAZ-lmHA'-) were prepared by mixing appropriate weighed + log( 1'1- i'HzAz-1i'HA'-) amounts of biochemicals, NaOH (GR, E. Merck) pK;(5' -UMP,5' -GMP) or III (GR, E. Merck) and KI solutions of known molality, and triply distilled water in well stop- = (E - EO)!k + log[m3(ml + mow )!(m2 - mow)) pered Jena bottles. The method of III distillation + 4Sr(.udO)ll2= pK3(5'-UMP,5'-GMP)+f(.u) was similar to that as described earlier". . .. (2) The emf's of the cells were measured usng a

Leeds and Northrup K4-potentiometer fitted with where .u = 3m! + 6m2 + ~ and log mow = 10gKw a moving coil galvanometer (Cambridge Instru- + PwH; PwH (Bates activi~ function) = -logaw ment Co.) as the null point detector. General ex- i'ow'" -logawi',- =(E-E )/k+logm,- (ref. 6), perimental procedures including the preparation mow stands for the molality of hydroxyl ion due of H2(Pt) and Ag-AgI electrodes and Pie measure- to hydrolysis, K; the ionic product of water. The ments of emf were similar to those described ear- required values for E~_Ag1at different tempera- lier", Ag-AgI electrodes were introduced after an tures were taken from the literature". The in- hour's discharge of hydrogen gas through the volved activity coefficients in water were obtained cells. Equilibrium was attained in 3-4 hours. The approximately by the Debye Hiickel Bronsted constancy of emf readings to about ± 0.1 mV for equation -log i'i = Srz?(.udo)lt2 + bi.u where .u- one hour was taken as the criterion of equilibri- molal ionic strength of the cell solutions: um. The readings were first taken at 15°C and z, = charge of the species involved, S, Debye then successively at higher temperatures of 5°C Hiickel constant and b, is a constant for the spe- intervals. The readings of 25°C when back: cies i depending upon the nature of the solvent checked after 35°C agreed within ±0.1 mY. and temperature. Since in all the cases the ob- served plots of pK' against .u 'were found to be Results and discussion linear, the pK values at different temperatures The measured emf of the ceUs corrected to were obtained from the linear expression PHz= 1 atm gave the value of E. The emf data (E j pK' = pK + b.u and these values with their stand- of the cells (A) and (B) at different temperatures ard deviations are listed in Table 2. For each of and the corresponding molalities of KI, NaH3A, the mononucleotide the pK values at different temperatures were fitted into Hamed Robinson- Na2HzA for cell (A) and KI, Na2H2A, Na3HA for cell (B) for each of the cell solutions are given in type expression Table 1. The pKz and pK3 of 5'-UMP and 5'- pK=JtJ-1 +B+CT ... (3) GMP were evaluated by using the functions pKi and pK; defined by Eqs 1 and 2. by the method of least squares. The thermody- namic parameters !l.Go, !l.S' and !l.Ir accompa- pK2(5'-UMP, 5'-GMP)= (E-EO)/k nying the deprotonation of the acids were evaluat- + 10g(ml-mH,A-ImHzA'-) ed using the following relations comprising the coefficients ofEq. (3) + log( 1'1- i'H,A-li'HzA'-) !l.Go= 2.3026R[A + BT + Cf2) pKi(5'-UMP,5'-GMP) ... (4) =(E- EO)/k+ log(~m/mz)+ 2S~.udo)lt2 !l.S' = - 2.3026R[B + 2CT) ... (5)

= pK2(5'-UMP,5'-GMP)+f(.u) ... (1) sir» 2.3026R[A-Gfl) ... (6)

where .u (ionic strength) = m1 + 3m2 + m3, The values of !l.CO, !l.S' and !l.Ir so obtained k= 2.3026RTIF, m and I' denote the molality and the values of constants A, B and C are pre- and molal activity coefficients of the species in- sented in Table 3. The maximum uncertainties in volved, EO the standard electrode potential of the dGo, dS and dJr are ± 0.01 kJ mol-t, Ag-AgI electrodes in water, R the universal gas ± 1 JK -I mol- 1and ± 0.3 kJ mol-I respectively. constant, F, Faraday, T absolute temperature in The probable sites of ionization for the monon- Kelvin, Sf (Debye Hiickel constant) = 1.824 x 106 ucleotides in the pH range 6-10 studied are dis- 1102 INDIAN J CHEM, SEe. A, DECEMBER 1994

Table 2-Values of pK with standard deviations of S'-UMP' and S'-GMP in pure water at different temperatures Nucleotides 15° 20° 25° 30· 35°C pKz 6,649±.002 6.662±.002 6.677±.002 6.692±.0035 6.709±.001 5'-UMP pKJ 9.694 ± .003 9.S82± .002 9.490 ± .003 9.419 ± .001 9.366 ± .002 pKz 6.660±.001 6.680±.001 6.700±.001 6.720±.001 6.740±.00l 5'-GMP pK) 9.781 ±.002 9.658 ±.002 9.543 ± .0015 9.438 ±.001 9.341 ±.OOIS

Correlation coefficient for pKU·.UMP/S'.GMP - pK 35.UMP/S·.QMP - I at different temperatures.

Table 3-Values of A, B and C and relevant energetics ~G', ~S" and~H"(molal scale) for S'-UMP and S'-GMP at 25°C Nucleotides A (K) B C (K - I) ~ G' (kJ mol- I) ~S" (JK-I mol- I) ~H" (kJmol")

2nd step dissociation

741 0.81 0.01134 38.11 -145.0 -5.1 (±9) (±.01) (±.OOOlO) 5'-UMP 3rd step dissociation 10528 -56.27 0.10214 S4'.}9 -88.75 27.7 (± 11) (±.O8) ( ±.OOO13)

2nd step dissociation 0 5.51 0.00400 38.26 -151.1 -6.8 (±O) (±O) (±O) 5'-GMP 3rd step dissociation 4553 - 14.455 0.02927 54.47 - 57.4 37.35 (±8) (±.O6) (±.0001O)

Entries in parenthesis are standard deviations.

o H,X H~~ pK~ 1..) 15 ~.) 15-H ~N~CI+z-O-~ o N~C~-o-i:-oH cs;- H H 0 H H 0 ft H HH (lHHO OHHO Conjugal. bast' ot 5'UMP(~A1 Oianion ot 5'UMP(~2-1 Dianion of !5'G""'I~A~ Jf o 9_ t)CH2~-r.-o H H 0 HH~ ()jHO Trianon ot 5'lJMPIHA~ SCHEME 2

SCHEME 1 group. Furthermore ultrasonic absorption studies 7 also provide the information that in the neutral cussed below and the equilibria involved in the solution the secondary phosphoric acid function deprotonation processes are depicted in Schemes - P03H- deprotonates. Moreover, as indicated 1 and 2. elsewhere's", the neutral base moieties exert no

Similarity of entropy of ionization values (65i:') detectable effect on the ionization of the secon- of di-anion of 5'-UMP, 5-GMP (H,N-) and con- dary phosphate and so that pKa values of all the jugate base of 5' -AMP (H2A -)(refs 2,3) suggests mononucleotides are virtually the same. Thus in that the second step deprotonation of the monon- the region of pH 6 the secondary phosphate dis- ucleotides occurs from secondary phosphate sociates and becomes doubly negatively charged. NOTES 1103

By analogy with the magnitude of ~ Si~n of neu- 3 Izatt R M, Christensen J C & Rytting J H, Chern Rev, 71 tral uracil, uridine and guanosine+!" with entropy (1971)439. change accompanying the third step deprotona- 4 Windholz M (Editor) The merck index (Merck and Co, Rahway, NJ) 1983, 10th edn. tion of 5'-UMP and 5' -GMP it appears that in the 5 (a) Kundu K K, Jana D & Das M N, Electrochim Acta, pH range 9~10 .deprotonation sites for the tri- 18 (1973) 75; (b) Kundu K I.< & Majumder K, } chem anion of 5'-UMP and 5'-GMP are N3C40 and Soc Trans J, 71 (1975) 1422. 6 Hetzer H. B, Robinson R A & Bates R G, } phys Chern, N1C20 groups respectively [vide Schemes 1 and 2}. The above notion is also supported by results 68 (1964) 1929. & J 8 7 Lang J, Strum J Zana R, (a) phys Chern, 77 (1973) based on Raman spectroscopy. and IR studies of 2329; (b) } phys Chern, 78 (1979) 80; (c) Biopolymers; uracil and guanine derivatives in solution. 10(1971)2639. 8 Ts'o POP, Basic principles in nucleic acid chemistry References (Academic Press, London) 1974, Vol. 1. 1 Pullman B & Pullman A, Quantum biochemistry (Intersci- 9 Phillips R, Eisenberg S J P, George P & Rutman R J, } . ence Publishers, New York) 1963, p. 185. bioi Chern, 240 (1965) 4393. 2 Ganguly S & Kundu K K,} Soln Chem(Press). 10 Ganguly S & Kundu K K, Can} Chern, 72 (1994) H20.