Anal. Chem. 1995, 67,2567-2574
Complete Resolution of the Microscopic Protonation Equilibria of D-myollnositol 1,2,6-Tris(phosphate) and Related Compounds by 31PNMR and Potentiometry Khalid Memissi-Arifi, Laurent Schmitt, Gilbert Schlewer, and Bemard Spiess" Laboratoire de Pharmacochimie Moleculaire, UPR 421 du CNRS, Facuke de Phamacie, 74, route du Rhin, B.P. 24, 67401 lllkirch Cedex, France
31P NMR and potentiometric titration experiments allowed the complete microconstant system to be resolved for D-myo-inositol 1,2,6-tris(phosphate) [Ins(1,2,6)P31 and DL-myo-inositol1,2-bis(phosphate), For comparison, DL-myo-inositol 1-mono(phosphate) and DL-myO-inOSitOl 2-mono(phosphate) were also considered. The studies have been performed in 0.1 M tetraethylammonium perchlorate or tetrabutylammonium bromide solutions at 25 "C (media 1) and, in addition, for Ins(1,2,6)P3 in a 0.2 M KCl medium at 37 "C (medium 2). The 31PNMR titration curves of the polyphosphates indicate that the protonation process of a given phosphate group is complex and far from that of a simple monoester. The interactivity parameters derived from the microconstants support the conclusion that two cis phosphates interact less than two trans phosphates. The influence of the presence of K+ on the microconstants determined in medium 2 can be explained by the competition between H+ and K+ for binding to the ligand. The distribution curves of the microspecies versus pH allow a direct determinationof the protonation state of each phosphate group. Among the inositol phosphates (IPS),phytic acid, the hexaphosphorylated derivative which is an abundant plant constituent,' and myo-inositol 1,4,5tris(phosphate) [Ins(1,4,5)P31, an intracellular m e s ~ e n g e r , ~are - ~ the major representatives. Nevertheless, another IP, D-myo-inositol1,2,6-tris(phosphate) [Ins(1,2,6)P31,has shown various promising pharmacological properties resulting from extracellular action.5 It is especially known to counteract various types of inflammations and to exert an analgesic effect through a mechanism which is still not fully understood. Previous studies carried out in our laboratory demonstrated the ability of the IPS to bind various cations, such as alkali,alkaline earth, or transition metallic Taking into account the (1) Graf, E. Phytic Acid, Chemistry and Applications; Pilatus Press: Minneapolis,
1986. Berridge, M. J.; Irvine, R. F. Nature 1984, 306, 67-69. Bemdge, M. J.; Irvine, R F. Nature 1984,312, 315-321. Bemdge, M. J. Nature 1993, 361, 315-325. Siren, M.; Linne, L.; Person, L. In Inositol-phosphates and deriuatiues; Reitz, A B., Ed.; ACS Symposium Series 463; American Chemical Society: Washington, DC, 1991; Chapter 7. (6) Bieth, H.; Jost, P.; Spiess, B.J. Inorg. Biochem. 1990,39, 59-73. (3Bieth, H.; Schlewer, G.; Spiess, B.J I n o q . Biochem. 1991, 41, 37-44. (8) Schmitt, L:Schlewer, G.; Spiess, B. J. Inorg. Biochem. 1992, 45, 13-19.
(2) (3) (4) (5)
0003-2700/95/0367-2567$9.00/0 0 1995 American Chemical Society
ionic environment of the IPS inside or outside the cell, metal ionIP interactions are likely to be involved in the biological activity of these compounds. Thus, a thorough investigation of the metal coordination and its structural and conformational consequences can be of prime importance in gaining reliable knowledge of the IPS' mechanisms of action. In such an attempt, the specific acidbase properties of each individual Coordination site are worth determining. In addition, such information can be of wider interest for biologically active organic compounds whose submolecular acid-base properties can provide useful structural and conformational information about the binding of the molecule to its receptor. The acid-base properties of a molecule are customarily determined and expressed as protonation or dissociation constants. The overwhelmingmajority of these equilibrium constants are macroconstants, i.e., constants which, in the case of polyfunctional molecules, do not refer to a unique donor site, but only characterize a molecule as a whole. Protonation or deprotonation processes are known to markedly change the electron density on the involved functional group, leading to significant modification in its chemical behavior. Very few chemical equilibrium systems are defined in terms of microconstants for molecules carrying spectroscopic three or more functional g r o u ~ s . ~ lCombined -~~ and pH metric methods were used in some cases, but due to the complexity of the systems, auxiliary ligands were considered or simpliiied treatments have been employed in which the microspe cies equilibrium constants of little or no consequence were neglected. Among the spectroscopic technics, NMR has proved, for a long time, to be a good probe to study prot~nation.'~-~~ Thus, 31P NMR is especially suited to monitor the proton ionization (9) Lapp, C.; Spiess, B.J. Inorg. Biochem. 1991, 42, 257-266. (10) Memissi-Arifi, IC;Wehrer, C.; Schlewer, G.; Spiess, B. J. Inorg. Biochem. 1994, 54, 263-277. (11) Martin, R B.; Edsall, J. T.; Wetlaufer, D. B.; Hollingworth, B. R. J. Biol. Chem. 1958,233, 1429-1435. (12) Ishimitsu, T.; Hirose, S.; Sakurai, H. Talanta 1978, 26, 74-78. (13) Burger, K. In Biocoordination Chemistry; Ellis Honvood Series in Inorganic Chemistry; Burger, IC, Ed.; k Jozef University: Szeged, Hungary, 1990. (14) Martell. A E.; Motekaitis, R J. Defemination and use ofstability constants, 2nd ed.; VCH Publishers, Inc.: New York, 1992. (15) Rabenstein, D. L.; Sayer, T. L. Anal. Chem. 1976, 48, 1141-46. (16) Sayer, T. L.; Rabenstein, D. L. Can. J. Chem. 1976, 54, 3392-400. (17) Samesski, J. E.; Reilley, C. N. In Essays on analytical chemistty; W&minen, E., Ed.; Pergamon: Oxford, 1977. (18) Cabral, M. F.; Costa, J.; Delgado, R; Frausto da Silva, J. J. R; Vilhena, F. M. Polyhedron 1990, 9 (23), 2847-2857. (19) Amorim, M. T. S.; Ascenso, J. R; Delgado, R; Frausto da Silva, J. J. R. J. Chem. Soc., Dalton Trans. 1993, 3449-3455.
Analytical Chemistty, Vol. 67,No. 15, August 1, 1995 2567
behavior of phosphoms-containing molecules. It has largely been used to determine macroprotonation constantsz0 as well as to measure intracellular pH.zl This work aims first to show the possibility of using simultaneously potentiometry and 31PNMR spectroscopy to determine the microprotonation states of a compound containing three phosphates. Thus, it describes for the first time the resolution and the quantization, without approximation, of the entire microprotonation equilibria system for a trifunctional system. This work also is part of a comprehensive study aimed at determining the submolecular acid-base properties of the inositol phosphates and their structural consequences. After having examined these properties for Ins(1,4,5)P3 and related compounds,2zwe report here the study of Ins(1,2,6)P3 and three of its less phosphorylated derivatives, Le., Ins(1,2)Pz, Ins(l)Pl, and Ins(2)P1. The studies OH
HO&oH
0PO.H.
OPO,H,
HO OH
OH
HO
HO
OH
were performed in 0.1 M tetrabutylammonium bromide (nBu4NBr) or tetraethylammonium perchlorate (Ef4NC104) solution at 25 "C (media 1). These supporting electrolytes should only weakly interact with the IPS and therefore lead to the "intrinsic" acid-base properties of the molecules. In addition, for Ins(1,2,6)P3, a 0.2 M KC1 medium at 37 "C (medium 2) was used to examine the influence of the alkali cations on the ionization state of this ligand.
Scheme 1
Lpa
LP,'
cannot be described by the above-mentioned macroconstants. In that case, a more detailed ionization scheme, involving microconstants at an inframolecular level, must be considered. Numerous papers have reported the microscopic constants for bifunctional ligands.15$16*23-z6 We have determined these constants for Ins(l,4)P2 and Ins(4,5)P~.~~ For organic phosphates such as inositol phosphates, 31PNMR spectroscopy gives easy access to the protonated fraction of each phosphate group. Indeed, the observed chemical shift for any resonance, dpbs, depends on the electronic effects accompanying the deprotonation of the phosphate groups and the variation of the 0-P-0 bond angle.27 If we assume that the phosphate groups freely move around the ester bond, averaging the latter effect, then dpbs corresponds to the weighted average of shifts for the possible protonated and deprotonated forms. Since each phosphate group carries only one proton in the pH range studied (2.5 < pH < lo), ~3;~' can be defined as in eq 1, wheref;,pandf;,d are respectively the protonated
and deprotonated fractions of the phosphate in position i, and di,p and 6i.d are the corresponding chemical shifts. The fraction of protonation, is defined in eq 2.
METHODS
Determination of Macroscopic and Microscopic Constants. The overall macroscopic protonation constants,
&,
characterize the general equilibrium, LC-
5 HyL(n-Y)-
+ yH+
in which n corresponds to the charge of the deprotonated ligand L and 0 < y 6 n. In the stepwise protonation process, the equilibrium shown below holds. K HY-1 L(n-Y+l)- + H+& H,L("-Y)-
However, for polybasic compounds such as inositol polyphosphates, the protonation process of each individual phosphate group (20) Costello, A J. R.; Glonek, T.; Myers, T. C. Carbohydr. Res. 1976,46 159-
171. (21) Robitaille, P. M. L.; Robitaille, P. A; Brown, G. G., Jr.; Brown, G. G. j.Magn. Reson. 1991,92.73-84. (22) Schmitt, L.; Bortmann, P.; Schlewer, G.; Spiess, B. J Chem. Soc., Perkin Trans. 2 1993.2257-2263.
2568 Analytical Chemisrry, Vol. 67,No. 75,August 7, 7995
The number of microspecies and microconstants rapidly increases with the number of functional groups of the compound.13 When three functional groups are present, the protonation scheme becomes complicated since it involves seven different protonated species and 12 microscopic equilibria. Scheme 1 depicts the protonation diagram of an inositol triphosphate. In the general case, the subscripts m, n, and 0 refer to the position of the phosphate on the inositol ring. Thus, for instance, for Ins(1,2,6)P3 studied here, m, n, and 0 will correspond to 1, 2 and 6, respectively. The k's are the microprotonation constants, and the associated subscripts indicate the number of protons and their binding sequence. Scheme 1considers only the &st protonation step of each phosphate, the second basicity of the phosphates being very weak for the inositol phosphates. (23) Kiss, T.; Toth, B. Talanta 1983,29,539-544. (24) Uguagliati, P.; Canovese, L. Talantu 1991,38, 697-704. (25) Kiss, T.; Balla, J.; Nagy, G.; Kozlowski, H.; Kowalik, J. Inorg, Chim. Acta 1987,138. 25-30. (26) Crisponi, G.; Nurchi, V.; Casu, M.;h i , A. Spectrochim. Acta 1993,49A, 1643-1649. (27) Cozzone, P.; Jardetzky, 0. Biochemistry 1976,15, 4853-4859.
a, and a, are similarly expressed. For step 2,
am,= k,k,,lH+12/D
= k,k,,[H+12/D
(13)
amo and an,are obtained from the same type of equations. For step 3, For Scheme 1,fm,p,fn,p, and& are the protonation fractions of the phosphates m,n,and o respectively (eqs 6-10). For example,
D is defined for eqs 12-14 as above in eq 11.
fm,p =
sum of the concns of microspecies protonated on site m total concn of all microspecies (6)
([IP,J&I + IIP,,H21 + [IPm,H21 + [IP,Hl)/ ([IP,,,HJ + [IP,,H,I + [IPmoH~l + [IP,,H~I + [IP,HI + [IP,Hl + [IP,HI + [IPI) (7)
fm,p =
Substitution of the microscopic dissociation constants into eq 7 gives eq 8,
Expressions for fn,p and
are obtained similarly, yielding eqs 9
Each protonation fraction expression includes three microscopic constants which can be obtained by fitting the data sets of a given 31PNMR titration curve. For the curve-fitting,values of ,&,Bz,and p3 previously determined by potentiometry under the same conditions were introduced into eqs 8-10, The remaining three microconstants are obtained from eq 5. It should be noted that, for instance, k,, and k,, cannot be mathematically unambiguoulsly calculated from eq 8. However, by considering eq 5, since k d k , = k n d k m n , the constants km, and km, can be successfully distinguished. Calculation of the Protonated Microspecies Distribution Curves. For the microspecies of Scheme 1, three types of equations were used according to the protonation step considered. For step 1, a,, the relative concentration of the IP,H species can be expressed as
a, = [IP,HI/([IPm,oH31 [IP,,H,I + [IP,Hl
+ [IPm,H,l + [IP,oH21 +
+ [IP,Hl + [IP,Hl + [IPI) = k,[H+l/D
(12)
EXPERIMENTAL SECTION
Materials. (f)-Ins(l)P1 and (f)-Ins(l,2)P~wereprepared as previously described.22,28Hydrated NaS HIns (1,2,6)P3,provided by Perstorp Pharma (Sweden), and Ins(2)Pl (Sigma) were used without further purification. Potentiometric Studies and NMR Determinations. A 3 mL aqueous solution of the IP as the cyclohexylammoniumor sodium salt was converted into the acidic form by ion exchange using a Amberlite IRN 7 7 0 resin column. The eluate was collected in a 5 mL flask containing 0.5 mL of DzO and the supporting electrolyte EbNC104 or BudNBr for media 1 (0.1 M). For the studies to be performed in 0.2 M KCl (medium 2), DzO was used as solvent in order to record in the same experiment the 31Pand 'H NMR spectra. Thus, the aqueous solution obtained after ion exchange was lyophilized, and the remaining IP was dissolved in DzO. These solutions, at a concentration of about 3 x moledm-3, were used the same day for both potentiometric and NMR measurements. First, 2 mL of the previous solution was titrated in a thermoregulated (25 or 37 f 0.1 'C) cell with a base. The titration reactant was, in media 1,tetramethylammonium hydroxide, and in medium 2, potassium deuterioxide. For the aqueous solutions, fresh, twice distilled water was used. The electrode and the automatic titration equipment were previously described.'j The potentiometric titration first allowed the determination of the concentration of the IP (e3and the total concentration of the acid (@d.Analysis of the pH measurements using the program Superquadzggave the macroscopic protonation constants. As the electrode was calibrated in concentration, pH corresponds to the cologarithm of the concentration of H+. Second, the same initial volume of solution and the same additions of the base were used for the NMR titration. The previously measured pH values were kept since there is only a small uncertainly in these values: two successive titrations showed that pH variations are less than 0.01 unit. 31PNMR spectra were recorded at 81.015 MHz on a Bruker AC200 fourier transform spectrometer. Chemical shifts were measured relative to an external 85% orthophosphoric acid reference. The sample temperature was regulated to 25 or 37 k 0.2 "C by a nitrogen flow using the standard Bruker temperature control unit. For the 1H-31P chemical shift correlative 2D NMRs, the spectral width in F1 was 2.2 kHz and in Fz was 746 Hz;16 scans (28) Schmitt, L. Ph.D. Thesis. Strasbourg, France, 1993. (29) Gans, P.; Sabatini, A; Vacca, A J. Chem. SOC.,Dalton Trans. 1985,11951200.
Analytical Chemistry, Vol. 67, No. 15, August 1, 1995
2569
P
n
t 2513-H5 H4
I/
H6
2.0 '
15.
1.01
.e,
*R
0.5. "PNM R 0
3
4
5
e
7
8
9
e.._u-.
bW.
lo
o-- l1
Potent10
0
0 1
___
,,o' 11
12
PH
Flgure 2. Mean number of protons bound per ligand (p)versus pH calculated from potentiometric (0)and 31NMR(H) measurements.
5
4 iP9m)
Figure 1. Two-dimensional iH-31P chemical shift correlation contour map showing connectivities via 3 J ~ - ~ ,
for each of 256 experiments with 1K data points were used. The 256 data points in 4 were zero-filled to 512 data points. The protonation fraction curves were analyzed by nonlinear regression using the iterative curve-fitting program E d t t e r (Elsevier-Biosoft). This program allows, by the use of the Marquart method, the calculation of the "best" microconstants as judged by the least-squares criterion ,yz. The uncertainties reported with the macro- and microconstantsare estimates of the standard deviation as calculated by Superquad and Enzfitter, respectively. RESULTS AND DISCUSSION
Assignment of Phosphorus Resonances. Resonance peaks of Ins(l,2,6)P3were assigned by performing phosphorus-proton 2D correlation experiments. The 'H NMR spectra of Ins(1,2,6)P3 show the presence of six protons whose assignment was previously made.30j31The proton-coupled phosphorus spectrum displays three resonances for the nonequivalent phosphates, split into doublets due to their coupling with the proton of the my5 inositol ring. At pH 6.5, the H1, H2, and H6 protons resonances are at 4.13, 4.74, and 4.32 ppm, respectively. The 1H-31P correlative 2D analysis of the contour map shown in Figure 1 indicates that these protons can be associated with the corresponding phosphate resonances at 1.95 (Pl), 4.60 (PZ), and 3.88 ppm (P6). Nevertheless, a major difficulty in the attribution of the phosphorus resonances occurs due to the crossing of the P2 (30) Johansson, C.;KiSrdel, J.; Drakenberg, T. Curbohydr. Res. 1990,207,177183. (31) Scholz, P.;Bergmann, G.; Mayr, G. W. In Methods in Inositide Research; Irvine, R F., Ed.; Raven Press: New York, 1965.
2570 Analytical Chemistry, Vol. 67, No. 15, August 7 , 7995
and P6 signals at high pH (see Figures 5 and 7). The pH at which the intersection point can be observed is largely dependent on the concentration of alkali cations present in the medium. For instance, it occurs at pH 9.50 in the absence of potassium and at pH 8.25 in the presence of this cation at a 0.2 M concentration. Thus, the pH values of the correlation experiments have to be chosen far enough from that point, Le., 7.5 < pH > 10. At pH 10.5 (contour map not shown), the H1, H2, and H6 proton resonances at 3.90, 4.60, and 4.15 ppm, respectively, can be correlated with the corresponding phosphate resonances at 5.03 (Pl), 5.17 (PZ), and 5.84 ppm (P6). For the latter pH conditions, it can be noted, as previously stated, that the chemical shifts of P2 and P6 are inverted with respect to those observed at the lower pH. The phosphorus resonances of Ins(l,Z)P*were assigned by analogy with those of Ins(1,2,6)P3. Significanceof bobsin 31PNMR. As stated in many studies on inositol phosphates and other organic phosphate^,^^^^^^^^^^^ the chemical shift of the phosphorus nuclei depends mainly on the protonation state of the phosphate groups. To check this statement, p = f@H) was considered, p being the mean number of protons bound per mole of inositol phosphates. Interestingly, p can be obtained by both potentiometry and 31PNMR Potentiometric measurements allow the p calculation according to eq 15, where CH and CLcorrespond to the analytical concentrations
of the acid and the ligand, respectively. On the other hand, if N is the number of nonequivalent phosphates in the molecule, p
can be derived from eq 16. i=N
P = %,p i=l
In Fgure 2, the3 curves are reported for Ins(l,2,6)P3calculated according to eqs 15 and 16. It can be seen that there is a very good agreement between both curves for pH < 11, Le., for the (32) Isbrandt, L. R; Oertel, R. P. J. Am. Chem. SOC.1980, 102,3144-3148. (33) Emsley, J.; Niazy, S. Phosphorus Sulfur Relat. Elem. 1981, 10,401-407.
6
6
.. ... .
..am.
3
2
2
3
4
5
6
7
8
9
10
11
12
I II
0 0
P2
P1
0
.
0 0 0
2
3
5
4
.. . 8
7
6
9
10
11
12
PH
Figure 3. Chemical shifts 6 from 31P NMR titrations as a function of pH for Ins(l)Pl and Ins(2)Pl.
entire pH range used in the calculation of the protonation constants. This undoubtedly shows that the protonation state of the phosphate groups predominantly governs the chemical shift of the phosphorus nuclei in the case of Ins(1,2,6)P3. Nevertheless, it can be observed that, for a phosphate in position i, differences in the dI,p- di,d values do exist, and these can be related to factors such as (i) the configuration of the phosphates, (ii) the number of vicinal phosphates, and (iii) the number and con6guration of the OH groups on the inositol ring. These factors are kept constant over the whole pH range where deprotonation occurs and therefore do not prevent the calculation of the protonation fraction for each phosphate. Accordingly, it must be considered that, by changing pH, the chemical shift variations are related to the ionization state variations. This assumption holds true only as long as no species other than H+ which are able to strongly bind to the phosphates are present and if no strong hydrogen bonding occurs between the phosphates and the vicinal OH groups. In the case of Ins(1,4,5)P~,previously we were unable to resolve the microequilibria system, presumably due to the formation of such hydrogen bonds. It can also be noted that above pH 11, both curves diverge when the potentiometrically calculated p curve is going to increase. Such an increase has no chemical meaning and probably results either from an interference of the inositol phosphates on the glass electrode at high pH or from the changes in the ionic strength in the presence of the highly negatively charged Ins(1,2,6)P3. Inositol Monophosphates and Diphosphates. The 31P NMR titration curves of Ins(l)P1 and Ins(2)Pl are displayed in Figure 3. From these curves it can be seen that the phosphate orientation on the ring largely influences the basicity of the phosphate group, as well as the chemical shift of the monoprotonated species. Indeed, the axially orientated phosphate group is significantly less basic (log K = 5.85) than the equatorial one (log K = 6.50), and for both phosphates is 1.4 ppm. On the other hand, whatever the orientation of the fully deprotonated groups, the d1,d values are nearly identical,which seems to indicate that both of the deprotonated phosphates experience approximately the same interactions with vicinal polar groups and with the solvent. Thus, the lower basicity of the axial phosphate cannot be attributed to a particular stabilization of its dianion with regard to that equatorially orientated. The differences in log K and di,, values should then be the result of the differences in the
PH
fip 0.8
P2
2
3
4
5
6
P1
7
8
9
10
11
12
PH
Figure 4. (a) Chemical shifts 6 from 31P NMR titrations for Ins(1,2)P2 and (b) the corresponding protonation fraction curves fi,pas a function of pH in nBudNBr, 0.1 M, at 25 "C. The least-squares fit of fi,pversus pH is shown in the solid line of (b).
orientation of the vicinal OH groups for both phosphates, and two cis OH groups exert a much larger effect than one cis and one trans. The importance of the presence of OH groups and its decreasing effect on the basic character of a neighboring phosphate has been r e p ~ r t e d . ~ It ~ ais~ likely that, for the inositol monophosphatesunder consideration, there might be a field effect affecting the phosphorus electron cloud either directly or via changing the solvent structure around the phosphate moiety, or both, rather than an inductive effect through u bonds. In Figure 4 are shown the 31PNMR titration curves and the corresponding protonation fractions of Ins(1,2)P2. Here again, it appears that the phosphates in positions 1and 2 behave differently, the latter being less basic than the former. The cis and trans hydroxyls of the axial and equatorial phosphates, respectively, might also partly account for such an observation. For Ins(1,2)Pz, the logarithm of the stepwise protonation constants log K1 and log K2 are respectively 7.97 and 5.74, leading to a KJK2 ratio of 162. Over the years, it has been shown that the ratio between 6rst and second protonation constants exhibited by various bifunctional bases (or dissociation constants of dibasic acids) depends upon a statistical factor, the electrostatic influence (34) Massoud, S.S.;Sigel, H.Inorg. Chem. 1988,27,14-17-1453,
Analytical Chemistry, Vol. 67, No. 15, August 1, 1995
2571
Table 1. Logarithms of the Macro- and Microprotonation Constants for Ins(l)PI, Ins(2)P1, Ins(1,2)Pz and lns(l,2,6)P3s
medium, t ("C) Et4NC104 (0.1 M), 25
ligand
Y
log K Y
Ins(1)Pl
1
nBu4NBr (0.1 M), 25
Ins(2)Pl
1
nBu4NBr (0.1 M)
Ins(1,B)Pz
1
6.50 (0.01) 5.83 (0.01) 7.97 (0.01) 5.74 (0.01) 9.53 (0.01) 7.31 (0.01) 5.85 (0.01)
25 (10%DzO)
EkNC104 (0.1 M), 25 (10%DzO)
2 Ins(1,2,6)P3
1 2 3
i
log k ,
ii'
log k g
1
7.95 (0.01) 7.39 (0.01) 9.44 (0.01) 7.32 (0.01) 8.80 (0.01)
12
5.76 (0.01) 6.32 (0.01) 6.76 (0.05) 7.17 (0.02) 8.94 (0.02) 8.66 (0.03) 7.78 (0.01) 7.20 (0.01) 6.20 (0.05) 6.47 (0.05) 7.83 (0.19) 7.70 (0.19) 6.69 (0.09) 6.48 (0.09)
2 1
2 6
21 12 16 21 26 61 62
KC1 (0.2 M), 37 (DzO)
Ins(1,2,6)P3
1
2 3
8.34 (0.01) 6.60 (0.01) 5.47 (0.01)
1 2 6
8.18 (0.01) 6.59 (0.01) 7.90 (0.01)
12 16 21 26
61 62
ii'i"
126 162 62 1
126 162 62 1
6.46 (0.03) 6.10 (0.01) 6.70 (0.01)
6.01 (0.02) 5.79 (0.03) 6.08 (0.04)
log ki, log kip, and log kip? represent general designations for respectively the logarithmsof the first, second, and third stepwise microprotonation constants. The uncertainties (in parentheses) are estimates of the standard deviations as calculated by Superquad and Enzfitter for the macro and microconstants, respectively. The interactivity parameters for Ins(1,2,6)P3are as follows. In media 1, A log k l - 2 , M = 0.53 f 0.03; A log k1-6,zd = 1.64 f 0.02; A log k2-6,ld = 0.13 f 0.02; A log kl-2,6 = 1.11 f 0.02; A log kl-6,2p = 2.21 f 0.04; A log k2-61p = 0.68 f 0.01. In medium 2, A log k l - 2 , ~= 0.37 f 0.03; A log kl-6,2d = 1.46 f 0.03; A fog k 2 - 6 , l d = 0.15 f 0.05; A log k1-2,ep = 0.65 f 0.05; A log k l - 6 , ~=~ 1.72 f 0.04; A log k2-6,1p = 0.44 f 0.03.
of the substituents and intramolecular hydrogen bonding in the monoprotonated ~ p e c i e s . 3 ~Eberson - ~ ~ et al.,38by studying a large number of dicarboxylic acids, argued that hydrogen bonding might predominantly be involved when KdKz > 104. It seems also likely, therefore, that for Ins(1,2)Pz, the electrostatic effects alone are sufticient to account for the observed KI/& ratio. Moreover, if by the addition of 1 equiv of protons, an H-bond would link P1 and P2, the second protonation would require the rupture of this bond, and thus a low log KZ value would be expected. Since log KZfor Ins(1,2)Pz is nearly the same as log K for Ins(2)P1, the formation of a strong H-bond can be ruled out. The calculation of microscopic protonation constants for Ins(1,2)P2 according to ref 22 (Table 1) provides a direct access to the quantification of the ionization state of each site. It can be noted that the calculated f2,p curve satisfactorily fits the experimental data with a slight divergence for fi,pat the highest pH values. The meaning of this difference remains unclear. As stated above, a given microscopic protonation constant accounts for both the intrinsic binding of the proton to the functional group and the perturbation deriving from the electrostatic forces of neighboring groups. Thus, for bifunctional compounds, interactivity (35) Bjer", N. Z. Z Phys. Chem. 1923,106, 219-242. (36) Kirkwood, J. G.; Westheimer, F. H. 1. Chem. Phys. 1938,6,506-517. (37) Westheimer, F. H.; Shookhoff, M. W. J. Am. Chem. SOC.1939,61, 555560. (38) Eberson, L.; Wadso, I. Acta Chem. Scand. 1963,17, 1552-1562.
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Analytical Chemistry, Vol. 67, No. 15, August 1, 1995
parameters are generally d e b e d in order to quantify the changes in basicity at one site when the other site takes up a proton and doing so modifies its charge. Thus, for Ins(1,2)Pz, this parameter can be defined as A log kl-2 = log kl - log kzl = log k2 - log klz = 1.64. This value lies in between A log kl-4 and A log k4-5, which were previouslyz2found to be 0.07 and 2.12, respectively. Such a result c o r h " the larger interaction between two vicinal phosphates than between two phosphates in para position on the ring, and shows that two cis phosphates interact less than two trans phosphates. Inositol 1,2,6-Tris(phosphate). The 31P NMR titration curves and the3 =f(pH) curves for Ins(1,2,6)P3 reported in Figure 5 differ largely from one phosphate to another in their shapes as well as in the position of their inflexion points. This indicates that the protonation process of a given phosphate group is far from that of a simple monoester, being markedly influenced by its neighboring phosphates. With the addition of the first equivalent of protons, phosphate P1 undergoes a large upfield shift resulting from protonation of about 80%of this group. Simultaneously, P6 takes the remaining protons, leaving P2 almost totally deprotonated. When the second equivalent of proton is added, mainly P2 and P6 are concerned, both taking about 45%-50%of the protons. Surprisingly, at the end of this addition, P1 remains at an ionization state close to that achieved about 2 pH units earlier. Finally, all three phosphates share the third equivalent
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Figure 6. Distribution curves of the protonated microspecies of Ins(1,2,6)P3 in EbNC104, 0.1 M, at 25 "C, plotted against pH (CL= 1 x 10-3 m ~ l - d m - ~ ) .
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Figure 5. (a) Chemical shifts 6 from 31P NMR titrations for Ins(1,2,6)P3 and (b) the corresponding protonation fraction curves fi,p as a function of pH in EL+NC104,0.1 M, at 25 "C. The least-squares fit of fj,p versus pH according to eqs 8-10 is shown in the solid line of (b). In (a), the vertical bars indicate the peak width at half-height of the phosphorus signals. In (b), the vertical lines correspond to the theoretical addition of 1-3 equiv of protons.
of protons to reach their monoanion state. The protonation sequence for Ins(1,2,6)P3 may be explained by considering that the protonation of a given site is primarily electrostatic in origin. P1 is located between P2 and P6; it is therefore likely that P1 will experience the highest density of negative charges and thus will be predominantly protonated. Upon its protonation, the electric charge density around P1 decreases until it reaches that of the neighboring phosphates. This then allows P2 and P6 to protonate, whereas the ionization state of P1 remains constant for statistical reasons. In the final step, the distribution of the protons is close to that expected for three binding sites of the same nature. Table 1contains the 12 microconstants calculated for Ins(1,2,6)P3 from the protonation fraction curves versus pH (Figure 5). The least-squaresfit of& versus pH according to eqs 8-10 shown in the solid line of Figure 5b can be considered as very satisfactory. The poorer match between the predicted and actual curves for P1 and P2 for the first equivalent of proton added may be attributed to the width of the NMR signals for steep chemical shift variations (see Figure 5a), the larger uncertainties of the pH measurements above pH 9 which can be estimated in our
experimental conditions, and the duration of the experiment (7-9 h) of 0.15 pH unit. The microconstants quantify the results previously reflected by the protonation fraction curves. Thus, log kl (9.44), very close to log K1 (9.53), indicates the high basicity of P1 with regard to P6 and particularly to P2. The log kii, and log kif? (see footnote of Table 1) values do not allow a direct and simple evaluation of the basicity of a given site since they are dependent upon the log ki values. Nevertheless, the calculation of microconstant ratios leads to concentration ratios of protonation isomers which are constant at any pH. For instance, kzdklz = [IP1H]/[IP2Hl = 151, ksl/k16 = [IPIH]/[IP&] = 4.1, and kZdk62 = [IPsHI/[IPzH]= 28.4. Such calculations clearly illustrate the relative basicity of the different phosphate groups. It is also possible to distinguish in Scheme 1 six subschemes involving the phosphates two-by-two leading to six interactivity parameters. In that case, these parameters account for the relative changes in the free energy of interaction of the phosphates on binding the proton. For instance, A log kl-6,Zd = log kl - log k61 = log k6 - log kl6 represents the interaction between P1 and P6, P2 being deprotonated. On the other hand, A log kl-6,Zp = log kzl - log k6Zl = log k26 - log klZ6 shows the interaction between P1 and P6 when P2 still carries a proton. These calculated interactivity parameters are given in the footnote of Table 1. From these values it can be seen that, in the presence of either a third dianionic phosphate or a third monoanionic phosphate, the interactivity parameters, and thus the interaction, for Pl-P6 are largely the highest, followed by those of Pl-P2 and P2-P6. Noteworthy is the fact that A log k1-6,Zp = A log k . 2 seems to indicate that the interactions between the two trans phosphates in Ins(4,5)Pz and Ins(1,2,6)Pg are nearly the same. In addition, for the latter, Pl-P2 interact to a lesser extent, and P2-P6 tend to respond independently. Moreover, the influence of the ionization state variation of the thiid phosphate on the interaction of the two other phosphates can be estimated from the difference A log ki-c,yj - A log ki-f,pd. By calculating these values for all systems, it appears that this influence is remarkably constant (mean value, 0.56 f 0.02). Figure 6 displays the distribution of the various microprotonated species versus the pH, allowing a direct observation of the protonation state of each phosphate group. Analytical Chemistry, Vol. 67,No. 75,August 7, 1995
2573
t
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Figure 7 . (a) Chemical shifts d from 31P NMR titrations for Ins(1,2,6)P3 and (b) the corresponding protonation fraction curves fi,p as a function of pH in KCI, 0.2 M, at 37 "C. The least-squares fit of fi,pversus pH according to eqs 8-10 is shown in the solid line of (b). In (b),the vertical lines correspond to the theoretical addition of 1-3 equiv of protons.
The 31PNMR titration curves and the protonation fractions for Ins(1,2,6)P3 in the presence of potassium cations are shown in Figure 7. It can be seen that the general shape of the curves remains the same as before, being only shifted toward the lower pH values. Such a shift is clearly a consequence of the competition between H' and K+ for the binding to the ligand. This confirms that alkali cations are readily complexed by In~(1,2,6)P3.~~ The & curves were also processed with eqs 13-15 in order to derive microconstants. The microconstants reported in Table 1 must be considered as conditional constants, taking into account the interaction with K+. By comparing the sets of microconstants obtained in both media, it appears that these constants are not equally affected by the presence of K+. The larger variations concern the protonation of Pl, then P6, and ultimately P2. Consequently, it seems likely that the potassium cation preferentially binds the phosphates in the same order, i.e., the binding ~~~
(39) White, A M.; Vamey, M. A; Watson, S. P.; Rigby, S.; Changsheng, L;Ward, J. G.; Reese C. B.; Graham, H. C.; Williams, R J. P. Biochem. I. 1991,278, 759-764. (40) Lit, E. S.; Mallon, F. IC;Tsai, H. Y.; Roberts, J. D. J. Am. Chem. SOC.1993, 115,9563-9567.
2574 Analytical Chemistry, Vol. 67, No. 15, August 1, 7995
ability increases when the phosphate basicity rises. Such a macroscopic complexation trend has been reported for various cations'O and phosphate^.^^ In addition, the A log kl-f,,l,,por A log kl-l.,t,,d values of Table 1, it can be seen that the interactivity parameters for Pl-P2 are mainly decreased in the presence of K+. This is in line with the previous conclusions, Le., the preferential binding of this cation between P1 and P6 leads to a more rigid structure than in the free ligand, which hinders the Pl-P2 interactions. The 'H NMR titration curves have also been performed (results not shown) in medium 2. In these curves, only small changes in the chemical shifts were observed over the pH range studied (2.5 < pH < 12). On going from the highest to the lowest pH limits, all resonances shift regularly to the lower frequencies of about 0.1 or 0.2 ppm, with the exception of H1. The signal corresponding to H1 moves in the opposite direction from the other resonances. As has already been shown for In~(1,4,5)P3,~~ the spectra of the protons bound to non-phosphorylated carbons are affected by the protonation state of the phosphates on the neighboring carbons. Therefore, it also appears for Ins(1,2,6)P3 that the protons of the phosphorylated positions (Hl, H2, and H6) will be influenced by the ionization of both the distal and the adjacent phosphates. The multicomponent character of these curves, which show in addition small chemical shift changes, prevents the deduction of the same information which can be obtained from 31PNMR titration curves. In summary, considering our results, it seems reasonable to suggest that, for the inositol phosphates under study, the chemical shift variations of the phosphorus nuclei during the titration reflect the changes of the ionization state of the corresponding phosphate. With respect to this, it becomes possible to determine microprotonation constants which reveal the nonequivalency in the protonation process of the different phosphates of the molecules. The reasons for the specific behavior of each phosphate rests in its relative position on the myo-inositol ring and its configuration, which determines the interactions with the neighboring phosphates, hydroxyls, and molecules of water in the solvent. Intramolecular hydrogen bonding seems not to be significantly involved in the stabilization of the protons and induction, and electrostatic effects are expected to play the major role. Nevertheless, other factors, such as external hydrogen bonds to water, steric hindrance, and rotational entropy changes,4Omay also partly account for the observations. ACKNOWLEWMENT
We express grateful acknowledgment to Perstorp Pharma (Sweden) for providing the Ins(1,2,6)P3 and for support for K.M. We wish also to acknowledge E. Kremp for technical assistance in the NMR measurements.
Received for review February 16, 1995. Accepted May 11, 1995.e AC950174C
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Abstract published in Advance ACS Abstracts, June 15, 1995.
