J. Phys. Chem. 1992, 96, 1961-1967 TABLE Iv: Rate and Equilibrium Coostants Obtained by Fitting Eq 3 (with m H = @)to Experimental Data for the Acid Denitroslltion of MNTS in Water-HDS-Alcohol Mixtures with Equal Concentrations of HDS and Alcohol
alcohol a
1-butanol isobutyl alcohol 2-butanol tert-butyl alcohol 1-pentanol tert-amyl alcohol 1-hexanol benzyl alcohol
K.. M-l 97 123 117
100 85 125 74 115 107
103w, M-l 3.78 3.66 4.69 4.41 4.34 3.64 4.55 5.62 3.82
s-1
Reference 10. was based on the fact that HDS and SDS have the same hydrocarbon chain, an important influence on the binding of amphiphiles to micelles,3ethe similarity between the measured aggregation numbers (and hence shape) of SDS-alcohol and HDSalcohol micelles (Table 11), and the similarity between the behaviors of HDS and SDS micelles in catalyzing the acid denitrosation of MNTS in the absence of alcohol (in particular, they both have the same affinity for MNTS and the same KNaHvalue).Io Table IV lists the values of K, and k2” obtained using an optimization program based on Marquardt’s2* algorithm, with
1961
values of /3 experimentally measured (Table I) and values of k, and the cmc the same as those for SDS. Both constants vary very little with the alcohol used and are very similar to the values obtained with SDS, so that again the effect of the alcohol is merely to increase the volume of reaction. This similarity between the two kinds of mixed micelles is quite as expected, since the two surfactants differ only as regards their counterion and also behave almost identically in the absence of alcohol.I0 The assumption that KRoHhas very similar values for SDS and HDS is supported by these quite reasonable findings. In conclusion, the addition of moderate quantities of mediumchain alcohols to anionic SDS and HDS micelles caused a reduction in reaction rate that can be explained as due purely to the resulting dilution of the reagents, with no change in kinetic or thermodynamic constants occurring.
Acknowledgment. C.B. thanks the Xunta de Galicia for the research training grant allowing him to take part in this research. Financial support from the Xunta de Galicia (Project XUGA 20903A90) is gratefully acknowledged. We also thank the referees for their comments. Rdstry NO. MNTS,80-11-5; HDS, 1510-16-3;SDS, 151-21-3; l-butanol, 71-36-3; 2-butanol, 78-92-2; isobutyl alcohol, 78-83-1; fertbutyl alcohol, 75-65-0; 1-pentanol,71-41-0; tert-amyl alcohol, 75-85-4; 1-hexanol, 11 1-27-3;benzyl alcohol, 100-51-6. (28) Marquardt, D. W. J . SOC.Ind. Appl. Math. 1963, 1 1 , 431.
Lanthanide Shift NMR Studies of Bile Salt Aggregates Steven M. Meyerhoffer: Thomas J. Wenze1,t and Linda B. McGown*Yt Department of Chemistry, P. M. Gross Chemical Laboratory. Duke University, Durham, North Carolina 27706, and Department of Chemistry, Bates College, Lewiston, Maine 04240 (Received: August 26, 1991; In Final Form: October IO, 1991)
The binding of Tb3+,Dy”, Eu”, Tm3+, and Yb3+ with sodium taurocholate (NaTC), 3-[(3-~holamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), and 3-[(3-~holamidopropyl)dimethylammonio] - 1-propanesulfonate (CHAPS) was studied by ’H NMR spectroscopy. Proton assignments are given for CHAPSO and CHAPS, and the proper selection of an internal reference for NMR shift studies is addressed. Results for NaTC indicate that the lanthanide ion binds asymmetrically with the anionic sulfonate group and the oxygen atom of the peptide carbonyl through a bidentate interaction. Association constants and bound shifts were calculated from the ‘H NMR shift data using a nonlinear least-squares regression analysis. In all cases, the shift data were fit to an equilibrium assuming 1:l lanthanidetaurocholate complexation. In micellar NaTC, however, the association constants calculated with the onestep model only approximate the overall binding interaction; the equilibrium may be influenced by the formation of a 1:4 stoichiometric complex as well as by the total concentrationof lanthanide salt present in solution. The magnitudes of the observed shifts and calculated association constants for Tb3+-taurocholate complexes were greater in micellar NaTC than in premicellar NaTC, indicating an increased binding strength in micellar solution. The Tb3+ion was also observed to bind with the anionic sulfonate group of CHAPSO and CHAPS, but the interactions were significantly weaker than with the taurocholate anion.
Introduction The bile salts are naturally occurring detergents which form micellar aggregates in aqueous solution.’ In addition to their important physiological role? bile salts have also been studied in recent years as alternatives to conventional detergents for chemical analysis. Specific areas of application include chemical separat i o n ~and ~ luminescence analy~is.~Bile salt micelles are smaller and more rigid than those of conventional detergents, resulting in unique aggregation behavior with respect to self-association as well as solubilization of hydrophobic molecules in aqueous soluti~n.~-~ *Author to whom correspondenceshould be addressed. ‘Duke University. *Bates College.
The effects of metal ions on the fluorescence intensity of molecular probes in aqueous solutions of the trihydroxy bile salt sodium taurocholate (NaTC)8,9and of 3-[(3-cholamidopropyl)dimethylammonio] -2-hydroxy- 1-propanesulfonate (CHAPSO)? (1) Small, D. M. In Bile Acids; Nair, P. P., Kritchevsky, D., Eds.; Plenum Press: New York, 1971; pp 249-357. (2) Hofmann, A. F.; Small, D. M. Annu. Reu. Med. 1967, 18, 333. (3) Buckley, J. J.; Wetlaufer, D. B. J . Chromnrogr. 1989, 464, 61. (4) McGown, L. B.; Kreiss, D. S. Fluorescence Detection 11. Proc. SPIE-Int. SOC.Opt. Eng. 1988, 910, 73-80. (5) Zana, R.; Guveli, G. J . Phys. Chem. 1985,89, 1687. (6) Mukerjee, P.; Moroi, Y.;Murata, M.; Yang, A. Y. S. Hepatology 1984,4, 61s. (7) Fisher, L.; Oakenfull, D. Aust. J . Chem. 1979, 32, 31. (8) Nithipatikom, K.; McGown, L. B. A n d . Chem. 1988, 60, 1043. (9) Meyerhoffer, S. M.; McGown, L. B. J . Am. Chem. SOC.1991, 113, 2 146.
0022-3654/92/2096-1961$03.00/00 1992 American Chemical Society
1962 The Journal of Physical Chemistry, Vol. 96, No. 4, 1992
a zwitterionic analogue of NaTC, have recently been described. In NaTC, the fluorescence of relatively soluble probes was quenched by the heavy lanthanide ions, whereas the fluorescence of highly insoluble probes was enhanced as much as 18-fold. The magnitude of the enhancement was found to depend primarily upon the charge of the metal ion. The enhancements were attributed, at least in part, to enhanced aggregation of NaTC in the presence of the metal ions. In contrast to the pronounced effects observed for NaTC, the metal ions were found to have little effect on the aggregation of CHAPSO and the fluorescence of probe molecules solubilized in aqueous CHAPSO solutions. In this work, we describe N M R shift studies of the binding of Tb3+,Dy3+, Eu3+,Tm3+,and Yb3+ with NaTC, CHAPSO, and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]l-propanesulfonate). In a previous study by Elgavish and co-workers, the Dy3+ ion was used as a shift reagent to investigate the binding of Na+ and Ca2+ with glycocholate and taurocholate bile salt monomers in premicellar aqueous solutions through competition studies.I0 The metal ions were found to bind with the carboxylate and sulfonate groups of glycocholate and taurocholate, respectively. An N M R shift reagent study of the structure of the Dy3+glycocholate complex in premicellar aqueous solution was subsequently desCribed.l1 Lanthanide shift NMR studies of a metal ion binding to bile salts in micellar solution have not been previously reported.
Theory The ability of the lanthanide ions to act as N M R shift reagents is well known.I2 The lanthanide ion, acting as a Lewis acid, will coordinate with Lewis base functional groups on organic substrates, thus inducing changes in the chemical shift positions of the nuclei of the substrate. Under conditions of rapid exchange, the observed chemical shift will be a weighted average of the N M R signals from the substrate in its free and bound f o m . l 3 The bound shift (A) is the change in chemical shift which would be observed for the complex in the absence of rapid exchange and is commonly used as a parameter in fitting lanthanide shift data to solution equilibria.IJ The solution equilibria describing the coordination process can be depicted by the following equations:
Meyerhoffer et al. TABLE I: h m p r Of~ Tb3++-M~~ed shifts In the NMR Spectra of M u m 2,2-Dimetbyl-2-silrpentnne-5-sulfon8te(DSS)' and the TetraethyhmmoniumCatioa (TEA+)b A6 (ppm) between resonances in
0 10 30
0.63 0.61 0.58
1.76 1.70 1.61
2.91 2.87 2.83
2.00 2.00 2.00
DSS: (CH3)3SiCH2nCH2fiCH2'S03-Na+ (100 mM). bTEA+: (CH3CH2)4N+(100 mM). contact and complexation shifts are usually small and the observed shift can be adequately described by the dipolar mechanism. The dipolar shift is given by the McConnell-Robertson equation21and in simplified form is
(3) where K is a temperature-dependent constant for a given LS complex, r is the distance between the lanthanide ion and the nucleus of interest, and 6 is the angle between the principal magnetic axis and the line drawn from the lanthanide ion to the nucleus of interest. If the dipolar shift mechanism predominates, resonance lines of those protons located closest to the lanthanide ion often exhibit the largest shifts. It is important to also consider the influence of the angle term on the observed dipolar shift in the interpretation of lanthanide shift data. In the present studies, equilibrium constants and bound shifts were calculated from the lanthanide-induced shift data using the nonlinear least-squares regression analysis (LISA) of Shapiro and J o h n s t ~ n . ' ~LISA fits one-step and two-step equilibria using lanthanide-induced shift (LIS) data obtained over a wide range of lanthanideto-substrate molar ratios. Both one-step and two-step equilibria were considered in this study; however, attempts to fit the shift data to a two-step model were unsuccessful.
Experimental Section The NaTC, CHAPSO, and CHAPS compounds (ULTROL grade, >98%) were purchased from Calbiochem (La Jolla, CA). The nitrate salts (>99.99%) of terbium(III), europium(III), L+S=LS (1) dysprosium(III), lanthanum(III), and thulium(II1) and the chloride salt of ytterbium(II1) were obtained from Aldrich. The LS + s = LS2 (2) internal references included sodium 2,2-dimethyl-2-silapentane5-sulfonate (DSS) (Aldrich), diethylamine hydrochloride and in which L and S represent the lanthanide ion and substrate, tetramethylammonium bromide (Kodak), and tetraethylrespectively. Several methods have been described for the calammonium chloride (Sigma). All of the compounds were used culation of equilibrium constants ( K I and K 2 ) and bound shifts as received. (Al and A,) from the shift data for either a one-step or two-step Stock solutions of the bile salt compounds (1 and 32 mM) were equilibrium (eq 1 only or eq 1 followed by eq 2, respecti~ely).l~-~~ prepared in D 2 0 (Cambridge Isotope Laboratories, 99.9% D). The shifts in the NMR spectra arise from contact, dipolar, and The acidity of the D 2 0was approximately neutral, and no attempts complexation shift mechani~ms.'~For 'H N M R spectra, the were made to control the pD of the solutions. The lanthanide ions (concentrations as high as 60 mM) were added to the bile salt solutions by dissolution of an appropriate amount of lanthanide (10) Mukidjam, E.; Barnes, S.; Elgavish, G. A. J . Am. Chem. SOC.1986, salt in bile salt stock solution. Bile salt solutions containing lower 108, 7082. concentrations of lanthanide ion were prepared by the con(11) Mukidjam, E.; Elgavish, G. A,; Barnes, S. Biochemistry 1987, 26, stant-substrate incremental dilution method,ls in which the most 6185. concentrated lanthanide ion-bile salt solution is diluted with (12) Wenzel, T. J. NMR Shift Reagents; CRC Press: Boca Raton, FL, 1987. original bile salt stock solution. (13) Morrill, T. C. In Lanthonide Shift Reagents in Stereochemical The IH N M R spectra were obtained on a Varian XL-300 Analysis; Morrill, T. C., Ed.; VCH Publishers, Inc.: New York, 1986; pp spectrometer operating at 299.9 MHz. Each spectrum is a Fourier 1-17. transform of 32-496 free induction decays accumulated at ambient (14) Raber, D. J. In Lanthanide Shift Reagents in Stereochemical Anulysis; Morrill, T. C., Ed.; VCH Publishers, Inc.: New York, 1986; pp temperature. Instrumental parameters include a pulse width of 55-105. 12 ps, acquisition time of 2 s, spectral width of 4000 Hz, and ( I S ) Shapiro, B. L.; Johnston, M. D., Jr. J . Am. Chem. Soc. 1972, 94, transmitter offset of 0 Hz. Two-dimensional spectra were obtained 8 185. on a General Electric QE-300 NMR spectrometer. Tetra(16) Reuben, J. J . Am. Chem. SOC.1973, 95, 3534. (17) Inagaki, F.; Tasumi, M.; Miyazawa, T. Bull. Chem. Soc. Jpn. 1975, methylammonium cation (TMA') (0.2 mM) or tetraethyl48, 1427. ammonium cation (TEA') (0.4 mM) was used as the internal (18) Inagaki, F.;Takahashi, S.; Tasumi, M.; Miyazawa, T. Bull. Chem. reference. SOC.Jpn. 1975, 48, 853. (19) Bouquant, J.; Chuche, J. Bull. SOC.Chim. Fr. 1977, 959. (20) Armitage, I.; Dunsmore, G.; Hall, L. D.; Marshall, A. G . J . Chem. SOC.,Chem. Commun. 1971, 1281.
(21) McConnell, H. M.; Robertson, R. E. J . Chem. Phys. 1958,29, 1361.
Lanthanide Shift N M R Studies of Bile Salt Aggregates
The Journal of Physical Chemistry, Vol. 96, No. 4, 1992 1963 O
TABLE II: Shifts in tbe NMR Spectrum of PremiceUnr NeTC with 10 mM Dv" using DSS and TMA+ Reference Compounds'
A& ppm' 'Hb DSS ref TMA+ ref 3 7 12 18 19
H
A 4 ppm' IHb DSS ref
+0.05 +0.04
-0.01
21
-0.02
+0.03
-0.03
+0.03 +0.04
-0.03 -0.02
23a 23b 25 26
+0.02 +0.17 +0.15 -0.14 -0.08
TMA'ref -0.04 +0.11 +0.09 -0.20 -0.14
"0.5 mM DSS, 0.2 mM TMA'. bSee Figures 1 and 2 for the chemical structure and proton assignments. 'Negative upfield, positive
sodium taurocholate: R = CH2CH2S03-
downfield.
25
Results Choice of Internal Refereme. In the lanthanide shift experiment, an internal reference is required to accurately measure N M R chemical shifts since the lanthanide ion will change the bulk magnetic susceptibility of the solution.22 Changes in chemical shift resulting from changes in the bulk magnetic susceptibility of the solution are identical for all of the protons, and the A6 values between resonances should therefore remain constant and easily calibrated with the use of the internal standard. It is essential, however, that the lanthanide ion not bind with the internal reference. Specific interactions between the lanthanide ion and the reference compound are indicated when differential shifts are observed for magnetically nonequivalent protons of the reference compound.23 A number of compounds, including DSS, tert-butyl alcohol, dioxane, acetone, and tetraalkylammonium cations, have been used as the internal reference in aqueous solution, and the relative merits of several of these compounds have been discussed.23 We evaluated the suitability of DSS and tetraalkylammonium cations for use as the internal reference in lanthanide shift experiments. The effects of 10 and 30 m M Tb3+ on the 'H N M R spectra of DSS and tetraethylammonium cation (TEA+) are listed in Table I. For DSS, small upfield shifts are observed for methylene protons a, 8, and y relative to CH3 (6 = 0.00 ppm) as the concentration of Tb3+ is increased to 10 and 30 mM. In comparison, the A6 between the CH2 and CH3 resonances of TEA+ remains constant at 2.00 ppm (assuming, of course, that the lanthanide-induced shifts of CH2 and CH3in TEA+ are not coincidentally the same). These results indicate that Tb3+ binds with DSS but not with TEA+. The Tb3+ ion is not expected to bind with the tetramethylammonium cation (TMA') either. Further evidence for the absence of binding between lanthanide ions and quaternary ammonium salts was provided by the 'H N M R spectrum of diethylamine hydrochloride in the presence of Tb3+. Previously, large shifts have been reported for diethylamine substrates with lanthanide tetrakis(j3diketonate) shift reagenkZ4 In this study, the Tb3+ion (50 mM) was observed to have no effect on the shift differential between the CH2 and CH3 groups in the N M R spectrum of the diethylamine hydrochloride compound (100 mM), supporting the results given in Table I. Additional comparison between DSS and tetraalkylammonium cations is shown in Table 11, where the effects of 10 mM Dy3+ on the 'H NMR chemical shifts of 1 mM NaTC are summarized. Those resonances which shift downfield relative to DSS show upfield shifts relative to TMA', with only H23a and H23b exhibiting downfield shifts in both cases. The observation of both upfield and downfield shifts in the resonances of a substrate is unusual. Contact shiftsz5or changes in the sign of the angle term of the dipolar shift equation for certain protons of substratesz6 have sometimes caused such behavior. Here, however, it is likely that (22) Reuben, J. Prog. Nucl. Magn. Reson. Spectrosc. 1973, 9, 1. (23) Horrocks, W. Dew., Jr.; Hove, E. G. J. Am. Chem. Soc. 1978,100, 4386. (24) Wenzel, T.J.; Zaia, J. J . Org. Chem. 1985, 50, 1322. (25) Lindoy, L. F.; Moody, W. E. J. Am. Chem. SOC.1977, 99, 5863. (26) Shapiro, B. L.; Hlubucek, J. R.;Sullivan, G. R.;Johnson,L. F. J . Am. Chem. Soc. 1971, 93, 3281.
26
i1 9" lz
CHAPSO: R = C H ~ C H Z C H ~ N ' C H ~ C H C H ~ S O ~ 25
27
26
29 30
28
31
I
CHAPS: A = C H ~ C H Z C H ~ N + C H ~ C H ~ C H ~ S O ~ 25
27
26
I
28
32
29
30
Figure 1. Chemical structures of NaTC, CHAPSO, and CHAPS.
i9 HOD
i
6
5
,
,
,
' ,
i
~
i
i
t3
,
i
,
2
,
i
r
~
1
,
,
~
i
,
,
PPm
TMA+
Figure 2. IH NMR spectrum of micellar NaTC (32 mM) in D20. The spectrum of premicellar NaTC (not shown) is similar.
the methyl resonance of DSS shifts further upfield than the resonances of H3, H7,Hlz, HI8,H19,and Hzl in NaTC, causing an apparent downfield shift for those protons. The A6 values obtained with DSS all differ from those obtained with TMA' by 0.06 ppm. The difference is attributed to the Dy3+-inducedupfield shift of the methyl resonance of DSS in this solution. The downfield shifts of Hz3aand H23bappear to be real; the origin of these "wrong way" shifts will be discussed later. All of the spectra reported hereinafter were referenced using either TMA+ or TEA'. Whereas TMA+ was used as a reference for NaTC, TEA+ had to be used for CHAPSO and CHAPS because of chemical shift overlap with the resoLance of TMA'. 'HNMR Shifts in Prdcellar NaTC Solutions. The chemical structures of NaTC, CHAPSO,and CHAPS are shown in Figure 1. The NaTC resonances considered in this N M R study include the protons geminal to the hydroxyl groups (H3, H,, H12),the methyl protons on the steroidal backbone (HI*, H19,Hzl),and the methylene protons on the hydrophilic tail (Hz3a,H23b, H25, H2& The 'HN M R spectrum of NaTC in D20 is shown in Figure 2. Assignments for NaTC are taken from published spectral data, 10,27928 The critical micelle concentration (cmc) of NaTC in water has been determined to be 8-12 mMaZ9 The chemical shifts in the (27) Small, D. M.; Penkett, S. A.; Chapman, D. Biochim. Biophys. Acto
1969, 176, 178.
(28) Barnes, S.; Geckle, J. M. J. Lipid Res. 1982, 23, 161. (29) Meyerhoffer, S. M.; McGown, L. B. Langmuir 1990, 6, 187.
~
1964 The Journal of Physical Chemistry, Vol. 96, No. 4, 1992 0.100,
I
h
E,
Meyerhoffer et al. h
0.000
a v -0.100
v
+
b-
LL -0.200
h wl I
I
-0.300
n 3
9-
2
e
P)
a W
-0.400
2) U
9 -
-0.500
1
-0.600
"P b-
-0.700 -0.6004
,
5
0
I
I
I
I
I
I
I
I
,
-0.800 7 0.000
1
~
-0.600-0.700.
0.400
1.200
0.800
10 15 20 25 30 35 40 45 50 55 60 65
1.600
2.000
[Tb3 C ] / [ N ~ l C ]
[Tb3+]/[NaTC]
Figure 3. Tb3+-induced shifts for premicellar NaTC (1 mM) vs HZI;U, H z ~V, ; H26. [Tbstl/[NaTCl: 0, H,; 0, HIZ;A, HI&A,Hl9; H3 was unresolved at most Tb" concentrations and is not shown.
*,
E,
0.300
a
TABLE III: Association Constants and Bound Shifts for Premicellar NaTC with the Tb3+ Ion IH K,,M-I A,, ppm" IH K,,M-I A,, ppm" 7 12 18 19
67 79
50 69
-0.09 -0.12 -0.18 -0.08
21 25 26
43 41 44
-0.3 1 -0.67 -0.69
Y
*
0.100
h
I VI 0
-0.100
W
-0.300
(8)
u 0 3
-0.500
i
0"
@Negativeupfield.
-0.700 -0.900 4 0.000
NMR spectrum of a premicellar NaTC solution (1 mM) are plotted as a function of [Tb3+]/[NaTC] ratio in Figure 3, where [Tb3+]and [NaTC] refer to the analytical concentrations of each species. All of the 'H resonances shift upfield with Tb3+. The magnitude of the shifts are in the order H26 Hz5 > Hzl > H I S > H l z > H,9 H,. The H3 resonance was unresolved at most Tb3+concentrations, and its shift is not reported. The shifts of H23aand H23b were obscured by the unresolved band of resonances due to the steroidal protons (ca. 1.3-2.3 ppm). Association constants (K,) and bound shifts (A1)were determined from the data sets assuming a 1:l complex of Tb3+taurocholate (Tb3+-TC) using nonlinear least-squares regression analysis (Table 111). Attempts to fit the shift data to a two-step model were unsuccessful; no minimum in the variance could be found when both K, and K2 were included in the regression analysis. IH NMR Shifts in Micellar NaTC Solutioos. The effects of Tb3+,Dy3+, and Eu3+ on the lH chemical shifts of a micellar solution of NaTC (32 mM) are shown in Figure 4. The magnitude of the shifts induced by Tb3+are in the order H26 Hzs > H2, > H I S> H12> H19 H7 H3. The shifts with Dy3+ and Eu3+follow a similar order with the exception of HZ5,which showed significantly larger shifts than HZ6. For H z and ~ ~H23b, fairly large downfield shifts were observed in the presence of Dy3+, while no significant shifts were seen with Eu3+. The shift behaviors of H23aand H23bwere then studied with Tm3+,which is a strong downfield shift reagent. In the presence of Tm3+,the resonances of H23aand H23bmove downfield of the steroidal band, allowing the magnitudes of the shifts to be easily discerned. The shifts obtained with 20 mM Tm3+were in the order (A6 values, in ppm downfield, are given in parentheses) H26 (+0.57) > H23a (+0.49) = H23b (+0.49) > H25 (+0.37) > H21 (+0.35) > Hi8 (+0.23) > HI2 (+0.20) > Hi9 (+0.17) = H7 (+0.17) > H3 (+0.16). Shift data were also collected with Yb3+, which generally produces the smallest contact shifts.12 The shifts obtained with 30 mM Yb3+were in the order H26(+0.18) > H25(+0.16) > H23a (+0.11) > H23b (+0.09) > Hzl (+0.08)> HI8 (+0.04) > HI9 (+0.03)= HI2 (+0.03) > H7 (+0.02) H3 (+0.02). Association constants and bound shifts for the Ln3+-TC complexes in micellar NaTC (assuming 1:1 complexation) using nonlinear least-squares regression analysis are listed in Table IV. As was the case for the premicellar NaTC solution, no minimum in the variance of the regression analysis could be found when a two-step model was considered.
-
-
- -
-
I
0.250
0.500
0.750
1.000
1.250
1.500
[oY3+l/[NIJTCl
a
-.,
0.050
v
0.040
5
/-
v-/
0.030
v
W 0
g a f n + a
W
0.020
0.010 0.000
I -0.0104 0.000
0.250
0.500
0.750
1.000
1.250
I
1.500
[Eu3+]/[NaTC]
Figure 4. Lanthanide-induced shifts for micellar NaTC (32 mM) as a function of [Ln3']/[NaTC] for (A) Tb", (B) Dy3+,and (C) E d + ; 0, H,; 0,HI; 0 , HIZ;A, Hit.; A, Hi9; Hzi; v, H23i 0 ,H23b; U, Hz5; V,
*,
H26.
TABLE Iv: Association Constants and B o d Shifts for Micellar NaTC with ,'bT Dy3+,and Eu3+Ioha Assuming 1:l Complexation
K,,M-' 'H 3 7
12 18
Tb3+ 166 161 157
19
c
*
145 154 5 av 163
AI? PP" Tb3+ Dy3+ Eu3+
Eu3'
b b b
174 169 162 163 171
152
160 21 152 25 152 26 153 av 157
Dy3'
*
139 142 156 138 147 10 av 144
*7
-0.30 -0.32 -0.43 -0.49 -0.33 -0.64 -1.12 -1.17
-0.18 -0.18 -0.23
b b b
-0.25 +0.02 -0.18 +0.02 c +0.04 -1.31 +0.08 -0.88 +0.05
@Negativeupfield, positive downfield. *Shift data could not be fitted for these protons. 'Hzl unresolved with the Dy3+ ion.
In previous studies: fluorescence intensity of a solubilized probe vs [Tb3+]was observed to increase up to an overall stoichiometry of 1:4 Tb3+-TC and then leveled off. In the present work, the same behavior is observed for the shifts, which asymptotically approach the bound shift values above 1:4 stoichiometry. Attempts were made to account for this apparent stoichiometry by fitting the shift data to a one-step model in which the effective concentration of substrate was taken to be 32 mM/4, or 8 mM. Here,
The Journal of Physical Chemistry, Vol. 96,No. 4, 1992 1965
Lanthanide Shift N M R Studies of Bile Salt Aggregates
,---
HOD
I
I I
I
CI
E
g
0.000
Y
t-
5
19
-0.030
I VI 0
-0.060
Y
V
a
0
z
n
-0.090 -0.120
t-
-0.150 0.000
0.250
0.500
0.750
1.000
1.250
1.500
[Tb3+]/[CHAPSO]
CI
E g
0.000
v
-0.030 I vl Z
M
0
(0)
-0.060
Y
Figure 5. 'H NMR spectrum of micellar CHAPSO (32 mM) in D20. The full spectrum is shown in the upper left-hand comer, while the region between 3.0 and 4.7 ppm is enlarged for greater detail.
2
0
-0.090
An
-0.120
z
c
-0.1soJ 0.000
0.250
0.500
0.750
1.000
1.250
I
1.500
[TbS+]/[CHAPS]
HOD
Figure 7. Plots of Tb3'-induced shifts vs [Tb3']/[CHAPSO] or [Tb"]/[CHAPS] for (A) 32 mM CHAPSO and (B) 32 mM CHAPS: 0,H7; 0 , HI^; A, HIS;4 HI^; H21; 0,H2s; m, H29; v, H3o; 0,H ~ I and H3> Only those resonances which were resolved and identified in
*,
each particular solution are shown.
31,32
3 28
I1
I
{
Figure 6. 'H NMR spectrum of micellar CHAPS (32 mM) in DzO.As in Figure 5, the full spectrum is shown in the upper left-hand corner, while the region between 2.8 and 4.6 ppm is enlarged for greater detail.
1 mol of lanthanide ion was assumed to interact simultaneously with 4 mol of taurocholate. Use of this model, however, proved unsatisfactory. The association constants calculated for the individual protons were in poor agreement, with values ranging from 213 to 440 M-' for Tb3+-TC, 204 to 3 15 M-I for Dy3+-TC, and 380 to 454 M-I for Eu3+-TC. IH NMR Assignments in CHApSo and CHAPS Spectra. The IH N M R spectra of micellar CHAPSO and CHAPS solutions (32 mM in both cases) are shown in Figures 5 and 6. To our knowledge, proton assignments have not been previously published for these compounds, although the I3CNMR spectrum of CHAPS has been described in a previous report in which the cmc and mean aggregation number of CHAPS were determinede30 The resonances of H3, H,, HI2, HI8, HI9, and H21were identified by comparison to the NaTC spectrum. Assignment of the proton resonances on the zwitterionic tails of CHAPSO and CHAPS required the use of several auxiliary N M R techniques, including homonuclear spin decoupling and I3C-IH phase-sensitive heteronuclear shift correlation (HETCOR) experiments. (30)Stark, R.E.;Leff, P.D.;Milheim, S. G.; Kropf, A. J . Phys. Chem. 1984, 88, 6063.
In CHAPSO, the resonances of HZ8(centered at 3.56 ppm) and H30 (a doublet at 3.18 ppm) were identified by IH-IH decoupling of HZ9(at 4.69 ppm). The methyl groups in CHAPSO (H31 and H32) are partially resolved at the field strength employed and were assigned to two singlets at 3.23 and 3.24 ppm. An impurity was present in CHAPSO and was suspected to contribute to the N M R spectrum. In CHAPS, the resonances of Hz8(part of the band at 3.4 ppm) and H30 (a triplet centered a t 3.02 ppm) were identified by heteronuclear correlation analyses. The H29methylene protons resonate upfield and overlap with resonances of the steroidal backbone protons. The resonances of H3I and H3*were assigned to the peak at 3.16 ppm. 'H NMR Shifts in Micellar CHAPSO and CHAPS Solutiolrs. The effects of Tb3+ on the IH NMR resonances of 32 mM CHAPSO and 32 mM CHAPS are shown in Figure 7. In contrast to the pronounced effects on the chemical shifts of NaTC, the 'H resonances of CHAPSO and CHAPS are only slightly shifted with Tb". Attempts at fitting the shift data to either a one-step or a two-step model using the nonlinear regression approach were unsuccessful because no minimum in the variance could be found. The linearity of the plots indicates that the association constants for Tb3+with CHAPSO and CHAPS are probably small.
Discussion
Our results demonstrate that tetraalkylammonium cations are preferable to DSS as internal reference compounds with lanthanide ions in water. The results shown in Table I1 indicate that the apparent downfield shifts of H,, H7, H12,HIS,HIP, and H21are artifacts resulting from binding of Dy3+ with DSS. The reported observation of downfield shifts for these protons in premicellar NaTC in the presence of Dy3+I0is probably a result of these binding interactions. The "wrong way" shifts of H23a and H 2 3 b observed in this work and by Elgavish et a1.I0 are real. One possibility is that they arise from a change in the sign of the angle term of the dipolar shift equation caused by structural changes across the series of lanthanides. Significant contact shift con-
Meyerhoffer et al.
1966 The Journal of Physical Chemistry, Vol. 96, No. 4, 1992 0.04
I
o, 0 2L s 0.00
h
N v)
-0.02
-
-0.04
-
-0.06
-
a
4
-0.08 -10
I
-8
-6
-2
-4
0
2
4
6
D/(S,)
Figure 8. Plots of A6/(S,)vs D / ( S , ) with 30 m M Ln3+for 0, H2];0, H25;and V, H2.+
tributions to the total shift may also be responsible. Finally, it is possible that the Ln3+-TC complexes do not possess the effective axial symmetry needed to apply the simplified form of the dipolar shift equation (eq 3). A quantitative separation of the contact and dipolar shift contributions may be accomplished using the method described by Reilley et al.31,32and Elgavish and R e ~ b e n . ~The ~ , ~A6~ resulting from contact and dipolar shift mechanisms is given by A6 = A ( $ )
+ GD
(4)
where A is the hyperfine coupling constant between a given nucleus and lanthanide ion (which provides a measure of the contact contribution to the A6), G contains the geometrical factors for the complex (which provides a measure of the dipolar contribution to the A6), and (Sz)35and D36are lanthanide ion-dependent constants. Rearrangement of eq 4 gives A6/(SZ) = A
+ G(D/(S,))
(5)
and a plot of A6/(S,) vs D/(S,) will allow determination of values for A and G, assuming the lanthanide complexes exhibit effective axial symmetry, isostructurality, and invariance in the hyperfine coupling constant across the lanthanide series.31,32,33-34 If the contact contribution is small, the least-squares line should pass through the origin. Plots of A6/(S,) vs D/(S,)for HZl, H25. and H26 in the presence of 30 mM Tb3+, Dy3+, Eu3+, and Yb3+ are shown in Figure 8. Complexation shifts were measured with 30 mM La3+. Linear least-squares regression analyses of the data gave values for A of 0.0015 f 0.0020,0.0035 f 0.0034, and -0.0008 f 0.0043 for H21rH25, and H26, respectively. The corresponding values of G were 0.0034 f 0.0003,0.0066 f 0.0004, and 0.0072 f 0.0005. Because the values of A are generally within one standard deviation of the origin, it is reasonable to assume that the contact contribution to the total shift of these protons is negligible. This is not surprising since contact shifts are usually minimized in 'H NMRZ2 and is especially true for weakly binding substrates such as sulfonate salts. The shift data for premicellar and micellar NaTC indicate that the lanthanide ions bind with the anionic sulfonate group on the taurocholate tail, which is consistent with previous reports.1°a3' When the dipolar shift mechanism predominates, those protons located closest to the binding site typically show shifts of greatest magnitude, and the significantly larger shifts of H26 and HZ5 compared to the other resonances lend support for the lanthanide ion-sulfonate binding interaction in NaTC. However, the fairly
large shifts of H23a and H23b with Tm3+indicate involvement of the peptide carbonyl oxygen atom in the binding as well. The larger shifts of H25 relative to H26 with Dy3+and Eu3+also support such an interaction. The lanthanide shift results can be explained by simultaneous binding of the lanthanide ion with the anionic sulfonate group and the oxygen atom of the peptide carbonyl through a bidentate interaction, forming an 8-membered chelate ring structure. In comparison, N M R shift studies of the Dy3+-glycocholate premicellar complex have indicated that Dy3+ binds with the proximal oxygen atom of the glycine carboxyl through a first-sphere interaction, while coordinating with the distal oxygen atom of the carboxyl and the oxygen of the peptide carbonyl through second-sphere interactions.]I Since the transition from outer- to inner-sphere complexation with lanthanides occurs at pKa values of about taurocholic acid (pK, = 1.8) is near this cross-over point, and the binding of the oxygen atoms of both the sulfonate and carboxyl groups is expected to be weak, mostly second-sphere interactions. In the absence of significant contact contributions, another explanation is needed for the "wrong way" shifts that are observed for H23a and H23b with Dy3+. Also, the shift reversals (e.g. Hz5 > HZ6with Dy3+and Eu3+;H26 Hz5 with Tb3+;and H26 > H23a = H23b > H25 with Tm3+)and the smaller relative shift of HZ1 with Dy3+,compared to those of Tb3+and Eu3+,provide further evidence to suggest that this system lacks either isostructurality or effective axial symmetry. The linearity of the plots (>0.996 in all cases) of As/(S,) vs D/(S,) (Figure 8) for Hzl,Hzs, and H,, indicates the absence of significant structural differences between the Ln3+-TC complexes, although the data is limited to only a few lanthanides. Further study is needed to address the questions raised by the irregular shift behavior. The binding equilibrium for the Tb3+-TC complex in premicellar solution was best fit to a one-step model. Using the shift data for each proton listed in Table 111, an average association constant of 56 f 15 M-I was calculated, with the absolute error (f15 M-I) reflecting deviations between individual proton data sets. If only those protons showing the largest shifts are considered (H26, Hz5,and HZI),an average association constant of 43 f 2 M-I is obtained. This value is closer to the value of 39.7 M-' reported by Elgavish et al. for Dy3+-TC at 25 OC.Io Average association constants calculated for the Ln3+-TC complexes in micellar solution, assuming 1:1 stoichiometry, are 157 f 5 M-I for Tb3+, 163 f 10 M-] for Dy3+,and 144 f 7 M-' for Eu3+. It is important to recognize, however, that association constants calculated with the one-step model probably only approximate the true binding equilibria in micellar NaTC. Previous fluorescence studies: as well as the N M R shift data presented here, strongly suggest a 1:4 Ln3+-TC overall binding stoichiometry; however, attempts to calculate association constants using this stoichiometry were unsuccessful. A further complication in the interpretation of the shift data arises because micellar NaTC solutions exhibit significant polydispersity with respect to aggregate size and structure,39and the effective concentration of micellized NaTC is expected to vary with the total Ln3+ion concentrati~n.~ The mechanism of counterion binding to conventional detergent micelles has been a subject of great interest and c o n t r o ~ e r s y , 4 ~ ~ ~ and the Ln3+-TC micellar binding interaction is being further studied. The magnitudes of the observed shifts and the calculated association constants for the Tb3+-TC complex in premicellar and micellar NaTC suggest that the binding strength is greater at micellar concentrations. These results are consistent with the general trend observed for counterion binding in solutions of
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(31) Reilley, C. N.; Good, B. W.; Allendoerfer, R. D. Anal. Chem. 1976, 48, 1446.
(32) Reilley, C . N.; Good, B. W.; Desreux, J. F. Anal. Chem. 1975, 47, 21 10. ( 3 3 ) Elgavish, G. A.; Reuben, J. J . Am. Chem. SOC.1977, 99, 5590. (34) Reuben, J.; Elgavish, G. A. J . Magn. Reson. 1980, 39, 421. (35) Golding, R. M.; Halton, M. P. Ausr. J . Chem. 1972, 25, 2577. (36) Bleany, B. J . Magn. Reson. 1972, 8 , 91. (37) Barnes, S.Hepatology 1984, 4, 9 8 s .
(38) Choppin, G.R.; Bertha, S.L. J . Inorg. Nucl. Chem. 1973, 35, 1309. (39) Kratohvil, J. P.; Hsu, W. P.; Jacobs, M. A,; Aminabhavi, T. M.; Mukunoki, Y.Colloid Polym. Sci. 1983, 261, 781. (40) Robb, I. D. J . Colloid Interface Sci. 1971, 37, 521. (41) Bunton, C. A.; Romsted, L. S. In Solution Behavior of Surfactants; Mittal, K. L., Fendler, E. J., Eds.; Plenum Press: New York, 1982; Vol. 2, pp 975-991. (42) Abuin, E. B.; Lissi, E. J . Colloid Interface Sci. 1983, 93, 562.
J. Phys. Chem. 1992,96, 1967-1972 conventional detergent^.^^ It is possible that the second-coordination sphere of Tb3+is less hydrated in the relatively hydrophobic environment of 32 mM NaTC than in the hydrophilic environment of 1 mM NaTC, resulting in the stronger binding interaction^.^^ In contrast to NaTC, the CHAPSO and CHAPS micellar solutions exhibited very little sensitivity to the paramagnetic shift effects of Tb3+. The shifts obtained for H2*,H29, and H30indicate that Tb3+ binds with CHAPSO and CHAPS at the anionic sulfonate group. The binding, however, is much weaker than with NaTC, resulting from reduced electrostatic interaction between Tb3+ and SO3- because of the positively charged ammonium center.9-44,4s Conclusions
The results of these studies lead to several important conclusions. First, it was demonstrated that the tetraalkylammonium cations are less likely than DSS to bind with free lanthanide ions and are therefore better internal references for NMR shift studies. Using TMA+ as a reference, it was then shown that the binding of (43)Prof. A. L.Crumbliss, Department of Chemistry, Duke University. Personal communication. (44) Roda, A.; Hofmann, A. F.; Mysels, K. J. J. Biol. Chem. 1983,258, 6362. (45)Hjelmeland, L.M.; Nebert, D. W.; Osborne, J. C., Jr. Anal. Biochem. 1983,130, 12.
1967
lanthanide ions with NaTC occurs through simultaneous interactions with the sulfonate and peptide carbonyl functional groups on the hydrophilic NaTC tail. The strength of the interaction increases above the cmc, which is similar to counterion binding behavior of conventional detergents. The association of lanthanide ions was found to be much weaker with zwitterionic CHAPSO and CHAPS relative to anionic NaTC. The weaker binding is attributed to the absence of net anionic charge on the zwitterionic tails as well as the increased distance between the sulfonate and peptide carbonyl groups, which reduces the likelihood of simultaneous chelate binding with the lanthanide ions. Contact contributions to the lanthanideinduced shifts of NaTC were shown to be negligible. Irregularities in the shift behavior of different lanthanide-taurocholate complexes may therefore reflect a lack of either effective axial symmetry in the complexes or isostructurality across the series. Further study is needed to determine the exact mechanism responsible for the irregular shift behavior.
Acknowledgment. L.B.M. is grateful for the support of this work from the US.Department of Energy, through Grant DEFG05-88ER13931. T.J.W. is grateful for the support of the National Science Foundation through Grant CHE-8921335. Registry No. NaTc, 145-42-6;CHAPSO, 82473-24-3;CHAPS, 75621-03-3.
Temperature- Induced Phase Transition of Pyrene-Labeled (Hydroxypropy1)cellulose in Water: Picosecond Fluorescence Studies F. M. Winnik,* Xerox Research Centre of Canada, 2660 Speakman Drive, Mississauga, Ontario, Canada L5K 2Ll
N. Tamai,+ J. Yonezawa, Y. Nishimura, and I. Yamazaki* Faculty of Engineering, Hokkaido University, Sapporo 060, Japan (Received: July 22, 1991; In Final Form: October 1 , 1991)
Picosecond time-resolved fluorescence experiments were performed with solutions in water of pyrene-labeled (hydroxypropy1)celluloses with three different levels of pyrene incorporation (1 pyrene per 26, 56,and 438 glucopyranose units), but identical molecular weights. These solutions exhibit a lower critical solution temperature (LCST) at ca 42 OC. The kinetics of pyrene excimer formation for solutions of these polymers (26.4 ppm) were monitored below and above the LCST. At 10 and 25 OC, the time-resolved spectra show two types of excimer emissions with maxima (1) at 420 nm (0-7 ns time scale), assigned to a one-center-type excimer similar to that observed in pyrene single crystals and (2) at 470 nm (after 50 ns), assigned to a sandwich-typeexcimer. Both excimers originate from preformed ground-state pyrene aggregates. By contrast, at 45 O C only one excimer emission was observed with a maximum at 470 nm. It originates predominantly from the dynamic encounter of excited- and ground-state pyrenes.
Introduction
The work described here originates from our previous investigations on the use of fluorescence techniques to explore on a molecular scale the properties of solutions in water of hydrophobic polymers. One of the most fascinating properties exhibited by many such polymers is a reverse temperature-dependent phase behavior. They form isotropic, one-phase systems in water at or below room temperature, but when their solutions are heated above a critical temperature, often called the cloud point, they separate into two phases, as indicated visually by a sudden transition from clear solutions to opaque suspensions. The phase transition is *To whom correspondence should be addressed. 'Present address: Microconversion Project, ERATO, Research Develop ment Corporation of Japan, Kyoto Research Park, Shimogyo-ku, Kyoto 600, Japan.
0022-365419212096-1967$03.00/0
reversible: cooling the solutions below their cloud point results in a sudden clarification of the twephase system. Like many other cellulosic ethers, (hydroxypropy1)cellulose (HPC, Figure 1) in water exhibits this phenomenon.' In an earlier study we used the photophysical properties of pyrene-labeled (hydroxypropyl)cellulases (HPC-Py, Figure 1) to investigate the phase transition behavior of this polymer in water.2 Through the use of several fluorescence techniques we were able to demonstrate that (1) below the LCST the polymers form interchain aggregates even in extremely dilute solution^;^ (2) interchain aggregation below the (!) See for example: Handbook of Water-Soluble Gums and Resins; Davidson, R. L., Ed.; McGraw Hill: New York, 1980; Chapter 13. WaterSoluble Polymers; Glass, E. D., Ed.; ACS Symp. Ser. 213; American Chemical Society: Washington DC, 1986. (2) Winnik, F. M. Macromolecules 1987, 20, 2145.
0 1992 American Chemical Society
