Stepwise aggregation of cholate and deoxycholate dictates the

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Stepwise aggregation of cholate and deoxycholate dictates the formation and loss of surface-available chirally selective binding sites Adam R. Meier, Jenna B Yehl, Kyle W Eckenroad, Gregory A Manley, Timothy G Strein, and David Rovnyak Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00467 • Publication Date (Web): 07 May 2018 Downloaded from http://pubs.acs.org on May 8, 2018

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Guest binding with processive bile aggregation early R

cholate

deoxycholate

R

= bile salt monomer

primary S

R

S

R

secondary S

R

S

R

R , S = R,S-binaphthyl-1,1’-diylhydrogenphosphate

ACS Paragon Plus Environment

S

S

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Stepwise aggregation of cholate and deoxycholate dictates the formation and loss of surface-available chirally selective binding sites

Adam R. Meier, Jenna B. Yehl, Kyle W. Eckenroad, Gregory A. Manley, Timothy G. Strein* and David Rovnyak*

Department of Chemistry, Bucknell University, 1 Dent Drive, Lewisburg, PA 17837

*Corresponding Authors, to whom correspondence [email protected], [email protected]

TOC Figure


should

be

addressed:

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Abstract Bile salts are facially amphiphilic, naturally occurring chemicals that aggregate to perform numerous biochemical processes.

Because of their unique intermolecular

properties, bile salts have also been employed as functional materials in medicine and separation science (e.g. drug delivery, chiral solubilization, purification of single-walled carbon nanotubes). Bile micelle formation is structurally complex, and remains a topic of considerable study. Here, the exposed functionalities on the surface of cholate and deoxycholate micelles are shown to vary from one another and with micelle aggregation state. Collectively, data from NMR and capillary electrophoresis reveal preliminary, primary and secondary stepwise aggregation of the salts of cholic (CA) and deoxycholic (DC) acid in basic conditions (pH 12, 298 K), and address how the surface-availability of chirally selective binding sites depends on these sequential stages of aggregation. Prior work has demonstrated sequential CA aggregation (pH 12, 298 K) including a preliminary CMC at ca. 7 mM (no chiral selection) followed by a primary CMC at ca. 14 mM that allows chiral selection of binaphthyl enantiomers. In this work, DC is shown to also form stepwise preliminary and primary aggregates (ca. 3 mM DC and 9 mM DC respectively, pH 12, 298 K) but the preliminary 3 mM DC aggregate is capable of chirally selective solubilization of the binaphthyl enantiomers. Higher-order, secondary bile aggregates of each of CA and DC show significantly degraded chiral selectivity. Diffusion NMR reveals that secondary micelles of CA exclude the BNDHP guests, while secondary micelles of DC accommodate guests but with a loss of chiral selectivity. These data lead to the hypothesis that secondary aggregates of DC have an exposed binding site, possibly the 7α -edge of a bile dimeric unit, while secondary CA micelles do not present binding edges to the solution, potentially instead exposing the three alcohol groups on the hydrophilic α-face to the solution.


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Introduction Bile acids are steroidal compounds that have remarkable and tunable properties including step-wise aggregation[1-3], self-assembly[4-6], solubilization of carbon nanotubes[7, 8], chiral recognition of small molecules[9-16] and nanomaterials[17, 18], and hydrogel formation. [19-22]

The useful and unusual chemical and physical

properties of some bile salt aggregates certainly result from the surface chemistry exhibited by particular aggregation states. Yet, a detailed molecular understanding of bile aggregation processes has been elusive, and even the size, structure and surface chemistry of bile aggregates remains poorly characterized.

Consequently, many

applications of bile salts have developed via a line of serendipitous experimental observations and characterization, rather than through rational design on optimization. This work builds on prior work with cholate[13] to systematically characterize the differences in bile salt aggregation behavior of cholate and deoxycholate (Figure 1ab), with an eye towards exploiting useful intermolecular interactions, enabled by structural elements formed during stepwise aggregation events, that give rise to surface-exposed hydrophobic binding sites for binaphthyl compounds. Through a combination of NMR and micellar electrokinetic capillary chromatography (MEKC) experimentation, the surfaces of stepwise bile aggregates are characterized for bile aggregates with chiral binaphthyl probes and in probe free conditions. Bile acids are synthesized in the liver, and have diverse roles in homeostasis. [2, 23-27] Usually studied in the deprotonated or “salt” form, bile structures are composed of a saturated ring system with alpha oriented hydroxyl groups (Figure 1a-b).

In

aqueous solution, bile salt monomers are known to self-aggregate and to solubilize hydrophobic compounds via non-covalent interactions. Examples of such guest-host binding include pharmacologically active compounds,[28-32] and single walled nanotubes. [17, 18] Bile salt derivatives are characterized by the number and location of their α-hydroxyl groups. For example, cholic acid (CA, Figure 1a) is a trihydroxy bile acid, while deoxycholic acid (DC, Figure 1b) is a dihydroxy bile acid, lacking the hydroxyl group found in cholic acid at position 7. Trihydroxy bile acids have higher aqueous solubility than dihydroxy bile salts and self-aggregate at higher concentrations


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than their dihydroxy counterparts. concentration.

Bile salt monomers aggregate as a function of

A general model for step-wise aggregation holds that a premicellar

aggregate forms first, followed by a primary micelle, and then by growth of higher-order (a.k.a. secondary) micelles, but bile aggregate bulk and surface structures remain unclear. [2, 23, 24, 28, 33-35] Primary micelles are likely dominated by hydrophobic contacts, while secondary micelles are distinguished by likely having hydrophilic interactions in the aggregates. [1, 36-38]

The parameter space influencing bile

aggregation is also quite large, including concentration, temperature, pH, and ionic strength dependence. Moreover, because different analytical methods may report on particular aspects or steps of bile aggregation, comparisons of results from different methods is challenging. Bile salts such as cholate and deoxycholate are used as pseudostationary phases for resolving chiral analytes in separation methods such micellar electrokinetic capillary chromatography (MEKC), [10, 12, 13, 16, 30, 31, 34, 39, 40] and also as chiral solvating agents. [41, 42] MEKC was initially developed to aid in the separation of neutral analytes [43, 44], but has also shown utility in chiral separations, where bile salts are particularly effective for separating chiral analyte molecules containing a rigid planar substructure such as a naphthyl ring system. [45-47] A great deal of evidence shows that bile aggregates can accommodate planar guest molecules, [29, 40, 47, 48] consistent with a widely held model in which a hydrophobic binding pocket forms the interior of an anti-parallel bile salt dimer. [49] Intermolecular NOE (nuclear Overhauser effect) analysis, an NMR technique which reports on inter-nuclear spatial proximity, has provided evidence for such dimers [40, 50, 51] and for the insertion of hydrophobic ring systems into these dimers. [40] The use of a probe may even stabilize or promote the formation of bile aggregates. [29] Precisely determining fundamental properties such as aggregation number, polydispersity, surface composition, critical micelle concentrations (CMC), and even the nature of processive aggregation events has proven elusive. [1, 13, 35, 42, 52-55]

To better understand and harness the capabilities of bile salt

micelles, a detailed understanding of the broader aggregation and solubilization properties of bile salt aggregates is needed.


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Figure 1. Bile acids may be categorized as trihydroxy, such as (a) cholic acid, or dihydroxy such as (b) deoxycholic acid. Planar guest molecules, particularly those featuring binaphthyl ring systems such as (c,d) R,S-1,1′-binaphthyl-2,2′diylhydrogenphosphate (BNDHP) are differentially solubilized by aggregates of cholate[13]or of deoxycholate (this work).

Binaphthyl compounds such as (R,S)-1,1′-binaphthyl-2,2′-diylhydrogenphosphate ((R,S)-BNDHP) (Figure 1c-d) are planar, chiral molecules that have been shown to act as guest molecules in hydrophobic binding pocket(s) within cholate micelles. [13, 39-41, 47] The atomic scale resolution of NMR applied to bile salts, [13, 40, 41, 47, 56] indicates that the H5-H7 protons on the naphthyl rings are particularly sensitive to chirally selective interactions of the analyte with cholate micelles. [13] The 1H NMR shifts of R,S-BNDHP (2.5 mM) guest molecules unambiguously detected an early cholate aggregate at about 7 mM, but no chiral resolution of isomers was observed in the MEKC analysis with cholate at 7 mM. However, at 14 mM cholate, MEKC and NMR each detected a subsequent cholate aggregate forming, and the MEKC results require that this aggregate presents a surface-exposed binding element, identified as an antiparallel dimer[40] for enantioselective interactions with the R,S-BNDHP guest molecules. A qualitative structural model that explains how bile micelles discriminate between R,S-BNDHP was determined with a combination of 2D-NOE and 1D chemical


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shift analysis, showing that S-BNDHP preferentially interacts with the 12α-hydroxy moiety of bile dimers, while the R-BNDHP enantiomer preferentially samples the opposite edge of the dimer near the C7 position. [40] These results suggest that the C7 and C12 edges are each accessible on the primary micellar surface of cholate and deoxycholate. Localizing the enantiomeric guest molecules to distinct surfaces on the bile aggregates implies different binding mechanisms, but prior data do not sufficiently restrain or define the interactions between BNDHP and the bile aggregates to explain the different binding affinities. In this work, we seek to expand the understanding of how bile surface chemistry gives rise to chiral selection with different bile salts and how discrete steps within processive aggregation affects these guest-host interactions.

Specifically, this work

describes the chiral selection behavior of bile acids in two areas: (i) detailed 1D NMR data for binaphthyl guest molecules interacting with deoxycholate micelles reveal multiple deoxycholate CMC values and localize guest-host interactions with atomic resolution, and (ii) hydrodynamic measurements of cholate and deoxycholate bile salt aggregates interacting with R,S-BNDHP guest molecules shed light on the surprising loss of chirally selective interactions when bile acids undergo higher order (a.k.a. secondary) aggregation to form much larger micelles. Galantini, et al. have previously demonstrated the utility of the diffusion (e.g. pulsed field gradient spin echo or PFGSE) NMR technique for probing high order aggregation of glycodeoxycholate. [57] Briefly, with PFGSE the translational diffusion constant is directly measured by applying a magnetic field gradient along the tube axis and recording the degree of refocusing of the magnetization, which is dependent on location, via spin echoes.

We apply this

approach to first confirm continuous growth in the average bile aggregate size as a function of increasing bile concentration, and further to discover that the loss of chiral resolution that accompanies aggregate size occurs by distinct mechanisms for cholate and deoxycholate. The data reveal that secondary deoxycholate micelles still bind R,SBNDHP, but that these interactions lose enantioselectivity, while secondary micelles of cholate exclude the guests altogether.


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Materials and Methods Reagents. Sodium deoxycholate monohydrate, and sodium cholate were obtained from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). Individual enantiomers of R/S -1,1 ′- binaphthol and R/S-1,1′-binaphthyl-2,2′-dihydrogenphosphate were obtained from Aldrich Chemical Company (St. Louis, MO, USA). Sodium hydroxide was obtained from Fisher Scientific (Fairlawn, NJ, USA). Deuterium oxide (D2O, 99%) was obtained from Cambridge Isotope Laboratories. All chemicals were used without further purification. Solutions for capillary electrophoresis. Stock solutions of 200 mM deoxycholate and/or cholate were prepared as needed by dissolving the sodium salt with sonication in 18 MΩcm H2O and adjusted to pH 12.0 by the dropwise addition of 1 M NaOH while monitoring the pH with an Accumet pH meter (Fisher Scientific). Background electrolyte solutions of varying bile salt concentration were prepared by diluting the above stock solutions, and adjusted to the desired pH via the dropwise addition of 1 M NaOH while monitoring the pH with an Accumet pH meter (Fisher Scientific). Solutions of racemic mixtures, as well as solutions of individual enantiomers of (S)- and/or (R)-binapthyl-2,2′dihydrogenphosphate were prepared in 18 MΩcm water with a small amount (ca. 1 equivalent) of 1 M NaOH and sonicated until dissolved. Each solution was filtered through a 0.45 µm PTFE membrane filter using a syringe into a 1 mL polypropylene vial (Agilent Technologies, Palo Alto, CA, USA) for capillary electrophoretic study. Capillary Electrophoresis Equipment and Procedures. An Agilent (Palo Alto, CA) CE system and Chemstation software were used for all micellar electrokinetic capillary chromatographic separations.

Fused silica capillaries from PolyMicro Technologies

(Phehonix, AR) with an inner diameter of 50 µm were employed in all experiments. Capillary length was either 35.0 or 41.0 cm with the detector placed 8.5 cm from the outlet end. Prior to each separation, the capillary was preconditioned with either three (cholate) or ten (deoxycholate) minute flushes at ca. 900 mbar with i) 1 M NaOH, ii) 18 MΩ-cm H2O, and iii) the background electrolyte buffer. Hydrodynamic sample injection was performed by applying a pressure of 50 mbar for a total of three seconds. The


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separation potential was 10.0 kV, and the capillary held at 25.0 + 0.1oC. Photodiode array UV/Vis absorption detection was employed at 214 nm and 232 nm. Solutions for NMR. Stock solutions of 200 mM cholate and deoxycholate were prepared by dissolving the sodium salts with sonication in 18 MΩcm H2O. The NMR sample solutions were prepared from dilutions of bile salt stock solutions in 18 MΩcm water and 10% D2O. Analyte solutions contained 2.5 mM of a single enantiomer of one analyte [(R,S)-binaphthyl-2,2’-dihydrogenphosphate], and a small amount (~5 drops) of 1M NaOH was required for dissolution. The pH of each sample was adjusted to 12 using 1 M NaOH.

Cholate and deoxycholate sample solutions were also prepared

without a guest analyte molecule. Diffusion NMR. Diffusion coefficients were calculated from cholate protons H12, H7 and H3 along with four arbitrarily chosen aliphatic cholate resonances located between 0.1 ppm and 2.25ppm. Deoxycholate diffusion coefficients were calculated from resonances H12, H3 along with five arbitrarily chosen aliphatic resonances between 0.1 ppm and 2.25 ppm.

PFGSE-NMR spectra[58-60] were obtained at

Bucknell University and were acquired with a Varian Direct Drive (TM) (Palo Alto, CA) 400 MHz spectrometer using a vendor-supplied pulse sequence and data analysis package; WATERGATE was used in suppression of water signals.

Spectra are

referenced to an external standard of 25 µM 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS) in 18 MΩ•cm H2O. The deoxycholate and cholate series employed 150 ms and 100 ms refocusing periods respectively; the longer time may have resulted in somewhat less uncertainty for large bile concentrations, but uncertainties were acceptably small in both cases.

The deoxycholate diffusion experiments utilized 64 transients per

increment. The cholate series utilized 72 transients per increment for 1 mM cholate concentrations, and 32 transients per increment at higher cholate concentrations. All diffusion experiments used a 10 ppm window, 1.5 s acquisition, and 15 increments with 2 ms gradients spanning 2-52 G cm-1. Typical 1H pulse widths were 8.5 µs. Gradient strengths were externally calibrated with a plug (courtesy Prof. J Maneval, Bucknell University) and verified by us on known standards. Diffusion constants were computed from signal integrations following 3 Hz line broadening and baseline correction with the


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vendor-supplied Diffusion-PackTM software.

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The viscosity of each solution was

measured in triplicate with an Ostwald viscometer. 1

H NMR Chemical Shift Titrations.

One series of samples (Series 1) was

prepared with 2.5 mM R- or S-BNDHP and spanned deoxycholate concentrations from 3-100 mM.

The Series 1 spectra were acquired on a Bruker Avance 600 MHz

spectrometer (Pennsylvania State University, XWin V3.5) equipped with a room temperature inverse triple-resonance probe (TXI), and using the vendor-supplied zgpr pulse sequence to perform low power presaturation of the water resonance. Samples contained 10% D2O by volume. For Series 1, sixteen transients were collected per 1H NMR spectrum.

An independent series of samples (Series 2, see Supporting

Information Section S.5) was prepared by a different person, also using 2.5 mM R- or SBNDHP and also spanning 3-100 mM deoxycholate. Series 2 spectra were acquired on a Bruker Avance 900 MHz spectrometer (MIT/Harvard center for magnetic resonance, TopSpin V1.3), equipped with an inverse triple-resonance cryoprobe (CPTCI). Samples contained 10% D2O by volume and proton spectra were obtained with the vendorsupplied zggpw5 pulse sequence which implements the WATERGATE principle for solvent suppression.

For Series 2, sixteen transients were collected per 1H NMR

spectrum. In Series 1 and 2, the temperature was controlled at 25 ˚C. Other conditions for the 1H NMR spectra were 10 ppm (Series 1) and 12 ppm (Series 2) windows, 16 (Series 1) and 32 (Series 2) transients, and 1.4 s (Series 1) and 3.0 s (Series 2) signal acquisitions.

Background and Theory Chemical shift and through-space NOE measurements are remarkably sensitive to changes in local solvation and inter-molecular interactions that accompany aggregation. [13, 40, 51]

When modeling the aggregation of surfactants to form

micelles, data are treated with either a phase transition model[61, 62] or a mass-action model. [63] The former assumes that the micelles are considered a distinctly separate phase and are therefore not quantified in the equilibrium expression. The mass action


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model describes all aggregation steps as independent equilibria, and includes the monomers as well as the individual aggregates as quantifiable solutes in the equilibrium expression(s). In most cases, the phase-transition approach is easier to apply, but is best suited for micellization processes that involve large aggregation numbers (N), where N could be on the order of tens or hundreds of monomers. When aggregation numbers are small, the mass action model, which explicitly considers the individual equilibria governing all species, could more accurately describe the system. Aggregation numbers of various bile acids reported in the literature vary, but are generally on the order of 2-20 monomers for the types of micelles that form at millimolar concentrations of bile acid. A practical problem with applying the mass action model is that with polydisperse micelle systems no single tractable equilibrium expression can be used in isolation. It is interesting that in this and prior work, the phase transition model is found to be a good descriptor of the observed experimental data. [13] Even when N is small for these bile systems, the phase transition model may still be appropriate if the process is highly discretized. [64] The phase transition model applies well under the specific conditions of interest here, which include basic solution (pH 12) and the presence of probe (a.k.a. guest) molecules at relatively high concentrations, e.g. 2.5 mM. Parts of this work focus on the NMR signals of the R,S-BNDHP analytes, which have previously been found to be good reporters on the micellization steps of the bile salts. [13] An extensively documented trend is that protons that are desolvated and experience more hydrophobic interactions are shifted upfield (lower ppm values). [13, 40, 65] The phase transition model may be applied to the chemical shifts of the signals of the probe molecules R,S-BNDHP interacting with bile aggregates, or to the signals from the bile molecules themselves.

The observed chemical shift for the probe

molecule takes on a constant value if the surfactant concentration is below the critical micelle concentration, meaning that the probe molecule is in an unbound state at all times. At or above the critical micelle concentration, the observed chemical shift is a weighted average of the free and micelle-bound states of the molecule. Importantly, the concentration of surfactant monomer takes on the value of the CMC. In other words, it


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is assumed that the concentration of free monomer is unaffected by the micellization. In summary: If [surfactant] < CMC , δ = δfree , or if [surfactant] ≥ CMC , δ = ffreeδfree + fmicelleδmicelle ,

(Eqn 1)

where ffree and fmicelle are the fraction of the probe molecules found in the free and bound state, respectively, and are computed based on the assumption that the monomer concentration is equal to the CMC. The chemical shift of the unbound state is known, so fitting this model to experimental data involves two adjustable parameters: the CMC and the chemical shift of the micelle-bound state, δbound. The application of the phase transition model is not suited to automated nonlinear least squares fitting for two general reasons. First, in this and prior work with bile salts, [13, 41] the data commonly reflect the behavior of multiple, sequential aggregation steps such that they cannot be adequately modeled by a single CMC. Secondly, even with very clean data, nonlinear least squares fitting routines will select poor values for the CMC, a value that is well defined in the experimental data. Specifically, the steep change in the experimental data immediately following the CMC is characterized by only a very small number of data points, and automated fitting tends to ignore the importance of these few data points immediately following the CMC in favor of over-fitting the chemical shifts at very high bile concentrations. The difficulties with fitting sequential CMCs to the data are ameliorated by restricting the modeling process to treat only the initial aggregation event reflected in the data.

Note that data sets are not truncated in any fashion.

Determining such a

truncation point would be subjective, risks introducing bias, and does not allow for identifying the stage at which the single CMC model ultimately deviates from the data. Rather, here the phase transition model of Eqn (1) is applied to the first aggregation event in a data series following a simple and objective rubric (see section S.1 in Supplemental Information). Use of rubrics and a human rater to model experimental data is an approach that has been previously established for the analysis of NMR metabolomics data, [66] where automated fitting routines are just emerging.


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In some cases, the signal of a particular proton is strongly affected by multiple aggregation steps such that a single aggregation event is not clearly reflected in the data. Such data are not treated here, but can still be modeled very effectively with a sequential fitting procedure that will be described in a separate work.


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Results and Discussion MEKC with cholate and deoxycholate Separations using bile salt micelles of cholate and deoxycholate as a pseudostationary phase assume a rapid exchange of the analyte molecules between free and bound states such that equilibrium populations of the free and bound analyte are present throughout the experiment. Successful separations require that the guest isomers have different binding equilibria with surface sites of the negatively-charged micelle, creating a difference in their respective electrophoretic mobilities (µep , velocity per unit field, cm2 V-1 s-1) which, under the influence of a constant voltage field (V cm-1), leads to different net migration rates (cm s-1). [43, 44] It is observed that the (S)-isomer has a longer migration time than the (R)-isomer in MEKC, indicating that the (S)-isomer spends more time interacting with the anionic bile salt micelle.

The difference in

migration behavior varies with bile salt concentration, indicating which concentrations of the bile salt represent significant structural changes in the bile micelle structure and/or size. Processive aggregation over large concentration ranges is a known property of bile acids. [1, 2, 13, 42, 45, 55, 67] When using cholate as the pseudostationary phase, the chiral resolution of R,S-BNDHP is first observed at about 13 mM (Figure 2), consistent with prior MEKC work wherein chiral resolution was observed for solutions containing ca. 14 mM cholate. [13] A 7 mM cholate aggregate, previously observed by NMR [18], is not capable of enantio-selective solubilization as a pseudostationary phase under the conditions used here. These observations support assigning the ca. 7 mM cholate CMC to a premicellar aggregate and the 13-14 mM cholate CMC to the primary micelle. Another interesting cholate concentration is observed at approximately 30 mM in Figure 2 where resolution of R,S-BNDHP is maximized. A degradation of chiral resolution occurs progressively with increasing cholate concentration above 30 mM. While the small change in phase ratio at higher bile concentration could contribute to this trend, the sequential formation of a different type of aggregate, one that is less capable of chiral discrimination above 30 mM cholate, appears to be responsible for the significant observed loss in chiral selectivity (see NMR diffusion data, vide infra).


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Figure 2. Plots of the difference in electrophoretic mobility (∆µep) of R-, S-BNDHP versus (a) cholate and (b) deoxycholate concentration. In (a) the separation conditions are Lt=35.0 cm, Ld=26.5 cm, separation potential=10 kV, 25.0 °C. Separations performed on 0.5 mM of each analyte individually. In (b) the separation conditions are Lt=41.0 cm, Ld=32.5 cm, separation potential=10 kV, 25.0 °C. Separations performed on 1.5 mM of each analyte individually. In (a) and (b) three trials were performed for each bile concentration, and error bars are standard deviations. These data are representative of trials with different BNDHP concentrations, which have minimal effects on observed mobility differences. (insensitivity to concentration separately confirmed in section S.2 in the Supplemental Information).

Enantio-resolution in MEKC with deoxycholate shows trends similar to those seen with cholate, but the onset of separation for R,S-BNDHP in deoxycholate occurs at a significantly lower bile salt concentration of ca. 3 mM DC (Figure 2b), which NMR will show to be a premicellar DC aggregate. Specifically, 1H NMR data show a premicellar aggregate at 3 mM DC as well as a primary DC micelle at ca. 9 mM (vide infra, Figure 4). The MEKC data are insensitive to the primary 9 mM deoxycholate CMC, requiring that the accessible surfaces must be the same for the premicellar and primary DC micelles. As with cholate, it appears that a distinct secondary aggregate is forming above about 20 mM DC that is responsible for decreasing the chiral resolution of R,SBNDHP. Importantly, the MEKC data in Figure 2 indicate that high cholate concentrations nearly abolish chiral resolution by MEKC, but high deoxycholate concentrations retain some chiral resolution. How secondary micellization, specifically the availability of binding sites on the aggregate surface, harms MEKC resolution will be further developed with diffusion NMR data.


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1

H NMR of deoxycholate micelles Finely grained chemical shift titration experiments were carried out by varying

deoxycholate concentration in the presence of R- or S-BNDHP (2.5 mM) at basic pH (pH 12), and are summarized in Figure 3 and further analyzed in Figures 4-6. Chemical shifts follow prior assignments and the original spectra are provided in the Supplemental Information (section S.3). [41, 68] From these one-dimensional chemical shift data, the following discussion points arise. (i) Correspondence between NMR and MEKC. The chemical shifts of S-BNDHP are more strongly perturbed in the presence of increasing bile concentration, confirming MEKC results that S-BNDHP exhibits higher anionic mobility and experiences stronger interactions with the deoxycholate micelles, helping to define the nature of intermolecular interactions that give rise to aggregate formation and enantio-specific resolution of guests. (ii) Localizing the BNDHP binding edge.

The chemical shift perturbations in

Figure 3 show that protons H4-H7 undergo the greatest changes with increasing bile concentrations, such that the H4-H7 edge of the naphthyl ring must be sampling the surface-exposed binding pocket most strongly, consistent with a model demonstrated also by NOE analysis in which the aromatic naphthyl rings insert into a hydrophobic pocket lined by the methyl groups of the bile micelles. [13, 40] Notably, the chemical shift of H3 is weakly perturbed in the opposite direction, and is apparently not sampling the hydrophobic micelle interior. (iii) Conserving the deoxycholate surface binding site. The onset of the BNDHP chemical shift perturbations occurs at a very low DC concentration of 3 mM, and continues up to about 10 mM DC, after which the chemical shifts are largely unperturbed. A surface-accessible binding pocket sampled by the BNDHP probe is therefore established at these low bile concentrations and is relatively unchanged even as DC micelles undergo higher order aggregation between 20-100 mM DC.


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Figure 3. Proton chemical shifts (600 MHz) obtained for 2.5 mM R- or S-BNDHP in the presence of increasing concentrations of deoxycholate (DC) at pH 12.0. Below an initial CMC of about 3 mM, free DC monomers do not perturb BNDHP chemical shifts, while at higher concentration, a binding pocket that perturbs the shift of the S-isomer more than the R-isomer is formed, and this pocket persists at higher [DC]. Data at 900 MHz agree closely and are shown in section S.5 of the Supplemental Information. See section S.3 of the Supplemental Information for a stacked plot of the spectra from which these data are derived.

(iv) H3 reports on primary and secondary aggregate surfaces at binding sites. The onset of an early aggregate at about 3 mM DC and a subsequent aggregate at 9 mM DC is unambiguously demonstrated by applying the phase transition model to the chemical shift data, shown for H4 (preliminary aggregate) and H3 (primary aggregate) of R-BNDHP in Figure 4b. These data further confirm prior NOE data[40] that H3 of BNDHP does not sample the interior of the binding site. Specifically, the H3 chemical shift data reported here are not affected by the initial aggregation at 3 mM, and instead H3 reports solely on a later aggregation step occurring discretely at about 9 mM DC. In contrast, H4-H7 each report strongly on the premicellar aggregate at ca. 3 mM DC. Two discretized aggregation steps were also observed for cholate (7 mM and 14 mM), reproduced in Figure 4a, with permission. [13]

Therefore, in both cholate and


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deoxycholate, H3 is insensitive to the premicellar aggregate, and instead samples the surfaces of the subsequent primary aggregates, but with a key difference that H3 reports on a change in the surface composition of higher order DC aggregates ca. 3040 mM (Figure 4b).

Figure 4. Fitting (solid lines) of BNDHP chemical shifts (data points) in the presence of (a) cholate and (b) deoxycholate at pH 12.0. Similar to prior results in the presence of cholate (part a*), in the presence of deoxycholate, the H3 and H4 protons of R-BNDHP (600 MHz) are sensitive to two distinct aggregation behaviors. With increasing deoxycholate concentration, the H4 proton is perturbed at about 3 mM DC whereas, in the same spectra, the H3 proton is unperturbed until about 9 mM DC. Single-step phase transition modeling of each data set, employing a CMC = 3 mM for the H4-BNDHP data and a CMC = 9 mM for the H3-BNDHP data, is shown with superimposed red lines. For the H3-BNDHP data in deoxycholate, the single CMC model deviates from the experimental data at roughly 30 mM DC, consistent with the onset of higher order aggregation behavior. *Part (a) of this figure is reproduced with permission from earlier work. [13]

(v) The DC premicelle is enantioselective.

Chiral resolution in MEKC data

(Figure 2) is observed at about 3 mM DC, which corresponds with the initial DC aggregate seen by NMR in Figure 4. In contrast, the early 7 mM CA aggregate does not correlate with chiral resolution in MEKC data. Instead, resolution of R,S-BNDHP


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with cholate correlates with the later aggregate at 14 mM. This and previous work have shown that DC and CA utilize very similar binding pockets and mechanisms to perform chiral recognition of R,S-BNDHP, yet the relative stability of these DC and CA premicelles may explain the difference in MEKC results. As DC is a dihydroxy bile salt, its aggregates form at lower overall concentrations than the tri-hydroxy CA bile salt, and therefore the DC aggregate ca. 3 mM may be substantially more stable than the ca. 7 mM early CA aggregate.

Figure 5. Fitting of R-and S-BNDHP H5-H7 signals to a single-stage phase transition (red lines). Results are shown for the 600 MHz spectra, while data at 900 MHz correspond closely and are given in Supporting Information Section S.5. The R-BNDHP data deviate from the model at about 30 mM DC, which is taken to be the onset of higher order (a.k.a. secondary) aggregation. However, the S-BNDHP data deviate from the model at about 20 mM [DC], indicating that higher order (a.k.a. secondary) aggregation starts at lower DC concentration in the presence of S-BNDHP, and that S –BNDHP is promoting secondary aggregation. Note also that the magnitude of shift perturbation with the S-isomer is greater than for the R-isomer.


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(vi) The S-BNDHP guest slightly decreases observed CMC values.

The

chemical shifts of H5-H7 of BNDHP are the most strongly perturbed by the binding of BNDHP to bile aggregates, and are analyzed in Figure 5. Each of H5, H6 and H7 in RBNDHP is seen to be sensitive to the early aggregation event at about 3 mM. Specifically, the data (600 MHz) for R-BNDHP are well modeled with a CMC of 3.6 mM, but the S-isomer data are best fit with a CMC of 2.5 mM. Similar data at 900 MHz were modeled with a CMC of 3 mM and 2.6 mM, respectively (see S.5 in Supporting Information).

The phase transition model was applied by manually fitting the data

following a rubric discussed in the Background and Theory section. Interestingly, the chemical shift titration results for S-BNDHP (Figure 5) in the presence of increasing DC in basic solution (pH = 12) all report a slightly lower value for the premicellar CMC, resulting in an average value of about 2.5 mM DC. Since SBNDHP interacts more strongly with bile aggregates (Figure 2), a potential explanation for the difference in results is that S-BNDHP may stabilize the formation of initial aggregates, resulting in slightly lower CMC values. (vii) Localizing Chiral Interactions with BNDHP.

The chemical shift data

represented in Figure 3 have been further analyzed to reveal chirally selective interactions by examining the difference in the chemical shifts of R- versus S-BNDHP (Figure 6). The greatest difference in the degree of chemical shift perturbation between these enantiomers occurs for the protons along the H4-H7 edge, indicating that H4-H7 are undergoing interactions with the bile micelles that correlate with the observed chiral selectivity. Moreover, the maximum difference between the R- and S-isomer shifts with these protons is observed at about 9-10 mM DC, the lower end of the optimal range for MEKC enantio-separations using deoxycholate. It is interesting that the chemical shift data for H4-H7 indicate that R- and S-BNDHP interact extremely similarly with bile micelles, yet a requirement for chiral discrimination is that the enantiomers must experience distinct interactions with the bile aggregates. Extensive NOE and chemical shift analysis support a model in which R,S-BNDHP utilize the same binding pocket of bile micelles, but approach that binding pocket preferentially from opposite sides. [40] Specifically, S-BNDHP interacts more strongly with the micellar edge lined with 12α-OH


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moieties, whereas the R-BNDHP isomer interacts preferentially with the opposite edge, which bears a 7α-OH moiety in the case of cholate. [40] 0.020

δ(1H,S-BNDHP)- δ(1H,R-BNDHP) / ppm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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H8

H3

0.0 H4 H5 H6 H7

-0.020

-0.040

5

4

3

-0.060

O

6 7

O

O P

8

O-

O

O O

P

O-

-0.080 0

20

40

60

deoxycholate (mM)

80

100

0

20

40

60

80

100

deoxycholate (mM)

Figure 6. Plot of the difference in chemical shift perturbation observed between S- and R-isomers as a function [DC]. The H4-H7 protons are all differentially perturbed for R- vs S-BNDHP, showing that this binding edge is experiencing different local environment. The H6-BNDHP and H7-BNDHP shifts exhibit the largest differences, indicating that the chirally selective solubilization of the R,S-BNDHP enantiomers results in very different local environments particularly for these two protons. These results are computed from the data in Figure 3.

Overall, the points (i-vii) above are consistent with a model in which the H4-H7 edge inserts into a hydrophobic pocket of the bile aggregate and experiences chirally selective interactions with the bulk of the bile micelle, but H3 is localized to the surface of such aggregates and does not directly participate in chiral recognition. Interestingly, the H8 chemical shifts (see section S.4 of Supplemental Information) are perturbed significantly by both the premicellar and primary aggregation steps and cannot be modeled satisfactorily with a single CMC. The H8 chemical shift trends qualitatively mean that H8 is in an intermediate local environment that interacts weakly with the hydrophobic pocket, similar to H4-H7, but is also exposed to the micelle surface, similar to H3. Under the conditions studied here, the premicellar DC aggregate (ca. 3 mM) is capable of chirally selective solubilization of R,S-BNDHP, but the premicellar 7 mM CA


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aggregate is not.

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The 7 mM CA aggregate could still possess a chirally selective

binding pocket, but may be too unstable to effect chiral resolution in MEKC. Table 1. Representative cholate and deoxycholate CMCs as measured by various methods. The scope of bile CMC determinations exceeds this table, and some more comprehensive summaries have been prepared. [33, 35, 45] (ITC: isothermal titration calorimetry, LS: light scattering, EPR: electron paramagnetic resonance, UV/Vis: absorption spectroscopy, HPLC: high pressure liquid chromatography, FLUOR: fluorescence emission, MEKC: micellar electrokinetic capillary chromatography, NMR: nuclear magnetic resonance)

Bile Salt

Method

Conditions

CMC (mM)

NaDC

ITC [69] LS [70] EPR[42] UV/Vis[45] LS[45] FLUOR [71] MEKC (this work) NMR (this work)

pH 7.5, 0.10 M NaCl, 298 K pH 9, 0.10 M NaCl pH 7.8, 0.06 M Na+, 298 K pH 7, 0.10 M NaCl , 298 K pH 7, 0.10 M NaCl , 298 K pH 7, 298 K pH 12, 298 K pH 12 , 298 K

4.0 2.5-3.0 2.0-3.0 2.6 2.4 2.4 & 6.5 3 3&9

NaC

ITC [69] EPR [42] UV/Vis [72] POT[45] FLUOR [71] MEKC [72] MEKC (this work) NMR[13]

pH 7.5, 0.10 M NaCl, 297 K pH 7.8, 0.06 M Na+, 298 K pH 7, 20 mM Pi , 298 K pH 7, 0.10 M NaCl , 298 K pH 7, 298 K pH 7, 20 mM Pi , 298 K [13], pH 12 , 298 K pH 12 , 298 K

10 5.0-8.0 12-13 7.3 6.2 & 12.8 14-15 13-14 7 & 14

This work measured DC CMC values using NMR and MEKC methods. These data, together with representative CMC values for CA and DC determined by others are summarized in Table 1. Other studies appear to report a CA aggregate at ca. 12-18 mM, consistent with the CMC we observe at 13-14 mM CA. The preliminary CA aggregate observed here by NMR at 7 mM CA is not always observed, but similar premicellar CMC’s for cholate have been obtained with both probe-based and probefree methods. [13, 42, 45, 71] The preliminary 7 mM CA aggregate may be less stable and difficult to observe by other methods, while the use of a relatively high probe concentration of 2.5 mM BNDHP may also partly stabilize the 7 mM CA aggregate, making it more amenable to observation.


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PFGSE-NMR of cholate and deoxycholate With these bile salts, enantio-resolution with MEKC is degraded at bile concentration above about 30 mM for CA or 20 mM for DC (Figure 2), which prompted a study of micelle aggregate growth and surface properties.

In this concentration

regime, bile salts are well-known to undergo additional aggregation beyond the two CMCs reported in Figure 4, meaning that the accessibility of binding sites on the surfaces of these higher-order (aka ‘secondary’) bile aggregates must be altered. Indeed, with cholate as the chiral selector, chiral resolution in MEKC is almost completely abolished upon reaching 90 mM CA (Figure 2). A finely grained NMR diffusion study of bile aggregates in the presence and absence of R- and S-BNDHP was performed. Pulsed field gradient spin echo (PFGSE) NMR yields translational diffusion constants of solution species, making it useful to characterize surfactant aggregation, [47, 73] including bile systems. [57] Here we show that PFGSE-NMR is not only sensitive to aggregation in cholate and deoxycholate systems, but is particularly sensitive to the modulation of aggregate size by accommodating the chiral guests. For each bile system three series of 15 samples each were prepared with increasing bile concentration. One series was probe-free, and the others contained 2.5 mM R- or S-BNDHP. The effective diffusion constants derived from selected regions of the bile signals (see Materials and Methods) for all 45 cholate and 45 deoxycholate samples are summarized in Figure 7. Viscosities were measured in triplicate for each solution and increased by about 30% over each series and therefore do not account for the changes observed in Figure 7 (see also section S.6 in Supplemental Information for D20,w plots). There are several important shared characteristics in the diffusion results for DC and CA aggregates (Figure 7). First, as the concentration of bile salt increases up to 100 mM, the effective diffusion coefficient monotonically decreases, clearly showing that the average aggregate size is increasing with increasing bile concentration for each of


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these bile systems. Next, the presence of (R,S)-BNDHP probe molecules results in a significant decrease in the diffusion coefficient of the bile molecules, particularly apparent over the bile salt concentration range which confers chiral selectivity in MEKC. In other words, incorporation of these guest molecules shows the expected increase in average aggregate size. Importantly, the difference between the diffusion of the bile aggregates in the presence of S- vs R-BNDHP is greatest in the same concentration regime where MEKC confirms maximal resolution (Figure 2). Finally, the aggregate size is larger in the presence of S-BNDHP than R-BNDHP, consistent with the greater migration time of the S- isomer in MEKC separations.

Figure 7. The observed diffusion coefficient of bile aggregates, as obtained by PFGSE-NMR, as a function of the (a) cholate or (b) deoxycholate concentration in the presence of R-BNDHP, S-BNDHP, and with no probe. In (a) diffusion coefficients are obtained as averages of several signals, including those of bile salt protons H3, H7 and H12, representing the steroidal backbone. In (b) the inset shows the region where the onset of chiral resolution in MEKC is first observed with DC. In (a) and (b) the aggregate size is significantly different in the presence of the different enantiomers and in the concentration regimes where chiral resolution by MEKC is observed. Error bars represent the standard deviation. The dashed grey arrow in (b) highlights that the presence of R,S-BNDHP still significantly increases the deoxycholate aggregate size at high DC concentrations. Viscosities were measured in triplicate for each solution and plots of D20,w values may be found in section S.6 of the Supplemental Information.

While the diffusion coefficients measured in this work could be converted to Stokes radii of equivalent spheres to suggest approximate aggregate sizes, we choose to not report such radii for several reasons. First, some reports suggest that the higher order bile aggregates could be ellipsoidal or rod-like rather than spherical and may show dynamic complexity. [46, 74-76] Since there is no objective determination of


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shape factors, there is a risk of inaccurately correlating the diffusion properties to the size of the aggregate. Next, the lifetimes of the aggregates are not known, but since narrow lines are observed for the proton resonances, it appears that the micelles are in a fast dynamic equilibrium. Thus, the diffusion behavior seen in Figure 7 is actually the weighted average of the diffusion of free particles, early micelles at 3 mM DC (or 7 mM CA), micelles forming at 9 mM DC (or 14 mM CA), and higher order aggregates. The measured diffusion coefficients therefore do not report on a specific micellar aggregate. The fact that the micelle diffusion data converge at high cholate concentration (Figure 7a), but do not converge at high deoxycholate concentration (Figure 7b) indicates that the higher order aggregates have distinct surfaces that now offer dramatically different guest-binding capacity and selectivity. In the presence of R- and S-BNDHP, DC aggregates are consistently larger than probe-free aggregates up to 100 mM, indicating a surface availability of binding sites is preserved in higher order DC aggregates. However the diffusion data also show that the difference in the effective diffusion constant of DC (inset Figure 7b) aligns with MEKC separations (Figure 2), and converges to the same value in the presence of R- and S-BNDHP with increasing DC, confirming the degradation of resolution at these high concentrations (Figure 2). It is necessary then to consider how the preservation of a binding site on the aggregate surface is also accompanied by a loss of chiral resolution. Given that the difference in binding enthalpy of R- and S-BNDHP enantiomers to the primary aggregates of DC is small, [77] it can be proposed that the complex surface of the higher order DC aggregates sufficiently changes the weak noncovalent guest-host interactions to degrade the difference in overall affinities. But before discussing models further, it is important to consider a strikingly different situation that arises with cholate. The diffusion data for higher order aggregates of cholate (CA) converges to the probe-free data, regardless of the presence of BNDHP, indicating exclusion of the guest molecule by high order CA aggregates. In the case of cholate, the loss of guest binding demonstrates inaccessibility of the binding pocket on the surface of higher order CA aggregates.


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The loss of guest binding in secondary cholate aggregates is in fact also indicated by the distinct behavior of the H3 chemical shift in Figures 4a and 4b, which is a reporter of the micelle surface. Specifically, The H3 shift of BNDHP is very well modeled by a single CMC throughout the 100 mM concentration range of cholate (Figure 4a). That is, even though the diffusion data clearly show cholate undergoing higher order (secondary) aggregation (Figure 7a), the H3-BNDHP shift is insensitive to this secondary aggregation of cholate (Figure 7a).

In contrast, in the presence of

deoxycholate, the H3-BNDHP chemical shift model (Figure 4b) deviates from the data over the same concentration range that secondary aggregation begins to take place (Figure 7b). The H3 proton of BNDHP is an ideal reporter on secondary aggregation since H3 is exposed to the environment on the surface of the DC aggregates. When smaller bile micelles aggregate along their surfaces to form higher order (a.k.a. secondary) micelles, then the changes in the local environments on the micelle surfaces will be reflected in the H3 chemical shift.

In other words, the H3 chemical shift

demonstrates that the surface of the secondary DC micelle differs from the surface of the primary micelle. The diffusion data have shown that BNDHP binds with secondary micelles of deoxycholate but not of cholate, so H3 is only a reporter of secondary structure with deoxycholate.

Likewise, H3 of BNDHP reports only on the 14 mM cholate CMC,

independently showing that BNDHP has no interaction with the higher-order cholate aggregates. When combined with intermolecular NOEs measured recently, [40] the data presented here appear to reveal the orientation of monomers on the surfaces of secondary cholate and deoxycholate aggregates, depicted schematically in Figure 8. Prior work has shown that binding sites for binaphthyl compounds are composed of antiparallel bile dimers with hydrophobic interior lined by methyl groups C18 and C19, and that these dimers present two distinct edges for guest binding, one defined by the C12 edge, and the other defined by the C7 edge. The accessibility of an edge of a dimeric unit is presumed to be necessary for binding the binaphthyl guests. [40] Since the diffusion and chemical shift data presented here indicate that secondary micelles of


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cholate exclude binaphthyl guests, then one potential model is that any cholate dimers in the secondary cholate structure may be oriented such that they display the hydrophilic α-face to the solvent, preventing access to the edge of the binding pocket. The diffusion data indicate that secondary micelles of deoxycholate, which is less hydrophilic than cholate owing to the lack of a hydroxyl at carbon-7, retain the ability to bind binaphthyls throughout the concentration ranges probed here. These data indicate then that the edge(s) of dimeric DC units are exposed on the surface of the secondary deoxycholate micelle.

Figure 8. Proposed differences in surface accessibility of dimeric binding sites (paired green ellipsoids) on cholate and deoxycholate secondary micelles (large grey ellipsoids). The combined data herein along with prior NOE work[40] provides insight on the difference in surface orientation of bile monomers in cholate and deoxycholate secondary micelles. Since binding of the BNDHP guest is abolished in 2’ cholate micelles, the binding edge of cholate dimers is inaccessible on the micelle surface. One model is that bile monomers may display their α-faces on the surface. In contrast, 2’ deoxycholate micelles can still interact with guests, but more weakly and with greatly decreased chiral selection, which can be accomplished if the deoxycholate dimer is oriented to expose the binding edge on the surface of its secondary micelle.


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It remains unclear why chiral resolution is diminished with DC secondary micelles, as the diffusion data indicate each BNDHP isomer is able to bind to the DC micelle. The DC structure could dictate that just one dimeric binding pocket edge (e.g. the C7 edge or the C12 edge) is exposed. Prior data have shown that S-BNDHP prefers to bind from the C12 edge; it is possible that if only the C7 edges of dimers are present on the surface, that binding can occur without selectivity. [19] The H3-BNDHP chemical shift data demonstrate that the local chemical environment on the surface of the secondary and primary DC micelles differs, but these data are insufficient to define the orientation of the bile dimer in the DC secondary aggregate. Still, the ability to unambiguously demonstrate unique surface compositions of bile salts motivates continuing work to better understand how to characterize and selectively design these surface features. The NMR data in this report cannot shed light on the exchange rates of R- and SBNDHP with each of the bile aggregates. The different diffusion rates for micelle guesthost aggregates seen in the time frame of the NMR measurement are time-averages of solution species. The observed trends in these data could be due to either a difference in the thermodynamic stability of the guest-host complexes, or to a difference in exchange rates. Thus, kinetic factors must also be considered as plausible reasons for the some of the behaviors seen here. A broader outcome is that the data in this work support the step-wise aggregation noted by Small[1] of an initial formation of dimers, followed by more stable primary micelles containing multiple dimers, and then higher order (a.k.a. secondary) micelles containing assemblies of the early and primary micellar structures. In this work, the highly discretized early aggregate observed at very low bile concentration (ca. 3 mM for DC, and ca. 7 mM for CA) is capable of solubilizing the binaphthyl guests, so that this early aggregate must at least contain a dimer, [40] and could itself be a dimer. The next aggregate (ca. 9 mM for DC, and ca. 14 mM for CA) can be identified as a more persistent primary micelle, and the BNDHP guests must still be sampling dimeric units in these primary aggregates. [40] Next, MEKC and NMR chemical shift data show a subsequent aggregation process (ca. 20 mM DC and 30 mM CA) which is corroborated


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by the diffusion data that show continued growth in micelle size, consistent with secondary aggregation. The distinct solubilization and chiral recognition properties of early, primary and secondary bile aggregates observed here supports an emerging understanding that the sequential aggregates in these and related systems have unique solubilization properties. [78, 79]


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Conclusions This work provides new insight into bile salt micelle formation and the surface availability of chirally selective interactions with a model analyte R,S-BNDHP. Combining MEKC and 1H NMR precisely correlates step-wise aggregation of bile salts with chirally selective guest-host solubilization. Premicellar, primary and higher order (a.k.a. secondary) micellization processes for each of cholate (CA) and deoxycholate (DC) are clearly established for the conditions used here. The premicellar aggregate of DC is shown to be an effective chiral selector while the premicellar aggregate of CA is not. Chiral selection is observed for primary micelles of both DC and CA, requiring surface availability of both edges of bile dimers[40], but higher order aggregates of either bile salt result in the loss of chiral resolution, owing to different mechanisms. Specifically, diffusion NMR and MEKC data together support that higher order aggregates of CA exclude guests while those of DC allow guest binding with nearly equal affinities for the enantiomers. The exclusion of the binaphthyl guest by cholate secondary micelles but not by deoxycholate secondary micelles is independently supported by H3-BNDHP chemical shift data, which is shown to be an effective reporter of the local environment of the micelle surface. Secondary micelles of CA likely display α-faces of the bile acids, whereas secondary deoxycholate micelles offer a binding pocket which is not capable of chiral selection.


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Figure Captions Figure 1. Bile acids may be categorized as trihydroxy, such as (a) cholic acid, or dihydroxy such as (b) deoxycholic acid. Planar guest molecules, particularly those featuring binaphthyl ring systems such as (c,d) R,S-1,1′-binaphthyl-2,2′diylhydrogenphosphate (BNDHP) are differentially solubilized by aggregates of cholate[13]or of deoxycholate (this work). Figure 2. Plots of the difference in electrophoretic mobility (∆µep) of R-, S-BNDHP versus (a) cholate and (b) deoxycholate concentration. In (a) the separation conditions are Lt=35.0 cm, Ld=26.5 cm, separation potential=10 kV, 25.0 °C. Separations performed on 0.5 mM of each analyte individually. In (b) the separation conditions are Lt=41.0 cm, Ld=32.5 cm, separation potential=10 kV, 25.0 °C. Separations performed on 1.5 mM of each analyte individually. In (a) and (b) three trials were performed for each bile concentration, and error bars are standard deviations. These data are representative of trials with different BNDHP concentrations, which have minimal effects on observed mobility differences. (insensitivity to concentration separately confirmed in section S.2 in the Supplemental Information). Figure 3. Proton chemical shifts (600 MHz) obtained for 2.5 mM R- or S-BNDHP in the presence of increasing concentrations of deoxycholate (DC) at pH 12.0. Below an initial CMC of about 3 mM, free DC monomers do not perturb BNDHP chemical shifts, while at higher concentration, a binding pocket that perturbs the shift of the S-isomer more than the R-isomer is formed, and this pocket persists at higher [DC]. Data at 900 MHz agree closely and are shown in section S.5 of the Supplemental Information. See section S.3 of the Supplemental Information for a stacked plot of the spectra from which these data are derived. Figure 4. Fitting (solid lines) of BNDHP chemical shifts (data points) in the presence of (a) cholate and (b) deoxycholate at pH 12.0. Similar to prior results in the presence of cholate (part a*), in the presence of deoxycholate, the H3 and H4 protons of R-BNDHP (600 MHz) are sensitive to two distinct aggregation behaviors. With increasing deoxycholate concentration, the H4 proton is perturbed at about 3 mM DC whereas, in the same spectra, the H3 proton is unperturbed until about 9 mM DC. Single-step phase transition modeling of each data set, employing a CMC = 3 mM for the H4-BNDHP data and a CMC = 9 mM for the H3-BNDHP data, is shown with superimposed red lines. For the H3-BNDHP data in deoxycholate, the single CMC model deviates from the experimental data at roughly 30 mM DC, consistent with the onset of higher order aggregation behavior. *Part (a) of this figure is reproduced with permission from earlier work. [13] Figure 5. Fitting of R-and S-BNDHP H5-H7 signals to a single-stage phase transition (red lines). Results are shown for the 600 MHz spectra, while data at 900 MHz correspond closely and are given in Supporting Information Section S.5. The R-BNDHP data deviate from the model at about 30 mM DC, which is taken to be the onset of higher order (a.k.a. secondary) aggregation. However, the S-BNDHP data deviate from the model at about 20 mM [DC], indicating that higher order (a.k.a. secondary) aggregation starts at lower DC concentration in the presence of S-BNDHP, and that S –BNDHP is promoting secondary aggregation. Note also that the magnitude of shift perturbation with the S-isomer is greater than for the R-isomer. Figure 6. Plot of the difference in chemical shift perturbation observed between S- and R-isomers as a function [DC]. The H4-H7 protons are all differentially perturbed for R- vs S-BNDHP, showing that this binding edge is experiencing different local environment. The H6-BNDHP and H7-BNDHP shifts exhibit the largest differences, indicating that the chirally selective solubilization of the R,S-BNDHP enantiomers results in very different local environments particularly for these two protons. These results are computed from the data in Figure 3.


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Figure 7. The observed diffusion coefficient of bile aggregates, as obtained by PFGSE-NMR, as a function of the (a) cholate or (b) deoxycholate concentration in the presence of R-BNDHP, S-BNDHP, and with no probe. In (a) diffusion coefficients are obtained as averages of several signals, including those of bile salt protons H3, H7 and H12, representing the steroidal backbone. In (b) the inset shows the region where the onset of chiral resolution in MEKC is first observed with DC. In (a) and (b) the aggregate size is significantly different in the presence of the different enantiomers and in the concentration regimes where chiral resolution by MEKC is observed. Error bars represent the standard deviation. The dashed grey arrow in (b) highlights that the presence of R,S-BNDHP still significantly increases the deoxycholate aggregate size at high DC concentrations. Viscosities were measured in triplicate for each solution and plots of D20,w values may be found in section S.6 of the Supplemental Information. Figure 8. Proposed differences in surface accessibility of dimeric binding sites (paired green ellipsoids) on cholate and deoxycholate secondary micelles (large grey ellipsoids). The combined data herein along with prior NOE work[40] provides insight on the difference in surface orientation of bile monomers in cholate and deoxycholate secondary micelles. Since binding of the BNDHP guest is abolished in 2’ cholate micelles, the binding edge of cholate dimers is inaccessible on the micelle surface. One model is that bile monomers may display their α-faces on the surface. In contrast, 2’ deoxycholate micelles can still interact with guests, but more weakly and with greatly decreased chiral selection, which can be accomplished if the deoxycholate dimer is oriented to expose the binding edge on the surface of its secondary micelle.


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Acknowledgements: This work was supported by the National Science Foundation (NSF RUI 1153052) and the ACS Petroleum Research Fund (47262-B6). We thank Bucknell University and the Department of Chemistry for supporting research infrastructure and for additional student summer stipends. We are grateful to Juliette Lecomte and the Pennsylvania State University for the use of their 600 MHz spectrometer, and to the MIT/Harvard Center for Magnetic Resonance for the use of their 900 MHz spectrometer. We thank Mr. Brian Breczinski for support of the Bucknell 400 MHz spectrometer. Literature Cited 1. Small, D. M. Size and structure of bile salt micelles Influence of structure, concentration, counterion concentration, pH, and temperature. Adv. Chem. Ser. 1968, 84, 31-52. 2. O'Connor, C. J.; Wallace, R. G. Physico-Chemical Behavior of Bile Salts. Adv. Colloid Interface Sci. 1985, 22, 1-111. 3. Galantini, L.; di Gregorio, M. C.; Gubitosi, M.; Travaglini, L.; Tato, J. V.; Jover, A.; Meijide, F.; Soto Tellini, V. H.; Pavel, N. V. Bile salts and derivatives: Rigid unconventional amphiphiles as dispersants, carriers and superstructure building blocks. Current Opinion in Colloid & Interface Science 2015, 20, 170-182. 4. Madenci, D.; Egelhaaf, S. U. Self-assembly in aqueous bile salt solutions. Current Opinion in Colloid & Interface Science 2010, 15, 109-115. 5. Schefer, L.; Sanchez-Ferrer, A.; Adamcik, J.; Mezzenga, R. Resolving self-assembly of bile acids at the molecular length scale. Langmuir 2012, 28, 5999-6005. 6. Zhang, M.; Fives, C.; Waldron, K. C.; Zhu, X. X. Self-Assembly of a Bile Acid Dimer in Aqueous Solutions: From Nanofibers to Nematic Hydrogels. Langmuir 2017, 33, 1084-1089. 7. Wenseleers, W.; Vlasov, I. ; Goovaerts, E.; Obraztsova, E. ; Lobach, A. ; Bouwen, A. Efficient Isolation and Solubilization of Pristine Single-Walled Nanotubes in Bile Salt Micelles. Advanced Functional Materials 2004, 14, 1105-1112. 8. Gubitosi, M.; Trillo, J. V.; Alfaro Vargas, A.; Pavel, N. V.; Gazzoli, D.; Sennato, S.; Jover, A.; Meijide, F.; Galantini, L. Characterization of carbon nanotube dispersions in solutions of bile salts and derivatives containing aromatic substituents. J Phys Chem B 2014, 118, 1012-1021. 9. Nishi, H.; Fukuyama, T.; Matsuo, M.; Terabe, S. Chiral separation of optical isomeric drugs using micellar electrokinetic chromatography and bile salts. J. Microcolumn Sep. 1989, 1, 234-240. 10. Terabe, S.; Shibata, M.; Miyashita, Y. Chiral separation by electrokinetic chromatrography with bile salt micelles. J. Chromatogr. 1989, 480, 403-411.


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11. Crego, A. L.; Gonzalez, M. J.; Marina, M. L. Chiral separation of polychlorinated biphenyls by micellar electrokinetic chromatography with sodium cholate. Electrophoresis 1998, 19, 2113-2118. 12. Vaton-Chanvrier, L.; Oulyadi, H.; Combret, Y.; Coquerel, G.; Combret, J. C. Chiral recognition of binaphthyl derivatives: a chiral recognition model on the basis of chromatography, spectroscopy, and molecular mechanistic calculations for the enantioseparation of 1,1'-binaphthyl derivatives on cholic acidbonded stationary phases. Chirality 2001, 13, 668-674. 13. Hebling, C. M.; Thompson, L. E.; Eckenroad, K. W.; Manley, G. A.; Fry, R. A.; Mueller, K. T.; Strein, T. G.; Rovnyak, D. Sodium cholate aggregation and chiral recognition of the probe molecule (R,S)-1,1'-binaphthyl-2,2'-diylhydrogenphosphate (BNDHP) observed by 1H and 31P NMR spectroscopy. Langmuir 2008, 24, 13866-13874. 14. D'Alagni, M.; Delfini, M.; Galantini, L.; Giglio, E. A study of the interaction of bilirubin with sodium deoxycholate in aqueous solutions. J. Phys. Chem. 1992, 96, 10520-10528. 15. Maeder, C.; Beaudoin, G. M.,3rd; Hsu, E.; Escobar, V. A.; Chambers, S. M.; Kurtin, W. E.; Bushey, M. M. Measurement of bilirubin partition coefficients in bile salt micelle/aqueous buffer solutions by micellar electrokinetic chromatography. Electrophoresis 2000, 21, 706-714. 16. Bielejewska, A.; Duszczyk, K.; Kwaterczak, A.; Sybilska, D. Comparative study on the enantiomer separation of 1,1′-binaphthyl-2,2′diyl hydrogenphosphate and 1,1′-bi-2-naphthol by liquid chromatography and capillary electrophoresis using single and combined chiral selector systems. Journal of Chromatography A 2002, 977, 225-237. 17. jain, R. M.; Ben-Naim, M.; Landry, M. P.; Strano, M. S. Competitive binding in mixed surfactant systems for single-walled carbon nanotube separation. J. Phys. Chem. C 2015, 119, 22737-22745. 18. Green, A. A.; Duch, M. C.; Hersam, M. C. Isolation of single-walled carbon nanotube enantiomers by density differention. Nano Research 2009, 2, 69-77. 19. Valenta, C.; Nowack, E.; Bernkop-Schnurch, A. Deoxycholate-hydrogels: novel drug carrier systems for topical use. Int. J. Pharm. 1999, 185, 103-111. 20. Senyigit, T.; Tekmen, I.; Sönmez, U.; Santi, P.; Ozer, O. Deoxycholate hydrogels of betamethasone17-valerate intended for topical use: In vitro and in vivo evaluation. Int. J. Pharm. 2010, Epub (advance article). 21. McNeel, K. E.; Das, S.; Siraj, N.; Negulescu, I. I.; Warner, I. M. Sodium Deoxycholate Hydrogels: Effects of Modifications on Gelation, Drug Release, and Nanotemplating. J Phys Chem B 2015, 119, 8651-8659. 22. McNeel, K. E.; Siraj, N.; Negulescu, I.; Warner, I. M. Sodium deoxycholate/TRIS-based hydrogels for multipurpose solute delivery vehicles: Ambient release, drug release, and enantiopreferential release. Talanta 2018, 177, 66-73. 23. Plass, J. R.; Mol, O.; Heegsma, J.; Geuken, M.; Faber, K. N.; Jansen, P. L.; Muller, M. Farnesoid X receptor and bile salts are involved in transcriptional regulation of the gene encoding the human bile salt export pump. Hepatology 2002, 35, 589-596.


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24. Small, D. M.; Bourges, M.; Dervichian, D. G. Ternary and quaternary aqueous systems containing bile salt, lecithin, and cholesterol. Nature 1966, 211, 816-818. 25. Burnat, G.; Majka, J.; Konturek, P. C. Bile Acids Are Multifunctional Modulators of the Barrett's Carcinogenesis. J. Phys. Pharm. 2010, 61, 185-192. 26. Rust, C.; Wild, N.; Bernt, C.; Vennegeerts, T.; Wimmer, R.; Beuers, U. Bile acid-induced apoptosis in hepatocytes is caspase-6-dependent. J. Biol. Chem. 2009, 284, 2908-2916. 27. Wang, Y.; Nordhues, B. A.; Zhong, D.; De Guzman, R. N. NMR characterization of the interaction of the Salmonella type III secretion system protein SipD and bile salts. Biochemistry 2010, 49, 4220-4226. 28. Mithani, S. D.; Bakatselou, V.; TenHoor, C. N.; Dressman, J. B. Estimation of the increase in solubility of drugs as a function of bile salt concentration. Pharm. Res. 1996, 13, 163-167. 29. Rinco, O.; Nolet, M. C.; Ovans, R.; Bohne, C. Probing the binding dynamics to sodium cholate aggregates using naphthalene derivatives as guests. Photochem. Photobiol. Sci. 2003, 2, 1140-1151. 30. Hu, S.; Guo, X.; Shi, H.; Luo, R. Separation mechanisms for palonosetron stereoisomers at different chiral selector concentrations in MEKC. Electrophoresis 2015, 36, 825-829. 31. Sanchez-Lopez, E.; Salgado, A.; Crego, A. L.; Marina, M. L. Investigation on the enantioseparation of duloxetine by capillary electrophoresis, NMR, and mass spectrometry. Electrophoresis 2014, 35, 28422847. 32. Reis, S.; Moutinho, C. G.; Pereira, E.; de Castro, B.; Gameiro, P.; Lima, J. L. Beta-blockers and benzodiazepines location in SDS and bile salt micellar systems. An ESR study. J. Pharm. Biomed. Anal. 2007, 45, 62-69. 33. Coello, A.; Meijide, F.; Nunez, E. R.; Tato, J. V. Aggregation behavior of bile salts in aqueous solution. J. Pharm. Sci. 1996, 85, 9-15. 34. Blanco, M.; Valverde, I. Electrophoretic behaviour of pharmacologically active alkylxanthines. J. Chromatogr. A 2002, 950, 293-299. 35. Natalini, B.; Sardella, R.; Gioiello, A.; Ianni, F.; Di Michele, A.; Marinozzi, M. Determination of bile salt critical micellization concentration on the road to drug discovery. J. Pharm. Biomed. Anal. 2014, 87, 62-81. 36. Venkatesan, P.; Cheng, y.; Kahne, D. Hydrogen bonding in micelle formation. J. Am. Chem. Soc. 1994, 116, 6955-6956. 37. Ju, C.; Bohne, C. Dynamics of Probe Complexation to Bile Salt Aggregates. J. Phys. Chem. 1996, 100, 3847-3854. 38. Sen, S.; Dutta, P.; Mukherjee, S.; Bhattacharyya, K. Solvation dynamics in bile salt aggregates. J. Phys. Chem. B 2002, 106, 7745-7750.


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39. Cole, R. O.; Sepaniak, M. J.; Hinze, W. L.; Gorse, J.; Oldiges, K. Bile salt surfactants in micellar electrokinetic capillary chromatography. Application to hydrophobic molecule separations. J. Chromatogr. 1991, 557, 113-123. 40. Eckenroad, K. W.; Manley, G. A.; Yehl, J. B.; Pirnie, R. T.; Strein, T. G.; Rovnyak, D. An Edge Selection Mechanism for Chirally Selective Solubilization of Binaphthyl Atropisomeric Guests by Cholate and Deoxycholate Micelles. Chirality 2016, 28, 525-533. 41. Eckenroad, K. W.; Thompson, L. E.; Strein, T. G.; Rovnyak, D. Proton NMR assignments for R,S1,1'-binaphthol (BN) and R,S-1,1'-binaphthyl-2,2'-diyl hydrogen phosphate (BNDHP) interacting with bile salt micelles. Magn. Reson. Chem. 2007, 45, 72-75. 42. Kawamura, H.; Murata, Y.; Yamaguchi, T.; Igimi, H.; Tanaka, M.; Sugihara, G.; Kratohvil, J. P. Spin-label studies of bile salt micelles. J Phys Chem 1989, 93, 3321-3326. 43. Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T. Electrokinetic separations with micellar solutions and open-tubular capillaries. Anal. Chem. 1984, 56, 111-113. 44. Terabe, S.; Otsuka, K.; Ando, T. Electrokinetic chromatography with micellar solution and opentubular capillary. Anal. Chem. 1985, 57, 834-841. 45. Reis, S.; Moutinho, C. G.; Matos, C.; de Castro, B.; Gameiro, P.; Lima, J. L. F. C. Noninvasive methods to determine the critical micelle concentration of some bile acid salts. Analyt. Biochem. 2004, 334, 117-126. 46. Santhanalakshmi, J.; Lakshmi, G. S.; Aswal, V. K.; Goyal, P. S. Small-angle neutron scattering study of sodium chjolate and sodium deoxycholate interacting micelles in aqueous medium. Proc. Indian Acad. Sci. (Chem. Sci.) 2001, 113, 55-62. 47. Morris, K. F.; Froberg, A. L.,.; Becker, B. A.; Almeida, V. K.; Tarus, J.; Larive, C. K. Using NMR to Develop Insights into Electrokinetic Chromatography. Anal. Chem. 2005, 77, 254A-263A. 48. Waissbluth, O. L.; Morales, M. C.; Bohne, C. Influence of planarity and size on guest binding with sodium cholate aggregates. Photochem. Photobiol. 2006, 82, 1030-1038. 49. Small, D. M.; Pemkett, S. A.; Chapman, D. Studies on simple and mixed bile salt micelles by nuclear magnetic resonance spectroscopy. Biochimica Et Biophysica Acta 1969, 176, 178-189. 50. Funasaki, N.; Fukuba, M.; Kitagawa, T.; Nomura, T.; Ishikawa, S.; Hirota, S.; Neya, S. Twodimensional NMR study of the structures of micelles of sodium taurocholate. J. Phys. Chem. B 2004, 108, 438-443. 51. Matsuoka, K.; Yamamoto, A. Study on Micelle Formation of Bile Salt Using Nuclear Magnetic Resonance Spectroscopy. J. Oleo Sci. 2017, 66, 1129-1137. 52. Gouin, S.; Zhu, X. X. Fluorescence and NMR Studies of the Effect of a Bile Acid Dimer on the Micellization of Bile Salts. Langmuir 1998, 14, 4025-4029.


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53. Burattini, E.; D'Angelo, P.; Giglio, E.; Pavel, N. V. EXAFS study of probe molecules in micellar solutions. J. Phys. Chem. 1991, 95, 7880-7886. 54. Campanelli, A. R.; De Sanctis, S. C.; Chiessi, E.; D′Alagni, M.; Giglio, E.; Scaramuzza, L. Sodium glyco- and taurodeoxycholate: possible helical models for conjugated bile salt micelles. J Phys Chem 1989, 93, 1536-1542. 55. Carey, M. C.; Small, D. M. Micelle formation by bile salts. Physical-chemical and thermodynamic considerations. Arch. Intern. Med. 1972, 130, 506-527. 56. Cruz, J. R.; Becker, B. A.; Morris, K. F.; Larive, C. K. NMR characterization of the host-guest inclusion complex between beta-cyclodextrin and doxepin. Magn. Reson. Chem. 2008, 46, 838-845. 57. Galantini, L.; Giampaolo, S. M.; Mannina, L.; Pavel, N. V.; Viel, S. Study of intramicellar interactions and micellar sizes in ionic micelle solutions by comparing collective diffusion and selfdiffusion coefficients. J. Phys. Chem. B 2004, 108, 4799-4805. 58. Stejskal, E. O.; Tanner, J. E. Spin Diffusion Measurements: Spin Echoes in the Presence of a Time‐ Dependent Field Gradient. J. Chem. Phys. 1965, 42, 288-292. 59. Jerschow, A.; Müller, N. Suppression of Convection Artifacts in Stimulated-Echo Diffusion Experiments. Double-Stimulated-Echo Experiments. Journal of Magnetic Resonance 1997, 125, 372-375. 60. Loening, N. M.; Keeler, J. Measurement of Convection and Temperature Profiles in Liquid Samples. Journal of Magnetic Resonance 1999, 139, 334-341. 61. Pluckthun, A.; Dennis, E. A. 31P nuclear magnetic resonance study on the incorporation of monomeric phospholipids into nonionic detergent micelles. J. Phys. Chem. 1981, 86, 678-683. 62. Söderman, O.; Stilbs, P.; Price, W. S. NMR studies of surfactants. Concepts in Magnetic Resonance Part A 2004, 23A, 121-135. 63. Persson, B. -.; Drakenberg, T.; Lindman, B. Carbon-13 NMR of micellar solutions. Micellar aggregation number from the concentration dependence of the 13C chemical shifts. J. Phys. Chem. 1979, 83, 3011-3015. 64. O'Connor, C. J.; Ch'ng, B. T.; Wallace, R. G. Studies in bile salt solutions: 1. Surface tension evidence for a stepwise aggregation model. Journal of Colloid and Interface Science 1983, 95, 410-419. 65. O'Farrell, C. M.; Hagan, K. A.; Wenzel, T. J. Water-soluble calix[4]resorcinarenes as chiral NMR solvating agents for bicyclic aromatic compounds. Chirality 2009, 21, 911-921. 66. Tredwell, G. D.; Behrends, V.; Geier, F. M.; Liebeke, M.; Bundy, J. G. Between-person comparison of metabolite fitting for NMR-based quantitative metabolomics. Anal. Chem. 2011, 83, 8683-8687. 67. Hao, L.; Lu, R.; Leaist, D. G.; Pulin, P. R. Aggregation number of aqueous sodium cholate micelles from mutual diffusion measurements. J. Solution Chemistry 1997, 26, 113-125.


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68. Mak, K. K. W. Synthesis and resolution of the atropisomeric 1,1'-bi-2-naphthol: an experiment in organic synthesis and 2D NMR spectroscopy. J. Chem. Ed. 2004, 81, 1636-1640. 69. Garidel, P.; Hildebrand, A.; Neubert, R.; Blume, A. Thermodynamic characterization of bile salt aggregation as a function of temperature and ionic strength using isothermal titration calorimetry. Langmuir 2000, 16, 5267-5275. 70. Roda, A.; Cerre, C.; Fini, A.; Sipahi, A. M.; Baraldini, M. Experimental evaluation of a model for predicting micellar composition and concentration of monomeric species in bile salt binary mixtures. J. Pharm. Sci. 1995, 84, 593-598. 71. Matsuoka, K.; Moroi, Y. Micelle formation of sodium deoxycholate and sodium ursodeoxycholate (part 1). Biochim. Biophys. Acta 2002, 1580, 189-199. 72. Fuguet, E.; Ràfols, C.; Rosés, M.; Bosch, E. Critical micelle concentration of surfactants in aqueous buffered and unbuffered systems. Analytica Chimica Acta 2005, 548, 95-100. 73. Furó, I. NMR spectroscopy of micelles and related systems. J. Molecular Lipids 2005, 117, 117-137. 74. Partay, L. B.; Sega, M.; Jedlovszky, P. Morphology of bile salt micelles as studied by computer simulation methods. Langmuir 2007, 23, 12322-12328. 75. Mangiapia, G.; D'Errico, G.; Capuano, F.; Ortona, O.; Heenan, R. K.; Paduano, L.; Sartorio, R. On the interpretation of transport properties of sodium cholate and sodium deoxycholate in binary and ternary aqueous mixtures. Phys. Chem. Chem. Phys. 2011, 13, 15906-15917. 76. Warren, D. B.; Chalmers, D. K.; Hutchison, K.; Dang, W.; Pouton, C. W. Molecular dynamics simulations of spontaneous bile salt aggregation. Colloids and Surfaces A: Phyicochem. Eng. Aspects 2006, 280, 182-193. 77. Anderson, S. L.; Rovnyak, D.; Strein, T. G. Direct Measurement of the Thermodynamics of Chiral Recognition in Bile Salt Micelles. Chirality 2016, 28(4), 290-298. 78. Barnadas-Rodriguez, R.; Cladera, J. Steroidal Surfactants: Detection of Premicellar Aggregation, Secondary Aggregation Changes in Micelles, and Hosting of a Highly Charged Negative Substance. Langmuir 2015, 31, 8980-8988. 79. Amundson, L. L.; Li, R.; Bohne, C. Effect of the guest size and shape on its binding dynamics with sodium cholate aggregates. Langmuir 2008, 24, 8491-8500.


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Page 39 of 46

Langmuir

a) Cholic Acid (CA)

1 OH 2 3 4 5 6 HO 7 8 9 OH 10 11 CH3 CH3 12 13 14 15 OH 16 OH 17 α-face 18 OH 19 20 21 22 c) S-BNDHP 23 24 25 26 27 28 P 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

b) Deoxycholic Acid (DC)

OH

OH 19

O

2

HO

CH3

1

3

10

11 12 13 9

5

6

8

23

18 20

OH

24

22

O

17 16 15

7

CH3

OH

14

21

CH3

CH3

OH O

O OH

OH

α-face

d) R-BNDHP 6

5

4 3 2

7

*

8


1

*

O O

O P

–

O

O O

O

O–

Langmuir b)

a)

∆µep (x10-6 cm² / V s)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.0

-5.0

-10.0 O-

OH

-15.0

O-

OH

O

O HO

HO

-20.0

OH

0

20

40

60

cholate (mM)

80

100

0

20


40

60

deoxycholate (mM)

80

100

Page 41 of 46

8.2 H4 8.0

H5 6

δ(1H) / ppm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

7

7.8

7' 6'

5

4

3 2

8 8'

5'

1'

1

*2

4'

O O

P

OO

R-BNDHP S-BNDHP

3'

7.6

H3

7.4

H6 H8

7.2

H7 0

20

40

60

Deoxycholate (mM)


80

100

Langmuir

a) cholate (Hebling et al.) 0.912

b) deoxycholate Exp Model

H4

4

H4

0.907

3 O-

O P

* 0.910

O

O

3' 4'

0.905

log(b(1H)/ppm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 46

0.908

7 mM

3 mM 0.903

0.906

14 mM

9 mM 0.8770

0.882 0.8760

H3

H3

0.880

30 mM 0

0.5

1.0

1.5

2.0

0

log([CA]/mM)


0.5

1.0

1.5

log([DC]/mM)

2.0

Page 43 of 46 0.904

log((1H) / ppm)

log((1H) / ppm)

log((1H) / ppm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

R-BNDHP

Langmuir

S-BNDHP

H5

H5

0.900

3.6 mM

2.5 mM

0.896

0.872

H6

H6

0.868

3.6 mM

0.864

2.5 mM

0.860

H7

H7 0.856

0.852

0.848

3.5 mM 0

0.5

2.4 mM 1.0

1.5

log( [deoxycholate] / mM)

2.0

0

0.5

1.0

1.5

2.0

log ([deoxycholate] / mM)


Langmuir

δ(1H,S-BNDHP)- δ(1H,R-BNDHP) / ppm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 46

0.020

H8

H3

0.0

H4 H5

H6 H7

-0.020

-0.040

5

4

3 O

-0.060

-0.080

O

0

20

40

60

deoxycholate (mM)

80

6 7

O P

O-

100

0

20

40

60

8

deoxycholate (mM)


O

O O

80

P

O-

100

Page 45 of 46

Langmuir

a)

diffusion coefficient (x10-6 cm²/s)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

b)

5.0

No probe R-BNDHP S-BNDHP

5.0

No probe R-BNDHP S-BNDHP

4.0

4.0

4.0

3.5

3.0

3.0

0

4

8

2.0 2.0 0

20

40

60

cholate (mM)

80

100

1.0

0

20

40

60

deoxycholate (mM)


80

100

= bile monomer Langmuir

OH OH

HO

O

O

CH3

C7

CH3

HO

C7

OH

O OH

cholate

2’ aggregate

OH

OH

deoxycholate 2’ aggregate

R ,S-BNDHP

R ,S-BNDHP

α-face

surface-exposed dimer face

*

*

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 46

surface-exposed dimer edge


Stepwise aggregation of cholate and deoxycholate dictates the

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