Nanoconfinement's Dramatic Impact on Proton Exchange between

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Nanoconfinement's Dramatic Impact on Proton Exchange Between Glucose and Water Benjamin Paul Wiebenga-Sanford, Joseph A. DiVerdi, Christopher D. Rithner, and Nancy E. Levinger J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.6b01651 • Publication Date (Web): 25 Oct 2016 Downloaded from http://pubs.acs.org on October 25, 2016

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The Journal of Physical Chemistry Letters

Nanoconfinement's Dramatic Impact on Proton Exchange between Glucose and Water Benjamin P. Wiebenga-Sanford, Joseph DiVerdi, Christopher D. Rithner, and Nancy E. Levinger* Department of Chemistry, Colorado State University, Fort Collins, CO 80523-1872 AUTHOR INFORMATION Corresponding Author *[email protected]

ABSTRACT Glucose nanoconfined by solubilization in water-containing AOT (sodium bis(2ethylhexyl)sulfosuccinate) reverse micelles has been investigated using 1H NMR. NMR spectra reveal well-defined signals for the glucose hydroxyl groups that suggest slow chemical exchange between them and the water hydroxyl group. Using the EXSY (ZZ-exchange) method, the chemical exchange rate from water to glucose hydroxyl groups was measured for glucose in reverse micelles as a function of size (water pool diameter of ~1-5 nm) at 25˚ C. The chemical exchange rates observed in the nanoconfined interior are dramatically slower (5-20 times) than observed for glucose in bulk aqueous solution at the same concentration as the micelle interior. Exchange rate constants are calculated via a mechanism that accounts for these observations, and implications of these results are presented and discussed.

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TOC GRAPHIC

KEYWORDS. Reverse micelle, NMR exchange spectroscopy, glucose, water, kinetics.


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Sugars and carbohydrates are ubiquitous in biology. Sugars usually serve as metabolic fuel sources, or generic polymer subunits for structural elements like cellulose in plant cell walls. In addition, sugars play an active role in biological processes, including cell-cell communication and signaling,1 as a forest of glycoproteins on cell surfaces, particularly cancer cells,1-3 modulating cell function,4 or acting as cryo- or lyoprotectants.5 In bulk aqueous solution, sugars can easily interact with water. Researchers have explored many different properties of carbohydrate-water solutions finding, for example, that interactions with sugars strongly affects water dynamics,6-10 but not water structure.8,11 However, in the crowded environment inside cells or on the surface of proteins, the nature of the water-sugar interaction is much less clear. In crowded systems, including biologically relevant ones like cells, cell organelles, and hydrophilic pockets in macromolecular structures (like proteins), molecular dynamics - translation and rotation - can differ significantly from bulk solution and impact reaction rates.12 Thus, measuring the interaction of sugars with water in confined environments provides insight to help us understand the role of sugars in biology. D-glucose, perhaps the most significant sugar in biological systems, is intimately involved in fundamental metabolic cellular physiology. This highly hydrophilic molecule contains five hydroxyl groups making it highly soluble in water and DMSO. Glucose has long been studied using NMR methods13,14 and its proton spectrum exhibits several interesting features. When dissolved in aprotic polar solvent, like DMSO, the ambient temperature NMR spectrum displays spectral features from both aliphatic and hydroxyl protons; in deuterated aqueous solution only signals from aliphatic protons are present.15 It is well established that glucose hydroxyl groups exchange rapidly in room temperature aqueous solution with a rate constant of approximately 2000 s-1 16 leading to coalescence of the glucose and water hydroxyl NMR signals (fast exchange


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regime).17 Lowering the temperature to -6 ˚C reduces the exchange rate enough to permit observation of signals from the glucose hydroxyl groups (slow exchange regime).13 Chemical exchange represents one of the earliest processes studied using magnetization transfer NMR methods.18 Here, experimentally induced perturbation of a particular spin magnetization propagates through the network of various couplings present in a chemical system, resulting in disturbance of distant yet coupled spins. Application of this method has elucidated structures and structural fluctuations in biochemical systems and is often termed ZZexchange, ZZ-spectroscopy, or EXSY.19 In our case, coupling arises through the mass exchange of protons from the water reservoir to the far smaller reservoir of glucose hydroxyl protons. We prepare the systems' spins so that only the water protons are polarized and aligned parallel or anti-parallel to the magnetic Z-axis; these spin polarized water hydroxyl protons exchange with glucose hydroxyl protons transferring polarization that we observe in the resultant NMR spectra. Analysis of the glucose signal amplitude reveals chemical exchange rates for water hydroxyl with individual glucose hydroxyl groups. The interior of a cell organelle can be challenging to study. Thus, we use a model confined system to explore the behavior of sugar, specifically glucose, in a confined aqueous environment. Our model system utilizes reverse micelles (RMs), i.e. spontaneously self-assembled nanoscopic polar droplets stabilized by a surfactant in a bulk non-polar liquid.20 RMs provide a convenient and flexible platform to explore the effect of confinement. Here, we use a common microemulsion system consisting of water and AOT surfactant (Fig. 1) in isooctane. We characterize the RM size with a parameter w0=[H2O]/[AOT]; because the particles are spherical on average, w0 is directly proportional to the particle diameter. Water pool diameters range from approximately 0.5 to 5 nm.


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OH 4 6 O HO 3 1 R1 2 HO OH R2 glucose α: R1= H; R 2 = OH β: R1= OH; R 2 = H

O O AOT O

O SO3- Na +

Figure 1: Chemical structures of d-glucose and AOT molecule. Numeric labels are used to identify signals in NMR spectra.

AOT RMs have been widely used and characterized through a range of different experimental and theoretical methods. For example, RM sizes measured directly using SAXS21, SANS,22 and light scattering23,24 reveal relatively monodisperse size distributions over a wide range of conditions, e.g., concentration, w0, nanodroplet composition, and/or nonpolar solvent. Other properties, such as dynamics of water confined in the RMs, have been measured using ultrafast laser spectroscopy.25,26 The stability of the AOT-nonpolar interaction facilitates RM formation with polar solvents other than water, such as ethylene glycol and formamide.27

NMR

spectroscopy has also been applied to examine water structure,28,29 dynamics,30,31 as well as the interactions of water and macromolecules32-35 in reverse micelles. In the study reported here, we have encapsulated aqueous d-glucose in AOT RMs, which allows us to explore the effect of a confined environment on glucose-water exchange. We present results from a series of 1D-EXSY NMR experiments that measure the rate of chemical exchange between water molecules and glucose hydroxyls groups in the water pool of AOT RMs. Results from these experiments demonstrate that the exchange rate between water and glucose hydroxyl groups is substantially slower than it is in bulk aqueous solution.


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Qualitatively, Figure 2 shows that the exchange rate for glucose in RMs is slower than bulk aqueous glucose because the RM hydroxyls are well defined (slow exchange) and the aqueous is coalesced (fast exchange). The interaction between exchange rate, instrument frequency, and the frequency difference in chemical shift36 allows us to estimate rate constants for exchange of >1500 s-1 for aqueous and

Nanoconfinement's Dramatic Impact on Proton Exchange between

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