Molecular Structural Transformations Induced by Spatial Confinement

Dec 3, 2013 - R. Vijay and Prasad L. Polavarapu*. Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37235, United States. J. Phys...
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Molecular Structural Transformations Induced by Spatial Confinement in Barium Fluoride Cells R. Vijay† and Prasad L. Polavarapu* Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37235, United States ABSTRACT: Confinement of peptides and surfactants in narrow spaces of the order of 50 μm between BaF2 plates is found to induce new structural transformations. Optical microscopy, transmission electron microscopy, and vibrational spectroscopic studies have been used to uncover the evidence for these structural transformations. These studies indicate that, using spatial confinement, small micelles can be converted to larger aggregates and peptide monomers can be converted to β sheets even in monomer-promoting 1,1,1,3,3,3hexafluoro-2-propanol solvent.

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INTRODUCTION Applications involving molecular response to external perturbations, such as electric field,1 magnetic field,2 pH,3 temperature,4 and light5 have been known for a long time. Similar applications involving molecular response to spatial confinement between surfaces are not known. Chiral compounds are best characterized using chiroptical spectroscopy.6−10 The robustness of chiroptical spectroscopy is reflected in its role in the reassignment of structures of some natural products which were incorrectly assigned using routine characterization techniques, including X-ray diffraction.11 Advances in experimental and quantum chemical methods have rendered chiroptical spectroscopy a unique role in determining 3D structures of a variety of molecules.6 Among the widely used chiroptical spectroscopy techniques, namely, optical rotation (OR)6,7,12−16 electronic circular dichroism (ECD),6,7,17,18 vibrational circular dichroism (VCD),6−10 and vibrational Raman optical activity (ROA),6−8,10 VCD and ROA have advantages in view of the sensitivity of vibrational transitions to molecular structure and the vast number of molecular vibrations that can be studied in the accessible spectral regions. This sensitivity has led to their applications in studying the structures of a variety of compounds.6−10,17−21 Inspired by several advances in the area of chiroptical spectroscopy, investigations of novel materials such as chiral nanoparticles22 and self-organized assemblies23−27 are just beginning. For instance, it was found recently that the changes in the specific optical rotation of spherical micelles as a function of concentration and/or temperature manifest corresponding changes in the micelle size.27 Although each of the chiroptical spectroscopic methods has advantages specific to that method, there are also limitations, which when not recognized or properly taken into account, may lead to incorrect conclusions.28 Therefore, it is paramount © 2013 American Chemical Society

to bring forth any limitations to facilitate the development of better methodologies. In this work we report for the first time that spatial confinement between surfaces can also provide a source for structural perturbations in chiral surfactants and peptides. The evidence for structural changes induced by spatial confinement has been gathered using multiple techniques, namely optical microscopy, transmission electron microscopy (TEM), and infrared vibrational absorption (VA) and VCD spectroscopies.

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EXPERIMENTAL SECTION The synthesis of lauryl ester of phenylalanine hydrochloride (LEP) is reported elsewhere.29 Diacetyl-L-tartaric acid mono lauryl ester (designated as T12O) was synthesized by reacting (+)-O,O′-diacetyl-L-tartaric anhydride (DATA) with 1 equiv of lauryl alcohol for 24 h in chloroform at room temperature. The product was purified by the extraction of the impurities in water, dried over magnesium sulfate, and converted to sodium salt (T12ONa) by the addition of concentrated sodium hydroxide. DATA, lauryl alcohol, sodium hydroxide, deuterium oxide, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), all with purity over 99%, and chloroform ACS grade were purchased from Aldrich. Trifluoroethanol (TFE) with purity over 99% was purchased from Acros Organics. The amyloid peptides Aβ(34− 42) and Aβ(25−35) were purchased from AnaSpec Inc. For measurements in HFIP, peptides were dissolved and equilibrated until the solutions were transparent. For measurements in TFE, peptides were reconstituted first by dissolving in HFIP until the solutions were transparent. Then HFIP was evaporated and peptide redissolved in TFE. VA and VCD Received: October 1, 2013 Revised: December 2, 2013 Published: December 3, 2013 14086

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spectra were measured using ChiralIR spectrometer. TEM images were obtained using a CM20, operated at an acceleration voltage of 200 kV. A drop of surfactant/peptide solution was placed onto an ultrathin carbon-coated TEM grid (Ted Pella, Inc.). About 1 min after deposition, the grid was blotted with filter paper to remove surface solution. Negative staining was performed by using a droplet of a 1 wt % phosphotungstic acid solution. Optical microscope images were obtained using an Olympus BX41 microscope equipped with a Pixera camera. Stainless steel sealed cells with BaF2 windows and fixed path length (50, 100 or 200 μm; Model SL-3; International Crystal Laboratories), and a demountable BaF2 cell with 6 μm spacer, were used for liquid solution phase VA and VCD measurements. Control solutions were stored in polypropylene microcentrifuge vials (Model UP2054N, United Laboratory Plastics).

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RESULTS AND DISCUSSION Two each of surfactants and peptides studied in the current work are displayed in Scheme 1. Figure 1. VCD (top panel) and VA (bottom panel) spectra of LEP (100 mM in D2O) in 50 μm BaF2 cell. The spectra (a)−(d) were recorded at different time intervals while the solution was aging inside the BaF2 cell undisturbed throughout the course of the experiment. The spectrum (e) is for control solution aged outside BaF2 cell (in a polypropylene vial). Traces (a) and (e) overlap and are almost indistinguishable.

Scheme 1. Schematic Representation of (a) LEP, (b) T12ONa, (c) Aβ(34−42), and (d) Aβ(25−35)

onward, VA spectrum of the solution left inside the BaF2 cell exhibits a decrease in the intensity of the 1739 cm−1 band with concurrent increase in the intensity of the 1620 cm−1 band; also a new band is seen at 1686 cm−1. These changes are not present for the control LEP solution that was stored outside the BaF2 cell (in a polypropylene vial) for a period of 4 days. Furthermore, the spectrum of LEP solution aged outside the BaF2 cell overlays with that obtained immediately after injecting the freshly made solution into the BaF2 cell. The VA spectral changes mentioned above are accompanied by changes in the physical appearance of the sample. The LEP solutions recovered from the BaF2 cell, even within hours, appear turbid in contrast to the control solutions which appear transparent after several days (see Figure 2). The solution recovered from the 50 μm BaF2 cell is more turbid than that recovered from the 200 μm BaF2 cell. The solution stored inside the demountable BaF2 cell with 6 μm spacer (not shown) turned into a solid filmlike material within 30 min. Note that the increase in the turbidity of the surfactant solution Surfactants. Two different surfactants, one with a positively charged and the other with a negatively charged headgroup, have been used to demonstrate structural transformations induced by spatial confinement. Lauryl Ester of Phenylalanine Hydrochloride (LEP). LEP is a well-characterized surfactant,27,29−31 with low CMC (∼1 × 10−4 M),29 a positively charged headgroup, and prominent vibrational bands in the mid-infrared region. The solventsubtracted VA and VCD spectra of LEP in D2O at a concentration of 100 mM measured using a 50 μm BaF2 cell is presented in Figure 1. No significant VCD features are associated with vibrational bands of LEP,27 so our focus here will be on the VA spectra. The VA spectrum of LEP measured immediately after injecting the sample solution into the BaF2 cell holder exhibits a band at 1739 cm−1. From the second day

Figure 2. Pictures of LEP solutions (100 mM in D2O) aged in (a) 50 μm and (c) 200 μm BaF2 cells for 1 h and corresponding 4 day old control solutions (b) and (d) aged outside BaF2 cell. 14087

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is indicative of the formation of larger aggregates.32−35 The formation of solidlike material inside the demountable BaF2 cell with 6 μm spacer, turbid solution in the 50 μm BaF2 cell, and less cloudy solution in the 200 μm BaF2 cell indicate the growth of aggregates whose size is inversely proportional to the path length of the BaF2 cell. Because the above-mentioned changes are not present for the control solutions, it is apparent that the noted changes of enhanced aggregation of LEP in the confined space between BaF2 windows could be directed by either the charged surfaces of or surface adsorption on, and space between, BaF2 windows of the cell. To establish further evidence for enhanced growth of LEP aggregates induced by spatial confinement inside BaF2 cells, the BaF2 cell containing LEP solution was viewed under an optical microscope. The optical micrographs were taken in lowcontrast dark field mode, where any material deposited on the BaF2 cell window will appear bright relative to the dark background. If no material is deposited on the surface of the window, the optical microscope images will appear dark. The optical micrograph of the BaF2 cell with LEP solution aged in the same BaF2 cell shows deposition of material on the surface of the window (Figure 3b). In contrast, the control LEP solution, aged in a polypropylene vial that was transferred into

the BaF2 cell just before imaging under the microscope, shows absence of any material on the surface of the window (Figure 3a). For TEM studies, the solutions were deposited on carboncoated TEM grids. The TEM image of 4 day old control solution aged outside a BaF2 cell (Figure 3c) shows spherical micellar aggregates as small as ∼100 nm. TEM images of the LEP solution aged inside the 50 μm BaF2 cell (Figure 3d−f) show different types of morphologies, viz., larger spherical aggregates (∼500 nm) (Figure 3d), sheetlike structures (Figure 3e), and micrometer size rods (Figure 3f). The changes in the VA spectra of LEP with time (Figure 1) in contrast to control solution are attributed to the transformation of simple spherical micelles to large higher-order aggregates. The TEM images substantiate the transformation of simple micelles to larger aggregates inside the BaF2 cell. The increase in the turbidity of the solution inside the BaF2 cell with decrease in the path length demonstrates the growth of smaller micelles to larger aggregates induced by spatial confinement. Two explanations for the transformation of LEP to larger aggregates inside BaF2 cells are possible: (a) Attraction of the positively charged headgroup of LEP toward the negatively charged F− ions on the BaF2 window surface leads to diminished electrostatic repulsion between the head groups of the surfactant, inducing the growth in the aggregation. The addition of an electrolyte to the aqueous solutions of ionic surfactants is known to increase the aggregation number because of the compression of the electrical double layer surrounding the headgroup.36−38 Once larger aggregates are formed, they do not tend to revert back to smaller aggregates spontaneously. (b) Material is deposited on the surface of the window due to preferential adsorption (due to weak van der Walls interactions) of LEP on the surface of the window. However, it is difficult to separately isolate the adsorption and electrostatic interactions, especially because there can be equilibrium between the surfactant molecules that form micelles and those that adsorb at the interface.36 It is known that LEP efficiently and effectively adsorbs at the interface and so was used as a high-performance reactor to generate nanoparticles.39,40 Sodium Salt of Diacetyl-L-tartaric Acid Mono Lauryl Ester (T12ONa). Analogous experiments were performed for a tartaric acid based surfactant, T12ONa (Scheme 1), with a negatively charged headgroup. The solvent-subtracted VCD and VA spectra of T12ONa in D2O at a concentration of 100 mM measured using a 50 μm BaF2 cell are presented in Figure 4. The VA spectrum of fresh T12ONa solution exhibits bands at 1736, 1620, and 1562 cm−1. From the second day onward, a decrease in the intensities of the 1736 and 1620 cm−1 bands is seen for the T12ONa solution aged inside the cell. The band at 1562 cm−1 exhibits no significant change in its intensity with time. The VA spectrum of the control solution aged outside the BaF2 cell, as in the case of LEP, matches the VA spectrum measured immediately after injecting fresh T12ONa solution into the BaF2 cell, again pointing to the role of spatial confinement between BaF2 windows in inducing the noted changes. There is no significant VCD associated with the absorption bands at 1620 and 1562 cm−1. The only significant feature in the VCD spectrum is a weak positive VCD couplet (negative at higher and positive at lower frequency) associated with the 1736 cm−1 absorption band, and its VCD intensity also changes slightly with time for the solution aged inside the BaF2 cell. This positive VCD couplet, although weak, is above the

Figure 3. Microscope images of LEP. Optical micrographs of 50 μm BaF2 cell with (a) control solution aged outside BaF2 cell and (b) solution aged in the same BaF2 cell. TEM images of (c) LEP control solution aged outside BaF2 cell and (d)−(f) LEP solution aged inside 50 μm BaF2 cell. Scale bars in (c), (d), (e), and (f) are 500, 500, 100, and 500 nm respectively. 14088

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Figure 4. VCD (top panel) and VA (bottom panel) spectra of T12ONa (100 mM in D2O) in a 50 μm BaF2 cell. Spectra (a)−(c) were recorded at different time intervals while the solution was aging inside BaF2 cells undisturbed throughout the course of the experiment. Spectrum (d) is for a control solution aged for 3 days outside a BaF2 cell. Traces (a) and (d) overlap and are almost indistinguishable.

noise level and was reproduced in three different measurements. The CMC of T12ONa (∼1 mM) is higher than that of LEP, so one would not expect to see changes in the texture of T12ONa solution upon aggregation as readily as in LEP. Accordingly, unlike in the case of the LEP solution, no distinct difference in the texture of the control solution and the solutions recovered from the 50 μm BaF2 cell could be seen for T12ONa. However, the T12ONa solution inside the demountable BaF2 cell with 6 μm spacer turned into a filmlike material on the surface of the window, indicating path length (or space) dependent changes in the aggregation properties of T12ONa. The optical micrographs of a BaF2 cell filled with control T12ONa solutions aged outside the BaF2 cell show darker images (Figure 5a), whereas that with T12ONa solution aged in the same BaF2 cell (Figure 5b) shows deposition of material on the surface of the window. The TEM image of the control T12ONa solution, aged outside the BaF2 cell, shows wormlike features (Figure 5c) with ∼100−500 nm length and ∼10 nm width. Upon aging inside the BaF2 cell, larger (∼100 nm both in length and width) aggregates (Figure 5d) are formed. Figures 5e and 5f are higher magnification images of the aggregates shown in Figures 5c and 5d, respectively. These results suggest that both T12ONa and LEP solutions aged inside the BaF2 cell behave similarly and that spatial confinement contributes to the structural transformation in the aggregates. The structural transformation of T12ONa could be attributed to the electrostatic attraction between negatively charged headgroups and positively charged barium ions. The barium ions are expected to diminish electrostatic repulsion between the negatively charged headgroups, thereby facilitating growth of aggregates. Alternately, the deposition of T12ONa on the surface of the window may also suggest preferential adsorption of T12ONa at the solid−liquid interface.

Figure 5. Microscope images of T12ONa. Optical micrographs of a 50 μm BaF2 cell with (a) control solution aged outside the BaF2 cell and (b) solution aged inside the BaF2 cell. TEM images of 4 day old (c, e) control solution and solutions aged inside the 50 μm BaF2 cell (d, f). Enlarged views of panels (c) and (d) are shown in panels (e) and (f), respectively. The scale bars shown in (c), (d), (e), and (f) are 500, 500, 100, and 100 nm respectively.

Peptides. Two different amyloid peptides, Aβ(34−42) and Aβ(25−35), the former being hydrophobic and the latter hydrophilic, have been investigated. Aβ(25−35) adopts different structures, viz., helix, random coil, and β sheet depending on the solvent and pH environments.41 HFIP solvent is known to promote helical conformations.42 Aβ(34−42). The solvent-subtracted VCD and VA spectra of Aβ(34−42) peptide in HFIP at a concentration of 20 mg/mL measured using a 100 μm BaF2 cell are presented in Figure 6A. The VA spectrum of Aβ(34−42) peptide in HFIP as a function of time and that of control solution on day 4 exhibit amide I and amide II bands at 1652 and 1528 cm−1, respectively. The VA spectrum also exhibits a small shoulder in the amide I region at ∼1632 cm−1. The VCD spectrum exhibits a negative VCD band at 1652 cm−1. The band position in the VCD and VA spectra did not change over a period of 4 days. The VA absorption band at ∼1632 cm−1 is considered to be characteristic of β-sheet structures. The appearance of a small shoulder at 1632 cm−1, besides a stronger VA band at 1652 cm−1, may be viewed as an indication of the existence of a small fraction of Aβ(34−42) monomers with β-sheet structures. There is a small increase in the intensity of the shoulder at 1632 cm−1 on day 4, but there is no significant growth of the ∼1632 cm−1 band, indicating the lack of tendency to form a large amount of β-sheet structures in the 100 μm BaF2 cell. However, 14089

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Figure 6. VCD (top panels) and VA (bottom panels) spectra of Aβ(34−42) peptide (20 mg/mL in HFIP) in (A) 100 and (B) 50 μm BaF2 cells. Traces (a)−(d) were recorded at different time intervals while the solution was sitting inside the cell undisturbed throughout the course of the experiment. Trace (e) was recorded for control solution aged outside the BaF2 cell.

when the peptide solution aged in the 100 μm BaF2 cell was transferred at the end of fourth day into a 50 μm BaF2 cell, the band characteristic of β-sheet structure started to grow within 24 h. To confirm this space-induced aggregation of Aβ(34−42), freshly prepared Aβ(34−42) peptide solution (in HFIP) at a concentration of 20 mg/mL was injected into a 50 μm BaF2 cell. The solvent-subtracted VCD and VA spectra of the Aβ(34−42) in the 50 μm BaF2 cell are shown in Figure 6B. The bands in the VA and VCD spectra of Aβ(34−42) in HFIP immediately after injecting into the 50 μm BaF2 cell match those obtained with the 100 μm BaF2 cell. However, from the second day onward, the VA band intensity at 1632 cm−1 increased significantly with time, along with some increase in the band intensity in the amide II region at ∼1528 cm−1. The VA spectrum of the control Aβ(34−42) solution in HFIP, however, is remarkably similar to the VA spectrum of Aβ(34− 42) in the 50 μm BaF2 cell on day 1. The increase in relative intensity of the VA band at ∼1632 cm−1 in the 50 μm BaF2 cell is an indication of space-induced aggregation of Aβ(34−42) peptide, as the confinement of Aβ(34−42) in the 50 μm space between BaF2 windows appears to have promoted the β-sheet formation, unlike that in the 100 μm BaF2 cell. These reproducible observations are further substantiated with optical micrographs and TEM images of Aβ(34−42) solutions (Figure 7). The optical micrographs of the BaF2 cell imaged immediately after filling with control solution did not show any deposits on the surface of the windows (Figure 7a), while that containing solution aged in the same BaF2 cell indicates significant deposition of the peptide on the surface of the window (Figure 7b). For TEM studies, when Aβ(34−42) solution is deposited on copper-coated grids, lack of solvent can induce aggregation. Peptides under such conditions can form usual fiberlike aggregates that some of the amyloid peptide fragments form in dilute solutions. However, if the peptide solution contains larger aggregates to begin with, the deposition

Figure 7. Microscope images of Aβ(34−42). Optical micrographs of the 50 μm BaF2 cell (a) filled with control solution aged outside BaF2 cell and (b) containing solution aged in the same BaF2 cell. TEM images of (c) control solution aged outside the BaF2 cell and (d) solution aged inside the 50 μm BaF2 cell. The scale bars shown for TEM images in (c) and (d) are both 100 nm.

of aggregates on copper-coated grids is not expected to alter their nature during the evaporation of the solvent. The TEM image of the 4 day old control solution of Aβ(34−42) in HFIP (Figure 7c) shows usual fiberlike features as anticipated. The TEM image of Aβ(34−42) solution aged in 50 μm BaF2 cell for 4 days shows aggregated structures (Figure 7d) demonstrating growth of fibers into larger aggregates inside 50 μm BaF2 cell. Aβ(25−35). Aβ(25−35) peptide was dissolved in HFIP at a concentration of 10 mg/mL. About 100 μL of this solution was 14090

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Figure 8. VCD (top panels) and VA (bottom panels) spectra of Aβ(25−35) peptide (10 mg/mL in HFIP) measured in (A) 100 and (B) 50 μm BaF2 cells. Traces (a)−(d) were recorded at different time intervals while the solution was aging inside the BaF2 cell undisturbed throughout the course of the experiment. The inset in (B) shows the VCD and noise for Aβ(25−35) peptide solution on the second day.

Figure 9. Microscope images of Aβ(25−35). Optical micrographs of solutions aged for 4 days outside the BaF2 cell (a) and inside (b) 100 and (c) 50 μm BaF2 cells. TEM images of control solution aged outside the BaF2 cell (d) and solution aged inside (e) 100 and (f) 50 μm BaF2 cells. The scale bar shown for TEM images is 100 nm.

HFIP adopts a small proportion of β sheets regardless of its aging in the 100 μm BaF2 cell or polypropylene vial. VA spectra of Aβ(25−35) peptide solution in the 50 μm BaF2 cell (Figure 8B) on the first day are very similar to those obtained in the 100 μm BaF2 cell (Figure 8A) except for onehalf decrease in overall absorption intensity (due to one-half path length difference). However, the VA spectra measured with the 50 μm BaF2 cell from the second day onward display the growth of a shoulder at 1628 cm−1 into a well-developed band. Note that the growth of the band at 1628 cm−1 is so significant that the band at 1670 cm−1 appears as a shoulder from the second day onward.

injected into each of the two 100 and 50 μm BaF2 cells at the same time, and the rest of the solution was stored as a control solution at room temperature in a polypropylene vial. The VCD/VA spectra recorded at regular intervals of time for solutions in 100 and 50 μm BaF2 cells are shown in Figures 8A and 8B, respectively. The VCD/VA spectrum of the control solution was recorded after 4 days. The spectra of Aβ(25−35) peptide in HFIP recorded with the 100 μm BaF2 cell on the first day displayed a VA band at 1670 cm−1 (Figure 8A). On the second day, a weak shoulder at 1628 cm−1 is observed in the VA spectrum. The VCD/VA spectra recorded from the second day onward are very similar to those of the aged control solution. These results indicate that Aβ(25−35) peptide in 14091

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The VA band at 1628 cm−1 has a corresponding weak bisignate VCD couplet (positive on the lower frequency and negative on the higher frequency side) centered at ∼1628 cm−1. The VA spectrum of the 4 day old control solution displays a band at 1670 cm−1 and a small shoulder at 1628 cm−1, similar to those seen for the solution aged in the 100 μm BaF2 cell (Figure 8A). The phenomenal growth of the 1628 cm−1 band from the second day onward indicates the formation of a larger proportion of β-sheet structures in the 50 μm BaF2 cell. Because larger proportions of β-sheet structures are not observed for the solutions aged inside the 100 μm BaF2 cell or aged outside in a polypropylene vial for a period of 4 days, these observations clearly demonstrate another stark proof of structural changes induced by spatial confinement. The optical micrograph of the BaF2 cell containing the solution aged outside (Figure 9a) shows no deposition on the surface of the window, but those containing the solution aged inside 100 and 50 μm BaF2 cells (Figure 9b and 9c, respectively) do indicate deposition on the cell windows. The size of the particles deposited on the windows of the 100 μm BaF2 cell (Figure 9b) is relatively smaller than that deposited on the windows of the 50 μm BaF2 cell (Figure 9c). TEM images also provide evidence for enhanced aggregation of Aβ(25−35) peptide inside the BaF2 cell, indicating surfaceand space-induced effects. The TEM image of Aβ(25−35) control solution in HFIP, aged for 4 days outside a BaF2 cell, has very different features (Figure 9d) in comparison to those of equally aged recovered solutions from both 100 μm (Figure 9e) and 50 μm (Figure 9f) BaF2 cells, demonstrating the influence of spatial confinement in 50 and 100 μm BaF2 cells toward directing structural transformation. On the basis of the chemical structures of peptides (see Scheme 1), Aβ(25−35) has three different NH2 groups that can exist in NH3+ ionic form, whereas Aβ(34−42) has only one NH2 group that can exist in NH3+ ionic form. In this context, Aβ(25−35) can anchor to the surface of the BaF2 window in more ways than is possible for Aβ(34−42). As a consequence, it is speculated that Aβ(25−35) in HFIP in the 50 μm cell could form larger amounts of β-sheet structures, in comparison to Aβ(34−42), because of multiple forms through which Aβ(25−35) can deposit on the surface of the BaF2 window. The formation of larger amounts of β sheets due to spatial confinement, opposing HFIP’s propensity for promoting monomers, is a novel observation. The initial deposition of peptide on the surface of the window due to either electrostatic attraction or preferential adsorption is believed to be directing the structure in the solution. The spatial confinement between the windows in the 50 μm BaF2 cell and the ease with which the peptide deposits on the surface of the window synergistically act against the solvent’s ability to bring the peptide back to its native state. A closer analysis of the behavior of surfactants and peptides reveals important information. Self-assembly of surfactants above CMC typically requires interaction of the hydrophilic and hydrophobic segments, located within the surfactant molecule, with similar segments of another surfactant molecule.36,37 Aggregation of surfactants takes place above their CMCs36,37 and Kraft temperatures. Although peptides can self-assemble like surfactants, the operating mechanism for peptides is different from that for surfactants. Despite these differences, a common theme for aggregation processes induced by spatial confinement of the studied molecules can be attributed to (a) electrostatic interactions, (b) surface

adsorption, or (c) both. Surfactants have charged head groups and the peptides are in zwitterionic form, in view of the existence of amino and carboxylic acid groups in the same peptide (see Scheme 1). In a BaF2 window, every barium cation is surrounded by two fluoride anions. The charged group of the surfactant or peptide would be attracted toward oppositely charged ions on the BaF2 window surface and vice versa. We speculate that the initial attraction to the surface of the window, due to either electrostatic interactions or surface adsorption, could direct the structural transformation in the rest of the surfactant or peptide solution inside the cell. The solvent is expected to resist the structural changes directed from the surface of the window. However, when the space available for the solution per unit area of the BaF2 surface between the BaF2 windows is decreased, the ability of the solvent to reverse the effect induced by the surfaces of the cell windows back to solvent-mediated structure diminishes significantly, leading to the domination of surface- and space-induced changes. For this reason, the availability of less solvent per unit surface area of BaF2 windows in a smaller space BaF2 cell, in comparison to that in a larger space BaF2 cell, facilitates the dominance of structural transformation induced by the spatial confinement over that of solvent-mediated influence. As a consequence, larger changes are seen in the smaller space (∼50 μm and less) BaF2 cell compared to those in the larger space (∼100 μm and greater) BaF2 cell, and the extent of the changes are inversely proportional to the path length of the BaF2 cell. Thus, the structural changes induced by spatial confinement become more prominent for smaller path length cells. In the event of dominance of electrostatic interactions, the spatial confinement effect should be noticeable for most peptides that can exist in zwitterionic form. Because spatial confinement effects reported here were not known before, no particular attention was given to such possibility in analyzing the time-dependent spectral changes noted for peptides in the literature. It is advisable to revisit those studies in light of the current observations. If surface adsorption is the determining factor, then the spatial confinement effect may be noticeable for neutral surfactants and even nonaggregating organic compounds, although consequential effects for the latter would be extremely small. In either case, the current observations are likely to incite many more future studies. BaF2 is sparingly soluble in pure water (at