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. 2021 Sep 23;6(39):25227–25234. doi: 10.1021/acsomega.1c03016

Understanding the Molecular-Level Interactions of Glucosamine-Glycerol Assemblies: A Model System for Chitosan Plasticization

Danielle R Smith 1, Austin P Escobar 1, Melissa N Andris 1, Brycelyn M Boardman 1,*, Gretchen M Peters 1,*
PMCID: PMC8495686  PMID: 34632182

Abstract

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Glycerol is the most widely used plasticizer for the biopolymer chitosan. However, there remains a lack of understanding of the molecular-level interactions between chitosan and glycerol. Here, we describe an in-depth spectroscopic study of the intermolecular interactions between the monomeric repeating unit of chitosan, glucosamine, and the plasticizer glycerol. Infrared and nuclear magnetic resonance spectroscopy were used to probe glucosamine assembly at high and low concentrations to establish diagnostic signals for intra- and intermolecular glucosamine interactions. Systematic addition of glycerol was found to disrupt intramolecular glucosamine hydrogen bonds and promote glucosamine self-assembly. Furthermore, we observed a significant preference for glycerol binding to the amine functionality of glucosamine. These findings indicate that the plasticization of chitosan with glycerol requires a specific binding motif and likely occurs via the gel theory mechanism.

Introduction

The biopolymer chitosan is promising for a wide range of applications. Chitosan is derived from chitin, the second most abundant polysaccharide in nature, and has been a popular alternative to petroleum-based polyolefins due to its biodegradable and nontoxic properties.1−3 Its biocompatible nature and antibacterial4 properties make chitosan an exciting material for a variety of utilizations from food packaging5 and sensing to pharmaceutical6−8 and orthopedic applications.9−11 However, due to the superior thermal, optical, and mechanical properties of petroleum-based polymers, chitosan has not been able to replace its petroleum-based counterparts. In its natural state, chitosan is rigid and brittle due to strong intermolecular hydrogen bonding between polymer chains. The literature is rich with investigations into plasticizers and their improvement of the mechanical properties of chitosan through the disruption of the interchain polymer interactions.12−17 Glycerol and other polyols are most commonly used as plasticizers for chitosan films, resulting in films that have improved flexibility (Figure 1A).13,14,18 While many theories exist that attempt to rationalize the plasticization effect, such as the gel, lubricity, and free volume theories,19 there has been little exploration into the specific molecular-level interactions present between chitosan and these polyols. This lack of understanding remains a roadblock for the future development of new plasticizers for biopolymers and ultimately for the use of these materials in commercial applications.

Figure 1.

Figure 1

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(A) Structures of chitosan and glycerol (Glyc) and corresponding pure (right) and plasticized (left) films. (B) IR spectral overlay of chitosan powder (blue), 2 wt % chitosan film (green), 1 wt % chitosan film (fuchsia), 2 wt % chitosan +50.0 mM Glyc film (dotted green), and 1 wt % chitosan +50.0 mM Glyc film (dotted fuchsia). Top: OH and CH stretching region from 3700 to 2500 cm–1. Bottom: CN/NH and CO–H combination band region from 1700 to 1450 cm–1 (for full spectra see Figure S1).

Recent studies investigating plasticized chitosan films with glycerol and larger polyols, such as xylitol and maltitol, have attempted to elucidate the interactions between the plasticizers and the polymer using FT-IR. Slight shifts in the OH stretching region were reported for all of the polyols, which resulted in the conclusion that these polyols were disrupting the hydrogen bonding between chitosan chains via the OH functionality.14 In these studies, no change in the region from ∼1680 to 1500 cm–1 was observed for the N–H stretch. Interestingly, chitosan films plasticized with glycerol had the optimal mechanical properties when compared to the larger polyols, suggesting that either the plasticization mechanism in each polyol system is different or that molecular shape and size of the plasticizer contribute to the efficiency of plasticization.

In addition to mechanistic considerations, the role of the chitosan’s amine in plasticization remains a topic of debate. In two separate FT-IR studies involving chitosan/poly(vinyl) alcohol systems with glycerol as a plasticizer, one group observed changes in the bands of chitosan in the region from ∼1680 to 1500 cm–1, suggesting that the amine functionality of chitosan is involved in plasticization,20 while the other study reported no shift in this band and suggested that the amine does not participate.21 While both of these studies propose that glycerol and other polyols interact with the OH functionality of chitosan, there is a clear divergence in the conclusions regarding the role of the NH functionalities in plasticization. These disparities are highly motivating for a more fundamental understanding of these complex systems. These studies also highlight the critical need to understand specific plasticizer–polymer interactions as a “one size fits all” approach hinders the implementation of these environmentally responsible materials.

Herein, we describe the molecular level investigation of glucosamine with glycerol using FT-IR and NMR spectroscopy. Glucosamine, the repeating unit of chitosan, is an ideal model system to evaluate how plasticizers such as glycerol interact with specific biopolymers. Glucosamine is both a smaller and simpler system than chitosan, allowing for solution-based studies, and the specific stretching and bending IR frequencies of interest have been well-defined both theoretically and experimentally.22 The development of new biopolymeric materials, as well as the consumer switch to environmentally responsible alternatives hinge on our ability to optimize the properties of these materials for specific applications. Therefore, it is essential that we elucidate the molecular-level interactions between glucosamine (Gluc) and glycerol (Glyc) and the structures of Gluc–Glyc assemblies, and, in turn, verify the plasticization mechanism of chitosan.

Results and Discussion

Previous reports have provided a wealth of characterization data on chitosan films plasticized with glycerol. Thermal, mechanical, and crystallographic data have consistently shown that glycerol disrupts the crystalline domains of pure chitosan to improve its overall properties.13,14,18 Infrared studies, however, have been less consistent, and therefore, we prepared chitosan films (1 and 2 wt %) in the presence and absence of Glyc (50.0 mM) and analyzed them using ATR-FTIR as an initial investigation in this study. Previously reported results indicated significant shifts in the OH and NH stretching frequency of chitosan powder when compared to the formation of chitosan films, which we also observe from 3356 cm–1 in pure chitosan to 3270 and 3180 cm–1 for 1 and 2 wt % films, respectively.13,23 These shifts to a lower wavenumber are the result of a reduction in the number of hydrogen bonds between chitosan chains when the films are formed. In the presence of Glyc, both 1 and 2 wt % films show a shift to ∼3280 cm–1, indicating an increase in hydrogen bonding (Figure 1B, top). However, this shift does not elucidate the plasticization mechanism that occurs in the system. An increase in hydrogen bonding as a result of interchain interactions between chitosan chains via crowding by the Glyc molecules would suggest the free volume theory model. However, interactions between the polymer chains and Glyc could indicate that gel or lubricity theory was at play. The inability to distinguish between interchain polymer–polymer and polymer–plasticizer interactions results from our inability to deconvolute the specific molecular-level interactions occurring in the OH/NH stretching region. The OH functionality is not a good diagnostic signal for understanding the intermolecular interactions because we cannot differentiate between the OH groups on chitosan from those on Glyc. The region from ∼1680 to 1500 cm–1 includes the C=O stretch (from the 15–25% residual acetylation) as well as the C–N stretch, CN–H bend, and the CO–H bend (1590 cm–1), which have also been used diagnostically in the study of chitosan and Glyc interactions. As mentioned, some previous studies suggest that there is no change in the bands in this region, indicating that no significant hydrogen bonds formed between chitosan and Glyc via the N–H functionality of chitosan.14 However, our results show that there is a significant shift in this region from 1590 to 1534 cm–1 and 1537 cm–1 for 1 and 2% films, respectively, and a decrease in the intensity of the band at ∼1650 cm–1 (Figure 1B, bottom).

When Glyc is added to the films, there is a shift in the region from ∼1680 to 1500 cm–1 to a higher wavenumber for both concentrations and a reappearance of the band at ∼1650 cm–1. The 1% chitosan film showed a more significant shift from 1534 to 1581 cm–1 as compared to a shift from 1537 to 1558 cm–1 for the 2% film. Since both films contain 50.0 mM Glyc, the ratio of chitosan/plasticizer is higher in the 1% film, resulting in a more dramatic shift of the bands in this region. While this does suggest that there are changes in the hydrogen bonding with respect to the NH2/NH3+ moiety, it again does not distinguish whether this increase in hydrogen bonding is a result of interchain interactions and crowding triggered by the Glyc molecules or the introduction of chitosan–Glyc interactions. Additionally, since this region also contains the CO–H bend, it is challenging to definitively deconvolute which functionalities are participating in these hydrogen bonds. Because of this inability to draw specific conclusions about the polymeric system directly, we turned instead to the model system using Gluc, the repeat unit of chitosan, with Glyc to understand this system at a more fundamental level.

Films of Gluc were formed on the ATR crystal of the FTIR spectrometer by drying aqueous Gluc solutions (12.5 and 50.0 mM) under N2 gas. The resulting spectra were then compared to the spectrum of pure Gluc powder (Figure 2). The IR region from 1680 to 1480 cm–1 has clearly defined bands for the CN stretching, NH, and CO–H bending frequencies of Gluc compared to the broad bands in this region previously discussed for chitosan (Figure 1B). Gluc powder displays well-resolved stretching and bending bands, indicating a highly ordered, self-assembled system with strong intermolecular hydrogen bonding. The films formed from low concentrations of Gluc (12.5 mM) show a loss of resolution in both the CN/NH combination band and the CO–H band, as well as a shift in frequency to a lower wavenumber. This indicates that at this low concentration, the Gluc molecules are in randomly oriented conformations and that the bulk system is overall disordered, consisting mainly of isolated Gluc molecules with intramolecular hydrogen bonds. In contrast, films formed from higher concentrations of Gluc (50.0 mM) display resolved CN/NH and CO–H bands at wavenumbers nearly identical to those of pure Gluc powder. At higher concentrations of Gluc, the system is more ordered, and Gluc is highly aggregated, resulting in the presence of mainly intermolecular hydrogen bonding interactions. This agrees well with the previously reported self-association constant of Gluc in DMSO (490 M–1), as determined by NMR titration.24

Figure 2.

Figure 2

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IR of CN/NH and the CO–H combination band region from 1700 to 1450 cm–1 (full spectra in Figure S2), Gluc powder (blue), 50.0 mM dried Gluc film (fuchsia), and 12.5 mM dried Gluc film (green). Films were formed from aqueous stock solutions on the ATR crystal under N2 flow.

Similar trends were observed by NMR. For these studies, we compared the same concentrations of Gluc in DMSO-d6 using nuclear Overhauser effect spectroscopy (NOESY). NOESY allows us to probe through space and chemical exchange (EXSY) interactions between protons in close proximity. Shown in Figure 3 are selected cross sections of the 2D NOESY spectra at 12.5 and 50.0 mM Gluc, respectively (see Figures S3–S4 for full spectra). The OH at C3 and NH of Gluc were chosen as representative protons as both are available for hydrogen bonding in the chitosan polymer. Notably, we observed signals for the alpha anomer of Gluc in all NMR spectra recorded. Mutarotation of Gluc has been well-established and extensively studied by NMR in both D2O and DMSO-d6.25,26 While anomeric mixtures have been observed in both solvent systems, the ratio of alpha to beta is much smaller in DMSO. In our spectra, slight variations in this anomeric ratio are likely due to differences in wait time prior to recording the NMR spectra. In the NOESY spectra, all OH/OH and OH/NH appear as positive cross-peaks, suggesting that these are the results of chemical exchange. In contrast, the signs for OH/CH and NH/CH cross-peaks are negative and thus are attributed to NOE interactions. At 12.5 mM Gluc, we observe positive cross-peaks for OH3 with OH4 and OH6. We attribute these signals to chemical exchange and hydrogen bonding between OH3–OH4 and OH3–OH6. Because the IR data indicate that Gluc molecules are highly isolated at this concentration, we can infer that these OH–OH cross-peaks are the results of intramolecular hydrogen bonding interactions. Importantly, at this low concentration of Gluc, NH “sees” only the proton on C2 (the carbon to which the amine is bound), as well as C1 and C3 (both of which are neighboring carbons) and has no detectable intramolecular hydrogen bonding interactions (Figures 3 and S4). As we increase the concentration of Gluc to 50.0 mM, we still see cross-peaks for the intramolecular Gluc/Gluc interactions (i.e., OH3–OH4, OH3–OH6, and NH–CH1) but now also observe additional OH–OH and NH–OH cross-peaks. Specifically, OH3 now exchanges with OH1, while the NH functionality of Gluc has new exchange cross-peaks with OH1, OH3, OH4, and OH6. Based on our initial IR findings and the observed aggregation at this concentration, we attribute these new cross-peaks to intermolecular Gluc/Gluc interactions. In combination, these IR and NMR results give us baseline metrics for assigning intra- and intermolecular Gluc interactions.

Figure 3.

Figure 3

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Representative cross sections (OH3 and NH) of the 2D NOESY spectra for 12.5 mM Gluc (top) and 50.0 mM Gluc (bottom) recorded at 400 MHz (9.4 T). While the cross-peaks in the 12.5 mM Gluc spectrum are consistent with intramolecular (blue, top right) Gluc interactions, the 50.0 mM spectrum suggests that intermolecular (red, bottom right) Gluc/Gluc interactions occur. Small peaks for the alpha anomer of glucosamine are observed in the spectra, and the corresponding peaks are denoted with an asterisk (*).

With these trends established, we next turned our attention to probing the intermolecular interactions between Gluc and Glyc and the impacts of adding Glyc to Gluc/Gluc assemblies. Solutions were prepared of Gluc (12.5 and 50.0 mM) with increasing molar ratios of Glyc, from 1:0.25 Gluc/Glyc to 1:2 Gluc/Glyc, and the solutions were then dried under N2 flow on the ATR crystal and were compared to pure Gluc powder and Gluc films containing no Glyc. As was discussed previously, increasing concentrations of Glyc result in an increase in the OH stretching frequency, and it is therefore difficult to draw molecular-level conclusions about Gluc/Glyc interactions from changes in the OH/NH stretching band. Therefore, we focused on the CN/NH combination band and CO–H bending frequency window of 1700–1450 cm–1 (Figure 4,top). Glyc does have a very weak band in this window at 1651 cm–1; control experiments were performed on neat glycerol and aqueous solutions of glycerol dried onto the ATR crystal at concentration to mimic those experiments performed on the 1:1 mol ratio of the Gluc/Glyc systems (12.5 and 50 mM Glyc) (Figure S5).

Figure 4.

Figure 4

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IR spectral overlay of CN/NH and CO–H combination band region from 1700 to 1450 cm–1 of Gluc powder (blue) and Gluc films with increasing molar ratio of Glyc. Gluc (top: 12.5 mM green, bottom: 50.0 mM fuchsia) with increasing molar ratio Glyc (yellow-1:0.25, light blue-1:0.5, purple-1:1, orange-1:2); for full spectra, see Figures S6–S7.

In the 12.5 mM Gluc experiment, increasing the concentration of Glyc in the film shifts both the CN/NH and CO–H bands to a higher wavenumber compared to the pure 12.5 mM Gluc film (Figure 4, top). This shift toward Gluc powder indicates an increase in hydrogen bonding. The broadness of the peaks remains unchanged, suggesting that there are a number of unique hydrogen bonding states in the system. Interestingly, in the presence of Glyc, both the CN/NH and CO–H frequencies are impacted. In contrast, in the absence of Glyc, the 12.5 mM Gluc film only displays a significant shift in the CO–H bending frequency. The presence of free Glyc cannot be ruled out based on the control experiments; however, the intensity of pure Glyc compared to that of the CN/NH band in the Gluc/Glyc system indicates that these shifts are a result of Glyc interacting with both the OH and NH2/NH3+ functionalities via hydrogen bonding.

In the 50.0 mM Gluc system, a 1:0.25 Gluc/Glyc ratio leads to a loss of resolution of the CN/NH combination band, indicating the Glyc disrupts the ordered structures present at this concentration of Gluc (Figure 4, bottom). There is also a slight shift to a higher wavenumber (centered at 1600–1607 cm–1) of this combination band, suggesting an increase in the overall number of hydrogen bonding states involving the NH2/NH3+ functionality. While a continuous shift to a higher wavenumber is observed in the CN/NH band with increasing Glyc, there is a significant shift in the CO–H band to 1511 cm–1 at the Gluc/Glyc ratio of 1:0.25 (compared to 1531 cm–1 for the pure Gluc film). This shift to a lower wavenumber indicates a disruption of the OH interactions occurring between Gluc/Gluc aggregates. As the molar ratio of Glyc increases, a shift to higher wavenumber is observed for the CO–H band, reaching 1520 cm–1 at the Gluc/Glyc ratio of 1:2 but never returning to the frequency of the pure Gluc film (1531 cm–1). When comparing these results back to the Gluc concentration study, we can infer that at higher concentrations of Gluc, Glyc acts to disrupt Gluc/Gluc hydrogen bonds.

While these IR studies have provided some insights into the association between Gluc and Glyc, there remains uncertainty in the specific binding motifs. Thus, we again turned to NMR to further probe the hydrogen bonding interactions between Gluc and Glyc. Notably, these NMR studies emphasized the importance of water in forming Gluc/Glyc assemblies. Shown in Figure 5 are two 1H NMR spectra of 50.0 mM Gluc with 1 eq. Glyc in DMSO-d6 with differing amounts of water present (see Figure S8 for full spectra).

Figure 5.

Figure 5

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1H NMR of Gluc in DMSO-d6 with high and low water content recorded at 400 MHz (9.4 T). Top: 50.0 mM Gluc with 1 eq. Glyc in DMSO-d6: high water content, [H2O] = ∼0.647 M (1 wt %). Bottom: 50.0 mM Gluc with 1 eq.Glyc in DMSO-d6: low water content, [H2O] = ∼0.073 M (0.1 wt %).

The top spectrum (“high water content”) has clearly defined and well-resolved signals for both Gluc and Glyc. In contrast, in the bottom spectrum (“low water content”), the OH signals for Gluc and Glyc have broadened substantially, and no splitting was observed. Glyc has been known to form extended hydrogen bonding networks, which can be controlled by varying water content in the system.27−31 These complex assemblies have not been investigated with respect to their role and impact in the plasticization process. However, since chitosan films are commonly prepared from aqueous solution, we chose to focus on the “high water content” data as it is the most representative of the conditions present during polymer film formation.

NOESY data were collected at 1 eq. of Glyc for both 12.5 mM and 50.0 mM Gluc. Cross sections (OH3 and NH) of these NOESY spectra are shown in Figure 6 (see Figures S9–S10 for full spectra). At 12.5 mM Gluc and 1 eq. Glyc (Figure 6, top), OH3 has strong positive exchange cross-peaks with NH and OH. Additionally, the cross-peak for OH3–OH6 exchange is significantly weaker than the system without Glyc, and the OH3–OH4 exchange cross-peak is lost entirely. Similar trends were observed with the amine functionality. Although the NH of Gluc originally has only one NOE in this cross section (an intramolecular interaction with CH1), in the presence of 1 eq. Glyc, NH now exchanges with OH1, OH3, OH4, and OH6 of Gluc exclusively and no longer has a NH–CH1 cross-peak. Glyc is also capable of disrupting intramolecular Gluc/Gluc hydrogen bonds when Gluc is already aggregated. At 50.0 mM Gluc and 1 eq. of Glyc (Figure 6, bottom), OH3 loses both exchange cross-peaks designated as intramolecular interactions (i.e., OH3–OH4 and OH3–OH6) while maintaining its intermolecular hydrogen bonds with NH and OH1. Likewise, the NH of Gluc still has exchange cross-peaks with all the OHs of Gluc, suggesting that it participates in intermolecular hydrogen bonding with other Gluc molecules. However, this NH loses its diagnostic cross-peak for intramolecular interactions (NH–CH1, see Figures S9–S10 for full spectra). This indicates that at both low and high concentrations of Gluc, added Glyc disrupts intramolecular Gluc/Gluc interactions and promotes the formation of intermolecular Gluc/Gluc hydrogen bonds (Figure 7).

Figure 6.

Figure 6

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Representative cross sections (OH3 and NH) of the 2D NOESY spectra for 12.5 mM Gluc (top) and 50.0 mM Gluc (bottom) with 1 eq. of Glyc recorded at 400 MHz (9.4 T). Strong interactions are observed between the NH of Gluc and OHs of Glyc at both concentrations. Small peaks for the alpha anomer of glucosamine are observed in the spectra, and the corresponding peaks are denoted with an asterisk (*). These spectra contain ∼0.65 M water.

Figure 7.

Figure 7

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Representative structures of the intra- (blue) and intermolecular (red) interactions present in the isolated Gluc (12.5 mM) and preaggregated Gluc (50.0 mM). In the presence of Glyc, both initial structures lose all intramolecular (blue) interactions to form assemblies containing intermolecular (red) Gluc/Gluc and Gluc/Glyc interactions.

In addition to these Gluc/Gluc interactions, we also observe cross-peaks between Gluc and Glyc. Namely, at both concentrations, all of the OHs and the NH of Gluc have exchange cross-peaks with the OHs of Glyc. Of these cross-peaks, the NH-Glyc cross-peak is the most intense signal, indicating that the amine of Gluc has a particularly strong intermolecular interaction with Glyc. This result is consistent with our observations in the IR experiments, as discussed above. Namely, the CN/NH combination band continuously shifts to a higher wavenumber with increasing amounts of Glyc. From our combined NMR and IR experiments, we can conclude that the Gluc/Glyc assemblies result from prominent hydrogen bonds formed between the NH of Gluc and the OHs of Glyc (Figure 7).

Conclusions

In conclusion, we have demonstrated the ability to differentiate intra- and intermolecular interactions of Gluc, our chitosan model system. The combined IR and NMR spectroscopic studies indicate that Glyc can both bind to Gluc and impede Gluc’s intramolecular interactions. Specifically, data obtained from IR and NMR indicate that increasing amounts of Glyc result in an increase in the number of intermolecular hydrogen bonds in the form of Gluc/Gluc and Gluc/Glyc assemblies. While previous studies of chitosan and glycerol have left the role of the NH functionality poorly defined, our results strongly indicate that the NH functionality is definitively involved in the Gluc/Glyc interactions. Differentiating and deciphering these interactions is critical to understanding the plasticization mechanism of chitosan with Glyc. In the context of this monomeric model system, these interactions can begin to shed light on the mechanism of the plasticization of chitosan with Glyc. For example, if lubricity theory was the dominant mechanism, at high concentrations of Glyc, no Gluc/Gluc interactions should be observed as an isolated Gluc molecule should be completely surrounded by Glyc. However, this is not observed for either concentration of Gluc (12.5 or 50.0 mM) in the presence of Glyc. Similarly, free volume theory can be ruled unlikely because we observe specific hydrogen bonding motifs via the NH while the remaining Gluc/Glyc interactions are less defined. These well-defined hydrogen bonding motifs and the persistence of Gluc/Gluc interactions suggests that gel theory is likely the best model for the plasticization of chitosan. However, as this monomeric model may not reflect the associations in polymeric networks, we will continue to explore the molecular-level interactions in oligomeric Gluc systems. Density functional theory calculations are currently being employed to further explore these mechanisms in more depth. Understanding the molecular-level interactions responsible for beneficial polymer properties will allow for the development of new tunable plasticizers for biopolymeric materials, opening up new possibilities in the applications of these environmentally responsible materials.

Experimental Section

Materials

Chitosan with a degree of deacetylation DD = 75–85% and a molecular weight range of 190,000–310,000 was used as received from Sigma Aldrich. d-Glucosamine hydrochloride (Gluc), glycerol (Glyc), and deuterated NMR solvents were also used as received from Sigma Aldrich.

Film Formation

Chitosan films were prepared by dissolving chitosan powder (0.100 and 0.200 g) in 1% aqueous acetic acid (100.0 mL) to obtain 1 and 2 wt % chitosan solutions, respectively. Glycerol-containing films were prepared in a similar fashion from chitosan powder, 1% aqueous acetic acid (50.0 mL), and a stock solution of glycerol in 1% acetic acid (100.0 mM, 50.0 mL). The resulting solutions contained 1 or 2 wt % chitosan with a final concentration of glycerol of 50.0 mM. All of the solutions were stirred at 80 °C for 1 h to ensure complete homogeneity of the casting solutions. All films were cast into polystyrene dishes using 5 mL of the prepared solutions. The films were dried at 60 °C overnight. Gluc and Gluc/Glyc solutions for IR experiments were prepared from aqueous stock solutions of Gluc (0.100 M), Glyc (0.200 M), and deionized water to a final volume of 1.000 mL.

Infrared and Nuclear Magnetic Resonance Spectroscopy

FT-IR spectroscopy of the chitosan films was performed on a Thermo Scientific Nicolet iS10 spectrometer, equipped with a SMART iTX attenuated total-reflection (ATR) accessory. The FTIR spectra were recorded from 400 to 4000 cm–1 with a resolution of 4 cm–1. For drying experiments, glucosamine and glucosamine/glycerol solutions were added directly to the ATR crystal under constant N2 flow. Spectra were recorded every 1 min until no change was observed in the spectra (see Supporting Information for representative spectra Figures S11–S12). The same FTIR parameters were used for all spectral collections.

Nuclear magnetic resonance (1H NMR, COSY, and NOESY) spectra were obtained using a Bruker AVANCE DPX-400 NMR spectrometer in DMSO-d6; chemical shifts are reported in parts per million downfield from tetramethylsilane (δ scale) (see Figures S13-S17 for full 1H and COSY spectra). NOESY measurements were recorded using the NOESYGPPHPP pulse protocol with a mixing time of 300 ms. Data were obtained with a 90° pulse of 10 μs and a relaxation delay of 2 s. A total of 24 scans were performed with a spectral width of 3998 Hz in each dimension. Cross-peaks for NOE interactions and chemical exchange (EXSY) were distinguished by the phase (i.e., NOE = negative, EXSY = positive).

Acknowledgments

The authors are grateful to James Madison University Department of Chemistry and Biochemistry for their financial support.

Glossary

Abbreviations

Gluc

glucosamine

Glyc

glycerol

IR

infrared

ATR

attenuated total reflectance

NMR

nuclear magnetic resonance

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c03016.

  • Full IR spectra, full 1D NMR spectra, and full COSY and NOESY spectra (PDF)

Author Contributions

B.M.B. and G.M.P. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.

Supplementary Material

ao1c03016_si_001.pdf (656.8KB, pdf)

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