Thematic Use of Ribavirin To Illustrate NMR Principles and

Chapter 2

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Thematic Use of Ribavirin To Illustrate NMR Principles and Techniques Brant L. Kedrowski* and William F. Wacholtz Department of Chemistry, University of Wisconsin Oshkosh, 800 Algoma Blvd., Oshkosh, Wisconsin 54901, United States *E-mail: [email protected]

The molecule ribavirin serves as an ideal example for illustrating a wide variety of NMR principles and instrumental techniques. Ribavirin was used in a thematic fashion as a recurring example throughout a semester-long interpretive spectroscopy course to introduce new concepts and new experimental techniques in NMR spectroscopy. The topics progressed from the fundamental to the advanced with ribavirin serving as a common thread to tie them together within a familiar molecular framework.

Introduction Teaching NMR spectroscopy in an undergraduate setting typically follows a logical pedagogical progression of concepts from the fundamental to the more advanced. High quality examples are needed at every stage of the process to introduce students to theory and instrumental techniques. Ribavirin has been used previously as an example to illustrate the 1H-1H COSY NMR experiment (1). It is reported here that ribavirin can also serve as an ideal example for numerous topics at all levels of the NMR curriculum. It was used thematically as a recurring example throughout a semester-long interpretive spectroscopy course to introduce new concepts and new experimental techniques in NMR spectroscopy. The goal of using a consistent example across the arc of the undergraduate NMR curriculum was to promote a sense of continuity, to connect unfamiliar new concepts with previously mastered material, and to demonstrate how much can be learned about one molecule’s structure and temperature dependent behavior using the tools of NMR spectroscopy. © 2016 American Chemical Society

Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Ribavirin’s structure and 1H NMR spectrum acquired at 270 MHz are shown in Figure 1. It has a number of structural and spectral features that make it a useful example in NMR education. Ribavirin contains multiple functional groups with protons in diverse chemical environments. It has four contiguous stereogenic centers, indicated by stars (*). All of ribavirin’s protons and carbons are chemically non-equivalent at room temperature, and when d6-DMSO is used as the solvent, the signals in ribavirin’s 1H NMR spectrum are remarkably well-resolved, with the more shielded signals of water and residual non-deuterated DMSO being located away from the ribavirin signals. Furthermore, all split signals appear as first order multiplets. Unfortunately, ribavirin has very low solubility in the most commonly used NMR solvent CDCl3 (30 mg/mL). This makes it straightforward to prepare samples that are concentrated enough to allow both 1H and 13C NMR spectra to be acquired in a timely manner. Furthermore, it is also quite stable in DMSO solution and NMR samples showed no noticeable changes in spectra over multiple months. Therefore, students 18 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


can use the same NMR tube sample over and over again as needed to save on costs. Another useful feature of ribavirin is that it has real world significance and an interesting backstory, which helps to increase student interest. It is a broad-spectrum antiviral agent that is used to treat serious viral infections, particularly in combination with other drugs to treat hepatitis (3–5). Ribavirin was used as an example in the undergraduate NMR curriculum to discuss: 1) The number of unique signals in 1H and 13C NMR spectra 2) Chemical shift differences between the signals of protons with subtle structural differences 3) Observing coupling between OH protons and neighboring CH protons, which is absent in the 1H NMR spectra of many molecules 4) Simple splitting patterns including doublets, triplets and quartets 5) More advanced splitting including a threefold doublet (ddd) and a doublet of triplets (dt), as well as inverted splitting tree analysis of these multiplets 6) Deuterium-proton exchange of the OH and NH protons in ribavirin, including its simplifying effect on the splitting of coupled CH protons 7) Selective decoupling experiments to unambiguously assign the proton signals of ribavirin, and its simplifying effect on the splitting of coupled CH protons 8) 1D NOE difference experiments to study the conformations of ribavirin 9) Temperature dependent NMR experiments to study the restricted rotation about the amide bond of ribavirin, and observe chemical shift temperature dependence of OH and NH protons 10) Proton decoupled carbon NMR and DEPT experiments to distinguish the carbon signals of ribavirin, and 11) Two dimensional NMR experiments such as COSY (1) and HETCOR to further elucidate the connectivity in the molecule

Experimental All spectra were acquired using a JEOL GSX-270 NMR spectrometer and processed using JEOL’s Delta software. Ribavirin was purchased from TCI America. Deuterated DMSO (99.9 atom % D) was purchased from Acros Organics in single use 0.75 mL ampules. To address safety concerns, students were informed that ribavirin is a powerful antiviral drug that can have serious side effects. They were also informed that DMSO solvent provides a vehicle for the drug’s direct entry into the body via skin exposure. Students were therefore required to wear full coverage goggles and disposable gloves when preparing their samples. For perspective, a daily adult dose of ribavirin for the treatment of hepatitis C is between 800-1400 mg (6). The amount used in the experiments described in this manuscript (5 mg) represents less than 1% of this amount of material, meaning that the risk of exposure to ribavirin in these experiments is quite small. 19



Students prepared samples for 1H NMR experiments by dissolving 5 mg of ribavirin in 0.75 mL of d6-DMSO. At this concentration, eight scans were sufficient to obtain high quality 1H NMR spectra. To prepare a sample for 13C NMR experiments, 25 mg of ribavirin was dissolved in 0.75 mL of d6-DMSO. To save on costs, this single sample was shared and used for all 13C NMR experiments. At this concentration, a 1H decoupled 13C NMR spectrum with a good signal to noise ratio was obtained with 128 scans in just over six minutes to acquire. Alternatively, 13C NMR spectra could be obtained at the same ribavirin concentrations used in the proton NMR experiments (5 mg / 0.75 mL), provided enough scans were used. Overnight experiments of 20,000 scans over 16 hours gave excellent results. Several general safety considerations were highlighted in procedures for variable temperature (VT) experiments. These included staying at least 10 °C above the freezing point and at least 10 °C below the boiling point of the NMR solvent, never leaving a VT experiment unattended, always cooling a heated sample back to near room temperature before ejecting it, and turning off the VT controller before leaving the instrument. Additionally, students were advised not to exceed 80 °C in any experiment that used a plastic spinner and air as the spinning and eject gas.

Results and Discussion Chemically Non-Equivalent Protons and Chemical Shifts After a basic introduction to non-equivalent protons and chemical shifts has been covered, using simple monofunctional molecules as examples, ribavirin is introduced as an advanced example. Each of the protons in ribavirin is chemically non-equivalent and generates a signal with a non-equivalent chemical shift. As students work to understand this, they encounter amide protons Hb and Hc that are non-equivalent at room temperature due to restricted rotation about the amide bond. They are also confronted with the non-equivalent diastereotopic methylene protons Hk and Hl, which illustrates how stereogenic centers desymmetrize molecules. Ribavirin contains four methine (CH) protons that all have different chemical shifts, and students may initially struggle to understand why the signal for proton Ha appears far downfield at nearly 9 ppm, which they are more likely to associate with aldehyde protons. They’ll learn that ribavirin’s triazole ring is aromatic, and like benzene, it has a deshielding effect on attached protons. The close proximity of Ha to electron-withdrawing nitrogen atoms further deshields the proton, which explains why it is even further downfield than a typical aromatic proton. To explain why the remaining methine protons Hd, Hh, Hi, and Hj appear at different chemical shifts, students need to consider their proximity to the deshielding aromatic ring and anomeric carbon, which are both located on the right side of the molecule as it is drawn in Figure 1. Students can use the same logic to explain the chemical shift ordering of the three hydroxyl protons He, Hf, and Hg. 20



Spin-Spin Splitting Ribavirin’s 1H NMR spectrum can be used to discuss spin-spin splitting between neighboring protons. An expansion of ribavirin’s 1H NMR spectrum showing splitting of signals d-l is shown in Figure 2. In an interesting coincidence, this splitting starts off simply on the downfield side of the spectrum and becomes more complex and nuanced toward the upfield side of the spectrum. This is convenient from a pedagogical perspective as it allows one to simply move from left to right across the spectrum in a discussion of splitting that progresses from simple examples of the n+1 rule to more complex splitting involving inverted splitting tree analysis. When students study splitting in the 1H NMR spectrum of ribavirin, one of the first things that they’ll observe is that all of the hydroxyl (OH) protons are coupled to neighboring CH protons via three bond coupling.

Figure 2. Expansions of ribavirin’s 1H NMR spectrum showing splitting. Students learn early on in their NMR studies that OH and NH protons often exchange rapidly enough that their proton signals appear as singlets and don’t split the signals of neighboring protons. The splitting observed in ribavirin’s 1H NMR spectrum serves as a good reminder that this isn’t always the case. The coupling of hydroxyl protons is reliably observed in ribavirin dissolved in d6-DMSO and 21 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


does not depend on careful sample preparation to exclude water or other potential exchange catalysts. It is common to observe coupling of hydroxyl protons in d6-DMSO, but much less common in CDCl3. The solvent DMSO forms strong hydrogen bonds with hydroxyl protons, which slows their exchange rate. Also, CDCl3 often contains acidic impurities that act as proton exchange catalysts. In contrast to what is observed for OH protons, the amide (NH) protons in ribavirin Ha and Hb are chemically non-equivalent at room temperature, and do not split each other, appearing as singlets. Signals a-j appear as singlets, doublets, a triplet, and several approximate quartets. These are good examples for illustrating the n+1 splitting rule, which states that a proton coupled to “n” neighbor protons will be split into n+1 peaks. Signals d-g exhibit splitting patterns that closely follow the n+1 rule, giving sharp, symmetrical multiplets with peak area ratios that closely follow Pascal’s triangle. Protons Hh, Hi, and Hj each have three vicinal neighbor protons and give split signals that appear as quartets or apparent quartets. The signal for Hh appears as a slightly distorted quartet with its two middle peaks broadened. This can provide a useful example to illustrate the limitations of the n+1 rule. Proton Hh has three coupled neighbor protons Hd, He and Hi, but these three protons are not identical to one another. The coupling constants between Hh and its coupled neighbors are slightly different at JHh-Hd = 3.8 Hz, JHh-He = 5.7 Hz, and JHh-Hi = 5.1 Hz, which causes the signal for Hh to appear as an imperfect quartet. Signals k and l appear as a threefold doublet (ddd) and a doublet of triplets (dt), respectively, and serve as good examples for discussing more complex splitting. The inset in Figure 2 shows how inverted splitting tree analysis of these signals can be used to determine the coupling constants associated with these signals. A useful protocol for building inverted splitting trees has appeared in the literature (7).

Proton-Deuterium Exchange Experiments Figure 3 shows a deuterium exchange experiment with ribavirin. Structural changes that occur in ribavirin when D2O is added are shown as a scheme at the top of the figure with affected atoms shaded. The middle of Figure 3 shows stacked 1H NMR spectra of ribavirin before addition of D2O below and after the addition of one drop of D2O, with thorough mixing, to the NMR sample above. The signals of exchangeable protons are shaded in the spectra. The bottom of Figure 3 contains an expansion of the upfield portion of the spectrum that shows the splitting of signals h-l before and after D2O addition. By comparing the before and after spectra students observe the disappearance of signals b-c and e-g and can definitively assign these to OH and NH protons. By comparing the splitting in the before and after spectra students also observe that signals h and i, which were initially apparent quartets, have collapsed to triplets. Additionally, signals k and l have each collapsed from a more complex multiplet to a simpler doublet of doublets. They can then conclude that protons Hh, Hi, Hk and Hl must be coupled with exchangeable protons in ribavirin.

22 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 3. Proton-deuterium exchange in ribavirin with affected protons and signals shaded (top and middle). Expansion of 1H NMR showing signals Hh-Hl before and after addition of D2O with splitting changes (bottom). Selective 1H-1H Decoupling Experiments Ribavirin was used to illustrate selective proton-proton decoupling experiments. Students can use these experiments to definitively assign the split signals in the molecule. It is also useful for simplifying complex multiplets to make them easier to analyze. This experiment is nearly identical to a simple single pulse experiment used to acquire a standard 1H NMR spectrum, but it adds continuous irradiation at the frequency of the signal to be analyzed. This homonuclear decoupling saturates the spin states of the targeted nuclei to eliminate their coupling to other nuclei. An example of one such experiment is provided in Figure 4, which shows the selective decoupling of signal g and its effect on signals k and l. The bottom of 23



Figure 4 shows the original 1H NMR spectrum, with a spectrum after irradiation of signal g stacked on top. The upper 1H NMR spectrum was generated by adding a low power, long duration Rf pulse at the specific frequency of signal g. This resulted in the loss of signal g as well as the loss of all couplings associated with it. The affected atoms and signals are highlighted gray in Figure 4. In the upper spectrum, students observe that signals k and l have each collapsed from a more complex multiplet (ddd and dt) to give a simpler doublet of doublets. They can then conclude that Hg must be coupled to both Hk and Hl, which supports the assignments of these signals. The simplified multiplets for signals k and l in the upper spectrum are also much easier for students to analyze. The remaining coupling constants associated with these simplified multiplets can be determined by inverted splitting tree analysis as was described previously.

Figure 4. Selective decoupling experiment example establishing that Hg is coupled to Hk and Hl. The original 1H NMR spectrum is shown below and a spectrum with Hg decoupled is stacked above. Affected protons and signals are shaded gray.

Students were instructed to carry out additional experiments to systematically decouple all of the split signals in ribavirin. This allowed them to definitively assign signals d-l to protons in ribavirin. The unsplit signal Ha can be rigorously assigned based on the proton-deuterium exchange experiments described previously because it is the only non-exchangeable singlet in ribavirin. The remaining exchangeable singlets b and c must therefore, by process of elimination, belong to the NH protons. In this way, students are able to prove that their proton assignments are correct completely through 1D 1H NMR experiments.


NOE Difference Experiments


Conformations about the bond between the ribose and triazole rings of ribavirin can be studied using a one-dimensional NOE difference experiment. Figure 5 shows two conformations of ribavirin that place proton Ha in close spatial proximity to Hg and Hh in the left structure and in close spatial proximity to proton Hd in the right structure.

Figure 5. NOE Difference experiment showing Ha is close in space to Hd, Hg, and Hh. The original 1H NMR spectrum is shown below and an NOE difference spectrum with Ha irradiated is stacked above. Affected atoms and signals are shaded gray. Figure 5 shows the original 1H NMR spectrum on the bottom with an NOE difference spectrum stacked above it. The difference spectrum was generated by acquiring a spectrum with selective irradiation of Ha and then subtracting the original spectrum from the irradiated spectrum. Students observe that the signals shaded in gray have been enhanced in intensity in the difference spectrum. Signals d, g, and h increased in integration intensity by 9.2%, 1.4% and 2.7%, respectively. Signal Hi may also be displaying a very small enhancement, but it is difficult to discern from noise. This experiment provides students with further evidence to confirm their signal assignments in the 1H NMR spectrum of ribavirin. It also offers them some insight into the molecule’s conformational behavior, with both conformations shown in Figure 5 being important. From the enhancement of signal g it is also clear that the hydroxyl group containing proton Hg must be rotated in toward the ribose ring, as shown in Figure 5, rather than outward and away from it. This is likely due to intramolecular hydrogen bonding between Hg and the oxygen within the ribose ring, which is indicated by the dotted lines in Figure 5. 25



Variable Temperature Experiments Dynamic processes in ribavirin can be studied using variable temperature NMR experiments. The restricted rotation about its amide bond affects the signals of Hb and Hc. These are unique well-resolved singlets at room temperature, but coalesce into a single signal at higher temperatures. Students were instructed to study this behavior by varying temperatures incrementally from 22 °C to 80 °C, and the results are summarized in Figure 6. They observed that at room temperature (22 °C) the rate of amide bond rotation is slow on the NMR timescale, leading to unique signals for Hb and Hc. However, heating the sample increases the bond rotation rate, which causes signals b and c to eventually merge to form one broad peak with a flat top at 74 °C. This is the coalescence temperature (Tc) for ribavirin’s amide bond. At higher temperatures this single peak representing Hb and Hc sharpens even more. Once students determined the value of Tc, they were able to calculate the rate of rotation at the coalescence temperature in hertz, as well as the energetic barrier to rotation. The equations and theory associated with this line shape analysis have been described in the chemical education literature (8). Another noticeable trend in the spectra in Figure 6 is that the averaged chemical shift of signals b and c moves significantly upfield with increasing temperature. When students examine the entire spectrum they observe that OH proton signals are shifted in a similar manner. This is a general feature of protons that participate in hydrogen-bonding. As the temperature increases the hydrogen bonds are broken, this causes the OH and NH protons to become more shielded (9).

Figure 6. Variable temperature experiments studying amide bond rotation. The chemical shifts of the CH protons in ribavirin also show temperature dependence. This is observed as a subtle shift downfield with increasing temperature for all CH protons except Ha, which shifts upfield. 26 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Carbon NMR and DEPT Experiments Ribavirin can also be used to illustrate carbon NMR concepts and techniques. All of the carbon atoms in ribavirin are unique and appear as well-resolved signals in its proton-decoupled 13C NMR spectrum, which is shown at the bottom of Figure 7. To assist with assigning the carbon signals in this spectrum, a DEPT 135 experiment (Distortionless Enhancement of Polarization Transfer) provides additional helpful information. It is often the case that 13C NMR and DEPT experiments are conducted earlier in the assignment process; however we chose to introduce these techniques later for pedagogical reasons. In this experiment, CH and CH3 signals appear as positive peaks, CH2 signals appear as negative peaks, and quaternary carbons are absent. The DEPT 135 spectrum of ribavirin is shown in Figure 7 stacked above the 13C NMR spectrum. Students can assign signals A and B to the two quaternary carbons in the molecule. Signal H is also clearly due to the one CH2 in the molecule. The remaining signals C-G are from methine (CH) carbons. Based on its chemical shift, signal C can be safely assigned to the methine carbon of the triazole ring. It is also logical to assume that the next most deshielded signal D is likely the anomeric carbon, and this turns out to be correct.

Figure 7. Proton decoupled 13C NMR spectrum (bottom) and DEPT 135 spectrum (top). However, it is difficult to assign the remaining signals E, F, and G based on chemical shifts alone since they don’t follow the pattern of chemical shifts observed in the proton spectrum. This problem can set the stage for a discussion of two dimensional heteronuclear correlation experiments in NMR, which students can use to definitively assign these signals. It should also be pointed out that other versions of the DEPT experiment exist including DEPT 45 and DEPT 90. 27 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

However, they don’t provide any additional information for ribavirin since there are no CH3 signals to distinguish from CH signals.


2D NMR Experiments Two-dimensional (2D) NMR experiments are among the most powerful structural elucidation techniques covered in the undergraduate curriculum. The first such experiment usually discussed is the proton-proton correlation spectroscopy (COSY) experiment. Ribavirin can be used as an effective example (1) to introduce this technique and begin a discussion of 2D NMR experiments. Students learn how to acquire COSY data, process it, and plot it on an example that they should feel quite comfortable with from previous 1D NMR experiments.

Figure 8. COSY spectrum highlighting correlations between signal l and signals g, j, and k with dotted lines. This is helpful when students first start manipulating 2D NMR data because it is significantly more involved than working with 1D data. The most common way to view COSY data is using a contour type plot as shown in Figure 8. To generate this plot, students need to be able to adjust the threshold of the contours such that all correlations can be seen but spots due to noise are avoided. This is 28 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


accomplished in the Delta software using the level tool. Students also learn to plot high-resolution projections of ribavirin’s 1D 1H NMR spectrum along the X and Y-axes. Once the 1H-1H COSY spectrum has been plotted, students can observe its basic features. This includes a mirror plane of symmetry about its diagonal, which is shown as a dashed diagonal line in Figure 8. Students can also look for correlations as off diagonal spots in the field of the spectrum. An example of one set of correlation spots is highlighted by horizontal and vertical dotted lines between Hl and its coupled neighbors Hg, Hj and Hk. By systematically identifying all such off-diagonal spots the students confirm the signal assignments that they determined using 1D NMR techniques. Students also observe the power of the COSY experiment to provide the same information in one convenient spectrum. In addition to its utility in demonstrating 2D homonuclear NMR correlation spectroscopy (COSY), ribavirin can also be used to introduce 2D heteronuclear NMR correlation techniques. One example is the HETCOR (heteronuclear correlation spectroscopy) experiment. The HETCOR spectrum of ribavirin is shown in Figure 9 with the molecule’s 13C NMR spectrum plotted on the X-axis and its 1H NMR spectrum plotted on the Y-axis.

Figure 9. HETCOR spectrum highlighting correlations between carbon CH and protons Hk and Hl with dotted lines. Proton and carbon signal assignments are shown using lower and upper case letters, respectively. 29 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Students observe that in contrast to homonuclear correlation experiments like COSY, heteronuclear correlation experiments like HETCOR lack spectral symmetry. This leads to all of the spots in the spectrum representing unique correlations between a carbon atom and a proton atom. An example of one set of correlation spots is highlighted in Figure 9 by horizontal and vertical dotted lines between carbon signal H and its coupled proton signals k and l. Interpreting the spots in the HETCOR spectrum allows students to confirm a number of conclusions from their 1D NMR experiments. For example, the assignment of signals b-c and e-g to NH and OH protons in the D2O exchange experiment is confirmed in the HETCOR experiment by the absence of carbon signals correlating to these proton signals. The classification of carbon signals in the DEPT experiment as quaternary, methine, methylene or methyl can also be confirmed in the HETCOR experiment by counting the number of protons correlated to each carbon. Additionally, the HETCOR spectrum provides new information about the assignment of carbon signals D-G. The interesting feature here is the assignment of signals E and G, which are flipped from what might be predicted based on the chemical shifts of the attached protons. Specifically, the more downfield methine carbon E is attached to the more upfield proton j, while the more upfield methine carbon G is attached to the more downfield proton h. Other examples of heteronuclear correlation experiments include HMQC and HSQC, which are the preferred methods for determining one bond correlations between carbon and proton atoms (10). The HMBC experiment (10) can be used to determine longer range correlations between carbon and proton atoms. However, the data from this technique is often not fully analyzed but is used to determine individual assignments when other approaches do not provide a definitive answer.

Conclusions The molecule ribavirin is useful for illustrating numerous concepts and techniques throughout the undergraduate NMR spectroscopy curriculum. It was used at a fundamental level to discuss the uniqueness of atoms, chemical shifts, splitting, and exchangeable protons. At an intermediate level, selective decoupling, NOE difference experiments, variable temperature NMR, and DEPT experiments were illustrated. Finally, advanced 2D NMR experiments including COSY and HETCOR were introduced using ribavirin as an example. While each example is useful on its own, they can be even more effective when used together to provide a molecular theme for the undergraduate NMR curriculum. This strategy provides a thread of continuity across the curriculum and can serve as a conceptual bridge for students as they move through NMR content of increasing complexity. Using ribavirin thematically also allows students to analyze one molecule from multiple experimental angles, which can provide a big picture perspective of the power and versatility of NMR spectroscopy for structural analysis.


Acknowledgments We thank the Department of Chemistry and the College of Letters and Science at the University of Wisconsin Oshkosh for supporting this work. We thank the NSF-ILI Program (USE-9153034) for funds to purchase the JEOL GSX-270 NMR spectrometer used in this study.


References 1.

Williams, K. R.; King, R. W. The Fourier transform in chemistry—NMR: Part 4. Two-dimensional methods. J. Chem. Educ. 1990, 67, A125–A137. 2. There are over one hundred distributors of ribavirin in the US. Examples include Sigma-Aldrich and TCI America. 3. Soota, K.; Mailiakkal, B. Ribavirin induced hemolysis: a novel mechanism of action against chronic hepatitis C virus infection. World J. Gastroenterol. 2014, 20, 16184–16190. 4. Paeshuyse, J.; Dallmeier, K.; Neyts, J. Ribavirin for the treatment of chronic hepatitis C virus infection: a review of the proposed mechanism of action. Curr. Opin. Virol. 2011, 1, 590–598. 5. Harrabi, H.; Maaloul, I. Ribavirin for chronic heptatitus E virus infection. N. Engl. J. Med. 2014, 370, 2446. 6. Merck Corporation. Rebetol (ribavirin) full prescribing information. https:// www.merck.com/product/usa/pi_circulars/r/rebetol/rebetol_pi.pdf (accessed August 26, 2015). 7. Hoye, T. R.; Hanson, P. R.; Vyvyan, J. R. A practical guide to first-order multiplet analysis in 1H NMR Spectroscopy. J. Org. Chem. 1994, 59, 4096–4103. 8. Gasparro, F. P.; Kolodny, N. H. NMR Determination of the rotational barrier in N,N-dimethylacetamide. A physical chemistry experiment. J. Chem. Educ. 1977, 54, 258–261. 9. Huelsekopf, M.; Ludwig, R. Temperature dependence of hydrogen bonding in alcohols. J. Mol. Liq. 2000, 85, 105–125. 10. Acronyms include: HMQC (heteronuclear multiple quantum correlation spectroscopy), HSQC (heteronuclear single quantum correlation spectroscopy), and HMBC (heteronuclear multiple bond correlation spectroscopy). For a discussion of these 2D heteronuclear correlation experiments see: Richards, S. A.; Hollerton, J. C. Essential Practical NMR for Organic Chemistry; Wiley: West Sussex, 2011; pp 130−138.


Thematic Use of Ribavirin To Illustrate NMR Principles and

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