Unequivocal Proof of Structure Using NMR Spectroscopy in an

Chapter 11

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Unequivocal Proof of Structure Using NMR Spectroscopy in an Organic Laboratory Project Christopher R. Butler, Allen M. Schoffstall,*,1 and Richard K. Shoemaker2 1Department

of Chemistry and Biochemistry, University of Colorado, Colorado Springs, Colorado 80918, United States 2Department of Chemistry and Biochemistry, NMR Spectroscopy Facility, University of Colorado, Boulder, Colorado 80309, United States *E-mail: [email protected]

This organic laboratory project requires synthesis of an organic azide using an SN2 reaction, followed by either a thermal cycloaddition to form a ketodiester or a Cu(I)-catalyzed cycloaddition giving a 1,4-disubstituted triazole ketoester. Each of these esters is subsequently treated with excess NaBH4 in methanol solvent yielding an alcohol or diol, depending on whether an ester group is reduced or not. Two-dimensional NMR techniques are pivotal in proving the structures of the regioselective cycloaddition product and the reduction products. This project provides an opportunity to introduce the utility of these routine 2D NMR methods. The project, or parts of it, may fit into the latter part of a sophomore organic lab course. The entire project may be more appropriate for an advanced lab course.

© 2016 American Chemical Society Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: First Year and Organic Chemistry Courses Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Introduction Many organic laboratory texts include multi-period projects involving two or more experiments. These projects are often included because they give a student the feeling of being a researcher working on a multi-step sequence of experiments. Sometimes it is possible to also make such projects discovery-based, which adds an additional element of surprise for the student. The project described here fits best during the second half of a two-semester laboratory sequence, after students have been schooled in interpreting many NMR spectra. For some, an advanced lab may be better suited for this project. The experiments are straightforward and require a reasonable time for completion. The experiments may be performed using the types of glassware found in most organic teaching labs. No inert atmospheric conditions are required. It is generally possible for students to characterize their solid products using melting behavior and a combination of IR and NMR spectroscopic analyses. Products prepared via regioselective methods often require more sophisticated analyses to distinguish between structurally similar regioisomers. The NMR spectra useful for structure proof in this project require modern high-field NMR instrumentation on site or within easy access. Also desirable is a local NMR expert who can help to explain details of complex 2D NMR spectra such as Heteronuclear Single Quantum Coherence (HSQC) and Heteronuclear Multiple Bond Coherence (HMBC) spectroscopy. The HSQC experiment is a 2D NMR experiment and is used in the project to tie particular proton resonances to their attached carbons. Two-dimensional HMBC spectroscopy is a method for associating protons through multiple bonds via long range coupling with carbons located two or three atoms away. The spectra obtainable for this project demonstrate the power of modern NMR techniques in facilitating structure proof of possible isomeric reaction products. It will become clear to the student that sometimes it is necessary to acquire more sophisticated NMR spectra than the simple 1H and 13C NMR spectra that appear in introductory organic textbooks. An alternate way for an instructor to use this project is to ask students to do the experiments, isolate and purify the products, and to acquire the usual 1H and 13C NMR spectra. It is left to the instructor how far to proceed with further NMR analysis, one choice being to use the already acquired HSQC and HMBC spectra shown in this chapter. Other adoptive avenues are to have students prepare and characterize only compounds 1 and 2, which illustrates the regioselective reduction of one of the two ester groups or to prepare only compounds 3 and 4 to demonstrate chemoselectivity.

Discussion Cycloaddition reactions and sodium borohydride reductions are two commonly encountered experiments assigned to students in the first-year organic laboratory. These reaction types form the basis of a project outlined in Figure 1. 152 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: First Year and Organic Chemistry Courses Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 1. Synthesis of triazole derivatives. Legend: (a) DMAD, heat; (b) NaBH4, CH3OH; (c) CuSO 4, sodium ascorbate, methyl propiolate, tert-butyl alcohol/water (1:1); (d) NaBH4, CH3OH. The conversion of 2-bromo-1-phenylethanone to an oily 2-azido-1phenylethanone is a modification of a literature preparation (1). The organic azide is then allowed to react with purified dimethyl acetylenedicarboxylate (DMAD) to afford the triazole ketodiester 1. The 4- and 5- carbomethoxy groups are non-equivalent, but are assignable using special NMR spectroscopic techniques. Reduction of 1 with excess sodium borohydride in methanol affords a diol 2 in good yield. Structure proof of 2 requires use of special NMR methods. In separate experiments, the 2-azido-1-phenylethanone is allowed to react with methyl propiolate using Sharpless/Meldal conditions (2, 3), using Cu(I) catalysis. The widely used technique is known to give a 1,4-disubstituted product 3. However, NMR spectroscopic analysis can be used to validate the structural assignment. Attempted reduction of the ketoester 3 results only in reduction of the ketoester 3 to an alcohol 4, leaving the carbomethoxy group at the 4- position untouched. Differentiating 2-azido-1-phenylethanone from 2-bromo-1-phenylethanone is difficult using TLC because the Rf values of the two compounds are very similar. The carbonyl absorptions in the IR spectra are also very close, but each compound shows a single, distinct carbonyl absorption at 1691 cm-1 (bromo compound) and 1692 cm-1 (azide). The azide spectrum shows a strong absorption for the azido group at 2096 cm-1. The 1H NMR spectrum of the bromide shows a methylene chemical shift at 4.5 ppm, whereas the azide methylene chemical shift is at 4.6 ppm. We have found complete conversion to the azido product after the reaction has proceeded for 2 hr. However, the instructor may wish to use a two-fold excess of sodium azide to ensure complete reaction. Monitoring the reaction for preparation of 1 can be done by observing the disappearance of DMAD by TLC. A similar approach is not possible when preparing 3 because methyl propiolate is very volatile and not possible to visualize by TLC. Therefore, it is better to check for absence of the starting azido compound when preparing 3. Students may question why 1 is so easily reduced to 2 since most organic textbooks state that aldehydes and ketones are reduced using NaBH4 and that stronger reducing agents are necessary to reduce esters. They might also ask why only one ester group is reduced. We offer a couple of insights on these issues. Since only the 5-carbomethoxy group of 1 is reduced might suggest that a reactive 153

Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: First Year and Organic Chemistry Courses Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


lactone (5) may be formed as an intermediate leading to reduction of the ester moiety as indicated in Figure 2. Earlier work has shown that 4-aryl-4-oxoesters are reduced to diols by NaBH4 in methanol under conditions where 4-aryl esters are unaffected. Under similar conditions 4-alkyl-4-oxoesters are converted to lactones (4). Alkoxyborate intermediates of the alcohol formed from the ketone reduction have been proposed to facilitate ester reduction in certain ketoesters via Lewis acid activation by the borate on the ester carbonyl group (5).

Figure 2. Formation of a possible intermediate (5) during reduction of (1) to (2).

Reduction of esters appears to be facilitated by an ancillary substituent, such as a carbonyl moiety in 1 or a strategically placed carbonyl group in aromatic esters (4), or an alpha N substituent in peptidyl esters (6). Reduction of ordinary esters without nearby functionality is more successful when a more powerful reducing agent than sodium borohydride is used (4). However, simple esters are known to react with excess NaBH4 (7). Generally, aldehydes and ketones are reduced more readily than esters and an example of a chemoselective reduction applicable to sophomore organic lab curricula has been reported (8).

Synthetic Procedures All melting points were measured with a Mel-Temp Electrothermal apparatus and are uncorrected. IR analyses were performed on a Perkin Elmer Spectrum One FT-IR Spectrometer. Reagent grade DMAD was chromatographed on silica gel using ethyl acetate/hexanes (1:1) and stored in the freezer prior to use to prevent decomposition. All compounds synthesized gave satisfactory elemental analyses. TLC plates were from Agela Technologies Silica MF254 with alumina back. Personal protective equipment (lab coats, nitrile gloves, and goggles) must be worn at all times when handling hazardous chemicals. Avoid contact with skin, eyes, and clothing. Use adequate ventilation. Sodium azide is a rapidly acting, potentially deadly chemical. When mixed with acid, sodium azide changes rapidly to hydrazoic acid, a toxic gas with a pungent (sharp) odor. The mixing of sodium azide with any acidic solution must be avoided at all times. Use of a plastic spoon is advised when measuring out sodium azide. Sodium azide reacts with many heavy metals to form explosive-sensitive compounds. Aqueous sodium azide-containing waste solutions should be collected separately from other chemical wastes and neutralized (9). 154



Preparation of 2-Azido-1-phenylethanone To a 50 mL round-bottomed flask were added 398 mg of 2-bromo-1phenylethanone (2.0 mmol) and 20 mL of tert-butyl alcohol/H2O (1:1), followed by 195 mg of sodium azide (3.0 mmol). The mixture was stirred for 2 hr at room temperature or 0.5 hr at reflux. The organic product was extracted with ethyl acetate (3 x 25 mL). The combined organic extracts were washed with brine and dried over anhydrous sodium sulfate. The drying agent was filtered and the solution was evaporated to dryness in vacuo on a rotary evaporator. Residual solvent was removed by passing a gentle stream of air over the resulting oil for 1 hr. This afforded the desired pure product as a straw-colored oil in 95% yield. This product was stored at 0 °C to prevent decomposition. It was used directly in further experiments. NMR (ppm): 4.6 (methylene signal of the azido compound); FTIR (cm-1): 2096, 1692. (Time: 3-3.5 hr) Preparation of Dimethyl 1-(2-Oxo-2-phenylethyl)-1H -1,2,3-triazole-4,5dicarboxylate (1) To a 50 mL round-bottomed flask were added 338 mg of 2-azido-1phenylethanone (2.1 mmol), 20 mL of tert-butyl alcohol /H2O (1:1) and 284 mg of purified dimethyl acetylenedicarboxylate (2.0 mmol). The reaction mixture was heated at reflux with stirring and monitored by TLC using ethyl acetate/hexanes (1:1). After DMAD was no longer detectable by TLC analysis (ethyl acetate/hexanes (1:1) (~3 hr), the product was extracted with ethyl acetate (3 x 25 mL). The combined organic extracts were washed with brine and dried over anhydrous sodium sulfate. The drying agent was filtered and the solution was evaporated to dryness in vacuo on a rotary evaporator. The resulting crude solid product was recrystallized from ethanol. This afforded the desired pure product, mp 179.6 °C in 84% yield. FTIR (cm-1): 1731, 1698. (Time prior to recrystallization: 4 hr) Preparation of Methyl 1-(2-Hydroxy-2-phenylethyl)-1H-1,2,3-triazole-5(methan-2-ol)-4-carboxylate (2) To a 50 mL round-bottomed flask were added 607 mg of 1 (2.0 mmol), 20 mL of cold methanol and 151 mg of sodium borohydride (4.0 mmol). The reaction mixture was stirred at room temperature and monitored by TLC using ethyl acetate/hexanes (70:30). When 1 was no longer detectable by TLC (~1 hr), the reaction mixture was quenched with 10 mL of saturated aqueous ammonium chloride solution and stirred for 5 min. The reaction mixture was evaporated in vacuo on a rotary evaporator to remove methanol. The resulting aqueous mixture was extracted with ethyl acetate (3 x 25 mL). The combined organic extracts were washed with brine and dried over anhydrous sodium sulfate. The drying agent was filtered and the solution was evaporated to dryness in vacuo on a rotary evaporator. This afforded the pure product in 98% yield, mp 98.5 °C. FTIR (cm-1): 3700-3100 with 3369 and 3313; 1719. (Time: 3-4 hr) 155 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: First Year and Organic Chemistry Courses Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Preparation of Methyl 1-(2-Oxo-2-phenylethyl)-1H-1,2,3-triazole-4carboxylate (3) To a 50 mL round-bottomed flask were added 338 mg of 2-azido-1phenylethanone (2.0 mmol), 20 mL of tert-butyl alcohol /H2O (1:1), 168 mg of methyl propiolate (2.1 mmol) and 40 mg of sodium ascorbate (0.2 mmol). Then 100 µL of a 1.0 M solution of CuSO4 . 5H2O was added. The reaction mixture was heated to reflux with stirring and monitored by TLC using ethyl acetate/hexanes (60:40). When the azido compound was no longer detectable by TLC analysis (~2.5 hrs.), the reaction was quenched with 5 mL of 10% ammonia solution and stirred for 5 min. The solution was extracted with ethyl acetate (3 x 25 mL). The combined organic extracts were washed with brine and dried over anhydrous sodium sulfate. The drying agent was filtered and the solution was evaporated to dryness in vacuo on a rotary evaporator. The product was then recrystallized using an ethanol/water solvent pair. This afforded the pure product, mp 98.5 °C in 86% yield. FTIR (cm-1): 1719, 1700. (Time prior to recrystallization: 4 hr) Preparation of Methyl 1-(2-Hydroxy-2-phenylethyl)-1H-1,2,3-triazole-4carboxylate (4) To a 50 mL round-bottomed flask were added 490 mg of 3 (2.0 mmol), 20 mL of cold methanol and 151 mg of sodium borohydride (4.0 mmol). The reaction mixture was stirred at room temperature and monitored by TLC. Upon completion (~1 hr), the reaction mixture was quenched with 10 mL of saturated aqueous ammonium chloride solution and stirred for 5 min. The reaction mixture was evaporated in vacuo to remove methanol. The resulting aqueous mixture was extracted with ethyl acetate (3 x 25 mL). The combined organic extracts were washed with brine and dried over anhydrous sodium sulfate. The drying agent was filtered and the solution was concentrated to dryness in vacuo on a rotary evaporator. The product was then purified by silica flash column chromatography using ethyl acetate/hexanes (1:1). This afforded the pure product in a 68% yield, mp 85.3 °C. FTIR (cm-1): 3600-3200 with 3401; 1732. (Time prior to chromatography: 2.5 hr)

Experimental NMR Spectroscopy One-dimensional 1H and 13C NMR spectra were acquired, along with twodimensional heteronuclear 1H-13C correlation experiments HSQC (1-bond H-C correlation) and HMBC (multiple-bond H-C correlation). 2D COSY spectroscopy was also used with compounds 2 and 4 to assist in the 1H resonance assignments. Compounds 1 and 3 contain no useful homonuclear proton couplings, other than those within the phenyl ring that can be assigned by inspection. In the spectra shown, gradient-selected 2D experiments were used, along with adiabatic pulses for the HSQC and HMBC, which provides more uniform polarization transfer and less sensitivity to slight pulse-calibration errors; however, any HSQC and HMBC pulse sequence should be suitable to characterize these compounds. All data were processed, assigned, and presented using the Mestrenova 10.1 Software 156



Suite (Mestrelab, Inc.). For simplicity the experiments will hereafter be referred to as simply COSY, HSQC, and HMBC. The NMR spectra presented here were acquired on Varian INOVA spectrometers operating at 400.16 MHz, or 500.37 MHz for 1H observation, using the Agilent VnmrJ 3.2A software; however, any modern NMR spectrometer with 2D NMR capability would be quite suitable for these experiments. To verify this, these experiments were repeated using a 2009 Bruker AVANCE-III 300 instrument with the Topspin 2.1b software, and ICON NMR automation, in less than 1 hr and the data were comparable to those presented here. To observe the long-range correlations from the methyl protons of the methyl-ester to the triazole ring carbons, the delay for the evolution of C-H coupling in the HMBC pulse-sequence was optimized for a long-range 13C-1H coupling constant of 3.0 Hz (vs. the typical default 8.0 Hz value). In the Varian/Agilent VnmrJ software, this parameter is normally called “jnxh”, and the delay is set to 1/2J based on this value. In the Bruker Topspin software, this is a constant normally called “CNST13”, entered as the J-value in Hz. Subsequently a delay (usually d6) will be set to 1/2J (or 0.167 sec for a 3Hz C-H coupling). One should check the user’s manual for their own instrument to verify this parameter for their particular instrument setup. For the data shown here, the samples were generally 15-20 mg, dissolved in 0.6 ml of deuterated chloroform, and experiments were acquired using the parameters below. Bear in mind, however, that any standard acquisition parameters for a properly calibrated modern NMR spectrometer should work just as well. For most samples all 1D and 2D NMR experiments required less than 1 hr to complete, with the 1D 13C NMR being the time-limiting experiment. 1H:

30-degree acquisition pulse of 2.5 μs, 3.0 sec acquisition time, 1.5 sec. relaxation delay, with 16 scans. Total time: less than 2 min. 13C:

45-degree acquisition pulse of 7.0 μs, 1.3 sec. acquisition time, 1.0 sec relaxation delay, broadband 1H decoupling using the standard WALTZ-16 1H decoupling scheme applied throughout the experiment. Between 256-1024 scans were acquired (depending on solubility). Total acquisition time: 10-40 min. 2D COSY: 90-degree pulses of 6.7 μs, 512 points acquired in t2 (0.151 sec acquisition time), 2 scans per increment, 256 FIDs acquired in t1. Processed to 1024x1024 points using linear prediction in F1, and sine-squared apodization functions in both dimensions. Spectrum was presented as magnitude-mode. Total acquisition time: 10 min. 2D HSQC: Pulse Sequence (gHSQCAD) using 146 Hz JCH coupling constant (j1xh). Acquired 2 scans per increment, with 512 points in t2 (0.151 sec acquisition time) and 128 increments in t1. Spectra were acquired using broadband 13C decoupling during f2, using an adiabatic WURST (W40) broadband decoupling scheme. Data was processed and presented in phase-sensitive mode using cosine-squared apodization in both dimensions with linear-prediction to 1024 points in the 13C(F1) dimension. Total acquisition time = 6 min. 157



2D HMBC: Spectra were either acquired using the gHMBC or gHMBCAD (adiabatic) pulse sequences with similar results. Acquired as magnitude-mode data in t2/F2, and phase-sensitive data in t1/F1 using 4 scans per t1 increment, with long-range coupling constant set to either 8.0 Hz (default) or 3.0 Hz (to enhance longer-range, smaller CH couplings). Spectra were also acquired using a 2-step filter (centered around 146 Hz) to minimize contributions from 1-bond CH correlations. No 13C decoupling was performed. Data were processed as magnitude-mode in F2 using a sine-squared apodization function, and phase-sensitive in F1 using a cosine-squared apodization function and zero-filling to 1024 complex points in F1. The magnitude-mode calculation was performed along the F2 dimension after processing. Total acquisition time = 19 min. For each of compounds 1-4, spectra are provided showing peak assignments that indicate how every part of each molecule is unambiguously assigned using 1H, 13C, COSY (when applicable), HSQC and HMBC. The HSQC is performed to provide CH-multiplicity information, as presented the CH2 signals are “down” (blue) and the CH & CH3 signals are “up” (red). Refer to the numbered atoms in the structure shown for each compound. For an introduction on how to analyze and interpret HSQC and HMBC NMR spectra, please refer to Chapter 8 of Volume 1 of this ACS Symposium Series (10).

Spectral Interpretation Compound 1: The 1H and 13C NMR spectra of 1 afford assignment of most elements of the structure. However, the methyl ester groups appear at different chemical shifts due to their chemical non-equivalence. To assign the NMR signals for the C4 and C5 methyl groups and for further NMR analysis of 1 and assignment of all protons and carbons, refer to the numbered structure shown in each figure. The interpretation and assignment of the proton NMR signals in the 1H NMR spectrum (Figure 3) is reasonably straightforward, with H10 and H14 appearing as equivalent doublets, at ~8.0 ppm, integrating to two hydrogens. The triplet at 7.56 ppm, two protons, corresponds to H11 and H13, and the final aromatic triplet integrates as a single hydrogen and is found at 7.69 ppm and corresponds to H12. The singlet at 6.18 ppm is the CH2 (H6), as confirmed by the phase of the signal (blue) in the gHSQC spectrum. The methyls, H18 and H22, remain at 4.01 and 3.91 ppm respectively, and cannot be unambiguously distinguished using the 1D NMR spectrum. The 13C NMR (Figure 4) contains more signals, including the unprotonated carbons. Note that all protonated carbons are assigned directly in the HSQC spectrum, so nothing further needs to be added here (Figure 5). The carbon at 189.24 ppm arises from the ketone carbonyl, and the two carbons at 160.34 and 158.92 ppm represent the ester carbonyls (C15 and C19), but cannot be distinguished using the 1-dimensional data. Likewise, C4, C5 and C9 can be presumed to account for 13C resonances at 140.09, 133.65, and 129.20 ppm respectively; however, without the 2D HMBC (Figure 6), they cannot be unambiguously assigned. 158



Figure 3. 1H NMR spectrum of Compound 1.

Figure 4.

13C

NMR spectrum of Compound 1. 159



Figure 5. HSQC spectrum of Compound 1. (see color insert)

The key to assigning all resonances is to perform the HMBC using a polarization-transfer evolution delay (1/2J) that corresponds to a very weak coupling (i.e., 3.0 Hz). This allows detection of the long-range (4-bond) correlation between the methyl protons of the methyl esters to the ring carbons (C4 and C5). Given that correlations between H6 and C5, C7 and C9 are readily observable, the corresponding long-range correlation between H22 and C5 (and H18 to C4) distinguishes the two similar methyl-ester moieties, and provides a complete, unambiguous assignment of all hydrogens and carbons in this molecule, including C19 vs. C15, and C4 vs. C5. Note that it can be educational to acquire the HMBC using a coherencetransfer delay corresponding to ~8Hz (the default value on most commercial spectrometers), to show that these long-range correlations are not observed (or greatly attenuated) because of the small long-range coupling constant. For this compound we acquired three gHMBC spectra corresponding to 8.0 Hz, 5.0 Hz, and 3.0 Hz (see Figure 6). Note that if the long-range C-H coupling constant is exactly 2x the value used to calculate the evolution delay, the signal would be exactly zero, so this should be avoided.

160 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: First Year and Organic Chemistry Courses Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 6. HMBC spectrum of Compound 1.

Compound 2: We will use the numbered structure here to discuss the assignment of the hydrogens and carbons for 2: The interpretation and assignment of the proton NMR signals in the 1H NMR spectrum (Figure 7) are straightforward; however, in this case the 2D-COSY spectrum (Figure 8) provides some very useful supporting information. It should be noted that the 1H NMR spectrum of this compound is very solvent dependent, with the geminal CH2 protons at H6 and H15 being inequivalent and well-separated in CDCl3, but nearly equivalent in DMSO-d6. Intramolecular hydrogen bonding (most likely between H8-N1 and H16-O18) is the best explanation for this solvent effect. The aromatic region of 2 is quite crowded, with hydrogens H10 and H14 appearing as an equivalent second-order doublet at ~7.44 ppm, integrating to 2 hydrogens. The complex triplet at 7.40 ppm, 2 protons, corresponds to hydrogens H11 and H13, and the final aromatic second-order triplet, integrates as 1 hydrogen is found at 7.35 ppm and corresponds to H12. The broad signal at 5.25 ppm arises from H7, with COSY correlations to the coalesced OH signal (H8,H16), and to both well separated signals from the H6 protons. The signals from the geminal pairs H15’ and H15” (4.98 and 4.82 ppm respectively), and H6’ and H6” (4.66 and 4.48 ppm respectively) are easily identified in the HSQC spectrum, appearing as blue CH2 signals that align with the separated 1H resonances and their commonly shared 13C resonances.




Figure 8. Selected regions of 2D-COSY spectrum of Compound 2.



The 13C NMR spectral assignments (Figure 9) are readily discernible for this compound. Note that all protonated carbons are assigned directly in the HSQC spectrum via their attached protons (Figure 10). As described above, the HSQC spectrum is particularly useful in assigning geminal, diastereotopic CH2 protons to their shared carbon (note again the two pairs of blue cross-correlation signals in the HSQC spectrum). Based on chemical shift, the carbon at 162.1 ppm can be assigned as the ester carbonyl (C17), further supported by a strong HMBC correlation to the methyl protons (H20, Figure 11). Carbon C4 is assigned to the peak at 136.0 ppm, confirmed by the 3-bond HMBC correlations to the H15 protons, as well as the long-range 4-bond correlation to the CH3 (H20) protons due to a very high signal:noise ratio. Carbon C5 at 142.01 ppm may be located through HMBC correlations to both sets of CH2 protons (H6 and H15). Carbon C9 has strong 3-bond HMBC correlations to the aromatic protons H11 and H13, thereby completing all of the 13C assignments.

Figure 9.

13C

NMR spectrum of Compound 2.



Figure 10. 2D HSQC spectrum of Compound 2. (see color insert)

Figure 11. 2D HMBC spectrum of Compound 2. 164 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: First Year and Organic Chemistry Courses Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Compound 3: We will use the numbered structure here to discuss the assignment of the hydrogens and carbons for 3: The interpretation and assignment of the proton NMR signals in the 1H NMR spectrum (Figure 12) is reasonably discernible, with hydrogens 10 and 14 appearing as equivalent, primarily doublets, at ~8.0 ppm, integrating to 2 hydrogens. The apparent triplet at 7.57 ppm, 2 protons, corresponds to hydrogens 11 and 13, and the final aromatic triplet, integrates as 1 hydrogen and is found at 7.70 ppm, corresponding to H12. The singlet at 5.94 ppm is the CH2 (H6), as confirmed by the gHSQC experiment (blue resonance). The CH at position H5 on the triazole ring appears at 8.30 ppm and directly correlates to carbon C5 in the 13C NMR at 129.6 ppm in the 1-bond correlation (HSQC) spectrum , verifying the triazole CH that is distinguishing in 3 (Figure 1). The methyl of the methyl ester, H18, appears a singlet integrating to 3 protons at 3.98 ppm in the 1H NMR spectrum.

Figure 12. 1H NMR spectrum of Compound 3. The 13C NMR spectral assignments (Figure 13) are readily explained, while noting that all protonated carbons are assigned directly via the HSQC correlations to their attached protons (Figure 14), so nothing further needs to be added here. Based on chemical shift, the carbon at 189.3 ppm is the ketone carbon (C7), and there also exists a strong HMBC correlation to equivalent ring protons 10 and 14, as well as to the CH2 hydrogens at position 6 (Figure 15). This confirms the connectivities that are consistent with this part of the overall structure. The carbon peak at 161.03 ppm arises from the ester carbonyl (C18), with a strong HMBC correlation to the methyl protons (H18). Carbon C4 is assigned to the peak at 140.35 ppm, and can be assigned by the very strong correlation to H3 in 165



both the default (Jnxh=8Hz, not shown) and the small-J optimized (Jnxh=3Hz) experiment in Figure 15. As discussed above for Compound 1, we also see a correlation between H18 and C4 in the 3 Hz HMBC experiment shown here.

Figure 13.

13C


Figure 14. 2D HSQC spectrum of Compound 3. (see color insert) 166 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: First Year and Organic Chemistry Courses Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 15. 2D HMBC spectrum of Compound 3 (jnxh = 3 Hz).

Compound 4: We will use the numbered structure shown to discuss the NMR spectral assignment of the hydrogens and carbons for 4. The aromatic region within the 1H NMR spectrum of 4 (Figure 16) poses a challenge, with H10, 11, 13 and 14 presenting as overlapping multiplets, even at 500 MHz. H12 is slightly shifted to lower frequency, and with the help of the HSQC spectrum can be assigned as the multiplet at 7.33 ppm. The 2D-COSY spectrum (Figure 17) is useful in assigning the spin-system from the OH (H8) at 3.65 ppm through H7 at 5.19 ppm, to the non-equivalent pair of H6 hydrogens presenting as apparent doublet of doublets at 4.69 and 4.43 ppm respectively. Of course the CH3 hydrogens at 3.90 ppm can be assigned by inspection.




Figure 17. Selected regions of 2D COSY spectrum of Compound 4.



Figure 18.

13C


The 13C NMR spectral assignments (Figure 18) are complicated by the overlap in the 1H aromatic region, but carbons C5, C7, C6, and C18 are readily assigned via the HSQC spectrum (Figure 19). Also, the HMBC spectrum (Figure 20) allows the correlation of H7 to C10 and 14 (3-bond coupling) providing unambiguous assignment of C10 and 14 at 125.80 ppm. This allows assignment of 128.88 ppm to C11 and 13, leaving C12 at 128.63 ppm. Carbons C9, C4, and C15 may also be assigned via the HMBC spectrum as follows: C15, 161.09 ppm can be assigned by chemical shift and HMBC correlation to H18. C4 at 139.36 ppm is directly assignable via the HMBC correlation to H5. Carbon C9 at 139.82 ppm can be located via many HMBC correlations to the aromatic hydrogens, H7, H6’, H6”, and even to the OH proton (H8). Note also that in Figure 20, the correlation between the CH3 protons (H18) and carbon C4 is not observed because this HMBC spectrum was acquired using an evolution delay corresponding to a 8 Hz CH coupling constant with relatively low signal-to-noise.



Figure 19. 2D HSQC spectrum of Compound 4. (see color insert)

Figure 20. 2D HMBC spectrum of Compound 4 (jnxh=8 Hz). 170 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: First Year and Organic Chemistry Courses Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Conclusions This project requires six laboratory periods for synthesis and characterization using physical properties, such as melting point and TLC behavior, and FTIR spectroscopy. NMR spectroscopy can be worked into the schedule since many of the experiments require two to three hours for the reactions to complete. Purifications of previously synthesized products can also be done during these waiting periods. Obtaining HSQC and HMBC spectra will require assistance of an experienced spectroscopist and NMR instrument user. Spectra are provided here in case it is not feasible to acquire spectra or if time does not allow. In any event, interpretation of the HSQC, COSY and HMBC spectra is provided in this article to serve as a “solutions key”. As noted in the Introduction, the instructor may choose to assign only parts of the synthetic project. We suggest making at least compounds 1 and 2, owing to the unusual ester reduction and the regioselectivity of the reduction. However, an instructor could choose synthesis of only compounds 3 and 4 to demonstrate special NMR techniques for a chemoselective reduction. Either of these two selections would cut the time required for the project to four lab periods. The novelty of this project for the undergraduate student is based upon a combination of modern synthetic procedures and selective reaction pathways, paired with crucial NMR analyses, resulting in precise structure elucidation of the products.

References 1.

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