Passage
Chloramphenicol (Compound 1) is an antibiotic that acts against gram-positive and gram-negative bacteria by blocking peptidyl transfer, which inhibits protein synthesis by bacterial ribosomes. However, it tastes bitter, so most patients cannot tolerate it. As a result, synthesis of hydroxyl-substituted chloramphenicol analogues has become of interest because ester chloramphenicol analogues taste better than chloramphenicol. Substituents on the primary hydroxyl group of chloramphenicol analogues are typically hydrolyzed quickly in vivo to regenerate Compound 1. Compound 3, a chloramphenicol analogue, was synthesized by the transesterification of Compound 1 and vinyl propionate (Compound 2) , catalyzed by the enzyme Lip_BA lipase (Figure 1) .
Figure 1 Transesterification of chloramphenicol catalyzed by Lip_BAAlthough enzyme catalysis allows for efficient regiospecific and stereospecific bond formation, the reaction conditions for the conversion efficiency of Compound 1 to Compound 3, as well as the purity of Compound 3, must also be optimized. The reaction solvent, temperature, and time were studied to find the conditions that gave the highest conversion rate and purity. Each parameter was altered individually while all others were kept constant, and the enzyme concentration was 4.5 g/L in each trial. The results of this study are shown in Table 1. The purity of Compound 3 was found to be over 90% when 1,4-dioxane was used and over 70% when dichloromethane was used.Table 1 Transesterification Conditions Optimization
The transesterification reaction was monitored by thin-layer chromatography (Figure 2) , and the product was confirmed by ¹H NMR. The ¹H NMR spectra of Compounds 1 and 3 can be distinguished by the shift of H_e and H_f due to the addition of the electronegative ester functional group.
Figure 2 Thin-layer chromatography of reaction mixture
Adapted from F. Dong et al., "Transesterification Synthesis of Chloramphenicol." Molecules. ©2017 MDPI.
-What is the number of signals corresponding to aromatic hydrogen atoms present in the ¹H NMR spectrum of Compound 3?

Question

Passage
Chloramphenicol (Compound 1) is an antibiotic that acts against gram-positive and gram-negative bacteria by blocking peptidyl transfer, which inhibits protein synthesis by bacterial ribosomes. However, it tastes bitter, so most patients cannot tolerate it. As a result, synthesis of hydroxyl-substituted chloramphenicol analogues has become of interest because ester chloramphenicol analogues taste better than chloramphenicol. Substituents on the primary hydroxyl group of chloramphenicol analogues are typically hydrolyzed quickly in vivo to regenerate Compound 1. Compound 3, a chloramphenicol analogue, was synthesized by the transesterification of Compound 1 and vinyl propionate (Compound 2) , catalyzed by the enzyme Lip_BA lipase (Figure 1) .

Passage Chloramphenicol (Compound <strong>1</strong>) is an antibiotic that acts against gram-positive and gram-negative bacteria by blocking peptidyl transfer, which inhibits protein synthesis by bacterial ribosomes. However, it tastes bitter, so most patients cannot tolerate it. As a result, synthesis of hydroxyl-substituted chloramphenicol analogues has become of interest because ester chloramphenicol analogues taste better than chloramphenicol. Substituents on the primary hydroxyl group of chloramphenicol analogues are typically hydrolyzed quickly in vivo to regenerate Compound <strong>1</strong>. Compound <strong>3</strong>, a chloramphenicol analogue, was synthesized by the transesterification of Compound <strong>1</strong> and vinyl propionate (Compound <strong>2</strong>) , catalyzed by the enzyme Lip<sub>BA</sub> lipase (Figure 1) . <strong>Figure 1</strong> Transesterification of chloramphenicol catalyzed by Lip<sub>BA</sub>Although enzyme catalysis allows for efficient regiospecific and stereospecific bond formation, the reaction conditions for the conversion efficiency of Compound <strong>1</strong> to Compound <strong>3</strong>, as well as the purity of Compound <strong>3</strong>, must also be optimized. The reaction solvent, temperature, and time were studied to find the conditions that gave the highest conversion rate and purity. Each parameter was altered individually while all others were kept constant, and the enzyme concentration was 4.5 g/L in each trial. The results of this study are shown in Table 1. The purity of Compound <strong>3</strong> was found to be over 90% when 1,4-dioxane was used and over 70% when dichloromethane was used.<strong>Table 1</strong> Transesterification Conditions Optimization The transesterification reaction was monitored by thin-layer chromatography (Figure 2) , and the product was confirmed by <sup>1</sup>H NMR. The <sup>1</sup>H NMR spectra of Compounds <strong>1</strong> and <strong>3</strong> can be distinguished by the shift of H<sub>e</sub> and H<sub>f</sub> due to the addition of the electronegative ester functional group. <strong>Figure 2</strong> Thin-layer chromatography of reaction mixture Adapted from F. Dong et al., Transesterification Synthesis of Chloramphenicol. Molecules. ©2017 MDPI. -What is the number of signals corresponding to aromatic hydrogen atoms present in the <sup>1</sup>H NMR spectrum of Compound <strong>3</strong>? A) 1 B) 2 C) 3 D) 4

Figure 1 Transesterification of chloramphenicol catalyzed by Lip_BAAlthough enzyme catalysis allows for efficient regiospecific and stereospecific bond formation, the reaction conditions for the conversion efficiency of Compound 1 to Compound 3, as well as the purity of Compound 3, must also be optimized. The reaction solvent, temperature, and time were studied to find the conditions that gave the highest conversion rate and purity. Each parameter was altered individually while all others were kept constant, and the enzyme concentration was 4.5 g/L in each trial. The results of this study are shown in Table 1. The purity of Compound 3 was found to be over 90% when 1,4-dioxane was used and over 70% when dichloromethane was used.Table 1 Transesterification Conditions Optimization

The transesterification reaction was monitored by thin-layer chromatography (Figure 2) , and the product was confirmed by ¹H NMR. The ¹H NMR spectra of Compounds 1 and 3 can be distinguished by the shift of H_e and H_f due to the addition of the electronegative ester functional group.

Figure 2 Thin-layer chromatography of reaction mixture
Adapted from F. Dong et al., "Transesterification Synthesis of Chloramphenicol." Molecules. ©2017 MDPI.
-What is the number of signals corresponding to aromatic hydrogen atoms present in the ¹H NMR spectrum of Compound 3?

Quizplus · Accepted Answer

11ecba2d_129d_5c64_a3bf_13524135fb09_MD0008_00 Proton nuclear magnetic resonance spectroscopy (¹H NMR) detects protons in a molecule when an external magnetic field and radio frequency are applied to a sample. The protons align their magnetic field either with (low-energy α spin state) or against (high-energy β spin state) the external magnetic field. Radio waves excite the protons from the α to the β spin state, and the peaks on the spectrum represent the energy difference between the spin states, known as the effective magnetic field. Hydrogen atoms that are in identical magnetic environments within a molecule exhibit rotational or planar symmetry. These atoms are said to be chemically equivalent because they have the same atoms surrounding them, have the same chemical shift, and appear as a single signal. Compound 3 contains four aromatic hydrogen atoms (H_a-H_d). H_a and H_d are chemically equivalent because they are both adjacent to an aromatic C-NO₂ and an aromatic C-H, which is adjacent to an aromatic carbon with a side chain. Similarly, H_b and H_c are equivalent, being adjacent to identical chemical groups. Because there are two sets of equivalent aromatic hydrogen atoms and no other aromatic protons, there are two aromatic hydrogen signals in the ¹H NMR spectrum. (Choice A) There would be one signal in the ¹H NMR spectrum for the aromatic hydrogens in Compound 3 if the four aromatic protons were all equivalent to one another. (Choice C) There are not three distinct (nonequivalent) aromatic hydrogen atoms in Compound 3; therefore, there cannot be three signals for aromatic protons in the ¹H NMR spectrum. (Choice D) Although there are four aromatic protons in the structure of Compound 3, some are chemically equivalent to one another, so fewer than four signals appear. Educational objective: Hydrogen atoms in a molecule that have the same types of atoms surrounding them and that exhibit rotational or planar symmetry are chemically equivalent and experience identical magnetic environments. Therefore, equivalent hydrogen atoms have the same chemical shift and appear as a single signal in the ¹H NMR spectrum.

Passage Chloramphenicol (Compound 1) Is an Antibiotic That Acts Against Gram-Positive