Passage
The protozoan Euglena gracilis expresses glycosyltransferases (enzymes that add carbohydrates to molecules) and glycosidases (enzymes that remove carbohydrates) within its membrane. These enzymes were previously studied using radiolabeled sugars. To avoid the use of radioactive materials, fluorescence-based assays were investigated. Derivatives of the fluorescent molecule coumarin were attached to carbohydrates (Figure 1) for detection in fluorescence assays.Compound 1, a carbohydrate derivative with hydroxyl groups protected by benzoate (Bz) , reacted with Compound 2 to form Compound 3. ¹³C NMR confirmed the formation of Compound 3 by observing a distinct C-H coupling at the anomeric carbon. Hydroxyl deprotection and addition of Compound 4 (a coumarin analogue) gave Compound 5, a fluorescent coumarin derivative. Compound 6, another coumarin derivative, was made following a similar sequence except an additional sugar was attached before the addition of the coumarin group.
Figure 1 Synthesis of fluorescent coumarin derivatives (Note: HCT denotes the fluorescent butyl hydroxycoumarin triazole group.) Small vesicles called microsomes were isolated from Euglena gracilis membranes, and microsomal glycosyltransferase activities were investigated using a hexose donor substrate and either Compound 5 (Figure 2) or Compound 6 (Figure 3) as the acceptor substrate. Thin-layer chromatography (TLC) analysis indicated the formation of a new product in each assay. These products were purified and characterized by liquid chromatography-mass spectrometry (LC-MS) . The molecular ion for each product was subjected to another round of MS, in which individual hexose units (MW) = 162) were cleaved from the coumarin derivatives that were formed during the assay. The MW of hydroxycoumarin triazole (HCT) is 301.
Figure 2 Analysis of Compound 5 as the acceptor substrate: (A) TLC analysis of fluorescent products; (B) Mass spectrum of product A fragmented molecular ion peak
Figure 3 Analysis of Compound 6 as the acceptor substrate: (A) TLC analysis of fluorescent products; (B) Mass spectrum of product B fragmented molecular ion peak
Adapted from: I. M. Ivanova et al., "Fluorescent mannosides serve as acceptor substrates for glycosyltransferase and sugar-1-phosphate transferase activities in Euglena gracilis membranes." Carbohydrate Research. © 2017 Elsevier.
- Based on the passage, which carbon gives a distinct C-H coupling in the ¹³C NMR spectrum that confirms formation of Compound 3?

Question

Passage
The protozoan Euglena gracilis expresses glycosyltransferases (enzymes that add carbohydrates to molecules) and glycosidases (enzymes that remove carbohydrates) within its membrane. These enzymes were previously studied using radiolabeled sugars. To avoid the use of radioactive materials, fluorescence-based assays were investigated. Derivatives of the fluorescent molecule coumarin were attached to carbohydrates (Figure 1) for detection in fluorescence assays.Compound 1, a carbohydrate derivative with hydroxyl groups protected by benzoate (Bz) , reacted with Compound 2 to form Compound 3. ¹³C NMR confirmed the formation of Compound 3 by observing a distinct C-H coupling at the anomeric carbon. Hydroxyl deprotection and addition of Compound 4 (a coumarin analogue) gave Compound 5, a fluorescent coumarin derivative. Compound 6, another coumarin derivative, was made following a similar sequence except an additional sugar was attached before the addition of the coumarin group.

Figure 1 Synthesis of fluorescent coumarin derivatives (Note: HCT denotes the fluorescent butyl hydroxycoumarin triazole group.) Small vesicles called microsomes were isolated from Euglena gracilis membranes, and microsomal glycosyltransferase activities were investigated using a hexose donor substrate and either Compound 5 (Figure 2) or Compound 6 (Figure 3) as the acceptor substrate. Thin-layer chromatography (TLC) analysis indicated the formation of a new product in each assay. These products were purified and characterized by liquid chromatography-mass spectrometry (LC-MS) . The molecular ion for each product was subjected to another round of MS, in which individual hexose units (MW) = 162) were cleaved from the coumarin derivatives that were formed during the assay. The MW of hydroxycoumarin triazole (HCT) is 301.

Figure 2 Analysis of Compound 5 as the acceptor substrate: (A) TLC analysis of fluorescent products; (B) Mass spectrum of product A fragmented molecular ion peak

Figure 3 Analysis of Compound 6 as the acceptor substrate: (A) TLC analysis of fluorescent products; (B) Mass spectrum of product B fragmented molecular ion peak
Adapted from: I. M. Ivanova et al., "Fluorescent mannosides serve as acceptor substrates for glycosyltransferase and sugar-1-phosphate transferase activities in Euglena gracilis membranes." Carbohydrate Research. © 2017 Elsevier.
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Based on the passage, which carbon gives a distinct C-H coupling in the ¹³C NMR spectrum that confirms formation of Compound 3?

Quizplus · Accepted Answer

11ecba2d_12ac_c5ee_a3bf_f5312de28cd9_MD0008_00 Sugars can interconvert between linear and cyclic forms. In the linear form, the carbonyl carbon (anomeric carbon) undergoes intramolecular nucleophilic attack by one of the hydroxyl groups on the sugar to cyclize and form a hemiacetal or hemiketal. The carbonyl oxygen of the anomeric carbon (C-1 in aldoses and C-2 in ketoses) becomes a hydroxyl oxygen in the cyclic form and can form glycosidic bonds with other biological molecules. In both the linear and cyclic forms, the anomeric carbon is characterized as the carbon in a carbohydrate with two bonds to oxygen. The passage states that ¹³C NMR confirmed that Compound 3 was formed because the anomeric carbon has a distinct C-H coupling in the product. The carbonyl carbon (C-1 in aldehydes and labeled IV in the question stem) is bonded to the C-5 hydroxyl oxygen and can be identified as the anomeric carbon because it has two bonds to oxygen. (Choices A, B, and C) These carbon atoms only have one bond to oxygen and do not correspond to the carbonyl carbon in the linear form. Therefore, they cannot be the anomeric carbon and do not show a distinct C-H coupling in the ¹³C NMR spectrum. Educational objective: The carbonyl carbon (C-1 in aldoses and C-2 in ketoses) of a sugar in linear form and the carbon bonded to two different oxygen atoms in the cyclic form of a sugar is known as the anomeric carbon. This carbon is characterized by two bonds to oxygen and can form glycosidic bonds with biological molecules.

Passage The Protozoan Euglena Gracilis Expresses Glycosyltransferases (Enzymes That Add Carbohydrates