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 mass spectrometry data given in the passage, how many hexose units were cleaved from molecular ions of products A and B, respectively?

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.
-Based on the mass spectrometry data given in the passage, how many hexose units were cleaved from molecular ions of products A and B, respectively?

Quizplus · Accepted Answer

11ecba2d_12af_8512_a3bf_3dcb765005b2_MD0008_00 Mass spectrometry (MS) is a technique that ionizes molecules and detects their molecular weight; the ions are accelerated toward a magnet and deflected according to mass. Molecular ions can be broken into fragments, each of which can be analyzed by another round of MS. The ions are then detected and a plot (mass spectrum) of the ion mass abundance vs. the mass-to-charge ratio (m/z) is generated. The mass spectrum can be used to identify the mass of a molecule's fragments by taking the m/z difference between peaks. After the purification of products A and B with LC-MS, the molecular ion products A and B were run through another round of MS and individual hexose units were cleaved by fragmentation. The m/z difference between each major peak in the mass spectra is 162, which corresponds to a single hexose unit. The mass spectrum for the molecular ion of product A has two major m/z peaks (464 and 302), indicating that one hexose unit was cleaved. The mass spectrum for the molecular ion of product B has three major m/z peaks (626, 464, and 302), indicating that two hexose units were cleaved. (Choice A) Because there are more than two peaks with a m/z difference of 162 in the product B mass spectrum, more than one hexose unit must have been cleaved. (Choices C and D) Because there is only one pair of peaks with a m/z difference of 162 in the product A mass spectrum, more than one hexose unit could not have been cleaved. Educational objective: Mass spectrometry is a technique that ionizes molecules in a sample, and the ions can fragment. The ions are accelerated toward a magnet, deflected according to mass, and detected. A plot of ion mass abundance vs. m/z ratio is generated; fragments of the sample can be identified by the m/z difference between two peaks in the mass spectrum.

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