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.
-Consider the cyclic form of the sugar from which Compound 1 is derived. Can this sugar be classified as a pyranose and a hemiacetal?

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.
-Consider the cyclic form of the sugar from which Compound 1 is derived. Can this sugar be classified as a pyranose and a hemiacetal?

Quizplus · Accepted Answer

11ecba2d_12ac_029c_a3bf_87385b37dbd1_MD0008_00 Sugars are linear carbohydrates containing a carbonyl and at least two other carbon atoms bonded to hydroxyl groups. If the carbonyl is an aldehyde (at the end of the chain), the sugar is an aldose; if the carbonyl is a ketone (within the chain), the sugar is a ketose. A linear sugar can cyclize to adopt either a hemiacetal (aldose) or a hemiketal (ketose) form. A hemiacetal originates from an aldehyde and consists of a carbon bonded to a hydroxyl group (-OH), an alkyl group, an -OR group (where R is any carbon chain), and a hydrogen atom. A cyclic sugar can be classified as a furanose or a pyranose based on its structural similarity to the heterocycles furan or pyran, respectively. Sugars that are five-membered rings (four carbon atoms and one oxygen atom) are furanoses, and sugars that are six-membered rings (five carbon atoms and one oxygen atom) are pyranoses. Compound 1 is a six-membered ring containing one oxygen and five carbon atoms; therefore, Compound 1 is derived from a pyranose. The structure from which Compound 1 is derived from is a hemiacetal because C-1 is bonded to two oxygen atoms (a hydroxyl group and an -OR group), a hydrogen atom, and an alkyl group that formed from an aldose. (Choices B, C, and D) Hemiacetals are formed from aldoses whereas hemiketals are formed from ketoses. Pyranoses are sugars that are six-membered rings whereas furanoses are sugars that are five-membered rings. Educational objective: The cyclic structure of a sugar is classified by the size of the ring as well as whether the linear form contains an aldehyde (aldose) or a ketone (ketose). Furanoses are sugars with five-membered rings (four carbons and one oxygen), and pyranoses are sugars with six-membered rings (five carbons and one oxygen). Aldoses cyclize to form hemiacetals, and ketoses cyclize to form hemiketals.

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