Reactions of Monosaccharides 12:44 PM 1 Reactions of Monosaccharides Introduction • Even though, monosaccharide sugars are multifunctional compounds, they undergo reactions typical of the functional groups they contain, but with a few modifications brought about by the co-existence of the functional groups in the same molecule. H O OH H OH O HO HO H HO OH OH H OH D-Glucopyranose H OH CH2OH D-Glucose • Most monosaccharides exist in cyclic hemiacetals, yet in solution they are in equilibrium with their open-chain aldehyde or ketone forms. • Thus, monosaccharides undergo most of the usual reactions 12:44 PM of aldehydes and ketones, alcohols and hemiacetals. 2 Epimerization of Monosaccharides • One of the most serious limitations of carbohydrate chemistry is the inability to transform monosaccharide sugars using basic reagents because of the tendency of these reagents to trigger base-catalysed epimerization to epimeric monosaccharides or isomeric ketoses. H O H O H O H O H-OH H O HO H H OH OH HO H HO H HO H H OH H OH H OH H OH H OH H OH CH2OH CH2OH CH2OH D-Glucose Enolate D-Mannose Base-catalysed epimerization of glucose • The proton a to the aldehyde group is reversibly deprotonated resulting in an enolate. Since C-2 is no longer chiral, its stereochemistry is lost. Reprotonation on either 12:44 PM face of the enolate, gives either configuration at this carbon. 3 Isomerization of Monosaccharides • A base-catalysed enediol rearrangement culminates in the migration of the carbonyl group up and down the monosaccharide carbon chain. • If the enolate ion formed by removal of a proton on C-2 reprotonates on the C-1 oxygen, an enediol intermediate results. • Keto-enol tautomerism of the enediol gives D-fructose, a 2- ketose. 12:44 PM 4 Reduction of Monosaccharides •Aldoses and ketoses can be reduced to the corresponding alcohols (polyols), called sugar alcohols or alditols and typically have a sweet taste. •Glucitol, mannitol and xylitol are widely used as sweeteners and moisturizers in a number of cosmetic products. They do not promote tooth decay. • The reaction occurs by reduction of the small amount of aldehyde that is in equilibrium with the cyclic hemiacetal. • As the aldehyde is reduced, the equilibrium shifts to the right, 12:44 PM so that eventually all of the sugar is reduced. 5 Oxidation of Monosaccharides • Since the cyclic hemiacetal forms of sugars are in equilibrium with a small but finite amount of the open-chain aldehyde, they can be easily oxidised to carboxylic acids. • The products are called aldonic acids. Consequently, monosaccharide sugars act as reducing agents. They are often referred to as reducing sugars. • The oxidation of aldoses is so easy that they react with such mild oxidizing agents as: (a)Tollens reagent (Ag+ in aqueous ammonia) (b)Fehling’s reagent (Cu2+ complexed with tartrate ion) (c)Benedict’s reagent (Cu2+ complexed with citrate ion) (d)Oxidases (Enzymes that catalyse oxidation) 12:44 PM 6 Oxidation of Monosaccharides with Tollens Reagent • The Tollens reagent (silver(1)ammonical hydroxide) oxidizes aldehydes to carboxylate ions. • The Ag(I) complex which is soluble in ammonium hydroxide is reduced to metallic silver, which is insoluble in ammonium hydroxide. This results in the formation of a silver mirror on the inside of the test-tube. R O R O + - + Ag + Ag(NH3)2 OH H O– Aldehyde Tollens reagent Acid anion Silver mirror 12:44 PM 7 Oxidation of Monosaccharides with Tollens Reagent • In its open form, an aldose has an aldehyde group, which reacts with the Tollens reagent to give an aldonic acid and a silver mirror. • Sugars that reduce the Tollens reagent are called reducing sugars. 12:44 PM 8 Oxidation of Monosaccharides with Tollens Reagent • The Tollens test cannot distinguish between aldoses and ketoses because the strongly basic solution in which theTollens reagent is dissolved promotes enediol rearrangements. • Under the basic conditions, the open-chain form of a ketose is converted to an aldose, which reacts to give a positive Tollens test. H O H OH COOH CH2OH H O H O + - H OH O H OH Ag(NH ) OH OH 3 2 + Ag HO H HO H HO H HO H Tollens reagent H OH H OH Positive Tollens H OH H OH Test H OH H OH H OH H OH CH OH CH OH CH2OH 2 2 CH2OH D-Glucose D-Fructose Gluconic acid Ketose Aldose 12:44 PM D-Fructose thus gives a positive test with the Tollens reagent 9 Oxidation of Monosaccharides with Fehlings Reagent •Fehling’s solution, a tartrate complex of copper (II) sulphate, has also been used as a test for reducing sugars. • Why does D-Fructose give a positive test with the Fehlings 12:44 PM reagent? 10 Oxidation of Monosaccharides with Benedict’s Reagent •Benedict’s reagent, an alkaline solution of copper (II) sulphate as its citrate complex oxidizes aliphatic aldehydes, aldoses and ketoses to the corresponding carboxylic acid. •In this test, the deep-blue colour of the solution is discharged to give a red precipitate of cuprous oxide, Cu2O. •A carbohydrate that gives a positive test with Benedict’s reagent is termed a reducing sugar because the reduction of the metal accompanies oxidation of the aldehyde group. 12:44 PM 11 Oxidation of Monosaccharides with Benedict’s Reagent • When done quantitatively, this test can be used to estimate the level of reducing sugar (i.e. glucose) in blood or urine. • Diabetics tend to have unusually high glucose levels in their urine and blood and must monitor their blood sugar carefully. • A variety of over-the-counter diagnostic test kits utilizing this reaction are available for those suffering from diabetes mellitus. • Benedict’s solution is the key reagent in the test kit available from drugstores that permits individuals to monitor the glucose levels in their urine. 12:44 PM 12 Enzymatic Oxidation of Monosaccharides • Enzymes, being chiral catalysts, are very specific with respect to the substrates they react with and do heavily discriminate against any other close variants. • For example, the enzyme glucose oxidase isolated from the mould Penicillium notatum is known to catalyze the oxidation of only b-D-glucopyranose to D-glucono-d-lactone. • This enzyme is very specific to the oxidation of the b-anomer of glucose and does not affect the a-anomer. • In spite of this specificity, the reaction is commonly used in the clinical assay for total blood glucose, containing both a- and b- D-glucopyranose. How could this be heavenly possible? 12:44 PM 13 Enzymatic Oxidation of Glucose with Glucose Oxidase • The oxidation of the entire glucose content is possible due to the fact that as b-D-glucopyranose is oxidised by glucose oxidase, more of it is generated from the a-D-glucopyranose component through the equilibrium shown below. OH H OH OH H OH H H H HO O + + H H H OH H HO O HO H OH HO H HO H H O HO OH OH HO H H H OH OH HO H OH H O OH H H H H a-D-Glucopyranose (36%) Open chain form b-D-Glucopyranose (64%) of D-Glucose Glucose Glucose oxidase oxidase OH No reaction H H HO O HO H OH O H Glucono-d-lactone 12:44 PM 14 Enzymatic Oxidation of Glucose with Glucose Oxidase • Glucose oxidase, coupled to a peroxidase reaction that visualizes colorimetrically the formed H2O2, is widely used as a diagnostic tool to quantify the amount of free glucose in sera or blood plasma. • Glucose oxidase converts glucose to gluconic acid and hydrogen peroxide. In the presence of peroxidase and o- dianisidine, a yellow color is generated that can be quantified colorimetrically by spectrophotometry. This forms the basis for the measurement of urinary and blood glucose. 12:44 PM 15 Oxidation of Monosaccharides with Bromine- Water •Bromine-water oxidizes the aldehyde group of an aldose to a carboxylic acid. Bromine water does not oxidize the alcohol groups or the ketoses. •Bromine-water is also acidic and does not cause epimerization or movement of the carbonyl group. •In the acidic media, the sugar exists as cyclic hemiacetals and reactions proceed through the cyclic hemiacetals. Aldehyde Acid H O OH O Br C 2 C (CHOH)n H2O (CHOH)n CH2OH CH2OH Aldose Aldonic acid (glyconic acid) Example O H O OH C C H OH H OH Br2 HO H HO H H2O H OH H OH H OH H OH CH OH CH OH 12:44 PM 2 2 16 D-Glucose Gluconic acid Mechanism of Oxidation of Aldoses with Bromine-Water • Bromine reacts with water to form a mixture of bromic acid and hypobromous acid (a weak acid). • The formation of hypobromous acid proceeds through an electrophilic bromonium species. 12:44 PM 17 Mechanism of Oxidation of Aldoses with Bromine-Water •In the acidic media, the sugar exists and reacts through the cyclic hemiacetals. •The bromonium ion then reacts with the cyclic hemiacetal leading to the formation of the lactone. 12:44 PM 18 Oxidation of Monosaccharides with Bromine- Water COOH CH2OHO O HO O Br d O H OH OH H OH 2 HO H HO HO a HO H b a b O H OH OH H2O OH H OH b-D-Xylopyranose D-Xylono--lactone D-Xylono-d-lactone CH2OH or or D-Xylonic acid D-Xylono-1,5-lactone D-Xylono-1,4-lactone open-chain form •Since the product of bromine-water oxidation is an aldonic acid and no epimerization occurs under these conditions, bromine- water serves as a convenient reagent for the conversion of aldoses to aldonic acids.