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Home , Aldaric acid, Aldonic acid, Aldose, Osazone

Reactions of

12:44 PM 1 Reactions of Monosaccharides Introduction • Even though, 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- • Most monosaccharides exist in cyclic , yet in solution they are in equilibrium with their open-chain or forms. • Thus, monosaccharides undergo most of the usual reactions 12:44 PM of and , alcohols and hemiacetals. 2 Epimerization of Monosaccharides • One of the most serious limitations of 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 .

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-

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 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- tautomerism of the enediol gives D-, a 2- .

12:44 PM 4 Reduction of Monosaccharides • and ketoses can be reduced to the corresponding alcohols (polyols), called 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 . • 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 has an aldehyde group, which reacts with the Tollens reagent to give an 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 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 . •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 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- 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.

12:44 PM 19 Oxidation of Monosaccharides with Bromine- Water • Because bromine-water oxidizes aldoses but not ketoses, it serves as a useful test reagent for distinguishing aldoses from ketoses. • This difference is conveniently observed in the colour changes that accompany these oxidation reactions. Bromine-water is red in colour, but the product of its reduction is colourless. Consequently, aldoses decolourize bromine-water, while ketoses do not decolourize bromine-water.

12:44 PM 20 Oxidation of Monosaccharides with Nitric Acid

•Nitric acid is a stronger oxidizing agent than bromine water,

oxidizing both the aldehyde group and the terminal –CH2OH group of an aldose to carboxylic acid groups. The resulting dicarboxylic acid is called an . Aldehyde Acid

H OH O OH O C C HO2C O HNO3 OH (CHOH)n (CHOH)n HO OH HO CO2H C CH2OH OH OH O OH Aldose Aldaric acid (glycaric acid or saccharic acid)

•This oxidation proceeds through nitrate ester intermediates formed from the reactive aldehyde group and also the terminal

CH2OH group which is easily accessible to engage in a

12:44 PM nucleophilic attack to an activated nitric acid molecule. 21 Oxidation of Monosaccharides with Nitric Acid Examples

• Note that the oxidation of and yield the same aldaric acid (altraric acid), and so does the oxidation of glucose and , both of which provide glucaric acid. • Note that the aldaric acid assumes, as its derived name, the 12:44 PM name of the aldose that comes first alphabetically. 22 Reaction of Monosaccharides with Phenylhydrazine

• One of the best methods of derivatizing ketones and aldehydes is conversion to hydrazones, especially phenylhydrazones and 2,4-dinitrophenylhydrazones.

R R H H N N + H2O O + H2N N R R1 1 Aldehyde or ketone Phenylhydrazine Phenylhydrazone

•Aldoses and ketoses react with excess phenylhydrazine to form products known as osazones, which contain two phenylhydrazine residues at C-1 and C-2; a third molecule of the reagent is turned into aniline and ammonia.

12:44 PM 23 Reaction of Monosaccharides with Phenylhydrazine • The term osazone is derived from the –ose suffix of a sugar and the suffix of the word hydrazone.

H H O H C + H N N C N NH Ph 3 2 + NH3 + H2N H OH C N NH Ph H OH Phenylhydrazine H OH Phenylamine R R (Aniline) Aldose Osazone

H H CH2OH + H N N C N NH Ph 3 2 + NH3 + H2N O C N NH Ph H OH Phenylhydrazine H OH R R Ketose Osazone

•Sugars that are epimeric at C-2 yield the same osazone. Consequently, the melting points of osazone derivatives are 12:44 PM valuable clues for identification and comparison of sugars. 24 Mechanism of Osazone Formation

12:44 PM 25 Mechanism of Osazone Formation

12:44 PM 26 Chain Shortening of Monosaccharides: Ruff Degradation • The most commonly used method of shortening sugar chains is the Ruff degradation, developed by Otto Ruff, a prominent German chemist.

• The Ruff degradation is a two-step process that begins with oxidation of the aldose to its aldonic acid.

• Treatment of the aldonic acid with hydrogen peroxide and

ferric sulphate oxidizes the carboxyl group to CO2 and gives an aldose with one less carbon.

• The Ruff degradation is used both for structure determination and synthesis of new sugars.

12:44 PM 27 Structure Determination of Monosaccharides: Ruff Degradation

12:44 PM 28 Structure Determination of Monosaccharides: Ruff Degradation •L-sugars are relatively rarer in nature and can only be obtained via synthesis from L-, the most abundant L-sugar in nature. It possesses the correct configuration at its three chiral centres for elaboration to the relatively rare L- and L- .

12:44 PM 29 Chain Extension of Monosaccharides: Kiliani- Fischer Synthesis

•In 1886, Heinrich Kiliani (at the Technische Hochshule in Munich) showed that an aldose can be converted into two diastereomeric cyanohydrins of the next higher carbon number by addition of HCN.

•The resulting diastereomeric cyanohydrins can be partially reduced to imines and then hydrolysed to diastereomeric sugars.

•The Kiliani-Fischer synthesis therefore extends an aldose carbon chain by adding one carbon atom at a time.

•This synthesis is useful both for determining the structure of existing sugars and for synthesizing new sugars.

12:44 PM 30 Chain Extension of Monosaccharides: Kiliani- Fischer Synthesis •L-Arabinose is abundant in nature and possesses the correct configuration at its three chiral centres for elaboration to the relatively rare L-glucose and L-mannose. H NH CHO CN C HO H HO H H2 HO H H2O H OH H OH H OH Pd/BaSO4 HO H HO H HO H HO H HO H HO H CH2OH CH2OH CH2OH L-Glucononitrile L-(-)-Glucose

CHO H OH Imines Epimers HCN Epimeric cyanohydrins HO H HO H

CH2OH H NH C CHO L-(+)-Arabinose CN H OH H OH H OH H OH H OH H OH H2O HO H H2 HO H HO H HO H HO H HO H Pd/BaSO4 CH OH CH2OH CH2OH 2 12:44 PM L-Mannononitrile L-(+)-Mannose 31