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�1. INTRODUCTION �

Carbohydrates are the most abundant biomolecules on earth. In general, carbohydrates are white solids, sparingly soluble in organic solvents, but soluble in water. They are widely distributed in plants and animals. They serve as sources of energy (e.g. from sugars) as well as stores of energy (e.g. starch and glycogen) and are synthesized in plants via photosynthesis. Carbohydrates are components of supportive structures in plants (cellulose), crustacean shells (chitin) and connective tissues in animals [acidic polysaccharide and are essential components of nucleic acids (deoxyribose and ribose)].

Most microorganisms and animals depend on the very existence of carbohydrates produced by plants. Carbohydrates are the fi rst products formed in photosynthesis, from which plants derive their food reserves and other chemical constituents that are used as foodstuff by other organisms. Secondary metabolites are then derived from carbohydrate metabolism. It is worth mentioning that many of the medicinally important secondary metabolites contain signifi cant amount of carbohydrate portion in their structures (e.g. glycosides).

Carbohydrates are defi ned as optically active polyhydroxy aldehydes or ketones or those substances that yield such compounds on hydrolysis. Many, but not all, carbohydrates have the empirical formula (CH

2O)

n (e.g. glucose: C

6H

12O

6; sucrose: C

12H

22O

11). But certain carbohydrates

do not correspond to this formula (e.g. rhamnose: C6H

12O

5; rhamnohexose: C

7H

14O

6; digitoxose:

C6H

12O

4) while some of the noncarbohydrates (e.g. formaldehyde: CH

2O; acetic acid: C

2H

4O

2; lactic

acid: C3H

6O

3; inositol: C

6H

12O

6) correspond to the above formula. Apart from carbon, hydrogen

and oxygen, some of the carbohydrates are found to contain nitrogen, phosphorus or sulphur. The simpler members of carbohydrate family are often referred to as saccharides because of their sweet taste (Latin word saccharum means ‘sugar’).

Earlier, carbohydrates were defi ned as compounds containing carbon, hydrogen and oxygen. As hydrogen and oxygen are present in the same ratio as in water, they were regarded as hydrates of carbon (carbo = carbon; hydrate = adding water).

Six-carbon sugars (hexoses) and fi ve-carbon sugars (pentoses) are the most important carbohydrate units (monosaccharides) in nature. Initially, a three-carbon sugar

Carbohydrates

��2

CHAPTER

Chapter-02.indd 11Chapter-02.indd 11 6/2/2012 5:24:13 PM6/2/2012 5:24:13 PM

12 �� Pharmaceutical Chemistry of Natural Products

3-phosphoglyceraldehyde is formed during photosynthesis, two molecules of which combine to synthesize glucose-6-phosphate by a sequence of reactions. By the complex reactions of the Calvin cycle, 3-phosphoglyceraldehyde is used in the formation of the pentoses such as ribose-5-phosphate, ribulose-5-phosphate and xylulose-5-phosphate by adopting fundamental biochemical reactions.

Photosynthesis H

CH OPO2 4

OH

CHOOH

H

OH

OH

H

HO

H

H

CHO

CH OPO2 4

D-Glucose-6-phosphate

D-3-Phospho glyceraldehyde

Calvin cycle

-Xylulose-5-phosphateDD-Ribose-5-phosphate D-Ribulose-5-phosphate

H

H

H

OH

OH

OH

CH OPO2 4

CHO CH OH2

CH OPO2 4

H

H

OH

O

OH

CH OH2

CH OPO2 4

HO

H OH

O

H

In the nomenclature of carbohydrates, the suffi x -ose is used for naming sugars. The portion of the name preceding this often refl ects the origin and history of the compound [e.g. lactose (milk sugar) is derived from the Latin word lactis which means milk].

1.1 Physiological Importance of Carbohydrates 1. Carbohydrates serve as a chief source of energy. 2. They are constituents of compound lipids and conjugated proteins. 3. The degradation products of carbohydrates act as ‘promoters’ or ‘catalysts’. 4. Certain derivatives of carbohydrates such as cardiac glycosides (e.g. digoxin, digitoxin)/

antibiotics (e.g. aminoglycosides) are used as drugs. 5. Derangement of glucose metabolism is seen in diabetes mellitus. 6. Polysaccharide glycogen found in liver and muscle cells is a polymer of glucose—a stored

form of energy.

�2. CLASSIFICATION OF CARBOHYDRATES �

Carbohydrates may be classifi ed into two types: sugars and nonsugars (polysaccharides).


Carbohydrates �� 13

Sugars: They are water soluble, sweet and crystalline substances (e.g. glucose, fructose and sucrose). They are further classifi ed into two types.

(a) Monosaccharides (b) Oligosaccharides

Monosaccharides: The term monosaccharide means ‘polyhydroxy aldehydes’ or ‘ketones’, which cannot be hydrolysed to simpler sugars. Their general formula is C

nH

2nO

n. The hexoses, the aldohexose

D-glucose and the ketohexose D-fructose, are the most common monosaccharides in nature. The aldopentoses, D-ribose and 2-deoxy-D-ribose, are components of nucleotides and nucleic acids.

Monosaccharides of more than four carbons tend to have cyclic structures. Monosaccharides are further subdivided according to the nature of the carbonyl group as follows:

(i) Aldoses: Monosaccharides with aldehyde group. (ii) Ketoses: Monosaccharides with keto group.

Based on the number of carbon atoms in the molecule, aldoses and ketoses are further divided as shown in Table 1.1.

Table 1.1 Classifi cation of aldoses/ketoses

Aldose/ketose (Generic names) Number of carbon atoms Example (found in humans)

Trioses 3 Glyceraldehyde

Tetroses 4 Erythrose

Pentoses 5 Ribose

Hexoses 6 Glucose

Heptoses 7 Sedoheptulose

Nonoses 9 Neuraminic acid

Oligosaccharides: The term oligosaccharide is derived from the Greek words oligos (means ‘a few’) and sacchar (means ‘sugar’). They consist of short chains of monosaccharide units, or residues, linked by characteristic linkages called glycosidic bonds. These carbohydrates yield 2–10 monosaccharide molecules on hydrolysis, and therefore, they are further classifi ed into various types based on the number of monosaccharide units formed on hydrolysis.

(i) Disaccharides: Disaccharides (e.g. maltose, lactose and sucrose) consist of two monosaccharides linked covalently by an O-glycosidic bond, which is formed when a hydroxyl group of one sugar reacts with the anomeric carbon of the other.

These sugars on hydrolysis give two molecules of monosaccharides. For example, sucrose yields one molecule of glucose and fructose, while maltose on hydrolysis yields two molecules of glucose.

(ii) Trisaccharides: They yield three molecules of monosaccharides on hydrolysis (e.g. raffi nose, C

18H

32O

16). Most oligosaccharides consisting of three or more units do not occur as free

entities but are linked to nonsugar molecules (lipids or proteins) in glycoconjugates. (iii) Tetrasaccharides: They yield four molecules of monosaccharides on hydrolysis (e.g. stachyose,

C24

H42

O21

).

Nonsugars or polysaccharides: These types of carbohydrates consist of large number of monosaccharide units bonded together by glycosidic bonds, which yield a large number of monosaccharide units upon hydrolysis. Chemically, they are long chain or polymers of monosaccharides. Some



polysaccharides, such as cellulose, are linear chains, while others such as glycogen are branched. Both glycogen and cellulose consist of recurring units of D-glucose, but they differ in the type of glycosidic linkage and therefore, they have different properties and biological roles. According to the type of monosaccharide unit present in polysaccharides (single type or different types of monosaccharides), they are further divided as homopolysaccharides (e.g. starch, cellulose and inulin) and heteropolysaccharides (e.g. chondroitin sulphate A and heparin).

Starch is the major form of stored carbohydrate in plant cells. Its structure is identical to glycogen, except for a much lower degree of branching (about every 20–30 residues). Unbranched starch is called amylose and branched starch is called amylopectin.

The above classifi cation of the carbohydrates is schematically represented as given:

Carbohydrates

Sugars Nonsugars (Polysaccharides)

Homopolysaccharides HeteropolysaccharidesMonosaccharides

Aldoses Ketoses Disaccharides Trisaccharides Tetrasaccharides

Oligosaccharides

Carbohydrates may also be classifi ed as (1) reducing sugars and (2) nonreducing sugars. This classifi cation is based on the ability of the carbohydrate to reduce Fehling’s solution and Tollens’ reagent.

�3. QUALITATIVE CHEMICAL TESTS FOR CARBOHYDRATES �

Molisch’s Test (general test): To 3 mL of aqueous extract add few drops of α-naphthol solution in alcohol, shake and add concentrated sulphuric acid along the sides of the test tube. Violet- or purple-coloured ring is formed between the junctions of the two liquids.



3.1 Tests for Reducing Sugars

1. Fehling’s test: Mix 1 mL of Fehling’s solution A and 1 mL of Fehling’s solution B, and boil it for 1 min. To this mixture, add an equal volume of test solution. Heat the contents on a boiling water bath for 5–10 min. A yellow precipitate followed by brick red precipitate is formed.

2. Benedict’s test: Mix an equal volume of Benedict’s reagent and test solution in a test tube. Heat the contents on a boiling water bath for 5 min. The solution appears green, yellow or red depending on amount of reducing sugar present in the test solution.

3.2 Tests for Monosaccharides

Barfoed’s test: Mix equal volume of Barfoed’s reagent (potassium cupric tartrate) and test solution. Heat it for 1–2 min on a boiling water bath and cool. Red-coloured precipitate is formed.

3.3 Test for Pentose Sugars

1. Bial’s orcinol test: To boiling Bial’s reagent add few drops of test solution. Green or purple colour appears.

2. Aniline acetate test: Boil the test solution in a test tube. Hold a fi lter paper soaked in aniline acetate on the vapour. Filter paper turns pink.

3. Mix equal amount of test solution and hydrochloric acid, heat it and add a crystal of phloroglucinol. Red colour appears.

3.4 Tests for Hexose Sugars

1. Seliwanoff’s test (for keto hexose such as fructose): Mix 3 mL of Seliwanoff’s reagent and 1 mL of test solution and heat it on a water bath for 1–2 min. Red colour is formed.

2. Tollens’ phloroglucinol test for galactose: Mix 2.5 mL of concentrated hydrochloric acid and 4 mL of 0.5% phloroglucinol, add 1–2 mL of test solution and heat it on a water bath. Yellow to red colour appears.

3. Cobalt chloride test: Mix 3 mL of test solution with 2 mL of cobalt chloride, boil and cool. To this add few drops of sodium hydroxide solution. The solution appears greenish blue (indicating the presence of glucose) or purplish (indicating the presence of fructose) or upper layer greenish blue and lower layer purplish (indicating a mixture of glucose and fructose).

3.5 Tests for Nonreducing Sugars

1. Test solution does not give response to Fehling’s and Benedict’s reagents. 2. After hydrolysis, test solution gives response to Fehling’s and Benedict’s tests.

3.6 Tests for Nonreducing Polysaccharides (Starch)

1. Iodine test: Mix 3 mL of test solution and few drops of dilute iodine solution. Blue colour appears; the colour disappears on boiling and reappears on cooling.

2. Tannic acid test for starch: With 20% tannic acid, test solution forms precipitate.

Each class of carbohydrates is discussed in detail in the following sections.



�4. MONOSACCHARIDES �

Monosaccharides are colourless, crystalline solids, freely soluble in water but insoluble in nonpolar solvents. Most of them have a sweet taste. The monosaccharide molecules have unbranched carbon chains in which all the carbon atoms are single bonded. However, one of the carbon atoms is double bonded to an oxygen atom in the open-chain to form a carbonyl group, while the other carbon atoms have a hydroxyl group. When the carbonyl group is at the end of the carbon chain and has an aldehyde group, the monosaccharide is an aldose. But if the carbonyl group is found within the carbon chain with a ketone group then the monosaccharide is called as ketose.

4.1 Configuration of Monosaccharides

Monosaccharides are classifi ed according to the nature of the carbonyl group and the number of carbon atoms. All monosaccharides have one or more asymmetric (chiral) carbon atoms and thus occur in optically active isomeric forms. The simplest monosaccharide, aldose glyceraldehyde, contains one chiral centre and therefore has two different optical isomers or enantiomers. By convention, one of these two forms is designated as D-isomer and the other as L-isomer.

Aldobiose

CHO

CH OH2

CHO

CHOH

CH OH2

CHO

(CHOH)2

CH OH2

CHO

(CHOH)3

CH OH2

CHO

(CHOH)4

CH OH2

C = O

(CHOH)3

CH OH2

CH OH2

Aldotriose Aldotetrose Aldopentose Aldohexose Ketohexose

The general formula is 2n, where n is the number of dissimilar asymmetric carbon atoms used to indicate the number of optically active stereoisomers for different types of aldoses and ketoses. Monosaccharides may have two or more than two stereoisomers due to different confi gurations (e.g. aldotriose having one chiral carbon atom may have two isomers, viz. D-and L-glyceraldehyde).

OH

D-Glyceraldehyde L-Glyceraldehyde

CHO

CH OH2

H

CHO

OH

CH OH2

H

Rosanoff in 1906 studied the confi guration of carbohydrates by taking the simplest sugar, i.e. glyceraldehyde (aldotriose) as the arbitrary standard. The glyceraldehyde molecule having hydroxyl group (OH group) at the right side of the penultimate carbon atom (last but one carbon atom) belongs to D-series, while the one having OH group to the left side is L-series. Thus, D and L indicate the stereochemical relationship but the actual rotation of optical activity is indicated by the signs + and –. Thus, when glucose is mentioned as D (+) glucose, it means the sugar belongs to D-glyceraldehyde confi guration with the dextrorotatory optical activity.

It is inconvenient to use the general R/S system of nomenclature for carbohydrates. For example, D (+) glucose must be mentioned as 2(R), 2(S), 4(R), 5(R), 2, 3, 4, 5, 6-(+) pentahydroxyhexanal. Despite the limitations of D and L system of nomenclature, it is still used in the designation of carbohydrates.



HO

HO

HH OH

OH

HH

CH

O

CH

OH

2

HO

HO

HO

HHH OH

H

CH

O

CH

OH

2

AL

DO

SE

Ery

thro

se

D-F

AM

ILY

OF

SU

GA

RS

CH

OK

ET

OS

E

Gly

coal

deh

yd

e

CH

OH

2

Gly

cera

ldeh

yde

CH

O

C CH

OH

2

OH

H

CH

O

C C

OH

H

OH

H

CH

OH

2

Rib

ose

CH

O

C C

OH

H

OH

H

C CH

OH

2

OH

H

Ara

bin

ose

CH

O

C C C

HO

H

HO

H

HO

H

CH

OH

2

Th

reo

se

CH

O

C C CH

OH

2

HO

H

HO

H

Xyl

ose

HO

CH

O

C C

OH

H

H

C CH

OH

2

OH

H

Lyx

ose

HO

CH

O

C C

HH

O

H

C CH

OH

2

OH

H

H OH

OH

OH

HO H H H

CH

O

CH

OH

2

OH

H OH

OH

H

HO H H

CH

O

CH

OH

2C

HO

H2H H O

H

OH

HO

HO HH

CH

O

OH

OH

OH

OH

H H H H

CH

O

CH

OH

2

OH

OH

HH

HO

CH

OH

2

CO

CH

OH

2 H

OH

H

HO

HO

CH

OH

2

CO

CH

OH

2 H HO

H

OH

OH

HHH

CH

OH

2

CO

CH

OH

2

OH

OH

HH

HO

CH

OH

2

CO

CH

OH

2 H

Ery

thru

lose

CO

C CH

OH

2

OH

H

CH

OH

2

HO H

CO

CH

C CH

OH

2

OH

CH

OH

2

Xyl

ulo

se

H

CO

CO

H

C CH

OH

2

OH

H

CH

OH

2

Rib

ulo

se

OH

OH

H OH

H H

HO H

CH

O

CH

OH

2

HO

HO

OH

HH OH

H H

CH

O

CH

OH

2

Dio

se

Trio

se

Tetr

ose

Pen

tose

Hex

ose

Allose

Alt

rose

Glu

cose

Man

nose

Gu

lose

Idose

Gal

acto

seTa

lose

Allu

lose

Fru

tose

Sorb

ose

Taga

tose

CO

CH

OH

2

CH

OH

2

Dih

ydro

xya

ceto

ne



4.2 Ascending and Descending in Monosaccharides

It is possible to convert one monosaccharide into another by this. It is important in the carbohydrate chemistry due to the following reasons:

1. These are used in the determination of relative confi gurations of monosaccharide. 2. These also provide route for the synthesis of rare compounds and compounds that are not

yet known.

Methods of ascending in sugar series

The following are the methods employed to convert sugars into their higher homologues.

1. Kiliani’s synthesis: This is one of the most important synthetic methods for the preparation of sugars and is used to prepare aldoses up to 10 carbon atoms. In this the aldose is fi rst treated with hydrogen cyanide and the cyanohydrin formed is hydrolysed to the higher aldonic acid. This is then converted into two isomeric sugars by lactonization followed by reduction with Na–Hg.

CHO

(CHOH)3

CH OH2

*CHOH

(CHOH)3

CH OH2

CN

*CHOH

(CHOH)3

CH OH2

COOH

*CHOH

(CHOH)3

CH OH2

CHO

HCN (i) Ba(OH)2

(ii) H SO2 4

(i) Lactonization

(ii) Na–Hg

Arabinose Glucose + Mannose

As the addition of hydrogen cyanide to an aldose introduces a new chiral carbon atom, two cyanohydrins and hence two isomeric higher aldoses are formed. But, in practice, only one cyanohydrin predominates because a new chiral centre is introduced into an already chiral molecule.

2. Kochetkov method: In this method, the aldose is condensed with carbethoxymethylene phosphorane (Wittig reaction) and the product is further treated suitably to obtain sugars with two carbon atoms more.

CHO

CHOH + Ph P=CH.COOC H3 2 5

Carbethoxy methylenephosphorane

COOC H2 5

CH

CH

CHOH

Wittigreaction

(–Ph P=O)3

OsO4

HClO4

COOH

CHOH

CHOH

CHOH

[O]CO

CHOH

CHOH

CH

O

Na–HgCHO

CHOH

CHOH

CHOH

�-Lactone

3. Sowden or nitromethane synthesis: This method is based on the condensation of aldose with nitromethane in the presence of methanolic sodium methoxide to form 1-nitro-1-deoxy compound, which is separated and converted into corresponding sodium salts. The sodium salts on hydrolysis with cold dilute sulphuric yield the higher sugar.



OHH

CHO

CH NO3 2+CH ONa3

OHH

CH NO2 2

*CHOH

OHH

CH

CHOHNaOH H SO2 4

OHH

CHO

CHOH

NO Na2

Methods of descending in sugar series

The following are the methods employed to convert sugars into their lower homologues, which involves removal of C

1 and conversion of C

2 to an aldehyde group. The various methods used for

degrading the aldoses by one carbon atom are given below. All these methods start from aldoses. In the same way, these methods can also be applied to descending the ketoses but it must be fi rst converted into the corresponding aldose.

1. Wohl’s method: This is one of the commonly applied methods for shortening the sugar into its lower homologue and involves the heating of aldose oxime with acetic anhydride in presence of zinc chloride or sodium acetate. In this method, the oxime group is dehydrated to the nitrile group with the simultaneous acetylation of the hydroxyl groups. The acetylated nitrile compound is warmed with ammonical silver nitrate solution, the nitrile group is eliminated as silver cyanide and the acetyl group as acetamide with the formation of a new aldose.

CHOH

(CHOH)3

CH OH2

CHO

H NOH2

CHOH

(CHOH)3

CH OH2

CH

AgNOAc O/ZnCl

3

2 2

( H O)2�

NOH

CHOAc

(CHOAc)3

CH OAc2

C N Ag (NH ) OH3 2

AgNO NH3 3�

�AgCN

CH(OH)2

(CHOH)3

CH OH2

�H O2CHO

(CHOH)3

CH OH2

Aldohexose Aldoxime Aldopentose

The newly formed aldopentose actually condenses with the by-product acetamide to yield a diacetamido derivative, which is then hydrolysed to the aldehyde, i.e. aldopentose. However, if sodium ethoxide is used in place of the ammonical silver nitrate solution, the aldopentose is obtained directly.

CHO

(CHOH)3

CH OH2

Aldopentose

2 CH CONH3 2

CH(NHCOCH )3 2

(CHOH)3

CH OH2

Diacetamido derivative

HOH

dil HCl

CHO

(CHOH)3

CH OH2

Aldopentose

2. Ruff’s method: As the Kiliani–Fischer synthesis is used for the synthesis to obtain higher homologue sugars, the Ruff’s degradation method can be used to shorten the chain. The steps involved are as follows:

(a) Oxidation of aldose to the corresponding aldonic acid electrolytically or using bromine water.



(b) Oxidative decarboxylation of the aldonic acid to its corresponding lower homologue sugar, which is performed by treating the aldonic acid with calcium acetate to form a calcium salt, which is then oxidized by hydrogen peroxide in the presence of ferrous salt as a catalyst (Fenton’s reagent: H

2O

2 + Fe3+) to the α-keto acid which is readily decarboxylated

to an aldose.

CHO

(CHOH)4

CH OH2

COOH

(CHOH)4

CH OH2

Br -water2 lime

water

COO�

(CHOH)4

CH OH2

H O –Fe2 23+

C

(CHOH)3

CH OH2

O

COOH

HeatCHO

(CHOH)3

CH OH2

Aldohexose Aldonic acid Calcium aldonate Keto acid Aldopentose

( CO )� 2

Ca2+

3. Weygand’s method: Aldose oxime is reacted with 1-fl uoro-2,4-dinitrobenzene and the resultant product is degraded with warm sodium bicarbonate.

CHO

CHOHH NOH2

CH

CHOH

NOH

O N2

F NO2

Na CO2 3

HC

CHOH

N O

NO2

NO2

Warm withaq. NaHCO3

NO2

HO NO +2 HCN+CHO

�HF

Aldose with 1carbon lesser

2,4-Dinitrophenol

4.3 Chemical Reactions of Monosaccharides

The monosaccharides are either aldehydes or ketones with two or more hydroxyl groups; the six-carbon monosaccharides, glucose and fructose, have fi ve hydroxyl groups. They answer most of the reactions but not all the reactions of the carbonyl group as well as of the alcoholic group. In addition, they may give typical reactions as it contains alcoholic and carbonyl group in the same molecule. As glucose and fructose are the most important examples of aldoses and ketoses, respectively, the properties will be studied with their reference.

1. Epimerization: Epimers are the optical isomers, which differ in the confi guration at only one asymmetric carbon atom (generally α-carbon, i.e. carbon 2). Glucose and mannose are examples of epimers.

The process of inversion of confi guration at one asymmetric centre, i.e. converting one epimer into another in a molecule containing several asymmetric centres is known as epimerization.

In this method, aldose is converted into its epimer via aldonic acid. Aldose is fi rst oxidized to aldonic acid, then the aldonic acid is heated to about 150°C in aqueous pyridine or quinoline (to prevent the formation of lactone of the aldonic acid) to yield an equilibrium mixture of



the original acid and its epimer. The two epimeric acids are separated and converted into their δ-lactones and then reduced to the original aldose and its epimer. Epimerization proceeds similar to that of racemization. The epimerization of glucose to mannose is depicted below:

O

CHO

(CHOH)3

OHH

CH OH2

D(+) Glucose�

Brominewater

COOH

(CHOH)3

OHH

CH OH2

Gluconic acid

COOH

(CHOH)3

HOH

CH OH2

PyridineC

(CHOH)3

OHH

CH OH2

O

O� �H+

C

(CHOH)3

OH

CH OH2

O�

�C

C

(CHOH)3

OH

CH OH2

O�

O�

C�H+

�H–

Mannonic acid

(i) Evaporation

(ii) Reduction

CHO

(CHOH)3

HHO

CH OH2

D(+) Mannose�

�H+

2. Reduction: Methods adopted for reducing sugars are electrolytic reduction, high-pressure hydrogenation over a nickel or copper chromite catalyst, or sodium borohydride. By these methods, sugars are reduced to their corresponding alditols.

On such reductions an aldose gives only one type of alcohol (glucose gives sorbitol and mannose gives mannitol), but a ketose gives a mixture of two alcohols (fructose gives a mixture of two alcohols, namely sorbitol and mannitol) due to the introduction of a new asymmetric carbon atom (shown by asterisk), which may have two different confi gurations to give two different alcohols.

OH

H

OH

OH

H

HO

H

H

CH OH2

CHO

Glucose

OH

H

OH

OH

H

HO

H

H

CH OH2

Sorbitol

CH OH2

NaBH4

O

H

OH

OH

HO

H

H

CH OH2

CH OH2

Fructose

NaBH4

�

OH

H

OH

OH

H

HO

H

H

CH OH2

CH OH2

Sorbitol

*

HO

OH

H

H

CH OH2

CH OH2

Mannitol

*

H

H

OH

OH

Reduction with hydroiodic acid–red phosphorus leads to the conversion of hydroxyl groups to methylene.

3. Oxidation: Depending upon the nature of oxidizing agents, monosaccharides are oxidized to a variety of products. The various important oxidizing agents are as follows:

(a) Benedict’s, Fehling’s and Tollens’ reagents

(b) Bromine water

(c) Nitric acid

(d) Lead tetra-acetate and periodic acid



(a) Benedict’s, Fehling’s and Tollens’ reagents: Benedict’s reagent [an alkaline solution of copper sulphate complexes with citrate ion (cupric citrate ion complex)], Fehling’s reagent (mixture of copper sulphate and potassium sodium tartrate) and Tollens’ reagent (ammoniacal silver nitrate solution) are mild oxidizing agents and oxidize the aldoses to the corresponding aldonic acid. These reagents are, therefore, used for detecting the presence of a reducing sugar in qualitative analysis. The sugars that are oxidized by these reagents are known as reducing sugars.

Because α-hydroxy ketones are also oxidized to the acids, these reagents also oxidize ketoses having α-hydroxy ketonic group. This explains why fructose reduces Fehling’s reagent, Benedict’s reagent and Tollens’ reagent though it does not have any aldehyde group.

(b) Bromine water: This mild oxidizing agent selectively oxidizes the –CHO group of aldose and gives carboxylic acid called aldonic acid, which on evaporation gives a stable δ-lactone. By using sodium amalgam in the presence of a trace of acid the lactone may be reduced to the original aldose; the lactone may also be reduced to alcohol by adding aqueous lactone to aqueous sodium borohydride. However, ketoses are not oxidized by bromine water.

CHOH

CH OH2

CHO

CHOH

CHOH

CHOH

CHOH

CH OH2

COOH

CHOH

CHOH

CHOH

CH OH2

CO

CHOH

CHOH

HC

CHOH

CHOH

CH OH2

CHOH

CHOH

CHOH

CH OH2

[O] Evaporate NaBH4

Alditol (Sorbitol)� Lactone(Glucose- -Lactone)

-�

Aldonic acid (Gluconic acid)Aldose (Glucose)

Na/Hg; H�

O

(c) Nitric acid: It is a strong oxidizing agent, and therefore, it oxidizes the aldehydic and primary alcoholic groups of aldose to corresponding dicarboxylic acid known as aldaric acid. Stronger oxidizing agents usually cleave the carbon chain of ketoses to form a mixture of acids having fewer carbon atoms.

CHOH

CH OH2

CHO

CHOH

CHOH

CHOH

CHOH

COOH

COOH

CHOH

CHOH

CHOH

HNO3

Aldose(Glucose)

Aldaric acid(Saccharic acid)

CO

CH OH2

CH OH2

CHOH

CHOH

CHOH

Ketose(Fructose)

HNO3

Glycolic acid

CH OH2

COOH

COOH

COOH

CHOH

CHOH

�

Tartaric acid

+



(d) Lead tetra-acetate and periodic acid: These reagents are frequently used in carbohydrate analysis. These reagents oxidize the 1,2-glycolic portions of the monosaccharide molecules with the cleavage of the C–C bond. Between every two 1,2-glycolic groups, one molecule of the reagent is consumed.

R’

R

CHOH

CHOH R’

CHO

CHO

R’�

For convenient handling, periodic acid is preferred over lead tetra-acetate. For determining the size of the ring in aldoses, oxidation by periodic acid has been proved to be the best method (refer to page XXX for detailed discussion).

4. Glycoside formation: It is one of the important properties of monosaccharide. Upon reaction with methanolic hydrogen chloride, monosaccharide forms two isomeric compounds known as methyl glycosides. In this reaction, the conversion of an aldehydic group to an acetal group occurs.

Strong methylating agents (e.g. dimethyl sulphate) are required for the methylation of hydroxyl groups of sugars. For example, the remaining four hydroxyl groups of methyl glucosides can be methylated with dimethyl sulphate or methyl iodide to give methyl tetramethyl glucoside. Acetals (and thus glycosides) are acid labile but stable in alkaline medium. When treated with acid they undergo hydrolysis only at the hemiacetal linkage, and not at other linkages to form tetramethyl glucose. This reaction is used in establishing the structure of the glucose.

CH OH2

CHO

(CHOH)3

CHOH

CH3OH

Aldose(Glucose)

�

�-Methyl glycoside

(CHOH)3

C

HC

CH OH2

H OCH3

O(CHOH)3

HC

CH OH2

H CO3 H

O

�-Methyl glycoside

Methylglycoside(� �or )

(CHOH)3

CHOCH3

HC

CH OH2

O (CH ) SO3 2 4 (CHOCH )33

CHOCH3

HC

CH OCH2 3

O

Methyl tetramethylglucoside

(CHOCH )33

CHOH

HC

CH OH2

O

Tetramethylglucose

H�

HCl

5. Acetylation: Monosaccharides react with acetic anhydride and form the corresponding polyacetate esters. The formation of sugar esters indicates the presence of alcohols in the



sugars. The total number of acyl groups that can be taken up by a molecule of sugar is the measure of the number of hydroxyl groups. Both glucose and fructose form penta-acetyl derivatives indicating the presence of fi ve hydroxyl groups in glucose and fructose.

OH

H

OH

OH

H

HO

H

H

CH OH2

CHO

Glucose

OAc

OAc

OAc

H

H

AcO

H

H

CH2OAc

Glucose pentaacetate

CHO

5(CH CO) O3 2

�5CH COOH3

H

OH

OH

HO

H

H

CH OH2

CH OH2

Fructose

O O

H

OAc

OAc

AcO

H

H

CH OAc2

CH OAc2

Fructose pentaacetate

5(CH CO) O3 2

�5CH COOH3

6. Reaction with phenylhydrazine: As the usual carbonyl group, aldoses and ketoses react with one molecule of hydroxylamine to form oxime but they react with three molecules of phenylhydrazine to yield osazones through phenylhydrazones.

The mechanism in this reaction has been studied by different scientists, but no defi nite conclusions have been obtained. Therefore, it is concluded that different mechanisms may apply under different conditions. Actually the mechanisms for osazone formation are of two types.

(a) Fischer type mechanism: According to this mechanism, a molecule of phenylhydrazine fi rst reacts with the aldose or ketose molecule to form phenylhydrazone. Then the second molecule of phenylhydrazine oxidizes either the C

2 hydroxyl group (in case of

aldoses, e.g. glucose) or the C1 hydroxyl group (in case of ketoses, e.g. fructose) of the

phenylhydrazone to a carbonyl group. The newly formed carbonyl group reacts with the third molecule of the phenylhydrazine to yield osazone.

OH

H

OH

OH

H

HO

H

H

CH OH2

CHO

Glucose

C H NHNH6 5 2

�H O2

OH

H

OH

OH

H

HO

H

H

CH OH2

HC

Glucose phenylhydrazone

NNHC H6 5

C H NHNH6 5 2

( C H NH , NH )� 6 5 2 3�

H

OH

OH

HO

H

H

CH OH2

HC

Phenylhydrazone ofglucosone

O

NNHC H6 5

Glucosazone

H

OH

OH

HO

H

H

CH OH2

HC

NNHC H6 5

NNHC H6 5

C H NHNH6 5 2

�H O2

C

CH OH2

(CHOH)3

CH OH2

O

Fructose

C H NHNH6 5 2

�H O2 (CHOH)3

C

CH OH2

NNHC H6 5

Fructosoephenylhydrazone

C H NHNH6 5 2 C H NHNH6 5 2

�H O2

Fructosazone

C

CH OH2

(CHOH)3

CH NNHC H6 5

NNHC H6 5

CH OH2

( C H NH , NH )� 6 5 2 3� (CHOH)3

C

CH OH2

NNHC H6 5

Phenylhydrazoneof fructosone

CHO



This reaction reveals that even though glucose and fructose are different sugars they both yield the same osazone.

Fischer’s mechanism of osazone formation suffers from the following objections:

(i) We know that phenylhydrazine is a powerful reducing agent, then how does it behave as an oxidizing agent in this reaction?

(ii) It also does not explain why the reaction stops as soon as the two phenylhydrazine residues are introduced at C

1 and C

2 of aldoses and ketoses.

(b) Weygand’s mechanism: In this mechanism, the phenylhydrazone (I) formed initially undergoes Amadori rearrangement to give an intermediate (II), which loses aniline through an intramolecular process to form iminoketone (III). The iminoketone (III) reacts with two molecules of phenylhydrazine forming osazone accompanied with the loss of ammonia.

Shortcomings of the Fischer’s mechanism are suitably explained by this mechanism. When phenylhydrazone (prepared by the reaction of glucose with 15N-labelled phenylhydrazine) is treated with ordinary phenylhydrazine, unlabelled osazone is obtained with the expulsion of labelled ammonia. This reaction supports Fischer’s mechanism. Osazone from fructose formation can also be written in the same way.

C

C

R

H

H O

O

H

C

C

R

H

H OH

C

C

R

H

O H

II

H NNHC H2 6 5*

NNHC H6 5*

(N = N)15*

I

HN* NHC H6 5

�C H NH6 5 2

C

C

R

H

O

NH*

H NNHC H2 6 5

C

C

R

H NH*

NNHC H6 5�NH3

*

H NNHC H2 6 5

C

C

R

H

Osazone

NNHC H6 5

NNHC H6 5

III

But Weygand’s mechanism also fails to explain why only the fi rst two carbon atoms are involved in osazone formation. However, in 1944, Fieser and Fieser suggested that as soon as the osazones are formed they are stabilized by internal hydrogen bonding (chelation) and hence no other (except C

1 and C

2) carbon atom takes part in osazone

formation. Thus, for comparing confi gurations at asymmetric centres below C2 in aldoses

and ketoses, osazone formation is an important tool.



A convenient way of identifying a pair of epimers is by the preparation of osazones. In the formation of osazones, asymmetry at C

2 of the aldose is lost and thus epimers (e.g. glucose

and mannose) yield identical osazones.

D-Glucose D-Mannose D-Fructose

CHO

OHH

HHO

OHH

OHH

CH OH2

CHO

HHO

HHO

OHH

OHH

CH OH2

C O

HHO

OHH

OHH

CH OH2

CH OH2

Identical configurationsat C ,C ,C3 4 5

3C H NH.NH6 5 23C H NH.NH6 5 23C H NH.NH6 5 2

CH

C

HHO

OHH

OHH

D-Glucosazone

NNHC6H5

NNHC6H5

CH OH2

7. Action of alkali: Monosaccharides react with alkalis in different ways.

Upon warming with concentrated alkalis, the sugar fi rst turns yellow, then brown and fi nally resinifi es.

In the presence of dilute alkali or amines, these sugars undergo rearrangement to form a mixture of more than one sugar. For example, in the presence of very dilute alkali (0.02 N NaOH), glucose is converted into a mixture of glucose, mannose and fructose (Lobry-de Bruyn–van Ekenstein rearrangement).

8. Reaction with hydrogen cyanide: Aldose reacts with hydrogen cyanide and the cyanohydrins formed can be hydrolysed to the higher aldonic acid, which is then converted into two isomeric sugars.

This is one of the most important synthetic reactions for the preparation of sugars, and it is used to prepare aldoses of up to 10 carbon atoms (refer to Kiliani’s synthesis under Page XXX for details).



CHO

OHH

HHO

OHH

OHH

D( )-Glucose�

C

C OH

HHO

OHH

OHH

CHO

HHO

HHO

OHH

OHH

OHH

Enediol D( )-Mannose�

0.02 N NaOH

CH OH2 CH OH2 CH OH2

C

HHO

OHH

OHH

O

D( )-Fructose�

CH OH2

CH OH2

4.4 Ring Size of Monosaccharides

The fi ve- and six-member oxygen-containing heterocyclic rings (furan and pyran, respectively) of monosaccharides bear a normal relationship to the heterocyclic compounds furan and pyran, respectively.

OFuran

O

Pyran

O

1

23

4

5O

1

23

4

Hence, the fi ve- and six-member rings in the structures of sugars (e.g. glucose) are indicated by α-D-glucofuranose and α-D-glucopyranose, respectively, which indicates the oxygen heterocyclic ring (furanose = furan and pyranose = pyran).



Thus, the two glucopyranose and two glucofuranose may be represented as below.

O

OH

OH

H

H

OH

OH

H

HCOH

1

23

4

O

OH

OH

H

H

OH

H

OH

HCOH

1

23

4OH

OH

H OH

OH

H

OHH

H

OH

OH

H OH

H

HO

OHH

H

CH OH2CH OH2

CH OH2

6

5

CH OH2

6

5

�- -GlucopyranoseD �- -GlucopyranoseD �- -GlucofuranoseD �- -GlucofuranoseD

Structures are represented by thin lines throughout the book for the sake of simplicity.

Ring structure of aldoses

The monosaccharides, polyhydroxy aldehydes (e.g. glucose) or ketones (e.g. fructose) with open chain formula failed to explain the following properties:

1. Glucose does not yield a bisulphite (upon reaction with NaHSO3/KHSO

3) and aldehyde–

ammonia compound (upon reaction with NH3).

2. Glucose does not answer for aldehyde with Schiff’s reagent. 3. When glucose is dissolved in water its specifi c rotation gradually changes until it reaches

a constant value. The phenomenon known as mutarotation is exhibited by almost all the reducing sugars (except few ketoses).

4. Two isomeric penta-acetates are obtained upon acetylation of glucose, neither of which reacts with carbonyl reagents.

5. Glucose reacts with only one molecule of methanol in presence of hydrogen chloride, to form two isomeric compounds, each containing one methoxy group exhibiting the properties of acetals. They have different melting points, specifi c rotations and enzymatic hydrolyses. Normal aldehyde usually reacts with methanolic hydrogen chloride to yield hemiacetal (I) and acetal (II).

R C H

O

� CH OH3 R C H

OH

OCH3

H�

CH OH3

R C H

OCH3

OCH3

Aldehyde Hemiacetal (I) Acetal (II)

H�

One of the ethers called the α-form undergoes ready hydrolysis by maltase, and the β-form is hydrolysed by emulsin. Hydrolysis of both the ethers, whether carried out with enzymes or acids (but not by alkali), always yields only D(+) glucose. These glucosides do not undergo mutarotation, and does not react with Tollens’ or Fehling’s reagent.

D( )-Glucose�CH OH3

HClMethyl- − -glucoside� D �

m.p.[ ]D�

165°C�157°

Methyl- − -glucoside� D

m.p.[ ]D�

101°C�33°



This behaviour can be demonstrated on the basis that the carbonyl group is not free in glucose but bound up in a form that destroys its reducing properties. On this basis and the stability of γ-lactones of the aldonic acid, Tollens proposed a cyclic hemiacetal structure (γ-oxide ring structure) for D(+) glucose.

CHOH

OHH

HHO

H

OHH

O

*

Ring structure

CHO

OHH

HHO

OHH

OHH

Open-chain structure

CH OH2 CH OH2

D(+)– Glucose

The objections against the open-chain structure are explained by the ring structure for glucose in the following ways:

1. As the ring structure of glucose is not having free aldehyde group, it does not react with certain weak carbonyl reagents (does not restore Schiff’s reagent colour, does not form a bisulphite or an aldehyde–ammonia compound), which are characteristic for aldehydes. But it gives certain reactions of the carbonyl group, such as the formation of cyanohydrins, oxime and phenylhydrazone, because the strong carbonyl reagents such as HCN, NH

2OH, C

6H

5NHNH

2

are able to open the ring to a free aldehydic group that reacts with these reagents. 2. One new chiral centre (C

1, marked by asterisk) has been introduced in the ring structure and

hence gives two different isomeric glucoses and its derivatives. Practically, glucose, methyl glucoside and glucose penta-acetate are found to exist in two forms, i.e. α and β. By studying the various aldoses it has been found and also confi rmed by both physical and chemical methods that in the α-form the hydroxyl group or substituted hydroxyl group is attached to the right, and in the β-form the hydroxyl group is attached to the left.

C

OHH

HOH

H

OHH

OHH

O

CHO

OHH

HOH

H

OHH

OH

CH OH2 CH OH2

C

OHH

HOH

H

OHH

HOH

O

CH OH2

�-D Glucose �-D GlucoseAldehydic form



The two isomers differing only in the confi guration of C1 (in aldoses) or C

2 (in ketoses) are

known as anomers, while such carbon atom is known as anomeric carbon atom. The existence of two methyl glucosides and penta-acetates can be explained in same way.

H

RO

H

H

H

OR

OR

OR

H

O

RO

RO

H

H

H

OR

OR

H

H

CH OR2CH OR2

O

��

Methyl glucoside when R = CHGlucose penta-acetates when R = COCH

3

3

3. Mutarotation: The cyclic structure can open and reclose, which allows the rotation to occur about the carbon bearing the reactive carbonyl to yield two distinct confi gurations (α and β) of the hemiacetals and hemiketals. The carbon about which this rotation occurs is called anomeric carbon and the two forms are called anomers. Carbohydrates can change spontaneously between α and β confi gurations. This process is known as mutarotation.

When glucose crystallized from water below 50°C is dissolved in water, its initial specifi c rotation of +113° falls gradually to a constant value of +52.5°; similarly, when the glucose crystallized from water above 95°C is dissolved in water, its initial specifi c rotation of +19.5° gradually rises to +52.5°. This phenomenon is known as mutarotation in glucose.

OHH

HHO

H

OHH

OHH

O

CHO

OHH

HHO

OHH

OHH

CH2OH

OHH

HHO

H

OHH

HHO

O

Aldehydic form

(36%) (64%)

CH OH2 CH OH2

��-Glucose

[ ] 113°D � ��-Glucose

[ ] 19.5°D � �

[ ] 52.5°D � ��



The ring structure for glucose explains that the change in specifi c rotation is due to interconversion of the α-form [(α)

D = +113°] of glucose to β-form [(α)

D = +19.5°) and vice versa

through the aldehydic structure till an equilibrium is reached between the two structures. The specifi c rotation value of this equilibrium mixture corresponds to +52.5°.

The question at our hand is how the open-chain form of glucose is obtained from the ring form. It can be explained that this happens because protonation by acid HA and deprotonation by base B takes place at the same time.

C

C OHH

C HHO

C OHH

C OHH

H O C

C OHH

C HHO

CH

C OHH

O H

O

H

Aldehydic form�- D( )-Glucose �-( )-GlucoseD

C

C OHH

C HHO

CH

C OHH

H O H :B

O H A

CH OH2 CH OH2 CH OH2

The concentration of aldehydic form can be increased by adding methanethiol and esterifi cation, followed by the removal of methanethiol.

CHO

OHH

HHO

OHH

OHH

Glucose

OHH

HHO

OHH

OHH

Pyridine

CH(SCH3)2

OAcH

HAcO

H

H

CHO

OAcH

HAcO

OAcH

OAcH

CH2OH

� 2CH SH3 –H O2

CH2OH

CH(SCH )3 2

Glucose dimethyl mercaptal

Ac O2

CH OAc2

HgCl2

H O/CdCO2 3

2,3,4,5,6-Penta-acetylglucose

CH OAc2

OAc

OAc

Two types of ring structures: Fischer isolated a new D-glucoside, which differs from that of already known α- and β-glucosides and called it γ-D-glucoside. But later on Haworth explained that this γ-glucoside was found to be a mixture of two different glucosides (α and β) different from that of previously known α and β-glucosides. These observations reveal that the sugars have two different ring structures, namely γ- and δ-oxide ring structures.



CHOH

OHH

HHO

H

OHH

O

CHOH

OHH

HHO

OHH

H

O

CH OH2 CH OH2

�-Oxide(5-membered ring structure)

�-Oxide(6-membered ring structure)

4.5 Determination of the Ring Size

The following methods are generally employed for the determination of the ring size in sugars.

1. Methylation method 2. Periodic acid oxidation method

1. Methylation method: This method was fi rst reported by Hirst. The sugar is fi rst completely methylated, which is then hydrolysed with dilute hydrochloric acid. When only the glycosidic methyl group is hydrolysed, the product is fi nally oxidized. On the basis of the oxidation product obtained, the ring size of the sugar is determined. This method is based on the assumption that during the reactions there is neither a change in the size of the ring nor in the position of any methyl group. The fi rst step of methylation can be effected by different methods.

CHOH

CHOH

HClO

OO � AgI� CH OH3

CHOCH3

CHOH

CH3I

Ag O2

CHOCH3

CHOCH3

(i) Purdie method: Upon reaction of the sugar with methanolic hydrogen chloride the sugar is converted into its methyl glycoside. The product is then treated with methyl iodide in the presence of dry silver oxide to afford fully methylated product.

Advantages

As the free sugar is fi rst methylated by methanolic hydrogen chloride the oxidation of the free �sugar by silver oxide is prevented.



As the methylation is performed at mild condition, the structural alterations in the molecule �are avoided.This method is free from untoward effects such as Walden inversion, racemization or glucosidic �interconversion.

Disadvantages

Silver oxide causes oxidation of a free reducing sugar. Therefore, this method is applicable only �to glycosides and other derivatives in which reducing group is either absent or protected by substitution.This method can be used only for the sugars of which the suitable solvents are known. �Reagents used in this method (e.g. methyl iodide and silver oxide) are expensive. �

(ii) Haworth and Hirst method: The sugar is fully methylated by using dimethyl sulphate in the presence of aqueous sodium hydroxide.

Advantages

This method is applicable to all kinds of reducing sugars. �Methylation occurs smoothly in a step-wise and uniform manner, and therefore, the �intermediates can be isolated by interrupting the process.Unreacted and partly reacted sugars are not formed; therefore, the purifi cation is carried out �easily.Yield is good and the reagents used are cheap. �

Disadvantage

Dimethyl sulphate is a poisonous compound; therefore, it should be used with great care under �controlled conditions.

(iii) Diazomethane method: Sugars can also be methylated by the use of diazomethane in the presence of alcoholic ethereal solution.

Advantages

Nitrogen is the only by-product and it can be eliminated easily. �The product obtained is very pure. �

Disadvantages

Diazomethane is very poisonous. �This method is applicable for methylation of acidic compounds. �

(iv) p-Toluene sulphonate method: Hydroxy compounds are methylated by reacting the compounds with p-toluene sulphonate in sodium hydroxide. This method is also called as tosylation.

Advantage

It is preferred over the above methods, as p-toluene sulphonate is harmless.



(v)Sodium and methyl iodide method: By using sodium and methyl iodide in liquor ammonia, the carbohydrates can be methylated.

Let us consider the example of D-glucose and apply the methylation method on both of the possible forms (pyranose and furanose).

Pyranose structure: Pyranose structure is also called δ-oxide, amylene oxide or six-membered ring. Using dimethyl sulphate in the presence of sodium hydroxide (Haworth and Hirst process of methylation), the methyl glucoside (I) (obtained by refl uxing the D-glucose with methanolic hydrochloric acid) is methylated to give methyl tetramethyl-D-glucoside (II), which upon hydrolysis gives a tetramethyl-D-glucose (III). Oxidation with nitric acid gives a dicarboxylic acid (V). The dicarboxylic acid (V) was identifi ed as xylotrimethoxy glutaric acid by the fact that it was also obtained by the oxidation of methylated xylose.

D-GlucoseHCl

Methyl glucosideNaOH

HClD-Tetramethyl glucose

Lactone

COOH

CH

H

H

COOH

V

I II III

IV

CH OH3(CH ) SO3 2 4 Methyl tetramethyl

-glucosideD

Br water2 HNO3

H CO3

OCH3

OCH3

Xylotrim ethoxyglutaric acid

The dicarboxylic acid structure, which helped in determining the structure of the parent compound glucose, is described here.

1. From the fi rst carbon atom of the lactone (IV), one of the –COOH groups in acid (V) must be derived and the second –COOH group from the carbon atom is involved in the ring formation in the sugar. As only three hydroxyl groups are present inside the ring of glucose, the ring must be δ-oxide in sugar.

2. Presence of three methoxy groups in the dicarboxylic acid (V) indicates the presence of only three methoxy groups in between the lactone (IV) and hence three hydroxyl groups inside the ring of glucose, i.e. the lactone formation involves C

1 and C

5 and hence the lactone (IV)

must be δ-lactone and the sugar must be six membered, which explains all the reactions in the following manner:

3. Finally, the formation of (V) also eliminates the possibility of an ε-ring (when C1 and C

6

are involved in ring formation) because if at all the case was ε-ring, the product 2,3,4,5-tetramethyl gluconic acid or tetramethylsaccharic acid (VI) would have been formed.

It is important to note the Fischer’s fi rst methyl glucosides were actually the pyranosides, which he called furanosides only on the basis of Tollens’s assumption that the γ-oxide ring is stable by analogy to γ-lactones.



Glucose

CHOCH3

OHH

HHO

OHH

H

OReflux

CHOCH3

OCH3H

H

OCH3H

H

O

I II

HCl

Brominewater, 90°C

IIIIV

CO

H

H

H

H

O

COOH

H

OH

CHOH

H

H

H

H

O

COOH

COOH

H

H

H

COOH

V VI

COOH

H

H

H

H

CHOH

OH

H

OH

O

H

HO

H

H

H

H

H

CH OH2CH OH2 CH OH2 CH OCH2 3

OCH3

OCH3

H CO3H CO3H CO3

CH OCH2 3CH OCH2 3CH OCH2 3

OCH3

OCH3

HNO3

OCH3

OCH3

HNO3

H CO3

OCH3

OCH3

OCH3

OCH3

OCH3

H CO3

CH OH3 –HCl

NaOH

(CH ) SO3 2 4

H CO3

;



Furanose structure: Fischer prepared a new methyl glucoside (actually which was later on found to be a mixture of two glucosides, α and β) by dissolving D-glucose in methanolic hydrochloric acid and then keeping the product at 0°C for some time. The size of the ring in these two new isomeric glucosides was found to be γ-1,4-butylene oxide. The steps used for establishing the size of the ring are same. For example, the methyl glucoside (I, prepared at 0°C as earlier) is completely methylated using methyl sulphate to yield methyl tetra-O-methyl-D-glucoside (II), which is hydrolysed with dilute hydrochloric acid to yield tetra-O-methyl-D-glucose (III), which is then oxidized fi rst with bromine water at 90°C to yield crystalline lactone (IV) and fi nally with nitric acid to yield dimethyl-D-tartaric acid (V, dimethoxysuccinic acid) of well-known structure.

The formation of dimethyltartaric acid from D-glucose indicates that within the ring of the sugar and its derivatives, (II), (III) and (IV), there are only two hydroxyl or methoxy groups and hence the ring must involve C

1 and C

4 which are fi nally oxidized during the set of reaction to the

acidic groups. The scheme for conversion of glucose to dimethyltartaric acid may be represented as shown below:

CHOH

OHH

HHO

H

OHH

O

Glucose I II

NaOH

CH OH2

CH OH-HCl3

0° C

OHH

HHO

H

OHH

O

CHOCH3

CH OH2

(CH ) SO3 2 4H CO3

H

H

H

H

O

OCH3

OCH3

CH OCH2 3

CHOCH3

HClH CO3

CHOH

H

H

H

H

III

OCH3

OCH3

CH OCH2 3

Brominewater

CO

H

H

H

H

IV

OCH3

OCH3

H CO3

CH OCH2 3

HNO3 COOH

H

H

COOH

V

OCH3

H CO3

OO



From the above contradictory statements it is concluded that the methyl glucoside prepared at refl ux temperature has 1,5-oxide (δ-oxide) ring structure. Hence, with the help of this method it is not possible to say whether glucose itself originally exists in the pyranose (1,5) or furanose (1, 4) form or whether the two forms are in equilibrium.

However, X-ray analysis of all the normal monosaccharides indicates the presence of six-membered ring in a manner similar to which α-D-glucose is found to have pyranose structure. Using periodic acid oxidation, the existence of glucose as pyranoside has been confi rmed.

2. Periodate oxidation method: This is a simple elegant method introduced by Malaprade. It involves the use of periodic acid at about pH 4 in dark to avoid overoxidation and decomposition of the oxidant.

The general principles of periodic acid oxidation have been discussed already. As shown in the example, between every 1,2-glycolic groups one molecule of periodate is consumed.

(i) (ii)

CHOH

CHOH

CHOH

R’

R

RCHO�

HCOOH

�

R’CHO

CHOH

R

CHOH

R’

RCHO

�

R’CHO

H IO5 6 2 H IO5 6

Principles useful in structural investigation by periodic acid may be summarized as below:

1. When a –CHOH group is attacked on one side, i.e. only by 1 mole of the reagent, it is converted into –CHO group.

2. One molecule of periodic acid is consumed between every two adjacent hydroxyl groups (1,2-glycolic groups).

3. When a –CH2OH group is attacked by periodic acid, it is converted into HCHO.

4. When –CHOH group is attacked on both the sides, i.e. one –CHOH group is attacked by 2 moles of periodic acid, it is converted into HCOOH.

5. The reagent does not open the oxide ring of the sugar or its derivatives.

Thus, by estimating the number of moles of the periodic acid consumed and the number of moles of HCHO and HCOOH produced along with the investigation of the oxidation product of the glycoside, this method has been successfully used to determine the size of the ring in glycosides.

Using this method, Hudson proved that the normal glycosides are pyranosides the oxidation of methyl α-D-glucopyranoside. The compound (I), for example, on oxidation with periodic acid consumes 2 moles of the acid along with the formation of 1 mole of formic acid and a dialdehyde (II) indicating beyond doubt that the compound (I) has three adjacent hydroxyl groups, which is possible only when the glycoside has δ-ring structure.



O

H

HO

H

H

OH

C

CH OH2

H OCH3

H

OH

2H IO5 6HCOOH �

H C OCH3

CHO

CHO

H

CH OH2

O i) Bromine water

ii) SrCO3

I II

H

OO

O

C

CO

CO

Sr

CH OH2

H

OCH3

i) H SO2 4

ii)Bromine water

COOH

C OH

CH OH2

H �COOH

COOH

III

D-Glyceric acid Oxalic acid

It has been proved by the above scheme that the structure (II) on oxidation with bromine water in the presence of strontium carbonate gives a crystalline strontium salt (III). The strontium salt (III) on hydrolysis followed by oxidation yields oxalic and D-glyceric acids.

O

H

HO

H

H

OH

C

CH OH2

H CO3 H

H

OH

2H IO5 6HCOOH �

H CO3 C H

CHO

CHO

H

CH OH2

O i) Bromine water

ii) SrCO3

I II

H CO3

OO

O

C

CO

CO

Sr

CH OH2

H

H

i) H SO2 4

ii)Bromine water

COOH

C OH

CH OH2

H �COOH

COOH

III

D-Glyceric acid Oxalic acid



From similar experiments it is also found that all methyl α-D-aldohexopyranosides of the normal type consume 2 moles of periodic acid and produce 1 mole of formic acid along with formation of the compounds (II), (III), oxalic acid and D-glyceric acid. Hence, all hexosides have δ-oxide rings.

Ring structure of ketoses

H

CO

OH

H

H

OH

HO

CH OH2

CH OH2

Open chain

O

CH2

HO H

OH

OH

OH

H

H

HOH C2 C

C

C

C

1 2

3

4

5

6

α− -FructopyranoseD

O

CH2

HO

HO

H

OH

OH

H

H

C

C

C

C

12

3

4

5

6

CH OH2

β− -FructopyranoseD

OH

OH

HO

H

H

OH

C

C

C

C

1 2

3

4

5

6CH OH2

HOH C2

α− -FructofuranoseD

OHHO

H

H

OH

C

C

C

C

CH OH2

HO CH OH2

β− -FructofuranoseD

The open-chain reactions of fructose fail to explain some of the facts. They are as follows:

1. It does not react with NaHSO3, indicating that the keto group is not free.

2. Like glucose, fructose also shows the property of mutarotation. 3. When treated with methanol in hydrogen chloride, it yields fructoside. This compound

does not exhibit the property of carbonyl group and can exist in two forms, namely α- and β-methyl fructosides.

4. Penta-acetyl fructose does not exhibit the property of carbonyl group, which indicates that the carbonyl group is not free.

The above facts can be explained if fructose forms a ring between carbonyl and hydroxyl groups as in hemiacetals.

1. Ring originating from C-2 and closing at C-6 (pyranose ring). 2. Ring originating from C-2 and closing at C-5 (furanose ring).

Each of these can exist as α- and β-anomers, and hence ketoses (e.g. D-fructose) may also exist in four isomeric ring structures similar to aldoses. The open-chain and various ring structures of D-fructoses may be drawn as shown below.

The size of the ring in particular fructose is determined by methylation. The method is the same as for aldoses, except that oxidation by bromine water is replaced by dilute nitric acid followed by acid permanganate. Let us apply the method on both types of fructoses (pyranose and furanose).

Pyranose Structure

Fructose at refl ux temperature is methylated to methyl-β-D-fructoside (I), which was completely methylated by using dimethyl sulphate in the presence of alkali to methyl-tetra-O-methyl-D-fructoside (II). The methyl-tetra-O-methyl-D-fructoside was hydrolysed with dilute hydrochloric acid to tetramethyl-D-fructose (III), which on oxidation with nitric acid gave trimethyl-β-D-fructonic acid (IV). This acid on oxidation with acidic permanganate afforded a lactone, trimethyl-



D-arabinolactone (V), which on oxidation with nitric acid gave D-arabinotrimethoxy glutaric acid (VI) of well-known structure.

H

COOH

OCH3

H

H

OCH3

H CO3

COOH

VI

Two acidic groups on C1 and C

5 formation indicate that the ring in fructoside (I) involves C

1

and C5, i.e. the ring is δ-oxide, and hence the scheme reaction is depicted as below.

O

HO

H

CH OH2

OH

CH2

HO

H

H

OH

O

H CO3

H

CH OH2

OH

CH2

HO

H

H

OH

CH OH–HCl3 (CH SO)3 2 4

NaOH

β−D-Fructopyranose Methyl- -Fructopyranosideβ-D

O

H CO3

H

CH OC2 3H

OCH3

CH2

H

H

OCH3

H CO3 HCl O

HO

H

CH OC2 3H

OCH3

CH2

H

H

OCH3

H CO3 HNO3

Methyl-1,3,4,5-Tetramethyl- -fructopyranosideβ-D 1,3,4,5-Trimethyl- -fructoseβ-D

HO

H

COOH

OCH3

CH2

H

H

OCH3

H CO3O KMnO4

H SO2 4H

CO

OCH3

CH2

H

H

OCH3

H CO3O HNO3

H

COOH

OCH3

H

H

OCH3

H CO3

COOH

3,4,5-Trimethyl- -fructonic acid (as lactol)β-D 2,3,4-Trimethyl- -arabinolactoneD D-Arabinotrimethoxy glutaric acid

I

II III

IV V VI



Furanose Structure

Fructose at room temperature is methylated to methyl fructoside (I) and is then methylated completely by using dimethyl sulphate. The product is hydrolysed with dilute hydrochloric acid, then oxidized with dilute nitric acid, acidic permanganate and concentrated nitric acid to give dimethyl-L-tartaric acid of known structure.

H CO3

H

H

OCH3

COOH

COOH

The dimethyl-L-tartaric acid formation indicates that in fructose ring structure C1 and C

4 are

involved, i.e. the ring is γ- or 1,4-oxide. Thus, the above reactions can be represented as below.

CH OH2

HO

OH

H

H

H

OH

O

CH OH2

H

H

H

OCH3

O

CH OCH2 3CH O3

CH OCH2 3

H CO3

H

H

H

OCH3

O

COOHHO

CH OCH2 3

H CO3

H

H

H

OCH3

O

CO

CH OCH2 3

I) CH OH–HClat 18°C

3

ii) (CH ) SOin NaOH

3 2 4

β D-Fructofuranose- Methyl-1,3,4,6-tetramethyl

-fructofuranosideβ D-

i) HCl

ii) Dil. HNO3

KMnO4

H SO2 4

3,4,6-Trimethyl- -fructonic acidβ D- 2,3,5-Trimethyl -arabinolactone(It is a slow changing lactone)

-β D-

H CO3

H

H

OCH3

COOH

COOH

Conc. HNO3

Dimethyl- -tartaric acidL

CH O3



4.7 Conformations of Monosaccharides

The arrangement of atoms and groups in space of a single chemical structure is termed as conformation. Similar to cyclohexane, the pyranose sugars and their derivatives assume a chair conformation in preference to any boat form. The chair form is more stable compared to boat form because of the minimum hydrogen interactions in the chair conformation. The chair conformation of α- and β-glucose is depicted below.

HO

OH

H

OCH OH2

HO

OH

H

H

H

HHO

H

H H

H HOH

HO

CH OH2

OH

O

α D-Glucose (36%)- β D-Glucose (64%)-

As the β-form is more stable, it is predominant which in turn is due to the fact that the OH or CH

2OH groups on C

1, C

2, C

3, C

4 and C

5 are all equatorial than in the α-form in which although

hydroxyl groups on C2, C

3 and C

4 and the CH

2OH group on C

5 are all equatorial, the hydroxyl

group at C1 is axial.

4.8 Monosaccharides of Pharmaceutical Importance

Among the natural hexose sugars, D-glucose, D-mannose, D-galactose and D-fructose are the important members. It is also interesting to note that these four sugars are also fermented by yeast.

D(+) Glucose (dextrose)

HO

H

H

H

OH

OH

CH OH2

C

CHO

C

C

C

*

*

*

*

OHH

1

2

3

4

5

6

The D-glucose is a six-carbon containing aldose sugar and is the most important and widely distributed monosaccharide. It is also known as dextrose of grape sugar and occurs in many plants in free as well as combined state. It is the component of glycosides, disaccharides and polysaccharides. The important sources of glucose are ripe grapes (20–30%), cane sugar (in which glucose occurs in combination with fructose), honey, human blood and urine of diabetic patients.



Human blood contains 0.15% of glucose. Calcium salt of gluconic acid is used as an intravenous calcium supplement.

Biological importance 1. It is the chief sugar present in the normal blood. 2. Erythrocytes and brain cells utilize glucose solely for energy purposes. 3. It is stored mainly as glycogen in liver and muscles.

Chemical tests: Apart from answering for the general reactions of the reducing monosaccharides, it gives red precipitate with cupric tartaric solution.

Preparation of glucose: Commercially, glucose is obtained by the acidic hydrolysis (using dilute mineral acids) of starch, usually corn starch. Glucose is obtained as glucose monohydrate by crystallization from water.

(C6H

10O

5)

n + nH

2O C

6H

12O

6

Structural Elucidation

1. Molecular formula: Molecular formula of glucose is found to be C6H

12O

6.

2. Presence of 5-OH groups: Glucose gives penta-acetate on acetylation with acetic anhydride and sodium acetate, indicating the presence of fi ve hydroxyl groups.

3. Presence of an aldehydic group: With hydrogen cyanide, glucose forms cyanohydrins; phenylhydrazone with phenyl hydrazine and a mono-oxime with hydroxylamine, indicating the presence of a carbonyl group.

Glucose reduces Fehling’s solution and Tollens’ reagent, which suggests that carbonyl group may be aldehydic or ketonic. Glucose on oxidation yields gluconic acid having the same number of carbon atoms, which confi rms the presence of carbonyl group as an aldehyde group. As the aldehydic group is monovalent, it must be present at the end of the chain.

C5H

11O

5CHO C

5H

11O

5COOH

Glucose Gluconic acid

4. Presence of straight chain (of carbon atoms): Glucose upon reaction with hydrogen cyanide yielded cyanohydrins, which on hydrolysis followed by reduction with hydriodic acid and red phosphorus yielded n-heptanoic acid. The formation of n-heptanoic acid from glucose indicates that the glucose has a straight chain of six carbon atoms.

C H O5 11 5CHOH O2

C H O5 11 5CH(OH)COOHC H O5 11 5CH(OH)CN C H6 13COOHHI – P

n-Heptanoic acid

5. Open-chain structure of glucoseGlucose contains a straight chain of six carbon atoms. �Of the two ends, one end is having the aldehyde group and the other end is having the �primary alcoholic group.The remaining four carbon atom bear one hydroxyl each. �

From the above points, it is suggested that glucose is a straight-chain pentahydroxy aldehyde of the following structure.



HO

H

H

H

OH

OH

CH OH2

C

CHO

C

C

C

*

*

*

*

OHH

Glucose

6. Confi guration of glucose: As the above structure of glucose possesses four asymmetric carbon atoms (marked by asterisks), sixteen (24 = 16) optical isomers are possible. The confi guration of glucose (arrangement of hydrogen and hydroxyl groups around the four asymmetric carbon atoms) is determined by the use of Kiliani’s synthesis (discussed earlier; refer to page XXX).

The open-chain structure of glucose failed to explain some of the characteristic reactions of glucose. (refer to page XXX).

The objections against the open chain structure are explained by the ring structure for glucose in the following ways (refer to page XXX).

7. Mutarotation: When glucose prepared by crystallization from water below 50°C is dissolved in water, its initial specifi c rotation of +113° falls gradually to a constant value of +52.5°. Similarly when the glucose crystallized from water above 95°C is dissolved in water its initial specifi c rotation of +19° gradually rises to +52.5°. This phenomenon is known as mutarotation.

The ring structure for glucose explains that the change in specifi c rotation is due to interconversion of the α-form [(α)

D = +113°] of glucose to β-form [(α)

D = +19.5)] and

vice versa through the aldehydic structure till an equilibrium is reached between the two structures. The specifi c rotation value of this equilibrium mixture corresponds to +52.5°.

8. Two types of ring structures: Glucose is found to possess two different ring structures, namely γ- and δ-oxide ring structures.

Determination of the ring size of the glucose ring is described in page XXX.

D(–) Fructose

HO

H

H

H

OH

OH

CH OH2

C O

CH OH2

C

C

C

*

*

*

*

1

2

3

4

5

6



The D-(levo)-fructose is a six-carbon containing ketose sugar. It is also known as levulose or fruit sugar and is found in many fruits and honey. Fructose is obtained from invert sugar after separating it from glucose. It is used as a food and sweetener for patients who cannot tolerate glucose, e.g. patients suffering from diabetes. It has the sweetness of sucrose, and about twice that of glucose. High-fructose containing corn syrup produced by enzymic hydrolysis/isomerization of starch is used as a food sweetener. It is the most important sugar of ketoses and occurs as free sugar along with glucose in honey and sweet fruits, and therefore, it is called fruit sugar. It also occurs in combination with glucose in cane and beet sugars. The most important source of fructose is the polysaccharide inulin.

It exhibits mutarotation; the specifi c rotation values of α, β and equilibrium mixtures are 21°, 133.5° and 92.3°, respectively. Fructose shows most of the usual properties of the ketonic and hydroxyl groups. It reduces Fehling’s solution and Tollens’ reagent, due to the presence of α-hydroxyl ketonic group, which is readily oxidized by Fehling’s and Tollens’ reagents.

Preparation: Commercially, fructose is obtained by the hydrolysis of inulin (a polysaccharide of fructose).

Biological importanceSeminal fl uid is rich in fructose and sperms utilize fructose for energy. Fructose is formed in the seminiferous fi bular epithelial cells from glucose.


1. Molecular formula: Molecular formula of fructose is found to be C6H

12O

6.

2. Presence of 5-OH groups: Fructose gives penta-acetate on acetylation with acetic anhydride, indicating the presence of fi ve hydroxyl groups.

3. Presence of a ketonic group: Fructose forms cyanohydrin and oxime, which reveals the presence of a carbonyl group. On oxidation with nitric acid, fructose gives a mixture of tartaric acid and glycolic acids (each having lesser number of carbon atoms), which reveals that the carbonyl group is ketonic in nature. The formation of glycolic and tartaric acids reveals that the ketonic group is present at position 2.

CH OH

CO

CHOH

2

2

CHOH

CHOH

CH OH

CH OH

COOH

2

COOH

CHOH

CHOH

COOH

�

Glycolic acid

Fructose Tartaric acid

4. Presence of straight chain of carbon atoms: Upon reduction with sodium amalgam and ethanol, fructose yields hexahydric alcohols, sorbitol and mannitol, which on further reduction with hydriodic acid and red phosphorus yield 2-iodohexane and n-hexane which indicates that in fructose molecule the six carbon atoms are present in a straight chain.



Fructose on treatment with hydrogen cyanide gives cyanohydrins, which upon hydrolysis followed by reduction with hydriodic acid and red phosphorus yields n-butylmethylacetic acid, CH

3CH

2CH

2CH

2CH(CH

3)COOH (I). Formation of the above acid (I) also reveals the

presence of ketonic group at position 2. 5. Open-chain structure of fructose: Based on the above points the straight chain structure

of fructose may be written as below.

CH

CHCOOH

CH

3

2

2

2

3

CH

CH

CH

CH OH

CO

CHOH

2

2

CHOH

CHOH

CH OH

*

*

*

CH OH

COOH

2

COOH

CHOH

CHOH

COOH

�i) HCN

ii) H O2

iii) HI–P

HNO3

(I)

6. Confi guration of fructose: As the above structure has three asymmetric carbon atoms, it may exist in eight (23 = 8) optical isomers. The exact confi guration of the fructose is recognized by the fact that it yields the same osazone as glucose, thereby revealing that the confi gurations of C

3 to C

6 atoms of fructose are same as that of glucose. Hence, the complete

structural formula of fructose may be written as shown below.

CH OH2

CO

HO

OHH

H

H

H

CH OH2

3C H NHNH6 5 2

HO

H

H

H

OH

OH

CH OH2

C

CH N.NHC H6 5

N.NHC H6 5

3C H NHNH6 5 2

H

HO

H

H

OH

H

OH

OH

CH OH2

CHO

Fructose Osazone Glucose

The open-chain structure of fructose fails to explain some of the facts (described in detail on to page XXX).

7. The objections against open-chain structure are explained by the ring structure of fructose: Like glucose, fructose also exhibits mutarotation and forms two isomeric methyl fructosides. Hence, fructose is assumed to exist in two isomeric (α and β) forms. The ring structure of fructose is already discussed on page XXX.

Fructose shows levorotatory properties and has D-confi guration of glucose, and therefore, it is denoted as D(–) fructose.

The commercial and natural fructose is the β-isomer with a pyranose ring. But the fructose found in sucrose (a disaccharide) and inulin (a polysaccharide) is present in the furanose ring, which upon hydrolysis is converted into the more stable pyranose form.



�5. DISACCHARIDES �

Covalent bonds between the anomeric hydroxyl of a cyclic sugar and the hydroxyl of a second sugar (or another alcohol containing compound) are termed as glycosidic bonds and the resultant molecules are called glycosides. The linkage of two monosaccharides to form disaccharides involves a glycosidic bond.

C H O C H O6 �12 6 6 12 6 C H O H O12 �22 11 2

Hexose Hexose Dihexoseor

Disaccharide

The reverse reaction yields two individual monosaccharides (hydrolyses by enzymes and dilute mineral acids), which is a characteristic reaction of all the disaccharides. Physiologically important disaccharides are sucrose, lactose and maltose.

5.1 Biological Importance of Disaccharides

1. Various food preparations such as baby foods are produced by hydrolysis of grains and contain large amounts of maltose. From nutritional point of view, disaccharides are easily digestible.

2. In lactating mammary gland, the lactose is synthesized from glucose by the duct epithelium. Lactose present in breast milk is a good source of energy for the new born baby.

3. Lactose is fermented by coliform bacilli (Escherichia coli), which is usually nonpathogenic, and not by typhoid bacillus, which is pathogenic (lactose nonfermenter). This test is used to distinguish the two microorganisms.

4. Many organisms that are found in milk (e.g. E. coli, Aerobacter aerogenes) convert milk to lactic acid.

5. Sucrose, if administered parenterally, cannot be utilized by human body but it can change the osmotic condition of the blood and cause fl ow of water from the tissues into blood.

5.2 Classification and Nomenclature of Disaccharides

In the formation of disaccharide at least one monosaccharide molecule must be linked through the glycosidic carbon (C

1). The second monosaccharide is linked to the C

1 of the fi rst monosaccharide

either through C1, C

4 or C

6 of the second monosaccharide. Thus, on the basis of these linkages the

disaccharides are classifi ed into three groups. They are as follows:

Nonreducing disaccharides

Disaccharides linked through the C1 of one monosaccharide to C1 of other monosaccharide

Like simple glycosides these disaccharides, which do not have free carbonyl group, are called nonreducing disaccharides. The reducing carbon of each monosaccharide is involved in the glycosidic linkage of the two monosaccharides. Common examples are sucrose and trehalose.



Reducing disaccharides

Disaccharides linked through C1 of the first monosaccharide and C4 of the second monosaccharide componentOne monosaccharide unit is linked by the reducing carbon at C

1 to the nonreducing carbon C

4 of

the second monosaccharide unit. Hence, the reducing carbon at C1 of the second monosaccharide is

free, i.e. it can be easily opened to hemiacetal function and thus such disaccharides reduce Fehling’s solution, undergo mutarotation in solution and form osazones. Therefore, such disaccharides are known as reducing disaccharides. Examples are lactose and maltose.

They are called glycosylaldoses or glycosylketoses. Either of the anomers (α or β) of the glycosidic unit may be involved, and therefore, two isomers arise.

Disaccharides linked through C1 of the first monosaccharide and C6 the second monosaccharide componentOne monosaccharide unit is linked by the reducing carbon at C

1 to the nonreducing carbon C

6

of the second monosaccharide unit. Hence, like the above type of disaccharides, they are called reducing disaccharides. Examples are gentiobiose and melibiose.

Nomenclature of disaccharides

The main use of this nomenclature is to emphasize structural and confi gurational relationship.

A simple way of naming the disaccharides is as follows:

1. The reducing sugars are named as glycosylaldoses or glycosylketoses. Thus, maltose is named as 4-O-α-D-glucopyranosyl-D-glucopyranose and gentiobiose is named as 6-O-β-D-glucopyranosyl-D-glucopyranose.

2. The nonreducing sugars are named as glycosylaldosides or glycosylketosides. Trehalose is named as α-D-glucopyranosyl-α-D-glucopyranoside.

The conventions in the systemic nomenclature of the oligosaccharides are described below: 1. Give the confi guration (or) at the anomeric carbon linking the fi rst monosaccharide unit (on

the left) to the second. 2. Name the nonreducing residue, and to distinguish fi ve- and six-membered ring structures,

insert furano or pyrano into the name.

Disaccharides

ReducingNonreducing

(C – C glycosidic linkage)11

Example: Sucrose

glycosidic linkage)(C – C1 4 glycosidic linkage)(C – C1 6

Example: Lactose Example: Gentiobiose



3. Indicate in parentheses the two carbon atoms joined by the glycosidic bond, with an arrow connecting the two numbers; for example, (1→4) shows that C-1 of the fi rst named sugar residue is joined to C-4 of the second.

4. Name the second residue. If there is a third residue, describe the second glycosidic bond by the same convention (to shorten the description of complex polysaccharides, abbreviations for the monosaccharides are often used).

Following this convention for naming oligosaccharides, maltose is α-D-glucopyranosyl-(1→4)-D-glucopyranose. In the abbreviated nomenclature, maltose is Glc(α1→4)Glc.

5.3 Determination of the Structure of Disaccharides

The normal procedure for determining the structure of disaccharides can be summarized as follows:

1. Determination of the nature of the disaccharides (reducing or nonreducing). 2. Acid hydrolysis of the disaccharide to yield two monosaccharides. 3. Determination of the size of the ring (pyranose or furanose) of each of the monosaccharide

obtained upon hydrolysis. 4. The point of linkage of one monosaccharide with the other carried out by methylating the

diasaccharide, hydrolysing it into monosaccharide followed by oxidation. From the oxidation products points (3) and (4) are settled.

5. The anomeric confi guration (α or β) of the monosaccharides is performed on the basis of the specifi city of the enzymes. The enzyme maltase (α-glucosidase) hydrolyses only the α-D-glycosides while the enzyme emulsin (β-glycosidase) hydrolyses only β-glycoside linkage.

6. Finally, the proposed structure is confi rmed by synthesis.

5.4 Disaccharides of Pharmaceutical Importance

Sucrose (synonyms: invert sugar, α-D-glucopyranosyl-β-D-fructofuranoside, beet sugar, cane sugar, sugar, saccharose, refi ned sugar):

OH

CH OH2C

C

C

C

C

H

H

H

HO

OHH

OH

O

HO

H

H

H

OH

O

CH OH2CH OH2

C

C

C

C

1

2

3

4

5

6

1

2

3

4

5

6



Sucrose is a disaccharide with nonreducing property and is composed of glucose and fructose through an α-(1,2)-β-glycosidic bond. It is also called table sugar obtained from variety of sources such as sugar cane (Saccharum offi cinarum, Linn, Gramineae), sugar beet (Beta vulgaris, Linn, Chenopodiaceae) and other sources. It is optically active and found to exist in two forms. The high sweetness of fructose combined with that of glucose means invert sugar provides a cheaper, less calorifi c food sweetener than sucrose. It is a white, dry, monoclinic, sphenoid crystals, crystalline mass or powder, odourless and sweet taste. Melts with charring and emits characteristic odour of caramel. Well soluble in water, slightly soluble in alcohol, moderately soluble in glycerol, pyridine and practically insoluble in chloroform.

Sucrose on hydrolysis yields invert sugar, which is equimolar amount of glucose and fructose. It is used as a sweetening agent and is a starting material in fermenting, production of glycerol, citric acid, butanol, ethanol and levulinic acid. In pharmaceuticals, it is used as preservative, antioxidant excipients, granulating agent and coating agent for tablets.

Sucrose has a specifi c rotation of +66.5°, which on hydrolysis yields D(+) glucose, which has specifi c rotation of +52.5° and D(–)fructose with a specifi c rotation of –92° giving the net negative value of –20°. Now, as the specifi c rotation value of fructose is high as compared to glucose as well as the parent compound sucrose, the mixture after hydrolysis will be on the whole, levorotatory. Furthermore, because the direction of rotation is reversed (or inverted), the mixture of sugars formed on hydrolysis of sucrose with a specifi c rotation of –20°, the sugar sucrose is known as invert sugar.

1. First is the stable form called sucrose A (melting point: 184–185°C). 2. Second is the unstable form called sucrose B (melting point: 169–170°C).

Chemical tests 1. Molisch’s test: Sucrose gives a purple colour when treated with α-naphthol and concentrated

sulphuric acid. 2. When sucrose is heated it melts, swells up and burns, giving an odour of burnt sugar and

leaving a bulky carbonaceous residue.


1. Molecular formula: Molecular formula of sucrose is found to be C12

H22

O11

. 2. Acid hydrolysis: Upon hydrolysis with acids or enzymes, sucrose gives equal parts of

D-glucose and D-fructose, which thus constitute the two monosaccharide units of hexose. 3. Linkage between two monosaccharide units: Carbonyl group of both the

monosaccharides is involved in linkage, i.e. the glucose is linked via its C1 (–CHOH) to the

C2 (CH

2OH C–OH) of fructose. This is indicated by the inability of sucrose, which neither

reacts with phenyl hydrazine nor reduces Fehling’s solution. 4. Enzyme hydrolysis: Sucrose is hydrolysed by maltase but not by emulsin, thus indicating the

presence of α-D-glucose unit. On the other hand, sucrose is also hydrolysed by an enzyme taka-invertase, which is believed to be specifi c for β-fructofuranosides, thus indicating a β-D-fructofuranose unit in sucrose. The above stereochemical aspects have been confi rmed by Hudson’s isorotation rule.



5. Ring size of glucose and fructose: The size of glucose and fructose units is to be established to assign the complete structure of sucrose. It is performed by usual methods. Complete methylation of sucrose with dimethyl sulphate in basic solution followed by hydrolysis gives 2,3,4,6-tetra-O-methylglucose and 1,3,4,6-tetra-O-methylfructose. The structure of these products indicates that glucose residue in sucrose is a glucopyranose, while fructose residue is in the form of fructofuranose.

6. Structure of sucrose: The following structure has been proposed for sucrose which accounts for all the above facts.

H

OH

H

OH

H

H

OH

OH

O

CH OH2O

H

OH

OH

H

H

CH OH2

CH OH2

Haworth structure

OH

CH OH2CH

H

H

HO

OHH

OH

O

HO

H

H

H

OH

O

CH OH2CH OH2

Fischer structure

HOOH

HOH C2H

HOH

O

HH

H

O

HOH C2

O

H

OH

OH

H

H

CH OH2

Conformational structure

Methylation followed by oxidation of sucrose is outlined below:

OH

CH OH2CH

H

H

HO

OHH

OH

O

HO

H

H

H

OH

O

CH OH2CH OH2

OCH3

OCH3

H CO3

CH OCH2 3H

H

H

H

H

C

O

O

H CO3

H

H

H

OCH3

O

CH OCH2 3CH OCH2 3

Sucrose

(CH ) SO3 2 4

NaOH

Octa- -methyl sucroseO



CH OCH2 3

HO

H CO3

H

H

H

OCH3

O

CH OCH2 3

�

H

H

H CO3

H

H

OH

H

OCH3

OCH3

C

CH OCH2 3

O

2,3,4,6 - Tetra- -methyl- -glucose

O

D

1,3,4,6- Tetra- -methyl- -fructose

O

D

dil. HCl

(Hydrolysis)

This structure for sucrose is confi rmed by several physical and chemical evidences.

Periodic acid oxidation: Sucrose (I) consumes 3 moles of periodic acid and forms 1 mole of formic acid and a tetra-aldehyde (II). Oxidation with bromine followed by acid hydrolysis gives glyoxalic acid (III), glyceric acid (IV) and hydroxypyruvic acid (V). This confi rms the structure of sucrose (except the nature of the glycosidic link).

3H IO5 6 HCOOH

CHO

CHO

C

CH OH2

H

H

O

O

H

CH OH2

CHO

CHO

O

CH OH2

�

I) Br –waterii) Hydrolysis

2

CHO

COOH

�C

COOH

OHH

CH OH2

� 2

CH OH2

COOH

CO

I II

IIIIVV

CH OH2

OHO

H

OHH

H

H

H

OH

1

2 3

C

O

HO

H

H

H

OH

CH OH2

CH OH2

O

X-ray analysis: The X-ray analysis of sucrose sodium bromide dihydrate confi rms the stereochemical confi guration and also the fi ve-membered ring of fructose.



Synthesis of sucrose: Further, the above structure of sucrose is confi rmed by the following synthesis.

CH OAc2

H

OAc

Pcl5

O

H

OAc

H

H

OAc

OAc

H

H

OAc

CH OAc2

H

OAc

H

H

OAc

OCl

H

H

OAc

CH OAc2

H

OAc

H

O

O

H

OHi) NH inether

3

ii) NH inbenzene

3

Opening ofthe oxide ring

CH OAc2O

OH H

OAc

OAc

H

H

CH OAc2

H

OAc

H

OAc

H

H

OH

O

OAcCH2

�

�

Heat in asealed tubeat 100°C for104 h

CH OAc2

H

OAc

H

OAc

H

H

OH

O

O

OH CH OAc2 H

OAc

H

H

OAc

CH OAc2

Removal ofacetyl group

Sucrose

1,3,4,6-Tetra- -acetyl--fructofuranose

O D

1,2-Anhydro- − -glucopyranose3,4,6-triacetate

α DTetra- -acetyl- − -glucoseO β D

H

Lactose (Synonyms

Lactin, milk sugar, sugar of milk, saccharum lactis, 4-β-D-galactopyranosyl-D-glucopyranose):

Lactose

Or

H

OHO

CH OH2

H

OH

H

H

OH

H H

O H

OH

CH OH2

H

OH

OH

H

H

O

1

23

4

5

6

1

23

4

5

6CH OH2

HC

C

C

C

C

CHHO

H

H

OH

O

H

H

HO O

H

HO

HO

H

H

H

O

CH OH2

OH

1

2

3

4

5

6

C

C

C

C

1

2

3

4

5

6



Lactose is a natural disaccharide found exclusively in the milk of mammals and consists of galactose and glucose in a β-(1,4) glycosidic bond. It is an odourless, white or almost white, crystalline powder, having faintly sweet taste. It is stable in air but readily absorbs odours. Several varieties of lactose are available such as (1) anhydrous α-lactose, (2) α-lactose monohydrate, and (3) anhydrous β-lactose. Cow’s milk contains 4–6% and human milk contains 5–8% of lactose.

It is used as fi ller or diluent in tablets, capsules, infant feed formulae and in dry powder inhalations. To prepare sugar-coating solutions, it is used in combination with sucrose. It is also used in the preparation of tablet by wet granulation method.

Chemical tests

1. On warming a mixture of 5 mL of a hot saturated solution of lactose and 5 mL of sodium hydroxide, the liquid becomes yellow and fi nally brownish red. Upon adding several drops of cupric sulphate, a red precipitate of cuprous oxide forms.

2. Dissolve 0.25 g of lactose in 5 mL of water and add 5 mL of 10 M ammonia; then heat the mixture in water bath at 80°C for 10 min. Colour develops.

Structural elucidation 1. Molecular formula: Molecular formula of lactose is found to be C

12H

22O

11.

2. Presence of free carbonyl group: Lactose reduces Fehling’s reagent, Benedict’s reagent and Tollens’ reagent. It also reacts with phenyl hydrazine and forms osazone. Therefore, lactose must possess at least one carbonyl group that is not involved in the disaccharide linkage.

3. Hydrolysis: Upon enzymatic (lactase) hydrolysis or acidic hydrolysis, lactose gives equimolar amounts of glucose and galactose.

4. Nature of linkage between the monosaccharides: Because lactose is hydrolysed only by lactase (identical with emulsin), the two monosaccharide units are linked through β-glycosidic linkage. This is also indicated by its two specifi c rotations.

5. Reducing half of lactose: Methylation of lactose yields methyl heptamethyl lactoside, which on vigorous hydrolysis yields 2,3,4-tri-O-methyl-D-glucose and 2,3,4,6-tetra-O-methyl galactose indicating that the glucose unit constitutes the reducing half of lactose.

6. Oxidation: Lactose on oxidation with bromine water yields lactobionic acid, which upon methylation followed by hydrolysis gives 2,3,5,6-tetra-O-methyl-D-gluconic acid and 2,3,4,6-tetra-O-methyl-D-galactose. This reveals that in lactose, C

4 of glucose is linked to C

1

of galactose. 7. Point of linkage: Further, the point of linkage (C

4) of glucose is confi rmed by osazone

formation and Zemplen degradation.

Or

H

OHO

CH OH2

H

OH

H

H

OH

H H

O H

OH

CH OH2

H

OH

OH

H

H

O

Lactose

CH OH2

HC CHHO

H

H

OH

O

H

H

HO O

H

HO

HO

H

H

H

O

CH OH2

OH

Lactose



HO

OH

HOH C2

O

H

OH

H

O

HH O

HH

HO

H

HOH C2

H H

OH H

OH

Conformational structure of lactose

8. Synthesis: Lactose has been synthesized by the condensation of acetobromogalactose and 1,2,3,6-tetra-O-acetyl-β-D-glucopyranoside.

Maltose

[Malt sugar, 4-(α-D-glucopyranosyl)-α- or β-glucopyranose]:

CHOH

OH

HO

C

O

H

H

H

H

CH OH2CH OH2

C

C

C

C

C

C

C

C

OO

H

H

HO

H

H

OH

OH

H

1

2

3

4

5

6

1

2

3

4

5

6

Maltose, also known as malt sugar, is composed of two glucose monomers in an α-(1,4) glycosidic bond. Acid hydrolysis of starch gives maltose as an intermediate, which upon further hydrolysis, yields glucose. The action of diastase (an enzyme in malt) upon starch also produces maltose. Fermentation of sucrose to ethanol at one stage also produces maltose.

Structural elucidation 1. Molecular formula: Molecular formula of maltose is found to be C

12H

22O

11.

2. Hydrolysis:

(a) Acid hydrolysis: Hydrolysis of maltose yields only D-glucose indicating that glucose is the only monosaccharide unit present.

As maltose reduces Fehling’s, Benedict’s and Tollens’ reagents, it is oxidized by bromine water to maltonic acid (or maltobionic acid), reacts with phenyl hydrazine, forms osazone and exhibits mutarotation. This revels that it must have an aldehyde group in one of the units.

(b) Enzymatic hydrolysis: Maltose is hydrolysed by maltase and by emulsin, which reveals that the linkage joining the reducing half of the maltose to the nonreducing half is an α-linkage.



3. Ring size of the monosaccharides: The size of the ring of monosaccharide units is established as follows:

Maltose is oxidized to maltonic acid (II) (i.e. the latent aldehyde group is oxidized to carboxyl group) upon oxidation with bromine water. Complete methylation of maltonic acid to methyl octa-O-methyl maltonate (III) and then by hydrolysis gives 2,3,4,6-tetra-O-methyl-D-glucopyranose (IV) (from the glucoside component) and 2,3,5,6-tetra-O-methyl gluconic acid (V) (from the alcohol component). The existence of free hydroxyl groups at positions C

1

and C4 in these products reveals that the glycosidic linkage in maltose is involved between

these carbon atoms, and that the original ring size in maltose was pyranose. Therefore, maltose may be written as (I), which explains all the above degradations.

CHOH

H

HO

OH

O

C

H

H

H O

H

H

HO

H

H

OH

H

OH

O

CH OH2CH OH2

H

HO

H

H

OH

H

OH

COOH

CH OH2

O

CH

H

H

H

HO

OH

H

OH

O

CH OH2

COOH

H

CH OCH2 3

O

CCHOH

OH

H CO3

H

H

OCH3

H

OCH3

H

H CO3

H

H

OCH3

H

OCH3

CH OCH2 3

H

H CO3

H

H

COOCH3

OCH3

H

OCH3

CH OCH2 3

O

H

H

H CO3

H

H

O

OCH3

H

OCH3

CH OCH2 3

HC1

Methyl ester of octa- -methylmaltonic acid (III)

O

(CH ) SO3 2 4

Maltonic acidMaltose

Br –water2



CHOH

O

H

HO

OH

O O

H

H

H

H

H

H

H

HO

OH

H

OH

CH OH2CH OH2

C CHOH

HO

H

H OH

H

O

O

H

H

H

H

HO

C

O

CH OH2CH2

CHOH

H

H OH

O O C

O

H

H

H

H

HO

OH

H

OH

(Wohl degradation)(i) NH OH2

Maltose (forms osazone) Glucosidoarabinose (forms osazone)

(i) NH OH(ii) Ac O(iii) C H ONa

2

2

2 5

(Wohldegradation)

Glucosidoerythrose (does not form osazone)

CH2

CH OH2

(ii) Ac O(iii) C H ONa

2

2 5

OH

H

OH

4. Anomeric nature: Lastly, it must be noted that the presence of free CHOH in maltose causes it to exist in two different anomers (α and β).

H

OH

O

H, OH

CH OH2

H

OH

H

H

OH

CH OH2

H H

O

H

OH

OHH

H

O

Nonreducing half Reducing half

CH OH2CH OH2

H OH

C

O

CHOH

HO H

H

H

O O

H

H

H

H

HO

OH

H

OH

Reducing half Nonreducing half

Or



HOOH

HOH C2H

HOH

O

HH

H

O

HHOH C2

O

H

OHOH

HHO

HH

Maltoseor

4-( -Glucopyranosyl)- -glucopyranose

1-(4 -Glucopyranosyl)- -glucopyranose

� �

�

D- -

- -

D

D D

5. Mutarotation: The existence of two anomeric maltoses explains the phenomenon of mutarotation.

6. Synthesis: Lemient et al. have synthesized maltose exactly in the same way as sucrose.

CH OAc2

H

OAc

O

H

HOAc

H O

H

+

H

OH

CH OAc2

H

OAc

H OAc

H

O

OAc

H

CH OAc2

H

OAc

H

OAc

H

H

OAc

O

O

H H

CH OAc2

H

OAc

H

H

OAc

OOAc

H

Ac O2

H

OAc

CH OAc2 CH OAc2

H

OAc

H

O

H

OH

H H

O

H

OAc

H

O

H

OAc

OAc

H

1,2-Anhydro-3,4,6-tri-O-

acetyl- -D-glucopyranoseα1,2,3,6-tetra-O-

acetyl- -D-glucoseβ

Deacetylation

Maltose

Heat at 120°for 13 h

Octa-acetyl- - -maltoseD� Hepta-acteyl- - -maltose� D

Trehalose

(α-D-glucopyranosyl-α-D-glucopyranoside):



H

HO

C

C

C

C

C

H

H

H

OH

H

OH

O

O

C

C

C

CC

C

O

OH

H

OH

H

H

HO

H

H

CH OH2 CH OH2

1

2

3

4

5

6

1

2

3

4

5

6

H

OH

CH OH2

H

OH

H

H

OH

H H

O

O

H OH

OH H

O

H

H

OH

HOH C2

1

23

4

5

6

1

2 3

4

5

6

It is a disaccharide found in mushrooms, yeasts and fungi.

Structural elucidation

1. Molecular formula: Molecular formula of trehalose is found to be C12

H22

O11

. 2. Linkage between two glucose units: It shows the properties of nonreducing

monosaccharides. Thus, the two glucose units are linked to each other through their C-1 hydroxyl groups.

3. Hydrolysis: Upon hydrolysis with dilute hydrochloric acid, it yields two molecules of D-glucose.

4. Acetylation: Upon acetylation it forms octa-acetate, which indicates the presence of eight hydroxyl groups.

5. Nature of ring structure of glucose: On complete methylation followed by hydrolysis, it gives 2 moles of 2,3,4,6-tetra-O-methyl-D-glucopyranose, thus indicating that both of the glucose units are present as pyranose rings in trehalose. These pyranose rings are further confi rmed by periodic acid oxidation (which shows that 4 moles of the acid are used to yield 2 moles of formic acid (for details, refer to page XXX).

6. Nature of linkage: The high specifi c rotation [α]D= +197° indicates that two glucose

residues are linked by α,α′-linkages. 7. Structure of trehalose: Thus, trehalose may be proposed as follows.

H

OH

CH OH2

H

OH

H

H

OH

H H

O

O

H OH

OH H

O

H

H

OH

HOH C2

Haworth structure of trehalose

H

OH

CH

H

H

OH

H

OH

O

O

C

OOH

H

OH

H

H

OH

H

H

CH OH2 CH OH2

Trehalose ( −D-Glucopyranosyl- '-D-Glucopyranoside)α α

This structure has been fi nally confi rmed by synthesis.



�6. TRISACCHARIDES �

These are sugars that upon hydrolysis give three units of monosaccharides. Some trisaccharides occur naturally (gentianose, raffi nose and manninotriose), whereas the others are obtained by partial hydrolysis of polysaccharides (e.g. cellotriose from cellulose and maltotriose from starch).

Trisaccharides may be reducing or nonreducing in nature. Their structures are elucidated as the disaccharides. Some of the trisaccharides, their composition and nature are listed in Table 1.2.

Table 1.2 Trisaccharides, their types and composition

Trisaccharide Constituent monosaccharides Type

Raffi nose Fructose–glucose–galactose Nonreducing sugar

Gentianose Fructose–glucose–glucose Nonreducing sugar

Mannotriose Galactose–galactose–glucose Reducing sugar

Galactose–rhamnose–rhamnose Reducing sugar

6.1 Gentianose

O

H

CH2

H

OH

H

OH

H

H

OH

OH

O

CH OH2O

H

OH

OH

H

H

CH OH2

D-Fructofuranose

OH

H

H OH

CH OH2

H

OH H

O

1α

1α 2β

D-GlucopyranoseD-Glucopyranose

Gentiobiose

Sucrose

1

23

4

5

6

1

23

4

5

6

1

2

3 4

5

6

It is a nonreducing sugar that gives 2 moles of D-glucose and 1 mole of D-fructose upon hydrolysis. It occurs in gentian roots. Its structure is established exactly in the same manner as that of disaccharides.


1. Molecular formula: Molecular formula of gentianose is found to be C18

H32

O16

. 2. Enzymatic hydrolysis: Upon hydrolysis with invertase it gives D-fructose and gentiobiose,

while with emulsin it yields D-glucose and sucrose. Thus, in gentianose two glucose and one fructose units are combined so as to give gentiobiose and sucrose, i.e. the arrangement of the three monosaccharide molecules is as glucose–glucose–fructose.



3. Structure of gentianose: As the structure of glucose and fructose are known, gentianose structure is drawn as below:

O

H

CH2

H

OH

H

OH

H

H

OH

OH

O

CH OH2O

H

OH

OH

H

H

CH OH2

D-Fructofuranose

OH

H

H OH

CH OH2

H

OH H

O

1α

1α 2β

D-GlucopyranoseD-Glucopyranose

Gentiobiose

Sucrose

�7. TETRASACCHARIDES �

These are sugars that on hydrolysis give four units of monosaccharides. Some tetrasaccharides occur naturally (stachyose), whereas the others are obtained by partial hydrolysis of polysaccharides. Tetrasaccharides may be reducing or nonreducing in nature. Their structure is elucidated similar to disaccharides.

7.1 Stachyose

OH

H

H OH

12

3

4 5CH OH2

H

OH H

O

123

4

5

6

H

O

1α

CH2

H

OHH

H

OH

OH

H

O

6

O

H

CH2

H

OH

H

OH

H

H

OH

O

1

23

4

5

6

H

O

CH OH2O

H

OH

OH

H

H

CH OH21

2

34

5

6

D-Galactopyranose D-Galactopyranose D-Glucopyranose D-Fructofuranose

Stachyose occurs in the roots of several plant species and is an important example of tetrasaccharides.




1. Molecular formula: Molecular formula of stachyose is found to be C24

H42

O21

. 2. Enzymatic hydrolysis: On enzymatic hydrolysis, stachyose gives sucrose and raffi nose. 3. Structure of stachyose: Its exact structure has been established by methylation and

hydrolysis, when it gives 2,3,4,6-tetra-O-methyl-D-galactose, 2,3,4-tri-O-methyl-D-galactose, 2,3,4-tri-O-methyl-D-glucose and 1,3,4,6-tetra-O-methyl D-fructose. Thus the structure of stachyose is as proposed as follows.

OH

H

H OH

12

3

4 5CH OH2

H

OH H

O

123

4

5

6

H

O

1α

CH2

H

OHH

H

OH

OH

H

O

6

O

H

CH2

H

OH

H

OH

H

H

OH

O

1

23

4

5

6

H

O

CH OH2O

H

OH

OH

H

H

CH OH21

2

34

5

6

D-Galactopyranose D-Galactopyranose D-Glucopyranose D-Fructofuranose

�8. POLYSACCHARIDES �

The term polysaccharide is usually applied to polymers having more than 10 monomer units. Polysaccharides usually contain 100–90,000 monosaccharide units. Polysaccharides are defi ned as high molecular weight (16,000–14,000,000) carbohydrates obtained by condensation of monosaccharides. They yield ultimately monosaccharides on complete hydrolysis. Some polysaccharides are linear polymers, while others are highly branched. They are also called glycans.

In living organisms, polysaccharides function as food reserves and structural elements. Plants accumulate starch as their main food reserve, a material that is composed entirely of glucopyranose units. The mammalian carbohydrate storage molecule is glycogen.

8.1 Classification and Nomenclature of Polysaccharides

Polysaccharides may be classifi ed into two groups according to their constituent sugars.

Homopolysaccharides

It contains only one kind of monosaccharide unit and they are also called homoglycans. Some homopolysaccharides serve as storage forms of monosaccharides that are used as fuels (e.g. starch and glycogen). Other homopolysaccharides, e.g. cellulose and chitin serve as structural elements in plant cell walls and animal exoskeletons.



Biological importance 1. Inulin is used in physiological investigation for determination of the rate of glomerular

fi ltration rate. 2. Inulin has also been used for estimation of body water extracellular fl uid volume. 3. Dextrin solutions are often used as ‘mucilages’. 4. Dextran solutions having molecular weight of approximately 75,000 have been used as

plasma expander. 5. Agar is used as laxative in constipation. 6. Agar is used in agar plate for media culture.

Heteropolysaccharides

These are known as heteroglycans, and contain more than one kind of monosaccharide units (e.g. α-heparin, chondroitin sulphate A and hyaluronic acid). Jeanloz coined the name ‘glycosaminoglycans’ to describe this group of substances. They are usually composed of amino sugar and uronic acid units as the principal components.

Heteropolysaccharides provide extracellular support for organisms of all kingdoms. For example, the rigid layer of the bacterial cell envelope (the peptidoglycan) is composed in part of a heteropolysaccharide built from two alternating monosaccharide units. In animal tissues, the extracellular space is occupied by several types of heteropolysaccharides, which form a matrix that holds individual cells together and provides protection, shape and support to cells, tissues and organs.

Biological importance

1. The invasive power of some pathogenic organisms may be increased because they secrete hyaluronidase. In the testicular secretions, it may dissolve the viscid substances surrounding the ova to permit penetration of spermatozoa.

2. Clinically, the enzyme is used to increase the effi ciency of absorption of solutions administered by clysis.

Polysaccharides may also be classifi ed into two groups according to their biochemical importance.

1. Reserve polysaccharides: Those which contain reserve carbohydrates stored up by plants or animals (e.g. glycogen, starch and inulin).

2. Skeletal polysaccharides: Those which serve as skeleton in plants and animals (e.g. cellulose).

Nomenclature

Earlier, polysaccharides were named on the basis of their source, which is not systemic. Now the suffi x ‘an’ is used after the name of the sugar which is a monomeric unit, i.e. ‘ose’ is replaced by an. For example, fructans are the polysaccharides containing fructose units. The general name for polysaccharides is glycans.

Starch (Synonyms: amylum, amido, amidon, amilo)

Starch is the most important source of carbohydrates in the human diet. It occurs as a reserve food material of plants, i.e. it is the form in which glucose is stored. The latter is mobilized for metabolic uses by enzymatic breakdown. The different sources of starch are grains of maize (Zea mays, Linn, Gramineae/Poaceae), rice (Oryza sativa, Linn, Gramineae/Poaceae), wheat (Triticum aestivum, Linn,



Gramineae/Poaceae), tubers of potato (Solanum tuberosum, Solanaceae) and arrow root (Maranta arundinacea, Marantaceae).

It is a very fi ne powder, white or slightly yellowish powder or irregular, angular white masses readily reducible to powder, which cracks when pressed between the fi ngers. It is odourless and tasteless. In addition to its use as a source of heat and energy to body it is also used for the manufacture of glucose, dextrin. It is also used in oral solid dosage formulations as an excipient (as a binder, diluent and disintegrant).

Starch contains chemically two different polysaccharides, amylose [α-amylose (about 25%)] and amylopectin [α-amylose (about 75%)]. Amylose is insoluble in water and amylopectin is water soluble, but swells in water and is responsible for the gelatinizing property of the starch. Soluble starch is obtained by partial acid hydrolysis and is completely soluble in hot water. α-Amylose is insoluble in water and gives blue colour. Amylopectin is soluble in water and gives red to violet colour with iodine. The blue colour is due to the entrapping of iodine molecules between the chains of α-amylose. As the blue colour of ordinary starch is entirely due to the adsorptive power of the α-amylose present, this property is used to estimate the proportion of amylose in starch.

Chemical tests 1. Boil about 10 gm of starch with 50 mL of water and cool. All starches produce thin and

cloudy mucilage except potato starch which gives thick and more transparent mucilage. 2. The above prepared mucilage upon addition of 0.05 mL of iodine turns deep blue. The blue

colour disappears on warming and reappears on cooling. 3. On water bath heat the starch solution with dilute hydrochloric acid for 10 min. Neutralize

the excess of acid then by adding 2–3 drops of sodium hydroxide solution. To this mixture add 2 mL of Fehling’s solution and heat it on water bath for 15 min. A brick red-coloured precipitate is formed.

Structural elucidation 1. Molecular formula: Molecular formula of starch is found to be (C

6H

10O

5)

n.

2. Presence of two structurally different polysaccharides (amylose and amylopectin): The starch solution when treated with water at 70°C, the water soluble fraction diffuses out from starch. When n-butanol is added to the hot solution of starch and the mixture is allowed to cool to room temperature, the α-amylose fraction is precipitated as complex with n-butanol. The β-amylose fraction (amylopectin) remains in the mother liquor from which it can be precipitated by adding methanol. α-Amylose is insoluble in water and amylopectin is soluble in water. This proves that the starch consists of two structurally different polysaccharides.

3. �-Amylose/A-fraction/amylose and �-amylose/B-fraction/amylopectin: Most starches consist of nearly 20% amylose and 80% amylopectin.

To establish the structure of starch, let us discuss the constitution of amylose and amylopectin.

Constitution of �-amylose

1. Molecular formula: Molecular formula of amylose is found to be (C6H

10O

5)

n.

2. Hydrolysis:

(a) Acid hydrolysis: Hydrolysis of amylose with acids yields D-glucose in quantitative manner. This indicates that amylase is composed of only D-glucose units.



(b) Enzymatic hydrolysis: Enzymatic hydrolysis of amylose by diastase yields the quantitative yield of maltose. As maltose is 4-O-α-D-glucopyranosyl-D-glucopyranose, all the glucose units in starch are linked through C

1 and C

4. Therefore, the following structure is proposed

for amylose, which explains the hydrolysis products.

O

H

OH

HH H

OH

HO

O

H

OH

HH H

OH

HO

CH OH2

O

H

OH

HH

OH

HO

O

H

OH

H

OH

H

O

H

OH

H

O

H

OH

H

O

H

OH

H

OH

H H

OH

H

O

H

OH

HH

OH

HO

OH

O

H

OH

H

OH

H

OH

HH, OH

Diastase H+C1

Starch

C1 C4

CH OH2 CH OH2 CH OH2CH OH2

H H

O

H H

O

C4

CH OH2 CH OH2

H O2 H O2

CH OH2

Linear chain nature of amylose: Hydrolysis of the fully methylated derivative to 2,3,6-tri-O-methyl-D-glucopyranose and the acetolysis of its methylated derivative to malto-triose and malto-tetrose derivatives confi rms the structure of amylose. Moreover, the percentage (0.32%) of 2,3,4,6-tetra-O-methyl-D-glucopyranose reveals that amylose has a chain length of 300–350 glucose units. As no dimethyl glucose is obtained, the chain must be linear and not branched.

The linear structure of the amylose is indicted by the high positive rotation; it is also further indicated by the fact that amylose acetate forms fi lms and fi bres like cellulose. The chain length (300–350) of glucose is confi rmed by the molecular weight determination by mass spectra.

Constitution of amylopectin

1. Molecular formula: Molecular formula of amylopectin is found to be (C6H

10O

5)

n.

2. Hydrolysis:

(a) Acid hydrolysis: Amylopectin upon acid hydrolysis yields D-glucose units quantitatively; it indicates that it is also composed of D-glucose units.

(b) Enzymatic hydrolysis: Hydrolysis of amylopectin by β-amylase yields 55% of maltose and high molecular weight compound called limit dextrin. This reveals that amylopectin has some other bonds (which are not attacked by β-amylase) in addition to 1,5-linkages (which are attacked by δ-amylase).

(c) Hydrolysis of methylated amylopectin: Upon hydrolysis, fully methylated amylopectin gives the following:

(i) 90% of 2,3,6 tri-O-methyl glucose; (ii) 4% of 2,3,4,6-tetra-O-methyl glucose; (iii) 4% of 2,3-di-O-methyl glucose.



This reaction leads to the following important conclusions:

1. Chain length of branches: The yield (4%) of tetra-O-methyl glucose reveals that the average chain length of branches is about 25 glucose units.

2. Number of branches:

The formation of 2,3-dimethyl glucose suggests that amylopectin is a branched polymer. The �branch chain is linked through C

6 of glucose unit.

The branched structure is further indicated by the fact that osmotic pressure measurements of �amylopectin affords a molecular weight of about 500,000, which corresponds to nearly 3000 glucose units.The total number of glucose units (3000) and the average number of glucose units (25) in chain �length of amylopectin further indicate that amylopectin has nearly 120 branches.

Nature of linkage: The formation of 2,3-di-O-methyl glucose reveals the presence of branched structure of amylopectin. This also indicates that the branch chain is linked through C

6 carbon

atom.

The branched structure of amylopectin is supported by the following evidences:

1. Amylopectin acetate does not form fi bres, a characteristic feature of linear molecules (unlike cellulose and amylase acetates).

2. Upon hydrolysis with β-amylase, amylopectin gives only 55% of maltose. Thus, there are block points which are not attacked by this enzyme. These block points occur at the branch points.

3. As the tetramethyl glucose and the dimethyl glucose are formed almost in equal amounts there would be one branch point for each end group.

4. Partial enzymatic hydrolysis of amylopectin also yields a small amount of isomaltose (1,6-α-linked diglucose) in addition to the normal product maltose (1,4-α-linked diglucose), which indicates that C

1 of one glucose unit and C

6 of the other glucose unit are involved in

branching, i.e. branching involves 1,6-linkages.

O

H

OH

H

OH

H

H

O

H

OH

H

H

OH

OH

H

OH

H, OH

Maltose

O

H

OH

H

OH

H

H

OH

H

O

O

CH2

H

OH

H

OH

H

H

OH

H, OH

Isomaltose

CH2OH CH2OH CH2OH



5. The 1,6-linkages are further confi rmed by periodate oxidation method. 6. From the foregoing points, structure of amylopectin may be represented as follows:

Amylopectin

O

H

OH

H

OH

H H

OH

H

CH2OH

O

H

OH

HH H

OH

HO

O

H

OH

HH H

OH

HO

O

H

OH

HH

O

H

OH

HO

O

O

H

OH

HH H

OH

HO

O

H

OH

HH H

OH

HO

CH2OH CH2OH CH2

CH2OH CH2OH

7. Origin points: The points of origin of the branches are still not settled in amylopectin. Various structures have been suggested and the two most acceptable structures are described below:

Haworth laminate structure: According to this, every chain is composed of nearly 24–25 glucose units and the branch arises from the middle of the chain.

Thus, β-amylase hydrolyses only half of the total glucose units of amylopectin to form maltose and the residue as limit dextrin. Because after this the 1,6-linkages start to which β-amylase is not effective. Using end group assay it has been observed that dextrin has a unit chain length of 11–12 glucose units to support the above explanation.

O

O

O

O

O

1212

1212

1212

121212

12

12

12

12

= Non reducing groups or end groups giving 2,3,4,6-tetramethyl glucopyranoses

= Chains of α-1,4 linked glucopyranoses giving 2,3,6-trimethyl glucopyranoses= Reducing end groups

= -1,6-Linkages giving 2,3-dimethyl glucopyranosesβ

O

Amylopectin

Maltose +

Limit dextrin

β–amylase



Meyer random structure: According to this, amylopectin is a multibranched structure and which explains the behaviour of amylases on amylopectin in a better way.

O

O

O

OOO

O O

O

Enzymatic hydrolysis of starch: By attacking the 1,4-linkages of nonreducing end, β-amylase hydrolyses the amylopectin of the outer chains to about 60% maltose and the residual polysaccharide is known as limit dextrin. But the enzyme α-amylase attacks 1,4 glycosidic linkages at random, i.e. indiscriminately in both inner and outer chains of amylopectin.

Lastly, the enzyme isoamylase from yeasts or R-enzyme from higher plants attacks the 1,6-glycosidic linkages only. The hydrolytic scheme of starch and its reaction with iodine are shown below:

Course of Hydrolysis: Reaction with IodineStarch Blue

Soluble starch Blue

Amylodextrin Purple

Erythrodextrin Red

Achrodextrin Colourless

Maltose

Cellulose

It is reputedly the most abundant organic material on earth, being the main constituent in plant cell walls. It is composed of glucopyranose units linked β1→4 in a linear chain.



�PROBABLE QUESTIONS �

1. Defi ne and classify carbohydrates with suitable examples. Enumerate the various chemical reactions exhibited by the reducing sugars and explain any two of them.

2. Explain Kiliani’s synthesis of a higher monosaccharide from the lower one. 3. How is the cyclic nature of glucose established? 4. Write in detail about the chemistry and structure of glucose. 5. How many isomers of D-glucose are possible? Draw their confi gurational formulae and explain the methods used to

determine the size. 6. How will you determine the structure of disaccharides? 7. Defi ne and classify polysaccharides and write their biological importance. 8. Write in brief about the structural elucidation of starch. 9. Explain the steps in the development of complete structure of D-glucose. 10. Briefl y describe the methods for determination of ring structure of hexose sugar. 11. Describe the preparation of glucosazones. 12. Defi ne mutarotation and explain it with chemical reaction. 13. Describe the experimental evidence for the ring structure of glucose. 14. Write the general methods used in establishing the cyclic structure of hexose. 15. How is the confi guration of monosaccharide determined? 16. Mention some examples for disaccharides and describe the structural determination of any one of the disaccharides. 17. Write in detail about the following reactions:

a. Acetylation of hexoseb. Preparation of osazone

18. What are the different disaccharides? Describe the structural elucidation of maltose. 19. Describe the confi gurational formula of fructose. Indicate the evidence leading to the ring structure of fructose. 20. How will you prove the structure of sucrose? 21. Write in detail the general methods for synthesis of glycosides. 22. Outline the constitution of starch. 23. How are amylose and amylopectin separated from natural starch? Describe the constitution of amylose and

amylopectin. 24. Write a brief note on the following:

a. Epimerizationb. Photosynthesis of carbohydratesc. Mutarotation

25. Answer any three of the following:a. Why glucose and mannose form the same osazone?b. How will you convert D(+)-glucose into mannose?



c. What are the stages involved in the conversion of glucose to arabinose?d. How can you show whether a given hexose is pyranose or furanose?e. How will you prove that fructose is a 2-ketohexose and not a 3-ketohexose?

26. Write equations to show how D(+)-glucose can be converted into 2,3,4,6-tetramethyl-D-glucose. 27. Describe the importance of methylation and periodate oxidation in the structural determination of carbohydrate

chemistry. 28. Describe the evidence in support of the accepted structure of maltose. 29. What is the other name for sucrose? Explain why this name has been given. 30. Write the various methods adopted for synthesis of monosaccharides by descending in sugar series. 31. Write a note on confi guration of monosaccharides. 32. Write the various methods adopted for synthesis of monosaccharides by ascending in sugar series. 33. Enumerate the methods adopted for the determination of the ring size in sugars and explain any one of them with suitable

example. 34. How will you prove that glucose exists as ring structure and not as open chain structure? 35. How will you classify disaccharides? Mention few disaccharides and write the biological importance of disaccharides. 36. Explain the following with suitable examples:

a. Kiliani’s synthesisb. Osazone reactionc. Epimerizationd. Mutarotatione. Invert sugar

�SUGGESTED READINGS �

Apsimon, J (ed) (1973). The Total Synthesis of Natural Products, Vol I. Wiley-Interscience, New York, pp. 1–80.

Aspinall, GO (ed) (1982). The Polysaccharides, Vol 1. Academic Press, Inc., New York.



Bernfeld, P (ed) (1967). Biogenesis of Natural Compounds, 2nd Ed. Pergamon, Oxford.

Bourne, EJ and Finch, P (1970). Polysaccharides—enzymic synthesis and degradation. R Inst Chem Rev 3 (1), 45–60.

Budzikiewicz, H, Djerassi, C and Williams, DH (1964). Structure Elucidation of Natural Products by Mass Spectrometry, Vol 2. Holden-Day, Inc., San Francisco.

Capon, B (1969). Mechanism in carbohydrate chemistry. Chem Rev 69: 407–498.

Chaplin, MF and Kennedy, JF (eds) (1994). Carbohydrate Analysis: A Practical Approach, 2nd Edn. IRL Press, Oxford.

Coffey, S (1967). Rodd’s Chemistry of Carbon Compounds, Vol. I, Part F, 2nd Ed. Elsevier, Amsterdam.

Collins, PM and Ferrier, RJ (1995). Monosaccharides: Their Chemistry and Their Roles in Natural Products. John Wiley & Sons, Chichester, England.

Cornejo, CJ, Winn, RK and Harlan, JM (1997). Antiadhesion therapy. Adv Pharmacol 39: 99–142.

de Mayo, P (ed.) (1964). Molecular Rearrangements. Part II, Interscience, New York.



Eeliel, E et al. (1965). Conformational Analysis. Interscience, New York.

Ferrier, RJ and Overend, WG (1959). Newer aspects of the stereochemistry of carbohydrates. Quart Rev 13: 265.

Florkin, M and Stotz, EH (eds) (1963). Comprehensive Biochemistry, Vol 5. Elsevier.

Franz, G (1989). Polysaccharides in pharmacy: current applications and future concepts. Planta Med 55: 493–497.

Geissman, TA and Grout, DHG (1969). Organic Chemistry of Secondary Plant Metabolism. Freeman, Cooper and co., San Francisco.

Gilman, H (1943). Organic Chemistry: An Advanced Treatise, Vol 1. Wiley, New York.



Guthrie, VRD and Honeyman, J (1964). An Introduction to the Chemistry of Carbohydrates. Clarendon Press, Oxford, pp. 72–76.

Guthrie, VRD and Honeyman, J (1968). An Introduction to the Chemistry of Carbohydrates, 3rd Ed. Clarendon Press, Oxford.

Honeyman, J (1959). Recent Advances in the Chemistry of Cellulose and Starch. Interscience, New York-London.

Hopkinson, SM (1969). The chemistry and biochemistry of phenolic glycosides. Quart Rev 23: 98.

Hudson, CS (1941). Emil Fischer’s discovery of the confi guration of glucose. J Chem Educ 18: 353–357.

Honeyman, J (1959). Recent Advances in the Chemistry of Cellulose and Starch. Heywood and Co, London.

Jay, A (1996). The methylation reaction in carbohydrate analysis. J Carbohydr Chem 15: 897–923.

Morrison, RT and Boyd, RN (1992). Organic Chemistry, 6th Ed. Benjamin Cummings, San Francisco.

Opdenakker, G and Dwek, RA (1997). Oligosaccharide sequencing technology. Nature 388: 205–207.

Perciva, EGV (1962). Structural Carbohydrate Chemistry. J. Garnet Miller, London.

Pigman, WW and Goepp, RG (1957). Chemistry of the Carbohydrates. Academic Press, New York.

Pigman, W and Horton, D (eds) (1970, 1972, 1980). The Carbohydrates: Chemistry and Biochemistry, Vols IA, IB, IIA and IIB. Academic Press, Inc., New York.

Rees, DA and Scott, WE (1971). Polysaccharide conformation part VI. J Chem Soc (B), 469.

Weymouth-Wilson, AC (1997). The role of carbohydrates in biologically active natural products. Nat Prod Rep 14: 99–110.

Wolfe, S et al. (1971). A Theoretical study of the Edward-Lemieux effect (the Anomeric Effect). J Chem Soc (B), 136.

(1969). Progress in Stereochemistry, Vol 4. Butterworths.


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