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CARBOHYDRATES
Carbohydrates were once considered to be “hydrates of carbon” because their molecular formulas in many (but not all)
cases correspond to Cn(H2O)m. More realistic is to define a carbohydrate as a polyhydroxy aldehyde or polyhydroxy
ketone

A monosaccharide is a simple carbohydrate, one that on attempted hydrolysis is not cleaved to smaller carbohydrates.
Glucose.(C6H12O6), for example, is a monosaccharide. They can be grouped according to the number of carbon atoms

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they contain and whether they are polyhydroxy aldehydes or polyhydroxy ketones. Monosaccharides that are polyhydroxy
aldehydes are called aldoses; those that are polyhydroxy ketones are ketoses. Aldoses and ketoses are further classified

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according to the number of carbon atoms in the main chain. ALDOTETROSES
ALDOPENTOSES AND ALDOHEXOSES.

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A MNEMONIC FOR CARBOHYDRATE CONFIGURATIONS:

Aldohexoses: A systematic way to set down all the D-hexoses (as in Fig. 25.2) is to draw skeletons of the necessary eight
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Fischer projections, placing the hydroxyl group at C-5 to the right in each so as to guarantee that they all belong to the D
series. Working up the carbon chain, place the hydroxyl group at C-4 to the right in the first four structures, and to the left
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in the next four. In each of these two sets of four, place the C-3 hydroxyl group to the right in the first two and to the left
in the next two; in each of the resulting four sets of two, place the C-2 hydroxyl group to the right in the first one and to
the left in the second.

All altruists gladly make gum in gallon tanks.

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Aldopentoses:(RAXL is an easily remem-bered nonsense word that gives the correct sequence.)

With four stereogenic centers, 16 stereoisomeric aldohexoses are possible; 8 belong to the D series and 8 to the L series.

CYCLIC FORMS OF CARBOHYDRATES Haworth formulas: FURANOSE FORMS: Haworth contributed to the
discovery that sugars exist as cyclic hemiacetals rather than in open-chain forms. Aldoses incorporate two functional
groups, C=O and OH, which are capable of reacting with each other. nucleophilic addition of an alcohol function to a

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carbonyl group gives a hemiacetal. When the hydroxyl and carbonyl groups are part of the same molecule, a cyclic
hemiacetal. Cyclic hemiacetal formation is most common when the ring that results is five- or six-membered. Five-
membered cyclic hemiacetals of carbohydrates are called furanose forms; six-membered ones are called pyranose forms.

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Aldoses exist almost exclusively as their cyclic hemiacetals; very little of the open-chain form is present at
equilibrium.

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The ring carbon that is derived from the carbonyl group, the one that bears two oxygen substituents, is called the
anomeric carbon. The two stereoisomeric furanose forms of D-erythrose are named α-D-erythrofuranose and β-D-

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erythrofuranose. The prefixes α and β describe relative configuration. The configuration of the anomeric carbon is α when
its hydroxyl group is on the same side of a Fischer projection as the hydroxyl group at the highest numbered stereogenic
cen-ter. When the hydroxyl groups at the anomeric carbon and the highest numbered stereo-genic center are on opposite

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sides of a Fischer projection, the configuration at the anomeric carbon is β.

Substituents that are to the right in a Fischer projection are “down” in the corre-sponding Haworth formula.
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Furanose forms: Ribose, Erythrose
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PYRANOSE FORMS:

All the ring substituents other than hydrogen in β -D-glucopyranose are equatorial in the most stable chair conformation.
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Only the anomeric hydroxyl group is axial in the α isomer; all the other substituents are equatorial. Normally the CH2OH
group is the bulkiest, most conformationally demanding substituent in the pyranose form of a hexose. CH2OH substituent
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to occupy an equatorial orientation.

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Galactose differs from glucose in configuration at C-4. The C-4 hydroxyl is axial in β -D-galactopyranose, but it is
equatorial in β -D-glucopyranose.

In spite of their easy interconversion in solution, α and β forms of carbohydrates are capable of independent existence,
and many have been isolated in pure form as crys-talline solids. When crystallized from ethanol, D-glucose yields α -D-
glucopyranose, mp 146°C, [α]D + 112.2°. Crystallization from a water–ethanol mixture produces β -D-glucopyranose, mp
148–155°C, [α]D + 18.7°.

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Glucose was isolated from raisins in 1747 and by hydrolysis of starch in 1811. Its structure was determined, in work
culmi-nating in 1900, by Emil Fischer.

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MUTAROTATION: On standing, the rotation of the solution containing the α isomer decreases from +112.2° to +52.5°;

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the rotation of the solution of the β isomer increases from +18.7° to the same value of +52.5°. This phenomenon is called
mutarotation.

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Reason: Each solution, initially containing only one anomeric form, undergoes equilibration to the same mixture of α -
and β -pyranose forms. The open-chain form is an intermediate in the process. The distribution between the α (36%) and β
(64%) anomeric forms at equilibrium.

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NOTE: the β-pyranose form is the major species present in an aqueous solution of D-glucose. α-pyranose form
predominates in a solution of D-mannose. IN
The relative abundance of α -and β -pyranose forms in solution is a complicated issue and depends on several factors. One
is solvation of the anomeric hydroxyl group. An equatorial OH is less crowded and better solvated by water than an axial
one. This effect stabilizes the β -pyranose form in aqueous solution. A second factor, called the anomeric effect, involves
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an electronic interaction between the ring oxygen and the anomeric substituent and preferentially stabilizes the axial OH
of the α-pyranose form. operate in different directions but are comparable in magnitude in aqueous solution, the α -
pyranose form is more abundant for some carbohydrates and the β -pyranose form for others.
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KETOSES: a large number of ketoses are known, and several of them are pivotal inter-mediates in carbohydrate
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biosynthesis and metabolism. Examples of some ketoses include D-ribulose, L-xylulose, and D-fructose
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DEOXY SUGARS: In deoxy sugars the hydroxyl group is replaced by hydrogen.

AMINO SUGARS: replacement of a hydroxyl group in a carbohydrate by an

amino group to give an amino sugar. N-Acetyl-D-glucosamine is the main

component of the polysaccharide in chitin, the substance that makes up the tough

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outer skeleton of arthropods and insects.

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anticancer drug doxorubicin hydrochloride (Adriamycin),

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contains the amino sugar L-daunosamine

GLYCOSIDES: (Reaction with alcohol) Glycosides are a large and very important class of carbohydrate derivatives
character-ized by the replacement of the anomeric hydroxyl group by some other substituent. Glycosides are termed O-

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glycosides, N-glycosides, S-glycosides, and so on, according to the atom attached to the anomeric carbon.
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The preparation of glycosides in the laboratory is carried out by simply allowing a carbohydrate to react with an alcohol in
the presence of an acid catalyst:
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NOTE: 1 In glycoside formation despite the presence of a number of other hydroxyl groups in the carbohydrate, only the
anomeric hydroxyl group is replaced. This is because a carbocation at the anomeric position is stabilized by the ring
oxygen and is the only one capable of being formed under the reaction conditions. Once the carbocation is formed, it is
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captured by the alcohol acting as a nucleophile. Attack can occur at either the α or β face of the carbocation. Attack at the
α face gives methyl α -D-glucopyranoside: Attack at the β face gives methyl β -D-glucopyranoside.
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2 All of the reactions, from D-glucose to the methyl glycosides via the carbocation, are reversible. The overall reaction is
thermodynamically controlled and gives the same mixture of glycosides irrespective of which stereoisomeric pyranose

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form of D-glucose we start with. Nor does it matter whether we start with a pyranose form or a furanose form of D-
glucose. Glucopyranosides are more stable than glucofuranosides and pre-dominate at equilibrium.

3 Under neutral or basic conditions glycosides are configurationally stable; unlike the free sugars from which they are
derived, glycosides do not exhibit mutarotation. Converting the anomeric hydroxyl group to an ether function
(hemiacetal → acetal) prevents its reversion to the open-chain form in neutral or basic media. In aqueous acid, acetal
formation can be reversed and the glycoside hydrolyzed to an alcohol and the free sugar.

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Diasccharide: A disaccharide on hydrolysis is cleaved to two monosaccharides, which may be the same or different.
Structurally, disaccharides are glycosides in which the alkoxy group attached to the anomeric carbon is derived from a

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second sugar molecule.

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Maltose, obtained by the hydrolysis of starch, and cellobiose, by the hydrolysis of cellulose, are isomeric disaccharides.
In both maltose and cellobiose two D-glucopyranose units are joined by a glycosidic bond between C-1 of one unit and C-
4 of the other. The two are diastereomers, differing only in the stereochemistry at the anomeric carbon of the glycoside

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bond; maltose is an α-glycoside, cellobiose is a β-glycoside. The configuration at the free anomeric center is variable and
may be either α or β. Indeed, two stereoisomeric forms of maltose have been isolated: one has its anomeric hydroxyl
group in an equatorial orientation; the other has an axial anomeric hydroxyl.

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A different enzyme, emulsin, produces the opposite result: emulsin catalyzes the hydrolysis of cellobiose (β -glucosidase)
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but not of maltose. maltase catalyzes the hydrolytic cleavage of the α-glycosidic (α -glucosidase)bond of maltose

Lactose is a disaccharide constituting 2–6% of milk and is known as milk sugar. one of its monosaccharide units is D-
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glucose. The other monosaccharide unit, the one that contributes its anomeric carbon to the glycoside bond, is D-
galactose. lactose is a β-glycoside. Digestion of lactose is facilitated by the β-glycosidase lactase. A deficiency of this
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enzyme makes it difficult to digest lactose and causes abdominal discomfort. Lactose intolerance is a genetic trait; it is
treatable through over-the-counter formulations of lactase and by limiting the amount of milk in the diet.
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Sucrose—common table sugar—is a disaccharide that yields one molecule of glucose and one of fructose on hydrolysis.
Sucrose is a disaccharide in which D-glucose and D-fructose are joined at their anomeric carbons by a glycosidic bond. Its
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chemical composition is the same irrespective of its source; sucrose from cane and sucrose from sugar beets are chemi-
cally identical. Since sucrose does not have a free anomeric hydroxyl group, it does not undergo mutarotation.

Oligosaccharide (oligos is a Greek word that in its plural form means “few”) yields 3–10 monosaccharide units on
hydrolysis.
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Polysaccharides are hydrolyzed to more than 10 monosaccharide units.

Cellulose is a polysaccharide molecule that gives thousands of glucose molecules when completely hydrolyzed. Cellulose
is the principal structural component of vegetable matter. Wood is 30–40% cellulose, cotton over 90%. Photosynthesis in
plants is responsible for the formation of 109 tons per year of cellulose. Structurally, cellulose is a polysaccharide
composed of several thousand D-glucose units joined by β (1,4)-glycosidic linkages. Complete hydrolysis of all the
glycosidic bonds of cellulose yields D-glucose. The disaccharide fraction that results from partial hydrolysis is cellobiose.

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Animals lack the enzymes necessary to catalyze the hydrolysis of cellulose and so can’t digest it. Cattle and other
ruminants use cellulose as a food source in an indirect way. Colonies of microorganisms that live in their digestive tract

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consume cellulose and in the process convert it to other substances that the animal can digest.

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Starch Starch is a plant’s way of storing glucose to meet its energy needs. It is a mixture of a water-dispersible fraction
called amylose and a second component, amylopectin. Amylose is a polysaccharide made up of about 100 to several
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thousand D-glucose units joined by α(1,4)-glycosidic bonds. Like amylose, amylopectin is a polysaccharide of α(1,4)-
linked D-glucose units. Instead of being a continuous length of α (1,4) units, however, amylopectin is branched. Attached
to C-6 at various points on the main chain are short polysaccharide branches of 24–30 glucose units joined by α (1,4)-
glycosidic bonds.
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Glycogen When more glucose is available than is needed as fuel, animals store it as glycogen. is similar to amylopectin in
that it is a branched polysaccharide of α (1,4)-linked D-glucose units with subunits connected to C-6 of the main chain.
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Cellulose Derivatives:

1 Treating cellulose with acetic anhydride produces the triacetate known as “Arnel” or “acetate,” used widely in the textile
industry.

2 Cellulose trinitrate, also called “gun cotton” or nitrocellulose, is used in explosives.

3 Rayon: is made by treating cellulose (from cotton or wood pulp) with carbon disulfide in a basic solution. The solution

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of cellulose xanthate is then passed through a small orifice or slit into an acidic solution. This operation regenerates the
OH groups of cellulose, causing it to precipitate as a fiber or a sheet. The fibers are rayon; the sheets, after softening with

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glycerol, are cellophane.

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Reactions of carbohydrates:

REDUCTION OF CARBOHYDRATES: A) Wth NaBH4 or Na-Hg or catalytic hydrogenation: The carbonyl group

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of carbohydrates can be reduced to an alcohol function. Typical procedures include catalytic hydrogenation and sodium
borohydride reduction. Lithium aluminum hydride is not suitable, because it is not compatible with the solvents
(water, alcohols) that are required to dissolve carbohydrates. The products of carbohydrate reduction are called
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alditols. Since these alditols lack a carbonyl group, they are, of course, incapable of forming cyclic hemiacetals and exist
exclusively in noncyclic forms.
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Another name for glucitol, obtained by reduction of D-glucose is sorbitol; it is used as a sweetener, especially in special
diets required to be low in sugar. Reduction of D-fructose yields a mixture of glucitol (sorbitol) and mannitol,
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corresponding to the two possible configurations at the newly generated stereogenic center at C-2.

B) With HI and red P at 373 K: Glucose gives n-hexane and 2-iodohexane

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Enolization, Tautomerization, and Isomerization (Lobry de Bruyn–Alberda van Ekenstein transformation):

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Dissolving monosaccharides in aqueous base causes them to undergo a series of enolizations and keto–enol
tautomerizations that lead to isomerizations. For example, if a solution of D-glucose containing calcium hydroxide is
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allowed to stand for several days, a number of products can be isolated, including D-fructose and D-mannose called the
Lobry de Bruyn–Alberda van Ekenstein transformation after the two Dutch chemists who discovered it in 1895.

NOTE: When carrying out reactions with monosaccharides, it is usually important to prevent these isomerizations and
thereby to preserve the stereochemistry at all of the chirality centers. One way to do this is to convert the monosaccharide
to the methyl glycoside first. We can then safely carry out reactions in basic media because the aldehyde group has been
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converted to an acetal and acetals are stable in aqueous base. Preparation of the methyl glycoside serves to “protect” the
monosaccharide from undesired reactions that could occur with the anomeric carbon in its hemiacetal form.

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OXIDATION REACTIONS OF MONOSACCHARIDES

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A Benedict’s or Tollens’ Reagents: Reducing Sugars: Benedict’s reagent (an alkaline solution containing a cupric
citrate complex ion). Tollens’ solution [Ag+(NH3)2OH] oxidize and thus give positive tests with aldoses and ketoses. The
tests are positive even though aldoses and ketoses exist primarily as cyclic hemiacetals. Benedict’s solution and the related

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Fehling’s solution (which contains a cupric tartrate complex ion) give brick-red precipitates of Cu2O when they oxidize an
aldose. Since the solutions of cupric tartrates and citrates are blue, the appearance of a brick-red precipitate is a vivid and
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unmistakable indication of a positive test. It is reaction of open chain form of sugars

Sugars that give positive tests with Tollens’ or Benedict’s solutions are known as reducing sugars, and all carbohydrates
that contain a hemiacetal group (Free aldehyde or keto group) give positive tests. The sugars reduce the tollens reagent to
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metallic silver and benedicts or fehlings solution to Cu2O brick red color are reducing sugars. Only carbonyl group is
oxidized.
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All monosaccharides are reducing sugars. And all polysaccharides non reducing.

In diasaccharides Sucrose is non reducing maltose (glucose is the reducing part), lactose (glucose is the reducing part) are
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reducing
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Note: simple ketones do not react with tollens reagent, benedicts reagent, fehlings solution but ketoses do react. This is
because ketones with alpha OH group isomerizes to aldehyde group in basic conditions employed under these reagents as
discussed in (Lobry de Bruyn–Alberda van Ekenstein transformation).
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2 Although Benedict’s and Tollens’ reagents have some use as diagnostic tools [Benedict’s solution can be used in
quantitative determinations of reducing sugars (reported as glucose) in blood or urine], neither of these reagents is useful
as a preparative reagent in carbohydrate oxidations. Oxidations with both reagents take place in alkaline solution, and in
alkaline solutions sugars undergo a complex series of reactions that lead to isomerizations
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B Bromine Water: The Synthesis of Aldonic Acids: Monosaccharides do not undergo isomerization and fragmentation
reactions in mildly acidic solution. Bromine water is a general reagent that selectively oxidizes the CHO group to a CO2H
group, thus converting an aldose to an aldonic acid. Bromine water specifically oxidizes the β anomer, and the initial
product that forms is a δ -aldonolactone. This compound may then hydrolyze to an aldonic acid, and the aldonic acid may
undergo a subsequent ring closure to form a γ-aldonolactone

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Glucose to Gluconic acid

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C Nitric Acid Oxidation: Aldaric Acids: oxidizes both the CHO group and the terminal CH2OH group of an aldose to
CO2H groups, forming dicarboxylic acids are known as aldaric acids. It is not known whether a lactone is an intermediate

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in the oxidation of an aldose to an aldaric acid; however, aldaric acids form γ- and δ-lactones readily.

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D Periodate Oxidations: Oxidative Cleavage: Compounds that have hydroxyl groups on adjacent atoms undergo
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oxidative cleavage when they are treated with aqueous periodic acid (HIO4). The reaction breaks carbon–carbon bonds
and produces carbonyl compounds (aldehydes, ketones, or acids). Periodate oxidations are thought to take place through a
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cyclic intermediate.
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Note 1 When three or more CHOH groups are contiguous, the internal ones are obtained. as formic acid. Periodate
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oxidation of glycerol, for example, gives two molar equiva-lents of formaldehyde and one molar equivalent of formic
acid.
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2 Oxidative cleavage also takes place when an OH group is adjacent to the carbonyl group of an aldehyde or ketone (but
not that of an acid or an ester). Glyceraldehyde yields two molar equivalents of formic acid and one molar equivalent of
formaldehyde.

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while dihydroxyacetone gives two molar equivalents of formaldehyde and one molar equivalent of carbon dioxide.

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3 Periodic acid does not cleave compounds in which the hydroxyl groups are separated. by an intervening CH2 group, nor
those in which a hydroxyl group is adjacent to an ether or acetal function.
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Conversion to Esters (Acetylation): Treating a monosaccharide with excess acetic anhydride and a weak base (such as
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pyridine or sodium acetate) converts all of the hydroxyl groups, including the anomeric hydroxyl, to ester groups. If the
reaction is carried out at a low temperature (e.g., 0-8C), the reaction occurs stereospecifically; the α anomer gives the α-
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acetate and the β anomer gives the β-acetate.

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Conversion to Cyclic Acetals: formation of the cyclic acetals occurs only when the vicinal hydroxyl groups are cis to
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each other. When acetals such as these are formed from acetone, they are called acetonides. Cyclic acetals are commonly
used to protect vicinal cis hydroxyl groups of a sugar while reactions are carried out on other parts of the molecule.

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REACTIONS OF MONOSACCHARIDES WITH PHENYLHYDRAZINE: OSAZONES The aldehyde group of an
aldose reacts with such carbonyl reagents as hydroxylamine and phenylhydrazine. With hydroxylamine, the product is the
expected oxime. With enough phenylhydrazine, however, three molar equivalents of phenylhydra-zine are consumed and
a second phenylhydrazone group is introduced at C2. The product is called a phenylosazone. Phenylosazones crystallize
readily (unlike sugars) and are useful derivatives for identifying sugars.

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Note: Osazone formation results in a loss of the chirality center at C2 but does not affect other chirality centers; d-glucose

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and d-mannose, for example, yield the same phenylosazone. This experiment, first done by Emil Fischer, established that
d-glucose and d-mannose have the same configurations about C3, C4, and C5.

SYNTHESIS AND DEGRADATION OF MONOSACCHARIDES

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Kiliani–Fischer Synthesis: In 1885, Heinrich Kiliani Ruff degradation: can be used to shorten the chain by a
discovered that an aldose can be converted to the epimeric similar unit. involves (1) oxidation of the aldose to an
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aldonic acids having one additional carbon through the aldonic acid using bro-mine water and (2) oxidative
addition of hydrogen cyanide and subsequent hydrolysis of decarboxylation of the aldonic acid to the next lower aldose
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the epimeric cyanohydrins. Fischer later extended this using hydrogen peroxide and ferric sulfate. d-(-)-Ribose,
method by showing that aldonolactones obtained from the for example, can be degraded to d-(-)-erythrose:
aldonic acids can be reduced to aldoses.
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Note On the basis of the Kiliani–Fischer synthesis, we cannot know just which aldotetrose has both OH groups on the
right and which has the top OH on the left in the Fischer projection. However, if we oxidize both aldotetroses to aldaric
acids, one [d-(-)-erythrose] will yield an optically inactive (meso) product while the other [d-(-)-threose] will yield a
product that is optically active.

Formation of Ethers: Hydroxyl groups of sugars can be converted to ethers using a base and an alkyl halide by a version
of the Williamson ether synthesis. Benzyl ethers are commonly used to protect hydroxyl groups in sugars. Sodium or

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potassium hydride is typically used as the base in an aprotic solvent such as DMF or DMSO. The benzyl groups can later
be easily removed by hydrogenolysis using a palladium catalyst.

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Methyl ethers can also be prepared. The pentamethyl derivative of glucopyranose, for example, can be synthesized by
treating methyl glucoside with excess dimethyl sulfate in aqueous sodium hydroxide. Sodium hydroxide is a competent
base in this case because the hydroxyl groups of monosaccharides are more acidic than those of ordinary alcohols due to

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the many electronegative atoms in the sugar, all of which exert electron- withdrawing inductive effects on nearby
hydroxyl groups. In aqueous NaOH the hydroxyl groups are all converted to alkoxide ions, and each of these, in turn,
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reacts with dimethyl sulfate in an SN2 reaction to yield a methyl ether. The process is called exhaustive methylation.
Although not often used as protecting groups for alcohols in carbohydrates, methyl ethers have been useful in the structure
elucidation of sugars. Because the C2, C3, C4, and C6 methoxy groups of the pentamethyl derivative are ethers, they are
not affected by aqueous hydrolysis. (To cleave them requires heating with concentrated HBr or HI, Section 11.12.) The
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methoxyl group at C1, however, is part of an acetal linkage, and so it is labile under the conditions of aqueous hydrolysis.
Hydrolysis of the pentamethyl derivative of glucose gives evidence that the C5 oxygen was the one involved in the cyclic
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hemiacetal form because in the open-chain form of the product (which is in equilibrium with the cyclic hemiacetal) it is
the C5 oxygen that is not methylated:
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Silyl ethers, including tert-butyldimethylsilyl (TBS) ethers and phenyl-substituted ethers, are also used. tert-
Butyldiphenylsilyl (TBDPS) ethers show excellent regioselectivity for primary hydroxyl groups in sugars, such as at
C6 in a hexopyranose.

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Proteins: are polyamides, and their monomeric units are composed of about 20 differentα-
amino acids. Although hydrolysis of naturally occurring proteins may yield as many as 22
different. amino acids, the amino acids have an important structural feature in common: with the
exception of glycine (whose molecules are achiral), almost all naturally occurring amino acids
have the l configuration at the a carbon.* Only 20 of the 22 α-amino acids in Table are actually
used by cells when they synthesize proteins. Two amino acids are synthesized after the polyamide chain is intact.
Hydroxyproline (present mainly in collagen) is synthesized by

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oxidation of proline, and cystine (present in most proteins) is
synthesized from cysteine. The conversion of cysteine to cystine

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requires additional comment. The SH group of cysteine makes
cysteine a thiol. One property of thiols is that they can be

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converted to disulfides by mild oxidizing agents. This conversion, moreover, can be reversed by mild reducing agents

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a
Single-letter abbreviations are now the most commonly used form in current biochemical literature. bAn essential amino
acid. cpKa is of protonated amine of R group.

Essential Amino Acids or indispensable amino acids: Amino acids can be synthesized by all living organisms, plants
and animals. Many higher animals, however, are deficient in their ability to synthesize all of the amino acids they need for
their proteins. Thus, these higher animals require certain amino acids as a part of their diet. For adult humans there are
eight essential amino acids.

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Non-Essential Amino Acids or dispensable amino acids: Amino acid which can be synthesized in our body.

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Amino Acids as Dipolar Ions: Amino acids contain both a basic group ( NH2) and an acidic group ( CO2H). In the dry
solid state, amino acids exist as dipolar ions, a form in which the carboxyl group is present as a carboxylate ion, CO2-, and

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the amino group is present as an aminium ion, NH3+. (Dipolar ions are also called zwitterions.) In aqueous solution, an
equilibrium exists between the dipolar ion and the anionic and cationic forms of an amino acid. The predominant form of
the amino acid present in a solution depends on the pH of the solution and on the nature of the amino acid. In strongly

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acidic solutions all amino acids are present primarily as cations; in
strongly basic solutions they are present as anions.

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The isoelectric point (pI): is the pH at which the concentration of the
dipolar ion is at its maximum and the concentrations of the anions and
cations are equal. Each amino acid has a particular isoelectric point.
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Proteins have isoelectric points as well this property of proteins is
important for their separation and identification.

Enhanced acidity of carboxyl group in α-amino acids reason for this

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enhanced acidity of the carboxyl group in an a-amino acid is the inductive

effect of the neighboring aminium cation, which helps to stabilize the
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carboxylate anion formed when it loses a proton. Loss of a proton from the
carboxyl group in a cationic α-amino acid leaves the molecule electrically
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neutral (in the form of a dipolar ion). The state of an α-amino acid at any
given pH is governed by a combination of two equilibria, as shown in the above
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equation for alanine

When a base is added to a solution of the net cationic form of alanine (initially at
pH 0, for example), the first proton removed is the carboxylic acid proton, as we
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have said. In the case of alanine, when a pH of 2.3 is reached, the carboxylic acid
proton will have been removed from half of the molecules.
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If an amino acid contains a side chain that has an acidic or basic group, the
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equilibria become more complex. Consider lysine, for example, an amino acid
that has an additional NH2 group on its ɛ carbon. In strongly acidic solution,
lysine is present as a dication because both amino groups are protonated. The first proton to be lost as the pH is raised is a
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proton of the carboxyl group (pKa1 = 2.2), the next is from the α-aminium group (pKa2 = 9.0), and the last is from the ɛ-
aminium group (pKa3 = 10.5):

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Note: At isoelectric point an amino acid has least solubility in water.

Neutral Amino acids: Amino acid having one amine and carboxylic acid group. They have isoelectric point at less than
pH 7.

Acidic Amino acids: Amino acid having one amine and two carboxylic acid group. They have isoelectric point at pH
between 3.4-5.4.

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Basic Amino acids: Amino acid having two amine and one carboxylic acid group. They have isoelectric point at pH
between 7.6-10.8.

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SYNTHESIS OF α-AMINO ACIDS:

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From Potassium Phthalimide: This method, a modification of the Gabriel synthesis of amines, uses potassium
phthalimide and diethyl α-bromomalonate to prepare an imido malonic ester.

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N
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The most widely used method for the laboratory synthesis of α-amino acids is a modification of the malonic ester
synthesis. The key reagent is diethyl acetamidomalonate, a derivative of malonic ester that already has the critical nitrogen
substituent in place at the α-carbon atom. The side chain is introduced by alkylating diethyl acetamidomalonate in the
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same way as diethyl malonate itself is alkylated. Hydrolysis removes the acetyl group from nitrogen and converts the two
ester functions to carboxyl groups. Decarboxylation gives the desired product.
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T
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B The Strecker Synthesis Treating an aldehyde with ammonia and

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hydrogen cyanide produces an α-aminonitrile. Hydrolysis of the

nitrile group of the α-aminonitrile converts the latter to an α-amino
acid. This synthesis is called the Strecker synthesis. The first step of
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this synthesis probably involves the initial formation of an imine from the aldehyde and ammonia followed by the
addition of hydrogen cyanide.
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C a nucleophilic substitution in which ammonia reacts with an α-halo carboxylic acid. α-halo acid is normally prepared by
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the Hell–Volhard–Zelinsky reaction

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A molecule formed by joining amino acids together is called a peptide, and the amide linkages in them are called peptide
bonds or peptide linkages. Each amino acid in the peptide is called an amino acid residue. Peptides that contain 2, 3, a
few (3–10), or many amino acids are called dipeptides, tripeptides, oligopeptides, and polypeptides, respectively. Proteins
are polypeptides consisting of one or more polypeptide chains.

Polypeptides are linear polymers. One end of a polypeptide chain

terminates in an amino acid residue that has a free NH3+ group; the other

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terminates in an amino acid residue with a free CO2- group. These two
groups are called the N-terminal and the C-terminal residues,

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respectively. By convention, we write peptide and protein structures with
the N-terminal amino acid residue on the left and the C-terminal residue on the

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right:

Hydrolysis: When a protein or polypeptide is refluxed with 6 M hydrochloric acid

for 24 h, hydrolysis of all the amide linkages usually takes place, liberating its

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constitutent amino acids as a mixture. Chromatographic separation and quantitative
analysis of the resulting mixture can then be used to determine which amino acids
composed the intact polypeptide and their relative amounts.

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One chromatographic method for separation of a mixture of amino acids is based on the use of cation-exchange resins
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which are insoluble polymers containing sulfonate groups. If an acidic solution containing a mixture of amino acids is
passed through a column packed with a cation-exchange resin, the amino acids will be adsorbed by the resin because of
attractive forces between the negatively charged sulfonate groups and the positively charged amino acids. The strength of
the adsorption varies with the basicity of the individual amino acids; those that are most basic are held most strongly. If
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the column is then washed with a buffered solution at a given pH, the individual amino acids move down the column at
different rates and ultimately become separated.
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The presence of amino acids can be detected by the formation of a purple color on treatment with ninhydrin. The same
compound responsible for the purple color is formed from all amino acids in which the α-amino group is primary.
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Proline, in which the α-amino group is secondary, gives an orange compound on reaction with ninhydrin.
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REACTIONS OF AMINO ACIDS: Amino acids undergo reactions characteristic of both their amine and carboxylic acid
functional groups. Acylation is a typical reaction of the amino group.
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C

Ester formation is a typical reaction of the carboxyl group.

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PARTIAL HYDROLYSIS OF PEPTIDES

Whereas acid-catalyzed hydrolysis of peptides cleaves amide bonds indiscriminately and eventually breaks all of them,
enzymatic hydrolysis is much more selective and is the method used to convert a peptide into smaller fragments.

The enzymes that catalyze the hydrolysis of peptides are called peptidases, proteases, or proteolytic enzymes. One group
of pancreatic enzymes, known as carboxypeptidases, catalyzes only the hydrolysis of the peptide bond to the C-terminal

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amino acid, for example. Trypsin, a digestive enzyme present in the intestine, catalyzes only the hydrolysis of peptide
bonds involving the carboxyl group of a lysine or arginine residue. Chymotrypsin, another digestive enzyme, is selective
for peptide bonds involving the carboxyl group of amino acids with aromatic side chains (phenylalanine, tryrosine,

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tryptophan). Chemical cleavage at specific sites can be done with cyanogen bromide (CNBr), which cleaves peptide
bonds on the C-terminal side of methionine residues.

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END GROUP ANALYSIS: An amino acid sequence is ambiguous unless we know the direction in which to read it—left
to right, or right to left. Carboxypeptidase-catalyzed hydrolysis cleaves the C-terminal amino acid and so can be

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used to identify it.

N terminus Several chemical methods have been devised for identifying the N-terminal amino acid. They all take

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advantage of the fact that the N-terminal amino group is free and can act as a nucleophile. The α-amino groups of all the
other amino acids are part of amide linkages, are not free, and are much less nucleophilic.
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Sanger’s method for N-terminal residue analysis: treating a peptide with 1-fluoro-4-nitrobenzene in the presence of a
weak base such as sodium carbonate, which is very reactive toward nucleophilic aromatic substitution. The base abstracts
a proton from the terminal H3N+ group to give a free amino function. The nucleophilic amino group attacks 1-fluoro-2,4-
dinitrobenzene, displacing fluoride.
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R
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E
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Edman Degradation: most widely used procedure for identifying the N-terminal amino acid in a peptide. reaction
between the N-terminal amino group and phenyl isothiocyanate, C6H5N=C=S. Phenyl isothiocyanate reacts with the N-
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terminal amino group to form a phenylthiocarbamyl derivative, which is then cleaved from the peptide chain by acid. The
result is an unstable anilinothioazolinone (ATZ), which rearranges to a stable phenylthiohydantoin (PTH) derivative of the
amino acid. In the automated process, the PTH derivative is introduced directly to a high-performance liquid
chromatograph and identified by comparison of its retention time with known amino acid PTH derivatives.

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C
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C
N
IN
Y

POLYPEPTIDE AND PROTEIN SYNTHESIS: Fragment method To form a peptide bond between two suitably
protected amino acids, the free carboxyl group of one of them must be activated so that it is a reactive acylating agent. In
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one method, treatment of a solution containing the N-protected and the C-protected amino acids with N,N’-
dicyclohexylcarbodiimide (DCC) leads directly to peptide bond formation.
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E
H
C

Protective groups for amine A number of reagents have been developed to meet these requirements. Three that are often
used are benzyl chloroformate, di-tert-butyl dicarbonate (sometimes abbreviated Boc2O, where Boc stands for tert-
butyloxycarbonyl), and 9-fluorenylmethyl chloroformate.
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The benzyloxycarbonyl group can be removed with catalytic hydrogenation or cold HBr in acetic acid. The tert-

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butyloxycarbonyl group can be removed with trifluoroacetic acid (CF 3CO2H) in acetic acid. The 9-
fluorenylmethoxycarbonyl (Fmoc) group is stable under acid conditions but can be removed under mild basic conditions

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using piperidine (a secondary amine). The easy removal of the Z and Boc groups in acidic media results from the excep-
tional stability of the carbocations that are formed initially. The benzyloxycarbonyl group gives a benzyl carbocation; the
tert-butyloxycarbonyl group yields, initially, a tert-butyl cation. Removal of the benzyloxycarbonyl group with hydrogen

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and a catalyst depends on the fact that benzyloxygen bonds are weak and subject to hydrogenolysis at low temperatures,
resulting in methylbenzene (toluene) as one product:

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to activate a carboxyl group is to convert it to an acyl chloride. This method was used in early peptide syntheses, but acyl
chlorides are actually more reactive than necessary. As a result, their use leads to complicating side reactions. A much
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better method is to convert the carboxyl group of the “protected” amino acid to a mixed anhydride using ethyl
chloroformate
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T

Disdvantage of fragment method of peptide synthesis: The methods that we have described thus far have been used to
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synthesize a number of polypeptides, including ones as large as insulin. They are extremely time-consuming and tedious,
however. One must isolate the peptide and purify it by lengthy means at almost every stage. Furthermore, significant loss
of the peptide can occur with each isolation and purification stage.
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Merrifield’s method, for which he received the 1984 Nobel Prize in Chemistry, is called solid-phase peptide
synthesis (SPPS): synthesis of the peptide residue by residue while one end of the peptide remains attached to an
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insoluble plastic bead. Protecting groups and other reagents are still necessary, but because the peptide being synthesized
is anchored to a solid support, by-products, excess reagents, and solvents can simply be rinsed away between each
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synthetic step without need for intermediate purification. After the very last step the polypeptide is cleaved from the
polymer support and subjected to a final purification by HPLC. The method works so well that it was developed into an
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automated process. Solid-phase peptide synthesis begins with attachment of the first amino acid by its carboxyl group to
the polymer bead, usually with a linker or spacer molecule in between. Each new amino acid is then added by formation
of an amide bond between the N-terminal amino group of the peptide growing on the solid support and the new amino
acid’s carboxyl group. Although Merrifield’s initial method for solid-phase peptide synthesis used the Boc group to
protect the a-amino group of residues being coupled to the growing peptide, several advantages of the Fmoc group have

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since made it the group of choice. The reasons have mainly to do with excellent selectivity for removing the Fmoc group
in the presence of other protecting groups used to block reactive side chains along the growing peptide and the ability to
monitor the progress of the solid-phase synthesis by spectrophotometry as the Fmoc group is released in each cycle.

Configuration and Conformation of peptide bond in polypeptides: An important feature is the planar geometry
associated with the peptide bond, and the most stable conformation with respect to this bond has the two α-carbon atoms
anti to each other. Rotation about the amide linkage is slow because delocalization of the unshared electron pair of

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nitrogen into the carbonyl group gives partial double-bond character to the carbon–nitrogen bond. A peptide bond has
about 40% double-bond character because of electron delocalization. Steric hindrance causes the trans configuration to be more
stable than the cis configuration,

C
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C
Ramachandran angles: Name after Indian Biophyscist G.N.A Ramachandran. Conformation of protein can be described
on the basis of. Angle ɸ between R-CH-NH and Angle ᴪ R-CH-CO.

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Protein STRUCTURE: IN
A Primary structure: The primary structure is the amino acid sequence plus any disulfide links. With the 20 amino acids
as building blocks, 202 dipeptides, 203 tripeptides, 204 tetrapeptides, and so on, are possible. Clearly, one of the most
important things to determine about a protein is the sequence of its amino acids. Fortunately, there are a variety of
methods available to determine the sequence of amino acids in a polypeptide. We shall begin with terminal residue
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analysis techniques used to identify the N- and C-terminal amino acids. the determination of the amino acid sequence of
insulin by Frederick Sanger of Cambridge University (England). Sanger was awarded the 1958 Nobel Prize in chemistry
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for this work. Importance of primary structure can be determined for fact that the genetics based disease sickle-cell
anemia results from a single amino acid error in the β chain of hemoglobin. In normal hemoglobin, position 6 has a
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glutamic acid residue, whereas in sickle-cell hemoglobin position 6 is occupied by valine. Red blood cells (erythrocytes)
containing hemoglobin with this amino acid residue error tend to become crescent shaped (“sickle”) when the partial
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pressure of oxygen is low, as it is in venous blood. These distorted cells are more difficult for the heart to pump through
small capillaries. They may even block capillaries by clumping together; at other times the red cells may even split open.
Children who inherit this genetic trait from both parents suffer from a severe form of the disease and usually do not live
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past the age of two.

B Secondary Structure of Proteins: the conformation of segments of the backbone chain of a peptide or protein. To
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minimize energy, a polypeptide chain tends to fold in a repeating geometric structure such as an α-helix or a β-pleated
sheet. Three factors deter-mine the choice of secondary structure:
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•the regional planarity about each peptide bond (as a result of the partial double-bond character of the amide bond), which
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limits the possible conformations of the peptide chain.

•maximization of the number of peptide groups that engage in hydrogen bonding (i.e., hydrogen bonding between the
carbonyl oxygen of one amino acid residue and the amide hydrogen of another)

•adequate separation between nearby R groups to avoid steric hindrance and repulsion of like charges
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α-helix: The backbone of the polypeptide coils around the long axis of the protein molecule. The helix is stabilized by
hydrogen bonds: Each hydrogen attached to an amide nitrogen is hydrogen bonded to a carbonyl oxygen of an amino acid
four residues away. The substituents on the α-carbons of the amino acids protrude outward from the helix, thereby minimizing
steric hindrance. Because the amino acids have the L-configuration, the α-helix is a right-handed helix; that is, it rotates in a
clockwise direction as it spirals down. Each turn of the helix contains 3.6 amino acid residues and 13 membered ring is formed,
and the repeat distance (pitch) of the helix is 5.4 Å. The percentage of amino acid residues coiled into an α-helix varies from
protein to protein, but, on average, about 25% of the residues in globular proteins are in α-helix. Many fibrous proteins α
keratin in hair, nail, wool, skin, beaks, claws and myosin in muscles. During stretching of hairs hydrogen bond in helix are

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broken and are reformed when stretching is removed.
Note: Not all amino acids are able to fit into an α-helix A proline residue, for example, forces a bend in a helix because the
bond between the proline nitrogen and the α-carbon cannot rotate to enable it to fit readily into a helix. Similarly, two adjacent

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amino acids that have more than one substituent on a β-carbon (valine, isoleucine, or threonine) cannot fit into a helix because
of steric crowding between the R groups. Finally, two adjacent amino acids with like-charged substituents cannot fit into a helix

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because of electrostatic repulsion between the R groups.

β-pleated sheet: the polypeptide backbone is extended in a zigzag structure resembling a series of pleats. A β-pleated sheet is

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almost fully extended—the average two-residue repeat distance is 7.0 Å. The hydrogen bonding in a β-pleated sheet occurs
between neighboring peptide chains. The adjacent hydrogen-bonded peptide chains can run in the same direction or in opposite
directions. In a parallel β-pleated sheet, the adjacent chains run in the same direction. In an antiparallel β-pleated sheet, the
adjacent chains run in opposite directions.

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IN
Y
R
T
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Because the substituents (R) on the α-carbons of the amino acids on adjacent chains are close to each other, the chains can
nestle closely together to maximize hydrogen bonding interactions only if the substituents are small. Silk, for example, a protein
with a large number of relatively small amino acids (glycine and alanine), has large segments of β-pleated sheets. The number
of side-by-side strands in a β-pleated sheet ranges from 2 to 15 in a globular protein. The average strand in a β-pleated sheet
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section of a globular protein contains six amino acid residues. Wool and the fibrous protein of muscle are examples of proteins
with secondary structures that are almost all α-helix consequently, these proteins can be stretched. In contrast, the secondary
structures of silk (fibroin) and spider webs are predominantly β-pleated sheets. Because the sheet is a fully extended structure,
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these proteins cannot be stretched.

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Coil Conformation Generally, less than half of a globular protein is in an α-helix or β-pleated sheet. Most of the rest of the
protein is still highly ordered but is difficult to describe. These polypeptide fragments are said to be in a coil conformation or a
loop conformation.
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C Tertiary Structure of Proteins: is the three-dimensional arrangement of all the atoms in the protein. Proteins
fold spontaneously in solution in order to maximize their stability. Every time there is a stabilizing interaction between two
atoms, free energy is released. The more free energy released (the more negative the ∆G), the more stable the protein. So a
protein tends to fold in a way that maximizes the number of stabilizing interactions. The stabilizing interactions include
covalent bonds, hydrogen bonds, electrostatic attractions (attractions between opposite charges), salt bridges and hydrophobic

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(van der Waals) interactions. Stabilizing interactions can occur between peptide groups (atoms in the backbone of the protein),
between side-chain groups and between peptide and side-chain groups. Because the side-chain groups help determine how a
protein folds, the tertiary structure of a protein is determined by its primary structure.
Most proteins exist in aqueous environments. Therefore, they tend to fold in a way that exposes the maximum number of polar
groups to the aqueous environment and that buries the nonpolar groups in the interior of the protein, away from water. The
interactions between nonpolar groups are known as hydrophobic interactions. These interactions increase the stability of a
protein by increasing the entropy of water molecules. Water molecules that surround nonpolar groups are highly structured.
When two nonpolar groups come together, the surface area in contact with water decreases, decreasing the amount of structured

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water. Decreasing structure increases entropy, which in turn decreases the free energy, which increases the stability of the
protein. (Recall that ∆G° = ∆H° - T∆S°.)

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Fibrous protein have almost same secondary structure (an α-helix or a β-pleated sheet) Eg an α-keratin has an α-helix
structure. This folds back to ropes or rods. Collagen has triple helix structure. Globular proteins have some part α-helix, some

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part β-pleated sheet and some part random coils
NOTE: Disulfide bonds are the only covalent bonds that can form when a protein folds. The other bonding interactions that
occur in folding are much weaker, but because there are so many of them they are the important interactions in determining how
a protein folds.

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D Quaternary Structure of Proteins: Proteins that have more than one peptide chain are called oligomers.
The individual chains are called subunits or protomers. A protein with a single subunit is called a monomer, one with two

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subunits is called a dimer; one with three subunits is called a trimer, and one with four subunits is called a tetramer. The
subunits are held together by the same kinds of interactions that hold the individual protein chains in a particular three-
dimensional conformation: hydrophobic interactions, hydrogen bonding, and electrostatic attractions. The quaternary structure
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of a protein describes the way the subunits are arranged in space. Hemoglobin is an example of a tetramer. Hemoglobin is the
oxygen-carrying protein of blood. It binds oxygen at the lungs and transports it to the muscles, where it is stored by myoglobin.
Hemoglobin binds oxygen in very much the same way as myoglobin, using heme as the prosthetic group. Hemoglobin is much
larger than myoglobin, however, having a molecular weight of 64,500, whereas that of myoglobin is
17,500; hemoglobin contains four heme units, myoglobin only one. Hemoglobin is an assembly of
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four hemes and four protein chains, including two identical chains called the alpha chains (141
amino acids) and two identical chains called the beta chains (146 amino acids).
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The iron of the heme group is in the 2+ (ferrous) oxidation state and it forms a coordinate bond to a
nitrogen of the imidazole group of histidine of the polypeptide chain. This leaves one valence of the
ferrous ion free to combine with oxygen as follows:
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What is remarkable about hemoglobin is that when the heme combines with oxygen the ferrous ion does not become
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readily oxidized to the ferric state. Studies with model heme compounds in water, for example, show that they undergo a
rapid combination with oxygen but they also undergo a rapid oxidation of the iron from Fe2+ to Fe3+. When these same
compounds are embedded in the hydrophobic environment of a polystyrene resin, however, the iron is easily oxygenated
and deoxygenated, and this occurs with no change in oxidation state of iron. In this respect, it is especially interesting to
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note that X-ray studies of hemoglo-bin have revealed that the polypeptide chains provide each heme group with a
similar hydrophobic environment.
Some substances, such as CO, form strong bonds to the iron of heme, strong enough to displace O2 from it. Carbon
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monoxide binds 30–50 times more effectively than oxygen to myoglobin and hundreds of times better than oxygen to
hemoglobin. Strong binding of CO at the active site interferes with the ability of heme to perform its biological task of
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transporting and storing oxygen, with potentially lethal results.

Types of proteins:
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1 On the basis of molecular structure:

A Fibrous protein: contain long chains of polypeptides that occur in bundles. Polypeptide chains are held together by H bonds
and disulphide linkages These proteins are insoluble in water. These proteins are stable to moderate changes in pH and temp.
These are chief structural materials of animal tissue. Eg keratin (hair, nail, wool, skin), collagen (in tendons), fibroin (in silk)
and myosin (in miscles).

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B Globular proteins: Polypeptied chains fold in such a manner to have roughly spherical shapes with lipophilic parts inside
and hydrophilic parts outside. Thus they are soluble in water and sensitive to moderate changes in pH and temp. Essentially all
enzymes, many hormones insulin (from pancreas), thyroglobulin from thyroid gland (which produces thyroxine), antibodies,
haemoglobin, fibrinogen that is converted to fibrin at blood clot time, albumin in eggs are globular proteins. Interactions in
globular priteins are: Disulphide bridging, inramolecular H bond, Vander waals, dipolar interactions.

2 On the basis of chemical composition or nature of products they give on hydrolysis:

A Simple Proteins: Give only amino acids on hydrolysis. Eg albumin in egg white, glutenin in wheat, oxyzenin in rice, keratin

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in hair, nail, horns etc
B Conjugated proteins: Gives in addition to amino acids a non-protein part called Prosthetic group. Function of prosthetic
group is to control biological functions.

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Types of conjugated protein
Name of the protein Prosthetic group

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Nucleoprotein Nucleic acid
Glycoproteins or Mucoproteins Sugars
Lipoproteins Lipids such as lecithin
Phosphoproteins Phosphoric acid residue

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Chromoproteins (haemoglobin, myoglobin) Heme (red coloured Protoporphyrin)

Protein Denaturation

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Destroying the highly organized tertiary structure of a protein is called denaturation. Anything that breaks the bonds
responsible for maintaining the three-dimensional shape of the protein will cause the protein to denature (unfold). Because these
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bonds are weak, proteins are easily denatured. The totally random conformation of a denatured protein is called a random coil.
The following are some of the ways that proteins can be denatured:
• Changing the pH denatures proteins because it changes the charges on many of the side chains. This disrupts electrostatic
attractions and hydrogen bonds.
• Certain reagents such as urea and guanidine hydrochloride denature proteins by forming hydrogen bonds to the protein groups
that are stronger than the hydrogen bonds formed between the groups.
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• Detergents such as sodium dodecyl sulfate denature proteins by associating with the nonpolar groups of the protein, thus
interfering with the normal hydrophobic interactions.
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• Organic solvents denature proteins by disrupting hydrophobic interactions.

• Proteins can also be denatured by heat or by agitation. Both increase molecular motion, which can disrupt the attractive forces.
A well-known example is the change that occurs to the white of an egg when it is heated or whipped. Soluble Globular proteins
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albumin unfold to insoluble fibrous proteins. Cudling of milk due to formation of lactic acid by bacteria.
During formation of cheese globular protein lactalbumin become fibrous.
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NOTE: During denaturation secondary and tertiary structures are destroyed but primary structure remains intact.
Denaturation may be reversible or irreversible
Irreversible denaturation: Coagulation of egg white, curdling of milk.
Reversible denaturation: Renaturation: When temp and pH of denatured proteins are brought back to normal conditions
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native structure of protein is restored. Secondary and tertiary structures are reformed.

Nucleosides, Nucleotides, and Nucleic Acids

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Nucleic acids are Polynucleotides made up of nucleotides Nuclic acid consist of chains of five-membered-ring sugars linked by
phosphate groups. The anomeric carbon of each sugar is bonded to a nitrogen of a heterocyclic compound (amines), they are
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commonly referred to as bases in a β-glycosidic linkage. In RNA the five-membered-ring sugar is D-ribose. In DNA it is 2-
deoxy-D-ribose (D-ribose without an OH group in the 2-position).
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Phosphoric acid links the sugars in both RNA and DNA. The acid has three dissociable OH groups with values pKa of 1.9, 6.7,
and 12.4. Each of the OH groups can react with an alcohol to form a phosphomonoester, a phosphodiester, or a phosphotriester.
In nucleic acids the phosphate group is a phosphodiester

There are only five nitrogen bases in two catagories two are substituted purines (adenine and guanine), and two are substituted
pyrimidines (cytosine, Uracil and thymine)

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The purines and pyrimidines are bonded to the anomeric carbon of the furanose ring------purines at N-9 and pyrimidines at N-

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1—in a β-glycosidic linkage.
DNA—two are substituted purines (adenine and guanine), and two are substituted pyrimidines (cytosine and thymine).
RNA also contains only four bases. (adenine, guanine, and cytosine, uracil).

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A compound containing a base bonded to D-ribose or to 2-deoxy-D-ribose is called a nucleoside. adenine is the base, whereas
adenosine is the nucleoside. Similarly, cytosine is the base, whereas cytidine is the nucleoside, and so forth.
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A nucleotide is a nucleoside with either the 5’or 3’ the -OH group bonded in an ester linkage to phosphoric acid. The
nucleotides of RNA—where the sugar is D-ribose—are more precisely called ribonucleotides, whereas the nucleotides of
DNA—where the sugar is 2-deoxy-D-ribose—are called deoxyribonucleotides.

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ATP: The Carrier of Chemical Energy. All cells require energy to ensure their survival and reproduction.
They get the energy they need by converting nutrients into a chemically useful form of energy. The most important form of
chemical energy is adenosine 5’-triphosphate (ATP). ATP is known as the universal carrier of chemical energy because the
energy of hydrolysis of ATP converts endergonic reactions into exergonic reactions. Two reactions in which the energy of one
is used to drive the other are known as coupled reactions.
The transfer of a phosphate group from ATP to D-glucose is an example of a phosphoryl transfer reaction.
Why is the hydrolysis of a phosphoanhydride bond so exergonic?
1. Greater electrostatic repulsion in ATP. 3 Greater resonance stabilization in the products.

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2. More solvation in the products.
Structure: The primary structure of a nucleic acid is the sequence of bases in the strand. Nucleic acids are composed of long
strands of nucleotide subunits linked by phosphodiester. These linkages join the group 3’-OH of one nucleotide to the 5’-OH

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group of the next nucleotide. Notice that the nucleotide at one end of the strand has an unlinked 5’-triphosphate group, and the
nucleotide at the other end of the strand has an unlinked 3’-hydroxyl group. Nucleotide triphosphates are the starting materials

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for the biosynthesis of nucleic acids. DNA is synthesized by enzymes called DNA polymerases, and RNA is synthesized by
enzymes called RNA polymerases. The nucleotide strand is formed as a result of nucleophilic attack by a 3’-OH group of one
nucleotide triphosphate on the α-phosphorus of another nucleotide triphosphate, breaking a phosphoanhydride bond and

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eliminating pyrophosphate.
Secondary structure Double Helix: Watson and Crick concluded that DNA consists of two strands of nucleic acids with the
sugar–phosphate backbone on the outside and the bases on the inside. The chains are held together by hydrogen bonds between
the bases on one strand and the bases on the other strand. The width of the double-stranded molecule is relatively constant, so a

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purine must pair with a pyrimidine. If the larger purines paired, the strand would bulge; if the smaller pyrimidines paired, the
strands would have to contract to bring the two pyrimidines close enough to form hydrogen bonds. Critical to Watson and
Crick’s proposal for the secondary structure of DNA were experiments carried out by Erwin Chargaff. These experiments
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showed that the number of adenines in DNA equals the number of thymines and the number of guanines equals the number of
cytosines. Chargaff also noted that the number of adenines and thymines relative to the number of guanines and cytosines is
characteristic of a given species but varies from species to species. For man it is 1.52 and in Ecoli it is 0.93.
Chargaff’s data showing that A= T and G= C could be explained if adenine (A) always paired with thymine (T) and guanine (G)
always paired with cytosine (C). This means the two strands are complementary—where there is an A in one strand, there is a T
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in the opposing strand, and where there is a G in one strand there is a C in the other strand. Adenine forms two hydrogen bonds
with thymine but would form only one hydrogen bond with cytosine. Guanine forms three hydrogen bonds with cytosine but
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would form only one hydrogen bond with thymine.

The two DNA strands are antiparallel—they run in opposite directions, with the sugar–phosphate backbone on the outside and
the bases on the inside. The DNA strands are not linear but are twisted into a helix around a common axis. The base pairs are
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planar and parallel to each other on the inside of the helix. Hydrogen bonding between base pairs is just one of the forces
holding the two strands of the DNA double helix together. The bases are planar aromatic molecules that stack on top of one
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another. Each pair is slightly rotated with respect to the next pair, like a partially spread-out hand of cards. There are favorable
van der Waals interactions between the mutually induced dipoles of adjacent pairs of bases. These interactions, known as
stacking interactions, are weak attractive forces, but when added together they contribute significantly to the stability of the
double helix. Stacking interactions are strongest between two purines and weakest between two pyrimidines. Confining the
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bases to the inside of the helix has an additional stabilizing effect—it reduces the surface area of the relatively nonpolar residues
exposed to water. This increases the entropy of the surrounding water molecules
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The two DNA strands are antiparallel—they run in opposite directions, with the sugar–phosphate backbone on the outside and
the bases on the inside. One strand runs from 5’-3’ and the other from 3’-5’.

Naturally occurring DNA can exist in the three different helical forms. The B- and A-helices are both right-handed. The B-helix
is the predominant form in aqueous solution, while the A-helix is the predominant form in nonpolar solvents. Nearly all the
DNA in living organisms is in a B-helix. The Z-helix is a left handed helix. It occurs in regions where there is a high content of
G-Cbase pairs. The A-helix is shorter (for a given number of base pairs) and about 3% broader than the B-helix, which is
shorter and broader than the Z-helix. Helices are characterized by the number of bases per 360° turn and the distance (the

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rise) between adjacent base pairs. A-DNA has 11 base pairs per turn and a 2.3 Å rise; B-DNA has 10 base pairs per turn
and a 3.4 Å rise (diameter 2 nm); and Z-DNA has 12 base pairs per turn and a 3.8 Å rise. There are two kinds of
alternating grooves in DNA. In B-DNA the major groove is wider than the minor groove. Cross sections of the double helix

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show that one side of each base pair faces into the major groove and the other side faces into the minor groove.
Only for IIT: Proteins and other molecules can bind to the grooves. The hydrogen-bonding properties of the functional groups

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facing into each groove determine what kind of molecules will bind to the groove. Mitomycin is a naturally occurring
compound that has been found to have both antibacterial activity and anticancer activity. It works by binding to the minor
groove of DNA. It binds at regions rich in A’s and T’s
The phosphate OH group has pKa of about 2, so it is in its basic form (negatively charged) at physiological pH. The

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negatively charged backbone repels nucleophiles, thereby preventing cleavage of the phosphodiester bonds
NOTE : Unlike DNA, RNA is easily cleaved because the 2’-OH group of ribose can act as the nucleophile that cleaves the
strand This explains why the 2’-OH group is absent in DNA. To preserve the genetic information, DNA must remain intact

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throughout the life span of a cell. Cleavage of DNA would have disastrous consequences for the cell and for life itself. RNA, in
contrast, is synthesized as it is needed and is degraded once it has served its purpose.
Besides primary and secondary structure, DNA have higher level structures in which it is bound to proteins, folded to form
chromatins and chromosomes.
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Melting: On heating two strands of DNA separate from each other
Annealing: On cooling two separate strands of DNA hybridize again.
Melting Temperature (Tm): Temperature at which two strands of DNA separate from each other. It is specific for
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specific sequence.
DNA: Replication: Synthesis of DNA is catalysed by enzymes.
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Two strands are complementary, both carry the same genetic

information. Both strands serve as templates for the synthesis of
complementary new strands. The new (daughter) DNA. molecules are
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identical to the original (parent) molecule—they contain all the original

genetic information. The synthesis of identical copies of DNA is called
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replication. The synthesis of DNA takes place in a region of the

molecule where the strands have started to separate, called a replication
fork. Because a nucleic acid can be synthesized only in 5’-3’ direction,
only the daughter strand on the left in is synthesized continuously in a
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single piece (leading strand because it is synthesized in the 5’-3’

direction). The other daughter strand needs to grow in the 3’-5’direction,
so it is synthesized discontinuously in small pieces (Okazaki fragment or
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lagging strand). Each piece is synthesized in the 5’-3’ direction and the
fragments are joined together by an enzyme called DNA ligase. Each of
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the two resulting daughter molecules of DNA that result contains one of
the original strands plus a newly synthesized strand. This process is
called semiconservative replication.
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Only for IIT Biosynthesis of RNA: Transcription: The sequence of DNA bases provides the blueprint
for the synthesis of RNA. The synthesis of RNA from a DNA blueprint, called transcription, takes place in the nucleus of the
cell. The newly synthesized RNA leaves the nucleus, carrying the genetic information into the cytoplasm (the cell material
outside the nucleus), where translation of this information into proteins takes place. DNA contains sequences of bases known as
promoter sites. The promoter sites mark the beginning of genes. An enzyme recognizes a promoter site and binds to it,

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initiating RNA synthesis. The DNA at a promoter site unwinds to give two single strands, exposing the bases. One of the
strands is called the sense strand or informational strand. The complementary strand is called the template strand or
antisense strand. The template strand is read in the direction, so that RNA can be synthesized in the direction (Figure 27.12).
The bases in the template strand specify the bases that need to be incorporated into RNA, following the same base pairing found
in DNA. For example, each guanine in the template strand specifies the incorporation of a cytosine into RNA, and each adenine
in the template strand specifies the incorporation of a uracil into RNA. Because both the sense strand and RNA are
complementary to the template strand, the sense strand and RNA have the same base sequence, except that RNA has a uracil
wherever the sense strand has a thymine. Just as there are promoter sites that signal the places to start RNA synthesis, there are

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sites in DNA that signal that no more bases should be added to the growing strand of RNA, at which point synthesis stops.
Often the bases of a gene are interrupted by bases that appear to have no informational content. A stretch of bases representing a
portion of a gene is called an exon, while a stretch of bases that contains no genetic information is called an intron. So after the

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RNA is synthesized, but before it leaves the nucleus, the so-called nonsense bases (encoded by the introns) are cut out and the
informational fragments are spliced together, resulting in a much shorter RNA molecule. This RNA processing step is known as

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RNA splicing. Scientists have found that only about 2% of DNA contains genetic information, while 98% consists of introns. It
has been suggested that the purpose of introns is to make RNA more versatile. The originally synthesized long strand of RNA
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Biosynthesis of Proteins: Translation: A protein is synthesized from its N-terminal end to its C-terminal
end by reading the bases along the mRNA strand in 5’-3’direction. There are three kinds of RNA—messenger RNA (mRNA)
whose sequence of bases determines the sequence of animo acids in a protein, ribosomal RNA (rRNA), a structural component

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of ribosomes, and transfer RNA (tRNA), the carriers of amino acids for protein synthesis. A sequence of three bases, called a
codon, specifies a particular amino acid that is to be incorporated into a protein. The bases are read consecutively and are never
skipped. A codon is written with the 5’-nucleotide on the left. Each amino acid is specified by a three-base sequence known as
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the genetic code. For example, UCA on mRNA codes for the amino acid serine, whereas CAG codes for glutamine.

Hormones are chemical messengers—organic compounds synthesized in ductless glands (endocrine glands) and delivered by
the bloodstream to target tissues in order to stimulate or inhibit some process. They cause their action at site other than site of
origin. They are not stored but are produced as and when required. Many hormones are steroids. Because steroids are nonpolar
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compounds, they are lipids. Their nonpolar character allows them to cross cell membranes, so they can leave the cells in which
they are synthesized and enter their target cells.
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In mammals the secretion of hormones is controlled by anterior lobe of pituitary gland located at base of brain. These hormones
are then carried to other gonads such as adrenal cortex, thyroid and sex glands to secrete other hormones. The disease caused by
deficiency of hormone is cured by administration of hormone.
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