Carbohydrates 2

Carbohydratesand

their Structures

BIOCHEM LABORATORY

Maria Salvacion A. Esmalla, M. Sc.University of Santo Tomas

Faculty of PharmacyBiochemistry Department

What are carbohydrates?What are carbohydrates?

BIOCHEM LABORATORY

- a biological compound containing carbon, hydrogen, and oxygen that is an important source of food and energy

- the formula of most of these compounds may be expressed as Cm(H2O)n

- the most abundant organic compounds found in nature

According to Number of Sugar UnitsAccording to Number of Sugar Units

BIOCHEM LABORATORY

Monosaccharide

Disaccharide

Polysaccharide

CH2OHO

According to Functional GroupAccording to Functional Group

BIOCHEM LABORATORY

Aldose Ketose

According to Number of CarbonsAccording to Number of Carbons

BIOCHEM LABORATORY

pentose

hexose

According to Number of CarbonsAccording to Number of Carbons

BIOCHEM LABORATORY

Triose

Tetrose

CH2OH3

CH2OH4

CH2OH5

CH2OH6

PentoseHexose

BIOCHEM LABORATORY

Fischer ProjectionFischer Projection

D-Glucose

CH2OH6

CH2OH1

CH2OH6

D-Fructose

two-dimensional representation of the stereochemistry of three-dimensional molecules

BIOCHEM LABORATORY

D, L NotationD, L Notation

●D,L tells which of the two chiral isomers we are referring to.

● If the –OH group on the next to the bottom carbon atom points to the right , the isomer is a D-isomer; if it points left, the isomer is L.

● The D form is usually the isomer found in nature.

BIOCHEM LABORATORY

D NotationD Notation

Right = D

BIOCHEM LABORATORY

What are optical isomers? also called stereoisomers

molecules that differ from each other only in configuration of structures

usual source of optical isomerism is a chiral carbon

D-Glyceraldehyde

L-Glyceraldehyde

BIOCHEM LABORATORY

What are enantiomers?

D-Glyceraldehyde

L-Glyceraldehyde

molecules that are mirror images but not superimposable

BIOCHEM LABORATORY

What are diastereomers?

nonsuperimposable, non-mirror image stereoisomers

D-ThreoseD-Erythrose

BIOCHEM LABORATORY

What are epimers?

D-Ribose D-Xylose

diastereomers that differ from each other in configuration at only one chiral carbon

BIOCHEM LABORATORY

What are epimers?

BIOCHEM LABORATORYBIOCHEM LABORATORY

D-Glucose

D-Fructose

Important MonosaccharidesImportant Monosaccharides

D-galactose

BIOCHEM LABORATORY

Does monosaccharide forms cyclic structure?

CH2OH6

D-Glucose

CH2OH6

Hemiacetal form

BIOCHEM LABORATORY

Does monosaccharide forms cyclic structure?

Hemiketal formD-Fructose

CH2OH1

CH2OH6

CH2OHH

CH2OH6

Mutarotation: A small amount of open chain is in equilibrium with the cyclic

forms.The most stable form of glucose is β-D-

glucose .

-D-glucose D-glucose (open) β-D-glucose

(36%) (trace) (64%)

Mutarotation

BIOCHEM LABORATORY

CH2OH6

Anomeric carbon

CH2OH6

CH2OHAnomeric

carbon

Are there new features in the cyclic structure of monosaccharides?

CH2OH6

α-D-Glucose β-D-Glucose

Are there new features in the cyclic structure of monosaccharides?

Furanose structures

Pyranose structures

perspective representation of the cyclic forms of sugars

Haworth Projection

CH2OH6

OCH2OH

OHOHOH

D-Glucose

α -D-Glucose

(α-D-Glucopyranose)

Fischer to Haworth Projection

Conformation

Haworth to Chair Conformation

CH2OH6

α -D-Glucose

(α-D-Glucopyranose)

Draw the Fischer, Haworth and Chair conformation of α-D-mannopyranose and α-D-galactopyranose

How are glycosidic bonds formed?

+ CH3OH

OCH2OH

OHOHOH

OCH2OH

OHOCH3OH

Glycosidic bond

Glycosidic linkage among sugars

OCH2OH

α(1-4) Glycosidic bond α(1-6) Glycosidic bond

β,β(1-1) Glycosidic bond

Important Disaccharides

Maltose = Glucose + Glucose

Lactose = Glucose + Galactose

Sucrose = Glucose + Fructose

Important Disaccharides

OCH2OH

OH O CH2OH

OCH2OH

Sucrose

,β(1-2) glycosidic bond

OCH2OH

OH OCH2OH

–D-Lactose

β(1-4) glycosidic bond

-D-Maltose

(1-4) glycosidic bond

β–D-Cellobiose

OCH2OH

Sucrose

oCH2OH

α, -1,2-glycosidic bond

Lactose

OCH2OH

OH OCH2OH

–D-Lactose

Maltose

OCH2OH

-D-Maltose

(1-4) glycosidic bond

Cellobiose

β–D-Cellobiose

OCH2OH

Learning CheckIdentify the monosaccharides in each of the following:

A. lactose

(1) glucose (2) fructose (3) galactose

B. maltose

C. sucrose

Solution

A. lactose

(1) glucose and (3) galactose

B. maltose

(1) glucose and (1) glucose

C. sucrose

(1) glucose and (2) fructose

Important Polysaccharides

Starch

Amylose

Amylopectin

Glycogen

Cellulose

Polysaccharides

CH2OH6

Polymers of D-glucose

Amylose

Polymer with α-1,4 bonds

α-1,4 bonds

Amylopectin

Polymer with α-1,4 and α-1,6 bonds branches

α-1,6 bond

α-1,4 bonds

Cellulose

Polymer with ß-1,4 bonds

ß-1,4 bonds

Glycogen

Learning CheckIdentify the polysaccharide in each as:

1) starch 2) glycogen 3) cellulose

A. B. C.

Solution

3) cellulose 1) starch 2) glycogen

Physical Properties

Carbohydrates

● Carbohydrates are solid at room temp

● Mono- and disaccharides are white crystalline substances and soluble in H2O

● Starches are amorphous and slighlty soluble in H2O

● Cellulose is fibrous and insoluble in H2O

Physical Properties

Chemical Properties

Carbohydrates

1. All aldoses are reducing sugars because they contain free aldehyde functional group.

The aldehydes are oxidized by a mild oxidizing agent (e.g. Benedict’s or Fehling’s reagent) to the corresponding carboxylates.

Reducing Property

2. All ketoses are reducing sugars because they have a ketone functional group next to an alcohol functional group.

The reactivity of the -hydroxyketone is due to the formation of -hydroxyaldehyde in basic media.

Reducing Property

3. Carbohydrates with hemiacetal functional groups can reduce mild oxidizing agent because hemiacetals can form aldehydes through the following equilibrium equation.

Reducing Property

- Sucrose is a non-reducing sugar because it does not contain a hemiacetal functional group.

- Starch is a non-reducing sugar because the effect of the hemiacetal

functional group is insignificant in a very large starch molecule.

1. Disaccharides and polysaccharides can be converted into monosaccharide by hydrolysis

Hydrolysis of acetal groups

Isolation

Carbohydrates

Glycogen from Chicken liver

- energy storage polysaccharide in animals- a branched-chain polysaccharide (homoglycan)- consists entirely of linear chains of glucose units connected by -1,4-

glycosidic bonds and branched points every 1 in 10 glucose units linked by -1,6-glycosidic bonds

Homogenization – disrupts membranes0.1% acetic acid – removes protein via precipitationGlycogen is insoluble in ethanol.Method: Protein denaturation and solubility of glycogen

Starch from Potato

- energy storage polysaccharide in plants- a homoglycan mixture of amylose and amylopectin

1. Amylose – linear homoglycan of glucose connected by -1,4 linkages

2. Amylopectin – branched homoglycan with approximately 1 in 25 glucose units linked in both -1,4 and -1,6 fashion

Starch is slightly soluble in H2OMethod: Selective dissolution

Hydrolysis

Carbohydrates

Acid Hydrolysis

- 2 N HCl- complete hydrolysis- efficient method to obtain monosaccharides

starch/glycogen

glucose

Enzymatic Hydrolysis

- salivary amylase (-amylase)- very specific - an endoglycosidase hydrolyzing -1,4 linkages at random to yield a mixture of glucose, maltose, dextrin & other hydrolysates

starch/glycogen

dextrin, amylodextrin, maltose,glucose

Enzymatic Hydrolysis

Thin layer chromatography

Carbohydrates

Thin Layer Chromatography

● based on adsorption phenomenon● normal phase chromatographystationary phase: silica G-250mobile phase: butanol: acetic acid:ether:H2O (9:6:3:1)visualizing agent: p-anisaldehyde● appeared as dark green spotsstandards: dextrin, maltose and glucose

Rf polar

Quantitative Determination

Carbohydrates

Nelson’s Test

REAGENTS: Nelson’s A – Na2CO3, NaKtartrate, NaHCO3, Na2SO4

Nelson’s B – CuSO4●5H2O, H2SO4

Arsenomolybdate reagent - CuSO4 serves as the oxidizing agent- Na2CO3 & NaHCO3 makes sugars more reactive-

NaKtartrate prevents the precipitation of Cu(OH)2

- NaKtartrate would tend to reduce Cu2+ in time

- Arsenomolybdate is added to dissolve Cu2O & is converted into molybdenum blue

- color intensity of molybdenum blue A480

y = mx + b

Characterization

Carbohydrates

General Test:General Test:

Molisch TestREAGENTS: conc. H2SO4; α-naphthol in 95% ethanol

PURPOSE: confirm presence of carbohydrates

POSITIVE RESULT: violet interphase

PRINCIPLE INVOLVED:

hydrolysis, dehydration forming either a furfural (pentoses) or a 5- hydroxufurfural (hexoses); condensation with α-naphthol

REACTION:

5-hydroxyfurfural

O CH2OHH

hexose

naphthol

purple-colored dye+-

Iodine Test

REAGENTS: iodine reagent

PURPOSE: distinguish starch and glycogen from other polysaccharides

POSITIVE RESULT: Starch gives a blue-black color Glycogen gives a red color

PRINCIPLE INVOLVED: complexation

REACTION:

Specific Test:Specific Test:

Anthrone Test

REAGENTS: conc. H2SO4; anthrone reagent

PURPOSE: confirm presence of carbohydrates

POSITIVE RESULT: blue-green solution

PRINCIPLE INVOLVED:

Hydrolysis, dehydration forming either a furfural or a 5- hydroxyfurfural; condensation of anthrone via anthranol intermediate

REACTION:

OR CHOO OH O

H+ [O]

Benedict’s Test

REAGENTS: CuSO4; Na3C6H5O7; Na2CO3 (Benedict’s reagent)

PURPOSE: detect presence of reducing sugars

POSITIVE RESULT: brick red precipitate

PRINCIPLE INVOLVED:

oxidation in less basic media

REACTION:

+ Cu2O

Na2CO3, Na citrate

-D-glucose sodium gluconate

Barfoed’s Test

REAGENTS: Cu(Ac)2; Acetic acid (Barfoed’s reagent)

PURPOSE: detect reducing sugars; distinguishes between reducing monosaccharide and reducing disaccharide

POSITIVE RESULT: brick red precipitate

PRINCIPLE INVOLVED:

oxidation of a reducing monosaccharide or disaccharide in acidic condition

REACTION:

Bial’s Orcinol TestREAGENTS: orcinol in conc. HCl; FeCl3 (Bial’s reagent)

PURPOSE: detect presence of pentoses

POSITIVE RESULT: blue-green solution

PRINCIPLE INVOLVED:

dehydration and condensation reaction with orcinol

REACTION:

Seliwanoff’s Test

REAGENTS: resorcinol; HCl

PURPOSE: differentiate ketohexoses from aldohexoses

POSITIVE RESULT: rapid formation of cherry red solution

PRINCIPLE INVOLVED:

rapid dehydration and condensation reaction with resorcinol

REACTION:

OHOH2C

Mucic acid Test

HNO3 12

REAGENTS: conc. HNO3

PURPOSE: detect presence of galactose (moiety)

POSITIVE RESULT: rhombic crystal

PRINCIPLE INVOLVED:

1,6 -oxidation of sugars whereby galactose-containing carbohydrates form a meso compound (galactaric acid)

REACTION:

Osazone/Phenylhydrazone TestSucrose – does not form osazone unless hydrolyze

Osazone/Phenylhydrazone TestGlucosazone - needle-like/feathery, 4-5min

Osazone/Phenylhydrazone TestFructosazone – needle-like/feathery, 2 min

Mannosazone – needle-like/feathery, 0.5min

Osazone/Phenylhydrazone Test

Lactosazone – fine needles; grouped in balls; soluble in hot H2O

Galactosazone – broad/flat crystals (15-20 min)

Maltosazone – broad needle-like crystals; soluble in hot H2O

Xylosazone – long, fine needle-like crystals (7 min)

Starch - does not form osazone unless hydrolyze

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