Carbohydrates

transcript

Biochemistry

Mohamed Mostafa Omran

Faculty of Sciences, Helwan University

Introduction to Biochemistry

Biochemistry

• Chemistry of living organisms.• Each second/ 8,000 trillion reactions in our

bodies

Biomolecules

• Carbohydrates• Proteins• Lipids• Enzymes• Nucleic acid• Vitamins

Carbohydrates

• Composed of carbon, hydrogen, and oxygen.• Sugars • Monosaccharides– Ex. Glucose

• Disaccharides– Ex. Sucrose

• Polysaccharides– Ex. Cellulose, Starch, amylopectin, Glycogen, Chitin

Proteins• Made up of polymers of amino acids.• 20 primary amino acids exist. • A polymer of 3 or more amino acids

forms a polypeptide.

Proteins

• Primary Structure– Linear sequence of amino acids.

• Secondary Structure– Form helices or sheets due to their structure.

• Tertiary Structure– A folded protein.

• Quaternary Structure– 2 or more polypeptide chains bonded together.

Lipids• Fatty Acids– The building blocks

of lipids.

Lipids• Examples are:– Waxes– Fats and oils– Phospholipids– Steroids

Phospholipid Cell Membrane. www.williamsclass.com

Enzymes• Are proteins.• Are considered

biological catalysts.– Speed up a chemical

reaction without being altered.

• Names often end in “-ase.”– Ex. Lipase, carbohydrase.

• Act on a substrate.• Proteins, including

enzymes, can be denatured.

Nucleic Acids

• DNA and RNA.

– (DNA - deoxyribonucleic acid,

– (RNA - ribonucleic acid).

• Is the “hereditary molecule.”

• Contains genes that code for a certain product.

• DNA is translated into RNA which is used to produce a

protein or other product.

Nucleic Acid Structure

• DNA nucleotides– Building blocks of DNA.

• RNA nucleotides– Building blocks of RNA.

Nucleic Acid Structure

Nitrogenous Bases

• Adenine (A) • Guanine (G)• Cytosine (C)• Thymine (T) – only DNA• Uracil (U) – only RNA

DNA and RNA

Nitrogenous Bases

• A and G – Purines (double-

ring structures)• C, T, and U– Pyrimidines

(single-ring structures)

hyperphysics.phy-astr.gsu.edu

Vitamins

water -soluble lipid-soluble

Vit C Vit B A, D, E, K

B1, B2, B6, B12, PP, patothenic acid，folic acid,biotin ,lipoic acid

CARBOHYDRATES

The term carbohydrate is derived from the French term : hydrate de carbone

Compounds composed of C, H, and O

Empirical formula (CH2O)n when n = 5 then C5H10O5

Not all carbohydrates have this empirical formula: e.g. deoxysugars, amino

sugars etc

Carbohydrates are the most abundant compounds found in nature (cellulose:

100 billion tons annually).

CARBOHYDRATES

A - Methods of Classification: 1-One method of classification is based on whether the

carbohydrate can be broken down into smaller units.Monosaccharides: simple sugars cannot be broken down

into smaller units by hydrolysis .1 .Disaccharides: can be broken down into two

monosaccharide units.2 .Oligosaccharides: can be broken into three to six

monosaccharide units.3. Polysaccharides: composed of 7 or more

monosaccharide units.

Carbohydrates Classification

2-Another method is based on the number of carbons found in a

simple sugar.

If it has 3 carbons it is called a triose.

If it has 4 carbons it is called a tetrose.

If it has 5 carbons it is called a pentose.

If it has 6 carbons it is called a hexose.

erythrose

fructose

3-Another method uses the kind

of carbonyl group.

A- Aldose: A monosaccharide

with an aldehyde group.

B- Ketose: A monosaccharide

with a ketone group.

Carbohydrates Classification

Stereoconfigurations of simple sugars

-Asymmetric carbon atom: It Is the carbon atom that is

attached to 4 different groups.

- All monosaccharides have it except dihydroxyacetone.

- Different isomers are possible based on the presence of

number of asymmetric carbon atoms

Optical Isomerism

The presence of asymmetric carbon

atoms also confers optical activity on

the compound. When a beam of plane-

polarized light is passed through a

solution of an optical isomer, it rotates

either to the right, dextrorotatory (+),

or to the left, levorotatory (–).

• Isomerism in Monosaccharides

Carbohydrate AnomersAnomers are carbohydrates that differ by the stereo-configuration of

the carbon involved in ring formation.

The α anomer always has the OH group oriented in a downward

fashion on the anomeric carbon of a D-sugar.

β anomer always has the OH group oriented in an upward fashion on

the anomeric carbon of a D-sugar.

Important Carbohydrates

Monosaccharides: composed of three

to seven carbon atoms.

1- Glucose: The most abundant

hexose in our diet.

•The building block of complex

carbohydrates.

•Component of the disaccharides:

sucrose, maltose and lactose.

•Found in the polysaccharides: starch,

cellulose and glycogen.

Diabetes mellitus (DM)

Clinical correlate: Diabetes mellitus (DM) in which a person

has high blood glucose level.

There are three main types of diabetes mellitus.

1. Type 1 DM results from the body's failure to produce

insulin.

2. Type 2 DM results from insulin resistance, a condition in

which cells fail to use insulin.

3. Gestational diabetes

Glucose determination using urine dipstick

• The dipstick has the enzyme glucose oxidase on its

surface – this oxidises the glucose into hydrogen

peroxide and gluconic acid.

• The hydrogen peroxide will oxidise an indicator

chemical on the dipstick, which changes color.

• The exact color change is related to the

concentration of glucose in the urine sample.

• The color can be compared with a color chart to

give a reading for the glucose concentration.

• Fasting and postprandial or random blood sugar

• Glycated hemoglobin: It is formed in a non-

enzymatic pathway by hemoglobin's exposure to

plasma glucose.

• In diabetes mellitus, higher amounts of glycated

hemoglobin, indicating poorer control of blood

glucose levels.

Blood Glucose determination

Galactose

• Galactose is the C-4 epimer of glucose.

• Found in the disaccharide, lactose.

Clinical correlate: Galactose is a component of the antigens

present on blood cells that determine blood type within the

ABO blood group system. In O and A antigens, there are two

monomers of galactose on the antigens, whereas in the B

antigens there are three monomers of glucose.

Fructose

Fructose is a keto sugar.

Fructose is sweetest of the

carbohydrates (Fructose is

regarded as being 1.73 times as

sweet as sucrose).

Fructose is the energy source for

sperm motility

1-Amino sugars: They are formed by replacing the hydroxyl group (at C2 usually) of monosaccharides by amino group.

Important derivatives of monosaccharides

-Are formed by removal of an oxygen atom usually

from 2 nd carbon atom

-one quite ubiquitous deoxy sugar is 2’-deoxy ribose

which is the sugar found in DNA

2-Deoxy sugars

Sugar acids are formed by the oxidation of

aldehyde C1 (Aldonic acid) or terminal hydroxyl

group at C6 of aldose sugar (uronic acid)

-or both (saccharic) to form carboxylic group.

-L-ascorbic acid(vitamin C) is a sugar acid.

Note: Glucuronic acid is involved in detoxification

of bilirubin and other foreign compounds.

3-Sugar acids

Are formed by the reduction of the carbonyl group (aldehyde or ketone ) of monosaccharide

4-Sugar alcohols

-Hydroxyl groups of sugars can be esterified to form Acetates,

phosphates, benzoates etc.

-Sugars are phosphorylated at terminal C1 hydroxyl group or at

other places .

At terminal hydroxyl: Glucose-6-P or ribose-5-P.

At C1 hydroxyl group: Glucose-1-P .

At both places: Fructose 2,6 bisphosphate

- Metabolism of sugars inside the cells starts with phosphorylation.

-Sugar phosphates are also components of nucleosides and

nucleotides.

5-Sugar Esters

They are formed by the reaction of the hydroxyl group of

anomeric carbon with the hydroxyl group of any other

molecule with the elimination of water.

A glycosidic bond is formed.

6-Glycosides

1 .Osazone formation

2. Reduction

3. Oxidation

4. Action of alkali

5. Action of acid

6. Glycoside formation

7. Ester formation

Reactions of monosaccharides

-Used for the identification of sugars

-Consists of reacting the monosaccharide with phenyl hydrazine -A crystalline compound with a sharp melting point and a characteristic shape is obtained

D-fructose and D-mannose give the same needle shaped osazone crsytals as D-glucose

-Seldom used these days for identification

-HPLC or mass spectrometry is used now a days for the

identification of sugars

Formation of osazones

Aldoses may be oxidized to 3 types of acids

1. Aldonic acids: aldehyde group is converted to a

carboxyl group e.g. Glucose --- Gluconic acid

2. Uronic acids: aldehyde is left intact and primary

alcohol at the other end is oxidized to COOH e.g.

Glucose --- Glucuronic acid

3) Saccharic acids: (glycaric acids) – oxidation at both

ends of monosaccharide) e.g. Glucose ---- Gluco

saccharic acid

Oxidation reactions

-Either done catalytically (hydrogen and a catalyst) or enzymatically

-The resultant product is a polyol or sugar alcohol -Glucose forms sorbitol (glucitol)

-Mannose forms mannitol

-Fructose forms a mixture of mannitol and sorbitol

-Glyceraldehyde forms glycerol

Reduction

-Monosaccharides are normally stable to dilute acids, but are dehydrated by

strong acids

-D-ribose when heated with concentrated HCl yields furfural

-D-glucose under the same conditions yields 5-hydroxymethyl furfural

- Forms the basis of Molisch test, Seliwanoff test and Bial’s Test

Action of strong acids on monosaccharides

-In mild alkaline conditions Enediols are formed

- Enediols are highly reactive sugars and are powerful reducing agents.

-This allows the interconversion of D-mannose, D-fructose and D-glucose

- Strong alkalis cause Decomposition of sugars.

Action of Alkalies on sugars

Important Carbohydrates * Disaccharides

composed of 2 monosaccharide units .

1. Maltose - malt sugar. • Used in cereals, candies and the brewing of

beverages.• Composed of two D-glucose sugars joined by an α-

1,4 linkage.

HOHOH H H

CH2OH CH2OH

2. Lactose - milk sugar.• Found in milk and milk products.• Composed of one galactose and one glucose unit

joined by a β-1,4 linkage.

3. Sucrose - table sugar.• Product of sugar cane and sugar beets.• Composed of one glucose and one fructose unit.• Linkage is at both anomeric carbons.

HOHCH2OHOH

Important Carbohydrates * Polysaccharides

composed of many (more than 10) monosaccharide units. • 1- Cellulose:• Major structural material of plant cells.• Consists of many glucose units joined by β-1,4

linkages.

composed of many (more than 10) monosaccharide units. 2. Starch:• Storage form of glucose found in rice wheat, potatoes, grains

and cereals.• Consists of many glucose units joined by α-1,4 linkages.• Maltose is the disaccharide starting material.

composed of many (more than 10) monosaccharide units. 3. Glycogen:• Animal starch. Storage form of glucose found in the liver

and muscle of animals.• Contains many highly branched glucose units.• Joined by α-1,4 linkages and branched by α-1,6 linkages.

composed of many (more than 10) monosaccharide units. 4. Dextrin:• Mixture of branched and un-branched soluble

polysaccharides produced by partial hydrolysis of starch by acids or amylases.

Reducing sugars• Any sugar that contains either:1- A free aldehyde group.2- An α-hydroxy ketone group.3- A hemiacetal linkage

• The presence of any of these groups allows the carbohydrate to undergo easy oxidation.

• If the sugar gets oxidized it causes reduction.• Thus the name “reducing sugar”.

QUALITATIVE TESTS FOR CARBOHYDRATES

Exp. 1 Molisch Test

• It is the general test for all carbohydrates.

• Monosaccharides give a rapid positive test. Disaccharides and polysaccharides react slower.

Exp. 1 Molisch Test

• The Molisch reagent dehydrates pentoses to form furfural.

• dehydrates hexoses to form 5-hydroxymethyl furfural.

• The furfurals further react with -naphthol present in the test reagent to produce a purple product.

Exp. 1 Molisch Test■ Method:• 1ml test solution + 2 drops of α-naphthol • mix well • add conc. H2SO4 down the side of the tube to form the

ring at the interface of the two layers.

-ve +ve

Exp. 2 Fehling's Test

• This test is used to differentiate between reducing and non reducing sugars.

• A reducing sugar reacts with Fehling's reagent in alkaline medium to form an orange to red precipitate.

• Fehling's reagent is commonly used for reducing sugars but is known to be not specific for aldehydes.

• Positive result is detected by reduction of the deep blue solution of cupric (II) to a red precipitate of insoluble cuprous oxide (Cu2O).

Exp. 2 Fehling's Test

• Positive result is detected by reduction of the deep blue solution of cupric (II) to a red precipitate of insoluble cuprous oxide (Cu2O).

Exp. 2 Fehling's Test• The sucrose does not react with Fehling's reagent. Sucrose is

a disaccharide of glucose and fructose. Most disaccharides are reducing sugars (e.g. lactose and maltose)

• Sucrose is non-reducing sugar because the anomeric carbon of glucose is involved in the glucose- fructose bond and hence is not free to form the aldehyde in solution.

Exp. 2 Fehling's Test• Fehling's Reagent: 2solutions are required:

*Fehling's “A" uses 7 g CuSO4.5H2O dissolved in distilled watercontaining 2 drops of dilute sulfuric acid.

*Fehling's "B" uses 35g of potassium tartrate and 12g of NaOH in 100

ml of distilled water. .

Exp. 2 Fehling's Test■ Method: • 1ml test solution + 1ml Fehling's reagent

• heat the mixture in BWB (5min)

• Reddish brown ppt

Exp. 3 Benedict's Test• This test is used also to differentiate between

reducing and non reducing sugars.• It works on the same principle but Benedict is more

stable than Fehling's reagent.• Benedict's reagent contains blue copper(II) sulfate (

CuSO4) · 5H2O which is reduced to red copper(I) oxide by aldehydes, thus oxidizing the aldehydes to carboxylic acids.

• The copper oxide is insoluble in water and so precipitates. The colour of the final solution ranges from green to brick red depending on how many of the copper (II) ions are present.

Exp. 3 Benedict's Test■ Method: • 1ml test solution + 1ml Benedict's reagent • heat the mixture in BWB

(5min)• Reddish brown ppt

Exp. 4 Barfoid Test• It is a test used to differentiate between

monosaccharides and disaccharides. This reaction will detect reducing monosaccharides in the presence of disaccharides. This reagent uses copper ions to detect reducing sugars in an acidic solution. Barfoed's reagent is copper acetate in dilute acetic acid (pH 4.6)

• Reducing monosaccharides are oxidized by the copper ions in a weak acidic medium to form a carboxylic acid and a reddish ppt of Cu2O (cuprous oxide).

• Reducing disaccharides (lactose but not sucrose) undergo the same reaction but at slower rate.

Exp. 4 Barfoid Test

■ Method:• 1 ml of the solution to be tested + 2 ml of freshly

prepared Barfoed's reagent. • Place test tubes into a boiling water bath and heat for

2 minutes. • Remove the tubes from the bath and allow to cool. • Formation of a green, red, or yellow precipitate is a

positive test for reducing monosaccharides. • Do not heat the tubes longer than 3 minutes, as a

positive test can be obtained with disaccharides if they are heated long enough.

Exp. 5 Seliwanoff Test

• The test reagent dehydrates ketohexoses to form 5-hydroxymethylfurfural.

• 5-hydroxymethylfurfural further reacts with resorcinol present in the test reagent to produce a red product within two minutes.

• Aldohexoses react to form the same product, but do so more slowly.

Exp. 5 Seliwanoff Test■ Method:• 1/2 ml of a sample + 2ml of Seliwanoff's reagent (a solution of resorcinol

and HCl) is added. • The solution is then heated in a boiling water bath for two minutes.• A positive test is indicated by the formation of a red product.• In case of sucrose, avoid over-boiling because sucrose may be hydrolyzed

to its component (glucose and fructose) and gives false positive result.

+ve -ve

Exp. 6 Hydrolysis Test

• Sucrose is the only non-reducing sugar so it does not reduce the alkaline Cu solutions (Fehling and Benedict). Sucrose must first be hydrolyzed to its component and then test for.

Exp. 6 Hydrolysis Test

■ Method: • 6 ml of 1% sucrose in a test tube + 2 drops of

concentrated hydrochloric acid (HCl). • Heat the tube in a boiling water bath for 15

minutes.• Then test for Fehling, Benedict and all the

previous tests.

Exp. 7 Iodine Test (Test for Polysaccharides) 1- Starch: • 1/2 mL of the fresh starch solution + 1 drop of

the iodine solution.• A dark blue color indicates a positive test for

starch. • If the yellow color of the iodine reagent simply

becomes diluted, no starch is present. • Record the observation as positive (blue) or

negative (yellow).

Exp. 7 Iodine Test (Test for Polysaccharides)

2- Dextrin:• 1/2 mL of the fresh dextrin solution + 1 drop

of the iodine solution.• A violet color indicates a positive test for

dextrin. • If the yellow color of the iodine reagent simply

becomes diluted, no dextrin is present. • Record the observation as positive (violet) or

negative (yellow).

Exp. 8 The preparation of osazone

• Phenyl hydrazine reacts with normal carbonyls to produce phenyl hydrazones.

• 1- Sugars undergo a variation of this reaction in which 3 molecules of phenylhydrazine react with the sugar to produce a 1,2-diphenylhydrazone.

• These 1,2-diphenylhydrazones are known as osazones.

glucose

osazone

• Because both carbons 1 and 2 are involved in the reaction C-2 epimers produce the same osazone.

glucose

osazone

mannose

Exp. 8 The preparation of osazone• Ketoses with configurations identical to aldoses below

C-2 give the same osazones e.g. glucose and fructose.• Explain glucose and fructose form the same osazone?

glucose

osazone

fructose

■ Characteristics of osazones: • Have a characteristic shape.• Have characteristic melting points.• Specific time and whether the osazone is

formed from hot solutions or only on cooling.

• Glucose + Phenyl hydrazine

Glucosazone (broomed shape)

• Fructose + Phenyl hydrazine

Fructosazone (broomed shape)

• Maltose + Phenylhydrazine

Maltosazone (spherical shape)

• Lactose + Phenyl hydrazine

Lactosazone

• Sucrose + Phenyl hydrazine

-ve (WHY?)

Exp. 8 The preparation of osazone■ Method: • 1/2 g of phenyl hydrazine +

1 spoon sodium acetate +

2ml of glucose solution

• BWB (45 min) until yellow crystals appear

• cold and examine a sample of crystals under microscope.

• Compare the crystal shapes with the supplied photographs.

Carbohydrates

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