carbohydrates 2
DESCRIPTION
CarbohydratesTRANSCRIPT
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
OH
OH
H
OH
H
OHH
OH
CH2OH
H
O
OHOH
OH
CH2OHO
OOH
OH
CH2OH
OH
O
OOH
OH
CH2OHO
OOH
OH
CH2OHO
OOH
OH
CH2OHO
OOH
OH
CH2OH
According to Functional GroupAccording to Functional Group
BIOCHEM LABORATORY
Aldose Ketose
CH
C
CH2OH
O
OHH
CH2OH
C
CH2OH
O
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
CHO1
C2
CH2OH3
OHH
CHO1
C2
C3
OHH
CH2OH4
H OH
CHO1
C2
C3
OHH
C4
H OH
CH2OH5
H OH
CHO1
C2
C3
OHH
C4
H OH
C5
H OH
CH2OH6
H OH
PentoseHexose
BIOCHEM LABORATORY
Fischer ProjectionFischer Projection
D-Glucose
C1
C2
OHH
C3
HHO
C4
OHH
C5
OHH
CH2OH6
OH
CH2OH1
C2
O
C3
HHO
C4
OHH
C5
OHH
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
C
C
CH2OH
OHH
OHH
CO
H
Right = D
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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
C OHH
CHO
CH2OH
C HHO
CHO
CH2OH
L-Glyceraldehyde
BIOCHEM LABORATORY
What are enantiomers?
D-Glyceraldehyde
C OHH
CHO
CH2OH
C HHO
CHO
CH2OH
L-Glyceraldehyde
molecules that are mirror images but not superimposable
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What are diastereomers?
nonsuperimposable, non-mirror image stereoisomers
C
C OHH
C OHH
CH2OH
OHC
C HHO
C OHH
CH2OH
OH
D-ThreoseD-Erythrose
BIOCHEM LABORATORY
What are epimers?
C
C OHH
C OHH
C
OH
CH2OH
OHH
C
C OHH
C HHO
C
OH
CH2OH
OHH
D-Ribose D-Xylose
diastereomers that differ from each other in configuration at only one chiral carbon
BIOCHEM LABORATORY
What are epimers?
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C
C
CH2OH
H OHC
OHH C
HHO
H OH
C OH
D-Glucose
C
C
CH2OH
H OHC
OHH C
HHO
O
CH2OH
D-Fructose
Important MonosaccharidesImportant Monosaccharides
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Important MonosaccharidesImportant Monosaccharides
D-galactose
C
C
C
C
C
CH2OH
OH
OHH
HO H
HHO
OHH
BIOCHEM LABORATORY
Does monosaccharide forms cyclic structure?
C1
C2
OHH
C3
HHO
C4
OHH
C5
OHH
CH2OH6
OH
D-Glucose
C5 O
C4
C3
C1
C 2
OH
H
H
OH
CH2OH6
H
OHO
H
H
HC5 O
C4
C3
C 1
C 2
OH
H
H
OH
CH2OH6
H
OH OH
H
H
Hemiacetal form
BIOCHEM LABORATORY
Does monosaccharide forms cyclic structure?
Hemiketal formD-Fructose
CH2OH1
C2
O
C3
HHO
C4
OHH
C5
OHH
CH2OH6
O
5C
4C
3
C
1
C
2
H
OH
OH
H
CH2OH6
H
O
CH2OHH
O
5C
4C
3
C
1
C
2
H
OH
OH
H
CH2OH6
H OH
CH2OH
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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%)
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Mutarotation
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Mutarotation
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C5 O
C4
C3
C 1
C 2
OH
H
H
OH
CH2OH6
H
OH OH
H
H
Anomeric carbon
O
5C
4C
3
C
1
C
2
H
OH
OH
H
CH2OH6
H OH
CH2OHAnomeric
carbon
Are there new features in the cyclic structure of monosaccharides?
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C5 O
C4
C3
C 1
C 2
OH
H
H
OH
CH2OH6
H
OH OH
H
H
C5 O
C4
C3
C 1
C 2
OH
H
H
OH
CH2OH6
H
OH H
OH
H
α-D-Glucose β-D-Glucose
Are there new features in the cyclic structure of monosaccharides?
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O
CC
CO
C
O C
C
C C
C
O
Furanose structures
Pyranose structures
O
O
Furan
Pyran
perspective representation of the cyclic forms of sugars
Haworth Projection
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C1
C2
H OH
C3
OHH
C4
HO H
OHH
C5
H
CH2OH6
O
5
4
3 2
1
OH
CH2OH6
H
OH
OH
H
H
OH
H
OH
5
4
3 2
1
OCH2OH
6
OH
OHOHOH
D-Glucose
α -D-Glucose
(α-D-Glucopyranose)
Fischer to Haworth Projection
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Conformation
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Haworth to Chair Conformation
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Haworth to Chair Conformation
5
4
3 2
1
OH
CH2OH6
H
OH
OH
H
H
OH
H
OH
α -D-Glucose
(α-D-Glucopyranose)
?
Draw the Fischer, Haworth and Chair conformation of α-D-mannopyranose and α-D-galactopyranose
BIOCHEM LABORATORYBIOCHEM LABORATORY
How are glycosidic bonds formed?
+ CH3OH
OCH2OH
OH
OHOHOH
OCH2OH
OH
OHOCH3OH
Glycosidic bond
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Glycosidic linkage among sugars
OCH2OH
OH
OH
OOH
OCH2OH
OH
OHOH
OCH2OH
OH
OH
OH
O
H2C
OH
OHOHO
OCH2OH
OH
OH
OH
O
OH
CH2OH
OH
OH
O
α(1-4) Glycosidic bond α(1-6) Glycosidic bond
β,β(1-1) Glycosidic bond
OH
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Important Disaccharides
Maltose = Glucose + Glucose
Lactose = Glucose + Galactose
Sucrose = Glucose + Fructose
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Important Disaccharides
OCH2OH
OH
OH
OH O CH2OH
CH2OH
OH
OHO
OCH2OH
OH
OH
OOH
OCH2OH
OH
OHOH
Sucrose
,β(1-2) glycosidic bond
OCH2OH
OH
OH
OH OCH2OH
OH
OHOH
O
–D-Lactose
β(1-4) glycosidic bond
-D-Maltose
(1-4) glycosidic bond
β–D-Cellobiose
β(1-4) glycosidic bond
OCH2OH
OH
OHOH
OCH2OH
OH
OH
OH
O
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Sucrose
O
CH2OH
OH
OH
CH2OH
oCH2OH
OHOH
OH
O
α, -1,2-glycosidic bond
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Lactose
OCH2OH
OH
OH
OH OCH2OH
OH
OHOH
O
–D-Lactose
β(1-4) glycosidic bond
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Maltose
OCH2OH
OH
OH
OOH
OCH2OH
OH
OHOH
-D-Maltose
(1-4) glycosidic bond
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Cellobiose
β–D-Cellobiose
OCH2OH
OH
OHOH
OCH2OH
OH
OH
OH
O
β(1-4) glycosidic bond
β
39
Learning CheckIdentify the monosaccharides in each of the following:
A. lactose
(1) glucose (2) fructose (3) galactose
B. maltose
(1) glucose (2) fructose (3) galactose
C. sucrose
(1) glucose (2) fructose (3) galactose
40
Solution
A. lactose
(1) glucose and (3) galactose
B. maltose
(1) glucose and (1) glucose
C. sucrose
(1) glucose and (2) fructose
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Important Polysaccharides
Starch
Amylose
Amylopectin
Glycogen
Cellulose
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Polysaccharides
C5 O
C4
C3
C 1
C 2
OH
H
H
OH
CH2OH6
H
OH OH
H
H
Polymers of D-glucose
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Amylose
Polymer with α-1,4 bonds
α-1,4 bonds
OOH
OH
CH2OH
O
O
OH
OH
CH2OH
O
O
OH
OH
CH2OH
O
O O
OH
OH
CH2OH
O
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Amylopectin
Polymer with α-1,4 and α-1,6 bonds branches
α-1,6 bond
α-1,4 bonds
OOH
OH
CH2OH
O
O
OH
OH
CH2OH
O
O
OH
OH
CH2
O
O O
OH
OH
CH2OH
O
OOH
OH
CH2OH
O
O
OH
OH
CH2OH
O
O
OH
OH
CH2OH
O
O
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Cellulose
O
O
OH
OH
CH2OH
O
O
OH
OH
CH2OH
O
O
OH
OH
CH2OH
O
Polymer with ß-1,4 bonds
ß-1,4 bonds
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Glycogen
47
Learning CheckIdentify the polysaccharide in each as:
1) starch 2) glycogen 3) cellulose
A. B. C.
48
Solution
3) cellulose 1) starch 2) glycogen
Physical Properties
of
Carbohydrates
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● 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
of
Carbohydrates
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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
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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
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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.
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1. Disaccharides and polysaccharides can be converted into monosaccharide by hydrolysis
Hydrolysis of acetal groups
Isolation
of
Carbohydrates
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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
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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
of
Carbohydrates
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Acid Hydrolysis
- 2 N HCl- complete hydrolysis- efficient method to obtain monosaccharides
starch/glycogen
glucose
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Enzymatic Hydrolysis
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- 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
of
Carbohydrates
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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
of
Carbohydrates
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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
of
Carbohydrates
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General Test:General Test:
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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
O
C
OHH
HHO
OHH
OHH
CH2OH
H2SO4
OH
hexose
OH
naphthol
O
OH
OH
HO
purple-colored dye+-
General Test:General Test:
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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:
I2
I2
I2I2
HEAT
I2
COOL
Specific Test:Specific Test:
BIOCHEM LABORATORYBIOCHEM LABORATORY
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
HHO
O
H
R
O
O
OH
R
H+ [O]
Specific Test:Specific Test:
BIOCHEM LABORATORYBIOCHEM LABORATORY
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
OH
CH2OH
H
H
OH
OH
H
O
O-Na
+
OH
H
O
CH2OH
H
H
OH
OH
H
OH
H
OH
H
Cu+2
Na2CO3, Na citrate
-D-glucose sodium gluconate
Specific Test:Specific Test:
BIOCHEM LABORATORYBIOCHEM LABORATORY
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:
OH
CH2OH
H
H
OH
OH
H
O
OH
OH
H
O
CH2OH
H
H
OH
OH
H
OH
H
OH
H
Cu+2
H+
Specific Test:Specific Test:
BIOCHEM LABORATORYBIOCHEM LABORATORY
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:
HO OH
OH
O
O
H
HO
OH
OH
OH
O
O
OH
OH
OH
H+
O
O
O-
O-
O-
Fe+3
[O]
+--
Specific Test:Specific Test:
BIOCHEM LABORATORYBIOCHEM LABORATORY
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:
C
C
O
C
OHH
C
H OH
CH2OH
- H2O
HO OH
OH
O
HOH2C
OHOH2C
H
HO
OH
OH
OH
OHOH2C
O
OH
OH
OH
H+[O]
General Test:General Test:
BIOCHEM LABORATORYBIOCHEM LABORATORY
Mucic acid Test
HNO3 12
HO
3
OH
4
HO
5
OH
6
O
HO
O
OH
C 1
C2
OHH
C3
OHHO
C4
OH
C5
OHHO
H2C6
H OH
OH
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:
Specific Test:Specific Test:
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Osazone/Phenylhydrazone TestSucrose – does not form osazone unless hydrolyze
Specific Test:Specific Test:
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Osazone/Phenylhydrazone TestGlucosazone - needle-like/feathery, 4-5min
Specific Test:Specific Test:
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Osazone/Phenylhydrazone TestFructosazone – needle-like/feathery, 2 min
Mannosazone – needle-like/feathery, 0.5min
Specific Test:Specific Test:
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Osazone/Phenylhydrazone Test
Lactosazone – fine needles; grouped in balls; soluble in hot H2O
Specific Test:Specific Test:
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Osazone/Phenylhydrazone Test
Galactosazone – broad/flat crystals (15-20 min)
Maltosazone – broad needle-like crystals; soluble in hot H2O
Xylosazone – long, fine needle-like crystals (7 min)
Specific Test:Specific Test:
BIOCHEM LABORATORYBIOCHEM LABORATORY
Osazone/Phenylhydrazone Test
Starch - does not form osazone unless hydrolyze