I. DEFINITION & ROLES
II. STRUCTURE OF CARBOHYDRATES
A. Simple saccharides
V. STUDY OF SOME GLYCOSIDES AND DERIVATIVES
VI. References and Internet links
I. Definition and roles
Carbohydrates are a group of substances whose basic units are simple sugars called monosaccharides.
Carbohydrates were defined as polyhydroxy aldehydes or ketones. They are water-soluble compounds and reducing agents.
Carbohydrates are present everywhere in the biosphere and represent the prominent weight class among organic molecules. The largest part of carbohydrates comes from photosynthesis, a process that incorporates CO2 into carbohydrates.
Carbohydrates play many critical roles in cells :
Polymerized form of carbohydrates are called saccharides. They can be made of :
A. Linear structure
It should be noted that in their phosphorylated form, these two compounds represent an important step in the glycolytic pathway , since it is the cleavage of a C6 sugar (fructose 1, 6 diphosphate) to 2 C3 sugars.
Glyceraldehyde has a carbon with four different substituents, so it is an asymmetric or chiral carbon.
Glyceraldehyde can therefore exist in two different forms (image of one another in a mirror, therefore non-superimposable) which correspond to opposite configurations around the chiral carbon: the two compounds are called enantiomers.
In 1906, Emil Fischer and Rosanoff chose glyceraldehyde as a reference compound to study sugars configuration.
Emil Fischer arbitrarily chose the symbol D for the dextrorotatory enantiomer, that is to say the compound which deflects the plane of polarized light to the right or rather in the direction of clockwise.
It was not until 1954 that Bijvoet showed by crystallographic studies that the arbitrary Fischer corresponded to the absolute configuration of monosaccharides.
All monosaccharides derived from dextrorotatory glyceraldehyde were said to belong to the D series and those from the glyceraldehyde levorotatory were said to belong to the L series.
All aldoses can be synthesized from glyceraldehyde.
In the Fischer projection , all monosaccharides whose hydroxyl carried by the penultimate carbon is on the right belong to D series.
When moving from one carbohydrates to a higher one, H-C-OH group is added between the chiral carbon bearing the terminal primary alcohol and the adjacent carbonyl carbon.
With each addition, there are two possibilities:
Example of glucose : it is a carbohydrate with 6 carbons or hexose. There are therefore 16 stereoisomers, 8 of D series and 8 of L series.
The natural sugars are mostly of D series.
Example : D-mannose and D-galactose are epimers of D-glucose but are not epimers among them.
Ascending series of D-ketoses and epimers of ketohexoses.
B. Cyclic structure
The linear structure or open-chain structure of carbohydrates do not realize all their properties as soon as the number of atoms is greater than 4.
First reducing properties that are not quite those of aldehydes and ketones:
This phenomenon has been called mutarotation by Lowry (1889).
The specific rotation [α]20D is measured with a device called a polarimeter.
It is defined by specifying the temperature, the wavelength at which the measurement is made (it is usually the sodium D line 589 nm).
In addition, the concentration is expressed in g/ml and the length of the polarimeter tube is expressed in decimeters.
Knowing the specific rotation of a compound, BIOT's law determines the concentration of a solution of that compound.
This law is additive, that is to say that the rotatory power of a mixture is the sum of the optical rotations of compounds that constitute the mixture.
2. Cyclization mechanism and representation of Norman HAWORTH.
The mutarotation phenomenon implies the existence of an additional asymmetric carbon.
In addition, the formation of hemiacetal implies that the reducing function has already established a connection with an alcohol.It was in 1884 that Bernhard Tollens provided explanation of the cyclic structure of monosaccharides:
The hydroxyl group carried by the carbon 4 can also react and get a 5 vertices or furanose ring.
The names of pyranose and furanose have been adopted by analogy with hydrocarbon containing 6 and 5 summits, respectively.
Studies of the conformational stability of cyclohexane showed that the spatial arrangements that are not subject to steric constraints adopt a so-called chair conformation and others, less stable, are in the so-called boat conformation.
The position of the hydrogen substituents may be either in an axis perpendicular to the plane defined by the six carbon-carbon bonds, these are so-called axial substituents, or conversely directed outwardly of the ring and they are said equatorial.
In the case of glucopyranose is essentially the chair form exists.
III. Chemical properties
A. Properties related to reductive group
Carbohydrates are lower seducers than true aldehydes or ketones. The result of oxidation depends on the conditions of oxidation.
a) aldonic acids are obtained by mild oxidation of aldoses using Br2 or I2 in alkaline condition :
b) further oxidation with nitric acid leads to aldaric acids, diacids having a carboxylic function on carbon 1 and carbon 6:
c) Finally, if the aldehyde function is protected during oxidation, uronic acids are obtained which are oxidized only on the primary alcohol function:
These compounds are involved in cell recognition in bacteria.
The glucuronic acid is the precursor of the synthetic pathway of vitamin C or L-ascorbic acid.
Vitamin C is an enediol form of a lactone derived from an aldonic acid . The pKa of the C3 hydroxyl group of Vitamin C is relatively low due to the resonance stabilization of its conjugate base.
The reactions are reduced by catalytic hydrogenation or by the action of an alkali metal borohydride such as NaBH4 or LiBH4.
It is worth mentioning other reduction reactions used for the determination of sugars and their characterization, including:
a) with cyanide and hydroxylamine
Condensation reactions include the Kiliani's synthesis reaction and the Wohl-Zemplen degradation' reaction. These two paths transform a carbohydrate to the upper one and the lower one, respectively.
They both helped establishing the parentage of carbohydrates with glyceraldehyde.
Kiliani's synthesis : glucose reacts with cyanhidric acid to form a cyanhidrine (2 stereoisomers) which, after hydrolysis, gives a hexahydroxylated acid.
It is reduced by IH (in the presence of red phosphorus) and gives heptanoic acid.
The same reaction from fructose acid gives methyl-2-hexanoic acid.
b) with alcohols and phenols
This reaction is particularly important : the substances obtained are saccharides or glycosides. The bond joining the carbohydrate to the alcohol or the phenol is the O-glycoside bond or glycosidic bond.
It is important to note that this bond formation is accompanied by the loss of the reducing power of the carbohydrate and locks the configuration of the cycle.
B. Properties related to alcoholic functions
They are used to perform electrophoresis of carbohydrates, which otherwise would not be possible since carbohydrates are not naturally charged.
These complexes were used to demonstrate that the hydroxyl group of the anomeric carbon of α-D-glucose is in the cis position relative to the hydroxyl group carried by carbon 2, so that it is located below the ring. Indeed, the complex is formed more easily with the cis anomer.
The anomeric sugar affect the formation of complexes with boron and thus their electrophoretic mobility.
Methylating agents such as methyl sulfate [(SO4(CH3)2] in the presence of sodium hydroxide (Haworth) or methyl iodide (ICH3) with Ag2O (Purdie) act by substituting all hydrogen of hydroxyl groups with a -CH3 group, thereby forming an ether group.
If the reducing group of carbohydrates is free, it will react by forming a O-methylated derivative.
However, this bond is a glycosidic bond which does not have the same stability in an acidic medium where it is easily hydrolyzed.
This bond must be therefore distinguished from ether bond by specifying it in the nomenclature of carbohydrates.
Methylation is an important technique that has two main applications:
a) Determination of the structure of cycles
A saccharide ring is completely methylated, then the glycosidic bond is hydrolyzed in a dilute acid medium.
The compound is then oxidized using nitric acid. The oxidation breaks the cycle and eliminates the carbons which are not part of the cycle, carbon 6 in the case of a pyranose and carbons 5 and 6 in the case of a furanose.
The remainder of the cycle becomes a tri-O-methylated diacid in the case of a pyranose and a di-O-methylated diacid in the case of a furanose.
b) Determination of the sequence in polysaccharides
A glycoside is completely methylated and then glycosidic bonds are cut in dilute acid medium.
The example of amylose shows that the non-reducing terminal compound (hence the compound whose hemiacetalic hydroxyl group is involved in the glycosidic bond but, conversely, whose carbon 4 is not involved in this bond) leads to a tetra-O-methylated derivative while all other elements leads to a tri-O-methylated derivative.
In the case of a branched structure, one obtains a di-O-methylated derivative for each carbohydrate involved in the connection (in this case, the monosaccharide whose carbon 6 is involved in the glycosidic bond).
IV. Study of several monosaccharides and derivatives
1. Glucose (aldohexose - pyranose)
It is extremely widespread in the plant kingdom and the animal kingdom under a free state or combined with other saccharides, either phosphorylated or not.
From a structural view, concerning monosaccharides, it is necessary to pay attention to the position of the hydroxyl group of the penultimate carbon (C5 for a hexose) in the Fischer projection and the position of the resulting C6 in Haworth's representation : in Haworth's representation, C6 indicates either a D or L series since the hydroxyl group of C5 is engaged to form a hemiacetal and therefore does not further indicates the series of carbohydrates.
2. Arabinose (aldopentose - pyranose)
Arabinose is abundant in the plant world. It contributes to the formation of the supporting tissues.
From a structural point of view, it is necessary to pay attention to the position of the hydroxyl group of C4.
3. Fructose (ketohexose - furanose)
This ketose is obtained by interconversion of glucose and mannose, e.g. by epimerization of C2 of glucose.
Fructose is a monosaccharide which emphasizes the importance of the linear form: there is always a high concentration of the linear form and there is also a balance with furan forms, e.g., the most stable forms.
4. Galactose and mannose (aldohexose - pyranose)
These two monosaccharides are much less abundant in cells than glucose but they are found as constituents of glycoproteins and glycolipides.
Osamines are synthesized from fructose-6-phosphate and are obtained by substitution by a NH2 group of the hydroxyl group of carbon 2. The amino group is most often acetylated.
These are compounds characteristic of glycoproteins. They are linked by a α-glycosidic bond.
The COOH function is free, which imparts a marked acid character to glycoproteins.
N-acetyl-mannosamine-6-phosphate is the precursor of sialic acids.
Sialic acids are all derived from neuraminic acid, whose the most common is N-acetylneuraminic acid.
The substituents vary according to the animal species (N-acetyl in the case of sheep / N-glycolyl in the case of pigs; ...).
Their synthesis is obtained from N-acetyl-mannosamine-6-phosphate and phosphoenolpyruvate.
In glycoproteins, the sialic acids are arranged at regular intervals along the chain. They thus form a electronegative cloud that, by electrostatic repulsion, keeps the chain in a elongated stick form. The consequence is a high viscosity.
The N-acetylated muramic acid is a component of murein, a high moleular weigth polymer type glycopeptide which forms the support core of the bacterial walls. The N-acetylated muramic acid derives from N-acetyl-glucosamine. It is also biosynthesized from phosphoenolpyruvate.
There is formation of phosphoric esters by the action of kinases which transfer the terminal phosphate group of ATP.
Used as an energy source, it is in their phosphorylated forms that carbohydrates are interconverted and therefore metabolized (glycolytic pathway and pentose phosphate pathway, for example).
The α-D-ribose-5-phosphate and 2-deoxy-D-ribose-5-phosphate are the two monosaccharides constituent of nucleic acids.
V. Study of some saccharides and derivatives
Osides or glycosides are substances in which the hydroxyl group of the hemiacetal anomeric carbon of a saccharide is condensed with a hydroxyl group (alcoholic or phenolic).
The bond between the carbohydrate and the alcohol or phenol function is called O-osidic or glycoside. Osides hydrolysis yield at least two sugars.
a) Determination of the nature of the constituent monosaccharides
Osides are hydrolyzed enzymatically or in the presence of acid , in order to break the osidic bonds. In the case of glycosides, the nature of the aglycone part must be identified.
Then they are separated by chromatographic techniques and identified and assayed individually.
b) Determination of the binding mode between the constituent monosaccharides
All free hydroxyl functions are marked by methylation and periodic acid. Further acid hydrolysis differ ether-oxides bonds from osidic bonds.
The determination of the osidic bond anomery needs the use of enzymes specific of each type of bond, or in the simplest case, needs the study of the mutarotation after hydrolysis.
c) Determination of the reducing power
Reduction by Na borohydride is used to characterize the terminal reducing monosaccharide in the case of an oligosaccharide or dioside.
In the case of a polysaccharide, the proportion of the terminal reducing sugar is so low that it is necessary to use borohydride marked by tritium.
d) Determination of the molecular weight and chain length in the case of polysaccharides
This is obtained by usual physical techniques (osmometry, ultracentrifugation, light scattering, viscometry, molecular gel filtration , electrophoresis of complexes with boron...), biochemical techniques (specific enzymes for degradation, ...) and immunochemical techniques.
This diholoside is released by amylose hydrolysis (see below), which is a polymer of glucose residues : maltose is the α-D-glucopyranosyl-(1,4)-D-glucopyranose.
Glucose residues are released by chemical hydrolysis or by an enzyme : the α-D-glucosidase.It is a reducing sugar since the hydroxyl group of the anomeric carbon of the second glucose residue is free.
Methylation followed by hydrolysis lead to 2,3,6 tri-O-methyl-glucose and 2,3,4,6 tetra-O-methyl-glucose.
It is the sugar in milk synthesized in the mammary glands. Lactose is the β-D-galactopyranosyl-(1,4)-D-glucopyranose.
This is the only reducer diholoside found naturally.
This is an extremely represented sugar in the plant kingdom and especially in sugar cane and sugar beet.
Sucrose and trehalose are the only non-reducer diholoside found naturally (the hydroxyl group of the anomeric carbon of fructose is involved in the osidic bond with glucose).
Sucrose is the α-D-glucopyranosyl-β-D-or β fructofuranoside-α-D-fructofuranosyl-D-glucopyranoside.
Methylation followed by hydrolysis lead to 3,4,6 tri-O-methyl-fructose and 2,3,4,6 tetra-O-methyl-glucose.
Cellobiose results from cellulose degradation.
Cellobiose is the β-D-glucopyranosyl-(1,4)-D-glucopyranose.
This is an epimer of lactose (C4 epimer of the first glucose residue).
These trisaccharides are sucrose derivatives :
This is the reserve polysaccharide of plants. The starch in food and non food industries is mainly produced from cereals (corn, wheat...).
Starch is actually a mixture of two polysaccharides: amylose and amylopectin. The relative proportions of amylose and amylopectin influences the physical properties of starch. Starch is insoluble in cold water, and form a paste (viscous dispersion) when the mixture is heated.
a. Amylose represents 15 to 30% of the weight of the starch. It is a linear polymer of glucose residues (roughly 500 to 20000 residues - MM = 105 to 106 Da) linked by a α-(1,4)-D-osidic bond.
The ramifications [α-(1,6)-D-osidic bond] represent only about 1%.
Amylose form a long chain form as a single left-handed helix :
Chain is stabilized by hydrogen bonds between the hydroxyl groups and water molecules.
This structure confers two properties to amylose :
b. The amylopectin : it represents 70 to 85% of the weight of the starch. It is a branched polymer :
Several results have identified the arrangement of amylopectin :
These results and the study of some models have shown that there is an average branch every 25 residues and branches contain twenty residues. There are more connections on the reducing end of the chain. Finally, some branches are themselves branched.
Visualization of the β-amylase of Glycine max at a resolution of 1.90 Å.
Cellulose is of vegetable origin only. This is a support substance, since it is the main constituent of the cell wall of young plants.
This is the most important biomolecule in term of biomass on the surface of the earth and it contains half the carbon available on earth.
It consists of long linear chains (roughly 300 to 1500 residues - MM = 5 104 to 2.5 106 Da) of glucose residues linked by a β-(1,4)-D-osidic bond.
The repeating unit is cellobiose (see above).
Cellulose is characterized by a high chemical inertness. It is insoluble in water. However it has a hydrophilic character (it is a hydrocolloid) : attaching many water molecules results in the swelling of cellulose.
Cellulose is not attacked by the digestive juices of omnivores : man is unable to digest cellulose because it is devoid of enzymes active on β-glucosidic bonds. Enzymes that degrade cellulose are called cellulases (ruminants, snails and some bacteria).
3. Industrial applications
Go to the website: " Polysaccharides food "- USTL
The food industry uses polysaccharide with thickening and stabilizing properties (e.g., starch, cellulose, hemicellulose, xanthan).
This is the reserve polysaccharide of animals. The main stock is located in the liver (200 g for an adult) and muscle (100 to 300 g).
The brain is a major user of glucose : 100 mg / min, but it has a limited supply of glycogen (10 to 20 g).
Glycogen is very similar to amylopectin : it is a chain of glucose residues linked by a α-(1,4)-D-osidic bond and connection between chains is a α-(1,6)-D-osidic bond. However, the chains are much shorter and the glycogen molecule is denser.
Glycogen is degraded by amylases like starch.
See a course on regulation of glycogenolysis.
5. Glucan degrading enzymes
a) Amylases that hydrolyse specifically α-1, 4 bonds :
b) Phosphorylases : they hydrolyse chains from the non-reducing ends, by phosphorolysis of α-1, 4 bonds, with the release of α-D-glucose-1-phosphate.
c) Enzymes catalyzing chain disconnection: they hydrolyse α-1, 6 bonds of branch points in different modes according to their origin.
These are heterogeneous compounds that result from the condensation of a high number of disaccharide subunits. This unit consists of :
Mucopolysaccharides are very acidic molecules. They are always associated with some proteins. However, in the final compound, carbohydrates represent roughly 95%.
Its main function, due to the high viscosity it provides to solutions, is to oppose the spread of foreign substances.
These compounds consist of a carbohydrate and a protein. The carbohydrate part varies from 1 to 50% of the weight of the assembly. The polysaccharide chains are often branched.
There are O-linked polysaccharides, such as galactose bound to the hydroxyl group of hydroxylysine in collagen. However, the amino acids involved are often serine or threonine.
N-linked polysaccharides are covalently joined to the nitrogen of the peptide bond of some asparagines.
Determining the structure of the glycoproteins is currently one of the most difficult jobs. Every carbohydrates has several free hydroxyl groups and any of them can establish a bond with another monosaccharide or another compound. Thus, the number of polysaccharides that can be formed is immense. For example, with only three monosaccharides, there are hundreds of configurations.
|VI. Liens Internet et références bibliographiques|
|"Principles of Biochemistry" Horton, Moran, Ochs, Rawn and Scrimgeour (1994) - Ed DeBoeck Universities - ISBN: 2-8041-1578-X|
"The Course of Organic Chemistry General C41" (Exercises and corrected)
Online University (RUCA realization): "Carbohydrates"
"Chapter 25: Carbohydrates" - University of Calgary
"Polysaccharides food" (very good educational site with quiz)