740143P Biomolecules for Biochemists (8 op)

740147P Biomolecules for Bioscientists (8 op)

740148P Biomolecules (5 op)

CARBOHYDRATES

Docent Tuomo Glumoff

Faculty of Biochemistry and Molecular Medicine

CARBOHYDRATES lectures 1

- carbohydrates = saccharides ≈ sugars

- What is a sugar? How to define a sugar?

Typical properties of sugars?

• sweet taste

• crystallizes

• water-soluble

• polyhydroxy aldehydes or

polyhydroxy ketones

• contains a chiral carbon

• optically active (stereoisomers)

• for monosaccharides the molecular

formula is (CH2O)n, where 3 n 9

- most abundant class of biological

molecules on earth by mass

- all organisms can synthesize, but

most important is photosynthesis:

solar energy to chemical energy

- mono- and disaccharides are

usually metabolites

- oligosaccharides are usually linked

to proteins or lipids

- polysaccharides are usually storage

forms or have structural function

PROBLEM 1. Why a 2-carbon compound cannot be a sugar although it would fulfill the formula

(CH2O)n?

Answers to problems will be given at the lectures and they will also be available after the lectures in NOPPA.

O

OH

OH

O

O

O

H

HH

H

O

HH O

H

H

O

H

H

O

H

H

O

H

H

O

H

HO

H

H

O

H

H

O

H

Figures under © taken from the following sources: Devlin: Textbook of Biochemistry with Clinical Correlations

Horton et al., Principles of Biochemistry Nelson & Cox: Lehninger Principles of Biochemistry

Mathews et al., Biochemistry Voet & Voet: Biochemistry Berg et al., Biochemistry

Campbell et al., Biochemistry Illustrated Varki et al. (ed.) Essentials of Glycobiology

Cooper & Hausman: The Cell – A Molecular Approach Rawn: Biochemistry Brown: Biochemistry

Appling et al., Biochemistry – concepts and connections

Recommended text books for further reading: any standard biochemistry text book, and certainly one of the following:

Mathews et al., Biochemistry, 4th edition (2012), (Wiley,) chapter 9

Appling et al., Biochemistry – concepts and connections (Pearson Education Limited, 2016), chapter 9

1. Basics of carbohydrates

*) (CH2O)n is an ”oversimplification”, since many saccharides are modified and some contain

also atoms such as S and N. Nevertheless, all compounds herewith either have this formula

or can be derived from substances that do. (Mathews et al. Biochemistry 4th edition)

Monosaccharides

- trioses, 3 carbons

- ketoses and aldoses are tautomers

PROBLEM 2. Please check that the formula (CH2O)n hold for both triose tautomers!

(an aldotriose) (a ketotriose)

2. Monosaccharides 2

- D- and L-enantiomers = isomers differing at the chiral carbon configuration

• mirror images

- in nature D-monosaccharides dominate

- D-L vs. R-S naming system:

• R- and S- would be absolute, but becomes difficult with multiple chiral carbons

• +/- and d/l (to right vs. to left) could be used with respect to ability to rotate plane-

polarized light

3

PROBLEM 3. You will be given molecular models of an R-isomer and an S-isomer. Which is which?

red = blue =

4- tetroses, 4 carbons

- may have 2 chiral carbons, and thus are diastereomers

- D- or L- is assigned based on the chiral carbon farthest from the carbonyl group (= highest

numbered chiral C) and additional names are given for diastereomers (an arbitrary naming system)

- epimers = multiple D/L possibilities, but a difference in only one of them

- pentoses, 5 carbons

• for example riboses in nucleotides

- hexoses, 6 carbons

• typical energy molecules, like glucose

PROBLEM 4. Observe the aldose and ketose structures on page 5 and answer the following questions:

4.1 Why are there a fewer number of different ketoses?

4.2 Identify each of the following:

a) two aldoses whose configuration at carbons 3, 4 and 5 matches that of D-fructose

b) the enantiomer of D-galactose

c) an epimer of D-galactose that is also an epimer of D-mannose

d) a ketose that has no chiral centers

e) a ketose that has one chiral center

D-sugars have the same absolute

configuration at the asymmetric center

furthest from their carbonyl group as does

D-glyceraldehyde

Examples of monosaccharides with their occurrence and physiological role:

5Fischer projections of C3 to C6 aldoses

Fischer projections of C3 to C6 ketoses

carbon

numbering:

1

2

3

4

6- the open chain form of a monosaccharide is in equilibrium with the ring form

- under physiological conditions ≥ 5-carbon monosaccharides are ≥ 99% in the ring form

- interconversion

between a and b forms

= mutarotation

- catalyzed by specific

mutarotases

- Haworth projection: thick edge

towards the viewer, thin edge

towards the rear

a = on the opposite side b = on the same side

Pyranose formation:

The open-chain form of glucose

cyclizes when the C-5 hydroxyl

group attacks the oxygen atom

of the C-1 aldehyde group to

form an intramolecular

hemiacetal. Two anomers,

designated a and b, can result.

trans cis

7

- hemiacetal or hemiketal bond is formed

- pentoses and hexoses form pyran and

furan rings => pyranoses and

furanoses

- anomers = upon cyclization the former

carbonyl carbon becomes asymmetric =>

anomeric carbon

1

2

34

5

6

8- note that pentoses and hexoses may both form 5- and 6-rings (size of ring not dependent on

number of carbons!)

- distribution between furanose and pyranose depends on the sugar, pH, solvent and temperature

*

* glucose: open chain form 0.2 %

9

chair and boat conformations:

- same stereochemistry, difference in three-dimensional shape

- conformations with bulky substrates in equatorial positions are favored, while steric hindrance of

axial substituents renders the boat conformation less favored

PROBLEM 5. On page 10 you will find the

Haworth projection of b-D-N-

acetylglucosamine. Draw it using the

Fischer projection (open-chain form).

PROBLEM 6. Take mannose from the tables

on page 5 and draw it in a ring form.

e = equatorial

a = axial

- conformations: interchangeable by simple deformation of the molecule

- configurations: interchangeable only by breaking and reformation of covalent bonds

10- monosaccharides may contain substituents or modifications that make them derivatives

- examples:

- aminosugars:

- reduction of the carbonyl

group => alditol

(xylitol is accordingly made

from xylose)

- sugar phosphates are very

important derivatives

- activated molecules in energy

metabolism

glyceraldehyde-3-phosphate

glucose-1-phosphate

glucose-6-phosphate

fructose-6-phosphate

11- glycosides:

a glycosidic bond is formed

- glycosidic bond is the bond

between two sugar units in

di- and polysaccharides,

but as seen here the

glycosidic bond can also

form between a sugar and

another group

- glycosidic bond is also

present in nucleotides in

DNA and RNA

- common abbreviations used for monosaccharides

12

- two monosaccharides are joined with a glycosidic bond

=> hydroxyl group of one sugar reacts with the hydroxyl group of the anomeric carbon of another sugar

NOTE!!

- equilibrium to

left

- polysaccharides

are metastable

compounds

- hemiacetal becomes acetal

- in common disaccharides O-glycosidic bond; in glycoproteins and nucleotides N-glycosidic bond

- glycosidic bond is readily hydrolyzed in acid, but resists bases

3. Disaccharides 13

- oxidation of the anomeric carbon means that the sugar is a reducing sugar (remember:

oxidation and reduction are always coupled – the compound that gets oxidized will leave

another compound reduced)

- a reducing sugar has a reducing end and a nonreducing end

- reducing end is the one with a free anomeric carbon (and thus also a free aldehyde group)

- only the open-chain form can undergo oxidation, not the hemiacetal (ring) form

- in a disaccharide only the rightmost monomer can adopt the open chain form

PROBLEM 7. One of the disaccharides on the following page is not a reducing sugar? Which

one and why?

14

- mutarotation may also take place in disaccharides, namely the reducing end monosaccharide

- a,a form is simply “a”, while “a,b” is simply b

- if the disaccharide ends in a nonreducing end, it cannot mutarotate (such as sucrose)

Practical example of using Copper reduction by a reducing sugar in clinical

biochemistry:

- amount of glucose in blood or urine (Fehling´s test)

- e.g. insulin dosage control

- but modern tests are based on specific enzymes acting on glucose!

15

Nomenclature of disaccharides (systematic names):

Reducing disaccharides (contain a free

hemiacetal):

"glycosylglycose"

example:

α-D-Glucopyranosyl-(1 4)-β-D-glucopyranose

(trivial name β-maltose)

Non-reducing disaccharides (without a free

hemiacetal):

"glycosyl glycoside"

example:

α-D-Glucopyranosyl- (1 1)- α-D-glucopyranoside

(trivial name α,α-trehalose)

Distinguishing features of different disaccharides:

1. The two monomers may be of the same kind, or

they may be different

2. The most common linkages between two

monosaccharides are:

1 1 1 2 1 4 1 6

(at least one anomeric hydroxyl is always involved

in the bond)

3. The order of the two monomers (if different)

determines if the disaccharide can be a reducing

sugar or not.

4. The anomeric configuration of the hydroxyl group

on carbon 1 of each monomer determine which

enzyme can hydrolyze the glycosidic bond

(maltose and cellobiose are both made up from

two glucoses, but cannot serve as a substrate for

the same hydrolytic enzyme) - Why?

Disaccharides in 3D for example from here:

www.biotopics.co.uk/JmolApplet/maltosejdisplay.htm

Compare maltose with cellobiose!

16Names and writing of disaccharides:

- sequence starts from the left

(nonreducing end)

- anomeric and enantiomeric forms can be

designated

- the ring configuration can be given

- the atoms between which the glycosidic

bond is formed are given

(the arrow shows the direction from the

anomeric carbon)

maltose: a-D-Glcp(14)-b-D-Glcp

or Glc(a1b4)Glc

sucrose: a-D-Glcp(1↔2)-b-D-Fruf

"invert sugar" = hydrolysis of sucrose to glucose

and fructose

= glycans

- storage polysaccharides like, starch and glycogen, as well as stability, biosynthesis and degradation

of polysaccharides will be postponed to Aineenvaihdunta I (Metabolism I) course later in the spring

- here we will study other types of oligo- and polysaccharides and their functions

- homopolysaccharides – repeating one type of units

- heteropolysaccharides – repeating usually two kinds of units

- complex polysaccharides – contain more than two kinds of units

4. Polysaccharides

PROBLEM 8. Write below a systematic name for lactose.

17- structural elements in plant cell walls and animal exoskeletons

- extracellular support – bacterial cell envelope and in animals a matrix that holds cells together and

supports tissues and organs

- no defined molecular weight = saccharide chains may grow to become “shorter or longer”

(But NOTE: saccharide chains with specific function e.g. in recognition must be of certain length and

composition; see later blood group antigens)

18Cellulose:

- linear polymer of b-D-glucose,

10.000 to 15.000 units

- most abundant polysaccharide

molecule on earth

Chitin:

- linear polymer of b-N-acetylglucosamine

- found in surface armor of insects

Glycosaminoglycans:

- polymers of repeating disaccharide units

- one of the sugars is either N-acetylgalactosamine or N-acetylglucosamine or a derivative thereof

- are acids through either a sulfate or carboxylate group present

- examples of modified sugar residues that gain novel properties and functions

- heparin is a natural anticoagulant in body fluids

(here only the repeating unit shown!)

- inhibits blood clotting enzymes through binding

to anti-prothrombin III protein

But NOTE: saccharide chains with specific function e.g. in recognition must be of

certain length and composition; e.g. blood group antigens

specific

difference

PROBLEM 9. Can you think of a practical

application for heparin in clinical

biochemistry?

GlcUA

19

GlcUA

20

PROBLEM 10. Chain length of a polysaccharide (number of units in a chain) is not known in

the beginning of its synthesis, i.e. it is not predefined. See p. 19: the numbers are not precise

– sometimes the glycan chain is “shorter”, sometimes “longer”. Please explain!

- hyaluronic acid is noted for its much longer chain than most other glycosaminoglycans

21

Cooper & Hausman: The Cell

crosslinked

tetrapeptides

Voet et al.: Principles of Biochemistry

- peptidoglycan is an important part of bacterial cell walls

22

Left: Gram negative bacilli

Right: Gram positive cocci

Glycoconjugates: proteoglycans, glycoproteins and glycolipids

- proteoglycan is a protein-carbohydrate complex found in extracellular space

23

- for comparison, the cell wall of mycobacteria is even more complicated in structure (figure below)

- on top of the peptidoglycan layer there are arabinogalactan (carbohydrate) and mycolic acid

(lipid) layers

- this makes the cell wall more resistant and contributes to the difficulty of eliminating tuberculosis

causing mycobacteria

24

galectin = galactoside-binding lectin

lectin = sugar-binding protein

- an example, how membrane proteins and carbohydrate structures attached to them form part of

the cell´s contact network

- cell to cell

- cell to extracellular matrix

- protein-protein interactions

- protein-carbohydrate interactions

- cell surface structures mediated via carbohydrates can be part of intracellular signaling

25

26- glycoproteins are proteins that contain certain types of carbohydrate chains attached to them

- O-linked glycoproteins and N-linked glycoproteins

- N-linked glycans are attached to asparagine residues in a sequence –Asn – X – Ser/Thr –

- O-linked glycans are attached to threonines or serines

- glycan chains can be of various composition and type of branching

- glycans are attached to protein cotranslationally in the ER (endoplasmic reticulum)

- glycans are further modified in the Golgi apparatus

- glycan chains give the cellular machinery extra possibilities for recognizing proteins and cells

• immunoglobulin tissue distribution and interaction with phagocytic cells

• intracellular targeting and excretion

• cellular identification

• control of body fluid viscosity

• blood groups

- other functions of glycans include:

• stabilize protein fold (ready-made protein)

• stabilize protein folding (during the folding!) by binding to intermediate conformations

• stiffens and extends the polypeptide chain

27

- oligosaccharide chains attached to membrane proteins

at the surface of red blood cells

- to attach the terminal monosaccharide to get either

A or B blood group, one needs specific enzymes;

heterozygotes have both to get AB

- type O oligosaccharides are nonantigenic

- note that saccharide chains are actually surprisingly large in size compared to proteins

– a chain of a couple of sugar residues easily make a structure with some dimensions

comparable to a protein molecule

PROBLEM 11. E. coli is often used in the

lab to produce cloned eukaryotic proteins.

Sometimes the proteins cannot be obtained

in good amounts (there can be many

reasons). Can you identify or guess one

possible reason from the material we just

discussed?

PLEASE NOTE!!!!

(refer to page 18)

Whenever a glycan

chain has a specific

recognition function, the

number and the sugar

structure must be

precise!!!!!

- glycolipids and lipopolysaccharides are membrane components, where carbohydrates are

attached to lipids28

© W

.W. C

hristie

Th

e S

cott

ish C

rop R

esearc

h I

nstitu

te, D

undee

- lipopolysaccharides (LPS) can be found on the

surface of bacteria, like E. coli and Salmonella

- antibodies are raised in the body against LPS

to fight bacterial infections

- LPS of some bacteria are toxic

PROBLEM 12. Write down the distinctive differences of the cell wall structures between Gram-positive

and –negative bacteria.

http://www.chem.qmul.ac.uk/iupac/2carb/

INTERNATIONAL UNION OF PURE AND APPLIED CHEMISTRY (IUPAC)

and

INTERNATIONAL UNION OF BIOCHEMISTRY AND MOLECULAR BIOLOGY (IUBMB) Joint

Commission on Biochemical Nomenclature (JCBN)

Symbols for Specifying the Conformation of Polysaccharide Chains:

http://www.chem.qmul.ac.uk/iupac/misc/psac.html

Lipid layer

Good to be aware of for possible further need:

Nomenclature of Carbohydrates:

295. Briefly about importance of carbohydrate-acting enzymes

- glycan binding proteins are abundant due to large amount of different carbohydrate structures

- for the synthesis or degradation of various glycan

molecules a large amount of specific enzymes are

needed

- specific for a type of bond and sugar molecules

connected by the bond

- wrong type of carbohydrate structures are oftentimes

found in cancer cells

30

(taken here just as examples of some practically important and interesting applications, but not

for exam):

- the hydroxy group in a sugar ring may also be derivatized

by another sugar ring, like in melezitose, which is a sugar

found as a minor component in honey

- sugar rings may also form ring-like structures

= cyclodextrins

- a-, b- and g-cyclodextrins have 6, 7 or 8 glucose units,

respectively

- cyclodextrins find use in applications like protecting

aroma molecules in foodstuffs or transferring and

delivering (slowly) drugs in the body due to complex

formation with such smaller molecules

- streptomycin, an antibiotic, interferes with bacterial protein synthesis

6. Some extraordinary di- and oligosaccharides

31- bacterially produced dextrans

can be cross-linked to form very

hydrophilic preparations that

swell and form gels

- for example Sephadex, which is

used as a chromatographic

support media in gel filtration

(protein purifications)

- Sucralose is an artificial sweetener

Stevioside (steviol glycoside)

- natural sweetener extracted from

leaves of stevia plant

- 250-300 times sweeter than glucose

- both react with the taste receptors of the

tongue like sugars do, i.e. is sweet

- zero calories, because cannot be

metabolized in the body