27-1 amino acids & proteins chapter 27. 27-2 27.1 a. amino acids amino acid: amino acid: a...

27-27-11

Amino AcidsAmino Acids

& Proteins& Proteins

Chapter 27Chapter 27

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27.1 27.1 A.A. Amino AcidsAmino Acids

Amino acid:Amino acid: a compound that contains both an amino group and a carboxyl group.• -Amino acid:-Amino acid: an amino acid in which the amino

group is on the carbon adjacent to the carboxyl group. There are 20 common -amino acids.

• although -amino acids are commonly written in the unionized form, they are more properly written in the zwitterionzwitterion (internal salt) form.

RCHCOH

NH2

O

RCHCO-

NH3+

O

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B.B. Chirality of Amino Acids Chirality of Amino Acids

With the exception of glycine, the 20 common amino acids have at least one stereocenter (the -carbon) and so are chiral.• All 20 have the L-configuration at their -carbon.

COO-

CH3

HH3N

L-Alanine

COO-

CH3

H NH3+

D-Alanine

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C.C. Table 27.1, Nonpolar side chains Table 27.1, Nonpolar side chains

NH3+

COO-

NH3+

COO-

NH H

COO-

NH

COO-

NH3+

Glycine (Gly, G)

Phenylalanine (Phe, F)

Proline (Pro, P)

Tryptophan (Trp, W)

NH3+

COO-

NH3+

COO-

NH3+

COO-

NH3+

COO-S

NH3+

COO-

Alanine (Ala, A)

Isoleucine (Ile, I)

Leucine (Leu, L)

Methionine (Met, M)

Valine (Val, V)

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Table 27.1, Polar side chainsTable 27.1, Polar side chains

NH3+

COO-H2N

O

NH3+

COO-H2N

O

NH3+

COO-HO

NH3+

COO-OH

Asparagine (Asn, N)

Glutamine (Gln, Q)

Serine (Ser, S)

Threonine (Thr, T)

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Table 27.1, Ionizable Side ChainsTable 27.1, Ionizable Side Chains

NH3+

COO--O

O

NH3+

COO--O

O

NH3+

COO-

HS

NH3+

COO-

HO

NH3+

COO-

NH

H2N

NH2+

NH3+

COO-

N

NH

NH3+

COO-H3NCysteine

(Cys, C)

Tyrosine (Tyr, Y)

Glutamic acid (Glu, E)

Aspartic acid (Asp, D)

Histidine (His, H)

Lysine (Lys, K)

Arginine (Arg, R)

+

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D.D. Other Amino Acids Other Amino Acids

NH3+

COO-

NH

H2N

O

NH3+

COO-

H3N

HO O CH2CHCOO-

NH3+

I I

I I

NH3+

-O

O

L-CitrullineL-Ornithine

L-Thyroxine, T4 4-Aminobutanoic acid

(-Aminobutyric acid, GABA)

+

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27.2 27.2 A.A. Acid-Base Properties, Table 27.2Acid-Base Properties, Table 27.2

pKa ofpKa of

valine 2.29 9.72tryptophan 2.38 9.39

9.102.09threonineserine 2.21 9.15

10.602.00prolinephenylalanine 2.58 9.24

9.212.28methionine9.742.33leucine

isoleucine 2.32 9.76glycine 2.35 9.78

9.132.17glutamine8.802.02asparagine9.872.35alanine

Nonpolar &polar side chains NH3

+ COOH

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Acid-Base Properties, Table 27.2Acid-Base Properties, Table 27.2

pKa ofpKa ofpKa of

10.079.112.20tyrosine

lysine 2.18 8.95 10.536.109.181.77histidine

glutamic acid 2.10 9.47 4.078.0010.252.05cysteine

aspartic acid 2.10 9.82 3.86

arginine 2.01 9.04 12.48

Side Chain

AcidicSide Chains NH3

+COOH

pKa ofpKa ofpKa ofSide Chain

BasicSide Chains NH3

+COOH

carboxylcarboxylsufhydrylphenolic

guanidinoimidazole1° amino

SideChainGroup

SideChainGroup

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Acidity: Acidity: -COOH Groups -COOH Groups

The average pKa of an -carboxyl group is 2.19, which makes them considerably stronger acids than acetic acid (pKa 4.76).

• the greater acidity is accounted for by the stability offered by the zwitterion formed on ionization

• and by the electron-withdrawing inductive effect of the -NH3

+ group.

NH3+

RCHCOOH H2O

NH3+

RCHCOO- H3O++ pKa = 2.19+

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Acidity: side chain -COOHAcidity: side chain -COOH

This electron-withdrawing inductive effect of the -NH3

+ group, decreases with increasing separation of the –COOH from the -NH3

+.

• Compare:

-COOH group of alanine (ppKKaa 2.35 2.35)

-COOH group of aspartic acid (ppKKaa 3.86 3.86)

-COOH group of glutamic acid (ppKKaa 4.07 4.07)

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Acidity: Acidity: -NH-NH33++ groups groups

The average value of pKa for an -NH3+ group is

9.47, compared with a value of 10.76 for a 1° alkylammonium ion.• Here there is competition between the electron-

withdrawing inductive effect of the –COOH and stability offered by the zwitterion.

NH3+

RCHCOO-

H2O

NH3+

CH3CHCH3 H2O

NH2

RCHCOO-

NH2

CH3CHCH3

H3O+

H3O+ pKa = 10.60++

+ pKa = 9.47+

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The Guanidine Group of ArgThe Guanidine Group of Arg

• basicity of the guanidine group is attributed to the large resonance stabilization of the protonated form relative to the neutral form.

CRNH

NH2+

NH2

C

NH2+

NH2

RNH

CRN

NH2

NH2

NH2

CRNH

NH2

H3O+

H2O

+

:

: :

:

::

::

+

pKa = 12.48

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Basicity- Imidazole GroupBasicity- Imidazole Group

• the imidazole group is a heterocyclic aromatic amine.

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B.B. Titration of Amino Acids Titration of Amino Acids

Figure 27.3 Titration of glycine with NaOH.

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C.C. Isoelectric Point Isoelectric Point

Isoelectric point (pI):Isoelectric point (pI): the pH at which an amino acid, polypeptide, or protein has no net charge.• the pH for glycine, for example, falls between the

pKa values for the carboxyl and amino groups.

• Average the two pKas that involve the zero net charge form.

pI = 12 (pKa COOH + pKa NH3

+)

= 21 (2.35 + 9.78) = 6.06

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Isoelectric Point, Table 27.2Isoelectric Point, Table 27.2

6.115.415.656.066.046.045.745.916.305.685.605.886.00

pKa ofpKa ofpKa of

pI

----------------

----------------------------

--------

valine 2.29 9.72tryptophan 2.38 9.39

9.102.09threonineserine 2.21 9.15

10.602.00prolinephenylalanine 2.58 9.24

9.212.28methionine9.742.33leucine

isoleucine 2.32 9.76glycine 2.35 9.78

9.132.17glutamine8.802.02asparagine9.872.35alanine

Side Chain

Nonpolar &polar side chains NH3

+ COOH

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Isoelectric Point, Table 27.2Isoelectric Point, Table 27.2

10.76

2.98

5.023.08

7.649.74

5.63

pKa ofpKa ofpKa ofpI

10.079.112.20tyrosine

lysine 2.18 8.95 10.536.109.181.77histidine

glutamic acid 2.10 9.47 4.078.0010.252.05cysteine

aspartic acid 2.10 9.82 3.86

arginine 2.01 9.04 12.48

Side Chain

AcidicSide Chains NH3

+ COOH

pKa ofpKa ofpKa of

pISide Chain

BasicSide Chains NH3

+ COOH

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D.D. Electrophoresis Electrophoresis

Electrophoresis:Electrophoresis: the process of separating compounds on the basis of their charge & mass.• electrophoresis of amino acids can be carried out

using paper, starch, polyacrylamide and agarose gels, and cellulose acetate as solid supports.

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ElectrophoresisElectrophoresis

• a sample of amino acids is applied as a spot on the paper strip or other solid support.

• an electric potential is applied to the electrode vessels and amino acids migrate toward the electrode with charge opposite their own.

• molecules with a high charge density move faster than those with low charge density.

• for molecules with the same charge, the heavier molecules move slower than lighter ones.

• molecules at their isoelectric point (no charge) do not move so remain at the origin.

• after separation is complete, the strip is dried and developed to visualize the separated amino acids.

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Detection of Amino AcidsDetection of Amino Acids

• a reagent commonly used to detect amino acid is ninhydrin, 19 of the 20 amino acids give the same purple colored product; proline produces a yellow-orange colored compound.

RCHCO-

O OHO

OOH

NH3+

O

O-

N

O

O

O

RCH CO2 H3O++

An -amino acid

Purple-colored anion

+ +

2+

Ninhydrin

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27.3 27.3 Polypeptides & ProteinsPolypeptides & Proteins

In 1902, Emil Fischer proposed that proteins are long chains of amino acids joined by amide bonds to which he gave the name peptide bonds.

Peptide bond:Peptide bond: the special name given to the amideamide bond between the - carboxyl group of one amino acid and the -amino group of another.

Note the analogy in carbohydrates where a glycosidic bond is an acetal.

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PeptidesPeptides

• peptide:peptide: the name given to a short polymer of amino acids joined by peptide bonds; they are classified by the number of amino acids in the chain.

• dipeptide:dipeptide: a molecule containing two amino acids joined by a peptide bond.

• tripeptidetripeptide: a molecule containing three amino acids joined by peptide bonds.

• polypeptidepolypeptide: a macromolecule containing many amino acids joined by peptide bonds.

• proteinprotein: a biological macromolecule of molecular weight 5000 g/mol of greater, consisting of one or more polypeptide chains.

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Serylalanine (Ser-Ala)Serylalanine (Ser-Ala)

H2N HO

O

HHOCH2

H2NO

OH

H CH3

H2NN

OH

HOCH2

H

H

CH3O

H O

Serine(Ser, S)

Alanine(Ala, A)

+

Serylalanine(Ser-Ala, (S-A)

peptide bond

Figure 27.5

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Writing PeptidesWriting Peptides

• by convention, peptides are written from the left, beginning with the free -NH3

+ group and ending with the free -COO- group on the right.

H3N

OH

NH O

HN

COO-

O-

OC6H5O

+

C-terminalamino acid

N-terminalamino acid

Ser-Phe-Asp

Serylphenylalanylaspartic acid

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Writing PeptidesWriting Peptides

• the tetrapeptide Cys-Arg-Met-Asn

• at pH 6.0, its net charge is +1.

HN

NH O

HN

O-

OO

O

NH2

SCH3

NH

NH2+H2N

OH3N

SH C-terminalamino acid

N-terminalamino acid

pKa 12.48

pKa 8.00

+~ pK 8.5

~ pK 3.5

~not ionizable

~not ionizable

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Levels of Protein StructureLevels of Protein Structure

Primary structure:Primary structure: the sequence of amino acids in a polypeptide chain.

Secondary structure: Secondary structure: conformations from rotation about bonds to the -carbon.

Tertiary structure: Tertiary structure: three dimensional folding of the chain.

Quaternary structure: Quaternary structure: assembly of tertiary structures (dimers, trimers etc.).

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27.4 27.4 A.A. Primary StructurePrimary Structure

Primary structure:Primary structure: the sequence of amino acids in a polypeptide chain; read from the N-terminal amino acid to the C-terminal amino acid.

Amino acid analysis:• hydrolysis of the polypeptide to its constituent

amino acids is commonly carried out using 6M HCl at 100oC.

• separation of the amino acids in the hydrolysate is by ion-exchange chromatography.

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Ion Exchange ChromatographyIon Exchange Chromatography

Figure 27.6

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Cleavage of proteinsCleavage of proteins

• Cyanogen bromide, BrCN, is used to cleave peptide bonds at the carboxyl group of methionine.

PN-C-NH CH C NH-PC

O O

CH2

CH2-S-CH3

cyanogen bromide isspecific for the cleavageof this peptide bond

from theN-terminalend

from theC-terminal end

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Enzymatic CleavageEnzymatic Cleavage

A group of protein-cleaving enzymes can be used to catalyze the hydrolysis of specific peptide bonds.

Phenylalanine, tyrosine, tryptophanChymotrypsin

Arginine, lysineTrypsin

Catalyzes Hydrolysis of Peptide Bond Formed by Carboxyl Group ofEnzyme

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Edman Degradation (Sequential)Edman Degradation (Sequential)

Edman degradation:Edman degradation: a reaction used for sequencing that cleaves the N-terminal amino acid of a polypeptide chain.

S=C=N-Ph

H2N COO-HN

NO

R

SPh

H3NNH

R

O

COO- +

+

N-terminalamino acid

A phenylthiohydantoin

Phenyl isothiocyanate

+

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Primary StructurePrimary Structure

Example 27.6 Example 27.6 Deduce the 1° structure of this pentapeptide.

pentapeptideEdman Degradation

Hydrolysis - Chymotrypsin

Fragment AFragment B

Hydrolysis - TrypsinFragment CFragment D

Arg, Glu, His, Phe, SerGlu

Glu, His, PheArg, Ser

Arg, Glu, His, Phe

Ser

Experimental ProcedureAmino Acid Composition

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27.5 27.5 A.A. Polypeptide SynthesisPolypeptide Synthesis

The problem is to join the -carboxyl group of aa-1 by an amide bond to the -amino group of aa-2, and not vice versa.

aa1

H3NCHCO-

O

aa2

H3NCHCO-

O

aa1 aa2

H3NCHCNHCHCO-

OOH2O

? +++

++

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B.B. Polypeptide Synthesis Polypeptide Synthesis

• protect the -amino group of aa-1.

• activate the -carboxyl group of aa-1.

• protect the -carboxyl group of aa-2.

+

+

form peptide bond

protectinggroup

activatinggroup

protectinggroup

O O

aa2aa1

Z-NHCHC-Y H2NCHC-X

Z-NHCHCNHCHC-X H-Y

O Oaa1 aa2

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C.C. Amino-Protecting Groups Amino-Protecting Groups

• the most common strategy for protecting amino groups and reducing their nucleophilicity is to convert them to amides.

O

(CH3)3COCOCOC(CH3)3

O O

(CH3)3COC-

O

PhCH2OC-

O

PhCH2OCCl

Di-tert-butyl dicarbonate

Benzyloxycarbonylchloride

tert-Butoxycarbonyl (BOC-) group

Benzyloxycarbonyl(Z-) group

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E.E. Peptide Bond Formation Peptide Bond Formation

The reagent most commonly used to bring about peptide bond formation is DCC.• DCC is the anhydride of a disubstituted urea and,

when treated with water, is converted to DCU.

1,3-Dicyclohexylcarbodiimide (DCC)

+

N,N' -dicyclohexylurea (DCU)

C NN

H

N NC

H

O

H2O

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Peptide Bond FormationPeptide Bond Formation

• DCC acts as dehydrating in bringing about formation of a peptide bond.

Carboxyl-protectedaa2

Amino-protectedaa1

++CHCl3Z-NHCHC-OH H2 NCHCOCH3

Amino and carboxyl protected dipeptide

+Z-NHCHC-NHCHCOCH3

DCC

DCU

R1 R2

R1 R2

O O

O O

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F.F. Solid-Phase Synthesis Solid-Phase Synthesis

Bruce Merrifield, 1984 Nobel Prize for Chemistry.• solid support: a type of polystyrene in which

about 5% of the phenyl groups carry a -CH2Cl group.

• the amino-protected C-terminal amino acid is bonded as a benzyl ester to the support beads.

• the polypeptide chain is then extended one amino acid at a time from the N-terminal end.

• when synthesis is completed, the polypeptide is released from the support beads by cleavage of the benzyl ester.

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Peptide Bond GeometryPeptide Bond Geometry

• two conformations are possible for a planar peptide bond.

• virtually all peptide bonds in naturally occurring proteins studied to date have the s-trans conformation.

C

C

O

C N

H

• • ••

••

CC

O

C N

H• • ••

••

s-trans s-cis

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Peptide Bond GeometryPeptide Bond Geometry

• to account for this geometry, Linus Pauling proposed that a peptide bond is most accurately represented as a hybrid of two contributing structures.

• the hybrid has considerable C-N double bond character (about 40%) and rotation about the peptide bond is restricted.

C

C

N

H

C

C

COO

C N

H

+

-: :

:

: : :

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Secondary StructureSecondary Structure

On the basis of model building, Pauling and Corey proposed that two types of secondary structure should be particularly stable.• -helix

• antiparallel -pleated sheet -Helix:-Helix: a type of secondary structure in which a

section of polypeptide chain coils into a spiral, most commonly a right-handed spiral.

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Secondary StructureSecondary Structure

• Figure 27.13 hydrogen bonding between amide groups, C=O • • • • H-N.

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The The -Helix-Helix

A segment of an -helix.

This is a 3.613 helix.

Note: text figure 27.14 is a 310 helix not an - helix.

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The The -Helix-Helix

In a section of -helix:• there are 3.6 amino acids per turn of the helix• each peptide bond is s-trans and planar.• N-H groups of all peptide bonds point in the same

direction, which is roughly parallel to the axis of the helix.

• C=O groups of all peptide bonds point in the opposite direction, and also parallel to the axis of the helix.

• the C=O group of each peptide bond is hydrogen bonded to the N-H group of the peptide bond four amino acid units away from it

• all R- groups point outward from the helix.

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-Pleated Sheet-Pleated Sheet

• Figure 27.15-pleated sheet with three polypeptide chains running in opposite directions.

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C.C. Tertiary Structure Tertiary Structure

Tertiary structure:Tertiary structure: the three-dimensional arrangement in space of all atoms in a single polypeptide chain.• disulfide bonds between the side chains of

cysteine play an important role in maintaining 3° structure.

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Tertiary StructureTertiary Structure

• Figure 27.18 A ribbon model of myoglobin.

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D.D. Quaternary Structure Quaternary Structure

Quaternary structure:Quaternary structure: the arrangement of polypeptide chains into a noncovalently bonded aggregation.• the major factor stabilizing quaternary structure is

the hydrophobic effect. Hydrophobic effect:Hydrophobic effect: the tendency of nonpolar

groups to cluster together in such a way as to be shielded from contact with an aqueous environment.

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Quaternary StructureQuaternary Structure

• Figure 27.19 The quaternary structure of hemoglobin.

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Amino Acids Amino Acids

& Proteins& ProteinsEnd of Chapter 27End of Chapter 27

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NucleicNucleicAcidsAcids

Chapter 28Chapter 28

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Nucleic AcidsNucleic Acids

Nucleic acid:Nucleic acid: a biopolymer containing three types of monomer units.• 1. heterocyclic aromatic amine bases derived

from purine and pyrimidine.

• 2. the monosaccharides D-ribose or 2-deoxy-D-ribose.

• 3. phosphoric acid.

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Comparison of BiomoleculesComparison of Biomolecules

Polysaccharides: Polysaccharides: --or or -glycosidic bonds -glycosidic bonds

• connecting -MS-MS-MS-MS-, connecting -MS-MS-MS-MS-,

• written non-reducing end ---> reducing endwritten non-reducing end ---> reducing end Membranes:Membranes: non-covalent bonds non-covalent bonds

• between phospholipids, between phospholipids, Proteins:Proteins: peptide bonds peptide bonds

• connecting -AA-AA-AA-AA-, connecting -AA-AA-AA-AA-,

• written N-term ---> COO termwritten N-term ---> COO term Nucleic acids:Nucleic acids: phosphodiester bondsphosphodiester bonds

• connecting –S-P-S-P-S-P-connecting –S-P-S-P-S-P-

• written 5’ end ---> 3’ endwritten 5’ end ---> 3’ end

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28.1 28.1 Purine/Pyrimidine Bases, Fig. 28.1Purine/Pyrimidine Bases, Fig. 28.1

HN

N

O

O

H

N

N

NH2

O

H

HN

N

O

O

H

CH3N

N

HN

N N

N

O

HH2N

N

N N

N

NH2

H

N

N N

N

H

Uracil (U) Thymine (T) Cytosine (C)Pyrimidine

1

2

34

5

6

Guanine (G)Adenine (A)Purine

1

2

34

56 7

8

9

Following are names and one-letter abbreviations for the five heterocyclic aromatic amine bases most common to nucleic acids.

27-27-5656

Nucleosides, Fig. 28.2Nucleosides, Fig. 28.2

Nucleoside:Nucleoside: a building block of nucleic acids, consisting of D-ribose or 2-deoxy-D-ribose bonded to a heterocyclic aromatic amine base by a -glycosidic bond (base + sugar).

HH

HOHOCH2

HO OH

O

O

HN

N

H anomericcarbon

a -N-glycosidicbond

Uridine

-D-riboside

uracil

1'

2'3'

4'

5'

1

Sugar numbers are primed to distinguish from base numbers.

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Nucleotides, Fig. 28.3Nucleotides, Fig. 28.3

Nucleotide:Nucleotide: a nucleoside in which a molecule of phosphoric acid is esterified with an -OH of the monosaccharide, most commonly either the 3’ or the 5’ OH (base + sugar + phosphate).

N

NN

N

NH2

O

OHOH

HH

H

CH2

H

OP

O-

O-O

5'

Adenosine 5'-monophosphate(AMP)

27-27-5858

NucleotidesNucleotides

Example 28.1Example 28.1 identify these nucleotides.

NO

N

O

HH

H

H

HOH

-O-P-O-P-O-CH2

NH2

(b)(a)-O -O

OO

O

HH

H

H

OH

HOCH2

HN

N N

NO

H2N

PO-

-O O

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28.2 28.2 A.A. DNA - 1° StructureDNA - 1° Structure

Deoxyribonucleic acids (DNA):• a backbone of alternating units of 2-deoxy-D-

ribose and phosphate in which the 3’-OH of one 2-deoxy-D-ribose is joined by a phosphodiester bond to the 5’-OH of another 2-deoxy-D-ribose.

Primary Structure:Primary Structure: the sequence of bases along the pentose-phosphodiester backbone of a DNA molecule (or an RNA molecule).• read from the 5’ end to the 3’ end.

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B.B. DNA - 2° Structure DNA - 2° Structure

Secondary structure:Secondary structure: the ordered arrangement of nucleic acid strands.

The double helix model of DNA 2° structure was proposed by James Watson and Francis Crick in 1953.

Double helix:Double helix: a type of 2° structure of DNA molecules in which two antiparallel polynucleotide strands are coiled in a right-handed manner about the same axis.

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DNA - 2° Structure, Table 28.1DNA - 2° Structure, Table 28.1

• Base composition in mole-percent of DNA for several organisms.

A C TOrganism G A/T G/CPurines/Pyrimidines

HumanSheepYeast

E. coli

30.4 19.9 19.9 30.1 1.01 1.00 1.0129.3 21.4 21.0 28.3 1.04 1.02 1.0331.7 18.3 17.4 32.6 0.97 1.05 1.0026.0 24.9 25.2 23.9 1.09 0.99 1.04

Purines Pyrimidines

27-27-6262

Base Pairing, Fig. 28.7Base Pairing, Fig. 28.7

• Base pairing between adenine and thymine and between guanine and cytosine.

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Double Helix, Fig. 28.9Double Helix, Fig. 28.9

• An idealized model of B-DNA.

27-27-6464

Forms of DNAForms of DNA

B-DNA: • the predominant form in dilute aqueous solution

• a right-handed helix.

• 2000 pm thick with 3400 pm per ten base pairs.

• minor groove of 1200pm and major groove of 2200 pm.

A-DNA:• a right-handed helix, but thicker than B-DNA.

• 2900 pm per 10 base pairs. Z-DNA:• a left-handed double helix.

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C.C. DNA - 3° Structure DNA - 3° Structure

Tertiary structure:Tertiary structure: the three-dimensional arrangement of all atoms of nucleic acid, commonly referred as supercoiling.

Circular DNA:Circular DNA: a type of double-stranded DNA in which the 5’ and 3’ ends of each stand are joined by phosphodiester bonds (Fig 28.10).

Histone:Histone: a protein, particularly rich in the basic amino acids lysine and arginine, that is found associated with DNA molecules.

27-27-6666

DNA - 3° Structure, Fig. 28.10DNA - 3° Structure, Fig. 28.10

Relaxed and supercoiled DNA.

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A.A. rRNA, Table 28.2 rRNA, Table 28.2

RNA molecules are classified according to their structure and function.

Ribosomal RNA (rRNA):Ribosomal RNA (rRNA): a ribonucleic acid found in ribosomes, the site of protein synthesis.

Molecular WeightRange (g/mol)

Number ofNucleotides

Percentageof Cell RNA

mRNA 25,000 - 1,000,000 75 - 3,000 2tRNA 23,000 - 30,000 73 - 94 16rRNA 35,000 - 1,100,000 120 - 2904 82

Type

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B.B. tRNA tRNA

Transfer RNA (tRNA):Transfer RNA (tRNA): a ribonucleic acid that carries a specific amino acid to the site of protein synthesis on ribosomes.

OBase

OHO

CH

RNH3

+

tRNA-O-P-O-CH2

amino acid, boundas an ester to itsspecific tRNA

HH H

H

C=O

O

O-

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C.C. mRNA mRNA

Messenger RNA (mRNA):Messenger RNA (mRNA): a ribonucleic acid that carries coded genetic information from DNA to the ribosomes for the synthesis of proteins.• present in cells in relatively small amounts and

very short-lived.

• single stranded.

• their synthesis is directed by information encoded on DNA.

• a complementary strand of mRNA is synthesized along one strand of an unwound DNA, starting from the 3’ end.

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Sequencing DNASequencing DNA

Polyacrylamide gel electrophoresis:Polyacrylamide gel electrophoresis: a technique so sensitive that it is possible to separate nucleic acid fragments differing from one another in only a single nucleotide.• Maxam-Gilbert method:Maxam-Gilbert method: a method developed by

Allan Maxam and Walter Gilbert; depends on base-specific chemical cleavage.

• Dideoxy chain termination method:Dideoxy chain termination method: developed by Frederick Sanger, depends on synthesis.

• Gilbert and Sanger shared the 1980 Nobel Prize for biochemistry for their “development of chemical and biochemical analysis of DNA structure.”

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D.D. Dideoxy Chain Termination Dideoxy Chain Termination

• the key to the chain termination method is addition to the synthesizing medium of a 2’,3’-dideoxynucleotide triphosphate (ddNTP).

• because a ddNTP has no 3’-OH, chain synthesis is terminated where a ddNTP becomes incorporated.

-O-P-O-P-O-P-O-CH2

O-

O

O- O-

O

H

Base

H H

H HO

H

A 2',3'-dideoxynucleoside triphosphate (ddNTP)

O

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Dideoxy Chain TerminationDideoxy Chain Termination

In this method the following are mixed together:

1. ssDNA fragment to be sequenced

2. small primer piece to initiate synthesis (5’-P32)

3. all 4 NTPs

Divide this mixture into four samples.

4. to each sample add one of the four ddNTPs

5. add DNA polymerase to eachPolymerization begins in each sample and continues

until incorporation of a ddNTP.

This procedure allows for random incorporation of ddNTP where bases pair.

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Dideoxy Chain TerminationDideoxy Chain Termination

Then subject the samples to side-by-side gel electrophoresis.

• since all strands are negative they move toward the positive electrode.

• afterwards a piece of film is placed over the gel.

• gamma rays released by P-32 darken the film and create a pattern of the resolved oligonucleotide.

• the base sequence of the complement to the original strand is read directly from bottom to top of the developed film.

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Dideoxy Chain Termination, Fig. 28.12Dideoxy Chain Termination, Fig. 28.12

• The primer-DNA template is divided into four separate reaction mixtures.

• To each is added one of the four ddNTPs and

polymerase.

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Dideoxy Chain Termination, Fig. 28.12 cont.Dideoxy Chain Termination, Fig. 28.12 cont.

• The mixture is separated by polyacrylamide gel electrophoresis and the gel read.

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Nucleic AcidsNucleic Acids

End Chapter 28End Chapter 28

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Organic Organic PolymerPolymerChemistryChemistryChapter 29Chapter 29

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29.1 29.1 Organic Polymer ChemistryOrganic Polymer Chemistry

Polymer:Polymer: from the Greek, polypoly ++ merosmeros, many parts.• any long-chain molecule synthesized by bonding

together single parts called monomers. MonomerMonomer: from the Greek, monomono ++ merosmeros,

single part.• the simplest nonredundant unit from which a

polymer is synthesized. Plastic:Plastic: a polymer that can be molded when hot

and retains its shape when cooled.

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Organic Polymer ChemistryOrganic Polymer Chemistry

Thermoplastic:Thermoplastic: a polymer that can be melted and molded into a shape that is retained when it is cooled.

Thermoset plastic:Thermoset plastic: a polymer that can be molded when it is first prepared but, once it is cooled, hardens irreversibly and cannot be remelted.

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29.5 29.5 Step-Growth Polymers Step-Growth Polymers

Step-growth polymerization (Condensation):Step-growth polymerization (Condensation): a polymerization in which chain growth occurs in a stepwise manner between difunctional monomers.

we discuss five types of step-growth polymers:• polyamides

• polyesters

• polycarbonates

• polyurethanes

• epoxy resins

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A.A. Polyamides Polyamides

Nylon 66 (from two six-carbon monomers).

• during fabrication, nylon fibers are cold-drawncold-drawn about 4 times their original length, increasing crystallinity, tensile strength, and stiffness.

O

HOOH

OH2N

NH2

Hexanedioic acid(Adipic acid)

1,6-Hexanediamine(Hexamethylenediamine)

+

O HN

NO H

heat

n

Nylon 66

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PolyamidesPolyamides

Nylons are a family of polymers, the two most widely used of which are nylon 66 and nylon 6.

• nylon 6 is synthesized from a six-carbon monomer

• nylon 6 is fabricated into fibers, brush bristles, high-impact moldings, and tire cords.

Caprolactam

1. partial hydrolysis2. heat n

nNH

O

NOH

Nylon 6

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PolyamidesPolyamides

Kevlar is a polyaromatic amide (an aramid).

• cables of Kevlar are as strong as cables of steel, but only about 20% the weight.

• Kevlar fabric is used for bulletproof vests, jackets, and raincoats.

+

1,4-Benzenediamine(p-Phenylenediamine)

1,4-Benzenedicarboxylic acid

(Terephthalic acid)

nKevlar

+

O

NH

COHnHOC

O O

nH2N NH2

CNHC

O2nH2O

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B.B. Polyesters Polyesters

Poly(ethylene terephthalate), abbreviated PET or PETE, is fabricated into Dacron fibers, Mylar films, and plastic beverage containers.

heatHO

O

OH

O

HOOH

O O

OO

n

1,4-Benzenedicarboxylic acid(Terephthalic acid)

+

1,2-Ethanediol(Ethylene glycol)

+ 2nH2O

Poly(ethylene terephthalate)(Dacron, Mylar)

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C.C. Polycarbonates Polycarbonates

• to make Lexan, an aqueous solution of the sodium salt of bisphenol A is brought into contact with a solution of phosgene in CH2Cl2 in the presence of a phase-transfer catalyst.

Phosgene

+

Disodium saltof Bisphenol A

+Na-O

CH3

CH3

O-Na+

Lexan(a polycarbonate)

+

Cl Cl

O

nO

CH3

CH3

O

O

2NaCl

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PolyurethanesPolyurethanes

• the more flexible blocks are derived from low MW polyesters or polyethers with -OH groups at the ends of each polymer chain.

CH3N=C=OO=C=N nHO-polymer-OH

CNH NHCO-polymer-OCH3 OO

Low-molecular-weightpolyester or polyether

2,6-Toluenediisocyanate

+

n

A polyurethane

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29.6 29.6 Chain-Growth Polymers (Addition)Chain-Growth Polymers (Addition)

Chain-growth polymerization:Chain-growth polymerization: a polymerization that involves sequential addition reactions, either to unsaturated monomers or to monomers possessing other reactive functional groups.

Reactive intermediates in chain-growth polymerizations include radicals, carbanions, carbocations, and organometallic complexes.

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Chain-Growth PolymersChain-Growth Polymers

We concentrate on chain-growth polymerizations of ethylene and substituted ethylenes.

• on the following two screens are several important polymers derived from ethylene and substituted ethylenes, along with their most important uses.

R

An alkene

R

n

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PolyethylenesPolyethylenes

CH2=CH2

CH2=CHCH3

CH2=CHCl

CH2=CCl2

MonomerFormula

Common Name

Polymer Name(s) andCommon Uses

Ethylene

Propylene

Vinyl chloride

1,1-Dichloro-ethylene

Polyethylene, Polythene;break-resistant containersand packaging materials

Polypropylene, Herculon;textile and carpet fibers

Poly(vinyl chloride), PVC;construction tubing

Poly(1,1-dichloroethylene), Saran; food packaging

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PolyethylenesPolyethylenes

CH2=CHCN

CF2=CF2

CH2=CHC6H5

CH2=CHCOOEt

CH3

CH2=CCOOCH3

Acrylonitrile

Tetrafluoro-ethylene

Styrene

Ethyl acrylate

Methylmethacrylate

Polyacrylonitrile, Orlon;acrylics and acrylates

Poly(tetrafluoroethylene), PTFE; nonstick coatings

Polystyrene, Styrofoam;insulating materials

Poly(ethyl acrylate); latex paintsPoly(methyl methacrylate), Plexiglas; glass substitutes

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C.C. Polymer Stereochemistry Polymer Stereochemistry

There are three alternatives for the relative configurations of stereocenters along the chain of a substituted ethylene polymer.

HR RH HR RH HR

Syndiotactic polymer(alternating configurations)

HR HR HR HR HR

Isotactic polymer (identical configurations)

HR HR HR HR RH

Atactic polymer(random configurations)

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Polymer StereochemistryPolymer Stereochemistry

In general, the more stereoregular the stereocenters are (the more highly isotactic or syndiotactic the polymer is), the more crystalline it is.• the chains of atactic polyethylene, for example, do

not pack well and the polymer is an amorphous glass.

• isotactic polyethylene, on the other hand, is a crystalline, fiber-forming polymer with a high melt transition.