amino acids df05

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Chem 437

Amino acids

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Chem 437

Bio-Polymer Chemistry• DNA

– Made up of four monomer units (adenine, guanine, thymine and cytosine)– In general, forms one structure (double helix).– Functional role: storing information

• RNA– Made up of four more abundant monomer units (adenine, guanine, uracil

and cytosine)– Many less abundant bases also exist– Simple to very complex structures– Functional role: template for protein synthesis, catalytic activity, protein

synthesis

• Proteins– Made up of 20 monomer units (α-amino-acids)– Vast array of different types of structure– Functional role: catalysts (organic synthesis), structural, regulation of all

intracellular and extracellular events, signaling, etc etc etc etc etc

• Polymers are an energetically efficient way to construct a wide range of macromolecules with the same machinery

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Chem 437

Basic structure of α-amino acids

Cα

R

CO2H3N+

HCα

R

CO2H3N+

H

Ball and stick model(Tetrahedral arrangement)

R R-group, varies with each amino acid

-CO2- Carboxyl group

-NH3+ Primary amino group

Zwitterion form

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Chem 437

Formation of the peptide bond

Peptide bond

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Chem 437

The α-amino acids

Cα

H

CO2H3N+

HGlycine, Gly, G

( not-chiral)

Cα

CH3

CO2H3N+

Alanine, Ala, A(non-polar/hydrophobic)

Cα

CH2

CO2H3N+

HSerine, Ser, S

(polar)

Cα

CH2

CO2H3N+

HCysteine, Cys, C

(polar)

Cα

C

CO2H3N+

HThreonine, Thr, T

(polar, two chiral centers)

Cα CO2H3N+

HValine, Val, V

(non-polar/hydrophobic)

H CH3

OH

CH CH3

CH3SH

OH

H

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Chem 437

The α-amino acids

Cα CO2H3N+

H

CH CH3

CH3

CH2

Leucine, Leu, L(non-polar/hydrophobic)

Cα CO2H3N+

H

CH2

CH3

C

Isoleucine, Ile, I(non-polar/hydrophobic

Two chiral centers)

H CH3

Cα CO2H3N+

H

CH2

S

CH2

Methionine, Met, M(non-polar/hydrophobic)

CH3

Proline, Pro, P(non-polar/hydrophobic,

α-imino acid)

Cα CO2H2N+

H

CH2CH2

CH2

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Chem 437

H

The α-amino acids

Cα CO2H3N+

H

CH2

Phenylalanine, Phe, F(non-polar/hydrophobic)

Cα CO2H3N+

H

CH2

Tryptophan, Trp, W(non-polar/hydrophobic)

Cα CO2H3N+

H

CH2

Tyrosine, Tyr, Y(polar)

OHN

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Chem 437

The α-amino acids

Lysine, Lys, K(polar, charged)

Cα CO2H3N+

H

CH2

CH2

CH2

NH3+

CH2

Arginine, Arg, R(polar, charged)

Cα CO2H3N+

H

CH2

CH2

CH2

CNH

NH2+NH2

Histidine, His, H(polar, charged)

Cα CO2H3N+

H

CH2

N

N

H

H

+

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Chem 437

CCH2

The α-amino acids

Asparagine, Asn, N(polar)

Cα CO2H3N+

H

CH2

CO NH2

Glutamine, Gln, Q(polar)

Cα CO2H3N+

H

CH2

O NH2

Cα CO2H3N+

HAspartic acid, Asp, D

(polar, charged)

CH2

CO2

Glutamic acid, Glu, E(polar, charged)

Cα CO2H3N+

H

CH2

CO2

CH2

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Chem 437

Labeling of the side chain atoms


Cα CO2H3N+

H

CH2

CH2

CH2

NH3+

CH2

βγδεζ

Cα

C

CO2H3N+

HThreonine, Thr, T

(polar, two chiral centers)

H CH3

OH

βγ

γ

• The carboxyl group and amino group attached to the Cα atom are referred to as the α−carboxy group and the α−amino group respectively

• The second amino group on lysine is referred to as the ε-amino group

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Chem 437

CH2

Representations of α-amino acids

Cα CO2H3N+

H

Phenylalanine, Phe, F

ball and stick

Space filling

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Chem 437

Uncommon amino acids found in proteins

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Chem 437

Biologically important derivatives of amino acids

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Chem 437

Acid-base properties

Net Charge +1 Net Charge 0 Net Charge -1

pH = 1 pH = 7 pH = 13

pKa 1.8-2.6pKa 8.8-10.4

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Chem 437

Acid-base properties

Cα

H

CO2H3N+

H

Cα

H

CO2HH3N+

H

Cα

H

CO2H2N

H

Glycine(A, V, I, L, P, F, M, N, Q, W)

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Chem 437

Acid-base properties : acidic amino acids

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Chem 437

Acid-base properties : basic amino acids

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Chem 437

Table of pKa values for amino acids

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Chem 437

Questions on histidine

• Draw all the ionization states of histidine in the order that they would be deprotonated

• Draw the titration curve of histidine as it is titrated with hydroxide ions (ensuring that the axis are correctly labeled and the pKa values are shown).

• Indicate on the curve the region where each of the above ionization states is the major species.

• Calculate the isoelectric point of histidine

• What percentage of histidine is in the protonated form at pH 7.0

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Chem 437

Answer slide

Cα CO2HH3N+

H

CH2

N

N

H

H

+

Cα CO2-H3N+

H

CH2

N

N

H

H

+

Cα CO2-H3N+

H

CH2

N

NH

Cα CO2-H2N

H

CH2

N

NH

pI = 7.6

[His][His+]

= 10

% His+ is 9.1%

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Chem 437• Substituents on the amino acids can alter their pKa values

• Anserine (N-β-alanyl-3-methyl-L-histidine) has a pKa of imidazole group is 7.04

• When amino acid residues occur within folded protein structures, the pKa values can be very different from the free amino acid (alters function)

Altering the pKa

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Chem 437

Properties of α-amino acids: Chemistry

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Chem 437

Properties of α-amino acids : Chemistry

α-amino acids give purple productα-imino acids give yellow product

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Chem 437

Reactivity of side chains

Cα CO2H3N+

HAspartic acid, Asp, D

(polar, charged)

CH2

CO2

Glutamic acid, Glu, E(polar, charged)

Cα CO2H3N+

H

CH2

CO2

CH2


Cα CO2H3N+

H

CH2

CH2

CH2

NH3+

CH2

Cα

CH2

CO2H3N+

HCysteine, Cys, C

(polar)

SH

Cα

CH2

CO2H3N+

HSerine, Ser, S

(polar)

OH

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Chem 437

Reactivity of side chains

-Phe-Ser-Tyr-Gly-Val-

Part of the amino acid sequence of Green Fluorescent Protein (GFP)

NN

O

HOOHN

Phe, etc.

Val, etcO2

Cα CH2

CO2

NH3+

H

Cysteine, Cys, C(polar)

SH2 Cα CH2

CO2

NH3+

H S CαCH2

CO2

NH3+

HS

Cystine(disulfide bond)

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Chem 437

Optical activity of amino acids• All amino acids are chiral except for glycine.

Two enantiomeric forms

Cα

R

2OCNH3

+H

Cα

R

CO2

H3N+ H

Mirror plane

α-alanine

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Chem 437

Rotation of polarized light by chiral molecules

Schematic diagram of a polarimeter

Sodium

Chiral molecules are optically active, i.e. they rotate plane polarized light

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Chem 437

Animation of rotation of polarized light

QuickTime™ and a Sorenson Video decompressor are needed to see this picture.

http://cwx.prenhall.com/petrucci/medialib/media_portfolio/28.html

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Chem 437

Rotation of polarized light by chiral molecules

• Clockwise rotation– Dextrorotatory (to the right)– Assigned a positive value

Observed rotation (º)

Optical path length (dm) × concentration (gcm-3)[α]25D

=

D D-line in the spectrum of sodium used to generate monochromatic light

25 Experiment carried out at 25ºC

• Anti-clockwise rotation– Levorotatory (to the left)– Assigned a negative value

Amino acid

Alanine +1.8Arginine +12.8Leucine -11.0Lysine +13.5Methionine -10.0

[α]25D

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Chem 437

Stereochemistry of α-amino acids - The D, L system

Cα

H

CH2OH

CHOHO

Cα

H

OHCH2OH

CHO

L-glyceraldehyde (-9.4) D-glyceraldehyde (+9.4)

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Chem 437

Stereochemistry of α-amino acids - The D, L system

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Chem 437

Stereochemistry of α-amino acids - CORN law

Clockwise direction

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Chem 437

Stereochemistry of α-amino acids

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Chem 437

Stereochemistry of α-amino acids - R, S system

Cα

R

CO2H3N+

HCα

H3N+ CO2

R

Hydrogen atom points to back

Cα

H3N+CO2

R

SH > OH > NH2 > CO2H > CHO > CH2OH > CH3

R(clockwise,

goes to the Right)

S(anti-clockwise,goes to the left)

• Atoms of higher atomic number bonded to a chiral center are ranked above those of lower atomic number

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Chem 437

Stereochemistry of α-amino acids - R, S system

• (2S,3S) and (2R,3R) are enantiomers• (2S,3R) and (2R,3S) are enantiomers• (2S,3S) and (2S,3R) are diastereoisomers• (2R,3R) and (2R,3S) are diastereoisomers

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Chem 437

Spectroscopic properties of α-amino acids

Beer/Lamberts Law

A = = εcl

A - absorbance (A.U.)Io = initial intensityI = emitted intensityε - molar extinction coefficient

(molar absorptivity, M-1cm-1)c - concentration (M)l - pathlength (cm)

logIIo

Detector

Io Il

Cuvette

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Chem 437

NMR spectroscopy of amino acids• The sample under study is placed in a strong magnetic field and a series of radio

frequency (rf) pulses are applied to it.

• Certain atoms (such as 1H) when they are exposed to these radio frequency pulses, emit an rf pulse of their own.

• The emitted rf gives information about the environment of that atom

• The specific emitted rf is represented relative to a rf from a standard (e.g. for a proton NMR, tetramethylsilane is used) in an NMR spectra

• Each peak in the spectra (referred to as the chemical shift) represents an atom (or set of atoms in identical environments) in a unique environment that give a unique rf

• Each amino acid will give a unique proton NMR as their hydrogens exist in unique environments

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Chem 437

1H NMR examples of amino acids

• The type of group the hydrogen is attached to will effect its position (the carboxyl hydrogen has a chemical shift > 10ppm and is not shown)

• The presence of hydrogens on a neighboring carbon will cause splitting of the chemical shift of a hydrogen

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Chem 437

Variation in chemical shift of 13C peaks with pH

• Atoms in groups that can be ionized show pronounced changes in chemical shift as the pH changes

• NMR can be used to study these changes in proteins

Cα CO2H3N+

H

CH2

CH2

CH2

NH3+

CH2

βγδεζ

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Chem 437

Analysis/separation of amino acid mixtures

• Required in the analysis of– Physiological fluids (imbalance in amino acid levels)– Hydrolyzed proteins (determine relative amounts of amino

acids)– Foodstuffs for nutritional value

• Properties of amino acids used to separate them chromatographically– Ionic properties– hydrophobicity

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Chem 437


SampleSO3

-

SO3-

SO3-

O3S-SO3

-SO3

-

Cation exchangecolumn

(CH3)3+N

Anion exchangecolumn

HydrophobicInteraction column(reverse phase)

(CH3)3+N

N(CH3)3+N(CH3)3

+

N(CH3)3+

N(CH3)3+

(CH2)17CH3

(CH2)17CH3

(CH2)17CH3(CH2)17CH3

CH3(CH2)17

CH3(CH2)17

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Chem 437


SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

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Chem 437


SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

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Chem 437


SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Cα CO2H3N+

H

CH2

CO2H

Cα CO2H3N+

H

(CH2)4

NH3+

Cα

CH3

CO2H3N+

H

Mixture (pH ~ 3.3)

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Chem 437


SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

Na+

Na+

Na+

Na+

Na+

Cα CO2H3N+

H

CH2

CO2H

Cα CO2H3N+

H

(CH2)4

NH3+

Cα

CH3

CO2H3N+

H

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Chem 437

Analysis of amino acid mixtures

Column

A BSolution ASolution B

Time/volume

Peak100% Solution B

50% Solution B

0% Solution B

Pumps anddetector

HPLC(High Performance

Liquid Chromatography)

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Chem 437

Analysis/separation of amino acid mixturesSO3

-

SO3-

SO3-

O3S-SO3

-SO3

-

Cation exchangecolumn

(CH3)3+N

Anion exchangecolumn

HydrophobicInteraction (C18) column(reverse phase)

(CH3)3+N

N(CH3)3+N(CH3)3

+

N(CH3)3+

N(CH3)3+

(CH2)17CH3

(CH2)17CH3

(CH2)17CH3(CH2)17CH3

CH3(CH2)17

CH3(CH2)17

Na+Cl-

or pH

CH3CN(acetonitrile)

Na+Cl-

or pH

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Chem 437


SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

Na+

Na+

Na+

Na+

Na+

Peak

100% B

50% B

0% B

Solution A : 0M NaClSolution B : 1M NaCl

Solution A : pH 3.3Solution B : pH 11.0

or

or

Mixture of both variation in pHand salt concentration

Cα CO2H3N+

H

CH2

CO2H

Cα CO2H3N+

H

(CH2)4

NH3+

Cα

CH3

CO2H3N+

H

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Chem 437


Peak100% B

50% B

0% B

Solution A : 0M NaClSolution B : 1M NaCl

Asp Ala Lys

A B

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

Na+

Na+

Na+

Na+

Na+

Cα CO2H3N+

H

CH2

CO2H

Cα CO2H3N+

H

(CH2)4

NH3+

Cα

CH3

CO2H3N+

H

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Chem 437

Analysis of amino acid mixtures

Ion exchange column

Post-column derivatization by,– Ninyhdrin– o-pthaldialdehyde (OPA)

C

O

H

CO

H

OPA

• Ninhydrin detected byvisible light absorption

• OPA detected through fluorescence

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Chem 437

Phenylisothiocyanate reaction with amino acids

C18 hydrophobic Interaction column

(Detection at 254nm)

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Chem 437

Things to do

• Garrett and Grisham, chapter 4• Problem : 1-13

amino acids df05

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