IVERS I TY UN OF

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

Amino acids

IVERS I TY UN OF

Bio-Polymer Chemistry
DNA

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

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 (-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

IVERS I TY UN OF

Basic structure of -amino acids

R H3 N+ C CO2 R C H H3 N+ CO2

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

H Zwitterion form

Ball and stick model (Tetrahedral arrangement)

R-group, varies with each amino acid

-CO2- Carboxyl group -NH3+ Primary amino group

IVERS I TY UN OF

Formation of the peptide bond

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

Peptide bond

IVERS I TY UN OF

The -amino acids

OH H H3N+ C H
Glycine, Gly, G ( not-chiral)

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

CH3 CO2 H3N+ C H

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

CH2 CO2 H3N+ C H

Serine, Ser, S (polar)

CO2

SH CH2 H3N+ C H
Cysteine, Cys, C (polar)

OH H CO2 H3N+ C C H
Threonine, Thr, T (polar, two chiral centers)

CH3 CH3 CO2 H H3N+ C C H

Valine, Val, V (non-polar/hydrophobic)

CH3 CO2

IVERS I TY UN OF

The -amino acids

CH3 H H3N+ C CH2 C H CO2 H3N+ CH3 CH2 H H3N+ C C H CH3 CO2 CH3 CH3 S CH2 CH2 C H
Methionine, Met, M (non-polar/hydrophobic)

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

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

CO2

CH2

CH2

CH2 CO2 H

H2N+

Isoleucine, Ile, I (non-polar/hydrophobic Two chiral centers)

Proline, Pro, P (non-polar/hydrophobic, -imino acid)

IVERS I TY UN OF

The -amino acids

OH N

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

CH2 H3N+ C H
Phenylalanine, Phe, F (non-polar/hydrophobic)

CH2 CO2 H3 N+ C H
Tyrosine, Tyr, Y (polar)

CH2 CO2 H3N+ C H

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

CO2

IVERS I TY UN OF

The -amino acids

NH2
+

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

NH2+

NH3+ CH2 CH2 CH2 CH2

C NH CH2 CH2 CH2 CO2 H3N+ C H

Arginine, Arg, R (polar, charged)

N CH2 H3N+

H3

N+

C H

CO2

C H

CO2

Histidine, His, H (polar, charged)

Lysine, Lys, K (polar, charged)

IVERS I TY UN OF

The -amino acids

CO2 CH2 H3 N+ C H
Aspartic acid, Asp, D (polar, charged)

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

O C CO2 CH2 H3N+ C H

NH2

CO2 O C CH2 CH2 H3N+ C H

Glutamine, Gln, Q (polar)

Asparagine, Asn, N (polar)

NH2

CO2 CH2 CH2 H3 N+ C H

Glutamic acid, Glu, E (polar, charged)

CO2

CO2

IVERS I TY UN OF

Labeling of the side chain atoms

NH3 CH2 CH2 CH2 CH2 H3 N+ C H
Lysine, Lys, K (polar, charged)
+

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

OH H CO2 H3N+ C C H

CH3 CO2

Threonine, Thr, T (polar, two chiral centers)

The carboxyl group and amino group attached to the C atom are referred to as the carboxy group and the amino group respectively The second amino group on lysine is referred to as the -amino group

IVERS I TY UN OF

Representations of -amino acids

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

ball and stick

CH2 H3N+ C H
Phenylalanine, Phe, F

CO2

Space filling

IVERS I TY UN OF

Uncommon amino acids found in proteins

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

IVERS I TY UN OF

Biologically important derivatives of amino acids

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

IVERS I TY UN OF

Acid-base properties
pKa 8.8-10.4 pKa 1.8-2.6

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

Net Charge +1

Net Charge 0

Net Charge -1

pH = 1

pH = 7

pH = 13

IVERS I TY UN OF

Acid-base properties
H H2N C H CO2

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

H H3N+ C H CO2

H H3N+ C H
Glycine (A, V, I, L, P, F, M, N, Q, W)

CO2H

IVERS I TY UN OF

Acid-base properties : acidic amino acids

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

IVERS I TY UN OF

Acid-base properties : basic amino acids

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

IVERS I TY UN OF

Table of pKa values for amino acids

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

IVERS I TY UN OF

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

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

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

IVERS I TY UN OF

Answer slide
+

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

N HN

H N HN HN CH2 H3N+ C CO2H H2N CH2 N

1 96 6

Chem 437

HN CH2 H3N+ C CO2H H

CH2 H3N+ C CO2H

C CO2

pI = 7.6 [His] = 10 [His+] % His+ is 9.1%

IVERS I TY UN OF

Altering the pKa

Substituents on the amino acids can alter their pKa values Anserine (N--alanyl-3-methyl-L-histidine) has a pKa of imidazole group is 7.04

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

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

IVERS I TY UN OF

Properties of -amino acids: Chemistry

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

IVERS I TY UN OF

Properties of -amino acids : Chemistry

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

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

IVERS I TY UN OF

Reactivity of side chains

CO2 CO2 CH2 H3N+ C H
Aspartic acid, Asp, D (polar, charged)

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

NH3+ CH2 CH2 CO2 H3N+ CH2 CH2 C H

Lysine, Lys, K (polar, charged)

Chem 437

CH2 CH2 CO2 H3N+ C H

Glutamic acid, Glu, E (polar, charged)

CO2

SH CH2 H3N+ C H
Cysteine, Cys, C (polar)

OH CH2 CO2 H3N+ C H

Serine, Ser, S (polar)

CO2

IVERS I TY UN OF

Reactivity of side chains

CO2 CO2 CH2 SH H C NH3+ CH2 S S

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

CO2 CH2 C H NH3+

2 H

C NH3+

Cysteine, Cys, C (polar)

Cystine (disulfide bond)

O O2 N HO N N Phe, etc. OH Val, etc

-Phe-Ser-Tyr-Gly-ValPart of the amino acid sequence of Green Fluorescent Protein (GFP)

IVERS I TY UN OF

Optical activity of amino acids

All amino acids are chiral except for glycine. Mirror plane

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

R C H3 N+ H CO2 H
2OC

R C NH3+

Two enantiomeric forms

-alanine

IVERS I TY UN OF

Rotation of polarized light by chiral molecules

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

Sodium

Schematic diagram of a polarimeter Chiral molecules are optically active, i.e. they rotate plane polarized light

IVERS I TY UN OF

Animation of rotation of polarized light

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

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

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

IVERS I TY UN OF

Rotation of polarized light by chiral molecules

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

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

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

Chem 437

[]25 D
D 25

Observed rotation () Optical path length (dm) concentration (gcm-3)

D-line in the spectrum of sodium used to generate monochromatic light Experiment carried out at 25C

Amino acid Alanine Arginine Leucine Lysine Methionine

[]25
D

+1.8 +12.8 -11.0 +13.5 -10.0

IVERS I TY UN OF

Stereochemistry of -amino acids - The D, L system

H C HO CHO CH2OH CHO CH2OH H C OH

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

L-glyceraldehyde (-9.4)

D-glyceraldehyde

(+9.4)

IVERS I TY UN OF

Stereochemistry of -amino acids - The D, L system

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

IVERS I TY UN OF

Stereochemistry of -amino acids - CORN law

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

Clockwise direction

IVERS I TY UN OF

Stereochemistry of -amino acids

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

IVERS I TY UN OF

Stereochemistry of -amino acids - R, S system

Hydrogen atom points to back

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

R C H H3 N+ CO2 H3 N+

R C CO2 CO2

R C H3 N+

S
(anti-clockwise, goes to the left)

R
(clockwise, goes to the Right)

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

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

IVERS I TY UN OF

Stereochemistry of -amino acids - R, S system

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

(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

IVERS I TY UN OF

Spectroscopic properties of -amino acids

Beer/Lamberts Law I A = log o = cl I

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

A - absorbance (A.U.) Io = initial intensity I = emitted intensity - molar extinction coefficient (molar absorptivity, M-1cm-1) c - concentration (M) l - pathlength (cm) l

Io

I Detector Cuvette

IVERS I TY UN OF

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.

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

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

IVERS I TY UN OF

1H

NMR examples of amino acids

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

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

IVERS I TY UN OF

Variation in chemical shift of

13C

peaks with pH

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

NH3+ CH2 CH2 CH2 CH2 H3N+ C H CO2

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

IVERS I TY UN OF

Analysis/separation of amino acid mixtures

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

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

1 96 6

Chem 437

IVERS I TY UN OF

Analysis/separation of amino acid mixtures

Sample
SO3 -O S 3 SO3 SO3 SO3 SO3 -

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

Cation exchange column

(CH3)3

+N

N(CH3)3+ N(CH3)3+
Anion exchange column

(CH3)3+N

N(CH3)3+ CH3(CH2)17

N(CH3)3+

(CH2)17CH3 (CH2)17CH3
Hydrophobic Interaction column (reverse phase)

CH3(CH2)17 (CH2)17CH3 (CH2)17CH3

IVERS I TY UN OF

Analysis/separation of amino acid mixtures

SO3 SO3 SO3 SO3 SO3 SO3 SO3 SO3 SO3 SO3 SO3 SO3

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

IVERS I TY UN OF

Analysis/separation of amino acid mixtures

SO3 Na+ SO3 Na+ SO3 Na+ SO3 Na+ SO3 Na+ SO3 Na+ SO3 Na+ SO3 Na+ SO3 Na+ SO3 Na+ SO3 Na+ SO3 Na+

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

IVERS I TY UN OF

Analysis/separation of amino acid mixtures

SO3 Na+ SO3 Na+ SO3 SO3 Na+ CO2H CH2 H3N+ C H NH3+ (CH2)4 H3N+ C H CO2 H3N+ CH3 C H CO2 CO2

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

Na+

SO3 Na+ SO3 Na+ SO3 Na+ SO3 Na+ SO3 Na+ SO3 Na+ SO3 Na+ SO3 Na+ -

Mixture (pH ~ 3.3)

IVERS I TY UN OF

Analysis/separation of amino acid mixtures

SO3 Na+ SO3 H N+ 3 SO3 + SO3 Na SO3 SO3 + SO3 H3N SO3 SO3 SO3 CO2H CH2 C H CO2

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

NH3+ (CH2)4 C H CH3 H3N+ C H CO2 CO2

Na+

SO3 Na+ SO3 Na+

IVERS I TY UN OF

Analysis of amino acid mixtures

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

Column
Solution A Solution B Pumps and detector

A B
HPLC (High Performance Liquid Chromatography)

100% Solution B Peak 50% Solution B Time/volume 0% Solution B

IVERS I TY UN OF

Analysis/separation of amino acid mixtures

SO3 -O S 3 SO3 SO3 SO3 SO3 Cation exchange column

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

Na+Clor pH

(CH3)3

+N

N(CH3)3+ N(CH3)3+
Anion exchange column

(CH3)3+N

Na+Clor pH

N(CH3)3+ CH3(CH2)17

N(CH3)3+

(CH2)17CH3 (CH2)17CH3
Hydrophobic Interaction (C18) column (reverse phase)

CH3(CH2)17 (CH2)17CH3 (CH2)17CH3

CH3CN (acetonitrile)

IVERS I TY UN OF

Analysis/separation of amino acid mixtures

SO3 Na+ SO3 H N+ 3 SO3 + SO3 Na SO3 SO3 + SO3 H3N SO3 SO3 SO3 CO2H CH2 C H CO2

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

100% B

Peak
50% B

NH3+ (CH2)4 C H CH3 H3N+ C H CO2 CO2

0% B
Solution A : 0M NaCl Solution B : 1M NaCl or Solution A : pH 3.3 Solution B : pH 11.0 or Mixture of both variation in pH and salt concentration

Na+

SO3 Na+ SO3 Na+

IVERS I TY UN OF

Analysis/separation of amino acid mixtures

SO3 Na+ SO3 H N+ 3 SO3 + SO3 Na SO3 SO3 + SO3 H3N SO3 SO3 SO3 CO2H CH2 C H CO2

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

A B

NH3+ (CH2)4 C H CH3 H3N+ C H CO2 CO2 Peak Asp Ala Lys 100% B 50% B

Na+

0% B

SO3 Na+ SO3 Na+

Solution A : 0M NaCl Solution B : 1M NaCl

IVERS I TY UN OF

Analysis of amino acid mixtures

Ion exchange column
O

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

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

H H

C O
OPA

Ninhydrin detected by visible light absorption OPA detected through fluorescence

IVERS I TY UN OF

Phenylisothiocyanate reaction with amino acids

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437

C18 hydrophobic Interaction column (Detection at 254nm)

IVERS I TY UN OF

Things to do
Garrett and Grisham, chapter 4 Problem : 1-13

YLA ND BA L AR TI M

UMBC

E OR
C OU NTY

1 96 6

Chem 437