Amino Acids Polypeptides and Proteins An a amino acid is a carboxylic acid which has an amino group attached to the carbon a to the COOH Polypeptides and proteins are composed either completely or partially of a amino acid fragments called residues which are joined to each other to form chains Twenty different amino acids are commonly found in proteins Nineteen of them are 1o amino acids having this structure H H2N C R COOH where the R group is alkyl or substituted alkyl The 20th proline has a 2o amino group H H N C COOH 1 Properties of Amino Acids Non volatile high mp glycine 262oC Insoluble in non polar solvents somewhat soluble in water Large dipole moments Glycine 14D cf propylamine 1 4D propanoic acid 1 7D Ka and Kb are much smaller than expected eg for glycine H2NCH2COOH Ka 1 6 x 10 10 Kb 2 5 x 10 12 cf Ka 10 5 for typical COOH Kb 10 4 for typical NH2 All of this suggests the existence of a zwitterion eg for glycine H3N CH2COO In this zwitterionic form the acid is the H3N which can donate a proton to become H2N not the COOH Also the base is the COO which can accept a proton to become COOH not the NH2 2 In base In acid H3NCH2COO OH H2NCH2COO H2O H3NCH2COO H3O H3NCH2COOH H2O H3NCH2COO because of the equlibria which exist may react as if it were H2NCH2COOH II III H2NCH2COO H3NCH2COO H3NCH2COOH H3O H2O OH In other words in solution there are glycine molecules that have a COOH group and others that have an NH2 There are even a very few that are completely unionized The hydrogen ion concentration at which II III is known as the isoelectric point of the amino acid If the amino acid is placed in an electric field at this pH it will show no net migration toward anode or cathode I 3 Remember Chemical equilibrium is a dynamic process and Lowry Bronsted acid base equilibria involving nitrogens and oxygens are usually fast So a given amino acid molecule will move toward the anode when it is in anionic II form but a moment later it will be in zwitterionic form I and will not move and a moment later it will be in cationic form III and move back toward the cathode So the molecule does a little dance in place back and forth but there is no net motion toward either electrode If the pH is increased II III and a given amino acid molecule spends more time in state II than in state III so there will be net migration toward the anode If the pH is decreased III II and there will be net migration toward the cathode Amino acids with neutral R groups are more acidic than basic Therefore the isoelectric point is acidic ie pI 7 for these compounds For amino acids with a basic R pI 7 For amino acids with an acidic R pI 7 Different amino acids have different isoelectric points Consequently they can be separated from each other by electrophoresis 4 Gel or paper pH buffer 5 97 A N O D E C A T H O D E H3N CH2 C pI 5 97 H3N CH2 C H2N CH2 C O O O OH O O O O O OH CH2SH CH C O O O H3N pI 5 07 H2N CH2 C pI 6 30 H3N CH C OH CH2SH H2N CH C CH2SH O O H2N CH2 C HN CH2 C O O 5 Amino acids usually are least soluble in water at their isoelectric point Configuration of amino acids Specification of configuration of stereocenter based on D and L glyceraldehyde H C HO O H CH3 H H O C OH CH3 S glyceraldehyde R glyceraldehyde L glyceraldehyde D glyceraldehyde Amino Acids HO H2N O H C R HO O H NH2 C R 6 L amino acid D amino acid Almost all naturally occurring amino acids except glycine no stereocenter have the L configuration Synthesis of Amino Acids in the Laboratory There are several ways to synthesize amino acids One of these ways is shown here Note that this syntheses will lead to racemic mixtures except for glycine Hell Volhard Zelinskii method CH3CH 2COOH CH 3CHCOOH Br Br CH3CHCOOH NH2 CH3CHCOOH Since it is usually the L amino acids that are of interest the racemic amino acids synthesized above would have to be resolved The classic method is to form diastereomeric salts One could form the carboxylate salts of the racemic amino acids using enantiomerically pure alkaloids eg brucine or strychnine or one could form the ammonium salts using tartaric acid In any case the salts are diasteriomeric and can be separated by ordinary means Once separated the amino acids can be 1 Br2 PBr3 2 H2O NH3 excess 7 regenerated from the salts as enantiomerically pure compounds An alternative method of resolution is to acetylate the amino group of the racemic mixture using acetic anhydride The acetylated racemic mixture is then hydrolyzed using an enzyme carboxypeptidase hog kidney acylase that will hydrolyze only one of the enantiomers actually the L isomer This is OK because the enzyme is chiral and enantiomerically pure The hydrolyzed amino acid can then be separated from the unhydrolyzed material by ordinary means A generic example follows HO H2N O H C R C R H NH2 D amino acid HO CH3CHN O O H C R C R H NHCCH3 O L amino acid acetic acylase L amino acid HO O anhydride HO O HO H2N O H C R HO O C R H NHCCH3 O The above compounds are easily separated Extraction with aqueous acid would likely be successful 8