Organic Chemistry William H. Brown & Christopher S. Foote

Amino Acids & Proteins Chapter 27

Amino 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 zwitterionalthough  -amino acids are commonly written in the unionized form, they are more properly written in the zwitterion (internal salt) form

Chirality of Amino Acids  With the exception of glycine, all protein-derived amino acids have at least one stereocenter (the  -carbon) and are chiral the vast majority have the L-configuration at their  - carbon

Nonpolar side chains

Polar side chains

Acidic & basic side chains

Other Amino Acids

Acid-BaseProper-ties

Acid-Base Properties

Acidity:  -CO 2 H Groups  The average pK a of an  -carboxyl group is 2.19, which makes them considerably stronger acids than acetic acid (pK a 4.76) the greater acidity is accounted for by the electron- withdrawing inductive effect of the adjacent -NH 3 + group

Acidity: side chain -COOH  Due to the electron-withdrawing inductive effect of the  -NH 3 + group, side chain -COOH groups are also stronger than acetic acid the effect decreases with distance from the  -NH 3 + group. Compare: pK a 2.35  -COOH group of alanine (pK a 2.35) pK a 3.86  -COOH group of aspartic acid (pK a 3.86) pK a 4.07  -COOH group of glutamic acid (pK a 4.07)

Acidity:  -NH 3 + groups  The average value of pK a for an  -NH 3 + group is 9.47, compared with a value of for a 1° alkylammonium ion

Basicity-Guanidine Group basicity of the guanidine group is attributed to the large resonance stabilization of the protonated form relative to the neutral form

Basicity- Imidazole Group the imidazole group is a heterocyclic aromatic amine

Ionization vs pH  Given the value of pK a of each functional group, we can calculate the ratio of each acid to its conjugate base as a function of pH consider the ionization of an  -COOH writing the acid ionization constant and rearranging terms gives

Ionization vs pH substituting the value of pK a (2.00) for the hydrogen ion concentration at pH 7.0 (1.0 x ) gives at pH 7.0, the  -carboxyl group is virtually 100% in the ionized form and has a net charge of -1 we can repeat this calculation at any pH and determine the ratio of [  -COO - ] to [  -COOH] and the net charge on the  -carboxyl at that pH

Ionization vs pH  We can also calculate the ratios of acid to conjugate base for an  -NH 3 + group; for this calculation, assume a value 10.0 for pK a writing the acid ionization constant and rearranging gives

Ionization vs pH substituting values for pK a of an  -NH 3 + group and the hydrogen ion concentration at pH 7.0 gives thus at pH 7.0, the ratio of  -NH 3 + to  -NH 2 is approximately 1 to 1000 at this pH, an  -amino group is 99.9% in the protonated form and has a charge of +1

Henderson-Hasselbalch for the ionization of any weak acid HA taking the log and rearranging gives substitution pH and pK a gives the Henderson- Hasselbalch equation

Henderson-Hasselbalch using the Henderson-Hasselbalch equation we see that when pH = pK a, the concentrations of weak acid and its conjugate base are equal when pH < pK a, the weak acid predominates when pH > pK a, the conjugate base predominates

Isoelectric Point  Isoelectric point, pI, of an amino acid:  Isoelectric point, pI, of an amino acid: the pH at which the majority of its molecules in solution have no net charge the pH for glycine, for example, falls between the pK a values for the carboxyl and amino groups

Electrophoresis  Electrophoresis:  Electrophoresis: the process of separating compounds on the basis of their electric charge electrophoresis of amino acids can be carried out using paper, starch, agar, certain plastics, and cellulose acetate as solid supports  In paper electrophoresis a paper strip saturated with an aqueous buffer of predetermined pH serves as a bridge between two electrode vessels

Electrophoresis a sample of amino acids is applied as a spot on the paper strip 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 molecules at their isoelectric point remain at the origin after separation is complete, the strip is dried and developed to make the separated amino acids visible

Electrophoresis a reagent commonly used to detect amino acid is ninhydrin

Polypeptides & 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 amide bond between the  -carboxyl group of one amino acid and the  -amino group of another

Serylalanine (Ser-Ala)

Peptides 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

Writing Peptides by convention, peptides are written from the left, beginning with the free -NH 3 + group and ending with the free -COO - group on the right

Primary 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, most commonly carried out using 6M HCl at elevated temperature quantitative analysis of the hydrolysate by ion- exchange chromatography

Cyanogen Bromide, BrCN cleavage of peptide bonds formed by the carboxyl group of methionine

Cyanogen Bromide, BrCN

Cyanogen Bromide, BrCN Step 1: nucleophilic displacement of bromine

Cyanogen Bromide, BrCN Step 2: internal nucleophilic displacement

Cyanogen Bromide, BrCN Step 3: hydrolysis of the imino group

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

Edman Degradation  Edman degradation:  Edman degradation: cleaves the N-terminal amino acid of a polypeptide chain

Edman Degradation Step 1: nucleophilic addition to the C=N group of phenylisothiocyanate

Edman Degradation Step 2: nucleophilic addition of sulfur to the C=O of the adjacent amide group

Edman Degradation Step 3: isomerization of the thiazolinone ring

Primary Structure Example 27.8 Example 27.8 Deduce the 1° structure of this pentapeptide

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

Polypeptide Synthesis protect the  -amino group of aa-1 activate the  -carboxyl group of aa-1 protect the  -carboxyl group of aa-2

Amino-Protecting Groups the most common strategy for protecting amino groups and reducing their nucleophilicity is to convert them to amides

Amino-Protecting Groups treatment of an amino group with either of these reagents gives a carbamate (an ester of the monoamide of carbonic acid)

Amino-Protecting Groups a carbamate is stable to dilute base but can be removed by treatment with HBr in acetic acid

Amino-Protecting Groups  The benzyloxycarbonyl group is removed by hydrogenolysis (Section 20.6C) the intermediate carbamic acid loses carbon dioxide to give the unprotected amino group

Carboxyl-Protecting Grps  Carboxyl groups are most often protected as methyl, ethyl, or benzyl esters methyl and ethyl esters are prepared by Fischer esterification, and removed by hydrolysis in aqueous base under mild conditions benzyl esters are removed by hydrogenolysis (Sect. 20.6C); they are also removed by treatment with HBr in acetic acid

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

Peptide Bond Formation DCC acts as dehydrating in bringing about formation of a peptide bond

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 -CH 2 Cl 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

Peptide Bond Geometry the four atoms of a peptide bond and the two alpha carbons joined to it lie in a plane with bond angles of 120° about C and N

Peptide 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 and rotation about the peptide bond is restricted

Peptide 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

Secondary Structure  Secondary structure:  Secondary structure: the ordered arrangements (conformations) of amino acids in localized regions of a polypeptide or protein  To determine from model building which conformations would be of greatest stability, Pauling and Corey assumed that 1. all six atoms of each peptide bond lie in the same plane and in the s-trans conformation 2. there is hydrogen bonding between the N-H group of one peptide bond and a C=O group of another peptide bond as shown in the next screen

Secondary Structure hydrogen bonding between amide groups

Secondary 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

The  -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

The  -Helix

 -Pleated Sheet  The antiparallel  -pleated sheet consists of adjacent polypeptide chains running in opposite directions each peptide bond is planar and has the s-trans conformation the C=O and N-H groups of peptide bonds from adjacent chains point toward each other and are in the same plane so that hydrogen bonding is possible between them all R- groups on any one chain alternate, first above, then below the plane of the sheet, etc.

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

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

Quaternary Structure if two polypeptide chains, for example, each have one hydrophobic patch, each patch can be shielded from contact with water if the chains form a dimer

Prob From what amino acid is histamine derived? By what type of reaction is the precursor amino acid converted to histamine?

Prob From which protein-derived amino acid are norepinephrine and epinephrine synthesized? What types of reactions are involved in each biosynthesis?

Prob From which amino acid are serotonin and melatonin derived? What types of reactions are involved in the biosynthesis of each?

Prob Do you expect the modified guanidino group of cimetidine to be more basic or less basic that the guanidino group of arginine?

Prob Draw a structural formula for the product formed by treating alanine with each reagent.

Prob A tetradecapeptide (14 amino acids) gives these fragments on partial hydrolysis. From this information, deduce the primary structure of this polypeptide.

Prob Name the amino acids in glutathione. What is unusual about the peptide bond formed by the N-terminal amino acid?

Prob Name the amino acids in aspartame. Estimate the isoelectric point of this dipeptide.

Prob Write a structural formula for the product formed by treating the N-terminal amino acid of a polypeptide chain with 2,4-dinitrofluorobenzene, and for the derivatized amino acid formed when the polypeptide chain is hydrolyzed in acid.

Amino Acids & Proteins End of Chapter 27