Amino Acids, Peptides, and Proteins Help (page 2)

Introduction

This study guide deals with the very important α-amino acids, the building blocks of the proteins that are necessary for the function and structure of living cells. Enzymes, the highly specific biochemical catalysts, are proteins.

Amino acids are zwitterions: they have both + and - charges in them at neutral pH. The natural amino acids have different side-chains (R groups), but the same α-aminocarboxylic acid backbone. With the exception of glycine (R=H), the natural amino acids are chiral, with the S configuration at the α-carbon.

The 20 most common naturally occurring amino acids are listed below. They all are primary amines, except for proline which is a 2° amine. They are grouped into different catagories, depending on the chemical nature of the side-chain group. Each amino acid has a 3-letter abbreviation.

Chemical Properties of Amino Acids

Acid-Base (Amphoteric) Properties. The pH at which [anion] = [cation] is called the isoelectric point. At this pH, there is no net charge on the molecule. The different amino acids have different isoelectric points, depending on whether the side-chain is acid, basic, or neutral.

Peptides

Peptides are polyamides composed of the different amino acids. The amide bond between two amino acid residues is often called a peptide bond. Peptides can range from simple 2-amino acid residue compounds up to long chains of 50 to 100 residues, at which point they approach the size of proteins.

The artificial sweetener aspartame is a synthetic dipeptide, the methyl ester of aspartylphenylalanine, or AspPheOCH₃.

Structure Determination

It is possible to completely hydrolyze a peptide to its component amino acids by heating in aqueous acid. While this can, in principal, provide the composition of the peptide, it does not reveal the order in which the amino acids occur in the chain. This information (the sequence) is determined by other methods.

Sequencing. By cutting off one terminal amino acid at a time from either the free amino end (N-terminus) or the free COOH end (C-terminus), sequencing techniques give the linkage order (primary structure) of the amino acids in the peptide.

The N-terminal residue can be identified with either the Sanger method or the Edman method. In the Sanger method, the free NH₂ group of the N-terminal residue reacts with 2,4-dinitrofluorobenzene (DNFB) in an unusual example of nucleophilic aromatic substitution. After complete hydrolysis of the peptide, the DNFB-labelled amino acid can be isolated and identified.

The Edman degradation is similar, although phenylisothiocyanate is used to label the N-terminal residue, and a phenylthiohydantoin is isolated. The Edman degradation can be repeated to identify each residue in turn.

Carboxypeptidase, an enzyme, selectively cleaves the C-terminal amino acid from a peptide chain, and so can be used to identify each residue in turn.

Peptide Synthesis

The synthesis of peptides from amino acids requires the use of protecting groups to prevent random coupling of amino acids and polymerization. These groups can be installed to render a functional group unreactive, then removed to liberate the reactive functional group.

Blocking the N-Terminal Amino Group. This reaction is achieved by attaching a group (B) to N of NH2. After the desired peptide is constructed, the blocking group is removed without destroying the peptide linkages. A common group is the t-butoxycarbonyl, or "Boc" group:

Activating the COOH. The –COOH is not reactive enough on its own to form an amide. It is first activated to form a peptide bond with an N-terminal NH₂ by reacting the N-blocked amino acid with ethyl chloroformate (EtOC(O)Cl). A mixed anhydride of an alkyl carbonic acid is formed. This technique minimizes racemization of the chiral α-carbon

The COOH groups can also be activated in the presence of free N-terminal amino groups with N,N-dicyclohexylcarbodimide (DCC).

Coupling. A CO₂H-activated, Boc protected amino acid can be coupled with an amino acid with a free NH₂ group to make a dipeptide.

The synthesis can be continued, or the protecting groups can be hydrolyzed off with aqueous acid to liberate the free peptide.

Merrifield Solid-Phase Synthesis. A major advance in peptide synthesis is the solid-phase method. Beads of solid chloromethylated polystyrene [chloromethyl groups (–CH₂Cl) para to about every hundredth phenyl group] are used in an automated process. The sequence of steps is: (1) a C-terminal Boc-protected amino acid is attached by substitution for the Cl of the –CH₂Cl group; (2) the Boc group is removed; (3) a Boc-protected amino acid is added and the new peptide bond is formed with the aid of DCC. Steps (2) and (3) are repeated as many times as is necessary to make the desired peptide. In the final step, the completed peptide is detached by hydrolysis of the ester bond that holds the peptide to the solid surface. The average yield for each step exceeds 99%.