Premed · Premed · Organic Chemistry 2
Lecture 18: Amino Acids and Peptides
Organic Chemistry II
Learning Objectives
By the end of this lecture, students will be able to:
- Draw and name the 20 standard amino acids and classify them by side chain properties
- Explain the stereochemistry of amino acids (L and D designation, R and S configuration)
- Describe the acid-base behavior of amino acids (zwitterions, isoelectric point)
- Draw peptide bonds and determine peptide sequences
- Describe methods for peptide sequencing and synthesis
- Explain the concept of solid-phase peptide synthesis (Merrifield synthesis)
Lecture Content
I. Structure of Amino Acids
The twenty standard amino acids share a common structural framework: an alpha carbon bearing an amino group (-NH2), a carboxyl group (-COOH), a hydrogen atom, and a variable side chain (R group). Because four different groups are attached to the alpha carbon, it is a stereocenter in all amino acids except glycine, where R = H.
The twenty amino acids encoded by the genetic code are classified by the chemical properties of their side chains. Nonpolar, aliphatic amino acids include glycine, alanine, valine, leucine, isoleucine, and proline (whose cyclic side chain connects back to the nitrogen, making it technically an imino acid). Nonpolar, aromatic amino acids include phenylalanine and tryptophan. Polar, uncharged amino acids include serine, threonine, cysteine, asparagine, glutamine, tyrosine, and methionine. Positively charged (basic) amino acids include lysine, arginine, and histidine. Negatively charged (acidic) amino acids include aspartate and glutamate. Nine of these amino acids are essential, meaning humans cannot synthesize them and must obtain them from the diet: valine, leucine, isoleucine, phenylalanine, tryptophan, methionine, threonine, lysine, and histidine.
II. Stereochemistry
The naturally occurring amino acids in proteins are exclusively L-amino acids. In the Fischer projection, the amino group of an L-amino acid points to the left, analogous to the convention for L-sugars. By the Cahn-Ingold-Prelog priority rules, L-amino acids correspond to the S configuration at the alpha carbon, with one notable exception: L-cysteine is assigned the R configuration because the -CH2SH side chain has higher CIP priority than -COOH, owing to the higher atomic number of sulfur.
D-amino acids are rare in nature but do appear in certain bacterial cell walls and some antibiotics. Under harsh conditions such as prolonged heating or strong acid or base, amino acids can racemize, a process that has been exploited in amino acid racemization dating of archaeological specimens.
III. Acid-Base Properties of Amino Acids
At physiological pH (approximately 7.4), amino acids exist predominantly as dipolar ions called zwitterions. The amino group is protonated (-NH3+) and the carboxyl group is deprotonated (-COO-), giving a net charge of zero for amino acids with neutral side chains. This zwitterionic character accounts for the high melting points and strong water solubility characteristic of amino acids.
The titration behavior of a simple amino acid such as alanine reveals two buffering regions. The first pKa (approximately 2.3) corresponds to deprotonation of the carboxyl group (-COOH to -COO-). The second pKa (approximately 9.7) corresponds to deprotonation of the ammonium group (-NH3+ to -NH2). At very low pH, the fully protonated form carries a +1 charge. Between the two pKa values, the zwitterion predominates. At very high pH, the fully deprotonated form carries a -1 charge.
The isoelectric point (pI) is the pH at which the amino acid has zero net charge. For neutral amino acids, pI equals the average of pKa1 and pKa2. For acidic amino acids (aspartate, glutamate), pI equals the average of the two lowest pKa values. For basic amino acids (lysine, arginine, histidine), pI equals the average of the two highest pKa values. In electrophoresis, an amino acid migrates toward the cathode at pH values below its pI (because it carries a net positive charge), toward the anode above its pI (net negative charge), and does not migrate at pH equal to its pI.
<image>Panel A: Titration curve of alanine showing pH vs. equivalents of NaOH added. The curve shows two buffering regions at pKa1 (2.3) and pKa2 (9.7), with the predominant ionic species drawn at each region: fully protonated cation at low pH, zwitterion at intermediate pH, and fully deprotonated anion at high pH. The isoelectric point (pI = 6.0) is marked at the midpoint between the two pKa values. Panel B: Schematic of electrophoresis showing migration of amino acids at different pH values relative to their pI.</image>
IV. The Peptide Bond
The peptide bond forms by a condensation reaction between the alpha-amino group of one amino acid and the alpha-carboxyl group of another, releasing water and creating an amide (peptide) bond. Chains of two amino acids are dipeptides, three are tripeptides, and chains of more than about fifty residues are generally called proteins. By convention, peptide sequences are written from the N-terminus (free amino group) on the left to the C-terminus (free carboxyl group) on the right.
The peptide bond has several distinctive properties arising from its amide character. Resonance gives the C-N bond approximately 40 percent double-bond character, shortening it to about 1.33 angstroms compared to a typical C-N single bond at 1.47 angstroms. This partial double-bond character restricts rotation, forcing the six atoms of the peptide bond unit (C-alpha, C=O, N-H, and the adjacent C-alpha) into a planar arrangement. The trans configuration about the C-N bond is overwhelmingly favored (greater than 99.9%), with the notable exception of X-Pro bonds, which adopt the cis configuration about 10 percent of the time due to proline's cyclic structure. While rotation is restricted about the C-N bond, the phi (N-C-alpha) and psi (C-alpha-C) dihedral angles remain free to rotate, defining the backbone conformation.
V. Peptide Sequencing
Determining the amino acid sequence of a peptide begins with amino acid composition analysis. Complete acid hydrolysis (6 M HCl, 110 degrees C, 24 hours) breaks all peptide bonds, and the resulting mixture of free amino acids is quantified by an amino acid analyzer.
Edman degradation is the classical method for determining the order of residues. Phenylisothiocyanate (PITC, the Edman reagent) reacts with the free N-terminal amino group. Under mild acid conditions, the labeled N-terminal residue is selectively cleaved as a phenylthiohydantoin (PTH) derivative, which is identified by HPLC. The shortened peptide, now with a new N-terminus, undergoes another round of degradation. This process can be repeated sequentially, and automated protein sequencers can determine up to 50 to 60 residues. Sanger's method (using 2,4-dinitrofluorobenzene) and dansyl chloride labeling provide alternative approaches to N-terminal identification.
C-terminal analysis uses carboxypeptidase enzymes that sequentially remove residues from the C-terminus. For longer peptides, partial hydrolysis with different proteases (trypsin cleaves after Lys and Arg; chymotrypsin after Phe, Trp, and Tyr; cyanogen bromide after Met) generates overlapping fragments whose sequences can be assembled to reconstruct the full sequence. Modern peptide sequencing increasingly relies on tandem mass spectrometry (MS/MS).
<image>Edman degradation cycle illustrated in three steps. Step 1: Phenylisothiocyanate (PITC) reacts with the free N-terminal amino group of the peptide under mildly basic conditions to form a phenylthiocarbamoyl (PTC) derivative. Step 2: Treatment with anhydrous acid cleaves the N-terminal residue as an anilinothiazolinone derivative, leaving the remaining peptide intact with a new N-terminus. Step 3: The anilinothiazolinone is converted to the stable PTH-amino acid, which is identified by HPLC. The shortened peptide re-enters the cycle for the next round.</image>
VI. Peptide Synthesis
The central challenge of peptide synthesis is controlling which amino group reacts with which carboxyl group to avoid random polymerization. The solution lies in protecting group strategies. The N-terminus is commonly protected with a Boc group (tert-butoxycarbonyl, removed by TFA) or an Fmoc group (fluorenylmethyloxycarbonyl, removed by piperidine). The C-terminus is protected as an ester (methyl or benzyl, removed by hydrolysis or hydrogenolysis). Side chain functional groups require their own specific protecting groups.
Coupling reagents such as DCC (dicyclohexylcarbodiimide), EDC, and HATU activate the carboxyl group, converting it to a better electrophile so that amide bond formation proceeds efficiently. DCC, for example, reacts with the carboxylic acid to form an O-acylisourea intermediate, which the amine then attacks.
Solid-phase peptide synthesis (SPPS), developed by Robert Bruce Merrifield (Nobel Prize, 1984), revolutionized peptide chemistry. The C-terminus of the first amino acid is anchored to an insoluble polymer resin bead, and the peptide chain is built from C-terminus to N-terminus by repeated cycles of deprotection and coupling. After each step, excess reagents and byproducts are simply washed away by filtration. When the synthesis is complete, the finished peptide is cleaved from the resin (typically with HF or TFA). SPPS is fast, automatable, and can reliably produce peptides of up to 50 to 100 residues. For larger proteins, native chemical ligation allows separate peptide fragments to be joined.
<image>Schematic of the Merrifield solid-phase peptide synthesis cycle. A polymer resin bead is shown at the bottom. Step 1: The first Fmoc-protected amino acid is attached to the resin via its C-terminus. Step 2: The Fmoc protecting group is removed with piperidine (deprotection). Step 3: The next Fmoc-amino acid is coupled using a coupling reagent (DCC or HATU). Steps 2 and 3 are repeated for each additional amino acid. Final step: The completed peptide is cleaved from the resin with TFA or HF. The growing peptide chain is shown with each amino acid added one at a time, N to C direction (right to left on the bead).</image>
