Amino acids — Organic Chemistry Notes

Alanine is one of the twenty building blocks of proteins. Its middle carbon carries an amino group (–NH₂) and a carboxyl group (–COOH), along with a hydrogen and a methyl group. Any molecule built this way is an α-amino acid. This carbon is the α carbon. It sits right next to the carboxyl carbon. The fourth group on it is the side chain, written R. In alanine, R is CH₃.

Alanine. The highlighted α carbon holds all four parts: H, the amino group, the carboxyl group and the side chain R.

Living things build their proteins from twenty α-amino acids. All twenty share the H, the amino group and the carboxyl group on the α carbon (in proline the amino group is part of a ring), and they differ in the side chain. Most of what makes amino acids special comes from carrying an acid and a base in one small molecule.

In water, an amino acid carries two charges

Put alanine in water at pH 7 and ask what each group does. The carboxyl group has a pK_a of about 2.3. The pH is far above that, so the carboxyl loses its proton and becomes a carboxylate, –COO⁻. The amino group is a base, and its conjugate acid, the ammonium group –NH₃⁺, has a pK_a of about 9.7. The pH is below that, so the nitrogen holds on to a proton. This is the rule taught in pKa: below its pK_a a group is mostly protonated, and above it mostly deprotonated.

So in water, alanine carries a negative charge at one end and a positive charge at the other. The two charges cancel, and the molecule has no net charge. A molecule like this, with a positive and a negative charge on different atoms but neutral overall, is a zwitterion (from the German for "hybrid ion").

Dissolve alanine in pure water, with nothing holding the pH at 7, and the same thing happens. The carboxyl group (pK_a about 2.3) is a far stronger acid than the ammonium group (pK_a about 9.7), so the proton moves from the oxygen to the nitrogen. An acid–base equilibrium favors the weaker acid, here the ammonium group, and so the zwitterion wins by a wide margin. Solid amino acids are zwitterions too.

The form you might draw first (top) and the form actually present in water and in the crystal (bottom).

The two charges explain how amino acids behave in bulk. They are crystalline solids, and most of them decompose above 200 °C instead of melting cleanly. They dissolve in water but not in ether. Those are the properties of a salt, and an amino acid is a salt whose cation and anion are the two ends of one molecule.

Many books draw an amino acid as H₂N–CHR–COOH, with no charges. That is a shorthand. The uncharged form is never the main species in water, at any pH. When a question asks for the structure at a given pH, draw the charged groups.

Charge follows pH: three forms

Now change the pH. Start in strong acid, at pH 1. That pH is below both pK_a values, so both groups keep their protons: the carboxyl is COOH and the amino group is NH₃⁺. Alanine is a cation with a net charge of +1.

Add base. The carboxyl’s pK_a is 2.34. Above that pH it loses its proton, and alanine is mostly the zwitterion, with net charge 0. The ammonium group’s pK_a is 9.69. Above that pH the nitrogen loses its proton too. At pH 11 alanine is an anion, with COO⁻ and NH₂ and a net charge of −1.

Alanine at three pH values, read from left to right as base is added. The highlighted groups are the charged ones.

Each time the pH rises past a pK_a, the molecule loses one proton and its net charge drops by one. Alanine has two groups that can lose a proton, so it has two pK_a values and three forms. Sketching this ladder of forms is the first step in almost every amino acid question.

The isoelectric point

Between its two pK_a values, alanine is mostly the zwitterion, with a little cation or a little anion mixed in. At one pH in that range the cation and the anion are present in equal amounts, and the average charge is exactly zero. That pH is the isoelectric point, pI.

For alanine the pI lies halfway between the two pK_a values: ½(2.34 + 9.69) = 6.02. The same formula works for any amino acid whose side chain cannot gain or lose a proton:

pI = ½(pK_a1 + pK_a2)

Alanine across the pH scale. Each band shows the form that dominates in that range.

Glycine works the same way. Its pK_a values are 2.34 and 9.60, so its pI is 5.97. Notice that both answers are near 6, not 7. The pI is below 7 because the carboxyl’s pK_a (2.34) sits farther below 7 than the ammonium group’s (9.69) sits above it. "No net charge" describes the molecule, not a neutral solution.

Some side chains carry a group that gains or loses a proton of its own. Lysine’s side chain ends in a second amino group, and aspartic acid’s ends in a second carboxyl group. Each of these amino acids has three pK_a values. Its pI is the average of the two pK_a values on either side of the form with no net charge. For an acidic side chain those are the two acidic pK_a values, which gives a low pI. For a basic side chain they are the two basic ones, which gives a high pI.

Worked example — pI when the side chain ionizes

Lysine has pK_a values 2.18 (COOH), 8.95 (α-NH₃⁺) and 10.53 (side-chain NH₃⁺). Find its pI.

Write the forms in order of pH, with their charges. Below 2.18, the COOH is intact and both amino groups are protonated: net +2. Between 2.18 and 8.95, the carboxyl has become COO⁻: net +1. Between 8.95 and 10.53, the α-amino group is neutral and the side chain is still protonated: net 0. Above 10.53: net −1.

Find the zero and average its two neighbors. The form with no net charge sits between 8.95 and 10.53, so pI = ½(8.95 + 10.53) = 9.74.

Check that the answer points the right way. A basic side chain should push the pI up, and 9.74 sits well above the 6 or so of an amino acid with an inert side chain. Averaging the two lowest values out of habit gives 5.57, the usual wrong answer.

Now aspartic acid: pK_a 1.88 (α-COOH), 3.65 (side-chain COOH), 9.60 (NH₃⁺). The charges run +1, 0, −1, −2, and the form with no net charge sits between 1.88 and 3.65, so pI = ½(1.88 + 3.65) = 2.77. Two acidic groups pull the pI low. The procedure never changes: build the ladder, find the zero, and average the two pK_a values on either side of it.

The four forms of lysine and of aspartic acid, in order of rising pH. The green box has no net charge, and the bracket marks the two pKa values to average.

Which way it moves in an electric field

The sign of the net charge decides which way an amino acid moves in an electric field. In electrophoresis, a sample sits in a buffer between two electrodes. The negative electrode is the cathode, and the positive one is the anode.

Worked example — which way does it move in an electric field?

Alanine (pI ≈ 6.0) is placed in a buffer at pH 9. Which electrode does it move toward?

Compare the pH with the pI. The pH is above the pI, so alanine is mostly the anion. It carries a net negative charge.

Match the charge to an electrode. A negative molecule moves toward the positive electrode, the anode.

Alanine (pI 6.0) started at the center of the strip in three buffers. The small dot marks the start and the larger one where it ends up.

One comparison answers every electrophoresis question in this chapter. pH below the pI: net positive, moves to the cathode. pH above the pI: net negative, moves to the anode. pH equal to the pI: no net charge, no movement. Laboratories separate proteins on the same principle.

Side chains sort into four classes

Compare alanine with serine. Alanine’s side chain is CH₃, which has no attraction for water. Serine’s is CH₂OH, and its OH forms hydrogen bonds with water. When a protein folds, that difference largely decides where each one ends up: side chains that avoid water go inside, and side chains that attract it stay on the surface.

Sorting the twenty by what the side chain does is far more useful than memorizing twenty structures. Four classes cover them:

Nonpolar: side chains made mostly of carbon and hydrogen, either alkyl or aromatic, such as alanine, valine, leucine and phenylalanine. They are hydrophobic ("water-avoiding"), so they end up buried inside a folded protein.
Polar, uncharged: side chains with an OH, SH or amide group, such as serine, threonine, cysteine and asparagine. They form hydrogen bonds and often sit at the protein’s surface or in its active site, the pocket where an enzyme does its chemistry.
Acidic: a second carboxyl group, in aspartic acid and glutamic acid. At physiological pH (about 7.4) this side chain is deprotonated and negative, and the pI is low. The charged forms are called aspartate and glutamate.
Basic: a side chain with an amine or a nitrogen-containing ring that can take a proton, in lysine, arginine and histidine. Lysine and arginine are protonated and positive at pH 7.4, and their pI values are high. Histidine’s side chain has a pK_a near 6, so at pH 7.4 only a few percent of it is protonated, and its pI is about 7.6.

One amino acid from each class. The backbone is the same in all four; only the highlighted side chain changes.

Textbooks sort a few borderline members differently, among them glycine, cysteine, tyrosine and tryptophan. Learn why each class behaves as it does, rather than memorizing one book’s list.

Proline is the one amino acid whose side chain bonds back to its own nitrogen. Its three side-chain CH₂ groups close a five-membered ring through that nitrogen. The nitrogen is bonded to two carbons, so proline’s amino group is a secondary amine, while every other amino acid has a primary one. The ring holds the chain rigid at that point, so proline puts a kink in a protein chain and breaks the regular coil (the α-helix) described in Peptides and proteins.

Proline. The highlighted bond is where the side chain joins the nitrogen and completes the ring.

Cysteine’s side chain ends in a thiol, an SH group. Oxidation joins the sulfur atoms of two cysteines into a disulfide bond, S–S, and removes the two hydrogens. The two cysteines can sit far apart in the chain, so the disulfide ties distant parts of a folded protein together. It is the most common covalent cross-link in proteins.

Two cysteine side chains before and after oxidation. The new S–S bond (highlighted) ties two parts of a chain together.

Amino acids are colorless, so chemists use ninhydrin to see them. Ninhydrin reacts with a free α-amino group to give a deep purple dye. Chemists spray it on chromatography plates, and forensic scientists use it to reveal fingerprints. Proline is the exception again. Its nitrogen is secondary and locked in a ring, so it gives a yellow product instead of purple.

One stereocenter, and biology uses L

Look again at alanine’s α carbon. It carries four different groups: NH₂, COOH, H and CH₃. That makes it a stereocenter, so alanine exists as two enantiomers, mirror images that cannot be superimposed. The same is true of every amino acid but one. (Isoleucine and threonine also have a second stereocenter in their side chains; the α carbon is the one that matters here.)

Glycine is the exception. Its side chain is a second hydrogen, so its α carbon carries two identical groups. Glycine is achiral and has no enantiomers. With only H as its side chain, glycine also lets a protein chain bend where no other amino acid can.

Amino acids label their two enantiomers D and L, as sugars do. Draw the amino acid as a Fischer projection, as in Fischer projections, with the carboxyl at the top and the side chain at the bottom. Then look at the amino group on the horizontal line. If it is on the left, the amino acid is L; if it is on the right, it is D.

Alanine in Fischer projection, carboxyl at the top and side chain at the bottom. Only the side the amino group sits on differs.

Cells build their proteins from L amino acids only. D amino acids do occur in nature, for example in the cell walls of bacteria, but they are not built into proteins.

A second way to recognize L works on a wedge-and-dash drawing. It uses the word CORN: CO for the carboxyl, R for the side chain and N for the amino group. Here R means the side chain, not the R of R/S.

Turn the molecule so that the H on the α carbon points toward you. Then trace CO → R → N. For an L amino acid, that path runs clockwise.

The direction depends on where you look from. Turn the same molecule so that the H points away from you, and CO → R → N runs counterclockwise instead. Viewing from the opposite side reverses every rotation you see, just as in the flip trick of R/S configuration. Forgetting which way the H points is the usual CORN mistake.

L-alanine drawn twice: on the left with its H on a wedge, on the right turned so the H is on a hash.

The configuration is not permanent. The α hydrogen sits next to a carbonyl group, so a base can remove it, and acid speeds the same change through the enol, as in Alpha hydrogens & enolates. The α carbon then goes flat, and the proton can come back to either face. That is why amino acids slowly racemize, drifting toward a 50:50 mix of D and L, when a protein is hydrolyzed in hot acid or base.

The R/S system gives a separate label. For L-alanine the CIP priorities are NH₂ first, COOH second and CH₃ third. With the H pointing away, 1 → 2 → 3 runs counterclockwise, so L-alanine is (S). At the α carbon, every chiral L amino acid is (S) except one.

Cysteine is that exception. Its side chain is CH₂SH. At the first atom out, the CH₂SH carbon carries (S, H, H) and the carboxyl carbon carries (O, O, O). Sulfur outranks oxygen, so CH₂SH now ranks above COOH. The shape is the same as in L-alanine, but the priority order has changed, and L-cysteine is (R).

The same shape, two different R/S labels. The green numbers are CIP priorities; H is priority 4 and points away.

D/L and R/S are separate systems. D/L comes from where a group sits in a Fischer projection drawn by a fixed convention. R/S comes from CIP priorities. For most amino acids L happens to be S, only because the priorities usually fall in the same order. CORN and the Fischer rule never consult priorities, so they give L for cysteine even though it is (R).

Where amino acids come from in a flask

Chemists have three classical ways to make an α-amino acid in the laboratory. All three can make alanine, so this section uses alanine to compare them. Each route ends in strong acid or base, where alanine is the cation or the anion. Bringing the solution to about pH 6, alanine’s pI, turns it into the zwitterion, which is the form isolated.

Route 1: the Hell–Volhard–Zelinsky reaction, then ammonia. Br₂ with a little PBr₃ puts a bromine on the α carbon of a carboxylic acid, as in Alpha halogenation & the haloform reaction. Excess NH₃ then displaces the bromide by S_N2. The excess keeps the new amino group from reacting a second time. Starting from propanoic acid, the route gives alanine.

Route 1: bromine goes onto the α carbon, then ammonia replaces it.

Route 2: the Gabriel–malonic ester synthesis. The starting material is diethyl phthalimidomalonate. It is a malonic ester, as in Malonic and acetoacetic ester, whose central carbon also carries a phthalimide nitrogen. That nitrogen is protected as in the Gabriel synthesis (Making amines). The figure writes it PhthN.

NaOEt removes the one proton left on the central carbon, and the enolate attacks an alkyl halide, which supplies the side chain. With CH₃I, the side chain is a methyl. Hot aqueous acid then hydrolyzes both esters and the phthalimide, and one of the two carboxyl groups leaves as CO₂. What remains is alanine.

Route 2: the side chain arrives as an alkyl halide, and hot acid then strips off everything that is not alanine.

Route 3: the Strecker synthesis. An aldehyde and ammonia form an imine, as in Imines and enamines. Cyanide then adds to the imine carbon, just as it adds to a carbonyl carbon in Hydrates & cyanohydrins. The product is an α-amino nitrile. Hot aqueous acid hydrolyzes the C≡N group to a carboxyl group (Nitriles). From acetaldehyde, the route gives alanine. In practice, chemists use NH₄Cl and KCN as safer sources of NH₃ and HCN.

Route 3: the aldehyde and ammonia form an imine, cyanide adds to it, and hydrolysis turns the C≡N into the carboxyl.

All three routes give a racemate, a 50:50 mix of D and L. In each one the α carbon passes through a flat intermediate (the enol in route 1, the enolate and then the enol in route 2, the imine in route 3), and nothing chiral is nearby to favor one face. Biology’s amino acids are single enantiomers, so a laboratory route has to be followed by a resolution, the separation of the two enantiomers described in Enantiomers. For a manufacturer, that resolution step is a real added cost.

What carries forward

The acid–base reasoning here is the reasoning of Acids & Bases, applied to a molecule with two or three groups that can gain or lose a proton. The stereochemistry is the same as in Stereochemistry. What is new is mostly vocabulary, plus a habit: for any amino acid at any pH, ask which form is present. Peptides and proteins joins amino acids into chains.