Functional groups in peptides: amines, amides, carboxylic acids and thiols

A functional group is a small cluster of atoms, such as –NH₂ or –COOH, that reacts the same way in whatever molecule it appears. A peptide's sequence is a list of amino acids, and its chemistry is a list of functional groups. Knowing the groups explains why a peptide carries a charge, why it dissolves in one solvent and not another, and which parts of it change over time. This guide names every functional group found in the standard amino acids and says what each one does.

Terms are defined in the glossary.

What is a functional group?

Most of a peptide is carbon and hydrogen, which are fairly unreactive. The interesting chemistry happens at groups that contain oxygen, nitrogen or sulfur. These groups are called functional groups.

Each has a name and a typical behaviour:

• Charge. Some groups give up or take on a proton (H⁺) depending on pH, and so become charged.
• Hydrogen bonding. Groups with an O–H or N–H can form hydrogen bonds with water and with each other.
• Reactivity. Some groups oxidise, hydrolyse or react with other molecules more readily than others.

Chemists recognise a group in an unfamiliar molecule and can predict a lot about it straight away. For the bonds that hold these groups together, see chemical bonds and polarity.

Functional groups in a peptide at a glance

The pKa values that decide each charge are listed in the 20 amino acids.

Amines

An amine is a nitrogen bonded to one or more carbons, with the rest of its bonds to hydrogen. A primary amine is –NH₂.

Amines are bases: the nitrogen picks up a proton to become –NH₃⁺. In peptides there are two kinds:

• The N-terminal amine, with a pKa of about 8.6 (EMBOSS value). It is mostly positive at neutral pH.
• The lysine side chain, with a pKa of about 10.8. It is positive across almost the whole working pH range.

Amines are what make many peptides basic and soluble in dilute acid. See peptide solubility. An N-terminal amine can be capped with an acetyl group, which removes its charge; see terminal modifications.

Proline's amine is part of a ring, so it is a secondary amine (–NH– between two carbons).

Amides

An amide is a carbonyl group (C=O) joined to a nitrogen. The peptide bond that links each amino acid to the next is an amide, so a peptide or polypeptide is, chemically, a chain of amides.

Amides behave very differently from amines, even though both contain nitrogen:

• They aren't basic. The nitrogen's lone pair is shared with the C=O next to it, so it doesn't pick up a proton. Amides stay neutral at any normal pH.
• They are flat and rigid. That sharing gives the C–N bond partial double-bond character, so the atoms around it lie in a plane. This shapes how peptide chains fold.
• They are stable. Breaking an amide bond with water needs strong acid or base and heat. Labs that want to break every peptide bond deliberately use 6 M hydrochloric acid at 105–110 °C for 16–24 hours (Thermo Fisher). See hydrolysis.

Asparagine and glutamine carry amides in their side chains. Over time these can lose ammonia and turn into carboxylic acids, a change called deamidation. See reading impurity peaks.

A C-terminal amide replaces the final –COOH with –CONH₂. It removes the negative charge at that end and is common in research peptides. It is shown as "-NH₂" at the end of a sequence.

Amine vs amide: what is the difference?

An amine has a nitrogen bonded only to carbon and hydrogen atoms, while an amide has its nitrogen bonded to a carbonyl carbon (C=O). That one neighbouring C=O changes how the nitrogen behaves:

In a peptide, the N-terminus and the lysine side chain are amines, and every peptide bond is an amide. The C=O and N–H of the amide also give a strong infrared band, the amide I band; see the IR spectrum table.

Carboxylic acids

A carboxylic acid is –COOH: a carbon double-bonded to one oxygen and single-bonded to an –OH.

Carboxylic acids are acids: they give up a proton to become carboxylate, –COO⁻. In peptides:

• The C-terminal carboxylic acid has a pKa of about 3.6.
• Aspartic acid and glutamic acid side chains have pKa values of about 3.9 and 4.1.

At neutral pH all of these are negative. Peptides with several of them are acidic and dissolve best in slightly basic solutions. Acetic acid, the common lab solvent, is a simple carboxylic acid; see acetic acid as a peptide solvent.

Thiols

A thiol is –SH, the sulfur version of an alcohol. Cysteine is the only standard amino acid with one.

Thiols are the most reactive groups in a peptide:

• They oxidise. Two thiols can join as a disulfide bond (–S–S–), either within one chain or between two. Air is enough to do this slowly, faster at neutral or basic pH.
• They are weakly acidic. With a pKa of about 8.5, cysteine's thiol is mostly uncharged at neutral pH but increasingly charged above it.
• They bind metals. Thiols hold metal ions such as copper and zinc tightly.

See oxidation and reduction for how disulfides form and are broken, and the 20 amino acids for more on cysteine.

Other groups in the side chains

• Hydroxyl (–OH). Serine and threonine carry an alcohol group. It makes the side chain polar and able to hydrogen-bond with water.
• Phenol. Tyrosine's –OH sits on a benzene ring. The ring absorbs UV light near 280 nm, which is used to measure concentration.
• Thioether (–S–). Methionine's sulfur sits between two carbons. It doesn't form disulfides, but it oxidises easily to a sulfoxide, adding 16 Da.
• Guanidinium. Arginine's side chain is strongly basic, with a pKa of about 12.5, so it is positive at almost any pH.
• Imidazole. Histidine's ring has a pKa of about 6.5, so it switches between charged and neutral close to neutral pH.
• Indole. Tryptophan's two-ring system is large and hydrophobic, absorbs strongly at 280 nm, and is sensitive to light and oxidation.
• Non-polar chains. Alanine, valine, leucine, isoleucine and phenylalanine carry plain hydrocarbon chains or rings. They have no reactive group, but they make a peptide hydrophobic.

Esters

An ester is a carbonyl group joined to an oxygen that links to another carbon (–C(=O)O–). It looks like an amide with the nitrogen swapped for oxygen.

None of the 20 standard amino acids carries an ester, but esters appear around peptide chemistry:

• In synthesis. Many resins hold the growing chain by an ester bond, and some side-chain protecting groups, such as tert-butyl esters on aspartic and glutamic acid, are esters. See solid-phase peptide synthesis.
• In depsipeptides, where one or more amide bonds in the backbone are replaced by ester bonds.

Esters hydrolyse much more easily than amides, in mild acid or base. That is why they are useful as linkers and protecting groups that need to come off cleanly.

Why the groups matter

Related guides

For how amino acids are joined, see what is a peptide. For the side chains in detail, see the 20 amino acids. Each product page lists the full sequence and modifications for that compound.