What Are Peptides? A Chemistry Guide

A peptide is a chain of two or more amino acids joined by amide bonds, called peptide bonds, each formed between the carboxyl group of one amino acid and the amino group of the next with the loss of one molecule of water. Chains of up to about 10–20 residues are often called oligopeptides and longer ones polypeptides; a defined chain of more than about 50 residues is normally called a protein, although chemists draw that line in different places.

This guide covers what peptides are built from, how the chain is described and weighed, and how a synthetic research peptide is made, specified and characterised.

Amino acids: the building blocks

An α-amino acid carries an amino group (–NH2) and a carboxylic acid group (–COOH) on the same carbon atom, the α-carbon. That carbon also holds a hydrogen atom and a side chain, written R, and the side chain is the only part that differs from one amino acid to the next. Twenty common amino acids have standard three-letter and one-letter symbols, from glycine (Gly, G), whose side chain is a single hydrogen atom, to tryptophan (Trp, W), which carries an indole ring.

Every common amino acid except glycine has a stereocentre at the α-carbon. The L form is the default, so the symbol Ala means L-alanine unless a D- prefix says otherwise. Synthetic peptides often contain residues outside the common twenty: D-amino acids, norleucine (Nle), α-aminoisobutyric acid (Aib) or 2,6-dimethyltyrosine (Dmt). The IUPAC-IUB recommendations ask for every such symbol to be defined in each document that uses it.

The peptide bond

When the carboxyl group of one amino acid condenses with the amino group of another, the two form an amide, –CO–NH–, and release one molecule of water. What remains of each amino acid in the chain is called a residue. Glycine (75.067 g/mol) and alanine (89.094 g/mol) give the dipeptide glycylalanine, written Gly-Ala, at 146.146 g/mol: the sum of the two, less 18.015 g/mol for the water released.

The same arithmetic works at any length. The molecular formula of a linear peptide is the sum of its residue formulas plus one H2O for the two free ends, which is why the fifteen residues of BPC-157 add up to exactly C62H98N16O22.

The repeating backbone unit is –NH–CH(R)–CO–. The carbon–nitrogen bond of each amide has partial double-bond character, so the atoms of each peptide unit lie close to one plane and rotation about that bond is restricted. Every linear chain has a free amino end, the N-terminus, and a free carboxyl end, the C-terminus. By convention the N-terminus is written on the left, as explained in how to read peptide sequence notation.

Peptides, polypeptides and proteins

The IUPAC-IUB definitions rest on length and are deliberately approximate:

The catalogue spans most of that range:

The order of residues, together with any covalent modification such as an acetyl group, an amide or a ring-forming bond, is the primary structure. Secondary structure describes local folding, such as α-helices and β-sheets held by hydrogen bonds between backbone C=O and N–H groups, and tertiary structure the overall three-dimensional fold. Quality documents for a synthetic peptide deal with the primary structure: whether the intended chain is present, and how much of the material it accounts for.

How synthetic research peptides are made

Most synthetic peptides are made by solid-phase peptide synthesis (SPPS), introduced by R. Bruce Merrifield in 1963 and recognised by the 1984 Nobel Prize in Chemistry. The chain is assembled on insoluble resin beads from the C-terminal residue towards the N-terminus, one protected amino acid at a time. Excess reagents and by-products are washed away after every step, and the finished chain is cleaved from the resin with trifluoroacetic acid (TFA). Fmoc chemistry, which removes each temporary protecting group with a mild base, is now the standard version of the method.

The crude product contains the target alongside shorter and modified chains, so it is purified by preparative reversed-phase HPLC, and the pooled fractions are freeze-dried to a dry powder. Very long chains are often produced by recombinant expression instead; IGF-1 LR3, at 83 residues, is listed in the catalogue as a recombinant analogue. Each stage is covered in solid-phase peptide synthesis explained.

How a synthetic peptide is specified

A peptide specification describes the substance in the vial, not only the chain drawn on paper:

The counter-ion is easy to overlook. TFA is used both to cleave the peptide from the resin and as an additive in the purification mobile phase, so a peptide with basic groups is normally isolated as its trifluoroacetate salt. The counter-ion can be exchanged for acetate by changing the mobile-phase additive, or for chloride by repeated dissolution in hydrochloric acid and freeze-drying.

Counter-ions and bound water add mass without adding peptide, so a weighed quantity of powder contains less peptide chain than its gross mass. That fraction is the net peptide content, explained in peptide purity vs net peptide content.

How peptides are characterised

HPLC for purity

Reversed-phase HPLC separates a peptide from closely related impurities by hydrophobicity. The column is packed with a non-polar stationary phase, typically C18- or C8-bonded silica, and the peptide is eluted with a gradient from water to acetonitrile, both containing a little TFA as an ion-pairing agent. Peptide bonds absorb strongly in the far ultraviolet, so detection is usually at 210–220 nm. Purity is reported as area percent: the area of the main peak divided by the total area of all integrated peaks. The method and its limits are covered in HPLC and peptide purity.

Mass spectrometry for identity

Mass spectrometry measures the mass-to-charge ratio of ions. Electrospray ionisation produces a series of multiply charged ions from one molecule, while MALDI mostly gives singly charged ions. Identity is supported when the observed mass matches the mass calculated from the sequence and its modifications. Which calculated mass to use matters: BPC-157 has an average molecular weight of 1419.53 g/mol, which is weighted by the natural abundance of every isotope, but a monoisotopic mass of 1418.70 Da, calculated from the most abundant isotope of each element (¹²C, ¹H, ¹⁴N, ¹⁶O). The monoisotopic figure is the one to compare with a high-resolution spectrum. See mass spectrometry and peptide identity.

Other measurements

Amino acid analysis hydrolyses the chain and quantifies each amino acid, confirming composition and supporting a net peptide content figure. Karl Fischer titration measures water, and ion chromatography or capillary electrophoresis measures the counter-ion. On this site the certificate status is shown on every product page, and certificates of analysis are published in the COA Library where available. Terms used here are defined in the glossary.