Peptide structure and synthesis are the foundation of every other topic in peptide research. Before a researcher can interpret a binding study, an animal model experiment, or a certificate of analysis, they need a working understanding of how peptides are built at the chemical level and how they are made in the laboratory. This article is a focused look at peptide structure and synthesis, written as a companion to the broader comprehensive guide to peptides in research. For researchers who want a survey of how the major classes of research peptides are organized, see the parallel article on peptide classification.
Amino acids and the peptide bond
Every peptide is built from amino acids. Each amino acid has a central carbon, called the alpha carbon, that bears four groups: a hydrogen atom, an amino group, a carboxylic acid group, and a side chain that distinguishes one amino acid from another. The side chain is what gives each amino acid its chemical personality. Glycine has a hydrogen as its side chain and is the smallest. Tryptophan has an indole ring and is one of the largest. Lysine has a four carbon chain ending in a primary amine and carries a positive charge at neutral pH. Aspartate and glutamate have carboxylic acid side chains and carry negative charges. Cysteine has a thiol group that can form disulfide bridges. There are twenty standard proteinogenic amino acids and each one is encoded by the genetic code.
When two amino acids meet, the carboxylic acid group of the first reacts with the amino group of the second, releasing a water molecule and forming a covalent bond between the carbonyl carbon and the nitrogen. This new bond is called the peptide bond. The peptide bond is planar, meaning the six atoms involved in the bond and its immediate neighbors lie in a single plane. The planarity is the result of partial double bond character that comes from resonance between the carbonyl and the nitrogen. This restricted rotation around the peptide bond is one of the key facts that allows peptides and proteins to fold into defined three dimensional shapes.
Peptide bond chemistry is well studied and is documented in standard biochemistry references. The Nature subject hub on peptides and the ScienceDirect topic page for peptide host a large body of primary literature on peptide bond formation and the chemistry of amino acid side chains.
Primary, secondary, and higher order structure
Peptide structure is conventionally described at four levels, although for short peptides only the first two are usually relevant.
Primary structure is the linear sequence of amino acids from the amino terminus to the carboxyl terminus. The amino terminus, often abbreviated as N terminus, is the free amino group at one end of the chain. The carboxyl terminus, often abbreviated as C terminus, is the free carboxylic acid at the other end. By convention, sequences are written from N to C. A short hand notation uses single letter codes such as G for glycine, H for histidine, and K for lysine. The tripeptide GHK is therefore glycine, histidine, lysine, with glycine at the N terminus.
Secondary structure refers to local folding patterns. Alpha helices, beta strands, and various beta turns are the canonical secondary structure elements. They are stabilized by hydrogen bonds between backbone atoms. Short peptides often do not have stable secondary structure in dilute aqueous solution because they are too small to support cooperative folding, but some short peptides do show defined secondary structure in particular environments such as membrane interfaces or in the presence of certain co solvents. For longer peptides and small proteins, secondary structure becomes important and is the basis for structural classification.
Tertiary structure refers to the full three dimensional fold of a single peptide chain. Quaternary structure refers to assemblies of multiple chains. Both are more relevant to proteins than to short peptides, but they can matter for cyclic peptides and for peptides that fold into a defined active conformation.
The American Chemical Society publication portal hosts journals such as the Journal of the American Chemical Society and the Journal of Peptide Science that publish primary research papers on peptide structure determination by methods such as nuclear magnetic resonance spectroscopy, circular dichroism, and X ray crystallography.
Solid-phase peptide synthesis
Solid-phase peptide synthesis, abbreviated as SPPS, is the dominant method for making research peptides. It was developed by Bruce Merrifield in the 1960s and earned him the Nobel Prize in Chemistry in 1984. SPPS replaced earlier solution phase methods that were slow and difficult to scale, and it remains the workhorse of peptide chemistry today.
The basic idea of SPPS is to anchor the growing peptide chain to a solid polymer bead and to add amino acids one at a time from the C terminus to the N terminus. The polymer is insoluble in the reaction solvent, which means that excess reagents and byproducts can be washed away after each step simply by filtering the resin and rinsing it with fresh solvent. This eliminates the need for slow purification steps between coupling cycles and makes the synthesis efficient and amenable to automation.
A typical Fmoc SPPS cycle has four steps. First, the amino group of the resin bound peptide is deprotected by treatment with a base such as piperidine, which removes the Fmoc protecting group. Second, the next amino acid in the sequence, also Fmoc protected on its amino group and with reactive side chains protected by orthogonal groups, is activated with a coupling reagent and added to the resin. The activated amino acid couples to the free amino group on the resin bound peptide, extending the chain by one residue. Third, the resin is washed thoroughly to remove unreacted reagents and byproducts. Fourth, the cycle repeats until the full sequence is built.
For a fifteen residue peptide, the synthesis involves fifteen coupling cycles. For a forty residue peptide, the synthesis involves forty coupling cycles. Each cycle has a coupling efficiency that is typically above ninety nine percent for standard amino acids, but the small inefficiency at each step can compound for longer sequences. Modern SPPS protocols use optimized coupling reagents, double couplings for difficult residues, and microwave heating to push longer sequences through with high yield.
After the full sequence is built, the peptide is cleaved from the resin by treatment with a strong acid, usually trifluoroacetic acid in a cocktail with scavengers that protect sensitive side chains during cleavage. The acid cleaves the bond between the peptide and the resin and at the same time removes the side chain protecting groups. The result is a crude peptide in solution that contains the target sequence along with byproducts, truncated sequences, and the cleavage cocktail components.
For longer peptides where stepwise SPPS becomes inefficient, native chemical ligation and other fragment based strategies can be used to assemble the full sequence from smaller fragments that are each made by SPPS. These methods extend the reach of synthetic peptide chemistry to small proteins of one hundred residues or more.
Purification by reversed-phase HPLC
The crude peptide from SPPS is rarely pure enough for research use. The standard purification method is reversed-phase high-performance liquid chromatography, abbreviated as RP HPLC. In RP HPLC, the crude peptide is dissolved in a mostly aqueous solvent and loaded onto a column packed with silica beads coated with hydrophobic chains, most commonly C18 chains that are eighteen carbons long. The column is then eluted with a gradient that starts mostly aqueous and gradually adds an organic solvent such as acetonitrile, with a small amount of an acidic modifier such as trifluoroacetic acid added to both the aqueous and the organic mobile phases.
As the gradient runs, peptides elute from the column at different times depending on their hydrophobicity. More hydrophilic peptides elute early, more hydrophobic peptides elute late. The eluting peptides are detected by ultraviolet absorbance at a wavelength such as two hundred fourteen or two hundred eighty nanometers, and the chromatogram shows a series of peaks. The fraction containing the target peptide is collected, the solvents are removed by rotary evaporation or lyophilization, and the purified peptide is recovered as a dry powder.
RP HPLC is also the standard method for measuring the purity of the final product. The purified peptide is reanalyzed on an analytical RP HPLC column under standard conditions and the area under the main peak is integrated and compared to the total integrated area to give a percent purity. Research peptides are typically supplied at greater than ninety eight or ninety nine percent purity by HPLC. The chromatogram is included on the certificate of analysis along with the calculated purity. The Journal of Biological Chemistry publishes many primary papers on peptide purification methods and on the analytical chemistry of peptide characterization.
Characterization by mass spectrometry
A peptide that elutes as a single peak by HPLC is not necessarily the right peptide. Two peptides with similar hydrophobicity can co elute, and a single peak can sometimes contain a mixture of closely related sequences. The standard identity check for a research peptide is mass spectrometry.
Electrospray ionization mass spectrometry, abbreviated as ESI MS, is the most common method. The peptide is dissolved in a solvent that promotes ionization, sprayed through a charged needle into a mass analyzer, and detected as charged ions. The mass to charge ratio of the ions is measured and the molecular weight of the peptide is calculated. The observed mass is compared to the theoretical mass calculated from the sequence, and a match within a few parts per million for high resolution instruments confirms that the peptide has the expected molecular weight.
For longer peptides, fragment ion analysis by tandem mass spectrometry, abbreviated as MS MS, can be used to confirm the actual sequence. In MS MS, the intact peptide ion is selected in a first stage, fragmented by collision with an inert gas, and the resulting fragment ions are analyzed in a second stage. The fragment ion pattern reveals the sequence of amino acids in the peptide and can distinguish between sequences that have the same molecular weight but different orderings.
Other characterization techniques include amino acid analysis, which hydrolyzes the peptide and quantifies each amino acid; nuclear magnetic resonance spectroscopy, which can confirm structural features; and Edman degradation, an older method that sequentially removes and identifies amino acids from the N terminus. Most research peptide COAs include at least the HPLC purity and the ESI MS molecular weight confirmation.
