Peptides and amino acids

Every research peptide is built from a small set of amino acids, and most of its chemistry (mass, charge, solubility, UV absorbance, oxidation sensitivity) can be read from the side chains in its sequence. This article covers the structure of an amino acid, what changes when it becomes a residue, and how side chains set the properties of the finished peptide. NuVion Health supplies synthetic research peptides as laboratory chemicals for in vitro research use only.

The structure of an amino acid

An alpha-amino acid has a central carbon bonded to an amino group, a carboxyl group, a hydrogen atom and a side chain, R. The twenty proteinogenic amino acids differ only in R, from a hydrogen in glycine (75.07 Da) to the indole of tryptophan (204.23 Da). In water at neutral pH the free amino acid is a zwitterion: the carboxyl group (pKa near 2) is deprotonated and the amino group (pKa near 9.5) is protonated. All except glycine have a chiral alpha-carbon and occur in ribosomal peptides as the L-enantiomer. A D-residue has the same mass and formula as its L counterpart, so it is invisible to mass spectrometry.

Side chain classes and what each contributes

• Aliphatic hydrophobic residues: glycine, alanine, valine, leucine, isoleucine, proline and methionine. They drive self-association in water and reversed-phase retention. Methionine oxidises to the sulfoxide, seen as a +16 Da peak.
• Aromatic residues: phenylalanine, tyrosine and tryptophan. Tyrosine and tryptophan absorb at 280 nm, with molar extinction coefficients of about 1,490 and 5,500 per M per cm, the basis of A280 concentration measurement. A peptide with neither residue is quantified at 214 nm instead.
• Polar uncharged residues: serine, threonine, asparagine, glutamine and cysteine. Asparagine deamidates to aspartate, especially before glycine, and the cysteine thiol pairs into a disulfide, which closes oxytocin between Cys1 and Cys6.
• Acidic residues: aspartate and glutamate, with side-chain pKa values near 4, so both carry a negative charge at neutral pH. Asp-Gly motifs form aspartimide during synthesis.
• Basic residues: lysine, arginine and histidine. The lysine epsilon-amine (pKa about 10.5) and the arginine guanidinium (pKa about 12.5) are positive at any working pH; the histidine imidazole (pKa about 6) is partly protonated at pH 7. The lysine amine is where lipids are attached, as in tirzepatide, and the histidine imidazole is a metal ligand, as in GHK-Cu.

From free amino acid to residue

When two amino acids condense, the carboxyl of one and the amino group of the other become an amide and a water molecule leaves. Each unit is now a residue, and the difference matters for two calculations. Mass first: a residue weighs 18.02 Da less than the free amino acid, so the molecular weight of a peptide is the sum of its residue masses plus one water. Thymogen, the dipeptide Glu-Trp, is 147.13 plus 204.23 minus 18.02, or 333.34 Da; KPV, Lys-Pro-Val, is 146.19 plus 115.13 plus 117.15 minus two waters, or 342.43 Da. A C-terminal amide subtracts 0.98 Da; an N-terminal acetyl adds 42.01 Da.

Then charge. The backbone amide is neither acidic nor basic in the working pH range, so once the alpha-amino and alpha-carboxyl groups are tied up in peptide bonds they contribute nothing. A residue in the middle of a chain is neutral unless its side chain is aspartate, glutamate, lysine, arginine or histidine, so the net charge and isoelectric point of a peptide are estimated by counting those residues and the two termini.

Reading a sequence

Given a sequence, most bench behaviour can be predicted. The counts of lysine, arginine, aspartate and glutamate give the net charge and pI, and so the solvent likely to work. The fraction of hydrophobic residues predicts water solubility and RP-HPLC retention. Methionine, cysteine or tryptophan flag oxidation sensitivity, asparagine flags deamidation and Asp-Pro flags an acid-labile bond. Lysine and arginine mark trypsin cleavage sites and aromatic residues mark chymotrypsin sites, which sets how long a peptide survives in serum-containing medium. The reconstitution calculator turns label mass and molecular weight into a working concentration.

Residues beyond the standard twenty

Solid-phase synthesis accepts any amino acid available as an Fmoc-protected building block, so synthetic peptides often contain residues the ribosome never uses. D-amino acids replace the L form where protease resistance or a set conformation is wanted: Melanotan 1 carries D-phenylalanine at position 7 and GHRP-6 carries D-tryptophan and D-phenylalanine. Norleucine, an unbranched isomer of leucine, replaces methionine at position 4 of Melanotan 1 to remove an oxidation site. 2-Aminoisobutyric acid (Aib), with two methyl groups on the alpha-carbon, favours helical conformation and blocks cleavage; tirzepatide carries it at positions 2 and 20. Each non-standard residue adds its own mass to the formula an identity test checks.

Characterisation and supply

Mass spectrometry confirms that a synthetic peptide has the intended formula, and analytical RP-HPLC reports how much of the sample is the main peak. NuVion Health has its peptides tested independently by Janoshik Analytical; most batches are tested and the Certificate of Analysis for a tested batch is published on the product page and in the Certificates of Analysis library. Manufacture is GMP-audited, and the peptides are supplied lyophilised in sealed vials and dispatched from within Australia.

Frequently asked questions

What is the difference between an amino acid and an amino acid residue?

An amino acid is the free molecule with its own amino and carboxyl groups. A residue is what remains inside a chain after condensation, lighter by one water and with its backbone groups tied up in amide bonds.

How is the molecular weight of a peptide calculated from its sequence?

Add the residue masses (each free amino acid mass minus 18.02 Da), add 18.02 Da for the terminal water, and adjust for modifications such as a C-terminal amide or an N-terminal acetyl group.

Why do some synthetic peptides contain D-amino acids?

Proteases recognise L-residues, so a D-residue at or near a cleavage site slows hydrolysis in culture medium or serum. It can also fix a backbone conformation the L form would not adopt.

Are research peptides therapeutic goods in Australia?

No. NuVion Health supplies them as laboratory chemicals for in vitro research use only, and they are not sold or presented as therapeutic goods. The notice below sets out their regulatory position.

Research use only. This product is a laboratory chemical supplied for in vitro research. It is not included in the Australian Register of Therapeutic Goods and has not been assessed by the Therapeutic Goods Administration for quality, safety or efficacy. It is not for human or veterinary use, and nothing on this page is a representation about therapeutic use.