What Is MT2? Structure and Chemistry

MT2, also known as Melanotan II (Melanotan 2, MT-II), is a synthetic cyclic heptapeptide with the sequence Ac-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-NH2. A lactam bond between the side chains of aspartic acid and lysine closes a ring of six residues, leaving N-acetyl-norleucine outside the ring at the N-terminus. The ring holds one D-configured residue, D-phenylalanine, and the C-terminal lysine ends in an amide. Its molecular formula is C50H69N15O9, its average molecular weight is 1024.18 g/mol and its CAS number is 121062-08-6.

This page covers MT2 as a molecule: the sequence and its notation, the ring and the amide terminus, the non-standard residues, the formula, mass and charge, how it is assembled and how its identity and purity are confirmed by HPLC and mass spectrometry.

Reading the MT2 sequence

Every part of the notation carries structure. "Ac-" is an acetyl group, CH3CO–, capping the N-terminal amine. "Nle" is norleucine, a non-proteinogenic amino acid. "cyclo[…]" encloses the residues that form the ring, and the bond that closes it runs between the side chains of the first and last residues inside the brackets. "D-Phe" marks the one D-residue. The closing "-NH2" is the C-terminal amide. In compact one-letter form the same molecule is written Ac-[Nle]DHfRWK-NH2, with a lower-case f for D-phenylalanine and the lactam noted separately. The conventions are covered in peptide sequence notation.

The lactam bridge

A lactam is a cyclic amide. In MT2 it joins the β-carboxyl group of aspartic acid to the ε-amino group of lysine. Following the ring from the aspartic acid α-carbon along the backbone through histidine, D-phenylalanine, arginine and tryptophan to the lysine α-carbon, then back through the lysine and aspartic acid side chains, gives a 23-membered macrocycle. The bridge holds the central His-D-Phe-Arg-Trp sequence, a four-residue motif shared across the melanocortin-ligand peptide class, in a constrained loop.

Closing the ring releases one molecule of water. The linear precursor, Ac-Nle-Asp-His-D-Phe-Arg-Trp-Lys-NH2, is C50H71N15O10 with a monoisotopic mass of 1041.5508 Da; the cyclic peptide is 1023.5403 Da, 18.0106 Da lighter. The cyclised and uncyclised forms are therefore easy to tell apart by mass.

The C-terminal amide

Most peptides end in a carboxylic acid. MT2 ends in a carboxamide, –CONH2, on the lysine α-carbon. The amide group carries no charge, so once the aspartic acid side chain is tied up in the lactam MT2 has no acidic group anywhere in the molecule.

The amide is also a precise mass marker. Swapping –CONH2 for –COOH replaces an NH with an O, a gain of 0.9840 Da. Because 0.984 Da is close to the spacing of the natural carbon-13 isotope peaks, confirming the amide terminus needs adequate mass resolution together with HPLC retention.

Norleucine and D-phenylalanine

Norleucine, 2-aminohexanoic acid, has a straight four-carbon side chain. It shares its formula and residue mass, 113.0841 Da, with leucine and isoleucine, so mass spectrometry alone cannot distinguish them, and identity at position 1 rests on chromatography and the synthesis record. As an unbranched hydrocarbon side chain, it has nothing to oxidise.

D-Phenylalanine is the mirror-image form of phenylalanine at its α-carbon. MT2 has seven stereocentres, one per residue, six L and one D. A peptide with L-Phe at position 4 is a diastereomer of MT2, with exactly the same formula and mass, and only chromatography separates the two.

Formula, molecular weight and charge

Near pH 7 the arginine guanidinium group is positively charged and the histidine imidazole is partly protonated, while there is no negative group at all. The net charge is about +1.2. At the acidic pH of reversed-phase HPLC both arginine and histidine are protonated, for a charge of +2, and the doubly charged ion at m/z 512.777 is typically the strongest signal in positive-ion electrospray.

That basic character shapes the solid as supplied. A basic peptide is isolated from purification as a salt, its positive groups paired with counter-ions such as acetate or trifluoroacetate, which add weight to the powder without adding peptide. Peptide purity vs net peptide content explains the arithmetic.

Why the ring matters for analysis

The lactam bridge does more than close a loop. A cyclic peptide has fewer free ends and a more compact shape than a linear chain of the same composition, so it usually elutes as a sharper, better-defined peak in reversed-phase HPLC. The linear precursor and the cyclic product differ in both mass and retention, which gives the analysis two independent ways to confirm that cyclisation is complete.

The ring also fixes which atoms are bonded to which. Because the bridge runs between the aspartic acid and lysine side chains, an alternative ring closure through the backbone ends would give a different molecule, a head-to-tail cycle, with its own retention time. The sequence notation, with the cyclo brackets placed around residues 2 to 7, records the intended connectivity precisely.

How a cyclic peptide like MT2 is assembled

A side-chain lactam is usually built on the resin during solid-phase peptide synthesis. The aspartic acid and lysine are introduced with side-chain protecting groups that can be removed selectively while every other protecting group stays in place. Once the linear chain is complete, those two groups are removed, the free carboxyl and amine are coupled to each other, and only then is the peptide cleaved from the resin with its remaining protecting groups.

Each step leaves a characteristic trace if it is incomplete. Material that never cyclised shows up 18 Da heavier than MT2. A dimer, formed when two chains link to each other instead of to themselves, appears near twice the mass. A missing residue lowers the mass by that residue's value. These are the related substances that HPLC and LC-MS are set up to resolve.

How MT2 is analysed

Cyclic peptides fragment in a distinctive way in MS/MS. One backbone cleavage inside the ring opens it without changing the mass, and a second cleavage is needed before any fragment separates, so the ring gives fewer and more complex sequence ions than a linear chain. The exocyclic Ac-Nle unit at the N-terminus fragments in the usual way. Mass spectrometry and peptide identity explains the fragment naming.

Certificates, specification and storage

Our MT2 is supplied at ≥99% purity (HPLC) as a lyophilised powder in a sealed 10 mg vial. When reading a certificate for MT2, check that the sequence is given as the cyclic, N-acetylated amide, that the observed mass matches 1023.5 Da or the ions above, that the HPLC method states its detection wavelength, that the salt form is stated, and that the report carries a reference and an analysis date. How to read a peptide certificate of analysis walks through each line of a report.

Store the sealed vial at 2–8 °C for short-term storage, or at −20 °C and below for long-term storage. Protect it from light, which matters for a tryptophan-containing peptide, and avoid repeated freeze-thaw cycles. Storing lyophilised peptides explains why.

MT2 belongs to the Melanocortin & Copper Peptides research area, together with PT-141 and the copper(II) complex GHK-Cu. Terms used on this page, such as lactam, diastereomer and counter-ion, are defined in the glossary.