What Is SS-31? The Aromatic-Cationic Tetrapeptide

SS-31 is a synthetic tetrapeptide with the sequence D-Arg-Dmt-Lys-Phe-NH2: D-arginine, 2′,6′-dimethyl-L-tyrosine (Dmt), L-lysine and L-phenylalanine, finished with a C-terminal amide. It is called an aromatic-cationic peptide because it alternates two positively charged residues with two aromatic ones. Its molecular formula is C32H49N9O5, its average molecular weight is 639.8 g/mol and its CAS number is 736992-21-5.

Three features set SS-31 apart from a typical short peptide: a D-amino acid, an amino acid that does not occur in proteins and an amidated C-terminus. This page covers each feature as a matter of structure, then works through the formula, the mass, the charge and what they mean for analysis.

The sequence, residue by residue

Written in full, with both termini shown, the peptide is H-D-Arg-Dmt-Lys-Phe-NH2. The "H-" is the free amine at the N-terminus and the "-NH2" is the C-terminal amide. PubChem gives the systematic name D-arginyl-2,6-dimethyl-L-tyrosyl-L-lysyl-L-phenylalaninamide, which names the same four residues in order. The conventions behind the three-letter notation are covered in peptide sequence notation.

The pattern is visible straight away: basic, aromatic, basic, aromatic. That alternation is what the description "aromatic-cationic" refers to.

Three structural features

A D-amino acid at position 1

Every standard amino acid except glycine has a stereocentre at its α-carbon, so it exists as two mirror-image forms, L and D. The amino acids encoded in proteins are the L forms. SS-31 opens with D-arginine, the mirror image of the usual residue at that one carbon.

In three-letter notation a D-residue is always marked with its prefix, while a residue written without one is L by convention, following the IUPAC-IUB recommendations for peptide symbols. The same fact appears in PubChem's IUPAC name for SS-31: the arginine carbon is labelled (2R) and the other three α-carbons (2S). For arginine, L corresponds to S and D to R, but the two systems are not interchangeable in general. The D/L label compares a molecule with glyceraldehyde, whereas R/S follows priority rules for the atoms attached to the stereocentre, and for cysteine, L is R.

The practical consequence is an isomer that cannot be told apart by mass. The all-L peptide, L-Arg-Dmt-Lys-Phe-NH2, has exactly the same formula and mass as SS-31. It is a diastereomer rather than a mirror image, so it has different physical properties and can be separated chromatographically, but a mass spectrum alone cannot distinguish the two.

2′,6′-Dimethyltyrosine (Dmt)

Dmt is tyrosine with two extra methyl groups on its aromatic ring. PubChem lists it as (2S)-2-amino-3-(4-hydroxy-2,6-dimethylphenyl)propanoic acid, C11H15NO3, 209.24 g/mol. The ring positions are numbered from the carbon that joins the ring to the side chain, so the hydroxyl group is at position 4, as in tyrosine, and the two methyl groups flank the ring's attachment point at positions 2 and 6. Primes (2′,6′) are often added to show that these numbers refer to the ring rather than the amino-acid chain. Compared with tyrosine, C9H11NO3 (181.19 g/mol), Dmt carries two more CH2 units, 28.05 g/mol in all.

Dmt is non-proteinogenic: no codon encodes it, so peptides containing it are made by chemical synthesis from a protected Dmt building block. The two methyl groups sit on either side of the bond that joins the ring to the β-carbon of the side chain, which makes this part of the side chain bulkier than it is in tyrosine.

A C-terminal amide

In most peptides the last residue ends in a carboxylic acid, –COOH, which is negatively charged near neutral pH. In SS-31 that group is an amide, –CONH2, written "-NH2" at the end of the sequence. The amide carries no charge, so the C-terminus contributes nothing to the peptide's negative charge. With three basic groups and no acidic one, SS-31 carries a high net charge for its size.

Formula, mass and charge

The formula can be checked by simple addition. The four residue formulas sum to C32H46N8O5. The free N-terminal hydrogen and the C-terminal NH2 together add NH3, giving C32H49N9O5.

Near pH 7, three groups carry a positive charge: the N-terminal amine, the guanidinium group of D-arginine and the side-chain amine of lysine. No group carries a negative charge, because the C-terminus is an amide and the phenol of Dmt stays protonated at neutral pH. The net charge is therefore +3, spread over a molecule of fewer than 50 heavy atoms.

That charge also affects how the material is supplied. A strongly basic peptide is isolated as a salt, with counter-ions such as acetate or trifluoroacetate paired with its positive groups. The counter-ions add to the weight of the powder without adding peptide. The table below shows the arithmetic, assuming exactly three counter-ions and no water; real material is described by its certificate.

This is why net peptide content can sit well below the gross weight of a lyophilised solid, a point covered in more depth in peptide purity vs net peptide content.

How SS-31 is analysed

The fragment ions in the table follow the standard b/y naming for peptide fragments. b ions contain the N-terminus, y ions the C-terminus. Because SS-31 ends in an amide, each y ion carries NH2 rather than OH, so the y series sits 0.984 Da lower than it would for the corresponding free acid.

Chiral analysis usually starts with acid hydrolysis of the peptide to its free amino acids, followed by derivatisation with a chiral reagent such as Marfey's reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide). Each D- or L-amino acid gives a different diastereomeric derivative, and reversed-phase HPLC separates them. Dmt is not part of the standard amino acid mixtures used as references, so a separate Dmt standard is needed if it is to be quantified.

Two other by-products are worth looking for. The first is the C-terminal free acid, D-Arg-Dmt-Lys-Phe-OH, which is 0.984 Da heavier than SS-31 and easy to miss at low resolution. The second is a set of deletion sequences from synthesis: a peptide missing lysine sits 128.095 Da below the parent mass, one missing Dmt 191.095 Da below and one missing phenylalanine 147.068 Da below. More detail on each method is in the guides to HPLC peptide purity and mass spectrometry for peptide identity.

Certificates, specification and storage

For our SS-31, the purity specification is ≥99% (HPLC, supplier specification), and certificate status is shown on every product page. When reading any certificate for SS-31, check that the sequence is written with the D-prefix on arginine and with Dmt at position 2, that the observed mass matches the values above, that the salt form or counter-ion content is stated, that the HPLC method gives its detection wavelength, and that the batch number and analysis date match the vial.

SS-31 is supplied as a lyophilised solid in a sealed vial. 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 and avoid repeated freeze-thaw cycles.

SS-31 belongs to the Mitochondrial Compounds research area. It is catalogued there with two compounds of different design: MOTS-c, a linear sixteen-residue peptide of standard L-amino acids, and NAD+, a dinucleotide that is not a peptide at all. Chemical terms used on this page are collected in the glossary.