Acetyl Hexapeptide-8 and Acetyl Octapeptide-3: 186 Daltons Apart
Acetyl hexapeptide-8 and acetyl octapeptide-3 are built on the same idea and share six of their residues. The second is the first with two more amino acids on the end, and the 186 dalton difference between them is the cleanest way to tell them apart.
The two sequences
Acetyl hexapeptide-8, widely sold under the trade name Argireline, is Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2. Acetyl octapeptide-3, sold as SNAP-8, is Ac-Glu-Glu-Met-Gln-Arg-Arg-Ala-Asp-NH2.
Both are acetylated at the N-terminus and amidated at the C-terminus, so neither has a free terminal charge. Both are fragments modelled on the N-terminal region of the protein SNAP-25, which is where the SNAP-8 name comes from.
Formulas, masses and registry numbers
- Acetyl hexapeptide-8 — C34H60N14O12S, monoisotopic 888.42 Da, average 889.0 Da, CAS 616204-22-9.
- Acetyl octapeptide-3 — C40H68N16O15S, monoisotopic 1074.49 Da, average 1075.2 Da, CAS 868844-74-0.
The gap is 186.06 daltons, which is exactly the sum of an alanine residue at 71.04 and an aspartate residue at 115.03. That arithmetic is worth doing once, because it means any spectrum showing both masses is showing a mixture of the two, not an impurity of one.
The terminal modifications are load-bearing
Neither compound is the free peptide. Remove the acetyl group and the mass drops 42 daltons; remove the amide and it rises one. Both are common outcomes of an incomplete synthesis step, and both produce species that a routine method may not separate from the parent.
A certificate that gives a sequence without stating the terminal modifications has described a different molecule from the one in the vial. The three-letter sequence, the acetyl prefix and the amide suffix are all part of the identity.
Methionine is the weak point
Both sequences carry a methionine at position three, and methionine oxidises readily to the sulfoxide under ambient conditions, in solution and even in the solid state given enough moisture and oxygen. The sulfoxide is sixteen daltons heavier and elutes earlier on a reversed-phase column because the added oxygen increases polarity.
A second oxidation to the sulfone adds another sixteen. In practice the sulfoxide is what appears, and a small plus-sixteen peak on a chromatogram of either compound is the expected finding rather than a surprising one. What matters is its size and whether it grows on storage.
Both are difficult to retain
Count the charged residues: two glutamates and two arginines in the hexapeptide, with an aspartate added in the octapeptide. Under acidic mobile-phase conditions the arginines are protonated and the carboxylates are not, giving a net positive charge and a very polar molecule with almost no hydrophobic surface.
The consequence is weak retention on conventional C18 packings. Both compounds elute early, sometimes close enough to the void that small polar impurities co-elute with them. Methods for these peptides generally start at very low organic content, use an aqueous-stable bonded phase, and rely on ion-pairing to generate retention.
Detection
Neither sequence contains tryptophan, tyrosine or phenylalanine, so there is no useful absorbance at 280 nm. Detection is at 214 nm, with the usual trade-off that the baseline is sensitive to mobile-phase quality.
The methionine sulfur does not contribute meaningful ultraviolet absorbance, so it offers no help here. Where a laboratory wants a second, orthogonal signal, mass spectrometric detection rather than a second wavelength is what provides it.
Naming, which is where most confusion starts
These compounds carry an unusual number of names each. Acetyl hexapeptide-8 appears as acetyl hexapeptide-3, as Argireline, and occasionally just as hexapeptide-8. Acetyl octapeptide-3 appears as SNAP-8 and as acetyl octapeptide-1. The numbering conventions come from cosmetic ingredient nomenclature rather than from chemistry, and they have been revised, which is why two numbers can refer to the same molecule.
The sequence and the mass are stable across all of that. A document giving both identifies the compound regardless of which trade name heads the page.
What confirms a lot
An accurate mass at 888.42 or 1074.49 daltons for the neutral species, with the acetyl and amide terminal states written out; a 214 nm chromatogram from a method that retains the peptide rather than eluting it in the void; and a stated figure for the oxidised species, because on a methionine-containing peptide that number is the one most likely to move between manufacture and analysis.
Related reading
Metal coordination and terminal modification both change what a mass measurement reports, and the copper tripeptide is the clearest illustration of the first. See GHK-Cu and copper complex coordination, and terminal modifications, acetylation and amidation for the second.
