Masses calculated from the elemental formula; the free dipeptide Asp-Phe can be entered into the MW calculator as DF.
Discovered by accident
In 1965 James Schlatter, a chemist at the pharmaceutical company G. D. Searle, was making peptides related to gastrin as part of a research programme. He had prepared the dipeptide methyl ester aspartyl-phenylalanine as an intermediate. Some of the powder got onto his hands, and when he later licked a finger to pick up a piece of paper, he noticed an intense sweetness.
That accident produced one of the most widely used sweeteners in the world. Aspartame was approved for dry foods in the United States in 1974, and after a long regulatory review for beverages in 1983. It is one of the few peptides most people consume regularly.
A dipeptide with a methyl ester
Aspartame is L-aspartyl-L-phenylalanine methyl ester. It is simply two amino acids joined by an ordinary peptide bond, with the C-terminal carboxyl of phenylalanine converted to a methyl ester.
The methyl ester is essential. The free dipeptide Asp-Phe, which the MW calculator returns for the sequence DF at 280.28 Da, is not sweet. Adding the methyl group, 14.016 Da, gives aspartame at 294.31 Da and creates the sweet taste. Two other structural requirements are just as strict:
- Both residues must be in the L configuration. The L-Asp–D-Phe isomer is bitter rather than sweet, and other stereoisomers are tasteless. This is a clear illustration of the principle discussed in the guide to D-amino acids: taste receptors are chiral, and a mirror-image molecule is a different molecule to them.
- The aspartate must be α-linked. The β-aspartame isomer, in which the linkage runs through the aspartate side chain, is not sweet. The distinction between α- and isopeptide linkages is described in the guide to the peptide bond.
Aspartame is roughly 200 times as sweet as sucrose by weight, so very small amounts are used. Its sweetness is often described as having a slower onset and longer duration than sugar, and it is frequently blended with other sweeteners such as acesulfame K, with which it is reported to act synergistically.
Why it breaks down
Aspartame is a good case study in peptide instability, because every one of its degradation routes is a reaction described in the guide to peptide stability and storage, and each one destroys the sweetness.
- Ester hydrolysis. The methyl ester is cleaved, releasing methanol and leaving the non-sweet dipeptide Asp-Phe.
- Diketopiperazine formation. The N-terminal amine attacks the ester, closing a six-membered ring and expelling methanol. This is exactly the cyclisation that troubles the first two residues of a peptide chain in solid-phase synthesis. The product is the main degradation compound found in stored beverages.
- Peptide bond hydrolysis, which splits the molecule into its two amino acids.
Stability is greatest around pH 4.3 and falls off sharply on either side, which is convenient, since many soft drinks sit near that range. It degrades quickly at high temperature, which is why aspartame is unsuitable for baking. Its gradual loss of sweetness in stored drinks, especially warm ones, is the everyday consequence of the same chemistry that makes peptide storage a technical problem.
Because aspartame contains phenylalanine, products containing it carry a declaration for people with phenylketonuria, an inherited condition in which phenylalanine cannot be metabolised normally. That labelling requirement is why the phrase about phenylalanine appears on so many packages.
How a peptide tastes sweet
Sweet taste is detected by a single receptor, a heterodimer of two proteins called T1R2 and T1R3, on taste cells of the tongue. It is a G protein-coupled receptor of an unusual kind, with a large extracellular "Venus flytrap" domain that closes around small molecules.
Different sweeteners bind at different sites on this receptor. Aspartame and the related sweetener neotame bind within the flytrap domain of T1R2, while others bind elsewhere, which explains why species differ in what they find sweet: rodents do not respond to aspartame, because their receptor differs at the relevant positions. This is a practical point for anyone reading the older literature, since it means rodent taste studies do not transfer.
Other peptide-based sweeteners exist and work differently. Neotame is aspartame with a bulky substituent on its N-terminus, which blocks the diketopiperazine route and makes it far more stable as well as much sweeter. Advantame is a related derivative. Several unrelated sweet proteins are known, among them thaumatin and brazzein from West African plants and monellin; these are proteins of 50 to 200 residues that bind a different part of the receptor, and they are thousands of times sweeter than sucrose by weight.
Peptides and taste more broadly
Aspartame is the most famous example of a peptide with a taste, but short peptides contribute to flavour throughout the food supply:
- Bitter peptides form when proteases break down proteins. Hydrophobic residues, especially at the C-terminus, correlate with bitterness, a relationship formalised by Ney's Q rule in the 1970s using the same kind of hydrophobicity values as the Kyte–Doolittle scale. Bitterness in aged cheese and in protein hydrolysates is a recurring problem in food processing.
- Kokumi peptides, above all γ-glutamyl peptides such as γ-Glu-Val-Gly, have little taste of their own but enhance the perceived richness and mouthfeel of savoury foods. They act on the calcium-sensing receptor. Glutathione itself is a γ-glutamyl peptide with kokumi activity.
- Umami peptides from aged cheese, fermented sauces and cured meats contribute savoury taste alongside free glutamate.
- Carnosine and anserine are present at high concentrations in meat and contribute to the flavour of broths, as described in their profile.
Calculating aspartame
| Compound | Formula | Average MW | Monoisotopic | Sweet? |
|---|---|---|---|---|
| Asp-Phe (free dipeptide) | C₁₃H₁₆N₂O₅ | 280.28 | 280.1059 | No |
| Aspartame (methyl ester) | C₁₄H₁₈N₂O₅ | 294.31 | 294.1216 | Yes |
| Diketopiperazine | C₁₃H₁₄N₂O₄ | 262.26 | 262.0954 | No |
Entering DF in the MW calculator gives the first row directly. Adding a methyl group (+14.016 average, +14.0157 monoisotopic) gives aspartame; losing methanol from aspartame (−32.026 average, −32.0262 monoisotopic) gives the cyclic degradation product. The whole chemistry of this molecule can be followed with nothing more than the residue masses and the modification shifts listed in the guide to post-translational modifications.
Frequently asked questions
Is aspartame really a peptide?
Yes. It is a dipeptide, two amino acids joined by a peptide bond, with its C-terminal carboxyl esterified. It is probably the peptide most people encounter most often.
Why does diet soda lose its sweetness over time?
Aspartame degrades, mainly by forming the cyclic diketopiperazine and by ester hydrolysis. Both products are tasteless. The process is faster at higher temperature and away from pH 4.3.
Why is aspartame not used in baking?
It decomposes rapidly at baking temperatures, so the sweetness is lost. Heat-stable alternatives, including neotame and non-peptide sweeteners, are used instead.
Why is the phenylalanine warning on the label?
Because digestion releases phenylalanine, which people with phenylketonuria must limit. The labelling allows them to account for it. It is a dietary declaration, not a general safety warning.
References
- Mazur RH, Schlatter JM, Goldkamp AH (1969) Structure-taste relationships of some dipeptides. Journal of the American Chemical Society 91:2684–2691.
- Li X, Staszewski L, Xu H, Durick K, Zoller M, Adler E (2002) Human receptors for sweet and umami taste. PNAS 99:4692–4696.
- Ney KH (1971) Voraussage der Bitterkeit von Peptiden aus deren Aminosäurezusammensetzung. Zeitschrift für Lebensmittel-Untersuchung und -Forschung 147:64–68.
- Ohsu T, Amino Y, Nagasaki H, et al. (2010) Involvement of the calcium-sensing receptor in human taste perception. Journal of Biological Chemistry 285:1016–1022.