Garry Hedgeslammer and Günther Schlonk
Specific: Scientists are very fond of talking about the importance of serendipity in research. This usually means one of two things. The first thing is “sure, my work appears to be incredibly niche and pointless, but don’t discount the possibility that I’ll stumble over a cure for cancer in the rectums of these worms I’m studying”. The second is “sometimes fuck-ups are salvageable, if you keep an open mind”. This paper is the latter sort of serendipity.
Introduction
Solid-phase peptide synthesis is humanity’s way of imitating chemistry that even amoeba can do. The difference is that amoeba are better at it than us. While most organisms can use their cellular machinery to string amino acids (AA’s) together ad infinitum, chemists must use blunter tools. Reagents like EDCI and DCC are capable of forming amides from carboxylic acids and amines, but if the desired peptide is more than a couple of AA’s long, the repeated solvent extractions and columns will make any chemist long for the numbing embrace of death.
Luckily for future generations of chemists, Robert Merrifield solved this problem in 1959 by sticking the first AA in the chain to tiny polystyrene beads. This meant that by simply filtering the beads out of a reaction mixture, all unwanted side-products and reagents could be removed. This system has been optimised to the point where machines can do it, by adding N-protected AA’s, coupling reagents and deprotecting agents in cycles, interspersed with filtrations. For peptides of >70 AA’s however, the method breaks down, and you have to ask an amoeba to make it for you.
As our lab has significant experience in this field, we were commissioned by members of a local gym to synthesise a gym- buddy from protein powder, by peptide coupling (Scheme 1). We tried to explain to them that it would be much easier to buy the amino acids separately, but they didn’t understand, and insisted we start with protein powder instead of “chemicals”.
Results and Discussion
We obtained 70 kg of Sigma Aldrich’s leading protein supplement (MillisigmaTM), and began the laborious process of separating out all 20 amino acids. This was a challenge in and of itself, but after more than twenty columns, a dozen recrystallizations and countless solvent extractions, we obtained pure (>99.5%) fractions of all twenty amino acids. With the acids in hand, we reacted tryptophan with DCC, and were just about to add the polystyrene beads when Garry dropped the flask onto our mid- century style wooden table, and smashed it (Figure 1).
As our tryptophan had already been activated with DCC, it immediately reacted with the hydroxyl groups on the surface of the table, and stuck fast. This precluded us from recovering our precious amino acid via a bench- extraction.1 There was no way we were gonna purify more protein powder, so we were pondering ways to chemically cleave the table from the tryptophan when the idea came to us: why don’t we make the whole protein while it’s bound to the table? (Figure 2).
While initially counterintuitive, tables have several advantages over plastic beads as solid-phase supports for peptide synthesis.
- Polystyrene beads are spheres: the shape with the worst surface area-to-volume ratio. Tables are table-shaped, and have far more surfaces on which to bind amino acids.
- Polystyrene is derived from petrochemical feedstocks, and as such every time you use it, a dolphin dies. Tables literally grow in trees, making them far greener (literally and environmentally) than plastics.
- Polystyrene beads are tiny, and have a tendency to block the filter paper during the filtration steps. Tables are significantly larger and faster to filter. Filter paper of ~0.025 mesh is sufficient to isolate a 2 m square dining table.
With these principles in mind, we developed a general method for solid-phase peptide synthesis on wooden-table supports (Scheme 2). We selected Semaglutide (Ozempic) as our test peptide for the purposes of optimisation, as it allowed us to make up a funding shortfall by selling the product outside KFC. We found that minimal alterations were necessary to the traditional reagents and solvents used in SPPS, but also that the choice of table was crucial (Table 1).
Plastic coatings and varnish were found to significantly impede the reaction, though these coatings did eventually dissolve in the reaction solvent. This resulted in the synthesis of peptides missing the first few residues. Non-wooden surfaces were also ineffectual, presumably due to a lack of available hydroxyl groups on the surface. Untreated pine proved to be the ideal substrate, and is also optimal from the perspectives of sustainability and cost.
Under these optimised conditions, solid-phase peptide synthesis has never been easier. One simply pours the reagents onto the table, waits a while, then tips the excess off the edge. When the desired peptide has been synthesised, it is cleaved from the table with trifluoroacetic acid (TFA). A facile filtration can then be used to remove the table and obtain pure peptide.
Conclusion
We have developed a novel strategy for solid-phase peptide synthesis that is both easier and greener than traditional methods, by replacing conventional polystyrene substrates with wooden tables. Future work will focus on leveraging this technique to create the ultimate gym-bro from protein powder.
Notes and references
1. “And then I threw it on the ground: a guide to bench- and floor-extractions” A. Samberg et al., 2011, Lonely J. Chem. 7, 11–15.