(324b) Crystallisation Process Design for the Separation of Dipeptides Based on Alanine and Glycine with Antisolvent Addition

Peptides are short chains of amino acids that are linked together by peptide bonds. They can range in length from a few amino acids to several hundred and are important building blocks of proteins. Peptides have a variety of functions in the body, such as acting as hormones, neurotransmitters, and signalling molecules.^1,2 Peptides are also used in various fields such as medicine, agriculture, and cosmetics. They can also be used as diagnostic tools to detect certain diseases or conditions³. Peptides are usually synthesised from amino acids, mainly in two approaches: solid-phase synthesis and liquid-phase synthesis⁴. Solid-phase synthesis involves attaching the first amino acid to an insoluble resin, and subsequently adding the remaining amino acids step-by-step⁵. Liquid-phase synthesis involves the use of solution-phase chemistry to assemble the peptide chain. In both cases, however, the synthesised peptides could be consisted of a mixture of different species.

In this research, we proposed a separation method design for the aqueous system involving model peptides based on alanine and glycine: dialanine (ala-ala), alanyl glycine (ala-gly), glycyl alanine (gly-ala) and diglycine (gly-gly). The difference in temperature dependency of solubility was utilise for the separation. Before the crystallisation started, all the peptide species were saturated at room temperature (298.15K). First, ala-ala was separated by increasing temperature from 298.15K to 313.15K, with a supersaturation ratio of 1.13. At the meantime, since the other 3 dipeptides’ supersaturation decreased, they stayed in the liquid phase. In this way, the pure ala-ala crystal could be obtained. Second, decrease temperature to 288.15K, where the supersaturation of gly-gly was 1.14, while gly-ala and ala-gly reached supersaturation ratio of 1.05 and 1.02 respectively. As the crystallisation driving force of gly-gly was higher than other, by kinetic reason, the solid obtained in this step would be gly-gly in majority. Third, as for the separation of ala-gly and gly-ala, the difference in temperature dependence of their solubilities were not obvious. However, the absolute solubility of gly-ala was higher than ala-gly, which gave gly-ala higher crystallisation rate. Therefore, when we drop temperature further to 283.15K, gly-ala would come out in large quantity first. Besides, silicas with surface function groups (-NH2, -COOH and so on) would be tested on there effect to improve the selectivity of gly-ala crystallisation. Finally, the temperature was lowered to 278.15 K for long enough time for getting ala-gly crystals out from solution.

This method enabled stepwise recovery of 4 different model dipeptide from mixtures. The work could potentially inspire us design a crystallisation protocol to purify and separated longer-chain peptides from its own fragments by taking advantage of differences in solubility behaviours.

1 Fosgerau, K. & Hoffmann, T. Peptide therapeutics: current status and future directions. Drug Discovery Today 20, 122-128 (2015). https://doi.org:https://doi.org/10.1016/j.drudis.2014.10.003

2 von Heijne, G. The signal peptide. The Journal of membrane biology 115, 195-201 (1990).

3 Reubi, J. C. Peptide receptors as molecular targets for cancer diagnosis and therapy. Endocrine reviews 24, 389-427 (2003).

4 Jung, G. & Beck‐Sickinger, A. G. Multiple peptide synthesis methods and their applications. New synthetic methods (87). Angewandte Chemie International Edition in English 31, 367-383 (1992).

5 Coin, I., Beyermann, M. & Bienert, M. Solid-phase peptide synthesis: from standard procedures to the synthesis of difficult sequences. Nature protocols 2, 3247-3256 (2007).