Greening the Process Presented by Beatriz G. de la Torre, PhD, Research Professor, Laboratory of Medicine, University of KwaZulu-Natal
Dr. Beatriz De La Torre began her talk at the TIDES US 2021 Conference by sharing three methods for monitoring solid phase peptide synthesis (SPPS). The generally accepted method for SPPS is colorimetric (Ninhydrin test). While it is fast and cheap, it suffers from its qualitative subjective nature. It is also subject to amino acid or sequence dependent false positives and negatives, and its reliability decreases as peptide length increases. Another method for SPPS monitoring requires removal of samples for cleavage and analysis, although more reliable it is very time consuming. The third method she discussed monitors the UV absorption upon the release of Fmoc into solution.
The Refractive Index (RI) is a physical property, based on the ratio between light speed in a vacuum and the speed it travels through a material. It is dependent on density, indirectly on temperature, and is expressed as nD20. Refractive indices are widely used in industries including food and beverage, pharma, chemical, cosmetic, and automotive. It is used mainly for testing purity of starting materials or to determine concentrations of solutions. RI may be applied to monitoring SPPS during the coupling, deprotection and washing steps as there are transfers of mass to and from solution at each step.
Dr. de la Torre shared results from her laboratory in which industrial refractometers were adjusted to use in a laboratory setting for some experiments. Teflon tubing were found to interfere with RI measurements unless they were covered in black material. While many of the solutions used in SPPS have reported RI values, there have not been values reported for Fmoc-AA-OH solutions. RI values were determined for increasing concentrations of a variety of protected Fmoc amino acid solutions, and linear responses were observed consistently. The range in RI values observed with increasing concentration was found to differ between amino acids due to mass differences between amino acids, not a function of the number of aromatic rings present. Sequential addition of a selection of different amino acids to a solution also demonstrated a linear response in RI values observed. Addition of solvent to the solution resulted in an expected drop in RI value due to amino acid concentration decrease. Addition of coupling reagents did not affect linearity of RI responses.
Coupling of individual amino acids to resin demonstrated a reduction of RI as the amino acid bound to the solid phase, decreases in RI values levelled off at the end of the reaction. RI monitoring demonstrated that increasing the equivalents of Fmoc-Gly-OH from 1.5 to 3 decreased the reaction time from 70 to 20 minutes. When monitoring the deprotection of amino acids identical RI responses are observed since the mass of Fmoc released is the same in the three reactions. The reactions of 20% piperidine (deprotection step) were too rapid to monitor on the RI setup unless the piperidine was slowly added to the solution, working up to the final concentration of 20%. Altering the piperadine concentration resulted in different reaction kinetics but ultimately a linear response was demonstrated. Monitoring wash steps confirmed that the Fmoc was fully removed by the fourth wash.
Synthesis of the pentapetide, Leu-enkephalin, was undertaken using RI for real time monitoring. Upon cleavage the correct peptide was identified and only a minor impurity was observed but it could be attributed due to a recognized secondary reaction of CTC resin loading (a double coupling of leucine). Thus real time monitoring of the full process during SPPS is possible with RI. Advantages include elimination of the need to remove samples and the ability to determine the reaction completion in real time.
Dr. de la Torre concluded that use of this method would lead to reduction in solvent and reagent consumption resulting in a greener synthetic process.
Beatriz de la Torre, PhD, is a Research Professor at the Laboratory of Medicine and Medical Sciences, at the University of KwaZulu-Natal. A paper with the same title as her talk was published in the Org. Process Res. Dev. 2021, 25, 4, 1047–1053
