Source of Resin cleavage

{{Infobox method | name = Resin cleavage | subtitle = Peptide synthesis | Reagent, Fmoc chemistry = Trifluoroacetic acid with scavengers | Simultaneous = Side-chain deprotection | Isolation = Precipitation into cold ether }} '''Resin cleavage''' is the step that releases an assembled peptide from the solid support. In [[Fmoc chemistry|Fmoc chemistry]] it also removes the acid-labile side-chain protecting groups, so a single treatment with concentrated trifluoroacetic acid accomplishes both.{{r|behrendt2016}} The removed protecting groups generate reactive carbocations, which will alkylate electron-rich side chains — tryptophan, methionine, cysteine and tyrosine — unless captured. Scavengers are included in the cleavage cocktail for that purpose, and their selection depends on which residues the sequence contains.{{r|behrendt2016}} Cleavage is therefore not a neutral release step. It is a chemical operation with its own characteristic impurities, and a substantial part of the difference between a clean and a dirty [[Crude peptide|crude]] is decided here.{{r|usp1503}} == Cocktails and scavengers == A typical cocktail is trifluoroacetic acid with a few per cent each of water, triisopropylsilane and, where cysteine or methionine is present, a thiol scavenger. Water and silane trap the tert-butyl and trityl cations; thiols address the more persistent species.{{r|behrendt2016}} Time and temperature are the other variables. Arginine's sulfonyl protecting groups are the slowest to remove, so a peptide rich in arginine requires longer treatment — during which more sensitive residues are exposed to acid for longer. The trade-off is direct: insufficient time leaves protected species in the crude, excessive time generates degradation. This is one of the process choices that a [[Certificate of analysis|certificate]] does not report but whose consequences it may show.{{r|usp1503}} == Isolation == After cleavage the peptide is precipitated by adding the acidic solution to a large volume of cold diethyl ether, in which peptides are insoluble and the scavengers and cleaved protecting groups are soluble. The precipitate is collected, washed with further cold ether, and dried.{{r|behrendt2016}} Precipitation is a crude purification and removes small-molecule material efficiently while doing nothing about sequence-related impurities, which remain and are the business of [[Preparative HPLC purification|preparative chromatography]]. Residual ether is a [[Residual solvent|residual solvent]] of Class 3, and residual trifluoroacetic acid becomes the [[Trifluoroacetate counterion|counterion]] of the isolated solid.{{r|usp1503}} == Signatures in the product == Cleavage-derived impurities are chemically distinctive. Alkylated residues carry a characteristic mass addition, detectable by [[Mass spectrometry|mass spectrometry]]; oxidised methionine adds 16 Da; incompletely deprotected species carry the mass of the retained group.{{r|aebersold2003}} Because these masses are large and distinctive, cleavage-derived impurities are among the easier ones to identify when a full mass spectrum is available — in contrast to deletion sequences and isomers, which need chromatographic resolution.{{r|usp1503}} An itemised related-substances table on a certificate that names such species is showing that the analysis looked for them. One reporting a single aggregate figure is not.{{r|usp621}} == References == {{reflist}} <ref name="behrendt2016">Behrendt R, White P, Offer J. "Advances in Fmoc solid-phase peptide synthesis." ''Journal of Peptide Science'' 22(1):4–27 (2016). PMID 26785684.</ref> <ref name="usp1503">United States Pharmacopeia, General Chapter <1503>, ''Quality Attributes of Synthetic Peptide Drug Substances''.</ref> <ref name="aebersold2003">Aebersold R, Mann M. "Mass spectrometry-based proteomics." ''Nature'' 422(6928):198–207 (2003). PMID 12634793.</ref> <ref name="usp621">United States Pharmacopeia, General Chapter <621>, ''Chromatography''.</ref> == See also == * [[Fmoc chemistry]] * [[Solid-phase peptide synthesis]] * [[Crude peptide]] * [[Trifluoroacetate counterion]] * [[Methionine oxidation]] {{DEFAULTSORT:Resin cleavage}} [[Category:Peptide synthesis]] [[Category:Purification]] [[Category:Impurities and residues]]