Solid-phase peptide synthesis (SPPS) is the standard method for building research peptides, residue by residue, on a solid resin support. Earlier articles on this blog covered the building blocks of that process: protecting groups, resin choice, coupling reagents and the cleavage that releases the crude peptide from the resin. What stays implicit in that chain is that SPPS has a practical length limit. As a chain grows longer, incomplete couplings, deletion sequences and on-resin aggregation of the growing peptide chain accumulate. Beyond roughly 50 amino acids the chance of a homogeneous end product becomes statistically ever smaller, even with an optimised protocol.<\/p>\n
For research into longer research peptides and small proteins that is a real bottleneck. The solution worked out in the literature is not one longer synthesis, but chemically joining several shorter fragments, each made by SPPS. The most widely used technique for this is native chemical ligation (NCL).<\/p>\n
NCL, originally described by Stephen Kent’s research group in the 1990s, uses two unprotected peptide fragments that react chemoselectively with one another in aqueous buffer at neutral pH. One fragment carries a thioester at its C-terminus, the other has a free cysteine at its N-terminus. Because both fragments are entirely unprotected, no separate deprotection step is needed after the ligation itself, which greatly simplifies the process compared with classical fragment condensation.<\/p>\n
The reaction proceeds in two steps. First a transthioesterification takes place: the thiol group of the cysteine attacks the thioester and forms a new, temporary thioester intermediate at the ligation site. That intermediate then undergoes an intramolecular S-to-N acyl shift, in which the acyl group migrates from the sulphur to the amine group of the same cysteine. The result is a native amide bond \u2014 chemically indistinguishable from a peptide bond formed during ordinary SPPS coupling.<\/p>\n
Each fragment is built up and purified separately via standard Fmoc-SPPS, exactly as described earlier on this blog for shorter peptides. The C-terminal fragment is converted into a thioester during or after synthesis, while the N-terminal fragment of the next segment requires a cysteine in the first position. For research purposes going beyond what two fragments can cover \u2014 target proteins of roughly one hundred to two hundred and fifty amino acids \u2014 several ligations are carried out one after another (sequentially) or from a central fragment (convergently). That requires a strict order of deprotection and ligation steps, because each fragment may act as a reactive partner only once before the next coupling takes its turn.<\/p>\n
The greatest practical limitation of classical NCL is the requirement for a cysteine at every ligation site. Not every research sequence has a cysteine there naturally, and building in an extra cysteine can change the properties of the object of study. The literature therefore describes removable thiol auxiliary groups and modified amino acids that mimic the reactivity of cysteine and can be removed again after ligation, so that the original sequence is preserved.<\/p>\n
Reaction conditions are equally critical. The ligation buffer is usually held at pH 7 and contains a thiol catalyst, such as MPAA, which accelerates thioester exchange and prevents the intermediate from stalling. Oxygen is excluded as far as possible, because free thiol groups would otherwise oxidise into unwanted disulfide bridges, lowering the ligation yield. Once ligation is complete, a further purification step via preparative RP-HPLC usually follows, separating unreacted fragment from the ligation product.<\/p>\n
Native chemical ligation has opened the door in peptide chemistry to research material that was inaccessible via classical SPPS: longer synthetic chains and even fully synthetic small proteins, built from pieces each of which can be checked and purified well on its own. For laboratories working with constructs of this kind, that means an extra layer of planning \u2014 which fragments, which ligation sites, which order \u2014 on top of the synthesis and purification already needed for each individual fragment.<\/p>\n
This information is intended solely for laboratory and research purposes. The products and information of Dutch Peptide Labs are not intended for use by humans or animals and not for diagnostic or therapeutic applications.<\/em><\/p>\n","protected":false},"excerpt":{"rendered":" Why SPPS does not scale indefinitely Solid-phase peptide synthesis (SPPS) is the standard method for building research peptides, residue by residue, on a solid resin support. Earlier articles on this blog covered the building blocks of that process: protecting groups, resin choice, coupling reagents and the cleavage that releases the crude peptide from the resin. […]<\/p>\n","protected":false},"author":0,"featured_media":0,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[1],"tags":[],"class_list":["post-1216","post","type-post","status-publish","format-standard","hentry","category-niet-gecategoriseerd"],"acf":[],"_links":{"self":[{"href":"https:\/\/dutchpeptidelabs.nl\/en\/wp-json\/wp\/v2\/posts\/1216","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/dutchpeptidelabs.nl\/en\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/dutchpeptidelabs.nl\/en\/wp-json\/wp\/v2\/types\/post"}],"replies":[{"embeddable":true,"href":"https:\/\/dutchpeptidelabs.nl\/en\/wp-json\/wp\/v2\/comments?post=1216"}],"version-history":[{"count":1,"href":"https:\/\/dutchpeptidelabs.nl\/en\/wp-json\/wp\/v2\/posts\/1216\/revisions"}],"predecessor-version":[{"id":1245,"href":"https:\/\/dutchpeptidelabs.nl\/en\/wp-json\/wp\/v2\/posts\/1216\/revisions\/1245"}],"wp:attachment":[{"href":"https:\/\/dutchpeptidelabs.nl\/en\/wp-json\/wp\/v2\/media?parent=1216"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/dutchpeptidelabs.nl\/en\/wp-json\/wp\/v2\/categories?post=1216"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/dutchpeptidelabs.nl\/en\/wp-json\/wp\/v2\/tags?post=1216"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}