Cysteine racemization during the Fmoc solid phase peptide synthesis of the Nav1.7‐selective peptide – protoxin II

Abstract

Protoxin II is biologically active peptide containing the inhibitory cystine knot motif. A synthetic version of the toxin was generated with standard Fmoc solid phase peptide synthesis. If N-methylmorpholine was used as a base during synthesis of the linear protoxin II, it was found that a significant amount of racemization (approximately 50%) was observed during the process of cysteine residue coupling. This racemization could be suppressed by substituting N-methylmorpholine with 2,4,6-collidine. The crude linear toxin was then air oxidized and purified. Electrophysiological assessment of the synthesized protoxin II confirmed its previously described interactions with voltage-gated sodium channels. Eight other naturally occurring inhibitory knot peptides were also synthesized using this same methodology. The inhibitory potencies of these synthesized toxins on Nav1.7 and Nav1.2 channels are summarized.

Identification, chemical synthesis, structure, and function of a new KV1 channel blocking peptide from Oulactis sp.

“…[63] Also, base-mediated couplings have been found to induce racemization of Cys during coupling steps. [57,64] Methods are optimized to reduce racemization, although they do not completely block the process. [65] Structurally, OspTx2a is more similar to BgK (Figures 3F, [6] ) than ShK.…”

Synthetic Cystine-Knot Miniproteins – Valuable Scaffolds for Polypeptide Engineering

“…Additionally, prolonged reaction times in Fmoc-SPPS are compensated through the advantage of fully automated synthesizers that can be utilized more regularly due to the usage of less aggressive reagents. In both methodologies racemization of the amino acid through deprotonation of the α-hydrogen with the activator base can be easily overcome by the usage of 2,4,6-trimethylpyridine or racemization-resistant cysteine protection [145,146]. To summarize, despite Fmoc-SPPS is to date the method of choice, Boc-SPPS is also a valuable back-up tool for aggregation-prone peptides or peptides with base-labile moieties which are not compatible with Fmoc-chemistry [139,141,142].…”

Chemical Synthesis, Backbone Cyclization and Oxidative Folding of Cystine-knot Peptides — Promising Scaffolds for Applications in Drug Design

“…Moreover, prolonged reaction times in Fmoc-SPPS are outweighed through the advantage of fully automated synthesizers that can be utilized more regularly due to the usage of less aggressive reagents (although peptide synthesizers compatible with Boc-SPPS are also commercially available). In both methodologies racemization of the amino acid through deprotonating the α-hydrogen with the activator base can be easily overcome by the usage of 2,4,6-tri-methylpyridine or racemization-resistant cysteine protection as e.g., the recently reported 4-methoxy-benzyloxymethyl group [ 50 , 67 ]. In summary, despite Fmoc-SPPS being to date the method of choice, Boc-SPPS is a valuable back-up tool for aggregation-prone peptides or peptides with base-labile moieties which are not compatible with Fmoc-chemistry [ 54 , 55 , 63 ].…”

“…RP-HPLC in combination with mass spectrometry, especially ESI-MS and MALDI-TOF, are commonly used for the routine analysis of cystine-knot peptides [ 23 , 31 , 48 , 50 , 55 ]. Therein, not only the polarity, but also the molecular weight are determined giving clear evidence of the quality and nature of the product [ 23 , 31 , 48 , 50 , 55 ].…”

“…RP-HPLC in combination with mass spectrometry, especially ESI-MS and MALDI-TOF, are commonly used for the routine analysis of cystine-knot peptides [ 23 , 31 , 48 , 50 , 55 ]. Therein, not only the polarity, but also the molecular weight are determined giving clear evidence of the quality and nature of the product [ 23 , 31 , 48 , 50 , 55 ]. For example, the progress of oxidative folding was determined through a shift in RP-HPLC retention time as well as a decreased molecular weight because of the loss of the respective number of hydrogens [ 23 , 31 , 48 , 55 ].…”