Methylene Analogues of Neopetrosiamide as Potential Antimetastatic Agents: Solid-Supported Syntheses Using Diamino Diacids for Pre-Stapling of Peptides with Multiple Disulfides

Abstract

Neopetrosiamide, a 28-residue peptide from Neopetrosia sp., contains three disulfide bonds and hinders mammalian tumor cell invasion. Proper connectivity of disulfide bonds is crucial for activity. Synthetic replacement of single disulfide bridges with methylene bridges gives active analogues. Pre-stapling of one ring enhances the correct formation of the remaining disulfides by reducing isomeric possibilities and possibly initiating the correct 3D fold. Cloning and expression of neopetrosiamide in E. coli affords access to the natural linear peptide.Letter pubs.acs.org/OrgLett

SPI “sandwich”: Combined SUMO‐Peptide‐Intein expression system and isolation procedure for improved stability and yield of peptides

“…The pET‐SUMO expression vector (Invitrogen) encoding neopetrosiamide A fused to the His‐tagged SUMO protein was described previously, 23 and cloning of genes for FaeMK, LeuA, and LcnA into this vector followed the described protocol (Appendix S1).…”

“…The lysis buffer was modified from previous SUMO fusion isolation procedures 19,23 to contain 300 mM NaCl (from 150 mM) because the higher salt content better prevented precipitation during isolation. We did not need to use Protease Inhibitor Cocktail tablets during any of the SPI isolations, as the expression system "sandwich" was found to be effective enough at preventing degradation during isolation.…”

“…Isolation procedures for SUMO-peptide fusion proteins have been previously described, 19,23 and the detailed procedure used in this study is included in Appendix S1.…”

“…Our group and others have routinely cloned, expressed, and isolated both proteins 19–21 and small peptides 22–24 as SUMO fusions in E. coli . While overproduction of proteins using this system has been successful in our group, we have observed, as described in this work, that SUMO fusions of small peptides including lactococcin A, leucocin A, faerocin MK, and neopetrosiamide A can be truncated during expression, resulting in low yield of the purified peptide.…”

“…18 While expression optimization can lead to improved yields, this can be extremely time consuming as there are a large number of factors to potentially modify, including induction temperature, inducer concentration, optical density, induction time, buffer conditions, isolation temperature, protease inhibitors, detergents, solubility additives, or isolation procedures. 13,18 Our group and others have routinely cloned, expressed, and isolated both proteins [19][20][21] and small peptides [22][23][24] as SUMO fusions in E. coli. While overproduction of proteins using this system has been successful in our group, we have observed, as described in this work, that SUMO fusions of small peptides including lactococcin A, leucocin A, faerocin MK, and neopetrosiamide A can be truncated during expression, resulting in low yield of the purified peptide.…”