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Understanding the process is difficult because they do not work as templates and are not directly controlled by genetics. The Macmillan group used organic synthetic chemistry, molecular biology, protein expression, and protein engineering to study post-transcriptional modification changes.
In particular, the Macmillan team discovered a novel process for the NCL key reagent polypeptide thioester: the peptide thioester can be prepared from a natural polypeptide with a Xaa-Cys sequence at its end (in the sequence Xaa is Gly, His or Cys) (Figure 1 , Ref 1A). The thioesters produced by this reaction have been used to synthesize biologically active human β-defensin 3 (Ref 1B) analogs and human Hepcidin. They have recently been shown to be compatible with O- and N-glycopeptide synthesis (Ref 2) and phosphorylated protein semi-synthesis.
Figure 1 shows the formation of a thioester by N→S acyl conversion. Typical reaction conditions: 10% v/v MESNa; 0.1 M Na phosphate buffer, pH 5.8; 0.5% v/v TCEP, 55 ° C, 48 h. (MESNa = sodium 2-mercaptoethanesulfonate, sodium sulfonate, TCEP = tris-carboxyethylphosphine, tris-carboxyethylphosphine).
Figure 2 shows a circular peptide that is connected end to end from a simple linear precursor. Typical reaction conditions: 10% v/v MESNa; 0.1 M Na phosphate buffer, pH 5.8; 0.5% v/v TCEP, 55 ° C, 48 h. (Note: TCEP can be used in the cyclization to replace sodium ascorbate.)
The Macmillan team focused on continuous research to understand how to improve the formation of cyclic peptides. In this process, the rearrangement of the first amino bond without intein was achieved by N→S acyl transfer in the native peptide sequence. This method should be applicable to the formation of various cyclic peptides. Future research efforts will focus on the preparation of various naturally occurring cyclic peptides and proteins derived from biologically derived linear precursors.
Figure 3. Short cyclic peptides and their mirror image isomers may have antimicrobial activity. Note: D peptide is significantly more stable in serum.
The recent research direction of the Macmillan research group is to develop new N→S acyl transfer reactions in peptides and proteins, directly using the thioesters of peptides to provide a key tool for protein synthesis and semi-synthesis (Ref 1-3).
references
1. A) J. Kang, JP Richardson, and D. Macmillan. "3-Mercaptopropionic acid-mediated synthesis of peptide and protein
2. B) J. Kang, NL Reynolds, C. Tyrrell, JR Dorin, and D. Macmillan. "Peptide thioester synthesis through N→S acyl-
3. B. Premdjee, AL Adams, and D. Macmillan. "Native N-glycopeptide thioester synthesis through N→S acyl transfer."
4. D. Macmillan, M. De Cecco, NL Reynolds, LFA Santos, PE Barran, and JR Dorin "Synthesis of Cyclic Peptides through an Intramolecular Amide Bond Rearrangement." Chembiochem 12 (2011) 2133-2136.
New technology for peptide thioester protein synthesis
The University of London Macmillan team is interested in the synthesis of modified peptides and proteins using chemical ligand technology. Protein synthesis and semi-synthesis - peptide fragments prepared from synthetic and recombinant sources - are important for the production of therapeutic proteins and for understanding the mechanisms underlying post-transcriptional modifications.
To better understand the reaction, it was found that increasing the relative small amount of excess cysteine ​​reverses the formation of thioester (Ref 2), indicating that thioester formation and NCL can occur under the same reaction conditions. In order to increase the efficiency of the NCL reaction, it is necessary to increase the concentration of cysteine. However, in the case of intramolecular NCL reaction (Fig. 2), when the effective concentration of cysteine ​​is high, the cyclic polypeptide product can be directly formed (Fig. 2C) without using any specific linker or protein treatment component, such as intein. (Ref 3).
The Macmillan team found that when the reaction was carried out at pH 2, the resulting thioesters dominated. However, when pH 5-6, the cyclic peptide was obtained without optimization (yield 40-60%). This is a continuous process in which a single amino bond at the C-terminus is selectively cleaved, while a new amino bond is formed in water without the use of a chemical coupling agent in the absence of an enzyme. It has been found that the prepared cyclic peptide fragment derived from β-defensins retains antimicrobial activity, and its mirror image isomer exhibits an increased resistance to proteolysis in serum (Fig. 3).
Dr. Derek Macmillan is Associate Professor of Organic Chemistry at the University of London (UCL). In 2005, he moved from the University of Edinburgh to the University of London and became a member of the Royal University Researchers Association. Previously, Dr. Macmillan received his master's and doctoral degrees from the University of Edinburgh. Postdoctoral research at the laboratory of Dr. Carolyn Bertozzi, University of California, Berkeley, 1999-2001. During the postdoctoral study, Dr. Macmillan began to take an interest in the use of natural chemical ligands for glycoprotein semisynthesis. Dr. Macmillan realized that therapeutic drugs require glycoproteins, but the need for glycoproteins cannot be met by biosynthesis alone. Therefore, his research team is working to develop an efficient way to incorporate glycans and oligosaccharide mimetics into synthetic and bacterial-derived polypeptides in a controlled manner. For the eternal work on glycoproteins, they continue to work with DexTra Laboratories.
Thioesters." Chem. Commun. (2009) 407-409.
Transfer: application to the synthesis of a β-defensin." Org. Biomol. Chem. 7 (2009) 4918-4923.
Bioorg. Med. Chem. Lett. 21 (2011) 4973-4975.