Bachem PEPTalk
A Newsletter from Bachem
October 2011

Lance Corey

Featured Lab

Derek Macmillan Group at University College London

The Research

The Macmillan group at University College London is interested in the synthesis of modified peptides and proteins using chemical ligation techniques. Protein synthesis and semi-synthesis—the making of peptide fragments from both synthetic and recombinant origin—are important for the production of therapeutic proteins and in deciphering the mechanisms of processes orchestrated by post-translational modifications.

Understanding such processes is difficult because they are not template driven and not under direct genetic control. The Macmillan group's research makes use of synthetic organic chemistry, molecular biology, protein expression, and protein engineering to study post-translational modifications.

Native chemical ligation (NCL) is an extremely important technique that facilitates preparation of large polypeptides and enables a better understanding of their functional significance through access to specific post-translational modifications using isotopic, fluorescent, or mechanistic probes.

In particular, the Macmillan group discovered a novel process where peptide thioesters, the key reagents for NCL, can be prepared from native peptide sequences terminating in an Xaa-Cys motif, where Xaa is Gly, His, or Cys (Fig 1, Ref 1A). Thioesters derived from this reaction have been applied to the synthesis of a biologically active analogue of human β-defensin 3 (Ref 1B), human hepcidin, and more recently they have demonstrated compatibility with O- and N- glycopeptide synthesis (Ref 2), and phosphoprotein semi-synthesis.

Studies aimed at better understanding the reaction revealed that the addition of relatively small excesses of cysteine can reverse thioester formation (Ref 2), indicating that thioester formation and NCL can take place under the same reaction conditions. High concentrations of cysteine would need to be added for the NCL reaction to become efficient. However, in the case of an intramolecular NCL reaction (Fig 2), where the effective concentration of cysteine is high (Fig 2A), a cyclic peptide product (Fig 2C) could be formed directly, without the use of any specialized linkers or protein-processing elements such as inteins (Ref 3).

The Macmillan group found that when conducting the reaction at pH 2, the thioester predominated (Fig 2B), but at a range of pH 5-6, cyclic peptides could be isolated in yields of 40-60% without optimization. The process is intriguing because a single C-terminal amide bond is selectively broken and a new amide bond is formed in water both in the absence of enzymes and without the use of chemical-coupling reagents. Cyclic peptide fragments derived from β-defensins were prepared and found to retain antimicrobial activity with their enantiomers also exhibiting increased resistance to proteolysis in serum (Fig 3). Since the cyclization reaction is inherently reversible, it is surprising that the cyclic peptide should accumulate.

Continuing studies by the Macmillan group are focused on understanding how cyclic peptide formation can be improved. The process constitutes the first example of intein-free amide bond rearrangement via N→S acyl transfer in native peptide sequences and should be applicable to a variety of cyclic peptide targets. Future work will be directed towards preparation of naturally occurring cyclic peptides and proteins from linear precursors of biological origin.

About Dr. Macmillan

Dr. Derek Macmillan is a Reader in Organic Chemistry at University College London (UCL). He moved to UCL from The University of Edinburgh as a Royal Society University Research Fellow in 2005. Prior to this, Dr. Macmillan obtained his B.Sc. and Ph.D. from The University of Edinburgh and pursued postdoctoral studies (1999-2001) in the laboratory of Professor Carolyn Bertozzi at UC Berkeley. It was during his postdoctoral studies that Dr. Macmillan became interested in the application of Native Chemical Ligation to glycoprotein semi-synthesis.

Dr. Macmillan realized that the therapeutic need for glycoproteins would not be met through biosynthetic means alone. Thus his group has pursued the development of efficient routes for the introduction of saccharides and oligosaccharide mimics into synthetic and bacterially-derived peptides in a controlled manner. Their work in the area of glycoproteins continues in collaboration with Dextra Laboratories.

More recent investigations by the Macmillan group have been aimed towards the development of a new N→S acyl transfer reaction in peptides and proteins that provides straightforward access to peptide thioesters, the key tools for protein synthesis and semi-synthesis (Ref 1-3).

Learn more about Dr. Macmillan's work on his home page at University College London.

Learn more about Dextra Laboratories.

References

1. A) J. Kang, J. P. Richardson, and D. Macmillan. "3-Mercaptopropionic acid-mediated synthesis of peptide and protein thioesters." Chem. Commun. (2009) 407-409. [RSC] [PubMed]
   B) J. Kang, N. L. Reynolds, C. Tyrrell, J. R. Dorin, and D. Macmillan. "Peptide thioester synthesis through N→S acyl-transfer: application to the synthesis of a β-defensin." Org. Biomol. Chem. 7 (2009) 4918-4923. [RSC] [PubMed]
2. B. Premdjee, A. L. Adams, and D. Macmillan. "Native N-glycopeptide thioester synthesis through N→S acyl transfer." Bioorg. Med. Chem. Lett. 21 (2011) 4973-4975.
   [Science Direct] [PubMed]
3. D. Macmillan, M. De Cecco, N. L. Reynolds, L. F. A. Santos, P. E. Barran, and J. R. Dorin "Synthesis of Cyclic Peptides through an Intramolecular Amide Bond Rearrangement." Chembiochem 12 (2011) 2133-2136. [Wiley Online] [PubMed]

Ask a Regulatory Affairs Question

Question: What are the tests and specifications that FDA requires for a peptide used as an injectable? —Anonymous

Answer: In general, peptide drug substance specifications are based on the guidance document ICH Q6A, "Specifications: Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances." Universal drug substance tests required by this guidance are description, identity, assay (content), and impurities tests.

For peptides, description tests generally include appearance and color; identity tests generally include mass spectral analysis, amino acid analysis, and identity by HPLC; assay tests generally include peptide content, counter-ion content, and water content; and impurities tests generally include related substances by HPLC, residual solvents, and residual counter-ion. Other tests that are not universal but are applicable to peptides include solubility and purity by HPLC.

For drug substances intended for use in an injectable drug product—including peptide drug substances—microbiological tests are applicable including USP<61> Microbial Enumeration Test and USP<85> Bacterial Endotoxins Test. —Bachem Regulatory Affairs

 

Bachem Amino Acid Sudoku

(Read the new rules below!)

This Month's Peptide:

Peptide YY (also known as PYY or Peptide Tyrosine Tyrosine) is a gut hormone which is primarily released from endocrine cells of the distal digestive tract in proportion to both the calorie content of a meal and its energy source composition. Peptide YY plays an important role in regulating gastrointestinal secretion and motility.

Peptide YY belongs to the pancreatic polypeptide (PP) family along with PP and neuropeptide Y (NPY). These peptides mediate their effects through the NPY receptors.

This month's amino acid sudoku is derived from near the N-terminal end of the Peptide YY preprotein. Note, a second serine residue has been replaced with Xxx.

Bachem provides research-grade Peptide YY in a variety of forms. We also provide Peptide YY as a generic active pharmaceutical ingredient (API) and immunology products for Peptide YY.

 

Reference

http://www.ncbi.nlm.nih.gov/protein/300068956

The NEW Rules

Each amino acid in the sequence listed above appears once in each of the nine rows, once in each of the nine columns, and once in each of the 3x3 boxes outlined with a double border.

Email your answers to puzzle@bachem.com. One winner will be chosen randomly from all correct entries received before 12:01am Pacific Time, October 10, 2011. (You have about one week to submit an entry!)

This month's prize is a Bachem Swiss Army Knife!