WARF: P00315US

Staudinger Ligation Method for Rapid and Reliable Chemical Synthesis of Proteins


Ronald Raines, Laura Kiessling, Bradley Nilsson

The Wisconsin Alumni Research Foundation (WARF) is seeking commercial partners interested in developing an improved method of synthesizing proteins.
OVERVIEWResearchers and drug development companies have traditionally obtained proteins by using recombinant DNA technology to express them in genetically engineered organisms.  However, this approach can take years to produce a given protein.  In addition, the proteins may not work correctly due to improper folding or biological contamination, or may not be produced in sufficient quantities. 

An alternative is the total chemical synthesis of proteins.  This approach is much faster, taking only days or weeks.  It produces homogeneous, correctly folded and biologically active proteins with nearly 100 percent reliability.  

However, current methods of chemically synthesizing proteins have major limitations. In particular, native chemical ligation and expressed protein ligation require the presence of a cysteine residue at the ligation junction.  But few peptides naturally have a cysteine at this junction, and adding a cysteine residue is often undesirable because cysteine reacts readily with disulfide bonds, O2 and other electrophiles.
THE INVENTIONUW-Madison researchers have developed an improved method of synthesizing proteins that does not require a cysteine residue at the ligation junction.  This method expands the utility of total protein synthesis by removing the limitation inherent in native chemical ligation and expressed protein ligation.  It is inspired by the Staudinger reaction, in which a phosphine molecule is used to reduce an azide to an amine. 

In this method, a phosphinothioester and an azide are united to form an amide bond.  A phosphinothiol reagent is used to efficiently generate a phosphinothioester from an amino acid, peptide or protein fragment.  An azido group then can be formed at the N-terminus or a basic side group of another amino acid, peptide or protein.  Then the phosphinothioester is reacted with the azido group to ligate the amino acids, peptides or proteins.

This reaction allows the formation of an amide bond among a wide variety of chemical species.  It can be used to repeatedly ligate natural and/or nonnatural amino acids to synthesize proteins or peptides.  Large peptides or proteins can be formed by ligating two or more small peptides or proteins.
  • Total chemical synthesis of proteins, which provides a rapid, reliable route to homogeneous, correctly folded proteins with full biological activity
  • Ligation of biological molecules, including amino acids, peptides and protein fragments
  • Synthesis of peptides and proteins, including the stepwise synthesis of peptides on solid supports
  • Eliminates need for cysteine residue at the ligation junction
  • Traceless - leaves no residual atoms in the ligated peptide product
  • Allows the formation of an amide bond between a wide variety of chemical species 
Contact Information
For current licensing status, please contact Jennifer Gottwald at or 608-960-9854.
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