Peptides, used to dislike the chemistry to obtain them, however reading the latest paper by the Danishefsky group  have made me appreciate it a lot more!. Note I’m not even going to attempt to draw the structures, not even a SMILES string will appear, I think ChemDraw would go on strike.

Anyway complementing his synthesis of erythropoietin (166 amino acids) at the end of last year Danishefsky has moved on to synthesise human luteinizing hormone (hLH), 121 amino acids and human chorionic gonadotropin (hCG), 145 amino acids. These compounds are two human glycoprotein hormones each consisting of two subunits, an identical α-subunit and a unique β-subunit, that form noncovalent heterodimers.  As was the case with erythropoietin these two hormones were prepared with all of the glycosidation sites occupied by glycans. 

For the preparation of hLH the amino acid chain was  split into fragments with the site of disconnection being at a cysteine residue. These fragments were all synthesised by Fmoc solid-phase methods using acid labile protecting groups. The  acetamidomethyl group was chosen to protect those cysteines not involved in the fragment coupling reactions. The following shows the yields for the last steps in the sequence leading to the peptide units required for NCL.

• Fragment 1: 37 amino acids, 22% yield.
• Fragment 2: 34 amino acids, 72% yield
• Fragment 3: 50 amino acids, 55% yield

Then NCL of fragments 2 & 3 followed by 1 gave the full hLH peptide in 15% yield with the acetamidomethyl protection still in place.

This was just a trial run to ensure everything worked! Now on to the hCG system containing the N/O glycans. Using similar disconnections they identified 5 fragments which could be coupled by NCL. This according to the authors: “This highly modular strategy would allow us to investigate the effect of introducing various defined carbohydrates at different sites on the protein scaffold.” As if this was not hard enough this statement appears: “a more challenging approach was also considered, consisting of the double incorporation, simultaneously, of the two N-linked carbohy- drates on a longer peptide backbone comprising both N- glycosylation sites.” No comment!

Finally “Thus, compound β-hCG(chitobiose)2(GalNAc)4[S1− Q145], bearing not only two N-linked carbohydrates but also, and even more challenging, four O-linked sugars, represents the largest human glycoprotein hormone to have been synthesized in homogeneous form using strictly chemical means. While the current work has involved chitobiose and GalNAc as representative N-/O-linked model glycans, this demonstration of feasibility through the modular glycoform assembly presented herein opens the door to the application of this technology to the synthesis of a library of homogeneous glycoproteins via chemical synthesis by installing alternative glycans on the corresponding peptide fragments.”

This is a monumental piece of work by this group, this and the previous synthesis of erythropoietin will stand out as examples of the power of chemical synthesis. I hope we see more like this. Congratulations.

2,909 total views, no views today