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A cut above the rest: the evolution and application of inteins

In this new Perspective, Chemical Science Associate Editor Tom Muir of Princeton University elucidates the biological role and evolutionary origin of inteins – a fascinating class of proteins which have the ability to “process”, or sever, their own peptide backbone. Usually, this type of post-translational modification is done enzymatically by proteases, but inteins contain a module which allows the spontaneous scission of peptide bonds with no external factor or energy source required. The only things needed are certain chemical functional groups in the neighbouring peptide residues, and the correct spatial folding of the domain.

Obviously, this is a truly interesting process, as the functions and actions of a protein are determined by its amino acid sequence and structure. By breaking peptide bonds and creating new ones, inteins essentially act as an on/off switch for the protein, the potential application for which is staggering.

The intein is flanked on either side by two exteins; during the “processing” reaction, the bonds between intein and exteins are broken, and a new bond between the exteins is created. Inteins share a common biochemical mechanism for this process, which is illustrated below. Crucially, all inteins must contain a cysteine or serine at their N-terminus which provides the nucleophile for the initial acyl shift. A subsequent trans(thio)esterification and an additional acyl shift forms the spliced product and the excised intein.

Mechanism of protein splicing

Mechanism of protein splicing

Muir and co-author Neel Shah investigate the evolutionary origin of inteins, which can be found in all domains of life – eukaryotes, bacteria, archaea and viruses. Although they are often found in proteins involved in genetic “housekeeping” – DNA replication, transcription, and maintenance – inteins have no obvious biological role, and do not provide any benefit to the host organism. As such, they are known as “selfish” genes. Despite the mystery of their purpose and origin, what is clear is that the future is bright for inteins.


The authors discuss a number of applications of inteins, the most exciting of which is conditional protein splicing (CPS) where inteins can be used as an on/off switch for the proteins they are splicing, even in vivo. CPS is currently achieved in a number of ways, shown below, in which the intein is kept in an inactive state – via (a) conformational distortion, (b) caging of the active site, or (c) the physical separation of a split intein – until activation is desired. Ligand binding, deprotection or dimerisation, respectively, then releases the active intein and triggers the peptide splicing. CPS is a promising tool for cell biology and should facilitate the development of “smart” protein therapeutics that are activated only at the target site.

All in all, this Perspective makes for an interesting read on a class of proteins with impressive potential. I think it’s a safe bet to predict that when it comes to future smart therapeutics, inteins will definitely make the cut.

Conditional protein splicing

Conditional protein splicing (CPS): a) Allosteric intein activation by ligand binding; b) Intein activation via deprotection of a photo-caged active site residue; c) Activation via chemically-induced dimerisation

For more, you can read Muir and Shah’s Chemical Science Perspective here:

Neel H. Shah and Tom W. Muir
Chem. Sci., 2014, Advance Article
DOI: 10.1039/C3SC52951G

Professor Muir serves as one of Chemical Science‘s Associate Editors, handling submission in chemical biology – read more about him and what useful advice he wishes someone had told him as an undergraduate.

Our Associate Editors Tom Muir and Ben Davis have highlighted their recommended chemical biology papers on Chemical Science – read their Editor’s Choice selection for FREE today!

Find many more excellent articles on chemical biology here: Online collection: Chemical biology

Ruth E. Gilligan is a guest web-writer for Chemical Science. She recently completed her PhD in the group of Prof. Matthew J. Gaunt at the University of Cambridge, and is currently pursuing an internship at Science Foundation Ireland.

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Resiniferatoxin– A Radical Approach

Resiniferatoxin (1) and daphnetoxin (2) are members of the daphnane diterpenoids—a class of diterpenoid orthoester compounds, many of which exhibit fascinating therapeutic activity. Resiniferatoxin, whose total synthesis has been achieved only once, is a strong analgesic possessing a complex and densely functionalised tetracyclic core. Now, the Inoue research group at the University of Tokyo have used a series of radical reactions to construct the tetracyclic skeleton of the daphnane diterpenoids (3).

The first radical process was a 3-component coupling of a cyclopentanone (4), O,Se-acetal 5 and an allylstannane (6), and proceeded via a bridgehead radical generated from the reactive O,Se-acetal species. This impressive coupling assembled the A and C rings of the cyclic skeleton, forming 5 consecutive stereocentres in one step. These stereocentres were generated with high stereoselectivity with regards to the tertiary centres at C4 and C10, while the reaction was stereospecific for the creation of the tetrasubstituted C9 centre.

Subsequently, the researchers performed a 7-endo radical cyclisation of xanthate 8 in order to construct the 7-membered B ring. The protected daphnane diterpenoid skeleton (9) was produced as a single isomer with the desired stereochemistry at C8.

Tetracyclic skeleton 3 represents a common intermediate which, following functional group manipulations, would allow the synthesis of naturally-occurring daphnane diterpenoids, including resiniferatoxin, in addition to artificial analogues. The approach demonstrated by the Inoue group showcases the power of radical reactions in the formation of highly congested carbon frameworks and reinforces their relevance in the field of total synthesis.

For more, read this ‘Hot’ Chemical Science article now:

Koichi Murai, Shun-ichiroh Katoh, Daisuke Urabe and Masayuki Inoue
Chem. Sci., 2013, Advance Article
DOI: 10.1039/C3SC50329A
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A possible new TB vaccine: Total Synthesis of Ac2SGL

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is responsible for the deaths of millions of people every year, while one third of the world’s population is infected with a dormant form of the bacteria. Researchers propose that better understanding of how the immune system copes with M. tuberculosis infection could aid the design of new vaccines to limit bacteria growth, and to eradicate any latent forms. Ac2SGL (1) is a sulfoglycolipid found in the outer membrane of M. tuberculosis which has been implicated in the modulation of TB growth and development, and as such, has potential for use in a new vaccine.

Structure of Ac2SGL with confirmed stereochemistry at C17 of the hydroxyphthioceranic acid residue

The first asymmetric total synthesis of Ac2SGL has been reported by Prof. Minnaard at the Stratingh Institute for Chemistry, University of Groningen. This elegant work resulted from a large international collaboration between researchers from the Stratingh Institute and the University of Toulouse, as well as a number of experimental immunologists from University Hospital Basel and the Singapore Immunology Network at the Agency for Science, Technology and Research (A*STAR).

To synthesise the hydroxyphthioceranic acid side chain, the researchers utilised an efficient and highly stereoselective iterative process for the construction of the 1,3-syn substituted fatty acid. The sequence was comprised of reduction, Horner–Wadsworth–Emmons olefination, and copper-catalysed asymmetric 1,4-addition (shown below).

Iterative sequence for the total synthesis of Ac2SGL

The side chain was completed using copper-catalysed asymmetric allylic substitution and platinum-catalysed diboration/oxidation to install the requisite functionality with excellent diastereoselectivity. This hydroxyphthioceranic acid residue was appended to the disaccharide trehalose. Finally, a regioselective sulfonation at the 2′-position completed the total synthesis.

The biological activity of the synthesised Ac2SGL was investigated, and found to be comparable with naturally isolated material. The researchers undertook modelling studies which shed light on Ac2SGL’s binding interactions and 3D conformation. This information, and the efficient total synthesis, facilitates further investigation into the use of Ac2SGL as a TB vaccine.

Read this ‘HOT’ Chemical Science article today:

Total synthesis, stereochemical elucidation and biological evaluation of Ac2SGL; a 1,3-methyl branched sulfoglycolipid from Mycobacterium tuberculosis

Danny Geerdink, Bjorn ter Horst, Marco Lepore, Lucia Mori, Germain Puzo, Anna K. H. Hirsch, Martine Gilleron, Gennaro de Libero and Adriaan J. Minnaard.

Chem. Sci., 2013, DOI: 10.1039/C2SC21620E

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