Adventurous combinations of research knowledge (ACORN)

Emergent catalytic properties from random peptide libraries and their relation to the “Origin of Life”. David TF Dryden, Mark Bradley, Michael Greaney, Perdita Barran and David Leigh
During this grant we explored 3 projects in this area. One was of a theoretical nature and a revised manuscript has now been submitted. The first experimental project was to start with fully folded proteins and then subject them to proteolysis to produce a peptide “library” and then to reverse the process to attempt to reassemble the active protein. We successfully repeated work on this in the literature but came to the conclusion that the reverse proteolysis was not due to macromolecular crowding effects as originally proposed but was due to the effects of ionic strength and the Hofmeister series. The second experimental project was to start with short peptides and investigate their self-assembly into larger structures under a variety of solution conditions including crowding and ionic strength. Crowding was found to have no effect but again the nature of the salt species was critical. The popular crowding agent, PEG, appears to actually simply separate out into two phases, an effect observed by physical chemists but not apparently by biological chemists whose samples are of too small a volume for the phase separation to be visible.
New ideas for catalytic Wittig, Mitsunobu and amide-bond forming reactions. Michael Greaney

The proposed research set out to develop greener, more atom-economical versions of some fundamental transformations in organic chemistry. We concentrated our efforts on the Wittig olefination, the principal method for converting a carbonyl compound into an alkene. The Wittig reaction is driven by the formation of a strong phosphorus-oxygen double bond, which generates a stoichiometric amount of triphenylphosphine oxide in the reaction. This by-product is soluble in organic solvents and presents an infamous purification problem, particularly when the chemistry is being carried out on a large scale.
Our strategy for improving the Wittig was based on making the reaction catalytic in phosphine. The addition of just 10 mol% of phosphine to the Wittig olefination, rather than the customary 100 mol%, would have an exponential impact on the environmental and economic footprint of the reaction. We attempted to reduce the triphenylphosphine oxide by-product in situ using a reducing agent. The challenge here is two-fold: First, reduction of the very stable phosphine oxide functional group is difficult and requires a strong reductant. Second, most reducing agents will easily reduce the carbonyl substrate faster than the triphenylphosphine oxide reagent.
We established a one pot Wittig reaction shown below in Scheme 1 using the bromoester 1, a benzaldehyde 2, a catalytic amount of triphenylphosphine oxide, a stoichiometric amount of trichlorosilane as the reductant and an amine base.

Scheme 1: Wittig reaction using catalytic triphenylphosphine oxide

Extensive optimisation of each reaction parameter established a workable reaction, producing good yields of the desired alkene product 3. However, control experiments established that the transformation was not in fact the sought-after catalytic Wittig reaction. The reaction was proceeding in the absence of any triphenylphosphine oxide, albeit in lower yield, implicating the silane as being the key player in activating the bromoester. Nonetheless, the reaction represented a novel example of using silanes, rather than phosphines, to mediate olefination. Given that the by-products of the silane are innocuous and easily removed upon purification, the Wittig reaction we developed met the objectives of the proposal.

Emergent catalytic properties from random peptide libraries and their relation to the “Origin of Life”. David TF Dryden, Mark Bradley, Michael Greaney, Perdita Barran and David Leigh
During this grant we explored 3 projects in this area. One was of a theoretical nature and a revised manuscript has now been submitted. The first experimental project was to start with fully folded proteins and then subject them to proteolysis to produce a peptide “library” and then to reverse the process to attempt to reassemble the active protein. We successfully repeated work on this in the literature but came to the conclusion that the reverse proteolysis was not due to macromolecular crowding effects as originally proposed but was due to the effects of ionic strength and the Hofmeister series. The second experimental project was to start with short peptides and investigate their self-assembly into larger structures under a variety of solution conditions including crowding and ionic strength. Crowding was found to have no effect but again the nature of the salt species was critical. The popular crowding agent, PEG, appears to actually simply separate out into two phases, an effect observed by physical chemists but not apparently by biological chemists whose samples are of too small a volume for the phase separation to be visible.
New ideas for catalytic Wittig, Mitsunobu and amide-bond forming reactions. Michael Greaney
The proposed research set out to develop greener, more atom-economical versions of some fundamental transformations in organic chemistry. We concentrated our efforts on the Wittig olefination, the principal method for converting a carbonyl compound into an alkene. The Wittig reaction is driven by the formation of a strong phosphorus-oxygen double bond, which generates a stoichiometric amount of triphenylphosphine oxide in the reaction. This by-product is soluble in organic solvents and presents an infamous purification problem, particularly when the chemistry is being carried out on a large scale.
Our strategy for improving the Wittig was based on making the reaction catalytic in phosphine. The addition of just 10 mol% of phosphine to the Wittig olefination, rather than the customary 100 mol%, would have an exponential impact on the environmental and economic footprint of the reaction. We attempted to reduce the triphenylphosphine oxide by-product in situ using a reducing agent. The challenge here is two-fold: First, reduction of the very stable phosphine oxide functional group is difficult and requires a strong reductant. Second, most reducing agents will easily reduce the carbonyl substrate faster than the triphenylphosphine oxide reagent.
We established a one pot Wittig reaction shown below in Scheme 1 using the bromoester 1, a benzaldehyde 2, a catalytic amount of triphenylphosphine oxide, a stoichiometric amount of trichlorosilane as the reductant and an amine base.

Scheme 1: Wittig reaction using catalytic triphenylphosphine oxide
Extensive optimisation of each reaction parameter established a workable reaction, producing good yields of the desired alkene product 3. However, control experiments established that the transformation was not in fact the sought-after catalytic Wittig reaction. The reaction was proceeding in the absence of any triphenylphosphine oxide, albeit in lower yield, implicating the silane as being the key player in activating the bromoester. Nonetheless, the reaction represented a novel example of using silanes, rather than phosphines, to mediate olefination. Given that the by-products of the silane are innocuous and easily removed upon purification, the Wittig reaction we developed met the objectives of the proposal.

a. How much of protein sequence space has been explored by life on Earth?
Revision being considered by Royal Society Interface Journal
David T. F. Dryden*, Andrew R. Thomson and John H. White
School of Chemistry, University of Edinburgh
Abstract
We suggest that the vastness of protein sequence space is actually completely explorable during the populating of Earth by life by considering upper and lower limits for the number of organisms, genome size, mutation rate and the number of functionally distinct classes of amino acids. We conclude that rather than life having explored only an infinitesimally small part of sequence space in the last 4 billion years, it is instead quite plausible for all of functional protein sequence space to have been explored and that furthermore, at the molecular level, there is no role for contingency.
b. Proteolysis and reverse proteolysis of Green Fluorescent Protein
In preparation
Andrew Thomson, Michael Millington, Kai Chen and David Dryden
School of Chemistry, University of Edinburgh
Abstract
GFP has a compact beta barrel structure that makes the native protein remarkably stable to proteolytic cleavage. We have shown that GFP can be proteolytically nicked at its loop regions, and that the proteolysed material retains the tertiary structure of the beta barrel despite the absence of a covalent connection. The nicked GFP remains fluorescent and is stable enough to be isolated. Furthermore the nicked GFP can be resynthesised simply by reducing the temperature of the protease reaction from 37oC to 4oC. This reassembly was a serendipitous discovery and may provide a facile way to manufacture novel GFP derivatives from peptide fragments of GFP.
c. Anion Effects on Coiled Coil Peptide Dimerisation
In preparation
Andrew Thomson and David Dryden
School of Chemistry, University of Edinburgh
Abstract:
The effect of different anions on the ability of model peptides to form homo- and heterodimers was investigated using pyrene excimer fluorescence as a probe. Depending on the charge of the peptide sequence different behaviours were observed, following either Hofmeister or anti-Hofmeister trends for the dimerization constant of the coils. These findings show that, for simple model systems at least, the nature of any co-solute salts can have a significant and unpredictable impact on the magnitude of intermolecular interactions.
Further notable outcomes
Dr Andrew Thomson has been appointed as laboratory manager for Professor Dek Woolfson, Dept. of Chemistry, University of Bristol. This will facilitate collaboration on further work on peptides between his lab and the Dryden lab.
New ideas for catalytic Wittig, Mitsunobu and amide-bond forming reactions. Michael Greaney
Smith, J. M.; Greaney, M. F. ‘A one-pot silyl-Reformatsky olefination’ Tetrahedron Lett., 2007, 48, 8687-8690.
Further notable outcomes
The PDRA working on the project, Dr James M. Smith, has been employed as a medicinal chemist at Cancer Research Technologies in Cambridge.