Exploiting Biocatalysis in the Synthesis of Supramolecular Polymers - Enzymatic Polymerisation - page 134

4.2 Enzyme-Triggered Self-Assembly Under

Thermodynamic Control

Proteases are well known for their ability to hydrolyse peptide bonds. The Gibbs
free energy change of amide synthesis/hydrolysis is small and the reaction is
readily reversed, for example by relative stabilisation of the peptide over the
hydrolysis products, as shown previously in organic solvent systems and het-
erogeneous reaction media [ 58 - 60 ]. By coupling protease catalysis with peptide
self-assembly, a similar reversal of hydrolysis to preferred synthesis occurs whereby
the self-assembly provides the thermodynamic driving force for peptide synthesis
[ 21 , 23 ] . The resulting thermodynamically controlled systems are fully reversible
and will proceed towards the lowest accessible free energy state. Interestingly,
when mixtures of starting materials are supplied, these systems should self-select
the most thermodynamically stable structures from dynamic mixtures, as discussed
below.
Dynamic combinatorial libraries (DCLs) are continuously interconverting li-
braries that eventually evolve to an equilibrium distribution [ 61 - 65 ]. This approach
has been used successfully in the discovery of stable supramolecular assemblies
from mixtures. Due to the nearly endless possible peptide sequences that can po-
tentially be synthesised, the DCL approach is attractive for the identification of
supramolecular peptide interactions. Indeed, disulfide exchange between cysteine
residues has been explored for this purpose [ 66 , 67 ] as has peptide-metal binding
[ 68 ]. We have recently demonstrated protease-catalysed amide exchange in this
context, which allows for the evolution of the self-assembled peptide structures,
and will therefore allow exploration of peptide sequence space for biomaterials
design.
Evolution of peptide nanostructures has been investigated for Fmoc-L peptides
(Fig. 6 ) . When Fmoc-L and LL were exposed to a non-selective protease, a Fmoc-
L n oligomer distribution results. Upon initiation of the reaction, Fmoc-L 3 is found
to be formed as the major component (because it is the direct coupling product
between the starting materials). Overtime, the system rearranges itself and eventu-
ally reaches an equilibrium distribution in which Fmoc-L 5 is predominant (Fig. 6 ) .
Analysis by atomic force microscopy (AFM) showed a drastic change in morphol-
ogy from fibres (Fmoc-L 3 ) to sheet-like structures (Fmoc-L 5 )(Fig. 6 a ) [ 21 ]and
suggests that the sheet-like pentapeptide structure represents the lowest accessible
folded state for this system. This enzymatic DCL approach was also explored for the
screening of a range of dipeptide sequences in Fmoc-dipeptide-methyl ester gela-
tors [ 69 ] .
Thus, dynamic peptide libraries offer the potential to identify the most stable
self-assembled supramolecular nanostructure from a mixture of several components.
This opens up the possibility of exploiting the versatility of peptides for the discov-
ery of new nanostructures.