Some chemistry examples of our abilities.
Example 1 – Small Molecule Design and Synthesis:
Methotrexate (MTx) is a leading drug over a number of clinical applications. MTx is broken down into DAMPA and glutamic acid and antibodies raised to MTx also recognise DAMPA, creating issues in specificity. Our challenge was to explore the functionality of the two molecules and to design and build two synthetic molecules that mimic MTx and DAMPA to enable targeted antibody generation whilst reducing the cross-reactivity.
The team hypothesised that the structural similarities between MTx and DAMPA are responsible for the high cross-selectivity between the antibodies generated to both molecules. Removing the pterin group and accessing the 4-methylamino aromatic with the appropriate C-terminal differentiation enabled the team to produce two synthetic equivalent molecules that have enabled more specific antibody generation.
Example 2 – Building Complexity at the Molecular Level: Rationally Designed Multi-Funtional Peptides:
Using rational design the team can incorporate multi-functional capability into target molecules. The example below exhibits four key areas integrated into one peptide: Fluorescein, biotin tag, a protease cleavable sequence for target protease and a small molecule epitope.
This complete molecule is incorporated into a simple lateral flow device for a sensitive positive read protease activity diagnostic. Without each component being carefully designed and synthesised, the resulting diagnostic products are insensitive or work in a negative read manner, which for the user is unfavourable.
Epitope Scanning Using Peptide Array, Optimising Specific Antibodies and their Targets:
Mologic develops polyclonal antibodies on route to recombinant monoclonal antibodies and characterise their specificity through peptide array mapping using linear epitopes of the target protein. This information can then be used to refine an immune response by affinity maturation or affinity purification / selection to generate the recombinant monoclonals through a targeted selection process.
The example below is an array using a sheep polyclonal antibody raised to a larger protein, and short peptide sequences covering the entire protein were displayed in an array. The darker spots show where the antibody representing greatest affinity to the displayed peptide have bound, and which sequences can be used in further selection / maturation experiments.
Example 3 – Overcoming Difficult Peptides:
The team often encounter a request for the synthesis and isolation of very hydrophobic peptides. The peptide depicted below was achieved only after the incorporation of fmoc-pseudoproline building blocks.
The insertion of these pseudoprolines into the sequence disrupts the formation of the secondary structure thought to be responsible for problems during peptide assembly, and therefore leading to improved de-protection kinetics, improved yields, purities and solubility from the crude mixture. This results in greater resolution in our HPLC purification as illustrated in the spectrum below.