subscribe
 

Use of fluorescent dyes increases in development of peptide products

1st April 2013


Dr. David Woodhouse looks at a range of peptide productsto design and satisfy the requirements of drug discoveryand development activities.

Radiolabelled peptides are at the extreme end of the weight range. One ­ 100µCi, tritium labelled neurokinin alpha (4,5-3H-Leu9-Neurokinin alpha), represents about 0.75µg of peptide at a specific activity of 150Ci/mmole. These highly specialised products are manufactured using a combination of peptide expertise and radioactive isotope handling technology.

The two most common isotopes used to radiolabel peptides are tritium (hydrogen-3) and carbon-14 since these can replace their natural isotope in the peptide to produce a material that is virtually identical in properties to the original peptide.

The results obtained from using these materials, such as in drug metabolism studies, can therefore be relied upon to be those of the non-labelled materials. This is not always the case with other isotopes; iodine-125 for example, a large unnatural isotope, often distorts the peptide molecule and causes it to behave differently to the non-labelled version.

Radiolabelled peptides are commonly prepared by one of two approaches. Peptide requiring labelling with tritium can often be conveniently obtained by treating a previously prepared aprecursor' peptide that contains an unsaturated or halogenated precursor amino acid with tritium gas in the presence of a catalyst. Under these conditions the precursor amino acid will be transformed into a natural amino acid containing tritium (for example, 4,5-dehydroleucine into 4,5-3H-leucine). With carbon-14 and some tritium labelled amino acids the radioactivity is not so readily introduced and a more involved direct synthesis of the peptide is needed. Special techniques are required to handle the high value amino acids and introduce them into the peptide.

Fluorescent peptides

Recently there have been developments that have allowed the replacement of radioactively labelled peptides with those labelled with fluorescent dyes for certain applications, principally high throughput screening. This technique, where thousands of compounds can be screened for activity very quickly, requires the availability of significant quantities of reagents and this can cause handling and disposal problems if the peptides are labelled with radioactivity. The use of fluorescent probes has been made possible by the development of highly intensely coloured dyes such as the CyDyes of Amersham Biosciences and those offered by Molecular Probes.

When a highly fluorescent dye is used in conjunction with an acceptor or quenching fluorophore the technique of FRET (fluorescence resonance energy transfer) can be used. This requires a material, such as a peptide, to be labelled at opposite ends with a donor fluorophore (such as Edans) and an acceptor fluorophore (such as Dabcyl). When the donor/acceptor pair is in close proximity little or no fluorescence is observed but if the peptide molecule is cleaved by an enzyme, for example, the acceptor is removed from the fluorophore and fluorescence is observed.

Fluorescent labelled peptides are usually prepared initially in relatively small quantities (1-2 mg) for evaluation purposes and then, once successful candidates have been identified, they may be required in larger quantities (10-100mg).

The preparation of these materials presents certain challenges principally associated with the need to use the minimum quantities possible of the (usually) expensive dye components. In this area, the skills acquired during the synthesis of radiolabelled peptides can be effectively employed. Another important factor is the selection of an appropriate strategy for the synthesis of doubly labelled peptides: which dye to introduce first, which type of protection for the assembled peptide and determination of suitable purification conditions.

The synthesis of quantities of peptides in the range 1­100g represents a significant undertaking for any peptide chemist. In the preparation of routine peptides on a mg scale it is possible to accept the presence of impurities in the crude peptide that an automated solid-phase approach will inevitably generate since these can usually be removed relatively easily during the subsequent purification stage. However the removal of these impunities from multi-gram quantities of peptides is more onerous and so the assembly of these peptides requires a much greater effort than is usually afforded to lower quantities. These usually require a ahands-on' assembly where the successful introduction of each amino acid into the elaborated peptide is followed by various analytical techniques to ensure that each amino acid is completely added. Similarly it is important to consider the use of various conditions to cleave the peptide from the solid support since this is a process that can lead to the introduction of significant impurities in the crude peptide.

The benefit of extra attention paid during the assembly and cleavage of larger quantities of peptide will be found during the purification process. Purifying larger quantities of peptides requires larger scale purification kit but even with this it is usually necessary to undertake multiple purification runs to achieve the desired quantities. In these cases it is useful to introduce automation into the purification process. HPLC systems can perform repetitive runs that allows them to operate unattended during the day and overnight and conditions can usually be chosen that minimise the amount of solvents consumed and fractions generated. u

ENQUIRY No 19

Dr. David Woodhouse is with Cambridge Research Biochemicals, Billingham, Cleveland, UK. www.crb.gb.com





Subscribe

Subscribe



Newsbrief

FREE NEWSBRIEF SUBSCRIPTION

To receive the Scientist Live weekly email NewsBrief please enter your details below

Twitter Icon © Setform Limited
subscribe