This project involves development of new and more efficient tritium labelling techniques of proteins and peptides.
Tritium-labelling of small-molecules have been thoroughly studied and various kinds of labelling techniques derived from synthetic chemistry have been developed. Though these don’t apply as general to proteins and peptides – The field of tritiation of peptides is narrow and the methods presented are sparse and mainly concentrated about the catalytic 3H-hydrogenation of unsaturated precursors or by detritioiodation of iodinated compounds – no one has at the moment published a good general method for tritiation of proteins that does not involve bioconjurgation.
The Project.
This project involves development of new and more efficient tritium labelling techniques of proteins and peptides.
We want to investigate and improve the accessibility and specificity of tritium incorporation into peptides and proteins. A few examples have been reported for the labelling of Proteins but no general methodology is present which result in the native, non-modified, protein.
Our initial focus and target will be on the native protein. This mean that we want to output a labelled compound with no chemical modification (eg. N-terminal tritioacylation or chemical modifications that interfere with protein activity) except for the X/3H exchange. This is why we at first want to focus on the dehydrohalogenation of iodated tyrosine residues incorporated into a peptide chain.
This project will include and has included:
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Identification of small peptides (substrates) of interest for radio-labelling for test and optimization studies.
Manually Solid Phase Peptide Synthesis (SPPS) of substrates for deuterium/tritium labelling. Eventually this could result in synthesizing peptide analogues for animal studies.
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Deuteriation / Tritiation of peptides and proteins using gaseous deuterium and tritium in a specialized manifold system. Screening different catalyst and solvents for optimum of labelling efficiency.
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With collaboration of our partners (Novo Nordisk A/S) we will initiate the tritiation of larger peptides and proteins which is difficult with traditional tritium-labelling methods. An example could be iodinated human insulin.
Current known methodologies for Tritium Labelling of Peptides.
(Halogen – Tritium exchange) Dehydrohalogenation of halogenated aromatic amino acid residues. This method is generally known for the labelling of peptides using transition state metals (mostly palladium) as heterogenous catalysts and tritium gas. The substrate for labelling is made by either synthesised peptides with halogenated amino acids or by halogenation of the peptide/protein directly.
(Hydrogenation) Catalytic Deuteriation/Tritiation of unsaturated precursors (amino acids or peptides) with gaseous tritium in solution.
(Bioconjurgation) Amine-tritio-acylating with highly active acylating reagents. This method can be done at any free amine group on peptide/protein surface either N-terminal or on lysine amine.
The Radioisotope
Tritium is an isotope of hydrogen with an atomic mass of approximately 3 units, it is a pure and weak electron (β-) emitter. With a energi of maximum 18.6 keV and an average of 5.7 keV, the detection of the radioactive decay, may be preformed only by Liqiud Scintillation Counting or by special Solid Scintillators. The triton nuclide has a halflife of 4500 days or 12.3 years, this is a relatively long halflife, which means that the shelflife of compounds labelled with tritium is also long compared with other nuclides.
The Hevesy Laboratory Tritium Manifold System
Our custom build tritium manifold system (RC Tritec) for the safehandling of multi-curie (Ci) amounts of tritium gas.
The tritium-gas is stored on metallic depleted uranium as uranium-tritride, and can be released as carrierfree T2 gas upon heating of the uranium bed to about 500oC.
In special glassware the tritium-gas can be used as in ordinary hydrogenation reactions or by the creation of different reducing and nuclephilic tritrides.
Extraordinary caution must be taken when preforming radiochemistry with gassious forms of radioactivity, so that’s why we have a tritium gas detector (from Canberra) at our disposal for our own protection, as well as a dedicated closed isotope fumehood.
With RP-HPLC we prepare and analyse tritiated compounds and with a radio-mass-spectrometer instrument we can also detect the incorporation of tritium in organic molecules.