Peptide-6-amino-D-luciferin conjugates can be used for measuring protease enzyme activity. Certain enzymes are tumour markers, the activity of which is usually detected with fluorescent systems, but no bioluminescent systems are used as yet. The significance of developing a bioluminescent detection method for these enzymes lies in the fact that with this the detection threshold is orders of magnitude lower than that of the fluorescent technique.
The published synthesis methods of these conjugates are complicated, mainly because of the unusually low reactivity of the amino function in position 6; furthermore, the commercially available conjugates are very expensive. Therefore, we set a goal: developing a new and more economical way of synthesis.
We tried to protect the amino function with Fmoc, but we found that the low reactivity of the amino function often leads to low yield; and due to the basic conditions of the Fmoc strategy, there is a heightened risk of dehydrogenation and racemisation.
We also worked out a fragment condensation strategy. During this, we coupled a suitable protected peptide to 6-amino-2-cyanobenzothiazole, which was followed by the addition of D-cysteine. Although this way it is possible to synthetize the desired conjugates, the method also has certain limitations: when attaching longer peptides, solubility problems occur.
During the Boc method we prepared Boc-protected amino-luciferin: with triphosgene and tert-butanol, we managed to produce the protected heterocycle, which was then reacted with D-cysteine, and finally we had the desired material. Although most coupling and cleaving reagents damage the product, we managed to find methods which make it possible to attach the Boc-amino-luciferin to resin, and at the moment we are working on building the peptide chain, which will be followed by cleaving the resulting peptide-heterocycle conjugate from the resin.
If successful, this third method will increase the possibilities for the synthesis of peptide-6-amino-D-luciferin conjugates.