Design and synthesis of an all-in-one 3-(1,1-difluoroprop-2-ynyl)-3H-diazirin-3-yl functional group for photo-affinity labeling

Abstract

A novel radioisotope-free photo-affinity probe containing the 3-(1,1-difluoroprop-2-ynyl)-3H-diazirin-3-yl functional group was designed and synthesized. This very compact functionality is envisaged to allow photochemically-induced coupling of a compound to its target followed by click reaction coupling with an azido-biotin reagent in order to facilitate purification of the labeled target. In a proof-of-concept study we have shown that 3-(1,1-difluoroprop-2-ynyl)-3H-diazirin-3-yl functional group could be photolyzed to efficiently furnish the methanol adduct 23 and that the generated highly unstable carbene does not react with the neighboring acetylene moiety. A subsequent click reaction with the azido-biotin derivative 25 proceeded smoothly to give triazole 26. This chemical probe should thus be of unique value for facilitating identification of the molecular structure of the target of a bioactive compound.

Introduction

Photo-affinity labeling (PAL) is an efficient and reliable tool used to identify, isolate and characterize novel biological molecules and potential drug targets, particularly when the target molecule is unknown.1, 2, 3, 4, 5, 6, 7, 8, 9, 10 In PAL, a ligand incorporates a photoreactive group in its structure which can be irradiated when it complexes with its target. Irradiation gives rise to a highly reactive intermediate, which in turn forms a covalent bond between the ligand and the target protein. Even though many types of photosensitive groups have been used such as azides, diazirine, and benzophenones that are suitable for PAL, aromatic diazirines have enjoyed widespread application and are considered to be the most effective PAL functional group because they generate carbenes, which react with many functional groups including C–H bonds11, 12 and they can be activated at a long wavelength (λ >300 nm), thus, preventing damage or competing activation of the examined biological system.

In order to identify and facilitate the isolation of the photo-labeled product, a reporter group such as a radioactive label, a biotin tag or a fluorescent tag is often incorporated into the PAL ligand.⁷ Radioactive labeling is often used for tracing the tagged molecules because of detection sensitivity and because radioactive labels do not need to employ sterically demanding tracer groups that may perturb interactions of the biological target and the labeled reagent. However, the hazardous nature of the radioactive ligand inhibits use and complicates the direct analysis of the tagged molecules. In addition, radioactive tagging methods do not offer a ready handle for target molecule enrichment and isolation. To address these disadvantages, biotin tagging has been developed which takes advantage of the high affinity between biotin for avidin to allow affinity purification of the probe-target adduct. However, the large and highly polar biotin unit often has unfavorable effects on the bioactivity of the probe.¹³ To address this issue, a number of ‘fishing’ techniques have been devised wherein ligands are made which incorporate either an alkyl azide or terminal alkyne instead of biotin. Once the PAL ligand–target complex is photo-reacted, the azide or the alkyne group can be used as a tag for the consecutive attachment of a detectable group (e.g., biotin) using either azido-targeting or alkyne-targeting bioconjugation reactions such as Cu(I) catalyzed 1,3-dipolar cycloaddition (click chemistry).14, 15, 16, 17, 18, 19, 20

Recently, Hosoya and co-workers have prepared a compact ‘all-in-one’ (3-azidodifluoromethyl-3H-diazirin-3-yl)benzene derivative 1 to be used for radioisotope-free PAL.²¹ Unfortunately, azido-containing compounds are potentially explosive, and in our hands a derivative (3) of compound 1 exploded when it was either treated with palladium, Cu(I), or Cu(II) catalyst (Fig. 1).²² In a separate attempt to make the 3-azidodifluoromethyl-3H-diazirin-3-yl group, the azido-O-tosyl-oxime 4 decomposed to give the undesired nitrile 5 when it was treated with ammonia under reaction conditions similar to those reported by Hosoya and co-workers. Given that 3-azidodifluoromethyl-3H-diazirin-3-yl group appeared to be unstable and more importantly, explosive in nature under our reaction conditions, we envisioned that a compound bearing an acetylene moiety instead of an azide should be more suitable. This is indeed the case and we report herein the first synthesis of the photo-affinity probe containing a 3-(1,1-difluoroprop-2-ynyl)-3H-diazirin-3-yl group (2).

In order to illustrate the potential use of the ‘all-in-one’ 3-(1,1-difluoroprop-2-ynyl)-3H-diazirin-3yl group we report the synthesis of a derivative (7) of a known 5-lipoxygenase (5-LO) inhibitor 6²³ and we also describe a sequence of carbene-trapping followed by click chemistry using an azido-biotin reagent 25.

Leukotrienes (LTs) are lipid mediators implicated in numerous diseases, including inflammation, atherosclerosis, and respiratory diseases such as asthma24, 25, 26, 27, 28, 29, 30, 31, 32 and 5-LO plays a key role in the biosynthesis of LTs from arachidonic acid. Therefore, blocking LT biosynthesis could lead to the development of novel therapies for such diseases. There are some indications of multiple protein complexes involved in LT biosynthesis in cells³³ and compounds such as 6 have shown anomalous LT inhibitory activity in cells³⁴ and may interact with other undefined protein targets. Hence, an affinity ligand such as compound 7 bearing the 3-(1,1-difluoroprop-2-ynyl)-3H-diazirin-3-yl group could be used to ‘fish out’ 5-LO or any other protein this molecule might interact with within the LT biosynthesizing cells. The results of these studies will be published elsewhere.