One of the "Golden Triangle" series of building blocks: acridine-based photoaffinity probes with "big" applications in a small body
Product Manager：Nick Wilde
Photoaffinity probes (PAPs) are nowadays the "magic weapons" in drug discovery and development, which can unlock the binding mechanism of drugs and target proteins and find new drug action sites. Among them, diazirine-based photoactivation reagents are the most widely used.
Introduction
Diazirine is a ternary ring structure containing two nitrogen atoms. It was initially studied spectroscopically as a carbene precursor and has since been widely used as photoaffinity probes (PAPs). When exposed to UV light, diazirine can generate highly reactive carbenes and form covalent bonds with surrounding molecules. It is this unique mechanism that allows it to label target proteins with high selectivity and map protein-ligand binding sites.
Currently, diazirine-based photoactivation reagents are classified into two main categories.
(1) aliphatic diazirines
(2) trifluoromethylphenyl diazirines (TPDs).
Compared with other PAPs such as phenylenedione and aromatic azide, bisacridines have the advantages of low photodamage and high selectivity, which are favored by pharmaceutical chemists.
● Early aliphatic diazolidines suffered from the limitation of 1,2-hydrogen rearrangement reaction, which limited their applications.
● In 1973, Knowles et al. found that the intramolecular rearrangement tendency of 3-phenylbisacridine was weakened, which improved its photolysis properties.However, its photolysis produces predominantly diazonium species that diffuse from the active site before reacting with nucleophiles, leading to nonspecific labeling.
● In 1980, Brunner et al. proposed trifluoromethylphenyl diazetidine (TPD) as a superior alternative.TPD reduces the occurrence of diffusive reactions due to the degenerate electronic properties of trifluoromethyl. Although the reactivity of the carbene spin state and the effect of various substituents is not fully understood, the diazo crosslinking properties are undeniable.
● Aliphatic and trifluoromethylphenyl diazirines are becoming increasingly popular and their applications are expanding.
Application
Target identification is a difficult and painful part of drug development. Diazirine-based photoaffinity probes show the way. It has been successfully applied to identify ligand targets, map binding sites, study protein-protein interactions, and live cell imaging. More excitingly, some diazirine amino acids can also be recognized by cellular synthesis systems and inserted into proteins for covalent labeling of targets.
1. Recognize ligand targets:
Recognition of target proteins through binding to specific ligands helps researchers to determine the target of action of a molecule and identify potential drug targets. Amino acids containing diazepines are also recognized by the native cellular transcription machinery.
2. Mapping binding sites:
By labeling binding sites in the protein structure, it helps researchers to map the sites of ligand-protein interactions, thus improving the understanding of substrate binding and catalytic mechanisms.
3. Study of protein interactions:
Used as a probe to study protein-protein interactions, such as protein-protein interactions, enzyme substrate binding, etc., to reveal the complex biomolecular networks in organisms.g
4. live cell imaging:
Used for live cell imaging to visualize intracellular biomolecules by labeling target substances or target proteins, helping researchers to study cellular functions, disease processes, and so on.
Synthesis
Although the unique formulation has attracted the favor of countless medicinal chemists, the synthesis process of diazirines is not so friendly. TPDs, for example, require at least a 4-step reaction with a low overall yield. The synthesis of aliphatic diazirines is relatively simple and can be accomplished by a one-pot method of ammonolysis and cyclization of ketones in liquid ammonia, etc. Wang's group optimized the reaction conditions to increase the yield to 81%, which provides more possibilities for the application of diazirines. The following is the synthesis and design of TPD and aliphatic diazirines.
Photoactivation of Diazirines
Typical Routes Used To Synthesize Aliphatic Diazirine
TPD Synthetic Routes
3-Azibutanol Was Used in Synthesis of Patented Phospholipid Photoaffinity Probes
Typical Functional Group Interconversions with Aliphatic Diazirines
Synthesis of Minimalist Alkyne-Diazirine Probes and Staurosporone Example
Synthesis of Cyclopropene Photolinkers and Their Use in Live Cell Imaging
Four Common Synthetic Routes To Access Photo-Leucine
Synthesis of Trifluoromethyl Aliphatic Diazirines
Synthesis of Boc and Fmoc Protected L-Photo-Proline
Reactions of TPD Aldehydes
Reactions of TPD Anilines
TPD-Compatible n-BuLi Reaction
TPD Reactions in Basic Conditions
TPD-Compatible Metal-Catalyzed Coupling Reactions
Summary
Earlier methods for synthesizing diazirine photocrosslinkers typically had low yields. However, the advent of new methods has greatly increased the yields and reduced the number of synthesis steps required. These methods will undoubtedly be widely used. A number of amino acids containing diazirine, both protein-derived and non-protein-derived, have been successfully synthesized. Among them, synthetic methods such as photoleucine and photomethionine have been well developed with short synthetic routes and high yields. Nevertheless, for high yields of bis-acridines, the possibility of improvement still exists and newly developed chemical methods are needed. Designing short synthetic strategies would be an attractive prospect.
The current applications of diazirine photoaffinity probes are exciting and the chemistry of these molecules has been rationally exploited, and in order to realize the full potential of the efficiency of this technology, scientists will continue to expand the synthetic methods and shorten the synthesis process, advancing the field of chemistry and drug discovery.
Reference
James R. Hill† and Avril A. B. Robertson. Fishing for Drug Targets: A Focus on Diazirine Photoaffinity Probe Synthesis. J. Med. Chem. 2018, 61, 6945−6963. https://doi.org/10.1021/acs.jmedchem.7b01561