A Focus on Diazirine Photoaffinity Probe Synthesis - American

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Cite This: J. Med. Chem. 2018, 61, 6945−6963

Fishing for Drug Targets: A Focus on Diazirine Photoaffinity Probe Synthesis James R. Hill† and Avril A. B. Robertson*,†,‡ †

Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland 4072, Australia School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia QLD4072, Australia

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‡

ABSTRACT: Target identification is a high-priority, albeit challenging, aspect of drug discovery. Diazirine-based photoaffinity probes (PAPs) can facilitate the process by covalently capturing transient molecular interactions. This can help identify target proteins and map the ligand’s interactome. Diazirine probes have even been incorporated by cellular machinery into proteins. Embarking on the synthesis of customized PAPs, containing either an aliphatic or trifluoromethyl phenyl diazirine, can be a considerable endeavor, particularly for medicinal chemists and chemical biologists new to the field. This review takes a synthetic focus, aiming to summarize available routes, propose new avenues, and illuminate recent advances in diazirine synthesis. Select examples of diazirine photoaffinity labeling applications have been included throughout to provide instructive definition of the advantages and limitations of the technology while simultaneously highlighting how these reagents can be applied in a practical sense. view on past diazirine summaries5,17 and complementing recent PAP reviews.18−21

1. INTRODUCTION Discovered in the 1960s, 3H-diazirines (diazirines) are unusually stable three-membered rings containing one carbon and two nitrogen atoms, with a double bond between the two nitrogen atoms.1−3 Diazirines were initially investigated as carbene precursors in spectroscopic studies and have since become widely used as photoaffinity probes (PAPs).4,5 Upon exposure to light, heat or ultrasonication6 diazirines liberate nitrogen to form carbenes, either directly or via slow photoisomerization to the linear diazo (Scheme 1).7 Carbenes can undergo a range of reactions depending on their spin state, either singlet or triplet.8 Crucially, singlet carbenes undergo C− H and heteroatom−H insertion to cross-link with proximal species.9,10 Early aliphatic diazirines (1) were known to undergo 1,2hydrogen rearrangements, initially limiting their utility.11 In 1973, Knowles et al. identified that 3-phenyl diazirine 2 exhibited a reduced tendency to undergo intramolecular rearrangement, improving its photolabeling utility.12 Unfortunately, photoactivation of 2 produced primarily the diazo species, causing nonspecific labeling as a result of dispersion from the active site before reacting with nucleophiles.12−14 In 1980, Brunner et al. proposed the trifluoromethyl phenyl diazirine 3 (TPD) as a superior alternative because the electron withdrawing nature of the trifluoromethyl group reduced the proportion and reactivity of the formed diazo species.14 Despite an incomplete understanding of the reactivity of carbene spin states and the effect of various substituents,15,16 there is no denying the utility of diazirines as PAPs due to their favorable cross-linking properties. This review will focus on the synthesis and design of TPDs and aliphatic diazirines, providing a fresh © 2018 American Chemical Society

2. PHOTOAFFINITY LABELING PAPs are multicomponent tool compounds useful for identifying ligand targets, conducting binding site mapping, investigating protein−protein interactions, and live cell imaging.18−22 PAPs contain a photo-cross-linking unit, a bioactive motif, and a reporter or purification tag (Figure 1), allowing spatioselective cross-linking with a biological target. Fundamental to this mechanism is the photo-cross-linker, which generates a highly reactive species upon ultraviolet (UV) irradiation, and the bioactive motif, which gives the PAP targetselectivity. When performing live cell photoaffinity labeling, a reporter/purification tag is included for identification of the PAP-target complex; these have been reviewed extensively elsewhere.18−22 With recombinantly expressed proteins, reporter/purification tags are not always required.23 Incorporating these three components into one biologically active compound requires a thorough understanding of the structure− activity relationships (SARs) to maintain target affinity and orientate the reactive motif in a manner allowing crosslinking.18 PAPs have a distinct advantage over electrophilic cross-linking agents because they can exist in dormant and active states, allowing them to reach equilibrium before being activated.24,25 Furthermore, PAPs can cross-link with functional groups unreactive toward electrophilic probes, making PAPs less biased toward nucleophilic protein side chains and avoiding the need for electrophile-nucleophile matching.25 Received: October 19, 2017 Published: April 23, 2018 6945

DOI: 10.1021/acs.jmedchem.7b01561 J. Med. Chem. 2018, 61, 6945−6963

Journal of Medicinal Chemistry

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Scheme 1. Photoactivation of Diazirines

heteroatom−H insertion.27 If the carbene is proximal to a protein, covalent cross-linking will occur; alternatively, the diazirine will be quenched by water.14 The propensity for diazirines to be quenched by water dictates that cross-linking yields are typically under 10% 28 and that positioning of the diazirine with respect to the active site is of heightened importance. Consequently, a number of diazirine-containing analogues should be synthesized to identify the optimal balance of target affinity and cross-linking yields. Although aliphatic diazirines can undergo undesirable rearrangements, these moieties have become increasingly popular due to their small size. Minimizing a photo-crosslinker’s steric demands facilitates accommodation within the active site of target proteins, leading to higher labeling yields. This also allows strategic placement on the ligand such that the diazirine moiety can probe specific ligand−protein interactions. Furthermore, by using a minimalist probe approach, the ligand’s physicochemical properties are not dramatically changed. In contrast to aliphatic diazirines, the effective size of TPDs and therefore their potential to maintain target affinity are dependent on whether biological activity requires or merely

Figure 1. PAPs covalently bind target proteins.

Diazirines have a number of advantages over other commonly employed photo-cross-linkers such as benzophenones, aryl azides, and the more recently discovered 2-aryl-5carboxytetrazole.26 Crucially, diazirines cross-link upon exposure to longwave (∼360 nm) UV irradiation, minimizing damage to proteins which occurs below 300 nm.14 Diazirines cross-link in a highly selective manner, reducing the risk of nonspecific binding that can occur when reactive intermediates diffuse from the site of activation, thus increasing the confidence that cross-linking has correctly identified the target protein. The high selectivity of diazirines is derived from their cross-linking mechanism, which involves the generation of a carbene upon photoactivation. This carbene exists for picoseconds before reacting with proximal species via C−H or

Figure 2. Biologically active compounds and their TPD derivatives: (A) nested TPDs; (B) appended TPD; (C) replacement TPD. 6946



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corresponding diazirine 17 can be achieved with several reagents including freshly prepared silver(I) oxide, iodine in the presence of triethylamine, or Hünig’s base.37 This methodology has been applied successfully to relatively complex aliphatic skeletons such as steroids and carbohydrates; however, reported literature yields of this three-step diazirine formation are typically 30−40%. Recent improvements provided a one-pot method for synthesizing aliphatic diazirines from ketones (Scheme 2). This was achieved by treating levulinic acid 14, where R = H, R′ = CH2CO2H, with HOSA in liquid ammonia at room temperature for 12 h, followed by the addition of potassium tert-butoxide affording the diazirine 17 in an impressive 81% yield.38 Further investigation, via systematic modification of the base used, ultimately identified a high-yielding, scalable methodology that avoided the use of costly and flammable potassium tert-butoxide.39 Sodium hydride gave an improvement over conventional three-step methods, yielding 47% product. The magnesium and calcium hydride bases were particularly poor (80% yield.48,49 Azialcohols 33 can be oxidized to the carboxylic acid 39, often in high yield, using common mild oxidants such as Jones reagent50 or Dess−Martin periodinane (DMP).33 The acid can be coupled directly to form amides 40 using typical coupling reagents, such as dicylcohexylcarbodiimide (DCC) and 1-ethyl-3-(3-

view to minimize both photo-cross-linker and alkyne click handle size, thereby maximizing the chance of efficient binding to the target protein. In this synthesis it should be possible to considerably shorten the route to the acid 43 from 45 using Jones oxidation in place of iodine and triethylamine to simultaneously form the diazirine and acid moieties from the diaziridine.50 42, 43, and 44 were appended to 12 different kinase inhibitors and used to probe for target proteins in cellbased proteomic studies. In direct comparison to the minimalist linker on staurosporone 46 to the more branched and therefore bulky design of 47, additional previously unknown protein targets were identified and later validated.52 This minimalist approach was extended in an elegant and unusual way to give the first set of PAPs for live cell imaging. Normally the alkyne click handle is used to attach a reporter/ purification tag via a copper-catalyzed dipolar cycloaddition, 6949



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Scheme 10. Synthesis of Cyclopropene Photolinkers and Their Use in Live Cell Imaging55

allowing purification of the target from the cellular milleau. However, if live cell imaging is intended, this approach is not ideal. Drawing on earlier work by Weissleder et al.,53,54 Li converted the alkyne of 42 to a cyclopropene 48 suitable for bioorthogonal ligation to a fluorescent tetrazine via an inverse electron demand Diels−Alder reaction (Scheme 10). Given that there are seven synthetic steps to access iodo 42, as shown in Scheme 9, improvement of the cyclopropene 48 yield of 21% would be desirable. Nevertheless, these innovative agents were conjugated with GW841819X (49), a protein−protein interaction inhibitor of the epigenetic protein bromodomaincontaining protein 4, affording cyclopropene-containing diazirines 50 and 51. These cyclopropenes were then utilized for bioimaging in live mammalian cells through photoactivation of the diazirine followed by in situ ligation to the tetrazine tag to give 52.22 Ligation of the cyclopropene ester derivative 50 was 10- to 2000-fold slower than with the alcohol variant 51 and therefore less favored. There are further developments in the tetrazine Diels−Alder reagents where these can be tuned according to pendent groups.55 Unfortunately these initial cyclopropene photo-cross-linkers caused high nonspecific background labeling.33 A further example of live-cell imaging was conducted with the most advanced clinical Aurora kinase cell cycle inhibitor, MLN8237.56 Three different probes were synthesized: an alkyne 53, an alkyldiazirine-alkyne 54, and an octene 55 (Figure 4). The alkynes were used for copper-catalyzed click chemistry, while the octene was reacted with a tetrazine tag for imaging. All three were used simultaneously in a multiplexed approach aiming to achieve small molecule bioimaging alongside target identification. Interestingly, the alkyldiazirine-alkyne performed as well as the octene-tetrazine upon bioimaging, and the authors indicate higher quality probes are required. These first generation live-cell small molecule imaging tools are an exciting development that will undoubtedly be improved upon in the future. Aliphatic diazirine probes have been used alongside other non-diazirine components in the generation of a chemical library based on the Ugi-azide multicomponent reaction (Scheme 11).57 Six isocyanides, eighteen carbonyl reagents

Figure 4. Three different linkers were attached to Aurora kinase inhibitor MLN8237 and used simultaneously in bioimaging and target identification.56

(three diazirine containing), and eight amines (six diazirine containing) were reacted together in a sparse matrix format to give sixty final products. The resulting molecules each contained a diazirine moiety and an alkyne. The probes were tested in a cell based assay, using the prostate cancer cell line PC-3, with photochemical activation to induce target capture and fully map the small molecule−protein interaction network. The alkyne handle was used for enrichment by reaction with fluorescent rhodamine azide via click chemistry. Proteomic studies then successfully identified target proteins. This library contained molecules with molecular weights 300−500, typical of many orally available drugs. Early stage fragment-based drug discovery typically involves screening of small molecular weight molecules against a particular target such that these molecules can achieve an optimal orientation when binding in a protein pocket. Larger compounds (molecular weight >300) have a greater chance of steric clash with the binding site or adopt a suboptimal binding pose. Once the binding site is identified, fragments can be chemically elaborated through extending outward (“growing”); alternatively, fragments that bind in close proximity may be 6950



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There is an increasing focus on identifying the nonspecific interactions of common photo-cross-linkers to help eliminate falsely identified target proteins. Indeed photo-cross-linkerbased hydrophobic or electrostatic interactions with nontarget proteins can obscure true targets, particularly if the activity of the probe is weak.19 Park et al. compared the protein binding profiles of an aliphatic diazirine, benzophenone, and arylazide photophore, each containing an alkyne click handle.58 Three cell types were investigated, HeLa, BV2, and 293T, and each photo-cross-linker was activated and then coupled to a common fluorescent azide moiety. Using both 1D and 2D gel electrophoresis and mass spectrometry to identify protein targets, it became evident each photo-cross-linker exhibited structure-dependent proteome labeling. Furthermore, this information helped to discern between target and nontarget proteins when these linkers were appended to different druglike molecules as proof-of-concept. The authors concluded that at least two different photo-cross-linkers should be used in any target identification process to distinguish between true targets common to both probes and off-target proteins common to specific photo-cross-linkers. Kleiner et al. compiled a useful inventory of off-target proteins common to specific photocross-linkers.50 It is recommended to take this protein profile into account when designing both probes and experiments. Diazirines and arylazides are considered more efficient photocross-linkers than benzophenones,50 and branched photo-crosslinkers tend to give less nonspecific protein labeling.59

Scheme 11. A 60 Member Diazirine Containing Screening Library Was Generated Using the Ugi Multicomponent Reaction57

linked together. In a novel approach, a library of 14 small fragments (molecular weight ∼176), based on known drugs, were coupled to a constant diazirine moiety further functionalized with an alkyne (Figure 5).32 These probes were used to

5. DIAZIRINE-CONTAINING AMINO ACIDS There have been several aliphatic and aromatic diazirine amino acid derivatives synthesized (Figure 6) and widely used. This review will focus on the aliphatic amino acids 56−62 since the aromatic derivatives 63−66 are reviewed elsewhere.21 On the basis of the work of Suchanek,60 photo-lysine 56 was incorporated into proteins using native mammalian translational systems in living cells.61 Post-translational modificationmediated protein interactions are ordinarily challenging to study. Such modifications can be dynamic, mediate weak/ transient interactions, and occur at very low levels. Diazirines make ideal tools to use in overcoming these difficult circumstances, as the light-initiated chemical reactivity allows trapping of these species. HeLa cells grown in the presence of photo-lysine were treated with UV light to activate the diazirine moiety before cell lysis and extraction of the histone content.61 Proteomic analysis of bands from Western blot allowed convincing identification of histone and chromatin binding proteins, many of which were already known giving confidence in the methodology. Photo-methionine (57) and photo-leucine (59) have been used in a study to map the protein interaction network of protein kinase D2.62 HeLa cells were grown for 24 h in media where methionine and leucine had been replaced by their diazirine equivalents prior to cell lysis. A GST-PKD2 fusion protein, immobilized on glutathione sepharose beads, was then incubated with the HeLa cell lysate and irradiated with 365 nm UV light. After washing the beads, enzymatic digestion was performed and proteomic analysis successfully identified both transient and high-affinity protein interaction partners. Synthesis of photo-lysine 56 was particularly lengthy (Scheme 12) taking 17 steps from methyl 3-hydroxypropanoate 67 and serine 68.61 Initial steps in the synthesis were conducted in parallel where serine was protected using Boc and methyl ester 69 before converting the side chain to an iodo leaving

Figure 5. Fragments of known drugs modified through appending a diazirine photocross linker.32

develop a particularly exciting fragment-based drug discovery screening approach in human cells (HEK293T and K562). The use of the diazirine allowed capture of transient and weak interactions mapping the entire ligand interactome in the native cellular environment, identifying over 2000 proteins. Further chemical elaboration of the identified ligands led to successful discovery of the first selective ligands for the enzyme PTGR2, the transporter SLC25A20, and the transmembrane protein PGRMC2. Throughout these studies the importance of control probes and competitors to the successful identification of protein targets was heavily emphasized. This novel platform may lead the way to discovery of ligands for orphan receptors, transcription factors, and adaptor proteins many of which were considered undruggable. 6951



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Figure 6. Common aliphatic and aromatic diazirine amino acid derivatives.

Scheme 12. Synthetic Route To Access Photo-Lysine61

group 70. The methyl 3-hydroxypropanoate 67 was protected using a tert-butyldimethylsilyl (TBDMS) group 71 and the methyl ester converted to a thioethyl ester 72. The iodo compound 70 was converted to the organozinc intermediate using activated zinc before using in a palladium-catalyzed crosscoupling with the thioester 72 giving the product 73 in an impressive 91% yield. After deprotection of the TBDMS and methyl ester, the ketone of 74 was converted to the diazirine using the conventional HOSA, iodine, and ammonia methodology in moderate 46% yield followed by carboxylic acid protection as the methyl ester to give 75. Intermediate 75 is in

itself an interesting nonproteinogenic amino acid that could be further elaborated in multiple ways through modification of the alcohol side chain. In this case, the alcohol of the side chain was converted via the azide 76 to the amine 56. Manipulation of the protecting groups provided a number of other interesting building blocks 77−79 which may be useful in peptide synthesis. The yields reported throughout this synthesis are, for the most part, very high; however, this length of route is a formidable undertaking, and shorter routes would be highly desirable. Moreover the yield of diazirine formation, in the middle of this synthesis, is 46%. This is a good yield for the 6952



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Scheme 13. Four Common Synthetic Routes To Access Photo-Leucine

methodology (Scheme 13, route 2) involved ozonolysis of 4,5dehydroleucine 84, in excellent yield, followed by conversion of the resulting ketone 85 to the diazirine moiety 86 (55%).64 Two alternative routes to photo-leucine have been devised that avoid the use of expensive dehydroleucine or enzymatic resolution. The first of these routes commenced by conversion of the Boc L-aspartic acid α-methyl ester 87 side chain to the corresponding β-ketoester 88 (Scheme 13, route 3).65 A simultaneous hydrogenolysis/decarboxylation was then used to give the ketone 89. Reaction to form the diazirine 86 via usual HOSA methodology proceeded in only 30% yield, but the subsequent deprotection/protection chemistry proceeded smoothly to give 90. Another approach (Scheme 13, route 4) was pursued from Boc D-aspartic acid α-tert-butyl ester 91 where the acid side chain was converted to the Weinreb amide 92.66,67 Degradation to the ketone 93 using methyl Grignard reagent did not proceed to completion, whereas the use of methyllithium was much more successful, proceeding in 90% yield. As in the previous synthesis, conversion to the diazirine 94 proceeded in low yield. Deprotection of the Boc and tertbutyl ester followed by reprotection of the amine with Boc was

transformation using these reagents; nevertheless, with newer methods available (see Diazirine Synthesis section), this could conceivably be improved. There have been several approaches to synthesis of photoleucine 59; one of the earliest was patented in 2006 (Scheme 13, route 1; yields were not well reported).63 4,4′-Azi-pentanoic acid 81 was synthesized from levulinic acid 80 using the method of Church and Weiss,37 then brominated in a threestep one-pot protocol. This involved chlorination of the acid with thionyl chloride, bromination of the α-carbon using Nbromosuccinimide/hydrogen bromide with heating at 55−80 °C for 4 h, then hydrolysis of the acid chloride back to the carboxylic acid 82. Aminolysis using ammonia in methanol for 5 days at 55 °C produced the racemic amino acid 59 in relatively poor yield (13% from the 4,4′-azi-pentanoic acid). It is perhaps useful to note that the diazirine 82 was heated for an extended period and did not completely degrade. The racemate 58 was acetylated to give 83 and then enzymatically deacetylated to give the pure L-photo-leucine 59. A very similar procedure was employed without enzymatic resolution to access photo-isoleucine 61 (Figure 5).63 A much simpler 6953



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successful in producing (Boc)-D-photo-leucine (95) in high yield.66 Identical methodology was successful for synthesis of the related (Boc)-D-photo-methionine as had been previously published.66−68 These routes would be high yielding, but the diazirine formation is only 30−35% yield. Perhaps the methods of Wang39 can be adapted to effect this conversion more efficiently. Photo-methionine can also be constructed using Strecker synthesis from the corresponding aldehyde 96 to give 57; acetylation and enzymatic resolution successfully gave the pure L-photo-methionine (57, Scheme 14).63 The starting aldehyde 96 was synthesized in two synthetic steps from levulinaldehyde dimethyl acetal.37

Boc protection and Jones oxidation were used to give the precursor ketone 102 followed by standard methodology to form the diazirine 103 in 31% yield from the ketone. Subsequent reactions changed the Boc protecting group to Fmoc 104. Yields in the diazirine formation step were typically low but with application of new methodology may be improved;39 moreover, the protecting group chemistry was nonoptimized and again might be improved with further investigation. These syntheses show that the diazirine-containing amino acids are readily accessible and they are also tolerant of typical peptide synthesis conditions. L-Photo-leucine was used in place of leucine in the synthesis of the cyclic lipopeptide antibiotic polymyxin B3 in which the lipid tail is replaced by an alkyne click handle (Figure 7).71 The polymyxin derivative retained its biological activity and was successfully used in mode of action studies. Other studies have illustrated that these novel amino acids can be recognized by native cellular systems where they are incorporated into proteins60 or can be used in site-specific native protein ligation.68

Scheme 14. Strecker Synthesis of Photo-Methionine

6. TPD BUILDING BLOCKS AND FGIs Much like aliphatic diaizirines, TPDs are typically incorporated into bioactive molecules via several key building blocks amenable to numerous FGIs. Popularlized by Nakashima et al., TPD aldehyde 105 and 106 are widely used because they are easily derivatized (Scheme 17).72 Aldehydes maybe converted to acrylonitrile derivative 107 with the corresponding Wittig reagent in excellent yield (98%) and good stereospecificity (77% trans/23% cis),73 reduced to benzyl alcohol 108 in quantitative yield with sodium borohydride,74 oxidized to carboxylic acid 109 with sodium chlorite and sulfamic acid,75 converted to ester 110 by dissolving in methanol and treating with a suspension of ammonium persulfate and sulfuric acid, refluxed with hydroxylamine hydrochloride in pyridine and ethanol to form oxime 111,76 or treated with D- or L-cysteine to form thiazolidine 112.77 Benzyl bromide TPDs (Scheme 18) are employed in highyielding alkylation reactions under basic conditions. For example Admas et al. used 113 to alkylate an amine giving 114 in 91% yield,78 while Woll et al. alkylated propargyl alcohol with benzyl bromide 115 to give 116 in 77% yield.79 Hashimoto et al. converted benzyl bromide 117 to azide 118 using sodium azide in 66% yield.80 Azide 118 was later used in a Staudinger−Bertozzi ligation to a phosphine. In a more

Trifluoromethyl-photo-leucine 60 and methionine 58 have been synthesized (Scheme 15) in their protected form (100), and their photoactivation was compared to their methyl counterparts.66 The trifluoromethyldiazirines were found to be more efficient at cross-linking but required a much lower 300−330 nm wavelength light to activate than is typical for aliphatic diazirine probes. Although these trifluoromethyl-photo probes are more efficient, synthetic routes are complicated by the electron withdrawing nature of the trifluoromethyl group and the related propensity to form stable hemiaminal intermediates. The trifluoromethyl derivatives were accessed from aldehyde 97 by reaction with Rupert−Prakash reagent69 followed by mild oxidation with DMP. The aliphatic ketone 98 was converted to the diazirine 99 via the tosyloxime methodology usually employed with TPD photoprobes. HOSA methodology was not successful, most likely due to the formation of stable hemiaminal as has previously been reported;41 in addition, the amine moiety required bis-Boc protection throughout to prevent formation of stable aminal. At the end of the synthesis, the bis-Boc was converted to the mono-Boc to give 100. Boc and Fmoc protected L-photo-proline 103 and 104 can be synthesized from L-trans-4-hydroxyproline 101 (Scheme 16).70 Scheme 15. Synthesis of Trifluoromethyl Aliphatic Diazirines66

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Scheme 16. Synthesis of Boc and Fmoc Protected L-Photo-Proline

Aniline 125 can react with thiophosgene to form an isothiocyanate 126 (yield not reported),83 be oxidized to the nitroarene 127 with zirconium tert-butoxide in 83% yield,84 or treated with triphosgene to form urea 128 in 50% yield via an in situ isocyanate.85 Using para TPD aniline 129, urea formation under the same conditions afforded 130, also in 50% yield (Scheme 20).86 This section has detailed how aldehyde, benzyl bromide, phenol, and aniline containing TPDs can undergo a large range of FGIs, giving a suite of building blocks and also inspiration as to how these could be incorporated into bioactive molecules.

7. TPD AROMATIC SUBSTITUTION Aromatic substitution reactions in the presence of TPDs is challenging due to the perceived low thermal stability and poor phenyl reactivity. Advances in this area are highly sought after and would aid synthetic versatility, increase the affordability of commercially available intermediates, and ultimately encourage the use of TPDs as PAPs. The poor reactivity of the TPDs toward electrophilic aromatic substitution (SEAr) is attributed to the highly electron withdrawing nature of the trifluoromethyl group and the poor reactivity of the aromatic C−H bonds. Presently, only a select few SEAr reactions can be performed without electron donating substitutents; this includes Lewis acid-catalyzed (SbF5 or TiCl4) formylation of phenyl 3 to give aldehyde 105 and formation of benzyl bromide 113 with bromomethyl methyl ether in triflic acid (Scheme 21A).72 In both instances, regiochemistry is inductively directed to the para position. To circumvent the poor reactivity of TPDs toward SEAr, Hatanaka et al. demonstrated the advantage of electron donating methoxy substituents (Scheme 21B).87 The meta methoxy-substituent (131) was preferred over the para analogue because demethylation to the phenol proceeded in higher yields.87 The electron density added by an alkoxy substituent improved the scope of SEAr, allowing the formation of aldehyde 106 in 62% yield,74 iodo 132 in 86% yield with iodine and iodobenzene di(trifluoroacetate),88 nitro 133 in 75% yield with nitric and acetic anhydride,89 ketone 134 in 93% yield with acetyl chloride and aluminum trichloride (AlCl3),90 and ester 135 in 57% yield upon thallation with thallium(III) trifluoroacetate and subsequent carbonylation with palladium(II) chloride and methanol.87 These strategies have been widely adopted, making it the predominant means of performing aromatic substitutions on the electron deficient TPDs. Diazonium salts provide a known, but under-utilized, means of performing aromatic substitutions in the presence of TPDs. In 1981, Brunner et al. showed that 3-iodo TPD 132 could be formed from the 3-diazonium TPD without the need for additional activating groups.91 This particular nucleophilic aromatic substitution (SNAr) (Scheme 22A) is not widely used, since it is more common to use electron donating substituents to promote iodination of phenyl C−H bonds. However, it hints at the plausibility of using diazonium salts to perform radical−nucelophilc aromatic substitution (SRN1). This

Figure 7. L-Photo-leucine was used in place of leucine in solid phase peptide synthesis of polymyxin B.

Scheme 17. Reactions of TPD Aldehydes

intricate example, benzyl bromide 113 was alkylated with tertbutyl 2-((diphenylmethylene)amino)acetate in the presence of the cinchonidine-based asymmetric catalyst and 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine (BEMP) as a sterically hindered base to afford 119 in 95% yield. Subsequent deprotection with trifluoroacetic acetic (TFA) afforded photo-phenyl alanine 120 in 97% yield.72 The meta-phenol TPDs are less widespread than the para counterpart and have to our knowledge only been employed in Williamson ether syntheses, such as the alkylation of phenol 121 with 1,6-dibromohexane to give 122 (Scheme 19).81 The para-phenol TPDs on the other hand are frequently used in Mitsunobu-style reactions with primary alcohols. Shigenari et al. applied this method to phenol 123 and hexadecan-1-ol (and 10 other primary alcohols) affording TPD ether 124 in 86% (53−97%) yield.82 6955



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Scheme 18. Reactions of TPD Benzyl Bromides

has been published using a TPD diazonium salt, in the presence of CuCl and SO2, to form a sulfonyl chloride via a Meerwein chlorosulfodediazotization reaction.92 The viability of this reaction highlights the potential to use other Sandmeyer-style reactions to perform FGIs in the presence of the TPDs, expanding synthetic access. Given the propensity to avoid heating TPDs, there are several unexplored means by which aromatic reactivity could be improved at low temperatures. Arynes are one such example, generated from aryl halides when treated with a strong base, allowing the introduction of various nucleophiles (Scheme 22B).93 Importantly, the basic conditions required to generate arynes have previously been tolerated by TPDs.42 Alternatively, electron deficient azines such as pyridine could be used to improve electrophilicity at the C-2 and C-4 positions and promote SNAr at TPD-compatible temperatures (Scheme 22C).94 The successful utilization of these strategies would improve synthetic access to a range of substituted trifluoromethyl aryl diazirines.

Scheme 19. Reactions of TPD Phenols

Scheme 20. Reactions of TPD Anilines

8. TPD-COMPATIBLE REACTION CONDITIONS TPDs are known to tolerate a wide range of reaction conditions including reduction, oxidation, acid, base, hydrolysis, nucleophiles, electrophiles, thermal heat, metal catalysis, and ambient light. Here we provide examples of some of the more harsh conditions used in TPD synthesis. This is intended to provide a more comprehensive understanding of TPD stability, inspiring synthetic chemists to develop more efficient synthetic routes. The exposure of TPDs to acidic conditions (Scheme 23) is well tolerated at low temperatures. For example, in the aforementioned formylation of unsubstituted TPD 3, exposure to dichloromethyl methyl ether, TiCl4, and triflic acid at 0 °C was required to afford the aldehyde 105 in good (80%) yield. Furthermore, TPD-containing aniline 125 can undergo diazotization in concentrated hydrochloride acid and acetic acid below 5 °C.91,92 Subsequent chlorosulfo-de-diazotization afforded the TPD sulfonyl chloride 136 in 52% yield.92 Attempts to chlorosulfonate TPD 137 in chlorosulfonic acid required the temperature to stay below −20 °C to prevent decomposition.40 After 1 h the reaction was warmed to room temperature, affording sulfonyl choride 138.40 6956



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Scheme 21. TPD SEAr Facilitated by Electron Donating Alkoxy Substituients

Scheme 24. TPD-Compatible n-BuLi Reaction

Scheme 22. Potential Aromatic Substitution Strategies Amenable to TPDs

Highly soluble, strong bases may be used, provided they are non-nucleophilic and the temperature is sufficiently low. Kumar et al. elegantly used lithium diisopropylamine (LDA) to lithiate bromothiazole 141, causing halogen migration (Scheme 25). The subsequent addition of tetrahydropyran-4-one enabled the formation of an alcohol, which was then methylated to give compound 142.98 Scheme 23. TPD Reactions in Acidic Conditions Scheme 25. TPD-Compatible LDA Reaction

Wang et al. used the poor solubility of various nucleophilic bases to their advantage (Scheme 26). Lithium amide in liquid Scheme 26. TPD Reactions in Basic Conditions

ammonia/diethyl ether at room temperature gave diazirine 143 from the corresponding tosyl-oxime 144 in 96% yield. By changing the base to sodium amide or sodium hydride, diazirine ring formation was observed; however, ester ammonolysis meant the desired ester was not isolated and only amide 145 was formed. TPDs can withstand an array of noncatalytic (Scheme 27) and catalytic reductions. Hashimoto et al. used sodium methoxide with diethyl phosphite to reduce TPD-containing β,β-dibromostyrenes 146 to the corresponding β-bromostyrene 147 in good yield (56−96%) (Scheme 27), whereas the use of sodium ethoxide under microwave (μW) irradiation caused

TPDs tolerate a range of basic conditions including hydrides, oxides, and amides; however, they are certainly far from impervious. Grignard reagents are known to attack the nitrogen−nitrogen double bond of diazirines,95 while highly nucleophilic, soluble, and sterically unencumbered bases, such as n-butyllithium, can cause degradation via nucleophilic attack on the diazirinyl ring.95,96 n-Butyllithium can be used in circumstances where it is consumed before the TPD component is added. An example of this is shown in Scheme 24, where the 2-oxazolidone 139 was lithiated before combining with an in situ acid chloride to give 140.97 6957



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preferential hydrogenation of iodoarenes 152, typically within 1 h; however, once hydrodeiodination is complete, TPDs (108) will reduce to the diaziridine 153.102 Sulfided platinum on carbon (5 mol %), however, enabled hydrogenation or deuteration of a TPD-containing iodothiazole 154 over 18 h to give a good yield of 155 or 156 (70−78%).98 Unsurprisingly, TPDs are stable to weak oxidants given the multitude used for diaziridine to diazirine conversion, but they also exhibit stability toward a number of stronger oxidants at low temperatures (Scheme 29). Potassium permanganate was

Scheme 27. TPD-Compatible Noncatalytic Reductions

Scheme 29. TPD-Compatible Oxidations

diazirine degradation.99 Reduction of the carbonyl in 148 to methylene 149, in the presence of a TPD, can be achieved with triethylsilane and TFA.100 However, attempts to perform the same reduction with lithium aluminum hydride/aluminum trichloride, p-tosylhydrazine/sodium cyanoborohydride, or Wolff−Kishner conditions caused TPD decomposition.100 The reduction of nitroarene 127 to the corresponding aniline is another TPD-compatible transformation. This is achieved using fresh sodium dithionite, affording moderate yields of 125 (47−58%).92,101 With careful consideration, catalytic hydrogenations are also possible in the presence of a TPD (Scheme 28). The hydrogenation of an alkene 150 with Wilkinson’s catalyst, RhCl(PPh3)3, was selective for the carbon−carbon bonds giving 151.73 In contrast, more caution was required in palladium-catalyzed reactions. Palladium on carbon facilitated

used to oxidize benzyl alcohol 108 to the corresponding benzoic acid 157 in 91% yield at room temperature.103 Oxidation of a tolyl motif 137 to the benzoic acid 157 with potassium permanganate occurred in excellent yield (95%) when performed at 50 °C over 6 h.104 The same conditions over 15 h resulted in a significantly lower yield (31%), perhaps owing to destruction of the diazirine ring.105 In another example of TPDs withstanding strong oxidants, ortho-periodic acid (8 equiv), chromium trioxide (10 mol %), and acetic anhydride were used to perform an oxidative cyclization on 138 (synthesized by chlorosulfonylation of 137) via a benzoic acid intermediate to give a low yield (28%) of 158 over the three steps.40 The multistep nature of this transformation, including use of chlorosulfonic acid, makes it difficult to identify whether diazirine decomposition contributed to the low yield. Although metal-catalyzed coupling reactions see widespread use in medicinal chemistry,106 this has not translated to the synthesis of TPD probes, potentially impacting the length of synthetic routes. In the context of TPD-metal-catalyzed coupling, there are several obstacles to consider, namely, the diazirine thermal instability and the incompatibility with several metallic reagents.95,107,108 In spite of these issues, there are examples of both Pd- and Cu-catalyzed coupling (Scheme 30). Kumar et al. used tris(dibenzylideneacetone)dipalladium(0) (Pd2dba 3) and 1,1′-ferrocenediyl-bis(diphenylphosphine) (dppf) to perform cross-coupling between thiols 159 and 160 and an iodoarene 161, albeit giving low yield of 155 or 142 (22−29%).108 Rennhack et al. investigated iron, indium, gold, and copper catalysts in an attempt to perform an aldehyde− amine−alkyne three-component coupling.109 Only copper(I) proved applicable to their TPD-containing aldehyde (Scheme 28). This reaction required exposure of the TPD aldehyde 105 to 60 °C for 48 h in a sealed vial under nitrogen atmosphere, with subsequent ester cleavage affording TPD 162 in 55% yield, while the same reaction at 90 °C for 24 h caused diazirine decomposition.108 Interestingly, the previously mentioned (Scheme 3) one-step synthesis of TPDs from tosyloximes

Scheme 28. TPD-Compatible Catalytic Reductions

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Scheme 30. TPD-Compatible Metal-Catalyzed Coupling Reactions

tolerated 4 h at 80 °C in a sealed tube. In the absence of a more systematic study, such examples provide valuable insight into the thermal stability of TPDs and their compatibility with various metal catalysts.

of trifluoromethyl diazirines, heterocycles may provide opportunities for chemical derivatization not accessible via the phenyl variant.

10. CONCLUDING REMARKS Aliphatic and trifluoromethyl phenyl diazirines have become increasingly popular, and the range of applications is ever expanding. These useful photo-cross-linkers can assist in target identification, mapping the ligand interaction landscape within the cellular milleau, fragment based drug discovery, live cell imaging. Moreover, diazirine containing amino acids have been incorporated into proteins as they can be recognized by native cellular transcriptional machinery. Aliphatic diazirines have seen a recent increase in popularity due to their small size in comparison to the TPD unit. However, with a nested strategy where an existing aromatic ring is modified with the trifluoromethyl diazirine unit, the size is not significantly increased. These are important considerations when thinking about the activity of the final probe as it may need to traverse membranes to reach its site of action and the physicochemical properties of the probes can vary significantly in comparison to the parent bioactive unit. Furthermore, additional moieties such as alkyne click handles are often incorporated, and these too can affect the physicochemical properties. A number of studies are also reporting that there are “off target” proteins that are specific to the photo-cross-linker selected, and therefore more than one probe is required to conduct target identification alongside control probes. Given these recommendations, it is more important than ever to increase the efficiency of the synthetic routes employed to access these useful probes. Early methods to synthesize diazirine photo-cross-linkers were often low yielding; however, newer methods are emerging that significantly improve yields and reduce the number of synthetic steps required. These will undoubtedly be widely applied in the field. The TPD is a remarkably stable moiety; however, more comprehensive understanding of TPD thermal and chemical stability would encourage increased confidence to use this powerful photo-cross-linker. The diazirine unit can survive an extensive array of reagents required for functional group interconversion. For the most part, conditions used are typically mild and surprisingly the diazirine ring can even survive hydrogenation with careful control of the reduction conditions. A variety of metal-catalyzed cross-coupling reactions have been successfully employed; however, there is still much scope to expand this chemistry. Diazirines have also been subjected to some very harsh reagents such as

9. NOVEL TPD-DERIVED PHOTO-CROSS-LINKERS Although the core structure of the TPD has remained relatively unchanged since inception, replacing one fluorine for an additional functionality has been explored (Figure 8). This led

Figure 8. Novel TPD-derived photo-cross-linkers.

to “all-in-one” diazirines 163 and 164 containing both an azide and an alkyne, intended as handles for Hüisgen 1,3-dipolar cycloadditions.109,110 Both all-in-one PAPs have been validated by successfully forming methanolic adducts upon UV irradiation and demonstrating click cycloadditions. Burkard et al. reported the synthesis of the two fluorous-tagged diazirines 165 and 166 to aid protein-bound PAP identification via fluorous affinity chromatography.111 Unfortunately, no purification or cross-linking data have yet been reported. Furthermore, significantly less is known about CF2 phenyl carbenes than their CF3 counterparts,112 which may have implications on the protein cross-linking efficiency of the fluorous-tagged and all-in-one diaizirines. Moreover, these additional functionalities may also add steps to already long syntheses. Leveraging the favorable properties of aryl and trifluoromethyl diazirine substituents, Kumar et al. demonstrated pyridine 167 and pyrimidine 168 diazirine analogues exhibited improved ambient light stability, with only marginal effects on UV irradiation times and the diazo−carbene ratio.113 This outcome was attributed to the introduction of electron withdrawing nitrogen atoms, causing a decrease in the molar absorption coefficient (ε) and a shift in the maximum UV absorption wavelength (λmax). Compared to phenyl derivative, the ε of pyridine 167 and pyrimidine 168 was reduced by 15% and 30%, respectively, while the λmax decreased to 345 and 350 nm, respectively. The effect on thermal stability was not determined. In addition to improving the ambient light stability 6959



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chlorosulfonic acid (at low temperature), and in some cases this has proven to be successful. Moreover, some probes have survived heating for several days. Aromatic substitution reactions of TPDs are challenging due to the poor phenyl reactivity in the presence of the electron withdrawing trifluoromethyl diazirine substituent. To mitigate this, many synthetic routes employ an electron donating group on the ring such as methoxy; however this may not suit the probe design or could hinder binding of a bioactive unit to its target. Advances in synthetic methodology in this area are highly attractive in order to aid versatility. A number of diazirine containing amino acids have been synthesized, both proteinogenic and nonproteinogenic. Some syntheses such as photo-leucine and photo-methionine have been well developed with short high-yielding synthetic routes. Consequently, these two photoactivatable amino acids are commercially available. Nevertheless improvements are still possible if the newly developed chemistry to access diazirines in high yield is employed. In contrast, the 17 step photo-lysine synthesis is highly likely to be a deterrent to widespread use and also to the likelihood of ever becoming commercially viable for compound suppliers. To devise short synthetic strategies to access these valuable tools would be an attractive prospect. The current applications of diazirine photoaffinity probes are exciting, and the chemistry of these intriguing molecules is reasonably developed. However, to fully exploit this technology in an efficient manner, it is highly desirable to expand the synthetic methodology and shorten synthetic routes through new innovative approaches. This review provides the synthetic foundation required for medicinal chemists entering the field for the first time but also an opportunity for those more experienced to think about how best to advance this technology into the future.

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research group is focused on targeting inflammatory pathways (particularly inflammasomes), anticancer and antifungal drug discovery. She has published over 50 papers and is inventor on multiple patents. Her work on sulfonylurea based NLRP3 inflammasome inhibitors was recently commercialized to found Inflazome Ltd.

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ACKNOWLEDGMENTS J.R.H is supported by a Research Advancement Award from the Institute for Molecular Bioscience and a University of Queensland Research Scholarship. A.A.B.R. is supported by The School of Chemistry and Molecular Bioscience at The University of Queensland, Michael J. Fox Foundation Grant, and NHMRC Development Grant (APP1118973).

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ABBREVIATIONS USED ε, molar absorption coefficient; 1D, one-dimensional; 2D, twodimensional; λmax, Aabsorption wavelength; BEMP, 2-tertbutylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine; CLogP, octanol−water partition coefficient; DCC, dicyclohexylcarbodiimide; DMAP, 4-dimethylaminopyridine; DMP, Dess−Martin periodinane; Dppf, 1,1′-ferrocenediyl-bis(diphenylphosphine); DSC, N,N′-disuccinimidyl carbonate; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; FGI, functional group interconversion; HOSA, hydroxylamineO-sulfonic acid; LDA, lithium diisopropylamide; MS, mass spectrometry; MSH, O-mesitylenesulfonylhydroxylamine; μW, microwave irradiation; PAL, photoaffinity labeling; TPD, trifluoromethylphenyldiazirines; PAP, photoaffinity probe; Pd2dba3, tris(dibenzylideneacetone)dipalladium(0); PL, phospholipase; SAR, structure−activity relationship; SEAr, electrophilic aromatic substitution; SNAr, nucleophilic aromatic substitution; SRN1, radical−nucleophilic substitution; t-BuOK, potassium tert-butoxide; TBDMS, tert-butyldimethylsilyl; TFA, trifluoroacetic acid; TPD, trifluoromethyl phenyl diazirine; tPSA, topological polar surface area; UV, ultraviolet

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Telephone: +61 7 3346 2204.

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ORCID

Avril A. B. Robertson: 0000-0002-9652-8357

REFERENCES

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Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest. Biographies James R. Hill is a Ph.D. Candidate at The University of Queensland’s Institute for Molecular Bioscience, where he also completed his B.Sc. (Hons) in chemistry. James’s research focuses the synthesis of tool compounds, namely, photoaffinity probes and positron emission tomography (PET) tracers, which he applies to inflammatory disease drug discovery. In 2018, James was awarded the Fulbright Queensland Scholarship to continue his PET chemistry research at the University of Michigan. Avril A. B. Robertson is Professor of Biotechnology and Biotechnology Program Director at the University of Queensland. She completed her Ph.D. (2000) at St. Andrews University and has worked 10 years as medicinal chemist in the UK drug discovery industry, most recently as project leader in oncology at Cyclacel Ltd. In 2011, she moved to The University of Queensland as head of medicinal chemistry in the Center for Drug Design and Discovery. Her 6960



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