Recent Advances in Chemical Biology Using Benzophenones and Diazirines As Radical Precursors

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Review Recent Advances in Chemical Biology Using Benzophenones and Diazirines as Radical Precursors

1,2, , 1,2, Muhammad Murtaza Hassan * † and Olasunkanmi O. Olaoye † 1 Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road North, Mississauga, ON L5L 1C6, Canada; [email protected] 2 Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, ON M5S 3H6, Canada * Correspondence: [email protected]; Tel.: +1-905-569-4588 These authors contributed equally to this work. † Academic Editor: Edward Lee-Ruﬀ Received: 18 April 2020; Accepted: 9 May 2020; Published: 13 May 2020

Abstract: The use of light-activated chemical probes to study biological interactions was ﬁrst discovered in the 1960s, and has since found many applications in studying diseases and gaining deeper insight into various cellular mechanisms involving protein–protein, protein–nucleic acid, protein–ligand (drug, probe), and protein–co-factor interactions, among others. This technique, often referred to as photoaﬃnity labelling, uses radical precursors that react almost instantaneously to yield spatial and temporal information about the nature of the interaction and the interacting partner(s). This review focuses on the recent advances in chemical biology in the use of benzophenones and diazirines, two of the most commonly known light-activatable radical precursors, with a focus on the last three years, and is intended to provide a solid understanding of their chemical and biological principles and their applications.

Keywords: photoaﬃnity labelling; benzophenone; diazirine; radical precursors; interactome; SABRE; hyperpolarizing agents; crosslinking; photochemistry

1. Introduction The use of radicals or photoactivatable radical precursors has become ubiquitous in the fields of medicinal chemistry and chemical biology in the past three decades. Their main application revolves around studying the interactions of biomolecule targets such as proteins with ligands containing photoactivatable handles. These photoreactive chemical groups (PCG) can be added to a probe, ideally with minimal perturbation of the intrinsic activity of the probe, which is then subsequently introduced into a biological system. After a given incubation period of the photolabelled probe in the biological system, the probe can be activated by light to reveal its biomolecular interactions, thus providing spatial and temporal information. This technique is referred to as photoaffinity labelling (PL). Such applications of chemical systems to study biological systems have transformed the chemical biology field by providing insights into protein interactomes, protein–ligand binding pocket identification, and studying the promiscuity of drugs in biological systems, enabling us to answer the fundamental “what, when, and where” questions of biological interactions. This review focuses on the chemistry of two commonly used PCGs, benzophenone and diazirines, as representatives of two classes of radical precursors (1,2-diradical and carbene radical, respectively; Scheme1), and their applications in biological systems with an emphasis on the last three years.

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SchemeScheme 1. 1. TwoTwo commonly commonly used radical precursors inin chemicalchemical biology.biology. ((aa)) Benzophenones Benzophenones generate generate a a1,2-diradical 1,2-diradical upon upon irradiation irradiation with with 330–365 330–365 nm nm light. light. ( b(b)) Diazirines Diazirines liberate liberate nitrogen nitrogen and and generate generate a acarbene carbene radical radical upon upon irradiation irradiation with with 340–380 340–380 nm nm light. light. 2. Chemical Principles 2. Chemical Principles 2.1. Benzophenones 2.1. Benzophenones The use of benzophenones (BPs) in PL was first recorded in 1974, when it was shown that they couldThe effi useciently of benzophenones label amino acids (BPs [)1 in]. PL BPs, was like first other recorded ketone in functionalities, 1974, when it was consist shown of a that carbonyl they couldcarbon efficiently that undergoes label amino intersystem acids crossing[1]. BPs, inlike high other yields, ketone making functionalities, it a robust tripletconsist photosensitizer of a carbonyl carbonfor use that in organic undergoes and biologicalintersystem chemistry. crossing Itsin high extensive yields chemical, making and it a physicalrobust triplet properties photosensitizer have been forreviewed use in organic elsewhere and[ biological2] and are chemistry. not the main Its extensive focus of thischemical review; and however, physical properties notable physical have been and reviewedchemical featureselsewhere pertinent [2] and toare this not review the main are discussedfocus of this herein. review BPs; however, undergo notable two transitions, physical πand–π*, chemicala high-energy features transition, pertinent and to this n– πreview*, a lower-energy are discussed transition. herein. BPs The undergo and n– twoπ* transitiontransitions, occurs π–π*, ata high330–365-energy nm transition, and is well and separated n–π*, a fromlower the-energyπ–π* transition. [The2,3]. and High-energy n–π* transition UV irradiation occurs at has330– a 365tendency nm and to damage is well proteins separated due from to the the spectral π–π* overlaptransition of endogenous[2,3]. High-energy fluorophores UV irradiation (e.g., tryptophan, has a tendencytyrosine, histidineto damage etc.); proteins thus, the lower-energy due to the spectraln–π* transition overlap of BPs of endogenousallows them to fluorophores be more applicable (e.g., tryptophan,to biological tyrosine, settings [histidine4]. Although etc.); the thus, n– πthe* transition lower-energy of BPs n– hasπ* transition a lower absorptive of BPs allows spectral them overlap to be morewith biologicalapplicable fluorophores, to biological itsettings has a much [4]. Although weaker absorption the n–π* transition than the π of–π BPs* transition, has a lower as indicated absorptive by spectralits low molar overlap absorptivity with biological coefficient fluorophores, (εmax < 300) it[5 ]. has This a can much be explained weaker absorption by the poor than orbital the overlap π–π* transition,of the orthogonal as indicated p-orbital by its containing low molar the absorptivity non-bonding coefficient electron (ε lonemax < pair 300) and [5]. πThis* orbital can be reducing explained the byprobability the poor oforbital excitation overlap (Figure of the1). orthogonal The hybridization p-orbital of containing the lone pairs the non on- thebonding carbonyl electron oxygen lone can pair be anddepicted π* orbital in one reducing of two the ways: probability both lone of pairsexcitation are sp (Figure2-hybridized, 1). The hybridization or one lone pair of the is sp-hybridized lone pairs on thewhile carbonyl the other oxygen is an can unhybridized be depicted p-orbital in one of [2, 6two,7]. ways: The non-bonding both lone pairs orbital are containingsp2-hybridized the lone, or one pair loneis depicted pair is below sp-hybridized as an unhybridized while the other p-orbital is an (Figure unhybridized1)[ 2,6]. The p-orbital n–π* transition[2,6,7]. The breaks non-bondi the C=ngO orbitaldouble containing bond and generatesthe lone pair a diradical, is depicted which below can as exist an unhybridized in a singlet or p triplet-orbital state (Figure and is1) capable[2,6]. The of ne–ffiπ*ciently transition crosslinking breaks the via C=O covalent double bonding bond and with generates nearby chemo-moieties. a diradical, which The can breaking exist in a of singlet the C= orO tripletdouble state bond and lengthens is capable the bond of efficiently distance, crosslin since theking promotion via covalent of one bonding non-bonding with nearby electron chemo to the- moieties.π* orbital The decreases breaking the of bond the C=O order double of the bond C=O lengthens bond from the 2 tobond 1.5. distance This also, since results the inpromotion a change of in onethe hybridizationnon-bonding electron of the carbon to the atom,π* orbital resulting decreases in a change the bond in theorder geometry of the C=O to a bond non-planar from 2 sp to3 -like1.5. Thispyramidal also results structure in a [ 8change]. in the hybridization of the carbon atom, resulting in a change in the geometry to a non-planar sp3-like pyramidal structure [8]. Excitation of the BP chromophore first yields a diradical in a singlet state (S1), which undergoes Excitation of the BP chromophore first yields a diradical in a singlet state (S1), which undergoes intersystem crossing to quantitatively yield the triplet state (T1). The exact mechanism of the intersystem intersystem crossing to quantitatively yield the triplet state (T1). The exact mechanism of the crossing is controversial [9]. El-Sayed et al. proposed a direct mechanism between the S1 and T1 intersystemexcited states crossing as indicated is controversial by the low-temperature [9]. El-Sayed et Zeeman al. proposed effect a observed direct mechanism in spin alignment between (ZESA) the S1 and T1 excited states as indicated by the low-temperature Zeeman effect observed in spin alignment experiments [8]. Presumably, although the direct S1 to T1 transition violates the El-Sayed selection rules, (ZESA)the BP aromatic experiments rings [8] lose. Presumably, planarity upon although excitation, the direct allowing S1 to for T1 the transition direct transition violates the(Figure El-Sayed1)[ 8]. selectionLater studies rules, however, the BP aromatic showed rings that lose the processplanarity is upon mediated excitation initially, allowing through for intersystem the direct transition crossing (Figure 1) [8]. Later studies however, showed that the process is mediated initially through to a higher-energy excited triplet state (T2) tunable through solvent polarity, which then relaxes via intersystem crossing to a higher-energy excited triplet state (T2) tunable through solvent polarity, which then relaxes via internal conversion to T1 [9,10]. Furthermore, recent studies in the dynamics

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MoleculesMolecules 20 202020, ,2 255, ,x x FOR FOR PEER PEER REVIEW REVIEW 33 ofof 2929 internal conversion to T1 [9,10]. Furthermore, recent studies in the dynamics of BP triplet formation ofof BPBP triplettriplet formationformation inin gasgas--phasephase conditionsconditions havehave suggestedsuggested thatthat uponupon excitationexcitation toto itsits SS22 state,state, itit in gas-phase conditions have suggested that upon excitation to its S2 state, it undergoes internal undergoesundergoes intinternalernal conversionconversion toto SS11 whichwhich thenthen decaysdecays toto TT11 [11][11].. conversion to S1 which then decays to T1 [11].

FigureFigureFigure 1. 1.1. Simpliﬁed SimplifiedSimplified depictiondepiction ofof thethe n–nn––ππ*π** transition;transition;transition; thethe π π bond bondbond and andand other otherother non-bonding nonnon--bondingbonding lonelonelone pairpairpair ininin thethethe sp-orbitalsp sp--orbitalorbital ofof of oxygenoxygen oxygen isis is omittedomitted omitted forf foror simplicity.simplicity. simplicity. ExcitationExcitation Excitation ofof of aa a BPBP BP yieldsyields yields anan an excitedexcited excited singletsinglet singlet statestate state (S(S (S111)) withwithwith a aa distorted distorteddistorted non-planar nonnon--planarplanar geometry geometrygeometry andandand a aa longer longerlonger C–O CC––OO bond bondbond (bond (bond(bond order orderorder = == 1.5), 1.5),1.5), which whichwhich undergoes undergoesundergoes

intersystemintersystemintersystem crossingcrossing crossing toto to yieldyield yield thethe the excitedexcited excited triplettriplet triplet statestate state (T(T (T11).1).).

TheTheThe T T T111 statestate reacts reacts as as an an alkoxy al alkoxykoxy radical radical radical and and and yields yields yields an an an electron-deficient electron electron--deficientdeficient oxygen oxygen oxygen thatthatthat reactsreactsreacts favorablyfavorablyfavorably withwithwith C–HCC––HH bondsbonds tottoo formformform relativelyrelativelyrelatively stablestablestable carboncarboncarbon radicalsradicalsradicals (e.g.,((e.g.e.g.,, α α-carbons--carboncarbonss ofof aminoamino acids)acids) bybyby hydrogenhydrogenhydrogen abstraction abstractionabstraction (Figure (Figure(Figure2 2).).2).The TheThe resulting resultingresulting carbon carboncarbon radical radicalradical formed formedformed in inin the thethe hydrogen hydrogenhydrogen abstraction abstractionabstraction processprocessprocess recombinesrecombinesrecombines withwithwith thethethe BPBPBP ketyl ketylketyl radical radicalradical to toto form formform a aa photo-crosslinked photophoto--crosslinkedcrosslinked adduct. adduct.adduct. Thus,Thus, itit isisis the thethe T TT111 statestatestate resultingresultingresulting fromfrom thethethe n–nn––ππ*π** transitiontransitiontransition thatthatthat isisis mainlymainlymainly responsibleresponsibleresponsible forforfor thethethe formationformationformation ofofof aaa covalentccovalentovalent bond,bondbond,, havinghaving a a quantumquantum efficiency efficiency efficiency ofof of ~1~1 ~1 for for for CC–– C–HHH abstraction abstraction abstraction and and and 0.10.1 0.1forfor theforthe πtheπ––π*π*π –transition transitionπ* transition [10][10].. [ The10The]. Theαα--carboncarbonα-carbon radical radical radical of of amino ofamino amino acids acids acids formed formed formed upon upon upon C C C–H––HH abstraction abstraction abstraction is is is synergistically synergistically stabilized stabilized by by captodativecaptodativecaptodative substituents,substituents substituents,, which which allowsallows eefficientefficientfficient hydrogenhydrogen hydrogen abstractionabstraction abstraction andand and itsi itsts subsequentsubsequent subsequent labellinglabelling labelling of of virtuallyvirtuallyvirtually all allall amino aminoamino acids acidsacids (Figure (Figure(Figure2 2))[2) 12 [12,13[12,13,13].]]..

FigureFigureFigure 2. 2.2. General GeneralGeneral depiction depictiondepiction of ofof photolabelling photophotolabellinglabelling of of proteinof proteinprotein residues residuesresidues (R corresponds(R(R correspondscorresponds to the toto amino thethe aminoamino acid sideacidacid chains)sideside chains) chains) by a BP.by by a Thea BP. BP. excited The The excited excited triplet triplet triplet BP 1,2-diradical BP BP 1,2 1,2--diradicaldiradical undergoes undergoes undergoes H-abstraction, H H--abstractionabstraction leading, ,leading leading to a stable to to a a stable ketylstable radicalketylketyl radical radical and an andα and-carbon an an α α- radical-carboncarbon that radical radical is stabilized that that is is stabilized bystabilized the captodative by by the the captodative captodative effect of the effect electron-withdrawingeffect of of the the electron electron-- carbonylwithdrawingwithdrawing group carbonyl carbonyl and the group group electron-donating and and the the electron electron amino--donatingdonating group. amino amino The group. group. ketyl The The radical ketyl ketyl and radical radicalα-carbon and and α α- radical-carboncarbon recombineradicalradical recombine recombine to form to theto form form crosslinked the the crosslinked crosslinked protein adducts.proteinprotein adducts. adducts. In addition to reacting with the α-carbon, the side chain functionalities of amino acids may also InIn additionaddition toto reactingreacting withwith thethe αα--carbon,carbon, thethe sideside chainchain functionalitiesfunctionalities ofof aminoamino acidsacids maymay alsoalso partake in photolabelling. For instance, the C–H bond energies for a series of stabilized radicals partakepartake in in photo photolabellinglabelling.. For For instance, instance, the the C C––HH bond bond e energiesnergies for for a a series series of of stabilized stabilized radicals radicals formed by amino acid side chains (tertiary, benzylic, and α radicals to the heteroatoms or carbonyl formedformed byby aminoamino acidacid sideside chainschains (tertiary,(tertiary, benzylic,benzylic, andand αα radicalsradicals toto thethe heteroatomsheteroatoms oror carbonylcarbonyl groups) allows for the potential labelling of all amino acid side chains through hydrogen abstraction groups)groups) allowsallows forfor thethe potentialpotential labellinglabelling ofof allall aminoamino acidacid sideside chainschains throughthrough hydrogenhydrogen abstractionabstraction reactions [4]. The 1,2-diradical reacts preferentially with C–H bonds over water, which affords it a reactionsreactions [4][4].. TheThe 1,21,2--diradicaldiradical reactsreacts preferentiallypreferentially withwith CC––HH bondsbonds overover waterwater,, whichwhich affordsaffords itit aa higher photo-linking efficiency compared to other photo-crosslinkers such as diazirines [14,15]. higherhigher photophoto--linkinglinking efficiencyefficiency comparedcompared toto ootherther photophoto--crosslinkerscrosslinkers suchsuch asas diazirinesdiazirines [14,15[14,15]].. BP derivatives can be red-shifted by electron-withdrawing appendages due to the lowering of their BPBP derivativesderivatives cancan bebe redred--shiftedshifted byby electronelectron--withdrawingwithdrawing appendagesappendages duedue toto thethe loweringlowering ofof π* orbital energy. The chemistry of BPs is also sufficiently advanced that incorporation into chemical theirtheir π*π* orbitalorbital energy.energy. TheThe chemistrychemistry ofof BPsBPs isis alsoalso sufficientlysufficiently advancedadvanced thatthat incorporationincorporation intointo and biological probes is easy to achieve, and their inertness to solvents makes them an attractive class chemicalchemical andand biological biological probes probes is is easy easy toto achieve, achieve, andand their their inertness inertness to to solventsolventss makesmakes them them an an of PCGs for PL. Some of the many advantages that allow widespread use of BPs in chemical biology attractiveattractive classclass ofof PCGPCGss forfor PL.PL. SomeSome ofof thethe manymany advantagesadvantages thatthat allowallow widespreadwidespread uusese ofof BPsBPs inin include: low spectral overlap of the n–π* (330–365 nm) with endogenous chromophores (<300 nm), chemicalchemical biology biology include: include: low low spectral spectral overlap overlap of of the the n n––π*π* (33 (3300––365365 nm)nm) with with endogenous endogenous quantitative quantum yields of intersystem crossing to yield the crosslinking relevant excited state, chromophoreschromophores (<300 (<300 nm), nm), quantitative quantitative quantum quantum yields yields of of intersystem intersystem crossing crossing to to yield yield the the quantitative quantum efficiency of C–H abstraction of the excited state, high metabolic stability, crosslincrosslinkingking relevantrelevant excitedexcited state,state, quantitativequantitative quantumquantum efficienefficiencycy ofof CC––HH abstractionabstraction ofof thethe excitedexcited state,state, high high metabolic metabolic stability, stability, and and relative relative stability stability of of the the BP BP and and its its excited excited state state [3,5 [3,5]].. However, However, they they requirerequire long long irradiation irradiation periods periods,, whichwhich may may result result in in non non--specificspecific crosslincrosslinkk formation formation and and cell cell

Molecules 2020, 25, 2285 4 of 27 and relative stability of the BP and its excited state [3,5]. However, they require long irradiation periods, which may result in non-specific crosslink formation and cell damage. Furthermore, their mass (182 Da) and high lipophilicity may limit their use in biological systems. Addition of large photolabels such as BPs may significantly alter the mode of interaction being studied due to added steric bulk and the ability of its π-system to interact with other aromatic amino acids [16]. The lipophilicity of molecules being studied is a main factor in biological systems, where most studied interactions depend upon the cellular permeability of the compound by passive diffusion. Furthermore, proteins are folded such that hydrophobic amino acids are less water-exposed than hydrophilic; thus, the addition of a lipophilic BP moiety to a biological probe may alter the site of interaction on a protein [16]. Nonetheless, BPs continue to be used as a popular PCGs for PL despite their drawbacks, and despite the discovery of many other PCGs since the advent of BPs [17,18]. Their application extends beyond the common PL techniques of binding-site and biological-interactome mapping, target identification and proteome profiling, and surface conjugation techniques amongst others. They are also found in natural products that have broad-spectrum biological effects such as anticancer, antiviral, antimicrobial, and anti-inflammatory effects [2,19–22]. The phototoxicity of this class of bioactive natural products is a probable adverse effect that may limit the widespread utilization of this pharmacophore in drug design [23]. Some research groups [24–27] nonetheless continue to explore BP groups in their drug discovery efforts, and the interested reader may consult a recent review [28] for further insight into the medicinal properties of BP-containing natural products. The use of BPs in PL techniques, including their design and utility in biological investigations, is discussed in Section3.

2.2. Diazirines

2.2.1. Chemical and Physical Properties Diazirines (DAZs) were first discovered in 1960 upon the oxidation of diaziridines, and are quite stable unless exposed to light (~360 nm), heat, or ultrasonication [29–31]. DAZs are used as carbene precursors that liberate molecular nitrogen and a carbene upon longwave irradiation (~360 nm). Upon irradiation, DAZs can generate a carbene by liberating nitrogen directly, or by rearrangement to yield a diazoalkane which liberates nitrogen, thereby forming a carbene indirectly [32–34]. When aliphatic DAZs were first discovered, their utility was limited due to their tendency to undergo 1,2-H shifts [33,34]. Initial efforts in the 1960s aimed at using carbene precursors such as diazoacetates for PL [35] which were later superseded by phenyldiazirines and spiro-adamantane-2,20-diazirine, as introduced by Bayley and Knowles in 1978 [36]. This promising discovery allowed the use of photoactivated carbenes at long wavelengths that biological groups such as amino acids and DNA do not absorb, but, however, suffered from the rearrangement to a linear diazoisomer, which is highly sensitive to acids and nucleophiles [33]. Subsequently, Brunner et al. invented a 3-trifluoromethyl-3-phenyldiazirine (TPD) group that was resistant to such hydrogen rearrangements and reduced the yield and reactivity of the diazoisomer, thereby increasing its applicability for PL and exhibiting nearly quantitative (>95%) OH insertion yields in methanol [33]. Upon excitation of DAZs to the S1 state, they can decay via four main pathways (Figures3 and4): (1) internal conversion to the singlet ground state (S0, Figure3); (2) liberation of nitrogen and formation of closed-shell singlet carbene (Figures3 and4a); (3) formation of open-shell singlet carbene (Figures3 and4b); and (4) internal rearrangement to the diazoisomer (Figure3)[37]. Carbenes can exist in three possible states (Figure4). Presumably, carbenes resulting from homolytic cleavage are triplet carbenes (two unpaired electrons, each occupying a different orbital), whereas a heterolytic cleavage results in a singlet carbene (two paired electrons in same orbital), even though the triplet carbene is lower in energy (Figure4c). The carbenes generated from DAZs, like other carbenes, are usually closed-shell singlet carbenes (Figure4a) unless a photosensitizer is used to promote intersystem crossing to the triplet state (Figure4c) [34,38]. Molecules 2020, 25, x FOR PEER REVIEW 5 of 29

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Figure 3. Possible relaxation pathways of excited DAZs. Upon excitation of DAZs by long-wave UV irradiation, they can relax by internal conversion, isomerize to the diazoisomer, or form a carbene directly.

Carbenes can exist in three possible states (Figure 4). Presumably, carbenes resulting from homolytic cleavage are triplet carbenes (two unpaired electrons, each occupying a different orbital), whereas a heterolytic cleavage results in a singlet carbene (two paired electrons in same orbital), even though the triplet carbene is lower in energy (Figure 4c). The carbenes generated from DAZs, like other carbenes, are usually closed-shell singlet carbenes (Figure 4a) unless a photosensitizer is used FigureFigure 3.3. Possible relaxation pathways of excitedexcited DAZs.DAZs. Upon excitation of DAZs by long-wavelong-wave UV to promoteirradiation,irradiation, intersystem they they can can relaxcrossing relax by by internal tointernal the conversion, triplet conversion, state isomerize (Figure isomerize to 4c) the to diazoisomer,[34,38 the dia]. zoisomer, or form or a carbeneform a directly.carbene directly.

Carbenes can exist in three possible states (Figure 4). Presumably, carbenes resulting from homolytic cleavage are triplet carbenes (two unpaired electrons, each occupying a different orbital), whereas a heterolytic cleavage results in a singlet carbene (two paired electrons in same orbital), even though the triplet carbene is lower in energy (Figure 4c). The carbenes generated from DAZs, like otherFigureFigure carbenes, 4. 4.Possible Possibleare usually open/shell open closed/ shellstates-shell statesand spin singlet and multiplicities spin carbenes multiplicities of (Figure carbenes of4a) formed carbenes unless from a formed photosensitizer DAZs. from (a) Closed DAZs. is- used to promoteshell(a) Closed-shell singl intersystemet carbene. singlet crossing (b carbene.) Open -toshell (b the) Open-shell singlet triplet carbene. state singlet (Figure ( carbene.c) Triplet 4c) ( [34,38c carbene,) Triplet]. carbene,usually generated usually generated by the additionby the addition of a photosensitizer. of a photosensitizer.

CarbeneCarbene spin spin-states-states can can be be influenced influenced by by substituents substituents and and solvent solvent effects effects [39] [39].. For For instance, instance, Sheridan’sSheridan’s group showedshowed that that 2-benzothienyl(CF 2-benzothienyl(CF3)carbene3)carbene is a is ground-state a ground-stat triplet,e triplet, whereas whereas the Platz’s the Platz’sgroup group showed showed that in that polar in solvents, polar solvents, the 4,40 the-biphenyl(CF 4,4′-biphenyl(CF3) carbene3) carbene has a singlet has a singlet ground-state ground and-state the andsinglet-to-triplet the singlet-to- intersystemtriplet intersystem crossing crossing rate depends rate depends on the coordinativeon the coordinative capability capability of solvents of solvents [39,40]. [39,40ComputationalFigure]. Computational 4. Possible density open/shell functionaldensity states studiesfunctional and spin (DFT) multiplicitiesstudies studies (DFT) (B3LYP of carbenes studies/6-31 formed+G(d) (B3LYP/6 andfrom RI-CC2 DAZs.-31+G(d) (/aTZVP;) Closed and singlet - RI- CC2/TZVP;excitedshell singlet singl singletet states carbene. excited (S1,S (b2), Open and singlet S-shell3) optimized states singlet (S carbene.1, with S2, RI-CC2 and (c) Triplet S3/)TZVP) optimized carbene, of phenydiazirine usually with generatedRI-CC2/TZVP) predicted by the the of phenydiazirineexcitedaddition singlet of statepredicta photosensitizer. (S1ed) to the be excited as a result singlet of an state s–p* (S transition1) to be as [a37 result,41]. of an s–p* transition [37,41]. TheThe reactivity reactivity of of a a singlet singlet carbene carbene differs differs greatly greatly from from that that of of a a triplet triplet carbene. carbene. A A triplet triplet carbene carbene cancan be beCarbene detected detected spin via- electronstateselectron can paramagneticp aramagnetic be influenced resonance resonance by substituents (EPR) (EPR) and and and reacts reacts solvent as as a geminate a effects geminate [39] diradical .diradical For instance, that that can canundergoSheridan’s undergo H-abstraction group H-abstraction showed or be that or oxidized be 2 - oxidizedbenzothienyl(CF by molecular by molecular3)carbene oxygen oxygen to is a aketone, ground to a ketone but-stat ise incapable, but triplet, is incapable whereas of inserting theof insertingintoPlatz’s an group O–H into bondan showed O– [42H ,bond 43that]. Ain [42,43 singletpolar]. solvents,A carbene singlet reactsthecarbene 4,4′ with-biphenyl(CF reacts insertion with 3insertion into) carbene X–H into bondshas Xa –singlet (XH bonds= CR ground3 ,(X NR =2 -CR,state OR,3, NRorand SR)2, OR,the to singlet or form SR) photo-linked- to- formtriplet photo intersystem adducts,-linked crossingadducts, making the ratemaking singlet depends the state singlet onmore the state coordinative relevant more forrelevant capability PL (Figure for PL of5)[ solvents(Figure44,45]. 5)Although[39,40 [44,45]. ].Computational Although a singlet carbene a singlet density can carbene be eitherfunctional can an be open- eitherstudies or an a closed-shell (DFT)open- or studies a species,closed (B3LYP/6-shell the photo-crosslinking species,-31+G(d) the andphoto RI is-- crosslinkingmostCC2/TZVP; likely dueis singlet most to the likely excited closed-shell due singletto the singlet closed states species,- she (Sll1, singlet S since2, and thespecies S open-shell3) , optimized since the singlet, open with much- shellRI-CC2/TZVP) likesinglet, the much triplet of likecarbene,phenydiazirine the triplet acts as carbene, a predict biradical, edacts the whereas as excited a biradical the singlet closed-shell, whereas state (S singlet 1the) to closedbe owes as a- its shellresult reactivity singlet of an to sowes– ap* zwitterionic transition its reactivity [37,41 character to]. a zwitterioniccausedThe by reactivity one character fully of occupied a caused singlet orbital bycarbene one and fully differs another occupied greatly empty orbitalfrom orbital that and (Figureof anothera triplet5)[ 46 emptycarbene.,47]. orbital A triplet (Figure carbene 5) [46,47can beIt]. maydetected be worthwhile via electron toparamagnetic note, however, resonance that the (EPR) exact and mechanism reacts as a for geminate the formation diradical of that the closed-shellcan undergo carbene H-abstraction was speculated or be oxidized by Platz by and molecular co-workers oxygen to be to formed a ketone through, but is the incapable open-shell of singletinserting intermediate into an O–H [37 bond]. [42,43]. A singlet carbene reacts with insertion into X–H bonds (X = CR3, NR2, OR, or SR) to form photo-linked adducts, making the singlet state more relevant for PL (Figure 5) [44,45]. Although a singlet carbene can be either an open- or a closed-shell species, the photocrosslinking is most likely due to the closed-shell singlet species, since the open-shell singlet, much like the triplet carbene, acts as a biradical, whereas the closed-shell singlet owes its reactivity to a zwitterionic character caused by one fully occupied orbital and another empty orbital (Figure 5) [46,47].

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Figure 5. Photolabelling of proteins by 3-trifluoromethyl-3-phenyldiazirine (TPD) a commonly used Figure 5. Photolabelling of proteins by 3-trifluoromethyl-3-phenyldiazirine (TPD) a commonly used DAZ motif. Irradiation of TPD generates a closed-shell singlet carbene which carries out an insertion DAZ motif. Irradiation of TPD generates a closed-shell singlet carbene which carries out an insertion into polar X–H bonds to give the photolabelled covalent adduct. into polar X–H bonds to give the photolabelled covalent adduct. 2.2.2. Recent Synthetic Developments It may be worthwhile to note, however, that the exact mechanism for the formation of the closed- shell Carbenescarbene was generated speculated from DAZsby Platz have and a tendencyco-workers to beto be quenched formed by through water, therebythe open lowering-shell singl theiret crosslinkingintermediate yields [37]. (>10%) [29,43]. This is both a limitation and an advantage, since it ensures that all photo-crosslinked targets are those in immediate proximity to the DAZ at the instance of irradiation, as2.2.2. the Recent carbene Synthetic generated Developments by photoactivation is too short-lived (pico- to nanoseconds) for off-target activity [29,48]. TheCarbenes utility generated of DAZs from as probes DAZs forhave covalently a tendency mapping to be quenched transient by molecular water, thereby interactions lowering has promptedtheir crosslin syntheticking yields chemists (>10%) to create [29,43 easier,]. This safer,is both and a morelimitation efficient and methods an advantage for DAZ, since incorporation it ensures intothat biologicalall photoprobes.crosslinked Synthetic targets developments are those in and immediate reaction compatibilityproximity to ofthe DAZs DAZ have at the been instance reviewed of inirradiati detailon previously, as the carbene [29,45 ,generated49] and are by not photoactivation the focus of this is too review; short- brieflived mention(pico- to isnanoseconds) made of recent for improvementsoff-target activity and [29,48 developments]. that may aid the reader. InThe the utility synthesis of DAZs of probes as probes incorporating for covalently TPD or mapping aliphatic transient DAZ motifs, molecular the PCGs interactions are usually has installedprompted near synthetic the end chemists of the synthesis to create in order easier, to preventsafer, and exposure more to efficient harsh conditions methods (reagents,for DAZ acid,incorporation heat, microwaves, into biological etc.) [50 probes.]. Chemical Synthetic biologists developments and medicinal and reactionchemists compatibility are thus constrained of DAZs in termshave been of when reviewed and wherein detail PCGs previously can be installed[29,45,49] during and are synthesis. not the focus The of incorporation this review; brief of DAZs mention can beis made categorized of recent into improvements three main categories: and developments (1) appended, that may (2) replacement, aid the reader. and (3) nested (Figure6b). An idealIn the photolabelled synthesis of probe probes should incorporating be structurally TPD or and aliphatic physicochemically DAZ motifs, similar the PCGs to the are unlabelled usually probeinstalled in order near tothe provide end of minimal the synthesis perturbation in order to itsto pharmacodynamicprevent exposure (potency,to harsh interactome,conditions (reagents, etc.) and acid, heat, microwaves, etc.) [50]. Chemical biologists and medicinal chemists are thus constrained in physical (lipophilicity, pKa, ionization, etc.) properties. Nested DAZs provide minimal perturbation andterms replacement of when and o ffwhereers a slightPCGs perturbation,can be installed whereas during appended synthesis. DAZsThe incorporation provide the greatestof DAZs degreecan be ofcategorized perturbation into (Figure three main6c) [ 29categories:,50]. Imaginably, (1) appended the greater, (2) replacement the perturbation, and (3) to nested the probe (Figure (such 6b). as An for appendedideal photolabelled and replacement probe should DAZs), bethe structurally less representative and physi a studycochemically using the similar labelled to probe the unmaylabelled of the unlabelledprobe in order probe. to provide minimal perturbation to its pharmacodynamic (potency, interactome, etc.) and Because physical of (lipophilicity, the thermal stability pKa, ionization and incompatibility, etc.) properties. of DAZs withNested metallic DAZs reagents, provide the minimal current synthesisperturbation routes and mainly replacement (approximately offers a three-quarters)slight perturbation revolve, whereas around appended the use of DAZs robust provide and simple the methodsgreatest degr suchee as of amide perturbation formation, (Figure alkylation, 6c) [29,50 and]. esterificationImaginably, the (Figure greater6a). the Perhaps perturbation this synthetic to the limitationprobe (such is theas for reason appended why more and than replacement half of the DAZs) DAZ-based, the less chemical representative probes a described study using to date, the consistlabelled of probe appended may of DAZs the un (thelabelled category probe. with the greatest degree of perturbation) (Figure6c). Because of the thermal stability and incompatibility of DAZs with metallic reagents, the current synthesis routes mainly (approximately three-quarters) revolve around the use of robust and simple methods such as amide formation, alkylation, and esterification (Figure 6a). Perhaps this synthetic limitation is the reason why more than half of the DAZ-based chemical probes described to date, consist of appended DAZs (the category with the greatest degree of perturbation) (Figure 6c).

Molecules 2020, 25, 2285 7 of 27 Molecules 2020, 25, x FOR PEER REVIEW 7 of 29

Figure 6. The method of synthesis and mode of incorporation of DAZ probes based on a survey of Figure 6. The method of synthesis and mode of incorporation of DAZ probes based on a survey of literature, including 212 published DAZ photolabelled probes. (a) The percentage distribution of literature, including 212 published DAZ photolabelled probes. (a) The percentage distribution of common synthetic methods of attachment of the DAZ containing moiety to the probe. (b) The common synthetic methods of attachment of the DAZ containing moiety to the probe. (b) The percentage percentage distribution of the three categories of incorporation of DAZs (appended, replacement, distribution of the three categories of incorporation of DAZs (appended, replacement, nested). nested). (c) A schematic representation of the three categories of DAZ incorporation and the (c) A schematic representation of the three categories of DAZ incorporation and the corresponding corresponding level of perturbation caused (adapted from Ichiishi et al., 2019 [50]). level of perturbation caused (adapted from Ichiishi et al., 2019 [50]).

In 2019In 2019 Merck Merck and Co. and published Co. published a study addressing a study addressing this limitation this in limi probetation design in byprobe developing design by a Suzuki–Miyauradeveloping a Suzuki cross-coupling–Miyaura c reactionross-coupling that isreaction compatible that is with compatible TPD [50 with]. Suzuki–Miyaura TPD [50]. Suzuki– cross-couplingMiyaura cross screens-coupling of 8-haloquinolines screens of 8-haloquinolines with phenylboronic with phenylboronic acid and boronic acid ester and derivatives boronic ester combinedderivatives with combined varying reaction with varying conditions reaction such conditions as temperature, such as temperatupalladiumre, catalyst, palladium solvents, catalyst, andsolvents bases,, in and the bases, presence in the of presence DAZ-containing of DAZ-containing spectator spectator molecules, molecules, were used were to used determine to determine the mostthe DAZ-friendly most DAZ-friendly cross-coupling cross-coupling conditions conditions that gave that thegave highest the highest yields. yields. After After the identificationthe identification of theof the most most suitable suitable cross-coupling cross-coupling conditions conditions (least (least harmful harmful to DAZsto DAZs and and producing producing the the greatest greatest yields),yields), the the substrate substrate scope scope of of the the reactionreaction ofof a TPD TPD derivative derivative (DAZ (DAZ-containing-containing aryl aryl bromide bromide (3-(4- (3-(4-bromophenyl)-3-(trifluoromethyl)-3H-diazirine))bromophenyl)-3-(trifluoromethyl)-3H-diazirine)) was evaluated evaluated with with a a variety variety of of boronate boronate esters. esters. TheThe results results concluded concluded that that the the average average yield yield was was ~40% ~40% andand that the the conditions conditions were were compatible compatible with withsaturated saturated and and unsaturated unsaturated heterocyclic heterocyclic rings rings and andhydrogen hydrogen bond bond donors donors (aromatic (aromatic amines, amines, alcohols, alcohols,and amides, and amides, Scheme Scheme 2). Furthermore,2). Furthermore, the applicabili the applicabilityty of cross of cross-coupling-coupling conditions conditions beyond beyond the p- the bromophenyldiazirinep-bromophenyldiazirine synthon synthon was was demonstrated demonstrated by by reproducing reproducing similar similar yields yields with with the the m- m-bromophenyldiazirinebromophenyldiazirine synthon.synthon. Lastly, an improvement in in yield yield (from (from 20% 20% to to 45%) 45%) and and reaction reaction time time(6 (6 days days to to 3 3h) h) was was reported reported in in the the synthesis synthesis of of known known photoaffinity photoaffinity probe probe GSK GSK-3-3 [50,51 [50,51].].

Molecules 2020, 25, 2285 8 of 27 MoleculesMolecules 20202020,, 2255,, xx FORFOR PEERPEER REVIEWREVIEW 88 ofof 2929 Molecules 2020, 25, x FOR PEER REVIEW 8 of 29

SchemeSchemeScheme 2. 2. 2. Optimized OptimizedOptimizedOptimized Suzuki Suzuki Suzuki–Miyaura Suzuki––Miyaura–MiyauraMiyaura reaction reaction reaction reaction that that that that is is is is compatible compatible compatible compatible with with with with DAZs DAZs DAZs DAZs and and and a a a varie varietyvarie varietytyty of of of functionalitiesfunctionalitiesfunctionalities (heterocycles,(heterocycles,(heterocycles, (heterocycles, aromatic aromaticaromatic aromatic amines, amines,amines, amines, alcohols, alcohols,alcohols, alcohols, amides, amidesamides amides etc.),, ,etc.)etc.) etc.) and andand and requires requiresrequires requires a short aa a shortshort short reaction reactionreaction reaction time timeandtimetime mild andand and mildmild reaction mild reactionreaction reaction conditions. conditions.conditions. conditions. Furthermore, Wang et al. recently reported the improved synthesis of aliphatic DAZs from Furthermore,Furthermore,Furthermore, Wang Wang Wang et et et al. al. al. recently recently recently reported reported reported the the the improved improved improved synthesis synthesis synthesis of of of aliphatic aliphatic aliphatic DAZs DAZs DAZs from from from aliphatic ketones by using hydroxylamine-O-sulfonic acid (HOSA) in liquid ammonia for 12 h at room aliphaticaliphaticaliphatic ketonesketones ketones byby by usiusi usingngng hydroxylaminehydroxylamine hydroxylamine--OO-O--sulfonicsulfonic-sulfonic acidacid acid (HOSA)(HOSA) (HOSA) inin in liquidliquid liquid ammoniaammonia ammonia forfor for 1212 12 hh h atat at temperature, followed by treatment with potassium hydroxide (Scheme3). This method produced a roomroomroom temperature temperature temperature,, ,followed followedfollowed by by by treatment treatment treatment with with with potassium potassium potassium hydroxide hydroxide hydroxide (Scheme (Scheme (Scheme 3). 3). 3). This ThisThis method method method major improvement in yield (~30–40% to 67%) and simplicity (three steps to one pot, two steps; room producedproducedproduced aa a majormajor major improvementimprovement improvement inin in yieldyield yield (~3(~3 (~300–0–40–4040%%% toto to 67%)67%) 67%) andand and simplicitysimplicity simplicity (three(three (three stepssteps steps toto to oneone one pot,pot, pot, twtw twooo temperature; under air) allowing for gram-scale DAZ production [29,52]. steps;steps;steps; roomroom room temperature;temperature; temperature; underunder under air)air) air) allowingallowing allowing forfor for gramgram gram--scalescale-scale DAZDAZ DAZ productionproduction production [29,52[29,52 [29,52]]..] .

SchemeSchemeScheme 3. 3. 3. ImprovedImprovedImproved Improved oneone one-pot, one--pot,pot,-pot, twotwo two-step two--stepstep-step synthesissynthesis synthesis ofof of aliphaticaliphatic aliphatic DAZsDAZs DAZs from from from aliphatic aliphatic aliphatic ketones. ketones. ketones. Additionally, Wang et al. developed two methods for synthesizing TPD derivatives in a one-pot Additionally,Additionally,Additionally, WangWang Wang etet et al.al. al. developeddeveloped developed twotwo two methodsmethods methods forfor for synthesizingsynthesizing synthesizing TPDTPD TPD derivativesderivatives derivatives inin in aa a oneone one--potpot-pot synthesis directly from the tosyl oxime without isolating the diaziridine precursor (Scheme4). Kinetic synthesissynthesissynthesis diredire directlyctlyctly fromfrom from thethe the tosyltosyl tosyl oximeoxime oxime withoutwithout without isolatingisolating isolating thethe the diaziridinediaziridine diaziridine precursorprecursor precursor (Scheme(Scheme (Scheme 4).4). 4). KineticKinetic Kinetic NMR experiments suggested the reaction to proceed via a diaziridine intermediate which is rapidly NMRNMRNMR experimentsexperiments experiments suggestedsuggested suggested thethe the reactionreaction reaction toto to proceedproceed proceed viavia via aa a diaziridinediaziridine diaziridine intermediateintermediate intermediate whichwhich which isis is rapidlyrapidly rapidly formed in situ, and is followed by a gradual oxidation to the diazirine [53]. formedformedformed inin in situsitu situ,, and,and and isis is followedfollowed followed byby by aa a gradualgradual gradual oxidationoxidation oxidation toto to thethe the diazirdiazir diazirineineine [53][53] [53].. .

SchemeSchemeScheme 4. 4. 4. CondensedCondensed Condensed method method method for for for the the the one one-potone one--potpot-pot synthesis synthesis synthesis of of of trifluoromethylphenyltriﬂuoromethylphenyl trifluoromethylphenyl trifluoromethylphenyl DAZDAZ DAZss s without without without isolatingisolatingisolating the the the diaziridine diaziridine diaziridine intermediate. intermediate.intermediate. intermediate. Another one-pot metal-free synthesis of terminal DAZs from unprotected amino acids using AnotherAnotherAnother oneone one--potpot-pot metalmetal metal--freefree-free synthesissynthesis synthesis ofof of terminalterminal terminal DAZsDAZs DAZs fromfrom from unprotectedunprotected unprotected aminoamino amino acidsacids acids usingusing using ammonia and phenyliodonium diacetate (PIDA) was developed by Glachet et al. (Scheme5)[ 54]. This ammoniaammoniaammonia andand and phenyliodoniumphenyliodonium phenyliodonium diacetatediacetate diacetate (PIDA)(PIDA) (PIDA) waswas was developeddeveloped developed byby by GlachetGlachet Glachet etet et alal al.. .(Scheme(Scheme (Scheme 5)5) 5) [54][54] [54].. . convenient and robust method tolerates a wide range of natural amino acids and can be scaled up to ThisThisThis convenientconvenient convenient andand and robustrobust robust methodmethod method toleratestolerates tolerates aa a widewide wide rangerange range ofof of naturalnatural natural aminoamino amino acidsacids acids andand and cancan can bebe be scaledscaled scaled multigram quantities. The reaction proceeds via three successive one-pot reactions: (1) decarboxylation upupup to to to multigram multigram multigram quantities. quantities. quantities. TheTheThe reaction reaction reaction proceeds proceeds proceeds viaviavia threethreethree successive successive successive one one one--potpot-pot reactions: reactions: reactions: (1) (1) (1) decarboxylationdecarboxylationdecarboxylation of of of the the the amino amino amino acid acid acid C C C--terminatermina-terminall,,l , (2)(2)(2) formation formation formation of of of diaziridine diaziridine diaziridine upon upon upon insertion insertion insertion of of of

Molecules 2020, 25, 2285 9 of 27 Molecules 2020, 25, x FOR PEER REVIEW 9 of 29 ofiodonitrene the amino, and acid (3) C-terminal, oxidation to (2) form formation the diazirine. of diaziridine Surprisingly, upon this insertion reaction of was iodonitrene, also successful and (3)in oxidationdiazirination to form of two the diazirine. dipeptides Surprisingly, by replacing this its reaction C-terminal was also with successful a terminal in diazirination DAZ without of tworacemization dipeptides of by the replacing chiral its centers C-terminal [54], withsuggesting a terminal the DAZ possibility without of racemization it being applicable of the chiral to centerspolypeptides. [54], suggesting the possibility of it being applicable to polypeptides.

Scheme 5 5.. Optimized reaction conditions fo forr the one one-pot-pot three three-step-step diazirinationdiazirination reaction.

3. Applications of Benzophenone and Diazirine Radical Precursors to Chemical Biology 3.1. Benzophenones 3.1. Benzophenones 3.1.1. Understanding Biological Interactions and Mechanisms 3.1.1. Understanding Biological Interactions and Mechanisms PL has become an important technique in elucidating how drugs, proteins, and biological molecules PL has become an important technique in elucidating how drugs, proteins, and biological interact by identifying novel mechanisms and targets of therapeutic value. BPs have found extensive molecules interact by identifying novel mechanisms and targets of therapeutic value. BPs have found application in PL studies of complex biological systems such as protein–protein interactions (PPIs) [55]. extensive application in PL studies of complex biological systems such as protein–protein interactions Wu et al. [56] reported a BP-based multifunctional probe that incorporated two alkyne handles for (PPIs) [55]. Wu et al. [56] reported a BP-based multifunctional probe that incorporated two alkyne double-click chemistry to create a stapled peptide and a third alkyne handle for target enrichment handles for double-click chemistry to create a stapled peptide and a third alkyne handle for target and pulldown (Figure7, left). Stapled peptides are mostly hydrocarbon-braced peptides locked into enrichment and pulldown (Figure 7, left). Stapled peptides are mostly hydrocarbon-braced peptides their α-helixes for stabilization, permeability, improved activity, and protease resistance [57–59]. This locked into their α-helixes for stabilization, permeability, improved activity, and protease resistance strategy has been shown to enable very eﬃcient biological targeting and investigation. [57–59]. This strategy has been shown to enable very efficient biological targeting and investigation. In this study, the probe was attached to the peptide via Cu-catalyzed click chemistry using the In this study, the probe was attached to the peptide via Cu-catalyzed click chemistry using the double alkyne moieties, to crosslink two diﬀerent amino-acid residues of the peptide and create an double alkyne moieties, to crosslink two different amino-acid residues of the peptide and create an α-helix stabilized by the BP-containing synthetic brace (commonly referred to as stapling). Subsequent α-helix stabilized by the BP-containing synthetic brace (commonly referred to as stapling). irradiation at 365 nm allowed for crosslinking to interacting proteins in proximity, giving insight into Subsequent irradiation at 365 nm allowed for crosslinking to interacting proteins in proximity, giving protein-interacting partners of the peptide (Figure7). Following crosslinking, the third alkyne handle insight into protein-interacting partners of the peptide (Figure 7). Following crosslinking, the third allowed subsequent Cu-mediated click chemistry for sample enrichment with biotin. Subsequently, alkyne handle allowed subsequent Cu-mediated click chemistry for sample enrichment with biotin. pulldown and proteomic mass spectrometry (MS) analysis was used to identify the target proteins. Subsequently,Molecules 2020, 25 ,pulldown x FOR PEER and REVIEW proteomic mass spectrometry (MS) analysis was used to identif10y ofthe 29 target proteins. This design was adopted into a p53-derived peptide probe and was effective in crosslinking mouse double minute 2 homolog (MDM2), a negative regulator of the p53 tumor suppressor, thus validating the efficiency of the designed probe. The time-dependent labelling of MDM2 was also observed via SDS-PAGE (Figure 7). The simple design of these BP probes allows for their integration into several study designs. The core scaffolds are easily amenable templates that can be used for a broad range of applications.

FigureFigure 7.7.BP BP multifunctionalmultifunctional linkerlinker designeddesigned andand incorporatedincorporated intointo aa stapledstapled peptidepeptide andand usefuluseful inin studyingstudying protein protein interactions interactions with with biomolecules biomolecules (polyethylene(polyethylene glycolglycol isis abbreviatedabbreviated asas PEG;PEG; Sodium Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) adapted from Wu et al., 2016 [56]). dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) adapted from Wu et al., 2016 [56]).

Because BPs are a feature of some natural compounds, sometimes only minimal modifications may be required for their use in PL studies, which ensures that their innate activity is unperturbed, and the results obtained can be trusted to be reliable. Zhao et al. [60] used elegaphenone (ELP), a naturally occurring antibiotic for activity-based protein profiling (ABPP), which helped to elucidate ELP’s binding partners and hence its cellular pathway in Gram-positive and Gram-negative bacteria. They incubated the natural compound with bacterial cells, irradiated cells at 365 nm, lysed, and performed a biotin–azide click conjugation (facilitated by an alkyne tag initially introduced to the para position to the BP in ELP) for pulldown and isolation of crosslinked targets. Subsequent LC- MS/MS analysis afforded complete proteomic studies that validated already known targets and suggested novel ones of ELP in its antibiotic mode of action. Uncovering the sites that confer activity and mediate interactions in a biomolecule is one way of proposing its mechanism of action. The oxidation of cholesterol is linked to several cancers and pathologies, and it is believed to proceed via intramolecular hydrogen abstraction mediated by distinct enzymatic and free-radical pathways. The use of BP in hydrogen abstraction has already been reported [61,62], and hence may be easily applied to understanding possible sites of hydrogen abstraction in cholesterol. Palumbo et al. [63] thus modified cholesterol with a BP at the C7 position and investigated possible sites of hydrogen abstraction following photoirradiation. While confirming the already known location of hydrogen extraction at C4, they also observed a similar occurrence at C15, a location that is thermodynamically unfavorable (Figure 8). The authors suggested that based on their finding, the activity and mechanism of cholesterol may be highly dependent on the specific reactant topology, and that this finding may be of interest in studying the reactivity of surface membrane.

Molecules 2020, 25, 2285 10 of 27

This design was adopted into a p53-derived peptide probe and was effective in crosslinking mouse double minute 2 homolog (MDM2), a negative regulator of the p53 tumor suppressor, thus validating the efficiency of the designed probe. The time-dependent labelling of MDM2 was also observed via SDS-PAGE (Figure7). The simple design of these BP probes allows for their integration into several study designs. The core scaffolds are easily amenable templates that can be used for a broad range of applications. Because BPs are a feature of some natural compounds, sometimes only minimal modifications may be required for their use in PL studies, which ensures that their innate activity is unperturbed, and the results obtained can be trusted to be reliable. Zhao et al. [60] used elegaphenone (ELP), a naturally occurring antibiotic for activity-based protein profiling (ABPP), which helped to elucidate ELP’s binding partners and hence its cellular pathway in Gram-positive and Gram-negative bacteria. They incubated the natural compound with bacterial cells, irradiated cells at 365 nm, lysed, and performed a biotin–azide click conjugation (facilitated by an alkyne tag initially introduced to the para position to the BP in ELP) for pulldown and isolation of crosslinked targets. Subsequent LC-MS/MS analysis afforded complete proteomic studies that validated already known targets and suggested novel ones of ELP in its antibiotic mode of action. Uncovering the sites that confer activity and mediate interactions in a biomolecule is one way of proposing its mechanism of action. The oxidation of cholesterol is linked to several cancers and pathologies, and it is believed to proceed via intramolecular hydrogen abstraction mediated by distinct enzymatic and free-radical pathways. The use of BP in hydrogen abstraction has already been reported [61,62], and hence may be easily applied to understanding possible sites of hydrogen abstraction in cholesterol. Palumbo et al. [63] thus modified cholesterol with a BP at the C7 position and investigated possible sites of hydrogen abstraction following photoirradiation. While confirming the already known location of hydrogen extraction at C4, they also observed a similar occurrence at C15, a location that is thermodynamically unfavorable (Figure8). The authors suggested that based on their finding, the activity and mechanism of cholesterol may be highly dependent on the specific reactant topology, and that this finding may be of interest in studying the reactivity of surface membrane. Molecules 2020, 25, x FOR PEER REVIEW 11 of 29

FigureFigure 8. 8.BP-modiﬁed BP-modified cholesterol cholesterol was was used used to to identify identif possibley possible regions regions of hydrogen of hydrogen abstraction abstraction in the in cellularthe cellular oxidation oxidation mechanism mechanism of cholesterol. of cholesterol.

The signaling mechanisms that trigger host responses at the cellular level are sometimes vaguely understood, and, in the case of hormonally controlled virulence of pathogenic bacteria, a BP-based probe was used to reveal cellular interactions involved in this process [64]. The peptide hormone dynorphin-A (Dyn-A) is known to be involved in cellular responses to pain and stress via enhanced virulence of the pathogenic bacteria, Pseudomonas aeruginosa (PA), which is involved in cystic fibrosis and may be associated with lung infections. The challenge in making treatments for this pathogen is in its elevated resistance to antibacterial therapies, and a poor understanding of its cellular pathways. Wright et al. [64] used mimics of Dyn-A that involved a BP PCG and an alkyne tag to discover Dyn- A-associated proteins in PA. They incubated their probe with the bacterial cell lysate, conducted UV- mediated crosslinking, and analyzed the pulled-down samples by LC-MS/MS. The authors subsequently took the top hit from this study, a membrane sensor kinase known as Pars, and through mutation studies were able to validate its role in PA’s cellular mode of action. Substrate identification for enzymes provides key information on their cellular roles and mechanisms. PL approaches are one of the most widely applied techniques for identifying and validating new substrates for enzymes. Histone deacetylases are a protein family that enzymatically deacetylate lysine residues mediating a cascade of cellular remodeling that ends in suppression of gene transcription [65,66]. Histones were initially thought to be the only substrates HDACs act on, but a series of proteomic studies have elucidated and validated other HDAC substrates. Lopez et al. [67] were able to use a BP-mediated crosslinking technique to map proteomic interactions of one of the clinically relevant HDACs, HDAC8, in cell lysate, and identified an unbiased subset of substrates. Site-directed mutagenesis was used to incorporate BP at I94, Y100, and F191 in the HDAC8 plasmid. The plasmid was expressed using bacterial culture to obtain the mutant HDAC8, which was then subjected to an HDAC8 activity assay which validated that the mutant enzyme’s catalytic property was intact [68]. This mutant HDAC8 was then incubated in HEK293 cell lysate and UV-irradiated for 30 mins to probe for its interacting partners. The crosslinked samples were enriched and extracted using an affinity column, and then analyzed by MS to reveal crosslinked partners (Figure 9). This approach is simple and could reasonably be adapted to several proteins and biomolecules of choice.

Molecules 2020, 25, 2285 11 of 27

The signaling mechanisms that trigger host responses at the cellular level are sometimes vaguely understood, and, in the case of hormonally controlled virulence of pathogenic bacteria, a BP-based probe was used to reveal cellular interactions involved in this process [64]. The peptide hormone dynorphin-A (Dyn-A) is known to be involved in cellular responses to pain and stress via enhanced virulence of the pathogenic bacteria, Pseudomonas aeruginosa (PA), which is involved in cystic fibrosis and may be associated with lung infections. The challenge in making treatments for this pathogen is in its elevated resistance to antibacterial therapies, and a poor understanding of its cellular pathways. Wright et al. [64] used mimics of Dyn-A that involved a BP PCG and an alkyne tag to discover Dyn-A-associated proteins in PA. They incubated their probe with the bacterial cell lysate, conducted UV-mediated crosslinking, and analyzed the pulled-down samples by LC-MS/MS. The authors subsequently took the top hit from this study, a membrane sensor kinase known as Pars, and through mutation studies were able to validate its role in PA’s cellular mode of action. Substrate identification for enzymes provides key information on their cellular roles and mechanisms. PL approaches are one of the most widely applied techniques for identifying and validating new substrates for enzymes. Histone deacetylases are a protein family that enzymatically deacetylate lysine residues mediating a cascade of cellular remodeling that ends in suppression of gene transcription [65,66]. Histones were initially thought to be the only substrates HDACs act on, but a series of proteomic studies have elucidated and validated other HDAC substrates. Lopez et al. [67] were able to use a BP-mediated crosslinking technique to map proteomic interactions of one of the clinically relevant HDACs, HDAC8, in cell lysate, and identified an unbiased subset of substrates. Site-directed mutagenesis was used to incorporate BP at I94, Y100, and F191 in the HDAC8 plasmid. The plasmid was expressed using bacterial culture to obtain the mutant HDAC8, which was then subjected to an HDAC8 activity assay which validated that the mutant enzyme’s catalytic property was intact [68]. This mutant HDAC8 was then incubated in HEK293 cell lysate and UV-irradiated for 30 mins to probe for its interacting partners. The crosslinked samples were enriched and extracted using an affinity column, and then analyzed by MS to reveal crosslinked partners (Figure9). This approachMolecules 20 is20 simple, 25, x FOR and PEER could REVIEW reasonably be adapted to several proteins and biomolecules of choice.12 of 29

Figure 9. Mutant HDAC8 bearing BP-modified amino acids that can be used to identify novel Figure 9. Mutant HDAC8 bearing BP-modified amino acids that can be used to identify novel HDAC8 HDAC8 substrates. substrates. 3.1.2. Other Applications Incorporating BP Probes 3.1.2. Other Applications Incorporating BP Probes PL techniques based on BP-mediated photoactivation formation remain attractive due to their easy incorporation,PL techniques minimal based on eff BPects-mediated on biological photoactivation processes, simple formation and non-invasiveremain attractive designs, due andto their the abilityeasy incorporation, to control their minimal timing andeffects location on biological of activation processes, which simpl makese and them non suitable-invasive for adesigns, host of studies.and the Asability such, to BPs control have theirbeen appliedtiming and to studies location outside of activation the realm which of protein makes profiling. them suitable Photo-caging, for a host like of protectingstudies. As groups, such, isBPs one have such been application applied involving to studies the outside masking the of anrealm interacting of protein site onprofiling. a biomolecule Photo- suchcaging, that like time-specific protecting irradiation groups, is cleavesone such the application PCG and allowsinvolving restoration the masking of function of an interacting and activity site [69 on]. Methylationa biomolecule of such C7 on that guanine time-specific (m7G) isirradiation the most cleaves observed the nucleoside PCG and allows modification, restoration mediating of function the 7 functionsand activity and [69 interactions]. Methylation of several of C7 nucleicon guanine acids, (m andG) understandingis the most observed the molecular nucleoside interactions modification that, requiremediat thising the modification functions isand highly interactions desirable. of Anhäuserseveral nucleic et al. [70acids] recently, and understanding reported a photocaged the molecular m7G usinginteractions BP and that they require observed this light-controlled modification is uncaging, highly desirable. a feature Anhäu that hasser the et al potential. [70] recently to facilitate reported new a 7 studiesphotocaged of molecular m G using interactions BP and of they m7G observed and m1A (methylatedlight-controlled adenine) uncaging, and could a feature enable that the controlhas the potential to facilitate new studies of molecular interactions of m7G and m1A (methylated adenine) and could enable the control of biological functions using light [70,71]. They proposed an uncaging mechanism that is driven by the protonated N7 and involves the biradical BP species abstracting hydrogen and transferring a lone radical to nearby chemical groups (EDTA and glycerol were used in this study) to form a trivalent carbon radical, which subsequently tautomerizes, is terminated, and regains aromaticity to form the methyl BP product (Figure 10). This product was proposed based on NMR and UHPLC-MS/MS analysis of the byproducts following irradiation.

Figure 10. A BP-based photo-caging approach was used to study DNA interactions mediated by the m7G modification.

Molecules 2020, 25, x FOR PEER REVIEW 12 of 29

Figure 9. Mutant HDAC8 bearing BP-modified amino acids that can be used to identify novel HDAC8 substrates.

3.1.2. Other Applications Incorporating BP Probes PL techniques based on BP-mediated photoactivation formation remain attractive due to their easy incorporation, minimal effects on biological processes, simple and non-invasive designs, and the ability to control their timing and location of activation which makes them suitable for a host of studies. As such, BPs have been applied to studies outside the realm of protein profiling. Photo- caging, like protecting groups, is one such application involving the masking of an interacting site on a biomolecule such that time-specific irradiation cleaves the PCG and allows restoration of function and activity [69]. Methylation of C7 on guanine (m7G) is the most observed nucleoside modification, mediating the functions and interactions of several nucleic acids, and understanding the molecular interactions that require this modification is highly desirable. Anhäuser et al. [70] recently reported a photocaged m7G using BP and they observed light-controlled uncaging, a feature that has the Moleculespotential2020, 25to, 2285facilitate new studies of molecular interactions of m7G and m1A (methylated 12adenine) of 27 and could enable the control of biological functions using light [70,71]. They proposed an uncaging mechanism that is driven by the protonated N7 and involves the biradical BP species abstracting of biological functions using light [70,71]. They proposed an uncaging mechanism that is driven by hydrogen and transferring a lone radical to nearby chemical groups (EDTA and glycerol were used the protonated N7 and involves the biradical BP species abstracting hydrogen and transferring a lone in this study) to form a trivalent carbon radical, which subsequently tautomerizes, is terminated, and radical to nearby chemical groups (EDTA and glycerol were used in this study) to form a trivalent regains aromaticity to form the methyl BP product (Figure 10). This product was proposed based on carbon radical, which subsequently tautomerizes, is terminated, and regains aromaticity to form the NMR and UHPLC-MS/MS analysis of the byproducts following irradiation. methyl BP product (Figure 10). This product was proposed based on NMR and UHPLC-MS/MS analysis of the byproducts following irradiation.

FigureFigure 10. 10.A BP-basedA BP-based photo-caging photo-caging approach approach was was used used to studyto study DNA DNA interactions interactions mediated mediated by theby the 7 m7Gm modiﬁcation.G modification.

In a dual crosslinking and immobilization approach, Jakubovska et al. [72] functionalized N4-cytidines with BP and demonstrated the dual usefulness of the installed BP. Through BP-mediated crosslinking, they successfully (1) immobilized the modified oligonucleotides to polymeric solid supports and (2) covalently labelled interacting proteins of the oligonucleotide. The single-stranded DNA was also able to efficiently recognize and hybridize with its complementary sequence, further expanding the usefulness of this approach. This dual approach presents opportunity for reduced functionalization and retention of innate activity in oligonucleotide studies, as BP-modified cytidine nucleobases are efficiently read and incorporated by several DNA polymerases, and the terminal deoxynucleotide transferase, TdT, is able to generate multi-BP-modified single-stranded DNA oligonucleotides. These studies continue to highlight the utility and widespread applications of BP PCGs as a versatile tool in chemical biology.

3.2. Diazirines in Chemical Biology Diazirines (DAZ) are perhaps the smallest of all the PCGs, and this affords them the added advantage of nominal alteration of the properties of the probe into which they have been introduced. This singular feature distinguishes DAZs from other PCGs and has made them very attractive for use in the study of biological processes and interactions. Upon photoactivation, DAZ generates a transient divalent carbene intermediate that is highly reactive and unstable, and rapidly interacts through covalent bond formation with nearby chemical species. This super-reactive feature of this carbene intermediate is double-edged. On one hand, this affords DAZs high selectivity and reduces Molecules 2020, 25, 2285 13 of 27 their propensity to bind non-specifically, because they quickly react with proximal co-interacting biomolecules. This also minimizes the probability that they will react with any random diffusing species. Because irradiation occurs at a longer wavelength of 365 nm, and the reactive carbene intermediate generated has a lifetime of picoseconds [37,47], these species also cause the least damage to tissues and biological systems under study. However, they are very unstable and may not be suitable for long-term studies. They are also easily quenched by water, ensuring that their crosslink yields are very low [43,73]. Trifluoromethyl diazirines such as TPD produce the carbene insertion products in high yields and do not suffer from the internal rearrangement observed with non-fluorinated DAZs [33]. In studying biological systems, there is usually a matrix in which isolation of crosslinked target molecules is desired, so purification usually involves some rounds of target enrichment and then elution and analysis. Some of these designs and applications are discussed below.

3.2.1. Target Engagement and Discovery of Novel Bioactive Compounds DAZs have been widely used as probes for validating target engagement during the drug discovery process. A simple approach was designed by Saaidin et al. [73] that incorporated CF3-diazirine meta to the nitro group on a 2-nitrobenzosulfonamide (ortho-Nosyl) moiety to validate its target. Nosyl groups are excellent protective groups for amines because their deprotection is obtained in high yields following treatment with nucleophilic thiols in an SNAr reaction. The approach in this study is thus useful for elucidating a drug candidate interactome, as most feature secondary amines that can aﬀord easy installation of this PCG. Saaidin et al. installed this ortho-Nosyl-DAZ probe onto biotin for target validation, using bovine serum albumin (BSA) protein, a known biotin target, as an example. The biotin probe was incubated with BSA, and photoirradiation with 365 nm light (250 W) allowed detection of the BSA photoproduct via SDS-PAGE chemiluminescence method (Figure 11). Further treatment of this photolabelled BSA with bodipy-thiol allowed over 70% cleavage of the model compound via an SNAr approach, and this allowed enrichment of photolabelled complexes for easier detection and identiﬁcation. The authors claim that this method may be a simple approach in PL procedures that can be used to identify a probe’s interactome. This was a proof-of-concept approach which only highlighted the utility of DAZ-mediated crosslinking approaches to determine biological targets. Molecules 2020, 25, x FOR PEER REVIEW 14 of 29

FigureFigure 11.11. DesignDesign ofof orthoortho-Nosyl-Nosyl probeprobe thatthat incorporatesincorporates aa DAZDAZ forfor useuse asas ligand-tagligand-tag exchangeableexchangeable photoaphotoaffinityﬃnity probe (Coomassie (Coomassie brilliant brilliant blue blue is abbreviated is abbreviated as CBB as;g CBB;gelel image imageadapted adapted from Saaidin from Saaidinet al., 2019 et al., [73] 2019). [73]).

Similarly, a TPD-based photoaﬃnity probe, TPD-950-Br, was used to analyze the molecular Similarly, a TPD-based photoaffinity probe, TPD-950-Br, was used to analyze the molecular interactions of MCC950 (also known as CP-456773 and CRID3), a known inhibitor of NLRP3 (NOD-, interactions of MCC950 (also known as CP-456773 and CRID3), a known inhibitor of NLRP3 (NOD- LRR-, and pyrin domain-containing protein 3) [74]. This study highlighted the use of a replacement-DAZ , LRR-, and pyrin domain-containing protein 3) [74]. This study highlighted the use of a replacement- approach to generate a minimally perturbed photoaﬃnity probe of MCC950, which could enable DAZ approach to generate a minimally perturbed photoaffinity probe of MCC950, which could future studies investigating its molecular target and mechanism of action. enable future studies investigating its molecular target and mechanism of action. Soluble guanylyl cyclase (sGC) is a nitric oxide (NO) receptor that is severely implicated in cardiovascular diseases. Novel stimulators of this receptor show a lot of clinical promise, yet their precise modes of action are largely unknown. Using a DAZ-mediated diagnostic application, Wales and coworkers showed the precise location where stimulators interact with the sGC receptor [75]. The authors used a known stimulator, IWP-051, and functionalized it with a DAZ moiety, which ensured its irreversible binding affinity to sGC upon photoirradiation. They were able to localize stimulators binding to a highly conserved β1 heme domain in human and insect sGC. Through DAZ covalent crosslinking interaction, they were also able to replicate similar localized stimulator binding to the sGC heme domain of bacterial homologs, showing that binding at this site is conserved across species. The authors went further to characterize this heme binding domain, showing by MS and NMR data that the known stimulators bind to a conserved pocket lying between the two subdomains found in the heme domain of sGC. This work characterized and identified the specific location of binding, and thus permits better informed and structurally guided future design of NO stimulators. One possible limitation of the approaches described above is in the sheer size of the appendage groups, which may result in disruption of the probe’s function. Several methods thus require simple linkers that can facilitate similar studies with minimal disturbance of native activity. Jackson et al. [76] described a bifunctional isocyanide probe incorporating a DAZ group for target biomolecule capture and an aliphatic alkyne that allowed enrichment of photolabelled products via metal- catalyzed click chemistry. The isocyanide group permitted isocyanide-based multicomponent reactions (IMCR) that allowed installation of different small-molecule probes with diverse chemical scaffolds containing at least one aldehyde, amine, ketone, or carboxylic acid functionality (Figure 12). This approach thus introduced the added advantage of performing concurrent fragment-based screening and proteomic exploration under the same assay conditions. The outcome of this work was a clickable photoreactive isocyanide tool that is suited for combinatorial chemistry of IMCR fragments and can allow chemoproteomic-enabled drug discovery.

Molecules 2020, 25, 2285 14 of 27

Soluble guanylyl cyclase (sGC) is a nitric oxide (NO) receptor that is severely implicated in cardiovascular diseases. Novel stimulators of this receptor show a lot of clinical promise, yet their precise modes of action are largely unknown. Using a DAZ-mediated diagnostic application, Wales and coworkers showed the precise location where stimulators interact with the sGC receptor [75]. The authors used a known stimulator, IWP-051, and functionalized it with a DAZ moiety, which ensured its irreversible binding affinity to sGC upon photoirradiation. They were able to localize stimulators binding to a highly conserved β1 heme domain in human and insect sGC. Through DAZ covalent crosslinking interaction, they were also able to replicate similar localized stimulator binding to the sGC heme domain of bacterial homologs, showing that binding at this site is conserved across species. The authors went further to characterize this heme binding domain, showing by MS and NMR data that the known stimulators bind to a conserved pocket lying between the two subdomains found in the heme domain of sGC. This work characterized and identified the specific location of binding, and thus permits better informed and structurally guided future design of NO stimulators. One possible limitation of the approaches described above is in the sheer size of the appendage groups, which may result in disruption of the probe’s function. Several methods thus require simple linkers that can facilitate similar studies with minimal disturbance of native activity. Jackson et al. [76] described a bifunctional isocyanide probe incorporating a DAZ group for target biomolecule capture and an aliphatic alkyne that allowed enrichment of photolabelled products via metal-catalyzed click chemistry. The isocyanide group permitted isocyanide-based multicomponent reactions (IMCR) that allowed installation of different small-molecule probes with diverse chemical scaffolds containing at least one aldehyde, amine, ketone, or carboxylic acid functionality (Figure 12). This approach thus introduced the added advantage of performing concurrent fragment-based screening and proteomic exploration under the same assay conditions. The outcome of this work was a clickable photoreactive isocyanide tool that is suited for combinatorial chemistry of IMCR fragments and can allow chemoproteomic-enabled drug discovery. Molecules 2020, 25, x FOR PEER REVIEW 15 of 29

FigureFigure 12.12.Bifunctional Bifunctional isocyanideisocyanide probeprobe facilitatesfacilitates multicomponentmulticomponent reactionsreactions andand aaffordsﬀords versatilityversatility andand dualdual useuse asas fragment-basedfragment-based screening screening and and proteome proteome mapping. mapping.

SeveralSeveral methodologies methodologies aim aim to to improve improve the the effi ciencyefficiency of screening of screening procedures procedures for identifying for identifying novel proteinnovel prot binders.ein binders. DNA-encoded DNA-encoded chemical chemical libraries libraries (DECLs) (DECL provides) hugeprovide numbers huge ofnumbers diverse of chemical diverse fragmentschemical fragments that can bethat unambiguously can be unambiguously screened screened against against protein protein targets. targets. The e ffTheectiveness effectiveness of this of approachthis approach is limited, is limited as it involves, as it involves several several post-binding post-binding washes thatwashes restricts that therestricts retention the retention and recovery and ofrecovery medium of to medium low-affi nity to low (Kd-affinity) binders. (Kd One) binders. new approach One new that approach helps to that uncover helps these to uncover micromolar these bindersmicromolar with binders protein w complexesith protein that complexes exhibit that limited exhibit kinetic limited stability kinetic is stability via DAZ-mediated is via DAZ-mediated covalent crosslinkingcovalent crosslin interactions.king interacti Followingons. Following several reports several [77 reports,78] on [77,78 the use] on of the DAZ use crosslinking of DAZ crosslinking reactions forreaction DECLs screenings,for DECL screenings, Sannino et Sannino al. [79] describedet al. [79] described a novel approach a novel approach that involved that ainvolved single-stranded a single- oligonucleotide-encodedstranded oligonucleotide fragment-encoded library fragment of which library the of complementarywhich the complementary sequence carried sequence a DAZ carried PCG. a UsingDAZ PCG. carbonic Using anhydrase carbonic IX anhydrase (CAIX) as IX the (CAIX) model as target the protein,model target a combinatorial protein, a combinatorial DECL was screened DECL andwas irradiatedscreened and at 365 irradiated nm to initiate at 365 covalentnm to initiate crosslinking covalent of crosslinking the DAZ with of CAIX,the DAZ the with target CAIX, protein, the suchtarget that protein, transient such and that low-a transientffinity and interactions low-affinity were interactions locked in were (Figure locked 13). in Following (Figure 13). washes Following and washes and elution steps, the authors were able to show that this crosslinking capture mechanism was able to identify low-affinity carbonic anhydrase IX binders when compared to traditional solid support-based affinity protein capture.

Figure 13. DAZ-functionalized oligonucleotide probes for studying protein–DNA interactions. (Electrophoretic mobility shift assay, enzyme-linked immunosorbent assay, and quantitative polymerase chain reaction are abbreviated to EMSA, ELISA, and qPCR respectively)

PL has been very instrumental in the structural characterization of specific binding sites, mainly for small-molecule probes. Complementary MS-enabled enrichment and NMR studies allow global proteomic studies that can spatiotemporally report these binding hotspots in an unbiased analytical procedure. The challenge, however, has been in extending this approach to larger MW and complex molecules; hence, the binding modes for these class of compounds have not been properly clarified. Rapamycin is a 914 Da compound described as a molecular glue, as it stabilizes the FKBP12– mTOR protein complex. This ternary complex formation was previously described and characterized by biophysical methods such as crystallography [80]. In a bid to understand how PL can decode structural information on large-molecule binding locations, Flaxman et al. [81] designed photo-

Molecules 2020, 25, x FOR PEER REVIEW 15 of 29

Figure 12. Bifunctional isocyanide probe facilitates multicomponent reactions and affords versatility and dual use as fragment-based screening and proteome mapping.

Several methodologies aim to improve the efficiency of screening procedures for identifying novel protein binders. DNA-encoded chemical libraries (DECLs) provide huge numbers of diverse chemical fragments that can be unambiguously screened against protein targets. The effectiveness of this approach is limited, as it involves several post-binding washes that restricts the retention and recovery of medium to low-affinity (Kd) binders. One new approach that helps to uncover these micromolar binders with protein complexes that exhibit limited kinetic stability is via DAZ-mediated covalent crosslinking interactions. Following several reports [77,78] on the use of DAZ crosslinking reactions for DECL screenings, Sannino et al. [79] described a novel approach that involved a single- stranded oligonucleotide-encoded fragment library of which the complementary sequence carried a MoleculesDAZ PCG.2020, 25Using, 2285 carbonic anhydrase IX (CAIX) as the model target protein, a combinatorial15 DECL of 27 was screened and irradiated at 365 nm to initiate covalent crosslinking of the DAZ with CAIX, the target protein, such that transient and low-affinity interactions were locked in (Figure 13). Following elutionwashes steps, and elution the authors steps, were the authors able to showwere able that to this sho crosslinkingw that this capturecrosslinking mechanism capture wasmechanism able to identifywas able low-a to identifyﬃnity carbonic low-affinity anhydrase carbonic IX bindersanhydrase when IX comparedbinders when to traditional compared solid to traditional support-based solid asupportﬃnity protein-based capture.affinity protein capture.

Figure 13. DAZ-functionalized oligonucleotide probes for studying protein–DNA interactions. (ElectrophoreticFigure 13. DAZ mobility-functionalized shift assay, oligonucleotide enzyme-linked immunosorbent probes for studying assay, andprotein quantitative–DNA interactionspolymerase . chain(Electrophoretic reaction are abbreviated mobility shift to EMSA, assay, ELISA,enzyme and-linked qPCR immunosorbent respectively) assay, and quantitative polymerase chain reaction are abbreviated to EMSA, ELISA, and qPCR respectively) PL has been very instrumental in the structural characterization of specific binding sites, mainly for small-moleculePL has been very probes. instrumental Complementary in the structural MS-enabled characteri enrichmentzation andof specific NMR studiesbinding allowsites, globalmainly proteomicfor small-molecule studies that probes. can spatiotemporally Complementary reportMS-enabled these bindingenrichment hotspots and NMR in an unbiasedstudies allow analytical global procedure.proteomic Thestudies challenge, that can however, spatiotemporally has been report in extending these binding this approach hotspots to largerin an unbiased MW and analytical complex molecules;procedure. hence, The challeng the bindinge, however, modes has for thesebeen classin extending of compounds this approach have not to beenlarger properly MW and clarified. complex molecules;Rapamycin hence, is athe 914 binding Da compound modes describedfor these class as a molecularof compounds glue, ashave it stabilizes not been the properly FKBP12–mTOR clarified. proteinRapamycin complex. is This a 914 ternary Da compound complex formationdescribed as was a molecular previously glue, described as it stabilizes and characterized the FKBP12 by– biophysicalmTOR protein methods complex. such This as crystallography ternary complex [80 formation]. In a bid towas understand previously how described PL can decodeand characterized structural informationby biophysical on methods large-molecule such as binding crystallography locations, [80]. Flaxman In a bid et al.to [understand81] designed how photo-rapamycin PL can decode bystructural functionalizing information the C40 on (earlier large-molecule shown to binding be amenable locations, to functionalization) Flaxman et al. [81] of rapamycin designed with photo a- DAZ-containing bifunctional probe similar to that described by Jackson et al. [76]. Following incubation and photoirradiation of the photo-rapamycin, FKBP12, and mTOR, the authors showed formation of the ternary complex in vitro by MS fragmentation. They also showed that a McLafferty rearrangement of photo-rapamycin occurred at the ternary complex, and in conjunction with molecular dynamics studies, were able to characterize possible conformations of this ternary complex. This study thus further emphasized the utility of DAZ-based PCGs in PL studies.

3.2.2. Studying Complex Protein Interactions The use of PL techniques is not restricted to synthetic molecules alone. Mapping out complete protein interactomes is highly desirable for understanding cellular mechanisms and biological functions, and PL has been effectively used in elucidating some of these complex biomolecular interactions. Studies like these are highly relevant, as there are still a lot of unknown PPIs despite advancements in predictive computational tools and high-throughput mapping. These calculative techniques rely on pre-existing PPI databases for their algorithm-based predictions but are inadequate for elucidating novel PPIs with no prior knowledge of a relatable social protein network. In a simple demonstration of how PL can be used to characterize new PPIs, Horne et al. [82] described the use of a DAZ-modified residue in a target protein that crosslinks interacting partners, and subsequent enrichment and MS characterization revealed these binding partners. In this approach, the authors introduced a reactive cysteine into the target protein to allow future incorporation of a disulfide bridge through a methanethiosulfonate (MTS) installed on the DAZ probe. On exposure to a pool of possible targets and subsequent irradiation of UV light at 365 nm, a crosslink was formed with Molecules 2020, 25, x FOR PEER REVIEW 16 of 29

rapamycin by functionalizing the C40 (earlier shown to be amenable to functionalization) of rapamycin with a DAZ-containing bifunctional probe similar to that described by Jackson et al. [76]. Following incubation and photoirradiation of the photo-rapamycin, FKBP12, and mTOR, the authors showed formation of the ternary complex in vitro by MS fragmentation. They also showed that a McLafferty rearrangement of photo-rapamycin occurred at the ternary complex, and in conjunction with molecular dynamics studies, were able to characterize possible conformations of this ternary complex. This study thus further emphasized the utility of DAZ-based PCGs in PL studies.

3.2.2. Studying Complex Protein Interactions The use of PL techniques is not restricted to synthetic molecules alone. Mapping out complete protein interactomes is highly desirable for understanding cellular mechanisms and biological functions, and PL has been effectively used in elucidating some of these complex biomolecular interactions. Studies like these are highly relevant, as there are still a lot of unknown PPIs despite advancements in predictive computational tools and high-throughput mapping. These calculative techniques rely on pre-existing PPI databases for their algorithm-based predictions but are inadequate for elucidating novel PPIs with no prior knowledge of a relatable social protein network. In a simple demonstration of how PL can be used to characterize new PPIs, Horne et al. [82] described the use of a DAZ-modified residue in a target protein that crosslinks interacting partners, and subsequent enrichment and MS characterization revealed these binding partners. In this Moleculesapproach,2020, 25 the, 2285 authors introduced a reactive cysteine into the target protein to allow16 future of 27 incorporation of a disulfide bridge through a methanethiosulfonate (MTS) installed on the DAZ probe. On exposure to a pool of possible targets and subsequent irradiation of UV light at 365 nm, a bindingcrosslink proteins, was formed treatment with with binding reducing proteins, agents treatment cleaved with the disulﬁde reducing bonds, agents and cleave onlyd thethe bindingdisulfide proteinbonds was, and retained only the (Figure binding 14 protein). was retained (Figure 14).

Figure 14. Bifunctional MTS-DAZ is a simple probe that can be used to investigate PPIs. Figure 14. Bifunctional MTS-DAZ is a simple probe that can be used to investigate PPIs. Digestion and MS analysis revealed the identity of these interacting proteins. The authors believe that thisDigestion strategy couldand MS potentially analysis be revealed used to mapthe identity cellular PPIs of these in both interacting precise and proteins. dynamic The locations. authors believeA further that this extension strategy ofcould this potentially approach wasbe used used to tomap investigate cellular PPIs the morein both complicated precise and cellulardynamic proteinlocations. machinery that regulates post-translational modifications such as SUMOylation, ubiquitination, and otherA further ubiquitin-like extension (Ubl) of modifications.this approachThe was specific used to networking investigate interactions the more of complicated E1-activating cellular and E2-conjugatingprotein machinery enzymes that combined regulates with E3 post ligases-translational are modulated modifications by several PTMs such and as cellular SUMOylation, triggers thatubiquitination, make their investigation and other ubiquitin somewhat-like challenging.(Ubl) modifications. Although The the specific use of networking ABPP is a powerful interactions tool of thatE1- hasactivating been extensively and E2-conjugating used to study enzymes the specific combined interactomes with E3 ligases and pathways are modulated of these by PPIsseveral [83 –PTMs87], thisand method cellular is triggers limited that to E3make ligases, their whichinvestigation have a somewhat catalytic cysteine challenging. that Although is able to acceptthe use Ubof /ABPPUbl from the E2-conjugating enzyme and transfer them to the proteins present in their social network. The majority of E3 ligases, however, lack a reactive cysteine that can facilitate Ub/Ubl transfer and are reliant on transient non-covalent interactions to carry out their ubiquitination roles. To successfully investigate the vast abundance of Ub/Ubl-E2-E3 interactions that were excluded by the ABPP approach, Zhang et al. [88] constructed a trifunctional E2-conjugating enzyme probe that used an amide bond to link the SUMO-UBc9 (an E2-conjugating enzyme) system, which is known to have incredible affinity for E3 ligase. By functionalizing the Ubc9 with DAZ and photoactivation at 365 nm, any E3 ligase recruited is crosslinked covalently to form a SUMO-E2-E3 ternary complex (Figure 15). This approach should allow the validation of known E3 ligases and the discovery of novel E3 ligases hitherto unreported and should provide a strategy for the construction of other E2-Ub/Ubl conjugation frameworks. Molecules 2020, 25, x FOR PEER REVIEW 17 of 29

is a powerful tool that has been extensively used to study the specific interactomes and pathways of these PPIs [83–87], this method is limited to E3 ligases, which have a catalytic cysteine that is able to accept Ub/Ubl from the E2-conjugating enzyme and transfer them to the proteins present in their social network. The majority of E3 ligases, however, lack a reactive cysteine that can facilitate Ub/Ubl transfer and are reliant on transient non-covalent interactions to carry out their ubiquitination roles. To successfully investigate the vast abundance of Ub/Ubl-E2-E3 interactions that were excluded by the ABPP approach, Zhang et al. [88] constructed a trifunctional E2-conjugating enzyme probe that used an amide bond to link the SUMO-UBc9 (an E2-conjugating enzyme) system, which is known to have incredible affinity for E3 ligase. By functionalizing the Ubc9 with DAZ and photoactivation at 365 nm, any E3 ligase recruited is crosslinked covalently to form a SUMO-E2-E3 ternary complex (Figure 15). This approach should allow the validation of known E3 ligases and the discovery of novel MoleculesE3 ligases2020 hitherto, 25, 2285 unreported and should provide a strategy for the construction of other E2-Ub/Ubl17 of 27 conjugation frameworks.

Figure 15. Trifunctional E2-conjugating enzyme probe to identify novel E3 ligase components of the SUMO-E2-E3 ternary complex. Figure 15. Trifunctional E2-conjugating enzyme probe to identify novel E3 ligase components of the TheSUMO utility-E2-E3 of ternary DAZ-based complex. PL techniques has been extended into biological studies of protein interactions in live cells, where there is a myriad of highly complex, interconnected series of molecular interactionThe utility networks of DAZ occurring-based concurrently. PL techniques The has resolution been extended and identification into biological of these studies interactions of protein for ainteractions specific biomolecule in live cells of interest, where with there no is or a minimal myriad perturbation of highly complex, of these cellularinterconnected events is series highly of challenging.molecular interaction However, thisnetworks ability occurring increases ourconcurrentl understandingy. The resol of howution cellular and identif eventsication are organized of these ininteractions time and spacefor a specific and provides biomolecule insight of oninterest how with to eff noectively or minimal modulate perturbation these events, of these especially cellular forevents therapeutic is highly purposes. challenging. Current However, techniques this ability employed increases to studyour understanding these myriad of interactions how cellular include events aareffinity organized purification–mass in time and spectroscopy space and (AP-MS),provides chemicalinsight on/enzymatic-tagging-based how to effectively modulate LC-MS these/MS, events BioID,, andespecially proximal for enzymatictherapeutic labellingpurposes. [89 Current–94]. Whiletechniques some employed of these techniquesto study these have myriad been interactions successful, theyinclude also affinity come with purification some significant–mass spectroscopy limitations (AP such-MS), as bias chemic againstal/enzymatic weaker- interactions,tagging-based need LC- forMS/MS, co-factors, BioID, and and labelling proximal approaches enzymatic thatlabelling require [89 initiation–94]. While by some exogenous of these chemicals techniques which have have been thesuccessful, potential they to unsettlealso come normal with some cellular significant homeostasis limitations [95,96 su].ch McCutcheon as bias against et weaker al. [97] interactions, reported a photo-proximityneed for co-factors, PPI (PhotoPPI)and labelling profiling approaches method that that require allowed initiation the characterization by exogenous of chemicals PPI networking which mapshave inthe vitro potentialand in to live unsettle cells. normal The authors cellular employed homeostasis a SNAP-tagged [95,96]. McCutcheon protein of et interest al. [97] (POI)reported and a photo-proximity PPI (PhotoPPI) profiling method that allowed the characterization of PPI functionalized their DAZ-based probe with a benzylguanine and a photocleavable group (NO2-veratryl carbamate),networking such maps that in vitro the DAZ-functionalized and in live cells. The probe authors was employed covalently a SNAP attached-tagged to the protein POI (e.g., of interest redox regulated(POI) and sensor functionaliz protein,ed KEAP1)their DAZ by-based the benzylguanine probe with a benzylguanine moiety. Subsequently, and a photocleavable upon irradiation group at (NO2-veratryl carbamate), such that the DAZ-functionalized probe was covalently attached to the 365 nm, the NO2-veratryl-carbamate group was cleaved off and any proximal proteins to the POI were simultaneouslyPOI (e.g., redox labelled regulated by thesensor DAZ protein, group, KEAP1) thereby identifyingby the benzylguanine any nearby moiety. proteins Subsequently, with the POI viaupon a irradiation at 365 nm, the NO2-veratryl-carbamate group was cleaved off and any proximal proteins subsequentMolecules 20 enrichment20, 25, x FOR PEER and REVIEW quantitative LC-MS/MS analysis (Figure 16). 18 of 29 to the POI were simultaneously labelled by the DAZ group, thereby identifying any nearby proteins with the POI via a subsequent enrichment and quantitative LC-MS/MS analysis (Figure 16).

Figure 16. Workﬂow of photo-PPI that allows characterization of a protein’s social network in live cells. Figure 16. Workflow of photo-PPI that allows characterization of a protein’s social network in live cells.

This technique was used by the authors to elucidate the interactome for KEAP1, validate known interactions, and discover novel ones for KEAP1 with high temporal resolution in live cells [97].

3.2.3. Probing Membrane Proteins and Other Protein–Biomolecule Interactions DAZ-based PL is a highly versatile technique that has been applied to the study of several biomolecular interactions and processes. DAZ-based PL was recently applied to the study of membrane proteins, a very challenging field of study in chemical biology. Despite the important roles that membrane proteins play in critical cellular processes such as cellular transport, signaling, and reception, their structures and mechanisms are still poorly understood. Some of the current techniques applied to study them include X-ray crystallography [98–100], cryo-EM [100], solid-state NMR [101,102], and MS [103–106]. Studying membrane proteins using X-ray crystallography has been a tricky approach due to the hydrophobic nature of these proteins, which increases the likelihood that they will aggregate and precipitate out of solution. Cryo-EM and solid-state NMR, on the other hand, have shown a lot of promise in this regard, but MS is arguably the most effective method to date for studying membrane proteins. However, the number of techniques available to study membrane proteins is still small when compared to their soluble counterparts, underscoring the need for new techniques that can provide valuable information on this class of proteins. In a proof-of-concept design, Manzi et al. [107] described a DAZ-based label (Figure 17) that was able to efficiently label the β-barrel porin protein OmpF, which is an outer membrane protein responsible for small-molecule diffusion in E. coli. They were able to show that their probe, due to its amphipathic nature, could access the micelle formed by OmpF and discriminately label and distinguish the hydrophobic transmembrane sections, as opposed to the subunit interfacial binding sections. By using MS/MS analysis, mapping of the surface-accessible binding sections in OmpF was possible.

Molecules 2020, 25, 2285 18 of 27

This technique was used by the authors to elucidate the interactome for KEAP1, validate known interactions, and discover novel ones for KEAP1 with high temporal resolution in live cells [97].

3.2.3. Probing Membrane Proteins and Other Protein–Biomolecule Interactions DAZ-based PL is a highly versatile technique that has been applied to the study of several biomolecular interactions and processes. DAZ-based PL was recently applied to the study of membrane proteins, a very challenging field of study in chemical biology. Despite the important roles that membrane proteins play in critical cellular processes such as cellular transport, signaling, and reception, their structures and mechanisms are still poorly understood. Some of the current techniques applied to study them include X-ray crystallography [98–100], cryo-EM [100], solid-state NMR [101,102], and MS [103–106]. Studying membrane proteins using X-ray crystallography has been a tricky approach due to the hydrophobic nature of these proteins, which increases the likelihood that they will aggregate and precipitate out of solution. Cryo-EM and solid-state NMR, on the other hand, have shown a lot of promise in this regard, but MS is arguably the most effective method to date for studying membrane proteins. However, the number of techniques available to study membrane proteins is still small when compared to their soluble counterparts, underscoring the need for new techniques that can provide valuable information on this class of proteins. In a proof-of-concept design, Manzi et al. [107] described a DAZ-based label (Figure 17) that was able to efficiently label the β-barrel porin protein OmpF, which is an outer membrane protein responsible for small-molecule diffusion in E. coli. They were able to show that their probe, due to its amphipathic nature, could access the micelle formed by OmpF and discriminately label and distinguish the hydrophobic transmembrane sections, as opposed to the subunit interfacial binding sections. By using MS/MS analysis, mapping of the surface-accessible binding sections in OmpF was possible. Molecules 2020, 25, x FOR PEER REVIEW 19 of 29

Figure 17. DAZ-modiﬁed OmpF allows successful mapping of proteins’ binding regions. Protein in red indicates regions that were successfully labelled by the probe; protein in blue indicates unlabelled (masked)Figure 17. regions DAZ- ofmodified the protein. OmpF (Image allows showing successful labelled mapping and of masked proteins protein’ binding region regions. adapted Protein with in permissionred indicates from regions Manzi that et al., were 2017 successfully [107]). labelled by the probe; protein in blue indicates unlabelled (masked) regions of the protein. (Image showing labelled and masked protein region adapted with permission from Manzi et al., 2017 [107]).

There is a paucity of techniques available for studying protein–lipid interactions. Lipids are key building blocks in cells, involved in protein lipidation, modulate subcellular transport, and are major components of cellular membranes. The protein–lipid interaction is a key metabolic interaction that is responsible for a host of cellular signaling and function and designing techniques that can be used to investigate these interactions is highly desirable. The use of DAZ-based PL has been applied to the study of lipid–protein interactions by some research groups [108,109], but, because of the need to chemically synthesize and incorporate their probes into the lipid design, they could not efficiently replicate innate cellular metabolic strategies using solely biologically synthesized lipids. In 2017, Wang et al. [110] reported a semi-synthetic bifunctional probe design that could be incorporated into cellular phospholipid biosynthesis and used this strategy to identify many binding protein partners to the phospholipid phosphatidylcholine. Another complex protein interaction that has been challenging to efficiently characterize is the protein–RNA interaction, due to the low affinity of these interactions, and the presence of accompanying protein–protein complexes usually complicates these studies. Protein–RNA interactions help to mediate cellular control of RNA localization, stability, splicing, and translation. The N6-methyladenosine (m6A) RNA post-transcriptional modification is a newly identified RNA modification that is postulated to regulate gene expression via subjective recognition by specific proteins. In a study to identify protein partners that recognize the m6A RNA modification, Arguello et al. [111] described an RNA photoaffinity probe that contained the m6A modification adjacent to a non-perturbing DAZ PCG, and a biotin affinity handle for subsequent protein enrichment post- crosslinking. This RNA probe was introduced into HeLa cell lysate, and irradiation at 365 nm ensured crosslinking to interacting proteins. Streptavidin enrichment and LC-MS/MS analysis were used to identify and quantify protein interacting with the m6A RNA modification and their abundance (Figure 18). This study further established the utility of DAZ probes in mapping biological

Molecules 2020, 25, 2285 19 of 27

There is a paucity of techniques available for studying protein–lipid interactions. Lipids are key building blocks in cells, involved in protein lipidation, modulate subcellular transport, and are major components of cellular membranes. The protein–lipid interaction is a key metabolic interaction that is responsible for a host of cellular signaling and function and designing techniques that can be used to investigate these interactions is highly desirable. The use of DAZ-based PL has been applied to the study of lipid–protein interactions by some research groups [108,109], but, because of the need to chemically synthesize and incorporate their probes into the lipid design, they could not efficiently replicate innate cellular metabolic strategies using solely biologically synthesized lipids. In 2017, Wang et al. [110] reported a semi-synthetic bifunctional probe design that could be incorporated into cellular phospholipid biosynthesis and used this strategy to identify many binding protein partners to the phospholipid phosphatidylcholine. Another complex protein interaction that has been challenging to efficiently characterize is the protein–RNA interaction, due to the low affinity of these interactions, and the presence of accompanying protein–protein complexes usually complicates these studies. Protein–RNA interactions help to mediate cellular control of RNA localization, stability, splicing, and translation. The N6-methyladenosine (m6A) RNA post-transcriptional modification is a newly identified RNA modification that is postulated to regulate gene expression via subjective recognition by specific proteins. In a study to identify protein partners that recognize the m6A RNA modification, Arguello et al. [111] described an RNA photoaffinity probe that contained the m6A modification adjacent to a non-perturbing DAZ PCG, and a biotin affinity handle for subsequent protein enrichment post-crosslinking. This RNA probe was introduced into HeLa cell lysate, and irradiation at 365 nm ensured crosslinking to interacting proteins. Streptavidin enrichment and LC-MS/MS analysis were used to identify and quantify protein interacting Molecules 2020, 25, x FOR PEER REVIEW 20 of 29 with the m6A RNA modification and their abundance (Figure 18). This study further established the utilityinteractomes of DAZ, probesand the in p mappingrobe designed biological here should interactomes, be applicable and the to probe the investigation designed here of shouldinteracting be applicablepartners of to any the investigationpost-transcriptional of interacting RNA modification. partners of any post-transcriptional RNA modification.

Figure 18. DAZ-modified RNA close to established m6a RNA modifications allows elucidation of binding partners that recognize this modification. Figure 18. DAZ-modified RNA close to established m6a RNA modifications allows elucidation of DAZ-basedbinding partners techniques that recognize have this also modification. been applied to immobilization of biomolecules on solid support systems to enable straightforward and selective capture of biochemical materials of interest. MultifunctionalDAZ-based nanoparticles techniques have (MNPs) also such been as applied supra-paramagnetic to immobilization iron oxides of biomolecules have been on highly solid successfulsupport systems in surface to enable chemistry straightforward applications and for selective the controlled capture and of biochemical site-specific material immobilizations of interest. of biologicalMultifunctional species nanoparticles facilitated by (MNPs)amide bonding such as [112 supra]. This-paramagnetic strategy can iron sometimes oxides have jeopardize been highly the biologicalsuccessful function in surface of immobilized chemistry applications species, especially for the controlled antibodies and (Abs), site- duespecific to blockage immobilization of their of bindingbiological sites species [113]. facilitated Other conjugation by amide protocols bonding such [112] as. This thiol strategy reduction can or sometimes oxidationalso jeopardize fall short, the duebiological to harsh function chemical of treatments immobilized that species may alter, especially Ab structure antibodies and function. (Abs), due Fan et to al. blockage [113] reported of their anbinding MNP functionalizedsites [113]. Other with conjugation a boronic acidprotocols for Ab such capture as thiol and reduc a DAZtion to or e ffioxidationciently crosslinkalso fall short and , covalentlydue to harsh immobilize chemical the treatments Ab to the metalthat may surface. alter TheyAb structure also showed and thatfunction. this approach Fan et al was. [113] a superior reported immobilizationan MNP functionalized strategy comparedwith a boronic to other acid known for Ab approaches. capture and Thea DAZ immobilized to efficiently Ab crosslink maintained and excellentcovalently stability immobilize and an activethe Ab orientation to the metal that surface.allowed selectiveThey also binding showed for that the target this approach protein. Thesewas a studiessuperior continue immobilization to emphasize strategy the utility comp ofared DAZ-based to other PL known probes approaches. and the variations The immobilized in their design Ab thatmaintained make them excellent applicable stability to various and an biological active orientation investigations. that allowed selective binding for the target protein. These studies continue to emphasize the utility of DAZ-based PL probes and the variations in their design that make them applicable to various biological investigations.

3.2.4. Molecular Markers for Imaging Studies Imaging techniques such as NMR and MRI represent powerful analytical tools that have found widespread use in academic investigation and clinical diagnosis, respectively. The biggest pitfall of both applications is in their low sensitivities arising from their low nuclear spin polarization, a feature that describes the population of nuclear spins that can transit different energy levels. High-field NMR/MRI [114], aside from other challenges, cannot considerably increase nuclear spin polarization to the level that will significantly enhance technique sensitivity. The use of hyperpolarizing agents is one way to dramatically increase signal intensities and improve sensitivity. Some examples include dynamic nuclear polarization (DNP) [115], hyperpolarized gases [116,117], parahydrogen-induced polarization [118], and signal amplification by reversible exchange (SABRE) [119,120]. Hyperpolarization by SABRE improves nuclear spin polarization via rapid and reversible exchange of parahydrogen (p-H2) with the available sites of a metal complex (usually iridium, Ir), and at the microtesla (µT) magnetic field strength usually employed, p-H2 spin polarization transfer is possible all the way to substrate molecules such as 15N-pyridine. This technique has been shown to provide significant gain in signal intensities that can translate to 15N biomedical imaging and spectroscopy [121–123]. 15N-DAZ has been proposed as an alternative to 15N-pyridines as substrate for SABRE technique due to its small size, ease of incorporation, longer polarization lifetime, and non-perturbance of native biomolecule activity [124]. The potential of 15N-DAZ as an imaging tag was investigated by Shen et al. [124], showing a 15,000-fold signal enhancement (ε) over 14N-DAZ (Figure 19). They also found several-fold increases in the singlet spin order (Ts) and magnetization relaxation (T1) time constants of 15N2 when compared to normal polarization decay time constants. On incorporation of their 15N-DAZ into choline derivatives, they observed a 2000-fold increase in signal with T1 = 3 min. Their set-up also displayed extended polarization lifetimes and signal enhancement in D2O, suggesting the applicability of this tag for in vivo biomedical imaging

Molecules 2020, 25, 2285 20 of 27

3.2.4. Molecular Markers for Imaging Studies Imaging techniques such as NMR and MRI represent powerful analytical tools that have found widespread use in academic investigation and clinical diagnosis, respectively. The biggest pitfall of both applications is in their low sensitivities arising from their low nuclear spin polarization, a feature that describes the population of nuclear spins that can transit different energy levels. High-field NMR/MRI [114], aside from other challenges, cannot considerably increase nuclear spin polarization to the level that will significantly enhance technique sensitivity. The use of hyperpolarizing agents is one way to dramatically increase signal intensities and improve sensitivity. Some examples include dynamic nuclear polarization (DNP) [115], hyperpolarized gases [116,117], parahydrogen-induced polarization [118], and signal amplification by reversible exchange (SABRE) [119,120]. Hyperpolarization by SABRE improves nuclear spin polarization via rapid and reversible exchange of parahydrogen (p-H2) with the available sites of a metal complex (usually iridium, Ir), and at the microtesla (µT) magnetic field strength usually employed, p-H2 spin polarization transfer is possible all the way to substrate molecules such as 15N-pyridine. This technique has been shown to provide significant gain in signal intensities that can translate to 15N biomedical imaging and spectroscopy [121–123]. 15N-DAZ has been proposed as an alternative to 15N-pyridines as substrate for SABRE technique due to its small size, ease of incorporation, longer polarization lifetime, and non-perturbance of native biomolecule activity [124]. The potential of 15N-DAZ as an imaging tag was investigated by Shen et al. [124], showing a 15,000-fold signal enhancement 14 (ε) over N-DAZ (Figure 19). They also found several-fold increases in the singlet spin order (Ts) 15 and magnetization relaxation (T1) time constants of N2 when compared to normal polarization decay time constants. On incorporation of their 15N-DAZ into choline derivatives, they observed a 2000-fold increase in signal with T1 = 3 min. Their set-up also displayed extended polarization Molecules 2020, 25, x FOR PEER REVIEW 21 of 29 lifetimes and signal enhancement in D2O, suggesting the applicability of this tag for in vivo biomedical applications.imaging applications. In a subsequent In a subsequent follow-up follow-up paper [125] paper, the [125 same], the authors same authorsdescribe describedd the design the of design new 15 terminalof new terminal 15N-DAZN-DAZ-based-based tags that tags are that easier are easier to incorporate, to incorporate, can substitutecan substitute for formethyl methyl groups groups in biologicalin biological structures, structures, and and display display similar similar relaxation relaxation time time constants constants as as the the earlier earlier tag tag (Figure (Figure 19)19),, validating the robustness of this hyperpolarizedhyperpolarized imagingimaging tag.tag.

Figure 19. The use of DAZ as hyperpolarized markers for SABRE leads to signiﬁcant enhancement in signals. Figure 19. The use of DAZ as hyperpolarized markers for SABRE leads to significant enhancement in signals.Synthetic methods to introduce terminal DAZ into biomolecules are of interest as functionalization at this position should impose the least disturbance on activity. The installation of terminal DAZ is, however,Synthetic poorly methods exploited. to introduce Glachet et terminal al. [54] showed DAZ a into one-pot biomolecules three-step aremetal-free of interest synthetic as procedurefunctionalization for the synthesisat this position of terminal should DAZ imp directlyose the fromleast aminodisturbance acids. on The activity. scope ofThe this installation reaction was of terminalevaluated DAZ in several is, however natural, poorly and unnatural exploited. amino Glachet acids, et al showing. [54] showed good yieldsa one-pot in most three cases.-step metal These- freereaction synthetic scopes procedure provide a for template the synthesis for future of terminal functionalization DAZ directly of peptides from amino and proteins. acids. The scope of this reactionIn a validation was evaluated of the applicability in several natural of a 15N-DAZ-based and unnatural platform amino acids for hyperpolarization, showing good yields studies, in mostPark etcases. al. [126 These] constructed reaction scope an arrays provide of several a template15N-DAZ-tagged for future biomoleculesfunctionalization ranging of peptides from simple and proteins. In a validation of the applicability of a 15N-DAZ-based platform for hyperpolarization studies, Park et al. [126] constructed an array of several 15N-DAZ-tagged biomolecules ranging from simple sugars and amino acids to drug molecules. The authors initially used SABRE to demonstrate the feasibility of 15N-DAZ hyperpolarization which, despite showing excellent signal enhancement and extended hyperpolarized lifetimes, could not be carried forward as method of choice due to its preference for organic solvents over aqueous solutions. In this new study, the authors decided to use DNP as an alternative approach due to its versatility and broad applicability. They functionalized known molecules such as glucose, the drug colecoxib, and several amino acids with 15N-DAZ and were able to show signal enhancements up to 400,000-fold and relaxation times T1 of up to 4 min. These experiments were also conducted in D2O, suggesting the potential of the technique for application in clinical imaging research. These works highlight the promising potential of 15N-DAZ molecular tags as future hyperpolarized imaging agents for use in clinical diagnostics and biological research.

Molecules 2020, 25, 2285 21 of 27 sugars and amino acids to drug molecules. The authors initially used SABRE to demonstrate the feasibility of 15N-DAZ hyperpolarization which, despite showing excellent signal enhancement and extended hyperpolarized lifetimes, could not be carried forward as method of choice due to its preference for organic solvents over aqueous solutions. In this new study, the authors decided to use DNP as an alternative approach due to its versatility and broad applicability. They functionalized known molecules such as glucose, the drug colecoxib, and several amino acids with 15N-DAZ and were able to show signal enhancements up to 400,000-fold and relaxation times T1 of up to 4 min. These experiments were also conducted in D2O, suggesting the potential of the technique for application in clinical imaging research. These works highlight the promising potential of 15N-DAZ molecular tags as future hyperpolarized imaging agents for use in clinical diagnostics and biological research.

4. Future Directions BPs and DAZs have become two mainstay PCGs that are used today in PL studies due to intrinsic properties that make them amenable for use in studies of biological systems. DAZs such as TPD have gained significant interest because of their chemical stability and small size, which is expected to minimally alter functions of the system of study. However, the chemical compatibility of DAZs to reaction conditions limits the point of incorporation in the synthesis as well as the position on the probe. Additionally, the study performed by Ichishi et al. optimizing the reaction conditions for a Suzkui–Miyaura compatible with the TPD system [50] will enable the future application of biphenyl DAZ-labelled motifs, a commonly used motif in medicinal chemistry, to the study of drug–target interactions. Further studies aimed at the expansion of the synthetic compatibility of DAZs with common synthetic reactions may enable the incorporation of DAZs with minimal perturbation (nested DAZs, Figure6c) to the chemical system being studied. Moreover, the usual synthetic methods to synthesize DAZs are inefficient and low yielding, and attempts to discover more convenient and efficient routes, such as those discovered by Wang et al. [52,53] and Glachet et al. [54] will likely make DAZs attractive PCGs for PL and other applications. The utility of both classes of PCG has enabled their incorporation into traditional PL approaches in elucidating diverse biochemical interactions of synthetic and biological micro and macro molecules in both in vitro and in cellulo studies. The use of both BPs and DAZs in solid-support immobilization systems continues to increase, and more studies that integrates them as covalent conjugation systems are expected. Simple and versatile designs such as IMCR, the ortho-Nosyl-DAZ probe, and the SUMO-E2-DAZ probe allow multicomponent studies and effectively use DAZ for light-controlled crosslinking, and their further development, validation, and widespread use are expected. These tools allow multitarget installation such that several concurrent studies can take place in the same system. They can be used to study ternary and quaternary biological interactions, and can integrate drug screening, target identification, and proteomic studies into a single probe. Because these designs are simple and easy to install and modify, their adaptation to other multicomponent system studies should be anticipated. Imaging systems that rely on DAZs as hyperpolarizing agents continue to gain significant attention and this is expected to continue. The advancement of DAZ agents into signal enhancers and contrasting agents in NMR investigations and MRI diagnosis should allow for the combination of these diagnostic tools with proteomic studies that already use DAZ probes. DAZ and BP probes thus hold substantial promise as a one-system multifunctional tool to synchronously investigate and diagnose a myriad of biological processes.

Author Contributions: M.M.H. and O.O.O. both contributed equally. All authors have read and agreed to the published version of the manuscript. Funding: The APC was funded by the University of Toronto. Acknowledgments: We are thankful to our supervisor Patrick T. Gunning for all his guidance and help. Conﬂicts of Interest: The authors declare no conﬂict of interest. Molecules 2020, 25, 2285 22 of 27

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