Photochemical Methods for Peptide Macrocyclisation

Abstract Photochemical reactions have been the subject of renewed interest over the last two decades, leading to the development of many new, diverse and powerful chemical transformations. More recently, these developments have been expanded to enable the photochemical macrocyclisation of peptides and small proteins. These constructs benefit from increased stability, structural rigidity and biological potency over their linear counterparts, providing opportunities for improved therapeutic agents. In this review, an overview of both the established and emerging methods for photochemical peptide macrocyclisation is presented, highlighting both the limitations and opportunities for further innovation in the field.


Introduction
Peptides and small proteins( collectively,P SPs) represent an exciting and emerging frontier in drug discovery, combining the advantages of modularity and syntheticf lexibility of small molecules (< 500 Da) with the exquisite levels of selectivity and potencyo fb iologics (> 5000 Da). [1][2][3] PSPs are particularly prized for their capacity to engagei np otent ands electivei nteractions with protein surfaces, and hence their ability to modulate protein-protein interactions (PPIs)-therapeutic targets that wereo nce considered "undruggable" using small molecules. [4,5] The peptidic nature of PSPs provides them with beneficial attributes such as high binding affinity,b iocompatibility and easea nd modularity of synthesis, but it is also their Achillesh eel.L ow cell permeability,p oor metabolic stability and unstable secondary structure formation as ar esult of conformationalf lexibility,a ll hinder clinicala pplications of PSPs. [6][7][8][9] To overcome these limitations, the design and synthesiso f macrocyclic PSPs has become increasingly prominent. [1-4, 8, 10] Macrocyclisation (defined as cyclisation to form rings of ! 12 atoms) can occur throughh ead-to-tail, head-to-sidechain,s idechain-to-tail or sidechain-to-sidechain coupling (stapling) (Figure 1), delivering cyclical products,w hichp ossessa number of advantageous characteristics over their linear counterparts: [11] i) structures are rigidified, stabilising or enforcing peptidec onformations that mimic elements of protein secondary structure [12] , a-helices, [13][14][15][16][17][18][19][20] b-sheets [21] and b-hairpin turns [22] ,w hich would otherwise be unstablec an all be in-duced by cyclisation;i i) cyclised peptides exhibit increased stability to proteolysis, thus prolonging their biological activity and improving their pharmacokinetics. This stability can result from an umber of factors, including the poor fit of macrocycles into the active sites of endopeptidases, [23] resistancet ot he activity of exoproteases that preferentially cleave near the peptide N-or C-termini, [11] or the formation of a-helices that are resistant to proteolysis duet ot he presence of ar igidifying, intramolecular,h ydrogen-bonding network; [17] iii)binding efficiency for at arget is often improved, an effect classically attributed to cyclic structures being held in conformations better disposed towardsb inding, with ar esultant reduction in the entropic penalty to binding. [23,24] However,s tudies such as those by the groups of Martin and Spaller illustrate am uch more complex picture,w here pre-organisation through macrocyclisation may insteads trengthen the enthalpic component of binding at the expense of entropy, highlightingt he importance of treating macrocycle bindingt hermodynamics on ac ase by case basis; [25][26][27][28] and iv) cell membrane permeability mayb ei mproved, as sidechains can be oriented aroundt he macrocycle in am anner that shields polar atoms from the solvent medium, reducing the polar surface area of the peptide. [10] Given the benefits of PSP macrocyclisation, it is unsurprising that many such compounds occur naturally. Av ariety of macrocyclic linkages have been identified in natural products,i ncluding head-to-tail amide bonds, disulfide bridges,b iaryls and biaryl ethers. [29][30][31][32][33] Chemistsh ave subsequently followed suit, utilising analogous tactics to form cyclic PSPs, as well as developing ad iverset oolkit of novel synthetic strategies for macrocyclisation,i ncluding ring-closing metathesis, [34] azide-alkyne cycloadditions, [35,36] other transition-metal-catalysed methods, [37] conjugatea dditions, [38] nucleophilic aromatic substitutions [39] and multicomponent reactions. [40,41] These and other methodologies have been extensively reviewed. [3,18,19,23,37,[42][43][44][45][46][47] Althought he coupling chemistry of each of these strategies may differ greatly,c ommon to all of these approaches are a set of challenges that must be overcome, chiefly coaxing the ground-state trans geometrieso fm ultiple amide bonds along the PSP backbone into as uitable conformation for cyclisation, [48] and outcompeting deleterious oligomerisation reactions.
In contrast to the methods outlined above, the use of photochemical methods for PSP cyclisationh ad until recently been relativelyu nderexplored. However,i nl ine with aw ider renaissance of the fields of photo-a nd radicalc hemistry,r ecent developmentsh ave broughtt his area to the fore. Photochemical

Introduction
Photochemical macrocyclisation reactions can typically be grouped into two categories:i )redox-neutral processes where the photoexcited state either directly takes part in the macrocyclisation,o rp roduces reactive speciest hrough atom transfer or bond rearrangement,w hich then participate in cyclisation; and ii)those where the photoexcited state first engages in photoinduced electron transfer (PET) to provide radical, radical ion or organometallic species, which are necessary for cyclisation. In this section, we will focus on the first class of reactions, where photochemical macrocyclisation can be furtherc ategorised based on the specific behaviouro ft he photoexcited state:h ydrogen atom transfer (HAT);b ond reorganisation;o r cycloaddition.P hotoactivatable motifs are widespread in the bioconjugation field, however,i nt his review we will focus only on reactions with levels of chemoselectivity that allow their use for controlled cyclisations. Thus, widely used reactive handles in photoaffinity labelling,s uch as diazirines and aryl azides, which generate highly reactive speciesu pon activation that can react in ap romiscuous, largelyn on-selectivem anner, will not be discussed.

Macrocyclisation triggered by photoinitiatedh ydrogen atom transfer (HAT)
Thiol-"ene" reactions Thiols represent av ersatile reactive handle for peptide and protein modificationt hat can undergo aw ide range of different chemistries. As ar esult, the modification of cysteine residues has been widely used for peptide macrocyclisationv ia al-kylation, [55,56] arylation [39] and disulfide formation. [57] Conjugate addition reactions, [38,58] also referred to as nucleophilic thiol-"ene" reactions,a re particularly popular owing to their chemoselectivity,m ild reactionc onditions and rapid kinetics. [59] However,t he need to use activated,e lectrophilic alkenes can lead to possible side reactions with other nucleophilic amino acids, particularly lysine. [60] The highlys elective reaction of thiyl radi- cals with unactivated alkenes, via ap hoto thiol-"ene" mechanism, is therefore an attractive alternative. [61] Thiol radicalf ormation (1,F igure 2) can be initiated through direct photolysis of the thiol with UV light. Although thiyl radical generation is slow at wavelengths > 280 nm (e.g.,w hen using as unlamp with pyrex filter), this can be advantageous for chain processes where al ow steady-state concentration of radicals helps suppress deleterious processes. [61] More often, a photoinitiator is employed and thiyl radicals are generated by ar apid HATb etweent he thiol and either the photoexcited state initiator directly,o rd aughter radicals stemming from its photo-decomposition (e.g.,f or HATb etween CCH 3 and EtSH, k 298K % 5 10 7 m À1 s À1 ). [62] Thiyl radical 1 subsequently adds across an alkene C=Cb ond in an anti-Markovnikov manner, generating ac arbon-centred radical 2.A bstraction of ah ydrogen atom from another thiol generates the hydrothiolated product 3 anda na dditional thiyl radical 1,f acilitatingc hain propagation.
The steric and electronic nature of the alkene substituents play as ignificant role in governing the efficiency and outcome of this process. Af ine balance exists between the forward( and reverse) rates of thiyl radical (1)a ddition to an alkene, S-H abstraction from another thiol by the resultant carbon-centred radical 2 and competitive addition of 2 to another alkene to afford off-cycle oligomerisation/polymerisation products. [63][64][65] In general, electron-rich alkenes typically react more rapidly, with terminal alkenes being similarly more reactive than internal analogues. [63] Norbornenea nd vinyl ether derivatives undergo exclusively hydrothiolation over oligo-/polymerisation owing to particularly rapid thiyl radical additions, as ac onse-quenceo fs train release and polarity matching, respectively, and fast S-H abstraction steps. Although the competitive formationo fd isulfide bonds is typically slow,r ecent reports by the Bowman group have highlighted the potential role of thiolate anions in accelerating this side reaction, through the formationo fametastable disulfide radical anion. [66] However, althought his may be problematic in the context of thiol-"ene" polymerisations, the high effective concentration of alkene duringi ntramolecular macrocyclisation likely negates this effect.
In the earliest example of photochemical thiol-"ene" peptide macrocyclisation, Aimetti et al. demonstrated the on-resin synthesis of an integrin-binding cyclic Arg-Gly-Asp (RGD) peptide ( Figure 3). [67] Irradiationo fr esin-bound peptides 4,c ontaining unprotected cysteine residues,a t3 65 nm in dimethylformamide (DMF) led to cyclisation with either allyloxycarbonyl   . First example of peptide macrocyclisation by aphotoactivated thiol-"ene" reaction. On-resin cyclisation onto bothalloc and norbornene alkene partners was achieved in the presence of aD MPAp hotoinitiator. [67] (alloc; 4a)o ra mido-norbornene (4b)m odified lysine residues in the presence of the photoinitiator 2,2-dimethoxy-2-phenylacetophenone (DMPA). As expected, given the reactivity profiles of different alkenes discussed above,h ighery ields and shorter reactiont imes were reported for 4b than 4a (37 %v s. 24 %, respectively). Notably, photoactivation wass hown to be both quicker and more effective than the use of the thermally activated radical initiatora zobisisobutyronitrile( AIBN). As all other amino acids in the peptides equence were protected, potential side reactions were minimised and the cyclised peptides 5a and 5b were efficiently obtained following cleavage. Macrocyclisation of the solution-phase, fully deprotectedp eptide was also performed in methanol. Although conversions were comparable, the need for subsequent peptidep urificationl ed to overall lower yields.
Building upon this work, the same authors demonstrated that peptides could undergo further chain extension following on-resin thiol-"ene" macrocyclisation. [68] This allowed the installation of as econd reactive cysteinef or subsequent conjugation of ac yclic-RGD motif to am ultivalent polymer backbone. Importantly,acombination of cyclic and multivalent peptides was found to act synergistically to enhance inhibition of fibrinogen bindingt og lycoprotein IIb/IIIa, ak ey integrinf ound on the surface of platelets, by up to two orders of magnitude.
In 2015, Wang et al. reportedt he photo thiol-"ene" macrocyclisation of peptides composed solely of natural amino acids through an ovel solution-phase, two-component approach. [70] As eries of bifunctionald ienes were used to link two cysteine residues under 365 nm irradiation, in the presence of aD MPA photoinitiator.A lthough the reactionw as compatible with DMF,t he highest yields were obtainedi nN-methyl-2-pyrrolidone (NMP). This two-component approach offers synthetic versatility,e nabling the generation of al ibrary of peptides cyclised with different diene linkers from as inglep eptidep recursor.T his is particularly important given the strong influence of cross-linker structure on peptidep roperties. For example, changes in lipophilicity have been shown to impact the ability of cyclic peptides to cross phospholipid bilayers and therefore the potency of therapeutic peptides, [71] whereas hydrogenbondingi nteractions have been shown to influence peptide conformation and target binding. [72] As ar esult,t he authors were able to develop bis-thioether cyclised peptidei nhibitors of p53-HDM2 interactions, which were ablet oi nduce the apoptosis of colorectal carcinoma cells with ac omparable potency to previously reported hydrocarbon-linked peptides generated by ring-closing metathesis. [73] More recently,t he same authors have developed this twocomponent approachf urther to enable the macrocyclisation to be performed in water. [74] To overcome challenges with aqueous solubility previously encounteredb yo ther groups, the water-soluble photoinitiator 2,2'-azobis[2-(2-imidazolin-2-yl)propane]-dihydrochloride (also known as VA-044, 10)w as employed to induce thiol conjugation to water-soluble diallyl-urea 11 ( Figure 5). At pH 4i na queous solution,t he reducing agent TCEP (tris(2-carboxyethyl)phosphine)w as found to greatlye nhance the conversion of peptide 12 to cyclised 13 from 53 % to 95 %b yminimising competing disulfide formation.
Impressively, the macrocyclisationo fa9kDa, dicysteine, coiled-coil protein substrate was also demonstrated. Although the addition of TCEP wasf ound to be detrimental in this scenario owing to competitive desulfurisation,i ni ts absence the reaction was found to proceed effectively in mildly acidic (pH 4) acetate buffer containing denaturing guanidine hydrochloride (Gdn-HCl). This reduced disulfide formation and maximised cyclisation efficiency,r espectively,l eading to 90 %c onversion to stapled protein 14.D ouble cyclisation of at etra-cysteine mutant 15,c ontaining two separate i,i + 7c ysteinep airs to generate 16,w as also found to proceed with 80 %c onversion ( Figure 6). Although not reported by the authors, the presence of undesired linkages between the two separatec ysteine pairs cannot be ruled out. However,i ti sl ikely that the spatialp roximity of the i,i + 7r esidues would strongly favour cyclisation between adjacent cysteines, even under denaturing conditions.

Thiol-"yne"reactions
In an analogous fashion to thiol-"ene" reactions, thiols can also react with alkynes by both nucleophilica nd radical mecha-nisms. Photoinitiated thiol-"yne" reactions with unactivated alkynes therefore offer an alternative strategy for achievingP SP macrocyclisation. Conjugation proceeds through am echanism similar to that for thiol-"ene" reactions, comprising light-initiated formation of thiyl radical 17,a ddition to an alkyne to form 18 in an anti-Markovnikov manner and subsequent hydrogen atom abstraction by vinyl radical 18 ( Figure 7). However,t he thiol-"yne" reaction differs in that it generatesavinyl sulfide product 19,w hich is itself susceptible to furtherm odification in the presence of excesst hiol, through as ubsequent thiol-"ene" reaction. Although less widely used than the analogous "ene" reaction, thiol-"yne" conjugations therefore offer intriguing possibilities for achieving double modification or dual functionality, [75] although these properties have yet to be exploited by the PSP macrocyclisation community.I ndeed, to date there has only been as ingle reporto fp hotoactivated thiol-"yne" mediated peptidemacrocyclisation.
Tian et al. demonstrated the intramolecular photo thiol-"yne" cyclisation of peptides containing an unnatural amino acid bearing ap ent-5-yne sidechain, and either cysteine( 20 a) or homocysteine (20 b)a tt he i + 4p osition ( Figure 8). [71] A screen of photoinitiators found IHT-PI 659 (8)a nd 2,2-dimethoxy-2-phenylacetophenone( DMPA, 21)t op rovide the highest conversions, with as trong preference for the formationo f the Z-vinyl sulfide products 22 a and 22 b.T he resultant peptides were designed to modulate intracellular interactions between the oestrogen receptor and its coactivators, ak ey target for the treatment of certain cancers and osteoporosis. Importantly,t he vinyl sulfide-containing macrocycle generated was proposed to provide increased rigidity,a nd to enhancet he ahelical character over the alkyl sulfide linker,w hich would be generated by an analogous thiol-"ene" cyclisation. Furthermore, comparison to an all hydrocarbon linked analogueg enerated by ring-closing metathesis indicated that the vinyl sulfide contributed to greatly reduced membrane toxicity. Thus, this report highlights the importance of the cyclisation linker  Figure 6. Aqueousdi-cyclisationofat etra-cysteine protein mutant by photoinitiated thiol-"ene" chemistry. [74] Figure 7. Mechanism of the photoactivated thiol-"yne" reaction.Aphotoinitiatorc an be used to accelerate the formation of at hiyl radical by SÀHa bstraction. Figure 8. Photoactivated thiol-"yne" macrocyclisationt og enerate av inyl sulfide-linked cyclic peptide. [71] structurei nd etermining the properties, both physicala nd biological, of ac yclised peptide. Rather than being interchangeable with the thiol-"ene" reaction, photo thiol-"yne" reactions should be considered an important addition to the macrocyclisation toolbox that can generate adistinctvinyl sulfidelinkage, which mayp ossess uniquep roperties.

Benzophenone-methionine conjugation
The ability of certain ketones and aldehydes to form reactive speciesu nder UV irradiation has been widely exploitedi no rganic synthesis and chemical biology.B enzophenones undergo photoexcitation on irradiation with longerw avelength UV light (350-360nm), [76,77] and importantly,r elative to other photoactivatable groups such as diazirines and aryl azides,f orm intermediates that exhibit useful (albeit limited) levels of chemoselectivity.M echanistically,t he highly reactive diradical 23 generated following irradiation can abstract ah ydrogen atom from an accessible CÀHb ond to generate an a-hydroxy radical 24 and carbon-centred radical 25 ( Figure 9). Pairing of these two speciesl eads to the formationo fanew carbon-carbon bond in tertiaryalcohol 26.
In an attemptt ou nderstand and harness preferential reactivity,D eseke et al. studied the regio-and chemoselective CÀH abstraction of irradiated benzophenones with ap anel of Nacetyl amino acid methyle sters. [78] In acetonitrile,t he highest reactivity was observed with glycine owing to itsr eadiness to undergo hydrogen atom abstraction from the a-carbon (51 % conversion). Methionine was also found to be preferentially modified (40 %c onversion), with alkylation occurring either at the g-o re-carbon atoms adjacent to sulfur.C onversely,i na 4:1m ixture of pyridine/water,m ethioninem odification was found to be favoured( 45 %c onversion), with ac orresponding drop in glycine modification (16 %). The authors proposed that this change in selectivity was due to competitive base-catalysed degradation of adducts formed at the a-positiono fg lycine. Although modification of these two amino acids was dominant, low levels of reactivity with severalo ther amino acids was also observed. Indeed, only residues containing primary amides or carboxylic acids (aspartic acid, asparagine, glutamic acid and glutamine)werefound to be inert.
Building on this preferential reactivity at glycineand methionine, Morettoe tal. reportedt he first use of benzophenone photoactivation for intramolecular peptide cyclisation in 2009. [79,80] Hexapeptides 27 containing the unnatural benzophenone-based amino acid Bpa (28)a nd methionine were synthesised, and their positions in the peptidec hain varied to study the effects of distance on macrocyclisation efficiency ( Figure 10). To prevent unwanted reactions at other sites in the peptide, the di-a-substituted unnaturala mino acid2 -aminoisobutyric acid (Aib, or a-methylalanine) was installed at all other positions owing to its known inertness to CÀHa bstraction, and ability to promote a-helix formation.C yclisation wass uccessful when methionine was placed at the i + 1, i + 2, i + 3, or i + 4 positions relative to Bpa,g enerating cyclised peptides 29.F or an analogous nonapeptide 30,c yclisation to the iÀ3a nd i + 3 positions wasf ound to occur exclusivelya tt he e-carbon of methionine, with the two diastereomers generated by the new chiral tertiary alcohol being formed in an approximately 1:1 ratio.
Building upon this work, Wright et al. subsequently introduced an ew a-C-tetrasubstituted, cyclic, benzophenone-based amino acid BpAib (31), with increased structural rigidity ( Figure 11). [81] Although the photoactivated macrocyclisation of this amino acid with methionine was demonstrated, efforts to exploit the benefits of decreased conformational freedom impartedb yB pAib have yet to be reported.
Lewandowska-Andralojc et al. have subsequently demonstrated that Bpa-methionine cyclisation can take place between the two sidechains of ac yclic dipeptide to form ar igid bridged macrocycle. [82] This study highlighted that the CÀHa bstraction step to form an a-hydroxy radical is in fact reversible. Furthermore, CÀCb ond formation was found to take place se-  , potential side reactionsw ith a-hydrogensa re prevented. [80] lectively at the methionine d-carbont om inimise the significant ring strain imposedb yc yclisation. Thus, both steric and chemicalfactorsare at play in dictating CÀHs electivity.
The ability of benzophenones to cyclise with natural amino acids followingi rradiation is an advantage to their use. However,t he lack of specificity resulting from off-target reactions with alternative amino acids, particularlyg lycine, is severely restricting. This is highlighted by the limited number of reports of benzophenone PSP macrocyclisation, and the even more striking lacko fd iversity in the amino acids that have been integrated into the peptides ubstrates. Moreover,t he formation of diastereomeric products, resulting from the creation of a new chiral centre, may be problematic, potentially requiring separation and complex purification to generate ah omogeneous cyclised product. As such, the photoactivation of benzophenonesa sam eans to control PSP macrocyclisationisu nlikely to find increasingp rominence in the coming years, given the advantages of many of the other reactions presented in this review.
The propensity to undergo hydrolysis under irradiation makes Bni-derivatives useful protecting groups, but also severely impacts their bioconjugation efficiency in aqueous media. However,i nn on-nucleophilic organic solvents, nucleophilic attack by amines can be used to induce amide formation followingp hotoactivation. This chemistry has therefore been exploitedf or intramolecular photocyclisation and in particular as au seful method for macrolactamisation. [85] Indeed, compared with otherm acrolactamisation strategiest hat require the use of added couplinga gents, the irradiation of Bniamides provides af acile means to inducec yclisation without the need for additives.
The capacity of photoactivated Bni-amides to react with nucleophiles was exploitedb yM ifune et al. for PSP macrocyclisation. [86] Thisa pproachi sa ttractive as it generates an ative peptide bond and the resultant macrocycles are therefore able to mimic natural cyclic peptides. Indeed, the authors demonstrated that as ynthetic pentapeptide 37,b earing aC -terminal Bni amide, could be cyclised in a3 6% yield following irradiation at 365 nm under flow conditions, generating ap reviouslyr eported cyclic RGD sequence 38 in situ that can act as as elective antagonist of the a v b 3 integrin receptor ( Figure 13). [87][88][89][90][91] The absence of excessa ctivating agents or catalysts during cyclisation greatlyf acilitated purification of the cyclised peptide, and the ability of the Bni group to play ap arallel role as aC -terminal protecting group during peptide synthesis was also highlighted by the authors as an otable advantage. However,t he presence of aC -terminal Bni-amide also necessitatedt he use of solution-phase peptide synthesis. Although this was achieved by using micro-flow peptide synthesis, such technologies are not as widespreada ss olid-phase approaches and the generality and translatability of this approachm ay therefore be limited at present. [92] Post-synthesis derivatisation is another possible route to achieveC -terminal Bni-amide installation for head-to-tail or sidechain-to-tail cyclisationsb ut also presents synthetic challenges. In contrast, head-to-sidechaino rs idechain-to-sidechain couplings would not experience thesed ifficultiesa nd the installation of aB ni-motif could plausibly be achieved in as traightforward manner on-resin. However,t he need to also protect Asp, Lysa nd Arg residues to prevent side reactions remains as ignificant limitation of this strategy,a nd as ar esult there remains only as ingle report of Bni photoactivation in the peptidem acrocyclisation literature to date.
The chemical versatility of the 2,5-diaryl scaffold is attractive as it provides significant scope to tune the properties of the tetrazolea nd intermediate nitrile imine. For example, by variation of the aryl substituents Xa nd Y, the wavelength sensitivity can be tuned within the UV region. [94] Similarly,t he rate of cycloaddition can be increased by selecting substituents that serve to raise the highest occupiedm olecular orbital (HOMO) energy of nitrile imine 40. [95] Monoaryl tetrazoles are also able to eliminate nitrogen under irradiation and can undergo efficient cycloaddition,albeit at as ignificantlyr educed rate. [96] The first use of this chemistry for peptidem acrocyclisation was reported by Maddene tal. in 2009. [97,98] Te trazole-and (meth)acrylamide-based unnaturala mino acids were intro-duced into as ynthetic heptapeptide 43 and irradiated at 302 nm, to trigger nitrile iminef ormation and subsequent1 ,3dipolar cycloaddition to form 44 ( Figure 15). Nitrile imines generated from both mono-and di-aryltetrazoles were able to undergo macrocyclisation with comparable efficiency,a lthough sidechainf lexibility was found to be am ajor determinant of cyclisation efficiency. Functionalised amino acids based on a shorter chain ornithine core (43 a,b, n = 3, 15 %) underwent cyclisation with lower efficiency than those based on lysine (43 c-h, n = 4, 40 %). Similarly,m acrocyclisation efficiency was found to depend on the structure of both the tetrazole and alkene reactive partners. Electron-donating substituents on the aryl tetrazole( R 2 = OMe, 43 e,f)g reatlyi ncreasedt he conversion by increasing the HOMO energy of the intermediate nitrile imine,t herefore accelerating cycloaddition. Interestingly,m ethacrylamide derivatives (R 1 = Me, 43 f,h)w ere found to react more efficiently than the corresponding acrylamides (R 1 = H, 43 e,g)d espite previous small molecule studies to the contrary. [94] Acrylamides are known to possess increased reactivity towards dipolar cycloaddition,s uggesting that conformational freedom is likely to play as ignificant role in dictating peptide cyclisation efficiency.I nterestingly,t he conversion of monoaryl or diaryl tetrazoles was found to be comparable despite the lower reactivity of monoaryl derivatives, again suggesting a conformational influence on cyclisation. [96] The high selectivity reported for nitrile imine-alkene cycloadditions would appear to make 2,5-di-aryltetrazoles interesting motifsf or light-induced peptidem acrocyclisation. However, it is important to note that side reactions of the highly reactive nitrile imine with natural amino acid sidechains have been reported. [99] This mayl imit macrocyclisation efficienciesi ns ystems where the tetrazole and alkene reactive pair are unfavourablyp ositioned, but does open up the intriguing possibility of using tetrazoles as light-activatable reagents for cyclisation with proteinogenic amino acids.

Photoinitiated cycloaddition
In the previouss ection, photoactivation led to the generation of ar eactive intermediate that could subsequently undergo cy- Figure 13. Solution-phasehead-to-tail macrocyclisationofapeptide containing aC-terminal Bni-amide under UV irradiation. [86] Figure 14. Mechanism of 2,5-diaryl tetrazole photoactivation to form an itrile imine, and subsequent 1,3-dipolar cycloaddition with as uitable dipolarophile. Figure 15. Macrocyclisation of tetrazole-and alkene-containing peptides under UV irradiation,byt he formationo fa nintermediate reactive nitrile imine and subsequent 1,3-dipolar cycloaddition. [97] cloaddition. An alternative strategy is to exploit photoexcitedstate molecules that can themselves directly undergo cycloaddition. For example, followingp hotoexcitation with UV light, maleimide molecules readily undergo photochemical dimerisation through ac oncerted [2+ +2] cycloaddition. In 2012, Tedaldi et al. reported that functionalised thiomaleimides,f ormed through as equential addition-elimination reactiono ft hiols with bromomaleimides, were also able to undergo [2+ +2] cycloaddition under irradiation. [100] As predicted by frontier molecular orbitalt heory, exo head-to-head products are preferentially formed. The redshifted absorbance of thiomaleimides relative to the parent maleimidei sa dvantageous, enabling photoactivation with lower energy 365 nm light.T his reaction wass ubsequently exploited for photoinduced peptidem acrocyclisation, as well as the re-bridgingo fn ative disulfide bonds in an antibodyf ragment (Figure 16). [101] The utility of this chemistry was demonstrated by generating an analogue of the therapeutic cyclic peptide octreotide, asynthetic mimic of somatostatin. Ak ey disulfide bridge is essential to the biological activity of octreotide, and the authors showedt hat linear di-thiomaleimide peptide 45 indeed showed very low biological activity. Upon irradiation at 365 nm to generate cyclised product 46,a partial recovery of activity was observed, albeit at levels < 10 % of disulfide-bridged octreotide. Interestingly,i tw as noted that irradiation led to the formationo ff our major products of identical mass. This was proposed to be due to the formation of different diastereo-andr egioisomers, in starkc ontrast to the high selectivity observed in the intermolecular reactions reported by Te daldie tal. [100] This serves as another indication that steric restrictions imparted by ap eptide substrate can significantlyinfluence regioselectivity.

Photoinduced Electron Transfer in Peptide Macrocyclisation
Introduction Photochemical macrocyclisation can be initiated by quenching of aphotoexcited-state chromophorethrough electron transfer (ET). This reactionm anifold is distinct from the quenching mechanisms discussed above.P hotoinduced electron transfer (PET) processes can generater adicali ons from neutral starting materials, or neutral radicals from charged precursors. Reaction efficiency is governed by the propensity of these secondary reactive intermediates to undergo macrocyclisation, versus deleteriouss ide reactions or competing back electron transfer (BET) to reform the ground-states tartingm aterials. [102À104] In this section, we detail the emerging field of PET-mediated peptide macrocyclisation, from its origins in UV-driveni ntramolecular PET,t oa pplications of intermolecular,v isible-light driven photoredox catalysis, ar apidly developing strategy that is impactingmanya reas of organic and biomolecular synthesis.

Intramolecular PET-initiated peptide macrocyclisation
Intramolecular PET-initiated macrocyclisations on peptides ubstrates have exclusively exploited ET from donor functional groups to photoexcited phthalimides. Phthalimides are easily incorporated at the N-termini of peptidec hains during solidphase peptides ynthesis, andu pon photoexcitation become highly oxidising (E S1 =+2.1 V, E T1 =+1.6 V). [105] Griesbeck and co-workersp erformed important early work demonstrating the intramolecular cyclisation of N-phthaloyl w-aminoa cids under UV irradiation. [106] Substrate 47 is prototypical of this approach-on photoexcitation to generate 48,t he phthalimide chromophore underwentP ET with the terminal carboxylate, generating ak etyl radical anion on the phthalimide,a nd ac arboxyl radical at the end of the chain, 49 (Figure 17 a). Rapid decarboxylation to form ap rimary alkyl radical 50 was followed by subsequentb iradical intersystem crossing (a triplet state to singlet state transition via the spin flip of an electron) and ensuing cyclisation through radical-radical coupling, to give macrocyclica midol 51 in 81 %y ield. The authors crucially also illustrated the compatibility of this chemistry with substrates containing amide bonds (52)i nt he formation of 26-membered ring compound 53 (Figure17b).
The first true PET-mediatedP SP macrocyclisations were reported by Griesbecke tal. in 2002 ( Figure 18). [107,108] Under analogousc onditions to those described above,t ripeptides 54, composed of diglycinea nd al ong-chain unnatural amino acid at either the Co rN -termini, afforded 13-or 18-membered macrocycles 55 in synthetically useful yields. Interestingly,t he triglycine derivative 54 a successfully underwent decarboxylation but then failed to cyclise, insteada ffording solely the quenched linear N-methyl product in 28 %y ield. This behaviour was attributed to hydrogen bondingb etween the amide Figure 16. Thiomaleimide [2+ +2] cycloaddition under UV irradiation to generate amacrocyclic peptide. [101] proximal tot he N-terminus and the phthalimide unit, which was seeminglyd isrupted in substrates bearing long-chain residues, thus enabling cyclisation. By exchanging the central glycine for its N-methylated analogues arcosine (Sar), this hydrogen-bonding contribution could be removed anda Gly-Sar-Gly tripeptide underwent cyclisation to afford an ine-membered ring in 35 %y ield.
The approach of employing substrates with N-alkylated proximal amides was subsequently extendedt o the first example of PET macrocyclisation of ap eptide composed solelyo fp roteinogenic amino acids ( Figure 19). Cyclisation of the tetrapeptides (Pht)Gly-Sar-Gly-Gly 56 a and (Pht)Gly-Pro-Gly-Gly 56 b (Figure 19 a,b) produced 12-membered ring compounds 57 a and 57 b,r espectively.I nterestingly, 57 b was isolated as as ingle diastereoisomer,a ssigned through ac ombination of 1 HNMR analysis and analogy to previously reported benzodiazepines. [109] Finally, this methodology was applied to the macrocyclisation of pentapeptide 58 (Figure 19 c). Proline residues played av ital dualr ole in enabling cyclisation of this substrate, by both removing the deactivating hydrogen bond at the second residue, and by introducing ah airpin turn that facilitated cyclisationb ye nhancing the proximity of the N-and C-termini. Unfortunately,h owever,t he presence of prolyl amide bond rotamers andt he formation of diastereomers at the amidol position led to the isolation of cyclic tetramer 59 as ac omplex mixture of species.
Interestingly,i n2 003, Yoon, Mariano and co-workers reported as imilar example of PET-mediated decarboxylative macrocyclisation on an all-glycine [(Pht)Gly-Gly-Gly-Gly] substrate. [110] Althought he proximal amide was not N-alkylated,a nd therefore substrate cyclisation might not have been expected on the back of Griesbeck'sp reviouso bservations, ac yclic trimer was generated in 41 %y ield. However, the product was observed to be unstable,with complete decomposition being observed by 1 HNMR spectroscopy over the period of 1day.T his behaviour was attributedt oad eleterious amidol!amido ketone!intermoleculara midol pathway,u ltimately forming insoluble oligomers. This process was successfully suppressed through the use of N-alkylated tertiary amide substrates (see below).  Figure 18. PET-initiated macrocyclisation of phthalimide-containing peptide substrates underU Virradiation. [107,108] Figure 19. PET-induced macrocyclisation of phthalimide-containing peptides composed of proteinogenic amino acids. [107,108] Vazdar,B asarić,a nd co-workerss ubsequently applied PETmediated decarboxylative cyclisation to tetra-andp entapeptides 60 bearing N-terminal adamantyl phthalimides and C-terminal phenylalanines, or methoxylated analogues thereof ( Figure 20). [111] Products 61 were found to reside as open chain amides, rather than the amidol structures observed in previous reports, and hence are fully peptidic albeit containing an unusual, andp otentially metabolicallyv ulnerable, phenyl ketone unit. [112] Interestingly,t he macrocycles were formed as single diastereomers (with the exception of 61 d), with the newly formed chiral centre found to be inverted between 17-or 20membered ring sizes. The stereochemistry,a ssigned through a combination of NOESY spectra and molecular dynamics simulations, was determined to result from the conformation of the linear peptides, whichc ontrolled the facial approacho ft he radicals peciesd uring cyclisation.I ncorporation of additional Phe residues in 60 b,d,f induced turns in the linear chains,a nd hence an opposites ense of approach. Possible epimerisation post-cyclisation was excluded through both computational studies and chemicale valuation through deuterium-labelling. Substrate-induced conformational controlo fasimilar biradical macrocyclisation to form an 11-membered ring product was previously observed by the authors in as eparate study.I nt his instance, however,as ingle stereogenic centre in the precursor orchestrated the cyclisation with complete stereofidelity throughachiral memory effect. [113] Mechanistically,t he macrocyclisation of C-terminal Phe substrates 60 a and 60 b was proposed to proceed in as imilar manner to that outlined in Figure 17 a. Direct PET from the carboxylate to the photoexcited-state phthalimide 62 is followed by decarboxylation (63 to 64), triplet to singlet ISC (intersystem crossing), cyclisation (64 to 65)a nd af inal amidol to amido ketone ring expansion (65 to 66;F igure 21). However,a mechanistic divergence was suggested for the C-terminal Phe(OMe) and Phe(OMe) 2 substrates 60 c-f,w ith am ore facile initial PET from the electron-rich arenes to the photoexcitedstate phthalimide taking place, generating aryl radical cations of the type 67.S ubsequent ET from the carboxylate to the aryl Figure 20. PET-initiated cyclisation of N-adamantyl phthalimidestoC -terminal phenylalanine derivatives. The stereochemistry of the resultantp roducts was dictatedbyr ing size. [111] Figure 21. Mechanism of PET-initiatedm acrocyclisationofN-adamantyl phthalimides to C-terminal phenylalanine derivatives. Phenylalanine-based substrates undergoananalogous PET process to that outlined in Figure 17. In contrast, mono-and di-methoxy-substituted phenylalanines undergoanintermediate generation of an aryl radical cation. [111] group would generate carboxyl radicals 63,w hich can then follow the established reactivity pattern. ET from the carboxylate to the more stable dimethoxy radical cationsi n67 e and 67 f is slower than to the less stable monomethoxy variants 67 c and 67 d,r eflecting the differences in redox potentials between the two substrates and resultingi nl ower conversions to the cyclised products 61 e and 61 f.H ence, through ab alance of increasedr ates of initial PET from the arene to the photoexcited-state phthalimide 62,a nd al ess stable radical cation intermediate 67 promoting rapid ET from the carboxylate, the highest yields were observed for the monomethoxy substates 60 c and 60 d.G enerally,t he isolated yields of the cyclic peptides 61 a-d generated by this methodology are rather low,s eeminglyr eflecting difficulties in purification, which may call into question the synthetic utility (at least at scale) of as tudy that teaches us much about the nature of these cyclisations.
Yoon, Mariano and co-workers introduced C-terminal N-trimethylsilylmethyl amides as alternative precursors for PETmediated peptide macrocyclisation in 2003. [110,114] All-glycine tri-, tetra-and pentacyclic peptides 68 were prepared in high yields upon UV irradiationo ft he N-trimethylsilylmethyl amides 69 in either acetonitrile/water mixtures or methanol ( Figure 22). Protection of the peptideb ackbonea sN-alkyl tertiary amides was required forthe stability of the products.
The authors suggest am echanism of initial PET between the phthalimide and ap roximal amide donor group, followedb y amide cation radicalmigration (or hole transfer) along the peptide backbonet ot he N-trimethylsilylmethyl amide. Upon desilylation, triplet to singlet ISC and radical-radical cyclisation can then occur. [110,114,115] This radicalm igration mechanism is supported by studies conducted by the authors on polyether-and polymethylene-linked systems, with macrocyclisation yields being lower without electron-donor atoms in the linker chain. Furthermore, in competition experiments on substrates containing branched chains, and hence two potentials ites for reactivity,c yclisation of polyether chains along which radical migration could take place was found to be favoured over hydrocarbon chain cyclisation. [115] Desilylation of N-trimethylsilylmethyl amide radicalc ations is ar apid process, comparable in rate to the analogous decarboxylation and significantly faster than a-deprotonation of the intermediate amide radical cation. [116,117] Hence, cation radicalm igration is competitive with BET and a-deprotonation and therefore yields are largely independento fc hain length. It is suggested that electrostatic preorganisation of the phthalimide radical anion/N-trimethylsilylmethyl amide radical cation pair helps to overcome some of the entropic barrier to cyclisation, lowering this energetic cost.
Collectively,t hese reports demonstrate that PET-initiated macrocyclisation using excited-state phthalimide chemistry is potentially as imple ande ffective strategy to preparep eptide macrocycles. Synthetically useful yields andt he possibility to control stereochemistry with substrate conformation are particular advantages of this approach. However,t he use of UV light in combinationw ith intramolecular single-electron transfer (SET) limits the compositiono ft he precursor peptides to a subset of amino acids that do not significantlya bsorb light at the same wavelengths as phthalimide, and which bear sidechainsw ith oxidation potentials highert han that of the carboxylate donor at the C-terminus. This greatly limits the versatility and likelyp recludes the macrocyclisation of peptides con-tainingA sp, Cys, Glu, Met, Sec, Ser,T hr,T rp or Tyrr esidues,o r His and Lysintheir free-base forms.This can be seen in the relatively limited diversity of peptide substrates that have been used. Yield-limiting hydrogen bonding, substratei nstability, high dilution conditions and ar equirement for peptide N-terminal pre-functionalisation (along with C-terminal for N-trimethylsilylmethyl amides)a re also factors that move this strategy away from being an ideal photochemical strategy for peptide macrocyclisation.

Peptidemacrocyclisation under photoredox catalysis
Photoredox catalysis has seen rapid growth in organic synthesis in the last decade, both in terms of applicationa nd capability. [118] PET with ap hotoexcited photoredox catalyst PC* under this manifold can take one of two possible courses( Figure 23): i) an oxidative quenching cycle whereby catalyst PC* transfers an electron to an acceptors pecies A,i tself being oxidised to a form PC + + 1 ,w hichc an then accept an electron from ad onor D to return to the ground-state PC;o ra lternatively,i i) ar eductive quenching cycle proceeding by reduction of the photoexcitedstate catalyst( PC*!PC À À1 )b yd onor D,f ollowed by oxidation Figure 22. Peptide macrocyclisationvia PET from C-terminal N-trimethylsilylmethylamides to an excited-state phthalimide. [110,114]  back to the ground-state PC through ET to acceptor A. [119] Acceptor and donor molecules can be reagents, substrates or intermediates generated during the reaction. Whether PC* quenches oxidativelyo rr eductively depends on the best match of the redox potentials of the excited-state catalyst relative to the speciesp resenti nt he reaction. Various catalytic structures can be exploited, including transition metal polypyridyl complexes, [119,120] lanthanide ions, [121] organic compounds, [122] bulk semiconductors [123,124] or metal-organic frameworks. [125] When applied to PET-initiatedP SP macrocyclisation, photoredox catalysis offers several potential advantages over ad irect intramolecular PETa pproach. Separating the chromophore from the substrate removes the necessity to pre-functionalise the PSP with ap otentially disruptive unnatural motif. It also gives greaterf lexibility over chromophore structure, allowing the use of catalysts that absorbl ower energy visible light to which all natural amino acids are transparent. This makes photoredox catalysis more compatible with biological speciest hat may suffer damage under UV irradiation. [53,[126][127][128] Furthermore, with indirect (and therefore asynchronous)E Tf rom the donor to the acceptor through the intermediary of the photocatalyst, more complex and varied chemistries are accessible. [129][130][131] The first reports of peptidem acrocyclisation under photoredox catalysis came from the Nolg roup, as part of their development of photocatalysedt hiol oxidations to form disulfides. [132,133] The proposed mechanism for this photoredox catalysed transformation commences with photoexcitation of the catalystw ith white LEDs, followed by ar eductive quenching event through proton-coupled electron transfer (PCET)f rom a thiol 70 to PC*,aproposal supportedb yt he observation of increasedy ields in the presence of base ( Figure 24). [132,133] The re-sultant thiyl radical 71 then undergoes disulfide bond formation to form 72,w ith the formal loss of ah ydrogen atom. This is suggested to occur by sequential thiol deprotonation, thiyl radicala ddition to the thiolatea nd single-electron oxidation of the disulfide radical anion( potentially by superoxide radical anions). [61,132] Closure of the catalytic cycle occurs through ET from the reduced form of the photocatalyst PC À À1 to molecular oxygen, which acts as the terminal oxidant for this net-oxidative process. An alternative mechanism where thiyl radical formation occurs through ET to singlet oxygen ( 1 O 2 ,p roduced through catalyst-mediated photosensitisation of ground-state 3 O 2 )w as discounted owing to the observation of lowy ields with Ru and Ir photocatalysts, which are known to be capable of generating 1 O 2 . [132] This technology was applied to the synthesis of the cyclic peptide hormone oxytocin (73,F igure 25). Irradiation of the organic photoredox catalyst eosin Yunder an oxygen gas flow in ac ontinuous-flow photoreactor (200 sr esidence time) offered as ignificant rate acceleration over batch conditions, owing to betterl ight penetration and improved oxygen mixing, which helpeds uppress sider eactions ( Figure 25, conditions A). [132] Full conversion of linear precursor 74 was observed, with the formation of oxytocin 73 being accompanied with intermolecular disulfide peptide dimers in varying oxidised states, the formationo fw hich was minimised by running the process at higherd ilutions. The same group subsequently reported the use of the semiconductor photocatalyst TiO 2 in batch, to mediate the cyclisation of oxytocin 73 ( Figure 25, conditions B). This heterogeneous photocatalyst is attractive as the authors demonstrated that it could be removed by simple filtration or centrifugation, and reused up to ten times with no drop in yield. Although product formation was monitored by LC-MS,n of urther isolation or purification was performed and hence the question of synthetic viability remains unanswered in full. However,a samild method (room temperature, neutral buffer, visible light) employing an easily separated and reusable catalyst, to synthesise naturally occurring disulfide-bridged macrocycles directly from native peptides bearing two cysteines, this approachs hows much promise and merits further investigation.
In 2014, MacMillan and co-workers published an influential paper outlining the Giese reaction (conjugate addition) of small molecule alkyl radicals, generated through the decarboxylation of carboxylica cids under visible-light photoredox catalysis, to electron-deficient alkenes. [134] Notably,t his chemistry was shown to be well-suited for radical generation from both N-carbamoyl a-aminoa cids and dipeptides. This strategy was subsequently extended to peptidem acrocyclisation by the same group in 2017, by incorporatingaMichael acceptorr adical trap at the peptideN -terminus (e.g.,a crylamide 75, Figure 26). [135] Mechanistically, PET from the carboxylate form of the C-terminal acid to the photoexcited catalyst PC* generates ac arboxyl radical, which rapidly undergoes loss of CO 2 to produce a-amido radical 76.F ollowing an intramolecular Michael addition, a-carbonyl radical 77 is reduced to an enolate speciest hroughE Tf rom PC À À1 ,c losing the photoredox catalytic cycle and affording macrocycle 78 after protonation. The macrocyclic products of the type 78 are fully peptidic, containing an unnatural g-aminoa cid linkage. The non-canonical g-aminobutyric acidl inker is likely to be insensitive to the action of proteases,p roviding products with increased stability under physiological conditions. When as eries of N-acryloyl pentapeptidesw ith aC -terminal glycine residue, 79 a-g,w ere irradiated with ab lue LED in the presence of the oxidising iridium photocatalyst Ir[dF(CF 3 )ppy] 2 (dtbbpy)PF 6 ,t he corresponding macrocyclic peptides 80 a-g were produced efficiently,a lthough isolated yields varied, which is ac ommon observation in this field ( Figure 27). The methodology was applied to peptides containing ab road range of amino acids, includingn on-canonical propargylglycine (Pra) and N-methyl alanine (N-Me-Ala). Notably,f or examples where there were amino acids bearing polar sidechain, protecting groups were utilised to suppress undesired nucleophilic and/or redox reactions. This lack of functional group tolerance may limit the widespread applicabilityo ft his chemistry at the present time. For substrates 79 a and 79 c,t he addition of 10 mol %2 ,4,6-triisopropylthiophenol to the reaction mixture was found to be beneficial to the yield. This effect was attributed to interception of the a-carbonyl radical of the form 77 ( Figure 26) by Ha tom abstraction from the thiol at ar ate competitive with retro-Michael addition and unwanted intermolecular oligomerisation, negating the need for rate-limiting reduction by PC À À1 ,t op rovide products 80 a and 80 c.I nt hese instances, the catalytic cycle is instead closed by ET from PC À À1 to the resultant thiyl radical.
Expanding the scopeo ft he reaction further,t he authors explored alternative functionalities at the C-and N-termini. Peptides bearing C-terminal amino acids with a-substituents (81 a-f)w ere found to be well tolerated and underwent decarboxylativec yclisation,h owever, yields were reduced relative to terminal glycines (Figure28). This may be attributable to increasedr ates of retro-Michael addition resulting from the increaseds tability of the substituted a-amido radicals generated. The formation of diastereomeric product mixtures was found to occur with little control (81 b and 81 c). Notably,s ubstrate 81 c with aC -terminal glutamic acidu nderwent chemoselective a-aminod ecarboxylation on accounto ft he lower pK a and oxidation potential of the C-terminusrelative to the g-carboxylic acid. [136] Although not demonstrated, analogous selectivity over aspartic acid sidechains would also be expected for the same reasons. Unnatural di-a-substituted cyclic amino acidsa t the peptideC -terminusw ere able to form unusual spirocyclic peptidem acrocycles (82 d and 82 e). At the N-terminus, only a single functionalised acrylamide motif was investigated, with an electronically activating phenylg roup at the a-position leadingt ot he formation of macrocycle 82 f in high yield and with good diastereoselectivity.T he methodology performed well when appliedt ot he synthesis of larger ring sizes, with remarkably little yield variationo bserved acrosst he preparation of 8-, 10-and 15-membered cyclic peptides. Importantly,t he authors also demonstrated the straightforward post-cyclisation removal of acid-labile protecting groups,l eading to the generation of the somatostatinr eceptor agonistp eptideC OR-005 (83 in 47 %isolated yield over two steps).  [c] N-Me-Ala = N-methyl alanine. [135] Peptidem acrocyclisation underd ual photoredoxand transition-metalcatalysis Advancesi nt he field of photoredox catalysis have been mirrored in their application in PSP macrocyclisation. At the cutting edge of this area is the merger of photoredox and transition-metal catalysis. This extremely powerful combination opens up unprecedented chemical transformations that are well-suited to applications in complex settings on account of the mild reaction conditions. [131,137,138] Key to the success of this approachi st he activation of transition-metal complexes by a photocatalyst, either through redox modulation or energy transfer pathways, triggering mechanistic steps that would not be operative under transition-metal catalysis alone. [139][140][141][142][143][144] The Sciammetta group disclosed am ethodology for the etherification of peptidic alcohols with aryl bromides by C(sp 2 )ÀOc ross-coupling under dual photoredoxa nd nickel catalysis. [145] In addition to intermolecular ether formation, the reaction conditions were also shown to be amenable to macrocyclisation of as eries of N-terminal bromobenzamides, via aryl etherification of C-terminal serine derivatives. Thea uthors pro-posed this methodology to target underutilised ether macrocyclic linkages, which they anticipatedw ould overcome several of the shortcomings at times exhibited by otherm acrocycle chemistries, such as proteolytic instability (e.g.,t hioether linkages), and the poor cell-permeability of hydrogen bond donor containing linkages (e.g.,those with an NÀHb ond).
The proposed mechanismo ft his reaction was analogous to that previously reportedb yM acMillan and co-workersf or the dual photoredox-nickel catalysed arylation of small-molecule alcohols ( Figure 29). [139] Following the oxidative addition of a Ni 0 complex 84 into an aryl bromide 85,t he resultant aryl-Ni II species 86 can undergo ligand exchange to form cyclic Ni II alkoxide 87.S ingle-electron oxidation of complex 87 by ET to the excited-state photocatalyst PC* generates ak ey Ni III aryl alkoxide 88,w hich, unlike Ni II aryl alkoxide complex 87,i su nstable with respect to reductive elimination and the catalytic cycle therefore generates peptidem acrocycle 89 andt he Ni I species 90.F inally,E Tt o90 from PC À À1 then regenerates Ni 0 species 84 and simultaneously closes both catalytic cycles.
Using the isophthalonitrile-based organic photocatalyst 4DPAIPN 91 and aN iBr 2 ·glyme/dtbbpy transition-metal catalyst system,i nc oncert with quinuclidinea sayield boosting additive (an affect attributed to its capacity to act as an electron donoro rs huttle), the authorsw ere able to induce macrocyclisation of as eries of N-terminal m-bromobenzamide modified peptides 92 a-f ( Figure 30). All substrates contained a b-hairpin inducing d-Pro-l-Pro subunit to preorganise the linear peptide for cyclisation via rigidifying hydrogen bonds, leading to the formation of cyclised peptides 93.Inthe absence of this dipeptide, only trace yields of macrocycle formation were detected by UPLC-MS. The importance of theseh ydrogen bondsw as supported by in silico conformational sampling by ad istance geometry approach. For pentapeptide substrates with C-terminal serinamides( 92 a,b,e), competing O-and N-arylation was observedt op rovide ether-and amide-linkedm acrocycles, which were separableb yH PLC. This competitive cyclisation favoured the ether over amide linkages by ratios of 6:4-7:3 (see Figure 30). For substrates with aC -terminal ester (92 c)o r glycinamide (92 d)r esidues,p resenting as ingle nucleophilic position, macrocyclisation proceedede xclusively through etherification or amidation, respectively.V ariation of the peptide sequence to incorporate residues bearing protected polar functionality (92 e)o ri ncreasing the ring size (92 f)w as also successful. Interestingly,m acrocyclisation in the presence of a free carboxylica cid wasn ot demonstrated, potentially because of competing lactonisation. This transformation hasb een demonstrated in an intermoleculars ense by an energy-transfer mechanism under similar reaction conditions. [140] Notably,t he requirementf or the protection of polar residues, and more importantly the presence of at urn-enforcing d-Pro-l-Pro subunit to achieveg ood cyclisation efficiency are limitations that need to be addressed fort his methodology to achieve broad synthetic applicability in peptidemacrocyclisation.

Conclusion
In this review,w ehave detailed the photochemical strategies that have been exploited to induce PSP macrocyclisation. These methods benefit from mild reactionconditions, the ability to selectively introduceenergy and the sheer variety of reaction modes available. Developmentsi nt his fieldh avem irrored the recent renewed interest in photochemistry,a nd this looks set to continue as macrocyclic PSPs become increasingly prevalent in the drug discovery pipeline. The rapid expansion of new photochemical strategies in organic synthesis provides a rich source of potential new reactions to control cyclisation. Indeed, there remains significant scope for innovation in the area, with ap ressing need to both evolve existing strategies for cyclisation, and develop new ones.
In particular,w esee an urgent need for new reactions that can overcome limitations in amino acid tolerance. It is notable that in many of the examples presented within this review,t he linear peptide substrates are composed of av ery simple set of afunctional amino acids with hydrocarbon sidechains. The resultant methodologies, while synthetically interesting, provide little scope to generatem acrocyclic peptides with useful biological properties. Al ack of functional group tolerance is also highlighted by the commonr eliance on fully protected peptides as substrates. Exceptions, such as thiol-"ene" cyclisations, must proceed with exquisite chemoselectivity to avoid side reactions with the myriado fr eactive functionalities found within unprotected, canonical amino acids. Recent developments in photoredox catalysis, enabling site-selective modification of even complex proteins, are therefore particularly exciting, althoughi ts hould be noted that even then the presence of a subset of amino acids must be avoided. [136,146] New reactions that can increase this selectivity still furtherw ould be invaluable to the bioconjugatec ommunity,a nd would provide an important and generalisable tool for peptide macrocyclisation.
Moreover,w ea nticipate increased use of reactions that exploit lower energy visible light sources to induce macrocyclisation, with associated improvements in selectivity.T he reliance of many of the reactions described hereo nh ighe nergy UV irradiation is inherently limiting to functional group tolerance. [147] Photoredox catalysis driven by visible light is an important avenue to overcome this limitation, and we anticipate ag reater application of this technology to the macrocyclisation of PSPs. Other visible light-driven photochemical catalysis manifolds, such as energy transfer and photon upconversion, are also well-positioned for implementation in PSP macrocyclisation. With these innovations the prospecto fa chieving general strategies for protein macrocyclisation will becomem ore realistic, adding to the fairly limited toolkito fp hotochemical reactions that have been exploited for this ambitious goal to date. [74,101] Finally,t he development of photochemical reactions that can deliver unique macrocyclic linkages represents an important future challenge for the community.T he diversity of reaction manifoldst hat are accessible through photochemical approaches is attractive given the importance of even subtle dif-ferences in peptide linker structure on biological properties, such as membrane permeability and target binding. [71] Newr eactions that can be used to modulate the properties of the resultant cyclic peptides would therefore represent valuable tools in the search for novel peptide therapeutics. Important challenges that need to be overcome in this regard include the stereocontrol of newly formed chiral centres, ad ifficult task in such af unctionally dense environment, and ar educed relianceo nt urn-inducing residues to aid cyclisation.
We anticipate the benefits of light-mediated chemistry will be increasingly exploited in the future as the field matures. Exciting opportunities for spatiala nd temporal control over cyclisation may enablea pplications in advanced biomedical technologies, diversifying away from the traditional roles of cyclised peptides as therapeutic agents. As the journey towards more 'ideal' macrocyclisation techniques that are readily applicable to PSP substrates and that tolerate ever increasing functionality continues, the future for photochemical macrocyclisation methodsl ooks bright.