molecules

Review Current Synthetic Routes to Peptidyl Mono-Fluoromethyl (FMKs) and Their Applications

Carissa M. Lloyd 1, Neil Colgin 2 and Steven L. Cobb 1,*

1 Department of Chemistry, Faculty of Science, Durham University, Durham DH1 3LE, UK; [email protected] 2 Cambridge Research Biochemicals, 17-18 Belasis Court, Belasis Hall Technology Park, Billingham, Cleveland TS23 4AZ, UK; [email protected] * Correspondence: [email protected]

Academic Editor: Derek J. McPhee   Received: 26 October 2020; Accepted: 24 November 2020; Published: 28 November 2020

Abstract: Peptidyl mono-fluoromethyl ketones (FMKs) are a class of biologically active molecules that show potential as both inhibitors for the treatment of a range of diseases and as chemical probes for the interrogation of cellular processes. This review describes the current solid- and solution-phase routes employed for the synthesis of peptidyl mono-FMKs. In addition, it provides a brief overview of some of the key applications of FMKs in the fields of chemical biology and medicinal chemistry.

Keywords: peptide; fluoromethyl ; peptide synthesis; peptide modification; fluorine; therapeutic; probe

1. Introduction Peptidyl mono-fluoromethyl ketones (FMKs) are a class of biologically active compounds that have been developed as protease inhibitors [1–5] and as chemical probes for the interrogation of cellular processes [6–8]. Peptidyl FMKs were first reported in 1985 [9] and have since gained preference in the field over the corresponding peptidyl chloromethyl ketones (CMKs), largely due to their lower reactivity towards nucleophiles. For example, peptidyl CMKs have been developed as both cysteine and serine protease inhibitors (e.g., Figure1,( 1)), but their highly reactive nature often causes issues with regards to selectivity. As such, for many peptidyl CMKs, indiscriminate binding of nonproteolytic leads to undesirable side-effects, rendering them unsuitable for in vivo applications [10]. Peptidyl FMKs are much less prone to nonspecific alkylations due to the intrinsic strength of the C-F bond making them significantly more selective [9] (Figure1,( 2), (3)). Since their discovery, the number of synthetic routes that are available to access peptidyl mono-FMKs remains rather limited, particularly when compared to those that can be used to access the corresponding peptidyl CMKs [11–13] and even peptidyl tri-fluoromethyl ketone systems (Figure1,( 4)) [14–16].

Molecules 2020, 25, 5601; doi:10.3390/molecules25235601 www.mdpi.com/journal/molecules Molecules 2020, 25, x 5601 FOR PEER REVIEW 2 of 16 Molecules 2020, 25, x FOR PEER REVIEW 2 of 16

Figure 1. Representative examples of a peptidyl chloromethyl ketone (1) [12], peptidyl fluoromethyl Figure 1.1. Representative examples of a peptidylpeptidyl chloromethyl ketone (1)[) [12],12], peptidylpeptidyl fluoromethylfluoromethyl ketones (2 [3] and 3 [2]), and a peptidyl tri-fluoromethyl ketone (4) [15]. ketones (2 [[3]3] andand 33 [[2]),2]), andand aa peptidylpeptidyl tri-fluoromethyltri-fluoromethyl ketoneketone ((44)[) [15].15]. This review provides an overview of the current state-of-the-art with regards to solid- and This review provides an overview of the currentcurrent state-of-the-artstate-of-the-art with regards to solid-solid- and solution-phase synthetic routes that can be employed for the synthesis of peptidyl mono-FMKs. In solution-phase synthetic synthetic routes routes that that can can be be employ employeded for for the the synthesis synthesis of ofpeptidyl peptidyl mono-FMKs. mono-FMKs. In addition, a brief summary of the key applications of this class of compound within the fields of Inaddition, addition, a brief a brief summary summary of ofthe the key key applications applications of of this this class class of of compound compound within within the the fields fields ofof chemical biology and medicinal chemistry is given. For a more detailed overview of FMKs within the chemical biologybiology andand medicinalmedicinal chemistrychemistry isis given.given. For a more detailed overview of FMKs within the field of medicinal chemistry, an excellent recent review of this area was published by A. Citarella and fieldfield of medicinal chemistry, anan excellentexcellent recentrecent reviewreview ofof thisthis areaarea was published by A. Citarella and N. Micale [17]. N. Micale [[17].17]. 2. Current Current Synthetic Synthetic Routes Routes to to Peptidyl Peptidyl Mono-FMKs 2. Current Synthetic Routes to Peptidyl Mono-FMKs 2.1. Solution-Phase Solution-Phase Routes Routes 2.1. Solution-Phase Routes 2.1.1. Halogen-Exchange Halogen-Exchange 2.1.1. Halogen-Exchange An earlyearly attempted attempted solution-phase solution-phase peptidyl peptidyl mono-FMK mono-FMK synthesis synthesis looked to looked use the displacementto use the An early attempted solution-phase peptidyl mono-FMK synthesis looked to use the displacementof bromide or of chloride bromide with or a chloride source of with inorganic a sour fluoridece of inorganic in a halogen-exchange fluoride in a reaction.halogen-exchange However, displacement of bromide or chloride with a source of inorganic fluoride in a halogen-exchange reaction.this approach However, did not this produce approach the desired did not FMK produce (7), but the rather, desired resulted FMK in(7 a), nonfluorinatedbut rather, resulted compound in a reaction. However, this approach did not produce the desired FMK (7), but rather, resulted in a nonfluorinated(6) along with various compound unwanted (6) along side-products with various (Scheme unwanted1)[9 ].side-products (Scheme 1) [9]. nonfluorinated compound (6) along with various unwanted side-products (Scheme 1) [9].

Scheme 1. AttemptedAttempted formation formation of of a a peptidyl peptidyl mono-fluoromethyl mono-fluoromethyl ketone ketone (FMK) (FMK) ( (77)) through through a Scheme 1. Attempted formation of a peptidyl mono-fluoromethyl ketone (FMK) (7) through a halogen-exchange reaction [[9].9]. halogen-exchange reaction [9]. Since then,then, aa successfulsuccessful halogen-exchange halogen-exchange method method has has been been developed developed and and was was reported reported by Kolb by Since then, a successful halogen-exchange method has been developed and was reported by Kolbet al. et in 1986al. in (Scheme1986 (Scheme2)[ 18]. 2) Initial [18]. isolationInitial isolation of 3-phthalimido-1-bromo-4-phenyl-2-butanone of 3-phthalimido-1-bromo-4-phenyl-2-butanone (8) from Kolb et al. in 1986 (Scheme 2) [18]. Initial isolation of 3-phthalimido-1-bromo-4-phenyl-2-butanone (N8)-phthaloyl from N-phthaloyl phenylalanine phenylalanine was achieved was through achieved generation through of thegeneration corresponding of the diazoketonecorresponding via (8) from N-phthaloyl phenylalanine was achieved through generation of the corresponding diazoketonethe acid chloride via the (not acid shown). chloride Subsequent (not shown). bromination Subsequent of bromination the diazoketone of the according diazoketone to previously according diazoketone via the acid chloride (not shown). Subsequent bromination of the diazoketone according toknown previously conditions known [19 conditions,20] led to the[19,20] formation led to the of 8 formation. Further reactionof 8. Further of 8 with reaction KF/l8-crown-6 of 8 with KF/l8- led to to previously known conditions [19,20] led to the formation of 8. Further reaction of 8 with KF/l8- crown-6FMK 9 in led a 45% to FMK yield 9 (Schemein a 45%2 ).yield Reduction (Scheme by 2). sodium Reduction borohydride by sodium enabled borohydride removal enabled of the phthaloyl removal crown-6 led to FMK 9 in a 45% yield (Scheme 2). Reduction by sodium borohydride enabled removal of the phthaloyl protecting group, however, this simultaneously reduced the ketone functionality of the phthaloyl protecting group, however, this simultaneously reduced the ketone functionality

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Molecules 2020, 25, x FOR PEER REVIEW 3 of 16 protectingMolecules 2020 group,, 25, x FOR however, PEER REVIEW this simultaneously reduced the ketone functionality which had3 of to 16 be reinstalledwhich had by to Swern be reinstalled oxidation by (Swern10)[21 oxidation,22]. Vederas (10) et[21,22]. al. utilised Vederas a similar et al. utilised approach a similar for the approach synthesis 13 ofwhich afor theC-labelled had synthesis to be peptidyl reinstalled of a 13C-labelled FMK by probeSwern peptidyl which oxidation wasFMK ( used10 probe) [21,22]. to investigatewhich Vederas was used theet al. mode toutilised investigate of actiona similar ofthe theapproach mode HAV of 3C 13 enzymeforaction the [of 23synthesis the]. HAV of 3C a enzymeC-labelled [23]. peptidyl FMK probe which was used to investigate the mode of action of the HAV 3C [23].

SchemeScheme 2. 2.Formation Formation ofof FMKFMK 10 via a halogen-exchange halogen-exchange reaction reaction [18]. [18 ]. Scheme 2. Formation of FMK 10 via a halogen-exchange reaction [18]. 2.1.2.2.1.2. Synthesis Synthesis via via Direct Direct Fluorination Fluorination ofof aa DiazoDiazo Intermediate 2.1.2. Synthesis via Direct Fluorination of a Diazo Intermediate AnotherAnother initially initially unsuccessful attempt attempt involved involved the reaction the reaction of HF ofwith HF a diazomethyl with a diazomethyl ketone; ketone;a methodAnother a method analogous initially analogous unsuccessfulto that used to that attemptfor usedthe synthesisinvolved for the thof synthesise CMKsreaction (chloromethyl of HF CMKs with (chloromethyla diazomethylketones) and ketone; ketones)BMKs a method analogous to that used for the synthesis of CMKs (chloromethyl ketones) and BMKs and(bromomethyl BMKs (bromomethyl ketones). This ketones). reaction,This which reaction, was attempted which on was Cbz-Phe-CHN attempted2, on did Cbz-Phe-CHN not yield the 2, did(bromomethyldesired not yield FMK, the ketones).but desired instead This FMK, reaction,resulted but which insteadin the was formation resulted attempted inof theonthe Cbz-Phe-CHN formation cyclic product of2, thedid 1-oxa-3-aza-4- not cyclic yield product the desired FMK, but instead resulted in the formation of the cyclic product 1-oxa-3-aza-4- 1-oxa-3-aza-4-benzylcyclohexan-2,5-dionebenzylcyclohexan-2,5-dione [9,24]. It was [9, 24found]. It was that found the use that of the HF/ use of HF/pyridine [25] allowed [25] allowed the benzylcyclohexan-2,5-dione [9,24]. It was found that the use of HF/pyridine [25] allowed the thesuccessful successful isolation isolation of of the the desired desired product. product. Furthermore, Furthermore, the the use use of ofphthaloyl phthaloyl instead instead of ofCbz Cbz as asa a successful isolation of the desired product. Furthermore, the use of phthaloyl instead of Cbz as a protectingprotecting group group also also ensured ensured that that no no amideamide protonproton was present, present, thus thus preventing preventing unwanted unwanted internal internal protecting group also ensured that no amide proton was present, thus preventing unwanted internal cyclisation.cyclisation. Generation Generation of of the the diazo diazo compoundcompound (12) was first first achieved achieved through through the the reaction reaction of of1111 withwith cyclisation.diazomethane Generation in the presence of the diazo of triethylamine compound ( 12and) was isobutylchloroformate first achieved through (Scheme the reaction 3). Subsequent of 11 with diazomethanediazomethane in in the the presence presence of of triethylamine triethylamine andand isobutylchloroformateisobutylchloroformate (Scheme (Scheme 3).3). Subsequent Subsequent exposureexposure to to HF HF/pyridine/pyridine led led to to the the isolationisolation ofof FMKFMK 13. Removal Removal of of the the phthaloyl phthaloyl group group under under exposurereducing conditionsto HF/pyridine allowed led peptide to the isolationchain extension of FMK [24], 13 . however,Removal theof theFMK phthaloyl carbonyl groupfunctionality under reducing conditions allowed peptide chain extension [24], however, the FMK carbonyl functionality reducingneeded to conditions be regenerated allowed in the peptide final stepchain through extension oxidation. [24], however, the FMK carbonyl functionality neededneeded to to be be regenerated regenerated in in the the final final step step throughthrough oxidation.oxidation.

Scheme 3. Formation of peptidyl mono-FMK building block (17) via a diazo intermediate [24]. SchemeScheme 3. 3.Formation Formation of of peptidyl peptidyl mono-FMKmono-FMK building block block (17 (17) )via via a adiazo diazo intermediate intermediate [24]. [24 ]. 2.1.3. Dakin–West Modification 2.1.3.2.1.3. Dakin–West Dakin–West Modification Modification A commonly used approach for the synthesis of keto-amides, the Dakin–West reaction [26], was A commonly used approach for the synthesis of keto-amides, the Dakin–West reaction [26], modifiedA commonly [9] in order used to approach allow for forthe the synthesis synthesis of aof selection keto-amides, of peptidyl-FMKs, the Dakin–West as shownreaction in [26], Scheme was wasmodified modified [9] [in9] order in order to allow to allow for the for synthesis the synthesis of a selection of a selection of peptidyl-FMKs, of peptidyl-FMKs, as shown asin Scheme shown in 4 for Bz-DL-Ala-CH2F (19). The isolation of 19 in yields ranging from 20–25% was achieved through 2 19 19 Scheme4reaction for Bz-DL-Ala-CH4 for of Bz-DL-Ala-CH18 with fluoroaceticF (19). 2TheF( anhydrideisolation). The isolation of in 19 the in yieldspresence of inranging yieldsof triethylamine from ranging 20–25% from and was catalytic 20–25% achieved wasamounts through achieved of throughreaction reaction of 18 with of 18fluoroaceticwith fluoroacetic anhydride anhydride in the presence in the of presencetriethylamine of triethylamine and catalytic amounts and catalytic of

Molecules 2020, 25, 5601 4 of 16 Molecules 2020, 25, x FOR PEER REVIEW 4 of 16 Molecules 2020, 25, x FOR PEER REVIEW 4 of 16 4-dimethylaminopyridine.amounts of 4-dimethylaminopyridine. However, However,it was note it wasd that noted racemisation that racemisation was unavoidable, was unavoidable, and andthe methodthe4-dimethylaminopyridine. method failed failed to produce to produce the However,desired the desired product it productwas fornote the ford thevalinethat valine racemisation analogue. analogue. was unavoidable, and the method failed to produce the desired product for the valine analogue.

Scheme 4. FormationFormation of of peptidyl mono-FMK building block Bz-DL-Ala-CH 22FF( (1919)) via via a a modified modified Scheme 4. Formation of peptidyl mono-FMK building block Bz-DL-Ala-CH2F (19) via a modified Dakin–West reaction [9]. Dakin–WestDakin–West reaction reaction [9]. [9]. 2.1.4. Epoxide Ring Opening Approach with Fluoride Source 2.1.4.2.1.4. Epoxide Epoxide Ring Ring Opening Opening Approach Approach with with Fluoride Fluoride Source The synthesis of peptidyl FMKs was also reported by Funeriu and coworkers in 2012. This was TheThe synthesis synthesis of of peptidyl peptidyl FMKs FMKs was was also also reportreported by Funeriu andand coworkers coworkers in in 2012. 2012. This This was was achieved through epoxide ring opening of epichlorohydrin 20 with a fluoride source (KHF2) to achievedachieved through through epoxide epoxide ring ring opening opening of of epichlorohydrin epichlorohydrin 20 withwith aa fluoride fluoride source source (KHF (KHF2) 2to) to give give give 21, which was then converted to N-phthalimid-fluoro- 22 in the presence of potassium 21,21 which, which was was then then converted converted to to NN-phthalimid-fluoro-alcohol-phthalimid-fluoro-alcohol 2222 inin thethe presencepresence of of potassium potassium phthalimide [6] (Scheme5). Subsequent reaction with hydrazine allowed the generation of the free phthalimidephthalimide [6] [6] (Scheme (Scheme 5). 5). Subsequent Subsequent reaction reaction wiwithth hydrazine allowedallowed the the generation generation of of the the free free amine (23), which could then be Fmoc protected to give 24, before oxidation with DMP-afforded FMK amineamine (23 (),23 which), which could could then then be be Fmoc Fmoc protected protected to to givegive 24, before oxidationoxidation with with DMP-afforded DMP-afforded FMK FMK 25. Temporary protection of the ketone functionality as a 1,3-dithiane followed by Fmoc deprotection 25.25 Temporary. Temporary protection protection of of the the ketone ketone functionality functionality as a 1,3-dithiane followed followed by by Fmoc Fmoc deprotection deprotection was employed in order to allow peptide growth to occur. Regeneration of the ketone group was waswas employed employed in inorder order to to allow allow peptide peptide growthgrowth toto occur.occur. RegenerationRegeneration of of the the ketone ketone group group was was achieved using bis(trifluoroacetoxy)iodobenzene, giving peptidyl FMK 29 in a quantitative yield. achievedachieved using using bis(trifluoroacetoxy bis(trifluoroacetoxy)iodobenzene,)iodobenzene, givinggiving peptidyl FMKFMK 29 29 in in a a quantitative quantitative yield. yield. A A A modified version of this procedure was utilised by Ellis et al. in 2016 in order to access peptidyl modifiedmodified version version of of this this procedure procedure was was utilised utilised by by EllisEllis et al. in 20162016 in in order order to to access access peptidyl peptidyl FMKs FMKs FMKs to be studied as irreversible protease inhibitors [8]. to beto bestudied studied as asirreversible irreversible protease protease inhibitors inhibitors [8].[8].

SchemeScheme 5. 5.Synthesis Synthesis of of peptidyl peptidyl FMKFMK 2929 utilising an epoxide epoxide ring ring opening opening approach approach [6]. [6 ]. Scheme 5. Synthesis of peptidyl FMK 29 utilising an epoxide ring opening approach [6]. 2.1.5.2.1.5. Synthetic Synthetic Pathway Pathway Involving Involving Utilisation Utilisation ofof a Fluorinated Hemiacetal/ Hemiacetal/Aldehyde 2.1.5.The SyntheticThe application application Pathway of fluorinatedof Involving fluorinated hemiacetals Utilisation hemiacetals// ofaldehydes a Fluorinated has alsohas Hemiacetal/Aldehyde beenalso exploredbeen explored for accessing for accessing peptidyl FMKs.peptidyl One FMKs. particular One particular example ofexample this involves of this involves the formation the formation of β-nitroalcohol of β-nitroalcohol species species32 derived 32 The application of fluorinated hemiacetals/aldehydes has also been explored for accessing fromderived nitro from alkane nitro30 reactingalkane 30 with reacting a fluorinated with a fluorinated hemiacetal hemiacetal (31), catalysed (31), catalysed by potassium by potassium carbonate peptidyl FMKs. One particular example of this involves the formation of β-nitroalcohol species 32 (Schemecarbonate6)[ 14(Scheme]. Further 6) [14]. reduction Further with reduction Raney with nickel Raney at 50 nickel psi of at hydrogen 50 psi of pressurehydrogen followed pressure by derived from nitro alkane 30 reacting with a fluorinated hemiacetal (31), catalysed by potassium treatmentfollowed with by treatment concentrated with concentrated HCl allowed HCl generation allowed generation of 33 asa of mixture 33 as a mixture of DL, ofthreo DL,, andthreo,erythro and carbonate (Scheme 6) [14]. Further reduction with Raney nickel at 50 psi of hydrogen pressure diastereomers.erythro diastereomers. After liberation After liberation of the hydrochloride of the hydrochloride salt, peptide salt, peptide coupling coupling with DCC with as DCC theactivator as the followed by treatment with concentrated HCl allowed generation of 33 as a mixture of DL, threo, and affordedactivator35 .afforded The final 35 step. The infinal the step process in the involved process involved a Sarett oxidation,a Sarett oxidation, allowing allowing isolation isolation of FMK of36 . erythro diastereomers. After liberation of the hydrochloride salt, peptide coupling with DCC as the ThisFMK pathway 36. This to pathway36 proceeded to 36 withproceeded an overall withyield an overall of 70–75%. yield of A 70–75%. modified A version modified of thisversion method of this was activator afforded 35. The final step in the process involved a Sarett oxidation, allowing isolation of FMK 36. This pathway to 36 proceeded with an overall yield of 70–75%. A modified version of this

Molecules 2020, 25, 5601 5 of 16 Molecules 2020, 25, x FOR PEER REVIEW 5 of 16 Molecules 2020, 25, x FOR PEER REVIEW 5 of 16 employedmethod bywas Revesz employed et al. inby 1994 Revesz [27]. et Chatterjee al. in 1994 and [27]. coworkers Chatterjee described and coworkers the preparation described of peptidyl the method was employed by Revesz et al. in 1994 [27]. Chatterjee and coworkers described the FMKspreparation using a similarof peptidyl procedure FMKs [using2], and a alsosimilar introduced procedure a new[2], and approach also introduced which will a benew detailed approach in the preparation of peptidyl FMKs using a similar procedure [2], and also introduced a new approach nextwhich Section will 2.1.6be detailed[2]. in the next Section 2.1.6 [2]. which will be detailed in the next Section 2.1.6 [2].

SchemeScheme 6. 6.Isolation Isolation ofof peptidylpeptidyl FMK 36 viavia a a fluorinated fluorinated hemiacetal hemiacetal [14]. [14 ]. Scheme 6. Isolation of peptidyl FMK 36 via a fluorinated hemiacetal [14]. TheThe synthetic synthetic route route presented presented by by Cai Cai etet al.al. in 2 2006006 (Scheme (Scheme 7)7) closely closely re resemblessembles the the methodology methodology The synthetic route presented by Cai et al. in 2006 (Scheme 7) closely resembles the methodology alreadyalready discussed discussed (Scheme (Scheme6), 6), with with the the key key di differencfferencee being the the use use of of a afluorinated fluorinated aldehyde aldehyde instead instead already discussed (Scheme 6), with the key difference being the use of a fluorinated aldehyde instead ofof the the hemiacetal hemiacetal (31 (31)[)4 [4].]. Starting Starting fromfrom esterester 37, conversion to to amide amide 3838 waswas achieved achieved through through the the of the hemiacetal (31) [4]. Starting from ester 37, conversion to amide 38 was achieved through the additionaddition of of dimethylamine. dimethylamine. Further Further reactionreaction with 2-fluoroacetaldehyde, 2-fluoroacetaldehyde, which which was was accessed accessed through through addition of dimethylamine. Further reaction with 2-fluoroacetaldehyde, which was accessed through Swern oxidation of the corresponding alcohol, enabled isolation of nitro-alcohol 39. A subsequent SwernSwern oxidation oxidation of of the the corresponding corresponding alcohol,alcohol, enabledenabled isolation of of nitro-alcohol nitro-alcohol 3939. .AA subsequent subsequent hydrogenation step afforded 40 which could then be coupled to Cbz-Val-OH and finally oxidised to hydrogenationhydrogenation step step a ffaffordedorded 40 40which which could could thenthen be coupled to to Cbz-Val-OH Cbz-Val-OH and and finally finally oxidised oxidised to to FMK 42. Similar methodology was later employed by M. L. Forrest et al. in 2012 for accessing an FMK FMKFMK42 .42 Similar. Similar methodology methodology was was later later employed employed byby M. L. Forrest Forrest et et al. al. in in 2012 2012 for for accessing accessing an an FMK FMK with a fluorescent moiety attached for cellular imaging purposes [7]. withwith a fluorescent a fluorescent moiety moiety attached attached for for cellular cellular imagingimaging purposespurposes [7]. [7].

Scheme 7. Synthetic route to peptidyl FMK 42 utilising a fluorinated aldehyde species [4]. Scheme 7. Synthetic route to peptidyl FMK 42 utilising a fluorinated aldehyde species [4]. Scheme 7. Synthetic route to peptidyl FMK 42 utilising a fluorinated aldehyde species [4]. 2.1.6. Synthetic Methodology Employing Silyl Enol Ether Fluorination 2.1.6.2.1.6. Synthetic Synthetic Methodology Methodology Employing Employing SilylSilyl EnolEnol Ether Fluorination Chatterjee and coworkers described a novel approach for accessing peptidyl FMKs in 1997 Chatterjee and coworkers described a novel approach for accessing peptidyl FMKs in 1997 (SchemeChatterjee 8) [2]. and Starting coworkers from describedCbz-Val-Phe-OH a novel (43 approach), conversion for accessingto the Weinreb peptidyl amide FMKs (44)in was 1997 (Scheme 8) [2]. Starting from Cbz-Val-Phe-OH (43), conversion to the Weinreb amide (44) was (Schemeachieved8)[ 2].with Starting unavoidabl from Cbz-Val-Phe-OHe epimerisation ( 43at), conversionthe P1 position to the Weinreb in the amide presence (44) was of achievedN,O- achieved with unavoidable epimerisation at the P1 position in the presence of N,O- withdimethylhydroxylamine unavoidable epimerisation hydrochloride, at the P 1triethylaminposition ine theand presenceBOP. Further of N,O reaction-dimethylhydroxylamine with MeMgBr led hydrochloride,dimethylhydroxylamine triethylamine hydrochloride, and BOP. Further triethylamin reactione and with BOP. MeMgBr Further led reaction to the with successful MeMgBr isolation led

Molecules 2020, 25, 5601 6 of 16 Molecules 2020, 25, x FOR PEER REVIEW 6 of 16 Molecules 2020, 25, x FOR PEER REVIEW 6 of 16 to the successful isolation of methyl ketone 45. This could then be transformed into the corresponding toof the methyl successful ketone isolation45. This of couldmethyl then ketone be transformed45. This could into then the be correspondingtransformed into silyl the enolcorresponding ether and silyl enol ether and subsequently fluorinated with F-TEDA-BF4, affording dipeptide FMK 46. silylsubsequently enol ether fluorinated and subsequently with F-TEDA-BF fluorinated4, a withffording F-TEDA-BF dipeptide4, affording FMK 46. dipeptide FMK 46.

Scheme 8. Isolation of dipeptide FMK 46 through silyl enol ether fluorination [2]. Scheme 8. IsolationIsolation of of dipeptide dipeptide FMK FMK 46 throughthrough silyl enol ether fluorination fluorination [[2].2].

2.1.7. Synthetic Pathway Involving 1-amino-3-fluoro-propan-2-ol 1-amino-3-fluoro-propan-2-ol hydrochloride ((4848)) 2.1.7. Synthetic Pathway Involving 1-amino-3-fluoro-propan-2-ol hydrochloride (48) A literature literature procedure procedure for for accessing accessing peptidyl peptidyl FMKs, FMKs, which whichwas reported was reported by Tang et by al. Tang in 2016 et A literature procedure for accessing peptidyl FMKs, which was reported by Tang et al. in 2016 al.(Scheme in 20169) [5], (Scheme involves9)[ the5 ],couplin involvesg of thea Boc-protected coupling of aamino Boc-protected acid (47) with amino 1-amino-3-fluoro- acid (47) with (Scheme 9) [5], involves the coupling of a Boc-protected amino acid (47) with 1-amino-3-fluoro- 1-amino-3-fluoro-propan-2-olpropan-2-ol hydrochloride (48 hydrochloride), which was (synthesised48), which wasaccording synthesised to a two-step according procedure to a two-step (not propan-2-ol hydrochloride (48), which was synthesised according to a two-step procedure (not procedureshown). Amide (not shown). bond formation Amide bond between formation 47 and between 48 proceeds47 and through48 proceeds exposure through to HATU exposure under to HATU basic shown). Amide bond formation between 47 and 48 proceeds through exposure to HATU under basic underconditions basic (DIPEA). conditions Subseq (DIPEA).uent Subsequent Boc deprotection Boc deprotection of 49 is achieved of 49 is achievedby treatment by treatment with HCl with in ethyl HCl conditions (DIPEA). Subsequent Boc deprotection of 49 is achieved by treatment with HCl in ethyl inacetate, ethyl allowing acetate, allowing for the coupling for the coupling of the following of the following amino acid. amino The acid. final Thestep final in the step process in the involves process acetate, allowing for the coupling of the following amino acid. The final step in the process involves involvesthe oxidation the oxidation of alcohol of 51 alcohol to FMK51 52to in FMK the 52presencein the presenceof Dess–Martin of Dess–Martin periodinane periodinane (DMP). (DMP). the oxidation of alcohol 51 to FMK 52 in the presence of Dess–Martin periodinane (DMP).

Scheme 9. Formation of peptidyl FMK 52 starting from 1-amino-3-fluoro-propan-2-ol hydrochloride Scheme 9. Formation of peptidyl FMK 52 starting from 1-amino-3-fluoro-propan-2-ol hydrochloride (48) [5]. Scheme(48)[5]. 9. Formation of peptidyl FMK 52 starting from 1-amino-3-fluoro-propan-2-ol hydrochloride (48) [5]. 2.1.8. Synthetic Approach Involving Employment of a Magnesium Fluoromalonate 2.1.8. Synthetic Synthetic Approach Involving Employment of a Magnesium Fluoromalonate In 1991, Palmer patented a novel approach to access peptidyl mono-FMKs through the use of In 1991, Palmer patented a novel approach to access peptidyl mono-FMKs through the use of magnesium fluoromalonate 56 [28]. This method was later employed by Vederas et al. in 1997, as magnesium fluoromalonatefluoromalonate56 56[28 [28].]. This This method method was was later later employed employed by Vederas by Vederas et al. inet 1997,al. in as 1997, shown as shown in Scheme 10 [23]. After initial generation of magnesium benzyl fluoromalonate 56 from shownin Scheme in Scheme10[ 23]. After10 [23]. initial After generation initial generation of magnesium of magnesium benzyl fluoromalonate benzyl fluoromalonate56 from dimethyl 56 from dimethyl fluoromalonate 53 (Scheme 10), further reaction with Boc-N,N-dimethylglutamine (57) in dimethyl fluoromalonate 53 (Scheme 10), further reaction with Boc-N,N-dimethylglutamine (57) in

Molecules 2020, 25, 5601 7 of 16

Molecules 2020, 25, x FOR PEER REVIEW 7 of 16 fluoromalonateMolecules 2020, 2553, x FOR(Scheme PEER REVIEW 10), further reaction with Boc-N,N-dimethylglutamine (57) in the presence7 of 16 of 1,1’-carbonyldiimidazole (CDI) afforded 58 without racemisation at the P position (Scheme 10). the presence of 1,1’-carbonyldiimidazole (CDI) afforded 58 without racemisation1 at the P1 position the presence of 1,1’-carbonyldiimidazole (CDI) afforded 58 without racemisation at the P1 position Subsequent(Scheme 10). catalytic Subsequent hydrogenation catalytic hydrogenation gave the desired gave FMK the desired59. FMK 59. (Scheme 10). Subsequent catalytic hydrogenation gave the desired FMK 59.

SchemeScheme 10. 10.Synthesis Synthesis ofof FMKFMK 5959 using magnesium benzyl benzyl fluoromalonate fluoromalonate (56 (56) [23].)[23 ]. Scheme 10. Synthesis of FMK 59 using magnesium benzyl fluoromalonate (56) [23]. ImplementationImplementation ofof this this methodology methodology was was later later carried carried out by out Scott by and Scott coworkers and coworkers in their work in their to Implementation of this methodology was later carried out by Scott and coworkers in their work to workexplore to explore the use the of use peptidyl of peptidyl FMKs FMKs as covalent as covalent inhibitors inhibitors of ofMALT1 MALT1 [3]. [3 ].Magnesium Magnesium benzyl benzyl explore the use of peptidyl FMKs as covalent inhibitors of MALT1 [3]. Magnesium benzyl fluoromalonatefluoromalonate (56 (56) was) was coupled coupled to to aa Boc-arginineBoc-arginine unit prot protectedected with with a a2,2,5,7,8-pentamethylchroman 2,2,5,7,8-pentamethylchroman fluoromalonate (56) was coupled to a Boc-arginine unit protected with a 2,2,5,7,8-pentamethylchroman (Pmc) sulfonyl group on the side chain (6060) in the presence of CDI (Scheme 11). A one-pot benzyl ester (Pmc)(Pmc) sulfonyl sulfonyl group group onon the side side chain chain (60 ( ) in) inthe the presence presence of CDI of CDI (Scheme (Scheme 11). A 11 one-pot). A one-pot benzyl benzylester esterdeprotection deprotection and andsubsequent subsequent decarboxylation decarboxylation to the FMK to the was FMK then was achieved then achieved through throughthe use of the H2 useand of deprotection and subsequent decarboxylation to the FMK was then achieved through the use of H2 and Pd/C in ethanol. A final selective Boc deprotection step gave the desired protected arginine building H2Pd/Cand in Pd ethanol./C in ethanol. A final Aselective final selective Boc deprotection Boc deprotection step gave step the gavedesired the protected desired protectedarginine building arginine block (62) which could then be coupled to other amino acid units as required. Unfortunately, partial buildingblock (62 block) which (62) could which then could be thencoupled be coupled to other toamino other ac aminoid units acid as unitsrequired. as required. Unfortunately, Unfortunately, partial racemisation of the arginine residue was observed during the formation of 61. partialracemisation racemisation of the ofarginine the arginine residue residue was observed was observed during the during formation the formation of 61. of 61.

Scheme 11. Synthesis of peptidyl FMK building block 62 using magnesium benzyl fluoromalonate (56) [3]. SchemeScheme 11. 11.Synthesis Synthesis of of peptidyl peptidyl FMK FMK building building block block 62 using 62 using magnesium magnesium benzyl fluoromalonate benzyl fluoromalonate (56) [3]. (56)[3]. 2.1.9. Synthetic Approach Involving Magnesium Monobenzyl Fluoromalonate Enolate 65 2.1.9. Synthetic Approach Involving Magnesium Monobenzyl Fluoromalonate Enolate 65 2.1.9. SyntheticVan der Marel Approach et al. Involving[29] described Magnesium an approach Monobenzyl that made Fluoromalonate use of monobenzyl Enolate fluoromalonate65 Van der Marel et al. [29] described an approach that made use of monobenzyl fluoromalonate magnesium enolate 65 as opposed to the neutral magnesium salt (56). The procedure for accessing magnesiumVan der Marelenolate et 65 al. as [29 opposed] described to the an neutral approach magnesium that made salt use (56 of). The monobenzyl procedure fluoromalonate for accessing FMK 68 (Scheme 12) involves an adapted version of the patented methodology described by Palmer magnesiumFMK 68 (Scheme enolate 12)65 involvesas opposed an adapted to the neutralversion magnesiumof the patented salt methodology (56). The procedure described for by accessing Palmer et al. [30]. Initial side-chain esterification and subsequent debenzylation of 63 allowed isolation of FMKet al.68 [30].(Scheme Initial 12 side-chain) involves anesterifi adaptedcation version and subsequent of the patented debenzylation methodology of 63 described allowed isolation by Palmer of et acid 64. Further reaction with CDI followed by addition of enolate 65 and subsequent hydrogenation al.acid [30]. 64 Initial. Further side-chain reaction esterificationwith CDI followed and subsequent by addition debenzylationof enolate 65 and of subsequent63 allowed hydrogenation isolation of acid gave FMK 66 in a yield of 68%. This was then Boc-deprotected under acidic conditions and coupled 64gave. Further FMK reaction 66 in a yield with of CDI 68%. followed This was by then addition Boc-deprotected of enolate 65 underand subsequent acidic conditions hydrogenation and coupled gave to fragment 67 in order to generate the FMK (68). In this case, the Boc group in 68 was removed and FMKto fragment66 in a yield 67 in oforder 68%. to generate This was the then FMK Boc-deprotected (68). In this case, under the Boc acidic group conditions in 68 was andremoved coupled and to reacted with BODIPY TMR-OSu, which allowed for the preparation of a fluorescent probe for fragmentreacted 67within orderBODIPY to generateTMR-OSu, the which FMK allowed (68). In thisfor the case, preparation the Boc group of a influorescent68 was removed probe for and studying yeast peptide N-glycanase activity. reactedstudying with yeast BODIPY peptide TMR-OSu, N-glycanase which activity. allowed for the preparation of a fluorescent probe for studying yeast peptide N-glycanase activity.

Molecules 2020, 25, 5601 8 of 16 Molecules 2020, 25, x FOR PEER REVIEW 8 of 16

Molecules 2020, 25, x FOR PEER REVIEW 8 of 16

Scheme 12. Synthetic route to peptidyl FMK 68 using fluorinated fluorinated magnesium enolate 65 [[29].29]. Scheme 12. Synthetic route to peptidyl FMK 68 using fluorinated magnesium enolate 65 [29]. 2.2. Solid-Phase Route 2.2. Solid-Phase Route 2.2. Solid-Phase Route At present, only one solid-phase peptide synthesis (SPPS) method for accessing peptidyl At present, only one solid-phase peptide synthesis (SPPS) method for accessing peptidyl mono- mono-FMKsAt present, has only been one reported solid-phase in the peptide literature synthe [31sis]. (SPPS) The approach method for involves accessing initial peptidyl formation mono- of FMKs has been reported in the literature [31]. The approach involves initial formation of the FMK theFMKs FMK has moiety been through reported utilisation in the literature of a halogen-exchange [31]. The approach reaction involves in a initial similar formation manner toof thethe workFMK of moiety through utilisation of a halogen-exchange reaction in a similar manner to the work of Kolb et Kolbmoiety et al. through [18] described utilisation earlier of a halogen-exchange (Scheme2). This isreaction followed in a by similar attachment manner to to the the resin work and of Kolb peptide et al. [18] described earlier (Scheme 2). This is followed by attachment to the resin and peptide elongation.al. [18] described The first partearlier of the(Scheme process, 2). involvingThis is followed construction by attachment of the FMK to group the resin from theand amino peptide acid elongation.elongation. The The first first part part of of the the process,process, involvinginvolving construction ofof the the FMK FMK group group from from the the amino amino building block Fmoc-Asp(OtBu)-OH (69), is shown in Scheme 13. Firstly, the formation of diazoketone acidacid building building block block Fmoc-Asp(O Fmoc-Asp(OttBu)-OHBu)-OH (69), is shownshown inin SchemeScheme 13.13. Firstly, Firstly, the the formation formation of of derivative 70 is achieved via a two-step process involving diazomethane. 70 is converted to the diazoketonediazoketone derivative derivative 70 70 is is achieved achieved viavia aa two-steptwo-step processprocess involving involving diazomethane. diazomethane. 70 70 is isconverted converted bromomethyl ketone 71 with HBr and then subsequently transformed into the desired FMK (72) using toto the the bromomethyl bromomethyl ketone ketone 71 71 withwith HBrHBr andand then subsequently transformedtransformed into into the the desired desired FMK FMK TBAF as a fluoride source in the presence of p-toluenesulfonic acid (PTSA). Repeating these steps (72(72) using) using TBAF TBAF as as a afluoride fluoride source source in in thethe presencepresence of pp-toluenesulfonic-toluenesulfonic acid acid (PTSA). (PTSA). Repeating Repeating these these using Fmoc-Gly-OH to produce the corresponding FMK analogue resulted in purification difficulties. stepssteps using using Fmoc-Gly-OH Fmoc-Gly-OH toto prodproduceuce thethe corresponding FMKFMK analogueanalogue resulted resulted in in purification purification As a result, the diazoketone was instead converted to the FMK via the hydroxymethyl ketone instead difficulties.difficulties. As As a aresult, result, the the diazoketone diazoketone waswas instead convertedconverted toto the the FMK FMK via via the the hydroxymethyl hydroxymethyl ofketoneketone the bromomethyl instead instead of of the the ketone,bromomethyl bromomethyl as this ketone, ketone, proved as more this pr successfuloved moremore for successful successful this particular for for this this substrate particular particular (not substrate substrate shown in(not(not scheme). shown shown in in scheme). scheme).

SchemeScheme 13. 13.Generation Generation ofof buildingbuilding block 73 fromfrom Fmoc-Asp(O Fmoc-Asp(OtBu)-OHtBu)-OH (69 (69) [31].)[31 ]. Scheme 13. Generation of building block 73 from Fmoc-Asp(OtBu)-OH (69) [31]. InIn the the case case of of the the FMK FMK ( 72(72)) obtainedobtained fromfrom Fmoc-Asp(OttBu)-OH,Bu)-OH, because because of of the the acid acid side-chain, side-chain, attachmentattachmentIn the case to to the theof resinthe resin FMK could could (72occur occur) obtained inin aa straightforwardstraightfo from Fmoc-Asp(Orward manner, manner,tBu)-OH, allowing allowing because for for subsequent subsequentof the acid growth side-chain, growth of of theattachmentthe peptide peptide to chain chain the resin and and standard couldstandard occur acidolyticacidolytic in a straightfo cleavagecleavagerward from from manner,the the resin. resin. allowing For For use use infor in solid-phase solid-phasesubsequent synthesis, growth synthesis, of temporarythetemporary peptide protection chainprotection and of standardof the the FMK FMK acidolytic ketone ketone functionality functionality cleavage from was was the required. required. resin. For Thus,Thus, use FMKFMK in solid-phase 7272 was heated synthesis, for 5 h withtemporaryh with methanol methanol protection in the in the presence of presencethe FMK of PTSA ofketone PTSA to functionality giveto give dimethyl dimethyl was ketal ketal required.73 73with with Thus, concomitant concomitant FMK 72 removal wasremoval heated of of the thefortBu 5 side-chainh twithBu side-chain methanol protection. protection. in the presence of PTSA to give dimethyl ketal 73 with concomitant removal of the tBu side-chainAs not allprotection. amino acids possess a functional group in their side chain, an alternativeAs not approachall amino needed acids possessto be employed a carboxylic for am acidino acids functional such as group Gly. This in modifiedtheir side approach chain, an alternativeinvolved theapproach synthesis needed of a linker to be which employed could befor anchored amino acids to both such the as ketone Gly. Thisof the modified FMK using approach a 1,3- involved the synthesis of a linker which could be anchored to both the ketone of the FMK using a 1,3-

Molecules 2020, 25, 5601 9 of 16

As not all amino acids possess a carboxylic acid functional group in their side chain, an alternative approachMolecules 2020 needed, 25, x FOR to bePEER employed REVIEW for amino acids such as Gly. This modified approach involved9 of the 16 synthesis of a linker which could be anchored to both the ketone of the FMK using a 1,3-diol and to thediol resinand to via the a resin carboxy via group.a carboxy The group. linker The was linker made was in made such ain way such that a way it wasthat stableit was duringstable during SPPS, yetSPPS, capable yet capable of being of cleavedbeing cleaved under under acidic conditions.acidic conditions. Figure Figure2 shows 2 theshows FMK the moiety FMK moiety bound bound to the linkerto the linker (74). (74).

Figure 2. FMK moiety bound to linker for solid-phase peptide synthesis (SPPS) [[31].31].

3. Applications and Uses of Peptidyl Mono-FMKs in Medicinal Chemistry 3. Applications and Uses of Peptidyl Mono-FMKs in Medicinal Chemistry 3.1. Drug Discovery 3.1. Drug Discovery FMKs have shown potential for use in the development of drug therapeutics as well as chemical FMKs have shown potential for use in the development of drug therapeutics as well as chemical probes for interrogating cellular processes and the identification of protein targets [17,31]. In particular, probes for interrogating cellular processes and the identification of protein targets [17,31]. In particular, FMKs have proven to be effective covalent protease inhibitors for the treatment of a wide range of FMKs have proven to be effective covalent protease inhibitors for the treatment of a wide range of diseases. These include rheumatoid arthritis, where FMKs with the ability to reduce the severity of diseases. These include rheumatoid arthritis, where FMKs with the ability to reduce the severity of inflammation and inhibit the extent of bone and cartilage damage have been reported [1,32,33]. Peptidyl inflammation and inhibit the extent of bone and cartilage damage have been reported [1,32,33]. Peptidyl FMKs have also shown significant promise as therapeutic agents for combating neurodegenerative FMKs have also shown significant promise as therapeutic agents for combating neurodegenerative disorders such as Alzheimer’s disease, along with various other illnesses including cancer [5]. disorders such as Alzheimer’s disease, along with various other illnesses including cancer [5]. Proteolytic cleavage of peptides within living organisms by protease enzymes is a vital process for Proteolytic cleavage of peptides within living organisms by protease enzymes is a vital process for maintaining optimum bodily function, with protease enzymes playing a significant role in many maintaining optimum bodily function, with protease enzymes playing a significant role in many physiological processes including blood coagulation, digestion, and healing of wounds [34,35]. Despite physiological processes including blood coagulation, digestion, and healing of wounds [34,35]. Despite this, if cleavage occurs in a disorderly and uncontrollable manner, the onset of ill health can be this, if cleavage occurs in a disorderly and uncontrollable manner, the onset of ill health can be observed observed [36]. For this reason, the ability of certain inhibiting molecules to bind selectively to the [36]. For this reason, the ability of certain inhibiting molecules to bind selectively to the , proteases, preventing them from performing these disruptive cleavages, can prove hugely beneficial in preventing them from performing these disruptive cleavages, can prove hugely beneficial in the the treatment of the patient. In addition to this, protease inhibitors can also be used for combating treatment of the patient. In addition to this, protease inhibitors can also be used for combating viral viral particles [37], as these too rely on proteolysis to be able to function properly. Peptidyl FMKs are particles [37], as these too rely on proteolysis to be able to function properly. Peptidyl FMKs are capable capable of binding to these viral protease enzymes [38], blocking their activity and therefore preventing of binding to these viral protease enzymes [38], blocking their activity and therefore preventing the the maturation of infectious viral particles. This hinders replication, thus helping to combat disease. maturation of infectious viral particles. This hinders replication, thus helping to combat disease. 3.1.1. Peptidyl FMKs for the Treatment of Rheumatoid Arthritis 3.1.1. Peptidyl FMKs for the Treatment of Rheumatoid Arthritis The use of peptidyl FMKs for the treatment of rheumatoid arthritis has been studied and subsequentlyThe use reportedof peptidyl in the FMKs literature for the [1]. treatment FMKs have of been rheumatoid designed arthritis that are ablehas tobeen inhibit studied and B,subsequently a cysteineprotease, reported in thus the helping literature to [1]. reduce FMKs the have extent been of designed inflammation that are and able combat to inhibit cartilage cathepsin and boneB, a cysteine damage. protease, The extracellular thus helping matrix to reduce of cartilage the extent consists of ofinflammation proteoglycan and and combat collagen cartilage molecules and assembledbone damage. in such The a wayextracellular that a rigid matrix gel is of formed, cartilag enablinge consists joints of toproteoglycan function properly. and collagen The development molecules ofassembled arthritis hasin beensuch associateda way that with a rigid the release gel is of form cathepsinsed, enabling into inflamed joints tissues,to function causing properly. destruction The ofdevelopment the matrices of because arthritis of proteoglycanhas been associated and collagen with degradation.the release of This can have into detrimental inflamed eff tissues,ects on thecausing organism, destruction leading of tothe a matrices loss of optimal because joint of proteoglycan function. and collagen degradation. This can have detrimentalA selection effects of on peptidyl the organism, FMK inhibitors, leading to a each loss consistingof optimal ofjoint a Phe-Alafunction. moiety with variable N-terminalA selection blocking of peptidyl groups, wasFMK synthesised inhibitors, andeach found consisting to be eofff ectivea Phe-Ala irreversible moiety inhibitorswith variablein vitro N-. Theterminal treatment blocking of ratsgroups, with was a single synthesised oral dose and of found 25 mg to/kg be resulted effective in irreversible a 22–91% reductioninhibitors in vitro liver. andThe kidneytreatment cathepsin of rats with B levels a single over oral a period dose of 25 just mg/kg 4 h (Table resulted1). The in a change 22–91% in reduction inhibition in observed liver and askidney a result cathepsin of varying B levels the Nover-terminal a period blocking of just 4 group h (Table suggests 1). The that change it has in a inhibition significant observed effect on as the a interactionresult of varying of the inhibitorsthe N-terminal with cathepsin blocking B. group suggests that it has a significant effect on the interaction of the inhibitors with .

Molecules 2020, 25, 5601 10 of 16 MoleculesMolecules 2020 2020, 25, ,x 25 FOR, x FOR PEER PEER REVIEW REVIEW 10 of10 16 of 16 MoleculesMoleculesMolecules 2020 2020,, 25 25, ,x 25x FOR FOR, x FOR PEER PEER PEER REVIEW REVIEW REVIEW 10 of10 16 of10 16 of 16 MoleculesMoleculesMolecules 2020 2020 ,2020 25, ,x ,25 25FOR,, x x FOR FORPEER PEER PEER REVIEW REVIEW REVIEW 10 of1010 16 of of 16 16 TableTableTable 1. 1.1.% % % Inhibition1. Inhibition Inhibition% Inhibition of of cathepsin ofcathepsin cathepsin B B inB inB liverin in liver liver liver and and and and kidn kidney kidnkidney tissueey tissuetissue homogenates homogenateshomogenates treated treated treated with with 25with mg/kg 25 25mg/kg mg/kg mgof / kgof of of a TableTable 1. % 1. Inhibition % Inhibition of cathepsin of cathepsin B in B liver in liver and and kidn kidney tissueey tissue homogenates homogenates treated treated with with 25 mg/kg 25 mg/kg of of aTable selectionaTable selection 1. % 1.ofInhibition % peptidyl ofInhibition peptidyl of FMKs cathepsin of FMKs cathepsin [1]. [1]. B in Bliver in liver and andkidn kidney tissueey tissue homogenates homogenates treated treated with with 25 mg/kg 25 mg/kg of of selectiona selectiona selection of peptidylof of peptidyl peptidylof peptidyl FMKs FMKs FMKs FMKs [ 1[1].]. [1]. [1]. a selectionaa selectionselection of peptidyl ofof peptidylpeptidyl FMKs FMKsFMKs [1]. [1].[1].

% Inhibition% Inhibition % Inhibition% Inhibition % Inhibition% Inhibition % Inhibition% Inhibition % Inhibition% Inhibition % Inhibition% Inhibition % Inhibition% Inhibition % Inhibition% Inhibition %%% inInhibition Inhibition%Inhibition% Liver InhibitioninInhibition Liver % in %Inhibition% %%Kidney Inhibitionin Inhibition InhibitionInhibition Kidney % inInhibition%% %Liver InhibitioninInhibition InhibitionInhibition Liver % in Inhibition %%Kidneyin % InhibitionInhibition Kidney InhibitionInhibition R R in Liverin Liver in Kidneyin Kidney R R in Liverin Liver in Kidneyin Kidney R R HomogenateininHomogenatein Liver Liverin inLiver LiverLiver Homogenatein HomogenateinKidneyininin Kidney Kidney KidneyKidney R R HomogenateinHomogenate Liverinininin LiverLiver LiverLiver Homogenatein HomogenateKidneyinin in inKidneyKidney KidneyKidney RR RR HomogenateHomogenateHomogenateHomogenate R RR HomogenateHomogenateHomogenateHomogenate HomogenateHomogenatesHomogenateHomogenateHomogenates s HomogenateHomogenatesHomogenateHomogenateHomogenates s HomogenateHomogenateHomogenateHomogenatesHomogenates s HomogenateHomogenateHomogenateHomogenatessHomogenate s s s s s s s s s s s ss s ss s s ss s s ss s 70.570.5 39.039.0 81.881.8 75.475.4 70.570.570.5 39.0 39.039.0 81.881.8 81.8 75.475.4 75.4 70.570.5 70.5 39.039.0 39.0 81.881.8 81.8 75.475.4 75.4 70.5 39.0 81.8 75.4

86.986.9 57.357.3 22.222.2 29.829.8 86.986.9 57.357.3 22.222.2 29.829.8 86.986.986.9 86.9 57.3 57.357.3 57.3 22.222.2 22.222.2 29.829.8 29.8 29.8

86.9 57.3 22.2 29.8

28.328.3 24.224.2 91.291.2 78.878.8 28.328.3 24.224.2 91.291.2 78.878.8 28.328.328.3 28.3 24.2 24.224.2 24.2 91.291.2 91.291.2 78.878.8 78.8 78.8

28.3 24.2 91.2 78.8

3.1.2.3.1.2. Peptidyl Peptidyl FMKs FMKs for forthe theTreatment Treatment of Neurological of Neurological Disorders Disorders 3.1.2.3.1.2.3.1.2.3.1.2. Peptidyl Peptidyl PeptidylPeptidyl FMKsFMKs FMKsFMKs for forthefor the thetheTreatment Treatment TreatmentTreatment of of Neurological ofof Neurological NeurologicalNeurological Disorders Disorders Disorders 3.1.2.The PeptidylThe onset onset FMKsand and progression for progression the Treatment of certainof certain of neurologicalNeurological neurological diseases Disorders diseases such such as Alzheimer’s,as Alzheimer’s, stroke, stroke, and and TheTheTheThe onsetonset onsetonset and andand progression progression progressionprogression of certainofofof certain certain neurological neurologicalneurological neurological diseases diseasesdiseases diseases such suchsuch as Alzheimer’s,suchasas Alzheimer’s,Alzheimer’s, as Alzheimer’s, stroke, stroke,stroke, and andand stroke, epilepsyepilepsy has has been been associated associated with with the thpresencee presence of activated of activated calpain I in Ithe in thebody body [2]. [2].During During a stroke, a stroke, epilepsyepilepsyepilepsyThe hasonset has hasbeen beenbeenand associated 2+ associatedprogressionassociated 2+ with withwith th ofe thpresencethcertainee presencepresence neurologicalof activated ofof activatedactivated calpain diseases calpaincalpain I in IsuchIthe inin thebodythe as bodybody Alzheimer’s,[2]. [2].[2].During DuringDuring a stroke,stroke, aa stroke,stroke, and andthe epilepsy theintracellular intracellular has beenCa 2+Ca concentration associated2+ concentration with increases theincreases presence as asa resulta of result activated of variousof various calpain biochemical biochemical I in the events. body events. [ 2This]. DuringThis a the theintracellular intracellular Ca 2+Ca concentration2+2+ concentration increases increases as asa resulta result of ofvarious various biochemical biochemical events. events. This This epilepsythe thetheintracellular intracellularintracellular has been Ca associated CaCaconcentrationconcentrationconcentration2+ with th increasese presence increasesincreases as of as aasactivated resultaa resultresult of calpain ofvariousof variousvarious I inbiochemical the biochemicalbiochemical body [2]. events. During events.events. This a Thisstroke,This stroke,subsequentlysubsequently the intracellular leads leads to theto Ca theactivation activationconcentration of calpainof calpain I increases which I which is responsible asis responsible a result for of fordegradation various degradation biochemical of neuronalof neuronal events. thesubsequently subsequentlysubsequentlyintracellular leads leads leadsCa to2+ the to toconcentration thetheactivation activationactivation of calpain increasesofof calpaincalpain I which IasI whichwhich a isresult responsible isis responsibleresponsible of various for forfordegradation degradationbiochemicaldegradation of neuronal ofofevents. neuronalneuronal This Thisstructural subsequentlystructural proteins. proteins. leads For Forthis to thethis reason, activationreason, the thedevelopment of development calpain Iof which calpainof calpain is I responsible inhibitors I inhibitors isfor desirable is degradationdesirable in orderin order of to neuronal to structuralstructuralstructural proteins. proteins.proteins. For For Forthis this thisreason, reason,reason, the thethedevelopment developmentdevelopment of calpain ofof calpaincalpain I inhibitors II inhibitorsinhibitors is desirable isis desirabledesirable in order inin orderorder to toto subsequentlypreventprevent , neurodegeneration, leads to the activationthus thus combatting combatting of calpain neurol neurol I ogicalwhichogical disorders.is responsibledisorders. A selectionA forselection degradation of peptidylof peptidyl of FMK neuronal FMK structuralpreventpreventprevent neurodegeneration, proteins. neurodegeneration,neurodegeneration, For this thus reason, thusthus combatting combattingcombatting the development neurol neurolneurologicalogicalogical of disorders. calpain disorders.disorders. IA inhibitors selection AA selectionselection of is peptidyl desirableofof peptidylpeptidyl FMK in FMKFMK order to structuraldipeptidesdipeptides proteins.with with varying Forvarying this amine reason,amine protecting protectingthe development grou groups pswere ofwere calpainsynthesised synthesised I inhibitors and and found isfound desirable to beto bepotent in potentorder to preventdipeptidesdipeptidesdipeptides neurodegeneration, with withwith varying varyingvarying amine thusamineamine protecting combatting protectingprotecting grou neurologicalgrougroups pswereps werewere synthesised disorders.synthesisedsynthesised and Aandand selectionfound foundfound to oftobeto peptidylbepotentbe potentpotent FMK preventirreversibleirreversible neurodegeneration, inhibitors inhibitors of calpainof calpain thus I. Interestingly, combattingI. Interestingly, neurolchanging changingogical the disorders.theN-capping N-capping Agroup selection group could could of be peptidyl usedbe used to FMKto dipeptidesirreversibleirreversibleirreversible with inhibitors varyinginhibitorsinhibitors of amine calpainofof calpaincalpain protecting I. Interestingly, I.I. Interestingly,Interestingly, groups changing were changingchanging synthesised the thetheN-capping NN-capping-capping and foundgroup groupgroup could to becouldcould potentbe usedbebe usedused irreversible to toto dipeptidesenhanceenhance the thewithselectivity selectivity varying of the ofamine thecompound compound protecting in favourin favourgrou ofps inhibitingof wereinhibiting synthesised calpain calpain I over I andover cathepsin foundcathepsin Bto and Bbe and L, potent L, inhibitorsenhanceenhanceenhance the of calpainthetheselectivity selectivityselectivity I. Interestingly,of the ofof thethecompound compoundcompound changing in favour inin favourfavour the of N inhibiting ofof-capping inhibitinginhibiting calpain group calpaincalpain I couldover II overover cathepsin be cathepsincathepsin used B to and BB enhance andand L, L,L, the irreversiblealongalong with with altering inhibitors altering potency. potency.of calpain The The absence I. absenceInterestingly, of aof prot a protecting changingecting group group the was Nwas found-capping found to give togroup give poor poorcould inhibition, inhibition, be used to selectivityalongalongalong with of withwith altering the alteringaltering compound potency. potency.potency. in The favour TheThe absence absenceabsence of inhibiting of a ofof prot aa protprotecting calpainectingecting group Igroupgroup over was cathepsinwaswas found foundfound to B give toto and givegive poor L, poorpoor along inhibition, inhibition,inhibition, with altering whilstwhilst t-Boc t-Boc appeared appeared to giveto give the thebest best selectivity selectivity for forcalpain calpain I over I over cathepsin cathepsin B. Additionally,B. Additionally, a a enhancewhilstwhilstwhilst t-Boc the tt-Boc-Boc appearedselectivity appearedappeared to of givetoto the givegive thecompound thethebest bestbest selectivity selectivityselectivityin favour for forforcalpainof inhibitingcalpaincalpain I over II over overcalpain cathepsin cathepsincathepsin I over B. Additionally,B. B.cathepsin Additionally,Additionally, B aand aa L, potency.hydrophobichydrophobic The absence group group was of awas protectingpreferred preferred at group theat theP1 was positionP1 position found in to orderin give order for poor forpotent inhibition,potent inhibition inhibition whilst to occur.tto-Boc occur. appearedThe The to alonghydrophobichydrophobic with altering group group waspotency. was preferred preferred The absenceat theat theP 1of positionP 1a position prot ectingin orderin ordergroup for forpotent was potent found inhibition inhibition to give to occur.poorto occur. inhibition, The The hydrophobichydrophobichydrophobic group groupgroup was waswas preferred preferredpreferred at theatat thetheP1 position PP11 positionposition in orderinin orderorder for forforpotent potentpotent inhibition inhibitioninhibition to occur.toto occur.occur. The TheThe importanceimportance of Leu of Leu or Val or Val at P at2 was P2 was also also notable notable from from the theresults results reported. reported. The The most most potent potent compound compound give the best selectivity for calpain2 2 I over cathepsin B. Additionally, a hydrophobic group was preferred whilstimportanceimportance t-Boc of appeared Leu of Leu or Val or Val toat P giveat was P2 wasthe also alsobest notable notable selectivity from from the for theresults resultscalpain reported. reported. I over The cathepsinThe most most potent potent B. compoundAdditionally, compound a importanceimportanceimportance of Leu ofof LeuLeu or− 1Val oror−−1 Val1 Valat− 1P atat2 was PP2 waswas also alsoalso notable notablenotable from fromfrom the theresultsthe resultsresults reported. reported.reported. The TheThe most mostmost potent potentpotent compound compoundcompound testedtested (k = ( 276,000k = 276,000 M− 1M s−−11) s(76−1) )( 76along) along with with the theFMKs FMKs showing showing the thegreatest greatest calpain calpain I selectivity I selectivity (77 (and77 and attested thetestedP1 (kposition = ( 276,000k = 276,000 in M order M s −1) s(− for761) )( 76along potent) along with inhibition with the theFMKs FMKs to showing occur. showingThe the thegreatest importance greatest calpain calpain of I Leu selectivity I selectivity or Val ( at77 P(and772 wasand also hydrophobictestedtestedtested (k = ( (276,000kk == 276,000276,000group M− 1 M Mwass−1−)1 s(s 76−preferred1)) ) (( 76along76)) alongalong with at withwith the theFMKstheP1 FMKspositionFMKs showing showingshowing in theorder thegreatestthe greatestgreatestfor potentcalpain calpaincalpain inhibitionI selectivity II selectivityselectivity to (77 occur. (and(7777 andand The notable78) 78can) fromcan be seen be the seen in results Figure in Figure reported.3. In 3. addition In addition The to most exhibiti to exhibiti potentng goodng compound good activity activity in tested an in assay,an (assay,k = these276,000 these compounds compounds M 1 s were1)( were76 ) along importance78) 78can78)) can canbe seen bebe of seenseen Leuin Figure inin or FigureFigure Val 3. atIn 3.3. Paddition In2In was additionaddition also to exhibiti notable toto exhibitiexhibiting from goodngng goodgoodthe activity results activityactivity in reported. an inin assay, anan assay,assay, theseThe thesethese most compounds compoundscompounds potent− − compoundwere werewere foundfound to be to cellbe cell permeable permeable and and capable capable of inhibi of inhibitingting intracellular intracellular recombinant recombinant human human calpain calpain I. I. withtestedfound thefoundfound to( FMKsk =be toto 276,000 cell bebe showing cell cellpermeable permeableMpermeable−1 s the−1) and( greatest76 and)and capablealong capablecapable calpain with of inhibi ofof the Iinhibiinhibi selectivity FMKstingtingting intracellular showing intracellularintracellular (77 and the recombinant78 greatestrecombinantrecombinant) can be calpain seenhuman humanhuman in Icalpain Figure selectivity calpaincalpain3 I.. In I.I. ( addition 77 and to78 exhibiting) can be seen good in activityFigure 3. in In an addition assay, these to exhibiti compoundsng good were activity found in an to assay, be cell these permeable compounds and capable were offound inhibiting to be intracellularcell permeable recombinant and capable human of inhibi calpainting intracellular I. recombinant human calpain I.

FigureFigure 3. Peptidyl 3. Peptidyl FMKs FMKs capable capable of inhibiting of inhibiting calpain calpain I [2]. I [2]. FigureFigure 3. Peptidyl 3. Peptidyl FMKs FMKs capable capable of inhibiting of inhibiting calpain calpain I [2]. I [2]. FigureFigureFigure 3. Peptidyl 3.3. PeptidylPeptidyl FMKs FMKsFMKs capable capablecapable of inhibiting ofof inhibitinginhibiting calpain calpaincalpain I [2]. II [2].[2].

3.1.3.3.1.3. Peptidyl Peptidyl FMKs FMKs as Inhibitors as Inhibitors of MALT1 of MALT1 Paracaspase Paracaspase 3.1.3.3.1.3.3.1.3. Peptidyl PeptidylPeptidyl FMKs FMKsFMKs as Inhibitors asas InhibitorsInhibitors of MALT1 ofof MALT1MALT1 Paracaspase ParacaspaseParacaspase FigureFigure 3. 3.Peptidyl Peptidyl FMKsFMKs capable of inhibiting inhibiting calpain calpain I I[2]. [2].

3.1.3. Peptidyl FMKs as Inhibitors of MALT1 Paracaspase

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The3.1.3. paracaspase Peptidyl FMKs enzyme as Inhibitors MALT1 of plays MALT1 a Paracaspasekey role in immune response through the activation of lymphocytes and other immune cells [3]. Despite the importance of these processes for healthy bodily The paracaspase enzyme MALT1 plays a key role in immune response through the activation function,of if lymphocytes they occur and in other an immuneabnormal cells manner, [3]. Despite this the can importance lead to of the these development processes for healthy of lymphoid malignancies.bodily function,For this ifreason, they occur the in synthesis an abnormal of manner,inhibitors this that can leadtarget to the MALT1 development proteases of lymphoid has proved an attractivemalignancies. method for For the this treatment reason, the synthesisof these of tumours. inhibitors thatPeptidyl-FMKs target MALT1 proteases such as has 79 proved (Figure an 4) have shown potentialattractive methodfor this for purpose. the treatment The of P these1 Arg tumours. residue Peptidyl-FMKs was found suchto be as essential79 (Figure 4due) have to shownits interaction potential for this purpose. The P1 Arg residue was found to be essential due to its interaction with with the acidic residues in the target protein, whilst Leu at P4 helped by occupying a hydrophobic the acidic residues in the target protein, whilst Leu at P4 helped by occupying a hydrophobic pocket. pocket. Although potent potent inhibitors inhibitors have nowhave been now successfully been successfully developed fordeveloped targeting MALT1 for targeting enzymes MALT1 enzymesin in vivo, vivo the, the acquisition acquisition of orally of orally available available inhibitors inhibitors remains an remains ongoing challenge. an ongoing challenge.

Figure Figure4. Peptidyl 4. Peptidyl FMK FMK capable capable of of inhibiting inhibiting thethe paracaspase paracaspase enzyme enzyme MALT1 MALT1 [3]. [3]. 3.1.4. Dipeptidyl Glutaminyl FMKs as SARS-CoV Inhibitors 3.1.4. Dipeptidyl Glutaminyl FMKs as SARS-CoV Inhibitors In 2003, an outbreak of SARS (Severe Acute Respiratory Syndrome) occurred as a result of In 2003,the spread an outbreak of coronavirus of SARS pathogen (Severe SARS-CoV. Acute Respiratory The virus, which Syndrome) leads to respiratory occurred di asffi culties,a result of the was responsible for causing nearly 800 deaths; a value close to 10% of all confirmed cases in 2003. spread of coronavirus pathogen SARS-CoV. The virus, which leads to respiratory difficulties, was In 2006, S. X. Cai et al. synthesised a selection of dipeptidyl glutaminyl FMKs to be investigated responsibleas potential for causing SARS-CoV nearly inhibitors 800 deaths; [4]. The a value viral cysteine close to protease 10% of enzyme all confirmed Mpro [39 ]cases is essential in 2003. for In 2006, S. X. Caiviral et al. replication, synthesised and wasa selection thus identified of dipeptidyl as a possible glutaminyl drug target. FMKs Antiviral to be activity investigated was evaluated as potential SARS-CoVthrough inhibitors cytopathogenic [4]. The vi effralect cysteine (CPE) inhibition protease in enzyme SARS-CoV M infectedpro [39] is Vero essential and CaCo2 for viral cultures. replication, and wasCbz-Leu-Gln(NMe thus identified2)-FMK as a ( 80apossible) was found drug to beta therget. most Antiviral potent inhibitor, activity with was an ECevaluated50 value of through 2.5 µM in Vero cells (infected with strain 6109), low cellular toxicity, and a selectivity index of >40 cytopathogenic effect (CPE) inhibition in SARS-CoV infected Vero and CaCo2 cultures. Cbz-Leu- (Table2). FMKs 80b and 80c also showed some promising EC50 values (Table2), and 80c was also Gln(NMefound2)-FMK to exhibit (80a) low was toxicity found in mice,to be suggesting the most that potent inhibitors inhibitor, should possess with goodan EC safety50 value profiles of for2.5 µM in Vero cellsfurther (infected efficacy with studies strain to be 6109), performed low in cellular animals. toxicity, and a selectivity index of >40 (Table 2). FMKs 80b and 80c also showed some promising EC50 values (Table 2), and 80c was also found to exhibit low toxicity in mice, suggesting that inhibitors should possess good safety profiles for further efficacy studies to be performed in animals.

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Table 2. EC50, CC50, and Selectivity Index (SI) values for a selection of dipeptidyl glutaminyl FMKs in SARS-CoV-infected Vero and CaCo2 cultures [4].

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Molecules 2020, 25, x FOR PEER REVIEW 12 of 16 TableTable 2.2. ECEC5050,, CC CC50, ,and and Selectivity Selectivity Index Index (SI) (SI) values values for for a a selection selection of dipeptidyl glutaminyl FMKsFMKs inin SARS-CoV-infected VeroVero andand CaCo2CaCo2 cultures cultures [ 4[4].]. Table 2. EC50, CC50, and Selectivity Index (SI) values for a selection of dipeptidyl glutaminyl FMKs in SARS-CoV-infected Vero and CaCo2 cultures [4].

EC50 (µM) CC50 (µM) SI Vero CaCo2 Vero Entry Compound R FFM1 6109 FFM1

1 80a 3.6 ± 1.3 EC 2.550 (µM) µ± 0.4 2.4 ± 0.56CC50 (µM) µ > 100SI > 40 EC50 ( M) CC50 ( M) SI EC Vero50 (µM) CaCo2CC 50 (µM)Vero SI ECVero50 (µM) CaCo2CC50 (µM) VeroSI Entry Compound R FFM1 6109 FFM1 Vero CaCo2 Vero 2 Entry Compound80b R8.9 FFM1 ± 2.9 5.3 6109 ± 1.7 FFM1 8.8 ± 2.5 > 100 > 18 Entry Compound R FFM1 6109 FFM1 1 80a 3.6 ± 1.3 2.5 ± 0.4 2.4 ± 0.56 > 100 > 40 3 1 1 80c80a80a 3.63.6 ±6.2 1.3 1.3± 1.9 2.5 ± 0.4 2.5 6.6 2.4±0.4 3.0± 0.56 2.4 12.60.56 > ±100 4.1 > 10040 > 100 >40 > 15 1 80a 3.6 ± 1.3± 2.5 ± 0.4 ± 2.4 ± 0.56 ± > 100 > 40 2 80b 8.9 ± 2.9 5.3 ± 1.7 8.8 ± 2.5 > 100 > 18

2 2 80b80b 8.98.9 ± 2.9 2.9 5.3 ± 1.7 5.3 8.81.7 ± 2.5 8.8 >2.5 100 > 10018 >18 3 80c ±6.2 ± 1.9 6.6 ± 3.0 12.6 ± 4.1± > 100 > 15

3.2. Probes for Cellular3 3 Interrogation80c80c 6.26.2 ± 1.9 1.9 6.6 ± 3.0 6.6 12.63.0 ± 4.1 12.6 >4.1 100 > 10015 >15 ± ± ± In addition3.2. Probes to theirfor Cellular applications Interrogation in the field of drug discovery, peptidyl FMKs have also been widely 3.2.employed Probes3.2. Probes Infor addition Cellularas for chemical Cellular Interrogationto their Interrogation toolsapplications for the in theinterroga field of drugtion discovery, of biological peptidyl system FMKs haves [31]; also for been example, the elucidationIn widelyof addition theIn additionemployedstructure to their to as theirapplications andchemical applications binding tools in the for requirements in fieldthe the interroga of field drug of drugtiondiscovery, for of discovery, proteasebiological peptidyl peptidyl system receptors FMKss FMKs[31]; have forin havealso orderexample, been also to been the design drugs with greaterwidelywidelyelucidation selectivityemployed employed of as the chemicaland structure as chemicalpotency. tools and for toolsbi ndingUsefulthe forinterroga requirements the information interrogationtion of forbiological protease ofcan biological systemalso receptors bes systems[31]; gained in fororder example, [31 toin]; design forrelation example,the drugs to the active elucidationthewith elucidation greater of the structureselectivity of the and structure and bi ndingpotency. and requirements binding Useful information requirements for protease can for receptors also protease be gained in receptors order in torelation design in order to drugs the to design selectivityelucidation of of individual the structure proteasesand binding requirementswithin broader for protease protease receptors classes. in order This to design can bedrugs achieved through with greaterdrugssite selectivity with selectivity greater of individual and selectivity potency. proteases and Useful potency. within information Useful broader informationcan protease also be classes. gained can also Thisin be relation can gained be toachieved in the relation active through to the observingsite selectivitytheactiveobserving nature site of selectivitythe individual of nature the binding of proteases the individual binding interactions within proteasesinteractions broader within when whenprotease broader amino amino classes. acid protease acid Thisgroups cangroups classes. in be the achieved Thispeptide in the can through region bepeptide achieved of the region of the peptidyl-FMKobservingthroughpeptidyl-FMK chain the nature observing are chain of varied the the are binding nature varied until interactions ofuntil the the the binding binding when interactions requirementsaminorequirements acid when groups are amino fullyare in the fullysatisfied acid peptide groupssatisfied [24]. region Conjugation in the of[24]. the peptide Conjugation of of peptidyl-FMKdyes into chainthe FMK are variedstructure, until an the example binding of requirements which is shown are fullyin Figure satisfied 5 (81 [24].), has Conjugation also been explored of dyes intopeptidyl-FMK theregion FMK of chain thestructure, peptidyl-FMK are varied an until example chain the arebinding variedof which requirements until theis shown binding are fully requirementsin satisfied Figure [24]. 5 are ( Conjugation81 fully), has satisfied also of [been24]. explored dyes intoConjugationin order the FMKto allow of structure, dyes for intofluorescent an the example FMK imaging structure, of which of cellular an is exampleshown activity in of Figure whichsuch 5as is( 81apoptosis shown), has inalso [7]. Figure been 5 explored( 81), has also in orderin to order allowbeen to exploredallow for forfluorescentin fluorescent order to allow imagingimaging for fluorescentof cellularof cellular activity imaging activity such of cellular as apoptosissuch activity as [7]. such as apoptosis [7]. [7].

Figure 5. Structure of IR780-linker-Val-Ala-Glu(OMe)-FMK (81) [7]. Figure 5. Structure of IR780-linker-Val-Ala-Glu(OMe)-FMK (81) [7]. Ellis and coworkers describe another example in which peptidyl FMKs are useful for Ellisunderstanding and coworkers the structural describe binding another requirements example inand whic propertiesh peptidyl of a target FMKs enzyme are useful [8]. In thisfor case, understandinginhibitor 82the (Figure structural 6) was binding synthesised requirements and found and properties to be a potent of a target and selective enzyme irreversible[8]. In this case, inhibitor Figure 5.Figure Structure 5. Structure of IR780-linker of IR780-linker-Val-Ala-Glu(OMe)-FMK-Val-Ala-Glu(OMe)-FMK (81)[7]. (81) [7]. inhibitorof PKAC 82 (Figureα, a kinase 6) was enzyme. synthesised The structureand found of to the be inhibitor a potent consistsand selective of an irreversibleelectrophilic inhibitor fluoromethyl of PKACketoneα, a moiety kinase attachedenzyme. toThe a substrate-competitive structure of the inhibitor inhibitor consists scaffold. of an This electrophilic means that fluoromethyl if the FMK binds Ellisketone andin moiety a regioncoworkers attached in which to a describea substrate-competitive reactive cysteine another residue inexamplehibitor is present, scaffold. in the Thiswhic nucleophilic meansh peptidyl that cysteine if the FMK willFMKs binds attack are the useful for understandingin a regionelectrophilic the in which structural FMK, a reactive causing binding cysteine inhibition. requirements residue The isnature present, of and the the propertiespeptide nucleophilic scaffold ofcysteine aalso target plays will enzymeattacka key rolethe [8].as it In this case, electrophilicallows FMK,the peptide causing to inhibition. bind in the The vicinity nature ofof ththee peptidereactive scaffoldcysteine, also an playsessential a key requirement role as it for inhibitor 82 (Figure 6) was synthesised and found to be a potent and selective irreversible inhibitor allowssuccessful the peptide inhibition. to bind As in a the result, vicinity inhibitor of th 82e reactiveis capable cysteine, of covalently an essential binding requirement to and modifying for a of PKACsuccessfulα, cysteinea kinase inhibition. residue enzyme. Asat thea result, bindingThe inhibitorstructure site of 82the is ofenzyme. capable the inhibitor The of covalently incorporation consists binding of rhodamineof to anand electrophilic modifying B, a fluorescent a fluoromethyl ketone moietycysteine residue attached at the to binding a substrate-competitive site of the enzyme. The in incorporationhibitor scaffold. of rhodamine This B,means a fluorescent that if the FMK binds in a region in which a reactive cysteine residue is present, the nucleophilic cysteine will attack the electrophilic FMK, causing inhibition. The nature of the peptide scaffold also plays a key role as it allows the peptide to bind in the vicinity of the reactive cysteine, an essential requirement for successful inhibition. As a result, inhibitor 82 is capable of covalently binding to and modifying a cysteine residue at the of the enzyme. The incorporation of rhodamine B, a fluorescent

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Ellis and coworkers describe another example in which peptidyl FMKs are useful for understanding the structural binding requirements and properties of a target enzyme [8]. In this case, inhibitor 82 (Figure6) was synthesised and found to be a potent and selective irreversible inhibitor of PKAC α, a kinase enzyme. The structure of the inhibitor consists of an electrophilic fluoromethyl ketone moiety attached to a substrate-competitive inhibitor scaffold. This means that if the FMK binds in a region in which a reactive cysteine residue is present, the nucleophilic cysteine will attack the electrophilic FMK, causing inhibition. The nature of the peptide scaffold also plays a key role as it allows the peptide to bind in the vicinity of the reactive cysteine, an essential requirement for successful inhibition. As a result,Molecules inhibitor 2020, 25, x82 FORis PEER capable REVIEW of covalently binding to and modifying a cysteine residue at the binding13 of 16 site of the enzyme. The incorporation of rhodamine B, a fluorescent tag, was employed in order to allowtag, was the employed inhibitor-substrate in order to adduct allow tothe be inhibitor- observed,substrate thus helping adduct to to confirm be observed, that the thus mechanism helping ofto inhibitionconfirm that occurs the mechanism in an irreversible of inhibition manner. occurs In this in way, an irreversible the peptidyl manner. FMK developed In this way, was the found peptidyl to be aFMK useful developed tool for studyingwas found PKAC to beα aactivity. useful tool for studying PKACα activity.

Figure 6. Peptidyl FMK possessing rhodamine B dye for studying PKACα activity [[8].8]. 4. Conclusions 4. Conclusions As highlighted, peptidyl mono-FMKs have been reported to exhibit promising therapeutic activity As highlighted, peptidyl mono-FMKs have been reported to exhibit promising therapeutic against a range of diseases including Rheumatoid arthritis, neurological diseases such as Alzheimer’s, activity against a range of diseases including Rheumatoid arthritis, neurological diseases such as lymphoid malignancies, and the SARS-CoV viral pathogen. Furthermore, FMKs can also be used Alzheimer’s, lymphoid malignancies, and the SARS-CoV viral pathogen. Furthermore, FMKs can as chemical probes for studying a range of cellular processes. The multifunctional nature of these also be used as chemical probes for studying a range of cellular processes. The multifunctional nature biologically relevant FMK warheads, coupled with the fact that they are significantly more selective than of these biologically relevant FMK warheads, coupled with the fact that they are significantly more the analogous chloromethyl-ketone-based inhibitors, makes them attractive moieties with potential selective than the analogous chloromethyl-ketone-based inhibitors, makes them attractive moieties utility in a wide range of applications including in vivo. with potential utility in a wide range of applications including in vivo. Given their applications in these aforementioned areas, a range of synthetic approaches have Given their applications in these aforementioned areas, a range of synthetic approaches have been developed to access FMKs. The key synthetic solid- and solution-phase routes discussed in this been developed to access FMKs. The key synthetic solid- and solution-phase routes discussed in this review for accessing peptidyl mono-fluoromethyl ketones (FMKs) are summarised in Figure7. review for accessing peptidyl mono-fluoromethyl ketones (FMKs) are summarised in Figure 7. These These include halogen exchange methods (Figure7a [ 22,23], diazomethyl ketone fluorination include halogen exchange methods (Figure 7, a [22,23], diazomethyl ketone fluorination (Figure 7, b) (Figure7b) [ 24], a modified Dakin–West reaction (Figure7c) [ 9], epoxide ring opening with a [24], a modified Dakin–West reaction (Figure 7, c) [9], epoxide ring opening with a fluoride source fluoride source (Figure7d) [ 6,8], fluorination of silyl enol ethers (Figure7e) [ 2], utilisation of a (Figure 7, d) [6,8], fluorination of silyl enol ethers (Figure 7, e) [2], utilisation of a fluorinated fluorinated hemiacetal/aldehyde (Figure7f) [ 4,7,14,27], and the use of magnesium fluoromalonates hemiacetal/aldehyde (Figure 7, f) [4,7,14,27], and the use of magnesium fluoromalonates (Figure 7, g) (Figure7g) [ 3,23,28–30]. Additionally, the use of a linker has been employed where appropriate for [3,23,28–30]. Additionally, the use of a linker has been employed where appropriate for the synthesis the synthesis of peptidyl FMKs by solid-phase peptide synthesis (Figure7h) [ 31]. With many of these of peptidyl FMKs by solid-phase peptide synthesis (Figure 7, h) [31]. With many of these approaches approaches suffering from low overall yields, racemisation, and incompatibility with solid phase suffering from low overall yields, racemisation, and incompatibility with solid phase peptide peptide synthesis (SPPS) methodology, there is clearly still scope for the future development of new synthesis (SPPS) methodology, there is clearly still scope for the future development of new synthetic synthetic routes in this area. routes in this area.

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Figure 7. 7. SummarySummary of ofcurrent current solid- solid- and and solution-phase solution-phase synthetic synthetic routes routes for accessing for accessing peptidyl peptidyl mono- mono-fluoromethylfluoromethyl ketones ketones (FMKs). (FMKs).

Funding: SLC and CML would like to acknowledge PhD studentship support from Cambridge Research Funding: S.L.C. and C.M.L. would like to acknowledge PhD studentship support from Cambridge Research Biochemicals Ltd. and European Development Agency Funding (ERDF—Intensive Industrial Innovation Biochemicals Ltd. and European Development Agency Funding (ERDF—Intensive Industrial Innovation Programme—34R17P02148). ConflictsConflicts ofof Interest:Interest: TheThe authorsauthors declaredeclare nono conflictconflict ofof interest.interest.

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