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HighlightDirectedEvolutionofIridium-SubstitutedMyoglobinAffordsVersatileArtificialMetalloenzymesforEnantioselectiveC–CBond-Forming

ReactionsThomasR.Ward

DepartmentofChemistryUniversityofBasel,Spitalstrasse51CH-4051Basel,Switzerlandemail:thomas.ward@unibas.chAbstractThegroupofHartwigandcoworkersrecentlyreportedonaversatilemetal-substitution strategy applied to natural heme proteins that beautifullycomplements the more traditional approaches pursued for the creation ofartificialmetalloenzymes.Hereinwehighlight thenoveltyof thisapproachandsetitintothebroadcontextofartificialmetalloenzymes.In the past decade, artificialmetalloenzymes (ArMs) have attracted increasingattention as alternative to the more traditional homogeneous catalysts andenzymes.[1, 2] Such ArMs result from the incorporation of an abiotic metalcofactor within a host protein. Until recently, three complementary strategieshadsuccessfullybeenpursued for theassemblyofArMs: i)Covalentanchoringwhereby the ligand bound to the metal is covalently linked to the protein(Scheme 1a)[3] ii) Supramolecular anchoring which relies on an high affinityinteractionbetweenahostproteinanditsguest(orinhibitor)(Scheme1b)[4]andiii)Dativeanchoringwherebyaminoacidside-chainsbindandactivateametal(Scheme 1c).[5] The group of Hartwig and coworkers recently reported on aversatile metal-substitution strategy applied to natural heme proteins thatbeautifullycomplementsthesethreeapproaches.[6]

ArMs combine attractive features of homogeneous catalysts and enzymes. Thecofactor can contain any element of the periodic table, thus significantlyexpanding the accessible reaction repertoire of ArMs as compared to naturalbiocatalysts. Furthermore, both chemical- and genetic optimization schemeshave successfully been applied to improve the performance of ArMs.[1, 2]However, the coinage metals, so dear to the organometallic community, arefrequentlyinhibitedbyvariouscellularcomponents,includingglutathione.Asaconsequence,thehostproteinsmustbe(partially)purifiedpriortotheadditionof theabiotic cofactor, thus imposinga significantbottleneck towardsdirectedevolutionschemesthattypicallyrelyonscreeninglargelibrariesofmutants.

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Capitalizing on the planar structure of heme (iron-protoporphyrin IX Fe-PIXhereafter),Watanabe,LuandHayashihavesubstitutedthisversatilecofactorbyavarietyofplanarpolydentateanalogsorheme-derivativestoaffordArMsforavarietyoftransformations.Inthesestrategieshowever,mostoftheoptimizationeffortwasfocusedonchemicalmodificationofthecofactorratherthandirectedevolutionofthehostproteinScheme2a.[7]

Scheme 1 SelectedexamplesofdirectedevolutionofArMsbasedupon: covalent anchoring toazidophenylalanine engineered into Propyl OligoPeptidase (POP-ZA4) for enantioselectivecyclopropanationa);supramolecularanchoringbasedonthebiotin-streptavidintechnology for

N2

CO2Me

MeO

POP-ZA4–Rh2L CO2Me

MeO

N3

POP-ZA4

H

HM

N

POP-ZA4–Rh2L

NN

M

OOO

RhRh

OOOO

O

Me

Me

MeMe

Me Me

OO

O

H

H

Rh2L

POP-ZA4 : E104A-F146A-K199AD202A-S477Zer 69:31, 29 % yield

1 mol %

azidophenylalanine

Rh2L

a)

evolved POP-ZA4 :E104A-F146A-K199A-D202A-S477Z-L328H-G99F-G594Fer 96:4, 74 % yield

NH

OOPiv CO2Me

[RhCp*biotinCl2]2 (1 mol %)Sav Mutant (0.67 mol %)

MOPS Buffer or H2O / MeOH

NH

O

CO2Me

Cp*

Rh

HNBiotin

NOPiv

O

H OO

Sav2

Entry Sav Mutant er

1 - < 5 -

2 < 5 -

3 N118K-K121E 99 82:18

4 S112Y-K121E 95 91:9

WT

proposed transition-state

Conversion (%)

b)

Sav = streptavidin

Catalytic Zn Catalytic ZnStructural Zn H100 H89

E86D60

E57Y105

K104

N

S MeMe

CO2H

H

O

HN

O

NH2

Ph

ArtificialHydrolase

H2O

HN

S MeMe

CO2H

HNHO

PhNH2

OOH

[(kcat/Km)/kuncat] > 106

Artificial Hydrolase:Evolved Homotetrameric Cytochrome cb562 bearing structural- and catalytic Zn ions

c)

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enantioselective intermolecular C–H activation b) and dative anchoring of zinc in engineeredcytochromecb562forhydrolyticreactionsc).(Adaptedfromref.2withpermissionfromWiley)

Inspired by the isolobal analogy between the {Fe=O}- and the iron-carbene ornitrene fragments, the groups of Arnold and later of Fasan repurposedcytochrome P450s and myoglobin (Mb) to catalyse a variety of abiotictransformations: enantioselective cyclopropanation,[9, 10] nitrene insertion etc.Scheme2b,c.[2,8]InstarkcontrasttoArMs,suchrepurposedenzymesarefullycompatiblewith cellular components and can thusbe screenedandevolved invivoor incell lysates.Thiselegantstrategy is thusonly limitedby the intrinsicreactivity of the Fe-PIX cofactor, augmented by the fine-tuning achieved viageneticmeans.

Scheme2Repurposingheme-containingproteinsfornoveltransformations:C–Hhydroxylationrelyingonporphycene-reconstitutedmyoglobinMPc·Mba);cyclopropanationforthesynthesisofalevomilnacipranprecursorrelyingoncytochromeP450BM3bearinganaxialhistidineC400H

a)N N

N N

CO2H CO2H

MHO

MPc

MPc · Mb (20 μM)

H2O2 (10 mM)

Cl–

8 mM M = Mn(III) 13 TON 57:43 erM = Fe(III) 0 TON

Ph NEt2

O

N2

O

EtO

• Mutant Accessed After 3 Rounds of Engineering

T268A-C400H-T438WV78M-L181V (Hstar)

whole cellsCO2Et

PhO

NEt2

anaerobic

98% yield98:2 dr96:4 er

aerobic

90% yield98:2 dr99:1 er

+ PhO

NEt2

NH2

levomilnacipran

b)

n-Pr NHSO O

Me

n-PrNH

S

Et

O O

n-Pr

Me

SO2N3

P411BM3-CIS-T438S-I263F

P411BM3-F87A-T268A

97:3 rr99:1 er

30:70 rr99:1 er

P411BM3-CIS-T438S-I263F = V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, I263F, T268A, A290V, L353V, I366V, C400S, T438S, E442K.

c)

R

N2

CO2Et

H64V-V68A

- up to 46800 TON- up to 1000:1 dr- up to 1000:1 er

R

CO2Et

(Fe-PIX) · Mb

R = H, MeR' = H, OMe, Cl, CF3

R' R'

+

d)

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b) and regioselective nitrene insertion relying on cytochrome P411BM3bearing an axial serineC400Sc);cyclopropanationofstyrenederivativesbasedonmyoglobinmutantsd)(Adaptedfromref.2withpermissionfromWiley).To capitalize on the versatile chemistry of precious metal porphyrins, theHartwig group recently reported on a metal-substitution strategy applied toheme-proteins.[6]SubstitutionofironinprotoporphyrinIX(Fe-PIX)byavarietyofmetals(M-PIX)allowedthemtocreateandevolveartificialmetalloenzymesforenantioselectiveC–Cbond formingreactions.Reactionofadiazoalkanoatewith (M-PIX)affordsthe corresponding (RO2CR'=M-PIX). If the substrate possesses an ortho-alkylether moiety, an intramolecular C–H insertion occurs to afford thecorrespondingenantioenricheddihydrobenzofurans,Scheme3a.Additionofanalkene to (RO2CR'=M-PIX) leads to the formation of enantio- anddiastereoenrichedcyclopropanes,Scheme3b.Exploratory screens resulting from varying the metal moiety as well as thehemoprotein lead to the identification of {IrMe} and myoglobin as the mostpromising combination. Genetic optimization of the catalytic performancewasundertakennext.TheproximalHis93,originallybound toFe-PIXwasmutated,introducing either coordinating or non-coordinating amino acids. This lead tothe identification of His93Ala and His93Gly as the most promising mutants.Subsequent rounds of iterative mutagenesis were aimed at tailoring thesubstrate binding site above the porphyrin plane. For this purpose, the distalHis64 as well as positions Leu32, Phe33, Phe43, Val68, His97 and Ile99weremutatedwitha focused library includinghydrophobicandunchargedresidues.In total, nearly five hundred (MeIr-PIX) ·Mbmutantswere screened. SelectedresultsfortheC–HinsertionaswellasthecyclopropanationaresummarizedinScheme3aandbrespectively.

Scheme 3 Iron substitution in myoglobin (Mb) by platinum group metals affords versatilemetalloenzymes (MeIr-PIX) · Mb for C–C bond forming reactions a) and cyclopropanation ofchallengingsubstratesb).

a)

O

N2

OR'

OR

O

RO

OR'

(IrMe-PIX) · Mb

N N

N N

CO2H CO2H

Ir

Me

IrMe-PIX

Mb H93A-H64A-F43W-V68G : R= H; R' = Me; R"= OMeMb H93G-H64L-V68A-L32F-H97Y : R= H; R' = Me; R"= OMeMb H93G-H64L-F43L-I99F : R= Me; R' = Me; R" = H

R"R"

er84:1613:8792:8

TON726014492

b)

N2

O

OEt+0.5 mol % (IrMe-PIX) · Mb

H93A-H64V-F43Y-V68A-H97F

CO2Et

70:30 er; 33:1 dr; 20 TON

N2

O

OEt+0.5 mol % (IrMe-PIX) · Mb

H93A-H64V-V68F

91:9 er; 40:1 dr; 40 TON

CO2Et5

0.0025-0.5 mol %

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The contribution by Hartwig and coworkers presents several fascinatingfeatureswhicharehighlightedbelow.i)Inordertospeedupthescreeningprotocol,ultimatelyleadingtothedirectedevolution of (MeIr-PIX) · Mb, Escherichia coliwas grown under iron-depletedconditions at low temperatures. This allowed the straightforward isolation ofapo-Mbmutants,whichwere complementedby themetallatedM-PIX cofactor.Parallel purification was achieved by affinity chromatography on a Ni(NTA)column(NTA=Nitrilotriacetate).Thisstrategythusallowstocombinetherichchemistry of abiological PIX variants with the power of directed evolution invitro.ii) Thanks to the position of the "artificial" cofactor (MeIr-PIX) withinmyoglobin's natural active site, the abiotic substrate is completely embeddedwithinaproteincavity.Thiscontrastswithotheranchoringstrategies,wherebythecofactorisoftenmoreexposedtothesolvent,thusofferingsignificantlylesscontroloversecondcoordinationsphereinteractionsbetweenthesubstrateandthe host protein.[2-4] The use of focusedMb-libraries, bearingmutations in thevicinity of (MeIr-PIX) cofactor, allowed to tailor the active site to catalyse twoenantioselective reactions, for which no known natural enzyme has beenreportedtodate.iii)Startingfromanactivebutanessentiallynon-selective(MeIr-PIX) ·WT-Mb(WTiswildtype),bothenantiomersofthebenzodihydrofuranproductcouldbeaccessed by directed evolution. Importantly, up to > 7’000 turnovers could beachieved. This highlights the versatility of the site isolation of the reactive Ir-carbene moiety, preventing catalyst deactivation. Screening the focused Mblibraryallowedtoidentifypromising(MeIr-PIX)·Mbforavarietyofsubstrates:the ester, the arene and the alkoxy functionality could all be varied affordinggoodenantioselectivitiesinmostcases,Scheme3a.iv)Enantio-anddiastereoselectivecyclopropanationcouldbeachievedforbothinternal vinylarenes as well as unactivated alkenes. A key asset of theoptimization strategy relies on themodularity of the approach since the same(M-PIX)·Mbmutantlibrarycanbescreenedforavarietyofdifferentreactions,metalsandsubstrates,eventuallyleadingtotheidentificationofpromisingArMsforvariousreactions,Scheme3b.Although metal-substitution has been used in the past to generate artificialmetalloenzymes, their catalytic performance for new-to-nature reactions hasremainedlimited.ThiscontributionunambiguouslydemonstratestheversatilityofthisstrategytocreateandevolveArMsfornew-to-naturereactions.Inviewofthe vast number of heme-dependent proteins, this versatile approach offersfascinatingopportunitiestocomplementotherArMstrategies(Scheme1).Itmaybedesirabletoassembleandevolvetheartficialmetalloenzymesinvivoinorderto screen significantly larger mutant libraries or to implement ArMs in thecontextofsyntheticbiologyapplications.Thisinvivoapplicabilityisanessentialmilestone toward new-to-naturemetabolism and remains to be demonstratedforthenoblemetal-containingArMs.Literaturecited[1] A.Ilie,M.T.Reetz,Isr.J.Chem.2015,55,51.

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[2] T. K. Hyster, T. R. Ward, Angew. Chem. Int. Ed.2016, 55, 7344; Angew.Chem.2016,128,7468.

[3] P.Srivastava,H.Yang,K.Ellis-Guardiola, J.C.Lewis,Nat.Commun.2015,6,DOI:10.1038/ncomms8789.

[4] T.K.Hyster,L.Knörr,T.R.Ward,T.Rovis,Science2012,338,500.[5] W.J.Song,F.A.Tezcan,Science2014,346,1525.[6] H.M.Key,P.Dydio,D.S.Clark,J.F.Hartwig,Nat.2016,534,534.[7] K. Oohora, Y. Kihira, E.Mizohata, T. Inoue, T. Hayashi, J.Am.Chem.Soc.

2013,135,17282.[8] H.Renata,Z. J.Wang,F.H.Arnold,Angew.Chem.Int.Ed.2015,54,3351;

Angew.Chem.2015,127,3408.[9] P.S.Coelho,E.M.Brustad,A.Kannan,F.H.Arnold,Science2013,339,307.[10] M. Bordeaux, V. Tyagi, R. Fasan, Angew. Chem. Int. Ed.2015, 54, 1744;

Angew.Chem2015,127,1764.TOCCatch-phrase:Ugradingmyoglobinwithiridium!Ametal-substitutionstrategyaffordsarepurposedmyoglobinforchallengingcyclopropanationandintramolecularC–Hactivationreactions.Theperformanceoftheiridium-loadedmyoglobin(orangesphere)wasimprovedbydirectedevolutionofeightactivesiteresidues(yellowsurface).

O

N2

OR'

OR

R"

O

RO

OR'

R"Ir-substituted myoglobin+ directed evolution up to > 7'000 turnovers

up to 84 % ee