Construction of Synthetic Models for Nitrogenase-Relevant Nifb Biogenesis Intermediates and Iron-Carbide-Sulﬁde Clusters

catalysts

Perspective Construction of Synthetic Models for Nitrogenase-Relevant NifB Biogenesis Intermediates and Iron-Carbide-Sulﬁde Clusters

Chris Joseph , John Patrick Shupp , Caitlyn R. Cobb and Michael J. Rose *

Department of Chemistry, The University of Texas at Austin, Austin, TX 78712, USA; [email protected] (C.J.); [email protected] (J.P.S.); [email protected] (C.R.C.) * Correspondence: [email protected]

Received: 14 October 2020; Accepted: 10 November 2020; Published: 13 November 2020

Abstract: The family of nitrogenase enzymes catalyzes the reduction of atmospheric dinitrogen (N2) to ammonia under remarkably benign conditions of temperature, pressure, and pH. Therefore, the development of synthetic complexes or materials that can similarly perform this reaction is of critical interest. The primary obstacle for obtaining realistic synthetic models of the active site iron-sulfur-carbide cluster (e.g., FeMoco) is the incorporation of a truly inorganic carbide. This review summarizes the present state of knowledge regarding biological and chemical (synthetic) incorporation of carbide into iron-sulfur clusters. This includes the Nif cluster of proteins and associated biochemistry involved in the endogenous biogenesis of FeMoco. We focus on the chemical (synthetic) incorporation portion of our own efforts to incorporate and modify C1 units in iron/sulfur clusters. We also highlight recent contributions from other research groups in the area toward C1 and/or inorganic carbide insertion.

Keywords: nitrogenase; biomimetic; FeMoco; NifB-co; carbide; biogenesis

1. Introduction

1.1. Structural Determination of the Resting Nitrogenase Active Site The nitrogenases are a class of enzymes that catalyze ammonia synthesis from dinitrogen and are the only biological systems known to catalyze the N 2 NH conversion. In contrast to the H -consuming 2→ 3 2 Haber-Bosch process, dinitrogen fixation by nitrogenase (N2ase) is achieved via coupled proton/electron transfer directed toward the substrate [1]. Three variants of nitrogenase are known: Mo-dependent, V-dependent, and Fe-type. The Mo-dependent nitrogenase, however, exhibits the highest rate of catalytic activity and has been the focus of the majority of biocatalytic dinitrogen-reduction research [2]. The crystal structure of the molybdenum-containing nitrogenase was first reported in 1992 by Rees et al. to 2.9 Å resolution (Figure1). This structure revealed a homodimeric enzyme in which each monomer consists of two components: (1) nitrogenase (MoFe protein in Mo-dependent nitrogenase) in which the catalytic active site is contained, and (2) nitrogenase reductase (Fe protein), which is an electron-transfer protein [3,4]. The active site contained in the MoFe protein was initially described as a two-component active site comprised of two clusters: a 4Fe:3S cluster and a 1Mo:3Fe:3S cluster bridged by three non-protein ligands (designated as two sulfides and one unresolved site). This iron-molybdenum cofactor (FeMoco) is tethered to the protein by two ligands coordinated to the two distal metal sites: a cysteine residue (Cysα275) located at the distal iron site and a histidine residue (Hisα442) ligated to molybdenum. The molybdenum site is also coordinated by two oxygens from a bidentate homocitrate (HC) ligand. However, the six proximal Fe sites in this proposed structure appear to be supported by a non-planar, trigonal coordination sphere uncharacteristic for biological iron in a resting state enzyme.

Catalysts 2020, 10, 1317; doi:10.3390/catal10111317 www.mdpi.com/journal/catalysts Catalysts 2020, 10, x FOR PEER REVIEW 2 of 22

proposed structure appear to be supported by a non-planar, trigonal coordination sphere uncharacteristic for biological iron in a resting state enzyme. A decade following the 1992 report, a higher resolution structure (1.16 Å resolution) of FeMoco by Einsle, Rees et al., revealed the presence of a central, interstitial hexacoordinate light (2p) atom residing in the tetrahedral hole of the six proximal Fe sites [5,6]. Einsle and Rees initially postulated the site to be a nitride based on electron density integration studies. However, they suggested that carbon and oxygen could not be unambiguously discounted. In 2011, two independent research efforts concurrently ascertained the identity of the central atom to be a carbide rather than a nitride (Figure 1). Rees, Einsle et al. confirmed the carbide experimentally by utilizing two separate experiments: (1) improved structural resolution (1.0 Å) for electron density integration analysis and (2) comparative pulsed EPR-ESEEM (Electron Paramagnetic Resonance, Electron Spin Echo Envelope Modulation) on isotopically labeled (13C or 15N) MoFe protein (resting state S = 3/2) in which significant hyperfine coupling was observed in the 13C-labeled (I = ½) protein [7]. These monumental findings were printed onto a single page in the mid-November 2011 issue of Science. In the same issue, a publication of equal importance by DeBeer et al. utilized a combination of valence-to-core X-ray emission spectroscopy (V2C-XES) and computationally calculated spectra for cofactors bearing various interstitial atoms (C4−, N3−, and O2−) to determine the Catalystsidentity2020 of, 10the, 1317 interstitial atom [8]. The capability of V2C-XES to distinguish between light atoms2 of 22 within a multi-metallic cluster was demonstrated in a preceding study by DeBeer, Holland et al. comparing synthetic and computational model compounds bearing various interstitial atoms to A decade following the 1992 report, a higher resolution structure (1.16 Å resolution) of FeMoco by differentiate between Fe6 hexacoordinate C, N, and O sites [9]. The results of these studies identified p Einsle,the interstitial Rees et al.,atom revealed in FeMoco the presence as a purely of acentral, inorganic interstitial carbide. hexacoordinate Thus, the concurrent light (2 findings) atom residing by the inEinsle the tetrahedral and DeBeer hole research of the groups six proximal unambiguously Fe sites [5, 6esta]. Einsleblished and the Rees presence initially of an postulated interstitial the carbide site to be a nitride based on electron density integration studies. However, they suggested that carbon and in the N2ase cofactor. oxygen could not be unambiguously discounted.

FigureFigure 1.1. Progression of structure reﬁnementrefinement forfor thethe iron-molybdenumiron-molybdenum cofactorcofactor (FeMoco)(FeMoco) fromfrom structuralstructural X-rayX-ray didiffractionﬀraction (XRD)(XRD) datadata andand specializedspecialized ElectronElectron ParamagneticParamagnetic ResonanceResonance (EPR)(EPR) andand X-rayX-ray emissionemission (XES)(XES) datadata [[3–8].3–8].

1.2. BiologicalIn 2011, twoSynthesi independents of the FeMoco research Cluster eff orts concurrently ascertained the identity of the central atom to be a carbide rather than a nitride (Figure1). Rees, Einsle et al. confirmed the carbide experimentallyThe biogenesis by utilizing of nitrogenase two separate is accomplished experiments: by a (1)set improvedof enzymes structural coded in resolutionthe “nitrogen-fixing” (1.0 Å) for electron(Nif) genes. density NifS integration and NifU provide analysis the and initial (2) comparative conversion pulsedof cysteinyl EPR-ESEEM thiolates (Electron and iron Paramagneticincorporation Resonance,to produce Electron the two Spin inorganic Echo Envelope Fe4S4 iron-sulfur Modulation) clusters on isotopically to be transferred labeled (13 Conto or 15 NifBN) MoFe for proteincarbide insertion. The presence of a CXXXCXXC signature motif consistent with the13 presence of S- (resting state S = 3/2) in which significant hyperfine coupling was observed in the C-labeled (I = 1/2) proteinadenosylmethionine [7]. These monumental (SAM) led findings Ribbe were and printed Hu to onto postulate a single the page utilizat in theion mid-November of a SAM-dependent 2011 issue ofmechanismScience. In theby sameNifB issue, [10,11]. a publication Using isotope-labeling of equal importance experiments by DeBeer etin al.conjunction utilized a combination with CW of(Continuous-Wave) valence-to-core X-ray and emissionpulse EPR, spectroscopy the origin of (V2C-XES) the carbide and was computationally determined to be calculated a methyl spectra group from SAM during cofactor assembly by NifB [11–13].4 Following3 initial2 transfer from SAM onto a for cofactors bearing various interstitial atoms (C −,N −, and O −) to determine the identity of thesulfide interstitial in the (Fe atom4S4) [28 ].K-cluster The capability (Figure 2), of the V2C-XES CH3 group to distinguish undergoes between H-atom light abstraction atoms withinby an a multi-metallic cluster was demonstrated in a preceding study by DeBeer, Holland et al. comparing synthetic and computational model compounds bearing various interstitial atoms to differentiate between Fe6 hexacoordinate C, N, and O sites [9]. The results of these studies identified the interstitial atom in FeMoco as a purely inorganic carbide. Thus, the concurrent findings by the Einsle and DeBeer research groups unambiguously established the presence of an interstitial carbide in the N2ase cofactor.

1.2. Biological Synthesis of the FeMoco Cluster The biogenesis of nitrogenase is accomplished by a set of enzymes coded in the “nitrogen-fixing” (Nif) genes. NifS and NifU provide the initial conversion of cysteinyl thiolates and iron incorporation to produce the two inorganic Fe4S4 iron-sulfur clusters to be transferred onto NifB for carbide insertion. The presence of a CXXXCXXC signature motif consistent with the presence of S-adenosylmethionine (SAM) led Ribbe and Hu to postulate the utilization of a SAM-dependent mechanism by NifB [10,11]. Using isotope-labeling experiments in conjunction with CW (Continuous-Wave) and pulse EPR, the origin of the carbide was determined to be a methyl group from SAM during cofactor assembly by NifB [11–13]. Following initial transfer from SAM onto a sulfide in the (Fe4S4)2 K-cluster (Figure2), the CH3 group undergoes H-atom abstraction by an additional equivalent of SAM, which is followed by continued, iterative deprotonation/dehydrogenation to form the L-cluster. The utility of SAM as Catalysts 2020, 10, x FOR PEER REVIEW 3 of 22 Catalysts 2020, 10, 1317 3 of 22 additional equivalent of SAM, which is followed by continued, iterative deprotonation/dehydrogenation to form the L-cluster. The utility of SAM as a methyl transfer agent a methyl transfer agent toward sp2 and sp3 C sites has been previously reported, such as upon the toward sp2 and sp3 C sites has been previously reported, such as upon the tryptophanyl bicyclic ring tryptophanyl[14] or in thienamycin bicyclic ring biosynthesis [14] or in [15]. thienamycin Subsequent biosynthesis maturation by [15 the]. NifEN Subsequent assembly maturation protein leads by the NifENto substitution assembly proteinof a distal leads Fe site to substitution with Mo(HC) of abe distalfore final Fe site delivery with Mo(HC)into NifDK before to achieve final delivery the final into NifDKM-cluster to achieve [16–18]. the final M-cluster [16–18].

FigureFigure 2. 2. ProposedProposed biosynthetic mechanism mechanism for for carbide carbide formation, formation, insertion insertion of the of‘9th’ the sulfide, ‘9th’ sulﬁde, and andheterometal heterometal substitution substitution prior prior to maturation to maturation of the MoFe of the protein MoFe as protein proposed as proposed by Ribbe and by RibbeHu [11– and Hu13,16–18]. [11–13,16 Image–18]. Imageadapted adapted with permission with permission from fromJoseph, Joseph, C., Cobb, C., Cobb, C. R., C. Rose, R., Rose, M. M.J. Single-Step J. Single-Step InsertionInsertion of of Sulﬁdes Sulfides and Thiolate Thiolate into into Iron Iron Carb Carbide-Carbonylide-Carbonyl Clusters: Clusters: Unlocking Unlocking the Synthetic the Synthetic Door Doorto FeMoco to FeMoco Analogues. Analogues. Angew.Angew. Chemie ChemieInt. Ed. 2020 Int.. Ed.Accepted,2020 .notAccepted, yet published not yet. Copyright published Wiley-VCH. Copyright Wiley-VCHGmbH. Reproduced GmbH. Reproduced with permission. with permission.

InitialInitial studies studies withwith nitrogenase nitrogenase utilized utilized dithionite dithionite as the as reductant, the reductant, convoluting convoluting the origin the of originthe of“9th” the “9th” sulfide sulfide into the into cluster. the cluster. As such, As Ribbe such, et Ribbe al. surveyed et al. surveyed a suite of a biologically suite of biologically relevant sulfur- relevant containing sources—sulfides (S2−), sulfites2 (SO32−), sulfates2 (SO42−)—and2 substituted EuII-EGTAII as a sulfur-containing sources—sulfides (S −), sulfites (SO3 −), sulfates (SO4 −)—and substituted Eu -EGTA asreductant a reductant in intheir their biogenesis biogenesis studies studies to toestablish establish the the role role of ofan anexternal external sulfur sulfur source source for for M-cluster M-cluster maturation [18]. It was demonstrated that only2 SO32− led to the formation of the L cluster. maturation [18]. It was demonstrated that only SO3 − led to the formation of the L cluster. Additionally, Additionally, when35 radiolabeled2 35SO32− was utilized, the radiolabel could be detected downstream when radiolabeled SO3 − was utilized, the radiolabel could be detected downstream in the extracted in the extracted L-cluster. In biosynthetic experiments performed in the absence of SAM, no L-cluster. In biosynthetic experiments performed in the absence of SAM, no significant retention of significant retention of the radiolabel was detected, suggesting that sulfide transfer likely occurs after the radiolabel was detected, suggesting that sulfide transfer likely occurs after incubation with SAM. 2− incubation with SAM. Finally, incubation conditions in which SAM2 is present while SO3 is absent Finally, incubation conditions in which SAM is present while SO3 − is absent leads to the formation of leads to the formation of an undefined L*-cluster (tentatively assigned as an Fe8S8C cluster). an undefined L*-cluster (tentatively assigned as an Fe8S8C cluster). 1.3. Multimetallic Co-Operativity toward Dinitrogen Activation 1.3. Multimetallic Co-Operativity toward Dinitrogen Activation Initial insight into the nitrogenase mechanism was primarily founded upon extensive kinetic Initial insight into the nitrogenase mechanism was primarily founded upon extensive kinetic studies conducted throughout the 1970s to 1980s. Lowe and Thorneley famously proposed a studies conducted throughout the 1970s to 1980s. Lowe and Thorneley famously proposed rudimentary kinetic scheme whereby the resting-state N2ase cofactor (designated E0) undergoes four a rudimentary kinetic scheme whereby the resting-state N ase cofactor (designated E ) undergoes four iterative protonation/reduction events (E1–E4), at which point,2 the stage is set for substrate0 binding. iterativeConcurrent protonation with the/reduction liberation eventsof one equiv (E1–E H4),2, atN2 which binds and point, proceeds the stage to be is setreduced for substrate to two NH binding.3 as Concurrentthe cofactor with continues the liberation to undergo of one subsequent equiv H2 ,Nprot2 onationbinds and and proceeds reduction. to Liberation be reduced of to the two second NH3 as theequivalent cofactor continuesNH3 returns to the undergo cofactor subsequent to its resting protonation state, E0 [1]. and The reduction. precise geometric Liberation and of electronic the second equivalentstructural NH details3 returns of the theE1–E cofactor4 intermediates to its resting are still state, mostly E0 unknown.[1]. The precise However, geometric recent spectroscopic and electronic structuraland computational details of the information E1–E4 intermediates about the E are1 state still has mostly begun unknown. to emerge, However, suggesting recent that spectroscopicthe E1 step andinvolves computational reduction information of an Fe site in about conjunction the E1 state with hasprotonation begun to of emerge, a belt-sulfide suggesting [19,20]. that the E1 step involves reduction of an Fe site in conjunction with protonation of a belt-sulfide [19,20]. The location and mode of substrate-binding was a topic of considerable debate throughout the last two decades. The differences in N2-reducing activity between the M-cluster, V-cluster, and L-cluster Catalysts 2020, 10, 1317 4 of 22 nominated the heterometal (i.e., Mo in FeMoco) as a likely site for substrate binding. This conclusion gained additional support with the publication in 2003 of Schrock’s synthetic, Mo-centered complex as the first report of a synthetic and functional N2-reducing catalyst with an N2ase-relevant metal center [21]. In 2013, however, Peters published a synthetic Fe complex bearing a 2p donor atom (B) in its ligation sphere and capable of catalytic N2 conversion [22]. Experimental and computational evidence with FeMoco and FeVco began to implicate iron as the likely N2-binding site. Inhibition of FeMoco with carbon monoxide (CO) and crystallization of the resulting MoFe protein revealed a structure in which the S2B belt sulfide—which bridges Fe2 and Fe6—is substituted with a bridging CO [23]. Additionally, treatment of FeMoco with KSeCN demonstrated similar lability of the belt sulfides: 1 equiv KSeCN under argon atmosphere resulted in substitution of S2B with Se [24]. Subsequent catalytic turnover with N2 produced structures in which the Se migrated to the 5A or 3A positions. Crystallization and X-ray diffraction (XRD) of vanadium-dependent N2ase during turnover presented a FeVco structure in which the S2B position was occupied by a small bridging ligand (presumably N or NHx, even though O or OHx cannot be discounted) [25]. Finally, the computational work surrounding the Mo site suggests that one of the oxygen donors dissociates from Mo during the E2–E4 steps, leaving a possible open coordination site. However, energy calculations suggest that activation of N2 at this site results in a significantly higher increase in energy and that N2 binding at Fe2 and Fe6 is much more thermodynamically favorable (though still endothermic) [26]. Furthermore, in early 2020, a ground-breaking crystallographic structure by Ribbe and Hu under physiological turnover conditions depicted a substrate-bound nitrogenase intermediate, which revealed details regarding the putative substrate binding sites as well as binding modes during turnover [27]. While the exact identities of the substrates remain unconfirmed (N2 vs. N2H2 vs. N2H4), the structural data demonstrates displacement of the belt sulfides to allow for substrate binding at the medial Fe sites. The two FeMoco clusters in the structure exhibit distinct substrate-binding modes, such that the substrate presents in a ‘side-on’ binding mode in the chain A cluster or an ‘end-on’ mode in the chain C cluster. In either case, the dinitrogen substrate adopts a bridged binding mode between two Fe sites, highlighting the significant role of multi-iron co-operativity toward the mechanism of dinitrogen activation and reduction by nitrogenase.

1.4. Synthetic Model Compounds in FeMoco Biomimetics Throughout the past two decades of nitrogenase research, studies utilizing synthetic model compounds have contributed greatly to the early mechanistic and electronic discussions surrounding nitrogenase in lieu of highly sophisticated spectroscopic and computational techniques, which have only recently become available for studying the complicated active site. The Schrock catalyst, a Mo complex featuring a sterically protected substrate-binding site (see Figure3) initially invigorated the debate to determine the binding site of FeMoco [21]. Nishibayashi has reported pincer-based monomeric and dimeric Mo complexes, which catalytically reduce N2 [28,29]. A pincer motif was later used by the same group to develop a catalytically active Fe-centered complex as well [30]. The Peters research group has published a series of Fe complexes featuring a tripodal ligand set with various axial donor atoms. The Si-ligated complex was ﬁrst shown to bind N2, followed by reports of B-ligated and C-ligated complexes that could catalytically perform the reduction [22,31,32]. Spectroscopic and mechanistic studies with these compounds have demonstrated the axial donor atom modulates covalency with the Fe center throughout catalysis to support the various oxidation states and substrate coordination modes. Results from these studies were used to hypothesize a stabilizing role for the central carbide in FeMoco, performing similar modulations throughout turnover. The proposed hybrid distal-alternating mechanism determined for Peters’ compounds was similarly extended to FeMoco as a possible mechanistic pathway [33]. Moreover, the tripodal donor set was later adapted to include a structurally relevant S donor as a bridging thiolate [34]. Synthetic work pursued by the Holland group has demonstrated activation of dinitrogen by multi-metallic cooperation between two or four iron sites, presenting biomimetic complexes for the di-iron activation of N2 displayed in the Ribbe and Catalysts 2020, 10, 1317 5 of 22 Catalysts 2020, 10, x FOR PEER REVIEW 5 of 22

Hu crystal structure [27]. Additional synthetic work with an iron-carbonyl-thiolate complex utilizes activation of N2 displayed in the Ribbe and Hu crystal structure [27]. Additional synthetic work with a tridentate ligand set and is capable of N activation [35]. an iron-carbonyl-thiolate complex utilizes2 a tridentate ligand set and is capable of N2 activation [35].

Figure 3.3. SelectedSelected functional functional synthetic synthetic compounds compounds for Nfor2 activation N2 activation and/or and/or reduction reduction from the from Schrock, the Schrock,Nishibayashi, Nishibayashi, Peters, and Peters, Holland and researchHolland groupsresearch [21 groups,22,28– [21,22,28–35].35].

Functional syntheticsynthetic model model compounds compounds capable capable of catalytically of catalytica reducinglly reducing or activating or dinitrogenactivating dinitrogenin various modesin various were modes particularly were impactfulparticularly toward impactful early toward mechanistic early discussionsmechanistic surroundingdiscussions surroundingnitrogenase. nitrogenase. In recent years, In recent structural years, models structur (Figureal models4) have (Figure also 4) been have extensively also been utilizedextensively as utilizedsynthetic as referencessynthetic references in studies in probing studies forprobin geometricg for geometric and electronic and electronic structure structure information information about 2 aboutFeMoco FeMoco and FeVco. and TheFeVco. carbonyl-supported The carbonyl-supported hexa-iron hexa-iron cluster [Fe cluster6(µ6-C)(CO) [Fe6(16µ6]-C)(CO)−—which16]2−—which contains containsan interstitial, an interstitial, 6-coordinate 6-coordinate carbide and carbide has beenand has featured been featured prominently prominently throughout throughout our own our research own program—wasresearch program—was utilized along utilized with along its nitride with congenerits nitride [Fe congener6(µ6-N)(CO) [Fe6(16µ]6−-N)(CO)as a means16]− as of a calibrating means of X-raycalibrating emission X-ray spectra emission to spectra distinguish to distinguish between 2betweenp atoms 2 inp atoms multi-metallic in multi-metallic frameworks, frameworks, thereby, thereby,setting the setting stage the for stage identiﬁcation for identification of the interstitial of the interstitial carbide in carbide FeMoco in [FeMoco9]. [9]. In manymany respects, respects, FeMoco FeMoco is structurallyis structurally similar simila to otherr to other synthetic synthetic and biological and biological iron sulfur iron clusters. sulfur Theclusters. average The Fe–Feaverage distance Fe–Fe distance of 2.63 of0.04 2.63 Å ± places 0.04 Å individual places individual iron sites iron within sites thewithin range the to range induce to ± inducea bonding a bonding interaction interaction between betw metaleen centers. metal Similarly,centers. Similarly, the average the Fe–Moaverage distance Fe–Mo ofdistance 2.684 of0.008 2.684 Å ± ±is 0.008 typical Å foris typical reported for synthetic reported heterometal-iron-sulfursynthetic heterometal-iron-sulfur clusters [36 clusters]. Fe–S contacts [36]. Fe–S exhibit contacts an average exhibit andistance average of distance 2.25 0.03 of Å.2.25 Finally, ± 0.03 Å. the Finally, Fe–C bond the Fe–C is not bond a known is not motif a known in iron-sulfur motif in iron-sulfur clusters outside clusters of ± outsidethe nitrogenases. of the nitrogenases. Moreover, synthetic Moreover, iron-sulfur syntheti clustersc iron-sulfur found in clusters the Cambridge found Structuralin the Cambridge Database (CSD)Structural do featureDatabase Fe-coordinated (CSD) do feature N and Fe-coordinat O sites, buted examplesN and O sites, with but carbon examples have remainedwith carbon elusive. have remainedNonetheless, elusive. the average Nonetheless, Fe–C distancethe average of 2.00 Fe–C0.02 distance Å observed of 2.00 in± 0.02 FeMoco Å observed fall within in theFeMoco range fall of ± withindistances the displayed range of indistances compounds displayed with N in (Fe–N compounds range: 1.871–1.977with N (Fe–N Å) orrange: O (Fe–O 1.871–1.977 range: 2.052–2.190 Å) or O (Fe– Å) Oincorporated range: 2.052–2.190 donor atoms. Å) incorporated Synthetic iron donor sulfur atoms. clusters Synthetic developed iron bysulfur the Holmclusters group developed and featuring by the Holmheterometallic group and Mo featuring3+ and V3 +heterometallicsites assisted in Mo the3+ assignmentand V3+ sites of assisted spin states in atthe individual assignment metal of spin sites states in the atresting-state individual M-clusters metal sites and in the V-clusters resting-state [37–39 M-cl]. Notableusters workand V-clusters by the Agapie [37–39]. group Notable has demonstrated work by the Agapie group has demonstrated electronic and catalytic modulation of iron cluster sites performed by substitution of various interstitial light atoms (O, F) [40,41]. Synthetic structural modelling work

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Catalystselectronic 2020 and, 10, catalyticx FOR PEER modulation REVIEW of iron cluster sites performed by substitution of various interstitial6 of 22 light atoms (O, F) [40,41]. Synthetic structural modelling work from the Tatsumi group has presented froma highly-relevant the Tatsumi dicuboidal,group has presen vanadium-containingted a highly-relevant FeS cluster dicuboidal, and an vanadium-containing astonishingly remarkable FeS cluster andfeaturing an astonishingly belt-thiolates remarkable and an interstitial cluster oxide featuring [42,43 ].belt-thiolates Remarkably, and Rauchfuss an interstitial has recently oxide published [42,43]. Remarkably,the ﬁrst known Rauchfuss multi-iron has cluster recently to featurepublished an interstitialthe first known carbide multi-iron and one orcluster multiple to feature inorganic an interstitialsulﬁdes [44 carbide,45]. and one or multiple inorganic sulfides [44,45].

Figure 4.4. SelectedSelected synthetic synthetic structural structural models models for the for nitrogenase the nitrogenase active site,active including site, including works published works publishedby the Holm, by Agapie,the Holm, Tatsumi, Agapie, and Tatsumi, Rauchfuss and [Rauchfuss37,38,40–49 [37,38,40–49].].

Synthetic development of mono-metallic and bimetallic iron complexes with C-donors is of potential importanceimportance for for understanding understanding the rolethe ofrole the of Fe-C the bond Fe-C in controllingbond in controlling the electronic the interactions electronic interactionsbetween the between metal sites the in metal FeMoco. sites in These FeMoco. complexes These complexes can be isolated can be with isolated low-coordinate, with low-coordinate, high-spin high-spinferrous, and ferrous, ferric and iron ferric centers iron such centers as the such complexes as the complexes reported reported by Power, by Fürstner, Power, Fürstner, and Re [and50–52 Re]. Additionally,[50–52]. Additionally, Yogendra Yogendra et al. recently et al. reportedrecently areported set of dimeric, a set of yldiide-bridged dimeric, yldiide-bridged iron complexes iron complexesaccessed from accessed the known from the monomers. known monomers. Magnetic Magnet susceptibilityic susceptibility and spectroscopic and spectroscopic studies studies of these of thesedimers dimers show show intense intense antiferromagnetic antiferromagnetic coupling coup betweenling between iron sites iron and sites pronounced and pronounced negative negative charge chargelocalization localization on the Con ligand the C [53 ligand]. The [53]. Holland The groupHolland reported group areported series of βa -diketiminateseries of β-diketiminate complexes withcomplexes various with bridging various C-donors. bridging C-donors. Among these,Among an th alkylidene-bridgedese, an alkylidene-bridged and sulﬁde-bridged and sulfide-bridged dimer + wasdimer shown was shown to be competent to be competent toward toward N2 to NH N24 toconversion NH4+ conversion with dissociation with dissociation of the sulﬁdeof the sulfide ligand. ligand. The presence of the carbon donor ligand ostensibly facilitates improvement of the N2 reduction activity of the complex along with sulfide lability [54]. An alkyl-ligated Fe4S4 cluster published by Suess et al. demonstrates the dramatic electronic shift enforced upon the cubane cluster by the coordination of the electron-donating R group [48]. Notably, a variety of new tripodal chelating ligands were employed to enforce 3:1 site differentiation and

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The presence of the carbon donor ligand ostensibly facilitates improvement of the N2 reduction activity of the complex along with sulfide lability [54]. An alkyl-ligated Fe4S4 cluster published by Suess et al. demonstrates the dramatic electronic shift enforced upon the cubane cluster by the coordination of the electron-donating R group [48]. CatalystsCatalysts 20202020,, 1010,, xx FORFOR PEERPEER REVIEWREVIEW 77 ofof 2222 Notably, a variety of new tripodal chelating ligands were employed to enforce 3:1 site differentiation andstabilizestabilize stabilize aa rangerange a range ofof alkylatedalkylated of alkylated clustercluster cluster oxidationoxidation oxidation statesstates states (Figure(Figure (Figure 5)5) 5[48,49].[48,49].)[ 48,49 TheThe]. The successsuccess success andand and versatilityversatility versatility ofof oftheirtheir their approachapproach approach representrepresent represent newnew new progressprogress progress inin thethe in the rationalrational rational controlcontrol control ofof multi-metallicmulti-metallic of multi-metallic clustercluster cluster chemistry,chemistry, chemistry, forfor forwhichwhich which thethe the ligand-controlligand-control ligand-control toolboxtoolbox toolbox hashas has historicallyhistorically historically beenbeen been limitedlimited limited comparedcompared compared toto to monometallic monometallic andand bimetallicbimetallic inorganicinorganic syntheticsynthetic work.work. As As such, such, th thesetheseese findings findingsfindings openopen newnew doorsdoors towardtoward modelingmodeling andand understanding Fe4S4-alkyl intermediates of enzyme active site clusters, especially the radical SAM understanding Fe4SS44-alkyl-alkyl intermediates intermediates of of enzyme enzyme active active si sitete clusters, especially the radical SAMSAM familyfamily [[55].[55].55].

Figure 5. Fe4S4-alkyl clusters stabilized by chelating tripodal ligand scaffolds recently reported by Figure 5.5. Fe4SS44-alkyl-alkyl clusters clusters stabilized stabilized by by chelating chelating trip tripodalodal ligand ligand scaffolds scaﬀolds recently reported by SuessSuess etet al.al.

1.5. A Two-Fold Strategy toward Achieving FeMoco Construction 1.5.1.5. AA Two-FoldTwo-Fold StrategyStrategy TowardToward AchievingAchieving FeMocoFeMoco ConstructionConstruction Undoubtedly,Undoubtedly, the thethe field fieldfield of ofof structural structurstructuralal modelling modellingmodelling in inin the thethe context contextcontext of ofof nitrogenase nitrogenasenitrogenase has hashas been beenbeen invaluable invaluableinvaluable in advancinginin advancingadvancing the structural,thethe structural,structural, electronic, electronic,electronic, and mechanistic andand mechanismechanis understandingtictic understandingunderstanding of the nitrogenase ofof thethe nitrogenasenitrogenase active site. activeactive However, site.site. muchHowever,However, synthetic muchmuch work syntheticsynthetic remains workwork unaccomplished. remainsremains unaccomplished.unaccomplished. As of this writing, AsAs of theof thisthis synthetic writing,writing, ‘Holy thethe Grail’ syntheticsynthetic for FeMoco ‘Holy‘Holy modelingGrail’Grail’ forfor of FeMocoFeMoco achieving modelingmodeling a paramagnetic ofof achievingachieving iron cluster aa paramagneticparamagnetic with sulfides /ironironthiolates, clustercluster and withwith interstitial sulfides/thiolates,sulfides/thiolates, carbide continues andand tointerstitialinterstitial be pursued. carbidecarbideAdditionally, continuescontinues synthetic toto bebe pursued.pursued. modelling AddiAddi worktionally,tionally, applied syntheticsynthetic to the FeMoco modellingmodelling biogenesis—such workwork appliedapplied to asto thethe intermediateFeMocoFeMoco biogenesis—suchbiogenesis—such steps involved asas in thethe carbide intermediateintermediate formation—are stepssteps involvedinvolved scarce inin if notcarbidecarbide absent formation—areformation—are completely. Through scarcescarce ifif not thenot workabsentabsent summarized completely.completely. ThroughThrough in this perspective, thethe workwork summarizedsummarized we sought inin to thth expandisis perspective,perspective, upon the wewe compendium soughtsought toto expandexpand of structural uponupon thethe modelscompendiumcompendium in progression ofof structuralstructural toward modelsmodels incorporation inin progressionprogression of important totowardward structural incorporationincorporation and electronic ofof importantimportant attributes structuralstructural of FeMoco andand (Figureelectronicelectronic6) into attributesattributes synthetic ofof FeMocoFeMoco clusters. (Figure(Figure 6)6) intointo syntheticsynthetic clusters.clusters.

Figure 6. Target structural and electronic attributes of interest in FeMoco for biomimetic modelling. FigureFigure 6.6. TargetTarget structuralstructural andand electronicelectronic attributesattributes ofof interestinterest inin FeMocoFeMoco forfor biomimeticbiomimetic modelling.modelling.

ToTo thisthis end,end, aa two-foldtwo-fold strategystrategy waswas employedemployed inin ourour nitrogenasenitrogenase biomimeticsbiomimetics researchresearch programprogram (Scheme(Scheme 1).1). First,First, wewe tooktook inspirationinspiration fromfrom thethe biomechanisticbiomechanistic pathwaypathway proposedproposed byby RibbeRibbe andand HuHu for FeMoco biogenesis. A carbonyl-supported iron sulfide cluster K2[Fe3S(CO)9] was synthesized and for FeMoco biogenesis. A carbonyl-supported iron sulfide cluster K2[Fe3S(CO)9] was synthesized and alkylated with various alkyl halides (R = Me, iPr, Bz). The resulting μ3-thiolato cluster was alkylated with various alkyl halides (R = Me, iPr, Bz). The resulting μ3-thiolato cluster was subsequentlysubsequently treatedtreated withwith aa radicalradical initiatorinitiator TEMPOTEMPO ((2,2,6,6-tetramethylpiperidin-1-yl)oxyl)((2,2,6,6-tetramethylpiperidin-1-yl)oxyl) toto emulateemulate thethe HH atomatom abstractionabstraction mechanismmechanism propagatedpropagated byby SAMSAM uponupon thethe KK cluster.cluster. WeWe additionallyadditionally pursuedpursued aa converseconverse strategystrategy utilizingutilizing aa familyfamily ofof carbonyl-supportedcarbonyl-supported ironiron clustersclusters featuringfeaturing

Catalysts 2020, 10, 1317 8 of 22

To this end, a two-fold strategy was employed in our nitrogenase biomimetics research program (Scheme1). First, we took inspiration from the biomechanistic pathway proposed by Ribbe and Hu for FeMoco biogenesis. A carbonyl-supported iron sulfide cluster K2[Fe3S(CO)9] was synthesized i and alkylated with various alkyl halides (R = Me, Pr, Bz). The resulting µ3-thiolato cluster was subsequently treated with a radical initiator TEMPO ((2,2,6,6-tetramethylpiperidin-1-yl)oxyl) to emulate the H atom abstraction mechanism propagated by SAM upon the K cluster. We additionally pursuedCatalysts a 2020 converse, 10, x FOR strategy PEER REVIEW utilizing a family of carbonyl-supported iron clusters featuring interstitial8 of 22 carbides—the first of which was reported by Braye et al. in 1962 [56]. The hexa-iron cluster interstitial carbides—the first of which was reported by Braye et al. in 1962 [56]. The hexa-iron cluster (NEt4)2[Fe6(µ6-C)(CO)16] reported by Churchill et al. [46,47] serves as a synthetic starting point (NEt4)2[Fe6(µ6-C)(CO)16] reported by Churchill et al. [46,47] serves as a synthetic starting point toward toward highly FeMoco-relevant structural models, demonstrating flexibility between multiple redox highly FeMoco-relevant structural models, demonstrating flexibility between multiple redox states statesand and versatility versatility for heterometal for heterometal substitution substitution and sulf andide sulfide incorporation incorporation [44,57,58]. [44,57 Here,,58]. a Here, strategy a strategy was was explored for ligand CO S substitution by using electrophilic sulfur upon the soft anionic clusters. explored for ligand CO→S→ substitution by using electrophilic sulfur upon the soft anionic clusters.

SchemeScheme 1. 1.A A two-fold two-fold synthetic synthetic approachapproach toward achieving structural structural analogues analogues of ofthe the L-cluster, L-cluster, precursorprecursor to to FeMoco. FeMoco. First, First, inspirationinspiration was was taken taken from from the the biosynthetic biosynthetic conversion conversion of the of K-cluster the K-cluster (2 (2 × FeFe4SS4) )to to the the L-cluster L-cluster (Fe (Fe8S9C)S byC) NifB. by NifB. As such, As such,a methyl a methyl group groupwas installed was installed upon an uponFe3S cluster an Fe S × 4 4 8 9 3 clusterusing using MeI. MeI.TEMPO TEMPO was wasthen thenutilized utilized as a asradical a radical initiator initiator to emulate to emulate similar similar radical radical abstraction abstraction of

ofhydrogen observed observed during during L-cluster L-cluster sy synthesis.nthesis. Alternatively, Alternatively, an anionic an anionic Fe6C Fe cluster6C cluster featuring featuring an aninterstitial interstitial carbide carbide was was found found to to be be aa useful useful synt synthetichetic starting starting point point for for sulfur sulfur incorporation incorporation via via electrophilicelectrophilic sulfur sulfur reagents. reagents.

2. Synthetic2. Synthetic Manipulations Manipulations of of CO-Supported CO-Supported FeSFeS and FeC Clusters Clusters

2.1.2.1. Biogenesis-Inspired Biogenesis-Inspired Approach Approach to to FeSC FeSCCluster Cluster ConstructionConstruction AsAs discussed discussed above, above, the the biogenesis biogenesis ofof thethe M-clusterM-cluster in nitrogenase has has garnered garnered much much interest interest 4 sincesince the the identiﬁcation identification of of the the interstitial interstitial carbide carbide inin 20112011 [[7,8].7,8]. The unique unique motif motif of of a a 6-coordinate 6-coordinate C C4− − boundbound to to the the six six iron iron centers centers present present in in FeMoco FeMoco/FeV/FeVcoco are the only only reported reported examples examples in in biological biological systemssystems [59 [59].]. It It is is highlyhighly likely,likely, however, that that the the all-iron all-iron analogue analogue of ofthe the enzyme enzyme also also contains contains a carbide. Few examples exist in chemical systems as well with the notable exception of the synthetic carbide clusters (vide infra). It was shown that carbide formation at the K-cluster requires two equivalents of SAM including one for the methyl group donation (which eventually becomes the carbide) and a second one for the radical abstraction of a hydrogen atom [11]. The initial binding site of the methyl group was also traced by liquid and gas chromatographic methods HPLC and GCMS, revealing that the methyl group first binds to a sulfide in the native enzyme [12]. This is counterintuitive, as one would expect an all-iron ligated carbide to initially bind to an iron center. However, biological Fe oxidation states

Catalysts 2020, 10, x FOR PEER REVIEW 9 of 22 Catalysts 2020, 10, 1317 9 of 22 are likely not nucleophilic enough to form Fe–C bonds. Considering this, it is then understandable that nitrogenase utilizes a radical mechanism for carbide formation. Furthermore, the proposed a carbide. Few examples exist in chemical systems as well with the notable exception of the synthetic mechanism indicates that transfer of the methyl group occurs prior to radical H atom abstraction and carbide clusters (vide infra). subsequent deprotonation/dehydrogenation. It wasThese shown results that provided carbide the formation stimulus at for the our K-cluster initial biomimetic requires two approach equivalents for investigating of SAM including the onereactivity for the methylof methyl-substituted group donation iron-sulfur (which eventuallyclusters with becomes a radical the initiator. carbide) The and synthetic a second cluster one for theK radical2[(μ3-S)Fe abstraction3(CO)9] (Scheme of a hydrogen 2) was chosen atom [due11]. to The its initialversatility binding in substituting site of the methylR groups group using was alsocommon traced electrophiles by liquid and that gas can chromatographic be installed on the methods S2− ‘bridgehead,’ HPLC and making GCMS, it a revealing suitable model that the system methyl groupfor firstinvestigating binds to a–SCH sulfide3 reaction in the native mechanisms enzyme [associated12]. This is with counterintuitive, iron-containing as one clusters. would expectThe anmethylation all-iron ligated of K carbide2[SFe3(CO) to initially9] was achieved bind to an by iron a reaction center. However,with CH3I biologicalin THF atFe 0 °C, oxidation affording states the are likelymonoanionic not nucleophilic K[(CH3S)Fe enough3(CO) to9] form(1). The Fe–C resulting bonds. bond Considering metrics this,were itsimilar is then to understandable those previously that nitrogenaseobserved utilizesin NEt4 a[(CH radical3S)Fe mechanism3(CO)9] [60]. for It carbidewould formation.be expected Furthermore, that radicalthe hydrogen proposed abstraction mechanism indicatesreagents that would transfer readily of the abstract methyl an group H-atom occurs from prior the anion. to radical However, H atom because abstraction the low and valent subsequent Fe deprotonationclusters typically/dehydrogenation. demonstrate a proclivity toward forming bridging hydrides, we elected to utilize a relativelyThese resultsmild H-atom provided abstraction the stimulus reagent. for Thus, our initial (2,2,6,6-tetramethylpiperidin-1-yl)oxyl biomimetic approach for investigating (TEMPO) the reactivitywas selected of methyl-substituted due to its mild O–H iron-sulfur bond dissociation clusters energy with [61]. a radical initiator. The synthetic cluster The reaction of the methylated Fe3S cluster and crystallization of the charged portion revealed K2[(µ3-S)Fe3(CO)9] (Scheme2) was chosen due to its versatility in substituting R groups using common an aggregation product in which the methyl2 group had been displaced, yielding the formula electrophiles that can be installed on the S − ‘bridgehead,’ making it a suitable model system for [K(benzo-15-crown-5)2]2[(SFe2(CO)12)2Fe(CO)2] (2). This reactivity indicated the occurrence of two key investigating –SCH3 reaction mechanisms associated with iron-containing clusters. The methylation transformations: (i) S–CH3 bond breakage to generate the inorganic sulfide, and (ii) the combination of K2[SFe3(CO)9] was achieved by a reaction with CH3I in THF at 0 ◦C, affording the monoanionic of two Fe3 units (and extrusion of one Fe center) to form an S2Fe5 framework. Additionally, two- K[(CH S)Fe (CO) ](1). The resulting bond metrics were similar to those previously observed in electron3 oxidation3 9 of the Fe centers was observed (one electron from each progenitor cluster of 1). NEt [(CH S)Fe (CO) ][60]. It would be expected that radical hydrogen abstraction reagents would The4 resultant3 3 formation9 of 2 is unaffected by dramatic changes in reaction conditions including readily abstract an H-atom from the anion. However, because the low valent Fe clusters typically solvent identity (THF, MeCN, FPh) or temperature (−78 °C or ambient temperature), showcasing the demonstratelow kinetic a barrier proclivity present toward in formingreactions bridgingwith thes hydrides,e types of we iron elected clusters to utilize such athat relatively the same mild H-atomthermodynamically abstraction reagent. stable Thus,product (2,2,6,6-tetramethylpiperidin-1-yl)oxyl is recurringly generated, highlighting (TEMPO) the uniqueness was selected of the due tobiogenesis its mild O–H in FeMoco bond dissociation assembly. energy [61].

SchemeScheme 2. 2.Synthetic Synthetic scheme scheme depictingdepicting the formation of of 1 1 and and its its reaction reaction with with the the radical radical initiator, initiator, TEMPO,TEMPO, to to form form 2. 2.

TheThis reaction process of does the methylatedbear some resemblance Fe3S cluster to andthe biosynthesis crystallization of the of theFeMoco charged active portion site, wherein revealed anthe aggregation addition of productSAM causes in whichoxidation the of methyl the SAM-motif-bound group had been [Fe4 displaced,S4]1+ in NifB yielding to an oxidized the formula EPR 2− 4− [K(benzo-15-crown-5)silent intermediate 2[62].]2[(SFe In 2this(CO) process,12)2Fe(CO) S 2and](2 ).C This are reactivity ultimately indicated generated the as occurrence charged ofligands, two key transformations:implying oxidation (i) S–CH of the3 bond ironbreakage centers en to route generate to the the intermediate inorganic sulﬁde, L cluster. and ( iiUnfortunately,) the combination the of twoincorporation Fe3 units (and of extrusionany carbon of (or one carbide) Fe center) motif toform to the an synthetic S2Fe5 framework. cluster 1 was Additionally, not observed two-electron in the oxidationgeneration of the of 2 Fe, even centers though was it observed was demonstrated (one electron that from a radi eachcal electron progenitor can cluster aid in the of 1 fusion). The of resultant two formationiron-sulfur of 2 unitsis una andﬀected aid in by the dramatic gradual changes oxidation in of reaction the Fe conditionscenters. including solvent identity (THF, MeCN, FPh) or temperature ( 78 ◦C or ambient temperature), showcasing the low kinetic barrier 2.2. Sulfide Incorporation into Iron-C− arbide Clusters: Initial Attempts present in reactions with these types of iron clusters such that the same thermodynamically stable productOur is recurringly initial foray generated,into ligand substitution highlighting upon the uniqueness(NEt4)2[Fe6(µ of6-C)(CO) the biogenesis16] began inwith FeMoco the utilization assembly. of Thisa bidentate, process doesanthracene-scaffolded bear some resemblance bis-phenylthio to the biosynthesislate ligand ofset the (Scheme FeMoco 3). active The site,choice wherein of the the anthracene scaffold was motivated by the observation that the 5.11+ Å distance between the 1 and 8 addition of SAM causes oxidation of the SAM-motif-bound [Fe4S4] in NifB to an oxidized EPR silent positions on the anthracene backbone2 closely4 correlated with the 5.0 Å S···S distance between distal intermediate [62]. In this process, S − and C − are ultimately generated as charged ligands, implying 2– oxidationsulfide ofsites the ironin FeMoco. centers enHowever, route to thetreatment intermediate of the L cluster.[Fe6C] Unfortunately, cluster with thedisodium incorporation 1,8- ofbis(phenylthiolato) any carbon (or carbide) anthracene motif does to the not synthetic induce any cluster color change1 was notin the observed reactionin mixture the generation (Scheme 4). of 2, even though it was demonstrated that a radical electron can aid in the fusion of two iron-sulfur units and aid in the gradual oxidation of the Fe centers. Catalysts 2020, 10, 1317 10 of 22

2.2. Sulﬁde Incorporation into Iron-Carbide Clusters: Initial Attempts

Our initial foray into ligand substitution upon (NEt4)2[Fe6(µ6-C)(CO)16] began with the utilization of a bidentate, anthracene-scaffolded bis-phenylthiolate ligand set (Scheme3). The choice of the anthracene scaffold was motivated by the observation that the 5.1 Å distance between the 1 and 8 positions on the anthracene backbone closely correlated with the 5.0 Å S S distance between distal sulfide sites 2– ··· inCatalysts FeMoco. 2020, However, 10, x FOR PEER treatment REVIEW of the [Fe6C] cluster with disodium 1,8-bis(phenylthiolato) anthracene10 of 22 does not induce any color change in the reaction mixture (Scheme4). In 2015, Figueroa et al. reported onIn 2015, isocyanide Figueroa analogues et al. reported of iron on carbonyls, isocyanide noting analogues that of isocyanides—similar iron carbonyls, noting to that carbonyls—act isocyanides— as excellentsimilar toπ carbonyls—act-acceptors, but as additionally excellent π exhibit-acceptors, stronger but additionσ-donatingally abilityexhibit relative stronger to σ carbon-donating monoxide. ability Thisrelative increases to carbon the reactivitymonoxide. of theirThis hostincreases metal the toward reactivity substrates of their including host metal N2 [ 63toward]. Following substrates this 2 lineincluding of research, N2 [63]. [Fe Following6C] − was this treated line with of research, 2,6-dimethylphenyl [Fe6C]2− was isocyanide, treated with resulting 2,6-dimethylphenyl in a reaction solutionisocyanide, dominated resulting by in a a dark reaction green solution color—indicative dominated of decompositionby a dark green to Fecolor—indicative3(CO)12 and other of intractabledecomposition products. to Fe3(CO)12 and other intractable products.

Scheme 3.3. SS•••SS distances distances between between the the 1 and1 and 8 positions 8 positions on anon anthracene an anthracene scaﬀ oldscaffold (left) and(left) the and distal the ••• sulﬁdesdistal sulfides in FeMoco in FeMoco (right). (right).

Another attempted attempted effort effort to to incorporate incorporate sulfur sulfur into into carbidocarbonyl carbidocarbonyl iron ironcluster cluster was in was the indirect the directreaction reaction of the of nido the nido-Fe5 -Feneutral5 neutral cluster cluster [Fe [Fe5(µ55(-C)(CO)µ5-C)(CO)15] 15with] with elemental elemental sulfur sulfur (S (S88).). However, However, performingperforming thethe reactionreaction at at room room temperature temperature or or 60 60◦C °C results results in in the the formation formation of aof small a small amount amount of anof 0 0 insolublean insoluble and and ferromagnetic ferromagnetic black black powder powder (presumably (presumably metallic metallic iron) andiron) an and unconverted an unconverted [Fe5C] [Feband.5C] 0 0 Increasingband. Increasing the temperature the temperature to 90 ◦ Cto significantly 90 °C significantly decomposes decomposes [Fe5C] into[Fe5C] the into same the black same powder, black andpowder, reaction and reaction at 120 ◦C at results 120 °C inresults total in decomposition. total decomposition. Alternatively, Alternatively, a literature a literature report report found found that 0 0 elementsthat elements from from the pnictogen the pnictogen series series were successfullywere successfully reacted reacted with [Fe with5C] [Fein5 theC] presencein the presence of H2SO of4 atH2 100SO4 ◦atC for100 30 °C min for to 30 generate min to agenerate tri-iron methylidenea tri-iron methylidene cluster with cluster the formula with the Fe 3(formulaµ3-CH)E(CO) Fe3(µ39- (ECH)E(CO)= As, Sb,9 Bi)(E = [ 64As,]. Sb, Elemental Bi) [64]. arsenic Elemental and bismutharsenic and were bismuth foundto were work found for this to work reaction. for this Interestingly, reaction. theInterestingly, Sb cluster couldthe Sb only cluster be achievedcould only with be SbClachieved5.Eff ortswith to SbCl replicate5. Efforts these to conditionsreplicate these with conditions elemental sulfurwith elemental produced sulfur a dark produced red-orange a solution,dark red-orange which a ffsolution,orded only which crystals afforded of the only non-carbide-containing crystals of the non- clustercarbide-containing Fe3S2(CO)9. cluster Similarly, Fe3S using2(CO)9 selenium. Similarly, powder using selenium under the powder same conditionsunder the same gave crystalsconditions of Fegave3Se crystals2(CO)9. of Fe3Se2(CO)9. Finally, thethe utilizationutilization ofof triphenyltriphenyl antimonyantimony sulfidesulfide (Ph(Ph33SbSb=S)=S) as a thermoneutralthermoneutral sulfur atomatom donor waswas considered considered due due to to its its behavior behavior as aas weak a weak sulfur-donating sulfur-donating ability ability and the and absence the absence of S–S bondsof S–S suchbonds as such those as those seen inseen elemental in elemental sulfur sulfur (Scheme (Scheme5)[ 655)]. [65]. Preparation Preparation of of the the reagent reagent for for the the inin situsitu 22− and transfertransfer intointo anan ice-coldice-cold toluenetoluene suspensionsuspension ofof [Fe[Fe66C]C] − resultedresulted in a highly convoluted mixture of compounds, therebythereby obstructingobstructing thethe growthgrowth ofof crystalscrystals suitablesuitable forfor X-rayX-ray didiffraction.ffraction.

Catalysts 2020, 10, x FOR PEER REVIEW 11 of 22

Catalysts 2020, 10, 1317 11 of 22

Catalysts 2020, 10, x FOR PEER REVIEW 11 of 22

SchemeSchemeScheme 4.4. VariousVarious 4. Various reaction reaction reaction conditions conditions conditions attempted attempted attempted to pursue to pursuepursue controlled controlled controlled ligand ligand ligand substitution substitution substitution onto onto iron ontoiron carbide iron clusterscarbidecarbide (clusterstop) clusters and (top) schematic (top) and and schematic representation schematic representation representation of reaction of conditionsof reactionreaction conditions reported conditions inrepoReference reportedrted in Reference [in64 Reference] (bottom [64] ). [64] (bottom).(bottom).

Scheme 5. In situ synthesis of triphenyl antimony sulfide and reaction conditions for sulfur donation

into [Fe6C]2− cluster. SchemeScheme 5.5. In situ synthesis ofof triphenyl antimony sulﬁdesulfide andand reactionreaction conditionsconditions forfor sulfursulfur donationdonation 2 intointo [Fe[Fe66C]2− cluster.cluster.

Catalysts 2020, 10, 1317 12 of 22

Catalysts 2020, 10, x FOR PEER REVIEW 12 of 22 2.3. Electrophilic Sulfur Sources: Isolation of Sulfide-Containing Carbidocabonyl Iron Clusters 2.3. Electrophilic Sulfur Sources: Isolation of Sulfide-Containing Carbidocabonyl Iron Clusters Because of the undesirable results obtained from using conventional Lewis basic ligands (sulfides, thiolates)Because with of the the anionic undesirable and neutral results iron obtained clusters, fr weom sought using an conventional alternative strategyLewis basic for achieving ligands (sulfides,sulfide incorporation. thiolates) with At the time,anionic the and only neutral report ir ofon successful clusters, ligationwe sought by aan sulfur-based alternative donationstrategy for set achieving sulfide incorporation. At the time, the only report of successful ligation by a sulfur-based upon carbidocarbonyl iron clusters was a 1988 publication by Shriver et al. describing the synthesis donation set upon carbidocarbonyl iron clusters was a 1988 publication by Shriver et al. describing and isolation of SO2-ligated Fe6 and Fe5 clusters [66]. However, the use of these SO2 clusters as isolable the synthesis and isolation of SO2-ligated Fe6 and Fe5 clusters [66]. However, the use of these SO2 intermediates in pursuit of a sulfide-ligated iron-carbide clusters was a suspected ‘dead-end’ in the clusters as isolable intermediates in pursuit of a sulfide-ligated iron-carbide clusters was a suspected absence of a strategy for successful reduction and oxide removal from SO2. We do note, however, ‘dead-end’ in the absence of a strategy for successful reduction and oxide removal from SO2. We do that Rauchfuss et al. has reported achievement of an S-ligated cluster via an SO2 intermediate [44]. note, however, that Rauchfuss et al. has reported achievement of an S-ligated cluster via an SO2 Nonetheless, the observation that, upon ligation, SO2 behaves electrophilically (therefore, oxidizing the intermediate [44]. Nonetheless, the observation that, upon ligation, SO2 behaves electrophilically anionic cluster) and the additional observation that the gallery of reported heteroleptic carbidocarbonyl (therefore, oxidizing the anionic cluster) and the additional observation that the gallery of reported+ iron clusters in the CSD feature initial oxidation prior to ligand incorporation (NO, H H−, heteroleptic carbidocarbonyl iron clusters in the CSD feature initial oxidation prior to ligand→ Lewis acidic Cu-, and Au-bearing reagents) [36] prompted us to seek out similarly electrophilic incorporation (NO, H+→H−, Lewis acidic Cu-, and Au-bearing reagents) [36] prompted us to seek out sulfur-based ligand sets. similarly electrophilic sulfur-based ligand sets. As such, a synthetic strategy centered around S2Cl2 was deemed appropriate for sulfide As such, a synthetic strategy centered around S2Cl2 was deemed appropriate for sulfide incorporation [67]. While SCl2 is more structurally analogous to SO2 (Figure7), the reported incorporation [67]. While SCl2 is more structurally analogous to SO2 (Figure 7), the reported instability instability of the reagent and its spontaneous conversion to S2Cl2 marked it as less preferable. of the reagent and its spontaneous conversion to S2Cl2 marked it as less preferable. Treatment of the Treatment of the hexa-nuclear cluster (NEt4)2[Fe6(µ6-C)(CO)16] with S2Cl2 afforded a mixture of hexa-nuclear cluster (NEt4)2[Fe6(µ6-C)(CO)16] with S2Cl2 afforded a mixture of products (Scheme 6). products (Scheme6). We initially characterized the asymmetric, sulfide-bridged ‘dimer of clusters’ We initially characterized the asymmetric, sulfide-bridged ‘dimer of clusters’ (NEt4)2{[(CO)15(µ6-C)Fe6](µ4-S)[Fe5(µ5-C)(CO)13]} (3). The dimer was structurally identified by XRD (NEt4)2{[(CO)15(µ6-C)Fe6](µ4-S)[Fe5(µ5-C)(CO)13]} (3). The dimer was structurally identified by XRD as as featuring a four-coordinate bridging sulfide tethered between a six-iron and a five-iron cluster featuring a four-coordinate bridging sulfide tethered between a six-iron and a five-iron cluster including each cluster with an interstitial carbide. Crystal structure data (Figure8) reveals Fe–S distances including each cluster with an interstitial carbide. Crystal structure data (Figure 8) reveals Fe–S [2.203(3), 2.189(3), 2.188(3), 2.169(2) Å] that are noticeably short for a CO supported {Fe2(µ4-S)Fe2} motif. distances [2.203(3), 2.189(3), 2.188(3), 2.169(2) Å] that are noticeably short for a CO supported {Fe2(µ4- Exploration of this motif in the CSD [36] produces an average of 2.25 0.01 Å. The average Fe–S bond S)Fe2} motif. Exploration of this motif in the CSD [36] produces an± average of 2.25 ± 0.01 Å. The distance of 2.19 0.01 Å in 3 is, thus, remarkably short. The longest bond length in 3 at 2.203(3) Å is average Fe–S bond± distance of 2.19 ± 0.01 Å in 3 is, thus, remarkably short. The longest bond length shorter than the shortest recorded CSD Fe–S bond (2.215 Å) in an {Fe2(µ4-S)Fe2} motif. In comparison in 3 at 2.203(3) Å is shorter than the shortest recorded CSD Fe–S bond (2.215 Å) in an {Fe2(µ4-S)Fe2} to the bond metrics found in FeMoco [68], the Fe–S bond falls short of the average Fe–S bond found in motif. In comparison to the bond metrics found in FeMoco [68], the Fe–S bond falls short of the the active site (2.25 0.03 Å). The average Fe–Fe bond distance of 2.65 0.06 Å in 3 is unremarkable. average Fe–S bond found± in the active site (2.25 ± 0.03 Å). The average ±Fe–Fe bond distance of 2.65 ± Thus, the presence of the sulfide does not induce any notable elongation or compression of Fe–Fe bond 0.06 Å in 3 is unremarkable. Thus, the presence of the sulfide does not induce any notable elongation lengths in Fe5–Fe8. The average Fe–C bond distance in the six-iron unit of cluster 3 is 1.89 0.01 Å or compression of Fe–Fe bond lengths in Fe5–Fe8. The average Fe–C bond distance in the six-iron± unit when compared to 1.881 0.005 Å in the published Fe6 cluster [57]. In the 5-iron unit of cluster 3, of cluster 3 is 1.89 ± 0.01 Å± when compared to 1.881 ± 0.005 Å in the published Fe6 cluster [57]. In the the equatorial iron sites average an Fe–C bond distance of 1.86 0.01 Å, while the axial iron resides at 5-iron unit of cluster 3, the equatorial iron sites average an Fe–C± bond distance of 1.86 ± 0.01 Å, while an elongated bond distance of 1.95(1) Å. This is strikingly similar to the published Fe5 neutral cluster the axial iron resides at an elongated bond distance of 1.95(1) Å. This is strikingly similar to the [(Feeq–Cavg = 1.88 0.02 Å; Feax–C = 1.949(7) Å]. The Fe–C bonds found in 3, however, are shorter published Fe5 neutral± cluster [(Feeq–Cavg = 1.88 ± 0.02 Å; Feax–C = 1.949(7) Å]. The Fe–C bonds found than the FeMoco average of 2.00 0.02 Å. in 3, however, are shorter than the± FeMoco average of 2.00 ± 0.02 Å.

Figure 7. SchematicSchematic indication of the dipole in electropositive sulfur reagents.

Catalysts 2020, 10, 1317x FOR PEER REVIEW 13 of 22 Catalysts 2020, 10, x FOR PEER REVIEW 13 of 22

Scheme 6. Synthetic scheme depicting the electrophilic sulfurization of the Fe6C dianion cluster by SchemeScheme 6. 6.Synthetic Synthetic scheme scheme depicting depicting the the electrophilic electrophilic sulfurization sulfurization of theof the Fe6 CFe dianion6C dianion cluster cluster by eitherby either S2Cl2 or elemental sulfur to afford a µ4-S cluster (3 or 4) and4– a {CS}4– cluster (5 or 6). The non- S2Cleither2 or elementalS2Cl2 or elemental sulfur to sulfur afford to aaffordµ4-S clustera µ4-S cluster (3 or 4 )(3 and or 4 a) {CS}and a {CS}cluster4– cluster (5 or 6().5 or The 6). non-carbide The non- carbide cluster [Fe3S2(CO)9] (not depicted) was also characterized as a side product in both reactions. clustercarbide [Fe cluster3S2(CO) [Fe9]3S (not2(CO) depicted)9] (not depicted) was also was characterized also characterized as a side as a product side product in both in reactions.both reactions. Image ImageadaptedImage adapted withadapted permission with with permission permission from Joseph, from from C., Joseph,Joseph, Cobb, C.,C., C. R.,Cobb, Rose, C.C. M. R.,R.,J. Rose,Rose, Single-Step M.M. J. J. Single-Step InsertionSingle-Step of Insertion SulfidesInsertion of and of SulfidesThiolateSulfides and into and Thiolate Iron Thiolate Carbide-Carbonyl into into Iron Iron Carbide-Carbonyl Carbide-Carbonyl Clusters: Unlocking Clusters: the UnlockingUnlocking Synthetic the the Door Synthetic Synthetic to FeMoco Door Door to Analogues.to FeMoco FeMoco Analogues.Angew.Analogues. Chemie Angew. Angew. Int. Chemie Ed. Chemie2020 Int. ,Int.Accepted, Ed. Ed. 20202020 not, , Accepted,Accepted, yet published not .yet Copyright publishedpublished. .Wiley-VCH CopyrightCopyright Wiley-VCH GmbHWiley-VCH. Reproduced GmbH GmbH. . ReproducedwithReproduced permission. with with permission. permission.

Figure 8. Thermal ellipsoid plots (50% probability) of (NEt4)2{[(CO)15(µ6-C)Fe6](µ4-S)[Fe5(µ5-C)(CO)13]} Figure 8. ThermalThermal ellipsoid plots (50%(50% probability)probability) of of (NEt (NEt)4)2{[(CO){[(CO)15((µµ6-C)Fe-C)Fe6](µ 4-S)[Fe5(µ5-C)(CO)-C)(CO)13]}]} (3). Orange = Fe, Gray = C, Yellow = S, Maroon = O. Two NEt4 2 4+ cations15 have6 been6 removed4 5 for5 clarity.13 ++ (3).Image Orange Orange adapted = Fe,Fe, Gray Graywith = =permission C,C, Yellow Yellow =from= S,S, MaroonMaroon Joseph, = =C., O.O. Cobb,Two Two NEt NEtC. 4R.,4 cations cationsRose, M.have have J. Single-Stepbeen been removed removed Insertion for for clarity. clarity. of ImageSulfides adaptedadapted and withThiolate with permission permission into Iron from Carbide-Carbonyl from Joseph, Joseph, C., Cobb,C., Clusters: Cobb, C. R., C. Unlocking Rose, R., Rose, M. J. the Single-StepM. Synthetic J. Single-Step Insertion Door toInsertion ofFeMoco Sulfides of SulfidesandAnalogues. Thiolate and intoThiolate Angew. Iron into Carbide-CarbonylChemie Iron Int. Carbide-Carbonyl Ed. 2020 Clusters:, Accepted, UnlockingClusters: not yet Unlockingpublished the Synthetic. Copyright the DoorSynthetic to Wiley-VCH FeMoco Door Analogues.to GmbH FeMoco. Analogues.Angew.Reproduced Chemie Angew. with Int. permission.Chemie Ed. 2020 Int., Accepted, Ed. 2020 not, Accepted, yet published not .yet Copyright published.Wiley-VCH Copyright GmbHWiley-VCH. Reproduced GmbH. Reproducedwith permission. with permission. However, the generation of side products and the presence of chloride in the synthesis of the sulfide-bridged,However, the asymmetricgeneration dimerof side led products us to seek and more th thee presencestraightforward of chloride reaction in the conditions. synthesis Since of the sulfide-bridged,sulﬁde-bridged,this cluster can asymmetricasymmetric be thematically dimerdimer formulated led led us us to to seekas seek an more Fe more6 cluster, straightforward straightforward an Fe5 cluster, reaction reaction a sulfur conditions. conditions. bridge, Sinceand Sincean this thisclusteroverall cluster can 2 be–can charge, thematically be thematically we attempted formulated formulated to asstoichiometrica an as Fe 6ancluster, Fe6 llycluster, an construct Fe5 ancluster, Fe the5 cluster, acompound sulfur a bridge,sulfur by bridge, andintroducing an and overall an – overall2 elementalcharge, 2– wecharge, sulfur attempted intowe attempteda to solution stoichiometrically ofto (Et stoichiometrica4N)2[Fe construct6(µ6-C)(CO)lly the 16construct compound] and [Fe 5the(µ by5-C)(CO) introducingcompound15]. To elemental byour introducing surprise, sulfur elementalintocrystal a solution structural sulfur of into (Et data4N) a 2solutionfor[Fe 6this(µ6 -C)(CO) chargedof (Et4N) 16species]2[Fe and6(µ [Fein6-C)(CO) 5this(µ5 -C)(CO)reaction16] and15 revealed ].[Fe To5( ourµ5-C)(CO) a surprise,symmetric15]. crystalTo dimer our structuralsurprise, of Fe5 crystaldataclusters for structural this bridged charged data by speciesthe for same this in 4-coordinatecharged this reaction species su revealedlfide in motifthis a reaction symmetric(Figure 9)revealed with dimer the a of formulasymmetric Fe5 clusters (NEt dimer4) bridged2{[Fe 5of(µ 5-Fe by5 13 2 4 5 clusterstheC)(CO) same bridged 4-coordinate] (µ -S)} by ( 4the). sulﬁdeThe same Fe–C 4-coordinate motif bond (Figure distances 9su)lfide with of this themotif cluster formula (Figure are (NEt 9)similar with4)2{[Fe tothe 5the( formulaµ5 Fe-C)(CO) component (NEt13]24()µ2{[Fe4 -S)}of 53(,µ ( 45).- exhibiting an average Feeq–C bond distance of 1.87 ± 0.01 Å and an average Feax–C bond distance of C)(CO)The Fe–C13]2( bondµ4-S)} distances (4). The Fe–C of this bond cluster distances are similar of this to thecluster Fe5 componentare similar to of the3, exhibiting Fe5 component an average of 3, 1.961 ± 0.005 Å. The average Fe–S distance of 2.175 ± 0.005 Å, however, is notably shorter than the exhibitingFeeq–C bond an average distance Fe ofeq–C 1.87 bond0.01 distance Å and of an 1.87 average ± 0.01 FeÅ axand–C an bond average distance Feax–C of bond 1.961 distance0.005 of Å. already short distance found in± 3 and is far shorter than the Fe–S distances observed in FeMoco.± 1.961The average ± 0.005 Fe–SÅ. The distance average of Fe–S 2.175 distance0.005 of Å, 2.175 however, ± 0.005 is Å, notably however, shorter is notably than the shorter already than short the ± alreadydistance short found distance in 3 and found is far in shorter 3 and thanis far the shorter Fe–S than distances the Fe–S observed distances in FeMoco. observed in FeMoco.

Catalysts 2020, 10, 1317 14 of 22 Catalysts 2020,, 10,, xx FORFOR PEERPEER REVIEWREVIEW 14 of 22

Figure 9. 9. ThermalThermal ellipsoid ellipsoid plots plots (50% (50%probability) probability) of (NEt of44))22{[Fe (NEt{[Fe55((4µ)525-C)(CO)-C)(CO){[Fe5(µ51313-C)(CO)]]22((µ44-S)}-S)}13 ((]42).).(µ OrangeOrange4-S)} (4 =).= ++ + OrangeFe, Gray= = Fe,C, Yellow Gray = = C,S, Maroon Yellow = = S,O. MaroonTwo NEt=44 O. cationscations Two have NEthave4 beenbeencations removedremoved have forfor been clarity.clarity. removed ImageImage for adaptedadapted clarity. Imagewith permission adapted with from permission Joseph, C., from Cobb, Joseph, C. R., C., Rose, Cobb, M. C. J.J. Single-Step R.,Single-Step Rose, M. InsertionInsertion J. Single-Step ofof SulfidesSulfides Insertion andand of ThiolateThiolate Sulﬁdes andintointo ThiolateIronIron Carbide-CarbonylCarbide-Carbonyl into Iron Carbide-Carbonyl Clusters:Clusters: UnlockinUnlockin Clusters:g Unlockingthe Synthetic the Door Synthetic to FeMoco Door to Analogues. FeMoco Analogues. Angew. Angew.Chemie Int. Chemie Ed. Int. 2020 Ed.,, Accepted,2020, Accepted, not yet notpublished yet published.. CopyrightCopyright. Copyright Wiley-VCHWiley-VCH GmbH GmbH.. ReproducedReproduced. Reproduced withwith withpermission. permission.

Our eeffortsﬀorts toto fullyfully characterizecharacterize the product proﬁlesprofilesiles forfor the reactions described above prompted usus to to separate separate and and purify purify the the remaining remaining neutra neutralll compounds.compounds. compounds. TheThe majormajor The componentcomponent major component ofof thethe pentane-pentane- of the pentane-solublesoluble portion portionis a deep, is a deep,bright bright red compound. red compound. Isolation Isolation and and crystallization crystallization of of the the bright-red compoundcompound generated generated the the well-known well-known cluster cluster Fe Fe33S322S(CO)2(CO)99 [69,70].[69,70].9 [69,70 MoreMore]. More remarkabremarkab remarkably,ly,ly, crystalscrystals crystals fromfrom from thethe thered-orange red-orange Et22O Et solution2O solution revealed revealed a remarkable a remarkable CO-supported CO-supported iron-sulfur iron-sulfur cluster cluster featuring featuring a a“carbide-like” “carbide-like” site, site, multiple multiple sulfur sulfur atoms, atoms, and and a a ‘dangler’ ‘dangler’ iron iron (Figure (Figure 10),10), resulting resulting in the formulaformula [{Fe[{Fe44((κ22S–S–κκ444C)(CO)C)(CO)101010}(}(µµ33-S)(3-S)(µ3µ3-S3-S22)Fe(CO)2)Fe(CO)33] 3(5]().5 ).

Figure 10.10. Thermal ellipsoidellipsoid plotsplots (50%(50% probability) probability) of of [{Fe [{Fe4(44κ((κ2S–22S–κκ44C)(CO)4C)(CO)101010}(}(}(µµ333-S)(-S)(µµ333-S-S22)Fe(CO))Fe(CO)33]]]( ((55).). Orange == Fe, Gray = C,C, Yellow Yellow = S,S, Maroon Maroon == O.O. ImageImage Image adaptedadapted adapted withwith with permissionpermission permission fromfrom Joseph,Joseph, C.,C., Cobb, C.C. R., R., Rose, Rose, M. M. J. Single-StepJ. Single-Step Insertion Insertion of Sulfides of Sulfides and Thiolate and Thiolate into Iron into Carbide-Carbonyl Iron Carbide-Carbonyl Clusters: UnlockingClusters: Unlocking the Synthetic the Door Syntheti to FeMococ Door Analogues. to FeMocoAngew. Analogues. Chemie Angew. Int. Ed. Chemie2020, Accepted, Int. Ed. not2020 yet,, Accepted, published. Copyrightnot yet publishedWiley-VCH.. CopyrightCopyright GmbH Wiley-VCH. Reproduced GmbH with.. permission.ReproducedReproduced withwith permission.permission.

A thoroughthorough searchsearch throughthrough thethe CSDCSD forfor {Fe{Fe444((µµ44-C)} motifs exclusively returns clustersclusters inin whichwhich thethe ironsirons sitessites adoptadopt a ‘butterfly’ ‘butterﬂy’ geometry about the carbide, marking the planar geometry displayed in 5 as unique. While the average Fe–Fe bond distance (2.66 0.09 Å) is typical of iron-carbonyl-carbide inin 5 asas unique.unique. WhileWhile thethe averageaverage Fe–FeFe–Fe bondbond distancedistance (2.66(2.66± ±± 0.090.09 Å)Å) isis typicaltypical ofof iron-carbonyl-iron-carbonyl- clusters, the average Fe–C distance of 1.97 0.04 Å is remarkably longer than average for this type carbide clusters, the average Fe–C distance of± 1.97 ± 0.04 Å is remarkably longer than average for this of cluster and, instead, is very close to the average Fe–C distance found in FeMoco (2.00 0.02 Å). type of cluster and, instead, is very close to the average Fe–C distance found in FeMoco (2.00± ± 0.02 ThisÅ). This elongation elongation is—in is—in part—an part—an artifact artifact of of the the displaced displaced position position of of the the carbide carbide from from the the FeFe444 plane,plane, suchsuch thatthat thethe carbide actually resides 0.590.59 ÅÅ aboveabove the least-squares planeplane derivedderived from thethe positions ofof the four four Fe Fe atoms. atoms. Lastly Lastly,,, thethe the Fe–SFe–S Fe–S distancesdistances distances foundfound found inin 5 in areare5 are notablenotable notable asas thethe as average theaverage average distancedistance distance ofof 2.272.27 of ± 0.03 Å, which is considerably elongated when compared to 3 andand 4,, placingplacing itit closercloser toto thatthat ofof FeMoco (2.25 ± 0.03 Å). Of particular note, if ‘belt sulfides’ are excluded from the FeMoco average,

Catalysts 2020, 10, 1317 15 of 22

Catalysts 2020, 10, x FOR PEER REVIEW 15 of 22 2.27 0.03 Å, which is considerably elongated when compared to 3 and 4, placing it closer to that of ± FeMocothe similarity (2.25 is 0.03even Å). more Of pronounced particular note, as the if ‘belt aver sulfides’age Fe–S areof 2.27 excluded ± 0.03 fromÅ in FeMoco the FeMoco is exactly average, on ± thepar similaritywith that isof even 5. Two more of pronounced the S sites (S3, as the S4) average are bonded Fe–S of(2.0 2.2748(2) 0.03Å), resulting Å in FeMoco in the is exactlypresence on of par a ± withpersulfide that of S–S5. Two bond. of the S sites (S3, S4) are bonded (2.048(2) Å), resulting in the presence of a persulfide S–S bond.Purification of the nonpolar fractions collected during the synthesis of 4 provides a related crystalPurification structure of of the formula nonpolar [{Fe fractions4(κ2S–κ collected4C)(CO)10 during}(µ3-S)2 theFe(CO) synthesis3] (6) of(Figure4 provides 11). aAverage related crystal bond structuredistances ofare formula quite similar [{Fe4( κto2S– thoseκ4C)(CO) in 5, with10}(µ 3the-S) 2markedFe(CO) 3difference](6) (Figure being 11). the Average average bond Fe–S distances of 2.25 ± are0.03 quite Å in similar6, which to is those nearly in identical5, with thewith marked that of diFeMoco.fference The being key the difference, average however, Fe–S of 2.25 displayed0.03 Åin ± in6 is6 ,the which absence is nearly of a sulfur identical site with (S4 in that 5), oftherefore, FeMoco. designating The key di ffS3erence, as a second however, face-bridging displayed sulfide in 6 is 2– the(S2–) absence site (in addition of a sulfur to siteS2). (S4The in absence5), therefore, of the S4 designating site, however, S3 as does a second induce face-bridging a break in the sulfide Cs chemical (S ) sitesymmetry (in addition displayed to S2). in Thecluster absence 5, such of thethatS4 the site, Fe5–Fe3 however, distance does has induce shortened a break to in 2.616(1) the Cs chemicalÅ. While symmetrywe found displayedthat synthesis in cluster of cluster5, such 6 that has, the in Fe5–Fe3 fact, been distance previously has shortened reported to from 2.616(1) the Å. reaction While we of foundFe3(CO) that12 with synthesis CS2 at of 80 cluster °C [71],6 thehas, conspicuous in fact, been absence previously of this reported structure from from the the reaction CSD coupled of Fe3(CO) with12 withthe fact CS 2thatat 80 the◦C[ original71], the report conspicuous has seemingly absence remained of this structure in obscurity from from the CSD the bioinorganic coupled with synthetic the fact thatmodelling the original community report has has seemingly hindered remained its utilization in obscurity as a clear from candidate the bioinorganic for nitrogenase synthetic modellingstructural communitymodeling. has hindered its utilization as a clear candidate for nitrogenase structural modeling.

FigureFigure 11.11. ThermalThermal ellipsoidellipsoid plotsplots (50%(50% probability) probability) of of [{Fe [{Fe4(κ4(2κS–2S–κ4κC)(CO)4C)(CO)1010}(}(µµ33-S)-S)22Fe(CO)3]]( (6). OrangeOrange == Fe, Gray == C, Yellow == S, Maroon = O. Image adapted with permission from Joseph, C., Cobb,Cobb, C.C. R.,R., Rose,Rose, M.M. J.J. Single-StepSingle-Step InsertionInsertion ofof SulﬁdesSulfides andand ThiolateThiolate intointo IronIron Carbide-CarbonylCarbide-Carbonyl Clusters:Clusters: Unlocking thethe SyntheticSynthetic DoorDoor toto FeMocoFeMoco Analogues.Analogues. Angew.Angew. ChemieChemie Int.Int. Ed. 2020, Accepted, notnot yetyet publishedpublished.. CopyrightCopyright Wiley-VCHWiley-VCH GmbH.. ReproducedReproduced withwith permission.permission.

InIn additionaddition to to the the biologically biologically relevant relevant bond bond metrics metrics observed observed in the twoin the {CS}-containing two {CS}-containing clusters, weclusters, found we that found the dangler that the Fe dangler site in clusterFe site 5inalso cluster approaches 5 also approaches similarly relevant similarly electronic relevant properties electronic (i.e.,properties approaching (i.e., approaching a ferrous oxidation a ferrous state). oxidation X-ray photoelectronstate). X-ray photoelectron spectroscopy (XPS)spectroscopy of 5 could (XPS) be ﬁt of as 5 3could distinct be componentsfit as 3 distinct at bothcomponents the Fe 2 pat(3 both/2) region the Fe (711.6, 2p(3/2) 709.4, region and (711.6, 707.5 eV)709.4, and and the 707.5 Fe 2p eV)(1/2) and region the (724.6,Fe 2p(1/2) 722.2, region and 720.5(724.6, eV) 722.2, with and integrated 720.5 eV) peak with area integrated ratios of 1:2:2,peak respectivelyarea ratios of (Figure 1:2:2, respectively12). On the basis(Figure of symmetry,12). On the the basis Fe5 of site symmetry, could be assignedthe Fe5 site to thecould higher be assigned binding energyto the higher peak (highest binding valence energy statepeak Fe(highest site). Thisvalence assignment state Fe wassite). additionally This assignment supplemented was additionally by density-functional supplemented theory by density- (DFT) calculations,functional theory indicating (DFT) Fe5 calculations, to be the Feindicating site with Fe5 the to least be electronthe Fe site density with the on theleast basis electron of Mulliken density populationon the basis analysis of Mulliken and back-bondingpopulation analysis contribution and back-bonding into the coordinated contributi COs.on into The the assignment coordinated of XPSCOs. peaks The assignment at 711.6 eV of and XPS 724.6 peaks eV at additionally 711.6 eV an fallsd 724.6 within eV additionally the expected falls range within for a the ferrous expected and CO-supportedrange for a ferrous iron and site CO-supported [72,73]. The conclusions iron site [72, drawn73]. The from conclusions the XPS datadrawn are from also the consistent XPS data with are thealso higher consistent CO stretchingwith the higher frequencies CO stretching observed freq inuencies the IR spectrumobserved in the which IR spectrum all νCO values in which range all upward from 2002 cm 1. These are signiﬁcantly blue-shifted relative to 3 (ν 1973 cm 1) and νCO values range upward− from 2002 cm−1. These are significantly blue-shifted relativeCO ≥ to 3 (νCO− ≥ 1973 4 (ν 1991 cm 1) and is additionally consistent with the presence of a ferrous site, which is not cm−CO1) and≥ 4 (νCO ≥− 1991 cm−1) and is additionally consistent with the presence of a ferrous site, which typicallyis not typically observed observed in CO-supported in CO-supported FeC clusters. FeC clusters.

Catalysts 2020, 10, 1317 16 of 22 Catalysts 2020, 10, x FOR PEER REVIEW 16 of 22

Figure 12.12. ObservedObserved high-resolution high-resolution X-ray X-ray photoelectron photoelectro spectrumn spectrum (XPS) (XPS) and component and component fitting offitting the iron of the2p region iron 2 ofp region cluster of5. cluster Image adapted5. Image with adapted permission with permission from Joseph, from C., Cobb, Joseph, C. C., R., Rose,Cobb, M. C. J.R., Single-Step Rose, M. J.Insertion Single-Step of Sulfides Insertion and of Thiolate Sulfides into and Iron Thiolate Carbide-Carbonyl into Iron Carbide-Carbonyl Clusters: Unlocking Clusters: the Synthetic Unlocking Door the to SyntheticFeMoco Analogues. Door to Angew.FeMoco Chemie Analogues. Int. Ed. Angew.2020, Accepted, Chemie not Int. yet Ed. published 2020,. CopyrightAccepted, notWiley-VCH yet published GmbH.. CopyrightReproduced Wiley-VCH with permission. GmbH. Reproduced with permission.

In many respects, the construction of clusters 5 and 6 elevateselevates the the family family of of carbonyl-supported, carbonyl-supported, carbide-containing ironiron clusters clusters into into the realmthe realm of highly of structurallyhighly structurally relevant modelsrelevant for models the nitrogenase for the nitrogenaseactive site. Comparisonactive site. Comparison of bond metrics of bond between metrics the between clusters the with clusters FeMoco with (Table FeMoco1) conveys (Table the 1) conveysapproaching the approaching similarity in similarity5 and 6 toward in 5 and that 6 toward analogous that FeMoco analogous metrics. FeMoco However, metrics. the However, presence the of 4– presencea true C–S of bond a true [1.714(5) C–S bond Å] does[1.714(5) preclude Å] do thees preclude C from being the C an from authentic being Can authenticcarbide. Nonetheless, C4– carbide. Nonetheless,this unusual bondingthis unusual motif bonding (only three motif entries (only inthree the entries CSD for in transition-metal-bound the CSD for transition-metal-bound {C–S}—none {C–S}—noneof which contain of which iron) iscontain reminiscent iron) ofis thereminiscent proposed of biogenesis the proposed mode biogenesis of carbide insertionmode of intocarbide the 4 insertionM-cluster into as postulated the M-cluster by Wiig, as postulated Hu, and Ribbe by Wiig, depicted Hu, in and Figure Ribbe2[ 11 depicted,12]. The in presence Figure 2 of [11,12]. the {C–S} The− presencemotif (i.e., of tetra-deprotonated the {C–S}4− motif (i.e., methylthiol) tetra-deprotonated in 5 and 6, methylthiol)thus, serves asin 5 the and ﬁrst 6, rudimentarythus, serves as structural the first rudimentarymodel for intermediates structural model in M-cluster for intermediates biosynthesis. in M-cluster Such intermediates biosynthesis. would Such intermediates likely be diﬃ wouldcult to likelyisolate be for difficult crystallographic to isolate characterization. for crystallographic On the characterization. other hand, comparative On the other spectroscopic hand, comparative studies of spectroscopicsynthetic cluster studies compounds of synthetic have previouslycluster compou demonstratednds have utilitypreviously in the demonstrated determination utility of structural in the determinationaspects of nitrogenase, of structural such as aspect the assignments of nitrogenase, of the central such carbideas the a (whichssignment itself of utilized the central the Fe 6carbidecluster precursor(which itself described utilized above the Fe as6 cluster a reference precursor compound) described [8,9 ].above The assortmentas a reference of ironcompound) clusters [8,9]. that have The assortmentbeen developed of iron by clusters our lab nowthat presenthave been a range developed of reference by our compounds lab now present available a range for spectroscopic of reference compoundscharacterization available and comparisonfor spectroscopic against characterization the various stages and ofcomparison nitrogenase against biosynthesis: the various Clusters stages1 ofand nitrogenase2 provide examplesbiosynthesis: of comparable Clusters 1 and alkylated 2 provide and non-alkylatedexamples of comparable FeS clusters alkylated as appropriate and non- for alkylatedearly-stage FeS NifB clusters reactions, as appropriate while clusters for early-stage5 and 6 bearing NifB reactions, the {C–S} while motif clusters present model5 and 6compounds bearing the {C–S}for intermediates motif present in model late-stage compounds NifB activity. for intermediates in late-stage NifB activity.

Table 1. Selected bond distance averages of the FeCS clusters described in this work compared with the corresponding averages found in the nitrogenase cofactor (FeMoco).

3 4 5 6 FeMoco Fe–Fe 2.65 ± 0.06 2.64 ± 0.05 2.66 ± 0.09 2.7 ± 0.1 2.63 ± 0.04 Fe–C 1.88 ± 0.03 1.88 ± 0.04 1.97 ± 0.04 1.96 ± 0.02 2.00 ± 0.02 Fe–S 2.19 ± 0.01 2.175 ± 0.005 2.27 ± 0.03 2.25 ± 0.03 2.25 ± 0.03

Catalysts 2020, 10, 1317 17 of 22

Table 1. Selected bond distance averages of the FeCS clusters described in this work compared with the corresponding averages found in the nitrogenase cofactor (FeMoco).

3 4 5 6 FeMoco Fe–Fe 2.65 0.06 2.64 0.05 2.66 0.09 2.7 0.1 2.63 0.04 ± ± ± ± ± Fe–C 1.88 0.03 1.88 0.04 1.97 0.04 1.96 0.02 2.00 0.02 ± ± ± ± ± Fe–S 2.19 0.01 2.175 0.005 2.27 0.03 2.25 0.03 2.25 0.03 ± ± ± ± ±

3. Extension of Electrophilic Sulfur Ligation toward Fine-Tuning of Cluster Thiolates

3.1. Electronic Effects of Fe-Alkyl Bonding in Iron-Sulfur Cubane Clusters The alkylated clusters generated by the Suess group (vide supra) illustrate how binding of an alkyl fragment at a single iron site significantly alters the oxidation states of the irons within the cluster. 2+ Classically, a [Fe4S4] cluster with relatively weak-field and uniform ligands features an overall S = 0 spin state with four Fe2.5+ sites in high-spin configurations featuring double exchange within two pairs + of [Fe2S2] units that antiferromagnetically couple with each other. This holds true for the chloride 2+ 2+ precursor of the [Fe4S4] -ethyl cluster and for many thiolate-ligated protein-bound [Fe4S4] clusters. Suess et al. show that the introduction of a strong-field, electron rich alkyl ligand at the apical iron site polarizes the double-exchange interaction and introduces ferric character at the apical alkyl-bound iron. Correspondingly, it introduces ferrous character at its spin-aligned partner iron site with no overall change in charge [48]. 3+ This effect was replicated in the reported [Fe4S4] -methyl structure where the ferric character was found to be localized at the alkylated iron site. Furthermore, solution studies of this structure suggest rapid interconversion of the oxidation states at the three chelated iron sites, resulting in a shared oxidation state of essentially 2.67+. Overall, alkylation has a profound and unique effect on oxidation states of the irons within both the 2+ and the 3+ Fe4S4 clusters [49]. These two synthetic structures represent the first direct bioinorganic insight into Fe-alkyl bonding and the electronic attributes of Fe4S4-alkyl intermediates in nature. Perhaps most essential for the field, the range of chelating ligand scaffolds employed, and the demonstrated impact of remote steric effects on apical substrate binding in this class of clusters promise a drastic increase in the degree of synthetic control over iron-sulfur clusters [74]. This approach has already allowed for the isolation of increasingly elusive intermediates as well as the study of the reactivity of radical formation from alkyl intermediates [48,49,75], and suggests one possible route toward controlling the environment of reactive intermediates and active sites in other iron-sulfur metalloclusters as well.

3.2. New Routes toward Carbide-Containing Iron-Thiolato Clusters The structure and reactivity of these alkyl clusters present novel findings relevant to radical SAM, but not directly relevant to the NifB intermediate. However, the growing family of chelating ligands and the control they afford over the oxidation states and reactivities of these clusters suggest new directions in rational cluster design that may yield novel insights into nitrogenase biogenesis intermediates [48,49]. Potential for any ligand-directed, cluster-based nitrogenase modeling remains to be borne out, but the recent results from Suess et al. suggest the stabilization of more reactive metalloclusters may be accessible through the design of suitable chelating ligand systems. Historically, controlled ligation of sulfides and thiolates onto iron carbonyl carbide clusters has proven difficult to control and would generally result in cluster decomposition. However, upon the discovery that the [Fe6] dianion was amenable toward substitution by an electrophilic sulfur source, we elected to extend the utility of this synthetic strategy toward the incorporation of a thiolato ligand. 2 As a proof of concept, p-toluenesulfenyl chloride was reacted with the [Fe6] − cluster, yielding pink crystals of the first-ever reported thiolato-iron-carbide cluster (Figure 13)[67]. This controlled ligation of an iron-carbide cluster presents an as-yet-unrealized golden opportunity to finally engage this family of interstitial carbide clusters to be functionally “fine-tuned” with the introduction of an electronically Catalysts 2020, 10, 1317 18 of 22 diverse range of ligands. As such, a new branch has sprouted in our own research program, and a pursuit for elegantly designed, functionally active carbide-containing iron clusters, which have, for so long,Catalysts evaded 2020, 10 the, x FOR synthetic PEER REVIEW modeling community, may now be forthcoming. 18 of 22

Figure 13.13. ReactionReaction conditionsconditions (left)(left) toto obtain obtain (NEt (NEt4)[Fe4)[Fe5(5µ(µ55-C)(SC-C)(SC77H77)(CO)13]] and and thermal ellipsoid plots (50% probability) of thethe thiolate-bearingthiolate-bearing clustercluster anionanion (right).(right). Image adapted with permission from Joseph, C., Cobb, C. R., Rose, M. J. Single-StepSingle-Step Insertion of SulﬁdesSulfides and Thiolate into Iron Carbide-Carbonyl Clusters:Clusters: UnlockingUnlocking the the Synthetic Synthetic Door Door to to FeMoco FeMoco Analogues. Analogues.Angew. Angew. Chemie Chemie Int. Int. Ed. 2020Ed. 2020, Accepted,, Accepted, not yetnot publishedyet published. Copyright. CopyrightWiley-VCH Wiley-VCH GmbH GmbH. Reproduced. Reproduced with with permission. permission.

4. Closing Remarks The pursuit for model compounds of structural re relevancelevance to FeMoco continues to inspire many creative research avenuesavenues forfor syntheticsynthetic chemists.chemists. Still, Still, the the “Holy “Holy Grail” for synthetic FeMoco modeling—a paramagneticparamagnetic iron iron cluster cluster with with inorganic inorganic sulfides, sulfides, an interstitial an interstitial carbide, and carbide, heterometal and Mo—remainsheterometal Mo—remains unaccomplished. unaccomplished. Success in this Success pursuit in will this allow pursuit for will insight allow into for the insight role of into the the carbide role asof athe structural carbide as and a structural electronic and modulator electronic in nitrogenasemodulator in and nitrogenase understanding and understanding into how the electronicinto how propertiesthe electronic of properties NifB affect of carbide NifB affect incorporation. carbide incorporation. Thus far, synthetic Thus far, modelssynthetic focusing models focusing on FeMoco on haveFeMoco shown have that shown the carbidethat the likely carbide facilitates likely facilitate structurals structural flexibility flexibility of the cluster of the and cluster even promotesand even substratepromotes bindingsubstrate and binding reduction and reduction (vide supra). (vide Other supra). modelling Other modelling avenues have avenues given have insight given into insight how suchinto how a C-donor such a ligandC-donor may ligand promote may verypromote strong very antiferromagnetic strong antiferromagnetic coupling incoupling the cluster in the and cluster play aand significant play a significant electronic electronic role in the role cofactor. in the cofactor. In our own venture venture to progress ever closer towardtoward achieving this goal, we have utilized carbidocarbonyl iron iron clusters clusters as as a synthetic a synthetic starting starting point point for structural for structural modeling modeling of the nitrogenase of the nitrogenase cofactor. 2 Whilecofactor. [Fe While6(µ6-C)(CO) [Fe6(µ166-C)(CO)] − had16 previously]2− had previously served a served crucial a role crucial in the role assignment in the assignment of the interstitial of the carbideinterstitial in FeMococarbide (and in laterFeMoco FeVco), (and further later utilizationFeVco), further of carbonyl-clusters utilization forof nitrogenasecarbonyl-clusters modelling for isnitrogenase sparse. For modelling our own research,is sparse. a two-foldFor our strategy own research, toward pursuinga two-fold nitrogenase-relevant strategy toward pursuing synthetic clustersnitrogenase-relevant was employed. synthetic First, inspiration clusters was was employed. taken from First, the radical-initiated inspiration was biosynthetictaken from the mechanism radical- asinitiated proposed biosynthetic by Ribbe mechanism and Hu in as which proposed an initial by Ribbe SAM and methylates Hu in which the K-cluster. an initial SAM The methylatedmethylates FeSthe clusterK-cluster. is subsequentlyThe methylated dehydrogenated FeS cluster byis subsequently an additional dehy equivalentdrogenated of SAM. by Thean pursuitadditional of thisequivalent strategy of forSAM. carbide The pursuit insertion of ledthis us strategy to explore for carbide TEMPO-mediated insertion led reactions us to explore upon alkylatedTEMPO- Femediated3S clusters. reactions Although upon carbide alkylated insertion Fe3S ultimatelyclusters. Although proved unsuccessful, carbide insertion the methylated ultimately precursor proved clustersunsuccessful, were successfullythe methylated isolated precursor and characterized. clusters were Alongsuccessfully with the isolated Fe3S precursor and characterized. [Fe3(µ3-S)(CO) Along9], thesewith the clusters Fe3S precursor do provide [Fe a structurally3(μ3-S)(CO)9], interesting these clusters synthetic do provide comparison a structurally for early-stage interesting NifB synthetic reactions. Incomparison an alternative for early-stage synthetic NifB strategy, reactions. the carbide-containing In an alternative synthetic Fe6 cluster strategy, served the ascarbide-containing a starting point forFe6 sulfurizationcluster served reactions. as a starting The point reaction for withsulfurizat electropositiveion reactions. sulfur The reagents reactionproved with electropositive successful in 4 achievingsulfur reagents an FeCS proved cluster, successful and we were in achieving additionally an Fe pleasedCS cluster, to discover and we two were {C–S} additionally−-containing pleased clusters, to buildingdiscover upontwo {C–S} our series4−-containing of biogenesis-relevant clusters, building models upon with our appropriate series of biogenesis-relevant compounds for late-stage models NifBwith reactivityappropriate modeling. compounds Future for pursuits late-stage to close NifB the reactivi gap betweenty modeling. the early-stage Future andpursuits late-stage to close NifB the models gap wouldbetween see the us aspireearly-stage toward and partially late-stage protonated NifB/ hydrogenatedmodels would {SCH see2} andus {SCH}aspire structurestoward partially [76]. protonated/hydrogenated {SCH2} and {SCH} structures [76].

Author Contributions: Writing—Original draft, C.J., J.P.S., and C.R.C. Visualization, C.J. Funding acquisition, M.J.R. Supervision, M.J.R. All authors have read and agreed to the published version of the manuscript.

Funding: The research related in this perspective was funded by the National Science Foundation (NSF CHE- 1808311) and the Robert A. Welch Foundation (F-1822).

Catalysts 2020, 10, 1317 19 of 22

Author Contributions: Writing—Original draft, C.J., J.P.S. and C.R.C. Visualization, C.J. Funding acquisition, M.J.R. Supervision, M.J.R. All authors have read and agreed to the published version of the manuscript. Funding: The research related in this perspective was funded by the National Science Foundation (NSF CHE-1808311) and the Robert A. Welch Foundation (F-1822). Conﬂicts of Interest: The authors declare no conﬂict of interest.

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