Oxygen‐Depleted Calixarenes as Ligands for Molecular Models of Galactose Oxidase

Abstract A calix[4]arene ligand, in which two of the phenol functions are replaced by pyrazole units has been employed to mimic the His2–Tyr2 (His: histidine, Tyr: tyrosine) ligand sphere within the active site of the galactose oxidase (GO). The calixarene backbone forces the corresponding copper(II) complex into a see‐saw‐type structure, which is hitherto unprecedented in GO modelling chemistry. It undergoes a one‐electron oxidation that is centered at the phenolate donor leading to a copper‐coordinated phenoxyl radical like in the GO. Accordingly, the complex was tested as a functional model and indeed proved capable of oxidizing benzyl alcohol to the respective aldehyde using two phenoxyl‐radical equivalents as oxidants. Finally, the results show that the calixarene platform can be utilized to arrange donor functions to biomimetic binding pockets that allow for the creation of novel types of model compounds.

Abstract: Ac alix [4]arene ligand, in which two of the phenolf unctions are replaced by pyrazole units has been employed to mimic the His 2 -Tyr 2 (His:h istidine, Tyr: tyrosine) ligand sphere within the active site of the galactose oxidase( GO). The calixarene backbone forcest he corresponding copper(II) complex into as ee-saw-type structure, whichi sh itherto unprecedented in GO modelling chemistry.I tundergoes ao ne-electron oxidation that is centered at the phenolate donorl eading to ac opper-coordinated phenoxyl radical like in the GO. Accordingly,t he complex was tested as af unctional model and indeed proved capable of oxidizing benzyl alcohol to the respective aldehyde using two phenoxyl-radical equivalents as oxidants. Finally,t he results show that the calixarene platform can be utilized to arrange donor functions to biomimetic binding pocketst hat allow for the creationo f novel types of model compounds.
Macrocyclic polyphenols-coined calixarenes by C. D. Gutsche-have been known for almost8 0years now. [1] Their highyield preparation from cheap commercially availables tarting materials and easy post modifications have made them wellestablished and often utilized macromolecules in diversef ields of chemicalr esearch and applications. [2] Calixarenes were also employeda sl igands in transition-metal chemistry,f or instance, as am imic of oxidic surfaces and hencef or the modelling of the active sites of heterogeneous catalysts. [3] However,g iven that they provideacoordinationp latform that contains exclusively hard oxygen donors, they have hardly been used in biomimetics tudies. There are no enzymes with such binding pockets composed for obvious reasons:h ard metal centers in high oxidation states (e.g. Fe III or Fe IV )m ainly occur in reactive intermediatesa sp art of catalytic cycles and in the course of turnover these are reduced to softer,l ow-oxidation-state metal centers (e.g. Fe II ), which prefers oft ligands. Metal-binding sites of metalloenzymes have to balance these two different demandsa nd therefore often feature mixedl igand spheres, whichhave to be mimicked also in models. Hence, it is not surprisingt hat calixarenes, so far,h ave rarely been employed in bioinorganic chemistry.O nly modified forms in which the phenol units are functionalized by pendant N-donors were investigated, for instance,i nb iomimetic copper or zinc chemistry. [4] From the structural point of view,t he introductiono ft he pendant groups makes the metal complex more flexible. Even though not necessarily detrimental to the desired activity,t his flexibility eliminates one attractive feature of the calixarene platform,n amely its potential to direct donors to aw ell-defined binding pocket, which rendersc alixarenes appealing as three-dimensional analogueso fr igid multidentates ystems, such as salenso rp orphyrins.
We were interested in exploiting calixarene-shaped binding sites also in bioinorganic investigations,w hichr equired the introduction of soft donor atoms directly in place of one or more oxygena tomso ft he calixarene lower rim. Recently, some of us have developed the oxygen-depleted calixarene bispyrazolyl-tert-butyl-calix [4]arene ([H 2 (bpzCal)],F igure1)f eaturingt wo pyrazole moieties besidet wo phenolicd onors, [5] whichi sr eminiscent of aH is 2 -Tyr 2 coordination sphere found in certain enzymes. Oneo ft hose is the galactose oxidase( GO) and hence we decided to test the potentialo ft he [bpzCal] 2À ligandt oc onstruct molecular modelso ft he GO.  [5] Right:Similarities of the ligand's donorsw ith natural amino acid side chains.  The GO catalyzes the oxidation of primary alcohols to the corresponding aldehydes. In the restings tate it contains ac opper(II) ion coordinated by two tyrosine and two histidine amino acid residues ( Figure 2). [6] Previous attempts to mimic this coordination environmento ften used salen ligands, which, however, forces the central ion into an almost square-planar coordination environment. This rigidity has provent oi mpede changes in oxidation state, because the Franc-Condon barrier is high in energy as evidenced by modelc omplexes that were oxidizedm ost easily when ah igh degree of distortion at the metal center was given. [7] In contrast to salens,c alixarenes provide am ore variablec oordination site, which, however,i ss till well-defined and pre-organized for metal-ion complexation.
X-ray structure analyses of single crystalst hat were grown by slow evaporation of the volatiles from THF solutions revealed almostidenticalcoordination spheres for the metal centers in the two complexes,c omposed of two phenolate and two pyrazole donors ( Figure 3a nd Figure S14 in the Supporting Information).
In both cases, there are two crystallographically independent molecules in the unit cell, the metrical parameters of which are almosti dentical.R egarding the t d value,t he coordination geometry of the metal ions can be described best as intermediate between tetrahedral ands quare-planar,c ommonly called the seesaw coordination. [11] To the best of our knowledge, these two complexes are the first examples of structurally characterized mononuclear nickel and copperc alixarene complexes in which phenolic oxygen donors directly coordinatet he metal and therefore interactwithoutany spacers.
Both compounds are in high-spin configurations with magnetic moments of m eff = 3.16 and m eff = 1.91 m B for [Ni(bpzCal)] (two unpaired electrons)a nd [Cu(bpzCal)] (one unpaired electron) respectively,a sd etermined by the Evans methodo n CD 2 Cl 2 solutions of the complexes. Therefore only [Cu(bpzCal)] is X-band EPR active ( Figure S10 in the Supporting Information) in contrast to [Ni(bpzCal)] for which the large zero-field splitting (ZFS) of the S = 1s pin state results in the absence of any observable signal.  Cyclovoltammetric (CV) measurements on both complexes surprisingly revealed variations in the electrochemical behavior:i nc ontrast to [Ni(bpzCal)],w hich exhibits only one quasireversible oxidation wave at E1 = 2 = 130 mV,[ Cu(bpzCal)] can be oxidizedasecond time at ap otential of 2 E = 670 mV ( 1 E1 = 2 = 110mV) althought his event is highly irreversible (Figure 4). To understand the origin of the oxidation (metal-or ligand-based) and the differing behavior,t he corresponding complexo ft he redox-inert metal zinc [Zn(bpzCal)] was analyzed. Thisc ompound also exhibits oxidation events in the CV in as imilar region. However,i nc ontrast to the cyclic voltammograms of the lighter analogues,t he zinc complex exhibits two quasi-reversible oxidation waves ( 1 E1 = 2 = 135, 2 E1 = 2 = 340 mV) separated by only 205 mV (Figure 4). This indicatest hat the redox events found for the previously mentioned complexes likely are not metal-based but involve the phenolate functions.
The different appearances of the three CVs can then be explained by the different degrees of electronic delocalization and communication after the first oxidation event:t he electronic communicationb etween the oxidized and the remaining second phenolate cannotb em ediated effectively by the centrali on in the case of zinc because of its completely filled d 10 configuration, leading to two oxidations at potentials that do not differ significantly; [12] indeed there are also examples reported in whichb oth electrons are removed simultaneously. [13] The communication can be facilitated by the open-shell 3d metal ions nickel(II) and copper(II), so that the removal of a second electron is affected more significantly. [14] Hence, in the case of [Cu(bpzCal)] this second oxidation is shiftedt om uch higher potentials and is irreversible in nature. This shift will be even more pronouncedi nt he case of [Ni(bpzCal)] so that it is not observable anymore in the potentialw indow of the used solvent.
To confirm the inferences made above spectroscopically,w e performed spectroelectrochemicalm easurements. When col-lecting UV/Vis spectra whileg oing through the first oxidation waves of the neutralc omplexes with al ow scan rate, the formationo fnew bands was observed( Figure5).
The electronic absorption spectra of the oxidized species have an ew stronga bsorption band around 400 nm in common (402 for [Ni(bpzCal)] + + and 392 nm for [Cu(bpzCal)] + + ). Given that [Zn(bpzCal)] + + also shows such ab and at 401 nm, these absorptions ( Figures S8 and S9 in the Supporting Information) can be assigned to the p-p*t ransition of ap henoxyl radical. [15] For further investigations, we headed towards chemical oxidation of the complexes. AgSbF 6 (E1 = 2 = 650 mV vs. Fc/Fc + + ) [16] provedt ob eas uitable one-electrono xidant to realize the first oxidation. Addition of one equivalent of the silver salt to solutions of dark-blue [Cu(bpzCal)] or deep-red [Ni(bpzCal)] in CH 2 Cl 2 caused an immediate color change to dark-green or dark-orange, respectively,a ccompanied by precipitation of elementals ilver.T hese oxidation products gave UV/Vis spectra identicaltot hose of the electrochemically generated species.
In previous extensive studies resonanceRaman (rR) spectroscopy has provenapowerful tool fort he detectiono fp henoxyl radicals. When CH 2 Cl 2 solutions of the oxidized compounds are excited with a4 13 nm laser,w hich matches the electronic transitionso ft he radical at around 400 nm well, especially the modes n 7a and n 8a of the phenoxyl radicalsa re enhanced ( Figure 6).
In contrastt ot he nickel derivative, [Cu(bpzCal)] + + shows a strongb and at 1484 cm À1 ,w hich can be assigned to ap henolate vibration by comparison with the reduced complex ( Figure S6 in the Supporting Information). This points towardsalocalization of the radicali nt his case. Clearly,t he nickel compoundf eatures am ore delocalized electronic structure and therefore lacks ap henolate band in its rR spectrum.B oth complexese xhibit ab and at around1 520 cm À1 assignable to the n 7a mode of the CÀOv ibration. The frequencies of these bands are somewhathighert han those found for other known metal-  coordinated phenoxylr adicals pecies, suggesting al arger contribution of the quinonoid canonical form in our case. The n 8a mode of the C ortho ÀC meta vibration appearsata round1 600 cm À1 in both cases fitting wellw ith reported values. [17] Galactose oxidase in its activef orm has ad iamagnetic ground state through antiferromagnetic couplingo ft he ligand-radical spin with the unpaired d-electron of the copper(II) metal ion. [18] Therefore, it was of interest to determine which spin state the phenoxyl radicalc omplexes described above adopt. Given the limited stability of the oxidized species in the solid state, no reliable SQUID data could be obtained. However,m agnetic susceptibilities in solution could be determined by using Evans method: [Ni(bpzCal)] + + has am agnetic momento fm eff = 3.67 m B fitting well with the expected value for three unpairede lectrons, thereby suggesting a S = 3 = 2 spin state. In contrast [Cu(bpzCal)] + + has am oment of m eff = 2.66 m B which is closet ot wo unpaired electrons of a S = 1s pin state, definitely excluding adiamagnetic ground state in our case.
To furtherc orroborate the actual spin states,X -band EPR measurements of frozen solutions were performed: in contrast to its neutralp recursor,[ Ni(bpzCal)] + + is EPR active and exhibits ar hombic signalt hat was simulated best with g x = 2.36, g y = 2.34, and g z = 2.24 ( Figure S12 in the Supporting Information). The g-factors of typical Ni III speciesa re usually somewhat lower at around2 .1, thusametal centered oxidationi no ur case can again be excluded. The signals are not well resolved, which may be attributed to the general difficulty of collecting EPR spectra of S = 3 = 2 systems at liquid-nitrogent emperatures. The originalE PR signal of [Cu(bpzCal)] is attenuated in the course of the oxidation leaving only ar esidual signal fora no rganic radical( g % 2) togetherw ith one forc opper(II) (FigureS11). They account for less than 10 %oft he total spin concentration, likely originating from ad isproportionation reactiony ielding Cu II and at wofold oxidized species. [Zn(bpzCal)] + + is the most unstable complex of the series, thus only traces ( % 10 %) of an organic radicalw ith g iso = 2.00 were detected by EPR (Figure S13).
Given that no crystals could be grown to investigate the molecular structures of these compounds due to the high instabilityo ft he oxidized complexes, DFTcalculations were per-formed to gain insights into possible structures and spin states:f or [Cu(bpzCal)] + + two isomersw ere found, one with the same symmetry as its neutral precursor and one asymmetric version, both in as inglet and at riplet state ( Figure 7). With exception of the symmetric closed-shell singlet state, all structures and states are very close in energy (Table S1 in the Supporting Information), with the asymmetric triplet as ground state, matching the results described above.
Both in the symmetric and the asymmetric isomer of [Cu(bpzCal)] + + the Cu atom wast hen replaced by Ni followed by re-optimization.G iven that Ni has one electron less than Cu, doublet and quartet states result. For the doublet state, a symmetric and an asymmetric isomer were found, however,f or the quartets tate all optimizationsr esulted in as ymmetric structure, whichi st he ground state of the molecule (Table S2 and Figures S19-S21 in the Supporting Information), which is in agreement with the experimental findings.
Hence, from the above data it becomes very clear that a complex with copper(II) ion in ac oordination sphere resembling the one in the active site of the GO can be generated and that it can be oxidized at the phenolate donor to yield a radicalc omplex with at riplet ground state. This complex compares well with the actives tate of the GO, with the difference that the latter has as inglet ground state. To that extent it was now of much interestt ot est the reactivity of our complexes towardsaprimary alcohol.
Accordingly, chemically generated solutions of [Cu(bpzCal)] + + and, for comparison, also [Ni(bpzCal)] + + in CH 2 Cl 2 were treated with benzyl alcohol as the models ubstrate. The formationo f the two-electron oxidation product benzaldehyde was indeed detected through 1 HNMR spectroscopy;h owever,u nlike in case of the enzymatic paragon,f ormation of only half an equivalent of the aldehyde per equivalent complex was observed( Scheme2).
Although in the enzymatic catalytic cycle the Cu II center performs the second oxidation step (!Cu I )i ts oxidation potential in the complex is obviously not sufficiently positive, so that a second equivalent of the complex is needed. [19] Indeed, the CV of [Cu(bpzCal)] exhibits ar edox event assigned to the Cu 2+ + /  Cu + + couple ( Figure S7 in the Supporting Information) at ap otential ( red E = À1.53 V) that is not suitable to performt he alcohol oxidation.
In conclusion, we reported here am odel of the GO with a unique biomimetic donor sphere around the copperc enter that is provided by ac alixarene framework. In contrastt om ost model complexes known so far [Cu(bpzCal)] features an onplanar structure that resembles the one of the His 2 -Tyr 2 core found for the enzyme, which, however,c oordinates in the active state an additional water molecule and is able to release one of the Tyrd onors upon protonation. Like the enzyme, the model can be singly oxidized to yield ap henoxyl radical coordinating the copperc enter,m imicking the actives tate of the GO albeit with ad ifferents pin state. The latter,h owever,i sn ot decisivef or the reactivity:t he oxidizedc omplex was capable of converting benzyl alcohol to the corresponding aldehyde, like the GO does, but through ad ifferent mechanism involving two equivalents of the complex. To mimic the two-electron oxidation more faithfully,future attempts will focusont he stabilization of the copper(I) state through the ligand system so that the copper(II)s tate becomes moreo xidizing.