extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Chemistry Biology

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Charge-tagged ligands: useful tools for immobilising complexes and detecting reaction species during catalysis

A critical overview is presented on the use of charged tagged ligands (CTLs) as immobilising agents in organometallic catalysis and as probes for studying mechanisms through electrospray ionisation mass spectrometry (ESI-MS) based on the most recent literature.

Introduction
Undoubtedly, the choice of an appropriate solvent is vital for any organic transformation. 1 The reaction media can dramatically affect the yield and selectivity of a reaction, owing to several factors including the reagents, the solubility of the products and catalysts as well as charge stabilisation. The importance of this issue is increased in homogeneous biphasic catalysis. Indeed, an incorrect solvent pairing can lead to low conversions, poor product extraction, no phase separation and catalyst lixiviation. 2 Historically, one of the best strategies to avoid lixiviation is to insert organic moieties into the ligand structure with the same features for anchoring the media, 3 for example uorinated ligands in organouorinated solvents, 4,5 polyethyleneglycol (PEG)-substituted ligands in PEG media [6][7][8] and charged ligands in highly polar solvents. [9][10][11] The use of charged ligands has attracted special attention, because water can be used as an anchoring medium, thereby combining sustainability and efficiency. 11,12 The rst successful protocol was the application of monosulphonated phosphines in rhodium-catalysed propene hydroformylation. [13][14][15] With the emergence of ionic liquids (ILs), the insertion of an ion-tagged side chain into the molecular skeleton of a known ligand became a useful protocol for anchoring ligands, and consequently catalysts, in an IL phase. 16 These ligands are referred to as ionophilic ligands or charge-tagged ligands (CTLs). The ionic modication confers a particular solubility prole that makes catalyst/product recovery possible, and oen improves the activity of the catalytic species compared to the parent tag-free analogue. 17 Moreover, new selective processes can be envisaged by changing the solubility and diffusion of substrates and products in the active phase of a reaction. 18 In addition to the change in catalyst partition, the insertion of a cationic moiety into various reagents represents a powerful tool that can be used to detect reaction intermediates in organometallic catalysis through electrospray ionisation mass spectrometry (ESI-MS) experiments. 19 Taking into account the fact that ESI constitutes a mild ionisation technique, some neutral intermediates may not be ionised and, therefore, are not detected by MS. 19 To overcome the detection problem of these "ESI-MS-blind intermediates", CTLs have been used. 20,21 The insertion of an ionic tag ensures that the charge of an intermediate is not caused by the ESI. For this reason, these ligands have been used as ionic probes in mechanistic studies for several catalytic reactions. Scheme 1 illustrates the application of CTLs for both polar phase anchoring and ESI-MS detection.
There are extensive reviews covering both ESI-MS detection and the anchoring processes; a review of catalysts with ionic tags and their use in ILs was published in 2008. 16 In the same year, a perspective review of using charged ligands for catalyst immobilisation and analysis appeared. 21 In addition, it was shown that the use of a proper ionic tag can improve the performance of catalytic systems. 22 Herein, we summarise examples from our laboratory and, where appropriate, recent literature studies on the use of CTLs as immobilising agents in organometallic catalysis and as probes for studying mechanisms through ESI-MS.

CTLs as immobilising agents for organometallic catalyst precursors
Olen metathesis reactions. Our group has dedicated the recent years to synthesising imidazolium-based metathesis CTLs. We have used these ligands to immobilise homogeneous catalysts in ILs. CTLs 1 and 2, for example, were easily obtained using three-step routes. These ligands were reacted with a second-generation Grubbs catalyst to produce the ionophilic Hoveyda-type complexes 3, 4 and 5 (Scheme 2). 23 These complexes were successfully applied to ring-closing metathesis (RCM) reactions. Initially, it was observed that a judicious choice of anchoring IL is crucial for both activity and reusability. With the optimised conditions (3, BMI$PF 6 (BMI ¼ 1-butyl-3-methylimidazolium), 45 C, toluene co-solvent), turnover frequencies (TOFs) of up to 343 000 h À1 were obtained in the 1,7-octadiene RCM reaction, even at ppm concentration Scheme 1 CTLs for polar phase anchoring and ESI-MS detection. levels. The reusability of the system was tested in the RCM of diallyldiethylmalonate, and seven cycles could be performed without an apparent decrease in activity. Another noteworthy nding is the fact that a high degree of substitution at the carbon attached to the oxygen in the ether portion of the CTL is benecial to the catalytic activity.
We also successfully used CTL 3 in a tandem isomerisationmetathesis reaction of trans-3-hexene, affording heavier linear olens. 18 The reaction was performed in a biphasic system composed of BMI$PF 6 and toluene. In this way, the metathesis catalyst, 1, was anchored in the IL phase and the isomerisation catalyst, RuHClCO(PPh 3 ) 3 , was kept in the organic phase. The use of this biphasic system is essential for keeping a superior olen concentration in the toluene phase and, consequently, ensuring the correct isomerisation-metathesis rate. In this way, 80% of the initial trans-3-hexene was converted to an olenic C4-C17 mixture, with 49% of the linear olens being higher than C6.
As well as Hoveyda-type catalysts, we have also produced the charge-tagged Grubbs-type catalyst 6. In this case, the ionic tag was provided by the ionophilic phosphine 7. The synthesis of this CTL is easy, and could be achieved through a one-step radical chain addition of secondary phosphines to allyl imidazolium salts. 24 The charge-tagged ruthenium complex 6 was applied to the RCM of 1,7-octadiene. When this reaction was performed in BMI$PF 6 -toluene, it afforded cyclohexene in a 98% yield with only 0.25 mol% of the catalyst. Moreover, this system could be recycled eight times without loss of activity. This reusability was superior to that of the second-generation Grubbs catalyst, which exhibited a pronounced decrease in activity in the second cycle. Atomic absorption analysis of the toluene phase aer each cycle, when using complex 6, revealed a ruthenium content below the detection limits of the technique (<2 ppm, i.e., less than 1.5% of the initial Ru content). Unfortunately, low conversions were observed when the system was applied to the RCM of highly substituted dienes. Fig. 1 illustrates the distribution of 6 and 8 (Grubbs catalyst) over the IL and toluene phases. Note that the toluene phase is uncoloured when using complex 6.
In 2010, a fashionable system for the recovery of ruthenium metathesis catalysts was described using ligand 9. 25 This ligand, when reacted with a second-generation Grubbs catalyst, produces the Hoveyda-type light-controlled charge-tagged complex 10, which is a very active catalyst in the RCM of N-, Sand O-containing substituted dienes (yields ranging from 90 to 97% were obtained), even at low catalyst loadings. Moreover, this type of light-controlled tagged ligand can be switched between a non-polar and a polar phase through a tag-centred photoreaction. The photoreaction is reversible and results in drastic changes in the polarity and solubility of the ligand. In this way, aer the RCM reaction, the solvent could be evaporated and the residue dissolved in a mixture of cyclohexane and alcohols (1 : 1). Thus, the catalyst and products were dissolved in the upper (cyclohexane) layer. The system was then irradiated with light (l >380 nm) to transform the spiro form, 10, into the ionic tag 11, which completely shied into the lower layer (alcoholic phase). The products remained in the apolar phase, which was subsequently removed. Next, CH 2 Cl 2 was added and the mixture was kept in the dark for 3-5 min in order to reobtain the neutral species 10 (Scheme 3). The solvent mixture was then evaporated and CH 2 Cl 2 , in conjunction with a new bath of diene, was added; six RCM cycles could be performed, retaining almost the same activity.
Recently, ruthenium supported ionic liquid phase (SILP) catalysts based on alginates or chitosans were synthesised and applied in olen metathesis reactions with a high level of recyclability and reusability combined with a good reactivity. 26 The CTL 12 and the ruthenium supported ionic liquid catalyst (Ru bio-SILP) used in this work are illustrated in Scheme 4. The reactions were performed under both mono-and biphasic conditions using the ionic liquid BMI$PF 6 . The best results were obtained in the RCM of diethyl-2,2-diallylmalonate under heterogeneous conditions with the alginate based Ru bio-SILP (at least 15 cycles could be performed with high activity $ 87%). The detection of ruthenium leaching by ICP-MS in the extraction phases showed low leaching of Ru throughout the cycles (200 ppm). This system was also active in the cross-metathesis reaction, with a high level of reusability (at least 12 cycles with activities up to 90%). This reusability could be attributed to the good anchoring of the catalyst in the IL phase supported on the biopolymer.
Metal-catalysed cross-coupling reactions. Several examples of using substituted imidazolium salts as both the solvent and the ligand in cross-coupling reactions have been reported (Fig. 2). In these CTLs, a coordinating imidazole, pyrazole or pyridine group was attached to either C2 or C1 of the imidazolium moiety. [27][28][29][30] For instance, mono-charged ligand 13, in combination with PdCl 2 , was very active in both Suzuki (yields ranging from 80 to 90%) 29 and Heck (yields ranging from 70 to 94%) cross-couplings. 28 In terms of reusability, for the Heck reaction, the system could be reused 9 times, even when less active aryl chloride was used. For the Suzuki cross-coupling reaction, the system could be reused 14 times without an apparent loss of activity.
The complex resulting from the reaction of 14 and PdCl 2 was used in the Heck reaction of acrylates and styrenes with aryl iodides or bromides, using 14 as the solvent. 27 In the reaction of PhI with n-butyl acrylate, a 99% yield was obtained over nine Heck reaction cycles. Moreover, yields from 49 to 94% were obtained when the same catalytic system was reused 14 times whilst varying the coupling partners.
Pyrazolyl-substituted ionophilic compounds 15 and 16 were effective when used as the solvent and ligand to stabilise the palladium species in Heck, Suzuki and Sonogashira crosscoupling reactions. 30 In all cases, the catalysts could be reused several times without loss of activity.
In addition to nitrogen-based ligands, phosphorus CTLs have also been effectively employed in palladium-catalysed cross-coupling reactions. The tagged diphenylphosphine ligand 17, for example, was used to produce palladium complex 18 (Scheme 5). 31 This precursor was active in the Suzuki coupling of aryl bromides and arylboronic acids in a mixture of 1-butyl-1methyl-pyrrolidinium bis(triuoromethylsulfonyl)imide and water (2 : 1). With a catalyst loading of 1%, the biaryls were obtained with yields ranging from 84 to 99%. Surprisingly, when aryl iodides were used, lower yields were obtained. This result was attributed to IL anion exchange (from NTf 2 À to I À ).
This hypothesis was supported by the fact that the addition of KI inhibited the conversion, whereas the addition of KBr did not cause a signicant decrease in the reaction rate. In terms of reusability, six cycles could be performed without an apparent loss of activity. Moreover, no "Pd black" was observed, and <10 ppb of the metal was leached from the IL into the product during recycling.
It is important to note that, in the majority of these cases, the use of "activated" aryl iodides and bromides suggests the probable involvement of Pd(0) species, including low nuclearity Pd nanoparticles (NPs). 32 Moreover, in the cases involving the imidazolium nucleus, it is quite probable that N-heterocyclic (NHC) carbenes are involved, at least as transient species. 33 However, in each of these cases, the CTL is able to stabilise and retain the active catalytic species in the IL phase.
Another important aspect of Heck coupling reactions in ILs is the possibility to induce a higher degree of selectivity, because the neutral and ionic pathways of this reaction can lead to opposite regioselectivities. Indeed, the Pd complexes associated with P-containing ligands in BMI$BF 4 promote the regioselective arylation of butyl vinyl ether, affording the a-isomer almost exclusively. Conversely, reactions performed in organic solvents afford variable mixtures of the aand b-isomers. 34 Unfortunately, this process has not yet been investigated using CTLs.
A variety of CTLs/ILs has also been used to promote better NP stabilisation, as well as to extend the catalyst's lifetime during catalytic reactions. In this context, the catalytic activity of palladium complexes immobilised in alkyl-substituted and nitrilefunctionalised pyridinium ILs was tested in the coupling of tributylphenylstannane and iodobenzene. 35 However, the presence of [Pd(0)] n NPs was identied aer the Stille reaction. Moreover, recycling experiments showed that the functionalised IL was a superior reaction medium compared with a non-functionalised analogue, which may be associated with enhanced stabilisation of the formed NPs in the nitrile-pyridinium CTL/IL, as corroborated by transmission electron microscopy (TEM). Imidazolium-based CTL/ILs containing nitrile groups attached to the alkyl side chain were also employed in the Stille reaction (Scheme 6). 36 In this case, a detailed study showed the inuence of the relative coordination strengths of the cations and anions on the efficiency of the coupling reaction. Interestingly, NPs were only formed in some cases, and the superior conversion observed for the nitrile-functionalised IL compared with the non-functionalised one was assigned to the improved stabilisation of the NPs offered by the nitrile CTL.  Scheme 5 PdCl 2 -tagged phosphine used as a precursor in the Suzuki cross-coupling reaction.
Scheme 6 Stille reaction and the IL/ligand that was employed.
A novel bisphenol-functionalized benzimidazolium salt was synthesised and applied in the obtainment of the air-stable ionic iron(III) complexes 19 and 20 with 85 and 82% yields, respectively (Scheme 7). 37 Iron-based catalysts have been employed in the alkylation of Grignard reagents 38,39 in place of palladium and nickel complexes, because of the possibility of undesired hydride b-elimination in these complexes. However, in most reports, the more active alkyl bromides and iodides are employed as the electrophilic partner using catalyst loadings around 5 mol%. Both complexes 19 and 20 afforded crosscoupling products with reasonable to excellent yields using 1 mol% of iron and secondary alkyl chlorides as the electrophile.
On the other hand, the results using primary chlorides were unsatisfactory, considering that yields ranging from 5 to 45% were observed. In recycling experiments, 19 provided the coupling product with yields greater than 95% for six runs, however, in these experiments 5 mol% of iron (instead of 1 mol%) and a more active alkyl bromide were applied. 37 The authors also highlighted that 19 outperforms the catalytic activity of an imidazolium analogue, 40 especially in avoiding the formation of the b-elimination product.
Metal-catalysed enantioselective reactions. Several examples of enantioselective modications have been reported that were catalysed by complexes with ionically tagged ligands. For example, an imidazolium cation bound to an h6-arene ruthenium chiral complex was evaluated and compared to neutral h6p-cymene complexes in hydrogenation transfer reactions. 41 Acetophenone was used as the substrate and 2-propanol or formic acid was used as the proton source (Scheme 8). For 21, the enantiomeric excess (ee) was similar to the neutral counterpart (98%). In addition, for both 21 and 22, the leaching was lower than for the neutral complexes when the reaction was performed in 1-butyl-2,3-dimethylimidazolium hexauorophosphate. However, the reaction yield decreased upon recycling. This result was attributed to base-induced catalyst degradation.
For O-and S-containing nucleophiles, only modest results were obtained (41% yield with 62% ee and 81% yield with 10% ee, respectively). Moreover, when recycling experiments were performed, disappointing results were obtained. This was attributed to the high reactivity of the C-H bond at the C2 position in the imidazolium moiety of 23 under basic conditions. However, when CTL 24, with a methylated C2, was employed, no improvements were achieved in yield or enantioselectivity.
Recently, allyl-palladium complexes containing imidazolium-tagged chiral diaminophosphite ligands (S,S)-25 and (R)-26 were synthesised and also applied as catalysts in asymmetric allylic substitution reactions with heteroatom and carbon nucleophiles (Scheme 10). 43 Complexes containing one and two units of (S,S)-25/(R)-26 were tested in BMI$PF 6 and BMPyr$NTf 2 as reaction media. Only 1 mol% of palladium was enough to promote the substitution with up to 100% conversion for N and C nucleophiles and 85% conversion for S nucleophiles, in three hours at 35 C. Concerning the enantioselectivity, 74% ee was obtained using benzylamine, 72% ee was observed for sodium p-toluenesulnate and only 45% ee was obtained using sodium diethylmalonate. The best results, considering both reactivity and enantioselectivity, were attained using [PdCl(methallyl)26] [BF 4 ] in BMPyr$NTf 2. In recyclability tests, unimpressive results were observed, since the conversion decreased from 75 to 55% in four runs, while the ee decreased from 75 to 58% ee over 10 reaction cycles. These results were attributed to catalyst Scheme 7 Synthesis of ionic iron-benzimidazole complexes and their application in the cross-coupling of aryl-Grignard reagents with alkyl halides. deactivation, given that palladium was not detected in the ICP-MS analyses of the organic extractive phase. It is important to mention, however, that to the best of our knowledge, only one efficient IL-based recycling system has been reported for Pdcatalysed asymmetric allylic substitution. 44 Ionically tagged Josiphos analogues, 27, were applied in the rhodium-catalysed hydrogenation of the benchmark substrates methyl acetamidoacrylate (MAA) and dimethyl itaconate (DMI) (Scheme 11). 45 The cationic precursor, Rh(NBD) 2 BF 4 (NBD ¼ 2,5-norbornadiene), was used with a H 2 pressure of 1 bar at room temperature. Initially, it was observed that the use of the appropriate reaction media was crucial for both activity and enantioselectivity. By using a biphasic system composed of tertbutyl methyl ether and BMI$BF 4 , total conversion and a 99% ee were obtained. In the recycling experiments, the ionic ligands kept the TOF above 1000 h À1 for at least ve cycles, whereas with neutral Josiphos ligands the reaction rate decreased markedly upon each reuse.
A series of ionophilic monophosphites, 28, based on Dmannitol was synthesised and successfully applied in the rhodium-catalysed, enantioselective hydrogenation of enamides, dehydroamino acids and dimethyl itaconate (Scheme 12). 46 The ligands provided high yields and excellent enantioselectivity in either the CH 2 Cl 2 or biphasic IL/toluene media. The best results were obtained with the ligand bearing a sixcarbon alkyl chain between the mannitol and imidazolium moieties. The recyclability of the system was tested in the BMI$BF 4 , BMI$PF 6 and BMMI$BF 4 (BMMI ¼ 1-butyl-2,3-dimethylimidazolium) ILs. In all cases, up to eight recycles could be performed, keeping the yield and enantioselectivity almost constant.
In 2012, the use of the imidazolium-tagged bis(oxazoline) ligand 29 was reported in the copper-catalysed, asymmetric Henry reaction (Scheme 13). 47 Several substituted benzaldehydes were reacted with CH 3 NO 2 , providing the corresponding adducts. The reaction was effective in various ILs and organic solvents. However, the best enantioselectivity was obtained when the reaction was performed in MeOH. In this medium, 23 to 94% ee was obtained. Moreover, the catalyst could be reused six times, by evaporating the solvent and extracting the product with ethyl ether, without loss of activity or enantioselectivity.
Metal-catalysed oxidation of olens. An ionophilic iron(III) complex, 30, bearing three imidazolium moieties was synthesised (Scheme 14) by reacting 31 and FeCl 3 in methanol. 48 This tagged complex was used in the epoxidation of methyl oleate and vegetable oils. Yields ranging from 79 to 98% were obtained when the reactions were performed in BMI$NTf 2 , using synthetic air at 15 bar as the oxidising agent. In the oxidation of methyl oleate, the catalytic system was recycled ten times without an apparent decrease in activity. Moreover, no signicant iron leaching (<2 ppm) was observed by means of inductively coupled plasma atomic emission spectroscopy (ICP-AES).
The use of ionophilic manganese porphyrins (Fig. 3) in alkane and alkene oxidation reactions has also been reported. Pyridinium-substituted catalysts 32 and 33, for instance, were applied to ethylarene oxidation (Scheme 15). 49 Under optimised conditions (0.4 mol% of 32, BPy$BF 4 , and PhIO as the oxidising agent) ethylbenzene was oxidised to 1-phenylethanol and acetophenone with 71 and 29% selectivity, respectively. In recycling experiments, for both 32 and 33, the selectivity for the ketone increased with successive reuse. In terms of activity, for 32, the conversion decreased with reuse, whereas for 33 it remained constant over four oxidation cycles. When substituted arylethenes were used as substrates, a positive effect on the conversion was observed for the electron-donating groups. According to the authors, this result is due to the increased electron density at the a-carbon of ethylbenzene derivatives.
Catalyst 32 was also used in the oxidation of arylethenes in CH 3 CN and BPy$BF 4 . 50 The system was active and selective for the formation of epoxides, in both media. Moreover, the recyclability of 32 was superior to that of the non-ionophilic analogue.
The synthesis of manganese porphyrins, bound to one, three or four imidazolium units (34), has been described 51,52 and applied to olen epoxidation reactions, using H 2 O 2 as the oxidizing agent (Scheme 15). In an early investigation, 51 ligand 34c (olen-catalyst ¼ 75 : 1) was applied efficiently to the epoxidation of cyclooctene, cyclohexene, 1H-indene and styrene. The oxirane products were obtained with yields ranging from 95 to 100% in CH 3 CN, with acetic acid as the co-catalyst. Next, charge-tagged metalloporphyrins were compared to the neutral analogue [R 1 -R 4 ¼ 1,5-Cl(C 6 H 3 )] in arylethene epoxidation. 52 All catalysts (34a-34c) were active in the epoxidation of styrene with a S/C ratio of 300. High conversions were attained with catalysts 34b and 34c. However, the best selectivity was obtained with the mono-tagged complex 34a, which provided similar results to the neutral complex (selectivity >90%). By using substituted styrenes, the authors were able to obtain Hammett plots for the reaction. Again, the reaction was accelerated by electron-donating groups and, furthermore, the small r-values suggested no signicant charge separation in the transition state.
Metal-catalysed olen hydroformylation. The cationic phosphine CTL 35 was applied in the rhodium-catalysed tandem hydroformylation-acetalyzation of olens in ionic liquidalcohol media (Scheme 16). 53 Initially, 1-octene was used as the substrate model for the optimization of the ligand counteranion, catalyst loading, and ionic liquid medium. In this way, using 35$BF 4 and 0.1 mol% of rhodium in a medium composed Scheme 14 Fe(III) complex bearing imidazolium moieties that was applied in the olefin epoxidation reactions.  of BMI$BF 4 and methanol, the product of the hydroformylation was obtained in a 96% yield, with 95% acetalyzation and a linear-branched ratio of 75 : 25 aer 2 h at 80 C and CO-H 2 (1 : 1) at 50 bar. Following this, other alkenes (alkyl and aryl substituted) were also hydroformylated in the presence of methanol, ethanol, ethylene glycol and propylene glycol, affording the products observed in Scheme 16. The high levels of acetalyzation were attributed to the Brønsted-acid character of the ligand 35, since only 1% acetalyzation was observed when the CTL was substituted by PPh 3 . It is worth noting the recyclability results observed with this strategy. For a system composed of Rh(acac)(CO) 2 , 35$BF 4 , BMI$BF 4 and methanol, conversions greater than 98% and an average acetalyzation of 95% were observed over 12 runs. When methanol was replaced by propyleneglycol, at least 15 runs were performed without signicant loss of activity. With both alcohols, the lixiviation of rhodium from the IL phase was less than 0.1%.
CTLs as immobilising agents in NP-catalysed hydrogenation reactions. Metal NPs have been widely used in catalysis because of their unique properties. However, it is well known that "naked" NPs are thermodynamically unstable and must be stabilised to prevent agglomeration. In this context, ionophilic ligands have been employed in catalysis to improve NP activity and stability and, most importantly, to immobilise these NPs in the ionic phase. 54 The catalytic activity was strongly inuenced by the structure of the CTL stabiliser employed in arene hydrogenation with [Rh(0)] n NPs, following the trend 36 > 37 > 38 (Scheme 17). 55 The [Rh(0)] n NPs stabilised by 36, containing a C7 alkyl chain between the bipyridine and the imidazolium group, showed the highest activity compared to the ligand with only a methylenic group between the bipyridine and the ionophilic group 38. This may be caused by a weaker interaction between the ligand 38 and the NP surface, which decreases the binding affinity of this ligand and promotes the decomposition of the NPs under the reaction conditions. Similarly, bipyridinium CTLs (39 and 40) were employed in styrene hydrogenation using [Rh(0)] n NPs. 56 The total conversion of styrene into ethylcyclohexane was observed and the quantitative hydrogenation of the aromatic ring was attributed to the monodentate coordination of the ligand to the NP (Scheme 18). Furthermore, NPs with small sizes (around 2.0-2.5 nm) and good stabilities were obtained in the presence of charged ligands. Comparable results were obtained in arene hydrogenation using [Rh(0)] n NPs stabilised by imidazoliumfunctionalised bipyridine derivatives in BMI$PF 6 . 57 Conversions of up to 85% were obtained for the total hydrogenation of benzene and toluene.
In the same context, functionalised ILs have been employed as the CTL/solvent in catalysis to improve the stability of the NPs. [Ru(0)] n NPs synthesised in nitrile-functionalised ILs display unusual selectivities towards the hydrogenation of nitrile-containing aromatic compounds. 58  coordinated to the ruthenium surface, which only allows the nitrile group of the substrate access to the NP surface, thereby avoiding arene coordination. Thus, it is possible to state that the presence of a CTL, effectively, modulates the selectivity of the catalyst.
The selective hydrogenation of alkynes to (Z)-alkenes, catalysed by [Pd(0)] n NPs (7.3 nm) stabilised in the CTL/IL 41, was also recently reported by our group. 60 Notably, 3-hexyne was partially hydrogenated to the corresponding (Z)-olen without any trace of the isomerised by-product. The lack of isomerisation indicates that the [Pd(0)] n NPs exhibit typical behaviour and possess surface-like (multi-site) catalytic properties. Moreover, it is possible to tune the selectivity towards alkenes or alkanes, depending on the hydrogen pressure employed (Scheme 20). These results are probably related to the low hydrogen gas solubility in ILs and the associated mass-transfer/ diffusion limitations. It is expected that, at low pressures, there is a minimal amount of hydrogen dissolved in the IL, affording the alkene product; on the other hand, increasing the gas pressure results in more hydrogen being present in the IL phase to yield total hydrogenation (i.e., the alkane product).

CTLs as probes for detecting reaction intermediates through ESI-MS
In the last decade, ESI-MS has become a fundamental technique for the detection of intermediates in organometallic catalysis. 21,[61][62][63] This method permits the transfer of ions from solution into the gas phase, thus allowing the sampling of dissolved, charged organometallics in situ. By using this methodology, important information concerning the catalytic cycle of a plethora of reactions could be obtained (for instance crosscoupling reactions, 64-66 the Wacker oxidation, 67 hydrosilylation, 68 hydrogenation, 69 etc.), including air-and moisturesensitive ones. 70 However, ESI represents a "gentle" ionisation method, and only charged intermediates are detectable in ESI-MS. Therefore, reactions that proceed through a neutral mechanism are not likely to be detected properly. Moreover, the common ionisation techniques, protonation/deprotonation, can affect the nature of the catalytic system under investigation. To overcome this limitation, some strategies have been employed, including (i) the formation of charged adducts 71 and (ii) the use of charge-tagged substrates (this strategy has been applied successfully for olen metathesis, 20 cross-coupling reactions, 19,72,73 the Pauson-Khand [2 + 2 + 1] cycloaddition 74 and other catalytic processes 75 ). However, both (i) and (ii) limit the scope of the substrates employed. An alternative is to use ionically marked ligands. Here, we describe interesting examples of the use of CTLs as probes to detect intermediates in catalysis. Note that although charged metal compounds behave straightforwardly in ESI-MS, low fragmentation conditions should be used i.e. a low cone angle voltage in order to observe parent ions or derivatives. For the details, scope and limitations of the use of ESI-MS readers are invited to consult the specialized literature. [76][77][78][79] Olen metathesis. The pioneering work on CTLs to be used in the detection of intermediates through ESI-MS was published by Chen and co-workers in 1998. 80 The authors used the charged-tagged ruthenium complex 42 as a probe in crossmetathesis (with 1-butene) and ROM (with cyclobutene, cyclopentene and norbornene) reactions (Scheme 21). Initially, the signal of 43 was observed in the gas phase, corresponding to the de-coordination of a phosphine ligand. Next, aer the addition of the olens, the metathesis adducts 44-46 were observed in the positive-ion ESI-MS [ESI(+)-MS] spectra. Adducts with additional olen units (up to three) were also observed. The ROM of the rst cycloalkene unit was much easier than the subsequent additions. This nding was attributed to the p-complexation of the penultimate double bond to the ruthenium centre.
In another work with the CTL 43, the same authors evaluated the effect of the carbene substituent and the isotope effect on the metathesis rate. 81 Initially, the electron-sprayed intermediate, 43, was reacted with substituted styrenes, generating 47 (Scheme 22). The reaction rate of 47 with propene and norbornene was measured. A modest acceleration of the acyclic metathesis reaction was observed in the presence of the electron-withdrawing groups on the aryl group of the substituted ruthenium benzylidene. Moreover, the carbene complex formed from 47 and styrene-d 8 exhibited secondary deuterium kinetic isotope effects for the acyclic metathesis of 1-butene and the ROM of norbornene in the formation of 44 and 48, respectively.
Remarkable ndings, concerning the reversibility of ROM, were obtained with ESI(+)-MS experiments for bifunctional substrates, which offer the product of a ROM reaction the chance to undergo a non-degenerate RCM to form a complex that is isotopically distinguishable from the original complex in MS. The cyclopentane derivative does not react with 44 under the relatively mild conditions in this experiment; the cyclopentene derivative, instead, leads to approximately 10% conversion of 44 to 44-d6 (Scheme 23), indicating that the ROM of cyclopentene had occurred.
The same charge-tagged intermediate, 42, was also used to study RCM through ESI(+)-MS experiments. 82 Again, 43 was generated and, aer a gas-phase reaction with ethylene, a charged, 14-electron, methylene ruthenium species was detected. Concerning the reactivity of N(Boc)(allyl) 2 and other diallyl compounds in RCM, it could be conrmed that all the monomolecular steps in the catalytic cycle of the RCM, following olen coordination, are fast, and no intermediate can be accumulated to become detectable by ESI-MS. Additionally, alkylidene trialkylphosphane and phosphonium salts were easily detected and attributed to degradation products.
Cross-coupling reactions. The actual mechanism of the copper-catalysed coupling between phenols/amines and aryl/ vinyl halides (Ullmann coupling) is still under discussion. It Scheme 20 Hydrogenation of alkynes, which is controlled by a variable hydrogen gas pressure. 60 Scheme 21 CTLs in the ESI-MS detection of olefin metathesis intermediates. . Another point of discussion is the putative formation of a nucleophilic copper species, preceding the organic-halide activation. We recently described the synthesis and use of the phenanthroline-based ligands 49 and 50 in ESI(+)-MS experiments. 92 These cationic ligands were used as probes to detect the intermediates in the copper-catalysed coupling of (E)-bromostilbene with phenols (Scheme 24).
In this way, we were able to detect various intermediates (Fig. 4). Initially, intermediates 49a and 50a, corresponding to 49 and 50 coordinated to copper iodide, were observed. Aer the addition of phenol, base and vinyl bromide, various restingstate intermediates could be detected. Intermediates 49b, in which a benzylic cleavage of the ligands had occurred, were similar to those described as very active catalysts in the arylation of phenols, even at very low concentrations of copper (10 ppm). 93 Another important intermediate was 49c. In this species, the ionophilic probe was not cleaved and it contained a Cu-OAr bond. The formation of Cu-nucleophile species has been described in the arylation of phenols 94 and for other nucleophiles. 84,89,[95][96][97][98] These species have been reported to be capable of reacting with aryl halides under mild conditions. 84,89,[94][95][96][97][98] Moreover, 49c also indicates that the phenol moiety can act as a nucleophile as well as a ligand.
Based on the results obtained from ESI(+)-MS, preliminary kinetic studies and the radical-scavenger experiment, we could propose viable mechanistic pathways for the copper/1,10-phenanthroline-catalysed vinylation of phenols. At rst, the coordination of the ligand to CuI takes place, providing a (phen)CuI species, which was detected as 49a and 50a in the ESI(+)-MS. Thereaer, an intermediate [(phen) 2 Cu] + is formed. The formation of this intermediate was supported by the detection of 49b. Next, the Cu-OAr intermediate was detected as 49c, which is reported to be able to react with the organic halides. 94 In terms of vinyl halide activation, as the radical pathway is not operational, we could argue in favour of an oxidative addition to form a Cu(III) intermediate. Although these species are not common, arylcopper(III) complexes have recently been identi-ed, 85,99,100 and arylcopper(III) intermediates containing nitrogen ligands are calculated to be kinetically accessible under the reaction conditions. 89 Then, the [(phen)-CuOAr] intermediate would be reformed by a putative reductive elimination, followed by the attack of the phenoxide to form the copper species (Scheme 25).
The ion-tagged complexes were characterised by ESI(+)-MS and ESI(+)-MS/MS. For both the Cu and Ni complexes, the formation of metallic NHC carbenes was observed in the mass spectra. The palladium dicationic carboxylate was successfully used in the Heck and Suzuki reactions. In both reactions, the activity of 51f was greater than that of Pd(OAc) 2 . Although the palladium carbene was not observed in the ESI-MS experiments, the superior activity of the CTL was attributed to formation of this type of intermediate.
It must be mentioned that the same research group described the detection of this NHC carbene through negativeion ESI-MS [ESI(À)-MS] experiments of COOH-and SO 3 Hsubstituted IL. In the gas phase, the doubly deprotonated (imidazolium and COOH or SO 3 H) NHC carbene was detected. Moreover, the behaviour of these carbenes in the gas phase was similar to those in the liquid phase, because they can react with CO 2 and generate organic carboxylates at the C2 positions of imidazolium. 101,102 Key reaction intermediates associated with Pd catalysis could also be detected and characterized due to the presence of the charge tag on the Pd-complex (Scheme 27). 103 The new charge-tagged Pd-complex is also shown to function as an active catalyst "on water" with the advantage of using cheaper and less reactive aryl chloride substrates in a phosphine-free version of the Heck reaction.
The use of tagged substrates 19 and ligands 104  Olen hydrogenation. Scheme 29 has been studied in detail, [105][106][107][108] and both the in-cycle (B-E) and off-cycle (A, F-J) species have been characterised. For this reason, the reaction can be used to evaluate the reliability of ESI-MS for detecting intermediates during catalysis. However, because all of the species are neutral, it is necessary to charge-tag them.
Therefore, Ph 2 P(CH 2 ) n PPh 2 (n ¼ 1, 2, 4, 6) diphosphines were selectively monobenzylated to produce charge-tagged phosphonium/phosphine [Ph 2 P(CH 2 ) n PPh 2 CH 2 Ph] + ligands. These CTLs were used to dope RhCl(PPh 3 ) 3 under hydrogenation conditions in order to detect the reaction intermediates in ESI(+)-MS. 109 Ligand 53 (n ¼ 4) was added to a solution of RhCl(PPh 3 ) 3 in chlorobenzene. The reaction was analysed before H 2 was added, aer H 2 was added and aer the addition of cyclohexene. The results are summarised in Table 1 )] (G-type). In summary, by using this method, all the offcycle (A, F-I) species could be identied rapidly.
An iron(III) complex with three ionic tags, 54, was applied to the reduction of olens in imidazolium-based ILs under oxidative conditions. 110 The catalyst was very efficient in promoting reactions involving biomass derivatives. ICP analysis revealed that the use of this catalyst greatly prevented catalyst leaching, and as little as 2 ppm of Fe was detected in the organic phase of the reaction. This indicates the efficient anchorage of the complex in the BMI$NTf 2 IL. Moreover, the activity of this complex remained unchanged aer ten runs. Some important mechanistic insights for this new reaction were also provided, mostly based on ESI quadrupole time-of-ight MS (ESI-QTOF-MS), as can be seen in Scheme 30. Based on these experiments, a catalytic cycle that includes a high-valence Fe atom was proposed.
Metal-catalysed multicomponent Biginelli reactions. The Biginelli reaction 111 is an acid-catalyzed, three-component reaction between an aldehyde, a b-ketoester and urea that allows the rapid and facile synthesis of dihydropyrimidones, which are interesting compounds with potential for pharmaceutical applications. This reaction catalysed by a Lewis acid in an IL was evaluated by Neto and co-workers using ESI-MS. 112 This multi-component reaction was used to obtain the biologically active 3,4-dihydropyrimidin-2(1H)-one (DHPMs). As indicated by ESI-MS experiments, the ionic medium plays a fundamental role in this synthesis, owing to the stabilisation of the charged intermediates. Presently, there are three major pathways accepted for this reaction: (i) the iminium, (ii) the Knoevenagel and (iii) the enamine mechanisms. [113][114][115] Through this work, the authors could detect some key intermediates and products of the reaction by using ESI(+)-MS(/MS) (Fig. 5). The online monitoring of the Biginelli reaction allowed the authors to detect and characterise the so-called iminium intermediate (Fig. 5E), indicating that, under these conditions, the preferred mechanistic pathway is the iminium mechanism. No key intermediate of the enamine mechanism could be detected.
This reaction has also been studied using the new ion-tagged iron catalysts 55 and 56 (Fig. 6) in ILs. 116 High yields were obtained and the system could be recovered and reused up to eight times. High-resolution ESI-QTOF-MS experiments were performed to determine the reaction mechanism.
The three proposed mechanisms for the Biginelli reaction were investigated by ESI-QTOF-MS and MS/MS. Only the intermediates from the iminium mechanism were detected. No intermediates from the Knoevenagel or enamine pathways were found.
Iron-catalysed olen epoxidation. The ionically tagged imidazolium-based iron(III) complex 54 was used in the oxidation of alkenes in imidazolium-based ILs in air (also hydrogen peroxide). 48 The epoxidised olen was obtained in very good yields (84-91%) and at least ten recycling reactions could be performed. ESI-QTOF-MS experiments were performed to achieve a better understanding of the active catalytic species that promoted epoxidation. The results pointed rmly towards two different oxidation states of the metal centre [Fe(IV) and Fe(V)], depending mostly on the selection of the oxidising agent. Hydrogen peroxide oxidation may proceed through a radical pathway and may use a Fe(IV) species in its catalytically active form, whereas air oxidation may favour a concerted mechanism with an Fe(V)-based intermediate as the active species to promote epoxidation. Some important mechanistic insights could be provided based on the ESI-QTOF-MS results for the oxidation reaction, which clearly indicated that oxidation can  take place by two different pathways, depending on the reaction conditions, that is, a radical or a concerted mechanism.

Conclusions and perspectives
It is evident that, with the advent of IL-phase chemistry, a new class of organic charged ligands has emerged. Contrary to classical water-phase catalysis, for which negatively charged ligands (in most cases, P-containing ligands) are used, IL-phase catalysis is mainly based on positively charged ligands. This is necessary, because an incorrect solvent pair can lead to low conversions, poor product extraction, no phase separation and catalyst lixiviation. Using this approach, various classical homogeneous catalytic processes have been successfully transposed to liquid-liquid biphasic catalysis. However, so far, it is only in rare cases that the changes to the mechanism and selectivity of the transformations, imposed by the intrinsic ionic nature of the media, have been addressed. Moreover, even fewer insights have been gathered for the supramolecular organisation imposed by these highly organised uids on the catalytically active sites. Therefore, a more detailed investigation on the mechanistic pathway in ionic media is necessary to allow the design of more efficient catalysts (for improved conversion, selectivity and sustainable catalytic processes). Notably, CTLs have also been used with a high degree of success in colloidal catalytic process. It also appears that the use of CTLs has opened a new window of opportunities for the investigation of reaction pathways through ESI-MS. This approach has already proven to be one of the most important tools for detecting several species involved in various catalytic reactions directly from the solution. It is also expected that, in view of the virtually non-volatile nature of ILs, other techniques will be used to probe and monitor in situ reactions performed in solution, such as techniques requiring a high vacuum and those that are currently limited to solid-state chemistry, for example, X-ray photoelectron spectroscopy, low-energy ion scattering spectroscopy and TEM. This may also lead to important in situ solution-phase information that can be used for the design of new and more efficient catalysts.


text:
Formal (4+1) Cycloaddition of Methylenecyclopropanes with 7-Aryl-1,3,5-cycloheptatrienes by Triple Gold(I) Catalysis**

7-Aryl-1,3,5-cycloheptatrienes react intermolecularly with methylenecyclopropanes in a triple gold(I)-catalyzed reaction to form cyclopentenes. The same formal (4+1) cycloaddition occurs with cyclobutenes. Other precursors of gold(I) carbenes can also be used as the C1 component of the cycloaddition.

Carbenes have been widely used as one-carbon synthon in organic synthesis, particularly in the context of cyclopropanation reactions. [1] However, only a few (4+1) cycloadditions [2] have been reported mainly with Fischer alkoxy-(alkenyl)carbene complexes [3] and dialkoxycarbenes. [2,4] To the best of our knowledge, there is no report on the (4+1) cycloaddition of aryl carbenes with 1,3-dienes, probably because of the known propensity of carbenes to give cyclopropanation products with 1,3-dienes. [5] We postulated that due to their high strain and unique electronic properties, cyclobutenes [6] could be used as synthetic equivalents of 1,3dienes for the development of a formal (4+1) cycloaddition with metal carbenes.
We have recently found that 7-substituted 1,3,5-cycloheptatrienes 1 undergo gold(I)-catalyzed retro-Buchner reaction to form carbenes 2 (Scheme 1). [7] Herein, we report a novel and potentially general formal (4+1) cycloaddition by reaction of 1 with methylenecyclopropanes 3 [8] or cyclobutenes 4 to form cyclopentenes 5. In this transformation, methylenecyclopropanes 3 undergo an isomerization to form cyclobutenes 4 similar to that catalyzed by platinum or palladium. [9] Therefore, in the reaction between 1 and 3, gold(I) plays a triple catalytic role, isomerizing 3 into 4 and, in parallel, generating gold(I) carbenes 2 from 1, which cyclopropanate the cyclobutenes. Finally, gold(I) cleaves the internal C À C bond of the resulting bicyclo[2.1.0]pentanes to form the cyclopentenes. This reaction can be viewed as an insertion of one carbon into a double bond, a process that has only been achieved in rare cases with dihalocarbenes. [10,11] Methylenecyclopropanes (MCPs) 3 can be readily prepared in one step by the Wittig olefination of carbonyl compounds with commercially available 3-bromo-triphenylphosphonium bromide. We first examined the reaction of phenylmethylenecyclopropane (3 a) with 7-naphthyl-cyclohepta-1,3,5-triene (1 a) in the presence of gold(I) complexes (Table 1). Using cationic [(JohnPhos)Au(MeCN)]SbF 6 (A) in 1,2-dichloroethane at 120 8C, disubstituted cyclopentene 5 a was isolated in 76 % yield (Table 1, entry 1). Other phosphine or N-heterocyclic carbene gold(I) complexes B-E gave lower yields (entries 2-5), whereas complexes F and G failed to promote this transformation, presumably due to their instability at the temperature required for the retro-Buchner reaction. The reaction also failed with silver(I), copper(II), and platinum(II) catalysts (entries 8-10).
7-Aryl-cyclohepta-1,3,5-trienes containing groups with different electronic and steric effects at the ortho, meta, or para positions reacted with MCPs 3 a-h to yield the (4+1) cycloadducts 5 b-m ( Table 2). The (4+1) cycloaddition proceeds satisfactorily with MCP bearing arenes with fluoro-, chloro-, and bromo-substituents. However, the reaction with o-bromophenylmethylenecyclopropane (3 f) led to cycloadduct 5 k in lower yield. The structure of 5 k was confirmed by X-ray diffraction ( Figure 1). [12] To demonstrate the synthetic utility of this method, cyclopentene 5 l was prepared on a 500 mg scale using only 1 mol % gold catalyst A in 51 % yield after purification by column chromatography. Alkylmethylenecyclopropanes also reacted to give (4+1) cycloaddition products, although in this case the reactions led to mixtures of regioisomers 5 n/n'-5 p/p'.
Substrate 3 l reacted intramolecularly using catalyst E to form 2,3-dihydro-1H-cyclopenta[l]phenanthrene (5 q') by iso- merization of the initially formed adduct 5 q (Scheme 2). In addition, polyarene fragments can be obtained by photochemical cyclization. Thus, compound 5 f can be transformed into a cyclopenta derivative of benzo [g]chrysene (6) by a one pot photo-induced isomerization/oxidative Mallory cyclization. [13] Tetrasubstituted MCP 3 m reacted with 1 a to give only the product of cyclopropanation 7 (Scheme 3 and Figure 1), whose structure was confirmed by X-ray diffraction (Figure 1). [12] Given that 3 m does not undergo ring-expansion, the isolation of spiro derivative 7 strongly suggests that the cyclopropanation of MCP is not the initial step in the formal (4+1) cycloaddition and that cyclobutenes are likely intermediates in this transformation.
To confirm the hypothesis that cyclobutenes are intermediates in the (4+1) reaction of MCP, we performed the reaction of 1 a with cyclobutene 4 a, which was isolated from the reaction mixture of 1 a and 3 g. Under identical conditions, cycloadduct 5 l was isolated in 77 % yield. Trisubstituted cyclobutenes [14] also took part in the (4+1) cycloaddition reaction to afford cyclopentenes 5 r-z (Table 3).
Cyclobutenes also react with intermediate gold(I) carbenes generated by 1,2-acyloxy migration of propargylic acetates [15] under mild conditions with catalyst E to give two separable isomers 5 aa-ac and 5'aa-ac in good overall yields (Scheme 4). By performing the reaction at room temperature at only 60 % conversion, bicyclo[2.1.0]pentane 10 a [16] could be isolated and then transformed cleanly into 5 aa at 40 8C in the presence of gold(I) catalyst. The gold(I) carbene generated from phenyl diazomethane [17][18][19][20] reacted similarly at room temperature with cyclobutene 4 c to form the desired formal (4+1) product 5 ad, along with 10 b. [21] This bicyclo-[2.1.0]pentane was converted quantitatively into cyclopentene 5 ad by warming at 60 8C in the presence of gold complex A.

Angewandte Chemie
To shed additional light on the reaction mechanism, we performed the reaction of cycloheptatriene 1 a with MCP [D 1 ]-3 a in the presence of catalyst A (Scheme 5). In this experiment, [D 1 ]-5 a was obtained with the deuterium label transferred completely to C-3.
According to all experimental data, we propose a mechanism for this formal (4+1) cycloaddition of cycloheptatrienes 1 and MCP in which gold(I) plays a triple role (Scheme 6). In the first catalytic cycle, h 2 -MCP-gold(I) complex I undergoes ring expansion to form intermediate II, which gives h 2 -cyclobutene-gold(I) complex III. Associative ligand exchange with the 7-aryl-1,3,5-cycloheptatriene, followed by retro-Buchner reaction then leads to the highly reactive gold(I) carbene 2, [7] which reacts with cyclobutene 4 to form bicyclo[2.1.0]pentane-gold(I) complex IV. Cyclopropane opening by gold(I) forms the tertiary carbocation V, which leads to complex VI by a final 1,2-H shift. The cyclopropanation of 4 by 2, followed by electrophilic cleavage probably follows a pathway similar to that occurring in the gas phase for the cyclopropanation/retro-cyclopropanation of enol ethers with gold(I) carbenes. [22] Formation of cyclopentenes from bicyclo[2.1.0]pentanes, the presumed intermediates of these reactions, has been mechanistically examined in a few cases using Rh I , Zn II , and other catalysts. [23,24] Formation of regioisomeric 3-alkyl-3-arylcyclopent-1-enes Scheme 2. Intramolecular formal (4+1) cycloaddition and its application to the preparation of a polyarene fragment. Scheme 3. Probing the mechanism of the formal (4+1) cycloaddition. [a] Reaction at 120 8C, 0.2 m in 1,2-dichloroethane, 2 equiv of 4 a-g, catalyst A (5 mol %), 3 h. Yields are for isolated adducts. [b] Cyclobutene 4 a was isolated from the reaction mixture of 1 a and 3 g.
[c] 2 Equiv of 7-(4-chlorophenyl)cyclohepta-1,3,5-triene were used. together with 5 n-p in the reaction of alkyl-substituted MCP can be explained by the competitive migration of the aryl group in intermediates V.
In summary, we have developed a synthesis of substituted cyclopentenes by a formal (4+1) cycloaddition from methylenecyclopropanes or cyclobutenes with gold(I) carbenes generated under catalytic conditions by retro-Buchner reaction of 1,3,5-cycloheptatrienes or by other methods. Further work on the application of this cycloaddition in synthesis is underway. . Keywords: (4+1) cycloaddition · carbenes · cyclobutenes · gold catalysis · methylenecyclopropanes


extfieldsofstudy:
Materials Science
Medicine

text:
Specific Recognition of Breast Cancer Cells In Vitro Using Near Infrared-Emitting Long-Persistence Luminescent Zn3Ga2Ge2O10:Cr3+ Nanoprobes

In this paper, near-infrared emitting long-persistence luminescent Zn3Ga2Ge2O10:Cr3+ (ZGG) nanoparticles with diameters of 30–100 nm and bright luminescence were prepared by a sol–gel synthesis method. After the surface amination, the nanoparticles were further bioconjugated with breast cancer-specific monoclonal antibody (anti-EpCAM) to form ZGG-EpCAM nanoprobes which can specifically target breast cancer cell lines (MCF7) in vitro. The results of in vitro images show that the luminescence signals from the cells treated with ZGG-EpCAM nanoprobes are stronger than those from cells treated with ZGG-unconjugated antibody, indicating that the prepared ZGG-EpCAM nanoprobes possessed excellent specific recognition capability. Furthermore, due to their long afterglow properties, the imaging could persist more than 1 h. Therefore, these nanoprobes could not only provide a high specificity detection method for cancer cells but also realize the long-time monitoring. Developed near-infrared emitting long-persistence luminescent nanoprobes will be expected to find new perspectives for cell therapy research and diagnosis applications.

Introduction
Optical imaging techniques have excellent potential to help us further understand biological processes at the molecular level and provide useful tools for sensitive cancer diagnosis [1]. Biological optical imaging greatly relies on the use of sensitive and stable optical probes. To date, organic dyes [2,3], quantum dots [4][5][6], metal nanoparticles [7][8][9], and upconverting nanoparticles [10] are the most commonly used optical labels probes, but they also have intrinsic limitations. Organic dyes suffer from a short observation time, partly due to photobleaching, poor stability under physiological conditions, and limited Stokes shift. Quantum dots display good photostability, size-dependent emissions, and high quantum yield, while they still require constant excitation, which is responsible for undesirable autofluorescence and enhanced background signal that reduce the sensibility of the assay [11,12]. Such inherent disadvantages would affect their application for imaging in vitro or in vivo.
In order to overcome some of inherent problems encountered with optical imaging probes [13], persistent luminescence nanoparticles have been developed. It comes down to a particular optical phenomenon in which the excitation energy can be reserved by the material and then slowly released by a photonic emission, which can be persistent luminescence for some hours [14,15]. After long-persistence luminescent nanoparticles are excited in vitro, real-time in vivo and vitro imaging can be achieved without needing any external illumination source. Therefore, the signal-to-noise ratio (SNR) can be significantly improved by eliminating the background noise originating from in situ excitation [16,17]. Moreover, the long-persistent luminescence of near-infrared (NIR) emitting long-persistence luminescent nanoparticles (NLPLNPs) (the afterglow wavelength varies from 650-900 nm) pertain to the tissue transparency window, where light attenuation is largely due to scattering rather than absorption, which is advantageous for long-term in vivo and vitro imaging with deep penetration and a high SNR [18]. So far, large numbers of NIR persistent luminescence nanoprobes have been applied for imaging in vitro and in vivo. For example, they have been used to diagnose tumors and realize long-time monitoring of tumors [19] and have also been utilized to develop novel monodisperse NIR emitting long-persistence luminescent nanoparticles with a porous structure to realize not only tumor diagnosis but also the drug delivery [20]. Recently, non-toxic NLPLNPs with a narrow size distribution were utilized to realize tumor-targeting and efficient cell tagging to track the biological fate of cells in vivo in healthy tumor [21]. Although the NLPLNPs have displayed excellent advantages for long-time in vivo imaging with a high SNR, few researches focused on long-time cell imaging which is one of these important parts in bioimaging. In particular, up to now, NLPLNPs still no effectively realize specific recognition of cancer cells in vitro due to lack of suitable surface functional groups. Therefore, developing NLPLNPs with specific recognition agent is important for the long-time cell imaging.
In this paper, we utilized NIR emitting long-persistence luminescent Zn 3 Ga 2 Ge 2 O 10 :Cr 3? (ZGG) nanoparticles as targeted optical probes for labeling human breast cancer cells. The antibody anti-EpCAM, where corresponding antigen receptors are known to be overexpressed in both primary and metastatic breast cancer [22], were utilized as specific recognition agent to functionalize ZGG surface. ZGG-EpCAM can specifically recognize the long-term monitored breast cancer cells (MCF7) without persistent activation, which can obviously improve the SNR. With confocal microscopy and IVIS Lumina II imaging system, we demonstrate receptor-mediated recognition ZGG-Ep-CAM bioconjugates to breast cancer cells.

Surface Modifications of Long Afterglow NIR ZGG Nanoparticles
The surface modification of ZGG with APTES to get SiO 2 and NH 2 layers was performed according to previous report [19,20]. In detail, the ZGG nanoparticles were firstly hydroxylated by basic wet grinding of the solid (40 mg) for 30 min with a mortar and pestle in 5 mM NaOH solution.
The hydroxylation was performed overnight by dispersing the ground powder in 80 mL NaOH solution. After standing for 6 h, the supernatant was collected by centrifugal separation at 3,000 rpm for 10 min, and the sediment was dried at 80°C for 8 h. The hydroxyl-modified ZGG particles were obtained. The 40 mg of hydroxyl-ZGG particles was dispersed into the mixture of 66.7 mL ethyl ethanol, 13.3 mL ultrapure water, and 5 mL concentrated aqueous ammonia (28 wt%) and ultrasonically treated for 50 min. The obtained suspension was put into 10°C water bath with continuous magnetic stirring for 30 min, and then 0.1 % (by volume) TEOS alcoholic solution was dropwise added. After that another 0.1 % (by volume) alcoholic solution was added and stirred for 10 h. Finally, the ZGG-NH 2 product was obtained by centrifugally separation at 10,000 rpm and washed with absolute ethyl ethanol for three times and dried in vacuum at 60°C for 5 h. Functionalization of ZGG-NH2 conjugated with an anti-Ep-CAM antibody. 10 mg of ZGG-NH 2 was immersed in 10 mL of PBS and ultrasonically treated for 30 min to form the ZGG-NH 2 solution. 20 lL of anti-EpCAM (200 lg mL -1 ) was diluted by 10 mL of PBS. 15 mg of EDC and 10 mg of NHS powder were added into the diluted solution, and then the carboxyl anti-EpCAM solution was activated. The above two solutions were mixed thoroughly and then centrifuged and washed with PBS for three times to obtain the conjugation of ZGG-EpCAM.

Characterization
X-ray powder diffractions (XRD) were performed on Panalytical X'pert PRO diffractometer equipped with Cu Ka radiation (k = 1.5418 Å ) at room temperature. Transmission electron microscopy (TEM) experiment was conducted on HITACHI H-7650 system. Hydrodynamic size distribution and zeta potential were measured by dynamic light scattering method (DLS) using a Malvern Zeta-sizer 3000 HS. The infrared spectra were obtained on a Fourier transform infrared spectroscopy with KBr pellet techniques (FTIR, Nicolet AVATAR, FTIR360). Optical imaging in cancer cells was achieved in an in vivo imaging system (Caliper Life sciences, IVIS lumina II) and Confocal Laser Scanning Microscopy (ZEISS, LSM710). The spectral properties (excitation and emission spectra, decay curves, long afterglow emission, and excitation spectra) of the ZGG nanoparticles were measured using an Instrument FS920 spectrometer fluorescence spectrometer.

Cytotoxicity Test In Vitro
Cytotoxicity was measured by MTT assay. Briefly, MCF7 were seeded in 96-well plates (CORNING, USA) for 12 h. Then, after the old medium was removed, fresh medium containing ZGG-NH 2 nanoprobes with different concentrations was added and incubated for 24 h at CO 2 incubator (Thermo heracell 150i, USA). Subsequently, MTT was added and incubated for 4 h at CO 2 incubator. The medium was removed, and DMSO was added to dissolve the formazan for 20 min. Finally, the absorbance at 490 nm was measured using a microplate reader (Molecular Devices, Sunnyvale, CA, USA), with a reference at 650 nm.

In Vitro Imaging of Cells
For observing in vitro imaging of ZGG-EpCAM, MCF7 cells were cultured in RMPI-1640 medium containing 10 % FBS, 1 % penicillin, and 1 % amphotericin B. An appropriate number of MCF7 cells were firstly seeded in 35-mm culture dishes for 12 h before the treatment day. And then the seeded cells in serum-supplemented media were treated with 200 lL ZGG-EpCAM nanoprobes (50 lg mL -1 ) for 2 h at 37°C. After 2 h, the cells were washed several times with PBS and directly imaged by a confocal microscope. In order to confirm that anti-EpCAM of modified ZGG-NH 2 could selectively bound with Ep-CAM protein of the cells, MCF7 cells were incubated with 200 lL unmodified ZGG-NH 2 (50 lg mL -1 ) and 5 lL anti-EpCAM (1 mg mL -1 ) blocking solution before addition of 200 lL ZGG-EpCAM nanoprobes (50 lg mL -1 ) as control. Confocal microscopy images were obtained using a fluorescence imaging system with laser excitation at 405 nm. The in vitro images of these two samples were obtained using IVIS Lumina II imaging system with UV lamp laser excitation for 2 min at 254 nm. All images were taken at the same condition. The pictures were taken per every 5 min, and the detecting time is 20 min.

Results and Discussion
The design and preparation process of long afterglow luminescent ZGG-EpCAM nanoprobe for cellular targeting and imaging was illustrated in Scheme 1. Firstly, the functionalization of ZGG nanoparticles could enhance the amount of -OH groups in NaOH solution. Subsequently, after the reaction with TEOS and APTES, a thin layer of SiO 2 and NH 2 groups were anchored on the ZGG-OH nanoparticle surface. Finally, in the presence of EDC and NHS, anti-EpCAM could react with -NH 2 groups on the ZGG-NH 2 nanoparticle surface to acquire recognition site and then the ZGG-EpCAM nanoprobe was obtained. It will be used to identify the cancer cells which could overexpress EpCAM antigen. The ZGG nanoparticles were systematically characterized by powder XRD. The XRD result of the ZGG nanoparticles is shown in Fig. 1. It shows that the phase of ZGG is in good agreement with reference data for the cubic phase of ZnGa 2 O 4 (JCPDS card no. 86-0415), rhombohedral structure of Zn 2 GeO 4 (JCPDS card no. 011-0687), and hexagonal structure of GeO 2 (JCPDS card no. 34-1089), suggestive of the presence of a mixture of ZnGa 2 O 4 , Zn 2-GeO 4, and GeO 2 in ZGG. Transmission electron microscopy illustrates that ZGG nanoparticles possess diameters ranging from 30 to 100 nm and some nonspherical shapes with different aspect ratios (Fig. 2a). After capped with a layer of SiO 2 and NH 2 groups, the diameter of ZGG-NH 2 did not obviously changed compared with ZGG as shown in Fig. 2b. DLS analysis manifested the effective hydrodynamic size and size distributions of ZGG-EpCAM nanoparticles suspensions in ultrapure water. It demonstrated that the nanoparticles suspensions had effective hydrodynamic diameters ranging from 35 to 140 nm, which mainly distributed in the range of 50-70 nm (Fig. 2c). It is well known that narrow size distribution of nanoparticle is important for bioimaging. The diameter which is less of 100 nm is fit for application in vitro imaging.
In order to improve the biocompatibility, solubility, and biological specificity of ZGG, anti-EpCAM monoclonal antibody was grafted onto the surface of ZGG. The epithelial cell adhesion molecule (EpCAM) is a monomeric membrane glycoprotein that can be expressed in most normal human epithelium and overexpressed in most carcinomas. It has become a potential target for the visualization and therapy of human solid tumors [23][24][25]. The surface zeta potentials of the nanoparticles during different surface modification processes were used to monitor the change of surface functional group, as shown in Fig. 3a. After the reaction with NaOH, zeta potential of the sample became negative (-12.6 mV) which proved that hydroxyl groups were modified on the ZGG surface. However, the samples further treated with APTES gave the positive surface potential (?24.9 mV) as a result of the amino groups at the surface. Furthermore, the anti-EpCAM layer was grafted on the nanoparticles, zeta potential switched from positive to nearly values (-4.83 mV). In order to confirm the modification process, the FTIR spectra analysis was utilized, as shown in Fig. 3b. The strong FTIR band of ZGG-OH at 3,415 cm -1 shows the successful hydroxylation of the ZGG surface. The FTIR peaks of ZGG-NH 2 at 3270, 1670, and 1051 cm -1 could be attributed to the stretching vibration of N-H and -Si-O-Sibonds, which indicate the existence of amino groups on the surface of ZGG via modification of APTES. The peak of ZGG-EpCAM at 1,725 cm -1 could be attributed to the stretching vibration of C=O bonds, indicative of the success of surface modification with anti-EpCAM monoclonal antibody. All the results suggest that the anti-EpCAM had  been successfully conjugated on the surface of ZGG-NH 2 to form specific recognition ZGG-EpCAM nanoprobes. The emission spectrum of ZGG-EpCAM nanoprobes at room temperature (Fig. 4a) consists of a narrow-band emission peaked at 698 nm under excitation at 254 nm. This NIR emission was characteristic of Cr 3? ions and could be attributed to the spin-forbidden 2 E ? 4 A 2 transition. The excitation spectrum monitored at 698 nm consists of three main excitation bands originating from the inner transitions of Cr 3? located at 264, 426, and 575 nm, ascribed to the Cr 3? , 4 A 2 ? 4 T 1 (te 2 ) 4 A 2 ? 4 T 1 (t 2 e), and 4 A 2 ? 4 T 2 (t 2 e) transitions, respectively [26]. Furthermore, ZGG-EpCAM nanoprobes show excellent long afterglow NIR properties. The long afterglow intensity monitored at 698 nm after irradiation by 254 nm UV light for 5 min  Fig. 4 a Excitation (k em = 698 nm) and emission (k ex = 264, 426, and 575 nm) spectra of ZGG; b NIR long afterglow decay curve of ZGG-NH 2 monitored at 698 nm after irradiated by 254 nm light for 5 min. The inset showed long afterglow emission of ZGG-NH 2 nanoprobes recorded at 5 min after stopping irradiation which was found to decrease in the first several minutes, and then slowly decayed. After 3 h of persistent emission, the persistent luminescence intensity was still kept (Fig. 4b).
It is well known that low toxicity of nanoprobes is critical for in vivo or in vitro studies [19][20][21]. Herein, the cytotoxicity of ZGG-NH 2 to MCF7 cells was examined by employing MTT method. As shown in Fig. 5, there are no obvious changes in the viability of MCF7 cells with the increasing concentrations of ZGG-NH 2 after 24 h of incubation. Even though the concentration of ZGG-NH 2 reached up as high as 40 mg L -1 , the viability of MCF7 cells could keep in 98.9 %. This result shows that the ZGG-NH 2 has no obvious cytotoxicity and is suitable for in vitro imaging.
In the in vitro imaging study, the ZGG-NH 2 and ZGG-EpCAM were utilized to label human breast cancer cells (MCF7), as shown in Fig. 6a and b. Weak luminescence signal could be observed in MCF7 cells treated with ZGG-NH 2 solution, while strong luminescence signal could be clearly observed in the case of MCF7 cells treated with ZGG-EpCAM (Fig. 6b), which could confirm the specific recognition of ZGG-EpCAM to MCF7 cells. In addition, when the blocking solution (add anti-EpCAM before adding ZGG-EpCAM) was used to eliminate the ability of anti-EpCAM to recognize human breast cancer cells (MCF7), weaker luminescence signal was observed in MCF7 cells (as shown in Fig. 6c), which indicated that the targeting capability of ZGG-EpCAM to breast cancer cells can be ascribed to the anti-EpCAM conjugated on surface. These results support that the engineered ZGG-EpCAM could specially recognize EpCAM overexpressing cells. Therefore, the engineered ZGG-EpCAM bioconjugates could serve as a potential biocompatible targeted nanoprobe to specifically diagnose human breast cancer cells.
To further support the recognition ability of ZGG-Ep-CAM nanoprobes to breast cancer cells, the persistent luminescence properties were also employed to probe recognition capacity of ZGG-NH 2 modified or unmodified anti-EpCAM to MCF7 cancer cells. The IVIS Lumina II imaging system was used to quantify their luminescence change within 20 min from the different treated cells, and the luminescence intensity from treated cells with ZGG-EpCAM was stronger than that of treated cells with ZGG-NH 2 . We could detect the luminescence signals of both samples even after 20 min as shown in Fig. 7a. The luminescence intensity of the cells treated with ZGG-Ep-CAM nanoprobes was nearly 2.3 times higher than that of cells treated with ZGG-NH 2 as shown in Fig. 7b, which might further reveal that the ZGG-EpCAM nanoprobes possessed relatively strong specific binding capability to breast cancer cells. Therefore, with the help of IVIS Lumina II imaging system, ZGG-EpCAM nanoprobes could be employed to realize quantitative luminescence signal detection of labeled breast cancer.

Conclusion
In summary, we synthesized NIR emitting long-persistence luminescent ZGG nanoparticles by employing a sol-gel method. In order to enhance their specific capability to breast cancer cells, ZGG nanoparticles were modified with anti-EpCAM antibody to obtain ZGG-EpCAM nanoprobes. These nanoprobes not only possess a narrow-band emission peaked at 698 nm under excitation at 254 nm with excellent NIR long afterglow properties and low cytotoxicity but also possess targeting capability toward breast cancer cells (MCF7) which overexpressed EpCAM antigen. Further results of in vitro imaging confirm that ZGG-EpCAM possesses specific targeting capability toward breast cancer cells. More important, we could utilize ZGG-EpCAM long afterglow nanoprobes to realize the real-time monitored breast cancer with the help of IVIS Lumina II imaging system. This will improve the efficiency of detection in vivo at an early stage. We expect this study to provide a new perspective in the diagnosis of cancer.
Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.  Fig. 7 In vitro imaging of a ZGG-NH 2 -conjugated anti-EpCAM treated with MCF7 cells in the (ZGG-EpCAM) culture dish, and just ZGG-NH 2 treated with MCF7 cells in the (ZGG-NH 2 ) culture dish; two culture dishes were simultaneously exposure to UV light 2 min before collecting images; the interval time is 5 min between two pictures; b The ratio of persistent luminescence intensity from cells of (ZGG-EpCAM) culture dish divides by the persistent luminescence intensity from (ZGG-NH 2 ) culture dish


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Carborane-Based Carbonic Anhydrase Inhibitors: Insight into CAII/CAIX Specificity from a High-Resolution Crystal Structure, Modeling, and Quantum Chemical Calculations

Carborane-based compounds are promising lead structures for development of inhibitors of carbonic anhydrases (CAs). Here, we report structural and computational analysis applicable to structure-based design of carborane compounds with selectivity toward the cancer-specific CAIX isoenzyme. We determined the crystal structure of CAII in complex with 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane at 1.0 Å resolution and used this structure to model the 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane interactions with CAIX. A virtual glycine scan revealed the contributions of individual residues to the energy of binding of 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane to CAII and CAIX, respectively.

Introduction
Carbonic anhydrases (CAs) play important roles in many physiological and pathophysiological processes. For example, extracellular CAs participate in tumor growth and progression [1]. CAIX, which is selectively expressed in a range of hypoxic tumors, is a validated diagnostic and therapeutic target (recently reviewed in [2][3][4]). There are 15 human CA isoenzymes, and due to the ubiquity of these enzymes in human tissues, selective inhibition is a very important aspect of drug design.
CA inhibitors from the first class (metal ion binders) contain specific functional groups that interact with the catalytic Zn 2+ ion in the CA active site. These metal-binding functionalities are typically joined to a "ring" structure. This moiety is not necessarily aromatic; however, it is usually consisting of a 5-or 6-membered hydrocarbon ring or conjugated ring system containing nitrogen, oxygen, and/or sulfur. Numerous functional groups have been added to the ring structure scaffold to modify inhibitor properties such as specificity toward a particular CA isoenzyme, pK a , or solubility (reviewed in [6]). Recently, we reported design of CA inhibitors containing space-filling carborane clusters in place of the typical ring structure [7]. We showed that various carborane clusters act as CA inhibitors and that modifying these clusters with an appropriately attached sulfamide group and other substituents leads to compounds with selectivity toward the cancer-specific CAIX isoenzyme.
Boron is one of few chemical elements that can form binary hydrides composed of more than two atoms, which leads to formation of boron cluster compounds (boron hydrides or boranes). Their basic structural feature is formation of a polyhedron with triangular facets held together by 3center 2-electron bonds with an extensive electron delocalization [8]. A typical structural archetype is represented by the divalent closo-B 12 H 12 2− anion, an extremely stable compound with a symmetrical 12-vertex icosahedron structure [9]. Replacement of one or more {BH − } in borane cage with {CH} leads to series of carboranes and removal of BH vertices leads to various open-cage (nido-) species. Carboranes thus offer a large variety of structural archetypes that provide interesting counterparts to organic compounds [10].
Many features of icosahedral 12-vertex carboranes are useful in the design of biologically active compounds. Carboranes have high thermal and chemical stability; therefore, they generally do not undergo catabolism and are nontoxic to the host organism [11,12]. The basic closo-C 2 B 10 H 12 carborane cluster is highly hydrophobic [13]; however, its controlled deboronation can generate water soluble 11-vertex nido-C 2 B 9 H 12 − . These anions represent important intermediates in the synthesis of a family of mainly anionic metal bis(dicarbollides) accessible via metal insertion. Incorporation of carborane cages into the structures of certain substances of medicinal interest can enhance hydrophobic interactions between the boron cluster-coupled pharmaceuticals and their protein targets, increase in vivo stability, and facilitate uptake through cellular membranes [14,15]. The successful use of boron clusters as hydrophobic pharmacophores has recently been increasing [16,17]. Examples of carborane pharmacophores include boron-containing antifolates [18], HIV protease inhibitors [19,20], and estrogen receptor agonists and antagonists [21], among others [16,22,23].
Drug design efforts benefit greatly from knowledge of the 3D structures of protein-ligand complexes. X-ray crystallography has contributed considerably to the development of CA inhibitors; more than 500 structures of human CA isoenzymes (wild-type and mutant forms) in complex with various inhibitors have offered unprecedented insight into inhibitor binding modes (reviewed in [24]). Structural information coupled with experimental inhibition data can be used to validate various computational approaches to assess inhibitor binding strength. Once a particular theoretical approach reproduces the known data well, it can be used for prospective design. For studies involving metal ions and unusual compounds such as boranes, the use of quantum chemistry (QM) is warranted [25,26]. Indeed, we recently used a quantum mechanics/molecular mechanics (QM/MM) methodology to quantitatively describe the binding of two carboranebased sulfamides to CAII [7] and to explain fundamental differences in the binding modes of closo-and nido-cages [27].
Here, we report the X-ray structure of CAII with bound 1methylenesulfamide-1,2-dicarba-closo-dodecaborane (compound 1, Figure 1(a)) determined at 1.0Å resolution. This atomic-level resolution allowed us to assess in detail the positions of carbon and boron atoms in the carborane cage of 1. Additionally, we modeled the complex of 1 with CAIX. We employed a virtual glycine scan to analyze the differences between the interactions of 1 with CAII and CAIX.
The complex of CAII with 1 was prepared by adding a 1.1fold molar excess of 1 (in DMSO) to the 10 mg⋅mL −1 solution of CAII in water without pH adjustment (the final DMSO concentration did not exceed 5% v/v).
The best diffracting crystals were obtained using the hanging-drop vapor diffusion method under the following conditions: 2 L protein-inhibitor complex solution was For cryoprotection, the crystals were incubated in mother liquor supplemented with 25% glycerol for approximately 30 s, flash-frozen, and stored in liquid nitrogen. Diffraction data for the CAII complex were collected at 100 K at the X14.2 BESSY beamline in Berlin, Germany [29]. Data were collected in two passes: the high-resolution range (11.75-1.00Å) and the low-resolution range (21.08-1.20Å). The two datasets were integrated with iMOSFLM [30] and merged and scaled with SCALA [31]. Data collection and refinement statistics are summarized in Table 1.

Structure Determination, Refinement, and Analysis.
Crystal structures were solved by difference Fourier method using the CAII structure (PDB code 3IGP [34]) as a starting model. The model was refined using REFMAC5 [35], part of the CCP4 program suite [36]. The model was initially refined with isotropic atomic displacement parameters (ADPs); hydrogen atoms in riding positions were added later. For the final rounds of refinement, we used a mixed isotropicanisotropic model of ADPs: anisotropic ADPs were used for all atoms, and only atoms in alternative conformations were refined isotropically. Atomic coordinates for the structure of 1 were generated by quantum mechanics computation with DFT-D methodology [37] using the B-LYP functional and SVP basis set [38] in the Turbomole program [39]. A geometric library for 1 was generated using the Libcheck program from the CCP4 suite. Coot [40] was used for rebuilding. The quality of the refined model was assessed using MolProbity [33]. The coordinates and structure factors were deposited in the PDB under accession code 4Q78. Final refinement statistics are summarized in Table 1 [43]. Preparation of structure coordinate files for further calculations was performed as described before for CAII [27]. The complex was fully optimized using a QM/MM procedure. We used ONIOM-like subtractive scheme [44] with link atoms and mechanical embedding to be consistent with our previous studies [27,[45][46][47][48]. The QM part is described at the DFT-D TPSS/TZVP//BLYP/SVP level of theory [39] and comprises 218 atoms including the atoms present in 1 and 8 amino acids (Trp5, Asn62, His64, Gln67, Gln92, Val131, Leu135, and Pro202). The MM part constituted the remainder of the protein, and the surrounding solvent was approximated by a generalized Born (GB) implicit model. Detailed description of the procedure was published in [27]. One crystal water molecule (Wat272) bridging the inhibitors and CAII residues Thr199, Glu106, and Tyr7 was retained to maintain the integrity of the active site. Other water molecules present in the crystal structures were omitted.

Virtual Glycine Scan.
The contribution of the active site amino acids to inhibitor binding was examined by virtual glycine scanning. Individual amino acids in contact with 1 in the CAIX-1 model and CAII-1 crystal structure were substituted with glycine. The energy contributions (ΔΔG int ) were calculated as the difference between the original ΔG int at the QM/MM level with the wild-type amino acid and the new ΔG int with the mutated glycine residue [27].

Crystal Structure of CAII in Complex with 1 at Atomic Resolution.
The overall structure of CAII in complex with 1 was refined to 1.0Å resolution. This high resolution allowed us to observe details that could not be fully resolved in the complex structure determined previously at lower resolution. Atomic resolution was achieved by derivatization of CAII using the 4-(hydroxymercury)benzoic acid (abbreviated MBO in the cif library of small molecules) method described by [28]. The mercury atom of MBO covalently binds to S of Cys206. This modification allows formation of a hydrogen bond between the OZ1 oxygen of the MBO carboxyl group and the mainchain amino group of Tyr40 in the neighboring protein molecule, reinforcing the crystal lattice and increasing the diffraction quality of the crystal. In our structure, MBO is modeled in two alternative conformations with occupancies of 0.6 and 0.2.
When our atomic resolution structure is compared with the structure of the CAII-1 complex determined at 1.35Å resolution (PDB code 4MDG [7]), the RMSD value for The data in parentheses refer to the highest-resolution shell. a merge = ∑ ℎ ∑ (ℎ ) − ⟨ (ℎ )⟩ | / ∑ ℎ ∑ (ℎ ), where (ℎ ) is the individual intensity of the th observation of reflection ℎ and ⟨ (ℎ )⟩ is the average intensity of reflection ℎ with summation over all data. b value = || | − | ||/| |, where and are the observed and calculated structure factors, respectively. free is equivalent to value but is calculated for 5% of reflections chosen at random and omitted from the refinement process [32]. d as determined by Molprobity [33].
superposition of the C atoms of residues 4-261 is 0.142Å, a value typical for superposition of identical structures [51]. The N-terminal residue His3 is traced differently in the two structures; double conformations of numerous side chains (e.g., Glu14, His64, and Gln74) are resolved in the atomic resolution structure. We found an additional difference in the loop formed by amino acid residues 124-139, with a maximum difference of 0.738Å for the position of Gln136 C . Gln136 forms van der Waals contacts with the MBO covalently attached to Cys206. The positions of Phe131 and Val135, which form a hydrophobic rim at the active site, are also influenced by MBO binding. This results in a subtle positional shift of the inhibitor, with an RMSD of 0.145Å for superposition of 12 atoms in the carborane cage of 1 bound to CAII and CAII derivatized by MBO. This value is below the value observed for superposition of identical structures [51].
Atomic-level resolution allowed us to resolve the carbon and boron atom positions in the symmetrical carborane cage of 1. When analyzing the values of the electron density map at positions of atoms bonded to the C1 atom, we can assume that positions with higher density levels are more likely to be carbon than boron atoms. Similar analysis was done by others for boron-containing inhibitor of human dihydrofolate reductase [18]. The C2 atom of the carborane cage (Figure 1(a)) was modeled into the position with an electron density value of 1.16 e/Å 3 , which was approximately 0.15 e/Å 3 higher than those for the B3, B4, B5, and B6 atoms. To exclude the possibility that higher density is caused by model bias, we altered the composition of the cage by replacing the C2 atom with a boron atom. Electron density values did not change significantly after several rounds of refinement cycles.
Thus, we can conclude that the most probable position of the second carbon atom in the carborane cage of 1 is the position assigned to the C2 atom in our crystal structure. This is in good agreement with the recently published QM/MM modeling study [27].

Detailed Analysis of Inhibitor Interactions with CAII.
The crystal structure of human CAII in complex with 1 determined at 1.0Å resolution confirmed the key interactions that our group observed previously [7]. The compound fits very well into the CAII active site cavity and makes numerous polar and nonpolar interactions with the residues in the enzyme active site. The sulfamide moiety, which forms key polar interactions with the active site Zn 2+ ion, also makes polar interactions with Thr199 typical of other sulfamide inhibitors of CAII (Figure 2). The linker NH group forms an additional polar interaction with O of Thr200. The compound makes several van der Waals interactions with residues Gln92, His94, His96, His119, Val121, Phe131, Leu198, and Thr200 ( Figure 2). All interactions between the inhibitor and protein are summarized in Table 2. 3 The idea of designing CA inhibitors containing a carborane cluster moiety originated from our previous structural studies of isoquinoline-containing sulfonamide inhibitors (Figure 3(a)). Structural analysis of CAII in complex with 6,7dimethoxy-1,2,3,4-tetrahydroisoquinolin-2-ylsulfonamide (2, PDB code 3IGP, [34]) and 6,7-dimethoxy-1-methyl-1,2,3,4tetrahydroisoquinolin-2-ylsulfonamide (3, PDB code 3PO6, [52]) revealed two distinct binding modes that engage two opposite sides of the enzyme active site cavity (Figure 3(b)). Following this analysis, we hypothesized that the binding space within the enzyme active site cavity could be effectively filled with a bulky hydrophobic molecule with a spherical structure. This led to design of 1 which exhibited inhibitory property to CAII and CAIX with Ki values in submicromolar range. Structural analysis of CAII-1 indicates that our structure-based design was sound. We found that the carborane cluster interacts with both sides of the enzyme active site as predicted (Figure 3(c), Table 3) and that the position of 1 in the CAII active site superposes well with the two binding modes observed for 2 and 3 (Figure 3(d)).

Model of the CAIX-1 Complex.
The CAII-1 crystal structure was used to model binding of compound 1 into the CAIX active site using QM/MM methods (Figure 4).
The substrate binding sites of CAII and CAIX differ by only six amino acids: Asn67 of CAII is replaced by Gln in CAIX, Ile91 by Leu, Trp123 by Leu, Phe131 by Val, Val135 by Leu, and Leu204 by Ala. These variations result in a differently shaped active site cavity, which accommodated 1 in a slightly different pose (Figure 4). While the position of the sulfamide anchor remained unchanged, the carborane cluster shifted by 2.1Å (expressed as a difference in the position of B12) away from the central -sheet. In CAIX-1, the carborane interacts more with the opposite site of the active site, specifically with amino acid residues His94, His96, Glu106, Leu198, Thr199, Thr200, and Pro201 ( Figure 4, Table 3). All polar and van der a Atom labels correspond to those shown in Figure 1(a). b All contacts with a distance between ligand and protein (or Zn) atoms less than or equal to 4Å are listed. c Polar interactions are highlighted in bold.
Waals interactions between CAIX and 1 are summarized in Table 4. We used a virtual glycine scan to study the roles of individual amino acid side chains in the active sites of CAII and CAIX in binding of 1. The changes in free energy of   Thr199  Thr199  Thr199  Thr199  Thr200  Thr200  Thr200  Thr200  Pro201  Pro201  Pro202  Trp209  Trp209 Interacting residues were identified from a crystal structure 4Q78 (this work); b crystal structure 3IGP [34]; c crystal structure 3PO6 [52]; d computational model (this work); e residues making polar interactions are highlighted in bold.
interaction (ΔΔG int ) upon mutation of a given amino acid residue to glycine are shown in Figure 5. The largest energy change (2.6 kcal/mol) occurred for Trp5, which is positioned closer to 1 in CAIX-1 than in CAII-1. The side chain of Trp5 forms several dihydrogen bonds with the carborane cage of 1. The shortest one has a H ⋅ ⋅ ⋅ H distance of 2.3Å. The other major contributor to strong a Atom labels correspond to those shown in Figure 1(a). b All contacts with a distance less than or equal to 4Å between ligand and protein (and Zn) atoms are listed. c Polar interactions are highlighted in bold.
CAIX-1 binding was Asn62; the energy of binding exceeded that in CAII-1 by nearly 1 kcal/mol. These contributions were cancelled out by differences in binding energy contributions of amino acid residues 131 (Phe/Val) and 135 (Val/Leu), which were lower in CAIX by 0.7 and 0.9 kcal/mol, respectively. The energy changes of other residues were small. When we compared binding of 1 to CAII and CAIX, we noted that the favorable energy changes in CAIX-1 due to the binding of residues Trp5, Asn62, and His64 were slightly larger than the unfavorable changes in binding caused by the different amino acids at residues 131 and 135. This is in qualitative agreement with the experimental values, which are 700 ± 141 nm for inhibition of CAII and 380 ± 111 nM for inhibition of CAIX [7].

Conclusions
We determined to atomic resolution the crystal structure of CAII in complex with 1-methylenesulfamide-1,2-dicarbacloso-dodecaborane (1), a parent compound of a recently reported series of CA inhibitors containing carborane cages [7]. Comparing this crystal structure with those of CAII complexes with conventional organic inhibitors showed that the three-dimensional cluster fills the enzyme active site cavity. Atomic-level resolution allowed us to distinguish the positions of carbon and boron atoms in the carborane cage. The crystal structure also served as a model for construction of the CAIX-1 computational model. Virtual glycine scan enabled us to quantify the contributions of individual residues to the energy of binding of 1 to CAII and CAIX and uncover differences of the enzyme active site cavities. Structural and computational analysis will be used in future structure-based design of carborane compounds with selectivity toward the cancer-specific CAIX isoenzyme.