Enzyme inhibitionDOI: 10.1002/anie.201307583

Carborane-Based Carbonic Anhydrase Inhibitors**Jir� Brynda, Pavel Mader, V�clav S�cha, Milan F�bry, Kristyna Poncov�, Mario Bakardiev,Bohum�r Gr�ner, Petr C�gler, and Pavl�na Rez�cov�*

Human carbonic anhydrases (CAs) are zinc metalloenzymesthat play an important role in many physiological processes.To date, 15 human CA isozymes with different subcellularlocalization and tissue expression profiles have been identi-fied. Vast experimental evidence also suggests the involve-ment of CAs in various pathological processes (e.g., tumor-igenicity, obesity, and epilepsy). Many CA isozymes are thusrecognized as diagnostic and therapeutic targets.[1]

About 30 CA inhibitors are used clinically, for example, asanti-glaucoma drugs (targeting CAII, CAIV, and CAXII),anti-convulsants (targeting CAII, CAVII, and CAXIV) andanti-obesity agents (targeting CAVA and CAVB).[2] Recently,other isozymes, namely the neuronal CAVII and CAXIV, andthe cancer-associated forms CAIX and CAXII, have beenvalidated as targets for inhibitor development.[3]

The traditional CA inhibitors contain a sulfonamide orsulfamide moiety that coordinates the zinc cation located inthe CA catalytic site.[4] Most of the currently used CAinhibitors lack selectivity, and their use causes numerousunwanted side effects. A current challenge is the design ofcompounds that can inhibit specific isozymes. Although theconical active-site clefts of different human CA isoezymes areconserved, variations exist in the amino acid residues at theentrance to the active site. As a result of their differing inshape and hydrophobicity, these surface pockets can beexploited to design specific inhibitors.[5,6]

Structural analysis of CAII in complex with numerousinhibitors revealed two general binding modes, each involvinga distinct site within the enzyme active site cavity.[7] This led usto hypothesize that CA inhibitors could be designed more

effectively based on three-dimensional scaffolds rather thanflat structures.

Carboranes, icosahedral clusters containing boron,carbon, and hydrogen are bulky pharmacophores used toreplace various hydrophobic structures in biologically activemolecules.[8, 9] The 12-vertex carboranes increase the in vivostability and bioavailability of biologically active moleculesand enhance the hydrophobic interactions between them andtheir receptors.[10] They are an abiotic species that are verystable towards catabolism and degradation by enzymes andthus the use of boron clusters as components of newpharmacological agents has been increasing.[11–15]

With the help of manual molecular docking into the activesite of CAII, we designed 1a, which contains a sulfamidegroup connected to a carborane cluster intended to optimallyfill the enzyme active site. The length of the linker betweenthe sulfamide group and carborane cluster was chosen basedon comparison with the structures of isoquinoline sulfon-amide inhibitors.[7] Attachment of the sulfamide moiety wasaccomplished by using a transamination reaction betweenaminomethylcarborane 1b and sulfamide (Scheme 1).

Compound 1a showed inhibitory activity toward CAII(Ki value of 0.7 mm) and showed almost 2-fold higher activitytoward the tumor-associated isoform CAIX (Ki of 0.38 mm).

The crystal structure of 1a in complex with CAIIdetermined at 1.35 � resolution (PDB code 4 MDG) con-firmed that the inhibitor binds in the enzyme active site aspredicted (Figure 1) and revealed key interactions responsi-ble for inhibitor binding and enzyme inhibition.

The sulfamide moiety of 1a proved to be the anchoringgroup that completes the coordination sphere of Zn2+ in theactive site and makes the polar interactions with Thr199 thatare typical of other CA inhibitors.[4] An additional polarinteraction is a hydrogen bond between a linker NH groupand the side chain Og of Thr200 (Figure 1a). Furtherinteractions of the inhibitor with the active site cavity aremediated through van der Waals interactions between thecarborane cluster and amino acid residues Gln92, His94,Phe131, Leu198, and Thr200. For a complete list of inter-actions, see Table S1 in the Supporting Information. Com-pound 1a fills the proximal active site cavity of CAII, but

Scheme 1. Preparation of 1a by heating 1b with sulfamide in dioxane.Vertices represent BH, black spheres CH or C if substituted.

[*] Dr. J. Brynda, Dr. P. Mader, Dr. M. F�bry, Dr. P. Rez�cov�Institute of Molecular GeneticsAcademy of Sciences of the Czech Republic, v.v.i.V�densk� 1083, 142 20 Prague 4 (Czech Republic)E-mail: rezacova@uochb.cas.cz

Dr. J. Brynda, K. Poncov�, Dr. P. C�gler, Dr. P. Rez�cov�Institute of Organic Chemistry and BiochemistryAcademy of Sciences of the Czech Republic, v.v.i.Flemingovo n�m. 2, 16610 Prague 6 (Czech Republic)E-mail: cigler@uochb.cas.cz

Dr. V. S�cha, Dr. M. Bakardiev, Dr. B. Gr�nerInstitute of Inorganic ChemistryAcademy of Sciences of the Czech Republic, v.v.i.250 68 Rez u Prahy (Czech Republic)E-mail: gruner@iic.cas.cz

[**] This work was supported by Grant Agency of the Academy ofSciences of the Czech Republic (project IAAX00320901), TechnologyAgency of the Czech Republic (project TE01020028) and in part byresearch projects RVO 68378050, 61388963, and 61388980 awardedby the Academy of Sciences of the Czech Republic.

Supporting information for this article is available on the WWWunder http://dx.doi.org/10.1002/anie.201307583.

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some side pockets at the entrance of the active site couldpotentially be targeted by cluster substituents (Figure 1 b).

Next, we considered several modifications and prepareda series of novel boron cage compounds combining variouscarborane clusters with a polar sulfamide moiety (Figure 2).Compounds 2a–10 a were prepared in high yields using thetransamination reaction depicted in Scheme 1.

Our pilot series of ten CA inhibitors includes the parentcompounds 1a and 4a, which contain one alkyl sulfamidegroup and icosahedral ortho- and meta-carborane units,respectively. A structurally similar, charged derivative basedon the [CB11H12]

� cage was also prepared (6 a). Closo-carboranes containing either hydrophobic (2a and 3a) orhydrophilic (second sulfamide moiety; 5a) substituents andtheir charged 11-vertex nido counterparts (7a–10a) completethis structurally diverse series.

The compounds were tested for inhibition of the humanCA isozymes CAII and CAIX using a colorimetric enzymaticassay. Most of the compounds tested displayed good CAinhibition profiles, with Ki values in the low micromolar orsubmicromolar range (Table 1). Almost all of the compoundsshowed selectivity for the cancer-specific CAIX isozyme overthe more abundant variant CAII.

Analysis of crystal structures of CAII in complex with 4aand 7a (PDB codes 4MDL and 4MDM) confirmed that thesecompounds are positioned similarly in the enzyme active siteto 1 a. In fact, 4a binds to CAII identically to 1 a in terms ofconformation and interactions (Figure 3a).

Compound 7a binds CAII differently and, unlike 1a and4a, does not form a hydrogen bond between its linker NHgroup and the side chain Og of Thr200 (Figure 3b). Thedistance between the position of the linker NH group in 1aand 7 a is 0.18 �. The nido-carborane cluster interacts withHis64, Gln92, His94, Leu198, Thr200, and Pro201 (fora complete list of interactions see Table S1 in SupportingInformation).

In our pilot series of carborane-based CA inhibitors, weexploited four different carborane cluster types. The effect ofcluster type on inhibition can be deduced from comparison of

the Ki values obtained for 1a, 4a, and 7a. The o-carboranederivative 1a has slightly better inhibitory potency towardCA isozymes than the m-carborane derivative 4a. This couldresult from the presence of a slightly acidic CH group in theposition vicinal to the substituted carbon in 1a. Compound 7a(containing 7,8-nido-carborane) is a less efficient inhibitor ofCA isozymes than the closo-carborane derivatives; however,it shows a slightly different binding mode. As revealed by the

Figure 2. Structures of compounds used in this study. Amino groupsattached to the anionic clusters (6b–10b) are protonated, formingzwitterions. 3 was prepared as an inseparable mixture of two isomers(termed 3m and 3n). The substituent in 10b is located at boron B(10)sitting in the open face and not at a carbon as in 7.

Table 1: In vitro inhibition of selected carbonic anhydrase isozymes.

Compound Ki (CAII)[mm]

Ki (CAIX)[mM]

SelectivityIndex[a]

1a 0.70�0.14 0.38�0.11 1.82a 348�113 161�37 2.23ma + 3na 0.51�0.08 0.43�0.17 1.24a 1.16�0.24 1.12�0.20 1.05a 0.38�0.14 0.23�0.04 1.76a 8.57�2.24 2.20�0.48 3.97a 6.79�2.01 5.09�2.38 1.38a 9.00�2.23 2.32�1.02 3.99a 2.71�0.47 0.15�0.06 18.110a 132�19 26.04�2.93 5.1

[a] Selectivity index is the ratio between Ki (CAII) and Ki (CAIX).

Figure 1. Crystal structure of CAII in complex with 1a. a) The protein isshown as a ribbon diagram; residues involved in interactions with theZn2+ ion (gray sphere) and 1a are shown as stick representations.C green, B pink, S yellow, O red, and N blue atoms are shown. Polarinteractions are represented by blue dashed lines; Zn2+ ion coordina-tion is shown as black dashed lines. b) Top view into the active site,shown as a surface representation. 1a is shown as a space fillingmodel.

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co-crystal structure, the nido cluster 7a has the possibility toadjust its position within the active site, which confersa potential advantage in that it can accommodate additionalsubstituents that might affect selectivity toward a particularCA isozyme.

To explore a greater diversity of carborane clusters, wealso prepared 6 a, which contains a 1-carbadodecaborate ion[CB11H12]

� . The removal of one methylene group from thelinker and the presence of a negatively charged cluster led toa decrease in efficiency but a substantial increase in selectivityindex compared to 1a, 4 a, and 7a. This demonstrates thata relatively subtle change in structure can strongly influenceinhibitor interactions with a particular CA isozyme.

In our initial molecular design, we hypothesized thattuning compound selectivity to a certain CA isozyme could beachieved by cluster substitutions. In our series, we exploiteda phenyl ring as a substituent in synthetically accessiblepositions on the cluster. Analysis of the crystal structure of 1ain complex with CAII suggested that a phenyl ring in theortho position on the closo-carborane cluster would havesteric clashes with amino acids lining the entrance to theenzyme active site. Indeed, this detrimental effect oninhibitory efficiency was illustrated by the 400-fold greaterKi value obtained for inhibition of CAII with 2a. On the otherhand, the phenyl substituent placed in the meta-position, as in3ma and 3na, seems not to experience any steric clash withthe bottom of the active site, and the inhibitory potenciestoward CAII and CAIX remain virtually unchanged, withKi values close to those of parent compound 1a.

The nido-carborane cluster was substituted with a phenylring in the ortho position, leading to 8a. We did not observea loss of inhibitory potency as a result of the addition ofa phenyl ring in this position. Derivative 8a shows onlya slightly increased Ki value for CAIX, while the Ki value forCAII is lower than that of parent compound 7a. The smallernido cluster is apparently able to adjust its position in theactive site cavity; the phenyl ring can thus be accommodatedin a side pocket. Inspection of the crystal structure of CAII incomplex with 7a suggests that a pocket formed by Ile91 andPhe131 (Figure 1b) would be accessible for substituent

binding. In CAIX, this pocket contains Leu91 and Val131and is more spacious.

In 5 a, an additional sulfamide group was attached bya methylene linker to the meta position of the meta-carboranecluster. This increased the inhibitory potency compared tothat of the parent m-closo-carborane by a factor of approx-imately 2. This increase can be attributed to the presence oftwo sulfamide groups that can anchor the compound into thecatalytic site of the enzyme.

The effect of the linker connecting the cluster to thesulfamide group can be deduced from comparison of theinhibitory properties of 8a and 9a, and 7a and 10a ; two pairsof compounds with the same carborane cage but with linkersdiffering in length and nature.

Compound 8a contains the methylene linker designed inthe initial docking. Increasing the length of the aliphaticlinker to a propylene moiety improved the inhibitory potencyof 9a and made it almost 5-fold more selective toward CAIX.Increasing the length of the cluster-sulfamide linker by twomethylene groups probably allows accommodation of thenido cluster with its phenyl substituent in side pockets at theentrance of the enzyme active site (Figure S3 in the Support-ing Information). With a CAIX/CAII selectivity index of 18.1,Compound 9a showed the highest selectivity observed withinthe entire series.

For the parental nido cluster, we therefore tested an evenlonger, 7-atom linker (10a). This elongation, however, sub-stantially decreased the inhibitory efficiency of the compoundcompared to that of the cluster bearing a short methylenelinker (7a). A possible explanation for this decrease, based onthis compound�s similarity to ionic metallacarboranes bearinga similar linker, is that complexation with sodium cations maylead to a rigid, crown-ether-like arrangement.[16] This woulddecrease the compound�s solubility and its ability to interactwith the catalytic cleft. Our observations suggest that both thelength and the nature of the cluster-to-sulfamide linker arecrucial for inhibitory efficacy and should be carefully tuned infuture design.

In conclusion, our results suggest that carborane-basedcompounds are promising lead structures for the develop-ment of inhibitors of CA isozymes. Our experiments demon-strated that various types of hydrophobic, space-fillingcarborane clusters can be accommodated in the CA activesite and that substitution with an appropriately attachedsulfamide group and other substituents leads to compoundswith high selectivity for the cancer-specific CAIX isozymeover the widespread CAII isozyme. Crystal structures con-firmed our hypothesis that three-dimensional scaffolds couldbe efficiently used in CA inhibitors and provided structuralinformation that can be applied to the structure-based designof specific CAIX inhibitors.

Experimental SectionExperimental procedures including detailed synthetic procedures forthe sulfamide derivatives and their precursors are described in theSupporting Information. Briefly, the sulfamide compounds wereprepared from their respective amine or ammonium carboranederivatives (1b–10 b). Compounds 1b[17] and 6b[18] were prepared

Figure 3. Crystal structure of CAII in complex with 4a (a) or 7a (b).The protein is shown as ribbon diagram; residues involved ininteractions are shown as stick representations. Polar interactions arerepresented by dashed blue lines; Zn2+ ion coordination is representedby dashed black lines. The binding mode of 1a (black line) issuperimposed on the crystal structures.

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according to known procedures. 4b and 5b were prepared bylithiation of 1,7-carborane, reaction with bromomethyl phthalimide,and removal of the phtalimido group by hydrazine hydrate.[19–22]

Derivatives 2b and 3b were synthesized similarly, but the finalsteps involved reduction with NaBH4 and subsequent hydrolysis.Although this pathway has been described as failing for 1-amino-methyl-o-carboranes,[20] we have demonstrated the feasibility of thisapproach for compounds 2b and 3b bearing methylene connectors.During synthesis of 3b, an equilibrium mixture of two isomers formedafter lithiation of 9-C6H5-1,2-C2B10H11 and subsequent reaction withbromomethyl phthalimide as a result of the presence of two availableCH sites with similar reactivity. The isolated mixture of the 9- and 12-phenyl isomers (in a nearly 1:1 ratio) could not be separated by liquidchromatography either as phthalimido-protected amines or afterdeprotection. This isomeric mixture was therefore used for thesynthesis of sulfamide derivatives 3ma and 3na. Derivatives 8b and9b were obtained using hydrazine hydrate to convert 1-alkylphthal-imido-2-phenyl-closo-1,2-carboranes into their respective 11-vertexnido-[C2B9H12]

� ion species, as described in a previous report.[17] Forthe preparation of 7b, we used a new straightforward one-stepdeboronation[8]/amination of 1-BrCH2-1,2-C2B10H11. The reactionproceeds even with 24 % aqueous NH4OH, producing a mixture ofthe target nido-derivative 7b along with two other boron-substitutedisomeric species (7-CH3-9-NH3-1,7-C2B9H10, 7-CH3-11-NH3-1,7-C2B9H10) in lower yield (ca. 35 %). Compound 7b could be isolatedin good yield (62%) by column chromatography. Compound 10b wasprepared by cleavage of the dioxane derivative 10-O(CH2CH2)2O-C2B9H11 with ammonia, as described in a previous report.[23]

Addition of the sulfamide end group was accomplished with highyields by heating the respective amines with sulfamide in dioxane (seeScheme 1). Potassium carbonate was used for the deprotonation ofthe ammonium groups or to release the respective amines from theirhydrochlorides in situ. Nevertheless, the attempted synthesis of 1-H2NSO2NH-(CH2)2-2-C6H5-closo-1,2-C2B10H10 and [7-H2NSO2NH-(CH2)2-8-C6H5-nido-7,8-C2B9H10]

� (potential members of the serieswith intermediate chain lengths; the amines prepared analogously toa published procedure[22]) failed owing to quantitative elimination ofthe substituent in the reaction with sulfamide, thus resulting in onlythe starting phenyl carborane.

Received: August 28, 2013Published online: November 4, 2013

.Keywords: carbonic anhydrases · carboranes · drug discovery ·inhibitors · structure elucidation

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Supporting Information

� Wiley-VCH 2013

69451 Weinheim, Germany

Carborane-Based Carbonic Anhydrase Inhibitors**Jir� Brynda, Pavel Mader, V�clav S�cha, Milan F�bry, Kristyna Poncov�, Mario Bakardiev,Bohum�r Gr�ner, Petr C�gler, and Pavl�na Rez�cov�*

anie_201307583_sm_miscellaneous_information.pdf

1a 7a 4a

Figure S1: Structures of compounds 1a, 4a and 7a with atom labels used in the crystal structure coordinate file.

Figure S2: Crystal structure of CAII in complex with compounds 1a (panel a), 4a (panel b) and 7a (panel c). The 2Fo-Fc electron density maps contoured at 1.0 . The inhibitors are in stick representation with carbon atoms colored green for 1a, cyan for 4a, and yellow for 7a. Boron atoms are colored pink and other heteroatoms are colored according to standard color coding: oxygen red; nitrogen blue; sulfur yellow. The protein is shown in cartoon representation.

1

Figure S3: Comparison of CAIX and CAII crystal structures. In panel a, crystal structure of CAIX (PDB code 3IAI) is represented by the VdW surface coloured by its electrostatic potential at a scale from −5 kT (red) to +5 kT (blue). CAII surface is shown as green mesh with compound 1a in black lines. In panel b, crystal structure of CAII (PDB code 4MDG) is represented by the VdW surface coloured by its electrostatic potential and CAIX surface is shown as green mesh. In both panels, two putative binding sites for inhibitor substituents at the entrance to the active site that differ in shape and electrostatic potential are indicated by arrows and numbers. Putative binding site 1 is formed by following residues in CAII/CAIX isoforms: Ile91/Leu91, Trp123/Leu123, Phe131/Val131, and a loop containing residues Asp71-Lys76/Pro71-Gly76. Binding site 2 is formed by following residues in CAII/CAIX isoforms: Gly132/Asp132, Val135/Leu135, Gln136/Gly136, and Leu204/Ala204.

2

Table S1 List of contacts between CA II and compounds 1a, 4a and 7a. All contacts with a distance below 4.0 Å between inhibitor atoms and protein residues or coordinated Zn ion are listed. Polar interactions are highlighted in italic bold.

CAII 1a CAII 4a CAII 7a

Residue Atom Atom Distance

[Å] Residue Atom AtomDistance

[Å] Residue Atom Atom Distance

[Å]

ZN ZN N2 1.86 ZN ZN O2 3.13 ZN ZN O2 3.12

ZN ZN O2 3.17 ZN ZN S1 3.11 ZN ZN S1 3.06

ZN ZN S 3.11 ZN ZN N2 1.84 ZN ZN N2 1.86

GLN 92 NE2 B6 3.64 GLN 92 NE2 B10 3.65 ZN ZN N1 3.96

GLN 92 CD B6 3.92 GLN 92 NE2 B5 3.67 HIS 64 CE1 B10 3.66

GLN 92 NE2 B11 3.65 GLN 92 CD B5 3.88 HIS 64 NE2 B10 3.88

HIS 94 CE1 O2 3.08 HIS 94 NE2 O2 3.38 GLN 92 NE2 B11 3.47

HIS 94 NE2 O2 3.35 HIS 94 CE1 O2 3.00 GLN 92 NE2 B6 3.44

HIS 94 CE1 S 3.97 HIS 94 NE2 S1 3.86 GLN 92 NE2 B2 3.51

HIS 94 NE2 S 3.84 HIS 94 CE1 S1 3.87 HIS 94 NE2 O2 3.49

HIS 94 CE1 N2 3.92 HIS 94 NE2 N2 3.14 HIS 94 CE1 O2 3.18

HIS 94 NE2 N2 3.24 HIS 94 CE1 N2 3.74 HIS 94 NE2 S1 3.81

HIS 94 CE1 C3 3.79 HIS 94 NE2 C3 3.93 HIS 94 CE1 S1 3.81

HIS 94 NE2 C3 3.92 HIS 94 CE1 C3 3.61 HIS 94 NE2 N2 3.21

HIS 96 CE1 N2 3.59 HIS 96 CE1 N2 3.57 HIS 94 CE1 N2 3.79

HIS 96 NE2 N2 3.13 HIS 96 NE2 N2 3.19 HIS 94 CE1 N1 3.80

HIS 119 ND1 N2 3.36 HIS 119 ND1 O2 3.99 HIS 96 CE1 N2 3.51

HIS 119 CE1 N2 3.85 HIS 119 ND1 N2 3.38 HIS 96 NE2 N2 3.18

VAL 121 CG2 O2 3.78 HIS 119 CE1 N2 3.96 HIS 119 ND1 O2 3.80

PHE 131 CZ B7 3.77 VAL 121 CG2 O2 3.69 HIS 119 ND1 N2 3.34

PHE 131 CZ B8 3.82 PHE 131 CZ B11 3.99 HIS 119 CE1 N2 3.98

LEU 198 CD2 O1 3.61 LEU 198 CD2 O1 3.54 VAL 121 CG2 O2 3.82

LEU 198 CA O1 3.17 LEU 198 CA O1 3.15 LEU 198 CD2 O1 3.93

LEU 198 CB O1 3.70 LEU 198 CB O1 3.53 LEU 198 CA O1 3.07

LEU 198 C O1 3.46 LEU 198 C O1 3.47 LEU 198 CB O1 3.51

THR 199 OG1 S 3.80 THR 199 OG1 S1 3.86 LEU 198 C O1 3.35

THR 199 N S 3.89 THR 199 N S1 3.99 LEU 198 CD2 C3 3.98

THR 199 CA O1 3.91 THR 199 CA O1 3.96 THR 199 OG1 S1 3.87

THR 199 OG1 O1 3.59 THR 199 OG1 O1 3.62 THR 199 N S1 3.90

THR 199 N O1 2.80 THR 199 N O1 2.85 THR 199 CA O1 3.87

THR 199 OG1 N2 2.79 THR 199 OG1 N2 2.89 THR 199 OG1 O1 3.72

THR 200 OG1 N1 3.00 THR 200 CG2 N1 3.85 THR 199 N O1 2.70

THR 200 OG1 C3 3.19 THR 200 CB N1 3.94 THR 199 CB N2 4.00

THR 200 OG1 C1 3.71 THR 200 OG1 N1 2.91 THR 199 OG1 N2 2.79

THR 200 OG1 B3 3.50 THR 200 OG1 C1 3.93 THR 200 OG1 N1 3.35

THR 200 OG1 B4 3.35 THR 200 OG1 C3 3.46 THR 200 OG1 C3 3.93

THR 200 OG1 B2 3.65 THR 200 OG1 C7 3.78

THR 200 OG1 B3 3.48 THR 200 OG1 B9 3.50

THR 200 OG1 C8 3.28

PRO 201 O B9 3.84

3

Chemical synthesis

General

The starting carboranes were purchased from Katchem Ltd., Czech Republic. Solvents, i.e.

tetrahydrofurane (THF), ethylene glycol dimethyl ether (DME) and 1,4-dioxane were dried

with sodium diphenyl ketyl and distilled prior to use. BuLi in hexane, Br-alkyl phtalimides

were purchased from Aldrich, other chemicals and solvents were from Aldrich, Merck,

Lachema a.s. and Penta Ltd., Czech Republic, respectively, and used without purification.

Analytical TLC was carried out on TLC plates Silufol® (silica gel layer on aluminium foil

with starch as the binder) from Lachema in CH2Cl2-CH3CN (3: 1, b.v.). Unless otherwise

specified, column chromatography was performed on a high purity silica gel (Merck Grade,

Type 7754, 70-230 mesh, 60 Å).

All reactions were performed using standard Schlenk type vacuum or inert-atmosphere

techniques, although some operations, such as column chromatography and crystallization

were carried out in the air.

Melting points were determined in sealed capillaries on the BŰCHI Melting Point B-545

apparatus and are not corrected.

As previously verified, the data of elemental analyses of sodium or potassium salts of

carbaborate ions are often misleading, due to inclusion of water, and cannot be thus

considered as a reliable criterion of purity.[1,2] Nevertheless, the identity of all the reported

compounds has been unambiguosly proven by combination of the 11B, 1H and 13C NMR

spectral data (complete assignment of the resonances), with Mass Spectrometry (two decimal

digits resolution), melting points, TLC and other methods. The purity was also assessed by an

previously developed analytical HPLC methods with DAD detection[3], being better than 98%

for all compounds reported in this article.

Symbols in structural formulae of carboranes mean:

Instrumental Techniques 1H, 13C, and 11B NMR spectra were measured on a Varian Mercury 400Plus Instrument. The

spectra of all compounds were measured immediately after dissolution. 11B NMR (128 MHz)

chemical shifts are given in ppm to high-frequency (low field) to F3BOEt2 as the external

reference. Residual solvent 1H resonances were used as internal secondary standards.

Coupling constants 1J(11B–1H) are taken from resolution-enhanced 11B spectra with a digital

4

resolution of 2 Hz. The NMR data are presented in the text as follows: 11B NMR: 11B

chemical shifts δ (ppm), multiplicity, coupling J(11B–1H) constants given in Hz. Signal

assignments are based on [11B-11B] COSY NMR spectroscopy. 1H NMR (400 MHz) and 13C

(100 MHz): chemical shifts δ in ppm relative to Me4Si (0 ppm) as the external standard,

coupling constants J(H,H) in Hz. In 1H{11B} spectra the δB-H values and peak assignments

were analyzed from 1H{11Bselective} experiments.

Mass spectrometry measurements were performed on a Thermo-Finnigan LCQ-Fleet Ion

Trap instrument using electrospray ionization (ESI) for ionic species or atmospheric pressure

chemical ionization (APCI) for neural carborane derivatives with detection of negative or

positive ions, respectively. Samples dissolved in acetonitrile (concentrations approximately

100 ng.ml–1) were introduced in case of ESI to the ion source by infusion of 10 μL/min–1,

source voltage 5.48 kV, tube lens voltage -119.7 V, capillary voltage -32.0 V, capillary

temperature was 160°C, drying gas flow 11 arb, and in case of APCI by infusion of

20 μL/min–1, source voltage 3.89 kV, tube lens voltage 100.0 V, capillary temperature was

275°C, drying gas flow 30 arb, and auxilliary gas flow rate 9 arb. In most cases the negative

ions corresponding to the molecular ion were observed with 100% abundance for the highest

peak in the isotopic distribution plot. Molecular ions [M]- were detected for all univalent

anions as the base peaks in the spectra. Full agreement of the experimental and calculated

isotopic distribution pattern was observed for all isolated compounds. The isotopic

distribution in the boron plot of all peaks is in perfect agreement with the calculated spectral

pattern. The data are presented for the most abundant mass in the boron distribution plot

(100%) and for the peak corresponding to the m/e value.

HPLC: A Merck-Hitachi HPLC system LaChrom Series 7000 equipped with DAD 7450

detector and an Programmable Autosampler L7250 was used. Chromatographic procedure:

For the anions, a previously reported Ion-Pair RP method for the separation of hydrophobic

borate anions was used.[3] Column: RP Separon SGX C8, 7m (250x 4mm I. D.), Tessek

Prague, mobile phase: 4.5 mmol hexylamine acetate in 58% aqueous CH3CN (pH 6.0), flow

rate 1 mL/ min, detection DAD, fixed wavelengths 235, 260, 285 and 312 nm; samples of

concentration approximately 1 mg·mL-1 in the mobile phase were injected. For carborane

phtalimides mobile phases containing 65% aqueous CH3CN (parent substituted carboranes)

or 70% CH3CN (phenyl substituted carboranes) were used with detection at 210, 220 235 and

260 nm.

5

Synthesis of the carborane amine derivatives 1b-10b

The 1-aminomethyl-1,2-dicarba-closo-dodecaborate hydrochloride (1b) was prepared

according to Wilson et al.[4] by insertion of propargylamine into bis(acetonitrile) decaborane

adduct. The synthesis of 2b has been previously reported by a different procedures consisting

of reaction of lithiated 1-phenyl-o-carborane with ClCN followed by reduction with LiAlH4[5]

or by reaction of methylene carboranyl triflate with sodium amide.[6] Nevertheless, reaction

sequence proceeding via phtalimido derivative (see Scheme S1) seems to be easier avoiding

handling of hazardous chemicals. Thus, most of the aminoalkyl carboranes or their

hydrochlorides (compounds 3b-5b) were prepared in analogy to described methods[7-9]

directly from the respective lithiated carboranes by reactions with bromoalkyl phtalimides and

subsequent cleavage of the protective group (see Schemes S1 to S5). However, according to

literature[8], this direct method fails to produce 1-aminomethyl-1,2-carboranes. We have

proven feasibility of this direct approach for compounds with methylene connectors, at least

in here described cases of phenyl o-carborane derivatives 2b and 3b (Schemes 1 and 2). The

experimental details are therefore given below, though the carborane alkyamines have been

previously known[7-11] but here described phenyl substituted analogues as well as aminometyl-

1,7-carboranes have not been reported, yet.

Mono and dimethylene amine-1,7-dicarba-closo-dodecaborate derivatives (4b and 5b)

were prepared via the respective methylene-phtalimides in close analogy with the procedure

by Rendina et al.[7], described for propylamino-1,7-carboranes. The 1-ammonio-1-carba-

closo-dodecaborate zwitterion derivative 6b was prepared by procedure described by Plešek

et al.[12] A method different from literature was used for synthesis of 11-vertex

ammoniomethyl derivative of dicarbaundecaborate ion (7b, see Scheme S5). This consisted in

easy deboronation of known[11] and commercially available bromomethyl dicarba-closo-1,2-

carborane by aqueous ammonia. Two derivatives of 7-ammonioalkyl-8-phenyl-7,8-dicarba-

undecaborate with amino group attached by methylene or propylene linker, respectively (8b,

Scheme S6 and 9b, Scheme S7), were prepared via degradation of the respective phtalimide

derivatives by hydrazine in methanol or ethanol. Also here, the phenyl substituted compounds

have not been reported yet. The compound 10b was prepared by ring cleavage of zwitterionic

10-dioxane derivative of 7,8-dicarba-nido-undecaborate ion, obtained according to previously

repoted method[13], by ammonia in aprotic solvent (see Scheme 6).

1-aminomethyl-2-phenyl-1,2-dicarba-closo-dodecaborane hydrochloride (2b)

6

Scheme S1. i) BuLi, DME, ii) bromomethyl phthalimide, iii) NaBH4, i-PrOHaq, iv) HCl.

The starting carborane, 1-C6H5-1,2-C2B10H11 (1.00 g, 4.53 mmol), dried in vacuum, was

dissolved in DME (40 ml). The resulting solution was cooled down to -33 oC and BuLi

(2.5 M in hexane, 2.0 ml, 5.0 mmol) was added from a syringe through septum. The reaction

slurry was stirred for 15 min and then left to warm up to room temperature. After stirring for

1h, the reaction mixture was cooled again down to -33 oC and bromomethyl phtalimide

(1.20 g, 5.0 mmol) dissolved in 15 ml of DME was added dropwise from a syringe. The

reaction mixture was stirred for 15 min and then left slowly to warm up to room temperature

over 4 h. After stirring for additional 2 h and standing overnight, the solids were filtered off

under argon and washed with two portions of DME (10 mL). Diluted acetic acid (3 M,

0.5 mL) was added to the combined DME extracts and volatiles were removed under reduced

pressure. A solid residue was dissolved in benzene and poured atop of a silica gel column (3 x

25 cm I. D.). Elution with benzene led to isolation of the starting 1-phenylcarborane (240 mg,

25%) continuing with benzene-CH3CN (4:1) solvent mixture which afforded the expected

1-methylene phtalimide-2-phenyl-1,2-carborane (0.85 g, 49%) which was directly used in

subsequent reaction step.

This derivative (50 mg, 0.13 mmol) was dissolved in 2-propanol (25 mL) and water

(5 mL) was added. After addition of NaBH4 (75 mg, 1.97 mmol) the reaction mixture was

stirred for 24 h at room temperature. Volatiles were then removed under reduced pressure and

methanol and several drops of diluted HCl (3 mol.L-1) were added three times and solvents

were evaporated to dryness. Concentrated acetic acid (25 mL) and HCl (35%, 2.5 mL) were

added and the resulting solution was heated for 16 h at 96 oC (bath temperature). The volatiles

were then removed under reduced pressure, water (20 ml) was added and the crude product

was extracted into ether (3x 20 mL). Combined ether extracts were evaporated under reduced

pressure and the unseparable mixture of both isomers was purified by chromatography on a

silica gel column (25 x 1.5 cm I.D.) from the rest of uncleaved by-product and organic

impurities using CH2Cl2-CH3CN mixture (95: 5 to 3: 1) as the mobile phase. Fractions

corresponding to product were collected, evaporated and crystallized from CH2Cl2 (with a

7

drop of MeOH for dissolution)-hexane. The product in form of white semi-crystalline solid

was obtained by evaporation the mother liquors under reduced pressure (34 mg, 90 %); M. p.

221°C; TLC RF = 0.30; 1H{11B} NMR (Acetone-d6, Me4Si): δ 7.76-7.41 (5H, m, Ph),

5.15(2H, bt, NH2), 3.68 (2H, t, CH2NH2), 2.48 (2H, s, B(8,10)H), 2.35 (1H, s, B(9)H), 1.35

(1H, s, B(12)H ), 3.17 (4H, s, B(4,5,7,11)H), 2.92 (2H, s, B(3,6)H); 13C NMR (CH3CN,

Me4Si): δ 132.34-129.02 (6C, m, Ph), 63.66 (1C, s, Ccarborane), 42.36 (1C, t, Ccarborane), 30.35

(1C , t, CH2N); 11B NMR (CD3CN, BF3.Et2O): δ -3.60 (1B, d, J=104, B9), -4.31 (1B, d,

J=137, B12), -9.66 (2B, d, J=92, B8,10), -10.30 (4B, d, J=153, B4,5,7,11), -11.06 (2B, d,

J=217, B3,6); MS (APCI) m/z 249.17 (100 %), 252.17 (25 %) [M-H]-, calcd. 249.25 (100

%), 252.25 (5%).

1-aminomethyl-9(12)-phenyl-1,2-dicarba-closo-dodecaborane hydrochloride, equimolar

mixture of both isomers (3mb, 3nb)

Scheme S2. i) BuLi, DME, ii) bromomethyl phthalimide, iii) NaBH4, i-PrOHaq, iv) aq. HCl.

* Only one of the two sites, either B(9) or B(12) is affected by substitution.

The starting 9-phenyl-1,2-dicarba-closo-dodecaborane 1.29 g (5.9 mmol) dried in vacuum

was dissolved in DME (40 ml) and the solution was cooled down to -78°C (bath temperature)

and BuLi (1.6 M in hexane, 3.9 ml, 6.2 mmol) was added from a syringe through septum. The

content of the flask was stirred for 15 min and the reaction mixture was left to warm up

slowly to room temperature and then cooled again. Then solution of bromomethyl phtalimide

(1.50 g, 6.2 mmol) in 15 ml DME was added dropwise during 30 min and the reaction

mixture was stirred for additional 15 min at -78°C and then left to warm slowly up to ambient

temperature during 4 h. After standing overnight the solids were removed by filtration under

argon, washed with DME (2x 10 mL), and few drops of diluted acetic acid were added

(3 mol.L-1, 0.5 mL). The volatiles were then removed under reduced pressure. A solid residue

was dissolved in minimum volume of benzene and this solution was injected atop of a silica

8

gel column (3 x 25 cm I.D.). Elution with benzene led to isolation of unreacted starting

carborane (530 mg) identified by HPLC and NMR; continuing of the elution with benzene-

CH3CN (95:5 b.v.) led to isolation of the by-product substituted by methylene phtalimide

group, after removal of the solvent in vacuum and drying (1.01g, 60.2 %). A part of this

compound (330 mg, 1.15 mmol) was reduced in 2-propanol (25 mL) and water (5 mL)

mixture by NaBH4 (1.3 g, 34.1 mmol) and the reaction mixture was stirred for 24 h at room

temperature. Volatiles were then removed under reduced pressure and methanol and several

drops of diluted HCl (3 mol.L-1) were added three times and solvents were evaporated to

dryness. Concentrated acetic acid (25 mL) and HCl (35%, 2.5 mL) were added and the

resulting solution was heated for 16 h at 96 oC (bath temperature). The volatiles were then

removed under reduced pressure, water (20 mL) was added and the crude product was

extracted by ether (3 x 20 mL). Combined ether extracts were evaporated under reduced

pressure and the unseparable mixture of both isomers was purified by chromatography on a

silica gel column (25 x 1.5 cm I.D.) from the rest of uncleaved by-product and organic

impurities using CH2Cl2-CH3CN mixture (95: 5 to 3: 1) as the mobile phase. Fractions

corresponding to product were collected, evaporated and crystallized from CH2Cl2 (with a

drop of MeOH for dissolution)-hexane. The pure product that remained in solution was

obtained by evaporation the mother liquors under reduced pressure yielding 125 mg (58%) of

a white semi-crystalline solid. TLC RF = 0.13; 1H{11B} NMR (Acetone-d6, Me4Si): δ 7.42

(2H, bt, C6H5), 7.21 (3H, m, C6H5), 4.34 (2H, 2 bd, NH2), 4.42 a 4.30 (1H, 2 br. s,

CHcarborane), 3.35 (2H, d, J=6.0 Hz, CH2NH), 2.47, 1.80 (1H, 2s, B(9,12)H); 2.31 (2H, s,

B(8,10)H), 2.27 (2H, s, B(3)H), 2.27 (2H, 2s, B(4,5)H), 2.31, 2.11 (4H, 2s, B(8,11)H); 13C

NMR (CH3CN, Me4Si): δ 133.12-132.97 (6C, m, Ph), 128.27-128.04 (6C, m, Ph), 55.04

(2C, s, Ccarborane), 48.15 (2C, d, CHcarborane), 30.16 (2C, t, CH2N); 11B NMR (CD3CN,

BF3.Et2O): δ 6.60, 4.25 (1B, 2s, B9,12'), -3.25, -5.93 (1B, 2d, J=143, 150, B9',12), -9.12

(2B, d, J=146, B8,10), -11.90, -12.31 (2B, 2d, overlap, B4,5), -13.47 (4B, d, J=167,

B3,6,7,11); MS (APCI) m/z 250.42 (100 %), 253.33 (4%) [M+K]+, calcd. 250.26 (100%),

53.26 (4%).

2

9

1-aminomethyl-1,7-dicarba-closo-dodecaborane hydrochloride (4b)

N

O

ONH2.HCl

4b

i)

ii)

iii)

iv)

1

7

1

7

1

7

Scheme S3. i) BuLi, DME, ii) bromomethyl phthalimide, iii) hydrazine hydrate, iv) aq. HCl.

The starting 1,7-dicarba-closo-dodecaborane 1.80 g (12.5 mmol) dried in vacuum was

dissolved in DME (40 ml) and the solution was cooled down to -78°C (bath temperature).

BuLi (2.5 M in hexane, 5.0 ml, 12.5 mmol) was added from a syringe through septum. The

content of the flask was stirred for 15 min and the reaction mixture was left to warm up

slowly to room temperature and then cooled again. Then solution of bromomethyl phtalimide

(3.30 g, 13.7 mmol) in 25 ml DME was added dropwise during 30 min and the reaction

mixture was stirred overnight leaving to warm slowly up to ambient temperature. Then, the

solids were removed by filtration under argon, washed with DME (2x 5 mL), and few drops

of diluted acetic acid were added (3 mol.L-1, 0.5 mL) followed with 5 g of silica gel. The

volatiles were then removed under reduced pressure. A solid residue was poured atop of a

silica gel column (3 x 30 cm I.D.) and eluted by benzene–acetone 3:1 eluting the mixture of

products, which was evaporated in vacuum. Repeated chromatography using elution with

benzene led to isolation of unreacted starting carborane (185 mg) identified by HPLC and

NMR; continuing of the elution with benzene-acetone (97: 3 b.v.) led to isolation of the crude

by-product substituted by methylene phtalimide group (after removal of the solvent in

vacuum and drying). Further elution with benzene-acetone (3: 1 b.v.) led to isolation of a

small quantity of disubstituted compound. The crude monosubstituted phtalimide was further

purified by reflux in hot toluene (20 ml), decantation from insoluble impurities, cooling down

and layering by hexane. A white product that separated upon standing was collected by

filtration and dried in vacuum; yield 1.35 g (36 %). A part of this compound (1.0 g, mmol)

was reduced in methanol (50 mL) by excess of hydrazine hydrate (5 mL) under reflux for 3h.

After cooling down the solids which separated were filtered out, washed with methanol (2 x

10 mL) and discarded. The solution was treated with diluted HCl (3 mol.L-1), left to stay for

30 min and a white precipitate was removed by filtration and washed by MeOH (2 x 10 mL).

The combined methanol fractions were evaporated under reduced pressure and dried in

10

vacuum for 12h. The solid was then stirred with CHCl3 20 ml, then were filtered and washed

with CHCl3 (4x 5 ml) to remove yellow organic impurities. Solid was dried in vacuum and

then treated with acetone (3 x 10 mL). Acetone extracts were evaporated in vacuum, dried and

treated once more by CHCl3 leaving the pure solid product 4b. Yield 0.45 g (65%), TLC RF

= 0.10; 1H{11B} NMR (Acetone-d6, Me4Si): δ 7.93 (2H, br. s, NH2), 3.71 (1H, s,

CHcarborane), 3.60 (2H, br s, CH2-NH), 2.79 (2H, s, B(2,3)H); 2.25 (1H, s, B(5)H), 2.21 (1H, s,

B(12)H), 2.45, 2.02 (4H, 2s, B(4,6,9,10)H), 2.13 (1H, s, B(8,11)H ; )

9 %).

1,7-diaminomethyl-1,7-dicarba-closo-dodecaborane hydrochloride (5b)

11B NMR (Acetone-d6,

BF3.Et2O): δ -4.32 (1B, d, J=159, B5), -9.67 (1B, d, J=225, B12), -11.26 (4B, d, J=128,

B4,6,9,10), -13.59 (2B, d, J=171, B8,11), -15.21 (2B, d, J=183 Hz, B2,3); 13C NMR

(Acetone-d6, Me4Si): δ 68.27 (1 C, s, Ccarborane), 57.21 (1C, d, CHcarborane), 39.61 (1C, t,

CH2); m/z (APCI+) 174.25 (100 %), 176.25 (40 %) [M+H]+, calcd. 174.22 (100%), 176.22

(3

N

O

O NH2.HCl

i)

ii)

iii)

iv)

1

7

1

7

1

7N

O

5b

O

NH2.HCl

Scheme S4. i) BuLi, DME, ii) bromomethyl phthalimide, iii) hydrazine hydrate iv) aq. HCl.

The compound was prepared from 1,7-dicarba-closo-dodecaborane 0.90 g (6.25 mmol)

analogously to 4b, but using slightly more than two equivalents of BuLi (2.5 M in hexane, 5.8

ml, 14.5 mmol) and bromomethyl phtalimide (3.5 g, 14.5 mmol). After addition of BuLi a

precipitation of white solid occurred, which did not dissolve even after warming up. After

addition of momomethyl phtalimide and warming up to ambient temperature, the reaction was

stirred for 48 h. Dark solids were removed by filtration under argon and separated between

water (20 ml) and benzene (2x 20 ml) and ether (2x 20 ml). The organic extracts were

combined with the DME filtrate and evaporated in vacuum providing 610 mg of solids. The

products were isolated by chromatography on silica gel column (25 x 2.5 cm). Elution with

benzene-acetone (95:5 b.v.) gave traces of unreacted m-carborane followed by pure

monosubstituted phtalimide in the second fraction (90 mg) identified by HPLC and NMR.

11

Continued elution with benzene-acetone (4:1 b.v.) led to isolation of pure disubstituted

phtalimide 305 mg and fractions containing oligomeric impurities. Additional quantity of the

by-product 720 mg was obtained after repeated chromatography of the impure fractions after

the first chromatography. Overall yield of the diphtalimide 1.25g (43%). A part of this

compound (720 mg, 1.55 mmol) was reduced in ethanol (25 mL) by excess of hydrazine

hydrate (5 mL) under reflux for 5h. After cooling down ethanol was evaporated and the

residue was separated between ethylacetate and water (2x 10 mL) and then treated by HCl (3

mol.L-1, 2 x 10 mL). The organic layer was separated, evaporated under reduced pressure and

dried in vacuum for 12h. The solid was then stirred with CHCl3 20 ml, filtered and washed

with CHCl3 (4x 5 ml) to remove yellow organic impurities. Resulting solid was dried in

vacuum and then treated with acetone (3 x 10 mL). Acetone extracts were evaporated in

vacuum leaving the solid product 5b. Yield 220 mg (52%); TLC RF = 0.06; 1H{11B} NMR

(Acetone-d6, Me4Si): δ 7.90 (1H, bs, NH2), 3.59 (2H, br. s, -CH2-NH), 3.22 (2H, s,

B(2,3)H); 2.28 (2H, s, B(5,12)H), 2.23, 1.98 (6H, 2s, B(4,6,8,9,10,11)H), 13C NMR

(Acetone-d6, Me4Si): δ 75.94 (2 C, s, Ccarborane), 37.19 (2C, t, CH2); 11B NMR (Acetone-d6,

BF3.Et2O): δ -6.93 (2B, d, J=161, B5,12), -11.64 (6B, d, J=156, B4,6,9,10,8,11), -

13.16 (2B, d, J=205, B2,3); MS (APCI+) m/z 203.25 (100 %), 205.25 (35 %) [M+H]+, calcd.

03.25 (100%), 205.25 (39 %).

-ammoniomethyl-7,8-dicarba-nido-undecaborate (7b)

2

7

HNH3

7b (62%)

H CH3H

CH3

+

i)

ii)

Br

H3N

NH3

(35%)

1

2

8

71110

9

8

71110

9 8

71110

9

Scheme S5. i) NH4OH (aq), ii) separation by liquid chromatography.

To 1-Br-CH2-1,2-C2B10H11 (1.0 g, 4.2 mmol) dissolved in aqueous ethanol 10 mL (96%)

aqueous ammonia (24% b.v., 5 mL) was added. The reaction mixture was stirred under

nitrogen for 8 h, when a spot of the starting compound on TLC disappeared (RF 0.91, Silufol,

CHCl3-CH3CN, 2:1 b.v.; detection: iodine vapours and spray with 1% AgNO3 – a slow

12

reduction to a grey colour). Volatiles were then removed under reduced pressure. A solid

residue was dried in vacuum and then dissolved in acetone: CH2Cl2 mixture (4: 1 b.v.), and

the product was isolated by chromatography on a silica gel column (20 x 2 cm I.D.) as the

first main band. The course of chromatography was monitored by TLC using the same solvent

mixture. The solvents were immediately removed at cold under reduced pressure; the product

was then dried and crystallized by dissolution in CH2Cl2, layering with hexane and leaving to

stand for three days, the white solid was collected and dried. It should be noted that

continuous elution with the same mobile phase provides mixture of other two geometric

isomers 7-Me-9-NH3-C2B9H10 and 7-Me-11-NH3-C2B9H10 in approx. 35% overall yield

(details on their characterization are not reported here). This reaction can be alternatively

carried out with toluene (dry) solution of the starting compound and gaseous 100% ammonia

from pressure bottle which is bubbled through. Reaction time was then 3 h providing a similar

mixture of identical products, inclusive their mutual ratios (according to TLC and NMR).

Yield 043g, 62%, M. p. 268 °C, TLC RF 0.33 (Silufol), 1H{11B} NMR (Acetone-d6,

Me4Si): δ 2.88 (2H, t, J=13.6, CH2NH), 2.03 (2H, s, B(9,11)H), 1.86 (1H, s, CHcarborane),

1.83 (2H, bt, J=13.4, NH2), 1.74, 1.42 (2H, 2s, B(2,4)H), 1.38 (1H, s, B(6)H), 1.32, 1.11 (2H,

2s, B(3,5)H), 0.61 (1H, s, B(1)H), 0.14 (1H, s, B(10)H), -2.67 (1H, s, μ-B(10)H); 11B NMR

(CD3CN, BF3.Et2O): δ -11.11 (2 B, d, J=137, B9,11), -15.23 (2B, d, J=119, B2,4), -19.82

(2B, d, J=149, B3,5), -20.08 (1B, d, J=156, B6), -32.85 (1B, d, J=94, B10), -37.16 (1 B, d,

J=140, B1); 13C NMR (CH3CN, Me4Si): δ 55.09 (1C, m, Ccarborane), 49.33 (1C , t, J=146,

CH2N), 44.26 (1C, m, Ccarborane); MS (ESI) m/z 163.18 (100 %), 165.18 (5%) [M-H]-, calcd.

63.21 (100%), 165.21 (2%).

7-ammoniomethyl-8-phenyl-7,8-dicarba-nido-undecaborate (8b)

1

Scheme S6. i) N2H4.H2O, EtOH, ii) diluted HCl, Et2O.

1-phtalimidomethyl-2-phenyl-1,2-carborane was prepared by the above described procedure

for 2b (see Scheme S1). 160 mg (0.42 mmol) of this starting material was dissolved in

13

aqueous ethanol (20 ml, 80% b.v.) and an excess of hydrazine hydrate (2.0 mL, 32.1 mmol)

was added. The reaction mixture was stirred at room temperature for 16 h. The volatiles were

removed under reduced pressure and the crude product was extracted from semi-solid residue

into ether (3x 15 mL), the combined ether extracts were washed with diluted HCl (3M, 2x 15

mL), water (2x 10 mL) and then separated, evaporated to dryness and dried in vacuum. Yield

78 mg (78%); M. p. 148 °C; TLC (CH3CN: CH2Cl2, 1:3) RF = 0.33; 1H{11B} NMR

(Acetone-d6, Me4Si): δ 7.35 (5H, m, C6H5), 4.11 (3H, t, CNH3), 2.67 (2H, q, CH2N), 2.06

(1H, s, B(11)H), 2.05 (1H, s, B(9)H), 2.02 (1 H, s, B(2)H), 1.61 (1H, s, B(4)H), 1.40 (1 H, s,

B(5)H), 1.33 (1H, s, B(6)H), 1.17 (1H, s, B(3)H), 0.66 (1H, s, B(1)H), 0.22 (1H, s, B(10)H), -

2.17 (1H, s, B-H-B); 11B NMR (CD3CN, BF3.Et2O): δ -8.76 (1B, d, J=140, B9), -10.45 (1B,

d, J=134, B11), -14.23 (1B, d, J=147, B2), -15.15 (1B, d, J=125, B5), -18.51 (2B, d, J=143,

B3,4), -20.13 (1B, d, J=195, B6), -33.09 (1B, d, J=92, B10), -36.25 (1B, d, J=140, B1), 13C

NMR (CH3CN, Me4Si): δ 128.13 (4C, C6H5), 127.10 (2C, C6H5), 67.45 (1C, Ccarborane),

49.19 (1C, Ccarborane), 30.17 (1C, CH2); MS (ESI) m/z 238.25 (100 %), 241.17 (7 %) [M-H]-,

alcd. 238.24 (100%), 241.24 (7%).

7-(3-ammoniopropyl)-8-phenyl-7,8-dicarba-nido-undecaborate (9b)

c

Scheme S7. i) N2H4.H2O, EtOH.

The compound was prepared via the corresponding phtalimide derivative using similar

procedure as for 8b. 1-phenyl-1,2-dicarba-closo-dodecaborane (2.00 g, 9.06 mmol) was

dissolved in DME (40 mL), cooled down to -33 oC and BuLi (2.5 mol.L-1 in hexane, 4.0 mL,

10.0 mmol) was then added dropwise from a syringe. The reaction mixture was stirred for

additional 15 min. and then left to warm up to room temperature. After stirring for 1h and

cooling down again to -33 oC, solution of bromopropyl phtalimide (2.68g, 10.0 mmol) in

DME (15 mL) was added slowly, reaction mixture was then kept at low temperature for 15

min. and then left to warm up slowly under stirring to room temperature over 4 h and left to

stand overnight. Solids were removed by filtration under argon and washed with DME (2x 10

14

mL). Diluted acetic acid (3 mol.L-1, 0.5 mL) was added to filtrate and the volatiles were

removed in vacuum. Solids were dissolved in benzene and the respective propylene

phtalimide by-product was separated by chromatography on a silica gel column (25 x 3 cm

I.D.) from unreacted starting compound (440 mg, 22%) by elution with benzene followed by

(ESI) m/z 269.42 (10 %), 267.25 (100 %) [M-H]-,

alcd. 269.27 (10 %), 267.27 (100 %).

benzene-CH3CN (95:5); yield 1.95 g (53%).

The propylene phtalimide derivative (500 mg, 1.23 mmol) was dissolved in aqueous ethanol

(80%, 50 ml) and treated with hydrazine hydrate in excess (0.7 ml, 14.4 mmol) at room

temperature for 16 h and stirred for 4h under reflux. After cooling down the volatiles were

removed under reduced pressure, water was added (20 mL) and the crude product was

extracted into diethyl ether (3 x 20 mL). Combined ether extracts were evaporated under

reduced pressure and the compound was purified by chromatography on a silica gel column

(25 x 1.5 cm I.D.) using CH2Cl2-CH3CN mixture (95:5 to 3:1) as the mobile phase. Fractions

corresponding to product were collected, evaporated and crystallized from CH2Cl2-hexane.

The small fraction of solids was discarded. The product remaining in solution and was

obtained in form of white semi-crystalline solid by evaporation the mother liquors under

reduced pressure; yield 226 mg (69%); M. p. 115 °C; TLC RF (CH3CN:CH2Cl2 1:3) 0.17; 1H{11B} NMR (Acetone-d6, Me4Si): δ 7.70 (2H, d, J=0.02, Ph), 7.45 (1H, d, J=0.05, Ph),

7.32 (2H, t, J=0.02, Ph), 3.88 (3H, t, J=0.06, NH3), 2.42 (2H, m, CH2N), 2.15 (1 H, s,

B(11)H), 2.00 (1 H, s, B(9)H), 1.99 (1 H, s, B(2)H), 1.92 (2H, p, CH2), 1.50 (2 H, s, B(3,

6)H), 1.45 (2H, m, J=0.01, CH2), 1.21 (1H, s, B(4)H), 1.21 (1 H, s, B(5)H), 0.55 (1 H, s,

B(1)H), 0.10 (1 H, s, B(10)H), -2.25 (1 H, s, B-H-B); 11B NMR (CD3CN, BF3.Et2O): δ -

8.95 (1B, d, J=134, B9), -10.83 (1B, d, J=140, B11), -13.61 (1B, d, J=156, B2), -17.34 (1B,

d, J=122, B5), -17.91 (1B, d, J =92, B4), -19.05 (2B, d, J=150, B3, 6), -33.85 (1B, d, J=89,

B10), -36.60 (1B, d, J=137, B1); 13C NMR (CH3CN, Me4Si): δ 132.57 (2C, d, J=1.60, Ph),

132.04 (1C, t, J=1.69, Ph), 129.97 (1C, t, J=1.55, Ph), 128.05 (2C, t, J=1.60, Ph), 55.21 (1 C,

s, Ccarborane), 53.35 (1 C, t, Ccarborane), 40.79 (1C, m, J=1.44, CH2N), 33.40 (1C, m, J=1.24

,CH2), 30.47(1C, t, J=1.34, CH2); MS

c

15

10-[2-(2-ammonioethoxy)ethoxy]-7,8-nido-dicarbaundecaborate (10b)

H OO

NH3H O

O

10b

i)8

7

9

10 8

7

9

10

Scheme S8. i) NH3, toluene, DME.

Zwitterionic compound 10-dioxane-7,8-C2B9H11 (0.50 g, 2.20 mmol) was dissolved in dry

THF (20 cm3, 4°C) under dry nitrogen gas and then was introduced dry ammonia gas from a

gas cylinder (Messer). The reaction mixture was evaporated to dryness after 10 min of

bubbling of ammonia through the THF solution. The product was obtained pure without

further purifications; yield 0.54g (99 %). Recrystalization of the product is possible from the

mixture of CH2Cl2, CH3OH, hexane (20:8.5:80). M. p. 285°C TLC RF = 0.13;; 1H{11B}

NMR (Acetone-d6, Me4Si): δ 6.71 (3H, bt, NH3), 3.64 (2H, s, CHcarborane), 3.64 (2H, t,

BOCH2), 3.55 (2H, t, CH2O), 3.05 (2H, t, CH2O), 2.32 (2H, bs, B(9,11)H), 1.64 (2H, s,

CH2N), 1.38 (1H, s, B(3)H ), 1.24 (2H, s, B(5,6)H), 1.09 (2H, s, B(2,4)H), 0.33 (2H, s,

B(1)H), -0.60 (1H, s, µ-H); 13C NMR (CH3CN, Me4Si): δ 71.75 (2C, t, BOCH2), 70.79 (2C,

d, CH carborane), 66.76 (2C, t, CH2O), 40.36 (2C ,t , CH2NH2); 11B NMR (CD3CN,

BF3.Et2O): δ -10.57 (1B, s, B10), -12.66 (2B, d, J=134, B9,11), -17.44 (2B, d, J=131, B5,6),

-23.74 (2B, d, J=150, B2,4), -24.93 (1B, d, J=159, B3), -40.65 (1B, d, J=140, B1); MS

(APCI) m/z 236.25 (100 %), 239.33 (5%) [M-H]-, calcd. 236.24 (100%), 239.24 (5%) and

238.28 (100 %), 241.20 (5%) [M+H]+, calcd. 238.26 (100 %), 241.26 (5%).

Synthesis of the carborane sulfamide derivatives

All the sulfamide compounds (1a-10a) were prepared via corresponding amino carborane

derivatives prepared by procedures reported in the literature or by new methods described in

the above paragraph. These derivatives were reacted with sulfamide in dioxane under

prolonged heating. Though we tested other polar solvents, such as dimethoxy and diethoxy

ethane and DMF, we chose dioxane due to its sufficiently high boiling point and because it

afforded easy dissolution of the reactants and isolation of the products.

16

1-(sulfamido)methyl-1,2-dicarba-closo-dodecaborane (1a)

To a mixture of the starting amine 1b (144 mg, 0.83 mmol) and solid

sulfamide (H2NSO2NH2, 399 mg, 4.16 mmol) 1,4-dioxane (10 mL) was

added from syringe. The slurry was heated to a reflux under stirring refluxed

for additional 2 h. After cooling down to room temperature the solvent was

removed under reduced pressure and a solid residue was extracted with

mixture of ethyl acetate and diethyl ether (1:1 b.v.). The organic layer was

separated by decantation and then treated with saturated solution o KHSO4 (15 mL), twice

with brine (2 x 15 mL) and the organic layer was separated. After drying over MgSO4 and

filtration the organic solvents were evaporated under reduced pressure. The solid residue was

dissolved in a minimum volume of CH2Cl2 and CH3OH solvent mixture (95:5), injected atop

of a silica gel column (20 x 1.5 cm) and eluted with the same solvent as the mobile phase.

Fractions containing the product were combined, evaporated under reduced pressure and dried

in vacuum; yield: 168 mg (80%) of white solid; 1H{11B} NMR (Acetone-d6, Me4Si): δ 7.52

(1H, t, J=7.5, CH2NHSO2), 6.81 (2H, s, NHSO2NH2), 4.87 (1H, bs, CHcarborane), 3.63 (2H, d,

J=7.5 , -CH2-NH), 2.36 (2H, bs, H), 2.15 (3H, bs, H), 2.05 (4H, bs, H), 1.99 (1H, bs, H); 13C

NMR (DMSO-d6, Me4Si): δ 75.6, 59,7, 49.9; 11B NMR (DMSO-d6): δ -3.19 (1B, d, J=145),

-5.76 (1B, d, J = 120), -9.83 (2B, d), -11.56 (2B, d), -13.00 (4B, d); MS (HR ESI–), m/z

253.1788 [M-H]–, calcd. 253.1790 (for C3H15O2N2B10S–)

1-(sulfamido)methyl-2-phenyl-1,2-dicarba-closo-dodecaborane (2a)

1,4-dioxane (20 cm3) was added to the mixture of small white leafs of 2b

(0.50 g, 2.10 mmol), sodium carbonate (0.45 g, 4.20 mmol) and sulfamide

(1.01 g, 10.52 mmol). The reaction mixture was heated to reflux for 2 h and

reaction progress was monitored using TLC and MS. The 1,4-dioxane was

then lyophilized from the reaction mixture. Isolation of pure product was

conducted with liquid chromatography on silicagel column using

dichlormethan and acetonitrile as solvents for gradient elution. The yield of the product was

0.18 g (28%, 0.58 mmol). M. p. 88 °C. 1H{11B} NMR (Acetone-d6, Me4Si): δ 7.76-7.41 (5H,

m, Ph), 4.79(1H, bt, CH2NH), 3.73 (2H, d, CH2NH), 2.53 (2H, s, B(8,10)H), 2.48 (1H, s,

B(9)H), 2.18 (2H, s, SNH2), 1.35 (1H, s, B(12)H ), 3.14 (4H, s, B(4,5,7,11)H), 2.81 (2H, s,

B(3,6)H); 13C NMR (CH3CN, Me4Si): δ 132.34-129.02 (6C, m, Ph), 63.66 (1C, s, Ccarborane),

42.36 (1C, t, Ccarborane), 30.35 (1C , t, CH2N); 11B NMR (CD3CN, BF3.Et2O): δ -3.60 (1B, d,

J=104, B9), -4.31 (1B, d, J=137, B12), -9.66 (2B, d, J=92, B8,10), -10.30 (4B, d, J=153,

17

B4,5,7,11), -11.06 (2B, d, J=217, B3,6); MS (APCI, 5% aqueous K2CO3 was added to

acetonitrile solution to improve ionization) m/z 366.42 (100 %), 369.42 (8 %) [M+K]-,

calcd. 366.18 (100 %), 369.18 (16 %).

1-(sulfamido)methyl-9(12)-phenyl-1,2-dicarba-closo-dodecaborane, equimolar mixture

of both isomers (3ma, 3na)

Equimolar mixture (according to 11B NMR) of

isomeric hydrochlorides 3mb and 3nb (105 mg, 0.42

mmol) was dissolved in dry 1,4-dioxane (10 mL).

Solid, previously dried (5h in vacuum) sulfamide

(250 mg, 2,6 mmol) was added in one portion and the

reaction mixture was refluxed for 72 h. After removal

of the solvent in vacuum, ether was added (20 mL) to a semi-solid residue followed with

water (10 mL) and diluted HCl (1 mol.L-1, 20 mL). The ether layer was separated and the

water phase was extracted by additional portions of ether (2 x 20 mL). Combined organic

fractions were evaporated under reduced pressure and the residue was dissolved in a

minimum volume of mixture of CH2Cl2 and CH3CN (95:5 b.v.) injected atop of a silica gel

column (20 x 1.5 cm I.D.). The products were eluted by gradient increase of acetonitrile

content (up to 3:1). Fractions corresponding to the product were collected; according to NMR,

no separation of the two isomeric species occurred during the chromatography. The resulting

solid was crystallized from its concentrated solution by layering with hexane and leaving to

stand for 3 days. The polycrystalline solid deposited on glass walls was decanted, washed

with hexane and dried 6 h in vacuum; yield 85 mg (61%); M. p. 61.1°C. 1H{11B} NMR

(Acetone-d6, Me4Si): δ 7.46 (2H, bt, C6H5), 7.24 (2H, m, C6H5), 5.90 (1H, br. t,

CH2NHSO2), 5.35 (2H, s, NHSO2NH2), 4.38, 4.27 (1H, 2 bs, CHcarborane), 3.77 (2H, 2d,

J=7.6, -CH2-NH), 2.74, 1.81 (1H, 2s, B(9',12)H); 2.31 (2H, s, B(8,10)H), 2.30 (2H, 2s,

B(4,5)H), 2.42, 2.20 (4H, 2s, B(8,11)H); 13C NMR (Acetone-d6, Me4Si): 13C NMR δ 133.7-

133.0 (6C, m Ph), 128.31-128.04 (6C, m, Ph), 55.02 (1C, s, Ccarborane), 48.21 (1C, d,

CHcarborane), 30.14 (1C, CH2); 11B NMR (Acetone-d6, BF3.Et2O): δ 7.29, 4.91 (1B, 2s,

B9,12'), -2,60, -5,29 (1B, 2d, J=159, B9',12), -9.16 (2B, d, J=225, B8,10), -12.16 (2B, d,

J=128, B4,5), -13.11 (4B, d, J=171, B3,6,7,11); MS (APCI, 5% aqueous K2CO3 was

added to acetonitrile solution to improve ionization) m/z 366.40 (100 %), 369.38 (10%)

[M+K]+, calcd. 366.18 (100%), 369.17 (10%).

18

1-(sulfamido)me

2,3); m/z (APCI) 251.08 (100 %), 254.08 (5 %) [M-H]-, calcd. 251.19 (100%), 254.19

%).

1,7-Bis[(sulfamido)methyl]-1,7-dicarba-closo-dodecaborane (5a)

thyl-1,7-dicarba-closo-dodecaborane (4a)

The amine 4b (65 mg, 0.38 mmol) was dissolved in 1,4-dioxane (10 ml) and

dry sulfamide (187 mg, 2.0 mmol) followed with K2CO3 (55 mg, 0.4 mmol)

were added and the slurry was stirred under reflux for 72 h. After cooling

down, the volatiles were removed in vacuum, water was added (10 mL) and

the crude product was extracted into diethylether (3x 10 mL). The combined

extracts were shaken with diluted hydrochloric acid (3 mol.L-1, 3x 10 mL),

washed with water (3x 10 mL) and evaporated. The solid was dissolved in ether, injected atop

a silica gel column (20 x 1.5 cm I.D.) and eluted with benzene-ether 1:1 a then with ether.

Ether fractions containing the product (according to NMR) were combined, evaporated to

dryness and dried 6 h in vacuum. Yield 50 mg (53%); M. p. 164 °C. 1H{11B} NMR

(Acetone-d6, Me4Si): δ 6.44 (1H, br, t, CH2NHSO2), 6.08 (2H, s, NHSO2NH2), 3.78 (1H,

bs, CHkarboran), 3.53 (2H, d, J=7.6, CH2-NH), 2.86 (2H, s, B(2,3)H); 2.38 (1H, s, B(5)H), 2.27

(1H, s, B(12)H), 2.27, 2.09 (4H, 2s, B(4,6,9,10)H), 2.15 (1H, s, B(8,11)H); 13C NMR

(Acetone-d6, Me4Si): δ 77.45 (1 C, s, Ccarborane), 56.72 (1C, d, CHcarborane), 48.24 (1C, t,

CH2); 11B NMR (Acetone-d6, BF3.Et2O): δ -4.50 (1B, d, J=159, B5), -9.40 (1B, d, J=225,

B12), -11.13 (4B, d, J=128, B4,6,9,10), -13.40 (2B, d, J=171, B8,11), -15.30(2B, d, J=183

Hz, B

(5

Dihydrochloride of diamine 5b (105 mg, 0.51 mmol) was dissolved in

1,4-dioxane (10 mL) and reacted with sulfamide (250 mg, 2.6 mmol)

and K2CO3 (765 mg, 5.6 mmol) at reflux temperature during 4 d under

stirring. After cooling down and removal of volatiles under reduced

pressure the crude product was extracted into acetonitrile (3x 10 mL)

and combined extracts were evaporated in vacuum. The product was purified by

chromatography on a silica gel column (20 x 1,5 cm I.D.) using benzene-ether (1:1 b.v.) and

then ether-CH3CN (4:1 b.v.) as the mobile phase. Ether-acetonitrile fractions containing the

product (according to NMR) were combined, evaporated to dryness and dried 6 h in vacuum.

The product was further crystallized from ether-hexane. Yield 75 mg (40%); M. p. 144 °C; 1H{11B} NMR (Acetone-d6, Me4Si): δ 5.63 (1H, bt, CH2NHSO2), 5.23 (2H, s, NHSO2NH2),

3.38 (2H, d, J=7.6, -CH2-NH), 2.88 (2H, s, B(2,3)H); 2.20 (4H, 2s, B(4,6,9,10)H), 2.19 (2H,

19

s, B(5,12)H), 1.99 (1H, s, B(8,11)H); 13C NMR (CD3CN, Me4Si): δ 76.34 (2 C, s, Ccarborane),

47.83 (2C, t, CH2); 11B NMR (CD3CN, BF3.Et2O): δ -7.15 (2B, d, J=162, B5,12), -11.47

(6B, d, J=155, B4,6,9,10,8,11), -14.26 (2B, d, J=180, B2,3); MS (APCI) m/z 359.33 (100

), 362.17 (5 %) [M-H]-, calcd. 359.18 (100%), 362.18 (5%).

-sulfamido-closo-1-carbadodecaborate (1-), potassium salt (6a)

-6); m/z (ESI)

37.30, (100 %), 240.22 (5 %) [M-H]-, calcd. 237.19 (100 %), 240.22 (5 %).

7-(sulfamido)methyl-7,8-dicarba-nido-undecaborate (1-), sodium salt (7a)

%

1

A suspension of 6b (220 mg, 1.4 mmol) and anhydrous K2CO3 (382 mg,

2.8 mmol) in 1,4-dioxane (20 mL) was stirred and heated to 100 °C over

1 h period. Solid sulfamide (665 mg, 6.9 mmol) was then added in one

portion, and the reaction mixture was refluxed for 48 h. After cooling

down, the solvent was removed under reduced pressure and the oily

residue was dissolved in acetonitrile (5 mL). The solution was filtered

through a glass filter, which was washed by additional portion of the solvent (2 x 1 mL). The

filtrate was layered with ether (40 mL) and the solvent were left to diffuse slowly. Yellowish

crystals of 6a (188 mg) were obtained after three days of standing, which were sucked off and

dried in vacuum. Crystals for X-ray diffraction study were obtained from this crop. An

additional portion of the pure product (34 mg) was obtained by evaporation of the mother

liquors in vacuum and chromatography of the residue upon dissolution in solvent mixture of

CH2Cl2 and CH3CN (9:1 b.v.) on a silica gel column (20 x 1 cm) stepwise increasing the

content of acetonitrile to 1:1. Fractions containing the pure product were combined. Overall

yield: 222 mg (67%); M. p. < 25 °C.; 1H{11B} NMR (Acetone-d6, Me4Si): δ 5.09 (4 H, s,

NHSO2NH3), 1.94 (5 H, s, B(7-11)H), 1.40 (5 H, s, B(2-6)H), 1.39 (1H, s, B(12)H); 13C

NMR (CD3CN, Me4Si): δ 95.48 (1C, s, Ccarborane); 11B NMR (CD3CN, BF3.Et2O): δ -11.16

(1B, d, J=137, B12), -14.11 (5B, d, J= 82, B7-11), -14.56 (5B, d, J=67, B2

S

2

1,4-dioxane (2 mL) was added by syringe to a solid mixture

of the starting derivative 7b (50 mg, 0.3 mmol, prepared as

described above) and sulfamide (145 mg, 1.5 mmol). The

resulting slurry was stirred and heated under reflux for 72 h.

After cooling down, silica gel was added (ca. 5 mL) the volatiles were removed under reduced

HN

6a

O O

NH2

K

20

pressure and dried in vacuum. The solid residue was poured on a dry silica gel column (20 x

1.5 cm I.D.) and the products were eluted by chromatography starting from pure CH2Cl2 as

the mobile phase to CH2Cl2- CH3CN mixture 1:1 increasing stepwise the acetonitrile content.

Yield 56 mg (76 %), white solid. M. p. 45 °C; 1H{11B} NMR (CD3CN, Me4Si): δ 4.31 (1H,

s, CHcarborane), 3.14 (1H, t, J= 0.02, NH), 3.05 (2H, s, NH2), 1.89 (2H, s, B(9,11)H), 1.64 (1H,

s, B(2)H), 1.22 (2H, t, J=0.02, CH2), 1.20 (1H, s, B(3)H), 1.18 (1H, s, B(4)H), 1.17 (1H, s,

B(6)H), 1.07 (1 H, s, B(5)H), 0.42 (1H, s, B(1)H), -2,80 (1H, s, B(10)H); 13C NMR (CD3CN,

Me4Si): δ 68.24 (1C, s, Ccarborane), 52.46 (1C, d, CHcarborane), 30.14 (1C, t, CH2); 11B NMR

(CD3CN, BF3.Et2O): δ -10.97 (1B, d, J=143, B9), -11.31 (1B, d, J=142, B11), -14.90 (1B, d,

J=159, B2), -16.94 (1B, d, J=159, B4), -18.39 (1B, d, J=140, B5), -19.13 (1B, d, J=128, B3),

-22.22 (1B, d, J=147, B6), -33.33 (1B, d, J=164, B10), -37.47 (1B, d, J=140, B1); MS (ESI)

/z 242.17 (100 %), 244.17 (10 %) [M-H]-, calcd. 242.18 (100%), 244.18 (7%).

7-(sulfamido)methyl-8-phenyl-7,8-dicarba-nido-undecaborate (1-), potassium salt (8a)

, -36.72 (1B, d, J=137, B1); MS

SI) m/z 320.24 (7%), 318.26 (100%) [M-H]-, calcd. 320.21 (7%), 318.21 (100%).

m

The starting amine 8b (50 mg, 0.15 mmol) was dissolved in 1,4-

dioxane (25 mL) and solid sulfamide (0.100 g, 1.04 mmol) was

added under nitrogen followed with anhydrous K2CO3 (0.100 g,

0.73 mmol). Reaction mixture was heated up and stirred under

reflux for 24 h. After cooling down the volatiles were removed

in vacuum and the crude product was isolated by extraction of solids by acetonitrile (2 x 10

mL) and evaporation of the extracts. The pure compound was obtained by chromatography on

a silica gel (20 x 1.5 cm I.D.) column using chromatographic conditions identical with these

used for purification of 7a. Yield: 47 mg (70%), colourless solid. M. p. < 25 °C. 1H{11B}

NMR (CD3CN, Me4Si): δ 7.184 (5H, m, C6H5), 5.26 (1H, t, CH2NH), 3.31 (1H, s, B(9)H),

3.05 (2H, s, SO2NH2), 2.84 (2H, d, CH2N), 2.32 (1H, s, B(11)H), 1.58 (1 H, s, B(2)H), 1.55

(1H, s, B(3)H), 1.41 (1H, s, B(6)H), 1.30 (1 H, s, B(5)H), 1.20 (1H, s, B(4)H), 0.58 (1H, s,

B(1)H); 0.25 (1H, s, B(10)H), -2.22 (1H, bs, B-H-B); 13C NMR (CD3CN, Me4Si): δ 128.16

(4C, m, C6H5), 127.06 (2C, m, C6H5), 67.47 (1C, s, C(8)carborane), 49.23 (1C, s, C(7)carborane),

30.14 (1C, t, CH2); 11B NMR (CD3CN, BF3.Et2O): δ -8.62 (1B, d, J=164, B9), -10.59 (1B,

d, J=131, B11), -14.11 (1B, d, J=156, B2), -16.63 (1B, d, J=150, B5), -17.94 (1B, d, J=143,

B4), -18.91 (2B, d, J=143, B3,6), -33.54 (1B, d, J=92, B10)

(E

21

7-[3-(sulfamido)-propyl]-8-phenyl-7,8-dicarba-nido-undecaborate (1-), potassium salt

(9a)

A suspension of starting amine 9b (0.050 g, 0.14 mmol),

sulfamide (0.055 g, 0.57 mmol) and K2CO3 (0,075 g, 0,54

mmol) in 1,4-dioxane (25 mL) was stirred under reflux

24 h. After cooling down, the volatiles were removed under

reduced pressure. The crude product was extracted into

acetonitrile and purified on a silica gel column using CH2Cl2 and CH3CN (4:1 b.v.) as a

mobile phase. Yield 0.058 g (77 %), colourless solid; M. p. 13.3 °C, TLC RF

(CH3CN:CH2Cl2 1:3) 0.17; 1H{11B} NMR (CD3CN, Me4Si): δ 7.70 (2H, d, C6H5), 7.45

(1H, d, C6H5), 7.32 (2H, d, C6H5), 5.22 (4H, s, NHSO2NH3), 2.73 (2H, m, CH2N), 2.50 (2H,

m, CH2), 1.60 (2H, t, BCH2), 2.74 (1H, s, B(4)H), 2.32 (1H, s, B(5)H), 2.25 (1H, s, B(9)H),

2.16 (1H, s, B(11)H), 2.00 (1H, s, B(6)H), 1.45 (1H, s, B(3)H), 1.19 (1H, s, B(2)H), 0.54 (1H,

s, B(1)H), 0.07 (1H, s, B(10)H), -2.22 (1H, s, μ-B-H-B); 13C NMR (CD3CN, Me4Si): δ

132.05 (2C, d, C6H5), 129.91 (1C, d, C6H5), 127.91 (3C, m, C6H5), 44.16 (1C, s, Ccarborane),

42.71 (1C, s, Ccarborane), 33.82 (1C, t, NCH2), 32.73 (1C, t, CH2), 30.68 (1C, t, CH2); 11B

NMR (CD3CN, BF3.Et2O): δ -4.24 (1B, d, J=150, B9), -8.90 (1B, d, J=125, B11), -9.80

(1B, d, J=146, B5), -10.83 (1B, d, J=214, B4), -13.66 (1 B, d, J=156, B6), -17.77 (1 B, d,

J=137, B2), -18.91 (1B, d, J=204, B3), -33.94 (1B, d, J=137, B10), -36.70 (1B, d, J=137,

B1); MS (ESI) m/z, 346.28 (100 %), 348.26 (10 %) [M-H]-, calcd. 346.24 (100 %), 348.24

0 %).

(2-[(sulfamido)ethoxy]ethoxy}-7,8-nido-dicarbaundecaborate (1-), potassium salt

(10a)

(1

10-{2-

1,4-dioxane (20 cm3) was added to the mixture of

small white leafs of 10a (0.50 g, 2.10 mmol),

sodium carbonate (0.45 g, 4.20 mmol) and

sulfamide (1.01 g, 10.52 mmol). The reaction

mixture was heated to reflux for 2 h and reaction progress was monitored using TLC and MS.

The 1,4-dioxane was then lyophilized from the reaction mixture. Isolation of pure product

was conducted with liquid chromatography on silicagel column using dichlormethan and

acetonitrile as solvents for gradient elution. The yield of the product was 0.18 g (28%, 0.58

mmol). M. p. 48.6°C. 1H{11B} NMR (Acetone-d6): δ 6.33 (2H, s, NH2), 3.67 (2H, s,

22

CHcarborane), 3.62 (2H, t, BOCH2), 3.58 (2H, t, CH2O), 3.37 (2H, t, CH2O), 3.24 (2H, d,

CH2NH), 2.15 (2H, bs, B(9,11)H), 1.87 (1H, t, NHS), 1.56 (1H, s, B(3)H ), 1.36 (2H, s,

B(5,6)H), 1.13 (2H, s, B(2,4)H), 0.37 (1H, s, B(1)H), -0.49 (1H, s, µ-H); 13C NMR (CH3CN,

Me4Si): δ 71.02 (2C, t, BOCH2), 69.17 (2C, d, CHcarborane), 69.16 (2C, t, CH2O), 41.52 (2C ,t

, CH2N); 11B NMR (CD3CN, BF3.Et2O): δ -9.58 (1B, s, B10), -12.03 (2B, d, J=137, B9,11),

-16.92 (2B, d, J=134, B5,6), -23.30 (2B, d, J=150, B2,4), -24.39 (1B, d, J=159, B3), -40.23

(2B, d, J=143, B1); MS (APCI) m/z 316.25 (100 %) 318.21 (8 %) [M-H]-, calcd. 316.22

00 %), 318.21 (7%).

and purified by gel filtration on

(1

CA Inhibition Assay

For the inhibiton assay, human CAII purchased from Sigma (cat. no. 55540) was used. CAIX

was produced by heterologous expression in Drosophila Schneider 2 (S2) Cells. The

expressed fusion protein contained N-terminal 6xHis tag, short GlySerGlySer linker, tobacco

etch virus (TEV) protease recognition sequence followed by the sequence of CAIX carbonic

anhydrase domain (aminoacid residues 130-390 of Genbank ID NP_001207.2).

Glycosylation site at position 346 was eliminated by mutation Asn→Asp . The His-tagged

protein was purified from cell culture media by affinity chromatography on His-select Nickel

Affinity Gel (Sigma), then processed by TEV protease

Superdex 200 10/30 GL column (Amersham Bioscience).

Testing of CA enzyme activity and inhibition was performed using a classical colorimetric

assay[14] with some modifications.[15] The principle of the method is based on measurement of

the rate of CA-catalyzed CO2 hydration activity monitored by the color change of an acid-

base indicator (at 0 °C). In a typical experiment, a 10 µl aliquot of 5 µM enzyme (CAII or

CAIX) in 10 mM HEPES, pH 7.0, was mixed with inhibitor dissolved in DMSO [final DMSO

concentration lower than 2% (v/v)]. The volume was adjusted to 100 µl with 10 mM HEPES,

pH 7.0, and this solution was transferred into 800 µl 1 mM NaHCO3, 1 mM Na2CO3, pH

10.2, containing 0.3 mM phenol red. The solution was saturated with CO2 for 60 s (at

50 ml/min), causing the color of the phenol red indicator to change to yellow. Following

saturation, 100 µl of a solution containing 500 mM NaHCO3 and 500 mM Na2CO3, pH 10.2,

were added, and the color changed back to red-purple. The solution was then again saturated

with CO2 under the same conditions, and the time it took the color to change back to yellow

was measured. The endpoint of the reaction was determined by visual control with a reference

tube containing 0.3 mM phenol red, pH 7.0. Negative controls (blanks without enzyme)

23

usually reached the endpoint at 58 ± 2 s. When enzymatic activity was present, the time was

proportionally shorter.

Catalytic activity (Cact) was calculated from the formula Cact = log (tB/tS) / log 2, where tB is

re analyzed in triplicate. The catalytic activities of the

he selectivity of the inhibitor was calculated as the ratio between its Ki values for CAII and

d in Table 1.

olution was mixed

ere collected at 100 K at beamline X12 of DESY, Hamburg.

iffraction data were processed using MOSFLM[17] and SCALA.[18] Data collection statistics

re summarized in Table S2.

the time measured for the negative control without enzyme and tS is the time for the sample

with enzyme in the presence of inhibitor.

To determine apparent Ki values, series of activity assays were performed in the presence of

selected compounds. Inhibitor concentrations in the reaction mixtures were adjusted to fit a

range of relative enzyme activity (between 1 and the lowest measurable). For each compound,

5 or more inhibitor concentrations we

inhibited enzymes at varying inhibitor concentrations were fitted using the Williams-Morrison

equation to estimate the apparent Ki.

T

CAIX. The results are liste

Protein Crystallization

Complexes of human CAII with 1a and 7a were prepared by adding a 1.1-fold molar excess

of inhibitor (20 mM stock solution in dimethyl sulfoxide) to a 10 mg/ml solution of CAII

(Sigma, cat. no. C6165) in 100 mM Tris-HCl, pH 8.5. For crystallization of CAII in complex

with 4a, human CAII (concentration 5 mg/ml) was pre-treated with a 2-fold molar excess of

p-hydroxymercuribenzoate (Sigma, cat. no. 55540). Unbound p-hydroxymercuribenzoate was

removed by diluting the protein sample several times with pure water and concentrating it

back to 11 mg/ml using Amicon Ultra-4 concentrators (Merck-Millipore). The complex of the

p-hydroxymercuribenzoate-treated human CAII with 4a was prepared by adding a 1.2-fold

molar excess of the inhibitor dissolved in DMSO to 11 mg/ml protein solution in water

(without pH adjustment). Crystals of all complexes were obtained by the hanging-drop vapor

diffusion method under the following conditions: 2 μl protein-inhibitor s

with 2 μl precipitant solution [2.5 M (NH4)2SO4, 0.3 M NaCl, 100 mM Tris-Cl, pH 8.2] and

equilibrated over a reservoir containing 1 ml precipitant solution at 18 °C.

For cryoprotection, the crystals were soaked in precipitant solution supplemented with 25%

glycerol, flash frozen, and stored in liquid N2. Diffraction data for CAII in complex with 1a

and 4a were collected at 100 K at beamline X14.2 of BESSY, Berlin.[16] Diffraction data for

CAII in complex with 7a w

D

a

24

Structure Determination, Refinement, and Analysis

Crystal structures were solved by difference Fourier method using the CAII structure (PDB

code 3IGP[19]) as the starting model. The models were refined using REFMAC5,[20] part of the

CCP4 program suite.[21] The final refinement steps included TLS (translation/libration/screw)

refinement[22] for the protein molecule and anisotropic ADPs for the Zn2+ cation and whole

inhibitor molecule or sulfamide part of the inhibitor molecule for 4a and 7a, respectively. For

the CAII-1a inhibitor complex, the last round of refinement was done in a mixed (isotropic-

anisotropic) model of atomic displacement parameters (ADPs); anisotropic ADPs were used

for protein, Zn ion and inhibitor molecule, while solvent molecules were refined with

isotropic ADPs. Atomic coordinates for structures of carborane compounds were generated

by quantum mechanics computation with DFT-D methodology[23] using the B-LYP

functional and SVP basis set[24] in the Turbomole program.[25] A geometric library for

carborane inhibitors was generated using the Libcheck program from the CCP4 suite.[21]

Coot[26] was used for rebuilding. The quality of the refined model was assessed using

MolProbity.[27] Final refinement statistics are summarized in Table S2. All figures were

prepared using PyMOL 1.4.1.[28]

25

Table S2. Crystal data and diffraction data collection and refinement statistics.

Complex CA II + 1aa CA II + 4aa CA II + 7aa Data collection statistics Space group P21 P21 P21 a, b, c (Å) 42.1, 41.6, 72.1 42.1, 41.7, 72.0 42.0, 41.3, 72.1 α, β, γ (º) 90.0, 104.1, 90.0 90.0, 104.2, 90.0 90.0, 104.2, 90.0Wavelength (Å) 0.9184 1.5418 0.9157 Resolution range (Å) 26.76 - 1.35

(1.39 – 1.35) 17.90 - 1.51

(1.59 – 1.51) 26.67 - 1.55 (1.59 – 1.55)

No. of unique reflections 48024 (2019) 36956(4385) 34903 (2505) Multiplicity 2.5 (2.4) 2.1 (2.0) 2.9 (2.3) Completeness (%) 90.2 (51.5) 96.0 (79.1) 99.6 (97.2) Rmerge

b (%) 9.4 (34.2) 7.6 (24.7) 7.2 (44.5) Average I/σ (I) 5.7 (2.0) 4.6 (2.2) 7.9 (1.9) Wilson B (Å2) 11.4 15.7 13.3 Refinement statistics Resolution range 24.59 – 1.35

(1.39 – 1.35) 28.78 – 1.52 (1.56 – 1.52)

26.67 – 1.55 (1.59 – 1.55)

No. of reflections in working set 45580 (1853) 35339(1235) 33131 (2299) No. of reflections in test set 2430 (109) 1858(99) 1758 (117) R c (%) 14.4 (20.6) 15.75(22.5) 17.37 (24.6) Rfree

d (%) 17.90 (26.9) 18.36(24.1) 20.23 (31.8) RMSD bond length (Å) 0.012 0.013 0.014 RMSD bond angle (º) 1.50 1.73 1.52 Mean ADP (Å2) 13.5 22.0 16.03 PDB code 4MDG 4MDL 4MDM [a] Values in parentheses correspond to the highest resolution shell; [b] Rmerge = hkl iIi(hkl) - I(hkl)|/hkl i Ii(hkl), where the Ii(hkl) is an individual intensity of the ith observation of reflection hkl and I(hkl) is the average intensity of reflection hkl with summation over all data; [c] R = ||Fo| - |Fc||/|Fo|, where Fo and Fc are the observed and calculated structure factors, respectively; [d] Rfree is equivalent to R value but is calculated for 5 % of the reflections chosen at random and omitted from the refinement process.[29]

26

27

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