David Augros

Boubacar Yalcouye

Sabine Choppin

Matthieu Chessé

Armen Panossian
email: armen.panossian@unistra.fr

R Leroux

Transition metal-free synthesis of a known intermediate in the formal synthesis of (-)-steganacin

The formal synthesis of both enantiomers of a natural axially chiral biaryl, steganacin is reported. The previously developed atropo-diastereoselective coupling of an aryne and an aryllithium (the 'ARYNE coupling') allows for this synthesis. In each step, the axial configuration of the biaryl could be maintained. The key intermediate of literature was accessed without using transition metals, demonstrating the interest of the ARYNE coupling as a complement or an alternative to transition metal-catalyzed couplings.

Introduction

(-)-Steganacin (2) (Scheme 1) is a known chiral biaryl-based natural product, which showed potent antimicrotubule in vivo activity against mice cancer cell lines and in vitro activity against human cancer cell lines, but did not find any medicinal application; however, the early interest for this potential anticancer agent as well as the synthetic challenge explain why (-)-steganacin and its one-step precursor (-)-steganone (1), their enantiomers and racemates have been the target of many new synthetic methods. [1] We already described a palladium-catalyzed atropodiastereoselective Suzuki-Miyaura coupling as a key step to prepare a known synthetic intermediate (3) initially described by Harayama, Abe et al. [1ab,1ac] in the formal synthesis of (-)steganone. [2] In the present paper, we report on an alternative transition metal-free, lithium-based synthesis of Harayama and Abe's intermediate 3, in view of a transition metal-free total synthesis of (-)-steganacin or its derivatives. Indeed, despite their versatile use as catalysts in all sorts of reactions, transition metals might be a concern especially in the pharmaceutical industry, where recommended levels in active pharmaceutical ingredients are very low (permitted daily exposure of patients to palladium: 100 μg/day for oral doses, 10 μg/day for parenterally administered drugs, 1 μg/day by inhalation). [3a,3b] In comparison, the respective permitted daily exposures of patients to lithium are of 560, 280 and 25 μg/day, [3b] which, given the atomic mass of both elements, favours lithium even if it is used in stoichiometric amount while palladium is used in catalytic quantities. Accordingly, the use of transition metals in late-stage synthesis is problematic and requires efficient, but costly, removal of trace metals (e.g. scavenging agents or nanofiltration). [4] Additionally, the price per tonne of palladium, ca. 2000-3000 times more expensive than lithium, has also to be taken in consideration. Consequently, transition metal-free syntheses might offer an appealing solution.

We recently described the coupling between an in situ-generated aryne and an aryllithium nucleophile bearing a chiral oxazolinyl group ortho to lithium; the design of the coupling partners was directed towards the formal synthesis of (-)-steganacin as a model target. Although very moderate selectivity was achieved, we showed that the desired biaryl 4 could be produced with a satisfying 67% yield, given the steric bulk surrounding the newly formed, tetra-ortho-substituted aryl-aryl bond, and that both atropo-diastereoisomers could be separated by column chromatography and their absolute configuration determined by X-ray diffraction crystallography. [5] Having created the biaryl backbone of (-)-steganacin, we had now to transform it into intermediate 3, without using transition metals.

Scheme 1. Planned strategy for the formal synthesis of (-)-steganacin.

Results and Discussion

In order to do so, three kinds of transformations had to be carried out, independently of their order: 1) double desilylation, 2)

(-)-steganone 1 (-)-steganacin 2 (CR 1 R 2 = CO) (R 1 = H, R 2 = OAc) 1) 2 equiv. tBuLi, THF, -78 °C 2) 1.2 equiv.
-35 °C to 25 °C conversion of iodine into a formyl group, 3) reduction of the oxazoline into an alcohol. Starting from iodo-to-formyl conversion by halogen/lithium exchange appeared to be unfruitful (Scheme 2). Indeed, addition of 1 equiv. of butyllithium in THF at -78 °C to 4b (S,aS)-as a model in place of the desired 4a (S,aR)followed by trapping with DMF after 30 minutes only led to the undesired fluorenone 5 [5] in 69% yield. When reducing the time before DMF addition to 5 minutes, we surprisingly recovered 50% of starting material, i.e. with untouched iodine, while 5 was still formed in 39% yield. The recovery of unreacted iodobiphenyl can be ascribed to the high steric bulk around iodine, decreasing its reactivity. Anyway, in both cases, no desired aldehyde was obtained after work-up. The fluorenone formation at the detriment of the trapping by DMF was coherent with our previous findings. [5,6] Indeed, cyclisation of the biaryllithium intermediate is favored over trapping with an electrophile when the latter is either slowly reacting or not already present in the reaction medium for in situ trapping of the forming biaryllithium.

Scheme 2. Attempts of iodine/lithium exchange and trapping with DMF on iodobiaryl 4b.

Consequently, we rather decided to perform the formylation as a last step, and to investigate on the double desilylation of the biaryl moiety. This transformation was critical from a synthetic point of view, insofar as both TMS groups had to be removed efficiently and without epimerization of the chiral axis. Therefore, we wished to perform the protodesilylation at low temperature if possible. We again started first with a model bis(trimethylsilylated) biaryl, 6 (Scheme 3), that we already described. [5] and treated it with TFA, a known desilylating agent of electron-rich aromatic rings. [7] The reaction proceeded well in the presence of excess TFA at room temperature, although a long reaction time was required to fully remove the TMS group in position 6. Indeed, the latter is quite hindered by the dimethoxyphenyl ring, as observed by 1 H NMR where TMS-6 is shielded by 0.34 ppm with regard to TMS-3 due to the anisotropy cone generated by the aforementioned dimethoxyphenyl (Scheme 3). Since the TFA-promoted protodesilylation -by electrophilic aromatic substitution-requires tetragonalization of the ipso carbon, the higher steric hindrance around carbon-6 explains that it is less prone to the latter change of geometry. In the end, the reaction afforded the totally desilylated product 7 in 87% yield after 72 h of reaction at 25 °C (Scheme 3).

Scheme 3. Double protodesilylation of model compound 6.

However, when applying these conditions to the (S,aS) diastereomer 4b, where the TMS-substituted carbon-6 is much more congested than in 6, only the mono-desilylated product 8 was obtained in 61% yield, nevertheless with retention of axial stereo-enrichment (Scheme 4). Apart from the characteristic shielded 1 H NMR chemical shift of the untouched TMS group, the structure of 8 was confirmed by NOESY, showing a correlation between the signals of the TMS and of the methoxy groups, as well as another between the signals of the TMS and of the aromatic proton located ortho to the oxazoline. When other conditions or other acids (HCl, H2SO4) were tried, complex mixtures were obtained, from which biphenylcarboxylic acids, coming from hydrolysis of the oxazoline moiety, could sometimes be identified. On the other hand, using a 1M TBAF solution in THF to accomplish the protodesilylation -via a different, anion-based mechanistic pathway-revealed completely ineffective. Eventually, we succeeded in deprotecting both silylated carbons of 4b and 4a by employing TBAF-trihydrate in DMF at 50 °C for 2.5 days. Gratifyingly, no erosion of diastereomeric ratio was observed in product 9, even at this temperature and reaction time, and quantitative yields were obtained. More gratifyingly, we later found out that the same reaction was complete after 30 min, even at room temperature (Scheme 4). In view of further transformations towards (-)-steganacin or analogues, it is noteworthy that the desilylations can be performed stepwise with first TFA, and then TBAF•3H2O/DMF, making this sequence an interesting tool for controlled deprotection. Then, to reduce the oxazoline of 9a, we first hydrolyzed it under conditions similar to Meyers', [8] using TFA in a THF/water mixture, at room temperature to prevent potential epimerization of the chiral axis (Scheme 5). The crude biaryl ester with a pendant ammonium function was then treated with reducing agents. Lithium aluminum hydride was effective, but also reduced the carbon-iodine bond. We therefore turned to lithium borohydride in combination with methanol, which, at first, gave irreproducible results. We soon realized that this was related to the basic workup of the reaction of 9a, which was critical as it could either afford the ammonium 10a, the corresponding amine 11a, or a mixture of both. Indeed, when the hydrolysis of the oxazoline was followed by simple washing of the organic layer with saturated NaHCO3 and extraction with ether, either 10a or a mixture of 10a and 11a, where ammonium 10a was largely major, were obtained. On the other hand, when the reaction mixture was quenched with saturated NaHCO3 until evolution of CO2 had ceased, then extracted with diethyl ether, amine 11a was the sole product, as shown by the absence of fluorine signals in 19 F NMR of the crude. Then, as implied above, the nature of the ester to be reduced (10a/11a) proved influential, as 10a and 11a did not behave similarly upon treatment with LiBH4/MeOH [9] . While the crude ammonium 10a afforded the desired alcohol 12a in 69% yield, amine 11a led to a mixture of 12a (minor) and amide 13a (major) by rearrangement of the aminoalkyl ester. Accordingly, to prevent formation of the amide 13a and to increase the yield of the alcohol 12a, we first reacted the crude 11a with the borane-THF complex and then treated the mixture with LiBH4/MeOH. We reasoned indeed that borane would coordinate to the free amine, preventing it from attacking the ester. This strategy proved efficient, and we finally obtained 91% yield of alcohol 12a from oxazoline 9a (Scheme 5). We then quantitatively protected the compound as a silyl ether 14a, of which the enantiomeric ratio was measured by chiral HPLC analysis to be higher than 99:1, confirming retention of axial stereoenrichment (Scheme 6). The opposite atropoisomers 12b and 14b were also synthesized from 9b with similar yields (Scheme 5 and Scheme 6). Finally, the most critical step was the iodine/lithium exchange followed by trapping with DMF, which ought to proceed without epimerization of the biaryllithium intermediate to afford the desired aldehyde 3. We thus carried out the reaction at low temperature (-100 °C) and with tert-butyllithium as the base for a clean halogen/metal exchange. Here as well (see Scheme 2), the usual number of equivalents of base (2 in the case of tBuLi) appeared insufficient for an effective conversion of the biaryl iodide, as only 8% yield of aldehyde 3 were achieved (Scheme 6).

In the end, 5 equiv. of tBuLi managed to produce 3 in 85% yield. Again, the same strategy was applied to the opposite atropoisomer 14b, giving ent-3 with 81% yield. Both enantiomers were showed to be enantiopure by chiral HPLC (>99:1 e.r.).

Conclusions

In conclusion, we carried out the synthesis of Harayama and Abe's intermediate 3 in 9 steps, 22% overall yield, excellent control of chirality (>98% e.e., with retention of axial stereoenrichment at each stage of the process), and in the absence of transition metals (Scheme 7). Interestingly, the enantiomer ent-3 of the target compound could be obtained with similar efficiency. This transition metal-free synthetic pathway is an efficient alternative to Harayama and Abe's synthesis of 3 [1ab,ac] (10 steps from piperonyl alcohol, 25% overall yield, 83% e.e.) or our previous Pd-catalyzed strategy [2] (15 steps from piperonyl alcohol, 9% overall yield, 94% e.e.), which both involved palladium-promoted steps and stoichiometric chromiummediated transformations. The current work enforces the interest in heavy metal-free biaryl synthesis applied to the preparation of potential pharmaceutically relevant ingredients.

Scheme 7. Formal synthesis of (-)-steganacin.

Experimental Section

See the Supporting Information for further information. The preparation of compounds 4a, 4b, 5 and 6 was already described. [5]

5-Bromo-6-(2,6-dimethoxyphenyl)benzo[d][1,3]dioxole (7)

To a stirred solution of bis(trimethylsilyl)biarylbromide 6 (0.20 g, 0.42 mmol, 1 equiv.) in dry dichloromethane (9 mL), was added trifluoroacetic acid (1.68 mL) at 25 °C. The reaction mixture was stirred for 3 d at 25 °C. The mixture was concentrated and the residue was taken up in CHCl3 (40 mL), washed with NaHCO3 (40 mL), water (40 mL), then brine (40 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to afford 7 as a yellow solid; yield = 0.12 g (0.36 mmol, 87%); Rf = 0.38 (cyclohexane/CH2Cl2 = 1:1); M. p. = 119-121 °C; ṽ = 294.7, 2902.7, 2838.2, 1593.4, 1473.5, 1243.

(S)-2-(2-(aS)-(6-Iodo-4-(trimethylsilyl)benzo[d][1,3]dioxol-5yl)-3,4,5trimethoxyphenyl)-4-isoproyl-4,5-dihydrooxazole (8)

To a stirred solution of 4b (220 mg, 0.33 mmol, 1 equiv.) in dry dichloromethane (4 mL), was added trifluoroacetic acid (1.22 mL, 16.43 mmol, 50 equiv.) at 25 °C. The reaction mixture was stirred at 25 °C for 16 h. Saturated aqueous NaHCO3 was added portionwise to the stirred mixture until no more carbon dioxide evolution was observed and the mixture was extracted thrice with Et2O. The organic layers were combined, dried over Na2SO4, filtered and concentrated under vacuum to give the 2, 2933.0, 1653.9 (C=N), 1589.8, 1398.2, 1359.3, 1196.8, 1099.2, 1019.5, 839.7 (Si(CH3)3), 765.3, 692.8, 630.6 cm - 1 ; 1 H NMR (CDCl3, 400 MHz): δ = 7.25 (s, 1 H, Ar-H), 7.23 (s, 1 H, Ar-H), 5.95 (ABq, 2 H, ΔδAB = 0.01, JAB = 1.3 Hz, O-CH2-O), 4.13-4.05 (m, 1 H, O-CH2-C), 3.94 (s, 3 H, Ar-OCH3), 3.90 (s, 3 H, Ar-OCH3), 3.88-3.80 (m, 2 H, O-CH2-C + NCH), 3.72 (s, 3 H, Ar-OCH3), 1.57 (sept.d, 1 H, 3 J = 6.6 Hz, 3 J = 6.6 Hz, CH iPr ), 0.78 (d, 3 H, 3 J = 6.7 Hz, CH3 iPr ), 0.77 (d, 3 H, 3 The desired atropo-diastereomer of 4 (1 equiv.) was dissolved in DMF (0.3 M). Tetrabutylammonium fluoride trihydrate (2.5 equiv.) was added and the mixture was stirred for 30 min at RT. The reaction mixture was taken up in brine and extracted twice with Et2O. The organic layers were combined, washed thrice with H2O, dried over Na2SO4, filtrated and concentrated under vacuum to give the desired product. 9a (S,aR): light orange oil; yield = 572 mg (1.09 mmol, 99 %) from 4a (734 mg, 1.10 mmol); Rf = 0.1 (cyclohexane/AcOEt = 9/1); [α]D 20 = -7.72 (c = 1.05, CHCl3); ṽ = 2964.0, 2930. 5, 2901.2, 1635.6 (C=N), 1440.0, 1415.1, 1340.6 (C-O), 1205.3 (C-O), 1101.7, 1037.0, 970.6 cm -1 ; 1 H NMR (CDCl3, 400 MHz): δ = 7.26 (s, 1 H, Ar-H), 7.22 (s, 1 H, Ar-H), 6.69 (s, 1 H, Ar-H), 5.97 (s, 2 H, O-CH2-O), 4.09 (dd, 1 H, 3 J = 9.1 Hz, 2 J = 8.2 Hz, O-CH2-C), 3.93 (s, 3 H, Ar-OCH3), 3.91 (s, 3 H, Ar-OCH3), 3.86 (ddd, 1 H, 3 J = 9.1 Hz, 3 J = 8.2 Hz, 3 J = 6.7 Hz, N-CH), 3.77 (dd, 1 H, 3 J = 8.2 Hz, 2 J = 8.2 Hz, O-CH2-C), 3.70 (s, 3 H, Ar-OCH3), 1.66 (sept.d, 1 H, 3 J = 6.7 Hz, 3 J = 6.7 Hz, CH iPr ), 0.86 (d, 3 H, 3 J = 6.7 Hz, CH3 iPr ), 0.81 (d, 3 H, 3 (s, 3 H, Ar-OCH3), 3.91-3.84 (m, 1 H, N-CH), 3.82 (dd, 1 H, 3 J = 7.8 Hz, 2 J = 7.8 Hz, O-CH2-C), 3.68 (s, 3 H, Ar-OCH3), 1.62 (sept.d, 1 H, 3 J = 6.7 Hz, 3 J = 6.7 Hz, CH iPr ), 0.81 (d, 3 H, 3 J = 6.7 Hz, CH3 iPr ), 0.80 (d, 3 H, 3 The desired atropo-diastereomer of 9 (1 equiv.) was dissolved in THF (0.12 M). Trifluoroacetic acid (5 equiv.) and water (50 equiv.) were then added and the mixture was stirred overnight at r.t. (LCMS monitoring). Saturated aqueous NaHCO3 was added portionwise to the stirred mixture until no more carbon dioxide evolution was observed and the mixture was extracted thrice with DCM. The combined organic layers were dried over Na2SO4, filtrated and concentrated under vacuum to give the crude aminoester intermediate that was then directly engaged into the reduction step. Under an argon atmosphere, a solution of borane-THF complex (1.2 equiv., 1 M in THF) and methanol (5 equiv.) were added to a solution of the previous crude (1 equiv.) in dry THF (0.12 M). Lithium borohydride (5 equiv.) was then added in one portion as a solid (CAUTION: hydrogen evolution). The mixture was refluxed for 1 h and quenched with 2 M HCl once cooled down (CAUTION: hydrogen evolution). Once there was no more hydrogen degassing, the mixture was extracted thrice with DCM. The organic layers were combined, dried over Na2SO4, filtrated and concentrated under vacuum. The crude was purified by flash chromatography on silica gel, eluting with a cyclohexane/AcOEt mixture, to give the purified alcohol as a colorless foam. Rf = 0.25 (cyclohexane/AcOEt = 6/4); ṽ = 3423.9 (O-H), 2934. 6, 1598.0, 1471.8, 1456.8, 1413.2, 1317.1, 1223.6, 1141.7, 1117.9, 1034.6, 1014.8, 731.6, 556.1 (C-I) cm -1 ; 1 H NMR (CDCl3, 400 MHz): δ = 7.33 (s, 1 H, Ar-H), 6.91 (s, Under an argon atmosphere, the desired atropo-enantiomer of 12 (1 equiv.) was dissolved in dry DCM (0.1 M) and imidazole (2.5 equiv.) was added in one portion. Once dissolved, tert-butyldimethylsilyl chloride (1.2 equiv.) was added in one portion and the mixture was stirred at RT for 5 min. Saturated aqueous NH4Cl was added to the reaction mixture that was then extracted thrice with DCM. The organic layers were combined, dried over Na2SO4, filtrated and concentrated under vacuum. The crude product was purified by flash chromatography on silica gel, eluting with a cyclohexane/AcOEt mixture, to give the purified silylated alcohol as a colorless oil. The enantiomeric ratio of the product was determined by chiral HPLC on a Chiralpak IB column (hexane/isopropanol = 80/20; 0.5 mL/min; tR = 8.00 min (aS), 8.78 min (aR)).

MeO

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Entry for the Table of Contents

FULL PAPER

The formal synthesis of both enantiomers of a natural axially chiral biaryl, steganacin with excellent enantiomeric ratio and without transition metals is presented.

Transition metal-free synthesis

6 Scheme 4 .

 64 Scheme 4. Single and double protodesilylation of both atropo-isomers of 4.

Scheme 5 .Scheme 6 .

 56 Scheme 5. Reduction of the oxazoline moiety.

  was purified by column chromatography on silica gel, eluting with a cyclohexane/AcOEt gradient to yield 8 as a pale yellow oil; yield = 120 mg (0.20 mmol, 61%); Rf = 0.21 (cyclohexane/AcOEt = 90:10); [α]D 20 = -2.1 (c = 1.00, CHCl3); = 2956.

  J = 6.8 Hz, CH3 iPr ), -0.07(s, 9 H, Si(CH3)3) ppm;13 C NMR (CDCl3, 100 MHz): δ = 162.43 (C=N), 153.03 (C), 152.83 (C), 151.87 (C), 145.65 (C), 144.26 (C), 139.58 (C), 134.13 (C), 124.30 (C), 122.25(C), 118.14 (CH Ar ), 108.05 (CH Ar ), 100.69 (OCH2O), 91.59 (C-I), 72.77 (NCH), 70.27 (OCH2), 60.68 (Ar-OCH3), 60.56 (Ar-OCH3), 56.16 (Ar-OCH3), 33.12 (CH iPr ), 18.69 (CH3 iPr ), 18.61 (CH3 iPr ), -0.34 (Si(CH3)3) ppm; HRMS ES m/z [M+Na] + calcd. for C22H24INaNO6: 620.0941, found 620.0938.

  J = 6.7 Hz, CH3 iPr ) ppm; 13 C NMR (CDCl3, 100 MHz): δ = 163.09 (CIV, C=N), 152.99 (CIV, C Ar ), 151.36 (CIV, C Ar ), 147.70 (CIV, C Ar ), 147.38 (CIV, C Ar ), 144.33 (CIV, C Ar ), 135.99 (CIV, C Ar ), 131.97 (CIV, C Ar ), 123.61 (CIV, C Ar ), 117.63 (CH Ar ), 110.53 (CH Ar ), 108.52 (CH Ar ), 101.58 (O-CH2-O), 89.70 (CIV, C-I), 72.61 (N-CH), 70.42 (O-CH2-C), 61.11 (Ar-OCH3), 61.00 (Ar-OCH3), 56.15 (Ar-OCH3), 32.78 (CH iPr ), 18.95 (CH3 iPr ), 18.40 (CH3 iPr ) ppm; HRMS ES m/z [M+ Na] + calcd. for C22H24INaNO6: 548.0546, found 548.0541. 9b (S,aS): light yellow oil; yield = 440 mg (0.84 mmol, 99 %) from 4b (561 mg, 0.84 mmol); Rf = 0.1 (cyclohexane/AcOEt = 9/1); [α]D 20 = -5.60 (c = 0.94, CHCl3); ṽ = 2963.0, 2928.5, 2900.2, 1633.6 (C=N), 1440.0, 1415.1, 1340.6 (C-O), 1204.3 (C-O), 1100.7, 1036.0, 968.6 cm -1 ; 1 H NMR (CDCl3, 400 MHz): δ = 7.25 (s, 1 H, Ar-H), 7.19 (s, 1 H, Ar-H), 6.76 (s, 1 H, Ar-H), 5.97 (ABq, 2 H, ΔδAB = 0.01, JAB = 1.3 Hz, O-CH2-O), 4.15 (dd, 1 H, 3 J = 8.7 Hz, 2 J = 7.3 Hz, O-CH2-C), 3.93 (s, 3 H, Ar-OCH3), 3.92

  J = 6.7 Hz, CH3 iPr ) ppm; 13 C NMR (CDCl3, 100 MHz): δ = 162.93 (CIV, C=N), 152.96 (CIV, C Ar ), 151.38 (CIV, C Ar ), 147.80 (CIV, C Ar ), 147.44 (CIV, C Ar ), 144.38 (CIV, C Ar ), 135.86 (CIV, C Ar ), 132.02 (CIV, C Ar ), 123.78 (CIV, C Ar ), 117.70 (CH Ar ), 111.03 (CH Ar ), 108.44 (CH Ar ), 101.61(O-CH2-O), 89.31 (CIV, C-I), 72.80 (N-CH), 70.41 (O-CH2-C), 61.05 (Ar-OCH3), 61.04 (Ar-OCH3), 56.14 (Ar-OCH3), 32.98 (CH iPr ), 18.85 (CH3 iPr ), 18.54 (CH3 iPr ) ppm.

  12a (aR): yield = 255 mg (0.57 mmol, 91 %) from 9a (330 mg, 0.63 mmol; [α]D 20 = +8.5 (c = 1, CH2Cl2) 12b (aS): yield = 319 mg (0.72 mmol, 88 %) from 9b (430 mg, 0.82 mmol); [α]D 20 = -8.8 (c = 1, CH2Cl2)

  1 H, Ar-H), 6.70 (s, 1 H, Ar-H), 6.01 (s, 2 H, O-CH2-O), 4.30 (ABq, 2 H, ΔδAB = 0.06, JAB = 13.0 Hz, Ar-CH2-OH), 3.92 (s, 3 H, Ar-OCH3), 3.89 (s, 3 H, Ar-OCH3), 3.73 (s, 3 H, Ar-OCH3), 1.73 (bs, 1 H, OH) ppm; 13 C NMR (CDCl3, 100 MHz): δ = 153.7 (CIV, C Ar ), 151.1 (CIV, C Ar ), 148.5 (CIV, C Ar ), 148.0 (CIV, C Ar ), 141.5 (CIV, C Ar ), 135.0 (CIV, C Ar ), 134.5 (CIV, C Ar ), 130.1 (CIV, C Ar ), 118.3 (CH Ar ), 110.7 (CH Ar ), 106.6 (CH Ar ), 101.9 (O-CH2-O), 89.9 (CIV, C-I), 63.1 (Ar-CH2-OH), 61.1 (Ar-OCH3), 61.0 (Ar-OCH3), 56.1 (Ar-OCH3) ppm; C17H17IO6 (444,01): calcd. C 45.97, H 3.86, found C 45.57, H 3.99. (aR)-and (aS)-tert-butyl({[2-(6-iodo-2H-1,3-benzodioxol-5-yl)-3,4,5trimethoxyphenyl]meth-oxy})dimethylsilane (14a and 14b)

  14a (aR): yield = 293 mg (0.52 mmol, 99 %, e.r. > 99:1) from 12a (236 mg, 0.53 mmol); [α]D 20 = +15.0 (c = 1, CH2Cl2) 14b (aS): yield = 350 mg (0.63 mmol, 87 %, e.r. > 99:1) from 12b (319 mg, 0.72 mmol); [α]D 20 = -15.3 (c = 1, CH2Cl2)

Acknowledgements

This work was supported by the French Centre National de la Recherche Scientifique (CNRS). D. A., A. P. and F. L. thank the French Agence Nationale pour la Recherche (ANR) (grant number ANR-14-CE06-0003-01, ChirNoCat) for financial support.

Keywords: Aryne • Asymmetric synthesis • Transition metal-free • Organolithium • Chirality

Rf = 0.25 (cyclohexane/AcOEt = 9/1); ṽ = 2930. 1, 1598.6, 1472.4, 1457.3, 1224.6, 1142.9, 1119.2, 1084.9, 1039.3, 1017.4, 836.2 (Si(CH3)3), 776.3 (Si(CH3)3), 555.8 (C-I) cm -1 ; 1 H NMR (CDCl3, 400 MHz): δ = 7.33 (s, 1 H, Ar-H), 6.98 (s, 1 H, Ar-H), 6.68 (s, 1 H, Ar-H), 6.02 (ABq, 2H, ΔδAB = 0.02, JAB = 1.1 Hz, O-CH2-O), 4.30 (ABq, 2H, ΔδAB = 0.08, JAB = 14.0 Hz, Ar-CH2-OTBS), 3.92 (s, 3 H, Ar-OCH3), 3.88 (s, 3 H, Ar-OCH3), 3.73 (s, 3 H, Ar-OCH3), 0.92 (s, 9 H, C(CH3)3), 0.04 (s, 3 H, CH3) (CH3)3),C23H31IO6Si (558,09) Under an argon atmosphere, a solution of tert-butyllithium (5 equiv., 1.5 M in pentane) was added to dry THF (0.2 M) at -78 °C. This solution was cooled down to -100 °C after 10 min stirring. A solution of the desired atropoenantiomer of the silylated alcohol 14 (1 equiv.) in dry THF (0.2 M) was cooled down to -100 °C and was then cannulated onto the first one at -100 °C. The mixture was stirred for 10 min at this temperature before being quickly quenched with dry dimethylformamide (15 equiv.). The mixture was stirred for 30 min from -100 °C to RT. Saturated aqueous NH4Cl was added and the mixture was extracted thrice with Et2O. The organic layers were combined, washed thrice with water, dried over Na2SO4, filtrated and concentrated under vacuum. The crude was purified by flash chromatography on silica gel, eluting with a cyclohexane/AcOEt mixture, to give the purified aldehyde as a colorless oil. The enantiomeric ratio of the product was determined by chiral HPLC on a Chiralpak IB column (hexane/isopropanol = 95/5; 0.5 mL/min; tR = 11.40 min (aS), 11.94 min (aR)). Rf = 0.12 (cyclohexane/AcOEt = 9/1); ṽ = 2930. 8, 1682.5 (C=O), 1612.2, 1598.2, 1475.4, 1462.3, 1239.5, 1139.0, 1102.0, 1058.8, 1035.4, 996.4, 835.2 (Si(CH3)3), 776.7 (Si(CH3)3), 507.9 cm -1 ; 1 H NMR (CDCl3, 400 MHz): δ = 9.50 (s, 1 H, CHO), 7.46 (s, 1 H, Ar-H), 6.96 (s, 1 H, Ar-H), 6.67 (s, 1 H, Ar-H), 6.10 (ABq, 2 H, ΔδAB = 0.02, JAB = 0.9 Hz, O-CH2-O), 4.30 (ABq, 2 H, ΔδAB = 0.09, JAB = 13.2 Hz, Ar-CH2-OTBS), 3.92 (s, 3 H, Ar-OCH3), 3.87 (s, 3 H, Ar-OCH3), 3.61 (s, 3 H, Ar-OCH3), 0.87 (s, 9 H, C(CH3)3), -0.03 (s, 3 H, CH3), -0.04 (s, 3 H, CH3)


Fabien Despinoy
email: fabien.despinoy@univ-rennes1.fr

Sandrine Voros

Nabil Zemiti

Nicolas Padoy

Pierre Jannin

Towards unified dataset for Modeling and Monitoring of Computer Assisted Medical Interventions

2CAMI is a working group dedicated to Modeling and Monitoring of Computer Assisted Medical Interventions within the CAMI Labex. It aims at unifying data acquired from different surgical trainers and procedures for collaborative research. In this paper, we propose a generic structure for multimodal dataset that allows faster development and easier processing. With such formalization, our objective is to go beyond the state of the art by sharing various types of data between international institutions and merge methodological approaches for better detection and understanding of surgical workflows.

Introduction

For the past decade, the medical community has shown a growing interest in biomedical international competitions, named challenges. Since 2007, more than 120 challenges have been proposed on the grand-challenge platform 1 where various research fields are addressed such as image segmentation and registration, anatomical structure localization and tracking or surgical workflow detection and understanding. Through such platform, multiple types of data are shared going from point cloud depth image acquisition to CT or MRI segmented images. These challenges share the same goal: use pre-clinical and clinical data in order to validate and evaluate methodologies that are developed for biomedical analysis purposes. Focusing on surgical workflow detection and understanding, various datasets have been released over the last years, where multiple types of data are accessible.

For each dataset, data representation and storage are unique because of the acquisition setup and the personal requirements. However, this sharing model is not suitable for collaborative research due to the heterogeneous representation of the same data type. Moreover, such approach does not promote the use of multimodal data for multiscale processing because of the complex integration of various data types and formats.

In this paper, we address the need of a standard formalism for multimodal data representation and storage. By taking into account the available datasets in the M2CAMI community, we propose a generic structure and a first implementation for data conversion to a generic format that is readable and usable in the different applications developed into the M2CAMI group (fig. 1).

Methods

In the M2CAMI group, data acquisition and analysis are oriented towards surgical workflow detection and understanding. In order to build a unified framework to convert data into a generic representation, we proposed a two-step approach. The first step was the inventory of the different available data into the working group. Then, in the second step, we described a common formalism for data representation and storage.

From the M2CAMI community, two datasets are publicly available: NeuroSurgicalTools [START_REF] Bouget | Detecting Surgical Tools by Modelling Local Appearance and Global Shape[END_REF] and Cholec80 [START_REF] Twinanda | EndoNet: A Deep Architecture for Recognition Tasks on Laparoscopic Video[END_REF] where the latter has been already used for a previous scientific challenge 2 . To generalize our work, we used also three other available datasets: JIGSAWS [START_REF] Gao | The JHU-ISI Gesture and Skill Assessment Working Set (JIG-SAWS): A Surgical Activity Dataset for Human Motion Modeling[END_REF] as well as acquisitions performed during previous CAMI works [START_REF] Wolf | Quantification de la qualité d'un geste chirurgical à partir de connaissances a priori[END_REF][5].

Data inventory

These datasets contain various data types that can be sorted into four distinct categories. Videos are (fig. 2a) automatically captured from the surgical field by recording the endoscopic point of view, or from the entire operating room with additional cameras. They can also be expressed as a set of images. Kinematic data (fig. 2b) is automatically captured from robotic systems or additional tracking devices to describe the surgical instrument motions over the time. Transcription data (fig. 2c), or annotations, is most of the time manually generated by human observers and describes the different actions, instruments or events taking place during the procedure. Evaluation data (fig. 2d) quantifies performance of the surgeon to achieve a clinical task or procedure. It can be automatically calculated from surgical trainers or manually generated by human experts and is necessary for surgical workflow referenced-based evaluations. As an extension to these four categories, unusual clinical data types are emerging and combine annotations with kinematics such as human body pose annotations for medical staff analysis [START_REF] Kadkhodamohammadi | Articulated clinician detection using 3D pictorial structures on RGB-D data[END_REF]. To cover the full spectrum from training to interventional clinical data available into the M2CAMI group, we developed an inventory form based on a previous formalism [START_REF] Jannin | Model for defining and reporting reference-based validation protocols in medical image processing[END_REF] to make a list of the different data and structures available.

2 http://camma.u-strasbg.fr/m2cai2016/

Generic data representation

As previously mentioned, a same data type can be represented in different ways. For instance, video is a set of images that is embedded into a streaming container. However, depending on the format, the encoder and the output container it can be difficult to use it for processing purpose. Thus, our objective is to define a standard representation that rely on open source protocols (e.g. x265 for video codec, Boost.PropertyTree for configuration file) to ease broadcast of our developments. This standard representation can be developed at two different scales. At the global level, we provide guidelines and tools to uniformly structure dataset in order to ease sharing, processing and hard data storage. Additionally, standardized header files are integrated into datasets to regroup data properties for conversion management. At a second level, we propose a C++ language-based container that stores data synchronously into memory for processing (e.g. as an extended map for data series).

The main advantage of the presented structure is to be easily extended to add new types of data. The only assumption we rely on is the data synchronization where for each timestamp in the timeline there is at least one sample from each data type available in the dataset.

Conclusion and Perspectives

At the current stage, data conversion seems feasible to provide larger datasets for each partner of the M2CAMI group. Relying on open source protocols, the current implementation is shareable between members and improvements can be easily undertaken. The added value of the current work will be emphasized by the connection of various developments coming from different teams, including surgical context detection [START_REF] Twinanda | EndoNet: A Deep Architecture for Recognition Tasks on Laparoscopic Video[END_REF] that will provide entry frame for instrument tracking [START_REF] Agustinos | 2D/3D Real-Time Tracking of Surgical Instruments Based on Endoscopic Image Processing[END_REF]. The output information from the tool tracking will be used for gesture recognition [START_REF] Despinoy | Unsupervised Trajectory Segmentation for Surgical Gesture Recognition in Robotic Training[END_REF] in order to, at the end, obtain a complete comprehension of the surgical context. These are the first steps towards generation of new decision-making systems where multimodal data allows to capture and understand surgical events for training, teaching and surgical assistance purposes.

1Figure 1 :

 1 Figure 1: Pipeline for data conversion into a standardized output for M2CAMI applications.

( a )

 a Video data. (b) Kinematic data. (c) Transcription data. (d) Evaluation data.

Figure 2 :

 2 Figure 2: Multimodal data of surgical workflow available into the M2CAMI collaborative research group.


Barbara Leśniewska

Anthony W Coleman

Yannick Tauran

Florent Perret

Kinga Suwińska

Pseudopolymorphs -a variety of self-organization of para-sulphonato-calix[8]arene and phenanthroline in the solid state

The paper presents results concerning construction of inclusion complexes of para-sulphonato-calix[8]arene with 1,10phenanthroline. Structures of crystalline host-guest complexes were determined by means of X-ray diffraction. Intermolecular interactions were analyzed and the organization of ions and molecules in the crystal lattice was described.

INTRODUCTION

Supramolecular chemistry is an interdisciplinary field of science that deals with the design, formation and exploration of complex chemical systems formed from smaller building blocks joined together by non-covalent intermolecular interactions. [START_REF] Steed | Supramolecular Chemistry[END_REF] However, small here can be a relative term as supramolecular systems can vary in size from clathrate hydrates, [START_REF] Sloan | Clathrate hydrates of natural gases[END_REF] and Werner clathrates, [START_REF] Jóna | [END_REF] through organic systems, [START_REF] Sadjadi | Organic Nanoreactors: From Molecular to Supramolecular Organic Compounds[END_REF] metal organic frameworks, [START_REF] Macgillivray | Metal-Organic Frameworks: Design and Application[END_REF] to micellar and liposomal structures, [START_REF] Souza | Liposomes: Methods and Protocols (Methods in Molecular Biology)[END_REF] to proteins, [START_REF] Cortajarena | Protein-based Engineered Nanostructures[END_REF] or DNA [START_REF] Seeman | Structural DNA Nanotchnology[END_REF] or even virus assemblies. [START_REF] Mateu | Structure and Physics of Viruses: An Integrated Textbook (Subcellular Biochemistry)[END_REF] Yet strangely the simple assembly of water can generate extremely large and complex supramolecular assemblies. [START_REF] Franks | Water a matrix of life[END_REF] One major task is the search for new molecular receptors, and to acquire knowledge about molecular recognition involving selective binding between a receptor and a guest or ligand, and resulting in host-guest complex formation. Molecules will recognize each other when there are efficient intermolecular interactions between them, such as preferred spatial relationships or the complementarity of the electrostatic fields of their molecules. Suitably modified receptors may exhibit supramolecular reactivity and the spontaneous formation of complex well-defined structures such as, for example capsules, pseudo-capsules, columns and chains, that is, to self-assemble, into autoassociated complex supramolecular systems. [START_REF] Albrecht | Chemistry of Nanocontainers[END_REF] In terms of organic systems, artificial molecular receptors include the cyclodextrins, [START_REF] Jin | Cyclodextrin Chemistry: Preparation and Application[END_REF] crown ethers, cryptands and spherands, [START_REF] Hiraoka | Crown Ethers and Analogous Compounds[END_REF] cyclic polyamines, 14 and cucurbiturils [START_REF] Kim | Cucurbiturils: Chemistry, Supramolecular Chemistry and Applications[END_REF] and the class of systems based on the calix[n]arenes. [START_REF] Gutsche | Calixarenes : An Introduction : Edition 2 RSC[END_REF][START_REF] Boinski | [END_REF] One of the most important tasks of supramolecular chemistry is to attempt to understand the construction and functioning of self-assembled structures systems in nature. This may be achieved through their modeling by means of simpler biomimetic systems, for example enzyme models, [START_REF] Kirby | From Enzyme Models to Model Enzymes[END_REF] or artificial membranes. [START_REF] Catalá | Membrane Organization and Lipid Rafts in the Cell and Artificial Membranes[END_REF] The panoply of processes of molecular recognition and complex formation occurring in nature include, enzymatic reactions in living organisms, [START_REF] Rehm | Bionanotechnology: Biological Self-assembly and its Applications[END_REF] distinguishing fragrance by olfactory receptors and synthetic analogues, [START_REF] Kaushik | Nanobiotechnology for Sensing Applications: From Lab to Field[END_REF] active transport of compounds through the membrane of the cell walls, [START_REF] Krämer | Membrane Transport Mechanism: 3D Structure and Beyond[END_REF] (indeed back transport gives rise to multi-drug resistance [START_REF] Boumendjel | ABC Transporters and Multidrug Resistance[END_REF] ) the formation of the DNA double helix, [START_REF] Watson | The Annotated and Illustrated Double Helix[END_REF] and virus assembly. [START_REF] Mateu | Structure and Physics of Viruses: An Integrated Textbook (Subcellular Biochemistry)[END_REF] Recent work by ourselves in the area of the biochemistry of the para-sulphonato-calix[n]arenes, [START_REF] Perret | [END_REF]26 has shown that there are considerable differences between the four, six and eight membered macrocycles. In particular para-sulphonatocalix [8]arene has been shown to be a more effective endonuclease inhibitor than the smaller analogs 27 and interestingly shows the ability to transport across the CaCo-2 cell model of the human gut. 28 Thus structural information on the binding of para-sulphonato-calix [8]arene to substrates will have considerable relevance in the biological and pharmacological fields, but also for the construction of novel solid-state systems. The aim of this work was to obtain self-organizing systems of para-sulphonato-calix [8]arene (C8S) with 1,10phenanthroline (Phen) and to undertake structural analysis of the obtained solid-state supramolecular assemblies by X-ray diffraction on single crystals. The obtained structural data were the basis for a detailed analysis of the conformation of the para-sulphonato-calix [8]arene receptor and for analyzing interactions between the host and guest species. Also the analysis of the organization of ions and molecules in the crystal lattices has been carried out, allowing us to observe pseudo-polymorphism in two of the systems, where the core structure varied in form, as a result of the differences in solvation. Such behavior is common in large bio-molecular structures, but less well known in molecular supramolecular complexes. The ligand, 1,10-Phenanthroline is a flat and rigid molecule which is not subject to conformational changes, but which can influence conformational changes for other flexible molecules which co-create the supramolecular complex. Previously we have studied solid state complexes of 1,10-phenanthroline with para-sulphonato-calix [4]arene 29 and also para-sulphonato-calix [8]arene with flexible guest molecules 1,2-bis(4-pyridyl)-ethane and 1,3-bis(4-pyridyl)propane. 30

EXPERIMENTAL SECTION Crystal growth

Synthesis of para-sulphonato-calix[8]arene

para-Sulphonato-calix [8]arene was syntheised by the method previously used for para-sulphonato-calix [4]arene by Coleman. 31 The physical properties, in particular, the ES/MS accord with the structure, it should be noted that this method generates calix [8]arene-para-sulphonic acid, however the pKa of the sulphonic acids is around 0 so the compound is fully deprotonated.

Crystal growth

Suitable crystals for X-ray diffraction were grown by slow diffusion of a solution of the Phen in ethanol and methanol at 4 equiv. into 1 mL of a 100 mg mL -1 solution of parasulphonatocalix [8]arene in water. The crystallization experiments were carried out at 20 °C in sealed tubes. The solvent ratios were varied from 0.5 Alcohol:1 Water to 2 Alcohol:1 Water to screen crystallization conditions. The crystals are colourless.

X-ray structure determination

The X-ray data were collected at 100 K on a Nonius Kap-paCCD diffractometer using MoKα radiation for complex 1 and on an Agilent SuperNova diffractometer equipped with a CuKα micro-focus X-ray source for complex 2 and 3. Structures were solved by direct methods with SHELXS-97 [START_REF] Sheldrick | SHELX97: Program for Crystal Structure Analysis[END_REF] and refined against F 2 with full-matrix least-squares using SHELXL-97 [START_REF] Sheldrick | SHELX97: Program for Crystal Structure Analysis[END_REF] . Hydrogen atoms were calculated at their idealized positions and were refined as riding atoms with isotropic thermal parameters based upon the corresponding bonding carbon atom (U iso = 1.2U eq , U iso = 1.5U eq for CH 3 and OH hydrogens). Hydrogen atoms of methyl and hydroxyl groups were refined in geometric positions for which the calculated sum of the electron density is the highest (rotating group refinement). Where possible hydrogen atoms of amino groups and water molecules were located on Fourier difference maps and refined with positional parameters. Because of the poor diffracting quality and disorder, the R and wR of structures 1 and 2 are high. Crystal structures are deposited with the Cambridge Crystallographic Data Centre as CCDC 1437840-1437842. Crystal data for 1, 2 and 3 are shown in Table 1.

RESULTS AND DISCUSSION

Complex 1 Crystals of the complex 1 were obtained from a methanolwater crystallization medium. The asymmetric part of the unit cell contains one C8S octaanion, eight monoprotonated phenanthroline cations and 15 water molecules (Figure 1). The exact charge on the para-sulphonato-calix [8]arene anion is subject to debate, as one or more of the phenolic residues may be deprotonated, [START_REF] Danylyuk | [END_REF] and as with cationic complexes of DNA, [START_REF] Bagchi | Water in Biological and Chemical processes[END_REF][START_REF] Soler-López | [END_REF] at least some of the water molecules may be protonated.

Figure 1. The asymmetric unit of the complex 1.

The structure is characterized by a high degree of disorder. Six Phen ions are disordered, four of which are in two orientations Y/O, V/M, T/L, K/Q, with site occupation factors, (s.o.f.) = 0.5 each, and two other Phen R and J cations are located on the center of symmetry, each of the two symmetricallydependent orientation of these two cations is half-occupied. Additionally five of the eight calixarene sulphonate groups and almost all water molecules are disordered. The octaanion of para-sulphonato-calix [8]arene adopts a new, previously undescribed conformation (Figure 2). This conformation is different from the pleated loop and double cone which have been highlighted as two extreme conformations for eight-membered calixarenes. 36 One of the phenolic rings H of C8S lies flat in the plane of the macrocyclic ring and its sulphonate group pointing toward the inside of the macrocycle. The two neighboring to it phenolic rings A and G are oriented anti-parallel one to another and are perpendicular to the plane of the macrocycle, the remaining five phenolic rings are inclined to the plane of macrocycle with sulphonate groups facing outwards and forming a folded loop. para-Sulphonato-calix [8]arene in this untypical conformation has the cavity inside of macrocycle blocked by self-inclusion of the sulphonate group of the phenolic ring H (Figure 3). Angles between planes of phenolic rings to the reference plane of calixarene (the plane defined by eight carbon atoms of methylene groups of C8S) are presented in Table 2. 2) Å] interaction between carbon atoms of the methylene group of C8S and the central ring of Phen (Figure 5a and Figure 1). Selected interactions present in the structure of complex 1 are presented in Table S1 (supplementary information). S1). A similar stacked self-organization of phenanthroline cations was previously observed in crystal structures of complexes of smaller para-sulphonatocalix [4]arene with phenanthroline. 29,37 Figure 6. The infinite stack formed by Phen cations in structure of complex 1.

Each anion of C8S interacts directly with two neighboring calixarenes (Figure 7) through π-π interactions between two adjacent aromatic rings of C8S with distances between centroids: 3. The crystal packing of ions and molecules in the complex 1 is close to layered. Stacks and hexamers of phenanthroline are separated from each other by C8S anions (Figure 8). The structure is quite highly hydrated but the conventional hydrophilic or hydrophobic layers cannot be unambiguously distinguished. Similar crystal packing was observed in the already mentioned structures of complexes formed by parasulphonato-calix [4]arene with phenanthroline 29 , but the current structure of complex 1 is more "dense" due to the presence of stronger interactions. The self-assembly of C8S anions and Phen cations, in the crystal lattice of complex 1 leads to the formation of cages with approx. dimensions of 16 × 6 × 6 Å. The solvent accessible volume is 733 Å 3 , which is 8.3% of the volume of the unit cell (Figure 9). The two partial cones of calix [8]arene anion are oriented antiparallel to each other (Figure 11b) and each partial cone is formed by phenolic rings A, C, and D, while phenolic ring B is inverted with an angle of -36.73 (2)° to the reference plane of calixarene (the plane defined by the carbon atoms of methylene groups of C8S). Phenolic rings located between the two partial cones are at a considerable distance from each other and thus a cavity inside the macrocycle is formed. As a consequence, the conformation of para-sulphonato-calix [8]arene in complex 2 can be also considered as a distorted pleated loop and compared to this conformation 36 (Figure 13). Dihedral angles between the planes of the phenolic rings of C8S and the reference plane of calixarene are presented in Table 3. 8) Å] between oxygen atoms of the phenolic hydroxyl groups and by aromatic, electrostatic and hydrophobic ions between C8S anion and the surrounding Phen cations (Figure 14). Such conformation of C8S anion as described above results in a presence of two cone-like cavities and additional creation of four "folds" formed between twisted phenolic groups. Cations Phen X and U are located in these "folds", while cations Phen Z are located within the partial cone cavities of C8S (Figure 14).

Table 3. Dihedral angles between the planes of the phenolic rings of C8S and the reference plane of macrocycle (the plane defined by the carbon atoms of methylene groups of C8S) in the crystal of complex 2.

Ring

Figure 14. Host-guest interactions in complex 2: two cations Phen Z (orange) located in partial cone cavities (the second Phen Z cation is located in the second cavity of C8S and is invisible in this orientation) and cations Phen X (blue) and U (violet) located in "folds" of the calixarene anion.

Anion of C8S, four cations of Phen (two Phen Z and two Phen X), and two molecules of ethanol form an inclusionadditive complex (Figure 15). The depth of inclusion of cation Phen Z within the partial cone of C8S measured as the distance between the centroid of included phenanthroline protonated ring and the reference plane of C8S is 3.782(6) Å, the dihedral angle between the plane of Phen Z and the reference plane of C8S is 43.89(5)°. Cation Phen Z is not located parallel to the axis of the partial cone, as it was observed in the case of para-sulphonatocalix[4]arene 29 , but is tilted within the cavity to allow π-π interaction [estimated plane-to plane distance of 3.4 Å] between the phenanthroline protonated ring and ring C of C8S (similarly to the complexes of para-sulphonato-calix [6]arene with Phen). 40 The structure of complex 2 show a typical bilayer packing. One layer consists of the columns of C8S anions, cations Phen X and Z and ethanol molecules involved in inclusionaddition complex formation; the second layer consists of the remaining three Phen cations forming the infinite stacks and water molecules. In this structure the distinct hydrophilic and hydrophobic layers are not observed. Most of the water molecules present in the crystal, are located near the sulphonate groups of C8S and all of them are inside the elongated centrosymmetric S-shaped cages of approx. dimensions 6 × 8 × 22 Å and running parallel to Phen stacks (Figure 17b).

Complex 3

Further evaporation of solvent from the solution containing crystals of complex 2 led to crystallization of new crystalline phase named complex 3. The asymmetric part of the unit cell consists one C8S anion in which four sulphonate groups are disordered, 11.5 phenanthroline cations and 24 water molecules of which 7 are disordered (Figure 18). Also three Phen cations are disordered; the one marked as Z/L was found in two orientations with s.o.f. = 0.69 and 0.31, respectively for Z and L, the second one occurs in three orientations V/M/K rotated relative to each other in the plane of the molecule and with the site occupancy factors of 0.34; 0.33 and 0.33, respectively for V, M and K, and the third disordered Phen cation denoted as N is located on a crystallographic center of inversion and is half-occupied (Figure 19). 4. In each partial cone of C8S one cation of Phen is included -X in one and Y in the second partial cone (Figure 21). . However, in contrary to complex 2 where the protonated rings of included Phen cations was found to be inserted within the cavities of C8S, in complex 3 the nonprotonated heterocyclic rings of Phen are located within the partial cones. Such type of complex formation was found in two crystal structures of para-sulphonato-calix [4]arene with Phen. 29 The layers formed by the inclusion polymers C8S/Phen (X, Y) are arranged alternately with layers constructed of other Phen cations. The entire structure is strongly hydrated, therefore, the conventional hydrophilic and hydrophobic layers cannot be distinguished.

CONCLUSIONS

As a result of co-crystallization of para-sulphonatocalix [8]arene with phenanthroline from an alcohol/water solvent mixtures crystals of two pseudopolymorphs were obtained. The term pseudopolymorph is emphasized by the variation in the core helical structural generated by a small difference in the solvation, such behavior is more common for large biomolecules than the smaller molecules typical of supramolecular chemistry. All the described complexes are organic salts. Structural diversity of obtained crystals is caused by the wealth and competitiveness of mutual interactions involved in the complex formation. In all three structures calixarene anions interact with Phen cations mainly through π-π and C-H•••π interactions. Complex 1 does not form inclusion complex, while in complexes 2 and 3 inclusion in the partial cone cavities of the C8S anion takes place. In each of the presented crystal structures occurs an intercalation of Phen cations between C8S anions: in complex 1 between neighboring C8S anions single cations and hexamers of Phen are located. In complex 3 dimers are present. Furthermore infinite stacks of Phen exist in all the three complexes. On the basis of the obtained structural data it can be stated that para-sulphonatocalix [8]arene/1,10-phenanthroline systems are able to spontaneous generation of well-defined, structured supramolecular architectures. The obtained results show structural diversity of supramolecular assemblies which can be obtained using these compounds, a richness of their supramolecular chemistry, and demonstrate that they are able to construct supramolecular architectures of high complexity. The information gained about the crystal and molecular structures of para-sulphonatocalix[n]arenes complexes with aromatic amines can contribute significantly to the development of supramolecular chemistry and crystal engineering -and more importanatly to our understanding of how para-sulphonato-calix [8]arene interacts with biopolymers including endonucleases, that is to lead to a better understanding of an analogy between para-sulphonatocalix [8]arene and DNA. Current work is underway to study how far this analogy extends.

CrystEngComm Accepted Manuscript

Published on 31 October 2016. Downloaded by Tokyo Daigaku on 01/11/2016 10:54:54.

View Article Online DOI: 10.1039/C6CE01753C

Final

  R indices [I > 2σ(I)] R = 0.220, wR = 0.485 R = 0.137, wR = 0.149 R = 0.078, wR = 0.246 R indices (all data) R = 0.321, wR = 0.524 R = 0.371, wR = 0.375 R = 0.098, wR = 0.261

Figure 2 .

 2 Figure 2. The unusual conformation of the para-sulphonatocalix[8]arene in the complex 1; (a) view from above showing intermolecular O-H•••O hydrogen bonding, (b) side view.

Figure 3 . 1 .Table 2 .

 312 Figure 3. Self-Inclusion of one of the sulphonate group of C8S in complex 1.Table 2. Dihedral angles between the planes of the phenolic rings of C8S and the reference plane (the plane defined by the carbon atoms of methylene groups of C8S) in the crystal of complex 1.RingAngle (°) Ring Angle (°) A

Figure 4 .

 4 Figure 4. Cations of Phen (X -blue, Y -red, J -green, Vpink, T -purple and K -yellow) located in the immediate vicinity of the C8S anion and their influence on the calixarene conformation.Conformation of the calixarene anion in the crystal of complex 1 prevents the formation of an inclusion compound. Phen

Figure 5 .

 5 Figure 5. Phen cations intercalated between two adjacent C8S anions; (a) intercalation of a single cation Phen J (b) intercalation of Phen 6 hexamer (Phen cations are disordered, only one orientation of cations V/M, T/L and K/Q is shown for clarity). Phen cations K, T and V form a centrosymmetric π-stacked Phen 6 hexamer V-T-K-K-V-T where the interplanar distance for K•••K is 3.72(4) Å and estimated distances for K•••T and T•••V interactions are 3.4 and 3.2 Å, respectively (Figure 5b). The hexamer is located between two anions of C8S. External Phen ion V interacts with calixarene by C-H•••π [3.61(2) Å] interaction present between the carbon atom of the methylene group of C8S and the central ring of Phen V as well as by C-H•••O [3.06(2) Å] hydrogen bonding present between the carbon atom of the nonprotonated ring of Phen and oxygen of sulphonate group of calixarene, and also forms π-π interaction (with distances between centroids: 4.48(2) and 4.62(2) Å) between the aromatic ring of C8S and central and protonated rings of Phen cation V. The Phen cation T interacts with the calixarene anion by C-H•••O [3.21(3) Å] interaction between the carbon atom of no protonated Phen ring and the oxygen atom of a sulphonate group of C8S, and through C-H•••O 3.24(4) Å interaction between the carbon atom of the central ring of Phen and the oxygen atom of the hydroxyl group of C8S. Ion Phen K does not interact directly with calixarene. Other phenantroline cations marked with symbols Y, X, Z, W, R create infinite stack (Figure 6) stabilized by π-π interactions, the estimated distance between Phen cations in the stack is 3.4 Å. Phenanthroline cations forming the stack interact with

  69(2) and 3.98(2) Å and by O-H•••O hydrogen bonding [2.59(3), 2.82(3), 2.74(2) Å] in which H-donors are oxygen atoms of hydroxyl groups of one C8S and acceptors are oxygen atoms of sulphonate groups of adjacent C8S anions, as well as by C-H•••O interaction [3.23(3) Å] between the carbon atom of methylene group of one calixarene and the oxygen atom of the sulphonate group of adjacent calixarene. C8S anions are organized in double tapes shown in (Figure7). Between the adjacent tapes of calixarenes no interactions are observed.

Figure 7 .

 7 Figure 7. Adjacent anions of C8S interacting via π-π, O-H•••O and C-H•••O interactions -construction of a double tape.

Figure 8 .

 8 Figure 8. Packing of ions and molecules in the crystal of complex 1; (a) view along [010] direction; (b) view along [11-1] direction.

Figure 9 . 2

 92 Figure 9. Solvent accessible cages in the crystal lattice of complex 1; view along the [100] direction. Water molecules are omitted for clarity.The cages are filled with water molecules. Three walls of cage are formed by hydrophobic parts of Phen cations, one wall is formed by the sulphonate groups of C8S and protonated rings of Phen cations.Complex 2Crystals of complex 2 were obtained from an ethanol-water crystallization medium. The asymmetric part of the unit cell (Figure10) contains half of the C8S anion in which three sulphonate groups are disordered, five monoprotonated cations of phenanthroline, one molecule of ethanol, and 10.5 water molecules. In the structure one cation Phen Y/W is disordered with site occupation factors (s.o.f.) equal to 0.79 and 0.21 for orientations Y and W, respectively; 4.5 out of 10.5 water molecules are also disordered.

Figure 10 .Figure 11 .Figure 12 .

 101112 Figure 10. The asymmetric unit of the complex 2. Part of molecule which is marked in fuchsia is symmetry related.The presence of water molecules and an oxygen atom of sulphonate group of C8S near the nitrogen atoms of Phen at distances typical to hydrogen bonds indicates that all phenanthroline molecules are monoprotonated and bearing a total charge +5. Consequently, the charge balance in the unit cell indicates deprotonation of two hydroxyl groups of calixarene in addition to deprotonation of eight sulphonate groups. It is evidenced by the very short distances between oxygen atoms of the phenolic groups O4C and O4D [2.477 (5) Å] which indicates the presence of a hydrogen bond supported by electric charge. C8S anion is located on a crystallographic center of inversion and adopts centrosymmetric inverted double partial cone conformation (Figure11) similar, but not identical, to these observed for the structures [LUKBUP]38 and [QULJUD]39 [QULJUD01] 39 , [QULKAK] 39 and [VERBEA]36 . Superposition of conformations of para-sulphonatocalix[8]arene in complex 2 and VERBEA is shown on Figure12.(a)

Figure 13 .

 13 Figure 13. The superposition of pleated loop conformation of C8S in complex 2 (red) and [VERBAW] 36 (gray); (a) top view, (b) side view.

Figure 15 .

 15 Figure 15. Inclusion-additive complex formed by the anion of C8S, four phenanthroline cations and two molecules of ethanol (green).

Figure 16 .Figure 17 .

 1617 Figure 16. (a) Columns of inclusion-additive complexes comprising two dimers of Phen and two molecules of ethanol forming an inclusion-additive polymer; (b) π-π interaction between cations Phen X and Phen Z in the dimer.The height of the repeating unit in such an inclusion-additive polymer is 11.94 Å Similar structural motifs have been observed in crystals of para-sulphonato-calix[6]arene with Phen40,41 but in this case the pseudo-capsules are smaller and are able to accommodate in a single dimer of Phen only. The three other cations Phen U, V and Y/W present in the crystal of complex 2 form infinite stacks stabilized by π-π interactions along the [001] crystallographic axis. The estimated distances between planes of Phen molecules are the same and all equal to 3.4 Å. The stacks of Phen and form together with water molecules present in the crystal separate layers in the structure (Figure17). Phen ions belonging to the infinite stacks interact with calixarenes through the C-H•••O (Phen U) and π-π (Phen Y/W) interactions. A similar stacking self-organization of phenantroline cations takes place in the structures of parasulphonato-calix[4]arene with Phen 29,37 and in the complex 1. In the crystal of complex 2, no direct interactions between C8S anions are observed.

Figure 18 .Figure 19 .

 1819 Figure 18. The asymmetric unit of the crystal 3.

10

 10

Figure 20 .

 20 Figure 20. Superposition of pseudo-centrosymmetric C8S anion in complex 3 (magenta) and centrosymmetric C8S anion found complex 2 (blue); (a) top view, (b) side view. Partial cones of C8S are formed by three phenolic rings A, B, C, and E, F, G, while the phenolic groups of rings D and H between the two partial cones are directed outside of the macrocyclic ring. The dihedral angles between the planes of phenolic rings to the reference plane of C8S are listed in Table4.

Figure 21 .

 21 Figure 21. An inclusion host-guest complex formed by the C8S anion and two cations X and Y of phenanthroline.The depth of inclusion of Phen cations measured as the distance between centroids of included heterocyclic monoprotonated rings of phenantroline and reference plane of C8S is 3.926(5) and 3.979(5) Å for Phen X and Y, respectively, and the dihedral angles between the reference plane of calixarene and the planes of Phen X and Y are 40.87(3) and 46.82(3)°, respectively. Similarly to the complex 2, Phen cations are not included vertically in the calixarene cavities but are tilted within the cavity to allow π-π interaction between Phen X and ring E of C8S [estimated plane to plane distance of 3.4 Å], and between Phen Y and ring A of C8S [estimated plane to plane distance of 3.3 Å]. However, in contrary to complex 2 where the protonated rings of included Phen cations was found to be inserted within the cavities of C8S, in complex 3 the nonprotonated heterocyclic rings of Phen are located within the partial cones. Such type of complex formation was found in two crystal structures of para-sulphonato-calix[4]arene with Phen.29 Additionally, the inclusion complex is stabilized by C-H•••π interactions [3.342(2), 3.689(3) and 3.475(2) Å] between carbon atoms belonging to the heterocyclic nonprotonated rings of Phen and aromatic rings of C8S, and C-H•••O hydrogen bonds [3.180(5), 3.205(8), 3.180(7), 3.45(1), 3.34(1) Å] in which the proton donors are atoms belonging to the protonated rings of Phen cations and acceptors are the sulphonate oxygen atoms of the host molecule. Protonated nitrogen atoms of included Phen cations are also involved in N-H•••O hydrogen bonds [2.901(4) and 2.760(5) Å] to water molecules. Selected interactions present in crystals of complex 3 are listed in the Table S3 (supplementary information). Each of the included Phen cations form a π-stacking dimers (X•••X) and (Y•••Y) to the related by crystallographic centers of symmetry inclusion complexes (Figure 22b and c), and thereby forming one-dimensional inclusion polymer (Figure 22a) containing two types of pseudo-capsules of estimated height 9.7 and 10.1 Å for capsules containing dimers (X•••X) and (Y•••Y), respectively.

Figure 22 .

 22 Figure 22. (a) Inclusion polymer formed by C8S, Phen X and Phen Y in the crystal of complex 3; (b) a dimer formed by cations Phen X; (c) dimer formed by cations Phen Y.Chains of polymeric capsules in complex 3 are arranged parallel to the [011] crystallographic direction (Figure23).

Figure 23 .Figure 24 .

 2324 Figure 23. Crystal packing of ions in complex 3, the view along the [011] crystallographic direction. Water molecules are omitted for clarity.

Figure 25 .

 25 Figure 25. The stack composed of cations Phen W, U, T, R, Q, Z, V, N in complex 3; W -blue, U -white T -green, R -blue, Qpink, Z -yellow, V -magenta, N -purple. The dimer Phen (O•••P) shown on (Figure 24b) consists of two crystallographically independent, almost parallel cations of phenantroline with the estimated distance between the planes of Phen of 3.4 Å. Each cation of Phen participates in one C-H•••π interaction [3.360(2) and 3.284(3) Å for O and P, respectively] between the carbon atom of the heterocyclic protonated ring of Phen and an aromatic ring of one of the surrounding calixarene. Intercalation of dimer Phen (O•••P) is also stabilized by several C-H•••O interactions [range 3.03(2)-3.34(2) Å] to sulphonate groups of C8S. The remain-

Figure 26 .

 26 Figure 26. The crystal packing of ions in the crystal of complex 3, view along the [11-2] crystallographic direction. Phen W, U, T, R, Q, Z, V, N cations are marked in purple. Water molecules are omitted for clarity. Phenantroline cations forming the stack are located closed to calixarene anions and they interact with them by numerous N-H•••O, C-H•••O, C-H•••π and π-π interactions. A similar selforganization of Phen cations in stacks was observed in the structures of complexes of para-sulphonato-calix[4]arene with Phen29,37 and complexes 1 and 2 described above. The structure of complex 3 represents the layered-columnar type of packing (Figure27).

Figure 27 .

 27 Figure 27. The crystal packing of ions in the crystal of complex 3: (a) view along the [100] crystallographic axis; (b) view along the [010] crystallographic axis. Water molecules have been omitted for clarity.

Table 1 . Crystal data for 1, 2, 3.

 1

	Compound	Complex 1			Complex 2			Complex 3
	Molecular formula	C 56 H 40 O 32 S 8	8-•8C 12 H 9 N 2	+	C 56 H 38 O 32 S 8	10-•10C 12 H 9 N 2	+	C 56 H 36 O 32 S 8	11.5-•11.5C 12 H 9 N 2	+
		•15H 2 O			•2C 2 H 5 OH•21H 2 O		•24H 2 O
	Formula weight	3201.30			3761.94			7987.31
	Temperature, K	100(2)			100(2)			100(2)
	Wavelenght, Å	0.71073			1.5418			1.5418
	Space group	P	1			C2/c			P	1
	a, Å	18.0000(9)			34.8241(9)			14.8749(4)
	b, Å	18.933(1)			11.9401(2)			20.9698(8)
	c, Å	28.187(2)			41.747(1)			30.2225(9)
	α, °	76.131(2)			90			97.318(3)
	β, °	71.402(3)			98.010			99.924(2)
	γ, °	86.574(3)			90			95.529(3)
	Volume, Å 3	8837.5(9)			17189.2(7)			9142.7(5)
	Z	2				4			1
	Density (calculated) g/cm 3	1.203			1.454			1.450
	Absorption coefficient, mm -1	0.179			1.773			1.712
	Crystal size, mm	0.60 × 0.35 × 0.28		0.25 × 0.15 × 0.10		0.40 × 0.30 × 0.25
	Ɵ range for data collection,°	2.76 -18.84			4.55 -72.04			3.42 -71.38
	Reflections collected	48332			56227			67527

Table 4 . Dihedral angles between the planes of the phenolic rings of C8S and the reference plane of the macrocycle (the plane defined by the carbon atoms of methylene groups of C8S) in the crystal of complex 3.

 4

	Ring	Angle (°)	Ring	Angle (°)
	A	35.91(4)	E	33.51(4)
	B	28.70(4)	F	31.96(4)
	C	72.44(4)	G	67.26(4)
	D	-36.31(4)	H	-38.68(4)

Published on 31 October 2016. Downloaded by Tokyo Daigaku on 01/11/2016 10:54:54. View Article Online DOI: 10.1039/C6CE01753C

13

The scientific work was funded by the National Science Centre, number of decision-DEC-2013/11/N/ST5/01920.

AUTHOR INFORMATION

E-mail: blesniewska@ichf.edu.pl E-mail: k.suwinska@uksw.edu.pl E-mail: anthony.coleman@univ-lyon1.fr E-mail: yannick.tauran@univ-lyon1.fr E-mail : florent.perret@univ-lyon1.fr ACKNOWLEDGMENTS


E K Stephens

Y Tauran

A W Coleman

M Fitzgerald

Structural requirements for anti-oxidant activity of calix[n]arenes and their associated anti-bacterial activity

Treatment of neural cells with calix[n]arenes featuring sulphonate moieties and linked to Ag nanoparticles results in reduced reactive species. For gram+ bacteria there is an inverse correlation between anti-bacterial activity and ROS reduction whereas for gram-bacteria only calix[6]arenes bearing O-alkyl sulphonate functions act as ROS inhibitors and anti-bacterial agents.

Injury to the central nervous system is characterized by altered Ca 2+ flux and associated increases in reactive species, thought to be triggered by excess glutamate release 1, 2 . If reactive species are not controlled by endogenous anti-oxidants, oxidative stress ensues, and this is a feature of traumatic brain and spinal cord injury as well as secondary degeneration [3][4][5] . Anti-oxidants have been used in preclinical studies to treat injury to the central nervous system, but new agents are needed in order to generate complementary combinations of anti-oxidant therapies to control multiple facets of oxidative stress 3 . Furthermore, antioxidants often suffer from lack of solubility and resultant poor bioavailability 6,7 , limiting their clinical usefulness. Calix [4]arenes have been used as therapeutic delivery systems for anti-oxidants and have also been shown to have inherent anti-oxidant properties 8 . As such they have the potential to provide a combinatorial anti-oxidant strategy for neurotrauma. The sulphonated calix [n]arene derivatives all have solubilities above 100mM in water making them attractive as therapeutic agents. The para-sulphonato-calix [n]arenes are well documented for their biological activities, 9 while the silver nanoparticles capped with para-and O-sulphonated derivatives have shown interesting antibacterial activities with selective action against gram+ or grambacteria. 10 However the structural requirements for anti-oxidant and antibacterial activity are unknown, making elucidation of the mechanisms of action problematic. Here we assess the effects of a range of calix[n]arene structures on viability and production of reactive oxygen species (ROS) by neuronal cells stressed with an excitotoxic concentration of glutamate. We also have studied possible association between effects on ROS and anti-bacterial activity, as the presence of radicals and hence ROS is one of the proposed mechanisms for the anti-bacterial action of silver nanoparticles 11 .

Calix[n]arenes were synthesized by literature methods, see SI and the sulphated cyclodextrin is commercially available. (Scheme 1). Calix[n]arenes were linked to Ag nanoparticles (Ag_NP) by direct interaction during the reduction of Ag + to Ag 0 (Scheme S1, SI). The linking of calix[n]arenes to Ag nanoparticles may reduce the strongly negative charge of the calixarene tail groups, thereby aiding in membrane penetration and perhaps increasing antioxidant efficacy, compared to calix [n]arenes not linked to Ag nanoparticles. Toxicity of nanoparticle preparations in biological systems remains an ongoing concern. We therefore assessed the effects of the calix[n]arene and cyclodextrin preparations on viability of neuronal cell that had been stressed with an excitotoxic concentration of glutamate, using Pheochromocytoma (PC12) neuronal-like cells.

Calcein dye was used to selectively stain viable cells. Images were taken at 2 locations in triplicate culture wells at each concentration, using a Nikon Inverted Fluorescence microscope. Viable cells (green) were counted, expressed relative to unit area and mean ± S.E.M calculated. Data were assessed using ANOVA with a significance value of p ≤ 0.05, using SPSS statistical software (IBM). There were no significant differences in PC12 cell viability following glutamate stress and treatment with any of the calix[n]arene or cyclodextrin preparations at 100 µg mL -1 , compared to untreated, glutamate stressed control cells (F=1.42, p=0.12). Choice of calix[n]arene and cyclodextrin concentration was in line with our previously reported studies and higher than concentrations previously shown to exhibit antioxidant activity 8 . Representative images of cells treated with selected preparations demonstrate that treated PC12 cells displayed normal morphology, extending small neurites (Figure 1). Treatment with each concentration of Ag_NP_SC4a resulted in significantly reduced ROS production by PC12 cells (F=5.87, p≤0.0001: note p value for least effective concentration provided), whereas treatments with SC4a not linked to Ag_NP were not effective at reducing ROS (p=0.06, Figure 2A). Efficacy of Ag_NP_SC4b was similarly dependent on linkage to Ag_NP (p=0.001): SC4b alone had no significant effect on ROS (p=0.16).

In contrast, Ag_NP_SC4c and SC4c were both effective at reducing ROS at the tested concentrations (p=0.001, p=0.01 respectively).

Treatment with the SC6 preparations followed a similar pattern (Figure 2B). Reduction of ROS with SC6a required linkage to Ag_NPs (F=3.11, p=0.05). However, SC6b and Ag_NP_SC6b were both effective at reducing ROS (p=0.001, p=0.001 respectively), as were SC6c and Ag_NP_SC6c (p=0.002, p=0.02 respectively). Note that there were some concentration dependent effects following treatment with the SC6c preparations but differences were minor and unlikely to be biologically meaningful. No reductions in ROS were detected following treatment with the SC8 calixarene preparations regardless of linkage to Ag_NP (F=1.53, p=0.13), or with the C4diP or CD preparations (F=0.84, p=0.61).

Our data demonstrate that the length and orientation of the sulphated tail groups on calix[n]arenes, as well as their interaction with Ag_NP, impact upon their intrinsic anti-oxidant activity, and that cyclodextrins lack anti-oxidant activity at the tested concentrations.

The highly anionic groups of the calix[n]arenes may reduce interactions with the negative exterior of the plasma membrane 9 . Nanoparticle surface charge is a determining factor of cellular uptake 10,11 and those with cations present on their surface are more easily internalised due to electrostatic interactions with the negatively charged cell surface. Attachment of calix[4a,4b,6a]arenes to silver-capped nanoparticles resulted in reduced intracellular ROS, perhaps via Ag cations reducing the negative charge of the calix[n]arenes and increasing internalisation within cells. Alternatively, the functional groups of the calix[n]arenes may be sequestered within the Ag_NP surface (Scheme S1, SI). Differential efficacy was also observed when comparing calix[n]arene preparations of the same ring size, but with different tail structures present. In the cases of both 4-and 6-moeity ring sizes, the most effective structure was that of SC(n)c, which corresponds to sulphonic acid groups present above and below the plane of the macrocycle. Calix[n]arenes have been shown to selectively associate with, 12,13 and transport 14 various cations present in biological systems, readily forming anionic derivatives, although the electrostatic interactions are not well understood and are thought to vary largely depending on the degree of charge localisation within the ions 15 . While the antioxidant mechanism of the SC(n)c is not known, complex intracellular anionic interactions are likely to be involved, as supported by our previous demonstration of antioxidant activity of calix [4]arenes that also featured anionic groups above and below the ring 8 . The observation that the calix [8]arenes failed to reduce ROS production, regardless of concentration or linkage to Ag_NP, suggests that there is a size limit, as well as structural requirements, to calix[n]arene efficacy, due to size exclusion of the calix[n]arene cup structure or protein binding incompatibility. Note that Ag_NP without calixarene capping degrade rapidly, limiting any likely biological effects if dissociation occurs 16 .

ChemComm Accepted Manuscript

Antioxidant efficacy is often associated with antibacterial activity 17 , but the mechanism and structural requirements of the association are as yet unclear. We had previously assessed efficacy of calix[n]arene and cyclodextrin capped Ag nanoparticles at inhibiting growth of gram + bacteria and gram -bacteria, by measuring cell growth as a percentage of growth in untreated cultures (Table 1). 10 There exists an inverse relation between the antioxidant efficacy of Ag-NPs capped by para-sulphonato-calix[n]arenes and Gram+ antibacterial activities. Gram -antibacterial activity was significant in the cases of SC6b and SC6c and in both cases, associated with the reduction of ROS species. Molecules are active with regard to ROS reduction as the isolated molecule and also as the capped NPs (Table 1). France: e-mail antony.coleman@adm.univ-lyon1.fr

Fig. 2

 2 Fig. 2 Effects of calix[n]arene and cyclodextrin preparations on ROS production by glutamate stressed PC12 cells. Cells were stressed with 100mM glutamate and treated with 1, 10 or 100 µg mL -1 calixarene or cyclodextrin preparations. ROS production was quantified using DCFH-DA and results expressed as mean ± S.E.M fluorescence intensity (arbitrary units): * p<0.05, significantly different from glutamate only control. Experiments were conducted 2-3 times and representative results displayed.

  Published on 21 November 2014. Downloaded by Université Claude Bernard Lyon on 23/11/2014 23:49:56.

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DOI: 10.1039/C4CC08576K

Table 1 Summary of antioxidant activity of calix[n]arenes and antioxidant and antibacterial activities of calix[n]arene capped silver nanoparticles. ROS levels are expressed as a % of values from control. Similarly, antibacterial levels are expressed as a % of growth compared to an untreated control. The silver nanoparticles showing an inverse dependence between their Gram + anti-bacterial and ROS activities are highlighted in red. The silver nanoparticles showing a direct association between their Gram -anti-bacterial and ROS activities have been highlighted in blue.

 1 From the above it is possible to postulate that while the action of calix[n]arene capped nanoparticles with regard to gram+ positive bacteria involves radical species and is inhibited by ROS reducing systems, for gram -bacteria the action is highly specific, involving only calix[6]arene derivatives carrying O-alkyl sulphonate groups at the phenolic face and is not related to ROS reducing activity.ConclusionCalix[n]arenes featuring sulphonate groups above and/or below the plane of the macrocycle have intrinsic anti-oxidant capacity: less sulphonated calix[n]arenes require linkage to Ag_NPs to achieve similar efficacy. Calix[n]arenes may be used to carry additional therapeutic agents to provide a combinatorial antioxidant strategy for treatment of neurotrauma and other diseases. Associated antibacterial activity may enhance therapeutic potential in certain clinical scenarios featuring infection. The mechanism of the anti-bacterial action remains a quandary for gram-bacteria. Experimental and Regenerative Neurosciences, School of Animal Biology, The University of Western Australia, Crawley, 6009 WA Australia: lindy.fitzgerald@uwa.edu.au b LMI, CNRS UMR 5615, Université de Lyon 1, Villeurbanne, F69622,

	Molecule	ROS level (%)	Molecule capped on silver nanoparticles	ROS level (%)	Gram + Bacterial growth (%)	Gram -Bacterial growth (%)
	SC4a	68	SC4a_Ag_NP	42	81	100
	SC6a	72	SC6a_Ag_NP	61	55	100
	SC8a	93	SC8a_Ag_NP	87	33	99
	SC4b	73	SC4b_Ag_NP	48	97	99
	SC6b	43	SC6b_Ag_NP	53	98	46
	SC8b	102	SC8b_Ag_NP	93	81	89
	SC4c	51	SC4c_Ag_NP	51	99	84
	SC6c	51	SC6c_Ag_NP	49	98	62
	SC8c	101	SC8c_Ag_NP	111	ND*	ND*
	C4diP	88	C4diP_Ag_NP	86	ND*	ND*
	CD	86	CD_Ag_NP	76	ND*	ND*
	ND*: Not Determined

a

ChemComm Accepted Manuscript