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Cite this: Chem. Commun., 2014, 50, 5514 Received 14th December 2013, Accepted 19th March 2014
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Cyclo-oligo-(1 - 6)-b-D-glucosamine based artificial channels for tunable transmembrane ion transport† Tanmoy Saha,a Arundhati Roy,a Marina L. Gening,b Denis V. Titov,b Alexey G. Gerbst,b Yury E. Tsvetkov,b Nikolay E. Nifantiev*b and Pinaki Talukdar*a
DOI: 10.1039/c3cc49490j www.rsc.org/chemcomm
Unimolecular ion channels were designed by functionalization of a new type of cyclic oligosaccharides, cyclo-oligo-(1 - 6)-b-D-glucosamines, with pentabutylene glycol chains. Their ion transporting activity was tuned by varying oligomericity. A halide selectivity sequence, Cl 4 Br 4 I was observed.
Ion channels are membrane pervasive proteins in living systems that are capable of performing complex tasks by allowing the passage of ions through their hydrophilic pores.1 Ion selectivity during transport is crucial to a wide variety of biological processes, such as signal transduction and cellular regulation.2 Different cyclic organic compounds were studied as potential building blocks to design artificial ion transporters and channels.3 But over the last several years, cyclic oligosaccharides of the cyclodextrin (CD) group have played a pivotal role in these purposes4 by providing a rigid platform to withstand the implosion caused by lipid pressure. Unimolecular ion channels, as reported by Gin and co-workers, involve b-CD as a rigid scaffold which is connected to multiple membrane-spanning pentabutylene glycol tails to form an anion selective ion channel.4 However, CDs offer limited scope for modulating ion channel activity via ring-size variation. CDs because of their smaller ring size are synthetically inaccessible5 and are therefore, never employed to manipulate the ion channel activity. Although rigid CDs have been applied considerably in molecular recognition studies,6 the presence of hydrophobic indentations in these macrocycles contradicts the crucial requirement of polar pores present in ion channels. Due to these limitations, new types of cyclic functionalized agents are required to design tuneable and efficient artificial ion channels. Recently, we have reported an efficient synthetic strategy for easy access to a series of individual cyclic oligo-(1 - 6)-b-D-glucosamine
(GA) derivatives [GA]2–[GA]7 consisting of two to seven glucosamine units (Fig. 1A).7 These compounds were until recently used to design the most potent oligodentate blockers of adhesin LecA from Pseudomonas aeruginosa.8 The presence of free amino groups in the discussed cyclic compounds makes them suitable for conjugating to lipid residues required to produce artificial channel-forming agents. The molecular dynamics simulation studies conclude an interesting correlation between the oligomericity and rigidity of GAs. A proportional balance of pore-diameter versus rigidity is retained from dimeric to tetrameric cyclo-(1 - 6)-b-D-glucosamines [GA]2.Ac–[GA]4.Ac (Fig. 1B and C). However, with the larger macrocycles [GA]5.Ac–[GA]7.Ac (Fig. 1A, m 4 2), the rigidity of system was compromised resulting in a conformational disorder (Fig. 1B). Therefore, we envisaged that ion channels constructed from rigid [GA]2–[GA]4 would sustain membrane pressure and show
a
Department of Chemistry, Indian Institute of Science Education and Research Pune, Maharashtra 411008, India. E-mail: [email protected] b Laboratory of Glycoconjugate Chemistry, N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky prospect 47, Moscow 119991, Russia. E-mail: [email protected] † Electronic supplementary information (ESI) available: Experimental procedures, supplemental data, and 1H-, 13C-NMR spectra. See DOI: 10.1039/ c3cc49490j
5514 | Chem. Commun., 2014, 50, 5514--5516
Fig. 1 Structures of the oligo-(1 - 6)-b-D-glucosamine macrocycles [GA]2–[GA]7 (A). Representation of oligomericity versus rigidity (B). Representation of the hydrophilic (blue) and hydrophobic (red) indentation in [GA]4.Ac (C) and b-CD (D) respectively, shown in the space-filling model.7 Proposed models of unimolecular ion channels with tunable pore diameter (E). ‘‘Hybrid’’ cyclic tetrasaccharide based channel with fewer numbers of membrane spanning tails (F).
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Fig. 2 Structures of cyclo-oligo-(1 - 6)-b-D-glucosamine based synthetic ion channels [GA]2.L–[GA]5.L with varied pore size and ‘‘hybrid’’ cyclic tetrasaccharide based synthetic ion channel [GA]4.L0 with fewer membrane spanning tails.
an increasing order of ion transporting activity due to the increase in ring-size (3.5 to 5.8 Å) while more flexible macrocycles ([GA]5–[GA]7) would result in implosion due to lipid pressure and loss of ion transporting activity. Herein, we report the use of neoglycolipid derivatives based on the scaffolds [GA]2–[GA]57 and [GA]40 9 to design artificial ion channels [GA]2.L–[GA]5.L and [GA]4.L0 , respectively (Fig. 2). An additional important argument for choosing cyclo-oligo-(1 - 6)-b-D-glucosamines is the presence of hydrophilic indentations in these macrocycles7 unlike the hydrophobic cavities present in b-CD (Fig. 1C and D). This characteristic was considered to overcome the energy barrier presumably present during transport of ions through the b-CD based channels due to the hydrophobicity–hydrophilicity mismatch.10 A pentabutylene glycol tail (33 Å) was selected for anchoring the components of artificial channels due to its matching length with the thickness of the egg yolk phosphatidylcholine (EYPC) lipid membrane (35–40 Å).11 Additionally, repeating ether moieties were placed in the tail designed to achieve a proper hydrophilic–hydrophobic balance and ensuring partitioning of the lipid membrane.4b To form target conjugates, pentabutylene glycol 1 was first converted to either the acid 3 or the activated ester 4 via the methyl ester 2. Acylation of compounds [GA]2, [GA]40 and [GA]5 was carried out by treatment with ester 4, while the synthesis of conjugates [GA]3.L and [GA]4.L was accomplished by acylation with acid 3 in the presence of the condensing reagent DMTMM (Scheme 1).12 Ion transporting activity of the synthesized neoglycolipids was evaluated by a fluorescence assay using large unilamellar vesicles (LUVs) prepared from EYPC lipids with the entrapped 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) dye i.e. EYPC-LUVs * HPTS (Fig. S1, ESI†).13 A pH gradient, DpH = 0.8 (pHin = 7.0 and pHout = 7.8) was applied with the NaOH pulse followed by the addition of channel-forming molecules and finally vesicles were lysed by adding 10% Triton X-100 (Tx). The change of the HPTS emission intensity in this process was monitored in time (Fig. S1B, ESI†). Addition of channel-forming molecules resulted in destruction of the pH gradient via either a Na+/OH symport or a Na+/H+ antiport mechanism. Ion transport activities of [GA]2.L (Fig. S2A, ESI†), [GA]3.L (Fig. S2B, ESI†), [GA]4.L (Fig. S2C, ESI†), [GA]4.L 0 (Fig. S2D, ESI†) and [GA]5.L (Fig. S2E, ESI†) were evaluated at varied concentrations (0 to 5 mM) indicating concentration dependent responses during the transport process. When the fractional activities (Y) of all compounds ([GA]2.L–[GA]5.L, and [GA]4.L 0 ) were plotted against concentration (0 to 5 mM) by
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Scheme 1 Synthesis of ion channel molecules. Reagents and conditions: (a) N2CHCOOMe, BF3Et2O, CH2Cl2, rt, 3 h, 77%; (b) KOH (aq), MeOH, rt, 25 min, 100%; (c) CF3COOC6F5, Py, CH2Cl2 rt, 3 h, 64%; (d) i. 4 (3.3 eq.), Et3N, DMF, rt, 24 h; ii. MeONa, MeOH, CH2Cl2, rt, 1 h, 30%; (e) 3 (4.5 eq.), DMTMM, Et3N, MeOH, rt, 24 h, 63%; (f) i. 3 (6.5 eq.), DMTMM, Et3N, MeOH, rt, 24 h; ii. Ac2O, Py, rt, 3 h, 27%; (g) MeONa, MeOH, CH2Cl2, rt, 1 h, 46%; (h) i. 4 (10 eq.), Et3N, DMF, rt, 24 h; ii. MeONa, MeOH, CH2Cl2, rt, 1 h; iii. Ac2O, Py, rt, 3 h, 46%, (i) MeONa, MeOH, CH2Cl2, rt, 1 h, 100%; (j) i. 4 (3.3 eq.), Et3N, DMF, rt, 24 h; ii. MeONa, MeOH, CH2Cl2, rt, 1 h, 57%.
recording the normalized fluorescence intensity values at t = 100 s, the compound [GA]4.L displayed sharpest enhancement in transport activity in comparison to other compounds (Fig. 3A). From this plot, an effective concentration EC50 = 0.72 mM was calculated. The Hill coefficient, n = 0.96, calculated from this plot confirmed the formation of the unimolecular ion channel. The unimolecularity of [GA]4.L was also confirmed from the linearity of the rate constant versus concentration plot (Fig. S3, ESI†). EC50 values calculated for other channels forming molecules (i.e. [GA]2.L = 2.78 mM, [GA]3.L = 1.68 mM, [GA]5.L = 1.77 mM, [GA]4.L0 = 6.12 mM) indicated an activity sequence [GA]4.L 4 [GA]3.L 4 [GA]5.L 4 [GA]2.L 4 [GA]4.L0 . A further quantitative comparison of ion transporting activity was done by recording the fractional activities of all the synthesized molecules at a concentration c = 1.5 mM (Fig. 3B). Compound [GA]3.L displayed 1.8-fold higher activity compared to [GA]2.L, while for compound [GA]4.L activity increased by 3.1-fold. These data clearly demonstrate that an increase in oligomericity (from m = 0 to m = 2) is important for improving transport activity. However, with further increase in
Fig. 3 Concentration profiles of [GA]2.L–[GA]5.L and [GA]4.L 0 (A). Comparison of ion transporting activity of [GA]2.L–[GA]5.L and [GA]4.L 0 at 1.5 mM concentrations (B).
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Fig. 4 MD trajectories graphs for interatomic distances O5–O5 0 and O6– O6 0 in the structure [GA]4.L (A and B). Anion selectivity of [GA]4.L (C).
oligomericity (from m = 2 to m = 3), i.e. for [GA]5.L the transporting activity decreased by 0.5-fold compared to [GA]4.L (and only 1.5-fold compared to [GA]2.L). These data were in accordance with our assumption that the rigidity of oligo-(1 - 6)-b-D-glucosamine macrocycles is important for providing defined indentations for permeation of ions and once rigidity is perturbed the transporting activity is reduced dramatically due to implosion of macrocycles caused by lipid pressure (Fig. 1B). On the other hand, a 0.3-fold activity for [GA]4.L0 in comparison to [GA]4.L (i.e. 0.8-fold in comparison to [GA]2.L) demonstrated that a larger number of membrane spanning tails ensures better permeation of [GA]4.L into the lipid membrane. To obtain theoretical insight into ion transporting activities, molecular dynamics (MD) simulations were carried out using the MM3 force field for di-, tri-, tetra- and penta-(1 - 6)-b-D-glucosamines both with (compounds [GA]2.L, [GA]3.L, [GA]4.L and [GA]5.L) and without butylene glycol tails (compounds [GA]2.Ac, [GA]3.Ac and [GA]4.Ac). For [GA]4.L, the insignificant variations observed for interatomic distances of O5–O50 and O6–O60 (Fig. 4A and B and Fig. S12, ESI†) confirm that the cavity size is not affected by the introduction of pentabutyleneglycol arms. Similar observations were recorded for other smaller neoglycolipid derivatives (Fig. S7–S10, ESI†). The MD trajectory graphs of [GA]5.L displayed significant variation for its interatomic distances of O5–O50 and O6–O60 (Fig. S13, ESI†) concluding the flexibility of the molecule. Ion selectivity of neoglycolipid derivatives [GA]3.L (2.0 mM), [GA]4.L (0.75 mM), and [GA]5.L (0.75 mM) were evaluated using fluorescencebased competitive assays (Fig. S4, ESI†).13a Variation of cations (M+ = Li+, Na+, K+, Rb+ and Cs+) in the extravesicular matrix did not offer any difference in transport activity (Fig S5, ESI†). In the next stage, exchange of anions under isoosmolar Cl (intravesicular) to monovalent anions, X (extravesicular, X = Cl, Br, I) was studied.3j In this case, the Cl/X antiport is expected to be faster than the Cl/OH antiport due to the much higher concentration of X (100 mM) compared to that of OH (pH = 7.8). Upon equilibration of the Cl/X exchange, the rise in HPTS fluorescence intensity obtained indicates the X/OH antiport.3k A uniform anion selectivity sequence Cl 4 Br 4 I was observed for all neoglycolipid derivatives (Fig. 4C and Fig. S6, ESI†). This selectivity sequence3k is a consequence of increasing anionic radii, and strong NH Cl binding14 is responsible for the observed anion selectivity within the channel interior. In summary, artificial ion channels were designed based on a new type of functionalized cyclic oligosaccharide scaffolds, namely
5516 | Chem. Commun., 2014, 50, 5514--5516
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cyclo-oligo-(1-6)-b-D-glucosamines. Amenability of ring-size alteration and its associated correlation with the ring rigidity made it possible to manipulate the ion transporting activity. The observed trend of the transporting activity [GA]4.L 4 [GA]3.L 4 [GA]5.L 4 [GA]2.L 4 (i.e. EC50 = 0.72, 1.68, 1.77 and 2.78 mM) demonstrated the synergistic correlation of oligomericity and ring rigidity. The importance of the number of membrane spanning tails was established by the observed [GA]4.L 4 [GA]4.L0 (i.e. EC50 = 6.12 mM) trend. Active channel forming molecules [GA]3.L, [GA]4.L, and [GA]5.L exhibited anion selectivity via hydrogen bonding with amide NH groups. This work was supported in part by collaborative grants from RFBR (research project 11-03-92694-a) and DST (research projects INT/RFBR/P-96 and SR/S1/OC-65/2012), T.S. and A.R. thank the UGC for research fellowships.
Notes and references 1 K. Kano and J. H. Fendler, Biochim. Biophys. Acta, Biomembr., 1978, 509, 289. 2 (a) S. C. Froehner, Annu. Rev. Neurosci., 1993, 16, 347; (b) T. E. DeCoursey, K. G. Chandy, S. Gupta and M. D. Cahalan, Nature, 1984, 307, 465; (c) G. J. Augustine, M. P. Charlton and S. J. Smith, Annu. Rev. Neurosci., 1987, 10, 633. 3 (a) S. Matile, A. Vargas Jentzsch, J. Montenegro and A. Fin, Chem. Soc. Rev., 2011, 40, 2453; (b) T. M. Fyles, Chem. Soc. Rev., 2007, 36, 335; (c) A. L. Sisson, M. R. Shah, S. Bhosale and S. Matile, Chem. Soc. Rev., 2006, 35, 1269; (d) S. Matile, A. Som and N. Sorde, Tetrahedron, 2004, 60, 6405; (e) N. Maulucci, F. De Riccardis, C. B. Botta, A. Casapullo, E. Cressina, M. Fregonese, P. Tecilla and I. Izzo, Chem. Commun., 2005, 1354; ( f ) K. S. J. Iqbal and P. J. Cragg, Dalton Trans., 2007, 26; (g) W. M. Leevy, M. R. Gokel, G. B. Hughes-Strange, P. H. Schlesinger and G. W. Gokel, New J. Chem., 2005, 29, 205; (h) A. J. Wright, S. E. Matthews, W. B. Fischer and P. D. Beer, Chem. – Eur. J., 2001, 7, 3474; (i) C. L. Murray and G. W. Gokel, J. Supramol. Chem., 2001, 1, 23; ( j) S. Fernandez-Lopez, H.-S. Kim, E. C. Choi, M. Delgado, J. R. Granja, A. Khasanov, K. Kraehenbuehl, G. Long, D. A. Weinberger, K. M. Wilcoxen and M. R. Ghadiri, Nature, 2001, 412, 452; (k) V. Gorteau, G. Bollot, J. Mareda and S. Matile, Org. Biomol. Chem., 2007, 5, 3000. 4 (a) L. Bacri, A. Benkhaled, P. Guegan and L. Auvray, Langmuir, 2005, 21, 5842; (b) N. Madhavan, E. C. Robert and M. S. Gin, Angew. Chem., Int. Ed., 2005, 44, 7584; (c) P. V. Jog and M. S. Gin, Org. Lett., 2008, 10, 3693; (d) J. K. W. Chui and T. M. Fyles, Chem. Commun., 2010, 46, 4169; (e) D. Montesarchio, C. Coppola, M. Boccalon and P. Tecilla, Carbohydr. Res., 2012, 356, 62. 5 S. Immel, J. Brickmann and F. W. Lichtenthaler, Liebigs Ann., 1995, 929. 6 (a) M. Vinodh, F. H. Alipour, A. A. Mohamod and T. F. Al-Azemi, Molecules, 2012, 17, 11763; (b) S. Tilloy, C. Binkowski-Machut, S. Menuel, H. Bricout and E. Monflier, Molecules, 2012, 17, 13062. 7 M. L. Gening, D. V. Titov, A. A. Grachev, A. G. Gerbst, O. N. Yudina, A. S. Shashkov, A. O. Chizhov, Y. E. Tsvetkov and N. E. Nifantiev, Eur. J. Org. Chem., 2010, 2465. 8 (a) M. L. Gening, D. V. Titov, S. Cecioni, A. Audfray, A. G. Gerbst, Y. E. Tsvetkov, V. B. Krylov, A. Imberty, N. E. Nifantiev and S. Vidal, Chem. – Eur. J., 2013, 19, 9272; (b) N. R. Clement and J. M. Gould, Biochemistry, 1981, 20, 1534. 9 D. V. Titov, M. L. Gening, A. G. Gerbst, A. O. Chizhov, Y. E. Tsvetkov and N. E. Nifantiev, Carbohydr. Res., 2013, 381, 161. 10 M. Becuwe, F. Cazier, M. Bria, P. Woisel and F. Delattre, Tetrahedron Lett., 2007, 48, 6186. 11 A. D. Bedells, C. Booth and K. Viras, Makromol. Chem., 1991, 192, 2099. 12 M. Kunishima, C. Kawachi, J. Monta, K. Terao, F. Iwasaki and S. Tani, Tetrahedron, 1999, 55, 13159. 13 (a) P. Talukdar, G. Bollot, J. Mareda, N. Sakai and S. Matile, J. Am. Chem. Soc., 2005, 127, 6528; (b) N. Sakai and S. Matile, J. Am. Chem. Soc., 2003, 125, 14348. 14 (a) S. Jana, A. L. Whiting, A. Hazra, S. Sen, S. Goswami, G. Mehta, H.-K. Fun and F. Hof, Supramol. Chem., 2012, 24, 385; (b) S. K. Dey, B. K. Datta and G. Das, CrystEngComm, 2012, 14, 5305; (c) P. Bose, I. Ravikumar, B. Akhuli and P. Ghosh, Chem. – Asian J., 2012, 7, 2373.
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Cite this: Chem. Commun., 2014, 50, 5514 Received 14th December 2013, Accepted 19th March 2014
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Cyclo-oligo-(1 - 6)-b-D-glucosamine based artificial channels for tunable transmembrane ion transport† Tanmoy Saha,a Arundhati Roy,a Marina L. Gening,b Denis V. Titov,b Alexey G. Gerbst,b Yury E. Tsvetkov,b Nikolay E. Nifantiev*b and Pinaki Talukdar*a
DOI: 10.1039/c3cc49490j www.rsc.org/chemcomm
Unimolecular ion channels were designed by functionalization of a new type of cyclic oligosaccharides, cyclo-oligo-(1 - 6)-b-D-glucosamines, with pentabutylene glycol chains. Their ion transporting activity was tuned by varying oligomericity. A halide selectivity sequence, Cl 4 Br 4 I was observed.
Ion channels are membrane pervasive proteins in living systems that are capable of performing complex tasks by allowing the passage of ions through their hydrophilic pores.1 Ion selectivity during transport is crucial to a wide variety of biological processes, such as signal transduction and cellular regulation.2 Different cyclic organic compounds were studied as potential building blocks to design artificial ion transporters and channels.3 But over the last several years, cyclic oligosaccharides of the cyclodextrin (CD) group have played a pivotal role in these purposes4 by providing a rigid platform to withstand the implosion caused by lipid pressure. Unimolecular ion channels, as reported by Gin and co-workers, involve b-CD as a rigid scaffold which is connected to multiple membrane-spanning pentabutylene glycol tails to form an anion selective ion channel.4 However, CDs offer limited scope for modulating ion channel activity via ring-size variation. CDs because of their smaller ring size are synthetically inaccessible5 and are therefore, never employed to manipulate the ion channel activity. Although rigid CDs have been applied considerably in molecular recognition studies,6 the presence of hydrophobic indentations in these macrocycles contradicts the crucial requirement of polar pores present in ion channels. Due to these limitations, new types of cyclic functionalized agents are required to design tuneable and efficient artificial ion channels. Recently, we have reported an efficient synthetic strategy for easy access to a series of individual cyclic oligo-(1 - 6)-b-D-glucosamine
(GA) derivatives [GA]2–[GA]7 consisting of two to seven glucosamine units (Fig. 1A).7 These compounds were until recently used to design the most potent oligodentate blockers of adhesin LecA from Pseudomonas aeruginosa.8 The presence of free amino groups in the discussed cyclic compounds makes them suitable for conjugating to lipid residues required to produce artificial channel-forming agents. The molecular dynamics simulation studies conclude an interesting correlation between the oligomericity and rigidity of GAs. A proportional balance of pore-diameter versus rigidity is retained from dimeric to tetrameric cyclo-(1 - 6)-b-D-glucosamines [GA]2.Ac–[GA]4.Ac (Fig. 1B and C). However, with the larger macrocycles [GA]5.Ac–[GA]7.Ac (Fig. 1A, m 4 2), the rigidity of system was compromised resulting in a conformational disorder (Fig. 1B). Therefore, we envisaged that ion channels constructed from rigid [GA]2–[GA]4 would sustain membrane pressure and show
a
Department of Chemistry, Indian Institute of Science Education and Research Pune, Maharashtra 411008, India. E-mail: [email protected] b Laboratory of Glycoconjugate Chemistry, N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky prospect 47, Moscow 119991, Russia. E-mail: [email protected] † Electronic supplementary information (ESI) available: Experimental procedures, supplemental data, and 1H-, 13C-NMR spectra. See DOI: 10.1039/ c3cc49490j
5514 | Chem. Commun., 2014, 50, 5514--5516
Fig. 1 Structures of the oligo-(1 - 6)-b-D-glucosamine macrocycles [GA]2–[GA]7 (A). Representation of oligomericity versus rigidity (B). Representation of the hydrophilic (blue) and hydrophobic (red) indentation in [GA]4.Ac (C) and b-CD (D) respectively, shown in the space-filling model.7 Proposed models of unimolecular ion channels with tunable pore diameter (E). ‘‘Hybrid’’ cyclic tetrasaccharide based channel with fewer numbers of membrane spanning tails (F).
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Fig. 2 Structures of cyclo-oligo-(1 - 6)-b-D-glucosamine based synthetic ion channels [GA]2.L–[GA]5.L with varied pore size and ‘‘hybrid’’ cyclic tetrasaccharide based synthetic ion channel [GA]4.L0 with fewer membrane spanning tails.
an increasing order of ion transporting activity due to the increase in ring-size (3.5 to 5.8 Å) while more flexible macrocycles ([GA]5–[GA]7) would result in implosion due to lipid pressure and loss of ion transporting activity. Herein, we report the use of neoglycolipid derivatives based on the scaffolds [GA]2–[GA]57 and [GA]40 9 to design artificial ion channels [GA]2.L–[GA]5.L and [GA]4.L0 , respectively (Fig. 2). An additional important argument for choosing cyclo-oligo-(1 - 6)-b-D-glucosamines is the presence of hydrophilic indentations in these macrocycles7 unlike the hydrophobic cavities present in b-CD (Fig. 1C and D). This characteristic was considered to overcome the energy barrier presumably present during transport of ions through the b-CD based channels due to the hydrophobicity–hydrophilicity mismatch.10 A pentabutylene glycol tail (33 Å) was selected for anchoring the components of artificial channels due to its matching length with the thickness of the egg yolk phosphatidylcholine (EYPC) lipid membrane (35–40 Å).11 Additionally, repeating ether moieties were placed in the tail designed to achieve a proper hydrophilic–hydrophobic balance and ensuring partitioning of the lipid membrane.4b To form target conjugates, pentabutylene glycol 1 was first converted to either the acid 3 or the activated ester 4 via the methyl ester 2. Acylation of compounds [GA]2, [GA]40 and [GA]5 was carried out by treatment with ester 4, while the synthesis of conjugates [GA]3.L and [GA]4.L was accomplished by acylation with acid 3 in the presence of the condensing reagent DMTMM (Scheme 1).12 Ion transporting activity of the synthesized neoglycolipids was evaluated by a fluorescence assay using large unilamellar vesicles (LUVs) prepared from EYPC lipids with the entrapped 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) dye i.e. EYPC-LUVs * HPTS (Fig. S1, ESI†).13 A pH gradient, DpH = 0.8 (pHin = 7.0 and pHout = 7.8) was applied with the NaOH pulse followed by the addition of channel-forming molecules and finally vesicles were lysed by adding 10% Triton X-100 (Tx). The change of the HPTS emission intensity in this process was monitored in time (Fig. S1B, ESI†). Addition of channel-forming molecules resulted in destruction of the pH gradient via either a Na+/OH symport or a Na+/H+ antiport mechanism. Ion transport activities of [GA]2.L (Fig. S2A, ESI†), [GA]3.L (Fig. S2B, ESI†), [GA]4.L (Fig. S2C, ESI†), [GA]4.L 0 (Fig. S2D, ESI†) and [GA]5.L (Fig. S2E, ESI†) were evaluated at varied concentrations (0 to 5 mM) indicating concentration dependent responses during the transport process. When the fractional activities (Y) of all compounds ([GA]2.L–[GA]5.L, and [GA]4.L 0 ) were plotted against concentration (0 to 5 mM) by
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Scheme 1 Synthesis of ion channel molecules. Reagents and conditions: (a) N2CHCOOMe, BF3Et2O, CH2Cl2, rt, 3 h, 77%; (b) KOH (aq), MeOH, rt, 25 min, 100%; (c) CF3COOC6F5, Py, CH2Cl2 rt, 3 h, 64%; (d) i. 4 (3.3 eq.), Et3N, DMF, rt, 24 h; ii. MeONa, MeOH, CH2Cl2, rt, 1 h, 30%; (e) 3 (4.5 eq.), DMTMM, Et3N, MeOH, rt, 24 h, 63%; (f) i. 3 (6.5 eq.), DMTMM, Et3N, MeOH, rt, 24 h; ii. Ac2O, Py, rt, 3 h, 27%; (g) MeONa, MeOH, CH2Cl2, rt, 1 h, 46%; (h) i. 4 (10 eq.), Et3N, DMF, rt, 24 h; ii. MeONa, MeOH, CH2Cl2, rt, 1 h; iii. Ac2O, Py, rt, 3 h, 46%, (i) MeONa, MeOH, CH2Cl2, rt, 1 h, 100%; (j) i. 4 (3.3 eq.), Et3N, DMF, rt, 24 h; ii. MeONa, MeOH, CH2Cl2, rt, 1 h, 57%.
recording the normalized fluorescence intensity values at t = 100 s, the compound [GA]4.L displayed sharpest enhancement in transport activity in comparison to other compounds (Fig. 3A). From this plot, an effective concentration EC50 = 0.72 mM was calculated. The Hill coefficient, n = 0.96, calculated from this plot confirmed the formation of the unimolecular ion channel. The unimolecularity of [GA]4.L was also confirmed from the linearity of the rate constant versus concentration plot (Fig. S3, ESI†). EC50 values calculated for other channels forming molecules (i.e. [GA]2.L = 2.78 mM, [GA]3.L = 1.68 mM, [GA]5.L = 1.77 mM, [GA]4.L0 = 6.12 mM) indicated an activity sequence [GA]4.L 4 [GA]3.L 4 [GA]5.L 4 [GA]2.L 4 [GA]4.L0 . A further quantitative comparison of ion transporting activity was done by recording the fractional activities of all the synthesized molecules at a concentration c = 1.5 mM (Fig. 3B). Compound [GA]3.L displayed 1.8-fold higher activity compared to [GA]2.L, while for compound [GA]4.L activity increased by 3.1-fold. These data clearly demonstrate that an increase in oligomericity (from m = 0 to m = 2) is important for improving transport activity. However, with further increase in
Fig. 3 Concentration profiles of [GA]2.L–[GA]5.L and [GA]4.L 0 (A). Comparison of ion transporting activity of [GA]2.L–[GA]5.L and [GA]4.L 0 at 1.5 mM concentrations (B).
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Fig. 4 MD trajectories graphs for interatomic distances O5–O5 0 and O6– O6 0 in the structure [GA]4.L (A and B). Anion selectivity of [GA]4.L (C).
oligomericity (from m = 2 to m = 3), i.e. for [GA]5.L the transporting activity decreased by 0.5-fold compared to [GA]4.L (and only 1.5-fold compared to [GA]2.L). These data were in accordance with our assumption that the rigidity of oligo-(1 - 6)-b-D-glucosamine macrocycles is important for providing defined indentations for permeation of ions and once rigidity is perturbed the transporting activity is reduced dramatically due to implosion of macrocycles caused by lipid pressure (Fig. 1B). On the other hand, a 0.3-fold activity for [GA]4.L0 in comparison to [GA]4.L (i.e. 0.8-fold in comparison to [GA]2.L) demonstrated that a larger number of membrane spanning tails ensures better permeation of [GA]4.L into the lipid membrane. To obtain theoretical insight into ion transporting activities, molecular dynamics (MD) simulations were carried out using the MM3 force field for di-, tri-, tetra- and penta-(1 - 6)-b-D-glucosamines both with (compounds [GA]2.L, [GA]3.L, [GA]4.L and [GA]5.L) and without butylene glycol tails (compounds [GA]2.Ac, [GA]3.Ac and [GA]4.Ac). For [GA]4.L, the insignificant variations observed for interatomic distances of O5–O50 and O6–O60 (Fig. 4A and B and Fig. S12, ESI†) confirm that the cavity size is not affected by the introduction of pentabutyleneglycol arms. Similar observations were recorded for other smaller neoglycolipid derivatives (Fig. S7–S10, ESI†). The MD trajectory graphs of [GA]5.L displayed significant variation for its interatomic distances of O5–O50 and O6–O60 (Fig. S13, ESI†) concluding the flexibility of the molecule. Ion selectivity of neoglycolipid derivatives [GA]3.L (2.0 mM), [GA]4.L (0.75 mM), and [GA]5.L (0.75 mM) were evaluated using fluorescencebased competitive assays (Fig. S4, ESI†).13a Variation of cations (M+ = Li+, Na+, K+, Rb+ and Cs+) in the extravesicular matrix did not offer any difference in transport activity (Fig S5, ESI†). In the next stage, exchange of anions under isoosmolar Cl (intravesicular) to monovalent anions, X (extravesicular, X = Cl, Br, I) was studied.3j In this case, the Cl/X antiport is expected to be faster than the Cl/OH antiport due to the much higher concentration of X (100 mM) compared to that of OH (pH = 7.8). Upon equilibration of the Cl/X exchange, the rise in HPTS fluorescence intensity obtained indicates the X/OH antiport.3k A uniform anion selectivity sequence Cl 4 Br 4 I was observed for all neoglycolipid derivatives (Fig. 4C and Fig. S6, ESI†). This selectivity sequence3k is a consequence of increasing anionic radii, and strong NH Cl binding14 is responsible for the observed anion selectivity within the channel interior. In summary, artificial ion channels were designed based on a new type of functionalized cyclic oligosaccharide scaffolds, namely
5516 | Chem. Commun., 2014, 50, 5514--5516
Communication
cyclo-oligo-(1-6)-b-D-glucosamines. Amenability of ring-size alteration and its associated correlation with the ring rigidity made it possible to manipulate the ion transporting activity. The observed trend of the transporting activity [GA]4.L 4 [GA]3.L 4 [GA]5.L 4 [GA]2.L 4 (i.e. EC50 = 0.72, 1.68, 1.77 and 2.78 mM) demonstrated the synergistic correlation of oligomericity and ring rigidity. The importance of the number of membrane spanning tails was established by the observed [GA]4.L 4 [GA]4.L0 (i.e. EC50 = 6.12 mM) trend. Active channel forming molecules [GA]3.L, [GA]4.L, and [GA]5.L exhibited anion selectivity via hydrogen bonding with amide NH groups. This work was supported in part by collaborative grants from RFBR (research project 11-03-92694-a) and DST (research projects INT/RFBR/P-96 and SR/S1/OC-65/2012), T.S. and A.R. thank the UGC for research fellowships.
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