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Self-assembling asymmetrical tripodal-like peptides as anion receptors
Tetrahedron 72 (2016) 2900e2905
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Self-assembling asymmetrical tripodal-like peptides as anion receptors Ishanki Bhardwaj, V. Haridas * Department of Chemistry, Indian Institute of Technology Delhi (IITD), Hauz Khas, New Delhi, 110016, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 February 2016 Received in revised form 31 March 2016 Accepted 1 April 2016 Available online 4 April 2016
Aspartic and glutamic-acid based molecules T1aebeT5aeb appended with different moieties were designed and synthesized. These molecules displayed unique self-assembly behavior dependent upon the nature of appended units. Trp favored vesicles, while phenyl unit favored fibrillar assembly. These molecules also showed binding towards anions. Ó 2016 Elsevier Ltd. All rights reserved.
Keywords: Molecular recognition Fluorophore Tryptophan Self-assembled vesicles Encapsulation
1. Introduction Molecular self-assembly is a powerful method for fabricating supramolecular structures.1 The formation of well-ordered supramolecular structures through self-assembly of diverse organic and inorganic building blocks has drawn much attention owing to their potential applications in biology and chemistry.2 Peptides possess hydrogen bonding capability; hence adopt specific structures through self-assembly. The intermolecular non-covalent interactions are utilized in designing diverse supramolecular structures such as fibers, vesicles, spheres, rods and helical ribbons. The potential applications of these assemblies involve their use in drug delivery, tissue engineering and wound healing.3e10 External stimuli such as temperature, pH, electric field, chemicals and ions can bring changes in the structure and solubility characteristics of assembly.11,12 Such self-assembling molecules have several applications in the design of stimuli responsive materials.13e15 Designed peptides can act as good receptors for anions as they have ability to bind guest through hydrogen bonds.16 The binding efficiency of a host depends upon its geometry and the cavity size. Designing tripodal receptors containing asymmetrical binding arms is an interesting area of host-guest chemistry research.17,18 Amino acids with functionalizable side chain seem to be ideal candidates for the design of tripodal receptors. The arms of the
* Corresponding author. Tel.: þ91 011 26591380; e-mail address: haridasv@iitd. ac.in (V. Haridas). http://dx.doi.org/10.1016/j.tet.2016.04.002 0040-4020/Ó 2016 Elsevier Ltd. All rights reserved.
tripodal receptors could offer more binding sites for guest and thereby achieve high binding affinity and selectivity. Appending with fluorophore can help in fluorescence detection of guests. Amino acids such as threonine, serine, tyrosine, histidine and tryptophan have OH or NH groups in their side chains, hence are useful models for designing receptors for anions.19 2. Results and discussion We report aspartic acid and glutamic acid-cored peptides T1aebeT5aeb with and without tryptophan residues (Fig. 1). T1aeb contain benzyl amine appended on Boc-L-Asp and Boc-L-Glu amino acid. T2aeb and T3aeb are synthesized by coupling pnitrobenzoic acid and Boc-L-Tryptophan to the N-terminal of T1a and T1b respectively. T4aeb and T5aeb constitute another class of scaffolds where Boc-Asp and Boc-Glu amino acid are partially or fully functionalized with tryptophan residues (Scheme 1, SI, Schemes S1 and S2). These diverse classes of molecules with amide functionalities provide an interesting set of molecules for anion binding and selfassembling studies. The three arms of aspartic and glutamic acid serve as branches to functionalize with different moieties to generate molecules with tripodal-like topology. Tryptophan provides indole NH as H-bond donor for binding to the guest and also helps in fluorescent detection of guests. Keeping in mind this concept, the binding efficiencies of T1aebeT5aeb towards anions such as F, Cl, Br, I, H2PO 4 and HSO 4 were analyzed by using spectroscopic techniques such as
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Fig. 1. Structural representation of tripodal receptors T1aebeT5aeb.
Scheme 1. Synthesis of tripodal peptides T1aebeT5aeb. (i) DCC, HOSu, NEt3, Benzyl amine, dry CH2Cl2, 0 C, 24 h (ii) Trifluoroacetic acid, dry CH2Cl2, 0 C, 4 h (iii) p-nitrobenzoyl chloride, NEt3, dry CH2Cl2, 0 C, 12 h (iv) Trifluoroacetic acid, dry CH2Cl2, 0 C, 4 h (v) Boc-L-Trp-OH, DCC, HOSu, NEt3, dry CH2Cl2, 0 C, 24 h (vi) DCC, HOSu, NEt3, L-Trp-OMe, dry CH2Cl2, 0 C, 24 h (vii) saturated soln. of HCl in ethyl acetate, 0 C, 6 h (viii) Boc-L-Trp-OH, DCC, HOSu, NEt3, dry CH2Cl2, 0 C, 24 h.
UVevis fluorescence and NMR (Figs. 2 and 3, SI, Figs. S1eS4). The anion binding studies were carried out in chloroform. UVevis and NMR titrations showed that T2b acts as a receptor for phosphate and chloride ion (Table 1). The occurrence of one isosbestic point in UV titration profile of T2b (4.5105 M) with phosphate (4.5102 M) is a noteworthy observation (Fig. 2a). Surprisingly, T2a (with one methylene less than T2b) didn’t show any changes in the UV spectra when titrated with TBAH2PO4, TBABr, TBAI and TBAHSO4. This may be due to steric crowding around the two amide NHs in T2a as compared to T2b. Presence of one additional methylene in T2b relieves the steric strain and hence increases the binding. One of the three arms of T1aeb is not functionalized in comparison to T2aeb and T3aeb. Therefore, T1aeb show differences in binding selectivity compared to tripodal receptors T2aeb
Fig. 2. (a) UV titration profile for T2b (4.5105 M) with (0e40 equiv) TBAH2PO4 (4.5102 M) in CHCl3 (b) Fluorescence titration profile for T4a (1.2104 M) with 1 TBAHSO M) in CHCl3. 4 (1.0810
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and T3aeb (Table 1). T1b showed higher binding compared to T1a due to same steric reasons. Changing the N-terminal substituent of T2aeb from p-nitrobenzoyl to Boc-L-Tryptophan provided T3aeb. The introduction of tryptophan residue was expected to increase the binding affinity as it can provide an additional NH for binding. Interestingly, the binding studies showed no considerable increase in binding affinity (Table 1). This may be due to orientation of tryptophan group away from the binding cavity, thereby not participating in the binding. The stoichiometry of binding for T1aebeT3aeb and T4beT5aeb with all anions is 1:1, while T4a binds to sulfate in 1:2 fashion (G:H 1:2) as evident from Job’s plot (SI, Fig. S2). In order to enhance the binding, T4aeb and T5aeb with two and three tryptophan residues were synthesized. UVevis and fluorescence titration experiments carried out with anions revealed that T4a efficiently binds to phosphate and sulfate. Fluorescence titration showed increase in fluorescence intensity at 445 nm upon slot-wise addition of TBAHSO4 in chloroform (Fig. 2b). 1H NMR titration showed the downfield shift of NHs upon addition of anion salt (Fig. 3). It is evident from Fig. 3, that amide NH(b) showed downfield shift from 6.32 ppm to 6.78 ppm while indole NH’s (d and e) showed downfield shift from 8.11 ppm to 9.12 ppm indicating that these NHs are involved in binding with the guest. T4b has less binding affinity as compared to T4a (Table 1). The extra methylene group present in T4b moves the side chains away from each other, thus decreasing the binding. When the Nterminal of T4aeb was functionalized by another tryptophan residue to give T5aeb, the observed binding affinity is comparatively lesser or similar (Table 1). The contribution towards binding is majorly due to two tryptophan residues. None of these receptors showed binding towards Br and I ions. As T1aebeT5aeb contain amide bonds, they can associate by intermolecular hydrogen bonds. In addition, they contain amino acid residues with side chains capable of pep interactions due to presence of aromatic groups. The presence of these moieties in the scaffolds can provide distinct self-assembling features. Hence, these peptides provide interesting candidates for self-assembly studies. The self-assembling features of T1aebeT5aeb were studied in 1:1 CH3OH/CHCl3 by several microscopic techniques such as scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM). The studies revealed that T1aeb and T2aeb self-assembled into fibers, while T3aebeT5aeb self-assembled into vesicles (Fig. 4, SI, Figs. S5eS6). The backbone of T1aebeT5aeb consists of Asp and Glu amino acids, while the arms are functionalized with different units. Thus, the self-assembly can be controlled by appended units. A closer look at the SEM images showed that the average size of vesicles of T3aebeT5aeb is 0.3e0.8 mm (SI, Fig. S7). TEM (stained with 0.2% phosphotungstic acid) revealed that T3aebeT5aeb formed vesicles in the range of 0.3e0.9 mm. After discovering the distinct morphological features of these peptides, we turned our attention to evaluate the encapsulating potency of the vesicles formed from self-assembly of T3aebeT5aeb. Fluorescence microscopic analysis of T3aebeT5aeb revealed that the vesicles can be seen in blue color (lex¼380e450 nm), while the rhodamine B entrapped vesicles appeared red (lex¼510e560 nm) (Fig. 5a,b) clearly indicating the encapsulation potency of rhodamine B by the vesicles. Addition of 0.2 equiv of sulfate salt to the dye entrapped vesicles resulted in the disruption of vesicles (Fig. 5c), since we could observe the reappearance of blue fluorescence of T3b (Fig. 5d). The vesicular morphology is lost due to anion binding, thus disrupting the self-assembly. The influence of anion binding on self-assembly of T1aebeT5aeb was studied by microscopic imaging. Experiments were performed by adding the anion to the self-assembled system
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Fig. 3. Partial 1H NMR (300 MHz, CDCl3) of T4a (3.9103 M) with and without addition of various amounts of TBAHSO4 (1.5101 M) (0e7.2 equiv) in CDCl3. Table 1 Binding affinities of T1aebeT5aeb with TBA anions in chloroform (NB¼not binding) Receptor Anion
T1a
T1b
T2a T2b
T3a T3b T4a
T4b
T5a
T5b
F CI H2PO 4 HSO 4
2300 NB NB NB
NB 4800 NB NB
NB NB NB NB
NB 360 NB NB
61,900 NB 630 NB
15,478 290 NB NB
450 NB 1300 NB
NB 1000 1000 NB
NB NB 22 NB
640 NB 8400 2.6105
of T1aebeT5aeb in 1:1 CH3OH/CHCl3. Addition of 0.5 equiv of anion salt resulted in the formation of holes on the surface of vesicles. Addition of 1.0 equiv resulted in the formation of wiffleball like structure (Fig. 6aec). The formation of wiffle-ball like
morphology is due to the anion binding on the self-assembled T3b, resulting in the removal of some T3b molecules from vesicles resulting in voids, which is observed in SEM as wiffle-ball. Addition of 2.0 equiv of anion to the self-assembled system completely destroys the vesicular assembly and formation of particles of indefinite shape is observed (Fig. 6c). 3. Conclusions In summary, we have designed and synthesized aspartic and glutamic acid based branched peptides. They displayed varied selfassembling architectures, such as fibers and vesicles. The anion binding property of these molecules is used to fabricate wiffle-ball like assembly. The vesicles as well as wiffle-ball like supramolecular self-assemblies offer opportunities to utilize these systems as
Fig. 4. SEM images of (a) T1a (3.1 mM) (b) T1b (3.4 mM) (c) T2a (2.8 mM) (d) T2b (2.9 mM) (e) T3a (3.1 mM) (f) T3b (3.4 mM) (g) T4a (2.8 mM) (h) T4b (2.9 mM) (i) T5a (2.8 mM) (j) T5b (2.9 mM) in 1:1 CH3OH/CHCl3 respectively.
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(multiplet). High resolution mass spectra (HRMS) were recorded in Bruker MicrO-TOF-QII model using ESI technique. UV-visible spectra were recorded in Shimadzu double beam spectrophotometer, UV-2450. The emission spectra were recorded in HORIBA JOBIN YVON Scientific, fluoromax-4 spectrofluorometer with a slitwidth of 5 nm. The experimental procedure and spectral data of T4b and T5b are reported in ref. 2(b). 4.2. Microscopy methods 4.2.1. Optical microscopy. A drop of solution of the compound was put on a glass slide, the solvent was allowed to evaporate in air. It was then viewed using Nikon Eclipse TS100 optical microscope system.
Fig. 5. Fluorescence microscopic image (in 1:1 CH3OH/CHCl3) of (a) T3b (2.54 mM) alone (lex¼380e450 nm) (b) T3bþ0.02 equiv rhodamine B (lex¼510e560 nm) (c) (b) þ2.0 equiv TBAHSO4 (lex¼510e560 nm) (d) (b) þ2.0 equiv TBAHSO4 (lex¼380e450 nm).
4.2.2. Transmission electron microscopy. Around 3.0 mM solution of the sample in HPLC grade methanol/chloroform (1:1) was used for TEM. All the sample solutions were filtered through a nylon syringe filter (0.2 mm). About 2 ml aliquot of the sample solution was placed on a 200 mesh copper grid and stained with 2% wt. phosphotungstic acid in water for 2 min and the grid was allowed to dry in atmosphere. Samples were viewed using a Philips CM 12 transmission electron microscope. 4.2.3. Scanning electron microscopy. Around 3.0e4.0 mM solution of compound in HPLC grade methanol/chloroform (1:1) was prepared and one drop of the solution was put on the glass cover slip pasted on a carbon tape mounted on a stub, dried under sodium lamp and coated with w10 nm of gold. Samples were analyzed using scanning electron microscope ZEISS EVO 50 SEM. 4.2.4. Atomic force microscopy. Around 3.0e4.0 mM solution of compound in HPLC grade methanol/chloroform (1:1) was prepared and one drop of the solution was put on silicon wafer and dried in atmosphere. Samples were analyzed using Dimension Icon AFM operating at tapping mode in air. Images were recorded in air at room temperature and data analysis was performed using nanoscope 5.31r software. 4.3. Anion binding studies
Fig. 6. SEM images of T3b (a) 0.5 equiv (b) 1.0 equiv (c) 2.0 equiv of TBAHPO4. All experiments were done in 1:1 CH3OH/CHCl3.
containers for guest encapsulation and release. The present study also demonstrates the successful execution of the principle of molecular recognition to fabricate wiffle-ball like structure from vesicular assembly. 4. Experimental section 4.1. Materials and methods All organic solvents employed in the synthesis were distilled and dried from appropriate drying agents. Reactions were monitored by silica gel-based thin layer chromatography (TLC). Silica gel (100e200 mesh) was used for purification by column chromatography. Fisher-Scientific melting point apparatus was used for rege 460 spectrometer was used cording melting points. Nicolet, Prote for recording FT-IR. Compounds were used as KBr pellets. 1H NMR spectra were recorded on Brucker-DPX-300 spectrometer. Tetramethylsilane (1H) was used as an internal standard. Coupling constants are reported in Hz and data are reported as s (singlet), d (doublet), br (broad), t (triplet), dd (double doublet) and m
4.3.1. UVevis titration experiments. Stock solutions of compounds T1aebeT5aeb were prepared in UV spectroscopy grade chloroform with concentration (104e105 M). Solutions of anion salts were prepared in UV spectroscopy grade chloroform with concentration (101e102 M). The absorbance of blank compound solution and upon gradual addition of anion salt was recorded using Shimadzu double beam UVevis spectrophotometer model UV-2450. 4.3.2. Fluorescence titration experiments. Stock solutions of compounds T1aebeT5aeb were prepared in UV spectroscopy grade chloroform with concentration (104e105 M). Solutions of anion salts were prepared in UV spectroscopy grade chloroform with concentration (101e102 M). The fluorescence emission spectra of blank compound solution and upon gradual addition of anion salt were recorded using Horiba Jobin Yvon Scientific, fluoromax-4 spectrofluoremeter with slit width of 5 nm. 4.3.3. NMR titration experiments. Stock solutions of compounds T1aebeT5aeb were prepared in CDCl3 with concentration 103 M. Solutions of anion salts were prepared in CDCl3 with concentration 101 M. The spectra were collected on a Bruker DRX 300 (300 MHz) NMR spectrometer. Anion binding titrations were carried out by monitoring changes in the aromatic proton (ArH) and amide NH
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signal of receptors as a function of added tetrabutylammonium halide concentration. 4.3.4. Calculation of binding constants and stoichiometry. The binding constants were calculated by UVevis titration experiments using Benesiehildbrand plot as standardized for 1:1 and 1:2 stoichiometry. Stoichiometry of binding was calculated by Job’s plot from UVevis absorbance data. 4.4. Synthesis of T1a To an ice-cooled and stirred solution of 1a (0.500 g, 2.13 mmol) in 100 mL of dry CH2Cl2 and 0.3 mL DMF, was added N-hydroxysuccinimide (HOSu) (0.538 g, 4.68 mmol), DCC (0.965 g, 4.68 mmol) and stirred for 10 min. To the reaction mixture was added mixture of triethylamine (0.65 mL, 4.68 mmol), benzyl amine (0.51 mL, 4.68 mmol), dry CH2Cl2 (20 mL) and stirred for 24 h at room temperature. The reaction mixture was filtered and the filtrate was diluted with 100 mL of CH2Cl2, washed sequentially with 0.2 N H2SO4, saturated NaHCO3 solution and brine solution. The organic layer was dried over Na2SO4, and concentrated under reduced pressure. The crude product was chromatographed on a column of silica gel using ethyl acetate/hexane as eluents to obtain the pure product T1a as white solid. Mp: 160e162 C. Yield: 91%. Rf (60% ethyl acetate/hexane): 0.6. 1H NMR (300 MHz, DMSOd6) d 1.40 (s, 9H, eCH (CH3)3), 2.50e2.68 (m, 2H, Asp (CH2)), 4.28e4.36 (sþm, 5H, 4 ArCH2þ1 aeCH), 6.95 (d, 1H, J¼7.8 Hz, NH), 7.27e7.33 (m, 10H, ArH), 8.31 (br s, 2H, NH). 13C NMR (75 MHz, DMSO-d6) d 27.60, 37.03, 41.55, 51.14, 77.67, 126.01, 126.11, 126.33, 126.57, 127.53, 127.60, 138.75, 138.87, 154.53, 168.86, 170.79. IR (KBr) 1049, 1086, 1166, 1243, 1309, 1444, 1532, 1573, 1628, 1693, 2850, 2926, 3032, 3320 cm1. HRMS calcd for C23H29N3O4Na m/z 434.2050 found 434.2049. 4.5. Synthesis of T1b To an ice-cooled and stirred solution of 1b (0.250 g, 1.01 mmol) in 100 mL of dry CH2Cl2 and 0.3 mL DMF, was added HOSu (0.253 g, 2.2 mmol), DCC (0.453 g, 2.2 mmol) and stirred for 10 min. To the reaction mixture was added triethylamine (0.31 mL, 2.2 mmol) and benzyl amine (0.24 mL, 2.2 mmol) and stirred for 24 h. The reaction mixture was filtered and the filtrate was diluted with 100 mL of CH2Cl2, washed sequentially with 0.2 N H2SO4, saturated NaHCO3 solution and brine solution. The organic layer was dried over Na2SO4, and concentrated under reduced pressure. The crude product was chromatographed on a column of silica gel using ethyl acetate/hexane as eluents to obtain the pure product T1b as white solid. Mp: 123e125 C. Yield: 93%. Rf (100% ethyl acetate): 0.7. 1H NMR (300 MHz, CDCl3) d 1.41 (s, 9H, eCH (CH3)3), 1.68e2.10 (m, 2H, Glu (CH2)), 2.10e2.36 (m, 2H, Glu (CH2)), 4.13e4.30 (m, 1H, aeCH), 4.41 (d, 4H, J ¼ 5.4 Hz, 4 ArCH2), 5.71 (d, 1H, NH), 6.32 (br s, 1H, NH), 6.89 (t, 1H, J¼6 Hz, NH), 7.26e7.31 (m, 10H, ArH). 13C NMR (75 MHz, DMSO-d6) d 27.54, 31.40, 32.81, 41.51, 53.78, 77.59, 126.15, 126.47, 126.62, 127.65, 138.89, 139.04, 154.82, 171.03, 171.40. IR (KBr) 1051, 1087, 1166, 1243, 1275, 1312, 1445, 1530, 1574, 1629, 1688, 2851, 2926, 3032, 3323 cm1. HRMS calcd for C24H31N3O4Na m/z 448.2260 found 448.2197. 4.6. Synthesis of T2a To T1a (0.231 g, 0.53 mmol) in dry CH2Cl2 (4.0 mL) at 0 C, added trifluoroacetic acid (1.2 mL, 9.0 mmol) drop-wise and stirred for 4 h. Solvent was evaporated under high vacuum to give N-deprotected T1a (0.175 g). Yield: quantitative. To an ice-cooled and stirred solution of free amine (0.175 g, 0.53 mmol) in 100 mL of dry CH2Cl2 and 0.3 mL DMF, was added triethylamine (0.07 mL, 0.53 mmol)
followed by addition of p-nitrobenzoyl chloride (0.083 g, 0.44 mmol) in dry CH2Cl2 (20 mL) and stirred for 12 h. The reaction mixture was diluted with 100 mL of CH2Cl2, washed sequentially with 0.2 N H2SO4, saturated NaHCO3 solution and brine solution. The organic layer was dried over Na2SO4, and concentrated under reduced pressure. The crude product was chromatographed on a column of silica gel using ethyl acetate/hexane as eluents to obtain the pure product T2a as off-white solid. Mp: 223e225 C. Yield: 37%. Rf (4% methanol/chloroform): 0.6. 1H NMR (300 MHz, DMSO-d6) d 2.73 (m, 2H, Asp (CH2)), 4.27 (br s, 4H, ArCH2), 4.87 (m, 1H, aeCH), 7.20e7.24 (m, 10H, ArH), 8.08 (d, 2H, J¼7.5 Hz, ArH), 8.31 (d, 2H, J¼8.0 Hz, NH), 8.52 (br s, 1H, NH), 8.97 (br s, 1H, NH). 13 C NMR (75 MHz, DMSO-d6) d 22.14, 31.34, 42.91, 53.17, 121.05, 123.45, 126.60, 126.77, 126.98, 127.21, 128.29, 136.15, 139.32, 139.51, 140.89, 158.12, 170.96, 172.50. IR (KBr) 1000, 1025, 1641, 2258, 2360, 2851, 2921, 3433 cm1. HRMS calcd for C25H24N4O5Na m/z 483.1639 found 483.1630. 4.7. Synthesis of T2b To T1b (0.229 g, 0.53 mmol) in dry CH2Cl2 (4.0 mL) at 0 C, added trifluoroacetic acid (1.2 mL, 9.0 mmol) drop-wise and stirred for 4 h. Solvent was evaporated under high vacuum to give N-deprotected T1b (0.175 g). Yield: quantitative. To an ice-cooled and stirred solution of free amine (0.175 g, 0.53 mmol) in 100 mL of dry CH2Cl2 and 0.3 mL DMF, was added triethylamine (0.07 mL, 0.53 mmol) followed by addition of p-nitrobenzoyl chloride (0.083 g, 0.44 mmol) in dry CH2Cl2 (20 mL) and stirred for 12 h. The reaction mixture was diluted with 100 mL of CH2Cl2, washed sequentially with 0.2 N H2SO4, saturated NaHCO3 solution and brine solution. The organic layer was dried over Na2SO4, and concentrated under reduced pressure. The crude product was chromatographed on a column of silica gel using ethyl acetate/hexane as eluents to obtain the pure product T2b as white solid. Mp: 216e218 C. Yield: 30%. Rf (4% methanol/chloroform): 0.4. 1H NMR (300 MHz, DMSOd6) d 1.95e2.21 (m, 2H, Glu (CH2)), 2.28 (m, 2H, Glu (CH2)), 4.27 (dd, 4H, J1¼5.4 Hz, J2¼15 Hz, PhCH2), 4.43e4.46 (m, 1H, aeCH), 7.202e7.32 (m, 10H, ArH), 8.14 (d, 2H, J¼8.4 Hz, ArH), 8.31 (d, 2H, J¼8.7 Hz, ArH), 8.37 (t, 1H, J¼6 Hz, NH), 8.55 (t, 1H, J¼6 Hz, NH), 8.96 (d, 1H, J¼7.5 Hz, NH). 13C NMR (75 MHz, DMSO-d6) d 26.86, 31.45, 41.50, 53.21, 122.76, 126.10, 126.44, 126.59, 127.64, 128.50, 138.83, 138.92, 139.91, 148.50, 164.28, 170.60, 171.03. IR (KBr) 1027, 1076, 1234, 1641, 2258, 2360, 2851, 2921, 3433 cm1. HRMS calcd for C26H26N4O5Na m/z 497.1795 found 497.1794. 4.8. Synthesis of T3a To an ice-cooled and stirred solution of Boc-L-Tryptophan (0.122 g, 0.40 mmol) in 100 mL of dry CH2Cl2 and 0.3 mL DMF, was added HOSu (0.55 g, 0.48 mmol), DCC (0.100 g, 0.48 mmol) and stirred for 10 min. To the reaction mixture was added triethylamine (0.07 mL, 0.48 mmol) and N-deprotected T1a (0.151 g, 0.48 mmol) in dry CH2Cl2 (20 mL) and stirred for 24 h. The reaction mixture was diluted with 100 mL of CH2Cl2, washed sequentially with 0.2 N H2SO4, saturated NaHCO3 solution and brine solution. The organic layer was dried over Na2SO4, and concentrated under reduced pressure. The crude product was chromatographed on a column of silica gel using ethyl acetate/hexane as eluents to obtain the pure product T3a as off-white solid. Mp: 193e195 C. Yield: 34%. Rf (100% ethyl acetate): 0.7. 1H NMR (300 MHz, DMSO-d6) d 1.26 (s, 9H, eCH (CH3)3), 2.58 (br s, 2H, Asp (CH2)), 2.87e3.20 (m, 2H, Trp (CH2)), 4.02e4.26 (m, 5H, 4Ph (CH2)þ1 aeCH), 4.62 (m, 1H, aeCH), 6.90e7.32 (m, 15H, ArH), 7.55 (d, 1H, J¼7.5 Hz, NH), 8.12 (t, 1H, J¼3.9 Hz, NH), 8.31 (d, 1H, J¼7.9 Hz, NH), 8.42 (t, 1H, J¼3.9 Hz, NH), 10.80 (br s, 1H, indole NH). 13C NMR (75 MHz, DMSO-d6) d 27.23, 28.01, 36.89, 42.04, 50.00, 55.50, 78.26, 110.03, 111.19, 118.07, 118.28,
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120.72, 123.72, 126.50, 126.60, 126.79, 127.11, 127.60, 127.28, 128.04, 128.12, 136.01, 139.09, 139.20, 155.45, 169.40, 170.54, 171.74. IR (KBr) 1172, 1248, 1533, 1650, 1688, 2930, 2974, 3318, 3406 cm1. HRMS calcd for C34H39N5O5Na m/z 620.2843 found 620.2849. 4.9. Synthesis of T3b To an ice-cooled and stirred solution of Boc-L-Tryptophan (0.170 g, 0.56 mmol) in 100 mL of dry CH2Cl2 and 0.3 mL DMF, was added HOSu (0.69 g, 0.56 mmol), DCC (0.115 g, 0.56 mmol) and stirred for 10 min. To the reaction mixture, was added triethylamine (0.07 mL, 0.56 mmol) and N-deprotected T1b (0.152 g, 0.47 mmol) in dry CH2Cl2 (20 mL) and stirred for 12 h. The reaction mixture was diluted with 100 mL of CH2Cl2, washed sequentially with 0.2 N H2SO4, saturated NaHCO3 solution and brine solution. The organic layer was dried over Na2SO4, and concentrated under reduced pressure. The crude product was chromatographed on a column of silica gel using ethyl acetate/hexane as eluents to obtain the pure product T3b as light yellow solid. Mp: 155e157 C. Yield: 73%. Rf (100% ethyl acetate): 0.5. 1H NMR (300 MHz, DMSOd6) d 1.28 (s, 9H, eCH (CH3)3), 1.85e2.20 (m, 2H, Glu (CH2)), 2.21 (br s, 2H, Glu (CH2)), 2.58e2.94 (m, 2H, Trp (CH2)), 4.18e4.45 (m, 6H, 4Phe (CH2)þ2aeCH), 6.83e7.32 (m, 15H, ArH), 7.59 (d, 1H, J¼7.8 Hz, NH), 8.05 (d, 1H, J¼7.5 Hz, NH), 8.34 (m, 2H, NH), 10.89 (br s, 1H, indole NH). 13C NMR (75 MHz, DMSO-d6) d 25.14, 28.02, 31.64, 32.51, 41.99, 52.33, 55.31, 79.32, 110.13, 111.16, 118.05, 118.38, 120.70, 123.59, 126.58, 126.98, 127.07, 128.14, 135.99, 139.11, 139.52, 155.18, 171.43, 171.85, 172.60. IR (KBr) 1168, 1247, 1536, 1644, 1692, 2929, 2976, 3303, 3401 cm1. HRMS calcd for C35H41N5O5Na m/z 634.3000 found 634.3007. 4.10. Synthesis of T4a To an ice-cooled and stirred solution of 1a (0.200 g, 0.86 mmol) in 100 mL of dry CH2Cl2 and 0.3 mL DMF, was added HOSu (0.216 g, 1.88 mmol), DCC (0.388 g, 1.88 mmol) and stirred for 10 min. To the reaction mixture was added triethylamine (0.26 mL, 1.88 mmol) and Trp-OMe.HCl (0.477 g, 1.88 mmol) in dry CH2Cl2 (20 mL) and stirred for 24 h. The reaction mixture was filtered and the filtrate was diluted with 100 mL of CH2Cl2, washed sequentially with 0.2 N H2SO4, saturated NaHCO3 solution and brine solution. The organic layer was dried over Na2SO4, and concentrated under reduced pressure. The crude product was chromatographed on a column of silica gel using ethyl acetate/hexane as eluents to obtain the pure product T4a as white solid. Mp: 108e110 C. Yield: 55%. Rf (100% ethyl acetate): 0.5. 1H NMR (300 MHz, CDCl3) d 1.37 (s, 9H, eCH (CH3)3), 2.30e2.78 (m, 2H, Asp (CH2)), 3.22 (m, 4H, Trp (CH2)), 3.44e3.60 (sþs, 6H, eOCH3), 4.54 (m, 1H, aeCH), 4.80 (m, 2H, aeCH), 6.13 (d, 1H, J¼6.9 Hz, NH), 6.65 (d, 1H, J¼6.9 Hz, NH), 7.01e7.51 (m, 11H, 10ArHþ1NH), 8.33 (br s, 1H, indole NH), 8.54 (br s, 1H, indole NH). 13C NMR (75 MHz, DMSO-d6) d 24.84, 27.43, 28.14, 33.80, 49.10, 52.27, 52.89, 80.29, 109.20, 111.36, 118.31, 119.35, 121.88, 123.54, 127.30, 136.11, 155.61, 170.73, 171.16, 171.97. IR (KBr) 1100, 1169, 1215, 1342, 1438, 1524, 1658, 1736, 2852, 2930, 2952, 2977, 3004, 3057, 3386 cm1. HRMS calcd for C33H39N5O8Na m/z 656.2691 found 656.2682. 4.11. Synthesis of T5a To T4a (0.909 g, 0.25 mmol), added saturated solution of HCl in ethyl acetate (10.0 mL) at 0 C and stirred for 4 h. Solvent was evaporated under high vacuum to give N-deprotected T4a (0.160 g). Yield: quantitative. To an ice-cooled and stirred solution of Boc-LTrp-OH (0.76 g, 0.25 mmol) in 100 mL of dry CH2Cl2 and 0.3 mL DMF, was added HOSu (0.35 g, 0.30 mmol), DCC (0.62 g, 0.30 mmol) and stirred for 10 min. To the reaction mixture, was added
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triethylamine (0.04 mL, 0.30 mmol) and N-deprotected T4a (0.160 g, 0.30 mmol) in dry CH2Cl2 (20 mL) and stirred for 24 h. The reaction mixture was filtered and the filtrate was diluted with 100 mL of CH2Cl2, washed sequentially with 0.2 N H2SO4, saturated NaHCO3 solution and brine solution. The organic layer was dried over Na2SO4, and concentrated under reduced pressure. The crude product was chromatographed on a column of silica gel using ethyl acetate/hexane as eluents to obtain the pure product T5a as offwhite solid. Mp: 151e153 C. Yield: 50%. Rf (100% ethyl acetate): 0.6. 1H NMR (300 MHz, CDCl3) d 1.41 (s, 9H, eCH (CH3)3), 2.18e2.60 (m, 2H, Asp (CH2)), 3.11e3.49 (m, 6H, Trp (CH2)), 3.54e3.80 (sþs, 6H, eOCH3), 4.64e4.85 (m, 4H, aeCH), 5.30 (br s, 1H, NH), 6.80e7.65 (m, 18H, 15ArHþ3 NH), 8.14 (br s, 1H, NH), 8.46 (br s, 1H, NH), 8.57 (br s, 1H, NH). 13C NMR (75 MHz, DMSO-d6) d 22.51, 25.01, 26.99, 28.23, 29.63, 49.10, 52.27, 52.33, 52.89, 79.00, 109.34, 111.42, 118.31, 119.37, 121.89, 123.77, 127.10, 127.31, 136.08, 155.22, 170.30, 171.50, 172.29. 173.90, 174.090. IR (KBr) 1169, 1225, 1368, 1438, 1517, 1663, 1736, 2360, 2927, 3411 cm1. HRMS calcd for C44H49N7O9Na m/z 842.3484 found 842.3472. Acknowledgments We thank DST (Grant No. SB/S1/OC-23/2014), New Delhi for financial assistance. We acknowledge Department of Textile engineering, IITD for SEM images. We thank Dr. A. K. Panda, NII, New Delhi for providing fluorescence microscopic facility. IB thanks CSIR, New Delhi for the fellowship. Supplementary data Supplementary data (1H NMR, 13C NMR and HRMS spectral data, microscopic images and binding studies data is available in the supplementary file provided with this manuscript.) associated with this article can be found in the online version, at http://dx.doi.org/ 10.1016/j.tet.2016.04.002. References and notes 1. (a) Milroy, K. P.; Brunsveld, L. Org. Biomol. Chem. 2013, 11, 219e232; (b) Haridas, V.; Bijesh, M. B.; Chandra, A.; Sharma, S.; Shandilya, A. Chem. Commun. 2014, 50, 13797e13800. 2. (a) Zhang, S.; Marini, D. M.; Hwang, W.; Santoso, S. Curr. Opin. Chem. Biol. 2002, 6, 865e871; (b) Bhardwaj, I.; Jha, D.; Admane, P.; Panda, A. K.; Haridas, V. Bioorg. Med. Chem. Lett. 2016, 26, 672e676. 3. Tanner, P.; Baumann, P.; Enea, R.; Onaca, O.; Palivan, C.; Meier, W. Acc. Chem. Res. 2011, 44, 1039e1049. 4. Ghadiri, M. R.; Granja, J. R.; Buehler, L. K. Nature 1994, 369, 301e304. 5. Hoeben, F. J. M.; Jonkheijm, P.; Meijer, E. W.; Schenning, A. P. H. J. Chem. Rev. 2005, 105, 1491e1546. 6. Maggini, L.; Bonifazi, D. Chem. Soc. Rev. 2012, 41, 211e241. 7. Gudlur, S.; Sukthankar, P.; Gao, J.; Avila, L. A.; Hiromasa, Y.; Chen, J.; Iwamoto, T.; Tomich, J. M. PLoS One 2012, 7, e45374. 8. Naskar, J.; Roy, S.; Joardar, A.; Das, S.; Banerjee, A. Org. Biomol. Chem. 2011, 9, 6610e6615. 9. Tian, B.; Tao, X.; Ren, T.; Weng, Y.; Lin, X.; Zhang, Y.; Tang, X. J. Mater. Chem. 2012, 22, 17404e17414. 10. Fairman, R.; Akerfeldt, K. S. Curr. Opin. Strut. Biol. 2005, 15, 453e463. 11. Ghosh, S.; Singh, S. K.; Verma, S. Chem. Commun. 2007, 2296e2298. 12. Ghosh, S.; Singh, P.; Verma, S. Tetrahedron 2008, 64, 1250e1256. 13. Koley, P.; Pramanik, A. Soft Matter 2012, 8, 5364e5374. 14. Alarcon, C. D. H.; Pennadam, S.; Alexander, C. Chem. Soc. Rev. 2005, 34, 276e285. 15. Yao, Y.; Xue, M.; Chen, J.; Zhang, M.; Huang, F. J. Am. Chem. Soc. 2012, 134, 15712e15715. 16. (a) Haridas, V.; Sahu, S.; Venugopalan, P. Tetrahedron 2011, 67, 727e733; (b) Haridas, V.; Sahu, S.; Praveen Kumar, P. P. Tetrahedron Lett. 2011, 52, 6930e6934; (c) Haridas, V.; Sadanandan, S.; Hundal, G.; Suresh, C. H. Tetrahedron Lett. 2012, 53, 5523e5527. 17. (a) Berrocal, M. J.; Cruz, A.; Badr, I. H. A.; Bachas, L. G. Anal. Chem. 2000, 72, 5295e5299; (b) Kuswandi, B.; Nuriman; Verboom, W.; Reinhoudt, D. N. Sensors 2006, 6, 978e1017. 18. Reinoso-Garcia, M. M.; Dijkman, A.; Verboom, W.; Reinhoudt, D. N.; Malinoswka, E.; Wojciechowska, D.; Pietrzak, M.; Selucky, P. Eur. J. Org. Chem. 2005, 2131e2138. 19. Kubik, S. Chem. Soc. Rev. 2009, 38, 585e605.
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Self-assembling asymmetrical tripodal-like peptides as anion receptors Ishanki Bhardwaj, V. Haridas * Department of Chemistry, Indian Institute of Technology Delhi (IITD), Hauz Khas, New Delhi, 110016, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 February 2016 Received in revised form 31 March 2016 Accepted 1 April 2016 Available online 4 April 2016
Aspartic and glutamic-acid based molecules T1aebeT5aeb appended with different moieties were designed and synthesized. These molecules displayed unique self-assembly behavior dependent upon the nature of appended units. Trp favored vesicles, while phenyl unit favored fibrillar assembly. These molecules also showed binding towards anions. Ó 2016 Elsevier Ltd. All rights reserved.
Keywords: Molecular recognition Fluorophore Tryptophan Self-assembled vesicles Encapsulation
1. Introduction Molecular self-assembly is a powerful method for fabricating supramolecular structures.1 The formation of well-ordered supramolecular structures through self-assembly of diverse organic and inorganic building blocks has drawn much attention owing to their potential applications in biology and chemistry.2 Peptides possess hydrogen bonding capability; hence adopt specific structures through self-assembly. The intermolecular non-covalent interactions are utilized in designing diverse supramolecular structures such as fibers, vesicles, spheres, rods and helical ribbons. The potential applications of these assemblies involve their use in drug delivery, tissue engineering and wound healing.3e10 External stimuli such as temperature, pH, electric field, chemicals and ions can bring changes in the structure and solubility characteristics of assembly.11,12 Such self-assembling molecules have several applications in the design of stimuli responsive materials.13e15 Designed peptides can act as good receptors for anions as they have ability to bind guest through hydrogen bonds.16 The binding efficiency of a host depends upon its geometry and the cavity size. Designing tripodal receptors containing asymmetrical binding arms is an interesting area of host-guest chemistry research.17,18 Amino acids with functionalizable side chain seem to be ideal candidates for the design of tripodal receptors. The arms of the
* Corresponding author. Tel.: þ91 011 26591380; e-mail address: haridasv@iitd. ac.in (V. Haridas). http://dx.doi.org/10.1016/j.tet.2016.04.002 0040-4020/Ó 2016 Elsevier Ltd. All rights reserved.
tripodal receptors could offer more binding sites for guest and thereby achieve high binding affinity and selectivity. Appending with fluorophore can help in fluorescence detection of guests. Amino acids such as threonine, serine, tyrosine, histidine and tryptophan have OH or NH groups in their side chains, hence are useful models for designing receptors for anions.19 2. Results and discussion We report aspartic acid and glutamic acid-cored peptides T1aebeT5aeb with and without tryptophan residues (Fig. 1). T1aeb contain benzyl amine appended on Boc-L-Asp and Boc-L-Glu amino acid. T2aeb and T3aeb are synthesized by coupling pnitrobenzoic acid and Boc-L-Tryptophan to the N-terminal of T1a and T1b respectively. T4aeb and T5aeb constitute another class of scaffolds where Boc-Asp and Boc-Glu amino acid are partially or fully functionalized with tryptophan residues (Scheme 1, SI, Schemes S1 and S2). These diverse classes of molecules with amide functionalities provide an interesting set of molecules for anion binding and selfassembling studies. The three arms of aspartic and glutamic acid serve as branches to functionalize with different moieties to generate molecules with tripodal-like topology. Tryptophan provides indole NH as H-bond donor for binding to the guest and also helps in fluorescent detection of guests. Keeping in mind this concept, the binding efficiencies of T1aebeT5aeb towards anions such as F, Cl, Br, I, H2PO 4 and HSO 4 were analyzed by using spectroscopic techniques such as
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Fig. 1. Structural representation of tripodal receptors T1aebeT5aeb.
Scheme 1. Synthesis of tripodal peptides T1aebeT5aeb. (i) DCC, HOSu, NEt3, Benzyl amine, dry CH2Cl2, 0 C, 24 h (ii) Trifluoroacetic acid, dry CH2Cl2, 0 C, 4 h (iii) p-nitrobenzoyl chloride, NEt3, dry CH2Cl2, 0 C, 12 h (iv) Trifluoroacetic acid, dry CH2Cl2, 0 C, 4 h (v) Boc-L-Trp-OH, DCC, HOSu, NEt3, dry CH2Cl2, 0 C, 24 h (vi) DCC, HOSu, NEt3, L-Trp-OMe, dry CH2Cl2, 0 C, 24 h (vii) saturated soln. of HCl in ethyl acetate, 0 C, 6 h (viii) Boc-L-Trp-OH, DCC, HOSu, NEt3, dry CH2Cl2, 0 C, 24 h.
UVevis fluorescence and NMR (Figs. 2 and 3, SI, Figs. S1eS4). The anion binding studies were carried out in chloroform. UVevis and NMR titrations showed that T2b acts as a receptor for phosphate and chloride ion (Table 1). The occurrence of one isosbestic point in UV titration profile of T2b (4.5105 M) with phosphate (4.5102 M) is a noteworthy observation (Fig. 2a). Surprisingly, T2a (with one methylene less than T2b) didn’t show any changes in the UV spectra when titrated with TBAH2PO4, TBABr, TBAI and TBAHSO4. This may be due to steric crowding around the two amide NHs in T2a as compared to T2b. Presence of one additional methylene in T2b relieves the steric strain and hence increases the binding. One of the three arms of T1aeb is not functionalized in comparison to T2aeb and T3aeb. Therefore, T1aeb show differences in binding selectivity compared to tripodal receptors T2aeb
Fig. 2. (a) UV titration profile for T2b (4.5105 M) with (0e40 equiv) TBAH2PO4 (4.5102 M) in CHCl3 (b) Fluorescence titration profile for T4a (1.2104 M) with 1 TBAHSO M) in CHCl3. 4 (1.0810
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and T3aeb (Table 1). T1b showed higher binding compared to T1a due to same steric reasons. Changing the N-terminal substituent of T2aeb from p-nitrobenzoyl to Boc-L-Tryptophan provided T3aeb. The introduction of tryptophan residue was expected to increase the binding affinity as it can provide an additional NH for binding. Interestingly, the binding studies showed no considerable increase in binding affinity (Table 1). This may be due to orientation of tryptophan group away from the binding cavity, thereby not participating in the binding. The stoichiometry of binding for T1aebeT3aeb and T4beT5aeb with all anions is 1:1, while T4a binds to sulfate in 1:2 fashion (G:H 1:2) as evident from Job’s plot (SI, Fig. S2). In order to enhance the binding, T4aeb and T5aeb with two and three tryptophan residues were synthesized. UVevis and fluorescence titration experiments carried out with anions revealed that T4a efficiently binds to phosphate and sulfate. Fluorescence titration showed increase in fluorescence intensity at 445 nm upon slot-wise addition of TBAHSO4 in chloroform (Fig. 2b). 1H NMR titration showed the downfield shift of NHs upon addition of anion salt (Fig. 3). It is evident from Fig. 3, that amide NH(b) showed downfield shift from 6.32 ppm to 6.78 ppm while indole NH’s (d and e) showed downfield shift from 8.11 ppm to 9.12 ppm indicating that these NHs are involved in binding with the guest. T4b has less binding affinity as compared to T4a (Table 1). The extra methylene group present in T4b moves the side chains away from each other, thus decreasing the binding. When the Nterminal of T4aeb was functionalized by another tryptophan residue to give T5aeb, the observed binding affinity is comparatively lesser or similar (Table 1). The contribution towards binding is majorly due to two tryptophan residues. None of these receptors showed binding towards Br and I ions. As T1aebeT5aeb contain amide bonds, they can associate by intermolecular hydrogen bonds. In addition, they contain amino acid residues with side chains capable of pep interactions due to presence of aromatic groups. The presence of these moieties in the scaffolds can provide distinct self-assembling features. Hence, these peptides provide interesting candidates for self-assembly studies. The self-assembling features of T1aebeT5aeb were studied in 1:1 CH3OH/CHCl3 by several microscopic techniques such as scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM). The studies revealed that T1aeb and T2aeb self-assembled into fibers, while T3aebeT5aeb self-assembled into vesicles (Fig. 4, SI, Figs. S5eS6). The backbone of T1aebeT5aeb consists of Asp and Glu amino acids, while the arms are functionalized with different units. Thus, the self-assembly can be controlled by appended units. A closer look at the SEM images showed that the average size of vesicles of T3aebeT5aeb is 0.3e0.8 mm (SI, Fig. S7). TEM (stained with 0.2% phosphotungstic acid) revealed that T3aebeT5aeb formed vesicles in the range of 0.3e0.9 mm. After discovering the distinct morphological features of these peptides, we turned our attention to evaluate the encapsulating potency of the vesicles formed from self-assembly of T3aebeT5aeb. Fluorescence microscopic analysis of T3aebeT5aeb revealed that the vesicles can be seen in blue color (lex¼380e450 nm), while the rhodamine B entrapped vesicles appeared red (lex¼510e560 nm) (Fig. 5a,b) clearly indicating the encapsulation potency of rhodamine B by the vesicles. Addition of 0.2 equiv of sulfate salt to the dye entrapped vesicles resulted in the disruption of vesicles (Fig. 5c), since we could observe the reappearance of blue fluorescence of T3b (Fig. 5d). The vesicular morphology is lost due to anion binding, thus disrupting the self-assembly. The influence of anion binding on self-assembly of T1aebeT5aeb was studied by microscopic imaging. Experiments were performed by adding the anion to the self-assembled system
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Fig. 3. Partial 1H NMR (300 MHz, CDCl3) of T4a (3.9103 M) with and without addition of various amounts of TBAHSO4 (1.5101 M) (0e7.2 equiv) in CDCl3. Table 1 Binding affinities of T1aebeT5aeb with TBA anions in chloroform (NB¼not binding) Receptor Anion
T1a
T1b
T2a T2b
T3a T3b T4a
T4b
T5a
T5b
F CI H2PO 4 HSO 4
2300 NB NB NB
NB 4800 NB NB
NB NB NB NB
NB 360 NB NB
61,900 NB 630 NB
15,478 290 NB NB
450 NB 1300 NB
NB 1000 1000 NB
NB NB 22 NB
640 NB 8400 2.6105
of T1aebeT5aeb in 1:1 CH3OH/CHCl3. Addition of 0.5 equiv of anion salt resulted in the formation of holes on the surface of vesicles. Addition of 1.0 equiv resulted in the formation of wiffleball like structure (Fig. 6aec). The formation of wiffle-ball like
morphology is due to the anion binding on the self-assembled T3b, resulting in the removal of some T3b molecules from vesicles resulting in voids, which is observed in SEM as wiffle-ball. Addition of 2.0 equiv of anion to the self-assembled system completely destroys the vesicular assembly and formation of particles of indefinite shape is observed (Fig. 6c). 3. Conclusions In summary, we have designed and synthesized aspartic and glutamic acid based branched peptides. They displayed varied selfassembling architectures, such as fibers and vesicles. The anion binding property of these molecules is used to fabricate wiffle-ball like assembly. The vesicles as well as wiffle-ball like supramolecular self-assemblies offer opportunities to utilize these systems as
Fig. 4. SEM images of (a) T1a (3.1 mM) (b) T1b (3.4 mM) (c) T2a (2.8 mM) (d) T2b (2.9 mM) (e) T3a (3.1 mM) (f) T3b (3.4 mM) (g) T4a (2.8 mM) (h) T4b (2.9 mM) (i) T5a (2.8 mM) (j) T5b (2.9 mM) in 1:1 CH3OH/CHCl3 respectively.
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(multiplet). High resolution mass spectra (HRMS) were recorded in Bruker MicrO-TOF-QII model using ESI technique. UV-visible spectra were recorded in Shimadzu double beam spectrophotometer, UV-2450. The emission spectra were recorded in HORIBA JOBIN YVON Scientific, fluoromax-4 spectrofluorometer with a slitwidth of 5 nm. The experimental procedure and spectral data of T4b and T5b are reported in ref. 2(b). 4.2. Microscopy methods 4.2.1. Optical microscopy. A drop of solution of the compound was put on a glass slide, the solvent was allowed to evaporate in air. It was then viewed using Nikon Eclipse TS100 optical microscope system.
Fig. 5. Fluorescence microscopic image (in 1:1 CH3OH/CHCl3) of (a) T3b (2.54 mM) alone (lex¼380e450 nm) (b) T3bþ0.02 equiv rhodamine B (lex¼510e560 nm) (c) (b) þ2.0 equiv TBAHSO4 (lex¼510e560 nm) (d) (b) þ2.0 equiv TBAHSO4 (lex¼380e450 nm).
4.2.2. Transmission electron microscopy. Around 3.0 mM solution of the sample in HPLC grade methanol/chloroform (1:1) was used for TEM. All the sample solutions were filtered through a nylon syringe filter (0.2 mm). About 2 ml aliquot of the sample solution was placed on a 200 mesh copper grid and stained with 2% wt. phosphotungstic acid in water for 2 min and the grid was allowed to dry in atmosphere. Samples were viewed using a Philips CM 12 transmission electron microscope. 4.2.3. Scanning electron microscopy. Around 3.0e4.0 mM solution of compound in HPLC grade methanol/chloroform (1:1) was prepared and one drop of the solution was put on the glass cover slip pasted on a carbon tape mounted on a stub, dried under sodium lamp and coated with w10 nm of gold. Samples were analyzed using scanning electron microscope ZEISS EVO 50 SEM. 4.2.4. Atomic force microscopy. Around 3.0e4.0 mM solution of compound in HPLC grade methanol/chloroform (1:1) was prepared and one drop of the solution was put on silicon wafer and dried in atmosphere. Samples were analyzed using Dimension Icon AFM operating at tapping mode in air. Images were recorded in air at room temperature and data analysis was performed using nanoscope 5.31r software. 4.3. Anion binding studies
Fig. 6. SEM images of T3b (a) 0.5 equiv (b) 1.0 equiv (c) 2.0 equiv of TBAHPO4. All experiments were done in 1:1 CH3OH/CHCl3.
containers for guest encapsulation and release. The present study also demonstrates the successful execution of the principle of molecular recognition to fabricate wiffle-ball like structure from vesicular assembly. 4. Experimental section 4.1. Materials and methods All organic solvents employed in the synthesis were distilled and dried from appropriate drying agents. Reactions were monitored by silica gel-based thin layer chromatography (TLC). Silica gel (100e200 mesh) was used for purification by column chromatography. Fisher-Scientific melting point apparatus was used for rege 460 spectrometer was used cording melting points. Nicolet, Prote for recording FT-IR. Compounds were used as KBr pellets. 1H NMR spectra were recorded on Brucker-DPX-300 spectrometer. Tetramethylsilane (1H) was used as an internal standard. Coupling constants are reported in Hz and data are reported as s (singlet), d (doublet), br (broad), t (triplet), dd (double doublet) and m
4.3.1. UVevis titration experiments. Stock solutions of compounds T1aebeT5aeb were prepared in UV spectroscopy grade chloroform with concentration (104e105 M). Solutions of anion salts were prepared in UV spectroscopy grade chloroform with concentration (101e102 M). The absorbance of blank compound solution and upon gradual addition of anion salt was recorded using Shimadzu double beam UVevis spectrophotometer model UV-2450. 4.3.2. Fluorescence titration experiments. Stock solutions of compounds T1aebeT5aeb were prepared in UV spectroscopy grade chloroform with concentration (104e105 M). Solutions of anion salts were prepared in UV spectroscopy grade chloroform with concentration (101e102 M). The fluorescence emission spectra of blank compound solution and upon gradual addition of anion salt were recorded using Horiba Jobin Yvon Scientific, fluoromax-4 spectrofluoremeter with slit width of 5 nm. 4.3.3. NMR titration experiments. Stock solutions of compounds T1aebeT5aeb were prepared in CDCl3 with concentration 103 M. Solutions of anion salts were prepared in CDCl3 with concentration 101 M. The spectra were collected on a Bruker DRX 300 (300 MHz) NMR spectrometer. Anion binding titrations were carried out by monitoring changes in the aromatic proton (ArH) and amide NH
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signal of receptors as a function of added tetrabutylammonium halide concentration. 4.3.4. Calculation of binding constants and stoichiometry. The binding constants were calculated by UVevis titration experiments using Benesiehildbrand plot as standardized for 1:1 and 1:2 stoichiometry. Stoichiometry of binding was calculated by Job’s plot from UVevis absorbance data. 4.4. Synthesis of T1a To an ice-cooled and stirred solution of 1a (0.500 g, 2.13 mmol) in 100 mL of dry CH2Cl2 and 0.3 mL DMF, was added N-hydroxysuccinimide (HOSu) (0.538 g, 4.68 mmol), DCC (0.965 g, 4.68 mmol) and stirred for 10 min. To the reaction mixture was added mixture of triethylamine (0.65 mL, 4.68 mmol), benzyl amine (0.51 mL, 4.68 mmol), dry CH2Cl2 (20 mL) and stirred for 24 h at room temperature. The reaction mixture was filtered and the filtrate was diluted with 100 mL of CH2Cl2, washed sequentially with 0.2 N H2SO4, saturated NaHCO3 solution and brine solution. The organic layer was dried over Na2SO4, and concentrated under reduced pressure. The crude product was chromatographed on a column of silica gel using ethyl acetate/hexane as eluents to obtain the pure product T1a as white solid. Mp: 160e162 C. Yield: 91%. Rf (60% ethyl acetate/hexane): 0.6. 1H NMR (300 MHz, DMSOd6) d 1.40 (s, 9H, eCH (CH3)3), 2.50e2.68 (m, 2H, Asp (CH2)), 4.28e4.36 (sþm, 5H, 4 ArCH2þ1 aeCH), 6.95 (d, 1H, J¼7.8 Hz, NH), 7.27e7.33 (m, 10H, ArH), 8.31 (br s, 2H, NH). 13C NMR (75 MHz, DMSO-d6) d 27.60, 37.03, 41.55, 51.14, 77.67, 126.01, 126.11, 126.33, 126.57, 127.53, 127.60, 138.75, 138.87, 154.53, 168.86, 170.79. IR (KBr) 1049, 1086, 1166, 1243, 1309, 1444, 1532, 1573, 1628, 1693, 2850, 2926, 3032, 3320 cm1. HRMS calcd for C23H29N3O4Na m/z 434.2050 found 434.2049. 4.5. Synthesis of T1b To an ice-cooled and stirred solution of 1b (0.250 g, 1.01 mmol) in 100 mL of dry CH2Cl2 and 0.3 mL DMF, was added HOSu (0.253 g, 2.2 mmol), DCC (0.453 g, 2.2 mmol) and stirred for 10 min. To the reaction mixture was added triethylamine (0.31 mL, 2.2 mmol) and benzyl amine (0.24 mL, 2.2 mmol) and stirred for 24 h. The reaction mixture was filtered and the filtrate was diluted with 100 mL of CH2Cl2, washed sequentially with 0.2 N H2SO4, saturated NaHCO3 solution and brine solution. The organic layer was dried over Na2SO4, and concentrated under reduced pressure. The crude product was chromatographed on a column of silica gel using ethyl acetate/hexane as eluents to obtain the pure product T1b as white solid. Mp: 123e125 C. Yield: 93%. Rf (100% ethyl acetate): 0.7. 1H NMR (300 MHz, CDCl3) d 1.41 (s, 9H, eCH (CH3)3), 1.68e2.10 (m, 2H, Glu (CH2)), 2.10e2.36 (m, 2H, Glu (CH2)), 4.13e4.30 (m, 1H, aeCH), 4.41 (d, 4H, J ¼ 5.4 Hz, 4 ArCH2), 5.71 (d, 1H, NH), 6.32 (br s, 1H, NH), 6.89 (t, 1H, J¼6 Hz, NH), 7.26e7.31 (m, 10H, ArH). 13C NMR (75 MHz, DMSO-d6) d 27.54, 31.40, 32.81, 41.51, 53.78, 77.59, 126.15, 126.47, 126.62, 127.65, 138.89, 139.04, 154.82, 171.03, 171.40. IR (KBr) 1051, 1087, 1166, 1243, 1275, 1312, 1445, 1530, 1574, 1629, 1688, 2851, 2926, 3032, 3323 cm1. HRMS calcd for C24H31N3O4Na m/z 448.2260 found 448.2197. 4.6. Synthesis of T2a To T1a (0.231 g, 0.53 mmol) in dry CH2Cl2 (4.0 mL) at 0 C, added trifluoroacetic acid (1.2 mL, 9.0 mmol) drop-wise and stirred for 4 h. Solvent was evaporated under high vacuum to give N-deprotected T1a (0.175 g). Yield: quantitative. To an ice-cooled and stirred solution of free amine (0.175 g, 0.53 mmol) in 100 mL of dry CH2Cl2 and 0.3 mL DMF, was added triethylamine (0.07 mL, 0.53 mmol)
followed by addition of p-nitrobenzoyl chloride (0.083 g, 0.44 mmol) in dry CH2Cl2 (20 mL) and stirred for 12 h. The reaction mixture was diluted with 100 mL of CH2Cl2, washed sequentially with 0.2 N H2SO4, saturated NaHCO3 solution and brine solution. The organic layer was dried over Na2SO4, and concentrated under reduced pressure. The crude product was chromatographed on a column of silica gel using ethyl acetate/hexane as eluents to obtain the pure product T2a as off-white solid. Mp: 223e225 C. Yield: 37%. Rf (4% methanol/chloroform): 0.6. 1H NMR (300 MHz, DMSO-d6) d 2.73 (m, 2H, Asp (CH2)), 4.27 (br s, 4H, ArCH2), 4.87 (m, 1H, aeCH), 7.20e7.24 (m, 10H, ArH), 8.08 (d, 2H, J¼7.5 Hz, ArH), 8.31 (d, 2H, J¼8.0 Hz, NH), 8.52 (br s, 1H, NH), 8.97 (br s, 1H, NH). 13 C NMR (75 MHz, DMSO-d6) d 22.14, 31.34, 42.91, 53.17, 121.05, 123.45, 126.60, 126.77, 126.98, 127.21, 128.29, 136.15, 139.32, 139.51, 140.89, 158.12, 170.96, 172.50. IR (KBr) 1000, 1025, 1641, 2258, 2360, 2851, 2921, 3433 cm1. HRMS calcd for C25H24N4O5Na m/z 483.1639 found 483.1630. 4.7. Synthesis of T2b To T1b (0.229 g, 0.53 mmol) in dry CH2Cl2 (4.0 mL) at 0 C, added trifluoroacetic acid (1.2 mL, 9.0 mmol) drop-wise and stirred for 4 h. Solvent was evaporated under high vacuum to give N-deprotected T1b (0.175 g). Yield: quantitative. To an ice-cooled and stirred solution of free amine (0.175 g, 0.53 mmol) in 100 mL of dry CH2Cl2 and 0.3 mL DMF, was added triethylamine (0.07 mL, 0.53 mmol) followed by addition of p-nitrobenzoyl chloride (0.083 g, 0.44 mmol) in dry CH2Cl2 (20 mL) and stirred for 12 h. The reaction mixture was diluted with 100 mL of CH2Cl2, washed sequentially with 0.2 N H2SO4, saturated NaHCO3 solution and brine solution. The organic layer was dried over Na2SO4, and concentrated under reduced pressure. The crude product was chromatographed on a column of silica gel using ethyl acetate/hexane as eluents to obtain the pure product T2b as white solid. Mp: 216e218 C. Yield: 30%. Rf (4% methanol/chloroform): 0.4. 1H NMR (300 MHz, DMSOd6) d 1.95e2.21 (m, 2H, Glu (CH2)), 2.28 (m, 2H, Glu (CH2)), 4.27 (dd, 4H, J1¼5.4 Hz, J2¼15 Hz, PhCH2), 4.43e4.46 (m, 1H, aeCH), 7.202e7.32 (m, 10H, ArH), 8.14 (d, 2H, J¼8.4 Hz, ArH), 8.31 (d, 2H, J¼8.7 Hz, ArH), 8.37 (t, 1H, J¼6 Hz, NH), 8.55 (t, 1H, J¼6 Hz, NH), 8.96 (d, 1H, J¼7.5 Hz, NH). 13C NMR (75 MHz, DMSO-d6) d 26.86, 31.45, 41.50, 53.21, 122.76, 126.10, 126.44, 126.59, 127.64, 128.50, 138.83, 138.92, 139.91, 148.50, 164.28, 170.60, 171.03. IR (KBr) 1027, 1076, 1234, 1641, 2258, 2360, 2851, 2921, 3433 cm1. HRMS calcd for C26H26N4O5Na m/z 497.1795 found 497.1794. 4.8. Synthesis of T3a To an ice-cooled and stirred solution of Boc-L-Tryptophan (0.122 g, 0.40 mmol) in 100 mL of dry CH2Cl2 and 0.3 mL DMF, was added HOSu (0.55 g, 0.48 mmol), DCC (0.100 g, 0.48 mmol) and stirred for 10 min. To the reaction mixture was added triethylamine (0.07 mL, 0.48 mmol) and N-deprotected T1a (0.151 g, 0.48 mmol) in dry CH2Cl2 (20 mL) and stirred for 24 h. The reaction mixture was diluted with 100 mL of CH2Cl2, washed sequentially with 0.2 N H2SO4, saturated NaHCO3 solution and brine solution. The organic layer was dried over Na2SO4, and concentrated under reduced pressure. The crude product was chromatographed on a column of silica gel using ethyl acetate/hexane as eluents to obtain the pure product T3a as off-white solid. Mp: 193e195 C. Yield: 34%. Rf (100% ethyl acetate): 0.7. 1H NMR (300 MHz, DMSO-d6) d 1.26 (s, 9H, eCH (CH3)3), 2.58 (br s, 2H, Asp (CH2)), 2.87e3.20 (m, 2H, Trp (CH2)), 4.02e4.26 (m, 5H, 4Ph (CH2)þ1 aeCH), 4.62 (m, 1H, aeCH), 6.90e7.32 (m, 15H, ArH), 7.55 (d, 1H, J¼7.5 Hz, NH), 8.12 (t, 1H, J¼3.9 Hz, NH), 8.31 (d, 1H, J¼7.9 Hz, NH), 8.42 (t, 1H, J¼3.9 Hz, NH), 10.80 (br s, 1H, indole NH). 13C NMR (75 MHz, DMSO-d6) d 27.23, 28.01, 36.89, 42.04, 50.00, 55.50, 78.26, 110.03, 111.19, 118.07, 118.28,
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120.72, 123.72, 126.50, 126.60, 126.79, 127.11, 127.60, 127.28, 128.04, 128.12, 136.01, 139.09, 139.20, 155.45, 169.40, 170.54, 171.74. IR (KBr) 1172, 1248, 1533, 1650, 1688, 2930, 2974, 3318, 3406 cm1. HRMS calcd for C34H39N5O5Na m/z 620.2843 found 620.2849. 4.9. Synthesis of T3b To an ice-cooled and stirred solution of Boc-L-Tryptophan (0.170 g, 0.56 mmol) in 100 mL of dry CH2Cl2 and 0.3 mL DMF, was added HOSu (0.69 g, 0.56 mmol), DCC (0.115 g, 0.56 mmol) and stirred for 10 min. To the reaction mixture, was added triethylamine (0.07 mL, 0.56 mmol) and N-deprotected T1b (0.152 g, 0.47 mmol) in dry CH2Cl2 (20 mL) and stirred for 12 h. The reaction mixture was diluted with 100 mL of CH2Cl2, washed sequentially with 0.2 N H2SO4, saturated NaHCO3 solution and brine solution. The organic layer was dried over Na2SO4, and concentrated under reduced pressure. The crude product was chromatographed on a column of silica gel using ethyl acetate/hexane as eluents to obtain the pure product T3b as light yellow solid. Mp: 155e157 C. Yield: 73%. Rf (100% ethyl acetate): 0.5. 1H NMR (300 MHz, DMSOd6) d 1.28 (s, 9H, eCH (CH3)3), 1.85e2.20 (m, 2H, Glu (CH2)), 2.21 (br s, 2H, Glu (CH2)), 2.58e2.94 (m, 2H, Trp (CH2)), 4.18e4.45 (m, 6H, 4Phe (CH2)þ2aeCH), 6.83e7.32 (m, 15H, ArH), 7.59 (d, 1H, J¼7.8 Hz, NH), 8.05 (d, 1H, J¼7.5 Hz, NH), 8.34 (m, 2H, NH), 10.89 (br s, 1H, indole NH). 13C NMR (75 MHz, DMSO-d6) d 25.14, 28.02, 31.64, 32.51, 41.99, 52.33, 55.31, 79.32, 110.13, 111.16, 118.05, 118.38, 120.70, 123.59, 126.58, 126.98, 127.07, 128.14, 135.99, 139.11, 139.52, 155.18, 171.43, 171.85, 172.60. IR (KBr) 1168, 1247, 1536, 1644, 1692, 2929, 2976, 3303, 3401 cm1. HRMS calcd for C35H41N5O5Na m/z 634.3000 found 634.3007. 4.10. Synthesis of T4a To an ice-cooled and stirred solution of 1a (0.200 g, 0.86 mmol) in 100 mL of dry CH2Cl2 and 0.3 mL DMF, was added HOSu (0.216 g, 1.88 mmol), DCC (0.388 g, 1.88 mmol) and stirred for 10 min. To the reaction mixture was added triethylamine (0.26 mL, 1.88 mmol) and Trp-OMe.HCl (0.477 g, 1.88 mmol) in dry CH2Cl2 (20 mL) and stirred for 24 h. The reaction mixture was filtered and the filtrate was diluted with 100 mL of CH2Cl2, washed sequentially with 0.2 N H2SO4, saturated NaHCO3 solution and brine solution. The organic layer was dried over Na2SO4, and concentrated under reduced pressure. The crude product was chromatographed on a column of silica gel using ethyl acetate/hexane as eluents to obtain the pure product T4a as white solid. Mp: 108e110 C. Yield: 55%. Rf (100% ethyl acetate): 0.5. 1H NMR (300 MHz, CDCl3) d 1.37 (s, 9H, eCH (CH3)3), 2.30e2.78 (m, 2H, Asp (CH2)), 3.22 (m, 4H, Trp (CH2)), 3.44e3.60 (sþs, 6H, eOCH3), 4.54 (m, 1H, aeCH), 4.80 (m, 2H, aeCH), 6.13 (d, 1H, J¼6.9 Hz, NH), 6.65 (d, 1H, J¼6.9 Hz, NH), 7.01e7.51 (m, 11H, 10ArHþ1NH), 8.33 (br s, 1H, indole NH), 8.54 (br s, 1H, indole NH). 13C NMR (75 MHz, DMSO-d6) d 24.84, 27.43, 28.14, 33.80, 49.10, 52.27, 52.89, 80.29, 109.20, 111.36, 118.31, 119.35, 121.88, 123.54, 127.30, 136.11, 155.61, 170.73, 171.16, 171.97. IR (KBr) 1100, 1169, 1215, 1342, 1438, 1524, 1658, 1736, 2852, 2930, 2952, 2977, 3004, 3057, 3386 cm1. HRMS calcd for C33H39N5O8Na m/z 656.2691 found 656.2682. 4.11. Synthesis of T5a To T4a (0.909 g, 0.25 mmol), added saturated solution of HCl in ethyl acetate (10.0 mL) at 0 C and stirred for 4 h. Solvent was evaporated under high vacuum to give N-deprotected T4a (0.160 g). Yield: quantitative. To an ice-cooled and stirred solution of Boc-LTrp-OH (0.76 g, 0.25 mmol) in 100 mL of dry CH2Cl2 and 0.3 mL DMF, was added HOSu (0.35 g, 0.30 mmol), DCC (0.62 g, 0.30 mmol) and stirred for 10 min. To the reaction mixture, was added
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triethylamine (0.04 mL, 0.30 mmol) and N-deprotected T4a (0.160 g, 0.30 mmol) in dry CH2Cl2 (20 mL) and stirred for 24 h. The reaction mixture was filtered and the filtrate was diluted with 100 mL of CH2Cl2, washed sequentially with 0.2 N H2SO4, saturated NaHCO3 solution and brine solution. The organic layer was dried over Na2SO4, and concentrated under reduced pressure. The crude product was chromatographed on a column of silica gel using ethyl acetate/hexane as eluents to obtain the pure product T5a as offwhite solid. Mp: 151e153 C. Yield: 50%. Rf (100% ethyl acetate): 0.6. 1H NMR (300 MHz, CDCl3) d 1.41 (s, 9H, eCH (CH3)3), 2.18e2.60 (m, 2H, Asp (CH2)), 3.11e3.49 (m, 6H, Trp (CH2)), 3.54e3.80 (sþs, 6H, eOCH3), 4.64e4.85 (m, 4H, aeCH), 5.30 (br s, 1H, NH), 6.80e7.65 (m, 18H, 15ArHþ3 NH), 8.14 (br s, 1H, NH), 8.46 (br s, 1H, NH), 8.57 (br s, 1H, NH). 13C NMR (75 MHz, DMSO-d6) d 22.51, 25.01, 26.99, 28.23, 29.63, 49.10, 52.27, 52.33, 52.89, 79.00, 109.34, 111.42, 118.31, 119.37, 121.89, 123.77, 127.10, 127.31, 136.08, 155.22, 170.30, 171.50, 172.29. 173.90, 174.090. IR (KBr) 1169, 1225, 1368, 1438, 1517, 1663, 1736, 2360, 2927, 3411 cm1. HRMS calcd for C44H49N7O9Na m/z 842.3484 found 842.3472. Acknowledgments We thank DST (Grant No. SB/S1/OC-23/2014), New Delhi for financial assistance. We acknowledge Department of Textile engineering, IITD for SEM images. We thank Dr. A. K. Panda, NII, New Delhi for providing fluorescence microscopic facility. IB thanks CSIR, New Delhi for the fellowship. Supplementary data Supplementary data (1H NMR, 13C NMR and HRMS spectral data, microscopic images and binding studies data is available in the supplementary file provided with this manuscript.) associated with this article can be found in the online version, at http://dx.doi.org/ 10.1016/j.tet.2016.04.002. References and notes 1. (a) Milroy, K. P.; Brunsveld, L. Org. Biomol. Chem. 2013, 11, 219e232; (b) Haridas, V.; Bijesh, M. B.; Chandra, A.; Sharma, S.; Shandilya, A. Chem. Commun. 2014, 50, 13797e13800. 2. (a) Zhang, S.; Marini, D. M.; Hwang, W.; Santoso, S. Curr. Opin. Chem. Biol. 2002, 6, 865e871; (b) Bhardwaj, I.; Jha, D.; Admane, P.; Panda, A. K.; Haridas, V. Bioorg. Med. Chem. Lett. 2016, 26, 672e676. 3. Tanner, P.; Baumann, P.; Enea, R.; Onaca, O.; Palivan, C.; Meier, W. Acc. Chem. Res. 2011, 44, 1039e1049. 4. Ghadiri, M. R.; Granja, J. R.; Buehler, L. K. Nature 1994, 369, 301e304. 5. Hoeben, F. J. M.; Jonkheijm, P.; Meijer, E. W.; Schenning, A. P. H. J. Chem. Rev. 2005, 105, 1491e1546. 6. Maggini, L.; Bonifazi, D. Chem. Soc. Rev. 2012, 41, 211e241. 7. Gudlur, S.; Sukthankar, P.; Gao, J.; Avila, L. A.; Hiromasa, Y.; Chen, J.; Iwamoto, T.; Tomich, J. M. PLoS One 2012, 7, e45374. 8. Naskar, J.; Roy, S.; Joardar, A.; Das, S.; Banerjee, A. Org. Biomol. Chem. 2011, 9, 6610e6615. 9. Tian, B.; Tao, X.; Ren, T.; Weng, Y.; Lin, X.; Zhang, Y.; Tang, X. J. Mater. Chem. 2012, 22, 17404e17414. 10. Fairman, R.; Akerfeldt, K. S. Curr. Opin. Strut. Biol. 2005, 15, 453e463. 11. Ghosh, S.; Singh, S. K.; Verma, S. Chem. Commun. 2007, 2296e2298. 12. Ghosh, S.; Singh, P.; Verma, S. Tetrahedron 2008, 64, 1250e1256. 13. Koley, P.; Pramanik, A. Soft Matter 2012, 8, 5364e5374. 14. Alarcon, C. D. H.; Pennadam, S.; Alexander, C. Chem. Soc. Rev. 2005, 34, 276e285. 15. Yao, Y.; Xue, M.; Chen, J.; Zhang, M.; Huang, F. J. Am. Chem. Soc. 2012, 134, 15712e15715. 16. (a) Haridas, V.; Sahu, S.; Venugopalan, P. Tetrahedron 2011, 67, 727e733; (b) Haridas, V.; Sahu, S.; Praveen Kumar, P. P. Tetrahedron Lett. 2011, 52, 6930e6934; (c) Haridas, V.; Sadanandan, S.; Hundal, G.; Suresh, C. H. Tetrahedron Lett. 2012, 53, 5523e5527. 17. (a) Berrocal, M. J.; Cruz, A.; Badr, I. H. A.; Bachas, L. G. Anal. Chem. 2000, 72, 5295e5299; (b) Kuswandi, B.; Nuriman; Verboom, W.; Reinhoudt, D. N. Sensors 2006, 6, 978e1017. 18. Reinoso-Garcia, M. M.; Dijkman, A.; Verboom, W.; Reinhoudt, D. N.; Malinoswka, E.; Wojciechowska, D.; Pietrzak, M.; Selucky, P. Eur. J. Org. Chem. 2005, 2131e2138. 19. Kubik, S. Chem. Soc. Rev. 2009, 38, 585e605.