Urea-cored peptides for anion binding and vesicle formation

Urea and urea-triazole cored peptides were synthesized and characterized. These compounds were analyzed for their potency to sense anions such as F – , Cl – , Br – , I – , H 2 PO 4 – and HSO 4 – by UV-vis and 1 H NMR spectroscopy. Urea-cored compounds showed very weak binding, while the urea-triazole containing compound showed high binding towards H 2 PO 4 – and HSO 4 – . The urea-triazole containing compound also acts as colorimetric sensor for F – and H 2 PO 4 – . Both classes of compounds favored vesicular assembly in 1:1 CH 3 OH/CHCl 3 .


Introduction
2] Therefore, considerable effort has been devoted to the design of receptors for anion binding and sensing.Anion binding is a great challenge because anions have different size, shape and charge and anion complexation is also pH-dependent. 1 Anion receptors can be positively charged or neutral; they bind anions mainly through hydrogen bonding and/or electrostatic interactions.[5][6][7][8][9][10] The pioneering work of Wilcox 11 and Hamilton 12 showed that urea moiety can bind anions, in particular anion such as acetate.4][15][16][17] Therefore, the urea unit has been taken as an excellent motif for the construction of anion receptors.The success of urea as a binding motif is due to the fact that it possesses two close N-H units that can chelate a spherical anion in bifurcate hydrogen bonding mode, giving rise to a six-membered ring (Figure 1a).The two parallel H-bonds with two oxygen atoms of a carboxylate results in an eight-membered ring (Figure 1b).The high affinity of urea towards anion is due to spatially close H-bond donor sites. 12Appending amino acids on urea-based core can introduce additional H-bonding centers which can help in enhancing binding affinity towards anions.Amino acids containing functionalizable side chains provide an opportunity to design versatile receptors.Peptides with a urea core could be considered as a mimic of anion binding proteins.In addition to providing NHs for binding to anions, the urea motif can form a supramolecular arrangement as a result of the urea α-type hydrogen bonding pattern (Figure 1c) leading to distinct self-assembling features.Incorporation of amino acid residues can result in diverse supramolecular structures through additional non-covalent interactions.The hydrogen bonding capability of peptides can assist in forming specific supramolecular structures such as spheres, vesicles, fibers, helical ribbons and rods. 19,20The structurally modified urea derivatives have been extensively explored in organocatalysis, 21,22 crystal engineering, 23 supramolecular gelation, [24][25][26] and as membrane transporters for anions. 27ompound 10 was synthesized by a different scheme (Scheme 2).First, urea derivative of L-leucine methyl ester (8) was prepared from NH2-Leu-OMe (7) using Scheme 1. Compound 8 was subjected to basic hydrolysis followed by reaction with propargylamine to give the dialkyne 9. Compound 9 was then reacted with p-nitrophenyl azide in the presence of CuI to give 1,2,3triazole containing compound 10.Introduction of Trp in place of Leu in 10 resulted in an insoluble compound.

Anion binding studies
These urea-cored and urea-triazole compounds provide an interesting class of molecules for anion binding and self-assembling studies.The scaffolds are functionalized with amino acid residues leucine and tryptophan.Tryptophan and triazole provide additional H-bond donors in the form of indole NH and CH respectively.Keeping in mind this concept, the binding efficiencies of the designed receptors were determined by UV-vis.and NMR spectroscopic techniques.UV-vis.and NMR titrations of 2 showed a very weak binding towards biphosphate with 1:1 stoichiometry (Figures S1 and S2).Compound 2 did not show much affinity towards other anions due to steric crowding by two indole units of Trp.The steric crowding around urea NHs is reduced in 4, in which Trp was replaced by the dipeptide L-Leu-Trp-OMe.UV-vis.and 1 H NMR titrations showed that 4 binds to acetate anion in 1:1 fashion (Figure 2, Figure S1) and for other anions, no observable changes in spectra were seen.The binding constant with CH3COO -in CHCl3 was calculated using 1:1 non-linear fit analysis 28 (http://www.supramolecular.org)and was found out to be 115.53M -1 (Figure S2). 1 H NMR titration of 4 with acetate was carried out in CDCl3 (Figure 2).Addition of tetrabutylammonium acetate to 4 resulted in downfield shift of urea protons (2.4 ppm), while the indole NH showed a downfield shift of 1.78 ppm (Table S1) indicating the binding of 4 to acetate.The amide protons did not take part in binding as evident from negligible changes in the chemical shift values upon addition of anion salt, this could be due steric hindrance by Leu-CH3 groups.
Compound 6 also surprisingly did not show considerable affinity towards anions which could be due to steric crowding around urea along with distantly placed Trp side chain.Urea-triazole containing scaffold 10 shows highest affinity amongst all of the compounds tested.Compound 10 forms 1:1 complex with the anions (Figure S1).UV-vis.titration carried out with fluoride, biphosphate and bisulfate showed one isosbestic point at 300 nm (Figure 3, Figure S2).The binding constants (calculated by performing 1:1 non-linear fit analysis) 28 for fluoride, biphosphate and bisulfate ions are reported in Table 1. 1 H NMR titration of 10 showed that upon slot-wise addition of TBAH2PO4 (Figure 3), the urea NHs shifted downfield (~ 2.0 ppm) and amide NH shifted downfield by approximately 1.0 ppm.The triazole CH protons (c) showed a downfield shift of ~ 0.80 ppm, 10, 29-30 while aromatic protons showed a small downfield shift (~ 0.30 ppm).The triazole protons shifted downfield (~ 0.20 ppm), while the aromatic protons showed a slight downfield shift (~ 0.10 ppm) (Figure S3).The stronger binding of 10 towards anions as compared to other receptors can be correlated to its structure.The p-nitrophenyl group enhances the acidity of triazole CH, which strengthens the hydrogen bonding.The less steric crowding around urea unit allows all NHs to take part in binding along with triazole CHs, hence increases the binding.Compound 10 also showed colorimetric response towards anions in acetone.The color change was monitored by adding 10.0 equiv. of TBA salts of anions to 4.34 × 10 -3 M solution of 10 in acetone.A dark brown color was observed in the case of F -and H2PO4 -(Figure 4).Thus, 10 acts as colorimetric sensor for anions and enable naked eye detection of anions.The binding constants of 2, 4, 6 and 10 are summarized in Table 1.5).The morphological features of 2, 4, 6 and 10 were also confirmed by transmission electron microscopy (TEM) (stained with 0.2 % phosphotungstic acid) and atomic force microscopy (AFM) (tapping mode) (Figure S4).Even though, there is a high propensity for urea-based molecules to form fibrous or sheet-like morphology, yet it is surprising to observe that 2, 4, 6 and 10 formed vesicles.It is noteworthy that 1(precursor of 2) shows fiber-like morphology, while 3 and 5 (precursors of 4 and 6 respectively) formed vesicles (Figure S5).Compound 1 contains lesser number of amide bonds and aromatic rings, compared to 2, 4, 6 and 10.This might be the reason for fibrous morphology of 1.Therefore, it shows the importance of number of amide bonds as well as aromatic rings in the molecule.Compounds 8 and 9, which are precursors of 10, formed fibrous morphology (Figure S5), thus underlining the importance of triazole and aromatic rings present in 10 to give vesicular structure.Aromatic rings may facilitate π-π stacking, while urea and amide NHs can form hydrogen bonding which results in this vesicular morphology.The size of vesicles obtained from microscopic studies range from 0.3-1.3m (Figure S6).This size approximately matches with the size obtained from dynamic light scattering (DLS) measurements (Figure S7).The DLS studies show the size of the vesicles ranges from 0.3-0.8m.
The effect of addition of anions on vesicles of 10 was studied by SEM and DLS.Addition of 5.0 equiv. of TBA anions (biphosphate/bisulfate) to a solution of 10 (Figure 6) resulted in the formation of flattened vesicles with increased size.Addition of biphosphate salt resulted in the formation of pores on the surface of vesicles (Figure 6).The increase in the average size of vesicles upon addition of 5.0 equiv. of anion salt is also supported by DLS studies (Figure S7).] The presence of vesicular structure even after addition of 5.0 equiv. of anion salt demonstrates the robust nature of the vesicles.

Conclusions
In summary, we have designed and synthesized urea functionalized peptides 2, 4, 6 and 10.The binding property of these peptides towards tetrabutylammonium salts of different anions was analyzed in detail.It was found that urea-cored compounds 4 and 6 have very weak affinity, while urea-triazole containing 10 turned out to be the most efficient receptor.Compound 10 acts as a colorimetric sensor for fluoride and biphosphate.The self-assembling studies on these urea-cored and urea-triazole containing peptides revealed that 2, 4, 6 and 10 self-assembled into vesicles.Addition of anions to the self-assembled vesicles of 10 resulted in the change of vesicles size.The unison of Trp or triazole with urea enabled strong bias towards vesicular self-assembly.Incorporation of triazole improved the anion binding ability of the urea-cored amino acid appended molecule.These findings will help in the design of new generation of self-assembling and anion binding molecules.

General information
All organic solvents employed in the synthesis were distilled and dried using appropriate drying agents.Reactions were monitored by silica gel-based thin layer chromatography (TLC).Silica gel (100-200 mesh) was used for purification by column chromatography.A Fisher-Scientific apparatus was used for recording melting points.FT-IR spectra were measured using a Nicolet Protégé 460 spectrometer using KBr pellets. 1 H NMR spectra were recorded on Brucker-DPX-300 spectrometer.Tetramethylsilane ( 1 H) 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 (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.

Calculation of binding constants and stoichiometry
The binding constants were calculated by a non-linear fitting using Bindfit (http://www.supramolecular.org).UV fitting for 1:1 model was done using the Nelder-Mead method.Stoichiometry of binding was calculated by Job plot from UV-vis.absorbance data.

Microscopy methods
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.
Transmission electron microscopy: Around 3.0 mM solution of the sample in methanolchloroform (1:1) was used for TEM.All the sample solutions were filtered through a nylon syringe filter (0.2 µm).About 2 μl aliquot of the sample solution was placed on a 200 mesh copper grid and stained with 0.2 % 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.Scanning electron microscopy: Around 3.0-4.0mM solution of compound in 1:1 methanolchloroform 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 ~ 10 nm of gold.Samples were analyzed using scanning electron microscope ZEISS EVO 50 SEM.
Atomic force microscopy: Around 3.0-4.0mM solution of compound in 1:1 methanol-chloroform 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. 18

Figure 1 .
Figure 1.Mode of binding in urea-based molecules by formation of two parallel H-bonds (a) forming six-membered ring with spherical anion (b) forming eight-membered ring with a compound containing a carboxylate group (R-COO -) (c) Intermolecular hydrogen bonds in ureabased molecules.
1 H NMR titration of 10 with TBAHSO4 showed that the urea NHs and amide NHs shifted downfield by

Figure 4 .
Figure 4. Images of vials showing colorimetric response of 10 (4.34 × 10 -3 M) after addition of 10.0 equiv of TBA anions in acetone