Synthesis and complexing properties of diglycol resorcinarene podands

Synthesis of new podands from resorcinarene and diethylene glycols are reported. The binding properties of these podands with alkali metal cations was studied by means of ESI–MS. The experimental results for podands with long diethylene glycol arms show the stable inclusion complexes with one or two metal cations and high affinity for sodium and potassium ions. This podands under appropriate conditions can thus form a sufficiently long cavity to accommodate more than one metal ion inside without disturbance of the axial symmetry like an ion channel. Podand with shorter arms, obtained from ethylene glycol form complexes with 1:1 stoichiometry and also readily dimers or trimers. In the presence of alkali metal cations this podand selectively binds cesium ions. The significant affinity of synthesized podands for the biologically important alkali metal ions may affect living organisms. Antibacterial activities were tested with series of Gram-positive and Gram-negative bacteria.


Introduction
Podands which were originally simply ligands with two or more pendant polyether arms has not lost its importance today. The excellent binding properties of biological receptors are still assigned to self-assembly, molecular recognition and multivalency. Multiple-interaction ligands are useful both for cation binding and protein surface recognition, cell transfection or crystal engineering [1][2][3][4][5]. All these many possibilities are associated also with a wide panel of scaffolds used for the synthesis of modern podands. Good platforms for the construction of such ligands are calixarenes or cyclodextrins [6][7][8]. Macrocyclic oligomers with cavities, able to encapsulate guest molecules are often used alone as receptors but with the pendant polyether arms can give great results [9][10][11][12][13][14]. Resorcinarenes are similar, although the specific group of cavityshaped supramolecular compounds and basic structural units for cavity-shaped ligands. Eight hydroxyl groups of upper rim, easily form intermolecular hydrogen bonds and, depending on the pendant arms may determine shape of cavity podands.
In this paper we reported synthesis of some new podands from resorcinarene and diethylene glycols. NMR spectroscopy and mass spectrometry were used to determine structure and composition of alkali metal complexes with podands differ in arms. Affinities and efficacies of these podands for alkali metal cations were investigated and also anti-bacterial activities were tested.

Reagents and measurements
The 1H and 13C NMR spectra were measured by an NMR Bruker Avance II 600 MHz using chloroform-d as solvent. Elemental analysis was run on a Model 240 Perkin-Elmer CHN Analyzer. The ESI mass spectra were recorded on a micrO-TOF-Q II (Brucker) equipped with syringe pump. The dry gas flow rate was at 4.0 l/min; the dry heater operated at 180°C; the capillary voltage was set at 4,500 V and collision energy was at 10 eV. The sample solutions were prepared in acetonitrilewater mixture (1:1). Podands concentration was always 5 9 10 -4 mol dm -3 , concentrations of alkali metal chlorides were the same or larger (1 9 10 -3 and 1.5 9 10 -3 mol dm -3 ). Melting points were determined with a Büchi Melting Point B-540 and are uncorrected. Chromatographic separations were carried out by silica gel 60 (SiO 2 , Merck, particle size 0.040-0.063 mm, 230-240 mesh). All starting materials were purchased from Fluka and Merck and were all used as received. All solvents were of reagent grade and used without further purification.

Synthesis of diglycol resorcinarene podands
The synthesis were performed by Mannich reaction under previously described conditions [15] with one slight modification (Scheme 1).
Resorcinarene 1 with formaldehyde and iminodiacetic acid were heated in a mixture of equal volume of ethylene diglycol derivatives and acetonitrile. This large excess of ethylene diglycol derivatives significantly improves yields and, in the case of diglycol ethers causes precipitation of the product from the reaction mixture. Recrystallization from acetonitrile gave pure products 2b-e in 75-81 % yields. Purification of the product 2a obtained from the reaction with diethylene glycol was more difficult. Yields after crystallization or flash chromatography on silica did not exceed 36 %. Preparation of podand 3 was carried out according to the literature [

Antibacterial activity
The antibacterial activity of the reported podands was determined by agar plate disk diffusion method [16] against Staphylococcus epidermidis, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli ISO, Escherichia coli B, Proteus mirabilis R110 (mutant Ra type of S1959), Proteus mirabilis R45 (Re mutant of S1959 strain), Proteus mirabilis S1959. The podands were dissolved in DMSO at final concentration of 34 mg ml -1 . The cellulose disks (5 mm diameter) were sprayed 20 ll of each tested solution (680 lg/disk). Determination of the bacteria susceptibility of the new synthesized podands were performed on Mueller-Hinton agar cultivation medium (Biocorp). The following procedure was used: 100 ll of bacterial suspensions were poured onto Mueller-Hinton medium, next bacterial suspension was evenly distributed on the surface of the agar. In next step cellulose discs impregnated tested podands were laid on the Mueller-Hinton agar with bacterial cells. Petri dishes with bacterial cultures and discs with podands were incubated at 37°C for 18 h. After this time the bacteria growth inhibition zone (in mm) were measured. Each experiments were repeated at least three times.

Spectroscopic analyses
The 1 H-NMR spectra in CDCl 3 of podand 2a with terminal hydroxyl groups on the pendant diethylene glycol arms is shown in Fig. 1. Compared to the previously obtained from The 1 H-NMR spectra of the podands 2b-e with terminal alkoxy groups on the diethylene glycol arms are slightly different (Fig. 2). The signals are sharp as in the case of shorter podands synthesized from glycol ethers [15]. All podands obtained from resorcinarene and diglycols or their ether derivatives are thus in the crown conformation with C 4v symmetry. The high symmetry of the podands 2a and 2b is retained also in the presence of alkali cations during the titration experiments with potassium and cesium ions. This shows that the long diethylene glycol arms of podands does not interact independently with alkali metal cations by adjacent or opposite arms. All pendant diethylene glycol arms interact together and form a sufficiently long cavity to  (Fig. 3b) Similar analyses were performed also for other alkali metal cations. In each case, addition of equimolar amounts of ions resulted in the disappearance of the dimeric adduct signals and the base peak is the signal at m/z 724.448 [M?H?K] 2? . In the doubly charged group, adducts with the same two-metal ion [M?2c] 2? (c = Li, Na, K, Rb and Cs) are always formed but the relative abundance decreases with increase of the ion size (Fig. 4). At this concentrations, mixed adducts, containing e.g., sodium and potassium ions also were observed. The most intense signals of adducts with one metal cation was always observed for adduct with added cation and next for the sodium adduct at These data indicate that the podands 2b-e with terminal alkoxyl groups on the diethyl glycol moieties can form with their arm a long cavity able to accommodate a two metal ions.
The ESI-MS spectum of the podand 2a is completely different (Fig. 5). The most intense peaks are that for sodium [M?Na] ? and potassium [M?K] ? adduct ions at m/z 1,207.661 and 1,223.642, but dimeric or double charged adducts were not observed. When alkali metal chlorides were added to the sample solution spectra were difficult to interpret.
Quite different results were obtained for podand 3 with terminal pendant hydroxyl groups and shorter arms, obtained from ethylene glycol. In the mass spectrum of  The significant affinity of synthesized podands for the biologically important alkali metal ions inclined us to determine their effects on living organisms. The ability to form the including complexes with two ions, allows to assume that the glycol resorcinarene podands can acts as an artificial ion channels that may resulted in antibacterial activities. Resorcinarene podands antibacterial activities were tested with series of Gram-positive and Gram-negative bacteria ( Table 2).
Tested podands exhibited antibacterial activity against some Gram-positive (S. epidemidis, S. aureus) as well as Gram-negative (P. mirabilis) strains at dose 680 lg ml -1 . However comparison of podands 2a and 2e indicates that terminal groups on the pendant diether arms do not play a significant role in the interactions between podands and bacterial cells. More important, it seems, is length of the pendant arms. The podand 3, with ethylene glycol arms exhibit somewhat enhanced antibacterial activity than the podand 2e with longer diethylene glycol arms. This effect is even more pronounced when antibacterial tests are performed on Tryptic soy agar. When bacteria were cultivated in this medium, podand 3 have significant antibacterial properties, especially against P. mirabilis R45(Re). In contrary, podand 2e does not exhibit activity at all. The differences on antibacterial activities of two R mutants-Ra and Re and smooth S 1959 strain may suggests that presence of hydrophilic polysaccharides on bacterial cells surface may prevent anti-bacterial activities of podand 3. This result also suggested that hydrophilic surfaces like in Re mutant type (R45) are more prone towards podand 3. It will be interesting in future to test its activities against hydrophobic cell walls of Mycobaterium sp. The complexation of more than one alkali ion in the podand cavity observed in ESI-MS spectra does not significantly effect on bacteria. Studies with podand dose-dependent antibacterial effect are needed.

Conclusions
Summarizing, these results indicate that glycol resorcinarene podands have significant affinity for the biologically Table 2 The measurement of grow inhibition zone (mm) on Mueller-Hinton agar and tryptic soy agar Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.