A new 3,4-dihydroxypyrrolidine-based material for molecular recognition

The synthesis of a new pseudopeptide material based on a chiral pyrrolidine skeleton is described. One of these new compounds interacts, in chloroform solution, selectively with amines.


Introduction
Molecular recognition phenomena are the basis of any biological process and extensive effort has been spent to understand the mechanism of such processes and to discover new examples and applications.Since large part of recognition processes, particularly those in biological systems, are based on the formation of hydrogen bonds between host and guest molecules, 1 most effort are devoted to the construction of a host abundant of hydrogen bond donating groups as , aminoacids, carbohydrates units and sulfonamides. 2One direction chosen by researcher on the way to build up a synthetic enzyme, consists of the selection of scaffold onto which easily accessible optically pure units, containing elements necessary for recognition, are implemented. 2,3Trimesic acid (benzene-1,3,5-tricarboxylic acid) has been often used as a rigid scaffold for the grafting of proper substituents and, recently, it has been demonstrated that amide derivatives of trimesic acid can adopt, in the solid phase, a bell shaped arrangement with the three carbonyl group pointing towards the same side of the aromatic ring. 4Such arrangement give rise to a cavity that could host small molecules properly substituted. 5The potential for application in molecular recognition processes is evident.Along this track we propose now a new material 1 (figure 1) based on a pseudo peptidic branch, built up around a C 2 symmetric pyrrolidine skeleton, implemented in a rigid frame of trimesic acid.In this article the synthesis of three derivatives, compounds 1a-c, characterized by the presence of three different aminoacids, as well as the application of compound 1a to the selective recognition of amines are described.

Results and Discussion
The synthesis of 1a-c was devised in a convergent way, i.e. by the synthesis of branches that are inserted in the central scaffold.The approach is suitable for further application in combinatoriallike synthesis.
Again the overall C 3 symmetry of compounds 1a-c is confirmed by the simple 1 H and 13 C NMR spectra of these compounds.The spectra of compound 1a are representative of all three compounds.Concerning the 1 H NMR spectrum, compound 1a showed a single resonance at 7.89 ppm for the three equivalent protons of the central aromatic ring.The amide moieties that link the pyrrolidine rings to the core hamper the free rotation along the N-C bond disrupting the C 2 simmetry of the two substituents of the branches and the H3 and H4 of each pyrrolidine ring resonate as separate signals giving rise to a multiplet at 5.24 ppm.Nevertheless the three groups grafted onto the central aromatic ring result equivalent.Also the 13 C NMR spectrum confirmed the absence of C 2 simmetry for each pyrrolidine nucleus (different signal for the two methylene groups at 52.4 and 50.5 ppm) but the equivalence of the three branches.
Tertiary aromatic amides are not planar for steric reasons.In tertiary benzamides steric hindrance surrenders to conjugation when the amide is twisted at an angle 57° to the ring. 6For this reason the three group grafted around the aromatic core of trimesic acid do not lie in the same plane but stretch out in space with the possibility to build a network of hydrogen bonds that could give rise to a bell shaped structure in solution.The result of a molecular mechanics study (Monte Carlo method, Force field MMFF94, 7744 cycles) 8 on a model compound showed that the minimum energy conformation is the one depicted in figure 2. This bell shaped conformation is stabilized by four hydrogen bond which link together the three branches of the molecule.
Compound 1a-c do not self-aggregate in solution as it is shown by 1 H NMR spectroscopy: no variation of chemical shift was evidenced recording spectra at increasing concentration from 10 -3 to 10 -1 M in CDCl 3 .A FT-IR analysis for compound 1a did not confirm definitely the presence of intramolecular hydrogen bonds in chloroform.However the spectrum at dilute (10 -3 M) concentration presented two slightly defined NH bands at 3450 and 3440 cm -1 that collapsed in a single broader resonance at 3440 cm -1 by increasing the concentration to 10 -2 M due to intra and intermolecular hydrogen bonding.
To verify the possibility of molecular recognition towards substrates prone to form hydrogen bond interactions we registered 1 H NMR spectra of 1 : 1 mixtures of hosts 1a-c and various structurally differentiated guests.A marked variation (at least 0.2 ppm) in chemical shift of any signal was considered to indicate an interaction worthwhile to be deeper analysed.
Compound 1a revealed to interact with amines, aminoalcohols and, to a lower extent, with primary and secondary amides while no interaction was evidenced with carboxylic acids, ammonium salt, carboxylates, alcohols and tertiary amides.Compounds 1b-c did not show any appreciable interaction towards any of these substrates.A deeper analysis was performed to quantify the interaction of 1a towards piperidine for which the strongest interaction was evidenced (shift of 1 ppm for the NH proton at 1:1 piperidine/1a ratio).In figure 3 is reported the titration curve of piperidine (NH signal) with 1a.The graphic shown in figure 2 indicates the saturation obtained increasing the concentration of host 1a from 5x10 -4 to 2.4x10 -2 M. The non-linear regression analysis data for titration of piperidine indicated a K ass = 380 ± 80 M -1 together with a δ ∞ = 2.14 ppm. 9 A Job plot, showing a maximum at 0.5 for the system piperidine-1a confirms a 1 : 1 interaction between the two molecules (Figure 4).To verify if the presence of three group grafted onto the aromatic ring is crucial for the recognition process, the complexation ability of 1a was compared with that of compounds 6 and 7, similar to 1a, but lacking of its C 3 symmetry.Compounds 6 and 7 were obtained through reaction of 5 with benzoyl and terephtaloyl chloride respectively (figure 5).None of the substrates screened with 1a showed an interaction with 6 and 7, even increasing the concentration of the host, ruling out also the possible acid-base reaction between piperidine and compound 1a.

Figure 5
The C 3 symmetry of compound 1a, therefore, must induce an appropriate arrangement of hydrogen bond donating groups which lacks in compounds 6 and 7, unable to interact with any substrates, and reveals essential to build up the complexation.Such arrangement might be described on the basis of the structure depicted in Figure 2. The selectivity observed and the 1:1 interaction between piperidine and compound 1a must derive, therefore, from a positive balance between attractive and repulsive interactions.Steric factors are probably responsible for the absence of interactions found between amines and 1b or 1c.A molecular mechanic study, in analogy to what reported for 1a, showed that the presence of substituents bulkier than Me on the aminoacidic residue hampers the formation of the bell shape conformation.In conclusion we have described the synthesis of a new compound characterised by an overall C 3 symmetry and by six terminal aminoacidic residues which showed selective complexing ability towards amines, aminoalcohols and, to a minor extent, amides.The extension of the synthetic procedure to obtain combinatorial libraries of these compounds and dendrimeric derivatives is currently ongoing in our laboratories, as well as experiments to gain a deeper insight in the interaction process.

Experimental Section
General Procedures.All operations were carried out under inert gas and with anhydrous solvents where required.Rf values refer to TLC on 0.25 mm silica gel plates (Merck F254) with the same eluent used for the chromatographic separation of compounds.H and C NMR spectra (in CDCl solution) were recorded at 200 MHz and 50 MHz respectively, with a Varian Gemini; the chemical shifts for H and C NMR spectra are given in ppm from TMS. IR spectra were recorded with a Perkin-Elmer 881 spectrophotometer.Optical rotation measurements were carried out with a Jasco DIP-370 polarimeter.

General procedure of synthesis of diesters 4a-c
A solution of 375 mg (1.94 mmol) of pyrrolidine 2 in 30 ml of anydrous CH 2 Cl 2 at room temperature and under nitrogen atmosphere, was added with an N-t-Boc protected aminoacid (alanine 3a, phenyl alanine 3b and lysine 3c) (3.88 mmol) and DMAP (20 mg, 0.16 mmol).The solution was then added with DCC (825 mg, 4 mmol) and stirred for 24 h.The final suspension was filtered and the solution concentrated to afford the crude reaction mixture which was purified by flash column chromatography.

General procedures of reductive debenzylation of diesters 4a-c
A solution of the diester (1 mmol) in MeOH (5 mL) was added with 100 mg of 20% Pd(OH) 2 /C and the resulting suspension was stirred under H 2 atmosphere for 12 h.The suspension was then filtered and the solution concentrated to afford the crude secondary amine in quantitative yield.The crude amines were immediately used without futher purification.

General procedures of synthesis of triamides of trimesic acid 1a-c
A solution of the amine 5 (1 mmol) in dry CH 2 Cl 2 (12 mL) was added with DMAP (20 mg, 0.16 mmol) and DIPEA (1.06 mL, 6 mmol).The solution was cooled in an ice bath and added with benzene-1,3,5-tricarbonyl chloride (87 mg, 0.33 mmol, 1 eq).The solution was stirred under inert atmosphere for 2 h.The solution was then diluted with CH 2 Cl 2 (50 mL) and washed with 0.5 M HCl, saturated NaHCO 3 and finally with brine.The organic phase was dried with Na 2 SO 4 and concentrated to afford the crude reaction mixture which was purified by passage on a short pad of silica gel.
The solution was stirred under inert atmosphere for 2 h.The solution was then diluted with CH 2 Cl 2 (50 mL) and washed with 0.5 M HCl, saturated NaHCO 3 and finally with brine.The organic phase was dried with Na 2 SO 4 and concentrated to afford the crude reaction mixture which was purified by passage on a short pad of silica gel.Amide (6).93% yield.R f (ethyl acetate petroleum ether 1:1) = 0.34.