The new approach to organocatalysts. Synthesis of a library of N -lipidated oligopeptides immobilized on cellulose and screening of their catalytic activity

A library of N -lipidated tripeptides was prepared from amino acids characteristic of the active sites of hydrolytic enzymes: Ser, His, and Glu immobilized in the highly regular pattern on the cellulose surface according to TASP concept. It was found that such structures can accelerate the rate of hydrolysis of p-nitrophenyl esters of N -protected dipeptides.


Introduction
Structures formed by N-lipidated oligopeptides immobilized in a regular pattern on a cellulose surface via triazine linker are able to recognize the shape and properties of ligands, and then selectively bind guest molecules matching the requirements of the binding pocket. 1 Due to the conformational flexibility of both interacting partners, the relative direction of the functional groups of a ligand as well as that of the binding pockets could be readjusted to the most energetically favored orientation of both counterparts.Our previous studies documented that the process of binding guest molecules is reversible and competitive. 2Inspired with this observation, we assumed that under favorable circumstances the host structure could operate as a catalyst if suitable functional groups 3 were incorporated into the molecular fragments forming the binding pocket.In order to verify this hypothesis, we prepared a library of 36 supramolecular tripeptide structures with permutations of His, Glu, and Ser residues to study their catalytic activity.

Results and Discussion
According to the presented concept, catalytically active structures are expected to be formed by self-organization of N-lipidated oligopeptides immobilized on the cellulose surface.The synthesis commenced with modification of cellulose (1) surface by treatment with 2,4-dichloro-1,3,5-triazine (2) followed by reaction with m-phenylenediamine (3) according to the synthetic procedure described previously [1].The sub-libraries of tripeptides were prepared with the diversomer strategy using Fmoc-Glu(tBu)-OH (4), Fmoc-Ser(tBu)-OH (5) and Fmoc-His(Trt)-OH (6).Fmoc protection for the α-amino group was removed with 20% piperidine in DMF.In the synthesis of the peptide fragment, DMT/NMM/BF4 - (7) was used 4 as a coupling reagent.For the final acylation of immobilized tripeptides with carboxylic acid, DMT/NMM/TosO -(8) was found more suitable 5 as a coupling reagent.Acid-labile side-chain protecting groups were removed by treatment with 50% trifluoroacetic acid in dichloromethane.The final products of the synthesis was a library of 36 structures formed by permutations of Ser, Glu, His triade acylated with 6 long chain carboxylic acids 9-14.
In the preliminary experiments, p-nitrophenyl ester of Z-L-Leu-L-Leu-OH was applied as a substrate to determine the catalytic activity of N-lipidated tripeptides immobilized on cellulose.It is already known 6 that hydrolysis of this substrate proceeds effectively at pH ≥ 9.5 and a temperature exceeding 30 °C.Spectrophotometric determination of the amount of p-nitrophenol liberated confirmed that Z-L-Leu-L-Leu-ONp (15) is stable in buffered (pH = 7) aqueous methanol at 20 °C; however, all 36 structures designed as permutations of the catalytic triad catalyzed the hydrolysis of 15 under conditions described above (see Figure 1).
To disclose the structural fragments most important for hydrolytic activity, the progress of reaction reached after 418 min.at the final experiment of kinetic cycle measurements was compared for each carboxylic acid (Figure 3) and each amino acid sequence (Figure 4).It has been observed that both fragments control the reaction.Relatively poor reactivity was found for all peptides N-acylated with long chain, unsaturated (Z)-octadec-9-enoic acid 9 (Figure 3, set 9).The most uniform, yet still poor, reactivity has been shown by tripeptides N-acylated with long chain octadecanoic acid (14) (Figure 3, set 14).In both these cases catalytic activity was less depended on the sequence of peptidic fragment, but probably controlled by diffusion of substrate or products through the lipidic layer.Participation of peptide fragment in determining catalytic activity was depicted on Figure 4.The most diversified activity was obtained for structures prepared from relatively short chain undec-10-enoic acid (11) (Figure 4, set 11).Structure of peptide fragment was a factor dominating overall activity and undec-10-enoyl-HES-NH-C6H4-NH-1,3,5-triazinyl-cellulose (11,H,E,S), and undec-10-enoyl-HSE-NH-C6H4-NH-1,3,5-triazinyl-cellulose (11,H,S,E) were the catalysts selected from library as most efficient.The next in the rank were (12 ,H,S,E),  (13,H,S,E), and (10,S,H,E).In the four of them histidine occupied N-terminal position and in the one case, this residue was located in penultimate position.This strongly suggests the privileged peptide primary structure with histidine residue most exposed inside the binding pocket.For the most active catalysts the abundant in the second position was serine and in the third position glutamic acid residue.
The feature of the less reactive triad sequences was the presence of (Z)-octadec-9-enoic acid (9) and undec-10-enoic acid (11).The less reactive peptide sequences were SHE and ESH, but in both cases acylation with (9Z,12R)-12-hydroxyoctadec-9-enoic acid (10) substantially increased their catalytic effectiveness.All these results are consistent with our earlier observation indicating that for the efficient binding of ligands all the elements of the structure of N-lipidated peptides are necessary, and the most important factor determining binding and recognition of the ligand, is the structure of peptide fragment.
In order to check if the presence of all fragments of the designed structures were obligatory for their catalytic activity, tests for hydrolysis of Z-Ala-Aib-ONp (16) were conducted in the presence of: non-modified 1; cellulose modified with 2,4-dichloro-6-methoxy-1,3,5-triazine (DCMT) then with m-phenylenediamine (17); cellulose O-acylated with Fmoc-Ala-OH ( 18 All experiments confirmed that non-modified cellulose (1), incomplete structures 17-22 as well as structures 23-32 without amino acid residues characteristic of the catalytic triad did not promote the hydrolysis Z-Ala-Aib-ONp (16).

Conclusions
The preliminary data presented above unequivocally confirm that the new type of catalyst prepared by modification of cellulose surface with N-lipidated peptides promotes the hydrolysis of ester bonds under very mild conditions.In the case of the library formed by permutations amino acid residues of catalytic triad the most efficient catalysts were structures with the sequence His-Ser-Glu immobilized on cellulose via m-phenylenediamine and a 1,3,5-triazine scaffold one can thus hypothesize that the process proceeds inside the "binding pocket" and involves the side chain functionality of the peptide fragment.This is in agreement with our previous SAR studies, strongly suggesting that the process of ligand recognition depends mainly on the structure of amino acid residues. 7However, the observations collected so far there are not sufficient for mechanistic considerations of catalytic process.Nevertheless, this finding would open an entirely new prospect for designing organocatalysts, 8 especially if other catalytic structures could be mimicked with a degree of efficiency comparable to that observed in the case of hydrolysis.

Immobilization of m-phenylenediamine. Synthesis of (17)
Forty-four sheets of paper functionalized by treatment with 2,4-dichloro-6-methoxy-1,3,5triazine (DCMT) were immersed in 1M solution of m-phenylenediamine (14 g) in THF (100 ml) and gently shaken for 24 h at room temperature, then removed from m-phenylenediamine solution, blotted with dry filter paper and washed successively with THF (2 x 75 ml), DMF ( Fmoc-protecting groups were removed with 25% piperidine as described above. Incorporation of lipidic fragment.Every sub-library of tripeptides were labeled with graphite pencil.The vigorously stirred solution of DMT/NMM/TosO -(8) (8.26 g, 20 mmol) in DCM (50 ml) was cooled to 0-5 °C, treated with carboxylic acid (20 mmol) and NMM (0.75 ml, 6.7 mmol).Stirring was continued at 0-50°C for 4 h and a set of every sub-library was immersed into suspension and gently shaken at room temperature for 24 h.After this the plates were soaked and washed with DCM (4 x 100 ml), DMF (2 x 100ml) and DCM (2 x 100ml).

Figure 2 .
Figure 2. The rate of hydrolysis of Z-L-Ala-Aib-ONp (16) at 20 °C in aqueous methanol at pH7 in the presence of a library of N-lipidated tripeptides immobilized on cellulose.

Figure 3 .
Figure 3. Progress of hydrolysis of Z-Ala-Aib-ONp (16) reached at final kinetic cycle measurement after 418 min.related to carboxylic acid used for N-acylation of tripeptides.

Figure 4 .
Figure 4. Progress of hydrolysis of Z-Ala-Aib-ONp (16) reached at final kinetic cycle measurement after 418 min.related to amino acid sequence of tripeptide fragment.

,S; E,H; H,S; H,E; S,E; and S,H immobilized on 17. Removal of Fmoc-protecting group. Fmoc
Each chemset 17{S}, 17{E} and 17{H} was immersed separately in 25% solution of piperidine in DMF (100 ml) and gently shaken for 20 minutes, then washed with DMF (3 x 75 ml), DCM (1 x 75 ml) and immediately used in the next synthetic stage.Incorporation of the second amino acid.The second amino acid was incorporated as described above affording dipeptide chemset with following primary structures of peptide fragment: E protected amino acid 13 functionalized cellulose sheets were immersed in the mixture and gently shaken for 24 h.An excess of acylating reagent was removed, then sheets were successively washed by gentle shaking in DMF (3 x 50 ml) and DCM (3 x 25 ml), then dried in vacuum desiccator.This procedure was used for preparation of chemset 17{S} using Fmoc-Ser(tBu)-OH (4.8 g, 12.5 mmol), chemset 17{E} using Fmoc-Glu(tBu)-OH (5.38 g, 12.5 mmol) and chemset 17{H} using Fmoc-His(Trt)-OH (7.75 g, 12.5 mmol).Removal of Fmoc-protecting group.-protectinggroupswere removed with 25% piperidine as described above.Incorporation of the third amino acid.The third amino acid were incorporated as described above affording tripeptides:E,S,H; E,H,S; H,S,E; H,E,S; S,E,H and S,H,E immobilized on 17.