Synthesis of inhibitors of the lectin LecB from Pseudomonas Aeruginosa
2.7 Synthesis of new fucosylated peptide dendrimers with high proteolytic stability
dendrimer and the negatively charges found close to the fucose binding site in UEA-I which can improve the binding. Also inhibition lectin PA-IIL test with FD2 gave a high result of IC50 of 0.14±0.035µM that corresponds to a 80 fold relative potency. As for UEA-I, also in PA-IIL negatively charged residues are found close to the fucose binding site36.
ensure a complete conversion and the temperature that needs to be maintained between 0 and -18°
C for all the time of the reaction. In this way we could obtain only the α-allyl compound pure. The final step was the conversion of the allyl group of 3 in the corresponding acetic acid moiety. The reaction was performed at room temperature in aqueous mixture of CCl4 and acetonitrile with ruthenium(III) chloride and sodium meta periodate under vigorous stirring. After work-up and trituration of the crude product with ethyl ether very pure compound 4 was obtained and used without further purification. The C-fucosyl-acetic acid was therefore synthesized from L-fucose in 84% overall yield.
Ac2O Pyridine rt, overnight
(a) (b)
(a) 1) (CH3)3SiCH2CH=CH2 0°C, 5 min. 2) BF3-OEt2 TMSOTf 0°C 5h, -18°C overnight, 0°C 5h, CH3CN dry (b) NaIO4 RuCl3, CCl4/CH3CN/H2O overnight
O OAc OAc OAc
OH
O O
OAc OAc OAc
O OAc OAcOAc
O OAc OH OH OH
OH
(1) (2) (3) (4)
Scheme 3 Synthesis of C-fucosyl acetic acid 4
The dendrimer synthesis was performed through the Solid Phase Peptide Synthesis (SPPS) manual procedure, in plastic syringes. Nova Syn TGS RAM resin was used as solid support.(scheme 4) The F-moc protected resin (see scheme 5), after swelling with dichlorometane, was deprotected with a solution of piperidine/DMF 20/80 (a scheme 4). The deprotection was checked with Trinitrobenzenesulfonic Acid (TNBS) (b scheme 4) which is a reagent used in qualitative tests for free amine. In fact, one can see at the microscope the beads red coloured if the amine is free and colourless when the amine is still protected.
The resin was then coupled with the amino acid using PyBOP and di-isopropyl-ethylamine (DIEA) (c). Generally, in the synthesis of first generation dendrimers the reaction time is 1 h and 30 min, but one needs 3 h reaction time after the first generation. After each coupling the resin was checked again with the TNBS test. If the TNBS test indicated the presence of some free amino groups, the coupling was repeated. In any case after each coupling, the remaining free amino groups were capped with acetic anhydride/CH2Cl2 (c1). The Fmoc protecting groups of each amino acid were then removed with a solution of 20% piperidine in DMF and steps (c) and (c1) repeated until the desired peptide structure was obtained. At the end of the sequence the resin was coupled with fucose 4 in the presence of DIEA and HCTU (d) overnight. In this step a large excess of carbohydrate was used and the coupling was repeated two more times. It was observed that
conjugation of the sugar is hard to be quantitative and often dendrimer minus one fucose unit were obtained. The glycodendrimer was then deacetylated using a solution of MeOH/NH4/H2O (v/v 8:1:1) for 24 h for two times (step 1 in scheme 4) The resin was then dried and the dendrimer cleaved from the resin with TFA/TIS/H2O (95:2.5:2.5) for 4 h (step 2 in scheme 4) The peptide was precipitated with methyl tert-butyl ether and dissolved in a water/acetonitrile mixture.
Fmoc N H
a) Fmoc deprotection b) Testing
c) AA coupling c1) Capping b) Testing
d) Protected Fucose coup.
HN Fuc Fuc
Fuc Fuc
1) CH3OH/H2O/NH4OH,8:1:1 (deacetylation) 2x24 h 2) TFA-TIS-H2O, 95:2.5:2.5 (cleavage) 3h and HPLC (eluent: A, H2O-TFA 0.1%; B, CH3CN-H2O 3:2, TFA 0.1%).
NH2 Fuc Fuc
Fuc Fuc
SPPS of peptide dendrimer a) Piperidin/DMF 20:80 (2x10 min.) b) TNBS/DIEA, two drops of each c) amino acid, PyBop, DIEA, NMP c1) Ac2O/DCM 1:1, 10 min.
d) Fucose(prot.), HCTU, DIEA, NMP two times
NOVA Syn® TGS Ram loading 500 mg capacity 0.24 mmol/g
OMe
MeO
NH-Fmoc
O O
HN PEG
Scheme 4 Solid Phase Peptide Synthesis general procedure
All dendrimers were purified by preparative HPLC with detection at λ= 214 nm on a chromolith performance RP-C18, 4.6 x 100 mm column flow rate 3 mL min,-1 eluent A: water and TFA (0.1%); eluent B: acetonitrile, water and TFA (3/2/0.1%). After purification, the structure of the dendrimer was confirmed by ESI-MS. Lectin binding was assessed with an Enzyme-Linked Lectin assay (ELLA). As reported above, this assay measures, through absorbance (read at 415 nm), the competition between the ligand and a biotinylated polymeric fucose reagent, toward the lectin
(PA-IIL). The logarithm of the concentration of the dendrimer was plotted vs the % of inhibition. The sigmoidal curve was fitted and the concentration at 50% of inhibition (IC50) was determined.
NH2 Ile His Lys Ile Ile Lys
Lys Phe
Phe Lys
Lys Leu Leu
Leu Leu Pro Pro
Pro Pro Lys Lys
Lys Lys
NH2 Leu His Lys Leu Leu Lys
Lys Phe
Phe Lys
Lys Leu Leu
Leu Leu Pro Pro
Pro Pro Lys Lys
Lys Lys
Ile - GAR- L Leu - GAR- D
ES-MS = 3535.0
Y = 8% isolated after prep. HPLC
ES-MS = 3535.0
Y = 12% isolated after prep. HPLC L-Fuc
L-Fuc
L-Fuc L-Fuc
L-Fuc L-Fuc
L-Fuc L-Fuc
NH2 Leu His Lys Ile Ile Lys
Lys Phe
Phe Lys
Lys Leu Leu
Leu Leu Pro Pro
Pro Pro Lys Lys
Lys Lys L-Fuc L-Fuc
L-Fuc L-Fuc
ES-MS = 3535.0
Y =10% isolated after prep. HPLC Leu - GAR- L
Ile - GAR- L dendrimer with L-amino acid sequence and Ile as first Leu - GAR- L dendrimer with L-amino acid sequence and Leu as first
Leu - GAR- D dendrimer with D-amino acid sequence, L-Lys as branch and Leu as first Fig. 4 Glycopeptide dendrimers synthesized in Berne
The results of IC50 obtained by the ELLA test (see Tab. 4) indicate that Ile GAR-L and Leu GAR-L having all L-amino acids are the most potent ligands against PA-IIL lectin, with an IC50 = 0.14 µM and 0.15 µM respectively, which corresponds to a relative potency of 80 and 73 relative to L-fucose. No differences, within experimental errors, between the two amino acid based dendrimers was observed, indicating that the presence of a L-Ile or a L-Leu as first amino acid is not significant. Concerning Leu-GAR-D dendrimer, ELLA test against PA-IIL lectin shown an IC50 = 0.66 µM and a relative potency of 17 relative to L-fucose. As could be expected, Leu-GAR-D has a
different IC50 value than its L-amino acid counterparts, but is only 4-fold less active. In any case, Leu-GAR-D is 17 times (ca 4 times per sugar units) more potent than the monovalent L-fucose, thus showing that also for this ligand, a multivalent effect is operating.
No. IC50 (µM) r.pa. nb r.p./nc
L-fucose 11 1 1 1
Ile-GAR-L 0.14 ± 0.035 80 4 20
Leu-GAR-L 0.15±0.082 73 4 18
Leu-GAR-D 0.66±0.12 17 4 4.2
Table 4. IC50 Values with PA-IIL lectin as measured by ELLA test
a r.p. = relative potency to fucose = IC50 (fucose)/IC 50 (ligand). b n. = number of fucosyl units per dendrimer.
c Relative potency per fucose residue.
After having demonstrated that Leu-GAR-D is able to inhibit PA-IIL, we undertook studies aimed at evaluating its proteolytic stability. All dendrimers were incubated with two proteins, Trypsin and Chymotrypsin, with human serum and bacteria PAO1 at 37 °C. The stability of each dendrimer was monitored by HPLC injection at 10, 30, 60 min and after 24 h. (see Tab 5). The dendrimers Ile-GAR-L and Leu-Ile-GAR-L are, as expected, not stable under these proteolytic conditions. In the presence of the proteolytic Trypsin or Chymotrypsin, more than 95% of the dendrimer is decomposed after only 30 min. of incubation. These L-dendrimers are slightly more stable in the presence of human serum or bacteria PAO1, but also in these cases an extended decomposition can be observed. Especially in the presence of bacteria PAO1, after 1 h, 20-50% of the dendrimer is decomposed, and after 24 h no traces of the inhibitor could be found in the incubation liquor.
Remarkably different is the situation observed with the D-peptide dendrimer Leu-GAR-D, since it is resistant to proteolytic conditions in human serum, PAO1 bacterial strains and even enzymes. In all cases also after prolonged incubation time Leu-GAR-D can be found completely integer.
Cond. Leu-GAR-L Ile-GAR-L Leu-GAR-D
Trypsin 10 min 68 17 100
Trypsin 30 min 6 1 100
Trypsin 1h 1 0 100
Trypsin 24 h 0 - 100
Chymotrypsin 10 min 49 2 100
Chymotrypsin 30 min 1 0 100
Chymotrypsin 1h 0 0 100
Chymotrypsin 24 h - - 100
Serum 30 min 100 100 100
Serum 4h 100 ~90 100
Serum 24 h 57 49 Not determ.
PAO1 10 min 92 100 100
PAO1 30 min 90 65 100
PAO1 1h 78 54 100
PAO1 24 h 0 0 not determ.
Tab.5 Percentage (%) of integer fucosylated peptide dendrimer detected by HPLC after the delay of time indicated in different proteolytic conditions (T = 37° C).