Thiocarbamate-Linked Polysulfonate–Peptide Conjugates As Selective Hepatocyte Growth Factor Receptor Binders

The capacity of many proteins to interact with natural or synthetic polyanions has been exploited for modulating their biological action. However, the polydispersity of these macromolecular polyanions as well as their poor specificity is a severe limitation to their use as drugs. An emerging trend in this field is the synthesis of homogeneous and well-defined polyanion–peptide conjugates, which act as bivalent ligands, with the peptide part bringing the selectivity of the scaffold. Alternately, this strategy can be used for improving the binding of short peptides to polyanion-binding protein targets. This work describes the design and first synthesis of homogeneous polysulfonate–peptide conjugates using thiocarbamate ligation for binding to the extracellular domain of MET tyrosine kinase receptor for hepatocyte growth factor.


Peptide 5
Peptide 5 was synthesized as previously described for peptide 4 by replacing Fmoc-L-Tyr(OtBu)-OH by Fmoc-L-Ala-OH during the SPPS. 0.1 mmol scale, 32 mg of peptide 5 after RP-HPLC purification (yield 39%). Peptide Fmoc-K(Fmoc)GY(OtBu)K(Boc) was assembled on a NovaSyn TGR ® resin (0.14 mmol, loading 0.25 mmol/g) using standard Fmoc-SPPS. 1 The peptidyl resin was deprotected with a solution of piperidine 20% in DMF (v/v), washed with DMF and drained. Solution A (2.8 mmol, see general section) and TEA (390 µL, 2.8 mmol) were sequentially added to the beads. The resin beads were agitated at rt for 19 h and then washed with DMF (5 × 2 min). The absence of free primary amino groups was confirmed using TNBS assay. 2 The peptide was cleaved from the resin and deprotected using TFA/H 2 O/TIS (5 mL, 95/2.5/2.5, v/v/v) at rt for 90 min. The peptide was precipitated in cold Et 2 O/n-heptane (100 mL, 1/1 ; v/v) and centrifuged (10 min, 3500 rpm, 4 °C). The white pellet was dissolved in deionized water and lyophilized to give 87 mg (69%) of di-sulfonated-peptide 3 which was used without further purification.
RP  Coupling of taurine residues using 13a Solution A (2 mmol) and TEA (280 µL, 2 mmol) were sequentially added to the beads. The suspension was agitated overnight and then washed with DMF (5 × 2 min). The coupling and washing steps was repeated four times, after which the TNBS assay proved the lack of free amino groups.
Coupling of taurine residues using 13b Alternatively, solution B (8 mL, 0.8 mmol, 8 equiv.) and DIEA (66 µL, 0.4 mmol, 4 equiv.) were sequentially added to the beads which were agitated at rt for 19 h. The coupling step was repeated once after which the TNBS assay proved the lack of free amino groups.

Coupling of sulfonate groups
Then, solution C (4 mL of DMF solution, 0.4 mmol, 4 equiv.) and TEA (28 µL, 0.2 mmol, 2 equiv.) were sequentially added to the beads. The resin was agitated for 19 h and then washed with DMF (5 × 2 min). The coupling step was repeated after which TNBS assay proved the lack of free amino groups.

QHK T S I T GHH L E P GG
The microarrays were analyzed using a fluorescence microarray laser scanner (LS Reloaded, Tecan Group Ltd., Switzerland). 16 bits images were quantified using dedicated image analysis software (Array-Pro Analyzer, Switzerland). The data correspond to the median (interquartile range) of the spot fluorescence intensities after background subtraction.
To verify that conjugation does not modify the adsorption properties of the compounds to a significant extent we have prepared a series of short peptides and conjugates derived from peptide 22 with a biotin modification (Fig. S91). The peptides were microarrayed as described above and detected using cyanine 3-labelled streptavidin (1 mg/mL diluted 1/500 in PBS with 0.1% BSA, 2 h at rt, 50 µL per well). The signal detected at 532 nm was similar for all biotinylated peptides and conjugates present on the microarray, whereas the different non biotinylated controls displayed background intensities. Intensity (AU)
Because the peptides may scavenge singlet oxygen at high concentrations, typically > 10 µM, and thus artificially decrease the emitted signal intensity, the peptides were also incubated with biotinylated rabbit IgG (10 µM final concentration), streptavidin donor and protein A acceptor beads (10 µg/mL final concentration) for 30 min to determine the signal loss due to singlet oxygen quenching. The resulting data were used to correct the signal intensities collected in the first Alphascreen® experiment.
The results are presented as percentage of the signal obtained in the absence of peptide.