Universal Access to Protease Chemiluminescent Probes through Solid-Phase Synthesis

Protease chemiluminescent probes exhibit extremely high detection sensitivity for monitoring activity of various proteolytic enzymes. However, their synthesis, performed in solution, involves multiple synthetic and purification steps, thereby generating a major limitation for rapid preparation of such probes with diverse substrate scope. To overcome this limitation, we developed a general solid-phase-synthetic approach to prepare chemiluminescent protease probes, by peptide elongation, performed on an immobilized chemiluminescent enol-ether precursor. The enol-ether precursor is immobilized on a 2-chlorotrityl-chloride resin through an acrylic acid substituent by an acid-labile ester linkage. Next, a stepwise elongation of the peptide is performed using standard Fmoc solid-phase peptide synthesis. After cleavage of the peptide-enol-ether precursor from the resin, by hexafluoro-iso-propanol, a simple oxidation of the enol-ether yields the final chemiluminescent dioxetane protease probe. To validate the applicability of the methodology, two chemiluminescent probes were efficiently prepared by solid-phase synthesis with dipeptidyl substrates designed for activation by aminopeptidase and cathepsin-B proteases. A more complex example was demonstrated by the synthesis of a chemiluminescent probe for detection of PSA, which includes a peptidyl substrate of six amino acids. We anticipate that the described methodology would be useful for rapid preparation of chemiluminescent protease probes with vast and diverse peptidyl substrates.

. General synthetic scheme for preparing the protease probes APCP, CBCP and CLPSA using SPPS.

Compatibility of Met and Trp towards oxidation with singlet oxygen
The compatibility of the oxidation with the amino acids Met and Trp was tested by performing Fmoc SPPS with a single amino acid (either Fmoc-Met-OH or Fmoc-Trp(Boc)-OH) on resin 1, after which the oxidation was performed (as shown in Figure S2). The results are presented below.

Tryptophan:
Coupling:   In addition to the coupling tests, Fmoc-Trp-OH was exposed to the oxidation conditions to see whether or not the side-chain nitrogen is oxidized by singlet oxygen:

Compound 3a
Compound 3a was synthesized according to previously reported procedure 2 .

Compound 1b
Benzyl alcohol compound 1a (1.2 gr, 2.9 mmol, 1eq) and NaI (1.3 gr, 8.6 mmol, 3eq) were dissolved in ACN and cooled to 0°C in an ice bath. TMSCl (1.1 ml, 8.6 mmol, 3eq) was added and the reaction was brought to RT. The reaction was stirred for 30 minutes and monitored by TLC (EtOAc). Upon completion, the solvent was evaporated under reduced pressure. The residue was triturated with diethyl ether and the liquid was decanted off. The product was obtained as an orange solid (1.5 gr, 98%). 1

Compound 2b
Benzyl alcohol compound 2b (269 mg, 0.48 mmol, 1eq) and NaI (217 mg, 1.45 mmol, 3eq) were dissolved in ACN and cooled to 0°C in an ice bath. TMSCl (184 µl, 1.45 mmol, 3eq) was added and the reaction was brought to RT. The reaction was stirred for 30 minutes and monitored by TLC (Hex:EtOAc 50:50). Upon completion, the solvent was evaporated under reduced pressure. The residue was triturated with diethyl ether and the liquid was decanted off. The product was obtained as an orange solid (198 mg, 62%). 1

Compound 3b
Compound 3b was synthesized according to previously reported procedure 2 .

Compound 1c
Phenol enol-ether 4 1 (410 mg, 0.99 mmol, 1eq) was dissolved in anhydrous DMF and cooled to 0°C in an ice bath. Potassium carbonate (164 mg, 1.19 mmol, 1.2eq) was added, and the mixture stirred for several minutes at 0°C. Next, benzyl iodide compound 1b (780 mg, 1.48 mmol, 1.5eq) was added and the reaction was brought to RT. The reaction was stirred overnight and monitored by TLC (Hex:EtOAc 50:50). Upon completion, the reaction mixture was diluted with EtOAc and washed with NH $ Cl solution and brine. The organic layer was dried over Na " SO $ and evaporated under reduced pressure. The product was purified via column chromatography (Hex:EtOAc 50:50) to afford compound 1c as a yellow solid (629 mg, 78%). 1

Compound 2c
Phenol enol-ether 4 1 (106 mg, 0.26 mmol, 1.5eq) was dissolved in anhydrous DMF and cooled to 0°C in an ice bath. Potassium carbonate (47 mg, 0.34 mmol, 2eq) was added, and the mixture stirred for several minutes at 0°C. Next, benzyl iodide compound 2b (111 mg, 0.17 mmol, 1eq) was added and the reaction was brought to RT. The reaction was stirred overnight and monitored by TLC (Hex:EtOAc 50:50). Upon completion, the reaction mixture was diluted with EtOAc and washed with NH $ Cl solution and brine. The organic layer was dried over Na " SO $ and evaporated under reduced pressure. The product was purified via column chromatography (Hex:EtOAc 50:50) to afford compound 2c as a yellow solid (116 mg, 71%). 1

Compound 3c
Phenol enol-ether 4 1 (95 mg, 0.23 mmol, 1eq) was dissolved in anhydrous DMF and cooled to 0°C in an ice bath. Potassium carbonate (38 mg, 0.28 mmol, 1.2eq) was added, and the mixture stirred for several minutes at 0°C. Next, benzyl iodide compound 3b 2 (200 mg, 0.34 mmol, 1.5eq) was added and the reaction was brought to RT. The reaction was stirred overnight and monitored by TLC (EtOAc). Upon completion, the reaction mixture was diluted with EtOAc and washed with NH $ Cl and brine. The organic layer was dried over Na " SO $ and evaporated under reduced pressure. The product was purified via column chromatography (EtOAc) to afford compound 3c as a yellow solid (80 mg, 40%). 1

Compound 1
Compound 1c (527 mg, 0.65 mmol, 1eq) was dissolved in a minimal amount of DCM. DMBA (304 mg, 1.94 mmol, 3eq) and Pd(PPh # ) $ (75 mg, 0.07 mmol, 0.1eq) were added, and the reaction was heated to 35°C. Reaction was stirred for 1 hour and monitored by TLC (Hex:EtOAc 50:50). Upon completion, the reaction mixture was diluted with DCM and washed with NH $ Cl solution and brine. The organic layer was dried over Na " SO $ and evaporated under reduced pressure. The product was purified via column chromatography (Hex:EtOAc 50:50) to afford compound 1 as a yellow solid (456 mg, 91%).

General resin loading procedure
The resin 2-chlorotrityl chloride (5eq) was swelled by swirling in DCM for 2 hours. Next, a solution of ccompounds 1-3 (1eq) and DIPEA (6eq) in DCM were added to the resin and the reaction was swirled overnight. Then, the loading solution was removed, and the resin was washed with DCM three times. Next, the capping solution (9 ml DCM, 1 ml MeOH and 0.5 ml DIPEA) was added to the resin and swirled for 1 hour. Then, the capping solution was removed, and the resin was washed with DCM three times to give Resin 1-3.

Resin 1
Resin 1 was prepared according to the general loading procedure on 0.65 mmol scale, 64% yield.

Resin 2
Resin 2 was prepared according to the general loading procedure on 0.08 mmol scale, 57% yield.

Resin 3
Resin 3 was prepared according to the general loading procedure on 0.15 mmol scale, 76% yield.

Enol-ether 3
Enol-ether 3 was synthesized via Fmoc-Solid Phase Peptide Synthesis. Resin 3 (215 mg, 0.063 mmol, 1eq) was swirled in DCM for 30 min. Fmoc deprotection was done with 20% piperidine in DMF (15 minutes) followed by coupling of the next amino acid (4 eq) using HBTU (4 eq) and DIPEA (6 eq) in DMF (30 minutes). These two steps were repeated until the sequence is completed. After Fmoc deprotection of the histidine was completed, the peptide was capped with 4morpholinecarbonyl chloride (110 µl, 0.95 mmol, 15eq) and DIPEA (329 µl, 1.89 mmol, 30eq) in DMF. Finally, Next, the resin was added the cleavage cocktail (HFIP/DCM/TES [50:45:5]), which also provided Mmt and Trt deprotection after swirling for 2 hours. After filtration, the solvent was evaporated under reduced pressure, and the crude was triturated using diethyl ether followed by filtration to afford the title compound

APCP
Enol-ether 1 (9.8 mg, 0.015 mmol) and few milligrams of methylene blue were dissolved in 10 ml of DMF. Oxygen was bubbled through the solution while irradiating with yellow light for 10 minutes. The reaction was monitored by RP-HPLC. After completion, the reaction mixture was purified by preparative RP-HPLC (50-100% ACN). The product APCP was obtained as a white solid