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Bioorthogonal CRISPR/Cas9-Drug Conjugate: A Combinatorial Nanomedicine Platform
Beha, Marcel Janis ; Kim, Joo-Chan ; Im, San Hae , et al. Adv. Sci.,2023,10(27):2302253. DOI: 10.1002/advs.202302253 PubMed ID: 37485817
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Abstract: Bioconjugation of proteins can substantially expand the opportunities in biopharmaceutical development, however, applications are limited for the gene editing machinery despite its tremendous therapeutic potential. Here, a self-delivered nanomedicine platform based on bioorthogonal CRISPR/Cas9 conjugates, which can be armed with a chemotherapeutic drug for combinatorial therapy is introduced. It is demonstrated that multi-functionalized Cas9 with a drug and polymer can form self-condensed nanocomplexes, and induce significant gene editing upon delivery while avoiding the use of a conventional carrier formulation. It is shown that the nanomedicine platform can be applied for combinatorial therapy by incorporating the anti-cancer drug olaparib and targeting the RAD52 gene, leading to significant anti-tumor effects in BRCA-mutant cancer. The current development provides a versatile nanomedicine platform for combination treatment of human diseases such as cancer.
Keywords: bioorthogonal ; cancer therapy ; chemotherapeutic drugs ; combinatorial delivery ; CRISPR ; Cas9 ; gene editing ; nanomedicines ; unnatural amino acids
Purchased from AmBeed: 763111-47-3
CAS No. : | 763111-47-3 | MDL No. : | MFCD18251631 |
Formula : | C20H19FN4O2 | Boiling Point : | - |
Linear Structure Formula : | - | InChI Key : | MFFUYEOGICAKCK-UHFFFAOYSA-N |
M.W : | 366.39 | Pubchem ID : | 11726399 |
Synonyms : |
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Chemical Name : | 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one |
Signal Word: | Warning | Class: | N/A |
Precautionary Statements: | P261-P305+P351+P338 | UN#: | N/A |
Hazard Statements: | H302-H315-H319-H335 | Packing Group: | N/A |
GHS Pictogram: |
* All experimental methods are cited from the reference, please refer to the original source for details. We do not guarantee the accuracy of the content in the reference.
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
61% | EXAMPLE A.5. A solution of 4-iodophenyl acetic acid (6.5 mg, 0.048 mmol), EDC (10.5 mg, 0.055 mmol), NHS and 600 μ DMF was stirred for 30 min at room temperature. After this time, the 4-(4-fluoro-3- (piperazine-l-carbonyl)benzyl)phthalazin-l(2H)-one (10 mg, 0.0275 mmol) was added to the solution and the mixture was stirred at room temperature overnight. H20 (500 μ) was added, the mixture extracted with DCM (2 x 500 μ), and the combined extracts dried under vacuum. The crude mixture was purified by silica column chromatography (100% DCM), and the product obtained as a white solid (8.8 mg, 61%). 1H NMR (CDCI3) δ = 9.82 (s, 1H), 8.40-8.38 (m, 1H), 7.83-7.81 (d, 1H), 7.77- 7.75 (d, 1H), 7.70-7.69 (m, 2H), 7.63-7.56 (m, 3H), 7.00-6.89 (m, 3H), 4.20 (s, 2H), 3.63-3.11 (m, 8H), 2.84 (s, 2H). LC-ESI-MS (+) m/z = 632.9 [M+Na+]+. HRMS-ESI [M+H+]+ m/z calculated for [C28H24FIN403]+ 611.0955, found 611.0971. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 70℃; for 12h; | General procedure: Step 1 (N-alkylation): To a solution of alkyl tosylate (0.5 mmol,1.3 equiv) in DMF (1.5 mL) was added phthalazinone (0.37mmol, 1 equiv) and DIPEA (0.75 mmol, 2.0 equiv) and stirred for12 hours at 70 C. After the reaction was completed, water andEtOAc were added to the residue. The organic layer was separated,filtered and evaporated. The reaction mixture was purifiedby flash silica gel column chromatography using MeOH/DCM 8% as the eluent to afford the desired products. Step 2 (Boc deprotection): A solution of Boc-protected substratesin DCM was treated with 4 N HCl in 1,4-dioxane solution(1 mL). The reaction mixture stirred at room temperature for 30min. The reaction mixture was concentrated under reducedpressure to afford the product as a pale yellow solid that wasused in the next step without purification. Step 3 (Amide coupling): HATU (0.11 mmol,1.1 equiv) and DIPEA(0.4 mmol, 4 equiv) were added to a solution of amine (0.1mmol) and carboxylic acid (0.1 mmol, 1 equiv) in DMF andstirred for 12 hours at room temperature. After the reactionwascomplete, water and EtOAc were added to the residue. Theorganic layer was separated, filtered and evaporated. The reactionmixture was purified by flash silica gel column chromatographyusing MeOH/DCM 8% as the eluent to afford thedesired products. | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 70℃; for 12h; | General procedure: In the solution of alkyl tosylate (0.5 mmol, 1.3 equivalents) of DMF (1.5 mL), phthalazinone (0.37 mmol, 1 eq) and DIPEA (0.75 mmol, 2.0 equivalents) were added, and stirred for 70 degrees to 12 hours. After the reaction was finalized, water and EtOAc were added to the residue. The organic layer was separated, filtered, and evaporated. The reaction mixture was purified with flash silica gel column chromatography using MeOH / DCM 8% as an elution agent to obtain the target product. |
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