4-BroMo-N-(4-broMophenyl)-3-[[(phenylMethyl)aMino]sulfonyl]benzaMide Chemical Properties

Density 

1.680±0.06 g/cm3(Predicted)

storage temp. 

room temp

solubility 

DMSO: ≥10mg/mL

form 

powder

pka

10.16±0.30(Predicted)

color 

off-white to light tan

Stability:

Stable for 1 year from date of purchase as supplied. Solutions in DMSO may be stored at -20°C for up to 3 months.

InChI

InChI=1S/C20H16Br2N2O3S/c21-16-7-9-17(10-8-16)24-20(25)15-6-11-18(22)19(12-15)28(26,27)23-13-14-4-2-1-3-5-14/h1-12,23H,13H2,(H,24,25)

InChIKey

VBOWCZDVEOFGRY-UHFFFAOYSA-N

SMILES

C(NC1=CC=C(Br)C=C1)(=O)C1=CC=C(Br)C(S(NCC2=CC=CC=C2)(=O)=O)=C1

CAS DataBase Reference

312756-74-4

Safety Information

Hazard Codes 

Xi

Risk Statements 

36

Safety Statements 

26

WGK Germany 

3

4-BroMo-N-(4-broMophenyl)-3-[[(phenylMethyl)aMino]sulfonyl]benzaMide Usage And Synthesis

Description

RS-1 is an activator of DNA repair protein RAD51 (K_d = 48-107 for human RAD51 with different cofactors present). It stimulates homologous strand assimilation activity at least 5- to 11-old, enhancing homologous recombination activity of hRAD51. RS-1 is a potent enhancer of CRISPR-mediated genome editing, increasing homology directed repair 3- to 6-fold. It is used to increase CRISPR-mediated knock-in efficiencies in vitro and in vivo.

Uses

4-Bromo-N-(4-bromophenyl)-3-[[(phenylmethyl)amino]sulfonyl]benzamide is a stimulant of the human homologous recombination protein RAD51.

Uses

RS-1 has been shown to enhance CRISPR genome editing efficiency. To see other small molecule CRISPR enhancers, visit sigma.com/CRISPR-enhancers.

General Description

A cell-permeable sulfonamido-benzamide-based allosteric regulator that stimulates DNA binding and recombinase activities of hRAD51 by locking hRAD51 in an active conformation without affecting its active site ATP hydrolysis. Although RS-1 enhances hRAD51 filament formation on ssDNA with or without the cofactor NTP, active filaments and recombinase activity are only induced in the presence of ATP or AMP-PNP, but not with ADP or no cofactors. Shown to promote resistance of primary human neonatal dermal fibroblasts to Cisplatin- (Cat. No. 232120) induced death in a dose-dependent manner. RS-1 is inactive toward related DNA strand exchange proteins scRAD5 and scDMC1 of yeast origin or E. coli RedA.This HDR (homology-directed repair) enhancer, is shown to significantly increase both Cas9 & TALEN-mediated knock-in efficiencies.

Biological Activity

RS-1 is a RAD51-stimulatory compound, which increases the DNA binding activity of RAD51. It is an HDR(homology-directed repair) enhancer that enhances Cas9- and TALEN-mediated knock-in efficiency in rabbit embryos both in vitro and in vivo.

Biochem/physiol Actions

RS-1 is a sulfonamido-benzamide compound.

Synthesis

Procedure for the synthesis of 3-(N-benzylaminosulfonyl)-4-bromo-N-(4-bromophenyl)benzamide (RS-1): 3-benzylaminosulfonyl-4-bromobenzoic acid (455 mg, 1.23 mmol) was dissolved in 5 mL of tetrahydrofuran (THF) and 0.1 mL of N,N-dimethylformamide (DMF) was added. Oxalyl chloride (0.21 mL, 2.46 mmol) was slowly added to the reaction mixture at room temperature. The reaction mixture was heated to reflux for 15 min, cooled and the volatiles were removed under reduced pressure. The residue was redissolved in 5 mL of THF and a 1 mL THF solution of 4-bromoaniline (254 mg, 1.48 mmol) was added dropwise, followed by dropwise addition of triethylamine (0.17 mL, 1.23 mmol). The reaction mixture was stirred at room temperature for 2 h, after which it was diluted by adding 10 mL of ethyl acetate and 10 mL of water. The aqueous phase was further extracted with two 15 mL of ethyl acetate. The organic phases were combined, washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the residue was purified by preparative high performance liquid chromatography (HPLC) to afford 257 mg (32% yield) of the target compound, 3-benzylaminosulfonyl-4-bromo-N-(4-bromophenyl)benzamide, as a white solid.1H-NMR (400 MHz, DMSO-d6): δ 10.60 (s, 1H), 8.55 (t, J = 6.1 Hz, 1H), 8.45 (d, J = 2.1 Hz, 1H), 8.01 (dd, J = 2.1 Hz, J = 6.3 Hz, 1H), 7.95 (d, J = 8.2 Hz, 1H), 7.75 (d, J = 7 Hz, 2H), 7.57 (d, J = 8.8 Hz, 2H), 7.22 (m, 5H), 4.15 (d, J = 6.2 Hz, 2H).13C-NMR (100 MHz, DMSO-d6): δ 164.0, 140.7, 138.6, 137.8, 135.8, 134.5, 132.7, 132.0, 130.4, 128.5, 128.0, 127.6, 123.3, 122.9, 116.3, 46.5.

in vitro

RS-1 stimulates the binding of hRAD51 to ssDNA. Low-micromolar concentrations of this small molecule enhance DNA binding and result in longer protein–DNA complex lengths. In addition, RS-1 stabilizes the active form of hRAD51 filaments and this is reflected in an enhanced strand assimilation activity[1]. RS-1 enhances Cas9- and TALEN-mediated knock-in efficiency in rabbit embryos both in vitro and in vivo.

storage

Room temperature

References

[1] KRISHANTHI JAYATHILAKA. A chemical compound that stimulates the human homologous recombination protein RAD51.[J]. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105 41: 15848-15853. DOI:10.1073/pnas.0808046105
[2] JENNIFER M MASON. The RAD51-stimulatory compound RS-1 can exploit the RAD51 overexpression that exists in cancer cells and tumors.[J]. Cancer research, 2014: 3546-3555. DOI:10.1158/0008-5472.can-13-3220
[3] JORDAN PINDER  Graham D  Jayme Salsman. Nuclear domain “knock-in” screen for the evaluation and identification of small molecule enhancers of CRISPR-based genome editing.[J]. Nucleic Acids Research, 2015, 43 19: 9379-9392. DOI:10.1093/nar/gkv993
[4] JUN SONG. RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency[J]. Nature Communications, 2016, 7 1. DOI:10.1038/ncomms10548

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