Screening of metal ions and organocatalysts on solid support-coupled DNA oligonucleotides guides design of DNA-encoded reactions

Abstract

DNA-encoded compound libraries are a widely used technology for target-based small molecule screening. Generally, these libraries are synthesized by solution phase combinatorial chemistry requiring aqueous solvent mixtures and reactions that are orthogonal to DNA reactivity. Initiating library synthesis with readily available controlled pore glass-coupled DNA barcodes benefits from enhanced DNA stability due to nucleobase protection and choice of dry organic solvents for encoded compound synthesis. We screened the compatibility of solid-phase coupled DNA sequences with 53 metal salts and organic reagents. This screening experiment suggests design of encoded library synthesis. Here, we show the reaction optimization and scope of three sp³-bond containing heterocyclic scaffolds synthesized on controlled pore glass-connected DNA sequences. A ZnCl₂-promoted aza-Diels-Alder reaction with Danishefsky's diene furnished diverse substituted DNA-tagged pyridones, and a phosphoric acid organocatalyst allowed for synthesis of tetrahydroquinolines by the Povarov reaction and pyrimidinones by the Biginelli reaction, respectively. These three reactions caused low levels of DNA depurination and cover broad and only partially overlapping chemical space though using one set of DNA-coupled starting materials.

Fig. 1. Solid phase-initiated access to DNA-encoded compound libraries. (A) Encoded chemistry provides DNA-barcode tagged small molecules that are concatenated to building blocks (BBs). These can be screened on protein targets by selection. (B) A solid phase-based encoding strategy allowed for synthesis of pyrrolidines by Ag(i)-mediated cycloaddition. (C) Screening of metal ions and organic reagents on DNA sequences suggests selection of preparative organic reactions for encoded compound synthesis.

Fig. 2. Representative bioactive compounds based on (A) pyridinone, (B) pyrrolotetrahydroquinoline, or (C) dihydropyrimidinone scaffolds.

Fig. 3. Scope of a ZnCl₂-promoted aza-Diels–Alder reaction of CPG-coupled 10mer ATGC oligonucleotide–aldehyde conjugate 15a with Danishefsky's diene 13 and amines 12. Reaction conditions: Condensation of CPG-coupled ATGC aldehyde conjugate 15a (20 nmol) with amine 12 (500 equiv., 10 μmol) in 36 μL acetonitrile/triethyl orthoformate (2 : 1) at ambient temperature for 4 h, followed by addition of ZnCl₂ (100 equiv., 2 μmol) dissolved in 30 μL acetonitrile and Danishefsky's diene 13 (1000 equiv., 20 μmol) at ambient temperature for 1 h. DNA cleavage with 30% aqueous ammonia at 50 °C for 6 h. ^aDetermined by analytical RP-HPLC analysis. ^bDimethyl sulfoxide was used instead of acetonitrile. ^c1000 equiv. of amine 12 were used. ^dYb(OTf)₃ was used instead of ZnCl₂. ^eThe 2^nd step of the reaction was performed overnight at 35 °C. 10mer ATGC = 5′-GTC ATG ATC T-3′, ACN = acetonitrile.

Fig. 4. Scope of a ZnCl₂-mediated aza-Diels–Alder reaction of CPG-coupled 10mer ATGC oligonucleotide–aniline conjugate 17 with Danishefsky's diene 13 and aldehydes 18. Reaction conditions: Condensation of CPG-coupled ATGC aniline conjugate 17 (20 nmol) with aldehyde 18 (1500 equiv., 30 μmol) in 36 μL tetrahydrofuran/triethyl orthoformate (2 : 1) at ambient temperature for 4 h, followed by addition of ZnCl₂ (100 equiv., 2 μmol) dissolved in 30 μL tetrahydrofuran and Danishefsky's diene 13 (1000 equiv., 20 μmol) at ambient temperature for 1 h. DNA cleavage with 30% aqueous ammonia at 50 °C for 6 h. ^aDetermined by analytical RP-HPLC analysis. ^bDimethyl sulfoxide was used instead of acetonitrile. 10mer ATGC = 5′-GTC ATG ATC T-3′, THF = tetrahydrofuran.

Fig. 5. Scope of the (R)-(–)-BNDHP A-mediated Povarov reaction of CPG-coupled 10mer ATGC oligonucleotide–aldehyde conjugates 15 with N-Boc-2,3-dihydro-1H-pyrrole 20 and anilines 12. Reaction conditions: Condensation of CPG-coupled ATGC aldehyde conjugate 15 (20 nmol) with aniline 12 (500 equiv., 10 μmol) in 36 μL ethanol/triethyl orthoformate (2 : 1) at ambient temperature for 4 h, followed by addition of (R)-(–)-BNDHP A (100 equiv., 2 μmol) dissolved in 30 μL ethanol and N-Boc-2,3-dihydro-1H-pyrrole 20 (500 equiv., 10 μmol) at 50 °C for 16 h. Boc removal by repeated incubation with 75% TFA for 30 seconds. Afterwards AMA (30% aqueous ammonia/40% aqueous methylamine, 1 : 1 (vol/vol)) was added at ambient temperature for 4 h. ^aDetermined by analytical RP-HPLC analysis. ^bDimethyl sulfoxide was used instead of ethanol. 10mer ATGC = 5′-GTC ATG ATC T-3′.

Fig. 6. Scope of the (R)-(–)-BNDHP A-mediated Biginelli reaction on CPG-coupled 10mer ATGC oligonucleotide–aldehyde conjugates 15 with ureas 24 and ethyl acetoacetate 25. Reaction conditions: CPG-coupled ATGC aldehyde conjugate 15 (20 nmol) was suspended with urea 24 (500 equiv.) and (R)-(–)-BNDHP A (50 equiv., 1 μmol) each dissolved in 30 μL of ethanol and ethyl acetoacetate 25 (500 equiv.), and the reaction mixture was shaken at 50 °C for 20 h. Afterwards AMA (30% aqueous ammonia/40% aqueous methylamine, 1 : 1 (vol/vol)) was added at ambient temperature for 4 h. ^aDetermined by analytical RP-HPLC analysis. ^b200 equiv. of (R)-(–)-BNDHP A were used. ^cThe reaction was performed at 50 °C for 44 h. 10mer ATGC = 5′-GTC ATG ATC T-3′.

Fig. 7. Cheminformatics analysis: A = PCA plot of the DA-1(red), P (blue) and B (green) libraries. B = PMI plot of the DA-1(red), P (blue) and B (green) libraries.