WO2020047095A1 - Utilisation d'une immobilisation non covalente dans des banques codées par l'adn - Google Patents

Utilisation d'une immobilisation non covalente dans des banques codées par l'adn Download PDF

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WO2020047095A1
WO2020047095A1 PCT/US2019/048570 US2019048570W WO2020047095A1 WO 2020047095 A1 WO2020047095 A1 WO 2020047095A1 US 2019048570 W US2019048570 W US 2019048570W WO 2020047095 A1 WO2020047095 A1 WO 2020047095A1
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solid support
dna
resin
del
reaction
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Philip Dawson
Dillon FLOOD
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The Scripps Research Institute
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1068—Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/265—Adsorption chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3847—Multimodal interactions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
    • B01J31/02—Catalysts comprising hydrides, coordination complexes or organic compounds containing organic compounds or metal hydrides
    • B01J31/0234—Nitrogen-, phosphorus-, arsenic- or antimony-containing compounds
    • B01J31/0235—Nitrogen containing compounds
    • B01J31/0237—Amines
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
    • B01J31/16—Catalysts comprising hydrides, coordination complexes or organic compounds containing coordination complexes
    • B01J31/24—Phosphines, i.e. phosphorus bonded to only carbon atoms, or to both carbon and hydrogen atoms, including e.g. sp2-hybridised phosphorus compounds such as phosphabenzene, phosphole or anionic phospholide ligands
    • B01J31/2404—Cyclic ligands, including e.g. non-condensed polycyclic ligands, the phosphine-P atom being a ring member or a substituent on the ring
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/32—Bonded phase chromatography
    • B01D15/325—Reversed phase
    • B01D15/327—Reversed phase with hydrophobic interaction
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B01D15/361—Ion-exchange
    • B01D15/362—Cation-exchange
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B01D15/361—Ion-exchange
    • B01D15/363—Anion-exchange
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2231/00—Catalytic reactions performed with catalysts classified in B01J31/00
    • B01J2231/40—Substitution reactions at carbon centres, e.g. C-C or C-X, i.e. carbon-hetero atom, cross-coupling, C-H activation or ring-opening reactions
    • B01J2231/42—Catalytic cross-coupling, i.e. connection of previously not connected C-atoms or C- and X-atoms without rearrangement
    • B01J2231/4205—C-C cross-coupling, e.g. metal catalyzed or Friedel-Crafts type
    • B01J2231/4211—Suzuki-type, i.e. RY + R'B(OR)2, in which R, R' are optionally substituted alkyl, alkenyl, aryl, acyl and Y is the leaving group
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2231/00—Catalytic reactions performed with catalysts classified in B01J31/00
    • B01J2231/40—Substitution reactions at carbon centres, e.g. C-C or C-X, i.e. carbon-hetero atom, cross-coupling, C-H activation or ring-opening reactions
    • B01J2231/42—Catalytic cross-coupling, i.e. connection of previously not connected C-atoms or C- and X-atoms without rearrangement
    • B01J2231/4205—C-C cross-coupling, e.g. metal catalyzed or Friedel-Crafts type
    • B01J2231/4266—Sonogashira-type, i.e. RY + HC-CR' triple bonds, in which R=aryl, alkenyl, alkyl and R'=H, alkyl or aryl
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2531/00—Additional information regarding catalytic systems classified in B01J31/00
    • B01J2531/80—Complexes comprising metals of Group VIII as the central metal
    • B01J2531/82—Metals of the platinum group
    • B01J2531/824—Palladium

Definitions

  • the present disclosure relates to the systems and methods for immobilization of DNA encoded libraries on a solid support.
  • the inventors have now developed a new technique to bring DNA into organic solvents. While the dominant paradigm in this field of organic chemistry has been to bring organic reactions into water, the inventors have presented herein a simpler, clever approach to bring DNA into organic solvents.
  • Various embodiments disclosed herein include a method of modifying a DNA encoded library (DEL) in an organic solvent, comprising: immobilizing the DEL on an inert resin to form a DEL-resin complex; contacting the DEL-resin complex with at least one reagent in the organic solvent to produce a modified DEL-resin complex; and eluting the modified DEL from the resin by washing the DEL-resin complex with an aqueous buffer.
  • the organic solvent is a neat organic solvent.
  • the resin may be a strong anion exchange resin, such as a quaternary ammonium resin. In resin may also be incorporate both hydrophobic interactions and electrostatic interactions.
  • the DEL is immobilized on the inert resin by non-covalent adsorption.
  • the chemical library members undergo single or multiple synthetic reaction steps while being immobilized to the solid support for expanded DEL reactivities and/or expanded chemical space.
  • the chemical reactions contemplated herein comprise C(sp2)-C(sp3) decarboxyl ative cross coupling linkage between the DEL molecule and the at least one reagent, or electrochemical amination, or reductive amination.
  • the synthetic reaction steps may be in the same or in different solvents.
  • the method may further comprise washing away small molecules and/or reagents while leaving the polynucleotide encoded chemical library immobilized to the solid support.
  • Various embodiments disclosed herein also include a method of facilitating a chemical reaction in an organic solvent, comprising providing a polynucleotide encoded chemical library, wherein the polynucleotide encoded chemical library members are immobilized to a solid support by non-covalent adsorption.
  • the polynucleotide encoded chemical library member is immobilized by non-covalent adsorption of the polynucleotide moiety to the solid support.
  • the solid support comprises an anion exchange resin.
  • the solid support comprises a cation exchange resin.
  • the solid support comprises SDB-L and/or C18 silica gel.
  • the solid support comprises a hydrophobic resin.
  • the hydrophobic resin comprises a reversed phase, hydrophobic interaction, strong cation, weak cation, and affinity chromatography.
  • the solid support comprises silica based resin, crosslinked polystyrene, crosslinked glycan, crosslinked PEG, and combinations thereof.
  • the organic solvent comprises trifluoroethanol (TFE), hexafluoroisopropanol (HFIP), l,4-dioxane, teterahydrofuran (THF), dimethylacetamide (DMA), toluene, N-methylpyrrolidone (NMP), l,2-dichloroethane (DCE), dimethylfomamide (DMF), and ethanol (EtOH).
  • TFE trifluoroethanol
  • HFIP hexafluoroisopropanol
  • HFIP hexafluoroisoxane
  • THFIP hexafluoroisoxane
  • THFIP hexafluoroisoxane
  • THFIP hexafluoroisoxane
  • THFIP hexafluoroisoxane
  • THFIP hexafluoroisoxane
  • THFIP hexafluoroisoxane
  • the chemical modification comprises an amide coupling in methylene chloride. In one embodiment, the chemical modification comprises a Suzuki coupling in dimethylacetamide. In one embodiment, the chemical library members undergo single or multiple synthetic reaction steps while being immobilized to the solid support. In one embodiment, the synthetic reaction steps may be in the same or in different solvents. In one embodiment, the method further comprises washing away small molecules and/or reagents while leaving the polynucleotide encoded chemical library immobilized to the solid support. In one embodiment, the method further comprises a covalent tag that modulates binding to the solid support and removal from the solid support. In one embodiment, the method further comprises a linker between the polynucleotide and the chemical library member.
  • the linker modulates steric or conformational interactions between the site of reaction, polynucleotide, and the solid support. In one embodiment, the linker facilitates the desired chemical reaction. In one embodiment, the solid support is modified with ligands, catalysts etc. to affect the desired reaction.
  • Figure 1 depicts, in accordance with embodiments herein, a cartoon illustrating (A) use of a solid support to facilitate the transfer of DNA into organic solvents for subsequent chemical modification, and (B) workflow of the Reversible Adsorption to Solid Support (RASS) technique illustrated herein.
  • RASS Reversible Adsorption to Solid Support
  • Figure 2 depicts, in accordance with embodiments herein, expanding DEL chemical space by use of the RASS technique.
  • Figure 3 depicts, in accordance with embodiments herein, use of different types of solid supports and the adsorption of the polynucleotide in the solid support.
  • Figure 4 depicts, in accordance with embodiments herein, ultraviolet (UV) detection of compounds of formula (1) and (2) as depicted herein in scheme A.
  • Figure 5 depicts, in accordance with embodiments herein, DEL Synthesis via RASS.
  • A Aqueous vs RASS reactions for DEL.
  • B Resins selection considerations.
  • C Basic DEL RASS workflow. DNA binding and elution of DNA by HPLC.
  • Figure 6 depicts, in accordance with embodiments herein, on-DNA decarboxyl ative sp 2 - sp 3 cross-coupling.
  • A Reaction scheme.
  • B Optimization table; Reactions include small molecule reactions under DEL conditions and on-DNA DEL reactions.
  • C Scope table; protocol A (18 h), b protocol B (2 c 3 h), isolated RAE, d l :l desired product/reduced product, and e 250 mM Nal added to reaction.
  • Figure 7 depicts, in accordance with embodiments herein, on-DNA electrochemical amination.
  • A Reaction scheme.
  • B Optimization table.
  • C Scope table, Conditions from Entry 6, Conditions from Entry 6 + DBET (100 mM).
  • Figure 8 depicts, in accordance with embodiments herein, on-DNA reductive amination.
  • A Reaction scheme.
  • B Optimization table.
  • C Scope table, a on resin, b aqueous reaction.
  • Figure 9 depicts, in accordance with embodiments herein, DEL-rehearsal, graphical workflow representation and synthesis of compound of formula 90.
  • Figure 10 depicts, in accordance with embodiments herein, structural insights. Confocal microscopy image of resin with (A) double stranded DNA. (B) Single stranded DNA adsorbed and stained with SYBR green. (C) Quantification of resin fluorescence.
  • the inventors have developed a novel technology that makes use of non-covalent adsorption of DNA onto a solid support to facilitate solvent exchange from aqueous buffers, in which DNA is most soluble, into polar and non-polar organic solvents that are otherwise considered as DNA incompatible.
  • the term“organic solvent” refers to any solvent except aqueous solutions.
  • the term“organic solvent” refers to any solvent containing carbon compounds. Examples of organic solvents include, but are not limited to, aromatic compounds, chloroform, alcohols, phenols, esters, ethers, ketones, amines, and nitrated and halogenated hydrocarbons.
  • RASS Reversible Adsorption to Solid Support
  • the Reversible Adsorption to Solid Support (RASS) approach enabled the rapid development of C(sp 2 )-C(sp 3 ) decarboxylative cross-couplings with broad substrate scope, an electrochemical amination (the first electrochemical synthetic transformation performed in a DEL context), and improved reductive amination conditions.
  • the inventors have demonstrated the utility of these reactions through a DEL-rehearsal in which all newly developed chemistries were orchestrated to accord a compound rich in diverse skeletal linkages. It is believed that RASS will offer expedient access to new DEL reactivities, expanded chemical space, and ultimately more drug-like libraries.
  • a small molecule tethered to a DNA hairpin can be adsorbed to the solid support, transferred to organic solvent and undergo a range of chemical transformations in an organic solvent selected specifically for the chemical reaction, rather than to accommodate the encoding DNA.
  • Another advantage of the approach is that excess reagents and solvent can be washed away leaving the small molecule-DNA conjugate adsorbed to the resin. This property allows for sequential reactions such as two-step transformations and deprotection reaction sequences to be performed in a facile manner. Following the desired chemical modifications, the small molecule- DNA conjugate can be released from the solid support for subsequent mixing and pooling steps to create the desired DEL library.
  • the inventors have disclosed a method of facilitating a chemical reaction in an organic solvent, comprising: providing a polynucleotide encoded chemical library, wherein the polynucleotide encoded chemical library members are immobilized to a solid support by non-covalent adsorption.
  • the polynucleotide encoded chemical library member is immobilized by non-covalent adsorption of the polynucleotide moiety to the solid support.
  • the polynucleotide is a DNA
  • the polynucleotide encoded chemical library is a DNA encoded chemical library.
  • the solid support comprises an anion exchange resin.
  • the solid support comprises a cation exchange resin.
  • the solid support comprises SDB-L and/or C18 silica gel.
  • the solid support comprises a hydrophobic resin.
  • the hydrophobic resin comprises a reversed phase, hydrophobic interaction, strong cation, weak cation, and affinity chromatography.
  • the solid support comprises silica based resin, crosslinked polystyrene, crosslinked glycan, crosslinked PEG, and combinations thereof.
  • This procedure identified a strong-anion exchange resin to which aqueous DNA can be adsorbed, transferred, and retained in a variety of organic solvents including: trifluoroethanol (TFE), hexafluoroisopropanol (HFIP), l,4-dioxane, teterahydrofuran (THF), dimethylacetamide (DMA), toluene, N-methylpyrrolidone (NMP), l,2-dichloroethane (DCE), dimethylfomamide (DMF), and ethanol (EtOH).
  • TFE trifluoroethanol
  • HFIP hexafluoroisopropanol
  • HFIP hexafluoroisoxane
  • THFIP hexafluoroisoxane
  • THFIP hexafluoroisoxane
  • THFIP hexafluoroisoxane
  • THFIP hexafluoroisoxane
  • the organic solvent comprises trifluoroethanol (TFE), hexafluoroisopropanol (HFIP), l,4-dioxane, teterahydrofuran (THF), dimethylacetamide (DMA), toluene, N-methylpyrrolidone (NMP), l,2-dichloroethane (DCE), dimethylfomamide (DMF), and ethanol (EtOH).
  • TFE trifluoroethanol
  • HFIP hexafluoroisopropanol
  • HFIP hexafluoroisoxane
  • THFIP hexafluoroisoxane
  • THFIP hexafluoroisoxane
  • THFIP hexafluoroisoxane
  • THFIP hexafluoroisoxane
  • THFIP hexafluoroisoxane
  • THFIP hexafluoroisoxane
  • the immobilized polynucleotide as disclosed herein can undergo selective chemical modification while adsorbed to the solid support and exposed to organic solvent.
  • the chemical modification comprises an amide coupling in methylene chloride.
  • the chemical modification comprises a Suzuki coupling in dimethylacetamide.
  • the chemical library members undergo single or multiple synthetic reaction steps while being immobilized to the solid support.
  • the synthetic reaction steps may be in the same or in different solvents.
  • the method further comprises washing away small molecules and/or reagents while leaving the polynucleotide encoded chemical library immobilized to the solid support.
  • the method disclosed herein may be used in challenging reactions such as decarboxyl ative cross couplings and CH activations.
  • the chemical composition of the linker between the DNA and the encoded small molecule may also be used to enhance the performance of a specific reaction.
  • anion exchange resin is highly promising, the inventors have found the current method to be compatible with a wide range of resins (anion exchange and others).
  • distinct resins should be used to facilitate specific desired chemical transformations.
  • the technology would allow adaptation of previously incompatible reactants and chemistries to DEL in a time and cost-effective manner, facilitating access to previously inaccessible chemical and conformational diversity in DEL.
  • the instantly disclosed method further comprises a covalent tag that modulates binding to the solid support and removal from the solid support.
  • the method further comprises a linker between the polynucleotide and the chemical library member.
  • the linker modulates steric or conformational interactions between the site of reaction, polynucleotide, and the solid support.
  • the linker facilitates the desired chemical reaction.
  • the solid support is modified with ligands, catalysts etc. to affect the desired reaction.
  • the presently disclosed method is believed to merge seamlessly with existing DEL protocols.
  • Water soluble DEL library members can be easily adsorbed to the solid support. Washing with organic solvents removes the aqueous buffer, leaving the DNA conjugates ready for organic reactions. Excess reaction reagents can be removed through a subsequent organic wash, and the DNA can be eluted with a high salt aqueous buffer. At this point the DNA can be ethanol precipitated, and redissovled in aqueous enzyme buffer for encoding. The newly encoded DNA can undergo another round of diversification by classical DEL protocols, or be re-adsorbed to a resin for a second organic transformation, directly from the encoding mixture.
  • the solid support that employed herein is already competent for high throughput manipulation (it can be pipetted as a slurry and is commercially available in 96 well plates) and thus should be immediately applicable for DEL generation.
  • RASS Reversible Adsorption to Solid Support
  • the RASS strategy could dramatically expand the toolkit of available organic reactions for library construction by bringing DNA into organic solvents and protecting the backbone from reagent-induced degradation.
  • the ivnentors screened a series of commercially available inert resins/solid supports for their ability to selectively bind and elute a small DNA-fragment which mimics that typically employed in DEL ( Figure 5).
  • An important additional requirement was that the resin itself should not interject its own reactivity profile (e.g., acids, bases, or nucleophiles).
  • Hydrophobic resins such as reversed phase silica could competently bind DNA, but unfortunately, the weak binding led to premature elution in organic media (Figure 5B).
  • a mixed mode polystyrene strong anion exchange resin (for example, Phenomenex, Strata-XA) which contains a butyl quaternary ammonium moiety, proved to be an excellent platform for RASS on DNA (Figure 5B).
  • Such resins incorporate both hydrophobic interactions (polystyrene, butyl substituents) and electrostatic interactions (quaternary amine) and effectively anchor the DNA in a polyvalent manner.
  • PBS 100 mIUI DNA
  • the bound DNA could then be eluted into a high salt elution buffer, an approach that is widely applied in molecular biology.
  • the Strata-XA resin allows the DNA- resin complex to be transferred from aqueous solution into neat organic solvents. Following removal of the solvent from the resin, the DNA can be washed with aqueous buffer, eluted from the resin with salt, and isolated through ethanol precipitation. Failures encountered with numerous other resins that were tested can be attributed to a lack of strong initial DNA binding or lack of retention in organic solvents. This represents a robust platform for the controlled, reversible binding and elution of DNA fragments to facilitate manipulation in organic media in ways that are not possible using conventional aqueous DEL techniques.
  • Low valent Ni is competent to reduce RAEs and form transient alkyl radicals, while the combination of various silanes and base can reduce Ni 11 to low valent Ni.“RubenSilane” (isopropoxy(phenyl)silane, RS) was therefore employed but unfortunately did not improve the conversion (entry 3, Figure 6B). Numerous other attempts were pursued changing various reagent concentrations, solvent systems, and surfactants to no avail.
  • reaction time could be dramatically reduced by simply subjecting the substrate to two cycles of reagent addition (82% yield, 3 h total reaction time, entry 9, Figure 6B).
  • extremely limited DNA recovery was observed ( ⁇ 5%), and only trace product was detected when utilizing sepharose or polystyrene-based weak anion exchange systems, with the optimized reaction conditions.
  • the basic nature of the reaction led to deprotonation of the tertiary amino groups of the DEAE sepharose, which are critical to DNA retention, and ultimately led to premature DNA release. This trend held true with a variety of other weakly binding (weak anion exchangers and reversed phase) resins (Entry 10, Figure 6B).
  • oxidative reactions are rare. This is because chemical oxidants lack chemoselectivity, indiscriminately attacking the sensitive functional groups found on the purine portion of the DNA backbone leading to degradation. Such an event in the context of DEL is deleterious and irreversible as critical encoding information can be permanently lost.
  • Synthetic organic electrochemistry offers an opportunity to precisely control redox-potentials and provide superior selectivity but has never been applied in such a context. This is likely due to the charged nature of DNA that, upon exposure to an electrode surface, will irreversibly be adsorbed.
  • RASS offers a unique opportunity to site-isolate DNA thus rendering it amenable to redox transformations accessible electrochemically that might otherwise destroy it.
  • Ni-catalyzed electrochemical aryl amination due to its highly modular nature and potential to improve upon the conditions that are currently used in analogous Pd- and Cu-based reactions (Figure 6A). Indeed, Eillmann-Buchwald-Hartwig type aminations are workhorse reactions to generate diversity in medicinal chemistry and drug discovery yet robust applications in the context of DEL are lacking.
  • the DEL-compatible variants require exotic ligand systems, scavengers and high temperatures due to the dilute aqueous conditions employed.
  • the Ni-catalyzed system was therefore investigated to probe both the DEL-compatibility of electrochemistry and to explore potential synergies with the RASS approach.
  • Removing DBLT reduced the hydroxylated side product at the expense of slightly attenuated yields of compound of formula 45 (34%, Figure 7B, entry 3).
  • the addition of 4 A molecular sieves to the base-free protocol reduced the hydroxylated side product and moderately boosted conversion (50%, Figure 7B, entry 5).
  • increasing concentration of the Ni precatalyst brought yields to acceptable levels (74%, Figure 7B, entry 6).
  • various alkyl, and heteroaryl amines as well as one amide proved competent coupling partners with yields ranging from usable to good (Figure 7C).
  • aniline and most of the piperazines tested proved to be incompetent coupling partners.
  • the observed yields are in line (20-70%) with those previously seen using the conditions mentioned above. More importantly, this provided a proof of concept for the use of electrochemical methodologies in the demanding context of DEL.
  • the RASS-based DEL platform presented herein not only enables access to new reaction manifolds, but also can improve the scope of known DEL-compatible reactions.
  • reductive amination listed as among the top-three reactions used in medicinal chemistry, is often employed in DEL-library synthesis despite the fundamental limitation that the amine partner must be used in excess.
  • performing reductive aminations between carbonyl- containing compounds and an amine partner loaded on-DNA has proven difficult and inefficient. For example, within Pfizer’s DEL-based reductive amination screen using 218 different carbonyl compounds, fewer than 50 provided yields above 50%.
  • the encoding DNA tag increases in length.
  • the applicability of the RASS approach with an elongated molecule was demonstrated by selectively binding and eluting a double stranded 40 base pair oligo from the same solid support used in reaction development (Strata-XA) as well as a related solid support optimized for molecules larger than 10 kDa (Strata-XAL).
  • This larger DNA representative of DNA after the first encoding cycle, bound efficiently and eluted from the solid support with the same properties as the smaller DNA headpiece.
  • the inventors have shown that resins having larger“pores” to enable the adsorption of longer or larger DNA,
  • a reductive amination was performed on the resultant free amine, which allowed for the introduction of another aryl iodide moiety.
  • the aryl-iodide product was then utilized in an electrochemical amination to produce the final compound 90 in 9% yield over 4 steps. This provides an example that these diversity generating chemistries can be coupled in series, through multiple RASS cycles, to yield on-DNA products that are rich in therapeutically relevant functionality.
  • the RASS-DEL approach is distinguished by a significant resistance to DNA damage. This effect could be a result of the DNA maintaining significant double helix structure while adsorbed to the support.
  • the inventors used fluorescence microscopy to observe the dsDNA specific interaction of a DNA intercalator ( Figure 10).
  • a 40-base pair dsDNA was designed to mimic the encoding molecule in a growing DEL library. As a control, two nonhydridizing 40-mers of single stranded DNA were used.
  • the average fluorescence intensity of the double stranded DAN was much greater than that of the single stranded DNA and the stained resin.
  • the increase in fluorescence between the double stranded and single stranded samples was statistically significant (p ⁇ 0.0001) while the difference in fluorescence between the single stranded DNA and resin (without any DNA) was insignificant (p > 0.05). If the DNA was predominantly denatured upon adsorbing to the resin, then a reduced fluorescence output would have been observed, similar to that of the single stranded control.
  • the observed difference in average fluorescence intensity supports the notion that DNA is double stranded while adsorbed to the resin in the presence of organic solvent.44,45
  • the instant disclosure illustrates the use of RASS technique for adsorbing a chemically modified DNA hairpin to a variety of chromatography resins. While adsorbed, the inventors have demonstrated a number of classic organic reactions commonly used in DEL such as amide coupling free acid (Scheme A), amide coupling NHS ester (Scheme B), Reductive Amination (Scheme C), Oxime formation (Scheme D), Benzimidizole formation (Scheme E), Wittig reaction (Scheme F), Henry reaction (Scheme G), Sonogashira reaction (Scheme H), and Decarboxylative cross coupling with aryl iodide (Scheme I).
  • Scheme A was performed in various solvents such as Tetrahydrofuran (THF), Diehl oromethane (DCM), l,4-Dioxane (Diox), Dimethylsulfoxide (DMSO), Acetonitrile (ACN), and Dimethylacetamide (DMA).
  • THF Tetrahydrofuran
  • DCM Diehl oromethane
  • DMSO Dimethylsulfoxide
  • ACN Acetonitrile
  • DMA Dimethylacetamide
  • DNA hairpin (1) 100 uL at 250 uM was adsorbed to swelled Strate-AX resin (100 uL) and washed twice with the solvent that the reaction would be carried out in. A variety of solvents were tested (Table 1). In 500 pL of the specified solvent, 4-bromophenyacetic acid (25 mM), Diisopropylcarbodiimide (25 m M ), Oxyma pure (25 mM) and diisopropylethylamine (50 mM) dissolved and added to the decanted resin bed in a 2 ml Eppendorf tube. The reaction was stirred at room temperature for 2 hours at which point the tube was spun down and the supernatant removed and discarded. The resin bed was washed (500 pL twice) with the solvent that the reaction had been carried out in eluted and analyzed. The results are shown in Figure 4.
  • DNA-hairpin (Formula 1) 100 pL at 250 pM) in phosphate buffered saline (PBS) or Buffer A (100 mM HFIP, 20 mM TEA, at pH 8) was incubated (10 min) with lOOpL of swelled solid support (washed with the same buffer that compound of formula (1) was dissolved in). The mixture was spun in a benchtop centrifuge, and the supernatant was collected and analyzed by HPLC. The support with bound DNA was incubated (1 hour) with 500 pL of various organic solvents.
  • the DNA (Formula 1) was then eluted from the support with the addition of 100 pL (3 times) either 1 M NaCl0 4 (pH 8) or 1 : 1 MeOH:Buffer A.
  • the DNA was precipitated by the addition of cold ethanol, centrifuged, and suspended for HPLC analysis.
  • the HPLC integrations were compared to DNA bound to the resin and incubated in water, and the results are shown in Table 2. Table 2.
  • the approach disclosed herein can be applied to the synthesis of complex DNA encoded libraries.
  • the ability to synthesize complex libraries of organic molecules encoded by DNA is limited by the requirement of DNA to be in aqueous or mixed aqueous solvent.
  • the instant disclosure provides a method of bringing the modified DNA into organic solvents, enabling a broader set of reactions to be performed in their preferred media.
  • the non-covalent adsorption of DNA onto a solid support has advantages of workup and handling - small molecules, reagents, buffers and solvents can be washed away, while the DNA remains immobilized.
  • DNA can be immobilized at a higher concentration than free in solution, even in the case of aqueous buffers.
  • Immobilized DNA is site-isolated, preventing interactions between individual DNA and tethered small molecules which may reduce side reactions.
  • the non-covalent binding of the DNA may reduce chemical side reactions on the nucleic acid. Single or multiple synthetic steps can be performed on the immobilized DNA before eluting the DNA.
  • the RASS method disclosed herein is compatible with any encoding polymer.
  • Figure 3 illustrates the adsorption properties of the compound of Formula 1 to various supports.
  • DNA-hairpin (Formula 1) 100 pL at 250 mM) in phosphate buffered saline (PBS) or Buffer A (100 mM HFIP, 20 mM TEA, at pH 8) was incubated for 10 minutes with lOOpLof swelled solid support (washed with the same buffer that (1) was dissolved in). The mixture was spun in a benchtop centrifuge, and the supernatant was collected and analyzed by HPLC. The DNA (1) was then eluted from the support with the addition of 100 uL (3 times) either 1 M NaCl0 4 (pH 8) or 1 : 1 MeOH:Buffer A. The supernatants were combined and filtered. The DNA was precipitated by the addition of cold ethanol, centrifuged, and redissolved for HPLC analysis. The results are shown in Figure 3.
  • the reaction below illustrates one example of a on-DNA sulfone formation reaction. This is a photochemical reaction done in the presence of light.
  • the optimization table is also provided below.
  • On-DNA sulfide oxidation The reaction below illustrates one example of a on-DNA sulfide oxidation reaction. This is one example of an electrochemical oxidation reaction as disclosed throughout the present application. The optimization table is also provided.
  • the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term“about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
  • the terms“a,”“an,” and“the” and similar references used in the context of describing a particular embodiment of the invention can be construed to cover both the singular and the plural.
  • the recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

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Abstract

L'invention concerne des procédés de facilitation d'une réaction chimique dans un solvant organique, consistant à utiliser une banque de composés chimiques codés polynucléotidiques, les éléments de la banque de composés chimiques codés polynucléotidiques étant immobilisés sur un support solide par une adsorption non covalente. L'invention concerne en outre des procédés pour étendre la réactivité dans la synthèse de la banque codée par l'ADN par l'intermédiaire d'une liaison réversible de l'ADN à un support à base d'ammonium quaternaire inerte.
PCT/US2019/048570 2018-08-28 2019-08-28 Utilisation d'une immobilisation non covalente dans des banques codées par l'adn WO2020047095A1 (fr)

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CN113563265B (zh) * 2020-04-29 2023-08-04 成都先导药物开发股份有限公司 一种合成On-DNA N,N-单取代吲唑酮类化合物的方法

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