This application claims priority to ET.S. Application 62/723,893, filed August 28, 2018, which is incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present disclosure relates to the systems and methods for immobilization of DNA encoded libraries on a solid support.

BACKGROUND OF THE DISCLOSURE

All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

Sydney Brenner and Richard Lerner’s seminal 1992 report established a profound, new type of combinatorial chemistry. Brenner, S.; Lerner, R. A. Encoded Combinatorial Chemistry. Proc. Natl. Acad. Sci. U. S. A. 1992, 89, 5381-5383. They postulated that individual chemical transformations could be encoded in DNA, resulting in libraries of unprecedented size and chemical diversity. Since their proposal, many groups and pharmaceutical companies have invested heavily into DEL research and technology.

Modem, industrialized DEL libraries routinely contain billions of compounds that are screened for biological activity, all at once. Although many success stories have resulted from DEL-based discovery campaigns, including multiple therapeutic candidates in clinical trials, the synthetic pathways employed during DEL construction lag severely behind the unconstrained methodologies of modem organic and medicinal chemistry. This glaring disparity can be attributed to the idiosyncratic reaction requirements of the encoding molecule, DNA, which manifests in three confounding ways: (1) as DNA is insoluble in most organic solvents, reactions need to be conducted in the presence of water, (2) highly diluted conditions are required (<l mM, due to solubility considerations) making bimolecular reactions sluggish, and (3) a high degree of chemoselectivity is required so as not to disturb the functional -group rich nucleotide back-bone. The pragmatic result of these factors is that most modern DEL libraries, while exhibiting broad diversity from the monomers employed, are composed of a severely limited set of skeletal linkages. To be sure, these are mostly composed of amides, biaryls, and C-N linkages through l,3,5-triazine“hubs” which create planar libraries lacking significant 3D shape. Thus, although great numbers of compounds can be generated, often, drug likeness and implicit diversity suffer. Even with those caveats, such libraries have shown some success for lead identification, fueling a resounding call for the development of more interesting DEL compatible chemistries and ultimately more drug-like libraries.

Numerous laboratories have chosen to directly address this challenge by adapting reaction conditions to fit the unusually demanding requirements of DEL synthesis. Although this approach has encountered some success, it has often proven to be a time-consuming and laborious endeavor. For instance, the recently developed DNA-compatible Giese reaction for use in the construction of high value sp3-sp3 linkages in DEL required a unique method for kinetic evaluation and optimization involving hundreds of optimization experiments. Clearly, adapting organic reactions for use in dilute water presents many difficulties as many interesting bond forming reactions invoke water-incompatible reagents or intermediates.

Thus, as explained above, full potential of DNA Encoded Libraries (DEL) has yet to be realized due to the limited compatibility of the encoding molecule (DNA) with the organic solvents that are favored in synthetic chemistry. Thus there remains a need in the art for new techniques to facilitate chemical reactions of DNA encoded library members in organic solvents

SUMMARY OF THE DISCLOSURE

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. In preferred embodiments, 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. In preferred embodiments, 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. In some embodiments, 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. In one embodiment, the polynucleotide encoded chemical library member is immobilized by non-covalent adsorption of the polynucleotide moiety to the solid support. In one embodiment, the solid support comprises an anion exchange resin. In one embodiment, the solid support comprises a cation exchange resin. In one embodiment, the solid support comprises SDB-L and/or C18 silica gel. In one embodiment, the solid support comprises a hydrophobic resin. In one embodiment, the hydrophobic resin comprises a reversed phase, hydrophobic interaction, strong cation, weak cation, and affinity chromatography. In one embodiment, the solid support comprises silica based resin, crosslinked polystyrene, crosslinked glycan, crosslinked PEG, and combinations thereof. In one embodiment, 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). In one embodiment, the polynucleotide encoded chemical library can undergo solvent exchange from aqueous buffer to an organic solvent. In one embodiment, the immobilized polynucleotide can undergo selective chemical modification while adsorbed to the solid support and exposed to organic solvent. In one embodiment, 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. In one embodiment, 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.

Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various embodiments of the invention.

DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

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.

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.

DETAILED DESCRIPTION

All references, publications, and patents cited herein are incorporated by reference in their entirety as though they are fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Homyak, et ah, Introduction to Nanoscience and Nanotechnology, CRC Press (2008); Singleton et ah, Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley & Sons (New York, NY 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 7th ed., J. Wiley & Sons (New York, NY 2013); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

As disclosed herein, 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. As used herein, the term“organic solvent” refers to any solvent except aqueous solutions. In some embodiments, 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.

Furthermore, the inventors have disclosed herein a strategy that enables reactions involving DELs to be performed in organic solvents at near anhydrous conditions. This, in turn, opens previously inaccessible chemical reactivities to DEL. The Reversible Adsorption to Solid Support (RASS) approach, as described further herein, 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.

The technique disclosed herein has several advantages over currently available methods. For example, in the context of DEL, a small molecule tethered to a DNA hairpin (headpiece domain) 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. While many organic reactions can be performed in water, typically significant concessions are made with respect to reactant scope (low aqueous solubility), dilution, reaction time, yield and handling. The non-covalent adsorption technique disclosed herein would provide a solution to most of these problems, and significantly expand the tool kit of both reactants and reactions compatible with DEL. Additionally, in classical DEL construction the DNA molecule itself is directly exposed to reactive species that can result in degradation of the encoding molecule. Adsorbing the DNA onto a solid support has significant potential to stabilize and shield duplex DNA, protecting it from unwanted chemical reactions.

In one embodiment, 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. In one embodiment, the polynucleotide encoded chemical library member is immobilized by non-covalent adsorption of the polynucleotide moiety to the solid support. In one embodiment, the polynucleotide is a DNA, and the polynucleotide encoded chemical library is a DNA encoded chemical library. The inventors have demonstrated the feasibility of this approach through screening a variety of chromatography resins for the required properties of adsorbing a chemically modified DNA hairpin, retention of the DNA during washing with organic solvents, and subsequent elution to obtain the intact DNA without significant handling losses or chemical modifications. In one embodiment, the solid support comprises an anion exchange resin. In one embodiment, the solid support comprises a cation exchange resin. In one embodiment, the solid support comprises SDB-L and/or C18 silica gel. In one embodiment, the solid support comprises a hydrophobic resin. In one embodiment, the hydrophobic resin comprises a reversed phase, hydrophobic interaction, strong cation, weak cation, and affinity chromatography. In one embodiment, 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). In one embodiment, the DNA encoded chemical library (DEL) can undergo solvent exchange from aqueous buffer to an organic solvent, while being immobilized to the solid support. In one embodiment, 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).

While adsorbed, the inventors have demonstrated a number of classic organic reactions commonly used in DEL such as amide coupling, benzimidazole formation, reductive amination, and Wittig, Henry and oxime reactions in solvents varying from DMA to DCM. Further, the compatibility of the electrostatically adsorbed DNA with palladium catalysis including Sonogashira cross-coupling reactions has been demonstrated, as disclosed herein. Importantly, all these reactions were demonstrated through a sequential series of an initial amide bond formation on the amino-linker- DNA hairpin, washing, solvent exchange, followed by a second reaction, all while adsorbed on the resin.

In one embodiment, the immobilized polynucleotide as disclosed herein can undergo selective chemical modification while adsorbed to the solid support and exposed to organic solvent. In one embodiment, 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 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. While the current 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). Furthermore, 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. In one embodiment, the instantly disclosed 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. In one embodiment, 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.

To build a true DEL library, 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.

The concept of adsorbing biological macromolecules onto solid support to facilitate chemical transformations has been developed in the context of peptide modification. The observation that the polyvalent adsorption kinetics of biomacromolecules compared to monovalent small molecules allows for their selective binding and release from various solid supports, distinct from their physical properties such as hydrophobicity. Recently, this phenomenon was exploited to perform selective chemical transformation of peptides and proteins while adsorbed to reversed phase silica in aqueous and organic systems. This Reversible Adsorption to Solid Support (RASS) approach has enabled solvents to be exchanged, excess reagents to be washed away, and sequential reactions to be performed on the adsorbed macromolecules without the need for intermediate purification and isolation steps.

Recent work by the inventors exploited peptide and protein immobilization as a tool for synthetic chemistry. The Reversible Adsorption to Solid Support (RASS) approach leverages the multivalent binding kinetics of biomacromolecules to selectively bind to and elute from an inert solid support, such as reversed phased silica. See Cistrone, et al, Click-Based Libraries of SFTI-l Peptides: New Methods Using Reversed-Phase Silica. ACS Comb. Sci. 2016, 18, 139-143, which is incorporated by reference herein in its entirety. Critically, the differences in binding kinetics of biomacromolecules and small molecules allow for adsorbed biomolecules to react with small molecule reagents with the same logic as employed in conventional (covalently bound) solid phase organic synthesis strategies. See Flood, et al Post-Translational Backbone Engineering through Selenomethionine-Mediated Incorporation of Freidinger Lactams. Angew. Chem., Int. Ed. 2018, 57, 8697- 8701, which is incorporated by reference herein in its entirety. Thus, excess small molecule reagents are simply washed away while the polyvalent macromolecule remains adsorbed to the solid support. This allows for the use of organic solvents and reaction conditions that would be otherwise incompatible for the biomolecule.

Applied to DEL, 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. To identify an appropriate solid support, 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). Weak anion exchange resins were ruled out as well, because most bear basic, nucleophilic, or otherwise reactive moieties (Figure 5B). The potential of DNA immobilization has been demonstrated for peptide and peptoid synthesis on DNA using a weak anion exchange resin (DEAE Sepharose). However, the presence of abundant hydroxylated functionality and tertiary amines has limited the scope of employable reactions (vide infra).

In contrast, 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. When a model DEL headpiece with a pendent free amine, in PBS (100 mIUI DNA), was added to the resin, it efficiently adsorbs to the surface, as indicated by the lack of DNA detected in the binding supernatant (Figure 5C). The bound DNA could then be eluted into a high salt elution buffer, an approach that is widely applied in molecular biology. Importantly, 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. As an initial proof of concept, a simple amide bond formation reaction was performed in CH2CI2 on a model DEL headpiece (75% yield). Alternative solvents such as THF, DMF, Dioxane, DMSO, and MeCN were also compatible, with observed yields mirroring solvent dependencies for small molecule carbodiimide couplings.

Expanding the Reaction Space of DEL

To demonstrate the advantage of utilizing an inert support via RASS, a reaction that was previously recalcitrant in a DEL-setting was enabled: forging highly desirable sp ² -sp ³ linkages from ubiquitous building blocks and reagents. Specifically, the cross coupling between abundant (hetero)aryl halides and redox active esters (RAEs) derived from simple, ubiquitous, carboxylic acids was pursued. Numerous attempts at this reaction built off the success of the Giese reaction, by enlisting aryl iodides and RAEs in aqueous solution using Zn as a reductant under Ni- catalysis (SI). Recent findings have demonstrated the feasibility of such a bond formation, albeit only in the case of carboxylic acids bearing a radical stabilizing a-heteroatom. Aiming for a transformation without this inherent limitation, 4-iodobenzoic acid was coupled to a common DEL head piece resulting in DNA- Ar-I (Formula 1) (Figure 6A), setting the stage for a coupling with unstabilized RAE (Formula 2). Conventional DEL-based conditions were interrogated resulting in a maximum conversion of ca. 5% to 3 (entry 2, Figure 6B). It was reasoned that a more soluble reductant capable of reducing both the RAE and the Ni ¹¹ precatalyst to its active low valent Ni state would be superior. 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.

The protocols used to improve the yield of a DEL-based reaction are quite different due to the kinetics of highly diluted conditions in water. In stark contrast, the RASS-based approach enabled a more traditional optimization strategy that ultimately led to success. Thus, although application of Zn-based conditions to this system resulted in a heterogeneous dual surface reaction that could not be readily optimized, the homogeneous solution using RS as a soluble reductant proved immediately fruitful (entry 6, Figure 6B) providing trace product, the compound of Formula 3 (Figure 6B, entry 6). From this initial hit, the reaction was optimized to yield compound 3 in 84% after 18-24 h (entry 8) by increasing the concentration of Ni catalyst and reductant. The 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). In striking contrast, 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. Most likely, 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).

With optimized conditions in hand, the utility of the reaction was demonstrated with >40 different relevant carboxylic acids. Satisfactory to excellent yields were obtained by utilizing either isolated RAEs or in situ generated RAEs without altering conditions (Figure 6C). Multiple different (het)aryl iodides (>5), substituted at various positions, are also competent coupling partners affording similar yields. Finally, the scope could be further expanded to aryl bromides (which are more widely available) upon addition of 250 mM Nal forming coupled products, albeit in slightly attenuated yields. In accord with standard practice in DEL synthesis, yields are determined by integration of the total ETV absorbance at 260 nm via HPLC/MS of the crude reaction mixture. This protocol provides an accurate measurement of relative yield on nanomole scale as DNA is the dominant chromophore. To confirm the robustness of these measurements, all of experiments using a preferred Protocol B (Figure 6B, Entry 9) were conducted in triplicate.

Within the known realm of DNA-compatible chemistry, 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. To test this hypothesis, the inventors turned to 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.

As predicted, attempts to employ electrochemical amination with a traditional aqueous system led to no recoverable DNA (Figure 7B, entry 1). In striking contrast, the site-isolation garnered by the RASS approach led to promising results on the first attempt. Thus, adapting the published conditions employing a Ni(bpy)3Br2 precatalyst (50 mM), DBLT (300 mM), and 4 mA of current for 3 h, furnished 37% of the alkyl-aryl amine (formula 45) from aryl iodide (formula 1) (Figure 7B, entry 2). Although this initial result was encouraging, the major side product was the corresponding phenol, likely a result of hydroxide formation in situ. 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). Finally, increasing concentration of the Ni precatalyst (100 mM) brought yields to acceptable levels (74%, Figure 7B, entry 6). With optimized conditions in hand, various alkyl, and heteroaryl amines as well as one amide proved competent coupling partners with yields ranging from usable to good (Figure 7C). Interestingly, 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. Toward this end, 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. Specifically, 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%. Acyclic ketones, benzylic carbonyls, and sterically hindered carbonyls were all challenging substrates. Noticing this limitation in current DEL technology, the inventors have developed conditions to overcome this challenge. Reductive aminations are typically performed with the carbonyl on-DNA and a vast excess of amine in solution. These conditions force the equilibrium of the reducible imine species and are a common tactic in borohydride mediated strategies. In classical organic chemistry conditions, the use of excess carbonyl is generally avoided, as dialkylated products can result. After significant optimization, conditions were identified to couple 4-heptanone (compound of Formula 68) to DNA-headpiece (compound of Formula 66), a commercially available scaffold that is widely employed in DEL studies, to deliver 71 in 75% yield (Figure 8B, entry 8). Multiple ketones and aldehydes proved viable in this transformation with yields ranging from satisfactory to excellent. The conditions identified also allow for the use of other on-DNA primary and secondary amine building blocks in good yield. The enabling effect of boric acid could also be enlisted in a purely aqueous DEL system (Figure 8B, Entry 9).

ETnlike the cross-coupling and e-amination described above, reductive amination is not strictly enabled using a RASS approach. Releasing the shackles of conventional DEL reaction development, a classical organic approach inadvertently led us to a new set of B(OH) ₃ -mediated conditions that can function with or without RASS. As with prior examples, the reactions reported in Figure 8C were conducted in triplicate.

RASS-Enabled Del: Toward Creating Libraries

In order for new strategies to be adopted for use in DEL library construction, individual diversity generating steps must be sufficiently chemoselective and DNA recovery sufficiently efficient for at least 30% of the DNA to be recovered. While developing the above sp ² -sp ³ cross coupling, it became apparent that the reaction would result in suboptimal DNA recovery (~20%).

Surprisingly, this problem was addressed by optimizing the EtOH precipitation procedure. Increasing the ratio of EtOH: elute buffer ratio from 5: 1 to 10: 1 resulted in satisfactory DNA recovery of ~30%. The observed low DNA recovery is likely a result of reaction workup and precipitation conditions, as opposed to actual reactivity based degradation. Surprisingly, the inventors did not observe any DNA recovery problems in any of the other reactions outlined above.

To further establish compatibility with library construction, the viability of the product DNA was confirmed. After a completion of a full RASS cycle (bind, reaction, elute, EtOH precipitate) compound of Formula 3 was ligated to a 20-mer dsDNA primer, commonly used in DEL builds. The ligation efficiency was identical to compound of Formula 1, DNA that had not undergone a RASS cycle. This suggests that a DEL library member can be enzymatically encoded after an organic reaction in the RASS cycle.

As a DEL library is assembled, 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. In some embodiments, the inventors have shown that resins having larger“pores” to enable the adsorption of longer or larger DNA,

To illustrate the multistep process of a DEL build, the three reactions reported herein, were utilized sequentially (Figure 9). This three-cycle synthesis was devised using cross- coupling/deprotection, reductive amination, and finally electrochemical amination. A mock DEL build was performed using three cycles of chemistry, resulting in a skeleton, rich in linkage diversity (Figure 9). The DNA headpiece-small molecule intermediates were eluted from resin after each cycle, as would be requisite in a DEL build. Starting with 1 a decarboxylative sp2-sp3 cross coupling with Fmoc-proline was performed and the resulting product was deprotected. 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.

Structural Insights

In addition to facilitating reactions in neat organic solvents, 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. To investigate the conformation of the immobilized DNA, 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. Each DNA was adsorbed to Phenomenex Strata-XAL resin, and then treated with SYBR Green which increases in fluorescence when intercalated into dsDNA. The resins were washed in acetonitrile (5x) and suspended in ethanol for microscopy. The relative fluorescence observed by confocal fluorescence microscopy was consistent with the dsDNA construct remaining in the duplex form (strong relative fluorescence). All experiments were normalized to the total DNA content.

As shown in Figure 10, 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

As the use of DEL in drug discovery increases, there is a growing need to expand the tool box of organic trans-formations available to practitioners. In this disclosure, the inventors have outlined how strong-anion exchange resins based on quaternary ammonium moieties (RASS) can be used to facilitate reactions in organic solvents. Reactions previously outside the realm of DEL including, complex sp2-sp3 cross couplings and electrochemical aminations have been demon strated. The realization of a B(OH) ₃ -mediated reductive amination with expanded generality in both aqueous and RASS contexts was also discovered. Application of these three reactions in a DEL-rehearsal suggests that this technology is applicable to real-world library construction. Finally, structural studies have confirmed that DNA is still double-stranded while bound to the resin, a necessary proviso to protect the encoding molecule. As such, it is anticipated that this approach to DEL construction will find widespread utility.

Embodiments of the present disclosure are further described in the following examples. The examples are merely illustrative and do not in any way limit the scope of the invention as claimed.

EXAMPLES

Example 1

Reaction scope

In one embodiment, 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). A variety of conditions were used, for example 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). In Scheme H, the inventors demonstrate the compatibility of the electrostatically adsorbed DNA with palladium catalysis including Sonogashira cross-coupling reactions. Importantly, all these reactions were demonstrated through a sequential series of an initial amide bond formation on the amino-linker- DNA hairpin, washing, solvent exchange, followed by a second reaction, all while adsorbed on the resin.

Scheme A. m

25 mM Oxyma

Formula (1) 25 mM NEt ₃

2 hrs in Various Solvent

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.

Table 1.

Solvent Conversion (%)

T etrahydrofuran (THF) 31.5

Diehl orom ethane (DCM) 95.4

l,4-Dioxane (Diox) 63.7

Dimethylsulfoxide (DMSO) 37.1

Acetonitrile (ACN) 21.4

Dimethylacetamide (DMA) 69.8

Scheme B

Conversion: >95%

Conditions: 50 mM Diamine, 18 Hrs, DMA, 65 °C Scheme F.

Conversion: >95%

Conditions: 25 mM Alkyne, 50 mM Piperdine, 1 eq PPd(Ph ₃ ) ₄ , 4 Hrs, 65° C

Scheme I.

Conversion: 20% Prd, 20% Dehal, 60% SM

Conditions: 100 mM RAE, 200 mM Nano Zn, 1 eq (25 nMols) (dtbby)MBr2, DMA, 18 hrs, RT

Example 2

Suzuki coupling

Suzuki coupling was done as illustrated in Scheme J. DNA-hairpin (100 pL at 250 pM) adsorbed to swelled Strata-AX resin (100 pL) was washed (500 pL twice) with degassed DMA. In de- gassed DMA (1000 uL), phenylboronic acid (10 mM), tetrakis(triphenylphosphine)palladium (~1 mM), and triethylamine (20 mM) were dissolved and added to the decanted resin bed in a glass 1.5 ml HPLC vial fitted with a septum-screw top. The reaction vial was evacuated and backfilled with argon (3 times). The reaction was stirred at 65 °C for 18 hours at which point the tube was spun down and the supernatant removed and discarded. The resin bed was washed (500 pL twice) with DMA, eluted, and analyzed.

Scheme J

Example 3

Solvent compatibilities

In one embodiment, the inventors tested solvent compatibilities of different solid supports. 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.

Example 4

Utility and advantages

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.

In one embodiment, 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. Example 5

Adsorption properties

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.

Example 6

On-DNA sulfone formation

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.

Example 7

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 various methods and techniques described above provide a number of ways to carry out the invention. Of course, it is to be understood that not necessarily all objectives or advantages described may be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as may be taught or suggested herein. A variety of advantageous and disadvantageous alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several advantageous features, while others specifically exclude one, another, or several disadvantageous features, while still others specifically mitigate a present disadvantageous feature by inclusion of one, another, or several advantageous features. Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be mixed and matched by one of ordinary skill in this art to perform methods in accordance with principles described herein. Among the various elements, features, and steps, some will be specifically included and others specifically excluded in diverse embodiments.

Although the invention has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the invention extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.

Many variations and alternative elements have been disclosed in embodiments of the present invention. Still further variations and alternate elements will be apparent to one of skill in the art. Among these variations, without limitation, are the selection of constituent modules for the inventive compositions, and the diseases and other clinical conditions that may be diagnosed, prognosed or treated therewith. Various embodiments of the invention can specifically include or exclude any of these variations or elements.

In some embodiments, 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.

In some embodiments, the terms“a,”“an,” and“the” and similar references used in the context of describing a particular embodiment of the invention (especially in the context of certain of the following claims) 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. The use of any and all examples, or exemplary language (e.g.“such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the invention can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this invention include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above cited references and printed publications are herein individually incorporated by reference in their entirety. In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that can be employed can be within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present invention are not limited to that precisely as shown and described.