Article pubs.acs.org/bc
DNA-Compatible Nitro Reduction and Synthesis of Benzimidazoles Huang-Chi Du and Hongbing Huang* Center for Drug Discovery, Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas 77030, United States S Supporting Information *
ABSTRACT: DNA-encoded chemical libraries have emerged as a cost-effective alternative to high-throughput screening (HTS) for hit identification in drug discovery. A key factor for productive DNA-encoded libraries is the chemical diversity of the small molecule moiety attached to an encoding DNA oligomer. The library structure diversity is often limited to DNA-compatible chemical reactions in aqueous media. Herein, we describe a facile process for reducing aryl nitro groups to aryl amines. The new protocol offers simple operation and circumvents the pyrophoric potential of the conventional method (Raney nickel). The reaction is performed in aqueous solution and does not compromise DNA structural integrity. The utility of this method is demonstrated by the versatile synthesis of benzimidazoles on DNA.
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INTRODUCTION The use of a DNA-encoded library (DEL) has emerged as a cost-effective alternative technology for hit generation that addresses the limitations and economic shortcomings of highthroughput screening (HTS).1−5 DEL enables the exploration of chemical spaces 4 to 5 orders of magnitude greater than is achievable by traditional HTS methods, and it has delivered high-affinity ligands of disease targets.6−9 Furthermore, the advent of increasingly efficient next-generation sequencing technology for high-throughput DNA sequencing allows the simultaneous interrogation of hundreds of millions of compounds at a fraction of the cost of conventional HTS.10 As such, DNA-encoded chemistry technology represents an integrated and cost-effective platform for the discovery of novel chemical probes and drug candidates.11−13 The key to the success of DEL is the structural diversity of the small molecules displayed by the library. The smallmolecule moieties of DELs are generally synthesized through a multistep process, and each chemical step is accomplished on an unprotected DNA oligomer.14 Hence, library chemical diversity is limited to DNA-compatible synthetic reactions. The application of conventional synthetic methods, which are often optimized for organic solvents, is complicated by the requirement of compatibility with the attached DNA oligomer. For instance, compatible reactions must proceed under conditions that solubilize the DNA substrate, which requires aqueous solutions. Also, reagents must not cause degradation of the DNA oligomer.15 Additionally, reactions applicable to DEL synthesis must have a broad substrate scope and be operationally simple.16 Herein, we report a facile sodium dithionite−mediated nitro reduction in water and subsequent synthesis of benzimidazoles on DNA. © XXXX American Chemical Society
Benzimidazoles are a privileged scaffold in pharmaceuticals and biologically active molecules.17 Despite well-established methods for the formation of this useful heterocycle in organic solvents and widespread application in medicinal chemistry, there is only one reported protocol for the synthesis of benzimidazoles in the presence of DNA in aqueous media.14 DNA-bound o-nitroarylamine 1 was reduced to the corresponding diamine 2 using Raney nickel and hydrazine, and subsequent condensation with acetaldehyde offered benzimidazole 3 (Scheme 1). Whereas Raney nickel is a powerful reducing reagent, there are several practical limitations to its use in library synthesis: the limits to its utility owing to its pyrophoric nature, the unpredictable deactivation on aging, and the difficulty in determining accurately the weight of Ni used. In search of a safe and easy-to-handle alternative to Raney nickel, we turned our attention to sodium dithionite (Na2S2O4), an inexpensive and versatile reducing reagent.18 It has been reported to reduce aryl nitro groups to aryl amines.19,20 The mild reaction conditions, broad functional group tolerance, and good aqueous solubility (0.2 g/mL) rendered Na2S2O4 a prime choice for our studies.
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RESULTS AND DISCUSSION A primary amine 4, covalently linked to a double-stranded DNA via a PEG linker, was used as the starting material for this study. A variety of DNA-conjugated o-nitroarylamine 5 were synthesized in good yields by either the acylation or the sulfonylation of 4, followed by nucleophilic aromatic substitution (SNAr) (Scheme 2). Received: July 19, 2017 Revised: August 19, 2017 Published: August 25, 2017 A
DOI: 10.1021/acs.bioconjchem.7b00416 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry Scheme 1. Previous Report for Synthesis of Benzimidazole in the Presence of DNA
Scheme 2. Preparation of Starting Materials
Scheme 3. Initial Attempts of Nitro Reduction with Na2S2O4
Table 1. Optimization of Reaction Conditions for Nitro Reduction
entry 1 2 3 4 5 6 7 a
buffer pH pH pH pH pH pH pH
9.5 9.5 5.5 9.5 9.5 5.5 9.5
borate buffer borate buffer phosphate buffer borate buffer borate buffer phosphate buffer borate buffer
Na2S2O4 (equiv.)
viologen
temperature
6e
7
8
100 200 100 100 100 100 100
none none none none 10 equiv. 10 equiv. 10 equiv.
rta rt rt 80 °C rt rt 80 °C
29% 28% 29% 51% 57% 31% 78%
52% 56% 59% 33% 26% 52% 7%
19% 16% 12% 16% 17% 17% 15%
Room temperature.
When electron-deficient o-nitroarylamine 5e was subject to the same conditions, the reaction yielded only 29% of the desired product 6e, with amino sulfite 7 as major product (52%) as well as a small amount of diamine byproduct 8 (Table 1, entry 1). To improve the conversion to the desired product, we surveyed the effects of buffer pH, reaction temperature, and increasing the amount of the reducing reagent and additives. The buffer pH did not appear to influence the course of the reaction; similar results were observed using pH 9.5 borate
To our delight, when o-nitroarylamine 5a was treated with 100 equiv of aqueous Na2S2O4 at room temperature, a clean and complete conversion to diamine 6a was achieved within 20 min in pH 9.5 borate buffer. Likewise, closely related onitroarylamines 5b−d also underwent complete conversion under the same reaction conditions offering corresponding diamines 6b−d in high yields, as determined by liquid chromatography−mass spectrometry (Scheme 3). B
DOI: 10.1021/acs.bioconjchem.7b00416 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry Table 2. Evaluation of Nitro Reduction on Various Substrates
To develop DNA-compatible chemistry applicable to DEL synthesis, a practical consideration is that the purification should be operationally easy and sufficient to remove excess reagents and impurities that may interfere subsequent ligation of unprotected DNA oligomer. During the chemistry validation process, we routinely assess ligation efficiency of the attached DNA oligomer upon which a novel chemical reaction is carried out. In this case, a 58 base pair double-stranded DNA (dsDNA), 34, was used for the DEL compatibility test (Scheme 4). The DNA was attached to the acid 35, and subsequent SNAr gave N-benzyl o-nitroarylamine 36. The onepot procedure of nitro reduction and cyclization furnished benzimidazole 39 in good yield. Ethanol precipitation was the only purification method and was performed between each chemical step. Enzymatic ligation was conducted with a 12 base pair dsDNA. The benzimidazole-attached DNA ligated efficiently and offered the 70 base pair DNA product 40 with similar efficiency as naiv̈ e dsDNA (see the Supporting Information for details).
buffer or pH 5.5 phosphate buffer (Table 1, Entry 1 and 3). Using more Na2S2O4 had little effect on the reaction (Table 1, entry 2). However, the reaction temperature had a notable impact. At elevated temperature, the desired product was obtained as major product (Table 1, entry 4). Viologens are well-known electrotransfer catalysts and enhance the reactivity of Na2S2O4 in converting nitroarenes to the corresponding amines.19 In our hands, methyl viologen dichloride improved the reduction potential of Na2S2O4 at high pH, providing the desired product as major product at room temperature (Table 1, entry 5). Interestingly, it had a marginal effect on the reaction at low pH (Table 1, entry 6). Ultimately, the combination of elevated temperature and addition of methyl viologen furnished the desired product in good yield (Table 1, entry 7). With the optimal conditions in hand, the scope of the reaction was explored (Table 2). Various N-substituted onitroarylamines underwent complete conversion within 20 min, offering corresponding amines in moderate to excellent yields. Having established an efficient protocol for reducing nitro group, we then explored a one-pot synthesis of benzimidazoles on DNA. Various DNA-conjugated o-nitroarylamines were first treated with Na2S2O4 and methyl viologen; upon completion of the nitro-reduction reaction, aldehydes were added to the reaction mixture. After heating at 80 °C for 12 h, corresponding benzimidazoles were formed in high yields (Table 3). Both aromatic and aliphatic aldehydes effectively participated in the reaction. The desired DNA-conjugated products were easily purified by ethanol precipitation.
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CONCLUSIONS
In summary, we have developed a facile and versatile method by which to reduce DNA-conjugated aryl nitro compounds to corresponding amines in aqueous media. The utility of this methodology was demonstrated in one-pot synthesis of various benzimidazoles on DNA. With a broad substrate scope and C
DOI: 10.1021/acs.bioconjchem.7b00416 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry Table 3. One-Pot Synthesis of Benzimidazoles
a
Ph(OMe)2: 2,4-dimethoxylphenyl. bCy: cyclohexyl. cPhNO2: 4-nitrophenyl. dCyMe: cyclohexylmethyl.
Ethanol Precipitation and DNA Reconstitution. To a DNA reaction mixture was added 5% (V/V) 5 M NaCl solution and 2.5−3 times the volume of absolute ethanol. The colloidal solution was then sit at −20 °C overnight. After centrifugation, the supernatant was decanted, and the DNA pellet was dried in the air. Water was added to reconstitute the DNA in certain concentration. Generally, ethanol precipitation was performed after each chemical reaction. General Procedure for Acylation. To a solution of DNA HP (10 nmol, 10 μL, 1.0 mM, 1 equiv) in H2O was added pH 9.5 borate buffer (4000 nmol, 16 μL, 250 mM, 400 equiv), acid (1000 nmol, 5 μL, 200 mM in MeCN, 100 equiv), and 4-(4,6dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (1000 nmol, 5 μL, 200 mM in H2O, 100 equiv). The reaction was allowed to sit at room temperature overnight and then quenched by EtOH precipitation. General Procedure for SNAr Reaction. The reconstituted acylation product (10 nmol, 10 μL, 1.0 mM, 1 equiv) in H2O was treated with borate buffer (pH 9.5, 2500 nmol, 10 μL, 250
simple operation, the newly developed chemistry is expected to find wide use in the preparation of diverse DELs.
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EXPERIMENTAL PROCEDURES Materials and Instrumentation. All of the reagents were purchased from vendors and used without further purification. Borate and Tris−borate−EDTA (TBE) buffer was freshly prepared in-house. DNA headpiece (HP) (5′ d Phos− CTGCAT−spacer 9−amino C7−spacer 9−ATGCAGGT 3′) was obtained directly from Biosearch Technologies, Novato, CA. Reverse-phase ultraperformance liquid chromatography (UPLC) was utilized to analyze the data using Waters Acquity UPLC oligonucleotide BEH C18 column (130 Å, 1.7 μm, 2.1 mm × 50 mm). The analyses were executed with the following solvent systems: 8.6 mM TEA and 100 mM HFIP in H2O (A) and 8.6 mM TEA and 100 mM HFIP in acetonitrile/H2O (40:60) (B) over 2 min. The observed multiple charges signals were deconvoluted using ProMass (Novatia software) to estimate DNA-conjugate mass. D
DOI: 10.1021/acs.bioconjchem.7b00416 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry Scheme 4. DNA-Compatible Chemistry Validation Processe
mM, 250 equiv) and amine (1000 nmol, 5 μL, 200 mM in MeCN, 100 equiv) at 80 °C overnight. The reaction mixture was cooled to room temperature prior to EtOH precipitation. General Procedure for Nitro Reduction. To a solution of DNA conjugate (10 nmol, 10 μL, 1.0 mM, 1 equiv) in H2O was added borate buffer (pH 9.5, 2500 nmol, 10 μL, 250 mM, 250 equiv) and viologen (100 nmol, 1 μL, 100 mM in H2O, 10 equiv), followed by the addition of Na2S2O4 (1000 nmol, 5 μL, 200 mM in H2O, 100 equiv). The reaction mixture was heated at 80 °C for 20 min. The reaction mixture was cooled to room temperature prior to EtOH precipitation. General Procedure for Formation of Benzimidazoles. For the one-pot synthesis of benzimidazole, the crude reduction product (10 nmol) in borate buffer (pH 9.5, 2500 nmol, 10 μL, 250 mM, 250 equiv) was treated with aldehyde (1000 nmol, 5 μL, 200 mM in MeCN, 100 equiv) at 80 °C overnight. For the step-wise procedure, pH 9.5 borate buffer (2500 nmol, 10 μL, 250 mM, 250 equiv) was added to the reconstituted reduction product (10 nmol, 10 μL, 1.0 mM, 1 equiv). After aldehyde (1000 nmol, 5 μL, 200 mM in MeCN, 100 equiv) was added, the reaction was heated at 80 °C overnight. Both protocols provided the desired product after EtOH precipitation and reconstitution. DNA Ligation. To DNA 39 (2 nmol, 5.88 μL, 1.0 equiv) was added DNA_1 (5′-CTACCAGGCGGT-3′, 2.4 nmol, 2.4 μL, 1.2 equiv), DNA_2 (5′-CGCCTGGTAGGA-3′, 2.4 nmol, 2.4 μL, 1.2 equiv), and nuclease-free water (7.1 μL), followed by the addition of 10× HEPES buffer (2 μL) and T4 DNA ligase (0.2 μL). The reaction mixture was incubated at room temperature overnight before performing gel electrophoresis. The crude material was purified by EtOH precipitation. Gel Electrophoresis. Gel electrophoresis is usually executed using precast 10% TBE acrylamide gel from Invitrogen (12 wells). The gel box was filled with 1× TBE buffer until the gel was covered. The purified DNA (by EtOH precipitation) was diluted to the concentration at 12 ng/μL. To a tube was added 10 μL of one DNA sample and 2 μL of 6× DNA loading dye to make a DNA dye loading sample. The first
lane of the gel was loaded with a DNA molecular weight ladder, and 5 μL of DNA-dye mixed samples was loaded into each lane. Gel was ran at 160 V for 35 min and then stained in a container with 0.5 ng/mL ethidium bromide in 1× TBE buffer for 50 min. DNA fragments were visualized under a UV light device.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.7b00416. Additional details on the structure of DNA headpieces, oligonucleotide sequences, representative mass spectra, starting material preparation data, analytical data, and Sanger sequencing. (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: (+1)-713-7987693. ORCID
Hongbing Huang: 0000-0001-5103-0574 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
We thank Drs. John Nelson and Martin Matzuk for critical reading the manuscript. We also thank Zhifeng Yu and Sanchit Trivedi for preparing samples for Sanger sequencing. These studies were supported by grants from the Welch Foundation (H-Q-0042) and a Core Facility Support Award (RP160805) from the Cancer Prevention Research Institute of Texas (CPRIT). E
DOI: 10.1021/acs.bioconjchem.7b00416 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry
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DOI: 10.1021/acs.bioconjchem.7b00416 Bioconjugate Chem. XXXX, XXX, XXX−XXX
