Development of DNA-Compatible Suzuki-Miyaura Reaction in

Article Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Development of DNA-Compatible Suzuki-Miyaura Reaction in Aqueous Media Jian-Yuan Li and Hongbing Huang* Center for Drug Discovery, Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas 77030, United States

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S Supporting Information *

ABSTRACT: DNA-encoded chemical libraries (DELs) are a cost-effective technology for the discovery of novel chemical probes and drug candidates. A major limiting factor in assembling productive DELs is the availability of DNA-compatible chemical reactions in aqueous media. In an effort to increase the chemical accessibility and structural diversity of small molecules displayed by DELs, we developed a robust Suzuki-Miyaura reaction protocol that is compatible with the DNA structures. By employing a water-soluble Pd-precatalyst, we developed conditions that allow efficient coupling of DNA-linked aryl halides with a wide variety of boronic acids/esters including heteroaryl boronates.

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conjugated aryl halides. Omumi et al. reported the first SuzukiMiyaura cross-coupling of DNA-linked 8-Br-purine employing Pd(OAc)2 and a hydrophilic phosphine ligand, 3-tri(3sulfonatophenyl)phosphine trisodium (TPPTS).18 In 2015, Ding et al. at GlaxoSmithKline (GSK) published a Pd(PPh3)4 mediated Suzuki-Miyaura cross-coupling reaction involving DNA-linked aryl bromides.19 Later, the same group optimized the reaction for less reactive DNA-linked aryl chlorides using a combination of phosphinous acid/Pd catalyst and the sSPhos ligand (Scheme 1).20 This catalyst system expanded the substrate scope by promoting coupling between less reactive aryl chlorides and challenging heteroaryl boronates. The same study also showed that preformation of the catalyst was required for good activity and the ratio of Pd to the ligand was critical for consistent results. In this paper, we report an efficient and operationally simple Suzuki-Miyaura reaction that has broad substrate scope of both aryl halides and aryl boronates (Scheme 1).

INTRODUCTION DNA-encoded chemical libraries (DELs) have emerged as a complementary approach to high-throughput screening (HTS) for discovery of novel chemical probes and drug candidates.1−5 By merging the well-established split-and-pool combinatorial library synthesis with highly sensitive and reliable polymerase chain reaction and DNA sequencing technology, DEL screening allows the exploration of a broader chemical space and simultaneous interrogation of hundreds of millions of compounds in a cost-effective manner, compared to traditional HTS.6 During the past decade, novel small molecule ligands for a wide variety of protein targets have been identified through screening of DELs.7−9 The chemical and structural diversity of the small molecules displayed by DELs is critical to the successful discovery of drug-like chemical matters.10 Thus, the availability of efficient synthetic methods that enable facile derivatization and are applicable to a broad scope of substrates is key to the preparation of productive DELs.11,12 Reactions suitable for DEL synthesis must also be compatible with the DNA structure and be operationally simple. The Suzuki-Miyaura coupling is one of the most widely employed reactions in medicinal chemistry, mainly because of its mild reaction conditions, high tolerance toward functional groups, the stability of the requisite reagents, and the wide diversity of commercially available coupling partners.13 While various conditions have been developed for conducting SuzukiMiyaura reaction in water or water/organic biphasic solvent systems,14−17 there are few examples that involve DNA© XXXX American Chemical Society

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RESULTS AND DISCUSSION The efficient generation of a phosphine-ligated Pd(0) species is key to a successful Suzuki-Miyaura coupling reaction. A variety of palladacycle-based precatalysts are reported to readily form a ligated Pd(0) species in situ when exposed to base.21 The Received: September 25, 2018 Revised: October 18, 2018 Published: October 19, 2018 A

DOI: 10.1021/acs.bioconjchem.8b00676 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry Scheme 1. On-DNA Suzuki-Miyaura Coupling Reaction Development

Table 1. Optimization of Reaction Conditions for Suzuki-Miyaura Couplinga

Entry

[Pd]

Base (equiv)

T (°C)

Conversion

1 2 3 4 5 6 7 8 9

SPhos-Pd -G2 sSPhos-Pd-G2 sSPhos-Pd-G2 sSPhos-Pd-G2 sSPhos-Pd-G2 sSPhos-Pd-G2 sSPhos-Pd-G2 sSPhos-Pd-G2 sSPhos-Pd-G2

CsOH (250) CsOH (250) NaOH (200) Cs2CO3 (200) CsOH (150) CsOH (250) CsOH (250) CsOH (250) CsOH (250)

80 80 80 80 80 70 60 80 80

no rxn 99% 77% no rxn 5% 51% no rxn 84%b 99%c

a Reaction conditions: 1 equiv of AH1 (1 mM in H2O), 100 equiv of boronate (200 mM in 1,4-dioxane/H2O (1/1)), 2 equiv sSPhos-Pd-G2 (10 mM in DMA), CsOH (600 mM in H2O), borate buffer (pH 8.2, 125 equiv). b50 equiv of boroates (200 mM in 1,4-dioxane/H2O (1/1)) was used. c Water was used as the reaction medium.

Scheme 2. Suzuki Coupling of Representative Boronates with AH1

versatility of these precatalysts and the simple reaction setup prompted us to investigate commercially available Buchwald precatalysts for the Suzuki-Miyaura coupling reaction on-DNA. The initial screen for suitable catalysts was performed using a DNA-linked phenyl chloride AH1 and p-tolylboronic acid, with CsOH in a borate buffer (pH 8.2) at 80 °C. Interestingly,

for all the precatalysts that we tested, only the water-soluble sulfonated SPhos-G2 precatalyst provided the desired product in high analytical yield, and the parent SPhos-G2-precatalyst failed to promote the reaction (Table 1, entries 1−2). The presence of a strong base (CsOH) is required for good yields; less basic NaOH gave lower conversions and weak bases, such B


Article

Bioconjugate Chemistry

with AH4 and AH6 to give >50% conversion. As expected, aryl bromide AH7 and aryl iodide AH8 demonstrated superior reactivity, and the majority of the reaction went to completion (>90%). Table 2 illustrates representative challenging boronate substrates of the reaction. For less reactive aryl halides, the increased steric hindrance at 2 and 6 positions hampered reaction conversions (B19, B20, and B21). 2,6-Difluorophenyl boronic acid (B21) proved to be an especially difficult substrate, presumably due to a combination of steric hindrance and electron withdrawing properties. Nevertheless, it effectively coupled with AH6 and AH8 with complete conversion. Another type of difficult substrate for less reactive aryl halides is boronates that contain at least one chloride substituent (B22−26). Very low conversions or no reaction were observed with the least reactive aryl chlorides (AH1 and AH2). More reactive aryl chlorides (AH4 and AH6) provided good to excellent yields. Again, AH7 and AH8 demonstrated superior coupling efficiency with these difficult substrates. To ensure that our protocol was compatible with DEL synthesis, we assessed ligation efficiency of the attached DNA oligomers (Scheme 3). A 58-base pair double-stranded DNA (dsDNA) 1, was attached to 3-chlorobenzoic acid, and subsequent Suzuki coupling gave DNA-conjugated 3-(ptolyl)benzamide 3 in good yield. Ethanol precipitation was performed at each chemical step to purify the products. The 4tolyl-phenyl-attached DNA 3 was ligated to a 12-base pair dsDNA to generate the 70-base-pair DNA product 4 with ̈ dsDNA. Sanger sequencing of the similar efficiency as naive amplified DNA 4 confirmed its sequence and the structural integrity of the DNA oligomers.26

as Cs2CO3, proved to be completely ineffective (Table1, entries 3−4). The reaction is also sensitive to the amount of the base and boronates used in the reaction (Table1, entries 5 and 8) and is less efficient at lower temperatures (Table1, entries 6−7). The borate buffer can be substituted with water, in which the reaction gave similarly high conversion (Table1, entry 9). To determine the scope of this reaction, we investigated the cross-coupling of AH1 with a structurally diverse set of boronic acids/esters. Scheme 2 illustrates representative boronates which provided the desired coupling products in good yields. Compared to the previously reported methods, the reaction demonstrated improved reactivity and better tolerance to functional groups. For example, both electron-rich (B11) and electron-deficient (B3, B5, B6, and B15) arylboronic acids underwent efficient coupling with AH1 and gave high conversions. In contrast, B5 and B6 demonstrated low reactivity when the POPD/sSPhos system was used.20 Under the current conditions, sterically hindered substrates also efficiently participated in the reaction (B7); the more hindered 2,6-disubstituted boronates were less reactive (B8). Some functional groups are not compatible with the previously reported methods. For instance, a nitrile group is labile to hydrolysis under those conditions.19 In contrast, the nitrile group was well tolerated (B2) and 2-F pyridines (B1 and B14) also remained intact with the newly developed method. Nitrogen-containing heterocycles are prominent structure motifs in bioactive compounds, but they also have the potential to temper catalyst activity when palladium is used.22−24 Notably, the α-heteroatom in B14 did not interfere with the reaction. A variety of heteroaryl boronates effectively coupled with AH1 to provide the desired products in good to excellent yields. We next investigated the substrate scope regarding DNAconjugated aryl halides. The reaction proved to be highly efficient for the cross-coupling of a wide variety of aryl halides. Figure 1 summarizes the conversion distribution from the

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CONCLUSIONS In summary, we have demonstrated that the water-soluble precatalyst sSPhos-G2 promotes highly efficient on-DNA Suzuki-Miyaura cross-coupling with broad substrate scope of aryl halides and boronates. We have also shown that the protocol maintains the structural integrity and ligation activity of the attached DNA oligomers. The broad substrate scope and simple operation makes the method a valuable tool for the construction 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 were freshly prepared in-house. DNA headpiece (HP) (5′d PhosCTGCAT-spacer 9-amino C7-spacer 9-ATGCAGGT 3′) was obtained directly from Biosearch Technologies, Novato, CA. Thermo Vanquish reverse-phase ultraperformance liquid chromatography (UHPLC) system was integrated with LTQ XL ion trap mass spectrometer for LC/MS analysis of oligonucleotides. LC settings: Thermo DNAPac RP column (2.1 × 50 mm, 4 μm), 15 mM TEA/100 mM HFIP in water (solvent A), 15 mM TEA/100 mM HFIP in 50% methanol (solvent B), methanol (solvent C), 0.65 mL/min (flow rate), 2 min (run time, gradient), 100 °C (column temperature), and 40 °C (post column cooler). MS settings: ESI in negative mode (Source), 4100 V (Spray voltage), 390 °C (Source heater temperature), 28 (Sheath Gas, arbitrary units), 8 (Auxiliary Gas, arbitrary units), 2 (Sweep Gas, arbitrary units),

Figure 1. Conversion distribution of the Suzuki coupling of DNAlinked aryl halides and boronates.

reaction of 8 DNA-linked aryl halides AH1−8 with 84 boronates.25 Phenyl chlorides AH1 and AH2 demonstrated similar activity. About 70% of the boronates participated in the reaction with >50% conversion. Heteroaryl chlorides AH3, AH4, AH5, and AH6 proved to be more reactive and effectively coupled to provide the desired heterobiaryl products in good yields. For AH3 and AH5, 80% of the boronates offered >50% conversion; about 90% of the boronates coupled C


Article

Bioconjugate Chemistry Table 2. Coupling of Challenging Boronates to DNA-Conjugated Aryl Halides

Scheme 3. DNA-Compatible Chemistry Validation Process

350 °C (Capillary temperature), −33.0 V(Capillary voltage), −92.0 V(Tube lens), 500−2000 m/z (MS Scan).

Data Analysis. Samples were analyzed on a Thermo Vanquish UHPLC system coupled to an electrospray LTQ ion D


Bioconjugate Chemistry

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trap mass spectrometer. An ion-pairing mobile phase comprising of 15 mM TEA/100 mM HFIP in a water/ methanol solvent system was used in conjunction with an oligonucleotide column Thermo DNAPac RP (2.1 × 50 mm, 4 μm) for all the separations. All mass spectra were acquired in the full scan negative-ion mode over the mass range 500−2000 m/z. The data analysis was performed by exporting the raw instrument data (.RAW) to an automated biomolecule deconvolution and reporting software (ProMass) which uses a novel algorithm known as ZNova to produce artifact-free mass spectra. The following deconvolution parameters were applied: peak width 3.0, merge width 0.2, minimum and normalize scores of 2.0 and 1.0 respectively. The noise threshold was set at S/N 2.0. The processed data were directly exported to Microsoft Excel_ (.xls) worksheets for further data comparisons. Ethanol Precipitation and DNA Reconstitution. To a DNA reaction mixture was added 5% (V/V) 5 M NaCl solution and 2.5−3× the volume of absolute ethanol. The colloidal solution was then allowed to sit at −20 °C overnight. After centrifugation for 1 h, the supernatant was decanted, and the DNA pellet was dried in the air. Invitrogen UltraPure distilled 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 Suzuki Coupling Reaction. To the reconstituted acylation product (10 nmol, 10 μL, 1.0 mM in H2O, 1 equiv) was added CsOH (4000 nmol, 6.67 μL, 600 mM in H2O, 400 equiv) and boronates (1000 nmol, 5 μL, 200 mM 1,4-dioxane/H2O (1/1), 100 equiv), followed by adding sSPhos-Pd-G2 (20 nmol, 2 μL, 10 mM in DMA, 2 equiv). The reaction mixture was heated at 80 °C for 15 min and was cooled to room temperature prior to EtOH precipitation. DNA Ligation. To DNA 3 (4 nmol, 4.2 μL, 1.0 equiv) was added DNA_1 (5′ Phos-AATGCCTAAGGT 3′, 4.8 nmol, 4.8 μL, 1.2 equiv), DNA_2 (5′ Phos-CTTAGGCATTGA 3′, 4.8 nmol, 4.8 μL, 1.2 equiv), and nuclease-free water (21.8 μL), followed by the addition of 10× HEPES buffer (4 μL) and T4 DNA ligase (0.4 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.

Article

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00676. Structure of DNA headpiece, oligonucleotide sequences, experimental procedures, LCMS analysis and Sanger Sequencing result (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: (+1) 713-798-7693. ORCID

Hongbing Huang: 0000-0001-5103-0574 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Drs. Richard Lewis and Conrad Santini for proofreading the manuscript. We also thank Mr. 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).

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Development of DNA-Compatible Suzuki-Miyaura Reaction in

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