Article Cite This: J. Org. Chem. 2017, 82, 10803-10811
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Synthesis of Small-Molecule/DNA Hybrids through On-Bead AmideCoupling Approach Kenji D. Okochi,† Luca Monfregola,† Sarah Michelle Dickerson,† Ryan McCaffrey,† Dylan W. Domaille,‡ Chao Yu,† Glenn R. Hafenstine,‡ Yinghua Jin,† Jennifer N. Cha,‡ Robert D. Kuchta,† Marvin Caruthers,† and Wei Zhang*,† †
Department of Chemistry and Biochemistry and ‡Department of Chemical and Biological Engineering, University of Colorado at Boulder Boulder, Colorado 80309, United States S Supporting Information *
ABSTRACT: Small molecule/DNA hybrids (SMDHs) have been considered as nanoscale building blocks for engineering 2D and 3D supramolecular DNA assembly. Herein, we report an efficient on-bead amide-coupling approach to prepare SMDHs with multiple oligodeoxynucleotide (ODN) strands. Our method is high yielding under mild and user-friendly conditions with various organic substrates and homo- or mixed-sequenced ODNs. Metal catalysts and moisture- and air-free conditions are not required. The products can be easily analyzed by LC−MS with accurate mass resolution. We also explored nanometer-sized shapepersistent macrocycles as novel multitopic organic linkers to prepare SMDHs. SMDHs bearing up to six ODNs were successfully prepared through the coupling of arylenethynylene macrocycles with ODNs, which were used to mediate the assembly of gold nanoparticles.
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INTRODUCTION The introduction of solid-phase phosphoramidite chemistry has enabled DNA to become the material of choice for bottom-up self-assembly.1−3 The coupling of DNA or oligodeoxynucleotides (ODNs) with small organic molecules offers precise control over the structure and geometry of the DNA-attached product and potentially could enable supramolecular DNA assembly, utilizing both the base-pairing of the DNA and the self-assembly of the organic component.4,5 To access the full potential of such small molecule/DNA hybrids (SMDHs) as nanoscale building blocks in supramolecular DNA assembly,6,7 it is necessary to have a robust conjugation methodology as well as a diversity of organic building blocks. SMDHs have been typically prepared either via solution-phase coupling8−12 (amide,8 thiol-Michael,9 or click10) or by incorporating the organic component into the growing ODN strand during solidphase synthesis, usually through phosphoramidite chemistry.13−16 However, the preparation of multitopic organic− DNA hybrids through such approaches has been challenging due to low-yielding steps with tedious purification. Recently, the use of on-bead chemical elaboration has been reported © 2017 American Chemical Society
through which SMDHs containing multiple DNA strands can be synthesized.17,18 For instance, Nguyen and co-workers obtained excellent yields of SMDH4 (the subscript denotes the number of ODNs conjugated to the small molecule) using onbead click chemistry with a tetraphenylmethane-based small molecule.18 Click chemistry, however, relies on copper reagents which can irreversibly bind to the DNA backbone, making analysis difficult and possibly compromising the biological activity of the resulting SMDHs.19 In contrast to click chemistry, amide couplings17,20−24 using N-hydroxysuccinimide (NHS) esters do not require the use of copper, and unlike phosphoramidite-based linkers, NHS esters are water tolerant, eliminating the need for strict moisture-free conditions.25,26 Herein, we explored a reliable and high-yielding on-bead amide coupling methodology for preparing multi-ODN SMDHs with a variety of organic substrates. The other important component in constructing hybrid materials is the type of organic linkers used. Organic linkers Received: December 7, 2016 Published: March 10, 2017 10803
DOI: 10.1021/acs.joc.6b02942 J. Org. Chem. 2017, 82, 10803−10811
Article
The Journal of Organic Chemistry
possible solutions: (1) Omitting the capping steps during solidphase synthesis. However, such an approach would only be suitable for shorter ODNs with relatively high coupling efficiencies e.g. poly thymidines; (2) Washing with 2% morpholine in acetonitrile to remove acetyl capping groups prior to removal of the 5′-amino MMT protecting group. We successfully prepared NH2-modified 5′-AmMo-C6-T10, consisting of thymidines with high coupling efficiency, using the no capping strategy. Previous work showed no significant difference in the T10 synthesis between the conditions with and without the capping steps.42 We also prepared two mixedsequenced ODNs with 5′-NH2 moieties: 5′-AmMo-C6-CCAGATCGAAATAGTATTGC-3′ (MixSeq1), and its complement, 5′-AmMo-C6-GCAATACTATTTCGATCTGG-3′,43 referred to as MixSeq2, whose syntheses require capping steps. To prevent the above-mentioned side reaction, we removed acetyl capping groups by morpholine syringe wash (2% in acetonitrile) after attaching the MMT-protected 5′-amino modifier C6. The beads were then dried, and the column was replaced on the synthesizer and MMT was deprotected as normal.44 Both the no-capping and morpholine wash strategies proved to be successful. Optimization of Reaction Conditions Using Simple Model Compounds. With the successful preparation of CPGbound ODNs with 5′-amino functionality, we next explored an on-bead amide coupling approach to prepare SMDHs (Scheme 1b). In order to investigate optimal reaction conditions for such methodology, we used small rigid aromatic molecules as model substrates (1−4, Figure 1). Mono-, di-, or tritopic compounds
reported so far are mainly limited to small aromatic molecules, such as perylene-22 and porphyrin-based9,27 conjugates. Arylene−ethynylene macrocycles (AEMs) represent welldefined nanometer-sized building blocks that are intermediate in size between small molecules and larger, but less monodisperse, polymers. AEMs have attracted considerable attention as materials due to their self-assembling properties and possible formation of nanotubular structures,28−33 which have shown numerous applications in host−guest chemistry,34 in chemical sensing,35,36 as liquid crystalline materials,37,38 and as porous gas-adsorption materials.39,40 Given their well-defined architectures, self-assembly behavior through π−π stacking interactions, and the chemical robustness of acetylene-linked backbones, we envision that AEMs could serve as intriguing organic building blocks for ODN modification. However, AEM/DNA hybrids remain undeveloped due to the synthetic challenges associated with conjugating multiple ODNs to carbon-rich molecules.41 In this work, we elaborate the conjugation of shape-persistent AEMs with ODNs using the on-bead amide coupling approach. The resulting AEM/DNA hybrids were characterized by liquid chromatography mass spectrometry (LC−MS) and/or polyacrylamide gel electrophoresis (PAGE). We further demonstrate that the resulting AEM/DNA hybrids can be used to direct the assembly of gold nanoparticles into bulk aggregates.
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RESULTS AND DISCUSSION Preparation of Controlled Pore Glass-Bound ODNs with 5′-NH2 Groups. We prepared controlled pore glass (CPG)-bound ODNs through classic solid-phase phosphoramidite chemistry in 3′- to 5′- direction. The 5′-terminus was then modified with monomethoxytrityl (MMT) protected amino groups using 5′-amino modifier C6 (Glen Research). However, our initial attempts to prepare 5′-NH2 terminated CPG-bound 5′-AmMo-C6-CCAGATCGAAATAGTATTGC3′, referred to as MixSeq1, via final deprotection of MMT groups failed to provide the clean product. We found considerable competing side reactions occur between the amino groups introduced at the 5′-terminus and acetyl groups that are commonly introduced during solid-phase synthesis to cap failed sequences and ensure fidelity of the product ODNs (Scheme 1a). To preclude such side reaction, we explored two
Figure 1. Structures of small organic building blocks.
Scheme 1. Solid-Phase Synthesis of CPG-Bound ODNs with 5′-NH2 Groups (a) and Schematic Representation of OnBead Amide Coupling Reaction (b)
functionalized with carboxylic acid groups or activated carboxylic acid derivatives (e.g., acid chloride or NHS esters) were chosen to couple with ODNs. We initially examined the coupling of 5′-AmMo-C6-T10 with monotopic organic linker 1a. To generate an active ester in situ from the carboxylic acid, we added 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU).17 We were able to attach one ODN strand in nearly quantitative yield to obtain 1(T10)1 (hybrids are referred to using this notation, where the first number in bold denotes the organic substrate, the ODN sequence is in parentheses, and the subscript denotes the number of oligonucleotides attached) (Table 1, entry 1). As an alternative approach, the carboxylic acid can be converted to NHS ester 1b by reaction with N-hydroxysuccinimide and N(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC−HCl) in DMSO at room temperature. The isolated pure 1b was then reacted with 5′-AmMo-C6-T10 to form 10804
DOI: 10.1021/acs.joc.6b02942 J. Org. Chem. 2017, 82, 10803−10811
Article
The Journal of Organic Chemistry Table 1. Summary of Reaction Condition Optimization and Substrate Scope entrya
substrate
ODN
1 2 3 4 5 6 7 8 9 10 11 12 13
1a 1b 2a 2b 2b 2b 3a 3b 4 2b 2b 4 4
20 28 10 10 20 30 30 33 30 20 20 30 30
SMDH3b (%) T10 T10 T10 T20 T10 T10 T10 T10 T10 Mixseq1 Mixseq2 Mixseq1 Mixseq2
0 59 81
60 60
SMDH2b (%)
SMDH1b (%)
ODN conv. (%)
16 65 84 86 4 36 5 77 57 20 9
99 84 63 99 86 89 68 88 71
CPG-bound 5′-AmMo-C6-oligonucleotides (0.1 μmol), TEA or DIPEA (0.4−12 μL), and DMSO (1 mL) were used. For entries 1 and 3, 0.95 equiv of HATU was added. bYields were based on normalized integration of the diode array detector (DAD) trace of the LC at 257 nm. a
This on-bead amide-coupling approach is also applicable to coupling of mixed-sequenced ODNs and organic substrates. Conjugation of 2b with the MixSeq1 proceeds smoothly to provide the desired 2(MixSeq1) 2 in 77% yield with 2(MixSeq1)1 as minor product (11%). The coupling between 2b and MixSeq2 provided 2(MixSeq2)2 and 2(MixSeq2)1 in 57% and 9% yield, respectively. Tritopic linker 4 was also successfully coupled with MixSeq1 or MixSeq2 to provide the desired product containing three-strand ODNs, 4(MixSeq1)3 (60%) and 4(MixSeq2)3 (60%), in excellent yields. Our study shows the on-bead amide-coupling approach is generally applicable for preparing SMDHs consisting of multiple homo- or mixed-sequenced ODNs and various multitopic organic substrates. This solid-phase amide coupling approach can produce high yields of desired SMDHs that are readily analyzed by LC−MS. The reaction is easy to set up, requiring simple mixing of all components overnight, without need for moisture- or air-free conditions. Synthesis of AEM/DNA Hybrids. Given the efficient coupling strategy we had developed, we next explored its application toward SMDHs linked by more complex macrocyclic building blocks (5 and 6, Figure 2). AEMs 5 and 6 were prepared through a thermodynamically controlled cyclooligomerization approach using alkyne metathesis.46,47 5′AmMo-C6-T10 on CPG beads was coupled with either 5 or
1(T10)1 in good yield (84%). The amide-coupling procedure was simple without requiring precautions for moisture- and airfree conditions: The above ODN-bound CPG beads were placed in a glass vial with an excess of the organic substrate (10−30 equiv), dimethyl sulfoxide (DMSO), and a base, either triethylamine (TEA) or diisopropylethylamine (DIPEA). The mixture was shaken overnight at room temperature. The beads were then washed with dichloromethane, air-dried, and cleaved using standard ammonium hydroxide conditions (Scheme 1a). However, interestingly, when ditopic 2a containing two carboxylic acid groups was coupled with T10 in the presence of HATU, the targeted 2(T10)2 containing two ODN strands was only obtained as a minor product (16%), and the major product was monosubstituted 2(T10)1 (63%). On the contrary, when activated NHS ester 2b was used, 2(T10)2 was obtained as a major product (65%) with a trace amount of 2(T10)1. It appears that, when multiple coupling sites are present, NHS chemistry provides higher yield of the desired SMDH. Therefore, investigations using HATU were discontinued. We observed a higher yield (82%) of 2(T10)2 when the amount of organic linker was increased from 10 equiv to 20 or 30 equiv. When more than 30 equiv of 2b was used, the yield of the desired disubstituted 2(T10)2 began to decrease and the yield of 2(T10)1 was increased. Next, we tested more challenging tritopic organic linkers (3a, 3b, and 4) to attach three ODNs using the same on-bead amide-coupling approach. Coupling of compound 3b functionalized with NHS ester groups with 5′-AmMo-C6-T10 proceeds with high ODN conversion (>99%), providing 3(T10)3 in excellent yield (59%) with some 3(T10)2 (36%) and little 3(T10)1 (7%). In great contrast, compound 3a with acid chloride groups gave monoconjugated 3(T10)1 as the major product with only trace amounts of 3(T10)2 (4%). 3(T10)3 with three ODN strands was not observed. The dramatic decrease in efficiency is presumably due to the increased hydrolytic reactivity of the acid chloride as compared to the NHS ester. This result further suggests the superior reactivity of NHS esters in solid-phase amide coupling compared to other activated forms of carboxylic acids. Our study clearly shows that the choice of coupling reagents is critical for achieving high yields of organic−DNA hybrids, in particular, those containing multiple DNA arms.45 Extended tritopic linker 4 was also efficiently coupled with T10 to yield the desired 4(T10)3 in excellent yield (81%).
Figure 2. Structures of AEM 5 and 6. 10805
DOI: 10.1021/acs.joc.6b02942 J. Org. Chem. 2017, 82, 10803−10811
Article
The Journal of Organic Chemistry
Figure 3. (a) PAGE analysis of the crude reaction mixtures between macrocycle 5 and 5′-AmMo-C6-T10 (lane 2), macrocycle 6 and 5′-AmMo-C6T10 (lane 3), and ladder (lane 1). The marker and sample lanes are from the same gel. (b) ImageQuant analysis of intensity of gel bands from lane 2. (c) ImageQuant analysis of intensity of gel bands from lane 3.
6 using the procedures developed above. When tetratopic AEM 5 was used as the substrate, 5(T10)4 (∼49%) was the major product with a small amount of 5(T10)3 (∼14%). Since the LC trace of the crude mixture was unable to resolve the individual hybrids, the yields of various SMDHs were estimated on the basis of the integration of the LC peaks and by separating the products by PAGE followed by ImageQuant analysis (Figure 3). While macrocycle 5 gave the desired product in good yield, the total conversion was moderate (∼70%) likely due to its poor solubility in the reaction medium. By contrast, macrocycle 6, which has better solubility in DMSO, was more efficient in reacting with the CPG-bound ODNs leaving only 7% unreacted ODNs (Figure 4). The increased reactivity of 6 is presumably also due to the more reactive aliphatic esters compared to the aromatic esters in 5. We obtained hexasubstituted product, 6(T10)6, in 32% yield. We also observed five and four ODNcoupled SMDHs, 6(T10)5 (28%) and 6(T10)4 (21%), along with a small amount of 6(T10)3. Mono- or di-ODN-substituted products, 6(T10)2 and 6(T10)1, were not detected. We could isolate a small amount of pure products 5(T10)4 and 6(T10)6 through HPLC purification, which represent rare examples of highly symmetrical SMDHs with multiple ODNs and accurate mass characterization. It should be noted that solution-phase amide coupling of macrocycle 5 or 6 with ODNs failed to give any multicoupling products, thus indicating on-bead amide coupling approach is advantageous, presumably due to higher local concentration of ODNs (anchored on beads) around macrocycles. AEM/DNA/AuNP Materials. With the desired AEM/DNA hybrids in hand, we next explored whether they were capable of mediating the assembly of gold nanoparticles (AuNPs). As a simple proof of concept, we mixed 5(T10)4 with 5 nm AuNPs bound to 5′-modified A10 ODN, which was prepared from A10 (100 equiv) and 5 nm AuNPs coated with bis(psulfonatophenyl)phenylphosphine (BSPP) as previously reported.48 A mixture of the 5(T10)4 and A10-functionalized 5 nm AuNPs (1:1 molar ratio of T10:A10) was cooled at 4 °C in TBE (45 mM Tris, 45 mM boric acid, and 1 mM EDTA) and 5 mM MgCl2. We obtained stable aggregates of AuNPs with reasonably regular interparticle spacings (Figure 5a), presumably linked by complementary paring of A10 and T10. TEM
Figure 4. (a) LC trace of crude reaction mixture between macrocycle 6 and on-bead 5′-AmMo-C6-T10; (b) LC trace of the purified 6(T10)6; (c) Mass spectra of purified 6(T10)6. The m/z peaks at 2876.5937 and 2516.9779 respectively correspond to the −7 and −8 charged peak 6(T10)6.
images of the resulting mixture showed AuNPs with median interparticle spacings of 6.8 nm (Figure 5c,d). To prove the critical importance of 5(T10)4, we conducted a control experiment in the absence of 5(T10)4 under otherwise identical conditions. We now observed primarily single particles rather than aggregates (Figure 5b), demonstrating that the AEM/ DNA hybrids can serve as linking agents to mediate nanoparticle assemblies. Since in this preliminary study we used AuNPs loaded with multiple ODNs, a fairly polydisperse mixture of aggregates was observed ranging from aggregates of 10806
DOI: 10.1021/acs.joc.6b02942 J. Org. Chem. 2017, 82, 10803−10811
Article
The Journal of Organic Chemistry
filtered, and the filtrate was concentrated to give the crude product, which was purified by flash column chromatography (25% CH2Cl2/ hexane) to afford 7 as a colorless oil (9.28 g, 93%): 1H NMR (400 MHz, CDCl3) δ 7.82−7.78 (m, 2H), 7.75−7.72 (m, 2H), 4.24 (d, J = 5.7 Hz, 2H), 1.73−1.55 (m, J = 6.1 Hz, 1H), 1.44 (qd, J = 7.4, 6.2 Hz, 4H), 0.94 (t, J = 7.4 Hz, 6H); 13C NMR (75 MHz, CDCl3) δ 166.4, 137.8, 131.1, 130.1, 100.7, 67.4, 40.6, 23.6, 11.3; HRMS-ESI (m/z) [M + Li]+ calcd for C13H17IO2 339.0434, found 339.0427.
Compound 8. To a Schlenk tube were added 7 (552 mg, 3.30 mmol), carbazole (996 mg, 3.00 mmol), CuI (34.3 mg, 0.18 mmol), K3PO4 (764 mg, 3.60 mmol), and LiCl (76.3 mg, 1.80 mmol). The flask was evacuated and refilled with nitrogen, and the evacuation/refill process was repeated three times. DMF (40 mL) was added into the tube, and the mixture was stirred at 180 °C for 14 h. The solvent was removed, and the crude product was washed with hexanes (250 mL). The solvent was removed and the crude product was purified by flash column chromatography (30% CH2Cl2/hexane) to afford pure 8 as a colorless oil (964 mg, 87%): 1H NMR (400 MHz, CDCl3) δ 8.33− 8.27 (m, 2H), 8.16 (dt, J = 7.7, 1.0 Hz, 2H), 7.74−7.66 (m, 2H), 7.49 (dt, J = 8.3, 0.9 Hz, 2H), 7.44 (ddd, J = 8.2, 7.0, 1.2 Hz, 2H), 7.33 (ddd, J = 8.0, 7.0, 1.2 Hz, 2H), 4.35 (d, J = 5.7 Hz, 2H), 1.74 (hept, J = 6.2 Hz, 1H), 1.57−1.46 (m, 4H), 1.01 (t, J = 7.5 Hz, 6H); 13C NMR (101 MHz, CDCl3) δ 166.2, 142.0, 140.4, 131.4, 129.2, 126.5, 126.3, 123.9, 120.6, 120.6, 109.9, 67.3, 40.7, 23.7, 11.3; HRMS-ESI (m/z) [M + Na]+ calcd for C25H25NO2, 394.1783, found 394.1794. Compound 1a. Compound 8 (116 mg, 0.31 mmol), KOH (150 mg, 2.7 mmol), and THF (3.0 mL) were added to a Schlenk flask, and the mixture was heated at 75 °C for 18 h. The mixture was cooled to room temperature, and HCl (2 N) was added until pH
