Stability of Oligonucleotide–Small Molecule Conjugates to DNA

Feb 24, 2017 - Oligonucleotide conjugates of small molecules are widely used in chemical biology and have found increasing interest in the context of ...
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Stability of Oligonucleotide−Small Molecule Conjugates to DNADeprotection Conditions Lik Hang Yuen and Raphael M. Franzini* Department of Medicinal Chemistry, College of Pharmacy, University of Utah, 30 S 2000 E, Salt Lake City, Utah 84112, United States S Supporting Information *

ABSTRACT: Oligonucleotide conjugates of small molecules are widely used in chemical biology and have found increasing interest in the context of DNA-encoded chemical libraries for drug discovery. Attachment of molecules to DNA bound to the solid support is an attractive small-molecule conjugation method that permits the use of organic solvents, rigorous reaction conditions, and simple workup. However, the conjugated structures must be resistant to the harsh DNA deprotection/cleavage conditions and the stabilities of building blocks under various deprotection conditions are mostly unexplored. In the present study, we analyzed the stability of 131 structurally diverse fragments that contain amides and amide-like elements during DNA deprotection protocols. Structural features susceptible to decomposition in DNA deprotection conditions were identified and a protocol that enabled the synthesis of DNA conjugates with labile fragments on solid support was identified.

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quence25−28has spurred a renaissance of DNA conjugation chemistry. DECLs bear great promise for applications in chemical biology26,28 and drug discovery.25,27,29 The synthesis of DECLs requires high-yielding bioconjugation methods, and several on-DNA reactions have been described for this purpose16 including reductive amination,19,30 amide bond formation,18,19 Suzuki-Miyaura coupling,30,31 and nucleophilic addition.6,32 Despite the tremendous progress in the preparation of DECLs, coupling structurally diverse fragments to DNA with near-quantitative yields and minimal side products remains challenging. Most reported DECLs were synthesized in solution but on-CPG synthesis has also been described for library preparation.7,33−36 This strategy generally provides high coupling yields and consumes minimal quantities of building blocks but requires that all fragments in the library are inert to the harsh DNA deprotection/cleavage conditions (conc. aq. NH3, 65 °C, 8 h; 1:1 conc. aq. NH3: aq. MeNH2 (AMA), RT, 2 h). Information on compound stability that can guide building block selection for on-CPG preparation of DECLs is lacking. Here, we describe a systematic stability evaluation of building blocks during DNA deprotection. This study focuses on building blocks that contain amide groups which are ubiquitous

INTRODUCTION Conjugates of small molecules and short peptides attached to oligonucleotides find widespread use in chemical biology, for example, as sensing probes,1−3 RNA-folding study tools,4 for cellular DNA delivery,5 and in drug discovery.6,7 Several DNAmodifiers have been developed including amines,8−11 thiols,8,12 and aldehydes,13−15 some of which are commercially available. Typically, DNA conjugation of molecules to these modifiers is performed in solution using established protocols.16−18 However, low solubility of small molecules in aqueous buffer can limit the yield for solution-phase conjugation. Furthermore, side-reactions of activated carboxylic acids with nucleobases can accompany amide bond formation on DNA.19,20 Relatively mild coupling reagents (e.g., DMT-MM, EDC) are normally used,16,18,19 but the reaction yields can be unsatisfactory.18,19 Alternatively, conjugation can be performed with nucleobaseprotected DNA on the controlled pore glass (CPG) used for automated oligonucleotide synthesis.1,2,21−24 These protocols allow for the use of polar aprotic solvents (e.g., DMF, NMP) that can dissolve most small molecules, circumvent side reactions with the exocyclic amines of the nucleobases, tolerate stringent reaction conditions, and facilitate product purification by simple filtration of reagents. The growing interest in DNA-encoded chemical libraries (DECLs)combinatorial small molecule libraries in which each compound is encoded by an attached DNA se© XXXX American Chemical Society

Received: January 3, 2017 Revised: February 1, 2017

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DOI: 10.1021/acs.bioconjchem.7b00005 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 1. Schematic representation of the study design. (A) Synthesis of DNA conjugates using amide coupling condition on solid support (CPG) followed by cleavage and deprotection with AMA. (B) Evaluation of the stability of building blocks with amide functionalities to DNA deprotection/ cleavage conditions. Two types of amide structures were investigated in this study: linear amide linkers and heterocycles.

HPLC and mass-spectrometry (Q-TOF). This protocol only addressed building block stability but not incomplete coupling or impurities in the carboxylic acid precursors. Stability of Building Blocks with Amide Linkers. Nearly all tested amides and amide-like structures (Supporting Information, Tables S1 and S2) in this analysis were stable under AMA deprotection conditions. The correct mass was found for 29 out of the 31 tested structures and for 23 of them the integration of the HPLC product peaks was >85% (Figure 2). The analyzed fragments included three ureas (L5, L6, L31), one carbamate (L7), and one sulfonamide (L30) and the

in DECLs and potentially labile. The stability of a total of 131 DNA-attached amide linkers and heterocycles under DNAcleavage/deprotection conditions was analyzed by HPLC chromatography and mass spectrometry. Most building blocks were found to be inert to AMA deprotection conditions. Structures associated with lability to nucleophiles and deprotection conditions that enabled the on-CPG conjugation of sensitive compounds were identified.

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RESULTS AND DISCUSSION Amide groups are commonplace in DNA-conjugates and in DECLs.25 Several reported DECLs included an on-CPG synthesis step for their preparation,7,33,34 which required that the incorporated building blocks were inert to hydrolytic/ aminolytic DNA cleavage/deprotection conditions. To test the stability of diverse amides and related groups, we selected a set of 31 fragments with internal linkers (Tables S1 and S2 in Supporting Information) and assessed the stability during DNA deprotection/cleavage (AMA, 2 h, RT; Figure 1). The use of building blocks with internal amides avoided uncertainties that could have arisen from incomplete conjugations if carboxylic acids were directly linked to the 5′-terminal amine. To study the influence of inductive and resonance electronic effects, we selected aliphatic and aromatic amides with varied electron density at the amide carbon and nitrogen. To determine the stability boundary, the test set included several amides composed of electron-poor carboxylic acids and amines, which were expected to be susceptible to alkaline cleavage. A nucleobase-protecting-group-free hexathymidine sequence (5′-TTT TTT-3′) on CPG support was chosen for this study, which allowed us to test different deprotection/cleavage protocols without concerning about incomplete nucleobase deprotection. The 5′-terminus of the DNA was modified with a C6 amino modifier and the MMt amine protecting group was removed with 3% trichloroacetic acid in DCM. Carboxylic acid fragments were conjugated to the 5′-amine using a standard HATU/DIPEA peptide coupling protocol (∼25 nmol of DNA, 50 mM carboxylic acids, 50 mM HATU, and 150 mM DIPEA for 4 h at RT in NMP).7 To examine the stability of the building blocks, the DNA conjugates on CPG were treated with AMA at RT for 15 min in the reaction plate, filtered, and the solution was left in a sealed container for 2 h, mimicking the deprotection of synthetic DNA.37 The resulting DNA products were analyzed by RP-

Figure 2. Summary of linker stability. (A) Representation of fragment stability as determined by HPLC analysis and mass-spectrometry. (B) Stability of selected N-aryl amides. The black and red numbers shown below structures represent the yield from the integrated HPLC peak area of the desired and cleaved product, respectively. The red lines indicate the cleavage sites based on mass-spectrometry results. B

DOI: 10.1021/acs.bioconjchem.7b00005 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry product peak of the conjugates of these building blocks integrated to >90% (Supporting Information, Tables S1 and S2). Because unreacted amino hexa-T does not originate from the cleavage of the amide of interest, it was excluded from DNA integration to avoid artifacts from reaction batch variability. We further excluded an elution peak at t = 5.5 min that originated from the AMA-treated reaction plates and whose absorbance spectrum was uncharacteristic of DNA. All aliphatic amides showed high stability to AMA condition, and different substituents had minimal effects on amide stability. Similarly, all tested amides derived from aromatic carboxylic acids including those with electron-withdrawing substituents such as 4-nitrobenzoic acid (L15, L16, L17), nicotinic acid (L14), and isonicotinic acid (L25) were inert to AMA deprotection/cleavage (Figure 2B). In contrast, the stability profile of N-aryl acetamides depended on the electronic properties of the N-arylamines. Whereas some Narylacetamides were recovered in good yield (52−100%), acetamides of electron-deficient arylamines were more prone to nucleophilic cleavage. For example, AMA treatment of 4acetylamino-3-nitrobenzamide (L28) near-quantitatively provided the acetyl-cleaved conjugate. This result is in agreement with the complete removal of acetamide protecting groups from cytosine during DNA deprotection. One special case was the quantitative aminolysis of the exocyclic amide of 3,4-dihydro2(1H)-quinoxalinone (L29; Figure 2), for which we observed only the cleaved methylamine adduct. Stability of Heterocycles During DNA Deprotection. We selected 100 representative heterocycles with amides and related structural elements for stability tests. We categorized each structure as aromatic (1−63), nonaromatic (64−80) or fused (81−100) based on the position of the amide (Supporting Information, Tables S3 and S4). Aromatic structures included heterocycles in which the group to be tested was part of a delocalized π-system. Fused structures were defined as heterocycles in which the amide or related group was part of a nonaromatic ring but conjugated to an aromatic system. Figure 3 summarizes the outcome of the AMA-stability tests of the heterocycles (Supporting Information, Tables S3 and S4). A putative product peak with the correct mass was found for 83 of the tested 100 compounds and for 52 of the analyzed structures the product peaks integrated to >85% in the HPLC traces. For 13 structures in this series (Figure 3B), none of the collected fractions showed the desired product mass, yet most of the corresponding HPLC chromatograms showed one major elution peak. In the case of four fused building blocks (82−84, 91), the HPLC traces indicated complex mixtures of decomposed products and no fractions were suitable for mass analysis. Most aromatic structures (38 out of 63; 60%) were stable, providing clean conjugates with good product yields (>85%). Stable aromatic heterocycles included, for example, quinolones (4,5), quinazolinones (7−11), uracil derivatives (17−22), and 2,4-quinazolinediones (26−29) and benzimidazolones (49−52) (Figure 3B). In contrast, benzoxazolinone (59,60), isatins (61,62), phthalimide (63), and pyrazolinone derivatives (56,58) were unstable in the deprotection condition and no desired products were found in these cases (Figure 3C). Major HPLC elution peaks of the benzoxazolinones had masses of methylamine adducts, which suggests nucleophilic opening of the fivemembered ring. Isatin derivatives provided complex mixtures without identifiable major products and AMA treatment of

Figure 3. Analysis of stability of heterocycles with amide-like elements during AMA deprotection. (A) Summary of fragment stability as determined by HPLC analysis and mass-spectrometry. (B) Representative building blocks that showed high stability during AMA deprotection. (C) Heterocycles for which some of the fragments lacked stability in AMA and the correct mass of the DNA conjugate could not be found.

phthalimide resulted in clean release of the primary amine as previously reported.10 Most of the nonaromatic compounds (8 out of 17; 47%) were stable in AMA condition. For example, trimethyleneurea (74−76), imidazolidone (77,78), and pyrrolidinone (79,80) all showed high stability (>85% in HPLC with correct mass). Hydantoins displayed a diverse range of stabilities from mostly stable (65, 70) and moderately stable (64, 68, 69, 71) to unstable (66, 67) (Table 1). Based on mass spectrometry data, the dominant side products were methylamine ring-opening adducts. Similarly, two dihydrouracils were tested and one was inert (73), whereas for the second one (72), the correct mass could not be identified (+ MeNH2). No structure−stability trends were apparent for these heterocycles. Fused rings showed the lowest AMA stability among the three categories. Only 6 out of 20 structures were stable in AMA, whereas for 8 of them, the expected product peak was absent. All six evaluated indolinone structures (Table 2, 82−87) provided complex product mixtures in AMA, especially those with electron-withdrawing substituents. In contrast, an electron-poor isoindolinone (81) was stable with clean conversion under the same deprotection condition, reflecting the difference in stability of N-alkyl and N-aryl amides. Ring systems with 6-membered fused rings showed mixed stability profiles (Table 3). For instance, heterocycles with oxygen (92− C

DOI: 10.1021/acs.bioconjchem.7b00005 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry Table 1. Stability of Hydantoins to DNA-Deprotection/ Cleavage Conditionsa

Table 3. Stability of 6-Membered Fused Rings to DNADeprotection/Cleavage Conditionsa

a Green wavy line indicates the attachment site to DNA. †Correct mass not found.

a Green wavy line indicates the attachment site to DNA. †Correct mass not found. *No major product found.

Table 2. Stability of Indolinones to DNA-Deprotection/ Cleavage Conditionsa

provide the desired conjugates for heterocycles that decomposed in AMA. We selected five structures (Figure 4 and Supporting Information Table S5) for which the stability in AMA deprotection conditions varied from inert to unstable and compared their stability in four reported oligonucleotide deprotection protocols: AMA, 2h, RT (Protocol A);37 gaseous AMA, 85 °C, 30 min (Protocol B);38 ethylenediamine:toluene (1:1), 2 h, RT (Protocol C);39 conc. aq. NH3, 2 h, RT (Protocol D).40 Protocol D corresponds to “UltraMild” deprotection conditions that require special nucleobase protecting groups.40 Figure 4 shows the overlaid HPLC chromatograms for each compound exposed to the different deprotection conditions. The two structures that were stable in AMA (5, 54) were also inert to the other deprotection conditions, and conclusively all four deprotection strategies are suitable for on-CPG conjugation protocols. 61 and 84, for which we failed to recover the correct DNA-conjugated product under AMA deprotection, were also unstable in ethylenediamine/toluene and gas phase AMA, whereas 56 was found in moderate yield in gaseous AMA with 40% integration. Protocol D provided the desired product for all five compounds (50−80%) including those that were unstable in AMA (Protocol A). Based on this result, Protocol D will be the method of choice for compounds that are chemically labile in standard oligonucleotide deprotection/cleavage conditions.

Green wavy line indicates the attachment site to DNA. *No major product found. †Correct mass not found.

a

93), sulfur (94), and carbon (95−97) conjugated to the aromatic ring showed good stability in the AMA condition, but the stability of heterocycles with nitrogen at the same position depended on the substituents. Of the three seven-membered fused rings one was completely stable (100) and two of them (98,99) provided a mixture of the desired product and methylamine adducts. Comparison of Fragment Stability in Different Deprotection Conditions. We next examined different deprotection strategies to assess their effect on DNAconjugated amide structures. We were especially interested to see if alternative deprotection/cleavage conditions would

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CONCLUSIONS A diverse set of DNA-conjugated molecules containing amides and amide-like elements were tested for stability in AMA deprotection condition. Based on the experimental data, it can be concluded that most structures are stable during AMA deprotection/cleavage and the corresponding conjugates can be D

DOI: 10.1021/acs.bioconjchem.7b00005 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 4. Comparative stability of selected DNA−small molecule conjugates in four DNA-deprotection/cleavage conditions. The HPLC chromatograms were shifted in time and absorption for clarity. * indicate peaks that do not contain DNA absorption and the peaks were truncated for clarity (see main text). Arrows indicate peaks that contained the correct products based on mass spectrometry.

prepared by on-CPG synthesis. Structural patterns linked to stability could be identified; for example, acetamides of electron-withdrawing aryl-amines tend to be unstable. Additionally, aromatic amides are mostly stable and so are most nonaromatic structures. Heterocycles with limited AMA stability included benzoxazolinones, isatins, phthalimide, pyrazolinones, and certain hydantoins. Additionally, some fused heterocycles with nonaromatic amides were susceptible to alkaline cleavage, especially indolinones which showed low stability under these conditions. The use of UltraMild deprotection allowed isolating conjugates even for these compounds with good recovery yields, although specific nucleosides with more labile protecting groups are required for oligonucleotide synthesis.

DNA conjugation has been a common practice since the development of automated DNA synthesis and numerous examples of small peptides, 21,23 fluorophores,1,41 small molecules,6,7 and bio-15 or synthetic macromolecules42 have been conjugated to DNA. In addition, there are many unnatural nucleosides that are commercially available or custom-made in research laboratories.43 All these structures require stability during oligonucleotide deprotection if they were conjugated on solid supports. Despite the limited analyzed structural space, this study should provide a general guidance for assessing DNA conjugation of small molecules. Of course, ultimately each structure needs to be individually tested when DNAconjugation is needed. E

DOI: 10.1021/acs.bioconjchem.7b00005 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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EXPERIMENTAL SECTION Materials and Instrumentation. Oligonucleotides were synthesized by the University of Utah DNA/peptide synthesis core facility using a C6-amino-5′-modifier (Glen Research). The carboxylic acid building blocks were purchased from Sigma-Aldrich, Enamine, CombiBlock, and ChemBridge. DIPEA was purchased from Sigma-Aldrich and HATU was purchased from ChemPep. Other reagents and solvents were obtained from Sigma-Aldrich, Alfa Aesar, and Fisher Scientific. All chemicals were used without further purification. DNA conjugation was performed in PALL 1 mL AcroPrep 96-well filter plates (PN 5056) using a Pall vacuum manifold. Gas phase deprotection was performed using a Biolytic heated pressure chamber with temperature controller. A Dionex UltiMate 3000 LC system equipped with a diode array detector and an automated fraction collector with a Phenomenex LUNA 5u C18 column was used for RP-HPLC analysis. Peak integration was done using Chromeleon chromatography data system. QTOF analysis was performed by the mass spectrometry facility in the University of Utah Department of Chemistry. General Protocol for Compound Coupling on CPGSupport. DNAs with the sequence 5′-TTT TTT-3′ and a MMT-protected C6 amino modification attached to the CPG solid supports were used for conjugation. The CPG beads were suspended in DCM (1 mL) and distributed into individual wells of a 96-well filtration plate (∼25 nmol DNA/well). The MMT-protecting group was removed with 3% trichloroacetic acid in DCM (200 μL × 6) and washed with NMP (300 μL × 2). Coupling solutions (300 μL) containing 50 mM carboxylic acid, 50 mM of HATU, and 150 mM DIPEA in NMP were added to each well and the plate was agitated for 4 h at RT. The reaction mixture was removed by vacuum filtration and the CPG beads were washed with NMP (300 μL × 4) and MeCN (300 μL × 4). Analysis of DNA−Small Molecule Conjugates. The DNA was cleaved from the CPG support using four different deprotection conditions (Protocols A−D outlined below). The collected DNA was dissolved in 100 μL of water and purified by HPLC (Solvent A: 0.1 M TEAA buffer pH 7, solvent B MeCN; 15% to 25% B in 6 min, then to 60% B in 4 min). DNAcontaining peaks were collected, integrated, and submitted for Q-TOF analysis. Deprotection Conditions. Cleavage and Deprotection with AMA Solution (Protocol A).37 DNA modification was performed in 96-well plates using the general procedure described above. AMA (1:1 aqueous ammonia (∼30%):methylamine solution (∼40% methylamine in water)) was added to the DNA conjugates on CPG support and incubated for 10 min (150 μL × 2). The solution was collected using vacuum filtration. 100 μL of AMA solution was added to each of the filtrates and the resulting mixtures were sealed and incubated at RT for 2 h. Cleavage and Deprotection with Gaseous AMA (Protocol B).38 DNA modification was performed in synthesis columns (BioAutomation − ML 6030C) using the general procedure described above. The conjugated CPG beads were incubated in a heated pressure chamber with 50 mL AMA solution at 85 °C for 30 min. The DNA was eluted from the CPG beads with water (500 μL × 2). The DNA solutions were evaporated to dryness in a centrifugal evaporator. Cleavage and Deprotection with Ethylenediamine in Toluene (Protocol C).39 DNA modification was performed in

synthesis columns using the general procedure described above. DNA cleavage was executed according to a modified literature protocol.39 Briefly, the conjugated CPG beads were treated for 5 min with 10% diethylamine in MeCN (500 μL × 3) followed by rinsing with toluene (500 μL × 3). 1:1 toluene:ethylenediamine (500 μL) was added and the mixture was agitated at RT for 2 h. The reaction mixture was removed by vacuum filtration and the beads were washed with toluene (500 μL × 3) and briefly dried under vacuum. The DNA was eluted from the CPG beads with water (500 μL × 2). The solution was evaporated to dryness in a centrifugal evaporator. Cleavage and Deprotection with Aqueous Ammonia (Protocol D).40 DNA modification was performed in synthesis columns using the general procedure described above. The conjugated CPG beads were incubated with aqueous ammonia (∼30%; 500 μL) at RT for 2 h. The ammonia solution was collected and the beads were washed with 500 μL of water. The ammonia solution was evaporated to dryness in a centrifugal evaporator.

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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.7b00005. HPLC and mass spectrometry analysis of DNA-small molecule conjugates (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: (+1) 801-585-9051. ORCID

Raphael M. Franzini: 0000-0001-6772-5119 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge financial support from the University of Utah, the Huntsman Cancer Institute, and the Utah Science and Technology Research (USTAR) initiative. We thank the University of Utah Oligo and Peptide facility for DNA synthesis and assistance with deprotection condition experiments and the Department of Chemistry mass spectrometry facility for LC-MS experiments.

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ABBREVIATIONS AMA, 1:1 conc. aq. ammonia:aq. methylamine; CPG, controlled pore glass; DCM, dichloromethane; DECL, DNAencoded chemical libraries; DIPEA, N,N-diisopropylethylamine; DMF, dimethylformamide; DMT-MM, 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride; EDC, N-(3-(Dimethylamino)propyl)-N′-ethylcarbodiimide; HATU, 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; MeCN, acetonitrile; MMT, monomethoxytrityl; NMP, 1-methyl-2-pyrrolidinone

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DOI: 10.1021/acs.bioconjchem.7b00005 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.bioconjchem.7b00005 Bioconjugate Chem. XXXX, XXX, XXX−XXX