Efficient Synthetic Approach to Linear DasatinibâDNA Conjugates by

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Efficient Synthetic Approach to Linear Dasatinib-DNA Conjugates by Click Chemistry Nan-Sheng Li, Nathan Gossai, Jordan A. Naumann, Peter M Gordon, and Joseph A. Piccirilli Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00557 • Publication Date (Web): 30 Sep 2016 Downloaded from http://pubs.acs.org on October 2, 2016

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Bioconjugate Chemistry

Efficient Synthetic Approach to Linear Dasatinib-DNA Conjugates by Click Chemistry

Nan-Sheng Li1,*, Nathan P. Gossai3,4, Jordan A. Naumann3,4, Peter M. Gordon3,4,*, Joseph A. Piccirilli1,2, *

1

Department of Biochemistry & Molecular Biology, 2Department of Chemistry, University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA.

3

Department of Pediatrics, Division of Pediatric Hematology and Oncology, 4Masonic Cancer

Center, University of Minnesota, MMC 366, 420 Delaware Street SE, Minneapolis, MN 55455, USA.

Corresponding authors: Nan-Sheng Li, [email protected]; Peter M. Gordon, [email protected]; or Joseph A. Piccirilli, [email protected]

Abstract: Two synthetic approaches to linear dasatinib-DNA conjugates via click chemistry are described. One approach involves the reaction of excess azido dasatinib derivative with 5′-(5hexynyl) tagged DNAs and the other involves the reaction of excess alkynyl linked dasatinib with 5′-azido tagged DNA. The second approach using alkynyl derived dasatinib and 5′-azido tagged DNA yielded the corresponding dasatinib-DNA conjugates in higher yield (47% versus 10-33% for the first approach). Studies have shown these linear dasatinib-DNA conjugates derived gold nanoparticles exhibit efficacy against leukemia cancer cells with reduced toxicity toward normal cells compared to free dasatinib.

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Introduction Gold nanoparticles (AuNPs) have been widely used for drug delivery1,2 because of their ability to deliver anticancer drugs specifically to the tumor sites with minimal toxicity to the normal cells. Anticancer drug-DNA adducts linked via a methylene group between the amino group of the drug and 2′-amino group of deoxyguanosine in DNA3 have been reported to be nuclease-resistant and programmable for anticancer drug delivery.4 Dasatinib-DNA conjugated AuNPs have been evaluated as a novel treatment for leukemia (Figure 1).5 These AuNPs consist of two active drugs to improve the treatment: an anti-sense oligonucleotide covalently bound to the nanoparticle designed to target and silence a cancer gene and a small molecule drug linked to the oligonucleotide that base-pairs to the anti-sense oligonucleotide. The synthesis of oligonucleotide bonded AuNPs was achieved according to a previously reported procedure.6,7 Here, we report a new general and efficient synthetic approach to linear drug-oligonucleotide conjugates, dasatinib-DNA conjugates in this case, via click chemistry.

Figure 1. Model of Drug-DNA conjugated gold nanoparticles (AuNPs). Yellow: gold nanoparticles. Black: DNA strand covalently linked to gold particle. Blue Star-Red: dasatinib conjugated DNA strand annealed to covalently linked DNA strand.

Results and Conclusions


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Dasatinib (1) is a FDA-approved anti-cancer drug sold under the trade name Sprycel. It is a second-generation drug displaying increased potency as an inhibitor of BCR-ABL tyrosine kinase and Ser/Thr kinase that has been used for chronic myeloid leukemia therapy.8,9 It has been selectively modified at its free hydroxyl position without perturbing its binding affinity to Bcr/Abl kinase.8 Our strategy for the synthesis of dasatinib-DNA conjugates focused on click chemistry due to its mild reaction condition and high conjugation yields.10-12 First, attempt to introduce the alkynyl group to the free hydroxyl position of dasatinib to form a dasatinib propargyl ether via the direct reaction of dasatinib with propargyl bromide in the presence of NaH in DMF13 was not successful. Instead, we observed a propargylation of the amide group, which greatly diminished the efficacy of the reaction. To obviate this complication, we instead prepared the dasatinib azido derivative 2 and subsequent dasatinib amino derivative 3 from commercially available dasatinib according to a literature procedure (Scheme 1).14 We prepared the azido derivative 2 in 82% yield and the amino compound 3 in 80% yield after reduction with triphenylphosphane.

Scheme 1



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The 5-hexynyl tagged DNAs could be prepared by a nucleic acid synthesizer using commercially available 5-hexynyl phosphoramidite (for 4) or purchased from IDT directly (for 5 and 6). To facilitate the click reaction, we used excess dasatinib derivative (2) and found no 5hexynyl tagged DNAs left over after the reaction was kept at room temperature for one hour. The click reaction of azido derivative 2 with 5-hexynyl tagged DNAs (4, 5, or 6) yielded the corresponding linear dasatinib-DNA conjugates (I-III) after 20% dPAGE purification in 10-33% isolated yields (Scheme 2). The isolated yield of shortest DNA conjugate (III) was lower than that of the longer DNA conjugates (I and II) probably because its lower water solubility resulted in product loss during the extraction and purification.

Scheme 2


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Alternatively, the amino dasatinib derivative 3 was easily converted into the amidecontaining compound 7 by reaction with 4-pentynoic acid in the presence of DCC.15 The amino tagged DNA 8 was then prepared by coupling with the 5′-amino-modifier C3-TFA phosphoramidite. After full deprotection, the 5′-amino modified DNA 8 was subsequently converted into the 5′-azido DNA 9 by reaction with azidobutyrate NHS ester. The click reaction of excess dasatinib alkyne derivative 7 with 5′-azido DNA 9 yielded a dasatinib-DNA conjugate (IV) in 47% isolated yield after 20% dPAGE purification (Scheme 3). Reaction of 5′-azido tagged DNA with alkynyl derived dasatinib yielded the dasatinib-DNA conjugates in higher yield.

Scheme 3



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N

N

O

N

N

OH /DCC

H N

DMF/CH 2Cl 2 (1:1)

3

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Cl

rt, overnight

S

N

N H

NH

O O 7

OCH 2CH 2CN P N O N(i-Pr 2) T H O H 2N complete DNA synthesis, O cleavage and deprotection CA AGG AGC TGG AA TFA

HO

O-(TCA AGG AGC TGG AA) P O OH

O

8 O O

N3 O

N

O

H N

N3

O-(TCA AGG AGC TGG AA) P O O-

O

O

rt, 2 h

9

N

Cl

S

N N

N H N

7 (50 equiv in DMSO), aq. CuSO 4:THPTA (1:1) (25 equiv) 47% aq. sodium ascorbate (40 equiv.), degass solution with argon, rt, 1 h

N H

N NH

O O

N N N

H N O

O

O-(TCA AGG AGC TGG AA) P O O - Na +

IV

These dasatinib-DNA conjugates (I-IV) were further used to generate dasatinib-DNAAuNPs.5 To compare the efficacy of dasatinib-DNA conjugates (I and IV) as well as their derived AuNPs toward leukemia cells, we measured their inhibition activities of Src kinase in vitro (Figure 2, A) or in leukemia cells (Figure 2, B). We found both dasatinib-DNA conjugates (I and IV) and their derived AuNPs show similar efficacy without significant difference (Figure 2).


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Figure 2. A. Dasatinib-DNA conjugates I and IV inhibit Src kinase in vitro. Src activity was assessed using an in vitro Src Kinase Assay (Promega) over a range of dasatinib and dasatinibDNA (I and IV) concentrations. Reactions were performed in triplicate and IC50 values were calculated using GraphPad Prism 6.0. Dasatinib: IC50=7.1 nM and R2=0.99; Dasatinib-DNA (I): IC50=8.5 nM and R2=0.98; Dasatinib-DNA (IV): IC50=7.9 nM and R2=0.99. B. DasatinibDNA conjugates (I and IV) derived AuNPs inhibit Src kinase in leukemia cells. K562 leukemia cells were treated with either dasatinib or AuNPs functionalized with dasatinib-DNA (I) or dasatinib-DNA (IV). After 24 hours the cells were fixed/permeabilized, stained for



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intracellular phospho-Src, and assessed by flow cytometry. Median fluorescence intensity (MFI) for each condition relative to the untreated control was calculated. P < 0.0005 when comparing untreated and either dasatinib or dasatinib-DNA (I and IV) Au-NP treated cells. No significant difference when comparing dasatinib-DNA (I and IV) conjugates.

Conclusions In summary, we have developed a general and efficient method for the production of linear dasatinib-DNA conjugates in good isolated yields via click chemistry, which upon further conjugation with AuNPs generate therapeutically important bifunctional particles. Drug-DNA conjugates (I) and (IV) as well as the derived AuNPs show equal efficacy toward leukemia cells. These two approaches may be extended to the synthesis of other drug-nucleic acid conjugates for further biological studies.

Experimental

Synthesis of conjugate (I) To the solution of dasatinib (488 mg, 1.00 mmol) in a mixed solvent of dry DMF/THF (15 mL, 2:1, v/v) at 0 °C, triethylamine (0.28 mL, 2.0 mmol) was added and the mixture was stirred at 0 °C for 10 min. Methanesulfonyl chloride (155 µL, 2.0 mmol) was then added slowly and the solution was allowed to warm up to room temperature and stirred overnight. Sodium azide (152 mg, 2.33 mmol) was added and the mixture was heated up to 50 °C and stirred for 47 h. After cooling to room temperature, about 5 mL reaction mixture (~1/3 portion, 0.33 mmol) was taken out, quenched with water, extracted with dichloromethane. The organic layers were combined. The solvent was removed, the residue was isolated by silica gel chromatography,


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eluting with 5% methanol in dichloromethane to yield 214 as a white solid 139 mg (82% yield). MS (ES-API) calcd for C22H26ClN10OS [MH+] 513.2, found 513.2. To the remained reaction mixture (2/3 portion, 0.67 mmol), triphenylphosphine (869 mg, 3.31 mmol) was added and the mixture was heated at 50 °C for 6 h. Water (90 µl, 5.0 mmol) was added and the mixture was heated to 70 °C and stirred overnight. The mixture was acidified to pH 3, extracted with dichloromethane. The aqueous layers were combined, neutralized and evaporated. The residue was purified by reverse silica gel column chromatography (octadecylfunctionalized silica gel) eluting first with water, then with 90% CH3CN to give compound 314 as a white solid: 261 mg (80% yield). MS (ES-API) calcd for C22H28ClN8OS [MH+] 487.2, found 487.1. To the solution of 5′-alkynyl DNA 4 (71 nmol) in water (200 µL), azide 2 in DMSO (355 µL, 10 mM, 3.55 µmol), THPTA (tris(3-hydroxypropyltriazolylmethyl)amine)/CuSO4 (1:1) complex in water (27 µL, 66 mM, 1.78 µmol) and aqueous sodium ascorbate (28.4 µL, 100 mM, 2.84 µmol) were added. The mixture was degassed with argon. After the mixture was kept at room temperature for 1 h, PCI (pH 8) extraction, and the aqueous phase was loaded on 20% dPAGE gel and purified to give dasatinib-DNA conjugate (I) 23.6 nmol (33% yield). MALDITof MS calcd for MH+, 5008.0, found 5007.1.

Synthesis of conjugate (II) To the solution of 5′-alkynyl DNA 5 (200 nmol) in water (121 µL), azide 2 in DMSO (500 µL, 10 mM, 5.0 µmol), THPTA/CuSO4 (1:1) complex in water (38 µL, 66 mM, 2.5 µmol) and aqueous sodium ascorbate (40 µL, 100 mM, 4.0 µmol) were added. The mixture was degassed with argon. After the mixture was kept at room temperature for 1 h, PCI extraction, and



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the aqueous phase was loaded on 20% dPAGE gel and purified to give dasatinib-DNA conjugate (II) 64 nmol (32% yield). MALDI-Tof MS calcd for MH+, 5287.3, found 5286.4.

Synthesis of conjugate (III) To the solution of 5′-alkynyl DNA 6 (100 nmol) in water (65 µL), azide 2 in DMSO (500 µL, 10 mM, 5.0 µmol), THPTA/CuSO4 (1:1) complex in water (38 µL, 66 mM, 2.5 µmol) and aqueous sodium ascorbate (40 µL, 100 mM, 4.0 µmol) were added. The mixture was degassed with argon. After the mixture was kept at room temperature for 1 h, PCI extraction, and the aqueous phase was loaded on 20% dPAGE gel and purified to give dasatinib-DNA conjugate (III) 10.1 nmol (10.1% yield). MALDI-Tof MS calcd for MH+, 4381.9, found 4382.8.

Synthesis of conjugate (IV) The dasatinib alkynyl derivative 7 was prepared by the reaction of the corresponding 3 (71 mg, 0.146 mmol) with molar equivalent of 4-pentynoic acid and DCC in a mixed solvent of CH2Cl2/DMF (v/v 4/1) (25 mL) at room temperature overnight. The solvent was removed, the residue was purified by preparative TLC, eluting with 10% methanol in dichloromethane, to give product as a white solid (49 mg, 58% yield). 1H NMR (DMSO-d6) δ 11.48 (s, 1H), 9.89 (s, 1H), 8.23 (s, 1H), 7.88 (t, 1H, J = 5.6 Hz), 7.40 (m, 1H), 7.35-7.21 (m, 2H), 6.06 (s, 1H), 3.52 (m, 4H), 3.22 (m, 2H), 2.77 (t, 1H, J = 2.6 Hz), 2.49-2.20 (m, 16H); 13C NMR (DMSO-d6) δ 170.2, 165.2, 162.6, 162.4, 159.9, 156.9, 140.9, 138.8, 133.5, 132.5, 129.0, 128.2, 127.0, 125.7, 83.8, 82.6, 71.3, 57.0, 52.3, 43.6, 36.2, 34.2, 25.6, 18.3, 14.3. MS (ES-API) 567.2, found 567.2. HRMS calcd for C27H32ClN8O2S [MH+] 567.2057, found: 567.2062.


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The 5′-azido DNA derivative was prepared by the reaction of 5′-amino tagged DNA (8) with azidobutyrate NHS ester according to the Glen Research protocol. MALDI-Tof MS calcd for MH+, 4583.9, found 4584.0. To the solution of 5′-azido DNA 9 (100 nmol) in water (37 µL), excess alkynyl-dasatinib 7 in DMSO (500 µL, 10 mM, 5.0 µmol), THPTA/CuSO4 (1:1) complex in water (38 µL, 66 mM, 2.5 µmol) and aqueous sodium ascorbate (40 µL, 100 mM, 4.0 µmol) were added. The mixture was degassed with argon. After the mixture was kept at room temperature for 1 h, PCI (pH 8) extraction, and the aqueous phase was loaded on 20% dPAGE gel and purified to give dasatinibDNA conjugate (IV) 47.2 nmol (47.2% yield). MALDI-Tof MS calcd for MH+, 5150.1, found 5150.0.

In vitro kinase assays: Src in vitro ADP-Glo Kinase Assays (Promega) were used according to manufacturer’s instructions. Luminescence was measured in the presence of increasing concentrations of either dasatinib or dasatinib-conjugated oligonucleotides (I and IV) and 100 ng of recombinant enzyme (Promega). IC50 values were calculated using GraphPad Prism 6.0 and nonlinear regression log (inhibitor) vs. response model.

Flow cytometry phospho-SRC Assays: Antibody against phospho-SRC (clone SC1T2M3) was from eBiosciences. K562 leukemia cells (obtained from ATCC) were treated with 1 nM dasatinib or 1 nM gold nanoparticles functionalized with dasatinib-DNA (I) or dasatinib-DNA (IV). After 24 hours, cells were fixed, permeabilized (Intracellular fixation buffer and permeabilization buffer, eBiosciences) according to manufacturer’s instructions, and stained with



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antibody for 30 minutes in the dark. The cells were washed and then analyzed by flow cytometry on a BD FACSAria II.

Acknowledgement We thank Mr. Saurja Dasgupta, Mr. Benjamin P. Weissman and Dr. Sandip A. Shelke for helpful discussions and critical comments on the manuscript. This work was supported by an N.I.H. grant to J.A.P. (1R01AI081987) and a Leukemia and Lymphoma Society New Idea Award to P.M.G.

A list of abbreviations: AuNPs: gold nanoparticles; BCR: break point cluster; ABL: abelson; Ser: serine; Thr: threonine; THPTA: tris(3-hydroxypropyltriazolylmethyl)amine; MsCl: methanesulfonyl chloride; DCC: N,N′dicyclohexylcarbodiimide; NHS: N-hydroxysuccinimide; PCI: phenol/chloroform/isoamyl alcohol; dPAGE: denatural polyacrylamide gel electrophoresis.

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