Biological activity of N(4)-boronated derivatives of 2′-deoxycytidine, potential agents for boron-neutron capture therapy

Bioorganic & Medicinal Chemistry 23 (2015) 6297–6304

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Biological activity of N(4)-boronated derivatives of 20 -deoxycytidine, potential agents for boron-neutron capture therapy Joanna Nizioł, Łukasz Uram, Magdalena Szuster, Justyna Sekuła, Tomasz Ruman ⇑ ´ ców Warszawy Ave., 35-959 Rzeszów, Poland Rzeszów University of Technology, Faculty of Chemistry, Bioorganic Chemistry Laboratory, 6 Powstan

a r t i c l e

i n f o

Article history: Received 8 July 2015 Revised 4 August 2015 Accepted 25 August 2015 Available online 28 August 2015 Keywords: Boron-neutron capture therapy Boron nucleoside Boron nucleotide DNA fragmentation DNA mass spectrometry

a b s t r a c t Boron-neutron capture therapy (BNCT) is a binary anticancer therapy that requires boron compound for nuclear reaction during which high energy alpha particles and lithium nuclei are formed. Unnatural, boron-containing nucleoside with hydrophobic pinacol moiety was investigated as a potential BNCT boron delivery agent. Biological properties of this compound are presented for the first time and prove that boron nucleoside has low cytotoxicity and that observed apoptotic effects suggest alteration of important functions of cancer cells. Mass spectrometry analysis of DNA from cancer cells proved that boron nucleoside is inserted into nucleic acids as a functional nucleotide derivative. NMR studies present very high degree of similarity of natural dG–dC base pair with dG–boron nucleoside system. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Radiation therapy has a crucial role in the management of many types of cancer, either as the only treatment or as part of a multimodal approach that can include surgery, chemotherapy and/or immunotherapy.1 Radiation therapy uses several types of ionizing radiation (X-rays, c-rays, electron, proton or alpha beams) to treat tumors. Ionizing radiation damages nucleic acids of tumor cells resulting in complex biochemical processes and, in some cases, cell death. Radiation therapies require precise targeting and dose control. Particle beams utilizing protons and heavier ions offer improved dose distributions compared with photon beams and therefore enable dose escalation within the tumor while sparing normal tissue.2 Both problems mentioned in last sentence may be solved by binary tumor therapies that apply two non-toxic agents acting synergically in vivo to provide efficient anti-tumor effect unattainable without one of them. Boron neutron capture therapy is a binary therapy that stems from the phenomenon involving the nuclear capture and fission reactions with the participation of boron-10 (10B), being a non-radioactive constituent of natural boron. Irradiation of non-radioactive 10B with low energy thermal neutrons results in a nuclear reaction 10B(n,a)7Li producing high linear energy transfer alpha particles and lithium-7 nuclei. The penetrability of these particles is in the range of 4–10 lm, hence limited to the diameter of a sin⇑ Corresponding author. E-mail address: [email protected] (T. Ruman). http://dx.doi.org/10.1016/j.bmc.2015.08.026 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

gle cell, suggesting a possibility of selective irradiation of such cancer cells that accumulate sufficient amount of 10B. Selectivity requires 10B to be accumulated to a significantly lesser degree by normal, healthy cells. On the other hand, tumor treatment by BNCT depends on the use of nuclear reactors capable of delivering of high fluxes of thermal neutrons penetrating all sections of the tumor.3–5 A successful boron delivery agent should exhibit (i) low systemic toxicity, (ii) distinctly lower uptake by normal than tumor tissue, (iii) high tumor/brain and tumor/blood concentration ratios (>3– 4:1), (iv) tumor concentrations of 20 lg 10B/g tumor, and (v) much faster clearance from blood and normal tissues than from tumor during BNCT. However, no boron delivery agent is known so far to fulfill all of these criteria,6 and two drugs currently used in clinics, sodium borocaptate (BSH) and boronophenylalanine (BPA), are relatively non-selective.7 Therefore search for more selective BNCT boron delivery agents appears a very important task.8–10 Integration with DNA or location of the boron compound in close proximity to the nucleus is extremely desirable for BNCT, as the required cellular boron concentration has been assessed 2–5-fold lower with the boron compound located in the nuclear or perinuclear region.11 Application of nucleosides as BNCT agents have many advantages such as (i) low cytotoxicity, (ii) high rate of phosphorylation, (iii) nuclear localization and (iv) possible synergistic effect in case of inhibitory effect with enzymes crucial for cancer cells such as thymidylate synthase. One of disadvantages that may be pointed out is that transport of nucleoside depends on the degree of cell proliferation and nucleoside kinases activity.

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Moreover, the expression of important nucleoside phosphorylating enzyme—for example—thymidine kinase-1 is regulated tightly during the cell cycle, and the active enzyme is found only in S-phase cells.12,13 Previously, our group has shown that 1 is phosphorylated in vivo into mono-, di- and triphosphate derivatives.14 We have also shown previously that boron nucleoside 1 inhibits human colorectal adenocarcinoma C85 cell growth with EC50 value of 6.2 ± 2.3 mM.14 The results appears to be in accord with the requirement for a boron delivery agent to be used in BNCT to exhibit low toxicity.11 On the basis of previously published results, the question was raised if boron nucleoside N(4)-[B-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan)methyl]-20 -deoxycytidine (1) is capable of being integrated into newly in vivo synthesized nucleic acids. To answer this question, biological tests, laser desorption/ionization (LDI) high resolution mass spectrometry (HRMS) and NMR studies were performed. 2. Results and discussion 2.1. Biological tests 2.1.1. Cytotoxicity of 1 The biological effects of N(4)-boronated derivatives of 20 -deoxycytidine on normal and cancer cells were estimated by means of two assays: XTT concerning activities of mitochondrial oxidoreductases yielding cellular redox state and NR providing information about active transport mechanism. Both assays confirmed inhibitory action of compound 1 on different cellular metabolic pathways. Significantly higher effects in cancer cells than in fibroblasts were observed. IC50 for SCC-15 cells was found to be 16.4 and 18.8 mM for NR and XTT assays respectively. These results are in good agreement with former study reporting EC50 = 6.2 mM of 1 in human colorectal carcinoma C85 cells.14 In normal fibroblasts IC50 amounts to 49.5 and 45.1 mM for NR and XTT assays respectively (Fig. S1A and B in Supplementary materials). The matter of interest is that literature data concerning boronated nucleosides present half maximal effective concentration IC50 in micromolar range.15–19 This strongly suggests that 1 is a promising candidate for application in BNCT. 2.1.2. Apoptotic effect of boron compounds After 48 h incubation of SCC-15 and BJ cells with 1, apoptosis was detected only in SCC-15 cells. PARP-1 is a subject of many pro-apoptotic factors (caspases, cathepsins, calpains and matrix metalloproteinases) with proteolytic activity resulting in cleavage fragments witch are markers of an early stage apoptosis due to damage of DNA repair systems.19 The significantly higher level of PARP fragmentation was detected for 20 and 40 mM concentrations of 1 in human carcinoma cells, while in fibroblasts such an effect was not observed for all of applied concentrations (Fig. 1). This is confirmed by images obtained from laser scanning confocal microscopy, showing that the apoptotic signal was significantly more intensive at 20 and 40 mM concentrations in SCC-15 cells in contrast to BJ cell line. At 40 mM concentration the density of cancer cell population was drastically diminished due to the loss of apoptotic cells adhesion (Fig. 1). Similar research focusing on cytotoxicity and apoptotic effect of native nucleosides on the human hepatoma HepG2 cells was described.20 Viability of HepG2 cells after 12 h incubation with 30 mM cytidine was significantly decreased, but Fas ligand content did not increase and cytochrome C and TNF-a (tumor necrosis factor a) were undetectable. The observed apoptotic effect of 1 strongly suggest that 1 is responsible for alteration of important functions of cancer cells. It is well

known that the high linear energy transfer associated with BNCT can induce apoptosis in many types of cancer cells.21–23 Results of our study indicate, that 1 is not toxic for normal cells, and induce apoptosis only in cancer cells. 2.2. MS analysis of DNA samples The application of a MALDI-based DNA sequencing approach has been demonstrated by the analysis of synthetic24,25 and enzymatically produced Sanger sequencing mixtures.26,27 Krause and other authors have shown, that nucleic acid analysis by MALDI is presently characterized by a limited mass range, as demonstrated by a decrease in spectral peak intensities and mass resolution for longer polynucleotides. One major reason for this limited mass range appears to be fragmentation of the DNA occurring during the MALDI process.28,29 In case of present work, DNA fragmentation occurring during MS measurements30,31 can be considered beneficial as no additional hydrolysis/fragmentation steps were applied due to the necessity of elimination of any possible step in which boron moieties could be modified or cleaved off. The measurements were intentionally made in low mass region as highresolution and high-precision reflectron mode was required to obtain unquestionable data within 0–10 ppm mass determination accuracy. In order to determine if boron nucleoside 1 could possibly be up-taken by cells and integrated into nucleic acids, a laser desorption/ionization mass spectrometry experiments were performed. After five-day incubation of 1 at 1 mM concentration in cell cultures, standard DNA isolation was made followed by several additional DNA washing steps with the use of 2-propanol. DNA solution was then measured for several hours with NMR apparatus in order to estimate general purity of the sample. DNA–deuterium oxide solution was placed on AuNPET plate with additional DHB matrix. AuNPET plate was chosen as a perfect solution for desorption/ionization of analyte but also due to possibility of performing internal calibration on emitted gold ions. Acidic DHB/trifluoroacetic acid (TFA) matrix system was applied in order to facilitate fragmentation of relatively long chains of nucleic acids as measurements were made up to m/z 3000 in reflectron mode at maximum possible resolution.30 The MS experiments were performed in described way due to requirement of minimum operations to be made on DNA sample for the as fastest and simplest analysis as possible. Chosen portion of data obtained in described MS experiments is shown in Table 1 and Figure 2 (also in Supplementary data S2 and S3). As can be seen in Table 1, there are thirty five ions found in spectra made in 180–2000 and 480–3000 m/z ranges that are assigned to expected natural dinucleotides such as P-dA-P-dG-P, P-dG-P-dG-P and trinucleotides P-T-P-T-P-T-OH, OH-dA-P-T-PdC-P and OH-dG-P-dC-P-dC-P where P is bridging or terminal (at 50 - or 30 -position) phosphate moiety. Mentioned compounds were found as mononegative ions containing various combinations of additional sodium, potassium and hydrogen cations that are counter-ions for phosphates. The products of fragmentation with terminal hydroxyl groups may be a result of classical acid-catalyzed hydrolysis as described earlier,30 but in-source laser induced dissociation (LID) as a fragmentation mechanism cannot be ruled out as there are many terminal bi-phosphate species found in spectra. There are also many ions that are in perfect agreement with structures of mono- di- and trinucleotides with unnatural boroncontaining 20 -deoxycytidine moiety. Smallest compound found that contains boron is P-dCBOH-P (Fig. 3A) in which boron is in form of dihydroxyboryl moiety, a product of hydrolysis of pinacol boronate ester moiety. The mentioned hydrolysis process occurs in vitro and in vivo, and was characterized with the use of MS and NMR methods.14

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Figure 1. (A) Apoptotic effect of 1 for SCC-15 and BJ cells. Results are median of triplicate assays from three independent experiments. *P <0.05; Kruskal–Wallis test (against untreated control). (B) Images obtained by confocal microscopy presented cleaved poly(ADP-ribose)polymerase (PARP; Asp214) protein (pink) in nuclei (blue) in SCC-15 and BJ cells after 48 h incubation with 1. Cells were stained with AlexaFluor 647-conjugated anti-PARP antibody (639:665 nm) and 40 ,6-diamidino-2-phenylindole-DAPI (405:461 nm).

There are six boron-containing compounds of dinucleotide-type found in MS spectra as shown in Table 1. Only one compound contains pinacolboronic moiety of significant steric hindrance, while all other contain boron in form of dihydroxyboryl groups. All of them contain two terminal phosphates which is not unusual as in-source laser-induced dissociation (LID) fragmentation produces fragment ions of this kind.31 There are also five boron-containing trinucleotide-type compounds found in MS spectra—P-T-P-dC-PdCBOH-P, P-dC-P-dCBOH-P-dCBOH-P, P-dA-P-dA-P-dCBOH-P, P-dAP-T-P-dCBOH-OH and P-dA-P-dC-P-dCBOH-P. All of them contain exclusively dihydroxyboryl moieties. Apart from very good agreement of calculated and experimental m/z values, assignments of peaks of natural and boron-containing nucleotides were additionally confirmed by comparison of isotopic distributions, examples of which are shown in Supplementary materials S4. On the basis of above mentioned findings, it is highly reasonably to state that compounds of di- and trinucleotide structures must be of nucleic acid origin. It should be noted that above mentioned findings are the first known that proves that boron nucleosides or their metabolites are integrated into nucleic acid structures. 2.3. NMR studies Our group have previously shown that 11B NMR chemical shift of 1 in deuterium oxide was in 7–8 ppm range suggesting

negatively charged boron as a dominating species. Negative charge of boron moiety is believed to originate from additional OD ion coordinating to boron with hydrogen/deuterium counterion at N(3) or N(4)-nitrogen.14 It was also shown that acidic conditions, for example addition of acetic acid or N,C-protected cysteine leads to hydrolysis of pinacol moiety resulting in formation of dihydroxyboronic nucleoside of much lower steric hindrance in boron neighborhood. The relatively fast hydrolysis of pinacol fragment catalyzed by nucleophilic and acidic reagents that was shown in NMR-controlled experiments is the probable cause of the fact that almost all of boron oligonucleotides found in MS experiments are having hydrolyzed B (OH)2 moieties. The mentioned hydrolysis process is crucial for DNA-integration as bulky pinacol structure might interfere with base pairing which is critical for in vivo polymerase-catalyzed process. In order to estimate if boron nucleoside 1 is capable of forming of typical Watson–Crick base pairs, NMR-controlled experiments were conducted. Measurements of two similar systems containing 20 -deoxynucleosides in 9:1 (v/v) DMSO-d6/D2O environment were performed for two pairs of nucleosides—20 -deoxyguanosine–20 deoxycytidine (dG–dC) and 20 -deoxyguanosine–1 (dG–1; Supplementary materials S5 and S6). In theoretical situation in which base pairing between boron nucleoside and dG would have identical parameters such as energy, bond length etc., it is assumed that

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Table 1 Results of analysis of two LDI HRMS spectra of DNA from 1-treated cells in low m/z range

Dm/z (ppm)

Compound

Formula

Ion formula

m/zcalcda

m/zexp.b

Mononucleotide-type compounds P-dCBOH-P

C10H14BN3O12P2

[M+H+Na+K+H2O]

521.9864

521.9865c 521.9889d

Dinucleotide-type compounds P-dA-P-dG-P P-dG-P-dG-P P-dA-P-dCBOH-P

C20H22N10O15P3 C20H22N10O16P3 C20H25BN8O17P3

[M+H+3Na] [M+H+3Na] [M+H+3Na]

805.0250 821.0199 823.0415

P-dC-P-dCBOH-P

C19H25BN6O18P3

[M+D+2Na+K+D2O]

805.0190d 821.0234d 823.0356d 823.0293c 836.0413d 836.0323c 841.0497d 873.0103c 888.9810d 888.9785c 845.0210d 845.0168c 851.0448d 851.0338c 867.0100d 866.9970c 848.0194d 904.9692d 1005.0735d 1005.0618c

7.5 4.3 7.2 14.8 9.2 1.6 2.9 11.9 8.0 5.2 5.0 9.9 13.5 0.6 3.2 11.8 5.9 6.1 10.0 1.7

989.0761d 999.0541d 1000.0622d 1000.0492c 1159.0437d 1161.0597d 1161.0548c 1162.0742d 1166.0779d 1167.0810d 1167.0771c 1168.0570d 1168.0543c

2.6 10.4 2.4 10.6 0.8 3.8 0.4 0.9 8.8 0.8 2.5 2.3 9.7

BOH

P-dA-P-dC

-P

P-T-P-dCBOH-P



836.0336

C20H25BN8O17P3

[M+H+3Na+H2O] [M+H+2K+Na+H2O] [M+H+3K+H2O]

841.0521 872.9999 888.9739

C20H26BN5O19P3

[M+D+H+2K+D2O]

845.0252

[M+D+2Na+K+D2O]

851.0333



867.0072

[M+D+2K+Na+D2O]

P-dCBOH-P-dCBOH-P P-dCB-P-dCBOH-P

C20H28B2N6O20P3 C26H38B2N6O20P3

[M+H+2Na+K+H2O] [M+H+3K] [M+H+3K+H2O]

848.0144 904.9747 1005.0635

Trinucleotide-type compounds P-T-P-T-P-T-OH OH-dA-P-T-P-dC-P OH-dG-P-dC-P-dC-P

C30H37N6O22P3 C29H35N10O19P3 C28H34N11O19P3

[M+H+Na+K] [M+H+2K] [M+H+2K]

989.0787 999.0645 1000.0598

P-T-P-dC-P-dCBOH-P

C29H37BN8O25P4

[M+H+3Na+K+H2O] [M+H+3Na+K+D2O]

1159.0428 1161.0553

P-dC-P-dCBOH-P-dCBOH-P P-dA-P-dA-P-dCBOH-P P-dA-P-T-P-dCBOH-OH

C29H39B2N9O26P4 C30H36BN13O22P4 C30H37BN10O24P4

[M+2H+2Na+K] [M+3H+2K+D2O] [M+H+4Na+H2O]

1162.0732 1166.0881 1167.0800

P-dA-P-dC-P-dCBOH-P

C29H36BN11O23P4

[M+H+3Na+K+H2O]

1168.0543

0.2 4.8

Bolded symbols represent ions assigned to boron oligonucleotides. dCB—N(4)-[B-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan)methyl]-20 -deoxycytidinyl moiety; dCBOH—N(4)-[(dihydroxyboryl)-methyl]-20 -deoxycytidinyl moiety; P—terminal or bridging phosphate moiety; OH—30 or 50 -hydroxyl group; dA, dC, dG, T—20 -deoxynucleotide moieties with adenine, cytosine, guanine and thymine nucleobases, respectively. a m/zcalcd—calculated m/z for highest peak in isotopic distribution. b m/zexp.—experimental m/z. c Data from m/z 180–2000 range experiment. d Data from m/z 480–3000 range experiment (the same sample as in m/z 180–2000 experiment).

NMR parameters for hydrogen and nitrogen atoms in or near pairing region will be identical or very similar. Overlay of two 15N–1H HMBC spectra fragments of 20 -deoxyguanosine–20 -deoxycytidine (dG–dC) and 20 -deoxyguanosine–1 (dG–1) pairs is shown in Figure 4. Visible peaks of heteronuclear coupling of H(8) with N(9) and NH2 hydrogens with closest NH2 nitrogen of common constituent in those two pairs—dG are in almost identical positions. Observed chemical shift differences for mentioned 2D peaks are 0.1 and 0.5 ppm for N(9) and NH2 nitrogens respectively, while differences are lower than 0.01 in case of chemical shifts of protons. What is interesting and unexpected—there are visible and relatively high differences of nitrogen chemical shift regarding N(7)–H(8) coupling system. As can be seen in Figure 4, exchange of dC for 1 resulted in 5.5 ppm downfield shift of N(7) resonance. It should be noted that there was almost no H(8) proton peak shifting observed between dG–dC and dG–1 pairs. The explanation of this phenomena must be based on assumption that additional effect responsible for N(7) shifting do not affect base pairing as other 2D peaks are in perfect agreement. Possible cause is the interaction of bulky pinacol moiety that is in near surrounding of discussed nitrogen. Other possibility is that boron atom is relatively close

to N(7) and coordination bond that may be forming would lower electron density on nitrogen causing observed upfield shift. 3. Conclusions The optimized synthetic route applied to synthesize N(4)-[B(4,4,5,5-tetramethyl-1,3,2-dioxaborolan)methyl]-20 -deoxycytidine (1) shows a considerable potential due to high yield and high purity of the product. NMR investigation of samples containing dG–dC and dG–1 pairs suggested that there is a very high degree of similarity of base pair systems. The only one observable difference of dG in 1H–15N HMBC of dG–dC/dG–1 spectra concerns N(7) nitrogen that most probably interact with boron atom. LDI MS measurements optimized for fragmentation of nucleic acids suggest that there are peaks of six boron-containing compounds of dinucleotide-type and five of trinucleotide-type visible in spectra of DNA from cancer cells. Biological properties of N(4)-boronated derivatives of 20 -deoxycytidine in human carcinoma and normal fibroblast cells estimated by two commonly used assays—NR, XTT, CV and apoptotic tests suggest that 1 is a very good candidate for BNCT. However, in vitro—observed incorporation into nucleic acids could have serious consequences during clinical use, because

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Intens. [a.u.]

A x104

Au3+ Au4+

Au5+

6

Au6+

4 2 0 600

B

700

800

900

1000

1 523.0

7 842

844

805

846

848

850 m/z

997

998

999

1000

m/z

820

1002 m/z

848

1002

824

826

m/z

834

835

836

837

838

m/z

10

850

852

854 m/z

862

13 1001

822

11

12 14 996

806

8

9

6 840

804

m/z

m/z

5

3

522.0

1200

4

2 521.0

1100

1003

1004

1005

864

15 1006

1007

1008 m/z

1156

1158

866

868

870

m/z

16 1160

1162

887

17 1164

m/z

1162

888

889

18 1164

890

19

1166

1168

891

m/z

20 1170

1172 m/z

Figure 2. Reflectron negative mode LDI MS spectrum of DNA from 1-treated cells on AuNPET with additional DHB matrix made in m/z 480–3000 range (A). Insets in lower part (B) present spectrum fragments with peaks assigned to: 1—P-dCBOH-P, 2—P-dA-P-dG-P, 3—P-dG-P-dG-P, 4—P-dA-P-dCBOH-P, 5—P-dC-P-dCBOH-P, 6, 11—P-dA-P-dCBOHP, 7–10—P-T-P-dCBOH-P, 12—OH-dA-P-T-P-dC-P, 13—P-dCB-P-dCBOH-P, 14—OH-dG-P-dC-P-dC-P, 15,16—P-T-P-dC-P-dCBOH-P, 17—P-dC-P-dCBOH-P-dCBOH-P, 18—P-dA-PdA-P-dCBOH-P, 19—P-dA-P-T-P-dCBOH and 20—P-dA-P-dC-P-dCBOH-P.

of its potentially mutagenic, carcinogenic or even teratogenic effects. 4. Experimental section 4.1. Materials and methods All NMR spectra were obtained with Bruker Avance spectrometer operating in the quadrature mode at 500.13 MHz for 1H and 50.69 MHz for 15N nuclei. All 15N chemical shifts presented in this work are related to liquid ammonia (0.0 ppm). The external standard used in 15N NMR was sodium nitrite (609.6 ppm rel. to liquid NH3) and sodium nitrate (376.5 ppm rel. to liquid NH3). Base pair experiments was analyzed with gradient-enhanced COSY, NOESY, 1H–15N Heteronuclear Multiple Bond Correlation (HMBC) experiments and 1D methods. All 11B spectra were performed using 5 mm pure quartz NMR tube. The residual peaks of deuterated solvents were used as internal standards. Reagents and deuterated solvents of the highest commercially available grade were purchased from Aldrich. DMSO was dried by vacuum distillation over anhydrous magnesium sulfate. All procedures, including preparation of samples for the NMR measurements,

were carried out under nitrogen. All reagents, with the exception of boranes, were dried by triple azeotropic distillation from deuterated chloroform. High-resolution COSY spectra were prepared using 4096x4096 measurement points. 11B NMR spectra were referenced to external BF3OEt2 sample (0.0 ppm). All 1H NMR experiments were made with HDO-suppression method applied. LDI time-of-flight (TOF) mass spectrometry experiments were performed using a Bruker Autoflex Speed reflectron time-of-flight mass spectrometer, equipped with a SmartBeam II laser (352 nm). The laser impulse energy was approximately 60120 lJ, the laser repetition rate was 1000 Hz, and the deflection value was set on m/z <80 Da. The first accelerating voltage was held at 19 kV, and the second ion-source voltage was held at 16.7 kV. The reflector voltages used were 21 kV (first) and 9.55 kV (second). The data were recorded and analyzed using the software provided with the Autoflex instrument (FlexAnalysis version 3.3). All samples were measured on a AuNPET32 plate with additional DHB matrix (Section 4.4.1). All measurements were made in reflectron negative mode. Mass calibration (typically cubic calibration based on five to seven points) was performed using internal standards (gold ions emitted from AuNPET, from Au to Au 15 depending on m/z range).

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A

HO

B

OH

O

-

O

B

-

O

NH2

N

P O

O

NH

N

N N

N

O

O

-

N

O

P

O

O

-

P

O

HN O

O

O

O

O

O

P O

OH

HO

N

O O

B

-

N O

-

O O

-

P O

O

-

-

HO

C H 2N

B

H 2N

N

N HN

N

N

O

-

P

O

O

O -

O O

-

P

O

O O

O

O

O N

N

N

O

N

N

N

OH

O

-

P O

O O

O

-

P

O

-

O

Figure 3. Molecular structures of chosen compounds found in LDI MS spectrum of DNA from 1-treated cells (Table 1). (A)—P-dCBOH-P, (B)—P-dA-P-dCBOH-P, (C)—P-dA-P-dAP-dCBOH-P.

H(5)dC H(8)dG H(6)dC

dG-dC H(6)1

dG-1

H(1’)dG+dC

H(1’)dG+1 H(8)dG

OHdG+dC

NH2dG

H(5)1

NH(4)1

NH2dG

N(3)dG N(3)dC N(1)dG

H(4)1 H(3)1

N(9)dG

N(7)dG

Figure 4. Overlay of two 15N–1H HMBC spectra for 20 -deoxyguanosine–20 -deoxycytidine (dG–dC, red 2D peaks) and 20 -deoxyguanosine–1 (dG–1, blue 2D peaks) pairs.

Isotopic distributions were calculated with the online Molecular Mass Calculator by Christoph Gohlke33 and ChemCalc calculator.34 4.2. Optimized synthesis of N(4)-[B-(4,4,5,5-tetramethyl-1,3,2dioxaborolan)methyl]-20 -deoxycytidine (1) Synthesis presented below is a high-yield, large-scale, purityoptimized version of published one.14 20 -Deoxycytidine (0.6 g) was dried thrice by azeotropic distillation of acetonitrile (3 ml) at reduced pressure (1.5 mbar maximum vacuum pump) at 60 °C. After acetonitrile evaporation to dryness, sample was dried at high vacuum for 30 min. Anhydrous DMSO (6 ml) was then added to the reaction flask, followed by addition of anhydrous potassium carbonate (0.73 g) and 2-(iodomethyl)-4,4,5,5-tetramethyl-1,3,2dioxaborolane (0.943 ml). The reaction mixture was immersed in an oil bath at approx. 80–83 °C (oil temperature) for 71 h, then filtered through G4 glass filter and the solution evaporated to dryness using rotary vacuum evaporator (90 °C bath temperature for 72 h) and vacuum pump. The product was washed twice with toluene (2  2 ml) and then twice with acetone (2  2 ml), sonificated in ultrasound bath and mixed with hexane (6 ml). The resulting solid material was filtered, and vacuum dried. Yield: 813 mg (84%). Analytical data is in perfect good agreement with published one.14

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4.3. In vitro assays 4.3.1. Materials A human normal fibroblasts BJ (ATCC-CRL-2522) and human squamous cell carcinoma (SCC-15) cells (ATCC-CRL-1623), Eagle’s minimum essential medium (EMEM), Dulbecco’s Modified Eagle’s Medium and Ham’s (DMEM:F-12), fetal bovine serum (FBS), penicillin and streptomycin solution were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Trypsin–EDTA solution, phosphate-buffered saline (PBS) with and without magnesium and calcium ions, hydrocortisone, XTT sodium salt (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt), Phenazine methosulfate (PMS), Nmethyldibenzopyrazine methyl sulfate, 0.33% neutral red solution (NR) (3-amino-m-dimethylamino-2-methyl-phenazine hydrochloride), 0.4% trypan blue solution and reagents for DNA isolation (NaCl, Tris–HCl, EDTA, SDS, isopropanol) were provided by Sigma–Aldrich (St Louis, MO, USA). Proteinase K solution (20 mg/ mL) was from A&A Biotechnology (Gdynia, Poland). Fluorescent marker DAPI (40 ,6-diamidino-2-phenylindole, dihydrochloride) was purchased from Life Technologies (Carlsbad, CA, USA). Mouse antibody (immunoglobulin G [IgG] j) human-specific against cleaved poly(ADP-ribose) polymerase (PARP; Asp 214) conjugated with AlexaFluor 647 were from BD Pharmingen (BD Biosciences, San Jose, CA, USA). All other cell culture sterile materials were purchased from Corning Incorporated (Corning, NY, USA) or Greiner Bio-One (Greiner Bio-One GmbH, Germany). 4.3.2. Cell cultures Normal human skin fibroblast BJ (CRL-2522 ATCC), doubling time 1.9, were cultured in EMEM, 10% heat-inactivated FBS, 100 U/mL penicillin and 100 lg/mL streptomycin. Human squamous cell carcinoma SCC-15 (CRL-1623 ATCC), doubling time 5, were grown in DMEM:F-12, 10% FBS, 100 U/mL penicillin, 100 lg/mL streptomycin and hydrocortisone (400 ng/mL). Cells were grown at 37 °C in an atmosphere of 5% CO2 and 95% humidity, with medium changed every 2—3 days and passaged at 70–85% confluence using 0.25% trypsin–EDTA in PBS (calcium and magnesium free). Cell morphology was checked under Nikon TE2000S Inverted Microscope with phase contrast. Number and viability of cells were estimated by Trypan blue exclusion test using Neubauer chamber (BRAND GmbH+ Co KG, Germany) or Automatic Cell Counter TC20TM (Bio-Rad Laboratories, Hercules, CA, USA). Cytotoxicity experiments were performed in four replications in three independent experiments and apoptosis assays in three independent experiments in triplicate. 4.3.3. Cytotoxicity assays (a) XTT assay: BJ and SCC-15 cells were seeded in a flat-bottom 96-well culture plates in triplicate (200 lL cell suspension per well) at a density 2  104 cells/well. Cells were allowed to attach for 24 h. The stock solution of 40 mM 1 in EMEM and DMEM:F12 was filtered (sterile syringe filters, 0.22 lm). Cell monolayers were treated with 1 solutions within a range of increasing concentrations from 0 to 40 mM (100 lL/well) for 48 h in 37 °C. After that time, media were exchanged (100 lL/well), 50 lL/well of 5 mM XTT and 25 lM PMS mixture was added, and plates were returned to the incubator. After either 1 h (BJ cells) or 2.5 h (SCC-15), absorbance was measured at the 450-nm wavelength against 620 nm against a blank sample (200 lL of complete growth medium containing XTT and PMS), using a microplate spectrophotometer (lQuantTM, BioTek Instruments, Inc. Winooski, VT, USA). (b) NR assay: Cells (both lines) were cultured as described earlier. After exposure to 1, medium was removed, 100 lL NR solution (amount equal to 2% of the culture medium volume) was added per well, and cells were incubated for 2 h in a CO2 incubator. After

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washing once with PBS, 100 lL/well of fixative (50% ethanol, 49% H2O, and 1% glacial acetic acid) was added and plates were shaken at 500 rpm for 15 min until complete dissolution of dye was achieved. Absorbance was measured at 540 nm against 620 nm in a microtiter plate reader against blank (fixative mixture). 4.3.4. Apoptosis assay Both lines of cells were seeded in black 96-well clear-bottom microplates (Corning Inc., Corning, NY, USA) at 2  104 or 1  104 cells/well (SCC-15 and BJ, respectively), cultured for 24 h, and treated with increasing concentrations (0–40 mM) of 1 for 48 h. After each step of the procedure, cells were washed three times with PBS. Then cells were fixed with 3.7% formaldehyde (100 lL/well, 15 min), and permeabilized with 0.1% Triton X-100/PBS (100 lL/ well for 15 min). After washing, antibody (1:10 in PBS) against PARP (Asp 214) was added (50 lL/well), and incubation was performed in darkness for 1 h at rt. After washing, 300 nM DAPI in PBS solution was added (100 lL/well) and cells were incubated 15 min at rt. Then fluorescence signal was read for AlexaFluor 647-conjugated anti-PARP antibody at 650/670 nm (exc/em) and 358/361 nm for DAPI using Infinite M200 PRO Multimode Microplate Reader (TECAN Group Ltd., Switzerland). Results are presented as median of PARP/DAPI signal intensity ratio. Moreover, confocal microscopy images were collected with a Carl Zeiss Axio Observer Z1 LSM 710 confocal microscope from each canal (DAPI: 405 nm/461 nm; AlexaFluor: 639 nm/665 nm). 4.3.5. DNA isolation BJ and SCC-15 cell lines were seeded in 12-well culture plates (Greiner Bio-One GmbH, Germany) at a density of 5  104 (BJ) and 1  105 (SCC-15) cells/well for 24 h before treatment. 0.01, 0.1 and 1 mM 1 solution in medium culture in triplicates was added (500 lL/well). To provide one cell cycle, incubations were carried for 2 days (BJ) and 5 days (SCC-15) according to doubling time of each cell line. After three times washing in PBS the next steps were performed due to the protocol of Lair et al.35 Isolated DNA was further purified by three washings with isopropanol and vacuum dried. 4.3.6. Statistical analysis A statistical analysis was performed using the Kruskal–Wallis test to estimate the differences between the 1-treated and nontreated control samples.36 Calculations were performed using Statistica 10. 4.4. LDI HRMS analysis of DNA 4.4.1. Preparation of sample for LDI MS 1-treated DNA sample prepared from experiments of 1 mM 1 incubation with SCC-15 cell lines (Section 4.3.5) was used for LDI HRMS analysis. DNA stock solution for MS was prepared by dissolution of whole DNA portion in 0.6 mL of deuterium oxide. Spot preparation: volume of 0.3 ll of matrix solution was placed on the AuNPET plate32 and air-dried followed by placement of identical volume of sample solution on top of the dried matrix and airdrying. Matrix solution used in MS experiments: saturated DHB in 1:1 (v/v) H2O/acetonitrile with 0.1% trifluoroacetic acid (TFA). 4.4.2. LDI MS analysis parameters DNA samples were measured with the use of AuNPET/DHB system in reflectron negative mode. Spectra were recorded by accumulation of 10,000 shots in m/z 180–2000 and 460–3000 ranges with ion deflection turned on for ions of m/z lower than 180 and 460 for mentioned ranges, respectively. Calibration used was cubic calibration based on internal standards—gold ions emitted from AuNPET.

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4.5. NMR-controlled experiments Two pairs containing dG–dC and dG–1 nucleosides at approx. 17 mM concentration in 95% DMSO-d6 5% D2O were independently measured with the use of 1D (1H, 11B, 15N) and 2D NMR methods (COSY, NOESY, 15N–1H HMBC) at 298 K. Acknowledgments This work was supported by financed by Faculty of Chemistry, Rzeszow University of Technology DS budget. NMR spectra were recorded in the Laboratory of Spectrometry, Faculty of Chemistry, Rzeszow University of Technology and were financed from DS budget. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2015.08.026. References and notes 1. Glatstein, E.; Glick, J.; Kaiser, L.; Hahn, S. M. JCO 2008, 26, 2438. 2. Schulz-Ertner, D.; Tsujii, H. JCO 2007, 25, 953. 3. Soloway, A. H.; Tjarks, W.; Barnum, B. A.; Rong, F.-G.; Barth, R. F.; Codogni, I. M.; Wilson, J. G. Chem. Rev. 1998, 98, 1515. 4. Barth, R. F. Appl. Radiat. Isot. 2009, 67, S3. 5. Moss, R. L. Appl. Radiat. Isot. 2014, 88, 2. 6. Barth, R. F.; Coderre, J. A.; Vicente, M. G. H.; Blue, T. E. Clin. Cancer Res. 2005, 11, 3987. 7. Chadha, M.; Capala, J.; Coderre, J. A.; Elowitz, E. H.; Iwai, J.-I.; Joel, D. D.; Liu, H. B.; Wielopolski, L.; Chanana, A. D. Int. J. Radiat. Oncol. 1998, 40, 829. 8. Soloway, A. H.; Zhuo, J. C.; Rong, F. G.; Lunato, A. J.; Ives, D. H.; Barth, R. F.; Anisuzzaman, A. K. M.; Barth, C. D.; Barnum, B. A. J. Organomet. Chem. 1999, 581, 150. 9. Al-Madhoun, A. S.; Johnsamuel, J.; Barth, R. F.; Tjarks, W.; Eriksson, S. Cancer Res. 2004, 64, 6280. 10. Higashida, R.; Oka, N.; Kawanaka, T.; Wada, T. Chem. Commun. 2009, 2466. 11. Wolfenden, R. R. Annu. Rev. Biophys. Bioeng. 1976, 5, 271.

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