Glycoconjugated Site-selective DNA-Methylating Agent Targeting

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Glycoconjugated Site-selective DNA-Methylating Agent Targeting Glucose Transporters on Glioma Cells Mairin K. Buchanan, Chase N. Needham, Nina E. Neill, Maria C. White, Charles Brian Kelly, Kelly Mastro-Kishton, Lacie M. Chauvigne-Hines, Tyler J. Goodwin, Andrew L McIver, Libero J. Bartolotti, Arthur R. Frampton, Andrea J Bourdelais, and Sridhar Varadarajan Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b01075 • Publication Date (Web): 21 Dec 2016 Downloaded from http://pubs.acs.org on December 22, 2016

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Glycoconjugated Site-selective DNA-Methylating Agent Targeting Glucose Transporters on Glioma Cells Mairin K. Buchanan, † Chase N. Needham, † Nina E. Neill, ‡ Maria C. White, ‡ Charles B. Kelly, † Kelly Mastro-Kishton, † Lacie M. Chauvigne-Hines, † Tyler J. Goodwin, † Andrew L. McIver, † Libero J. Bartolotti,॥ Arthur R. Frampton, ‡ Andrea J. Bourdelais, § Sridhar Varadarajan,†. †

Department of Chemistry and Biochemistry, University of North Carolina Wilmington,

Wilmington, NC 28403, United States ‡

Department of Biology and Marine Biology, University of North Carolina Wilmington,

Wilmington, NC 28403, United States §

॥

MARBIONC, University of North Carolina Wilmington, Wilmington, NC 28409, United States Department of Chemistry, East Carolina University, Greenville, NC 27858, United States.

CORRESPONDING AUTHOR *

Telephone: 1-910-962-7350. E-mail: [email protected]

ACS Paragon Plus Environment

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ABBREVIATIONS 3MeA, N3-methyladenine; 7MeG, N7-methylguanine; A/T, adenine/thymine; ANOVA, Analysis of variance; CDCl3, deuterated chloroform; CHCl3, chloroform; DCM, dichloromethane; DI, deionized; DMAP, 4-Dimethylaminopyridine; DMEM, Dulbecco's Modified Eagle's Medium; DMF, dimethylformamide; DMSO, dimethylsulfoxide; DNA, deoxyribonucleic acid; EC50, half maximal effective concentration, EDCI, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide; EtOAc, ethyl acetate; EtOH, ethanol; FDA, Federal Drug Administration; FITC, fluorescein isothiocyanate; GAFF, general AMBER force field; GLUT, glucose transporters; GMB, glioblastoma multiforme; HBSS, Hank's Balanced Salt Solution; HCl, hydrochloric acid; HOBt, hydroxybenzotriazole; MD, molecular dynamics; MMS, methyl methane sulfonate; MTS, 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; NaHCO3, sodium bicarbonate; NaOH, sodium hydroxide; PDB, Protein Data Bank; TOF-Q, time-of-flight quadrupole; STZ, streptozotocin; TEA, triethylamine; THF, tetrahydrofuran; TLC, thin layer chromatography.


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ABSTRACT

DNA alkylating drugs continue to remain an important weapon in the arsenal against cancers. However, they typically suffer from several shortcomings due to the indiscriminate DNA-damage that they cause and due to their inability to specifically target cancer cells. We have developed a strategy to overcome the deficiencies in current DNA-alkylating chemotherapy drugs by designing a site-specific DNA-methylating agent that can target cancer cells due to its selective uptake via glucose transporters, which are overexpressed in most cancers. The design features of the molecule, its synthesis, its reactivity with DNA, and its toxicity in human glioblastoma cells, are reported here. In this molecule, a glucosamine unit, which can facilitate uptake via glucose transporters, is conjugated to one end of a bispyrrole triamide unit, which is known to bind to the minor groove of DNA at A/T rich regions. A methyl sulfonate moiety is tethered to the other end of the bispyrrole unit to serve as a DNA-methylating agent. This molecule produces exclusively N3-methyladenine adducts upon reaction with DNA, and is an order of magnitude more toxic to treatment resistant human glioblastoma cells than streptozotocin, an FDA approved, glycoconjugated DNA-methylating drug. Cellular uptake studies using a fluorescent analog of our molecule provide evidence for uptake via glucose transporters and localization within the nucleus of cells. These results demonstrate the feasibility of our strategy for developing more potent anticancer chemotherapeutics, while minimizing common side-effects resulting from off-target damage.

KEYWORDS: DNA methylation, DNA alkylation, DNA damage, minor groove, glioma, GLUT, glucose transporter, glycoconjugation, 3-methyladenine.


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Alkylating agents are one of the oldest and most widely used therapeutics for cancer1, 2 and function primarily by causing DNA damage in cells. Typically, these drugs inflict DNA damage in all cells, but are able to selectively destroy cancer cells by relying on the differences between normal cells and cancer cells in the efficiency of their DNA-repair mechanisms. Efficient functioning of cell-cycle checkpoints and repair mechanisms in normal cells ensures that DNAdamage is repaired before cells enter the S-phase, thus allowing DNA replication to proceed smoothly. On the other hand, in rapidly dividing cancer cells, inefficient repair of DNA-damage results in the continued presence of toxic DNA-adducts and repair intermediates when the cells approach S phase, which triggers cell-death mechanisms. This ability of alkylating agents to utilize the deficiencies of cancer cells in order to cause their selective destruction is the reason why they continue to be used as frontline chemotherapeutic drugs. While DNA-alkylating drugs have been effective in treating different cancers, they also possess several drawbacks. Since the major proportion of these drugs are wasted in inflicting DNAdamage in normal cells, larger doses are needed to effectively destroy all cancer cells, which increases the severity of the side-effects. Additionally, since these drugs rely on rapid celldivision for their effectiveness, normal cells that divide rapidly, such as hair cells, bone marrow cells, cells in the gastric lining, etc., also succumb to the treatment. Toxicity to these normal cells is what leads to the commonly seen side-effects of cancer such as hair loss, suppressed immunity, gastric irritation, etc. Also, these drugs are usually indiscriminate in the kind of DNAdamage they inflict, and mutagenic damage caused by these chemotherapeutic agents is believed to be responsible for the higher risk of secondary cancer observed in patients treated with DNAalkylating chemotherapeutics.3-7 Since alkylating agents continue to be an essential component of


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chemotherapy, improvements in the design of molecules are required in order to address the limitations outlined above. One strategy commonly investigated for improving the selective delivery of alkylating agents to cancer cells has been to conjugate them to ligands which target proteins that are overexpressed in cancers.8-15 There are several requirements that need to be satisfied for the successful design of such conjugates: (1) The ligand-alkylating agent conjugate should be reasonably soluble in water in order to be useful in an aqueous biological environment; (2) The alkylating agent should be relatively stable in order to survive hydrolysis in the aqueous environment until it reaches the target sites on DNA, but should be electrophilic enough in order to alkylate nucleophilic sites on DNA and form cytotoxic adducts; (3) The targeting ligand should not contain nucleophilic groups that can be easily alkylated by the alkylating agent; (4) The ligand component of the conujugate molecule should continue to maintain its specificity for the targeted protein; (5) The ligand-alkylating agent conjugate should be efficiently transported into, and retained within cancer cells, but exhibit limited uptake in non-cancer cells; and (6) The conjugate molecule (or at least the alkylating component) should be able to enter the nucleus and gain access to the DNA. This complex, and sometimes conflicting set of requirements makes the design of useful ligandconjugated alkylating drugs for targeted delivery to cancer cells quite challenging. A promising approach for targeting chemotherapeutics to a range of cancers is glycoconjugation.16 This approach is based on the observation by Warburg almost a century ago that most cancers exhibit elevated rates of glycolysis, and increased glucose uptake, in order to maintain their selective growth advantage.17, 18 Streptozotocin (STZ, Figure 1)19 and Glufosfamide9 are examples of clinically used DNA-alkylating chemotherapeutics that utilize sugar conjugates to target cancers by taking advantage of the Warburg effect. Glucose


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derivatives conjugated to fluorophores, such as in 2-NBDG (Figure 1) have also been used for imaging cancer cells due to their selective uptake in tumors.20, 21 Similarly, there are several reports in literature of conjugation of glucose derivatives to anticancer agents in order to improve properties such as selective uptake into cancer cells, minimization of collateral damage to normal cells, and enhancement of water solubility of anticancer agents.8, 22-35 Since the up-regulation of glycolysis, and consequent increased glucose consumption, is nearly ubiquitous in all cancers, and since glycoconjugation has been demonstrated to be an effective method for improving uptake into cancer cells, conjugating alkylating agents to glucose derivatives is an attractive strategy for selectively targeting cancer cells. While targeting alkylating agents to cancer cells would diminish adverse side-effects, the choice of alkylating agent is also important in order to maximize formation of cytotoxic adducts and minimize random DNA-alkylation. Most DNA-alkylating agents are indiscriminate in their damage, and form multiple DNA-adducts, some of which are cytotoxic (desired consequence in a cancer drug) while others may cause mutations which may lead to secondary cancer. Most alkylating agents target the easily accessible major groove, and primarily alkylate guanines at the N7-position (the most nucleophilic site on DNA base pairs) and to a lesser extent at the O6position.1, 2 Certain alkylating agents, especially those tethered to minor-groove binding agents, alkylate the N3-position of adenines.36, 37 The majority of these agents result in the attachment of large alkyl groups to DNA, and lead to both cytotoxic and mutagenic consequences. One important class of DNA-alkylating agents are methylating agents, which can transfer a methyl group to nucleophilic N and O atoms within the major and minor groove of duplex DNA.38 Typical methylating agents include methyl methane sulfonate (MMS), dimethyl sulfate, N-methyl-N’-nitro-N-nitrosoguanidine, N-methyl-N-nitrosourea, and the clinically used


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dacarbazine, procarbazine, Temozolomide39 and STZ.40 These methylating agents produce a range of methylated adducts, but the major adduct formed is N7-methylguanine (7MeG), while adducts such as N3-methyladenine (3MeA) and O6-methylguanine are formed to a much smaller extent.38, 41 The main adduct, 7MeG, is well tolerated by cells, can persist in the genome even after cell division, and is not known to have any direct toxic or mutagenic consequences. However, depurination of 7MeG adducts produces abasic sites which have toxic and mutagenic consequences.38, 42-45 O6-methylguanine adducts, produced at low levels by many methylating agents, are known to be both cytotoxic and mutagenic.46-48 On the other hand, 3MeA adducts are highly cytotoxic, and only weakly mutagenic,45 and are, therefore, ideal lesions to generate with chemotherapy drugs. Although these 3MeA adducts are not formed at high levels by most methylating agents, they are formed almost exclusively by a compound called Melex (Figure 1).37 The focus of the research in our laboratory is to develop new ligand-conjugated DNA-alkylating molecules that can address the limitations of currently used DNA-alkylating chemotherapeutics. The goal of this project was to design a new DNA-alkylating molecule that could target cancer cells due to its selective uptake via glucose transporters that are overexpressed in most cancers, and that could then destroy these cells by generating cytotoxic, non-mutagenic 3MeA DNA adducts. We designed 1a (Figure 1) for this purpose, which has three functional components. At one end of the molecule is the 2-deoxy-D-glucosamine unit which is expected to enable the molecule to enter cells via glucose transporters (GLUT). This is the same unit that is believed to be responsible for the selective uptake of STZ and 2-NBDG (see Figure 1) into cancer cells. This glucosamine unit is connected by a short linker to the C-terminus of a bispyrrole triamide core which is known to bind to the minor groove of DNA at A/T rich regions.49 This site for


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attachment of the glucosamine unit was selected based upon prior evidence from our laboratory that ligands can be attached to this site without significantly affecting the binding of the bispyrrole triamide unit to A/T-rich minor groove regions.50 Once the molecule enters cancer cells and binds to A/T-rich regions in the minor groove of DNA, the methyl sulfonate unit at the N-terminus of the molecule is expected to methylate N3-adenine sites, just like it does in Melex, to produce the desired 3MeA adducts. The molecule is designed to have relatively weak DNAbinding so that, upon transfer of the methyl group to DNA, the resultant negatively charged sulfonate compound would be expelled from DNA, and would not have any further biological effect on DNA. In this manuscript, we describe the successful development of 1a (Figure 1), and demonstrate its anti-cancer activity against a human glioblastoma multiforme (GBM) cell line. We report its synthesis, the computational analysis of its DNA-binding, characterization of its reactivity with genomic DNA, and provide evidence that implicates the role of glucose transporters in its bioactivity. MATERIALS AND METHODS Molecular Dynamics (MD) Simulations. MD simulations were conducted in order to evaluate the ability of 1a to bind to target sites on DNA. The MD simulations were conducted using the AMBER8 suite of programs,51 and parameters for 1a were derived using the general AMBER force field (GAFF).52 For these computations, the crystal structure of netropsin (Figure 1, a compound that bears the same DNA-binding core as 1a) bound to the duplex B-DNA dodecamer d(CGCGAATTCGCG) was downloaded from the Research Collaboratory for Structural Bioinformatics (PDB code: 6 BNA)53 into the INSIGHT II program package (Accelyrs Software,


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Inc, San Diego), and then altered to represent compound 1a. The simulation was initiated with the bis-pyrrole triamide unit located within its binding site at the A/T rich minor groove (where this unit was located for netropsin in the crystal structure) whereas the glucose unit was extended outside the DNA, into the bulk solvent. MD simulations were performed until equilibrium was achieved, and the structure was averaged from the leveled section after equilibration was achieved (last 11 ns) and used for analysis. Synthesis of molecules. All solvents and reagents were purchased from standard suppliers such as VWR International (West Chester, Pennsylvania) or Sigma-Aldrich (Atlanta, Georgia), and were of the highest grade available unless otherwise noted. Thin layer chromatography was performed on Silica Gel 50 F254 (EMD Chemicals USA) and visualization was accomplished with a 254 nm UV light. Column chromatography was carried out on EMD Chemicals Geduran® 60 silica gel (SiO2, 40 to 63 μm) purchased from VWR International. Rotary evaporations were carried out using a Buchi R-3000 equipped with a Welch Model 2025 vacuum pump, or a Buchi R-114 rotary evaporator equipped with a Welch DryFast vacuum pump. High pressure hydrogenations were performed using a Parr Hydrogenation Apparatus in a 500 mL Parr jar. All anhydrous reactions were carried out under positive pressure of nitrogen. Glassware used for anhydrous reactions was dried overnight at 110 °C or over a flame, assembled while still hot, and cooled to room temperature under nitrogen. Solvents and liquid reagents for anhydrous reactions were obtained in bottles with sure-seal caps and transferred by using oven-dried needles and glass syringes. NMR spectra were recorded on a Bruker instrument (400 MHz for 1H and 100 MHz for 13C, or 600 or MHz for 1H and 150 MHz for 13C). Deuterated (d6)-dimethyl sulfoxide (DMSO) or deuterated chloroform (CDCl3) was used as the NMR solvent. The spectra are reported in ppm and referenced to the DMSO peak (2.49 ppm for 1H, 39.5 ppm for 13C) or


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the chloroform peak (7.26 ppm for 1H, 77.0 ppm for 13C. Reported spin multiplicities are abbreviated as follows: s (singlet); d (doublet); t (triplet); q (quartet); p (pentet); m (multiplet), dd (doublet of doublets). Coupling constants are reported in hertz (Hz). The samples were contained in 5 mm Pyrex glass NMR tubes obtained from Wilmad-LabGlass (Buena, New Jersey). High resolution mass spectrometry data were acquired with a TOF-Q instrument via electron spray ionization (ESI, positive mode). 2-NBDG was purchased from Cayman Chemical, Ann Arbor, MI. Streptozotocin (STZ) was purchased from EMD Millipore (Biosciences), Billerica, MA.

2,2,2-trichloro-1-(1-methyl-4-nitro-1H-pyrrol-2-yl)ethanone (2). Compound 2 was synthesized according to previously reported procedures. Mp = 127-129 °C. TLC (1:1 EtOAc/Hexane) Rf = 0.56. 1H NMR (CDCl3): δ 8.56 (d, J = 1.7 Hz, 1H), 7.78 (d, J = 1.7 Hz, 1H), 3.98 (s, 3H). 13C NMR: δ 173.30, 134.73, 133.09, 121.09, 116.82, 95.02, 79.44. IR: 3367.4, 3145.4, 3133.6, 2963.3, 1693.8, 1531.1, 1515.6, 1494.5, 1423.2, 1406.0, 1314.2, 1183.0, 1112.6, 859.9, 813.7, 800.3, 750.1, 715.8, 684.6. HRMS (ESI) m/z for C7H5Cl3N2O3 [M+H]+ calcd 270.9439, found: 270.9439.

Ethyl 3-(1-methyl-4-nitro-1H-pyrrol-2-carboxamido)propanoate (3). Compound 2 (30.00 g, 0.11 mol) was taken in a 500mL round bottom flask followed by the addition of β-alanine ethyl


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ester hydrochloride (17.05 g, 0.11 mol) and EtOAc (100 mL), which had been dried over molecular sieves. TEA (35.5 mL, 0.25 mol) was dissolved in EtOAc (50 mL), dried over sieves, and this mixture was added dropwise to the reaction mixture, with stirring over a period of 15 hours under a nitrogen atmosphere. The reaction was then stirred for an additional 48 hours during which a white precipitate was formed. The precipitate was removed by filtration and the filtrate was washed with 1M HCl (2 x 100 mL) and then DI water (1 x 100 mL). The organic layer was then dried over anhydrous magnesium sulfate, filtered and the solvent was removed by rotary evaporation to give 3 as a yellow solid (25.00g, yield 89%): Mp 120-124 °C. TLC (1:1 EtOAc/hexane) Rf = 0.42. 1H NMR (CDCl3): δ 7.47 (d, J = 1.2 Hz, 1 H), 6.99 (d, J = 1.6 Hz, 1 H), 6.62 (s, 1 H), 4.12 (q, J = 7.2 Hz, 2 H), 3.91 (s, 3 H), 3.58 (q, J = 6 Hz, 2 H), 2.54 (t, J = 7.2 Hz, 2 H), 1.22 (t, J = 7.2 Hz, 3 H). 13

C NMR δ 171.63, 160.34, 134.23, 128.32, 126.71, 107.86, 60.42, 37.78, 35.41, 34.11, 14.51. IR:

3360.6, 3129.1, 1711.7, 1655.6, 1547.5, 1423.7, 1355.0, 1310.1, 1197.2, 1136.4, 1111.5, 1086.4, 1020.9, 986.5, 885.1, 848.4, 810.1, 821.0, 748.7, 700.1, 603.3, 595.5, 583.0. HRMS (ESI) m/z for C11H15N3O5 [M+H]+ calcd: 270.1084, found: 270.1084.

Ethyl

3-(1-methyl-4-(1-methyl-4-nitro-1H-pyrrol-2-carboxamido)-1H-pyrrole-2-

carboxamido propanoate (4). Compound 3 (13.0 g, 48.3 mmol) was added to 95% EtOH (50 mL) in a 500 mL Parr jar. Wet palladium on carbon (10%, 2.50 g) was added to this mixture and was shaken on a hydrogenation apparatus under pressurized hydrogen (70 psi). Once TLC (EtOAc)


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indicated that no starting material was present, the reaction mixture was filtered through celite. The celite was washed with ethanol (2 x 50 mL) and the solvent was removed by rotary evaporation. The resulting pale yellow solid was placed under vacuum for 24 hours. Then, this solid, and compound 2 (14.50 g, 53.0 mmol) were dissolved in EtOAc (250 mL, dried over sieves) in a 500 mL round bottom flask. The mixture was then allowed to stir for 48 hours during which time a yellow precipitate formed. After the reaction was complete by TLC (EtOAc), the precipitate was filtered and placed under vacuum to yield pure 4 as a yellow solid (28.20 g, yield 94%). Mp = 199-202 °C. TLC (EtOAc) Rf = 0.64. 1H NMR (DMSO-d6): δ 10.26 (s, 1H), 8.18 (d, J = 1.6 Hz, 1H), 8.06 (t, J = 5.6 Hz, 1H), 7.59 (d, J = 1.6 Hz, 1H), 7.23 (d, J = 1.6 Hz, 1H), 6.85 (d, J = 1.6 Hz, 1H), 4.10 (q, J = 7.2 Hz, 2H), 3.96 (s, 3H), 3.82 (s, 3H), 3.42 (q, J = 7.2 Hz, 5.6 Hz, 2H), 2.56 (t, J = 7.2 Hz, 2H), 1.21 (t, J = 7.2 Hz, 3H). 13C NMR: δ 171.84, 161.64, 157.31, 134.23, 128.68, 126.74, 123.42, 121.81, 118.57, 108.01, 104.58, 60.36, 37.93, 36.50, 35.28, 34.46, 14.56. IR: 3404.4, 3372.1, 3115.1, 2993.5, 2950.9, 1712.3, 1670.5, 1642.7, 1580.7, 1563.4, 1520.5, 1496.6, 1465.6, 1435.7, 1415.2, 1400.7, 1385.5, 1306.6, 1250.3, 1216.4, 1200.9, 1159.8, 1143.5, 1120.1, 1093.5, 1074.2, 1025.5, 986.5, 865.2, 815.3, 805.7, 770.7, 755.3, 667.8, 643.8, 609.2, 597.4 HRMS (ESI) m/z for C17H21N5O6 [M+H]+ calcd: 392.1565, found: 392.1565.


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Ethyl

4-(4-(4-acrylamido-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-pyrrole-2-

carboxamido)butanoate (5). 4 (2.51 g, 6.41 mmol) was added to a 500 mL Parr jar and dissolved in 45 mL of 95% EtOH. Wet palladium on activated carbon (10%, 0.480 g) was added to the jar. The jar was then pressurized under hydrogen (70 psi) and shaken. When TLC (EtOAc) indicated that 4 was no longer present, the solution was filtered through celite, which was washed with EtOH (2 × 50 mL). The solvent was removed by rotary evaporation and the resultant dark oil was placed under vacuum for 24 h. The dark oil was then dissolved in 20 mL of anhydrous THF, and stirred under a nitrogen atmosphere in a dry ice/acetone bath at -40 °C for 15 min. DIEA (2.8 mL, 16.9 mmol) was then added to the reaction mixture and stirred for 30 min at -40 °C, followed by the addition of acryloyl chloride (445 µL, 5.4 mmol). The flask covered with aluminum foil to protect it from light, and the reaction mixture was stirred under a nitrogen atmosphere for 24 h. Then, the solution was concentrated by rotary evaporation and the resulting oil was re-dissolved in 100 mL of EtOAc and washed with DI H2O (2 ×100 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered and the solvent was removed by rotary evaporation. The residue was protected from light by aluminum foil and placed under vacuum for 24 h to yield 3 as a brown solid (1.85 g, 91% yield). Mp = 87-90 °C. TLC (6:1 EtOAc/MeOH) Rf = 0.55. 1H NMR (DMSOd6): δ 10.10 (s, 1H), 9.89 (s, 1H), 8.03 (t, J = 5.6 Hz, 1H), 7.26 (d, J = 1.8 Hz, 1H), 7.18 (d, J = 1.8 Hz, 1H), 6.91 (d, J = 1.8 Hz, 1H), 6.85 (d, J = 1.8 Hz, 1H), 6.37 (dd, J = 17 Hz, J = 10.2 Hz, 1H), 6.20-6.15 (dd, J = 17 Hz, J = 2.1 Hz, 1H), 5.66 (dd, J = 10.2 Hz, 2.1 Hz, 1H), 4.04 (q, J = 7.2 Hz, 2H), 3.84 (s, 3H), 3.79 (s, 3H), 3.18 (q, J= 6 Hz, 5.6 Hz, 2H), 2.31 (t, J= 7.2 Hz, 2H), 1.73 (q, J= 7.2 Hz, 6 Hz, 2H), 1.17 (t, J = 7.2 Hz, 3H). 13C NMR: δ 173.18, 162.09, 161.75, 158.74, 131.95, 126.04, 123.47, 123.33, 122.46, 122.15, 118.89, 118.32, 104.63, 104.41, 60.22, 38.16, 36.65, 36.42, 31.50, 25.17, 14.57. IR: 3288.4, 2937.9, 1730.2, 1632.9, 1580.7, 1534.1, 1465.5,


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1436.8, 1405.2, 1384.2, 1351.1, 1266.1, 1233.7, 1205.7, 1062.6, 1027.3, 775.7. HRMS (ESI) m/z for C21H27N5O5 [M+H]+ calcd: 430.2085, found: 430.2087.

3-(4-(4-acrylamido-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-pyrrole-2carboxamido)propanoic acid (6). Compound 5 (0.100 g, 0.24 mmol) was dissolved in 4 mL reagent acetone in a 50 mL round bottom flask. A solution of NaOH (0.0387 g, 0.97 mmol) in 1 mL of DI H2O was added in one portion and the reaction was stirred and covered with aluminum foil to protect it from light. When TLC (5:2 EtOAc/MeOH) indicated that 5 was no longer present, the reaction mixture was concentrated to a volume of 1 mL by rotary evaporation, and then 2 mL of cold DI H2O was added to the flask, and the mixture was cooled in an ice bath for 15 min. The mixture was acidified to pH 1 with concentrated HCl, causing a yellow precipitate to form. After the flask was cooled in an ice bath for an additional 30 min, the precipitate was collected by vacuum filtration to yield 6 as a brown solid (0.050 g, 85% yield). Mp = 108-111 °C. TLC (1:1 EtOAc/MeOH) Rf = 0.48. 1H NMR (DMSO-d6): δ 12.09 (br s, 1H), 10.12 (s, 1H), 9.91 (s, 1H), 8.04 (t, J = 5.6 Hz, 1H), 7.26 (d, J = 1.6 Hz, 1H), 7.19 (d, J = 1.6 Hz, 1H), 6.91 (d, J = 1.6 Hz, 1H), 6.84 (d, J = 1.6 Hz, 1H), 6.38 (dd, J = 17 Hz, 10 Hz, 1H), 6.16 (dd, J = 17 Hz, 2 Hz, 1H), 5.65 (dd, J = 10 Hz, 2 Hz, 1H), 3.84 (s, 3H), 3.79 (s, 3H), 3.38 (m, 2H), 2.49 (m, 2H). 13C NMR: δ 173.48, 162.11, 161.72, 158.75, 131.98, 123.46, 123.16, 122.49, 122.15, 118.91, 118.42, 104.72,


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Biochemistry

104.45, 36.65, 36.44, 35.30, 34.49, 21.54. IR: 3297.7, 1717.7, 1635.6, 1581.9, 1539.1, 1465.1, 1437.0, 1403.8, 1268.5, 1205.1, 1101.8, 1064.4, 775.1, 667.9. HRMS (ESI) m/z for C18H21N5O5 [M+H]+ calcd: 388.1615, found: 388.1616.

(2S,3S,4S,5R,6S)-2,4,5-tris(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3-amine hydrochloride (BzG). BzG was synthesized following previously published procedures.54 Mp 222 °C. TLC (1:1 EtOAc:MeOH) Rf = 0.15. 1H NMR (DMSO-d6) δ 8.48 (s, 3H), 7.47-7.15 (m, 20H), 4.88-4.51 (m, 9H), 3.89 (m, 1H), 3.72-3.63 (m, 4H), 3.06 (t, J = 9.2 Hz, 1H). 13C NMR: δ 138.58, 138.48, 138.48, 137.38, 128.78, 128.77, 128.56, 128.31, 128.23, 128.17, 128.11, 128.01, 98.66, 79.78, 78.71, 74.78, 74.42, 74.16, 72.82, 71.07, 68.64, 55.03. IR: 3088.4, 3029.2, 2923.2, 2749.2, 2601.4, 2553.0, 2028.5, 1599.6, 1504.7, 1476.6, 1453.0, 1413.3, 1369.9, 1346.2, 1327.0, 1263.9, 1244.7, 1209.8, 1140.6, 1103.6, 1067.8, 1027.7, 1008.7, 939.1, 930.0, 912.9, 732.7, 693.8, 660.5, 618.1, 562.5, 459.4. HRMS (ESI) m/z for C34H38NO5 [M+H]+ calcd: 540.2744, found: 540.2768


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4-acrylamido-1-methyl-N-(1-methyl-5-((2-oxo-2-(((2R,3R,4R,5S,6R)-2,4,5-tris(benzyloxy)6-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)amino)propyl)carbamoyl)-1H-pyrrol-3yl)-1H-pyrrole-2-carboxamide (7). 6 (0.126 g, 0.32 mmol), EDCI (0.127 g, 0.66 mmol), DMAP (0.103 g, 0.84 mmol), and HOBt (0.132 g, 0.98 mmol) were combined in an oven-dried round bottom flask flushed with nitrogen and dissolved in anhydrous DMF (3 mL). The mixture was stirred under a nitrogen atmosphere for 30 min until all solids dissolved. BzG (0.193 g, 0.33 mmol) was then added and the reaction was stirred under a nitrogen atmosphere for 24 h, after which 50 mL of cold 1M HCl was added causing a white precipitate to form. The solution was stirred for an additional 2 h, and the precipitate was collected by vacuum filtration. The precipitate was dissolved in a minimal volume of DMF, and then 50 mL of cold 5% NaHCO3 was added, causing a white precipitate to form. The mixture was stirred for 30 min, and the precipitate was collected by vacuum filtration. The precipitate was again dissolved in a minimal volume of DMF, 50 mL of cold 1M HCl was added causing a white precipitate to form. The mixture was stirred for 30 min, and the precipitate was collected by vacuum filtration and rinsed with cold DI H2O to yield 7 as a pale white solid (0.240 g, 82% yield). Mp 120-124 °C TLC (5:2 EtOAc/MeOH) Rf = 0.76. 1H NMR (DMSO-d6): δ 10.13 (s, 1H), 9.93 (s, 1H), 8.16 (d, J = 9.2 Hz, 1H), 8.05 (t, J = 5.8 Hz, 1H), 7.37-7.19 (m, 23H), 6.92 (d, J = 2 Hz, 1H), 6.85 (d, J = 1.6 Hz, 1H), 6.38 (dd, J = 17 Hz, J = 10.2 Hz, 1H), 6.19 (dd, J = 17 Hz, J = 2.2 Hz, 1H), 5.67 (dd, J = 10.2 Hz, J = 2.2 Hz, 1H), 4.79-4.50 (m, 8H), 3.85 (s, 3H), 3.80 (s, 3H), 3.74-3.66 (m, 5H), 3.50 (d, J = 5.2 Hz, 2H), 3.39 (m, 2H), 2.39 (m, 2H). 13C NMR: δ 170.87, 162.09, 161.70, , 158.74, 138.97, 138.77, 138.67, 138.35, 131.95, 128.75, 128.71, 128.67, 128.62,128.25, 128.07, 127.94, 127.89, 127.75, 126.06, 123.48, 123.19, 122.54, 122.15, 118.89, 118.47, 104.55, 104.42, 100.93, 82.69, 78.42, 74.64, 74.44, 72.80, 70.28, 69.24, 54.99, 36.65, 36.45, 36.34, 35.83. IR: 3277.9, 3088.1, 3062.0, 3028.5, 2919.9, 2869.9,


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Biochemistry

1646.8, 1533.1, 1496.8, 1452.8, 1436.7, 1405.3, 1360.8, 1264.1, 1206.2, 1150.5, 1094.3, 1057.2, 1027.2, 735.4, 695.7. HRMS (ESI) m/z for C52H56N6O9 [M+Na]+ calcd: 931.4001, found: 931.4001.

3-((1-methyl-5-((1-methyl-5-((2-oxo-2-(((2R,3R,4R,5S,6R)-2,4,5-tris(benzyloxy)-6((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)amino)propyl)carbamoyl)-1H-pyrrol-3yl)carbamoyl)-1H-pyrrol-3-yl)amino)-3-oxopropane-1-sulfonic acid (8). Compound 7 (0.200 g, .022 mmol) was taken in a 250 mL round bottom flask and dissolved in 12 mL of 95% EtOH. NaHSO3 (0.138 g, 1.3 mmol) dissolved in 5 mL of DI H2O was added to the reaction mixture. The pH was adjusted to 8 with 5% NaOH and the reaction mixture refluxed until TLC (5:2 EtOAc/MeOH) indicated that 7 was no longer present. The reaction mixture was then concentrated to 1 mL by rotary evaporation and 2 mL of cold DI H2O was added. After cooling in an ice bath for 15 min., the mixture was acidified to pH 1 with concentrated HCl, causing a yellow precipitate to form. The flask was cooled in an ice bath for an additional 30 min, after which the precipitate was collected by vacuum filtration to yield 8 as a pale yellow solid (0.199 g, 92% yield). Mp = 142-146 °C. TLC (5:2 EtOAc/MeOH) Rf = 0.12. 1H NMR (DMSO-d6): δ 9.98 (s, 1H), 9.88 (s, 1H), 8.16 (d, J = 9.2 Hz, 1H), 8.04 (t, J = 5.2 Hz, 1H), 7.37-7.15 (m, 23H), 6.85 (dd, J = 3 Hz, J = 1.8


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Hz, 2H), 4.72-4.50 (m, 8H), 3.81 (s, 3H), 3.80 (s, 3H), 3.74-3.62 (m, 5H), 3.50 (m, 2H), 3.38 (m, 2H), 2.69 (m, 2H), 2.55 (m, 2H), 2.38 (q, J = 5.4 Hz, 2H). 13C NMR: δ 170.85, 168.90, 161.72, 158.82, 138.96, 138.77, 138.67, 138.34, 128.75, 128.71, 128.68, 128.62, 128.25, 128.06, 127.94, 127.90, 127.75, 123.15, 123.10, 122.61, 122.53, 118.57, 118.41, 104.35, 100.93, 82.68, 78.43, 74.64, 74.41, 72.80, 70.29, 69.25, 54.99, 48.03, 36.53, 36.42, 35.83, 32.90. IR: 3278.9, 3062.7, 3029.4, 2920.9, 1647.0, 1536.4, 1497.0, 1453.2, 1437.7, 1404.8, 1361.7, 1260.6, 1207.3, 1154.1, 1057.0, 1028.0, 736.7, 696.8. HRMS (ESI) m/z for C52H58N6O12S [M+H]+ calcd: 991.3906, found: 991.3902.

Methyl

3-((1-methyl-5-((1-methyl-5-((2-oxo-2-(((2R,3R,4R,5S,6R)-2,4,5-tris(benzyloxy)-6-

((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)amino)propyl)carbamoyl)-1H-pyrrol-3yl)carbamoyl)-1H-pyrrol-3-yl)amino)-3-oxopropane-1-sulfonate (9). Compound 8 (0.101 g, 0.10 mmol) was placed into an oven dried round bottom flask with a reflux condenser and the system was cooled to room temperature under a dry nitrogen atmosphere. Anhydrous THF (6 mL) was added and the mixture was refluxed with stirring until the solid dissolved. 3-Methyl-1-ptolyltriazene (0.076 g, 0.51 mmol) was added and the reaction stirred at reflux, while monitored by TLC (EtOAc). After 1 h, TLC indicated that further formation of the desired product ceased, and the reaction was cooled to room temperature. The solution was purified by flash column


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Biochemistry

chromatography (EtOAc). The desired fractions were collected and concentrated by rotary evaporation and washed with dry diethyl ether (dried over sieves). The diethyl ether was pipetted off and the remaining solid was put under vacuum to yield 9 as a white solid (0.048 g, 47% yield). Due to the reactive nature of this compound, it was utilized immediately for the next reaction after confirming its identity using a small sample for 1H NMR. TLC (5:2 EtOAc/MeOH) Rf = 0.71. 1H NMR (DMSO-d6): δ 10.07 (s, 1H), 9.89 (s, 1H), 8.15 (t, J = 5.8 Hz, 1H), 8.04 (d, J = 8.8 Hz, 1H), 7.36-7.18 (m, 23H), 7.17 (d, J = 1.6 Hz, 1H), 6.85 (d, J = 1.6 Hz, 1H), 4.80-4.51 (m, 8H), 3.86 (s, 3H), 3.84 (s, 3H), 3.78 (s, 3H), 3.79-3.67 (m, 5H), 3.62 (t, J = 7.2 Hz, 2H), 3.48 (d, J = 3.6 Hz, 2H), 2.74 (t, J = 7.2 Hz, 2H), 2.67 (m, 2H).

Methyl

3-((1-methyl-5-((1-methyl-5-((3-oxo-3-((2,4,5-trihydroxy-6-

(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)propyl)carbamoyl)-1H-pyrrol-3yl)carbamoyl)-1H-pyrrol-3-yl)amino)-3-oxopropane-1-sulfonate (1a). 9 (0.048 g, 0.05 mmol) was added to an oven dried flask flushed with nitrogen and then dissolved in 6 mL of anhydrous THF. Wet palladium on carbon (10%, 0.090 g) was then added to the flask. The flask was then flushed with hydrogen gas and kept at a constant hydrogen atmosphere with vigorous stirring. TLC (5:2 EtOAc/MeOH) was taken every 20 minutes along with a new addition of palladium (0.015 g each). After 3 hours, TLC indicated that all the starting material had disappeared and two spots


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(anomers) with very low Rf values remained. The solution was immediately purified using flash column chromatography (85:15 DCM/MeOH), yielding 1a as a pure yellowish/white solid (0.009 g, 29% yield). TLC (5:2 EtOAc/MeOH) Top Spot Rf = 0.17, Bottom Spot Rf = 0.07. 1H NMR (DMSO-d6): δ 10.06 (s, 1H), 9.87 (s, 1H), 7.97 (t, J = 3.8 Hz, 1H), 7.80 (d, J = 6 Hz, 0.3H), 7.73 (d, J = 5.6 Hz, 1H), 7.18 (d, J = 7.6 Hz, 2H), 6.84 (m, 2H), 6.52 (d, J = 4 Hz, 0.3H), 6.42 (d, J = 10.8 Hz, 1H), 4.94 (d, J = 2.8 Hz, 1H), 4.90 (d, J = 3.6 Hz, 1H), 4.63 (d, J = 3.6 Hz, 1H), 4.42 (m, 1H), 3.87 (s, 3H), 3.83 (s, 3H), 3.81 (m, 2H), 3.80 (s, 3H), 3.61 (m, 4H), 3.50 (m, 3H), 3.52 (m, 1H), 2.74 (t, J = 4.8 Hz, 2H), 2.38 (t, J = 5 Hz, 2H). 13C NMR: δ 171.70, 167.03, 162.11, 159.25, 123.78, 123.76, 122.96, 122.63, 119.13, 118.90, 118.87, 105.05, 104.80, 96.31, 91.52, 77.76, 75.25, 73.01, 72.06, 71.70, 71.41, 62.12, 62.06, 58.08, 55.21, 37.08, 36.90, 36.84, 36.38, 36.32, 36.27, 29.15, 26.43. HRMS (ESI) m/z for C25H36N6O12S [M+Na]+ calcd: 667.2009, found: 667.2020.

Tert-butyl 3-(1-methyl-4-nitro-1H-pyrrole-2-carboxamido)propanoate (10). The nitro pyrrole 2 (30.00 g, 0.11 mol) was taken in a 500 mL round bottom 1 neck flask followed by addition of βalanine tert-butyl ester hydrochloride (17.05, 0.11 mol) and EtOAc (100 mL) which had been dried over sieves. TEA (35.5 mL, 0.25 mol) was dissolved in EtOAc (50 mL) dried over sieves, and this mixture was added dropwise to the reaction mixture, with stirring over a period of 15 hours under a nitrogen atmosphere. The reaction was then stirred for an additional 48 hours during which


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Biochemistry

a white precipitate was formed. The precipitate was removed by filtration and the filtrate was washed with 1M HCl (2 x 100 mL) and DI water (1 x 100 mL). The organic layer was then dried over MgSO4, filtered and the solution concentrated by rotary evaporation to give 10 as yellow solid (0.197 g, 90% yield). Mp = 103-105 °C. TLC (EtOAc) Rf = 0.66. 1H NMR (DMSO-d6):  7.53 (d, J = 1.6 Hz, 1H), 7.05 (d, J = 1.6 Hz, 1H), 6.83 (t, J = 5.6 Hz, 1H), 3.95 (s, 3H), 3.56 (t, J = 6.4 Hz, 2H), 2.50 (t, J = 6.4 Hz, 2H), 1.42 (s, 9H). 13C NMR data:  171.29, 159.67, 134.28, 126.14, 125.80, 106.39, 80.83, 37.27, 34.50, 34.33, 27.50. IR: 3408.5, 3347.2, 3147.8, 3135.2, 3123.5, 3116.2, 3000.8, 2978.1, 2945.8, 1722.5, 1647.9, 1547.8, 1523.7, 1497.2, 1417.3, 1368.3, 1311.1, 1267.7, 1221.3, 1151.5, 1111.7, 986.4, 901.5, 873.8, 856.5, 842.6, 832.9, 821.5, 750.6, 596.3, 557.1. HRMS (ESI) m/z for C13H19N3O5 [M+Na]+ calcd: 320.1217, found: 320.1216.

Tert-butyl-3-(1-methyl-4-(1-methyl-4-nitro-1H-pyrrol-2-carboxamido)-1H-pyrrole-2carboxamido propanoate (11). Compound 10 (13.0 g, 48.3 mmol) was dissolved in 95% EtOH (50 mL) in a 500 mL Parr jar and wet palladium on carbon (10%, 1.0 g) was added to this mixture. The jar was pressurized with hydrogen gas (70 psi) and then shaken for two hours until reaction was complete by TLC (EtOAc). The solution was filtered through Celite and washed with EtOH (2 x 50 mL). The solvent was removed by rotary evaporation and the resulting pale yellow solid was kept under vacuum overnight. In a 500 mL round bottom flask, this pale yellow


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solid and compound 2 (30.00 g, 0.11 mol) were dissolved in EtOAc (250 mL dried over sieves). The mixture was then allowed to stir for 48 hours during which time a yellow precipitate formed. After the reaction was complete by TLC (EtOAc), the precipitate was filtered and dried under vacuum to yield pure 11 as a yellow solid (28.20 g, yield 94%). Mp = 149-150 °C. TLC (EtOAc) Rf = 0.63. 1H NMR (DMSO-d6): δ 10.27 (s, 1H), 8.18 (d, J = 1.6 Hz, 1H), 8.09 (t, J = 5.6 Hz, 1H), 7.59 (d, J = 1.6 Hz, 1H), 7.24 (d, J = 1.6 Hz, 1H), 6.84 (d, J = 1.6 Hz, 1H), 3.97 (s, 3H), 3.82 (s, 3H), 3.39 (t, J = 6.4 Hz, 2H), 2.36 (t, J = 6.4 Hz, 2H), 1.41 (s, 9H). 13C NMR: δ 170.59, 161.01, 156.73, 133.66, 128.07, 126.16, 122.89, 121.26, 117.97, 107.45, 103.87, 79.66, 37.35, 35.92, 35.10, 34.80, 27.61. IR: 3390.5, 3316.7, 3135.2, 1717.4, 1659.3, 1624.3, 1567.0, 1535.9, 1494.9, 1465.0, 1437.3, 1419.3, 1405.6, 1384.7, 1367.2, 1304.2, 1273.6, 1251.8, 1206.1, 1155.4, 1111.0, 811.8, 781.1, 752.6. HRMS (ESI) m/z for C19H25N5O6 [M+Na]+ calcd: 442.1697, found: 442.1694.

3-(methylsulfonyl)propanoic acid. 3-methylthiopropionic acid (14.30 g, 119 mmol) was dissolved in a 1:1 solution of acetic anhydride and acetic acid (154 mL) in a 500 mL flask and the mixture was cooled in an ice bath and stirred for 30 minutes. H2O2 (50%, 36 mL) was added 1 mL at a time in 5 mL installments every 15 minutes. Solution was allowed to stir overnight and warm to room temperature. Upon completion of the reaction as indicated by NMR, a trace of MnO2 was added to quench excess H2O2, and the mixture stirred for an additional two hours. The solution was filtered through celite, the organic solvents removed with rotary evaporation and the residue was placed under vacuum for 24 hours to yield the desired product as a white solid


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Biochemistry

(18.05 g, 99% yield). Mp 100-102 °C. 1H NMR (CDCl3): δ 12.59 (br s, 1H), 3.33 (t, J = 7.2 Hz, 2H), 2.99 (s, 3H), 2.68 (t, J = 7.2 Hz, 2H). 13C NMR: δ 172.72, 50.35, 41.15, 28.03. IR: 3026.5, 2689.6, 1696.2, 1415.9, 1295.1, 1210.1, 1160.4, 1125.1, 1058.8, 974.6, 957.7, 920.4, 809.7, 789.5, 768.6, 513.3, 466.8. HRMS (ESI) m/z for C4H8O4S [M+Na]+ calcd: 175.0036, found: 175.0035.

Tert-butyl 3-(1-methyl-4-(1-methyl-4-(3-(methylsulfonyl)propanamido)-1H-pyrrole-2carboxamido)-1H-pyrrole-2-carboxamido)propanoate (12). Compound 11 (3.50 g, 8.6 mmol) was dissolved in 95% EtOH (50 mL) in a 500 mL Parr jar. Wet palladium on carbon (10%, 0.50 g) was added to this mixture. The jar was pressurized under hydrogen gas (70 psi) and shaken for two hours until reaction was complete as indicated by TLC (EtOAc). The reaction mixture was filtered through celite, which was then washed with EtOH (2 x 50 mL). The solvent was removed by rotary evaporation and the resulting black solid was kept under vacuum overnight. This solid (3.1 g, 7.5 mmol), 3-(methylsulfonyl)propanoic acid (2.8 g, 18.0 mmol), EDCI (3.0 g, 18.00 mmol), DMAP (1.70 g, 11.26 mmol), and HOBt (2.90 g, 21.46 mmol) were combined in a 500 mL round bottom flask flushed with nitrogen gas. The solids were then dissolved in anhydrous DMF (10 mL). The mixture was then allowed to stir for 3 days. After the reaction was complete as indicated by TLC (EtOAc), the solution was diluted with 250 mL of DCM and washed, in sequence, with 5% NaHCO3 (2 x 250 mL), DI H2O (1 x 250 mL), 1M HCl (2 x 250


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mL) and DI H2O (1 x 250 mL). The organic layer was dried over anhydrous magnesium sulfate, the solvent was removed by rotary evaporation, and the residue was placed under vacuum for 24 h to yield 12 as a brown solid. (2.25 g, yield 60%): Mp = 175-176 °C. TLC (6:1 DCM:MeOH) Rf = 0.57. 1H NMR (DMSO-d6):  10.09 (s, 1H), 9.91 (s, 1H), 8.04 (t, J = 5.6 Hz, 1H), 7.21 (d, J = 1.6 Hz, 1H), 7.19 (d, J = 1.6 Hz, 1H), 6.89 (d, J = 1.6 Hz, 1H), 6.84 (d, J = 1.6 Hz, 1H), 3.84 (s, 3H), 3.81 (s, 3H), 3.40 (m, 4H), 3.03 (s, 3H), 2.76 (t, J = 7.6 Hz, 2H), 2.44 (t, J = 7.6 Hz, 2H), 1.41(s, 9H). 13C NMR:  171.20, 166.56, 161.70, 158.77, 123.29, 123.18, 122.50, 122.15, 118.65, 118.43, 104.62, 104.32, 80.25, 50.21, 40.86, 36.61, 36.43, 35.70, 35.36, 28.68, 28.20. IR: 3343.0, 3128.0, 2980.2, 2939.8, 1725.7, 1655.2, 1576.8, 1529.7, 1466.7, 1437.8, 1406.6, 1367.5, 1342.7, 1322.9, 1259.3, 1213.3, 1132.2, 1097.0, 1061.9, 1005.7, 958.7, 843.8, 801.2, 773.7, 661.5, 602.3, 500.7. HRMS (ESI) m/z for C23H33N5O7S [M+Na]+ calcd: 546.1993, found: 546.1998.

3-(1-methyl-4-(1-methyl-4-(3-(methylsulfonyl)propanamido)-1H-pyrrole-2-carboxamido)1H-pyrrole-2-carboxamido)propanoic acid (13). Compound 12 (0.300 g, 0.5 mmol) was dissolved in formic acid (97%, 3 mL) and allowed to stir while under a nitrogen atmosphere for 24 h until reaction was complete as indicated by TLC (9:1 DCM/MeOH). The solution was diluted with DCM (200 mL), the solvent was removed by rotary evaporation and the residue was placed under vacuum for 24 h to yield pure 13 as a white solid (0.250 g, yield 100%): Mp = 209-


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Biochemistry

211 °C. TLC (3:2 EtOAc/MeOH) Rf = 0.27. 1H NMR (DMSO-d6):  12.06 (s, 1H), 10.07 (s, 1H), 9.88 (s, 1H), 8.04 (t, J = 5.6 Hz, 1H), 7.17 (q, J = 1.6 Hz, 2H), 6.86 (q, J = 1.6 Hz, 2H), 3.83 (s, 3H), 3.79 (s, 3H), 3.39 (m, 4H), 3.01 (s, 3H), 2.73 (t, J = 7.6 Hz, 2H), 2.47 (m, 2H). 13C NMR:  173.54, 166.54, 161.71, 158.74, 123.28, 123.17, 122.48, 122.14, 118.64, 118.40, 104.70, 104.29, 50.21, 40.88, 36.62, 36.44, 35.33, 34.54, 28.66. IR: 3354.1, 3135.2, 2942.1, 1730.9, 1650.2, 1580.6, 1536.6, 1467.9, 1438.2, 1397.1, 1359.9, 1262.0, 1240.6, 1181.6, 1132.8, 959.1, 798.0, 766.1, 668.5, 606.5, 503.3. HRMS (ESI) m/z for C19H25N5O7S [M+H]+ calcd: 468.1547, found: 468.1547.

(2S,3R,4R,5R,6R)-2,4,5-tris((trimethylsilyl)oxy)-6-(((trimethylsilyl)oxy)methyl)tetrahydro2H-pyran-3-amine (TMSG). This compound was synthesized according to previously reported procedures55 by reacting glucosamine hydrochloride (2.50 g, 11.59 mmol) with chlorotrimethylsilane (8.25 mL, 65.0 mmol) and hexamethyldisilazane (17.5 mL, 82.4 mmol) in pyridine (52 mL). TMSG was obtained as a white solid (5.39 g, 99% yield). 1H NMR (CDCl3): δ 5.12 (d, J = 3.2 Hz, 1H), 3.73-3.61 (m, 2H), 3.56-3.44 (m, 3H), 2.54 (dd, J = 9.2 Hz, J = 3.2 Hz, 1H), 1.12 (s, 2H), 0.204 (s, 9H), 0.176 (s, 9H), 0.159 (s, 9H), 0.096 (s, 9H).


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Biochemistry

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1-methyl-4-(1-methyl-4-(3-(methylsulfonyl)propanamido)-1H-pyrrole-2-carboxamido)-N(3-oxo-3-(((2R,3R,4R,5S,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3yl)amino)propyl)-1H-pyrrole-2-carboxamide (1b). Compound 13 (0.428 g, 0.914 mmol), EDCI (0.523 g, 2.74 mmol), HOBt (0.494 g, 3.66 mmol), and TMSG (0.872 g, 1.87 mmol) were combined in a 100 mL flask and dissolved in anhydrous DMF (3 mL). The mixture was stirred under a nitrogen atmosphere for 24 h at which time 75 mL of cold isopropanol was added, resulting in a white precipitate. The mixture was then further stirred for 2 h, after which the precipitate was collected by vacuum filtration to yield 1b as a white solid (0.484 g, 84% yield). No further deprotection step was necessary as the trimethyl silyl protecting groups were not present in the reaction product. 1H NMR (DMSO-d6):  10.08 (s, 1H), 9.89 (s, 1H), 7.99 (t, J = 5.6 Hz, 1H), 7.79(m, 0.15H), 7.75 (d, J = 8 Hz, 0.85H), 7.19 (dd, J = 8.8 Hz, J = 1.6 Hz, 2H), 6.86 (d, J = 1.6 Hz, 1H), 6.82 (d, J = 1.2 Hz, 1H), 6.54 (d, J = 6.4 Hz, 0.15H), 6.44 (d, J = 4 Hz, 0.85H), 4.94 (m, 2H), 4.64 (d, J = 5.2 Hz, 1H), 4.44 (t, J = 5.8 Hz, 1H), 3.83 (s, 3H), 3.80 (s, 3H), 3.60 (m, 2H), 3.49 (m, 2H), 3.40 (m, 5H), 3.11 (m, 1H), 3.02 (s, 3H), 2.73 (m, 2H), 2.38 (m, 2H). 13C NMR: δ 171.2, 166.54, 158.75, 123.25, 122.47, 122.14, 118.65, 104.28, 91.03, 72.54, 71.57, 70.92, 61.57, 54.70, 50.19, 36.63, 36.44, 35.80, 28.65. IR: 3305.5, 2929.3, 1637.0, 1585.3, 1537.9, 1468.2, 1438.8, 1403.2, 1365.8, 1284.2, 1203.4, 1118.2, 1071.4, 1030.4. HRMS (ESI) m/z for C25H36N6O11S [M+Na]+ calcd: 651.2060, found: 651.2076.

3-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)propanoic acid (NBD CO2H). was synthesized according to previously reported procedures.55 In a 250 mL round bottom flask, β-alanine (3.125


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Biochemistry

g, 35.08 mmol) and NaHCO3 (2.946 g, 35.08 mmol) were dissolved in 15 mL DI H2O. After addition of 4-chloro-7-benzofurazan (1.00 g, 5.01 mmol) in methanol (60 mL) the mixture was stirred at 50 ºC until TLC (6:1 CHCl3/MeOH) indicated the disappearance of the starting material. The reaction mixture was then concentrated by rotary evaporation to a volume of about 10 mL. An additional 10 mL of cold DI H2O was added, and the solution was cooled in an ice bath for 15 min. The solution was then acidified to pH 1 with concentrated HCl, causing a brown precipitate to form. The flask was cooled in an ice bath for an additional 30 min, after which the precipitate was collected by vacuum filtration to yield NBD CO2H as a dark brown solid (1.084 g, 86% yield). TLC (6:1 CHCl3/MeOH) Rf = 0.12. 1H NMR ((DMSO-d6): δ 12.45 (s, 1H), 9.49 (s, 1H), 8.54 (d, J = 8.8 Hz, 1H), 6.46 (d, J = 8.8 Hz, 1H), 3.67 (s, 2H), 2.73 (d, J = 6.4 Hz, 2H). 13C NMR (DMSOd6): δ 172.87, 145.28, 144.76, 144.51, 138.30, 121.40, 99.79, 40.79, 40.52, 40.24, 39.96, 39.82, 39.68, 39.40, 39.13, 32.76. IR: 3379.4, 3080.6, 2929.8, 1700.3, 1577.0, 1525.9, 1490.2, 1217.5, 1127.7, 884.4, 515.5, 419.4. HRMS (ESI) m/z for C9H8N4O5 [M+Na]+ calcd: 275.0387, found: 275.0390.

Tert-butyl

3-(1-methyl-4-(1-methyl-4-(3-((7-nitrobenzo[c][1,2,5]oxadiazol-4-

yl)amino)propanamido)-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-


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carboxamido)propanoate (14). Compound 11 (0.767 g, 1.83 mmol) was taken in a 500 mL Parr jar and then dissolved in 95% EtOH (50 mL). Wet palladium on carbon (10%, 0.50 g) was added to this mixture. The jar was pressurized under hydrogen gas (70 psi) and shaken for two hours until reaction was complete, as indicated by TLC (EtOAc). The reaction mixture was filtered through celite, which was then washed with EtOH (2 x 50 mL). The filtrate and the washings were combined, and the solvent was removed by rotary evaporation, leaving behind a dark residue which was dried under vacuum overnight. This residue was then dissolved in anhydrous DMF (5 mL) along with EDCI (0.701 g, 3.66 mmol), DMAP (0.559 g, 4.57 mmol), HOBt (0.742 g, 5.49 mmol), and NBD CO2H (0.369 g, 1.46 mmol). The mixture was stirred for 24 h, and then the solution was diluted with 250 mL of DCM and washed, in sequence, with 5% NaHCO3 (2 x 250 mL), DI H2O (1 x 250 mL), 1M HCl (2 x 250 mL) and DI H2O (1 x 250 mL). The organic layer was dried over anhydrous magnesium sulfate, the solvent was removed by rotary evaporation, and the residue was placed under vacuum for 24 h to yield 14 as a brown solid (0.65 g, 57% yield). TLC (6:1 CHCl3:MeOH) Rf = 0.69. 1H NMR (DMSO-d6): δ 10.01 (s, 1H), 9.88 (s, 1H), 9.55 (s, 1H), 8.56 (d, J = 8.8 Hz, 1H), 8.02 (t, J = 5.6 Hz, 1H), 7.19 (q, J = 1.6 Hz, 2H), 6.87 (d, J = 1.6 Hz, 1H), 6.81 (d, J = 1.6 Hz, 1H), 6.49 (d, J = 9.2 Hz, 1H), 3.82 (s, 3H), 3.79 (s, 3H), 3.77 (m, 2H), 3.37 (m, 2H), 2.74 (t, J = 6.8 Hz, 2H), 2.44 (t, J = 7 Hz, 2H), 1.40 (s, 9H). IR: 3315.9, 1705.4, 1646.4, 1527.4, 1464.0, 1436.6, 1068.6, 904.2, 812.0, 737.5, 663.9, 508.0, 418.4. HRMS (ESI) m/z for C28H33N9O8 [M+Na]+ calcd: 646.2344, found: 646.2355


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Biochemistry

3-(1-methyl-4-(1-methyl-4-(3-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)propanamido)1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamido)propanoic acid (15). Compound 14 (1.09 g, 1.75 mmol) and formic acid (5 mL) were added to a 50 mL round bottom flask and stirred, resulting in the formation of an orange precipitate. When TLC (6:1 CHCl3/MeOH) indicated that 14 was no longer present, the residue was collected by vacuum filtration to yield 15 as a dark, orange solid (0.715 g, 72% yield). TLC (6:1 CHCl3/MeOH) Rf = 0.34. 1H NMR (DMSO-d6): δ 12.23 (s, 1H), 10.00 (s, 1H), 9.86 (s, 1H), 8.56 (d, J = 8.4 Hz, 1H), 8.03 (t, J = 5.6 Hz, 1H), 7.18 (d, J = 1.2 Hz, 2H), 6.85 (dd, J = 12.2 Hz, J = 1.4 Hz, 2H), 6.49 (d, J = 9.2 Hz, 1H), 3.82 (s, 3H), 3.79 (s, 3H), 3.76 (m, 2H), 3.37 (m, 2H), 2.74 (t, J = 6.8 Hz, 2H), 2.47 (m, 2H). 13C NMR (DMSOd6): δ 173.46, 167.47, 161.72, 158.77, 123.23, 123.16, 122.48, 122.25, 118.64, 118.43, 104.74, 104.31, 39.79, 39.51, 36.61, 36.43, 35.31, 34.50, 31.15. IR: 3253.8, 1620.7, 1575.3, 1527.3, 1463.5, 1433.9, 1399.6, 1289.7, 1251.9, 1178.8, 1123.2, 1045.0, 997.7. HRMS (ESI) m/z for C24H25N9O8 [M+Na]+ calcd: 590.1718, found: 590.1712


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1-methyl-4-(1-methyl-4-(3-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)propanamido)-1Hpyrrole-2-carboxamido)-N-(3-oxo-3-(((2R,3R,4R,5S,6R)-2,4,5-trihydroxy-6(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)propyl)-1H-pyrrole-2-carboxamide (1c). Compound 15 (0.712 g, 1.25 mmol), EDCI (0.722 g, 3.77 mmol), HOBt (0.678 g, 5.02 mmol), and compound TMSG (1.17 g, 2.51 mmol) were combined in a 250 mL round bottom flask and then dissolved in anhydrous DMF (4 mL). The reaction was covered with aluminum foil and the mixture stirred under a nitrogen atmosphere for 24 h. Cold isopropanol (100 mL) was added, resulting in a brown precipitate. The mixture was stirred for 30 min, after which the precipitate was collected by vacuum filtration. The precipitate was re-dissolved in a minimal amount of DMF and 100 mL of cold 1M HCl was added, resulting in a dark orange precipitate. The mixture was stirred for 30 min, after which the precipitate was collected by vacuum filtration. The precipitate was re-dissolved in a minimal amount of DMF and 100 mL of cold 5% NaHCO3 was added, resulting in a dark orange precipitate. The mixture was then further stirred for 30 min, after which the precipitate was collected by vacuum filtration. The precipitate was once again re-dissolved in a minimal volume of DMF and 100 mL of cold 1M HCl was added, resulting in a dark orange precipitate. The mixture was stirred for 2 h, after which the precipitate was collected by vacuum filtration to yield 1c as a dark orange solid (0.458 g, 50% yield). 1H NMR (DMSO-d6): δ 10.02 (s, 1H), 9.87 (s, 1H), 9.54 (s, 1H), 8.55 (m, 1H), 7.97 (d, J = 5.6 Hz, 1H), 7.81 (d, J = 8.8 Hz, 0.5H),


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Biochemistry

7.75 (d, J = 8.4 Hz, 0.8H), 7.19 (s, 2H), 6.86 (s, 1H), 6.81 (s, 1H), 6.50 (d, J = 8.8 Hz, 1H), 4.94 (d, J = 2.8 Hz, 1H), 4.46 (d, J = 8.0 Hz, 1H), 3.82 (s, 3H), 3.80 (s, 3H), 3.13 (m, 2H), 2.74 (m, 2H), 2.37 (m, 2H). 13C NMR: δ 171.24, 167.46, 161.63, 158.76, 138.38, 123.18, 122.47, 122.25, 121.32, 118.66, 118.41, 104.55, 104.28, 99.85, 95.83, 91.04, 72.54, 71.55, 70.90, 61.56, 57.57, 54.73, 36.63, 36.44, 35.80, 34.37, 2.49. IR: 3256.7, 1635.4, 1577.2, 1528.5, 1434.8, 1402.51, 1293.0, 1256.8, 1186.0, 1149.7, 1122.9, 1008.4, 949.9, 594.7. HRMS (ESI) m/z for C30H36N10O12 [M+Na]+ calcd: 751.2406, found: 751.2429.

1-methyl-4-(1-methyl-4-nitro-1H-pyrrole-2-carboxamido)-N-propyl-1H-pyrrole-2carboxamide (16). Compound 16 was synthesized by previously reported procedures.37 Mp = 238-240 °C. TLC (EtOAc) Rf = 0.66. 1H NMR: δ 10.26 (s, 1H), 8.19 (d, J = 1.6 Hz, 1H), 8.07 (t, J = 6.4 Hz, 1H), 7.59 (d, J = 1.6 Hz, 1H), 7.21 (d, J = 1.6 Hz, 1H), 6.85 (d, J = 1.6 Hz, 1 H), 3.96 (s, 3H), 3.81 (s, 3H), 3.13 (q, J = 6.4 Hz, 5.6 Hz, 2H), 1.49 (m, 2H), 0.87 (t, J = 7.2 Hz, 3H). 13C NMR: δ 160.96, 156.70, 133.64, 128.10, 126.17, 123.22, 121.17, 117.73, 107.44, 103.78, 37.37, 35.89, 22.47, 11.32. HRMS (ESI) m/z for C15H19N5O4 [M+Na]+ calcd: 356.1329 , found: 356.1330.


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1-methyl-4-(1-methyl-4-(3-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)propanamido)-1Hpyrrole-2-carboxamido)-N-propyl-1H-pyrrole-2-carboxamide ().1-methyl-4-(1-methyl-4-(3((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)propanamido)-1H-pyrrole-2-carboxamido)-Npropyl-1H-pyrrole-2-carboxamide (NBD-lex). Compound 16 (0.114 g, 0.341 mmol) was dissolved in 30 mL of 95% EtOH in a 500 mL Parr jar and wet palladium on carbon (10%, 0.072 g) was added to the jar. The jar was pressurized with hydrogen gas (70 psi) and shaken. When TLC (EtOAc) indicated that 16 was no longer present, the solution was filtered through celite, which was then washed with EtOH (3 x 50 mL). The solvent was removed by rotary evaporation and the resultant dark oil was placed under vacuum for 24 h. The oil was dissolved in anhydrous DMF (1.25 mL) and then EDCI (0.132 g, 0.689 mmol), HOBt (0.164 g, 1.07 mmol), and DMAP (0.108 g, 0.882 mmol) were added. The mixture was stirred under a nitrogen atmosphere for 15 minutes until all solids were dissolved at which time NBD CO2H (0.086 g, 0.342 mmol) was added. The mixture was stirred for 24 h, diluted with 75 mL of DCM and washed with 10% NaHCO3 (2 x 25 mL), 1 M HCl (2 x 25 mL), and then with DI H2O (3 x 25 mL). The organic layer was dried over anhydrous magnesium sulfate and filtered. The solvent of the filtrate was removed by rotary evaporation and the residue placed under vacuum for 24 h. The resultant black solid was purified by flash column chromatography (95:5 CHCl3/MeOH) to yield NBD-lex as a red solid (0.115 g, 65% yield). Mp = 207 – 208 °C. TLC (95:5 CHCl3/MeOH) Rf = 0.39. 1H NMR (DMSO-d6) δ 10.00 (s, 1H), 9.85 (s, 1H), 9.54 (s, 1H), 8.54 (d, J = 8.9 Hz, 1H), 8.00 (t, J =


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Biochemistry

5.8 Hz, 1H), 7.16 (dd, J = 4.6, 1.8 Hz, 2H), 6.87 – 6.79 (m, 2H), 6.48 (d, J = 9.0 Hz, 1H), 3.79 (d, J = 14.4 Hz, 7H), 3.10 (q, J = 6.7 Hz, 2H), 2.73 (t, J = 6.8 Hz, 2H), 1.47 (h, J = 7.3 Hz, 2H), 1.22 (s, 0H), 0.85 (t, J = 7.4 Hz, 4H), -0.07 (s, 0H). 13C NMR (DMSO-d6): δ 167.43, 161.65, 158.75, 123.53, 123.25, 122.40, 122.24, 118.61, 118.19, 104.54, 104.25, 36.62, 36.38, 23.05, 11.91. IR: 3269.3, 1715.7, 1528.7, 1461.0, 1375.8, 1350.5, 1044.9, 902.6, 777.8, 656.0, 504.2. HRMS (ESI) m/z for C24H27N9NaO6 [M+Na]+ calcd: 560.1977, found: 560.1955.

7-nitro-N-propylbenzo[c][1,2,5]oxadiazol-4-amine (NBD-propyl).4-Chloro-7-benzofurazan (0.287 g, 1.44 mmol) was taken in a 25 mL round bottom flask equipped with a dropping funnel, dissolved in 5 mL of MeOH and stirred in an ice bath for 30 min. The flask was covered with aluminum foil to protect the contents from light. Propylamine (0.145 mL, 2.45 mmol) and 5 mL of MeOH were combined into the dropping funnel. The mixture was added dropwise into the round bottom flask. The flask was then slowly brought to room temperature and stirred. When TLC (2:1 Hexane/EtOAc) showed that 4-chloro-7-benzofurazan was no longer present, the solvent was removed by rotary evaporation and then purified by flash column chromatography to give NBD-propyl a red solid (0.297 g, 93% yield). Mp = 189-190 °C. TLC (DCM) Rf = 0.23. 1H NMR (400 MHz, DMSO-d6) δ 9.59 (s, 1H), 8.50 (d, J = 9.0 Hz, 1H), 6.42 (d, J = 9.0 Hz, 1H), 3.43 (t, J = 7.2 Hz, 2H), 1.70 (h, J = 7.4 Hz, 2H) 0.96 (t, J = 7.4 Hz, 3H). 13C NMR (DMSO0d6): δ 145.64, 144.80, 144.56, 138.30, 120.90, 99.50, 45.47, 40.81, 21.53. IR: 3303.4, 2956.9,


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2928.2, 2871.5, 1541.5, 1499.5, 1466.0, 1369.7, 1036.6, 900.9, 888.8, 627.0, 523.9.HRMS (ESI) m/z for C9H10N4NaO3 [M+Na]+ calcd: 245.0645; found: 245.0652.

methyl

3-((5-((5-(ethylcarbamoyl)-1-methyl-1H-pyrrol-3-yl)carbamoyl)-1-methyl-1H-

pyrrol-3-yl)amino)-3-oxopropane-1-sulfonate (Melex). Melex was synthesized by previously reported procedures.37 Mp = 208-212 oC. TLC (2:1 EtOAc/MeOH) Rf = 0.52. 1H NMR (DMSOd6): δ 10.03 (s, 1H), 9.89 (s, 1H), 8.03 (br s, 1H), 7.18 (d, J = 1.6, 1H), 7.16 (d, J = 1.6 Hz, 1H), 6.89 (d, J = 1.6 Hz, 1H), 6.85 (d, J = 1.6 Hz), 3.82 (s, 3H), 3.79 (s, 3H), 3.12 (m, 2H), 2.74 (m, 2H), 2.58 (m, 2H), 1.48 (m, 2H), 0.86 (t, J = 7.2 Hz, 3H).

DNA Methylation Studies. The DNA-methylating compounds, 1a, Melex and MMS, were reacted with 1 mM genomic calf thymus DNA in 10 mM cacodylate buffer (pH 7.0) with 10% DMSO. Experiments with 100 µM 1a were conducted both in the presence and absence of 100 µM netropsin. When netropsin was used, DNA samples were incubated with netropsin for 30 min. prior to the addition of 1a. All reactions were conducted in triplicate. The compounds were reacted with DNA for 24 h at room temperature, after which the reaction mixture was subjected to a heat treatment (90 ºC for 20 min) in order to cleave the methylated adducts (7-MeG and 3-


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Biochemistry

MeA). Cold 0.1 N HCl was then added to the reaction causing DNA to precipitate out of solution. The supernatant, containing the methylated adducts was collected and analyzed using reverse phase HPLC with UV detection (270 nm). Analytical HPLC was conducted on a Sonoma C182 reverse-phase 5μ, 100 Å, 25 cm × 4.6 mm column. The mobile phase used was a 0.1 M NaOAc buffer with 4% methanol at a pH of 5. Reactions with STZ were performed in a separate experiment along with MMS, in duplicate, and adduct analysis by HPLC was conducted using a Synergi Fusion-RP (Phenomenex, Torrance, CA, USA) reverse phase 4 μm, 80 Å, 15 cm x 4.6 mm column. The mobile phase used in this case was a 50 mM ammonium formate buffer with 2% isopropanol at a pH of 5. Since 5 mM MMS was included in both experiments, the values of 3-MeA adducts formed by MMS were used to normalize the STZ adduct levels for comparison to the adducts formed by 1a and Melex. Cell Toxicity Studies. The human glioblastoma cell line U251 was purchased from ATCC (Manassas, VA, USA). U251 cells were maintained at 37 °C and 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were maintained in high glucose (4.5 g/L) conditions but assays were performed in low glucose (1.0 g/L) conditions. DMSO toxicity assay. The extent of glioma cell death mediated by DMSO alone was measured using an MTS assay. This assay assesses the metabolic activity of cells by measuring the bioreduction of the tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] to formazan. The MTS assay was performed using the Promega CellTiter 96® AQueous One Solution Cell Proliferation Assay kit (Madison, WI, USA). U251 glioma cells were seeded in a 96 well tissue culture plate at a density of 1 x 104 cells/well in triplicate. Twenty-four hours later, 50 µL of a


35

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two-fold dilution series of DMSO made in DMEM was added to the cells. In addition, one set of cells received 50 µL DMEM (0% DMSO) and served as the negative control. A second set of cells were lysed in 10% Triton X-100 detergent and served as the positive control for cell death at the end of the assay. Twenty-four hours post DMSO addition, the cells in the total cell death control wells were lysed with 5 µL lysis buffer (10% Triton X-100) for 5 minutes. The DMSO/media was then removed from all wells and the cells were washed with 100 µL serumand phenol red-free DMEM (Lonza, Walkersville, MD, USA). One hundred microliters of assay mixture containing five parts phenol red-free DMEM and one part MTS solution from the Promega kit was then added to each well. The plate was incubated for 60 minutes at 37 ºC and 5% CO2 and then read on a plate reader (ELx800 multiplate absorbance reader, Biotek, Winooski, VT, USA) at 490 nm. For each well, percent cell survival was calculated by determining the average absorbances of the mock treated wells and the total cell death wells and the following formula was applied:

abs ( well )  abs (total death)  100 . Three replicates for abs (mock )  abs (total death)

each treatment per cell line were performed and the overall averages for each cell line were calculated and graphed. Cell viability assay (STZ, 1a and 1b). To determine the effect of 1a, 1b, and STZ on glioma cell viability, an MTS cell viability assay was used. U251 glioma cells were seeded in a 96 well plate at a density of 10,000 cells/well in triplicate. Twenty-four hours later, 50 µL of 1% DMSO media (0 mM wells and total cell death wells) or 50 µL of compound dilutions made in 1% DMSO media was added to the cells. Twenty-four hours post compound addition, the cells in the total cell death control wells were lysed and the MTS assay was performed as described above.


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Fluorescence Uptake Studies. U251 Human glioblastoma cells (ATCC, Manassas, VA, USA)

were grown in DMEM (ATCC) supplemented with 10% fetal bovine serum (FBS, Invitrogen), 2 mM L-glutamine (Invitrogen), 100 µg/ml streptomycin, and 100 units/ml penicillin (Invitrogen) and maintained in a humidified incubator with 5% CO2 at 37 ºC. Upon reaching approximately 80% confluency, cells were plated at a density of 4,000 cells /100 μL onto poly-D-lysine-coated 96-well plates (Greiner) or into a 4-chamber, glass bottom, 2 mL cell culture dish (Greiner). Cells were harvested in 0.25% Trypsin-EDTA (Life Technologies) and suspended in fresh DMEM medium. Cell counts were acquired using a hematocytometer. Cells were then plated and incubated with 5% CO2 at 37 °C for at least 24 hours prior to use. Cellular Uptake. U251 cells were seeded onto 96-well plates in DMEM at a density of 4,000 cells/well and incubated with 5% CO2 at 37 °C. After 24 hours, the DMEM medium (4,500 mg/L glucose) was removed and replaced with either warm glucose-free DMEM, or fresh warm DMEM medium (4.5 g /L glucose) (ATCC). Cells in both high-glucose and glucose-free medium wells were then treated with 1µM dilutions of either NBD-propyl, 2-NBDG, NBD-lex, 1c, DMSO (vehicle control) or left untreated (maintained at 5% CO2, 37 °C). After a one hour

incubation period, the intracellular fluorescence in each treatment was assessed by rinsing the cells twice with HBSS warmed in a 37 °C water bath and imaging the cells using a 20x magnification objective on an Image Xpress Micro system equipped with an environmental control chamber warmed to 37 °C (Molecular Devices). Preset FITC filter settings were used to visualize the NBD fluorophore with 12 second exposure times. Transmitted light images were also collected to assess cell morphology. The integrated fluorescence intensity in each well above the background fluorescence was then calculated, in order to quantify the degree of fluorescence, and corresponding uptake, of the NBD-conjugates within the interior of the cells of


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each treatment. The mean intracellular fluorescence of each treatment was compared to quantify comparative uptake rates of each NBD compound. In order to determine whether any statistically significant differences were present among the treatments, ANOVAs were performed between treatments. Comparisons between the means were done using Tukey’s HSD. All statistics were performed using JMP 7.0 (SAS). Finally, maps of pixel intensity were generated in order to visualize the distribution and comparative concentrations of NBD within individual cells. Confocal Microscopy. U251 cells were harvested and seeded into 4-chambered, 2 mL glassbottom dishes. At the time of treatment, half of the cells were treated with 0.1 µg/mL Hoechst 33342 nuclear stain, which binds to adenine and thymine rich regions of the minor groove of DNA. Dishes were incubated for 24 hours before being treated to a final concentration of 1 M with NBD-propyl, 2-NBDG, NBD-lex, 1c, or left untreated as a control. Cells were incubated for 24 h with 5% CO2 at 37 °C before being rinsed with warm HBSS and imaged using an Olympus Fluoview FV1000 scanning confocal microscope, using the FITC dye settings (excitation: 498 nm, emission: 515 nm). Laser strength, binning, contrast and exposure were adjusted for each image in order to optimally enhance image quality. Nuclear Localization. To verify and compare the nuclear uptake of the NBD compounds, confocal images of U251 cells stained with the various NBD compounds were taken, with and without Hoechst 33342 dye, and assessed using Adobe Photoshop. Ten different cells per treatment were selected and the nuclear and whole cell regions were analyzed. A histogram of the pixels in each region was generated, and the mean pixel intensity was recorded for each region, per cell. The ratio of nuclear:whole cell fluorescence for each cell was calculated as:


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100*((mean nuclear fluorescence)/(mean whole cell fluorescence)). The resultant data was run through an ANOVA, with Tukey’s HSD test to compare means. RESULTS MD Simulations. Computational studies were conducted primarily to determine that attachment

of the glucosamine unit to Melex would not interfere with the binding of the bispyrrole triamide unit to the minor groove of DNA at A/T-rich regions before investing resources into the synthesis of molecules. The simulations were initiated with the bispyrrole unit already bound to its target site, and with the glucosamine unit extended into the bulk solvent medium. Simulations were conducted with several initial orientations of the glucosamine unit. In all simulations, when equilibrium was reached, the bispyrrole unit maintained its location within the minor groove at the A/T-rich region, and the glucosamine unit had also moved into the minor groove of DNA. The glucosamine nitrogen atom formed hydrogen bonds with acceptor atoms (N3 of adenine or O2 of cytosine) on the bases in its vicinity. Figure 2 shows the stereoscopic view (a) of a final equilibrated structure of 1a bound within the DNA dodecamer, along with the top view (b) and a snapshot of the molecule within the groove (c). These results confirmed that 1a was capable of binding within the minor groove of DNA at A/T-rich regions, and that the attachment of the glucosamine unit did not interfere with the binding, and may even contribute to enhance the binding by forming hydrogen bonds within the groove. Further details of these computational studies will be published elsewhere. Synthesis. The reaction sequence used for the synthesis of 1a is outlined in Scheme 1.

Commercially available 2 was condensed with β-alanine ethyl ester to form 3. High pressure catalytic hydrogenation of the nitro group on 3 followed by condensation of the resultant aryl


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amine with another unit of 2 resulted in the formation of 4. Reduction of the nitro group on 4 by high pressure catalytic hydrogenation, followed by reaction with acryloyl chloride resulted in the formation of 5 which now contains the DNA binding bispyrrole triamide core and provides functional handles at either end for conversion to the desired molecule. The ester on the Cterminus of 5 was hydrolyzed to the carboxylic acid with sodium hydroxide at room temperature, and condensed with the 2-deoxy-D-glucosamine unit which had its OH groups protected as benzyl ethers. Conjugate addition of bisulfite to the terminal alkene resulted in the formation of the sulfonic acid 8, and methylation of the sulfonic acid with 3-methyl-1-(p-tolyl)triazene yielded 9. Catalytic hydrogenation of 9 deprotected the alcohols and afforded the desired compound 1a. The final pure product was distributed into vials in 0.5 mg or 0.25 mg aliquots and stored under nitrogen at -20 C until needed for further experiments. The syntheses of compounds 1b and 1c are outlined in Scheme 2. These compounds were required as control and probe molecules to investigate the mechanism of action of 1a. The synthesis of 1b and 1c from 2 followed a sequence similar to that of 1a with a few differences. The β-alanine component used in the first step for this sequence was now a tert-butyl ester, instead of the ethyl ester used earlier. The bispyrrole intermediate 11 formed in this sequence was first reduced by high pressure catalytic hydrogenation, and the resultant aryl amine was used for the synthesis of both 1b and 1c. For preparing 1b, the amine was condensed with 3(methylsulfonyl)propanoic acid to produce intermediate 12, whereas for 1c, the amine was condensed with NBD CO2H to produce intermediate 14. In both cases, acid hydrolysis of the tert-butyl ester and condensation of the resultant carboxylic acid with trimethyl silyl protected glucosamine (TMSG), followed by acid work-up, afforded the desired final compounds 1b and


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1c. The final pure products were distributed into vials in 0.5 mg or 0.25 mg aliquots and stored

under nitrogen at -20 C until needed for further experiments. DNA methylation studies. Alkylation studies were conducted with genomic calf thymus DNA

in order to characterize the DNA methylation profile of 1a, and the quantities of the major groove adduct, 7MeG, and the desired minor groove adduct, 3MeA, produced were determined. Formation of N3-methylguanine adducts was monitored, but this adduct was not detected in our experiments with any of the methylating agents (detection limit ≥ 10 mol/mol DNA bp). The formation of O6-methylguanine was not measured since neither Melex56 nor MMS57 produce significant levels of this adduct. The methylation profile of 1a was compared to that of Melex, MMS, and STZ, and the ability of netropsin to inhibit DNA-methylation by 1a was also investigated. The results of these studies are shown in Table 1. Compound 1a predominantly forms 3MeA adducts (over 97%) similar to Melex, and forms only a small amount of 7MeG, indicating that the molecule preferentially alkylates in the minor groove of DNA. Preincubation of DNA with netropsin, which binds strongly in the minor groove of DNA at A/T-rich regions, almost completely prevented the formation of 3MeA adducts by 1a, indicating that the minor groove methylation of adenines by 1a occurs only at A/T-rich regions. Preincubation of DNA with netropsin had no effect on the formation of 7MeG by 1a. Both 3MeA and 7MeG adducts formed by 1a showed a dose response. The adduct levels formed by 1a were very similar to those formed by Melex. When compared on an equimolar basis (100 M), 1a produced over 300 times the amount of 3MeA produced by MMS, and 75 times that produced by STZ. and produced as much 7MeG as MMS. In contrast to 1a, the major adduct produced by MMS and STZ is the 7MeG adduct (over 85%).


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Cell toxicity studies. Cell toxicity studies of 1a were conducted in human glioblastoma U251

cells. The toxicity of 1a was compared to that of STZ, a clinically used anti-cancer drug, in order to determine if these two methylating agents with different DNA methylation profiles, would exhibit different potency in these cells. Additionally, the toxicity of 1a was compared to the toxicity of 1b, which is incapable of DNA alkylation, in order to assess the importance of methylation in any observed toxicity. In order to enhance the solubility of the compounds, 1% DMSO was used in the toxicity assays after determining that at this concentration, DMSO is not toxic to U251 cells (see Supplementary Materials, Figure S1). The results of the toxicity experiments are shown in Figure 3. Compound 1a was an order of magnitude more toxic (EC50 = 0.73 mM) to these glioma cells than STZ (EC50 > 8 mM). No toxicity was observed for the sulfone analog 1b, which is incapable of methylating DNA. Fluorescence Cell Transport Studies. Four different fluorescent compounds: NBD-propyl,

NBD-lex, 2-NBDG and 1c, all containing the same NBD fluorophore unit, were used in celltransport studies in order to investigate the cellular uptake and localization of the molecules and the influence of the glucosamine unit on the uptake. The results of these studies are shown in Figure 4 in the form of pixel intensity maps and a quantified bar chart. Of the four compounds, 2-NBDG, NBD-propyl, and NBD-lex showed poor uptake into U251 cells in normal medium containing glucose, while 1c showed better uptake. In glucose-free medium, only the two compounds bearing the glucosamine unit, 2-NBDG and 1c showed significant improvement in cellular uptake, indicating that at least a portion of these two compounds enters the cell through the glucose transporters. There was no difference between the uptake in glucose-free medium and the normal glucose medium for the two compounds lacking the glucosamine targeting unit, NBD-propyl and NBD-lex.


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Biochemistry

In order to evaluate the ability of the molecules to penetrate the nucleus of cells and approach the DNA, confocal images of U251 cells treated with NBD-propyl, NBD-lex and 1c were taken, and the results are shown in Figure 5. Of the three compounds tested, the fluorescence intensity within the nucleus for NBD-propyl was approximately half of the intensity of the whole cell, and this ratio was not significantly affected by the presence or absence of the Hoechst 33342 dye. By contrast, both compounds containing the bispyrrole triamide unit, NBD-lex and 1c, showed higher localization within the nucleus (fluorescence intensity inside the nucleus was approximately 85% of the fluorescence intensity of the whole cell). Additionally, when the nuclear stain Hoechst 33342 was absent, there was a significant increase in the proportion of NBD-lex and 1c within the nucleus (12% and 20% increase, respectively). DISCUSSION Since DNA-alkylating drugs continue to be an important weapon in the arsenal against cancers, the development of a new generation of drugs that are more potent against tumors, but less toxic to normal cells, is essential. However, progress in this area has been slow due to the complex set of properties required in molecules designed for this purpose, as discussed in the introduction. We have attempted to address these requirements in designing the methylating agent 1a, upon which we have placed several functional burdens, which include selective uptake via glucose transporters, site-specific binding to DNA, and preferential methylation at N3-adenine sites on DNA. In order to accomplish these functions, the different components of 1a have to be able to accomplish the following tasks : (1) the glucosamine unit, which is known to escort other moieties into cells via glucose transporters, has to be able to do so in 1a, with the bispyrrole unit attached to it; (2) the bispyrrole triamide unit, which is known to enable other molecules to bind to the minor groove of DNA at A/T-rich regions, has to maintain this DNA site-specificity in 1a,


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in spite of being tethered to the glucosamine unit; and (3) a significant proportion of the methyl sulfonate unit has to maintain its reactivity until it reaches its target sites on DNA in cancer cells without either getting hydrolyzed in the aqueous environment, or reacting with nucleophiles on other biomolecules. Our results presented in this article provide evidence that 1a performs all three of the above mentioned functions. The design of 1a incorporates several features to minimize damage to off-target sites. Firstly, 1a contains a glucosamine unit which can enable the uptake of the molecule preferentially into cancer cells which overexpress glucose transporters. Secondly, the molecule contains a DNAbinding unit which sequesters it specifically into the minor groove of DNA at A/T-rich sites and thus limits the DNA-methylation to N3-adenine, the most nucleophilic atom at these sites. Thirdly, the alkylating unit, a methyl sulfonate, is a relatively weak methylating agent, especially when tethered to the end of a large molecule as it is in 1a. For example, when reactions of 1a are conducted with single stranded DNA oligomers, to which 1a cannot bind, no 3-MeA and 7-MeG adducts are detected (detection limit ≥ 10 mol/mol DNA bp). At similar concentrations, reactions of 1a with duplex DNA produce several thousand µmol adduct/mol of DNA base pairs. Therefore, it is unlikely that 1a would cause significant collateral damage to other non-target biomolecules. The solubility and reactivity of 1a in an aqueous medium will be an important determinant of its utility as an anti-cancer drug candidate. The glucosamine unit, in addition to enabling the molecule to target cancer cells, also confers increased solubility in aqueous medium to the molecule. For example, 1b, the sulfone analog of 1a, has approximately an 8-fold higher water solubility than that of the sulfone analog of Melex, which lacks the glucosamine unit. At the same time, the molecule appears to have reasonable stability in water, with a half-life of over 24


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hours, as monitored by the decrease in area of the sulfonate methyl peak in the 1H NMR spectrum in D2O (containing 10% deuterated DMSO). Also, self-methylation of the celltargeting unit is not a concern with this molecule, since no methylation of the hydroxyl groups on the glucosamine unit was observed within 24 hours. One of the critical requirements for the success of our strategy was that the bispyrrole triamide core of 1a had to maintain its site-specific binding to the minor groove of DNA at A/T-rich regions. Therefore, we first undertook computational studies in order to determine whether tethering the glucosamine moiety to the pyrroles would interfere with the requisite DNAbinding, before initiating the synthesis of molecules, which is expensive in time and resources. MD studies were conducted using a DNA dodecamer which had only one A/T-rich binding site. These MD simulations were initiated with the bispyrrole unit bound to its target site on DNA, and the tethered glucosamine unit extended outside the minor groove into the bulk solvent. It was anticipated that, if the glucosamine unit experienced steric conflict in the vicinity of the DNA minor groove, and interfered significantly with the relatively weak DNA-binding of the bispyrrole unit, it would result in the expulsion of the entire molecule from DNA, as was seen with a test molecule in previous studies.58 In all simulations, each of which was conducted with a different initial orientation of the glucosamine unit in the bulk solvent, there was no evidence of any alteration in the DNA-binding of the bispyrrole triamide unit within the minor groove at its binding site. In fact, in all simulations, once equilibrium had been achieved, the glucosamine unit had migrated into the minor groove, and established several hydrogen bonds with DNA atoms in its vicinity, as shown schematically in Figure 6. The glucosamine amide hydrogen appears to be at the optimum location to establish hydrogen bonds similar to the other amide hydrogens of the molecule. In addition, hydroxyl groups on the glucosamine unit had also established hydrogen


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bonds with DNA atoms in its vicinity. While the base pair sequences flanking the A/T-rich binding sites for 1a may vary in genomic DNA, an analysis of the hydrogen bonding interactions of the glucosamine unit suggests that these interactions are likely to exist irrespective of the bases in its vicinity. Thus, the computational analysis engendered confidence that the glucosamine unit would not compromise the required DNA-binding of 1a at A/T-rich regions, and may even enhance this binding, and therefore, the synthesis of molecules was initiated. The synthesis of 1a required a ten-step sequence (see Scheme 1) starting from commercially available inexpensive compounds, and involved the sequential formation of the four amide bonds in the molecule by coupling together appropriate units bearing amines and activated carboxylic acids, followed by conjugate addition of bisulfite to the terminal alkene in 7. The first eight steps were all high yield reactions (all over 80%) with the overall yield for the eight steps being over 45%. The two challenging steps in the synthesis sequence were the final two steps, due to the presence of the reactive methyl sulfonate unit. Since the hydroxyls on the glucosamine unit were all protected as benzyl ethers, the methylation of the sulfonic acid proceeded well, but purification of the methylated compound, 9, using flash column chromatography on silica gel decreased the yield (47%) due to partial hydrolysis of the methyl sulfonate on the column. The final step, the deprotection of the hydroxyl moieties on the glucosamine unit, resulted in very polar products, and flash column chromatography to isolate pure 1a, as an anomeric mixture, also resulted in some loss of compound due to hydrolysis of the methyl sulfonate. While further optimization of the isolation procedure for 1a is likely possible, perhaps utilizing reverse phase chromatography, it was not attempted since the current procedure provided sufficient quantity of 1a to perform the studies described here.


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The syntheses of 1b and 1c were comparatively simpler than that of 1a, since these molecules did not contain any reactive functional groups. One difference in the initial step of the sequence was that the -CH2CH2- linker unit had to be introduced as a tert-butyl ester rather than as an ethyl ester (compare step 1 in schemes 1 and 2). This modification was because base hydrolysis of the ethyl ester equivalent of 12 (see Scheme 2) resulted in the elimination of the sulfone unit too, whereas the tert-butyl ester in 12 could be hydrolyzed quantitatively with formic acid without affecting the sulfone. For both 1b and 1c, the glucosamine unit, with the hydroxyl groups protected as trimethylsilyl ethers, was introduced in the final step of the sequence, and acid work-up of the reactions resulted in the formation of the desired products. Flash column chromatography on silica gel resulted in the isolation of pure 1b and 1c as a mixture of anomers, and again, no further optimization of the final step was attempted since this procedure produced all the material necessary for our experiments. While the ultimate effectiveness of 1a in a biological system depends upon multiple factors, the fruition of our strategy depends critically upon its ability to produce significant amounts of 3MeA upon reaching its target – the DNA. Therefore, we evaluated the DNA-methylating ability of 1a in experiments with genomic DNA, and compared its methylation profile to that of the following related molecules: (1) MMS, which represents the methylating terminus of 1a, and is a small molecule lacking any cell-targeting or DNA-binding ability; (2) Melex, which is known to efficiently produce 3MeA adducts and has DNA-binding ability, but lacks the cell-targeting glucosamine unit; and (3) STZ, which is the clinically used DNA-methylating anti-cancer compound that possesses the glucosamine cell-targeting unit which can facilitate STZ’s uptake into cells via glucose transporters, but lacks any site-specific DNA-binding ability. MMS, Melex


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and 1a methylate DNA by the SN2 mechanism, whereas STZ decomposes to form a methyldiazonium cation and methylates DNA by a “SN1-type” mechanism.38 The methylation studies gratifyingly showed that 1a produces 3MeA, almost exclusively, as seen in Table 1. The ratio of 3MeA:7MeG was 97:3. Remarkably, 1a produced only slightly less (8%) 3MeA than Melex when compared on an equimolar basis, and was as selective as Melex, whose 3MeA:7MeG was 96:4. These results indicate that the attachment of the glucosamine unit does not compromise the ability of the molecule to enter the narrow minor groove and bind to its target A/T-sites. The ability of 1a to produce the minor groove 3MeA adduct was over 300 times that of the much smaller MMS, and over 75 times that of STZ, when compared on an equimolar (100 M) basis. In contrast to 1a, MMS and STZ predominantly produce 7MeG (over 85%), as shown in Figure 7. Since pre-incubation of DNA with netropsin, a strong minor groove binder at A/T-rich regions, decreased the formation of 3MeA by 1a by 95%, the vast majority of the 3MeA adducts formed by 1a must be produced at A/T-rich regions. As expected, pre-incubation of DNA with netropsin had no effect on the production of 7MeG by 1a. Thus, these methylation studies with genomic DNA confirmed that 1a indeed interacts with DNA as designed, and is capable of producing the desired lethal 3MeA adducts in cells upon interacting with DNA. In order to evaluate the effectiveness of 1a in cancer cells, toxicity experiments were conducted with U251 glioblastoma cells. Glioblastoma multiforme (GBM) is a highly aggressive human cancer that is the most common form of primary brain tumor in adults, and is responsible for over 13,000 deaths per year in the United States.59 This tumor is highly resistant to current traditional methods of treatment largely due to both the heterogeneity of the tumor mass and the tendency of the tumor to infiltrate into the surrounding brain tissue, making complete resection and eradication of the tumor unlikely.60, 61 In order to survive in a hypoxic micro-environment,


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Biochemistry

these tumor cells activate hypoxia-inducible factor-1 (HIF-1) which in turn trans-activates hypoxia response genes including the glucose transporters.62 Glucose transporters GLUT1 and GLUT3 are significantly up-regulated in GBM compared to the adjacent non-cancerous cells,63-66 and the increased expression of the GLUTs allows the GBM cells to out-compete the normal tissue for the available glucose. All these features made the GBM cell line an excellent candidate for initial toxicity evaluation of 1a. The toxicity assay (see Figure 3) showed that 1a was toxic to U251 cells and had an EC50 value of 0.73 mM, whereas the non-methylating analog, 1b, was non-toxic. Since 1b is essentially identical in structure to 1a, containing both the cell-targeting glucosamine unit and the DNAbinding bispyrrole component, and lacks only an oxygen atom at one terminus when compared to 1a, it is likely to have similar uptake and binding properties as 1a. Therefore, the absence of toxicity with 1b implicates methylation of cellular components as the reason for the toxicity of 1a.

There are certain pieces of evidence that indicate that the biomolecule being methylated by 1a, and leading to toxicity, is cellular DNA. Firstly, 1a is a poor methylator unless it binds to biomolecules, since methylation studies with single stranded DNA oligomers containing A/T rich regions did not result in the production of methylated DNA adducts (neither 3MeA nor 7MeG was detected at treatment concentrations similar to those reported in Table 1). Therefore, it is unlikely that random methylation by 1a leads to high levels of damage of other biomolecules. Secondly, MMS, which is a small molecule representing the methylating terminus of 1a (see Figure 1), is non-toxic to U251 cells at the highest concentration tested (2 mM). If random methylation of other biomolecules by the methyl sulfonate unit of 1a was responsible for the observed toxicity, MMS should also have exhibited significant toxicity in these cells. Finally,


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it is possible that, if 1a targets glucose transporters on these U251 cells as designed, then specific damage to these transporters, due to methylation, may be responsible for the toxicity. However, if damage to glucose transporters played an important role in the observed toxicity of 1a, one would expect similar toxicity with the methylating agent STZ, which is known to enter cells via glucose transporters, but this was not the case. STZ was an order of magnitude less toxic (EC50 > 8 mM) to U251 cells than 1a. Taken together, the above results indicate that DNA-methylation mediates the toxicity of 1a in these cells. It is very promising that 1a is 10 times more toxic than STZ, an FDA approved alkylating anticancer drug. The toxicity of 1a (EC50 = 0.73 mM after 24 h exposure) in U251 glioma cells also compares very favorably with the reported toxicity of temozolomide67 (IC50 > 0.32 mM after 72 h exposure), which is currently is currently used as the first-line treatment for glioblastoma multiforme. This difference in toxicity of 1a and STZ in our experiments could be either due to differences in the uptake of the molecules into U251 cells, and/or differences in the DNAmethylation profile of the two molecules. As described earlier, 1a is a potent producer of 3MeA adducts upon reaction with DNA, whereas STZ is a weaker methylating agent producing 75 times less 3MeA than 1a, and primarily produces 7MeG adducts which are typically well tolerated by cells. Further experiments are ongoing in order to establish the causative role of DNA methylation, and particularly the formation of 3MeA within cells, in the observed toxicity of 1a, and the results of these studies will be published at a future date. The results of the toxicity studies described above are promising, but do not implicate the uptake of 1a via glucose transporters, which is an essential requirement for the success and further development of our strategy. Therefore, cellular uptake studies were conducted using the fluorescently labeled compound 1c, and other test molecules NBD-propyl, NBD-lex, and 2-


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NBDG (see Figure 1 for structures). In 1c, the methyl sulfonate unit of 1a has been replaced with the fluorophore NBD, but the rest of the molecule (the DNA-binding and glucose transporters targeting units) is identical to 1a. Therefore, the cellular uptake and localization properties of the two molecules are likely to be similar, and any conclusions drawn regarding the cell-transport properties of 1c are likely to apply to 1a as well. The test molecules used in this study were selected in order to determine the effect of the different individual components on the cellular uptake. Thus, NBD-propyl would reveal how the fluorophore unit itself would influence transport into the cell, NBD-lex would demonstrate the effect of the DNA-binding unit on cellular uptake and localization, and 2-NBDG would reveal the role of the glucosamine unit in enhancing cellular uptake via glucose transporters. Since the normal medium used for growing the cells contains high levels of glucose, which could inhibit the transport of compounds via glucose transporters, the uptake experiments were also performed in no-glucose media. The results of the fluorescence studies (Figure 4) showed that very little of NBD-propyl is taken up into U251 cells indicating that the fluorophore, NBD, does not contribute much to cellular uptake by itself. The fluorescently labeled DNA-binding bispyrrole unit, NBD-lex, showed a slightly higher uptake into the cells. For both these fluorescent molecules, the cellular uptake was identical both in normal-glucose and no-glucose media, indicating that glucose transporters play no significant role in the transport of these molecules into the cells. By contrast, 2-NBDG, in which the fluorophore is attached to the glucosamine unit, showed poor uptake in normal medium, but significantly higher uptake in no-glucose medium, indicating that glucose transporters play an important role in the transport of this molecule into cells. Compound 1c, which differs from NBD-lex only in the presence of the glucosamine unit, showed much higher uptake into cells than NBD-lex, and this uptake was significantly enhanced when glucose was


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eliminated from the medium. These results show that at least a proportion of 1c (and by inference, of 1a) is transported into U251 cells via its glucose transporters, and that attachment of the glucosamine unit to a compound is an effective strategy for enhancing its uptake into these cancer cells. The fluorescence experiments discussed above demonstrated that 1c is effectively transported into U251 cells, but for our strategy to succeed, the molecule has to be able to penetrate the nuclear membrane and bind to target sites on DNA. In order to determine whether the nuclear localization signal, the bispyrrole triamide unit, is effective in enhancing the nuclear concentration of the molecule, confocal images of U251 cells exposed to 1c, NBD-lex and NBDpropyl were taken. Since the nuclear stain, Hoechst 33342, which is typically used in these experiment to visualize the nucleus, binds to the same A/T-rich DNA-minor groove sites targeted by our molecule, these experiments were also repeated without using the Hoechst 33342 dye. The results (see Figure 5) showed that there was indeed an increase in the proportion of molecules found within the nucleus when the molecule contained the bispyrrole unit (1c and NBD-lex) than when it did not (NBD-propyl). Furthermore, the absence of Hoechst 33342 dye had no effect on the nuclear uptake of NBD-propyl, which is not expected to have any DNAbinding properties. By contrast, vacating the A/T-rich minor groove sites on DNA previously occupied by Hoechst 33342 resulted in a significant enhancement in the proportion of 1c and NBD-lex found within the nucleus, suggesting that these two molecules target the same sites preferred by the Hoechst 33342 dye. Taken together, the studies reported here indicate that 1a exhibits all the essential functions that it was designed to accomplish. The fluorescence studies indicated that the molecule can enter cells via glucose transporters and localize within the nucleus. The DNA-methylation studies


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showed that once in the vicinity of DNA, 1a can produce predominantly the desired lethal 3MeA adducts. The toxicity experiments showed that the molecule can indeed function as designed in a cellular environment, and exhibit significantly enhanced toxicity in the treatment-resistant GBM cells when compared to STZ, which is a glucose transporter targeting DNA-methylating agent currently approved for treatment of other cancers. CONCLUSION We have successfully designed a DNA-methylating molecule, 1a, that can be taken up into glioma cells via glucose transporters and can destroy these cells effectively. Studies are ongoing in order to determine the selectivity of 1a for cancer cells, to screen it against multiple cancer cell lines, and to establish the role of 3MeA adducts formed within cells in mediating the toxicity of the molecule. Since most types of cancer cells over-express glucose transporters to satisfy their increased energy needs, the studies reported here confirm that our strategy can be successful in developing treatments for multiple cancers. Additionally, treatment for a specific cancer can also be developed using this strategy by employing a targeting ligand that recognizes receptors overexpressed in that particular cancer, as has been reported elsewhere.50 Supporting Information. Toxicity of DMSO in U251 cells, and 1H NMR and 13C NMR spectra

of molecules. AUTHOR INFORMATION Corresponding Author

*Telephone: 910 962 7350. Fax: 910 962 3013. E-mail: [email protected] Funding Sources


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This work was supported by the Research Corporation (Grant CC6245), the North Carolina Biotechnology Center (Grant 2008BRG1214), UNCW (Charles L. Cahill Award), NSF (Grant CHE0821552, used to acquire the 600 MHz NMR used for the characterization of molecules) Notes

The authors declare no competing financial interest. REFERENCES

[1] Celkan, T. (2013) Alkylating agents in Chemotherapy, In Alkylating Agents as Environmental Carcinogen and Chemotherapy Agents (Yildiz, D., Ed.), pp 69-99, Nova Science Publishers, Inc., Hauppauge, N. Y. [2] Ralhan, R., and Kaur, J. (2007) Alkylating agents and cancer therapy, Expert Opin. Ther. Pat. 17, 1061-1075. [3] Ghalie, R. G., and Goodkin, D. E. (2001) Secondary leukemia after adjuvant chemotherapy for breast cancer, J. Clin. Oncol. 19, 1231-1233. [4] Rund, D., Krichevsky, S., Bar-Cohen, S., Goldschmidt, N., Kedmi, M., Malik, E., Gural, A., Shafran-Tikva, S., Ben-Neriah, S., and Ben-Yehuda, D. (2005) Therapy-related leukemia: clinical characteristics and analysis of new molecular risk factors in 96 adult patients, Leukemia 19, 1919-1928. [5] Allan, J. M., and Lois, B. T. (2005) Mechanisms of therapy-related carcinogenesis, Nat. Rev. Cancer 5, 943-955. [6] Boivin, J. F., Hutchison, G. B., Zauber, A. G., Bernstein, L., Davis, F. G., Michel, R. P., Zanke, B., Tan, C. T., Fuller, L. M., Mauch, P., and et al. (1995) Incidence of second cancers in patients treated for Hodgkin's disease, J. Natl. Cancer Inst. 87, 732-741. [7] Smith, M. A., McCaffrey, R. P., and Karp, J. E. (1996) The secondary leukemias: challenges and research directions, J. Natl. Cancer Inst. 88, 407-418. [8] Reux, B., Weber, V., Galmier, M. J., Borel, M., Madesclaire, M., Madelmont, J. C., Debiton, E., and Coudert, P. (2008) Synthesis and cytotoxic properties of new fluorodeoxyglucose-coupled chlorambucil derivatives, Bioorg. Med. Chem. 16, 50045020. [9] Pohl, J., Bertram, B., Hilgard, P., Nowrousian, M. R., Stuben, J., and Wiessler, M. (1995) D19575 - a Sugar-Linked Isophosphoramide Mustard Derivative Exploiting Transmembrane Glucose-Transport, Cancer Chemother. Pharmacol. 35, 364-370. [10] Descôteaux, C., Brasseur, K., Leblanc, V., Parent, S., Asselin, É., and Bérubé, G. (2012) Design of novel tyrosine-nitrogen mustard hybrid molecules active against uterine, ovarian and breast cancer cell lines, Steroids 77, 403-412.


54

Page 55 of 71

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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[11] Gupta, A., Saha, P., Descôteaux, C., Leblanc, V., Asselin, É., and Bérubé, G. (2010) Design, synthesis and biological evaluation of estradiol–chlorambucil hybrids as anticancer agents, Bioorg. Med. Chem. Lett. 20, 1614-1618. [12] Sharma, U., Marquis, J. C., Dinaut, A. N., Hillier, S. M., Fedeles, B., Rye, P. T., Essigmann, J. M., and Croy, R. G. (2004) Design, synthesis, and evaluation of estradiol-linked genotoxicants as anti-cancer agents, Bioorg. Med. Chem. Lett. 14, 3829-3833. [13] Ishiki, N., Onishi, H., and Machida, Y. (2004) Evaluation of antitumor and toxic side effects of mitomycin C-estradiol conjugates, Int. J. Pharm. 279, 81-93. [14] Mitra, K., Marquis, J. C., Hillier, S. M., Rye, P. T., Zayas, B., Lee, A. S., Essigmann, J. M., and Croy, R. G. (2002) A rationally designed genotoxin that selectively destroys estrogen receptor-positive breast cancer cells, J. Am. Chem. Soc. 124, 1862-1863. [15] Hillier, S. M., Mitra, K., Marquis, J. C., Zayas, B., Wogan, G. N., Wishnok, J., Essigmann, J. M., and Croy, R. G. (2001) Synthesis, biochemical properties, and pharmacokinetic profiles of DNA-damaging genotoxins with selective toxicity towards estrogen receptorpositive breast cancer cells and androgen receptor-positive prostate cancer cells., Abstr. Pap., Am. Chem. Soc. 222, U299-U299. [16] Calvaresi, E. C., and Hergenrother, P. J. (2013) Glucose conjugation for the specific targeting and treatment of cancer, Chem. Sci. 4, 2319-2333. [17] Warburg, O., Wind, F., and Negelein, E. (1927) The Metabolism of Tumors in the Body, J. Gen. Physiol. 8, 519-530. [18] Warburg, O. (1956) On the origin of cancer cells, Science 123, 309-314. [19] Murray-Lyon, I. M., Eddleston, A. L., Williams, R., Brown, M., Hogbin, B. M., Bennett, A., Edwards, J. C., and Taylor, K. W. (1968) Treatment of multiple-hormone-producing malignant islet-cell tumour with streptozotocin, Lancet 2, 895-898. [20] Beuthien-Baumann, B., Hamacher, K., Oberdorfer, F., and Steinbach, J. (2000) Preparation of fluorine-18 labelled sugars and derivatives and their application as tracer for positronemission-tomography, Carbohydr. Res. 327, 107-118. [21] O'Neil, R. G., Wu, L., and Mullani, N. (2005) Uptake of a fluorescent deoxyglucose analog (2-NBDG) in tumor cells, Mol. Imaging Biol. 7, 388-392. [22] Mandai, T., Okumoto, H., Oshitari, T., Nakanishi, K., Mikuni, K., Hara, K. J., Hara, K. Z., Iwatani, W., Amano, T., Nakamura, K., and Tsuchiya, Y. (2001) Synthesis and biological evaluation of water soluble taxoids bearing sugar moieties, Heterocycles 54, 561-+. [23] Lin, Y. S., Tungpradit, R., Sinchaikul, S., An, F. M., Liu, D. Z., Phutrakul, S., and Chen, S. T. (2008) Targeting the Delivery of Glycan-Based Paclitaxel Prodrugs to Cancer Cells via Glucose Transporters, J. Med. Chem. 51, 7428-7441. [24] Liu, D. Z., Sinchaikul, S., Reddy, P. V., Chang, M. Y., and Chen, S. T. (2007) Synthesis of 2'-paclitaxel methyl 2-glucopyranosyl succinate for specific targeted delivery to cancer cells, Bioorg. Med. Chem. Lett. 17, 617-620. [25] Halmos, T., Santarromana, M., Antonakis, K., and Scherman, D. (1996) Synthesis of glucose-chlorambucil derivatives and their recognition by the human GLUT1 glucose transporter, Eur. J. Pharmacol. 318, 477-484. [26] Halmos, T., Santarromana, M., Antonakis, K., and Scherman, D. (1997) Synthesis of Omethylsulfonyl derivatives of D-glucose as potential alkylating agents for targeted drug delivery to the brain. Evaluation of their interaction with the human erythrocyte GLUT1 hexose transporter, Carbohydr. Res. 299, 15-21.


55

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 56 of 71

[27] Kumar, P., Shustov, G., Liang, H., Khlebnikov, V., Zheng, W. Z., Yang, X. H., Cheeseman, C., and Wiebe, L. I. (2012) Design, Synthesis, and Preliminary Biological Evaluation of 6-O-Glucose-Azomycin Adducts for Diagnosis and Therapy of Hypoxic Tumors, Journal of medicinal chemistry 55, 6033-6046. [28] Cao, J., Cui, S. S., Li, S. W., Du, C. L., Tian, J. M., Wan, S. N., Qian, Z. Y., Gu, Y. Q., Chen, W. R., and Wang, G. J. (2013) Targeted Cancer Therapy with a 2-DeoxyglucoseBased Adriamycin Complex, Cancer Res 73, 1362-1373. [29] Cheng, H., Cao, X. H., Xian, M., Fang, L. Y., Cai, T. B., Ji, J. J., Tunac, J. B., Sun, D. X., and Wang, P. G. (2005) Synthesis and enzyme-specific activation of carbohydrategeldanamycin conjugates with potent anticancer activity, J. Med. Chem. 55, 6033-6046. 48, 645-652. [30] Lee, H. Y., Kwon, J. T., Koh, M., Cho, M. H., and Park, S. B. (2007) Enhanced efficacy of 7-hydroxy-3-methoxycadalene via glycosylation in in vivo xenograft study, Bioorg. Med. Chem. Lett. 17, 6335-6339. [31] Tietze, L. F., von Hof, J. M., Krewer, B., Mueller, M., Major, F., Schuster, H. J., Schuberth, I., and Alves, F. (2008) Asymmetric Synthesis and Biological Evaluation of Glycosidic Prodrugs for a Selective Cancer Therapy, ChemMedChem 3, 1946-1955. [32] Tietze, L. F., Major, F., and Schuberth, I. (2006) Antitumor agents: Development of highly potent glycosidic duocarmycin analogues for selective cancer therapy, Angew. Chem., Int. Ed. 45, 6574-6577. [33] Hwu, J. R., Hsu, C. I., Hsu, M. H., Liang, Y. C., Huang, R. C. C., and Lee, Y. C. (2011) Glycosylated nordihydroguaiaretic acids as anti-cancer agents, Bioorg. Med. Chem. Lett. 21, 380-382. [34] Simons, A. L., Ahmad, I. M., Mattson, D. M., Dornfeld, K. J., and Spitz, D. R. (2007) 2Deoxy-D-glucose combined with cisplatin enhances cytotoxicity via metabolic oxidative stress in human head and neck cancer cells, Cancer Res. 67, 3364-3370. [35] Maschek, G., Savaraj, N., Priebe, W., Braunschweiger, P., Hamilton, K., Tidmarsh, G. F., De Young, L. R., and Lampidis, T. J. (2004) 2-deoxy-D-glucose increases the efficacy of adriamycin and paclitaxel in human osteosarcoma and non-small cell lung cancers in vivo, Cancer Res. 64, 31-34. [36] Denny, W. A. (2001) DNA minor groove alkylating agents, Curr. Med. Chem. 8, 533-544. [37] Zhang, Y., Chen, F. X., Mehta, P., and Gold, B. (1993) Groove-Selective and SequenceSelective Alkylation of DNA by Sulfonate Esters Tethered to Lexitropsins, Biochemistry 32, 7954-7965. [38] Wyatt, M. D., and Pittman, D. L. (2006) Methylating agents and DNA repair responses: Methylated bases and sources of strand breaks, Chem. Res. Toxicol. 19, 1580-1594. [39] Newlands, E. S., Stevens, M. F. G., Wedge, S. R., Wheelhouse, R. T., and Brock, C. (1997) Temozolomide: A review of its discovery, chemical properties, pre-clinical development and clinical trials, Cancer Treat. Rev. 23, 35-61. [40] Bennett, R. A., and Pegg, A. E. (1981) Alkylation of DNA in rat tissues following administration of streptozotocin, Cancer Res. 41, 2786-2790. [41] Beranek, D. T. (1990) Distribution of Methyl and Ethyl Adducts Following Alkylation with Monofunctional Alkylating-Agents, Mutat. Res. 231, 11-30. [42] Lawley, P. D., and Warren, W. (1976) Removal of Minor Methylation Products 7-MethylAdenine and 3-Methylguanine from DNA of Escherichia-Coli Treated with Dimethyl Sulfate, Chem. -Biol. Interact. 12, 211-220.


56

Page 57 of 71

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

[43] Karran, P., Lindahl, T., Ofsteng, I., Evensen, G. B., and Seeberg, E. (1980) Escherichia-Coli Mutants Deficient in 3-Methyladenine-DNA Glycosylase, J. Mol. Biol. 140, 101-127. [44] Boysen, G., Pachkowski, B. F., Nakamura, J., and Swenberg, J. A. (2009) The formation and biological significance of N7-guanine adducts, Mutat. Res., Genet. Toxicol. Environ. Mutagen. 678, 76-94. [45] Fronza, G., and Gold, B. (2004) The biological effects of N3-methyladenine, J. Cell. Biochem. 91, 250-257. [46] Loechler, E. L., Green, C. L., and Essigmann, J. M. (1984) In vivo mutagenesis by O6methylguanine built into a unique site in a viral genome, Proc. Natl. Acad. Sci. U. S. A. 81, 6271-6275. [47] Meikrantz, W., Bergom, M. A., Memisoglu, A., and Samson, L. (1998) O6-alkylguanine DNA lesions trigger apoptosis, Carcinogenesis 19, 369-372. [48] Pauly, G. T., and Moschel, R. C. (2001) Mutagenesis by O(6)-methyl-, O(6)-ethyl-, and O(6)-benzylguanine and O(4)-methylthymine in human cells: effects of O(6)alkylguanine-DNA alkyltransferase and mismatch repair, Chem. Res. Toxicol. 14, 894900. [49] Kopka, M. L., Yoon, C., Goodsell, D., Pjura, P., and Dickerson, R. E. (1985) The Molecular-Origin of DNA Drug Specificity in Netropsin and Distamycin, Proc. Natl. Acad. Sci. U. S. A. 82, 1376-1380. [50] Kishton, R. J., Miller, S. E., Perry, H., Lynch, T., Patel, M., Gore, V. K., Akkaraju, G. R., and Varadarajan, S. (2011) DNA site-specific N3-adenine methylation targeted to estrogen receptor-positive cells, Bioorg. Med. Chem. 19, 5093-5102. [51] Pearlman, D. A., Case, D. A., Caldwell, J. W., Ross, W. S., Cheatham, T. E., Debolt, S., Ferguson, D., Seibel, G., and Kollman, P. (1995) Amber, a Package of ComputerPrograms for Applying Molecular Mechanics, Normal-Mode Analysis, MolecularDynamics and Free-Energy Calculations to Simulate the Structural and Energetic Properties of Molecules, Comput. Phys. Commun. 91, 1-41. [52] Wang, J. M., Wolf, R. M., Caldwell, J. W., Kollman, P. A., and Case, D. A. (2004) Development and testing of a general amber force field, J. Comput. Chem. 25, 11571174. [53] Kopka, M. L., Yoon, C., Goodsell, D., Pjura, P., and Dickerson, R. E. (1985) Binding of an Antitumor Drug to DNA Netropsin and C-G-C-G-a-a-T-T-Brc-G-C-G, J. Mol. Biol. 183, 553-563. [54] Medgyes, A., Farkas, E., Lipták, A., and Pozsgay, V. (1997) Synthesis of the monosaccharide units of the O-specific polysaccharide of Shigella sonnei, Tetrahedron 53, 4159-4178. [55] Skaddan, M. B., Wust, F. R., and Katzenellenbogen, J. A. (1999) Synthesis and binding affinities of novel re-containing 7 alpha-substituted estradiol complexes: Models for breast cancer imaging agents, J. Org. Chem. 64, 8108-8121. [56] Encell, L., Shuker, D. E. G., Foiles, P. G., and Gold, B. (1996) The in vitro methylation of DNA by a minor groove binding methyl sulfonate ester, Chem. Res. Toxicol. 9, 563-567. [57] Beranek, D. T., Weis, C. C., and Swenson, D. H. (1980) A Comprehensive QuantitativeAnalysis of Methylated and Ethylated DNA Using High-Pressure LiquidChromatography, Carcinogenesis 1, 595-606. [58] Kelly, C. B., Hill, H., Bartolotti, L., and Varadarajan, S. (2009) Molecular dynamics of d(CGCGAATTCGCG)(2) complexed with netropsin and its minor groove methylating


57

Biochemistry

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analog, Me-lex, using explicit and implicit water models, J. Mol. Struc.: THEOCHEM 894, 50-58. [59] Berger MS, W. C. (1999) The gliomas, 1st ed., WB Saunders, Philadelphia. [60] Arko, L., Katsyv, I., Park, G. E., Luan, W. P., and Park, J. K. (2010) Experimental approaches for the treatment of malignant gliomas, Pharmacol. Ther. 128, 1-36. [61] Singh, S. K., Clarke, I. D., Terasaki, M., Bonn, V. E., Hawkins, C., Squire, J., and Dirks, P. B. (2003) Identification of a Cancer Stem Cell in Human Brain Tumors, Cancer Res. 63, 5821-5828. [62] Bache, M., Rot, S., Kessler, J., Guttler, A., Wichmann, H., Greither, T., Wach, S., Taubert, H., Soling, A., Bilkenroth, U., Kappler, M., and Vordermark, D. (2015) mRNA expression levels of hypoxia-induced and stem cell-associated genes in human glioblastoma, Oncol. Rep. 33, 3155-3161. [63] Liu, Y., Li, Y. M., Tian, R. F., Liu, W. P., Fei, Z., Long, Q. F., Wang, X. A., and Zhang, X. (2009) The expression and significance of HIF-1alpha and GLUT-3 in glioma, Brain Res. 1304, 149-154. [64] Nagamatsu, S., Sawa, H., Wakizaka, A., and Hoshino, T. (1993) Expression of facilitative glucose transporter isoforms in human brain tumors, J. Neurochem. 61, 2048-2053. [65] Nishioka, T., Oda, Y., Seino, Y., Yamamoto, T., Inagaki, N., Yano, H., Imura, H., Shigemoto, R., and Kikuchi, H. (1992) Distribution of the glucose transporters in human brain tumors, Cancer Res. 52, 3972-3979. [66] Younes, M., Lechago, L. V., Somoano, J. R., Mosharaf, M., and Lechago, J. (1997) Immunohistochemical detection of Glut3 in human tumors and normal tissues, Anticancer Res. 17, 2747-2750. [67] Liu, Q., Xu, X., Zhao, M., Wei, Z., Li, X., Zhang, X., Liu, Z., Gong, Y., and Shao, C. (2015) Berberine induces senescence of human glioblastoma cells by downregulating the EGFR-MEK-ERK signaling pathway, Mol. Cancer Ther. 14, 355-363.


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Table 1: DNA adduct levels obtained upon reaction of compounds with calf thymus DNAa

adduct level µmol adduct/mol DNA bp 3MeA 7MeG 50 6070 ± 70 167 ± 72 1a 100 10580 ± 171 284 ± 61 100 100 540 ± 28 257 ± 10 200 15218 ± 213 486 ± 55 Melex 100 11539 ± 44 500 ± 78 MMS 5000 1700 ± 12 13591 ± 96 STZb 5000 6836 ± 2488 40623 ± 7225 a DNA (1 mM) was reacted with different methylating compounds, in the presence or absence of netropsin, for 24 h at room temperature in 10 mM sodium cacodylate buffer (pH 7.0) containing 10% DMSO. b STZ reactions were performed in a separate experiment along with MMS, and normalized using MMS 3MeA adduct levels for inclusion in this table. Compound

conc µM

netropsin µM


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O

O

O 2N

Cl H3N CCl3

N

1. H2, Pd/C EtOH, 75 psi

O NH

N

TEA, EtOAc, RT

2

11

2. EDCI, HOBt, O DMAP, DMF S CO2H O 60%

11

S O

O 2N

NH

O O H N

N

O2 N N

O

N

N

N H O N

NH

O

O

CO2H

NBD CO2H

O

12

NH

O 14

O

H N

N

NH N

NH

O

1. H2, Pd/C EtOH, 70 psi 2. EDCI, HOBt, DMAP, DMF

O

N

NH

O N 11

O

1. Formic acid 2. EDCI, HOBt, DMAP, DMF TMS = trimethyl silyl H N TMSO 2 OTMS OTMS O TMSG OTMS 1b 84%

O 1. H2, Pd/C EtOH, 70 psi

H N

N

94%

10

O O

2. 2, EtOAc, RT

O

90%

O

O 2N

O

O2N

1. Formic acid 2. EDCI, HOBt, DMAP, DMF TMS = trimethyl silyl H N TMSO 2 OTMS OTMS O TMSG OTMS 1c 3. HCl 36%

O

57% Scheme 2: Synthesis of 1b and 1c


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Figure Legends Figure 1. Structure of molecules used in this study. Figure 2. Results of the MD simulation of the binding of 1a to the minor groove of the duplex

DNA dodecamer 5’-d(CGCGAATTCGCG) . 3’-d(GCGCTTAAGCGC) after equilibration was achieved: (a) Stereoscopic view of 1a bound within the minor groove of DNA; (b) Top view of 1a bound within the minor groove of DNA; (c) Snapshot of 1b taken after equilibration to show

the curvature the molecule – the DNA strands have been removed for clarity. Figure 3. Toxicity of 1a, 1b and STZ in U251 glioma cells determined by the MTS assay. The

non-methylating analog (1b) was not toxic to these cells, while 1a (EC50 > 0.73 mM) was over an order of magnitude more toxic than STZ (EC50 > 8 mM). Figure 4. Intracellular fluorescence of compounds in U251 cells in normal glucose and no

glucose medium. * indicates statistical significance between the NBD-compound uptake in cells incubated in glucose-free DMEM medium versus cells incubated in normal-glucose DMEM. Figure 5. Confocal images (20X magnification) of U251 cells treated with NBD-propyl (A),

NBD-lex (B) and 1c (C), in the presence (I) or absence (II) of the nuclear stain Hoechst 33342. The cells treated with the NBD-conjugated compounds in the presence of the Hoechst 33342 dye (I) are visualized with (a) green fluorescence, representing the NBD-conjugated molecules, (b) blue fluorescence, representing the Hoechst 33342 nuclear stain, and (c) overlay of both green and blue fluorescence. III. Ratio of the fluorescence intensity of the NBD-conjugated compounds within the nucleus to the whole cell fluorescence in U251 cells in the presence and absence of Hoechst 33342. (error bars ± sem) (n = 10 cells). * indicates statistical significance


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Biochemistry

between the fluorescence intensity ratio for the NBD-conjugated compound in cells incubated in the presence of Hoechst 33342 versus cells incubated in the absence of Hoechst 33342. Figure 6. Schematic representation of hydrogen bonding distances (donor atom-acceptor atom,

in angstroms) between atoms of 1a and DNA. Hydrogen bonding to adenines are to the N3-atom, and to thymines and cytosines are to the O2-atom, and to G16 is to the exocyclic N2-atom. The complete dodecamer sequence is shown to the right. Figure 7. A comparison of the ratios of 3MeA and 7MeG produced by 1a, Melex, MMS and

STZ.


63

Biochemistry

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Figure 1


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Biochemistry

Figure 2

a

b

c


65

Biochemistry

Figure 3

Toxicity in U251 cells 120

1b

100

% cell viability

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STZ

80 60 40

1a

20 0 1 mM

0.5 mM

0.25 mM

0 mM

concentration


66

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Figure 4 Normal glucose medium

No glucose medium

Control

Control

2‐NBDG

2‐NBDG

NBD‐propyl

NBD‐propyl

NBD‐lex

NBD‐lex

1c

1c

Intracellular Fluorescence 2.50E+08

Fluorescence (RFU)

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Biochemistry

Normal Glucose

No Glucose

*

2.00E+08 1.50E+08

*

1.00E+08 5.00E+07 1.00E+00 Control

2-NBDG NBD-propyl NBD-lex

1c

Compound


67

Biochemistry

Figure 5 I. U251 cells pre-treated with Hoechst 33342 Dye

II. No Hoechst 33342

A

a

b

c

B

a

c

b

C

a

b

III

c

Nucleus: Whole Cell Fluorescence 120

With Hoescht Dye

Without Hoescht Dye

*

*

100

% Nucleus:Whole Cell

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80 60 40 20 0

NBD‐propyl

NBD‐lex

1c


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Biochemistry

Figure 6


69

Biochemistry

Figure 7

Relative amounts of 3MeA and 7MeG formed

Total Adduct

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3MeA 7MeG

100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

1a

Melex

MMS

STZ


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Biochemistry

For Table of Contents Use Only


71

Glycoconjugated Site-selective DNA-Methylating Agent Targeting

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