New Modified Deoxythymine with Dibranched Tetraethylene Glycol Stabilizes G-Quadruplex Structures

Methods for stabilizing G-quadruplex formation is a promising therapeutic approach for cancer treatment and other biomedical applications because stable G-quadruplexes efficiently inhibit biological reactions. Oligo and polyethylene glycols are promising biocompatible compounds, and we have shown that linear oligoethylene glycols can stabilize G-quadruplexes. Here, we developed a new modified deoxythymine with dibranched or tribranched tetraethylene glycol (TEG) and incorporated these TEG-modified deoxythymines into a loop region that forms an antiparallel G-quadruplex. We analyzed the stability of the modified G-quadruplexes, and the results showed that the tribranched TEG destabilized G-quadruplexes through entropic contributions, likely through steric hindrance. Interestingly, the dibranched TEG modification increased G-quadruplex stability relative to the unmodified DNA structures due to favorable enthalpic contributions. Molecular dynamics calculations suggested that dibranched TEG interacts with the G-quadruplex through hydrogen bonding and CH-π interactions. Moreover, these branched TEG-modified deoxythymine protected the DNA oligonucleotides from degradation by various nucleases in human serum. By taking advantage of the unique interactions between DNA and branched TEG, advanced DNA materials can be developed that affect the regulation of DNA structure.


Synthesis of 3.
To a dry pyridine (10 mL) solution of 2 (0.952 g, 8.97 mmol) was added 1 (1.23 g, 4.41 mmol) at 25 °C under A, and the resulting mixture was stirred overnight at 25 °C. To the reaction mixture was added water (100 mL), and the resulting mixture was extracted with EtOAc (100 mL, three times). The collected organic extract was washed with brine (50 mL) and dried over anhydrous Na2SO4, and filtered off from insoluble substances. The filtrate was evaporated to dryness under reduced pressure at 30 °C, and the residue was chromatographed on silica gel (Silica Gel 60) with a gradient of AcOEt/n-hexane (20/80 to 60/40 v/v) to allow isolation of 3 (1.79 g, 3.38 mmol) as white solid in 77% yield. 1

Synthesis of 5.
To a dry MeOH (60 mL) solution of 4 (1.48 g, 1.46 mmol) was added glacial acetic acid (10 mL) dropwise over 20 min at 25 °C under Ar, and the resulting mixture was refluxed for 3 h. Then, the reaction mixture was cooled to 25 °C followed by evaporation under reduced pressure at 40 °C. To the residue was added CH2Cl2 (200 mL), and the mixture was washed with brine (200 mL, three times). The collected organic extract was dried over anhydrous Na2SO4, and filtered off from insoluble substances.
The filtrate was evaporated to dryness under reduced pressure at 40 °C, and the residue was

Synthesis of 6.
To a dry THF (10 mL) suspension of NaH (washed twice with dry n-hexane to remove mineral oil just prior to use; 57 mg, 2.5 mmol) was added a dry THF (20 mL) solution of 5 (0.733 g, 0.950 mmol) dropwise over 30 min at 0 °C under Ar, and the resulting mixture was stirred for 10 min at 0 °C.
To the reaction mixture was added propargyl bromide (0.100 mL, 1.33 mmol) at 0 °C, and the resulting mixture was stirred overnight at 25 °C. Then, the reaction mixture was cooled to 0 °C followed by addition of water (1 mL) and CH2Cl2 (50 mL), and the resulting mixture was filtrated through celite 545. The filtrate was evaporated to dryness under reduced pressure at 40 °C, and the residue was chromatographed on silica gel (Silica Gel 60) with a gradient of AcOEt/n-hexane (50/50 to 70/30 v/v) to allow isolation of 6 (0.602 g, 4 0.745 mmol) as colorless oil in 78% yield. 1 H NMR (400 MHz, CDCl3 containing 0.03% TMS, 24 °C): which was then cooled to 25 °C followed by evaporation under reduced pressure at 50 °C. To the residue was added brine (40 mL) and the mixture was extracted with with EtOAc (50 mL, three times). The collected organic extract was dried over anhydrous Na2SO4, and filtered off from insoluble substances.
The filtrate was evaporated to dryness under reduced pressure at 40 °C, and the residue was

Synthesis of Tribranched TEG modified deoxythymine
Tribranched TEG modified deoxythymine 14 was synthesized by following the synthetic procedure of Dibranched TEG modified deoxythymine 9 .

The synthesis of oligonucleotides containing TEG-modified deoxythymines
The oligonucleotides with TEG-modified deoxythymines were synthesized using standard phosphoramidite methods on an automated DNA synthesizer. The amidites of TEG-modified deoxythymines were incorporated during synthesis, and the obtained oligonucleotides were purified by Japan Bio Services Co., LTD, Japan. To remove the protecting groups, the oligonucleotides were incubated in 30% (v/v) ammonia represented by violet balls. All atoms of (2X4) are shown in ball and stick representation with red for oxygen, blue for nitrogen, gray for carbon, and white for hydrogen. Figure S4. Denaturing gel electrophoresis of (a) F-Q1 and (b) F-Q1-(2X4)4,,7,13 after the addition of human serum at 37 °C. Samples incubated for 0, 1, 3, 5, and 24 hours were loaded on lanes 1 to 5, respectively. Lane M shows 10-nt size marker. After electrophoresis, the gels were stained by SYBR® Gold (PerkinElmer Life Sciences). 11 Figure S5. Denaturing gel electrophoresis of F-Q1-(3X4)4,,7,13 after the addition of human serum at 37 °C.