Synthesis and biophysical properties of carbamate-locked nucleic acid (LNA) oligonucleotides with potential antisense applications

Carbamate-LNA oligonucleotides have improved biophysical properties for theraputic applications.


General Synthetic Procedures
All reagents were purchased from Sigma-Aldrich, Acros Organics, Fluka and Fisher Scientific, FluoroChem, Jena Bioscience or Alfa Aesar and used without further purification. Dry solvents (pyridine, Et 3 N, CH 2 Cl 2 , THF, MeCN) were obtained using an MBraun SPS Bench Top solvent purification system (SPS). All air/moisture sensitive reactions were carried out under inert atmosphere (argon) in oven-dried glassware. Solvents used for phosphitylation reactions and purification were also degassed by saturating the solvents with argon for 20 min. Reactions were monitored by thin layer chromatography (TLC) using Merck Kieselgel 60 F24 silica gel plates (0.22 mm thickness, aluminium backed). The compounds were visualized by UV irradiation at 254/265 nm and by staining in p-anisaldehyde or ninhydrin solution followed by gentle heating. Silica column chromatography was performed using Merck Geduran® 60 Å (40-62 µm) or using a Biotage Isolera™ Prime auto column (10-100 g SNAP ultra-cartridges). 1 H NMR (400 MHz), 13 C NMR (101 MHz) and 31 P NMR (162 MHz) were recorded using a Bruker DPX 400 (AVIIIHD 400) or Bruker AVII 500 spectrometer ( 1 H-500 MHz, 13 C-126 MHz) and referenced to an appropriate deuterated solvent. Assignment was aided by DEPT-135, COSY, HSQC-DEPT and HMBC spectra. Data was then processed using MestreNova software. Low resolution mass spectrometry (LRMS) using electrospray ionisation positive (ESI+) and negative (ESI-) modes was recorded on a Waters ZMD quadrupole mass spectrometer in HPLC grade MeOH/MeCN. High resolution mass spectrometry (HRMS) using electrospray ionisation was recorded on a Bruker APEX III FT-ICR mass spectrometer. Oligonucleotide mass spectrometry was recorded on a UPLC-MS Waters XEVO G2-QTOF (ESI-) using an ACQUITY UPLC oligonucleotide BEH C18 column, 130 Å (1.7 µm, 2.1 mm × 50 mm). Data was then deconvoluted using MassLynx v4.1. A gradient of MeOH in Et 3 N and hexafluoroisopropanol (HFIP) was used (buffer A, 8.6 mM Et 3 N, 200 mM HFIP in 5% MeOH/H 2 O (v/v); buffer B, 20% buffer A in MeOH). Buffer B was increased from 0-70% over 8 min, at a flow rate of 0.2 mL min −1 .

Synthesis of DNA oligonucleotides
Standard phosphoramidites, solid supports and reagents were purchased from Link Technologies and Applied Biosystems. LNA phosphoramidites were obtained from Exiqon. Automated solid phase synthesis of oligonucleotides (trityl off) was performed on an Applied Biosystems 394 synthesiser. Synthesis was performed on 1.0 µmole scale involving cycles of acid catalysed detritylation, coupling, capping, and iodine oxidation. Standard DNA phosphoramidites were coupled for 60 s while extended coupling time of 10 min was used for LNA phosphoramidites. Coupling efficiencies and overall synthesis yields were determined by the inbuilt automated trityl cation conductivity monitoring facility and were ≥98.0% in all cases. The oligonucleotides were then cleaved from the solid support and protecting groups from the nucleobase and backbone were removed by exposure to conc. aq ammonium hydroxide for 60 min at room temperature, followed by heating in a sealed tube for 5 h at 55 °C.

Biophysical studies and assays
Ultraviolet melting studies UV DNA melting curves were recorded in a Cary 4000 Scan UV-Visible Spectrophotometer using 3 µM of each oligonucleotide in a 10 mM phosphate buffer containing 200 mM NaCl at pH 7.0. Samples were annealed by heating to 85 °C (10 °C/min) and then slowly cooling to 20 °C (1 °C/min). As these six successive cycles (heating and cooling) were performed at a gradient of 1 °C/min, the change in UV absorbance at 260 nm was recorded. The melting temperature was calculated from the first derivative of the melting curve using in built software.
Additional UV-data

Circular dichroism studies
A solution of 3 µM sample oligonucleotide and complementary sequence was added to 10 mM phosphate buffer with 200 mM NaCl (pH 7.0). Sample were incubated for 2 mins at 85 °C and cooled to rt. Circular dichroisms was performed on a Chirscan Plus spectrometer using a quartz cuvette (L = 10 mm). Scans were taken at 20 °C from 200-340 nm with 0.5 a step and 1.0 s time point intervals. The average of four scan were taken and smoothed to 20 points using a third order polynomial (OriginPro 2017). The spectra were then baseline corrected to the θ-value at 340 nm.

Snake venom assay
5 nmol of oligonucleotide was dissolved in 100 μL of buffer (50 mM Tris-HCl, 10 mM MgCl 2 , pH 9.0). 20 μL of sample was removed (t = 0 control) and 2 μL of snake venom phosphodiesterase 1 from C. adamanteus (Sigma-Aldrich, cat P3243, 0.45 units, dissolved in 7 mL H 2 O) was added to the remaining volume. The reaction was incubated at 37 °C and 20 μL aliquots were removed at set times, mixed with formamide (v/v) and stored at -80 °C. Samples were analysed by 20% denatured polyacrylamide gel electrophoresis (400 V) and viewed under short wave UV-light.

Foetal bovine serum (FBS) assay
5 nmol of oligonucleotide was dissolved in 50 μL of Dulbecco's PBS and 50 μL of FBS (Gibco, standard sterile filtered). This was then vortexed and 20 μL was removed (t = 0 control), mixed with formamide (v/v) and frozen at -80 °C. The remaining solution was incubated at 37 °C and 20 μL aliquots were removed at set times, mixed with formamide (v/v) and stored at -80 °C. Samples were analysed by 20% denatured polyacrylamide gel electrophoresis (400 V) and viewed under short wave UV-light.

A B
Gel quantification Figure S4. Enzymatic stability of ONs shown in Figure 3 in (A) snake venom and (B) foetal bovine serum. Data points were acquired from PAGE gels (Figure 3) by creating a plot profile for each lane and successive integration of the area under the curve from band corresponding to the highest molecular weight ONs using ImageJ. The intensities are presented as percentage in relation to t=0 controls.