Solid-phase synthesis and structural characterisation of phosphoroselenolate-modified DNA: a backbone analogue which does not impose conformational bias and facilitates SAD X-ray crystallography† †Electronic supplementary information (ESI) available: PDB ID: 6S7D. See DOI: 10.1039/c9sc04098f

Stable selenium-modified DNA which maintains the native tertiary structure has been prepared under automated conditions enabling SAD X-ray crystallography.

Section A. General Information CAUTION! Strict safety guidelines are required when handling selenium-containing compounds due to the potential for generating highly toxic materials. 1,2 Potassium selenocyanate and its adducts must always be handled in a fume hood and wearing the appropriate PPE to avoid exposure to airways, eyes and skin. Contaminated equipment must be treated with aqueous sodium hypochlorite (bleach) to inactivate the selenium species to a less harmful oxidised form. 3 Unless further purified (see below), reagents and solvents were of HPLC or analytical grade and used as supplied (Merck / Sigma-Aldrich / Chemgenes or LGC / Link Technologies). Anhydrous acetonitrile used in reactions was diluent grade (LGC / Link Technologies) which was stored under argon over 1 g or 5 g molecular traps (LGC Biosearch). Methyl-protected phosphoramidite derivatives used in the preparation of the corresponding H-phosphonates were purchased from LGC / Link Technologies (dA, dC and T) or ChemGenes (dG). Analytical grade dichloromethane used in reactions was dried and distilled from calcium hydride, stored over 3 Å activated molecular sieves for a minimum of 24 h in the absence of light, purged with argon for 30 min and used within 7 days. When used to co-evaporate trityl protected material the anhydrous dichloromethane was deacidified by passing over a plug of activated basic alumina prior to use. HPLC grade methanol >99% (Fischer) was rendered anhydrous following storage over 3 Å activated molecular sieves 48-72 h prior to use. N,Ndiisopropylethylamine 99% (TCI) was dried over 3 Å activated molecular sieves for at least 48 h prior to use. Column chromatography was performed using silica (Fluorochem 60 Å, 40 -63 µm) which had been dried at 150 °C. Mobile phases were prepared using HPLC grade acetone, analytical grade dichloromethane and rendered anhydrous following storage over 3 Å activated molecular sieves (minimum 24 h) and purged with argon for 30 min prior to their use to purify phosphoramidites 5a-d. Pyridine (Acros 99+% extra pure) was refluxed and distilled from potassium hydroxide immediately prior to use.

Mass Spectroscopy
For nucleosides, dinucleotides and dinucleotide phosphoramidites, mass spectrum were recorded using a VG Quattro II Triple Quadrupole Mass Spectrometer (Electrospray) or using a Waters Xevo G2-XS QTof Mass Spectrometer (Electrospray). Mass spectrometry was performed by Analytical Services and Environmental Projects (ASEP) at Queen's University Belfast.
For oligodeoxynucleotides mass spectrum were recorded using a Waters Xevo G2-XS QTof Liquid Chromatography For oligodeoxynucleotides MALDI-TOF spectra were acquired using an Ultraflex MALDI-TOF (Bruker-Daltonik, Germany) controlled using flex control 3.0 software (Bruker-Daltonik, Germany). The instrument was equipped with a nitrogen laser (λ = 337 nm) set to 32% power and triggered at 25 Hz for a total of 300 shots. Analysis was performed in positive ion reflection mode with reflector voltages of 26.30 kV and 13.75 kV respectively. Detector voltage was set at 1898V and all fragments were isolated as hydrogen adducts [M+H]. Spectra were analysed using Flex analysis 3.0 software (Bruker-Daltonik, Germany) and expressed as m/z. Samples were diluted to a concentration of 200 nmol/L in ultrapure water. Matrix solution (0.5 µL) was transferred to a clean ground steel MALDI sample plate and dried under reduced pressure. This was then overlaid with 0.5 µL of sample and dried under reduced pressure. This process was repeated for the peptide mix used for mass calibration. The 3-hydroxypicolinic acid matrix (3-HPA) was prepared by suspending 3-HPA (50 mg) and diammonium hydrogen citrate (10 mg) in 0.1% (v/v) TFA in 50% (v/v) MeCN : H2O.

HPLC:
HPLC was performed on a ThermoFisher SpectraSYSTEM modular HPLC system consisting of a P2000 binary gradient pump and UV1000 sample detector. Samples were injected manually via a Rheodyne injection valve. The HPLC was interfaced via an SN4000 controller (Thermo Scientific) to a Windows PC running ChromQuest 5.0 data acquisition software (Thermo Scientific). Buffers were prepared using H2O purified to 18.2 mΩ by reverse osmosis (Barnstead NANOpure Diamond water purification system), acetonitrile (Aldrich 34851) triethylamine (Aldrich 471283), acetic acid (Aldrich 320099) and CO2 generated by sublimation of the solid compound.
Triethylammonium acetate (TEAA) buffers were prepared from solutions of acetic acid in H2O following neutralisation with triethylamine to pH 6.5 and suitable dilution to give final concentrations of: 100 mM TEAA (aq.) (
Under a gentle stream of argon, a microwaveable test tube (10 mL) was sequentially charged, in quick succession, with 5′-O-tosylthymidine (396 mg, 1.0 mmol), potassium selenocyanate (216 mg, 1.5 mmol, 1.5 eq), anhydrous MeCN (3 mL) and a stir bar. The tube was sealed, and the suspension was stirred to achieve homogeneity and then subject to microwave irradiation (sealed vessel, 100 °C, 20 W, 1.5 h). The reaction mixture was extracted from the vessel in methanol (10 mL) and stored at -20 °C under inert conditions. This was repeated a total of twenty times (over 4 days), the extractates were then combined and stirred at ambient temperature during addition of benzyl bromide (1.40 mL, 11.8 mmol, 0.59 eq). After 60 min complete consumption of excess potassium selenocyanate was observed by tlc and the quenched reaction mixture was reduced in vacuo. After the volume had been reduced by half, silica gel (40 g) was added and residual solvent removed.
The crude material was purified by silica gel column chromatography eluting with a gradient of 5-15% (v/v) methanol in DCM. Fractions containing pure 2 were combined and reduced in vacuo to yield a cream, electrostatically-charged amorphous solid (4.94 g, 75%). Characterisation consistent with the literature. containing pure product (as a mixture of diastereoisomers) were combined, concentrated to a viscous oil in vacuo and diluted in a minimum volume of DCM (ca. 10 mL) and 50% (v/v) diethyl ether/n-hexane added until the first appearance of turbidity.

Oligodeoxynucleotide synthesis:
Modified oligodeoxynucleotides were synthesised trityl-on using the manual syringe method on 1 µmol scale in duplicate (ODN 1, ODN 2, ODN 3, ODN 4 40% (w/v) MeNH2 (aq)). The reaction mixtures were stored at ambient temperature for 2 h, the CPG removed by filtration using a Corning Co-star centrifuge tube filter (2 mL volume, 0.45 μm cellulose acetate filter) at 13,000 rpm, washed with water (2 x 1 mL) reduced in vacuo and analysed by RP-HPLC. The chromatographic profiles from the crude thiophenol and NaDEC-treated tritylated oligomers were essentially identical and were therefore combined and purified by RP-HPLC using gradient G1. Fractions containing pure material were pooled, reduced in vacuo to ca. 50 µL and to this solution was added 80% (v/v) aqueous acetic acid (1 mL) and stored at ambient temperature for 1 h. The reaction mixture was diluted with absolute ethanol (1 volume) and reduced in vacuo by half. This procedure was repeated but subsequently evaporated to dryness and the residues suspended in TEAB buffer A (1 mL) and centrifuge (10 min, 13,000 rpm). The solution of detritylated oligomer ODN 1 was subject to desalting by RP-HPLC using buffers derived from volatile salts (gradient G2) and subsequent

mL). This was followed by AMA deprotection (3 h). The DMTr-ON oligomer was purified by RP-HPLC
using gradient G7. Detritylation was effected using 80% (v/v) aqueous acetic acid (1 mL) and storage at ambient temperature for 1 hr. Desalting was achieved by RP-HPLC using gradient G5 followed by co-evaporation with H2O (6 x 500 L). Analytical RP-HPLC performed using gradient G6. Initial attempts resolved two peaks. The analytical chromatogram was repeated at 52 °C to resolve one peak. tR = 29.60 min. ESI mass spec is available in Table 2.

Enzyme Digest
Solutions containing 2 OD 260nm units of either ODN 5 (d(TSeTCCCGGGAA)) or its native congener (d(TTCCCGGGAA)) in 100 mM Tris HCl buffer (pH 8), and 100 mM NaCl were heated at 95 °C for 3 min and snap cooled on ice. To these solutions were then added 4.2 µL MgCl2 (1 M stock soln, final concentration = 14 mM) followed by 10 µL of a freshly prepared solution of snake venom (Sigma -V7000, 1 mg/mL aqueous stock soln, final concentration = 33 µg/mL) (total end volume of digest = 300 µL). After vortex mixing, digestions were incubated at 37 °C for 8 h. From each digest, an aliquot (150 µL) was removed and quenched following heating at 95 °C for 3 min. The reaction mixture was then analysed by RP-HPLC using gradient G8.

UV Thermal Denaturation Studies
ODN 2-6 along with their native analogues were diluted in sodium phosphate buffer (10 mM, pH 7) and NaCl (100 mM) to a final concentration of 10 µM ssDNA and a final volume of 600 µL. The sequences were annealed by heating to 90 °C for 3 min and allowed to slowly cool to room temperature. UV spectra were recorded on an Agilent technologies Cary 100/300 UV-vis spectrophotometer equipped with a 6 x 6 multicell block Peltier cuvette holder and a thermoelectric temperature controller. For the measurement the instrument was programmed to heat from 10-70 °C (ODN 2-5) or 10-90 °C (ODN 6) with a temperature change rate of 0.5 °C per minute in a 1 cm pathlength cuvette. UV absorbance was monitored at 260 nm and recorded at 0.5-min intervals. Melting temperatures (Tm) were determined by the maximum of the first derivative averaged over two runs (ODN 2-5). Tm for ODN 6 was determined as the midpoint of the normalised absorbance and averaged (Tm) over two runs.

CD Spectroscopy Studies
ODN 2-6 along with their native analogues were diluted in sodium phosphate buffer (10 mM, pH 7) and NaCl (100 mM) to a final concentration of 10 µM ssDNA and final volume of 600 µL. The sequences were annealed by heating to 90 °C and slowly cooling to room temperature. CD spectra were recorded at 10 °C between 220 and 350 nm with 1 nm wavelength increments in a 1 cm pathlength cuvette on a Chirascan spectrophotometer at Diamond Light Source. Data were collected on beamline I03 at Diamond Light Source. 3600 images were collected, using a 0.1° oscillation and 0.05 s exposure time. The data were collected just above the Se-edge, to facilitate anomalous phasing, using a wavelength of 0.9596 Å. The data was processed using xia2 7 with DIALS 8 and the structure solved using Phenix.autosol 9 , with the anomalous signal of Se. It was subsequently found that the crystal suffered radiation damage, so the dataset was cut to use the first 600 images. The model was built using Coot 10 and refined with Phenix.refine, to give a final Rfactor of 0.18 and Rfree of 0.23. The structure was initially refined in spacegroup P 6122 but, following analysis with Phenix.xtriage, it became apparent that the crystal was a near-perfect twin with true symmetry in a lower space group. This was assigned as P 3121 following attempted model building and refinement in all possible lower symmetry spacegroups. The twin law applied during refinement was -h,k,l and the crystal possessed merohedral twinning with a twin fraction of 0.47. Full data processing and refinement statistics can be found in Table 3.
The asymmetric unit of the structure contains two DNA strands (in an A-DNA double helix), two Na + , one Cl -, two Ba 2+ bifurcated to guanine bases, 64 water molecules and one spermine molecule bound between the phosphate backbones The two strands are coloured with carbon atoms in either yellow or green, to aid clarity. The carbon atoms of the spermine molecule are coloured magenta. All other atoms are coloured according to type with Na + black, Ba 2+ silver, Clyellow, nitrogen blue, hydrogen white, oxygen red and the phosphate backbone is drawn as an orange strand. Water molecules are drawn as red spheres.