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Compound '''12''' (2 mg, 2.6 μmol) was dissolved in DMSO (50μL) and added to a tube with amine modified DNA (2.5 nmol) in MilliQ water (50 μL). Precipitate was observed and MeCN (3∙50 μL) was added until the solution remained clear. Triethylamine (50 μL) was added and the reaction mixture incubated for 1 h at 22 ⁰C. To precipitate the DNA conjugate NaOAc (43 μL, 3M, pH=5.2), glycogen (1 μL) and cold ethanol (750 μL, 96%) was added. The mixture was incubated on dry ice for 15 min and centrifuged (4 ⁰C, 60 minutes 14000 rpm). The supernatant was decanted off and the DNA pellet redissolved in TEAA buffer (100 μL). The crude product was purified by RP-HPLC (10% to 70% MeOH in TEAA buffer over 30 min).  The product was lyophilized o.n. The light green product was estimated to 1.6 nmol (65%).
Compound '''12''' (2 mg, 2.6 μmol) was dissolved in DMSO (50μL) and added to a tube with amine modified DNA (2.5 nmol) in MilliQ water (50 μL). Precipitate was observed and MeCN (3∙50 μL) was added until the solution remained clear. Triethylamine (50 μL) was added and the reaction mixture incubated for 1 h at 22 ⁰C. To precipitate the DNA conjugate NaOAc (43 μL, 3M, pH=5.2), glycogen (1 μL) and cold ethanol (750 μL, 96%) was added. The mixture was incubated on dry ice for 15 min and centrifuged (4 ⁰C, 60 minutes 14000 rpm). The supernatant was decanted off and the DNA pellet redissolved in TEAA buffer (100 μL). The crude product was purified by RP-HPLC (10% to 70% MeOH in TEAA buffer over 30 min).  The product was lyophilized o.n. The light green product was estimated to 1.6 nmol (65%).


 
==Reference==
#Hussey Hussey, S. L. ''et al.''. Synthesis of chimeric 7α-substituted estradiol derivatives linked to cholesterol and cholesterylamine. ''Org. Lett.'' '''4''', 415–418 (2002). [http://dx.doi.org/10.1021/ol0171261}34]
#Horwitz Horwitz, J. P. ''et al.'' Nucleosides. IX. The formation of 2’,3'-unsaturated pyrimidine nucleosides via a novel beta-elimination reaction. ''J. Org. Chem.'' '''121''', 205–211 (1966). [http://dx.doi.org/10.1021/jo01339a045}32]
#Trybulski Trybulski, E. J. ''et al''. The synthesis and biochemical pharmacology of enantiomerically pure methylated oxotremorine derivatives. ''J. Med. Chem.'' '''36''', 3533–3541 (1993).[http://dx.doi.org/10.1021/jm00075a007]
#Simeone Simeone, L. ''et al''. Cholesterol-based nucleolipid-ruthenium complex stabilized by lipid aggregates for antineoplastic therapy. ''Bioconjugate Chem.'' '''23''', 758–770 (2012) [http://dx.doi.org/10.1021/bc200565v]
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Chemical modification

General experimental data

All reactions were monitored by thin-layer chromatography (TLC) analysis on Merck silica gel 60 F254 TLC plates. The TLC plates were visualized by exposure to UV (254 nm) or by staining with an acidic solution of either p-anisaldehyde or vanillin. Flash column chromatography was performed using Merck silica gel 60 (230-400 mesh) as stationary phase. Preparative TLC was performed on glass plates coated with silica gel 60 F254. All solvents were of HPLC grade quality. For inert reactions, the solvents DMF and THF were dried prior to use by a MBRAUN solvent purification system. Dry MeOH and anhydrous pyridine were purchased and used without further treatment. For the DNA-conjugation procedures the used water was of Milli-Q quality.

Cholesterol (3) (CAS: 57-88-5) was purchased from Sigma Aldrich. 2′-Deoxyuridine (13) (CAS: 951-78-0) was purchased from Apollo Scientific Limited. 5-Propargylamino-ddUTP (20) was purchased from Jena Bioscience. Terminal deoxynucleotidyl transferase (TdT) labeling kit was purchased from Roche Diagnostics.

RP-HPLC purification was performed on a Hewlett-Packard Agilent 1100 series with a RP stationary column (flow = 1 mL/min, T=25 °C). The TEAA buffer for RP-HPLC is 0.1 M, pH = 7. NMR spectra were recorded on a Bruker BioSpin GmbH AscendTM 400 at 400 MHz (1H NMR) and at 100 MHz (13C NMR) and calibrated to the residual solvent peak. Chemical shifts are reported in parts per million. In the interpretation of the 1H NMR spectra, the following abbreviations are used: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. Melting points were measured on a Büchi B-540 instrument and are reported uncorrected. IR spectroscopy was performed on a PerkinElmer Spectrum TwoTM UATR spectrometer. Optical rotation was obtained on an ADP 440+ Polarimeter from Bellingham and Stanley and is reported as the specific rotation. High-resolution mass spectrometry (HRMS) was performed using electrospray ionization on a Bruker Daltonics MicrOTOF. Low-resolution mass spectrometry (LRMS) was performed on a Bruker Autoflex Speed MALDI-TOF instrument.

Cholesterol derivative

Tert-butyl cholesteroxy acetate (4). [1]

Cholesterol (3) (0.310 g, 0.802 mmol) was added to a flame-dried round-bottomed flask and dissolved in dry THF (2 mL) under an argon atmosphere. The solution was cooled to 0 °C and NaH (60% in mineral oil, 0.065 g, 1.63 mmol, 2 equiv.) was added. The resulting mixture was stirred for 1 h before the electrophile tert-butyl bromoacetate (0.30 mL, 2.0 mmol, 2.5 equiv.) was added. This solution was heated to room temperature and left for 20 h. The solution obtained a slightly yellow color. The reaction was cooled to 0 °C and quenched with a small amount of MeOH. The solvent was evaporated and the residue dissolved in DCM (15 mL). The organic phase was washed with H2O (2x15 mL), brine (10 mL), dried over Na2SO4, filtered by suction and evaporated to dryness in vacuo. The product was purified by flash column chromatography (EtOAc/pentane 3:97). This afforded compound 4 (0.031 g, 0.062 mmol, 8%) as a white solid.

Rf 0.29 (EtOAc/pentane 3:97). mp (uncorr.) 144.1-146.3 °C (lit.[2] 148-149 °C). 1H NMR (400 MHz, CDCl3): δ 5.35 (d, J = 5.1 Hz, 1H), 4.00 (s, 2H), 3.28-3.18 (m, 1H), 2.42-2.20 (m, 2H) 2.05-1.75 (m, 5H), 1.64-0.80 (m, 42H), 0.67 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 170.1, 140.7, 121.8, 81.4, 79.8, 66.1, 56.8, 56.2, 50.2, 42.3, 39.8, 39.5, 38.8, 37.1, 36.8, 36.2, 35.8, 31.9, 31.9, 28.2, 28.1, 28.0, 24.3, 23.8, 22.8, 22.6, 21.1, 19.4, 18.7, 11.9. HRMS (ESI) m/z [M + Na]+ calcd for C33H56O3Na, 523.4127; found, 523.4127. IR (neat) νmax (cm-1): 2953, 2933, 2866, 1745, 1127. [α]D26 -31.6 (c 0.56, CHCl3).

Cholesteroxy-acetic acid (5). [1]

To a 25 mL round-bottomed flask containing 4 (0.091 g, 0.18 mmol) was added formic acid (9 mL) and the reaction was left to react for 20 h at rt. The solvent was co-evaporated with DCM. The residue was dissolved in Et2O (25 mL) and extracted with sat. aq. NaHCO3 (8x25 mL). The combined water phases were made acidic with 2 M HCl and extracted with DCM (6x20 mL). The combined organic phases were washed with H2O (25 mL), brine (25 mL), dried over MgSO4, filtered under suction and evaporated to dryness in vacuo, affording 5 (0.061 g, 0.14 mmol, 76%) as a white solid.

Rf 0.47 (EtOAc/pentane 1:1 + 1% formic acid). mp (uncorr.) 152.5-154.9 °C (lit.[2] 163-165 °C). 1H NMR (400 MHz, CDCl3): δ 5.38 (d, J = 5.0 Hz, 1H), 4.13 (s, 2H), 3.38-3.26 (m, 1H), 2.41-2.23 (m, 2H), 2.05-1.76 (m, 6H), 1.62-0.81 (m, 33H), 0.68 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 174.0, 140.1, 122.6, 80.6, 65.3, 56.9, 56.3, 50.3, 42.5, 39.9, 39.7, 38.8, 37.1, 36.9, 36.3, 35.9, 32.1, 32.0, 28.4, 28.2, 28.2, 24.4, 24.0, 23.0, 22.7, 21.2, 19.5, 18.9, 12.0. HRMS (ESI) m/z [M + Na]+ calcd for C29H48O3Na, 467.3501; found, 467.3498. IR (neat) νmax (cm-1): 3000 (broad), 2935, 2904, 2866, 1726, 1704, 1269, 1146. [α]D26 -30.9 (c 0.91, CHCl3).

2,5-dioxopyrrolidin-1-yl cholesteroxy acetate (6).

The two reagents EDC•HCl (0.044 g, 0.23 mmol, 2.1 equiv.) and NHS (0.019 g, 0.17 mmol, 1.7 equiv.) were added to a solution of 5 (0.048 g, 0.11 mmol) in DCM (3 mL) in a 10 mL round-bottomed flask. The resulting mixture was stirred for 3 h, after which it was transferred to a separatory funnel and washed with a 2.5% aq. solution of NaHSO4 (3x5 mL). The organic phase was dried over Na2SO4, filtered by suction and evaporated to dryness in vacuo. The resulting product was not purified further, yielding 6 (0.047 g, 0.087 mmol, 80%) as a slightly yellow solid.

Rf 0.39 (EtOAc/pentane 3:7). 1H NMR (400 MHz, CDCl3): δ 5.37 (d, J = 5.0 Hz, 1H), 4.47 (s, 2H), 3.37-3.28 (m, 1H), 2.88-2.80 (m, 4H) 2.43-2.36 (m, 1H), 2.33-2.23 (m, 1H), 2.04-1.77 (m, 5H), 1.61-0.82 (m, 33H), 0.67 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 168.9, 166.6, 122.5, 81.0, 63.7, 56.9, 56.3, 50.2, 42.5, 39.9, 39.7, 38.7, 37.1, 36.9, 36.3, 35.9, 32.1, 32.0, 28.4, 28.2, 25.7, 24.4, 24.0, 23.0, 22.7, 21.2, 19.5, 18.9, 12.0. HRMS (ESI) m/z [M + Na]+ calcd for C33H51NO5Na, 564.3659; found, 564.3630.

Photosensitizer

Extraction of pheophytin a (7). [3]

Spirulina powder (125.6 g) was mixed with acetone (450 mL) under stirring. Liquid nitrogen (approx. 400 mL) was slowly added until the suspension was frozen. The mixture was allowed to melt, and the resulting slurry was refluxed for 2 h. The slurry was filtered and the precipitate washed with acetone (2x10 mL) and discarded. Acetic acid (1.25 mL) was added to the filtrate, after which the solvent was evaporated and crude product dried in vacuo. The crude product was purified by flash column chromatography (pentane/DCM 1:20 to elute the cartenoids (red and yellow bands), then EtOAc/DCM 1:5 to elute the product).

The product was obtained as a black solid (1.205 g). Rf=0.65 (EtOAc/DCM 1:15); 1H NMR (appendix) (400 MHz, CDCl3): δ(ppm): 9.39 (s, 1H), 9.21 (s, 1H), 8.56 (s, 1H), 7.88 (dd, J=12.1, 17.7, 1H), 6.31 (s, 1H), 6.22 (d, J=17.9, 1H), 6.12 (d, J=11.6, 1H), 5.28-5.10 (m, 1H), 4.60-4.43 (m, 3H), 4.34-4.21 (m, 1H), 3.93 (s, 3H), 3.67 (s, 3H), 3.53 (q, J=7.6, 2H), 3.37 (s, 3H), 3.09 (s, 3H) 2.73-2.63 (m, 1H), 2.58-2.47(m, 1H), 2.43-2.30 (m, 1H), 2.30-2.15 (m, 1H), 1.64 (t, J=7.7, 3H), 1.61 (s, 3H), 1.58-0.94 (m, 24H), 0.87 (d, 6H), 0.82 (2∙d, 6H), 0.48 (s, 1H), -1.71 (s, 1H); 13C NMR (appendix) (100 MHz, CDCl3): δ(ppm): 189.8, 173.1, 172.4, 168.9, 161.4, 155.8, 151.1, 149.8, 145.3, 143.0, 142.2, 138.1, 136.6, 136.4, 136.3, 132.0, 129.2, 129.2, 129.1, 122.9, 117.9, 106.4, 104.5, 97.7, 93.3, 64.9, 61.6, 53.0, 51.3, 50.3, 39.9, 39.5, 37.5, 37.5, 37.4, 36.8, 32.9, 32.8, 31.3, 30.0, 28.1, 25.1, 24.9, 24.6, 23.2, 22.9, 22.8, 19.9, 19.8, 19.6, 17.5, 16.4, 12.3, 12.2, 11.4; ES-HRMS m/z calcd for C55H75N4O5 [M+H]+: 871.5732; found 871.5737; mp (uncorr.) 107.3-110.4 ⁰C (literature [4] 113-114 ⁰C).

Synthesis of Pheophorbide a allyl ester (8). [3]

Pheophytin a (7) (0.243 g, 279 μmol, 1 eq.) was dissolved in allyl alcohol (10 mL, 147 mmol) and conc. sulphuric acid (0.17 mL, 3.19 mmol, 11 eq.) was added. The mixture was stirred at rt. and after 28 h the solution was diluted with water (20 mL) and neutralized with aqueous saturated NaHCO3 (10 mL). The water phase was extracted with DCM (3x10 mL), and the combined organic phases were dried over Na2SO4, filtered, concentrated in vacuo and purified by flash column chromatography (EtOAc/DCM 1:17).

The product was obtained as a black solid (140 mg, 79%). Rf=0.42 (EtOAc/DCM 1:15); 1H NMR (appendix) (400 MHz, CDCl3): δ(ppm): 9.50 (s, 1H), 9.40 (s, 1H), 8.56 (s, 1H), 8.00 (dd, J=11.5, 18.0, 1H), 6.30 (d, J=18.3, 1H), 6.25 (s, 1H), 6.19 (d, J=11.7, 1H), 5.82-5.70 (m, 1H), 5.22-5.06 (m, 2H), 4.52-4.39 (m, 3H), 4.31-4.19 (m, 1H), 3.88 (s, 3H), 3.70 (q, J=7.6, 2H), 3.69 (s, 3H), 3.41 (s, 3H), 3.24 (s, 3H), 2.70-2.58 (m, 1H), 2.57-2.44 (m, 1H), 2.42-2.29 (m, 1H), 2.27-2.17 (m, 1H), 1.81 (d, J=7.1, 3H), 1.70 (t, J=6.7, 3H), 0.55 (s, 1H), -1.62 (s, 1H); 13C NMR (appendix) (100 MHz, CDCl3): δ(ppm): 189.8, 172.7, 172.3, 169.8, 161.3, 155.8, 151.1, 149.8, 145.3, 142.2, 138.1, 136.6, 136.4, 136.3, 132.0, 132.0, 129.2, 129.1, 129.1, 122.9, 118.6, 106.3, 104.6, 97.7, 93.2, 64.8, 63.4, 53.0, 51.2, 50.2, 31.2, 29.9, 23.2, 19.6, 17.0, 12.3, 12.3, 11.4; ES-HRMS m/z calcd for C38H41N4O5 [M+H]+: 633.3071; found 633.3076; mp (uncorr.) 184.4-188.2 ⁰C.

Synthesis of Pyropheophorbide a allyl ester (PPa allyl ester) (9). [3]

Pheophorbide a allyl ester (8) (125 mg, 198 μmol) was dissolved in 2,4,6-collidine (15 mL, 114 mmol) and stirred at 170 ⁰C under an argon atmosphere. After 90 min. the solution was cooled to rt. The solvent was evaporated in vacuo, and the crude product was purified by flash column chromatography (EtOAc/DCM 1:15).

The product was obtained as a black solid (103 mg, 91%). Rf=0.30 (EtOAc/DCM 1:15); 1H NMR (appendix) (400 MHz, CDCl3): δ(ppm): 9.44 (s, 1H), 9.33 (s, 1H), 8.55 (s, 1H), 7.97 (dd, J=11.7, 17.8, 1H), 6.27 (dd, J=17.9, 1.3, 1H), 6.16 (dd, J=1.3, 11.5, 1H), 5.88-5.76 (m, 1H), 5.31-5.06 (m, 4H), 4.58-4.45 (m, 3H), 4.34-4.26 (m, 1H), 3.64 (q, J=7.6, 2H), 3.63 (s, 3H), 3.40 (s, 3H), 3.20 (s, 3H), 2.77-2.65 (m, 1H), 2.64-2.53(m, 1H), 2.39-2.25 (m, 2H), 1.82 (d, J=7.3, 3H), 1.68 (t, J=7.3, 3H), 0.42 (s, 1H), -1.73 (s, 1H); 13C NMR (appendix) (100 MHz, CDCl3): δ(ppm): 196.4, 172.9, 171.5, 160.4, 155.3, 150.9, 149.1, 145.1, 141.7, 138.0, 136.3, 136.2, 136.0, 132.1, 131.7, 130.6, 129.4, 128.5, 122.7, 118.7, 106.2, 104.2, 97.3, 93.2, 65.4, 51.8, 50.1, 48.2, 31.2, 30.0, 23.3, 19.6, 17.6, 12.3, 12.2, 11.4; ES-HRMS m/z calcd for C36H39N4O3: 575.3017; found 575.3022; mp (uncorr.) 80.1-81.3 ⁰C; UV-Vis (appendix) (MeCN) λmax (log⁡ε ) = 666 (4.4), 410 (4.8), 274 nm (5.2).

Synthesis of Indium (pyropheophorbide a allyl ester) chloride (In(PPa-allyl ester)Cl) (10). [3]

PPa allyl ester (9) (88 mg, 153 μmol, 1 eq.) was dissolved in benzene (26 mL), followed by addition of InCl3 (541 mg, 2.45 mmol, 16 eq.), NaOAc (843 mg, 10.7 mmol, 70 eq.) and K2CO3 (865 mg, 6.43 mmol, 42 eq.). The reaction mixture was refluxed under an argon atmosphere for 90 min., then allowed to cool to rt., and neutralized with acetic acid. The organic phase was separated and washed with water (4x10 mL). The water phase (slightly blue) was extracted with DCM (3x10 mL) until colourless. The combined organic phases were dried over Na2SO4, concentrated in vacuo, and purified by flash column chromatography (MeOH/DCM 1:100 + 0.1% formic acid). The product contained significant impurities and a second purification was performed (MeOH/DCM 1:200 + 0.1 % formic acid).

The product was obtained as a dark blue-green solid (50 mg, 45%). Rf=0.72 (MeOH/DCM 1:10); 1H NMR (appendix) (400 MHz, CDCl3): δ(ppm): 9.69 (s, 1H), 9.49 (s, 1H), 8.54 (s, 1H), 7.94 (dd, J=11.7, 17.8, 1H), 6.25 (dd, J=17.8, 1.4, 1H), 6.16 (dd, J=1.4, 11.8, 1H), 6.00-5.67 (m, 1H), 5.36-4.92 (m, 4H), 4.70-4.23 (m, 4H), 3.79 (q, J=7.7, 2H), 3.65 (s, 3H), 3.35 (s, 3H), 3.30 (s, 3H), 2.89-2.42 (m, 3H), 2.24-2.11 (m, 1H), 2.00 (d, J=7.8, 1H (should be 3H)), 1.74 (t, J=7.8, 3H); 13C NMR (appendix) (100 MHz, CDCl3): δ(ppm): 195.4, 172.8, 169.1, 160.8, 156.1, 153.9, 150.7, 146.6, 146.4, 145.5, 144.6, 140.6, 136.8, 135.6, 135.0, 132.9, 132.1, 129.4, 122.8, 118.8, 107.9, 106.9, 100.5, 92.5, 65.5, 51.0, 48.3, 48.3, 30.4, 29.0, 23.4, 19.7, 17.4, 12.9, 12.4, 11.2; ES-HRMS m/z calcd for C36H37N4O3 [M+H]+: 723.1587; found 723.1581; mp (uncorr.) 119.2-121.1 ⁰C; UV-Vis (appendix) (MeCN) λmax (log⁡ε )= 657 (5.5), 423 (5.7), 230 nm (5.3).

Synthesis of Indium (pyropheophorbide a) chloride (In(PPa)Cl) (11). [3]

Pd(PPh3)4 (48 mg, 42 μmol, 0.2 eq.) and PPh3 (15 mg, 57 μmol, 0.3 eq.) were dissolved in DCM (8 mL) and added to a suspension containing In(PPa-allyl ester)Cl (10) (132 mg, 182 μmol, 1 eq.) and Et2NH∙HCl (119 mg, 1.09 mmol, 6 eq.) in DCM (15 mL). The reaction mixture was stirred at rt. PPh3 (32 mg, 122 μmol, 0.7 eq), Pd(PPh3)4 (61 mg, 53 μmol, 0.3 eq) and Et2NH∙HCl (66 mg, 602 μmol, 3.3 eq) was added until no further progress was observed with TLC analysis after 4 h. According to the TLC analysis the conversion was approximately 50%. The reaction mixture was concentrated to a smaller amount of liquid, which could be transferred directly to the column. The crude product was purified by flash column chromatography (MeOH/DCM from 5:200 + 2 drops of AcOH to 6:200 + 2 drops of AcOH). In the 1H-NMR spectrum signals from (4) were present and a second purification was performed (MeOH/DCM from 5:200 + 2 drops of AcOH to 9:100 + 3 drops of AcOH). The product was dried in vacuo o.n.

The product was obtained as a dark green solid (26 mg, 21%). Rf=0.52 (MeOH/DCM 1:10); 1H NMR (appendix) (400 MHz, CDCl3): δ(ppm): 9.68 (s, 1H), 9.45 (s, 1H), 8.18 (s, 1H), 7.94 (dd, J=11.7, 17.7, 1H), 6.21 (d, J=18.1, 1H), 6.10 (d, J=11.7, 1H), 4.67-4.48 (app. q, 2H), 3.91-3.54 (m, 3H), 3.63 (s, 3H), 3.44 (b, 1H), 3.30 (s, 3H), 3.25 (s, 3H), 1.73 (t, J=7.9, 3H), 1.33-1.19 (m, 7H); 13C NMR (appendix) (100 MHz, CDCl3): δ(ppm): 195.7, 169.2, 168.3, 160.8, 156.1, 153.9, 150.7, 146.6, 146.4, 145.5, 144.6, 140.5, 136.8, 135.7, 134.9, 132.8, 129.3, 122.8, 107.9, 106.8, 100.4, 92.6, 50.8, 48.3, 48.2, 30.2, 29.8, 23.3, 19.7, 17.4, 12.9, 12.4, 11.2; HRMS (ES) m/z calcd for C33H32InN4O3 [M-Cl]+: 647.1508; found 647.1512; mp (uncorr.) 220-230 ⁰C.

Synthesis of Indium (pyropheophorbide a NHS ester) chloride (12). [5]

Compound (11) (2.3 mg, 3.4 μmol, 1 eq.) was dissolved in dry DCM (5 mL) and EDC∙HCl (1.3 mg, 6.8 μmol, 2.1 eq.) and NHS (2.4 mg, 21 μmol, 6.2 eq.) were added. The mixture was stirred at rt. and after 65 min EDC∙HCl (1.6 mg, 36 μmol, 2.5 eq) was added. After 90 min. the reaction mixture was washed with NaHSO4 (2x5 mL, 2.5 m% aq.) and brine (5 mL). The organic phase was dried over Na2SO4, filtered, concentrated and dried in vacuo o.n.

The product was obtained as a dark green solid (2.5 mg, 91%). Rf=0.28 (MeOH/DCM 1:10); 1H NMR (appendix) (400 MHz, CDCl3): δ(ppm): 9.70 (s, 1H), 9.50 (s, 1H), 8.55 (s, 1H), 7.94 (dd, J=11.6, 17.9, 1H), 6.25 (d, J=17.4, 1H), 6.14 (d, J=11.6, 1H), 4.67-4.46 (m, 2H), 3.86-3.65 (m, 3H), 3.65 (s, 3H), 3.35 (s, 3H), 3.30 (s, 3H), 3.12-3.01 (m, 1H), 2.80 (s, 4H), 2.69-2.09 (m, 4H), 2.05-1.96 (m, 2H (should be 3H), 1.74 (app. s (3H)); 13C NMR (appendix) (100 MHz, CDCl3): δ(ppm): 195.5, 169.7, 169.2, 168.8, 168.4, 160.8, 155.5, 153.9, 150.7, 146.6, 146.4, 145.5, 144.6, 140.6, 136.8, 135.7, 135.0, 133.0, 129.3, 122.8, 108.0, 106.0, 100.5, 92.7, 50.3, 48.3, 48.1, 30.4, 27.8, 25.7, 25.7, 23.3, 19.7, 17.4, 12.9, 12.4, 11.2; HRMS (ES) m/z calcd for C37H35InN45O5 [M-Cl]+: 744.1671; found 744.1670; mp (uncorr.) 106.5-112.6 ⁰C.

5-propargylamino-ddUTP

1-[2,6-Dioxa-bicyclo [3.2.0]hept-3-yl]-1H-pyrimidine-2,4-dione (15). [6]

2′-Deoxyuridine (13) (1.099 g, 4.816 mmol) was added to a flame-dried 50 mL round-bottomed flask under an argon atmosphere and dissolved in anhydrous pyridine (8 mL). The solution was cooled to 0 °C before methanesulfonyl chloride (1.2 mL, 15.5 mmol, 3.2 equiv.) was added to the solution and the reaction was stirred at 0 °C for 2 h. The reaction was allowed to reach rt and left for 1.2 h. The solvent was evaporated in vacuo and the resulting brown oil was washed with ice water (10 mL) and EtOAc (10 mL). The water phase was extracted with EtOAc (100 mL) and the collected organic phases were added to the residual oil in the reaction flask. The organic solvent was evaporated to dryness before MeOH (25 mL) was added together with 6 M NaOH (5 mL, 30 mmol, 6.2 equiv.). When the mixture was heated to 80 °C the residual oil started to dissolve. The solution was left for 5 h at 80 °C before it was cooled to rt and neutralized with 1 M HCl (18 mL). The resulting mixture was transferred to a 100 mL flask and evaporated to dryness. The solid residue was refluxed in acetone (4x50 mL) for 10 min each time to extract the product. The combined organic phases were evaporated to dryness and the product purified by flash column chromatography (5% to 10% MeOH in DCM) yielding 15 as a white solid (0.483 g, 2.30 mmol, 48%).

Rf 0.63 (MeOH/DCM 1:9). mp (uncorr.) 209.5-211.3 °C (lit.[7] 206-210 °C). 1H NMR (400 MHz, d6-DMSO): δ 11.35 (s, 1H), 8.16 (d, J = 8.1 Hz, 1H), 6.51 (t, J = 5.3 Hz, 1H), 5.71 (dd, J = 2.3, 8.1 Hz, 1H), 5.48 (q, J = 3.3 Hz, 1H), 4.93-4.89 (m, 1H), 4.69 (dd, J = 4.1, 8.2 Hz, 1H), 4.01 (dd, J = 1.5, 8.1 Hz, 1H), 2.53-2.46 (m, 2H). 13C NMR (100 MHz, d6-DMSO): δ 163.1, 151.2, 141.1, 102.2, 88.5, 86.9, 80.1, 75.2, 37.2. HRMS (ESI) m/z [M + Na]+ calcd for C9H10N2O4Na, 233.0538; found, 233.0535. IR (neat) νmax (cm-1): 3158 (broad), 3042, 2990, 1713, 1682, 1461, 1269, 1088. [α]D26 -36.9 (c 1.06, MeOH).

2′,3′-Didehydro-2′,3′-dideoxyuridine (16). [6]

A solution of 15 (0.600 g, 2.85 mmol) in dry DMF (12 mL) placed in a flame-dried 25 mL round-bottomed flask under an argon atmosphere was cooled to 0 °C, after which NaH (60% mineral oil suspension, 0.363 g, 9.08 mmol, 3.2 equiv.) was added. The resulting mixture was heated to 100 °C and left to react for 7 h after which the reaction was quenched with H2O (8 mL) and the solvent was evaporated to dryness. The residue was dissolved in MeOH and neutralized with 1 M HCl (8 mL). The solvent was evaporated in vacuo, and the product purified by flash column chromatography (5% to 10% MeOH in DCM) affording 16 (0.351 g, 1.67 mmol, 58%) as a white solid.

Rf 0.30 (MeOH/DCM 1:9). mp (uncorr.) 152.1-153.2 °C (lit.[7] 150-152 °C). 1H NMR (400 MHz, d6-DMSO): δ 11.30 (s, 1H), 7.74 (d, J = 8.1 Hz, 1H), 6.82-6.80 (m, 1H), 6.40 (dt, J = 1.6, 6.0 Hz, 1H), 5.94-5.90 (m, 1H), 5.59 (d, J = 8.0 Hz, 1H), 4.97 (t, J = 5.3 Hz, 1H), 4.80-4.75 (m, 1H), 3.58 (dd, J = 3.6, 5.0 Hz, 2H). 13C NMR (100 MHz, d6-DMSO): δ 163.2, 150.8, 141.1, 135.1, 125.8, 101.6, 89.1, 87.4, 62.2. HRMS (ESI) m/z [M + Na]+ calcd for C9H10N2O4Na, 233.0538; found, 233.0536. IR (neat) νmax (cm-1): 3449, 3171, 3101, 3043, 1697, 1672, 1454, 1393, 1106. [α]D26 -6.7 (c 1.01, MeOH).

2′,3′-Dideoxyuridine (17). [6]

To a solution of 16 (0.304 g, 1.45 mmol) in dry MeOH (10 mL) placed in a flame-dried 25 mL round-bottomed flask was added palladium on carbon (10 w/w%, 0.024 g, 0.023 mmol, 0.016 equiv.) under an argon atmosphere. The resulting solution was purged with H2 before it was left with an H2 atmosphere for 4 h at rt. The catalyst was filtered off and the solvent was evaporated in vacuo, affording 17, without further purification, in a quantitative yield (0.306 g) as a white solid.

Rf 0.28 (MeOH/DCM 1:9). mp (uncorr.) 107.7-111.4 °C (lit.[7] 117.5-118.5 °C). 1H NMR (400 MHz, d6-DMSO): δ 11.24 (br. s, 1H), 7.94 (d, J = 8.1 Hz, 1H), 5.95 (dd, J = 3.6, 6.8 Hz, 1H), 5.58 (d, J = 8.1 Hz, 1H), 5.03 (br. s, 1H), 4.06-3.98 (m, 1H), 3.67 (dd, J = 3.3, 11.9 Hz, 1H), 3.53 (dd, J = 3.8, 11.9 Hz, 1H), 2.34-2.21 (m, 1H), 1.99-1.77 (m, 3H). 13C NMR (100 MHz, d6-DMSO): δ 163.3, 150.5, 140.6, 101.0, 85.1, 81.5, 62.0, 31.8, 24.8. HRMS (ESI) m/z [M + Na]+ calcd for C9H12N2O4Na, 235.0695; found, 235.0690. IR (neat) νmax (cm-1): 3370, 3170, 3050, 2908, 1655, 1618, 1468, 1397, 1275, 1100. [α]D26 +51.4 (c 0.89, MeOH).

2′,3′-Dideoxy-5-iodoridine (18). [6]

Compound 17 (0.082 g, 0.39 mmol) was placed in a flame-dried 25 mL round-bottomed flask and dissolved in glacial acetic acid (3 mL) under an argon atmosphere. Iodine (0.082 g, 0.32 mmol, 0.8 equiv.) and CAN (0.104 g, 0.19 mmol, 0.5 equiv.) was added to the solution and the reaction was stirred at 80 °C. After 37 min the mixture was cooled to rt. The solvent was evaporated and co-evaporated with MeOH (2x10 mL). The product was purified by flash column chromatography (3% to 5% MeOH in DCM) yielding 18 (0.096 g, 0.28 mmol, 72%) as a slightly yellow solid.

Rf 0.57 (MeOH/DCM 1:9). mp (uncorr.) 179.0-181.5 °C. 1H NMR (400 MHz, d6-acetone): δ 10.24 (br. s, 1H), 8.71 (s, 1H), 5.99 (dd, J = 2.9, 6.5 Hz, 1H), 4.40 (t, J = 4.8 Hz, 1H), 4.23-4.15 (m, 1H), 4.00-3.93 (m, 1H), 3.77-3.71 (m, 1H), 2.45-2.31 (m, 1H), 2.22-1.88 (m, 3H). 13C NMR (100 MHz, d6-acetone): δ 160.9, 151.0, 146.6, 87.4, 83.4, 67.2, 62.8, 33.8, 24.9. HRMS (ESI) m/z [M + Na]+ calcd for C9H11IN2O4Na, 360.9656; found, 360.9658. IR (neat) νmax (cm-1): 3414 (broad), 3158, 3043, 2923, 1674, 1603, 1435, 1275, 1094, 1063. [α]D30 +16.5 (c 0.71, MeOH).

5-[3-(Trifluoroacetyl amino)-prop-ynyl]-2’, 3’-dideoxyuridine (19).

To a round-bottomed flask were added compound 18 (30 mg, 0.089 mmol), CuI (2.8 mg, 0.015 mmol, 0.17 equiv.), PdCl2(PPh3)2 (9.0 mg, 0.013 mmol, 0.14 equiv.). The reagents were then dried for 30 min under high vacuum and subsequently placed under an Argon atmosphere. Dry DMF was degassed with argon and sonicated. Dry, degassed DMF (5 mL) was added to the solid reagents followed by addition of TEA (0.1 mL, 0.72 mmol, 8 equiv.) and compound 21 (94.9 mg, 0.628 mmol, 7 equiv.). The reaction was left at room temperature overnight. The mixture was diluted with brine (20 mL) and extracted with EtOAc (2x25 mL). The combined organic phases were dried over MgSO4 and evaporated to dryness. The product was purified by flash column chromatography (5% MeOH in DCM) yielding 19 (13.1 mg, 0.036 mmol, 41%) as a slightly yellow oil.

1H NMR (400 MHz, d6-Acetone) δ 10.19 (s, 1H), 9.03 (s, 1H), 8.47 (s, 1H), 6.00 (dd, J =3.2, 6.5 Hz, 1H), 4.38-4.28 (m, 2H), 4.23-4.14 (m, 1H), 3.93 (d, J = 11.9 Hz, 1H), 3.74 (d, J = 13.2 Hz, 1H), 2.49-2.29 (m, 1H), 2.28-1.84 (m, 4H).

5-[3-Aminoprop-1-ynyl]-2’, 3’-dideoxyuridine-5’-O-triphosphate (20).

Compound 19 (13.1 mg, 0.036 mmol),salicyl phosphorochloridite (22.9 mg, 0.113 mmol, 3 equiv.) and tributylammonium pyrophosphate (85.3 mg, 0.155 mmol, 2.1 equiv.) were dried overnight in separate flasks. Tributylamine (0.6 mL, 2.5 mmol, 35 equiv.) was dried under high vacuum for 1 h. Tributylammonium pyrophosphate was dissolved in dry DMF (0.5 mL) before Tributylamine was added. Salicyl phosphorochloridite was dissolved in dry DMF (0.25 mL) followed by addition of 0.55 mL of the pyrophosphate mixture. The solution was left for 40 min before it was transferred to the flask containing the nucleoside. The reaction was left for 3 h before an iodine solution (0.6 mL, 3% in pyridine/H2O 9:1) was added. After 15 min water (2 mL) was added and the reaction was left for another 1.5 h. The product was precipitated by addition of aq. NaCl (0.5 mL,3 M) and EtOH (15mL, 96%), cooled on dry ice for 1 h and centrifuged (4 °C, 20 min, 3000 rpm). The supernatant was discarded and the residue dissolved in water (5 mL). To the dissolved product was added NH4OH (5 mL, 24%) and the mixture was left for 3 h. The resulting mixture was freeze-dried and purification was attempted using a prep. TLC (i-propanol/NH4OH/H2O 48:32:20). No product was obtained.

Propargyl trifluoroacetamide (21). [8]

Propargylamine (0.990 g, 18.0 mmol) was dissolved in dry DMF (12mL). To the solution was added TEA (2.5mL) before the mixture was cooled to °0 C. A solution of Trifluoroacetic anhydride (2.80 mL, 20.0 mmol, 1.1 equiv.) in dry DMF (5mL) was added dropwise. The reaction was let for 50 min before water (5 mL) was added. The resulting mixture was washed with 1 M HCl (15 mL) and sat. NaHCO3 (15 mL), dried over Na2SO4, filtered and concentrated to dryness in vacuo. Compound 21 (0.7115 g, 4.7 mmol, 26%) was obtained without further purification as a yellow oil.

1H NMR (400 MHz, CDCl3) δ 6.63 (br. s, 1H), 4.24-4.09 (m, 2H), 2.34 (s, 1H).

DNA conjugation

DNA labeling with cholesterol

Mix of 6 DNA strands amine modified by the enzyme reaction, followed by amide formation.

The DNA strands (3.13 μL, 0.5 nmol of each) were mixed together. To the DNA batch were added buffer containing potassium cacodylate (20 μL), a solution of the co-factor CoCl2 (20 μL, 25 mM), 20 (4 μL, 5 mM, 20 nmol), TdT (5 μL, 400 U/ μL) and H2O (32 μL). The reaction was incubated at 37 °C for 15 min and then cooled on ice. The reaction was stopped by addition of EDTA (2 μL, 0.25 M) and glycogen (1 μL, 20 mg/mL). NaOAc (15 μL, pH = 5.2, 3 M) and cold EtOH (250 μL, 96%) were added and the mixture was put on dry-ice for 15 min. The mixture was centrifuged (4 °C, 30 min, 14000 rpm). The supernatant was discarded and the pellet dissolved in H2O (50 μL). The activated acid 6 (2 mg, 3.7 μmol, 1200 equiv.) was dissolved in DMF (50 μL) and added. MeCN (50 μL) and TEA (1 μL) were added to the solution and the reaction was incubated at rt overnight. NaOAc (21 μL, pH = 5.2, 3 M), Glycogen (1 μL, 20 mg/mL) and cold EtOH (375 μL, 96%) were added and the mixture was cooled on dry-ice for 15 min followed by centrifugation (4 °C, 45 min, 14000 rpm). The supernatant was discarded and the pellet dissolved in TEAA buffer (100 μL). The solution was centrifuged for 15 min and the supernatant was purified by RP-HPLC (10% to 70% MeCN in TEAA buffer over 30 min). The fractions containing the modified DNA strands were collected and freeze-dried. The DNA was dissolved in H2O (200 μL). The UV absorbance was measured to calculate a concentration using the average extinction coefficient for the 6 strands. The concentration obtained was 2.58 μM giving the modified DNA in 17% yield.

DNA labeling with photosensitizer

Amine modification of stable strands for the DNA origami with terminal dideoxynucleotidyl transferase.

Potassium cacodylate reaction buffer (20 μ, pH=6.6 [1 M potassium cacodylate, 0.125 M Tris-HCl, 1.25 mg/ml Bovine Serum Albumin]) and CoCl2 (20 μL, 25 mM) was transferred to a tube. Unmodified DNA stable strands (2.5 nm, 16 μL, 160 μM), the 5-propargylamino-ddUTP (4 μL, 5 mM), TdT (5 μL, 400 U/μL) and MilliQ water (35 μL) was added. The reaction mixture was incubated at 37 ⁰C for 15 minutes. To stop the reaction EDTA (2 μL, 0.25 M, pH=8) was added. The modified DNA was precipitated by adding NaOAc (15 μL, 0.3 M, pH=5.2), glycogen (1 μL) and cold ethanol (250 μL, 96%). The mixture was incubated on dry ice for 15 min and centrifuged (4 ⁰C, 30 minutes 14000 rpm). The supernatant was decanted off, and the modified DNA stable strands redissolved in MilliQ water (50 μL).

Conjugation of origami stable strands with In(PPa NHS)Cl

Compound 12 (2 mg, 2.6 μmol) was dissolved in DMSO (50μL) and added to a tube with amine modified DNA (2.5 nmol) in MilliQ water (50 μL). Precipitate was observed and MeCN (3∙50 μL) was added until the solution remained clear. Triethylamine (50 μL) was added and the reaction mixture incubated for 1 h at 22 ⁰C. To precipitate the DNA conjugate NaOAc (43 μL, 3M, pH=5.2), glycogen (1 μL) and cold ethanol (750 μL, 96%) was added. The mixture was incubated on dry ice for 15 min and centrifuged (4 ⁰C, 60 minutes 14000 rpm). The supernatant was decanted off and the DNA pellet redissolved in TEAA buffer (100 μL). The crude product was purified by RP-HPLC (10% to 70% MeOH in TEAA buffer over 30 min). The product was lyophilized o.n. The light green product was estimated to 1.6 nmol (65%).

Reference

  1. Hussey Hussey, S. L. et al.. Synthesis of chimeric 7α-substituted estradiol derivatives linked to cholesterol and cholesterylamine. Org. Lett. 4, 415–418 (2002). [1]
  2. Horwitz Horwitz, J. P. et al. Nucleosides. IX. The formation of 2’,3'-unsaturated pyrimidine nucleosides via a novel beta-elimination reaction. J. Org. Chem. 121, 205–211 (1966). [2]
  3. Trybulski Trybulski, E. J. et al. The synthesis and biochemical pharmacology of enantiomerically pure methylated oxotremorine derivatives. J. Med. Chem. 36, 3533–3541 (1993).[3]
  4. Simeone Simeone, L. et al. Cholesterol-based nucleolipid-ruthenium complex stabilized by lipid aggregates for antineoplastic therapy. Bioconjugate Chem. 23, 758–770 (2012) [4]

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</style> </head> <body> <div id="indexing"> <div id="sitemap"> <p id="sitemapTitle">SITEMAP | BIOMOD 2013 NANO CREATORS | Aarhus University</p> <div id="footer-contents"> <div class="footer-section"> <p class="footer-section-title">INTRODUCTION</p> <ul> <li><a href="/wiki/Biomod/2013/Aarhus">Home, abstract, animation and video</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Introduction">Introduction</a></li </ul> </div> <div class="footer-section"> <p class="footer-section-title">RESULTS AND DISCUSSION</p> <ul> <li><a href="/wiki/Biomod/2013/Aarhus/Results_And_Discussion/Origami">Origami</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Results_And_Discussion/Peptide_lock">Peptide lock</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Results_And_Discussion/Chemical_Modification">Chemical modification</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Results_And_Discussion/sisiRNA">sisiRNA</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Results_And_Discussion/System_In_Action">System in action</a></li> </ul> </div> <div class="footer-section"> <p class="footer-section-title">MATERIALS AND METHODS</p> <ul> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/Origami">Origami</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/Peptide_lock">Peptide lock</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/Chemical_Modification">Chemical modification</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/sisiRNA">sisiRNA</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/System_In_Action">System in action</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/Methods">Methods</a></li> </ul> </div> <div class="footer-section"> <p class="footer-section-title">SUPPLEMENTARY</p> <ul> <li><a href="/wiki/Biomod/2013/Aarhus/Supplementary/Team_And_Acknowledgments">Team and acknowledgments</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Supplementary/Optimizations">Optimizations</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Supplementary/Supplementary_Data">Supplementary data</a></li> 

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href="/wiki/Biomod/2013/Aarhus/Supplementary/Supplementary_Informations">Supplementary informations</a> <li><a href="/wiki/Biomod/2013/Aarhus/Supplementary/References">References</a></li> </ul> </div> </div> <div> <p id="copyright">Copyright (C) 2013 | BIOMOD Team Nano Creators @ Aarhus University | Programming by: <a href="mailto:pvskaarup@gmail.com?Subject=BIOMOD 2013:">Peter Vium Skaarup</a>.</p> </div> </div>

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