Controlled porous glass (CPG) is the standard solid substrate utilized in automated phosphoramidite sythesis of oligos. Alkylthiol modified oligos are only available com- mercially in either propyl or hexyl forms, neither of which can be effectively utilized with my current set of dodecylthiol molecules BAT and TEG. Thus, I set out to synthesize my own dimethoxytrityl-(DMT)-modified CPG to generate oligos with a 3’ disulfide modification. This asymmetric disulfide is comprised of the oligo and a propanol group. We expect that the propyl group will be quickly displaced by long- chain reagent TEG during SAM formation, and thus calculate the oligo reagent sulfur contribution as a thiolate. The synthesis scheme is given in Figure 4.1.
Figure 4.1Synthesis scheme of DMT-modified CPG. a) HBr, cyclohexane. Re- flux for 6 hrs, argon. b) DMT-Cl, TEA, CH2Cl2. Stir overnight at RT. c) C2H3OSK, DMF. Stir overnight at RT. d) Mercaptoethanol, NaOH, ACN. Stir in air overnight at RT. e) succinic anhydride, pyridine. Stir overnight at RT in air. f) BOP, DIPEA in CH2Cl2, amino-modified coarse porous glass. Swirl for 2 hrs at RT. g) Quench unreacted amine. TEA, acetic anhydride. Swirl 30 min.
4.3.1 Synthetic Methods
2. 12-bromododecan-1-ol
To a round bottom flask of 10.2 g (50.4 mmol) 1,12 dodecanediol (1) in 150 ml of cyclohexane was added 150 ml HBr (48%) and stirred under reflux for 6 hours.
The mixture was extracted three times wtih hexane (50 ml), washed with saturated NaHCO3, then washed with brine, and dried with Na2SO4. The solution was then concentrated to a yellow oil which was loaded onto a silica gel in hexane. The column was run with hexane:ethyl acetate (90:10 to 80:20). The product fraction was collected and concentrated to give white crystals. Yield 8.65 g (32.6 mmol); 64.7% yield. TLC Rf=0.33; 60% hexane: 40% ethyl acetate. 1H-NMR (DMSO-d6, 300 MHz): δ1.22- 1.37 (m, 18H), δ1.73 (q, 2H), δ3.45 (t, 2H), δ3.49 (t, 2H), δ4.09 (bs, 1H). Mass spec.
m/z [M+H]+: 265.2
3. 4,4’-((12-bromododecyloxy)(phenyl)methylene)bis(methoxybenzene) To 1.1 g (4.15 mmol) of 2 in a round bottom flask was added 15 ml chloroform solvent, 14.05 g (41 mmol) DMT-Cl, and 0.87 ml (6.26 mmol) triethylamine. The mixture was stirred overnight at room temperature, then extracted with hexane two times. The organic fraction was then washed with NaHCO3 and brine, then dried with Na2SO4, and concentrated to give a dark brown oil which was loaded onto a silica column in hexane. The column was run with hexane:ethyl acetate (90:10 to 80:20). The product fraction was collected and concentrated to give white crystals in high yield. TLC Rf=0.7; 6 hexane: 1 ethyl acetate. 1H-NMR (DMSO-d6, 300 MHz):
δ1.19 (m, 16H), δ1.51 (q, 2H), δ1.75 (q, 2H), δ2.91 (t, 2H), δ3.49 (t, 2H), δ3.7 (s, 6H), δ6.8-7.3 (m, 13H)
4. S-12-(bis(4-methoxyphenyl)(phenyl)methoxy)dodecyl ethanethioate To 4.71 g (8.47 mmol) 3 in a round bottom flask was added 50 ml DMF and 2.9 g (25 mmol) potassium thioacetate to give a brown solution. The reaction was stirred overnight at room temperature and took on a black color. The organic phase was extracted out of DMF by adding ethyl acetate and water, then it was washed with brine, dried with Na2SO4, and concentrated to give a dark oil which was loaded onto a silica gel in hexane. The column was run with hexane:ethyl acetate (95:5 to 85:15).
The product fraction was collected and concentrated to give a brown oil. Yield 4.03 g (7.17 mmol); 85% yield. 1H-NMR (DMSO-d6, 300 MHz): δ1.19 (m, 16H), δ1.48 (m, 4H), δ2.28 (s, 3H), δ2.79 (t, 2H), δ2.92 (t, 2H), δ3.7 (s, 6H), δ6.8-7.3 (m, 13H).
Mass spec. m/z [M+Na]+: 585.4
5. 2-((12-(bis(4-methoxyphenyl)(phenyl)methoxy)dodecyl)disulfanyl)ethanol In a round bottom flask, 0.89 g (1.58 mmol) of 4 was dissolved completely with ACN (40 ml) and 2.2 ml (32 mmol) mercaptoethanol and stirred very well. To this mixture was added 0.7 g (5.07 mmol) K2CO3 and stirred overnight. The reaction was concentrated to an oil and redissolved in CH2Cl2. This organic phase was washed with brine, dried with Na2SO4, and concentrated to give a pale pink oil which was loaded onto a silica column in hexane. The column was run with hexane:ethyl acetate (90:10 to 80:20). 1H-NMR (DMSO-d6, 300 MHz): δ1.19 (m, 16H), δ1.53 (m, 4H), δ2.67 (t, 2H), δ2.74 (t, 2H), δ2.91 (t, 2H), δ3.6 (q, 2H), δ3.71 (s, 6H), δ4.89 (t, 1H) δ6.8-7.3 (m, 13H). Mass spec. m/z [M+Na]+: 619.4
6. 4-(2-((12-(bis(4-methoxyphenyl)(phenyl)methoxy)dodecyl)disulfanyl)- ethoxy)-4-oxobutanoic acid
To a 10 ml round-bottom flask was added 0.47g (.0788 mmol)5 dissolved in pyridine (5 ml) with 0.157 g (1.57 mol) succinic anhydride and a catalytic amount of 0.19 g
(1.57 mmol) DMAP. The reaction was stirred overnight in air. The pyridine was co- evaporated with toluene three times to give a white power. This solid was redissolved in CH2Cl2 (10 ml), washed twice with ice cold citric acid and water, then dried with Na2SO4. The product was concentrated to give a white powder and loaded onto a silicon column in hexane. The column was run with hexane:ethyl acetate (90:10 to 80:20). The product fraction was collected and concentrated to give a white powder.
1H-NMR (DMSO-d6, 300 MHz): δ1.19 (m, 16H), δ1.58 (m, 4H), δ2.43 (s, 2H), δ2.54 (t, 2H),δ2.68 (t, 2H),δ2.89 (t, 2H), δ2.9 (t, 2H),δ3.71 (s, 6H),δ4.2 (t, 2H), δ6.8-7.3 (m, 13H). Mass spec. m/z [M+Na]+: 695.5
7. 3-((16-(bis(4-methoxyphenyl)(phenyl)methoxy)hexadecyl)disulfanyl)propyl 4-(CPG-amino)-4-oxobutanoate
To 0.03 g (43µmol) of 6 was added CH2Cl2 (5 ml) with 1 g of amino-modified coarse porous glass (Sigma, 130 µmol g−1 capacity) and swirled gently by hand. With gentle swirling, 0.02 g (43 µmol) BOP was added first, followed by 0.02 ml (129 µmol) DIPEA. The reaction was swirled gently on an elliptical rotator for 2 hours.
The CPG was then collected by vacuum filtration and soaked in CH2Cl2. To quench unreacted amines, to this slurry was added TEA (0.2 ml) and acetic anhydride (0.2 ml), and it was gently swirled for another 30 minutes. The slurry was collected again by vacuum filtration and washed with methanol three times. DMT loading on CPG was quantitated by adding µl of trichloroacetic acid to 2 mg of 7. The solvent turns orange once DMT deprotection is achieved and is quantified by UV-Vis spectrometry.
Quantitation reveals 1 µmol DMT per 25 mg CPG.
Figure 4.2Synthesis scheme of oligo-modified alkylthiols. a) Mercaptoethanol, ethanol, excess ammonia. Stirred overnight at RT. b) CPG-bound oligo (9), NH4OH, 8. Incubated overnight at 65◦C, then HPLC purified.