Organic & Biomolecular Chemistry
Paper
(
0.7 mL, 14 mmol) was added drop wise. After stirring over- CDCl
3
): δ 163.3, 141.1, 134.5, 131.4, 130.1, 128.6, 123.4,
night, the reaction was quenched with NaOH [1 M]. 119.0, 78.8. HRMS (positive mode) (m/z) calculated mass for
+
The organic layer was washed with H
2 × 30 mL), dried over MgSO and concentrated under mp = 163–164 °C.
reduced pressure. The crude solid was further purified by O-(4-Azidobenzyl) hydroxylamine (15). To a solution 2-((4-azido-
column chromatography (n-hexane : ethyl acetate, 7 : 3) to give benzyl)oxy)-isoindoline-1,3-dione 14 (3.2 g, 10.9 mmol) in
2
O (2 × 30 mL), brine
15 10 4 3
C H N O Na [M + Na] 317.06451, found mass 317.06436
(
4
1
1
0 as a colorless oil (0.83 g, 4.8 mmol. 69% yield). H NMR 45 ml of CH
2 2
Cl , hydrazine hydrate (1.04 mL, 33 mmol) was
(
400 MHz, CDCl ): δ 5.34 (br s, 2H –ONH ), 3.65 (t, J = 6.7 Hz, added drop wise. After stirring overnight the reaction was
3
2
2
H), 1.57 (m, 2H), 1.18–1.39 (s, 14H), 0.88 (t, J = 6.6 Hz, 3H). quenched with NaOH [1 M]. The organic layer was washed
1
3
3 2
C NMR (101 MHz, CDCl ): δ 76.3, 32.0, 29.7, 29.6, 29.6, 29.4, with H O (2 × 30 mL) and brine (2 × 30 mL). The organic
2
8.4, 26.1, 22.7, 14.5. HRMS (positive mode) (m/z) calculated phase was dried over MgSO and concentrated under reduced
4
+
mass for
10
C H24NO [M + H] 174.18524, found mass pressure to give 15 as yellow-brownish oil (1.1 g, 6.1 mmol,
1
1
74.18518.
3
56%). H NMR (400 MHz, CDCl ): δ 7.36 (d, J = 8.2 Hz, 2H),
The product was immediately converted to the corres- 7.03 (d, J = 8.0 Hz, 2H), 5.41 (br s, 2H –ONH ), 4.65 (s, 2H).
2
1
3
ponding hydrochloride salt by treatment with of HCl [6 M].
After filtration, the white solid was lyophilized.
C NMR (101 MHz, CDCl
3
): δ 139.7, 134.4, 129.9, 119.0, 77.2.
HRMS (positive mode) (m/z) calculated mass for C
7
9 4
H N O
+
1
-Azido-4-methylbenzene (12). Prepared following pub- [M + H] 165.07709, found mass 165.07690.
2
4
lished procedure. 10 g (92.8 mmol) of p-toluidine was dis-
solved in 46 mL of a solution of H O : HCl (1 : 1) at 0 °C. ponding hydrochloride salt by treatment with of HCl [6 M].
Sodium nitrite (6.3 g, 92.8 mmol) dissolved in 26 mL of cold After filtration, the yellow solid was lyophilized.
The product was immediately converted to the corres-
2
H
2
O was added drop wise, followed by drop wise addition of
sodium azide (6.1 g, 92.8 mmol) in 58 mL H O. After 1 h
CHCl (100 mL) was added. The aqueous phase was extracted
DNA modifications
2
Stock solutions of reagents. The hydrochloride salts (com-
3
twice with CHCl
washed with H O (2 × 30 mL) and dried over MgSO
trated under pressure to give 12 as a brown oil (11.1 g, solutions were stored at −20 °C.
3
(100 mL). The combined organic layers were pounds 1–7) were dissolved in double distilled (dd) H
2
O and
2
4
, concen- brought to the desired pH by using NaOH [6 M]. The stock
1
7
8
9.1 mmol, 85%). H NMR (400 MHz, CDCl
.0 Hz, 2H), 6.94 (d, J = 8.0 Hz, 2H), 2.35 (s, 3H). C NMR centrations of 200 µM). The concentrations of the oligonucleo-
tides were determined from their UV/Vis absorptions at
-Azido-4-(bromomethyl)benzene (13). Prepared following a 260 nm (Nanodrop) using the calculated extinction
3
): δ 7.17 (d, J =
2
The oligonucleotides were dissolved in ddH O (final con-
1
3
(101 MHz, CDCl ): δ 137.1, 134.86, 130.3, 118.7, 20.5.
3
1
2
5
published procedure. To a solution of 1-azido-4-methyl- coefficients.
benzene (12) (7.14 g, 53.7 mmol) in benzene 200 mL under a Modification of oligo 1 with CH ONH ·HCl; representative
3
2
nitrogen atmosphere, N-bromosuccinimide (10.51 g, 59 mmol) procedure. 100 µL of oligo 1 was mixed with 100 µL of the
and AIBN 1.0 g were added. The reaction was heated under desired solution of methoxyamine hydrochloride (for final
reflux for 3 days. The solvent was evaporated under reduced concentrations see Table 1) at the indicated temperatures for
pressure. The resulting solid was dissolved in CH
2
Cl
2
the indicated times. The products were purified by preparative
(
100 mL). The organic layer was washed with H O (2 × 30 mL) reversed phase HPLC (Xterra column gradient B was used:
2
and brine (2 × 30 mL), dried over MgSO4 and concentrated CH CN/TEAA buffer 50 mM pH = 7; gradient: 05/95 0 to
3
under reduced pressure to give the crude product which was 10 min, to 10/90 at 15 min, to 20/80 at 20 min to 30/70 at
further purified by column chromatography (petroleum ether) 30 min, to 50/50 at 50 min to 70/30 at 65 to 05/95 at 95 min for
1
−1
to give 13 as a brown oil (7.8 g, 37.1 mmol, 69%). H NMR 15 min); flow of 0.5 mL min for analytical runs and flow of
400 MHz, CDCl
3
): δ 7.39 (d, J = 12.4 Hz, 2H), 7.01 (d, J = 1.0 mL min− for the preparative column.) and the pure frac-
1
(
1
1
3
2 Hz, 2H), 4.48 (s, 2H). C NMR (101 MHz, CDCl3) δ = 139.9, tions were lyophilized.
34.2, 130.6, 119.3, 32.9. Purity of the samples was evaluated with analytical reversed
-((4-Azidobenzyl)oxy)-isoindoline-1,3-dione (14). To a solu- phase HPLC (gradient A) and the identity was confirmed with
tion of 1-azido-4-(bromomethyl)benzene 13 (3.4 g, 16 mmol) MALDI-TOF:
and N-hydroxyphtalamide (3.13 g, 19.2 mmol) in 50 mL DMF, M1A: Retention time 33.3 min. MALDI-TOF (m/z) found
CO (2.65 g, 19.2 mmol) was added in two portions. The 1507 (calculated 1506).
reaction mixture reacted overnight. The solvent was evaporated M1B: Retention time 34.2 min. MALDI-TOF (m/z) found
under reduced pressure. The resulting solid was partitioned in 1554 (calculated 1553).
CH Cl (150 mL) and H O (100 mL). The organic layer after Reaction of oligo 1 with 2. 100 µL of oligo 1 solution was
1
2
K
2
3
2
2
2
isolation, it was washed with brine (2 × 50 mL) and then it was mixed with the desired volume (for final concentrations, see
dried over MgSO , concentrated under reduced pressure to Table 2) of O-decylhydroxylamine hydrochloride 5 ([0.7 M],
4
1
give 14 as a brown solid (3.5 g, 12.16 mmol, 76%). H NMR pH = 4). The reaction was followed by RP-HPLC and purified using
(
7
400 MHz, CDCl
3 3
): δ 7.87–7.69 (m, 4H), 7.52 (d, J = 7.2 Hz, 2H), a preparative Xterra column. Gradient C was used: CH CN/
1
3
.02 (d, J = 7.4 Hz, 2H), 5.17 (s, 2H). C NMR (101 MHz, TEAA buffer 50 mM pH = 7; gradient: 05/95 0 to 10 min, to
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