Bioconjugate Chemistry
Article
resulting suspension was stirred for 1 h, then diluted with
saturated sodium acetate and stirred at 0−10 °C for 3 h. The
resulting solution was filtered, washed with warm water, and
crystallized in ethanol to afford the azo dye methyl ester 2.
solubility). NMR and ESI-MS indicate that this compound
degrades to the naphthalic anhydride during prolonged storage.
ESI MS: M + H+ = 467.50 (Calc 467.17).
1
MPQ3 (3c). (80% yield) H NMR (CDCl3, 500 MHz):
1
MPQ1 Methyl Ester (2a). (50% yield) H NMR (CDCl3,
8.62−8.66 (3H, m), 8.12 (1H, dd, J = 8.5, 2.5), 7.80 (1H, dd, J
= 7.0), 7.41 (1H, d, J = 9.5), 6.55 (1H, dd, J = 9.5, 2.5), 5.99
(1H, d, J = 2.5), 4.51 (2H, t, J = 7), 3.52 (4H, q, J = 7), 2.79
(2H, t, J = 7), 1.31 (6H, t, J = 7). 13C NMR (spectrum could
not be obtained due to poor solubility). ESI MS: M + H+ =
461.33 (Calc 461.18).
400 MHz): 9.25 (1H, d, J = 7.2), 8.63−8.67 (2H, m), 8.04 (2H,
d, J = 7.6), 7.96 (1H, d, J = 8.4), 7.83 (1H, dd, J = 7.2), 6.80
(2H, d, J = 7.6), 4.52 (2H, t, J = 7.4), 3.71 (3H, s), 3.17 (6H, s),
2.80 (2H, t, J = 7.4). 13C NMR (CDCl3, 100 MHz): 32.7, 36.2,
40.4, 51.9, 111.6, 112.3, 121.8, 122.3, 126.5, 126.8, 131.0, 131.6,
132.1, 144.7, 152.2, 153.5, 164.0, 164.4, 171.9. ESI MS: M + H+
= 431.42 (Calc 431.17).
1
MPQ5 (3d). (87% yield) H and 13C NMR data could not
be obtained as a result of very poor solubility in common NMR
solvents. ESI MS: M + H+ = 494.42 (Calc 494.18).
1
MPQ2 Methyl Ester (2b). (62% yield) H NMR (CDCl3,
MPQ4 (3e). A solution of 0.270 g (1.05 equiv, 3.90 mmol)
NaNO2 in 2 mL H2O was added dropwise to a mixture of 0.510
g (1.0 equiv, 3.72 mmol) 4-aminobenzoic acid in 12 mL 1 M
HCl at 0 °C. After 30 min, a solution of 0.657 g (1.05 equiv,
3.90 mmol) of 1-H-perimidine in 9 mL 1 M HCl was added,
and the resulting suspension stirred at 0 °C for 2 h. The
suspension was diluted with a solution of 2.15 g NaOAc in 15
mL H2O, stirred at room temperature for 2 h, then filtered and
washed with warm water to give a dark purple solid.
Crystallization from ethanol afforded 0.916 g (78%). A complex
1H NMR spectrum results from tautomerization in the
perimidine scaffold (see Supporting Information). 13C NMR
data could not be obtained as a result of poor solubility in
common NMR solvents. ESI MS: M + H+ = 317.25 (Calc
317.10).
3-(2-Methyl-2,3-dihydro-1H-perimidin-2-yl)propan-1-ol
(4). 6.19 g (39 mmol) 1,8-diaminonaphthalene was added to a
solution of 4.35 mL (1.1 equiv, 43 mmol) 5-hydroxy-2-
pentanone and 0.089 g (0.01 equiv, 0.4 mmol) p-
toluenesulfonic acid monohydrate in 15 mL ethanol. The
solution was stirred at 60 °C for 90 min, whereupon the
solution had solidified. The resulting solid was cooled to room
temperature, filtered, washed with ethanol, and crystallized
from aqueous ethanol to yield a pale gray solid (4.11 g, 43%).
1H (DMSO-d6, 400 MHz): 7.07 (2H, dd, J = 7.8), 6.83 (2H, d,
J = 8.4), 6.34 (2H, d, J = 7.2), 6.29 (2H, brs, NH), 4.37 (1H, t,
OH), 3.30 (2H, m), 1.53−1.62 (4H, m), 1.29 (3H, s). 13C
NMR (DMSO-d6, 100 MHz): 26.5, 27.1, 36.9, 61.2, 65.5,
103.6, 111.5, 114.0, 127.0, 134.2, 141.8. ESI MS: M + H+ =
243.50 (Calc 243.15).
MPQ6 (5). 0.291 g (1.20 mmol) of 4 was dissolved in a
solution of 20 mL 1:1 THF/acetone stirred at 0 °C. 0.600 g
(1.2 equiv, 1.44 mmol) Fast Corinth V was added in portions
to give a dark blue solution, which was stirred at 0 °C for 1 h.
The reaction was diluted with water and extracted 3 times with
CH2Cl2. The combined organic extracts were washed once with
water, once with brine, dried (MgSO4), filtered, and the solvent
removed in vacuo. Column chromatography (5% MeOH in
CH2Cl2 containing 0.5% Et3N) afforded 0.094 g (14%) of a
dark purple solid. 1H NMR (CDCl3, 400 MHz): 8.19 (1H, d, J
= 8.0), 7.84 (1H, d, J = 8.8), 7.66−7.69 (3H, m), 7.40−7.47
(3H, m), 6.53−6.56 (2H, m), 4.02 (3H, s), 3.66 (2H, t, J =
6.0), 2.74 (3H, s), 2.50 (3H, s), 1.88−1.92 (2H, m), 1.75−1.80
(2H, m), 1.52 (3H, s). 13C NMR (CDCl3, 100 MHz): 17.0,
21.3, 27.2, 27.6, 29.8, 38.6, 56.3, 62.7, 99.0, 112.8, 119.2, 124.4,
133.6, 141.3, 143.6, 147.4. ESI MS: M + H+ = 554.42 (Calc
554.25).
400 MHz): 9.34 (1H, d, J = 8.8), 9.11 (1H, d, J = 8.0), 8.67−
8.70 (2H, m), 8.22 (1H, d, J = 8.4), 8.10−8.14 (2H, m), 7.89
(1H, dd, J = 8.0), 7.69 (1H, dd, J = 8.0), 7.59 (1H, dd, J = 7.6),
7.11 (1H, d, J = 8.4), 4.54 (2H, t, J = 7.4), 3.71 (3H, s), 3.11
(6H, s), 2.81 (2H, t, J = 7.4). 13C NMR (CDCl3, 100 MHz):
32.7, 36.3, 44.8, 52.0, 112.8, 112.9, 114.0, 122.6, 123.8, 125.0,
125.6, 127.2. 127.6, 131.0, 131.7, 132.0, 143.3, 152.0, 156.4,
163.9, 164.3, 167.7, 171.9. ESI MS: M + H+ = 481.92 (Calc
481.19).
1
MPQ3 Methyl Ester (2c). (52% yield) H NMR (CDCl3,
400 MHz): 8.59−8.63 (3H, m), 8.09 (1H, d, J = 8.4), 7.78 (1H,
dd, J = 7.8), 7.38 (1H, d, J = 9.6), 6.53 (1H, dd, J = 9.6, 2.6),
5.97 (1H, d, J = 2.6), 4.50 (2H, t, J = 7.4), 3.70 (3H, s), 3.51
(4H, q, J = 7.2), 2.78 (2H, t, J = 7.4), 1.30 (6H, t, J = 7.2). 13
C
NMR (CDCl3, 100 MHz): 13.1, 32.7, 36.2, 45.6, 51.9, 98.1,
110.8, 111.6, 122.7, 126.9, 128.6, 131.6, 132.6, 154.9, 163.8,
171.9. ESI MS: M + H+ = 475.42 (Calc 475.20).
1
MPQ5 Methyl Ester (2d). (20% yield) H NMR (CDCl3,
400 MHz): 9.34 (1H, d, J = 8.4), 8.66−8.68 (2H, m), 8.40 (d,
1H, J = 8.4), 8.19 (d, 1H, J = 8.4), 8.09 (d, 1H, J = 8.0), 7.84
(dd, 1H, J = 7.6), 7.50 (dd, 1H, J = 7.6), 6.62 (d, 1H, J = 7.2),
6.55 (d, 1H, J = 8.4), 4.53 (t, 2H, J = 7.2), 3.71 (s, 3H), 2.81 (t,
2H, J = 7.2), 1.61 (s, 6H). 13C NMR: (spectrum could not be
obtained due to poor solubility). ESI MS: M + H+ = 508.42
(Calc 508.20).
General Procedure for Methyl Ester Hydrolysis. 500 mg
MPQ methyl ester was dissolved in anhydrous THF (at a
concentration of 0.085 M). To this solution, 2.02 equiv
potassium trimethylsilanolate was added, and the resulting
solution stirred overnight at room temperature, at which point
TLC indicated consumption of starting material. The solvent
was removed in vacuo and the resulting solid dissolved in a
minimal amount of water. Hydrochloric acid (1 M) was added
to precipitate the free carboxylic acid, which was filtered,
washed with water, and dried. The product 3 was used for DNA
conjugation without further purification. All free acids were
found to be poorly soluble in common deuterated solvents.
1
MPQ1 (3a). (80% yield) H NMR (CDCl3, 500 MHz):
9.26−9.35 (1H, m), 8.64−8.96 (2H, m), 7.96−8.05 (3H, m),
7.84−7.91 (1H, m), 6.80 (2H, d, J = 9.0), 4.54 (2H, t, J = 7.0),
3.18 (s, 6H), 2.87 (2H, t, J = 7.0). NMR indicates some
degradation to naphthalic anhydride during prolonged storage.
13C NMR: (spectrum could not be obtained due to poor
solubility). ESI MS: M + H+ = 417.33 (Calc 417.16).
MPQ2 (3b). (36% yield) 1H NMR (CDCl3, 500 MHz): 9.45
(1H, d, J = 8.5), 9.12 (1H, d, J = 7.5), 8.71−8.75 (2H, m), 8.24
(1H, d, J = 8.0), 8.14−8.18 (2H, m), 7.96 (1H, dd, J = 8.0),
7.73 (1H, dd, J = 7.0, 1.5), 7.63 (1H, dd, J = 7.0, 1.5), 7.14 (1H,
dd, J = 8.5), 4.59 (2H, t, J = 7.0), 3.17 (6H, s), 2.91 (2H, t, J =
7.0). 13C NMR: (spectrum could not be obtained due to poor
MPQ6 Phosphoramidite (6). 0.164 g (0.296 mmol) MPQ6
was dissolved in 2.5 mL anhydrous acetonitrile, 0.15 mL (3
equiv, 0.861 mmol) N,N-diisopropylethylamine was added and
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dx.doi.org/10.1021/bc200424r|Bioconjugate Chem. 2011, 22, 2345−2354