P.-Y. Renard, A. Romieu et al.
1006 cmꢀ1; MS (ESI+): m/z (%): 161.0 [M+H]+ (100); m/z calcd for
C10H8O2: 160.0.
1516, 1461, 1343, 1197, 1139, 1096 cmꢀ1; UV-Vis (CH3CN): lmax =636 nm;
+
MS (ESI+): m/z (%): 530.20 [M]+ (100); m/z calcd for C31H32N9
:
C
530.65.
General procedure for the synthesis of functionalized BHQ-3 dyes:
Methylene Violet 3RAX (50 mg, 132.5 mmol, 1 equiv) was dissolved in
dry CH3CN (1 mL) and the solution was stirred at 08C for 15 min. Solid
NOBF4 (17 mg, 145.5 mmol, 1.1 equiv) was added and the reaction mix-
ture was stirred at 08C for 15 min. The functionalized tertiary aniline
(1.2 equiv) was dissolved in dry CH3CN (0.5 mL) and this solution was
slowly added to the preformed N-nitrosamine/diazonium salt intermedi-
ate. The reaction mixture was stirred at 08C for a further 1 h. Reaction
completion was assessed by RP-HPLC (system A). CH3CN was removed
under reduced pressure and the crude product was purified by semi-prep-
arative RP-HPLC (system B). The product-containing fractions were
lyophilized to give the TFA salt of the BHQ-3 derivative as a blue amor-
phous powder.
Quencher 5b: Derivative 5a (7 mg, 13.2 mmol, 1 equiv) and 7 (2.5 mg,
15.8 mmol, 1.2 equiv) were dissolved in 1:1 DMSO/H2O (3 mL). Aqueous
solutions of sodium ascorbate (0.5 mg, 2.5 mmol, 0.19 equiv) and CuSO4
pentahydrate (0.3 mg, 1.25 mmol, 0.09 equiv) were sequentially added and
the reaction mixture was stirred at RT under an Ar atmosphere for 2 h.
The reaction was monitored by RP-HPLC (system A). On completion,
the mixture was diluted with an aqueous solution of TFA (0.1%, 4 mL)
and purified by semi-preparative RP-HPLC (system B). After lyophiliza-
tion, 5b was obtained as a dark-blue amorphous powder (6.7 mg, 75%).
HPLC (system A): tR =26.1 min, purity=95%; 1H NMR (300 MHz,
[D6]DMSO): d=1.00 (brt, 3H), 1.24 (brt, 3H), 2.90 (s, 3H), 3.36 (brd,
2H), 3.81 (brq, 2H), 3.99 (brt, 2H), 4.65 (brq, 2H), 5.23 (s, 2H), 5.71 (s,
1H), 6.71 (d, 3J
8.7 Hz, 2H), 7.25 (m, 1H), 7.68 (d, 3J
7.92 (m, 3H), 8.00 (d, 3J(H,H)=9.9 Hz, 1H), 8.20 (m, 2H), 8.28 (d, 4J-
(H,H)=2.4 Hz, 1H), 8.43 (dd, 4J(H,H)=2.4 Hz, 3J
(H,H)=9.0 Hz, 1H),
G
(H,H)=2.2 Hz, 3J
ACHUTGTNRNEUNG ACHTUNGTRENNUNG(H,H)=
Quencher 4a: Purification by semi-preparative RP-HPLC (system B),
yield=60%. HPLC (system A): retention time (tR)=25.9 min, purity=
99%; 1H NMR (300 MHz, [D4]MeOH): d=1.08 (brt, 3H), 1.32 (brt,
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
3
4
3
3H), 1.84 (q, J
6.8 Hz, 2H), 2.38 (t, 4J
3.62 (t, 3J(H,H)=7.3 Hz, 2H), 3.81 (brq, 2H), 5.81 (d, 4J
1H), 6.80 (d, 3J(H,H)=9.3 Hz, 2H), 7.26 (d, 4J
(H,H)=1.8 Hz, 1H), 7.68
(m, 3H), 7.72 (s, 1H), 7.86 (dd, J
7.12 (m, 2H), 8.28 ppm (d, 3J(H,H)=9.0 Hz, 1H); 13C NMR (75 MHz,
G
ACHUTGTNRENN(GU H,H)=2.6 Hz, JACHTUNGTERN(NUGN H,H)=
G
E
ACHTUNGTRENNUNG
9.83 ppm (s, 1H); 13C NMR (75 MHz, [D6]DMSO): d=11.1, 13.7, 38.2,
46.5, 47.1, 51.5, 61.3, 91.5, 111.1, 111.8, 115.2, 118.6, 119.8, 124.6, 125.6,
126.4, 127.72, 129.8, 131.6, 131.8, 132.5, 133.9, 134.3, 135.7, 138.0, 139.0,
142.2, 143.3, 144.2, 152.9, 154.8, 155.5, 157.7, 158.2, 162.9, 191.4 ppm; IR
(neat): n˜ =1689, 1628, 1595, 1513, 1349, 1242, 1139, 1099 cmꢀ1; UV-Vis
ACHTUNGTRENNUNG
E
ACHTUNGTRENNUNG
G
ACHTUNGTRENNUNG
4
3
ACHUTNGREN(NUG H,H)=2.5 Hz, JAHCTUNGTREN(NUNG H,H)=10.0 Hz, 1H),
AHCTUNGTRENNUNG
[D4]MeOH): d=16.4, 26.7, 39.1, 47.9, 50.1, 52.2, 70.6, 84.1, 93.2, 112.0,
112.9, 121.8, 124.9, 127.9, 128.9, 132.9, 132.9, 133.8, 135.1, 135.8, 137.4,
139.3, 140.7, 145.0, 145.2, 154.9, 156.7, 157.8 ppm; IR (neat): n˜ =2928,
(CH3CN): lmax =640 nm; MS (ESI+): m/z (%): 690.20 [M]+ (100); m/z
C
calcd for C41H40N9O2+: 690.81.
Quencher 5c: BHQ-3 derivative 5a (10 mg, 18.5 mmol, 1 equiv) and 8
(6.25 mg, 22.5 mmol, 1.2 equiv) were dissolved in 1:1 DMSO/H2O (3 mL).
Aqueous solutions of sodium ascorbate (0.7 mg, 3.7 mmol, 0.2 equiv) and
CuSO4 pentahydrate (0.1 mg, 1.85 mmol, 0.1 equiv) were sequentially
added and the reaction mixture was stirred under an Ar atmosphere at
RT for 2 h. The reaction was monitored by RP-HPLC (system A). On
completion, the mixture was diluted with an aqueous solution of TFA
(0.1%, 4 mL) and purified by semi-preparative RP-HPLC (system B).
After lyophilization, 5c was obtained as a dark-blue amorphous powder
(10 mg, 67%). HPLC (system A): tR =17.2 min, purity=99%; 1H NMR
(300 MHz, [D6]DMSO): d=0.98 (brt, 3H), 1.23 (brt, 3H), 2.90 (s, 3H),
1689, 1595, 1516, 1355, 1242, 1142, 1096 cmꢀ1; UV-Vis (CH3CN): lmax
=
658 nm; MS (ESI+): m/z (%): 527.33 [M]+ (100); m/z calcd for
C
C34H35N6+: 527.68.
Quencher 4b: Purification by semi-preparative RP-HPLC (system B),
yield=17%. HPLC (system A): tR =24.7 min, purity=93%; 1H NMR
(200 MHz, CD3CN): d=1.02 (brt, 3H), 1.27 (brt, 3H), 2.15 (s, 3H), 3.08
(s, 3H), 3.38 (brq, 2H), 3.69 (brq, 2H), 4.35 (s, 2H), 5.79 (d, 4J
ACHTUNGTRENNUNG
2.5 Hz, 1H), 6.69 (d, 3J
1H), 7.56 (m, 2H), 7.74 (d, J
3H), 8.05 (d, 3J
(H,H)=9.2 Hz, 2H), 7.29 (d, 4J
ACHUTGTNRENNUG CAHTUNGTRENNUNG
3
AHCTUNGTRENNUNG
G
ACHTUNGTRENNUNG
3
ACHTUNGTRENNUNG(H,H)=9.0 Hz, 1H), 8.30 ppm (d, JHCATUNGTRENN(UGN H,H)=9.0 Hz, 1H); too little of this
compound was obtained to be well-characterized by 13C NMR spectro-
3.5–3.8 (m, 10H), 4.02 (brq, 2H), 4.66 (brq, 2H), 5.69 (m, 1H), 6.78
3J(H,H)=9.0 Hz, 2H), 7.23 (m, 1H), 7.70 (d, 3J
(H,H)=8.9 Hz, 2H), 7.76
(H,H)=7.2 Hz, 2H), 7.95 (m, 3H), 7.98 (m, 1H), 8.18 (m, 3H), 8.25
(m, 1H), 8.40 (d, 3J(H,H)=9.0 Hz), 8.61 ppm (s, 1H); 13C NMR
ACHTUNGTRENNUNG(d,
A
ACHTUNGTRENNUNG
scopy; IR (neat): n˜ =2916, 1725, 1686, 1631, 1589, 1516, 1467, 1349, 1242,
3
1136, 1090 cmꢀ1; UV-Vis (CH3CN): lmax =640 nm (CH3CN); MS (ESI+):
(d, JACHTGNUTRENNUNG
+
m/z (%): 517.33 [M]+ (100); m/z calcd for C32H33N6O : 517.64.
AHCTUNGTRENNUNG
C
(75 MHz, [D6]DMSO): d=11.0, 13.6, 37.6, 41.1, 46.4, 47.2, 51.3, 63.7,
91.4, 111.1, 111.8, 119.8, 124.5, 126.3, 127.0, 127.6, 131.5, 132.4, 133.7,
137.2, 135.6, 137.8, 138.9, 142.6, 143.3, 144.0, 152.7, 154.6, 155.4, 158.9,
167.2, 170.9 ppm; IR (neat): n˜ =3315, 2959, 1737, 1662, 1597, 1517, 1467,
1354, 1241, 1143, 828 cmꢀ1; UV-Vis (PBS): lmax =610 nm; MS (ESI+):
Quencher 4c: Purification by semi-preparative RP-HPLC (system B),
yield=6%. HPLC (system A): tR =20.7 min, purity=95%; 1H NMR
(300 MHz, [D4]MeOH): d=1.08 (brt, 3H), 1.30 (brt, 3H), 3.10 (s, 3H),
3.32 (brq, 2H), 3.47 (d, 3J
1H), 3.67 (d, 3J
(d, 4J(H,H)=2.5 Hz, 1H), 6.82 (d, 3J
(H,H)=1.8 Hz, 1H), 7.62 (m, 2H), 7.72 (d, 3J
(m, 4H), 8.09 (d, 3J(H,H)=10.0 Hz, 1H), 8.16 (dd, 4J
(H,H)=9.0 Hz, 1H), 8.30 ppm (d, J
(H,H)=9.0 Hz, 2H), 3.62 (d, 3J
ACHUTGTNRENNUG CAHTUNGTRENNUNG
ACHTUNGTRENNUNG
m/z (%): 404.67 [M2H]2+
,
808.27 [M+H]+ (100); m/z calcd for
G
ACHTUNGTRENNUNG
C39H41N11O7S: 807.29.
E
ACHTUNGTRENNUNG
E
G
Quencher 6b: Quencher 6a was synthesized from Methylene Violet
3RAX and 3a by the general procedure described above, and rapidly pu-
rified by triturating the crude with a mixture of Et2O/MeOH (9:1). The
recovered blue powder was dissolved in dry acetone (2 mL) and NaI
(200 mg, 265 mmol, 10 equiv) was added. The reaction mixture was stirred
at RT for 1 h. The reaction was monitored by RP-HPLC (system A). On
completion, acetone was removed under reduced pressure and the crude
product was purified by semi-preparative RP-HPLC (system B). After
lyophilization, 6b was obtained as a dark-blue amorphous powder (5 mg,
42% over two steps). HPLC (system A): tR =25.1 min, purity=95%;
1H NMR (300 MHz, [D6]DMSO): d=0.98 (brt, 3H), 1.23 (brt, 3H), 3.36
3
R
ACHTUNGTRENNUNG
compound was obtained to be well-characterized by 13C NMR spectro-
scopy; IR (neat): n˜ =3244, 2916, 1680, 1586, 1510, 1467, 1349, 1300, 1239,
1178, 1121, 1066 cmꢀ1; UV-Vis (CH3CN): lmax =638 nm; MS (ESI+): m/z
(%): 535.40 [M]+ (100); m/z calcd for C32H35N6O2+: 535.66.
C
Quencher 4d: Due to its poor chemical stability, this compound was only
obtained in trace amounts and NMR characterization was not possible.
Quencher 5a: Purification by semi-preparative RP-HPLC (system B),
yield=64%. HPLC (system A): tR =24.8 min, purity=96%; 1H NMR
(300 MHz, [D6]DMSO): d=1.01 (brt, 3H), 1.26 (brt, 3H), 3.09 (s, 3H),
(brq, 2H), 3.60 (m, 8H), 5.69 (d, 4J
(H,H)=9.2 Hz, 2H), 7.26 (d, 4J
(H,H)=1.9 Hz, 1H), 7.76 (m, 4H), 7.92
(m, 3H), 8.02 (dd, 4J(H,H)=2.2 Hz, 3J
(H,H)=10.0 Hz, 1H), 8.19 (m,
2H), 8.43 ppm (d, 3J(H,H)=9.2 Hz, 1H); 13C NMR (75 MHz,
(H,H)=2.2 Hz, 1H), 7.07 (d, 3J-
ACHTUNGTRENNUNG
3.32 (brq, 2H), 3.51 (t, 3J
2H), 3.73 (brq, 2H), 5.75 (d, 4J
9.0 Hz, 2H), 7.21 (d, 4J(H,H)=1.8 Hz, 1H), 7.6 (m, 2H), 7.65 (d, 3J-
(H,H)=9 Hz, 2H), 7.8 (dd, 4J(H,H)=2.5 Hz, 3J
(H,H)=10.0 Hz, 1H),
7.89 (m, 3H), 8.04 (m, 2H), 8.22 ppm (d, 3J(H,H)=9 Hz, 1H); 13C NMR
A
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
[D6]DMSO): d=11.0, 13.7, 41.1, 45.6, 45.8, 46.5, 91.5, 111.6, 113.7, 119.7,
124.8, 126.3, 127.72, 131.63, 132.9, 133.8, 134.3, 135.7, 138.0, 139.0, 144.1,
144.5, 153.7, 154.8, 155.2, 157.7, 158.2, 166.7 ppm; IR (neat): n˜ =1962,
AHCTUNGTRENNUNG
(75 MHz, [D6]DMSO): d=11.6, 14.0, 39.4, 52.4, 93.2, 111.8, 112.4, 113.1,
121.5, 125.1, 127.7, 128.9, 132.9, 133.0, 133.8, 134.9, 135.8, 137.3, 139.2,
140.6, 145.4, 154.6, 156.8, 157.5 ppm; IR (neat): n˜ =2132, 2102, 1589,
1943, 1589, 1513, 1464, 1346, 1136, 1094 cmꢀ1; UV-Vis (CH3CN): lmax
=
1696
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 1686 – 1699