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<A NAME="RG03210ST-25">25</A>
Synthesis of Compound
2
Under N2, freshly distilled 2,3,3-trimethylindolenine
(1.5
mL, 9.34 mmol) was dissolved in CHCl3 (17 mL) and degassed
via freeze-pump-thaw (3 cycles). 3-Iodopropan-1-ol (1.00 g, 10.4
mmol) was added. The mixture was refluxed for 24 h and slowly cooled
to r.t. The solvent was evapo-rated. The remaining purple oil was
washed with PE and triturated with Et2O to afford 2 as a purple solid (3.22 g, 99%). ¹H
NMR (300 MHz, DMSO-d
6): δ = 8.01-7.91
(m, 1 H), 7.90-7.81 (m, 1 H), 7.67-7.57 (m, 2
H), 4.56 (t, 2 H, J = 6.9
Hz), 3.55 (t, 2 H, J = 5.5
Hz), 2.87 (s, 3 H), 2.11-1.97 (m, 2 H), 1.54 (s, 6 H). ¹³C
NMR (75 MHz, DMSO-d
6): δ = 196.4,
141.7, 141.0, 129.2, 128.8, 123.4, 115.4, 57.8, 54.1, 45.7, 29.6,
21.8, 14.3, 5.4. ESI-MS (CH2Cl2-MeOH + 10
mmol/L NH4Ac): m/z (%) = 218.0(100) [M+].
<A NAME="RG03210ST-26">26</A>
Synthesis of Compound
3
Compound 2 (3.09 g, 8.97
mmol) was suspended in degassed H2O (53 mL) and finely
ground KOH (1.23 g, 21.9 mmol) was added. The reaction mixture was
stirred at r.t. under N2 for 2 h. CH2Cl2 (50
mL) was added, and the mixture was stirred for additional 30 min.
The aqueous layer was separated and extracted with CH2Cl2 (2 × 45
mL). The combined organic layers were washed with brine and H2O and
dried over Na2SO4. The solvent was removed
under reduced pressure, and the residue was dried under vacuum. The
crude product was purified by gradient flash chromatog-raphy on
silica gel (PE-EtOAc, 4:1 to 1:1) to afford 3 as colorless
crystals (0.84 g, 43%). ¹H NMR (600
MHz, CDCl3): δ = 7.13
(t, 1 H, J = 7.7
Hz, CH arom.), 7.08 (d, 1 H, J = 7.3
Hz, CH arom.), 6.81 (t, 1 H, J = 7.2
Hz, CH arom.), 6.59 (d, 1 H, J = 7.8
Hz, CH arom.), 4.08 (dt, 1 H, J = 2.6,
12.4 Hz, CH2O), 3.72 (dd, 1 H, J = 5.2,
11.8 Hz, CH2O), 3.66 (dd, 1 H, J = 4.6,
14.6 Hz, CH2N), 3.54 (m, 1 H, CH2N), 2.02-1.92
(m, 1 H), 1.56 (s, 3 H, CH3), 1.30 (s, 3 H, CH3),
1.20 (d, 1 H, J = 13.4
Hz), 1.07 (s, 3 H, CH3).
¹³C
NMR (150 MHz, CDCl3): δ = 148.1
(Cquat.), 139.2 (Cq), 127.2 (+, CH),
122.0 (+, CH), 119.2 (+, CH), 108.6 (+,
CH), 98.3 (Cq), 61.0 (-, CH2), 48.0
(Cq), 39.1 (-, CH2), 26.7 (+, CH3),
21.7 (-, CH2), 18.6 (+, CH3),
13.0 (+, CH3). HRMS (EI-MS): m/z calcd
for C14H19NO [M+
]: 217.1467; found: 217.1472.
<A NAME="RG03210ST-27">27</A>
Synthesis of Compound
4
Under strictly degassed conditions, compound 3 (1.642 g, 7.56 mmol) was dissolved in
dry EtOH and degassed. 5-Nitrosalicylaldehyde (1.28 g, 7.64 mmol)
was added under N2, and the mixture was sonicated at
35 kHz for 1 h. EtOH was removed under reduced pressure. The residue
was taken up in CH2Cl2 and washed with H2O.
The organic layer was dried over Na2SO4 and
evaporated. The product was purified by flash chromatography on
silica gel (hexane-EtOAc = 1:1) to yield 4 (2.34 g, 84%) as a purple solid. ¹H
NMR (600 MHz, CDCl3): δ = 8.00
(td, 2 H, J = 2.7,
7.9 Hz), 7.19 (dt, 1 H, J = 1.2,
7.7 Hz), 7.09 (dd, 1 H, J = 0.8,
7.2 Hz), 6.91 (d, 1 H, J = 10.3
Hz), 6.88 (dt, 1 H, J = 0.8,
7.5 Hz), 6.75 (d, 1 H, J = 8.9
Hz), 6.65 (d, 1 H, J = 7.8
Hz), 5.88 (d, 1 H, J = 10.4
Hz), 3.71 (t, 2 H, J = 6.0
Hz), 3.40-3.34 (m, 1 H), 3.29-3.23 (m, 1 H), 1.98-1.90
(m, 1 H), 1.84-1.77 (m, 1 H), 1.29 (s, 3 H), 1.19 (s, 3
H). ¹³C NMR (150 MHz, CDCl3): δ = 159.6,
147.0, 141.0, 136.0, 128.2, 127.8, 125.9, 122.7, 121.8, 121.7, 119.6,
118.5, 115.5, 106.9, 106.8, 60.7, 52.6, 40.67, 31.6, 25.9, 19.9.
HRMS (EI-MS): m/z calcd for C21H22N2O4 [M+
]: 366.1580; found: 366.1572.
<A NAME="RG03210ST-28">28</A>
Synthesis of Compound
5
A solution of 4 (1.06 g,
2.89 mmol) in dry CH2Cl2 (22 mL) was cooled
to 0 ˚C, and dry DIPEA (2.2 mL, 12.936 mmol) was
added under Ar. 4-Nitrophenylchloroformate (1.765 g, 8.757 mmol)
was dissolved in dry CH2Cl2 (16.5 mL) and added
in small portions over 3 h. The reaction was slowly warmed up to
ambient temperature, and the solvent was evaporated. The crude product
was dried over night under vacuum and purified by flash chromatography
on silica gel (PE-EtOAc = 2:1) to afford a pale
pink solid that was repeatedly purified by flash chromatography
on silica gel(toluene). The product was triturated with Et2O
and obtained as a pale yellow solid (1.44 g, 93%). ¹H
NMR (400 MHz, CDCl3): δ = 8.28
(d, 2 H, J = 9.3
Hz), 8.05-7.98 (m, 2 H), 7.34 (d, 2 H, J = 9.3
Hz), 7.20 (dt, 1 H, J = 1.3,
7.7 Hz), 7.11 (dd, 1 H, J = 0.9,
7.3 Hz), 6.91 (t, 2 H, J = 8.6
Hz), 6.77 (d, 1 H, J = 8.8
Hz), 6.61 (d, 1 H, J = 7.7
Hz), 5.88 (d, 1 H, J = 10.3
Hz), 4.34 (dt, 2 H, J = 1.7,
5.9 Hz), 3.44-3.28 (m, 2 H), 2.19-1.98 (m, 2 H),
1.30 (s, 3 H), 1.20 (s, 3 H). ¹³C NMR (100
MHz, CD2Cl2): δ = 162.0
(Cq), 159.9 (Cq), 156.0 (Cq), 152.9
(Cq), 147.3 (Cq), 145.9 (Cq), 141.5
(Cq), 136.6 (Cq), 128.9 (+, CH),
128.2 (+, CH), 126.5 (+, CH), 126.3 (+,
CH), 125.7 (+, CH), 123.2 (+, CH), 122.3 (+,
CH), 122.3 (+, CH), 122.0 (+, CH), 120.2 (+,
CH), 119.1 (Cq), 116.1 (+, CH), 115.9 (+,
CH), 107.3 (Cq), 107.0 (+, CH), 67.6 (-,
CH2), 40.5 (-, CH2), 28.3 (-,
CH2), 26.1 (+, CH3), 20.0 (+,
CH3). HRMS (PI-EI): m/z calcd
for C28H25N3O8 [M+
]: 531.1642; found: 531.1639.
<A NAME="RG03210ST-29">29</A>
Synthesis of Compound
7
A solution of 5 (25 mg,
0.047 mmol) in dry DMF (5 mL) was cooled to 0 ˚C.
Dry DIPEA (50 µL, 0.30 mmol) was added under N2 and
stirred for 5 min. Compound 6 (37 mg, 0.09 mmol)
was added. The reaction mixtures was stirred at 0 ˚C over
5 h, was slowly warmed to r.t., concentrated in vacuo, and purified
by flash chromatography on silica gel (PE-EtOAc = 1:1 + 0.1% DIPEA).
Compound 7 (36 mg, 97%) was obtained
as a pale pink solid. ¹H NMR (400 MHz, CDCl3): δ = 8.05-7.93
(m, 2 H), 7.41 (d, 2 H, J = 7.3
Hz), 7.33-7.27 (m, 6 H), 7.24-7.15 (m, 2 H), 7.09
(dd, 1 H, J = 0.8,
7.2 Hz), 6.90-6.81 (m, 6 H), 6.74 (d, 1 H, J = 8.9 Hz),
6.57 (d, 1 H, J = 7.7
Hz), 5.84 (d, 1 H, J = 10.4
Hz), 4.96 (s, 1 H, OH), 4.15-4.01 (m, 3 H), 3.90-3.83
(m, 1 H), 3.78 (s, 6 H, 2 × OMe), 3.39
(s, 1 H, NH), 3.32-3.25 (m, 1 H), 3.25-3.12 (m,
4 H), 2.01-1.92 (m, 1H), 1.90-1.83 (m, 1 H), 1.28
(s, 3 H), 1.18 (s, 3 H). ¹³C NMR (100
MHz, CDCl3): δ = 159.5,
158.6, 146.9, 144.5, 141.0, 136.0, 135.7, 130.0, 128.2, 128.0, 128.0,
127.8, 126.9, 125.9, 122.7, 121.7, 119.6, 118.5, 115.5, 113.2, 106.7,
106.7, 86.3, 70.3, 70.3, 65.0, 64.9, 62.5, 60.4, 55.2, 52.6, 44.1,
40.3, 31.5, 28.2, 28.2, 25.9, 25.5, 21.0, 19.8, 14.2. HRMS (PI-MS): m/z calcd for C46H47N3O9 [M+
]: 785.3312; found: 785.3325.
<A NAME="RG03210ST-30">30</A>
Synthesis of Compound
8
Compound 7 (100 mg, 0.13
mmol) was dissolved in dry CH2Cl2 (5 mL).
Et3N (250 µL, 1.794 mmol) and 2-cyano-ethyl-N,N-diisopropylchlorophosphoramidite
(56.8 µL, 0.26 mmol) were added and the solution stirred
for 11 h at r.t. The solvents were evaporated in vacuo, CH2Cl2 was
added, the solution was poured into aq sat. NaHCO3, and
extracted with CH2Cl2 (2×). The combined
organic layers were dried over Na2SO4, the
solvent was evaporated, and the remaining solid was purified by
flash chromatography on silica gel (PE-EtOAc, 2:1 + 1% Et3N).
The product was resolved in benzene (3 mL) and lyophilized yielding 8 (119 mg, 95%) as pale purple
foam, which was dissolved in dry MeCN and applied directly for oligonucleotide
synthesis. ³¹P NMR(121 MHz, CDCl3): δ = 149.9,
149.8. ESI-MS (CH2Cl2-MeOH + 10
mmol/L NH4Ac): m/z (%) = 986.5
(62) [MH+], 303.2 (100) [DMT+].
<A NAME="RG03210ST-31">31</A>
The oligonucleotide DNA1 was
prepared using an Expedite 8909 DNA synthesizer (Applied Biosystems)
via standard phosphoramidite protocols using CPGs (1 µmol)
with a longer coupling time of 6.3 min and a higher concentration of 8 (0.1 M). After preparation, the trityl-off
oligonucleotide was cleaved off the resin and deprotected by treatment
with concd NH4OH at r.t. for 18 h, dried and purified
by RP HPLC using the following conditions: A = NH4OAc
buffer (50 mM), pH 6.8; B = MeCN;
gradient = 20% B over 45 min,
flow rate 2.5 mL/min. ESI-MS: DNA1: m/z calcd: 5408.0; found: 1802.7 [M - 3
H]³-, 1351.6 [M - 4
H]4-.
<A NAME="RG03210ST-32">32</A>
Weisbrod SH.
Marx A.
Chem. Commun.
2008,
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Bertozzi CR.
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109:
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Gramlich PM.
Wirges CT.
Manetto A.
Carell T.
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2008,
47:
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Huisgen R.
Angew.
Chem., Int. Ed. Engl.
1962,
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Tornoe CW.
Christensen C.
Meldal M.
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Finn MG.
Sharpless KB.
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Berndl S.
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Kele P.
Lachmann D.
Li X.
Wolfbeis OS.
Wagenknecht H.-A.
Bioconjugate
Chem.
2009,
20:
558
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Binder WH.
Sachsenhofer R.
Straif CJ.
Zirbs R.
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Zhang Q.
Ning Z.
Yan Y.
Qian S.
Tian H.
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<A NAME="RG03210ST-42">42</A>
Synthesis of Compound
9
Under strictly degassed conditions freshly distilled
2,3,3-trimethylindolenine (30 mL, 186.9 mmol) was dissolved MeCN
(40 mL) and degassed using freeze-pump-thaw method (3 cycles). Freshly
distilled 1,3-diiodopropane (75 mL, 653 mmol) was added, and the
reaction mixture was refluxed for 48 h. After cooling, the remaining
solid was filtered off, washed with MeCN and CHCl3 (2×)
and dried under vacuum yielding 9 (65.7
g, 77%) as a pale grey-yellow solid. ¹H
NMR (400 MHz, DMSO-d
6): δ = 8.02-7.94
(m, 1 H, arom. C8-H), 7.88-7.80 (m, 1 H, arom. C5-H), 7.69-7.59 (m,
2 H, arom. C6/7-H), 4.48 (t, 2 H, N-CH2), 3.43
(t, 2 H, ICH2), 2.86 (s, 3 H, C2Me), 2.41 (m, 2 H, CH2-propyl),
1.55 (s, 6 H, C3-Me). ¹³C NMR (100
MHz, DMSO-d
6): δ = 197.3,
141.8, 141.1, 129.4 (Carom), 129.3, 128.9 (Carom), 123.5
(Carom), 115.2 (Carom), 54.2, 48.4 (NCH2),
31.0 (CH2-propyl), 22.0, 14.3 (C2Me), 2.2 (ICH2).
ESI-MS (CH2Cl2-MeOH + 10
mmol/L NH4Ac): m/z (%) = 327.8
(100) [M+].
<A NAME="RG03210ST-43">43</A>
Synthesis of Compound
10
Under N2 9 (212
mg, 0.466 mmol) was suspended in degassed H2O (56 mL)
and finely ground NaOH (559 mg, 13.98 mmol) was added in one portion.
The mixture was heated at 80 ˚C for 15 min, cooled
to r.t., Et2O (60 mL) was added, stirred for additional
1 h, and extracted with Et2O and CH2Cl2 (2 × 20
mL). The organic layers were washed with H2O, combined,
dried over Na2SO4, and evaporated to give 10 (139 mg, 91%) as a pale pink
solid. ¹H NMR (300 MHz): δ = 7.18-7.08
(m, 2 H), 6.83-6.61 (m, 2 H), 3.93 (dd, 2 H, J = 1.9, 22.2
Hz), 3.63 (t, 2 H, J = 6.8
Hz), 3.23 (t, 2 H, J = 6.7
Hz), 2.21 (quint, 2 H, J = 6.8
Hz), 1.35 (s, 6 H).
¹³C NMR
(75 MHz): δ = 161.5 (Cq),
145.7 (Cq), 137.5 (Cq), 127.6 (+,
CH), 122.0 (+, CH), 118.7 (+, CH), 105.3 (+,
CH), 104.1 (+, CH3), 87.5 (+, CH3),
73.8 (-, CH2), 44.3 (Cq), 42.6 (-,
CH2), 30.1 (-, CH2), 3.6 (-,
CH2). MS (EI, 70 eV): m/z (%) = 184.0(100) [(M - HI - CH3)+
], 198.9 (42) [(M - HI)+
], 327.0(9) [M+
].
<A NAME="RG03210ST-44">44</A>
Synthesis of Compound
11
Freshly prepared 10 (2.78
g, 8.49 mmol) was dissolved in dry EtOH (85 mL) and degassed via
freeze-pump-thaw (3 cycles). 5-Nitrosalicylaldehyde (1.43 g, 8.53
mmol) was added. The mixture was sonicated at 35 kHz for 1 h. The solvent
was removed under reduced pressure, the residue was taken up in
CH2Cl2, and washed with H2O. The
organic layer was dried over MgSO4 and evoporated. The
raw product was dried under vacuum and purified by flash chormatography
on silica gel (CH2Cl2) yielding 11 (7.97 mmol, 94%) as golden
foam. ¹H NMR (400 MHz, CDCl3): δ = 8.06-8.01
(m, 2 H), 7.23 (dt, 1 H, J = 1.3,
7.7 Hz), 7.14 (dd, 1 H, J = 0.9,
7.3 Hz), 6.99 (d, 1 H, J = 10.2
Hz), 6.93 (dt, 1 H, J = 0.8,
7.5 Hz), 6.76 (d, 1 H, J = 8.5
Hz), 6.69 (d, 1 H, J = 7.7
Hz), 5.93 (d, 1 H, J = 10.3
Hz), 3.41-3.17 (m, 4 H), 2.34-2.21 (m, 1 H), 2.17-2.06
(m, 1 H), 1.34 (s, 3 H), 1.23 (s, 3 H). ¹³C
NMR (100 MHz, CDCl3): δ = 159.2,
146.6, 140.8, 135.8, 128.4, 127.6, 125.7, 122.6, 121.6, 121.6, 119.6,
118.3, 115.3, 106.6, 106.4, 52.4, 43.9, 32.3, 25.8, 19.7, 3.3. HRMS
(EI-MS): m/z calcd for C21H21N2O3I [M+
]: 476.0597; found: 476.0598.
<A NAME="RG03210ST-45">45</A>
Synthesis of Compound
12
Compound 11 (1.58 g, 3.31
mmol) was dissolved in dry DMF (62 mL). NaN3 (877 mg,
13.5 mmol) was added in one portion, and the reaction mixture was
stirred in the dark at r.t. for 19 h. The solvent was evaporated
under reduced pressure, and the crude product was purified by flash chromatography
on silica gel (CH2Cl2) yielding 12 (1.08 g, 83%) as a golden foam. ¹H
NMR (400 MHz, CDCl3): δ = 8.01
(m, 2 H), 7.20 (dt, 1 H, J = 1.3,
7.7 Hz), 7.10 (dd, 1 H, J = 0.9,
7.3 Hz), 6.94 (d, 1 H, J = 10.3
Hz), 6.90 (dt, 1 H, J = 0.9,
7.5 Hz), 6.75 (d, 1 H, J = 8.4
Hz), 6.60 (d, 1 H, J = 7.8
Hz), 5.86 (d, 1 H, J = 10.4
Hz), 3.39-3.18 (m, 4 H), 2.01-1.90 (m, 1 H), 1.88-1.75
(m, 1 H), 1.29 (s, 3 H), 1.19 (s, 3 H). ¹³C
NMR (100 MHz, CDCl3): δ = 159.4,
146.8, 141.1, 136.0, 128.4, 127.8, 125.9, 122.8, 121.8, 121.6, 119.8,
118.4, 115.5, 106.7, 106.6, 52.6, 49.0, 40.8, 28.1, 25.9, 19.9.
HRMS (PI-EI): m/z calcd for
C21H21N5O3 [M+
]: 391.1644; found: 391.1644.
<A NAME="RG03210ST-46">46</A>
The concentration of the DNA building
block was increased to 0.1 M and the coupling time extended to 6.3
min.³9 The trityl-off oligonucleotides were
cleaved from the resin and deprotected by treatment with concd NH4OH
at r.t. for 24 h. Compound 12 (1.5 mL,
10 mM), CuI (223.6 mL, 100 mM), TBTA (447 mL, 100 mM), each in DMSO-t-BuOH (3:1), and sodium ascorbate (125
mL, 400 mM) in H2O were added to the oligonucleotide
(1 mmol). The vial was vortexed, shaken 22 h at r.t., and then evaporated
to dryness using a SpeedVac. NaOAc (100 mL, 0.15 mmol) was added,
and the mixture was stored for 1 h r.t. After EtOH precipation,
the oligonucleotides were dissolved in H2O (500 mL),
desalted by NAP-5 column (GE Healthcare), dried, purified by RP HPLC
using the following conditions: A = NH4OAc
buffer (50 mM), pH 6.8; B = MeCN;
gradient = 15% B over 45 min,
flow rate 2.5 mL/min, lyophilized, and quantified by their
absorbance at 260 nm. Duplexes were prepared by heating of the modified
oligonucleotides in the presence of 1.2 equiv unmodified complementary
strand to 90 ˚C (10 min), followed by slow cooling
to r.t. ESI-MS: DNA2: m/z calcd
5550.0 [M]-, 1849.0 [M - 3
H]³-; found: 1849.2 [M - 3
H]³-, 1386.8 [M - 4
H]4-; DNA3: m/z calcd 5580.0 [M]-, 1859.0 [M - 3
H]³-; found: 1860.1 [M - 3
H]³-, 1395.0 [M - 4
H]4-; DNA4: m/z calcd 5598.0 [M]-,
1865.0 [M - 3 H]³-; found:
1866.2 [M - 3 H]³-,
1399.3 [M - 4 H]4-; DNA5: m/z calcd
5630.5 [M]-, 1875.8 [M - 3
H]³-; found: 1876.7 [M - 3
H]³-, 1407.5 [M - 4
H]4-.
<A NAME="RG03210ST-47A">47a</A>
Görner H.
Chem. Phys.
1997,
222:
315
<A NAME="RG03210ST-47B">47b</A>
Chibisov AK.
Görner H.
J.
Phys. Chem. A
1997,
101:
4305
