87
TRDRP 3 11/30/2008
Research Design and Methods
Chronological outline of proposed research (2 years of fellowship support are requested)
1. Synthesize and characterize a ruthenium-dye conjugate that exhibits observable acceptor emission through RET only in the presence of DNA base mismatches.
2. Synthesize and characterize a rhodium-dye conjugate that luminesces only in the presence of DNA base mismatches.
3. Synthesize and characterize non-conjugated ruthenium molecules that show luminescence enhancement in the presence of DNA base mismatches.
4. Evaluate the luminescence responses of these molecules to synthetic oligonucleotides containing base mismatches in physiologically relevant media.
5. Apply molecules with desired luminescence properties in confocal microscopy imaging and flow cytometry analysis with MMR-deficient and MMR-proficient human cell lines.
1. Design and synthesis of a ruthenium-dye conjugate molecule
As ruthenium complexes such as Ru(bpy)2(dppz)2+ fluoresce in the presence of both well-matched and mismatched DNA, albeit more strongly with the latter, a strategy to selectively register the signal from ruthenium bound to
mismatched DNA will effectively suppress the fluorescence signal from ruthenium bound to well-matched DNA, thus allowing much more sensitive detection of mismatches. Since matched DNA binding occurs from the major groove and mismatch binding occurs from the minor groove, we propose to tether a RET acceptor, which emits only when bound to DNA in the minor groove, to ruthenium, so that emission from the RET acceptor is observed only when ruthenium is also in the minor groove, i.e. only when there is a mismatch (Figure 2).
A good candidate for a RET acceptor is TO-PRO-3, a trimethine cyanine dye that binds DNA from the minor groove and exhibits substantially enhanced fluorescence in the bound form.13 It has been shown to effect RET with
Ru(bpy)2(dppz)2+ efficiently.14 We will tether a slightly modified TO-PRO-3 (herein termed TO-3) to ruthenium (Scheme 1). As illustrated in Figure 2, without mismatches, ruthenium binds to DNA nonspecifically from the major groove and the tethered TO-3 moiety is unable to bind DNA from the major groove. Models show that the tether is too short to allow simultaneous binding of ruthenium and TO-3 in adjacent major and minor grooves. As a result, little emission from TO-3 will occur when the conjugate is excited at the ruthenium absorption wavelength 440 nm. However, when a mismatch is present, ruthenium binds the mismatch from the minor groove, allowing the tethered TO-3 to bind in the minor groove as well. With excitation of ruthenium at 440 nm, RET occurs (based on spectral overlap of TO-3 and Ru(L2)(dppz)2+), and TO-3 will emit at 660 nm. At the same time, ruthenium emission at 620 nm will be greatly reduced. The shift in emission maximum of the resulting emission profile and altered intensity will be indicative of mismatch binding.
Fig. 2 Illustration of Ru-TO-3 conjugate binding to well-matched (left) and mismatched (right) DNA. TO-3 emission through RET is detectable only in the presence of a mismatch (mismatched bases shown in red) when both the ruthenium and TO-3 are bound in the minor groove.
Figure 4.1. Illustration of Ru-TO-3 conjugate binding to well-matched (left) and mis- matched (right) DNA. TO-3 emission through RET is detectable only in the presence of a mismatch (red) when both the ruthenium and TO-3 are bound in the minor groove.
ing ruthenium signal without TO-3. Second, ruthenium fluorescence has a long lifetime, which will be translated to a longer lifetime for TO-3 emission through RET. A longer lifetime allows better distinction of probe signal from cellular background fluorescence.5 Both stronger emission intensity and longer lifetimes have been observed in an untethered system of Ru(bpy)2dppz2+ and TO-PRO-3 simultaneously bound to DNA,4 and we expect these emission features to be preserved in our conjugated system. In this chapter, we report the synthesis and luminescence properties of four conjugates (1,2,3,4, Figure 4.2) in the presence of mismatch-containing oligonucleotides.
N Ru N N N
N N
O N NH
N S N
N
3+
N Ru N N N
N N
O N NH
N S N
N
3+
Ru N N N N N N
N
N N
3+
N O NH
N S
HN O
1
2
3
N Ru N N N
N N
3+
N N
N
NH2 NH
O NH
O
4
Figure 4.2. Chemical structures of Ru-dye conjugates: Ru-10-TO (1), Ru-1-TO (2), Ru- 7-TO (3), Ru-Eth (4).
components in all conjugate syntheses.11,12 Briefly, HBTU was added to the carboxylic acid-terminated moiety and DIEA was added to the amine-terminated moiety, both in a solution of DMF. The two parts were then mixed and the coupling reaction was allowed to proceed overnight at room temperature under a dry Ar atmosphere. The compounds5, 9,10 and 12were recrystallized by vapor diffusion of diethyl ether into a MeOH solution.
Purity of the final conjugates was checked by analytical HPLC. Characterization of the compounds are reported below.
TO-COOH 5. 1H-NMR (300 MHz, DMSO) δ 8.51 (d, J = 7.2 Hz, 1H), 8.46 (d, J = 8.6 Hz, 1H), 8.07 (dt, 2H), 7.97–7.89 (t, 1H), 7.83 (m, 2H), 7.73–7.63 (t, 1H), 7.52 (d, 1H), 7.45 (t, 1H), 7.26 (t, 1H), 7.09 (d, J = 13.1 Hz, 1H), 6.41 (d, J = 11.9 Hz, 1H), 4.68 (t, J
= 7.1 Hz, 2H), 3.68 (s, 3H), 2.41 (t, 2H). ESI-MS: calc. 389.13, obs. 389.2 (M+).
Ru(phen)(HDPA)dppz2+ 6. 1H-NMR (300 MHz, CD3CN) δ 11.64 (s, 1H), 9.77 (dd, J
= 8.2, 1.3 Hz, 1H), 9.45 (dd, J = 8.2, 1.3 Hz, 1H), 9.17–9.05 (m, 2H), 8.77 (d, J = 7.1 Hz, 1H), 8.53–8.41 (m, 3H), 8.30 (d, J = 8.9 Hz, 1H), 8.20–8.12 (m, 4H), 8.07–8.01 (m, 1H), 7.91 (dd, J = 5.3, 1.2 Hz, 1H), 7.79 (dd, J = 5.4, 1.3 Hz, 1H), 7.69–7.52 (m, 5H), 7.46 (dd, J = 8.2, 5.3 Hz, 1H), 7.30 (d, J = 5.8 Hz, 1H), 7.20 (d, J = 5.7 Hz, 1H), 6.65–6.50 (m, 2H).
ESI-MS: calc. 735.14, obs. 734.2 (M-H)+, 367.6 (M2+). UV-vis (H2O): 357 nm (32,000 M−1cm−1), 374 nm (36,000 M−1cm−1), 410 nm (23,000 M−1cm−1).
Ru(phen)(DPA-(CH2)6-COOH)dppz2+ 7. ESI-MS: calc. 863.23, obs. 431.7 (M2+).
UV-vis (H2O): 357 nm (32,000 M−1cm−1), 374 nm (35,000 M−1cm−1), 410 nm (24,000 M−1cm−1).
Ru(phen)(DPA-(CH2)6-CONH-(CH2)2-NH2)dppz2+8. ESI-MS: calc. 905.29, obs. 452.7 (M2+).
Ru-10-TO 1. ESI-MS: calc. 1276.41, obs. 425.8 (M3+).
Bpy-1-TO 9. 1H-NMR (500 MHz, CD2Cl2) δ 8.59 (d, J = 4.9 Hz, 1H), 8.56–8.47 (m, 2H), 8.38 (d, J = 7.4 Hz, 2H), 8.29 (d, J = 8.1 Hz, 2H), 7.98–7.85 (m, 3H), 7.69–7.59 (m, 1H), 7.53–7.46 (m, 1H), 7.39–7.27 (m, 5H), 6.87 (d, J = 13.4 Hz, 1H), 6.25 (d, J = 12.3 Hz, 1H), 4.86 (t, J = 6.8 Hz, 2H), 4.52 (d, J = 6.0 Hz, 2H), 3.71 (s, 3H), 3.15 (t, J = 6.8 Hz, 2H), 2.50 (s, 3H). ESI-MS: calc. 570.23, obs. 570.1 (M+).
Ru-1-TO 2. 1H-NMR (500 MHz, CD3CN) δ 9.63 (d, J = 8.3 Hz, 1H), 9.51 (d, J = 8.3 Hz, 1H), 8.83 (d, J = 19.5 Hz, 2H), 8.71 (d, J = 7.7 Hz, 1H), 8.58 (d, J = 8.0 Hz, 1H), 8.50 (d, J = 8.5 Hz, 1H), 8.44 (d, J = 7.8 Hz, 1H), 8.40 (d, J = 8.6 Hz, 1H), 8.37 (d, J = 7.3
7.32 (d, J = 6.2 Hz, 1H), 7.27 (d, J = 7.4 Hz, 1H), 7.22 (d, J = 7.5 Hz, 1H), 7.16–7.09 (m, 2H), 6.95-6.90 (m, 2H), 6.22 (d, J = 12.3 Hz, 1H), 4.86 (t, J = 6.2 Hz, 2H), 4.54–4.46 (m, 2H), 3.54 (s, 3H), 2.62 (s, 3H). ESI-MS: calc. 1134.30, obs. 378.3 (M3+), 566.3 (M-H)2+, 623.0 (M+TFA)2+.
Bpy-7-TO 10. 1H-NMR (500 MHz, CD2Cl2) δ 8.63–8.48 (m, 2H), 8.46–8.30 (m, 3H), 8.15 (d, 1H), 8.01 (t, 1H), 7.92–7.83 (m, 2H), 7.66-7.60 (m, 2H), 7.51–7.46 (m, 1H), 7.42 (d, 1H), 7.36–7.27 (m, 4H), 6.92 (d, J = 13.4 Hz, 1H), 6.25 (d, J = 12.3 Hz, 1H), 4.81 (t, J = 6.7 Hz, 2H), 3.68 (s, 3H), 3.18 (dd, J = 12.7, 6.9 Hz, 2H), 2.88 (t, J = 6.3 Hz, 2H), 2.79–2.63 (m, 2H), 2.51 (s, 3H), 1.68 (m, 2H), 1.46 (m, 2H), 1.34 (m, 4H), 1.27 (m, 2H).
ESI-MS: calc. 654.33, obs. 654.2 (M+), 327.8 (M+H)2+.
Ru-7-TO 3. 1H-NMR (500 MHz, CD3CN) δ 9.67 (d, J = 8.3 Hz, 1H), 9.58 (d, J = 8.3 Hz, 1H), 8.69 (d, J = 8.4 Hz, 1H), 8.59 (d, J = 8.2 Hz, 1H), 8.50 (m, 4H), 8.37 (d, J = 8.9 Hz, 1H), 8.28 (m, 3H), 8.23 (d, J = 6.4 Hz, 1H), 8.20–8.16 (m, 2H), 8.09 (d, J = 6.6 Hz, 2H), 7.98 (m, 2H), 7.92 (m, 2H), 7.84 (m, 1H), 7.70 (m, 4H), 7.62 (m, 1H), 7.50 (m, 2H), 7.46 (d, J = 7.2 Hz, 1H), 7.38 (d, J = 7.9 Hz, 1H), 7.30 (t, J = 7.5 Hz,1H), 7.22 (d, J = 5.6 Hz, 1H), 7.14–7.10 (d, J = 5.6 Hz, 1H), 6.94 (d, J = 13.5 Hz, 1H), 6.75–6.70 (m, 1H), 6.30 (d, J = 12.1 Hz, 1H), 4.73 (t, J = 6.5 Hz, 2H), 4.49 (s, 3H), 3.63 (s, 2H), 3.14–3.06 (m, 2H), 2.79 (m, 2H), 2.56 (s, 3H), 1.68 (m, 2H), 1.31 (m, 6H), 1.24–1.18 (m, 2H). ESI-MS:
calc. 1218.39, obs. 406.3 (M3+), 608.8 (M-H)2+, 665.0 (M+TFA)2+.
Ethidium-COOH 11. 1H-NMR (300 MHz, CD3OD)δ8.73 (d, J = 8.9 Hz, 2H), 8.26 (dd, J = 9.2, 2.3 Hz, 1H), 7.85–7.72 (m, 4H), 7.65–7.62 (m, 2H), 7.43–7.40 (m, 2H), 4.71–4.64 (m, 2H), 2.39–2.27 (m, 2H), 2.18 (t, J = 7.1 Hz, 2H), 1.93–1.83 (m, 2H), 1.53 (t, J = 7.2 Hz, 3H). ESI-MS: calc. 428.20, obs. 428.2 (M+).
Bpy-ethidium 12. 1H-NMR (300 MHz, CD3OD)δ 8.74 (d, J = 9.3 Hz, 2H), 8.55 (d, J
= 5.2 Hz, 1H), 8.43 (d, J = 4.9 Hz, 1H), 8.25 (dd, J = 9.1, 2.4 Hz, 1H), 8.18 (d, J = 14.8 Hz, 2H), 7.77 (m, 4H), 7.63 (m, 2H), 7.42 (m, 2H), 7.34 (d, J = 5.2 Hz, 1H), 7.26 (m, 1H), 4.67 (m, 2H), 4.47 (s, 2H), 2.44 (s, 3H), 2.38 (dd, J = 14.2, 7.2 Hz, 4H), 2.00 (m, 2H), 1.53 (t, J = 7.1 Hz, 3H). ESI-MS: calc. 609.30, obs. 609.3 (M+).
Ru-Eth 4. ESI-MS: calc. 1173.36, obs. 391.1 (M3+).
N
S CH3I
N S
HN N
N
S N
O
O O
N
S N
O
TEA, DCM
N
O OH N
S
65C 125C
5
N Br N
OH OH
O O
Figure 4.3. Scheme showing the synthesis of TO-COOH5.9 4.2.3 Steady state fluorescence
Luminescence spectra were recorded on an ISS-K2 spectrophotometer at ambient temper- ature in aerated solutions. Standard deviations in integrated luminescence intensity were calculated from three samples. Concentrations of Ru-TO conjugates were estimated using 630=102,000 M−1cm−1.9All DNA concentrations are reported as the duplex concentration.
Raman scattering of water at 520 nm (λex = 440 nm) is shown in all emission spectra.