Fluorescent nucleobase analogues for base-base FRET in nucleic acids: synthesis, photophysics and applications

Abstract

Förster resonance energy transfer (FRET) between a donor nucleobase analogue and an acceptor nucleobase analogue, base-base FRET, works as a spectroscopic ruler and protractor. With their firm stacking and ability to replace the natural nucleic acid bases inside the base-stack, base analogue donor and acceptor molecules complement external fluorophores like the Cy-, Alexa- and ATTO-dyes and enable detailed investigations of structure and dynamics of nucleic acid containing systems. The first base-base FRET pair, tC^O-tC_nitro, has recently been complemented with among others the adenine analogue FRET pair, qAN1-qA_nitro, increasing the flexibility of the methodology. Here we present the design, synthesis, photophysical characterization and use of such base analogues. They enable a higher control of the FRET orientation factor, κ², have a different distance window of opportunity than external fluorophores, and, thus, have the potential to facilitate better structure resolution. Netropsin DNA binding and the B-to-Z-DNA transition are examples of structure investigations that recently have been performed using base-base FRET and that are described here. Base-base FRET has been around for less than a decade, only in 2017 expanded beyond one FRET pair, and represents a highly promising structure and dynamics methodology for the field of nucleic acids. Here we bring up its advantages as well as disadvantages and touch upon potential future applications.

Keywords: B-to-Z-DNA transition; FRET; Z-DNA; fluorescent base analogues; netropsin; nucleic acid structure and dynamics; quadracyclic adenines; tricyclic cytosines.

Figure 1

a) Angles and unit vectors used to define the relative orientations of the donor and acceptor transition dipole moments (e₁, e₂) and the separating vector (e₁₂). b) Illustration of the external fluorophores covalently attached to a DNA and their transition dipole moments (e₁, e₂) with free rotation relative to the DNA, i.e., isotropic orientation, κ² = 2/3. R_DA is the separation between donor and acceptor. c) DNA top-view (left) and side-view (right) illustrating the typical situation for the virtually static transition dipole moments (e₁, e₂) of fluorescent base analogues in base–base FRET and the distance separating them (R_DA).

Figure 2

Notable recent examples of fluorescent base analogues. For ^cnA and ^dnA the attachment point to the substituted naphthalene moiety has been varied.

Scheme 1

Synthesis of the tricyclic cytosine aromatic core [39]. (a) Ethylene glycol, K₂CO₃, 120 °C, 1 h, 40%; (b) EtOH, 1 M HCl, reflux, 16 h, 75%.

Scheme 2

Synthesis of protected tC and tC^O deoxyribose phosphonates [41]. (a) Ac₂O, pyridine, rt; (b) 2-mesitylenesulfonyl chloride, TEA, then 2-aminothiophenol or 2-aminophenol, DBU, rt, 27% and 54% yield, respectively, over two steps; (c) t-BuOK in EtOH, reflux, 38%; (d) NH₃ in MeOH, rt, then 10 equiv of KF, EtOH, reflux; (e) 4,4´-dimethoxytrityl chloride, pyridine, rt, yielding 50% of compound 10 over three steps; (f) 2-chloro-4H-1,3,2,-benzodioxaphosphorin-4-one, pyridine, DCM, 0 °C, 71% over two steps and 80%, respectively.

Scheme 3

Synthesis of protected tC_nitro deoxyribose phosphoramidite [14]. a) aq NaOH, 24 h, reflux; b) EtOH, HCl, 24 h, reflux, 15% over two steps; c) DMF, toluene, 3,5-di-O-p-toluoyl-α-D-erythro-pentofuranosyl, NaH, 18 h, rt, 11%; d) MeONa, MeOH, 18 h, rt, 71%; e) pyridine, DMAP, DMTr-Cl, 18 h, rt, 74%; f) DCM, DIPEA, 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, 1 h, rt, 93%.

Scheme 4

Improved synthesis of tC and tC derivatives, where R = H, 7-MeO or 8-MeO [47]. a) H₂NNH₂ followed by H₂O₂, 20 h, 100 °C, 60–98%; b) PEt₃, H₂O, diglyme, then Na₂CO₃ and 5-bromouracil, rt to 120 °C, 2 h, 24–86%; c) HCl, butanol, 120 °C, 24–72 h, 27–86%; d) BSA (bis(trimethylsilyl)acetamide), Hoeffer´s α-chloro sugar, SnCl₄, 0 °C to rt, 2 h, 12–41%; e) NaOMe, MeOH, 30 min, 69–90%.

Scheme 5

Improved synthesis of tC^O derivatives [47]. a) Ac₂O, pyridine, 16 h, rt, 85%; b) PPh₃, CCl₄, DCM, 5 h, 44 °C; c) DBU, DCM, R = 4-MeO, 5-MeO, 4-F, 4-Cl, 5-Cl, 15 min, 0 °C; d) NaOMe, MeOH, 3–4 h, rt, 40%, 48%, 61%, 20%, and 41%, respectively, over three steps; e) KF, 18-crown-6, diglyme, 1–2 h, 120 °C, 20% for R = 8-MeO, 11% for R = 7-MeO, 11% for R = 7-F, 24% for R = 8-Cl, and 3% for R = 7-Cl.

Scheme 6

Synthesis of protected tC^O ribose phosphoramidite [50]. a) MesSO₂Cl, DIPEA, MeCN, 4 h, rt; b) 2-aminophenol, 30 min, rt, 71% over two steps; c) KF, EtOH, 2 h, MW 140 °C, 86%; d) DMTr-Cl, pyridine, 1.5 h, rt, 72%; e) AgNO₃, TBDMS-Cl, pyridine, THF, 4 h, rt, 76%; f) 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, DIPEA, THF, 1 h, rt, 93%.

Scheme 7

Synthesis of protected deoxyribose qA [51]. a) N-(tert-Butoxycarbonyl)-2-(trimethylstannyl)aniline, (Ph₃P)₂PdCl₂, DMF, 24 h, 60 °C, 68%; b) DABCO, DBU, DMF, 21 h, 75 °C; c) 25% TFA in CH₂Cl₂, 3 h, rt, 96% over two steps; d) NaOMe, MeOH, rt, 64%; e) DMTr-Cl, pyridine, 65%; f) 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one, pyridine, CH₂Cl₂, 30 min, 0 °C to rt; g) aq triethylammonium bicarbonate, 52% over two steps.

Scheme 8

Synthesis of protected deoxyribose qA for DNA SPS [53]. a) AcCl, MeOH, rt, 40 min; b) p-toluoyl chloride, pyridine, overnight, 0 °C to rt; c) AcCl, AcOH, H₂O, 0 °C, 36% over three steps; d) NaH, MeCN, 30 min, rt, then 2 h at 60 °C, 73%; e) t-BuLi, SnBuCl₃, THF, 2h, −78 °C, 65%; f) Pd(PPh₃)₄, CuI, CsF, DMF, 1 h, 100 °C, 55%; g) DABCO, DBU, DMF, 16 h, 75 °C; h) 25% TFA in CH₂Cl₂, 1.5 h, 0 °C to rt, 46% over two steps; i) NaOMe, MeOH, overnight, rt, 61%; j) DMTr-Cl, pyridine, 1 h, rt, 68%; k) 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, DIPEA, CH₂Cl₂, 1.5 h, rt, 79%.

Scheme 9

Synthesis of qA derivatives. a) EtI, Cs₂CO₃, DMF, 4 h, rt, 90%; b) HBPin, Pd(PPh₃)₄, Et₃N, 1,4-dioxane, 24 h, 80 °C, 86%; c) PdCl₂(PPh₃)₂, K₃PO₄, MeCN, H₂O, 80 °C, 2 h, 56–86%; d) TMS-Cl, THF, 30 min, rt; e) LiHMDS, THF, 100 °C, 3 h, 33–71%.

Scheme 10

Synthesis of quadracyclic adenine base–base FRET pair. a) HCHO, NaOH, MeCN, H₂O, 50 °C, 1 h; b) TBDMS-OTf, pyridine, 1 h, 0 °C to rt, 86% over two steps; c) HBPin, Pd(PPh₃)₄, Et₃N, 1,4-dioxane, 24 h, 80 °C, 91%; d) PdCl₂(PPh₃)₂, K₂CO₃, MeCN, H₂O, 80 °C, 2 h, 86–95%; e) AcCl, pyridine, CH₂Cl₂, 3 h, rt; f) LiHMDS, THF, 100 °C, 2–6 h, 87–89%; g) Boc₂O, DMAP, THF, 10 h, rt, 83–89%; h) ethane-1,2-diamine, TBAF, THF, 2 h, 0 °C to rt, 97–100%; i) NaH, Hoffer´s α-chloro sugar, MeCN, 2 h, 0 °C to rt, 55–69%; j) NaOMe, MeCN or MeOH, 1 h, 50 °C, 81–99%; k) DMTr-Cl, pyridine, 1.5 h, rt, 55–75%; l) 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, DIPEA, CH₂Cl₂, 2 h, rt, 87–90%.

Figure 3

Absorption and emission of tC (dashed line) and tC^O (solid line) in dsDNA. The absorption below 300 nm is divided by three to emphasize the absorption spectral features of the lowest energy absorption bands of tC and tC^O.

Figure 4

Spectral overlap between the emission of qAN1 (cyan) and the absorption of qA_nitro (black) in dsDNA. The shaded region constitutes the overlap integral (J integral).

Figure 5

Example of typical FRET efficiency as a function of number of base pairs separating the donor and acceptor (data, blue dots, is an average of steady-state and time-resolved measurements of the FRET pair qAN1 and qA_nitro). The line shows a curve fitted to the data based on FRET theory. The top graph shows how the orientation factor, κ², varies with number of base pairs separating the donor and acceptor.

Figure 6

FRET efficiency as a function of number of base pairs separating the donor (qAN1) and acceptor (qA_nitro). Red dots mark the measured FRET efficiency with netropsin bound. The black line shows the best fit to the data based on FRET theory. The blue line shows the curve for B DNA. The yellow area depicts the range possible if each netropsin molecule overwinds the DNA as stated in previous literature.