A Facile Synthesis of Red-Shifted Bis-Quinoline (BisQ) Surrogate Base

Forced intercalation peptide nucleic acids (FIT-PNAs) are DNA mimics that act as RNA sensors. The sensing event occurs due to sequence-specific RNA hybridization, leading to a substantial increase in fluorescence. The fluorophore in the FIT-PNA is termed a surrogate base. This molecule typically replaces a purine in the PNA sequence. BisQ is a surrogate base that connects two quinolines via a monomethine bond. BisQ-based FIT-PNAs have excellent biophysical features that include high brightness and red-shifted emission (λem, max = 613 nm). In this report, we detail two chemical approaches that allow for the facile synthesis of the BisQ PNA monomer. In both cases, the key compound used for the synthesis of BisQ-CH2COOH is the tBu-ester-modified quinoline synthon (compound 5). Subsequently, one method uses the Alloc acid-protected PNA backbone, whereas the other uses the tBu ester-protected PNA backbone. In the latter case, the overall yield for BisQ acid (compound 7) and BisQ PNA monomer syntheses was 61% in six synthetic steps. This is a substantial improvement to the published procedures to date (7% total yield). Lastly, we have prepared an 11-mer FIT-PNA with either BisQ or thiazole orange (TO) and studied their photophysical properties. We find superior photophysical properties for the BisQ FIT-PNA in terms of the brightness and selectivity, highlighting the added value of using this surrogate base for RNA sensing.


Introduction
Peptide nucleic acids (PNAs), DNA analogs developed in the early 90s by Nielsen and co-workers [1,2], have been used for the past three decades as potent antisense molecules for the down-regulation of RNA in vitro and in vivo [3][4][5][6][7][8][9][10][11][12].In addition, in the early 2000s, PNAs were designed as RNA-sensing molecules by a concept devised by the Seitz group termed forced intercalation PNA (FIT-PNA) [13].FIT-PNAs are designed by replacing one of the PNA monomers in a given PNA sequence with a fluorophore that is a mono-methine cyanine dye (also known as a surrogate base).In solution, the fluorescence of FIT-PNA, as a single strand, is quenched due to the free rotation of the monomethine bond [14].However, once hybridized to its complementary RNA/DNA strand, the FIT-PNA fluoresces at the appropriate wavelength of the cyanine dye.The most used surrogate base is thiazole orange (TO).This fluorophore emits at (λ em,max ≈ 535 nm).Other cyanine dyes used in FIT-PNAs include Benzothiazole Orange (BO), Oxazole Yellow (YO), and Oxazolopyridine (JO) [15,16].All three fluorophores are blue-shifted in comparison to TO. BisQ, developed by our lab for FIT-PNAs [17] and by the Seitz lab for RNA/DNA-based FIT probes (QB, Quinoline Blue) [18], is a red-shifted surrogate base (λ em,max ≈ 602 nm) that has high brightness and quantum yields [19][20][21].In this report, we describe two alternative synthetic routes to prepare the BisQ PNA monomer; one leading to a 10-fold improvement in the overall yield.We then prepared a model 11-mer FIT-PNA with either TO or BisQ as the surrogate base and compared their sensing properties in terms of the brightness (BR), mismatch discrimination, and binding affinity to complementary RNA.

Synthesis of PNA Backbone
The aeg (aminoethyl glycine) PNA backbone is an achiral backbone that may be prepared by several synthetic routes [22,23].The reported procedure for BisQ is based on eight synthetic steps where the PNA backbone is first coupled to BisQ acid, followed by the removal of both tBOC and tBu ester-protecting groups.The final step is the instillation of the Fmoc group on the primary amine.The overall yield for this synthetic route is ca.7% [17].Alternatively, Wickstrom and co-workers [24], reported an alternative route for PNA backbone synthesis (for TO) that is based on the use of the Alloc-protecting group on the C-terminal acid (Scheme 1, Compound 2).In the newly designed synthesis of BisQ, we have used either the Wickstrom route or a simpler route that is depicted in Scheme 2 that avoids the re-instillation of the Alloc-protecting group.Previously, the motivation for changing the protecting group on the carboxylic acid from tBu ester to Alloc was mainly due to difficulties in the purification of the final compound [24].
BisQ as the surrogate base and compared their sensing properties in terms of the brightness (BR), mismatch discrimination, and binding affinity to complementary RNA.

Synthesis of PNA Backbone
The aeg (aminoethyl glycine) PNA backbone is an achiral backbone that may be prepared by several synthetic routes [22,23].The reported procedure for BisQ is based on eight synthetic steps where the PNA backbone is first coupled to BisQ acid, followed by the removal of both tBOC and tBu ester-protecting groups.The final step is the instillation of the Fmoc group on the primary amine.The overall yield for this synthetic route is ca.7% [17].Alternatively, Wickstrom and co-workers [24], reported an alternative route for PNA backbone synthesis (for TO) that is based on the use of the Alloc-protecting group on the C-terminal acid (Scheme 1, Compound 2).In the newly designed synthesis of BisQ, we have used either the Wickstrom route or a simpler route that is depicted in Scheme 2 that avoids the re-instillation of the Alloc-protecting group.Previously, the motivation for changing the protecting group on the carboxylic acid from tBu ester to Alloc was mainly due to difficulties in the purification of the final compound [24].

Synthesis of BisQ-CH2-COOH
Previously, BisQ-CH2-COOH was synthesized by using the free acid form as bromo acetic acid (BrCH2COOH).In our subsequent syntheses of BisQ-CH2-COOH, we observed low yields in this step, presumably due to the polymerization of the starting material (BrCH2COOH).As an alternative approach, we used the protected acid, namely, BrCH2COOtBu.Using this starting material significantly improved the synthesis of the BisQ acid by both methods.BrCH2COOtBu was added to 4-methyl quinoline (compound 5, Scheme 3) and the tBu ester was removed either prior to BisQ acid formation (compound 7, Scheme 3) or after (compound 8, Scheme 3).In both methods, high yields were obtained for all synthetic steps.Scheme 1.Chemical synthesis of the Allyl-protected PNA backbone as reported by Wickstrom and co-workers [24].
BisQ as the surrogate base and compared their sensing properties in terms of the brightness (BR), mismatch discrimination, and binding affinity to complementary RNA.

Synthesis of PNA Backbone
The aeg (aminoethyl glycine) PNA backbone is an achiral backbone that may be prepared by several synthetic routes [22,23].The reported procedure for BisQ is based on eight synthetic steps where the PNA backbone is first coupled to BisQ acid, followed by the removal of both tBOC and tBu ester-protecting groups.The final step is the instillation of the Fmoc group on the primary amine.The overall yield for this synthetic route is ca.7% [17].Alternatively, Wickstrom and co-workers [24], reported an alternative route for PNA backbone synthesis (for TO) that is based on the use of the Alloc-protecting group on the C-terminal acid (Scheme 1, Compound 2).In the newly designed synthesis of BisQ, we have used either the Wickstrom route or a simpler route that is depicted in Scheme 2 that avoids the re-instillation of the Alloc-protecting group.Previously, the motivation for changing the protecting group on the carboxylic acid from tBu ester to Alloc was mainly due to difficulties in the purification of the final compound [24].Scheme 2. New route for the synthesis of BisQ monomer using the tert-butyl protected PNA backbone (compound 4, Scheme 1).

Synthesis of BisQ-CH2-COOH
Previously, BisQ-CH2-COOH was synthesized by using the free acid form as bromo acetic acid (BrCH2COOH).In our subsequent syntheses of BisQ-CH2-COOH, we observed low yields in this step, presumably due to the polymerization of the starting material (BrCH2COOH).As an alternative approach, we used the protected acid, namely, BrCH2COOtBu.Using this starting material significantly improved the synthesis of the BisQ acid by both methods.BrCH2COOtBu was added to 4-methyl quinoline (compound 5, Scheme 3) and the tBu ester was removed either prior to BisQ acid formation (compound 7, Scheme 3) or after (compound 8, Scheme 3).In both methods, high yields were obtained for all synthetic steps.Scheme 2. New route for the synthesis of BisQ monomer using the tert-butyl protected PNA backbone (compound 4, Scheme 1).

Synthesis of BisQ-CH 2 -COOH
Previously, BisQ-CH 2 -COOH was synthesized by using the free acid form as bromo acetic acid (BrCH 2 COOH).In our subsequent syntheses of BisQ-CH 2 -COOH, we observed low yields in this step, presumably due to the polymerization of the starting material (BrCH 2 COOH).As an alternative approach, we used the protected acid, namely, BrCH 2 COOtBu.Using this starting material significantly improved the synthesis of the BisQ acid by both methods.BrCH 2 COOtBu was added to 4-methyl quinoline (compound 5, Scheme 3) and the tBu ester was removed either prior to BisQ acid formation (compound 7, Scheme 3) or after (compound 8, Scheme 3).In both methods, high yields were obtained for all synthetic steps.

Synthesis of BisQ PNA Monomer
In the first synthetic route (Scheme 4), BisQ acid (compound 8) was directly coupled to the allyl-protected PNA monomer (compound 2, Scheme 4).After coupling, compound 9 was purified by normal phase column chromatography, and the purified material was subjected to Pd (Ph3)4 for the final removal of the Alloc group.The overall yield for this synthetic route (excluding the preparation of the backbone, compound 1, Scheme 1) was 23%.In the second synthetic route (Scheme 3), compounds 5 and 6 were added to a DCM solution with triethylamine, affording compound 7 that was purified by normal phase column chromatography.After the tBu ester removal, the TFA salt of BisQ acid was directly coupled to the tBu ester-protected PNA backbone (compound 1, Scheme 1).Subsequently, compound 4 (Scheme 2) was purified by normal phase column chromatography, followed by the final removal of the tBu ester with TFA.The overall yield for this synthetic route (excluding the preparation of the PNA backbone, compound 1, Scheme 1) was 61%.

Synthesis and Biophysical Studies on a Model 11-Mer FIT-PNA
We selected an 11-mer FIT-PNA sequence (Table 1) in order to compare the biophysical properties of BisQ with a well-documented cyanine dye, TO.These FIT-PNAs were prepared with a short cationic peptide (dK4) as a means to improve their water solubility [25].Both FIT-PNAs were prepared on the solid support as previously reported.[17] Scheme 3. Synthesis of BisQ-COOH (compound 8) or its tert-butyl ester (compound 3) via two synthetic routes using the tButyl-protected 4-methyl quinoline (compound 5).

Synthesis of BisQ PNA Monomer
In the first synthetic route (Scheme 4), BisQ acid (compound 8) was directly coupled to the allyl-protected PNA monomer (compound 2, Scheme 4).After coupling, compound 9 was purified by normal phase column chromatography, and the purified material was subjected to Pd (Ph 3 ) 4 for the final removal of the Alloc group.The overall yield for this synthetic route (excluding the preparation of the backbone, compound 1, Scheme 1) was 23%.

Synthesis of BisQ PNA Monomer
In the first synthetic route (Scheme 4), BisQ acid (compound 8) was directly coupled to the allyl-protected PNA monomer (compound 2, Scheme 4).After coupling, compound 9 was purified by normal phase column chromatography, and the purified material was subjected to Pd (Ph3)4 for the final removal of the Alloc group.The overall yield for this synthetic route (excluding the preparation of the backbone, compound 1, Scheme 1) was 23%.In the second synthetic route (Scheme 3), compounds 5 and 6 were added to a DCM solution with triethylamine, affording compound 7 that was purified by normal phase column chromatography.After the tBu ester removal, the TFA salt of BisQ acid was directly coupled to the tBu ester-protected PNA backbone (compound 1, Scheme 1).Subsequently, compound 4 (Scheme 2) was purified by normal phase column chromatography, followed by the final removal of the tBu ester with TFA.The overall yield for this synthetic route (excluding the preparation of the PNA backbone, compound 1, Scheme 1) was 61%.

Synthesis and Biophysical Studies on a Model 11-Mer FIT-PNA
We selected an 11-mer FIT-PNA sequence (Table 1) in order to compare the biophysical properties of BisQ with a well-documented cyanine dye, TO.These FIT-PNAs were prepared with a short cationic peptide (dK4) as a means to improve their water solubility [25].Both FIT-PNAs were prepared on the solid support as previously reported.[17] Scheme 4. BisQ monomer synthesis via the allyl-protected PNA backbone.
In the second synthetic route (Scheme 3), compounds 5 and 6 were added to a DCM solution with triethylamine, affording compound 7 that was purified by normal phase column chromatography.After the tBu ester removal, the TFA salt of BisQ acid was directly coupled to the tBu ester-protected PNA backbone (compound 1, Scheme 1).Subsequently, compound 4 (Scheme 2) was purified by normal phase column chromatography, followed by the final removal of the tBu ester with TFA.The overall yield for this synthetic route (excluding the preparation of the PNA backbone, compound 1, Scheme 1) was 61%.

Synthesis and Biophysical Studies on a Model 11-Mer FIT-PNA
We selected an 11-mer FIT-PNA sequence (Table 1) in order to compare the biophysical properties of BisQ with a well-documented cyanine dye, TO.These FIT-PNAs were prepared with a short cationic peptide (dK 4 ) as a means to improve their water solubility [25].Both FIT-PNAs were prepared on the solid support as previously reported [17].
Fluorescence spectra were recorded for both FIT-PNAs before and after annealing to a fully complementary RNA (Figure 1).As opposed to BisQ FIT-PNA, the fluorescence enhancement for TO-FIT-PNA is only ca.2-fold (I/I 0 = 1.85,Table 1).
Fluorescence spectra were recorded for both FIT-PNAs before and after annealing to a fully complementary RNA (Figure 1).As opposed to BisQ FIT-PNA, the fluorescence enhancement for TO-FIT-PNA is only ca.2-fold (I/I0 = 1.85,Table 1).We next examined the sequence selectivity of both FIT-PNAs by introducing a single mismatch at the flanking base (5′) to the surrogate base (Figures 2 and 3).BisQ-FIT-PNA shows a robust selectivity for all mismatches tested.However, TO-FIT-PNA has no selectivity to these mismatches.Moreover, for the TU mismatch, the We next examined the sequence selectivity of both FIT-PNAs by introducing a single mismatch at the flanking base (5 ′ ) to the surrogate base (Figures 2 and 3).
Fluorescence spectra were recorded for both FIT-PNAs before and after annealing to a fully complementary RNA (Figure 1).As opposed to BisQ FIT-PNA, the fluorescence enhancement for TO-FIT-PNA is only ca.2-fold (I/I0 = 1.85,Table 1).We next examined the sequence selectivity of both FIT-PNAs by introducing a single mismatch at the flanking base (5′) to the surrogate base (Figures 2 and 3).BisQ-FIT-PNA shows a robust selectivity for all mismatches tested.However, TO-FIT-PNA has no selectivity to these mismatches.Moreover, for the TU mismatch, the Fluorescence spectra were recorded for both FIT-PNAs before and after annealing to a fully complementary RNA (Figure 1).As opposed to BisQ FIT-PNA, the fluorescence enhancement for TO-FIT-PNA is only ca.2-fold (I/I0 = 1.85,Table 1).We next examined the sequence selectivity of both FIT-PNAs by introducing a single mismatch at the flanking base (5′) to the surrogate base (Figures 2 and 3).BisQ-FIT-PNA shows a robust selectivity for all mismatches tested.However, TO-FIT-PNA has no selectivity to these mismatches.Moreover, for the TU mismatch, the BisQ-FIT-PNA shows a robust selectivity for all mismatches tested.However, TO-FIT-PNA has no selectivity to these mismatches.Moreover, for the TU mismatch, the fluorescence of this duplex is higher by almost 2-fold in comparison to the fully matched duplex (Figure 3).One may speculate that the uridine base that is opposite and adjacent to TO does not quench TO fluorescence due to the lack of a primary amine on this base.This may be verified, in the future, by testing other TO-FIT-PNA sequences.From Table 1, it is clear that BisQ-FIT-PNA is a brighter and more selective RNA probe.The brightness (BR) is ca.4.3-fold higher for BisQ-FIT-PNA compared to TO-FIT-PNA.This is manifested by a lower extinction coefficient and a lower quantum yield.It is important to note that this comparison was studied for a single 11-mer FIT-PNA sequence and one may not conclude that these dramatic differences will be valid for any given FIT-PNA.
Given the broad use of FIT-PNAs for diagnostics in various diseases, including cancer [17,24,26], RNA editing [20], and infectious diseases [16,21,27], the ease of BisQ synthesis reported in this paper will allow research groups from a variety of fields to readily synthesize and explore BisQ FIT-PNAs and other BisQ-based dyes [28].
TFA salt of compound 3 was dissolved in 10 mL of dry DMF.Then, HATU (381 mg, 1.0 mmol), HOBt (135 mg, 1.0 mmol), and DIPEA (149 mg, 1.15 mmol) were The mixture was stirred at room temperature for 10 min.This reaction mixture was slowly added to a mixture of the HCl salt of Fmoc-aeg-OtBu (compound 1, 338 mg, 0.85 mmol) and DIPEA (149 mg, 1.15 mmol) in dry DMF (5 mL).The reaction mixture was stirred under an inert atmosphere (argon) for 12 h.Once the reaction was finished (followed by TLC), 10 mL of water was added to the reaction mixture and extracted with 3 × 40 mL DCM.The combined organic layers were then washed with NaHCO 3 (30 mL) and saturated brine (3 × 30 mL).The organic layers were dried using anhydrous Na 2 SO 4 , filtered, and then concentrated under vacuum.After obtaining the crude product, it was purified using silica gel chromatography (5-6% MeOH in DCM).This resulted in the formation of a blue sticky compound 4 (550 mg, 0.83 mmol, 77-83% yield).1-Methyl-chloroquinolinium iodide (compound 6).Compound 6 was synthesized as previously described [17], with a 92% yield.

Tm Analysis
Melting curves (Tm) of the FIT-PNA/RNA duplexes were estimated from UV melting curves measured on an Evolution One Plus UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).FIT-PNAs and their complementary RNAs (1:1.5 ratio) were prepared in PBS buffer (pH 7.0) and adjusted to a final duplex concentration of 2.5 µM.Prior to analysis, the samples were heated from 20 • C to 90 • C at a rate of 5 • C/min and then cooled to the starting temperature at a rate of 2 • C/min.The change in absorbance was monitored at 260 nm by increasing the temperature to 90 • C at a rate of 1 • C/min.

UV-Vis Titrations and Fluorescence Measurements for Determining Quantum Yields
The measurement of the fluorescence quantum yield of FIT PNA:RNA duplexes followed the same protocol as previously reported [30].The absorbance range of the FIT-PNA duplex determined the choice of reference dye.For example, cresyl violet was used as a reference dye for the BisQ PNA/RNA (1:1) duplex, while fluorescein was employed as a reference dye for the TO PNA/RNA (1:1) duplex.
A serial dilution of FIT-PNA/RNA duplex solutions with an OD smaller than 0.1 at the maximum wavelength was measured to verify that the absorbance maxima remained constant.FIT-PNA duplexes with similar heights of absorbance were used to determine the cut point with respect to the reference dye, and this information was used to gather the fluorescence emission spectra (see Supplementary Materials, Figures S9-S12).
The fluorescence quantum yield was calculated by considering the refractive index (RI) value of the solvents, using the integrated emission value and the OD value at the

Scheme 1 .
Scheme 1.Chemical synthesis of the Allyl-protected PNA backbone as reported by Wickstrom and co-workers[24].

Scheme 1 .
Scheme 1.Chemical synthesis of the Allyl-protected PNA backbone as reported by Wickstrom and co-workers[24].

Table 1 .
The photophysical properties and binding affinities of these FIT-PNAs are summarized.