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Solid- and solution-phase synthesis and application of R6G dual-labeled oligonucleotide probes
Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
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Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Solid- and solution-phase synthesis and application of R6G dual-labeled oligonucleotide probes Aleksander Yu. Skoblov a,y, Maxim V. Vichuzhanin b, Valentina M. Farzan b, Olga A. Veselova b, Tatiana A. Konovalova b, Alexander T. Podkolzin b, German A. Shipulin b, Timofei S. Zatsepin b,c,d,⇑ a
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, 117997 Moscow, Russia Central Research Institute of Epidemiology, Novogireevskaya 3a, Moscow 111123, Russia Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1-3, Moscow 119992, Russia d Skolkovo Institute of Science and Technology, 5 Innovation Center ‘‘Skolkovo”, Skolkovo 143026, Russia b c
a r t i c l e
i n f o
Article history: Received 26 June 2015 Revised 17 August 2015 Accepted 28 August 2015 Available online xxxx Keywords: Rhodamine 6G Phosphoramidite CuAAC Oligonucleotide Fluorescent dye qPCR
a b s t r a c t A novel N-TFA-protected carboxyrhodamine 6G (R6G) phosphoramidite was synthesized for use in an automated DNA synthesis to prepare 50 -labeled oligonucleotides. Deprotection and purification conditions were optimized for 50 -labeled and dual-labeled oligonucleotide probes. As an alternative we synthesized an azide derivative of R6G for CuAAC post-synthetic oligonucleotide labeling. Dual-labeled probes obtained by both methods showed the same efficacy in a quantitative PCR assay. R6G-labeled probes demonstrated superior properties in a qPCR assay in comparison with alternative HEX, JOE and SIMA dyes due to more efficient fluorescence quenching by BHQ-1. We successfully used R6G dual-labeled probes for rotavirus genotyping. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Nowadays quantitative polymerase chain reaction (qPCR or real-time PCR) has become a powerful instrument for the detection and measurement of nucleic acids in life science,1–3 biomedical research,4 diagnostics5 and forensic studies.6 qPCR is a rapid, sensitive, specific and reproducible PCR technique. In addition, the risk of carryover contamination is minimized in comparison to other PCR techniques.7 The multiplex variant of qPCR allows the detection of multiple targets within a single reaction and introduction of an internal control sample.2 Combining the use of multiple fluorophores with the discrimination of additional targets by postamplification melting gives us a simultaneous identification of a significantly larger number of targets.8 Today qPCR instruments allow us to work with 5–6 fluorophores (and probes, respectively) simultaneously in a large wavelength range (450–700 nm). This range is limited as is the case with shorter wavelengths a significant increase of fluorescence noises leads to a drop of sensitivity and the IR-range dyes are unstable at increased temperatures, thus ⇑ Corresponding author. Tel.: +7 495 939 3148; fax: +7 495 939 3181. E-mail address: [email protected] (T.S. Zatsepin). Present address: Metkinen Chemistry, Yliopistonranta 1B, FI-70210 Kuopio, Finland. y
having low quantum yields. The main problem for multiplication is cross-lighting of fluorophores that could be solved by choosing new dyes with single narrow peaks in emission spectra. Therefore, dyes and combinations of dyes for multiplex detection in qPCR are still under development. Rhodamine dyes are valuable tools for analytical9–11 and biochemical studies.12–15 They are stable over a wide pH range, photostable16,17 and have a high quantum yield in water solutions.18 Carboxyrhodamine 6G is a rarely used alternative to HEX (6-carboxy-4,7,20 ,40 ,50 ,70 -hexachlorofluorescein), SIMA (6-carboxy4,7-dichloro-20 ,70 -diphenylfluorescein) and JOE (6-carboxy-40 ,50 dichloro-20 ,70 -dimethoxyfluorescein) dyes in case qPCR probes, as automated solid-phase synthesis is unavailable for it at present. Fluorescence properties of 6-R6G are superior to HEX, JOE and more recently developed SIMA dyes (Table 1). To note, data from Table 1 should be used only for estimation of the dye applicability in qPCR. In most cases, data were obtained using a free dye solution in various solvent/pH conditions. It is well known that the absorbance and emission of fluorescent dyes are affected by the environment, including factors as solvent, pH, and conjugation to biomolecules, that is, the fluorescence of fluorescein drops in low pH due to protonation. Sjoback et al.23 have demonstrated that the conjugation of fluorescein to the oligonucleotide leads to a change of the pKa from 6.4 to 6.9 and the quantum yield
http://dx.doi.org/10.1016/j.bmc.2015.08.041 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
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Table 1 Fluorescence properties of 6-CR6G, HEX, JOE and SIMA Dye 11,19
6-R6G 6-HEX 6-JOE20,21 6-SIMA22
Absorbance (max), nm
Emission (max), nm
Molar extinction coefficient, L * cm1 * M1
Quantum yield
Brightness (Ec * QY)
520 535 522–529 536
557 556 548–560 551
116 000 96 000 75 000 64 000
0.95 0.7 0.58–0.8 Unknown
110 200 67 200 43 500–60 000 Unknown
decreases from 0.93 to 0.72. Also, Lilley and coauthors24 have shown that the extinction coefficient of fluorescein decreases by 30% after conjugation to DNA in close proximity to four-way DNA junctions. On the other hand, rhodamine dye does not undergo a similar change in absorption efficiency following conjugation. Marras25 has shown that the quantum yield of some xanthenes decreases with increasing temperature, whereas some others do not show a significant change in their quantum yield. However, such a comprehensive analysis is not available for most dyes. As conjugation to an oligonucleotide and the pH/buffer used can affect fluorescence behavior, these factors should be considered for choosing dye combinations, especially for multiplexed assays. Stronger brightness is preferable both in qPCR and end point PCR studies for the detection of low copy targets. FRET (Förster (fluorescence) energy transfer) primers26 bearing an R6G dye as the acceptor chromophore were successfully used for DNA sequencing27,28 and short tandem repeat (STR) analysis.29,30 However all labeled oligonucleotides were synthesized through post-synthetic acylation of amino oligonucleotides by a N-hydroxysuccinimide ester (NHS) of 5- or 6-carboxy-R6G. Postsynthetic acylation is a laborious, time and cost-consuming procedure for dye incorporation. Also, additional HPLC purification is needed for the synthesis of probes that lead to production of smaller amounts of labeled material. The phosphoramidite approach usually provides higher yields, takes less time and is more convenient in its synthesis. Today, an efficient and robust copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) reaction31 is widely used in oligonucleotide chemistry32 and is extremely valuable for small-scale synthesis. In most cases conjugates can be used immediately after gel-filtration or precipitation by acetone or ethanol. In this report we present the synthesis of a novel 6-carboxyrhodamine 6G phosphoramidite that is based on 6-aminohexanol and 6-carboxyrhodamine 6G 3-azidopropylamide. These derivatives were used for efficient labeling of oligonucleotides. The R6G dual-labeled probes with superior fluorescent properties were successfully used for rotavirus genotyping. 2. Results and discussion 2.1. Synthesis Many of rhodamines (R110 (rhodamine 110), R6G, TAMRA (5 (6)-carboxy N,N,N0 ,N0 -tetramethylrhodamine), ROX (5(6)-carboxyX-rhodamine), Texas Red (sulforhodamine 101)) have been used to label oligonucleotides by postsynthetic acylation33–38 or by application of dye-modified solid supports for automated oligonucleotide synthesis.39 Phosphoramidite derivatives were developed only for the TAMRA dye.40–42 The chemical synthesis of the phosphoramidite building block 4 is outlined in Figure 1. We have chosen the 6-isomer of CR6G as its fluorescence spectra fits better within the band-pass filters in qPCR machines—557 ± 5 nm (Fig. 2). In the beginning we have synthesized mixture of isomers—5(6)-carboxyR6G as reported previously with minor modifications.27 Protection of the secondary aromatic amino groups of 6-CR6G is required to fix the lactone ring of the molecule. The presence of ‘flipping’ lacton moiety can probably
lead to the self-activation of phosphoramidite followed by degradation (Fig. 3). It has been demonstrated previously that protonated weak bases can be used for phosphoramidite activation.43,44 So we blocked these amino functions from unwanted side-reaction by use of an amine-labile trifluoroacetic (TFA) group. Also a phosphoramidite should be soluble in acetonitrile, the most suitable solvent for oligo synthesis and presence of TFA groups increase such solubility. While compound 1 was allowed to react with trifluoroacetic anhydride in pyridine, derivative 2 was isolated in an only 15% yield after column chromatography. Low yield is a result of close Rf values for 5- and 6-isomers, so only a part of pure 6-R6G could be isolated easily; additional purifications of the isomer mixture could increase the yield. To solve the problem of poor isomer separation we developed an alternative procedure for 5(6)-carboxyR6G synthesis. We separated the stages of acylation of the first and the second molecules of substituted aminophenol that significantly improved the purity of products. Attempts to separate isomers of 5(6)-carboxyR6G by normal phase chromatography on silica gel or aluminum oxide led to disastrous losses of compounds so we used reverse-phase column chromatography to separate isomers. Separation of isomers was rather smooth and the summarized yield of two isomers was about 50%. As a result at the next stage—TFAA acylation was significantly improved. TFAA acylation was carried out in THF in presence of TEA at 20 °C and the yield was increased up to 70%. First, there was no need to separate isomers with close Rf values after acylation, second, due to thorough purification of carboxyR6G less amount of byproducts was formed. Carbodiimide-assisted acylation of 6-aminohexanol gave an excellent yield of amide 3. The subsequent phosphitylation of the hydroxy group in an inert atmosphere using (N,N-diisopropylamino)-2-cyanoethoxychlorophosphine in dichloromethane in the presence of N,N-diisopropylethylamine45 afforded the title phosphoramidite 4 (Fig. 1). Following that, the phosphoramidite 4 was evaluated in machine-assisted solid-phase oligonucleotide synthesis by the standard 2-cyanoethyl phosphoramidite method. The average coupling efficiency of the novel monomer in dry acetonitrile was found to be more than 98%. The phosphoramidite obtained was used in the synthesis of a number of 50 -modified oligonucleotides (Table 2). Oligomer I (50 -R6G-dT10) was synthesized as a model in order to choose and optimize deprotection conditions. Deprotection of the modified oligonucleotides was carried out by aq ammonia at RT (overnight), AMA (water solutions of ammonia and methylamine 1:1) at RT (2 h), 0.2 M NaOH in aq methanol46 or with an TAMRA cocktail (t-butylamine/methanol/water—1/1/2 (v/v/v)) at 55 °C (overnight) (Fig. 4). Both alkaline and t-butylamine deprotection gave excellent results without any side products (Fig. 4, traces 3, 4, peak a), in the case of ammonia deprotection a partial formation of colorless non-fluorescent spirolactame 8a47–49 (Fig. 5) was observed (Fig. 4, trace 1, peak b). AMA deprotection led only to spirolactame 8b (Fig. 5) formation (Fig. 4, trace 2, peak c). As in the case of alkaline deprotection additional neutralization and desalting are needed before purification, we used more convenient MeOH-free TAMRA cocktail for the deprotection of R6G-labeled oligonucleotides. Oligomers II-XVI were isolated by denaturing
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Figure 1. Synthesis of R6G phophoramidite 4 and 3-azidopropylamide 6: (a) TFAA, Et3N, THF; (b) (1) NHS, DCC, THF; (2) 6-aminohexanol, THF; (c) Pri2NP(Cl)O(CH2)2CN, Pri2NEt, DCM, (d) (1) NHS, DCC, THF; (2) 3-azidopropylamine, THF; (e) K2CO3, MeOH.
Figure 2. Normalized emission spectra of 5-carboxyR6G (solid line) and 6-carboxyR6G (dashed line) in a PBS buffer.
polyacrylamide gel electrophoresis (PAGE) followed by RP-HPLC, and their structure was confirmed by ESI-MS. MS data is presented Table 2, HPLC and MS profiles of purified products—in Support materials. However in the case of small-scale oligonucleotide synthesis (especially in 96-well plates) followed by post-conjugation of various dyes CuAAC showed to be a good alternative to phosphoramidite chemistry. Therefore, we synthesized a novel azidoalkyl derivative of R6G that was further conjugated to a 50 -alkyne oligonucleotide under CuAAC conditions (Fig. 6). As azide–alkyne click reaction usually proceeds smoothly and quantitatively we
Figure 3. Probable equilibrium of 6-carboxamide R6G phosphoramidite.
tried to minimize purification procedures. However simple precipitation by lithium perchlorate in acetone or conventional RP HPLC purification lead to a product with increased background fluorescence due to incomplete removal of the dye. Better results were obtained when heat denaturation before injection of the reaction mixture was carried out (6 M urea, 95 °C, 5 min). The only method of purification after CuAAC in our hands that gave the same level of
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Table 2 Modified oligonucleotides used in the study N
Name
Oligonucleotide
MALDI-MS or ESI-MS (calcd/observed)
I
R6G-T10
R6G-TTTTTTTTTT
3601.2/3599.0a 3600.2/3598.0b 3610.9/3610.9c
II III IV V VI
R6G-P4z R6G-G1z R6G-G3z SIMA-P4z SIMAG1z SIMAG3z JOE- P4z JOE-G1z JOE-G3z HEX- P4z HEX-G1z HEX-G3z -P4z -G1z -G3z
R6G-CAC TCT GAC TAC TAC CTT TAA ACA GAG CG-BHQ1 R6G-CTG AGC TTT AGT YAA GGC AAA TAA TGC TCA G-BHQ1 R6G-TAC CCA ACT GAA GCA GCA ACA GGG TA-BHQ1 SIMA-CAC TCT GAC TAC TAC CTT TAA ACA GAG CG-BHQ1 SIMA-CTG AGC TTT AGT YAA GGC AAA TAA TGC TCA G-BHQ1
9973.9/9974.3 10711.0; 10726.0/10711.4; 10725.4 9150.8/9151.1 10111.8/10113.1 10848.9; 10863.9/10850.6; 10865.6
SIMA-TAC CCA ACT GAA GCA GCA ACA GGG TA-BHQ1
9288.2/9289.9
JOE-CAC TCT GAC TAC TAC CTT TAA ACA GAG CG-BHQ1 JOE-CTG AGC TTT AGT YAA GGC AAA TAA TGC TCA G-BHQ1 JOE-TAC CCA ACT GAA GCA GCA ACA GGG TA-BHQ1 HEX-CAC TCT GAC TAC TAC CTT TAA ACA GAG CG-BHQ1 HEX-CTG AGC TTT AGT YAA GGC AAA TAA TGC TCA G-BHQ1 HEX-TAC CCA ACT GAA GCA GCA ACA GGG TA-BHQ1 Alkyne-CAC TCT GAC TAC TAC CTT TAA ACA GAG CG-BHQ1 Alkyne-CTG AGC TTT AGT YAA GGC AAA TAA TGC TCA G-BHQ1 Alkyne-TAC CCA ACT GAA GCA GCA ACA GGG TA-BHQ1
10019.7/10021.9 10756.8; 10771.8/10757.5; 10772.5 9196.6/9197.9 10105.1/10097.8 10842.6; 10857.6/10835.4; 10850.4 9281.6/9275.7 9625.7d (10166.0e)/9626.2d (10166.3)e 10362.9d; 10377.9 d (10903.1, 10918.1)e/10363.3d; 10378.4d (10903.4; 10918.4)e 8802.6d (9342.9e)/8803.2d (9343.2)e
VII VIII IX X XI XII XIII XIV XV XVI a
TAMRA cocktail deprotection,
b
Ammonia deprotection, c AMA deprotection,
d
Before click conjugation, e After click conjugation.
Figure 6. Synthesis of R6G-labeled oligonucleotides by CuAAC.
2.2. qPCR studies
Figure 4. HPLC analysis of deprotected oligonucleotide I at 280 nm. Peak at 25 min is R6G-labeled oligonucleotide. Deprotection was carried out with: (1) ammonia, (2) AMA, (3) NaOH, (4) TAMRA cocktail.
background fluorescence as application of R6G phosphoramidite was denaturative PAGE. Perhaps denaturative IE HPLC could be the method of choice. In sum, CuAAC is cost effective, but in the case of R6G derivation additional purification in denaturative conditions is needed. However this method is preferable for sporadic synthesis of R6G-labeled oligonucleotides in comparison to the phosphoramidite approach due to lower consumption of the dye derivative.
Recently, we developed an in-house qPCR kit for rotavirus genotyping.50 This study gave us an understanding of the specific role of rotaviruses in gastroenteritis which leads to hospitalization for both children and adults and shows the epidemiological significance of several rotavirus strains. To compare ‘yellow’ dyes we synthesized several dual-labeled probes labeled with R6G, HEX, JOE or SIMA dyes and BHQ1 (2,20 -((4-(2-methoxy-5-methyl-4((4-methyl-2-nitrophenyl)diazenyl)phenyl)diazenyl)phenyl)azanediyl)bis(ethan-1-ol)) as a quencher (Table 2) to find out if a ‘best’ fluorophore in qPCR exists. In case of these dyes BHQ1 and BHQ2 (2,20 -((4-((E)-(2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)phenyl)-azanediyl)bis(ethan-1-ol)) gave the same efficiency of quenching (data not shown). The crucial parameter in qPCR data is a threshold cycle (Ct)—the intersection between an amplification curve and a threshold line (Fig. 7B–D). It is a relative
Figure 5. Deprotection of R6G-modified oligonucleotides by primary amines.
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Figure 7. qPCR traces (R6G – red, JOE – black, HEX – green, SIMA – blue). (A) Raw data for P4z probes, (B) normalized data for P4z probes, (C) normalized data for G1z, (D) normalized data for G3z probes.
measure of the concentration of target DNA in the PCR reaction and is calculated after data normalization (Fig. 8). In case of end-point PCR relative increase of fluorescence is more important. Small difference between final and initial fluorescence lead to low sensitivity and increased number of false negative results due to a relatively large grey zone between a lower and an upper cut-off. If somebody is going to use a kit in both methods these parameters become significant. So the best probe should give the minimal Ct and a maximal relative increase of fluorescence. The R6G-labeled probes gave the lowest fluorescence background and the HEX/SIMA probes—the highest (Fig. 7A). Such additional quenching could be a result of the tight interactions of R6G moiety with heterocyclic bases.51,52 As a result higher relative increase of fluorescence after normalization in case of R6G allows the assured detection of low-copy DNA targets due to improvement of threshold cycle (Ct) (Fig. 8). We want to emphasize a significant difference in Ct at the lowest concentration of target DNA for all probes used in the study. Also a higher increase of fluorescence during amplification makes R6G a preferable dye for endpoint application. This is a combined result of R6G high brightness, considerable quenching by BHQ and congruence of fluorescence spectra with the band-pass filters in common qPCR machines. 3. Conclusions We have presented here a multi gram procedure that allows to do preparative scale synthesis of single isomer of carboxy-R6G derivatives. We developed an efficient and reliable method for the preparation of 50 -R6G labeled oligonucleotides using our newly developed modification reagents. Phosphoramidite derivative has significant advantages both for small- and medium-scale synthesis of fluorescent oligonucleotides in comparison to traditional NHSesters. Our method was shown to be a useful route for the synthesis of dual-labeled probes. R6G azide derivative is a good alternative to phosphoramidite, especially if somebody needs only few oligonucleotides on a small scale. We made a comprehensive comparison of several common ‘yellow’ dyes used in qPCR and have demonstrated that R6G is a preferable dye.
4. Experimental Chemicals were obtained from commercial suppliers and used without further purification unless otherwise noted. Chemical shifts (d, ppm) for 1H, 13C and 31P are referenced to internal solvent resonances and reported relative to SiMe4 and 85% aq H3PO4, respectively. Chemical shifts are accurate to within 0.01 ppm for 1 H, 13C and CSSI are accurate within 0.5 Hz. The compounds were visualised under short-wavelength UV. Fluorescence spectra were recorded as predescribed.53 4.1. N-(9-(2,5-Dicarboxyphenyl)-6-(ethylamino)-2,7-dimethyl3H-xanthen-3-ylidene) ethanaminium chloride (6-carboxyrhodamine-6G) (1) 3-Ethylamino-4-methylphenol (51.9 g 343 mmol) was dissolved CH2Cl2 (500 ml) and methanesulfonic acid (22 ml, 343 mmol) was added dropwise under vigorous mixing. After 1 h solvent was evaporated, the residue was suspended in toluene (1 L) and 1,2,4-benzenetricarboxylic anhydride (31.3 g, 162 mmol) was added. The mixture was refluxed overnight, then toluene was evaporated, the residue was dissolved in DMF (500 ml) and the reaction mixture was heated at 140 °C for 48 h. Then DMF was evaporated in vacuum, and gum residue was dissolved in 0.2 M NaOH (3 L). Title compound was purified by reverse-phase column chromatography (sorbent LPS-500-H, Purolite) with gradient of isopropanol (0 ? 15%) in water. 6-Isomer is elited in 3–8% of isopropanol (isolated yield—20.1 g, 25.0%), 5-isomer—in 8–12% of isopropanol (isolated yield—21.0 g, 26.1%). 6-Carboxyrhodamine-6G: 1H NMR (DMSO-d6) d 8.30 (s, 2H, H4, H5), 7.88 (s, 1H, H7), 6.86 (s, 2H, H40 , H50 ), 6.83 (s, 2H, H10 , H80 ), 3.50 (q, 4H, H2⁄), 2.14 (s, 6H, H2a0 , H7a0 ), 1.29 (t, 6H, H1⁄); 13C NMR (DMSO-d6) 166.3 (C3a), 166.1 (C6a), 157.3 (C4a0 , C5a0 ), 156.8 (C1), 156.4 (C30 , C60 ), 135.3 (C3), 134.9 (C6), 134.0 (C2), 131.7 (C7), 131.2 (C4), 131.1 (C5), 129.0 (C40 , C50 ), 126.0 (C9a0 , C8a0 ), 113.5 (C20 , C70 ), 94.1 (C10 , C80 ), 38.5 (C2⁄), 17.9 (C2a0 , C7a0 ), 14.0 (C1⁄). HRMS (ESI+): m/z [M+H]+ calcd for C27H27N2O+5: 459.1914, found: 459.1924.
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R6G HEX JOE SIMA
42
38
P4Z
34
30 1.0E+06
1.0E+05
1.0E+04
1.0E+03
target DNA, copies/ml
Ct 40
36
R6G HEX JOE SIMA
G1Z
32
28 1.5E+06
1.5E+05
1.5E+04
1.5E+03
target DNA, copies/ml
Ct 40
36
R6G HEX JOE SIMA
4.3. N,N0 -(6-((6-Hydroxyhexyl)carbamoyl)-20 ,70 -dimethyl-3-oxo3H-spiro[isobenzofuran-1,90 -xanthene]-30 ,60 -diyl)-bis(N-ethyl-2, 2,2-trifluoroacetamide) (6-(30 ,60 -N-ditrifluoroacetylrhodamine 6G-6-carboxamido)hexan-1-ol) (3)
G3Z
32
28 1.2E+06
1.2E+05
1.2E+04
4.2.2. Method B 6-Carboxyrhodamine-6G (1) (4.60 g, 10 mmol) was dissolved in THF (200 ml) in presence of triethylamine (7 ml, 50 mmol). The reaction mixture was cooled in an ice bath/sodium chloride to 20 °C with magnetic stirring and trifluoroacetic anhydride (3.5 ml, 25 mmol) was added during one hour in three portions. The mixture was then evaporated and product was precipitated by water (100 ml), then filtered and dried in vacuum over P2O5. The residue was purified by column chromatography on a silica gel with gradient of methanol (1 ? 10%) in dichloromethane/5% pyridine as the mobile phase. The product was obtained as a pale yellow solid in 69% yield (4.48 g). Rf value is 0.3 for 6-Carboxy(TFA)2R6G and 0.25 for 5-Carboxy (TFA)2R6G (10% MeOH in DCM). 1H NMR (MeCN-d3) d 8.46 (br s, 1H, H4), 8.24 (d, 1H, H5, J = 7.9 Hz), 7.33 (m, 3H, H10 , H7, H80 ), 6.93 (m, 2H, H40 , H50 ), 3.44 (m, 4H, H2⁄), 2.11 (s, 6H, H2a0 , H7a0 ), 1.46 (m, 6H, H1⁄). 13C NMR (MeCN-d3) d 168.28 (C3a), 165.15 (C6a), 156.07 (C(O)CF3), 154.81 (C2), 148.88 (C4a0 , C5a0 ), 139.80 (C60 , C30 ), 137.62 (C6), 132.68 (C3), 130.27 (C40 , C50 ), 126.39 (C4 or C5), 124.11 (C4 or C5), 123.92 (C7), 119.39 (C20 , C70 ), 118.74 (C8a0 , C9a0 ), 118.41 (C10 , C80 ), 117.15 (CF3), 80.97 (C1), 39.79 (C2⁄), 26.46 (C1⁄), 15.97 (C2a0 , C7a0 ). HRMS (ESI-): m/z [MH] calcd for C31H23F6N2NaO 7 : 649.1409, found: 649.1398.
1.2E+03
target DNA, copies/ml Figure 8. Results of qPCR of target DNA by probes used in this study.
5-Carboxyrhodamine-6G: 1H NMR (DMSO-d6) d 8.72 (s, 1H, H4), 8.35(d, 1H, H6, J = 7.9 Hz) 7.54 (d, 1H, H7, J = 7.9 Hz), 6.83 (s, 2H, H40 , H50 ), 6.82 (s, 2H, H10 , H80 ), 3.48 (m, 4H, H2⁄), 2.14 (s, 6H, H2a0 , H7a0 ), 1.30 (t, 6H, H1⁄). 13C NMR (DMSO-d6) 165.6 (C3a), 165.2 (C6a), 156.0 (C4a0 ,C5a0 ), 156.4 (C30 , C60 ), 152.0 (C1), 132.4 (C3), 132.3 (C5), 131.2 (C4), 130.7 (C6), 130.0 (C7), 128.0 (C40 , C50 ), 124.8 (C9a0 , C8a0 ), 111.9 (C20 , C70 ), 93.4 (C10 , C80 ), 37.6 (C2⁄), 16.9 (C2a0 , C7a0 ), 13.2 (C1⁄). HRMS (ESI+): m/z [M+H]+ calcd for C27H27N2O+5: 459.1914, found: 459.1921. 4.2. 30 -(N-Ethyl-2,2,2-trifluoroacetamido)-60 -(ethylamino)-20 ,70 dimethyl-3-oxo-3H-spiro[isobenzofuran-1,90 -xanthene]-6-carboxylic acid (30 ,60 -N-ditrifluoroacetyl-6-carboxyrhodamine-6G) (2) 4.2.1. Method A 5,6-Carboxyrhodamine-6G (4.60 g, 10 mmol) was dissolved in pyridine (50 ml). The reaction mixture was cooled in an ice bath with magnetic stirring and trifluoroacetic anhydride (4.2 ml, 30 mmol) was added during one hour in three portions. The mixture was then evaporated and product was precipitated by water (100 ml), then filtered and dried in vacuum over P2O5. The residue was purified by column chromatography on a silica gel with gradient of methanol (1 ? 10%) in dichloromethane/5% pyridine as the mobile phase. The product was obtained as a pale orange solid in 15% yield (0.98 g).
970 mg (1.5 mmol) of compound (2) was dissolved in 50 ml of dichloromethane and 412 mg (2 mmol) of dicyclohexylcarbodiimide were added, followed by 412 mg (2 mmol) of N-hydoxysuccinimide (230 mg, 2 mmol). The mixture was stirred overnight at room temperature, then filtered, solids were washed twice with dichloromethane (10 ml) and 176 mg (1.5 mmol) of 6-aminohexanol was added, then again the mixture was stirred overnight at room temperature. The mixture was extracted with H2O and then with 0.1 M HCl(aq), and organic phase is evaporated. The product was applied on a silica gel column and eluted with gradient of methanol (1 ? 10%) in dichloromethane. Next, the product-containing fractions were evaporated and 1.01 g of yellow solid was obtained. The yield is 90%, Rf value is 0.55 (10% MeOH in DCM). 1H NMR (MeCN-d3) d 8.46 (s, 1H, NH), 8.16 (d, 1H, H5, J = 7.9 Hz), 8.11 (d, 1H, H4, J = 8.1 Hz), 7.89 (s, 1H, H7), 7.4 (br s, 2H, H40 , H50 ), 6.96 (s, 2H, H10 , H80 ), 4.18(m, 2H, H600 ), 3.70 (m, 2H, H100 ), 3.44 (m, 4H, H2⁄), 3.29 (m, 2H, H500 ), 2.11 (s, 6H, H2a0 , H7a0 ), 1.66 (m, 2H, H200 ), 1.46 (m, 6H, H1⁄), 1.22 (m, 2H, H300 ), 1.21 (m, 2H, H400 ). 13C NMR (MeCN-d3) d 168.28 (C3a), 165.15 (C6a), 156.07 (C(O)CF3), 154.81 (C2), 148.88 (C4a0 , C5a0 ), 139.80 (C60 , C30 ), 137.62 (C6), 132.68 (C3), 130.27 (C40 , C50 ), 126.39 (C4 or C5), 124.11 (C4 or C5), 123.92 (C7), 119.39 (C20 , C70 ), 118.74 (C8a0 , C1a0 ), 118.41 (C10 , C80 ), 117.15 (CF3), 80.97 (C1), 46.65 (C600 ), 39.79 (C2⁄), 30.97 (C100 ), 29.25 (C200 ), 26.46 (C1⁄), 25.54 (C300 ), 15.97 (C2a0 , C7a0 ), 13.63 (C500 ), 11.21 (C400 ). HRMS (ESI+): m/z [M+Na]+ calcd for C37H37F6N3NaO+7: 772.2433, found: 772.2441. 4.4. (6-(30 -(N-Ethyl-2,2,2-trifluoroacetamido)-60 -(ethylamino)20 ,70 -dimethyl-3-oxo-3H-spiro[isobenzofuran-1,90 -xanthene]-6carboxamido)hexyl)-2-cyanoethyl-diisopropylphosphoramidite (6-(30 ,60 -N-ditrifluoroacetylrhodamine 6G-6-carboxamido)hexyl)1-O-(2-cyanoethyl)-(N,N-diisopropyl)phosphoramidite) (4) 375 mg (0.5 mmol) of compound (3) was dissolved in 5 ml of dichloromethane, 218 ll (1.25 mmol) N,N-diisopropylethylamine was added, and then 142 ll (0.6 mmol) of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite was added at room temperature.
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A. Yu. Skoblov et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
After one hour, reaction mixture was extracted with an aqueous 0.5 M phosphate buffer pH 8, and the organic phase was dried with anhydrous sodium sulfate and evaporated. The product was purified on a silica gel column with addition of 1% of Et3N and eluted with 1–5% MeOH in DCM (Rf value is 0.75 (10% MeOH in DCM)). Pale yellow solid of R6G-amidite was obtained (380 mg, yield— 70%). 1 H NMR (MeCN-d3) d 8.46 (br s, 1H, NH), 8.24 (d, 1H, H5, J = 7.9 Hz), 8.14(d, 1H, H4, J = 8.1 Hz) 7.77 (s, 1H, H7), 7.5 (br s, 2H, H40 , H50 ), 6.96 (s, 2H, H10 , H80 ), 4.19 (m, 2H, H600 ), 3.82 (m, 2H, P-OCH2), 3.70 (m, 2H, H100 ), 3.64 (m, 2H, PN-CH), 3.44 (m, 4H, H2⁄), 3.29 (m, 2H, H500 ), 2.66 (m, 2H, CH2-CN), 2.11 (s, 6H, H2a0 , H7a0 ), 1.66 (m, 2H, H200 ), 1.46 (m, 6H, H1⁄), 1.22 (m, 2H, H300 ), 1.21 (m, 2H, H400 ), 1.18 (m, 12H, (CH3)2-C). 13C NMR (MeCN-d3) d 168.3 (C3a), 165.2 (C6a), 156.0 (C(O)CF3), 154.8 (C2), 148.9 (C4a0 , C5a0 ), 139.8 (C60 , C30 ), 137.6 (C6), 132.7 (C3), 130.3 (C40 , C50 ), 126.4 (C4 or C5), 124.1 (C4 or C5), 123.92 (C7), 119.4 (C20 , C70 ), 118.7 (C8a0 , C1a0 ), 118.4 (C10 , C80 ), 117.2 (CF3), 81.0 (C1), 58.4 (P-OCH2), 45.6 (C600 ), 42.8 (N-CH), 39.8 (C2⁄), 30.97 (C100 ), 29.25 (C200 ), 26.5 (C1⁄), 25.5 (C300 ), 24.17 ((CH3)2-CH), 20.2 (CH2-CN), 16.0 (C2a0 , C7a0 ), 13.6 (C500 ), 11.2 (C400 ). 31P NMR (MeCN-d3) d 146.92. HRMS (ESI+): m/z [M+Na]+ calcd for C47H54F6N4NaO8P+ 972.3512, found: 972.3562. 4.5. N,N0 -(6-((3-Azidopropyl)carbamoyl)-20 ,70 -dimethyl-3-oxo3H-spiro[isobenzofuran-1,90 -xanthene]-30 ,60 -diyl)-bis(N-ethyl2,2,2-trifluoroacetamide) (6-((3-azidopropyl)carbamoyl)-(30 ,60 N-ditrifluoroacetylrhodamine 6G)) (5) Compound (4) (1 g, 1.5 mmol) was dissolved in THF (10 ml), cooled to 0 °C by ice bath and dicyclohexylcarbodiimide (800 mg, 3.8 mmol, 2.5 equiv) in THF (2 ml) was added, followed by 3-azidopropylamine (230 mg, 2.3 mmol, 1.5 equiv) in 1 h. Reaction mixture was warmed to room temperature and stirred additional 18 h, excess of carbodiimide was decayed by water (1 ml) for 1 h. Reaction mixture was evaporated, dissolved in DCM (20 ml), extracted twice with saturated aq NaHCO3 followed by 5% citric acid and organic phase was dried with anhydrous sodium sulfate and evaporated. The product was purified on the silica gel column and eluted with 15–50% EtOAc in toluene (Rf value is 0.67 (ethyl acetate/hexane 1:1, v/v)). Pale yellow solid was obtained (905 mg, yield—80%). 1H NMR (Me2SO-d6) d 8.46 (br s, 1H, NH), 8.26 (d, 1H, H5, J = 8.1 Hz), 8.16 (d, 1H, H4, J = 8.1 Hz), 7.77 (s, 1H, H7), 7.5 (s, 2H, H40 , H50 ), 6.96 (s, 2H, H10 , H80 ), 3.36 (m, 2H, H300 ), 3.29 (m 2H, H100 ), 3.21 (m, 4H, H2⁄), 2.08 (s, 6H, H2a0 , 7a0 ), 1.76 (m, 2H, H200 ), 1.14 (m, 6H, H1⁄). 13C NMR (Me2SO-d6) d 167.8 (C3a), 165.1 (C6a), 154.1 (C2), 150.9 (C4a0 , C5a0 ), 148.6 (C60 , C30 ), 140.0 (C6), 130.0 (C40 , C50 ), 128.8 (C3), 128.6 (C5), 124.3 (C4), 122.1 (C7), 118.5 (C70 , C20 ), 117.5 (C80 , C10 ), 117.2 (CF3), 104.7 (C8a0 , C1a0 ), 80.9 (C1), 48.4 (C300 ), 39.8 (C2⁄), 36.7 (C100 ), 27.9 (C200 ), 26.5 (C1⁄), 16.4 (C7a0 , C2a0 ). HRMS (ESI+): m/z [M+Na]+ calcd for C34H30F6N6NaO+6 775.2029, found: 775.2035. 4.6. N-(9-(5-((3-Azidopropyl)carbamoyl)-2-carboxyphenyl)-6(ethylamino)-2,7-dimethyl-3H-xanthen-3-ylidene)ethanaminium chloride (6-((3-azidopropyl)carbamoylrhodamine 6G) (6) Compound (5) (730 mg, 1 mmol) was suspended in 80% aq MeOH, potassium carbonate (280 mg, 2 mmol, 2 equiv) was added and reaction mixture was heated to 60 °C for 3 h. Mixture was evaporated, solid was filtered and washed 5 10 ml THF. Solvent was evaporated in vacuum dark red solid was obtained. (388 mg, yield—72%, Rf value is 0.2 (5% E3N in acetone). 1H NMR (Me2SOd6) d 8.56 (t, 1H, NH, J = 5.1 Hz), 8.14 (dd, 1H, H5, J = 1.5 Hz, 8.1 Hz), 8.03 (d, 1H, H4, J = 8.1 Hz), 7.60 (s, 1H, H7), 6.38 (s, 2H, H40 , H50 ), 6.29 (s, 2H, H10 , H80 ), 5.18 (br s, 2H, NH), 3.36 (t, 2H,
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H300 , J = 6.8 Hz), 3.29 (m, 2H, H100 ), 3.21 (q, 4H, H2⁄, J = 7.0 Hz), 1.93 (s, 6H, H2a0 , H7a0 ), 1.76 (m, 2H, H200 ), 1.24 (t, 6H, H1⁄, J = 7.1 Hz). 13C NMR (Me2SO-d6) d 167.8 (C3a), 165.1 (C6a), 154.1 (C2), 150.9 (C4a0 , C5a0 ), 148.6 (C60 , C30 ), 140.0 (C6), 128.8 (C3), 128.6 (C5), 127.5 (C80 , C10 ), 124.3 (C4), 122.1 (C7), 118.5 (C70 , C20 ), 104.7 (C8a0 , C1a0 ), 95.01 (C40 , C50 ), 80.9 (C1), 48.4 (C300 ), 37.15 (C2⁄), 36.7 (C100 ), 27.9 (C200 ), 16.4 (C7a0 , C2a0 ), 13.7 (C1⁄). HRMS (ESI+): m/z [M+H]+ calcd for C30H33N6O4 541.2563, found: 541.2560, m/z [M+Na]+ calcd for C30H32N6NaO4 563.2383, found: 563.2356. 4.7. Oligonucleotide synthesis and purification Oligonucleotides were assembled in an ABI 3400 DNA synthesizer by the phosphoramidite method according to the manufacturer’s recommendations. Protected 20 -deoxyribonucleoside 30 -phosphoramidites, Unylinker-CPG (500 Å) and S-ethylthio-1Htetrazole were purchased from ChemGenes; 50 -alkyne, SIMA and HEX phosphoramidites from Metkinen Chemistry Oy; JOE phosphoramidite from Primetech LLC; BHQ-1-CPG and BHQ-2-CPG from Glen Research. For couplings with modified phosphoramidite 3 a 0.1 M concentration in dry MeCN was used and the coupling time was 30 s. The phosphoramidite solution was treated with 3 Å molecular sieves for a day prior to use. A model oligonucleotide was cleaved from the support and deprotected using: (1) concentrated aqueous ammonia overnight at room temperature; (2) AMA—1:1 (v/v) concd aq ammonia and 40% aq methylamine for two hours at room temperature; (3) 0.2 M NaOH in MeOH/water 4:1 (v/v) overnight at room temperature (4) TAMRA cocktail: tbutylamine/MeOH/water 1:1:2 (v/v/v) or t-butylamine/water 1:3 (v/v) overnight at 55 °C. Other R6G-modified oligonucleotides were cleaved from the support and deprotected by the TAMRA cocktail as described for a model. 50 -alkyne and native oligonucleotides were deprotected by AMA for 2 h at room temperature. Unpurified or PAGE/RP-HPLC purified 50 -alkyne oligonucleotides were conjugated with R6G-azide as described previously.54 Oligonucleotide probes were double purified by denaturing PAGE followed by RPHPLC. The denaturing gel electrophoresis of oligonucleotides was performed in 15% PAGE containing 7 M urea in Tris–borate buffer (50 mM Tris HCl, 50 mM boric acid, 1 mM EDTA, pH 8.3). Oligonucleotides were recovered from the gel by electroelution with Elutrap (Whatman) in Tris–borate buffer (5 mM Tris HCl, 5 mM boric acid, 0.1 mM EDTA, pH 8.3). The HPLC analysis and the purification of oligonucleotides were carried out on a 4.6 250 mm Jupiter C18 column (5 mkm, Phenomenex); a buffer A: 0.05 M ammonium acetate (pH 7), 5% MeCN; a buffer B: 0.03 M ammonium acetate, 80% MeCN, pH 7; a gradient of B: 0 ? 15% (1 CV), 15 ? 50% (10 CV); a flow rate of 1 ml/min; temperature 45 °C ESI-MS spectra were recorded using Bruker Maxis Impact q-TOF system: R6G derivatives were analyzed by direct injection in 97% aq acetonitrile: oligonucleotides were analyzed by LC–MS as prescribed.55 4.8. PCR The PCR target region corresponded to the VP4 and VP7 genome segments of different P and G rotavirus types. The target regions were cloned into a pGem-t vector. The sequences of primers for the VP4 segment (889 bp) and the VP7 segment (947 bp) are presented in Table S2. For PCR, we used the reverse primers 2T1 and 9T3P described previously56,57 (Table S2). Forward primers and specific TaqMan probes were designed with Sarani software (Strand Genomics, USA) with some probes containing mixed nucleotides to improve their analytical characteristics. Each PCR assay was performed with a final reaction volume of 25 ll. The samples contained 5 ll PCR-mix-2-FRT (AmpliSens, Russia), 2.5 U
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of TaqF polymerase (AmpliSens, Russia), 176 lM dNTPs (Biosan, Russia). The final concentrations of primers and fluorescently labeled probes were 0.36 lM and 0.12 lM, respectively (Table S2). The amplification reactions were performed in the RotorGene 6000 (Corbett Research) under the following cycling conditions: 1 cycle at 95 °C 15 min; 45 cycles at 95 °C 10 s, 55 °C 25 s and 72 °C 10 s. The fluorescence measurements were recorded at the detection step (55 °C) during each of the 45 cycles. Acknowledgements This work has been supported by the Russian Scientific Foundation (Grant No. 14-14-00489). We are grateful to Dr. V. Korshun (Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry) for help with recording fluorescence spectra. Supplementary data Supplementary data (sequences of primers and probes, HPLC and MS profiles of purified oligonucleotides) associated with this article can be found, in the online version, at http://dx.doi.org/10. 1016/j.bmc.2015.08.041. References and notes 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
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Solid- and solution-phase synthesis and application of R6G dual-labeled oligonucleotide probes Aleksander Yu. Skoblov a,y, Maxim V. Vichuzhanin b, Valentina M. Farzan b, Olga A. Veselova b, Tatiana A. Konovalova b, Alexander T. Podkolzin b, German A. Shipulin b, Timofei S. Zatsepin b,c,d,⇑ a
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, 117997 Moscow, Russia Central Research Institute of Epidemiology, Novogireevskaya 3a, Moscow 111123, Russia Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1-3, Moscow 119992, Russia d Skolkovo Institute of Science and Technology, 5 Innovation Center ‘‘Skolkovo”, Skolkovo 143026, Russia b c
a r t i c l e
i n f o
Article history: Received 26 June 2015 Revised 17 August 2015 Accepted 28 August 2015 Available online xxxx Keywords: Rhodamine 6G Phosphoramidite CuAAC Oligonucleotide Fluorescent dye qPCR
a b s t r a c t A novel N-TFA-protected carboxyrhodamine 6G (R6G) phosphoramidite was synthesized for use in an automated DNA synthesis to prepare 50 -labeled oligonucleotides. Deprotection and purification conditions were optimized for 50 -labeled and dual-labeled oligonucleotide probes. As an alternative we synthesized an azide derivative of R6G for CuAAC post-synthetic oligonucleotide labeling. Dual-labeled probes obtained by both methods showed the same efficacy in a quantitative PCR assay. R6G-labeled probes demonstrated superior properties in a qPCR assay in comparison with alternative HEX, JOE and SIMA dyes due to more efficient fluorescence quenching by BHQ-1. We successfully used R6G dual-labeled probes for rotavirus genotyping. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Nowadays quantitative polymerase chain reaction (qPCR or real-time PCR) has become a powerful instrument for the detection and measurement of nucleic acids in life science,1–3 biomedical research,4 diagnostics5 and forensic studies.6 qPCR is a rapid, sensitive, specific and reproducible PCR technique. In addition, the risk of carryover contamination is minimized in comparison to other PCR techniques.7 The multiplex variant of qPCR allows the detection of multiple targets within a single reaction and introduction of an internal control sample.2 Combining the use of multiple fluorophores with the discrimination of additional targets by postamplification melting gives us a simultaneous identification of a significantly larger number of targets.8 Today qPCR instruments allow us to work with 5–6 fluorophores (and probes, respectively) simultaneously in a large wavelength range (450–700 nm). This range is limited as is the case with shorter wavelengths a significant increase of fluorescence noises leads to a drop of sensitivity and the IR-range dyes are unstable at increased temperatures, thus ⇑ Corresponding author. Tel.: +7 495 939 3148; fax: +7 495 939 3181. E-mail address: [email protected] (T.S. Zatsepin). Present address: Metkinen Chemistry, Yliopistonranta 1B, FI-70210 Kuopio, Finland. y
having low quantum yields. The main problem for multiplication is cross-lighting of fluorophores that could be solved by choosing new dyes with single narrow peaks in emission spectra. Therefore, dyes and combinations of dyes for multiplex detection in qPCR are still under development. Rhodamine dyes are valuable tools for analytical9–11 and biochemical studies.12–15 They are stable over a wide pH range, photostable16,17 and have a high quantum yield in water solutions.18 Carboxyrhodamine 6G is a rarely used alternative to HEX (6-carboxy-4,7,20 ,40 ,50 ,70 -hexachlorofluorescein), SIMA (6-carboxy4,7-dichloro-20 ,70 -diphenylfluorescein) and JOE (6-carboxy-40 ,50 dichloro-20 ,70 -dimethoxyfluorescein) dyes in case qPCR probes, as automated solid-phase synthesis is unavailable for it at present. Fluorescence properties of 6-R6G are superior to HEX, JOE and more recently developed SIMA dyes (Table 1). To note, data from Table 1 should be used only for estimation of the dye applicability in qPCR. In most cases, data were obtained using a free dye solution in various solvent/pH conditions. It is well known that the absorbance and emission of fluorescent dyes are affected by the environment, including factors as solvent, pH, and conjugation to biomolecules, that is, the fluorescence of fluorescein drops in low pH due to protonation. Sjoback et al.23 have demonstrated that the conjugation of fluorescein to the oligonucleotide leads to a change of the pKa from 6.4 to 6.9 and the quantum yield
http://dx.doi.org/10.1016/j.bmc.2015.08.041 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
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Table 1 Fluorescence properties of 6-CR6G, HEX, JOE and SIMA Dye 11,19
6-R6G 6-HEX 6-JOE20,21 6-SIMA22
Absorbance (max), nm
Emission (max), nm
Molar extinction coefficient, L * cm1 * M1
Quantum yield
Brightness (Ec * QY)
520 535 522–529 536
557 556 548–560 551
116 000 96 000 75 000 64 000
0.95 0.7 0.58–0.8 Unknown
110 200 67 200 43 500–60 000 Unknown
decreases from 0.93 to 0.72. Also, Lilley and coauthors24 have shown that the extinction coefficient of fluorescein decreases by 30% after conjugation to DNA in close proximity to four-way DNA junctions. On the other hand, rhodamine dye does not undergo a similar change in absorption efficiency following conjugation. Marras25 has shown that the quantum yield of some xanthenes decreases with increasing temperature, whereas some others do not show a significant change in their quantum yield. However, such a comprehensive analysis is not available for most dyes. As conjugation to an oligonucleotide and the pH/buffer used can affect fluorescence behavior, these factors should be considered for choosing dye combinations, especially for multiplexed assays. Stronger brightness is preferable both in qPCR and end point PCR studies for the detection of low copy targets. FRET (Förster (fluorescence) energy transfer) primers26 bearing an R6G dye as the acceptor chromophore were successfully used for DNA sequencing27,28 and short tandem repeat (STR) analysis.29,30 However all labeled oligonucleotides were synthesized through post-synthetic acylation of amino oligonucleotides by a N-hydroxysuccinimide ester (NHS) of 5- or 6-carboxy-R6G. Postsynthetic acylation is a laborious, time and cost-consuming procedure for dye incorporation. Also, additional HPLC purification is needed for the synthesis of probes that lead to production of smaller amounts of labeled material. The phosphoramidite approach usually provides higher yields, takes less time and is more convenient in its synthesis. Today, an efficient and robust copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) reaction31 is widely used in oligonucleotide chemistry32 and is extremely valuable for small-scale synthesis. In most cases conjugates can be used immediately after gel-filtration or precipitation by acetone or ethanol. In this report we present the synthesis of a novel 6-carboxyrhodamine 6G phosphoramidite that is based on 6-aminohexanol and 6-carboxyrhodamine 6G 3-azidopropylamide. These derivatives were used for efficient labeling of oligonucleotides. The R6G dual-labeled probes with superior fluorescent properties were successfully used for rotavirus genotyping. 2. Results and discussion 2.1. Synthesis Many of rhodamines (R110 (rhodamine 110), R6G, TAMRA (5 (6)-carboxy N,N,N0 ,N0 -tetramethylrhodamine), ROX (5(6)-carboxyX-rhodamine), Texas Red (sulforhodamine 101)) have been used to label oligonucleotides by postsynthetic acylation33–38 or by application of dye-modified solid supports for automated oligonucleotide synthesis.39 Phosphoramidite derivatives were developed only for the TAMRA dye.40–42 The chemical synthesis of the phosphoramidite building block 4 is outlined in Figure 1. We have chosen the 6-isomer of CR6G as its fluorescence spectra fits better within the band-pass filters in qPCR machines—557 ± 5 nm (Fig. 2). In the beginning we have synthesized mixture of isomers—5(6)-carboxyR6G as reported previously with minor modifications.27 Protection of the secondary aromatic amino groups of 6-CR6G is required to fix the lactone ring of the molecule. The presence of ‘flipping’ lacton moiety can probably
lead to the self-activation of phosphoramidite followed by degradation (Fig. 3). It has been demonstrated previously that protonated weak bases can be used for phosphoramidite activation.43,44 So we blocked these amino functions from unwanted side-reaction by use of an amine-labile trifluoroacetic (TFA) group. Also a phosphoramidite should be soluble in acetonitrile, the most suitable solvent for oligo synthesis and presence of TFA groups increase such solubility. While compound 1 was allowed to react with trifluoroacetic anhydride in pyridine, derivative 2 was isolated in an only 15% yield after column chromatography. Low yield is a result of close Rf values for 5- and 6-isomers, so only a part of pure 6-R6G could be isolated easily; additional purifications of the isomer mixture could increase the yield. To solve the problem of poor isomer separation we developed an alternative procedure for 5(6)-carboxyR6G synthesis. We separated the stages of acylation of the first and the second molecules of substituted aminophenol that significantly improved the purity of products. Attempts to separate isomers of 5(6)-carboxyR6G by normal phase chromatography on silica gel or aluminum oxide led to disastrous losses of compounds so we used reverse-phase column chromatography to separate isomers. Separation of isomers was rather smooth and the summarized yield of two isomers was about 50%. As a result at the next stage—TFAA acylation was significantly improved. TFAA acylation was carried out in THF in presence of TEA at 20 °C and the yield was increased up to 70%. First, there was no need to separate isomers with close Rf values after acylation, second, due to thorough purification of carboxyR6G less amount of byproducts was formed. Carbodiimide-assisted acylation of 6-aminohexanol gave an excellent yield of amide 3. The subsequent phosphitylation of the hydroxy group in an inert atmosphere using (N,N-diisopropylamino)-2-cyanoethoxychlorophosphine in dichloromethane in the presence of N,N-diisopropylethylamine45 afforded the title phosphoramidite 4 (Fig. 1). Following that, the phosphoramidite 4 was evaluated in machine-assisted solid-phase oligonucleotide synthesis by the standard 2-cyanoethyl phosphoramidite method. The average coupling efficiency of the novel monomer in dry acetonitrile was found to be more than 98%. The phosphoramidite obtained was used in the synthesis of a number of 50 -modified oligonucleotides (Table 2). Oligomer I (50 -R6G-dT10) was synthesized as a model in order to choose and optimize deprotection conditions. Deprotection of the modified oligonucleotides was carried out by aq ammonia at RT (overnight), AMA (water solutions of ammonia and methylamine 1:1) at RT (2 h), 0.2 M NaOH in aq methanol46 or with an TAMRA cocktail (t-butylamine/methanol/water—1/1/2 (v/v/v)) at 55 °C (overnight) (Fig. 4). Both alkaline and t-butylamine deprotection gave excellent results without any side products (Fig. 4, traces 3, 4, peak a), in the case of ammonia deprotection a partial formation of colorless non-fluorescent spirolactame 8a47–49 (Fig. 5) was observed (Fig. 4, trace 1, peak b). AMA deprotection led only to spirolactame 8b (Fig. 5) formation (Fig. 4, trace 2, peak c). As in the case of alkaline deprotection additional neutralization and desalting are needed before purification, we used more convenient MeOH-free TAMRA cocktail for the deprotection of R6G-labeled oligonucleotides. Oligomers II-XVI were isolated by denaturing
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Figure 1. Synthesis of R6G phophoramidite 4 and 3-azidopropylamide 6: (a) TFAA, Et3N, THF; (b) (1) NHS, DCC, THF; (2) 6-aminohexanol, THF; (c) Pri2NP(Cl)O(CH2)2CN, Pri2NEt, DCM, (d) (1) NHS, DCC, THF; (2) 3-azidopropylamine, THF; (e) K2CO3, MeOH.
Figure 2. Normalized emission spectra of 5-carboxyR6G (solid line) and 6-carboxyR6G (dashed line) in a PBS buffer.
polyacrylamide gel electrophoresis (PAGE) followed by RP-HPLC, and their structure was confirmed by ESI-MS. MS data is presented Table 2, HPLC and MS profiles of purified products—in Support materials. However in the case of small-scale oligonucleotide synthesis (especially in 96-well plates) followed by post-conjugation of various dyes CuAAC showed to be a good alternative to phosphoramidite chemistry. Therefore, we synthesized a novel azidoalkyl derivative of R6G that was further conjugated to a 50 -alkyne oligonucleotide under CuAAC conditions (Fig. 6). As azide–alkyne click reaction usually proceeds smoothly and quantitatively we
Figure 3. Probable equilibrium of 6-carboxamide R6G phosphoramidite.
tried to minimize purification procedures. However simple precipitation by lithium perchlorate in acetone or conventional RP HPLC purification lead to a product with increased background fluorescence due to incomplete removal of the dye. Better results were obtained when heat denaturation before injection of the reaction mixture was carried out (6 M urea, 95 °C, 5 min). The only method of purification after CuAAC in our hands that gave the same level of
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Table 2 Modified oligonucleotides used in the study N
Name
Oligonucleotide
MALDI-MS or ESI-MS (calcd/observed)
I
R6G-T10
R6G-TTTTTTTTTT
3601.2/3599.0a 3600.2/3598.0b 3610.9/3610.9c
II III IV V VI
R6G-P4z R6G-G1z R6G-G3z SIMA-P4z SIMAG1z SIMAG3z JOE- P4z JOE-G1z JOE-G3z HEX- P4z HEX-G1z HEX-G3z -P4z -G1z -G3z
R6G-CAC TCT GAC TAC TAC CTT TAA ACA GAG CG-BHQ1 R6G-CTG AGC TTT AGT YAA GGC AAA TAA TGC TCA G-BHQ1 R6G-TAC CCA ACT GAA GCA GCA ACA GGG TA-BHQ1 SIMA-CAC TCT GAC TAC TAC CTT TAA ACA GAG CG-BHQ1 SIMA-CTG AGC TTT AGT YAA GGC AAA TAA TGC TCA G-BHQ1
9973.9/9974.3 10711.0; 10726.0/10711.4; 10725.4 9150.8/9151.1 10111.8/10113.1 10848.9; 10863.9/10850.6; 10865.6
SIMA-TAC CCA ACT GAA GCA GCA ACA GGG TA-BHQ1
9288.2/9289.9
JOE-CAC TCT GAC TAC TAC CTT TAA ACA GAG CG-BHQ1 JOE-CTG AGC TTT AGT YAA GGC AAA TAA TGC TCA G-BHQ1 JOE-TAC CCA ACT GAA GCA GCA ACA GGG TA-BHQ1 HEX-CAC TCT GAC TAC TAC CTT TAA ACA GAG CG-BHQ1 HEX-CTG AGC TTT AGT YAA GGC AAA TAA TGC TCA G-BHQ1 HEX-TAC CCA ACT GAA GCA GCA ACA GGG TA-BHQ1 Alkyne-CAC TCT GAC TAC TAC CTT TAA ACA GAG CG-BHQ1 Alkyne-CTG AGC TTT AGT YAA GGC AAA TAA TGC TCA G-BHQ1 Alkyne-TAC CCA ACT GAA GCA GCA ACA GGG TA-BHQ1
10019.7/10021.9 10756.8; 10771.8/10757.5; 10772.5 9196.6/9197.9 10105.1/10097.8 10842.6; 10857.6/10835.4; 10850.4 9281.6/9275.7 9625.7d (10166.0e)/9626.2d (10166.3)e 10362.9d; 10377.9 d (10903.1, 10918.1)e/10363.3d; 10378.4d (10903.4; 10918.4)e 8802.6d (9342.9e)/8803.2d (9343.2)e
VII VIII IX X XI XII XIII XIV XV XVI a
TAMRA cocktail deprotection,
b
Ammonia deprotection, c AMA deprotection,
d
Before click conjugation, e After click conjugation.
Figure 6. Synthesis of R6G-labeled oligonucleotides by CuAAC.
2.2. qPCR studies
Figure 4. HPLC analysis of deprotected oligonucleotide I at 280 nm. Peak at 25 min is R6G-labeled oligonucleotide. Deprotection was carried out with: (1) ammonia, (2) AMA, (3) NaOH, (4) TAMRA cocktail.
background fluorescence as application of R6G phosphoramidite was denaturative PAGE. Perhaps denaturative IE HPLC could be the method of choice. In sum, CuAAC is cost effective, but in the case of R6G derivation additional purification in denaturative conditions is needed. However this method is preferable for sporadic synthesis of R6G-labeled oligonucleotides in comparison to the phosphoramidite approach due to lower consumption of the dye derivative.
Recently, we developed an in-house qPCR kit for rotavirus genotyping.50 This study gave us an understanding of the specific role of rotaviruses in gastroenteritis which leads to hospitalization for both children and adults and shows the epidemiological significance of several rotavirus strains. To compare ‘yellow’ dyes we synthesized several dual-labeled probes labeled with R6G, HEX, JOE or SIMA dyes and BHQ1 (2,20 -((4-(2-methoxy-5-methyl-4((4-methyl-2-nitrophenyl)diazenyl)phenyl)diazenyl)phenyl)azanediyl)bis(ethan-1-ol)) as a quencher (Table 2) to find out if a ‘best’ fluorophore in qPCR exists. In case of these dyes BHQ1 and BHQ2 (2,20 -((4-((E)-(2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)phenyl)-azanediyl)bis(ethan-1-ol)) gave the same efficiency of quenching (data not shown). The crucial parameter in qPCR data is a threshold cycle (Ct)—the intersection between an amplification curve and a threshold line (Fig. 7B–D). It is a relative
Figure 5. Deprotection of R6G-modified oligonucleotides by primary amines.
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Figure 7. qPCR traces (R6G – red, JOE – black, HEX – green, SIMA – blue). (A) Raw data for P4z probes, (B) normalized data for P4z probes, (C) normalized data for G1z, (D) normalized data for G3z probes.
measure of the concentration of target DNA in the PCR reaction and is calculated after data normalization (Fig. 8). In case of end-point PCR relative increase of fluorescence is more important. Small difference between final and initial fluorescence lead to low sensitivity and increased number of false negative results due to a relatively large grey zone between a lower and an upper cut-off. If somebody is going to use a kit in both methods these parameters become significant. So the best probe should give the minimal Ct and a maximal relative increase of fluorescence. The R6G-labeled probes gave the lowest fluorescence background and the HEX/SIMA probes—the highest (Fig. 7A). Such additional quenching could be a result of the tight interactions of R6G moiety with heterocyclic bases.51,52 As a result higher relative increase of fluorescence after normalization in case of R6G allows the assured detection of low-copy DNA targets due to improvement of threshold cycle (Ct) (Fig. 8). We want to emphasize a significant difference in Ct at the lowest concentration of target DNA for all probes used in the study. Also a higher increase of fluorescence during amplification makes R6G a preferable dye for endpoint application. This is a combined result of R6G high brightness, considerable quenching by BHQ and congruence of fluorescence spectra with the band-pass filters in common qPCR machines. 3. Conclusions We have presented here a multi gram procedure that allows to do preparative scale synthesis of single isomer of carboxy-R6G derivatives. We developed an efficient and reliable method for the preparation of 50 -R6G labeled oligonucleotides using our newly developed modification reagents. Phosphoramidite derivative has significant advantages both for small- and medium-scale synthesis of fluorescent oligonucleotides in comparison to traditional NHSesters. Our method was shown to be a useful route for the synthesis of dual-labeled probes. R6G azide derivative is a good alternative to phosphoramidite, especially if somebody needs only few oligonucleotides on a small scale. We made a comprehensive comparison of several common ‘yellow’ dyes used in qPCR and have demonstrated that R6G is a preferable dye.
4. Experimental Chemicals were obtained from commercial suppliers and used without further purification unless otherwise noted. Chemical shifts (d, ppm) for 1H, 13C and 31P are referenced to internal solvent resonances and reported relative to SiMe4 and 85% aq H3PO4, respectively. Chemical shifts are accurate to within 0.01 ppm for 1 H, 13C and CSSI are accurate within 0.5 Hz. The compounds were visualised under short-wavelength UV. Fluorescence spectra were recorded as predescribed.53 4.1. N-(9-(2,5-Dicarboxyphenyl)-6-(ethylamino)-2,7-dimethyl3H-xanthen-3-ylidene) ethanaminium chloride (6-carboxyrhodamine-6G) (1) 3-Ethylamino-4-methylphenol (51.9 g 343 mmol) was dissolved CH2Cl2 (500 ml) and methanesulfonic acid (22 ml, 343 mmol) was added dropwise under vigorous mixing. After 1 h solvent was evaporated, the residue was suspended in toluene (1 L) and 1,2,4-benzenetricarboxylic anhydride (31.3 g, 162 mmol) was added. The mixture was refluxed overnight, then toluene was evaporated, the residue was dissolved in DMF (500 ml) and the reaction mixture was heated at 140 °C for 48 h. Then DMF was evaporated in vacuum, and gum residue was dissolved in 0.2 M NaOH (3 L). Title compound was purified by reverse-phase column chromatography (sorbent LPS-500-H, Purolite) with gradient of isopropanol (0 ? 15%) in water. 6-Isomer is elited in 3–8% of isopropanol (isolated yield—20.1 g, 25.0%), 5-isomer—in 8–12% of isopropanol (isolated yield—21.0 g, 26.1%). 6-Carboxyrhodamine-6G: 1H NMR (DMSO-d6) d 8.30 (s, 2H, H4, H5), 7.88 (s, 1H, H7), 6.86 (s, 2H, H40 , H50 ), 6.83 (s, 2H, H10 , H80 ), 3.50 (q, 4H, H2⁄), 2.14 (s, 6H, H2a0 , H7a0 ), 1.29 (t, 6H, H1⁄); 13C NMR (DMSO-d6) 166.3 (C3a), 166.1 (C6a), 157.3 (C4a0 , C5a0 ), 156.8 (C1), 156.4 (C30 , C60 ), 135.3 (C3), 134.9 (C6), 134.0 (C2), 131.7 (C7), 131.2 (C4), 131.1 (C5), 129.0 (C40 , C50 ), 126.0 (C9a0 , C8a0 ), 113.5 (C20 , C70 ), 94.1 (C10 , C80 ), 38.5 (C2⁄), 17.9 (C2a0 , C7a0 ), 14.0 (C1⁄). HRMS (ESI+): m/z [M+H]+ calcd for C27H27N2O+5: 459.1914, found: 459.1924.
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R6G HEX JOE SIMA
42
38
P4Z
34
30 1.0E+06
1.0E+05
1.0E+04
1.0E+03
target DNA, copies/ml
Ct 40
36
R6G HEX JOE SIMA
G1Z
32
28 1.5E+06
1.5E+05
1.5E+04
1.5E+03
target DNA, copies/ml
Ct 40
36
R6G HEX JOE SIMA
4.3. N,N0 -(6-((6-Hydroxyhexyl)carbamoyl)-20 ,70 -dimethyl-3-oxo3H-spiro[isobenzofuran-1,90 -xanthene]-30 ,60 -diyl)-bis(N-ethyl-2, 2,2-trifluoroacetamide) (6-(30 ,60 -N-ditrifluoroacetylrhodamine 6G-6-carboxamido)hexan-1-ol) (3)
G3Z
32
28 1.2E+06
1.2E+05
1.2E+04
4.2.2. Method B 6-Carboxyrhodamine-6G (1) (4.60 g, 10 mmol) was dissolved in THF (200 ml) in presence of triethylamine (7 ml, 50 mmol). The reaction mixture was cooled in an ice bath/sodium chloride to 20 °C with magnetic stirring and trifluoroacetic anhydride (3.5 ml, 25 mmol) was added during one hour in three portions. The mixture was then evaporated and product was precipitated by water (100 ml), then filtered and dried in vacuum over P2O5. The residue was purified by column chromatography on a silica gel with gradient of methanol (1 ? 10%) in dichloromethane/5% pyridine as the mobile phase. The product was obtained as a pale yellow solid in 69% yield (4.48 g). Rf value is 0.3 for 6-Carboxy(TFA)2R6G and 0.25 for 5-Carboxy (TFA)2R6G (10% MeOH in DCM). 1H NMR (MeCN-d3) d 8.46 (br s, 1H, H4), 8.24 (d, 1H, H5, J = 7.9 Hz), 7.33 (m, 3H, H10 , H7, H80 ), 6.93 (m, 2H, H40 , H50 ), 3.44 (m, 4H, H2⁄), 2.11 (s, 6H, H2a0 , H7a0 ), 1.46 (m, 6H, H1⁄). 13C NMR (MeCN-d3) d 168.28 (C3a), 165.15 (C6a), 156.07 (C(O)CF3), 154.81 (C2), 148.88 (C4a0 , C5a0 ), 139.80 (C60 , C30 ), 137.62 (C6), 132.68 (C3), 130.27 (C40 , C50 ), 126.39 (C4 or C5), 124.11 (C4 or C5), 123.92 (C7), 119.39 (C20 , C70 ), 118.74 (C8a0 , C9a0 ), 118.41 (C10 , C80 ), 117.15 (CF3), 80.97 (C1), 39.79 (C2⁄), 26.46 (C1⁄), 15.97 (C2a0 , C7a0 ). HRMS (ESI-): m/z [MH] calcd for C31H23F6N2NaO 7 : 649.1409, found: 649.1398.
1.2E+03
target DNA, copies/ml Figure 8. Results of qPCR of target DNA by probes used in this study.
5-Carboxyrhodamine-6G: 1H NMR (DMSO-d6) d 8.72 (s, 1H, H4), 8.35(d, 1H, H6, J = 7.9 Hz) 7.54 (d, 1H, H7, J = 7.9 Hz), 6.83 (s, 2H, H40 , H50 ), 6.82 (s, 2H, H10 , H80 ), 3.48 (m, 4H, H2⁄), 2.14 (s, 6H, H2a0 , H7a0 ), 1.30 (t, 6H, H1⁄). 13C NMR (DMSO-d6) 165.6 (C3a), 165.2 (C6a), 156.0 (C4a0 ,C5a0 ), 156.4 (C30 , C60 ), 152.0 (C1), 132.4 (C3), 132.3 (C5), 131.2 (C4), 130.7 (C6), 130.0 (C7), 128.0 (C40 , C50 ), 124.8 (C9a0 , C8a0 ), 111.9 (C20 , C70 ), 93.4 (C10 , C80 ), 37.6 (C2⁄), 16.9 (C2a0 , C7a0 ), 13.2 (C1⁄). HRMS (ESI+): m/z [M+H]+ calcd for C27H27N2O+5: 459.1914, found: 459.1921. 4.2. 30 -(N-Ethyl-2,2,2-trifluoroacetamido)-60 -(ethylamino)-20 ,70 dimethyl-3-oxo-3H-spiro[isobenzofuran-1,90 -xanthene]-6-carboxylic acid (30 ,60 -N-ditrifluoroacetyl-6-carboxyrhodamine-6G) (2) 4.2.1. Method A 5,6-Carboxyrhodamine-6G (4.60 g, 10 mmol) was dissolved in pyridine (50 ml). The reaction mixture was cooled in an ice bath with magnetic stirring and trifluoroacetic anhydride (4.2 ml, 30 mmol) was added during one hour in three portions. The mixture was then evaporated and product was precipitated by water (100 ml), then filtered and dried in vacuum over P2O5. The residue was purified by column chromatography on a silica gel with gradient of methanol (1 ? 10%) in dichloromethane/5% pyridine as the mobile phase. The product was obtained as a pale orange solid in 15% yield (0.98 g).
970 mg (1.5 mmol) of compound (2) was dissolved in 50 ml of dichloromethane and 412 mg (2 mmol) of dicyclohexylcarbodiimide were added, followed by 412 mg (2 mmol) of N-hydoxysuccinimide (230 mg, 2 mmol). The mixture was stirred overnight at room temperature, then filtered, solids were washed twice with dichloromethane (10 ml) and 176 mg (1.5 mmol) of 6-aminohexanol was added, then again the mixture was stirred overnight at room temperature. The mixture was extracted with H2O and then with 0.1 M HCl(aq), and organic phase is evaporated. The product was applied on a silica gel column and eluted with gradient of methanol (1 ? 10%) in dichloromethane. Next, the product-containing fractions were evaporated and 1.01 g of yellow solid was obtained. The yield is 90%, Rf value is 0.55 (10% MeOH in DCM). 1H NMR (MeCN-d3) d 8.46 (s, 1H, NH), 8.16 (d, 1H, H5, J = 7.9 Hz), 8.11 (d, 1H, H4, J = 8.1 Hz), 7.89 (s, 1H, H7), 7.4 (br s, 2H, H40 , H50 ), 6.96 (s, 2H, H10 , H80 ), 4.18(m, 2H, H600 ), 3.70 (m, 2H, H100 ), 3.44 (m, 4H, H2⁄), 3.29 (m, 2H, H500 ), 2.11 (s, 6H, H2a0 , H7a0 ), 1.66 (m, 2H, H200 ), 1.46 (m, 6H, H1⁄), 1.22 (m, 2H, H300 ), 1.21 (m, 2H, H400 ). 13C NMR (MeCN-d3) d 168.28 (C3a), 165.15 (C6a), 156.07 (C(O)CF3), 154.81 (C2), 148.88 (C4a0 , C5a0 ), 139.80 (C60 , C30 ), 137.62 (C6), 132.68 (C3), 130.27 (C40 , C50 ), 126.39 (C4 or C5), 124.11 (C4 or C5), 123.92 (C7), 119.39 (C20 , C70 ), 118.74 (C8a0 , C1a0 ), 118.41 (C10 , C80 ), 117.15 (CF3), 80.97 (C1), 46.65 (C600 ), 39.79 (C2⁄), 30.97 (C100 ), 29.25 (C200 ), 26.46 (C1⁄), 25.54 (C300 ), 15.97 (C2a0 , C7a0 ), 13.63 (C500 ), 11.21 (C400 ). HRMS (ESI+): m/z [M+Na]+ calcd for C37H37F6N3NaO+7: 772.2433, found: 772.2441. 4.4. (6-(30 -(N-Ethyl-2,2,2-trifluoroacetamido)-60 -(ethylamino)20 ,70 -dimethyl-3-oxo-3H-spiro[isobenzofuran-1,90 -xanthene]-6carboxamido)hexyl)-2-cyanoethyl-diisopropylphosphoramidite (6-(30 ,60 -N-ditrifluoroacetylrhodamine 6G-6-carboxamido)hexyl)1-O-(2-cyanoethyl)-(N,N-diisopropyl)phosphoramidite) (4) 375 mg (0.5 mmol) of compound (3) was dissolved in 5 ml of dichloromethane, 218 ll (1.25 mmol) N,N-diisopropylethylamine was added, and then 142 ll (0.6 mmol) of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite was added at room temperature.
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After one hour, reaction mixture was extracted with an aqueous 0.5 M phosphate buffer pH 8, and the organic phase was dried with anhydrous sodium sulfate and evaporated. The product was purified on a silica gel column with addition of 1% of Et3N and eluted with 1–5% MeOH in DCM (Rf value is 0.75 (10% MeOH in DCM)). Pale yellow solid of R6G-amidite was obtained (380 mg, yield— 70%). 1 H NMR (MeCN-d3) d 8.46 (br s, 1H, NH), 8.24 (d, 1H, H5, J = 7.9 Hz), 8.14(d, 1H, H4, J = 8.1 Hz) 7.77 (s, 1H, H7), 7.5 (br s, 2H, H40 , H50 ), 6.96 (s, 2H, H10 , H80 ), 4.19 (m, 2H, H600 ), 3.82 (m, 2H, P-OCH2), 3.70 (m, 2H, H100 ), 3.64 (m, 2H, PN-CH), 3.44 (m, 4H, H2⁄), 3.29 (m, 2H, H500 ), 2.66 (m, 2H, CH2-CN), 2.11 (s, 6H, H2a0 , H7a0 ), 1.66 (m, 2H, H200 ), 1.46 (m, 6H, H1⁄), 1.22 (m, 2H, H300 ), 1.21 (m, 2H, H400 ), 1.18 (m, 12H, (CH3)2-C). 13C NMR (MeCN-d3) d 168.3 (C3a), 165.2 (C6a), 156.0 (C(O)CF3), 154.8 (C2), 148.9 (C4a0 , C5a0 ), 139.8 (C60 , C30 ), 137.6 (C6), 132.7 (C3), 130.3 (C40 , C50 ), 126.4 (C4 or C5), 124.1 (C4 or C5), 123.92 (C7), 119.4 (C20 , C70 ), 118.7 (C8a0 , C1a0 ), 118.4 (C10 , C80 ), 117.2 (CF3), 81.0 (C1), 58.4 (P-OCH2), 45.6 (C600 ), 42.8 (N-CH), 39.8 (C2⁄), 30.97 (C100 ), 29.25 (C200 ), 26.5 (C1⁄), 25.5 (C300 ), 24.17 ((CH3)2-CH), 20.2 (CH2-CN), 16.0 (C2a0 , C7a0 ), 13.6 (C500 ), 11.2 (C400 ). 31P NMR (MeCN-d3) d 146.92. HRMS (ESI+): m/z [M+Na]+ calcd for C47H54F6N4NaO8P+ 972.3512, found: 972.3562. 4.5. N,N0 -(6-((3-Azidopropyl)carbamoyl)-20 ,70 -dimethyl-3-oxo3H-spiro[isobenzofuran-1,90 -xanthene]-30 ,60 -diyl)-bis(N-ethyl2,2,2-trifluoroacetamide) (6-((3-azidopropyl)carbamoyl)-(30 ,60 N-ditrifluoroacetylrhodamine 6G)) (5) Compound (4) (1 g, 1.5 mmol) was dissolved in THF (10 ml), cooled to 0 °C by ice bath and dicyclohexylcarbodiimide (800 mg, 3.8 mmol, 2.5 equiv) in THF (2 ml) was added, followed by 3-azidopropylamine (230 mg, 2.3 mmol, 1.5 equiv) in 1 h. Reaction mixture was warmed to room temperature and stirred additional 18 h, excess of carbodiimide was decayed by water (1 ml) for 1 h. Reaction mixture was evaporated, dissolved in DCM (20 ml), extracted twice with saturated aq NaHCO3 followed by 5% citric acid and organic phase was dried with anhydrous sodium sulfate and evaporated. The product was purified on the silica gel column and eluted with 15–50% EtOAc in toluene (Rf value is 0.67 (ethyl acetate/hexane 1:1, v/v)). Pale yellow solid was obtained (905 mg, yield—80%). 1H NMR (Me2SO-d6) d 8.46 (br s, 1H, NH), 8.26 (d, 1H, H5, J = 8.1 Hz), 8.16 (d, 1H, H4, J = 8.1 Hz), 7.77 (s, 1H, H7), 7.5 (s, 2H, H40 , H50 ), 6.96 (s, 2H, H10 , H80 ), 3.36 (m, 2H, H300 ), 3.29 (m 2H, H100 ), 3.21 (m, 4H, H2⁄), 2.08 (s, 6H, H2a0 , 7a0 ), 1.76 (m, 2H, H200 ), 1.14 (m, 6H, H1⁄). 13C NMR (Me2SO-d6) d 167.8 (C3a), 165.1 (C6a), 154.1 (C2), 150.9 (C4a0 , C5a0 ), 148.6 (C60 , C30 ), 140.0 (C6), 130.0 (C40 , C50 ), 128.8 (C3), 128.6 (C5), 124.3 (C4), 122.1 (C7), 118.5 (C70 , C20 ), 117.5 (C80 , C10 ), 117.2 (CF3), 104.7 (C8a0 , C1a0 ), 80.9 (C1), 48.4 (C300 ), 39.8 (C2⁄), 36.7 (C100 ), 27.9 (C200 ), 26.5 (C1⁄), 16.4 (C7a0 , C2a0 ). HRMS (ESI+): m/z [M+Na]+ calcd for C34H30F6N6NaO+6 775.2029, found: 775.2035. 4.6. N-(9-(5-((3-Azidopropyl)carbamoyl)-2-carboxyphenyl)-6(ethylamino)-2,7-dimethyl-3H-xanthen-3-ylidene)ethanaminium chloride (6-((3-azidopropyl)carbamoylrhodamine 6G) (6) Compound (5) (730 mg, 1 mmol) was suspended in 80% aq MeOH, potassium carbonate (280 mg, 2 mmol, 2 equiv) was added and reaction mixture was heated to 60 °C for 3 h. Mixture was evaporated, solid was filtered and washed 5 10 ml THF. Solvent was evaporated in vacuum dark red solid was obtained. (388 mg, yield—72%, Rf value is 0.2 (5% E3N in acetone). 1H NMR (Me2SOd6) d 8.56 (t, 1H, NH, J = 5.1 Hz), 8.14 (dd, 1H, H5, J = 1.5 Hz, 8.1 Hz), 8.03 (d, 1H, H4, J = 8.1 Hz), 7.60 (s, 1H, H7), 6.38 (s, 2H, H40 , H50 ), 6.29 (s, 2H, H10 , H80 ), 5.18 (br s, 2H, NH), 3.36 (t, 2H,
7
H300 , J = 6.8 Hz), 3.29 (m, 2H, H100 ), 3.21 (q, 4H, H2⁄, J = 7.0 Hz), 1.93 (s, 6H, H2a0 , H7a0 ), 1.76 (m, 2H, H200 ), 1.24 (t, 6H, H1⁄, J = 7.1 Hz). 13C NMR (Me2SO-d6) d 167.8 (C3a), 165.1 (C6a), 154.1 (C2), 150.9 (C4a0 , C5a0 ), 148.6 (C60 , C30 ), 140.0 (C6), 128.8 (C3), 128.6 (C5), 127.5 (C80 , C10 ), 124.3 (C4), 122.1 (C7), 118.5 (C70 , C20 ), 104.7 (C8a0 , C1a0 ), 95.01 (C40 , C50 ), 80.9 (C1), 48.4 (C300 ), 37.15 (C2⁄), 36.7 (C100 ), 27.9 (C200 ), 16.4 (C7a0 , C2a0 ), 13.7 (C1⁄). HRMS (ESI+): m/z [M+H]+ calcd for C30H33N6O4 541.2563, found: 541.2560, m/z [M+Na]+ calcd for C30H32N6NaO4 563.2383, found: 563.2356. 4.7. Oligonucleotide synthesis and purification Oligonucleotides were assembled in an ABI 3400 DNA synthesizer by the phosphoramidite method according to the manufacturer’s recommendations. Protected 20 -deoxyribonucleoside 30 -phosphoramidites, Unylinker-CPG (500 Å) and S-ethylthio-1Htetrazole were purchased from ChemGenes; 50 -alkyne, SIMA and HEX phosphoramidites from Metkinen Chemistry Oy; JOE phosphoramidite from Primetech LLC; BHQ-1-CPG and BHQ-2-CPG from Glen Research. For couplings with modified phosphoramidite 3 a 0.1 M concentration in dry MeCN was used and the coupling time was 30 s. The phosphoramidite solution was treated with 3 Å molecular sieves for a day prior to use. A model oligonucleotide was cleaved from the support and deprotected using: (1) concentrated aqueous ammonia overnight at room temperature; (2) AMA—1:1 (v/v) concd aq ammonia and 40% aq methylamine for two hours at room temperature; (3) 0.2 M NaOH in MeOH/water 4:1 (v/v) overnight at room temperature (4) TAMRA cocktail: tbutylamine/MeOH/water 1:1:2 (v/v/v) or t-butylamine/water 1:3 (v/v) overnight at 55 °C. Other R6G-modified oligonucleotides were cleaved from the support and deprotected by the TAMRA cocktail as described for a model. 50 -alkyne and native oligonucleotides were deprotected by AMA for 2 h at room temperature. Unpurified or PAGE/RP-HPLC purified 50 -alkyne oligonucleotides were conjugated with R6G-azide as described previously.54 Oligonucleotide probes were double purified by denaturing PAGE followed by RPHPLC. The denaturing gel electrophoresis of oligonucleotides was performed in 15% PAGE containing 7 M urea in Tris–borate buffer (50 mM Tris HCl, 50 mM boric acid, 1 mM EDTA, pH 8.3). Oligonucleotides were recovered from the gel by electroelution with Elutrap (Whatman) in Tris–borate buffer (5 mM Tris HCl, 5 mM boric acid, 0.1 mM EDTA, pH 8.3). The HPLC analysis and the purification of oligonucleotides were carried out on a 4.6 250 mm Jupiter C18 column (5 mkm, Phenomenex); a buffer A: 0.05 M ammonium acetate (pH 7), 5% MeCN; a buffer B: 0.03 M ammonium acetate, 80% MeCN, pH 7; a gradient of B: 0 ? 15% (1 CV), 15 ? 50% (10 CV); a flow rate of 1 ml/min; temperature 45 °C ESI-MS spectra were recorded using Bruker Maxis Impact q-TOF system: R6G derivatives were analyzed by direct injection in 97% aq acetonitrile: oligonucleotides were analyzed by LC–MS as prescribed.55 4.8. PCR The PCR target region corresponded to the VP4 and VP7 genome segments of different P and G rotavirus types. The target regions were cloned into a pGem-t vector. The sequences of primers for the VP4 segment (889 bp) and the VP7 segment (947 bp) are presented in Table S2. For PCR, we used the reverse primers 2T1 and 9T3P described previously56,57 (Table S2). Forward primers and specific TaqMan probes were designed with Sarani software (Strand Genomics, USA) with some probes containing mixed nucleotides to improve their analytical characteristics. Each PCR assay was performed with a final reaction volume of 25 ll. The samples contained 5 ll PCR-mix-2-FRT (AmpliSens, Russia), 2.5 U
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of TaqF polymerase (AmpliSens, Russia), 176 lM dNTPs (Biosan, Russia). The final concentrations of primers and fluorescently labeled probes were 0.36 lM and 0.12 lM, respectively (Table S2). The amplification reactions were performed in the RotorGene 6000 (Corbett Research) under the following cycling conditions: 1 cycle at 95 °C 15 min; 45 cycles at 95 °C 10 s, 55 °C 25 s and 72 °C 10 s. The fluorescence measurements were recorded at the detection step (55 °C) during each of the 45 cycles. Acknowledgements This work has been supported by the Russian Scientific Foundation (Grant No. 14-14-00489). We are grateful to Dr. V. Korshun (Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry) for help with recording fluorescence spectra. Supplementary data Supplementary data (sequences of primers and probes, HPLC and MS profiles of purified oligonucleotides) associated with this article can be found, in the online version, at http://dx.doi.org/10. 1016/j.bmc.2015.08.041. References and notes 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
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Please cite this article in press as: Skoblov, A. Y.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.08.041