Direct labeling of nucleosides with 3-thiazolylcoumarin fluorescent dyes

© 2020 Ia. B. Kuziv et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 577.113+577.336+47.814


Introduction
Non-radioactive labeling of proteins, nucleic acids and other biomolecules allows their visualization and quantification to study the biological functions and dynamics. Appli ca tions of biomolecules carrying fluorescent reporter groups include research on their cellular transport, interactions with other molecules, bioimaging, medical diagnostics, etc. [1,2].
Selective modification of nucleoside amino groups has attracted great attention in early period of the development of nucleoside chemistry. Application of numerous active compounds was investigated. The first attempt to selectively block cytidine, 2'-deoxycytidine and their 5'-phosphates with active esters was performed with 2-chloromethyl-4-nitrophenyl benzoate [16]. It was noted that adenosine and guanosine containing less basic amino groups were unable to react. Other authors employed pentaflurophenyl [17] and p-nitrophenyl [18] benzoates to block 2'-deoxycytidine. Katritzky et al. performed the acylation of cytidine, adenosine and deoxyadenosine by benzotriazoleactivated carboxyalkyl-modified fluorescent dyes [14]. We have previously studied active esters of amino acids as acylating agents for the nucleoside amino functions. Cytidine, adenosine and guanosine conjugates with amino acids were obtained from O-protected nucleosides [19]. Thus, active esters can be used for the acylation of exocyclic amino groups of nucleosides to prepare their conjugates.
Direct introduction of reporter groups at nucleoside hydroxyls is much more challenging, as these groups are not highly nucleophilic and thus require very active acylating agents. Most methods that are commonly used for the preparation of nucleoside esters are based on acyl halides or anhydrides. However, such reagents are not suitable for the attachment of labile reporting groups. Other esterification methods include transesterification of esters by alcohols under acidic or basic catalysis [20] and alkylation of carboxylic acids via Mitsunobu reaction [21].
Mild conditions of ester bond formation can be achieved by active ester approach. Direct O-acylation with these reagents is complicated by relatively low reactivity of aliphatic OHgroups and a low acylation efficiency of active esters, although there are numerous reports on their use in this reaction [22][23][24][25][26][27]. Hydroxyl esterification with active esters is frequently used in multistep syntheses of various classes of compounds [28][29][30][31][32][33].
These esterification methods were used in the synthesis of amino acid esters [23,27], depsipeptides [25,28,30], dendrimers [29], carbohydrate and glycoside derivatives [32,33], and for the attachment of amino acids and nucleotides to polymer supports [24,31,34]. However, we were unable to find any papers describing the direct attachment of reporter groups to hydroxyl functions of nucleosides.
Previously we have synthesized a series of carboxyl-modified UV-excitable 7-substituted 3-hetarylcoumarins as biomolecular labeling reagents with bright blue fluorescence [35,36]. In the present work, we have attempted to obtain their nucleoside conjugates by active ester approach without nucleoside functionalization with aminoalkyl or other reactive group.
Solvents for synthesis were obtained from Macrochim (Ukraine). DMF was dried by distillation over CaO, P 2 O 5 and stored over 3A molecular sieves (Rathburn, UK). Dioxane was distilled over potassium hydroxide, DIPEA over sodium and stored over molecular sieves. Triethylamine was distilled over maleic anhydride and CaO. HOBT was dried in vacuum with P 2 O 5 . Methanol for spectroscopy (Labskan, Ireland) was additionally purified by distillation over KHSO 4 and K 2 CO 3 .
Phosphate buffers (PB) with certain pH were prepared by mixing solutions (0.1 M) of NaH 2 PO 4 , Na 2 HPO 4 and Na 3 PO 4 in appropriate ratios.
NMR spectra were obtained with Varian VXR-300 (300 MHz) and Varian Gemini-2000 (400 MHz) instruments in DMSO-d 6 using tetramethylsilane as an internal standard; chemical shifts are given in ppm.
Fluorescence spectra were recorded with Quanta Master 40 spectrofluorimeter (Photon Technology, Canada) in 1×1 cm quartz cuvette. The slit width was 2.0 nm for excitation and emission, point time detection 0.1 s, sample concentration was in the range (0.5-1.5)×10 -6 M. Emission was exited at absorption maximum and excitation was detected at emission maximum. The emission spectra of ionized forms of 7-hydroxycoumarin derivatives in methanol were recorded with excitation at 437 nm. UV-Vis spectra were recorded on UV-2802 spectrophotometer (Unico, USA) in 1 cm quartz cuvette in methanol or PB, with dyes and conjugates concentration in the range 4-35 µM (0.1-1.1 OD units). The stock solutions were prepared in methanol or DMSO. The fluorescence quantum yields of compounds (Φ) were determined by common procedure using Coumarin-1 (Em max 445 nm) and Coumarin-314 (Em max 480 nm) as standards; their Φ values in EtOH are 0.50 and 0.58, respectively [38].
The pK a values for 7-hydroxyxoumarin conjugates were obtained from Henderson-Hasselbach equation [39]: where pH i is pH of a given buffer, A i -absorbance of buffer with pH i , A A --absorbance of the phenolate anion form of conjugate (pH above 9.5), A HA -absorbance of conjugate's acid forms (pH below 5.0) at certain wavelength (in this case, 440 nm). UV-Vis spectra were recorded at various pH, then the plots of log((A i -A pH4.7 )/(A pH10 -A i )) vs. pH were built and the pK a values were calculated.

General protocol for the synthesis of deoxycytidine conjugates
Reagent 1a, 1b, 2a or 2b (dried in vacuum over Р 2 О 5 ) and anhydrous HOBT were dissolved in dry DMF (dye concentration ~0.1 M), and DCC was added. After 4-hour activation, dry triethylamine and 2'-deoxycytidine hydrochloride were added, and the mixture was stirred at room temperature for a day. Molar ratio between dC×HCl, NEt 3 , dye, HOBT and DCC was 1.0 : 1.0 : 1.2 : 1.45 : 1.38. When the coupling was complete (TLC control), the mixture was evaporated and dried in vacuum over Р 2 О 5 . The product was purified by silica gel column chromatography.

N-acylation of nucleosides
Our previous studies on N-acylation of O-protected nucleosides with active esters [have] indicated that HOBT derivatives were the most efficient reagents, whereas pentafluorophenyl esters were less active [19]. In the present work we have studied the acylation of unprotected deoxycytidine by HOBT esters of carboxy-coumarins 1-2 (1.2 eq.) in DMF at room temperature. Active esters were prepared using DCC as activating reagent (Scheme 1). They were found to be selective for exocyclic NH 2 over less nucleophilic sugar hydroxyls, so the level of O-acylation was low. Smooth coupling reaction allowed obtaining the conjugates 4-5 in good yields (65-72% for methoxycoumarin derivatives 4a,b, 50-62% for 7- hydroxy analogs 5a,b).
We have also tested the acylation of unprotected purine nucleosides by 2b under repor ted condition. In case of adenosine, a major product was formed, but the reaction was very slow (about 20% conversion in a week, by TLC). This is in agreement with the published data [17,19]. In attempted labeling of free guanosine the reaction was also slow and the mixture of products was formed containing O-acylated compounds. Some authors suggest that amino functions of purine nucleosides do not require protection in oligonucleotide synthesis due to their low reactivity [40]. We did not isolate labeled purine nucleosides because of their low yields.

5'-Hydroxyl acylation
O-acylation is usually carried out under basic catalysis with tertiary amine, such as triethylamine, DIPEA, 4-dimethylaminopyridine, etc. As it was mentioned above, carboxyl activation can be performed by phosphonium or uronium coupling reagents to form acyl intermediates able to react with hydroxyl groups. This approach provides high acylation yields (90-100%), but high cost of reagents limits their use. Activation by less expensive carbodiimides in the presence of nucleophilic additives like HOBT [22] is very popular, despite lower yields and slower acylation.
We have studied the interaction of HOBT esters of carboxyalkyl-coumarins with nucleoside hydroxyl. 3'-O-benzoylthymidine was used as a model nucleoside with a single OH group. Dyes were activated with DCC-HOBT system in DMF (Scheme 1). Then nucleoside and DIPEA base were added; 2-fold molar excess of the dye over nucleoside was used.
In addition to sugar hydroxyl, DIPEA can deprotonate also the phenolic OH of 7-hydroxycoumarins. As a result, 5'-O-labeling with coumarin reagents 1a,b was not very efficient (40% conversion in 1.5 days and ~60% in two weeks). The main reason of low yields could be the acylation of phenolic hydroxyl of compounds 1 and/or 7 by active ester upon DIPEA addition, with possible polycondensation. Compounds 7a,b were not isolated due to formation of complex reaction mixture and almost identical R f values of dyes and conjugates complicating their chromatographic separation.
Almost full conversion of nucleoside upon its reaction with active ester of 3b with acetylprotected phenolic OH was achieved in 1.5 days. However, partial loss of rather labile Ac group in the presence of such strong base as DIPEA was observed.
In contrast, coupling reactions between 7-methoxycoumarins 2a,b and dT(Bz) were smooth and efficient. Labeled nucleosides 6a and 6b were isolated in 65 and 70% yield, respectively.
All conjugates were characterized by NMR and LC-MS. Their purity was >95%.
In NMR spectrum of compound 6a the proton signals of methylene group of thiazole-CH 2 -CO fragment appear as a doublet with a large coupling constant (J = 16.8 Hz). Thus CH 2 protons in a short linker between dye and nucleoside are non-equivalent (AB system), perhaps due to restricted rotation around

Spectral properties of conjugates
The spectral characteristics of compounds are presented in Table 1. UV-Vis spectra of conjugates are superpositions of dye and nucleoside absorption bands. The spectra of thymidine conjugates in methanol and phosphate buffer have two maxima in UV region, whereas those of 2'-deoxycytidine have three (Fig. 1,  2). In general, spectral properties of conjugates (shape, position and intensity of long-wavelength absorption and emission bands, quantum yields) are close to those of corresponding free dyes. The absorption (main band), excitation and emission spectra of coumarin fragments in compound 4a-6a, 5b, 6b well coincide with the spectra of free dyes 1-2 and reference dyes 9-10 with blocked COOH group. However, in case of 4b there are spectral differences typical for intramolecular interactions (see below). The excitation of 7-hydroxycoumarin conjugates 4a,b in MeOH at small long-wavelength shoulder above 430 nm results in the emission band shift from 455-458 to 483-484 nm (Fig. 2). This is due to the presence of some amount of ionized forms with different spectral properties. These forms become predominant in basic aqueous medium (pH above 9-9.5); this is common for hydroxycoumarins [41].
Both UV-Vis and fluorescent spectra of hydroxycoumarins are pH-dependent, in contrast to methoxy-analogs ( Fig. 3-5). The shapes and positions of the main absorption bands of 4a and 1a are very close at pH 6.3 and 10. However, the main band of 4b is redshifted for 8 nm in comparison with 1b (Fig. 3) that can be due to the interaction of cytosine and dye fragments in 4b.
In phosphate buffer (pH 6.3 and 10) excitation and emission spectra of conjugate 4a are close to corresponding spectra of 1a. Excitation spectrum of 4a coincides with its UV-Vis spectrum. Its quantum yield is rather close to that of 1a at pH 6.3, but it is two times lower at basic pH (Table 1). A strong decrease of the quantum yield of 4b in comparison with 1b is observed (about 2-and 6-fold at pH 6.3 and 10, Table 1). The excitation spectrum of 4b at pH 10 is blue-shifted for ~8 nm relative to its absorption band (Fig. 4, top). This spectrum, however, is identical with the excitation spectrum of the dye 1b, for which the excitation and absorption spectra are identical. The emission bands of the conjugate and 1b coincide (Fig. 5, bottom). These effects could be explained by the existence of 4b in two main conformational forms: open and "contact pair" between coumarin and cytosine moieties. In the open form 4b behaves similarly to free dye or 4a containing shorter dye-nucleoside linker, whereas in the "contact pair" the fluorescence is quenched.

pK a determination
To determine the basicity of hydroxycoumarin conjugates we have used a Henderson-Hasselbach method [39]. In the titration experiments UV-Vis spectra were recorded in a broad range of pH (Fig. 5). From these data, the pK a values for 4a and 4b were obtained (7.3 and 7.2, respectively).
Thus, we have synthesized a series of dyenucleoside conjugates with intense blue fluo-   rescence. Direct coupling of thiazolylcoumarin reagents to NH 2 -group of 2'-deoxycytidine and 5'-hydroxyl of 3'-benzoylthymidine was performed under mild conditions. Active esters react selectively with dC amino group, whereas the acylation of OH group requires the presence of a base.