OF THE FLUORESCEIN REAGENT FOR SOLID-PHASE OLIGONUCLEOTIDE 5 '-LABELLING AND ITS USE FOR THE SYNTHESIS OF FLUORESCENTLY LABELLED PCR PRIMERS FOR HIV-1 DETECTION

Introduction. The progress in our understanding of gene structure and function has depended upon methods for detecting small amounts of nuc­ leic acids. The screening, isolation, structural and functional analysis of genes all involve the use and detection of labelled oligonucleotides. Ra­ dioactive labels are still very popular because they are easy to handle and provide a great sensitivity of detection. But an increasing number of highly sensitive non-radioactive bioanalytical systems are being developed to reduce or eliminate the problems associated with radioactivity [1, 2]. Non-isotopic reporter groups, in contrast to isotopic labels, are usually stable and have safety advantages. Fluorescent labelling is now widely used since fluorescent dyes are detected directly and at high sensitivity [3]. The latter is adequate for most applications in the nucleic acids stu­ dies except those which require ultimate sensitivity [1]. Oligonucleotides carrying fluorescent reporter groups are used as hybridization probes for the detection of specific nucleic acids, including diagnostic procedures in medicine [1—5], as primers for automated sequencing [6], in fluores­ cence microscopy [7], etc. We have recently started the studies of the fluorescent DNA labelling, particularly for AIDS detection. Polymerase chain reaction (PCR) is now used as diagnostic procedure in screening for HIV sequences [4]. It would be convenient for clinical laboratories to detect amplified fragments using a fluorescently labelled primers or probes. Here we describe the preparation of the reagent for solid-phase oligonucleotide labelling with fluorescein and its use for the synthesis of labelled primers for HIV-1 detection. Materials and Methods. 4,4-Dimethoxytrityl chloride (DMTrCl) and 1,2,4-trtazole (Tri) were purchased from Fluka (Switzerland), acrylamide and N,N'-methylene bisacrylamide for electrophoresis from Reanal (Hun­ gary). Another reagents and solvents were of home production. Solvents were dried according to [8]. Silicagel 60 F254 plates (Merck, Germany) were used for TLC analysis in the following solvent systems: CHC13СНзОН 9 : 1 (A), CHCI3 (B), iso-PrOH-conc. NH3-H20 7 : 2 : 1 (C). Trimethylsilyl silica (TMS-silica) was prepared as described [9]. Reversephase HPLC was performed on Pharmacia FPLC System (Sweden) using HR 5/2 column in the gradient of CH3CN in 0,1 M triethylammonium ace­ tate (pH 7.5) with elution rate 0,5 ml/min. Absorbance spectra were re­ corded on Specord UV-VIS spectrometer (Karl Zeiss Jena, Germany), fluorescence spectra were obtained on Hitachi MPF-4 spectrofluorimeter (Japan). Victoria-6M gene synthesizer (Novosibirsk, Russia) was used

for oligonucleotide synthesis by standard H-phosphonate method [10] except that couplings were performed in acetonitrile-pyridine (4:1) mix ture.Nucleoside H-phosphonates were prepared as described [10].Our own polymer support based on Silochrom-2 silica [11] was used for the solid-phase synthesis.
Synthesis of fluorescein-labelled oligonucleotides.After the last chain elongation steps the oligonucleotide bound to the polymer support was detritylated.The solution of (4a) (0,04 M in acetonitrile-pyridine 4 : 1, 200 [i\) and PivCl (0,2 M in the same solvent, 200 \xl) were added to the polymer simultaneously.After 4-5 min the support was washed with py ridine and after standard oxidation (2 % iodine in pyridine-water 98 : 2, 15-20 min) it was treated with cone.NH 3 at 50 °С overnight (or 3 days room temperature).Fluorescein-labelled oligonucleotides were isolated by standard polyacrylamide gel electrophoresis and desalted by gel-filtration on PD-10 cartridge (Pharmacia).
Discussion.Fluorescent dyes as well as other reporter molecules can be covalently linked to oligonucleotides by a wide variety of methods (see comprehensive reviews [12][13][14] for detailed discussion).Generally, oli gonucleotide labelling can be performed at both termini or internally du ring chemical synthesis or by the post-synthetic procedures.The post-syn thetic functionalization of deprotected oligonucleotide derivatives contai ning aminoalkyl or mercaptoalkyl linkers is more popular, and many reagents for this purpose are commercially available.But complex reac tion mixtures are usually formed in this case containing starting oligo nucleotide together with labelled product which should be purified by HPLC; moreover, removal of the excess dye can also be a serious problem [15].As for us, another general approach, namely direct labelling, is more attractive since it avoids laborious post-synthetic functionalization.In this approach, a suitably protected chemical moieties are used which can be coupled at the 5'-terminus of protected oligonucleotide directly during the solid-phase synthesis in a similar manner to the internucleotide con densation reaction.It is possible when the reporter molecule to be linked can be converted into phosphoramidite (H-phosphonate, phosphodiester) derivati\e and withstands the coupling and deprotection steps.There are several examples of the use of phosphorylating derivatives of fluorescent dyes and other reporter groups of the direct labelling [12][13][14].
As is generally known, the fluorescence intensity is proportional to extinction coefficient (є) and quantum yield of fluorescence (q) of the substance being irradiated.Thus, the detection limit of fluorescent label depends mainly on e-q value.We have choosen a well-known fluorescein as fluorescent marker since it has both intense absorbance (8496 = 75 000) and high quantum yield (q 0,6-0,9) and can be therefore detected in lo wer concentration that most of common fluorophores [16].Fluoresceinlabelled primers are used in automated DNA sequencing where a sensi tivity level was achieved (3-10~] S M per band) comparable to that for isotopic labels Oligonucleotides are usually labelled with fluorescein by post-synthe tic approach using fluoresceinisothiocyanate (FITC) or iodoacetamidofluorescein via amino-or mercaptoalkyl groups, respectively [6,[12][13][14][15][16][17].We elaborated a preparative synthesis of the fluorescein H-phosphonate deri vatives (4) for oligonucleotide 5'-labelling during solid phase synthesis.A similar fluorescein phosphitamide reagent was proposed previously by F. Schubert et al. in short communication [18], but experimental details are still unavailable, as far as we know.In present article we discribe a full synthetic protocol for the preparation of the reagent (4a).It was synthesized as illustrated in Scheme 1.

CF3COOH
Fluorescein methyl ester (1) was first prepared by the esterification of fluorescein with methanol in the presence of H 2 S0 4 .Its phenolic hydroxy! was then alkylated by modified Claisen method to introduce a linker group.The alkylation with l-(4,4 / -dimethoxytrityloxy)-4-chlorobuLane was performed in DMF in the presence of potassium carbonate and ca talyzed by iodide.O-Protected 4-chlorobutanol-l was easily prepared by tritylation of alcohol in pyridine.Intermediate (2a) was detritylated and the resulting 6-(4-hydroxybutyl) fluorescein methyl ester (3a) was phosphonylated with tris(l,2,4-triazolyl)phosphine according to [10].The lat ter reaction was performed in acetonitrile-pyridine mixture due to low solubility of (3a) in acetonitrile.H-phosphonate (4a) was isolated by si lica gel chromatography.Compounds (l)-( 4) were characterized by absorbance and fluorescence spectroscopy and elemental analysis.
Reagent (4b) containing an ethylene linker was also synthesized by the way described starting from ethylene chlorohydrin (data not presented; see below).
Reagent (4) can be used as P-component for the coupling to oligo nucleotide or nucleoside on solid phase or in solution in H-phosphonate coupling reaction, according to general Scheme 2. As in H-phosphonate method of oligonucleotide synthesis, the coupling is performed via activa tion of the phosphonate building block by condensing reagent (PivCl) and then H phosphonate diesters are converted to the native phosphodiesters by aqueous iodine oxidation: .

/I -nucleoside ОҐ oligonucleotide residue
To check the reagent (4a) 5'-fluorescein-labelled nucleoside (5a, R^= -thymidine) was first synthesized successfully in solution according to this scheme.Fluorescein H-phosphonate (4a) was coupled with a slight excess of З'-O-acetylthymidine in pyridine in the presence of PivCl as con-•densing reagent.TLC analysis showed that starting (4a) was disappea red immediately and H-phosphonate diester was formed quantitatively.The latter was oxidized with aqueous iodine without isolation, and after the deblocking the fluorescein-thymidine conjugate was purified by rever se-phase chromatography on TMS-silica and its properties were studied, The absorbance spectra of fluorescein intermediates (1) -( 4) are al most identical showing two bands in the visible region at 459-460 and 487-489 nm.Fluorescein has a similar spectrum too.S'-Fluorescein-labelled thymidine (5a) shows slightly different pattern of spectrum which is Fig. 1.The absorbance spectra of intermediate (3a) (/) and conjugate (5a, R = T) (//) in the visible region Fig. 2. Reverse-phase HPLC of unlabelled (/) and 5'-fluorescein-labelled (2) primer SK 68 after purification shifted to the short-wave region for 5-6 nm (Fig. 1).This difference is probably due to the conversion of methyl ester function present in ( 1) -( 4) into amide group during the ammonolysis of fluorescein conjugate.The limit of detection of S'-fluorescein-labelled thymidine by the na ked eye was determined as approximately 1-5 pmol by spotting various quantities of labelled nucleoside on filter paper and employing a light of the Chromatoscop lamp.Required sensitivity level usually depends on the ammount of nucleic acid sample to be studied.The PCR with its ability to amplify DNA exponentially would permit the use of even relatively insensitive non-radioactive labels [1,4].So, this detection limit should be adequate for use of fluorescein-labelled primers or probes in PCR.Of course, one could increase the sensitivity considerably using a suitable equipment.
Fluorescein H-phosphonate reagent (4a) was used as a building block for solid phase oligonucleotide synthesis at the last step.We have synthe sized two fluorescein-labelled primers for the amplification of env region of HIV-1 genome.These primers are known as SK 68 and SK 69 [19]: SK 68 5'AGCAGCAGGAAGCACTATGG 3' SK 69 5'CCAGACTGTGAGTTGCAACAG 3' Oligonucleotides were synthesized by H-phosphonate method using Victoria 6M gene synthesizer with average coupling yields 98-98,5 % per step.After the chain elongation was complete the fluorescein reagent (4a) was coupled to the 5'-hydroxy group of oligonucleotide.The coupling was carried out manually under standard conditions, as for nucleoside H-phosphonates, except that coupling time was prolonged (4-5 min).The coupling yields at this step were almost quantitative.After oxidation and deprotection the desired S'-fluorescein-labelled oligonucleotides were purified by polyacrylamide gel electrophoresis where the labelled primers were visible as bands with greenish fluorescence.The products were eluted and desalted by gel-filtration.The purity of the labelled oligonucleo tides was checked by reverse-phase HPLC (Fig. 2).
The UV VIS spectra of labelled primers showed two maxima at 260 and 454 nm.The contribution to the absorbance at 260 nm is due mostly to the oligonucleotide part of conjugate whereas the absorbance at 454 nm is due exclusively to dye moiety.
The biological part of work (the use of labelled PCR primers for HIV detection) is now in progress, as well as more detailed spectrosco pic studies.
We tried to use the fluorescein derivative (4b) for oligonucleotide la belling too, but it showed poor results.We could give the following ex planation.since fluorescein is electron withdrawing group, the conjugates (5b) containing an ethyl linker are probably susceptible to p-elimination and would therefore be cleaved at the ammonolysis step.This type of decomposition is however impossible in the conjugates (5a).
So, the use of the fluorescein reagent (4a) permits the efficient intro duction of the fluorescent label into oligonucleotides and nucleosides on solid-phase as well as in solution.This reagent can be used in solidphase oligonucleotide synthesis without changing current synthesis, de blocking and purification procedures, thus significantly simplifying the preparation of fluorescein-labelled oligonucleotides.It would be useful also for the fluorescent labelling of another classes of biologically active compounds.
This research was supportet by the Presidentis National anti-AIDS Commitee.