Synthesis and use of disulfide-based H-phosphonate reagent for 3 '-and / or 5 '-oligonucleotide labelling via mercaptoalkyl linker

Synthesis of new disulfide-based H-phosphonate reagent for solid-phase oligonucleotide functionalization with mercaptopropyl group at one or two termini is described. Using this single reagent, 3'and 3', 5'-disulfide-linked oligonucleotides were synthesized', disulfide bonds cleaved with dithiothreitol and generated thiol groups labelled with iodoacetamidofluorescein. Tis oligonucleotides containing one or two fluorescein residues at 3'and 5'-ends were prepared in high yields.


Introduction. The need for methods and reagents to modify oligonucleotides has increased in an extra
ordinary rate due to new applications of oligo nucleotide conjugates as potent research tools, diag nostic and therapeutic agents [1][2][3][4][5].Oligonucleo tides labelled with reporter groups, e. g. fluorescent dyes, are used in non-isotopic detection of nucleic acids, automated DNA sequencing, studies on nucleic acid structure and dynamics.A number of molecules, including intercalators, artificial nucleases, lipophilic carriers, peptides etc, has been covalently linked to oligonucleotides to improve their antisense activity by increasing cellular uptake, nuclease resistance or binding affinity.
A wide variety of methods for the preparation of oligonucleotide conjugates has been discussed in de tail in comprehensive reviews and books [2, 6-9 ].In principle, conjugation is based on the introduction of appropriate functional group into oligonucleotide, fol lowed by its specific coupling with another molecule.Chemical approach to the preparation of labelled DNA involves incorporation of modified nucleotide or non-nucleotide reagent during oligonucleotide syn thesis; once incorporated, these synthons are functionalized post-synthetically, although direct intro duction of some reporter molecules during solid-phase synthesis is also possible.The most common labelling methods use aminoalkyl linker groups.Another po pular approach is to employ the highly reactive thiol function introduced as mercaptoalkyl linker able to react with thiol-specific groups.
Certainly, the latter strategy seems to be more promising and flexible, as thiols may be used in greater variety of reactions than amines.The thiol is a group capable of selective derivatization in the presence of oligonucleotide functional groups, i. e. amines, phosphates and hydroxyls.Two types of thiol modification are available, to generate stable thiol ether linkage or easily cleavable disulfide bond.S-alkylation reaction, that is coupling of highly nucleophilic sulfhydryl group with molecules bearing electrophilic thiol-specific functions, yields oligonuc leotide conjugated via thioether bond.Iodo(bromo)acetamides, aziridinyl sulfonamides, y-bromo-a, /З-unsaturated carbonyls, maleimides and less reactive acrylates are the functionalities observed to selectively react with aliphatic mercapto group [10].
Besides that, thiol group can also form disulfides with other thiols, usually after activation with 2,2'dipyridyl disulfide or related reagent [11][12][13][14].This approach is especially important for oligonucleotide coupling with peptides and proteins under mild con ditions, although conjugation through thioether lin kage was also used for this purpose [13][14][15][16][17][18].An important feature of disulfide formation is that it is readily reversed by treatment with mercaptans like dithiothreitol.SH-oligonucleotides were reversibly bo und to thiol-modified solid supports [12,19], or to hydrophobic transport agents like thiocholesterol to be cleaved within the cell by cellular reducing agents releasing a free oligonucleotide [20].S-S bond clea vage is widely used to generate thiol linkers from disulfide precursors [11,13,[21][22][23].It should be noted that disulfide formation is a common problem on storage of thiol oligonucleotides.On the other hand, circular and looped oligonucleotides were pre pared by oxidative disulfide bond formation between mercaptoalkyl tether groups [23,24].An additional interesting property of mercapto group is its ability to bond readily to some metals, and thiol-modified oligonucleotides have been immobilized on metal electrode surfaces for use in hybridization assays, biosensors, etc. ( [25] and references therein).
Thus, the unique chemistry of sulfhydryl group makes thiol oligonucleotides very attractive for diverse applications, and at the same time the development of reagents and methods for their preparation and conjugation is still an important problem.Here we describe the synthesis and use of H-phosphonate reagent for solid-phase oligonucleotide functionali zation with aliphatic thiol groups at both 3'-and/or 5'-termini.Reagents for gel electrophoresis were from «Sigma» (USA).Solvents were dried as follows: pyridine was distilled over ninhydrin and then refluxed over cal cium hydride for 2 h and distilled; acetonitrile was distilled over phosphorus pentoxide and calcium hyd ride.TLC was performed on Kieselgel 60F 254 plates («Мегск») using the following solvent systems: chlo roform (A), chloroform-methanol 9:1 (B) and isopropanol -cone NH 3 -water 7:2:1 (C).To detect nucleoside derivatives, the plates were sprayed with a mixture 95 % EtOH -acetic acid -anisaldehydecone H 2 S0 4 9:0.1:0.5:0.5 (v/v) and heated at 110 °С.Nucleosides are revealed as blue to black spots [26].SH-containing compounds were detected by spraying TLC plates with 0.5 % ethanol solution of 5,5'dithiobis(2-nitrobenzoic acid) («Sigma») as yellow spots [27 ].Thiols were also detected by exposing TLC plates to iodine vapors (white spots on dark background), although this reagent is less specific producing similar results with all compounds that could be oxidized by iodine, e. g. dialkyl sulfides.
3,3'-D ithiodipropanol (3) was synthesized by adapting procedure described for the preparation of diethanoldisulfide [32].To 2.20 g of 3-mercaptopropanol-l (2, 2.5 mmol) 0.5 eq. of 30 % hydrogen peroxide was added dropwise with stirring and ice-cooling.The solution was allowed to stand overnight at room temperature until thiol had com pletely disappeared (test with thiol reagent).Water was removed under reduced pressure.2.17 g of colourless oil was obtained (quantitative yield).Purity of ( 3 [17,36,38 ] or H-phosphonates [37,38 ].These reagents can be introduced into oligonucleotides at the last coupling step of solid-phase synthesis, and then S-trityl group is removed by AgN0 3 /DTT.This approach has been reported to work well with rela tively short sequences (up to 12-mers), but yields decreased significantly for longer oligomers, espe cially with the use of amidite reagents [17,37].Low yields of thiol oligonucleotides were probably resulted from the chemical modification of 5'-thiol terminus during deprotection.So, there are several methods for oligonucleotide 5'-or З'-end functionalization with thiol linker groups, but these approaches are usually not highly efficient, and moreover they are not applicable for the synthesis of oligonucleotides with thiols at both termini.
Our intention was to achieve a simple technique for obtaining 3'-and/or 5'-thiol labelled oligonuc leotides using single reagent.As many methods for 3'-and 5'-labelling described above briefly are based on the cleavage of disulfide precursors, it was clear that properly designed disulfide-containing functionaiizing reagent could be able to label any end of oligonucleotide.Cystamine has been used for 3'-and 5'-labelling, however we would like to create a reagent for direct functionalization during solid-phase syn thesis.We decided that such a reagent has to be bifunctional, like cystamine, but with hydroxy groups instead of amines, containing one DMTr-protected hydroxyl to allow for chain elongation and one phosphorylating moiety for reagent introduction as P-component during oligonucleotide synthesis; these two units should be connected with cleavable disulfide linker.Dialcohol disulfides were an obvious сіюісе for our purpose, and reagent resulted from tritylation and phosphorylation of two hydroxy groups linked via S-S bond would be introduced at 3'-or 5'-end of oligo nucleotide to obtain 3'-or 5'-thiol derivatives after internal disulfide cleavage.Reagent under design was based on the same principle as oligonucleotide phosphorylating agent derived from the symmetric sulfodiethanol, with one hydroxyl being protected with DMTr group and another phosphorylated; it can be added at 3'-or 5'-end of oligonucleotide, and during ammonia deprotection central sulfonyl linkage is /?eliminated to produce 3'-or 5'~phosphate (or both) [391.
Our first attempts to prepare oligonucleotide functionalizing reagent based on the easily available diethanoldisulfide failed: when O-monotritylated in termediate was phosphorylated with tris(triazo-lyDphosphinc, starting material disappeared, but sig nificant cleavage of the product (R f ca.0.05 in system B) was observed during its isolation with formation of two compounds able to be oxidized with iodine, probably the result of S-S bond cleavage or another redox process.This reaction was not further inves tigated, as it was found in the literature that 2mercaptoethyl phosphates are anyway unstable under basic conditions cleaving ethylmercapto group to yield free phosphates.Nevertheless, it was demonstrated that mercaptopropyl and -hexyl phosphates were completely stable [21,36].
As a result, we have prepared thiol-introducing H-phosphonate reagent (5) based on 3,3'-dithiodipropanol (dipropanoldisulfide).Our first report on this reagent was presented at the conference in 1993 [40 ], however its synthesis and use still has not been described in detail.Recently, a similar phosphoramidite reagent based on 6,6-dithiodihexanol has been used to prepare circular oligonucleotides by oxidizing 5'-, З'-terminal thiol groups [24]; to our best knowledge, its synthesis was not yet reported too.
The preparation of the reagent (5) (Scheme 1) started from the synthesis of 3,3'-dithiodipropanol (3).S-acetylmercaptopropanol (1) was obtained by the addition of thiolacetic acid to allyl alcohol in the presence of benzoyl peroxide, and deacylated with 10 % NaOH to give 3-mercaptopropanol (2), ac cording to [31].Its subsequent oxidation with cal culated amount of hydrogen peroxide resulted in quantitative yield of the desired dipropanoldisulfide (3).The latter was tritylated with DMTrCl in pyri dine (yield 71 %); this method was found to be more efficient than previously described tritylation in the presence of DMAP and TEA (yield 48 %) [14].Monotritylated 3,3'-dithiodipropanol (4) has been previously attached to solid support via succinate linkage for the preparation of 3'-thiol oligonucleotides solely [14,23].In our study, this intermediate was phosphitylated with PTri 3 to produce H-phosphonate reagent (5) for solid-phase synthesis.No product cleavage was observed in this case, as was with diethanoldisulfide derivative.As 31 P NMR demon strated, reagent was completely stable: no additional signals were observed in the spectrum after one year storage at -18 °С.So, possible in theory selfoxidation of monoester P-H bond of reagent ( 5) by its S-S group didnot occur.Possible as well sulfurization of diester P-H bonds (including intra molecular reaction of З'-linked disulfide) during so lid-phase synthesis was not specially studied; how ever, this process seems to be unlikely to proceed under normal synthetic conditions, as dialkyl disul fides are not active sulfurizing agents since their S-S bond is not activated by electron-acceptor substituents.This type of side reaction has never been reported for phosphoramidite oligonucleotide synthe sis on disulfide-containing supports, although P(III) species are much more succeptible to sulfurization with disulfides than tetracoordinated H-phosphonates.Clear HPLC profiles of reaction mixtures from the syntheses of thiol oligoucieotides and their con jugates could probably confirm this conclusion to a certain degree (see below).
To test the efficasy of H-phosphonate reagent for thiol group introduction into oligonucleotides, model labelling of nucleoside (З'-O-acetylthymidine) was first carried out in the solution, according to Scheme 2, a. Nucleoside was phosphitylated with (5) using pivaloyl chloride as condensing reagent, and hydrophosphoryl group of coupling product was oxidized smoothly with iodine in aqueous pyridine to give phosphodiester containing disulfide linkage.The lat ter was cleaved by DTT in the presence of base (TEA) to result in the formation of nucleoside functionalized with mercapto group (6, J? = -dT(Ac)).This product was not however specially isolated and studied since model reaction was performed just to demonstrate that nucleoside could be converted to thiol-containing product by coupling with H-phos phonate reagent (5) followed by iodine oxidation and DTT treatment.
For oligonucleotide functionalization, proposed reagent can be added as P-component at the first and/or last coupling cycle of solid-phase synthesis, according to normal H-phosphonate synthetic protocol (Scheme 2).5'-Thiol oligonucleotides (6) are prepa red through the addition of reagent (5) at last H-phosphonate coupling step after the assembly of Scheme 1 desired oligonucleotide chain was completed.To pre pare З'-modified oligonucleotides (7), H-phosphonate reagent has to be introduced at the first addition to any nucleoside support, followed by the normal synthesis of target sequence.Post-synthetic reducing with DTT cleaves S-S bond leaving a thiol group at 5'-or 3-terminus.In such a way, oligonucleotides labelled at both 3'-and 5'-ends could be also easily prepared.
We have used H-phosphonate (5) to synthesize a 15-mer oligothymidylate containing one or two thiol groups at 3'-and 5'-termini on DNA synthesizer by conventional H-phosphonate approach.Coupling time for (5) was increased to 5 min, as is usually recommended for functionalizing reagents containing long alkyl chains.In this case, the yields were 92 and 95 % for the first and last coupling step, respectively, as determined by DMTr cation release, whereas common nucleoside H-phosphonates were coupled for 2 min with average yield about 98 %.After final oxidation with aqueous iodine, oligonucleotides were cleaved from the polymer support with concentrated NH 4 OH.Ammonolysis was carried out overnight at room temperature, as only thymidines were present in the sequence; otherwise common deblocking (e. g. at 55 °С for at least 6 h) should be performed.Disulfide bond cleavage could be performed simultaneously, by adding DTT to ammonia solution [19].In our case, S-S bonds were kept during oligonucleotide puri fication to avoid the oxidative dimerization of SHfunctionalized oligonucleotides [11,12,36].5-0-DMTr protecting groups were not removed to simplify the isolation of product.As HPLC analysis of crude reaction mixtures showed good chromatographic pat terns (Fig. 1, я), no special attempts were made to purify oligomers by gel electrophoresis or HPLC.DMTr-and disulfide-containing oligonucleotides were purified using Oligo-Pak cartridges.Purification is based there on the principle of reverse phase chroma tography: the desired oligomer with hydrophobic trityl group is retained by the support while the failure sequences without DMTr residue, as well as deprotection by-products, are removed from the reaction mixture by washing to deliver an oligomer of good purity sufficient for most applications.Detritylation of oligonucleotides was performed directly on the Oligo-Pak support after removing side products.All oligo nucleotides were of good quality after Oligo-Pak isolation, and no additional purification was carried out.Then 3 -and 5'-disulfide linkages in purified oligonucleotides were quantitatively cleaved with DTT to produce the corresponding thiol derivatives.The latter should be stored (if necessary) in the presence of a little amount of DTT (ca.5-10 mM) to avoid dimerization.DTT can be removed before conjugation by butanol or ethylacetate extraction.In principle, it is not essential to purify 5-thiol oligonucleotides at this step, as only SH-containing oligomer will react with labelling reagent and conjugation product could be easily purified.But in the synthesis of 3'-thiol oligonucleotides failure sequences also contain 3'-SH groups, and purification of starting oligomer would be desirable.Nevertheless, our thiol oligomers were of good purity, as HPLC showed, and additional puri fication seemed to be unnecessary.On reverse phase HPLC, thiol oligonucleotides had somewhat higher retention time than non-modified T I5 , but lower than corresponding disulfide precursors (Fig. 1, b).
To demonstrate the utility of our approach for oligonucleotide labelling, we have synthesized T I5 oligonucleotides bearing one or two fluorescein resi dues at 3 -and both 3'-and 5'-ends for the studies on fluorescence polarization.The coupling step invol ves reaction of oligonucleotide bearing a nucleophilic thiol linker(s) with dye reagent containing electrophilic thiol-specific iodoacetamide group (Scheme 3).
Labelling reactions were performed in sodium carbonate/bicarbonate buffer (pH 9) containing ca. 30 % DMF (where IAF was dissolved).Labelling was ) was confirmed by *H NMR: б (ppm) 3mixture was kept at room temperature for additional hour, poured into 100 ml of 5 % NaHC0 3 and extracted with chloroform (3 * 30 ml).Combined organic layer was washed with 0.25 M TEAB (pH 7.5, 50 ml) and water, dried over Na 2 S0 4 and evaporated.After coevaporation with toluene to remove remaining pyridine, product was isolated by silica gel chromatography in the gradient 40-80 % CHCI3 in hexane to give a light yellow oil after evaporation.Yield 1.20 g (2.48 mmol, 71 % based on DMTrCl).R f 0.10 (system A); 0.68 (B).*H NMR: б (procedure for nucleoside phosphitylation [28 | was used with some modifications.1,2,4-TriazoIe (1.86 g, 27 mmol) was evaporated twice with anhydrous acetonitrile and suspended in the same solvent (15 ml).N-methylmorpholine (5.5 ml, 50 mmol) and then phosphorus trichloride (718 /Л, 8.25 mmol) were added with cooling (5-10 °С), The mixture was stirred for 30 min at this temperature.Then solution of monotritylated dithiodipropanol (4, 0.80 g, L65 mmol in 10 ml of dry CH 3 CN) was added dropwise over 20 min with cooling, and the reaction mixture was stirred for further 20 min at room temperature.TLC (system B) showed almost complete conversion of starting mate rial into product with low mobility.The mixture was poured into 100 ml of 0.5 M TEAB (pH 7.5) and 10 min later extracted with chloroform (3x50 ml).Organic extract was washed with 0.25 M TEAB (2 x x 50 ml) and water (50 ml), dried over sodium sulfate and evaporated.Product (5) was isolated by flash silica gel chromatography.Column was washed with 5 % methanol in chloroform and then the desired H-phosphonate was eluted with 7-8 % MeOH in chloroform containing 1 % triethylamine.Eluate was washed with 0.25 M TEAB and water, dried over Na 2 S0 4 and evaporated.Product was dried with oil pump.0.72 g of viscous pale yellow oil was obtained (67 %).R f 0.05 (system B). l H NMR: б (mmol) were evaporated twice with anhydrous pyridine, dissolved in the same solvent (600 jul) and treated with pivaloyl chloride (24/Л, 0.2 mmol).3 min later TLC (system B) showed complete conversion of starting H-phospho nate (R f 0.05) into trityl-, nucleoside-containing coup ling product (R f 0.42) well separated from the slight excess of starting nucleoside (R f 0.33).Iodine (25 mg, 0.1 mmol) in 400 //1 of pyridine-water (20:1) was added, and 10 min later the complete oxidation of H-phosphonate diester into phosphodiester (trityl-, nucleoside-positive, R f 0) was observed.The mixture was diluted with 0.25 M TEAB (1 ml) and aqueous solution of Na 2 S0 3 was added dropwise carefully to neutralise excess iodine until its colour disappeared.No disulfide bond cleavage was observed.Further 2 ml of 0.25 M TEAB were added, and the mixture was extracted with chloroform (3x1 ml), extract was washed with 0.25 M TEAB and water (1 mi each) and evaporated.Disulfide-functionaiized dT(Ac) contai ning a little amount of starting nucleoside was dissol ved in 1 ml of CHCI3-TEA (9:1), and 0.25 mmol of DTT was added.In 2 hours TLC showed the complete disappearance of starting trityl-positive ma terial with R f 0, to be converted into 2 new products of S-S bond cleavage: (a) DMTr-negative, nucleosidepositive, SH-positive, Rf 0 (systems A, B); 0.73 (C), and (b) DMTr-positive, nucleoside-negative, SH-po sitive, R f 0.75 (A); 1.0 (B).Compound (b) was formed also as the only product upon treatment of 1,1 '-0-bis-dimethoxytrityl-3,3'-dithiodipropanol (see above) with DTT, it was trityl-positive product of l-0-dimethoxytrityl-3,3'-dithiodipropanol (4) cleava ge with DTT, and, finally, the same compound was formed upon deacylation of AcSCH 2 CH 2 CH 2 ODMTr with KOH/EtOH.Therefore, compound (b) could be considered as HSCH 2 CH 2 CH 2 ODMTr, whereas com pound (a) with low TLC mobility could be 5'mercaptopropylphosphate nucleoside derivative (6, R --dT(Ac)).Synthesis of oligonucleotide conjugates.Intro duction of disulfide reagent in to oligonucleotides.The solution of disulfide H-phosphonate reagent (5) was attached to the spare port on the synthesizer.For thiol intro duction at the 3'-terminus, reagent was directly coupled to thymidine-derivatized solid support (CPG) with coupling time extended to 5 min (coupling yield 92 %).Standard H-phosphonate synthesis of oligo-T, 5 chain was then performed, with average coupling yield 98.1 %.After sequence elongation was comp leted, the support containing З'-disulfide-linked T 15 was divided into 2 equal parts.The first one was used for the preparation of 3'-thiol oligomer, whereas from the other half of polymer 5',3'-dithiol derivative was obtained by repeating coupling with reagent (5) (coupling time 5 min, yield 95 %, as determined by detritylation of small weighed part of polymer).After standard oxidation (2 % iodine in pyridine-water 98:2, 10 min) oligonucleotides were cleaved from the support (concentrated ammonia, room temperature overnight).5'-DMTr-containing sequences were puri fied by Oligo-Pak cartridge (Milligen/Biosearch, USA) following manufacturer's protocol.Oligonuc leotides were detritylated directly on the cartridge, eluted and evaporated.(Note that at this step both oligomers contained additional thymidine residue lin ked at З'-end through disulfide tether).Normal T, 5 oligonucleotide was also synthesized by standard Hphosphonate method to be used as control.Disulfide bond cleavage.Disulfide-containing oligonucleotides were quantita tively reduced before labelling by treatment with 30 mM DTT (0.5 mg per 1 OD 260 of oligonucleotide) in 10 mM Tris-HCl buffer (pH 8.0) overnight at 37 °С under argon.Cleavage was monitored by HPLC.Most of the DTT was removed by extraction with butanol, and the reduced thiol oligonucleotides were desalted at PD-10 cartridge («Pharmacia»).Oligonucleotide labelling with fluorescein.5 OD 260 of З'-thiol or 3',5'-dithiol oligonucleotide (0.04 mmol) were dissol ved in 200 ml of 0.1 M sodium carbonate/bicarbonate buffer (pH 9.0).20 (for З'-thiol oligo-T l5 ) or 30 (for 3',5'-dithiol oligo-T 15 ) eq of 5-(iodoacetamido)fluorescein in 100 /Л of freshly distilled DMF were added with agitation, and the reaction mixture was incubated under argon at room temperature overnight in the dark.Labelling reaction was monitored by reverse phase HPLC.If necessary, a further 5-10 eq of IAF could be added.The reaction mixture was diluted with 500 /Л of water and passed through PD-10 gel filtration column to remove excess label.The desired product was eluted with 50 mM TEAB (pH 7.5) containing 5 % of ethanol, and eluate was evaporated.In the synthesis of З'-labelled T 15 oligo nucleotide, some non-labelled starting oligomer was removed efficiently at Oligo-Pak cartridge following the protocol for DMTr-oligonucleotides isolation, and 3',5'-bis-fluorescein-T 15 was purified by reverse phase HPLC Purity of products was confirmed by PAGE and HPLC 3'-Fluorescein-T I5 : yield 78 %.UV/.A number of methods for the preparation of thiol-modified oligonucleotides has been described [2, 6-9].SH groups can be intro duced into oligonucleotide using suitably protected nucleoside mercapto derivatives, e. g. 5'-mercaptonucleosides for 5'-derivatization [33] or 5-mercaptouridine for site-specific labelling [34].Cystamine is widely employed to prepare thiol oligonucleotides, as it has amino group to react with amine-specific entities, and cleavable disulfide bond generating thiol upon treatment with DTT.Cystamine linkers have been introduced at the internucleotide linkage via H-phosphonate approach [35], 5'-cystaminyl oligo nucleotides have been synthesized to yield 5'-thiol derivatives [13, 16, 23], and З'-thiol oligonucleotides were prepared by cleaving from the special support with cystamine with subsequent S-S bond cleavage [23 ].More common approaches to the introduction of thiols into 3'-or 5'-terminus have been also develo ped.З'-Thiol oligonucleotides are usually synthesized on specially designed disulfide-derivatized solid sup ports [11, 14, 18, 19, 22, 23].Preparation of these supports is rather laborious procedure, although they are now commercially available.5'-Thiol oligonuc leotide functionalization is more simple, and corres ponding reagents are also available.They are deriva tives of S-protected mercaptoalcohols, namely S-trityl mercaptopropanol or -hexanol phosphoramidites