Novel nucleic acid analogs with a chimeric phosphinate / phosphate backbone; synthesis and biophysical properties

Novel analogs of acyclic nucleosides based on a bis-hydroxymethylphosphinic acid (BHPA) backbone were incorporated into a thymidine-containing DNA strand by phosphoramidite methodology. The physicochemical properties of these constructs were evaluated. Melting temperature measurements demonstrate that chimeric oligomers with a phosphinate / phosphate backbone possess lower binding affinity towards complementary single stranded templates and slightly higher binding affinity towards double stranded DNA, as compared to non-modified reference oligomers. The polyanionic oligomers containing BHPA abasic residue were also synthesized by the same methodology. These oligomers show low cytotoxic activity toward HUVEC and HeLa cell lines and, as expected, are resistant to nucleolytic degradation at the modification site


Introduction
DNA analogs with improved stability to nucleolytic degradation and higher binding affinity toward complementary DNA or RNA strands are of interest due to their potential application as therapeutics in antisense 1 or antigene strategies. 2 Several acyclic analogs of DNA, including those derived from isosteric glyceronucleosides, have been synthesized for these purposes up to now. 3,4Despite their improved flexibility those oligomers in most cases form weaker duplexes with complementary DNA or RNA strands, except in the case when increasing flexibility of the carbohydrate portion resulted in triplex stability. 5he aim of reported studies was the design of novel antisense / antigene therapeutics based on the use of a bis-(hydroxymethyl)phosphinic acid (BHPA) unit as a scaffold molecule.We assumed that bis-(hydroxymethyl)phosphinic acid could replace the 3'-, 4'-and 5'-carbons of the sugar moiety and provide a site for attachment of nucleobases via a one-or two-carbon linker.Esterified or amidated BHPA, if successfully incorporated into the oligonucleotide chain, were expected to possess an additional hydrogen bond acceptor site at the phosphinyl oxygen atom.The conformational flexibility and neutral nature of such a unit as in 2 could enhance cellular uptake, while attached nucleobases were expected to interact via stacking and hydrogen bonds with complementary DNA or RNA.Moreover, it was anticipated that even an abasic bis-(hydroxymethyl)phosphinic unit, as in 1, could provide protection of chimeric constructs against nucleolytic degradation.The use of a bis-(hydroxymethyl)phosphinic acid for the synthesis of acyclic nucleoside analogs and their successful incorporation into short DNA oligomers has been disclosed. 6Such acyclic nucleoside analogs were successfully used for the protection of deoxyribozymes, directed towards the HIV-1 viral RNA sequence in in vitro HIV-1 infected cellular experiments, 7 as well as for deoxyribozymes designed to cleave bcr-abl mRNA fragments in in vitro experiments. 8n this paper we report on the synthesis of longer oligomers with a chimeric phosphinate / phosphate backbone and the evaluation of the biophysical properties of such constructs, including their affinity toward RNA and DNA templates, their stability in the presence of 3'-and 5'-exonucleases, and their cytotoxic activity in selected cell lines.

Synthesis of oligomers with a chimeric phosphinate / phosphate backbone Short oligomers
In our previous communication we described conditions for the incorporation of the modified BHPA units 9a-d into short DNA oligomers. 6The phosphoramidite monomers 9 and conventional phosphoramidite monomers were used as substrates for the synthesis of our constructs by automated phosphoramidite methodology. 14The synthesis was performed on an ABI 394 synthesizer (Applied Biosystem Inc., Foster City, CA) using succinyl-either oxalyllinked 15 LCA-CPG solid supports.The only difference in the procedure recommended by manufacturer was a prolonged coupling time of the modified monomers (up to 600 s).The coupling efficiency of 9a-d, as determined by DMT-ion assay, was in the range of 95-97 %.The 5'-terminal DMT group was usually removed before cleavage of the product from the solid support.
While the synthesis of trimers dApYpdC 10 (where Y is a modified BHPA unit as in 2, B = thymin-1-yl or thymin-3-yl, n=2) followed by a standard ammonia deprotection and RP HPLC purification led to the desired amidate derivatives 10c and 10d, this approach did not yield the esterified derivatives 10a and 10b (Table 2, a-d as in Scheme 1).Instead, as revealed by MALDI-TOF analysis, both isolated products in fact constituted the same compound 11, possessing a BHPA unit with depleted alkyl-nucleobase moiety (Y ab , see table 2).In this case hydrolytic deprotection conditions (concentrated aqueous ammonia, 16 h, 55 °C) resulted in cleavage of the ester bond of the phosphinic acid derivatives 10a and 10b, giving rise to anionic DNA analogs with a pseudo-abasic site (as presented in the structure 1).This instability of the BHPA ester bond of 10a and 10b was also observed under less harsh conditions, routinely used for the cleavage of oligomers from the solid support (28 % aq.NH 4 OH, 1 h, 20 °C).Despite the observed stability of the ester bond of 7a and 7b in concentrated aqueous ammonia (2 h at RTdata not shown), we considered that such compounds, as base-labile phosphotriester analogs, 16 might not survive the deprotection conditions during DNA synthesis.This result, albeit expected for those phosphotriester analogs, 17 convinced us that the routine phosphoramidite methodology could not be used for the synthesis of the modified oligomers containing nucleotide units with required protection of nucleobases.However, application of an oxalyl-LCA CPG solid support 15 and methyl-phosphoramidite methodology 14 allowed us to obtain homo-thymidylates TpTpYpYpTpT (12a and 12b). 6Removal of the phosphate protecting methyl groups was achieved by treatment of solid support-bound oligomers with a thiophenol / dioxane / triethylamine mixture (2:1:2, v/v) for 5 min at RT, followed by the release of oligomers from the solid support by treatment with 1 % aqueous triethylamine for 7-10 min at RT. Expected oligomers were also obtained by using an oxalyl-LCA CPG solid support and protection of the phosphate function with a 2-cyanoethyl group.In this case treatment of solid support-bound oligomers with 1 % aqueous triethylamine for 10 min at room temperature resulted in simultaneous release of the oligomer and removal of the 2-cyanoethyl protecting groups.The basic conditions used were sufficient for release of the oligomer from the solid support without hydrolytic cleavage of the P-O bond.Chimeric oligomers 10 and 12 were synthesized as diastereomeric mixtures by virtue of a chiral center at the phosphorus atom of the BHPA moiety.In spite of our efforts neither trimers 10c and 10d nor hexamers 12a and 12b could be separated by HPLC in the form of P-chiral pure diastereomers.

Substrates for the synthesis of abasic DNA analogs
A much simpler synthetic route to the anionic DNA analogs 1 could be used starting from bis-(hydroxymethyl)phosphinic acid methyl ester 4. Dimethoxytritylation of one of OH groups in 4 followed by phosphitylation of the resulting 8e led to phosphoramidite monomer 9e (Scheme 2).Monomer 9e could be used for the synthesis of pseudo-abasic DNA analogs, either those possessing a Y ab group within the oligomer chain, or at the 5'-end of the construct.For introduction of the abasic BHPA unit at the 3'-end of the oligomer derivative 8e was acylated with succinic anhydride in pyridine and then coupled with an aminoalkyl linker of controlled pore glass solid support (LCA CPG).This modified solid support 9f was loaded with the BHPA derivative up to 57.0 µmol /g, as determined by a DMT-cation assay and used for the synthesis of 3'-modified chimeric DNA analogs.

Synthesis of longer chimeric DNA analogs -compounds 13-21
Our preliminary experiments led us to the following conclusions: • the routine phosphoramidite strategy has proved to be successful for the synthesis of the short chimeric oligomers containing BHPA amidate units; • esterified BHPA analogs can be introduced only into homo-thymidylate oligomers exclusively via an oxalyl-linked solid support approach; • anionic abasic analogs of DNA can be obtained from an easily accessible monomer (DMT protected phosphoramidite of BHPA methyl ester) 4.
Therefore, this methodology was used for the synthesis of numerous longer oligomers with phosphinate / phosphate backbone.To study the properties of the abasic DNA analogs 18 containing ionic bis-(hydroxymethyl)phosphinic acid residues as in 1, one or two BHPA units (Y ab ) were introduced into the central domain of the longer homo-thymidylate chain, as in 13 and 14, respectively, or were used for 3'-and 5'-terminal protection of the DNA chain, as in 15 and 16.The polyanionic oligomer dCp(Y ab p) 17 dC 17 and its 5'-fluorescently labeled analog 18 were also synthesized by the same methodology.Compounds 13-17, due to their stability under acidic conditions, were synthesized as 5'-DMT-protected oligomers (DMT-ON) and separated from shorter by-products by reverse phase HPLC. 19 Purity of compounds 13 -18 was checked by 20 % polyacrylamide / 7 M urea gel electrophoresis (PAGE).For compounds 13, 14 and 17 PAGE analysis was done on the 5'-32 Plabeled oligomers.Representative PAGE gels of oligomers 13 and 14 as well as 15 and 16 are show at Figure 2a and b respectively.Nonadecadeoxyadenylate dA 19 and nonadecathymidylate T 19 are used as the reference oligomers.Electrophoretic mobility of oligomers 13 and 14 (Figure 2a) is slightly higher than that of the reference T 19 due to the presence of the Y ab units increasing total negative charge of the chimeric oligomers 13 and 14.The structure of the 5'-fluorescently labeled oligomer 18 was confirmed by MALDI-TOF analysis (Figure 3a) and its purity was checked by PAGE analysis.Band visualisation was achived by means of a Stains all reagent (Figure 3b).For comparison mobility of 5'-32 P-labeled oligomer 17 was checked by analogous PAGE analysis (Figure 3c).At this gel the spot of 5'fluorescently labeled oligomer 18 could be seen in the UV light and was marked at the autoradiogram film as shown.Oligomer 17 exhibits higher mobility at the polyacrylamide gel.This oligomer is contaminated in ca. 10 % of with the product of lower PAGE mobility.This impurity could be neither separated from oligomer 17 by RP HPLC nor identified by MALDI-TOF mass spectrometry.Monomers 9a and 9c, mimicking acyclic DNA nucleosides (N-1-substituted thymine), were used for the synthesis of nonadecathymidylate analogs 19, 20 and 21, containing incorporated one, two or three BHPA units, respectively.

DMT-ON
Oligonucleotides 19a-21a, possessing esterified BHPA units, due to their rather low stability either in basic and acidic conditions, were obtained as fully deprotected oligomers.The yields of these oligomers, after RP HPLC separation, were rather moderate (10-15 optical units from 1 µmol DNA synthesis).
For the synthesis of oligomers 19c-21c containing BHPA amidate units we used a two step deprotection / purification procedure. 19After the synthesis was completed, oligomers 19c-21c were released from the solid support as the 5′-DMT-protected derivatives.Shorter by-product oligomers were separated chromatographically from the desired, DMT-containing hydrophobic oligomers, which then were exposed to acidic conditions (3 % TFA in dichloromethane).These conditions were safe during the automated synthesis of oligomers and were supposed to be suitable for final deprotection of the terminal 5′-OH group of 19c-21c.Unfortunately, all our efforts to obtain the desired fully deprotected oligomers without cleavage of the P-N bond have failed, even if the time of the reaction was as long as required for the removal of the DMT group during the DNA solid phase synthesis.Thus, we concluded that the removal of the 5'-terminal DMT protecting group should be carried out before the release of the oligomer from the solid support, as we proved by the successful synthesis of the trimer dApYpdC 10c. 6No other methods of removal of DMT group as e.g. using ZnBr 2 20 were tested.
For evaluation of biophysical properties oligomers 19a-21a were used as fully deprotected species, and oligomers 19c-21c as 5′-DMT-protected constructs.The structures of all oligomers listed in Table 2 were confirmed by MALDI-TOF mass spectrometry, and their purity was confirmed by HPLC analysis.As determined by analytical RP HPLC the purity of oligomers 19-21 was ca.98-99 %.Representative analytical HPLC profile and MALDI-TOF mass spectrum of oligonucleotide 19a are shown in Figure 4a and 4b, respectively.In the mass spectrum molecular ion with m/z 5754 represents oligomer 19a (MW 5750), while a minute peak at m/z 5602 gives rise from [M-CH 2 CH 2 Thy] + fragmentation ion or decomposition product (Tp) 9 Y ab (pT) 9 .
Oligomers containing a 5'-DMT-protecting group and amidated BHPA units showed remarkable instability in MALDI-TOF experimental conditions due to the presence of the acid labile 5'-DMT protecting group and of the acid-labile amide P-N bond.A representative analytical HPLC profile and MALDI-TOF spectrum of oligonucleotide 19c are shown at Fig. 5a and 5b, respectively.The profile of analytical RP HPLC analysis shows exclusively one peak representing oligomer 19c and the MALDI-TOF mass spectrum confirms the presence of the expected product giving a signal at m/z 6053 (calc.6051).However, due to instability of compound 19c in the conditions used for the MS analysis, oligomer 5'-DMT-(Tp) 9 Y(pT) 9 , where Y is BHPA-NH-CH 2 CH 2 Thy moiety, undergoes an acidic hydrolysis resulting in the appearance the three other products of the following MS signals: at m/z 5901 for product 5'-DMT-(Tp) 9 Y ab (pT) 9 (calc.5900), at m/z 5751 for product (Tp) 9 Y(pT)9 (calc.5749) and at m/z 5599 for product (Tp) 9 Y ab (pT) 9 (calc.5598).Similar MALDI-TOF mass spectra were obtained for  Anionic groups present at the ends of oligomers, as in the case of compounds 15 and 16, have little influence on duplex stability.In this case the differences in melting temperatures of chimeric duplexes in comparison to the Tm of the non-modified duplex T 19 /dA 19 are up to 3 °C (Table 3).The presence of an anionic abasic site in the third strand of the DNA triplex [as in the complex of 13 with hairpin oligomer d(A 21 C 4 T 21 )] significantly decreased triplex stability resulting in lowering of the triplex-duplex transition Tm by about 15 °C per modification.No triplex formation was observed for complexes of hairpin DNA with oligomer 14 possessing two Y ab residues.Thus, as expected, the lack of either Watson-Crick or Hoogsteen interactions of Y ab units, located within the central domain of the modified oligomers, with their counterparts in corresponding duplexes and triplexes has dramatic consequences on the stability the these complexes.However, abasic BHPA units positioned at the ends of oligomers exhibit minimal influence on duplex stability (as in duplexes 15/dA 19 , 16/T 19 and 15/16) due to smaller participation of the terminal base pairs in duplex stability. 21ybridization affinity of chimeric phosphinate / phosphate backbone oligomers 19 -21, containing one, two or three BHPA units with alkylnucleobase moiety, to ssDNA and RNA templates is not improved in comparison to hybridization affinity of chimeric oligomers containing abasic units Y ab .The differences in Tm of non-modified complexes T 19 /dA 19 and T 19 /A 19 and duplexes of oligomers 19a, 19c, 20a, 20c, 21a and 21c with their DNA and RNA templates are within 2 to 6 °C.The lower affinity of these novel DNA analogs towards their complementary strands probably results from their higher flexibility and the loss of entropy in comparison to natural DNA molecule. 3,4It has been proven that the more rigid structure of DNA analog (like e.g.LNA) provides better affinity to a complementary RNA strand. 22From the other side peptide nucleic acid analogs (PNA) despite their structural flexibility exhibit extremely high affinity toward their DNA and RNA complements. 23n interesting hybridization feature was found for oligomers 19a,c -21a,c with respect to their ability to form stable triplexes with hairpin DNA [d(A 21 C 4 T 21 )].Melting temperatures of these triplexes (Table 3) are identical or slightly higher than that of the reference complexes.Probably the phosphinate / phosphate backbone flexibility of the third strand has less influence on triplex stability as compared to contribution of this feature in the duplex structure.

Enzymatic degradation of chimeric oligomers 13 and 14 with 3′-and 5′-exonucleases as analyzed by MALDI-TOF technique
Chimeric oligomers 13 and 14, possessing one or two abasic units (Y ab ) located in the central domain of the oligomer sequence (Table 2), were used to study their recognition by 3′-and 5′exonucleases.Chimeric oligonucleotides were incubated with snake venom (svPDE, PDE I) and with calf spleen phosphodiesterases (PDE II), respectively and the digestion products of each reaction were identified by MALDI-TOF mass spectrometry analysis.
Figure 6a presents four subsequent MALDI-TOF mass spectra of degradation products of oligonucleotide 13 with PDE I taken after 10 min, 1, 2 and 3 hours of the cleavage reaction.A ladder of products, which differ by m/z 304, corresponds to the products of subsequent removal ISSN 1424-6376 Page 164 © ARKAT USA, Inc of pT nucleotides from the parent oligonucleotide 13 (m/z 5601).After 3 hours of cleavage reaction an accumulation of oligonucleotide (Tp) 9 Y ab of m/z 2863 is observed.No further degradation of this product occurs even after 6 hours.These results demonstrate that cleavage of the parent oligomer undergoes up to the modification site.An analogous product (Tp) 8 Y ab pY ab of m/z 2747 is accumulated when oligomer 14 is treated with svPDE (Figure 6d).In contrary, both these oligomers, 13 and 14, when treated with PDE II nuclease, result in accumulation of the products containing one natural nucleotide upstream of the modification site.Thus, oligomer 13 is subsequently degraded to product TpY ab (pT) 9 of m/z 3167 (Figure 6b), while oligomer 14 results in the formation of compound TpY ab pY ab (pT) 9 of m/z 3354 (Figure 6d).Snake venom phosphodiesterase, which is a 3′-exonuclease, is active up to the modification site being able to cleave the phosphodiester bond between thymidine and BHPA units.In contrast, calf spleen phosphodiesterase, which is a 5′-exonuclease, is not able to cleave the phosphodiester bond between 5'-thymidine and BHPA units.This nuclease removes all the 5′terminal nucleotides but one upstream of the modification site.As expected, BHPA structural motives present in the DNA chain are resistant toward 3′-and 5′-exonucleases, however their recognition depends on the cleaving features of used nucleases. 24,25Y ab units introduced at the ends of the oligomers can be a novel class of protecting "clamps" against cellular exonucleases and therefore can be used for protection of antisense, TFO, ribozyme and deoxyribozyme oligonucleotides tested in vivo or in cellular systems for their gene down-regulation activity.The added value of such protection is that BHPA abasic units located at the ends of the oligonucleotides demonstrate minimal influence on duplex stability, as it is shown in thermal stability of duplexes 15/dA 19 , 16/T 19 and 15/16.We prepared several oligomers with 3′-and 5′terminal Y ab protecting groups for their screening as antisense agents (data not shown).Such phosphinic acid "clamps" were also successfully applied for the protection of deoxyribozymes, directed towards the HIV-1 viral RNA sequence in in vitro HIV-1 infected cellular experiments, 7 as well as for deoxyribozymes designed to cleave bcr-abl mRNA fragments in in vitro experiments. 8ligonucleotides 13 and 14 and the reference oligothymidylate T 20 were also given to the cleavage reaction with 3'-exonuclease from human plasma. 26For this assay, 5′-radiolabeled oligonucleotides were incubated with 50 % human plasma.Products of degradation were analysed by PAGE under denaturing conditions (20 % polyacrylamide, 7 M urea).The results show that the reference oligomer incubated for 8 hours at 37 °C afforded a ladder of products ranging from T 19 to T 2 (Figure 7a).The chimeric oligomers were degraded only partially under these conditions.Degradation of oligonucleotide 13 proceeded from the 3'-terminus and was arrested by the presence of the abasic BHPA motif.As a consequence, accumulation of the product (Tp) 9 Y ab was observed (Figure 7b).An analogous ladder of products, with accumulation of the product (Tp) 8 Y ab pY ab was observed for degradation of oligonucleotide 14 (results not shown).

Cytotoxicity of chimeric oligomers 17 and 18 towards HUVEC and HeLa cell lines
For determination of cytotoxicity of chimeric phosphinate / phosphate oligomers we chose two polyanionic oligomers 17 and 18.The cytotoxicity of the test oligomers was evaluated in tumor HeLa and endothelial HUVEC cell lines with an MTT assay. 27Endothelial cells (HUVEC) were isolated and cultured as described. 28HeLa cells were cultured according to the standard method.
As shown in Table 4 cytotoxicity of both polyanionic polymers toward tested cell lines was rather low.
(Tp) 9 Y ab a Toxicity of oligomers was determined as described 27 by an MTT method.Absorbance of a given sample was measured at 570 nm, with the reference wavelength 630 nm (Microplate Reader 450, BioRad).The percentage of living cells (P LC ) was calculated from the equation: P LC = (A S -A M )/(A C -A M ) x 100 %, where As is the absorbance of a given sample of cells treated with oligomers, AM is the absorbance of a cell medium, Ac is the absorbance of a control (untreated) cells.

Conclusions
A series of novel DNA analogs, containing chimeric phosphinate / phosphate backbone could be effectively synthesized by incorporation of acylic nucleoside analogs derived from bishydroxymethylphosphinic acid (BHPA) residue into an oligonucleotide chain.Structural flexibility and loss of entropy make these chimeric oligomers unable to form stable duplexes with their complementary RNA and DNA strands.However, these oligomers possess slightly higher binding affinity towards double stranded DNA, as compared to a non-modified reference.Their low cytotoxic activity toward HUVEC and HeLa cell lines and, as expected, excellent stability toward 3'-and 5'-exonucleases does not preclude their use as therapeutic agents either as 3'-and 5'-terminal protecting groups or as triplex forming oligomers in antigene approach.

Experimental Section
General procedure for the synthesis of compounds 7 MSNT procedure.MSNT (0.44 g, 1.5 mmol) was added to a solution of 6 (0.80 g, 1.0 mmol) and N-1or N-3-(2-hydroxyethyl)thymine (0.17 g, 1 mmol) in anhydrous pyridine (8 mL) and the mixture was stirred at RT for 24 h.The solvent was evaporated and the crude product was isolated by column chromatography on silica gel with a gradient of 0-5% MeOH in CHCl 3 to give pure 7a or 7b in the form of a white foam.Appel procedure.CCl 4 (0.54 mL, 5.6 mmol) was added with stirring under argon at RT to a solution of 6 (0.90 g, 1.1 mmol) and PPh 3 (0.88 g, 3.4 mmol) in anhydrous pyridine (8 mL).

General procedure for the synthesis of compounds 8a-d
To a solution of compound 7 (0.22 mmol) in MeOH (4 mL), a methanolic solution of toluene-4sulfonic acid (0.13 mL, 0.02 M) was added, with stirring.After 5 min the reaction was terminated by addition of pyridine (0.4 mL).The product and unreacted substrate were separated by means of preparative TLC on silica gel with CHCl 3 /MeOH (9:1, v/v).The recovered substrate again was reacted as above.After several-fold repetition of the procedure, pure product 8 was obtained as a white solid.Yields of the reactions, TLC R f values, FAB MS and 31 P NMR (CHCl 3 ) spectral data are given in Table 1.

ISSN 1424-6376
Page 170 © ARKAT USA, Inc General procedure for phosphitylation of derivatives 8a-d 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite (1.2 eq. ) was added under argon to the solution of 8a-d (1.0 eq.) and 2-ethylthio-1H-tetrazole (2.4 eq.) in anhydrous acetonitrile.The reaction mixture was stirred for 1 h at room temperature and then loaded under argon on to a silica gel column.The products 9a-d were eluted with a gradient (0-5%) of methanol in methylene chloride, concentrated in vacuo and stored under argon at -20 °C.
Yields of these reactions (%), TLC chromatographic mobility R f and 31 P NMR (CHCl 3 ) spectral data of 8a-d are given in Table 1.

Loading of 8e on a LCA CPG solid support
The solution of 8e (300 mg, 0.7 mmol), succinic anhydride (100 mg, 1.0 mmol) and 4dimethylaminopyridine (110 mg, 0.9 mmol) in anhydrous pyridine (10 mL) were stirred for 24 hours at room temperature.After this time an additional portion of succinic anhydride (21 mg, 0.05 mmol) was added and the reaction mixture was kept for an additional 4 hours.Then chloroform was added to the reaction mixture, the organic phase was washed twice with 2 % aqueous citric acid (2x20 mL) and once with water (20 mL).The organic layer was dried with anhydrous magnesium sulfate, filtered and then evaporated to dryness.The acyl derivative of 8e was loaded on a silica gel column, eluted with CHCl 3 /MeOH 9:1 (v/v) and dried in vacuo.Long chain aminoalkyl control pore glass (LCA CPG, CHEMGENES, Ashland Technology Center, Ashland, MA) (2g) and acyl derivative of 8e (340 mg, 0.65 mmol) were dried overnight in vacuo, then dissolved in anhydrous DMF (10 mL) and pyridine (1 mL).DCC (250 mg, 1.2 mmol) was added to this solution and the reaction mixture was shaken for 48 hours at room temperature.Then the glass was washed with anhydrous methanol/pyridine (10 mL, 1:1, v/v) solution, with anhydrous acetonitrile (10 mL) and dried in vacuo.The loading efficiency was determined by DMT-cation assay.The modified solid support 9f was loaded with the derivative 8e up to 57.0 µmol /g.

Oligonucleotide synthesis
The 3'-O-phosphoramidite building units 9a-e were used for the synthesis of chimeric oligomers 10-21 by automated solid phase methodology. 14The 1 µmole scale synthesis of oligomers was performed on an ABI 394 synthesizer (Applied Biosystems Inc., Foster City, CA) using succinyl-or oxalyl-linked LCAA-CPG solid support.The only difference in the manufacturer's recommended protocol was a prolonged coupling time (up to 600 s).The coupling efficiency was determined by DMT-ion assay.Oligomers 10c, 10d, 11 and 18 were synthesized on succinyl-linked LAC CPG as DMT OFF constructs, cleaved from the solid support by treatment with 28 % ammonium hydroxide (1mL) for 1 h at room temperature and purified by reverse phase semi-preparative HPLC.
Oligomers 12a, 12b, 19a-21a were synthesized on oxalyl-linked LCAA-CPG solid support as DMT OFF constructs.These oligomers were removed from the solid support by treatment with thiophenol / dioxane / triethylamine mixture (2:1:2, v/v) for 5 min at room temperature and then solid support was washes with 1 % aqueous triethylamine for 7-10 min at room temperature.After solvent evaporation oligomers were purified by semi-preparative or analytical HPLC (as indicated in table 2).
Oligomers 13-17, 19c-21c were obtained as the 5'-O-DMT protected constructs.These oligomers were purified by a standard RP HPLC method (DMT ON step).The removal of 5'-DMT group was achieved by treatment with 50 % acetic acid for 30 min at room temperature, followed by the RP HPLC semi-preparative purification (DMT OFF step) to produce fully deprotected oligonucleotides with 50-80 % yield.
The structure and purity of oligomers were confirmed by MALDI-TOF mass spectrometry, 20 % polyacrylamide / 7 M urea gel electrophoresis and RP HPLC analysis.

Melting temperature measurements
Samples for the melting temperature measurements (duplexes or triplexes) were prepared by hybridization of modified oligomers with the complementary single stranded DNA, RNA or double stranded DNA, as listed in Table 3. Melting profiles were recorded after heating up to 70 ºC, followed by annealing to 5 ºC with a temperature gradient of 0.5 ºC/min.Oligonucleotides were kept at 5 ºC for 5 min then heated at temperature gradient of 0.2 ºC/min to 86 ºC.The melting temperatures were calculated using the first order derivative method.Measurements were performed on Cintra 40 instrument (GBC Australia).

MALDI-TOF measurements
Samples (1 µL) withdrawn from the digestion reactions were put on a sample plate, mixed with the matrix solution [1 µL of an 8/1, v/v mixture of 2,4,6-trihydroxyacetophenone (10 µg/mL in ethanol) and diammonium citrate (50 µg/mL in water)] and left for crystallization.MALDI-TOF spectra were recorded on a Voyager-Elite instrument (PerSeptive Biosystems, CT) in the reflector mode, at a resolution of 2000.M/z negative ion peaks are shown at the spectra.

Cytotoxicity studies
The cytotoxicity of oligomers 17 and 18 was studied for HeLa and HUVEC cell lines using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO] assay (activity of the mitochondrial respiratory chain). 27Cells were trypsinized and diluted with appropriate culture medium to a density of 5000 cells/200 µL for tested cells.Cell suspensions were added to the 96-well plates (200 µL per well).After 24 hours of cell cultivation oligonucleotides were added to a final concentration of 2.5 µM.The cells were incubated for 72 hours in the presence of tested oligonucleotides.As controls, cultured cells were grown in the absence of oligonucleotides.After 72 hours of incubation, 25 µl of MTT solution (5 mg/mL) in PBS was added to each well and incubated for additional 2 hours at 37 °C.Finally, 95 µL of lysis buffer (20 % SDS, 50 % aqueous dimethylformamide, pH 4.5) was added into each well and incubated at 37 °C for additional 24 hours.Absorbance of a given sample was measured at 570 nm, with the reference wavelength 630 nm (Microplate Reader 450, BioRad).The percentage of living cells (P LC ) was calculated from the equation: where A S is the absorbance of a given sample of cells treated with oligomers, A M is the absorbance of a cell medium, A C is the absorbance of a control (untreated) cells.HeLa cells were cultured according to the standard method.Data points represent means of at least three measurements.

Scheme 2 .
Scheme 2. Synthesis of phosphoramidite derivative of BHPA methyl ester 9e and solid support bound BHPA methyl ester.

Figure 1 .
Figure 1.Semi-preparative purification of oligomer Y ab p(Tp) 17 Y ab (15) by the two step deprotection / purification procedure. 19RP HPLC profiles are shown for purification of the 5'-DMT-Y ab p(Tp) 17 Y ab oligomer, step DMT-ON (a) and fully deprotected oligomer Y ab p(Tp) 17 Y ab , step DMT-OFF (b).Collected were oligomers with retention time 19.54 min (a) and 9.19 min (b).HPLC deatils are described in Experimental.

Figure 2 .
Figure 2. Analysis of purity of oligomers 13 -16 by 20 % polyacrylamide / 7 M urea gel electrophoresis.dA 19 and T 19 oligomers are used as the reference oligomers; Figure a: oligomers 13 and 14 as well as the reference oligomers were used for analysis as 5'-32 P-labeled derivetives, Figure b: detection of tested oligomers was done with Stains all reagent.

Figure 3 .
Figure 3. Spectral and electrophoretic analysis of polyanionic oligomers 17 and 18. a: MALDI-TOF spectrum of oligomer 18 (m/z 4244, calc.4246); b: 20 % polyacrylamide / 7 M urea gel electrophoresis with band visualisation in Stains all reagent; c: PAGE of 5'-32 P-labeled oligomer 17 and its 5'-fluoresently labeled analog 18.The spot of the oligomer 18 visible in the UV light on the gel was as marked at the autoradiogram film as shown.

Figure 4 .Figure 5 .
Figure 4. Analytical RP HPLC profile (a) and MALDI-TOF spectrum (b) of oligonucleotide 19a.Chromatographic analysis conditions are given in Experimental.

Figure 6 .Figure 6 (
Figure 6.MALDI-TOF MS analysis of degradation products of chimeric phosphinate / phosphate oligomers 13 and 14 with snake venom and calf spleen phosphodiesterases (PDE I and PDE II, respectively).Oligomers were used at the concentration of 1 nM with the amount of enzyme and the reaction time described in Experimental.MALDI-TOF mass spectra show analysis of the products of the following reactions: a -(Tp) 9 Y ab (pT) 9 (13) / PDE I, b -(Tp) 9 Y ab (pT) 9 (13) / PDE II.

Table 1 .
Spectral and chromatographic data for bis-(hydroxymethyl)phosphinic acid derivatives 7 -9.Yields of the transformation reaction of the salt 6 to amidated or esterified BHPA derivatives 7a-d (in %) are also given a CHCl 3 / MeOH, 9:1, v/v.b The multiple resonances (four or eight signals) may be due to the presence of threo-erythro diastereoisomerism and/or P-C-O-P spin couplings.c methyl-protection of phosphoramidite residue.d 2-cyanoethyl-protection of phosphoramidite residue.

Inc Hybridization properties of chimeric phosphinate / phosphate backbone oligomers
The affinity of polymers 13-16 and 19-21 toward complementary DNA and RNA strands as well as double stranded DNA was determined by UV thermal melting measurements.The Tm values of investigated duplexes and triplexes are given in Table3.Introduction of anionic BHPAderived abasic sites (Y ab ) into the DNA chain, as in chimeric oligomers 13 and 14, causes remarkable destabilization of the corresponding duplexes, with similar effects on Tm for both DNA and RNA complexes.One abasic site present in the center of the oligomer sequence causes the respective Tm to decrease by 6 and 3 °C for DNA and RNA templates, respectively.The presence of a second Y ab group further decreases the affinity of chimeric DNA towards complementary strands of DNA or RNA by about 6-7 °C.

Table 3 .
Binding affinity of oligomers 13-16 and 19a,c-21a,c towards dA 19 , A 19 and double stranded region of DNA hairpin sequence d(A 21 C 4 T 21 ).All Tm values a were determined by the UV melting temperature measurements (details given in the Experimental section) Melting temperatures were measured at 10 mM Tris-HCl buffer pH 7.4, 10 mM MgCl 2 , 100 mM NaCl, with an error of ± 1.0 °C.For determination of Tm's of complexes with oligomers 19a-21a the 10 mM Tris-HCl buffer pH 7.0, 10 mM MgCl 2 , 100 mM NaCl was used.

Table 4 .
Cytotoxicity of oligomers 17 and 18 toward HUVEC and HeLA cells given as percentage of living cells a