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ISSN 10681620, Russian Journal of Bioorganic Chemistry, 2012, Vol. 38, No. 4, pp. 400–411. © Pleiades Publishing, Ltd., 2012. Original Russian Text © T.V. Abramova, M.F. Kassakin, Yu.V. Tarasenko, A.A. Lomzov, V.V. Koval, D.V. Pyshnyi, V.N. Silnikov, 2012, published in Bioorganicheskaya Khimiya, 2012, Vol. 38, No. 4, pp. 458–471.

Synthesis and Properties of Nucleic Acid Methylene Carboxamide Mimics Derived from Morpholine Nucleosides T. V. Abramova1, M. F. Kassakin, Yu. V. Tarasenko, A. A. Lomzov, V. V. Koval, D. V. Pyshnyi, and V. N. Silnikov Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, pr. Lavrent’eva 8, Novosibirsk, 630090 Russia Received July 11, 2011; in final form, December 12, 2011

Abstract—Uracyl and adenine containing oligomers derived from carboxymethyl derivatives of morpholine nucleoside analogues (MorGly) were synthesized using the methods of peptide chemistry. Capillary electro phoresis conditions were found for the analysis of the homogeneity of the nucleic acid mimics protonated at physiological pH. The thermal stability of complementary complexes formed by the MorGly oligomers was shown to depend dramatically on the heterocyclic base structure (uracil or adenine). Based on the study of tandem complexes it was demonstrated that the impact on the thermal stability of cooperative interactions at oligomer junctions was higher for modified oligomers than for native oligodeoxyriboadenylates. Adenine containing MorGly oligomers formed more stable complexes with poly(U) than native oligodeoxyriboade nylates of the same length. Complexes formed by modified oligomers with polyribonucleotides were more stable if compared with polydeoxyribonucleotides. Keywords: capillary electrophoresis, morpholine nucleosides, NA mimics, nucleosides, oligonucleotide analogues DOI: 10.1134/S1068162012040024 1

INTRODUCTION Huge efforts of molecular biologists at the end of the twentieth century directed to the identification and decoding of the human genome resulted particu larly in the development of a new direction in medi cine. Human health and diseases became to be ana lyzed on the molecular level, primarily, on the level of nucleic acid functioning. Basic courses of molecular medicine that have been developed in practice are molecular diagnostics, prophylactics, and gene ther apy. At the same time, a swift growth in gene therapy studies has required new tools able to influence the cell’s genetic apparatus. From the early studies of sitespecific modification of nucleic acids, and until now, the most promising candidates for the tools have been natural or synthetic oligonucleotide analogues [1]. To date, rich experimental data on the synthesis and properties of oligonucleotide analogues of vari ous types have been accumulated [2–4]. Oligonucle otide morpholine analogues can be considered as one of the most successful examples of the use of these compounds for gene expression regulation [5]. Abbreviations: MorGly, 4'carboxymethyl derivatives of nucleo side morpholine analogues; HOBt, 1hydroxybenzotriazole; HBTU, O(1Hbenzotriazole1yl)N,N,N',N'tetramethylu ronium hexafluorophosphate; DIPEA, diisopropylethylamine; Nb, pnitrobenzyl; PAM, phenylacetamidomethyl; PEI, poly ethyleneimine. 1 Corresponding author: fax: +7 (383) 3635182; phone: +7 (383) 3635183; email: [email protected].

Various conjugates with reporter or reactive groups have been synthesized based on the commercial dime thylamidomorpholidophosphate mimics (Ia). Photo reactive derivatives of morpholine oligonucleotides were obtained for the elucidation of molecular mech anisms of genome functioning during embryonic development [6]. New positively charged and proto nated (at physiological pH) diamidophosphate mor pholinebased oligonucleotides (Ib)–(Ic) were pro posed as potential viral and bacterial inhibitors [7, 8]. However, oligomers (Ia)–(Ic) contain an additional chiral phosphorus atom. They are normally used as a racemic mixture without separating into isomers [8]. The properties of these diastereomers differ dramati cally [9] and this can be a factor determining the effi cacy of interactions with nucleic acids, such as the for mation of complementary complexes and other more complicated structures [10]. Earlier, we reported oligonucleotide morpholine analogues lacking chiral atoms at monomer junctions [11]. However, oligouridylate analogues, in which monomer units are joined with a tight oxalate linker (Id), could not form stable complementary complexes with native nucleic acids [12]. Morpholine analogues of oligouridylates and oli goadenylates (II) with monomeric units joined with a flexible glycine (carboxamidomethyl) linker were first reported in [13]. However during the next decade the synthesis and properties of these analogues were not studied in detail.

400

SYNTHESIS AND PROPERTIES OF NUCLEIC ACID METHYLENE CARBOXAMIDE MIMICS

401

Table 1. The protocol of the solid phase synthesis of oligomers based on 4'carboxymethylmorpholine nucleoside ana logues (the BocpAlaPAM support, 0.05 g, capacity of 0.2 mmol/g) Operation

Reagents

Time

Removal of the Boc protection

CH2Cl2–TFA, 1 : 4, 2 × 1 mL

2 × 10 min

Washing

CH2Cl2, 1 mL; DMSO, 0.5 mL;

Successively, 3 min each

CH2Cl2, 1 mL; DMSO, 0.5 mL; CH2Cl2, 1 mL Drying in vacuum

5 min

Coupling

DMSO–DIPEA, 9 : 1, 2 × 0.5 mL

2 × 10 min

TEA (0.02 mL, 0.14 mmol) was added to Preliminary activation for 10 min a solution of monomer (IIIb) (0.015 g, Coupling for 4 h 0.039 mmol) and HBTU (0.01 g, 0.026 mmol) in DMSO (0.2 mL) and the mixture was added to the support Washing

Successively: DMSO, 0.5 mL; CH2Cl2, 1 mL; DMSO, 0.5 mL; CH2Cl2, 1 mL

3 min each

Drying in vacuum

5 min

+

1'

O

B

H2N

6'

2' 3'

O

B

4'

O

N

N

HN

N P O O

NH2

HN

5'

O

N P O O

B

N

O

B

N

N P O

O (Ia)

N

(Ib)

B

N

O

O

B

(Ic)

N O

HN

HN

B

O N

N

HN (Id)

B = Ura, Ade, Cyt, Gua

O

O

O

B

O

B

N

In this work we describe the synthesis of uracil and ade nine containing morpholine oligocarboxamides (II) in solution and on a polymer and compare the thermal stabil ity of their DNA and RNA complementary complexes. RESULTS AND DISCUSSION The synthesis of oligonucleotide analogues (II) was performed using 2'Bocaminomethyl4'carboxyme thylmorpholine monomers (IIIa,b) prepared as RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

HN

(II)

O

B

N

described earlier [14]. Since they belong to amino acids, whose amino group was protected with an acid labile Boc group, for the oligomer preparation we used the Boc strategy of solid phase synthesis in a manual mode. The sequence of amino group deblocking and coupling reactions is shown in Table 1. Carboxy com ponents were preliminary activated with HBTU using the procedures developed for the Boc strategy of PNA synthesis [15]. Vol. 38

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O O

N H

O

B

N Ade

O (IIIa,b)

O

O

OH

N

H2N

O

B

H2N

Ade O N H

Ade O

N

O N H

O N

O N H

N H

N

n

N O (VIa,b)

OH

NH2[Mor(Ade)Gly]5NH(CH2)2C(O)NH(CH2)2NMe2 (IV) n = 3, NH2[Mor(Ade)Gly]6NH(CH2)2C(O)NH(CH2)2NMe2 (V) n = 4.

B = Ura (a), AdeBz (b)

After the coupling and removal of a Boc protective group the oligomer was separated from the resin by treatment with N,Ndimethylaminoethylenedi amine. However, for extended adeninecontaining oligocarboxamides (more than 6 monomer units) this removal resulted in complex mixtures, from which we failed to isolate the target products. The stage monitoring demonstrated that the reaction yields of monomer addition to the growing chain were half as much at each subsequent coupling stage, even at the initial steps of the synthesis, although the addition of the first and second monomer units pro vided good yields (95%). Thus, using the protocol shown in Table 1, we obtained the adeninecontain ing pentamer NH2[Mor(Ade)Gly]5NH(CH2)2C(O) NH(СH2)2NMe2 (IV) and hexamer NH2[Mor(Ade) Gly]6NH(CH2)2C(O)NHСH2СH2NMe2 (V). The alternative to the solid phase synthesis is the strategy of the chain block construction (Schemes 1 and 2). For the maximal simplification of the synthesis we did not protect the terminal carboxyl group at the first stage (Scheme 1). Activation was carried out with DCC in the presence of Nhydroxysuccinimide, pen tafluorophenol, or 1hydroxybenzotriazole. The acti vated esters were used for the coupling reaction with out isolation and purification. The resulting dimers (VII) were deprotected to give compounds (VIII) and used as amino components for the tetramer synthesis (Scheme 1). The yield of tet ramers (IXa,b) was about 25, 50, and 10% when Nhydroxysuccinimide, pentafluorophenol, and HOBt, respectively, were used for the activation of dimers (VII). However, for the preparation of longer oligomers in reasonably good yields, a considerable excess of the amino component had to be used, which made the isolation of the target compounds extremely

difficult and labourintensive. However, using this approach with compound (IXb) as a carboxy compo nent and monomer (VIb) as an amino component we obtained pentamer NH2[Mor(Ade)Gly]5OH (X) (Scheme 1). Dramatically decreased yields upon increasing the length of the synthesized oligonucleotide analogues can be a result of several factors. The most significant are as follows: a considerable decrease in solubility of unprotected oligomers in organic solvents and the accumulation of side products due to the involvement of the free carboxyl group of the amino component. To overcome these difficulties we used monomers or oli gomers in the form of pnitrobenzyl carboxylates (Scheme 2). The use of pnitrobenzyl ester of the amino compo nents (XIIa,b) with increased solubility in organic sol vents allowed a smooth spectrophotometric control of the reaction course. At the same time, this protective group could be easily removed at pH 8.0 with sodium dithionite [16]. The efficacy of the coupling of uracilcontaining monomers, dimers, and tetramers was high enough in the presence of DCC and Nhydroxysuccinimide in DMF. For octamers BocNH[Mor(Ura)Gly]8OН and NH2[Mor(Ura)Gly]8ONb we used pentafluorophenyl activated esters of the carboxy components with the goal of increasing the coupling efficacy without a large excess of an amino component. Thus, using the block proce dure we obtained, after deprotection, uracilcontaining dodecamer NH2[Mor(Ura)Gly]12OH (XIV) and hexa decamer NH2[Mor(Ura)Gly]16OH (XV).

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O O

O

O

N H

B

O

O

N H

B

N

N

O

O (IIIa,b)

403

1) DCC, C6F5OH, DMF 2) (VIa,b), TEA

O

HN

1) DCC, C6F5OH, DMF

B

O

HN

2) (VIIIa,b), TEA

N

N

O

O

(VIIa,b)

CH2Cl2/TFA

TFA+ H3N

O

3

OH (IXa,b)

OH

−

B

B

N O O

HN (VIIIa,b)


B

N O OH Ade O

O (IXb)

1) DCC, C6F5OH, DMF 2) (VIa,b), TEA 3) EtOH/NH3/H2O 4) CH2Cl2/TFA

Ade

N H2N

O

Ade O

O N

N H

N H

O N

OH

3

NH2[Mor(Ade)Gly]5OH (X) Scheme 1. The synthesis of oligomers based on 4'carboxymethylmorpholine nucleoside analogues in solution without protection of carboxyl groups.

The structures of properly protected oligomeric amino and carboxy components were confirmed by 1H NMR spectra based on the ratio of integral inten sities of characteristic proton resonances related to Boc and pnitrobenzyl protective groups, and 6 and 2' protons. In addition, a ratio of А260 (for the uracil residue) to А300 (for the pnitrobenzyl group) in the UV spectrum was used (Table 2). Unfortunately, we failed to obtain extended ade ninecontaining oligomers using Scheme 2. Probably, the reported low efficacy of the coupling reactions in the polynucleotide synthesis on a polymer [17] is of a general character. Additional studies on the selection of condensing agents, solvents, protective groups, and reaction conditions are required to increase the effi cacy. At the same time, protonated or cationcontain ing 5–6mer oligonucleotide analogues were reported RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

to form complementary complexes with a high ther mal stability sufficient for comparative study of the oli gomer affinity toward DNA and RNA [18, 19]. We suggested some schemes for the synthesis of uracil and adeninecontaining oligomers MorGly based on morpholine nucleosides in solution and on a polymer support using methods of peptide chemistry. Oligomers bearing not more than 5 units can be pre pared using the block procedure in solution from Boc protected monomers with unprotected carboxyl groups. The use as amino components of monomers whose carboxyl group was protected with a pnitrobenzyl residue, made it possible to synthesize uracilcontaining oligomers bearing as many as 16 units. However, the synthesis of adeninecontain ing oligomers was ineffective. Vol. 38

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proper HPLC conditions for the separation of any reaction mixture. Taking into consideration the increased interest in the synthesis of positively charged nucleic acid mimics [8, 19], the development of a uni versal procedure for the analysis of reaction mixture composition and homogeneity of the isolated isomers is a topical issue.

At present, after the oligomer synthesis (for exam ple, peptide nucleic acid analogues or peptides) on a polymer support is completed, and the resin is removed, the target product is purified by HPLC in the presence of 0.1% TFA. Its structure is confirmed by MALDITOF massspectrometry [20]. However, for charged hydrophobic oligomers bearing more than four monomeric units, it is a nontrivial task to find O O

(IIIa,b)

O O

N H

DCC, NbOH, HONSu, DMF, TEA

B

O

N H

1) Na2S2O4/Na3CO3, H2O/EtOH

N O (XIa,b)

O

B

N O

2) DCC, C6F5OH, DMF, 3) (XIIa,b), TEA,

ONb

O

HN

CH2Cl2/TFA

−

N O

+

TFA H3N

O

(XIIIa,b)

B

ONb

N


O (XIIa,b)

Ura O N H2N

ONb

Ura O

O N H

B

Ura O

N

O N H n

O N

OH

NH2[Mor(Ura)Gly]12OH (XIV) n = 10, NH2[Mor(Ura)Gly]16OH (XV) n = 14. Scheme 2. The synthesis of oligomers based on 4'carboxymethylmorpholine nucleoside analogues in solution using pnitrobenzyl protective groups.

Similarly, the analysis of natural oligonucleotides, cationic oligonucleotides and, particularly, 4'carbox amidomethylmorpholine analogues (II), which are protonated at physiological pH (рКВН+ 7.38 for Nmethylmorpholine), can also be analyzed by capil lary electrophoresis according to their charges and masses. The advantages of this method over routine gel electrophoresis are the rate of the analysis and the small sample amounts required. At the same time, a widely used procedure for the analysis of oligonucle otide mixtures by capillary gel electrophoresis in PAAG [21] is unsuitable for positively charged oligo mers. A principal problem is the use of acid conditions for electrophoresis in PAAG as well as the adsorption of amino compounds on the surface of quartz capillar

ies in the absence of the stationary phase. It was pro posed earlier to treat quartz capillaries with positively charged polymers, for example, polyethyleneimine, with the goal of reducing the protein sorption [22]. This treatment results in the inversion of the electroos motic flow, which allows the analysis of positively charged compounds as a function of the number of charges or molecule size [23]. Based on this data we developed the protocol of capillary electrophoretic analysis of oligocarboxamide reaction mixtures and homogeneity of the resulting oligomers (IV), (V), (X), (XIV), and (XV) in conditions of reversed electroos motic flow (Fig. 1). The products synthesized according to Schemes 1 and 2 were purified by cationexchange chromatogra

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Table 2. Ratios of integral intensities of proton resonancesa characteristic for carboxy and amino components in the cou pling reactions (Scheme 2) Oligomer

H6(Ura)/Bocprotons/CH2protons Nb/aromatic protons Nb/H2' the oligomers NH2 end/other H2’ protons

NH2[Mor(Ura)Gly]ONb (XIIa)

The data are given in the experimental section

A300/A260 0.22

BocNH[Mor(Ura)Gly]2OH

1/4.6/–/–/0.53/0.55

–

NH2[Mor(Ura)Gly]2ONb

1/–/1.04/1.05 + 1.07/0.51/0.50

0.13


1/2.38/–/–/0.29/0.73


1/–/0.53/0.65 + 0.55/0.27/0.80


1/1.23/–/–/0.14/0.78


1/–/0.30/0.35 + 0.35/0.14/0.84

0.06


–

0.03

0.09

a 1H NMR spectra were registered in a D O–DMSOd mixture. 2 6

phy on a SP Sepharose column in a gradient of NaCl in the presence of 0.1% TFA, followed by desalting of the target fractions by reversephase HPLC. The structures of the resulting oligomers were confirmed by 1H NMR spectroscopy and MALDITOF mass spectrometry. We studied the complex formation properties of the modified oligomers under standard conditions

using the thermal denaturation procedure with the optical registration of signals in a multiwave detec tion mode (Table 3). The study of the thermal stabil ity of complementary complexes of natural oligo and polynucleotides and their synthetic analogues (1 M NaCl, 10 mM Na2HPO4, 1 mM Na2EDTA, pH 7.0) provided the comparison of parameters for the helix–globule transfer and the evaluation of

A254 0.0150 0.0125 0.0100

2

0.0075 65 0.0050

B

0.0025

А

0

4

3 1

acetone 3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

minutes Fig. 1. UV profiles of the products analyzed by capillary electrophoresis (for the conditions, see the Experimental section). A: purified oligomers (X); B: the reaction mixture of the solid phase synthesis of adeninecontaining oligomers after the support was cleaved (7 coupling stages); figures show the oligomer length. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

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Table 3. Temperatures of thermal denaturation of the tested complementary complexes The complex number

Oligomer

Complementaty chain

Tmax,°C

1

(dA)5

d(CT5C)