Solid Phase Synthesis and Chromatographic

Russian Journal of Bioorganic Chemistry, Vol. 29, No. 3, 2003, pp. 274–280. Translated from Bioorganicheskaya Khimiya, Vol. 29, No. 3, 2003, pp. 303–309. Original Russian Text Copyright © 2003 by Kachalova, Tashlitsky, Stetsenko, Romanova, Gait, Oretskaya.

Solid Phase Synthesis and Chromatographic Characteristics of Nucleophilic Agents Conjugated with Oligonucleotides Containing 5'-Terminal Carboxyl Group A. V. Kachalova*, V. N. Tashlitsky*, D. A. Stetsenko**, E. A. Romanova*, M. J. Gait**, and T. S. Oretskaya**1 *Faculty of Chemistry and Belozersky Institute of Physicochemical Biology, Moscow State University, Vorob’evy gory, Moscow, 119992 Russia **Laboratory of Molecular Biology, Medical Research Council, Hills Road, Cambridge, CB2 2QH, United Kingdom Received, March 6, 2002; in final form, June 7, 2002

Abstract—Conjugates of amines or short peptides with oligonucleotides containing 5'-terminal carboxyl group were prepared by solid phase chemical synthesis. A correlation between the physicochemical parameters and retention times of the synthesized conjugates was established by ion-pair reversed-phase HPLC. Key words: ion-pair reversed-phase HPLC, modified oligodeoxyribonucleotides, oligonucleotidopeptides 1

INTRODUCTION Conjugates of oligonucleotides with various molecules are currently extensively used for studying the regularities of protein and nucleic acid recognition and the problems of molecular biology.2 Reporter molecules, intercalators, biologically active peptides, and enzymes have covalently been linked to fragments of nucleic acids [1–3]. At present, the methods of chemical synthesis of oligonucleotide derivatives containing nucleophilic groups (amino [4] and mercapto groups [5]) and conjugates based on these oligonucleotide derivatives [6] have sufficiently been developed. On the other hand, the synthetic methods for the conjugates based on oligonucleotide derivatives with carboxyl or aldehyde function are few. The synthetic methods for these oligonucleotide derivatives described in [7, 8] imply the introduction of 3'-terminal carboxyl group in oligonucleotide through a modification of polymeric support by an additional nonnucleotide link. In [7], subsequent interactions with peptides and fluorescent labels were carried out in aqueous solution after removal of the oligonucleotide from the polymeric support. Hovinen, Guzaev, et al. [8] proposed an in situ method of synthesis of such conjugates when oligonucleotide was removed from polymeric support by the corresponding nucleophilic agent. Carboxyl group is introduced at 5'-terminus of an oligonucleotide either by attachment of a modified unit 1 Corresponding

author; phone: +7 (095) 939-3148; fax: +7 (095) 939-3181; e-mail: [email protected] 2 Abbreviations: HBTU, O-(1H-benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; HOBt, N-hydroxybenzotriazole; and rpHPLC, reversed-phase high performance liquid chromatography.

during automated synthesis [9] or by formation of the functional group after termination of the elongation of oligonucleotide chain and complete deprotection of the oligonucleotide [10]. Subsequent interaction with the nucleophilic component was carried out in solution, which could result in low yields, an increased reaction time, and formation of by-products. In this connection, the method of solid phase synthesis of oligonucleotide derivatives appears to be more convenient due to simplicity of the reaction procedure and the removal of excessive reagents. For example, Guzaev et al. [11, 12] introduced into 5'-terminus of oligonucleotide chain a modified unit containing an active ester of carboxylic acid. Then nucleophilic components were covalently attached to the modified oligonucleotides before their removal from the polymeric support. The disadvantages of this method are a long time of conjugation (6– 12 h) [11], instability of the modified phosphoamidites on storage, and formation of by-products during conjugation [12]. The goal of this study is the solid phase chemical synthesis of conjugates of oligonucleotides containing 5'-terminal carboxyl group with various amines and short peptides (Table 1). It was also of interest to find a correlation between the retention times of the resulting compounds in ion-pair rpHPLC and their physicochemical characteristics. This would enable a rapid and reliable estimate of the efficiency of a modified oligonucleotide coupling with the corresponding nucleophilic component and indirectly confirm the structure of the resulting compound.

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Table 1. Structures and yields of the synthesized oligonucleotide conjugates Nucleophilic component of synthesis

Structure of conjugate HO

Conjugate designation

Transformation extent upon conjugation*, %

A1

100

A2

100

A3

100

A4

98

A5

98

P1

90

P2

90

P3

80

O

2-(2-Hydroxyethoxy)ethylamine (1)

HN

O R

O

R

NH

Tetrahydrofurfurylamine (2)

O NH

2-Phenylethylamine (3)

R

O O H3C H-Leu-NH2 (4)

NH2 O

H3C HN R O

H-Phe-NH2 (5)

NH2 O NH R

H-Gly-Phe-NH2 (6)

O R

NH

O

NH

NH2

O

CH3

CH3

S

O H3C H-Gly-Leu-Met-NH2 (7)

R

NH

NH NH

O

O

O NH2 CH3 O H-Pro-Leu-Gly-NH2 (8)

CH3 NH

NH N

R

O NH2

O

O R = 5'-(CH2)2-CH(CH3)-O- oligonucleotide -OH3'. * Calculated from HPLC data. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

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Table 2. Molecular masses of the starting oligonucleotide and synthesized conjugates Compound

MR calculated

MR experimental

(I) A1 A2* A3* A4* A5* P1 P2 P3

4702.06 4789.2 4785.3 4805.02 4814.2 4848.2 4905.3 5003.5 4969.4

4700.89 4787.1 4783.1/4784.2 4801.6/4803.2 4812.2/4811.68 4847.8/4848.7 4904.71 5006.5 4973.0

*Molecular masses separated by solidus correspond to two separated diastereomers.

RESULTS AND DISCUSSION We modified oligonucleotide chain at 5'-terminus using O-[1-methyl-3-(2-chlorotrityloxycarbonyl)propyl]-O'-2-cyanoethyl-N,N-diisopropyl phosphoamidite, whose synthesis we described in [13]. Cl O

N P

O

CN

C O O

This reagent contains an o-chlorotrityl ester group [14], which can selectively be cleaved by dilute trichloroacetic acid. This enables the deprotection of carboxyl group during automatic solid phase synthesis without affecting other protective groups of oligonucleotide and perform subsequent transformations on the solid phase in organic medium. Phosphoamidite was introduced in the automatic oligonucleotide synthesis at its last stage, with no changes in the standard coupling protocol. The carboxyl function was deprotected by standard detritylation. For preparing conjugates and subsequent studying their chromatographic characteristics, the following oligodeoxyribonucleotide sequence was synthesized: O –O

5' 3' H O p GCTCCCAGGCTCAAA C CH3

(I)

Within its molecule, oligonucleotide (I) contains a fragment complementary to the TAR site of RNA for the HIV Tat protein (underlined in the formula). An analysis of reaction mixture of synthesis of (I) by rpHPLC revealed two peaks, which correspond to two different compounds. Their formation can be explained

by the presence of an asymmetric carbon atom in the 5'terminal linker. After the separation of the compounds by rpHPLC, it was shown that both compounds have the same retention times on ion-pair HPLC. The structure of (I) was also confirmed by mass spectrometry (Table 2). To confirm the presence of 5'-terminal carboxy group and test its reactivity, oligonucleotide (I), split off from the polymer and deprotected, was allowed to react with ethylenediamine dihydrochloride upon activation with a water-soluble carbodiimide. Yield of the reaction product was 98%. The protected oligonucleotide (I) with free carboxyl function was treated with nucleophilic components on the solid phase in organic medium. The conditions of coupling, analysis, and separation of conjugates were established using an aliphatic (1) and two aromatic (2) and (3) amines as models (Table 1). The yields of the corresponding conjugates Ä1, Ä2, and Ä3 were 98– 100%. The modified oligonucleotide was preliminarily activated for 35 min at 35°ë with 1 : 1 HBTU–HOBt mixture in anhydrous DMF. After the addition of the corresponding amine, the mixture was incubated for 1 h. Conjugates with amino acids and short peptides were similarly synthesized (Table 1). Yields of the compounds were close to quantitative (90–100%), except for the conjugate ê3 (80%), whose lower yield can be explained by a lower reactivity of secondary amino group of proline. After coupling, the conjugates were deprotected and removed from the support under standard conditions [15]. The reaction mixtures were analyzed by ion-pair rpHPLC (figure). In some cases, the analysis revealed the formation of two products. We think that these products are the conjugate diastereomers, which was confirmed by mass spectrometry after separation of the isomers by rpHPLC (Table 2). The purity and the coupling efficiency were checked by the rpHPLC with equidistant gradient. This method of equidistant separation of oligonucleotides was developed in our laboratory [16]. It is based on the fact that the extension of a nucleotide by one unit increases the retention time by a constant value, which is different for each of the mononucleotides and does not depend on the oligonucleotide length. The contribution of a nucleotide unit is additive and is determined by two components: the ion-pair interaction of the internucleoside phosphate with tetrabutylammonium cation followed by sorption on the surface of apolar solid phase and the direct hydrophobic interaction of heterocyclic base with the surface of the apolar solid phase. The first component is the same for all nucleotides, and the second determines a different partial contribution of a particular nucleotide unit. In this case, retention time of an oligonucleotide is determined by the sum of partial contributions of nucleotide units within this oligonucleotide molecule. Here we demonstrate the advantages of the ion-pair method of rpHPLC in the analysis of modified oligonu-

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(I)

57.18

A260 (a)

277

0.15

0

0.10

57.18

0.05

A3-1 A3-2

(b) 0.015

0

0.010 0.005

P3

57.18

0

(c) 0.05

0.03

0

0.04

0.02 0.01 2

4

6

8

10

12

14

16

18

20

min

Analysis of reaction mixtures by ion-pair rpHPLC: (a) automated synthesis of (I); (b) synthesis of conjugate A3; and (c) synthesis of conjugate P3. Conditions of HPLC are given in the Experimental section.

cleotides that differ in the structure of attached radicals. We suggest to relate the changes in retention time of an oligonucleotide to the physicochemical properties of compounds attached to it: hydrophobicity, acidic and basic characteristics, molecular mass, and molecular volume. The acidity and hydrophobicity constants of radicals corrected for their possible ionization were calculated and the correlation equations were constructed with the statistical analysis of experimental data using RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

a software package kindly provided by the ACDLabs company. The structures of attached radicals used for constructing the correlation equation were chosen so that the program predicted substantially different retention times for the corresponding substances and, therefore, different values of log D, the coefficient of substance distribution between organic and aqueous phases considering possible ionization (that depends on pH). Oli-

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Table 3. Chromatographic mobility of synthesized compounds tR Compound experimental (I) A1 A2-1 A2-2 A3-1 A3-2 A4-1 A4-2 A5-1 A5-2 P1 P2 P3

13.07 12.79 13.84 14.61 18.74 19.37 14.65 15.13 15.39 16.05 17.43 19.26 15.52

calculated calculated from series 1* from series 2** 13.17 12.78 13.53 – 18.22 – 14.75 – 15.98 – 15.85 17.77 15.96

13.23 12.95 – 13.9 – 18.9 – 15.28 – 16.78 16.28 19.32 17.11

*Series 1, the base mixture is composed of conjugates A1, A2-1, A3-1, A5-1, and oligonucleotide (I). **Series 2, the base mixture is composed of conjugates A1, A2-2, A3-2, A5-2, and oligonucleotide (I).

gonucleotide (I) and conjugates Ä1, Ä2, Ä3, and Ä5 were analyzed under standard conditions of ion-pair separation of extended oligonucleotides with a step of 0.5 min/unit. Compounds Ä2, Ä3, and Ä5 exhibited two peaks of putative diastereomers with equal molecular masses (Table 2). The peaks of isomeric Ä3 and Ä5 exhibited a rather good resolution on rpHPLC. The isomers were separated, and individual diastereomers were analyzed by ion-pair rpHPLC. The chromatographic results were exported to ACD/Chromman 4.5 and processed, and the structures of radicals attached to the oligonucleotides were assigned to the corresponding peaks. The retention times found and the radical structures were exported to ACD/LC Simulator 5.09. From the retention times of peaks corresponding to conjugates Ä1, Ä2-1, Ä3-1, Ä5-1, and oligonucleotide (I) (where Ä2-1, Ä3-1, and Ä5-1 are the first of the two isomer peaks), a correlation equation was constructed according to the model of reversed-phase separation: log ( t R – t 0 ) = 0.049 ( ± 8.2e – 3 ) log D + 2.90 ( ± 4.5e – 3 ),

( A)

n = 5, R = 0.9608, StD = 0.026, where n is the number of samples, tR is the retention time, logD is the coefficient of distribution of the substance between organic and aqueous phases corrected for possible ionization (depending on pH), which the program was calculated for each compounds; R is the correlation coefficient; and StD is the standard deviation.

The following additional parameters were taken into account in the computation of equation A: reversed phase; pH = 7.00; t0 = 2.50 min. A similar calculation was performed from the retention times of peaks corresponding to conjugates Ä1, Ä2-2, Ä3-2, Ä5-2, and oligonucleotide (I) (where Ä22, Ä3-2, and Ä5-2 are the second of two isomer peaks): log ( t R – t 0 ) = 0.054 ( ± 7.5e – 3 ) log D ( B) + 2.91 ( ± 4.1e – 3 ), n = 5, R = 0.9724, StD = 0.024. The following additional parameters were taken into account in the computation of equation B: reversed phase; pH = 7.00; t0 = 2.50 min. Despite good correlation coefficients, the use of only logD as a correlation parameter leads to systematic errors in predicting the retention time of (I) (12.4 and 12.5 according to Eqs. A and B, respectively, instead of 13.07 min) and, consequently, the violation of the predicted order of elution of peaks of conjugate A1 and oligonucleotide (I). The underrate predicted values of retention times for the modified oligonucleotide are due to a partial dissociation of carboxyl group at pH 7. The ionized carboxyl group can interact by the ion-pair mechanism with tetrabutylammonium ion. This leads to an experimentally observed increase in retention time of the oligonucleotide. It is possible to take into account the factor if the correlation equation were calculated for the combined mode of anionic exchange and reversed-phase interaction. The contribution from the anionic exchange to the retention mechanism is determined by the empiric parameter arbitrary charge (Z). We found that the correlation coefficient reaches its maximum and the predicted elution order of conjugate Ä1 and oligonucleotide (I) peaks corresponds to the experimentally observed order at Z = 4. Starting from this, new correlation equations have the form (C) and (D). The equation (C) was constructed from the retention times of peaks corresponding to conjugates Ä1, Ä2-1, Ä3-1, Ä5-1, and (I) according to the model of anionic exchange: log ( t R – t 0 ) = 0.052 ( ± 0.011 ) log D' + 7.6e (C) – 4 ( ± 2.9e – 4 )MV + 2.77 ( ± 0.015 ), n = 5, R = 0.9725, StD = 0.027, where logD' is the coefficient of substance distribution between organic and aqueous phases corrected for the electrostatic interaction of charged organic medium with charged particles in aqueous solution, and MV is the molecular volume. The following additional parameters were taken into account in the computation of equation C: anionic exchange; pH = 7.00; Z = 4.00; t0 = 2.50 min. The retention times for Ä4-1, ê1, ê2, and ê3 predicted by this equation correspond to the experimental values (Table 3).


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A similar calculation was performed using the retention times of peaks corresponding to conjugates Ä1, Ä2-2, Ä3-2, Ä5-2, and (I): log ( t R – t 0 ) = 0.053 ( ± 0.012 ) log D' + 9.3e – 4 ( ± 3.2e – 4 )MV + 2.76 ( ± 0.017 ),

( D)

n = 5, R = 0.9705, StD = 0.030. The following additional parameters were taken into account in the computation of equation D: anionic exchange; pH = 7.00; Z = 4.00; t0 = 2.50 min. One can see from these results (Table 3) that the predicted retention times of two isomers obtained after the coupling of an amino acid residue (4) agree well with the experimentally observed values. Greater errors in determining the retention times of conjugates with the residues of dipeptide ê1 and tripeptides ê2 and ê3 are likely due to more complex structures of these compounds and possible intramolecular interactions. Thus, we synthesized conjugates of oligonucleotides containing a 5'-carboxylic group with various amines by the solid phase method using the procedures we described in [13]. This approach can also be recommended for the synthesis of oligonucleotidopeptides, although this method is sometimes restricted by a low solubility of extended peptides in polar aprotic solvents (DMF, DMSO). The method of ion-pair rpHPLC with equidistant gradient in combination with the specially developed software package ACDLabs enables a rapid structure confirmation for the modified compound. EXPERIMENTAL The following preparations were used: 5'-Odimethoxytrityl-3'-(N,N-diisopropylamido)-β-cyanoethylphosphites of 2'-deoxyribonucleosides, 5-S-ethylthiotetrazole, and the polymeric support dN CPG-500 with the loading of the first nucleotide unit of 35– 40 µmol/g (Glen Research, United States); triethylamine, HBTU, HOBt, and DMF (Fluka, Switzerland); 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 2-(2hydroxyethoxy)ethylamine, ethylenediamine dihydrochloride, and lithium perchlorate (Sigma, United States); hydrochlorides of amides of amino acids and peptides (Bachem, UK); and tetrahydrofurfurylamine and 2-phenylethylamine (Reakhim, Russia). MALDI-TOF mass spectra were recorded on a Voyager DE spectrometer (PerSeptive Biosystems, United States). Automated synthesis of modified oligodeoxyribonucleotide (I) was carried out by the phosphoamidite method on an Applied Biosystems 380B synthesizer (United States) using commercial reagents and solvents according to the standard protocol. A solution of modified phosphoamidite in dry acetonitrile at a concentration of 0.18 M was used. The analysis of reaction mixtures and the monitoring of product purity after separation were performed RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

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by ion-pair rpHPLC on a Waters chromatograph (United States) when eluting two nucleotide units per minute. A column (4 × 250 mm) of Diasorb-130-ë16í (particle size 7 µm) was used. The conditions of analytical separation were as follows: column temperature 45°ë and the flow rate of the eluent 1 ml/min. The eluent was a 48 mM potassium phosphate buffer (pH 7.0) containing 2 mM tetrabutylammonium dihydrophosphate; gradient of acetonitrile concentration was 5– 20.4% (1 min), 20.4–21.6% (1 min), 21.6–23.2% (3 min), 23.2–24.4% (5 min), and 24.4–25.6% (10 min). The isolation of the synthesized conjugates and the separation of the corresponding diastereomers were carried out by rpHPLC on a Tracor chromatograph (Netherlands). A column (4 × 250 mm) packed with Diasorb-130-ë16í (particle size 7 µm) was used. The separation conditions were: temperature of column 45°ë, flow rate of the eluent 1 ml/min. The eluent was 0.1 M ammonium acetate, and the gradient of acetonitrile concentration was 0–40% (80 min). Reaction of modified oligonucleotide (I) with ethylenediamine dihydrochloride. Ethylenediamine dihydrochloride (5.3 mg) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (3 mg) were added to a solution of 0.4 OD260 units of oligonucleotide (I) in 40 µl of water, and the mixture was incubated for 3 h at room temperature. Oligonucleotides were precipitated by 2 M lithium perchlorate solution (100 µl) and acetone (1 ml), reprecipitated, and washed with 300 µl of acetone. The products were analyzed by ion-pair rpHPLC under conditions described above. Synthesis of conjugates. After the completion of automated synthesis and deblocking of carboxyl group, a solution of a mixture of HBTU (100 eqiv) and HOBt (100 eqiv) in dry DMF (150 µl) was added to the modified oligonucleotide (I) immobilized on a polymeric carrier, and the mixture was incubated for 35 min at 35°ë with occasional shaking. Then amine (100 eqiv) and, in the case of compounds (4)–(8), triethylamine (100 eqiv) were added. The mixture was kept for 1 h at 35°ë under stirring and centrifuged. The supernatant was removed, and the polymer was successively washed with DMF (2 × 200 µl), ethanol (2 × 200 µl), and water (2 × 100 µl). The removal of conjugates from the polymeric carrier and the deblocking of functional groups were carried out as described in [15]. The reaction mixtures were analyzed by ion-pair rpHPLC, and conjugates were separated by rpHPLC under conditions described above. Structures of the synthesized compounds were confirmed by mass spectrometry. ACKNOWLEDGMENTS The authors thank A.N. Murav’eva (Chemical Department, Moscow State University) for assistance in chromatographic separation of the compounds.

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The study was supported by a Wellcome Trust grant (no. 057361) and the program “Leading Scientific Schools” (project no. 00-15-97944). REFERENCES 1. Beaucage, S.L. and Iyer, R.P., Tetrahedron, 1993, vol. 49, pp. 10 441–10 488. 2. Stetsenko, D.A., Arzumanov, A.A., Korshun, V.A., and Geit, M., Mol. Biol. (Moscow), 2000, vol. 34, pp. 998– 1006. 3. Tung, C.-H. and Stein, S., Bioconj. Chem., 2000, vol. 11, pp. 605–618. 4. Agrawal, S., Protocols for Oligonucleotide Conjugates, Agrawal, S., Ed., Totowa, NJ: Humana, 1994, pp. 93– 120. 5. Fidanza, J.A., Ozaki, H., and McLaughlin, L.W., Protocols for Oligonucleotide Conjugates, Agrawal, S., Ed., Totowa, NJ: Humana, 1994, pp. 121–144. 6. Zubin, E.M., Romanova, E.A., and Oretskaya, T.S., Usp. Khim., 2002, vol. 71, pp. 273–302. 7. Kahl, J.D. and Greenberg, M.M., J. Org. Chem., 1999, vol. 64, pp. 507–510.

8. Hovinen, J., Guzaev, A., Azhayev, A., and Lonnberg, H., Tetrahedron, 1994, vol. 50, pp. 7203–7218. 9. Kremsky, J.N., Wooters, J.L., Dougherty, J.P., Meyers, R.E., Collins, M., and Brown, E.L., Nucleic Acids Res., 1987, vol. 15, pp. 2891–2909. 10. Gottikh, M., Asseline, U., and Thuong, N.T., Tetrahedron Lett., 1990, vol. 31, pp. 6657–6660. 11. Hovinen, J., Guzaev, A., Azhayev, A., and Lonnberg, H., J. Chem. Soc., Perkin Trans. 1, 1994, vol. 19, pp. 2745– 2750. 12. Guzaev, A. and Manoharan, M., Bioorg. Med. Chem. Lett., 1998, vol. 8, pp. 3671–3676. 13. Kachalova, A.V., Stetsenko, D.A., Romanova, E.A., Tashlitsky, V.N., Gait, M.J., and Oretskaya, T.S., Helv. Chim. Acta, 2002, vol. 85, pp. 2409–2416. 14. Athanassopoulos, P., Barlos, K., Gatos, D., Hatzi, O., and Tsavara, C., Tetrahedron Lett., 1995, vol. 36, pp. 5645–5648. 15. Oligonucleotides and Analogues: A Practical Approach, Eckstein, F., Ed., New York: IRL, 1991. 16. Tashlitskii, V.N. and Oretskaya, T.S., Bioorg. Khim., 1997, vol. 23, pp. 732–741.


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