ORGANIC LETTERS
A Simple Method for Electrophilic Functionalization of DNA
2002 Vol. 4, No. 21 3599-3601
Gregory P. Miller and Eric T. Kool* Department of Chemistry, Stanford UniVersity, Stanford, California 94305-5080
[email protected] Received July 10, 2002
ABSTRACT
An extremely simple and versatile method for placing an electrophilic functional group (iodide) at the 5′ end of oligodeoxyribonucleotides is described. The reaction is carried out while the protected oligodeoxyribonucleotide remains on a solid support and utilizes inexpensive iodination chemistry. We demonstrate that this reaction can be automated on a DNA synthesizer as the last step of DNA synthesis.
Molecular conjugates of oligonucleotides have numerous and diverse applications in biology, biotechnology, and medicine. While many methods for conjugation of DNAs are known,1 there remains a need for highly efficient methods that are simple to perform but widely applicable to varied types of molecules. By far, the majority of methods for functionalization of DNAs make use of nucleophilic amine or thiol derivatives. However, many molecules of interest such as peptides, proteins, and other nucleic acids are much more readily obtained in nucleophilic rather than electrophilic form, and therefore there is a mismatch in functionality that must be addressed by further steps if conjugation is to be carried out. Here we report an effective strategy for DNA chemical activation that takes the opposite approach, rendering the DNA electrophilic. The approach involves conversion of the 5′-hydroxyl group to a 5′-iodide while the oligodeoxyribonucleotide remains on a solid support. We show that the (1) (a) Naylor, R.; Gilham, P. T. Biochemistry 1966, 5, 2722. (b) Zoller, M. J.; Smith, M. Nucleic Acids Res. 1982, 10, 6487. (c) Sokolova, N. I.; Ashirbekova, D. T.; Kolinnaya, N. G.; Shabarova, Z. A. FEBS Lett. 1988, 232, 153. (d) Luebke, K. J.; Dervan, P. B. J. Am. Chem. Soc. 1989, 111, 8733. (e) Goodwin, J. T.; Lynn, D. G. J. Am. Chem. Soc. 1992, 114, 9197. (f) Gryaznov, S. M.; Letsinger, R. L. J. Am. Chem. Soc. 1993, 115, 3808. (g) Fujimoto. K.; Matsuda, S.; Takahashi, N.; Saito, I. J. Am. Chem. Soc. 2000, 122, 5646. 10.1021/ol0264915 CCC: $22.00 Published on Web 09/19/2002
© 2002 American Chemical Society
method is sequence-general, rapid, inexpensive, high-yielding, and able to be automated. We and others have previously described 5′-iodo and 5′tosylate derivatives of oligonucleotides.2 These have been shown to be useful in preparative assembly of unusual DNA structures, for joining of DNAs, and for detection of DNA and RNA sequences. All 5′-iodo or tosyl oligonucleotides used in previous studies were prepared by incorporating the electrophile in 5′-derivatized deoxythymidine phosphoramidites.2 For sequence generality, one would be required to separately prepare suitably protected 5′-modified derivatives of all four deoxyribonucleosides. Not only would that be costly and time-consuming, but there are serious synthetic difficulties in preparing 5′-iodo derivatives of the purine nucleosides, due in part to intramolecular reaction by N3 of purines with 5′-electrophiles during their synthesis.3 These limitations severely restrict the accessibility of the 5′ electrophilic functionalization strategy for many applications. Thus, a more general approach for 5′-iodination of DNA, (2) (a) Herrlein, M. K.; Nelson, J. S.; Letsinger, R. L. J. Am. Chem. Soc. 1995, 117, 10151. (b) Xu, Y.; Kool, E. T. Tetrahedron Lett. 1997, 38, 5595. (c) Xu, Y.; Kool, E. T. Nucleic Acids Res. 1998, 26, 3159. (d) Xu, Y.; Kool, E. T. Nucleic Acids Res. 1999, 27, 875. (e) Xu, Y.; Karalkar, N. B.; Kool, E. T. Nature Biotech. 2001, 19, 148. (3) Dimitrijevich, S. D.; Verheyden, J. P. H.; Moffatt, J. G. J. Org. Chem. 1979, 44, 400.
irrespective of sequence, would be useful, especially if it avoids preparation of individually functionalized nucleosides. To explore the possibility of a DNA 5′-end iodination reaction, two iodination reagents were tested: 1.0 M Ph3PI2 with 1.3 M imidazole in dioxane and 0.5 M (PhO)3PCH3I in DMF. Trinucleotides of the form 5′-dNTT-3′ (N ) A, G, T, C) were used to test and optimize the procedure. All oligonucleotides were prepared on a DNA synthesizer using standard phosphoramidite chemistry with removal of the terminal DMT group prior to iodination. In the initial tests, we used a manual procedure in which DNA synthesis columns were removed from the synthesizer and syringes were used to pass 1.0 mL of reagent from one syringe, through the column, and into the other syringe three or four times over 1 min before putting the columns on a shaker at room temperature. The reactions were completed by removing the iodination reagent and washing the columns with 10 mL of CH2Cl2, 5 mL of CH3CN, and then 5 mL of CH2Cl2. Cleavage and deprotection of the oligonucleotides was achieved by soaking the controlled pore glass beads in concentrated ammonia at room temperature for 24 h, a procedure that was previously shown to result in virtually no hydrolysis of the 5′-iodo group.2b In our hands, (PhO)3PCH3I proved to be a faster and more effective iodination reagent than Ph3P/I2 (Table 1 and Table
anticipated that this reaction might be practical for use on a DNA synthesizer. Under the conditions used, no precipitate had ever been observed, so no damage to the synthesizer was expected. A 15 min iodination cycle program was thus written for an Applied Biosystems DNA synthesizer. This “iodo cycle” (see Supporting Information) contains two 5 min iodination steps using a total of 0.8 mL of filtered iodination reagent (0.5 M) in DMF, followed by a series of rinses with DMF, CH3CN, and CH2Cl2. This cycle was used to synthesize all four d(NTT) oligomers and gave results comparable or superior to those obtained using the manual procedure (Figure 1, Table 2). We estimate the cost of the
Table 1. Reaction Conditions and Yields for Iodination of Short Oligodeoxynucleotides Using the Manual Procedure sequence
reagent
time (min)
absolute yielda
relative yielda
dTTT dTTT dTTT dCTT dCTT dCTT dATT dATT dATT dGTT dGTT dGTT
PPh3/I2 (PhO)3PCH3I (PhO)3PCH3I PPh3/I2 (PhO)3PCH3I (PhO)3PCH3I PPh3/I2 (PhO)3PCH3I (PhO)3PCH3I PPh3/I2 (PhO)3PCH3I (PhO)3PCH3I
240 5 15 240 5 15 900 5 15 870 5 15
63.7 73.1 76.7 55.1 62.4 65.9 54.1 68.2 66.4 35.3 63.3 56.5
93.0 89.8 94.3 89.5 82.3 91.8 93.4 91.8 92.1 77.7 94.0 93.4
a
See ref 4 in the main text.
1S (Supporting Information)). All reactions were essentially complete within 15 min using (PhO)3PCH3I, whereas Ph3P/ I2 required 4-15 h. Reactions using (PhO)3PCH3I resulted in the formation of fewer side-products and gave higher yields,4 particularly in the case of dGTT. Variation of solvent and temperature for reaction of dGTT did not improve yields further (see Supporting Information). Having found that the iodination reaction goes to completion within 15 min using (PhO)3PCH3I in DMF, we (4) We define “absolute yield” as the peak area of product divided by the area of all peaks (measured at 260 nm); “relative yield” is product area divided by the sum of starting material and product peak areas. 3600
Figure 1. HPLC chromatograms of starting materials and products from iodination reactions performed on DNA synthesizer.
iodination reaction to be 57¢ per oligonucleotide (1 µmol scale) using (PhO)3PCH3I obtained from Aldrich. Electrospray MS confirmed that the oligomers were iodinated only
Table 2. Yields Using “Iodo Cycle” on a DNA Synthesizer reaction
absolute yield
relative yield
dTTT dCTT dATT dGTT
94.6 67.3 76.7 77.5
96.6 93.2 94.7 96.6 Org. Lett., Vol. 4, No. 21, 2002
once and 1H NMR spectra indicated they had indeed been iodinated at the 5′-position (see Supporting Information). We also found that following storage of the iodination solution at -78 °C for 50 days, identical results were obtained. Finally, to test the method in longer oligodeoxynucleotides containing all four bases, a series of iodinated 13-mers of the sequence 5′-dNGTAGGCAAGAGT-3′ (N ) A, G, T, C) were synthesized using the automated iodo cycle. HPLC chromatograms of the cleaved and deprotected products showed very good conversion of >90% relative yield and 48-76% absolute yield (see Supporting Information). It should be noted when considering the absolute yields that the overall yields from the DNA synthesis steps ranged from 77 to 91%; therefore, many peaks not corresponding to starting material or iodination product are the result of incomplete phosphoramidite couplings during DNA synthesis as opposed to side-products from the iodination reaction itself. To test for electrophilic reactivity of these iodinated sequences, an assay to ligate these to a nucleophilic (3′phosphorothioated) oligodeoxynucleotide was performed. Ligation reaction mixtures containing phosphorothioated 7-mers, iodinated 13-mers, and complementary 28-mer templates (1.3 µM each) were incubated at 25 °C for 24 h in Tris-borate buffer containing 10 mM MgCl2. Denaturing PAGE gel analysis revealed nearly quantitative formation of the expected 20-mer products (>90% by visual inspection) in the presence of each of the four unpurified iodo-13-mers and no ligation with noniodinated 13-mers (Figure 2). It is noteworthy that the 5′-iodinated DNAs were easily purified by reverse-phase HPLC because of the apparent hydrophobicity of iodine. The functional group slowed mobilities (relative to unreacted DNA) by several minutes, allowing for ready separation. Thus, the iodide performs some of the useful function that terminal dimethoxytrityl groups have traditionally provided in DNA synthesis. Electrophilic derivatization of the 5′-terminus of DNA has also been carried out by bromoacetylation of terminal phosphorothioates.1f The present approach offers the advantages of being performed by automated methods and requiring no postsynthesis derivatization of the DNA. Moreover, alkyl iodides are readily converted to many other functional groups. Although more work will be needed to test this in the DNA context, we anticipate the possibility of using the terminal iodide to generate other reactivities. With the development of this automated procedure for producing 5′-iodinated DNAs, the 5′-iodo/3′-phosphorothioate ligation system is now available for general use in all sequences. Of the DNA self-ligation methods developed
Org. Lett., Vol. 4, No. 21, 2002
Figure 2. Denaturing polyacrylamide gel of ligations testing the reactivity of 5′-iodides in four sequences: lanes 1-4, reactions using unpurified 5′-iodinated 13-mers prepared on a DNA synthesizer; lanes 4-8, reactions using noniodinated 13-mer controls; lanes 9-12, 28-mer, 20-mer, 13-mer, and 7-mer lane markers, respectively.
to date, this system is the only one that gives a product nearly identical to natural DNA. The ligation products retain many of the properties of natural DNA, including abilities to hybridize and act as a substrate for polymerase enzymes.2d Beyond this, the bridging phosphorothioester also confers new properties that may be desirable, such as increased resistance to cleavage by some nucleases and the ability to be selectively cleaved by certain metal cations.2d,5 The ease with which 3′-phosphorothioate-modified and now 5′-iodomodified oligodeoxyribonucleotides can be produced makes this ligation system a viable alternative to enzymatic ligations in some applications. Acknowledgment. We thank the NIH (GM62658 and RR15054) for support. Supporting Information Available: Details of the manual iodination procedure, the iodination cycle on a DNA synthesizer, chromatograms of iodination products, NMR, and mass spectral characterization. This material is available free of charge via the Internet at http://pubs.acs.org. OL0264915 (5) Komiyama, M. J. Biochem. 1995, 118, 665. (b) Fitzsimons, M. P.; Barton, J. K. J. Am. Chem. Soc. 1997, 119, 3379. (c) Hettich, R.; Schneider, H.-J. J. Am. Chem. Soc. 1997, 119, 5638.
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