Bioconjugate Chem. 1997, 8, 757−761
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TECHNICAL NOTES Preparation and Properties of Oligodeoxynucleotides Containing 5-Iodouracil and 5-Bromo- and 5-Iodocytosine Elisenda Ferrer,† Marten Wiersma,† Bernard Kazimierczak,‡ Christoph W. Mu¨ller,‡ and Ramon Eritja*,† European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany, and European Molecular Biology Laboratory, Grenoble Outstation, c/o ILL, B.P. 156, 38042 Grenoble Cedex 9, France. Received March 26, 1997X
The behavior of oligonucleotides containing 5-iodouracil, 5-bromocytidine, and 5-iodocytidine in concentrated ammonia is described. 5-Aminouracil and 5-aminocytidine are obtained as side products when deprotection is performed at 60 °C. Small amounts, if any, of side products are obtained when ammonia deprotection is performed at room temperature. The base-pairing properties of these 5-halopyrimidines including triple helix are described.
Oligonucleotides containing 5-halopyrimidines are important tools for research into nucleic acid and nucleic acid-protein interactions. Specifically, they are used in the following areas: (a) in structural elucidation of mispairs with natural bases to explain their mutagenic properties (1-3); (b) in the study of cytosine methyltransferases (4-6); (c) for photo-cross-linking with proteins which bind DNA and RNA (7-9); (d) in X-ray diffraction experiments of nucleic acids (10, 11) and nucleic acid binding proteins (12); (e) as nonradioactive labels (13) and as alternatives for mixed probes (14); (f) for triple-helix stabilization (15, 16); (g) as antisense oligonucleotides (17, 18); and (h) in the study of the restriction endonucleases cleavage mechanism (19, 20). The preparation of these modified oligonucleotides can be performed by enzymatic methods (8, 9, 13) or preferably by chemical synthesis (1-7, 10, 14-20). Phosphoramidite derivatives of most of the 5-halopyrimidines are commercially available. The use of mild conditions during the ammonia treatment is recommended to avoid base modification (24-48 h, room temperature, refs 10 and 21), although, to our acknowledge, there is no detailed study on these modifications. During the preparation of oligonucleotides containing 5-bromo-2′-deoxyuridine (2, Figure 1), a side product produced during the ammonia treatment was isolated and characterized as 5-amino-2′-deoxyuridine (4, Figure 1) (16). The degree of modification of 5-bromouracil during the standard ammonia treatment (16 h, 50 °C) was near 20%, and no side product was detected if the deprotection was performed at room temperature. To ensure complete deprotection of natural bases at room temperature, phosphoramidites carrying more labile protecting groups such as phenoxyacetyl (22), tert-butylphenoxyacetyl (23), or FOD (24) are recommended (3, 16). 5-Fluorouracil was found to be stable during ammonia deprotection (3). * Address correspondence to this author at EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany (telephone 49-6221387210; fax 49-6221-387306; e-mail
[email protected]). † EMBL, Heidelberg. ‡ EMBL, Grenoble. X Abstract published in Advance ACS Abstracts, August 1, 1997.
S1043-1802(97)00042-6 CCC: $14.00
Figure 1. Chemical structures of 5-substituted pyrimidines.
In this paper we show that 5-iodo-2′-deoxyuridine (3), 5-bromo-2′-deoxycytidine (6), and 5-iodo-2′-deoxycytidine (7) (Figure 1) are also prone to ammonia degradation, giving 5-amino derivatives as described by 5-bromouracil. However, using optimal deprotection conditions, oligonucleotides containing these bases can be obtained with the high purity required for cocrystallization with proteins and subsequent X-ray diffraction studies. RESULTS AND DISCUSSION
To assess the stability of 5-halopyrimidines to oligonucleotide deprotection conditions, the corresponding 2′deoxypyrimidine nucleosides were treated with concentrated ammonia at 60 °C and analyzed by reversed-phase HPLC. 5-Bromo- (2) and 5-iodo-2′-deoxyuridine (3) gave, after ammonia treatment, a more polar product having the same retention time and the same UV absorption spectrum as 5-amino-2′-deoxyuridine (4) (25). The degree of base modification was more pronounced with 5-iodo2′-deoxyuridine (3) than with 5-bromo-2′-deoxyuridine (2). After 24 h, nearly 50% of the 5-iodo-2′-deoxyuridine (3) is converted to 5-amino-2′-deoxyuridine (4), and in the case of the 5-bromo derivative 2, 50% of modification is observed after 3 days. Some minor products were also observed in the 5-iodo derivative (3) eluting very closely to the peak of 5-amino-2′-deoxyuridine (4). Treatment of the nucleosides with ammonia at room temperature did not give any detectable side products. Similar results were obtained during the treatment of halogenated derivatives of 2′-deoxycytidine (6 and 7). A polar product appeared when 5-bromo- (6) and 5-iodo2′-deoxycytidine (7) were treated with concentrated am© 1997 American Chemical Society
758 Bioconjugate Chem., Vol. 8, No. 5, 1997
Ferrer et al.
Table 1. Modification of 5-Halogenated Pyrimidine Oligonucleotides in Concentrated Ammonia Solutions oligonucleotide sequence 5′IUT3′ 5′IUT3′ 5′BrCT3′ 5′BrCT3′ 5′ICT3′ 5′ICT3′
conditions
% 5-haloPyr oligonucleotide
HPLC retention time (min)
MS (Da)
% 5-aminoPyr oligonucleotide
60 °C, 16 h RT, 24 h 60 °C, 16 h RT, 24 h 60 °C, 16 h RT, 24 h
53 100 67 100 35 93
21.2 21.2 18.5 18.5 18.4 18.4
658.1 658.1 608.9; 611.1 608.9; 611.1 656.9 656.9
47 none 33 none 38a 1a
HPLC retention time (min)
MS (Da)
9.4
547.2
6.8
546.0
7.0 7.0
546.1
a In the case of the dimer ICT two more side products were observed: compound X, retention time ) 11.6 min, MS ) 531 Da; compound Y, retention time ) 13.9 min, MS ) 546.1 Da. After 16 h at 60 °C, compound X is present at 10% and compound Y at 16%. After 24 h at RT, compound X is present at 2% and compound Y at 4%.
Figure 2. Analytical HPLC of dimer ICT after ammonia treatment at room temperature and at 60 °C. HPLC conditions are given under Experimental Procedures.
monia at 50 °C which had the same retention time and UV absorption spectrum as 5-amino-2′-deoxycytidine (8) (26). However, in this case the modification was more severe than with the halogenated uracils. After 24 h, 5-bromo-2′-deoxycytidine (6) yielded near 90% of the amino nucleoside (8) and more than 95% modification was observed with 5-iodo-2′-deoxycytidine (7). Some minor products were also observed in the 5-iodo derivative. Treatment of the cytidine derivatives with concentrated ammonia at room temperature yielded only small amounts of the amino derivatives (near 5%). The behavior of oligonucleotides containing halogenated nucleosides in concentrated ammonia was studied on the dinucleotide 5′XT3′; X ) 5-iodo-2′-deoxyuridine (3) and 5-bromo- (6) and 5-iodo-2′-deoxycytidine (7). Dinucleotides were prepared using standard phosphoramidite techniques and commercially available phosphoramidites (21). The amino group of 5-bromo- and 5-iodo2′-deoxycytidine was protected with the benzoyl group (21). Oligonucleotides were prepared without the dimethoxytrityl group at the 5′ end, and aliquots of the dinucleotide support were treated with concentrated ammonia at room temperature (24 h) and at 60 °C (16 h). Products were analyzed by reversed-phase HPLC, and the different products were analyzed by UV, enzyme digestion, and mass spectrometry. Results are shown in Table 1 and Figure 2. Similar to what has been found at the nucleoside level, 5-halopyrimidine oligonucleotides were partially decomposed to the corresponding 5-amino derivatives when ammonia treatment was performed at 60 °C.
The desired 5-halopyrimidine dimer had a larger retention time than the side products (Figure 2). The rate of modification at 60 °C was different for each dimer. Onethird of the 5-bromocytosine dimer was transformed into 5-aminocytosine dimer (33%) after 16 h and, under the same conditions, half of the 5-iodouracil dimer was transformed into 5-aminouracil (47%). Base modification on 5-iodocytosine dimer was more complex. Only 35% of the UV-absorbing material was due to 5-iodocytosine dimer. The remaining 65% was divided in three different products. The major product was 5-aminocytosine dimer (38%), but two other minor products were observed. One of these side products (compound Y, 16%, retention time ) 13.9 min) had a mass similar to that of the 5-aminocytosine dimer but had a different UV absorption spectrum. This side product can be assigned either to a dimer containing 6-aminocytosine or to a dimer containing 5-hydroxycytosine. Previous studies have shown that 6-aminocytosine is produced as minor product when 5-bromocytosine is treated with liquid ammonia (26). Alternatively, if water acts as a nucleophile, 5-hydroxycytosine may be obtained. The molecular weights of these products differ by only 1 mass unit of difference. Finally, the less abundant side product (compound X, 10%, 11.6 min) had a molecular mass of 531 Da, corresponding to a dimer without iodine. Enzyme digestion of this product gave two nucleoside peaks that eluted similarly to T and dC; therefore, this side product was assigned to the CT dimer. The replacement of a 5-iodo group by hydrogen by treatment with hot ammonia solutions has been previously described (27). When deprotection was performed at room temperature for 24 h, complete elimination of the benzoyl group of BrC and IC was observed, as was the absence of base degradation. In the case of IC dimer it was possible to detect small amounts (1-4%) of base degradation products. The side products observed were the same as the products observed at high temperature, but the relative amounts were different. These results are in agreement with previous observations of degradation of 5-halopyrimidines during deprotection (10, 16, 21) and show the importance of using milder conditions for deprotection of these modified oligonucleotides. Once the deprotection conditions were established on model dinucleotides, longer oligonucleotides were prepared and base-pairing properties of 5-halopyrimidine oligonucleotides were analyzed. For this purpose, the following oligonucleotide sequences were prepared: 15mer, A, 5′ TAG AGG XTC CAT TGC 3′ (being X ) IU, BrC and IC); and 11-mers, B, 5′ CYY CCY CCY CT 3′ (being Y ) BrU and IU), and C, 5′ ZTT ZZT ZZT ZT 3′ (being Z ) BrC and IC). Phosphoramidites protected with the more labile, tert-butylphenoxyacetyl protecting groups were used for the natural bases to ensure complete deprotection with concentrated ammonia at room temperature. Oligonucleotides were separated from trun-
Bioconjugate Chem., Vol. 8, No. 5, 1997 759
Technical Notes
Table 3. Melting Temperaturesa (°C) for the Triplex h26:s11 Containing 5-Halouracil and Related Compounds
X,Y
pH 5.5
pH 6.0
pH 6.5
C,T C,BrU C,IU C,NU
49 53 40 31
20 33 27
