Bioconjugate Chem. 1993, 4, 319-325
319
Oligonucleotide Derivatives Bearing Reactive and Stabilizing Groups Attached to C5 of Deoxyuridine’ Asya S. Levina, David R. Tabatadse, Ludmila M. Khalimskaya, Tatjana A. Prichodko, Gennadii V. Shishkin, Ludmila A. Alexandrova,+ and Valentina P. Zarytova’ Institute of Bioorganic Chemistry, Siberian Division of the Russian Academy of Sciences, Prospect Lavrentjeva, 8 Novosibirsk 630090, Russia, and Institute of Molecular Biology, Russian Academy of Sciences, Vavilova, 32, Moskow, Russia. Received March 1, 1993
Oligonucleotides bearing an aliphatic amino group at the C5-position of deoxyuridine (ULNH2TCCCA, TULNH2CCCA,ULNH2CCACTT,where L = -CH2-, -CH20CH2CH2- or -CH2NHCOCH2CH2-) have been synthesized. The photoactive @-azidotetrafluorobenzamido, 2-nitro-5-azidobenzamido,or p azidobenzamido), alkylating [4-[N-(2-chloroethyl)-N-methylaminol benzyl], or intercalating [N-(2hydroxyethy1)phenaziniumlgroups were attached to the amino linker of oligonucleotides. The T, values were determined for the duplexes formed by the above oligonucleotide derivatives. The alkylating group does not change the melting temperature of the corresponding duplex. The duplex stability is increased a little in the case of photoactive groups. The influence of the phenazinium residue on the duplex stability strongly depends on its location in the oligonucleotide. The spacer length between the C5 atom of deoxyuridine and the photoactive or phenazinium group was shown to influence the complementary duplex stability.
INTRODUCTION
The oligonucleotide derivatives bearing different reactive groups and labels are widely used in molecular biology, biotechnology, and medicine. The common practice is to introduce chemical groups at the 5/-terminal phosphate of oligonucleotides (1). Over the last few years oligonucleotides bearing different labels (fluorescent, biotin, complexes with metals) and reactive groups in heterocyclic bases have been reported (2-7). If these derivatives have reactive groups, they may be used for site-specific modification of nucleic acid targets on the condition that these groups are at positions that do not take part in complex formation. The oligonucleotides bearing reactive groups at heterocyclic bases have some advantages. These groups can be introduced anywhere on the oligonucleotides; they can be brought closer to the complementary chain and have a stronger influence on it (or on the whole duplex), and the local site of their action on the targets may be more accurate. It has been shown that an alkylating group coupled to the C5-position of deoxyuridine disposed in the middle of oligonucleotide alkylates the complementary chain in only one base (4-6). Furthermore, introduction of any groups in the heterocyclic base of one of the nucleosides allows other groups to be attached to terminal phosphates. In all cases, amino spacers or SH groups were preliminarily introduced into the oligonucleotides. There are several ways of synthesis of oligonucleotide derivatives with different groups at the C5-position of deoxyuridine. The most common approach is the intro-
* Author to whom correspondence should be addressed. + Institute of
Molecule Biology, Russian Academy of Science. Abbreviations used: Phn, N-(2-hydroxyethyl)phenazinium residue; TFA-8-Ala-OSuc,N-hydroxysuccinimideester of N-(trifluoroacety1)-@-alanine; DMTr, dimethoxytrityl group; dRib, deoxyribose residue; T,, melting temperature of an oligonucleotide duplex determined at the point of curve bending. Only deoxyderivatives are used in this report, therefore the prefix “d” in the symbols of deoxyribonucleosides and oligonucleotides is omitted.
duction of an alkene or alkyne having amino groups into the heterocyclic bases of oligonucleotides by using palladium catalysts (8-10). In this paper we suggest a simple and convenient method for the synthesis of oligonucleotide derivatives bearing an aliphatic amino group at the CBposition of deoxyuridine. We used this method to prepare the oligonucleotide derivatives bearing reactive (alkylating or photoreactive) and intercalating groups. The ability of the compounds obtained for complex formation has been investigated. EXPERIMENTAL PROCEDURES
Chemicals. In the present work the followingchemicals were used. N,5/-Protected nucleosides and dimethoxytrityl chloride were from Olaine. N-(2-Hydroxyethyl)phenazinium chloride (111,N-(trifluoroacety1)aminoethan01 ( l l ) , N-hydroxysuccinimide ester of N-(trifluoroacetyl)-&alanine (12), 4-[N-(2-chloroethyl)-N-methylamino] benzaldehyde (131, and 3’,5‘-diacetyl-5-bromomethy1deoxyuridine)-N-methylaminolbenzaldehyde (131,and 3‘,5’-diacetyl-5-bromomethyldeoxyuridine (14 ) were synthesized according to the referenced methods. 3’-H-Phosphonates of protected nucleosideswere prepared according to ref 15. Oligonucleotides were synthesized by the H-phosphonate method (16). The amino linker was incorporated into the terminal phosphates of oligonucleotides as described in ref 17; p-azidotetrafluorobenzoic acid and N-hydroxysuccinimide esters of all azidobenzoic acids used were prepared as described in ref 18. The mixture of phosphodiesterase and 5’-nucleotidase from snake venom was a gift of Dr. V. I. Yamkovoi from Novosibirsk State University (Russia). Chromatography. Isolation and purification of unprotected oligonucleotides and their derivatives were performed by HPLC on a Waters system with Polisil SA (10 pm) anion exchange (a gift of Dr. S. I. Yastrebov from the Institute of Molecular Biology, Novosibirsk, Russia) and Lichrosorb RP 18 (5 pm) (Merck) reverse-phase columns. After enzymatic digestion, the oligonucleotide hydrolyzate was chromatographed on a Milichrom liquid chromatograph with Lichrosorb RP 18 (5 pm) using the
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Bioconjugste Chem., Vol. 4, No. 5, 1993
Table I. Absorption Spectral Data of Azidoarylamides RNH(CH2)aOH in a 1:9 Ethanol-Water Mixture
'co-
gradient of methanol in water. The products of the reaction of derivatives XIV with NazSz03 were analyzed in the same manner (19). Physical Measurements. UV-visible absorption spectra of oligonucleotide derivatives in water were recorded in the 200-600-nm range on a Specord M-40 instrument (Carl Zeiss, Jena, Germany). 'H NMR spectra of the C5modified derivatives of deoxyuridine were run on a Bruker WP-200 spectrometer; chemical shifts are reported in ppm downfield from Mersi. Tmvalues of the oligonucleotide complexes M) were measured in a buffer 0.16 M NaC1-0.02 M NazHP044.1 mM EDTA (pH 7.5), using the equipment for thermal denaturing based on a Milichrom spectrophotometer (20). Molar extinction coefficients (6) were estimated a t 260 nm for oligonucleotides according to ref 21. For the oligonucleotide derivatives, they were calculated as a s u m oft values for the unmodified oligonucleotide and the conjugated group. For 4-[N-(2chloroethy1)-N-methylamino]benzyl and 2- (alky1amino)10-(2-hydroxyethyl)phenaziniumgroups the values Of e260 14.7.103 M-lmcm-' (22)and 10.103 M-l.cm-l(I1) were used, respectively. The spectral data for the azidoarylamido groups are represented in Table I. To estimate the e values of the azidoarylamido groups, the N-hydroxysuccinimide esters of the azidobenzoicacids used were dissolved in a 1:9 ethanol-water mixture with 3 equiv of propanolamine, and the absorption spectra were recorded after 2 min (by that time the reaction was completed). The t values of the N-hydroxysuccinimide formed in these reactions was taken into account. The spectral data are represented in Table I. 5-(Azidomethyl)deoxyuridine,UCHzN3 (IVa). 3',5'Diacetyl-5-(bromomethyl)deoxyuridine(I)prepared from 3 mmol (Ac)T(Ac) (14) was dissolved in 5 mL of dry dimethylformamide; 200 mg of LiN3 was added. The reaction was monitored by TLC in ethyl acetate-hexane (8:2). After storage for 16 h, the reaction mixture was diluted with chloroform (30 mL) and washed with water (2 X 10 mL), the organic extracts were collected and evaporated in vacuo, and concentrated NH4OH (10 mL) and ethanol (2 mL) were added. The reaction mixture was stirred for 3 h a t 20 0C,2then it was evaporated and 5-(azidomethyl)deoxyuridine was isolated by silica gel chromatography. After evaporation, the desired product (IVa) was crystallized from ethanol with a yield of 80% (0.68 g, 2.4 mmol), relative to (Ac)T(Ac). The 'H NMR spectral data of IVa are presented in Table 11. Compound IVa has a band at 2130 cm-l in the IR spectrum (spectrometer Perkin-Elmer 180). Synthesis of UCH20CHKH2NHCOCF3 (IVb). 3',5'-Diacetyl-5-(bromomethyl)deoxyuridine(I) prepared from 1 mmol of (Ac)T(Ac) (14) was dissolved in 5 mL of dry Long treatment with ammonia or the heating of the reaction mixture at 50 "C causes substitution of an azido by an amino group.
Levlna et al.
dimethylformamide; 0.9 mL (5.4 mmol) of N-(trifluoroacety1)aminoethanol was added. The reaction was monitored by TLC with chloroform-ethanol (95:5). After storage for 16 h the reaction mixture was evaporated and treated with concentrated NHdOH (16 h, 20 "C) for 5'3'deblocking. The intermediate compound UCHzOCHzCHzNHz (11) was isolated by reverse-phase chromatography on a 30 X 350 mm Silasorb C8 column (Chemapol) using gradient of ethanol (0-50%) in water with a yield of 40% (0.4 mmol) (monitoring by TLC in ethanol-concentrated NHIOH, 8:2; the presence of an amino group in the product was confirmed by the ninhydrine reaction). Compound I1 was dissolved in 7 mL of methanol, and 0.6 mL (5mmol) of ethyl trifluoroacetate was added. After 16h (monitoring by TLC with chloroform-ethanol, l:l),the desired product (IVb) was isolated by silica gel chromatography in 70 % yield. The lH NMR spectral data of IVb are presented in Table 11. Synthesis of UCHzNHCOCHflHzNHCOCF3 (Ivc), 3'5'Diacetyl-5-(bromomethyl)deoxyuridine I prepared from 4 mmol of (Ac)T(Ac) (14) was dissolved in 20 mL of concentrated NH40H. After 30 min (monitoring by TLC with chloroform-ethanol, 8:2) 5-(aminomethyl)deoxyuridine (111) was isolated by chromatography on Dowex50x2 (H+) with a 55% yield (2.16 mmol). A 890-pmol portion of this compound was dissolved in 2 mL of dry dimethylformamide, and 282 mg (1mmol) of TFA-@-AlaOSuc was added. After storage for 2 h (monitoring by TLC with chloroform-methanol, 8 2 ) , the desired product (IVc) was isolated by silica gel chromatography in 65% yield (565 pmol). The 'H NMR spectral data are given in Table 11. Synthesis of (DMTr)UCH~NHCoCH~CH~NHcocF~ (Vc). A 582-mg (1 mmol) portion of 5'-0-(dimethoxytrityl)-5(azidomethy1)deoxyuridinewas prepared from derivative IVa by the reaction with dimethoxytrityl chloride in 70% yield and dissolved in a 2-mL mixture of pyridine-watertriethylamine (15:5:1), and HzS was passed through during 1h. The reaction was monitored by TLC with chloroformethanol (9:l). The presence of an amino group in product VI1 was c o n f i i e d by the ninhydrine reaction. Compound VI1 was isolated by silica gel chromatography in a yield of 80% (0.8 mmol). It was dissolved in 5 mL of dry dimethylformamide, and 300 mg (1mmol) of TFA-8-AlaOSuc was added. After 3 h (monitoring by TLC with chloroform-ethanol, 9:1), the desired product Vc) was isolated by gel filtration on LH-20 (column, 50 mL; eluent, chloroform-ethanol, 1:l) in 56% yield (0.45 mmol). The structure of the compound prepared was confirmed after deblocking. A DMTr group was removed by 1% CF3COOH in dry methylene chloride (1 min) followed by product isolation by gel chromatography on LH-20. After removal of the trifluoroacetyl protecting group (concentrated NH40H in a water-ethanol mixture, 1:3,1h, 50 "C) the product, 5-(N-(P-aminopropiony1)aminomethyl)deoxyuridine, was isolated by chromatography on Dowex-BOX2 (H+). Its 'H NMR spectrum was recorded (Table 11). Synthesis of Oligonucleotides with theC5-Modified Derivatives of Deoxyuridine (VIII-X). C5-modified compounds IV were converted by a standard method (15) to the corresponding 5'-0-(dimethoxytrityl) 3'-H-phosphonates VI by the reaction with Pc13 and imidazole described in ref 15. H-Phosphonates were isolated by silica gel chromatography using a gradient of ethanol in chloroform in a yield of 80-90%. They were used for the synthesis of oligonucleotides according to ref 16 on a polymer support (CPG-BOO, Sigma) with a capacity for DMTr-nucleoside about 50 pmol/g. The synthesis in-
Bloconjugate Chem., Vol. 4, No. 5, 1993 321
C5-Derhratlred Oligonucleotides
Table 11. 'H-NMR Data (6, ppm) for 5-Substituted Derivatives of Deoxyuridine
1'
OH H
R'
CH3O CH2Ng (IVa) a
b
c
CH(1') CH(6) t ( J = CH(3') CH(4') CH2(5') CH2(2') s 7Hz) m m m m CHda) 7.65 6.25 4.46 4.02 3.82 2.37 1.87 8d 8.00 6.25 4.46 4.05 3.82 2.40 2.40s
CH2(c)
NH(3) NH(e) NH(d) brs brs br s
d
CHZOCH~CH~NHCOCF~~ (IVb) 8.39 CH~OCH~CHZNHCOCF~' (IVb) 8.26 a e b c d CH2NHCOCH2CH2NH(COCF3)b 8.45 (IVC) CH2NHCOCH2CH2NH(COCF,q)' 8.22 (IVC) CHzNHCOCHzCHzNHz' 8.36 a
CHdb)
6.74 6.59
4.85 4.80
4.24 4.25
4.04 3.99
2.54 2.45
4.32 4.25
6.86
4.89
4.39
4.08
2.53
6.64
4.82
4.32
4.02
2.47
6.80
4.82
4.30
4.00
2.53
4.30 d (J= 2.77 t (J = 3.81 m 13.16 5 Hz) 7 Hz) 4.21s 2.76 t (J= 3.75 t (J= 7 Hz) 7 Hz) 4.30s 2.53 t (J= 3.06 t (J= 7 Hz) 7 Hz)
3.57m 3.60 m
13.07
10.37 9.24
10.59
DzO, * C&N, and C&N/DzO (91) were solvents. Signals of the 5-CH3group of thymidine.
Table 111. Molar Ratio of Products after Enzymatic Digestion of Oligonucleotides XCCACTT and YZCCCA Determined by HPLC Data
XinXCCACTT UCHzOCHzCH2NHz UCHzNHCOCHzCHzNHz UCHzNHRILI UCHzOCHzCHzNHR1 UCHzNHCOCHpCHzNHRl T YZ in YZCCCA
compd VIIIb VIIIc XIa XIb XIc
compd IXa Xa UCHzNHCOCHzCHzNHiT IXc TuCH~NHCOCH~CH~NH~ Xc UCHzWhT XVa UCHJWCOCH2NHPhT XVc T u C H ~ N H C O C H ~ C H ~ N H P ~ XVIc UCHzNHzT TUCHW~
T 2.0 2.1 2.0 2.0 2.0 3.0 T 1.0
C 3.0 3.0 3.2 3.0 3.1 3.2
X 1.0 1.0 0.9 0.9 0.9 YZ
Y 0.8
0.9
0.9
1.1) 1.1
1.0 1.0
1.1 1.3 1.1
C 2.6 3.0 2.9 2.4 2.9 2.8 2.7
A 0.8 1.1 1.0 0.8 1.1 1.1
A 1.0
1.0 0.9 1.0
1.2 1.2 1.5
R1as in Table I. cluded repeating the followingstages: 5'-deprotection with 2 % trifluoroacetic acid in methylene dichloride and the coupling reaction in the presence of pivaloyl chloride. The concentrations of H-phosphonate synthones and the condensing agent in the pyridine-acetonitrile mixture (1: 1)were 0.05 and 0.25 M, respectively. We used a 5-fold excess of H-phosphonates to the 5'-hydroxy group on the polymer support. The coupling efficiency and the average yield in each stage were the same as for standard monomers (more than 95% by DMTr cation and 60-80% according to the isolated oligonucleotide). The oligonucleotides containing UCHzN3were converted to compounds VIIIX,a by passing H2S through the oligonucleotide solution in a mixture of pyridine-water (1:l)for 1 h (the azido group was partially substituted by the amino group on treatment with concentrated NH4OH for 16 h at 50 "C). The oligonucleotides bearing the modified deoxyuridine residue were isolated by ion-exchange and reverse-phase chromatography. Their composition was confirmed by enzymatic digestion (Table 111). Synthesis of Arylazido Derivatives of Oligonucleotides XIa-d, XIIb, XIIIb. The amino derivatives of heptanucleotide VIIIa-c and NH2(CH&NHpTCCACTT (l.le7 mol) were dissolved in 15 pL of a water-dimethylformamide mixture (1:2); 6 pmol of the N-hydroxysuc-
cinimide ester of the corresponding azidobenzoic acid in 20 pL of dimethylformamide was added in lo-, 5-, and 5-pL portions at 30-min intervals. After 1.5h, the reaction products were precipitated with 2% LiC104 in acetone, washed with acetone, and isolated by reverse-phase chromatography (Figure lA,B). The yields of acylation products were 70-80%. The UV spectra of p-azidotetrafluorobenzamido and 2-nitro-Bazidobenzamido derivatives XIb and XIIb are presented in Figure 2. The enzymatic digestion data for compounds XIa-c are given in Table 111. Synthesis of the Alkylating Derivatives of Oligonucleotides XIVa-c. The heptanucleotide dULNH2CCACTT (VIIIa-c) (0.1-0.2 pmol) was dissolved in 4 pL of water; 3 pL (21 pmol) of triethylamine, 1mg (5 pmol) benzaldehyde in of 4-[N-(2-chloroethyl)-N-methylaminol 8 mL of ethanol, and in 15-25 min 1.5 mg (40pmol) of NaBH4 were added. After 30 min the products were precipitated with 2 % LiC104 in acetone and isolated by reverse-phase chromatography (Figure 1C)with a yield of 20-50 % , as follows from chromatographic data. Synthesis of Phn-ContainingOligonucleotides XVXVII. The Phn residue was attached to the amino linker of oligonucleotides IXa,c, Xa,c, and NHz(CH2)sNHpTTCCCA according to ref 11. A 0.2-0.5 pmol portion of an amino-containing oligonucleotide was dissolved in 80 p L of 0.05M N-(2-hydroxyethyl)phenaziniumchloride in 0.2 M aqueous Na2C03. After 10 min of incubation, the product was precipitated and isolated by reverse-phase chromatography with a yield of 80-90 % . The typical elution profile is shown in Figure 1D. The absorption spectrum of compound XVc is presented in Figure 2. The enzymatic digestion data for compounds XVa,c and XVIc are shown in Table 111. Enzymatic Hydrolysis of Modified Oligonucleotides. Modified oligonucleotides (1-1e2 pmol) were treated with a mixture of phosphodiesterase at 5'nucleotidase from snake venom in the buffer 0.5 M TrisHC1,0.025 M MgC12, pH 8.5, during 16h, then the reaction mixture was heated for 10 min at 100 OC and analyzed by reverse-phase chromatography. The digestion data are presented in Table 111. The hydrolysis of oligonucleotides results in a mixture of A, C, T, and ULNHR monomers for the compounds bearing the modified deoxyuridine residue
Levlna et el.
Figure 2. Electron absorption spectra of TTCCCA (l),ULNWhCCACTT (XIIb) (31, and ULNHR1 TCCCA (XVc) (2), ULNHR2 CCACTT (XIb) (4) in water (103-1W MI. R1 and R2 are the same as in Table I; Phn is the N-(2-hydroxyethyl)phenazinium residue.
Scheme I
J uCHZN~
uCH@H&HzNHCOCF3
m
IVa
UR
=
H N 5 R . 0 4 4
I
dRib
5
10
15
20
MlN
Figure 1. Results of isolation of modified oligonucleotides from the reaction mixture by a Lichrosorb RP 18 HPLC column (4 X 250 mm): (A) ULNHR1 CCACTT (XIb), (B)ULNHR2 CCACTT (XIIb), (C) ULNHR‘ CCACTT (XIVc), and (D) ULNHPhTCCCA (XVc). R*,RZ,and R4 are the same as in Tables I and IV. Phn is the N-(2-hydroxyethy1)phenazinium residue. (1) ULNH2CCACTT, ( 2 ) XIb, (3) XIIb, (4) XIVc, (5) ULNH2TCCCA,(6) ULNHPhTCCCA(XVc). The flow rate was 2 mL/min with a linear gradient of acetonitrile in 0.05 M LiC104.
at the 5’-end (VIII, IX, XI, XV) and dimers TULNHR and A and C nucleosides for the compounds TULNHRCCCA (X and XVI). The chromatographic mobility of ULNH2 is higher than that of T and is the same as that of control samples UCHzNHz (111), UCH20CH2CH2NH2,and UCH2NHCOCH2CH2NHz.The last two were obtained after removing the protecting group from compounds IVb and IVc. ULNHPhn and ULNHR’ are eluted after all nucleosides due to the high hydrophobicity of the aromatic residues. TULNHPhn has the maximum retention time. The E values for ULNHR were considered to be the same for T in the case of R = H or as a sum of spectra for T and Phn or R1if R was the corresponding residue. RESULTS AND DISCUSSION
The oligonucleotides containing an amino linker at the C5-position of deoxyuridine have been synthesized. For
CF3COOC&l5
J
TFA-kAla-OSuc
UCH~NHCOCH~~H~NHCOCF~
W C
R’ = CH2Br,CH2N3,CH2C€H2CH2NHz,CHzNH2, CHZWH~CH~NHCOCF~, CH2NHCOCH2CH2NHCOCF3
this purpose, the following deoxyuridine derivatives were preliminary obtained according to Scheme I. 5’,3’-Diacetylthymidine was brominated (14) and 5‘,3‘diacetyl-5-(bromomethyl)deoxyuridine (I) was used for the preparation of the modified derivatives of deoxyuridine (1Va-c). To prepare 5-(azidomethyl)deoxyuridine (IVa), compound I was sequentially treated with lithium azide and ammonia. In another experiment, the brominated product I reacted with N-(trifluoroacetyl)aminoethanol, ammonia, and the ethyl ester of trifluoroacetic acid, giving the derivative IVb. Compound IVc was synthesized via the formation of intermediate 111, which was converted to the desired product by reaction with the N-hydroxysuccinimide ester of (trifluoroacety1)-&alanine. The structures of the compounds obtained were confirmed by the IR and lH NMR spectra. In the NMR spectra of derivatives IVb,c, the signals of deoxyuridine and amino linker protons (R’) (Table 11) are recorded. In the NMR spectrum of product IVa,the displacement of signals of the 5-CHz protons (2.40 ppm) is observed downfield compared to the 5-CH3 protons of thymidine (1.87 ppm) (Table 11). This indicates that the proton in the CH3 group is substituted by an electron-accepting group. In the IR spectrum of this compound, there is a line at 2130 cm-’ corresponding to the antisymmetric valency oscillation of the azido group (23). Derivatives IVa-c were converted to the corresponding
Bbconjugate Chem., Vol. 4, No. 5, 1993 323
C5Derivatired Ollgonucleotkles
Scheme I1 UR'
N
-
-
0
ii
1. PCk. Im
DMTrCl
(DMTr)UR
(DMTr)Uy-O-P-H
2.Hz0
0-
H-phosphonate synthones (VIa-c) by a standard procedure (15, 16) (Scheme 11). Compound Vc can also be prepared in an alternative manner, by using the 5-(azidomethyl)deoxyuridine(IVa) and passing H2S through for the reduction of the N3 group to the NH2 group (Scheme 111). The modified and unmodified H-phosphonate synthones were used for the synthesis of amino-containing oligonucleotides VIII-X. UNH2CCACTT
TU~~~~CCCA
ULNHZTCCCA
VIIIa-c
Qkc
Xa,c UWHZ
a: L = - C H r b: L = -C:H20CH2C& C:L = -CH&ONHCH&&
= HN%LNHz OAN
I dRb
The composition of the modified oligonucleotides prepared was confirmed by enzymatic digestion with phosphodiesterase and 5'-nucleotidase from snake venom (Table 111). The presence of an amino group in oligonucleotides makes it possible to join them with different labels. We have synthesized oligonucleotides with the alkylating, photoreactive, and intercalating groups at the 5'-terminal phosphate or a t the C5-position of deoxyuridine. The photoactive derivatives of heptanucleotides XIXI11 were synthesized by the reaction of the amino group R' --NH(CHz),NHpTCCAClT
ULNHRCCACTT
XIa-c, XIIb, XIIIb
XId
R = R' (XI),R = R2 (XII),R = R3 (XIII) (RasinTable I)
a: L = -CH*
b L = -CH@HzC&
C: L = -CH&ONHCH&HT
NaBH,
(OLIG0)- N=CH
( O L I G O ) - N H C H 2 e (H2cH2c' CH3
XIVa-c (OLlG0)- NHZU LNHzCCACTT
VIIIa-c a: L = - C H r
b: L = -CH2OCH2CHT
C: L = -CH,CONHCH&Hr
of compounds VIIIa-c and NHz(CH2)3NHpTCCACTT
with N-hydroxysuccinimide esters of p-azidotetrafluorobenzoic, 2-nitro-5-azidobenzoic,or p-azidobenzoic acids. The alkylating 4- [N-(2-chloroethyl)-N-methylaminolbenzyl group was attached to the amino spacer of an oligonucleotide by the reaction of an amino group with 4- [N-(2-chloroethyl)-N-methylaminol benzaldehyde followed by the reduction of the Schiff base with sodium borohydride (Scheme IV). The covalent chlorine amount (80-90% ) was estimated by the reaction of compounds XIV with NazS203 and subsequent reverse-phase chromatography of the reaction products (19). The residue N-(2-hydroxyethy1)phenazinium (Phn) which stabilizes the complementary complexes, was attached to the amino group of oligonucleotide derivatives IXa,c, Xa,c and NHz(CH2)3NHpTTCCCA according to ref 11. The following Phn derivatives were obtained: U ~ ~ ~ ~ ~ T C C C T A U ~ ~ ~ ~ ~ ~ C CPhnNH(CH2)3NHpTTCCCA C A
XVa,c
XVIa,c
a: L = - C H r
C:
XVII
L = -CH@NHCH&Hr
The oligonucleotide derivatives XI-XVII were isolated by reverse-phase HPLC in a yield of 70-80% for compounds carrying the photoreactive groups (XIa-c, XIIb, and XIIIb), 20-50% for alkylating derivatives (XIVa-c), and 80-90 5% for Phn-containing oligonucleotides (XVa,c, XVIa,c, and XVII). Figure 1 shows typical chromatography elution profiles for reagents XIb, XIIb, XIVc, and XVc. The products bearing the groups mentioned above were eluted at a higher concentration of acetonitrile than the parent amino derivatives of oligonucleotides, which indicates hydrophobic residue coupling. The composition of some of the derivatives obtained was confirmed by the enzymatic digestion (see Table 111). As follows from Figure 2, the absorption spectra of compounds XI-XVII were the combination of the spectra of the oligonucleotide moiety and the corresponding attached group. In the case of the oligonucleotide derivative XIIb bearing the 2-nitro-5-azidobenzamidegroup, which has an absorption at 318 nm (Table I),there are two absorption bands in the spectrum. The spectra of compounds XIa-c and XIIIb are close to the spectrum of the parent oligonucleotideTCCACTT. In the electron spectra of compounds XV-XVII with the phenazinium residue, we have observed absorption at 237, 268, 290 (sh), 390, and 530 nm, in agreement with literature data (11). When the aromatic group and the oligonucleotide residue have absorption a t different wavelengths, they are in a 1:lratio. The interaction of oligonucleotides carrying different groups with nucleic acids depends on their ability to form the complementary complexes. Therefore the influence of the attached groups on complex formation was estimated using the oligonucleotide derivatives prepared (Table IV). To compare the properties of oligonucleotide derivatives bearing some group at the C5-position and at the terminal phosphate (the most well-investigated ones), we have synthesized compounds XId and XVII. It is shown that the alkylating group does not change the melting temperature of the corresponding duplex. The duplex stability does not decrease but, on the contrary, increases a little in the case of the reagents bearing photoactive groups; the effect of the incorporated residue depends on the spacer length (it is minimal for L = -CH2-)
Levlna et al.
Bioconjugate Chem., Vol. 4, No. 5, 1993
324
Table IV. Melting Temperatures (Tm, "C) of Duplexes A and Bn
"TAAGTGGAGTTTGGC?
'TGAATGGGAACA3' (duplex B)
(duplex A)
?T T c A c c x5 X
A ' CCCZ compd
T $H,NH,
UCH20CHzCH~NHp ~CHzNHCOCHzCHzNHz
R'NH(CH2)sNHpT UCHzNHRl UCHzOCHzCH,NHR' UCHzNHCOCHzCHzNHR1 UCHzNHCOCHzCHzNHRz UCHzNHCOCHzCHzNHR~ UCHzNHCOCHzCHINHR~
Tm, "C 25
VIIIa VIIIb VIIIC XId XIa XIb XIC XI10 XIIIC XIVcb
YZ
Y5' compd
TT
25 25 27 27
UCHzNHPhnT TUCHzNHPhn
30 26 29 29 29 29 26
PhnNH(CH2)zNHpTT
uCH~NHCOCH~CH~NHP~~T TuCH~NHCOCH~CH~NHP~U
XVa XVIa xvc XVIC XVII
Tmt O C 16 18
