Bioconjugate
Chemistry MAY/JUNE 1993 Volume 4, Number 3 0 Copyright 1993 by the American Chemical Society
ARTICLES Synthesis of Bleomycin A5 Oligonucleotide Derivatives and Site-Specific Cleavage of the DNA Target Valentina F. Zarytova,' Dmitrij S. Sergeyev, and Tatjana S. Godovikova Institute of Bioorganic Chemistry, Siberian Division of the Russian Academy of Sciences, Novosibirsk 630090, Russia. Received May 18, 1992
A method for coupling bleomycin Ag to oligonucleotides is proposed. The reaction was carried out between an amino group of a spermidine residue of the Cu(I1) complex of bleomycin A b (Cu(I1)Blm-RH) and a 5'-phosphate group of the oligonucleotides d(pCCAAACA) (I), d(pCGTCCTC) (II),d(pT), (III), d(pCAAACA) (IV), and d(pGCCAAACA) (V) activated with a mixture of triphenylphosphine and 2,2'-dipyridyl disulfide in the presence of 4-(N&-dimethylamino)pyridine 1-oxide. The yields of the products Cu(I1)Blm-R-d(pCCAAACA)(Ia), Cu(I1)Blm-R-d(pCGTCCTC)(IIa), Cu(II)Blm-R-d(pT)IG (IIIa), Cu(I1)Blm-R-d(pCAAACA)(IVa),and Cu(I1)Blm-R-d(pGCCAAACA) (Va) were 60-80%. After removal of the Cu(I1) ion from the bleomycin Ag oligonucleotide derivatives Ia-IIIa, compounds Ib-IIIb were obtained. Compounds Ia, IVa, Va, and Ib-IIIb were further used for modification of the target d(pTGT'M'GGCGAAGGA) in the presence of Fe(I1) ions and 2-mercaptoethanol. Site-specific cleavage of the target by Blm coupled to complementary oligonucleotides was demonstrated. It was shown that efficiency and position of cleavage of the complementary reagents Ia, Ib, IVa, and Va are determined by their oligonucleotide part while the action of the noncomplementary reagents IIb and IIIb was similar to that of the free antibiotic.
INTRODUCTION Reactive oligonucleotide derivatives are widely used for site-specificmodification of nucleic acids (1-5). They form complementary complexes with definite sequences of target nucleic acids and direct reactive groups to a certain nucleotide residue in the vicinity of these sequences. A number of oligonucleotides, bearing EDTA (1-31, 1,lOphenanthroline, or porphyrin ( 4 , 5 ) ,with chelated transition metal ions were prepared and used for sequencespecific oxidative cleavage of nucleic acids. One of the
* Correspondenceaddress: Instituteof BioorganicChemistry, SiberianDivision of the RussianAcademyof Sciences,Lavrentjev pr.8, Novosibirsk 630090, Russia. Phone: 007-3832-35-45-19. Fax: 007-3832-35-34-59.
reagents for DNA oxidative cleavage is the antitumor antibiotic bleomycin (Blm). It is a clinically important agent, which is generally thought to disrupt cell proliferation by causing DNA strand scission (6). Blm has the following structural characteristics: a bithiazole terminal residue that is believed to contribute to the binding to DNA, 8-aminoalanine,pyrimidine, and O-hydroxyhistidine moieties that can form metal-binding centers generating reduced oxygen to damage DNA (7),a dissaccharide moiety, and a linker moiety which connects each part of Blm at an appropriate distance and in desired orientation (Figure 1). The metal-binding center was reported to participate in recognition of nucleotide sequences (8). I t has been well-documented that Blm cleaves DNA preferentially at the GT and GC sequences in the presence of
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Bloconjugate Chem., Vol. 4, No. 3, 1993
Cui TI)B l m -R -pd ( C C A A A C A ) ( I a ) 0
RH = NH-NH-NH2 Figure 1. Structure of bleomycin A5 oligonucleotide derivative
Ia.
Fe(I1) ions and molecular oxygen (9). Blm itself is not a specific agent because all DNA, be it a target nucleic acid in cancer cell or a specific sequence of a viral genome in infected cell, have GT and GC sequences. To achieve specificity, Blm may be coupled to oligonucleotides which bind only to those nucleic acids that have complementary nucleotide sequences. In this respect Blm-conjugated oligonucleotide derivatives may be accurate weapons affecting unique target nucleotide sequences. However there are problems: the structure of bleomycin is complicated,having numerous nucleophilic groups able to react with activated phosphates of oligonucleotides, and there is a danger of affecting the metal-binding center of the antibiotic. Therefore the methods for designing bleomycin oligonucleotide derivatives have to be very special. Earlier we have reported the synthesis of a bleomycin derivative of an oligonucleotide and effective scission by this reagent of DNA (10). We describe here in detail a method for conjugation of bleomycin A5 to oligonucleotides and prove that sitespecific cleavage of a DNA target by the obtained bleomycin derivatives does take place. The results suggest that the bleomycin oligonucleotide derivatives could be important therapeutic drugs of the future. EXPERIMENTAL PROCEDURES General Procedures. All the oligonucleotides I-VI1 and the target d(pTGTTTGGCGAAGGA) were synthesized by the phosphotriester method (11). Ph3P1 and ( P y s ) were ~ from Fluka AG (Switzerland). DMAPO was prepared as described in ref 12. Bleomycin A5 was purchased from the Pilot Plant of the Institute of Organic Synthesis, Latvian Academy of Sciences. UV/vis absorption spectra were recorded on a Specord M40 spectrophotometer (Carl Zeiss Jena) in the buffer 0.2 M NaC1, 0.01 M Tris-HC1(pH 7.5). T,values of the oligonucleotide complexes were measured using equipment for thermal denaturing based on a Milichrom spectrophotometer (13). Molar extinction coefficients were estimated at X = 260 nm for oligonucleotides I-VI1 and the target according to ref 14and for their derivatives Ia-Va and Ib-IIIb as a sum 1 Abbreviations: Ph3P,triphenylphosphine; (PyS)z,2,2'-dipyridyldisulfide;DMAPO,4-(N,Wdimethylamino)pyridine l-oxide: Cu(I1)Blm-RH,Cu(I1) complex of bleomycin Ab.
of the oligonucleotide and the bleomycin molar extinction coefficients. Synthesis of Bleomycin ASOligonucleotideDerivatives Ia-Va. A cetyltrimethylammonium salt of oligonucleotide (1.10-7 mol) was dissolved in 40 mL of DMSO; 3.5 mg of Ph3P, 3 mg of (PyS)2, and 1.5 mg of DMAPO were added. After 10 min of incubation the zwitterionic oligonucleotide formed (Scheme I) was precipitated with 2 % LiC104 in acetone. The Cu(I1) complex of bleomycin A5 hydrochloride (1-10-6 mol or 1.5 mg) in 40 mL of 0.2 M NaHC03 (pH 8.5) was added to the precipitate and the solution was incubated for 6 h. The bleomycin oligonucleotide derivatives formed were isolated by revened-phase liquid chromatography (Figure 2) on a 4.6 X 250 mm Lichrosorb PR-18 10-pmcolumn (Merck, Germany) using a 0-3076 linear gradient of acetonitrile in 0.05 M LE104 using an Altex-332 chromatograph. The yield was 6080%. UV/vis absorption spectra of the compounds revealed an oligonucleotidemoiety and a bleomycin residue (Figure 3). The effect of the antibiotic on the T , of the complementary complex with Ia was studied (Figure 4). The phosphoramide bond in bleomycin oligonucleotide Ia was hydrolyzed in 1M CHOONa (pH 3.5) at 37 "C for 16 h; the oligonucleotide I formed was subject to ionexchange chromatography. To remove Cu(I1) ions from the antibiotic residues of oligonucleotide derivatives IaIIIa, the latter (1.10-8 mol) were absorbed on to a 1 X 50 mm 10-pmLichrosorb RP-18 column and washed with 7.5 mL of 0.1 M EDTA (pH 6.3) at a rate of 0.05 mL/min. The compounds Ib-IIIb so obtained were eluted by 0.4 mL of 20 7% acetonitrile in 0.05 M aqueous LiC104. The copper content of compounds Ib-IIIb was measured spectrophotometrically at X = 612 nm and shown to be less than 10% of the original. Target Degradation. The target d(pTGTTTGGCGAAGGA) was labeled at the 5'-end using [T-~~PIATP and T4 polynucleotide kinase (15)and isolated in a 20 % denaturing gel. DNA degradation reaction (total volume 20 mL) was performed in 0.2 M LiC1, 0.01 M Tris-HC1 (pH 7.5) at 0 or 20 "C. The concentrations were as follows: target and reagents, l.le5M; Fe(NH&&04)2, 1.104 M; 2-mercaptoethanol, 0.05 M. The reactions were initiated by addition of Fe(I1) ions. After the reaction, the oligonucleotide material was precipitated by addition of a 2 % LiC104 solution in acetone. The reaction mixtures with or without piperidine treatment (45 min, 95 OC) were electrophoresed in a 20 % polyacrylamide gel under denaturing conditions (Figures 5 and 6). The gels were visualized by autoradiography. Extents of modification were estimated in an Ultroscan laser densitometer (LKB). RESULTS AND DISCUSSION For the bleomycinoligonucleotide derivatives to be able to site-specifically damage a DNA target, it is necessary to preserve the cleaving capacity of bleomycin and not to lose the ability of the oligonucleotidetail to form complexes with complementary oligonucleotides. With this aim in view, the antibiotic was attached to the 5'-phosphate group of the oligonucleotide which had been activated with a mixture of triphenylphosphineand 2,2'-dipyridyl disulfide in the presence of 4-(N&-dimethylamino)pyridine l-oxide (Scheme I). These reagents activate exclusively terminal phosphate groups of oligonucleotides without interacting with internucleotide phosphate groups and deblocked heterocyclic bases. In this way the terminal phosphate is converted to a zwitterionic group readily reacting with aliphatic amino groups (16). Bleomycin A5 has two regions containing amino groups (Figure 1): the terminal sper-
Bloconjugate Chem., Vol. 4, No. 3, 1993
DNA Cleavage by Bleomycin OllgonucleotMes
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-4 -1 -1 Ex10 M . c m
Scheme I
Cu(1l)Blm-
(oligdl) = CCAAACA (I) CGTCCTC (II) T1 6
1
RH
0 II
- 2 -1 -1 Ex10 H.cm
Cu(1I)Blm-R-P-d(digoN) I
(III) (IV)
-0
CAAACA GCCAAACA (v)
( Ia-Va)
[CH: 41."/0
"251
30 ,
300
200
400
,,
.
600nm
500
Figure 3. UV/vis absorption spectra of oligonucleotide I, ita bleomycin derivative Ia, and Cu(I1) complex of bleomycin A5 (Cu(I1)Blm-RH). 15
60
120 mL
Figure 2. Profile of reversed-phase chromatography of the reaction mixture for synthesis of bleomycin A5 oligonucleotide derivative Ia. A 4.6 x 250 mm column was used with Lichrosorb RP-18 lO-pm, 0-30% gradient of acetonitrile in 0.05 M LiC104, flow rate 2 mL/min.
midine residue (RH) and a metal-binding domain. To preserve reactivity of the antibiotic, the reagent must be able to discriminate between these amino groups and not to react with the metal-binding center. A similar task was fulfilled by Umezawa (17) to obtain semisynthetic derivatives of a Cu(I1) complex of the antibiotic. Thus the Cu(I1) complex of bleomicin Ab was chosen for the reaction with the activated zwitterionic 5'-terminal phosphates of oligonucleotides I-V (Scheme I). A 10-fold excess of the antibiotic was used to stave off coupling of two or more molecules of an oligonucleotide to bleomycin. The obtained products Ia-Va were isolated by reversed-phase chromatography (Figure 2). They were eluted after the initial oligonucleotides and before the antibiotic. The isolated compounds were homogeneous by ion-exchange chromatography. Cu(I1) complexes IaVa were eluted as compounds having two or three charges less than parent oligonucleotides I-V. The yields of IaVa were at least 60 % UV/vis absorption spectra of the obtained compounds Ia-Va had maximal characteristics of both the oligonucleotide component (260 nm) and bleomycin (shoulder at 290-320 nm and band a t 612 nm, e = 1*102M-' cm-I) (Figure 3). This proves that the Cu(I1)-bleomycin complex was attached to the oligonucleotides. The experimental ratios ~600/~260 were quite similar to the theoretical ones estimted for the case when equal molar amounts of the oligonucleotide and antibiotic were coupled. The oligonucleotides and antibiotic were connected by a phosphoramide bond which was hydrolyzed at pH 3.5, 37 "C, during 16 h. There is not detectable depurination of the oligonucleotide part in these conditions. Data of ion-exchange and reversed-phase chromatographiesshowed that derivatives Ia-Va have been completely converted into compoundswith chromatographicbehavior equivalent to that of parent oligonucleotides. This and the ability of
.
10
20
30
io
50
60
T'C
Figure 4. Differential curves of thermal denaturing of com-
plementary complexesI + target (1) and Ia + target (2). Complex concentrationwas 2.5.10-5M in 0.16 M NaCl, 0.02 M Na2HP04, 0.1 mM EDTA (pH 7.4).
bleomycin oligonucleotide derivatives to cleave DNA (see below) favor the oligonucleotide's attachment to a spermidine residue and not to the metal-binding center. However, to be sure which nitrogen of the spermidine residue is involved, we needed some additional data which can be obtained using NMR spectroscopy. This study is in progress now. Bleomycin oligonucleotide derivatives Ia-Va are stable: their 3010-~ M aqueous solutions can be stored for a month at -10 "C without degradation. It is known that the efficiency of site-specific modification of DNA by oligonucleotide reagents depends very much on stability of complementary complexes formed. We investigated the effect of a bulky antibiotic (Blm) covalently attached to an oligonucleotide (Ia) on complex formation. It was found that the T, of the duplex formed by the target d(pTGTTTGGCGAAGGA) with Ia was 11 "C higher than that of its duplex with parent oligonucleotide I (Figure 4). This suggests that, due to a stabilizing effect of the antibiotic, bleomycin oligonucleotide derivatives should destroy functions of nucleic acids more effectively than parent oligonucleotides without bleomycin. I t was important to check this ability of the antibiotic covalently attached to an oligonucleotide. Earlier we demonstrated (IO)that, after removal of the Cu(I1) ion from Ia, Ib without metal ion was obtained. Further it was used for modification of the DNA target in the presence of Fe(I1) ions and 2-mercaptoethanol (Scheme 11). A 14-meric oligonucleotide was used as a target. The reaction of the reagent and the target was carried out in the presence of additional 7-meric oligo-
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192 Bloconlugate Chem., Vol. 4, No. 3, 1993 1 2 3 4 5 6 7 8 9101112
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a
b
1 2
1 2 3
C0
C7
T3
c Figure 5. Autoradiogram of the DNA target cleavage. Lane 1, -~ the target without reagents under reaction conditions ( l ~ l O M Fe(I1) ions, 0.05 M 2-mercaptoethanol in 0.2 M LiC1, 0.01 M Tris-HC1,pH 7.5); lanes 2-12, the target under reaction conditions in the presence of the following oligonucleotides and reagents: 2, Ib + VI; 3, Ib; 4, Ib + VI (l-lO-:) M Fe(I1) ions); 5, Ia + VI without Fe(I1) ions; 6, I + VI + Blm-RH; 7, VI1 + Blm-RH; 8, I + VI + IIb; 9, VI1 + IIb; 10, I + VI + IIIb; 11, VI1 + IIIb; 12, Ia + VI. Concentrations of the reagents (Ia, Ib-IIIb, Blm-RH), target, and complementaryoligonucleotides(VI,VII) were l ~ l O - ~ M. Reaction mixtures were incubated at 20 "C during 6 h and analyzed after piperidine treatment (30 min, 95 "C).
Figure 6. Autoradiogram of DNA target degradation: (a) lane
1, the target with reagent Ib; lane 2, the target with VI1 and reagent Ib; reaction mixtures were incubated at 20 "C for 2 h and analyzed; (b) the target was degraded by IVa (lane l), Ia (lane 2), and Va (lane 3); reaction mixtures were incubated at 0 "C for 4 h and analyzed. Scheme I11
..............
S d( pTGTTTGGCGA AGGA ) 3' 3'd( ACAAACCGCTTCCTp)S
+
3'd(ACAAACCp)5'
Scheme I1
.............. ....... .......
S d ( pTGTTTGGCGAAGGA) 3' 3'd(ACAAACCp)
d ( GCTTCCTp)5'
nucleotide VI complementary to the 3'-end of the target to imitate double-stranded DNA (10). Reagent Ib cleaved the target effectively both in the presence and absence of oligonucleotideVI (Figure 5, lanes 2 and 3, respectively). What is really important here are Fe ions: at 10-fold excess of Fe(I1) over reagent Ib, the target is degraded almost completely while at the equimolar ratio Fe(II)/Ib, a considerable part of the target remains intact (Figure 5, lanes 2 and 4, respectively). We have already mentioned that bleomycin derivatives of oligonucleotides are obtained in the form of copper complexes. It seems attractive to use such complexes for DNA degradation without removing the Cu(I1) ions. It is reported that the Cu(I1) complex of Blm is nearly as effective in vivo as the free Blm (18)and is able to degrade DNA in vitro (19). To demonstrate the possibility to use the Cu(I1) complexes of bleomycin oligonucleotide derivatives for effective DNA cleavage, it is necessary to compare its reactivity with that of analogous compounds without Cu(I1) ions. The latter were obtained by washing out the copper ions from Ia-IIIa absorbed on a reversedphase sorbent with a solution of strong chelating agent such as EDTA which has an association constant not lower than that of bleomycin (10,20). Decreased copper content in Ia-IIIa leads to decreased absorption a t X = 612 nm, which makes it possible to control the process. We show that the Cu(I1)complex of Blm coupled to oligonucleotide Ia does not damage DNA in the absence of the Fe(I1) ions (Figure 5, lane 5). In contrast, in the presence of Fe ions, the same complex attached to the same oligonucleotide does modify the DNA (Figure 5, lane 12) under conditions employed. The combination of the antibiotic with Fe ions is likely to be the key factor for degradation in this case. Since,in the presence of Fe(I1)ions and 2-mercaptoethanol, efficient degradation may be achieved by both the copper
Blm-R
I
(VII)
(W
complex and free reagents, we used both these types of reagents in our experiments. The products of degradation by free and tethered Blm were different for all cases investigated. As shown in Figure 5 (lane 2) and Figure 6a (lane 1)treatment of the target with reagent Ib having a complementary oligonucleotide part caused substantial cleavages of the target sites T3T4-T5and G7without affecting C8,which is characteristic of free bleomycin (Figure 5,lanes 6,7). These results are identical with those described in ref 10. On the other hand, reagent Ib was almost inactive in the presence of oligonucleotide VII, which is complementary to the target (Scheme 111) (Figure 6a, lane 2). These results clearly show that reagent Ib is active only in a complementary complex formed with the DNAtarget. Oligonucleotide VI1 is a competitive inhibitor of modification because it is longer than Ib and its duplex with the target is more stable. Furthermore noncomplementary reagents IIb and IIIb failed to affect the target sites T3T4-T5under conditions similar to those used for the cleavage with reagent Ib (Scheme 111). They cleaved the target at the same sites as free Blm but less efficiently (Figure 5, lanes 7,9, and 11). Similar results were obtained when oligonucleotide VI1was replaced by I + VI, as shown by Scheme I1 (Figure 5,lanes 6,8, and 10). It is suggested that both bleomycin coupled with uncomplementary oligonucleotides (compound IIb and IIIb) and free bleomycin damage the same sites of the target that are different from those cleaved by Blm coupled with the complementary oligonucleotide (compound Ib). A reason for the decrease of the cleavage efficiency of IIb and IIIb as compared with free Blm (Figure 5, lanes 7,9, and 11)may be a result of addition of the negatively charged phosphate groups of the oligonucleotide to the positively charged C-terminal side chain of free Blm. Previous studies have demonstrated strong dependence of the DNA cleavage (21) and the unwinding of plasmid DNA (22) on the presence of positively charged groups in the Blm molecules. Thus Cu(I1)-demethyl-Blm A2 was found to be a much
DNA Cleavage by Bleomycln Ollgonucleotkles
Scheme IV B’d(pTGTTTGGCGAAGGA)3’
-----t t ------t t t _ _ _ _ t_ _ _ _t
IVa Ia Va
* Sites of cleavage are indicated by arrows.
less effective unwinding and cleavage agent than Cu(I1)Blm A2, though it differs from Blm A2 only in that it lacks a methyl group and an associated positive charge at the C-terminus (21,22). Sequence-specificity is changed only if the oligonucleotide is complementary to the template. In this case it is just oligonucleotide that delivers the Blm “warhead”to the nucleosidesof the target that are adjacent to the oligonucleotide binding site (Figure 5, lanes 2-4 and Figure 6b, lanes 1-3). Scheme IV shows that the target was cleaved at different sites by the reagents with different length (also Figure 6, lanes 1-3). The principal sites of cleavage were deoxyribosesof C8and T5for octanucleotide reagent Va, G7,T5,and partially T3for heptanucleotide Ia, and T5 and T3for hexanucleotide IVa. It is worth mentioning that 7- and 8-meric reagents Ia and Va attack target bases that are complementary to their last base carrying Blm at the 5’-phosphate (G7and C8, respectively). On the contrary, 6-mer reagent IVa which modifies T5 and T3but not the last target base G6, is involved in complex formation with the reagent. Bases G6 and T4 did not react with any of the three reagents under study. The oligonucleotide moiety of the latter may target Blm to a definite complementary site of the DNA in the vicinity of which the Blm cleaves the nucleotide sequences in preference. In summary, the obtained results prove that the Blm linked to oligonucleotides cleaves the DNA target site-specifically and can be further used as site-directed agent in molecular biology studies. Also it can be considered as a candidate for future therapeutic use. ACKNOWLEDGMENT We are thankful to Dr. S. G. Lokhov, for measurements of melting temperatures of the duplexes, and Drs. V. V. Vlassov and N. N. Lomakina for helpful discussion. LITERATURE CITED (1) Boutorin, A. S., Vlassov, V. V., Kazakov,S. A., Kutyavin, I. V., and Podyminogin,M. A. (1984) Complementary addressed
reagents carrying EDTA-Fe(I1)groups for directed cleavage of single-stranded nucleic acids. FEBS Lett. 172, 43-46. (2) Chu,B. C. F., and Orgel,L. E. (1985)Nonenzymaticsequencespecific cleavage of single-stranded DNA. Proc. Natl. Acad. Sci. U.S.A. 82, 963-967. (3) Dreyer, G. B., and Dervan, P. B. (1985) Sequence specific cleavage of single stranded DNA. OligodeoxynucleotideEDTAsFe(I1). Proc. Natl. Acad. Sci. U.S.A. 82, 968-972. (4) Chen, C. B., and Sigman, D. S. (1986) Nuclease activity of l,l0-phenanthroiine-copper:Sequence-specifictargeting. Proc. Natl. Acad. Sci. U.S.A. 83, 747-7151.
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