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We have examined the binding of derivatives of TOTO with different substituents on the benzothiazole ring. The analogues are the following: 1,1'-(4,4,...
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Bioconjugate Chem. 1999, 10, 66−74

Bis-Intercalation of Homodimeric Thiazole Orange Dye Derivatives in DNA Michael Petersen, Atef A. Hamed, Erik B. Pedersen, and Jens Peter Jacobsen* Department of Chemistry, Odense University, Odense M, DK-5230 Denmark. Received June 20, 1998; Revised Manuscript Received November 17, 1998

The thiazole orange dye 1,1′-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)-bis-4-[(3-methyl-2,3dihydro-2(3H)-benzo-1,3-thiazolylidene)methyl]quinolinium tetraiodide (TOTO) binds to doublestranded DNA (dsDNA) in a sequence selective bis-intercalation. We have examined the binding of derivatives of TOTO with different substituents on the benzothiazole ring. The analogues are the following: 1,1′-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)-[4-[3-(benzyl-2,3-dihydro-2-(3H)-benzothiazolylidene)methyl]quinolinium]-[4-[3-(methyl-2,3-dihydro-2-(3H)-benzothiazolylidene)methyl]quinolinium]tetraiodide (TOTOBzl) and 1,1′-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)-bis-4[(3-ethyl-2,3-dihydro-2(3H)-benzo-1,3-thiazole)methyl]quinolinium tetraiodide (TOTOEt). In this paper, we report the synthesis of TOTOBzl and TOTOEt together with the one- and two-dimensional 1H NMR investigations of complexes between these TOTO analogues and the dsDNA oligonucleotide d(CGCTAGCG)2. Both analogues yield extremely stable complexes in which each chromophore is sandwiched between two base pairs in a (5′-CpT-3′):(5′-ApG-3′) site. The linker spans over two base pairs in the minor groove. The benzyl group in TOTOBzl and the ethyl groups in TOTOEt is pointing outward in the major groove.

INTRODUCTION

Interest in nonradioactive detection of nucleic acids has led to the search for DNA1 stains that are stable under gel electrophoretic conditions. Recently, this has led to the synthesis and characterization of a family of homoand heterodimeric DNA-binding dyes (Benson et al., 1993a,b; Rye et al., 1992). These dyes form highly fluorescent and stable noncovalent complexes with double-stranded DNA (dsDNA). Prominent among these are 1,1′-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)bis-4-[(3-methyl-2,3-dihydro-2(3H)-benzo-1,3-thiazolyl idene)methyl]quinolinium tetraiodide (TOTO, Scheme 1). The enhancement of the fluorescence quantum yield of TOTO upon complex formation with dsDNA is greater than 1000 (Rye et al., 1992), and t1/2 for the dissociation of the dsDNA-TOTO complex under gel electrophoretic * To whom correspondence should be addressed. E-mail: [email protected]. 1 Abbreviations: AMBER, assisted model building with energy refinement; DNA, deoxyribose nucleic acid; DMSO, dimethyl sulfoxide; dsDNA, double-stranded DNA; DSS, 2,2dimethyl-2-silapentane-5-sulfonate; EDTA, ethylenediaminetetraacetic acid; HSQC, heteronuclear single quantum coherence; MARDIGRAS, matrix analysis of relaxation for discerning the geometry of an aqueous structure; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser effect spectroscopy; RANDMARDI, randomized MARDIGRAS; RMD, restrained molecular dynamics; TOCSY, total correlation spectroscopy; TOTO, 1, 1′-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)-bis4-[(3-methyl-2,3-dihydro-2(3H)-benzo-1,3-thiazolylidene)methyl]quinolinium tetraiodide; TOTOBzl,1, 1′-(4,4,8,8-tetramethyl-4,8diazaundecamethylene)-[4-[3-(benzyl-2,3-dihydro-2-(3H)benzothiazolylidene)-methyl]quinolinium]-[4-[3-(methyl-2,3dihydro-2-(3H)-benzothiazolylidene)methyl]quinolinium] tetraiodide; TOTOEt, 1, 1′-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)-bis-4-[(3-ethyl-2,3-dihydro-2(3H)-benzo-1,3-thiazole)methyl]quinolinium tetraiodide; TPPI, time proportional phase incrementation.

Scheme 1. Numbering Scheme for TOTO, TOTOEt, and TOTOBzl

conditions is approximately 11 h (Benson et al., 1993a). This makes TOTO an excellent choice as a marker with a detection limit of ∼4 pg of DNA (Benson et al., 1993b). We have used 1H NMR spectroscopy to study the binding mode and sequence selectivity of TOTO with various dsDNA oligonucleotides and shown that TOTO binds via bis-intercalation preferentially to oligonucleotides containing the (5′-CTAG-3′)2 binding site (Faridi et al., 1997; Hansen et al., 1996; Jacobsen et al., 1995; Spielmann et al., 1995; Stærk et al., 1997). The solution structure of the complex of TOTO bisintercalated in the (5′-CTAG-3′)2 binding site of the d(5′CGCTAGCG-3′)2 oligonucleotide shows that the strong binding of TOTO is caused by the ability of the TOTO

10.1021/bc980073w CCC: $18.00 © 1999 American Chemical Society Published on Web 12/31/1998

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Bioconjugate Chem., Vol. 10, No. 1, 1999 67

Figure 1. Parts of the 200 ms NOESY spectrum of the TOTOBzl complex recorded in D2O. Note that the sample contains ca. 20% d(CGCTAGCG)2-TOTO complex. (a) The aromatic to H1′ region. The sequential H1′(n - 1) - H6/H8(n) - H1′(n) connectivity pattern is shown for the 5′-G2-G6-3′ and 5′-G10-G14-3′ dsDNA segments with solid lines. Interruption due to bis-intercalation is shown with broken lines. Some TOTOBzl resonances are marked with solid arrows. (b) The aromatic to methyl region.

chromophores to adapt to the propeller twist of the nucleobases (Spielmann et al., 1995). This is possible since the benzothiazole ring can twist relative to the quinolinium ring due to the flexible cyanine methine bond between. The benzothiazole is intercalated between two pyrimidine bases and the quinolinium ring between the two purine bases. In contrast to other intercalators, the sequence selectivity of TOTO arises directly from the interaction of the intercalated chromophores with the nucleobases. Although the linker of TOTO adds significantly to the binding strength, it is only a spectator with respect to the sequence selectivity. The charged amino groups most probably contribute to the binding free energy by a nonspecific polyelectrolytic contribution. In this paper, we present the results of one- and twodimensional 1H NMR studies of complexes between dsDNA and TOTO derivatives. We have synthesized 1,1′-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)-[4[3-(benzyl-2,3-dihydro-2-(3H)-benzothiazolylidene)methyl]quinolinium]-[4-[3-(methyl-2,3-dihydro-2-(3H)-benzothiazolylidene)methyl]quinolinium] tetraiodide (TOTOBzl, Scheme 1) and 1,1′-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)-bis-4-[(3-ethyl-2,3-dihydro-2(3H)-benzo-1,3thiazole)methyl]quinolinium tetraiodide (TOTOEt, Scheme 1) and investigated their complexes with the oligonucleotide d(CGCTAGCG)2. This oligonucleotide contains the (5′-CpT-3′):(5′-ApG-3′) and the (5′-ApG-3′):(5′-CpT-3′) sequences.

SYNTHESIS

The major problem in the synthesis of TOTOEt and TOTOBzl was to achieve proper 2-(methylthio)benzothiazolium salts to be used as starting materials for the synthesis of the intermediate monomeric cyanine dyes 6a and 6b. According to the procedure of Brooker et al. (1941, 1942), these 2-(methylthio)benzothiazolium salts should be condensed with the quinolinium salt 5 in the presence of triethylamine in methanol. For the synthesis of 6a, it was decided to use N-ethyl-2-(methylthio)benzothiazolium tetrafluoroborate (3) which could be easily obtained in a high yield from commercially available 2-(methylthio)benzothiazole (1) by alkylation with triethyloxonium tetrafluoroborate. When it was attempted to N-benzylate compound 1 by treatment with neat benzyl bromide at 100 °C for 12 h in order to obtain a proper benzothiazolium salt for the synthesis of 6b, a demethylation reaction occurred and compound 2 was isolated in 51% yield. However, S-ethylation with triethyloxonium tetrafluoroborate afforded the adequate starting material 4. In the final step, TOTOEt was obtained in 85% yield by condensing 2 equiv of the monomeric cyanine dye 6a with 1 equiv of N,N,N′,N′tetramethyl-1,3-propanediamine in dry methanol according to the procedure of Glazer and co-workers (Rye et al., 1992). Compound 7b, believed to be formed in situ in this reaction, was also synthesized in a preparative

68 Bioconjugate Chem., Vol. 10, No. 1, 1999

scale by reacting 6a with an excess of N,N,N′,N′-tetramethyl-1,3-propanediamine. The so formed 7b could then be used for an alternative synthesis of TOTOEt by condensation with 6a. TOTOBzl was obtained in a similar way by condensing the compounds 6b and 7a.

Petersen et al. Scheme 2. Synthetic Path for the TOTO Analogues

RESULTS

The d(CGCTAGCG)2-TOTOBzl Complex. Spectral Data. The line width observed in the 1D 1H spectrum of the d(CGCTAGCG)2-TOTOBzl complex is approximately 6-7 Hz indicating essentially no dynamic exchange between two or more complexes. The d(CGCTAGCG)2TOTOBzl complex arises from bis-intercalation in the (5′CTAG-3′)2 binding site. The amount of this complex is at least 100 times higher than the complex of TOTOBzl in any other site as seen from inspection of the spectra. The dyad symmetry of the d(CGCTAGCG)2 oligonucleotide is broken upon binding of TOTOBzl resulting in 16 spectroscopically distinct dsDNA residues and two distinct TOTOBzl chromophores henceforth designated as the Me-chromophore and the Bzl-chromophore. The protons of the dsDNA were assigned by conventional methods (Wu¨thrich, 1986; Feigon et al., 1983; Hare et al., 1983; Scheek et al., 1983, 1984; Boelens et al., 1985) while the TOTOBzl protons were assigned by the strategy usually employed for TOTO analogues (Spielmann et al., 1995). Figure 1 shows the aromatic to H1′ part of a NOESY spectrum. The sequential connectivity pathway of the H1′(n - 1)-H6/H8(n)-H1′(n) resonances in the intercalation sites is shown with solid lines in Figure 1. Interruption due to intercalation in the 5′-C3pT4-3′ and the 5′-A5pG6-3′ (numbering of the dsDNA in Scheme 3) intercalation sites and similarly at the 5′-C11pT12-3′ and the 5′-A13pG14-3 intercalation sites is indicated with broken lines. The assignment of a few TOTOBzl protons is also indicated. The interruption of the connectivity pathway combined with a multitude of intermolecular dsDNA-TOTOBzl cross-peaks unambiguously identifies binding in the (5′CTAG-3′)2 site. H13, H14, and H15 on TOTOBzl yield cross-peaks to A5 H2′/H2′′ while H1 and H3 on TOTOBzl have cross-peaks to C3 H2′/H2′′. These, and other, NOEs position the benzothiazole rings between C3/C11 and T4/ T12 while the quinolinium rings are sandwiched between A5/A13 and G6/G14. The methyl group protons of T4/ T12 have an upfield shift of 0.7 ppm and yield NOEs to H1-H4 on TOTOBzl implying that the methyl groups are positioned right above the benzothiazole rings. The CH2(6) protons on TOTOBzl give rise to two sharp resonances. It is only possible to identify three resonances from the phenyl ring of TOTOBzl; these resonances are identifiable as the ortho, meta, and para protons, respectively, by the magnitude of their NOESY cross-peaks to the methylene protons. The relative conformation of TOTOBzl in the complex is as shown in Scheme 1 as confirmed by prominent H8-H16 cross-peaks. The NOESY spectra of the complex recorded in H2O contain the normal Watson-Crick NOE connectivity pattern. Chemical shift values of labile protons are included in Table 1. Intermolecular contacts between the N-methyl groups and A5/G6 confirm that the linker crosses the minor groove. The cross-peak pattern observed for the d(CGCTAGCG)2-TOTBzl complex is very similar to that observed for the d(CGCTAGCG)2-TOTO complex (Spielmann et al., 1995). A 1H,13C-HSQC spectrum gave unambiguous assignment of the H3′ and H4′ protons of the dsDNA. However,

Scheme 3

not all H5′ and H5′′ resonances could be assigned due to spectral overlap. Chemical shift values of all protons are shown in Table 1. As we observe no exchange cross-peaks between the two strands of the dsDNA in the NOESY spectrum with a mixing time of 200 ms, we conclude that the d(CGCTAGCG)2-TOTOBzl complex has a lifetime on the order of, or above, 200 ms. Ligand-dsDNA NOE contacts. Interruption of the sequential connectivities in the C3pT4, A5pG6, C11pT12, and A13pG14 base pair steps are a clear indication that TOTOBzl bis-intercalates in the (5′-C3T4A5G6-3′):(5′C11T12A13G14-3′) site. Intermolecular NOEs between T4 CH3 and H1-H4(Me), T12 CH3 and H1-H4(Bzl), A5 H1′, H3′ and H13-H15(Me), G6 H1′ and H13(Me) A13 H1′, H3′ and H13-H15(Bzl), and G14 H1′ and H13(Bzl) places the two benzothiazoles in the C3pT4 and C11pT12 pyrimidine base pair steps while the quinolinium rings are placed in the A5pG6 and A13pG14 purine base pair steps. NOEs between a number of minor groove markers (H1′ and H2 of the adenosines in the intercalation site) and the polyamin(propylene)linker positions the linker of TOTOBzl in the minor groove of the dsDNA. Observation of NOE contacts between protons on the phenyl ring and the methyl groups of T4 and T12 unequivocally places the phenyl ring in the major groove of the dsDNA. NOESY cross-peaks between the amino

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Bioconjugate Chem., Vol. 10, No. 1, 1999 69

Table 1. 1H Chemical Shift Values (ppm) for the d(CGCTAGCG)2-TOTOBzl Complex Compared with Those of the Free Oligonucleotide in Parenthesesa C1 G2 C3 T4 A5 G6 C7 G8 C9 G10 C11 T12 A13 G14 C15 G16

H6/H8

H1′

H2′

H2′′

H3′

H4′

H5/H2/CH3

7.53(7.65) 7.80(1.99) 6.59(7.44) 7.20(7.41) 8.23(8.23) 7.82(7.79) 7.28(7.30) 7.89(7.93) 7.53(7.65) 7.75(7.99) 6.42(7.44) 6.96(7.41) 8.11(8.23) 7.82(7.70) 7.31(7.30) 7.89(7.93)

5.68(5.81) 5.76(5.97) 6.04(5.98) 5.35(5.60) 5.35(6.02) 5.82(5.71) 5.71(5.76) 6.09(6.16) 5.68(5.81) 5.65(5.97) 5.97(5.98) 4.62(5.60) 5.59(6.02) 5.82(5.71) 5.76(5.76) 6.09(6.16)

1.92(2.00) 2.45(2.71) 1.83(2.07) 1.85(2.14) 2.81(2.74) 2.64(2.50) 1.86(1.87) 2.60(2.60) 1.86(2.00) 2.54(2.71) 1.92(2.07) 1.91(2.14) 2.83(2.74) 2.64(2.50) 1.88(1.87) 2.60(2.60)

2.33(2.45) 2.45(2.77) 2.44(2.51) 2.05(2.44) 2.72(2.88) 2.70(2.60) 2.29(2.33) 2.32(2.37) 2.30(2.45) 2.44(2.77) 2.54(2.51) 1.97(2.44) 2.72(2.88) 2.67(2.60) 2.31(2.33) 2.32(2.37)

4.67(4.76) 4.86(5.00) 4.72(4.78) 4.67(4.88) 5.11(5.05) 4.87(4.95) 4.78(4.79) 4.66(4.66) 4.67(4.76) 4.84(5.00) 4.61(4.78) 4.62(4.88) 5.11(5.05) 4.87(4.95) 4.78(4.79) 4.66(4.66)

4.02(4.09) 4.27(4.38) 4.17(4.24) 4.16(4.14) 4.29(4.40) 4.57(4.37) 4.17(4.14) 4.06(4.16) 4.02(4.09) 4.24(4.38) 4.19(4.24) 4.18(4.14) 4.32(4.40) 4.53(4.37) 4.17(4.14) 4.06(4.16)

5.78(5.92)

H1/H3

H4/H6

H4/H6

6.27(6.58)

8.06(8.27)

6.68(6.54)

7.31(7.81)

6.84(6.47)

8.18(8.34)

6.57(6.58)

7.94(8.27)

6.84(6.47)

8.18(8.34)

12.78(13.02) 4.69(5.39) 1.00(1.70) 6.97(7.37)

13.10(13.76) 12.02(12.76)

5.32(5.34) 5.78(5.92) 12.75(13.02) 4.52(5.39) 0.96(1.70) 6.95(7.37)

13.49(13.76) 12.01(12.76)

5.32(5.34)

TOTOBzl

Meb

Bzlc

TOTOBzl

Meb

Bzlc

TOTOBzl

Meb

Bzlc

H1 H2 H3 H4 H8 H9 H10 H13 H14

7.62 7.45 7.59 7.48 6.15 6.49 7.98 7.11 7.01

7.59 7.51 7.59 7.54 6.10 6.47 7.98 6.87 6.87

H15 H16 H19′ H19′′ H20 H21′ H21′′ CH3(22) H23′

6.67 7.39 4.13 4.70 ∼2.5d 3.66 3.66 3.28 3.42

6.65 7.09 4.11 4.48 ∼2.5d 3.6 3.71 3.29 3.43

H23′′ H24 CH3(6) CH2′(6) CH2′′(6) Ph(6), ortho Ph(6), meta Ph(6), para

3.51 ∼2.5d 3.63

3.43 ∼2.5b 5.24 5.45 7.27 7.44 7.34

a The values are relative to DSS at 25 °C. b Values of the chromophore containg a methyl group on the benzothiazole ring. c Values of the chromophore containg a benzyl group on the benzothiazole ring. d Overlapping resonances at 2.5 ppm.

Table 2. Observed NOE Contacts between the Phenyl Ring and dsDNA and TOTOBzl Protons phenyl atom

DNA atom

TOTOBzl atom

ortho

T4 CH3, T12 CH3

meta para

T12 CH3, T4 CH3a T12 CH3

CH2′(6), CH2′′(6), H8(Bzl), H4(Bzl), H2(Bzl) CH2′(6), CH2′′(6)

a

Weak NOESY cross-peak.

protons of C3 and CH2(6) and between the para proton of the phenyl ring and the methyl group of T12 show that the phenyl ring points toward the center of the oligonucleotide. The observed NOE contacts between the dsDNA and the phenyl ring of TOTOBzl are shown in Table 2. Molecular Modeling. A total of 146 distance restraints were incorporated into RMD calculations; 87 of these restraints were found by the RANDMARDI procedure based on 97 NOESY cross-peak volumes; 33 restraints were qualitative restraints found by inspection of the NOESY spectrum with a mixing time of 200 ms and were conservatively chosen as strong (1.8-5.0 Å), medium (1.8-5.5 Å) or weak (1.8-6.0 Å). Four restraints were included to keep the ends of the dsDNA duplex in a B-DNA-like conformation. The remaining 22 of these restraints served to mimic Watson-Crick hydrogen bonding between the two DNA strands as justified by the NOESY spectrum recorded in H2O. As an evaluation of the precision of the distances returned by RANDMARDI, the covalently fixed distances in TOTOBzl were left out of the distance reference file for MARDIGRAS. Consequently, the magnetization transfer for these protons are calculated and the distances returned can be used to evaluate the precision of the RANDMARDI procedure. Four of the 97 intensities used in the calculations stemmed from covalently fixed proton pairs in TOTOBzl and the distances returned for these proton pairs by RANDMARDI were in all cases in error of less than 0.3 Å relative to the covalently known “real” distances. This

Table 3. Distance Restraints Incorporated in the RMD Calculations of the d(CGCTAGCG)2-TOTOBzl Complexa DNA-DNA

DNA-TOTOBzl

interresidue 16 C3-TOTOBzl 2 (0) intraresidue 25 T4-TOTOBzl 6 (0) A5-TOTOBzl 5 (13) G6-TOTOBzl 0 (5) C11-TOTOBzl 0 (0) T12-TOTOBzl 5 (0) A13-TOTOBzl 8 (11) G14-TOTOBzl 0 (4) total 41 total 26 (33)

TOTOBzl-TOTOBzl 20

total

20

a

Found by RANDMARDI calculations. Qualitative distance restraints found by inspection of the NOESY spectrum with a mixing time of 200 ms are tabulated in parentheses.

lends credibility to the distances returned by RANDMARDI despite the sparse experimental data and justifies the inclusion of the 87 proton-proton distances in the RMD calculations. A detailed survey of the distance restraints is shown in Table 3. Convergence. Twenty structures were generated for the dsDNA-TOTOBzl complex with B-DNA conformation as starting structure by RMD calculations. The 20 structures all converged to just one family of structures, and the pairwise root-mean-square (rms) deviation was 0.99 Å. We observed only two violations in excess of 0.2 Å but none in excess of 0.3 Å. A model structure as the one here presented should not be mistaken for a high-resolution structure which requires a much larger set of experimental data. Due to the rather large number of restraints incorporated in the RMD calculations, the consideration of spin-diffusion effects in the RANDMARDI procedure, and the convergence after the RMD calculations, we have confidence in the model structure obtained and believe it to show a fairly precise “picture” of the structure of the TOTOBzl complex. Hence, we believe that the model structure enables us to determine various aspects of the local

70 Bioconjugate Chem., Vol. 10, No. 1, 1999

Petersen et al.

Figure 2. Stereoview of a superposition of 10 structures of the d(CGCTAGCG)2-TOTOBzl complex looking into the major groove. Deoxyribose protons have been omitted for clarity.

Figure 3. The complexes between d(CGCTAGCG)2 and TOTO (left), TOTOEt (center) and TOTOBzl (right). The d(CGCTAGCG)2TOTO complex is reproduced from Spielmann et al. (1995). The d(CGCTAGCG)2-TOTOEt complex is reproduced from Petersen and Jacobsen (1998).

structure of the complex. Views of the model structure are shown in Figures 2 and 3. The d(CGCTAGCG)2-TOTOEt Complex. The line width observed in the 1D 1H NMR spectrum of the d(CGCTAGCG)2-TOTOEt complex is just as narrow as for the TOTOBzl complex, indicating essentially no dynamic exchange between two or more complexes. Furthermore, only one resonance frequency is observed

for the protons on each strand of the dsDNA and for each chromophore. This shows that the TOTOEt forms a single complex with d(CGCTAGCG)2 with dyad symmetry. The d(CGCTAGCG)2-TOTOEt complex arises from bisintercalation in the (5′-CTAG-3′)2 binding site. The amount of this complex is at least 100 times higher than any other complex as seen from inspection of the spectra. A full relaxation matrix analysis employing RAND-

Bis-Intercalation of Thiazole Orange Dye Derivatives

MARDI (Liu et al.,1995) followed by restrained molecular dynamics calculations using cross-peak intensities in NOESY build-up experiments yielded a high-resolution structure of the d(CGCTAGCG)2-TOTOEt complex. This work is reported elsewhere (Petersen and Jacobsen, 1998). DISCUSSION

The sequence selectivity and the binding mode of the TOTO analogues to the dsDNA oligonucleotide examined in this work are very similar to those of TOTO. In the d(CGCTAGCG)2 oligonucleotide, both TOTOEt and TOTOBzl bind exclusively to the (5′-CTAG-3′)2 site. Though the mode of binding and selectivity of TOTOEt, TOTOBzl, and TOTO is similar, there are interesting differences in the local structure of the dsDNA and the ligands. These differences are discussed below. Description of the d(CGCTAGCG)2-TOTOBzl Model Structure. The structure of the d(CGCTAGCG)2TOTOBzl complex shows that TOTOBzl bis-intercalates in the (5′-CTAG-3′)2 site with the benzothiazole ring systems sandwiched in the CpT base pair steps and the quinolinium ring systems sandwiched in the ApG base pair steps. The N-benzyl group on the benzothiazole is placed in the major groove orientated toward the center of the oligonucleotide, and the polyamin(propylene)linker is in the minor groove. The dsDNA is in an overall B-DNA like conformation. As can be gleaned from Figure 2 the nucleobases and the intercalating chromophores are fairly well-defined in the model structure while the N-benzyl group and the polyamin(propylene)linker cannot be accurately determined from the spectral data. The positions in the two grooves of the dsDNA are, however, beyond any doubt. In the complex, the intercalating chromophores stretch across the dsDNA helix; T4 and T12 unstack from A5 and A13 to maximize van der Waals contacts with the intercalating chromophores. The methyl groups of the thymines are placed right on top of the benzothiazoles of TOTOBzl. Cross-strand NOEs between T12 H3 and A5’s amino protons and between the adenosine H2s and the thymidine H1s reveal that normal Watson-Crick base pairing is present in the intercalation site even though the adenosines and thymidines are unstacked. For the uncomplexed d(CGCTAGCG)2, no cross-peaks are observed between the thymine imino proton and the base-paired adenine amino protons due to exchange with the solvent. In the complex, cross-peaks are observed between T12 H3 and A5’s amino protons. This is a testimony of the increased stability of the T12-A5 base pair due to intercalation of the Me-chromophore of TOTOBzl. Intriguingly, no cross-peaks are observed between T4 H3 and A13’s amino protons. This can be explained by analysis of the model structure: A5 and T12 stack well with the ring systems of the intercalating Mechromophore while T4 and A13 do not seem to make any appreciable stacking with the intercalating ring systems of the Bzl-chromophore. Therefore, the stability of the A5-T12 Watson-Crick base pair is enhanced relative to the stability of an A-T base pair in the uncomplexed dsDNA duplex. Comparison of the Complexes of d(CGCTAGCG)2 with TOTO, TOTOEt, and TOTOBzl. Bearing in mind that the model structure of the TOTOBzl complex is not a high-resolution structure, as are the structures determined of the TOTO and TOTOEt complexes, we refrain from discussing more detailed parameters such as helix

Bioconjugate Chem., Vol. 10, No. 1, 1999 71 Table 4. Observed Twist between the Ring Systems of the Aromatic Chromophores in the TOTO, TOTOBzl, and TOTOEt Complexes and the Chemical Shift Values of the Protons Discussed in the Text twist anglea (deg)

thymidine H1′ b

∆δc

thymidine H6b

∆δc

22 6 1 25

4.62 4.96 5.35 4.62

0.98 0.64 0.25 0.98

6.97 7.08 7.20 6.96

0.44 0.33 0.21 0.45

TOTO TOTOEt TOTOBzl TOTOBzl

a Twist angle between the two ring systems of the intercalating chromophore. b Chemical shift value of the specified proton on the thymidine adjacent to the intercalating chromophore, values are given (ppm). c Change in chemical shift relative to the free dsDNA.

parameters. Instead, we are making a qualitative comparison of the three complexes. In the three complexes, the dsDNA adopts an overall B-DNA-like geometry. A striking feature of the three complexes is the change in the angle around the cyanine methine bond of the intercalators (Figure 2). This angle defines the angle of the benzothiazole relative to the quinolinium ring. It appears that the substitution of ethyl and benzyl at the benzothiazole N-atom lowers this angle in the complexes relative to the complexes where a N-methyl is present at the benzothiazole (see Table 4). As for chemical shifts, pronounced changes from free dsDNA are observed for the thymidine H6, H1′, H2′, H2′′, and the adenosine H2 protons for the three complexes discussed. These protons are placed in the minor groove of dsDNA, and with the charged polyamin(propylene)linker of the bis-intercalators “intruding” in this groove, it is to be expected that the electrostatic potential of the minor groove is profoundly altered in the complexes, resulting in changes of chemical shift values of protons positioned herein. The thymidine H6 and H1′ protons, however, seem to occupy exceptional positions as shown by the much different chemical shifts values for the three complexes (see Table 4). The relationship between the value of the abovementioned twist angle and the thymidine H6 and H1′ chemical shifts is evident as seen from Table 4. Although it appears that no simple relationship can account for the observations, the qualitative trend is clear that a large twist angle between the chromophores of the bisintercalator leads to a large change in chemical shift for the thymidine H6 and H1′ protons relative to free dsDNA. Most striking are the results obtained for the TOTOBzl complex where each of the two intercalating chromophores display profoundly different behavior. As the twist angle between the ring systems of the chromophores is enlarged, the overlap of the π-orbitals of the two ring systems diminishes, resulting in a quenching of the flow of π-electrons between the two ring systems, thus changing the ring current of the intercalating chromophore. As neither the H2′ nor H2′′ protons of the thymidine experience these different changes in chemical shifts it would seem that the change in “environment” for the H6 and H1′ protons is a local feature. This can be rationalized by the intercalating aromatic chromophore inducing a change in the ring current of the thymine ring and should be expected only to affect the protons in close proximity to this ring (Davies, 1978). CONCLUSION

We have examined the complexes between d(CGCTAGCG)2 and the bis-intercalators TOTOEt and TOTOBzl

72 Bioconjugate Chem., Vol. 10, No. 1, 1999

resulting in a high-resolution structure of the d(CGCTAGCG)2-TOTOEt complex and a model structure of the d(CGCTAGCG)2-TOTOBzl complex. We have compared these complexes with the previously published highresolution structure of the d(CGCTAGCG)2-TOTO complex (Spielmann et al., 1995). Though the overall binding of the bis-intercalators are alike with nearest neighbor bis-intercalation in the (5′-CTAG-3′)2 site, we are able to point out pronounced changes in the structure of the dsDNA upon binding of the different bis-intercalators. There are no indications whatsoever that TOTOBzl or TOTOEt make less stable complexes than TOTO when bound to dsDNA. Our long-term objective of this work is to move the linker of the bis-intercalator from the minor groove to the major groove while retaining TOTO’s sequence selectivity. The structure of the TOTOEt complex reveals that the benzothiazole N-ethyl group adopts a geometry near to perfect for the linking of the two chromophores in the major groove while the TOTOBzl complex shows that there is ample room to accommodate a benzyl group. This, thus, renders our goal a distinct future possibility. EXPERIMENTAL PROCEDURES

Synthesis. 3-Ethyl-2-(methylthio)benzothiazolium Tetrafluoroborate (3). Triethyloxonium tetrafluoroborate (1.90 g, 0.01 mol) in CHCl3 (10 mL) is added with stirring to 2-(methylthio)benzothiazole (1.81 g, 0.01 mol) in CHCl3 (50 mL) at -10 °C. The temperature was allowed to increase to 20 °C, and stirring was continued for 5 h. The precipitate was collected by filtration; yield 2.26 g (75%); mp 172-175 °C. 3-Benzyl-2-(ethylthio)benzothiazolium Tetrafluoroborate (4). A mixture of 2-(methylthio)benzothiazole (18.1 g, 0.1 mol) and benzyl bromide (51.1 g, 0.3 mol) was heated at 100 °C for 12 h. After cooling to roomtemperature, ether (200 mL) was added under stirring and the precipitate of 2 was collected by filtration; yield 13.2 g (51%); mp 150-152 °C; lit. mp 149 °C (Reed et al., 1939). Et3OBF4 (1.90 g, 0.01 mol) in 50 mL CH2Cl2 was added dropwise to 2 (2.6 g, 0.01 mol) in 50 mL of CH2Cl2 at -10 °C with stirring. The temperature was allowed to increase to room temperature during 1 h and the mixture was stirred for 5 h. Compound 4 precipitated as pale yellow crystals on addition of ether; yield 3.3 g (88%); mp 165-168 °C. 1H NMR (DMSO-d6): δ 1.56 (t, 3H, J ) 7.3 Hz, CH3), 3.68 (q, 2H, J ) 7.3 Hz, CH2), 5.95 (s, 2H, CH2), 7.31-8.45 (m, 9H, Ar). FAB MS: m/z 286 (M - BF4). Anal. Calcd for C16H16NS2‚BF4: C, 51.49; H, 4.32; N, 3.75. Found: C, 51.65; H, 4.09; N, 3.81. 4-[(3-Ethyl-2(3H)-benzothiazolylidene)methyl]-1-(3iodopropyl)quinolinium Tetrafluoroborate (6a). Equimolar amounts of 3 and 5 were dissolved in absolute ethanol by heating. One equivalent of triethylamine was added, and the mixture was stirred at room temperature for 30 min. The title compound precipitated on addition of ether; yield 70%; recrystallization from acetone/ether; mp 250252 °C. 1H NMR (DMSO-d6): δ 1.40 (t, 3H, J ) 7.0 Hz, CH3), 2.39 (quint, 2H, J ) 7.0 Hz, CH2), 3.35 (t, 2H, J ) 7.0 Hz, CH2), 4.65 (m, 4H, 2 × CH2), 6.94 (s, 1H, dCH), 7.37-8.80 (m, 10H, Ar). FAB MS m/z 473 (M - BF4). Anal. Calcd for C22H22IN2S‚BF4‚0.5H2O: C, 46.42; H, 4.07; N, 4.92. Found: C, 46.65; H, 3.91; N, 4.89. 4-[(3-Benzyl-2(3H)-benzothiazolylidene)methyl]-1-(3iodopropyl)quinolinium Tetrafluoroborate (6b). The title compound was synthesized from equimolar amounts of 4 and 5 in a similar way as 6a; yield 65%; mp 237-247 °C (dec). 1H NMR (DMSO-d6): δ 2.38 (quint, 2H, J ) 7.0

Petersen et al.

Hz, CH2), 3.33 (t, 2H, J ) 7.0 Hz, CH2), 4.64 (t, 2H, J ) 7.0 Hz, CH2), 5.96 (s, 2H, CH2), 6.99 (s, 1H, dCH), 7.298.64 (m, 15H, Ar). FAB MS: m/z 535 (M - BF4). Anal. Calcd for C27H24IN2S‚BF4‚0.5H2O: C, 51.37; H, 3.99; N, 4.44. Found: C, 51.56; H, 3.91; N, 4.44. 1,1′-[1,3-Propanediylbis[(dimethylimino)-3,1-propanediyl]]bis[4-[(3-ethyl-2(3H)-benzothiazolylidene)methyl]quinolinium Tetraiodide, TOTOEt. A suspension of 6a and 0.5 equiv of N,N,N′,N′-tetramethyl-1,3-propanediamine in anhydrous methanol was heated at reflux for 24 h. Recrystallization from DMF/MeOH afforded the title compound as an orange-red solid: yield 85%; mp 248-251 °C (dec). 1H NMR (DMSO-d6): δ 1.42 (t, 6H, J ) 6.8 Hz, 2 × CH3), 2. 38 (m, 6H, 3 × CH2), 3.19 (s, 12H, 4 × CH3), 3.36 (m, 4H, 2 × CH2), 3.71 (m, 4H, 2 × CH2), 4.68 (m, 8H, 4 × CH2), 6.93 (s, 2H, 2 × dCH), 7.35-8.82 (m, 20H, Ar). FAB MS: m/z 1203 (M - I). Anal. Calcd for C51H62I4N6S2: C, 46.03; H, 4.70; N, 6.31. Found: C, 46.28; H, 4.85; N, 6.10. 1-[3-[[3-[Dimethyl[3-[4-[(3-methyl-2(3H)-benzothiazolylidene)methyl]quinolinio]-propyl]ammonio]propyl]dimethylammonio]propyl]-4-[(3-benzyl-2(3H)-benzothiazolylidene) - methyl]quinolinium Tetraiodide, TOTOBzl. Equivalent amounts of 6b and 7a in anhydrous methanol were refluxed for 12 h. TOTOBzl was precipitated by addition of ether and recrystallized from acetone/ether: yield 90%; mp 228-230 °C (dec). 1H NMR (DMSO-d6): δ 2.38 (m, 6H, 3 × CH2), 3.19 (s, 12H, 4 × CH3), 3.40 (m, 4H, 2 × CH2), 3.72 (m, 4H, 2 × CH2), 4.01 (s, 3H, CH3), 4.70 (m, 4H, 2 × CH2), 5.97 (s, 2H, CH2), 6.90 (s, 1H, dCH), 6.97 (s, 1H, dCH), 7.30-8.90 (m, 25H, Ar). FAB MS: m/z 1251 (M - I). Anal. Calcd for C55H62I4N6S2‚4H2O: C, 45.53; H, 4.86; N, 5.79. Found: C, 45.49; H, 4.89; N, 5.81. Sample Preparation. The purified oligonucleotide d(CGCTAGCG)2 was purchased from DNA technology, Århus, Denmark, and used without further purification. TOTOEt and TOTOBzl are almost insoluble in water and the complexes with dsDNA was therefore made by the procedure described earlier (Jacobsen et al., 1995). The NMR samples were prepared by dissolving the complexes in 0.5 mL of 10 mM sodium phosphate buffer (pH 7.0), 0.05 mM NaEDTA, 0.01 mM NaN3, and 0.1 mM DSS. For experiments carried out in D2O the solid complexes, lyophilized three times from D2O, were redissolved in 99.96% D2O (Cambridge Isotope Laboratories). A mixture of 90% H2O and 10% D2O (0.5 mL) was used for experiments examining exchangeable protons. The samples were kept in NMR tubes under nitrogen. The final concentration of the complexes were ca. 2 mM. NMR Experiments. NMR experiments were performed on a Varian Unity 500 spectrometer at 25 °C for the TOTOBzl complex and at 27 °C for the TOTOEt complex. NOESY spectra of the complexes were acquired in D2O using 1024 complex points in t2 and a spectral width of 5000 Hz. A total of 512 t1 experiments were recorded using the States phase cycling scheme. The residual signal from HOD was removed by presaturation. For the TOTOEt complex, spectra with mixing times of 50, 100, 150, and 200 ms were recorded sequentially without removing the sample from the magnet. NOESY spectra of the TOTOBzl complex with mixing times of 100 and 200 ms were acquired at 750 MHz using 2048 complex points in t2 and a spectral width of 7500 Hz on a Varian INOVA 750 spectrometer. NOESY spectra of the complexes in H2O were obtained by using a NOESY pulse sequence where the last 90° pulse is replaced by a 1-1 jump-return pulse to suppress the solvent signal (Gue´ron and Plateau, 1982). The mixing times were 160 ms. In addition, a Watergate NOESY spectrum in H2O

Bis-Intercalation of Thiazole Orange Dye Derivatives

with a mixing time of 50 ms was recorded of the TOTOEt complex on a Varian Unity+ 500 spectrometer. The NOESY spectra in H2O were acquired with a spectral width of 10 000 Hz in 2048 complex points using 512 t1 experiments with 64 scans acquired in each t1 experiment. NOESY spectra with mixing times of 500 ms were recorded of the free TOTOBzl and TOTOEt in DMSOd 6. TOCSY spectra of the complexes with mixing times of 30 and 90 ms were obtained in the TPPI mode using 1024 complex points in t2 and 512 t1 experiments with 64 scans each. DQF-COSY spectra of the complexes were recorded using the States phase cycling scheme with 4096 complex points in t2 and 1024 t1 experiments with 64 scans each. Removal of the residual HOD signal was done by presaturation in all spectra. 1 H,13C HSQC spectra in D2O were recorded for both the complexes: the spectrum of the TOTOEt complex on a Varian Unity+ 500 spectrometer and the spectrum of the TOTOBzl complex on a Varian INOVA 750 spectrometer. The acquired data were processed using FELIX (version 2.35, Biosym/MSI, San Diego, CA). The spectra were assigned by conventional methods (Wu¨thrich, 1986; Feigon et al., 1983; Hare et al., 1983; Scheek et al., 1983, 1984; Boelens et al., 1985). Restrained Molecular Dynamics. The d(CGCTAGCG)2 oligonucleotide and the TOTOBzl molecule were built in INSIGHTII (version 95.0, Biosym/MSI, San Diego, CA). TOTOBzl was manually docked into the model of B-form DNA to obtain the starting model for the structure refinement. The potentials for both DNA and TOTOBzl were assigned by the AMBER force field, modified to accommodate the benzothiazole sulfur atom of TOTOBzl. The distance restraints obtained from RANDMARDI were incorporated into an energy minimization and a RMD procedure using DISCOVER (version 95.0, Biosym/ MSI, San Diego, CA). An initial energy minimization was followed by 28 ps of restrained molecular dynamics: dynamics at 600 K for 4 ps, followed by cooling to 200 K in 50 K steps of 3 ps each. The final structure was then energy minimized to a maximum derivative of 0.01 Å. The pseudo energy terms used to enforce the restraints were

{

k1(r - r1)2 when r < r1 when r1 e r e r2 ENOE ) 0 k2(r - r2)2 when r > r2 where r1 and r2 are the lower and upper distance bounds determined from the cross-peak volumes; k1 and k2 are the force constants. A lower force constant of k1 ) 50 kcal/ (mol Å2) and upper force constant of k2 ) 25 kcal/(mol Å2) were employed. The width of the flat region of the potential well reflects the accuracy of a given constraint. A distance dependent dielectric,  ) r, was employed to account for solvent effects. Distance Restraints. The Initial Model. An initial model of the d(CGCTAGCG)2-TOTOBzl complex was made with restraints found from the NOESY spectrum with a mixing time of 100 ms. Cross-peak volumes were converted to proton-proton distances by the isolated spin pair approximation (ISPA) using the volumes of covalently fixed dsDNA protons as references (cytosine H5-H6, 2.45 Å; deoxyribose H2′-H2′′, 1.78 Å). All lower bounds were set to 1.8, and 1 Å was added to the

Bioconjugate Chem., Vol. 10, No. 1, 1999 73

distances determined to obtain the upper bounds. Distance restraints involving methyl and methylene protons were further loosened. In this manner, a total of 81 distance restraints were obtained (25 of these were intermolecular restraints). A further 22 distance restraints were included to mimic Watson-Crick base pairing. Three hydrogen bonds were included for each of the six C/G base pairs and two for both of the T/A base pairs. Lower and upper bounds were 1.7 and 2.1 Å, respectively. Thus, a total of 103 distance restraints were included in the RMD calculations. A total of 20 structures were generated by the RMD protocol described above, all structures converged to just one family of structures. A representive structure of this family was chosen for further refinement by including restraints obtained by including a relaxation matrix analysis. Relaxation Matrix Analysis. A total of 97 cross-peaks on the upper diagonal of the NOESY spectrum with a mixing time of 200 ms were integrated. The RANDMARDI variation of MARDIGRAS was employed to transfer the cross-peak volumes to proton-proton distance restraints taking spin diffusion into account (Borgias and James, 1990; Borgias et al., 1990; Liu et al., 1995). The RANDMARDI procedure generated 50 peak intensity sets and MARDIGRAS calculations were performed on each of those. A “noise level” of five times the measured volume of the weakest cross-peak was used in the RANDMARDI calculaions. Upper and lower bounds were calculated as the average interproton distance ( one standard deviation as found from the MARDIGRAS calculations. A total of 97 cross-peak volumes were measured in FELIX; of those, 88 cross-peak volumes stemmed from interproton distances not detemined by the covalent geometry of neither the dsDNA nor the TOTOBzl. RANDMARDI returned 87 interproton distances for use in the RMD calculations, and 0.6 Å was added to all upper bounds for these distances before implementation as restraints. The average flat well potential width was 1.34 Å. ACKNOWLEDGMENT

We thank The Instrumentcenter for NMR spectroscopy of Biological Macromolecules at The Carlsberg Laboratory, Copenhagen, granted by The Danish Natural Science Research Council for providing spectrometer time at the 750 MHz spectrometer. LITERATURE CITED Benson, S. C., Singh, P., and Glazer, A. N. (1993a) Heterodimeric DNA-binding dyes designed for energy transfer: synthesis and spectroscopic proporties. Nucleic Acids Res. 21, 5727-5735. Benson, S. C., Mathies, R. A., and Glazer, A. N. (1993b) Heterodimeric DNA-binding dyes designed for energy transfer: stability and applications of DNA complexes. Nucleic Acids Res. 21, 5720-5726. Boelens, R., Scheek, R. M., Dijkstra, K., and Kaptein, R. (1985) Sequential Assigment of Imino- and Amino-Proton Resonances in 1H NMR Spectra of Oligonucleotides by TwoDimensional NMR Spectroscopy. Application to lac Operator Fragment. J. Magn. Reson. 62, 378-386. Brooker, L. G. S., White, F. L., Keyes, G. H., Smyth, C. P., and Oesper, P. E. (1941) Color and constitution. II. Absorptions of some related vinylene-homologous series. J. Am. Chem. Soc. 62, 3192-3203. Brooker, L. G. S., Keyes, G. H., and Williams, W. W. (1942) Color and constitution. V. The absorption of unsymmetrical cya-

74 Bioconjugate Chem., Vol. 10, No. 1, 1999 nines. Resonance as a basis for a classification of dyes. J. Am. Chem. Soc. 63, 199-210. Davies, D. B. (1978) Conformations of Nucleosides and Nucleotides. Prog. NMR Spectrosc. 12, 135-225. Faridi, J., Nielsen, K. E., Stein, P. C., and Jacobsen, J. P. (1997) Bis-Intercalation of a Homodimeric Thiazole Orange Dye in DNA: Dynamic Exchange between Two Sites. J. Biomol. Struct. Dyn. 15, 321-331. Feigon, J., Leupin, W., Denny, W. A., and Kearns, D. R. (1983) Two-Dimensional Proton Nuclear Magnetic Resonance Investigation of the Synthetic Deoxyribonucleic Acid Decamer d(ATATCGATAT)2. Biochemistry 22, 5943-5951. Gue´ron, M., and Plateau, P. (1982) Exchangeable Proton NMR without Base-Line Distortion, Using New Strong-Pulse Sequences. J. Am. Chem. Soc. 104, 7310-7311. Hansen, L. F., Jensen, L. K., and Jacobsen, J. P. (1996) Bisintercalation of a homodimeric thiazole orange dye in DNA in symmetrical pyrimidine-pyrimidine-purine-purine oligonucleotides. Nucleic Acids Res. 24, 859-867. Hare, D. R., Wemmer, D. E., Chou, S.-H., Drobny, G., and Reid, B. R. (1983) Assignment of the nonexchangeable proton resonances of d(C-G-C-G-A-A-T-T-C-G-C-G) using two-dimensional nuclear magnetic resonance methods. J. Mol. Biol. 171, 319-336. Jacobsen, J. P., Pedersen, J. B., Hansen, L. F., and Wemmer, D. E. (1995) Site selective bis-intercalation of a homodimeric thiazole orange dye in DNA oligonucleotides. Nucleic Acids Res. 23, 753-760. Petersen, M., and Jacobsen, J. P. (1998) Solution Structure of a DNA Complex with the Fluorescent Bis-Intercalator TOTO Modified on the Benzothiazole Ring. Bioconjugate Chem. 9, 331-340.

Petersen et al. Reed, F. P., Robertson, A., and Sexton, W. A. (1939) Reaction of benzthiazole derivatives. Part II. The Conversion of 1-Alkylthiobenzthiazoles into 1-Thio-2-alkyl-1: 2-dihydrobenzthiazoles. J. Chem. Soc. 473-476. Rye, H. S., Yue, S., Wemmer, D. E., Quesada, M. A., Haugland, R. P., Mathies, R. A., and Glazer, A. N. (1992) Stable fluorescent complexes of double-stranded DNA with bisintervalating asymmetric cyanine dyes: proporties and applications. Nucleic Acids Res. 20, 2803-2812. Scheek, R. M., Russo, N., Boelens, R., and Kaptein, R. (1983) Sequential Resonance Assigments in DNA 1H NMR Spectra by Two-Dimensional NOE Spectroscopy. J. Am. Chem. Soc. 105, 2914-2916. Scheek, R. M., Boelens, R., Russo, N., Van Boom, J. H., and Kaptein, R. (1984) Sequential Resonance Assigments in 1H NMR Spectra of Oligonucleotides by Two-Dimensional NMR Spectroscopy. Biochemistry 23, 1371-1376. Spielmann, H. P., Wemmer, D. E., and Jacobsen, J. P. (1995) Solution Structure of a DNA Complex with the Fluorescent Bis-Intercalator TOTO Determined by NMR Spectroscopy. Biochemistry 34, 8542-8553. Stærk, D., Hamed, A. A., Pedersen, E. B., and Jacobsen, J. P. (1997) Bis-Intercalation of Homodimeric Thiazole Orange Dyes in DNA: The Effect of Modifying the Linker. Bioconjugate Chem. 8, 869-877. Wu¨thrich, K. (1986) NMR of Proteins and Nucleic Acids, John Wiley & Sons, New York.

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