Synthesis of Naphthyridine Carbamate Dimer (NCD) Derivatives

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Synthesis of Naphthyridine Carbamate Dimer (NCD) Derivatives Modified with Alkanethiol and Binding Properties of G−G Mismatch DNA Takeshi Yamada, Shouta Miki, Anisa Ul’Husna, Akiko Michikawa, and Kazuhiko Nakatani* Department of Regulatory Bioorganic Chemistry, The Institute of Scientific and Industrial Research (ISIR), Osaka University, Mihogaoka 8-1, Ibaraki 567-0047, Japan S Supporting Information *

ABSTRACT: A series of new DNA binding molecules NCD−Cn−SH (n = 3, 4, 5, and 6) is reported, which possesses the NCD (naphthyridine carbamate dimer) domain selectively binding to the G−G mismatch in the 5′-CGG-3′/5′-CGG-3′ sequence and a thiol moiety, which undergoes spontaneous dimerization to (NCD−Cn−S)2 upon oxidation under aerobic conditions. The S− S dimer (NCD−Cn−S)2 produced the 1:1 binding complex with improved thermal stability. The dimer binding to the CGG/ CGG DNA showed higher positive cooperativity than the binding of monomer and previously synthesized NCTn derivative. The dimerization of NCD−Cn−SH was selectively accelerated on the CGG repeat DNA but not on the CAG repeat DNA.

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molecular tool to investigate fragile X syndrome (FXS),19 FXTAS,20 and FXPOI,21 which were associated with pathological expansion of CGG repeats in FMR1.22 Despite the improved binding properties, the large molecular size and hydrophobicity of Z-NCTS and other NCT derivatives are disadvantages to utilize them for the biological studies. In the development of low molecular weight drugs, it is known that highly hydrophobic compounds often have problems on ADME properties, particularly absorption.23,24 To take advantage of Z-NCTS and NCTn derivatives25,26 with higher affinity than the monomer and to circumvent the issues of large molecular size and low hydrophilicity, we planned in situ dimerization of thiol derivative of NCD by spontaneous oxidation. Thus, we anticipated that the oxidation of thiol derivatives of NCD under aerobic conditions in aqueous solution should provide a NCD dimer through a disulfide linkage, which might show comparable binding properties to those characteristics of NCT derivatives. It was also anticipated that CGG/CGG triad would serve as a reaction template to accelerate the oxidative dimerization of NCD−Cn−SH (Figure 1b). Based on these assumptions, a series of NCD−Cn−SH (n = 3, 4, 5, or 6) derivatives (Figure 1a) that have an alkyl thiol group were designed. Molecular modeling simulations of the complex consisted of two NCD−C4−SHs, and a G−G mismatch DNA (Figure S1b) revealed that two thiol groups

rinucleotide repeat diseases are neurodegenerative disorders caused by excessive expansion of genetically unstable 5′-CNG-3′ (N = A, C, G, and T) sequences in genome.1−3 Several small molecules binding to those trinucleotide repeats DNAs and RNAs were reported.4−12 We previously reported a small molecule named naphthyridine carbamate dimer NCD (Figure 1a), which strongly bound to the G−G mismatch in the 5′-CGG-3′/5′-CGG-3′ triad,13 possibly produced in a hairpin secondary structure of CGG repeat DNAs.14 In the CGG/CGG triad, two NCD molecules bound to four guanines by disrupting Watson−Crick hydrogen bonding of two C−G base pairs and a G−G mismatch. NMR15 and cold spray ionization (CSI) TOF-MS analysis,16 and the increased reactivity of flipped out cytosines toward hydroxylamine17 revealed that two NCD domains bound to four guanines in the CGG/CGG triad to force the widowed cytosine to flip out. A series of naphthyridine tetramer NCTn16 and ZNCTS18 (Figure 1a), in which two NCD moieties were connected by an aliphatic linker or Z-stilbene, were synthesized to improve the affinity, and Z-NCTS was found to show stronger binding to the G−G mismatch than NCD,18 most likely due to the positive cooperative binding of four naphthyridine units in two NCD domains to the CGG/CGG triad by the spatial arrangement of two NCD domains in close vicinity to the binding site. This speculation was supported by the failure of detection of 1:1 bound complex, which underwent subsequent binding of a second NCD molecule to form the 2:1 complex (Figure 1b). The strong binding property of NCD and Z-NCTS to the CGG repeat DNAs implicates the potential as a © XXXX American Chemical Society

Received: May 31, 2017

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DOI: 10.1021/acs.orglett.7b01632 Org. Lett. XXXX, XXX, XXX−XXX

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(retention time 15 min) (Figure S2) spontaneously started to produce a product at the retention time of 18 min, which was identified as the disulfide (NCD−C4−S)2 by MS (m/z = 1181.9, calcd [M + H]+ 1181.5). The rate for the dimerization was determined by assuming the second order kinetics and described in the Supporting Information. Among four derivatives, NCD−C3−SH undergoes dimerization the most rapidly and NCD−C5−SH was the slowest under the conditions, although the difference in the rate is within 5-fold. Next, the oxidative dimerization of NCD−C4−SH (40 μM) was investigated in the presence of repeat DNAs 5′-(CXG)9-3′ (X = G or A, 5 μM), which could form a hairpin secondary structure involving X−X mismatch in the CXG/CXG triad.14 The decrease in the amount of NCD−C4−SH was significantly faster in the presence of the CGG repeat DNA (Figure 2c, blue

Figure 1. (a) Hydrogen bonding pattern of NCD derivatives to two guanines in the G−G mismatch DNA and compounds used in these studies. (b) Schematic illustration of cooperative binding of two NCD derivatives and template assisted S−S dimer formation.

of two NCD−C4−SHs were close enough to form a disulfide linkage, and the dimer (NCD−C4−S)2 is likely to form a stable 1:1 complex with the G−G mismatch DNA (Figure S1c). We here describe the preparation and the binding properties of these NCD−Cn−SH (n = 3, 4, 5, or 6) derivatives under redox conditions. These studies revealed accelerated dimerization of NCD−Cn−SH selectively on the CGG/CGG triad and the formation of the 1:1 complex with high cooperativity as judged by the tangent angle in the UV-melting profile. These in situ dimerizations of ligands on the target repeat DNA could be a potentially useful strategy for the molecular design in repeat targeting. Preparation of NCD−Cn−SH is summarized in Scheme 1. Aldehydes having trityl-protected thiol (see Supporting

Figure 2. RP-HPLC profiles of NCD−C4−SH in the presence of 5′(CGG)9-3′ repeat DNA in aerobic conditions at 37 °C after (a) 0 and (b) 4 h detected at 260 nm. Reaction conditions: NCD−C4−SH (40 μM), 5′-(CGG)9-3′ (5 μM), sodium cacodylate buffer (10 mM, pH 7.0), NaCl (100 mM), internal standard (*, adenosine 20 μM), and 0.1% Tween20. HPLC: 0.1% TFA and MeCN were eluted 1 mL/min through an ODS-end-capped column. The percentage of MeCN was changed from 0 to 40 gradually for 20 min. The eluted compounds were detected at 260 nm by PDA. (c) Plot of [NCD−C4−SH] in the absence of DNA (red) and in the presence of NaCl (100 mM) and 5′(CGG)9-3′ (5 μM, blue) or 5′-(CAG)9-3′ (5 μM, green).

Scheme 1

line) than the reaction without DNA (Figure 2c, red line; Figure S2), while no significant acceleration was observed in the presence of nontarget CAG repeat DNA (Figure 2c, green line; Figure S5). In addition to the accelerated rate for the dimerization, the efficiency of the reaction is also improved. Most of NCD−C4−SH was transformed into the dimer after 6 h in the presence of the CGG repeat DNA, whereas about 50% of the monomer remained without DNA and with the CAG repeat DNA. These results clearly indicated that the oxidative dimerization of NCD−C4−SH was accelerated selectively by the presence of the CGG repeat DNA. It is highly likely that the biding of two NCD−C4−SHs at the CGG/CGG site arranged two thiols in close vicinity with the geometry suitable for the oxidative dimerization. The hydrophilicity of molecules was assessed by the retention time on RP-HPLC using a ODS-end-capped column and 0.1% TFA and CH3CN as eluents.28 The retention time of

Information) were coupled with NCD under reductive amination conditions with NaBH3CN. Subsequent deprotection of the trityl group was carried out with triethylsilane to finish the synthesis.27 First, the oxidation reactions of NCD− C4−SHs leading to the corresponding S−S dimer were investigated under aerobic conditions by reverse phase HPLC (RP-HPLC) equipped with a photo diode array (PDA) and analyzed by electrospray ionization mass spectrometry (ESIMS) or MALDI-TOF-MS. The solution of freshly prepared NCD−C4−SH (40 μM, m/z = found 592.2694, calcd [M + H]+ 592.2700) in an aqueous buffer solution (pH 7) was left exposed to the air at 37 °C. Dimerization of NCD−C4−SH B

DOI: 10.1021/acs.orglett.7b01632 Org. Lett. XXXX, XXX, XXX−XXX

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−S−S− dimer during the measurements (Figure 4). The reported melting temperatures (tm) were the average of three independent measurements and determined as the temperature showing the highest tangent angle (TA), which was determined by plotting the slope of the line connecting every two data points (Table 1).

NCD−C4−SH, (NCD−C4−S)2, NCT8, and Z-NCTS were about 16, 18, 23, and 18 min, respectively (Figure S6), showing that NCD−C4−SH is more hydrophilic than NCT8 and ZNCTS. The stoichiometry for the binding of NCD−Cn−SH and (NCD−Cn−S)2 to the G−G mismatch in the 5′-CGG-3′/5′CGG-3′ sequence was determined by CSI-TOF-MS. The CSITOF-MS analysis of the mixture of NCD−C4−SH and the G− G mismatch DNA 5′-d(TCA ACG GTT GA)-3′/3′-d(AGT TGG CAA CT)-5′ showed the 5− ion of the ligand-bound DNA complex in 2:1 binding stoichiometry [2NCD−C4−SH· DNA]5− (m/z 1578.0593). The CSI-MS analysis of the solution of the dimer (NCD−C4−S)2 and the DNA also showed the 5− ion (m/z 1577.6612). The difference between two 5− ions in m/z was 0.3981, suggesting the formation of the 1:1 complex (NCD−C4−S)2·DNA having a difference of two hydrogens (in theory, 0.4 at the 5− state) from the 2:1 bound complex 2NCD−C4−SH·DNA of the monomer (Figure 3).

Table 1. TA and Δtm Values for the DNA Containing a G−G Mismatch in the Absence or the Presence of Compoundsa compd

TAb (°C−1)

tm (°C)

SD

Δtmc (°C)

NCD NCT8d Z-NCTSd NCD−C3−SHe (NCD−C3−S)2d NCD−C4−SHe (NCD−C4−S)2d NCD−C5−SHe (NCD−C5−S)2d NCD−C6−SHe (NCD−C6−S)2d

0.010 0.013 0.013 0.031 0.015 0.011 0.012 0.027 0.013 0.019 0.014 0.027

32.6 54.6 53.0 54.7 51.6 34.3 55.7 58.0 51.4 59.1 51.2 59.1

2.5 0.3 2.7 0.2 0.1 0.8 0.1 0.7 0.3 1.3 0.3 1.1

22.0 20.4 22.1 19.0 1.7 23.1 25.4 18.8 26.5 18.6 26.5

a UV-melting profiles for the 5 μM DNA 5′-(TCAA CGG TTGA)-3′/ 3′-(AGTT GGC AACT)-5′ in the absence or the presence of each compound were measured in 10 mM sodium cacodylate buffer (pH 7.0) containing 100 mM NaCl, and 0.1% Tween 20. bThe calculation method for tangent angle (TA) was described in Supporting Information. cΔtm = tm − tm (w/o ligand). dCompound concentration was 20 μM. eCompound concentration was 40 and 60 μM TCEP was added in addition to the shown above.

Figure 3. CSI-TOF-MS analysis of the G−G mismatch DNA (5′TCAA CGG TTGA-3′/3′-AGTT GGC AACT-5′) (10 μM) in the presence of NCD−C4−SH (40 μM) (red) and (NCD−C4−S)2 (20 μM) (blue) measured in 1:1 MeOH/H2O (v/v) containing NH4OAc (100 mM). The region of the 5− ion of 2:1 adducts was shown. The full range spectra are shown in Figure S6.

The effects of each compound on the stability of the CGG/ CGG DNA were evaluated by the Δtm value. The Δtm value obtained for NCD was 22.0 °C, and those for NCD−Cn−SH were 19.0 (n = 3), 23.1 (n = 4), 18.8 (n = 5), and 18.6 (n = 6) °C, respectively, suggesting that modification at the secondary amino group in NCD showed small effects on the thermodynamic stability of the 2:1 complex. In contrast to the monomer, the dimer showed marked structure-dependent stability of the 1:1 complex. While (NCD−C3−S)2 demonstrated only a negligible Δtm value (1.7 °C), possibly due to the thermal degradation of (NCD−C3−S)2 under the measurements, the other three dimers showed higher Δtm values 25.4 (n = 4), 26.5 (n = 5), and 26.5 (n = 6) as compared with 22.0, 20.4, and 22.1 °C obtained for NCD, NCT8, and Z-NCTS, respectively (Table 1).

NCD−Cn−SH and (NCD−Cn−S)2 were also determined by CSI-TOF-MS to produce 2:1 and 1:1 binding complexes with the CGG/CGG DNA, respectively (Figure S8). All m/z values of complexes were summarized in Table S1. Having confirmed the superior hydrophilicity of NCD−Cn− SH and (NCD−Cn−S)2 to NCT8 and Z-NCTS and the 1:1 binding stoichiometry to the CGG/CGG DNA, we then analyzed the cooperativity of four naphthyridine heterocycles in the binding to the guanine base from the UV-melting profiles. For the UV-melting measurements of NCD−Cn−SH, a reducing agent tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added to the solution to avoid the oxidation to the

Figure 4. UV-melting profiles of the G−G mismatch DNA (open circle) and tangent angle (filled circle) of the UV-melting profiles in the presence or absence of the ligand. The highest TA is represented by a bar. (a) DNA only (black) and DNA with NCD (green); Z-NCTS (orange); (b) NCD−C4−SH (blue), (NCD−C4−S)2 (red); (c) NCD−C5−SH (blue), (NCD−C5−S)2 (red); (d) NCD−C6−SH (blue), (NCD−C6−S)2 (red). C

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The characteristic aspects of the UV-melting profiles for the S−S dimer gave steeper increase in the absorbance against temperature than the monomer, suggesting positive cooperativity for the binding of four naphthyridine units in two NCD moieties. TAs for the monomer were almost the same as that for NCD and NCT8 of 0.013, suggesting the mode of the binding is not affected by the modification as we speculated from stoichiometry analysis. In contrast, those for S−S dimers were 0.027 (n = 4), 0.019 (n = 5), and 0.027 °C−1 (n = 6), showing about 1.5−2.1 times larger TA than that for NCD, NCT8, and monomers. Z-NCTS showed the largest TA value (0.031 °C−1) among molecules we investigated. These data strongly supported that the dimerization of NCD−Cn−SH is positively effective for the binding of four naphthyridine units to the four guanine bases in the CGG/CGG DNA. In conclusion, NCD−Cn−SH showed superior hydrophilicity to NCT8 and Z-NCTS and efficient binding to the CGG/CGG DNA upon spontaneous dimerization under aerobic conditions. The oxidative dimerization was selectively accelerated in the presence of the CGG repeat DNA. While these results would be one of marked examples of DNA− template organic synthesis,29−31 much more important aspects of NCD−Cn−SH were disclosed by these studies. Thus, the monomeric NCD−Cn−SH may have a chance to accumulate on the CGG repeat DNA by the accelerated oxidation to the S−S dimer leading to the formation of the complex with higher thermal stability. These scenario would be attractive for chemical biology studies on the Fragile X syndrome and related diseases caused by CGG repeat expansion not only in vitro but also in more diseases relevant cell models.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b01632. Experimental section and additional figures, schemes, and tables (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Takeshi Yamada: 0000-0003-3275-415X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by JSPS KAKENHI Grant-in-Aid for Specially Promoted Research (26000007) to K.N. and by Young Scientist (B) (15K17885, 17K14516) to T.Y., performed under the Research Program of “Dynamic Alliance for Open Innovation Bridging Human, Environment and Materials” in “Network Joint Research Center for Materials and Devices”. The authors would like to thank Dr. Chikara Dohno and Mr. Yihuan Lu for providing Z-NCTS.

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REFERENCES

(1) Pearson, C. E.; Edamura, K. N.; Cleary, J. D. Nat. Rev. Genet. 2005, 6, 729−742. (2) Mirkin, S. M. Nature 2007, 447, 932−940. D

DOI: 10.1021/acs.orglett.7b01632 Org. Lett. XXXX, XXX, XXX−XXX