Synthesis and Characterization of Oligodeoxyribonucleotides Modified

Article pubs.acs.org/joc

Synthesis and Characterization of Oligodeoxyribonucleotides Modified with 2′-Amino-α‑L‑LNA Adenine Monomers: High-Affinity Targeting of Single-Stranded DNA Nicolai K. Andersen,†,§ Brooke A. Anderson,‡,§ Jesper Wengel,† and Patrick J. Hrdlicka‡,* †

Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, 5230 Odense, Denmark Department of Chemistry, University of Idaho, Moscow, Idaho 83844-2343, United States

‡

S Supporting Information *

ABSTRACT: The development of conformationally restricted nucleotide building blocks continues to attract considerable interest because of their successful use within antisense, antigene, and other gene-targeting strategies. Locked nucleic acid (LNA) and its diastereomer α-L-LNA are two interesting examples thereof. Oligonucleotides modified with these units display greatly increased affinity toward nucleic acid targets, improved binding specificity, and enhanced enzymatic stability relative to unmodified strands. Here we present the synthesis and biophysical characterization of oligodeoxyribonucleotides (ONs) modified with 2′-amino-α-L-LNA adenine monomers W−Z. The synthesis of the target phosphoramidites 1−4 is initiated from pentafuranose 5, which upon Vorbrüggen glycosylation, O2′-deacylation, O2′-activation and C2′-azide introduction yields nucleoside 8. A one-pot tandem Staudinger/intramolecular nucleophilic substitution converts 8 into 2′-amino-α-L-LNA adenine intermediate 9, which after a series of nontrivial protecting-group manipulations affords key intermediate 15. Subsequent chemoselective N2′-functionalization and O3′-phosphitylation give targets 1−4 in ∼1−3% overall yield over 11 steps from 5. ONs modified with pyrene-functionalized 2′-amino-α-L-LNA adenine monomers X−Z display greatly increased affinity toward DNA targets (ΔTm/modification up to +14 °C). Results from absorption and fluorescence spectroscopy suggest that the duplex stabilization is a result of pyrene intercalation. These characteristics render N2′-pyrene-functionalized 2′-amino-α-L-LNAs of considerable interest for DNA-targeting applications.

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INTRODUCTION Major efforts have been devoted over the past 20 years to the development of conformationally restricted nucleotides. Oligonucleotides that are modified with these building blocks often display markedly increased affinity toward nucleic acid targets, improved discrimination of nontargets, and greater resistance against enzymatic degradation relative to reference strands.1 Such oligonucleotides are accordingly widely used for nucleic acid-targeting applications in molecular biology, biotechnology, and medicinal chemistry.2 Locked nucleic acid (LNA) is one of the most prominent members of this compound class because of its extraordinary affinity toward DNA and RNA complements (Figure 1); thus, increases in the thermal denaturation temperature (Tm) of up to +10 °C per modification have been observed.3−5 The diastereomeric α-LLNA (Figure 1) shares many characteristics with LNA, © 2013 American Chemical Society

including very high affinity toward DNA/RNA targets, but is less well characterized as a result of its more limited commercial availability.6 The interesting properties of LNA, α-L-LNA, and other conformationally restricted nucleotides have spurred the development of many analogues.1,7 As part of our ongoing interest in LNA chemistry and diagnostic applications of pyrene-functionalized oligonucleotides,2c,8 we recently pursued the development of N2′-pyrenefunctionalized 2′-amino-α-L-LNA thymine monomers (Figure 1).9 Oligodeoxyribonucleotides (ONs) modified with these monomers display remarkable affinity toward complementary DNA, as the pyrene moieties are preorganized to intercalate and engage in stacking with neighboring nucleobases upon Received: October 13, 2013 Published: December 4, 2013 12690

dx.doi.org/10.1021/jo4022937 | J. Org. Chem. 2013, 78, 12690−12702

The Journal of Organic Chemistry

Article

Figure 1. Structures of LNA, α-L-LNA, and the 2′-amino-α-L-LNA monomers studied herein. T = thymin-1-yl.

Scheme 1. Retrosynthetic Analysis of 2′-Amino-α-L-LNA-adenine Monomers W−Za

a

Abbreviations: PG = PN(iPr)2OCH2CH2CN; R = H, CH2Py, COPy, and COCH2Py for W−Z, respectively; py = pyren-1-yl; ABz = 6-Nbenzoyladenin-9-yl.

duplex formation.9b Thus, increases in Tm of up to 20 °C per modification have been observed for short ONs modified with these building blocks. We have taken advantage of these characteristics and have developed probes for a variety of diagnostic applications. For example, ONs modified with two next-nearest-neighbor incorporations of 2′-N-(pyren-1-yl)acetyl-2′-amino-α-L-LNA-T monomers are promising tools for the detection of single-nucleotide polymorphisms, which are the most prevalent type of genetic mutation in the human genome.10 These probes efficiently discriminate between complementary and single-base-mismatched DNA/RNA targets through differences in pyrene excimer emission levels. In another example, the fluorescence of ONs modified with 2′-N(pyren-1-yl)acetyl-2′-amino-α-L-LNA-T monomers was found to increase 16-fold upon hybridization with DNA targets featuring abasic sites (i.e., DNA lesions that result in genomic mutations and emergence of cancers if unrepaired).11 We have also utilized N2′-pyrene-functionalized 2′-amino-α-L-LNA-T monomers as the key activating components of the so-called Invader probes, which recognize mixed-sequence doublestranded DNA.12 Unfortunately, the synthesis of N2′-pyrenefunctionalized 2′-amino-α-L-LNA-thymine phosphoramidites is challenging (∼20 steps, Z ≥ X)

ΔTm/°C ON B1 D2 B2 D2 B3 D2 D1 B4 D1 B5 D1 B6

duplex 5′-GTG 3′-CAC 5′-GTG 3′-CAC 5′-GTG 3′-CAC 5′-GTG 3′-CAC 5′-GTG 3′-CAC 5′-GTG 3′-CAC

BTA TAT ATB TAT BTB TAT ATA TBT ATA TAT ATA TBT

TGC ACG TGC ACG TGC ACG TGC ACG TGC BCG TGC BCG

B=W

B=X

B=Y

B=Z

−0.5

+5.0

+11.0

+6.0

+0.5

+7.0

+14.0

+7.5

−1.0

+5.0

+13.5

+16.0

±0.0

+6.5

+11.5

+7.5

±0.0

+5.5

+12.0

+6.0

−2.0

+5.0

+13.5

+14.5

ΔTm = change in Tm relative to the value for the unmodified reference duplex D1:D2 (Tm ≡ 27.5 °C). Tm was determined as the maximum of the first derivative of the melting curve (A260 vs T) recorded in medium-salt buffer ([Na+] = 110 mM, [Cl−] = 100 mM, pH 7.0 (NaH2PO4/Na2HPO4)) using a concentration of 1.0 μM for each strand. Tm values are averages of at least two measurements within 1.0 °C. A/C/G/T = adenin-9-yl/cytosin-1-yl/guanin-9-yl/ thymin-1-yl DNA monomers. For the structures of monomers W−Z, see Figure 1. Data for the B1, B2, B4, and B5 series for monomers X, Y, and Z were previously reported in ref 12b. a

indicate that (i) monomers in which the pyrene moiety is attached to the 2-oxo-5-azabicyclo[2.2.1]heptane skeleton via an alkanoyl linker induce greater thermostabilization than the corresponding monomers employing alkyl linkers (N2′-PyCO 12694

dx.doi.org/10.1021/jo4022937 | J. Org. Chem. 2013, 78, 12690−12702

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PyCO Y-modified duplexes being moderately stabilized (ΔTm/ modification between +1.5 and +6.5 °C; Table 2). N2′-PyAc Zmodified ONs also displayed lower affinities toward RNA than DNA targets but resulted in more thermostable heteroduplexes (ΔTm/modification between +4.0 and +8.0 °C; Table 2). These differences may again reflect the greater flexibility of monomer Z, which allows the pyrene moieties to adopt more favorable positions for affinity-enhancing intercalation (vide infra). DNA-selective hybridization (Table S3 in the Supporting Information), as seen for X/Y/Z-modified ONs and ONs modified with the thymine counterparts,9b is often observed for ONs modified with intercalating moieties,24 as intercalators favor the B-type helix geometry of DNA:DNA duplexes over the more compressed A/B-type helix geometry of DNA:RNA duplexes.25 Mismatch Discrimination. The binding specificities of centrally modified ONs against DNA/RNA strands with mismatched nucleotides opposite the W−Z monomers were determined. W4 displayed improved discrimination of mismatched DNA targets relative to reference strand D2, with the AG mismatch being discriminated particularly well (Table 3).

Y > N2′-PyMe X) and (ii) monomers with short alkanoyl linkers result in more thermostable DNA duplexes than the corresponding monomers with longer alkanoyl linkers (N2′PyCO Y > N2′-PyAc Z). We previously observed similar structure−property relationships with the analogous thymine monomers, which on the basis of UV−vis spectroscopy and molecular modeling results were explained by differential placement of the pyrene moiety for affinity-enhancing intercalation.9b Similar structural underpinnings are likely in effect for monomers X−Z (vide infra). However, the electron density of the pyrene moieties, which differs among the three monomers, may also be a contributing factor to the differential duplex stabilization. An interesting trend was observed for the doubly modified ONs: incorporation of a second N2′-PyMe X or N2′-PyCO Y monomer did not result in additionally increased thermal affinity against DNA targets, whereas a second incorporation of the more flexible N2′-PyAc Z monomer did (compare the ΔTm values for the B1−B3 and B4−B6 series in Table 1). We hypothesize that the former observation is due to violation of the “nearest-neighbor exclusion principle”, which states that free intercalators at most bind to every second base pair of a DNA duplex because of limits on the local expandability of duplexes.23 If the principle is extrapolated to tethered intercalators and 3′-intercalative binding modes of the pyrene moieties of monomers X−Z are assumed (vide infra), the resulting duplexes involving B3 and B6 would feature a localized region with two intercalators in an area defined by four base pairs, which is at the saturation threshold. We speculate that the greater flexibility of the N2′-PyAc Z monomer allows the modified DNA duplexes to structurally compensate for any stress induced by the high intercalator density. Interestingly, the modified ONs displayed rather different thermal denaturation characteristics with RNA targets. ONs modified with 2′-amino-α-L-LNA adenine monomer W displayed significantly higher affinity against RNA than DNA targets (ΔTm/modification between +1.5 and +7.0 °C; Table 2). Further, X- and Y-modified ONs displayed markedly lower thermal affinities toward RNA than DNA targets, with N2′PyMe X-modified DNA:RNA duplexes displaying similar thermostabilities as unmodified reference duplexes (ΔTm/ modification between −0.5 and +2.5 °C; Table 2) and N2′-

Table 3. Discrimination of Mismatched DNA Targets by B4 Series and Reference ONsa DNA: 5′-GTG ABA TGC

D2 W4 X4 Y4 Z4

duplex 5′-GTG 3′-CAC 5′-GTG 3′-CAC 5′-GTG 3′-CAC 5′-GUG 3′-CAC 5′-GUG 3′-CAC 5′-GUG 3′-CAC

BTA UAU ATB UAU BTB UAU AUA TBT AUA TAT AUA TBT

TGC ACG TGC ACG TGC ACG UGC ACG UGC BCG UGC BCG

B=W

B=X

B=Y

B=Z

+2.5

−1.0

+1.5

+4.0

+2.5

+1.0

+5.0

+4.5

+3.0

−1.5

+4.5

+11.0

+4.5

−0.5

+2.5

+6.5

+7.0

+2.5

+6.5

+8.0

+7.0

±0.0

+7.0

+14.5

3′-CAC 3′-CAC 3′-CAC 3′-CAC 3′-CAC

TAT TWT TXT TYT TZT

ACG ACG ACG ACG ACG

B=T

B=A

B=C

B=G

27.5 27.5 34.0 39.0 35.0

−17.0 −20.0 −16.5 −21.0 −21.5

−15.5 −17.0 −7.5 −12.0 −14.0

−9.0 −16.0 −17.0 −17.0 −14.5

For conditions of the thermal denaturation experiments, see Table 1. Tm values for the fully matched duplexes are shown in bold. ΔTm = change in Tm relative to the value for the fully matched DNA:DNA duplex.

X4/Y4/Z4 displayed surprisingly efficient discrimination of mismatched DNA targets, particularly of AG mismatches (Table 3), considering the likely intercalative binding mode of the pyrene moieties, which is known to typically decrease base-pairing fidelity.9b,26 However, ONs with X/Y/Z monomers positioned as next-nearest neighbors displayed poor discrimination of the centrally mismatched DNA target (B3 series; Table S4 in the Supporting Information), indicating that the modification pattern has a major influence on the binding specificity. Discrimination of RNA mismatches was generally less efficient with W4/X4/Y4 than with reference ONs but improved with Z4 (Table 4). Optical Spectroscopy. UV−vis absorption and steadystate fluorescence emission spectra of X/Y/Z-modified ONs and the corresponding duplexes with DNA/RNA targets were recorded to gain additional insight into the binding modes of the pyrene moieties of monomers X/Y/Z. Bathochromic shifts of pyrene absorption maxima, which are indicative of strong interactions between pyrenes and nucleobases,27 were generally observed upon hybridization with complementary DNA/RNA (Δλmax = 0−6 nm; Table 5, Figure 3, and Figures S2 and S3 in the Supporting Information), with the most pronounced increases being observed for duplex formation of Y-modified ONs with DNA. These results are consistent with intercalative binding modes for the pyrene moieties of monomers X/Y/Z

ΔTm/°C B1 R2 B2 R2 B3 R2 R1 B4 R1 B5 R1 B6

sequence

a

Table 2. ΔTm Values for Duplexes between B1−B6 and Complementary RNA Targetsa ON

ΔTm/°C

Tm/°C ON

a ΔTm = change in Tm relative to the value for the unmodified reference duplex D1:R2 (Tm ≡ 26.0 °C) or R1:D2 (Tm ≡ 24.5 °C); for conditions of the thermal denaturation experiments, see Table 1.

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To further substantiate the proposed intercalative binding mode of N2′-PyCO monomer Y, we synthesized three additional Y-modified ONs in which the 3′-flanking nucleotide was varied systematically (Y7−Y9, Table 6). ONs with 3′flanking purines displayed greater relative increases in thermal affinity against DNA targets than the corresponding ONs with 3′-flanking pyrimidines (compare the ΔTm values for Y7:D3 and Y9:D5 with those for Y8:D4 and Y4:D1 in Table 6). This is consistent with the proposed binding mode, as 3′intercalating pyrenes are expected to interact more strongly with large purines. Steady-state fluorescence emission experiments provided further evidence for an intercalative binding mode of the pyrenes, as the emission levels of these four DNA duplexes decreased in the anticipated order of nucleobase quenching efficiency (from least to most quenched: 3′-flanking A > T > C > G; Figure 4).28b,29

Table 4. Discrimination of Mismatched RNA Targets by B4 Series and Reference ONsa RNA: 5′-GUG ABA UGC ΔTm/°C

Tm/°C ON D2 W4 X4 Y4 Z4

sequence 3′-CAC 3′-CAC 3′-CAC 3′-CAC 3′-CAC

TAT TWT TXT TYT TZT

ACG ACG ACG ACG ACG

B=U

B=A

B=C

B=G

24.5 29.0 24.0 27.0 31.0

−15.0 −13.5 −10.5 −10.5 −19.0

−15.0 −13.0 −8.0 −11.0 −15.5

−11.0 −11.5 −10.0 −8.5 −14.5

a

For conditions of the thermal denaturation experiments, see Table 1. Tm values for the fully matched duplexes are shown in bold. ΔTm = change in Tm relative to the value for the fully matched RNA:DNA duplex.

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and in agreement with our previous observations for the corresponding thymine analogues.9b Steady-state fluorescence emission spectra were recorded at 5 °C using an excitation wavelength of λex = 350 nm. Hybridization of N2′-PyMe X-modified or N2′-PyAc Zmodified ONs with complementary DNA/RNA resulted in decreased emission (Figure 3 and Figures S4 and S7 in the Supporting Information), which is consistent with intercalationinduced quenching by flanking nucleobases.27,28 The resulting duplexes were only weakly fluorescent and exhibited defined vibronic bands at ∼380 nm and ∼400 nm along with a shoulder at 420−425 nm. On the other hand, a range of responses was observed upon hybridization of N2′-PyCO Y-modified ONs with complementary DNA/RNA, varying from ∼50% reduction to ∼3-fold enhancement of emission (Figure S5 in the Supporting Information). The resulting duplexes were strongly fluorescent (nearly 2 orders of magnitude more fluorescent than X-modified duplexes) with little or no vibronic fine structure (λem,max ≈ 400 nm, broad band). The lack of a consistent response upon hybridization to DNA/RNA presumably is not due to different pyrene binding modes in the resulting duplexes but rather reflects different fluorescence intensities of the single-stranded probes, possibly due to different sequence-dependent rotational barriers around the Py−CO bond. Thus, singly modified DNA duplexes displayed similar emission intensities (Figure S6 in the Supporting Information), suggesting that the pyrene moieties are in similar microenvironments in the duplex, which is consistent with an intercalative binding mode.

CONCLUSION ONs modified with N2′-pyrene-functionalized 2′-amino-α-LLNA-adenine monomers X−Z display very high affinity toward DNA targets. The DNA-selective hybridization, together with hybridization-induced bathochromic shifts of pyrene absorption maxima and quenching of pyrene fluorescence, is indicative of intercalative binding modes for the pyrene moieties. ONs with such characteristics are likely to be of significant interest for applications in nucleic acid diagnostics and biotechnology.30 However, the synthesis of the corresponding phosphoramidites is very challenging because of nontrivial protecting-group manipulations (90% unless stated otherwise) was verified either by the ion-pair reversedphase HPLC system running in analytical mode or ion-exchange HPLC using a Tris-Cl/EDTA−NaCl gradient. Protocol for Thermal Denaturation Studies. Concentrations of ONs were calculated using the following extinction coefficients (OD260/μmol): G, 12.0; A, 15.2; T, 8.4; U, 10.0; C, 7.1; pyrene, 22.4. ONs (each strand at 1.0 μM) were thoroughly mixed, denatured by heating, and subsequently cooled to the starting temperature of the experiment. Quartz optical cells with a path length of 10.0 mm were used. The thermal denaturation temperature (Tm/°C) was measured on a temperature-controlled UV−vis spectrophotometer and determined as the maximum of the first derivative of the thermal denaturation curve (A260 vs T) recorded in medium-salt buffer (Tm buffer) (100 mM NaCl, 0.1 mM EDTA, and pH 7.0 adjusted with 10 mM NaH 2 PO 4 /5 mM Na 2 HPO 4 ). The temperature of the denaturation experiments ranged from at least 15 °C below Tm to 20 °C above Tm (although not below 3 °C). A temperature ramp of 0.5 °C/min or 1.0 °C/min was used in the experiments. The reported thermal denaturation temperatures are averages of two measurements within ±1.0 °C. Protocol for UV−Vis Absorption Spectroscopy. UV−vis spectra were recorded on a spectrophotometer at 5 °C (except for single-stranded X/Y-modified probes, which were recorded at room temperature) using each strand at a concentration of 1 μM in Tm buffer and quartz cells with 1 cm path lengths. Protocol for Fluorescence Emission Spectroscopy. Steadystate fluorescence spectra were recorded at 5 °C using an excitation wavelength of λex = 350 nm, each strand at 1 μM concentration in Tm buffer (except with Y-modified ONs, where strands were used at 0.15 μM concentration), and quartz cells with 1 cm path lengths.

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material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions §

N.K.A. and B.A.A contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS P.J.H. appreciates financial support through Award R01 GM088697 from the National Institute of General Medical Sciences, National Institutes of Health, and from the Institute of Translational Health Sciences (ITHS) (supported by Grants UL1 RR025014, KL2 RR025015, and TL1 RR025016 from the National Center for Research Resources, NIH). J.W. appreciates financial support from The Danish National Research Foundation and the Danish Agency for Science Technology and Innovation. We thank Dr. T. Santhosh Kumar (University of Southern Denmark) for providing us with starting material and Dr. Lee Deobald (EBI Murdock Mass Spectrometry Center, University of Idaho) for mass spectrometric analyses.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

General experimental section; additional synthetic strategies; NMR spectra for all new compounds; MS data for all modified ONs; representative thermal denaturation curves; additional Tm data; representative absorption and fluorescence emission spectra; and evaluation of SNP-discriminatory potential. This 12701

dx.doi.org/10.1021/jo4022937 | J. Org. Chem. 2013, 78, 12690−12702

The Journal of Organic Chemistry

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dx.doi.org/10.1021/jo4022937 | J. Org. Chem. 2013, 78, 12690−12702