Guanine and 8-Azaguanine in Anomeric DNA Hybrid Base Pairs

May 17, 2018 - A gradual fluorescence change takes place in duplex DNA when the ... Bifacial Nucleobases for Hexaplex Formation in Aqueous Solution...
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Guanine and 8-Azaguanine in Anomeric DNA Hybrid Base Pairs: Stability, Fluorescence Sensing and Efficient Mismatch Discrimination with #-D-Nucleosides Jiang Liu, Sachin Asaram Ingale, and Frank Seela Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00261 • Publication Date (Web): 17 May 2018 Downloaded from http://pubs.acs.org on May 18, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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Bioconjugate Chemistry

Guanine and 8-Azaguanine in Anomeric DNA Hybrid Base Pairs: Stability, Fluorescence Sensing and Efficient Mismatch Discrimination with α-D-Nucleosides

Jiang Liu,†,‡,§ Sachin A. Ingale,‡,§ and Frank Seela*‡,§ ‡

Laboratory of Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology, Heisenbergstrasse 11, 48149 Münster, Germany, and †State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & Dept. of Oral Medicine of West China Hospital of Stomatology, Sichuan University, 610041 Chengdu, Sichuan (P. R. China) and §Laboratorium für Organische und Bioorganische Chemie, Institut für Chemie neuer Materialien, Universität Osnabrück, Barbarastrasse 7, 49069 Osnabrück, Germany

To the memory of Professor Jean-Louis Imbach and his pioneering work on alpha-nucleosides and alpha nucleic acids

Corresponding author: Prof. Dr. Frank Seela Phone: +49 (0)251 53 406 500; Fax: +49 (0)251 53 406 857 E-mail: [email protected] Homepage: www.seela.net

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Table of Contents

Key words. 8-azaguanine, α nucleosides, DNA base pair stability, fluorescence, mismatch discrimination

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ABSTRACT The α-anomers of 8-aza-2’-deoxyguanosine (αGd*) and 2’-deoxyguanosine (αGd) were sitespecifically incorporated in 12-mer duplexes opposite to the four canonical DNA constituents dA, dG, dT and dC. Oligodeoxyribonucleotides containing αGd* display significant fluorescence at slightly elevated pH (8.0). Oligodeoxyribonucleotides incorporating βanomeric 8-aza-2’-deoxyguanosine (Gd*) and canonical dG were studied for comparison. For αGd* synthesis, an efficient purification of anomeric 8-azaguanine nucleosides was developed on the basis of protected intermediates and a new αGd* phosphoramidite was prepared. Differences were observed for sugar conformations (N vs. S) and pKa values of anomeric nucleosides. Duplex stability and mismatch discrimination was studied employing UVdependent melting and fluorescence quenching. A gradual fluorescence change takes place in duplex DNA when the α-nucleoside αGd* was positioned opposite to the four canonical βnucleosides. The strongest fluorescence decrease appeared in duplexes incorporating αGd*-Cd base pair matches. Decreasing fluorescence corresponds to increasing Tm values. For mismatch discrimination the α-anomers αGd* and αGd are more efficient than the corresponding β-nucleosides. Duplexes with single “purine-purine” αGd*-αGd* or αGd-αGd base pairs are significantly more stable than those displaying β-D configuration. CD-spectra indicate that single mutations by α-anomeric nucleosides do not affect the global structure of B-DNA.

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INTRODUCTION Naturally occurring nucleosides display β-D-configuration for the linkages between the nucleobases and sugar moieties. Very few of them contain α-D-glycosylic connectivity at the anomeric carbon.1 Also, nucleoside residues of canonical DNA or RNA show β-D-configuration. However, γ irradiation under anaerobic conditions can cause DNA lesions resulting in the formation of DNA with α-nucleotides.2-4 Substantial levels of αGd (α-D-2’deoxyguanosine) were detected in mammalian DNA.5 These α-anomeric lesions can be bypassed by human polymerases. Canonical nucleoside triphosphates can be incorporated opposite to α-anomeric nucleosides and also misincorporation is observed.6,7

Concerning nucleoside synthesis α-D-anomers are often formed as side products of glycosylation or by anomerization of β-D-nucleosides.8-10 The Imbach group assembled α-Dnucleosides with canonical bases to oligonucleotides and a few studies exist by this group and others on their base pairing properties.11-20 Recently, our laboratory reported on the enhanced stability of silver mediated base pairs between anomeric 2'-deoxycytidines incorporated in duplex DNA.21 Earlier, the base pairing properties were studied of anomeric 5-aza-7deazaguanine nucleosides in DNA with parallel and antiparallel chain orientation.22

The 8-azapurine heterocycle is a useful scaffold for applications in chemistry, chemical biology, diagnostics and in the development of drugs.23-29 There is a large body of information devoted to synthesis, tautomerism and fluorescence of 8-azapurines.23-25,29-31 8-Aza-2’deoxyadenosine and 8-aza-2’-deoxyguanosine, which can be considered as purine shape mimics of the canonical DNA constituents, show fluorescence induced by altered nucleobase. This photophysical behavior is rare in the class of modified nucleosides32-36 with only one atom nucleobase modification. Our laboratory reported on the syntheses, base pairing and 4

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fluorescence properties of 8-aza-2’-deoxyguanosine, 8-aza-2’-deoxyadenosine and 8-aza-2’deoxyisoguanosine and corresponding oligodeoxyribonucleotides.37-43 However, the use of the β-nucleosides was not satisfying to sense unknown base composition as discrimination was not sufficient.39 The fluorescence properties of the 8-azapurine skeleton are now combined with the αnucleoside connectivity. This is the first report on a fluorescent α-nucleoside shape mimic that contains Watson-Crick and Hoogsteen binding sites. Earlier, α-etheno-2’-deoxyadenosine was studied.44 However, in this case the Watson-Crick binding site of nucleoside was blocked. Now, the α-anomer of 8-aza-2’-deoxyguanosine (1, αGd*, Figure 1) was incorporated in oligodeoxyribonucleotides using the phosphoramidite 3 and applying solid-phase synthesis. Base pairing, mismatch recognition, and fluorescence properties were investigated and αGd* was used to build up a powerful sensor for DNA mismatch recognition. Thermal stabilities and photophysical properties of α-modified oligodeoxyribonucleotides were studied and compared with those of the β-anomer of 8-aza-2’-deoxyguanosine (2, Gd*, Figure 1). Also, base pairing of the anomeric 2’-deoxyguanosines was examined to compare the purine and 8azapurine system in the environment of canonical B-DNA. As the glycosylation of the 8azaguanine system leads to mixtures of anomers an effective separation protocol was developed to access the pure anomer αGd* as well as the phosphoramidite building block 3.

Figure 1. Structures of anomeric nucleosides (αGd* and Gd*) and phosphoramidite 3 of 8aza-2’-deoxyguanosine.

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RESULTS AND DISCUSSION Monomers. Synthesis. The 8-azapurine base 4 has been synthesized chemically but is also naturally occurring showing antiviral and anticancer activity.23-25,28-31,37-45 Various purine nucleoside phosphorylases accept 8-azaguanine as substrate and convert it into the nucleoside.46 Nucleobase-anion glycosylation was performed on 8-azaguanine derivatives with Hoffer’s sugar 2-deoxy-3,5-di-O-p-toluoyl-α-D-erythro-pentofuranosyl chloride (5) or with a 2,3dideoxy-D-glycero-pentofuranosyl chloride. 47,48,49 By these routes, 8-aza-2’-deoxyguanosine (Gd*) as well as regioisomeric analogues were prepared under kinetically controlled conditions,37 and were fully characterized. Tolman et al.29 used a modification of the fusion reaction employing silylated 8-azaguanine 4 and the halogenose 5 (Scheme 1) at elevated temperature resulting in a mixture of protected α-D- and β-D-2'-deoxyribonucleosides of guanine. Separation was accomplished by crystallization and regioisomeric glycosylation products were not observed due to thermodynamic control. The synthetic route developed by Tolman et al.29 was now used for the synthesis of αGd*. However, separation of anomeric 8-aza-2'-deoxyguanosines by crystallization was laborious and low yielding. Therefore, the separation of anomers was transferred to a later stage of the synthesis. To this end, the amino group of the anomeric αGd*/Gd* mixture was protected with N,N-di-n-butylformamide dimethyl acetale to give a mixture of the amidines 6/7. This mixture was converted to the 5'-O-4,4'-dimethoxytrityl (5'-O-DMT) derivatives (8/9). On this stage the anomers 8 and 9 could be easily separated by silica gel column chromatography. The α-D-anomer (8) was isolated in 36% yield and the β-D-compound (9) in 32% (Scheme 1). From the protected α-D-anomer 8 the free nucleoside αGd* was obtained in 28% yield in two steps by DMT deprotection in CH3COOH/CH3OH and removal of the amidine group with 6

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Bioconjugate Chemistry

saturated methanolic ammonia. Phosphitylation of 8 furnished the phosphoramidite 3 (Scheme 1). Scheme 1. Synthesis of Nucleoside αGd* and Phosphoramidite 3a

a

Reagents and conditions: i) a) hexamethyldisilazane (HMDS), reflux, 4 h; b) fusion reaction

of the silyl derivative with halogenose 5 at 130 °C, 1 h;29 ii) NH3/CH3OH, 35 °C, 2 days; iii) N,N-di-n-butylformamide dimethyl acetal, CH3OH, rt, 24 h; iv) DMT-chloride, pyridine, rt, 2 h; v) 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, N,N-diisopropylethylamine ((iPr)2NEt), CH2Cl2, rt, 20 min; vi) CH3COOH/CH3OH (4:1, v/v), rt, overnight; vii) NH3/CH3OH, rt, 2 days. Although the α-nucleoside αGd* has been described, its anomeric configuration was not assigned unambiguously. To this end, NOE measurements were performed. Irradiation of

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H-1' of αGd* resulted in the strongest NOE's at H-2'β and H-3' confirming α-D-configuration; regarding the β-D-nucleoside Gd* irradiation on H-1' gave strongest NOE's at H-2'α and H-4' corroborating β-configuration (Figure 2).

Figure 2. Assignment of anomeric nucleosides by NOE measurements.

All synthesized compounds were characterized by NMR spectra as well as ESI-TOF mass spectra (Experimental section). 1H-13C correlated (HMBC and HSQC) NMR spectra were used to assign the 13C NMR signals. For details see the Experimental section (for spectra, see the Supporting Information).

Sugar Conformation of Anomeric Nucleosides. The sugar conformation of anomeric nucleosides influences the stability of the DNA double helix.50,51 Whereas sugar residues in DNA prefer S-conformation, the conformation in RNA is N (Figure 3). To detect conformational changes in the series of anomeric nucleosides used in this study, 600 MHz 1HNMR spectra were measured of the anomers αGd* and Gd* and their purine counterparts αGd and Gd in DMSO-d6. The population of S vs N conformers were calculated using the program PSEUROT (version 6.3).52 The input used the following coupling constants: 3J(H1′, H2′), 3

J(H1′, H2′′), 3J(H2′, H3′), 3J(H2′′, H3′), and 3J(H3′, H4′). The coupling constants are

summarized in Table 1 (Table S1, Supporting Information).

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Figure 3. Twist conformations (N and S) for β-D and α-D nucleosides. B corresponds to nucleobase. ax: pseudoaxial; eq: pseudoequatorial.

The sugar puckering is described relative to the exocyclic atom C(5′), and is defined as C2′endo, if the puckered atom is at the same side of the plane as C(5′), otherwise it is C2′-exo. The north (N) conformation is centered around the C(3′)-endo conformer, while the south (S) conformation is centered around the C(2′)-endo species (Figure 3). The conformer equilibrium is driven by various stereoelectronic gauche and anomeric effects.50 According to Table 1, the replacement of carbon-8 by nitrogen changes the N/S conformational equilibrium of the pentofuranose moiety substantially. Differences exist between the series of purine and 8-azapurine nucleosides.53 The S-sugar pucker dominates in the purine compounds whereas in the 8-azapurine compounds both anomers display more equal distribution. These changes are caused by the higher electron deficiency of the 8-azagaunine system compared to the guanine base. Stereoelectronic effects are transmitted from the base to the sugar moiety.

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Bioconjugate Chemistry 1 2 3 4 5 Table 1. Chemical Shift of the 1H Signals and Proton-Proton Vicinal and Geminal Coupling Constants of α- and β-Anomers of 8-Aza-2′6 deoxyguanosines and 2′-Deoxyguanosinesa 7 8 9 Conformation Chemical shift/ppm Coupling constant/Hz [J3(HH)] 10 11 12 H-1’ H-2’ H-2” H-3’ H-4’ H-5’ H-5’’ OH-3’ OH-5’ NH2 NH 1’2’ 1’2’’ 2’2’’ 2’,3’ 2’’3’ 3’4’ 4’5’ 4’5’’ 5’5’’ %N %S 13 2.73 2.78 4.17 3.96 3.60 3.43 5.37 4.75 6.96 11.03 14 αG * 6.23 6.0 7.5 -13.2 7.0 7.3 6.8 3.0 5.0 -12.0 59 41 d (dd) (ddd) (dt) (p) (ddd) (dt) (dt) (t) (s) (s) (d) 15 16 4.74 6.94 11.00 6.29 2.90 2.32 4.46 3.84 3.51 3.38 5.32 6.0 6.8 -13.3 6.0 4.4 4.0 5.7 5.8 -11.4 41 59 17 Gd* (t) (s) (s) (t) (dt) (ddd) (dq) (td) (dt) (dt) (d) 18 19 4.82 6.48 6.10 2.68 2.20 4.28 4.06 3.45 3.41 5.47 -b 7.9 3.0 -14.3 6.7 3.1 3.5 4.7 4.8 -11.7 26 74 20 αGd (dd) (ddd) (dt) (dq) (td) (dt) (dt) (t) (s) (d) 21 4.95 6.46 10.65 6.11 2.50 2.19 4.33 3.80 3.55 3.50 5.26 22 G 7.9 6.0 -13.1 5.5 2.8 2.6 4.5 4.5 -11.6 23 77 d (t) (s) (s) (dd) (ddd) (ddd) (dq) (td) (dt) (dt) (d) 23 24 a Measured in DMSO-d6 at 298 K; b not detected; rms ˂ 0.4 Hz. H-2’ = H-2′β; H-2’’ = H-2′α 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 10 40 41 42 43 44 ACS Paragon Plus Environment 45 46 47

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Bioconjugate Chemistry

pKa Values of Anomeric Nucleosides. The pKa values of nucleobases are of utmost importance for the stability of DNA and RNA base pairs.54 Structural modification of nucleobases from purines to 7-deazapurines e.g. from the canonical 2'-deoxyguanosine (pKa = 9.1) to 7-deaza-2'-deoxyguanosine (pKa = 10.3)55 demonstrates this behavior. Due to stereoelectronic effects an interplay exists between nucleobases and sugar residues that includes response to the configuration. Regarding this, it was anticipated that a change in the sugar configuration and/or conformation can change the pKa value of the nucleobase. Such changes were reported for anomeric imidazole, 1,2,4-triazole and benzimidazole nucleosides.56 Consequently, the pKa values of the pairs of anomers αGd* vs Gd* and αGd vs Gd were measured UV spectrophotometrically in 0.1 M sodium phosphate buffer (Figure 4, Figure S1, Supporting Information). According to Figure 4, the pKa value for deprotonation for αGd* with 7.9±0.1 is slightly lower than for Gd* (pKa = 8.1±0.1). The corresponding values for deprotonation are 9.2±0.1 for αGd and 9.1±0.1 for Gd. Earlier, pKa values for protonation of the anomeric 2’-deoxyguanosines were determined from 1H NMR spectra as 2.6 for αGd and 2.3 for Gd.57 Our measurements performed by UV titration resulted in similar values (pKa = 2.7±0.1 for αGd and pKa = 2.4±0.1 for Gd). Therefore, the 8-azaguanine nucleoside anomers are more acidic than their purine counterparts. As discussed for changes in the sugar conformation the higher electron deficiency of the triazole system in αGd* and Gd* in comparison to the imidazole system of purine nucleosides is responsible for the changes.

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Figure 4. pKa Values of nucleosides determined by UV titration in 0.1 M sodium phosphate buffer; a) αGd*; b) Gd*; c) αGd and d) Gd (red lines correspond to first derivatives)

Oligodeoxyribonucleotides. Synthesis and Characterization. DNA and RNA fragments containing Gd* and its ribofuranosyl derivative were already synthesized enzymatically31,41 and chemically29,32. Also corresponding oligonucleotides were prepared and their properties were studied.33,34 Oligodeoxyribonucleotides (ODNs) used in this study contain the anomeric pairs of 8azaguanine (αGd* and Gd*) and guanine (αGd and Gd) nucleosides (Table 2). They were prepared by solid-phase synthesis using the corresponding phosphoramidites of αGd*, Gd*,39 αGd and Gd together with standard building blocks.

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To investigate the influence of the anomeric nucleosides on duplex stability and mismatch recognition the duplex 5'-d(TAG GTC AAT ACT) (ODN-7) • 3'-d(ATC CAG TTA TGA) (ODN-8) was used which represents a standard duplex used in our laboratory for many years. Single strands and duplex show a normal behavior. ODN-8 was modified by replacing a single near central Gd residue by αGd* (ODN-1) or by αGd (ODN-4). Corresponding oligodeoxyribonucleotides with the β-constituents Gd* (ODN-2) and Gd (ODN-8) were prepared for comparison (Table 2). Also (ODN-7) was modified by replacing a single near central dC residue by αGd (ODN-3), αGd* (ODN-5) and Gd* (ODN-6). After solid-phase synthesis, the oligodeoxyribonucleotides were cleaved from the solid support and deprotected in concentrated 28% aqueous ammonia at 55 °C for 2 h and then at rt overnight. The coupling yields of the modified building blocks were always higher than 95%. All synthesized oligodeoxyribonucleotides were purified by reversed-phase HPLC (RP-18), detritylated with 2.5% dichloroacetic acid in dichloromethane and again purified by HPLC. The contents of single peaks were isolated in all cases (Figure S2, Supporting Information). Subsequently, the molecular masses were determined by MALDI-TOF mass spectrometry. Table 2 displays all modified oligodeoxyribonucleotides used in this study together with their mass data.

Table 2. Molecular Masses Measured by MALDI-TOF Mass Spectrometrya

Molecular Weight Oligodeoxyribonucleotides

Calc. [M+1]+

Found [M+1]+

5'-d(AGT ATT αG*AC CTA) (ODN-1)

3646.5

3644.8

5'-d(AGT ATT G*AC CTA) ( ODN-2)

3646.5

3646.5

5'-d(TAG GTαG AAT ACT) ( ODN-3)

3685.5

3683.5

5'-d(AGT ATT αGAC CTA) ( ODN-4)

3645.5

3645.8

5'-d(TAG GTαG* AAT ACT) ( ODN-5)

3686.5

3687.2

5'-d(TAG GTG* AAT ACT) ( ODN-6)

3686.5

3685.9

5'-d(TAG GTC AAT ACT) ( ODN-7)

58

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5'-d(AGT ATT GAC CTA) ( ODN-8)58 5'-d(TAG GTG AAT ACT) ( ODN-9)

3685.5

3685.1

5'-d(TAG GTT AAT ACT) ( ODN-10)

3660.5

3657.9

5'-d(TAG GTA AAT ACT) ( ODN-11) a Measured in the positive linear mode.

3669.5

3668.3

Oligodeoxyribonucleotide Duplex Stability. It has been reported that oligonucleotides with α-D configuration incorporating 2'deoxyribonucleosides with all four canonical nucleobases form stable duplexes with complementary strands displaying β-D configuration.59 These anomeric hybrid duplexes show parallel chain orientation. However, much less is known on the behavior of a single αnucleoside mutation within canonical DNA with antiparallel chains. Studies regarding base pair stability and recognition were performed by Imbach, Sugimoto, Germann and others.11-20 As α-nucleosides might be valuable as sensors to detect mismatches in DNA, thermal melting as well as fluorescent quenching experiments were performed. Stability changes and nucleobase recognition were detected within the canonical (antiparallel) environment of DNA. Studies used the environmental changes of the α-nucleoside αGd* as well as of the corresponding αGd caused by base pairing or mismatch formation. The β-anomers Gd* and Gd were examined for comparison. To this end, a series of complementary 12-mer oligodeoxyribonucleotides with single α-mutation were hybridized and Tm values as well as thermodynamic data were determined (Table 3, Figure S3, Supporting Information). According to the Tm data of Table 3, the anomeric nucleosides show similar mismatch discrimination in the series of guanine- and 8-azaguanine-modified oligodeoxyribonucleotides. Stability differences exist also within the series of α-anomeric series. Surprisingly, a similar stability order was found for the β-series with Gd*-Cd >> Gd*14

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Td > Gd*-Ad > Gd*-Gd compared to the α-series (αGd*-Cd >> αGd*-Td > αGd*-Gd > αGd*Ad). Only the purine changes the places. The same stability order observed for 8-azaguanine nucleosides was observed in the series of guanine nucleosides (Table 3). Thermodynamic data of duplex formation support these dependencies. Data were calculated using the program MELTWIN (Table 3).60 Changes in ∆G values reflect the stability of oligodeoxyribonucleotide duplexes. Enthalpic and entropic changes are small between the series of guanine and 8-azaguanine oligodeoxyribonucleotide duplexes.

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Table 3. Tm Values and Thermodynamic Data of Anomeric Duplexesa Duplexes

5'-d(TAG GTC AAT ACT) (ODN-7) 3'-d(ATC CAαG TTA TGA) (ODN-4) 5'-d(TAG GTG AAT ACT) (ODN-9) 3'-d(ATC CAαG TTA TGA) (ODN-4) 5'-d(TAG GTT AAT ACT) (ODN-10) 3'-d(ATC CAαG TTA TGA) (ODN-4) 5'-d(TAG GTA AAT ACT) (ODN-11) 3'-d(ATC CAαG TTA TGA) (ODN-4)

Tm b [°C]

∆H° [kcal /mol]

∆S° [cal/K-1 mol-1]

∆G°310 [kcal/ mol]

37.5

-73

-209

-8.1

31.0

-58

-165

-6.8

36.5

-68

-195

-7.9

27.0

-65

-191

-5.8

Duplexes

5'-d(TAG GTC AAT ACT) (ODN-7) 3'-d(ATC CAαG* TTA TGA) (ODN-1) 5'-d(TAG GTG AAT ACT) (ODN-9) 3'-d(ATC CAαG* TTA TGA) (ODN-1) 5'-d(TAG GTT AAT ACT) (ODN-10) 3'-d(ATC CAαG* TTA TGA) (ODN-1) 5'-d(TAG GTA AAT ACT) (ODN-11) 3'-d(ATC CAαG* TTA TGA) (ODN-1)

Tm b [°C]

∆Tm c [°C]

∆H° [kcal/ mol]

∆S° [cal/K-1 mol-1]

∆G°310 [kcal/ mol]

42.0

+4.5

-75

-211

-9.2

32.0

+1.0

-59

-167

-7.0

34.0

-2.5

-65

-186

-7.3

28.0

+1.0

-63

-184

-6.0

5'-d(TAG GTC AAT ACT) (ODN-7) 5'-d(TAG GTC AAT ACT) (ODN-7) 47.5 -78 -220 -10.5 48.5 +1.0 -79 -221 -10.8 3'-d(ATC CAG TTA TGA) (ODN-8) 3'-d(ATC CAG* TTA TGA) (ODN-2) 5'-d(TAG GTG AAT ACT) (ODN-9) 5'-d(TAG GTG AAT ACT) (ODN-9) 27.0 -57 -165 -6.1 27.5 +0.5 -57 -164 -6.2 3'-d(ATC CAG TTA TGA) (ODN-8) 3'-d(ATC CAG* TTA TGA) (ODN-2) 5'-d(TAG GTT AAT ACT) (ODN-10) 5'-d(TAG GTT AAT ACT) (ODN-10) 32.0 -67 -193 -6.9 30.5 -1.5 -64 -184 -6.6 3'-d(ATC CAG TTA TGA) (ODN-8) 3'-d(ATC CAG* TTA TGA) (ODN-2) 5'-d(TAG GTA AAT ACT) (ODN-11) 5'-d(TAG GTA AAT ACT) (ODN-11) 30.5 -67 -195 -6.5 29.0 -1.5 -66 -192 -6.2 3'-d(ATC CAG TTA TGA) (ODN-8) 3'-d(ATC CAG* TTA TGA) (ODN-2) a b Measured at 260 nm in 0.1 M NaCl, 10 mM MgCl2, 10 mM Na-cacodylate (pH 7.0) with 5 µM + 5 µM single strand concentration. Tm values were determined from the melting curves by using the software MELTWIN 3.0. c ∆Tm was calculated as Tm modified duplex − Tm unmodified duplex. The thermodynamic data for the duplex ODN7 • ODN-8 were determined by MELTWIN and by concentration-dependent Tm values and were consistent within a range of 10%.61

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Next, the self-pairing of anomeric 8-azaguanine and guanine nucleosides was studied as many investigations have shown that a positioning of identical residues in opposite positions within the DNA double helix are more stable as in other combinations.62 Purine-purine base pairs have been reported for all-purine DNA,62,63 including those with guanine-guanine interactions. A single crystal X-ray structure by Neidle describes the influence on the DNA backbone conformation and the formation of Hoogsteen base pairs.64 However, nothing is known about base pairing of anomeric “purine” nucleosides with identical or nearly identical nucleobases. Table 4 shows that duplex combinations in which α-nucleosides facing each other are much more stable than those with β-anomeric connectivity (Figure S4, Supporting Information). Combinations of α with β nucleosides in opposite location show stabilities in between. We anticipate that possible stress on the DNA backbone induced by 2’deoxyguanosine-2’-deoxyguanosine interaction, when both are in β-D configuration, is partially released when both purine residues are α-D anomers. It stays to proof if WatsonCrick type or Hoogsteen base pairs are formed. The latter would be rather unstable for αGd*αGd* pairs as nitrogen-8 reduces the electron density of nitrogen-7 required for Hoogsten base pairing. Table 4. Tm Values of Oligodeoxyribonucleotide Mismatch Duplexes Containing αGd*, αGd, Gd* and Gda Tm [°C]

Duplexes

Duplexes

Tm [°C]

5'-d(TAG GTαG AAT ACT) (ODN-3) 3'-d(ATC CAαG TTA TGA) (ODN-4)

36.5

5'-d(TAG GTG AAT ACT) (ODN-9) 3'-d(ATC CAG TTA TGA) (ODN-8)

26.5

5'-d(TAG GTαG* AAT ACT) (ODN-5) 3'-d(ATC CAαG* TTA TGA) (ODN-1)

33.0

5'-d(TAG GTG* AAT ACT) (ODN-6) 3'-d(ATC CAG* TTA TGA) (ODN-2)

28.0

5'-d(TAG GTαG AAT ACT) (ODN-3) 3'-d(ATC CAαG* TTA TGA) (ODN-1)

35.0

5'-d(TAG GTG AAT ACT) (ODN-9) 3'-d(ATC CAG* TTA TGA) (ODN-2)

27.0

5'-d(TAG GTαG* AAT ACT) (ODN-5) 3'-d(ATC CAαG TTA TGA) (ODN-4)

35.5

5'-d(TAG GTG* AAT ACT) (ODN-6) 3'-d(ATC CAG TTA TGA) (ODN-8)

28.5

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5'-d(TAG GTG AAT ACT) (ODN-9) 3'-d(ATC CAαG TTA TGA) (ODN-4)

31.0

5'-d(TAG GTG* AAT ACT) (ODN-6) 3'-d(ATC CAαG TTA TGA) (ODN-4)

30.0

5'-d(TAG GTαG AAT ACT) (ODN-3) 3'-d-(ATC CAG TTA TGA) (ODN-8)

33.5

5'-d(TAG GTαG AAT ACT) (ODN-3) 3'-d(ATC CAG* TTA TGA) (ODN-2)

30.5

5'-d(TAG GTG* AAT ACT) (ODN-6) 3'-d(ATC CAαG* TTA TGA) (ODN-1)

30.0

5'-d(TAG GTG AAT ACT) (ODN-9) 3'-d(ATC CAαG* TTA TGA) (ODN-1)

32.0

5'-d(TAG GTαG* AAT ACT) (ODN-5) 3'-d(ATC CAG* TTA TGA) (ODN-2)

31.0

5'-d(TAG GTαG* AAT ACT) (ODN-5) 3'-d(ATC CAG TTA TGA) (ODN-8)

34.0

[a] Measured at 260 nm with 5 µM + 5 µM single-strand concentration at a heating rate of 1.0 °C/min in 100 mM NaCl, 10 mM MgCl2, 10 mM Na-cacodylate buffer, pH 7.0. Tm values were calculated from the heating curves.

Next, CD spectra of oligodeoxyribonucleotide duplexes were measured. Figure 5 shows the CD spectra of duplexes 3'-d(ATC CAG TTA TGA) (ODN-8) • 5'-d(TAG GTC AAT ACT) (ODN-7) containing the αGd* and αGd as well as the Gd* and Gd in the marked dG position. The data show that the modification with the α-anomers did not lead to major changes of the CD spectra. It indicates that the global B-DNA structure is retained when one hybrid base pair was incorporated.

Figure 5. CD spectra of oligodeoxyribonucleotide duplexes containing αGd* (ODN-1), Gd* (ODN-2), αGd (ODN-4) and Gd (ODN-8) opposite to dC (ODN-7) respectively. The spectra were measured at 5 °C in 100 mM NaCl, 10 mM MgCl2, 10 mM Na-cacodylate buffer (pH 7.0) with 5 µM + 5 µM single strand concentration.

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Bioconjugate Chemistry

Photophysical Properties and Mismatch Discrimination by Fluorescence Quenching. 8-Azapurines and their nucleosides display fluorescence and develop excited state phototautomerism.30,31,65,66 The 8-azapurines fluorescence is sensitive to solvent changes and responds on changes occurring within the DNA double helix. Recently, the fluorescence properties of 8-azaguanine and 8-azaisoguanine 2'-deoxy-β-D-ribonucleosides were studied in single and double stranded DNA.39,42,43 The fluorescence emerges at slightly elevated pH as the anionic 8-azaguanine base is the fluorescent species. The UV spectra of αGd* in water show a strong absorption at 255 nm (ε255 = 12700) and a shoulder at 278 nm (ε260 = 11300; Figure S5 in Supporting Information). The same was observed for the β-anomer Gd* but the extinction coefficient was slightly different.39 In methanol the molecules show one absorption at 255 nm (neutral molecule) whereas in aqueous solution at pH 12 only one band at 278 nm exists (nucleoside anion). Then, UV spectra of αGd* and Gd* were measured at a 40 µM nucleoside concentration in phosphate buffer at pH 8.0. Both anomers show nearly identical UV spectra with absorptions at 255 nm and 278 nm (Figure 6a). Also the extinction coeffients are similar at pH 8.0 in phosphate buffer with ε = 10200 (278 nm) for αGd* and 9800 (278 nm) for Gd*. As the measurements were performed near to the pKa value only about 50% of the molecules are in the fluorescent (anionic) state. When the pH value was increased to 9.0 or 10.0 the absorption band of the neutral species at 255 nm disappeared and only the absorption of the nucleoside anion at 278 nm was present (Figure 6b). Next, fluorescence spectra were recorded for both anomers αGd* (Figure 6c) and Gd* (Figure 6d) at pH 8.0 and 10.0 with excitation at 278 nm and fluorescence emission peaks centered around 360 nm. From this and according to the pKa

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values near 8.0 it is obvious that at pH 8.0 only half of the molecules display fluorescence as they are in the anionic state. The quantum yields of αGd* and Gd* were determined in phosphate buffer (pH 8.0). The fluorescence quantum yields (Φ) were calculated using quinine sulfate in 0.5 M H2SO4 (Φfl = 0.55).67 The quantum yield of αGd* was 0.61, which is higher compared to the value for the β-nucleoside Gd* (0.53). A similar quantum yield as for Gd* was reported earlier for the 8azaguanine ribonucleoside (0.55 at pH 8.0).31 Please note, that quantum yields obtained at pH 8.0 are caused by the fluorescent anionic molecules, which are in a mixture with equal amounts of the non-fluorescent neutral species. A pH value of 8.0 was chosen as higher pH values can open canonical base pairs in oligodeoxyribonucleotide duplexes.

Figure 6. a) UV-spectra of anomeric 8-aza-2'-deoxyguanosines αGd* and Gd* in 0.1 M sodium phosphate buffer at pH 8.0 at 40 µM nucleoside concentration; b) UV-spectra of the 20

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Bioconjugate Chemistry

α-nucleoside αGd* at pH 9.1 and 10.0; c) fluorescence excitation and emission spectra of αGd* (5 µM concentration) at pH 8.0 and pH 10.0. Excitation wavelength is 278 nm. d) fluorescence excitation and emission spectra of Gd* (5 µM concentration) at pH 8.0 and pH 10.0. Excitation wavelength is 278 nm.

Subsequently, the pKa value of deprotonation for the αGd* nucleoside was determined by fluorescence (Figure 7) and was found to be similar to the values measured by UV (pKa = 8.0 and 7.9) (Figure 7c). From Figure 7 it is apparent that the fluorescence intensity increase from pH 4.4 to pH 9.7 and decrease between pH 9.7 to pH 12.5. The curves imply that another deprotonation pKa might exist around pKa =11.7. Phototautomerism which has been reported for 8-azapurine nucleosides might cause this phenomenon (Figure 7c).65

Figure 7. pH-dependent fluorescence spectra of αGd* measured in 0.1 M sodium phosphate buffer, a) emission spectra at pH values from 4.4 to 9.7; b) emission spectra at pH values from 9.7 to 12.5; c) graph of fluorescence emission against pH value (sigmoidal curve) and its first derivative (lines marked in red) using data from (a, b).

Then, the fluorescence spectra of single-stranded (ss) oligodeoxyribonucleotides and oligodeoxyribonucleotide duplexes (ds) incorporating the anomeric 8-azaguanine nucleosides αGd* and Gd* were measured (see Figure 8, Figure S6, Supporting Information). The highest fluorescence intensity was observed for single stranded oligodeoxyribonucleotides incorporating αGd* and Gd*. Then, duplexes were studied which were already utilized in Tm 21

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experiments (Table 3). Fluorescence decreased after hybridization (Figure 8, Figure S6, Supporting Information). The strongest fluorescence decrease appeared in duplexes incorporating αGd*-Cd or Gd*-Cd matches. This shows that both anomers form stable base pairs with dC although the sugar connectivity is different. However in both cases an additional low fluorescence emission appears around 450 nm. The source of this phenomenon is unclear.

In mismatches a gradual fluorescence change takes place in both series of α- and βnucleosides. In the α-series the fluorescence of duplexes increases in the following order: αGd*-Cd