Anomeric DNA: Functionalization of α‐d Anomers of 7‐Deaza‐2′‐deoxyadenosine and 2′‐Deoxyuridine with Clickable Side Chains and Click Adducts in Homochiral and Heterochiral Double Helices

Abstract Anomeric base pairs in heterochiral DNA with strands in the α‐d and β‐d configurations and homochiral DNA with both strands in α‐d configuration were functionalized. The α‐d anomers of 2′‐deoxyuridine and 7‐deaza‐2′‐deoxyadenosine were synthesized and functionalized with clickable octadiynyl side chains. Nucleosides were protected and converted to phosphoramidites. Solid‐phase synthesis furnished 12‐mer oligonucleotides, which were hybridized. Pyrene click adducts display fluorescence, a few of them with excimer emission. T m values and thermodynamic data revealed the following order of duplex stability α/α‐d≫β/β‐d≥α/β‐d. CD spectra disclosed that conformational changes occur during hybridization. Functionalized DNAs were modeled and energy minimized. Clickable side chains and bulky click adducts are well accommodated in the grooves of anomeric DNA. The investigation shows for the first time that anomeric DNAs can be functionalized in the same way as canonical DNA for potential applications in nucleic acid chemistry, chemical biology, and DNA material science.


Introduction
Anomeric DNA is formed when one strand of a duplex is in the α-d and the other in the β-d-configuration. [1,2] When DNA is constructed from two strands in the α-d configuration, homochiral α/α-DNA is generated; the anomeric counterpart to canonical DNA. The strand orientation in heterochiral α/β-DNA is parallel, whereas homochiral α/α-DNA displays an antiparallel alignment. [1d] Here, the terms homochiral and heterochiral correspond solely to the stereochemistry at the anomeric center and not to d/l configuration. Recently, 2-aminoadenine and the related 8-aza-7-deazaadenine nucleobases (purine numbering is used throughout the results and discussion section) were used as replacements of the adenine moiety in an adenine-thymine pair. As a result, a significant stabilization of modified duplexes over that with canonical bases was observed. Also, the capability of anomeric nucleosides to form silver-mediated base pairs was demonstrated on the basis of an anomeric α-d/β-d dC-dC metal base pair. [3] Herein, we report on the functionalization of the 2'deoxyadenosine-2'-deoxythymidine base pair. The 2'-deoxyadenosine moiety was replaced by the α-d anomer of 7-deaza-2'deoxyadenosine and the 2'-deoxythymidine residue was substituted by anomeric 2'-deoxyuridine moieties. Both nucleosides were functionalized; the 7-deazapurine base at the 7-position and the pyrimidine base at the 5-position. Modifications were performed at one site of the base pair and double modifications at both sites. We anticipated that the positions of functionalization occurring in the major groove of canonical DNA will be also suitable for anomeric DNA and bulky residues might be well accommodated in anomeric double helices.
Octadiynyl residues with terminal triple bonds were introduced in the α-d anomer of 7-deaza-2'-deoxyadenosine (c 7 A d ) and 2'-deoxyuridine (dU) as they can be clicked to almost any other azide by the Huisgen-Meldal-Sharpless cycloaddition. Bulky pyrene azide was employed to form click adducts. To this end, nucleosides 2 and 9 were protected and converted to phosphoramidites and 12-mer oligonucleotides were synthesized. The synthesis of the oligonucleotides with α-d configuration made use of phosphoramidites of the α-d anomers of 7-deaza-2'-deoxyadenosine 12 and 2'-deoxyuridine 4 together with those of the four α-d nucleoside phosphoramidites with canonical bases. Then Heisenbergstrasse 11,48149 Münster (Germany) combinations to form heterochiral and homochiral duplexes ( Figure 1). Within the anomeric strands, single incorporations of base-modified α-nucleosides were performed at the purine or the pyrimidine site and double modifications on both sites. Finally, copper(I)-catalyzed click reactions were executed and bulky pyrene substituents were introduced. In addition, the sequential order of heterochiral DNAs was altered to evaluate sequence dependencies between anomeric and canonical DNAs.
With duplexes in hand, temperature-dependent melting profiles were recorded and T m values and thermodynamic data were calculated. CD-spectra were measured to detect global helical changes. Fluorescence studies with DNA pyrene adducts gave information on DNA dye conjugates. Finally, AMBER force field energy minimization were undertaken on the functional-ized DNA. These studies visualize the size of bulky substituents and the available space of the grooves.
Next, the pyrene functionalized α-dU and β-dU nucleosides 17 and 18 were synthesized by using the Huisgen-Meldal-Sharpless click reaction (Scheme 2). [10,11] For this, the 5-octadiynylated compounds 2 and 13 were treated with an excess of pyrene azide in the presence of Cu II sulfate pentahydrate and ascorbic acid as reducing agent in THF/tBuOH/H 2 O at RT overnight. By this, the functionalized nucleosides 17 and 18 were obtained in 62 % (α-d) and 70 % (β-d) yield. All new synthesized compounds were characterized by 1 H, 13 C NMR spectra as well as ESI-TOF mass spectra. The 1 H, 13 C correlated (HMBC and HSQC) NMR spectra were used to assign the 13 C Figure 1. Schematic view of duplex structures with oligonucleotide strands in the α/β-d, α/α-d and β/β-d configurations and with α/β-d base pairs. Anomeric 5-substituted 2'-deoxyuridines and 7-substituted 7-deaza-2'-deoxyadenosines with clickable side chains and click adducts used in this study. NMR signals (Tables S1 and S2 in the Supporting Information). Then, fluorescence spectra were recorded in various solvents to determine solvent dependent changes ( Figure S7A and B). Both anomeric dU click conjugates show almost identical spectra and the same solvent dependence. Fluorescence was high in DMSO but was low in water. Monomer emission occurred in all cases and the compounds did not show excimer emission.
Typical melting profiles are shown in Figure 2. All curves show cooperative melting. In Tables 2 and 3, T m values and thermal hypochromicity data are summarized and duplex stability of α/β-DNA is compared to β/β-DNA.
According to the T m data only small stability changes occur in homochiral DNAs, whereas significantly stronger changes are observed for heterochiral DNA. For heterochiral DNA, already the stabilities of the nonfunctionalized duplexes differ and the same effect is observed for duplexes with single or double modifications within the base pairs ( Table 2 vs. Table 3). Apparently, heterochiral duplexes with parallel chain orientation are more sensitive to sequence changes than their homochiral antiparallel counterparts.
In more detail, when side chains were introduced in the 5position of dU or 7-position of c 7 A d in canonical homochiral β-DNA (both series of duplexes, Tables 2 and 3) modifications have only a slight impact on the duplex stability with respect to the unmodified duplexes. For heterochiral duplexes incorporating dU or c 7 A d side-chain derivatives the T m values are usually around 4 to 8°C lower compared to homochiral DNA. This is valid for single or double modifications.
Furthermore, pyrene side chains contribute extra stability to both homo-and heterochiral duplexes. However, in homochiral DNA stabilization by pyrene residues is particularly strong which might be related to the intercalation of the pyrene moiety. Thermal hypochromicities are similar for heterochiral and Double functionalization Double functionalization [a] Measured at 260 nm at a concentration of 5 μM + 5 μM single strand at a heating rate of 1.0°C min À 1 in 100 mM NaCl, 10 mM MgCl 2 , and 10 mM Na-cacodylate (pH 7.0).
[b] T m values were calculated from the heating curves using the program Meltwin 3.0. [12] [c] H = hypochromicity.
[d] For duplexes containing pyrene click adducts, a concentration of 2 μM + 2 μM single strand was used. homochiral duplexes and values between 15 to 20 % were measured in both series. Next, thermodynamic data were determined. To this end, the program Meltwin 3.0 [12] was used and data were extracted from melting curve shape analyses. ΔH°values were higher for homochiral DNA duplexes with both strands in β-d configuration with respect to heterochiral α/β DNA. Data indicate that loss of duplex stability might be due to altered stacking forces and/or weaker H-bonding. [13] The global helix conformation of duplex DNA can be monitored by CD spectra. [14,15] Various factors contribute to the shape of the CD-spectra: i) the conformation of the monomers which can be syn or anti, ii) the conformation of the sugar residues (N vs. S), iii) the hydrogen-bonding network formed between nucleobases, iv) stacking interactions between nucleobases or base pairs and v) the helicity of the duplex (+ or À ). Previous CD experiments have shown that CD spectra of αanomeric single strands display spectra with mirror-like Cotton effects with respect to the β-strand. [1,2,3] Complete mirror images are not expected as diastereoisomers are compared and not enantiomeric molecules with strands in d-and l-configuration. The phenomenon has been already discussed for nonfunctionalized α-d hexamer oligonucleotide duplexes with all-purine or all-pyrimidine bases in either the αor the β-strand. [1b] Our DNA fragment represent a full helix turn that contains all four DNA bases in random composition with and without clickable side chains and click adducts.
According to Figure 3 remarkable strong negative Cotton effects are observed for the α-strands. These negative CD signals around 280 nm disappear when the α-strand is hybridized with the β-strand to form a duplex. Now, the Cotton effect becomes positive and displays a similar shape as the βstrand. These strong changes of the CD spectra are typical for all heterochiral DNA duplexes used in this study -modified or not. It shows that the strands adopt the conformation of the βstrand and a strong conformational change occurs during hybridization. It has been already known for canonical DNA that single strands are more flexible than duplexes. Single strands show a persistence length of a few nanometers whereas double stranded helices form stiff rods with a persistence length of around 40 nm. Here, the β-strand dictates the conformation of the final duplex. Duplex DNA is then much more difficult to bent as single stranded DNA. This was shown for canonical DNA but is now also anticipated for anomeric DNA.
Also, experimentally determined CD spectra of duplex DNAs were compared with calculated spectra (sum of the CD spectra of the single strands, Figure 3). The shape of CD curves of homochiral β/β duplexes from calculated and measured spectra is similar, whereas completely different spectra were obtained for the measured heterochiral α/β duplexes with respect to the calculated spectra. Measured α/βduplexes exhibit a similar shape as homochiral β/β-d duplexes, calculated spectra of α/βduplexes display curves similar to α-d single strands. Apparently, upon duplex formation the β-anomer seems to dictate the sign of CD spectra and therefore the structure of the heterochiral duplexes. This phenomenon is observed for all α/β duplexes with and without side chains. All data indicate that base pair overlaps and stacking interactions are similar in Figure 2. Melting curves of heterochiral parallel α/β-d duplexes and the corresponding homochiral antiparallel β/β-d ones measured at 260 nm at a concentration of 5 μM + 5 μM single strand at a heating rate of 1.0°C min À 1 in 100 mM NaCl, 10 mM MgCl 2 , and 10 mM Na-cacodylate (pH 7.0). For duplexes containing pyrene click adducts, a concentration of 2 μM + 2 μM single strand was used. Table 3. T m values and thermodynamic data for antiparallel-and parallel-strand duplexes containing α-7-octadiynyl-c 7 A d 9, β-5-octadiynyl-dU 13, β-7-octadiynyl-c 7 A d 14 and pyrene click conjugates. [a] Heterochiral (α/β) duplexes

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Research Article doi.org/10.1002/chem.202103872 heterochiral (α/β) DNA with respect to homochiral (β/β) DNA. From the spectra of duplexes one can conclude that clickable side chains of moderate size (octadiynyl) and more space demanding residues (pyrene) are well accommodated in the grooves of heterochiral α/β DNA. Thus, data confirm that the position-5 for pyrimidines and position-7 for 7-deazapurines are ideally suited for functionalization.

Functionalized homochiral DNA with both side chains in α-d configuration
Next, duplexes were studied with both strands in an α-d configuration. [16] To determine the strand stoichiometry, socalled mixing experiments of a series of homochiral duplexes were performed. For each synthetic duplex, a series of mixtures were prepared with varying ratios of oligonucleotide and a constant total oligonucleotide concentration. The absorbance of each mixture was measured three times at a wavelength of 260 nm resulting in a titration graph (Figures 4 and S2). According to Figure 4, all duplexes showed one to one strand stoichiometry confirming that only duplexes are formed and that the formation of other assemblies, for example triplexes, is excluded. Figure 5 shows melting curves for α/α-d duplexes measured at 260 nm. According to this figure, all curves showed cooperative melting with high T m values around 60°C. From that thermodynamic data were calculated (Table 4). Single modification of a dT residue by the side-chain derivative 2 shows a significantly higher T m value than replacement of dA by the derivative 9. On the contrary, the α-anomeric dU click adduct 17 retains the T m value of the nonfunctionalized duplex ODN-1 · ODN-5. Apparently, side-chain derivatives with α-dU modification have no negative impact on the stability of homochiral α/α DNA.
Compared to that 7-deazapurine functionalized duplexes are generally less stable. This phenomenon is also apparent from duplexes with double modifications and is in line with previous results on homochiral DNA formed by both strands in β-d configuration. [3,17] Hypochromicities were low for α/α duplexes (ca. 12 %) with respect to α/β or β/β duplexes (ca. 20 %). Obviously, the base overlap is significantly different in homochiral α/α DNA compared to β/β or α/β DNA. Nevertheless, base pairing is extremely strong.
From the thermodynamic data it is obvious that a favorable enthalpy is responsible for this phenomenon. Encouraged by these results, we synthesized the α-d anomeric counterpart of the self-complementary Dickerson Drew dodecamer [18] β-5'd(CGC GAA TTC GCG) 2 (ODN-18), namely α-5'-d(CGC GAA TTC GCG) 2 (ODN-9). The T m of this α-anomeric Dickerson duplex was extremely high under buffer conditions used in this manuscript and a complete melting profile could not be recorded. Consequently, a low salt buffer was chosen. Now, a complete sigmoidal melting curve was observed with a T m value of 75°C

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Research Article doi.org/10.1002/chem.202103872 ( Figure S4). The β-configurated Dickerson dodecamer showed a T m value of 40°C for duplex melting. It is obvious that non-selfcomplementary and also self-complementary α/α duplexes exhibit a much higher stability than those with β/β-configuration.
To get information on global changes of oligonucleotide duplexes formed by two α-d strands CD spectra were measured. In Figure 6A, B, the measured and calculated spectra of oligonucleotide duplexes with α/α-configuration as well as spectra of the corresponding single strands are displayed. All CD spectra showed maxima with negative Cotton effects around 278 nm. This is different to the CD spectra of the heterochiral α/β duplexes all showing positive lobes. Temperature dependent CD spectra were used to determine T m values ( Figure 6C, D). The T m data obtained by CD spectra (Figure 6E, F) were almost identical to those measured by UV showing high T m values around 60°C.
A few homochiral and heterochiral duplexes reported in this study contain pyrene residues linked to the α-d or the β-d oligonucleotide strands. As discussed above, pyrene residues contribute stability to duplexes. Pyrene is a fluorescent molecule with five emission peaks (375-405 nm) and an additional band (excimer) when two pyrenes are in proximal position. [19] As excimer emission is sensitive to environmental changes, it can be used to determine intermolecular interactions. [20,21] Thus, differences might exist among the various duplexes with oligonucleotides in anomeric configuration. To this end, three duplexes were chosen with homochiral α/α, β/β strands and heterochiral α/β configuration and fluorescence measurements were performed. According to Figure 7, all duplexes show monomer fluorescence but only the homochiral duplexes ODN-3 · ODN-8 (α/α) and ODN-21 · ODN-22 (β/β) show significant excimer emission.

Molecular models of anomeric DNA and impact of nucleobases and side chains
Molecular models of anomeric DNA were constructed by Amber force field incorporated in the software package HyperChem 8.0 for Windows (Hypercube, Inc.). Original Amber parameters were used (see the Experimental Section) and no water or counter Table 4. T m values and thermodynamic data for antiparallel-and parallel-strand duplexes containing α-7-octadiynyl-c 7 A d 9, α-5-octadiynyl-dU 2 and pyrene click conjugates. [ [a] Measured at 260 nm at a concentration of 5 μM + 5 μM single strand at a heating rate of 1.0°C min À 1 in 100 mM NaCl, 10 mM MgCl 2 , and 10 mM Nacacodylate (pH 7.0).
[b] T m values were calculated from the heating curves using the program Meltwin 3.0. [12] [c] H % corresponds to hypochromicity in %.
[d] For duplexes containing pyrene click adducts, a concentration of 2 μM + 2 μM single strand was used. A few single stranded α-d oligonucleotides showed weak cooperative melting which disappeared after hybridization (data are shown in Table S3 and Figure S4).
ions were added. All duplexes were energy minimized but not refined. To this end, the β-nucleosides from one strand of the standard β/β duplex ODN-12 · ODN-13 were replaced by αnucleosides including those with functionalized side chains. For homochiral duplexes with both strands in α/α-d configuration and antiparallel strand alignments, all β-nucleoside residues were substituted by α-nucleosides including the nucleosides with clickable side chains or pyrene click adducts. Figure Figure S9A-I provides information on the available space in the major grooves of the different DNA structures. From that it is obvious, that the clickable octadiynyl residues as well as the bulky pyrene click adducts are well accommodated in heterochiral and homochiral double helices and do not disturb the global double-helix structure. Anomeric DNA with strands in α-d and β-d configurations form duplexes with parallel strand orientation. The same strand alignment was reported for duplexes containing iG d -dC, iC d -dG and reversed Watson-Crick base pairs (Donohue pair; iG d = 2'deoxyisoguanosine, iC d = 2'-deoxy-5-methylisocytidine). [22] According to Figure 8, heterochiral duplexes with parallel strand alignment form adenine-thymine and guanine-cytosine base pairs in the Watson-Crick mode. Furthermore, Hoogsteen pairing involving nitrogen-7 of purine bases can be excluded due to the absence of this purine nitrogen. This principle has been already reported for heterochiral α/β DNA in which all purine bases of the β-strand are replaced by nonfunctionalized 7-deazapurines. [3] The absence of purine nitrogen-7 and the replacement of the electronegative nitrogen atom of dA by an electropositive CH group (!c 7 A d ) affect the electrostatic potential in the major groove. Base stacking interactions are reduced and as a result a slight enthalpy destabilization of the c 7 A d -dT base pair is observed with respect to the dA-dT pair. Nevertheless, base pairing geometry of the Watson-Crick mode was retained. [17] Our T m measurements and thermodynamic data indicate that this is also valid for anomeric DNA.

Conclusion
DNA possesses an intrinsic polymorphism that depends on the sequence, the structure of nucleobases, and the sugar-phosphodiester backbone. [23][24][25] Base recognition and helix conformation play vital roles. This work reports the impact of clickable side chains and click adducts on heterochiral DNA with complementary strands in the α-d and β-d configurations and their homochiral counterparts with both strands in an α-d or βd configuration. Strand communication occurs in anomeric DNA. To this end, the α-anomer of 2'-deoxyuridine was functionalized with clickable octadiynyl side chains at the nucleobase 5-position, and the α-anomer of 7-deaza-2'-deoxyadenosine at position-7. Functionalized nucleosides were protected and converted to phosphoramidite building blocks, and oligonucleotides were synthesized. They were clicked to a bulky fluorescent pyrene azide. Heterochiral and homochiral duplexes were formed after hybridization. According to the T m values and thermodynamic data, alkynyl side chains of moderate size and with bulky pyrene residues are well accommodated in homochiral and heterochiral DNAs. Side-chain functionalization has only a minor effect on the stability of α/β-DNA or on DNA with both strands in the α/α configuration. Remarkably, α/ α-DNAs are much more stable than their α/β and β/β counterparts. The order of duplex stability was α/α-d @ β/β-d � α/β-d. CD spectra of all α-d single strands show mirror-like behavior with respect to β-d oligomers. After hybridization, α/β duplexes exhibit positive Cotton effects similar to those of their β/β counterparts, whereas α/α-d duplexes display negative signs. The global changes in the α-strands can be attributed to conformational helix adaption during base-pair formation. In all of our cases, the β-strands dictated the sign of the CD spectrum in the final duplex. The functionalized DNAs were modeled and energy minimized. HyperChem 8.0 dynamic simulations followed by AMBER force-field energy minimization showed that heterochiral and homochiral duplexes containing clickable side chains or click adducts form stable duplex structures. Side chains have sufficient space in double helices. With the knowledge from this investigation, almost any functionality or covalent label can be incorporated into anomeric DNAs by base modification without disturbing the helix structure. Clickable DNAs [26] can be used as hybridization probes in nucleic acids diagnostics, chemical biology, and material science to expand the toolbox of nucleic acid applications beyond canonical DNA. [7,9] Experimental Section General: All chemicals and solvents were of laboratory grade as obtained from Acros Organics or Sigma Aldrich and were used without further purification. Flash column chromatography (FC): silica gel 60 from VWR (40-60 μM) at 0.4 bar. UV-spectra were recorded on a Hitachi U-3000 UV spectrophotometer: λ max (ɛ) in nm, ɛ in dm 3 mol À 1 cm À 1 . 13   (HMBC, HSQC) NMR spectra were used for the assignment of the 13 C signals (Table S1). The J values are given in Hz; δ values in ppm relative to Me 4 Si as internal standard. For NMR spectra recorded in [D 6 ]DMSO, the chemical shift of the solvent peak was set to 2.50 ppm for 1 H NMR and 39.50 ppm for 13 C NMR. ESI-TOF mass spectra of nucleosides were recorded on a Micro-TOF spectrometer.
Oligonucleotide syntheses and characterization: Solid-phase oligonucleotide syntheses were performed on an ABI 392-08 synthesizer at 1 μmol scale (trityl-on mode) employing the phosphoramidites 4, 12, 15 [9] and 16 [7] as well as the standard building blocks with an average coupling yield over 95 %. After cleavage from the solid support, the oligonucleotides were deprotected in 28 % aqueous ammonia at 55°C for 2 h. The DMT-containing oligonucleotides were purified by reversed-phase HPLC (RP-18) with the gradient system at 260 nm: A) MeCN, B) 0.1 M (Et 3 NH)OAc (pH 7.0)/ MeCN, 95 : 5; gradient I: 0-3 min 10-15 % A in B, 3-15 min 15-50 % A in B; flow rate 0.7 mL/min. The purified "trityl-on" oligonucleotides were treated with 2.5 % CHCl 2 COOH/CH 2 Cl 2 for 2 min at 0°C to remove the 4,4'-dimethoxytrityl residues. The detritylated oligomers were purified again by reversed-phase HPLC with gradient II: 0-20 min 0-20 % A in B; 20-25 min, 20 % A in B; flow rate 0.7 mL/min. The oligonucleotides were desalted on a reversed-phase column (RP-18) using water for elution of salt, while the oligonucleotides were eluted with H 2 O/CH 3 OH (2 : 3). The oligonucleotides were lyophilized on a Speed-Vac evaporator to yield colorless solids which were frozen at À 24°C. The molecular masses of the oligonucleotides were determined by MALDI-TOF mass spectrometry on a Bruker Autoflex Speed in the linear positive mode with 3hydroxypicolinic acid (3-HPA) as a matrix. The thermal melting curves were measured with an Agilent Technologies Cary 100 Bio UV-vis spectrophotometer equipped with a thermoelectrical controller. The temperature was measured continuously in the reference cell with a Pt-100 resistor with a heating rate of 1°C min À 1 . T m values were determined from the melting curves using the software Meltwin, version 3.0. [12] CD spectra were recorded at 25°C on a Jasco J-815 spectrometer.

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Research Article doi.org/10.1002/chem.202103872 octadiynyl-dU (13) 3800, [7] β-7-octadiynyl-c 7 A d (14) 5300, [7] α-7octadiynyl-c 7 A d (9) 6400 and their pyrene click conjugates 17 14 600, 18 13 800 and 20 19 400 mol À 1 dm À 3 cm À 1 . The extinction coefficients of the oligonucleotides were calculated from the sum of the extinction coefficients of nucleoside constituents considering the hypochromic change for the particular single strands. Click reactions were made by post-modification of modified oligonucleotide single strands. The DNA synthesizer cycle was the same for α-d oligonucleotides as for their β-d counterparts. Fluorescence spectra were recorded on a fluorescence spectrophotometer (Hitachi, Tokyo, Japan) in the wavelength range between 350 and 600 nm. Molecular modeling was performed with the Amber force field as incorporated in the software package HyperChem 8.0 (Hypercube Inc., Gainesville, FL, USA). Only original Amber parameters were used and no counter ions or water were included. A distance dependent scale factor of ɛ = 1 was used. One to four non-bonded interactions were scaled by 0.5. No cutoffs were applied. All duplex structures were built on the basis of a B-DNA energy minimized but not refined.