C5-alkynyl-functionalized α-L-LNA: synthesis, thermal denaturation experiments and enzymatic stability.

Major efforts are currently being devoted to improving the binding affinity, target specificity, and enzymatic stability of oligonucleotides used for nucleic acid targeting applications in molecular biology, biotechnology, and medicinal chemistry. One of the most popular strategies toward this end has been to introduce additional modifications to the sugar ring of affinity-inducing conformationally restricted nucleotide building blocks such as locked nucleic acid (LNA). In the preceding article in this issue, we introduced a different strategy toward this end, i.e., C5-functionalization of LNA uridines. In the present article, we extend this strategy to α-L-LNA: i.e., one of the most interesting diastereomers of LNA. α-L-LNA uridine monomers that are conjugated to small C5-alkynyl substituents induce significant improvements in target affinity, binding specificity, and enzymatic stability relative to conventional α-L-LNA. The results from the back-to-back articles therefore suggest that C5-functionalization of pyrimidines is a general and synthetically straightforward approach to modulate biophysical properties of oligonucleotides modified with LNA or other conformationally restricted monomers.

Introduction

Significant efforts have been devoted to the development of conformationally restricted nucleotides.[1−3] Oligonucleotides that are modified with such building blocks often display high affinity toward nucleic acid targets and are accordingly used for a variety of applications in molecular biology, biotechnology, and medicinal chemistry.[4] Locked nucleic acid (LNA),[5−7] which is also known as bridged nucleic acid (BNA),[8] is one of the most promising members of this compound class, as it displays some of the highest affinities toward complementary DNA/RNA targets reported to date (increases in duplex thermal denaturation temperatures, Tm’s, of up to +10 °C per modification have been observed). One of the diastereoisomers of LNA, i.e., α-L-LNA (α-l-ribo configuration; Figure 1) displays similar hybridization characteristics[9] and lower hepatotoxicity[10a] and has accordingly been studied as a potential modification for antisense, antigene, and decoy oligonucleotides.[10] The interesting properties of α-L-LNA have spurred the development of many analogues, all of which have focused on further improving the biophysical properties of α-L-LNA through modification or expansion of the oxymethylene bridge spanning the C2′ and C4′ positions of α-L-LNA and/or introduction of small branching substituents onto the conformationally restricted furanose skeleton.[10a,11]

Figure 1

Structures of nucleotide monomers studied herein.

Our continued interest in LNA chemistry and C5-functionalized pyrimidine DNA building blocks[4c,12] prompted us to pursue the synthesis of C5-alkynyl-functionalized LNA uridine (U) monomers.[13] We hypothesized that C5 substituents could be used to modulate the characteristics of LNA pyrimidines. Our preliminary results on a small set of C5-alkynyl-functionalized LNA-U were promising and supported this hypothesis.[13] Thus, oligodeoxyribonucleotides (ONs) modified with LNA-U monomers that are conjugated to small alkynes display increased target affinity and binding specificity along with moderately improved protection against 3′-exonucleases. ONs modified with large C5-functionalized LNA-U monomers display very high enzymatic stability, albeit at the expense of target affinity. Motivated by these results, we set out to (i) study a greater number of C5-functionalized LNA-U monomers[14] and (ii) explore if this strategy for modulation of biophysical properties can be applied to other conformationally restricted nucleotides.

Here, we present the synthesis of six different C5-alkynyl-functionalized α-L-LNA-U phosphoramidites, their incorporation into ONs, and the characterization of these modified ONs via thermal denaturation, enzymatic stability, and steady-state fluorescence emission experiments. These monomers were selected to ensure a representation of C5 substituents with different sizes and polarities (Figure 1) and to facilitate direct comparison with corresponding C5-alkynyl-functionalized LNA-U.[14]

Results and Discussion

Synthesis of α-L-LNA Key Intermediate 9

Our synthetic route to C5-functionalized α-L-LNA-U phosphoramidites 11S–Y is inspired by the optimized routes to LNA[15] and α-L-LNA,[9b] as well as our recent synthesis of C5-alkynyl-functionalized LNA nucleotides.[13,14] Thus, fully protected glycosyl donor 1, which is obtained from diacetone-α-d-glucose in six steps and ∼30% overall yield,[9b,16] was used as a starting material (Scheme 1). One-pot glycosylation of 1 with persilylated uracil under Vorbrüggen conditions afforded nucleoside 2 in 85% yield via anchimeric assistance. Treatment of 2 with hydrogen chloride in methanol afforded O2′-deacetylated nucleoside 3 in 97% yield. We found these conditions to be preferable to the use of cold dilute methanolic ammonia,[9b] which results in the formation of small amounts of xylo-LNA byproducts. Subsequent O2′-mesylation of 3 provided activated nucleoside 4 in 98% yield. Treatment of 4 with aqueous sodium hydroxide resulted in a cascade reaction,[17] i.e., formation of an O2,O2′-anhydronucleoside, hydrolysis of the anhydronucleoside, and ring formation via intramolecular nucleophilic displacement, to afford α-L-LNA nucleoside 5 in quantitative yield. Subsequent protecting group manipulations entailing nucleophilic substitution of the O5′-mesylate of 5 (98%), O5′-debenzoylation of 6 (95%), and O3′-debenzylation of 7 (85%), using catalytic transfer hydrogenation conditions known to minimize undesired uracil C5–C6 reduction (formic acid and Pd(OH)2/C),[18] proceeded smoothly to afford diol 8. Next, a reaction sequence entailing O3′,O5′-diacetylation, C5-iodination, O3′,O5′-deacylation, and O5′-dimethoxytritylation converted diol 8 into key intermediate 9 in high yield without purification of intermediates. Direct C5-iodination of 8 and subsequent O5′-dimethoxytritylation to directly afford key intermediate 9 in only two steps is also possible but less attractive, due to lower overall yield and more complicated purification (see Scheme S1, Supporting Information). Hence, key intermediate 9 is obtained in ∼45% overall yield and four chromatographic purification steps from glycosyl donor 1 (Scheme 1).

Scheme 1

Synthesis of Key Intermediate 9

Abbreviations: BSA, N,O-bis(trimethylsilyl)acetamide; U, uracil-1-yl; CAN, ceric ammonium nitrate; DMTr, 4,4′-dimethoxytrityl.

Synthesis of C5-Alkynyl-Functionalized α-L-LNA Phosphoramidites

Sonogashira reactions[19] between key intermediate 9 and different terminal alkynes[20] provided C5-alkynyl-functionalized LNA uridines 10 in moderate to excellent yield (Scheme 2). Desilylation of 10S′ using TBAF afforded 10S in 78% yield. Finally, O3′-phosphitylation of nucleosides 10 using 2-cyanoethyl-N,N′-diisopropylchlorophosphoramidite provided target phosphoramidites 11S–11Y in 52–84% yield.

Scheme 2

Synthesis of C5-Alkynyl-Functionalized α-L-LNA-U Phosphoramidites 11S–Y

Abbreviation: PCl-reagent, 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite.

Structural Verification of α-L-LNA Nucleosides

As expected,[9b] the 1H NMR signals of H1′, H2′, and H3′ of the α-L-LNA nucleosides appear as singlets or narrow doublets (J < 2 Hz),[21] since the torsion angles defined by H1′–C1′–C2′_-H2′ and H2′–C2′–C3′–H3′ are restricted to +gauche and −gauche conformations, respectively. Moreover, the ROESY spectrum of α-L-LNA diol 8 exhibits through-space couplings between (i) H6 and H5″, (ii) H1′, H2′ and H3′, and (iii) H5′ and H3′, whereas no through-space coupling between H2′ and H6 is observed (Figure S1, Supporting Information). These observations are fully consistent with the proposed stereochemical configuration.

Incorporation of C5-Alkynyl-Functionalized α-L-LNA Monomers into ONs

Novel phosphoramidites 11S–Y (and the known phosphoramidite of monomer Z(22)) were used to incorporate monomers S–Z into 9-mer mixed-sequence ONs via machine-assisted DNA synthesis. Standard procedures were applied except for the use of extended hand-coupling times during incorporation of C5-alkynyl-functionalized α-L-LNA monomers (generally 15 min with 4,5-dicyanoimidazole as an activator—see the Experimental Section). The composition and purity of all modified ONs was verified by MALDI-MS (Table S1, Supporting Information) and ion-pair reversed-phase HPLC, respectively. Unmodified reference DNA and RNA strands are denoted D1/D2 and R1/R2, respectively, while ONs containing a single incorporation of a modified nucleotide in the 5′-GTG ABA TGC context are denoted S1, V1, W1, and so on. Similar conventions are used for ONs in the B2–B4 series.

Thermal Denaturation Studies

The thermostabilities of duplexes between singly or doubly modified 9-mer ONs and DNA/RNA complements were determined by thermal denaturation experiments conducted in medium salt phosphate buffer ([Na+] = 110 mM). Thermal denaturation temperatures of modified duplexes are discussed relative to unmodified reference duplexes unless otherwise mentioned (Table 1).

Table 1

Thermal Denaturation Data for C5-Alkynyl-Functionalized α-L-LNA and Reference Strands against Complementary DNA/RNAa

		ΔTm/mod (°C)
ON	duplex	B =	L	S	V	W	X	Y	Z
B1	5′-GTG ABA TGC		+4.0	+7.5	+7.0	+9.5	+1.0	–2.5	–1.0
D2	3′-CAC TAT ACG
D1	5′-GTG ATA TGC		+2.0	+3.5	+4.0	+5.5	–0.5	–10.0	–10.5
B2	3′-CAC BAT ACG
D1	5′-GTG ATA TGC		+8.5	+8.5	+7.0	+9.5	+0.5	–2.0	±0.0
B3	3′-CAC TAB ACG
D1	5′-GTG ATA TGC		+4.0	+4.0	+4.0	+6.5	nd	nd	+0.5
B4	3′-CAC BAB ACG
B1	5′-GTG ABA TGC		+9.5	+11.0	+8.5	+12.5	+3.5	+1.0	+1.5
R2	3′-CAC UAU ACG
R1	5′-GUG AUA UGC		+4.0	+5.0	+5.0	+7.0	+2.5	+0.5	–8.5
B2	3′-CAC BAT ACG
R1	5′-GUG AUA UGC		+9.0	+9.0	+8.5	+12.0	+2.5	–0.5	+2.5
B3	3′-CAC TAB ACG
R1	5′-GUG AUA UGC		+5.5	+6.0	+6.0	+7.5	nd	nd	+2.0
B4	3′-CAC BAB ACG

ΔTm = change in Tm values relative to unmodified reference duplexes D1:D2 (Tm ≡ 29.5 °C), D1:R2 (Tm ≡ 27.0 °C), and D2:R1 (Tm ≡ 27.0 °C). Tm values are determined as the first-derivative maximum of denaturation curves (A260 vs T) recorded in medium salt phosphate buffer ([Na+] = 110 mM, [Cl–] = 100 mM, pH 7.0 (NaH2PO4/Na2HPO4)), using 1.0 μM of each strand. Tm values are averages of at least two measurements within 1.0 °C. See Figure 1 for structures of monomers. nd = not determined. Data for L1–L4 were previously reported in ref (11b).

As previously noted,[11b] ONs modified with conventional α-L-LNA-T monomer L form very thermostable duplexes, especially with RNA targets, although considerable sequence variation is observed (ΔTm = +2.5 to +9.5 °C, Table 1). ONs modified with α-L-LNA uridines that are conjugated to small alkynes at the C5 position generally result in the formation of even more thermostable duplexes (compare ΔTm values for S/V/W-modified ONs with L-modified ONs, Table 1). The effect is most pronounced with W-modified ONs, which result in additional duplex stabilization on the order of 1.0–5.5 °C relative to ONs modified with conventional α-L-LNA-T. We initially attributed these effects to improved base stacking (larger aromatic surface area due to extended conjugation) and reduced electrostatic repulsion (partially positively charged aminopropynyl shielding negatively charged strands).[23] However, analysis of thermodynamic parameters for the formed duplexes indicates that the structural underpinnings accounting for these results are more complex (vide infra). In contrast, α-L-LNA-U monomers that are conjugated to large hydrophobic entities reduce duplex thermostability relative to conventional α-L-LNA, presumably due to unfavorable steric interactions and/or disruption of the hydration sphere in the major groove (note ΔTm values for X/Y/Z-modified ONs; Table 1). Nevertheless, many of the X/Y/Z-modified duplexes display thermostabilities similar to those of the unmodified reference duplexes.

The binding specificities of ONs with a single central modification (B1 series) were evaluated against centrally mismatched DNA/RNA targets (Table 2). As previously reported,[11b] ONs modified with conventional α-L-LNA monomer L display significantly improved binding specificity relative to the unmodified reference strand, as evidenced by the greater drops in Tm values of mismatched duplexes (compare ΔTm values for L1 and D1, Table 2). Interestingly, the high-affinity ONs S1/V1/W1 display similar or slightly improved binding specificity in comparison to conventional α-L-LNA L1 (compare ΔTm values for S1/V1/W1 and L1, Table 2), whereas improvements are less pronounced for ONs modified with hydrophobic C5-functionalized α-L-LNA-U monomers (compare ΔTm’s for X1/Y1/Z1 and L1, Table 2).

Table 2

Discrimination of Mismatched DNA/RNA Targets by Singly Modified C5-Alkynyl-Functionalized α-L-LNA and Reference ONsa

		DNA: 3′-CAC TBT ACG				RNA: 3′-CAC UBU ACG
		Tm	ΔTm			Tm	ΔTm
ON	sequence	A	C	G	T	A	C	G	U
D1	5′-GTG ATA TGC	29.5	–16.5	–8.0	–15.5	27.0	<−17.0	–4.5	<−17.0
L1	5′-GTG ALA TGC	33.5	–23.5	–13.5	–17.5	36.5	–22.5	–8.0	–22.5
S1	5′-GTG ASA TGC	37.0	–24.0	–17.0	–23.0	38.0	–27.0	–11.0	–25.0
V1	5′-GTG AVA TGC	36.5	–23.5	–15.5	–22.0	35.5	–21.0	–8.5	–21.0
W1	5′-GTG AWA TGC	39.0	–25.0	–17.0	–22.5	39.5	–23.5	–11.0	–24.5
X1	5′-GTG AXA TGC	30.5	–18.0	–15.5	–18.5	30.5	–15.0	–7.0	–20.0
Y1	5′-GTG AYA TGC	27.0	<−17.0	<−17.0	<−17.0	28.0	<−18.0	–11.0	<−18.0
Z1	5′-GTG AZA TGC	28.5	–13.5	–13.5	–8.5	28.5	<−18.5	–13.0	<−18.5

For experimental conditions and sequences see Table 1. ΔT = change in T value relative to fully matched ON:DNA or ON:RNA duplex ( = A). Data for L1 previously reported in reference 11b.

The binding specificities of ONs with two next-nearest neighbor modifications (B4 series) were determined using DNA/RNA targets with a mismatched nucleotide opposite the central 2′-deoxyriboadenosine (Table 3). α-L-LNA-T modified L4 displays improved binding specificity relative to the unmodified reference D2. Unlike the observations in the B1 series, ONs modified with C5-alkynyl-functionalized monomers do not result in additional improvements in mismatch discrimination (compare ΔTm values for the B4 series, Table 3). This suggests that C5-alkynyl-functionalized α-L-LNA should be designed in a manner that places likely single-nucleotide polymorphism (SNP) sites directly opposite the modified nucleotide for optimal thermal discrimination of singly mismatched targets.

Table 3

Discrimination of Mismatched DNA/RNA Targets by Doubly Modified C5-Alkynyl-Functionalized α-L-LNAa

		DNA: 5′-GTG ABA TGC				RNA: 5′-GUG ABA UGC
		Tm	ΔTm			Tm	ΔTm
ON	sequence	T	A	C	G	U	A	C	G
D2	3′-CAC TAT ACG	29.5	<−19.5	–16.5	–7.5	27.0	–16.0	–16.0	–11.0
L4	3′-CAC LAL ACG	37.0	–23.0	–18.0	–17.0	38.0	–17.5	–19.0	–14.0
S4	3′-CAC SAS ACG	37.0	–18.0	–16.5	–12.5	39.0	–15.0	–17.0	–13.5
V4	3′-CAC VAV ACG	37.5	–23.0	–15.5	–12.0	38.5	–14.0	–15.5	–14.0
W4	3′-CAC WAW ACG	42.0	–21.5	–21.5	–14.5	41.5	–14.5	–16.5	–12.5
Z4	3′-CAC ZAZ ACG	30.5	<−20.5	–14.5	<−20.5	30.5	–15.5	<−20.5	–16.0

For experimental conditions and sequences see Table 1. ΔTm = change in T value relative to fully matched duplexes ( = T).

Thermodynamic Parameters for Duplex Formation

Thermodynamic parameters for formation of duplexes modified with C5-functionalized α-L-LNA-U monomers were derived from thermal denaturation curves via curve fitting.[24] In agreement with the Tm data, duplexes modified with conventional α-L-LNA thymidines are 3–12 kJ/mol more stable than unmodified reference duplexes (see ΔΔG298 values for L1–L3, Table 4). Duplexes that are modified with α-L-LNA-U monomers conjugated to small alkynes display duplex stabilities comparable to or slightly higher than those of duplexes modified with conventional α-L-LNA-U monomers, while X/Y-modified duplexes are less stable (compare ΔΔG298 values for S/V/W series and X/Y series vs L series, Table 4).

Table 4

Thermodynamic Parameters for Formation of Duplexes Modified with Select C5-Functionalized α-L-LNA Monomersa

		+complementary DNA			+complementary RNA
ON	sequence	ΔG298 [ΔΔG298] (kJ/mol)	ΔH [ΔΔH] (kJ/mol)	–T298ΔS [Δ(T298ΔS)] (kJ/mol)	ΔG298 [ΔΔG298] (kJ/mol)	ΔH [ΔΔH] (kJ/mol)	–T298ΔS [Δ(T298ΔS)] (kJ/mol)
D1	5′-GTG ATA TGC	–42	–314	271	–36	–278	241
D2	3′-CAC TAT ACG	–42	–314	271	–39	–293	254
L1	5′-GTG ALA TGC	–49 [−7]	–349 [−35]	300 [+29]	–48 [−12]	–308 [−30]	260 [+19]
L2	3′-CAC LAT ACG	–45 [−3]	–311 [+3]	266 [−5]	–44 [−5]	–306 [−13]	262 [+8]
L3	3′-CAC TAL ACG	–48 [−6]	–302 [+12]	254 [−17]	–46 [−7]	–295 [−2]	249 [−5]
S1	5′-GTG ASA TGC	–49 [−7]	–333 [−19]	284 [+13]	–47 [−11]	–290 [−12]	243 [+2]
S2	3′-CAC SAT ACG	–45 [−3]	–276 [+38]	231 [−40]	–45 [−6]	–299 [−6]	254 [±0]
S3	3′-CAC TAS ACG	–48 [−6]	–325 [−11]	275 [+4]	–48 [−9]	–318 [−25]	270 [+16]
V1	5′-GTG AVA TGC	–51 [−9]	–406 [−92]	354 [+83]	–50 [−14]	–302 [−24]	252 [+11]
V2	3′-CAC VAT ACG	–49 [−7]	–391 [−77]	342 [+71]	–46 [−7]	–340 [−47]	293 [+39]
V3	3′-CAC TAV ACG	–51 [−9]	–343 [−29]	292 [+21]	–49 [−10]	–289 [+4]	240 [−14]
W1	5′-GTG AWA TGC	–49 [−7]	–317 [−3]	267 [−4]	–50 [−14]	–302 [−24]	252 [+11]
W2	3′-CAC WAT ACG	–46 [−4]	–312 [+2]	266 [−5]	–46 [−7]	–340 [−47]	293 [+39]
W3	3′-CAC TAW ACG	–49 [−7]	–271 [+43]	222 [−49]	–49 [−10]	–289 [+4]	240 [−14]
X1	5′-GTG AXA TGC	–44 [−2]	–304 [+10]	260 [−11]	–44 [−8]	–319 [−41]	275 [+34]
X2	3′-CAC XAT ACG	–41 [−3]	–294 [+20]	253 [−18]	–41 [−2]	–245 [+48]	204 [−50]
X3	3′-CAC TAX ACG	–43 [−1]	–287 [+27]	244 [−27]	–42 [−3]	–283 [+10]	241 [−13]
Y1	5′-GTG AYA TGC	–39 [+3]	–313 [+1]	274 [+3]	–40 [−4]	–313 [−35]	273 [+32]
Y2	3′-CAC YAT ACG	–36 [+6]	–305 [+9]	269 [−2]	–40 [−1]	–325 [−32]	285 [+31]
Y3	3′-CAC TAY ACG	–39 [+3]	–324 [−10]	285 [−14]	–39 [±0]	–310 [−17]	271 [+17]

Parameters were determined from thermal denaturation curves, which were recorded as described in Table 1. ΔΔG298, ΔΔH, and Δ(T298ΔS) are calculated relative to reference duplexes D1:D2, D1:R2, and D2:R1.

The underlying structural underpinnings accounting for the additional stabilization of S/V/W-modified duplexes are not as clear as those for the corresponding C5-alkynyl-functionalized LNA, which are stabilized by more favorable enthalpy, a phenomenon that we ascribed to improved base stacking.[14] For example, the additional stabilization of V-modified DNA duplexes is enthalpic in nature, whereas the situation is more ambiguous with V-modified DNA:RNA duplexes (compare ΔΔH and Δ(T298ΔS) values for V vs L series, Table 4). In contrast, S/W-modified duplexes are generally stabilized by more favorable entropy (compare ΔΔH and Δ(T298ΔS) values for S/W series vs L series, Table 4). The lower stability of DNA duplexes modified with hydrophobic monomers X and Y is due to unfavorable enthalpic contributions, whereas Y-modified duplexes with RNA are destabilized by unfavorable entropic contributions. Additional studies are clearly needed to fully delineate the underlying reasons that govern these observations. However, the mechanisms through which the C5-alkynyl substituents exert their influence on duplex thermostability appear to be different between LNA-U and α-L-LNA-U nucleotides. This is not necessarily surprising, since incorporation of LNA and α-L-LNA nucleosides is known to have different effects on global duplex geometries, i.e., LNA nucleotides tune duplexes toward more RNA-like geometries,[25] whereas α-L-LNA nucleotides leave duplexes globally unperturbed.[26]

3′-Exonuclease Stability of C5-Alkynyl-Functionalized α-L-LNA

Encouraged by the promising hybridization properties of S- and W-modified ONs, we set out to study their stability in the presence of snake venom phosphodiesterase (SVPDE), a 3′-exonuclease (Figure 2). As expected, unmodified D2 is degraded rapidly (>95% degradation after 15 min), while conventional α-L-LNA L1 displays moderate resistance against SVPDE-mediated degradation (∼95% degradation after 2 h). Interestingly, C5-ethynyl- and C5-aminopropynyl-functionalized α-L-LNA S1 and W1 confer additional protection against SVPDE (<80% and <60% degradation after 2 h, respectively), which strongly suggests that the C5 substituents interfere with SVPDE’s mode of action. Expectedly, these trends are more pronounced with doubly modified ONs (B4 series). Thus, considerable amounts of conventional L4 and C5-ethynyl-functionalized α-L-LNA S4 remain after 2 h (<70% and <40% cleavage, respectively). It is noteworthy that C5-aminopropynyl-functionalized α-L-LNA W4, following a brief period of degradation of the 1–3 nucleotides closest to the 3′-end, is inert to further degradation.

Figure 2

3′-Exonuclease degradation of singly (top, 3′-CAC AT ACG) and doubly modified (bottom, 3′-CAC A ACG) C5-functionalized α-L-LNA and reference strands. Nuclease degradation studies were conducted in magnesium buffer (50 mM Tris-HCl, 10 mM Mg2+, pH 9.0) using [ON] = 3.3 μM and 0.03 U of snake venom phosphodiesterase.

Fluorescence Properties of Z-Modified ONs

Steady-state fluorescence emission spectra of Z-modified ONs and the corresponding duplexes with complementary or mismatched DNA targets were recorded to evaluate the diagnostic potential of these probes. Hybridization of singly Z-modified ONs with complementary DNA/RNA generally results in significantly increased fluorescence emission and the formation of duplexes with two broad emission maxima at ∼388 and ∼401 nm (Figures 3 and Figures S2 and S3 (Supporting Information)). In contrast, hybridization of doubly modified Z4 with DNA/RNA complements results in decreased monomer emission along with increased excimer emission (λem ∼510 nm, Figures S4 and S5 (Supporting Information)), which is consistent with the formation of pyrene–pyrene dimers in the major groove.[12c,27]

Figure 3

Steady-state fluorescence emission spectra of single-stranded Z1 and corresponding duplexes with complementary or mismatched DNA/RNA strands (mismatched nucleotide opposite to modification in parentheses). Conditions: λex 344 nm, T = 5 °C, each oligonucleotide used in 1 μM concentration. Note that different axis scales are used.

Interestingly, the fluorescence intensity of Z1 is sensitive to the nature of the nucleotide opposite to the modification; hybridization with matched DNA/RNA targets results in the formation of highly fluorescent duplexes, whereas incubation with centrally mismatched targets results in much lower fluorescence intensities (Figure 3). Presumably this is due to different positioning of the pyrene moiety in matched vs mismatched duplexes in a similar manner as proposed for the corresponding DNA analogue of monomer Z.[28] According to this hypothesis, the pyrene moiety is directed into the nonquenching environment of the major groove in matched duplexes, whereas it is intercalating in mismatched duplexes, leading to nucleobase-mediated quenching[29] of pyrene fluorescence. In contrast, hybridization of doubly modified Z4 with centrally mismatched DNA/RNA targets results in a less intense excimer signal but more pronounced monomer emission (Figures S4 and S5 (Supporting Information)). This suggests that the presence of mismatches in the vicinity of two Z monomers positioned as next-nearest neighbors perturbs pyrene–pyrene stacking in a manner similar to that observed with other pyrene array forming probes.[12a,30]

We have recently studied the fluorescent properties of longer Z-modified probes in detail.[22] In comparison to ONs modified with the analogous DNA monomer,[28]Z-modified probes (i) display slightly larger increases in fluorescence intensity upon hybridization with complementary DNA, (ii) result in formation of more brightly fluorescent duplexes, and (iii) discriminate single-nucleotide polymorphisms more efficiently in AT-rich sequence contexts. In summary, Z-modified ONs are interesting probes for the discrimination of single-nucleotide polymorphisms for applications in nucleic acid diagnostics.

Conclusion

Attachment of small alkynyl entities (ethynyl, hydroxypropynyl, aminopropynyl) to the C5 position of α-L-LNA uridines significantly increases the target affinity, binding specificity, and enzymatic stability of oligonucleotides modified with these building blocks in comparison to conventional α-L-LNA uridines. On the other hand, attachment of larger alkynyl groups (derivatives of stearic acid, cholesterol, and pyrene) counteracts the stabilization provided by the extended conjugation. Suitably designed C5-functionalized α-L-LNA uridines are therefore interesting oligonucleotide modifications for nucleic acid targeting applications in molecular biology, biotechnology, and medicinal chemistry. The results from the back-to-back articles strongly suggest that C5 functionalization of pyrimidines is a general and synthetically convenient approach for improving the pharmacodynamic properties of oligonucleotides modified with LNA or other conformationally restricted monomers.

Experimental Section

1-[2-O-Acetyl-3-O-benzyl-5-O-(methanesulfonyl)-4-C-(methanesulfonyloxymethyl)-α-l-threo-pentofuranosyl]uracil (2)

Glycosyl donor 1 (6.10 g, 12.0 mmol) and uracil (2.70 g, 24.0 mmol) were coevaporated with anhydrous CH3CN (100 mL) and resuspended in anhydrous CH3CN (150 mL). To this was added N,O-bis(trimethylsilyl)acetamide (BSA; 10.4 mL, 41.9 mmol), and the solution was refluxed until homogeneous. After the mixture was cooled to room temperature, trimethylsilyl triflate (TMSOTf, 5.5 mL, 29.9 mmol) was added and the reaction mixture was refluxed for 28 h, whereupon it was concentrated to near dryness. The resulting residue was taken up in EtOAc (200 mL), and the organic phase was washed with saturated aqueous NaHCO3 (2 × 100 mL) and brine (100 mL). The aqueous phase was back-extracted with EtOAc (100 mL), and the combined organic layers were dried (Na2SO4) and evaporated to dryness. The resulting residue was purified by silica gel column chromatography (0–2% MeOH in CH2Cl2, v/v) to afford nucleoside 2 (4.80 g, 85%) as a white foam: Rf = 0.5 (2% MeOH in CH2Cl2, v/v); FAB-HRMS m/z 563.0998 ([M + H]+, C21H25N2O12S2·H+, calcd 563.1005); 1H NMR (DMSO-d6) δ 11.40 (d, 1H, ex, J = 2.0 Hz, NH), 7.76 (d, 1H, J = 8.0 Hz, H6), 7.33–7.39 (m, 5H, Ph), 6.09 (d, 1H, J = 6.0 Hz, H1′), 5.69 (dd, 1H, J = 8.0 Hz, 2.0 Hz, H5), 5.54 (t, 1H, J = 6.0 Hz, H2′), 4.68–4.71 (d, 1H, J = 12.0 Hz, CH2Ph), 4.65–4.68 (d, 1H, J = 12.0 Hz, CH2Ph), 4.58–4.62 (d, 1H, J = 11.0 Hz, H5′), 4.39–4.47 (m, 4H, H3′, H5′, 2 × H5″), 3.27 (s, 3H, CH3SO2), 3.20 (s, 3H, CH3SO2), 2.02 (s, 3H, CH3); 13C NMR (DMSO-d6) δ 169.6, 162.7, 150.4, 140.2 (C6), 137.1, 128.3 (Ar), 127.91 (Ar), 127.86 (Ar), 102.7 (C5), 84.6 (C1′), 81.8, 80.7 (C3′), 77.3 (C2′), 72.5 (CH2Ph), 68.3 (C5″), 67.9 (C5′), 36.7 (CH3SO2), 36.6 (CH3SO2), 20.3 (CH3).

1-[3-O-Benzyl-5-O-(methanesulfonyl)-4-C-(methanesulfonyloxymethyl)-α-l-threo-pentofuranosyl]uracil (3)

Method A

A 1 M solution of HCl in MeOH (50 mL) was added to a solution of nucleoside 2 (2.81 g, 5.00 mmol) in MeOH (30 mL). and after the reaction mixture was stirred at room temperature for 24 h, the solvent was evaporated off. The resulting residue was dissolved in CH2Cl2 (100 mL), and the organic phase was washed with saturated aqueous NaHCO3 (2 × 100 mL). The aqueous phase was then back-extracted with CH2Cl2 (2 × 50 mL). The combined organic phase was dried (Na2SO4) and evaporated to dryness to afford analytically pure nucleoside 3 (2.52 g, 97%) as a white solid material: Rf = 0.4 (4% MeOH in CH2Cl2, v/v); FAB-HRMS m/z 521.0900 ([M + H]+, C19H24N2O11S2·H+, calcd 521.0894); 1H NMR (DMSO-d6) δ 11.42 (d, 1H, ex, J = 2.0 Hz, NH), 7.76 (d, 1H, J = 8.0 Hz, H6), 7.29–7.40 (m, 5H, Ph), 6.12 (d, 1H, ex, J = 5.0 Hz, 2′-OH), 5.92 (d, 1H, J = 7.5 Hz, H1′), 5.68 (dd, 1H, J = 8.0 Hz, 2.0 Hz, H5), 4.74–4.77 (d, 1H, J = 12.0 Hz, CH2Ph), 4.67–4.69 (d, 1H, J = 12.0 Hz, CH2Ph), 4.33–4.52 (m, 5H, H2′, 2 × H5′, 2 × H5″), 4.19–4.21 (d, 1H, J = 6.5 Hz, H3′), 3.23 (s, 3H, CH3SO2), 3.17 (s, 3H, CH3SO2); 13C NMR (DMSO-d6) δ 162.8, 150.8, 140.4 (C6), 137.5, 128.2 (Ar), 127.7 (Ar), 127.6 (Ar), 102.5 (C5), 86.0 (C1′), 82.8 (C3′), 80.9, 75.7 (C2′), 72.3 (CH2Ph), 68.9 (C5″), 68.2 (C5′), 36.7 (CH3SO2), 36.6 (CH3SO2).

Method B

To a solution of nucleoside 2 (1.50 g, 2.66 mmol) in MeOH (50 mL) was added saturated methanolic ammonia (50 mL). The solution was stirred for 2 h at room temperature, whereupon solvents were evaporated off. The resulting residue was purified by silica gel column chromatography (0–3% MeOH in CH2Cl2, v/v) to afford nucleoside 3 (1.11 g, 80%) as a white solid material with physical data as reported above.

1-[3-O-Benzyl-2,5-O-bis(methanesulfonyl)-4-C-(methanesulfonyloxymethyl)-α-l-threo-pentofuranosyl]uracil (4)

Nucleoside 3 (10.0 g, 19.2 mmol) was coevaporated with anhydrous pyridine (2 × 75 mL) and redissolved in anhydrous pyridine (120 mL). To this was added methanesulfonyl chloride (MsCl, 1.8 mL, 23.3 mmol), and the reaction mixture was stirred for 4 h at room temperature, whereupon it was poured into saturated aqueous NaHCO3 (200 mL) and extracted with CH2Cl2 (2 × 200 mL). The organic phase was dried (Na2SO4) and concentrated to dryness to afford analytically pure nucleoside 4 (11.3 g, 98%) as a slightly brown solid material: Rf = 0.4 (2% MeOH in CH2Cl2, v/v); FAB-HRMS m/z 599.0675 ([M + H]+, C20H26N2O13S3·H+, calcd 599.0670); 1H NMR (DMSO-d6) δ 11.48 (d, 1H, ex, J = 2.0 Hz, NH), 7.77 (d, 1H, J = 8.0 Hz, H6), 7.32–7.43 (m, 5H, Ph), 6.25 (d, 1H, J = 7.5 Hz, H1′), 5.70 (dd, 1H, J = 8.0 Hz, 2.0 Hz, H5), 5.54 (t, 1H, J = 7.0 Hz, H2′), 4.72 (s, 2H, CH2Ph), 4.58–4.64 (2d, 2H, J = 11.0 Hz, J = 7.0 Hz, H5′, H3), 4.41–4.49 (m, 3H, H5′, 2 × H5″), 3.28 (s, 3H, CH3), 3.25 (s, 3H, CH3), 3.18 (s, 3H, CH3); 13C NMR (DMSO-d6) δ 162.7, 150.5, 140.3 (C6), 136.8, 128.3 (Ar), 128.1 (Ar), 128.0 (Ar), 102.8 (C5), 83.6 (C1′), 81.1 (C2′), 81.0, 80.7 (C3′), 73.0 (CH2Ph), 68.5 (C5″), 67.7 (C5′), 37.8 (CH3SO2) 36.7 (CH3SO2), 36.6 (CH3SO2).

(1S,3R,4S,7R)-7-Benzyloxy-1-methanesulfonyloxymethyl-3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (5)

To a solution of nucleoside 4 (9.87 g, 16.5 mmol) in 1,4-dioxane/H2O (60 mL, 1/1, v/v) was added aqueous NaOH (2 M, 50 mL, 0.10 mol). After it was stirred at room temperature for 4 h, the reaction mixture was neutralized by 10% aqueous AcOH and diluted with EtOAc (300 mL) and the phases were separated. The organic phase was washed with saturated aqueous NaHCO3 (100 mL) and H2O (100 mL), dried (Na2SO4), and evaporated to dryness to afford nucleoside 5 (7.00 g, quantitative) as a slightly brown solid material, which was used in the next step without further purification: Rf = 0.5 (80% EtOAc in petroleum ether, v/v); FAB-HRMS m/z 425.1018 ([M + H]+, C18H20N2O8S·H+, calcd 425.1013); 1H NMR (DMSO-d6) δ 11.39 (br s, 1H, ex, NH), 7.80 (d, 1H, J = 8.0 Hz, H6), 7.35–7.41 (m, 5H, Ph), 5.97 (s, 1H, H1′), 5.63 (d, 1H, J = 8.0 Hz, 1.2 Hz, H5), 4.55–4.73 (m, 5H, 2 × CH2Ph, H2′, 2 × H5′), 4.50 (s, 1H, H3′), 4.04–4.08 (d, 1H, J = 8.5 Hz, H5″), 3.99–4.03 (d, 1H, J = 8.5 Hz, H5″), 3.23 (s, 3H, CH3); 13C NMR (DMSO-d6) δ 163.0, 150.2, 140.3 (C6), 137.5, 128.3 (Ar), 127.7 (Ar), 127.5 (Ar), 100.6 (C5), 86.7, 86.6 (C1′), 79.2 (C3′), 76.4 (C2′), 71.9 (C5″), 71.2 (CH2Ph), 65.4 (C5′) 36.9 (CH3SO2). A trace impurity of 1,4-dioxane was identified at 66.3 ppm in the 13C NMR spectrum.[31]

(1S,3R,4S,7R)-1-Benzoyloxymethyl-7-benzyloxy-3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (6)

To a solution of nucleoside 5 (7.00 g, 16.5 mmol) in anhydrous DMF (300 mL) was added NaOBz (7.00 g, 48.6 mmol), and the reaction mixture was stirred at 90 °C for 20 h, whereupon it was cooled to room temperature and poured into ice-cold water (500 mL). The solution was extracted with EtOAc (2 × 300 mL) and the organic phase washed with H2O (2 × 150 mL) and brine (100 mL). The organic phase was dried (Na2SO4) and concentrated to dryness to afford analytically pure nucleoside 6 (7.24 g, 98%) as a slightly brown solid material: Rf = 0.6 (80% EtOAc in petroleum ether, v/v); FAB-HRMS m/z 451.1505 ([M + H]+, C24H22N2O7·H+, calcd 451.1500); 1H NMR (DMSO-d6) δ 11.38 (br s, 1H, ex, NH), 7.98 (dd, 2H, J = 8.5 Hz, 1.0 Hz, Ar), 7.82 (d, 1H, J = 8.0 Hz, H6), 7.66–7.71 (t, 1H, J = 7.5 Hz, Ar), 7.51–7.55 (m, 2H, Ar), 7.25–7.38 (m, 5H, Ar), 6.00 (s, 1H, H1′), 5.62 (dd, 1H, J = 8.0 Hz, 8.0 Hz, 1.5 Hz, H5), 4.66–4.76 (m, 3H, 2 × CH2Ph, H5′), 4.62 (s, 1H, H2′), 4.59 (s, 1H, H3′), 4.54–4.58 (d, 1H, J = 12.5 Hz, H5′), 4.14–4.17 (d, 1H, J = 8.5 Hz, H5″), 4.07–4.10 (d, 1H, J = 8.5 Hz, H5″); 13C NMR (DMSO-d6) δ 165.2, 163.1, 150.3, 140.5 (C6), 137.6, 133.5 (Ar), 129.4 (Ar), 129.1, 128.7 (Ar), 128.2 (Ar), 127.6 (Ar), 127.5 (Ar), 100.5 (C5), 87.0, 86.6 (C1′), 79.3 (C3′), 76.3 (C2′), 72.1 (C5″), 71.1 (CH2Ph), 59.8 (C5′).

(1R,3R,4S,7R)-7-Benzyloxy-1-hydroxymethyl-3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (7)

To a solution of nucleoside 6 (5.00 g, 11.1 mmol) dissolved in MeOH (100 mL) was added saturated methanolic ammonia (100 mL). The reaction mixture was stirred at room temperature for 14 h in a sealed flask. The solvent was then evaporated and the resulting residue purified by silica gel column chromatography (0–7% MeOH in CH2Cl2,v/v) to afford nucleoside 7 (3.66 g, 95%) as a white solid material: Rf = 0.4 (7% MeOH in CH2Cl2, v/v); FAB-HRMS m/z 347.1230 ([M + H]+, C17H18N2O6·H+, calcd 347.1243); 1H NMR (DMSO-d6) δ 11.36 (s, 1H, ex, NH), 7.80 (d, 1H, J = 8.0 Hz, H6), 7.30–7.40 (m, 5H, Ph), 5.88 (s, 1H, H1′), 5.62 (d, 1H, J = 8.0 Hz, H5), 5.05 (t, 1H, ex, J = 5.5 Hz, 5′-OH), 4.63–4.71 (2d, 2H, J = 12.0 Hz, CH2Ph), 4.52 (s, 1H, H2′), 4.33 (s, 1H, H3′), 3.92–3.98 (2d, 2H, J = 8.5 Hz, H5″), 3.73–3.78 (m, 2H, 2 × H5′); 13C NMR (DMSO-d6) δ 163.1, 150.3, 140.4 (C6), 137.9, 128.3 (Ar), 127.6 (Ar), 127.4 (Ar), 100.3 (C5), 90.1 (C4′), 86.5 (C1′), 79.3 (C3′), 76.3 (C2′), 72.4 (C5″), 71.1 (CH2Ph), 57.1 (C5′).

To a solution of nucleoside 6 (1.50 g, 3.33 mmol) in THF/H2O (25 mL, 1/1, v/v) was added aqueous NaOH (2 M, 10.0 mL, 20.0 mmol), and the reaction mixture was stirred at room temperature for 4 h, whereupon it was carefully neutralized with 10% aqueous AcOH at 0 °C and diluted with EtOAc (50 mL). The organic phase was washed with saturated aqueous NaHCO3 (50 mL) and the combined aqueous phase extracted with EtOAc (2 × 50 mL). The combined organic layers were dried (Na2SO4) and concentrated to dryness to afford nucleoside 7 (1.05 g, 91%) as a slightly brown solid.

(1R,3R,4S,7R)-7-Hydroxy-1-hydroxymethyl-3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (8)

To a solution of nucleoside 7 (1.50 g, 4.32 mmol) in THF/MeOH (100 mL, 9/1, v/v) were added Pd(OH)2/C (20 wt %, 0.60 g) and 88% aqueous formic acid (2.3 mL, 61.1 mmol) from a freshly opened bottle. The reaction mixture was refluxed for 24 h, whereupon it was cooled to room temperature. The catalyst was filtered off and washed with excess MeOH, and the combined filtrates were concentrated to dryness. The resulting crude residue was purified by silica gel column chromatography (0–16% MeOH in CH2Cl2, v/v) to afford nucleoside 8 (0.94 g, 85%) as a white solid material: Rf = 0.4 (15% MeOH in CH2Cl2, v/v); FAB-HRMS m/z 257.0760 ([M + H]+, C10H12N2O6·H+, calcd 257.0768); 1H NMR (DMSO-d6) δ 11.34 (s, 1H, ex, NH), 7.80 (d, 1H, J = 8.0 Hz, H6), 5.87 (s, 1H, H1′), 5.85 (d, ex, J = 4.0 Hz, 3′-OH), 5.63 (d, 1H, J = 8.0 Hz, H5), 4.93 (t, ex, J = 5.5 Hz, 5′-OH), 4.27 (d, 1H, J = 4.0 Hz, H3′), 4.21 (s, 1H, H2′), 3.88–3.94 (2d, 2H, J = 8.5 Hz, H5″), 3.73 (d, 2H, J = 5.5 Hz, 2 × H5′); 13C NMR (DMSO-d6) δ 163.2, 150.3, 140.4 (C6), 100.2 (C5), 90.9, 86.5 (C1′), 78.7 (C2′), 72.5 (C3′), 71.9 (C5″), 57.4 (C5′).

(1S,3R,4S,7R)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-3-(5-iodoracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (9)

Ac2O (0.21 mL, 2.20 mmol) was added to a solution of nucleoside 8 (0.25 g, 1.00 mmol) in anhydrous pyridine (10 mL) and the reaction mixture was stirred at 60 °C for 14 h. After it was cooled to room temperature, the reaction mixture was diluted with saturated aqueous NaHCO3 (30 mL) and CH2Cl2 (30 mL) and the phases were separated. The organic phase was washed with saturated aqueous NaHCO3 (20 mL) and the combined aqueous phase back-extracted with CH2Cl2 (2 × 20 mL). The combined organic layers were dried (Na2SO4), evaporated to dryness, and coevaporated with toluene/absolute EtOH (2 × 30 mL, 1/2, v/v). The resulting residue, tentatively assigned as the O3′,O5′-diacetylated nucleoside, was used in the next step without further purification (Rf = 0.5 (2% MeOH in CH2Cl2, v/v); FAB-MS m/z 341 ([M + H]+)).

To a solution of the crude O3′,O5′-diacetylated α-L-LNA uridine in glacial AcOH (10 mL) were added iodine (160 mg, 0.62 mmol) and ceric ammonium nitrate (CAN, 235 mg, 0.50 mmol), and the reaction mixture was stirred at 80 °C for 50 min. After it was cooled to room temperature, the reaction mixture was evaporated to dryness and taken up in CH2Cl2 (50 mL). The organic phase was washed with saturated aqueous NaHCO3 (2 × 20 mL) and H2O (20 mL). The combined aqueous phase was back-extracted with CH2Cl2 (2 × 20 mL). The combined organic layers were dried (Na2SO4) and evaporated to dryness. The resulting residue, tentatively assigned as the C5-iodo-O3′,O5′-diacetylated nucleoside, was used in the next step without further purification (Rf = 0.5 (3% MeOH in CH2Cl2, v/v); FAB-HRMS m/z 466.9966 ([M + H]+, C14H15IN2O8·H+, calcd 466.9946)).

The crude C5-iodo-O3′,O5′-diacetylated nucleoside was dissolved in saturated methanolic ammonia (30 mL) and stirred in a sealed flask at room temperature for 12 h. The reaction mixture was evaporated to dryness, affording a residue that was tentatively assigned as the C5-iodo α-L-LNA diol and used in the next step without further purification (Rf = 0.4 (15% MeOH in CH2Cl2, v/v); FAB-HRMS m/z 382.9735 ([M + H]+, C10H11IN2O6·H+, calcd 382.9740)).

The crude C5-iodo α-L-LNA diol was dried through coevaporation with anhydrous pyridine (10 mL) and redissolved in anhydrous pyridine (10 mL). To this was added 4,4′-dimethoxytrityl chloride (DMTrCl, 0.40 g, 1.20 mmol) and the reaction mixture was stirred at room temperature for 16 h, whereupon it was diluted with saturated aqueous NaHCO3 (20 mL) and CH2Cl2 (25 mL). The phases were separated, and the organic phase was washed with saturated aqueous NaHCO3 (20 mL). The aqueous phase was back-extracted with CH2Cl2 (2 × 20 mL). The combined organic layers were dried (Na2SO4), evaporated to near dryness, and coevaporated with toluene/absolute EtOH (2 × 30 mL, 1/2, v/v). The resulting residue was purified by silica gel column chromatography (0–4.5% MeOH in CH2Cl2, v/v) to afford nucleoside 9 (0.48 g, 70%, over four steps) as a slightly yellow solid material: Rf = 0.5 (5% MeOH in CH2Cl2, v/v); FAB-HRMS m/z 684.0980 ([M]+, C31H29IN2O8+, calcd 684.0969); 1H NMR (DMSO-d6) δ 11.78 (s, 1H, ex, NH), 8.16 (s, 1H, H6), 7.23–7.43 (m, 9H, Ar), 6.92 (d, 4H, J = 8.5 Hz, Ar), 5.92 (d, 1H, ex, J = 4.5 Hz, 3′-OH), 5.89 (s, 1H, H1′), 4.35 (d, 1H, J = 4.5 Hz, H3′), 4.27 (s, 1H, H2′), 3.98–4.02 (d, 1H, J = 8.5 Hz, H5″), 3.92–3.96 (d, 1H, J = 8.5 Hz, H5″), 3.75 (s, 6H, 2 × CH3O), 3.34–3.37 (d, 1H, J = 10.5 Hz, H5′), 3.28–3.31 (d, 1H, J = 10.5 Hz, H5′ - partial overlap with H2O); 13C NMR (DMSO-d6) δ 160.6, 158.134, 158.126, 149.9, 144.7, 144.3 (C6), 135.2, 135.1, 129.64, 129.62 (Ar), 127.9 (Ar), 127.5 (Ar), 126.7 (Ar), 113.3 (Ar), 89.3, 87.2 (C1′), 85.3, 78.8 (C2′), 72.9 (C3′), 72.3 (C5″), 67.7, 60.0 (C5′), 55.0 (CH3O).

Representative Protocol for Sonogashira Coupling Reactions (10S′–Z)

The key intermediate 9, Pd(PPh3)4, CuI, and alkyne were added to anhydrous DMF (quantities and volumes specified below), and the reaction chamber was degassed and placed under an argon atmosphere. To this was added Et3N, and the reaction mixture was stirred in the dark at room temperature (unless otherwise mentioned) for 6–12 h, whereupon the solvents were evaporated. The resulting residue was dissolved in EtOAc (100 mL), and the organic phase was washed with brine (2 × 50 mL) and saturated aqueous NaHCO3 (50 mL). The combined aqueous phase was back-extracted with EtOAc (100 mL). The combined organic phase was dried (Na2SO4) and evaporated to dryness and the resulting residue purified by silica gel column chromatography (0–5% MeOH in CH2Cl2, v/v) to afford the desired product.

(1S,3R,4S,7R)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-3-[5-(trimethylsilylethynyl)uracil-1-yl]-2,5-dioxabicyclo[2.2.1]heptane (10S′)

Nucleoside 9 (0.68 g, 1.00 mmol), Pd(PPh3)4 (120 mg, 0.10 mmol), CuI (40 mg, 0.20 mmol), trimethylsilylacetylene (0.42 mL, 3.00 mmol), and Et3N (0.60 mL, 4.27 mmol) in anhydrous DMF (10 mL) were reacted as described in the representative Sonogashira protocol, and the mixture was stirred at room temperature for 12 h. After work-up and purification, nucleoside 10S′ (0.55 g, 84%) was obtained as a slightly brown solid material: Rf = 0.5 (5% MeOH in CH2Cl2, v/v); FAB-HRMS m/z 655.2462 ([M + H]+, C36H38N2O8Si·H+, calcd 655.2476); 1H NMR (DMSO-d6) δ 11.75 (s, 1H, ex, NH), 7.93 (s, 1H, H6), 7.21–7.43 (m, 9H, Ar), 6.90 (d, 4H, J = 8.5 Hz, Ar), 5.94 (d, 1H, ex, J = 4.5 Hz, 3′-OH), 5.92 (s, 1H, H1′), 4.38 (d, 1H, J = 4.5 Hz, H3′), 4.26 (s, 1H, H2′), 3.99–4.02 (d, 1H, J = 8.5 Hz, H5″), 3.93–3.96 (d, 1H, J = 8.5 Hz, H5″), 3.74 (s, 6H, 2 × CH3O), 3.34 (s, 2H, H5′), 0.21 (s, 9H, Me3Si); 13C NMR (DMSO-d6) δ 161.3, 158.1, 149.1, 144.6, 143.7 (C6), 135.2, 135.1, 129.7 (Ar), 129.6 (Ar), 127.8 (Ar), 127.6 (Ar), 126.7 (Ar), 113.2 (Ar), 97.8, 97.1, 96.9, 89.4, 87.3 (C1′), 85.3, 78.6 (C2′), 72.7 (C3′), 72.3 (C5″), 60.0 (C5′), 55.0 (CH3O), −0.12 (Me3Si).

(1S,3R,4S,7R)-1-(4,4′-Dimethoxytrityloxymethyl)-3-(5-ethynyluracil-1-yl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptane (10S)

TBAF in THF (1M, 1.2 mL, 1.2 mmol) was added to a solution of nucleoside 10S′ (0.53 g, 0.81 mmol) in THF (20 mL) and the reaction mixture was stirred at room temperature for 2 h. EtOAc (50 mL) was added, and the solution was washed with brine (2 × 30 mL) and H2O (30 mL). The combined aqueous phase was back-extracted with EtOAc (30 mL). The combined organic layer was dried (Na2SO4) and concentrated to dryness and the resulting residue purified by silica column chromatography (0–5% MeOH in CH2Cl2, v/v) to afford nucleoside 10S (0.37 g, 78%) as a light brown solid material: Rf = 0.5 (5% MeOH in CH2Cl2, v/v); ESI-HRMS m/z 605.1918 ([M + Na]+, C33H30N2O8·Na+, calcd 605.1894); 1H NMR (DMSO-d6) δ 11.74 (s, 1H, ex, NH), 8.03 (s, 1H, H6), 7.23–7.43 (m, 9H, Ar), 6.90 (d, 4H, J = 8.5 Hz, Ar), 5.90–5.96 (m, 2H, 1ex, H1′, 3′-OH), 4.40 (s, 1H, H3′), 4.28 (s, 1H, H2′), 4.18 (s, 1H, HC≡C), 4.01–4.04 (d, 1H, J = 8.5 Hz, H5″), 3.91–3.94 (d, 1H, J = 8.5 Hz, H5″), 3.75 (s, 6H, 2 × CH3O), 3.32 (br s, 2H, H5′); 13C NMR (DMSO-d6) δ 161.6, 158.1, 149.2, 144.7, 143.7 (C6), 135.2, 135.1, 129.7 (Ar), 128.9 (Ar), 127.8 (Ar), 127.6 (Ar), 126.7 (Ar), 113.2 (Ar), 96.4, 89.4, 87.3 (C1′), 85.3, 83.4 (HC≡C), 78.7 (C2′), 76.3, 72.8 (C3′), 72.2 (C5″), 59.8 (C5′), 55.0 (CH3O).

(1S,3R,4S,7R)-3-[5-(3-Benzoyloxypropyn-1-yl)uracil-1-yl]-1-(4,4′-dimethoxytrityloxymethyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptane (10V)

Nucleoside 9 (0.50 g, 0.73 mmol), Pd(PPh3)4 (90 mg, 0.07 mmol), CuI (30 mg, 0.14 mmol), prop-2-ynyl benzoate[32] (180 mg, 1.12 mmol), and Et3N (0.40 mL, 2.84 mmol) in anhydrous DMF (10 mL) were reacted as described in the representative Sonogashira protocol, and the mixture was stirred at room temperature for 12 h. After workup and purification, nucleoside 10V (0.40 g, 76%) was obtained as a slightly brown solid material: Rf = 0.5 (5% MeOH in CH2Cl2, v/v); ESI-HRMS m/z 739.2234 ([M + Na]+, C41H36N2O10·Na+, calcd 739.2262); 1H NMR (DMSO-d6) δ 11.79 (s, 1H, ex, NH), 8.03 (s, 1H, H6), 7.98 (dd, 2H, J = 8.3 Hz, 1.2 Hz, Ar), 7.65–7.69 (td, 1H, J = 7.5 Hz, 1.2 Hz, Ar), 7.50–7.54 (m, 2H, Ar), 7.17–7.44 (m, 9H, Ar), 6.90 (d, 4H, J = 9.0 Hz, Ar), 5.94 (br s, 2H, 1 ex, H1′, 3′-OH), 5.20 (s, 2H, CH2OBz), 4.42 (d, 1H, J = 4.5 Hz, H3′), 4.28 (s, 1H, H2′), 4.01–4.04 (d, 1H, J = 8.0 Hz, H5″), 3.90–3.94 (d, 1H, J = 8.0 Hz, H5″), 3.73 (s, 6H, 2 × OCH3), 3.29 (s, 2H, H5′); 13C NMR (DMSO-d6) δ 165.0, 161.4, 158.1, 149.2, 144.7, 144.0 (C6), 135.2, 135.1, 133.6 (Ar), 129.68 (Ar), 129.66 (Ar), 129.2 (Ar), 129.0, 128.8 (Ar), 127.8 (Ar), 127.5 (Ar), 126.6 (Ar), 113.2 (Ar), 96.1, 89.4, 87.3 (C1′), 86.6, 85.3, 79.2, 78.6 (C2′), 72.7 (C3′), 72.2 (C5″), 59.7 (C5′), 55.0 (CH3O), 53.2 (CH2OBz).

(1S,3R,4S,7R)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-3-[5-(3-trifluoroacetylaminopropyn-1-yl)uracil-1-yl]-2,5-dioxabicyclo[2.2.1]heptane (10W)

Nucleoside 9 (0.50 g, 0.73 mmol), Pd(PPh3)4 (90 mg, 0.07 mmol), CuI (30 mg, 0.14 mmol), 2,2,2-trifluoro-N-(prop-2-ynyl)acetamide[33] (180 mg, 1.46 mmol), and Et3N (0.4 mL, 2.84 mmol) in anhydrous DMF (10 mL) were reacted as described in the representative Sonogashira protocol and stirred at room temperature for 12 h. After workup and purification, nucleoside 10W (0.43 g, 84%) was obtained as a slightly brown solid material: Rf = 0.5 (5% MeOH in CH2Cl2, v/v); MALDI-HRMS m/z 730.1976 ([M + Na]+, C36H32F3N3O9·Na+, calcd 730.1983); 1H NMR (DMSO-d6) δ 11.76 (s, 1H, ex, NH(U)), 10.04 (t, ex, 1H, J = 5.5 Hz, NHCH2), 7.94 (s, 1H, H6), 7.21–7.43 (m, 9H, Ar), 6.91 (d, 4H, J = 9.0 Hz, Ar), 5.94–5.96 (m, 2H, 1ex, H1′, 3′-OH), 4.44 (d, 1H, J = 4.0 Hz, H3′), 4.25–4.29 (m, 3H, H2′, CH2NH), 3.98–4.00 (d, 1H, J = 8.5 Hz, H5″), 3.92–3.95 (d, 1H, J = 8.5 Hz, H5″), 3.74 (s, 6H, 2 × OCH3), 3.28–3.32 (m, 2H, H5′, overlap with H2O); 13C NMR (DMSO-d6) δ 161.5, 158.1, 156.0 (q, J = 36.3 Hz, COCF3) 149.2, 144.7, 143.4 (C6), 135.2, 135.1, 129.72 (Ar), 129.69 (Ar), 127.8 (Ar), 127.6 (Ar), 126.7 (Ar), 115.7 (q, J = 287 Hz, CF3), 113.2 (Ar), 96.5, 89.4, 87.3, 87.1 (C1′), 85.3, 78.6 (C2′), 75.4, 72.7 (C3′), 72.3 (C5″), 59.7 (C5′), 55.0 (CH3O), 29.5 (CH2NH).

(1S,3R,4S,7R)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-3-[5-(3-octadecanoylaminopropyn-1-yl)uracil-1-yl]-2,5-dioxabicyclo[2.2.1]heptane (10X)

Nucleoside 9 (0.34 g, 0.50 mmol), Pd(PPh3)4 (60 mg, 0.05 mmol), CuI (20 mg, 0.10 mmol), N-(prop-2-ynyl)stearamide,[14] (0.28 g, 1.00 mmol) and Et3N (0.30 mL, 2.13 mmol) in anhydrous DMF (10 mL) were reacted as described in the representative Sonogashira protocol, and the mixture was stirred at 40 °C for 6 h. After workup and purification, nucleoside 10X (0.24 g, 55%) was obtained as a brown solid material: Rf = 0.5 (5% MeOH in CH2Cl2, v/v); ESI-HRMS m/z 900.4813 ([M + Na]+, C52H67N3O9·Na+, calcd 900.4770); 1H NMR (CDCl3) δ 7.89 (s, 1H, H6), 7.24–7.46 (m, 9H, Ar), 6.87 (d, 4H, J = 9.0 Hz, Ar), 5.97 (s, 1H, H1′), 5.88 (t, ex, 1H, J = 5.0 Hz, NHCH2), 4.55 (s, 1H, H2′), 4.48 (s, 1H, H3′), 4.27–4.29 (m, 2H, CH2NH), 4.10–4.13 (d, 1H, J = 9.0 Hz, H5″), 3.97–4.01 (d, 1H, J = 9.0 Hz, H5″), 3.81 (s, 6H, 2 × CH3O), 3.50–3.57 (2d, 2H, J = 11.0 Hz, H5′), 2.16–2.19 (m, 2H, CH2CONH), 1.60–1.64 (m, 2H, CH2CH2CONH), 1.20–1.25 (m, 28H, 14 × CH2), 0.89 (t, 3H, J = 7.0 Hz, CH3CH2); 13C NMR (CDCl3) δ 172.8, 161.6, 158.77, 158.76, 149.0, 144.3, 142.7 (C6), 135.23, 135.21, 130.04 (Ar), 130.03, (Ar), 128.04 (Ar), 127.98 (Ar), 127.1 (Ar), 113.4 (Ar), 98.5, 89.8, 89.6, 88.0 (C1′), 86.7, 78.9 (C2′), 74.5, 74.3 (C3′), 72.8 (C5″), 59.5 (C5′), 55.3 (CH3O), 36.5 (CH2CONH), 31.9 (CH2), 30.1 (CH2NH), 29.69 (CH2), 29.68 (CH2), 29.66 (CH2), 29.65 (CH2), 29.63 (CH2), 29.5 (CH2), 29.353 (CH2), 29.345 (CH2), 29.33 (CH2), 25.5 (CH2CH2CONH), 22.7 (CH2), 14.1 (CH3).

(1S,3R,4S,7R)-3-[5-(3-Cholesterylcarbonylaminopropyn-1-yl)uracil-1-yl]-1-(4,4′-dimethoxytrityloxymethyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptane (10Y)

Nucleoside 9 (0.34 g, 0.50 mmol), Pd(PPh3)4 (60 mg, 0.05 mmol), CuI (20 mg, 0.10 mmol), cholesterylprop-2-ynyl-carbamate amine[34] (0.47 g, 1.00 mmol), and Et3N (0.30 mL, 2.13 mmol) in anhydrous DMF (8 mL) were reacted as described in the representative Sonogashira protocol and the reaction mixture was stirred at room temperature for 12 h. After workup and purification, nucleoside 10Y (0.39 g, 76%) was obtained as a slightly yellow solid material: Rf = 0.5 (5% MeOH in CH2Cl2, v/v); ESI-HRMS m/z 1046.5533 ([M + Na]+, C62H77N3O10·Na+, calcd 1046.5501); 1H NMR (CDCl3) δ 8.92 (bs, 1H, ex, NH(U)), 7.89 (s, 1H, H6), 7.23–7.46 (m, 9H, Ar), 6.87 (d, 4H, J = 9.0 Hz, Ar), 5.98 (s, 1H, H1′), 5.36 (d, 1H, J = 5.0 Hz, HC=C-Chol), 5.12 (t, ex, 1H, J = 5.0 Hz, NHCH2), 4.47–4.56 (m, 3H, H2′, HC-O-Chol, H3′), 4.20 (d, 2H, J = 5.0 Hz, CH2NH), 4.11–4.15 (d, 1H, J = 9.0 Hz, H5″), 3.98–4.02 (d, 1H, J = 9.0 Hz, H5″), 3.81 (s, 6H, 2 × CH3O), 3.50–3.58 (2d, 2H, J = 11.0 Hz, H5′), 0.87–2.36 (m, 40H, Chol), 0.69 (s, 3H, CH3-Chol); 13C NMR (CDCl3) δ 161.6, 158.74, 158.73, 149.0, 144.3, 142.7 (C6), 139.8, 135.3, 132.1 (Ar), 132.0 (Ar), 130.04 (Ar), 130.03 (Ar), 128.5 (Ar), 128.4 (Ar), 128.02 (Ar), 128.00 (Ar), 127.1 (Ar), 122.5 (CH=C-chol), 113.4 (Ar), 98.6, 89.9, 88.0 (C1′), 86.6, 78.9 (C2′), 74.9 (CH-O-chol), 74.4, 74.3 (C3′),[35] 72.8 (C5″), 59.5 (C5′), 56.7 (CH-chol), 56.2 (CH-chol), 55.2 (CH3O), 50.0 (CH-chol), 42.3, 39.8 (CH2-chol), 39.5 (CH2-chol), 38.5 (CH2–chol), 37.0 (CH2–chol), 36.6, 36.2 (CH2–chol), 35.8 (CH-chol), 31.9 (CH-chol), 31.8 (CH2NH and CH2-chol overlap), 28.2 (CH2-chol), 28.1 (CH2-chol), 28.0 (CH-chol), 24.3 (CH2-chol), 23.8 (CH2-chol), 22.8 (CH3-chol), 22.5 (CH3-chol), 21.0 (CH2-chol), 19.3 (CH3-chol), 18.7 (CH3-chol), 11.9 (CH3-chol).

Representative Protocol for Phosphitylation

Unless otherwise mentioned, the following protocol was used. Alcohol 10 was dried through coevaporation with anhydrous 1,2-dichloroethane (2 × 10 mL) and dissolved in anhydrous CH2Cl2. To this solution was added anhydrous EtN(iPr)2 (DIPEA) and 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (PCl-reagent), and the reaction mixture was stirred at room temperature until analytical TLC showed full conversion of the starting material (2–3 h) (quantities and volumes specified below). The reaction mixture was diluted with CH2Cl2 (25 mL) and washed with 5% aqueous NaHCO3 (2 × 10 mL) and the combined aqueous phase back-extracted with CH2Cl2 (2 × 10 mL). The combined organic layers were dried (Na2SO4) and evaporated to dryness, and the resulting residue was purified by silica gel column chromatography (0–3% MeOH in CH2Cl2, v/v) and subsequently triturated from CH2Cl2 and petroleum ether to provide phosphoramidite 11.

(1S,3R,4S,7R)-7-[2-Cyanoethoxy(diisopropylamino)phosphinoxy]-1-(4,4′-dimethoxytrityloxymethyl)-3-(5-ethynyluracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (11S)

Nucleoside 10S (0.35 g 0.60 mmol), DIPEA (0.50 mL, 2.88 mmol), and PCl-reagent (0.20 mL, 0.87 mmol) in anhydrous CH2Cl2 (10 mL) were reacted, worked up, and purified as described in the representative phosphitylation protocol to provide 11S (0.39 g, 83%) as a white foam: Rf = 0.5 (2% MeOH in CH2Cl2, v/v); ESI-HRMS m/z 805.2943 ([M + Na]+, C42H47N4O9P·Na+, calcd 805.2958); 31P NMR (CDCl3) δ 150.2, 149.9.

(1S,3R,4S,7R)-3-[5-(3-Benzoyloxypropyn-1-yl)uracil-1-yl]-7-[2-cyanoethoxy(diisopropylamino)phosphinoxy]-1-(4,4′-dimethoxytrityloxymethyl)-2,5-dioxabicyclo[2.2.1]heptane (11V)

Nucleoside 10V (0.35 g, 0.49 mmol) was coevaporated with anhydrous 1,2-dichloroethane (2 × 7 mL) and redissolved in anhydrous CH2Cl2 (7 mL). To this solution were added DIPEA (425 μL, 2.44 mmol) and N-methylimidazole (31 μL, 0.39 mmol), followed by dropwise addition of the PCl-reagent (220 μL, 0.98 mmol). The reaction mixture was stirred at room temperature for 3 h, whereupon it was evaporated to near dryness. The resulting residue was purified by silica gel column chromatography (0–3% MeOH in CH2Cl2) and subsequently triturated from CH2Cl2 and petroleum ether to provide 11V (0.28 g, 62%) as a white foam: Rf = 0.5 (3% MeOH in CH2Cl2); ESI-HRMS m/z 939.3332 ([M + Na]+, C50H53N4O11P·Na+, calcd 939.3341); 31P NMR (CDCl3) δ 150.1, 149.8.

(1S,3R,4S,7R)-7-[2-Cyanoethoxy(diisopropylamino)phosphinoxy]-1-(4,4′-dimethoxytrityloxymethyl)-3-[5-(3-trifluoroacetylaminopropyn-1-yl)uracil-1-yl]-2,5-dioxabicyclo[2.2.1]heptane (11W)

Nucleoside 10W (0.25 g 0.35 mmol), DIPEA (0.30 mL, 1.7 mmol), and PCl-reagent (0.10 mL, 0.45 mmol) in anhydrous CH2Cl2 (10 mL) were reacted, worked up, and purified as described in the representative phosphitylation protocol to provide 11W (0.27 g, 84%) as a white foam: Rf = 0.5 (2% MeOH in CH2Cl2, v/v); ESI-HRMS m/z 930.3068 ([M + Na]+, C45H49F3N5O10P·Na+, calcd 930.3080); 31P NMR (CDCl3) δ 150.2, 149.9.

(1S,3R,4S,7R)-7-[2-Cyanoethoxy(diisopropylamino)phosphinoxy]-1-(4,4′-dimethoxytrityloxymethyl)-3-[5-(3-octadecanoylaminopropyn-1-yl)uracil-1-yl]-2,5-dioxabicyclo[2.2.1]heptane (11X)

Nucleoside 10X (170 mg, 0.19 mmol), DIPEA (145 μL, 0.83 mmol), and PCl-reagent (61 μL, 0.27 mmol) in anhydrous CH2Cl2 (2 mL) were mixed and reacted as described in the representative phosphitylation protocol. After it was stirred at room temperature for 3 h, the mixture was diluted with EtOAc (20 mL) and washed with H2O (2 × 30 mL). The organic phase was dried (Na2SO4) and evaporated to dryness, and the resulting residue was purified by silica gel column chromatography (0–70% EtOAc in petroleum ether, v/v) and subsequently triturated from CH2Cl2 and petroleum ether to provide 11X (107 mg, 52%) as a white foam: Rf = 0.4 (5% MeOH in CH2Cl2, v/v); ESI-HRMS m/z 1100.5859 ([M + Na]+, C61H84N5O10P·Na+, calcd 1100.5848); 31P NMR (CDCl3) δ 150.2, 149.9.

(1S,3R,4S,7R)-3-[5-(3-Cholesterylcarbonylaminopropyn-1-yl)uracil-1-yl]-7-[2-cyanoethoxy(diisopropylamino)phosphinoxy]-1-(4,4′-dimethoxytrityloxymethyl)-2,5-dioxabicyclo[2.2.1]heptane (11Y)

Nucleoside 10Y (150 mg, 0.15 mmol), DIPEA (105 μL, 0.59 mmol), and PCl-reagent (50 μL, 0.21 mmol) in anhydrous CH2Cl2 (1.5 mL) were mixed and reacted as described in the representative phosphitylation protocol. After it was stirred at room temperature for 3 h, the reaction mixture was diluted with EtOAc (20 mL) and washed with H2O (2 × 30 mL). The organic layer was dried (Na2SO4) and evaporated to dryness, the resulting residue was purified by silica gel column chromatography (0–70% EtOAc in petroleum ether, v/v); subsequent trituration from CH2Cl2 and petroleum ether provided 11Y (102 mg, 57%) as a white foam: Rf = 0.4 (5% MeOH in CH2Cl2, v/v); ESI-HRMS m/z 1246.6519 ([M + Na]+, C71H94N5O11P·Na+, calcd 1246.6579); 31P NMR (CDCl3) δ 150.2, 149.9.

Alternative Route to (1S,3R,4S,7R)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-3-(5-iodoracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (9)

Nucleoside 12(36) (200 mg, 0.52 mmol) was coevaporated with anhydrous pyridine (10 mL) and redissolved in anhydrous pyridine (10 mL). To this was added 4,4′-dimethoxytrityl chloride (DMTrCl, 230 mg, 0.68 mmol), and the reaction mixture was stirred at room temperature for 16 h. At this point, saturated aqueous NaHCO3 (20 mL) and CH2Cl2 (25 mL) were added and the phases were separated. The organic phase was washed with saturated aqueous NaHCO3 (20 mL). The combined aqueous phase was back-extracted with CH2Cl2 (2 × 20 mL). The combined organic layer was dried (Na2SO4), concentrated to near dryness, and coevaporated with toluene/absolute EtOH (2 × 30 mL, 1/2, v/v). The resulting crude product was purified by silica gel column chromatography (0–4.5% MeOH in CH2Cl2, v/v) to afford nucleoside 9 (250 mg, 70%) as a light yellow solid material.

(1R,3R,4S,7R)-7-Hydroxy-1-hydroxymethyl-3-(5-iodoracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (12)

To a solution of nucleoside 8 (200 mg, 0.78 mmol) in glacial AcOH (10 mL) were added iodine (119 mg, 0.47 mmol) and ceric ammonium nitrate (213 mg, 0.39 mmol), and the reaction mixture was stirred at 80 °C for 50 min. After it was cooled to room temperature, the mixture was evaporated to dryness and the resulting residue purified by silica gel column chromatography (0–16% MeOH/CH2Cl2, v/v) to afford 12(36) (240 mg, 80%) as a white solid material: Rf = 0.4 (15% MeOH in CH2Cl2, v/v); FAB-HRMS m/z 382.9735 ([M + H]+, C10H11IN2O6·H+, calcd 382.9740); 1H NMR (DMSO-d6) δ 11.76 (s, 1H, ex, NH), 8.09 (s, 1H, H6), 5.88 (d, 1H, ex, J = 4.5 Hz, 3′-OH), 5.84 (s, 1H, H1′), 4.97 (t, 1H, ex, J = 5.4 Hz, 5′-OH), 4.25 (d, 1H, J = 4.5 Hz, H3′), 4.23 (s, 1H, H2′), 3.93–3.96 (d, 1H, J = 8.5 Hz, H5″), 3.78–3.81 (d, 1H, J = 8.5 Hz, H5″), 3.70–3.77 (m, 2H, 2 × H5′); 13C NMR (DMSO-d6) δ 160.5, 149.9, 144.2 (C6), 91.2, 87.1 (C1′), 78.7 (C2′), 72.4 (C3′), 72.0 (C5″), 67.7, 57.5 (C5′).

Synthesis of Oligodeoxyribonucleotides and Biophysical Characterization Studies

Unmodified DNA and RNA strands were obtained from commercial suppliers and used without further purification. L1–4 were prepared and characterized with respect to identity (MALDI-MS) and purity (>80%, ion-pair reverse-phase HPLC) in a previous study.[11b] ONs modified with C5-alkynyl-functionalized α-L-LNA monomers were synthesized, purified, structurally characterized, and utilized in biophysical experiments essentially as described for the corresponding C5-alkynyl-functionalized LNA in the preceding article.[14] The following hand-coupling conditions (coupling time; activator; phosphoramidite solvent) were used for incorporation of monomers L-Z into ONs: monomers S/V/W/X (15 min; 0.25 M 4,5-dicyanoimidazole in CH3CN; CH3CN), monomer Y (15 min; 0.25 M 5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole[37] in CH3CN; CH3CN), and monomer Z (30 min; 0.25 M 4,5-dicyanoimidazole in CH3CN; CH2Cl2).