Synthesis of antisense oligonucleotides containing acyclic alkynyl nucleoside analogs and their biophysical and biological properties

Accepted Manuscript Synthesis of antisense oligonucleotides containing acyclic alkynyl nucleoside analogs and their biophysical and biological properties Aya Ogata, Yusuke Maeda, Yoshihito Ueno PII: DOI: Reference:

S0968-0896(16)31227-5 http://dx.doi.org/10.1016/j.bmc.2017.01.051 BMC 13560

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

19 November 2016 12 January 2017 13 January 2017

Please cite this article as: Ogata, A., Maeda, Y., Ueno, Y., Synthesis of antisense oligonucleotides containing acyclic alkynyl nucleoside analogs and their biophysical and biological properties, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.01.051

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Synthesis of antisense oligonucleotides containing acyclic alkynyl nucleoside analogs and their biophysical and biological properties

Aya Ogata[a], Yusuke Maeda[b] and Yoshihito Ueno[a,b,c]*

a

United Graduate School of Agricultural Science, Gifu University, bFaculty of Applied

Biological Sciences, Gifu University, and cCenter for Highly Advanced Integration of Nano and Life Sciences (G-CHAIN), Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.

*To whom correspondence should be addressed. Phone & Fax: +81-58-293-2919 E-mail: [email protected]

Keywords: acyclic nucleoside; antisense oligonucleotide; alkyne; RNase H; thermal stability; nuclease resistance

1

Abstract: The synthesis of oligonucleotide (ON) analogs, which can be used as antisense molecules, has recently gained much attention. Here, we report the synthesis and properties of an ON analog containing acyclic thymidine and cytidine analogs with a 4-pentyl-1,2-diol instead of the

D-ribofuranose

moiety. The incorporation of these

analogs into the ON improved its nuclease resistance to 3′-exonucleases. Furthermore, it was found that the incorporation of the acyclic thymidine analog into a DNA/RNA duplex accelerates the RNA cleavage of a DNA/RNA duplex by Escherichia coli RNase H.

2

1. Introduction A large number of chemically modified oligonucleotides (ONs) have been synthesized and used as antisense molecules to regulate the functions of specific mRNA targets.1,2 For ONs to be effective as antisense molecules, it is necessary that they form thermally and thermodynamically stable Watson-Crick hybrids with complementary RNAs, while being sufficiently resistant to degradation by ubiquitous nucleases. Recently, ONs composed of conformationally restricted nucleosides such as 2′-O,4′-C-methylene-bridged nucleic acid (2′,4′-BNA,3 also called LNA4) have attracted much attention as antisense molecules because some of them have been shown to form thermally and thermodynamically stable duplexes with complementary RNAs and be resistant to degradation by nucleases. On the other hand, the synthesis of ONs comprising acyclic nucleosides has not received much attention because of the low stability of the duplexes containing these nucleoside analogs.5 However, Meggers et al. recently reported that an ON composed of acyclic nucleoside analogs 1 containing glycol (GNA) instead of the D-ribofuranose moiety can form a thermally stable duplex with complementary RNA but not with complementary DNA (Fig. 1).6 Inouye and his coworkers also reported a 2′-deoxyribonucleoside analog whose sugar moiety linked with a nucleobase by the ethynyl group. 7 The modification of ONs with alkynyl residues at the 5-position of the pyrimidine moieties is known to stabilize the corresponding duplexes.8 This stabilization is reported to be mainly due to a ΔH° contribution based on the π−π interactions between the alkynyl residues and the bases either above or below it. Recently, we reported the synthesis of small interfering RNAs (siRNAs) containing the acyclic alkynyl thymidine analog 2.9 It was found that the incorporation of analog 2 into siRNA enhances its 3

silencing activity. Based on these findings and experimental results, we designed an antisense ON containing acyclic alkynyl thymidine and cytidine analogs 2 and 3. We envisioned that the modification of GNA by addition of alkyne moieties might enhance the thermal stability of the duplex between this acyclic ON and a complementary RNA sequence. In this paper, we report the synthesis and properties of the ON analogs containing acyclic thymidine and/or cytidine analogs 2 and 3.

Fig. 1.

Structure of acyclic nucleoside analogs

2. Results and discussion 2.1. Synthesis In order to incorporate the acyclic alkynyl nucleotide analogs into ONs, their phosphoramidite derivatives were needed. The phosphoramidite of thymidine analog 2 was synthesized according to a reported method. 9 The synthetic routes towards phosphoramidite 10 of cytidine analog 3 are shown in Schemes 1 and 2. First, 2-amino-5-iodo-4-pyrimidinone (4)10 was treated with N,N-dimethylformamide dimethyl acetal to give 1-methyl-N2-dimethylaminomethylene derivative 5 in 66% yield. 4

Then, 4-pentyne-1,2-diol derivative 6, which was synthesized according to a reported method,8 was coupled with 5 in presence of CuI, Pd(PPh3)4, and Et3N in DMF to give cytidine

analog

7

in

67%

yield.

Subsequently,

7

was

desilylated

with

tetrabutylammonium fluoride (TBAF) in THF to produce diol derivative 8 in 45% yield. The primary hydroxy group of 8 was then protected with a 4,4′ -dimethoxytrityl (DMTr) group to afford mono-DMTr derivative 9 in 69% yield. Mono-DMTr derivative 9 was phosphitylated

using

a

standard

procedure

to

produce

the

corresponding

phosphoramidite 10 in 89% yield. All ONs containing analogs 2 and 3 were synthesized by using a DNA/RNA synthesizer. The ON sequences used in this study are depicted in Table 1. The ONs were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS), and the observed molecular weights obtained from these analyses were in agreement with the ON structures.

Scheme 1.

Synthesis of cytosine derivative 5

5

Scheme 2.

Synthesis of the phosphoramidite of cytidine analog 3

6

2.2. Thermal stabilities of duplexes The thermal stabilities of DNA/RNA and ON/RNA duplexes containing analogs 2 and 3 were investigated by measuring their melting temperatures (Tms) in a 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl (Table 1). The Tm value of the DNA 1/RNA 1 duplex (Duplex 1) was 56.5 °C, whereas that of the ON 1/RNA 1 duplex (Duplex 2) containing three thymidine analogs at each side of the 5′- and 3′-regions of the ON was 52.0 °C [ΔTm (Tm of Duplex 1–Tm of Duplex 2) = –4.5 °C]. In addition, the Tm value of the DNA 2/RNA 2 duplex (Duplex 3) was 56.3 °C, while that of the ON 2/RNA 2 duplex (Duplex 4) containing five consecutive thymidine analogs at each side of the 5′- and 3′-regions of the ON was 47.1 °C [ΔTm (Tm of Duplex 3–Tm of Duplex 4) = –9.2 °C]. Other ON/RNA duplexes containing analog 2 (Duplexes 8 and 10), which were composed of the different sequence from Duplexes 2 and 4, also showed similar results. Thus, it was found that the incorporation of analog 2 into DNA/RNA duplexes decreased the thermal stability of the duplexes (ΔTm = –0.4 °C ~ –1.3 °C/analog). The Tm value of the ON 3/RNA 3 duplex containing thymidine and cytidine analogs 2 and 3 (Duplex 6) was also lower than that of the parent DNA 3/RNA 3 duplex (Duplex 5). The ON/RNA duplex containing analogs 2 and 3 (Duplex 12), which was composed of the different sequence from Duplex 6, also showed similar results. In general, the incorporation of a dC:rG instead of a T:rA base pair increases the thermal stability of a duplex, since the dC:rG base pair is composed of three hydrogen bonds, while the T:rA base pair comprises two hydrogen bonds. Actually, the Tm value of Duplex 5 with two dC:rG base pairs instead of the two T:rA base pairs of Duplex 1 was 3.9 °C greater than that of the parent duplex. On the other hand, the increment in Tm value observed by 7

replacing a 2:rA base pair with a 3:rG base pair was smaller than that of the natural duplex. The ΔTm value between Duplex 6 and Duplex 2 was 1.1 °C. Furthermore, the Tm value of Duplex 12 with two 3:rG base pairs instead of the two 2:rA base pairs of Duplex 8 was slightly smaller than that of the parent duplex. These results suggest that the strength of the hydrogen bonds in the 3:rG base pair, namely, the ability to form base pairs of cytidine analog 3, is weaker than that of the 2:rA base pair in the DNA/RNA duplexes. Schlegel et al. also reported that the incorporation of a G:C base pair into a GNA/RNA duplex decreases the thermal stability of the duplex. 6

8

Table 1.

Sequences of DNAs, RNAs, ONs, and duplexes, and Tm values of the duplexes. a

Abbreviation

Abbreviation

of duplex

of ON

Duplex 1

DNA 1

5′-d(TTTCACTACTCCTATTT)-3′

RNA 1

3′-r(AAAGUGAUGAGGAUAAA)-5′

Duplex 2

Duplex 3

Duplex 4

Duplex 5

Duplex 6

Duplex 7

Duplex 8

Duplex 9

Duplex 10

Duplex 11

Duplex 12

ON 1

Sequence

5′-d(222CACTACTCCTA222)-3′

RNA 1

3′-r(AAAGUGAUGAGGAUAAA)-5′

DNA 2

5′-d(TTTTTCTACTCCTTTTT)-3′

RNA 2

3′-r(AAAAAGAUGAGGAAAAA)-5′

ON 2

5′-d(22222CTACTCC22222)-3′

RNA 2

3′-r(AAAAAGAUGAGGAAAAA)-5′

DNA 3

5′-d(TCTCACTACTCCTATCT)-3′

RNA 3

3′-r(AGAGUGAUGAGGAUAGA)-5′

ON 3

5′-d(232CACTACTCCTA232)-3′

RNA 3

3′-r(AGAGUGAUGAGGAUAGA)-5′

DNA 4

5′-d(TTTCACCGACGGCGTTT)-3′

RNA 4

3′-r(AAAGUGGCUGCCGCAAA)-5′

ON 4

5′-d(222CACCGACGGCG222)-3′

RNA 4

3′-r(AAAGUGGCUGCCGCAAA)-5

DNA 5

5′-d(TTTTTCCGACGGTTTTT)-3′

RNA 5

3′-r(AAAAAGGCUGCCAAAAA)-5′

ON 5

5′-d(22222CCGACGG22222)-3′

RNA 5

3′-r(AAAAAGGCUGCCAAAAA)-5

DNA 6

5′-d(TCTCACCGACGGCGTCT)-3′

RNA 6

3′-r(AGAGUGGCUGCCGCAGA)-5′

ON 6

5′-d(232CACCGACGGCG232)-3′

Tm (°C)

ΔTm (°C)b

56.5

–

52.0

-4.5

56.3

–

47.1

-9.2

60.4

–

53.1

-7.2

66.1

–

63.8

-2.3

57.6

–

44.2

-13.4

69.3

–

61.9

-7.4

RNA 6

3′-r(AGAGUGGCUGCCGCAGA)-5

–

RNA 7

F-5′-r(AAAUAGGAGUAGUGAAA)-3′

–

–

–

RNA 8

F-5′-r(AAAAAGGAGUAGAAAAA)-3

–

–

–

RNA 9

5′-r(AAGAAGGAGUAGAAGAA)-3

–

–

–

RNA 10

F-5′-r(AAACGCCGUCGGUGAAA)-3′

–

–

–

RNA 11

F-5′-r(AAAAACCGUCGGAAAAA)-3′

–

– 9

–

RNA 12

F-5′-r(AGACGCCGUCGGUGAGA)-3′

–

–

–

DNA 7

F-5′-d(TTTTTTTTTT)-3′

–

–

–

ON 7

F-5′-d(TTTTTTTTT2)-3′

–

–

a

F: fluorescein. bΔTm = Tm (DNA/RNA duplex) – Tm (corresponding ON/RNA duplex). Experimental conditions are shown in the Experimental section.

10

To study the base discriminating abilities of analogs 2 and 3 in a DNA/RNA duplex, we measured the Tms of duplexes containing mismatched base pairings (Table 2). The Tm value of the ON 4/RNA 6 duplex (Duplex 8m) containing a 2:G mismatched base pair was 61.8 °C, whereas that of the ON 4/RNA 4 duplex (Duplex 8) composed of a complementary sequence was 63.8 °C. Similarly, the Tm value of the ON 2/RNA 10 duplex (Duplex 4m, Tm = 44.7 °C) containing the 2:rG mismatched base pair in five consecutive 2 analogs was smaller than that of the ON 2/RNA 2 duplex (Duplex 4, Tm = 47.1 °C) composed of a complementary sequence. These results suggest that analog 2 possesses a base discriminating ability in a DNA/RNA duplex. On the other hand, the Tm value of the ON 6/RNA 4 duplex (Duplex 12m, Tm = 63.0 °C) containing a 3:rA mismatched base pair was slightly higher than that of the ON 6/RNA 6 duplex (Duplex 12, Tm = 61.9 °C) comprising a complementary sequence. Similarly, the Tm values of the duplexes (Duplex 15m and 16m) containing a 3:rC or 3:rU mismatched base pair were slightly higher than that of Duplex 12 composed of a complementary sequence (Table S1). The results suggest that analog 3 does not have a base discriminating ability in a DNA/RNA duplex.

11

Table 2.

Tm values of duplexes including mismatched base pairs.

Abbreviation

Abbreviation

of duplex

of ON

Duplex 7

DNA 4

5′-d(TTTCACCGACGGCGTTT)-3′

RNA 4

3′-r(AAAGUGGCUGCCGCAAA)-5′

DNA 4

5′-d(TTTCACCGACGGCGTTT)-3′

RNA 6

3′-r(AGAGUGGCUGCCGCAGA)-5′

Duplex 7m

Duplex 8

ON 4 RNA 4

Duplex 8m

Duplex 3

Duplex 3m

Duplex 4

ON 4

Duplex 11

Duplex 11m

Duplex 12

5′-d(TTTTTCTACTCCTTTTT)-3′

RNA 2

3′-r(AAAAAGAUGAGGAAAAA)-5′

DNA 2

5′-d(TTTTTCTACTCCTTTTT)-3′

RNA 10

3′-r(AAGAAGAUGAGGAAGAA)-5′

ON 2

5′-d(22222CTACTCC22222)-3′

5′-d(22222CTACTCC22222)-3′ 3′-r(AAGAAGAUGAGGAAGAA)-5′

DNA 6

5′-d(TCTCACCGACGGCGTCT)-3′

RNA 6

3′-r(AGAGUGGCUGCCGCAGA)-5′

DNA 6

5′-d(TCTCACCGACGGCGTCT)-3′

RNA 4

3′-r(AAAGUGGCUGCCGCAAA)-5′

ON 6

ON 6

ΔTm (°C)b

65.6

–

65.0

-0.6

63.8

–

61.8

-2.0

56.3

–

51.1

-5.2

47.1

–

44.7

-2.4

66.9

–

63.2

-6.1

61.9

–

63.0

+1.1

3′-r(AAAAAGAUGAGGAAAAA)-5′

RNA 10

RNA 4 a

5′-d(222CACCGACGGCG222)-3′

DNA 2

ON 2

Tm (°C)

3′-r(AAAGUGGCUGCCGCAAA)-5

3′-r(AGAGUGGCUGCCGCAGA)-5

RNA 6 Duplex 12m

5′-d(222CACCGACGGCG222)-3′

RNA 6

RNA 2 Duplex 4m

Sequencea

5′-d(232CACCGACGGCG232)-3′ 3′-r(AGAGUGGCUGCCGCAGA)-5 5′-d(232CACCGACGGCG232)-3′ 3′-r(AAAGUGGCUGCCGCAAA)-5

Underlined letters indicate the mismatched bases. bΔTm = Tm (duplex containing a mismatch base

pair) – Tm (duplex composed of a matched sequence).

12

In order to examine the stability of the duplexes in further detail, we calculated the thermodynamic parameters of duplex formation based on the slope of the plot of 1/Tm vs. ln (CT/4), where CT is the total concentration of single-stranded ON and RNA. The corresponding results are depicted in Table 3. ON/RNA duplexes containing the modified analogs were thermodynamically less stable than the parent DNA/RNA duplexes. It was found that the thermodynamic destabilization of the duplexes was mainly caused by disadvantages in terms of enthalpy (ΔH°). However, the entropy (ΔS°) terms were favorable for the duplexes containing the analogs except for Duplex 2. These results suggest that the incorporation of an ethynyl group into the analogs weakens the formation of hydrogen bonding between the analogs and the natural nucleosides in the DNA/RNA duplexes, despite the advantages in terms of ΔS°.

13

Table 3.

Thermodynamic parameters of the duplexes.

Abbreviation

Abbreviation

of duplex

of ON

Duplex 1

Duplex 2

Duplex 3

Duplex 4

Duplex 7

Duplex 8

Duplex 9

Duplex 10

Duplex 11

Duplex 12

Sequence

DNA 1

5′-d(TTTCACTACTCCTATTT)-3′

RNA 1

3′-r(AAAGUGAUGAGGAUAAA)-5′

ON 1

5′-d(222CACTACTCCTA222)-3′

RNA 1

3′-r(AAAGUGAUGAGGAUAAA)-5′

DNA 2

5′-d(TTTTTCTACTCCTTTTT)-3′

RNA 2

3′-r(AAAAAGAUGAGGAAAAA)-5′

ON 2

5′-d(22222CTACTCC22222)-3′

RNA 2

3′-r(AAAAAGAUGAGGAAAAA)-5′

DNA 4

5′-d(TTTCACCGACGGCGTTT)-3′

RNA 4

3′-r(AAAGUGGCUGCCGCAAA)-5′

ON 4

5′-d(222CACCGACGGCG222)-3′

RNA 4

3′-r(AAAGUGGCUGCCGCAAA)-5

DNA 5

5′-d(TTTTTCCGACGGTTTTT)-3′

RNA 5

3′-r(AAAAAGGCUGCCAAAAA)-5′

ON 5

5′-d(22222CCGACGG22222)-3′

RNA 5

3′-r(AAAAAGGCUGCCAAAAA)-5

DNA 6

5′-d(TCTCACCGACGGCGTCT)-3′

RNA 6

3′-r(AGAGUGGCUGCCGCAGA)-5′

ON 6

5′-d(232CACCGACGGCG232)-3′

RNA 6

3′-r(AGAGUGGCUGCCGCAGA)-5

ΔG°310

ΔH°

ΔS°

[kcal/mol]

[kcal/mol]

[cal/K∙mol]

-13.6

-100.3

-279.8

-13.5

-118.8

-339.5

-14.5

-106.8

-297.4

-10.9

-73.3

-236.2

-15.4

-83.8

-220.5

-13.7

-71.2

-185.2

-15.1

-103.0

-283.3

-11.0

-71.6

-195.5

-19.1

-111.4

-297.7

-14.0

-75.6

-198.5

14

2.3. Conformation of duplexes In order to compare the global conformation of the natural DNA/RNA duplexes with that of the ON/RNA duplexes containing analogs 2 and 3, circular dichroism (CD) spectra of the duplexes were measured (Fig. 2). The ON/RNA duplexes (Duplexes 8 and 12) containing the modified analogs exhibited CD spectra with positive and negative CD bands at 260 and 210 nm, respectively, that were similar to those of the natural DNA/RNA duplexes (Duplexes 7 and 11). Thus, this suggested that the ON/RNA duplexes containing the modified analogs formed an A-type duplex.

15

Fig. 2.

CD spectra of the duplexes.

Experimental conditions are shown in the Experimental section. Concentration of duplexes = 6 μM.

16

2.4. Nuclease resistance of ONs The nuclease resistance of synthetic ONs is an important factor in view of their therapeutic application, since unmodified DNA is easily degraded by nucleases that are present both inside and outside the cells. Thus, the susceptibility of a modified ON to snake venom phosphodiesterase (SVPD), a 3′-exonuclease, was examined. DNA 7 and ON 7 containing analog 2 at their 3′-ends were labeled with fluorescein at their 5′-ends, and subsequently incubated with SVPD. The reactions were analyzed using polyacrylamide gel electrophoresis (PAGE) under denaturing conditions. As shown in Fig. 3, DNA 7 was immediately hydrolyzed after 1 min of incubation, whereas ON 7 was resistant to the enzyme for 3 h. Therefore, it could be concluded that the incorporation of analog 2 into the ON improved its nuclease resistance against a 3′-exonuclease.

17

Fig. 3.

PAGE of DNA and ON hydrolyzed by SVPD. (a) DNA 7. (b) ON 7.

(a) DNA 7

F-5'-d(TTTTTTTTTT)-3'

(b) ON 7

F-5'-d(TTTTTTTTT2) - 3'

F: fluorescein. Experimental conditions are shown in the Experimental section.

18

2.5. Ability to elicit the activity of RNase H It has been postulated that the antisense activity of ONs to suppress gene expression is caused, at least in part, by the cleavage of a target RNA by RNase H. Thus, we examined the ability of a modified ON to elicit the RNase H activity. Duplexes consisting of RNA labeled with fluorescein at the 5′-end and ON were incubated with Escherichia coli RNase H. The reaction mixtures were then analyzed by PAGE under denaturing conditions (Fig. 4). The initial velocities of the reactions are depicted in Table 4. The ON/RNA duplexes containing analog 2 (Duplexes 2f and 4f) were more rapidly hydrolyzed by the enzyme than the corresponding parent DNA/RNA duplexes (Duplexes 1f and 3f). The initial velocities for Duplexes 2f and 4f were 70 and 86 pmol/min, whereas those for Duplexes 1f and 3f were 42 and 71 pmol/min, respectively. Thus, it was suggested that the incorporation of thymidine analog 2 into a DNA/RNA duplex accelerates the RNA cleavage reaction by E. coli RNase H. The incorporation of analog 2 into a DNA/RNA duplex decreased the duplex thermal stability. Thus, we considered that the RNA fragments, which were cleaved by the enzyme, could be released from the ON/RNA duplex containing analog 2 more rapidly than from the natural DNA/RNA duplex. This might accelerate the turnover of the enzyme in the cleavage reaction, leading to an increase of the reaction rate. Next, the cleavage reaction promoted by E. coli RNase H was assessed by using a duplex with high GC-content as a substrate. When three thymidine analogs were incorporated into each side of the 5′- and 3′-regions of the ON (Duplex 8f), the rate of the cleavage reaction by E. coli RNase H decreased compared to that of the corresponding parent duplex (Duplex 7f). However, when a duplex containing five analogs at each side of the 5′- and 3′-regions of the ON (Duplex 10f) was used as 19

substrate, the initial velocity of the cleavage reaction increased in comparison with that of the corresponding parent duplex (Duplex 9f). Finally, we examined the cleavage reaction by using a duplex containing cytidine analog 3. It was found that the incorporation of cytidine analog 3 into a DNA/RNA duplex decreased the rate of the cleavage reaction by E. coli RNase H. The initial velocity for Duplex 11f was 56 pmol/min, whereas that for Duplex 12f containing analogs 2 and 3 was 31 pmol/min, which was smaller than that of Duplex 8f containing only analog 2 (initial velocity: 45 pmol/min). When the mismatched base pairs were incorporated into the natural DNA/RNA duplex (Duplex 11f), the rate of the cleavage reaction decreased, while the incorporation of the mismatched base pairs into Duplex 12f containing analogs 2 and 3 substantially did not influence the rate of the cleavage reaction by the enzyme (Fig. S6). The thermal denaturation study of the duplexes indicated that the ability of cytidine analog 3 to form a base pair with a complementary base was weaker than that of thymidine analog 2 in the DNA/RNA duplex. Thus, it can be reasoned that the properties of analog 3 could, at least in part, influence the ability of the duplex containing analog 3 to elicit the RNase H activity.

20

Fig. 4.

PAGE of DNA/RNA duplexes hydrolyzed by E. coli RNase H.

Duplex 1f DNA 1 RNA 7

Duplex 2f 5'-d(TTTCACTACTCCTATTT)-3' 3'-r(AAAGUGAUGAGGAUAAA)-5'-F

Duplex 3f DNA 2 RNA 8

ON 1 RNA 7

5'-d(222CACTACTCCTA222)-3' 3'-r(AAAGUGAUGAGGAUAAA)-5'-F

Duplex 4f 5'-d(TTTTTCTACTCCTTTTT)-3' 3'-r(AAAAAGAUGAGGAAAAA)-5'-F

ON 2 RNA 8

5'-d(22222CTACTCC22222)-3' 3'-r(AAAAAGAUGAGGAAAAA)-5'-F

21

Duplex 7f DNA 4 RNA 10

Duplex 8f 5'-d(TTTCACCGACGGCGTTT)-3' 3'-r(AAAGUGGCUGCCGCAAA)-5'-F

Duplex 9f 5'-d(TTTTTCCGACGGTTTTT)-3' DNA 5 RNA 11 3'-r(AAAAAGGCUGCCAAAAA)-5'-F

ON 4 RNA 10

5'-d(222CACCGACGGCG222)-3' 3'-r(AAAGUGGCUGCCGCAAA)-5'-F

Duplex 10f ON 5 RNA 11

5'-d(22222CCGACGG22222)-3' 3'-r(AAAAAGGCUGCCAAAAA)-5'-F

22

Duplex 11f DNA 6 RNA 12

Duplex 12f 5'-d(TCTCACCGACGGCGTCT)-3' 3'-r(AGAGUGGCUGCCGCAGA)-5'-F

ON 6 RNA 12

5'-d(232CACCGACGGCG232)-3' 3'-r(AGAGUGGCUGCCGCAGA)-5'-F

F: fluorescein. Experimental conditions are shown in the Experimental section.

23

Table 4.

Initial velocities for the cleavage of RNAs in DNA/RNA duplexes by E. coli RNase H. Abbreviation

Abbreviation of

of duplex

ON

Duplex 1f

Duplex 2f

Duplex 3f

Duplex 4f

Duplex 7f

Duplex 8f

Duplex 9f

Duplex 10f

Duplex 11f

Duplex 12f

Sequence

DNA 1

5′-d(TTTCACTACTCCTATTT)-3′

RNA 7

3′-r(AAAGUGAUGAGGAUAAA)-5′-F

ON 1

5′-d(222CACTACTCCTA222)-3′

RNA 7

3′-r(AAAGUGAUGAGGAUAAA)-5′-F

DNA 2

5′-d(TTTTTCTACTCCTTTTT)-3′

RNA 8

3′-r(AAAAAGAUGAGGAAAAA)-5′-F

ON 2

5′-d(22222CTACTCC22222)-3′

RNA 8

3′-r(AAAAAGAUGAGGAAAAA)-5′-F

DNA 4

5′-d(TTTCACCGACGGCGTTT)-3′

RNA 10

3′-r(AAAGUGGCUGCCGCAAA)-5′-F

ON 4

5′-d(222CACCGACGGCG222)-3′

RNA 10

3′-r(AAAGUGGCUGCCGCAAA)-5′-F

DNA 5

5′-d(TTTTTCCGACGGTTTTT)-3′

RNA 11

3′-r(AAAAAGGCUGCCAAAAA)-5′-F

ON 5

5′-d(22222CCGACGG22222)-3′

RNA 11

3′-r(AAAAAGGCUGCCAAAAA)-5′-F

DNA 6

5′-d(TCTCACCGACGGCGTCT)-3′

RNA 12

3′-r(AGAGUGGCUGCCGCAGA)-5′-F

ON 6

5′-d(232CACCGACGGCG232)-3′

RNA 12

3′-r(AGAGUGGCUGCCGCAGA)-5′-F

initial velocity [pmol/min] 42

70

71

86

57

45

192

245

56

31

24

3. Conclusions We have demonstrated the synthesis of ONs containing acyclic alkynyl nucleoside analogs 2 and 3, and examined their biophysical and biological properties. The incorporation of analogs 2 and 3 into a DNA/RNA duplex decreased its thermal stability. However, the incorporation of analog 2 into an ON improved its nuclease resistance against a 3′-exonuclease. Furthermore, it was found that the incorporation of thymidine analog 2 into a DNA/RNA duplex tends to accelerate the RNA cleavage in a DNA/RNA duplex by E. coli RNase H. Thus, we believe that these information can be useful for the future design of novel antisense molecules.

4. Experimental section 4.1. General remarks CDCl3 (CIL) or DMSO-d6 (CIL) was used as a solvent for obtaining NMR spectra. Chemical shifts (δ) are given in parts per million (ppm) downfield from (CH3)4Si (δ 0.00 for 1H NMR in CDCl3), or a solvent (for 13C NMR and 1H NMR in DMSO-d6) as an internal reference with coupling constants (J) in Hz. The abbreviations s, d, dd and m signify singlet, doublet, double doublet and multiplet, respectively. 4.1.1.

2-[(N,N-Dimethylamino)methylene]amino-5-iodo-N1-methyl-4-pyrimidinone

(5).

2-Amino-5-iodo-4-pyrimidinone (4) (1.30 g, 5.49 mmol) was dissolved in DMF (5.5 mL), added N,N-dimethylformamide dimethylacetal (3.64 mL, 27.4 mmol) and stirred at 40 °C for 5 h. The mixture was concentrated and the residue was purified by column chromatography (SiO 2, 10% MeOH in CHCl3) to give 5 (1.11 g, 66%). 1H NMR (400 MHz, DMSO-d6)  8.64 (s, 1H), 8.08 (s, 1H), 3.47 (s, 3H), 3.19 (s, 3H), 3.09 (s, 3H); 13C NMR (100.5 MHz, DMSO-d6)  160.0, 159.8, 158.5, 158.3, 76.3, 40.9, 35.0, 30.3; HRMS (ESI) m/z; calcd. for C8H12IN4O [M + H]+ 307.0056, found 307.0043. 4.1.2. (S)-5-[5-(tert-Butyldiphenylsilyloxy)-4-(tert-butyldimethylsilyloxy)-1-pentynyl]-2-[(N,N-dimethyla mino]methylene]amino-N1-methyl-4-pyrimidinone

(7).

A

mixture

of

(S)-2-O-(tert-butyldimethylsilyl)-1-O-(tert-butyldiphenylsilyl)-4-pentyne-1,2-diol (6) (0.73 g, 1.61 mmol), 5 (0.41 g, 1.34 mmol), CuI (27 mg, 0.14 mmol), Pd(PPh3)4 (162 mg, 0.14 mmol) and Et 3N (0.98 mL, 7.05 mmol) in DMF (7.0 mL) was stirred at 40 °C for 12 h. The mixture was partitioned between EtOAc and aqueous NaHCO3 (saturated). The residue was purified by column chromatography (SiO2, 50% EtOAc in hexane) to give 7 (0.56 g, 67%) . 1H NMR (400 MHz, CDCl3)  8.56 (s, 1H), 7.79 (s, 1H), 7.69-7.68 (m, 4H), 7.40-7.34 (m, 6H), 3.98-3.95 (m, 1H), 3.70 (dd, 1H, J = 10.1 and 4.6), 3.62 (dd, 1H, J = 10.1 and 6.0), 3.57 (s, 3H), 3.18 (s, 3H), 3.14 (s, 3H), 2.90 (dd, 1H, J = 17.0 and 5.5), 2.59 (dd, 2

1H, J = 16.5 and 6.9), 1.05 (s, 9H), 0.86 (s, 9H), 0.08 (s, 3H), 0.00 (s, 3H);

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C NMR (100.5 MHz,

CDCl3)  163.4, 158.8, 158.1, 155.5, 135.8, 135.7, 134.0, 133.9, 133.8, 133.7, 129.7 (2C), 128.8, 128.7, 127.8 (2C), 104.9, 92.4, 75.6, 72.3, 67.4, 41.5,35.4, 29.9, 27.0 (3C), 26.0 (4C), 19.4, 18.2, -4.5 (2C); HRMS (ESI) m/z; calcd. for C35H50KN4O3Si2 [M + K]+ 669.3059, found 669.3030. 4.1.3. (S)-5-(4,5-Dihydroxy-1-pentynyl)-2-[(N,N-dimethylamino)methylene]amino-N1-methyl-4-pyrimidi none (8). Compound 7 (0.52 g, 0.83 mmol) was dissolved in THF (4.2 mL). 1 M TBAF in THF (1.78 mL) and a few drops of acetic acid were added to the solution, and the mixture was stirred at room temperature for 24 h. The mixture was concentrated, and the residue was purified by column chromatography (SiO2, 20% MeOH in CHCl3) to give 8 (0.10 g, 45%). 1H NMR (500 MHz, DMSO-d6)  8.67 (s, 1H), 7.82 (s, 1H), 4.83 (d, 1H, J = 4.6), 4.59 (t, 1H, J = 5.7 and 5.2), 3.62-3.59 (m, 1H), 3.44-3.36 (m, 2H), 3.42 (s, 3H), 3.20 (s, 3H), 3.09 (s, 3H), 2.54 (dd, 1H, J = 16.6 and 6.3), 2.41 (dd, 1H, J = 16.6 and 6.3);

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C NMR (100.5 MHz, DMSO-d6)  162.2, 158.7, 158.7, 155.5, 102.6, 91.4, 76.0,

70.4, 64.7, 41.0, 35.0, 29.2, 24.6; HRMS (ESI) m/z; calcd. for C13H18N4NaO3 [M + Na]+ 301.1277, found 301.1291. 4.1.4. (S)-5-[4-(4,4′-Dimethoxytrityloxy)-5-hydroxy-1-pentynyl]-2-[(N,N-dimethylamino)methylene]amin o-N1-methyl-4-pyrimidinone (9). A mixture of 8 (37.6 mg, 0.14 mmol) and DMTrCl (53.0 mg, 0.16 mmol) in pyridine (1.4 mL) was stirred at room temperature for 4 h. The mixture was partitioned between EtOAc and aqueous NaHCO3 (saturated). The organic layer was washed with brine, dried (Na2SO4), and concentrated. The residue was purified by column chromatography (a neutralized SiO 2, 2% MeOH in CHCl3) to give 9 (54.2 mg, 69%). 1H NMR (500 MHz, CDCl3) ：8.56 (s, 1H), 7.77 (s, 1H), 7.45-7.43 (m, 2H), 7.34-7.25 (m, 6H), 7.21-7.17 (m, 1H), 6.82-6.80 (m, 4H), 4.01-3.99 (m, 1H), 3.77 (s, 6H), 3.55 (s, 3H), 3.28 (dd, 1H, J = 9.2 and 5.7), 3.24 (dd, 1H, J = 9.8 and 5.2), 3.17 (s, 3H), 3.13 (s, 1H), 2.77 (dd, 1H, J = 16.6 and 5.7), 2.70 (dd, 1H, J = 16.7 and 6.9);

13

C NMR (125 MHz,

CDCl3)  163.5, 158.9, 158.5, 158.2, 155.3, 145.0 (3C), 136.2, 130.2 (4C), 128.3 (2C), 127.9 (2C), 126.8, 113.2 (4C), 104.2, 91.1, 86.2, 69.7, 66.1, 55.3 (2C), 41.5, 35.4, 29.8, 25.8; HRMS (ESI) m/z; calcd. for C34H36N4NaO5 [M + Na]+ 603.2583, found 603.2572. 4.1.5. (S)-5-{5-[(2-Cyanoethoxy)(N,N-diisopropylamino)phosphanyloxy]-4-(4,4′-dimethoxytrityloxy)-1-p entynyl}-2-[(N,N-dimethylamino)methylene]amino-N1-methyl-4-pyrimidinone (10). A mixture of 9 (0.10

g,

0.17

mmol),

N,N-diisopropylethylamine

(0.11

mL,

0.63

mmol),

and 3

choro(2-cyanoethoxy)(N,N-diisopropylamino)phosphine (80 μL, 0.36 mmol) in THF (1.0 mL) was stirred at room temperature for 1.0 h. The mixture was partitioned between CHCl3 and aqueous NaHCO3 (saturated). The organic layer was washed with brine, dried (Na2SO4), and concentrated. The residue was purified by column chromatography (a neutralized SiO2, 33% EtOAc in hexane) to give 10 (0.12 g, 89%). 31P NMR (158.6 MHz, CDCl3)  149.57, 149.19.

4.1.6. Oligonucleotide synthesis Synthesis was carried out with a DNA/RNA synthesizer by phosphoramidite method. Deprotection of bases and phosphates was performed in concentrated NH4OH at 55 °C for 12 h. The reaction was quenched with 0.1 M triethylammonium acetate (TEAA) buffer (pH 7.0) and desalted on Sep-Pak C18 cartridge. Deprotected ONs were purified by 20% PAGE containing 7 M urea to give the purified ON 1 (2), ON 2 (2), ON 3 (0.1), ON 4 (3), ON 5 (2), ON 6 (5) and ON 7 (7). The yields are indicated in parentheses as OD units at 260 nm starting from 0.2 µmol scale.

4.1.7. MALDI-TOF/MS analysis of oligonucleotides The Spectra were obtained with a time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). A solution of 3-hydroxypicolinic acid (3-HPA) and diammonium hydrogen citrate in H2O was used as the matrix. Data of synthetic ONs: ON 1 m/z = 4953.6 ([M–H]–, calcd 4950.8; C165H202N48O100P16); ON 2 m/z = 4876.9 ([M–H]–, calcd 4875.7; C166H197N41O101P16); ON 3 m/z = 4950.7 ([M–H]–, calcd 4948.8; C165H204N50O98P16); ON 4 m/z = 4880.3 ([M–H]–, calcd 4873.8; C166H199N43O99P16); ON 5 m/z = 5044.7 ([M–H]–, calcd 5041.8; C165H199N57O98P16); ON 6 m/z = 4959.5 ([M–H]–, calcd 4965.8; C167H165N49O99P16); and ON 7 m/z = 5034.6 ([M–H]–, calcd 5039.8; C165H201N59O96P16).

4.2. Thermal denaturation study The solution containing the duplex in a buffer comprising 10 mM sodium phosphate (pH 7.0) and 100 mM NaCl was heated at 100 °C for 5 min, cooled gradually to an appropriate temperature, and then used for the thermal denaturation study. The thermally induced transition of each mixture was monitored at 260 nm on UV-Vis spectrophotometer fitted with a temperature controller in quartz cuvettes with a path length of 1.0 cm and a 3.0 μM duplex concentration in a buffer of 10 mM sodium phosphate (pH 7.0) and 100 mM NaCl. The sample temperature was increased by 0.5 °C/min. The thermodynamic parameters of the duplexes on duplex formation were determined by calculations based on the slope of a 4

1/Tm vs. ln (CT/4) plot, where CT (3, 6, 9, 12, 15, 18 and 21 μM) is the total concentration of single strands.

4.3. Circular dichroism (CD) spectroscopy The solution containing the duplex in a buffer comprising 10 mM sodium phosphate (pH 7.0), 100mM NaCl and 10 mM MgCl2 was heated at 100 °C for 5 min. The reaction mixture (6 μM) were cooled gradually to an appropriate temperature, and then used for the measurement of CD spectra by a spectropolarimeter.

4.4. Hydrolysis of RNA with E. coli RNase H The solution containing ON (300 pmol) and RNA (1500 pmol) labeled with fluorescein at the 5′-end in a buffer comprised of 50 mM Tris-HCl (pH 8.0), 75 mM KCl, 3 mM MgCl2 and 10mM dithiothreitol was heated at 100 °C for 5 min. The reaction mixture were then cooled gradually to an appropriate temperture. Then, E. coli Rnase H (3.0 unit) was added to the solution, and the mixture was incubated at 37 °C. Aliquots (5 μL) were taken at 0, 1, 3, 5, 10, 15, 30 and 60 min, and mixed with a solution comprised of formamide (14 μL) and 0.1M EDTA (1 μL) on ice. Each sample was analyzed by 20% denaturing PAGE at room temperature at 5 mA for 5h. The gel was visualized by use of a Luminescent Image analyzer LAS-4000 (Fujifilm).

4.5. Partial Hydrolysis of ONs with Snake Venom Phosphodiesterase (SVPD) Each ON (1200 pmol) labeled with fluorescein at the 5′-end was incubated with SVPD (0.006 unit) in a buffer (150 μl) comprised of 250 mM Tris-HCl and 50 mM MgCl2 at 37 °C. At appropriate periods, aliquots (5 μL) of the reaction mixture were separated and added to the loading buffer (15 μL), comprising Tris-borate-EDTA (TBE) buffer and 20% glycerol, on ice. The mixture were analyzed by electrophoresis on 20% polyacrylamide gel containig 7M urea. The labeled ON in the gel was visualized by use of a Luminescent Image analyzer LAS-4000 (Fujifilm).

A. Supplementary data Supplementary data associated with this article can be found in the online version, at http://

References and notes

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1. (a) Zamecnik, P. C.; Stephenson, M. L. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 280−284; (b) Stephenson, M. L.; Zamecnik, P. C. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 285−288. 2. Muthiah, M. Antisense Nucleic Acid Drug. Dev. 2002, 12, 103−128; (b) Aboul-Fadl, T. Curr. Med. Chem. 2005, 12, 2193−2214. 3. (a) Obika, S.; Nanbu, D.; Hari, Y.; Morio, K.; In, Y.; Ishida, T.; Imanishi, T. Tetrahedron Lett. 1997, 38, 8735−8738; (b) Obika, S.; Nanbu, D.; Hari, Y.; Andoh, J.; Morio, K.; Doi, T.; Imanishi, T. Tetrahedron Lett. 1998, 39, 5401−5404. 4. (a) Sing, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J. Chem. Commun. 1998, 455−546; (b) Koshkin, A. A.; Nielsen, P.; Meldgaard, M.; Rajwanshi, V. K.; Sing, S. K.; Wengel, J. J. Am. Chem. Soc. 1998, 120, 13252−13253. 5. (a) Schneider, K. C.; Benner, S. A. J. Am. Chem. Soc. 1990, 112, 453−455; (b) Augustyns, K.; Van Aerschot, A.; Van Schepdeal, A.; Urbanke, C.; Herdewijin, P. Nucleic Acids Res. 1991, 19, 2587−2593; (c) Azymah, K.; Chavis, C.; Lucas, M.; Morvan, F.; Imbach, J.–L. Nucleosides & Nucleotides 1992, 11, 1241−1255; (d) Nielsen, P.; Kirpekar, F.; Wengel, J. Nucleic Acids Res. 1994, 22, 703−710. (e) Obika, S.; Takashima, Y.; Matsumoto, Y.; Shimoyama, A.; Koishihara, Y.; Doi, T.; Imannishi, T. Bioorg. Med. Chem. Lett. 1996, 6, 1357−1360; (f) Peng, L.; Roth, H.-J. Helv. Chim. Acta 1997, 80, 1494−1512; (g) Rana, V. S.; Kumar, V. A.; Ganesh, K. N. Tetrahedron 2001, 57, 1311−1321. 6. (a) Zhang, L.; Peritz, A.; Meggers, E. J. Am. Chem. Soc. 2005, 127, 4174−4175; (b) Zhang, L.; Peritz, A. E.; Carroll, P. J.; Meggers, E. Synthesis 2006, 645−653; (c) Schlegel, M. K.; Peritz, A. E.; Kittigowittana, K.; Zhang, L.; Meggers, E. ChemBioChem 2007, 8, 927−932; (d) Schlegel, M. K.; Xie, X.; Meggers, E. Angew. Chem. Int. Ed. 2009, 48, 960–963. 7. Doi, Y.; Chiba, J.; Morikawa, T.; Inouye, M. J. Am. Chem. Soc. 2008, 130, 8762–8768. 8. (a) Froehler, B. C.; Wadwani, S.; Terhorst, T. J.; Gerrard, S. R. Tetrahedron Lett. 1992, 33, 5307−5310; (b) Sagi, J.; Szemo, A.; Ebinger, K.; Szaboles, A.; Sagi, G.; Ruff, E.; Otvos, L. Tetrahedron Lett. 1993, 34, 2191−2194; (c) Graham, D.; Parkinson, J. A.; Brown, T. J. Chem. Soc, Perkin Trans. 1 1998, 1131−1138. 9. Ogata, A.; Ueno, Y. Bioorg. Med. Chem. Lett. 2015, 25, 2574−2578. 10. Mayer, A.; Haeberli, A.; Leumann, C. J. Org. Biomol. Chem. 2005, 3, 1653−1658.

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Legends

Fig. 1.

Structure of acyclic nucleoside analogs.

Scheme 1.

Synthesis of cytosine derivative 5. Reagents and conditions: (a) N,N–dimethylformamide dimethyl acetal, DMF, 40 °C, 5 h, 66%.

Scheme 2.

Synthesis of the phosphoramidite of cytidine analog 3. Reagents and conditions: (a) CuI, Pd(PPh3)4, Et3N, DMF, 40 °C, 12 h, 67%; (b) TBAF, AcOH, THF, r.t., 24 h, 45%; (c) DMTrCl,

pyridine,

r.t.,

4

h,

69%;

(d)

chloro(2-cyanoethoxy)(N,N-diisopropylamino)phosphine, i-Pr2NEt, THF, r.t., 1 h, 89%. Table 1.

Sequences of DNAs, RNAs, ONs, and duplexes, and Tm values of the duplexes. aF: fluorescein. bΔTm = Tm (DNA/RNA duplex)–Tm (corresponding ON/RNA duplex). Experimental conditions are shown in the Experimental section.

Table 2.

Tm values of duplexes including mismatched base pairs. aUnderlined letters indicate the mismatched bases. bΔTm = Tm (duplex containing a mismatch base pair)–Tm (duplex composed of a matched sequence).

Table 3.

Thermodynamic parameters of the duplexes.

Fig. 2.

CD spectra of the duplexes. Experimental conditions are shown in the Experimental section. Concentration of duplexes = 6 μM.

Fig. 3.

PAGE of DNA and ON hydrolyzed by SVPD. (a) DNA 7. (b) ON 7. F: fluorescein. Experimental conditions are shown in the Experimental section.

Fig. 4.

PAGE of DNA/RNA duplexes hydrolyzed by E. coli RNase H. F: fluorescein. Experimental conditions are shown in the Experimental section.

Table 4.

Initial velocities for the cleavage of RNAs in DNA/RNA duplexes by E. coli RNase H. 7

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Synthesis of antisense oligonucleotides containing acyclic alkynyl nucleoside analogs and their biophysical and biological properties

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Aya Ogata, Yusuke Maeda and Yoshihito Ueno

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