Synthesis of 2′-N-methylamino-2′-deoxyguanosine and 2′- N, N-dimethylamino-2′-deoxyguanosine and their incorporation into RNA by phosphoramidite chemistry

DOI: 10.1021/jo201364x

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Journal of Organic Chemistry p. 8718 - 8725 (2011)

Update date:2022-07-30

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Authors:

Dai, Qing

Sengupta, Raghuvir

Deb, Shirshendu K.

Piccirilli, Joseph A.

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Article abstract of DOI:10.1021/jo201364x

The 2′-hydroxyl groups within RNA contribute in essential ways to RNA structure and function. Previously, we designed an atomic mutation cycle (AMC) that uses ribonucleoside analogues bearing different C-2′-substituents, including -OCH₃, -NH View More

Full text of DOI:10.1021/jo201364x

Article

pubs.acs.org/joc

Synthesis of 2′-N-Methylamino-2′-deoxyguanosine and 2′-N,N-

Dimethylamino-2′-deoxyguanosine and Their Incorporation into

RNA by Phosphoramidite Chemistry

Qing Dai,^†,‡Raghuvir Sengupta,^‡,§Shirshendu K. Deb,^‡and Joseph A. Piccirilli*^,†,‡

^†Department of Biochemistry & Molecular Biology and ^‡Department of Chemistry, The University of Chicago, 929 East 57th Street,

MC 1028, Chicago, Illinois 60637, United States

* Supporting Information

ABSTRACT: The 2′-hydroxyl groups within RNA contribute in essential ways to RNA structure and function. Previously, we

designed an atomic mutation cycle (AMC) that uses ribonucleoside analogues bearing different C-2′-substituents, including

−OCH₃, −NH₂, −NHMe, and −NMe₂, to identify hydroxyl groups within RNA that donate functionally significant hydrogen

bonds. To enable AMC analysis of the nucleophilic guanosine cofactor in the Tetrahymena ribozyme reaction and at other

guanosines whose 2′-hydroxyl groups impart critical functional contributions, we describe here the syntheses of 2′-methylamino-

2′-deoxyguanosine (G_NHMe) and 2′-N,N-dimethylamino-2′-deoxyguanosine (G_NMe) and their corresponding phosphoramidites.

The key step in obtaining the nucleosides involved S_N2 displacement of 2′-β-triflate from an appropriate guanosine derivative by

methylamine or dimethylamine. We readily obtained the G_NMephosphoramidite and incorporated it into RNA. However, the

G_NHMephosphoramidite posed a significantly greater challenge due to lack of a suitable −2′-NHMe protecting group. After

testing several strategies, we established that allyloxycarbonyl (Alloc) provided suitable protection for 2′-N-methylamino group

during the phosphoramidite synthesis and the subsequent RNA synthesis. This work enables AMC analysis of guanosine’s 2′-

hydroxyl group within RNA.

−NHMe. When the energetic penalty for the 2′-OH to 2′-

INTRODUCTION

■

OCH₃substitution (ΔΔG_OH→OCH) exceeds that for the 2′-

Folded RNAs rely upon their 2′-OH groups to confer stability,

often through hydrogen-bond interactions.¹For many catalytic

RNAs, including the group I and II introns, the ribosome, and

the spliceosome, multiple 2′-OH groups on the substrates and

ribozyme make important energetic contributions to function

as 2′-H substitution at these positions results in a substantial

loss of activity.²However, defining the physiochemical role of

these relevant 2′-OH groups poses significant challenges and

frequently requires the design and synthesis of chemically

modified nucleosides in conjunction with their application in

functionally meaningful ways.³

To investigate hydrogen-bond donation by 2′-OH groups

within RNA, we previously developed atomic mutation cycle

(AMC) analysis (Figure 1).⁴This approach requires the

synthesis and functional characterization of three analogues

bearing modifications at the 2′-position: −OCH₃, −NH₂, and

NH₂to 2′-NHCH₃substitution (ΔΔG_{NH →NHCH}), we attribute

the difference to the absence of a hydrogen atom (ΔG_{H removal}

)

on the 2′-OCH₃analogue and infer that the 2′-hydroxyl group

under investigation imparts function by donating a hydrogen

bond. A nucleoside bearing a 2′-N(CH₃)₂substitution enables a

further test of this conclusion, based on the prediction that the

absence of a hydrogen atom on the 2′-amine would engender a

greater energetic penalty than the 2′-NHCH₃derivative.

As part of our effort to conduct AMC analysis on the

guanosine nucleophile 2′-hydroxyl group in the Tetrahymena

ribozyme reaction,⁵we herein describe the syntheses of 2′-N-

methylamino-2′-deoxyguanosine and 2′-N,N-dimethylamino-2′-

Received: June 30, 2011

Published: September 26, 2011

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the presence of the N-methyl group increases the liability of the

trifluoroacetamide derivative.

As an alternative, we turned to our previously reported

strategy to prepare substituted guanosine derivatives by S_N2

reaction from the 2′-O-triflate derivative 1⁷(Scheme 1).

Treatment of 1 with dimethylamine (2 M in THF) or

methylamine (2 M in THF) overnight at 60 °C in a sealed

pressure tube generated the corresponding silyl-protected 2′-

N,N-dimethylamino-2′-deoxyguanosine (2) and 2′-N-methyl-

amino-2′-deoxyguanosine (3) in 55% and 60% yield,

respectively. In both cases, we observed byproduct resulting

from elimination. Treatment of 2 and 3 with ammonium

fluoride in MeOH gave free nucleosides 4 and 5, respectively,

which were purified by reversed-phase column chromatography

eluting with water.

Figure 1. Atomic mutation cycle for analysis of the guanosine

nucleophile 2′-hydroxyl.

2. Synthesis of the Phosphoramidite of 2′-N,N-

Dimethylamino-2′-deoxyguanosine and Its Incorpora-

tion into RNA. We proceeded with synthesis of the

deoxyguanosine (herein referred to as G_NHMeand G_NMe

respectively) and their incorporation into RNA. The availability

of these two novel nucleosides expands the arsenal of modified

nucleosides to better understand the role of the RNA’s 2′-OH

group.

phosphoramidite for G_NMe(4) without protection of the

Scheme 2. Synthesis of the Phosphoramidite of 2′-N,N-

Dimethylamino-2′-deoxyguanosine (9)

RESULT AND DISCUSSION

■

1. Syntheses of 2′-N,N-Dimethylamino-2′-deoxygua-

nosine (4) and 2′-N-Methylamino-2′-deoxyguanosine

(5). Unlike 2′-N-amino-2′-deoxyguanosine (G_NH) and its

phosphoramidites, whose syntheses and incorporation into

oligonucleotides are well documented,⁶there are no reports

describing the syntheses of G_NHMeand G_NMeand their

phosphoramidites. Since G_NHhas been successfully synthesized

from 2′-N-trifluoroacetylamido-2′-deoxyuridine by transglyco-

sylation,^6awe first attempted to synthesize G_NHMein an

analogous manner. Thus, 2′-N-methyl-N-trifluoroacetylamido-

2′-deoxyuridine was synthesized from 2′-amino-2′-deoxyuridine

by trifluoroacetylation followed by methylation with methyl

iodide. However, subsequent transglycosylation with N²-

palmitoylguanine in the presence of BSA and TMSOTf

afforded only a byproduct, 2′-N-methylamino-2′-deoxyuridine,

indicating loss of the 2′-N-trifluoroacetyl (TFA) protecting

group. Premature deacylation from 2′-N-methyl-N-trifluoroace-

tylamido-2′-deoxyuridine but not 2′-N-trifluoroacetylamido-2′-

deoxyuridine under the same reaction conditions suggests that

Key: (a) DMF−DMA, MeOH, 88%; (b) 1.0 M TBAF in THF, 78%;

CH₂Cl₂, 82%.

tertiary amine (Scheme 2). Treatment of 2 with DMF−DMA

in methanol to protect the exocyclic amino group generated

intermediate 6 in 88% yield. Removal of the 3′,5′-silyl

protecting group with TBAF provided intermediate 7 in 78%

yield. 4,4′-Dimethoxyltritylation of 7 gave 8 in 86% yield and

Scheme 1. Syntheses of 2′-N,N-Dimethylamino-2′-deoxyguanosine (4) and 2′-N-Methylamino-2′-deoxyguanosine (5)

Key: (a) 2 M Me₂NH in THF, 60 °C, 55%; (b) 2 M MeNH₂in THF, 60 °C, 60%; (c) 0.5 M NH₄F in MeOH, 60 °C, 74%; (d) 0.5 M NH₄F in

MeOH, 60 °C, 72%.

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subsequent phosphitylation of 8 under standard conditions

produced phosphoramidite 9 in 82% yield. Incorporation of 9

anticipated that the greater stability of the acetamide relative to

the trifluoracetamide could balance the liability conferred by

the presence of the methyl group, allowing the N-

methylacetamide to remain intact during solid-phase synthesis

but undergo postsynthetic deacetylation smoothly. To test this

hypothesis, we treated 3 with excess acetyl anhydride in

pyridine and isolated a bisacetamide I-2 bearing an acetyl group

on both 2′-NHMe and exocyclic amine (Figure 2). TBAF

treatment removed the 3′,5′-silyl protecting group readily while

retaining the acetyl groups to generate II-2. When II-2 was

treated with ammonium hydroxide at 55 °C, the acetyl group

on the exocyclic amino group was readily removed within 2 h,

but the removal of the acetyl group on the 2′-methylamino

required 48 h to reach completion. Subsequent 4,4′-

dimethoxytritylation of the 5′-OH afforded III-1 (Figure 2),

but its polar character and poor solubility in dicholoromethane

made the corresponding phosphoramidite difficult to isolate.

We abandoned this strategy and turned our attention the

phenoxyacetyl group (PhOAc).

into an RNA sequence CUCG_mA (G_m= G_NMe) by solid-phase

synthesis occurred as efficiently as that of the commercial

guanosine phosphoramidite. After standard deprotection and

reversed-phase HPLC purification, the structure of the

oligonucleotide was confirmed by MALDI-TOF MS ([M −

H]⁻= 1554).

3. Synthesis of the Phosphoramidite for 2′-N-Methyl-

N-phenoxyacetylamido-2′-deoxyguanosine and Its In-

corporation into an Oligonucleotide (14). In contrast to

the phosphoramidite 9, the synthesis of a phosphoramidite

suitable for G_NHMeincorporation into RNA presented

significant challenges because of the need to establish an

appropriate protecting group for the 2′-methylamino group (2′-

NHMe). For the synthesis of the phosphoramidite of G_NH

Eckstein et al. protected the 2′-amino group (2′-NH₂) as a

trifluoroacetamide (TFA), which remains stable during

phosphoramidite and oligonucleotide synthesis but releases

the free amine after oligonucleotide synthesis upon exposure to

ethanolic ammonia.⁸Intending to follow the analogous strategy

for G_NHMe, we generated the trifluoroacetamide derivative of 3

(Figure 2) by treatment with 1-(trifluoroacetyl)imidazole⁹in

Two features of PhOAc protection made it seem attractive:

(1) the electron-withdrawing character of the phenoxy group

causes the PhOAc ester to undergo hydrolysis 50 times faster

than the corresponding Ac ester;¹²(2) the large, hydrophobic

phenyl ring could reduce the polarity and thus improve the

solubility of the DMTr derivative in dichloromethane, thereby

facilitating synthesis of the phosphoramidite.

PhOAc has been used to protect the exocyclic amines of

adenosine and guanosine in phosphoramidite chemistry¹³but

not the 2′-amino group. To test the suitability of PhOAc as a

protecting group for the 2′-amine, we first treated 5 with

DMF−DMA in methanol to protect the exocyclic amine with

dimethylaminomethylene group. Subsequent treatment with

phenoxyacetyl chloride in pyridine generated I-3 (Figure 2).

Desilylation with TBAF gave II-3. Unlike the N-methyltri-

fluoroacetamide, II-3, the 2′-N-methylphenoxyacetamide re-

mained intact during this treatment. To test whether the

ethanolic ammonia treatment that follows oligonucleotide

synthesis would remove the PhOAc, we performed a control

reaction by treating II-3 with ethanolic ammonia at 55 °C. Both

dimethylaminomethylene and PhOAc groups were cleanly

removed within 4 h to give 5. Thus, we prepared the

corresponding phosphoramidite IV-1 by treating II-3 with

DMTr-Cl in pyridine to generate III-2 followed by 3′-O-

phosphitylation (Figure 2). Under standard coupling con-

ditions, phosphoramidite IV-1 was incorporated into a RNA

sequence CUCGmA as efficiently as commercial guanosine

phosphoramidite. After ethanolic ammonia (55 °C, 4 h) and

fluoride treatment, we purified the modified oligonucleotide by

HPLC. MALDI-TOF MS analysis ([M − H]⁻= 1674)

indicated that the oligonucleotide retained the PhOAc group,

however.

We suspected that loss of the PhOAc group from the

nucleoside but not the oligonucleotide upon ethanolic

ammonia treatment might reflect participation of the free 3′-

OH (Figure 3). Possibly the 3′-OH group attacks the carbonyl

directly to form a five-membered-ring tetrahedral intermediate.

Subsequent expulsion of the nitrogen would generate the 3′-O-

acetyl ester, which would undergo deacylation. The absence of

a free 3′-OH in the oligonucleotide may allow the PhOAc

group to survive ethanolic ammonia treatment. We also

attempted to remove the PhOAc group using harsher

conditions (stronger base methylamine at 65 °C, 4 h), but

Figure 2. Structures of some intermediates with TFA, Ac, PhOAc, or

Cbz as 2′-NHMe protecting group.

pyridine followed by protection of the exocyclic amino group as

the isobutyrylamide to give I-1 in 75% yield as a 1:5 mixture of

isomers (cis- and trans-2′-deoxy-2′-N-methylamides).¹⁰How-

ever, attempts to remove the silyl protecting group from I-1

with TBAF or ammonium fluoride also removed the TFA

protecting group to yield only byproduct II-1 (Figure 2). In

contrast, the corresponding derivative of G_NH, which lacks the

methyl group, retains the TFA group under the same

desilylation conditions. The greater liability of the N-

methyltrifluoracetamide parallels our observations from the

transglycosylation reaction noted above.

As an alternative protection strategy for the methylamino

group, we considered acetylation. Acetylation is less useful for

protection of 2′-aminonucleosides because postsynthetic

deacetylation from the 2′-amine occurs too slowly.¹¹We

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Figure 3. Possible mechanism for removal of PhOAc from II-3.

Scheme 3. Synthesis of the 2′-N-Methyl-N-allyloxycarbonylamido-2′-deoxyguanosine Phosphoramidite (14)

Key: (a) DMF−DMA, MeOH, 88%; (b) allyloxycarbonyloxybenzotriazolyl, i-Pr₂NEt, THF, 86%; (c) TBAF, THF, 84%; (d) DMTr-Cl, Py, 81%;

(e) (i-Pr)₂NP(Cl)OCH₂CH₂CN, (i-Pr)₂NEt, 1-methylimidazole, CH₂Cl₂, 78%.

MALDI TOF MS indicated that no desired deprotected

oligonucleotide formed.

without affecting Cbz or Alloc groups to give 12 and II-4,

which were converted to the corresponding phosphoramidites

14 and IV-2, respectively, using standard tritylation and

phosphitylation reactions (Scheme 3 and Figure 2).

During solid-phase synthesis of the RNA sequence CUCG_mA

(G_m= G_NHMe), 14 and IV-2 coupled under standard conditions

as efficiently as commercial guanosine phosphoramidite.

However, attempts to remove the Cbz-protecting group from

the oligonucleotide by Pd-C-catalyzed hydrogenation failed

either with the oligonucleotide attached to the CPG or

removed from the CPG (with 2′-TBDMS groups still present

or removed).¹⁷

In contrast to Cbz, removal of Alloc groups on the

nucleobases requires only a solution-phase catalyst. For

example, Hayakawa et al. reported that tris-

(dibenzylideneacetone)dipalladium(0)−chloroform complex

[Pd₂(dba)₃−CHCl₃] catalyzes removal of Alloc protecting

groups from CPG-supported DNA oligonucleotides (32−

60mer).¹⁸We treated 5′-CUCG_mA-CPG similarly followed by

standard ethanolic ammonia and fluoride. MS analysis of the

resulting oligonucleotide showed only partial removal of the

4. Synthesis of 2′-N-Methyl-N-benzyloxycarbonylami-

do- and 2′-N-Methyl-N-alloxycarbonylamido-2′-deoxy-

guanosine Phosphoramidites (IV-2 and 14). Having

encountered problems with each of the amide protecting

groups (TFA, Ac, and PhOAc), we decided to test carbamate

protecting groups for 2′-NHMe, which utilize hydrogenation

for removal. Both benzyloxycarbonyl (Cbz)¹⁴and allylox-

ycarbonyl (Alloc)¹⁵groups have been used for protection of the

exocyclic amines of the nucleobases in phosphoramidites of dA,

dG, and dC. Following DNA synthesis and cleavage of the

oligonucleotide from the solid support by ammonium

hydroxide treatment, the Cbz groups are removed by Pd−C-

catalyzed hydrogenation while Alloc groups are removed using

a soluble palladium catalyst.

Treatment of 3 with DMF−DMA in methanol gave

intermediate 10, which was converted to 11 and I-4 by

treatment with allyloxycarbonyloxybenzotriazolyl¹⁶or benzy-

loxycarbonyl chloride in pyridine, respectively (Scheme 3 and

Figure 2). TBAF treatment removed the silyl protecting groups

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18.64, 18.60, 18.58, 18.4, 18.3, 18.25, 18.24, 14.2, 14.1, 14.0, 13.9.

HRMS: calcd for C₂₃H₄₃N₆O₅Si₂[MH]⁺539.2834, found 539.2811.

2′-N,N-Dimethylamino-2′-deoxyguanosine (4). To a solution

of 2′-dimethylamino-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-

2′-deoxyguanosine (2, 110 mg, 0.2 mmol) in MeOH (8 mL) was

added ammonium fluoride (0.5 M solution in MeOH, 0.1 mL) under

argon. The mixture was heated to 60 °C for 16 h. After removing the

solvent under reduced pressure, the residue was coevaporated with

deionized water (5 × 30 mL). The residue was purified by C18

reversed-phase column chromatography, eluting with water to give 4

Alloc group (∼50%). We tested other conditions and identified

two ways to remove the Alloc group more effectively:

Pd(Ph₃P)₄in the presence of HCO₂H/Et₃N¹⁹or Pd(Ph₃P)₄

in the presence of AcOH and Bu₃SnH.²⁰Both conditions

removed the Alloc group completely to allow access to the

desired oligonucleotide.²¹

SUMMARY AND IMPLICATIONS

We have synthesized two guanosine analogues, G_NHMeand

■

(46 mg, 74%) as a white solid. H NMR (500.1 MHz) (DMSO-d₆) δ:

_NMe, with the key reaction involving S_N2 displacement of

10.75 (br, 1H), 8.08 (s, 1H), 6.60 (br, 2H), 6.05 (d, J = 7.8 Hz, 1H),

5.16 (br, 2H), 4.33 (m, 1H), 3.96 (m, 1H), 3.59 (m, 2H), 3.32 (m,

1H), 2.22 (s, 6H). ¹³C NMR (125.8 MHz) (DMSO-d₆) δ: 157.1,

154.2, 151.4, 135.8, 116.7, 87.0, 84.0, 72.0, 70.4, 62.2, 43.7. HRMS:

calcd for C₁₂H₁₉N₆O₄[MH]⁺311.1468, found 311.1480.

suitably protected 2′-β-triflate derivatives of guanosine with

methylamine and dimethylamine, respectively. The tertiary

amine of G_NMerequired no protection, allowing straightfor-

ward synthesis of the phosphoramidite. The synthesis of the

corresponding G_NHMephosphoramidite proved to be more

challenging because of the need to identify a suitable protecting

group for the 2′-N-methylamino group. After testing several

strategies, we established that the allyloxycarbonyl moiety

provided suitable protection for the 2′-N-methylamino group,

being easily introduced, stable during the phosphoramidite

preparation and subsequent RNA synthesis, and readily

removed from the synthetic oligonucleotide by reduction

2′-N-Methylamino-2′-deoxyguanosine (5). To a solution of 2′-

methylamino-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2′-de-

oxyguanosine (3, 82 mg, 0.17 mmol) in MeOH (8 mL) was added

ammonium fluoride (0.5 M solution in MeOH, 0.1 mL) under argon.

The mixture was heated to 60 °C for 16 h. After removing the solvent

under reduced pressure, the residue was coevaporated with deionized

water (5 × 30 mL). The residue was purified by C18 reversed-phase

column chromatography, eluting with water to give 5 (36.5 mg, 72%)

as a white solid. H NMR (500.1 MHz) (DMSO-d₆) δ: 10.96 (br,

1H), 7.92 (s, 1H), 6.72 (br, 2H), 5.55 (d, J = 7.5 Hz, 1H), 5.52 (br,

1H), 5.12 (br, 1H), 4.38 (m, 1H), 3.91 (m, 1H), 3.54 (m, 2H), 3.46

(m, 1H), 2.24 (s, 3H). ¹³C NMR (125.8 MHz) (DMSO-d₆) δ: 157.2,

154.4, 151.7, 135.8, 117.0, 86.9, 85.9, 68.8, 66.1, 62.0, 34.9. HRMS:

calcd for C₁₁H₁₇N₆O₄, [MH]⁺297.1311, found 297.1302.

using a soluble palladium catalyst. Both G_NMeand G_NHMe

phosphoramidites enabled successful synthesis of oligonucleo-

tides containing these analogues.

The availability of G_NHMeand G_NMe, in conjunction with the

other guanosine analogues required for AMC analysis (Figure

1), permits further investigation of functionally important

guanosine 2′-hydroxyl groups within structured RNAs. Addi-

2′-Dimethylamino-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-

1,3-diyl)-N²-dimethylaminomethylene-2′-deoxyguanosine

(6). To a solution of 2′-dimethylamino-3′,5′-O-(1,1,3,3-tetraisopropyl-

disiloxane-1,3-diyl)-2′-deoxyguanosine (2, 242 mg, 0.44 mmol) in

methanol (10 mL) was added DMF-DMA (1 mL). After stirring at rt

for 3 h, all the volatile components were removed under reduced

pressure. The residue was purified by silica gel chromatography,

eluting with 3−5% methanol in dichloromethane, to give 6 (234 mg,

88%) as white solid. ¹H NMR (500.1 MHz) (CDCl₃) δ: 10.27 (s, 1H),

8.48 (s, 1H), 7.83 (s, 1H), 6.17 (s, 1H), 4.74 (m, 1H), 4.23 (m, 1H),

4.06 (s, 1H), 3.39 (m, 1H), 3.17 (s, 3H), 3.05 (s, 3H), 2.65 (s, 6H),

1.06 (m, 28H). ¹³C NMR (125.8 MHz) (CDCl₃) δ: 158.2, 158.0,

156.9, 149.6, 135.4, 120.2, 84.9, 83.5, 72.4, 70.3, 61.9, 43.3, 41.4, 35.0,

17.4, 17.29, 17.26, 17.22, 17.0, 16.94, 16.88, 13.3, 13.02, 12.96, 12.5.

HRMS: calcd for C₂₇H₅₀N₇O₅Si₂[MH]⁺608.3412, found 608.3414.

2′-Dimethylamino-N²-dimethylaminomethylene-2′-deoxy-

guanosine (7). To a solution of 2′-dimethylamino-3′,5′-O-(1,1,3,3-

tetraisopropyldisiloxane-1,3-diyl)-N²-dimethylaminomethylene-2′-de-

oxyguanosine (6) (220 mg, 0.36 mmol) in THF (10 mL) was added

TBAF (1 M, 72 μL), and the mixture was stirred at rt for 15 min. All

the volatile components were removed under reduced pressure. The

residue was purified by silica gel chromatography, eluting with 0−12%

methanol in dichloromethane, to give 7 (103 mg, 78%) as white solid.

¹H NMR (500.1 MHz) (MeOD) δ: 8.65 (s, 1H), 8.22 (s, 1H), 6.26

tionally, G_NHMeand G_NMemay serve as useful analogues to

define the functional contribution of hydroxyl groups by

application of Quantitative Structure Activity Relationship

(QSAR) analysis^2gor to evaluate the packing density around

individual hydroxyl groups.^4a,22

EXPERIMENTAL SECTION

■

2′-Dimethylamino-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-

1,3-diyl)-2′-deoxyguanosine (2). To a pressure tube (35 mL)

under argon was added 2′-deoxy-2′-β-triflate-3′,5′-O-(1,1,3,3-tetraiso-

propyldisiloxane-1,3-diyl)guanosine¹¹(1, 623 mg, 0.95 mmol) and

dimethylamine in THF (2 M, 20 mL). The mixture was stirred

overnight at 60 °C. After being cooled to rt, the reaction mixture was

concentrated to dryness under reduced pressure. The residue was

purified by silica gel chromatography, eluting with 5−7% methanol in

dichloromethane, to give 2 (287 mg, 55%) as a white solid. H NMR

(500.1 MHz) (DMSO-d₆) δ: 10.58 (br 1H), 7.78 (s, 1H), 6.38 (br,

2H), 5.84 (d, J = 6.0 Hz, 1H), 4.56 (m, 1H), 3.90 (m, 1H), 3.80 (m,

2H), 3.54 (m, 1H), 2.35 (s, 6H), 0.85−0.97 (m, 28H). ¹³C NMR

(125.8 MHz) (DMSO-d₆) δ: 158.1, 155.3, 152.3, 136.5, 118.0, 85.6,

83.8, 75.0, 69.4, 64.5, 43.9, 18.8, 18.67, 18.65, 18.61, 18.43, 18.41,

18.35, 18.34. HRMS: calcd for C₂₄H₄₅N₆O₅Si₂[MH]⁺553.2990,

found 553.2967.

2′-Methylamino-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-

1,3-diyl)-2′-deoxyguanosine (3). To a pressure tube (35 mL)

under argon was added 2′-deoxy-2′-β-triflate-3′,5′-O-(1,1,3,3-tetraiso-

propyldisiloxane-1,3-diyl)-guanosine (1, 580 mg, 0.88 mmol) and

methylamine in THF (2 M, 18 mL). The mixture was stirred overnight

at 60 °C. After cooling to rt, the reaction mixture was concentrated to

dryness under reduced pressure. The residue was purified by silica gel

chromatography, eluting with 5−8% methanol in dichloromethane, to

give 3 (284 mg, 60%) as white solid. ¹H NMR (500.1 MHz) (DMSO-

d₆) δ: 10.55 (br 1H), 7.71 (s, 1H), 6.34 (br, 2H), 5.52 (d, J = 3.5 Hz,

1H), 4.44 (m, 1H), 3.90 (m, 1H), 3.81 (m, 1H), 3.26 (m, 2H), 2.30 (s,

3H), 0.90−0.99 (m, 28H). ¹³C NMR (125.8 MHz) (DMSO-d₆) δ:

158.1, 155.2, 152.2, 135.9, 118.1, 87.2, 84.2, 71.2, 67.3, 63.2, 36.2, 18.8,

(d, J = 8.5 Hz, 1H), 4.42 (d, J = 5.0 Hz, 1H), 4.10 (t, J = 5.0 Hz, 1H),

3.73 (m, 2H), 3.44 (m, 1H), 3.19 (s, 3H), 3.10 (s, 3H), 2.21 (s, 6H).

¹³C NMR (125.8 MHz) (MeOD) δ: 158.6, 158.4, 157.9, 150.4, 137.5,

119.0, 87.2, 85.5, 72.2, 71.5, 62.2, 42.9, 40.0, 33.8. HRMS: calcd for

C₁₅H₂₄N₇O₄, [MH]⁺366.1890, found 366.1899.

2′-Dimethylamino-5′-O-4,4′-dimethoxytrityl-N²-dimethyla-

minomethylene-2′-deoxyguanosine (8). 2′-Dimethylamino-N²-

dimethylaminomethylene-2′-deoxyguanosine (7, 88 mg, 0.24 mmol)

was dissolved in pyridine (3 mL), and 4,4′-dimethoxytrityl chloride (98

mg, 0.29 mmol, 1.2 equiv) was added while stirring the solution. After

being stirred overnight at rt, the reaction mixture was diluted with

MeOH (1 mL) and stirred for an additional 5 min. The reaction

mixture was concentrated to dryness under vacuum. Water was added

to the resulting residue, and the mixture was extracted with CH₂Cl₂.

The organic phase was washed consecutively with 5% sodium

bicarbonate, water, and brine and then dried over sodium sulfate.

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After the organic phase was concentrated to dryness, the residue was

purified by silica gel chromatography, eluting with 4% MeOH in

CH₂Cl₂containing 0.2% Et₃N, to give 8 (143 mg, 86%) as white foam.

¹H NMR (500 MHz) (CD₃CN) δ: 10.05 (br, 1H), 8.65 (s, 1H), 7.76

removed under reduced pressure. The residue was purified by silica gel

chromatography, eluting with 0−10% methanol in dichloromethane,

to give 12 (112 mg, 92%) as a white solid. H NMR (500.1 MHz)

(DMSO-d₆) δ: 11.35 (br, 1H), 8.55 (s, 1H), 8.03 (s, 1H), 6.31 (m,

1H), 5.85 (br, 1H), 5.78 (br, 1H), 5.20 (m, 1H), 5.14 (m, 2H), 4.44

(m, 2H), 4.34 (m, 1H), 3.97 (m, 1H), 3.62 (m, 2H), 3.34 (m, 1H),

3.14 (s, 3H), 3.01 (s, 6H). ¹³C NMR (125.8 MHz) (DMSO-d₆) δ:

158.6, 158.5, 157.9, 157.8, 150.1, 149.5, 137.2, 133.5, 120.4, 120.2,

117.3, 116.4, 88.0, 87.5, 87.0, 82.7, 77.1, 71.1, 66.6, 65.9, 62.1, 61.6,

61.2, 49.0, 41.1, 35.0, 32.9, 30.2. ¹³C NMR (125.8 MHz) (CDCl₃) δ:

158.4, 158.0, 157.9, 157.0, 156.5, 156.0, 150.0, 135.8, 134.9, 132.6,

132.2, 120.8, 120.6, 118.1, 117.5, 85.8, 85.2, 83.7, 72.6, 71.9, 66.5, 66.4,

64.6, 64.0, 62.3, 61.1, 41.3, 35.1, 33.2, 32.7, 17.4, 17.3, 17.23, 17.21,

16.90, 16.86, 16.78, 13.21, 13.16, 12.8, 12.5. HRMS: calcd for

C₁₈H₂₆N₇O₆[MH]⁺436.1945, found 436.1940.

(s, 1H), 7.51 (d, J = 7.5 Hz, 2H), 7.31−7.46 (m, 7H), 6.91 (m, 4H),

6.27 (d, J = 8.5 Hz, 1H), 4.38 (d, J = 5.0 Hz, 1H), 4.21 (m, 1H), 3.82

(s, 6H), 3.46 (m, 2H), 3.28 (m, 1H), 3.13 (s, 3H), 3.08 (s, 3H), 2.26

(s, 6H). ¹³C NMR (125.8 MHz) (CD₃CN) δ: 158.6, 158.4, 157.9,

157.6, 150.3, 144.9, 135.9, 135.7, 135.6, 130.0, 127.9, 127.8, 126.8,

119.8, 113.0, 86.3, 84.8, 84.2, 71.7, 71.0, 64.4, 54.8, 43.1, 40.6, 34.3.

HRMS: calcd for C₃₆H₄₁N₇O₆Na [MNa]⁺690.3011, found 690.3022.

2′-Dimethylamino-5′-O-4,4′-dimethoxytrityl-N²-dimethyla-

minomethylene-2′-deoxyguanosine 3′-O-(2-Cyanoethyl-N,N-

diisopropyl)phosphoramidite (9). 2′-Dimethylamino-5′-O-4,4′-di-

methoxytrityl-N²-dimethylaminomethylene-2′-deoxyguanosine (8)

(120 mg, 0.17 mmol) was dissolved in dry CH₂Cl₂(5 mL) and 1-

methylimidozale (2.90 mg, 35.0 μmol). N,N-Diisopropylethylamine

(126 mg, 0.68 mmol) was added to the stirring solution followed by 2-

cyanoethyl N,N-(diisopropylchloro)phosphoramidite (2.0 equiv).

After being stirred at room temperature for 1 h, the reaction mixture

was diluted with CH₂Cl₂(50 mL) and washed with 5% NaHCO₃and

brine. After dried over sodium sulfate and filtered, the filtrate was

concentrated to dryness. The residue was purified by silica gel

chromatography, eluting with 12% acetone in CH₂Cl₂containing 0.2%

Et₃N, to give 9 (115 mg, 78%) as a colorless oil. ¹H NMR (500 MHz)

(CD₃CN) δ: 10.03 (br, 1H), 8.62 (m, 1H), 7.78 (m, 1H), 7.50 (m,

2H), 7.35−7.40 (m, 7H), 6.91 (m, 4H), 6.36 (m, 1H), 4.92 (m, 1H),

4.01−4.42 (m, 2H), 3.82 (m, 6H), 3.55 (m, 2H), 3.50 (m, 1H), 3.24

(m, 1H), 3.09−3.06 (m, 6H), 2.78 (m, 1H), 2.72 (m, 1H), 2.32 (m,

6H), 1.30 (m, 12H). ³¹P NMR (202.4 MHz) (CD₃CN) δ: 141.3,

141.5. HRMS: calcd for C₄₅H₅₉N₉O₇P [MH]⁺868.4275, found

868.4268.

2′-N-Methyl-N-alloxycarbonyl-N²-dimethylaminomethy-

lene-5′-O-4,4′-dimethoxytrityl-2′-deoxyguanosine (13). 2′-N-

Methyl-N-alloxycarbonyl-N²-dimethylaminomethylene-2′-deoxyguano-

sine (12) (100 mg, 0.23 mmol) was coevaporated with anhydrous

pyridine (10 mL × 20) and then dissolved in pyridine (8 mL), and

4,4′-dimethoxytrityl chloride (116 mg, 0.34 mmol, 1.5 equiv) was

added. After being stirred overnight at room temperature, the reaction

mixture was diluted with MeOH (1 mL) and stirred for an additional 5

min. The reaction mixture was concentrated to dryness under vacuum

and coevaporated with toluene (20 mL × 2). Water was added to the

resulting residue, and the mixture was extracted with CH₂Cl₂. The

organic phase was washed consecutively with 5% sodium bicarbonate,

water, and brine and then dried over sodium sulfate. After the organic

phase was concentrated to dryness, the residue was purified by silica

gel chromatography, eluting with 0−3% MeOH in CH₂Cl₂containing

0.2% Et₃N, to give 13 (137 mg, 81%) as white foam. ¹H NMR (500.1

MHz) (CD₃CN) δ: ¹³C NMR (125.8 MHz) (CD₃CN) δ: 10.06 (br,

1H), 8.57 (s, 1H), 7.73 (m, 1H), 7.42 (m, 2H), 7.15−7.30 (m, 7H),

6.79 (m, 4H), 6.41 (m, 1H), 5.85 (br, 1H), 4.955.30 (m, 3H), 4.53 (m,

2H), 4.44 (m, 1H), 4.20 (m, 1H), 3.72 (s, 6H), 3.35 (m, 1H), 3.27 (m,

1H), 3.08 (s, 3H), 3.00 (s, 3H), 2.96 (s, 3H). ¹³C NMR (125.8 MHz)

(CD₃CN) δ: 158.6, 158.5, 158.3, 157.9, 157.4, 150.4, 144.9, 135.70,

135.67, 133.2, 130.0, 129.9, 127.9, 127.8, 126.8, 120.1, 116.4, 113.0,

86.2, 85.5, 83.1, 65.9, 64.2, 54.8, 40.6, 34.2, 32.7. HRMS: calcd for

C₃₉H₄₄N₇O₈[MH]⁺738.3251, found 738.3232.

2′-Methylamino-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-

1,3-diyl)-N²-dimethylaminomethylene-2′-deoxyguanosine

(10). To a solution of 2′-methylamino-3′,5′-O-(1,1,3,3-tetraisopropyl-

disiloxane-1,3-diyl)-2′-deoxyguanosine (3, 264 mg, 0.50 mmol) in

methanol (10 mL) was added DMF−DMA (1 mL). After being stirred

overnight at rt, all the volatile components were removed under

reduced pressure. The residue was purified by silica gel chromatog-

raphy, eluting with 5% methanol in dichloromethane, to give 10 (234

mg, 88%) as white solid. ¹H NMR (500.1 MHz) (CDCl₃) δ: 9.94 (br,

1H), 8.63 (s, 1H), 7.94 (s, 1H), 6.02 (s, 1H), 4.65 (m, 2H), 4.20 (m,

1H), 4.08 (m, 1H), 3.23 (r. 1H), 3.20 (s, 3H), 3.14 (s, 3H), 2.70 (s,

3H), 1.02−1.16 (m, 28H). ¹³C NMR (125.8 MHz) (CDCl₃) δ: 158.0,

158.0, 156.7, 149.2, 135.5, 120.6, 86.9, 82.3, 68.7, 67.8, 60.6, 41.4, 35.1,

17.4, 17.3, 17.2, 17.0, 16.95, 16.88, 16.8, 13.3, 12.91, 12.86, 12.47.

HRMS: calcd for C₂₆H₄₇N₇O₅Si₂[MH]⁺594.3272, found 594.3256.

2′-N-Methyl-N-alloxycarbonyl-3′,5′-O-(1,1,3,3-tetraisopro-

pyldisiloxane-1,3-diyl)-N²-dimethylaminomethylene-2′-deox-

yguanosine (11). To a solution of 2′-methylamino-3′,5′-O-(1,1,3,3-

tetraisopropyldisiloxane-1,3-diyl)-N²-dimethylaminomethylene-2′-de-

oxyguanosine (10, 220 mg, 0.37 mmol) in THF (10 mL) was added

allyloxycarbonyloxybenzotriazolyl (122 mg, 1.5 equiv) and N,N-

diisopropylethylamine (0.2 mL). The mixture was stirred at rt for 1

h, and MeOH (0.5 mL) was added to quench the reaction. All of the

volatile components were removed under reduced pressure. The

residue was purified by silica gel chromatography, eluting with 4%

2′-N-Methyl-N-alloxycarbonyl-N²-dimethylaminomethy-

lene-5′-O-4,4′-dimethoxytrityl-2′-deoxyguanosine (2-Cya-

noethyl-N,N-diisopropyl)phosphoramidite (14). 2′-N-Methyl-N-

alloxycarbonyl-N²-dimethylaminomethylene-5′-O-4,4′-dimethoxytrityl-

2′-deoxyguanosine (13, 120 mg, 0.16 mmol) was dissolved in dry

CH₂Cl₂(5 mL) and 1-methylimidazole (2.90 mg, 35.0 μmol). N,N-

Diisopropylethylamine (120 mg, 0.64 mmol) was added to the stirring

solution followed by 2-cyanoethyl N,N-(diisopropylchloro)-

phosphoramidite (2.0 equiv). After being stirred at room temperature

for 1 h, the reaction mixture was diluted with dichloromethane (50

mL), and the mixture was washed with 5% aqueous sodium carbonate

and brine, dried over sodium sulfate, and concentrated. The residue

was purified by silica gel chromatography, eluting with 12% acetone in

CH₂Cl₂containing 0.2% Et₃N, to give 14 (119 mg, 78%) as a colorless

oil. ³¹P NMR (202.4 MHz) (CD₃CN) δ: 149.95 and 149.66, 149.34.

HRMS: calcd for C₄₈H₆₀N₉O₉P, [M]⁺937.4252, found 937.4288.

2′-N-Methyl-N-phenoxyacetyl-3′,5′-O-(1,1,3,3-tetraisopro-

pyldisiloxane-1,3-diyl)-N²-dimethylaminomethylene-2′-deox-

yguanosine (I-3). To a solution of 2′-methylamino-3′,5′-O-(1,1,3,3-

tetraisopropyldisiloxane-1,3-diyl)-N²-dimethylaminomethylene-2′-de-

oxyguanosine (10, 178 mg, 0.30 mmol) in CH₂Cl₂(10 mL) and

pyridine (10 mL) at 0 °C under argon were added phenoxyacetyl

chloride (47 μL, 1.1 equiv) and N,N-diisopropylethylamine (0.2 mL).

The mixture was stirred at 0 °C for 1 h, and MeOH (0.5 mL) was

added to quench the reaction. All of the volatile components were

removed under reduced pressure. The residue was coevaporated with

toluene. CH₂Cl₂was added to the resulting residue, and the mixture

was washed consecutively with 5% sodium bicarbonate, water, and

brine and then dried over sodium sulfate. After the organic phase was

concentrated to dryness, the residue was purified by silica gel

methanol in dichloromethane, to give 11 (216 mg, 86%). H NMR

(500.1 MHz) (CDCl₃) δ: 9.75 (br, 1H), 8.61 (s, 0.75H), 8.50 (s,

0.25H), 8.04 (s, 1H), 6.16 (m, 1H), 5.86 (m, 0.75H), 5.75 (m, 0.25H),

5.42 (m, 0.75H), 5.26 (m, 0.25H), 5.15 (m, 2H), 4.62 (m, 2H), 4.57

(m, 1H), 4.09 (m, 2H), 3.82 (m, 0.25H), 3.80 (m, 0.75), 3.17 (s,

2.25H), 3.12 (s, 0.75H), 3.07 (s, 6H), 0.93−1.03 (m, 28H). HRMS:

calcd for C₃₀H₅₁N₇O₇Si₂[MH]⁺678.3467, found 678.3458.

2′-N-Methyl-N-alloxycarbonyl-N²-dimethylaminomethy-

lene-2′-deoxyguanosine (12). To a solution of 2′-N-methyl-N-

alloxycarbonyl-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-N²-di-

methylaminomethylene-2′-deoxyguanosine (11) (190 mg, 0.28 mmol)

in THF (10 mL) was added TBAF (1 M, 0.28 mL), and the mixture

was stirred at rt for 15 min. All of the volatile components were

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chromatography, eluting with 0−4% MeOH in CH₂Cl₂, to give I-3

δ: 152.2, 151.1, 150.51, and 150.46. HRMS: calcd for C₅₂H₆₂N₉O₉P

[MNa]⁺1010.4300, found 1010.4344.

(164 mg, 75%) as a white foam. H NMR (500.1 MHz) (CDCl₃) δ:

2′-N-Methyl-N-benzyloxycarbonyl-3′,5′-O-(1,1,3,3-tetraiso-

propyldisiloxane-1,3-diyl)-N²-dimethylaminomethylene-2′-

deoxyguanosine (I-4). To a solution of 2′-methylamino-3′,5′-O-

(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-N²-dimethylaminomethy-

lene-2′-deoxyguanosine (10, 210 mg, 0.35 mmol) in anhydrous THF

(10 mL) under argon were added N,N-diisopropylethylamine (122 μL,

2.0 equiv) and benzyl chloroformate (68 μL, 1.2 equiv). The mixture

was stirred at rt for 2 h and MeOH (0.5 mL) was added to quench the

reaction. All the volatile components were removed under reduced

pressure. CH₂Cl₂(50 mL) was added to the resulting residue, and the

mixture was washed consecutively with 5% sodium bicarbonate, water,

brine, and then dried over sodium sulfate. After the organic phase was

concentrated to dryness, the residue was purified by silica gel

chromatography, eluting with 0−4% MeOH in CH₂Cl₂, to give I-4

9.86 (br, 1H), 8.67 (s, 0.75H), 8.40 (s, 0.25H), 7.78 (s, 0.25H), 7.76

(s, 0.75H), 7.30 (m, 2H), 7.25 (m, 1H), 7.00 (m, 1.5H), 6.86 (m,

0.5H), 6.20 (m, 1H), 6.05 (m, 0.75H), 5.25 (m, 0.25H), 4.74−4.80

(m, 3H), 4.25 (m, 1H), 4.18 (m, 1H), 4.05 (m, 0.25H), 3.90 (m,

0.75H), 3.08−3.30 (m, 9H), 1.01−1.06 (m, 28H). ¹³C NMR (125.8

MHz) (CDCl₃) δ: 158.8, 157.9, 157.8, 157.6, 157.4, 157.0, 149.9,

149.6, 136.0, 135.9, 129.6, 129.4, 121.7, 121.5, 120.8, 114.3, 114.0,

86.3, 85.8, 84.6, 84.0, 72.5, 72.3, 68.0, 66.9, 64.5, 63.9, 62.3, 58.8, 41.3,

35.1, 33.0, 32.1, 17.4, 17.3, 17.2, 17.1, 16.97, 16.95, 16.8, 13.21, 13.19,

12.8, 12.5, 12.4. HRMS: calcd for C₃₄H₅₃N₇O₇Si₂[MH]⁺728.3618,

found 728.3584.

2′-N-Methyl-N-phenoxyacetyl-N²-dimethylaminomethy-

lene-2′-deoxyguanosine (II-3). To a solution of 2′-N-methyl-N-

phenoxyacetyl-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-N²-di-

methylaminomethylene-2′-deoxyguanosine (I-3) (150 mg, 0.21 mmol)

in THF (10 mL) was added TBAF in THF (1.0 M, 0.21 mL), and the

mixture was stirred at rt for 15 min. Silica gel (1.0 g) was added, and

the volatile components were removed under reduced pressure. The

residue was purified by silica gel chromatography, eluting with 0−9%

methanol in dichloromethane, to give II-3 (95 mg, 95%) as a white

(216 mg, 85%) as a white foam. H NMR (500.1 MHz) (CD₃CN) δ:

9.95 (br, 1H), 8.68 (s, 0.63H), 8.35 (s, 0.37H), 7.73 (s, 0.63H), 7.68

(s, 0.37H), 7.25 (m, 5H), 7.01 (s, 0.37H), 6.12 (d, J = 5.5 Hz, 0.63H),

5.56 (m, 1H), 5.05 (m, 2H), 4.61 (m, 1H), 4.06 (s, 1H), 3.84 (m, 2H),

3.08−3.17 (m, 9H), 0.89−1.03 (m, 28H). ¹³C NMR (125.8 MHz)

(CDCl₃) δ: 158.0, 157.8, 156.9, 156.3, 149.7, 136.4, 136.0, 128.6,

128.4, 128.2, 127.9, 127.8, 121.1, 98.5, 86.1, 85.3, 84.4, 72.8, 72.1, 67.8,

67.6, 67.4, 64.8,64.1, 61.2, 41.8, 35.8, 33.3, 32.9, 23.6, 17.6, 17.44,

17.40, 17.08, 17.04, 16.9, 13.3, 13.0, 12.6. HRMS: calcd for

C₃₄H₅₃N₇O₇Si₂[MH]⁺728.3623, found 728.3609.

solid. H NMR (500.1 MHz) (CD₃OD) δ: 8.59 (s, 0.75H), 8.40 (s,

0.25H), 8.20 (s, 0.25H), 8.18 (s, 0.75H), 7.16−7.22 (m, 2H), 6.91 (m,

1H), 6.79 (d, J = 8.0 Hz, 1.5H), 6.73 (d, J = 8.5 Hz, 0.5H), 5.75 (m,

0.75H), 5.25 (m, 0.25), 4.85 (m, 2H), 4.75 (m, 1H), 4.62 (m, 1H),

4.22 (m, 1H), 3.83 (m, 2H), 2.98−3.30 (m, 9H). ¹³C NMR (125.8

MHz) (CD₃OD) δ: 170.6, 169.8, 158.6, 158.1, 157.7, 157.6, 150.3,

150.0, 137.8, 129.1, 128.9, 121.1, 120.9, 119.8, 119.5, 114.1, 114.0,

113.8, 87.6, 87.4, 85.0, 83.4, 71.0, 66.6, 65.9, 64.4, 62.5, 61.9, 61.5,

60.0, 58.0, 40.0, 33.81, 33.75, 32.3, 31.2. HRMS: calcd for C₂₂H₂₇N₇O₆

[MH]⁺486.2101, found 486.2092.

2′-N-Methyl-N-benzyloxycarbonyl-N ²-dimethylamino-

methylene-2′-deoxyguanosine (II-4). To a solution of 2′-N-

methyl-N-benzyloxycarbonyl-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-

1,3-diyl)-N²-dimethylaminomethylene-2′-deoxyguanosine (I-4) (198

mg, 0.27 mmol) in THF (10 mL) was added TBAF in THF (1 M,

0.27 mL), and the mixture was stirred at rt for 10 min. Silica gel (1.0 g)

was added, and volatile components were removed under reduced

pressure. The residue was purified by silica gel chromatography,

eluting with 0−8% MeOH in CH₂Cl₂, to give II-4 (126 mg, 96%) as a

2′-N-Methyl-N-phenoxyacetyl-N²-dimethylaminomethy-

lene-5′-O-4,4′-dimethoxytrityl-2′-deoxyguanosine (III-2). 2′-N-

Methyl-N-phenoxyacetyl-N²-dimethylaminomethylene-2′-deoxyguano-

sine (II-3) (95 mg, 0.20 mmol) was coevaporated with anhydrous

pyridine (10 mL × 20) and then dissolved in pyridine (8 mL), and

4,4′-dimethoxytrityl chloride (99 mg, 0.29 mmol, 1.5 equiv) was