Ethynyl side chain hydration during synthesis and workup of "clickable" oligonucleotides: Bypassing acetyl group formation by triisopropylsilyl protection

Article abstract of DOI:10.1021/jo401780u

Clickable oligonucleotides with ethynyl residues in the 5-position of pyrimidines (^ethdC and ^ethdU) or the 7-position of 7-deazaguanine (^ethc⁷G_d) are hydrated during solid-phase oligonucleotide synthesis and workup conditions. The side products were identified as acetyl derivatives by MALDI-TOF mass spectra of oligonucleotides and by detection of modified nucleosides after enzymatic phosphodiester hydrolysis. Ethynyl → acetyl group conversion was also studied on ethynylated nucleosides under acidic and basic conditions. It could be shown that side chain conversion depends on the nucleobase structure. Triisopropylsilyl residues were introduced to protect ethynyl residues from hydration. Pure, acetyl group free oligonucleotides were isolated after desilylation in all cases.

Full text of DOI:10.1021/jo401780u

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Article
Ethynyl Side Chain Hydration During Synthesis and
Work-up of “Clickable” Oligonucleotides: Bypassing
Acetyl Group Formation by Triisopropylsilyl Protection
Sachin Asaram Ingale, Hui Mei, Peter Leonard, and Frank Seela
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 18 Oct 2013
Downloaded from http://pubs.acs.org on October 26, 2013
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Ethynyl Side Chain Hydration During Synthesis and Work-up of “Clickable”
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Oligonucleotides: Bypassing Acetyl Group Formation by Triisopropylsilyl Protection
Sachin A. Ingale,†‡ Hui Mei,†‡ Peter Leonard† and Frank Seela*†‡
†Laboratory of Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology,
Heisenbergstraße 11, 48149 Münster, Germany and ‡Laboratorium für Organische und
Bioorganische Chemie, Institut für Chemie neuer Materialien, Universität Osnabrück,
Barbarastraße 7, 49069 Osnabrück, Germany
Corresponding author:
Prof. Dr. Frank Seela
Phone: +49 (0) 251 53406 500; Fax: +49 (0) 251 53406 857
E-mail: Frank.Seela@uni-osnabrueck.de
Homepage: www.seela.net
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ABSTRACT
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Clickable oligonucleotides with ethynyl residues in the 5-position of pyrimidines (ethdC and
ethdU) or the 7-position of 7-deazaguanine (ethc7Gd) are hydrated during solid-phase
oligonucleotide synthesis and work-up conditions. The side products were identified as acetyl
derivatives by MALDI-TOF mass spectra of oligonucleotides and by detection of modified
nucleosides after enzymatic phosphodiester hydrolysis. Ethynyl → acetyl group conversion
was also studied on ethynylated nucleosides under acidic and basic conditions. It could be
shown that side chain conversion depends on the nucleobase structure. Triisopropylsilyl
residues were introduced to protect ethynyl residues from hydration. Pure, acetyl group free
oligonucleotides were isolated after desilylation in all cases.
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INTRODUCTION
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Alkynyl groups are currently used in nucleoside and oligonucleotide synthesis to make these
compounds applicable for functionalization with different reporter groups by the copper
promoted Huisgen-Meldal-Sharpless click chemistry.1,2 As ethynyl groups are less space
demanding than octadiynyl residues, they have been preferred over long side chain derivatives
in enzyme assisted DNA synthesis catalyzed by polymerases.3 Compounds 2-4 (Figure 1)
were successfully incorporated into DNA in form of their triphosphates by a template
controlled polymerase chain reaction. By this means, the intracellular detection of cytosine
was studied with the triphosphate of 5-ethynyl-2’-deoxycytidine (ethdC) in genomic DNA.4
The 7-ethynyl-7-deaza-2’-deoxyguanosine 1 (ethc7Gd) and 7-ethynyl-7-deaza-2’-
deoxyadenosine 4 (ethc7Ad) were used for the metabolic labelling of DNA in vivo.5a The
triphosphate of 5-ethynyl-2’-deoxyuridine 3 (ethdU) has found application in place of 5-
bromo-2’-deoxyuridine in a cell proliferation assay.5b
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The situation is different when phosphoramidite chemistry6 is employed for the synthesis of
oligonucleotides with alkynyl side chains on the nucleobases. While those with octadiynyl
residues are commonly in use,7 we noticed serious problems when phosphoramidites of
ethynyl nucleosides were utilized.8 Side products were formed, and they often became the
major components instead of the target oligonucleotides. As chromatographic mobility of
contaminated oligonucleotides is often the same as that of the ethynylated oligonucleotides,
formation of the former can be easily overseen, and clean oligonucleotides are not accessible.
We were able to circumvent this problem partially for the synthesis of 5-ethynyl-dU
oligonucleotides, when short deprotection times were used.8 Therefore, for functionalization
of ethynyl side chains, the click reaction was performed on solid support or already
functionalized phosphoramidites were used.9 Thus, either ethdU oligonucleotides could not be
obtained efficiently or were isolated after cumbersome purification in only moderate yield.
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In this situation, we started a careful inspection of the side products formed during solid-phase
synthesis and the work-up procedure using oligonucleotide deprotection in conc. aq.
ammonia. This manuscript reports on the incorporation of nucleosides 1-4 (Figure 1) into
oligonucleotides by using their phosphoramidites (9, 10, 13 and 14), evaluates the structures
of the major side products and provides a protocol to circumvent these difficulties with
phosphoramidites that are protected with triisopropylsilyl (TIPS) groups on the ethynyl side
chains.
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Figure 1. Structures of 7-ethynyl-7-deazapurine and 5-ethynylpyrimidine nucleosides.
RESULTS AND DISCUSSION
1. Synthesis of Phosphoramidites 9 and 13 with Ethynyl Side Chains. For this study, four
different phosphoramidites based on the structures 1-4 were prepared. The building blocks 108
and 1410 are known, while the syntheses of phosphoramidites 9 and 13 are described below.
Recently, a two-step synthesis without isolation of the trimethylsilyl (TMS) intermediate 6
was reported for nucleoside 1 in small scale with low yield (19%).5a Now, the synthesis of
nucleoside 1 was performed including the isolation of intermediate 6. Toward this end, the
palladium-catalyzed Sonogashira cross-coupling reaction was performed on nucleoside 511
with trimethylsilylacetylene giving 6 in 77% yield. Deprotection in MeOH/K2CO3 gave
nucleoside 1 in 77% yield. The overall yield over two steps was 59%. Then compound 1 was
protected in the 2-position with an isobutyryl residue (→ 7, 75%), converted to the 5’-O-
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DMTr derivative 8 (57%) and was finally phosphitylated to afford 9 in 75% yield (Scheme 1).
Phosphoramidite 10 was synthesized according to an already published procedure8.
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Scheme 1. Synthesis of the 7-Ethynyl-7-deazapurine Phosphoramidite 9a
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aReagents and conditions: i) trimethylsilylacetylene, [Pd0[P(Ph3)4], CuI, DMF, Et3N, rt, 12 h;
ii) K2CO3, MeOH, rt, 12 h; iii) iBu2O, TMSCl, anhydrous pyridine, rt, 3 h; iv) 4,4’-
dimethoxytriphenylmethyl chloride, anhydrous pyridine, rt, 12 h; v) NC(CH2)2OP(Cl)N(i-
Pr)2, (i-Pr)2NEt, anhydrous CH2Cl2, rt.
Next, phosphoramidite 13 was prepared from 5-ethynyl-2’-deoxycytidine12 (2). The 5’-DMTr
residue was introduced under standard conditions to give compound 11 (79%). Acetylation of
the 4-amino group gave 12 (73%). Final phosphitylation furnished phosphoramidite 13 in
63% yield (Scheme 2). Phosphoramidite 14 was prepared as described.10 All compounds were
characterized by elemental analyses or ESI-TOF mass spectra as well as by their 1H and 13C
NMR spectra (see Supporting Information). The 13C NMR chemical shifts are listed in the
Experimental Section (Table 3) and were assigned by 1H-13C coupling constants (Tables S1
and S2, Supporting Information) and DEPT-135 NMR spectra.
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Scheme 2. Synthesis of the 5-Ethynyl Pyrimidine Phosphoramidite 13a
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aReagents and conditions: i) 4,4’-dimethoxytriphenylmethyl chloride, anhydrous pyridine, rt,
16 h; ii) acetic anhydride, DMF, rt, 15 h; iii) NC(CH2)2OP(Cl)N(i-Pr)2, (i-Pr)2NEt, anhydrous
CH2Cl2, rt.
2. Syntheses of Oligonucleotides Using Ethynylated Phosphoramidites 9, 10, 13 and 14.
Following monomer synthesis, oligonucleotides (ODNs) were prepared on solid-phase using
the phosphoramidites 9, 10, 13 and 14 together with standard building blocks. Nucleosides 1-
4 were incorporated into central positions of the oligonucleotides 5’-d(TAGGTCAATACT)
(15) or 3’-d(ATCCAGTTATGA) (16), thereby replacing particular dG, dC, dT or dA residues
(Table 1). After solid-phase synthesis, the oligonucleotides were cleaved from the solid
support and deprotected in conc. aq. ammonia at 55°C for 16 h. The oligonucleotides were
purified by reversed-phase HPLC (RP-18), detritylated with 2.5% dichloroacetic acid in
dichloromethane and again purified by HPLC. Materials of the single peaks were isolated in
all cases (Figure 2). Subsequently, the molecular masses of the single peak contents were
determined by MALDI-TOF mass spectrometry. Unexpectedly, the mass spectra of the
isolated oligonucleotides 17-19 containing nucleosides 1-3 showed two masses (Figure 2a-c).
The lower masses in the MALDI-TOF spectra correspond to the calculated values for the
ethynylated oligonucleotides (3668.5 for ODN 17; 3668.3 for ODN 18, 3653.5 for ODN 19),
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while the higher masses indicate the presence of “side products” (3686.4 for ODN 17; 3686.4
for ODN 18; 3671.6 for ODN 19) (Table 1, Experimental Section and Figures S1-S4,
Supporting Information). The mass difference between the expected product masses and the
masses for the side product amounts to ~18 mass units. In all cases (ODNs 17-19), the “side
product” masses gave the major mass peaks, and the expected ethynylated oligonucleotides
were only the minor components. Unfortunately, the mobility on reversed-phase HPLC of the
expected oligonucleotide and side products are the same, and the product content could only
be verified by their mass differences (Figure 2). Oligonucleotide 20 incorporating the 2’-
deoxyadenosine derivative 4 was an exception. It showed a single HPLC peak with the
calculated mass and without contamination (Figure 2d).
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Figure 2. Reversed-phase HPLC elution profiles of purified oligonucleotides (a) 5’-d(AGT
ATT 1AC CTA) ODN 17; (b) 5’-d(TAG GT2 AAT ACT) ODN 18; (c) 5’-d(AGT AT3 GAC
CTA) ODN 19 and (d) 5’-d(TAG GTC 4AT ACT) ODN 20. Monitored at 260 nm using
gradient system II.
The presence of side products was further evidenced by enzymatic hydrolysis of the
synthesized oligonucleotides. Toward this end, the nucleoside composition of ethynylated
ODNs 17-20 containing 1-4 was determined by tandem enzymatic hydrolysis with snake
venom phosphodiesterase and alkaline phosphatase in 0.1 M Tris-HCl buffer (pH 8.3) at 37
°C (for details, see Experimental Section). The mixtures obtained from the digest were
analyzed by reversed-phase HPLC (RP-18, at 260 nm) showing the peaks for the canonical
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nucleosides and the ethynylated nucleosides 1-4 (Figure 3). For oligonucleotides
incorporating nucleosides 1-3, additional peaks were found indicating formation of side
products (Figure 3a-c). The ratio of ethynyl to side product was roughly 1:3 for ODN 17 and
18. In case of ODN-19, two side products were formed. Only the digest of oligonucleotide 20
containing 4 gave a clean pattern without showing side product formation and thereby
confirming the mass spectrometric data.
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Peak heights corresponding to the ethynylated nucleosides and side products are small
compared to those of the canonical nucleosides. This is a result of single incorporations of the
modified residues and their low 260 nm extinction coefficients (see Table 2, Experimental
Section, for UV-spectra see Figure S6, Supporting information). According to their mass
spectra, side products were probably formed by hydration as they contain 18 mass units more
than the ethynyl compounds. However, it was to examine whether these reactions occurred
during oligonucleotide synthesis (iodine oxidation in water and/or detritylation with
trichloroacetic acid) and/or work-up (conc. aq. ammonia treatment). Therefore, additional
information on the behaviour of monomeric nucleosides 1-3 under acidic and alkaline
conditions was collected.
Figure 3. HPLC profiles of the enzymatic hydrolysis products of (a) ODN 17; (b) ODN 18;
(c) ODN 19; (d) ODN 20. Monitored at 260 nm using gradient system III and IV.
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3. Acid-Catalyzed Hydration of Ethynyl Modified Nucleosides 1-4. It is expected that side
product formation occurs under acidic or oxidative aqueous conditions during solid-phase
oligonucleotide synthesis of compounds 1-3. Consequently, the nucleosides were subjected to
acid treatment. Hydration of 5-ethynyl-2’-deoxyuridine was already reported by Walker13
using 0.1 M aq. H2SO4 (7 days at rt) and the compound was also prepared by Mertes14 by
glycosylation of the acetylated nucleobase. Water addition follows the Markovnikov rule and
yields acetyl compounds.15 According to these observations and the molecular weight
increase of the oligonucleotides (corresponding to one water molecule), the acid catalyzed
hydration reaction of nucleosides 1-4 in an MeOH/H2O (9:1 mixture) containing H2SO4 was
studied first. Only nucleosides 1 and 3 provided new nucleosides (21 and 24) which were
isolated in 70-80% yield. The reaction product of 3 was already described as 24,13 while the
hydration product of 1 was assigned to 21 on the basis of mass spectra, and 1H NMR and 13C
NMR chemical shifts. The signals of a methyl group appeared in the 1H NMR spectrum of 21,
and the side chain gave 13C NMR signals similar to those of nucleoside 24 (Table 3,
Experimental Section). For the conversion of the ethynylated 7-deaza-2’-deoxyguanosine (1),
only a catalytic amount of acid (0.1 eq. of H2SO4 relative to alkyne) was required to yield the
7-acetyl nucleoside 21, while the conversion of the dU analog 3 to the acetyl compound 24
required 1 eq. of H2SO4. In case of ethdC (2), the situation is different. Here, acid treatment led
to cleavage of the N-glycosylic bond and formation of a 1:1 mixture of 5-ethynylcytosine16
(22) and 5-acetylcytosine17 (23) in 84% overall yield. Both components were identified by
NMR spectra and mass data. Treatment of 7-deaza-2’-deoxyadenosine derivative 4 with
H2SO4 did not yield any product. So, the reactivity of the ethynyl side chains towards H2SO4
in MeOH/H2O strongly depends on the nucleobase structure. 7-Deazaguanine nucleoside 1 is
the most sensitive nucleoside towards water addition, while the ethdC 2 is the most sensitive
nucleoside towards acid-catalyzed glycosylic bond hydrolysis. The 7-deazaadenosine analog
4 was stable under these conditions.
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Scheme 3. Acid-Catalyzed Hydration Reactions of Ethynyl Modified Nucleosides 1-4
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4. Stability of Nucleosides 1-4 in Concentrated Aqueous Ammonia. Next, the effect of
conc. aq. ammonia on the ethynylated nucleosides 1-4 was investigated at elevated
temperature (55 °C, 16 h). These conditions correspond to those used for oligonucleotide
deprotection. First, the reactions were performed on analytical scale and were monitored by
TLC. The crude mixtures of nucleosides 1 and 4 did not show any additional spot, while those
of compounds 2 and 3 showed new products migrating faster than the starting materials. The
formation of these products was further evidenced by HPLC analysis shown in Figure 4.
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Figure 4. Reversed-phase HPLC elution profiles of the crude reaction mixtures of nucleosides
1-4 treated with conc. aq. ammonia at elevated temperature (55 °C for 16 h). (a) ethc7Gd 1; (b)
ethdC 2; (c) ethdU 3 and (d) ethc7Ad 4. Monitored at 260 nm using gradient system III and IV.
The ratio of the ethynyl nucleoside 2 and side product 25 (after 16 h treatment with conc. aq.
ammonia at 55 °C) was approximately 2:1, and for nucleoside 3 and side product 24 a ratio of
4:1 was observed by HPLC. The mobility of the side product formed by ethdU (3), which was
obtained by ammonia treatment, was identical to that of side product 24 obtained from 3 by
acid treatment (Scheme 4, Figure S7, Supporting Information). This confirmed that hydration
of the side chain of 3 (→ 24) occurs under acidic as well as alkaline conditions. For
identification of the side product of ethdC, compound 2 was treated with conc. aq. ammonia at
55 °C for 20 h (Scheme 4). The product was isolated and identified as 5-acetyl-dC on the
basis of its mass and the typical 1H and 13C NMR chemical shifts of the acetyl side chain (see
Experimental Section and Table 3). As hydration of a triple bond is uncommon under alkaline
conditions, we searched in the literature for similar observations. Indeed, the silylated
arabinofuranosyl-5-ethynylcytosine nucleoside was partially converted to the acetyl
compound (5%) upon treatment with potassium carbonate in methanol.18
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Scheme 4. Base-Catalyzed Hydration Reaction on Ethynyl Modified Nucleosides 2 and 3
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5. Comparison of Side Products Obtained from the Enzymatic Hydrolysis of
Oligonucleotides with Those Obtained by Synthesis. For the identification of side products
formed during oligonucleotide synthesis, ODNs 17-20 were digested by tandem enzymatic
hydrolysis using snake venom phosphodiesterase and alkaline phosphatase. The enzymatic
hydrolysis mixtures of these oligonucleotides were co-injected together with the ethynylated
nucleosides 1-3 and the newly synthesized acetylated compounds 21, 24 and 25 (Figure 5).
Figures 5a-c clearly indicate that the co-injected nucleosides match the hydrolysis products
(compare Figures 3 and 5). A minor side product whose signal appeared between the peaks
for nucleosides 3 and 24 was not identified. These results prove that the side products formed
during oligonucleotide synthesis and work-up are the acetyl nucleosides 21, 24 and 25. As
expected, the HPLC profile of the enzymatic hydrolysis of ODN 20 (containing nucleoside 4)
showed the presence of ethynyl nucleoside 4 exclusively without any detectable amounts of
side products (Figure 5d). We noted that side product formation, occurring during
oligonucleotide synthesis, takes place on protected ethynyl nucleosides (except for 3). Side
products are deprotected under work-up conditions. While we are able to correlate side
product formation to the process of deprotection under alkaline conditions, we cannot provide
evidence at what stage of oligonucleotide synthesis hydration takes place.
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Figure 5. HPLC profiles of mixtures of the enzymatic hydrolysis products of
oligonucleotides, ethynyl modified nucleosides and acetyl modified nucleosides. (a) ODN 17
+ nucleosides 1 and 21; (b) ODN 18 + nucleosides 2 and 25; (c) ODN 19 + nucleosides 3 and
24 and (c) ODN 20 + nucleoside 4. Monitored at 260 nm using gradient system III and IV.
6. Synthesis of Triisopropylsilyl Protected Phophoramidites 28, 31 and 34. From the
results described above, it is obvious that the ethynylated nucleosides 1-3 are not stable,
neither during oligonucleotide synthesis nor under work-up conditions or both. Although
solid-phase oligonucleotide synthesis is performed in anhydrous acetonitrile (10 ppm water
content), water is present during iodine oxidation of the phosphorous (III) to phosphorous (V)
residues. We noticed that the extent of side chain modification increases with the number of
coupling steps and ethynyl group transformation is more pronounced when the modified base
is incorporated near the 3’-end of the oligonucleotide (data not shown). Another source for
ethynyl group hydration is aq. ammonia treatment. Therefore, we silylated the ethynyl group
by a triisopropylsilyl (TIPS) residue. This bulky and hydrophobic protecting group is
sufficiently stable during oligonucleotide synthesis and under work-up conditions. A similar
protection strategy was used by Gramlich et al for the protection of the terminal triple bond of
octadiynyl-dC utilized in sequential click reactions.2e Earlier experiments using the
trimethylsilyl group failed as this group got lost during oligonucleotide synthesis.10 The 5- or
7-TIPS-ethynylated phosphoramidite building blocks 28, 31 and 34 were synthesized from the
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5- or 7-iodinated nucleosides 2619, 29 and 3220 by the palladium catalyzed Sonogashira cross-
coupling reaction with triisopropylsilylacetylene. Triisopropylsilyl ethynyl nucleosides 27, 30
and 33 were obtained in 59% to 79% yield. Phosphitylation under standard conditions
furnished the phosphoramidites 28, 31 and 34 (61-80% yield) (Scheme 5).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
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59
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Scheme 5. Synthesis of Phosphoramidite Building Blocks 28, 31 and 34a
aReagents and conditions: i) triisopropylsilylacetylene, [Pd0[P(Ph3)4], CuI, DMF, Et3N, rt, 12
h; ii) NC(CH2)2OP(Cl)N(i-Pr)2, (i-Pr)2NEt, anhydrous CH2Cl2, rt.
6.1 Synthesis of Oligonucleotides with TIPS-Phosphoramidites 28, 31 and 34. The
phosphoramidites 28, 31 and 34 were used together with unmodified building blocks in the
synthesis of TIPS ethynyl oligonucleotides by solid-phase synthesis. Oligonucleotides were
characterized by MALDI-TOF mass spectrometry (Table 1, Figure S8, Supporting
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Information), and the masses matched the calculated values for ODNs 35-37 (Figure 6). No
cleavage of the TIPS group or hydration of the ethynyl side chain was detected by MALDI-
TOF, even after deprotection of the oligonucleotides in concentrated aq. ammonia at elevated
temperature. Only the dC derivative was an exception when rigorous conditions were used.
Partial cleavage (~ 20%) of the TIPS group was observed during deprotection with conc. aq.
ammonia at elevated temperature. This problem could be overcome by application of mild
deprotection conditions and the use of 4-tert-butylphenoxyacetyl-protected canonical
phosphoramidites together with 4-tert-butylphenoxyacetic anhydride as capping reagent (see
Experimental Section). Subsequently, deprotection of the oligonucleotides was performed
with conc. aq. ammonia at rt, thus preventing cleavage of the TIPS group.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Finally, the TIPS groups of the oligonucleotides 35-37 were removed with
tetrabutylammonium fluoride (TBAF) in CH3CN/DMF (4:1) at 45 °C for 16 h (Scheme 6).
After completion of the reaction, the oligonucleotides were precipitated by adding sodium
acetate buffer and isopropanol (for details see Experimental Section). The deprotected
oligonucleotides were purified by reversed-phase HPLC. Figure 6 shows the HPLC profiles of
TIPS protected oligonucleotides and oligonucleotides with deprotected intact ethynyl groups.
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Scheme 6. Deprotection of the TIPS Group from Oligonucleotides (35-37)
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 6. Reversed-phase HPLC elution profiles of purified ethynylated and silylated
oligonucleotides. (a) ODN 17 and ODN 35; (b) ODN 18 and ODN 36; (c) ODN 19 and ODN
37. Monitored at 260 nm using gradient system II.
The TIPS ethynyl oligonucleotides (35-37) can be easily distinguished from the ethynylated
oligonucleotides (17-19) due to their different HPLC mobility (Figure 6). The purity of
ethynyl modified oligonucleotides (prepared after deprotection of TIPS group) was confirmed
by enzymatic hydrolysis and MALDI-TOF spectra (Figure S9 and S10, Supporting
Information). This method was also applied for the synthesis of ODN 38 containing two
consecutive 7-TIPS ethynyl-7-deazaguanine residues, which after deprotection gave the pure
ethynylated ODN 39 (Table 1).
As discussed above, protection of ethynyl groups with TIPS residues prevents hydration of
the side chain and results in the formation of noncontaminated oligonucleotides.
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CONCLUSION AND OUTLOOK
5
6
7
8
Oligonucleotides with ethynyl groups in the 5-position of dC or dU or 7-position of 7-deaza-
dG are in part hydrated during their synthesis and work-up. The major side products were
identified as acetyl derivatives formed by Markovnikov hydration of ethynylated nucleobase
side chains. To confirm the structures of the hydration products, the corresponding acetylated
nucleosides were prepared from ethynylated nucleosides by acid or base treatment. The
efficacy of hydration depended strongly on the structure of the nucleobase moieties. Only the
7-ethynyl-7-deazaadenine base was stable under the conditions of oligonucleotide synthesis
and deprotection, while 7-ethynyl-7-deazaguanine was extremely labile. These side reactions
were circumvented by triisopropylsilyl protection of the ethynyl groups. Pure oligonucleotides
were isolated after removal of the TIPS residues with tetrabutylammonium fluoride. By use of
TIPS protected phosphoramidites, clean ethynylated oligonucleotides are now accessible
which are ready to be used in the post-synthetic modification by the Huisgen-Meldal-
Sharpless “click” chemistry.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
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49
50
51
52
53
54
55
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59
60
EXPERIMENTAL SECTION
General Methods and Materials. All chemicals and solvents were of laboratory grade as
obtained from commercial suppliers and were used without further purification. Thin-layer
chromatography (TLC) was performed on TLC aluminium sheets covered with silica gel 60
F254 (0.2 mm). Flash column chromatography (FC): silica gel 60 (40-60 μM, for flash
chromatography) at 0.4 bar. UV-spectra were recorded on a spectrophotometer: λmax (ε) in
nm, ε in dm3 mol-1 cm-1. NMR spectra were measured at 300.15 MHz for 1H, 75.48 MHz for
13C and 121.52 MHz for 31P. The 13C NMR signals were assigned on the basis of DEPT-135
and 1H-13C gated-decoupled NMR spectra (for coupling constants see Tables S1-2,
Supporting Information). The J values are given in Hz; δ values in ppm relative to Me4Si as
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internal standard. For NMR spectra recorded in DMSO-d6, the chemical shift of the solvent
peak was set to 2.50 ppm for 1H NMR and 39.50 ppm for 13C NMR. Reversed-phase HPLC
was carried out on a 4 × 250 mm RP-18 (10 μm) LiChrospher 100 column with a HPLC
pump connected with a variable wavelength monitor, a controller and an integrator. ESI-TOF
mass spectra of nucleosides were recorded on a Micro-TOF spectrometer. Molecular masses
of oligonucleotides were determined by MALDI-TOF mass spectrometry in the linear positive
mode with 3-hydroxypicolinic acid (3-HPA) as a matrix.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
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44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis, Purification, and Characterization of Oligonucleotides. The oligonucleotides
were synthesized on an automated DNA synthesizer on a 1 µmol scale employing standard
phosphoramidites as well as the phosphoramidites 9, 10, 13, 14, 28, 31 and 34. After cleavage
from the solid support, the oligonucleotides were deprotected in concentrated aqueous
ammonia solution for 16 h at 55 °C. ODN 36 was prepared by solid-phase synthesis using 4-
tert-butylphenoxyacetyl protected canonical phosphoramidites as well as phosphoramidite 31.
In addition, the capping reagent 4-tert-butylphenoxyacetic anhydride instead of acetic
anhydride was used. After cleavage from the solid support, ODN 36 was deprotected in
concentrated aqueous ammonia solution for 16 h at room temperature. The purification of the
“trityl-on” oligonucleotides was carried out on reversed-phase HPLC using the following
gradient system at 260 nm: (A) MeCN, (B) 0.1 M (Et3NH)OAc (pH 7.0)/MeCN, 95:5;
gradient I: 0-3 min 10-15% A in B, 3-15 min 15 -50% A in B; flow rate 0.8 mL/min. The
purified “trityl-on” oligonucleotides were treated with 2.5% CHCl2COOH/CH2Cl2 for 2 min
at 0 °C to remove the 4,4′-dimethoxytrityl residues. The detritylated oligomers were purified
again by reversed-phase HPLC with gradient II: 0-20 min 0-20% A in B; 20-25 min, 20% A
in B; flow rate 0.8 mL/min (ODNs 17-20, 39) or 0-20 min 0-25% A in B; 20-25 min, 25% A
in B; flow rate 0.8 mL/min (ODNs 35-37). The oligonucleotides were desalted on a short
column (RP-18) using water for elution of salt, while the oligonucleotides were eluted with
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H2O/MeOH (2:3). The oligonucleotides were lyophilized on a Speed-Vac evaporator to yield
colorless solids which were frozen at -24 °C. Extinction coefficients ε260 of the unmodified
nucleosides (H2O): dA 15400, dG 11700, dT 8800, dC 7300.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
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44
45
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47
48
49
50
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Table 1. Molecular Masses of Oligonucleotides Measured by MALDI-TOF Mass
Spectrometrya
Molecular Weight
Oligonucleotides
Calc.
Found
5’-d(AGT ATT 1AC CTA) (17)
3667.4
3668.5
3654.4
3667.4
3823.8
3824.8
3810.7
3667.4
3668.5
3654.4
4003.1
3690.5
3668.5, 3686.4
3668.3, 3686.4
3653.5, 3671.6
3667.4
5’-d(TAG GT2 AAT ACT) (18)
5’-d(AGT AT3 GAC CTA) (19)
5’-d(TAG GTC 4AT ACT) (20)
5’-d(AGT ATT 1TIPSEAC CTA) (35)
5’-d(TAG GT2TIPSE AAT ACT) (36)
5’-d(AGT AT3TIPSE GAC CTA) (37)
5’-d(AGT ATT 1AC CTA) (17) after deprotection
5’-d(TAG GT2 AAT ACT) (18) after deprotection
5’-d(AGT AT3 GAC CTA) (19) after deprotection
5’-d(TA1TIPSE 1TIPSETC AAT ACT) (38)
5’-d(TA1 1TC AAT ACT) (39) after deprotection
3824.7
3824.2
3810.0
3667.1
3668.1
3654.0
4002.5
3689.5
a Measured in the positive linear mode. TIPSE = triisopropylsilylethynyl.
Tandem Enzymatic Hydrolysis of Oligonucleotides. The enzymatic hydrolysis of
oligonucleotides 17-20 was performed using snake-venom phosphodiesterase (EC 3.1.15.1,
Crotallus adamanteus) and alkaline phosphatase (EC 3.1.3.1, Escherichia coli) in 0.1 M Tris
HCl buffer (pH 8.5) at 37 °C. The resulting mixtures were analyzed by reversed-phase HPLC
(RP-18). The enzymatic digestion products were analyzed by reversed-phase HPLC using
gradient III (ODNs 17, 20): 25 min 100% B, 25–60 min 0–40% A in B and gradient IV
(ODNs 18, 19): 25 min 100% B; flow rate 0.7 mL/min. (A) MeCN, (B) 0.1 M (Et3NH)OAc
(pH 7.0)/MeCN, 95:5.
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Table 2. UV Maxima and Extinction Coefficients of Ethynyl and Acetyl Nucleosidesa,b
7
8
9
λ260 (nm),
ɛ (dm3 mol-1cm-1)
λmax (nm),
Comp.
ɛ (dm3 mol-1cm-1)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
270,
9000
7-Ethynyl-c7Gd (1)
7400
307,
5800
7-Acetyl-c7Gd (21)
5-Ethynyl-dC (2)
5-Acetyl-dC (25)
6600
3600
4600
292,
8100
285,
8900
287,
10300
5-Ethynyl-dU (3)
3800
282,
12700
5-Acetyl-dU (24)
7-Ethynyl-c7Ad (4)
4900
7400
277,
12100
a Data were determined in water. b For the corresponding UV-spectra see
Figure S6, Supporting Information.
Conversion of Ethynyl Nucleosides to Acetyl Nucleosides. Ethynyl modified nucleosides
(1-4, 5 mg each) were dissolved in of 28% aq. ammonia (1 mL), and the reaction mixture was
heated at 55 °C in a closed vessel for 16 h. The solvent was removed, and the residue was
dissolved in water (200 µM). The formation of the new product was monitored by TLC
(CH2Cl2/MeOH, 90:10) and HPLC. For HPLC elution profiles see Figure 4.
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Table 3. 13C-NMR Chemical Shifts of 7-Deazapurine and Pyrimidine Derivativesa
5
6
7
8
9
C2b
C2c
C2d
C6 b
C4c
C6d
C5 b
C4ac
C5d
C7 b
C5c
-
C8 b
C6c
-
C4b
C7ac
C4d
C≡C
CH/CH2/CH3/OCH3
C=O
C1’
C2’
C3’
C4’
C5’
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
f
1
153.2
153.2
147.7
147.7
153.2
147.8
157.8
157.7
155.8
155.9
158.2
155.7
99.5
97.9
98.5
98.5
98.7
120.5
99.6
122.7
123.1
125.0
124.9
123.4
124.5
150.3
150.3
147.2
147.3
152.1
147.2
80.7, 78.1
99.2, 93.6
81.6, 77.2
81.6, 77.0
-
-
-
82.3
82.3
82.7
82.8
82.6
82.8
-
70.9
70.9
70.8
70.5
70.9
70.7
87.2
87.2
87.4
85.6
87.3
85.7
61.8
61.8
61.7
64.1
61.7
64.1
f
6
100.1
103.5
103.9
96.9
-/-/0.15/-
-
-
f
7
34.8/-/18.9/-
34.8/-/18.9/55.1
-/-/30.3/-
180.1
180.2
193.4
180.1
-
f
8
-
f
-
21
27
f
103.8
100.1, 90.7
34.8/-/10.9,18.9/55.0
-
2
153.4e 145.4
88.8
-
-
164.2e
85.9, 75.9
-
-
85.4
40.9
69.9
87.5
60.9
11
12
153.2e 144.7
152.3e 147.2
89.1
92.4
-
-
-
-
164.2e
161.0e
85.9, 75.3
87.2, 74.5
-/-/-/55.0
-
-
85.8 e
87.1
41.0
40.9
70.6
70.3
85.8 e
86.4
63.7
63.5
-/-/25.0/55.0
22 + 154.9
147.2
153.0
87.3
102.1
164.9
163.6
85.5, 75.8
-
-/-/-/-
-/-/26.0/-
-
-
-
-
-
-
-
23
25
29
30
3g
154.5
-
-
-
-
-
195.7
152.9e 150.2
152.9e 149.5
152.1e 146.4
103.1
86.9
-
-
-
-
-
-
163.0e
161.6e
160.9e
161.6
161.6
160.9
-
-/-/26.2/-
195.9
86.3
86.4
87.3
84.7
85.5
84.8
41.4
40.9
40.7
69.1
70.4
70.3
70.9
70.2
69.9
87.7
85.9
86.5
87.5
87.9
85.3
60.2
61.1
63.4
61.8
61.0
63.1
-
-/-/24.5/55.0
-/-/10.6, 18.3, 24.9/54.9
-
-
97.7
97.4, 93.3
83.6, 76.4
-
-
f
149.5
149.9
148.7
144.5
146.5
144.1
97.5
-
-
f
24
33
112.1
98.3e
-/-/30.2/-
193.4
-
-
-
-
f
98.3e, 93.3
-/-/10.1,17.8/54.4
-
a Measured in DMSO-d6 at 298 K. b Purine numbering for 7-deazapurine derivatives. c Systematic numbering for 7-deazapurine derivatives.
d Systematic numbering for pyridmidine derivatives. e Tentative. f Superimposed by DMSO. g Reference 21.
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2-Amino-7-(2-deoxy-β-D-erythro-pentofuranosyl)-3,7-dihydro-5-(trimethylsilylethynyl)-
4H-pyrrolo[2,3-d]pyrimidin-4-one (6). To a suspension of 511 (2.0 g, 5.10 mmol) and CuI
(0.194 g, 1.02 mmol) in anhydrous DMF (20 mL) was added successively [Pd(PPh3)4] (0.589
g, 0.51 mmol), anhydrous Et3N (1.239 g, 12.2 mmol) and trimethylsilylacetylene (5.01 g, 51.0
mmol). The reaction mixture was stirred under inert atmosphere and allowed to proceed until
the starting material was consumed (TLC monitoring). The solvent was evaporated and the
residue was adsorbed on silica gel and subjected to FC (silica gel, column 15 x 4 cm,
CH2Cl2/MeOH, 90:10) to give the product 6 (1.42 g, 77%) as a yellowish foam. TLC
(CH2Cl2/MeOH, 90:10) Rf 0.40. UV λmax (MeOH)/nm 243 (ε/dm3 mol-1 cm-1 24800), 276
(11500), 294 (11200). 1H NMR (DMSO-d6, 300 MHz) (δ, ppm): 0.18 (s, 9H, 3 x CH3), 2.03-
2.10 (m, 1H, H-2’), 2.27-2.29 (m, 1H, Hβ -2’), 3.44-3.53 (m, 2H, 2 x H-5’), 3.74-3.75 (m,
1H, H-4’), 4.26-4.27 (m, 1H, H-3’), 4.92 (t, J = 5.4 Hz, 1H, HO-5’), 5.21 (d, J = 3.6 Hz, 1H,
HO-3’), 6.26 (dd, J = 6.0, 5.7 Hz, 1H, H-1’), 6.36 (s, 2H, NH2), 7.31 (s, 1H, H-8), 10.45 (s,
1H, NH). Anal. Calcd. for C16H22N4O4Si (362.46): C, 53.02; H, 6.12; N, 15.46. Found: C,
53.02; H, 6.00; N, 15.30.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2-Amino-7-(2-deoxy-β-D-erythro-pentofuranosyl)-5-ethynyl-3,7-dihydro-4H-pyrrolo[2,3-
d]pyrimidin-4-one (1). To a solution of 6 (1.2 g, 3.31 mmol) in MeOH (50 mL) was added
K2CO3 (1.464 g, 10.6 mmol). After stirring for 12 h at room temperature, the suspension was
filtered, adsorbed on silica gel and applied to FC (silica gel, column 15 x 3 cm,
CH2Cl2/MeOH, 90:10) to give the product 1 (0.740 g, 77%) as a reddish solid. TLC
(CH2Cl2/MeOH, 90:10) Rf 0.35. UV λmax (H2O)/nm 260 (ε/dm3 mol-1 cm-1 7400), 270 (9000).
1H NMR (DMSO-d6, 300 MHz) (δ, ppm): 2.04-2.11 (m, 1H, H-2’), 2.27-2.36 (m, 1H, Hβ-
2’), 3.45-3.54 (m, 2H, 2 x H-5’), 3.74-3.77 (m, 1H, H-4’), 3.88 (s, 1H, C≡CH), 4.27-4.28 (m,
1H, H-3’), 4.93 (t, J = 5.4 Hz, 1H, HO-5’), 5.22 (d, J = 3.9 Hz, 1H, HO-3’), 6.27 (dd, J = 6.0,
5.7 Hz, 1H, H-1’), 6.35 (s, 2H, NH2), 7.30 (s, 1H, H-8), 10.50 (s, 1H, NH). Anal. Calcd for
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C13H14N4O4 (290.27): C, 53.79; H, 4.86; N, 19.30. Found: C, 53.65; H, 5.00; N, 19.21. The
obtained NMR data correspond to earlier reported literature values.5a
7
8
9
10
11
12
13
14
15
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7-(2-Deoxy-β-D-erythro-pentofuranosyl)-5-ethynyl-3,7-dihydro-2-(isobutyrylamino)-4H-
pyrrolo[2,3-d]pyrimidin-4-one (7). Compound 1 (0.500 g, 1.72 mmol) was dried by repeated
co-evaporation with anhydrous pyridine (3 x 8 mL) and dissolved in anhydrous pyridine (12
mL). Then, trimethylsilyl chloride (0.936 g, 8.61 mmol) was added to the solution. The
reaction mixture was stirred for 15 min at rt, then isobutyric anhydride (1.362 g, 8.61 mmol)
was added and the solution was stirred for additional 3 h at rt. Then, the reaction mixture was
cooled in an ice-bath, H2O (1.5 mL) and subsequently (5 min later) 28-30% aq. NH3 solution
(1 mL) were added, and stirring was continued for 30 min at room temperature. The solvent
was evaporated to near dryness, co-evaporated with toluene (3 x 10 mL), and the residue was
purified by FC (silica gel, column 15 x 4 cm, CH2Cl2/MeOH, 90:10) to give 7 (0.467 g, 75%)
as a colorless solid. TLC (CH2Cl2/MeOH, 90:10) Rf 0.60. UV λmax (MeOH)/nm 235 (ε/dm3
mol-1 cm-1 17600), 280 (14700). 1H NMR (DMSO-d6, 300 MHz) (δ, ppm): 1.10, 1.12 (2s, 6H,
2 x CH3), 2.11-2.18 (m, 1H, H-2’), 2.33-2.42 (m, 1H, Hβ-2’), 2.69-2.78 (m, 1H, CH), 3.49-
3.54 (m, 2H, 2 x H-5’), 3.78-3.81 (m, 1H, H-4’), 4.00 (s, 1H, C≡CH), 4.31-3.32 (m, 1H, H-
3’), 4.96 (t, J = 5.1 Hz, 1H, HO-5’), 5.26 (d, J = 3.3 Hz, 1H, HO-3’), 6.37 (dd, J = 6.0, 8.1
Hz, 1H, H-1’), 7.62 (s, 1H, H-8), 11.57 (s, 1H, NH), 11.82 (s, 1H, NH). Anal. Calcd for
C17H20N4O5 (360.36): C, 56.66; H, 5.59; N, 15.55. Found: C, 56.42; H, 5.72; N, 15.49.
7-[2-Deoxy-5-O-(4,4’-dimethoxytrityl)-β-D-erythro-pentofuranosyl]-5-ethynyl-3,7-
dihydro-2-(isobutyrylamino)-4H-pyrrolo[2,3-d]pyrimidin-4-one (8). Compound 7 (0.390
g, 1.08 mmol) was dried by repeated co-evaporation with anhydrous pyridine (3 x 8 mL). The
residue was dissolved in anhydrous pyridine (12 mL) and stirred with 4,4’-dimethoxytrityl
chloride (0.706 g, 1.84 mmol) at room temperature for 5 h. The solution was poured into 5%
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aq. NaHCO3 solution and extracted with CH2Cl2 (3 x 50 mL). The combined extracts were
dried (Na2SO4) and the solvent was evaporated. The residue was purified by FC (silica gel,
column 15 x 3 cm, CH2Cl2/acetone, 80:20) to give the product 8 (0.41 g, 57%) as a colorless
foam. TLC (CH2Cl2/acetone, 80:20) Rf 0.44. UV λmax (MeOH)/nm 234 (ε/dm3 mol-1, cm-1
41300), 281 (19600). 1H NMR (DMSO-d6, 300 MHz) (δ, ppm): 1.10, 1.12 (2s, 6H, 2 x CH3),
2.19-2.26 (m, 1H, H-2’), 2.46-2.55 (m, 1H, Hβ-C2’), 2.71-2.80 (m, 1H, CH), 3.07-3.19 (m,
2H, 2 x H-5’), 3.72 (s, 6H, 2 x OCH3), 3.90-3.91 (m, 1H, H-4’), 4.02 (s, 1H, C≡CH), 4.33-
4.34 (m, 1H, H-3’), 5.32 (d, J = 3.9 Hz, 1H, HO-3’), 6.38 (t, J = 6.3 Hz, 1H, H-1’), 6.82-6.86
(m, 4H, Ar-H ), 7.17-7.37 (m, 9H, Ar-H), 7.48 (s, 1H, H-8), 11.61 (s, 1H, NH), 11.86 (s, 1H,
NH). Anal. Calcd for C38H38N4O7 (662.73): C, 68.87; H, 5.78; N, 8.45. Found: C, 68.93; H,
5.87; N, 8.32.
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7-[2-Deoxy-5-O-(4,4’-dimethoxytrityl)-β-D-erythro-pentofuranosyl]-5-ethynyl-3,7-
dihydro-2-(isobutyrylamino)-4H-pyrrolo[2,3-d]pyrimidin-4-one 3’-(2-Cyanoethyl)-N,N-
diisopropylphosphoramidite (9). To a solution of 8 (0.1 g, 0.15 mmol) in dry CH2Cl2 (10
mL), (i-Pr)2NEt (0.049 g, 0.38 mmol) and 2-cyanoethyl diisopropylphosphoramidochloridite
(0.064 g, 0.27 mmol) were added. After stirring for 45 min at rt, the solution was diluted with
CH2Cl2 (30 mL) and extracted with 5% aq. NaHCO3 solution (20 mL), the combined organic
layer was dried over Na2SO4 and evaporated. The residue was purified by FC (silica gel,
column 10 x 2 cm, CH2Cl2/acetone, 95:15) affording product 9 (0.098 g, 75%) as a colorless
foam. TLC (CH2Cl2/acetone, 85:15) Rf 0.75. 31P NMR (CDCl3, 121 MHz) (δ, ppm): 148.1,
147.4. ESI-TOF m/z calcd for C47H55N6O8P [M + Na]+ 885.3711, found 885.3688.
1-(2-Deoxy-β-D-erythro-pentofuranosyl)-5-ethynylcytosine (2). To a solution of 5-
trimethylsilylethynyl-2’-deoxycytidine9c (4.0 g, 12.4 mmol) in MeOH (50 mL) was added
K2CO3 (0.060 g, 0.43 mmol). The reaction mixture was stirred at room temperature for 1 h
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(TLC monitoring). Then, the solvent was evaporated and the remaining residue was purified
by FC (silica gel, column 20 x 4 cm, CH2Cl2/MeOH, 80:20). Evaporation of the solvent gave
nucleoside 2 (2.65 g, 85%) as amorphous solid. TLC (CH2Cl2/MeOH, 80:20) Rf 0.40. UV λmax
(H2O)/nm 260 (ɛ/ dm3 mol-1cm-1 3600), 292 (8100). 1H NMR (DMSO-d6, 300 MHz) (δ,
ppm): 1.94-2.03 (m, 1H, Hα-2’), 2.12-2.19 (m, 1H, Hβ-2’), 3.53-3.65 (m, 2H, H-5’), 3.78-3.81
(m, 1H, H-4’), 4.19-4.22 (m, 1H, H-3’), 4.34 (s, 1H, C≡CH), 5.10 (t, J = 5.1 Hz, 1H, 5’-OH),
5.22 (d, J = 4.2 Hz, 1H, 3’-OH), 6.09 (t, J = 6.3 Hz, 1H, H-1’), 6.83 (s, 1H, NHa), 7.71 (s, 1H,
NHb), 8.26 (s, 1H, H-6). The obtained NMR data correspond to earlier reported literature
values.9c,12
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1-[2-Deoxy-5-O-(4,4’-dimethoxytrityl)-β-D-erythro-pentofuranosyl]-5-ethynylcytosine
(11). Compound 2 (2.5 g, 9.95 mmol) was dried by repeated co-evaporation with anhydrous
pyridine (2 x 10 mL) before dissolving in anhydrous pyridine (20 mL). Then 4,4’-
dimethoxytrityl chloride (4.4 g, 12.99 mmol) was added and the solution was stirred at room
temperature for 5h. The reaction mixture was diluted with CH2Cl2 (50 mL) and extracted with
5% aq. NaHCO3 solution (100 mL), the organic layer was dried over Na2SO4, and then
concentrated. Purification by FC (silica gel, column 15 x 3 cm, CH2Cl2/acetone, 90:10) gave
11 as colorless foam (4.36 g, 79%). TLC (CH2Cl2/MeOH, 90:10) Rf 0.5. λmax (MeOH)/nm 235
(ɛ/ dm3 mol-1cm-1 34600), 283 (8000). 1H NMR (DMSO-d6, 300 MHz): 2.08-2.15 (m, 1H, Hα-
2’), 2.21-2.24 (m, 1H, Hβ-2’), 3.12-3.25 (m, 2H, H-5’), 3.74 (s, 6H, 2 x OCH3), 3.93-3.94 (m,
1H, H-4’), 4.20-4.22 (m, 2H, H-3’, C≡CH), 5.30 (d, J = 4.2 Hz, 1H, 3’-OH), 6.11 (t, J = 6.6
Hz, 1H, H-1’), 6.87-6.90 (m, 5H, 4 x Ar-H, 1 x NHa), 7.19-7.41 (m, 9H, Ar-H), 7.77 (s, 1H,
NHb), 7.95 (s, 1H, H-6). ESI-TOF m/z calcd for C32H31N3O6 [M + Na]+ 576.2105, found
576.2108.
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N4-Acetyl-1-[2-deoxy-5-O-(4,4’-dimethoxytrityl)-β-D-erythro-pentofuranosyl]-5-
ethynylcytosine (12). To a solution of compound 11 (4.3 g, 7.77 mmol) in N,N-
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dimethylformamide (20 mL) was added acetic anhydride (900 µL, 9.41 mmol) and the
reaction mixture was stirred at room temperature for 24 h. Then, another portion of acetic
anhydride (200 µL, 2.09 mmol) was added, and the mixture was stirred for additional 12 h
(TLC monitoring). After evaporation of DMF under reduced pressure, the residue was applied
to FC (silica gel, column 15 x 4 cm, CH2Cl2/acetone, 80:20). After evaporation of the solvent
from the main zone, compound 12 was isolated as colorless foam (3.38 g, 73%). TLC
(CH2Cl2/acetone, 80:20) Rf 0.53. λmax (MeOH)/nm 235 (ɛ/ dm3 mol-1cm-1 38500), 282 (6700).
1H NMR (DMSO-d6, 300 MHz): 2.15-2.24 (m, 1H, Hα-2’), 2.30 (s, 3H, CH3), 2.37-2.41 (m,
1H, Hβ-2’), 3.17-3.27 (m, 2H, 2 x H-5’), 3.73 (s, 6H, 2 x OCH3), 4.00 (bs, 1H, H-4’), 4.23 (bs,
1H, H-4’), 4.36 (s, 1H, C≡CH), 5.35 (d, J = 4.2 Hz, 1H, 3’-OH), 6.05 (t, J = 6.3 Hz, 1H, H-
1’), 6.87-6.90 (m, 4H, Ar-H), 7.19-7.39 (m, 9H, Ar-H), 8.26 (s, 1H, H-6), 9.36 (s, 1H, NH).
ESI-TOF m/z calcd for C34H33N3O7 [M + Na]+ 618.2211, found 618.2196.
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N4-Acetyl-1-[2-deoxy-5-O-(4,4’-dimethoxytrityl)-β-D-erythro-pentofuranosyl]-5-ethynyl
cytosine 3’-(2-Cyanoethyl)-N,N-diisopropylphosphoramidite) (13). A stirred solution of
12 (1.0 g, 1.67 mmol) in anhydrous CH2Cl2 (15 mL) was treated with (i-Pr)2NEt (400 µL, 2.4
mmol) and 2-cyanoethyl-N,N-diisopropylphosphoramido chloridite (520 µL, 2.4 mmol). The
reaction mixture was stirred for 15 min (TLC monitoring) at room temperature, then the
solution was diluted with CH2Cl2 (30 mL) and extracted with 5% aq. NaHCO3 solution (20
mL). The organic layer was dried over Na2SO4, concentrated and purified by FC (silica gel,
column 10 x 4 cm, CH2Cl2/acetone, 80:20). Evaporation of the solvent from the main zone
gave 13 (0.850 g, 63%) as colorless foam. TLC (CH2Cl2/acetone, 80:20) Rf 0.7. 31P NMR
(CDCl3, 121 MHz) (δ, ppm): 149.2, 148.6. ESI-TOF m/z calcd for C43H50N5O8P [M + Na]+
818.3289, found 818.3281.
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5-Acetyl-2-amino-7-(2-deoxy-β-D-erythro-pentofuranosyl)-3,7-dihydro-4H-pyrrolo[2,3-
d]pyrimidin-4-one (21). To a solution of compound 1 (0.073 g, 0.25 mmol) in MeOH (9 mL)
was added water (1 mL) and H2SO4 (0.025 mmol). The reaction mixture was stirred for 1 h at
75 °C. The reaction mixture was brought to pH 7 by the use of a strongly basic ion-exchange
resin. The resin was filtered-off and washed with MeOH (20 mL). The filtrate was evaporated
and the remaining residue was purified by FC (silica gel, column 10 x 2 cm, CH2Cl2/MeOH,
80:20) to give the product 21 (0.054 g, 70%) as a colorless powder. TLC (CH2Cl2/MeOH,
75:25) Rf 0.26. λmax (H2O)/nm 260 (ε/dm3 mol-1, cm-1 6600), 307 (5800). 1H NMR (DMSO-d6,
300 MHz) (δ, ppm): 2.12-2.15 (m, 1H, H-2’), 2.29-2.38 (m, 1H, Hβ-2’), 2.62 (s, 3H, CH3),
3.47-3.53 (m, 2H, 2 x H-5’), 3.76-3.80 (m, 1H, H-4’), 4.29-4.30 (m, 1H, H-3’), 4.94 (t, J = 5.4
Hz, 1H, HO-5’), 5.23 (d, J = 3.6 Hz, 1H, HO-3’), 6.32 (dd, J = 6.0, 5.7 Hz, 1H, H-1’), 6.43 (s,
2H, NH2), 7.63 (s, 1H, H-8), 10.61 (s, 1H, NH). ESI-TOF m/z calcd for C13H16N4O5 [M +
Na]+ 331.1013, found 331.1008.
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5-Ethynylcytosine (22) and 5-Acetylcytosine (23). As described for 21, using compound 212
(0.063 g, 0.25 mmol) in MeOH (9 mL), water (1 mL) with H2SO4 (0.25 mmol). FC (silica gel,
column 10 x 2 cm, CH2Cl2/MeOH 85:15) gave an inseparable mixture of products 22 and 23
(1:1) (0.030 g, 84%) as a colorless powder. TLC (CH2Cl2/MeOH, 85:15) Rf 0.34. 1H NMR
(DMSO-d6, 300 MHz) (δ, ppm): 2.35 (s, 3H, CH3), 4.28 (s, 1H, C≡CH), 6.69, 7.85, 8.39 (3br
s, 4H, 2 x NH2), 7.74, 8.45 (2s, 1H, 2 x H-6), 11.33 (br s, 2H, 2 x NH). ESI-TOF m/z calcd
for 22 C6H5N3O [M + Na]+ 158.0325, found 158.0331 and for 23 C6H7N3O2 [M + Na]+
176.0430, found 176.0438. The obtained NMR data correspond to earlier reported literature
values.16-17
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5-Acetyl-1-(2-deoxy-β-D-erythro-pentofuranosyl)uracil (24). As described for 21
compound 316a (0.063 g, 0.25 mmol) in MeOH (9 mL) and water (1 mL) with H2SO4 (0.25
mmol) and FC (silica gel, column 10 x 2 cm, CH2Cl2/MeOH, 85:15) to give the product 24
(0.054 g, 80%) as a colorless powder. TLC (CH2Cl2/MeOH, 85:15) Rf 0.36. UV λmax
(H2O)/nm 260 (ɛ/ dm3 mol-1cm-1 4900), 282 (12700). 1H NMR (DMSO-d6, 300 MHz) (δ,
ppm): 2.11-2.22 (m, 2H, H-2’), 2.43 (s, 3H, CH3), 3.52-3.58 (m, 2H, H-5’), 3.83-3.87 (m, 1H,
H-4’), 4.21-4.23 (m, 1H, H-3’), 5.04 (t, J = 4.5 Hz, 1H, 5’-OH), 5.27 (d, J = 4.2 Hz, 1H, 3’-
OH), 6.11 (t, J = 6.3 Hz, 1H, H-1’), 8.64 (s, 1H, H6), 11.62 (s, 1H, NH). The obtained NMR
data correspond to earlier reported literature values.13,14
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5-Acetyl-1-(2-deoxy-β-D-erythro-pentofuranosyl)cytosine (25). Compound 212 (0.150 g,
0.60 mmol) was dissolved in 28% aqueous NH3 (200 mL) and stirred at 55 °C for 20 h in an
autoclave. The resulting solution was concentrated, and the residue was applied to FC (silica
gel, column 15 x 3 cm, CH2Cl2/MeOH, 90:10) to give 25 as a colorless solid (0.03 mg, 19%).
TLC (CH2Cl2/MeOH, 90:10) Rf 0.40. UV λmax (H2O)/nm 260 (ɛ/ dm3 mol-1cm-1 4600), 285
(8900). 1H NMR (DMSO-d6, 300 MHz) (δ, ppm): 2.08-2.16 (m, 1H, Hα-2’), 2.24-2.31 (m,
1H, Hβ-2’), 2.36 (s, 3H, CH3), 3.59-3.73 (m, 2H, H-5’), 3.86-3.87 (m, 1H, H-4’), 4.24-4.26
(m, 1H, H-3’), 5.24-5.28 (m, 2H, 5’-OH and 3’-OH), 6.10 (t, J = 5.7 Hz, 1H, H-1’), 7.94 (s,
1H, NHa), 8.33 (s, 1H, NHb), 9.06 (s, 1H, H-6). ESI-TOF m/z calcd for C11H15N3O5 [M +
Na]+ 292.0904, found 292.0903.
7-[2-Deoxy-5-O-(4,4’-dimethoxytrityl)-β-D-erythro-pentofuranosyl]-3,7-dihydro-2-
(isobutyrylamino)-5-(triisopropylsilylethynyl)-4H-pyrrolo[2,3-d]pyrimidin-4-one (27).
To a suspension of 26 (0.400 g, 0.52 mmol) and CuI (0.020 g, 0.10 mmol) in anhydrous DMF
(5 mL) was added successively [Pd(PPh3)4] (0.090 g, 0.08 mmol), anhydrous Et3N (0.131 g,
1.30 mmol) and triisopropylsilylacetylene (0.382 g, 2.10 mmol). The reaction mixture was
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