Tetrahedron Letters 54 (2013) 1869–1872
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Efficient microwave-assisted solid phase coupling of nucleosides, small library
generation, and mild conditions for release of nucleoside derivatives
Hanumantharao Paritala a, Yuta Suzuki b, Kate S. Carroll a,⇑
a
b
Department of Chemistry, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 33458, USA
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA
a r t i c l e
i n f o
Article history:
Received 22 January 2013
Accepted 24 January 2013
Available online 1 February 2013
Keywords:
Solid phase synthesis
Microwave assisted
Nucleoside
Combinatorial library
a b s t r a c t
Nucleosides are essential bio-molecules that participate in a wide array of biological processes involved
in maintaining physiologic homeostasis. Recent efforts geared toward the synthesis of nucleoside analogues and development of nucleoside combinatorial libraries using solid phase synthesis have contributed invaluable information toward drug design and development. These studies have provided
information concerning the structural requirements of substrate binding pockets of enzymes and evaluation of enzyme kinetics. However, the synthesis of nucleosides and its corresponding analogues remains
a challenging and time consuming process. Herein, we report an efficient, microwave assisted solid phase
coupling of nucleosides, combinatorial chemistry on the coupled nucleosides to generate small library,
and mild cleavage conditions to release nucleoside derivatives from its solid support. We anticipate these
findings will accelerate the development of synthetic methods or combinatorial library design of nucleoside analogues in similar settings.
Ó 2013 Elsevier Ltd. All rights reserved.
Introduction
Nucleosides and its derivatives are important components and
hold key roles in numerous biological processes and signal transduction. These bio-molecules are utilized as genomic building
blocks, enzyme substrates, and cellular energy sources.1 Additionally, nucleoside analogues have been successfully utilized in anticancer, -bacterial, and—viral therapeutics.2 In this context, the synthesis and testing of bio-mimetic nucleoside derivatives has
emerged as a widely used approach in drug discovery campaigns.
The design of nucleoside analogues and combinatorial libraries
using solid phase based techniques has garnered large momentum
due to the importance of their therapeutic applications.3 However,
the synthesis of nucleosides remains a challenge due to long reaction times, harsh cleavage conditions, and poor product yields.4
Redwan et al. recently reported a novel solid phase strategy that
enables the synthesis of structurally different 5-O-[N-(acyl)-sulfamoyl] adenosines from a protected ribo-purine starting material.
This approach applied cleavage conditions using trifluoroacetic
acid (TFA) and ammonium formate, which resulted in poor yields
of the nucleoside derivatives.5 In a separate study, Caroline et al.
reported the synthesis of solid phase poly-phosphorylated nucleoside analogues with decent yields and mild cleaving conditions,
but extremely slow reaction times.6 Therefore, we have explored
microwave assisted solid phase nucleoside synthesis in order to
⇑ Corresponding author. Tel.: +1 561 228 2460; fax: +1 561 228 2919.
E-mail address: kcarroll@scripps.edu (K.S. Carroll).
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.01.109
optimize reaction conditions and to create a platform to perform
combinatorial chemistry on solid phase nucleosides. Herein, we
describe an efficient microwave assisted solid phase coupling of
nucleosides, small chemical library generation on the resin bound
nucleosides, and the mild cleavage conditions to release the nucleoside derivatives from its resin support.
Results and discussion
For the synthesis of the adenosine scaffold, we initially used an
existing protocol developed for generating inosine and uridine
analogues3a. This procedure required purification after formation
of the benzylidene intermediate 4, gave low yields of the scaffold
(10–25%), and was insufficient for further solid phase synthesis
of adenosine analogues. Collectively, this synthetic procedure
was time consuming and required 8–10 days to achieve the final
product. Optimization of the reported literation conditions would
result in a facile synthesis of nucleoside derivatives with improved
yield and decreased reaction times. The following key factors were
included during the development of our approach: (1) employ
microwave conditions to enhance reaction rates, and (2) utilize
mild reaction conditions (Scheme 1). Generation of the poly styrene bound nucleoside scaffold started with the synthesis of Aldehyde 2 as previously reported3a, and activated by the formation of
dimethylacetal 3. Compound 3 was immediately converted into
benzylidene 4 via transacetylation to prevent reversion back to
compound 2. The acetal forming reaction produced a mixture of
diastereomers due to non-selective formation of the acetal
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H. Paritala et al. / Tetrahedron Letters 54 (2013) 1869–1872
Scheme 1. Synthesis of solid-supported nucleoside derivatives.
Table 1
Optimization of reaction conditions for alkylation of aromatic amine on the nucleosides
a
b
S. No
Microwave temperature (°C)
Time (min)
Equivalents of propargylbromide
Equivalents of base (DIEA)b
Yielda (%)
1
2
3
4
80
100
150
150
10
15
10
15
1
1
1
2
1
1
1
2
0
0
45
90
% yield was determined by NMR using alanine as an internal standard.
N,N-Diisopropylethylamine.
carbon.7 Compound 4 was hydrolyzed to the carboxylic acid 5 with
48% overall yield in four steps. Finally, compound 5 was coupled to
aminomethyl resin using O-(Benzotriazol-1-yl)-N,N,N0 ,N0 -tetramethyluroniumtetrafluoroborate (HBTU) activation to obtain the
corresponding solid phase nucleoside scaffolds 6. When compared
to solution chemistry, MW irradiation provides an overall higher
yield and shorter reaction times.
Using the resin bound nucleosides 6 as platform we started
exploring possible chemistry on the aromatic amine and 50 -hydroxyl functionalities to construct four member library of resin bound
nucleosides. In order to achieve this 50 -hydroxyl group of the resin
bound nucleosides 6 was protected by using tert-butyldimethylsilyl chloride and imidazole with quantitative yields and this protection allowed us to selectively explore the alkylation reactions on
the aromatic amine of 7. We have tested variety of conditions to
install propargyl group on the aromatic amine as shown in Table 1.
The ideal conditions to conduct this reaction using microwave are
achieved by using two equivalents of propargylbromide and two
equivalents of N,N-diisopropylethylamine at 150 °C with reasonable yields. Using the alkyne functional group on 8 we have
H. Paritala et al. / Tetrahedron Letters 54 (2013) 1869–1872
1871
Scheme 2. Click chemistry on the solid-supported adenosine derivative and simultaneous 50 -TBDMS deprotection and release of nucleoside from solid phase.
Scheme 3. Click chemistry on the solid-supported cytidine derivative and simultaneous 50 -TBDMS deprotection and release of nucleoside from solid phase.
conducted click reaction with benzyl azide and methyl 2-azioacetate to create a four member library of solid phase nucleoside
derivatives 9a, 10a, 9b, and 10b, as shown in scheme 2 and scheme
3. This chemistry demonstrated the facile synthesis of solid phase
nucleoside derivatives using microwave conditions.
Our next objective was to determine the mildest conditions required to cleave the polystyrene bound adenosine moiety (Table 2).
We envisioned the development of cleavage conditions that would
efficiently release the resin-bound nucleoside, but be able to
tolerate the stability of nucleoside derivatives. This approach
would contribute to the development of combinatorial libraries
composed of nucleoside analogues with diverse structural motifs.
We tested various cleavage conditions against resin 6a and 7a to
yield adenosine as shown in Table 2. The product yield of each
reaction condition was determined by NMR using alanine as an
internal standard, which was chosen based on its solubility and
stability.8 HPLC was used to analyze the integrity of the nucleoside
after cleavage since adenosine can undergo decomposition to adenine and ribose sugar3a. In order to identify optimal cleavage conditions, we screened various conditions beginning with TFA as
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H. Paritala et al. / Tetrahedron Letters 54 (2013) 1869–1872
Table 2
Cleavage conditions to cleave adenosine from solid phase adenosine scaffold
Type
Acidic
Acidic
Acidic
Oxidation
Oxidation
Oxidation
Catalyst
Catalyst
Catalyst
Catalyst
Catalyst
Catalyst
Catalyst
Catalyst
Acidic
Acidic
a
b
c
d
e
Condition
c
TFA 80%, 15 min, RT
TFA 80%, 30 min, RTc
TFA 5%, 30 min, RTc
DDQ 1.5 equiv, 3 h, RTc
DDQ 3 equiv, 3 h, RTc
DDQ 3 equiv, 24 h, RT
8 mol % In(OTf)3, 6 min, 100 °C (MW assisted)c
8 mol % In(OTf)3, 30 min, 100 °C (MW assisted)c
8 mol % In(OTf)3, 30 min, 130 °C (MW assisted)c
8 mol % In(OTf)3, 30 min, 150 °C (MW assisted) c
40 mol% In(OTf)3, 30 min, 100 °C (MW assisted)c
8 mol% In(OTf)3, 1 h, 50 °C (MW assisted)c
8 mol% In(OTf)3, 24 h, RTc
40 mol% In(OTf)3, 24 h, RTc
Acetic acid/TBAF (1:1), DMSO/water, rt, 2 he
Acetic acid 10% v/v, 2 h, RTe
Yielda (%)
(%) Of adenosine7b
91
67
24
11
14
41
2
6
NAd
NAd
15
6
5
22
88
89
88
80
90
78
77
93
99
99
—
—
91
99
99
98
82
99
% Yield was determined by NMR using alanine as an internal standard.
% Of adenosine is the ratio between adenosine and adenine (decomposed product from adenosine).
R = OH.
NA: adenosine was not observed.
R = TBDMS.
shown in Table 2. TFA is commonly used as a reagent to cleave the
ribose acetal linker, and was found to efficiently cleave the nucleoside in 15 min with 80% TFA. Extending the reaction time resulted
in cleavage of the glycosidic bond and formation of free adenine
and ribose moieties. Mild acidic conditions (5% TFA, 24 h) were
tested in hopes of improving adenosine cleavage while maintaining the glycosidic bond. These conditions reduced product yield
and did not prevent adenosine decomposition. Oxidative cleavage
conditions
(e.g.,
2,3-dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ)9, 3 equiv) afforded 41% yield of nucleoside coupled with
the formation of adenine. Furthermore, catalytic cleavage of the
acetal linker with indium trifluoromethanesulfonate (In(OTf)3)10
did not prove as efficient as acidic or oxidative conditions, but preserved the glycoside bond. Finally, nucleoside release from the resin was tested using acetic acid to cleave the acetal bond. Acetic
acid (10% v/v) in water cleaved the nucleoside with simultaneous
50 -TBDMS deprotection with 89% yield without any detectable
decomposition.
In conclusion, we have developed a microwave-assisted synthetic route for coupling of nucleosides to aminomethylated polystyrene resin and showed using microwave conditions generations
of four member solid phase nucleoside library coupled with mild
cleavage conditions. This procedure offers decent product yields,
shorter reaction times, and bypasses lengthy purification steps in
comparison to previous reports. We anticipate that the present
synthetic scheme should increase the overall efficiency of ribosebased combinatorial nucleoside libraries and is amendable to the
synthesis of other derivatives.
Acknowledgement
This work was supported by the National Institutes of Health
(GM087638 to K.S.C.).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2013.01.
109.
References and notes
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2004, 6, 717–723; (b) Ramasamy, K. S.; Amador, R. B.; Habib, Q.; Rong, F.; Han,
X.; Li, D. Y.; Huang, J.; Hong, Z.; An, H. Nucleosides Nucleotides Nucleic Acids
2005, 24, 1947–1970; (c) Rodenko, B.; Detz, R. J.; Pinas, V. A.; Lambertucci, C.;
Brun, R.; Wanner, M. J.; Koomen, G.-J. Bioorg. Med. Chem. 2006, 14, 1618–1629;
(d) Oliviero, G.; Amato, J.; D’Errico, S.; Borbone, N.; Piccialli, G.; Mayol, L.
Nucleosides Nucleotides Nucleic Acids 2007, 26, 1649–1652; (e) Poon, K. W. C.;
Liang, N.; Datta, A. Nucleosides Nucleotides Nucleic Acids 2008, 27, 389–407.
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Chem. 2012, 2012(19), 3665–3669.
6. Tonn, V. C.; Meier, C. Chem. Eur. J. 2011, 17, 9832–9842.
7. (a) Douglass, J. G.; Patel, R. I.; Yerxa, B. R.; Shaver, S. R.; Watson, P. S.; Bednarski,
K.; Plourde, R.; Redick, C. C.; Brubaker, K.; Jones, A. C.; Boyer, J. L. J. Med. Chem.
2008, 51, 1007–1025; (b) Bakthavachalam, V.; Lin, L.-G.; Cherian, X. M.;
Czarnik, A. W. Carbohydr. Res. 1987, 170, 124–135.
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1998, 63(3), 708–718.
9. (a) McDonald, C. E.; Dugger, R. W. Tetrahedron Lett. 1988, 29, 2413–2415; (b)
Oikaw, Y.; Yoshioka, T.; Yonemitsu, O. Tetrahedron Lett. 1982, 23, 889–892.
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Efficient microwave-assisted solid phase coupling of nucleosides, small library
generation and mild conditions for release of nucleoside derivatives.
Hanumantharao Paritalaa, Yuta Suzukib, Kate S. Carrolla,*
a
Department of Chemistry, The Scripps Research Institute, Jupiter, Florida, 33458, USA
Department of Chemistry, University of Michigan, Ann Arbor, Michigan, 48109, USA
*
Corresponding author
b
Address:
Department of Chemistry, The Scripps Research Institute, 130 Scripps Way, Jupiter, Florida,
33458, USA
email: kcarroll@scripps.edu
Phone: (561) 228-2460
Fax: 561-228-2919
Supplementary Information
General Experimental:
Reagents and solvents were purchased from Sigma or other commercial sources and used
without further purification.
Aminomethyl polystyrene HL (100 – 200 mesh) and 4-
benzyloxybenzaldehyde polystyrene HL were purchased from Novabiochem.
Microwave
reactions were conducted in sealed vessels using a microwave synthsizer (Initiator One Point,
Biotage) with a power output ranging from 0 to 300W. Experiments were performed in 0.5-2
mL, 2-5 mL, or 10-20 mL glass vials with constant mixing. NMR spectra were obtained on a
bruker 400 MHz for 1H and
13
C. 1H and
13
C NMR chemical shifts are reported in parts per million
(ppm) referenced to the residual solvent peak. To determine percent yield of adenosine, the
molarity of adenosine was calculated from the 1H NMR integral of the nucleoside pentose sugar
ring relative to alanine (10 mg). Mass spectra were obtained with a agilent LC/MS mass
spectrometer.
Reverse-phase HPLC was performed on a Agilent HPLC system with a UV
detector (λ = 260 nm) using a C18 (4.6 × 250 mm) Beckman Coulter Ultrasphere with a gradient
of 0.1% TFA in H2O and CH3CN as the mobile phase.
6-(4-dimethoxymethyl-phenoxy)-hexanoic acid ethyl ester (3). A mixture of 2 (6.0 g, 25.4
mmol), trimethylorthoformate (8.78 mL, 80.2 mmol), and PTSA (216 mg, 1.14 mmol) in MeOH
(3.15 mL) was irradiated by MW for 25 min at 60 oC. Triethylamine (158 µL, 1.14 mmol) was
added to neutralize the reaction mixture. The reaction was concentrated in vacuo, diluted with
EtOAc (30 mL), and washed with H2O (2 × 30 mL) and brine (1 × 30 mL). The organic layer
was dried over Na2SO4, filtered, and concentrated in vacuo to provide a white solid (7.05 g, 25.4
mmol, 100%). The analytical data matched previously reported data3a.
General procedure for the synthesis of (4). To a solution of adenosine/cytidine (7.5 mmol) in
DMF (15 mL), 3 (5.17 g, 16.7 mmol) was added, followed by PTSA (144 mg, 0.757 mmol). The
reaction mixture was subjected to MW irradiation for 1 hr at 130 oC, concentrated in vacuo,
washed with methanol, and filtered to give a white solid. 4a: (2.06 g, 53.0 %). 1H NMR
((CD3)2SO, 400 MHz): δ = 8.37 (s, 1H), 8.36 (s, 1H´), 8.16 (s, 1H), 8.16 (s, 1H´), 7.48 (d, J = 9.0,
2H), 7.41 (d, J = 8.4, 2H´), 7.35 (s, 2H, 2H´), 6.98 (d, J = 9.0, 2H), 6.94 (d, J = 9.0, 2H´), 6.28 (d,
J = 2.4, 1H), 6.25 (d, J = 3.0, 1H´), 6.17 (s, 1 H´), 5.96 (s, 1H), 5.46 (m, 1H, 1H´), 5.25 (t, J =
6.3, 1H), 5.13 (t, J = 5.7, 1H´), 5.05 (m, 1H, 1H´), 4.36 (q, J = 2.4, 1H), 4.26 (q, J = 4.8, 1H´),
4.03 (m, 2H, 2H´), 3.98 (m, 2H, 2H´), 3.58 (m, 2H, 2H´), 2.30 (q, J = 6.6, 2H, 2H´), 1.71 (m, 2H,
2H´), 1.60 (m, 2H, 2H´), 1.41 (m, 2H, 2H´), 1.16 (m, 3H, 3H´).13C NMR ((CD3)2SO, 400 MHz): δ
= 172.8, 159.8, 159.6, 156.1, 156.1, 152.7, 152.6, 149.0, 148.8, 139.8, 139.7, 128.5 (2C), 128.4
(2C), 128.0, 127.9, 119.1, 119.0, 114.2 (2C), 114.1 (2C), 106.5, 102.9, 89.5, 87.9, 86.3, 84.4,
83.6, 82.8, 82.5, 80.4, 67.4, 67.3, 61.5, 59.6, 33.4, 28.3, 25.0, 24.2, 14.1. Mass calculated for
C25H31N5O7 is 513.5429, found (M+H) = 514.62. 4b:(1.5 g, 42.5 %) 1H NMR ((CD3)2SO, 400
MHz): δ = 9.16 (d, J = 8.5, 1H), 7.28 (d, J = 9.0, 2H), 7.28 (d, J = 8.4, 2H´), 7.35 (s, 2H, 2H´),
6.17 (d, J = 9.0, 2H), 6.94 (d, J = 9.0, 2H´), 6.28 (d, J = 2.4, 1H), 6.25 (d, J = 3.0, 1H´), 6.17 (s, 1
H´), 5.96 (s, 1H), 5.46 (m, 1H, 1H´), 5.25 (t, J = 6.3, 1H), 5.13 (t, J = 5.7, 1H´), 5.05 (m, 1H,
1H´), 4.36 (q, J = 2.4, 1H), 4.26 (q, J = 4.8, 1H´), 4.03 (m, 2H, 2H´), 3.98 (m, 2H, 2H´), 3.58 (m,
2H, 2H´), 2.30 (q, J = 6.6, 2H, 2H´), 1.71 (m, 2H, 2H´), 1.60 (m, 2H, 2H´), 1.41 (m, 2H, 2H´),
1.16 (m, 3H, 3H´).13C NMR ((CD3)2SO, 400 MHz): δ = 173.5, 160.2, 158.6, 154.1, 155.2, 151.8,
151.1, 149.5, 147.6, 139.8, 139.7, 127.5 (2C), 129.4 (2C), 128.0, 127.9, 119.1, 119.0, 114.2
(2C), 114.1 (2C), 106.5, 102.9, 89.5, 87.9, 86.3, 84.5, 83.6, 83.1, 82.8, 80.9, 67.5, 67.2, 61.2,
59.7, 33.6, 28.3, 25.2, 24.1, 14.2. Mass calculated for C24H31N3O8 is 489.5182. Found (M+H) =
490.34.
General procedure for synthesis of (5). A solution of NaOH (195 mg, 4.87 mmol) in H2O (1
mL) and MeOH (3 mL) was added to a suspension of 4a/4b (1.0 mmol) in MeOH (4.5 mL),
irradiated by MW for 25 min at 100 oC, and concentrated in vacuo. The remaining residue was
dissolved in H2O, and was treated dropwise with 1 M HCl to pH 4. The white precipitate was
filtered and dried in vacuo to yield a white powder. 5a: (461 mg, 97.6 %); 1H NMR (CDCl3, 400
MHz): δ = 8.38 (s, 1H), 8.37 (s, 1H´), 8.16 (s, 1H), 8.15 (s, 1H´), 7.47 (d, J = 8.4, 2H), 7.40 (d, J
= 8.4, 2H´), 7.34 (s, 2H, 2H´), 5.98 (d, J = 8.4, 2H), 6.94 (d, J = 8.4, 2H´), 6.28 (d, J = 3.0, 1H),
6.26 (d, J = 3.6, 1H´), 6.16 (s, 1H), 5.94 (s, 1H), 5.45 (m, 1H, 1H´), 5.06 (m, 1H, 1H´), 4.36 (m, J
= 2.4, 1H), 3.96 (m, 2H, 2H´), 3.57 (m, 2H, 2H´), 2.15 (q, J = 6.6, 2H, 2H´), 1.70 (m, 2H, 2H´),
1.53 (m, 2H, 2H´), 1.40 (m, 2H, 2H´).13C NMR (CDCl3, 400 MHz): δ = 175.1, 159.8, 159.7,
156.1, 156.1, 152.7, 152.6, 149.0, 148.8, 139.8, 139.7, 128.5 (2C), 128.4 (2C), 128.0, 127.9,
119.1, 119.0, 114.2 (2C), 114.1 (2C), 106.5, 102.9, 89.5, 87.9, 86.3, 84.5, 83.6, 82.8, 82.6, 80.5,
67.5, 67.4, 61.5, 34.7, 28.5, 25.3, 24.7. Mass calculated for C23H27N5O7 is 485.4898, found
(M+H) 486.54. 5b: (461 mg, 97.6 %); 1H NMR (CDCl3, 400 MHz): δ = 9.16 (d, J= 6.9, 1H), 8.6
(s, 1H), 8.16 (s, 1H), 8.15 (s, 1H´), 7.27 (d, J = 8.4, 2H), 7.29 (d, J = 8.4, 2H´), 6.92 (d, J= 8.2,
2H, 2H´), 5.98 (d, J = 8.4, 2H), 6.94 (d, J = 8.4, 2H´), 6.28 (d, J = 3.0, 1H), 6.26 (d, J = 3.6, 1H´),
6.16 (s, 1H), 5.94 (s, 1H), 5.45 (m, 1H, 1H´), 5.06 (m, 1H, 1H´), 4.37 (m, J = 2.4, 1H), 3.96 (m,
2H, 2H´), 3.57 (m, 2H, 2H´), 2.17 (q, J = 6.6, 2H, 2H´), 1.72 (m, 2H, 2H´), 1.54 (m, 2H, 2H´),
1.41 (m, 2H, 2H´).13C NMR (CDCl3, 400 MHz): δ = 178.4, 165.5, 159.8, 155.1, 143, 129.6,
127.4, 115.1, 113.1, 100.2, 95.2, 88.1, 87.1, 84.8, 69.6, 62.3, 34.2, 28.7, 25.3, 24.3. Mass
calculated for C22H27N3O8 is 461.4651, found (M+H) 462.54.
General procedure for resin-bound nucleoside scaffold (6). Aminomethyl resin (864 mg,
0.50 mmol) was swelled in DMF (2 mL) for 20 minutes. A solution of 5a/5b (0.6 mmol), HBTU
(198 mg, 0.62 mmol), DIEA (108 µL, 0.62 mmol) in DMF (6 mL) was added to the swollen resin
in DMF and subjected to MW irradiation for 1.5 hr at 60 oC. Then the Resin was washed with
DMF and MeOH, and lyophilized to remove excess residual solvent. Nucleoside coupling to
solid phase resin was confirmed by LC/MS analysis. Resin bound nucleoside (6a/6b) 20mg
was taken and washed with 10%V/V acetic acid in water for 2 hours and the solid phase resin
was filtered with help of glass wool. Then the filtrate was lyophilized, added with 10mg alanine
as internal standard, solubilised in 1ml of CD3OD and NMR spectra were recorded (6a, 75%;
6b, 63%).
General procedure for synthesis of (7). Solid phase nucleoside (6a/6b) (0.4 mmol) was dried
under high vacuum and swelled in DMF (5 ml) for 20 minutes. Imidazole (68 mg, 1 mmol),
TBDMS-Cl (150mg, 1mmol) added to the reaction at 0C. The contents were stirred for 15 min
then switched to microwave and conducted the reaction at 45C, for 20min. Then the resin was
washed with DMF followed by methanol to remove residual imidazole and unreacted TBDMSCl. The resin was dried and under high vaccum. Resin bound 5’-TBDMS protected nucleoside
(7a/7b) 20mg was taken and washed with 10%V/V acetic acid in water for 2 hours and the solid
phase resin was filtered with help of glass wool. Then the filtrate was lyophilized, added with
10mg alanine as internal standard, solubilised in 1ml of CD3OD and NMR spectra were
recorded. 7a (85%) 1H NMR (CD3OD, 400 MHz): δ = 8.58 (s, 1H), 8.35 (s, 1H), 6.12 (s, 1H),
4.71 (m, 1H), 4.52(m, 1H), 4.40 (m, 1H), 1.01(s, 9H), 0.26(s, 6H.13C NMR (CD3OD, 400 MHz): δ
= 156.2, 152.4, 149.8, 140.3, 119.4, 97.5, 88.0, 74.2, 70.1, 63.2, 30.6, 25.9, 3.2. Mass
calculated for C16H27N5O4Si is 381.5022, found (M+H) 382.27; 7b (79%)
13
C NMR (CD3OD, 400
MHz): δ = 9.11(d, J=8.2, 1H), 5.93 (s, 1H), 4.52(s, 1H), 4.40(m, 1H), 0.99(s, 9H), 0.24(s, 6H.13C
NMR (CD3OD, 400 MHz): δ= 165.5, 155.2, 143.8, 100.3, 95.2, 86.7, 73.2, 70.6, 63.1, 30.6, 25.9,
2.4. Mass calculated for C15H27N3O5Si is 357.4775, found (M+H) 358.53.
General procedure for synthesis of (8). Resin bound 5’-TBDMS nucleoside (7a/7b) (0.35
mmol) was dried under high vacuum and swelled in DMF (6ml) for 20 minutes, added propargyl
bromide(238 mg, 2 mmol), N,N-Diisopropylethylamine (260mg, 2mmol) at room temperature.
The contents were stirred for 5 min then switched to microwave and conducted the reaction at
150 oC, for 15minutes. Then the resin was washed with DMF followed by methanol to remove
residual N,N-Diisopropylethylamine and un reacted prpargylbromide. The resin was dried and
under high vaccum. The resin bound 5’-TBDMS, N-alkylated nucleoside (8a/8b) 20mg was
taken and washed with 10%V/V acetic acid in water for 2 hours and the solid phase resin was
filtered with help of glass wool. Then the filtrate was lyophilized, added with 10mg alanine as
internal standard, solubilised in 1ml of CD3OD and NMR spectra were recorded. 8a (65%) 1H
NMR (CD3OD, 400 MHz): δ = 8.35 (s, 1H), 8.16 (s, 1H), 6.16 (d, J=6.3, 1H), 4.75 (m, 1H), 4.51
(m, 1H), 4.40 (m, 1H), 3.8 (s, 2H), 2.65 (t, J=5.8, 1H), 1.01(s, 9H), 0.26(s, 6H.13C NMR (CD3OD,
400 MHz): δ = 154.7, 152.4, 149.8, 140.3, 119.4, 97.3, 87.0, 79.5, 73.7, 73.2, 70.5, 63.0, 30.7,
30.4, 25.9, 3.2. Mass calculated for C19H29N5O4Si is 419.5502, found (M+H) 420.41.8b (54%) 1H
NMR (CD3OD, 400 MHz): δ = 9.16 (d, J=8.2, 1H), 5.93 (d, J=5.7, 1H), 5.4 (d, J=8.4, 1H),
4.51(m, 1H), 4.40 (m, 1H), 4.28 (m, 1H), 4.04 (m, 2H), 3.66 (m, 2H), 2.65 (s, 1H), 1.01(s, 9H),
0.26(s, 6H.13C NMR (CD3OD, 400 MHz): δ =158.9, 155.1, 143.8, 100.2, 95.7, 86.7, 73.4, 73.2,
70.6, 63.0, 25.9, 5.1. Mass calculated for C18H29N3O5Si is 395.5255, found (M+H) 396.73.
General procedure for synthesis of (9 and 10). Solid supported 5’O-TBDMS, N-alkyl
nucleoside, 8a/8b (0.1 mmol), was swelled in DMF for 20 mins. Added CuI (19mg, 0.1 mmol),
N,N-Diisopropylethylamine (130mg, 1mmol) and Benzyl azide/methyl 2-azidoacetate, (134mg,
1mmol/115mg, 1mmol respectively) to the reaction mixture and stirred for 10 min at room
temperature. Then the reaction was transferred to microwave reactor heated at 100oC for 15
minutes. Then the resin was washed with DMF followed by methanol to remove residual
reagents. The resin was dried and under high vaccum. The resin bound 5’-TBDMS, N-alkyl
trazolyl nucleoside (9a/10a/9b/10b) 20mg was taken and washed with 10%V/V acetic acid in
water for 2 hours and the solid phase resin was filtered with help of glass wool. Then the filtrate
was lyophilized, added with 10mg alanine as internal standard, solubilised in 1ml of CD3OD and
NMR spectra were recorded.
Optimization of conditions for the cleavage of nucleoside derivatives from the resin (11
and 12).
1. Cleavage under acidic or catalytic conditions:
The resin bound scaffold (100 µmol) was treated TFA, In(OTf)32,or acetic acid in D2O (total
volume: 2 mL) and subjected to MW irradiation under the conditions indicated in Table 1. The
reaction was then filtered through glass wool and washed with its respective solution (1mL).
The filtrates were combined and transferred to a NMR tube for direct evaluation of the cleaved
product yield as described in the general experimental section. The sample was evaporated in
vacuo and/or lyophilized overnight. The sample was characterized using analytical RP-HPLC
and ESI-LRMS.
2. Cleavage under oxidative conditions:
The resin bound adenosine scaffold (0.1 mmol) was treated with DDQ3 in 1:1 H2O/acetonitrile (2
mL) and subjected to the conditions indicated in Table 1. The reaction was then filtered through
glass wool and washed with 1:1 H2O/acetonitrile (1mL). The sample was evaporated in vacuo
and lyophilized overnight. The residue was dissolved in D2O (2 mL) and analyzed by NMR as
described in the general experimental section. The sample was characterized using analytical
RP-HPLC and ESI-LRMS.
Optimized procedure for the release of nucleoside derivative from resin (11 and 12).
The resin bound 5’-TBDMS, N-alkyl trazolyl nucleoside (9a/10a/9b/10b) (0.1mmol) was taken
and washed with 10%V/V acetic acid in water for 2 hours and the solid phase resin was filtered
with help of glass wool. Then the filtrate was lyophilized, added with 10mg alanine as internal
standard, solubilised in 1ml of CD3OD and NMR spectra were recorded. 11a (45%) 1H NMR
(CD3OD, 400 MHz): δ = 8.35 (s, 1H), 8.16 (s, 1H), 7.63 (s, 1H), 7.33 (m, 5H), 6.16 (d, J=8.2,
1H), 5.48 (s, 2H), 4.75 (s, 1H), 4.51 (m, 1H), 4.40 (m, 1H), 3.79 (m, 2H).
13
C NMR (CD3OD, 400
MHz): δ = 154.7, 152.4, 149.8, 140.3, 133.7, 130.7, 128.6, 125.7, 122.9, 119.4, 97.3, 87.4, 73.7,
70.5, 61.6, 57.3, 42.3. Mass calculated for C20H22N8O4 is 438.4399, found (M+H) 439.51. 12a
(52%) 1H NMR (CD3OD, 400 MHz): δ = 8.35 (s, 1H), 8.16 (s, 1H), 7.5 (s, 1H), 6.16 (d, J= 6.4,
1H), 5.12 (s, 2H), 4.75 (m, 1H), 4.51 (m, 1H), 4.4 0 (m, 1H), 4.36 (s, 2H), 3.79 (m, 2H), 3.68 (s,
3H).
13
C NMR (CD3OD, 400 MHz): δ = 166.1, 154.7, 152.4, 149.8, 140.3, 130.7, 122.9, 119.4,
97.3, 87.4, 73.7, 70.5, 61.6, 52.9, 51.6, 42.3. Mass calculated for C16H20N8O6 is 420.38, found
(M+H) 421.22. 11b (65%) 1H NMR (CD3OD, 400 MHz): δ = 9.16 (d, J=8.2, 1H), 7.63 (s, 1H),
7.23 (m, 5H), 5.93 (d, J=5.3, 1H), 5.48(s, 2H), 5.39 (d, J=7.9, 1H), 4.52 (m, 1H), 4.4(m, 1H),
4.28(s, 1H), 3.8 (m, 2H), 3.59 (s, 2H).
13
C NMR (CD3OD, 400 MHz): δ = 158.9, 155.1, 143.8,
133.7, 130.7, 128.6, 127.6, 125.7, 122.9, 100.2, 95.7, 87.0, 73.2, 70.6, 61.6, 57.3, 43.1. Mass
calculated for C19H22N6O5 is 414.4152, found (M+H) 415.38. 12b (62%) 1H NMR (CD3OD, 400
MHz): δ = 9.16 (d, J=7.8, 1H), 7.5 (s, 1H), 5.93 (d, J=5.4, 1H), 5.4 (d, J=8.1, 1H), 5.12 (s, 2H),
4.51 (m, 1H), 4.40 (m, 1H), 4.28 (m, 1H), 3.79 (m, 2H), 3.68 (s, 3H).
13
C NMR (CD3OD, 400
MHz): δ = 170.1, 158.9, 155.1, 143.8, 130.7, 123.1, 100.5, 96.1, 87.3, 73.2, 70.6, 2.9, 51.6,
44.1.Mass calculated for C15H20N6O7 is 396.3553, found (M+H) 397.42.