The synthesis of a menthol derivative of 2-aminopurine as a fluorescent DNA lesion

Article abstract of DOI:10.1016/j.tet.2009.10.023

An efficient synthetic route to the phosphoramidite of a menthol functionalized guanosine analog is presented. Two procedures were executed for the key introduction of the 6′-allyl menthyl moiety. Stille vinylation on 6-O-tosylguanosine followed by cross-

Full text of DOI:10.1016/j.tet.2009.10.023

Tetrahedron 65 (2009) 10430–10435
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The synthesis of a menthol derivative of 2-aminopurine as a fluorescent
DNA lesion
Wouter F.J. Hogendorf, Carlo P. Verhagen, Erik Malta, Nora Goosen, Herman S. Overkleeft,
*
*
Dmitri V. Filippov , Gijsbert A. Van der Marel
Leiden Institute of Chemistry, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient synthetic route to the phosphoramidite of a menthol functionalized guanosine analog is
presented. Two procedures were executed for the key introduction of the 60-allyl menthyl moiety. Stille
vinylation on 6-O-tosylguanosine followed by cross-metathesis using an excess of allyl menthyl ether
proved to be less efficient than a Stille coupling on the same tosylate using an advanced menthyl-allyl
stannane derivative. Incorporation of the modified nucleoside using the phosphoramidite method into
a DNA 50-mer proceeded uneventfully.
Received 28 May 2009
Received in revised form
18 September 2009
Accepted 8 October 2009
Available online 13 October 2009
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
2-aminopurine via an allyl spacer. Herein we present a synthetic
route toward suitably protected phosphoramidite 2 and its in-
Modified nucleic acids play an important role in biochemistry
and medicinal chemistry.1 Amongst the modifications, fluorescent
nucleosides are frequently exploited as reporters in structural
studies and for the elucidation of fundamental biological processes,
such as DNA repair.2 In this context we are engaged in a study on
nucleotide excision repair (NER), an evolutionary highly conserved
DNA repair mechanism, which recognizes a range of structurally
unrelated DNA lesions, including artificially made modifications.3,4
NER is a complicated process, in which recognition of a DNA lesion,
and subsequent DNA incision is accomplished by the action of three
proteins: UvrA, UvrB, and UvrC. A synthetic oligonucleotide,
equipped with a non-natural cholesterol modified deoxyribose as
a damage mimic and 2-aminopurine as fluorescence reporter was
recently applied to elucidate the DNA repair mechanism.5 It was
demonstrated that upon UvrB binding to the cholesterol lesion
both the base adjacent to the 30-end of the lesion and the base
opposite to the lesion in the undamaged strand are flipped out of
the DNA helix. To further investigate the function of base flipping in
the damage specific binding of UvrB in the NER process, the
availability of a fluorescent nucleotide that is recognized as
a damage site would be advantageous. Based on the fluorescent
properties of 2-aminopurine and our finding that a menthol-thy-
mine adduct is recognized as a damage,4 nucleoside 1 was
designed, in which a menthyl moiety is attached at C-6 of
corporation into DNA 50-mer I (Fig. 1).
2. Results and discussion
Key in the synthetic route of phosphoramidite 2 is an efficient
procedure to install the allyl menthyl moiety at C-6 of an easily
available nucleoside to obtain a suitably protected fluorescent
2-aminopurine derivative. Deoxyguanosine (dG) was selected as
a convenient starting compound as installation of a 6-O-tosyl
function in dG allows a number of transformations such as
palladium catalyzed cross-couplings.6 In this context, Sasaki and
co-workers reported in 1997 that 6-vinyl-2-aminopurine can be
prepared via Stille vinylation of 6-O-tosyl dG.6b It was envisaged
that implementation of this procedure would allow elongation of
* Corresponding authors.
E-mail addresses: filippov@chem.leidenuniv.nl (D.V. Filippov), marel_g@chem.
leidenuniv.nl (G.A. Van der Marel).
Figure 1. Chemical structures of nucleoside 1, phosphoramidite 2, and oligonucleotide I.
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.10.023

W.F.J. Hogendorf et al. / Tetrahedron 65 (2009) 10430–10435
10431
Scheme 1. Synthesis of adduct 6.
the vinyl entity at the C-6 of the purine base by means of cross-
metathesis reactions. Up to now, only productive metathesis re-
actions involving the ribose moiety of purine nucleosides have
been reported.7
give protected guanosine 3. The 6-O-position was subsequently
tosylated to provide 6-O-tosyl 4 in 70% over three steps.
Stille vinylation of 4 in refluxing dioxane, using 10 mol % of
Pd(PPh3)4 and 5 equiv of tributylvinyl stannane under inert atmo-
sphere gave 6-vinylpurine 5 in 84% yield. The crucial cross-me-
tathesis of 6-vinylpurine 5 with 5 equiv of allyl menthyl ether
(prepared from sodium menthoxide and allyl bromide), using
Grubbs second generation catalyst9,10 (10 mol %) gave E-adduct 6 in
22% yield while the corresponding Z-adduct could not be detected.
The low yield of target compound 6 together with the lack of
starting alkene 5 and formation of baseline material, as monitored
by TLC analysis, suggested substantial dimerization of 5. Un-
fortunately, additional treatment of the reaction mixture, after total
consumption of starting compound 5, with both 10 mol % of Grubbs
catalyst and 5 equiv of allyl menthyl ether did not improve the
outcome.
Our route of synthesis started with the protection of the hy-
droxyls of 20-deoxyguanosine by tert-butyldimethylsilyl (TBS)
groups, that were considered to be sufficiently stable to survive
upcoming transformations (Scheme 1). The relatively base labile
phenoxyacetyl (PAC) protecting group was selected to protect the
exocyclic amine function of the purine to avoid prolonged ammonia
treatment and, therefore, reduce possible side reactions at the final
deprotection stage of the solid-phase synthesis of the
oligonucleotide.8 Introduction of the PAC group with phenoxyacetyl
chloride in a mixture of pyridine and DMF proved to be troublesome.
Contrary, when performed in DCM at 0 ꢀC using the less reactive
phenoxyacetic anhydride, the reaction proceeded uneventfully to
Scheme 2. Improved route to phosphoramidite 2.

10432
W.F.J. Hogendorf et al. / Tetrahedron 65 (2009) 10430–10435
The disappointing outcome of the cross-metathesis reaction and
4. Experimental
4.1. General
the favorable preceding Stille coupling with tributylvinyl stannane
stimulated us to explore the introduction of the allyl menthyl moiety
by a Stille coupling of tosylate 4 and the required tributyl stannane
derivative of menthol (8, Scheme 2). Stannane 8 can be obtained from
All chemicals (Acros, Fluka, Merck, Schleicher & Schuell, Sigma–
Aldrich) were used as received. Reactions were carried out dry,
under an argon atmosphere and at ambient temperature, unless
stated otherwise. The commercially available reagents for DNA
synthesis were all from Proligo. Column chromatography was
performed on Merck silica gel 60 (0.040–0.063 mm). TLC analysis
was conducted on DC-fertigfolien (Schleicher & Schuell, F1500,
LS254) or HPTLC aluminum sheets (Merck, silica gel 60, F245).
Compounds were visualized by UV absorption (245 nm), by
spraying with 20% H2SO4 in ethanol or with a solution of
(NH4)6Mo7O24$4H2O 25 g/l, (NH4)4Ce(SO4)4$2H2O 10 g/l,10% H2SO4
in H2O followed by charring at ꢂ140 ꢀC. Some unsaturated com-
L
-(ꢁ)-menthol by propargylation and subsequent hydrostannylation
of intermediate alkyne 7. Propargylation4 of
L-(ꢁ)-menthol using so-
dium hydride and propargylbromide led to the isolation of prop-
argylmenthyl ether 7 in modest yield (42%) and purity (90–95%).
Performing this transformation using a twofold excess of commer-
cially available (3-bromoprop-1-ynyl)trimethylsilane and subsequent
desilylation of the intermediate with K2CO3 in MeOH furnished
propargyl ether 7 of higher purity (>99%), albeit in lower yield (26%).
Hydrostannylationof alkyne 7 (purity>99%), usingtributyltinhydride
and azobisisobutyronitrile (AIBN) as initiator provided 8 in 82% yield
(E/Z ratio: w5/1). When the less pure alkyne 7 was used, the stannane
product was acquired in a substantially lower yield (50–55%).
Gratifyingly, Stille reaction of stannyl derivative 8 (1.5 equiv)
and tosylguanosine 4 for 2.5 h in boiling dioxane gave target
E-adduct 6 as the sole product in 75% yield. To obtain phosphor-
amidite 2, suitable for application in automated solid-phase oli-
gonucleotide synthesis, the following sequence of reaction was
executed. Desilylation of 6 with tetrabutylammonium fluoride
(TBAF) to diol 9 was followed by regioselective dimethoxy-
tritylation giving 10, which was subsequently functionalized using
2-cyanoethyl-(N,N-diisopropylamino)-chlorophosphite in the
presence of DIPEA, yielding phosphoramidite 2 in 78% over three
steps. For fluorescence studies, a small part of diol 9 was depro-
tected with aqueous NH3, yielding compound 1. Nucleoside 1
was compared with 2-amino-9-(20-deoxyribofuranosyl)-purine
(2-AP).16 The results are summarized in Table 1.
pounds were visualized by spraying with a solution of KMnO4 (2%)
20
and K2CO3 (1%) in water. Optical rotation measurements ([a] )
D
were performed on a Propol automated polarimeter (Sodium D-
line,
¼589 nm) in CHCl3 with a concentration of 10 mg/ml (c¼1),
unless stated otherwise. Infrared spectra were recorded on a Shi-
madzu FTIR 8300 and data are reported in cmꢁ1 1H, 13C and 31P
l
.
NMR spectra were recorded with a Bruker AC200 (200, 50 and
80.7 MHz, respectively), a Bruker AV 500 (500 and 125 MHz, re-
spectively) or a Bruker DMX 600 (600 and 150 MHz, respectively).
NMR spectra were recorded in CDCl3 with chemical shift (
to tetramethylsilane, unless stated otherwise. High resolution mass
spectra were recorded by direct injection (2 l of a 2 M solution in
d) relative
m
m
water/acetonitrile; 50/50; v/v and 0.1% formic acid) on a mass
spectrometer (Thermo Finnigan LTQ Orbitrap) equipped with an
electrospray ion source in positive mode (source voltage 3.5 kV,
sheath gas flow 10, capillary temperature 250 ꢀC) with resolution
R¼60,000 at m/z 400 (mass range m/z¼150–2000) and dioctylph-
thalate (m/z¼391.28428) as a lock mass. The high resolution mass
spectrometer was calibrated prior to measurements with a cali-
bration mixture (Thermo Finnigan).
Table 1
Fluorescence parameters of 1 compared to 2-APa
2-AP (H2O)b
2-AP (MeOH)
1 (MeOH)
lmax abs.
lmax exc.
lmax emi.
305
307
367
0.68
309
312
366
0.44
323
343
439
0.20
4.2. Experimental procedures
F
ꢂ0.01
a
Compound 1 was prepared by treating 9 with 20% NH3 in dioxane/H2O (1/4),
4.2.1. 30,50-Di-O-t-butyldimethylsilyl-20-deoxyguanosine. 20-Deox-
yguanosine (14.2 g, 53.1 mmol) and imidazole (10.0 g, 149 mmol)
were suspended in DMF (450 ml) and cooled to 0 ꢀC. After addition
of TBS-Cl (20.0 g, 133 mmol) and a catalytic amount of DMAP, the
reaction was allowed to stir for 6 h. The solvents were evaporated
and the residue taken up in DCM (400 ml). The solution was washed
with aq HCl (1 M), until the pH became slightly acidic. The organic
layer was washed once with satd aq NaHCO3 (150 ml), and dried
with MgSO4. The solution was concentrated in vacuo and yielded an
amber waxy solid, that was washed with Et2O yielding the title
followed by column chromatography (68% yield).
b
Results are comparable with literature values.16
Phosphoramidite 2 was applied in automated solid-phase DNA
synthesis and proved to be as efficient in the phosphitylation step
as the commercially available phosphoramidites of the common
bases, allowing the assembly of the fully protected and immobi-
lized progenitor of 50-mer I by a standard solid-phase protocol.11,12
Removal of all the protecting groups and concomitant release from
the solid support was effected by treatment with aqueous ammonia
to give, after purification with anion exchange HPLC and desalting
by gel filtration, homogeneous 50-mer I. In a first study with 50-
mer I, it was shown that upon binding of UvrB the modified and
fluorescent base of nucleoside 1 is not flipping but retains its
original intra-helical position, as reported elsewhere.13
compound (22.9 g, 87%) as
a
white solid. Mp >294–295 ꢀC
0.07 (s, 6H, 2ꢃCH3 TBS),
(decomp.)6b; 1H,1H-COSY NMR (500 MHz):
d
0.11 (s, 6H, 2ꢃCH3 TBS), 0.85 (s, 18H, 2ꢃt-butyl TBS), 2.34–2.39 (m,
1H, H-20), 2.43–2.49 (m, 1H, H-20), 3.74–3.82 (m, 2H, H-50, H-50), 3.98
(dd, 1H, J¼3.5 Hz, 7.0 Hz, H-40), 4.57 (m, 1H, H-30), 6.23 (t, 1H,
J¼6.5 Hz, H-10), 7.84 (s, 1H, H-8).
3. Conclusion
4.2.2. 2-N-Phenoxyacetyl-30,50-di-O-t-butyldimethylsilyl-20-deoxy-
guanosine (3). Silylated dG (2.48 g, 5.00 mmol) was dissolved in
DCM (250 ml) and cooled to 0 ꢀC. Et3N (5.55 ml, 40.0 mmol) and
phenoxyacetic acid anhydride (PAC2O, 4.29 g, 15.0 mmol, made
from phenoxyacetic acid and DCC) were subsequently added to
the reaction mixture. After stirring for 10 min, a catalytic amount
of DMAP was added and the reaction was stirred for another 72 h
at ambient temperature. The solution was washed with aq HCl
(1 M) until the pH was slightly acidic. After washing with satd aq
NaHCO3 (100 ml), the organic phase was dried with MgSO4 and
In summary, a novel modified nucleoside has been prepared and
incorporated in an oligonucleotide by the application of phos-
phoramidite 2. Two routes of synthesis, using deoxyguanosine as
starting compound were explored to obtain building-block 2. The
first route entailed eight steps, including a Stille vinylation and
a cross-metathesis reaction to give amidite 2 in 10% overall yield.
The second route avoided the inefficient metathesis reaction and
used advanced stannane 8 in the crucial Stille coupling to afford
amidite 2 in 41% overall yield.

W.F.J. Hogendorf et al. / Tetrahedron 65 (2009) 10430–10435
10433
concentrated in vacuo. Silica gel column chromatography (EtOAc/
(C-40), 114.9, 122.2 (CHarom), 126.8 (CH2 vinyl), 128.9 (Cq PAC), 129.8
(CHarom), 131.4 (CH vinyl),142.9 (C-8),151.6,152.4–152.6,154.3 (C-2,
C-4, C-5, C-6); HRMS: C32H49N5O5Si2þHþ calculated 640.3345,
found 640.3346.
PE) afforded 3 (2.84 g, 90%) as a yellow foam. [
a
]
20: þ1.0; IR:
D
1096, 1115, 1250, 1607, 1678; 1H,1H-COSY NMR (500 MHz):
d 0.11
(s, 6H, 2ꢃCH3 TBS), 0.13 (s, 6H, 2ꢃCH3 TBS), 0.92 (s, 18H, 2ꢃt-
butyl TBS), 2.41–2.44 (m, 1H, H-20), 2.48 (ddd, 1H, J¼6.5 Hz,
6.5 Hz, 13.0 Hz, H-20), 3.75–3.81 (m, 2H, H-50, H-50), 3.99 (dd, 1H,
J¼3.5 Hz, 6.5 Hz, H-40), 4.59–4.61 (m, 1H, H-30), 4.71 (s, 2H, CH2
PAC), 6.29 (t, 1H, J¼6.5 Hz, H-10), 7.00–7.12 (m, 3H, Harom), 7.36–
7.39 (m, 2H, Harom), 8.02 (s, 1H, H-8), 9.22 (br s, 1H, NHPAC); 13C
4.2.5. 2-N-Phenoxyacetyl-6-(3-O-
t-butyldimethylsilyl-20-deoxy-
-ribofuranosyl)purine (6). A solu-
L
-menthyl-propenyl)-9-(30,50-di-O-
b-D
tion of 5 (115 mg, 0.180 mmol) and allyl menthyl ether (177 mg,
0.902 mmol) in DCM (6.0 ml) was stirred under argon for 30 min.
Grubbs second generation catalyst (30 mg, 0.035 mmol) was added
and the resulting mixturewas gently refluxed (w45 ꢀC) for 1 h. After
the addition of a second portion of catalyst (30 mg, 0.035 mmol), the
mixture was refluxed for another hour. Evaporation of the solvent
and purification by silica gel column chromatography (EtOAc/PE)
NMR (125 MHz):
d 17.9 (Cq t-butyl TBS), 18.3 (Cq t-butyl TBS),
25.7–25.9 (4ꢃCH3 TBS, 2ꢃt-butyl CH3 TBS), 41.4 (C-20), 62.6 (C-
50), 67.0 (CH2 PAC), 71.6 (C-30), 83.5 (C-10), 87.9 (C-40), 114.8, 123.0,
129.2 (CHarom), 129.2 (C-8), 146.0, 147.5, 155.3, 156.4 (C-2, C-4, C-
5, Cq PAC), 169.5 (C-6); HRMS: C30H47N5O6Si2þHþ calculated
630.3138, found 630.3140.
yielded 6 (32 mg, 22%) as a colorless oil. [
a
]
D
20: ꢁ12.3; IR: 777, 833,
1068, 1213, 1495, 1587, 1709, 2928; 1H,1H-COSY NMR (600 MHz):
4.2.3. 2-N-Phenoxyacetyl-6-O-p-toluenesulphonyl-30,50-di-O-t-
butyldimethylsilyl-20-deoxyguanosine (4). To a cooled (0 ꢀC) solu-
tion of 3 (3.15 g, 5.00 mmol) and Et3N (1.40 ml, 10.1 mmol) in
DCM (100 ml) was added tosylchloride (1.91 g, 10.0 mmol), fol-
lowed by a catalytic amount of DMAP. After TLC analysis showed
conversion to a less polar material, the reaction was quenched
with H2O (10 ml). The organic phase was washed twice with H2O
(30 ml) and once with satd aq NaCl (20 ml) before it was dried
with MgSO4. After concentration in vacuo, silica gel column
chromatography (EtOAc/PE) yielded 4 (3.50 g, 89%) as a slightly
d
0.08 (s, 6H, 2ꢃCH3 TBS), 0.12 (s, 6H, 2ꢃCH3 TBS), 0.78 (d, 3H,
J¼7.2 Hz, CH3 menthol), 0.89–1.00 (m, 27H, 2ꢃt-butyl TBS, 2ꢃCH3
menthol, 3ꢃCHH menthol),1.28–1.36 (m, 2H, 2ꢃCH menthol),1.62–
1.66 (m, 2H, 2ꢃCHH menthol), 2.14 (m,1H, CHH menthol), 2.30–2.33
(m, 1H, CH menthol), 2.44–2.48 (m, 1H, H-20), 2.71–2.73 (m, 1H, H-
20), 3.17 (ddd, 1H, J¼3.9 Hz, 10.7 Hz, 14.7 Hz, CH menthol), 3.79 (dd,
1H, J¼3.3 Hz,11.1 Hz, H-50), 3.88 (dd,1H, J¼4.2 Hz,11.4 Hz, H-50), 4.02
(dd, 1H, J¼3.3 Hz, 6.9 Hz, H-40), 4.23 (ddd, 1H, J¼1.5 Hz, 4.3 Hz,
15.3 Hz, CH2 propenyl), 4.45 (ddd, 1H, J¼1.8 Hz, 4.8 Hz, 15.0 Hz, CH2
propenyl), 4.64–4.65 (m, 1H, H-30), 4.80 (br s, 2H, CH2 PAC), 6.48 (t,
1H, J¼6.3 Hz, H-10), 7.02–7.06 (m, 3H, Harom), 7.18 (d, 1H, J¼16.2 Hz,
CH-200 propenyl), 7.33–7.36 (m, 2H, Harom), 7.64 (ddd, 1H, J¼4.8 Hz,
4.8 Hz, 15.6 Hz, CH-100 propenyl), 8.28 (s, 1H, H-8); 13C NMR
yellow foam. [
a
]
20: þ23.8; IR: 1026, 1177, 1383, 1495, 1587, 1711,
D
2856, 2928; 1H,1H-COSY NMR (500 MHz):
d
0.10 (s, 6H, 2ꢃCH3
TBS), 0.11 (s, 6H, 2ꢃCH3 TBS), 0.89 (s, 18H, 2ꢃt-butyl TBS), 2.42 (s,
3H, CH3 tosyl), 2.43–2.48 (m, 1H, H-20), 2.66 (ddd, 1H, J¼6.4 Hz,
6.4 Hz, 12.8 Hz, H-20), 3.78 (dd, 1H, J¼3.3 Hz, 11.3 Hz, H-50), 3.89
(dd, 1H, J¼4.0 Hz, 11.5 Hz, H-50), 4.01 (dd, 1H, J¼3.5 Hz, 7.0 Hz, H-
40), 4.62–4.64 (m, 1H, H-30), 4.78 (br s, 2H, CH2 PAC), 6.43 (t, 1H,
J¼6.5 Hz, H-10), 7.02–7.08 (m, 3H, Harom), 7.27–7.38 (m, 4H,
Harom), 8.09 (d, 1H, J¼8.5 Hz, Harom), 8.09 (d, 1H, J¼8.5 Hz, Harom),
(150 MHz):
d 16.2 (CH3 menthol), 18.0 (Cq t-butyl TBS), 18.4 (Cq t-
butyl TBS), 21.0 (CH3 menthol), 22.3 (CH3 menthol), 23.3 (CH2
menthol), 25.5 (CH menthol), 25.8–25.9 (4ꢃCH3 TBS, 2ꢃt-butyl CH3
TBS), 31.5 (CH menthol), 34.5 (CH2 menthol), 40.3 (CH2 menthol),
41.0 (C-20), 48.2 (CH menthol), 62.8 (C-50), 68.0 (CH2 propenyl), 68.3
(CH2 PAC), 71.9 (C-30), 79.2 (CH menthol), 84.2 (C-10), 88.0 (C-40),
114.9 (CHarom), 122.2 (CHarom), 124.6 (CH-10 propenyl), 128.7 (Cq
PAC),129.8 (CHarom),141.5 (CH-20 propenyl),142.6 (C-8),151.3,152.3,
154.4, 157.2 (C-2, C-4, C-5, C-6); HRMS: C43H69N5O6Si2þHþ calcu-
lated 808.4859, found 808.4865.
8.31 (s, 1H, H-8); 13C NMR (125 MHz):
d 17.9 (Cq t-butyl TBS), 18.3
(Cq t-butyl TBS), 25.7–25.9 (4ꢃCH3 TBS, 2ꢃt-butyl CH3 TBS), 41.1
(C-20), 62.6 (C-50), 67.9 (CH2 PAC), 71.6 (C-30), 84.6 (C-10), 88.1 (C-
40), 114.5 (CHarom), 120.2 (Cq tosyl), 122.2, 129.1, 129.6, 129.7
(CHarom), 133.4 (Cq tosyl), 143.2 (C-8), 145.8, 150.3, 154.0, 154.5,
157.1 (C-2, C-4, C-5, C-6, Cq PAC); HRMS: C37H53N5O8SSi2þHþ
calculated 784.3226, found 784.3233.
4.2.6. L L-Menthol (7.81 g,
-Menthyl propargyl ether (7)4. Method a:
50.0 mmol) and tetra-n-butyl ammonium iodide (1.40 g,
3.79 mmol) were dissolved in DMF (300 ml) and cooled to 0 ꢀC.
NaH (60% dispersion in mineral oil, 2.4 g, 60 mmol) and prop-
argylbromide (80% solution in toluene, 21.5 ml, 200 mmol) were
slowly added. The mixture was allowed to stir at room temperature
for 96 h. The reaction was quenched with MeOH (20 ml) and con-
centrated in vacuo. The oily residue was taken up in Et2O (400 ml)
and washed twice with H2O (200 ml) and once with satd aq NaCl
(100 ml), before it was dried over MgSO4. After evaporation of the
solvent, silica gel column chromatography (toluene/PE) gave 7
(4.06 g, 42%) as a slightly yellow oil. Method b: To a flask equipped
4.2.4. 2-N-Phenoxyacetyl-6-vinyl-9-(30,50-di-O-t-butyldimethylsilyl-
20-deoxy-
b-D-ribofuranosyl) purine (5). A solution of LiCl (165 mg,
3.90 mmol), tri-n-butyl-vinylstannane (3.00 ml, 10.2 mmol), and 4
(1.53 g, 1.95 mmol) in dioxane (26 ml) was stirred under argon for
30 min. After the addition of Pd(PPh3)4 (0.25 g, 0.22 mmol), the
mixture was heated under reflux for 1.5 h. After concentration in
vacuo, the residue was taken up in EtOAc (100 ml), and washed
successively with aqueous NH3 (2.5% v/v, 20 ml) and satd aq NaCl
(20 ml). The organic layer was dried with MgSO4 and concentrated
to give the crude product, which was purified by silica gel column
chromatography (EtOAc/PE) to yield 5 (1.25 g, 84%) as an amor-
with L-menthol (0.312 g, 2.00 mmol) in THF (10 ml) was added NaH
(60% dispersion in mineral oil, 0.12 g, 3.0 mmol). After stirring at
room temperature for 30 min TMS-propargylbromide (0.627 ml,
4.00 mmol) was added. After 96 h K2CO3 (5.0 g, 36 mmol) and
MeOH (5.0 ml) were added and the mixture stirred for another 3 h.
The mixture was then diluted with Et2O (40 ml) and washed with
H2O (20 ml) and satd aq NaCl (20 ml) before it was dried with
MgSO4. The organic layer was concentrated under reduced pressure
and the resulting oil purified by silica gel column chromatography
(Et2O/pentane). Menthyl propargyl ether 7 (99 mg, 26%) was
phous transparent yellow solid. [
a
]
20: þ16.6; IR: 1070, 1215, 1252,
D
1497, 1587, 2361, 2856, 2928; 1H,1H-COSY NMR (500 MHz):
d 0.09
(s, 6H, 2ꢃCH3 TBS), 0.12 (s, 6H, 2ꢃCH3 TBS), 0.92 (s, 18H, 2ꢃt-butyl
TBS), 2.44–2.49 (m, 1H, H-20), 2.72 (ddd, 1H, J¼6.4 Hz, 6.4 Hz,
12.8 Hz, H-20), 3.79 (dd, 1H, J¼3.3 Hz, 11.3 Hz, H-50), 3.89 (dd, 1H,
J¼4.0 Hz, 11.5 Hz, H-50), 4.02 (dd, 1H, J¼3.5 Hz, 7.0 Hz, H-40), 4.65
(ddd, 1H, J¼3.8 Hz, 3.8 Hz, 6.5 Hz, H-30), 4.80 (br s, 2H, CH2 PAC),
5.96 (dd, 1H, J¼1.5 Hz, 11.0 Hz, CH2 vinyl), 6.49 (t, 1H, J¼6.5 Hz, H-
10), 6.99–7.07 (m, 3H, Harom, CH2 vinyl), 7.23–7.37 (m, 4H, Harom, CH
obtained as an colorless oil. [
a]
20: ꢁ116.6; IR: 1072, 1084, 1454,
D
vinyl), 8.30 (s, 1H, H-8); 13C NMR (125 MHz):
d
18.0 (Cq t-butyl TBS),
2920, 3310; 1H,1H-COSY NMR (200 MHz):
d
0.78–0.97 (m, 12H,
18.4 (Cq t-butyl TBS), 25.7–25.9 (4ꢃCH3 TBS, 2ꢃt-butyl CH3 TBS),
3ꢃCH3 menthol, 3ꢃCHH menthol), 1.10–1.42 (m, 2H, 2ꢃCH men-
41.0 (C-20), 62.8 (C-50), 68.0 (CH2 PAC), 71.9 (C-30), 84.2 (C-10), 88.0
thol), 1.57–1.69 (m, 2H, 2ꢃCHH menthol), 2.07–2.30 (m, 2H, CH

10434
W.F.J. Hogendorf et al. / Tetrahedron 65 (2009) 10430–10435
menthol, CHH menthol), 2.36–2.42 (m, 1H, CH propyn), 3.27 (ddd,
1H, J¼4.4 Hz, 11.0 Hz, 14.6 Hz, CH menthol), 4.09–4.28 (m, 2H, CH2
propyn).
(br s, 2H, CH2 PAC), 6.48 (t, 1H, J¼6.3 Hz, H-10), 7.02–7.06 (m, 3H,
Harom), 7.18 (d, 1H, J¼16.2 Hz, CH-200 propenyl), 7.33–7.36 (m, 2H,
Harom), 7.64 (ddd,1H, J¼4.8 Hz, 4.8 Hz,15.6 Hz, CH-100 propenyl), 8.28
(s, 1H, H-8); 13C NMR (150 MHz):
d 16.2 (CH3 menthol), 18.0 (Cq t-
4.2.7. 3-Tri-n-butyltin-2-propenyl menthyl ether (8). To a flask,
charged with 7 (486 mg, 2.50 mmol) in toluene (7.5 ml), were
added tri-n-butyltin hydride (800 mg, 2.75 mmol) and 2,20-azo-
bis(2-methylpropionitrile) (50 mg, 0.30 mmol). The mixture was
stirred overnight at 85 ꢀC before it was allowed to cool to room
temperature. The reaction mixture was diluted with Et2O (40 ml)
and washed with H2O (20 ml) and satd aq NaCl (20 ml). The organic
layer was dried with MgSO4 and concentrated in vacuo. The crude
product was then obtained as a mixture of regioisomers (w9/1, 3-
stannyl (8)/2-stannyl) that could be separated by silica gel column
chromatography (Et2O/PE/pentane). 8 (996 mg, 82%, w5/1 E/Z) was
butyl TBS), 18.4 (Cq t-butyl TBS), 21.0 (CH3 menthol), 22.3 (CH3
menthol), 23.3 (CH2 menthol), 25.5 (CH menthol), 25.8–25.9 (4ꢃCH3
TBS, 2ꢃt-butyl CH3 TBS), 31.5 (CH menthol), 34.5 (CH2 menthol), 40.3
(CH2 menthol), 41.0 (C-20), 48.2 (CH menthol), 62.8 (C-50), 68.0 (CH2
propenyl), 68.3 (CH2 PAC), 71.9 (C-30), 79.2 (CH menthol), 84.2 (C-10),
88.0 (C-40), 114.9 (CHarom), 122.2 (CHarom), 124.6 (CH-10 propenyl),
128.7 (Cq PAC), 129.8 (CHarom), 141.5 (CH-20 propenyl), 142.6 (C-8),
151.3, 152.3, 154.4, 157.2 (C-2, C-4, C-5, C-6); HRMS:
C43H69N5O6Si2þHþ calculated 808.4859, found 808.4865.
4.2.9. 2-N-Phenoxyacetyl-6-(3-O-
b-D-ribofuranosyl)purine (9). A solution of 6 (1.00 g, 1.24 mmol) in
L
-menthyl-propenyl)-9-(20-deoxy-
acquired as a colorless oil. [
a]
20: ꢁ41.6; IR: 758, 1084, 1456, 2920,
D
2955; 1H,1H-COSY NMR (500 MHz): E-product:
d
0.77–0.98 (m, 27H,
THF (50 ml) was cooled to 0 ꢀC. After adding TBAF (1 M solution in
THF, 3.7 ml) the mixture was stirred for 2 h at room temperature.
After evaporation of the solvent, silica gel column chromatography
(MeOH/DCM) afforded 9 (681 mg, 95%) as an amorphous green
3ꢃCH3 menthol, 3ꢃCHH menthol, 3ꢃCH3 Bu3Sn, 3ꢃCH2 Bu3Sn),
1.22–1.34 (m, 8H, 2ꢃCH menthol, 3ꢃCH2 Bu3Sn), 1.43–1.52 (m, 6H,
3ꢃCH2 Bu3Sn), 1.59–1.65 (m, 2H, 2ꢃCHH menthol), 2.08–2.10 (m,
1H, CHH menthol), 2.23–2.27 (m, 1H, CH menthol), 3.08 (ddd, 1H,
J¼4.3 Hz, 10.8 Hz, 14.8 Hz, CH menthol), 3.91–3.95 (m, 1H, CH2 al-
lyl), 4.12–4.15 (m, 1H, CH2 allyl), 6.06–6.09 (m, 1H, CH allyl), 6.16 (d,
solid. [
a
]
D
20: ꢁ21.6; IR: 748, 1088, 1227, 1350, 1497, 1589, 1666, 2924;
1H,1H-COSY NMR (600 MHz):
d
0.78–0.99 (m, 12H, 3ꢃCH3 menthol,
3ꢃCHH menthol), 1.26–1.36 (m, 2H, 2ꢃCH menthol), 1.62–1.67 (m,
2H, 2ꢃCHH menthol), 2.12–2.14 (m, 1H, CHH menthol), 2.28–2.30
(m, 1H, CH menthol), 2.42–2.45 (m, 1H, H-20), 3.07 (ddd, 1H,
J¼6.9 Hz, 6.9 Hz, 13.5 Hz, H-20), 3.17 (ddd, 1H, J¼4.2 Hz, 10.8 Hz,
14.4 Hz, CH menthol), 3.86–3.88 (m, 1H, H-50), 3.96–3.98 (m, 1H, H-
50), 4.17 (m, 1H, H-40), 4.22 (dd,1H, J¼3.6 Hz,15.6 Hz, CH2 propenyl),
4.45 (dd, 1H, J¼3.0 Hz, 15.6 Hz, CH2 propenyl), 4.73 (br s, 2H, CH2
PAC), 5.02 (br s, 1H, H-30), 6.38 (t, 1H, J¼6.9 Hz, H-10), 7.03–7.08 (m,
3H, Harom), 7.16 (d, 1H, J¼15.6 Hz, CH-100 propenyl), 7.34–7.37 (m,
2H, Harom), 7.63 (ddd, 1H, J¼4.8 Hz, 4.8 Hz, 15.6 Hz, CH-200 pro-
1H, J¼19.0 Hz, CH allyl terminus); Z-product:
d 0.77–0.98 (m, 27H,
3ꢃCH3 menthol, 3ꢃCHH menthol, 3ꢃCH3 Bu3Sn, 3ꢃCH2 Bu3Sn),
1.22–1.34 (m, 8H, 2ꢃCH menthol, 3ꢃCH2 Bu3Sn), 1.43–1.52 (m, 6H,
3ꢃCH2 Bu3Sn), 1.59–1.65 (m, 2H, 2ꢃCHH menthol), 2.08–2.10 (m,
1H, CHH menthol), 2.23–2.27 (m, 1H, CH menthol), 3.06–3.11 (m,
1H, CH menthol), 3.84–3.88 (m, 1H, CH2 allyl), 4.06–4.09 (m, 1H,
CH2 allyl), 6.02–6.05 (d, 1H, J¼11.5 Hz, CH allyl terminus), 6.60–6.64
(m, 1H, CH allyl); 13C NMR (125 MHz): E-product:
d
9.5 (3ꢃCH2
Bu3Sn), 13.7 (3ꢃCH2 Bu3Sn), 16.2 (3ꢃCH3 menthol), 21.0 (CH3
menthol), 22.4 (CH3 menthol), 23.4 (CH2 menthol), 25.5 (CH men-
thol), 27.3 (3ꢃCH2 Bu3Sn), 29.1 (3ꢃCH2 Bu3Sn), 31.6 (CH menthol),
34.6 (CH2 menthol), 40.6 (CH2 menthol), 48.3 (CH menthol), 72.5
(CH2 allyl), 78.3 (CH menthol), 130.4 (CH allyl terminus), 145.8 (CH
penyl), 8.11 (s, 1H, H-8); 13C NMR (150 MHz):
d 16.2 (CH3 menthol),
21.0 (CH3 menthol), 22.3 (CH3 menthol), 23.3 (CH2 menthol), 25.5
(CH menthol), 31.5 (CH menthol), 34.5 (CH2 menthol), 40.3 (CH2
menthol), 40.8 (C-20), 48.2 (CH menthol), 62.7 (C-50), 67.5 (CH2
PAC), 68.2 (CH2 propenyl) 72.1 (C-30), 79.4 (CH menthol), 86.3 (C-
10), 88.4 (C-40), 114.7 (CHarom), 122.4 (CHarom), 124.2 (CH-10 pro-
penyl), 129.7 (Cq PAC), 129.9 (CHarom), 142.1 (CH-20 propenyl), 145.0
(C-8), 151.0, 151.8, 155.0, 157.0 (C-2, C-4, C-5, C-6); HRMS:
C31H41N5O6þHþ calculated 580.3130, found 580.3128.
allyl); Z-product:
d
9.5 (3ꢃCH2 Bu3Sn), 13.7 (3ꢃCH2 Bu3Sn), 16.2
(3ꢃCH3 menthol), 21.0 (CH3 menthol), 22.4 (CH3 menthol), 23.4
(CH2 menthol), 25.5 (CH menthol), 27.3 (3ꢃCH2 Bu3Sn), 29.1
(3ꢃCH2 Bu3Sn), 31.6 (CH menthol), 34.6 (CH2 menthol), 40.6 (CH2
menthol), 48.3 (CH menthol), 71.9 (CH2 allyl), 78.7 (CH menthol),
131.1 (CH allyl terminus), 145.6 (CH allyl); HRMS: C25H50OSnþHþ
calculated 487.2961, found 487.2953.
4.2.10. 2-N-Phenoxyacetyl-6-(3-O-
L
-menthyl-propenyl)-9-(50-O-
-ribofuranosyl)purine
[4,40-dimethoxytrityl]-20-deoxy-
b-D
4.2.8. 2-N-Phenoxyacetyl-6-(3-O-
L
-menthyl-propenyl)-9-(30,50-di-O-
(10). Purine 9 (406 mg, 0.700 mmol) was dissolved in DCM (25 ml)
and cooled to 0 ꢀC. Subsequently, Et3N (0.20 ml, 1.4 mmol) and
DMT-Cl (356 mg, 1.05 mmol) were added and the reaction was
stirred for 3 h. After quenching with MeOH (5 ml) the mixture was
concentrated in vacuo. Purification of the crude product by silica gel
column chromatography (MeOH/DCM/Et3N) gave 10 (603 mg, 98%)
t-butyldimethylsilyl-20-deoxy-
b-D-ribofuranosyl)purine (6). A mixture
of tosylate 4 (430 mg, 0.886 mmol), stannyl derivative 8 (688 mg,
1.42 mmol), and LiCl (82.6 mg, 1.95 mmol) in dioxane (8.0 ml) was
stirred under argon for 30 min. After addition of Pd(PPh3)4 (102 mg,
0.0886 mmol), the mixture was heated under reflux for 2.5 h. The
mixture was then diluted with EtOAc (40 ml) and washed succes-
sively with aqueous NH3 (2.0% v/v, 20 ml), H2O (20 ml), and satd aq
NaCl (20 ml). The organic layer was dried with Na2SO4 and con-
centrated in vacuo. Silica gel column chromatography (EtOAc/PE)
delivered 6 (334 mg, 75%, pure E) as a lime-colored transparent oil.
as an amorphous off-white solid. [
a
]
D
20: ꢁ6.4; IR: 756, 826, 1034,
1173, 1242, 1504, 1589, 1697; 1H,1H-COSY NMR (600 MHz):
d 0.78–
1.00 (m, 12H, 3ꢃCH3 menthol, 3ꢃCHH menthol), 1.23–1.31 (m, 2H,
2ꢃCH menthol), 1.63–1.67 (m, 2H, 2ꢃCHH menthol), 2.13–2.15 (m,
1H, CHH menthol), 2.30–2.32 (m, 1H, CH menthol), 2.63–2.65 (m,
1H, H-20), 2.82 (ddd, 1H, J¼6.6 Hz, 6.6 Hz, 13.2 Hz, H-20), 3.17 (ddd,
1H, J¼4.2 Hz, 10.5 Hz, 14.7 Hz, CH menthol), 3.37 (dd, 1H, J¼4.2 Hz,
10.2 Hz, H-50), 3.45 (dd, 1H, J¼4.8 Hz, 10.2 Hz, H-50), 3.75 (s, 6H,
2ꢃCH3 DMT), 4.19–4.24 (m, 2H, H-40, CH2 propenyl), 4.45 (dd, 1H,
J¼3.0 Hz, 16.2 Hz, CH2 propenyl), 4.67 (br s, 2H, CH2 PAC), 4.86 (br s,
1H, H-30), 6.61 (m,1H, H-10), 6.76–6.78 (m, 4H, Harom), 7.00–7.08 (m,
3H, Harom), 7.14–7.24 (m, 4H, CH-10 propenyl, Harom), 7.26–7.29 (m,
4H, Harom), 7.34–7.39 (m, 3H, Harom), 7.63 (ddd, 1H, J¼4.8 Hz, 4.8 Hz,
15.6 Hz, CH-20 propenyl), 8.14 (s, 1H, H-8); 13C NMR (150 MHz):
[
a
]
20: ꢁ12.3; IR: 777, 833, 1068, 1213, 1495, 1587, 1709, 2928; 1H,1H-
D
COSY NMR (600 MHz):
d
0.08 (s, 6H, 2ꢃCH3 TBS), 0.12 (s, 6H, 2ꢃCH3
TBS), 0.78 (d, 3H, J¼7.2 Hz, CH3 menthol), 0.89–1.00 (m, 27H, 2ꢃt-
butyl TBS, 2ꢃCH3 menthol, 3ꢃCHH menthol), 1.28–1.36 (m, 2H,
2ꢃCH menthol), 1.62–1.66 (m, 2H, 2ꢃCHH menthol), 2.14 (m, 1H,
CHH menthol), 2.30–2.33 (m, 1H, CH menthol), 2.44–2.48 (m, 1H, H-
20), 2.71–2.73 (m, 1H, H-20), 3.17 (ddd, 1H, J¼3.9 Hz, 10.7 Hz, 14.7 Hz,
CH menthol), 3.79 (dd, 1H, J¼3.3 Hz, 11.1 Hz, H-50), 3.88 (dd, 1H,
J¼4.2 Hz, 11.4 Hz, H-50), 4.02 (dd, 1H, J¼3.3 Hz, 6.9 Hz, H-40), 4.23
(ddd, 1H, J¼1.5 Hz, 4.3 Hz, 15.3 Hz, CH2 propenyl), 4.45 (ddd, 1H,
J¼1.8 Hz, 4.8 Hz, 15.0 Hz, CH2 propenyl), 4.64–4.65 (m,1H, H-30), 4.80
d
16.3 (CH3 menthol), 21.0 (CH3 menthol), 22.3 (CH3 menthol), 23.3
(CH2 menthol), 25.5 (CH menthol), 31.5 (CH menthol), 34.5 (CH2

W.F.J. Hogendorf et al. / Tetrahedron 65 (2009) 10430–10435
10435
menthol), 40.3 (CH2 menthol), 40.7 (C-20), 48.2 (CH menthol), 55.2
(2ꢃCH3 DMT), 64.1 (C-50), 67.8 (CH2 PAC), 68.3 (CH2 propenyl) 72.5
(C-30), 79.3 (CH menthol), 84.1 (C-10), 86.6 (C-40), 113.1 (CHarom),
114.9 (CHarom), 122.3 (CHarom), 123.4 (CH-10 propenyl), 124.5, 126.9,
127.8, 128.1 (4ꢃCHarom), 128.8 (Cq PAC), 129.8 (CHarom), 130.0
(CHarom), 135.7 (2ꢃCq DMT), 141.5 (CH-20 propenylþC-8), 144.6 (Cq
DMT), 151.0, 151.8, 154.5, 158.5 (C-2, C-4, C-5, C-6); HRMS:
C52H59N5O8þHþ calculated 882.4436, found 882.4447.
modified oligonucleotide was cleaved from the resin by 25%
ammonium hydroxide solution at room temperature (2 h). The
resulting oligonucleotide was purified on a Q-Sepharose column at
pH 12 applying a gradient of buffer B (0.01 M NaOHþ2 M NaCl) in
buffer A (0.01 M NaOH). Fractions containing the pure product were
combined, desalted on Sephadex G-25 column (0.15 M ammonium
bicarbonate), and lyophilized. The identity and purity of the product
were confirmed by MALDI TOF mass spectrometry (Voyager DE PRO,
PerSeptive Biosystems, positive ionization mode, matrix 3-hydrox-
ypicolinic acid/ammonium hydrogen citrate), and IE HPLC (DNA-Pac
PA200 analytical column, Dionex). For MALDI TOF analysis calcd
15,528 (average); found 15,578 (approx.) broad signal because of
sodium clusters.
4.2.11. 2-N-Phenoxyacetyl-6-(3-O-
[4,40-dimethoxytrityl]-30-O-[{N,N-diisopropylamino}-2-cyanoethoxy-
phosphite]-20-deoxy-
-ribofuranosyl)purine (2). A mixture of 10
L
-menthyl-propenyl)-9-(50-O-
b-D
(450 mg, 0.510 mmol) and DIPEA (0.135 ml, 0.815 mmol) in DCM
(7.0 ml) was cooled to 0 ꢀC. 2-Cyanoethoxy-(N,N-diisopropyla-
mino)-chlorophosphine (0.148 ml, 0.663 mmol) was slowly added
and the mixture was allowed to stir for 3 h. After quenching with
H2O (1.0 ml), the mixture was diluted with DCM (20 ml) and
washed twice with satd aq NaHCO3 (15 ml), and once with satd aq
NaCl (15 ml). The organic phase was dried (Na2SO4) and concen-
trated under reduced pressure. After silica gel column chroma-
Acknowledgements
The authors thank Nico Meeuwenoord for technical assistance.
The Netherlands Organization of Scientific Research (NWO) is
gratefully acknowledged for financial support.
tography (EtOAc/PE/Et3N),
a colorless oil. 31P NMR (80.7 MHz, CD3CN):
(200 MHz, CD3CN):
0.72–0.88 (m, 12H, 3ꢃCH3 menthol, 3ꢃCHH
2
(462 mg, 84%) was afforded as
d
150.1; 1H NMR
References and notes
d
1. For recent reviews, see: (a) Cobb, A. J. A. Org. Biomol. Chem. 2007, 5, 3260–3275;
(b) Silverman, A. P.; Kool, E. T. Chem. Rev. 2006, 106, 3775–3789.
2. Wilson, J. N.; Kool, E. T. Org. Biomol. Chem. 2006, 4, 4265–4274.
3. For reviews, see: (a) Truglio, J. J.; Croteau, D. L.; Van Houten, B.; Kisker, C. Chem.
Rev. 2006, 106, 233–252; (b) Goosen, N.; Moolenaar, G. F. DNA Repair 2008, 7,
353–379.
4. Verhoeven, E. E. A.; Van Kesteren, M.; Turner, J. J.; Van der Marel, G. A.; Van
Boom, J. H.; Goosen, N. Nucleic Acids Res. 2002, 30, 2492–2500.
5. Malta, E.; Moolenaar, G. F.; Goosen, N. J. Biol. Chem. 2006, 281, 2184–2194.
menthol), 1.00–1.31 (m, 14H, 2ꢃCH menthol, 4ꢃCH3 iso-
propylamino), 1.52–1.65 (m, 2H, 2ꢃCHH menthol), 2.06–2.25 (m,
2H, CHH menthol, H-20), 2.43–2.60 (m, 3H, CH menthol, CH2 cya-
noethoxy), 2.99–3.81 (m, 14H, CH menthol, H-20, 2ꢃH-50, CH2
cyanoethoxy, 2ꢃCH isopropylamino, 2ꢃCH3 DMT), 4.08–4.47 (m,
3H, H-40, CH2 propenyl), 4.79–4.89 (m, 3H, H-30, CH2 PAC), 6.35 (t,
1H, J¼6.2 Hz, H-10), 6.62–6.71 (m, 4H, Harom), 6.93–6.97 (m, 3H,
Harom), 7.06–7.32 (m, 11H, CH-10 propenyl, Harom), 7.56 (ddd, 1H,
J¼5.1 Hz, 5.1 Hz, 16.0 Hz, CH-20 propenyl), 8.17 (s, 1H, H-8), 8.87 (br
s, 1H, NHPAC).
6. (a) Czernecki, S.; Hoang, A.; Vale
´ry, J. Tetrahedron Lett. 1996, 37, 8857–8860; (b)
Nagatsugi, F.; Uemura, K.; Nakashima, S.; Maeda, M.; Sasaki, S. Tetrahedron
1997, 53, 3035–3044; (c) Lakshman, M. K.; Thomson, P. F.; Nuqui, M. A.; Hilmer,
J. H.; Sevova, N.; Boggess, B. Org. Lett. 2002, 4, 1479–1482; (d) Mitsui, T.; Kimoto,
M.; Harada, Y.; Yokoyama, S.; Hirao, I. J. Am. Chem. Soc. 2005, 127, 8652–8658;
(e) Lakshman, M. K.; Gunda, P.; Pradhan, P. J. Org. Chem. 2005, 70, 10329–10335.
7. (a) Batoux, N.; Benhaddou-Zerrouki, R.; Bressolier, P.; Granet, R.; Laumont, G.;
Aubertin, A.; Krausz, P. Tetrahedron Lett. 2001, 42, 1491–1493; (b) Wnuk, S. F.;
Sacasa, P. R.; Lewandowska, E.; Andrei, D.; Cai, S.; Borchardt, R. T. Bioorg. Med.
Chem. 2008, 16, 5424–5433; (c) Roy, V.; Kumamoto, H.; Berteina-Raboin, S.;
Nolan, S. P.; Topalis, D.; Deville-Bonne, D.; Balzarini, J.; Neyts, J.; Andrei, G.;
Snoeck, R.; Agrofoglio, L. A. Nucleosides Nucleotides Nucleic Acids 2007, 26, 1399–
1402; (d) Kaucher, M. S.; Harrell, W. A., Jr.; Davis, J. T. J. Am. Chem. Soc. 2006, 128,
38–39.
8. Schulhof, J. C.; Molko, D.; Teoule, R. Tetrahedron Lett. 1987, 28, 51–54.
9. No conversion of the nucleoside starting material was observed when Grubbs
first generation catalyst was used, while allyl menthyl ether readily dimerized.
10. Choi, T.; Chatterjee, A. K.; Grubbs, R. H. Angew. Chem., Int. Ed. 2001, 40, 1277–
1279.
11. de Kort, M.; de Visser, P. C.; Kurzeck, J.; Meeuwenoord, N. J.; van der Marel, G. A.;
Ruger, W.; van Boom, J. H. Eur. J. Org. Chem. 2001, 2075–2082.
12. de Kort, M.; Ebrahimi, E.; Wijsman, E. R.; van der Marel, G. A.; van Boom, J. H.
Eur. J. Org. Chem. 1999, 2337–2344.
4.2.12. Synthesis of oligonucleotide
I
(50-GGGATTACTTACGGG-
CACATTACAAAXAAACCTCAGAACGACCTCACACG-30). The solid-phase
synthesis of the oligonucleotide was performed on a fullyautomated
Expedite instrument (PerSeptive Biosystems) starting from con-
trolled pore glass functionalized with the appropriate nucleoside.
The synthesis was performed ona 1-mmol scale via phosphoramidite
methodology11,12 but using mildly removable N-t-butylpheno-
xyacetyl (tac) protection for nucleobases.14 Elongation was per-
formed by coupling of the 30-phosphoramidite derivatives of DMT-
protected nucleosides (50-DMT-dAtac, 50-DMT-dCtac, 50-DMT-dGtac
,
and 50-DMT-T, 10 equiv, 10
m
mol, 0.1 M stock) with 4,5-dicyanoimi-
dazole15 as the activator (50 equiv, 50
mmol 0.25 M stock), for 3 min.
The phosphoramidites of the fluorescent nucleoside analogs
(15 equiv) were coupled for 5 min with 75 equiv of 4,5-dicyanoi-
midazole (75 m
mol). The50-DMTgroup was removed using 3% TCA in
DCM. After each coupling, remaining free 50-hydroxyls were blocked
using a mixture of cap A (t-butylphenoxyacetic anhydride, 0.2 M in
THF) and cap B (1-methylimidazole in THF/pyridine) followed by
oxidation of the phosphite linkage to the phosphate using 0.05 M of
I2 in THF/pyridine/water (1 min). After final DMT removal the
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