Conjugation at the oligonucleotide level based on isoxazole phosphoramidites generated by click chemistry

Article abstract of DOI:10.1016/j.tet.2011.06.096

The versatility of the isoxazole generating nitrile oxide-alkyne Huisgen cycloaddition for provision of chemically modified oligonucleotides has been extended; in a novel approach isoxazole conjugated oligodeoxyribonucleotides have been constructed by pho

Full text of DOI:10.1016/j.tet.2011.06.096

Tetrahedron 67 (2011) 7860e7865
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Conjugation at the oligonucleotide level based on isoxazole phosphoramidites
generated by click chemistry
*
Colin Freeman, Aisling Ni Cheallaigh, Frances Heaney
Department of Chemistry, National University of Ireland, Maynooth, Co. Kildare, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
The versatility of the isoxazole generating nitrile oxideealkyne Huisgen cycloaddition for provision of
chemically modified oligonucleotides has been extended; in a novel approach isoxazole conjugated
oligodeoxyribonucleotides have been constructed by phosphoramidite chemistry of isoxazole derivatives
previously generated by nitrile oxideealkyne click chemistry. The conjugation involves manual solid
phase synthesis at room temperature in aqueous ethanol and proceeds in high yield.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 2 February 2011
Received in revised form 9 June 2011
Accepted 24 June 2011
Available online 23 July 2011
1. Introduction
a complement to solid phase oximation or CuAAC (copper catalysed
azide alkyne cycloaddition) approaches to DNA conjugates and, in
Huisgen cycloaddition reactions performed on DNA substrates
represent a powerful approach to chemically modified oligonu-
cleotides, in particular the Cu(I) catalysed azideealkyne triazole
forming reaction has received much attention.1 However, we and
others have reported attributes of the [3þ2]-cycloaddition reaction
of nitrile oxides and nitrones as a viable, catalyst free, regioselective
alternative for the formation of functionalised nucleosides and
oligonucleotides.2e6 In previously reported work the alkyne part-
ner was introduced to the resin supported oligonucleotide, and
subsequent cycloaddition was conducted directly on the solid
phase. The objective of the current work was to develop an alter-
native, modular strategy, whereby modified oligonucleotides could
be constructed by coupling of isoxazole phosphoramidite building
blocks previously generated by click chemistry. The alternative
strategies, where the key cycloaddition reaction is conducted on-,
or off-resin, are shown retrosynthetically, in Fig.1. The attractions of
on-resin click chemistry for oligonucleotide conjugation are al-
ready well known.1 Potential benefits of the off-resin approach, (a),
may include ease of operation, particularly with regard to scale-up
and functional group compatibility. Dipolar cycloadditions present
a robust synthetic strategy for heterocycle formation, and it is the
hypothesis of this paper that if either the dipole or the dipolar-
ophile have a pendant hydroxy group, then following cycloaddition
and phosphoramidite formation oligonucleotide conjugation can
be achieved in a catalyst free environment. Certainly off-resin cy-
cloaddition will serve to increase the versatility of ‘click chemistry’
for oligonucleotide conjugation and it could be used as
theory, be applied for the reliable introduction of a variety of
reporting or targeting groups into DNA.
Approaches to chemically modified oligonucleotides using
building blocks previously prepared by CuAAC chemistry have been
reported. In one application, oligodeoxyribonucleotide glyco-
conjugates have been generated by solid-supported oximation of
aminooxy terminated oligonucleotides with glycoclusters con-
structed by click chemistry.7 In other applications phosphoramidite
derivatives of click chemistry derived triazole monomers have been
utilised, most frequently the triazole moiety is borne at the C-5
position of functionalised pyrimidine bases including deoxy-
uridines,8e10 cytidines11 and thymidines,12 however, the triazole
unit has also been sited at the 50-position of the sugar13 and as
a spacer between the base and sugar moieties.14 Finally, non-ca-
nonical15 and non-nucleosidic16 triazole phosphoramidites have
also been reported. In contrast, whilst isoxazoles are an interesting
heterocyclic family with diverse applications, isoxazole bearing
phosphoramidites are not well known. To the best of our knowl-
edge, the -threoninol derived 1, Fig. 2, prepared for inclusion in
D
a library of cyclic oligonucleotides targeted to the Hepatitis C virus,
NS5B, represents the only known example.17
2. Results and discussions
The retrosynthetic approach set out in Fig. 1(a) requires iso-
xazole building blocks equipped with a pendant hydroxy func-
tionality to act as
a handle for ultimate conversion to
phosphoramidite monomers. We intended to prepare the isoxazole
from an in situ generated, nitrile oxideealkyne cycloaddition,
(NOAC), and believed the isomeric hydroxyethoxybenzaldehyde
oximes 2aec represented an ideal platform to explore the concept
* Corresponding author. Tel.: þ353 1 708 3802; fax: þ353 1 708 3815; e-mail
address: mary.f.heaney@nuim.ie (F. Heaney).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.06.096

C. Freeman et al. / Tetrahedron 67 (2011) 7860e7865
7861
Fig. 1. Retrosynthetic approaches to chemically modified oligonucleotides involving (a) off-resin and (b) on-resin nitrile oxideealkyne cycloaddition (NOAC) chemistry.
7.50e8.50 ppm,18e20 indeed, in each case low intensity signals, at
w8.5 ppm in the 1H NMR spectrum of the crude cycloaddition
products provides evidence for minor amounts of the 4-substituted
regioisomers 5. Further, singlet resonances appearing at w4.9 ppm,
are deemed characteristic of the methylene protons adjacent to the
isoxazole ring. The regioselectivity of the reaction ranges from 44:1
to 27:1 in favour of the 5-substituted isomers 4.
The regioisomeric phosphoramidite monomers 6 were pre-
pared, in reasonable yields, from the corresponding isoxazole
building blocks 4 following reaction with 2-cyanoethyl-N,N,N0,N0-
tetraisopropylphosphorodiamidite in anhydrous acetonitrile. Ben-
zylmercaptotetrazole (BMT) was employed as activator in the
presence of diisopropylamine (Scheme 1). Following 30 min at
room temperature, TLC analysis indicated complete consumption of
the starting alcohol. 31P NMR spectroscopy demonstrated phos-
phorus resonances at w148e149 ppm, supporting formation of the
desired monomers. Purification of phosphoramidites by flash col-
umn chromatography can be tricky; to avoid decomposition and
erosion in yield, rapid elution under a stream of an inert gas is re-
quired. For synthetic purposes we have found 6aec were suitable
for manual solid phase coupling without purification. The unpuri-
fied isoxazole phosphoramidite monomers, 6, were introduced to
resin bound nucleoside/oligonucleotide substrates (Scheme 2).
Initial experimentation focused on reaction between CPG-(con-
trolled pore glass)-supported thymidine and the o-substituted
isoxazole phosphoramidite 6a. Reaction was achieved by manual
Fig. 2. Isoxazole phosphoramidite, 1.17
since all three regioisomers, with their varying steric demands,
could be obtained.
The desired oximes
2 were prepared in excellent yields
(88e96%) from commercially available aldehyde precursors 3 fol-
lowing reaction with hydroxylamine hydrochloride in the presence
of sodium acetate. For each oxime, in situ generation of the corre-
sponding nitrile oxide was achieved through reaction with
chloramine-T in aqueous ethanol. The transient dipole was trapped
with propargyl phenyl ether and the cycloaddition products, 4aec,
were obtained in 81e85% isolated yield following stirring at room
temperature, Scheme 1. In each case reaction was, as expected,
highly selective for formation of the 5-substituted isomer. Regio-
chemical assignment of the major isomer follows from the reso-
nance position of the isoxazole ring proton, which presents as
a singlet in the range 6.59e6.76 ppm. This chemical shift is typical
of an isoxazole H-4 proton; a proton at the C-5 position is expected
to resonate more downfield, somewhere in the region of
solid phase coupling on a 1 mmol scale. The thymidine loaded resin,
Scheme 1. Synthesis of the phosphoramidite monomers 6.

7862
C. Freeman et al. / Tetrahedron 67 (2011) 7860e7865
7, was placed in a DNA synthesis column and a 1 mL syringe con-
taining an acetonitrile solution of the required phosphoramidite
was attached to one end. A second syringe, containing a solution of
the activator, benzylmercaptotetrazole, was attached to the other
end. The solutions were passed through the column over a period of
15 min with mixing between the two syringes. To ensure complete
reaction the procedure was repeated with a second portion of each
of a new solution of the phosphoramidite and the activator. Fol-
lowing the second 15 min period of mixing at room temperature
oxidation preceeded cleavage of the reaction product from the resin
8a. The coupling yield of crude product 9, Fig. 3, was judged from
the HPLC profile of the raw material to be higher than 95%.
four DNA bases with their standard protecting groups, was selected
to establish the general applicability of the concept. As described
above, during a 30 min period, separate solutions of the phos-
phoramidite monomer 6a and the activator were manually passed
over the resin-supported material 13 contained in a DNA synthesis
column. The oligonucleotide was then deprotected and removed
from the solid support 14a following treatment with ammonium
hydroxide (24 h, room temperature). Significantly, HPLC analysis of
the unpurified reaction product indicated quantitative conversion
of 13 to 15a, Fig. 4.
In a further examination of the generality of this approach the
regioisomeric phosphoramidites 6b and 6c were successfully in-
Scheme 2. Solid phase synthesis of chemically modified oligonucleotides using previously clicked isoxazole phosphoramidite monomers.
Fig. 3. Structures of isoxazole modified oligonucleotides.
Fig. 4. HPLC profiles of (a) reference sample of 30-CGCACACACGCT-OH-50, (b) (c) and (d), conjugates 15a, 15b and 15c derived, respectively, from isoxazole phosphoramidites 6aec
and prepared by manual solid phase synthesis (e) 15c prepared on a DNA synthesizer.
To establish compatibility with an oligonucleotide, reaction with
CPG-loaded decathymidine, 10, was examined. Gratifingly, under
the same conditions, reaction with the 10-mer substrate was found
to proceed smoothly, and following cleavage of the DNA from 11a
HPLC analysis of the unpurified reaction product indicated 12 was
present in essentially quantitative yield.
corporated into the resin-supported mixed base 12-mer 13 by
manual solid phase synthesis. In each case, cleavage and depro-
tection of the supported products, 14b,c was effected by treatment
with ammonium hydroxide. HPLC analysis, Fig. 4, confirmed com-
plete consumption of starting material and selective formation of
the products 15b,c.
To further probe the scope of the reaction a CPG-supported 12-
mer oligonucleotide 13 was examined. The sequence, bearing all
The potential to automate the coupling was established on an
Expedite DNA synthesizer using purified samples of the isoxazole

C. Freeman et al. / Tetrahedron 67 (2011) 7860e7865
7863
phosphoramidites. The conjugate 15c was formed in excess of 90%
yield from reaction between 6c and resin supported 13 (0.2 mol)
(30 mL), washed with H2O (3ꢁ20 mL) and dried over anhydrous
magnesium sulfate. Removal of the solvent under reduced pressure
yielded the pure product as a white solid in excellent yield.
m
using the standard instrument protocol, viz. 37 equiv of phos-
phoramidite and 1.5 min coupling time. However, to achieve the
same level of conjugation with the sterically more demanding
substrates 6a and 6b more generous conditions were required, viz.
50 equiv of phosphoramidite and 15 min coupling time. HPLC
analysis shows a favourable comparison between the manual and
automated approaches; in all cases, greater than 90% incorporation
of the non-natural phosphoramidites was observed; comparative
data are shown for samples of 15c prepared by both approaches,
Fig. 4, (d) and (e).
4.1.1. 2-(2-Hydroxyethoxy)benzaldehyde oxime, 2a. White solid
(96%), mp 98e100 ꢀC, Rf¼0.44 (hexane/EtOAc, 3:7); 1H NMR
d
8.39 (s, 1H, HC]N), 7.54 (dd, J¼7.7, 1.7 Hz, 1H, ArH), 7.38e7.32
(m, 1H, ArH), 7.04e6.95 (m, 2H, ArH), 4.17 (t, J¼4.2 Hz, 2H, CH2),
3.98 (t, J¼4.2 Hz, 2H, CH2); 13C NMR
d 157.0, 147.8, 131.2, 128.9,
121.6, 121.2, 113.7, 70.7, 61.2; IR (KBr) 3388, 3219, 1601, 1251,
764 cmꢂ1; HRMS (ESI) calcd for [MþNa]þ, C9H11NO3Na, 204.0631,
found 204.0640.
All new conjugates, 9a,12a and 15aec derived from the isomeric
phosphoramidites 6 were characterized by mass spectrometry (ESI-
MS or MALDI-TOF MS), and most importantly, in all cases yields of
conjugates derived from isoxazoles generated off-resin, Fig. 1 (a),
compare favourably with those obtained for similar conjugates
constructed by on-resin click cycloaddition chemistry with alkyne
modified DNA substrates, Fig. 1, path (b).4
4.1.2. 3-(2-Hydroxyethoxy)benzaldehyde oxime, 2b. White solid
(86%), mp 94e96 ꢀC, Rf¼0.44 (hexane/EtOAc, 3:7); 1H NMR
d 8.10 (s,
1H, HC]N), 7.59 (br s, 1H, OH), 7.33e7.28 (m, 1H, ArH), 7.19e7.13
(m, 2H, ArH), 6.96 (dd, J¼9.0, 2.4 Hz, 1H, ArH), 4.12 (t, J¼4.2 Hz, 2H,
CH2), 3.98 (t, J¼4.2 Hz, 2H, CH2); 13C NMR
d 156.9, 147.7, 131.2, 128.8,
121.6, 121.2, 113.6, 70.6, 61.2; IR (KBr) 3350, 3154, 1595, 1262, 907,
795 cmꢂ1; HRMS (ESI) calcd for [MþH]þ, C9H12NO3, 182.0812,
found 182.0809.
3. Conclusion
Novel isoxazole building blocks were generated by Cu-free ni-
trile oxideealkyne cycloadditions. Following conversion to phos-
phoramidites, incorporation into oligonucleotides has been
achieved manually on the solid phase and on a DNA synthesizer
employing a standard protocol. The coupling reaction is efficient
both at the monomeric level (CPG-Thymidine) and for oligonucle-
otides (CPG-12-mer DNA). Whilst on-resin functionalisation of al-
kyne modified oligonucleotides by nitrile oxide or CuAAC click
chemistry is already established as a powerful method for oligo-
nucleotide conjugation, for some applications it may be more at-
tractive to assemble the conjugate from phosphoramidite building
blocks previously prepared by click chemistry. Possible advantages
may ensue in terms of cost, ease of operation and enhanced po-
tential for scale-up.21
4.1.3. 4-(2-Hydroxyethoxy)benzaldehyde oxime, 2c. White solid
(88%), mp 109e111 ꢀC, Rf¼0.40 (hexane/EtOAc, 3:7); 1H NMR
d 8.09
(s, 1H, HC]N), 7.51 (d, J¼8.8 Hz, 2H, ArH), 7.32 (br s, 1H, OH), 6.93
(d, J¼8.8 Hz, 2H, ArH), 4.12 (t, J¼4.8 Hz, 2H, CH2), 3.98 (t, J¼4.8 Hz,
2H, CH2); 13C NMR
d 160.1, 149.9, 128.5, 125.2, 114.8, 69.3, 61.4; IR
(KBr) 3262, 1605, 1259, 836 cmꢂ1
; HRMS (ESI) calcd for
C9H11NO3Na, [MþNa]þ, 204.0631, found 204.0622.
4.2. General procedure for preparation of cycloadducts 4
The required oxime 2aec (500 mg, 2.76 mmol) was dissolved in
EtOH (2.5 mL) and to this solution was added propargyl phenyl
ether (182 mg, 1.38 mmol), chloramine T (785 mg, 3.45 mmol) and
H2O (2.5 mL) The mixture was stirred for 2 h at room temperature
after which analysis by TLC (hexane/EtOAc, 3:7) indicated complete
reaction. After removal of the solvent under reduced pressure, the
residue was taken up in EtOAc (30 mL) and the solution was
washed with 5% NaOH (3ꢁ10 mL). The organic layer was dried over
anhydrous magnesium sulfate and the solvent was removed under
reduced pressure. The crude products were purified by flash col-
umn chromatography (hexane/EtOAc, 3:7) to give the cycloadduct
in good yield.
4. General experimental
Analytical TLC was performed on precoated (250 mm) silica gel
60 F254 plates from Merck. All plates were visualized by UV irra-
diation, and/or staining with 5% H2SO4 in ethanol followed by
heating. Flash chromatography was performed using silica gel
ꢀ
32e63
m
m, 60 A. Mass analysis was performed on an Agilent
Technologies 6410 Time of Flight LC/MS. MALDI-TOF mass data was
acquired on an Applied Biosystem Voyager instrument or a LASER-
TOF LT3 from Scientific Analytical Instruments with 3-
hydroxypicolinic acid or 20,40,60-trihydroxyacetophenone as ma-
trix. NMR spectra were obtained on a Bruker instrument at 25 ꢀC
(1H at 300 MHz; 13C at 75 MHz; 31P at 121 MHz). Chemical shifts are
reported in parts per million downfield from TMS as standard. All
NMR spectra are recorded in CDCl3. HPLC was carried out using
a Gilson instrument equipped with a diode array detector and
a Nucleosil C18 column (4.0ꢁ250 mm). Automated oligonucleotide
synthesis was carried out on an Expedite nucleic acid synthesis
system.
4.2.1. 2-{2-[5-(Phenoxymethyl)isoxazol-3-yl]phenoxy}ethanol,
4a. Colourless oil (81%), Rf¼0.54 (hexane/EtOAc, 3:7); 1H NMR
d
7.71 (dd, J¼7.6, 1.3 Hz, 1H, ArH), 7.42e7.29 (m, 3H, ArH), 7.08e6.97
(m, 5H, ArH), 6.76 (s, 1H, isox-H), 5.20 (s, 2H, CH2), 4.18 (t, J¼4.2 Hz,
2H, CH2), 3.92 (br s, 2H, CH2), 3.24 (br s, 1H, OH); 13C NMR
d
167.4,
160.4, 157.8, 156.7, 131.4, 129.8, 129.7, 121.8, 121.6, 118.4, 114.8, 113.9,
104.2, 70.9, 61.3, 61.1; IR (film) 3400, 1601, 1245, 755 cmꢂ1; HRMS
(ESI) calcd for C18H18NO4, [MþH]þ, 312.1230, found 312.1242.
4.2.2. 2-{3-[5-(Phenoxymethyl)isoxazol-3-yl]phenoxy}ethanol,
4b. White solid (81%), mp 70e72 ꢀC, Rf¼0.52 (hexane/EtOAc, 3:7);
1H NMR
d 7.39e7.28 (m, 5H, ArH), 7.03e6.96 (m, 4H, ArH), 6.61 (s,
4.1. General procedure for preparation of oximes 2aec
1H, isox-H), 5.17 (s, 2H, CH2), 4.12 (t, J¼4.2 Hz, 2H, CH2), 3.96 (br s,
2H, CH2) 2.42 (br s, 1H, OH); 13C NMR
d
168.7, 162.4, 159.1, 157.8,
A
solution of the required aryl aldehyde 3aec (1.00 g,
130.1, 130.0, 129.7, 121.9, 119.8, 116.8, 114.8, 112.5, 101.5, 69.4, 61.4,
61.3 cmꢂ1; IR (KBr) 3208, 1600, 1260, 1231, 885, 755 cmꢂ1; HRMS
(ESI) calcd for C18H18NO4, [MþH]þ, 312.1230, found 312.1233.
6.02 mmol) and hydroxylamine hydrochloride (1.26 g, 18.1 mmol)
in EtOH (35 mL) was stirred at room temperature for 10 min, after
which a solution of sodium acetate (1.69 g, 24.1 mmol) in H2O
(15 mL) was added. The mixture was heated under reflux for
40 min. Following solvent removal under reduced pressure the
crude product, obtained as a white solid, was taken up in EtOAc
4.2.3. 2-{4-[5-(Phenoxymethyl)isoxazol-3-yl]phenoxy}ethanol,
4c. White solid (85%), mp 95e97 ꢀC, Rf¼0.49, (hexane/EtOAc, 3:7);
1H NMR
d
7.74 (d, J¼8.6 Hz, 2H, ArH), 7.32 (t, J¼7.7 Hz, 2H, ArH),

7864
C. Freeman et al. / Tetrahedron 67 (2011) 7860e7865
7.04e6.98 (m, 5H, ArH), 6.59 (s,1H, isox-H), 5.20 (s, 2H, CH2), 4.13 (t,
J¼4.2 Hz, 2H, CH2), 3.99 (t, J¼4.2 Hz, 2H, CH2), 2.04 (br s, 1H, OH);
protocols. All reactions were conducted on a scale involving CPG-
DNA (0.2 mol). For coupling 6a,b 50 equiv of phosphoramidite
were used and 15 min coupling time. For coupling 6c 37 equiv of
m
13C NMR
d
168.4, 162.1, 160.1, 157.8, 129.7, 128.3, 121.9, 121.7, 114.9,
114.8, 101.1, 69.3, 61.5, 61.4; IR (KBr), 3438, 1613, 1258, 1238,
842 cmꢂ1; HRMS (ESI) calcd for C18H18NO4, [MþH]þ, 312.1230,
found 312.1245.
phosphoramidite were used and 1.5 min coupling time.
4.5. General deprotection procedure, preparation of 9, 12 and
15aec
4.3. General procedure for preparation of phosphoramidites 6
For analytical purposes a portion of the DNA was deprotected
and cleaved from the CPG by incubating the CPG-DNA in either
The required cycloadduct 4aec (100 mg, 0.321 mmol), and
benzylmercaptotetrazole (31 mg, 0.159 mmol) were placed in
a dried round bottomed flask under an argon atmosphere.
Anhydrous acetonitrile (5 mL) was added to the flask followed
i
40% aqueous CH3NH2 (500 m
L) at 65 ꢀC for 30 min (for sub-
strates 8 and 11)
ii. 28% aqueous NH4OH (1 mL) at 25 ꢀC for 24 h (for substrates
by diisopropylamine (23
m
l, 0.162 mmol) and 2-cyanoethyl-
N,N,N0,N0-tetraisopropylphosphorodiamidite (112
ml, 0.353 mmol).
14aec)
The reaction mixture was stirred for 30 min at room temperature
after which TLC analysis (hexane/EtOAc, 3:7) showed complete
consumption of the starting alcohol. The reaction mixture was di-
luted with ethyl acetate (25 mL) and washed with aqueous sodium
bicarbonate (10ꢁ3 mL). The organic layer was dried over anhydrous
sodium sulfate and the solvent was removed under reduced pres-
sure to give the crude phosphoramidite, which was purified by
flash column chromatography (hexane/EtOAc, 3:7) with elution
under a positive pressure of nitrogen gas.
As appropriate, CH3NH2/NH4OH was evaporated using a con-
centrator. The CPG was washed with H2O (3ꢁ150
mL aliquots), all
solutions and washings were combined to afford an aqueous so-
lution of DNA-alkynes 9, 12 and 15aec. On analysis by HPLC re-
tention times (tR) of the starting nucleosides/oligonucleotides and
the modified conjugates are as follows.
Thymidine tR¼22.7 min, 9 tR¼16.5 min.
Decathymidine tR¼12.3 min, 12 tR¼16.0 min.
12-mer 30-CGCACACACGCT-OH-50 tR¼10.4 min, 15a tR¼13.8 min,
15b tR¼13.9 min, 15c tR¼14.0 min.
4.3.1. 2-Cyanoethyl 2-{2-[5-(phenoxymethyl)isoxazol-3-yl]phenoxy}
Compound 9 HRMS (ESI) m/z calcd for 638.1510 [MþNa]þ; found
638.1534.
ethyl N,N-diisopropylphosphoramidite, 6a. Colourless oil, Rf¼0.83
(hexane/EtOAc, 3:7); 1H NMR
d
7.94 (dd, J¼7.7, 1.7 Hz, 1H, ArH),
7.43e7.31 (m, 3H, ArH), 7.07e6.98 (m, 6H, ArH and isox-H), 5.19 (s,
2H, CH2), 4.25e4.22 (m, 2H), 4.16e3.54 (m, 6H), 2.55e2.51 (m, 2H),
Compound 12 MALDI-TOF-MS m/z: calcd 3353; found 3353.
Compound 15a MALDI-TOF-MS m/z: calcd 3948; found 3949.
Compound 15b MALDI-TOF-MS m/z: calcd 3948; found 3949.
Compound 15c MALDI-TOF-MS m/z: calcd 3948; found 3949.
1.19e1.14 (m, 12H); 31P NMR
d 148.8.
4.3.2. 2-Cyanoethyl 2-{3-[5-(phenoxymethyl)isoxazol-3-yl]phenoxy}
ethyl N,N-diisopropylphosphoramidite, 6b. Colourless oil, Rf¼0.83
4.6. General method for HPLC analysis
(hexane/EtOAc, 3:7); 1H
d NMR 7.39e7.32 (m, 5H, ArH), 7.05e6.98
(m, 4H, ArH), 6.64 (s, 1H, isox-H), 5.21 (s, 2H, CH2), 4.20 (t, J¼5.1 Hz,
Nucleoside and oligonucleotide conjugates were analyzed by
reverse-phase HPLC under the following conditions; 200 mL in-
jection loop; buffer A: 0.1 M TEAAc, pH 6.5, 5% (v/v) MeCN; buffer B:
0.1 M TEAAc, pH 6.5, 65% (v/v) MeCN; Gradient: 0e4.3 min, 5% B;
4.3e16.6 min, 5e100% B; flow rate: 1.0 mL/min and detection at
260 nm.
2H), 4.10e3.49 (m, 6H), 2.63 (t, J¼6.4 Hz, 2H),1.20 (dd, J¼6.8, 2.1 Hz,
12H); 31P NMR
d 149.1.
4.3.3. 2-Cyanoethyl 2-{4-[5-(phenoxymethyl)isoxazol-3-yl]phenoxy}
ethyl N,N-diisopropylphosphoramidite, 6c. Colourless oil, Rf¼0.83
(hexane/EtOAc, 3:7); 1H NMR
d
7.73 (d, J¼8.7 Hz, 2H, ArH), 7.32 (t,
J¼7.6 Hz, 2H, ArH), 7.04e6.95 (m, 5H, ArH), 6.59 (s, 1H, isox-H) 5.20
(s, 2H, CH2), 4.29 (t, J¼5.1 Hz, 2H, CH2), 4.10e3.77 (m, 4H),
3.70e3.57 (m, 2H), 2.63 (t, J¼6.5 Hz, 2H), 1.20 (dd, J¼6.8, 2.9 Hz,
Acknowledgements
Financial support from the Science Foundation of Ireland
(Programme code 05/PICA/B838) is gratefully acknowledged. C.F.
is grateful to the Irish Research Council Science and Engineering
for receipt of an Embark Postgraduate Research Scholarship
(Programme code RS/2007/48). A.N.C. acknowledges support
from the Science Foundation Ireland UREKA Programme (SFI/08/
UR/F1353).
12H); 31P NMR
d 149.0.
4.4. General procedure for the phosphitylation of CPG-DNA-
OH 50, preparation of 8, 11 and 14aec
Option 1. To manually couple the phosphoramidites 6aec to the
resin supported nucleoside/oligonucleotide, 7, 10 or 13, separate
solutions of the phosphoramidite (500
CH3CN) and of benzylmercaptotetrazole (500
CH3CN), both in 1 mL syringes were attached to either end of a DNA
synthesis column containing CPG-DNA (1 mol). The mixture was
m
L, 100 mM in anhyd
Supplementary data
mL, 0.3 mM in anhyd
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2011.06.096. These data in-
clude MOL files and InChiKeys of the most important compounds
described in this article.
m
reacted for 15 min at room temperature with mixing between the
two syringes. This reaction was repeated with a second portion of
each of a new solution of the phosphoramidite and benzylmer-
captotetrazole. The CPG was washed with CH3CN (5ꢁ2 mL) prior to
References and notes
treatment with oxidizer (700 mL, 0.1 M Iodine solution in THF/pyr-
idine/water; 78:20:2). Further washing with CH3CN (2ꢁ5 mL) and
drying yielded CPG-DNA-isoxazole conjugates 8, 11 and 14aec.
Option 2. Automated coupling of the phosphoramidites 6aec to
the resin supported oligonucleotide, 13, was achieved using an
Expedite Nucleic Acid Synthesis System using standard instrument
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