Efficient synthesis of DNA conjugates by strain-promoted azide-cyclooctyne cycloaddition in the solid phase

Article abstract of DOI:10.1002/ejoc.201101045

Conjugation of ligands to DNA oligonucleotides has been achieved in the solid phase by strain-promoted azide-alkyne cycloaddition (SPAAC). The oligonucleotide, modified with a simple nonfluroinated, monocyclic octyne, efficiently forms conjugates with a r

Full text of DOI:10.1002/ejoc.201101045

FULL PAPER
DOI: 10.1002/ejoc.201101045
Efficient Synthesis of DNA Conjugates by Strain-Promoted Azide–Cyclooctyne
Cycloaddition in the Solid Phase
Ishwar Singh,*[a] Colin Freeman,[a] and Frances Heaney*[a]
Keywords: Medicinal chemistry / Oligonucleotides / DNA / Solid-phase synthesis / Click reaction / Azides / Alkynes /
Cycloaddition / Bioconjugation
Conjugation of ligands to DNA oligonucleotides has been
achieved in the solid phase by strain-promoted azide–alkyne
cycloaddition (SPAAC). The oligonucleotide, modified with a
simple nonfluroinated, monocyclic octyne, efficiently forms
conjugates with a range of azide dipoles with varying steric
and electronic characteristics. The reaction is clean and eas-
ily executed in a copper free environment at room tempera-
ture. It provides a variety of triazole-linked nucleic acid con-
jugates and is potentially useful in biotechnology and cell
biology.
incorporation of a ring heteroatom, steric encumberments
or electron-attracting substituents; more recent rate en-
hancements have been observed with diaryl fused cyclo-
octynes and their aza analogues.[17–25] Although azides have
been the dipole of choice in most cases, more attractive ki-
netics have been observed with nitrile oxides.[26] Applica-
tions of strain-promoted conjugation are numerous and
span diverse areas including bioimaging,[17,27,28] quantum
dot formation,[29] peptide conjugation,[30] drug discovery,[18]
drug delivery,[31] synthetic chemistry[23] and surface and ma-
terials science.[22,24]
We have previously reported the postsynthetic modifica-
tion of oligonucleotides by strain-promoted nitrile oxide cy-
cloaddition chemistry (SPNOC),[8] and Filippov[32] and
Manoharan[33] independently demonstrated conjugate for-
mation by cycloaddition of azides to oligonucleotide cyclic
alkynes. In their elegant studies, Filippov and Manoharan
have concentrated on solution rather than solid phase con-
jugation. The attractions of solid-phase synthesis include
the ease of purification and the possibility of automation,
thus, we wished to develop resin-supported conjugation as
a robust, technically accessible platform for postsynthetic
oligonucleotide modification. As an additional improve-
ment to existing methods, we wished to demonstrate the
synthetic utility of a simple nonfluorinated, monocyclic oc-
tyne, in preference to an enhanced diarylcyclooctyne, as the
strained alkyne partner. From a synthetic standpoint, the
attractions of a cyclooctyne substrate include ease of ac-
cess[8] and the possibility for superior kinetics with bulky
azides. The reduced lipophilicity and the limited steric bulk
are potentially attractive for applications in drug delivery,
biomedicine and imaging where aqueous solubility and a
reduced tendency to interact with hydrophobic proteins are
important. Finally, advanced applications involving living
systems, e.g. genetic encoding of alkynes are expected to
proceed more smoothly.[34] The objective of the current
Introduction
The application of functionalised oligonucleotides in key
areas including diagnostics and therapeutics has created the
need for reliable chemical methods for postsynthetic conju-
gation.[1] The high chemical stability and orthogonal reac-
tivity of azides and alkynes has made the copper-catalysed
1,3-dipolar cycloaddition a highly attractive conjugation
strategy in materials science and biotechnology.[2] However,
in spite of its vast successes, this reaction it is not ideal for
oligonucleotide applications; CuI-mediated oxidative DNA
degradation can be problematic, and copper ion contami-
nation of the final product can culminate in issues of cyto-
toxicity and nucleic acid hydrolysis.[3,4] Recent develop-
ments indicate that careful selection of CuI-stabilizing li-
gands can avoid DNA degradation and even facilitate Cu-
promoted azide–alkyne click (CuAAC) modifications in liv-
ing organisms.[5] However, toxicity may remain an issue
when oligonucleotide therapeutics are desired, and, in se-
arch of alternative bioconjugation methodologies, we and
others have developed catalyst-free nitrone and nitrile oxide
cycloadditions.[6–11] Additional, metal-free bioorthogonal
conjugation strategies include Diels–Alder cycloadditions
and photoinduceable cycloadditions of tetrazines or nitrile
imines to alkenes.[12–14] More recent advances in this area
include strain-promoted azide–alkyne cycloadditions
(SPAAC). These reactions, first observed more than 50
years ago,[15] exploit the intrinsic ring strain of cyclooc-
tynes.[16] First generation substrates had relatively sluggish
reactivity, and incremental improvements followed from the
[a] Department of Chemistry, National University of Ireland
Maynooth, Co. Kildare, Republic of Ireland
Fax: +353-1-708-3815
E-mail: ishwar.singh@nuim.ie
mary.f.heaney@nuim.ie
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201101045.
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I. Singh, C. Freeman, F. Heaney
FULL PAPER
Scheme 1. Resin-supported postsynthetic oligonucleotide conjugation by SPAAC.
work, summarised in Scheme 1, was to develop postsyn-
thetic oligonucleotide conjugation on the solid phase by
strain-promoted cycloaddition between azide dipoles and
simple monocyclic octynes.
Results and Discussion
The design and synthesis of the resin-supported DNA
cyclooctynes 1 and 2 has been detailed elsewhere.[8] We se-
lected a range of azide partners, Figure 1, including small
organic azides for proof of concept studies viz. benzyl azide
(3) and cinnamyl azide (4) as well as those with potential
for biomedical[35] or imaging[36] applications viz. glucose
azide (5) as a mixture of α- and β-isomers, coumarin azide
(6), biotin azide (7), cholesterol azide (8) and fluorescein
azide (9).
Azides 3 and 5 are commercially available, and 4[37] and
6[38] were prepared according to literature methods. 2-Azi-
doacetohydrazide (10), prepared from ethyl 2-azidoacet-
ate[37] by reaction with hydrazine hydrate, was coupled with
biotin NHS ester (11) to yield 7. The analogous 8 was ac-
cessed from a parallel route starting from cholesterol chlo-
roformate (12, Scheme 2). Direct coupling of fluorescein
methyl ester (13) and 1-azido-4-bromobutane[39] furnished
9 in 69% yield (Scheme 3).
The attendant advantages of solid over solution phase
chemistry[40] guided our research, and we initially explored
the reaction between CPG (controlled pore glass)-sup-
ported T10-cyclooctyne 1 with 3 and 4. Reactions were con-
ducted on a scale employing 0.2 μm 1 and, in the interest
of expediency, we initially employed 90 equiv. of azide in an
effective dipole concentration of ca. 133 mm. To assist with
azide solubility, aqueous dimethylsulfoxide (DMSO) was
selected as the solvent and reactions were conducted at
room temp. (Scheme 4). Work up of the supported products
14, involving deprotection and cleavage from the resin, af-
forded 16, Figure 2. HPLC analysis indicated complete con-
sumption of 1 within 30 min and the formation of two new
products. In each case, the retention time of the products
was only marginally greater than cleaved 15. SPAAC is not
a perfect reaction and the formation of regioisomeric tri-
azoles is deemed as an acceptable trade off for the avoid-
ance of a copper catalyst in azide–alkyne bioconjugations.
Consistent with triazole formation with little regard for re-
gioselectivity,[32,35] two product peaks were observed for
both 16a and 16b; a minimal bias was seen for the re-
gioisomer with slightly longer retention time.
Figure 1. Resin supported DNA–cyclooctynes and azide reaction
partners for solid-phase SPAAC.
To optimise the reaction, the ratio of the reacting dipole
to the CPG-alkyne was reduced to 20:1 maintaining the di-
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Synthesis of DNA Conjugates by Azide–Cyclooctyne Cycloaddition
Scheme 2. Synthesis of 8 and 9; i. anhydr. DMF, NEt3, Ar, 18 h, room temp., 86%; ii. CH2Cl2, NEt3, Ar, 18 h, room temp., 34%.
Figure 2. Structures of cleaved and deprotected oligonucleotides
T10-cyclooctyne 15 and T10-conjugates 16a and 16b.
Scheme 3. Synthesis of 9.
pole concentration at ca. 140 mm. Aqueous DMSO (70%)
was an ideal solvent for reaction with 3, however, because
of limited solubility, 90% DMSO was preferable for reac-
tion with 4. Gratifyingly, despite the reduced number of di-
pole equivalents, reaction with 3 was complete within
20 min as indicated by HPLC. An additional 10 min reac-
tion time was required for the full consumption of starting
material by 4 (Table 1). The products were readily purified
by HPLC and MALDI-TOF mass spectrometry confirmed
the structural integrity of the benzyl- and cinnamyl-conju-
gated T10-derivatives 16a and 16b. The optimised experi-
ments indicate that it is possible to compensate for the slug-
gish kinetics of strain-promoted cycloaddition of azides,
with respect to nitrile oxide dipoles,[26] simply by increasing
the effective concentration of reacting dipole from about 30
to 140 mm.[8]
Having proven that resin-supported DNA bearing a cy-
clooctyne is a suitable substrate for SPAAC we turned to
structurally complex, yet potentially more valuable, azides
5, 6 and 7. As a consequence of limited dipole solubility in
Scheme 4. Solid-supported SPAAC of 1 with 3 and 4.
aqueous DMSO, reactions with 5 and 6 were conducted in
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I. Singh, C. Freeman, F. Heaney
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Table 1. Reaction conditions for 1 with 3–8.
pared to 3, 4 and 6, to account for the fall in the rate of
reaction (Table 1). However, it is significant that the rates
of formation of 16a–e, by cycloaddition to cyclooctyne,
compare favourably with those observed for conjugates
formed by cycloaddition to RNAs bearing strained alkynes
of enhanced reactivity, viz. dibenzocyclooctyne.[32,33]
Azide
Solvent
Reaction time
Product
3
4
5
6
7
8
70% aq. DMSO
90% aq. DMSO
DMSO
DMSO
90% aq. DMSO
CHCl3
20 min
30 min
4 h
25 min
4 h
16a
16b
16c
16d
16e
16f
It is known that lipid conjugation facilitates both cellular
uptake and intracellular delivery of oligonucleotides and so
provides hope for a nontoxic alternative to the cationic lipo-
philic or polymeric siRNA delivery systems,[41,42] and we
wished to extend the scope of the SPAAC reaction to in-
clude steroidal examples. However, despite the advances in
methodologies for the postsynthetic modification of oligo-
nucleotides, the synthesis of steroidal conjugates remains
nontrivial. In two recent reports, CuAAC modifications
have been demonstrated. In one, conjugation at the mono-
meric level preceded oligonucleotide synthesis,[43] and in the
other, microwave-assisted conjugation to a resin-supported
oligonucleotide–alkyne has been reported (60 °C,
45 min).[41] For pharmaceutical applications the minimal
acceptable copper concentration in the final product is
15 ppm,[44] thus syntheses avoiding copper catalysis have
obvious advantages. Solubilisation of 8 was optimal in
CHCl3, which was employed as the solvent for its reaction
with 1, CHCl3 was also used in place of the traditional
MeCN for washing excess reagents from the resin during
the work up. HPLC analysis of the raw products indicates
complete consumption of 1 after 16 h at room temp.
(Table 1). Due to the lipophilicity of the steroidal moiety,
HPLC analysis of the crude products and separation of the
cholesterol conjugates (16f, Figure 3) were performed on a
Spherisorb C8 column.
16 h
the absence of water, and aqueous DMSO (90%) was effec-
tive for solubilisation of 7. Reactions were conducted at
room temp., with 20 equiv. of dipole. HPLC analysis of the
cleaved reaction products indicate that a reaction time of
between 25 min and 4 h was required for complete con-
sumption of 1 (Table 1). For the conjugates, the peak split-
ting of 16c, arising from the reaction with 5, is consistent
with a mixture of anomers in the starting azide. Products
arising from 6 and 7, 16d and 16e, respectively, are repre-
sented by two sharp peaks. The DNA conjugated triazoles
were easily separated from impurities in the starting mate-
rial. MALDI-TOF mass analysis of 16c indicated deacetyl-
ation of the glucose moiety during the standard protocol
for cleavage of the oligonucleotide from the resin; mass data
confirmed the structural integrity of 16d and 16e (Figure 3).
Having verified the potential of resin-supported 1 as a
click partner with azide dipoles, we wished to demonstrate
compatibility with the CPG-supported dodecamer 2 (DNA
= 5Ј-TCG CAC ACA CGC-3Ј), which carries all four nu-
cleobases with their standard protecting groups. Exposure
of 2 to 3 or 4 (20 equiv., 140 mm) in aqueous DMSO (50%)
resulted in the formation of conjugates 17a and 17b (Fig-
ure 4) as judged by HPLC. As observed for all the T10-cy-
clooctyne–azide conjugates 16, except 16f, similarities in the
polarity characteristics of the parent 15/18 and the conju-
gated oligonucleotides 16a–e/17a–b resulted in HPLC pro-
files with relatively little difference between the retention
time of reacted and unreacted cyclooctynes. However, coin-
jection of a reference sample of 18 with the reaction prod-
ucts indicated almost complete conversion to the conjugates
17a and 17b after 20 min at room temperature. As expected,
the reactions proceeded almost without regiochemical pref-
erence and MALDI-TOF mass spectrometry confirmed the
structural integrity of the new conjugates. Conjugation to 7
required 4 h to reach completion in aqueous DMSO,
whereas reaction with 8 progressed smoothly in CHCl3 af-
ter agitation overnight at room temp. (Table 2). In each
case, HPLC analysis confirmed virtually full consumption
Figure 3. Triazole-ligated conjugates 16c–f, prepared by solid-sup-
ported SPAAC.
It is reported that strain-promoted cycloaddition is rela- of 2. Purified samples of the regioisomeric triazole conju-
tively insensitive to the electronic structure of the azide[19] gates 17c and 17d were obtained following separation by
and we judge the increased steric demands of 5 and 7, com- HPLC.
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Synthesis of DNA Conjugates by Azide–Cyclooctyne Cycloaddition
Fluorescent tags have largely taken over from radioactive
labels in unravelling molecular pathways and we wished to
demonstrate the potential of resin-supported SPAAC with
9 to furnish fluorescent oligonucleotide probes. A range of
solvents, including aqueous N,N-dimethylformamide
(DMF), DMSO and CHCl3 (90%), were selected for experi-
ments between 9 and 2. In all cases, following washing of
excess reagents, the resin-supported products retained an
intense yellow colour and displayed fluorescence. However,
during the cleavage/deprotection protocol, the colour began
to fade before disappearing completely. We supposed that
this concurred with a prevalence of the nonfluorescent lac-
tone form of the hydrolysed ester.[45] Although the HPLC
traces of the crude reaction products indicated significant
consumption of the starting material (≈ 85–100%) and the
emergence of a double peak characteristic of regioisomeric
triazole conjugates, MALDI-TOF mass analysis of a puri-
fied product sample found m/z = 4262, which is consistent
with the spirolactone structure 17e (Figure 5).
The failure of the fluorescein methyl ester to remain inert
under the standard ammomium hydroxide mediated cleav-
age/deprotection protocol of solid phase oligonucleotide
synthesis suggested that conjugation to 9 may be more
suited to solution chemistry. DNA-cyclooctyne 18, ob-
tained from 2 following deprotection and cleavage from the
resin, was exposed to an aqueous DMF solution of 9. The
reaction progressed cleanly, judged by HPLC, however,
even after 18 h at room temp. consumption of the starting
oligonucleotide was modest (≈ 35%). This rose to an ac-
ceptable 80% by doubling the number of azide equivalents.
The identity and integrity of the conjugate, which retained
the yellow colour characteristic of molecules incorporating
a tetracyclic fluorescein skeleton, was confirmed as 17f by
HPLC and MALDI-TOF mass analysis (17f m/z requires
4278; found 4277).
Figure 4. Triazole-ligated DNA-conjugates 17a–d prepared by so-
lid-supported SPAAC.
Table 2. Reaction conditions for 2 with 3, 4, 7, 8 and 9.
Azide
Solvent
Reaction time
Product
3
50% aq. DMSO
50% aq. DMSO
90% aq. DMSO
CHCl3
90% aq. DMF
90% aq. DMF
30 min
30 min
4 h
16 h
16 h
17a
17b
17c
17d
17e
17f
4
7
8
Conclusions
9
9[a]
16 h
We have demonstrated that SPAAC in the solid phase
offers a robust and reliable method for oligonucleotide con-
[a] This reaction was conducted in the solution phase.
Figure 5. Triazole-ligated 17e and 17f prepared from the reaction of 9 with 2 or in the solution phase with DNA-cyclooctyne 18,
respectively.
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I. Singh, C. Freeman, F. Heaney
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55.4, 49.3, 32.9, 28.0, 27.9, 25.0 ppm. HRMS (ESI): calcd. for
C12H20N7O3S [M + H]+ 342.1343; found 342.1352.
jugation. It has been shown that, for reaction in the solid
phase, it is not necessary to have an alkyne partner with
enhanced reactivity and that a simple DNA-cyclooctyne is
a suitable substrate for bioconjugation. Our success with
the monocyclic strained alkyne will be deemed beneficial
by those interested in conjugates of large azido partners
(8S,9S,10R,13R,14S,17R)-10,13-Dimethyl-17-[(R)-6-methylheptan-
2-yl]-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclo-
penta[a]phenanthren-3-yl 2-(2-Azidoacetyl)hydrazinecarboxylate (8):
To a solution of 10 (0.62 g, 5.39 mmol) and triethylamine (620 μL)
or conjugates with reduced lipophilicity. Compatibility is in CH2Cl2 (50 mL) was added a solution of 12 (2.00 g, 4.45 mmol)
in CH2Cl2 (100 mL). The resulting mixture was stirred at room
temperature overnight. The solvent was removed under reduced
pressure and the crude product was purified by flash column
chromatography (hexane/ethyl acetate, 7:3) to yield a white solid
demonstrated with azides of varying steric bulk and elec-
tronic demands, including biotin and cholesterol, the conju-
gates of which have potential application in cell biology and
drug delivery. In circumstances where the azido partner car-
ries functionalities incompatible with solid phase chemistry,
it has been demonstrated that conjugation to DNA-cyclo-
octynes can be efficiently executed in solution.
1
(800 mg, 34%). H NMR: δ = 8.19 (s, 1 H), 6.75 (s, 1 H), 5.38 (d,
J = 5.1 Hz, 1 H), 4.62–4.51 (m, 1 H), 4.08 (s, 2 H), 2.40–2.33 (m,
2 H), 2.03–0.85 (m, 38 H), 0.68 (s, 3 H) ppm. 13C NMR: δ = 166.2,
155.5, 139.3, 123.0, 56.7, 56.1, 51.5, 50.0, 42.3, 39.7, 39.5, 38.2,
36.9, 36.5, 36.2, 35.8, 31.9, 31.8, 28.2, 28.0, 27.9, 24.3, 23.8, 22.8,
22.6, 21.0, 19.3, 18.7, 11.9 ppm. C30H49N5O3 (527.38): C 68.27,
H9.36, N 13.27; found C 68.02, H 9.39, N 13.33.
Experimental Section
Methyl 2-[6-(4-Azidobutoxy)-3-oxo-3H-xanthen-9-yl]benzoate (9):
To a solution of 1-azido-4-bromobutane[39] (270 mg, 1.26 mmol) in
anhydrous DMF (5 mL) was added 13[47] (314 mg, 0.90 mmol) and
potassium carbonate (170 mg, 1.23 mmol). The mixture was heated
to 70 °C under an argon atmosphere for 2 h after which TLC analy-
sis (CH2Cl2/MeOH, 9:1) indicated complete reaction. The mixture
was diluted with EtOAc (20 mL) and washed with water (3ϫ
10 mL). The organic layer was dried with MgSO4, and the solvent
was removed under vacuum to yield an orange-brown solid. This
was washed with hexane (6ϫ 10 mL) to remove excess starting az-
ide, yielding the product as an orange-brown solid, which was used
without further purification (276 mg, 69%). 1H NMR: δ = 8.25 (d,
J = 7.5 Hz, 1 H), 7.77–7.65 (m, 2 H), 7.31 (d, J = 7.2 Hz, 1 H),
6.95–6.83 (m, 3 H), 6.73 (dd, J = 2.1, 8.7 Hz, 1 H), 6.54 (dd, J =
1.8, 9.6 Hz, 1 H), 6.45 (d, J = 1.8 Hz, 1 H), 4.10 (t, J = 6.0 Hz, 2
H), 3.64 (s, 3 H), 3.39 (t, J = 6.6 Hz, 2 H) 1.98–1.73 (m, 4 H) ppm.
13C NMR: δ = 185.5, 165.6, 163.3, 158.9, 154.2, 150.3, 134.5, 132.7,
131.1, 130.5, 130.2, 130.2, 129.7, 129.7, 128.9, 117.4, 114.8, 113.6,
105.6, 100.8, 68.1, 52.3, 51.03, 26.2, 25.6 ppm. HRMS (ESI): calcd.
for C25H22N3O5 [M + H]+ 444.1554; found 444.1574.
General: Analytical TLC was performed on precoated (250 μm) sil-
ica gel 60 F-254 plates from Merck. All plates were visualised by
UV irradiation and/or staining with 5% H2SO4 in ethanol followed
by heating. Flash chromatography was performed using silica gel
32–63 μm, 60 Å. Mass analysis was performed with a LASER-TOF
LT3 or Applied Biosystem Voyager with 3-hydroxypicolinic acid or
2Ј,4Ј,6Ј-trihydroxyacetophenone as matrix or recorded by Meta-
bion, Germany. NMR spectra were obtained with a Bruker instru-
ment at 25 °C (1H at 300 MHz; 13C at 75 MHz). Chemical shifts
are reported in ppm downfield from TMS as standard. NMR spec-
tra are recorded in CDCl3 unless otherwise stated. UV analysis
was performed with a Jasco V-630BIO spectrophotometer at 25 °C.
HPLC was carried out with a Gilson instrument equipped with a
diode array detector and a Nucleosil C18 column (4.6ϫ 250 mm)
or a Phenonmex C8 column (4.6ϫ250 mm); a Dionex Ultimate
3000 instrument equipped with a Clarity Oligo RP C18 (4.6ϫ
250 mm) or a Spherisorb C8 (4.6ϫ 250 mm) column was also used.
DNA was desalted with illustra™ NAP™-10 Sephadex™ G-25
DNA grade columns purchased from GE Healthcare.
2-Azidoacetohydrazide (10): To a solution of ethyl 2-azidoacetate[37]
(2.0 g, 15.5 mmol) in ethanol (10 mL) was added hydrazine hydrate
(1.3 mL, 25.3 mmol). The mixture was allowed to stir at room tem-
perature for 2 h after which TLC analysis (CH2Cl2/MeOH, 9:1)
indicated complete consumption of the starting material. Following
removal of the solvent under reduced pressure, the crude product
was purified by flash column chromatography (CH2Cl2/MeOH,
General Procedure for the Azide Click Reactions on 1. Preparation
of Conjugates 16: To solid-supported 1[8] (0.2 μmol) in an Eppen-
dorf tube was added a solution of the azide in DMSO (20 μL of a
200 mm stock solution, 4 μmol, 20 equiv.) and the final volume was
adjusted to 30 μL with DMSO and water according to the solubil-
ity of azide (Table 1). The mixture was agitated at room tempera-
ture. After completion of the conjugation (Table 1), as monitored
by HPLC, the supernatant liquid was removed by syringe and the
CPG was washed with CH3CN (5ϫ 300 μL) and H2O (5ϫ
300 μL). In the reaction with 8, chloroform was used as the reac-
tion solvent and in place of CH3CN during the work up. Cleavage
from the resin, deprotection (method i) and HPLC analysis (condi-
tions A) followed according to the procedures described below.
1
9:1) to yield a colourless oil (1.6 g, 90%). H NMR: δ = 8.14 (br.
s, 1 H), 4.06 (br. s, 2 H), 4.01 (s, 2 H) ppm. 13C NMR: δ = 167.5,
51.6 ppm. HRMS (ESI): calcd. for C2H5N5ONa [M + Na]+
138.0386; found 138.0390.
NЈ-(2-Azidoacetyl)-5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-
yl)pentanehydrazide (7): To a solution of 11[46] (870 mg, 2.55 mmol)
and 10 (352 mg, 3.06 mmol) in anhydrous DMF (20 mL) was
added triethylamine (1.5 mL, 20.4 mmol). The resulting mixture
was stirred overnight at room temperature under an argon atmo-
sphere. Following removal of particulate matter by filtration, the
filtrate was evaporated to yield the crude product as a white solid,
General Procedures for Deprotection and Cleavage of DNA on Solid
Phase: For analytical purposes a portion of the DNA was depro-
tected and cleaved from the CPG by incubating the CPG-DNA
under conditions i) 28% aqueous NH4OH (500 μL) at 25 °C for
which was washed with CH2Cl2 (5 ϫ 10 mL) to yield pure 7 30 min (for substrates 16), or ii) 28% aqueous NH4OH (500 μL)
(745 mg, 86%). 1H NMR ([D6]DMSO): δ = 10.05 (br. s, 1 H), 9.86
diluted to 750 μL with EtOH at 25 °C for 24 h (for substrates 17).
(s, 1 H), 6.43 (s, 1 H), 6.36 (s, 1 H), 4.33–4.29 (m, 1 H), 4.16–4.12 NH4OH was evaporated using a concentrator. The CPG was
(m, 1 H), 3.89 (s, 2 H), 3.14–3.07 (m, 1 H), 2.86–2.80 (m, 1 H), washed with H2O (3ϫ 150 μL aliquots), all solutions and washings
2.60–2.56 (m, 1 H), 2.14 (t, J = 7.2 Hz, 2 H), 1.64–1.30 (m, 6 H)
were combined to afford an aqueous solution of DNA, which was
analysed and purified by reversed-phase HPLC.
ppm. 13C NMR ([D6]DMSO): δ = 170.9, 166.3, 162.7, 61.0, 59.2,
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Synthesis of DNA Conjugates by Azide–Cyclooctyne Cycloaddition
General Methods for HPLC Analysis and Purification of Oligo-
nucleotide Conjugates: DNA cycloaddition products 16 and 17 were
analyzed by reverse-phase HPLC under either conditions A (for
products 16) or B (for products 17). Conditions A: 20 μL injection
loop. Buffer A: 0.1 m TEAAc, pH 7.5, 1% (v/v) MeCN; Buffer B:
0.1 m TEAAc, pH 7.5, 80% (v/v) MeCN. Gradient for 16a–e: 0–
3 min, 5% B; 3–23 min, 5Ǟ95% B. Gradient for 16f: 0–3 min 10%
B, 3–13, 10Ǟ95% B. Flow rate: 1.0 mL/min. Detection at 260 nm.
Column: Clarity Oligo RP C18 (4.6ϫ 250 mm) for 16a–e and a
Spherisorb C8 (4.6ϫ 250 mm) column for 16f. Conditions B:
200 μL injection loop. Buffer A: 0.1 m TEAAc, pH 7.5, 5% (v/v)
MeCN; Buffer B: 0.1 m TEAAc, pH 7.5, 65% (v/v) MeCN. Gradi-
ent: 0–4.3 min, 5% B; 4.3–16.6 min, 5Ǟ100% B. Flow rate: 1.0 mL/
min. Detection at 260 nm. Column: Nucleosil C18 column (4.6ϫ
250 mm) for 17a–c,e,f or Phenonmex C8 column (4.6ϫ 250 mm)
for 17d.
Acknowledgments
Financial support from the Science Foundation of Ireland (Pro-
gramme code 05/PICA/B838) is acknowledged. C. F. is grateful to
NUI Maynooth and the Irish Research Council Science and Engi-
neering for receipt of an Embark Postgraduate Research Scholar-
ship (Programme code RS/2007/48).
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Supporting Information (see footnote on the first page of this arti-
cle): Copies of 1H and 13C NMR spectra, HPLC data and
MALDI-TOF MS data.
Eur. J. Org. Chem. 2011, 6739–6746
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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6746
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Eur. J. Org. Chem. 2011, 6739–6746