1996 Oxford University Press
Nucleic Acids Research, 1996, Vol. 24, No. 2
361–366
Photocleavable biotin phosphoramidite for
5′-end-labeling, affinity purification and
phosphorylation of synthetic oligonucleotides
Jerzy Olejnik, Edyta Krzymanska-Olejnik and Kenneth J. Rothschild*
Department of Physics and Molecular Biophysics Laboratory, Boston University, Boston, MA 02215, USA
Received August 17, 1995; Revised and Accepted November 20, 1995
ABSTRACT
We report the design, synthesis and evaluation of a
non-nucleosidic photocleavable biotin phosphoramidite (PCB-phosphoramidite) which provides a simple
method for purification and phosphorylation of oligonucleotides. This reagent introduces a photocleavable
biotin label (PCB) on the 5′-terminal phosphate of
synthetic oligonucleotides and is fully compatible with
automated solid support synthesis. HPLC analysis
shows that the PCB moiety is introduced predominantly on full-length sequences and is retained during
cleavage of the synthetic oligonucleotide from the
solid support and during subsequent deprotection
with ammonia. The full-length 5′-PCB-labeled oligonucleotide can then be selectively isolated from the
crude oligonucleotide mixture by incubation with
immobilized streptavidin. Upon irradiation with
300–350 nm light the 5′-PCB moiety is cleaved with
high efficiency in 3 h) under acidic conditions (80% acetic acid).
We report here the synthesis of a photocleavable biotin
phosphoramidite (PCB-phosphoramidite). This phosphoramidite
incorporates a recently described photocleavable biotin moiety
(PCB) (18) on the 5′-end of a synthetic oligonucleotide. We show
that: (i) the PCB-phosphoramidite reagent is fully compatible
with automated DNA/RNA synthesizers using phosphoramidite
chemistry; (ii) the 5′-PCB moiety is retained during cleavage
from the solid support and deprotection of the oligonucleotide
with ammonia; (iii) the 5′-PCB moiety allows streptavidin
affinity purification of the oligonucleotide from failure
sequences; (iv) the PCB moiety is rapidly and quantitatively
photocleaved from the 5′-end upon irradiation with near-UV light
(300–350 nm) to give a 5′-phosphorylated oligonucleotide.
Importantly, PCB-phosphoramidite provides a rapid method for
the purification, isolation and phosphorylation of synthetic
oligonucleotides. As an example, we have used PCB-
whom correspondence should be addressed at: Department of Physics, Boston University, 590 Commonwealth Avenue, Boston, MA 02215, USA
362
Nucleic Acids Research, 1996, Vol. 24, No. 2
phosphoramidite to synthesize, purify and phosphorylate 50- and
60mer oligonucleotides.
MATERIALS AND METHODS
All chemicals used in the synthesis were purchased from Aldrich
Chemical Co. (Milwaukee, WI). 1H NMR spectra were recorded
in CDCl3 on a Varian (Palo Alto, CA) Unity Plus spectrometer at
400 MHz with chemical shifts (δ, p.p.m.) reported relative to a
tetramethylsilane internal standard. 31P NMR spectra were
recorded in CDCl3 on a JEOL (Peabody, MA) JNM-GSX270
spectrometer at 109.36 MHz with chemical shifts (δ, p.p.m.)
reported relative to an 85% H3PO4 external standard. Oligonucleotide synthesis was performed on an Applied Biosystems
(Foster City, CA) DNA/RNA synthesizer model 392. Samples
were irradiated with a Blak Ray XX-15 UV lamp (Ultraviolet
Products Inc., San Gabriel, CA) at a distance of 15 cm (emission
peak 365 nm, 300 nm cut-off, 1.1 mW intensity at 31 cm).
UV-visible spectra were recorded on a Shimadzu 2101PC
spectrophotometer. HPLC analysis was performed on a Waters
(Milford, MA) system consisting of a U6K injector, 600
Controller, Novapak C18 (3.9 × 150 mm) column and a 996
photodiode array detector. Buffer A, 0.1 N triethylamine acetate,
pH 6.0; buffer B, acetonitrile. Elution was performed using a
linear gradient (8–45%) of buffer B in buffer A over 45 min at a
flow rate of 1 ml/min. Preparative purification of 5′-PCB-(dT)7
was achieved on a Waters Novapak C18 RCM cartridge (8 × 100
mm) using conditions as specified above, except for flow rate,
which was increased to 2 ml/min. Fractions were then analyzed,
pooled and freeze dried. No special precautions were necessary
to protect the reagent and the 5′-PCB-labeled oligonucleotides
from light.
PCB-phosphoramidite synthesis
1-N-(4,4′-dimethoxytrityl)-5-(6-biotinamidocaproamidomethyl)2-nitroacetophenone (compound 2). 5-(6-Biotinamidocaproamidomethyl)-2-nitroacetophenone (1) (18) (0.5 g, 0.94 mmol) was
dried by co-evaporation with anhydrous pyridine (3 × 2 ml) and
then dissolved in 5 ml of the latter. To this solution was added
4,4′-dimethoxytrityl chloride (DMTr-Cl) (0.634 g, 1.87 mmol)
followed by 4-dimethylaminopyridine (0.006 g, 0.046 mmol).
The reaction mixture was stirred at room temperature for 5 h and
then an additional 0.317 g DMTr-Cl was added. After 24 h the
reaction was quenched with methanol (1 ml), poured into 100 ml
0.1 M sodium bicarbonate and extracted with methylene chloride
(3 × 50 ml). Evaporation of the combined extracts gave a yellow
oil, which was further purified on a silica gel column using a step
gradient of methanol in dichloromethane, 0.2% triethylamine.
Appropriate fractions were pooled and evaporated to give
compound 2 as a white foam (0.73 g, 93% yield). TLC,
CHCl3:MeOH 9:1 v/v; Rf = 0.45. 1H NMR: 7.87–7.85 (d,2H),
7.23–7.20 (m,5H), 7.17–7.15 (d,1H), 7.11–7.05 (m,5H),
6.75–6.71 (m,4H), 5.92–5.86 (t,1H), 5.61 (s,1H), 4.43–4.36
(m,2H), 4.05–3.85 (m,2H), 3.73 (s,6H), 3.70 (s,3H), 3.40–3.30
(m,1H), 3.12–3.02 (m,2H), 2.98–2.89 (m,1H), 2.48 (s,1H),
2.32–2.20 (m,2H), 2.11–2.03 (m,4H), 1.63 (s,3H), 1.60–1.34
(m,7H), 1.26–1.23 (m,2H). Elemental analysis (%): calculated
(C46H53N5O8S), C 66.09, H 6.39, N 8.38; found, C 65.85, H 6.23,
N 8.05.
1-N-(4,4′-dimethoxytrityl)-5-(6-biotinamidocaproamidomethyl)1-(2-nitrophenyl)-ethanol (compound 3). 1-N-(4,4′-Dimethoxytrityl)-5-(6-biotinamidocaproamidomethyl)-2-nitroacetophenone
(2) (0.85 g, 1.016 mmol) was dissolved in 7 ml ethanol and
sodium borohydride (0.028 g, 0.74 mmol) was added with
stirring. After 1 h the reaction was quenched with 4 ml acetone
and evaporated under reduced pressure to give a yellow oil, which
was redissolved in 10 ml methanol and the solution added to 120
ml water. The precipitate was isolated by centrifugation (7000
r.p.m., 45 min) and dried in vacuo over KOH to give compound
3 (0.7 g, 82%). TLC, CHCl3:MeOH 9:1 v/v; Rf = 0.39. 1H NMR:
7.83–7.74 (m,2H), 7.32 (t,1H), 7.24–7.17 (m,5H), 7.13–7.00
(m,4H), 6.98 (d,1H), 6.73–6.69 (m,4H), 5.99–5.91 (d,1H),
5.86–5.78 (m,1H) (OH), 5.49–5.45 (m,1H), 4.50–4.24 (m,4H),
3.74 (s,6H), 3.58–3.25 (m,1H), 3.07–3.01 (m,1H), 3.00–2.85
(m,1H), 2.29–2.12 (m,2H), 2.01–1.96 (m,2H), 1.80–1.75 (m,1H),
1.64 (s,3H), 1.53–1.43 (m,12H), 1.38–1.11 (m,1H). Elemental
analysis (%): calculated (C46H55N5O8S), C 65.93, H 6.62, N
8.36; found, C 65.97, H 6.52, N 8.10.
[1-N-(4,4′-dimethoxytrityl)-5-(6-biotinamidocaproamidomethyl)1-(2-nitrophenyl)-ethyl]-2-cyanoethyl-N,N-diisopropylaminophosphoramidite (compound 4). 1-N-(4,4′-Dime t h o x y trityl)5-(6-biotinamidocaproamidomethyl)-1-(2-nitrophenyl) ethanol (3)
(0.186 g, 0.22 mmol) was placed in an oven-dried flask with a
magnetic stirring bar, sealed with a septum and dried for at least
6 h in vacuo. Anhydrous acetonitrile (0.003% water) (1 ml) was
added through a septum under argon. Subsequently N,N-diisopropylethylamine (0.15 ml, 0.88 mmol) was added, followed by
2-cyanoethoxy-N,N-diisopropylchlorophosphine (0.052 g, 0.22
mmol). After 1 h another 0.5 eq. phosphine was added. After an
additional 2 h at room temperature the reaction mixture was
treated with 0.3 ml ethyl acetate, followed by a saturated saline
solution (10 ml), and extracted with methylene chloride (3 × 10
ml). The organic layer was washed with water, dried over sodium
sulfate and evaporated under reduced pressure and purified on a
silica gel column using a step gradient (0–3%) of triethylamine
in acetonitrile. Appropriate fractions were pooled and evaporated
to give compound 4 as a white foam (0.144 g, 62% yield). TLC,
MeCN:Et3N, 95:5 v/v; Rf = 0.48. 1H NMR (p.p.m.): 7.79–7.32
(m,1H), 7.65–7.61 (m,1H), 7.25–7.19 (m,5H), 7.13–7.05 (m,4H),
6.91–6.85 (m,1H), 6.75–6.73 (m,4H), 5.75–5.66 (br s,1H),
5.54–5.43 (m,1H), 5.22s, 5.12d (1H), 4.38–4.26 (m,3H),
4.23–4.11 (m,2H), 3.88–3.77 (m,1H), 3.73 (s,1H), 3.66–3.54
(m,2H), 3.46–3.37 (m,1H), 3.29–3.21 (m,1H), 3.10–3.02 (m,2H),
2.65–2.60 (m,1H), 2.54–2.44 (m,1H), 2.40–2.32 (m,1H),
2.26–2.20 (dd,1H), 2.10–2.11 (app. t,1H), 2.08–2.01 (m,2H),
1.62–1.58 (m,6H), 1.55–1.45 (m,4H), 1.39–1.35 (t,2H),
1.31–1.27 (t,2H), 1.16–1.07 (m,9H), 0.87–0.83 (dd,3H). 31P
NMR (p.p.m.): 146.7, 147.9. Elemental analysis (%): calculated
(C55H72N7O9PS), C 63.63, H 6.99, N 9.44; found, C 63.11, H
6.79, N 9.20.
5′-PCB-oligonucleotide synthesis
A 0.1 M solution of the PCB-phosphoramidite (4) in anhydrous
acetonitrile was attached to the extra port of the Applied
Biosystems 392 DNA/RNA synthesizer. The syntheses were
carried out on a 0.2 µmol scale using cyanoethyl phosphoramidites. For the last coupling (introduction of 4) the coupling time
was increased by 120 s, as recommended for conventional biotin
phosphoramidite (19). Typical coupling efficiency (as deter-
363
Nucleic Acids
Acids Research,
Research,1994,
1996,Vol.
Vol.22,
24,No.
No.12
Nucleic
363
mined by trityl cation conductance) was between 95 and 97%.
Standard detritylation (‘trityl-off’ option) as well as cleavage and
deprotection procedures were used. Control 5′-phosphorylated
sequences were synthesized using chemical phosphorylation
reagent Phosphalink (Applied Biosystems) according to the
manufacturer’s instructions (20).
Affinity purification and photocleavage
Crude 5′-PCB-oligonucleotide (16 nmol) was added to a
suspension of streptavidin–agarose beads (700 µl, 24 nmol)
(Sigma, Milwaukee, WI) and the suspension incubated at room
temperature for 1 h. It was then spin-filtered (5 min, 5000 r.p.m.)
using a 0.22 µm Ultrafree MC filter (Millipore, Bedford, MA).
Beads on the filter were washed with 100 µl phosphate buffer, pH
7.2, and spin-filtered (three times). Finally, the beads were
resuspended in 700 µl phosphate buffer and irradiated for 5 min.
After irradiation the suspension was spin-filtered, the beads were
washed with phosphate buffer (3 × 100 µl) and the combined
filtrate volume was adjusted to 1 ml and analyzed by UV
absorption spectroscopy or HPLC.
Time dependence of the photocleavage
In order to calculate the time dependence of the photocleavage,
HPLC-purified 5′-PCB-(dT)7 (48 nmol) was incubated with 1.5
eq. streptavidin–agarose beads for 1 h. The beads were spinfiltered, washed, resuspended in phosphate buffer, pH 7.2, and
irradiated. Aliquots (200 µl each) were withdrawn after 0, 0.25,
0.5, 1, 2, 4, 6 and 10 min irradiation, spin-filtered and washed as
described above. The filtrate volume was adjusted to 1 ml and the
absorbance at 260 nm measured. A sample of 700 µl streptavidin
which had not been incubated with oligonucleotide was spinfiltered and the UV absorption measured, serving as background.
A similar measurement was made on a sample of oligonucleotide
not incubated with streptavidin (16 nmol, 700 µl phosphate
buffer, serving as 100% control). The molar extinction coefficient
at 260 nm for the PCB moiety was determined separately
(4700/M/cm) and this value subtracted from the estimated
(assuming a molar extinction coefficient equal to 12 000 for each
dT) molar extinction coefficient of 5′-PCB-(dT)7 (88 700) for
photorelease efficiency calculations. In order to determine the
time course of photocleavage in solution (dT)7-5′-PCB (1 OD260)
was dissolved in 1 ml phosphate buffer and irradiated at 300–350
nm. Aliquots (10 µl) were withdrawn after 0, 0.25, 0.5, 1, 2, 4, 6
and 10 min irradiation and injected onto an HPLC column. The
percent conversion was calculated from the ratio of the area of the
particular peak (i.e. 5′-PCB-(dT)7 or 5′-p-(dT)7) over the sum of
the areas of the component peaks, with molar extinction
coefficients of the components adjusted as described above.
RESULTS
Design and synthesis of PCB-phosphoramidite
The synthesis of PCB-phosphoramidite (4) is depicted in Scheme
1 (see Materials and Methods for more details). The compound
consists of a protected biotin moiety linked through a spacer arm
(6-aminocaproic acid) to a photoreactive 1-(2-nitrophenyl)ethyl
moiety (21), which is derivatized with N,N′-diisopropyl-2-cyanoethyl-phosphoramidite. The starting material, 5-(6-biotinamidocaproamidomethyl)-2-nitroacetophenone (1) was synthesized as
Scheme 1.
described previously (18). The 4,4′-dimethoxytrityl (DMTr)
group was introduced selectively onto the N1 nitrogen of biotin
(3,6). The intermediate (2) was then selectively reduced using
sodium borohydride to give compound 3 and, finally, the
resulting hydroxyl group was phosphitylated using 1.5 eq.
2-cyanoethoxy-N,N-diisopropylchlorophosphine. No phosphitylation of biotin nitrogen N2 was observed under the reaction
conditions.
PCB-phosphoramidite (4) was designed for direct use in any
automated DNA/RNA synthesizer employing standard phosphoramidite chemistry. As shown in Scheme 2, the selective reaction
of compound 4 with the free 5′-OH group of a full-length
oligonucleotide results in the introduction of a phosphodiester
group linked to a photocleavable biotin moiety. In contrast, all
capped failure sequences which lack a free 5′-OH group do not
react with the PCB-phosphoramidite. The biotinyl moiety thus
allows selective isolation of only full-length sequences through
streptavidin affinity media. Upon irradiation with near-UV light
the phosphodiester bond between the PCB moiety and the
phosphate is cleaved, resulting in the formation of a 5′-monophosphate on the released oligonucleotide. The 1-(2-nitrophenyl)ethyl moiety is converted into a 2-nitrosoacetophenone
derivative.
Synthesis and evaluation of PCB-oligonucleotides
The heptamer 5′-PCB-(dT)7 was assembled using PCB-phosphoramidite (4) in an automated DNA/RNA synthesizer. The
unmodified sequence 5′-OH-(dT)7 and a 5′-phosphorylated
sequence, 5′-p-(dT)7, were prepared using standard procedures
(see Materials and Methods). Figure 1 shows the HPLC trace of
364
Nucleic Acids Research, 1996, Vol. 24, No. 2
Figure 1. HPLC traces of (a) 5′-PCB-(dT)7, (b) 5′-PCB-(dT)7 complexed with
streptavidin after 4 min irradiation, (c) 5′-p-(dT)7 and (d) 5′-OH-(dT)7. See
Materials and Methods for more details.
Scheme 2.
5′-PCB-(dT)7 (trace a). Two main peaks are observed in this trace,
with retention times of 23.7 and 24.3 min. These two peaks can
be attributed to the two diastereoisomers generated by introduction of the PCB moiety onto the 5′-end of the oligonucleotide
(22). Compared with the unmodified oligonucleotide
5′-OH-(dT)7 (trace d, retention time 14.5 min) the PCB-modified
oligonucleotide (trace a) shows an increased retention time,
which is typical for biotinylated oligonucleotides (5,16). We
conclude from these data that the 5′-PCB moiety is retained
during cleavage and deprotection of the oligonucleotide with
ammonia [5′-phosphorylated oligonucleotide is not present in the
5′-PCB-(dT)7 sample].
Interaction of the PCB-modified oligonucleotide with streptavidin and photorelease of the oligonucleotide were evaluated by
incubating 5′-PCB-(dT)7 with streptavidin–agarose beads, separating the beads from the solution by spin-filtering and irradiating
the resuspended beads with 300–350 nm light. The effects of
irradiating the resuspended beads for 4 min are shown in Figure
1. The two peaks assigned to 5′-PCB-(dT)7 (trace a) disappear
and a single peak appears (trace b) with a retention time of 12.5
min. Importantly, the retention time of this peak is almost
identical to that of the reference 5′-phosphorylated sequence, i.e.
5′-p-(dT)7 (trace c, retention time 12.6 min). These data
conclusively show that irradiation causes cleavage of the PCB
moiety and release of 5′-phosphorylated oligonucleotide into
solution.
Time dependence of photocleavage and oligonucleotide
release
We measured the time dependence of the photoconversion of
5′-PCB-(dT)7 into 5′-p-(dT)7 in solution. For this purpose a
5′-PCB-(dT)7 solution was subjected to irradiation with 300–350
nm light and the reaction mixture was analyzed by reversed phase
HPLC after different irradiation times (Fig. 2). It can be seen from
the decrease in the intensity of peaks at 23.7 and 24.3 min,
assigned to 5′-PCB-(dT)7, and the increase in the intensity of the
single peak at 12.6 min, assigned to 5′-p-(dT)7, that the
photoreaction is complete in ∼4 min. The appearance of
additional small peaks with a retention time of ∼33 min can be
attributed to formation of the biotinyl-2-nitrosoacetophenone
derivative and other minor photoproducts identified previously
(23,24).
The time dependence and efficiency of photocleavage of
5′-PCB-(dT)7 complexed with streptavidin–agarose beads was
determined by measuring the absorbance of the supernatant at
260 nm, which reflects the amount of 5′-p-(dT)7 released into
solution. The initial A260 value at 0 min (Fig. 2, inset) corresponds
to
