Fluorescent labeling of (oligo)nucleotides by a new fluoride cleavable linker capable of versatile attachment modes

Article abstract of DOI:10.1021/bc900542f

The development of a fluoride cleavable linker 1 for reversibly labeling (oligo)nucleotides is described here. The linker allows different ways of chemical attachment of a reporter molecule, for example, click chemistry or amide formation. The versatile attachment modes of labels are demonstrated by derivatizations with pyrene and fluorescein. Besides the synthesis of the new linker, we also show the derivatization of iodobenzene as a model compound and a nucleoside to demonstrate the applicability. Further, cleavability studies in solution and on a solid-supported oligonucleotide are shown. The linker can be applied in the synthesis of reversible terminators, useful for new DNA sequencing technologies like cyclic reversibly terminating (CRT) sequencing.

Full text of DOI:10.1021/bc900542f

Bioconjugate Chem. 2010, 21, 1043–1055
1043
Fluorescent Labeling of (Oligo)Nucleotides by a New Fluoride Cleavable
Linker Capable of Versatile Attachment Modes
Diana C. Knapp, Jennifer D’Onofrio, and Joachim W. Engels*
Institut fu¨r Organische Chemie und Chemische Biologie, Johann Wolfgang Goethe Universita¨t, Max-von-Laue Strasse 7,
60438 Frankfurt am Main, Germany. Received December 9, 2009; Revised Manuscript Received March 31, 2010
The development of a fluoride cleavable linker 1 for reversibly labeling (oligo)nucleotides is described here. The
linker allows different ways of chemical attachment of a reporter molecule, for example, click chemistry or amide
formation. The versatile attachment modes of labels are demonstrated by derivatizations with pyrene and fluorescein.
Besides the synthesis of the new linker, we also show the derivatization of iodobenzene as a model compound
and a nucleoside to demonstrate the applicability. Further, cleavability studies in solution and on a solid-supported
oligonucleotide are shown. The linker can be applied in the synthesis of reversible terminators, useful for new
DNA sequencing technologies like cyclic reversibly terminating (CRT) sequencing.
INTRODUCTION
The fluorescent labeling of nucleotides is an important tool
in biochemistry (1, 2). So far a very important application of
fluorescently labeled nucleotides is the Sanger DNA-sequencing
method (3). Novel sequencing approaches (4, 5) for future
demands on “individualized” diagnostic genome analysis also
require reversibly labeled nucleotides. This creates a need for
cleavable linkers to reversibly attach a dye to a nucleotide.
These new sequencing techniques include methods for whole
Figure 1. Core structure 1 of the new cleavable linker.
genome sequencing (6-8), as well as for the detection of
nucleotides to the reporter moiety (fluorescent dye). This linker
should be efficiently cleavable under the same conditions as
the 3′-blocking group to allow the regeneration of the 3′-OH
group and the removal of the linker-dye system in a single
deprotection step. The dye will be attached to the base moiety
of the nucleosides because Sanger sequencing has already
demonstrated that modifications at the 7-position of purines and
the 5-position of pyrimidines are well tolerated by DNA
polymerases.
In this publication, we present the design, the synthesis, and
the evaluation of a new fluoride cleavable linker for reversible
labeling of (oligo)nucleotides. In Figure 1, the core structure 1
of the new linker is displayed.
The linker design was derived from the well-known acidity
of protons vicinal to a nitrile function. This motif also plays a
key role in the cleavage of other fluoride cleavable protecting
groups that are not based on silicon, like the above-mentioned
CE (12, 13), the 1-(2-cyanoethoxy)methyl (14), and the l-(2-
cyanoethoxy)ethyl (15) protecting groups.
The connection of the substrate (nucleotide) is planned at
the secondary alcohol function via a prop-2-ynylcarbamate
moiety. The azide group at the end of the triglycol spacer then
serves as a juncture for reporter molecules. These molecules
can either be attached by click reactions to the existing azide
moiety or via amide or carbamate bonds after reduction of the
azide to the amine.
mutations (mainly SNPs) in minisequencing approaches (9, 10).
One of these new approaches is the array-based cyclic reversibly
terminating (CRT) sequencing (7, 11). This technique does not
need traditional gel electrophoretic separation but affords direct
readout of the sequence by fluorescence detection. The labeled
nucleotides are added sequentially to oligonucleotide templates
that are immobilized on an array platform, thus sequencing can
be performed in a highly parallel fashion. CRT sequencing is
based on the use of so-called “reversible terminators” to achieve
the defined sequential addition of one base after the other. The
structural requirements for these reversible terminators include
a reversibly terminating moiety at the 3′-position and a reporter
molecule (i.e., a fluorescent dye) reversibly attached via a
cleavable linker.
During development of such reversible terminators, one issue
is to find a 3′-modification that meets the following demands:
reliable termination of the polymerase reaction, quantitative and
fast cleavage under reasonable conditions, and acceptance by a
DNA polymerase. In a recent publication, we presented the
fluoride cleavable 2-cyanoethyl (CE)1 group as a very promising
candidate for a 3′-reversibly terminating group (12). The next
step is to develop a suitable cleavable linker to connect the
* To whom correspondence should be addressed. Phone number:
+49-69-79829150. Fax number: +49-69-79829148. E-mail address:
joachim.engels@chemie.uni-frankfurt.de.
First the synthesis of the linker will be shown. For the
evaluation of the cleavage properties, a simple model compound
9 was synthesized where the nucleotide moiety is replaced by
a phenyl moiety. After defining the cleavage conditions of the
model compound a 5-,3′-modified 2′-deoxyuridine derivative
17 was synthesized, bearing the 3′-O-(2-cyanoethyl) (CE) group
and the cleavable linker at position 5 of the base moiety. Finally
1Abbreviations: ACN, acetonitrile; Boc, tert-butoxycarbonyl; Bz,
benzoyl; CE, 2-cyanoethyl; CPG, controlled pore glass; DIPEA,
diisopropylethylamine; DMF, N,N-dimethylformamide; DMTr, 4,4′-
dimethoxytrityl; EDTA, ethylenediaminetetraacetate; TBAF, tetrabu-
tylammonium fluoride; TCA, trichloroacetic acid; TFA, trifluoroacetic
acid; THF, tetrahydrofuran; TLC, thin layer chromatography; TMS(Cl),
trimethylsilyl (chloride).
10.1021/bc900542f  2010 American Chemical Society
Published on Web 05/28/2010

1044 Bioconjugate Chem., Vol. 21, No. 6, 2010
Knapp et al.
the optimized cleavage conditions were applied to a CPG-bound
8-mer bearing the fluorescently labeled and 3′-O-modified 2′-
deoxyuridine at the 3′-end.
3.07-3.11 (1H, m, CH oxirane ring), 3.268 (2H, dd, J ) 6.4
and 11.6 Hz, CH2 adjacent oxirane), 3.395 (2H, “t”, N3-CH2),
3.52-3.63 (10H, m, 5 × CH2 glycol), 3.714 (1H, dd, J ) 2.8
and 11.6 Hz, CH2 adjacent oxirane) ppm. 13C-NMR (100 MHz,
DMSO-d6, 300 K): δ ) 43.39 (CH2 oxirane ring), 50.03 (N3-
CH2), 50.29 (CH oxirane ring), 69.27, 69.72, 69.82, 69.84, 69.97
(5 × CH2 glycol), 71.53 (CH2 adjacent oxirane) ppm. FT-IR
(NaCl): ν ) 3050 (m, epoxide C-H), 2900 (s, >CH2), 2120 (s,
-N3) cm-1. ESI(+)-MS (m/z) Calcd: 231.2 [C9H17N3O4]. Found:
254.0 [M + Na]+, 249.0 [M + H2O]+, 232.0 [M]+. Anal.
(C9H17N3O4) C, H, N.
EXPERIMENTAL PROCEDURES
Materials and Methods. All reagents were of the highest
commercially available quality and were used as received.
Benzoyl chloride and acrylonitrile were distilled before use.
Oligonucleotide synthesis was accomplished using an Expedite
nucleic acid synthesis system from PerSeptive Biosystems. The
5′-O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite-3′-(4,4′-
dimethoxytrityl)-2′-deoxyribonucleosides building blocks were
purchased from Pharmacia; the solid support (dT5′-CPG) was
purchased from Glen Research. NMR spectra were recorded
on Bruker AM, DPX, and AV instruments at 250, 300, and
400 MHz and 300 K. Chemical shifts (δ) are reported in ppm
relative to the solvent signal. The fine structure of proton signals
was specified with s (singlet), d (doublet), t (triplet), q (quartet),
m (multiplet), dd (doublet of doublet), br (broad), and in
quotations for pseudo-fine structure. Assignments were made
by DEPT, COSY, HSQC, and HMBC experiments. Thin layer
chromatograms (TLC) were recorded on Polygram Sil G/UV254
by Macherey Nagel & Co., Dueren (thickness of layer 0.2 mm)
or 60 F254 by Merck KGaA, Darmstadt (thickness of layer 0.2
mm). Flash column chromatography was carried out on silica
gel 60 (15-40 µm) by Merck KGaA, Darmstadt at a pressure
of 2-3 bar. Reversed phase (RP) HPLC was performed on a
Jasco LC-2000Plus HPLC system equipped with a Jasco UV
2075Plus detector (detection at 254 nm) and a Phenomenex
Jupiter 4u Proteo 90A 4 µm column (250 mm × 4.6 mm). One
molar triethylammonium acetate (TEAA) buffer, pH ) 6.5, (A)/
water (B)/acetonitrile (C) were used as eluents. Ion-exchange
HPLC was performed on a Jasco LC-900 HPLC system
equipped with a Jasco UV-970 detector (detection at 254 nm)
and a Dionex BioLC DNAPac PA-100 column (250 mm × 9
mm) using water/0.25 M tris(hydroxymethyl)aminomethane
hydrochloride (Tris-Cl) buffer, pH ) 8/1 M sodium chloride
solution as eluent. ESI mass spectrometry was performed on a
Fisons instrument equipped with a VG platform II with
quadrupole analyzer. MALDI mass spectrometry was performed
on a VG TOFSpec from Fisons using 2,5-dihydroxybenzoic acid
as matrix for single molecules and 6-aza-2-thiothymin as matrix
for oligonucleotides. Fluorescence spectroscopy was performed
on a Hitachi F4500 fluorescence spectrometer using 0.3 cm
cuvettes. Elemental analyses were recorded on a Foss-Heraeus
CHN-O Rapid instrument. The numbering of the atoms in the
nucleoside parts is according to the common numbering
convention of nucleosides.
2-{2-[2-(2-Azidoethoxy)ethoxy]ethoxymethyl}oxirane, 4.
To a suspension of 151 mg of sodium hydride (6.28 mmol, 1.1
equiv) in 6 mL of dry THF, 1 g of compound 3 (5.7 mmol, 1.0
equiv) was added dropwise, and the resulting mixture was stirred
for 2 h at room temperature. (()-Epichlorohydrin (2.3 mL, 28.54
mmol, 5.0 equiv) was added dropwise, and the resulting mixture
was stirred for 16 h at room temperature. The reaction mixture
then was neutralized with 30% methanolic H2SO4 and poured
into 40 mL of saturated NaCl solution. The aqueous layer was
extracted two times with 30 mL of ethyl acetate and two times
with 30 mL of methylene chloride. The combined organic layers
were dried over Na2SO4 and concentrated under reduced
pressure. The resulting oily residue was purified by flash column
chromatography using a gradient from n-hexane/ether ) 1:2 +
1% NH3 to n-hexane/ether ) 1:5 + 1% NH3. Title compound
2 was obtained as colorless oil (884 mg, 67%). RF ) 0.13 (n-
hexane/ether ) 1:2 + 1% NH3). 1H NMR (400 MHz, DMSO-
d6, 300 K): δ ) 2.540 (1H, dd, J ) 2.7 and 5.1 Hz, CH2 oxirane
ring), 2.724 (1H, dd, J ) 4.2 and 5.1 Hz, CH2 oxirane ring),
4-{2-[2-(2-Azidoethoxy)ethoxy]ethoxy}-3-hydroxybutyroni-
trile, 1. To a solution of 500 mg of compound 4 (2.16 mmol,
1.0 equiv) in 2 mL of ethanol, 10 mL of water was added. After
stirring for 5 min, 153 mg of sodium cyanide (3.03 mmol, 1.4
equiv) was added, and stirring was continued for 16 h at room
temperature. The reaction mixture was then concentrated to
about half the volume under reduced pressure, extracted two
times with 20 mL of ethyl acetate and one time with 20 mL of
methylene chloride, dried over Na2SO4 and concentrated. The
oily residue was purified by flash-column-chromatography using
CH2Cl2/MeOH ) 97:3 as eluent. The desired compound 1 was
obtained as colorless oil (498 mg, 89%). RF ) 0.37 (CH2Cl2/
1
MeOH ) 95:5). H NMR (400 MHz, DMSO-d6, 300 K): δ )
2.532 (1H, dd, J ) 6.7 and 16.9 Hz, NC-CH2), 2.655 (1H, dd,
J ) 4.4 and 16.8 Hz, NC-CH2), 3.321 (1H, dd, J ) 6.2 and
10.0 Hz, NC-CH2-CH-CH2), 3.382 (2H, “t”, N3-CH2), 3.404
(1H, dd, J ) 5.3 and 10.0 Hz, NC-CH2-CH-CH2), 3.52-3.62
(10H, m, 5 × CH2 glycol), 3.872 (1H, m, NC-CH2-CH), 5.455
(1H, d, J ) 5.2 Hz, OH) ppm. 13C-NMR (100 MHz, DMSO-
d6, 300 K): δ ) 22.52 (NC-CH2), 49.99 (N3-CH2), 65.24
(NC-CH2-CH), 69.27, 69.70, 69.71, 69.83, 70.14 (5 × CH2
glycol), 73.37 (NC-CH2-CH-CH2), 118.86 (NC) ppm. FT-IR
(NaCl): ν ) 3520-3300 (s, O-H), 2900 (s, >CH2), 2250 (m,
-Ct N), 2108 (s, -N3) cm-1. ESI(+)-MS (m/z) Calcd: 258.3
[C10H18N4O4]. Found: 280.9 [M + Na]+, 275.9 [M + NH4]+,
299.9 [M + K]+. Anal. (C10H18N4O4) C, H, N.
(2-{2-[2-(3-Cyano-2-hydroxypropoxy)ethoxy]ethoxy}ethyl)-
carbamic Acid tert-Butyl Ester, 5. Compound 1 (5 g, 19.36
mmol, 1.0 equiv) was dissolved in dry dioxane, 10.47 g of
triphenylphosphine (38.72 mmol, 2.0 equiv) was added, and the
mixture was stirred at room temperature. After 8 h, TLC control
indicated complete consumption of the starting material, and
100 mL of 25% aqueous ammonia was added. The reaction
was left at room temperature for 12 h and subsequently dried
in vacuum. The residue was dissolved in 100 mL of methylene
chloride and extracted three times with 25 mL of 1 M HCl
solution. The water phase was neutralized with solid NaHCO3,
and 40 mL of dioxane and another 2.4 g of NaHCO3 (29.00
mmol, 1.5 equiv) were added. The mixture was cooled to 0 °C,
and 8.7 g of Boc2O (38.72 mmol, 2.00 equiv) was added in
portions over 20 min. The reaction mixture was allowed to warm
to room temperature and stirred for 10 h. After concentration
in vacuum, the residue was divided between 100 mL of water
and 100 mL of ethyl acetate. The aqueous layer was extracted
two times with 70 mL of ethyl acetate. The combined organic
layers were dried over Na2SO4, and the solvent was removed
under reduced pressure. The oily residue was purified by flash
column chromatography using CH2Cl2/MeOH ) 99:1 + 1%
EtMe2N as eluent. Compound 5 was obtained as a slightly
yellow oil (6.45 g, 100%). RF ) 0.51 (CH2Cl2/MeOH/NH3 )
1
90:10:1). H NMR (300 MHz, DMSO-d6, 300 K): δ ) 1.368
(9H, s, 3 × CH3), 2.518 (1H, dd, J ) 6.6 and 18.6 Hz,
NC-CH2), 2.645 (1H, dd, J ) 4.4 and 16.8 Hz, CN-CH2),
3.058 (2H, q, J ) 5.9 Hz, NH-CH2), 3.27-3.45 (4H, m,
NC-CH2-CH-CH2, NH-CH2-CH2), 3.48-3.56 (8H, m, 4
× CH2 glycol), 3.864 (1H, m, NC-CH2-CH), 5.443 (1H, d, J

New Fluoride Cleavable Linker for Dye Labeling
Bioconjugate Chem., Vol. 21, No. 6, 2010 1045
) 5.2 Hz, OH), 6.703 (1H, m, NH) ppm. 13C NMR (75 MHz,
DMSO-d6, 300 K): δ ) 22.47 (NC-CH2), 28.19 (3 × CH3),
39.69 (NH-CH2), 65.25 (NC-CH2-CH), 69.18 (NH-CH-
CH2), 69.50, 69.67, 69.75, 70.14 (4 × CH2 glycol), 73.37
(NC-CH2-CH-CH2), 77.56 ((CH3)3C), 118.71 (NC), 155.54
(CO) ppm. ESI(+)-MS (m/z) Calcd: 332.4 [C15H28N2O6]. Found:
355.2 [M + Na]+, 333.1 [M + H]+, 350.2 [M + NH4]+. Anal.
(C15H28N2O6) C, H, N.
0.1 equiv) were mixed and irradiated for 30 min at 250 W in
the microwave. Afterward, the residue was dissolved in meth-
ylene chloride and washed two times with 10 mL of 5% EDTA
solution. The organic layer was dried over Na2SO4, and the
solvent was evaporated in vacuum. The crude product was
purified by flash column chromatography (CH2Cl2/MeOH )
99:1 to CH2Cl2/MeOH ) 98:2), yielding 110 mg (60%) of the
product 7 as yellow oil. RF ) 0.22 (CH2Cl2/MeOH ) 95:5). 1H
NMR (400 MHz, DMSO-d6, 300 K): δ ) 2.459 (1H, dd, J )
6.7 and 16.8 Hz, NC-CH2), 2.575 (1H, dd, J ) 4.3 and 16.9
Hz, NC-CH2), 3.226 (1H, dd, J ) 6.3 and 10.1 Hz,
NC-CH2-CH-CH2), 3.30-3.35 (1H, m, NC-CH2-
CH-CH2), 3.45-3.66 (8H, m, 4 × CH2 glycol), 3.807 (“sextet”,
NC-CH2-CH), 3.992 (2H, t, J ) 4.9 Hz, triazole-CH2-CH2),
4.728 (2H, t, J ) 5.1 Hz, triazole-CH2), 5.437 (1H, d, J ) 5.4
Hz, OH), 8.115 (1H, t, J ) 7.9 Hz, pyrene CH), 8.20-8.45
(7H, m, 7 × pyrene CH), 8.742 (1H, s, triazole CH), 8.884
(1H, d, J ) 9.5 Hz, pyrene CH) ppm. 13C-NMR (100 MHz,
3-Hydroxy-4-(2-{2-[2-(4-phenyl-1,2,3-triazol-1-yl)ethoxy]eth-
oxy}ethoxy)butyronitrile, 6. Linker 1 (500 mg, 1.94 mmol, 1.0
equiv) was dissolved in 10 mL of methylene chloride and 5
mL of methanol. To the resulting solution, first 31 mg of CuSO4
(0.19 mmol, 0.1 equiv) and then 775 mg of sodium ascorbate
(3.87 mmol, 2 equiv) were added, followed by the addition of
1.5 equiv of phenylacetylene (329 mg, 2.90 mmol). The mixture
was stirred at room temperature. After 18 h, another 0.5 equiv
of phenylacetylene (110 mg, 0.97 mmol) was added, and the
mixture was stirred at room temperature for additional 24 h.
The reaction mixture then was concentrated under reduced
pressure, and the residue was dissolved again in 50 mL of
methylene chloride. The solution of the crude product was
washed two times with 25 mL of 5% EDTA solution. The
organic layer was dried over Na2SO4, and the solvent was
evaporated in vacuum. The crude product was purified by flash
column chromatography (CH2Cl2/MeOH ) 98:2 to CH2Cl2/
MeOH ) 95:5), yielding 670 mg (96%) of the product 6 as
DMSO-d6, 300 K):
δ
)
22.43 (NC-CH2), 49.67
(triazole-CH2), 65.17 (NC-CH2-CH), 68.68 (triazole-
CH2-CH2), 69.63, 69.64, 69.68, 70.06 (4 × CH2 glycol), 73.32
(NC-CH2-CH-CH2), 118.77 (NC), 124.83, 124.87 (triazole
CH, pyrene CH), 125.12, 125.15, 125.52, 126.46, 126.98, 127.33
127.64, 127.96 (8 × pyrene CH), 123.93, 1242.31, 125.46,
127.46, 130.36, 130.52, 130.94, 145.98 (triazole quaternary C,
7 × pyrene quaternary C) ppm. ESI(+)-MS (m/z) Calcd: 484.5
[C28H28N4O4]. Found: 485.5 [M + H]+, 507.6 [M + Na]+. HR-
ESI(-)-MS (m/z) Calcd: 483.2038 [C28H27N4O4]-. Found:
483.2043. Fluorescence: excitation 352 nm, emission 390 nm,
408 nm.
1
colorless oil. RF ) 0.40 (CH2Cl2/MeOH ) 90:10). H NMR
(400 MHz, DMSO-d6, 300 K): δ ) 2.498 (1H, dd, J ) 7.0 and
16.9 Hz, NC-CH2), 2.619 (1H, dd, J ) 4.3 and 16.9 Hz,
NC-CH2), 3.271 (1H, dd,
J ) 6.2 and 9.8 Hz,
NC-CH2-CH-CH2), 3.371 (1H, dd, J ) 5.5 and 9.8 Hz, NC-
CH2-CH-CH2), 3.46-3.58 (8H, m, 4 × CH2 glycol), 3.79-3.90
(3H, m, NC-CH2-CH, triazole-CH2-CH2), 4.571 (2H, t, J
) 5.1 Hz, triazole-CH2), 5.449 (1H, d, J ) 5.1 Hz, OH),
7.30-7.36 (1H, m, phenyl CHp), 7.42-7.49 (2H, m, 2 × phenyl
CHm), 7.81-7.87 (2H, m, 2 × phenyl CHo), 8.522 (1H, s,
triazole CH) ppm. 13C NMR (100 MHz, DMSO-d6, 300 K): δ
Carbonic Acid 1-{2-[2-(2-Azidoethoxy)ethoxy]ethoxymethyl}-
2-cyanoethylester 2,5-dioxo-pyrrolidin-1-yl ester, 10. Compound
1 (750 mg, 2.90 mmol, 1.0 equiv) was dissolved in 25 mL of
dry ACN and cooled to 0 °C. Then 223 mg of anhydrous K2CO3
(1.60 mmol, 0.55 equiv) was added, and the suspension was
stirred at 0 °C for 1 h before 1.96 g of di(N-succinimidyl)-
carbonate (7.26 mmol, 2.5 equiv) was added and the mixture
was stirred for another 20 h at 0 °C. The solvent was removed
in vacuum, and the residue was divided between 50 mL of ethyl
acetate and 50 mL of saturated NaHCO3 solution. The water
phase was additionally extracted with 50 mL of ethyl acetate
and 50 mL of methylene chloride. The combined organic layers
were dried over Na2SO4 and concentrated under reduced
pressure. The crude product was obtained as yellow oil 10 (1.15
g, 99%) and used without further purification. RF ) 0.35
(CH2Cl2/MeOH ) 99:1). 1H NMR (400 MHz, CDCl3, 300 K):
δ ) 2.853 (4H, s, 2 × succinimidyl CH2), 2.902 (1H, dd, J )
5.4 and 17.1 Hz, NC-CH2), 2.968 (1H, dd, J ) 6.0 and 17.0
Hz, NC-CH2), 3.38-3.42 (2H, m, N3-CH2), 3.65-3.74 (10H,
m, 5 × CH2 glycol), 3.805 (1H, dd, J ) 5.3 and 11.0 Hz,
NC-CH2-CH-CH2-O), 3.851 (1H, dd, J ) 4.7 and 11.0 Hz,
NC-CH2-CH-CH2-O), 5.100 (1H, “quintet”, NC-CH2-CH)
ppm. 13C-NMR (100 MHz, DMSO-d6, 300 K): δ ) 19.77
(NC-CH2), 25.50 (succinimidyl CH2), 50.74 (N3-CH2), 69.36,
70.09, 70.62, 70.73, 70.74, 71.35 (NC-CH2-CH-CH2, 5 ×
CH2 glycol), 74.91 (NC-CH2-CH), 115.23 (NC), 150.86
(carbonate CO), 168.28 (succinimidyl CO) ppm. ESI(+)-MS
(m/z) Calcd: 399.4 [C15H21N5O8]. Found: 417.00 [M + NH4]+,
422.00 [M + Na]+, 372.00 [M - N2+H]+.
)
22.47 (NC-CH2), 49.58 (triazole-CH2), 65.19
(NC-CH2-CH), 68.64 (triazole-CH2-CH2), 69.61, 70.07 (4
× CH2 glycol), 73.33 (NC-CH2-CH-CH2), 118.79 (NC),
121.71 (triazole CH), 125.07 (2 × phenyl CHo), 127.76 (phenyl
CHp), 128.86 (2 × phenyl CHm), 130.82 (triazole quaternary
C), 146.16 (phenyl quaternary C) ppm. ESI(+)-MS (m/z) Calcd:
360.4 [C18H24N4O4]. Found: 361.3 [M
(C18H24N4O4) C, H, N.
+
H]+. Anal.
3-Hydroxy-4-(2-{2-[2-(4-pyren-1-yl-1,2,3-triazol-1-yl)ethoxy]-
ethoxy}ethoxy)butyronitrile, 7. Method a. Linker 1 (110 mg,
0.43 mmol, 1.0 equiv) was dissolved in 2 mL of methylene
chloride and 1 mL of methanol. To the resulting solution, first
7 mg of CuSO4 (0.04 mmol, 0.1 equiv) and then 170 mg of
sodium ascorbate (0.85 mmol, 2 equiv) were added. In a separate
flask, 146 mg of 1-ethynylpyrene (0.64 mmol, 1.5 equiv) was
dissolved in 4 mL of methylene chloride and 2 mL of methanol.
The pyrene solution was added to the linker solution via a
syringe, and the resulting mixture was stirred for 20 h at room
temperature. Because the reaction seemed to be quite slow, it
was heated to 40 °C for another 24 h. Afterward the solvents
were removed under reduced pressure, and the residue was
dissolved in 20 mL of methylene chloride. The solution of the
crude product was washed two times with 10 mL of 5% EDTA
solution. The organic layer was dried over Na2SO4, and the
solvent was evaporated in vacuum. The crude product was
purified by flash column chromatography (CH2Cl2/MeOH )
99:1 to CH2Cl2/MeOH ) 98:2), yielding 85 mg (41%) of the
product 7 as a yellow oil.
Carbonic Acid 1-{2-[2-(2-tert-Butoxycarbonylaminoethoxy)-
ethoxy]ethoxymethyl}-2-cyano-ethylester 2,5-Dioxopyrrolidin-
1-yl Ester, 11. Alcohol 5 (5.27 g, 15.86 mmol, 1.0 equiv) was
dissolved in 150 mL of dry ACN and cooled to 0 °C. Anhydrous
K2CO3 (1.11 g, 7.93 mmol, 0.5 equiv) was added, and the
mixture was left at 0 °C for 2 h. Then 10.69 g of di(N-
succinimidyl)carbonate (39.64 mmol, 2.5 equiv) was added, and
stirring at 0 °C was continued for 24 h. The solvent was
Method b. Linker 1 (100 mg, 0.38 mmol, 1.0 equiv), DIPEA
(130 µL, 0.77 mmol, 2.0 equiv), and CuI (7.2 mg, 0.038 mmol,

1046 Bioconjugate Chem., Vol. 21, No. 6, 2010
Knapp et al.
evaporated, and the residue was divided between 75 mL of water
and 75 mL of ethyl acetate. The aqueous layer was extracted
two times with 50 mL of methylene chloride. The combined
organic layers were dried over Na2SO4 and concentrated under
reduced pressure. To the solid residue, first 30 mL of methylene
chloride was added and the insoluble parts were filtered off,
followed by the addition of 60 mL of ether. The resulting
suspension was stored at 4 °C for 2 h and filtered again. The
filtrate was concentrated in vacuum to give 6.73 g (90%) of
the activated linker 11 as yellow oil. It was used as obtained,
without further purification. RF ) 0.03 (CH2Cl2/MeOH/NH3 )
(3-Phenylprop-2-ynyl)carbamic Acid 1-{2-[2-(2-Azidoethoxy-
ethoxy]ethoxymethyl}-2-cyanoethyl Ester, 9. Iodobenzene (300
mg, 1.44 mmol, 1.00 equiv) was dissolved in 7 mL of dry
methylene chloride and degassed three times using the
freeze-pump-thaw technique. Then 28 mg of CuI (0.14 mmol,
0.10 equiv) and 84 mg of Pd(PPh3)4 (0.07 mmol, 0.05 equiv)
were added to the shaded flask. The alkyne 8 (572 mg, 11.69
mmol, 1.17 equiv) was added dropwise in two portions; one-
half was added immediately and the other half after 1 h stirring
at room temperature. After another 2 h stirring at room
temperature, the reaction mixture was diluted by adding 20 mL
of methylene chloride and washed two times with 50 mL of
5% EDTA solution and one time with 50 mL of saturated
NaHCO3 solution. The organic layer was dried over Na2SO4,
filtered and the solvent was removed in vacuum. The crude
product was purified by flash column chromatography (n-hexane
+ 1% EtMe2N to n-hexane/ethyl acetate ) 1:1 + 1% EtMe2N).
Compound 9 was obtained as yellow oil (549 mg, 92%). RF )
0.18 (n-hexane/ethyl acetate/EtMe2N ) 1:1:0.02). 1H NMR (300
MHz, DMSO-d6, 300 K): δ ) 2.80-2.96 (2H, m, NC-CH2),
3.379 (2H, “t”, N3-CH2), 3.51-3.62 (12H, m, NC-CH2-
CH-CH2, 5 × CH2 glycol), 4.064 (2H, d, J ) 5.8 Hz,
NH-CH2), 4.988 (1H, “quintet”, NC-CH2-CH), 7.34-7.44
(5H, m, 5 × phenyl CH), 7.964 (1H, t, J ) 5.7 Hz, NH) ppm.
13C NMR (75 MHz, DMSO-d6, 300 K): δ ) 19.70 (NC-CH2),
30.57 (NH-CH2), 49.98 (N3-CH2), 67.88 (NC-CH2-CH),
69.21, 69.65, 69.66, 69.79, 70.19, 70.33 (NC-CH2-CH-CH2,
5 × CH2 glycol), 81.79 (alkyne CC-CH2), 86.89 (alkyne
CC-CH2), 117.70 (NC), 122.22 (phenyl quaternary C), 128.47,
128.57, 131.28 (5 × phenyl CH), 154.90 (carbamate CO) ppm.
FT-IR (NaCl): ν ) 3332 (s br., >N-H), 3056 (m, C-H arom.),
2872 (s br., >CH2), 2251 (m, -Ct N, -Ct C-), 2108 (s, -N3),
1730 (s, -CdO) cm-1. MALDI(+)-MS (m/z) Calcd: 415.44
[C20H25N5O5]. Found: 439.83 [M + Na + H]+. Anal.
(C20H25N5O5) C, H, N.
1
90:10:1). H NMR (400 MHz, DMSO-d6, 300 K): δ ) 1.372
(9H, s, 3 × CH3), 2.817 (4H, s, 2 × succinimidyl CH2),
3.01-3.11 (4H, m, NC-CH2, NH-CH2), 3.374 (2H, t, J )
6.1 Hz, NH-CH2-CH2), 3.47-3.61 (8H, m, 4 × CH2 glycol),
3.654 (1H, dd, J ) 6.4 and 11.7 Hz, NC-CH2-CH-CH2),
3.701 (1H, dd, J ) 3.8 and 11.6 Hz, NC-CH2-CH-CH2),
5.16-5.23 (1H, m, NC-CH2-CH), 6.68-6.77 (1H, m, NH)
ppm. 13C NMR (100 MHz, DMSO-d6, 300 K): δ ) 19.12
(NC-CH2), 25.34 (succinimidyl CH2), 28.19 (3 × CH3), 39.65
(NH-CH2), 69.21 (NH-CH2-CH2), 69.45, 69.57, 69.68, 69.74,
70.27 (5 × CH2 glycol), 75.57 (NC-CH2-CH), 77.55
((CH3)3C), 116.79 (NC), 150.63 (carbonate CO), 155.54 (car-
bamate CO), 169.61 (succinimidyl CO) ppm. ESI(+)-MS (m/
z) Calcd: 473.5 [C20H31N3O10]. Found: 491.0 [M + NH4]+, 496.2
[M + Na]+, 474.1 [M]+.
Prop-2-ynylcarbamic Acid 1-{2-[2-(2-Azidoethoxy)ethoxy]-
ethoxymethyl}-2-cyanoethyl Ester, 8. Compound 1 (130 mg,
0.5 mmol, 1.0 equiv) was dissolved in 10 mL of dry ACN, and
the solution was cooled to 0 °C before 70 mg of anhydrous
K2CO3 (0.5 mmol, 1.0 equiv) was added and the suspension
was stirred at 0 °C for 2 h. Afterward 190 mg of di(N-
succinimidyl)carbonate (0.7 mmol, 1.4 equiv) was added
together with another 5 mL of dry ACN. The suspension was
stirred at 0 °C for 20 h. After the activation process, 100 mg of
NaHCO3 (1.0 mmol, 2.0 equiv) and 72 µL of propargylamine
(1.1 mmol, 2.2 equiv) were added. The mixture was stirred at
0 °C for 1 h and at room temperature for 2 h. The solvent was
removed under reduced pressure, and the residue was divided
between 20 mL of saturated NaHCO3 solution and 20 mL of
methylene chloride. The organic layer was washed with 20 mL
of saturated NaHCO3 solution and 20 mL of saturated NH4Cl
solution. The combined aqueous layers were extracted two times
with 20 mL of methylene chloride, and the combined organic
layers were then dried over Na2SO4. The solvent was removed
under reduced pressure, and the crude product was purified by
flash column chromatography (n-hexane + 1% EtMe2N to
n-hexane/ethyl acetate ) 1:1 + 1% EtMe2N). Compound 8 was
obtained as slightly yellowish oil (129 mg, 76%). RF ) 0.27
(E/Z)-4-{2-[2-(2-Azidoethoxy)ethoxy]ethoxy}but-2-eneni-
trile, 13. Model compound 9 (440 mg, 1.06 mmol, 1.0 equiv)
was dissolved in 5 mL of dry THF, and 4.71 g of 1 M TBAF
in THF (5 equiv) was added. The solution was stirred at room
temperature for 10 min; then the solvents were evaporated in
vacuum, and the residue was purified by flash column chroma-
tography using a gradient from n-hexane/ethyl acetate ) 3:1 to
CH2Cl2/MeOH ) 90:10 + 1% EtMe2N. The elimination product
13 was obtained in quantitative yield (258 mg) in a 1:1 ratio of
1
E and Z product (determined by H NMR). Also cleavage
product 12 was isolated in quantitative yield (145 mg); the
analytical data for 12 correspond to literature values (16). RF
1
) 0.35 (CH2Cl2/MeOH ) 99:1). H NMR (300 MHz, CDCl3,
300 K): δ ) 3.395 (4H, t, J ) 5.0 Hz, N3-CH2,E, N3-CH2,Z),
3.63-3.72 (20H, m, 5 × CH2,E glycol, 5 × CH2,Z glycol), 4.191
(2H, dd, J ) 2.4 and 3.8 Hz, NC-CHdCH-CH2,E), 4.373 (2H,
dd, J ) 1.7 and 6.0 Hz, NC-CHdCH-CH2,Z), 5.461 (1H, dt,
J ) 1.7 and 11.3 Hz, NC-CHdCHZ), 5.735 (1H, dt, J ) 2.3
and 16.3 Hz, NC-CHdCHE), 6.617 (1H, dt, J ) 6.0 and 12.0
Hz, NC-CHZ), 6.737 (1H, dt, J ) 3.7 and 16.3 Hz, NC-CHE)
ppm. 13C NMR (75 MHz, CDCl3, 300 K): δ ) 50.73 (N3-CH2,E/
Z), 69.33, 69.58, 70.09, 70.13, 70.49, 70.59, 70.64, 70.71, 70.74,
70.76, 70.78 (2C) (NC-CHdCH-CH2,E, NC-CHdCH-CH2,Z,
5 × CH2,E glycol, 5 × CH2,Z glycol), 99.75 (NC-CHdCHE),
100.60 (NC-CHdCHZ), 115.15 (NCZ), 117.31 (NCE), 150.77
(NC-CHZ), 150.87 (NC-CHE) ppm. ESI(+)-MS (m/z) Calcd:
240.3 [C10H16N4O3]. Found: 258.0 [M + NH4]+, 262.9 [M +
Na]+, 240.9 [M + H]+. Anal. (C10H16N4O3) C, H, N.
1
(n-hexane/ethyl acetate ) 1:2). H NMR (400 MHz, DMSO-
d6, 300 K): δ ) 2.832 (1H, dd, J ) 6.3 and 17.2 Hz, NC-CH2),
2.900 (1H, dd, J ) 4.8 and 17.2 Hz, NC-CH2), 3.120 (1H, t,
J ) 2.4 Hz, alkyne CH), 3.390 (2H, t, J ) 5.2 Hz, N3-CH2),
3.50-3.62 (12H, m, NC-CH2-CH-CH2, 5 × CH2 glycol),
3.782 (2H, dd, J ) 2.4 and 5.8 Hz, NH-CH2), 4.947 (1H,
“quintet”, NC-CH2-CH), 7.891 (1H, t, J ) 5.7 Hz, NH) ppm.
13C-NMR (100 MHz, DMSO-d6, 300 K): δ ) 19.72 (NC-CH2),
29.82 (NH-CH2), 50.00 (N3-CH2), 67.89 (NC-CH2-CH),
69.26, 69.67, 69.69, 69.83, 70.20, 70.32 (NC-CH2-CH-CH2,
5 × CH2 glycol), 73.17 (alkyne CCH), 81.08 (alkyne CCH),
117.70 (NC), 154.88 (carbamate CO) ppm. FT-IR (NaCl): ν )
3297 (s br., >N-H, t C-H), 2900 (s br., >CH2), 2252 (m,
-Ct N), 2108 (s, -N3), 1730 (s, -CdO) cm-1. ESI(+)-MS
(m/z) Calcd: 339.3 [C14H21N5O5]. Found: 362.0 [M + Na]+,
357.0 [M + NH4]+, 340.0 [M]+, 679.3 [2M]+. Anal.
(C14H21N5O5) C, H, N.
N3-Benzoyl-5-iodo-5′-O-benzoyl-2′-deoxyuridine, 15. To a
solution of 1.5 g of compound 14 (3.27 mmol, 1.0 equiv) in 15
mL of dry pyridine were added dropwise 1.68 mL of DIPEA
(9.82 mmol, 3.0 equiv) and 0.63 mL of TMSCl (4.91 mmol,

New Fluoride Cleavable Linker for Dye Labeling
Bioconjugate Chem., Vol. 21, No. 6, 2010 1047
1.5 equiv). The reaction mixture was stirred 45 min at room
temperature before 0.84 mL of benzoyl chloride (4.91 mmol,
1.5 equiv) was added. The red solution was stirred for another
15 min at room temperature and then poured into 100 mL of
ice water and stirred for 10 min, and the organic layer was
extracted two times with 40 mL of chloroform, dried over
Na2SO4, and evaporated to dryness. The crude brown oil was
dissolved in 60 mL of CHCl3/MeOH ) 1:1, and 75 µL portions
of TFA were added every 20 min until the TMS group was
cleaved quantitatively (TLC control). TFA (375 µL, 4.89 mmol,
1.5 equiv) was added within 2 h. The reaction mixture was
diluted with 30 mL of chloroform and washed two times with
50 mL of 5% NaHCO3 solution. The water phase was extracted
three times with 30 mL of chloroform; the combined organic
layers were dried over Na2SO4 and concentrated in vacuum.
The crude product was filtered through silica gel and eluted
with a gradient from pure CH2Cl2 to CH2Cl2/MeOH ) 95:5.
The product 15 was obtained as yellow foam (1.85 g, 98%). RF
) 0.43 (CH2Cl2/MeOH ) 95:5). 1H NMR (400 MHz, DMSO-
d6, 300 K): δ ) 2.271 (1H, ddd, J ) 3.7, 6.5, and 13.9 Hz,
2′-CH2), 2.39-2.47 (1H, m, 2′-CH2), 4.14-4.18 (1H, m, 4′-
CH), 4.37-4.42 (1H, m, 3′-CH), 4.493 (1H, dd, J ) 5.8 and
12.1 Hz, 5′-CH2), 4.558 (1H, dd, J ) 3.6 and 12.1 Hz, 5′-CH2),
5.487 (1H, d, J ) 4.4 Hz, 3′-OH), 6.114 (1H, t, J ) 6.5 Hz,
1′-CH), 7.54-7.62 (4H, m, 2 × 5′-O-benzoyl CHm, 2 × N3-
benzoyl CHm), 7.67-7.72 (1H, m, N3-benzoyl CHp), 7.77-7.81
(1H, m, 5′-O-benzoyl CHp), 8.01-8.05 (4H, m, 2 × 5′-O-
benzoyl CHo, 2 × N3-benzoyl CHo), 8.202 (1H, s, 6-CH) ppm.
13C NMR (100 MHz, DMSO-d6, 300 K): δ ) 39.36 (2′-CH2),
64.31 (5′-CH2), 68.54 (5-CI), 70.04 (3′-CH), 84.43 (4′-CH),
86.06 (1′-CH), 128.88, 129.26, 129.45, 130.56 (5′-O-benzoyl
CHo, 5′-O-benzoyl CHm, N3-benzoyl CHo, N3-benzoyl CHm),
130.64 (5′-O-benzoyl quaternary C, N3-benzoyl quaternary C),
133.53 (N3-benzoyl CHp), 135.65 (5′-O-benzoyl CHp), 145.28
(6-CH), 148.66 (2-CO), 159.18 (4-CO), 165.61 (N3-benzoyl
CO), 168.67 (5′-O-benzoyl CO) ppm. ESI(+)-MS (m/z) Calcd:
562.3 [C23H19IN2O7]. Found: 579.8 [M + NH4]+, 584.6 [M +
Na]+, 563.2 [M + H]+, 601.1 [M + K]+. Anal. (C23H19IN2O7)
C, H, N.
CHo, 2 × N3-benzoyl CHo), 8.219 (1H, s, 6-CH) ppm. 13C NMR
(100 MHz, DMSO-d6, 300 K): δ ) 18.19 (NC-CH2), 36.66
(2′-CH2), 63.77 (NC-CH2-CH2), 64.45 (5′-CH2), 68.69 (5-
CI), 78.68 (3′-CH), 81.97 (4′-CH), 86.13 (1′-CH), 119.56 (NC),
128.88, 129.28, 129.45, 130.56 (5′-O-benzoyl CHo, 5′-O-benzoyl
CHm, N3-benzoyl CHo, N3-benzoyl CHm), 130.62 (5′-O-benzoyl
quaternary C, N3-benzoyl quaternary C), 133.56 (N3-benzoyl
CHp), 135.67 (5′-O-benzoyl CHp), 145.22 (6-CH), 148.66 (2-
CO), 159.15 (4-CO), 165.56 (N3-benzoyl CO), 168.61 (5′-O-
benzoyl CO) ppm. ESI(+)-MS (m/z) Calcd: 615.4
[C26H22IN3O7]. Found: 633.2 [M + NH4]+, 616.1 [M + H]+,
638.1 [M + Na]+. Anal. (C26H22IN3O7) C, H, N.
The crude benzoyl-protected product N3-benzoyl-5-iodo-3′-
O-(2-cyanoethyl)-5′-O-benzoyl-2′-deoxyuridine (1.78 g) was
dissolved in 150 mL of methanol, and 30 mL of concentrated
(25%) aqueous ammonia was added. The mixture was left
stirring for 20 h at room temperature, evaporated, and purified
by flash column chromatography using a gradient from CH2Cl2
to CH2Cl2/MeOH ) 98:2. 3′-Modified nucleoside 16 was
obtained as colorless foam (765 mg, 65%, over two steps). RF
) 0.4 (CH2Cl2/MeOH ) 90:10). 1H NMR (400 MHz, DMSO-
d6, 300 K): δ ) 2.18-2.30 (2H, m, 2′-CH2), 2.773 (2H, t, J )
6.1 Hz, NC-CH2), 3.634 (2H, t, J ) 6.1 Hz, CN-CH2-CH2),
3.55-3.68 (2H, m, 5′-CH2), 3.94-3.98 (1H, m, 4′-CH),
4.12-4.17 (1H, m, 3′-CH), 5.19-5.25 (1H, m, 5′-OH), 6.061
(1H, “t”, 1′-CH), 8.359 (1H, s, 6-CH), 11.680 (1H, s, N3H) ppm.
13C-NMR (100 MHz, DMSO-d6, 300 K): δ ) 18.25 (NC-CH2),
37.05 (2′-CH2), 61.06 (5′-CH2), 63.56 (NC-CH2-CH2), 69.51
(5-CI), 78.99 (3′-CH), 84.80 (1′-CH), 84.82 (4′-CH), 119.17
(NC), 144.88 (6-CH), 150.09 (2-CO), 160.44 (4-CO) ppm.
ESI(-)-MS (m/z) Calcd: 407.2 [C12H14IN3O5]. Found: 406.1 [M
- 1]-. Anal. (C12H14IN3O5) C, H, N.
5-(3-Aminoprop-1-ynyl)-3′-O-(2-cyanoethyl)-2′-deoxyuri-
dine, 17. Compound 16 (500 mg, 1.23 mmol, 1.0 equiv) was
dissolved in 15 mL of dry DMF, and 855 µL of triethylamine
(6.14 mmol, 5.0 equiv) was added. The solution was degassed
three times by using the freeze-pump-thaw technique. After
the solution warmed up, 48 mg of CuI (0.25 mmol, 0.2 equiv)
and 143 mg of Pd(PPh3)4 (0.12 mmol, 0.1 equiv) were added.
2,2,2-Trifluor-N-(prop-2-ynyl)acetamid (371 mg, 2.46 mmol, 2.0
equiv) was added in two portions; one-half was injected directly,
and the other half was added after stirring at room temperature
for 1 h. The resulting mixture was stirred at room temperature
in a shaded flask. After 5 h, the solvent was removed under
reduced pressure, and the residue was dissolved in 25 mL of
methylene chloride and washed two times with 25 mL of 5%
EDTA solution. The organic layer was dried over Na2SO4 and
filtered, and the solvent was removed in vacuum. The crude
product, 5-[(2,2,2-trifluoro-acetylamino)-ethynyl]-3′-O-(2-cya-
noethyl)2′-deoxyuridine, was filtered over silica gel (CH2Cl2/
MeOH ) 98:2), and 344 mg (65%) of the target compound
was obtained as a yellow oil. RF ) 0.21 (CH2Cl2/MeOH ) 90:
5-Iodo-3′-O-(2-cyanoethyl)-2′-deoxyuridine, 16. Compound 15
(1.70 g, 3.02 mmol, 1.0 equiv) was dissolved in 4.5 mL of
freshly distilled acrylonitrile (68.02 mmol, 22.5 equiv) in a dry
100 mL Erlenmeyer flask, equipped with a big triangle stirring
bar and a septum connected to an argon line. The solution was
stirred vigorously for 2 min before 9 mL of t-BuOH was added,
and stirring continued for another 5 min; 990 mg of Cs2CO3
(3.02 mmol, 1.0 equiv) was added quickly, and the resulting
suspension was stirred vigorously for 2.5 h. The reaction solution
was separated from solid components by filtration over silica
gel (CH2Cl2/MeOH ) 95:5) and concentrated in vacuum.
Because the purification of compound 16 is difficult because
of loss of protecting groups on the column, only a small amount
was purified by flash column chromatography for analysis of
the fully protected compound N3-benzoyl-5-iodo-3′-O-(2-cya-
noethyl)-5′-O-benzoyl-2′-deoxyuridine (CH2Cl2/MeOH ) 99.6:
0.4 to CH2Cl2/MeOH ) 99:1). The rest of the crude product
(1.78 g) was directly used for the subsequent deprotection with
ammonia. RF ) 0.27 (n-hexane/ethyl acetate/EtMe2N ) 1:2:
0.03). 1H NMR (400 MHz, DMSO-d6, 300 K): δ ) 2.428 (1H,
ddd, J ) 2.6, 6.1, and 14.0 Hz, 2′-CH2), 2.47-2.55 (1H, m,
2′-CH2), 2.793 (2H, t, J ) 6.0 Hz, NC-CH2), 3.695 (2H, t, J
) 6.0 Hz, NC-CH2-CH2), 4.33-4.38 (2H, m, 3′-CH, 4′-CH),
4.518 (1H, dd, J ) 5.2 and 12.0 Hz, 5′-CH2), 4.578 (1H, dd, J
) 3.6 and 12.0 Hz, 5′-CH2), 6.090 (1H, t, J ) 6.8 Hz, 1′-CH),
7.54-7.62 (4H, m, 2 × 5′-O-benzoyl CHm, 2 × N3-benzoyl
CHm), 7.67-7.72 (1H, m, N3-benzoyl CHp), 7.76-7.82 (1H,
m, 5′-O-benzoyl CHp), 8.01-8.05 (4H, m, 2 × 5′-O-benzoyl
1
10). H NMR (250 MHz, DMSO-d6, 300 K): δ ) 2.15-2.34
(2H, m, 2′-CH2), 2.777 (2H, t, J ) 5.9 Hz, NC-CH2),
3.52-3.68 (2H, m, 5′-CH2), 3.632 (2H, t, J ) 6.0 Hz,
NC-CH2-CH2), 3.91-3.99 (1H, m, 4′-CH), 4.11-4.17 (1H,
m, 3′-CH), 4.234 (1H, d, J ) 5.5 Hz, NH-CH2), 5.177 (1H, t,
J ) 5.0 Hz, 5′-OH), 6.068 (1H, t, J ) 6.5 Hz, 1′-CH), 8.182
(1H, s, 6-CH), 10.067 (1H, t, J ) 5.3 Hz, NH), 11.676 (1H, s,
N3H) ppm. 13C NMR (63 MHz, DMSO-d6, 300 K): δ ) 18.22
(NC-CH2), 29.42 (NH-CH2), 36.93 (2′-CH2), 61.08 (5′-CH2),
63.54 (NC-CH2-CH2), 75.29 (alkyne CC-CH2), 79.01 (3′-
CH), 84.70 (1′-CH), 84.90 (4′-CH), 87.53 (alkyne CC-CH2),
97.80 (5-C), 119.13 (NC), 144.00 (6-CH), 149.27 (2-CO),
161.47 (4-CO) ppm, the trifluoroacetate signals are not visible
due to their low intensity (quartet structure). ESI(+)-MS (m/z)
Calcd: 430.3 [C17H17F3N4O6]. Found: 430.9 [M + H]+.

1048 Bioconjugate Chem., Vol. 21, No. 6, 2010
Knapp et al.
5-[(2,2,2-Trifluoroacetylamino)ethynyl]-3′-O-(2-cyanoethyl)2′-
deoxyuridine (300 mg, 0.70 mmol, 1.0 equiv) was dissolved in
40 mL of methanol, and 8.5 mL of concentrated (25%) aqueous
ammonia solution was added. The resulting mixture was stirred
at room temperature for 12 h. Then the solvent was removed
under reduced pressure, and 230 mg (99%) of compound 17
was obtained. The crude product was used without further
purification. RF ) 0.03 (CH2Cl2/MeOH ) 90:10). 1H NMR (400
MHz, DMSO-d6, 300 K): δ ) 2.15-2.24 (1H, m, 2′-CH2), 2.278
(1H, ddd, J ) 2.7, 8.7, and 13.6 Hz, 2′-CH2), 2.776 (2H, t, J )
5.8 Hz, NC-CH2), 3.547 (2H, s, NH2-CH2), 3.59-3.66 (4H,
m, 5′-CH2, NC-CH2-CH2), 3.94-3.99 (1H, m, 4′-CH),
4.13-4.18 (1H, m, 3′-CH), 5.04-6.50 (4H, br. s, 5′-OH, N3H,
NH2), 6.079 (1H, dd, J ) 6.3 and 7.5 Hz, 1′-CH), 8.153 (1H,
s, 6-CH) ppm. 13C NMR (100 MHz, DMSO-d6, 300 K): δ )
18.22 (NC-CH2), 30.63 (NH2-CH2), 37.02 (2′-CH2), 61.13 (5′-
CH2), 63.52 (NC-CH2-CH2), 74.76 (alkyne CC-CH2), 79.10
(3′-CH), 84.66 (1′-CH), 84.89 (4′-CH), 92.88 (alkyne CC-CH2),
98.29 (5-C), 119.15 (NC), 143.43 (6-CH), 149.39 (2-CO),
161.50 (4-CO) ppm. HR-ESI(-)-MS (m/z) Calcd: 333.1204
[C15H17N4O5]-. Found: 333.1204.
5-(Prop-2-ynyl)carbamic Acid 1-{2-[2-(2-tert-Butoxycarbo-
nylaminoethoxy)ethoxy]ethoxymethyl}-2-cyanoethyl Ester 3′-
O-(2-Cyanoethyl)-2′-desoxyuridine, 19. Activated linker 11 (2.24
g, 4.74 mmol, 2.2 equiv) was dissolved in 15 mL of dry ACN
and cooled to 0 °C. After cooling, 433 mg of KHCO3 (4.31
mmol, 2.0 equiv) was added. In a separate flask, 1.0 g of
compound 17 (2.15 mmol, 1.0 equiv) was dissolved in another
10 mL of dry ACN and added via a syringe dropwise to the
solution of compound 11. The resulting mixture was stirred at
0 °C for 1 h and at room temperature for 3.5 h. The solvent
then was removed under reduced pressure, and the residue was
divided between 75 mL of water and 100 mL of methylene
chloride. The aqueous layer was extracted two times with 75
mL of methylene chloride. The combined organic layers were
dried over Na2SO4 and filtered, and the solvent was removed
in vacuum. The crude product 19 was purified by flash column
chromatography using a gradient from CH2Cl2/MeOH ) 99:1
to CH2Cl2/MeOH ) 90:10, yielding 1.15 g (77%) of compound
1
19 as colorless foam. RF ) 0.34 (CH2Cl2/MeOH ) 90:10). H
NMR (300 MHz, DMSO-d6, 300 K): δ ) 1.370 (9H, s, 3 ×
CH3), 2.16-2.31 (2H, m, 2′-CH2), 2.774 (2H, t, J ) 5.9 Hz,
nucleoside NC-CH2), 2.876 (2H, m, linker NC-CH2), 3.055
(2H, q, J ) 6.0 Hz, Boc-NH-CH2), 3.370 (2H, t, J ) 6.1 Hz,
Boc-NH-CH2-CH2), 3.48-3.65 (14H, m, 5′-CH2, nucleoside
NC-CH2-CH2, linker NC-CH2-CH-CH2, 4 × CH2 glycol),
3.93-3.97 (1H, m, 4′-CH), 4.022 (2H, d, J ) 5.9 Hz,
NH-CH2), 4.138 (1H, quintet, J ) 2.7 Hz, 3′-CH), 4.953 (1H,
quintet, J ) 5.5 Hz, linker NC-CH2-CH), 5.13-5.19 (1H, m,
5′-OH), 6.071 (1H, t, J ) 7.3 Hz, 1′-CH), 6.68-6.77 (1H, m,
5-(Prop-2-ynyl)carbamic Acid 1-{2-[2-(2-Azidoethoxy)-
ethoxy]ethoxymethyl}-2-cyanoethyl ester]-3′-O-(2-cyanoethyl)-
2′-desoxyuridine, 18. Activated linker 10 (806 mg, 2.02 mmol,
2.5 equiv) was dissolved in 4.5 mL of dry ACN and cooled to
0 °C. After cooling, 162 mg of KHCO3 (1.62 mmol, 2.0 equiv)
was added. In a separate flask, 270 mg of compound 17 (0.81
mmol, 1.0 equiv) was dissolved in another 4 mL of dry ACN
and added via a syringe dropwise to the solution of compound
10. The resulting mixture was stirred at 0 °C for 30 min and at
room temperature for 3.5 h. The solvent then was removed under
reduced pressure, and the residue was divided between 25 mL
of saturated NH4Cl solution and 25 mL of methylene chloride.
The organic layer was washed with 20 mL of saturated NaCl
solution, and the combined aqueous layers were extracted two
times with 30 mL of methylene chloride. The combined organic
layers were dried over Na2SO4 and filtered, and the solvent was
removed in vacuum. The crude product 18 was purified by flash
column chromatography using a gradient from CH2Cl2/MeOH
) 98:2 to CH2Cl2/MeOH ) 95:5, yielding 328 mg (66%) of
compound 18 as a yellow oil. RF ) 0.39 (CH2Cl2/MeOH )
90:10). 1H NMR (400 MHz, DMSO-d6, 300 K): δ ) 2.17-2.31
(2H, m, 2′-CH2), 2.773 (2H, t, J ) 6.1 Hz, nucleoside
NC-CH2), 2.839 (1H, dd, J ) 6.3 and 17.3 Hz, linker
NC-CH2), 2.878 (1H, dd, J ) 5.0 and 17.2 Hz, linker
NC-CH2), 3.387 (2H, t, J ) 5.0 Hz, N3-CH2), 3.51-3.65 (16
H, m, 5′-CH2, nucleoside NC-CH2-CH2, linker
NC-CH2-CH-CH2, 5 × CH2 glycol), 3.93-3.97 (1H, m, 4′-
CH), 4.022 (2H, d, J ) 5.6 Hz, NH-CH2), 4.12-4.16 (1H, m,
3′-CH), 4.954 (1H, “quintet”, linker NC-CH2-CH), 5.169 (1H,
t, J ) 5.0 Hz, 5′-OH), 6.071 (1H, “t”, 1′-CH), 7.934 (1H, t, J
) 5.5 Hz, NH), 8.139 (1H, s, 6-CH), 11.642 (1H, s, N3H) ppm.
13C NMR (100 MHz, DMSO-d6, 300 K): δ ) 18.25 (nucleoside
NC-CH2), 19.73 (linker NC-CH2), 30.69 (NH-CH2), 36.90
(2′-CH2), 50.00 (N3-CH2), 61.14 (5′-CH2), 63.55 (nucleoside
NC-CH2-CH2), 67.91 (linker NC-CH2-CH), 69.25, 69.66,
69.68, 69.82, 70.21 (5 × CH2 glycol), 70.30 (linker
NC-CH2-CH-CH2), 74.49 (alkyne CC-CH2), 79.09 (3′-CH),
84.62 (1′-CH), 84.88 (4′-CH), 89.61 (alkyne CC-CH2), 98.23
(5-C), 117.71 (linker NC), 119.17 (nucleoside NC), 143.60 (6-
CH), 149.42 (2-CO), 154.88 (carbamate CO), 161.53 (4-CO)
ppm. FT-IR (NaCl): ν ) 3650-3120 (m br., -O-H, >N-H),
3056 (m br., C-H arom.), 2874 (m br., >CH2), 2252 (m,
-Ct N, -Ct C-), 2103 (s, -N3), 1780-1630 (m br., -CdO)
cm-1. ESI(+)-MS (m/z) Calcd: 618.6 [C26H34N8O10]. Found:
636.4 [M + NH4]+, 641.5 [M + Na]+, 619.5 [M]+. Anal.
(C26H34N8O10) C, H, N.
N
BocH), 7.933 (1H, t, J ) 5.7 Hz, NH), 8.137 (1H, s, 6-CH),
11.643 (1H, s, N3H) ppm. 13C NMR (75 MHz, DMSO-d6, 300
K): δ ) 18.23 (nucleoside NC-CH2), 19.72 (linker NC-CH2),
28.21 (3 × CH3), 30.68 (NH-CH2), 36.90 (2′-CH2), 39.69 (Boc-
NH-CH2), 61.14 (5′-CH2), 63.56 (nucleoside NC-CH2-CH2),
67.91 (linker NC-CH2-CH), 69.17 (Boc-NH-CH2-CH2),
69.49, 69.60, 69.73, 70.21, 70.30 (linker NC-CH2-CH-CH2,
4 × CH2 glycol), 74.49 (alkyne CC-CH2), 77.58 ((CH3)3C),
79.08 (3′-CH), 84.64 (1′-CH), 84.89 (4′-CH), 89.57 (alkyne
CC-CH2), 98.23 (5-C), 117.64 (linker NC), 119.26 (nucleoside
NC), 143.40 (2-CO), 143.56 (6-CH), 154.84 (carbamate CO),
155.54 (Boc CO), 161.49 (4-CO) ppm. ESI(+)-MS (m/z) Calcd:
692.7 [C31H44N6O12]. Found: 710.5 [M + NH4]+, 715.4 [M +
Na]+. Anal. (C31H44N6O12) C, H, N.
5-(Prop-2-ynyl)carbamic Acid 1-{2-[2-(2-Aminoethoxy)-
ethoxy]ethoxymethyl}-2-cyanoethyl Ester 3′-O-(2-Cyanoethyl)-
2′-deoxyuridine 5′-Phosphoramidite, 21. The well-dried nucle-
oside 19 (vacuum over 2 days) (200 mg, 0.29 mmol, 1.0 equiv)
was dissolved in 5 mL of dry methylene chloride, 138 µL (0.44
mmol, 1.5 equiv) of N,N,N,N-tetraisopropyl-O-cyanoethyl-
phosphoramidite and 40 mg (0.33 mmol, 1.15 equiv) of 4,5-
dicyanoimidazole were added, and the reaction mixture was
stirred at room temperature for 4 h. The mixture was diluted
with methylene chloride to a volume of 25 mL and poured into
25 mL of saturated NaHCO3 solution. After careful mixing, the
phases were separated immediately, and the organic layer was
washed quickly with saturated NaCl solution, dried over sodium
sulfate for 5 min without stirring, filtered, and concentrated in
vacuum. The crude product was purified over a short column
by flash column chromatography (first CH2Cl2/EE/EtMe2N )
8:2:0.2 then CH2Cl2/MeOH/EtMe2N ) 98:2:0.02). The phos-
phoramidite 21 was obtained as a slightly yellowish foam (260
mg, 100%). The two steroisomers caused by the chiral phos-
phorus atom (21a and 21b) can be distinguished in the NMR
spectra. RF ) 0.17 (CH2Cl2/MeOH/EtMe2N ) 9:1:0.1). 1H NMR
(400 MHz, acetone-d6, 300 K): δ ) 1.24-1.31 (24H, m,
N-(CH-(CH3)2)2,a, N-(CH-(CH3)2)2,b), 1.45 (18H, s, Boc-
C(CH3)3,a, Boc-C(CH3)3,b), 2.26-2.41 (2H, m, 2′-CH2,a, 2′-

New Fluoride Cleavable Linker for Dye Labeling
Bioconjugate Chem., Vol. 21, No. 6, 2010 1049
Scheme 1. Synthesis of the Core Structure 1 of the Cleavable
Linker and Its Reduced, Boc-Protected Form 5a
CH2,b), 2.47-2.57 (2H, m, 2′-CH2,a, 2′-CH2,b), 2.82 (4H, t, J )
6.2 Hz, nucleoside NC-CH2,a, nucleoside NC-CH2,b), 2.85-2.99
(8H, m, linker NC-CH2,a, linker NC-CH2,b, phosphorus
NC-CH2,a, phosphorus NC-CH2,b), 3.26 (4H, q, J ) 5.7 Hz,
Boc-NH-CH2,a, Boc-NH-CH2,b), 3.54 (4H, “t”, Boc-
NH-CH2-CH2,a, Boc-NH-CH2-CH2,b), 3.61-3.80 (24H, m,
linker NC-CH2-CH-CH2,a, linker NC-CH2-CH-CH2,b, 4
× CH2,a glycol, 4 × CH2,b glycol, N-(CH)2,a, N-(CH)2,b),
3.82-3.88 (4H, m, nucleoside NC-CH2-CH2,a, nucleoside
NC-CH2-CH2,b), 3.91-4.05 (8H, m, 5′-CH2,a, 5′-CH2,b, phos-
phorous NC-CH2-CH2,a, phosphorous NC-CH2-CH2,b),
4.17-4.24 (4H, m, NH-CH2,a, NH-CH2,b), 4.28/4.31 (each 1H,
each “q”, 4′-CHa, 4′-CHb), 4.34-4.38/4.39-4.43 (each 1H, each
m, 3′-CHa, 3′-CHb), 5.09 (2H, quintet, J ) 5.4 Hz, linker
NC-CH2-CHa, linker NC-CH2-CHb), 5.92 (2H, br. s, Boc-
NHa, Boc-NHb), 6.24/6.29 (each 1H, each dd, J ) 5.7 and 8.6
Hz/J ) 5.9 and 8.1 Hz, 1′-CHa, 1′-CHb), 7.04 (2H, m, carbamate
NHa, carbamate NHb), 8.04/8.13 (each 1H, each s, 6-CHa, 6-CHb,
10.24 (2H, br. s, N3Ha, N3Hb) ppm. 13C NMR (100 MHz,
acetone-d6, 300 K): δ ) 19.18/19.20 (nucleoside NC-CH2,a,
nucleoside NC-CH2,b), 20.54 (linker NC-CH2,a, linker
NC-CH2,b), 20.84/20.87 (each d, J ) 7.1 Hz/J ) 7.1 Hz,
phosphorus NC-CH2,a, phosphorus NC-CH2,b), 24.9-25.1 (4C,
m, 2 × N-(CH-(CH3)2)2,a, 2 × N-(CH-(CH3)2)2,b), 28.65
(Boc-C(CH3)3,a, Boc-C(CH3)3,b), 31.87/31.93 (NH-CH2,a,
NH-CH2,b), 38.32/38.47 (2′-CH2,a, 2′-CH2,b), 41.05 (Boc-
NH-CH2,a, Boc-NH-CH2,b), 43.86 (d, J ) 12.4 Hz, N-(CH)2,a,
N-(CH)2,b), 59.63/59.77 (each d, J ) 19.9 Hz/J ) 19.9 Hz,
phosphorus NC-CH2-CH2,a, phosphorus NC-CH2-CH2,b),
64.03/64.81 (each d, J ) 8.3 Hz/J ) 8.4 Hz, 5′-CH2,a, 5′-CH2,b),
64.99/65.04 (nucleoside NC-CH2-CH2,a, nucleoside
NC-CH2-CH2,b), 69.30 (linker NC-CH2-CHa, linker
a Reagents and conditions: (a) NaN3, NaI, EtOH, reflux, 4 d, 94%;
(b) (i) NaH, THF, rt, 2 h, (ii) (()-epichlorohydrin, rt, 16 h, 70%; (c)
NaCN, EtOH/H2O ) 5:1, rt, 16 h, 89%; (d) (i) triphenylphosphine,
dry dioxane, rt, 8 h, (ii) 25% aqueous ammonia, rt, 12 h; (e) Boc2O,
NaHCO3, water/dioxane, 0 °C to rt, 10 h, 100%.
Deprotection of the CPG-Supported Oligonucleotide 23. The
CPG-support 23 was suspended in 6 mL of DMF and treated
with 6 mL of 1 M TBAF/THF solution. The support was shaken
for 15 min at 45 °C. Then the supernatant was collected, and
its fluorescence was qualitatively analyzed under a UV lamp at
366 nm. The support was washed with methylene chloride and
methanol.
Cleavage of Oligonucleotide 24 from the CPG Support. The
support was treated with 2 mL of 25% aqueous ammonia at 50
°C for 18 h. The supernatant was filtered, and the support was
washed with H2O. The combined filtrates and washings were
concentrated under reduced pressure, redissolved in H2O, and
analyzed and purified by ion exchange HPLC as described in
the Materials and Methods using the gradient given below.
The collected fractions were evaporated. The detached
oligonucleotide 25 was then dissolved in 1 mL of Millipore
water and desalted on a PD-10 desalting column. The desalted
oligonucleotide 25 was analyzed by ESI-MS. Ion exchange
HPLC: flow rate 5 mL/min, gradient 0 min 85% A, 10% B,
5% C f 20 min 65% A, 10% B, 25% C f 23 min 0% A, 0%
B, 100% C f 25 min 0% A, 0% B, 100% C f 27 min 85% A,
10% B, 5% C f 30 min 10% A, 90% B, 0% C; retention time
16.36 min (UV detection at 254 nm). ESI(-)-MS (m/z) Calcd:
NC-CH2-CHb),
70.63
(Boc-NH-CH2-CH2,a,
Boc-
NH-CH2-CH2,b), 70.91, 71.07, 71.18, 71.31, 71.65 (5 × CH2,a
glycol, 5 × CH2,b glycol), 75.36/75.40 (alkyne CC-CH2,a,
alkyne CC-CH2,b), 78.65 (C(CH3)3,a, C(CH3)3,b), 80.91/81.20
(3′-CHa, 3′-CHb), 85.09/85.11 (each d, J ) 8.3 Hz/J ) 8.4 Hz,
4′-CHa, 4′-CHb), 86.18/86.50 (1′-CHa, 1′-CHb), 90.06/90.20 (5-
Ca, 5-Cb), 99.81/99.92 (alkyne CC-CH2,a, alkyne CC-CH2,b),
117.72 (linker NCa, linker NCb), 118.96/118.97 (nucleoside NCa,
nucleoside NCb), 119.15/119.16 (phosphorus NCa, phosphorus
NCb), 143.74/143.94 (6-CHa, 6-CHb), 150.22/150.29 (2-COa,
2-COb), 155.85/155.88 (carbamate COa, carbamate COb), 156.62
(Boc COa, Boc COb), 162.02 (4-COa, 4-COb) ppm. 31P NMR
(121 MHz, D2O, 300 K): δ ) 148.23, 148.43 (s, a-isomer,
b-isomer) ppm. ESI(+)-MS (m/z) Calcd: 892.9 [C40H61N8O13P].
Found: 910.9 [M + NH4]+, 915.8 [M + Na]+, 893.8 [M +
H]+.
2423.6. Found: 807.3 [M]3-, 1211.2 [M]2-, 814.5 [M + Na]3-
,
1222.3 [M + Na]2-, 1233.4 [M + 2Na]2-, 1244.5 [M + 3Na]2-.
Calculated masses: 2424.4 [M], 2446.4 [M + Na], 2468.8 [M
+ 2Na], 2490.8 [M + 3Na].
RESULTS AND DISCUSSION
Synthesis of the Core Structure 1 and the Reduced Form
5. The synthesis of the basic compound 1 and the reduced and
Boc-protected form of the linker 5 are shown in Scheme 1. They
can easily be synthesized with good to very good yields from
inexpensive starting materials.
Synthesis of Oligonucleotide 22. The oligonucleotide synthesis
was carried out on an Expedite PerSeptive Biosystems DNA
synthesizer, using commercially available 5′-O-(2-cyanoethyl)-
N,N-diisopropylphosphoramidite 2′-deoxyribonucleosides and
21 as building blocks and dT5′-CPG as solid support. The
protocol was a standard 1 µmol synthesis protocol with
prolonged detritylation and coupling times. The sequence 5′-
TATGACCT-3′ was assembled, observing in all cases coupling
yields greater than 98% as evaluated by DMTr analysis.
Starting from the commercially available triglycol monochlo-
ride 2, the azido-modified triglycol 3 could be obtained with
94% yield following a literature procedure (17). In the next step,
racemic epichlorohydrin was introduced using sodium hydride
in dry THF yielding 70% of compound 4. The epoxide was
finally opened using sodium cyanide to build up the cleavage
site, yielding 89% of compound 1. Compound 1 was obtained
with an overall yield of 58% over three steps. From the core
structure 1, the reduced linker 5 can easily be obtained in two
additional steps. It was synthesized by applying the Staudinger
reaction and subsequently protecting the free amino group as
tert-butyl carbamate without intermediate purification of the free
amine. A quantitative yield of compound 5 was obtained over
these two steps. By the use of racemic epichlorohydrin, the
linker is obtained as racemic mixture. This does not cause any
problems as also in further derivatization where diasteroisomers
Coupling with Fluorescein to Obtain Oligonucleotide 23.
CPG-supported oligonucleotide 22 was treated several times
with 3% TCA solution in methylene chloride to remove the
Boc protecting group and afterward washed several times with
ACN. After drying the support in vacuum, 10 equiv of
fluorescein-NHS ester (5 mg, 10 µmol) and 15 equiv of DIPEA
(2.6 µL, 15 µmol) in 400 µL of DMF were added to the support,
and the reaction was left at room temperature for 20 h. After
20 h, the support was washed thoroughly with DMF, methylene
chloride, methanol, and diethyl ether and dried in vacuum.

1050 Bioconjugate Chem., Vol. 21, No. 6, 2010
Knapp et al.
Scheme 2. Modification of the Linker 1 by the Use of Click
Reactionsa
Scheme 3. Synthesis of the Model Compound 9 and the
Activated Compounds 10 and 11a
a Reagents and conditions: (a) phenylacetylene (6) or 1-ethynylpyrene
(7), CuSO4, sodium ascorbate, CH2Cl2/MeOH ) 2:1, for 6 rt, 42 h,
96%, for 7 rt, 20 h, 40 °C, 24 h, 41%; (b) 1-ethynylpyrene, CuI, DIPEA,
30 min, 250 W, 60%.
result, no difference in chemical or physical behavior (reactivity,
on TLC, or in NMR spectra) was observed.
Click Reactions on the Azido-Modified Linker 1. The core
linker 1 was coupled to phenylacetylene in a click reaction to
evaluate the effectiveness of this method of attachment. The
reaction is shown in Scheme 2. It was accomplished using
catalytic amounts of copper(II)sulfate and (L)-ascorbic acid
sodium salt in a 2:1 mixture of methylene chloride and methanol
as solvents. The product 6 was obtained with 96% yield and
thereby confirms the very good qualities of the linker in click
reactions.
a Reagents and conditions: (a) (i) di-(N-succinimdyl)carbonate,
anhydrous K2CO3, dry ACN, 0 °C, 20 h, (ii) propargylamine, KHCO3,
rt, 3 h, 76%; (b) iodobenzene, Pd(PPh3)4, CuI, Et3N, dry CH2Cl2, rt,
3.5 h, 92%; (c) di(N-succinimidyl)carbonate, anhydrous K2CO3, dry
ACN, 0 °C, 10, 20 h, 99%; 11, 24 h, 90%.
Table 1. Expected Cleavage Mechanism and Cleavage Experiments
Using Model Compound 9 under Various Conditionsa
After this successful test reaction, we wanted to introduce a
fluorescent label via a click reaction. Because often used dyes
like fluorescein or TexasRed are not commercially available as
their alkyne derivatives, we used 1-ethynylpyrene as a model
dye (7 in Scheme 2). The conversion was accomplished applying
two different methods (a, b). Using very similar conditions as
for derivative 6, we observed that the reaction with pyrene went
much slower. After stirring for 20 h at room temperature, there
was still starting material left, so we heated it to 40 °C for
another 24 h. We could isolate the labeled product 7 with a
yield of 41%. An improved yield of 60% could be achieved by
performing the reaction in the microwave (30 min, 250 W)
without solvents according to a literature procedure (18). This
demonstrates that the linker is useful for attaching labels by
click reactions.
exp. no.
reagent (equiv)/solvent
temp
cleavage/time [min]
1
2
3
4
5
6
1 M TBAF (50)b/dry THF
1 M TBAF (50)b/dry THF
1 M TBAF (5)b/dry THF
60 °C
rt
rt
quant/<1
quant/<1
quant/ca. 10
ca. 60%/>1200
no cleavage
no cleavage
28% aq. NH3 (100)b/MeOH rt
Et3N·3HF (5)b/dry THF
rt
rt
aq. TFA/methanol
Synthesis and Cleavage of Model Compound 9. To prove
the cleavability with fluoride and to investigate the cleavage
mechanism, model compound 9 was synthesized where the
nucleotide is replaced by a phenyl moiety. The cleavage
mechanism is supposed to follow a ꢀ-elimination as is also
suggested for the above-mentioned fluoride cleavable protecting
groups. Fluoride therefore has to act as a base, not as a
nucleophile. The synthesis of the model compound 9 is shown
in Scheme 3.
Compound 1 was converted to the propargylamine carbamate
8 with 76% yield. In a one-pot procedure, compound 1 was
first activated as N-succinimidyl carbonate at 0 °C with K2CO3
as base followed by the addition of propargylamine and KHCO3
as additional base. The model compound 9 was then synthesized
via Sonogashira coupling of the modified linker 8 to iodoben-
zene with a yield of 92%. From the starting materials 1 and 5,
the N-succinimidyl carbonates 10 and 11 were isolated as well
by applying the same conditions as described for the transient
activation of compound 1, followed by an aqueous workup.
They were used later on for derivatization of a nucleosidic
compound (see Scheme 4).
a For following the reaction, 100 µL probes were taken out, diluted
with 450 µL of methanol and 50 µL of Millipore water and applied to
reversed-phase HPLC column. b Numbers in parentheses are the
corresponding equivalents of TBAF used.
the first experiment was accomplished at elevated temperature
because this is required for effective 3′-O-CE cleavage (12). A
high excess of fluoride is chosen because this mimics the
conditions on the chip in a CRT approach. The result for the
designed linker was very good because it was cleaved in less
than 1 min. Because of this very fast cleavage at 60 °C, the
temperature was reduced to room temperature (experiment no.
2, Table 1). Comparing entries 1 and 2 demonstrates that no
elevated temperature is needed for this ultrafast cleavage (see
also Figure 2). Further the reduction of equivalents decelerated
the reaction, but it was still complete in less than 10 min
(experiment no. 3 in Table 1). One explanation for the ultrafast
cleavage is the release of carbon dioxide.
The linker is also expected to be cleavable by other bases.
So the cleavage with high excess of concentrated ammonia was
investigated (entry 4, Table 1). The HPLC chromatogram in
Figure 3 shows that there is a big difference in effectiveness
between fluoride and ammonia. The linker is also cleavable with
concentrated ammonia, but it is not complete after 20 h at room
temperature (approximately 60% cleavage).
The expected cleavage mechanism and products as well as
several cleavage conditions examined using compound 9 are
summarized in Table 1. The experiments were performed in a
small scale (max. 30 mg of starting material) and analyzed by
RP-HPLC where the UV visible compounds were detected (9
and 12).
Entry 5 in Table 1 shows that the linker is stable against the
slightly acidic fluoride reagent Et3N·3HF (Figure 3); no
cleavage occurs during 18 h. We further found excellent stability
even after 48 h under acidic conditions (TFA solution, entry 6
To make a comparison between the cleavage of the linker
and our results found for the cleavage of the 3′-O-CE group,

New Fluoride Cleavable Linker for Dye Labeling
Bioconjugate Chem., Vol. 21, No. 6, 2010 1051
temperature to -18 °C we could improve the yield to 77% of
5′-mono protected product as compared with the yield reported
in literature (70%). Following conditions described in the
literature (20), the next step was the benzoyl protection of N3
by transient TMS protection of the 3′-OH function in a one-pot
procedure. The yield of 75% of completely protected nucleoside
15 suggests that two of the three steps mentioned were
quantitative, including isolation and purification. The Michael
addition was accomplished following the procedure described
for the RNA 2′-position (13) using freshly distilled acrylonitrile
and Cs2CO3 as base. The crude product of the Michael addition
was directly deprotected and 65% of the 3′-O-modified nucleo-
side 16 was obtained after two steps. To enable the attachment
of the cleavable linker, the propargylamine moiety was intro-
duced by the well-known Sonogashira coupling with propar-
gylamine-N-(1,1,1-trifluoro)acetate. After deprotection, com-
pound 17 was obtained over the two steps in 65% yield. The
coupling of the linker was readily accomplished by adding the
activated linker moiety 10 to the modified nucleoside 17 in dry
ACN with KHCO3 as base giving 67% of the final compound
18. Adding the corresponding activated linker 11 to compound
17 yields 77% of compound 19.
Figure 2. HPLC chromatogram of cleavage experiment nos. 1 and 2
in Table 1.Conditions: Phenomenex Jupiter 4u Proteo 90A 4 µm column
250 mm × 4.6 mm, flow rate 1 mL/min, gradient 0 min 10% A, 70%
B, 20% C f 14 min 10% A, 15% B, 75% C f 20 min 10% A, 0% B,
90% C.
The cleavage experiments using compound 18 were ac-
complished in the same manner as with model compound 9.
Approximately 30 mg of starting material was used, and the
reactions were followed by RP-HPLC. In Table 2, the experi-
ments are summarized.
With fluoride, the cleavage proceeds successively as shown
in Table 2. For the linker, we observe a very fast cleavage
according to the results obtained with the model compound 9.
For the 3′-O-CE group, we found a cleavage time of more than
1 h under the conditions applied in entry 1 of Table 2 (12).
Estimated from these observed times, the linker is cleaved about
100 times faster than the 3′-modification. With ammonia, the
cleavage of the linker occurs more slowly, and no cleavage is
monitored for the 3′-O-CE group.
Figure 3. HPLC chromatograms of exp. no. 4, 5, and 6 in Table 1.
Condition as described in Figure 2.
and Figure 3). This stability is important for the use of the Boc-
protected linker because it has to be deprotected under acidic
conditions after attachment to the nucleoside to enable the
labeling.
The HPLC chromatograms provide a first hint of the expected
cleavage mechanism because they show the appearance of
cleavage product 12. The glycol cleavage product is not UV-
active and therefore not detectable by this HPLC system.
Another indicator of the ꢀ-elimination mechanism is the stability
of the linker against Et3N·3HF. The final proof of the cleavage
and its mechanism was achieved by repeating the reaction in a
bigger scale to be able to isolate both cleavage products. The
reaction was performed using an excess of 5 equiv of TBAF in
dry THF at room temperature for 10 min. Both expected
products 12 and 13 could be isolated in quantitative yields. The
olefin is obtained in a 1:1 ratio of cis and trans isomers as
In Figure 4, the three reference compounds are shown. In
Figures 5 and 6, the chromatograms of the two entries in Table
2 at different points of time are displayed.
The results of the HPLC analysis of experiment no. 1 are in
accordance with the expectations. Figure 5 shows a very fast
cleavage (less than 1 min) of the linker and a complete cleavage
after 2 h of the 3′-O-CE group. Hence the cleavage is equally
effective with the nucleoside compound 18 and the model
compound 9. The cleavage of the linker from the nucleoside
18 with ammonia (entry 2 in Table 2 and Figure 6) is faster
than that from the model compound 9. However it is not
completely clear whether this is due to the nucleoside moiety
because the conditions were not identical. In experiment no. 2
in Table 2, no additional solvent was used, and the excess was
much higher compared with entry 4 in Table 1.
1
determined by H NMR.
These experiments show that the linker is extraordinarily
effectively cleaved with 1 M TBAF in THF following a
ꢀ-elimination mechanism and that it is completely stable under
acidic conditions.
The fluoride cleavage experiment (exp. no. 1 in Table 2)
shows that both parts are cleavable under the same conditions.
For our future aim to use this combination of 3′-O-blocking
group and cleavable linker as dye-labeled reversible terminator,
it is important that both parts are efficiently cleaved within
minutes. At room temperature, the speed of cleavage of the 3′-
O-CE group does not meet the demands for CRT sequencing.
However this problem we already solved and the optimal
conditions for cleavage of the 3′-blocking group were found to
be at 40 °C with DMF or DMSO as cosolvent (cleavage within
3 min) (12).
The future idea for the use of these reversible terminators is
to elongate oligonucleotide templates, immobilized on a chip.
To mimic these conditions and to show that both modifications
can be cleaved in a reasonable time, a final deprotection
experiment on a CPG-supported oligonucleotide was ac-
Synthesis and Cleavage of the 5-,3′-Modified 2′-Deoxyuri-
dine Derivatives 18 and 19. To ensure that the cleavage works
equally efficiently if the linker is attached to a nucleoside and
to analyze the difference in cleavage rate between the 3′-O-CE
group and the linker, the 5-,3′-modified compound 18 was
synthesized (see Scheme 4). To enable the final cleavage
experiment on a labeled, CPG-bound 8-mer, compound 19 was
also synthesized, bearing the Boc-protected linker at the
5-position (see Scheme 4).
The introduction of the CE group to 5-iodo-2′-deoxyuridine
14 was accomplished in a similar way as described for the
thymidine derivative (12). The only difference is that for the 5′-
O-protection the benzoyl group was used instead of the MMTr
group to just have one single deprotection step in the end.
According to this, the first step was the selective introduction
of the benzoyl group at the 5′-OH (19). By lowering the

1052 Bioconjugate Chem., Vol. 21, No. 6, 2010
Knapp et al.
Scheme 4. Synthesis of the 5- and 3′-Modified Nucleosides 18 and 19a
a Reagents and conditions: (a) BzCl, dry CH2Cl2, dry pyridine, -18 °C, 3 h, 77%; (b) (i) TMSCl, DIPEA, dry pyridine, rt, 45 min, (ii) BzCl,
rt, 15 min, 98%; (c) CHCl3/MeOH ) 1:1, TFA, rt, 2 h; (d) acrylonitrile, Cs2CO3, t-BuOH, rt, 2.5 h; (e) 25% aq. ammonia, rt, 20 h, 65% over two
steps; (f) propargylamine-N-(1,1,1-trifluoro)acetate, Pd(PPh3)4, CuI, Et3N, dry DMF, rt, 20 h, 65%; (g) 25% aq. ammonia, rt, 12 h, 99%; (h) 10,
KHCO3, dry ACN, 0 °C, 30 min, rt, 3.5 h, 66%; (i) 11, KHCO3, dry ACN, dry DMF, 0 °C, 1 h, rt, 3.5 h, 77%.
Table 2. Cleavage Experiments Using the Modified Nucleoside 18a
exp. no.
reagent (equiv)/solvent
1 M TBAF (55)b/dry THF
28% aq. NH3 (450)b/no additional solvent
temp
cleavage/time [min]
1
2
rt
rt
linker: quant./<1 3′-O-CE: quant/>60
linker: quant./120 3′-O-CE: no cleavage
a For following the reaction, 50 µL probes were taken out, diluted with 450 µL of methanol and 50 µL of Millipore water, and applied to
reversed-phase HPLC column. b Numbers in parentheses are the corresponding equivalents of TBAF used.
Figure 5. Chromatograms of the fluoride-mediated cleavage of
Figure 4. HPLC chromatogram of the reference products for the
compound 18. Conditions as described in Figure 4.
nucleoside cleavage experiments. Conditions: Phenomenex Jupiter
4u Proteo 90A 4 µm column 250 mm × 4.6 mm, flow rate 1 mL/
reagent and 4,5-dicyanoimidazole as base. The 3′-modified 5′-
min, gradient 0 min 10% A, 90% B, 0% C f 20 min 10% A, 60%
phosphoramidite 21 was obtained in quantitative yield.
B, 30% C f 25 min 10% A, 0% B, 90% C f 30 min 10% A, 90%
As a model oligonucleotide, we chose the following 8-mer:
5′-TCCAGTATmod-3′, 22, where Tmod is the 5-,3′-O-modified
nucleotide. The oligonucleotide synthesis had to be carried out
in the inverse way (direction 5′ to 3′, instead of the conventional
3′ to 5′) with commercially available 5′-O-(2-cyanoethyl)-N,N-
diisopropylphosphoramidite-2′-deoxyribonucleosides building
blocks. After assembly, the oligonucleotide was left at the CPG
support, and the amino group of the linker was deprotected by
washing the column 10 times with deblock solution (3% TCA
in methylene chloride). To this free amino group, fluorescein
was coupled in order to be able to detect the cleavage product
of the linker by fluorescence (Scheme 6). The solid phase bound
labeled oligonucleotide 23 was washed and dried thoroughly
before the treatment with 1 M TBAF in THF and dry DMF as
cosolvent at 45 °C for 15 min. After this time, the supernatant
B, 10% C.
complished. We chose an 8-mer bearing the complete 2′-
deoxyuridine-derived reversible terminator at the 3′-end. To be
able to detect the cleavage of the linker moiety from the
oligonucleotide, the linker was labeled with fluorescein after
assembly of the oligonucleotide. For this experiment, nucleoside
19 was used, and the dye was applied as its N-hydroxysuccin-
imidyl active ester.
In order to use the complete reversible terminator 19 in an
automated oligonucleotide synthesis, the 5′-O-phosphoramidite
was synthesized. In Scheme 5, the synthesis of the phosphora-
midite 21 is shown. It was carried out using N,N,N,N-
tetraisopropyl-O-cyanoethyl-phosphoramidite as phosphorylating

New Fluoride Cleavable Linker for Dye Labeling
Bioconjugate Chem., Vol. 21, No. 6, 2010 1053
Scheme 6. Synthesis of the Labeled Oligonucleotide 23 and
Cleavage of the 5- and 3′-Modifications from the CPG-Bound
Oligonucleotidea
Figure 6. Chromatograms of the ammonia-mediated cleavage of
compound 18. Conditions as described in Figure 4.
Scheme 5. Synthesis of the 5′-Phosphoramidite 21a
a Reagents and conditions: (a) N,N,N,N-tetraisopropyl-O-cyanoethyl-
phosphoramidite, 4,5-dicyanoimidazol, dry CH2Cl2, rt, 4 h, 100%.
was recovered, concentrated, redissolved in methanol, and
irradiated under a UV lamp at 366 nm. At this wavelength, the
solution showed a very strong fluorescence (small picture in
Scheme 6). This demonstrates the cleavage of the linker-dye
system during the fluoride treatment. To prove the cleavage of
the 3′-O-CE group as well, the oligonucleotide was deprotected
and detached from the solid support by treatment with concen-
trated aqueous ammonia at room temperature. After purification
of the oligonucleotide 24 by ion exchange HPLC and desalting
by gel filtration, the ESI-MS analysis indeed proved the presence
of the completely deprotected oligonucleotide 24 (Scheme 6).
We designed a linker that is rapidly cleaved by fluoride
through a ꢀ-elimination mechanism. The linker is useful for
different modes of attachment, and therefore it is a versatile
tool for reversibly labeling (oligo)nucleotides. We showed
examples of successful attachment of labels by click reactions
and by amide formation.
To prove the use of this linker for a reversible terminator,
applicable in a DNA-sequencing approach, the modified nucleo-
sides 18 and 19 were synthesized, and the cleavage properties
were analyzed. Initial experiments were performed in solution
but with the final cleavage experiment from the CPG-bound
oligonucleotide, we could show that also under heterogeneous
conditions TBAF efficiently removes the linker-dye system and
the 3′-O-blocking group in one single step. Further this setup
confirmed that oligonucleotides are stable under the applied
chemical conditions. These results demonstrate that the devel-
oped linker, together with the 3′-O-blocking group meets the
demands for a reversible terminator.
a Reagents and conditions: (a) TCA deblock solution; (b) 5(6)-
carboxy-fluorescein-NHS ester, DIPEA, DMF, rt, 20 h; (c) 1 M TBAF/
THF, dry DMF, 45 °C, 15 min; (d) conc. aq. NH3, rt, 18 h.
3-Hydroxy-4-(2-{2-[2-(4-phenyl-1,2,3-triazol-1-yl)ethoxy]-
ethoxy}ethoxy)butyronitrile, 6. C: calcd, 59.99; found, 59.89.
H: calcd, 6.71; found, 6.68. N: calcd, 15.55; found, 15.28.
Prop-2-ynylcarbamic Acid 1-{2-[2-(2-Azidoethoxy)ethoxy]-
ethoxymethyl}-2-cyanoethyl Ester, 8. C: calcd, 49.55; found,
49.68. H: calcd, 6.24; found, 6.30. N: calcd, 20.64; found, 20.78.
(3-Phenylprop-2-ynyl)carbamic Acid 1-{2-[2-(2-Azidoethoxy-
ethoxy]ethoxymethyl}-2-cyanoethyl Ester, 9. C: calcd, 57.82;
found, 58.04. H: calcd, 6.07; found, 6.01. N: calcd, 16.86; found,
16.92.
(E/Z)-4-{2-[2-(2-Azidoethoxy)ethoxy]ethoxy}but-2-eneni-
trile, 13. C: calcd, 49.99; found, 49.83. H: calcd, 6.71; found,
6.76. N: calcd, 23.32; found, 23.46.
N3-Benzoyl-5-iodo-5′-O-benzoyl-2′-deoxyuridine, 15. C: calcd,
49.13; found, 49.01. H: calcd, 3.41; found, 3.67. N: calcd, 4.98;
found, 4.86.
NUMERICAL RESULTS OF ELEMENTAL ANALYSES
2-{2-[2-(2-Azidoethoxy)ethoxy]ethoxymethyl}oxirane, 4. C:
calcd, 46.74; found, 46.52. H: calcd, 7.41; found, 7.37. N: calcd,
18.17; found, 18.45.
4-{2-[2-(2-Azidoethoxy)ethoxy]ethoxy}-3-hydroxybutyroni-
trile, C: calcd, 46.26; found, 46.50. H: calcd, 7.18; found, 7.02.
N: calcd, 21.81; found, 21.69.
(2-{2-[2-(3-Cyano-2-hydroxypropoxy)ethoxy]ethoxy}ethyl)-
carbamic Acid tert-Butyl Ester, 5. C: calcd, 54.20; found, 54.19.
H: calcd, 8.49; found, 8.57. N: calcd, 8.43; found, 8.25.
5-Iodo-3′-O-(2-cyanoethyl)-2′-deoxyuridine, 16. C: calcd,
35.40; found, 35.51. H: calcd, 3.47; found, 3.62. N: calcd, 10.32;
found, 10.29.
N3-Benzoyl-5-iodo-3′-O-(2-cyanoethyl)-5′-O-benzoyl-2′-deox-
yuridine. C: calcd, 50.75; found, 50.94. H: calcd, 3.60; found,
3.82. N: calcd, 6.83; found, 6.67.

1054 Bioconjugate Chem., Vol. 21, No. 6, 2010
Knapp et al.
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ACKNOWLEDGMENT
We would like to thank the EU FP6 CRAFT project
“ArraySBS” for financial support.
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