DNA conjugation by the staudinger ligation: New thymidine analogues

Article abstract of DOI:10.1055/s-2007-983728

Two novel modified 2′-deoxyuridine triphosphates carrying an azide functionality linked to the nucleobase were synthesized. For probing the sterical influence on enzymatic incorporation and Staudinger ligation, differently sized flexible linkers were chosen. Both nucleotides can completely replace natural thymidine in primer extension as well as polymerase chain reaction (PCR) using Pyrococcus woesei DNA polymerase. For PCR with larger gene fragments as template, however, the longer linker disturbs the DNA polymerase and yields less product. For azide-labeled primer extension products, subsequent conjugation of suitably functionalized phosphines via Staudinger ligation was achieved, for example for the conjugation of biotin as an affinity tag. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2007-983728

PAPER
1949
DNA Conjugation by the Staudinger Ligation: New Thymidine Analogues
D
NA Conjugation
n
by the
S
taud
n
inger Ligatio
a
n
Baccaro, Samuel H. Weisbrod, Andreas Marx*
Department of Chemistry, Chair of Organic Chemistry & Cellular Chemistry, University of Konstanz, 78457 Konstanz, Germany
Fax +49(7531)885140; E-mail: Andreas.Marx@uni-konstanz.de
Received 18 March 2007; revised 2 May 2007
Dedicated to Prof. Dr. Wolfgang Pfleiderer on the occasion of his 80th birthday
cently, we presented the synthesis of 7-azide-modified 7-
deaza-2¢-deoxyadenosine, its incorporation in a growing
DNA strand by enzymatic primer extension reaction, and
subsequent biotin labeling by Staudinger ligation.13 Here,
Abstract: Two novel modified 2¢-deoxyuridine triphosphates car-
rying an azide functionality linked to the nucleobase were synthe-
sized. For probing the sterical influence on enzymatic incorporation
and Staudinger ligation, differently sized flexible linkers were cho-
sen. Both nucleotides can completely replace natural thymidine in we report the synthesis of two new azide-modified thymi-
primer extension as well as polymerase chain reaction (PCR) using
Pyrococcus woesei DNA polymerase. For PCR with larger gene
fragments as template, however, the longer linker disturbs the DNA
was then investigated using primer extension reactions as
polymerase and yields less product. For azide-labeled primer exten-
sion products, subsequent conjugation of suitably functionalized
dine analogues with different linker lengths and their con-
version into triphosphates. Their incorporation into DNA
well as PCR and the resulting DNA was subsequently
conjugated with biotin using Staudinger ligation.
phosphines via Staudinger ligation was achieved, for example for
the conjugation of biotin as an affinity tag.
Our strategy to label DNA site-specifically involves two
steps (Scheme 1): First the azide-modified triphosphate
should replace the corresponding natural 2¢-deoxynucleo-
side-5¢-O-triphosphate (dNTP) in a DNA polymerase re-
action. For this the polymerase has to accept the unnatural
nucleoside and ideally can use it as a template for incor-
poration of the canonical base in PCR. Then the obtained
azide-labeled DNA should serve as substrate for further
modifications by Staudinger ligation with a suitably func-
tionalized phosphine. Since it has been shown that 5-mod-
ified thymidine derivatives are accepted by DNA
polymerases,7,14,15 we focused our synthesis on these
kinds of analogues. A sterically encumbered moiety might
disturb the acceptance of the DNA polymerase; however,
a short linker might decrease the yield of the subsequent
Staudinger ligation. So we designed two nucleotides bear-
ing a flexible linker with different lengths towards the
azide moiety to test which is best suited for our endeavor
(4a and 4b, Scheme 2).
Key words: azides, conjugation, Staudinger ligation, DNA,
nucleotides
To enable investigation of complex biological systems,
efficient labeling techniques for the involved biopolymers
are often required. Conjugation of functional molecules
like dyes or affinity tags to these biopolymers allows fur-
ther and better investigations of such systems. Ideally, the
coupling reaction has to be site-specific, bioorthogonal,
high yielding, and for in vivo experiments biocompatible
and nontoxic. There is no standard conjugation protocol
which fulfills all of these conditions so far.1
Recently, two promising reactions based on the reactivity
of the azide moiety were reported.2 One method makes
use of the [2+3] cycloaddition reaction of an azide and an
alkyne first reported by Huisgen.3 Copper catalysis pro-
motes the reaction to proceed at room temperature4 and
led to widespread applications especially in bioconjugate
chemistry.5 Recently, the method has been extended for
conjugation of DNA.6,7 On the other hand, the so-called
Staudinger ligation developed by Bertozzi readily occurs
between an azide and a phosphine without the need for
further reagents.8 The intermediate aza-ylide gets thereby
trapped by an acyl group to form a stable amide bond.
This reaction has been applied, for example, in the conju-
gation of carbohydrates, protein and phage particles, pep-
tide ligation, and immobilization.9 Few examples for the
conjugation of DNA by Staudinger ligation are known.
The conjugation of a fluorescence dye to the 5¢ terminus
of single stranded DNA10 as well as the sequence specific
conjugation of phenanthroline to DNA for subsequent
Cu(I)-induced strand scission has been reported.11,12 Re-
The synthesis started with a Sonogashira reaction from
fully TBS protected 5-iodo-2¢-deoxyuridine 1 with two
different unprotected alkynyl alcohols (pent-4-yn-1-ol or
O-propargyl triethylene glycol) to afford the different
linker lengths (Scheme 2).16 The alcohols 2 were then
converted into the mesylates and afterwards substituted
with NaN3 to form the azides 3. After deprotection of the
hydroxyl groups of the sugar moiety, the corresponding 5¢
triphosphates 4 were obtained using the reported meth-
ods.17 The yields of each step were high in both cases, but
overall slightly lower for the nucleoside bearing the trieth-
ylene glycol linker.
With these triphosphates in hand, we next investigated the
properties of 4a and 4b towards their action on DNA poly-
merases. In order to test the ability of DNA polymerases
to accept the modified triphosphates and to incorporate
the respective nucleotide into a nascent DNA strand, we
set up a primer extension reaction. A 35-nucleotide (nt)
template was designed in a way to contain a single A res-
SYNTHESIS 2007, No. 13, pp 1949–1954
x
x.
x
x
.
2
0
0
7
Advanced online publication: 18.06.2007
DOI: 10.1055/s-2007-983728; Art ID: T05607SS
© Georg Thieme Verlag Stuttgart · New York

1950
A. Baccaro et al.
PAPER
DNA template
DNA primer
DNA polymerase
dATP, dCTP, dGTP, dXTP
N3
DNA1
O
R
N
H
O
N
H
N3
OMe
3
Staudinger
ligation
5
Ph2P
O
O
DNA2
R
N
H
O
O
N
H
3
NH
Ph2P
O
HN
NH
O
R = H
H
H
Figure 1 (A) Sequences of primer template complex used to study
incorporation of modified triphosphates 4a and 4b. (B) Denaturing
PAGE analysis of primer extension reactions. Lane 1: 5¢-32P-labeled
23nt primer strand; Lane 2: primer template complex including
dATP, dCTP, dGTP, and Pwo DNA polymerase; Lane 3: same as in
lane 2 including dTTP; Lane 4: same as in lane 2 including 4a; Lane
5: same as in lane 2 including 4b. (C) Staudinger ligation employing
enzymatically synthesized double-stranded DNA (dsDNA) as shown
in Figure 1B lane 4 (upper autoradiogram) or lane 5 (lower autoradio-
gram) with 25% DMF in aqueous buffer and phosphine 5a at various
temperatures as indicated. nt = nucleotide.
S
O
5a
5b
DNA2b
DNA2a
Scheme 1 Schematic depiction for labeling of DNA with phos-
phines 5a and 5b.
idue, coding for insertion of a modified dTTP analogue af-
ter extending the 23nt primer strand by three residues
(Figure 1A). The reactions were analyzed by denaturing
polyacrylamide gel electrophoresis (PAGE). We tested
Thermus aquaticus (Taq), Thermococcus litoralis [Vent
(exo-)] and Pyrococcus woesei (Pwo) DNA polymerase,
whereas Pwo DNA polymerase accepted the modified
triphosphates best as has been reported by us13 and Famu-
lok et al. for generation of highly functionalized DNA.14
Reactions lacking dTTP predominantly abort before the
template A after extension by three nucleotides, while re-
actions including dTTP gave rise to full-length products
(Figure 1B). In case of substitution of natural dTTP by 4a
or 4b full-length product was also obtained in a similar
fashion. However, the reaction product is somewhat shift-
ed on the gel due to its higher molecular weight and there-
fore lower mobility in the polyacrylamide gel.
studied using phosphine 5a as a model (Figure 1C).11 The
ligation reaction was carried out in aqueous buffer with
25% of DMF to increase the solubility of the phosphine
compound. Increasing temperatures led to higher conver-
sion within four hours reaction time. Interestingly, DNA
derived from building block 4b formed the reaction prod-
uct in higher yields compared to 4a. However, best results
were obtained after increasing reaction time to 12 hours at
60 °C. For further ligation experiments we used phos-
phine 5b bearing a biotin at its end. Biotin is often used in
biochemistry for labeling issues and can be further conju-
gated with streptavidin functionalized materials.18 In this
reaction, again, DNA with building block 4b generated
higher yields (70%) than 4a (60%, Figure 2A). The yield
was determined by quantifying the radioactivity of both
the product and starting material DNA bands on the poly-
acrylamide gel by conventional phosphorimaging.
Next, we investigated whether the double-stranded enzy-
matically synthesized azide-labeled DNA is suitable for
Staudinger ligation. First the temperature dependence was
O
O
O
O
I
d,e
a
b,c
NH
NH
NH
NH
TBSO
N
O
TBSO
N
O
TBSO
N
O
4– O9P3O
N
O
O
O
O
O
HO
N3
N3
3a
4a
4b
2a
2b
HO
N3
N3
OTBS
OTBS
2
OTBS
3
OH
O
O
O
3
3
3
1
4
3b
Scheme 2 Synthesis of azide modified triphosphates. Reagents and conditions: Et3N, CuI, Pd(PPh3)4, DMF, 20 °C, 2a: pent-4-yn-1-ol, 81%,
2b: O-propargyl triethylene glycol, 83%; (b) MsCl, DIPEA, CH2Cl2, 0 °C; (c) NaN3, DMF, 35 °C, 3a: 70% over 2 steps, 3b: 50% over 2 steps;
(d) TBAF, THF, 0 °C; (e) proton sponge, POCl3, PO(OMe)3, 0 °C, then (Bu3NH)2H2P2O7, Bu3N then TEAB buffer, 4a: 28% over 2 steps, 4b:
23% over 2 steps.
Synthesis 2007, No. 13, 1949–1954 © Thieme Stuttgart · New York

PAPER
DNA Conjugation by the Staudinger Ligation
1951
gene fragments with 4b instead of dTTP under standard
reaction conditions. Further development of the reaction
conditions might lead to improvements along these lines.
However, Staudinger ligation with large azide-labeled
DNA fragments could lead to some new applications in
nanobiotechnology as well as material science.
All melting and boiling point values quoted are uncorrected. All re-
agents are commercially available and were used without further
purification. TBS protected nucleoside 1,19 O-tosyl triethylene gly-
col,20 and phosphines 5a and 5b11 were prepared according to liter-
ature. Solvents were stored over molecular sieves (Fluka) and used
directly without further purification, unless otherwise noted. All re-
actions were conducted under rigorous exclusion of air and mois-
ture. Elemental analyses were carried out by the microanalysis
facility of the University of Konstanz. NMR spectra were recorded
on a Jeol JNA-LA-400 (1H: 400 MHz, 13C: 101 MHz, 32P: 162
MHz), a Bruker Avance DRX 600 (1H: 600 MHz, 13C: 151 MHz).
The solvent signals were used as references and the chemical shifts
converted to the TMS scale and are given in ppm (d). ESI-IT mass
spectra were recorded on a Bruker Daltonics esquire 3000+. Flash
chromatography was done using Merck silica gel G60 (230–400
mesh), and Merck precoated plates (silica gel 60 F254) were used for
TLC. Both technical solvents were distilled prior to use. Petroleum
ether (PE) used refers to the fraction boiling in the range 35–80 °C.
Figure 2 (A) Denaturing PAGE analysis of Staudinger ligation with
phosphine 5b with 25% DMF for 12 h at 60 °C employing dsDNA,
which contains 4a (left gel) or 4b (right gel). (B) 2.5% agarose gel
stained with ethidium bromide showing PCR products of 98nt tem-
plate. Lane 1: Marker; Lane 2: PCR with dTTP; Lane 3: PCR without
TTP; Lane 4: PCR with 4a instead of dTTP; Lane 5: PCR with 4b in-
stead of dTTP. (C) 0.8% agarose gel stained with ethidium bromide
showing PCR products of 1.9k nt template. Lanes as in Figure 2B.
nt = nucleotide; bp = base pair.
Finally, we tested the suitability of building blocks 4a and
4b for substituting dTTP in polymerase chain reactions
(PCR). In this case we only tested Pwo DNA polymerase,
first with a short template of 98nt in size. As with the
primer extension tests we conducted one control reaction
with all four natural dNTPs leading to a single PCR prod-
uct with the desired length. In another control we per-
formed the same reaction in the absence of dTTP leading
to no observable PCR product formation (Figure 2B). Us-
ing standard PCR conditions the DNA polymerase was
able to substitute compounds 4a and 4b for TTP and am-
plify the modified DNA in a similar fashion as with all
four natural dNTPs. Again a small shift of the product
bands to lower mobility was observed for the modified an-
alogues, which is more pronounced with the larger modi-
fication of 4b. Encouraged by these results we switched to
a larger genomic DNA fragment as template for the PCR.
Here, compound usage of 4a resulted in similar amounts
of PCR product compared to the PCR with all natural
dNTPs. PCR including compound 4b instead of dTTP
produced less but observable product. Presumably the
larger modification interfered with the DNA polymerase.
Nevertheless only one PCR product was observed, and so
we were able to generate large azide-labeled DNA frag-
ments with both compounds.
O-Propargyl Triethylene Glycol
To a suspension of NaH (1.5 equiv, 0.69 g, 17.2 mmol) in THF (5
mL), was added propargyl alcohol (1.5 equiv, 1.0 mL, 17.2 mmol)
at 0 °C. After 30 min, a solution of O-tosyl triethylene glycol (1.0
equiv, 3.5 g, 11.5 mmol) in THF (10 mL) was added and the mixture
stirred for 3 h at 0 °C. Then it was combined with sat. aq NaHCO3
(15 mL) and extracted with EtOAc (6 × 10 mL). The combined or-
ganic layers were dried (MgSO4), and concentrated in vacuo. The
residue was purified by flash chromatography [silica gel (EtOAc–
PE, 2:3 to 5:1); Rf = 0.3 (EtOAc–PE, 3:1)]; yield: 1.0 g (46%).
4
1H NMR (400 MHz, CDCl3): d = 2.43 (t, J = 2.2 Hz, 1 H, CH),
3.57–3.64 (m, 2 H, CH2OH), 3.64–3.75 (m, 10 H, OCH2CH2O),
4.19 (d, 4J = 2.2 Hz, 2 H, CCH2O).
13C NMR (101 MHz, CDCl3): d = 58.2, 61.5, 68.9, 70.1, 70.2, 70.5,
72.4, 74.6, 79.5.
ESI-MS: m/z = 211.0 [M + Na]+.
Compounds 2a and 2b; General Procedure
To a solution of 1 (1.0 equiv) and CuI (0.2 equiv) in degassed DMF,
were added with stirring the corresponding alkynol (2a: pent-4-yn-
1-ol, 2b: O-propargyl triethylene glycol, 3.0 equiv), (PPh3)4Pd (0.1
equiv) and Et3N (2.0 equiv). The mixture was protected from light
and stirred at r.t. (for 2a, 16 h; for 2b, 18 h). The mixture was com-
bined with sat. aq NaHCO3 (10 mL) and extracted with EtOAc
(3 × 10 mL). The combined organic layers were washed with brine
(10 mL), dried (MgSO4), and concentrated in vacuo. The residue
was purified by flash chromatography.
In conclusion, a straightforward synthesis of azide modi-
fied thymidines was developed. The corresponding triph-
osphates were tested with different DNA polymerases
indicating that Pwo DNA polymerase was most suited for
our endeavor. Even amplification of large DNA fragments
could be accomplished by PCR using this DNA poly-
merase and the modified triphosphates. The azide-labeled
DNA in turn can be conjugated with phosphines that bear
several functional groups, such as biotin. For Staudinger
ligation reactions DNA generated by building block 4b
seems to be superior over DNA derived from 4a due to
smaller sterical hindrance. On the other hand, 4b leads to
smaller PCR product formation, when amplifying large
3¢,5¢-Bis-O-(tert-butyldimethylsilyl)-5-(5-hydroxypent-1-ynyl)-
2¢-deoxyuridine (2a)
The reaction was carried out with deoxyuridine 1 (1.0 g, 1.72
mmol), CuI (65 mg, 0.34 mmol), pent-4-yn-1-ol (476 mL, 5.16
mmol), (PPh3)4Pd (27 mg, 0.17 mmol), and Et3N (482 mL, 3.43
mmol) in DMF (5 mL). Purification: silica gel (EtOAc–PE, 1:1 to
3:2); Rf = 0.3 (EtOAc–PE, 3:2); yield: 750 mg (81%).
1H NMR (600 MHz, CDCl3): d = 0.09 (s, 3 H, CH3Si), 0.1 (s, 3 H,
CH3Si), 0.15 (s, 3 H, CH3Si), 0.16 (s, 3 H, CH3Si), 0.91 (s, 9 H, t-
Synthesis 2007, No. 13, 1949–1954 © Thieme Stuttgart · New York

1952
A. Baccaro et al.
H, t-C4H9Si), 1.77–1.86 (m,
PAPER
C4H9Si), 0.95 (s,
9
2
H,
CH2CH2CH2OMs), 3.07 (s, 3 H, OSO2CH3), 3.77 (dd, 2J5¢b,5¢a = 11.5
2
CH2CH2CH2OH), 2.00–2.07 (m,
1
H, H-2¢a), 2.31 (ddd,
Hz, 3J5¢b,4¢ = 2.1 Hz, 1 H, H-5¢b), 3.89 (dd, J5¢a,5¢b = 11.5 Hz,
2J2¢b,2¢a = 13.1 Hz, 3J2¢b,1¢ = 5.7 Hz, 3J2¢b,3¢ = 2.5 Hz, 1 H, H-2¢b), 2.51
3J5¢a,4¢ = 2.4 Hz, 1 H, H-5¢a), 3.96–3.98 (m, 1 H, H-4¢), 4.37–4.41
3
3
(t, J = 6.8 Hz, 2 H, CH2CH2CH2OH), 3.76–3.81 (m, 3 H, H-5¢b,
(m, 3 H, H-3¢, CH2CH2CH2OMs), 6.27 (dd, J1¢,2¢a = 7.3 Hz,
CH2CH2CH2OH), 3.91 (dd, 2J5¢a,5¢b = 11.4 Hz, J5¢a,4¢ = 2.0 Hz, 1 H,
3J1¢,2¢b = 6.0 Hz, 1 H, H-1¢), 7.94 (s, 1 H, H-6), 8.05 (br s, 1 H, NH).
3
H-5¢a), 3.97–3.99 (m, 1 H, H-4¢), 4.40–4.43 (m, 1 H, H-3¢), 6.30 (t,
3J1¢,2¢a = 3J1¢,2¢b = 5.7 Hz, 1 H, H-1¢), 7.93 (s, 1 H, H-6), 8.28 (br s, 1
H, NH).
13C NMR (151 MHz, CDCl3): d = –4.5, –4.4, –3.8, –3.6, 17.4, 19.0,
19.5, 26.7, 27.0, 32.8, 43.0, 62.7, 64.0, 73.0, 73.3, 86.7, 89.3, 95.6,
101.6, 142.5, 150.0, 162.7.
13C NMR (151 MHz, CDCl3): d = –4.5, –4.4, –3.8, –3.6, 16.9, 19.0,
19.5, 26.7, 27.0, 28.8, 38.2, 42.9, 49.3, 64.0, 69.5, 73.3, 86.7, 89.4,
93.4, 101.1, 143.0, 150.0, 162.6.
ESI-MS: m/z = 639.4 [M + Na]+.
Anal. Calcd for C27H48N2O8SSi2: C, 52.57; H, 7.84; N, 4.54. Found:
C, 52.62; H, 7.77; N, 4.50.
ESI-MS: m/z = 561.5 [M + Na]+.
Anal. Calcd for C26H46N2O6Si2: C, 57.96; H, 8.60; N, 5.20. Found:
C, 57.98; H, 8.62; N, 5.14.
3¢,5¢-Bis-O-(tert-butyldimethylsilyl)-5-(5-azidopent-1-ynyl)-2¢-
deoxyuridine (3a)
Prepared from mesylate derived from 2a (70 mg, 0.11 mmol), and
NaN3 (46 mg, 0.71 mmol) in DMF (2 mL). Purification: silica gel
(EtOAc–PE, 1:3 to 1:1); Rf = 0.8 (EtOAc–PE, 1:1); yield: 61 mg
(95%).
1H NMR (600 MHz, CDCl3): d = 0.09 (s, 3 H, CH3Si), 0.1 (s, 3 H,
CH3Si), 0.15 (s, 3 H, CH3Si), 0.16 (s, 3 H, CH3Si), 0.91 (s, 9 H, t-
C4H9Si), 0.95 (s, 9 H, t-C4H9Si), 1.85 (dddd, 3J = 6.8, 6.8, 6.8, 6.8
Hz, 2 H, CH2CH2CH2N3), 2.03 (ddd, 2J2¢a,2¢b = 13.3 Hz, 3J2¢a,1¢ = 7.7
3¢,5¢-Bis-O-(tert-butyldimethylsilyl)-5-[12-hydroxy-(4,7,10-tri-
oxadodec-1-ynyl)]-2¢-deoxyuridine (2b)
The reaction was carried out with deoxyuridine 1 (0.5 g, 0.86
mmol), CuI (33 mg, 0.17 mmol), O-propargyl triethylene glycol
(0.48 g, 2.50 mmol), (PPh3)4Pd (13 mg, 0.09 mmol), and Et3N (242
mL, 1.72 mmol) in DMF (5 mL). Purification: silica gel (CH2Cl2–
MeOH, 20:1); Rf = 0.4 (CH2Cl2–MeOH, 20:1); yield: 460 mg
(83%).
3
2
Hz, J2¢a,3¢ = 6.3 Hz, 1 H, H-2¢a), 2.31 (ddd, J2¢b,2¢a = 13.3 Hz,
3J2¢b,1¢ = 5.9 Hz, 3J2¢b,3¢ = 2.7 Hz, 1 H, H-2¢b), 2.51 (t, 3J = 6.8 Hz, 2
H, CH2CH2CH2N3), 3.45 (t, 3J = 6.8 Hz, 2 H, CH2CH2CH2N3), 3.78
1H NMR (400 MHz, CDCl3): d = 0.05 (s, 3 H, CH3Si), 0.06 (s, 3 H,
CH3Si), 0.10 (s, 3 H, CH3Si), 0.11 (s, 3 H, CH3Si), 0.86 (s, 9 H, t-
2
2
C4H9Si), 0.90 (s, 9 H, t-C4H9Si), 1.99 (ddd, J2¢a,2¢b = 13.2 Hz,
(dd, J5¢b,5¢a = 11.5 Hz, 3J5¢b,4¢ = 2.2 Hz, 1 H, H-5¢b), 3.91 (dd,
3J2¢a,1¢ = 7.5 Hz, 3J2¢a,3¢ = 5.9 Hz, 1 H, H-2¢a), 2.31 (ddd, 2J2¢b,2¢a = 13.0
Hz, 3J2¢b,1¢ = 5.9 Hz, 3J2¢b,3¢ = 2.5 Hz, 1 H, H-2¢b), 3.57–3.77 (m, 13
H, H-5¢b, OCH2CH2O), 3.87 (dd, 2J5¢a,5¢b = 11.4 Hz, 3J5¢a,4¢ = 2.5 Hz,
1 H, H-5¢a), 3.94–3.96 (m, 1 H, H-4¢), 4.34–4.39 (m, 3 H, H-3¢,
CH2O), 6.27 (t, 3J1¢,2¢a = 7.5 Hz, 3J1¢,2¢b = 5.9 Hz, 1 H, H-1¢), 8.00 (s,
1 H, H-6), 8.50 (br s, 1 H, NH).
2J5¢a,5¢b = 11.5 Hz, 3J5¢a,4¢ = 2.3 Hz, 1 H, H-5¢a), 3.98 (ddd,
3J4¢,5¢b = 2.2 Hz, J4¢,5¢a = 2.3 Hz, J4¢,3¢ = 2.2 Hz, 1 H, H-4¢), 4.40–
4.43 (m, 1 H, H-3¢), 6.3 (dd, 3J1¢,2¢a = 7.7 Hz, 3J1¢,2¢b = 5.9 Hz, 1 H, H-
1¢), 7.93 (s, 1 H, H-6), 8.27 (br s, 1 H, NH).
3
3
13C NMR (151 MHz, CDCl3): d = –4.5, –4.4, –3.8, –3.6, 18.0, 19.0,
19.5, 26.8, 27.0, 28.7, 43.0, 51.3, 64.0, 73.4, 73.5, 86.7, 89.4, 94.1,
101.4, 142.9, 150.0, 162.5.
13C NMR (101 MHz, CDCl3): d = –5.3, –5.2, –4.6, –4.4, 18.1, 18.6,
25.8, 26.1, 42.0, 59.0, 61.6, 62.9, 68.9, 70.2, 70.3, 70.5, 72.2, 72.5,
77.5, 85.7, 88.2, 89.7, 99.3, 142.7, 148.9, 161.3.
ESI-MS: m/z = 586.6 [M + Na]+.
ESI-MS: m/z = 665.5 [M + Na]+.
3¢,5¢-Bis-O-(tert-butyldimethylsilyl)-5-[12-azido-(4,7,10-trioxa-
dodec-1-ynyl)]-2¢-deoxyuridine (3b)
Prepared from 2b (0.1 g, 0.16 mmol), DIPEA (40 mL, 0.23 mmol),
MsCl (38 mL, 0.18 mmol) in CH2Cl2 (5 mL) followed by the addi-
tion of NaN3 (50 mg, 0.78 mmol) and DMF (2 mL). Purification:
silica gel (CH2Cl2–MeOH, 20:1); Rf = 0.8 (CH2Cl2–MeOH, 20:1);
yield: 53 mg (50% over two steps).
Compounds 3a and 3b; General Procedure
To a solution of 2 (1.0 equiv) in CH2Cl2 at 0 °C was added DIPEA
(2a: 1.8 equiv, 2b: 1.5 equiv) and stirred for 2a: 30 min, 2b: 10 min,
then MsCl (1.2 equiv) was added slowly and the mixture stirred; for
2a: 40 min, 2b: 20 min at 0 °C (in the case of 2b, the mixture was
evaporated and the crude product was used directly without purifi-
cation). For 2a, the mixture was combined with sat. aq NaHCO3 (10
mL) and extracted with Et2O (3 × 15 mL). The combined organic
layers were washed with brine (15 mL), dried (MgSO4), and con-
centrated in vacuo. The residue was purified by flash chromatogra-
phy [silica gel (EtOAc–PE, 1:2); Rf = 0.8 (EtOAc–PE, 3:1)]. To a
solution of the respective mesylate (1.0 equiv) in DMF was added
NaN3 (2a: 6.5 equiv, 2b: 5.0 equiv). The mixture was warmed to
35 °C for 20 h, then combined with sat. aq NaHCO3 (5 mL) and ex-
tracted with EtOAc (3 × 5 mL). The combined organic layers were
dried (MgSO4), and concentrated in vacuo. The residue was purified
by flash chromatography.
1H NMR (600 MHz, CDCl3): d = 0.08 (s, 3 H, CH3Si), 0.09 (s, 3 H,
CH3Si), 0.14 (s, 3 H, CH3Si), 0.15 (s, 3 H, CH3Si), 0.90 (s, 9 H, t-
2
C4H9Si), 0.94 (s, 9 H, t-C4H9Si), 2.03 (ddd, J2¢a,2¢b = 13.2 Hz,
3J2¢a,1¢ = 7.6 Hz, 3J2¢a,3¢ = 6.0 Hz,
1 H, H-2¢a), 2.33 (ddd,
2J2¢b,2¢a = 13.2 Hz, 3J2¢b, 1¢ = 5.9 Hz, 3J2¢b,3¢ = 2.6 Hz, 1 H, H-2¢b), 3.40
(t, 3J = 5.1 Hz, 2 H, OCH2CH2N3), 3.67–3.71 (m, 8 H, OCH2CH2O),
2
3.72–3.75 (m, 2 H, OCH2CH2N3), 3.77 (dd, J5¢b,5¢a = 11.4 Hz,
3J5¢b,4¢ = 2 Hz, 1 H, H-5¢b), 3.90 (dd, 2J5¢a,5¢b = 11.4 Hz, J5¢a,4¢ = 2.4
3
Hz, 1 H, H-5¢a), 3.97–4.00 (m, 1 H, H-4¢), 4.39 (s, 2 H, CH2O),
4.40–4.43 (m, 1 H, H-3¢), 6.29 (dd, 3J1¢,2¢a = 7.6 Hz, 3J1¢,2¢b = 5.9 Hz,
1 H, H-1¢), 8.02 (s, 1 H, H-6), 8.40 (br s, 1 H, NH).
13C NMR (151 MHz, CDCl3): d = –4.5, –4.4, –3.8, –3.6, 19.0, 19.4,
26.7, 27.0, 43.0, 51.7, 60.0, 64.0, 70.2, 71.0, 71.5, 71.6, 71.7, 73.4,
78.4, 86.9, 89.4, 90.6, 100.5, 144.1, 150.0, 162.2.
3¢,5¢-Bis-O-(tert-butyldimethylsilyl)-5-(5-methanesulfonate-
pent-1-ynyl)-2¢-deoxyuridine
Prepared from 2a (0.32 g, 0.59 mmol), DIPEA (182 mL, 1.07
mmol), and MsCl (55 mL, 0.71 mmol) in CH2Cl2 (5 mL); yield: 270
mg (74%).
ESI-MS: m/z = 690.3 [M + Na]+.
Compounds 4a and 4b; General Procedure
1H NMR (600 MHz, CDCl3): d = 0.07 (s, 3 H, CH3Si), 0.08 (s, 3 H,
CH3Si), 0.13 (s, 3 H, CH3Si), 0.14 (s, 3 H, CH3Si), 0.89 (s, 9 H, t-
C4H9Si), 0.93 (s, 9 H, t-C4H9Si), 1.97–2.05 (m, 3 H, H-2¢a,
To a solution of 3 (1.0 equiv) in THF, was added TBAF (1 M solu-
tion in THF; 2.2 equiv) at 0 °C. The mixture was allowed to warm
to r.t. and stirred; for 3a: 16 h, 3b: 20 h, and evaporated in vacuo to
give the crude unprotected sugar, which was purified by flash chro-
matography. This product (1.0 equiv) and proton sponge [1,8-
2
3
CH2CH2CH2OMs), 2.31 (ddd, J2¢b,2¢a = 13.1 Hz, J2¢b,1¢ = 6.0 Hz,
3J2¢b,3¢ = 2.7 Hz, 1 H, H-2¢b), 2.55 (dd, 3J = 6.8 Hz, 3J = 6.8 Hz, 2 H,
Synthesis 2007, No. 13, 1949–1954 © Thieme Stuttgart · New York

PAPER
DNA Conjugation by the Staudinger Ligation
1953
bis(dimethylaminonaphthalene)] (1.5 equiv) were dried overnight
protected from light in vacuo, dissolved in trimethyl phosphate, and
cooled to 0 °C. Freshly distilled POCl3 (4a: 1.3 equiv, 4b: 2.2
equiv) was added to the mixture and stirred; for 4a: 3.5 h, 4b: 4.5 h.
A 0.5 M soln of (Bu3NH)2H2P2O7 in anhyd DMF (4a: 5 equiv, 4b:
10 equiv) and Bu3N (4a: 10 equiv, 4b: 20 equiv) were added simul-
taneously to the mixture. After 5 min, 1 M aq triethylammonium bi-
carbonate (TEAB buffer, pH 7.5) was added and the aqueous layer
was washed with EtOAc (2 × 10 mL). The aqueous layer was lyo-
philized and the resulting residue purified by ion-exchange chroma-
tography [DEAE-Sephadex A-25, linear gradient of TEAB buffer
(0.1 M to 1 M, 1000 mL), flow 2 mL/min] and further purified by
RP-MPLC (RP-18, 40-63 mm) using a gradient of 5% (200 mL),
20% (200 mL) and 40% (200 mL) MeCN in 50 mM aq triethylam-
monium acetate (TEAA buffer, pH 7.0). The triphosphates 4a and
4b were eluted with 20% MeCN in 50 mM aq TEAA buffer.
5-[12-Azido-(4,7,10-trioxa-1-dodecynyl)]-2¢-deoxyuridine-5¢-
triphosphate (4b)
Prepared from 5-[12-azido-(4,7,10-trioxadodec-1-ynyl)]-2¢-deoxy-
uridine (26.5 mg, 60 mmol), proton sponge (19.4 mg, 90 mmol),
POCl3 (17 mL, 0.19 mmol), (Bu3NH)2H2P2O7 solution (1.2 mL, 0.6
mmol), Bu3N (0.32 mL, 1.2 mmol), and trimethyl phosphate (1
mL); yield: 15.4 mg (24%).
1H NMR (600 MHz, MeOD): d = 1.27 [t, 3J = 7.0 Hz, (HNEt3)+ ],
4
2.27 (m, 2 H, H-2¢b, H-2¢a), 3.07–3.12 [m, (HNEt3)+ ], 3.37 (t,
4
3J = 4.9 Hz, 2 H, OCH2CH2N3), 3.64–3.69 (m, 8 H, OCH2CH2O),
3.71–3.74 (m, 2 H, OCH2CH2N3), 4.06–4.10 (m, 1 H, H-4¢), 4.14–
4.19 (m, 1 H, H-5¢b), 4.24–4.3 (m, 1 H, H-5¢a), 4.40 (s, 2 H, CH2O),
4.58–4.61 (m, 1 H, H-3¢), 6.22 (t, 3J1¢,2¢a = 3J1¢,2¢b = 6.8 Hz, 1 H, H-
1¢), 8.08 (s, 1 H, H-6).
31P NMR (162 MHz, MeOD): d = –21.6 (m, 1 P, Pb), –10.4 (d,
2Ja,b = 21.6 Hz, 1 P, Pa), –8.6 (d, 2Jg,b = 20.8 Hz, 1 P, Pg).
ESI-MS: m/z = 677.8 [M – H]–.
5-(5-Azidopent-1-ynyl)-2¢-deoxyuridine
Prepared from 3a (58 mg, 0.10 mmol), TBAF soln (226 mL, 0.23
mmol) in THF (2 mL). Purification: silica gel (CH2Cl2–MeOH,
20:1); Rf = 0.6 (CH2Cl2–MeOH, 20:1); yield: 32 mg (93%).
Primer Extension
DNA primer strands were purchased from IBA, dNTPs were from
Roche, Pwo DNA polymerase, 10 × Pwo reaction buffer (100 mM
Tris·HCl (pH 8.8), 250 mM KCl, 20 mM MgSO4) were from
Peqlab. A typical primer extension reaction (20 mL) contained
1 × Pwo reaction buffer, 150 nM 32P-labeled primer, 200 nM tem-
plate, 100 mM each of 4a or 4b, 100 mM each of dATP, dCTP,
dGTP, and 0.12 U/20 mL Pwo DNA polymerase. First primer and
template were annealed in 1 × Pwo reaction buffer by heating the
probes to 95 °C and allowing to cool down to 20 °C. Afterwards the
primer template complex, nucleotides, and Pwo DNA polymerase
were incubated at 72 °C for 30 min. The reactions were quenched
by addition of 20 mL PAGE gel loading buffer (80% formamide, 20
mM EDTA, 0.1% bromophenol blue, 0.1% xylene cyanole FF) and
the product mixtures were analyzed by 12% denaturing polyacryl-
amide gel, and subjected to autoradiography.
1H NMR (400 MHz, MeOD): d = 1.75 (dddd, 3J = 6.8, 6.8, 6.8, 6.8
Hz, 2 H, CH2CH2CH2N3), 2.10–2.26 (m, 2 H, H-2¢a, H-2¢b), 2.43 (t,
3J = 6.8 Hz, 2 H, CH2CH2CH2N3), 3.40 (t, 3J = 6.8 Hz, 2 H,
2
3
CH2CH2CH2N3), 3.67 (dd, J5¢b,5¢a = 12.0 Hz, J5¢b,4¢ = 3.4 Hz, 1 H,
2
3
H-5¢b), 3.75 (dd, J5¢a,5¢b = 12.0 Hz, J5¢a,4¢ = 3.0 Hz, 1 H, H-5¢a),
3.84–3.88 (m, 1 H, H-4¢), 4.31–4.36 (m, 1 H, H-3¢), 6.18 (t,
3J1¢,2¢a = 3J1¢,2¢b = 6.6 Hz, 1 H, H-1¢), 8.19 (s, 1 H, H-6).
13C NMR (101 MHz, CDCl3): d = 17.3, 28.8, 41.6, 51.3, 62.7, 72.2,
73.7, 87.1, 89.4, 93.8, 101.3, 145.1, 151.9, 165.5.
ESI-MS: m/z = 357.9 [M + Na]+.
5-(5-Azidopent-1-ynyl)-2¢-deoxyuridine-5¢-triphosphate (4a)
Prepared from 5-(5-azidopent-1-ynyl)-2¢-deoxyuridine (29 mg, 86
mmol), proton sponge (28 mg, 0.13 mmol), POCl3 (10.5 mL, 0.11
mmol), (Bu3NH)2H2P2O7 solution (0.86 mL, 0.43 mmol), Bu3N
(229 mL, 0.86 mmol), and trimethyl phosphate (1 mL); yield: 25.7
mg (30%).
Staudinger Ligation on DNA 1
The primer extension product was used for the Staudinger ligation.
The modified DNA was prepared according to the procedure men-
tioned above. The concentrations of dNTPs and Pwo DNA poly-
merase were adjusted to 200 mM and to 0.5 U/20 mL, respectively.
After incubation at 72 °C for 60 min, the 20 mL reactions were
quenched with 1 mL of 210 mM EDTA solution. The reaction prod-
uct was purified using Sephadex G-25 spin columns. For the
Staudinger ligation, 2.5 mL of 1 M NaHCO3/Na2CO3 buffer (pH
9.0), 2.5 mL of 5a or 5b (10 mM in DMF) and 5 mL of the primer
extension product (~1 mM) were mixed and incubated either at
60 °C for 12 h or at various temperatures for 4 h in the case of tem-
perature-dependence experiments. The reactions were stopped by
adding 10 mL of PAGE-gel-loading buffer and the product mixtures
were analyzed by 12% denaturing polyacrylamide gel.
1H NMR (400 MHz, MeOD): d = 1.20–1.35 [m, 36 H, (HNEt3)+ ],
4
1.8–1.93 (m, 2 H, CH2CH2CH2N3), 2.30–2.40 (m, 2 H, H-2¢b, H-
2¢a), 2.47–2.55 (m, 2 H, CH2CH2CH2N3), 3.13–3.25 [m, H-5¢a, H-
5¢b, (HNEt3)+ ], 3.25–3.46 (m, 2 H, CH2CH2CH2N3), 4.1–4.2 (m, 1
4
H, H-3¢), 4.56–4.62 (m, 1 H, H-4¢), 6.26 (m, 1 H, H-1¢), 8.0 (s, 1 H,
H-6).
31P NMR (162 MHz, MeOD): d = –22.5 (m, 1 P, Pb), –10.6 (d,
2Ja,b = 20.2 Hz, 1 P, Pa), –9.3 (d, 2Jg,b = 20.2 Hz, 1 P, Pg).
ESI-MS: m/z = 575.0 [M – H]–.
5-[12-Azido-(4,7,10-trioxadodec-1-ynyl)]-2¢-deoxyuridine
Prepared from 3b (43 mg, 64 mmol), TBAF soln (142 mL, 0.14
mmol) in THF (2 mL). Purification: silica gel (CH2Cl2–MeOH,
20:1); Rf = 0.5 (CH2Cl2–MeOH, 10:1); yield: 26.5 mg (94%).
PCR
Typical PCR reactions (20 mL) contained 1 × Pwo reaction buffer,
200 mM each of 4a or 4b, 200 mM each of dATP, dCTP, dGTP, 500
nM primer I, 500 nM primer II, 1.2 pg/mL template (98 bp), and 0.02
U/mL Pwo DNA polymerase. A typical PCR cycling protocol began
with an initial denaturation at 95 °C for 60 s and was followed by
35 cycles of 95 °C, 30 s; 55 °C, 35 s; 72 °C, 40 s. The reactions were
quenched by addition of 4 mL agarose-gel-loading buffer (10 mM
Tris·HCl, 60 mM EDTA, 30% glycerol, 0.1% bromophenol blue,
0.1% xylene cyanole FF) and the product mixtures were analyzed
by agarose gel electrophoresis.
2
1H NMR (600 MHz, MeOD): d = 2.23 (ddd, J2¢a,2¢b = 13.5 Hz,
3J2¢a,1¢ = 6.3 Hz, 3J2¢a,3¢ = 6.5 Hz,
1 H, H-2¢a), 2.32 (ddd,
2J2¢b,2¢a = 13.5 Hz, 3J2¢b,1¢ = 6.3 Hz, 3J2¢b,3¢ = 3.7 Hz, 1 H, H-2¢b), 3.38
(t, 3J = 4.9 Hz, 2 H, OCH2CH2N3), 3.65–3.70 (m, 8 H, OCH2CH2O),
2
3.72–3.76 (m, 3 H, H-5¢b, OCH2CH2N3), 3.90 (dd, J5¢a,5¢b = 12.0
Hz, 3J5¢a,4¢ = 3.0 Hz, 1 H, H-5¢a), 3.92–4.00 (m, 1 H, H-4¢), 4.38 (s,
2 H, CH2O), 4.39–4.42 (m, 1 H, H-3¢), 6.24 (t, 3J1¢,2¢a = 3J1¢,2¢b = 6.3
Hz, 1 H, H-1¢), 8.35 (s, 1 H, H-6).
13C NMR (151 MHz, CDCl3): d = 41.8, 51.8, 59.7, 62.5, 70.2, 71.1,
71.4, 71.5, 71.6, 71.9, 78.8, 87.1, 89.2, 90.2, 100.0, 145.7, 151.2,
164.3.
The PCR reaction using the 1.9 kB plasmid as template was done
according to the procedure mentioned above. The concentrations of
plasmid DNA and Pwo DNA polymerase were adjusted to 0.4 ng/
mL and 0.04 U/mL, respectively. The PCR cycling protocol was de-
ESI-MS: m/z = 463.2 [M + Na]+.
Synthesis 2007, No. 13, 1949–1954 © Thieme Stuttgart · New York

1954
A. Baccaro et al.
PAPER
natured at 95 °C for 120 s, followed by 35 cycles of 95 °C, 60 s;
63 °C, 60 s; and 72 °C, 240 s. The reactions were quenched by the
addition of 4 mL agarose-gel-loading buffer and the product mix-
tures were analyzed by agarose gel electrophoresis.
(9) (a) Liu, L.; Hong, Z.-Y.; Wong, C.-H. ChemBioChem 2006,
7, 429. (b) Grandjean, C.; Boutonnier, A.; Guerreiro, C.;
Fournier, J.-M.; Mulard, L. A. J. Org. Chem. 2005, 70,
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H.; Soulère, L.; Breinbauer, R.; Niemeyer, C. M.;
Waldmann, H. Angew. Chem. Int. Ed. 2003, 42, 5830.
(f) Kim, H.; Cho, J. K.; Aimoto, S.; Lee, Y.-S. Org. Lett.
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Kohn, M.; Wacker, R.; Schroeder, H.; Breinbauer, R.;
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S.; Waldmann, H. Bioorg. Med. Chem. 2006, 14, 6288.
(h) Watzke, A.; Köhn, M.; Gutierrez-Rodriguez, M.;
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Acknowledgment
The assistance of Dr. Karl-Heinz Jung in manuscript preparation is
kindly acknowledged.
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Synthesis 2007, No. 13, 1949–1954 © Thieme Stuttgart · New York