Azidopropylvinylsulfonamide as a New Bifunctional Click Reagent for Bioorthogonal Conjugations: Application for DNA-Protein Cross-Linking

Article abstract of DOI:10.1002/chem.201502209

N-(3-Azidopropyl)vinylsulfonamide was developed as a new bifunctional bioconjugation reagent suitable for the cross-linking of biomolecules through copper(I)-catalyzed azide-alkyne cycloaddition and thiol Michael addition reactions under biorthogonal conditions. The reagent is easily clicked to an acetylene-containing DNA or protein and then reacts with cysteine-containing peptides or proteins to form covalent cross-links. Several examples of bioconjugations of ethynyl- or octadiynyl-modified DNA with peptides, p53 protein, or alkyne-modified human carbonic anhydrase with peptides are given.

Full text of DOI:10.1002/chem.201502209

DOI: 10.1002/chem.201502209
Full Paper
&
Bioconjugation
Azidopropylvinylsulfonamide as a New Bifunctional Click Reagent
for Bioorthogonal Conjugations: Application for DNA–Protein
Cross-Linking
Jitka Dadovµ,[a] Milan Vrµbel,[a] Matej Adµmik,[b] Marie Brµzdovµ,[b] Radek Pohl,[a]
Miroslav Fojta,[b, d] and Michal Hocek*[a, c]
Abstract: N-(3-Azidopropyl)vinylsulfonamide was developed
as a new bifunctional bioconjugation reagent suitable for
the cross-linking of biomolecules through copper(I)-cata-
lyzed azide–alkyne cycloaddition and thiol Michael addition
reactions under biorthogonal conditions. The reagent is
easily clicked to an acetylene-containing DNA or protein and
then reacts with cysteine-containing peptides or proteins to
form covalent cross-links. Several examples of bioconjuga-
tions of ethynyl- or octadiynyl-modified DNA with peptides,
p53 protein, or alkyne-modified human carbonic anhydrase
with peptides are given.
Introduction
enabled the introduction of many reactive groups into DNA
(e.g., aldehyde,[7] alkyne,[8,9] alkene,[10] diene,[11] azide,[12] or tetra-
zole[13]). These reactive DNA probes were subsequently further
modified by reductive amination,[14] [2+3]-dipolar cycloaddi-
tions,[8,9] Diels–Alder reactions,[15] or Staudinger ligation.[16] Re-
active base-modified DNA has also enabled the crystal struc-
tures of several proteins with DNA targets to be solved by co-
valent trapping of protein cysteine (Cys) mutants by using di-
sulfide cross-linking.[17] Recently, two new DNA–protein conju-
gation strategies were developed: reductive amination of
aldehyde-modified DNA with lysine or arginine[18] and photo-
cross-linking of DNA containing diazirine with various nucleo-
philic amino acids.[19] The former method requires the use of
sodium borohydride as an external reducing agent, which
limits its application in cells, whereas the latter is nonspecific.
We developed vinylsulfonamide (VS) as a reactive group for
the construction of modified DNA probes by enzymatic synthe-
sis with the corresponding VS-modified dNTP (dCVSTP).[20] This
Michael acceptor modified DNA was successfully utilized for se-
lective conjugation with tumor suppressor protein p53
through the thiol group of Cys by taking advantage of the
proximity effect. Although this highly reactive group can be in-
corporated by polymerase synthesis, it would not be compati-
ble with the phosphoramidite protocol for the chemical syn-
thesis of modified ONs on a solid support. Therefore, we envis-
aged the possibility of installing the Michael acceptor moiety
postsynthetically through a suitable bifunctional reagent. The
copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC)[21] is
the most versatile bioconjugation method and has been exten-
sively used for the modification of oligonucleotides and
DNA,[22–25] RNA,[26] proteins,[27,28] or carbohydrates.[29–31] The only
known example of an azido-linked Michael acceptor reagent,
N-3-(azidopropyl)maleimide, was recently described[32] for the
cross-linking of two proteins to create a lipase–bovine serum
Biorthogonal conjugation reactions are an indispensable tool
for the modification of biomolecules (i.e., proteins, nucleic
acids, lipids, or glycans).[1] Many of these bioconjugation reac-
tions can even be performed in living cells to monitor biologi-
cal processes in real time.[2] In particular, bioconjugation of nu-
cleic acids with peptides, proteins, or lipids has found many
applications in medicinal chemistry, chemical biology, and
nanotechnology.[3] These biomolecules are usually connected
to either the 3’- or the 5’-terminus of an oligodeoxynucleotide
(ON),[4] but it is highly desirable to attach them to nucleobases
to allow control of the position(s) and number of modifica-
tions. Recent advances in oligonucleotide synthesis (either
chemical phosphoramidite[5] or enzymatic synthesis by using
base-modified 2’-deoxynucleoside triphosphates[6] (dNTPs)) has
[a] J. Dadovµ, Dr. M. Vrµbel, Dr. R. Pohl, Prof. Dr. M. Hocek
Institute of Organic Chemistry and Biochemistry
Academy of Sciences of the Czech Republic
Gilead Sciences & IOCB Research Center
Flemingovo nµm. 2, 16610 Prague 6 (Czech Republic)
E-mail: hocek@uochb.cas.cz
[b] M. Adµmik, Dr. M. Brµzdovµ, Prof. Dr. M. Fojta
Institute of Biophysics
Academy of Sciences of the Czech Republic
Kralovopolska 135, 61265 Brno (Czech Republic)
[c] Prof. Dr. M. Hocek
Department of Organic Chemistry
Faculty of Science, Charles University in Prague
Hlavova 8, 12843 Prague 2 (Czech Republic)
[d] Prof. Dr. M. Fojta
Central European Institute of Technology
Masaryk University, Kamenice 753/5
625 00 Brno (Czech Republic)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201502209.
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albumin (BSA) heterodimer, but such a reagent would inher-
ently suffer from limited stability of the conjugate, which is
susceptible to hydrolysis.[33] Therefore, we designed N-(3-azido-
propyl)vinylsulfonamide (3) as a general bifunctional reagent
for the postsynthetic labeling of biomolecules with a reactive
VS group.
Results and Discussion
Compound 3 was synthesized in two steps starting from 1-
chloro-3-aminopropane (1; Scheme 1a). Nucleophilic substitu-
tion with sodium azide[34] followed by reaction with 2-chloroe-
thanesulfonyl chloride afforded the desired compound 3 in ac-
ceptable yield (34% over two steps). Then we intended to test
the reactivity of 3 as a new reagent for the CuAAC in a reaction
with alkyne-bearing DNA, which could be prepared either on
a solid support[8b,9,35] or by enzymatic incorporation of modi-
fied dNTPs.[36,37] Therefore, the study required the preparation
of 2’-deoxycytidine triphosphates that contained an alkyne
moiety at position 5 (dCETP and dCOTP) as substrates for the
polymerase synthesis of alkyne-modified DNA. They were pre-
pared from 5-iodo-2’-deoxycytidine (Scheme 1b) by Sonoga-
shira coupling with trimethylsilylacetylene (followed by depro-
tection) or octa-1,7-diyne in a modification of literature proto-
cols.[9,37,38] The reaction temperature for the coupling was low-
ered to 258C (in contrast to previously used[37] 558C for dCE);
this resulted in full conversion in 1 h (compared with previous-
ly published times of 3.5 and 24 h[37,38]) and a cleaner reaction
mixture. Both alkyne-modified nucleosides were ob-
tained in excellent yields (92% over two steps for
dCE and 70% for dCO). The triphosphorylation of dCE
and dCO with POCl3 in trimethylphosphate followed
by treatment with tributylammonium pyrophospha-
te[36a,37] gave the corresponding compounds dCETP
(45%) and dCOTP (56%).
Scheme 1. Syntheses of a) 3 and b) alkyne-modified 2’-deoxynucleosides
and nucleotides. Reagents and conditions: i) NaN3, H2O, 808C, 16 h; ii) 2-
chloroethanesulfonyl chloride, diisopropylethylamine (DIPEA), CH2Cl2, 08C,
1 h; iii) 1) trimethylsilylacetylene, [PdCl2(PPh3)2], CuI, trimethylamine, DMF,
258C, 1 h; 2) potassium fluoride, methanol/dioxane, 258C, 1.5 h; iv) octa-1,7-
diyne, [PdCl2(PPh3)2], CuI, trimethylamine, DMF, 258C, 1 h; v) POCl3,
PO(OMe)3, 08C, 1 h; vi) 1) POCl3, PO(OMe)3, 08C, 1 h; 2) (NHBu3)2H2P2O7, Bu3N,
DMF, 08C, 1 h; 3) tetraethylammonium bicarbonate (TEAB; 2m).
To test the bioconjugations, we also needed the
corresponding modified 2’-deoxycytidine monophos-
phates (dCEMP and dCOMP) as model compounds.
They were prepared by phosphorylation of dCE and
dCO in yields of 80 and 76%, respectively
(Scheme 1b). The model click reactions were then
performed with a twofold excess of bifunctional re-
agent 3 in the presence of copper(II) sulfate and
sodium ascorbate at room temperature (Scheme 2a).
In both cases, quantitative conversions to the target
Michael acceptor modified dCMPs were observed in
3 h. Final purification by reverse-phase HPLC afforded
dCEVSMP in good yield (86%). On the other hand, the
yield of pure dCOVSMP was lower (55%). Therefore,
compound dCEVSMP was used as a model substrate
for Michael additions of Cys and glutathione (GSH)
(Scheme 2b). The reactions were performed in trie-
thylammonium acetate (TEAA) buffer (pH 8.3) at
378C. In the case of Cys, the conjugation reaction
Scheme 2. a) Model click reaction of azide 3 to alkyne-modified dCMPs (dCEMP, dCOMP);
b) Michael addition of l-cysteine and GSH to dCEVSMP. Reagents and conditions: i) CuSO4,
sodium ascorbate, H2O/tBuOH, 258C, 3 h; ii) triethylammonium acetate (TEAA) buffer
was completed with 1.1 equivalents of Cys in 2 h,
whereas a larger excess of GSH (3 equiv) was neces-
sary to reach 90% conversion after overnight incuba- (0.3m, pH 8.3), 378C.
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tion. The products dCEVSMP_Cys (50%) and dCEVSMP_GSH
(42%) were purified by reverse-phase HPLC. In the case of
dCEVSMP_GSH, product of sulfonamide hydrolysis (dCENH2MP,
11 %) was isolated as a byproduct.
isolation of the corresponding single-stranded ON (ssON)
ON1XVS by magnetoseparation[39] for MALDI-TOF analysis. The
PEX products were purified by using a QIAquick nucleotide re-
moval kit (QIAGEN) to remove unincorporated dNTPs. At first,
the click reaction of DNA1X with azide 3 was performed under
commonly used conditions,[35] that is, in water/DMSO/tBuOH,
with CuBr as a source of CuI, TBTA as a CuI-stabilizing ligand,[40]
and sodium ascorbate, which was proven to have significant
effect to achieve the best re-
Alkyne-modified DNA was prepared by enzymatic incorpora-
tion of dCXTPs into DNA in a primer extension experiment
(PEX; Scheme 3). Both dCETP and dCOTP were described to be
good substrates for various DNA polymerases (e.g., Vent(exo-),
sults[25] (Scheme 3). MALDI-TOF
analysis confirmed full conver-
sion to ON1XVS, but the product
was always accompanied by cer-
tain
amount
of
ON1XNH2
(Scheme 3) as a product of par-
tial hydrolysis of sulfonamide
(for copies of the MALDI-TOF
mass spectra, see Figures S14
and S15 in the Supporting Infor-
mation). To evaluate whether the
product was hydrolyzed during
the click reaction or in the mass
spectrometer, VS-modified cyti-
dine triphosphate, dCEVSTP, was
prepared through the click reac-
tion of dCETP with
me S3b in the Supporting Infor-
mation). After incorporation of
dCEVSTP into DNA by PEX with
3 (Sche-
Scheme 3. Enzymatic synthesis of alkyne-bearing DNA (DNAX) followed by the click reaction of azide 3 and mag-
netoseparation. Reagents and conditions: i) CuBr/tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA; 1:5),
sodium ascorbate, 378C, 1 h.
Taq, Pwo, KOD XL) in both PEX and PCR.[36a,37] For this study,
three DNA polymerases were selected (KOD XL, Vent(exo-), and
Pwo) and the incorporation was monitored by denaturing
polyacrylamide gel electrophoresis (PAGE; Figure S1 in the
Supporting Information). All sequences of primer and tem-
plates used in this study are listed in Table 1.
a biotinylated template and subsequent magnetoseparation,
the resulting ON1EVS was analyzed by MALDI-TOF MS (for
a copy of the spectrum, see Figure S18 in the Supporting Infor-
mation). No signal of the hydrolyzed product (ON1ENH2) was ob-
served, which indicated that partial decomposition occurred
during the click reaction.
KOD XL polymerase gave clean full-length DNA products
that contained either one or two modifications (DNA1X,
DNA2X), which were characterized by MALDI-TOF MS (Table S2
and Figures S10–S13 in the Supporting Information). The modi-
fied DNA1E and DNA1O were then prepared in a larger amount
(3 nmol) by PEX with biotinylated template to obtain enough
material for the subsequent click reaction, denaturation, and
Therefore, the conditions of CuAAC were further optimized
(all examined conditions are summarized in Table S4 in the
Supporting Information). The ratio of DNA to azide 3, and CuI
to ligand (either commercially available TBTA or next genera-
tion, water-soluble bis[(tert-butyltriazoyl)methyl][(2-carboxyme-
thyltriazoyl)methyl]amine (BTTAA);[41] for structures, see
Scheme S1 in the Supporting Information), were varied for
both DNA1E and DNA1O. In parallel, the influence of reaction
time, temperature, and solvent effect were tested (water or
phosphate-buffered saline (PBS), pH 7.4 were used instead of
a mixture of water/DMSO/tBuOH).[12b,25] Unfortunately, the for-
mation of ON1XNH2 byproduct was observed in all cases (Table
S4 in the Supporting Information). Based on all of these obser-
vations, all further click reactions were performed with 5 mm
DNA1E, an excess of 3 (400 equiv), CuBr/TBTA (1:5, 4 equiv),
and sodium ascorbate (2 equiv) in water/DMSO/tBuOH (9:3:1)
at 378C for 1 h; under these conditions, the hydrolysis byprod-
ucts were formed in an apparently lowest ratio (as determined
by MALDI-TOF MS).
Table 1. List of oligonucleotides used or synthesized for this study.
Oligonucleotide
Sequence[a]
prim
5’-TCAAGAGACATGCCT-3’
temp1C
temp2C
ON1X
5’-ATAATAAACATGTCTAGGCATGTCTCTTGA-3’
5’-ATAATAGACATGTCTAGGCATGTCTCTTGA-3’
5’-TCAAGAGACATGCCTAGACATGTTTATTAT-3’
5’-TCAAGAGACATGCCTAGACATGTCTATTAT-3’
3’-AGTTCTCTGTACGGATCTGTACAAATAATA-5’
5’-TCAAGAGACATGCCTAGACATGTTTATTAT-3’
3’-AGTTCTCTGTACGGATCTGTACAGATAATA-5’
5’-TCAAGAGACATGCCTAGACATGTCTATTAT-3’
ON2X
DNA1X
DNA2X
Because mass spectrometry is not a quantitative method,
we decided to quantify sulfonamide hydrolysis during the click
reaction on ethynyl-modified DNA by using HPLC. To achieve
[a] Italics: p53 recognition sequence. Bold: nucleotides containing modifi-
cation.
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good separation of all three potential click reaction mixture
components (ONE, ONEVS, and ONENH2), it was necessary to use
short (ca. 10–12 nucleotides (nt)) starting ssONs (ONE). The
nicking enzyme amplification reaction (NEAR) was used for
their preparation (Scheme S5 in the Supporting Information).
This method was recently developed[42] for the enzymatic syn-
thesis of larger amounts (up to 10 nmol) of short base-modi-
fied ssONs (10–22 nt). It has been already successfully applied
for the preparation of alkyne-modified[25] and fluorescent[43] pri-
mers for the polymerase chain reaction (PCR). The template for
NEAR was designed to obtain the 10 nt ssON containing one
modified cytidine (for sequences of both primer and template,
see Table S1 in the Supporting Information). Then NEAR was
conducted on a semipreparative scale (750 mL) in the presence
of Vent(exo-) DNA polymerase and Nt.BstNBI nicking enzyme
with dCETP, dCENH2TP (for preparation, see Scheme S6 in the
Supporting Information), or dCEVSTP to obtain ON standards
for the product of both the click reaction, ONnickEVS, and hy-
drolysis, ONnickENH2 (for copies of MALDI-TOF mass spectra,
see Figures S21–23 in the Supporting Information). The prod-
uct ONnickE was then used as a model for the click reaction
with 3 under standard conditions. The products were analyzed
by HPLC and compared with the HPLC chromatogram of all
three authentic standards (Figures S4 and S5 in the Supporting
Information). The amount of hydrolyzed byproduct (ON-
nickENH2) was determined to be about 12% based on signal in-
tegral intensity. The formation of hydrolyzed byproduct could,
in principle, be overcome by using a copper-free click reaction,
for example, stain-promoted azide–alkyne cycloaddition
(SPAAC),[2a,44] for DNA modification with bifunctional reagent 3.
Recently, DNA containing cyclooctyne modification was pre-
pared both by enzymatic synthesis[8c] and on a solid sup-
port.[8d]
A double-stranded DNA1EVS containing Michael acceptor
label was then prepared by the click reaction of DNA1E with 3
under the above-described conditions and reacted with Cys or
model Cys-containing undecapeptide (Scheme 4a). After mag-
netoseparation, the desired conjugates ON1EVS_Cys and
ON1EVS_pept were successfully characterized by MALDI-TOF MS
(for copies of spectra, see Figure S16 and S17 in the Support-
ing Information). Analogously to our previous study,[20] we
tested DNA2XVS prepared by the click reaction of either ethynyl-
or octadiynyl-modified DNA with 3 in conjugation with the
GST-tagged core domain of tumor suppressor p53 (GSTp53CD;
Scheme 4b–d).[45] Protein p53 in a complex with its target DNA
contains two free Cys residues in close proximity to the oligo-
nucleotide.[46] Cys 277 can be selectively trapped by Michael
addition to VS-bearing DNA when the modification is connect-
ed to the cytosine through a propargyl linker (DNA2VS).[20] The
DNA probe was designed to contain two modifications in the
p53 binding sequence[47] (Table 1). GSTp53CD was expressed
and purified according to a literature protocol.[48]
Scheme 4. Conjugation of DNA containing Michael acceptor modification
with a) l-cysteine and model undecapeptide; b–d) GST-tagged core domain
of p53 (GSTp53CD). Reagents and conditions: i) TEAA buffer (0.3m, pH 8.3),
378C; ii) Tris buffer (pH 7.6), 08C, 30 min, and then 258C, 2 h. (Protein repre-
sentations generated from PDB 3Q05, ref. [46a].)
ty of p53 protein to recognize modified DNA was monitored
by 5% native PAGE (Figure 1a), which confirmed that none of
the modifications prevented p53 binding (lanes 3–7). Denatur-
ing SDS-PAGE was then performed to investigate whether co-
valent DNA–protein cross-links had been formed (Figure 1b).
The bands with lower mobility confirmed the formation of co-
valent conjugates (Figure 1b, lanes 3, 5, and 7). The reactivity
of new DNA probes prepared by CuAAC (Figure 1b, lanes 5
and 7) was slightly lower than that of the previously used[20]
DNA2VS (Figure 1b, lane 3), which was probably caused by
longer linkers in the clicked adduct (the CVS was designed[20]
based on the crystal structure of DNA-p53 complex). Probe
Then six different DNA probes were prepared: natural DNA;
DNA containing alkyne modification (DNA2E, DNA2O); DNA con-
taining a reactive handle with different linkers, that is, DNA2VS
(ref. [20]; Scheme 4d), DNA2EVS (Scheme 4b), and DNA2OVS
(Scheme 4c), which were incubated with GSTp53CD. The abili-
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Figure 1. Gel analysis of GSTp53CD binding to various DNA2X. a) Native
PAGE; b) denaturing sodium dodecyl sulfate (SDS) PAGE. Lane: 1) Natural
DNA, 2) natural DNA+GSTp53CD, 3) DNA2VS +GSTp53CD,
4) DNA2E +GSTp53CD, 5) DNA2EVS +GSTp53CD, 6) DNA2O +GSTp53CD,
7) DNA2OVS +GSTp53CD.
DNA2EVS was selected as a candidate suitable for trapping p53
from cellular lysates.
Recently, the group of Davis described a method for the
analysis of cellular DNA-binding proteins with respect to the
presence of C or 5-hydroxymethyl-C when using diazirine-con-
taining reactive DNA probes.[19] These probes were incubated
with nuclear extract of HeLa cells and nonspecifically photo-
cross-linked to DNA-binding proteins. Herein, we aimed to de-
velop a complementary method, in which only proteins that
bound to DNA and contained a free Cys side chain in close
proximity to the Michael acceptor modification were trapped.
We selected three different human nonsmall lung carcinoma
cell lines, p53-deficient H1299, and derived stable Tet-inducible
cell lines for the expression of wild-type p53 (p53wt) or p53
mutant R273H, which does not specifically bind to DNA.[49]
Total cell lysates were incubated with 5’-FAM-modified DNA2EVS
(Figure 2a; FAM=fluorescein) and the formation of DNA–pro-
tein cross-links was monitored by denaturing PAGE. Fluores-
cence scanning by Typhoon FLA 9500 (Figure 2b) proved the
conjugation of many various cellular proteins from all cell lines
to our reactive DNA2EVS probe (Figure 2b, lanes 3, 6, and 9).
This opens the door to many applications of these probes in
proteomics or in the synthesis of irreversible inhibitors of some
DNA-processing enzymes. On the other hand, western blotting
followed by immunodetection of p53 (Figure 2c) confirmed
the DNA2EVS_p53 conjugate only in cells with induced p53wt
(lane 6). Mutant R273H is not trapped by DNA2EVS (Figure 2c,
lane 9), since the proximity effect is crucial for cross-linking.[20]
The new bifunctional reagent 3 could, in principle, be used
for the introduction of the Michael acceptor moiety to any
other alkyne-containing biomolecule. To verify its potential in
protein modification, human carbonic anhydrase II (hCA)-con-
taining terminal acetylene in a specific position was selected
as a model protein.[50] In the past few years, several methods
have been developed to introduce unnatural amino acids
(UAA) into proteins.[51] One of the most powerful approaches
for incorporating UAA site-specifically into proteins expressed
in cells is genetic code expansion.[52] A wide range of amino
Figure 2. a) Conjugation of p53 from human cancer cells by using reactive
DNA2EVS probe; b) fluorescence (5’-FAM-labeled DNA) scanning of SDS-PAGE
gel; c) western blotting of SDS-PAGE by using anti-p53 antibody. Human
nonsmall lung carcinoma cell line H1299 (lanes 2–4): p53 null; Hwt (lanes 5–
7): wild-type p53; H273 (lanes 8–10): p53 mutant R273H. Lane L: protein
ladder; 1, 3, 6, 9: DNA2EVS; 2, 5, 8: no DNA; 4, 7, 10: natural DNA. Conditions:
cell lysate (total protein amount of 2.5 mg/mL), DNA (2.5 ng/mL), Tris buffer
(pH 7.6), 08C, 30 min, and then 258C, 2 h.
acids can be inserted into proteins by using this methodology,
which enables the expansion of chemical biology.[53] Herein,
the noncanonical amino acid containing alkyne in its side
chain, that is, N6-[(prop-2-yn-1-yloxy)carbonyl]-l-lysine (4), was
introduced into position 131 of hCA by following the proce-
dure described by Carell and co-workers.[50] The modified hCA
(hCA*) was expressed in Escherichia coli by using the amber
codon suppression and orthogonal pyrrolysyl-tRNA/pyrrolysyl-
tRNA synthetase system (Figure 3).[54,55] The modified amino
acid 4 was prepared according to a previously published pro-
cedure from a-Boc-protected lysine (Boc=tert-butyloxycarbon-
yl; Scheme S2 in the Supporting Information).[56] The structure
of the modified protein was confirmed by measuring MALDI-
TOF mass spectra (m/z calcd for [M+H]+: 29236 Da; found:
29238 Da; Figure S27 in the Supporting Information). The
CuAAC reaction with hCA* was first optimized by using azido-
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Figure 3. a) Preparation of hCA containing alkyne-modified amino acid 4 at position 131 (hCA*) followed by the click reaction with coumarin azide 5 or new
bifunctional reagent 3. b) MALDI-TOF mass spectrum of hCA*VS. c) MALDI-TOF mass spectrum of hCA*_coum; SDS-PAGE gel analysis of reaction of hCA* with
coumarin 5 visualized by both standard Coomassie staining and fluorescence scanning. Conditions: i) CuSO4/3-[4-({bis[(1-tert-butyl-1H-1,2,3-triazol-4-yl)methy-
l]amino}methyl)-1H-1,2,3-triazol-1-yl]propanol (BTTP; 1:2), sodium ascorbate, 308C, 1 h. (Protein representations generated from PDB 2VVB.[58]
)
coumarin[57] 5 as a substrate (Figure 3). The resulting triazolyl–
coumarin is fluorescent, and therefore, the formation of hCA*_
coum can be easily monitored by in-gel fluorescence analysis.
The click reaction with azide 5 was performed under the opti-
mized conditions (for details, see Figures S6–S9 in the Support-
ing Information), that is, with copper(II) sulfate and BTTP[31]
ligand (1:2) in the presence of sodium ascorbate at 308C for
1 h. The obtained hCA*_coum was characterized by measuring
MALDI-TOF mass spectra and gel electrophoresis with fluores-
cent scanning (Figure 3c). Finally, the above-described condi-
tions for the click reaction on protein hCA* were applied to
the reaction with bifunctional azide 3. The reaction proceeded
smoothly and the structure of target protein hCA*VS containing
the Michael acceptor handle was proven by mass spectrometry
(Figure 3b).
Reactive hCA*VS was then incubated with GSH or a model
undecapeptide at 378C in TEAA buffer (pH 8.3; Figure 4). When
GSH was used, complete conversion to Michael adduct
hCA*VS_GSH was observed and confirmed by mass spectrome-
try (Figure 4). On the other hand, the longer peptide did not
give full conversion to the conjugate hCA*VS_pept, which was
accompanied by a residual amount of starting hCA*VS even
after overnight incubation (Figure 4).
Figure 4. a) Conjugation of hCA containing Michael acceptor modification
(hCA*VS) with GSH and model undecapeptide. b) MALDI-TOF mass spectrum
of hCA*_GSH. c) MALDI-TOF mass spectrum of hCA*_pept. Conditions:
i) TEAA buffer (0.3M, pH 8.3), 378C, overnight.
Conclusion
cules to attach VS as a Michael acceptor. Because alkyne (eth-
ynyl or octadiynyl)-linked ONs and/or DNA are easily accessible
and even commercially available, this postsynthetic modifica-
tion is significantly more practical than the direct synthesis of
ONs/DNA with reactive nucleotides. Also, the expression of
alkyne-modified proteins is now a routine procedure and their
conversion into a Michael acceptor handle is simple and
We developed N-(3-azidopropyl)vinylsulfonamide 3 as a new
bifunctional bioconjugation reagent. It could be clicked to an
alkyne-modified DNA or protein by the CuAAC reaction. Al-
though we showed that partial hydrolysis (ca. 12%) of the sul-
fonamide occurred in the click modification of DNA, it could
still be used for efficient biorthogonal modification of biomole-
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dure reported in the literature.[48] 1-Amino-3-azidopropane (2),[34]
dCE,[37,38] dCO,[9] dCETP,[37] and dCOTP,[36a] were synthesized according
to procedures reported in the literature, as described in the Sup-
porting Information.
useful. The VS-linked DNA or protein reacts specifically with
Cys-containing peptides and/or proteins to form stable cova-
lent cross-links. We believe that this approach will also work
for any other combination of two biomolecules when one con-
tains an alkyne and the other one a thiol. Clearly, there is great
potential in pull-down experiments for the identification and
isolation of DNA-binding proteins containing Cys near to rec-
ognition sequences. Studies in this direction are under way in
our laboratories.
N-(3-Azidopropyl)vinylsulfonamide (3)
1-Amino-3-azidopropane (2, 250 mg, 2.50 mmol) and N,N-diisopro-
pylethylamine (1 mL, 6.25 mmol) were dissolved in dry dichlorome-
thane (7 mL) on ice under an argon atmosphere. 2-Chloroethane-
sulfonyl chloride (0.25 mL, 2.50 mmol) was added dropwise and
the resulting mixture was stirred at 08C for 1 h. The reaction was
quenched by the addition of brine (5 mL). The organic phase was
washed with brine (10 mL) and water (10 mL) and then dried over
MgSO4. The crude product was purified by column chromatogra-
phy (hexane/ethyl acetate, 1:1) to give 3 as a colorless oil (187 mg,
Experimental Section
General
1
NMR spectra were recorded on a 600 (600.1 MHz for H, 150.9 MHz
1
1
for 13C), 500 (499.8 or 500.0 MHz for H, 202.3 or 202.4 MHz for 31P,
40%). The product was photolabile and stored in the dark. H NMR
1
125.7 MHz for 13C), or 400 MHz (400.0 MHz for H, 162 MHz for 31P,
(400 MHz, CD3OD): d=1.77 (p, 2H, J=6.7 Hz), 3.02 (t, 2H, J=
6.7 Hz), 3.41 (t, 2H, J=6.6 Hz), 5.97 (d, 1H, J=10.0 Hz), 6.14 (d, 1H,
J=16.6 Hz), 6.63 ppm (dd, 1H, J1 =16.5, J2 =10.0 Hz); 13C NMR
(100 MHz, CD3OD): d=30.4, 41.0, 49.6, 126.5, 137.6 ppm; MS (ESI+):
m/z (%): 213.0 (30) [M+Na]+, 403.1 (100) [2M+Na]+, 593.2 (25)
[3M+Na]+; HRMS (ESI+): m/z calcd for C5H10O2N4NaS [M+Na]+:
213.04167; found: 213.04143.
100 MHz for 13C) spectrometer from solutions in D2O, CDCl3, or
CD3OD. Chemical shifts (in ppm, d scale) were referenced as fol-
lows: D2O (referenced to dioxane as an internal standard: d=
3.75 ppm for 1H NMR spectroscopy and d=69.30 ppm 13C NMR
spectroscopy); CD3OD (referenced to solvent signal: d=3.31 ppm
1
for H NMR spectroscopy and d=49.00 ppm for 13C NMR spectros-
copy), CDCl3 (referenced to solvent signal: d=7.26 ppm for
1H NMR spectroscopy and d=77.16 ppm for 13C NMR spectrosco-
py). Coupling constants (J) are given in Hz. Complete assignment
of all NMR spectroscopy signals was achieved by using a combina-
tion of H,H-COSY, H,C-HSQC, and H,C-HMBC experiments. Mass
spectra and high-resolution mass spectra were measured by using
the ESI ionization technique. Chemicals were purchased from
Sigma-Aldrich, Stem Chemicals, Berry&Associates, or Acros Organ-
ics and used without further purification. The water used in syn-
thetic procedures was of HPLC quality; in biochemical experiments
of ultrapure quality (18 MWcm). The MALDI-TOF spectra were mea-
sured on an UltrafleXtreme MALDI-TOF/TOF mass spectrometer
(Bruker Daltonics, Germany) with a 1 kHz smartbeam II laser. The
measurements were done in reflectron mode by means of the
droplet technique, with a mass range up to 30 kDa. The matrix
consisted of 3-hydroxypicolinic acid (HPA)/picolinic acid (PA)/am-
monium tartrate in a ratio of 9/1/1. The matrix (1 mL) was applied
on the target (ground steel) and dried at room temperature. The
sample (1 mL) and matrix (1 mL) were mixed and added on the top
of the dried matrix preparation spot and dried at room tempera-
ture. UV/Vis spectra were measured on a NanoDrop1000 (Thermo-
Scientific) spectrometer at room temperature. Samples were con-
centrated on a CentriVap Vacuum Concentrator system (Labconco).
Synthetic oligonucleotides (primers, templates, and biotinylated
templates; for sequences see Table S1 in the Supporting Informa-
tion) were purchased from Generi Biotech. Natural nucleoside tri-
phosphates (dATP, dGTP, dTTP, dCTP), Vent(exo-) DNA polymerase,
and nickase Nt.BstNBI were purchased from New England Biolabs;
KOD XL DNA polymerase from Merck; streptavidine magnetic parti-
cles from Roche; QIAquick nucleotide removal kit from Qiagen;
and BioTrace NT nitrocellulose transfer membrane from Pall. Mono-
clonal mouse anti-p53 antibody DO-1 was kindly provided by Dr.
Vojtesek (Masaryk Memorial Cancer Institute, Brno, Czech Republic),
antimouse IgG horseradish peroxidase (HRP)-linked antibody was
purchased from Sigma-Aldrich. Amersham ECL western blotting
detection reagent was purchased from GE Healthcare. Syntheses
and characterization data for 3-azido-7-hydroxycoumarin,[57] 4,[56]
BTTAA,[41] BTTP,[31] and dCVSTP[20] were reported previously. GSTp53_
CD was expressed in E. coli cells and purified according to a proce-
General procedure A: Preparation of alkyne-modified 2’-de-
oxycytidine 5’-O-phosphates (dCXMP)
Compound dCX was dried at 808C for 2 h in vacuo. After cooling,
dry PO(OMe)3 and POCl3 (1.2 equiv) were added and the reaction
mixture was stirred under an argon atmosphere for 1 h on ice.
Phosphorylation was stopped by the addition of TEAB (2m, 1 mL)
and water (2 mL). After purification, several codistillations with
water and conversion to sodium salt (Dowex 50WX8 in Na+ cycle),
followed by freeze-drying from water, gave the desired dCXMP as
a white powder.
5-Ethynyl-2’-deoxycytidine 5’-O-phosphate (dCEMP): Compound
dCEMP was prepared according to general procedure A from dCE
(100 mg, 0.398 mmol) and POCl3 (45 mL, 0.478 mmol) in PO(OMe)3
(1 mL). After purification by C18 reverse-phase HPLC with water/
methanol (5 to 100%) containing 0.1m TEAB buffer as the eluent,
product dCEMP was isolated in 80% yield (107 mg). 1H NMR
(400 MHz, D2O): d=2.28 (m, 1H), 2.41 (m, 1H), 3.76 (s, 1H), 3.98
(dd, 2H, J1 =5.3, J2 =4.2 Hz), 4.16 (m, 1H), 4.50 (m, 1H), 6.23 (t, 1H,
J=6.6 Hz), 8.20 ppm (s, 1H); 31P{1H} NMR (162 MHz, D2O): d=
2.11 ppm; MS (ESIÀ): m/z (%): 331.1 (100) [M+H]À, 353.1 (10) [M+
Na]À; HRMS (ESIÀ): m/z calcd for C11H13O7N3P [M+H]À: 330.04966;
found: 330.04877.
5-(Octa-1,7-diynyl)-2’-deoxycytidine 5’-O-phosphate (dCOMP):
Compound dCOMP was prepared according to general procedur-
e A from dCO (150 mg, 0.453 mmol) and POCl3 (55 mL, 0.550 mmol)
in PO(OMe)3 (1.5 mL). After purification on a DAE Sephadex
column (water/2m TEAB gradient 0–60%), product dCOMP was iso-
lated in 76% yield (170 mg, contained 30 mol% of inorganic
PO43À). 1H NMR (400 MHz, D2O): d=1.57–1.79 (m, 4H), 2.19–2.35
(m, 3H), 2.37 (m, 1H), 2.39–2.43 (m, 3H), 4.00–4.10 (m, 2H), 4.19
(m, 1H), 4.48–4.60 (m, 1H), 6.26 (t, 1H, J=6.7 Hz), 8.03 ppm (s,
1H); 31P{1H} NMR (162 MHz, D2O): d=3.36 ppm; MS (ESIÀ): m/z (%):
410.1 (100) [M+H]À; HRMS (ESIÀ): m/z calcd for C17H21O7N3P [M+
H]À: 410.11226; found: 410.11191.
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dCEVSMP_Cys: Compound dCEVSMP_Cys was prepared according to
general procedure C by stirring dCEVSMP (10.0 mg, 19 mmol) with l-
cysteine (2.6 mg, 21.2 mmol) for 2 h; full conversion was confirmed
by TLC (iPrOH/NH4OH/H2O, 11:7:2). The product was isolated as
a white powder (6 mg, 50%). 1H NMR (600.1 MHz, D2O): d=2.24
(brquin, J2’“,1’” =J2’“,3’” =6.4 Hz, 2H; H-2’“), 2.39 (brddd, Jgem =13.9,
General procedure B: Copper-catalyzed click reactions to
dCXMP
Alkyne-modified nucleotide, azido derivative (2 equiv), sodium as-
corbate (0.4 equiv), and copper(II) sulfate pentahydrate (0.1 equiv)
were dissolved in a mixture of water/tert-butanol (1:1, 0.5 mL). The
reaction mixture was stirred at room temperature for 3 h (complete
consumption of the starting material was proved by TLC with
propan-2-ol/water/aqueous ammonia (11:2:7) as the eluent). The
products were purified by C18 reverse-phase HPLC with a mixture
of water/methanol (5 to 100%) containing 0.1m TEAB buffer as
eluent. Several codistillations with water and conversion to the
sodium salt (Dowex 50WX8 in Na+ cycle), followed by freeze-
drying from water, gave the desired click reaction products.
J
2’b,1’ =6.5, J2’b,3’ =6.0 Hz, 1H; H-2’b), 2.43 (ddd, Jgem =13.9, J2’a,1’ =
5.9, J2’a,3’ =3.2 Hz, 1H; H-2’a), 2.96 (brm, 3H; H-2”“, 3Cb), 3.08 (brm,
1H; H-3Ca), 3.15 (t, J3’”,2’“ =6.4 Hz, 2H; H-3’”), 3.48 (brm, 2H; H-1“”),
3.91 (brm, 1H; H-2C), 3.98 (brt, JH,P =J5’,4’ =4.4 Hz, 2H; H-5’), 4.21
(brtd, J4’,5’ =4.4, J4’,3’ =3.2 Hz, 1H; H-4’), 4.58 (dt, J3’,2’ =6.0, 3.2,
J
J
3’,4’ =3.2 Hz, 1H; H-3’), 4.61 (t, J1’“,2’” =6.4 Hz, 2H; H-1’“), 6.35 (dd,
1’,2’ =6.5, 5.9 Hz, 1H; H-1’), 8.29 (s, 1H; H-6), 8.71 ppm (s, 1H; H-
5”); 13C NMR (125.7 MHz, D2O): d=27.52 (CH2-2“”), 32.32 (CH2-2’“),
35.35 (CH2-3C), 42.34 (CH2-3’”), 42.38 (CH2-2’), 50.55 (CH2-1’“), 53.66
(CH2-1”“), 56.35 (CH-2C), 66.40 (d, JC,P =4.3 Hz; CH2-5’), 73.99 (CH-
3’), 89.08 (CH-1’), 89.24 (d, JC,P =8.5 Hz; CH-4’), 101.98 (C-5), 126.53
(CH-5”), 143.05 (CH-6), 143.92 (C-4“), 159.20 (C-2), 165.99 ppm (C-4),
(C-1C not detected); 31P{1H} NMR (202.4 MHz, D2O): d=3.89 ppm;
MS (ESIÀ): m/z (%): 641.1 (100) [M+H]À, 520.1 (35) [M+HÀCys]À,
663.1 (30) [M+Na]À, 685.1 (10) [M+2Na]À; HRMS (ESIÀ): m/z calcd
for C19H30O11N8PS2 [M+H]À: 641.12185; found: 641.12146.
dCEVSMP: According to general procedure B, dCEVSMP was pre-
pared from dCEMP (19.0 mg, 53 mmol), 3 (20.0 mg, 105 mmol),
sodium ascorbate (4.2 mg, 21 mmol) and copper(II) sulfate pentahy-
drate (1.3 mg, 5 mmol) as white solid (24 mg, 86%). 1H NMR
(500.0 MHz, D2O): d=2.22 (quin, J2’“,1’” =J2’“,3’” =6.6 Hz, 2H; H-2’“),
2.39 (ddd, Jgem =14.0, J2’b,1’ =7.4, J2’b,3’ =6.1 Hz, 1H; H-2’b), 2.45
(ddd, Jgem =14.0, J2’a,1’ =6.3, J2’a,3’ =3.4 Hz, 1H; H-2’a), 3.02 (t, J3’”,2’“
=
6.6 Hz, 2H; H-3’”), 3.97 (dd, JH,P =5.1, J5’,4’ =3.8 Hz, 2H; H-5’), 4.21
(qd, J4’,3’ =J4’,5’ =3.8, JH,P =1.1 Hz, 1H; H-4’), 4.58 (m, 1H; H-3’), 4.60
(t, J1’“,2’” =6.6 Hz, 2H; H-1’“), 6.09 (dd, J2”“b,1”“ =10.1, Jgem =0.7 Hz, 1H;
H-2”“b), 6.19 (dd, J2”“a,1”“ =16.5, Jgem =0. Hz, 1H; H-2”“a), 6.36 (dd,
dCEVSMP_GSH: Compound dCEVSMP_GSH was prepared according
to general procedure C by stirring of dCEVSMP (15.0 mg, 29 mmol)
with GSH (26.6 mg, 86.7 mmol) overnight; conversion of around
90% was confirmed by TLC (iPrOH/NH4OH/H2O, 11:7:2). Product
dCEVSMP_GSH was isolated as a white powder (10 mg, 42%) and
compound dCENH2MP was obtained as a byproduct (3 mg, 11 %).
J
1’,2’ =7.4, 6.1 Hz, 1H; H-1’), 6.66 (dd, J1”“,2”“ =16.5, 10.1 Hz, 1H; H-
1”“), 8.30 (s, 1H; H-6), 8.72 ppm (s, 1H; H-5”); 13C NMR (125.7 MHz,
D2O): d=32.04 (CH2-2’“), 42.29 (CH2-3’”), 42.38 (CH2-2’), 50.62 (CH2-
1’“), 66.34 (d, JC,P =4.5, CH2-5’), 74.04 (CH-3’), 89.08 (CH-1’), 89.31 (d,
1
Spectral data for dCEVSMP_GSH: H NMR (500.0 MHz, D2O): d=2.07
J
C,P =8.4, CH-4’), 101.99 (C-5), 126.52 (CH-5”), 131.05 (CH2-2“”),
(m, 2H; H-3E), 2.24 (brquin, J2’“,1’” =J2’“,3’” =6.5 Hz, 2H; H-2’“), 2.32
(brddd, Jgem =14.0, J2’b,1’ =7.4, J2’b,3’ =6.0 Hz, 1H; H-2’b), 2.44 (ddd,
136.75 (CH-1“”), 143.06 (CH-6), 143.93 (C-4“), 159.23 (C-2),
165.99 ppm (C-4); 31P{1H} NMR (202.4 MHz, D2O): d=3.91 ppm; MS
(ESIÀ): m/z (%): 520.3 (100) [M+H]À, 542.1 (60) [M+Na]À, 564.1
(25) [M+2Na]À; HRMS (ESIÀ): m/z calcd for C16H23O9N7PS [M+H]À:
520.10211; found: 520.10193.
J
gem =14.0, J2’a,1’ =6.2, J2’a,3’ =3.4 Hz, 1H; H-2’a), 2.44 (m, 2H; H-4E),
2.82 (dd, Jgem =14.1, J3b,2 =8.9 Hz, 1H; H-3bC), 2.84 (m, 2H; H-2”“),
3.03 (m, 1H; H-3Ca), 3.07 (t, J3’”,2’“ =6.5 Hz, 2H; H-3’”), 3.37 (m, 2H;
H-1“”), 3.68 (m, 3H; H-2E, 2G), 3.90 (dd, JH,P =5.2, J5’,4’ =3.9 Hz, 2H;
H-5’), 4.13 (tdd, J4’,5’ =3.9, J4’,3’ =3.2, JH,P =1.2 Hz, 1H; H-4’), 4.47–
4.55 (m, 4H; H-3’,1’“,2C), 6.27 (dd, J1’,2’ =7.4, 6.2 Hz, 1H; H-1’), 8.21
(s, 1H; H-6), 8.62 ppm (s, 1H; H-5”); 13C NMR (125.7 MHz, D2O): d=
27.5 (CH2-2“”), 29.0 (CH2-3E), 32.3 (CH2-2’“), 34.1 (CH2-4E), 35.9 (CH2-
3C), 42.4, 42.4 (CH2-2’,3’”), 46.1 (CH2-2G), 50.6 (CH2-1’“), 53.8 (CH2-
1”“), 55.7 (CH-2C), 56.9 (CH-2E), 66.5 (d, JC,P =4.7 Hz; CH2-5’), 74.0
(CH-3’), 89.1 (CH-1’), 89.2 (d, JC,P =8.2 Hz; CH-4’), 102.0 (C-5), 126.5
(CH-5”), 143.1 (CH-6), 143.9 (C-4“), 159.3 (C-2), 166.1 (C-4), 174.5 (C-
1C), 176.9 (C-1E), 177.7 (C-5E), 179.0 ppm (C-1G); 31P{1H} NMR
(202.4 MHz, D2O): d=3.68 ppm; MS (ESIÀ): m/z (%): 520.3 (100)
[MÀGSH+H]À, 554.1 (98) [MÀGSH+SH+H]À, 871.1 (80) [M+
2Na]À, 827.2 (70) [M+H]À, 849.2 (50) [M+Na]À, 542.1 (40)
[MÀGSH+Na]À; HRMS (ESIÀ): m/z calcd for C26H40O15N10PS2 [M+
H]À: 827.18591; found: 827.18469.
dCOVSMP: According to general procedure B, compound dCOVSMP
was prepared from dCOMP (50.0 mg, 151 mmol),
3 (57.4 mg,
302 mmol), sodium ascorbate (12.0 mg, 60 mmol), and copper(II) sul-
fate pentahydrate (3.8 mg, 15 mmol) as a white solid (43 mg, 55%).
1H NMR (400.0 MHz, D2O): d=1.62 (m, 2H), 1.78 (m, 2H), 2.12 (m,
2H), 2.29 (m, 1H), 2.48 (m, 3H), 2.76 (t, J=7.2 Hz, 2H), 2.90 (t, J=
6.7 Hz, 1H), 3.09 (s, 1H), 3.99 (m, 2H), 4.16 (s, 1H), 4.59–4.42 (m,
3H), 6.09 (m, 1H), 6.25 (m, 1H), 6.60 (dd, J=16.5, 10.1 Hz, 1H), 7.81
(s, 1H), 7.98 ppm (s, 1H); 13C NMR (100 MHz, D2O): d=18.5, 23.9,
26.9, 27.9, 29.2, 39.1, 39.3, 47.2, 64.0, 70.5, 71.0, 85.8, 86.2, 93.3,
97.9, 123.5, 128.4, 133.9, 143.4, 148.3, 156.0, 165.2 ppm;
31P{1H} NMR (161 MHz, D2O): d=5.35 ppm; MS (ESIÀ): m/z (%):
600.2 (100) [M+H]À; HRMS (ESIÀ): m/z calcd for C22H31O9N7PS [M+
H]À: 600.16471; found: 600.16437.
Spectral data for dCENH2MP: 1H NMR (600.1 MHz, D2O): d=2.35–
2.49 (m, 4H; H-2’,2’“), 2.92 and 2.97 (2”m, 2”1H; H-3’”), 3.99 (ddd,
General procedure C: Michael addition of Cys and GSH to
dCEVSMP
J
J
gem =11.8, JH,P =5.5, J5’b,4’ =3.3 Hz, 1H; H-5’b), 4.03 (ddd, Jgem =11.8,
H,P =3.6, J5’a,4’ =2.6 Hz, 1H; H-5’a), 4.23 (m, 1H; H-4’), 4.60–4.64 (m,
Modified nucleoside monophosphate analogue (dCXVSMP) and l-
cysteine (1.1 equiv) or GSH (3 equiv) were dissolved in TEAA buffer
(0.3m, pH 8.3, 0.5 mL) and the mixture was stirred (300 rpm) at
378C. The products were purified by C18 reverse-phase HPLC with
a mixture of water/methanol (0 to 100%) containing 0.1m TEAB
buffer as the eluent. Several codistillations with water and conver-
sion to the sodium salt (Dowex 50WX8 in Na+ cycle), followed by
freeze-drying from water, gave the desired conjugates.
3H; H-3’,1’“), 6.39 (dd, J1’,2’ =6.9, 6.4 Hz, 1H; H-1’), 8.45 (s, 1H; H-6),
8.93 ppm (s, 1H; H-5”); 13C NMR (125.7 MHz, D2O): d=29.7 (CH2-
2’“), 38.8 (CH2-3’”), 42.9 (CH2-2’), 49.9 (CH2-1’“), 66.2 (d, JC,P =4.2,
CH2-5’), 73.8 (CH-3’), 89.0 (CH-1’), 89.5 (d, JC,P =8.6 Hz, CH-4’), 101.9
(C-5), 126.0 (CH-5”), 143.0 (CH-6), 144.6 (C-4“), 159.1 (C-2),
165.8 ppm (C-4); 31P{1H} NMR (202.4 MHz, D2O): 3.85 ppm; MS
(ESIÀ): m/z (%): 430.1 (100) [M+H]À.
Chem. Eur. J. 2015, 21, 16091 – 16102
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Incorporation of dCXTP into DNA by PEX
was purified with a QIAquick nucleotide removal kit (QIAGEN) and
eluted with water. The concentration of DNA1XVS product was de-
termined from the absorbance at l=260 nm and the extinction
coefficient was obtained from an online calculator by IDT Biophys-
ics.[59]
The reaction mixture (20 mL) contained primer (0.5 mm), template
(0.75 mm), DNA polymerase (0.05 U KOD XL, 0.16 U Vent(exo-), or
0.2 U Pwo), dNTPs (either all natural or 3 natural and 1 modified;
20 mm) in enzyme reaction buffer supplied by the manufacturer.
Primer was labeled on the 5’-end by 6-carboxyfluorescein (6-FAM).
The reaction mixture was incubated for 30 min at 608C in a thermal
cycler. Primer extension was stopped by the addition of stop solu-
tion (2”, 80% (v/v) formamide, 20 mm ethylenediaminetetraacetic
acid (EDTA), 0.025% (w/v) bromophenol blue, 0.025% (w/v) xylene
cyanol) and heated for 5 min at 958C. Samples were separated by
12.5% PAGE (acrylamide/bisacrylamide 19:1, 25% urea) under de-
naturing conditions (TBE buffer 1”, 42 mA, 1 h). Visualization was
performed by fluorescence imaging by using a Typhoon FLA 9500
instrument from GE Healthcare (Figure S1 in the Supporting Infor-
mation).
To obtain single-stranded ON1XVS for MALDI-TOF mass analysis,
water (60 mL) was added to the click reaction mixture and magne-
toseparation was performed. Streptavidine magnetic particles
(Roche, 50 mL) were washed with binding buffer (3”200 mL, 10 mm
Tris, 1 mm EDTA, 100 mm NaCl, pH 7.5). The diluted click reaction
mixture (100 mL) and binding buffer (100 mL) were added. The mix-
ture was incubated for 30 min at 158C and 1200 rpm. The magnet-
ic beads were collected on a magnet (DynaMagTM-2, Invitrogen)
and washed with washing buffer (3”500 mL, 10 mm Tris, 1 mm
EDTA, 500 mm NaCl, pH 7.5) and water (4”500 mL). Then water
(30 mL) was added and the sample was denatured for 2 min at
508C and 900 rpm. The beads were collected on a magnet and the
solution was transferred into a clean vial. The product was evapo-
rated to dryness, dissolved in a mixture water/acetonitrile (1:1,
5 mL), and analyzed by MALDI-TOF MS (for copies of mass spectra
see Figures S14 and S15 in the Supporting Information).
MALDI-TOF analysis of alkyne-modified oligonucleotides
(ON1E, ON1O, ON2E, ON2O)
The PEX solution (50 mL) contained KOD XL DNA polymerase (0.5
U), primer (4 mm), 5’-biotinylated template (4 mm), and dNTPs
(either natural or modified, 264 mm) in KOD XL reaction buffer sup-
plied by the manufacturer. The reaction mixture was incubated for
40 min at 608C in a thermal cycler. The reaction was stopped by
cooling to 48C. Streptavidine magnetic particles (Roche, 60 mL)
were washed with binding buffer (3”200 mL, 10 mm Tris, 1 mm
EDTA, 100 mm NaCl, pH 7.5). The PEX solution and binding buffer
(50 mL) were added. The mixture was incubated for 30 min at 158C
and 1200 rpm. The magnetic beads were collected on a magnet
(DynaMagTM-2, Invitrogen) and washed with washing buffer (3”
500 mL, 10 mm Tris, 1 mm EDTA, 500 mm NaCl, pH 7.5) and water
(4”500 mL). Then water (50 mL) was added and the sample was de-
natured for 2 min at 508C and 900 rpm. The beads were collected
on a magnet and the solution was transferred into a clean vial. The
product was evaporated to dryness, dissolved in a mixture water/
acetonitrile (1:1, 5 mL), and analyzed by MALDI-TOF MS (the results
are summarized in Table S2 in the Supporting Information; for
copies of mass spectra, see Figures S10–13 in the Supporting Infor-
mation).
Addition of Cys and Cys-containing peptide to DNA1EVS pre-
pared by a click reaction (DNA1EVS_Cys, DNA1EVS_pept)
The reaction mixture (20 mL) contained DNA1EVS (0.4 nmol) and l-
cysteine or an undecapeptide (1 mmol) in TEAA buffer (0.3m,
pH 8.3). The solution was incubated overnight at 378C in a thermal
cycler. Water (40 mL) was added. Streptavidine magnetic particles
(Roche, 100 mL) were washed with binding buffer (3”200 mL,
10 mm Tris, 1 mm EDTA, 100 mm NaCl, pH 7.5). The conjugation re-
action mixture (60 mL) and binding buffer (60 mL) were added. The
mixture was incubated for 30 min at 158C and 1200 rpm. The mag-
netic beads were collected on a magnet (DynaMagTM-2, Invitro-
gen) and washed with washing buffer (3”500 mL, 10 mm Tris,
1 mm EDTA, 500 mm NaCl, pH 7.5) and water (4”500 mL). Then
water (30 mL) was added and the sample was denatured for 2 min
at 508C and 900 rpm. The beads were collected on a magnet and
the solution was transferred into a clean vial. The product was
evaporated to dryness, dissolved in a mixture water/acetonitrile
(1:1, 5 mL), and analyzed by MALDI-TOF MS (for copies of mass
spectra see Figures S16 and S17 in the Supporting Information).
Click reaction of azide 3 on DNA1E and DNA1O: Optimized
conditions
Conjugation of reactive DNA probes (DNA2VS, DNA2EVS
DNA2OVS) with protein p53
,
The DNA1X was prepared by means of a PEX (300 mL) containing
KOD XL DNA polymerase (1.25 U), primer (3.3 mm), 5’-biotinylated
template temp1C (3.3 mm), and dNTPs (either natural or modified,
264 mm) in KOD XL reaction buffer supplied by the manufacturer.
The reaction mixture was incubated for 40 min at 608C in a thermal
cycler. The products were purified on QIAquick nucleotide removal
kit (QIAGEN) and eluted with water. The concentration of DNA
product was determined from the absorbance at l=260 nm and
the extinction coefficient obtained from an online calculator by IDT
Biophysics.[59] The solution of CuI catalyst was freshly prepared im-
mediately prior to the reaction by mixing CuBr (0.5 mL, 10 mm in
DMSO/tBuOH 3:1), TBTA ligand (2.5 mL, 10 mm in DMSO/tBuOH
3:1), and DMSO/tBuOH (3:1, 47 mL).
The click reaction mixture (40 mL) containing DNA1X (5 mm), azide 3
(2 mm), CuBr/TBTA (1:5; 20 mm CuI), and sodium ascorbate (10 mm)
in water/DMSO/tBuOH 9:3:1 was incubated for 1 h at 378C in
a thermal cycler.
To use the obtained DNA1XVS in further bioconjugations (with Cys,
peptide, p53, or cellular proteins), the product of the click reaction
Natural DNA, DNA2VS, DNA2E, and DNA2O were prepared by means
of a PEX (300 mL) containing KOD XL DNA polymerase (1.25 U), 5’-
FAM-labeled primer (3.3 mm), template temp2C (3.3 mm), and dNTPs
(either natural or modified, 264 mm) in KOD XL reaction buffer sup-
plied by the manufacturer. The reaction mixture was incubated for
40 min at 608C in a thermal cycler. The products were purified
with a QIAquick nucleotide removal kit (QIAGEN) and eluted with
water. The concentration of DNA product was determined from
the absorbance at l=260 nm and the extinction coefficient was
obtained from an online calculator by IDT Biophysics.[59]
The DNA2EVS and DNA2OVS were prepared by the click reaction of
DNA2E or DNA2O with azide 3 under the optimized conditions de-
scribed above. The products were purified with a QIAquick nucleo-
tide removal kit (QIAGEN) and eluted with water. The concentra-
tion of DNA product was determined from the absorbance at l=
260 nm and the extinction coefficient was obtained from an online
calculator by IDT Biophysics.[59]
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The reaction mixtures for GSTp53CD protein binding (20 mL) were
prepared from purified PEX or click reaction products (10 mL, 6 ng/
mL), KCl (500 mm, 2 mL), DTT (2 mm, 2 mL), VP buffer (50 mm Tris,
0.1% Triton-X100, pH 7.6, 2 mL), and GSTp53CD stock solution
(700 ng/mL in 25 mm Hepes pH 7.6, 200 mm KCl, 10% glycerol,
1 mm DTT, 1 mm benzamidine; 1 mL). The control sample was pre-
pared analogously without GSTp53CD. All samples were incubated
for 30 min on ice, glycerol was added (80%, 2 mL), and part of the
reaction mixture (3.5 mL) was separated by use of a 5% native
PAGE (acrylamide/bisacrylamide 37.5:1; 0.5”TBE, 48C, 80 V/1 h).
The rest of the reaction mixture in vials was incubated for 2 h at
258C. Loading buffer (5”, 0.3m Tris·HCl, 5% SDS, 50% glycerol,
2.5% b-mercaptoethanol, 0.05% bromophenol blue) was added
and the mixture was heated at 658C for 10 min. The samples
(10 mL) were separated by 10% SDS-PAGE (0.025m Tris, 0.192m gly-
cine, 0.1% SDS) at room temperature (100 V/30 min then 150 V/
1 h). Visualization was performed by fluorescence imaging with
a Typhoon FLA 9500 instrument from GE Healthcare (Figure 1).
was incubated with Amersham ECL western blotting detection re-
agent (GE Healthcare) for 1 min. Chemiluminescence was mea-
sured (Figure 2c) on an ImageQuant LAS 4000 Mini luminescence
analyzer (GE Healthcare).
Expression and purification of hCA II containing alkyne at
position 131
The plasmids pACA_HCA_G131amber (hCA under control of the T7
promoter; ampicillin resistance) and pACyc_pylSwt_pylTTT
(wtPylRS and three copies of pylT under control of the glutamine
promoter from E. coli; chloramphenicol resistance) were a kind
donation from Prof. Thomas Carell (Ludwig Maximilians University,
Munich, Germany).[50] Both plasmids were amplified in E. coli DH5a
and isolated by using a Zyppy Plasmid Miniprep kit (Zymo Re-
search). The plasmids pACA_HCA_G131amber and pACyc_pylSwt_
pylTTT were transformed together into E. coli strain BL21(DE3).
Cells were grown in LB medium (1 L, 378C, 220 rpm) containing
modified amino acid 4 (2 mm), carbenicillin (100 mgLÀ1) and chlor-
amphenicol (25 mgLÀ1) until OD600 =0.9 was reached. The expres-
sion of the HCA G131amber gene was induced by the addition of
ZnSO4 (1 mm) and isopropyl b-d-1-thiogalactopyranoside (IPTG;
0.1 mm). After further incubation (10 h, 378C, 220 rpm), cells were
harvested (3.900 rpm, 10 min, 48C) and stored at À208C until fur-
ther use. Cell pellets were resuspended in washing buffer (80 mL;
25 mm Tris, 50 mm Na2SO4, 50 mm NaClO4, pH 8.0). After ultrasonic
lysis (4”30 s), cell debris was removed by centrifugation
(14000 rpm, 45 min, 48C). The supernatant was loaded on p-ami-
nomethylbenzenesulfonamide–agarose resin (3 mL; Sigma-Aldrich
A0796) equilibrated with washing buffer. After protein binding, the
column was washed with 10 column volumes of washing buffer.
HCA protein was eluted by low-pH buffer (100 mm NaOAc,
200 mm NaClO4, pH 5.6). The fractions were analyzed by 15% SDS-
PAGE. The hCA-containing fractions were combined, buffer was ex-
changed to water by means of a VivaSpin 4 centrifugal concentra-
tor (Vivaproducts), and the sample was freeze-dried. The yield of
pure modified hCA protein was 17 mg and its structure was con-
firmed by measuring of MALDI-TOF mass spectrum (M (calcd)=
29236 Da, M (found)=29238 Da [M+H]+; Figure S27 in the Sup-
porting Information).
Cell culture and lysis
Human p53 null nonsmall lung carcinoma cell line H1299 (NCI-
H1299, ATCC), and derived stable Tet-inducible cell lines for expres-
sion of wtp53 or mutp53 (R273H; described in ref. [49]) were
grown in Dulbecco’s modified eagle medium (DMEM; Invitrogen)
supplemented with 5% fetal bovine serum (FBS; FCS; PAA Labora-
tories GmbH) and penicillin/streptomycin (Gibco) on a 10 cm plate.
All cultures were incubated at 378C in 5% CO2. At about 70% con-
fluency, tetracyclin was added to a final concentration of 1 mgmLÀ1
for 24 h to induce p53 prior to cell harvest. Total cell lysates were
prepared by cell pellet lysis in 1xPLB, according to the manufactur-
er’s protocol (Promega). The concentration of protein in the lysates
was determined by a standard Bradford assay.
Conjugation of DNA2EVS with cellular proteins
The samples (20 mL) containing natural DNA or DNA2EVS (5 mL,
6 ng/mL), KCl (500 mm, 2 mL), DTT (2 mm, 2 mL), Tris buffer (50 mm,
pH 7.6, 2 mL), and total cell lysates (50 mg of total proteins in 1xPLB
buffer). Control samples were prepared analogously without DNA
or cellular extracts. All samples were incubated for 30 min on ice,
and then for 2 h at 258C. Loading buffer (5”, 0.3m Tris·HCl, 5%
SDS, 50% glycerol, 2.5% b-mercaptoethanol, 0.05% bromophenol
blue) was added and the mixture was heated at 958C for 5 min.
The samples (25 mL) were separated by 10% SDS-PAGE (0.025m
Tris, 0.192m glycine, 0.1% SDS) at room temperature (100 V/
30 min then 150 V/1 h). Visualization was performed by fluores-
cence imaging with a Typhoon FLA 9500 instrument from GE
Healthcare (Figure 2b).
Copper-catalyzed click reaction on alkyne-modified hCA*:
Optimized conditions
The solution of CuII with BTTP ligand was freshly prepared immedi-
ately prior to the reaction by mixing CuSO4 (3.8 mL, 10 mm in
water) and BTTP ligand (0.75 mL, 100 mm in DMSO). The click reac-
tion mixture (15 mL) containing hCA* (50 mm), azide (1.25 mm),
CuSO4/BTTP (1:2; 200 mm CuII), and sodium ascorbate (500 mm) in
water was incubated for 1 h at 308C in a thermal cycler.
The gel was blotted (0.025m Tris, 0.192m glycine, 20% methanol;
150 V/1.5 h at 48C) on BioTrace NT nitrocellulose transfer mem-
brane (Pall). The membrane was washed with PBS (137 mm NaCl,
2.7 mm KCl,10 mm Na2HPO4, 2 mm KH2PO4, pH 7.4), blocked with
5% nonfat milk (in PBS-T, 137 mm NaCl, 2.7 mm KCl, 10 mm
Na2HPO4, 2 mm KH2PO4, 0.5% Tween-20, pH 7.4) for 45 min, and
then washed with PBS-T (15 mL, 5 min). The membrane was incu-
bated with monoclonal mouse anti-p53 antibody DO-1 (superna-
tant provided by Dr. Vojtesek) at a 1:10 dilution in 5% nonfat milk
(in PBS-T) at 48C overnight. The unbound antibodies were re-
moved by washing with PBS-T (15 mL, 3”5 min). The membrane
was incubated with antimouse IgG HRP-linked antibody (Sigma Al-
drich) at a 1:10000 dilution in 5% nonfat milk (in PBS-T) at 258C
for 1 h. The remaining unbound antibody was washed away by
TBS-T (20 mL, 4”5 min) and TBS (20 mL, 5 min). The membrane
For gel analysis of hCA*_coum formation, part of the reaction mix-
ture (3 mL) was diluted with water (10 mL) and then loading buffer
(5”, 0.3m Tris·HCl, 5% SDS, 50% glycerol, 2.5% b-mercaptoetha-
nol, 0.05% bromophenol blue) was added. The mixture was
heated at 958C for 5 min. The samples (16.5 mL) were separated by
15% SDS-PAGE (0.025m Tris, 0.192m glycine, 0.1% SDS) at room
temperature (100 V/1 h then 160 V/30 min). Visualization was per-
formed by fluorescence imaging with a Typhoon FLA 9500 instru-
ment from GE Healthcare and the protein was visualized by stan-
dard Coomassie staining (Figure 3c).
To obtain hCA*VS or hCA*_coum for MALDI-TOF analysis, the sam-
ples were dialyzed against water by using a Slide-A-Lyzer MINI dial-
ysis unit (3500 MWCO, Thermo Scientific) and then Amicon Ultra
Chem. Eur. J. 2015, 21, 16091 – 16102
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centrifugal filters (0.5 mL, 3 K, Merck Millipore). The products were
evaporated to dryness, dissolved in water (10 mL), and analyzed by
MALDI-TOF MS (for copies of mass spectra, see Figures S28 and
S29 in the Supporting Information).
To use the obtained hCA*VS in further bioconjugations with pep-
tides, the product of the click reaction was dialyzed against water
by using a Slide-A-Lyzer MINI dialysis unit (3500 MWCO, Thermo
Scientific) and then Amicon Ultra centrifugal filters (0.5 mL, 3 K,
Merck Millipore).
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The reaction mixture (250 mL) contained hCA*VS (2 nmol) and pep-
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(3500 MWCO, Thermo Scientific) and then Amicon Ultra centrifugal
filters (0.5 mL, 3 K, Merck Millipore). The products were evaporated
to dryness, dissolved in water (10 mL), and analyzed by MALDI-TOF
MS (for copies of mass spectra, see Figures S30 and S31 in the Sup-
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We would like to thank Prof. Thomas Carell from Ludwig Maxi-
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plasmids for alkyne-modified carbonic anhydrase expression.
This work was supported by institutional support from the
Academy of Sciences of the Czech Republic (RVO 61388963
and 68081707), the Czech Science Foundation (P206/12/G151),
and Gilead Sciences, Inc.
Keywords: biotransformations · click chemistry · conjugation ·
nucleic acids · proteins
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Received: June 5, 2015
Published online on September 17, 2015
Chem. Eur. J. 2015, 21, 16091 – 16102
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