A cleavable amino-thiol linker for reversible linking of amines to DNA

Article abstract of DOI:10.1021/jo0619736

(Chemical Equation Presented) A cleavable heterobifunctional cross-linker for the reversible conjugation of amines to thiol-modified DNA has been developed and tested. The succinimidyl 2-(vinylsulfonyl)-ethyl carbonate (SVEC) was prepared in three steps and tested for its ability to react with amines and thiols. The linker was efficient for binding leucine to a thiol-modified DNA sequence and for releasing the amino acid at pH 11.8.

Full text of DOI:10.1021/jo0619736

SCHEME 1. Chemistry of the Bifunctional Linker
BSOCOES2 and Structure of the New Heterobifunctional
SVEC Linker 1a
A Cleavable Amino-Thiol Linker for Reversible
Linking of Amines to DNA
Jonas W. Højfeldt,†,‡ Peter Blakskjær,§ and
Kurt V. Gothelf*,†
Center for Catalysis and Interdisciplinary Nanoscience Center
(iNANO), Department of Chemistry, UniVersity of Aarhus,
Langelandsgade 140, 8000 Aarhus C, Denmark, Department of
Chemistry, UniVersity of Michigan, 930 N. UniVersity, Ann
Arbor, Michigan 48109-1055, and Vipergen, FruebjergVej 3,
DK-2100, Copenhagen OE, Denmark
kVg@chem.au.dk
ReceiVed September 24, 2006
under DNA compatible conditions to release the free amino
group and, (iii) after cleavage it should leave a nonreactive
functional group at the DNA strand. Several commercially
available heterobifunctional linkers fulfill the first demand. They
typically contain a N-hydroxysuccinimide ester for reaction with
the amine and a maleimide, vinylsulfone, pyrimidyl disulfide,
R-haloester, or R-haloamide for reaction with the thiol. However,
to the best of our knowledge, none of the existing cross-linkers
also fulfill the second requirement. The bifunctional linker
BSOCOES which is commercially available can almost do the
job (Scheme 1).2 In spite of being an amine-amine linker it is
capable of subsequently liberating a free amine by base-
catalyzed elimination at a pH of 11-12. However, cleavage of
this linker liberates two free amino groups which have to be
separated before further chemical functionalization. In work by
Liu et al. the BSOCOES linker was applied for attachment of
amines to amino-modified oligonucleotide-sequences.4 To cir-
cumvent the presence of two amino groups after cleavage, the
oligonucleotide contained a biotin label for removal of the
sequence from solution by avidine beads.
Described herein is the preparation and application of a new
amino-thiol cross-linker, the succinimidyl 2-(vinylsulfonyl)-
ethyl carbonate 1a (SVEC), which fulfills the three requirements
listed above (Scheme 1). The linker contains an N-hydroxysuc-
cinimide carbonate for coupling to the amine and a vinylsulfone
for subsequent coupling to the thiol. We demonstrate that it
can be used for the reversible linking of amines and amino acids
to thiol-modified DNA sequences.
The linker was synthesized in a straightforward manner
starting from 2-mercaptoethanol and vinyl bromide (Scheme
2). The first step of the synthesis was performed according to
a method described by Scho¨berl and Biedermann.5 The reaction
between 2-mercaptoethanol and vinyl bromide involves a
A cleavable heterobifunctional cross-linker for the reversible
conjugation of amines to thiol-modified DNA has been
developed and tested. The succinimidyl 2-(vinylsulfonyl)-
ethyl carbonate (SVEC) was prepared in three steps and
tested for its ability to react with amines and thiols. The linker
was efficient for binding leucine to a thiol-modified DNA
sequence and for releasing the amino acid at pH 11.8.
Introduction
Bifunctional chemical linkers are important for covalently
attaching molecules to organic supports such as resins or
biomolecules.1,2 In a part of our ongoing interest in DNA-
programmed synthesis,3 a cleavable amino-thiol cross-linker
with the following properties was required: (i) it should be
capable of straightforward conjugating an amino functionality
to a thiol modified DNA sequence, (ii) it should be cleavable
† University of Aarhus.
‡ Present address: University of Michigan.
§ Vipergen.
(1) Hermanson, G. T. Bioconjugate Techniques; Academic Press: San
Diego, CA, 1996.
(2) See www.piercenet.com
(3) (a) Gothelf, K. V.; Brown, R. S. Chem. Eur. J. 2005, 11, 1062-
1069. (b) Gothelf, K. V; Thomsen, A. H.; Nielsen, M.; Clo´, E.; Brown, R.
S. J. Am. Chem. Soc. 2004, 126, 1044. (c) Nielsen, M.; Thomsen, A. H.;
Clo´, E.; Kirpekar, F.; Gothelf, K. V. J. Org. Chem. 2004, 69, 2240. (d)
Clo´, E.; Snyder, J. W.; Voigt, N. V.; Ogilby, P. R.; Gothelf, K. V. J. Am.
Chem. Soc. 2006, 128, 4200-4201. (e) Hansen, J. A.; Mukhopadhyay, R.;
Hansen, J. Ø; Gothelf, K. V. J. Am. Chem. Soc. 2006, 128, 3860- 3861. (f)
Gothelf, K. V.; LaBean, T. H. Org. Biomol. Chem. 2005, 3, 4023-4037.
(g) Brown, R. S.; Nielsen, M.; Gothelf, K. V. Chem. Commun. 2004, 1464.
(h) Nielsen, M.; Dauksaite, V.; Kjems, J.; Gothelf, K. V. Bioconjugate
Chem. 2005, 16, 681-685. (i) Blakskjær, P.; Gothelf, K. V. Org. Biomol.
Chem. 2006, 4, 3442-2447.
(4) Gartner, Z. J.; Tse, B. N.; Grubina, R.; Doyon, J. B.; Snyder, T. M.;
Liu, D. R. Science 2004, 305, 1601-1605.
10.1021/jo0619736 CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/15/2006
9556
J. Org. Chem. 2006, 71, 9556-9559

SCHEME 2. Synthesis of the Linkers 1a and 1b
has a greater solubility in the aqueous phase. The two sulfone
products, 1a and 1b, are crystalline, and stable at 4 °C for several
months. The synthesis can easily be performed on a 2 g scale.
To investigate the performance of the prepared linkers,
reactions with amines and thiols have been carried out. Initial
experiments showed that if the linkers were first mixed with a
thiol, the thiols preferently react with the carbonate. Thus, the
linkers must be reacted with the amine first and subsequently
with the thiol. Furthermore, it was observed that the succinimidyl
carbonate 1a was much more reactive toward amines such as,
for example, t-butyl phenylalanine 4a, than p-nitrophenyl
carbonate 1b, and 1a allows reactions to be completed in just
15 min at room temperature. For comparison the reaction of
1b with 4a under similar conditions only led to 50% conversion
1
after 2 h as monitored by H NMR. Therefore, the succeeding
experiments have been performed exclusively with linker 1a.
Experiments also showed that the amines do not react with the
vinylsulfone moiety of 1a. Base is required for the reactions to
proceed and it was observed that the linkers and their carbamate
products are stable in the presence of weak bases such as
diisopropylamine and sodium bicarbonate.
To demonstrate the ability of the linker to attach an amine
functionality to a thiol and to provide well-defined products,
the linking of three amino acids to benzylthiol was demonstrated
(Scheme 3). The goal is to link free amino acids to DNA via
the linker as shown in the last part of this Note; however, for
the initial experiments the amino acid was t-butyl protected at
the acid moiety to improve solubility in organic solvents.
Reactions of 1a with a slight excess of t-butyl protected
phenylalanine 4a, leucine 4b, and tyrosine 4c on a ∼100 mg
scale for 15 min at room temperature were performed (Scheme
substitution at an unsaturated carbon to give 2. The 2-mercap-
toethanol is first added to an excess of sodium ethanoate. Vinyl
bromide, which is a gas at room temperature, was added
dissolved in THF, and the reaction was heated in an autoclave
at 105 °C. The product was obtained in 84% yield.
1
3). Analysis of the products by H NMR revealed quantitive
In the second step the carbonate is prepared by reaction with
N,N′-disuccinimidyl carbonate.6-8 The carbonate reacts with 2
in acetonitrile to give 3a in 76% yield. This is comparable to
yields seen in the literature for analogous reactions.6-8 For
comparison we also prepared the corresponding p-nitrophenyl
carbonate 3b by a slightly modified procedure.9,10
Finally, the sulfides 3a and 3b are oxidized to the respective
sulfones 1a and 1b. The oxidation is performed by treatment
of the sulfides with peracetic acid at 0 °C for 30 min and
subsequently 2 h at room temperature.10 The extracted crude
products are pure, and no further purification is required.
Oxidation of the olefin to the epoxide is not observed. In our
first attempt to prepare 1a, compound 2 was oxidized to the
corresponding (2-hydroxyethyl)vinylsulfone; however, the re-
sulting sulfone was unstable and tended to polymerize. By the
present procedure, the yield of p-nitrophenyl 2-(vinylsulfonyl)-
ethyl carbonate (PNVEC) 1b is nearly quantitative (97%), while
SVEC 1a is obtained in a slightly lower yield of 82%. This is
likely due to loss of compound during the extraction, since the
succinimidyl group is more polar than the p-nitrophenol and
conversion. Excess of the amines was removed by acidic
extraction to provide the products 5a-c in 76-94% yield after
purification by column chromatography.
The three amino acid linker conjugates 5a-c were next
subjected to reaction with benzylthiol to test the performance
of the vinylsulfone. 1H NMR spectra of the three reaction
mixtures of the thiol additions showed quantitative reactions
with the vinylsulfone after 1 h at room temperature in aceto-
nitrile. Reactions were performed on a ∼50 mg scale, and after
purification the products 6a-c were obtained in 67-95% yields.
As shown in the following, the linker is also very useful for
attachment of an amino acid to a thiol-modified DNA sequence
and for subsequent cleavage to release the amino acid. The 21
bp DNA sequence 8 is obtained from a commercially purchased
sequence containing an internal amine spacer at a thymine base
(Scheme 4). The amine was extended with a disulfide modifier,
and after cleavage of the disulfide the thiol modified sequence
8 was obtained.
The conjugation procedure is straightforward and the first
two steps performed in one pot (Scheme 4). The linker 1a is
mixed with 1.5 equiv of leucine to form the linker-amino acid
intermediate 7. After 15 min the DNA sequence 8 is added in
a ratio of 7 and 8 of 200:1 and incubated at room temperature
for 16 h. The resulting conjugate 9 was purified by HPLC. The
shift in mass from 8 to conjugate 9 is verified by MALDI-TOF
mass spectrometry. The conjugation of leucine with a 5′-thiol
modified oligonucleotide sequence using linker 1a was also
performed (data not shown).
(5) Scho¨berl, A.; Biedermann, M. Liebigs Ann. Chem. 1968, 716, 37-
46.
(6) Ghosh, A. K.; Duong, T. T.; McKee, S. P.; Thompson, W. J.
Tetrahedron Lett. 1992, 33, 2781-2784.
(7) Manoharan, M.; Prakash, T. P.; Barber-Peoc’h, I.; Bhat, B.; Vasquez,
G.; Ross, B. S.; Cook, P. D. J. Org. Chem. 1999, 64, 6468-6472.
(8) Gosh, A. K.; Hussain, K. A. Tetrahedron Lett. 1998, 39, 1881-
1884.
(9) Hojo, K.; Maeda, M.; Smith, T. J.; Kita, E.; Yamaguchi, F.;
Yamamoto, S.; Kawasaki, K. Chem. Pharm. Bull. 2004, 52, 422-427.
(10) Hojo, K.; Maeda, M.; Kawasaki, K. Tetrahedron 2004, 60, 1875-
1886.
Finally, it was demonstrated that the amino acid can be
cleaved from the DNA sequence. Incubating conjugate 9 at pH
J. Org. Chem, Vol. 71, No. 25, 2006 9557

SCHEME 3. Testing of Linker 1a’s Ability to Conjugate Amino Acids to Benzylthiol
SCHEME 4. Demonstration of the Attachment of Leucine to a Thiol-Modified DNA Sequence and Subsequent Cleavage of the
Linker
11.8 for 2 h in the presence of 2-mercaptoethanol led to cleavage
of the carbamate. The intermediately formed reactive Michael
acceptor, the vinyl sulfone, is converted by reaction with
2-mercaptoethanol into the corresponding sulfide. Simulta-
neously, liberation of carbon dioxide from the carbamate leads
to liberation of the free amino acid. Cleavage of the carbamate
and reaction of the remainder with 2-mercaptoethanol was
verified by mass spectrometry after precipitation of 10.
It is our belief that the SVEC 1a linker reported here can be
useful for conjugation of amines to thiols and biomolecules
containing thiols. It may indeed be useful for the transfer of
amino acids from one biomolecule to another by reaction of,
for example, the carboxylic acid group in 9 (Scheme 4) with
an amino group on another sequence followed by cleavage of
the linker. In another aspect the vinylsulfone, which is liberated
after basic cleavage of the carbamate may, in the absence of
2-mercaptoethanol, be used for conjugation with another thiol.
In conclusion, we have prepared a new type of cleavable
amino-thiol linkers SVEC (1a) and PNVEC (1b). Both linkers
are synthesized in three steps from commercially available
starting materials. In particular 1a was efficient for linking amino
acids to a thiol as demonstrated in three examples. Most
importantly it is straightforward and efficient to conjugate amino
acids to thiol-modified DNA by the use of 1a as demonstrated
for leucine. It was furthermore demonstrated that the linker is
efficently cleaved at pH 11.8 and that the reactive vinylsulfone
residue on the DNA sequence is passivated by reaction with
2-mercaptoethanol.
Experimental Section
Succinimidyl 2-Vinylsulfanylethyl Carbonate (3a). To a stirred
solution of 25 (1.100 g, 10.56 mmol) in dry acetonitrile (40 mL),
N,N′-disuccinimidylcarbonate (3.953 g, 15.43 mmol) is added at
room temperature. To the reaction mixture, which is a cloudy
suspension, triethylamine (3.206 g, 31.7 mmol) is added, and after
a couple of minutes the reaction mixture turns clear. Analysis by
TLC (eluent: diethyl ether) shows that the reaction is complete
after 30 min. The solvent is removed in vacuo. The residue is
dissolved in 50 mL CH2Cl2 and washed with 3 × 25 mL saturated
NaHCO3. The organic phase is washed with 30 mL brine and dried
with anhydrous MgSO4, and the solvent is removed in vacuo. The
crude product is purified with flash column chromatography to yield
1
3a (1.868 g, 7.62 mmol, 72%). H NMR (400 MHz, CDCl3): δ
6.29 (dd, Jc ) 10.0 Hz, Jt ) 16.8 Hz, 1H), 5.29 (d, Jt ) 10.0 Hz,
1H), 5.25 (d, Jt ) 16.8 Hz, 1H), 4.45 (t, J ) 7.2 Hz, 2H), 3.03 (t,
J ) 7.4 Hz, 2H), 2.84 (s, 4H). 13C NMR (100 MHz, CDCl3): δ
25.7, 29.3, 69.2, 113.3, 130.7, 151.6, 168.9.11
Succinimidyl 2-Vinylsulfonylethyl Carbonate (SVEC) (1a).
To a stirred solution 3a (1.868 g, 7.62 mmol) in glacial acetic acid
(11) We were not able to obtain HRMS or MS of 3a or 3b
9558 J. Org. Chem., Vol. 71, No. 25, 2006

(4 mL), peracetic acid (3.20 mL of 32% in dilute acetic acid, 15.24
mmol) is added dropwise at 0 °C. After 30 min the cooling bath is
removed, and the reaction is continued at ambient temperature. The
reaction is followed by TLC (eluent: EtOAc). The sulfone product
has a higher retention factor than the sulfoxide intermediate. After
2 h, the only spot seen on the TLC plate is from the sulfone product,
and the solvent is removed in vacuo. The residue is dissolved in
45 mL water and extracted with 3 × 45 mL dichloromethane. The
organic phase is washed with brine and dried over sodium sulfate,
and the solvent is evaporated off. To remove remaining acetic acid,
toluene is added to the crude product and evaporated off as an
azeotropic mixture to yield the crystalline product 1a (1.737 g, 6.26
with Na2SO4 and concentrated in vacuo. The crude product is
purified by flash column chromatography (eluent: EtOAc/pentane
1:1) to give 6a as a colorless oil in 71% yield (44.1 mg, 0.087
1
mmol). H NMR (400 MHz, CD3Cl): δ 7.29 (m, 8H), 7.16 (d, J
) 6.8 Hz, 2H), 5.28 (d, J ) 8.00 Hz, 1H), 4.50 (q, J ) 6.67 Hz,
1H), 4.44 (dt, J ) 2.22 Hz, J ) 5.78 Hz, 2H), 3.74 (s, 2H), 3.24
(dt, J ) 2.26 Hz, J ) 5.78 Hz, 2H), 3.08 (m, 3H), 2.83 (m, 2H),
1.40 (s, 9H). 13C NMR (100 MHz, CD3Cl): δ 23.5, 28.2, 36.9,
38.6, 53.2, 54.3, 55.4, 58.5, 82.8, 127.3, 127.8, 128.8, 129.0, 129.1,
129.7, 136.1, 137.6, 154.8, 170.5. HRMS m/z calcd for C25H33-
NNaO6S2 (M + Na)+, 530.1647; found, 530.1626.
Conjugation of Sequence 8 with SVEC-Leu to Form 8-(SVEC)-
Leu (9). A 20 mM solution of leucine attached to the linker 1a
(SVEC) was prepared by mixing the 1a (80 µL of a 50 mM solution
in MeCN) with leucine (100 µL of a 60 mM aqueous solution) in
the presence of N,N-diisopropyl ethylamine (20 µL of a 400 mM
solution in MeCN) for 15 min at room temperature. This solution
can be stored for several days at 4 °C. Conjugation to an
oligonucleotide was achieved by mixing the SVEC-Leu solution
(50 µL) with the thiol functionalized oligonucleotide 8 dissolved
in 333 mM HEPES buffer pH 7.0. The solution was incubated at
room temperature for 16 h. The conjugate 9 was purified by EtOH
precipitation, purified by HPLC, and identified by MALDI -TOF
mass spectrometry: m/z calcd for 9 (M)+, 6891.3; observed, 6890.8.
Cleavage of Conjugate 9 under Formation of 10. The linker
was cleaved by treating the conjugate 9 (80 pmol) with CAPS buffer
pH 11.8 (100 mM) and 2-mercaptoethanol (40 mM) in a total
reaction volume of 10 uL. After incubation for 2 h at room
temperature, the mixture was diluted to 40 uL with water. EtOH
precipitated from ammonium acetate buffer pH 8.6 (2.5 M), and
the cleaved conjugate was identified by MALDI-TOF mass
spectrometry: m/z calcd for 10 (M)+, 6794.6; found, 6795.1.
1
mmol, 82%). H NMR (400 MHz, CDCl3): δ 6.64 (dd, Jc ) 9.6
Hz, Jt ) 16.4 Hz, 1H), 6.45 (d, Jt ) 16.4 Hz, 1H), 6.22 (d, Jc )
9.6 Hz, 1H), 4.65 (t, J ) 5.8 Hz, 2H), 3.37 (t, J ) 5.8 Hz, 2H),
2.78 (s, 4H). 13C NMR (100 MHz, CD3CN): δ 26.1, 53.1, 65.1,
131.7, 137.5, 152.0, 170.5. HRMS: m/z calcd for C9H11NNaO7S
(M + Na)+, 300.0154; found, 300.0127.
Reaction of O-tBu-Leu with 1a (5a). To a solution of SVEC
1a (102.3 mg, 0.37 mmol) in acetonitrile (5 mL), is added 4a‚HCl
(101.0 mg, 0.392 mmol) and water (5 mL). Sodium bicarbonate
(66 mg, 0.784 mmol) (1 equiv if the free amino acid is used and 2
equiv if the hydrochloride salt of the amino acid is used) is added,
and the reaction is complete within minutes according to TLC
analysis. The CH3CN is removed in vacuo, and the remaining
aqueous solution is diluted with brine (5 mL). The aqueous phase
is extracted two times with DCM (10 mL). The organic phases are
combined, washed with brine, dried with Na2SO4, and concentrated
in vacuo. The crude product is purified by flash column chroma-
tography (eluent: ethylacetate/pentane 3:2) to give 5a as a colorless
oil in 76% yield (107 mg, 0.28 mmol). 1H NMR (400 MHz, CD3-
Cl): δ 7.32-7.15 (m, 5H), 6.61 (dd, Jc ) 10.00 Hz, Jt ) 16.40
Hz, 1H), 6.42 (d, Jt ) 16.80 Hz, 1H), 6.01 (d, Jc ) 9.80. Hz, 1H),
5.21 (d, J ) 8.00 Hz, 1H), 4.46 (m, 3H), 3.31 (t, 5.60 Hz, 1H),
3.07 (dt, J ) 6.00 Hz, J ) 10.00 Hz, 2H), 1.42 (s, 9H). 13C NMR
(100 MHz, CD3Cl): δ 28.2, 38.5, 54.1, 55.3, 58.5, 82.8, 127.3,
128.7, 129.7, 130.8, 136.1, 136.9, 154.9, 170.6. HRMS: m/z calcd
for C18H25NNaO6S (M + Na)+, 406.1300; found, 406.1301.
Addition of Benzylthiol to 5a (6a). To a stirred solution of 5a
(47.2 mg, 0.123 mmol) in acetonitrile (6 mL) and water (1 mL),
benzylthiol (18 mg, 0.145 mmol) and triethylamine (14.67 mg,
0.145 mmol) are added. After 1 h acetonitrile is removed in vacuo.
Additional water (5 mL) is added and extracted two times with the
double volume of diethyl ether (10 mL). The organic phase is dried
Acknowledgment. This work has been supported by grants
from the Danish Technical Research Council, the Danish
National Research Foundation, and the Carlsberg Foundation.
Supporting Information Available: General experimental
procedures, full characterization of all new compounds, procedures
for DNA-modifications and purifications, MALDI-TOF spectra,
HPLC chromatogram, and copies of 1H NMR and 13C NMR spectra.
This material is available free of charge via the Internet at
http://pubs.acs.org.
JO0619736
J. Org. Chem, Vol. 71, No. 25, 2006 9559