Thiol-selective fluorogenic probes for labeling and release

Article abstract of DOI:10.1021/ja809345d

Thiol alkylation is a powerful technique for the labeling of proteins. We report a new class of highly reactive, selective, and fluorogenic probes for thiols in aqueous solution at neutral pH, based on the 7-oxanorbornadiene (OND) framework. The maleate m

Full text of DOI:10.1021/ja809345d

Published on Web 07/06/2009
Thiol-Selective Fluorogenic Probes for Labeling and Release
Vu Hong, Alexander A. Kislukhin, and M. G. Finn*
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research
Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
Received November 30, 2008; E-mail: mgfinn@scripps.edu
Abstract: Thiol alkylation is a powerful technique for the labeling of proteins. We report a new class of
highly reactive, selective, and fluorogenic probes for thiols in aqueous solution at neutral pH, based on the
7-oxanorbornadiene (OND) framework. The maleate moiety in 7-oxabicyclo[2.2.1]hept-2,5-diene-2,3-
dicarboxylic acid esters serves as both a tunable electrophile and an intramolecular quencher of an attached
dansyl fluorophore. Thiols have been found to add with high rates (second-order rate constants of 40-200
M-1 s-1) to give adducts that exhibit enhancements of fluorescence intensity up to 180-fold. The resulting
adducts are also versatile with respect to cleavage (release) reactions by two mechanisms. First, retro-
Diels-Alder fragmentation occurs with half-lives from days to weeks at room temperature, and an epoxide
derivative is also reported that is incapable of cycloreversion cleavage. Second, monoamide OND derivatives
undergo rapid closure to succinimides upon thiol addition, providing a thiol-triggered mechanism for
immediate alcohol release. Peptides and proteins containing free thiol groups were labeled with OND
electrophiles with high chemoselectivity. Since the system is so easily assembled from readily accessible
modules, various functional groups can be added to OND linkers to allow the attachment of other molecules
of interest.
Introduction
amides and ring-opened amic acids, respectively) can compete
significantly with thiol modification, particularly above pH
The unique nucleophilicity of the thiol group makes cysteine
side chains a popular site for selective bond formation to
proteins. The alkylating agents normally used for such reactions
are haloacetamides, maleimides, benzylic halides, ꢀ-chloro-
acrylamides, bromomethylketones, and epoxides, with the first
two being the most popular.1 However, these agents are often
not optimal in terms of selectivity, stability, or synthetic
accessibility. Haloacetamides can react with other nucleophilic
residues of proteins, such as N-terminal amine, lysine, methion-
ine, histidine, and tyrosine, in addition to cysteine thiol.2
Maleimides are preferred in this regard, since they are more
reactive with thiols3,4 and more selective with respect to other
nucleophiles.4 It is commonly assumed that the relative reactivity
of maleimides for thiol over amine is on the order of 1000:1 at
pH 7, although we have observed less selective examples,5 and
cross-linking with amines can occur.6 Maleimide synthesis can
be challenging in some cases,7-10 and the deactivation of
haloacetamides and maleimides by water (giving R-hydroxy-
8.11-13
For these reasons, the development of selective thiol-reactive
linkers is a subject of much current interest, especially systems
that provide an optical signal upon conjugation. The fluorogenic
bromobimanes14-17 are popular as analytical reagents for
identifying cellular thiols and metabolites. Chromogenic vinylic
sulfones,18 dye-maleimides,19 and cyanine-type linker electro-
philes20 have been recently described, all of them highly reactive
and selective. Michael acceptors generated in situ via Knoev-
enagel condensation have also been elegantly used to modify
thiols, serving as convenient photocleavable linkages or protect-
(9) Weh, J.; Duerkop, A.; Wolfbeis, O. S. ChemBioChem 2007, 8, 122–
128.
(10) Lu, Z. K.; Weber, R.; Twieg, R. J. Tetrahedron Lett. 2006, 47, 7213–
7217.
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25, 2739–2743.
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33–44.
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(3) Schelte´, P.; Boeckler, C.; Frisch, B.; Schuber, F. Bioconjugate Chem.
2000, 11, 118–123.
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386.
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2007, 8, 2883–2889.
(16) Kosower, E. M.; Kosower, N. S. Methods Enzymol. 1995, 251, 133–
148.
(5) When performing thiol alkylation reactions on cowpea mosaic virus
capsids bearing surface cysteine residues, we have observed as much
as 25% reactivity with lysines having diminished pKa values by virtue
of their unique microenvironment. The assumption of high thiol
selectivity for maleimides may therefore be invalid on occasion.
(6) Ishii, Y.; Lehrer, S. S Biophys. J. 1986, 50, 75–80.
(7) Keller, O.; Rudinger, J. HelV. Chim. Acta 1975, 58, 531–541.
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references therein.
(17) Sen, C. K.; Roy, S.; Packer, L. Methods Enzymol. 1999, 299, 247-
258, and references therein.
(18) Holler, M.; Sin, H. S.; James, A.; Burger, A.; Tritsch, D.; Biellmann,
J.-F. Chem.-Eur. J. 2000, 6, 2053–2062.
(19) Matsumoto, T.; Urano, Y.; Shoda, T.; Kojima, H.; Nagano, T. Org.
Lett. 2007, 9, 3375–3377.
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S. A. Org. Lett. 2008, 10, 37–40.
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9986 J. AM. CHEM. SOC. 2009, 131, 9986–9994
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Fluorogenic Electrophiles for Thiols
A R T I C L E S
Figure 1. Preparation and reactions of electrophiles 5-9. Reagents and conditions: (a) Et3N, CH2Cl2, 3 h, room temp, 96-97%; (b) R2O2CC≡CCO2R2,
neat, 60-80 °C, 2-4 h; (c) NaOH, THF/H2O, 0 °C, 1 h, 80-85%; (d) DMT-MM, THF, room temp, 1 h, 37-43%; (e) MeI, Cs2CO3, DMF, 20 min, 84%.
ing groups.21 We describe here a system that is significantly
easier to make than maleimides, more stable in aqueous solution,
highly reactive with thiols, and which undergoes controllable
fragmentation after thiol addition.
acceptable yields of propargylamides 8a and 8b. Oxanorbor-
nadienes 6 and 925 were synthesized in a similar manner. The
propargyl substituents of 5c, 6, 8a, and 8b were chosen to
facilitate further functionalization by azide-alkyne “click”
cycloaddition,29-32 which is compatible with the electrophilic
nature of the OND core.
Results and Discussion
Synthesis and Properties of Thiol-Reactive Reagents. It has
been previously reported that the attachment of a maleimide
group to dye fragments such as dansylamide (5-dimethylamino-
1-naphthalenesulfonamide) results in quenching of the chro-
mophore, which is relieved when the π-system of the maleimide
is disrupted.8,19,22 We applied the same strategy to electron-
deficient oxanorbornadienes, which are readily obtained by [4
+ 2] cycloaddition of electron-deficient alkynes with furans,
and are known to react with thiolates23,24 and organic azides,25
much like the alkynes from which they are derived.
Reaction of furfurylamines 1 and 2 with dansyl chloride gave
sulfonamides 3 and 4, which underwent smooth Diels-Alder
cycloaddition with acetylenedicarboxylates to give adducts 5
and 6 (Figure 1). All of the components are inexpensive and
the reactions can be performed on gram scale without difficulty.
The compounds were characterized by standard methods, and
the structure of 5c was confirmed by X-ray crystallographic
analysis. The ester group distal to the sulfonamide was
selectively hydrolyzed to monoacids 7,26 which proved resistant
to coupling with several amines and alcohols under the influence
of carbodiimide/base reagents. However, 4-(4,6-dimethoxy-
1,3,5-triazin-2-yl)-4-methylmorpholinum chloride (DMT-
MM),27 previously employed in a similar setting,28 provided
As expected, furans 3 and 4 were highly fluorescent and
compounds 5, 6 and 9 were significantly quenched, presumably
due to photoinduced electron transfer from the fluorophore
excited state to the LUMO of the maleate moiety of the
Diels-Alder adducts, analogous to the quenching mechanism
of maleimide-dye pairs.22,33 Dansyl fluorescence was restored
upon conjugate addition of thiol, resulting in high signal-to-
noise ratios for the fluorogenic sensing of thiol nucleophiles
(Figure 2 and Table 1).
OND reagents were found to be highly selective for thiols.
Compound 5a was used as a representative electrophile (0.1
mM) in reactions with 1 mM of a selection of potentially
nucleophilic amino acids at pH 7 (10% DMSO in 0.1 M
phosphate buffer). Lysine and histidine gave a very small (<2%)
increase in fluorescence after 60 min relative to the signal
generated by thiol within seconds after mixing. The other amino
acids, as well as the disulfide cystine, induced even less
fluorescence (Figure 3). The subsequent addition of excess thiol
to these samples gave rise to an immediate and strong increase
in fluorescence, verifying that no reaction had occurred in the
presence of nonthiol nucleophiles.
The rates of glutathione (0.11 mM) conjugate addition to
OND probes (0.1 mM) were conveniently followed by the
increase in fluorescence at 550 nm (Figure 2). Second-order
kinetic behavior was observed with rate constants in the range
of 40-200 M-1s-1 at pH 7 (Table 1), comparable to the reported
values for the most reactive maleimides.12 (In a competition
(21) Clarke, K. M.; LaClair, J. J.; Burkart, M. D. J. Org. Chem. 2005, 70,
3709–3711.
(22) Girouard, S.; Houle, M. H.; Grandbois, A.; Keillor, J. W.; Michnick,
S. W. J. Am. Chem. Soc. 2005, 127, 559–566.
(23) Albert, S.; Soret, A.; Blanco, L.; Deloisy, S. Tetrahedron 2007, 63,
2888–2900.
(24) Mahajna, M.; Quistad, G. B.; Casida, J. E. Chem. Res. Toxicol. 1996,
9, 241–246.
(29) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
2001, 40, 2004–2021.
(25) Van Berkel, S. S.; Dirks, A. J.; Debets, M. F.; van Delft, F. L.;
Cornelissen, J. J. L. M.; Nolte, R. J. M.; Rutjes, F. P. J. T.
ChemBioChem 2007, 8, 1504–1508.
(30) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew.
Chem., Int. Ed. 2002, 41, 2596–2599.
(31) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67,
3057–3062.
(26) Niwayama, S. J. Org. Chem. 2000, 65, 5834–5836.
(27) Kunishima, M.; Kawachi, C.; Morita, J.; Terao, K.; Iwasaki, F.; Tani,
S. Tetrahedron 1999, 55, 13159–13170.
(32) Rodionov, V. O.; Presolski, S.; Gardinier, S.; Lim, Y.-H.; Finn, M. G.
J. Am. Chem. Soc. 2007, 129, 12696–12704.
(28) Baran, P. S.; Li, K.; O’Malley, D. R.; Mitsos, C. Angew. Chem., Int.
Edit. 2006, 45, 249–252.
(33) Guy, J.; Caron, K.; Dufresne, S.; Michnick, S. W.; Skene, W. G.;
Keillor, J. W. J. Am. Chem. Soc. 2007, 129, 11969–11977.
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for reactions with organic azides,25,34 was the least stable
electrophile tested.35
The rates of thiol addition to the OND electrophiles and their
aqueous decomposition were therefore not directly proportional
to each other (Table 1). With the exception of 7c, the OND
compounds all reacted with glutathione at similar rates, but
differed widely in their aqueous phase stabilities. Those reagents
that are highly reactive toward thiols while being most stable
toward decomposition in aqueous buffers are the most useful;
5b/8b and 5a/8a fit these criteria best. The difference in
reactivity between 5c and 6 is also instructive. The introduction
of a methyl group at the furan bridgehead (adjacent to the site
of thiol attack) strongly inhibited reaction with glutathione but
did not change the stability of the electrophile very much. This
suggests that decomposition may not occur by a similar
conjugate addition of water; the mechanism of this process is
under investigation. For practical purposes, all of the OND
electrophiles may be used in water or buffer if the aqueous
solutions are freshly prepared, and some of these solutions are
stable for many days or weeks (Table 1).
Linear pH-log(rate) profiles in aqueous buffers and a strong
correlation of rate with solvent polarity in nonaqueous solvents
(Supporting Information, Figures S4 and S5) support the
assumption that thiolate anions are far more reactive with OND
electrophiles than neutral thiols.36,37 For this reason, reactions
in anhydrous acetonitrile were sluggish even at high millimolar
concentrations, but the addition of trace amounts of triethylamine
or diisopropylethylamine resulted in very fast reactions signaled
by the immediate appearance of strong yellow-green fluores-
cence. Compound 7c, bearing a deprotonated carboxylate at pH
7, presented a special case. Its relatively electron-rich core does
not engage in photoinduced electron transfer quenching of the
fluorophore, so the molecule was highly fluorescent throughout,
requiring HPLC of aliquots to determine rate constants for both
thiol addition and aqueous decomposition. Its pH-reactivity
profile was distinguished by compensating trade-offs between
accelerating increases in thiolate concentration at higher pH and
deactivating increases in the percentage of OND electrophile
in the anionic carboxylate form (see Supporting Information).
Adducts 10a and 10b, resulting from syn-addition of thiol to
the less-hindered site of the electrophile,23,24 were isolated as
single diastereomers in high yield (Figure 4). Under the same
conditions, monoamide derivative 8a underwent additional ring
closure upon thiol addition to give succinimide 13, without
observation of adduct 12. This process is likely triggered by
the conversion of the electrophilic sp2 centers to sp3, making
ring closure less strained. It has the additional and very useful
feature of inducing the release of an alcohol fragment in
response to thiol attack (see below). The identity of 13 was
confirmed by standard mass and NMR spectra as well as the
presence of cross-peaks between propargyl protons and both
Figure 2. Excitation and emission spectra of 5a (100 µM) treated with
glutathione at the indicated concentrations (0.1 M phosphate buffer, pH 7,
containing 1% DMSO). Intensity (y-axis) is plotted in arbitrary units.
for limiting glutathione, 5a and N-ethylmaleimide were found
to be equally reactive; see Supporting Information.) Methyl,
ethyl, and propargyl ester substituents, having similar steric and
electronic properties, did not change thiol reactivities very much
(propargyl 5c > methyl 5a > ethyl 5b, but within a factor of 3
for the series), and the installation of a bridgehead methyl group
was inhibitory (6 vs 5c). A single amide group had little
influence, with compounds 8a and 8b being as reactive toward
thiol as their ester analogues 5a and 5b. Thus, additional
functional groups such as alkyne can be introduced via an amide
linkage without sacrificing the desired electrophilic reactivity
of the OND core.
OND adducts may lose activity by conjugate addition of water
or by hydrolysis of a conjugated ester group to the less electron-
deficient carboxylate anion. (Carboxylic acid derivative 7c was
by far the most sluggish electrophile in the series, being nearly
600 times less reactive with glutathione than its propargyl ester
precursor 5c.) To gauge OND stability toward such deactivation,
each compound was incubated in aqueous buffer and the amount
remaining in solution was determined by HPLC as a function
of time (Table 1). The observed trends were also reproduced
by a different method, in which aliquots were removed and
fluorogenic signals measured after addition of excess glutathione,
compared to the same treatment of freshly prepared solutions
(data not shown). Methyl and ethyl diesters 5a and 5b were
found to be significantly more stable (half-lives in excess of 9
days at room temperature and pH 7) than propargyl esters 5c
and 6 (half-lives of 2-3 days), which are somewhat more
electron-deficient. Both methylation of the sulfonamide nitrogen
(Me-5a) and epoxidation of the electron-rich double bond
(epoxy-5a) made the electrophile moderately more sensitive to
decomposition (half-lives of 5.6 and 3.2 days vs 9.3 days for
5a), without appreciably changing reactivity with thiol. As
would be expected from the diminished electron-withdrawing
power of carboxylate anions and amides relative to esters, 7c,
8a, and 8b were also very stable toward aqueous deactivation
at pH 7. The stable members of the OND family are therefore
longer lived in water than most maleimides;11,12 an example
(5a vs N-ethylmaleimide) is shown in Supporting Information.
Trifluoromethyl derivative 9, similar to ONDs previously used
(34) Van Berkel, S. S.; Dirks, A. J.; Meeuwissen, S. A.; Pingen, D. L. L.;
Boerman, O. C.; LaVerman, P.; van Delft, F. L.; Cornelissen,
J. J. L. M.; Rutjes, F. P. J. T. ChemBioChem 2008, 9, 1805–1815.
(35) This elegant report by Cornelissen, Rutjes, and co-workers (ref 25,
see also ref 34) describes the azide reactivity of OND derivatives in
the context of bioconjugation. The rate of this reaction is far slower
than condensation with thiol. For example, we found 9 to engage
glutathione with a rate constant of approximately 78 M-1 s-1 (Table
1, room temperature) and benzyl azide at a rate of 2.3 × 10-3 M-1
s-1 in methanol (37 °C), the latter in excellent agreement with the
value reported in ref 25 for the analogous oxanorbornadiene lacking
the dansylaminomethyl substituent.
(36) Schmidt, T. J.; Ak, M.; Mrowietz, U. Bioorg. Med. Chem. 2007, 15,
333–342.
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Table 1. Reactivity, Fluorogenicity, Aqueous Stability, And Adduct Stability of OND Electrophiles
a R ) CH2NHDansyl. b Second-order rate constant for addition of compound to glutathione. c Ratio of emission intensity (550 nm, excitation 332
nm) observed 5 min after addition of 1 mM glutathione to 0.1 mM compound, vs the emission intensity before addition, at room temperature.
d Calculated from the first-order rate constant for the inactivation of the indicated electrophile by incubation of a 0.1 mM solution at room temperature.
e Calculated from first-order rate constants for the retro Diels-Alder reaction of the ꢀ-mercaptoethanol adduct of the indicated compound. f R-Me )
CH2NMeDansyl. g No cleavage is observed: the adduct maintains a covalent connection between thiol and electrophile fragments while decomposing by
route(s) other than retro-Diels-Alder fragmentation, with a half-life of 16.9 ( 0.5 days. The details are being explored and will be described elsewhere.
h Rates of reaction determined by HPLC of aliquots; see text. i 8a and 8b give the same imide thiol adduct, and so show identical rates of
retro-Diels-Alder cleavage. All addition and decomposition measurements were performed in 0.1 M potassium phosphate buffer, pH 7, containing 10%
DMSO.
tion.23,39-42 We measured the cycloreversion rates of the
ꢀ-mercaptoethanol addition products of most of the OND
electrophiles described here (Table 1). Four categories of
stability were identified: (a) cleavable but highly stable (thiol
adducts of 5a, 6, and 9, half-lives greater than 20 days at room
temperature), (b) intermediate in stability (thioethers from 5b
and Me-5a, half-lives approximately 4.5 days), (c) rapidly
cleaved (adducts from 5c and 8a/8b, half-lives 12-36 h), and
(d) noncleavable (thioether from epoxy-5a, which underwent
one or more reactions with half-life of approximately 17 days
at room temperature, but without cleaving into two fragments43).
(37) Khatik, G. L.; Kumar, R.; Chakraborti, A. K. Org. Lett. 2006, 8, 2432–
2436.
(38) The reactivity of a representative thiomaleate of structure 11 is
discussed in Supporting Information. We are currently exploring the
electrophilic and potential thiol cross-linking and exchange capabilities
Figure 3. Fluorogenic activity of 5a with various amino acids. Relative
fluorescence intensity of 0.1 mM 5a (1% DMSO in 0.1 M phosphate buffer,
pH 7) at 550 nm (λex ) 332 nm) after incubation with 1 mM analyte (1
min for cysteine and glutathione; 60 min for all others).
of thiomaleates and thiomaleimide adducts such as 14 and 17.
(39) Takaguchi, Y.; Tajima, T.; Ohta, K.; Motoyoshiya, J.; Aoyama, H.;
Wakahara, T.; Akasaka, T.; Fujitsuka, M.; Ito, O. Angew. Chem., Int.
Ed. 2002, 41, 817–819.
(40) Chen, X.; Dam, M. A.; Ono, K.; Mal, A.; Shen, H.; Nutt, S. R.; Sheran,
K.; Wudl, F. Science 2002, 295, 1698–1702.
1
carbonyl groups in a H-13C HMBC spectrum. Each of these
adducts underwent retro-Diels-Alder fragmentation to furan 3
and either thiomaleate 11 or thiomaleimide 14 (Figure 4).24,38
Thiol addition and retro-Diels-Alder fragmentation of electron-
deficient oxanorbornadiene adducts has been used as a cleavable
linkage strategy for solid-phase synthesis and polymer derivatiza-
(41) Gheneim, R.; Perez-Berumen, C.; Gandini, A. Macromolecules 2002,
35, 7246–7253.
(42) Keller, K. A.; Guo, J.; Punna, S.; Finn, M. G. Tetrahedron Lett. 2005,
46, 1181–1184.
(43) The synthesis and reactivities of epoxy-5a and related compounds will
be described in detail elsewhere.
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Hong et al.
Figure 4. Structure and retro-Diels-Alder decomposition of thiol adducts. (a) 200 mM 5a, 180 mM nucleophile in CH3CN, iPr2NEt (cat.), 5 min, RT,
>80% isolated yield; (b) 10% DMSO in aqueous buffer, 20 °C.
Figure 5. Peptide labeling with OND electrophiles.
The thiol adduct 10a and its dansylsulfonamide N-methylated
analogue Me-10a exhibited a 6-fold difference in retro-
Diels-Alder decomposition rate in water (Table 1), with the
latter structure being the less stable. This suggests that an
intramolecular hydrogen bond between the sulfonamide NH and
the adjacent ester group may help to stabilize compounds such
as 10a. The four categories of thioethers each include members
that exhibited high rates of thiol addition, so OND systems can
be efficiently assembled while being tuned to provide variable
rates of release.
fragmentation induced in the MALDI analysis.44 When the
peptide was pretreated with N-ethylmaleimide (1 mM), no
reaction was observed with the OND electrophiles, confirming
the thiol as the site of reaction.
Treatment of bovine serum albumin (BSA, 1 mg/mL, 15 µM)
with 50 µM of reagents 5b, 8b, or 16 (derived from 8b) under
mild conditions provided efficient labeling of the single free
cysteine residue at position 34 in 2 h as revealed by the
appearance of strong dansyl fluorescence in the protein band
following denaturing gel electrophoresis (Figure 6). The dena-
turing protocol had some effect on the intensity of the fluorescent
signal, consistent with the expected lability of the adduct. Thus,
treatment of the BSA-5b conjugate with dithiothreitol (DTT)
and urea at 95 °C gave a significantly weaker fluorescent band
than a room temperature protocol using the same denaturing
reagents (lane 7 vs 3). As observed with their ꢀ-mercaptoethanol
conjugates (Table 1), the adduct of BSA with 8b was much
less stable than with 5b, completely losing its fluorescence upon
heating due to its faster retro-Diels-Alder fragmentation (lane
6 vs 7). If one places a dye on the electrophilic fragment,
however, the label is permanently attached. Thus, when treated
with bis(dansyl) OND probe 16, BSA remained labeled even
Peptide and Protein Labeling. To test the bioconjugation
reactivity of representative OND labels, a 1 mM solution of
the nonapeptide 15 (KCIRGDTFG*, where G* represents
C-terminal N-propargylglycine) containing amine, carboxylate,
guanidine, and alkyne functional groups in addition to thiol,
was mixed with an equimolar amount of 5a, 8a, or 8b in pH 7
buffer containing 10% DMSO (Figure 5). A strong fluorescent
signal was generated immediately in each case, reaching
completion within 1 min. Analysis by HPLC and MALDI-TOF
mass spectrometry of the crude reaction mixtures showed no
unreacted starting material (Supporting Information, Figures S6
and S7) and a major product corresponding to the addition of
one electrophile in each case, along with some retro-Diels-Alder
(44) Meng, J.-C.; Averbuj, C.; Lewis, W. G.; Siuzdak, G.; Finn, M. G.
Angew. Chem., Int. Ed. 2004, 43, 1255–1260.
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A R T I C L E S
Figure 6. (Top) SDS-PAGE of BSA (1 mg/mL) labeled with the indicated OND reagents (50 µM, pH 7.0, 2 h, room temperature), followed by denaturation
with DTT and urea at room temperature for 1 h or at 95 °C for 10 min, as indicated. The upper images show the gels visualized under ultraviolet irradiation
before staining; the lower images show staining with Coomassie blue. (Bottom) Sequence of bond forming and breaking events with electrophile 16. The
thiomaleimide adduct 17 may take on an additional nucleophile such as DTT or protein thiol.38
ether, dichloromethane, and toluene were dried by passage through
activated alumina columns.53,54 Routine mass spectra were obtained
on an Agilent 1100 (G1946D) ESI-MSD instrument, using a standard
C18 reversed-phase column eluted with 9:1 CH3CN/H2O containing
0.1% CF3CO2H. Elemental analyses were performed by Midwest
MicroLab, LLC. Flash chromatography was performed on 230-400
mesh silica or SINGLE StEP prepacked MPLC columns (Thomson
Instrument Company, Oceanside, CA). Dye-containing materials were
protected from light by wrapping the reaction and storage glassware
in aluminum foil. Reactions of OND reagents with thiols were
conducted in air rather than under inert atmosphere; for long-term
storage, the exposure of thiol reagents to air was minimized. Disulfide
bond formation was not found to be significant under our reaction
conditions.
Synthesis. Dipropargyl acetylenedicarboxylate. A slight modi-
fication of the published procedure55 was used. A suspension of
acetylenedicarboxylic acid (5.15 g, 45.1 mmol), propargyl alcohol (5.20
g, 92.7 mmol) and p-toluenesulfonic acid monohydrate (0.5 g) in
benzene (50 mL) was refluxed with a Dean-Stark trap for 24 h,
collecting approximately 1.8 mL of water. The resulting clear brown
mixture was cooled, diluted with diethyl ether (50 mL), and washed
with saturated aqueous sodium bicarbonate (50 mL) and water (2 ×
50 mL). The organic phase was dried over magnesium sulfate,
concentrated, and purified by flash chromatography (silica, 10-20%
(v/v) ethyl acetate/hexanes) to provide the pure compound as a pale-
yellow oil that solidified on storage at +4 °C to colorless crystals (4.78
g, 56%, mp 26-29 °C). Rf 0.56 (25% EtOAc/hexanes). Spectral data
was in accordance with the literature.56 1H NMR (CDCl3, 400 MHz)
δ 4.82 (d, 4H, J ) 2.5), 2.57 (t, 2H, J ) 2.5). 13C NMR (CDCl3, 100
MHz) δ 150.9, 76.7, 75.8, 74.9, 54.2.
after denaturation at high temperature. The fluorescence intensity
of the protein diminished by approximately one half (lane 5 vs
1) because cycloreversion left one of the two dansyl units
attached to the protein (17) and allowed the other to diffuse
away, as shown.38 A noncleavable linkage was also made with
the epoxide derivative epoxy-5a. When analyzed after room
temperature treatment, both 5a and epoxy-5a showed complete
labeling (Figure 6, lanes 9 and 10); in contrast, only epoxy-5a
provided a heat-stable linkage (lane 12 vs 11).
Conclusions. Oxanorbornadienedicarboxylates (OND), elec-
trophilic adducts of furans and electron-deficient alkynes, have
long been known as synthetic intermediates in Diels-Alder/
retro-Diels-Alder sequences45,46 and more recently as reactive
compounds toward cycloaddition47-49 and ene reactions.28,50,51
Their polar reactivity as Michael acceptors has, with one
exception,23 received little attention. We show here that these
readily available compounds provide excellent water stability
while retaining rapid, selective, and fluorogenic reactivity toward
thiols in small molecules, peptides, and proteins. Among fluoro-
and chromogenic thiophiles recently described,18-20,52 the OND
molecules are notable for their ease of synthesis and tunability
in both addition and fragmentation processes. It is envisioned
that other dyes with appropriate redox potentials can be used
to provide better fluorogenic properties. The utility of these
compounds for protein modification in Vitro and in ViVo, drug
release, and polymer cross-linking is the subject of ongoing
research in our laboratory.
N-Dansylfurfurylamine 3. To a stirred solution of 5-dimethy-
laminonaphthalenesulfonyl chloride (1.00 g, 3.70 mmol) and triethy-
lamine (1.0 mL, 7.2 mmol) in CH2Cl2 (15 mL) was added a solution
of furfurylamine (378 mg, 3.89 mmol) in CH2Cl2 (5 mL) via syringe
under nitrogen. The resulting solution was stirred for 3 h at room
temperature and poured into 1.0 M pH 7 phosphate buffer (30 mL).
Experimental Section
General. Reagents and solvents were purchased from commercial
sources and used without further purification. THF, acetonitrile, diethyl
(45) Grigg, R.; Roffey, P.; Sargent, M. V. J. Chem. Soc. C 1967, 2327.
(46) Mavoungou-Gomes, L. Bull. Soc. Chim. Fr. 1967, 1753–1758.
(47) Allen, A.; Le Marquand, P.; Burton, R.; Villeneuve, K.; Tam, W. J.
Org. Chem. 2007, 72, 7849–7857.
(53) Alaimo, P. J.; Peters, D. W.; Arnold, J.; Bergman, R. G. J. Chem.
Educ. 2001, 78, 64.
(48) Burton, R. R.; Tam, W. J. Org. Chem. 2007, 72, 7333–7336.
(49) Burton, R. R.; Tam, W. Tetrahedron Lett. 2006, 47, 7185–7189.
(50) Choony, N.; Kuhnert, N.; Sammes, P. G.; Smith, G.; Ward, R. W.
J. Chem. Soc., Perkin Trans. 1 2002, 1999–2005.
(54) Pangborn, A. B.; Giardello, M. A.; Grubbs, R. H.; Rosen, R. K.;
Timmers, F. J. Organometallics 1996, 15, 1518–1520.
(55) Tom Dieck, H.; Munz, C.; Muller, C. J. J. Organomet. Chem. 1990,
384, 243–255.
(51) Choony, N.; Sammes, P. G.; Smith, G.; Ward, R. W. Chem. Commun.
2001, 2062–2063.
(56) Hashmi, A. S. K.; Grundl, M. A.; Nass, A. R.; Naumann, F.; Bats,
J. W.; Bolte, M. Eur. J. Org. Chem. 2001, 4705–4732.
(52) Pires, M. M.; Chmielewski, J. Org. Lett. 2008, 10, 837–840.
9
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A R T I C L E S
Hong et al.
The organic phase was separated, dried over magnesium sulfate,
concentrated, and purified by flash chromatography (silica, 0-25%
EtOAc/hexanes) to provide the title compound as a yellow-green oil
which solidified on storage (1.18 g, 96%). Rf 0.35 (25% EtOAc/
hexanes). 1H NMR (CDCl3, 400 MHz) δ 8.51 (app d, 1H, J ) 8.5),
8.22 (m, 2H), 7.57-7.52 (m, 1H), 7.52-7.47 (m, 1H), 7.17 (app d,
1H, J ) 7.6), 7.03 (dd, 1H, J ) 1.8, 0.9), 6.04 (dd, 1H, J ) 3.2, 1.8),
5.89 (dd, 1H, J ) 3.2, 0.9), 4.98 (t, 1H, J ) 6.0), 4.13 (d, 2H, J )
6.0), 2.88 (s, 6H). 13C NMR (CDCl3, 100 MHz) δ 152.1, 149.5, 142.4,
134.8, 130.7, 130.0, 129.78, 129.7, 128.6, 123.3, 118.7, 115.3, 110.3,
108.1, 45.6, 40.4. ESI-MS (C17H18N2O3S) 331.0, [M + H]+. Note:
the crude material may be precipitated by cooling a solution in 1:5
EtOAc/hexanes to obtain crystalline material of satisfactory purity.
N-Dansyl(5-methylfurfuryl)amine (4). To a stirred solution of
5-dimethylaminonaphthalene-sulfonyl chloride (472 mg, 1.75 mmol)
and triethylamine (0.39 mL, 2.81 mmol) in CH2Cl2 (5 mL) was added
a solution of furfurylamine (211 mg, 1.90 mmol) in CH2Cl2 (1 mL)
via syringe under nitrogen. The resulting solution was stirred for 3 h
at room temperature and poured into 1.0 M pH 7 phosphate buffer
(15 mL). The organic phase was washed with water (15 mL), dried
over magnesium sulfate, concentrated, and purified by flash chroma-
tography (silica, 0-25% EtOAc/hexanes) to provide the title compound
as a yellow-green oil which solidified on storage (576 mg, 97%). Rf
0.40 (25% EtOAc/hexanes). 1H NMR (CDCl3, 400 MHz) δ 8.50 (app
d, 1H, J ) 8.5), 8.25-8.19 (m, 2H), 7.58-7.52 (m, 1H), 7.51-7.45
(m, 1H), 7.17 (app d, 1H, J ) 7.6), 5.71 (d, 1H, J ) 3.0), 5.55 (dq,
1H, J ) 3.0, 1.0), 4.97 (t, 1H, J ) 5.9), 4.08 (d, 2H, J ) 6.0), 2.88 (s,
6H), 1.91 (app s, 3H). 13C NMR (CDCl3, 100 MHz) δ 152.2, 147.5,
135.0, 130.6 130.1, 129.9, 129.8, 128.5, 123.3, 118.9, 115.3, 109.1,
106.1, 45.6, 40.6, 13.3. ESI-MS (C18H20N2O3S) 345.1, [M + H]+.
OND Structures 5. A mixture of N-dansylfurfurylamine (331 mg,
1.00 mmol) and the appropriate acetylenedicarboxylate (1.5-1.8 equiv)
was dissolved in CH2Cl2 (0.5 mL). The solvent was removed under a
stream of nitrogen at 50 °C and the reaction mixture was heated with
stirring at 60-65 °C until the dye was consumed (ca. 3 h). The reaction
mixture was dissolved in a minimal amount of hot toluene and loaded
directly on a prepacked silica gel chromatography column and eluted
with the appropriate solvent gradient to provide the pure product.
Dimethyl 1-((5-(Dimethylamino)naphthalene-1-sulfonamido)-
methyl)-7-oxanorborna-2,5-diene-2,3-dicarboxylate (5a). Yellow
foam, 81% yield. Rf 0.23 (25% EtOAc/hexanes). 1H NMR (CDCl3,
400 MHz) δ 8.55 (app d, 1H, J ) 8.5), 8.28-8.22 (m, 2H),
7.59-7.51 (m, 2H), 7.19 (d, 1H, J ) 7.6), 7.13 (dd, 1H, J ) 5.2,
1.8), 6.89 (d, 1H, J ) 5.3), 5.57 (d, 1H, J ) 1.9), 5.17 (app t, 1H,
J ) 6.3), 3.78 (s, 3H), 3.71 (s, 3H), 3.71 (dd, 1H, J ) 13.5, 7.0),
3.60 (dd, 1H, J ) 13.5, 5.7), 2.88 (s, 6H). 13C NMR (CDCl3, 100
MHz) δ 163.9, 162.8, 153.7, 152.2, 152.1, 145.0, 142.8, 134.4,
130.8, 130.1, 129.9, 129.7, 128.7, 123.32, 118.8, 115.5, 95.9, 84.1,
52.6, 52.6, 45.6, 42.1. ESI-MS 473.1, [M + H]+. Anal. Calcd for
C23H24N2O7S: C, 58.46; H, 5.12; N, 5.93. Found: C, 58.26; H, 5.16;
N, 5.77.
Dimethyl 1-((5-(Dimethylamino)-N-methylnaphthalene-1-sul-
fonamido)methyl)-7-oxanorborna-2,5-diene-2,3-dicarboxylate (Me-
5a). To a suspension of 5a (135 mg, 0.286 mmol) and cesium
carbonate (186 mg, 0.573 mmol, 2.0 equiv) in dimethylformamide
(0.6 mL) was added methyl iodide (57 mg, 0.40 mmol, 1.4 equiv)
and the mixture was stirred for 20 min at room temperature.
Aqueous potassium dihydrogen phosphate (5%, 1.5 mL) was added
and the mixture was extracted with EtOAc (4 × 2 mL). The
combined organic solutions were dried over magnesium sulfate,
concentrated, and purified by flash chromatography (silica 30%
EtOAc/hexanes) to provide the desired product as an orange foam
(117 mg, 84%). 1H NMR (CDCl3, 400 MHz) δ 8.55 (app d, 1H, J
) 8.5), 8.42 (app d, 1H, J ) 8.7), 8.15 (app d, 1H, J ) 7.3),
7.58-7.51 (m, 2H), 7.19 (d, 1H, J ) 7.6), 7.17-7.14 (m, 2H),
5.64 (d, 1H, J ) 2.2), 4.19 (d, 1H, J ) 15.1), 3.91 (d, 1H, J )
15.1), 3.87 (s, 3H), 3.79 (s, 3H), 2.88 (s, 6H), 2.86 (s, 3H). 13C
NMR (CDCl3, 100 MHz) δ 164.2, 162.9, 154.0, 152.5, 151.9, 144.7,
143.8, 133.8, 130.6, 130.5, 130.3, 130.0, 128.3, 123.3, 119.8, 115.4,
98.0, 84.1, 52.8, 52.5, 48.3, 45.6, 36.6. ESI-MS 487.1, [M + H]+.
Anal. Calcd for C24H26N2O7S: C, 59.25; H, 5.39; N, 5.76. Found:
C, 59.26; H, 5.45; N, 5.63%.
The synthesis of 1-((5-(dimethylamino)-naphthalene-1-sulfona-
mido)methyl)-5,6-epoxy-7-oxanorborna-2-ene-2,3-dicarboxylate (ep-
oxy-5a) is not optimized and will be described separately.43 Rf 0.10
(40% EtOAc/hexanes)1H NMR (CDCl3, 400 MHz) δ 8.55 (app d,
1H, J ) 8.5), 8.27-8.21 (m, 2H), 7.59-7.50 (m, 2H), 7.18 (app
d, 1H, J ) 7.6), 5.14 (dd, 1H, J ) 8.2, 4.7), 5.03 (s, 1H), 3.81 (s,
3H), 3.78 (s, 3H), 3.74 (dd, 1H, J ) 13.7, 8.2), 3.74 (d, 1H, J )
3.5), 3.59 (d, 1H, J ) 3.5), 3.41 (dd, 1H, J ) 13.7, 4.7), 2.88 (s,
6H). 13C NMR (CDCl3, 100 MHz) δ 163.1, 162.0, 152.2, 148.5,
147.4, 134.2, 130.9, 130.1, 129.9, 129.7, 128.8, 123.3, 118.6, 115.5,
90.6, 78.7, 57.4, 56.1, 53.0, 52.8, 45.6, 41.4. ESI-MS (C23H24N2O8S)
489.1, [M + H]+.
Diethyl 1-((5-(Dimethylamino)naphthalene-1-sulfonamido)
methyl)-7-oxanorborna-2,5-diene-2,3-dicarboxylate (5b). Yel-
1
low foam, 80% yield. Rf 0.26 (25% EtOAc/hexanes). H NMR
(CDCl3, 400 MHz) δ 8.55 (app d, 1H, J ) 8.5), 8.27 (d, 1H, J )
8.5), 8.28-8.22 (m, 2H), 7.59-7.51 (m, 2H), 7.18 (d, 1H, J )
7.5), 7.12 (dd, 1H, J ) 5.3, 1.9), 6.89 (d, 1H, J ) 5.3), 5.55 (d,
1H, J ) 1.9), 5.21 (t, 1H, J ) 6.3), 4.22 (2 × dq, 2H, J ) 7.2,
2.0), 4.18 (q, 2H, J ) 7.1), 3.72 (dd, 1H, J ) 13.5, 6.9), 3.60 (dd,
1H, J ) 13.5, 5.8), 2.88 (s, 6H), 1.30 (t, 3H, J ) 7.1), 1.28 (t, 3H,
J ) 7.2). 13C NMR (CDCl3, 100 MHz) δ 163.6, 162.7, 153.5, 152.2,
151.6, 145.0, 142.8, 134.5, 130.8, 130.1, 129.9, 129.7, 128.7, 123.3,
118.8, 115.4, 95.9, 84.1, 61.9, 61.7, 45.6, 42.1, 14.2, 14.1. ESI-
MS 501.1, [M + H]+. Anal. Calcd for C25H28N2O7S: C, 59.99; H,
5.64; N, 5.60. Found: C, 59.24; H, 5.62; N, 5.31.
Dipropargyl 1-((5-(Dimethylamino)naphthalene-1-sulfonami-
do)methyl)-7-oxanorborna-2,5-diene-2,3-dicarboxylate (5c). A mix-
ture of N-dansylfurfurylamine (331 mg, 1.00 mmol), dipropargyl
acetylenedicarboxylate (350 mg, 1.84 mmol), and toluene (0.5 mL)
was heated at 60-65 °C with stirring for ca. 3 h. After the reaction
was complete, the resulting brown oil was loaded directly on a
silica gel column and purified by flash chromatography (20-40%
EtOAc/hexanes) to provide the title compound as an oil that
solidified to yellow crystalline powder on trituration with Et2O and
1
hexanes (428 mg, 82%). Rf 0.13 (25% EtOAc/hexanes). H NMR
(CDCl3, 400 MHz) δ 8.56 (d, 1H, J ) 8.5), 8.28-8.23 (m, 2H),
7.60-7.51 (m, 2H), 7.19 (d, 1H, J ) 7.6), 7.14 (dd, 1H, J ) 5.2,
1.9), 6.90 (d, 1H, J ) 5.2), 5.60 (d, 1H, J ) 1.9), 5.15 (t, 1H, J )
6.4), 4.77 (d, 2H, J ) 2.5), 4.67 (2 × dd, 2H, J ) 15.5, 2.5), 3.73
(dd, 1H, J ) 13.7, 7.1), 3.63 (dd, 1H, J ) 13.7, 5.8), 2.89 (s, 6H),
2.50 (m, 2H). 13C NMR (CDCl3, 100 MHz) δ 162.6, 161.6, 153.9,
152.3, 152.2, 145.1, 142.9, 134.4, 130.9, 130.1, 130.1, 129.8, 128.8,
123.4, 118.8, 115.6, 96.2, 84.2, 76.0, 75.9, 53.3, 53.1, 45.7, 42.0.
ESI-MS 520.1, [M + H]+. Anal. Calcd for C27H24N2O7S: C, 62.30;
H, 4.65; N, 5.38. Found: C, 62.37; H, 4.71; N, 5.28.
Dipropargyl 1-((5-(Dimethylamino)naphthalene-1-sulfonami-
do)methyl)-4-methyl-7-oxanorborna-2,5-diene-2,3-dicarboxylate (6).
Prepared according to the general procedure above; the reactants
were heated for 5 h at 80 °C. Yellow foam, 40% yield. Rf 0.27
1
(40% acetone/hexanes). H NMR (CDCl3, 400 MHz) δ 8.55 (app
d, 1H, J ) 8.5), 8.28-8.23 (m, 2H), 7.59-7.50 (m, 2H), 7.19 (app
d, 1H, J ) 7.1), 6.88 (app s, 2H), 5.28 (t, 1H, J ) 6.4), 4.79 (d,
2H, J ) 2.4), 4.62 (2 × dd, 2H, J ) 15.6, 2.4), 3.72 (dd, 1H, J )
13.8, 6.4), 3.67 (dd, 1H, J ) 13.8, 6.6), 2.89 (s, 6H), 2.50 (m, 2H),
1.69 (s, 3H). 13C NMR (CDCl3, 100 MHz) δ 163.2, 162.1, 158.0,
152.2, 150.0, 147.6, 144.3, 134.6, 130.8, 130.1, 130.0, 129.8, 128.7,
123.4, 119.0, 115.5, 94.3, 93.2, 77.0, 75.9, 75.8, 53.1, 53.0, 45.7,
42.2, 15.1. ESI-MS 535.2, [M + H]+. Anal. Calcd for C28H26N2O7S:
C, 62.91; H, 4.90; N, 5.24. Found: C, 62.65; H, 4.99; N, 5.10%.
Ethyl 1-((5-(Dimethylamino)naphthalene-1-sulfonamido)methyl)-
3-trifluoromethyl-7-oxanorborna-2,5-diene-2-carboxylate (9). A
mixture of N-dansylfurfurylamine (370 mg, 1.12 mmol), ethyl 4,4,4-
trifluoro-2-butynoate (250 mg, 1.50 mmol), toluene (2 mL) was
heated at 80 °C with stirring for 18 h. The mixture was concentrated
to half its original volume, loaded directly on a column of silica
9
9992 J. AM. CHEM. SOC. VOL. 131, NO. 29, 2009

Fluorogenic Electrophiles for Thiols
A R T I C L E S
gel, and purified by flash chromatography (0-3% isopropanol/
toluene) to give an oil which contained a minor fluorescent impurity,
inseparable by chromatography. Two recrystallizations from Et2O/
hexanes (2:3 (v/v), 50 mL) afforded the pure compound as a yellow
chromatography (20-40% acetone/hexanes) to yield the desired
product as a yellow solid (52 mg, 37%). Rf 0.40 (40% acetone/
hexanes). 1H NMR (CDCl3, 400 MHz) δ 8.75 (br t, 1H, J ) 4.8),
8.57 (app d, 1H, J ) 8.5), 8.29-8.21 (m, 2H), 7.59-7.51 (m, 2H),
7.22-7.17 (m, 2H), 6.75 (d, 1H, J ) 5.2), 5.65 (d, 1H, J ) 2.0),
5.00 (dd, 1H, J ) 8.3, 4.1), 4.09 (ddd, 2H, J ) 6.2, 4.8, 2.5), 3.96
(dd, 1H, J ) 13.0, 8.3), 3.72 (s, 3H), 3.49 (dd, 1H, J ) 13.0, 4.1),
2.89 (s, 6H), 2.25 (t, 1H, J ) 2.6). 13C NMR (CDCl3, 100 MHz)
δ 164.7, 161.8, 161.4, 152.2, 146.3, 145.8, 141.5, 134.4, 130.9,
130.1, 129.8, 129.8, 128.9, 123.2, 118.6, 115.6, 95.9, 84.6, 79.0,
71.8, 53.0, 45.6, 42.9, 29.4. ESI-MS 496.1, [M + H]+. Anal. Calcd
for C25H25N3O6S: C, 60.59; H, 5.08; N, 8.48. Found: C, 60.52; H,
5.18; N, 8.33%.
1
microcrystalline powder (350 mg, 63%). H NMR (CDCl3, 400
MHz) δ 8.56 (app d, 1H, J ) 8.5), 8.28-8.23 (m, 2H), 7.59-7.51
(m, 2H), 7.21-7.15 (m, 2H), 7.10 (dd, 1H, J ) 5.2, 2.0), 6.95 (d,
1H, J ) 5.2), 5.51 (d, 1H, J ) 2.0), 5.16 (t, 1H, J ) 6.4), 4.17 (q,
1H, J ) 7.0), 4.11 (q, 1H, J ) 7.0), 3.76 (dd, 1H, J ) 13.7, 7.1),
3.62 (dd, 1H, J ) 13.7, 5.8), 2.89 (s, 6H), 1.25 (t, 3H, J ) 7.1).
13C NMR (CDCl3, 100 MHz) δ 162.1, 152.0, 151.5, [150.21,
150.17, 150.12, 150.07] (q), 144.2, 143.1, 134.1, 130.7, 129.8,
129.8, 129.5, 128.5 [125.26, 122.54, 119.86, 117.18] (q, CF3), 123.1,
118.5, 115.2, 96.0, 82.7, 62.0, 45.4, 41.7, 13.7. ESI-MS 497.1, [M
+ H]+. Anal. Calcd for C23H23F3N2O5S: C, 55.64; H, 4.67; N, 5.64.
Found: C, 55.71; H, 4.71; N, 5.54%.
Monosaponification of Dialkyl Oxanorbornadienedicarboxy-
lates 5 (General). To a stirred solution of oxanorbornadiene 5 (1
mmol) in THF (8 mL) was added freshly prepared aqueous NaOH
(0.5 M, 4 mL) dropwise over 10 min at 0 °C. The reaction was
monitored by TLC until the starting material was consumed (ca.
1 h), at which point the solution was acidified with 1 M HCl to pH
3-4 and extracted with ethyl acetate (3 × 10 mL). The combined
organic layers were washed with brine, dried over magnesium
sulfate, and concentrated to obtain the desired pure product.
Ethyl 1-((5-(Dimethylamino)naphthalene-1-sulfonamido)methyl)-
3-propargylcarbamoyl-7-oxanorborna-2,5-diene-2-carboxylate (8b).
The procedure is the same as for 8a, using 7b (87 mg, 0.184 mmol),
propargylamine (11 mg, 0.200 mmol), and DMT-MM (56 mg, 0.202
1
mmol); yield 40 mg (43%). H NMR (CDCl3, 400 MHz) δ 8.76
(br t, 1H, J ) 4.9), 8.56 (d, 1H, J ) 8.5), 8.28-8.21 (m, 2H),
7.59-7.51 (m, 2H), 7.19 (d, 1H, J ) 7.7), 7.17 (dd, 1H, J ) 5.3,
2.0), 6.75 (d, 1H, J ) 5.3), 5.65 (d, 1H, J ) 2.0), 4.99 (dd, 1H, J
) 7.9, 4.5), 4.17 (2 × dq, 2H, J ) 7.1, 3.6), 4.09 (ddd, 2H, J )
6.5, 5.4, 2.6), 3.91 (dd, 1H, J ) 13.1, 7.9), 3.53 (dd, 1H, J ) 13.1,
4.5), 2.89 (s, 6H), 2.24 (t, 1H, J ) 2.6), 1.30 (t, 3H, J ) 7.1). 13
C
NMR (CDCl3, 100 MHz) δ 164.1, 161.2, 161.1, 151.9, 146.4, 145.4,
141.4, 134.1, 130.7, 129.8, 129.6, 129.5, 128.6, 123.0, 118.5, 115.3,
95.7, 84.3, 78.8, 71.6, 62.5, 45.3, 42.6, 29.2, 13.7. ESI-MS
(C26H27N3O6S) 510.1, [M + H]+.
1-((5-(Dimethylamino)naphthalene-1-sulfonamido)methyl)-2-
methoxycarbonyl-7-oxanorborna-2,5-diene-3-carboxylic Acid (7a).
1
Yellow solid, 85%. H NMR (CDCl3, 400 MHz) δ 8.55 (d, 1H, J
) 8.5), 8.27 (d, 1H, J ) 8.5), 8.22 (d, 1H, J ) 8.6), 7.59-7.50
(m, 2H), 7.22 (d, 1H, J ) 7.6), 7.21-7.17 (m, 1H), 6.77 (d, 1H, J
) 5.6), 5.64 (d, 1H, J ) 1.8), 5.14 (t, 1H, J ) 4.8), 3.99 (dd, 1H,
J ) 13.2, 8.4), 3.78 (s, 3H), 3.50 (dd, 1H, J ) 13.2, 4.0), 2.88 (s,
6H). 13C NMR (CD3OD, 100 MHz) δ 165.9, 165.8, 157.8, 153.0,
151.4, 145.9, 143.9, 136.8, 131.3, 131.1, 131.0, 130.2, 129.2, 124.3,
120.6, 116.5, 97.4, 85.3, 52.9, 45.9, 42.9. ESI-MS (C22H22N2O7S)
459.1, [M + H]+.
Thiol Adducts (General). To a solution of oxanorbornadiene
(0.100 mmol) in acetonitrile (0.5 mL) was added a thiol (0.090
mmol), followed by diisopropylethylamine (1 µL) to give an instant
strong increase in fluorescence. On smaller scale, the amine was
introduced as a vapor from a Pasteur pipet. After 5 min, the reaction
was quenched by the addition of acetic acid (5 µL), concentrated,
and purified by flash chromatography or preparative TLC.
Compound 10a. Yellow foam, yield 95%, >90% purity. Rf 0.17
(1:1 EtOAc/hexanes). 1H NMR (CDCl3, 400 MHz) δ 8.56 (d, 1H,
J ) 8.5), 8.28-8.21 (m, 2H) (m, 2H), 7.58-7.51 (m, 2H), 7.19
(d, 1H, J ) 7.5), 6.42 (d, 1H, J ) 5.7), 6.36 (dd, 1H, J ) 5.7, 1.7),
5.04 (t, 1H, J ) 6.4), 4.83 (d, 1H, J ) 1.7), 3.72 (m, 2H), 3.63 (s,
3H), 3.61 (s, 3H), 3.48 (dd, 1H, J ) 13.8, 6.0), 3.38 (dd, 1H, J )
13.8, 6.8), 3.18 (s, 1H), 2.94 (dt, 2H, J ) 11.7, 5.5), 2.89 (s, 6H),
1.97 (m, 1H). 13C NMR (CDCl3, 100 MHz) δ 169.9, 169.7, 152.2,
137.7, 135.5, 134.4, 130.9, 130.1, 129.9, 129.7, 128.8, 129.7, 128.8,
123.3, 118.8, 115.5, 91.2, 84.7, 64.7, 60.9, 57.5, 52.8, 52.5, 45.6,
43.3, 34.7. ESI-MS (C25H30N2O8S2) 551.1, [M + H]+.
1-((5-(Dimethylamino)naphthalene-1-sulfonamido)methyl)-2-
ethoxycarbonyl-7-oxanorborna-2,5-diene-3-carboxylic Acid (7b).
1
Yellow solid, 80%. H NMR (CDCl3, 400 MHz) δ 8.55 (d, 1H, J
) 8.5), 8.27 (d, 1H, J ) 8.5), 8.22 (d, 1H, J ) 8.6), 7.60-7.53
(m, 2H), 7.22 (d, 1H, J ) 7.6), 7.18-7.21 (m, 1H), 6.82 (d, 1H, J
) 5.3), 5.66 (d, 1H, J ) 2.0), 4.96 (t, 1H, J ) 4.8), 4.32 (dd, 1H,
J ) 10.9, 7.2), 4.24 (dd, 1H, J ) 10.8, 7.2), 3.95 (dd, 1H, J )
13.2, 7.9), 3.56 (dd, 1H, J ) 13.2, 4.7), 2.91 (s, 6H), 1.36 (t, 1H,
J ) 7.2). 13C NMR (MeOD, 100 MHz) δ 165.5, 165.3, 156.8, 152.8,
152.75, 145.9, 143.9, 136.8, 131.2, 131.0, 130.9, 130.2, 129.2,
124.4, 120.7, 116.5, 97.45, 85.2, 12.9, 45.9, 42.8, 14.2. ESI-MS
(C23H24N2O7S) 473.1, [M + H]+.
Compound 10b. Yellow solid, yield 80%, >90% purity. 1H NMR
(CDCl3, 400 MHz) δ 8.56 (d, 1H, J ) 8.5), 8.32-8.26 (m, 2H),
7.58-7.51 (m, 2H), 7.19 (d, 1H, J ) 7.3), 6.41 (d, 1H, J ) 5.7),
6.36 (dd, 1H, J ) 5.6, 1.3), 4.99 (t, 1H, J ) 6.4), 4.83 (d, 1H, J )
1.4), 4.15 (q, 2H, J ) 7.2), 3.63 (s, 3H), 3.60 (s, 3H), 3.47 (dd,
1H, J ) 13.8, 6.2), 3.34 (dd, 1H, J ) 13.8, 6.6), 3.18 (s, 1H), 2.94
(dt, 2H, J ) 11.7, 5.5), 2.89 (s, 6H), 2.55 (t, 2H, 7.3), 1.27(t, 3H,
7.2). 13C NMR (CDCl3, 100 MHz) δ 171.3, 169.8, 169.3, 152.0,
137.5, 135.3, 134.1, 130.7, 129.9, 129.8, 129.7, 128.6, 123.1, 118.6,
115.3, 90.9, 84.2, 64.7, 60.8, 57.3, 52.5, 52.2, 45.4, 43.1, 33.7, 26.3,
14.2. ESI-MS (C28H34N2O9S2) 607.1, [M + H]+.
1-((5-(Dimethylamino)naphthalene-1-sulfonamido)methyl)-2-
propargyloxycarbonyl-7-oxanorborna-2,5-diene-3-carboxylic Acid
1
(7c). Yellow solid, 85%. H NMR (CDCl3, 400 MHz) δ 8.55 (d,
1H, J ) 8.5), 8.35 (d, 1H, J ) 8.7), 8.32 (d, 1H, J ) 7.3), 7.64-7.59
(m, 2H), 7.34 (d, 1H, J ) 7.6), 7.18-7.21 (m, 1H), 6.82 (d, 1H, J
) 5.3), 5.61 (d, 1H, J ) 2.0), 5.07 (t, 1H, J ) 4.8), 4.83 (dd, 1H,
J ) 15.5, 2.5), 4.69 (dd, 1H, J ) 15.5, 2.5), 3.98 (dd, 1H, J )
13.8, 8.0), 3.62 (dd, 1H, J ) 13.8, 4.9), 3.03 (s, 6H), 2.63 (t, 1H,
J ) 2.4). 13C NMR (CD3OD, 100 MHz) 165.2, 164.2, 151.6, 145.9,
143.9, 137.2, 131.0, 130.5, 130.4, 129.1, 124.9, 122.0, 117.1, 97.7,
85.3, 78.0, 77.0, 53.8, 46.1, 42.7. ESI-MS (C24H22N2O7S) 483.1,
[M + H]+.
Compound Me-10a. Yellow foam, yield 90%, >90% purity. Rf
1
0.30 (2:1 EtOAc/hexanes). H NMR (CDCl3, 400 MHz) δ 8.55
(app d, 1H, J ) 8.5), 8.32 (app d, 1H, J ) 8.7), 8.12 (app d, 1H,
J ) 7.3), 7.59-7.49 (m, 2H), 7.19 (d, 1H, J ) 7.5), 6.61 (d, 1H,
J ) 5.6), 6.42 (dd, 1H, J ) 5.6, 1.2), 4.89 (d, 1H, J ) 1.2), 3.90
(d, 1H, J ) 5.6), 3.86 (d, 1H, J ) 4.4), 3.79-3.74 (m, 2H), 3.71
(s, 3H), 3.66 (s, 3H), 3.41 (s, 1H), 3.08-3.03 (m, 2H), 2.92 (s,
3H), 2.88 (s, 6H). 13C NMR (CDCl3, 100 MHz) δ 170.1, 169.9,
151.9, 138.5, 135.1, 133.9, 130.7, 130.4, 130.3, 130.0, 128.4, 123.3,
119.7, 115.4, 93.0, 85.0, 64.5, 61.0, 57.9, 52.8, 52.5, 49.6, 45.6,
36.7, 34.8. ESI-MS (C26H32N2O8S2) 565.2, [M + H]+.
Methyl 1-((5-(Dimethylamino)naphthalene-1-sulfonamido)methyl)-
3-propargylcarbamoyl-7-oxanorborna-2,5-diene-2-carboxylate (8a).
To a suspension of acid 7a (130 mg, 0.283 mmol) and 4-(4,6-
dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride hy-
drate (87 mg, 0.314 mmol) in THF (1.5 mL) was added propar-
gylamine (16 mg, 0.29 mmol). The reaction mixture was stirred
for 4 h at room temperature, concentrated to dryness, taken up in
CH2Cl2, loaded on a column of silica, and purified by flash
9
J. AM. CHEM. SOC. VOL. 131, NO. 29, 2009 9993

A R T I C L E S
Hong et al.
Compound 12. Yellow solid, yield 83%, >90% purity. 1H NMR
(CDCl3, 400 MHz) δ 8.56 (app d, 1H, J ) 8.5), 8.33-8.26 (m,
2H), 7.60-7.53 (m, 2H), 7.19 (app d, 1H, J ) 7.5), 6.40 (dd, 1H,
J ) 5.7, 1.5), 6.19 (d, 1H, J ) 5.8), 5.36 (t, 1H, J ) 6.3), 5.09 (d,
1H, J ) 1.5), 4.05 (d, 2H, J ) 2.4), 3.84-3.73 (m, 2H), 3.66 (dd,
1H, J ) 13.8, 6.1), 3.53 (dd, 1H, J ) 13.8, 6.6), 3.06-3.01(m,
2H), 2.91 (s, 1H), 2.89 (s, 6H), 2.17 (t, 1H, J ) 2.5). 13C NMR
(CDCl3, 100 MHz) δ 173.5, 171.2, 152.2, 136.6, 135.9, 134.2,
131.0, 130.3, 130.1, 129.7, 128.8, 123.4, 118.7, 115.4 91.0, 84.3,
75.6, 72.0, 61.8, 59.8, 55.6, 45.6, 43.8, 33.7, 28.2. ESI-MS
(C26H27N3O6S2) 542.2, [M + H]+.
Compound 16. To a solution of tris-(benzyltriazolylmethyl)
amine (11 mg, 0.02 mmol), 5-dimethylamino-naphthalene-1-
sulfonic acid (3-azidopropyl) amide (23 mg, 0.069 mmol), and 8b
(35 mg, 0.069 mmol) in DMSO (1.6 mL) was added CuSO4 (0.2
mL, 50 mM) and sodium ascorbate (0.2 mL, 100 mM). After 2 h,
water was added to the reaction to form a pale-yellow solid which
was filtered, washed with water, and purified by preparative TLC
(3% MeOH/CHCl3) to yield the desired product (44 mg, 77%). Rf
0.35 (3% MeOH/CDCl3). 1H NMR (CDCl3, 500 MHz) δ 7.63-7.53
(m, 4H), 7.44 (s, 1H, triazole), 7.38-7.35 (m, 1H, CONH),
7.28-7.22 (m, 2H), 7.12 (dd, J ) 5.2, 1.9, 1H, OND-H5), 6.82 (d,
J ) 5.2, 1H, OND-H6), 5.97-5.90 (m, 2H, SO2NH), 5.49 (d, J )
1.9, 1H, OND-H4), 4.44 (dd, J ) 15.9, 5.3, 1H, CH2NHCO), 4.41
(dd, J ) 15.9, 5.3, 1H, CH2NHCO), 4.21 (t, J ) 6.9, 2H), 4.00 (m,
2H), 3.72 (dd, J ) 14.0, 7.0, 1H, OND-CH2NH), 3.72 (dd, J )
14.1, 5.6, 1H, OND-CH2NH), 2.85 (s, 6H), 2.84 (s, 6H), 2.79 (m,
2H), 1.88 (quint, J ) 7.8, 2H) 1.08 (t, J ) 7.1, 3H). 13C NMR
(CDCl3, 125 MHz) δ 165.0, 162.7, 161.6, 153.1, 153.1, 146.8,
145.8, 145.0, 143.2, 136.1, 136.1, 131.3, 131.2, 130.7, 130.4, 130.3,
130.3, 129.2, 124.4, 124.3, 123.6, 119.8, 116.3, 100.6, 96.9, 85.3,
80.5, 63.0, 47.9, 45.7, 43.3, 40.9, 35.7, 30.8, 14.0. ESI-MS
(C39H42N8O8S2) 843.2, [M + H]+.
Peptide Synthesis. The peptide 15 was prepared by standard Fmoc
methods using Rink amide MBHA resin with an initial loading of
0.70 mmol/g. The resin was swollen in DMF for 45 min prior to
synthesis. For sequence extension, the Fmoc-protected amino acid (4
equiv) was activated by treatment with HCTU (3.9 equiv) and N,N-
diisopropylethylamine (500 µL per mmol of amino acid) in DMF (4
mL per mmol of amino acid) for 2 min. This solution was added to
the free amine on resin, and the coupling reaction was allowed to
proceed for 15 min (longer for difficult couplings) with intermittent
stirring. After washing with DMF, Fmoc deprotection was achieved
with 20% 4-methylpiperidine in DMF (2 × 7 min). The resin was
washed again and the process was repeated for the next amino acid.
The symmetric anhydride of 2-bromoacetic acid (5 equiv of
anhydride relative to free amine) was prepared by mixing the acid
(10 equiv) with N,N′-diisopropylcarbodiimide (DIC, 5 equiv) in DMF
at room temperature for 15 min. This mixture was then added to the
resin and allowed to react for 1 h with intermittent stirring. The resin
was washed and then treated with propargylamine (20 equiv) in DMF
for 12-16 h. After washing, coupling of the next residue to the
resultant secondary amine was achieved by preparing the symmetrical
anhydride of that protected residue (5 equiv of anhydride) with DIC
in DMF at 0 °C for 15 min, and reacting this mixture with the amine
for 1 h with occasional stirring. The following Fmoc-protected amino
acids with side chain protecting groups were used as received from
NovaBiochem: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp-
(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Ile-OH,
Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH.
The peptide was cleaved from the resin with 2% triisopropylsilane
(TIS) in trifluoroacetic acid (TFA, approximately 1 mL TFA/TIS per
g resin) for 2.5 h. The cleavage mixture (including resin) was mixed
with cold ether to precipitate the peptide and then filtered. The filtrate
was washed with cold ether, and 15 was extracted from the residue
with H2O/MeCN mixtures (typically 5-20% MeCN in water) contain-
ing 0.1% TFA. The resulting solution was frozen and lyophilized to
afford a colorless, solid product.
Reactivity. Stability Study at pH 7 (Table 1). One milliolar
stock solutions of each OND compound in DMSO (100 µL) were
diluted with 0.1 M phosphate buffer (pH 7, 900 µL) at time 0 to initiate
the experiment. At different time points, aliquots (50 µL) were injected
on the HPLC, with peak detection at 332 nm.
Stability Study of the ꢀ-Mercaptoethanol Adducts (Table
1). A solution of each adduct was created by incubating 0.1 mM of
the OND electrophile with an equimolar amount of ꢀ-mercaptoethanol
in 100 mM phosphate buffer, pH 7, for 1 h. HPLC analysis (peak
detection at 332 nm) verified the complete formation of adduct in each
case. These solutions were then allowed to stand at room temperature
with periodic HPLC analysis, following the disappearance of the adduct
peak with time. First-order rate constants were obtained by a standard
linear plot of ln[adduct] vs time.
Selectivity of 5b with Amino Acids (Figure 3). One milliolar
stock solutions of 5b in DMSO (100 µL) were diluted with 100 mM
phosphate buffer (pH 7, 800 µL). Each amino acid (0.1 mmol) was
dissolved in 100 mM phosphate buffer (pH 7, 10 mL), and 10 µL of
this solution was added to a solution of 5b (90 µL). The fluorescence
intensity (λex 332 nm, λem 550 nm) was monitored every 30 s over 60
min.
Rate Constant Determination of 5, 6, 8, and 9 with Glu-
tathione at pH 7 (Table 1). Stock solutions of each OND compound
(1 mM) in DMSO (100 µL) were diluted with 100 mM phosphate
buffer (pH 7, 800 µL). Glutathione (1.1 mM, 10 µL) was added to
these reagent solutions (111 µM, 90 µL) to final concentrations of
110 µM and 100 µM, respectively. The fluorescence intensity (λex 332
nm, λem 550 nm) was recorded every 30 s. Each reaction reached the
same maximum emission intensity, which was the same as displayed
by a freshly prepared 100 µM solution of the authentic adduct of 5a
under the same conditions, and was therefore assumed to represent
100% completion. Rate constants were determined by a standard fit
of % completion (ratio of fluorescence intensity at time t to the
maximum intensity) versus time to the integrated second-order rate
equation; error values represent the standard deviation of three
independent runs.
Rate Constant Determination of 7c with Glutathione, Cys-
teine, And Cysteine Methyl Ester. One millimolar stock solutions
of 7c in DMSO (100 µL) were diluted with buffer (800 µL) (100 mM
phosphate buffer at pH 7, 25 mM citrate at pH 4, 5, or 6). Thiol (1.1
mM, 100 µL) was added to these solutions of 7c (111 µM, 900 µL)
to final concentrations of 110 µM and 100 µM, respectively. Aliquots
(150 µL) withdrawn at different time points were quenched with ethyl
maleimide (1 µL, 100 mM), diluted with water containing 0.1% TFA
(250 µL), and injected on the HPLC, with peak detection at 332 nm.
Peptide and BSA Labeling (Figures 5, and 6). The OND
electrophile (10 mM in DMSO, 10 and 5 µL, respectively) was added
to peptide 15 (1 mM in 0.1 M phosphate buffer, pH 7, 90 µL) or
BSA (1 mg/mL, 1 mL). Assuming a molar absorptivity similar to that
of the corresponding ꢀ-mercaptoethanol adduct, a labeling density of
1.0 ( 0.2 attachments per BSA molecule was found in each case.
Pretreatment of the protein with N-ethylmaleimide eliminated the
reaction with OND reagent.
Acknowledgment. We thank The Skaggs Institute for Chemical
Biology and the National Institutes of Health (RR021886) for financial
support, Dr. Reshma Jagasia for the synthesis of peptide 15, and Dr.
Michal Sabat (University of Virginia) for the X-ray structural analysis
of 5c.
Supporting Information Available: X-ray crystal structure of
5c, kinetic data for 7c, HPLC and MALDI data for the reactions
with peptide 15, and 1H NMR spectra of 3, 5a, and thiol adduct
10a. This material is available free of charge via the Internet at
http://pubs.acs.org.
JA809345D
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9994 J. AM. CHEM. SOC. VOL. 131, NO. 29, 2009