New tetramethylthiepinium (TMTI) for copper-free click chemistry

Article abstract of DOI:10.1039/c2cc35034c

A new derivative of the strained 3,3,6,6-tetramethylthiacycloheptyne (TMTH) bearing a functional handle is reported. Following an optimized synthesis, the handle was introduced by mild alkylation of the sulphur atom. The resulting functionalized strained 4,5-didehydro-3,3,6,6-tetramethyl-2,3,6,7- tetrahydrothiepinium (TMTI) proved to be stable and underwent extremely fast [3+2] cycloaddition reaction with benzyl azide in both organic and aqueous solvents. The reaction was equally efficient in cell lysate and serum and therefore opens interesting prospects for chemical-biology applications.

Full text of DOI:10.1039/c2cc35034c

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New tetramethylthiepinium (TMTI) for Copper-Free Click Chemistry
Mathias King,a Rachid Baati*a and Alain Wagner*a
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
45 introduction, two strategies seemed feasible to us, modification of
the sterically protective methyl groups or direct alkylation of the
sulphur atom of TMTH. This last strategy appealed to us most not
only for the conservation of the molecular symmetry, increased
water solubility due to the charged molecule and the converꢀ
50 gence of its synthesis, but also for the prospect that this slight
change in CꢀS bond length between thioether and sulfonium
should not strain the structure past its limit and yield a stable
class of compounds.7
When we set to the task of synthesizing TMTH, we
55 predominantly followed the route set out by Krebs and Kimling.6
In the progress of this synthesis we came up with a slight
improvement, when we were able to replace the tedious Deanꢀ
Stark conditions by a swift microwave assisted setup while
maintaining the high yield (see supplementary informations). The
60 first synthesis strategies we followed to prepare the desired
sulfoniums were based on literature procedures originating for
alkyl bromides8 and iodides9 in mild room temperature conditions
in order to reduce the stress on the volatile TMTH. As these
conditions proved ineffective even after heating to 50°C for
65 several hours, we turned to strategies based on replacing the
halide counter ion by triflate10 or tetrafluoroborate.11 While the
triflate strategy with LiOTf relies mainly on the triflates superior
leaving group characteristics,12 the tetrafluoroborate reaction with
AgBF4 is driven by the insolubility of the generated silver
5
A
new
derivative
of
the
strained
3,3,6,6-
tetramethylthiacycloheptyne (TMTH) bearing a functional
handle is reported. Following an optimized synthesis, the
handle was introduced by mild alkylation of the sulphur
atom. The resulting functionalized strained 4,5-didehydro-
10 3,3,6,6-tetramethyl-2,3,6,7-tetrahydrothiepinium
(TMTI)
proved to be stable and underwent extremely fast [3+2]
cycloaddition reaction with benzyl azide in both organic and
aqueous solvents. The reaction was equally efficient in cell
lysate and serum and therefore opens interesting prospects
15 for chemical-biology applications.
Recently bioorthogonal chemistry moved into the focus of
attention in the scientific community for its possibility to insert
desirable probes into a variety of biological targets in vitro as
well as in vivo. As the spearhead of this development serves the
20 Copperꢀfree click reaction and its impressive collection of
strained cycloalkynes designed especially for this purpose1–3
shown in a variety of applications4,5. To date overwhelming
number of reactive probes contains the 8ꢀmembered cyclooctyne
as it is the reagent best balanced between stability and reactivity.
25 Our bibliographic survey revealed that more than 40 years ago
Krebs and Kimling described an isolatable 7ꢀmembered
thiacycloheptyne6, as well as its “explosive” reactivity towards
benzyl azide. This attracted our attention even though the
described thiacycloheptyne has one drawback for the modern
30 application of bioorthogonal ligation, which is the absence of any
anchor point where a probe for any kind of purpose (fluorescence
imaging, ABPP, surface immobilization etc) could be attached to
the reactive moiety. The currentness of this problem was
highlighted, when during our preparation of this article two works
35 were published confirming the interest of thiacyclooctynes and ꢀ
heptynes as dipolarophile partner in biocompatible SpAAC like
reactions. Nonetheless, Bertozzi concluded her work with the
deduction that the original thiacycloheptyne remains
insurmounted in its balance between reactivity and stability.
40 Therefore the need for attaching an anchor point to this molecule
in order to make it utilizable in modern application while
maintaining its delicate balance appeared even more pressing to
us.
Scheme 1: Sulfonium synthesisa
When we turned to the problematic of anchor point
a Laboratory of Functional ChemoꢀSystems UMR 7199, 74 Route du
Rhin, 67401, Illkirch, France. Eꢀmail: alwag@unistra.fr;
Fax: +33 368854306
Electronic supplementary information (ESI) available. See DOI:
This journal is © The Royal Society of Chemistry [year]
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bromide. Finally the triflate strategy was successful and yielded
the desired TMTI 3.
We would like to thank the European Union (FP7ꢀITNꢀ238434
BioChemLig) for the funding that supported this work.
Two different substrates bearing potential anchor points were
investigated, an electron rich pꢀmethoxy benzyl bromide and an
electron deficient pꢀmethoxycarbonyl benzyl bromide (table 1).
While the electron rich benzyl bromide proofed to yield only very
modest product quantities, the electron poor aromatic system
allowed us to isolate the desired product in a reasonable yield of
64%. Noteworthy about the purification of 3a-b is the fact that
1.
60 2.
3.
J. Tummatorn, P. Batsomboon, R. J. Clark, I. V. Alabugin, and
G. B. Dudley, Journal of Organic Chemistry, 2012, 77, 2093ꢀ7.
M. R. Karver, R. Weissleder, and S. a Hilderbrand, Angew.
Chem., Int. Ed., 2012, 51, 920ꢀ2.
G. de Almeida, E. M. Sletten, H. Nakamura, K. K. Palaniappan,
and C. R. Bertozzi, Angew. Chem., Int. Ed., 2012, 1ꢀ6.
K. E. Beatty, J. Szychowski, J. D. Fisk, and D. a Tirrell,
ChemBioChem, 2011, 12, 2137ꢀ9.
5
4.
65
10 they were both isolated and purified by conventional silica gel
column chromatography and exhibited reasonable stability in
aqueous solution and oxygen containing atmosphere (see SI).
Furthermore we were also able to proof good solubility of 3b at
5mM in a 60% PBS solution and a reasonable stability versus
15 acids, amines and alcohols.
5.
T. Plass and C. Schultz, Advanced Fluorescence Reporters in
Chemistry and Biology III, Springer Berlin Heidelberg, Berlin,
Heidelberg, 2011, vol. 113.
A. Krebs and H. Kimling, Tetrahedron Lett., 1970, 761ꢀ764.
F. H. Allen, O. Kennard, D. G. Watson, L. Brammer, A. G.
Orpen, and R. Taylor, J. Chem. Soc., Perkin Trans. 2, 1987, S1.
I. Stahl, S. Schomburg, and H. O. Kalinowski, Chem. Ber.,
1984, 117, 2247ꢀ2260.
H. Paulsson, M. Berggrund, E. Svantesson, A. Hagfeldt, and L.
Kloo, Sol. Energy Mater. Sol. Cells, 2004, 82, 345ꢀ360.
M. a Lowe, M. Ostovar, S. Ferrini, C. C. Chen, P. G. Lawrence,
F. Fontana, A. a Calabrese, and V. K. Aggarwal, Angew.
Chem., Int. Ed., 2011, 50, 6370ꢀ4.
6.
70 7.
8.
In the next step we investigated the reactivity of the pꢀ
methoxycarbonyl derivative 3b versus benzyl azide. The results
of the kinetic experiments proofed that the standard NMR setup
is not suited to determine the second order rate constant of this
20 particular reagent as the experiment after 1:30 minutes revealed a
conversion of >80%. Nonetheless we were able to ascertain that
the reaction speed of TMTI 3b and benzyl azide is comparable to
TMTH and benzyl azide by direct injection of azide into the
NMR sample inside the NMR instrument. By this method we
9.
75
10.
11.
12.
Y. Miyake, A. Oyamada, Y. Nishibayashi, and S. Uemura,
Heteroat. Chem., 2002, 13, 270ꢀ275.
A. Streitwieser, Jr, C. L. Wilkins, and E. Kiehlmann, J. Am.
Chem. Soc., 1968, 90, 1598ꢀ1601.
80
25 received a second order rate constant for TMTI of 1.8 ꢀ1sꢀ1,
M
which makes it comparable to TMTH, recently published by
Bertozzi3. In order to insure the suitability of the reagent for
applications in chemical Biology we investigated the reaction in
various aqueous conditions. A 5 mM solution of 3b in 60% PBS
30 and 40% DMSO was treated with an equimolar amount of benzyl
azide and monitored via HPLC. We were pleased to see that the
reaction is neither poisoned nor hampered by water nor were any
significant traces of side products detectable. Injection into HPLC
showed complete disappearance of the starting material and
35 conversion into the corresponding product after 10 min.
Encouraged by this result we turned to investigate the reaction in
biological media in a similar setup. Again we were pleased to
find that neither foetal bovine serum nor HaCaT cell lysate
interfered with the reaction, while the speed of the reaction was
40 unchanged high so that injection into HPLC after 10 min proofed
total conversion.
Conclusions
We herein reported the synthesis of
a sulfonium based
thiacycloheptyne derivative TMTI containing an anchor point for
45 derivatisation. We proved its good stability toward amines,
alcohols and carboxylic acids as well as high reactivity toward
benzyl azide. The kinetic of the cycloaddition was found to be at
similar level that of the parent TMTH structure. We furthermore
were able to proof the suitability of the reagent for aqueous
50 conditions as well as the tolerance of the reaction to biological
media. Therefore we conclude that 3b appears to be a very
promising candidate for bioorthogonal ligation reactions as it
exhibits good stability towards oxygen and biological media like
cyclooctyne derivatives while it exceeds them in reactivity.
55 Notes and references
2
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