Thiacycloalkynes for copper-free click chemistry

Article abstract of DOI:10.1002/anie.201106325

The heteroatom helps! The introduction of an endocyclic sulfur atom enables fine-tuning of the reactivity and stability of thiacycloalkynes for copper-free click chemistry. The stabilizing effect of the endocyclic sulfur atom allows the use of highly activated seven-membered rings as reagents for bioorthogonal copper-free click chemistry. Copyright

Full text of DOI:10.1002/anie.201106325

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DOI: 10.1002/anie.201106325
Bioorthogonal Reagents
Thiacycloalkynes for Copper-Free Click Chemistry**
Gabriela de Almeida, Ellen M. Sletten, Hitomi Nakamura, Krishnan K. Palaniappan, and
Carolyn R. Bertozzi*
Bioorthogonal chemistry enables the interrogation of bio-
molecules and physiological processes that are inaccessible by
using conventional research tools.[1] A common experimental
protocol starts by labeling a target biomolecule in cells or live
organisms with a bioorthogonal functional group. Then,
a probe molecule bearing complementary functionality is
added to the system and the ensuing bioorthogonal chemical
reaction delivers the probe specifically to the targets of
interest. For many applications, rapid reaction kinetics are
essential. This is particularly true for labeling experiments in
live animals, in which reagent concentrations are limited (i.e.,
nm to low mm), or in which the process that is probed occurs on
a fast time scale. Consequently, methodologists interested in
the development of bioorthogonal reactions are increasingly
focused on kinetic optimization.[2]
The strain-promoted cycloaddition reaction of azides and
cyclooctynes, also called Cu-free “click” chemistry, is a bio-
orthogonal reaction that is well-tolerated by cells and the
model organisms zebrafish, Caenorhabditis elegans, and mice
(Scheme 1a).[3] The development of this reaction was inspired
by the classic work of Wittig and Krebs in the 1960s.[4] They
observed that, in contrast to unactivated linear alkynes, which
undergo 1,3-dipolar cycloaddition with azides only at elevated
temperatures, cyclooctyne readily reacts with the same
substrates at room temperature. The heightened reactivity
of cyclooctyne was attributed to approximately 19 kcalmolÀ1
Scheme 1. Copper-free click-chemistry probes. a) Azide-labeled biomol-
of ring strain resulting from deformation of the bond angles of
ecules can be selectively labeled with cyclooctyne reagents; b) an array
the alkyne from 1808 to 1588.[5]
of cyclooctynes for copper-free click chemistry; c) thiacycloalkynes
reported herein.
Capitalizing on this classic work, we and other research
groups have reported numerous cyclooctyne analogues, which
in comparison to linear alkynes display accelerated reaction
rates with benzyl azide because of two major rate-enhancing
modifications. Starting from the prototype OCT (1,
Scheme 1b),[6] electronic activation was achieved through
the addition of a gem-difluoro group as exemplified by DIFO
(2). This compound reacts with benzyl azide with a second-
order rate constant of k = 7.6 ꢀ 10À2 mÀ1 sÀ1, which is 33 times
higher than that exhibited by the parent compound OCT
(1).[7] The reactivity of cyclooctyne can also be enhanced by
augmenting ring strain through the addition of fused phenyl
or cyclopropyl rings as demonstrated by DIBO (3, k = 5.7 ꢀ
10À2 mÀ1 sÀ1)[8] and BCN (4, k = 0.14mÀ1 sÀ1), respectively.[2i]
The further addition of sp2-like centers as in DIBAC (5, k =
0.31mÀ1 sÀ1)[2d] and BARAC (6, k = 0.96mÀ1 sÀ1)[2c] has led to
reagents that are three orders of magnitude more reactive
than OCT (1).
[*] G. de Almeida, E. M. Sletten, H. Nakamura, K. K. Palaniappan,
Prof. C. R. Bertozzi
Department of Chemistry, University of California
Berkeley, CA 94720 (USA)
E-mail: crb@berkeley.edu
Prof. C. R. Bertozzi
Department of Molecular and Cell Biology
and Howard Hughes Medical Institute, University of California
Berkeley, CA 94720 (USA)
These experimental results, as well as a number of
computational studies performed in parallel,[9] have paved
the way for the design of highly reactive cyclooctynes through
a combination of activating factors. Unfortunately, unwanted
side reactivity can result from such modifications. As an
example, we sought to combine electronic activation with
ring-strain enhancement by designing DIFBO (7).[10]
[**] E.M.S. was supported by a predoctoral fellowship from the
American Chemical Society Division of Organic Chemistry (Gen-
entech). H.N. was partially supported by the Amgen Scholars
Program. This work was supported by a grant to C.R.B. from the
NIH (GM58867).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201106325.
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Although the compound reacted with azides quite rapidly
(k = 0.22mÀ1 sÀ1), it also underwent spontaneous trimerization
in solution. Notably, compounds such as BARAC react even
more rapidly with azides, yet are otherwise quite stable.
Goddard and coworkers proposed that the ortho-flagpole
hydrogen atoms of biarylcyclooctynes such as BARAC can
engage in unfavorable steric interactions with reaction
partners to disfavor certain reaction pathways.[9a] Thus, the
unusual combination of reactivity with azides and stability
toward oligomerization may reflect a finely-tuned balance
between the activating and stabilizing effects of the fused aryl
rings of BARAC.
From this perspective, another approach to designing fast
but stable cycloaddition substrates is to start with compounds
such as DIFBO (7) and selectively eliminate their unwanted
reactivities through the introduction of stabilizing modifica-
tions. One type of stabilizing perturbation that, unlike fused
aryl rings, would not introduce steric bulk near the reaction
center is an expansion of ring size. Indeed, the stabilities of
cycloalkynes are stongly influenced by ring size, with cyclo-
heptynes being unisolable at room temperature because of
rapid oligomerization, and cyclononynes being several orders
of magnitude less reactive with azides than cyclooctynes.[11]
We thought that a more subtle expansion of the cyclooctyne
ring with a concomitant reduction in ring strain might be
achieved by replacing a carbon atom with a larger sulfur
Scheme 2. Synthesis of thiaOCT. AIBN=2,2’-azobisisobutyronitrile,
DMDO=2,2-dimethyldioxirane, HMDS=1,1,1,3,3,3-hexamethyldisil-
azide, LDA=lithium diisopropylamide, Piv=pivaloyl, Tf=trifluorome-
thylsulfonyl, THF=tetrahydrofuran, TIPS=triisopropylsilyl.
3
3
À
atom. The length of a canonical C(sp ) C(sp ) bond is 1.54 ꢁ,
3
3
À
whereas the length of a C(sp ) S(sp ) bond is closer to 1.81 ꢁ;
this difference is significant, but more conservative than ring-
size augmentation.
than that of the parent compound OCT. The small ring
expansion achieved by the substitution with a sulfur atom
caused a significant reduction in dipolar-cycloaddition reac-
tivity.[14]
Herein, we explore the reactivities and stabilities of
thiacycloalkynes. We found that incorporation of a sulfur
atom into the cyclooctyne ring improves overall reagent
stability but also reduces azide cycloaddition kinetics. We
then combined this stabilizing modification with ring con-
traction, an activating feature previously unexplored in the
context of Cu-free click chemistry, and identified a known
thiacycloheptyne that is stable under ambient conditions but
reacts with model azides faster than any reported cyclooctyne.
These results suggest that thiacycloheptynes constitute
a promising class of reagents for Cu-free click chemistry.
To assess the effects of an endocyclic sulfur atom on
cyclooctyne reactivity, we first synthesized thiaOCT (8), the
sulfur-containing analogue of OCT (1), from 1,6-dienyl-4-
oxysilane 12 in eight steps (Scheme 2). Diene 12 was
monoepoxidized by treatment with dimethyldioxirane[12]
and the resulting monoalkene 13 underwent a thiol-ene
reaction with thioacetic acid to give 14. Treatment of 14 with
sodium hydride resulted in deprotection of the thiol and
subsequent intramolecular cyclization to afford compound 15.
Protection of the free hydroxy group in 15 as a pivaloyl ester
followed by removal of the TIPS group and oxidation with
Dess–Martin periodinane gave ketone 17.[13] Compound 17
was transformed to the desired thiacyclooctyne 8 by syn
elimination of vinyl triflate 18.
We next explored whether an endocyclic sulfur atom
could improve the stability of DIFBO (7) by tempering the
activating effects of the fused aryl ring and propargylic gem-
difluoro group. ThiaDIFBO (9) was prepared similarly to
DIFBO (7) and relied on a key ring expansion of 19 by using
TMSCHN2 (see Scheme 3, and Scheme S1 in the Supporting
Information). We were pleased to find that, unlike DIFBO,
thiaDIFBO did not oligomerize during concentration and
storage, thus confirming the dramatic stabilizing effect of the
endocyclic sulfur atom. The second-order rate constant for
cycloaddition with benzyl azide in CD3CN was found to be
1.4 ꢀ 10À2 mÀ1 sÀ1, almost twenty times lower than that of
DIFBO (see Figure S2 in the Supporting Information).[10]
Notably, this rate constant is comparable to that of previously
reported monobenzocyclooctyne MOBO,[10] which is identical
We measured the second-order rate constant for the
cycloaddition reaction of thiaOCT and benzyl azide in
CD3CN (3.2 ꢀ 10À4 mÀ1 sÀ1, see Figure S1 in the Supporting
Information), and it was almost one order of magnitude lower
Scheme 3. Retrosynthetic analysis of thiaalkynes 9 and 11.
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to DIFBO but lacks the fluorine atoms. The similar reac-
tivities of these two compounds suggest that the substantial
activating effect of the gem-difluoro group is directly bal-
anced by the stabilizing influence of the sulfur atom.
We next tested the reactivity of TMTH with azide-
functionalized biomolecules. Indirect evidence of the ability
of TMTH to selectively label azide-modified glycoproteins
was obtained in an azide-blocking experiment with TMTH
and phosphine–FLAG, which reacts with azides through
a Staudinger ligation.[18] Jurkat cells were treated with
peracetylated N-azidoacetylgalactosamine (Ac4GalNAz),
which metabolically labels cell-surface glycoproteins with
GalNAz residues and intracellular proteins with GlcNAz
residues.[19] Cell lysates were generated and incubated with
TMTH at concentrations ranging from 1 nm to 1 mm for
1.5 hours at room temperature. The progress of the reaction
was determined by the detection of unreacted azides with
500 mm phosphine–FLAG (Figure 1a). Western-blot analysis
The stability of thiaDIFBO provided an opportunity to
enhance reactivity through a more dramatic structural
modification: the contraction to a seven-membered thiacy-
cloheptyne ring, a motif that has not yet been explored in the
context of bioorthogonality. Krebs and Kimling reported in
the 1970s that 3,3,6,6-tetramethylthiacycloheptyne (TMTH,
10) is indeed a stable cycloalkyne, and that it is known to react
with phenyl azide and other 1,3 dipoles in a manner that
correlates with its increased ring strain, although specific rate
constants for the reaction with 1,3 dipoles were not
reported.[15] Furthermore, although acetic acid adds to 10
over 10000 times faster than to cyclooctyne, the rate constant
for this addition is 1.41 ꢀ 10À5 mÀ1 sÀ1,[5] which makes the
reaction significantly slower than the azide–alkyne cyclo-
addition reaction with the more temperate reagent OCT.
Given this precedent, we presumed that the smaller ring size
of TMTH would promote the cycloaddition with azides at
faster rates than exhibited by cyclooctyne, and that the methyl
groups at the propargylic positions would shield the alkyne
from unwanted side reactions.
We synthesized TMTH in a manner similar to that
reported previously (see Scheme S2 in the Supporting Infor-
mation).[15] Its reaction with benzyl azide in CD3CN pro-
ceeded cleanly with a second-order rate constant of (4.0 Æ
0.4)mÀ1 sÀ1, the fastest reported cycloalkyne–azide reaction to
date (see Figures S3 and S4 in the Supporting Information).
During the preparation of this manuscript, Banert and Plefka
reported that TMTH was more reactive than a DIBO-like
cyclooctyne in a 1,3-dipolar cycloaddition with nitrous
oxide,[16] a result that is consistent with our findings.
Motivated by our results, we sought to further enhance the
reactivity of thiacycloheptynes through the addition of a fused
aryl ring. We attempted to synthesize the seven-membered
thiaDIFBO analogue 11 in which the activating propargylic
gem-difluoro group was replaced with the stabilizing prop-
argylic gem-dimethyl group, but we were unable to isolate 11
or any identifiable oligomerization or degradation products
thereof (see Scheme S3 in the Supporting Information).
Compound 11 could be formed transiently, as evidenced by
the formation of triazole products when the final alkyne-
generating reaction was performed in the presence of benzyl
azide, but 11 was clearly too unstable for practical purposes.
We thus returned to TMTH, whose unique combination of
azide reactivity and stability prompted us to explore its
potential as a bioorthogonal reagent. Importantly, TMTH
proved to be stable in water and phosphate-buffered saline
(PBS) and far less reactive with biologically relevant thiols
than with azides (see Figures S5 and S6 in the Supporting
Information). TMTH is known to undergo oxidation in air to
form the corresponding diketone,[17] but we saw no evidence
for this particular side reaction in our experiments under
aqueous conditions. Some degradation of TMTH in water was
observed, giving a complex product mixture, but the process
was slow on the time scale of most biological labeling
experiments (see Figure S5 in the Supporting Information).
Figure 1. Pretreatment of Ac4GalNAz-labeled cell lysates with TMTH
(10) results in inhibition of phosphine–FLAG (PHOS–FLAG) labeling.
Jurkat cells were incubated with Ac4GalNAz (50 mm) or vehicle
(DMSO) for 3days, and then lysed. a) The lysates were treated with
TMTH (concentration 1 nm–1 mm) for 1.5 h followed by 500 mm
phosphine–FLAG overnight; b) the lysates were analyzed by Western
blot by using a primary anti-FLAG antibody and a secondary antibody
conjugated to horseradish peroxidase. India Ink staining confirmed
equal protein loading (see Figure S7 in the Supporting Information).
DMSO=dimethyl sulfoxide.
indicated that pretreatment with TMTH diminished phos-
phine–FLAG labeling of cell lysates in a dose-dependent
manner (Figure 1b), thus implying that TMTH reacts with
azide-bearing glycoproteins. Similar results were obtained
through a direct competition experiment between TMTH and
phosphine–FLAG (see Figures S8 and S9 in the Supporting
Information).
To obtain direct evidence of the reactivity of TMTH with
azides, we sought to demonstrate selective labeling of an
azide-functionalized protein. By expressing the small protein
barstar from B. cenocepacia (11.7 kDa) in a methionine
auxotrophic E. coli cell line, we generated a variant in
which both methionine (MET) residues were replaced with
azidohomoalanine (AHA) residues (barstar-AHA). The
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barstar-AHA protein and its methionine counterpart (bar-
star-MET) were treated with TMTH or vehicle in PBS at
room temperature for 120 hours. The intact proteins were
analyzed by LC-MS (see the Supporting Information). When
reacted with TMTH, only the barstar-AHA sample showed
the shift in mass (168 Da) expected for TMTH-modified
barstar (Figure 2), thus confirming that the cycloaddition
reaction occurred in a selective manner. The reaction yield
was unexpectedly modest, considering the intrinsic kinetics of
the TMTH–azide cycloaddition. This may indicate that steric
future, other large heteroatoms in various oxidation states
could provide improved precision when tuning reactivity and
stability.[20]
Received: September 7, 2011
Revised: November 30, 2011
Published online: January 26, 2012
Keywords: bioorthogonal chemistry · click chemistry ·
.
protein modifications · strained alkynes · sulfur heterocycles
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showed the expected mass shift of 168 Da. The mass shifts of 43 Da
correspond to the N-terminal acylated barstar species.
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reaction so that some TMTH, the half-life of which is
approximately 48 hours in aqueous media (see Figure S5 in
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was complete.
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In conclusion, thiacycloheptynes are a promising new
class of reagents for bioorthogonal Cu-free click chemistry.
More broadly, the use of endocyclic heteroatoms to fine-tune
ring strain offers a new mechanism to balance reactivity and
stability when designing new bioorthogonal reagents. One of
the greatest challenges in the design of these reagents is that,
unlike for conventional chemistry, in which unwanted side
reactivities can be mitigated by changing the reaction
conditions, bioorthogonal reagents must be stable while
reacting rapidly and selectively in a highly functionalized
environment in which many reaction pathways are possible.
An emerging theme in the design of bioorthogonal reagents
for Cu-free click chemistry involves optimization for the
desired reaction pathway through activating and deactivating
modifications. Herein, we have identified the decreased ring
strain that resulted from substitution with an endocyclic sulfur
as a new tool for such precise reagent engineering. In the
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[20] Oxidizing the sulfide to a sulfoxide or sulfone may increase the
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