Acylazetine as a dienophile in bioorthogonal inverse electron-demand Diels-Alder ligation

Article abstract of DOI:10.1021/ol501049c

A new bioorthogonal N-acylazetine tag, suitable for tetrazine mediated inverse electron-demand Diels-Alder conjugation, is developed. The tag is small and achiral. We demonstrate the usefulness of N-acylazetine-tetrazine based bioorthogonal chemistry in two-step activity-based protein profiling. The performance of the new tetrazinophile in the labeling of catalytically active proteasome subunits was comparable to that of the more sterically demanding norbornene tag.

Full text of DOI:10.1021/ol501049c

Letter
pubs.acs.org/OrgLett
Acylazetine as a Dienophile in Bioorthogonal Inverse Electron-
Demand Diels−Alder Ligation
Sander B. Engelsma, Lianne I. Willems, Claudia E. van Paaschen, Sander I. van Kasteren,
Gijsbert A. van der Marel, Herman S. Overkleeft,* and Dmitri V. Filippov*
Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands
S
* Supporting Information
ABSTRACT: A new bioorthogonal N-acylazetine tag, suitable
for tetrazine mediated inverse electron-demand Diels−Alder
conjugation, is developed. The tag is small and achiral. We
demonstrate the usefulness of N-acylazetine-tetrazine based
bioorthogonal chemistry in two-step activity-based protein
profiling. The performance of the new tetrazinophile in the labeling of catalytically active proteasome subunits was comparable to
that of the more sterically demanding norbornene tag.
ioorthogonal chemistry strives to chemoselectively modify
Bbiomolecules, such as proteins, carbohydrates, and lipids,
within the complex environments of cell lysates, living cells,
and, ultimately, in living animals. Bioorthogonal chemistry has
pushed forward various fields of research, such as activity-based
protein profiling, in vitro and in vivo imaging, and modified
Figure 1. Known bioorthogonal dienophilic moieties (A−C) and the
azetine tag (D) described here.
metabolite labeling.1 Ideally, in a bioorthogonal process a
reactive group incorporated into the biomolecule of interest
reacts selectively with a complementary reactive group modified
with a reporter moiety, such as a fluorescent label to enable
visualization of the target or an affinity probe for postlabeling
purification.2 To enable this, a bioorthogonal tag should be
inert to the broad spectrum of functional groups that reside in a
biological system, while exerting sufficiently fast reaction
kinetics for conjugation to occur at nanomolar concentrations.
Additives to promote the reaction between the tag and the
reactive functionality of the reporter group are best avoided.
Finally, the tag should be sterically compact to minimize
unfavorable steric interactions with the biological system being
studied and be synthetically accessible.
In the past decade, several bioorthogonal ligation strategies
have been developed. Among these, prominent transformations
are the Staudinger−Bertozzi ligation,3 the copper-catalyzed,4
and the strain-promoted azide−alkyne [2 + 3] cycloaddition.5
An important recent development is the inverse-electron
demand Diels−Alder reaction (IEDDA), which exhibits the
fastest reaction kinetics among all commonly used bioorthog-
onal transformations, while being chemoselective, efficient, and
additive-free.6 In its most typical setup, IEDDA uses a strained
alkene tag, while its reaction partner is a reporter-group
functionalized tetrazine derivative. Well-established strained
alkene handles are norbornene (Figure 1A) and trans-
cyclooctene (Figure 1B). Although these alkenes exhibit
exceptionally fast reaction kinetics, their steric bulk and
lipophilicity may induce a biological response, bringing a
paradoxical problem, known as the observer effect, into
chemical biology. Examples of an influence of a lipophilic
reporter groups, either favorable or otherwise, on the behavior
of biomolecules has been noted by us7a and others.7b Hence,
there is room for the development of more compact
dienophiles for tetrazine mediated bioorthogonal cycloaddi-
tions. Recently, Devaraj et al. introduced the methylcyclopro-
pene core (Figure 1C) as a dienophile for IEDDA that
combines a small size (“minitag”) with fast reaction kinetics.8
Other laboratories developed related cyclopropene based
bioorthogonal tags.9 The reactivity of cyclopropenes in a
normal Diels−Alder reaction has been recently highlighted as
well.10
After perusing the literature, we observed that all dienophiles
used in IEDDA-based bioorthogonal chemistry rely on ring
strain to attain the desired reactivity toward tetrazines. These
contemporary dienophilesnorbonene, trans-cylcooctene, and
cyclopropene (Figure 1A−C)are already described in the
seminal 1990 report by Sauer et al. on the reactivity of electron-
rich dienophiles in IEDDA processes. In the same paper, these
authors also showed that alkenes activated by an electron-
donating heteroatom adjacent to the double bond exhibit a
higher reactivity toward tetrazine relative to their non-
conjugated analogues.11 Introduction of a heteroatom into
the cyclobutene scaffold should therefore lead to a viable
dienophile for IEDDA-based bioorthogonal chemistry. Follow-
ing this reasoning, a 2-azetine would be the smallest viable core
that could utilize this effect in addition to ring strain, with the
Received: April 10, 2014
© XXXX American Chemical Society
A
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Letter
ring nitrogen amenable for substitution to yield a new and
compact bioorthogonal tag. Although N-alkylazetines are the
more electron-rich species, we considered N-acylazetines as
more suitable candidates because of their reported stability.12
We here report on the development of N-acylazetines as
(Figure 1D) as IEDDA dienophiles suitable for bioorthogonal
chemistry.
The synthesis of N-acylazetines 6 and 7, equipped with an
activated ester for further modification (Scheme 1), com-
Scheme 1. Synthesis of Active Ester Functionalized N-
Acylazetines 6 and 7
Figure 2. (A) Preparation of model acylazetine 8 and an overview of
the kinetics experiment. (B) Data plots following the tetrazine
absorption band over time in a reaction between N-acylazetine 8 (6
mM) and tetrazine 9 (0.6 mM) in 12% DMSO/water. The kinetics
experiment was performed four times. Compound 10 is formed as a
mixture of regioisomers and probably tautomers.
was determined by monitoring the absorbance of the tetrazine
at 517 nm (Figure 2). We established the pseudo-first-order
reaction rate constant to be 4.5 × 10−3 8.2 × 10−5 s−1 at 20
°C in a solution of 12% DMSO in water. Next, in a separate
experiment, we determined a second-order rate constant k2 to
be approximately 0.39 0.1 s−1 M−1 (Supporting Information
(SI)). The mass-spectroscopic data of the reaction product,
formed as a mixture of isomers, were fully consistent with
putative structure 10. The characterization of 10 by NMR was
complicated by the two tertiary amide bonds, which appear to
exist as rotamers on the NMR time scale. In order to obtain a
more spectroscopically tractable product and to investigate the
course of the N-azylazetine cycloaddition in more detail we
performed the reaction of 11 with symmetrical tetrazine 12
(Scheme 2). We observed the clean formation of rearranged
menced with the preparation of the azetidine core.13
Epichlorohydrin was reacted with benzhydrylamine in a two-
step one-pot procedure to afford protected azetidine 2. The
hydroxy group of 2 was mesylated, affording methanesulfonate
3, which was subsequently deprotected using chloroethyl
chloroformate and methanol. The route from 1 to 4 was
optimized in such a way that each intermediate could be
purified through crystallization and washing steps (in 48% yield
over three steps). With free amine 4 in hand, the ring opening
of glutaric anhydride could commence. Initially this was carried
out under the agency of triethylamine. However, during the
reaction, a displacement reaction took place, wherein the
methanesulfonate was substituted by chloride. Triethylamine
proved to interfere with the column purification of the free
carboxylic acid formed upon the opening of glutaric anhydride.
Switching the base to potassium carbonate, and premixing 4
with silver methanesulfonate to precipitate the chloride as
AgCl, solved these issues, and 5 could be obtained in 79% yield.
The following elimination−esterification sequence was carried
out as a one-pot procedure. First, methanesulfonic acid was
eliminated with KOtBu to afford the N-acylazetine moiety.
Next, the carboxylate was converted to either pentafluorophe-
nol (PFP) ester (6) or a para-nitrophenyl (PNP) counterpart
(7) via EDC mediated condensation with the appropriate
alcohol. Both esters were readily obtained; however, the PFP-
ester (6) proved to be unstable and could not be stored for
extended periods of time. Another disadvantage was its gel-like
consistency at rt, making it difficult to handle. On the other
hand, the PNP-ester (7) was isolated as a crystalline solid that
is stable at rt for at least 3 months.
Scheme 2. A Model Cycloaddition of Simple N-Acylazetine
11 to Symmetric Disubstituted Tetrazine 12
compound 14 that was formed as a single product via opening
of the azetidine ring of the immediate IEDDA adduct 13. We
observed a trace amount of the product of the same
rearrangement after the reaction of 8 with 9, as evidenced by
the resonance of the tertiary carbon of pyridazine at 123 ppm in
the 13C NMR spectrum of 10.
To investigate the kinetics of the IEDDA reaction of N-
acylazetine with tetrazine, 7 was coupled with morpholine to
make the water-soluble N-acylazetine 8. A 10-fold excess of
compound 8 was reacted with tetrazine 9 (Figure 2).14 The rate
In order to evaluate the applicability of the novel N-
acylazetine ligation handle for biological labeling strategies, the
B
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Letter
tag was incorporated into an activity-based proteasome probe
to enable two-step activity-based protein profiling (ABPP)
through ligation with a fluorescently labeled tetrazine (17,
Figure 3).15 As a model target enzyme we selected the
Figure 4. (A) Labeling of proteasome activity in HEK cell extracts by
exposure to 5 μM of ABP 15 or ABP 16 for 1 h followed by reaction
with 0.3−100 μM of Bodipy-tetrazine 17 for 1 h. 12.5% SDS-PAGE
with fluorescent readout followed by coomassie brilliant blue staining.
“ctrl”: control sample labeled with 1 μM MV151. Proteasome β-
subunits are designated on the basis of reported labeling by MV151.
‘M’: protein marker. B: Labeling of proteasome activity in HEK cell
extracts by exposure to 5 μM of ABP 15 or ABP 16 for 1 h followed by
reaction with 10 μM of Bodipy-tetrazine 17 for 5−240 min. “ep”: 100
μM epoxomicin added to incubation with ABP 15. Proteasome β-
subunits are designated on the basis of reported labeling by MV151.
‘M’: protein marker.
Figure 3. Structures of acylazetine- (15) and norbornene (16)-
functionalized proteasome ABPs and tetrazine-BODIPY reporter
reagent (17).
constitutive proteasome, a multisubunit protein complex
containing three different catalytically active subunits (β1, β2,
and β5). These β-subunits each have a different substrate
preference and can be targeted with either subunit-selective16
or with broad-spectrum17 activity-based probes (ABPs). The
designed ABP 15 is based on the broad-spectrum irreversible
proteasome inhibitor epoxomicin18 functionalized at the N-
terminus with the acylazetine moiety. Compound 15 (Figure 3)
was readily prepared from protected peptide epoxyketone (see
SI).
The ability of the azetine-functionalized ABP 15 to target the
catalytically active proteasome β-subunits was confirmed by
performing competition experiments against the fluorescent
broad-spectrum proteasome ABP MV151 in human embryonic
kidney (HEK) cell extracts (SI, Figure S1).17 These experi-
ments revealed that 3 μM of ABP 15 was required to
completely block the fluorescent labeling by MV151. Therefore,
for all ensuing two-step labeling experiments a probe
concentration of 5 μM was used. The utility of the azetine
tag for two-step labeling of proteasome activity in cell extracts
was next established by exposure of HEK cell extracts to ABP
15 followed by ligation with varying concentrations of tetrazine
17 (Figure 3). As a control, the same procedure was performed
using the previously reported norbornene-functionalized ABP
16.15 Analysis of the labeled proteins on gel with fluorescent
readout (Figure 4A) revealed that tetrazine ligation of cell
extracts treated with azetine-functionalized ABP 15 results in
the specific fluorescent labeling of three bands which
correspond to the proteasome β-subunits labeled by fluorescent
ABP MV151. The labeling is dependent on the concentration
of tetrazine in a similar manner as that for norbornene-
functionalized ABP 16. With both ABPs a similar increase in
fluorescent labeling was observed when the ligation step was
performed for increasing reaction times (Figure 4B).
tetrazine 17. The absence of labeling in samples in which the
proteasome activity was inhibited by an excess of epoxomicin
shows that the labeling indeed reflects labeling of the
catalytically active proteasome β-subunits. In order to test
whether N-acylazetine in 15 may cross-react with other
commonly used ligation reagents, two-step proteasome labeling
procedures were performed in HEK cell extracts using alkyne-,
azide-, and phosphine-functionalized reagents instead of
tetrazine for the ligation step (SI, Figure S4). No proteasome
labeling was detected when using Bodipy-alkyne,19 Bodipy-
azide,15 biotin-phosphine,20 and biotinylated dibenzocyclo-
octene21 reagents, demonstrating that these do not react with
the azetine-tagged probe. Together these results demonstrate
the utility of the new compact N-acylazetine ligation handle for
bioorthogonal labeling of proteins or other biomolecules via
tetrazine ligation.
In conclusion, we designed and synthesized a novel N-
acylazetine tag for the tetrazine ligation strategy and showed its
applicability in an ABPP experiment. The acylazetine was
incorporated into a compact linkable tag that is accessible
through an efficient five-step synthesis. Determination of the
reaction kinetics between the N-acylazetine and tetrazine
showed a reaction rate constant in line with that of the
methylcyclopropene mini-tag. We functionalized epoxomicin, a
broad-spectrum proteasome inhibitor, with our novel ligation
handle and could successfully label the proteasome through
tetrazine ligation. Based on these results, we anticipate that this
small tag is a valuable addition to the current arsenal of IEDDA
dienophiles for application in chemical biology research.
These results demonstrate that in this experimental setup the
N-acylazetine moiety reacts with equal efficiency as the
norbornene ligation handle in the IEDDA reaction with
C
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(16) Verdoes, M.; Willems, L. I.; van der Linden, W. A.;
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ASSOCIATED CONTENT
* Supporting Information
Experimental details, spectra, and biological experiments. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: h.s.overkleeft@lic.leidenuniv.nl.
*E-mail: filippov@chem.leidenuniv.nl.
Notes
The authors declare no competing financial interest.
(19) Verdoes, M.; Hillaert, U.; Florea, B. I.; Sae-Heng, M.; Risseeuw,
M. D. P.; Filippov, D. V.; van der Marel, G. A.; Overkleeft, H. S.
Bioorg. Med. Chem. Lett. 2007, 17, 6169−6171.
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Marel, G. A.; Overkleeft, H. S. ChemBioChem 2008, 9, 1735−1738.
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ACKNOWLEDGMENTS
■
This research was financially supported by The Netherlands
Organisation for Scientific Research (NWO).
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