Exploring isonitrile-based click chemistry for ligation with biomolecules

Article abstract of DOI:10.1039/c1ob06424j

We show here that isonitriles can perform click reactions with tetrazines in aqueous media, making them promising candidates for ligation reactions in chemical biology and polymer chemistry. This is the first time that a [4+1] cycloaddition has been used as a biocompatible ligation reaction.

Full text of DOI:10.1039/c1ob06424j

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Exploring isonitrile-based click chemistry for ligation with biomolecules†
Henning Sto¨ckmann,a,b Andre´ A. Neves,b Shaun Stairs,a Kevin M. Brindleb and Finian J. Leeper*a
Received 19th August 2011, Accepted 24th August 2011
DOI: 10.1039/c1ob06424j
We show here that isonitriles can perform click reactions with
tetrazines in aqueous media, making them promising candi-
dates for ligation reactions in chemical biology and polymer
chemistry. This is the first time that a [4+1] cycloaddition has
been used as a biocompatible ligation reaction.
which is usually followed by a secondary reaction with a suitable
anion (Scheme 1). In addition, activated carbonyl compounds such
as 1,1,1,5,5,5- hexafluoropentane-2,4-dione can add isonitriles in
a 1 : 1 manner.11 Isonitriles can also react with carboxylates at
elevated temperatures and function as ligands for a number of
transition metals.12
Ligation reactions are of great importance in areas such as
modification of biomolecules, supramolecular chemistry, material
science and nanotechnology.1 Examples of bioorthogonal ligation
reactions are (a) the condensation of ketones and aldehydes with
amine nucleophiles such as hydrazines and hydroxylamines, (b)
the Staudinger ligation of azides and phosphines and the [3
+ 2] cycloaddition of azides and alkynes2 and (c) the inverse
electron demand Diels–Alder reaction between tetrazines and
trans-cyclooctenes.3 The relatively small number of bioorthogonal
ligation reactions reflects the pressing need for the exploration of
new orthogonal chemical reporter groups.
Here we investigate the possibility of using the isonitrile group
for ligation reactions. The isonitrile group is a two-atom functional
group that has been known since 18674 and it is one of the
few examples of a stable chemical compound with a divalent
carbon. Its rate of hydrolysis into the corresponding formamide
is negligible around neutral pH values.5 Its small size may have
distinct advantages for labelling
[R–N C: ↔ R–N+ C-]
biomolecules, due to the minimal perturbation of the biological
system being studied. Isonitriles were monitored toxicologically by
Bayer AG in the 1960’s and shown to exhibit no appreciable toxi-
city for mammals, with oral and subcutaneous doses of 500–5000
mg kg-1 being tolerated by rodents.6 Like the widely used azido
group, the isonitrile group is not found in mammals. However,
numerous isonitrile-bearing natural products have been found in
marine and terrestrial sources.7 Isonitriles are weak nucleophiles
lacking reactivity towards simple ketones, aldehydes or imines.
Suitable reaction partners for isonitriles are resonance-stabilized
electrophilic carbocations. Iminium ions,8 N-alkylquinolinium
ions9 and tropylium ions10 all react with isonitriles by a-addition,
Scheme 1 Candidate reactions with isonitriles.
Ugi reactions are water-compatible13 and a few reports exist
where Ugi reactions have been used to label biomolecules under
physiological conditions.5,14 A bifunctional azide-isonitrile conju-
gating agent combining [3 + 2] cycloaddition click chemistry and
Ugi chemistry has recently been reported.15 However, biomolecu-
lar functional groups such as amines and carboxylic acids, which
are abundant on proteins, could interfere with Ugi-type reactions
and thus decrease their efficiency. Instead we investigated a [4
+ 1]-cycloaddition between a selection of isonitriles and 3,6-di-
2-pyridyl-1,2,4,5-tetrazine (1, R1 = 2-pyridyl) (Scheme 2). This
[4 + 1]-cycloaddition has only been reported in two papers by
Seitz and co-workers, who used it to prepare pharmacologically
interesting aminopyrazoles from benzyl isonitrile 2a and a range
of substituted tetrazines (1). The first step of the reaction sequence
is the formation of non-isolable tetraazanorbornadienimine
aUniversity of Cambridge, Department of Chemistry, Lensfield Road,
Cambridge, UK, CB2 1EW. E-mail: fjl1@cam.ac.uk; Fax: +44 (0) 1223
336 362; Tel: +44 (0) 1223 336 403
bCancer Research UK Cambridge Research Institute, Li Ka Shing Centre,
Robinson Way, Cambridge, UK, CB2 0RE
† Electronic supplementary information (ESI) available: Experimental
data and NMR spectra. See DOI: 10.1039/c1ob06424j
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Scheme 2 The [4 + 1] cycloaddition between isonitriles and tetrazines 1.
Table 1 Rate constants for [4 + 1] cycloadditions and half-lives of adducts
derivatives 3 which spontaneously undergo a [4 + 2]-cycloreversion
with release of N2 to result in 4H-pyrazol-4-imine derivatives 4.
These intermediates could not be isolated as they tautomerized to
the aromatic pyrazoles 5, which then hydrolysed to give the desired
aminopyrazoles 6.16
in PBS–CD3CN (1 : 1)
k (MeOH)
k (THF–H2O (1 : 1))
(¥ 10-2 M-1 s-1)
Isonitrile
(¥ 10-2 M-1 s-1)
t1/2 (h)
Because of the hydrolytic lability of imine 5, this reaction cannot
be used in its reported form for chemical ligation reactions in water.
Thus, we investigated the possibility of modulating the course of
the reaction by modifying the structure of the isonitriles being used
and thus turning it into an effective aqueous ligation reaction. We
also investigated water-compatibility and reaction rates, neither of
which have been reported in the literature.
When isonitriles 2a to 2d were used in the reaction sequence,
the 4H-pyrazol-4-imine could not be isolated. Instead, rapid
tautomerisation to imines 5 occurred, followed by spontaneous
hydrolysis to aminopyrazole 6 by traces of moisture. For the case
of the simple alkyl isonitrile 2b the reaction was followed by NMR
spectroscopy in CDCl3 (Figure S2, ESI†). The intermediate imine
5b was the main component after 10 min but some hydrolysis by
traces of water in the solvent had already occurred and aldehyde
and aminopyrazole were major components after 24 h. In MeOH–
H2O (1 : 1) hydrolysis to give the aldehyde is rapid. Secondary
isonitrile 2d was converted into the corresponding ketone 7d in
the same manner. Although the fast hydrolysis limits the reaction’s
usefulness for conjugation reactions, it does provide a novel way
to mask carbonyl groups as isonitriles and unmask them with
tetrazines, effectively providing a new protecting group strategy
for aldehydes and ketones.
2e
2f
5.2 0.9
7.7 0.3
12.4 0.7
57.5 1.5
16
63
as difluorocyclooctyne (DIFO), with benzyl azide, (4.2 0.1) ¥
10-2 M-1 s-1 in CD3CN.18 Adduct 8 showed reasonable stability in
buffered aqueous systems with a half-life of ca. 16 h in phosphate
buffered saline (PBS, pH 7.4)–CD3CN (1 : 1) as determined by
1H-NMR spectroscopy.
Next tert-isonitrile 2f was tested as the imine 4f cannot
tautomerize. The reaction rate for the [4 + 1]-cycloaddition in
MeOH at 25 C was determined to be (7.7 0.3) ¥ 10-2 M-1 s-1,
◦
slightly higher than that of primary isonitrile 2e. In H2O–THF
(1 : 1), a 7-fold rate-enhancement was found (Table 1). Despite its
electron-deficient character, imine 4f was found to be surprisingly
stable in aqueous systems with a half-life of ca. 63 h in PBS–
CD3CN (1 : 1). Having shown that both tertiary isonitriles and
isocyanopropionic acid-based isonitriles are, in principle, suitable
for ligation reactions with tetrazine 1 in aqueous systems, the [4 +
1]-cycloaddition was next tested in a protein system to show that
the chemistry is biocompatible.
A convenient method to prepare a cysteine-reactive isonitrile
linker started with 4,4-dimethyl-2-oxazoline 9 (Scheme 3), which
was deprotonated with n-BuLi and readily fragmented into
the isonitrile-alkoxide.19 This alkoxide was reacted with para-
nitrophenylchloroformate 10 to give the corresponding carbonate
11, which is stable indefinitely at room temperature and readily
forms carbamates upon reaction with primary amines such as
12. The resulting hetero-bifunctional linker 13 was reacted with
a mutant of the C2A domain of synaptotagmin-I having a single
cysteine residue at position 7820 (C2Am, MW = 16222 Da) via
conjugate addition of its cysteine to the maleimide unit of 13 (full
conversion, Scheme 3). The identity of C2Am–isonitrile adduct 14
was confirmed by mass spectrometry (MW = 16576 Da, Scheme
4) and no byproducts were detected.
In the case of isonitrile 2e, imine 5e did not hydrolyse (even
when the reaction was performed in THF–H2O, 1 : 1), but instead
tautomerized again into the stable a,b-unsaturated system 8
(a vinylogous urethane). Although the reaction predominantly
yielded the trans-olefin, this slowly isomerised into the cis-olefin
in CDCl3 due to the intramolecular hydrogen bond.17 In D2O–
CD3CN the proton adjacent to the ester exchanges for deuterium,
showing that enamine–imine tautomerisation does occur. The
reaction rate for the [4 + 1]-cycloaddition was determined by
UV–vis spectroscopy in MeOH at 25 ◦C to be (5.2
0.9) ¥
10-2 M-1 s-1 for isonitrile 2e. This rate is comparable to that for
the [3 + 2]-cycloaddition reaction of strained cyclooctynes, such
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secondary isonitriles tautomerization and hydrolysis converts the
isonitrile into a carbonyl group, but isocyanopropanoates and
tertiary isonitriles form adducts with tetrazines that only hydrolyse
slowly in buffered aqueous systems. This chemistry has been shown
to be biocompatible by using it for fluorophore conjugation to a
tertiary isonitrile-tagged protein. This is the first time that a [4 + 1]
cycloaddition has been used as a biocompatible ligation reaction.
It could potentially be used for the ligation of other biomolecules,
such as sugars, nucleic acids or natural products, in addition to
proteins.
We wish to thank Cancer Research UK for funding this work
and Dr Len Packman, Protein and Nucleic Acid Chemistry
Facility, Department of Biochemistry, Cambridge, UK, for expert
support in MS analysis.
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Scheme 3 Synthesis of an isonitrile linker and attachment to the protein
C2.
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Scheme 4 Conjugation of C2A–isonitrile (top) with tetrazine–rhodamine
(bottom). R1 = rhodamine (see ESI† for structural details).
In summary, a biocompatible conjugation reaction of isonitriles
has been investigated. The tetrazine-reagent 1 underwent [4 + 1]
cycloadditions with primary, secondary and tertiary isonitriles in
aqueous solution. The final outcome of the reaction depended
on the substituents on the isonitrile. With ordinary primary and
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The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 7303–7305 | 7305
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