Sterically Shielded, Stabilized Nitrile Imine for Rapid Bioorthogonal Protein Labeling in Live Cells

Article abstract of DOI:10.1021/jacs.8b00126

In pursuit of fast bioorthogonal reactions, reactive moieties have been increasingly employed for selective labeling of biomolecules in living systems, posing a challenge in attaining reactivity without sacrificing selectivity. To address this challenge, here we report a bioinspired strategy in which molecular shape controls the selectivity of a transient, highly reactive nitrile imine dipole. By tuning the shape of structural pendants attached to the ortho position of the N-aryl ring of diaryltetrazoles-precursors of nitrile imines, we discovered a sterically shielded nitrile imine that favors the 1,3-dipolar cycloaddition over the competing nucleophilic addition. The photogenerated nitrile imine exhibits an extraordinarily long half-life of 102 s in aqueous medium, owing to its unique molecular shape that hinders the approach of a nucleophile as shown by DFT calculations. The utility of this sterically shielded nitrile imine in rapid (～1 min) bioorthogonal labeling of glucagon receptor in live mammalian cells was demonstrated.

Full text of DOI:10.1021/jacs.8b00126

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A sterically shielded, stabilized nitrile imine for
rapid bioorthogonal protein labeling in live cells
Peng An, Tracey Lewandowski, Tugce Gulsen Erbay, Peng Liu, and Qing Lin
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b00126 • Publication Date (Web): 22 Mar 2018
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A sterically shielded, stabilized nitrile imine for rapid bioorthogonal protein labeling in live cells
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Peng An,† Tracey M. Lewandowski, † Tuğçe G. Erbay, ‡ Peng Liu ‡ and Qing Lin†,*
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†Department of Chemistry, State University of New York at Buffalo, Buffalo, New York 14260-3000,
United States
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‡Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States
RECEIVED DATE (automatically inserted by publisher)
E-mail: qinglin@buffalo.edu
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Abstract: In pursuit of fast bioorthogonal reactions, reactive moieties have been increasingly employed
for selective labeling of biomolecules in living systems, posing a challenge in attaining reactivity without
sacrificing selectivity. To address this challenge, here we report a bioinspired strategy in which molecular
shape controls the selectivity of a transient, highly reactive nitrile imine dipole. By tuning the shape of
structural pendants attached to the ortho position of the N-aryl ring of diaryltetrazoles―precursors of
nitrile imines, we discovered a sterically shielded nitrile imine that favors the 1,3-dipolar cycloaddition
over the competing nucleophilic addition. The photo-generated nitrile imine exhibits an extraordinarily
long half-life of 102 sec in aqueous medium, owing to its unique molecular shape that hinders the
approach of a nucleophile as shown by DFT calculations. The utility of this sterically shielded nitrile
imine in rapid (~1 min) bioorthogonal labeling of glucagon receptor in live mammalian cells was
demonstrated.
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Introduction
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Harnessing chemical reactivity for covalent modification of biomolecules in their native environment
is currently attracting a lot of interest.1 The success of this ‘in vivo’ chemistry approach depends critically
on robust bioorthogonal ligation reactions that proceed between a pair of reactants at low concentrations
without cross-reactivity with the endogenous functional groups.2 Because of their concerted reaction
mechanism, fast reaction kinetics, and operational simplicity, cycloaddition-based bioorthogonal ligation
reactions have received the most attention. These include the azide-alkyne cycloaddition,3 the nitrone-
alkyne cycloaddition,4 the inverse electron demand Diels-Alder reaction5 and the tetrazole-alkene
cycloaddition.6 Analogous to the optimization of small molecule ligands with higher affinity and
selectivity toward their macromolecular targets, the optimization of bioorthogonal ligation reactions,
particularly their reaction rate and chemo-selectivity, i.e., bioorthogonality, is of paramount importance
for their use in biological systems.
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In optimizing bioorthogonal reactions, two general strategies have been developed: one uses a metal
catalyst to accelerate the ligation reaction, e. g., copper-catalyzed azide-alkyne cycloaddition (CuAAC)3;
the other activates substrates directly through the use of strain. Because of the well-known copper
toxicity,7 recent efforts by many groups have largely focused on the design of strained cycloalkenes and
cycloalkynes such as trans-cyclooctene,8 norbornene,9 cyclopropene,10 cyclooctyne,11 thiacycloheptyne,12
and bicyclo[6.1.0]nonyne (BCN).13 For example, a cyclopropane-fused trans-cyclooctene was designed
by Fox and co-workers that adopts the high-energy ‘half-chair’ conformation and gives a second-order
rate constant, k2, of 22,000 M‒1 s‒1 in the inverse electron demand Diels-Alder reaction with 3,6-di(2-
pyridyl)-s-tetrazine in methanol.14 A major challenge for substrate activation strategy is that it is generally
difficult to achieve high reactivity while maintain high stability and selectivity in cellular systems because
these factors counteract each other in most circumstances.
We recently reported the design of strained cycloalkenes such as 3,3-disubstituted cyclopropene15 and
spiro[2.3]hex-1-ene16 that show drastic rate acceleration in the photoinduced tetrazole-alkene
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cycloaddition reaction with k2 values approaching 10,000 M‒1 s‒1. Compared to other fast bioorthogonal
reactions such as tetrazine ligation, the tetrazole-alkene cycloaddition requires photoactivation, which is
a very attractive attribute to biological applications where the spatiotemporal control is of paramount
importance. Because the photo-generated nitrile imine dipoles also exhibit electrophilic character, they
are known to undergo nucleophilic thiol and water additions in the absence of a suitable dipolarophile.17,18
To harness the nitrile imine reactivity for bioorthogonal reactions in living systems, it is critical that we
can selectively tune up the cycloaddition reactivity while suppress the competing nucleophilic additions.
Here, we report a bioinspired strategy whereby an exquisite pendant group was identified that dramatically
stabilizes the nitrile imine in aqueous medium, increases its reactivity in the desired cycloaddition
reaction, suppresses the undesired nucleophilic additions, and allows robust bioorthogonal labeling of
GCGR—a member of class B G protein-coupled receptors—with a small organic fluorophore in live
mammalian cells.
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Results and discussion
Synthesis and evaluation of sterically shielded diaryltetrazoles. To control the reaction selectivity
of the in situ generated nitrile imine, we were inspired by how the bacterial enzymes UbiD/UbiX catalyze
the decarboxylation step in bacterial ubiquinone biosynthesis pathway, a rare example of biocatalytic 1,3-
dipolar cycloaddition involving azomethine ylide in nature.19 Crystal structures of A. niger Fdc1UbiX (Fdc1
is a fungal homolog of UbiD) revealed that the enzyme utilize a unusual isoprene ring-fused flavin
mononucleotide (prFMN) cofactor for this transformation.20 In addition to providing the necessary
azomethine ylide, the extra isoprene ring may prevent the competing hydride and carbanion addition to
the isoalloazine ring21 through steric effect. Inspired by Nature’s elaborate design, we envisioned that the
reactivity of the nitrile imine dipole can be directed through adjacent structural pendants away from the
nucleophilic addition and toward the desired 1,3-dipole cycloaddition (Scheme 1).
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A series of ortho-substituted diphenyltetrazoles were synthesized (Scheme S1-S8 in SI) and their
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reactivities were assessed in an HPLC-based competition assay using equal amount of excess acrylamide
and glutathione (GSH) in mixed acetonitrile/phosphate buffer (Scheme 2 and Table S1). In principle, the
in situ photo-generated nitrile imines can produce four different adducts: (i) pyrazoline a from 1,3-dipolar
cycloaddition, (ii) hydrazone b from nucleophilic GSH addition, (iii) hydrazide c from water quenching
and subsequent tautomerisation, and (iv) adduct d from intramolecular nucleophilic addition (Table 1
scheme). Four series of tetrazoles (Scheme 2) were evaluated in this assay: (i) mono-aliphatic substituted
tetrazoles 2-10; (ii) mono-methoxymethyl-substituted tetrazoles 11-14; (iii) dimethyl, dimethoxy, and
dimethoxymethyl-substituted tetrazoles 15-17; and (iv) monoaryl and diaryl-substituted tetrazoles 18-29.
The di(o-2’-N-Boc-pyrrole)-substituted tetrazole 26 showed the highest cycloaddition selectivity with
a:b:c:d ratio of 92:2:6:0 among all the tetrazole analogs (Table S1). Both the N-substituent and the
proximity to the nitrile imine center appear to be important as lack of either feature resulted in erosion of
this high selectivity (compare tetrazole 26 to 27‒29 in Table S1).
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Reactivity comparison of tetrazole 26 versus tetrazole 1. With the highly selective tetrazole 26 in
hand, we assessed its reactivity toward a range of alkene dipolarophiles (Table 1). Compared to tetrazole
1, the sterically shielded tetrazole 26 displayed outstanding selectivity (92-99%) toward the conjugated
alkenes including acrylamide, dimethyl fumarate and styrene, as well as the highly strained cycloalkenes
including spiro[2.3]hex-1-ene (Sph) and trans-cyclooctene (TCO). For these reactive alkenes, GSH
adducts were either undetectable or negligible. A reversal of selectivity was observed for less reactive
alkenes including norbornene, 1,3- and 3,3-disubstituted cyclopropenes (entries 4-6 in Table 1). The
selectivity for photoinduced cycloaddition with tetrazole 26 is robust as the reaction with Sph proceeded
in the presence of 10 mM glutathione, 10 mM glutamic acid, or a mixture of nucleophilic amino acids
(Figs. S1-S3). Kinetic measurements revealed that the second-order rate constants are lower for tetrazole
26 than those of tetrazole 1 by about two-fold (Table 1 and Figs. S4-S11), suggesting that the increased
selectivity for cycloaddition with tetrazole 26 is due to the greater inhibition of GSH addition. For styrene
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and 1,3-cyclopropene, the cycloadducts were either negligible or non-detectable with tetrazole 1, but
became dominant with tetrazole 26 when nucleophilic GSH addition is inhibited (entries 3 and 5 in Table
1). Interestingly, the amount of water adducts remained low and essentially unchanged for all alkene
dipolarophiles, indicating that the steric shielding does not suppress the water-quenching process as
effectively as the GSH addition presumably due to the small size of water.
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Effect of steric shielding on transition state energies. To understand how the steric shield
influences the reaction selectivity of the in situ generated nitrile imine, we computed the transition states
of the 1,3-dipolar cycloaddition and the nucleophilic addition reactions involving the sterically shielded
nitrile imine derived from tetrazole 26 and the unhindered nitrile imine from 1 using density functional
theory (Figure 1). In these calculations, 5-methyl-spiro[2.3]hex-1-ene and methyl thiolate were used as
the alkene dipolarophile and a model nucleophile, respectively. The 1,3-dipolar cycloadditions of 5-
methyl-spiro[2.3]hex-1-ene to the unhindered nitrile imine (TS1) and to the sterically shielded nitrile
imine (TS2) occur in a concerted manner. The N-Boc-pyrrole substitution led to increases of activation
free energy (∆G‡) and activation enthalpy (∆H‡) of the cycloaddition, by 2.2 kcal/mol and 0.4 kcal/mol,
respectively. On the other hand, the reactivity difference between the nucleophilic addition of methyl
thiolate22 to the unhindered nitrile imine (TS3) and to the sterically hindered nitrile imine (TS4) is much
greater (ΔΔG‡ = 5.2 kcal/mol; ΔΔH‡ = 2.8 kcal/mol). In TS4, the N-Boc-pyrrole group perpendicular to
the C-phenyl ring effectively creates a steric shield surrounding the electrophilic side of nitrile imine, and
therefore blocks the approach of the methyl thiolate nucleophile. As a result, the activation energies for
the nucleophilic addition reactions increase from 12.2 kcal/mol for TS3 to 17.4 kcal/mol for TS4. This
staggering 5.2 kcal/mol increase in ∆G‡ corresponds to >6000-fold reduction in reaction rate, which is
consistent with the low reactivity to form the GSH adduct in reactions with tetrazole 26. The greater
sensitivity to steric shielding effect in the nucleophilic addition compared with cycloaddition is attributed
to the preferred orientation of the nucleophile, which places the methyl thiolate in close proximity to the
C-phenyl ring on the nitrile imine (see TS3 and Fig. S12). In TS4, the bulky N-Boc-pyrrole substitution
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forces the methyl thiolate to adopt the higher energy orientation. Together, these computational studies
indicate that the steric shield formed by the two N-Boc-pyrrole groups in tetrazole 26 causes a dramatic
slowdown of the competing thiolate addition, thereby shifting the selectivity toward the 1,3-dipolar
cycloaddition (Scheme 1).
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Structures of tetrazoles in solid state and in solution. To corroborate computational results, we
solved the crystal structures of the sterically shielded tetrazole 26 and its analog 27 (Tables S3 and S4).
Figure 2b shows that the pyrrole rings in 26 are nearly perpendicular to the C-phenyl ring with the t-butyl
group at one N-Boc-pyrrole extending to the other pyrrole ring. The closest distance between the H1 of t-
butyl and H4 of the other pyrrole is 3.1 Å (Figure 2b). This type of unusual side contact generates a
continuous bowl-shaped shield on the C-aryl side of the tetrazole, similar to what we observed in TS2. In
contrast, when the Boc groups are removed as in tetrazole 27, the pyrrole rings are coplanar with the C-
phenyl ring, forcing the tetrazole ring to twist out of the co-planarity with the C-phenyl ring (Figure 2d).
The collapse of the three-dimensional bowl-shaped shield likely causes the destabilization of nitrile imine-
27, leading to predominant water quenching (Table S1). To probe whether the steric shield exists in
solution, we performed a 2D NMR experiment with tetrazole 26 in CDCl3. The proton signals of tetrazole
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26 were assigned based on the H-1H COSY spectrum (Fig. S13). The spatial proximity between two
pyrrole rings was assessed by examining the NOE effect. Strong NOE cross-peaks were detected between
H1 of one pyrrole, and H4 of the other pyrrole, and between H1 and H5 of the C-phenyl ring in the
ROESY spectrum (Figure 2e), indicating the steric shield remains intact in solution.
Stability of nitrile imine-26 in aqueous medium. Because the in situ generated nitrile imines also
undergo water-quenching, we compared the water-quenching rate of nitrile imine-26 to that of nitrile
imine-1 by subjecting the corresponding tetrazole to a 15-sec photoirradiation at 302 nm and quantifying
the residual nitrile imines post-photoirradiation by intercepting them with excess dimethyl fumarate. To
our surprise, despite complete photoinduced tetrazole ring rupture we could not detect residual nitrile
imine-1 under these conditions (Fig. S14b, c), suggesting that the half-life of nitrile imine-1 is less than
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7.5 sec. In contrast, the half-life of nitrile imine-26 was determined to be 102 sec (Fig. S14d, e),
reminiscent of the sterically crowded 2,5-ditritylnitrilimine reported in the literature.23 To probe structural
basis of the extraordinarily long half-life of nitrile imine-26, we also examined the nitrile imine derived
from Moc-pyrrole-tetrazole 28 and found its half-life to be 21 sec (Fig. S14f, g), 5-times shorter than that
of nitrile imine-26, indicating that the Boc groups are crucial to the formation of an effective steric shield.
The reduced protection from the less bulky Moc groups may also explain the modest selectivity for the
cycloaddition with tetrazole 28 (Table S1).
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In situ generation of QBP conformation sensor. Because the photoinduced cycloaddition reaction
produces an environmentally sensitive pyrazoline fluorophore24-26 whose emission intensity increases
when placed in less polar solvents (Fig. S15), we investigated whether tetrazole 26 is capable of generating
a fluorescent sensor in situ for studying protein conformational dynamics. To this end, we selected
glutamine binding protein (QBP) because its active site closes after binding to glutamine with reduced
polarity near the binding cleft.27-29 Indeed, environmentally sensitive probes have been installed at Asn160
position to sense this conformational transition by fluorescence.30 Accordingly, we expressed the QBP
mutants encoding SphK at position-160 and reacted this mutant with a water-soluble tetrazole 30 (Figure
3a). In-gel fluorescence analysis revealed the pyrazoline band for QBP-N160SphK but not for the control
mutant QBP-N160BocK, indicating that the cycloaddition-mediated pyrazoline formation is highly
specific (Fig. S16). Upon titration with glutamine, the QBP-pyrazoline fluorescence showed
concentration-dependent increases with a maxima reached when the glutamine concentration was 10 mM
(Figure 3b), indicating reduced polarity of the environment surrounding the pyrazoline fluorophore
following the closure of the QBP active site. As a control, the small-molecule pyrazoline did not show
the glutamine-dependent fluorescence change (Fig. S15c). Fitting the data to a one-site specific binding
model gave a dissociation constant, Kd, of 3.3 mM for glutamine (Figure 3c), significantly higher than the
reported Kd values for other fluorophore-modified QBP-N160C mutants (Kd = 0.3 ~ 6.6 M),28
presumably due to perturbation by the larger pyrazoline. Nevertheless, the result demonstrates that the
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sterically shielded nitrile imine is capable of generating a fluorescent sensor in situ for monitoring protein
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conformational change.
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Fluorescent labeling of GCGR in live mammalian cells. To probe whether the sterically shielded
nitrile imine reacts selectively with an alkene-encoded protein in cellular environment, we synthesized a
Cy5-conjugated tetrazole 31 and verified its selective reactivity in fluorescent labeling of the Sph-encoded
sfGFP in bacterial cell lysates (Fig. S17). We then investigated the efficiency of tetrazole 31 in fluorescent
labeling of a glucagon receptor (GCGR) mutant encoding SphK at position-372 in mammalian cells. We
chose GCGR because: (1) it is a member of class B G protein-coupled receptor family and a validated
drug target for diabetes;31,32 (2) the study of GCGR activation is crucial to the design of GCGR
antagonists;33 and (3) bioorthogonal fluorescent labeling of GCGR via tetrazine ligation was reported
recently.34 Accordingly, HEK 293T cells expressing GCGR-H372SphK-GFP was treated with 500 nM
tetrazole 31 followed by a brief exposure to 302-nm photoirradiation (Figure 4a). Strong Cy5 fluorescence
was detected at the plasma membrane by confocal microscopy as early as 30 sec with maximum intensity
reached at 1 min (Figure 4b). The Cy5 fluorescence co-localized with that of GFP, indicating that the
tetrazole 31 mediated GCGR labeling is highly specific. For comparison, HEK 293T cells expressing
GCGR-H372SphK-GFP were similarly labelled with a Cy5-conjugated tetrazine,34 though a higher
tetrazine concentration (1 M) and longer incubation time (30 min) were needed to reach maximum
fluorescence (Fig. S18). Based on fluorescence-activated cell sorting, 89% of GCGR-H372SphK-GFP-
expressing cells were labelled by tetrazole 31 after 1 min (Figure 4c-e; Fig. S19). The labeling specificity
is particularly striking as cells without expression of GCGR-GFP show negligible Cy5 signals (Figure 4b,
4e). Together, the confocal and FACS analysis results confirmed that the sterically shielded tetrazole
offers a new class of reagents for fast (~1 min) and highly specific bioorthogonal labeling of membrane
proteins with a small organic fluorophore in live mammalian cells.
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In summary, we have adopted a bioinspired strategy in the design of sterically shielded tetrazoles for
bioorthogonal photoinduced cycloaddition with a suitable alkene dipolarophile. The photo-generated
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nitrile imine from the sterically shielded tetrazole 26 exhibited dramatically enhanced selectivity for the
cycloaddition over the nucleophilic addition. The DFT calculations and structural studies showed that the
N-Boc-pyrrole groups adjacent to the in situ generated nitrile imine provide a steric shield to prevent the
thiolate addition by raising the corresponding activation energy. Indeed, the photoactivationinterception
study of the in situ generated, sterically shielded nitrile imine revealed a remarkably long half-life of 102
sec, compared to less than 7.5 sec for the unshielded parent nitrile imine. Because the tetrazole-alkene
cycloaddition produces an environmentally sensitive pyrazoline fluorophore, the sterically shielded
tetrazole 26 was used to successfully generate in situ a fluorescent sensor for monitoring ligand-induced
conformational transition of QBP. Due to its fast kinetics and extraordinary selectivity, the sterically
shielded nitrile imine was also utilized to fluorescently label an alkene-encoded GCGR in live mammalian
cells at a concentration as low as 500 nM within 1 min, which is significantly faster than the optimized
tetrazine reagent. While the present work focuses on the N-aryl ring of the 2,5-diaryltetrazoles for
enhanced selectivity, we expect the half-life, reactivity, and selectivity of this class of sterically shielded
nitrile imines can be further tuned through optimization of the C-aryl rings26,35 for their expanded use in
chemical biology. In addition, the use of molecular shape to control reaction selectivity could provide a
general strategy for optimization of other cycloaddition-based bioorthogonal reactions.
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Experimental Section
Expression and purification of SphK or BocK-encoded QBP. BL21(DE3) competent cells were
transformed with the pETtrio-QBP160TAG and pEVOL-TCOKRS (for SphK) or pEVOL-PylT-PylRS
(for BocK) plasmids. The transformants were recovered in 1 mL SOC medium at 37 ºC for 1 h before
plating onto LB agar plate containing 100 μg/mL ampicillin and 34 μg/mL chloramphenicol. A single
colony was picked and allowed to grow in 5 mL LB medium supplemented with 100 μg/mL ampicillin
and 34 μg/mL chloramphenicol at 37 °C overnight. This starter culture was used to inoculate 25 mL LB
medium supplemented with 100 μg/mL ampicillin and 34 μg/mL chloramphenicol. Cells were allowed to
grow at 37 ºC with shaking at 280 rpm until OD600 reached 0.6~0.8. Protein expression was then initiated
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by adding 1 mM IPTG, 0.2% arabinose, and 1 mM SphK or BocK. After 6 hours of incubation, cells were
harvested by centrifugation, resuspended in binding buffer (300 mM NaCl, 50 mM Na2HPO4, 10 mM
imidazole, pH 8.0), and lysed by sonication. The lysate was clarified by centrifugation. Afterwards, 150
μL Ni-NTA beads (Thermo Scientific) was added to the supernatant and the mixture was rocked at room
temperature for 2 h. Beads were collected by centrifugation and washed with washing buffer (300 mM
NaCl, 50 mM Na2HPO4, 50 mM imidazole, pH 8.0). The bound proteins were eluted with elution buffer
(300 mM NaCl, 50 mM Na2HPO4, 250 mM imidazole, pH 8.0). A buffer exchange was then carried out
with DPBS using a 10 kD MWCO spin column (Thermo Scientific). The protein mass was determined
by deconvoluting the charge ladder in the LC-MS analysis. The protein concentration was determined by
the BCA assay.
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Measurement of ligand-dependent QBP-pyrazoline fluorescence change. A concentrated solution of
QBP-N160SphK and tetrazole 30 (1 mM dissolved in DMSO) were added to 50 L of DPBS/ACN (4:1)
to obtain a final protein concentration of 10 M and tetrazole concentration of 20 M. The mixture was
irradiated with a handheld 302 nm UV light for 1 min. An equal volume of L-glutamine solution in
DPBS/ACN (4:1) (concentration = 200 M, 2 mM, 10 mM, 20 mM, or 30 mM) was added to the mixture,
and the fluorescence spectra were measured using Horiba FluoroMax-4 spectrofluorometer at 25 ºC with
the excitation wavelength set at 405 nm.
HEK 293T cell culture and transfection. HEK 293T cells were maintained in a growth medium
containing Dulbecco’s modified eagle medium (DMEM, Life Technologies) supplemented with 10%
(v/v) fetal bovine serum (FBS, Life Technologies) and 10 g/mL gentamicin (Life Technologies).
Transfection was performed at 70-80% confluency using 3:1 reagent/DNA ratio of Lipofectamine 2000
(Life Technologies) with 2.5 g total DNA or polyethylenimine (PEI, Polysciences Inc.) with 3 g total
DNA per 35-mm dish. For imaging experiments, cells were maintained in 35-mm glass bottom culture
dishes using FluoroBriteTM DMEM medium (Life Technologies) supplemented with 10% FBS, 4 mM L-
glutamine, and 25 mM HEPES.
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Bioorthogonal labeling of SphK-encoded GCGR-GFP and confocal microscopy. HEK 293T cells
were co-transfected with 4:1 ratio of pCMV-MmPylRS-U6-tRNA and pCMV-GCGR-H372TAG-GFP
plasmids. A solution of 100 mM SphK in DMSO was diluted in growth medium to obtain a final
concentration of 1 mM SphK and the resulting medium was filtered through 0.2 m polyethersulfone
membrane. The medium was added to the cells followed by the transfection mixture. The cells were
incubated for additional 24-48 h, and then washed with the growth medium prior to labeling. A fresh
solution of tetrazole 31 and DpTz-Cy5 was prepared from 1 mM stock solution in DMSO to a final
concentration of 500 nM and 1 M, respectively, in 1 mL growth medium. For the photoclick chemistry
mediated labeling, HEK 293T cells expressing GCGR-H372SphK-GFP were treated with 500 nM
tetrazole 31 in 1 mL DMEM medium containing 10% FBS, and the culture was exposed to 302 nm UV
light for a brief period (30 sec, 1 min, or 2 min). The tetrazole-containing medium was removed and the
cells were washed with growth medium before incubation in 1 mL growth medium at 37 °C and 5% CO2
for additional 30 min. For the tetrazine ligation mediated labeling, HEK 293T cells expressing GCGR-
H372SphK-GFP were treated with 1 M DpTz-Cy5 in 1 mL DMEM medium containing 10% FBS, and
the culture was incubated at 37 °C and 5% CO2 for various time (5 min, 10 min, 30 min, or 60 min). The
DpTz-Cy5 containing medium was removed, and the cells were washed with growth medium before
incubation at 37 °C and 5% CO2 for additional 30 min. The medium was then switched to FluoroBriteTM
DMEM before laser scanning confocal microscopy. The confocal images were acquired using a Zeiss
LSM 710 equipped with Plan-Apochromat 20/0.8 M27 or 40/1.3 Oil DIC M27 objective with ex. 488
/em. 493-598 nm for GFP channel and ex. 640/em. 645-759 nm for Cy5 channel.
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Flow cytometry analysis of fluorescent labelled HEK 293T cells. HEK 293T cells expressing GCGR-
H372SphK, GCGR-H372SphK-GFP, or Cy5-labeled GCGR-H372SphK-GFP (~106 cells each) were
detached from the culture dish using trypsin and washed successively with PBS (2) and FACS buffer
(DPBS, 1% BSA, 0.03% NaN3; 2) at room temperature. The cells were resuspended in FACS buffer,
and 10,000 cells were recorded in each fluorescence activated cell sorting.
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Computational Details. All calculations were carried out with the Gaussian 09 program package.
Geometry optimizations were performed with the B3LYP functional with Grimme’s DFT-D3 dispersion
correction, the 6-31+G(d) basis set, and the SMD solvation model with water as the solvent. Frequency
analysis was conducted at the same level of theory to verify the stationary points to be minima or saddle
points and to obtain zero-point energy (ZPE) and thermal energy corrections at 298.15 K. Single-point
energy calculations on the B3LYP-D3-optimized geometries were performed with the ωB97XD
functional, using 6-311++G(d,p) basis set and the SMD solvation model with water as the solvent.
Computed structures are illustrated using CYLView.
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ASSOCIATED CONTENT
Supporting Information
Supplemental figures, tables and methods, synthetic schemes, characterization of new compounds. This
material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*qinglin@buffalo.edu
ACKNOWLEDGMENTS
We gratefully acknowledge the NIH (R01GM085092) for generous financial support. We thank Prof.
Wenshe Liu at Texas A&M University for providing the pETtrio-1-AcKRS-pylT(TAA)-QBP141TAA
plasmid, Alan Siegel at SUNY Buffalo North Campus Imaging Facility for assistance with confocal
microscopy, Alan Connor at SUNY Buffalo for assistance with 2D NMR analysis, and Alan Friedman at
SUNY Buffalo School of Engineering and Applied Sciences Instrumentation Center for assistance with
high-resolution mass spectrometry.
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(17) Zahra, J. A.; Abu Thaher, B. A.; El-Abadelah, M. M.; Boese, R. Org. Biomol. Chem. 2003, 1, 822-
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be lower than those of the thiolate additions with glutathione. However, the use of methyl thiolate should
have a smaller impact on the relative reactivity (ΔΔG‡) of the two nitrile imines in thiolate addition
reactions.
(23) Réau, R.; Veneziani, G.; Dahan, F.; Bertrand, G. Angew. Chem. Int. Ed. 1992, 31, 439-440.
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(31) Wootten, D.; Miller, L. J.; Koole, C.; Christopoulos, A.; Sexton, P. M. Chem. Rev. 2017, 117, 111-
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Scheme 1. Strategy of employing structural pendants to stabilize the in situ generated nitrile imine and
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direct the reaction toward 1,3-dipolar cycloaddition over the competing nucleophilic addition.
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Scheme 2. Potential products from the substituted tetrazoles under the competitive conditions. Four
types of substituted tetrazoles were evaluated in this competition assay; see Table S1 in SI for structural
details. PB = phosphate buffer; ACN = acetonitrile.
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Table 1. Product distribution and reaction kinetic of tetrazole 26 vs. 1 in 1,3-dipolar cycloaddition
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reaction with the various alkene dipolarophiles a
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Tetrazole 1
Tetrazole 26
Entry
1
Dipolarophile
Product distribution (%)
k2
M1 s1
Product distribution (%)
k2
M1 s1
a
b
c
a
b
c
47
51
2
2,000 ± 300
92
2
6
860 ± 110
2
3
4
95
5
5
0
4
5
4,700 ± 500
NA b
99
96
89
0
2
3
1
2
8
2,400 ± 300
1,000 ±100
1,000 ± 250
91
75
20
2,200 ± 700
5
6
7
0
4
93
90
35
7
6
3
NA b
85
86
96
6
2
0
9
12
4
520 ± 140
1,400 ± 130
2,800 ± 200
2,800 ± 300
3,100 ± 600
62
8
60
37
3
3,800 ± 700
97
0
3
2,300 ± 300
a For determination of product distribution, a solution of 25 M tetrazole and 500 M each of alkene
dipolarophile and glutathione (reduced form; GSH) in 500 L phosphate buffer/acetonitrile (1:1), pH 7.4,
was photoirradiated with a handheld 302-nm UV lamp at room temperature for 1 min. The product
distribution was analyzed by reverse-phase HPLC with absorbance set at 254 nm. For kinetic
measurement, a mixture of 10 M tetrazole and 100 M alkene dipolarophile (50 M for dimethyl
fumarate, Sph, and TCO) in 1 mL phosphate buffer/acetonitrile (1:1), pH 7.4, in a quartz cuvette was
photoirradiated with a handheld 302-nm UV lamp at room temperature for a period of 30-40 s, and the
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reactions were monitored by a fluorimeter. The measurements were repeated three times to derive the
mean and standard deviation. b NA = not available because pyrazoline fluorescence was not detected under
kinetics conditions.
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Figure 1. Computational studies of the steric shielding effect of the ortho N-Boc-pyrrole
substituents. Computed transition states and activation energies involving nitrile imine-1 and -26 for (a)
1,3-dipolar cycloaddition with 5-methyl-spiro[2.3]hex-1-ene, and (b) nucleophilic addition with methyl
thiolate, respectively. DFT calculations were performed at the 휔B97x-D/6-311++G(d,p)/SMD(Water)
level of theory using geometries optimized by B3LYP-D3/6-31+G(d)/SMD(Water). R = N-Boc. The
distances shown are in Å, and energies are in kcal/mol.
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b
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y
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90 CW
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x
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90 CW
5
3.098
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3.189
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90 CW
x
90 CW
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4
CDCl3
1
Figure 2. Structural characterization of tetrazoles 26 and 27. (a) Chemical structure of tetrazole 26
with the proton numbering marked on the structure. (b) X-ray crystal structure of tetrazole 26 with depth
cue showing co-planarity of the diphenyltetrazole (left) and perpendicular arrangement of N-Boc-pyrrole
relative to the diphenyltetrazole (right). CW, clockwise. (c) Chemical structure of tetrazole 27. (d) Crystal
structure of 27 with depth cue showing co-planarity between the C-phenyl and pyrrole rings, and
perpendicular twisting of the tetrazole and the C-phenyl ring. The structures are viewed from the same
angles as in tetrazole 26. (e) Partial 1H-1H ROESY spectrum of tetrazole 26 in CDCl3 showing prominent
cross-peaks between tert-butyl protons and proton-4, -5 and -7 as marked on the chemical structure.
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a
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480 nm
480 nm
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405 nm
405 nm
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60000
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20000
0
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20000
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10
10000
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0
500
600
700
0
5
10
15
Wavelength (nm)
Glutamine Concentration (mM)
Figure 3. Application of the sterically shielded nitrile imine to generate in situ a fluorescent sensor
for probing ligand-induced QBP conformational change. (a) Scheme for in situ synthesis of a
pyrazoline-based sensor from QBP-N160SphK mutant and a water-soluble tetrazole 30. The glutamine-
free QBP (PDB code: 1GGG) adopts an open conformation27 while the glutamine-bound QBP (PDB code:
1WDN) adopts a closed conformation29. (b) Fluorescence spectra of QBP-pyr-30 upon titration of
glutamine (final concentration = 0.1 mM, 1 mM, 5 mM, 10 mM or 15 mM in 4:1 DPBS/acetonitrile). ex
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= 405 nm; QBP concentration was set at 5 M. (c) Plot of the change of fluorescence intensity at 480 nm
vs. glutamine concentration. The data were fitted to one-site specific binding model with the following
equation: ∆Fobs = ∆FmaxS /(Kd + S).
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GCGR-H372SphK-GFP
GCGR-H372Cy5-GFP
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GFP
DIC
Overlay
0.5 min
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Figure 4. Application of the stabilized nitrile imine to bioorthogonal labeling of GCGR in live cells.
(a) Scheme for bioorthogonal labeling of a GCGR-GFP mutant encoding SphK at position-372 with a
Cy5-funtionalised water-soluble tetrazole 31. The C-terminal GFP is not shown for clarity. (b) Confocal
micrographs of HEK 293T cells expressing GCGR-H372SphK-GFP after photoirradiation with a
handheld 302-nm UV lamp for a period of 0.5, 1, or 2 min in DMEM medium containing 500 nM
tetrazole-Cy5. Scale bar = 20 m. Flow cytometry analysis of HEK 293T cells expressing (c) GCGR-
H372SphK, (d) GCGR-H372SphK-GFP before labeling, and (e) GCGR-H372SphK-GFP after labeling
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with tetrazole 31. Cells were treated with 500 nM of tetrazole 31 and photoirradiated for 1 min. A total of
10,000 cells were analyzed in each measurement. X-axis, GFP channel; y-axis, Cy5 channel; both axes
are in log scale.
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