Site-Specific Antibody Functionalization Using Tetrazine-Styrene Cycloaddition

Article abstract of DOI:10.1021/acs.bioconjchem.8b00114

Biologics, such as antibody-drug conjugates, are becoming mainstream therapeutics. Consequently, methods to functionalize biologics without disrupting their native properties are essential for identifying, characterizing, and translating candidate biologics from the bench to clinical practice. Here, we present a method for site-specific, carboxy-terminal modification of single-chain antibody fragments (scFvs). ScFvs displayed on the surface of yeast were isolated and functionalized by combining intein-mediated expressed protein ligation (EPL) with inverse electron-demand Diels-Alder (IEDDA) cycloaddition using a styrene-tetrazine pair. The high thiol concentration required to trigger EPL can hinder the subsequent chemoselective ligation reactions; therefore, the EPL reaction was used to append styrene to the scFv, limiting tetrazine exposure to damaging thiols. Subsequently, the styrene-functionalized scFv was reacted with tetrazine-conjugated compounds in an IEDDA cycloaddition to generate functionalized scFvs that retain their native binding activity. Rapid functionalization of yeast surface-derived scFv in a site-directed manner could find utility in many downstream laboratory and preclinical applications.

Full text of DOI:10.1021/acs.bioconjchem.8b00114

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Site-Specific Antibody Functionalization
Using Tetrazine–Styrene Cycloaddition
Benjamin Umlauf, Kalie Ann Mix, Vanessa A. Grosskopf, Ronald T. Raines, and Eric V. Shusta
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00114 • Publication Date (Web): 25 Apr 2018
Downloaded from http://pubs.acs.org on April 26, 2018
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Site-Specific Antibody Functionalization Using Tetrazine–Styrene Cycloaddition
Benjamin J. Umlauf,† Kalie A. Mix,‡,§ Vanessa A. Grosskopf, † Ronald T. Raines,‡,#,§ and Eric V.
Shusta*,†
‡
†Department of Chemical and Biological Engineering, Department of Biochemistry, and
#Department of Chemistry, University of Wisconsin–Madison, Madison, Wisconsin 53706, United
States
*Corresponding author:
Eric V. Shusta
Department of Chemical and Biological Engineering
University of WisconsinꢀMadison
1415 Engineering Drive
Madison, WI 53706
eshusta@wisc.edu
Ph: (608) 265ꢀ5103
Fax: (608) 262ꢀ5434
§Present Address: Department of Chemistry, Massachusetts Institute of Technology, 77
Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
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ABSTRACT
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Biologics, such as antibody–drug conjugates, are becoming mainstream therapeutics.
Consequently, methods to functionalize biologics without disrupting their native properties are
essential for identifying, characterizing, and translating candidate biologics from the bench to
clinical practice. Here, we present a method for siteꢀspecific, carboxyꢀterminal modification of
singleꢀchain antibody fragments (scFvs). ScFvs displayed on the surface of yeast were isolated and
functionalized by combining inteinꢀmediated expressed protein ligation (EPL) with inverse
electronꢀdemand Diels–Alder (IEDDA) cycloaddition using a styrene–tetrazine pair. The high thiol
concentration required to trigger EPL can hinder the subsequent chemoselective ligation reactions;
therefore, the EPL reaction was used to append styrene to the scFv, limiting tetrazine exposure to
damaging thiols. Subsequently, the styreneꢀfunctionalized scFv was reacted with tetrazineꢀ
conjugated compounds in an IEDDA cycloaddition to generate functionalized scFvs that retain
their native binding activity. Rapid functionalization of yeast surfaceꢀderived scFv in a siteꢀ
directed manner could find utility in many downstream laboratory and preꢀclinical applications.
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INTRODUCTION
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Antibodies are a rapidly growing class of therapeutic agents with significant clinical
success.51 In addition, imaging, diagnostics, and separation technologies often employ antibodies
due to the high specificity and affinity for their cognate antigens. Functionalization of antibodies
with chemical probes such as fluorophores,1,2 smallꢀmolecule drugs,3ꢀ5 or other biomolecules6ꢀ8 is
used to customize these reagents for many applications. Still, a growing number of studies indicate
it is essential to append these probes in a siteꢀspecific manner that does not disrupt antibody
function.3,6,9ꢀ12
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We previously developed a method for siteꢀspecific, carboxyꢀterminal antibody
modification by employing yeast surface display in combination with expressed protein ligation
(EPL).13,14 In this system, the carboxyꢀterminus of the scFv is fused to an engineered, non–selfꢀ
cleavable intein (202ꢀ08) and expressed on the surface of yeast cells. Our laboratory previously
generated 202ꢀ08 by evolving the Mxe GyrA intein to improve yeast surface display of scFvꢀintein
fusions.14,36 Addition of a thiol, 2ꢀmercaptoethanesulfonic acid (MESNA), liberates the scFv from
the yeast surface and activates intein splicing to undergo transthioesterification and produce a
carboxyꢀterminal thioester. Subsequently, a cysteine (Cys) amide is used to link the scFv to a Cysꢀ
modified probe of interest via an amide bond. Of note, this system enables rapid protein
modification specifically at the carboxyꢀterminus of the released protein, without requiring protein
purification.14,36 As a result, the yeast display EPL system is ideal for the rapid, highꢀthroughput
functionalization of antibodies.
While other systems for siteꢀspecific labeling including FLASH, CLIP tags, and genetic
code expansion (GCE) can also facilitate antibody functionalization, nonꢀselfꢀcleaving inteins are
beneficial in that they exhibit traceless appendage of the functional group upon EPL.13,14,36
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Although one can append a variety of functional groups to proteins using EPL, including postꢀ
translationally modified peptides,13 nonꢀcanonical amino acids,18 and biophysical probes,19 it can
be advantageous to employ the expanding catalog of bioorthogonal reagents to append peptides,
fluorophores, nanoparticles, and purificationꢀenhancing moieties.45ꢀ48 To this end, we previously
employed EPL with surfaceꢀdisplayed proteins to append an azide as a reactive handle for copperꢀ
catalyzed azide–alkyne cycloaddition (CuAAC).14 Although the rapid rate of this reaction makes it
useful for many in vitro applications, CuAAC can have more limited utility in vivo due to the
oxidative stress induced by Cu(I) and crossꢀreactivity of ascorbate with biological
nucleophiles.20,21 Additionally, the multiꢀcomponent nature of the reaction, requiring a copper
catalyst, activating ligand, and reducing agent in addition to the azide and alkyne reagents often
requires optimization to apply the reaction to different molecules and conditions. Further, the high
concentration of thiols can impair certain CuAAC reactions.22ꢀ24 As an alternative, strained
cyclooctynes (e.g., DBCO) could be used in copper free reactions with azides. A DBCOꢀcysteine
reagent cannot be effectively used for the EPLꢀmediated scFv modification because strained
cyclooctynes interact with thiols at concentrations that are orders of magnitude lower than those
used in the EPL reaction.25 Instead, we have successfully used EPL to first append an azide that is
subsequently reacted with a DBCOꢀconjugated probe to modify scFvs.36 However, certain
applications such as in vivo targeting with an azideꢀmodified scFv, followed by a DBCO probe
would be limited since strained cyclooctynes suffer from poor bioavailability.52
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In order to address the aforementioned chemical compatibility issues with the yeast display
EPL system, and to expand its utility, we explored the possibility of replacing Cuꢀcatalyzed and
strainꢀbased biorthogonal reactions with an inverse electronꢀdemand Diels–Alder (IEDDA)
cycloaddition reaction. Styrene was shown to be inert to millimolar thiol concentration, enabling
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EPL driven carboxyꢀterminal modification of scFv with styrene. Subsequently, a styreneꢀmodified
scFv can be covalently linked to tetrazineꢀcontaining probes via IEDDA cycloaddition, and the
functionalized scFvs retain their antigenꢀbinding capacity.
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RESULTS
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A Styrene is Compatible with Both EPL and IEDDA Reactions. The reaction of a tetrazine with
transꢀcyclooctene (TCO) has become a wellꢀestablished and useful tool in chemical biology as a
result of its aqueous, rapid, and twoꢀcomponent nature.27 For compatibility with yeast surface
display EPL, either tetrazine or TCO must be inert to high thiol concentrations (Figure 1, Steps A–
C). To measure stability in the presence of free thiols, transꢀcyclooctenol was incubated with
FmocCysOH. After only 4 h, the transꢀcyclooctenol had isomerized completely to the unreactive
cis isomer (Figure 2a). The dienophile in an IEDDA cycloaddition can also be activated by
electronꢀdonating groups instead of strain.28 One such activated alkene, 4ꢀaminostyrene,29
possesses an amino group to both activate the alkene by donating electrons into the aryl system
and act as a handle for derivatization. To test the compatibility of styrene with a high concentration
of thiols, 4ꢀaminostyrene was incubated with FmocCysOH and in contrast to transꢀcyclooctenol,
no degradation of the styrene was detectable after 12 h (Figure 2b). NMR spectroscopy was next
used to examine the reaction kinetics of 4ꢀacetamidostyrene (4) with a phenyltetrazine (Figure
S1).27 The secondꢀorder rate constant was found to be k = (4.0 ± 0.1) × 10–3 M–1s–1.
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Step A
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Step B
Step C
.
Step D
Figure 1. Route for the siteꢀspecific functionalization of a yeast surfaceꢀdisplayed scFv. A fourꢀ
step process is used to functionalize the carboxyꢀterminus of an scFv by coupling expressed
protein ligation (EPL) and inverse electronꢀdemand Diels–Alder (IEDDA) cycloaddition. Step A is
the release of the scFv from the yeast surface by thiol–disulfide exchange with MESNA. Step B is
the cleavage of the intein and formation of a thioester with MESNA at the carboxyꢀterminus. Step
C is reaction of the thioester with CysꢀPEG3ꢀstyrene (3). Steps A–C occur in a oneꢀpot reaction
with yeast, MESNA and CysꢀPEG3ꢀstyrene (3). After removal of yeast and MESNA, Step D is the
IEDDA conjugation between scFv–styrene and a tetrazineꢀcontaining probe. Inset: structures of
two tetrazine probes used for IEDDA cycloaddition
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Figure 2. Stability of candidate reagents for EPL/IEDDA. (a) NMR spectra of vinyl protons of
transꢀcyclooctenol over the course of 4 h of incubation with FmocCysOH. (b) NMR spectra of 4ꢀ
aminostyrene, FmocCysOH, and the reaction mixture after a 12ꢀh incubation. (c) Absorbance at
525 nm of 5 mM tetrazineꢀamine with or without 100 µL of yeast cell culture incubated for 45
min. Mean and standard deviation are plotted from a minimum of three replicates. Inset: images
showing the loss of the characteristic pink color of a tetrazine in the presence of yeast cells (*p <
0.01 using two tailed tꢀtest). (d) Absorbance at 525 nm of a 5 mM tetrazine–amine solution
containing various reducing agents used for yeast surface cleavage and EPL incubated for 45 min.
Mean and standard deviation are plotted from a minimum of three replicates. Inset: images
demonstrate loss of the characteristic pink color of a tetrazine (&p < 0.01 using ANOVA).
A Tetrazine is Not Compatible with a Yeast Display EPL Reaction. While a styrene appeared
to be an appropriate dienophile for IEDDA in the context of a yeast display EPL reaction, the
feasibility of using tetrazine in an EPL reaction was also explored. The tetrazine would have to
withstand incubation with yeast and thiol for it to be compatible with the yeast display EPL
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reaction (oneꢀpot reaction of Figure 1, Steps A–C) since it has been reported that a reduced
tetrazine is no longer capable of an IEDDA reaction.30 Incubation of a tetrazine with live yeast
cells resulted in a significant decrease in reactive tetrazine as measured by tetrazine absorbance
(Figure 2c), indicating that the tetrazine is destroyed, sequestered, or reduced by the yeast cells.30
Tetrazine was also affected by common reducing agents used in inteinꢀmediated EPL reactions
(Figure 2d). Thiolꢀbased reducing agents, including 2ꢀmercaptoethanol, dithiothreitol, and
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MESNA, all drove tetrazine reduction. Incubation of
a
tetrazine with tris(2ꢀ
carboxyethyl)phosphine (TCEP) at only 4 mM, the minimal amount required to release scFv from
the yeast surface, also resulted in tetrazine reduction. Given that tetrazine reduction would hinder
an IEDDA reaction with a styreneꢀprobe reactant (Figure 1, Step D), its use in the EPL stages of
the conjugation scheme was not pursued further.
Styrene Modification of scFv by EPL Followed by IEDDA Cycloaddition with a Tetrazine.
Based on the results above, we chose to modify the scFv with the styrene reagent using EPL, and
then derivatize the modified scFv with a tetrazine probe after removal of yeast and reducing agents
(Figure 1). To test the strategy, an scFv that binds to fluorescein (4ꢀ4ꢀ20) was used. 4ꢀ4ꢀ20 scFv–
intein fusion protein was displayed on the yeast surface, and the yeast was pelleted to remove
growth medium. The yeast cells were resuspended, and 50 mM of MESNA was added, followed
by 5 mM Cys–PEG3–styrene (3) to promote the EPL reaction. The EPL reaction proceeded for 3–
18 h at room temperature with gentle shaking. MESNA released the Aga2p fusion and triggered
intein cleavage, yielding an scFv with a carboxyꢀterminal thioester (Figure 1, Steps A and B).
Subsequently, the Cys–PEG3–styrene reacted with the scFvꢀthioester to yield an scFv modified at
its carboxyꢀterminus with styrene (Figure 1, Step C). After removal of yeast cells by pelleting,
sterileꢀfiltering through 0.22ꢀµm spin columns, and dissolution in MESNA via buffer exchange
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with 10,000 Da MWCO filters, styreneꢀmodified scFv was reacted with tetrazine–biotin in an
IEDDA reaction to generate scFv functionalized with carboxyꢀterminal biotin (Figure 1, Step D).
Western blotting demonstrated that biotin functionalization of scFv was dependent both on styrene
modification and reaction with tetrazine–biotin (Figure 3a). MESNA concentrations of 200 mM
yielded the most robust combined EPL/IEDDA reaction as measured by the maximal amount of
biotinꢀfunctionalized scFv (Figure 3b). The efficiency of the combined EPL/IEDDA reactions, in
terms of the fraction of scFv released from the yeast surface that was successfully biotinylated,
was 78% ± 10% (Figure 3c).
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Figure 3. Functionalization of 4ꢀ4ꢀ20 scFv by EPL followed by IEDDA. (A) Western blot
examining the biotin modification of a 4ꢀ4ꢀ20 scFv with an EPL reaction followed by reaction with
5 mM tetrazine–biotin overnight at 30 °C with gentle shaking. Total released and inteinꢀcleaved
scFv (αꢀFLAG) and functionalized scFv (αꢀBiotin) were probed. (B) Western blot showing
EPL/IEDDA reaction yields as a function of MESNA concentration. αꢀFLAG detection is
indicative of total antibody available for functionalization while αꢀBiotin detection is used to
compare the combined EPL and IEDDA efficiency. (C) Fraction of 4ꢀ4ꢀ20 scFv released from
yeast that is biotinylated through combined EPL/IEDDA reactions. Total scFv released from yeast
and the fraction of this scFv that was biotinylated as determined by binding to streptavidin beads
were quantified by western blotting with αꢀFLAG antibody. The mean and standard deviation for a
minimum of three replicates is plotted. 78% ± 10% of the total scFv is modified using the
combined EPL/IEDDA reaction (p < 0.05, tꢀtest).
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ScFv Modification with Styrene–Tetrazine EPL/IEDDA Yields Functional scFvs. Next,
styreneꢀmodified scFvs were conjugated to a fluorophore in an IEDDA reaction with tetrazine–
Cy5 (Figure 1, Step D and inset). As demonstrated for the 4ꢀ4ꢀ20 scFv, antibodyꢀassociated Cy5
fluorescence required both styrene and tetrazine reactants (Figure 4a). To test that the scFv
antigenꢀbinding region retains function following modification, 4ꢀ4ꢀ20–Cy5 was applied to
microtiter wells coated with antigen in the form of FITCꢀdextran. There was a 50ꢀfold increase in
Cy5 signal for 4ꢀ4ꢀ20ꢀCy5 compared with an antibody that does not bind fluorescein, scFvA49,
indicating that the Cy5ꢀmodified 4ꢀ4ꢀ20 retained antigenꢀbinding capability (Figure 4b). To
confirm that antigen binding was not affected by EPL/IEDDA, the antigen binding affinities for
unmodified 4ꢀ4ꢀ20 and 4ꢀ4ꢀ20ꢀCy5 scFvs were measured, and the Kd values for 4ꢀ4ꢀ20–Cy5 (1.52
± 0.05 nM) and unmodified 4ꢀ4ꢀ20 scFv (1.65 ± 0.04 nM) were indistinguishable (Figure 3c; p =
0.38, nonꢀpaired, two tailed tꢀtest).
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Figure 4. Evaluation of 4ꢀ4ꢀ20 scFv function after EPL/IEDDA with styreneꢀtetrazine. (a) 4ꢀ
4ꢀ20 scFvꢀassociated Cy5 signal after EPL/IEDDA modification with Cys–PEG3–styrene (3) and
tetrazine–Cy5. Unreacted tetrazineꢀCy5 is removed from the scFv fraction using buffer exchange
and the remaining Cy5 fluorescence measured (&p < 0.01 using ANOVA). The mean and standard
deviation for a minimum of three replicates for each group is plotted. (B) 4ꢀ4ꢀ20 and scFvA
modified with Cy5 are incubated with immobilized FITC–dextran antigen. Unbound scFv was
removed and wells quantified for Cy5 signal (*p < 0.01, tꢀtest). The mean and standard deviation
for a minimum of three replicates is plotted. (C) An equilibrium binding analysis by fluorescence
quench affinity titration was used to compare the binding affinity of 20 nM unmodified 4ꢀ4ꢀ20 or
Cy5ꢀmodified 4ꢀ4ꢀ20. Calculated Kd values (mean ± SD) are 1.65 ± 0.04 nM and 1.52 ± 0.05 nM,
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respectively (p = 0.38 using nonꢀpaired, twoꢀtailed tꢀtest).
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As further verification of scFv functionalization and to demonstrate the utility of the
EPL/IEDDA approach for cellꢀbased assays, scFvA, a brain endothelial cell binding and
internalizing antibody,49 was functionalized with Cy5. The scFvAꢀCy5 conjugate was then used in
flow cytometry and immunofluorescence based internalization assays. Rat brain endothelial cells
(RBE4) were incubated with scFvA–Cy5 or control 4ꢀ4ꢀ20–Cy5, for 1 h at 37 °C to allow for scFv
internalization. Internalization was quantified using flow cytometry, with 96% ± 3% of RBE4 cells
having internalized scFvA–Cy5, whereas only ~1% of cells internalized 4ꢀ4ꢀ20 scFv–Cy5 (Figure
5a and b), indicating a specific interaction of scFvAꢀCy5 with RBE4 cells. The internalization of
scFvA was also evaluated using fluorescence microscopy. ScFvAꢀCy5 was found localized in
intracellular puncta (Figure 5c) as previously described.49 Staining patterns of scFvAꢀCy5 and
unmodified scFvA appear similar (Figure 5d), and a strong coꢀlocalization between antiꢀmyc and
Cy5 signal was observed (Figure 5c) indicating internalization of intact scFvAꢀCy5 conjugates.
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(a)
Myc
Merge
Cy5
(c)
(d)
(e)
FSC
(b)
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0
*
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y
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Figure 5. Internalization of Cy5ꢀlabeled scFv into rat brain endothelial (RBE4) cells. (a)
Representative flow cytometry dot plot after RBE4 cell internalization of scFvAꢀCy5 (blue) or 4ꢀ4ꢀ
20ꢀCy5 (red), with sample gate used for quantification. (b) Quantification of Cy5ꢀpositive cells
across three independent experiments for each group, with mean and standard deviation plotted (*p
< 0.01, tꢀtest). (c) Fluorescence microscopy images of RBE4 cells after allowing for internalization
of scFvA–Cy5 (red). Cells were fixed, permeabilized and stained for a Myc tag on the scFv
(green). Nuclei were stained with Hoechst 33342 (blue) (d) RBE4 cells treated with scFvA–styrene
that was not reacted with tetrazine–Cy5 and is stained as in panel F. (e) RBE4 cells not treated
with any scFv, and stained as in panel c. Scale bars = 20 µm.
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DISCUSSION
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We have described a facile method for functionalizing yeastꢀdisplayed scFvs at their carboxyꢀ
terminus by combining EPL and IEDDA reactions. EPL modification presented challenges for the
subsequent IEDDA reactions because of the high concentration of sulfur nucleophiles required for
the EPL reaction.23 As a result, TCO was not an effective dienophile in our system due to its
sensitivity to thiol reduction, but styrene was stable under the high thiol concentrations required of
EPL. Styrenes are known to undergo cycloaddition with tetrazines;27,28,31,32 and have been reported
for the bioconjugation of DNA.31 However, taking advantage of the inertness of styrene to thiols in
an EPL/IEDDA proteinꢀlabeling system has not yet been described previously. One shortcoming is
that the rate of reaction between styrene and tetrazine (Figure S1, 4.0 ± 0.1 × 10–3 M–1s–1) is quite
slow compared to the reported reaction of TCO and tetrazine (1–10 × 103 M–1s–1).27 Using the
system described here, the lowered reaction rate was overcome by adding an excess of tetrazine
and performing the reaction at 30 °C.22 Further tuning the electronics of each reagent could
potentially enhance the reaction rates, and thereby enhance functionalization of low concentrations
of styreneꢀmodified proteins.29,33 For instance, the rate could be accelerated by incorporation of
electronꢀdonating groups on the aryl ring of the styrene reagent, and by using dipyridylꢀtetrazine in
place of phenyltetrazine.
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There exist several constraints with respect to the tetrazine moiety in the yeast display
EPL/IEDDA system. Both live yeast cells and reducing agents, including the nonꢀthiol based
TCEP, resulted in reduction and subsequent inactivation of a tetrazine. To circumvent this
problem, Cys–PEG3–styrene (3) rather than Cys–PEG3–tetrazine was used as the EPL reagent. It
may be possible, however, to modify the scFv with Cys–PEG3–tetrazine if reducing reagents were
removed postꢀreaction and tetrazine reꢀoxidized. A recent study described incubation of tetrazines
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with methylene blue or using intense red light to reꢀoxidize a reduced tetrazine and restore
reactivity.30 Both of these routes would, however, require additional steps; and thus the addition of
styrene, rather than tetrazine, by EPL is preferable in this system. Given the relative differences in
size between an scFv and styrene (~30 kDa versus ~100 Da), a PEG spacer between the cysteine
amide used for EPL and the styrene moiety was added. In the experiments described here with
biotin or fluorophore modification, the relatively small size of the tetrazineꢀprobe is unlikely to
cause major steric hindrance. Nevertheless, in applications that seek to modify the scFv–styrene
with a tetrazineꢀprobe of larger size (e.g., nanoparticulate substrates), the PEG spacer will likely
have a beneficial impact.34,35
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Another important factor in method optimization was the concentration of MESNA employed
in the EPL reaction. Interestingly, there exists an optimal EPL concentration of MESNA at 200
mM for maximum yields from the IEDDA reaction, despite the fact that additional scFv is released
from the yeast surface when up to 800 mM MESNA is used. While one might expect a plateau
rather than optimum in MESNA concentration, the presence of an optimum could be due to high
concentrations of MESNA preventing the Sꢀ to Nꢀacyl switch required for EPL modification with
the styrene handle, or a result of competition between MESNA and cysteine for nucleophilic attack
on the inteinꢀgenerated thioester. At this optimum, we observed 78% ± 10% modification of 4ꢀ4ꢀ
20 scFv using the EPL/IEDDA protocol, which is sufficient for downstream applications as
demonstrated here.
Finally, the utility of the yeast display EPL/IEDDA antibody modification method was
demonstrated using a variety of fluorescenceꢀbased assays. Modification of 4ꢀ4ꢀ20 scFv with
styrene enabled carboxyꢀterminal conjugation to tetrazine–Cy5 without affecting antigen
binding.40,41 Such siteꢀspecific modification distal to the binding site can provide a distinct
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advantage over nonꢀspecific, amineꢀ or thiolꢀbased antibody functionalization protocols that often
result in a large percentage of inactive antibody because probe functionalization occurs near to or
within the antigenꢀbinding domain. The utility of this method was also demonstrated using brain
endothelial cell internalization assays with scFvAꢀCy5,42 where the scFvA–Cy5 conjugate retains
its ability to internalize into RBE4 cells. We believe that the facile, modular, siteꢀdirected
antibodyꢀlabeling protocol demonstrated herein is a powerful means to facilitate the development
and assessment of antibodies and biologics for laboratory and preclinical use.
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Materials and Methods
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Conditions. All procedures were performed in air at ambient temperature (~22 °C) and
pressure (1.0 atm) unless indicated otherwise.
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Chemical Synthesis. Materials. Unless noted otherwise, reagents and solvents were from
Sigma−Aldrich (Milwaukee, WI) and were used without further purification. Reagentꢀgrade
solvents: acetonitrile, dichloromethane (DCM), tetrahydrofuran (THF), and triethylamine (TEA)
were dried over a column of alumina and were removed from a dry still under an inert atmosphere.
Flash column chromatography was performed with 40−63 Å silica (230−400 mesh) from Silicycle
(Québec City, Canada), and thinꢀlayer chromatography was performed with EMD 250 ꢁm silica
gel 60 F254 plates.
Solvent Removal. The phrase “concentrated under reduced pressure” refers to the removal of
solvents and other volatile materials using a rotary evaporator at water aspirator pressure
(<20 Torr) while maintaining a water bath below 40 °C. Residual solvent was removed
from samples at high vacuum (<0.1 Torr).
1. N-hydroxysuccinimide
DCC, THF
O
O
H
H
2.
N
N
H2N
DCM
O
NH2
Boc
OH
Boc
N
O
NH2
3
H
3
S
S
S
S
1
Compound 1. BocꢀSꢀtertꢀbutylthioꢀ
Lꢀcysteine (500 mg, 1.6 mmol) from ChemꢀImpex
International (Wood Dale, IL) was dissolved in THF (5 mL). NꢀHydroxysuccinimide (186 mg, 1.6
mmol) and N,N′ꢀdicyclohexylcarbodiimide (DCC; 363 mg, 1.7 mmol) were added, and the
resulting solution was stirred overnight. The reaction mixture was filtered, and the filtrate was
concentrated under reduced pressure. The residue was dissolved in DCM (15 mL). 4,7,10ꢀTrioxaꢀ
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1,13ꢀtridecanediamine (0.9 mL, 4.3 mmol) was added, and the resulting solution was stirred
overnight. The reaction mixture was filtered, and the filtrate was concentrated under reduced
pressure. The residue was purified by reverseꢀphase HPLC on a C18 column using a gradient of
water–acetonitrile containing trifluoroacetic acid (0.1% v/v) to yield compound 1 as a clear oil (64
mg, 10% for 2 steps).
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O
O
O
O
TEA, CH2Cl2
HO
N
H2N
H
O
2
Compound 2. 4ꢀAminostyrene (100 mg, 0.8 mmol) was dissolved in DCM (8 mL). Succinic
anhydride (84 mg, 0.84 mmol) and TEA (0.24 mL, 1.7 mmol) were added, and the resulting
solution was stirred overnight. The reaction mixture was concentrated under reduced pressure. The
residue was dissolved in EtOAc and washed twice with 1 M HCl. The organic layer was dried over
Na2SO4(s) and concentrated under reduced pressure to yield compound 2 as a white solid (99 mg,
1
54%). H NMR (500 MHz, CD3OD, δ): 7.52 (d, 2H, J = 8.6 Hz), 7.37 (d, 2H, J = 8.6 Hz), 6.65–
6.71 (dd, 1H, J = 10.9 Hz, 17.7 Hz), 5.71 (d, 1H, J = 17.6 Hz), 5.15 (d, 1H, J = 11.0 Hz), 2.66 (s,
13
4H). C NMR (125 MHz, CD3OD, δ): 176.4, 172.8, 139.6, 137.6, 137.5, 134.7, 127.6, 120.9,
32.3, 30.0. HRMS–ESI (m/z): [M – H]– calcd for C12H13NO3, 218.0823; found, 218.0821.
1.
O
HO
N
H
O
2
O
O
O
N-hydroxysuccinimide
DCC, CH2Cl2
2. HCl/dioxane
H
H
N
H2N
N
N
N
O
Boc
N
O
NH2
H
H
3
H
3
O
S
S
S
S
1
3
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Cys(StBu)–PEG3–styrene (3). Compound 1 (361 mg, 0.71 mmol) was dissolved in DCM
(7 mL). Compound 2 (156 mg, 0.71 mmol), Nꢀhydroxysuccinimide (82 mg, 0.71 mmol), and DCC
(146 mg, 0.71 mmol) were added, and the resulting solution was stirred overnight. The reaction
mixture was filtered, and then concentrated under reduced pressure. The residue was dissolved in
acetonitrile and purified by reverseꢀphase HPLC on a C18 column using a gradient of water–
acetonitrile containing trifluoroacetic acid (0.1% v/v). The residue was then dissolved in 4.0 M
HCl in dioxane, and the resulting solution was stirred for 1 h. The solution was sparged with N2(g)
for 10 min to remove HCl and then concentrated under reduced pressure to yield compound 3 as a
white solid (61 mg, 12% for 2 steps).
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O
Cl
O
TEA, CH2Cl2
N
H2N
H
4
4ꢀAcetamidostyrene (4). 4ꢀAminostyrene (100 mg, 0.84 mmol) was dissolved in DCM
(8.4 mL). Acetyl chloride (0.18 mL, 0.84 mmol) and TEA (0.24 mL, 1.68 mmol) were added, and
the resulting solution was stirred overnight. The reaction mixture was concentrated under reduced
pressure, and the residue was dissolved in EtOAc. The solution was washed twice with 1 M HCl
and twice with saturated aqueous NaHCO3. The organic layer was dried over Na2SO4(s), and then
concentrated under reduced pressure. The residue was purified further by chromatography on silica
gel, eluting with 1:1 EtOAc/hexanes to yield compound 4 as a white solid (39 mg, 29%). 1H NMR
(500 MHz, CDCl3, δ): 7.47 (d, 2H, J = 8.6 Hz), 7.37 (d, 2H, J = 8.5 Hz), 7.14 (s, 1H), 6.70–6.64
(dd, 1H, J = 10.9, 17.6 Hz), 5.68 (d, 2H, J = 17.6 Hz), 5.20 (d, 2H, J = 10.9 Hz), 2.19 (s, 3H). 13C
NMR (125 MHz, CDCl3, δ): 168.1, 137.4, 136.1, 133.7, 126.8, 119.7, 113.1, 24.7. HRMS–ESI+
(m/z): [M + H]+ calcd for C10H11NO, 162.0913; found, 162.0912.
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Styrene and trans-Cyclooctene Stability. Stock solutions were prepared by dissolving
FmocCysOH, 4ꢀacetamidostyrene (4), and transꢀcyclooctenol in CD3OD at a concentration of
200 mM. The solutions were combined in an NMR tube to give an equimolar ratio and mixed, and
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the tube was inserted immediately into an NMR spectrometer. A 16ꢀscan H NMR spectrum was
acquired every 60 min.
O
O
O
O
O
O
N
OH
N
OH
N
OH
H
H
H
N
N
N
N
N
N
N
+
N
CD3OD
+
NH
O
O
N
H
O
NH
4
5
5'
Tetrazine–Styrene NMR Kinetics. Stock solutions (6.25 mM in CD3OD) were prepared of 5ꢀ
[4ꢀ(1,2,4,5ꢀtetrazinꢀ3ꢀyl)benzylamino]ꢀ5ꢀoxopentanoic acid and 4ꢀacetamidostyrene (4). The
solutions were combined in an NMR tube at an equimolar ratio and mixed, and the tube was
inserted immediately into an NMR spectrometer. A 16ꢀscan 1H NMR spectrum was acquired every
5 min. Conversion was monitored by disappearance of the tetrazine as determined by integration of
the peak at 10.300–10.275 ppm. The integrity of the cycloaddition product (5) and its regioisomer
(5′) was assessed by LC–MS. The value of the secondꢀorder rate constant was determined by
linear regression analysis of a plot of 1/[tetrazine] versus time.
Yeast Surface Display. Two distinct scFv’s were used in this work: 4ꢀ4ꢀ20 scFv (which binds
to FITC and has an Nꢀterminal FLAG tag) and scFvA (which binds to an antigen on the surface of
RBE4 cells and has an Nꢀterminal Myc tag). Fusions of these scFv’s to the 202ꢀ08 intein in the
pCTre vector, were transfected into EBY100 yeast cells as described previously.14 Transfected
cells were grown at 30 °C in SD–CAA medium (20.0 g/L dextrose, 6.7 g/L yeast nitrogen base, 5.0
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g/L casamino acids, 10.19 g/L Na2HPO4·7 H2O, 8.56 g/L NaH2PO4·H2O) as described
previously.14,36,43 Cultures were induced with SG–CAA medium, that is identical to SDꢀCAA
medium except dextrose is substituted for galactose at an OD600nm of 0.8–0.9. Cultures were
induced for 48 h at 20 °C before harvesting the scFv from the yeast surface.
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EPL and IEDDA Cycloaddition of Yeast Surface-Displayed Proteins. Yeast cultures were
pelleted, and the cells were washed two times with 50 mM HEPES buffer, pH 7.2. For a typical
reaction, 50 mL of yeast were pelleted and reꢀsuspended in 800 µL of 50 mM HEPES buffer, pH
7.2. One hundred microliters of 2 M MESNA solution was added to bring the final MESNA
concentration to 200 mM. Immediately after MESNA addition, 100 µL of 50 mM Cys–PEG3–
styrene is added. These proportions can be scaled as needed to achieve the amount of modified
scFv needed for downstream assays. The yeast reaction slurry was incubated for 1 h with gentle
shaking at room temperature. Yeast cells were removed by centrifugation and secondary filtration
through a 0.22ꢀµm filter, to recover the styreneꢀmodified scFvꢀcontaining supernatant. The
reaction proceeds for an additional 2–17 h. After incubation, the supernatant was buffer exchanged
four times with 50 mM HEPES buffer, pH 7.2, using 10,000ꢀDa MWCO filters, to remove
MESNA and excess styrene reactant (total exchange is 500 µL/wash). The scFv is concentrated
using 10,000ꢀDa MWCO filter to <50 µL. Next, 50 mM of 10× tetrazine probe (final concentration
of 5 mM) is added to scFvꢀstyrene (typically 45 µL scFv with 5 µL of tetrazine probe). The
bioorthogonal conjugation step requires incubating the reaction overnight at 30 °C with gentle
shaking. Finally, the reaction solution was then bufferꢀexchanged using 10,000 Da MWCO filters
to remove excess tetrazine reactant. Two tetrazine probes were used in this work (Figure 1):
tetrazine–biotin (Product number CPꢀ6001 from ConjuꢀProbe, San Diego, CA) and tetrazine–Cy5
(Product number 1189 from Click Chemistry Tools, Scottsdale, AZ).
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Incubation of Tetrazine with Live Yeast and Reducing Agents. Live yeast were incubated
with 5 mM of 3ꢀ(4ꢀbenzylamino)ꢀ1,2,4,5ꢀtetrazine HCl (product number CPꢀ6021ꢀ25MG from
Conjuꢀprobe). After 45 minutes yeast was pellet and tetrazine absorbance was quantified at 525 nm
using microplate reader. Similarily, 5 mM of phenyltetrazine functionlized with an amine was
incubated with MESNA (50 mM), TCEP (4 mM), BME (5%), or DTT (50 nM) for 45 minutes.
After incubation, tetrazine absorbance at 525 nm was quantified using microplate reader.
SDS–PAGE and Immunoblotting of Reacted Proteins. scFv fractions were resolved using
4–12% w/v BisꢀTris acrylamide gels. Proteins were reduced and denatured by boiling samples in
SDSꢀcontaining loading buffer with 5% v/v βꢀmercaptoethanol for 10 min. Proteins were
transferred to nitrocellulose membranes. An antiꢀFLAG M2 monoclonal FLAG antibody (Sigma–
Aldrich) and a BTN.1 antiꢀbiotin antibody (Biolegend) were used to probe membranes for scFv.
Antiꢀmouse HRP secondary antibody (Jackson Laboratory, Bar Harbor, ME) was detected via
ECL, and imaged using a BioꢀRad imaging system. A protein standard that is similar in size and
that contained an Nꢀterminal FLAG tag (Human IL17RB from Sino Biological, Beijing, China)
was used to determine concentrations of scFv by immunoblotting.
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Quantifying Percentage of EPL/IDEAA modified scFv. 4ꢀ4ꢀ20 scFv was modified with
biotin as described above. Next, scFv was incubated with magnetic streptavidin beads (Pierce) for
1 h at room temperature. For the total scFv samples the entire slurry was mixed with SDS loading
buffer and boiled. Magnetic beads were removed and sample was resolved on 4–12% w/v bisꢀTris
acrylamide gel, transferred to nitrocellulose, and probed for scFv using antiꢀFlag antibody (Sigma).
For the biotinꢀmodified samples, magnetic beads were pulled down and washed 3 times to remove
nonꢀbound scFv. Beads were then reꢀsuspended, boiled, and quantified for scFv as described for
the total scFv group. To account for background scFv binding of strepꢀbeads to scFv, unmodified
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4ꢀ4ꢀ20 was used and subtracted from each replicate to generate specific modification percentages.
Western blot band intensity was quantified using Image J and normalized to total scFv signal.
Solution-phase determination of scFv modification with Cy5. To determine
modification of scFv by tetrazineꢀCy5, 4ꢀ4ꢀ20 scFv was modified with styrene as described above
in EPL/IDEAA section. Unreacted tetrazineꢀCy5 was removed by buffer exchanging four times
with 10,000 Da MWCO filters. Groups were then directly assessed for Cy5 fluorescence using a
plate reader (Tecan).
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Solid-phase determination of scFv modification with Cy5. 4ꢀ4ꢀ20 and scFvA were
modified with Cy5 using the approach described above. FITCꢀdextran was immobilized onto
maxiꢀsorp ELSIA plates (Nunc) by O.N. incubation at 4 °C. The next day, plates were washed two
times with PBS + 0.05% w/v tween 20 and blocked for 2 h with PBS + 1% w/v BSA. Plates were
washed two times PBS + 0.05% w/v tween 20 and 100 nM of scFv was added to each well and
allowed to incubate at room temperature for 1 h. Plates were washed 5 times PBS + 0.05% w/v
tween 20 and Cy5 signal determined using a microtiter plate reader (Tecan).
Fluorescein Quench Assay. Modified and unmodified 4ꢀ4ꢀ20 scFv were titrated with
fluorescein as described previously.14,40,44 Briefly, a 200ꢀmL yeast display culture of 4ꢀ4ꢀ20 was
split into two groups to generate 4ꢀ4ꢀ20 ± Cy5. The 4ꢀ4ꢀ20 concentration was determined using
comparative western blotting with known amounts of protein containing an Nꢀterminal Flag tag.
Samples were then incubated with fluorescein dilutions ranging from 0.08–40 nM. Fluorescein
fluorescence was quantified using FluoroMaxꢀ3 fluorimeter (Horiba). Values of Kd were
determined as described previously.50
Flow Cytometry. RBE4 cells were cultured on collagen type Iꢀcoated tissue culture flasks in
45% v/v Alpha Minimum Essential Medium, 45% v/v Ham’s F10 medium, 10% v/v fetal calf
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serum, 100 mg/L streptomycin, 100,000 units/L penicillin G, 0.3 g/L geneticin, and 1 ꢁg/L basic
fibroblast growth factor (bFGF) as described previously.42 Cells were incubated with scFvAꢀCy5
or unmodified scFvA (0.5 µM) for 1 h at 37 °C and 5% CO2. Cells were washed three times with
PBS (5ꢀmin washes) and trypsinized for 5 min at 37 °C and 5% CO2. Cultures were diluted 1:1
with serumꢀcontaining growth medium to quench trypsin, and the cells were pelleted by
centrifugation. The cell pellet was resuspended in PBS containing 10 mM EDTA. Internalized
fluorescence was measured with a BD FACSCaliber cytometer by quantifying 10,000
events/group using software from FlowJo (Ashland, OR).
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Fluorescence Microscopy. RBE4 cells were cultured on glass coverslips.42 RBE4 cells were
incubated with modified or unmodified scFvA (2 µM) for 1 h at 37 °C and 5% CO2. Cells were
then washed three times with PBS, fixed in 4% v/v paraformaldehyde for 10 min, and
permeabilized with 0.1% v/v Triton Xꢀ100. Permeabilized RBE4 cells were stained with 9E10
antiꢀMyc antibody (1:200), goat antiꢀmouseꢀAF488 antibody (1:200), and Hoechst 33342 (1:800).
Slides were washed, mounted, and imaged on a Nikon upright fluorescence microscope.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
10.1021/acs.xxx.
1
Figure S1 demonstrates disappearance of a tetrazine in the presence of 4ꢀacetamidostyrene. H and
13C NMR spectra as well as LC–MS chromatograms of synthetic compounds used in this study are
presented.
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Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
K.A.M. was supported by Molecular Biosciences Training Grant T32 GM007215 (NIH) and a
fellowship from the University of Wisconsin–Madison College of Agricultural and Life Sciences.
This work was funded by National Science Foundation grant CBET1403350.
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