Site-selective protein modification: Via disulfide rebridging for fast tetrazine/ trans -cyclooctene bioconjugation

Article abstract of DOI:10.1039/c9ob02687h

An inverse electron demand Diels-Alder reaction between tetrazine and trans-cyclooctene (TCO) holds great promise for protein modification and manipulation. Herein, we report the design and synthesis of a tetrazine-based disulfide rebridging reagent, which allows the site-selective installation of a tetrazine group into disulfide-containing peptides and proteins such as the hormone somatostatin (SST) and the antigen binding fragment (Fab) of human immunoglobulin G (IgG). The fast and efficient conjugation of the tetrazine modified proteins with three different TCO-containing substrates to form a set of bioconjugates in a site-selective manner was successfully demonstrated for the first time. Homogeneous, well-defined bioconjugates were obtained underlining the great potential of our method for fast bioconjugation in emerging protein therapeutics. The formed bioconjugates were stable against glutathione and in serum, and they maintained their secondary structure. With this work, we broaden the scope of tetrazine chemistry for site-selective protein modification to prepare well-defined SST and Fab conjugates with preserved structures and good stability under biologically relevant conditions.

Full text of DOI:10.1039/c9ob02687h

Organic &
Biomolecular Chemistry
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Site-selective protein modification via disulfide
rebridging for fast tetrazine/trans-cyclooctene
bioconjugation†
Cite this: DOI: 10.1039/c9ob02687h
Lujuan Xu,a,b Marco Raabe, a,b Maksymilian M. Zegota,a,b João C. F. Nogueira,c
a,b
Vijay Chudasama, c Seah Ling Kuan
*
a,b and Tanja Weil
*
An inverse electron demand Diels–Alder reaction between tetrazine and trans-cyclooctene (TCO) holds
great promise for protein modification and manipulation. Herein, we report the design and synthesis of a
tetrazine-based disulfide rebridging reagent, which allows the site-selective installation of a tetrazine
group into disulfide-containing peptides and proteins such as the hormone somatostatin (SST) and the
antigen binding fragment (Fab) of human immunoglobulin G (IgG). The fast and efficient conjugation of
the tetrazine modified proteins with three different TCO-containing substrates to form a set of bioconju-
gates in a site-selective manner was successfully demonstrated for the first time. Homogeneous, well-
defined bioconjugates were obtained underlining the great potential of our method for fast bioconjuga-
tion in emerging protein therapeutics. The formed bioconjugates were stable against glutathione and in
serum, and they maintained their secondary structure. With this work, we broaden the scope of tetrazine
chemistry for site-selective protein modification to prepare well-defined SST and Fab conjugates with
preserved structures and good stability under biologically relevant conditions.
Received 18th December 2019,
Accepted 8th January 2020
DOI: 10.1039/c9ob02687h
rsc.li/obc
challenges in fundamental biological and medical appli-
cations.4 In this manner, protein therapeutics can be syntheti-
Introduction
Peptides and proteins are emerging as powerful treatment cally customized to program their properties for envisaged
options, as exemplified by the application of antibodies and applications.
antibody fragments (Fab) in immunotherapy and antibody–
Over the past decades, several bioorthogonal reactions,
drug conjugates in targeted cancer therapy.1 Nevertheless, including the [3 + 2] azide–alkyne cycloaddition, photoclick
protein stability, immunogenicity and the time required to 1,3-dipolar cycloaddition and the inverse electron demand
engineer recombinant antibodies could limit their develop- Diels–Alder (IEDDA) reactions, have been developed and
ment for in vivo studies.2 In this regard, nature has evolved an applied for protein modifications.5 Among these, the IEDDA
optimal synthetic factory in the form of posttranslational pro- reaction of 1,2,4,5-tetrazine with trans-cyclooctene (TCO)
cesses, in which diverse functionalities can be attached to pro- stands out, providing fast reaction kinetics (rate constant of up
teins in a site-directed fashion.3 Inspired by this, chemists to 106 M−1 s−1), excellent orthogonality, catalyst-free con-
strive to develop methodologies to impart diverse functional- ditions and good biocompatibility, and is a widely employed
ities to native proteins with similar levels of precision.
bioorthogonal approach in chemical biology.6 Previously, the
Site-selective modification of therapeutically relevant pep- IEDDA reaction has been applied for the preparation of anti-
tides and proteins to introduce reactive bioorthogonal handles body conjugates, mainly via statistical modifications using
has emerged for post-modification in an “on-demand” fashion N-hydroxysuccinimide (NHS) ester chemistry for cell imaging,7
to expand the features and functions of proteins to address the nanoparticle functionalization,8 and antibody targeted
therapy.6a,9 In the literature, two common approaches to site-
selectively modify proteins utilizing the IEDDA reaction are,
aMax Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz,
Germany. E-mail: weil@mpip-mainz.mpg.de, kuan@mpip-mainz.mpg.de
bInstitute of Inorganic Chemistry I, University of Ulm, Albert-Einstein-Allee 11,
89081 Ulm, Germany
cDepartment of Chemistry, University College London, London, UK
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9ob02687h
the introduction of a diene or dienophile by genetic code
expansion methods via the incorporation of noncanonical
amino acids10 and tag-based posttranslational attachment
strategies using enzymes (e.g. lipoic acid protein ligase A).11
Nevertheless, the genetic code expansion method suffers from
low yields and tedious synthesis. This in turn limits its scal-
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ability and accessibility for further applications, while tag- phosphine to the electron-poor alkene moiety.18 The versatility
based posttranslational attachment strategies first require the of the method was demonstrated by modifying two model sub-
genetic fusion of a specific peptide sequence (e.g. lipoate strates: SST and IgG Fab under mild conditions which yielded
acceptor peptide) to the protein of interest.11 A single tetrazine a set of well-defined protein conjugates (Fig. 1). The formed
or TCO group has also been introduced site-selectively into protein conjugates showed good stability and maintained
peptides or proteins at an unpaired cysteine site via the thiol- their secondary structures after bioconjugation. The reported
maleimide reaction.6a,12
methodology provides a rapid, robust and straightforward
However, very few native proteins contain free cysteine resi- strategy for site-selective protein labeling and expands the scope
dues.13 Furthermore, thiol–maleimide chemistry easily under- of using IEDDA chemistry in the functionalization of thera-
goes the retro-Michael reaction resulting in a maleimide peutically relevant peptides and proteins in a precise manner.
exchange with other reactive thiols present, for example gluta-
thione, under physiological conditions causing off-target
effects.4a Therefore, other methods, which can introduce a
tetrazine or TCO group in a site-selective fashion into proteins
for subsequent IEDDA reactions are highly desirable to expand
the scope of their applications. Since many therapeutically
Results and discussion
Synthesis and characterization of the tetrazine-based disulfide
rebridging reagent
relevant peptides and proteins contain at least one disulfide
bond close to the protein surface, the disulfide rebridging
strategy provides a versatile technique to modify the solvent
accessible disulfide bonds on these proteins and peptides to
install suitable bioorthogonal tags.13,14 Smith and coworkers
presented an elegant approach to introduce the tetrazine
group at the disulfide site of proteins/peptides via dichloro-
tetrazine.15 However, a significantly reduced activity of the
protein was reported after the modification. In this context, di-
sulfide modification based on bis-alkylating reagents to form
bisthioether conjugates gained wide applications for site-selec-
tive protein modifications without compromising their bio-
activity. Furthermore, it has been reported that bisthioether
conjugates formed by disulfide rebridging are more stable
than thiol-maleimide conjugates16 suggesting that it could be
a viable strategy for the installation of IEEDA reaction handles.
Herein, we present a simple and straightforward method for
the site-selective incorporation of a tetrazine group into native
peptides and proteins through disulfide rebridging with the
allyl sulfone scaffold reported previously.17 In comparison
with known bis-sulfone disulfide rebridging reagents, allyl
sulfone reagents provide improved reactivity and higher water
solubility, which greatly facilitates protein bioconjugations.17
The disulfide rebridging reactions of allyl sulfones proceed
in situ without side reactions e.g. with reducing agents
such as tris(2-carboxyethyl)-phosphine hydrochloride (TCEP).
In comparison, disubstituted maleimide disulfide rebridging
reagents, such as 3,4-dibromomaleimide, show side reactions
with TCEP, presumably due to the nucleophilic addition of
For the site-selective installation of bioorthogonal handles on
the proteins, a longer incubation time (usually overnight) is
often required.13 Therefore, the diene “6-methyl-1,2,4,5-tetra-
zine” instead of the dienophile “TCO” was introduced into the
protein. This strategy fully utilizes the fast kinetics of the
IEDDA reaction but minimizes the inherent isomerization
issues of TCO to nonreactive cis-cyclooctene, which occurs at a
longer incubation time or in the presence of thiols.4c,19 In this
context, a tetrazine-based allylsulfone disulfide rebridging
reagent (IC-Tetrazine, Scheme 1) was designed and readily
Scheme 1 Synthesis of the tetrazine-containing disulfide rebridging
reagent (IC-Tetrazine). (a) Di-tert-butyl dicarbonate, CH2Cl2, overnight.
(b) Methacryloyl chloride, Et3N, CH2Cl2, 90%. (c) 1. I2, sodium p-toluene-
sulfinate, CH2Cl2, 3 days. 2. Et3N, CH2Cl2, overnight. 3. Et3N, ethyl
acetate, 95 °C, overnight, final yield: 60%. (d) Trifluoroacetic acid (TFA),
CH2Cl2, 98%. (e) Methyltetrazine NHS ester, Et3N, CH2Cl2, overnight,
40%.
Fig. 1 Schematic overview of site-selective incorporation of a reactive tetrazine tag into a solvent-accessible disulfide site of a cell-targeting
peptide or antibody fragment. The tetrazine-modified peptide/protein is used for post-functionalization to construct a small set of protein bioconju-
gates with a (1) dye, (2) polymer and (3) protein in a fast and highly selective manner with high conversions (one isomer is shown as a
representation).
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obtained through a four-step synthesis. As shown in Scheme 1, chemical structure of the fragmentation species is shown in
tetraethylene glycol monoamine (1) was protected to afford 2- Fig. S3†), respectively. Notably, SST-Tetrazine is stable for more
(2-boc-aminoethoxy)ethanol (2) that underwent condensation than one year when stored as a solid at −20 °C (Fig. S4†).
with methacryloyl chloride yielding the corresponding meth-
Next, we applied the modification method to an antibody
acrylate derivative (3). After a tandem iodosulfonylation–dehy- fragment (Fab) from human immunoglobulin G (IgG). Fab
droiodination reaction, the tosyl group was introduced to form fragments have been successfully used for constructing anti-
allyl-sulfone (4). The tert-butoxycarbonyl group was sub- body–drug and antibody–nanoparticle conjugates providing
sequently removed in an acidic medium to afford the primary several advantages compared to the complete antibody.22 The
amino group, which then reacted with methyl-tetrazine NHS conjugate retains the antigen-binding region which is crucial
ester to afford the IC-Tetrazine reagent.
for active targeting, but circumvents the non-specific binding
of the Fc region of antibodies.22a Since the interchain disul-
fides are much more exposed to the solvent and less stable
Site-selective protein modification
Subsequently, two model substrates (somatostatin and human relative to the intrachain disulfides that are buried between
IgG Fab) were selected for site-selective modification with the two layers of anti-parallel β-sheet structures,23 IgG Fab was
IC-Tetrazine. Somatostatin (SST) regulates the endocrine incubated with 50 equivalents of TCEP (optimization of the
system and mediates signal transduction via five G protein- amounts of TCEP is shown in Fig. S5†) to reduce the solvent
coupled receptors (SSTR) that are overexpressed in high levels accessible interchain disulfide bonds and subsequently
in various kinds of cancer cells and tumor blood vessels.20 SST reacted with IC-Tetrazine overnight (Fig. 2a). The tetrazine
conjugates have been widely investigated for targeted drug modified Fab fragment (Fab-Tetrazine) was purified by ultrafil-
delivery into SSTR-expressing cancer cells.20,21 IC-Tetrazine tration to remove residual TCEP and IC-Tetrazine. The
was reacted with SST after its single accessible disulfide bond MALDI-TOF-MS analysis of Fab-Tetrazine revealed a molecular
was reduced to generate free thiol groups by the addition of weight of 48.5 kDa (Fig. 2c), showing an increase of ∼500 Da
two equivalents of TCEP at room temperature (Fig. 2a). The compared to that of the native Fab (48.0 kDa, Fig. S5B†)
product (SST-Tetrazine) was purified by high-performance demonstrating its successful site-selective modification.
liquid chromatography (HPLC) yielding SST-Tetrazine in 30% Sodium dodecyl sulfate polyacrylamide gel electrophoresis
yield, which is comparable to that reported in the literature (SDS-PAGE) of Fab-Tetrazine revealed a single band at ∼48 kDa
(20% to 40%).13 Characterization by matrix assisted laser de- showing that only a slight reduction occurred after site-selec-
sorption/ionization time of flight mass spectrometry tive modification even in the presence of 100 equivalents of
(MALDI-TOF-MS) revealed two signals corresponding to the TCEP after one hour (Fig. 2d, band 2). In contrast, under the
desired SST-Tetrazine (m/z = 2110.95 [M + H]+, Fig. 2b, the same conditions, the native IgG Fab was almost completely
molecular weight of native SST is 1637 g mol−1) and the laser reduced exhibiting a single band appearing at ∼24 kDa on
fragmentation species of SST-Tetrazine (m/z = 2041.93, the SDS-PAGE (Fig. 2d, band 1). Based on the concentration calcu-
Fig. 2 (a) Site-selective modification of SST and IgG Fab with IC-Tetrazine. (b) MALDI-TOF-MS spectrum of SST-Tetrazine (found: 2110.95 [M + H]+,
calculated: 2110.95 [M + H]+). The m/z peak at 2041.93 corresponds to the fragmentation of SST-Tetrazine. (c) MALDI-TOF-MS spectrum of Fab-
Tetrazine (Fab-Tetrazine: found: 48.5k [M + H]+, calculated: 48.5k [M + H]+). (d) SDS-PAGE analysis of native Fab and Fab-Tetrazine (M: Applichem
Protein Marker VI, 1: Fab (native) + 100 eq. TCEP incubation for one hour, and 2: Fab-Tetrazine + 100 eq. TCEP incubation for one hour).
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lated from gel densitometry (Fig. 2d), about 80% of the Fab the desired conjugate SST-Cy5 in 90% yield. MALDI-TOF-MS
fragment was successfully modified. A thiol quantification showed the m/z signal at 3041.17 (Fig. 3c).
experiment using the thiol reagent 4,4′-dithiodipyridine was
Due to the much larger molecular weight of Fab-Tetrazine
performed and the result showed that about 73% of IgG Fab compared to SST-Tetrazine, a longer PEG chain with a mole-
were successfully modified, which is consistent with the result cular weight of 5000 Da containing 113 repeating units on
obtained from gel densitometry (details are shown in the ESI, average (TCO-PEG113) was incubated with Fab-Tetrazine in
Fig. S5c†). Taken together, the results clearly indicated that the phosphate buffer (pH = 7.4) for 30 minutes to afford Fab-
disulfide bonds in IgG Fab were successfully rebridged using PEG113. The characteristic band of Fab-Tetrazine at ∼48 kDa
IC-Tetrazine.
almost entirely disappeared after PEGylation and a new band
emerged, which was broader due to the polydispersity of the
PEG chain (Fig. 3d). A control experiment of mixing native Fab
and TCO-PEG113 did not show any unspecific absorption on
SDS-PAGE (Fig. 3d). MALDI-TOF-MS spectra revealed a signal
at 53.5 kDa indicating the successful conjugation (Fig. S11D†).
Under similar conditions, the widely applied chromophore
Cy5 was also attached to Fab-Tetrazine to afford Fab-Cy5 (mole-
cular weight: 49.5 kDa, Fig. 3e) in 73% yield (the calculation is
shown in the ESI†). Successful conjugation was confirmed by
an increase in the molecular weight of ∼1.5 kDa in the
MALDI-TOF-MS spectra in comparison with those of native
Fab (molecular weight: 48 kDa, Fig. 3e).
CytC is a 12 kDa hemeprotein typically located in the mito-
chondria of viable cells.24 Iso-yeast CytC has a single thiol
group on its surface for modification via thiol-maleimide
chemistry.25 TCO-PEG3-maleimide was reacted with CytC to
introduce the TCO group via thiol-maleimide chemistry
affording CytC-PEG3-TCO (Scheme S4†). MALDI-TOF-MS data
revealed the successful modification of CytC (Fig. S6, ESI†). In
Bioconjugation with a chromophore, PEG and enzyme
The versatility of the tetrazine-modified SST and IgG Fab for
the construction of well-defined bioconjugates was demon-
strated through post-modification with a fluorophore (cyanine-
5, Cy5) and a PEG chain. In addition, SST-Tetrazine was also
conjugated to a protein enzyme (cytochrome C, CytC) to form
a peptide–protein conjugate with high conversion. A TCO-ter-
minated PEG chain of a precise chain length with 12 repeating
units (TCO-PEG12) was selected to facilitate the characteriz-
ation by MALDI-TOF-MS. TCO-PEG12 was incubated with
SST-Tetrazine in phosphate buffer (PB, 50 mM, pH = 7.4) and
the pink color of the tetrazine group disappeared immediately
after mixing, indicating that the reaction took place instan-
taneously. The bioconjugation reaction was carried out for
30 minutes to ensure completion. Thereafter, the reaction
mixture was injected into the HPLC column and a predomi-
nant peak of SST-PEG12 was obtained (Fig. S7, ESI†).
SST-PEG12 was isolated in nearly quantitative yield (95%) and
characterized by MALDI-TOF-MS (Fig. 3b). SST-Tetrazine was
also conjugated to TCO-Cy5 under similar conditions yielding
addition,
a
tetrazine-based dye (tetrazine-5-fluorescein,
Tetrazine-FAM, excitation wavelength: 492 nm and emission
Fig. 3 (a) Bioconjugation between (i) SST-Tetrazine with TCO-PEG12 and TCO-Cy5. (ii) Fab-Tetrazine with TCO-Cy5 and TCO-PEG113 via a fast
click reaction. (b) MALDI-TOF-MS spectrum of SST-PEG12 (found: 2852.21 [M + H]+, calculated: 2852.28 [M + H]+). (c) MALDI-TOF-MS spectrum of
SST-Cy5 (found: 3041.17 [M + H]+, calculated: 3041.28 [M + H]+). Sinapic acid was used as the matrix for all MALDI-TOF-MS measurements. (d)
SDS-PAGE analysis of Fab-PEG113 (M: Applichem Protein Marker VI, 1: Fab-Tetrazine, and 2: Fab + TCO-PEG113, 3: Fab-PEG113). (e) MALDI-TOF-MS
spectra of Fab-Cy5 (found: 49.5k [M + H]+, calculated: 49.5k [M + H]+) and native Fab (found: 48.0k [M + H]+, calculated: 48.0k [M + H]+).
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wavelength: 517 nm) was used to react with CytC-PEG3-TCO to also performed. However, MALDI-Tof-MS data showed almost
assess the modification yield of CytC. Based on the absorbance no conjugation product, presumably due to the steric hin-
of CytC at 280 nm and the FAM dye at 490 nm, about 90% of drance of the two bulky macromolecules.
CytC was successfully modified with the TCO group (experi-
mental details are shown in the ESI†). Thereafter, the targeting
Stability studies of SST-Tetrazine, Fab-Tetrazine and Fab-Cy5
peptide SST-Tetrazine was conjugated to CytC-PEG3-TCO by
mixing and shaking for 30 minutes.
The stability of the bioconjugates after disulfide modification
is an important consideration for their intended applications
in cellular environments. Therefore, the stability of the modi-
fied protein with the newly synthesized disulfide rebridging
reagent in glutathione (GSH) was investigated using liquid
chromatography mass spectrometry (LC-MS). SST-Tetrazine
(10 µM) was incubated with two-fold excess of GSH at biologi-
cally relevant concentrations (20 μM)26 at 37 °C and
SST-Tetrazine and the degradation product (GSH-Tetrazine,
1085 [M + H]+; the chemical structure is shown in Scheme S9†)
were identified by simultaneous detection by UV-Vis spec-
troscopy at an absorption wavelength of 254 nm and selective
ion monitoring (SIM). The amount of SST-Tetrazine in each
sample was determined as a ratio of the integration of the
chromatogram at 254 nm of SST-Tetrazine to the standard
(Fmoc-L-phenylalanine) (Fig. 5a and b). LC-MS data showed
that SST-Tetrazine remained intact after 24 hours of incu-
bation and no degradation products were detected in the SIM
profile (Fig. S12C†). Next, the stability of the Fab conjugates
was investigated based on the literature protocol.22b Fab-
Tetrazine and Fab-Cy5 were incubated with 1% fetal bovine
serum (FBS) at 37 °C and monitored using SDS-PAGE.
SDS-PAGE data revealed no change in Fab-Tetrazine after
24 hours, indicating no obvious degradation (Fig. 5c). The
stability of Fab-Cy5 was also investigated based on the quanti-
fication of the fluorescence of the conjugate (shown in the
ESI,† page 15). The data indicated a good stability of Fab-Cy5
after incubation with FBS for 24 hours (Fig. 5d). Taken
together, these experiments showed that the resultant peptide
and protein bioconjugates formed by the disulfide rebridging
strategy and the subsequent IEDDA reaction remained stable
under physiological conditions, e.g. in the presence of GSH or
in media containing serum proteins.
After ultrafiltration to remove the residual SST-Tetrazine,
SST-PEG3-CytC was obtained and characterized by
MALDI-TOF-MS and SDS-PAGE. In the MALDI-TOF-MS spectra,
the signal at 15 315 Da corresponds to the desired SST-PEG3-
CytC conjugate (Fig. 4b), whereas the signal for CytC-PEG3-
TCO (13 233 Da) completely disappeared. On SDS-PAGE, a
slight band shift was observed indicating the successful conju-
gation between SST-Tetrazine and CytC-PEG3-TCO (Fig. 4c).
The conjugation of Fab-Tetrazine with CytC-PEG3-TCO was
Fig. 4 (a) Bioconjugation between SST-Tetrazine and CytC-PEG3-TCO.
(b) MALDI-TOF-MS spectrum of SST-PEG3-CytC (found: 15 315 [M +
H]+, calculated: 15 314 [M + H]+) matrix: sinapic acid. (c) SDS-PAGE ana-
lysis of SST-PEG3-CytC (M: Applichem Protein Marker VI, 1: CytC-PEG3-
TCO, and 2: SST-PEG3-CytC).
Fig. 5 (a) Stability study of SST-Tetrazine via LC-MS. (b) Area ratio of SST-Tetrazine at 254 nm compared to Fmoc-L-phenylalanine (standard) at
three time points (n = 3, values are given as mean SD). (c) Stability study of Fab-Tetrazine by SDS-PAGE (M: Applichem Protein Marker VI, 1: Fab-
Tetrazine, 2: 1% FBS, 3: Fab-Tetrazine + 1% FBS incubated for 12 hours, and 4: Fab-Tetrazine + 1% FBS incubated for 24 hours), (d) Stability study of
Fab-Cy5 by SDS-PAGE with (left) and without (right) Coomassie blue (M: Applichem Protein Marker VI, 1: 1% FBS, 2: Fab-Cy5, 3: Fab-Cy5 + 1% FBS
incubated for 12 h, 4: Fab-Cy5 + 1% FBS incubated for 24 h, 5: fluorescence of Fab-Cy5, 6: fluorescence of Fab-Cy5 after incubation with 1% FBS for
12 h, and 7: fluorescence of Fab-Cy5 after incubation with 1% FBS for 24 h).
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Fig. 6 (a) CD spectra of SST, SST-Tetrazine and SST-PEG12 (SST, SST-Tetrazine and SST-PEG12 were diluted to 0.1 mg mL−1 in PBS, the data are the
average of three runs). (b) CD spectra of Fab, Fab-Tetrazine and Fab-PEG113 (Fab, Fab-Tetrazine and Fab-PEG113 were measured at 0.1 mg mL−1 in
PBS, the data are the average of three runs). (c) ELISA data of Fab and Fab-Tetrazine (five different concentration points: 1 nM, 10 nM, 50 nM, 100
nM, and 200 nM). Data are plotted as mean SD (n = 3).
Structural and functional studies
two-step procedure as the disulfide rebridging reaction is
greatly limited by the steric demand of the reagent. Bulky sub-
stituents attached to the rebridging reagent such as polymers,
proteins or drug molecules have only limited access to the
reduced disulfide residues resulting in complex product mix-
tures with large amounts of unreacted starting materials and
very low product yield.29 Therefore, the two-step approach pre-
sented herein based on the site-selective installation of the
tetrazine tag first followed by the IEDDA reaction provides con-
venient access to compound libraries, in which a broad range
of small and bulky TCO-modified functionalities could be
attached to the corresponding tetrazine-modified proteins in a
convenient, fast, and efficient way.
Notably, the modified proteins exhibited good stability
under biologically relevant conditions and their secondary
structure was preserved after modification. We envision that
this method would enable the preparation of the desired bio-
conjugates “on site”, e.g. in hospitals for the desired appli-
cations. In this way, the final conjugate would be applied to
the patient immediately after preparation, preventing long
term storage, quality control and loss of bioactivity of the final
conjugate. The approach presented herein is a valuable
addition to the chemical toolbox for site-selective protein lab-
elling and manipulation, offering a fast, robust and straight-
forward method to be easily adapted in any laboratory.
Circular dichroism (CD) is a versatile technique to determine
the secondary and tertiary structures of proteins. According to
Fig. 6a, SST, SST-Tetrazine and SST-PEG12 showed typical
random coil structures with a negative band at 201 nm. The
peak at 225 nm of SST is attributed to the presence of a higher
degree of polyproline (PPII) conformation.27
Both native IgG Fab, Fab-Tetrazine and Fab-PEG113 conju-
gates, possess well-defined antiparallel β-pleated sheets con-
firmed by the peaks at 218 nm and 202 nm consistent with the
literature report,23 which confirmed that the secondary struc-
ture of Fab was preserved after modification (Fig. 6b). Next, we
assessed whether the modification of IgG Fab affects its func-
tion to bind to protein L, a bacterial protein known to interact
with Fab fragments of immunoglobulins.28 The binding and
recognition of native Fab and Fab-Tetrazine were thus investi-
gated by enzyme-linked immunosorbent assay (ELISA).
Different concentrations (1 nM, 10 nM, 50 nM, 100 nM, and
200 nM) of IgG Fab and Fab-Tetrazine were added to the
protein L coated well plate and incubated for one hour. After
removing the unbound protein, anti-human IgG (Fab specific)-
peroxidase antibody was added to the well plate and incubated
for one hour. Enhanced chemiluminescence solution was
added to the well plate after washing three times with PBS. As
shown in Fig. 6c, the binding affinities between IgG Fab and
Fab-Tetrazine are comparable, suggesting that the binding of
Fab-Tetrazine to protein L was preserved.
Conflicts of interest
There are no conflicts to declare.
Conclusions
Herein, we report a straightforward and broadly applicable bio-
conjugation method for introducing a single tetrazine group
into a targeting peptide and antibody fragment at a pre-
Acknowledgements
defined, distinct site in aqueous media through disulfide The authors are grateful to the Max Planck Society, the
modification. The tetrazine functionalized peptide or Fab was Deutsche Forschungsgemeinschaft (DFG, German Research
further conjugated with a series of TCO-modified functional- Foundation) – Projektnummer 316249678-SFB 1279 (C01), and
ities, such as dyes, polymers, or proteins/enzymes yielding a to the financial support from the European Union’s Horizon
small library of stable bioconjugates within a short reaction 2020 Research and Innovation Program under grant agreement
time. Such bulky functionalities could only be introduced by a no. 667192 (“Hyperdiamond”). LX is grateful to the China
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Scholarship Council for a scholarship. MMZ and JCFN are
grateful to the Marie Curie International Training Network
Protein Conjugates for research scholarships. We thank Astrid
Heck, Siyuan Xiang and Zhixuan Zhou for their technical
support, Nicole Kirsch-Pietz for proofreading and the MPIP
mass spectrometry group for the MALDI-TOF-MS measure-
ments. Open Access funding provided by the Max Planck
Society.
D. J. Cox, M. L. Blackman, J. M. Fox and J. W. Chin, J. Am.
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