Site-specific glycoconjugation of protein via bioorthogonal tetrazine cycloaddition with a genetically encoded trans -cyclooctene or bicyclononyne

Article abstract of DOI:10.1021/acs.bioconjchem.5b00101

Efficient access to proteins modified site-specifically with glycans is important in glycobiology and for therapeutic applications. Herein, we report a biocompatible protein glycoconjugation by inverse demand Diels-Alder reaction between tetrazine and tra

Full text of DOI:10.1021/acs.bioconjchem.5b00101

Communication
pubs.acs.org/bc
Site-Specific Glycoconjugation of Protein via Bioorthogonal
Tetrazine Cycloaddition with a Genetically Encoded trans-
Cyclooctene or Bicyclononyne
†
‡
§
⊥
,†
Takuya Machida, Kathrin Lang, Lin Xue, Jason W. Chin, and Nicolas Winssinger*
†Department of Organic Chemistry, NCCR Chemical Biology, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva,
Switzerland
‡
Technical University Munich, Institute for Advanced Study, Department of Chemistry, 4 Lichtenbergstraße, 85748 Garching,
Germany
§
́ ́ ́
Ecole Polytechnique Federale de Lausanne (EPFL), Institut des sciences et ingenierie chimiques, NCCR Chemical Biology, 1015
Lausanne, Switzerland
⊥
Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0GH, United Kingdom
S Supporting Information
*
ABSTRACT: Efficient access to proteins modified site-specifically with glycans is important in glycobiology and for therapeutic
applications. Herein, we report a biocompatible protein glycoconjugation by inverse demand Diels−Alder reaction between
tetrazine and trans-cyclooctene. Tetrazine functionalized glycans were obtained in one step by CuAAC (Cu-catalyzed alkyne
azide cycloaddition) between glycosyl azide and an alkyne-tetrazine adduct. Site-specific glycoconjugation was performed
chemoselectively on a target protein in which a trans-cyclooctene derivatized lysine was genetically encoded. Glycoconjugation
proceeded to completion on purified protein and was shown to be selective for the target protein in E. coli.
rotein glycosylation constitutes an important post-transla-
a tyrosine-selective conjugation was used to introduce an alkyne
into a target protein that was subsequently coupled to an azide-
derivatized glycan via CuAAC. In parallel, a chemoenzymatic
P
tional modification and the glycans of glycoproteins have
1
13
been associated with numerous biological processes. For
instance, cell surface glycoproteins are important in cellular
recognition and have been implicated in embryonic develop-
method has also been employed to convert a cysteine residue
within a defined protein sequence to obtain an aldehyde that
2
−4
14
ment, lymphocyte trafficking, and cancer metastasis,
and
was subsequently conjugated to an aminooxy glycan.
5
there is resurging interest in glycoconjugates as therapeutics.
Accordingly, there is a strong interest in technologies to access
homogeneous glycoconjugates and several methods have been
reported to derivatize protein with functionalized glycan
However, current technologies have been restricted to purified
proteins. Performing a selective glycoconjugation in more
complex systems (lysates or in cellulo) would require a
bioorthogonal conjugation without toxic reagent that proceeds
at a rate compatible with low or sub μM concentration of target
protein. The recent development of the inverse demand
cycloaddition of a tetrazine with strained alkenes and alkynes
6
−8
(
Figure 1).
Davis and co-workers pioneered the use of
9
10
glycosylselenylsufide and glycosyl thiols for glycoconjugation
to cysteine residues on a target protein. A free radical
hydrothiolation using glycosyl thiol and a target protein
containing an alkene (such as homoallyl glycine) was also
reported. More recently, the CuAAC was harnessed to
conjugate glycodendrons to a capsid protein genetically
15−19
fits these requirements.
Furthermore, progress in genetic
1
1
Received: February 17, 2015
Revised: March 30, 2015
12
engineered to contain a homopropargyl glycine. Alternatively,
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.bioconjchem.5b00101
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Communication
Figure 2. CuAAC conjugation of glycosyl azide (1) with alkyne-
tetrazine (2).
glucosyl azide affording the desired product in 81% isolated
yield (Entry 1). However, applying the same conditions to
oligosaccharides proved more problematic with significant
formation of side products arising from oxidative degradation
(
entry 2, iododerivative 3b and homodimer 3c). We thus
turned to the reductive conditions using CuSO /sodium
4
ascorbate. Controlling the amounts of copper and sodium
ascorbate was critical to suppress tetrazine reduction (entry 3 vs
4
). Conditions C (Figure 2) were found to yield the desired
cycloaddition without detectable tetrazine degradation, afford-
ing the disaccharide−tetrazine adduct in 76% isolated yield.
The practicality of these conditions coupled to the
expediency led us to explore these conditions with a broader
set of substrates (Table 1). Glycosyl azides 1 were prepared
from native carbohydrates using 2-chloro-1,3-dimethylimidazo-
lium chloride (DMC) and sodium azide as previously
Figure 1. Schematic representation of glycoconjugation technologies.
26
encoding of unnatural amino acid through genetic code
reported. Next, the CuAAC reaction according to the
optimized procedure afforded the cycloaddition adduct in
64−80% isolated yield. Gratifyingly, performing the reaction at
lower glycan concentration (10−30 mM, entries 10−12 vs 200
mM, entries 1−9) still afforded useful yield of the desired
product after HPLC purification (required based on the
polarity of the product formed).
2
0−22
expansion
has now yielded an aminoacyl-tRNA synthe-
23−25
tase/tRNA pair to introduce strained alkenes and alkynes.
The fast kinetics and bioorthogonality of the tetrazine-strained
alkene reaction make it ideal for live cell experiments. Herein
we report a straightforward method to access tetrazine glycans
and demonstrate their conjugation proteins containing
genetically encoded unnatural amino acids.
We next assessed the reactivity of the glycan−tetrazine
conjugate in reaction with trans-cyclooctene (TCO). It has
been shown that subtle change in the steric and electronic
nature of the tetrazine can have notable impact on the reaction
To be broadly applicable, the synthesis of tetrazine glycan
conjugates would ideally be achieved in a few steps directly
from native (oligo)sacharides. Recently, Shoda reported a
remarkable reaction to selectively activate the anomeric
position of unprotected oligosaccharide providing simple and
practical access to glycans with an azide at the anomeric
18
rate. Using Cy-3 labeled glucosamine-tetrazine conjugate (3-
GluNAc-Cy3, see SI for full experimental details), we
calculated the kinetics of cycloaddition using second-order
and pseudo-first-order conditions, measuring the change in
fluorescence over time (1 μM of the glycan and 1 or 10 equiv of
2
6
position. Leveraging on this powerful reaction, we sought to
conjugate a tetrazine via a CuAAC reaction to the glycans of
interest. Surprisingly, there are no precedents for the combined
use of these two powerful conjugation technologies (CuAAC/
−
1
TCO, respectively). A second-order rate constant of 8649 M
−1
s
was calculated which is consistent with previous analysis of
27
18
tetrazine cycloaddition). To evaluate the potential of the
related structures.
reaction we used alkyne 2, obtained by coupling of propargyl
amine with commercially available tetrazinylphenylacetic acid,
and glucosyl azide 1 (Figure 2). Based on the potential
reduction of tetrazine under the action of sodium ascorbate and
copper, we started our investigation using a source of Cu(I),
performing the reaction under oxygen-free conditions. As
shown in Figure 2, the reaction proceeded smoothly with
Site-Specific Protein Glycoconjugation. We then
evaluated the suitability of this chemistry to achieve site-
selective glycan conjugation. First, purified sfGFP-TCOK and
sfGFP-BocK (both proteins were prepared by incorporation of
unnatural amino acid bearing TCO and Boc by MbPylRS/
tRNACUA pair into sfGFP overexpressed in E. coli) at 13.5 μM
were incubated with 3-GluNAc-Cy3 (10 equiv) for 12 h in Tris
28
24
B
DOI: 10.1021/acs.bioconjchem.5b00101
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Table 1. CuAAcC Conjugation of Alkyne-Tetrazine (2) with a Panel of Glycans
Table 2. Glycoconjugation of sfGFP
Figure 3. Glycoconjugation of 3-GluNAc-Cy3 to purified sfGFP-
TCOK or purified sfGFP-BocK. Bottom left panel: SDS-PAGE
analysis of reactions; lower right panel: MALDI analysis of the reaction
from lane 1.
buffer at 37 °C. As controls, the same reaction was performed
with sfGFP-BocK and in the absence of tetrazine 3-GluNAc-
Cy3. As shown in Figure 3, SDS-PAGE analysis of the
conjugation reaction showed a strong fluorescent band
corresponding to the conjugation of sfGFP-TCOK with
tetrazine−glycan adduct (lane 1) but not in the controls
illustrate the high chemoselectivity of the reaction and stability
of the product (no degradation of the product is observed after
12 h).
(
lanes 2−4). Analysis of the crude reaction mixture by MALDI-
TOF showed a complete conversion with a mass gain
corresponding the cycloaddition product and N extrusion.
Considering the rate constant of the TCO−tetrazine
conjugation, these conditions are very forceful; however, they
We next investigated the kinetics of the reaction with
genetically encoded bicyclononyne (sfGFP-BCNK, prepared
according to the same procedure as sfGFP-TCOK). Tetrazine
2
2
4
C
DOI: 10.1021/acs.bioconjchem.5b00101
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Bioconjugate Chemistry
Communication
(
lanes 2−4). Taken together, the data is consistent with a
highly specific glycoconjugation of sfGFP-TCOK (lane5)
within E. coli.
In summary, we have developed a simple method to rapidly
access tetrazine functionalized glycans from native carbohy-
drates. The work reported establishes the compatibility of
CuAAC with inverse electron demand Diels−Alder reaction. It
is noteworthy that tetrazine adduct can be prepared with
complex glycans (hexasaccharide) and the more challenging
sialyl-type oligosaccharides. Rapid, site-specific glycoconjuga-
tion was achieved using genetically encoded trans-cyclooctene
(
TCO) and bicyclononyne (BCN) modified unnatural amino
acids. The reaction was shown to be suitable for performing
glycoconjugation in E. coli. We anticipate that the fast kinetics
of the tetrazine cycloaddition coupled to the bioorthogonality
of this reaction will facilitate the preparation of tailored
glycoproteins.
ASSOCIATED CONTENT
■
*
S
Supporting Information
Details on the experimental protocols and characterization data.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Figure 4. Glycoconjugation of 3-GluNAc-Cy3 in E. coli. SDS-PAGE
analysis of the reaction with silver staining (left) and fluorescence
scanning (Cy3, right).
AUTHOR INFORMATION
2
9
■
conjugation with bicyclononyne was reported to be 10−15
2
4
Corresponding Author
E-mail: Nicolas.Winssinger@unige.ch. Phone: +41 22 379 61
5. Fax: +41 22 379 32 15.
times slower than with TCO and should provide a more
stringent test for the reactivity of tetrazine conjugates 3. As
shown in Table 2, conjugation of 3-GluNAc-Cy3 with sfGFP-
BCNK at the same concentrations as used in sfGFP-TCOK
afforded the desired glycoconjugation after 10 min (entry 1,
reactions were quenched with 100 equiv of TCO). Reducing
protein concentration to 1 μM or 100 nM and glycan
equivalence to 5 equiv still afforded the desired glycoconjugates
*
0
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Swiss National Science
Foundation and NCCR Chemical Biology.
(
4
entry 2−4). At 100 nM concentration of sfGFP-BCNK (entry
), traces of starting material are still present after 10 min
indicating that these conditions approach the limit of reactivity.
BCN is known to also undergo clycoaddition with azide-
functionalized substrates, albeit with slower rates than with
tetrazine. To compare the reactivity of the two conjugation
methods, sfGFP-BCNK was reacted with glucosyl azide 1-
GluNAc-Cy3 under the same forceful conditions as used in
entry 1, namely, 13.5 μM of protein with 10 equiv of glycan.
After 10 min, the reaction was quenched with tetrazine 2
REFERENCES
■
(
1) Varki, A. (1993) Biological roles of oligosaccharides: all of the
theories are correct. Glycobiology 3, 97−130.
2) Borsig, L., Wong, R., Hynes, R. O., Varki, N. M., and Varki, A.
(
(
2002) Synergistic effects of L- and P-selectin in facilitating tumor
metastasis can involve non-mucin ligands and implicate leukocytes as
enhancers of metastasis. Proc. Natl. Acad. Sci. U.S.A. 99, 2193−2198.
(3) Crocker, P. R., Paulson, J. C., and Varki, A. (2007) Siglecs and
their roles in the immune system. Nat. Rev. Immunol. 7, 255−266.
4) Hudak, J. E., Canham, S. M., and Bertozzi, C. R. (2014)
Glycocalyx engineering reveals a Siglec-based mechanism for NK cell
immunoevasion. Nat. Chem. Biol. 10, 69−75.
5) Hudak, J. E., Bertozzi, C. R. Glycotherapy: new advances inspire a
reemergence of glycans in medicine. Chem. Biol. 21, 16−37.
6) Adamo, R., Nilo, A., Castagner, B., Boutureira, O., Berti, F., and
Bernardes, G. J. L. (2013) Synthetically defined glycoprotein vaccines:
current status and future directions. Chem. Sci. 4, 2995−3008.
(7) Chalker, J. M., Bernardes, G. J. L., and Davis, B. G. (2011) A “tag-
and-modify” approach to site-selective protein modification. Acc.
Chem. Res. 44, 730−741.
(8) Gamblin, D. P., Scanlan, E. M., and Davis, B. G. (2008)
Glycoprotein synthesis: an update. Chem. Rev. 109, 131−163.
9) Gamblin, D. P., Garnier, P., van Kasteren, S., Oldham, N. J.,
(
Entry 5) yielding the quenched product without notable
glycoconjugation. Extending the reaction to 3 h afforded ca.
0% of the conjugation product. Thus, while glycoconjugation
(
3
with glycosyl azide 1 with genetically BCN proteins is possible
over extended time, the data in Table 2 clearly demonstrates
the superiority of glycosyl terazine 3 over glycosyl azide 1 in
glycoconjugations.
(
(
We then investigated the specificity of the glycoconjugation
in E. coli expressing sfGFP-TCOK. Cells were pelleted, washed
with PBS to remove excess TCOK present in the medium, and
incubated with 3-GluNAc-Cy3 (25 μM; the Cy-3 is not
30
sulfated and cell permeable ) for 8 h at 37 °C. As controls, the
same reaction was performed on E. coli expressing sfGFP-BocK
and in the absence of tetrazine 3-GluNAc-Cy3. After the
reaction, lysis buffer (LDS) was added, and the crude mixture
was analyzed by SDS-PAGE (silver stain and fluorescence scan
for Cy-3) showing that a single protein migrating at the
molecular weight of GFP underwent conjugation (lane 1,
Figure 4), whereas the control reaction showed no conjugation
(
Fairbanks, A. J., and Davis, B. G. (2004) Glyco-SeS: Selenenylsulfide-
mediated protein glycoconjugation - A new strategy in post-
translational modification. Angew. Chem., Int. Ed. 43, 828−833.
(
10) Bernardes, G. J. L., Gamblin, D. P., and Davis, B. G. (2006) The
direct formation of glycosyl thiols from reducing sugars allows one-pot
protein glycoconjugation. Angew. Chem., Int. Ed. 45, 4007−4011.
D
DOI: 10.1021/acs.bioconjchem.5b00101
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Communication
(
11) Floyd, N., Vijayakrishnan, B., Koeppe, A. R., and Davis, B. G.
2009) Thiyl glycosylation of olefinic proteins: S-linked glycoconju-
gate synthesis. Angew. Chem., Int. Ed. 48, 7798−7802.
12) Ribeiro-Viana, R., Sanchez-Navarro, M., Luczkowiak, J., Koeppe,
(30) Zambaldo, C., Sadhu, K. K., Karthikeyan, G., Barluenga, S.,
Daguer, J. P., and Winssinger, N. (2013) Selective affinity-based probe
for oncogenic kinases suitable for live cell imaging. Chem. Sci. 4, 2088−
2092.
(
(
́
J. R., Delgado, R., Rojo, J., and Davis, B. G. (2012) Virus-like
glycodendrinanoparticles displaying quasi-equivalent nested polyva-
lency upon glycoprotein platforms potently block viral infection. Nat.
Commun. 3, 1303.
(
13) Hu, Q.-Y., Allan, M., Adamo, R., Quinn, D., Zhai, H., Wu, G.,
Clark, K., Zhou, J., Ortiz, S., Wang, B., et al. (2013) Synthesis of a well-
defined glycoconjugate vaccine by a tyrosine-selective conjugation
strategy. Chem. Sci. 4, 3827−3832.
(
14) Smith, E. L., Giddens, J. P., Iavarone, A. T., Godula, K., Wang,
L.-X., and Bertozzi, C. R. (2014) Chemoenzymatic Fc glycosylation via
engineered aldehyde tags. Bioconjugate Chem. 25, 788−795.
(
15) Blackman, M. L., Royzen, M., and Fox, J. M. (2008) Tetrazine
ligation: Fast bioconjugation based on inverse-electron-demand Diels-
Alder reactivity. J. Am. Chem. Soc. 130, 13518−13519.
(
16) Devaraj, N. K., and Weissleder, R. (2011) Biomedical
applications of tetrazine cycloadditions. Acc. Chem. Res. 44, 816−827.
(
17) Devaraj, N. K., Weissleder, R., and Hilderbrand, S. A. (2008)
Tetrazine-based cycloadditions: application to pretargeted live cell
imaging. Bioconjugate Chem. 19, 2297−2299.
(
18) Karver, M. R., Weissleder, R., and Hilderbrand, S. A. (2011)
Synthesis and evaluation of a series of 1,2,4,5-tetrazines for
bioorthogonal conjugation. Bioconjugate Chem. 22, 2263−2270.
(
19) Liu, F., Liang, Y., and Houk, K. N. (2014) Theoretical
elucidation of the origins of substituent and strain effects on the rates
of Diels−Alder reactions of 1,2,4,5-tetrazines. J. Am. Chem. Soc. 136,
1
(
1483−11493.
20) Chin, J. W., Cropp, T. A., Anderson, J. C., Mukherji, M., Zhang,
Z. W., and Schultz, P. G. (2003) An expanded eukaryotic genetic code.
Science 301, 964−967.
(
21) Wang, L., Brock, A., Herberich, B., and Schultz, P. G. (2001)
Expanding the genetic code of Escherichia coli. Science 292, 498−500.
(
22) Lang, K., and Chin, J. W. (2014) Cellular incorporation of
unnatural amino acids and bioorthogonal labeling of proteins. Chem.
Rev. 114, 4764−4806.
(
23) Lang, K., Davis, L., Torres-Kolbus, J., Chou, C., Deiters, A., and
Chin, J. W. (2012) Genetically encoded norbornene directs site-
specific cellular protein labelling via a rapid bioorthogonal reaction.
Nat. Chem. 4, 298−304.
(
24) Lang, K., Davis, L., Wallace, S., Mahesh, M., Cox, D. J.,
Blackman, M. L., Fox, J. M., and Chin, J. W. (2012) Genetic encoding
of bicyclononynes and trans-cyclooctenes for site-specific protein
labeling in vitro and in live mammalian cells via rapid fluorogenic
Diels−Alder reactions. J. Am. Chem. Soc. 134, 10317−10320.
(
25) Wang, K. H., Sachdeva, A., Cox, D. J., Wilf, N. W., Lang, K.,
Wallace, S., Mehl, R. A., and Chin, J. W. (2014) Optimized orthogonal
translation of unnatural amino acids enables spontaneous protein
double-labelling and FRET. Nat. Chem. 6, 393−403.
(
26) Tanaka, T., Nagai, H., Noguchi, M., Kobayashi, A., and Shoda,
S.-i. (2009) One-step conversion of unprotected sugars to [small
beta]-glycosyl azides using 2-chloroimidazolinium salt in aqueous
solution. Chem. Commun., 3378−3379.
(
27) For an example combining a strained alkyne and a strained
alkene for conjugation to azide and tetrazine respectively, see: Neves,
A. A., Stockmann, H., Wainman, Y. A., Kuo, J. C. H., Fawcett, S.,
̈
Leeper, F. J., and Brindle, K. M. (2013) Imaging cell surface
glycosylation in vivo using “double click” chemistry. Bioconjugate
Chem. 24, 934−941.
(
28) Yang, J., Karver, M. R., Li, W. L., Sahu, S., and Devaraj, N. K.
(
2012) Metal-catalyzed one-pot synthesis of tetrazines directly from
aliphatic nitriles and hydrazine. Angew. Chem., Int. Ed. 51, 5222−5225.
29) Dommerholt, J., Schmidt, S., Temming, R., Hendriks, L. J. A.,
(
Rutjes, F. P. J. T., van Hest, J. C. M., Lefeber, D. J., Friedl, P., and van
Delft, F. L. (2010) Readily accessible bicyclononynes for bioorthog-
onal labeling and three-dimensional imaging of living cells. Angew.
Chem., Int. Ed. 49, 9422−9425.
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