Electrochemically Promoted Tyrosine-Click-Chemistry for Protein Labeling

Article abstract of DOI:10.1021/jacs.8b09372

The development of new bio-orthogonal ligation methods for the conjugation of native proteins is of particular importance in the field of chemical biology and biotherapies. In this work, we developed a traceless electrochemical method for protein bioconju

Full text of DOI:10.1021/jacs.8b09372

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Article
Electrochemically promoted tyrosine-click-chemistry for protein labelling
Dimitri Alvarez-Dorta, Christine Thobie, Mikaël Croyal, Mohammed Bouzelha,
Mathieu MEVEL, David Deniaud, Mohammed Boujtita, and Sébastien G. Gouin
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b09372 • Publication Date (Web): 13 Nov 2018
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Electrochemically promoted tyrosine-click-chemistry for pro-
tein labelling.
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Dimitri Alvarez-Dorta,†* Christine Thobie-Gautier,† Mikael Croyal,‡├ Mohammed Bouzelha, Mathieu
Mével, David Deniaud,† Mohammed Boujtita,†* and Sébastien G. Gouin.†*
† Université de Nantes, CEISAM, Chimie Et Interdisciplinarité, Synthèse, Analyse, Modélisation, UMR CNRS 6230, UFR
des Sciences et des Techniques, 2 rue de la Houssinière, BP 92208, 44322 Nantes Cedex 3, France.
‡CRNHO, West Human Nutrition Research Center, F-44000 Nantes, France.
├ INRA, UMR 1280 PhAN, F-44000 Nantes, France.
 INSERM UMR1089, Université de Nantes, CHU de Nantes, France.
ABSTRACT: The development of new bioorthogonal ligation methods for the conjugation of native proteins is of particular im-
portance in the field of chemical biology and biotherapies. In this work, we developed a traceless electrochemical method for protein
bioconjugation. The electrochemically promoted tyrosine-click (e-Y-CLICK) allowed the chemoselective Y-modification of peptides
and proteins with labeled urazols. A low potential is applied in an electrochemical cell to activate PTAD anchors in situ and on
demand, without affecting the electroactive amino-acids from the protein. The versatility of the electrosynthetic approach was shown
on biologically relevant peptides and proteins such as oxytocin, angiotensin 2, serum bovine albumin and epratuzumab. The fully
conserved enzymatic activity of a glucose oxidase observed after e-Y-CLICK further highlights the softness of the method. The e-Y-
CLICK protocols were successfully performed in pure aqueous buffers, without the need for co-solvents, scavenger or oxidizing
chemicals, and should therefore significantly broaden the scope of bioconjugation.
INTRODUCTION
the presence of water.21 Phenylisocyanate (Ph-NCO) side-prod-
uct resulting from PTAD decomposition leads to the unwanted
modification of lysines.20–22 The isocyanate could be scavenged
in high concentrations of Tris (tris-(hydroxymethyl)amino-
methane) buffer (100mM) to prevent the lysine conjugation,21
but these conditions were not always fully effective and amino
groups modifications were observed.17 This cross-reactivity
limited the scope of the Y-click reaction that should be per-
formed with acetonitrile as co-solvent or in Tris buffer with ex-
cess Ph-Ur.
Bioorthogonal ligation (BL) methods are extensively explored
for the development of protein conjugates,1,2 which are of con-
siderable importance in the therapeutic fields and in the biotech-
nology industry. Direct protein modifications are generally per-
formed onto amino group of the abundant lysine amino-acid
with N-hydroxysuccinimide-activated esters, sulfonyl chlorides
or iso(thio)cyanates. Alternatively, the relatively rare cysteine
residues can also be modified for single-site functionalization
through disulfide exchange and Michael addition.3 In compari-
son, the remaining 18 amino-acid have been much less ex-
ploited.4
To date, only a few studies reported the electrochemical activa-
tion of urazols,23 and all of them deal with the electrosynthesis
of chemicals or the functionalization of electrode surfaces.24,25,26
In this work, we explored the possibility of electrochemically
activating Ph-Ur species in the presence of proteins such as a
traceless Y-labeling method. We applied an appropriate electro-
chemical potential to activate the Ph-Ur dormant species in situ,
on demand, without oxidizing the sensitive amino-acids from
the protein or the ligands linked to the Ph-Ur (Figure 1A).
Recently, click-like reactions specifically targeting the elec-
tron-rich tyrosine (Y) residues on native proteins have been de-
veloped. Y-labelling selectivity was achieved with π-allyl pal-
ladium complexes,5,6 Mannich-type reactions,7 metal-catalyzed
oxidations,8 rhodium salts and boronic acids,9 single-electron-
transfer catalysts,10 ruthenium photocatalysts,11 and small ben-
zenediazonium species.12,13,14
Ene-like Y-modifications with cyclic diazodicarboxyamide15,16
anchors such as N-methylluminol17,18 or phenyl-urazol (Ph-Ur)
derivatives19,20 were also investigated by the groups of Naka-
mura and Barbas, respectively. While these approaches for Y-
labeling are efficient and very promising, there is still a need to
extend their potential scope. The use of chemical oxidants to
generate the reactive intermediates may not be compatible with
sensitive conjugates or proteins. 4-phenyl-3H-1,2,4-triazole-
3,5(4H)-diones (PTAD) anchors obtained after Ph-Ur oxidation
were also shown to be stable in acetonitrile but to decompose in
RESULTS AND DISCUSSION
In a first assay (Figure 1B), we studied the electrochemical ox-
idation behavior of Ph-Ur analytically, in the presence of L-ty-
rosine (Y). The study was performed in a neutral media (pH =
7.4) for physiological relevance and in a mixture of Tris-ace-
tonitrile (ACN) buffer as the electrolyte. As previously men-
tioned, ACN was shown to limit the degradation of chemically
formed PTAD into isocyanate, and Tris is a scavenger of this
side-product which limits lysine modification.21
Cyclic voltammograms of the isolated reagent phenylurazol 1
(Ph-Ur, quasi-reversible system A1/C1 0.36V/0.27V), Y 2 (non-
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reversible system A2) and clicked compound 3 (chemically pre-
laminar or crucible) electrode (see Table S1) was used as the
working electrode, a platinum as the counter-electrode and a
saturated calomel electrode (SCE) as the reference electrode.
The e-Y-click was stopped after the effective consumptions of
2 mol e- per mol of PTAD (around 2h, Figure S1B). Phosphate
buffer (PB), PB-ACN and Tris-ACN mixtures were used as
electrolytes (pH = 7.4). We were pleased to see that the highest
conversion yields were obtained in pure PB without the need
for a Ph-NCO scavenger. The reaction yields were even higher
in this medium compared to Tris-ACN. This could be explained
by a better transfer of charge with the phosphate ions. The Ph-
NCO side-product was not observed, even when conducting the
e-Y-click with one equivalent of L-lysine in the media as a nu-
cleophilic trap (Entry 11). The influence of the Y:Ph-Ur stoi-
chiometry and of the carbon working electrode (reticulated,
laminar or crucible, see details in Table S1) were also evaluated,
with the best yields (96%) obtained for the crucible with Y: Ph-
Ur = 1:2 (Entries 7, 8 and 10). The small yield improvement
observed with the crucible electrode may be the result of a
higher available area.
pared, non-reversible system A3 (0.55V) were recorded (Figure
1C). When the same experiment was performed mixing
equimolar quantities of Ph-Ur/Y the new anodic peak A3 corre-
sponding to the conjugated oxidation product 3 appears at a
more positive potential of 0.55V and the cathodic peak C1 at
0.27V, corresponding to the PTAD reduction, disappears during
the reverse scan, indicating a chemical reaction on the surface
of the electrode. The anodic peak A2 for Y 2 also disappeared
in the presence of Ph-Ur 1.
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Fortunately, the oxidation of both the phenol moiety present on
the Y (A2) and the clicked product 3 (A3) appear at even more
positive potential from Ph-Ur oxidation (A1), making the selec-
tive electrochemically activation of the PTAD possible. Thus,
this analytical study shows that the selection of an appropriate
potential of 0.36V (A1 peak), accomplishes the e-Y-Click reac-
tion without oxidizing the Y reagent or the clicked product 3.
The electrosynthesis experiments were optimized and carried
out using coulometry technique (Figure 1D) at controlled-po-
tential (0.36 V vs SCE). A conventional three-electrode system
was employed under stirring conditions. A graphite (reticulated,
Figure 1 A) The electrochemical activation of urazol species in situ (e-Y-click). B) e-Y-click protocol with Ph-Ur 1 and L-Tyr 2 C) Cyclic
voltammograms of 1.0 mM Ph-Ur (1) in the absence (green) and in the presence (red) of 1 mM Y (blue) and of 1 mM isolate conjugated
product 3 (purple) at a glassy carbon electrode, in 50 mM TRIS / ACN (50/50), pH 7.4. Scan rate: 100 mVs-1, reference electrode SCE. D)
Conversion yields after 2h of controlled-potential coulometry as determined by 1H NMR (Figure S1C). The electrolysis was performed with
1.0 mM of Y in the presence of 1.0 or 2.0 mM Ph-Ur (1) using different graphite forms as the working electrode. [b] 1.0 mM of L-lysine
was added in the media to potentially scavenge the Ph-NCS side product (not observed). Elec = Electrode, Ret = Reticular, Lam = Laminar,
Cru = Crucible.
Next we investigated the chemoselectivity of the reaction with
regards to other potentially oxidizable amino-acids bearing ar-
omatic or heteroaromatic residues (Figure 2A). Cyclic volt-
ammograms of Ph-Ur 1 were recorded in the presence of a
stoichiometric amount of commercially available protected
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(p) L-tyrosine, L-phenylalanine, L-tryptophan, L-histidine or
The electrolysis was performed in the presence of 2 eq Ph-Ur
1 in PB using a crucible carbon electrode at 0.36V and moni-
tored by cyclic voltammetry showing the complete Y conver-
sion in less than 2 hours (a decrease in the anodic peak). The
kinetic reaction was also followed by HPLC-MS (Figure S2)
and the labeled peptide was obtained as a pure compound in
approximately 99% conversion yield after 2h.
L-lysine. Only L-tyrosine (AY = 0.6 V) and L-tryptophan (AW
= 0.7 V) are electroactive at the studied potential range (-0.3
V – 1 V). As seen in Figure S1F the two amino-acids start to
oxidize at around 0.45 and 0.5V, respectively, which is not
problematic considering the lower potential of 0.36 V (Ph-Ur
oxidation peak) applied to generate the PTAD species. Nota-
bly, all aminoacids except L-tyrosine were inert with PTAD
and no electrochemical-chemical mechanisms were detected.
The occurrence of a chemical reaction would spawn the dis-
appearance of the cathodic peak C1, indicating that the PTAD
formed on the surface of the electrode is consumed. This re-
sult opens perspectives for the chemoselective in situ electro-
chemical Y-labeling of peptides and proteins.
The e-Y-click was applied in optimized conditions to label an-
giotensin II, a hormone regulating blood pressure (Figure 2C).
When commercially available Ph-Ur (1) was used, the conju-
gated compound was obtained in 83% conversion, with 17%
of starting material recovered, and no degradation during the
reaction (see kinetics by HPLC, Figure S3). More interest-
ingly, when the e-Y-click reaction was carried out using 4-(4-
(2-Azidoethoxy)phenyl)-3H-1,2,4-triazole-3,5(4H)-dione
(4),20 the conjugated peptide was obtained in 73% conversion.
This azide-armed peptide can be later functionalized by cop-
per-free click cycloaddition reactions (SPAAC), giving other
perspectives to biorthogonal tagging.
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The e-Y-click protocol was subsequently evaluated on the
neuropeptide hormone oxytocin (Figure 2B) in various bio-
logical roles in the childbirth process, human behaviors and
social bonding. Oxytocin was selected as a nonapeptide bear-
ing a single Y and a disulfide bridge.
Figure 2 A) L-Tyr chemoselectivity of the e-Y-click. Cyclic voltammogram of 1.0 mM Ph-Ur (1) in the presence of 1mM of a) L-
tyrosine, b) L-phenylalanine, c) L-tryptophane, d) L-histidine and e) L-lysine, at a glassy carbon electrode, in 50 mM TRIS / ACN
(50/50), pH 7.4. Scan rate: 50 mV s-1. B) The e-Y-click protocol for 0.5 mM oxytocin labelling in presence of 1 mM Ph-Ur (1). B)
Cyclic voltammograms at a glassy carbon electrode during controlled-potential coulometry at 0.36V versus SCE. After 0, 1, 1.5 and
2 h. Scan rate: 100 mV s-1. C) The e-Y-click protocol for angiotensin II labeling. In the presence of 2 equiv of Ph-Ur (1) or 2 equiv
of N3-Ph-Ur (4) by controlled-potential coulometry at 0.36V versus SCE. c) HPLC analysis of the reaction media after two hours
showed 83 and 73% conversion after 2h respectively (Figure S3).
bridges (Figure 3A). The electrochemical reaction was con-
ducted in the presence of a varied amount of Ph-Ur 1 (0, 1, 4,
10 eq per insulin) for 2h. Samples were then reduced, alkyl-
The e-Y-click was next evaluated on human insulin, a two
chain polypeptide bearing 51 amino acids including four tyro-
ated and trypsin digested prior to UPLC-MS and UPLC-
sines, one lysine (putative side reaction) and three disulfide
MS/MS analyses (Figures S4, S5). UPLC-MS analyses were
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also performed on undigested samples to estimate the number
of Ph-Ur grafted to the whole peptide for each condition.
Then, UPLC-MS analyses were achieved on samples after
proteolysis to confirm and refine observations. Trypsin diges-
tion led to the formation of
3
major peptides
(GIVEQCCTSICSLYQLENYC,
GFFYTPK, and
NFVNQHLCGSHLVEALYLVCGER) carrying two Y, one
Y and one K, or one Y residues, respectively. Fragmentation
patterns obtained in MS/MS mode ascertained which amino-
acid residue was modified with Ph-Ur as well as the quantifi-
cation of the number of Y modified in function of the amount
of Ph-Ur (0, 1, 4 or 10 eq). The proportion of the average num-
ber of modified Y (0 to 4) in function of Ph-Ur 1 is shown
Figure 3B. Insulin proportion was shown to decrease with in-
creasing amounts of 1 to reach less than 10% in the media
when using 10 equivalents of 1 (0xY, Figure 3B). The high
concentration of mono-clicked peptides (1xY in green) rap-
idly reached a stable threshold of around 40% suggesting a
higher reactivity and availability of a specific Y. In that re-
spect, our MS/MS data suggested that Y108 and Y40 could
respectively be the most and the least reactive Y residues (Fig-
ure S5). The reaction showed a complete Y chemoselectivity
and unspecific labeling of lysine (K53) was not observed.
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Figure 3 A) Structure of native insulin. B) % of mono- to
tetra-clicked PTAD adducts in function of Ph-Ur equiva-lents.
A penta-clicked product (4Y + 1K = 5) was not observed fur-
ther highlighting the e-Y-Click chemoselectivity for the 4 Y.
Bovine Serum Albumin (BSA), a 66 kDa protein was chosen
as a more complex system bearing 21 Y. The e-Y-click proto-
col could easily be scaled to functionalize up to 100 mg of
modified BSA in a single batch using the 50 mL electrochem-
ical cell. However, a limitation of the method would be the
functionalization of small quantities of material (< 1 mg)
which would require specific miniaturized electrochemical
cells or flow cells. Previous Y-BSA modifications with lumi-
nol derivatives (100 equiv) in the presence of H2O2 (100
equiv) and hemin (1 equiv) showed site-specificity in Y label-
ling.17 Modifications were shown to occur at 8 specific Y out
of the 21 available Y. The authors also observed a double
modification at Y400 which is one of the most exposed resi-
dues on BSA surface. Here, the BSA : Ph-Ur 1 ratio was var-
ied from 1:0 (reference) to 1:30 The gradual increase in BSA:
Ph-Ur 1 ratio led to a higher BSA modification up to an aver-
age of 9.1 clicked Y (Table 1). The level of Y modifications
can therefore be tuned by adjusting the amount of Ph-Ur 1.
When the labeling experiment was performed in strictly sim-
ilar conditions using a chemical oxidant (1,3-Dibromo-5,5-di-
methylhydantoin) instead of the electrochemical potential, the
number of labeled Y on the BSA more than halved (from 9.1
to 3.7). Thus, high levels of Y modification, surpassing the
chemical approach, were reached with the e-Y-Click protocol.
The BSA samples were further studied by protein mapping to
detect the modified Y. Sixteen peptide candidates with at least
one Y were identified in silico (web.expasy.org/pep-
tide_mass/) but only two of them (HPEY374AVSVLL R and
LGEY424GFQNAL IVR) could be found experimentally by
LC-ESI-HRMS analysis after protein proteolysis. The ratio of
functionalization for Y374 and Y424 was 30% and 10%, respec-
tively, which is indicative of site-selectivity in function of Y
exposition at the protein surface.
Table 1. e-Y-click optimization during controlled potential
coulometry.
Entry
BSA: 1
1:0
Method
MW
Mod Y[a]
0
1
2
3
4
5
6
e-Y-Click
e-Y-Click
e-Y-Click
e-Y-Click
e-Y-Click
Chemistry
66443
66769
66894
67058
68035
67084
1:5
1.9
1:10
1:20
1:30
1:30
2.6
3.5
9.1
The faradaic efficiency ꢀꢁ was calculated for the Ph-Ur 1
ꢂ
3.7
oxidation using the faraday law ( ꢀꢁ ꢃ ꢄ.ꢅ.ꢂ ) where n is the
ꢂ
ꢆ
[a] the average of Y modified was determined by LC-ESI-
HRMS analysis.
number of electrons exchanged, N the number of moles for
Ph-Ur 1, F Faraday's constant (96485 C/mol) and Q the charge
passed (C). A high ꢀꢁ of around 90-100% was observed in-
ꢂ
Next, we investigated the possibility of grafting two different
chemical functionalities on the BSA protein in a sequential
approach. We carried out the e-Y-click using mannosyl deriv-
ative 5 (synthesis in Scheme S1) and/or the azido-functional-
ized compound 4 (Figure 4). Carbohydrates are of primary
importance in the design of conjugated vaccines,27 and N3-
functionalized proteins (N3-BSA here) are interesting plat-
forms for anchoring epitopes, ligands, chelators, or probes by
Staudinger reactions,28 conventional copper-catalyzed cy-
clization,29,30 or copper-free SPAAC with cyclooctyne deriv-
atives.31,32 BSA protein was first efficiently labeled with N3-
dependently of the Ph-Ur 1 concentration (5 to 100µmol)
which is indicative of the absence of surface passivation dur-
ing the experimental time (two hours, Figure S6A).
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PTAD 4 with an average of x = 3.7 modified Y by N3-BSA.
(x+y = 4.4). In all cases, SDS-PAGE followed by silver stain-
ing, showed that BSA was modified without inducing protein
degradation. Secondly, N3-BSA and Man-N3-BSA could by
labeled by a commercially available fluorescent FAM probe
(DBCO-PEG4-5/6-FAM) by SPAAC. The formation of the
FAM-BSA and FAM-Man-BSA was unambiguously de-
tected in the western blot using an anti-FAM antibody.
In the same way, Man-PTAD 5 was successfully anchored to
the BSA to form the glycoconjugated Man-BSA bearing 2.7
mannose residues as evidenced by mass spectrometry and
western blotting (indirect detection with labeled concanavalin
A, Figure 4). The small difference observed in the grafting
levels (x and y) with 4 and 5 may be explained by different
diffusion coefficients of the compounds at the electrode sur-
face. The bi-functional conjugate Man-N3-BSA was obtained
using a 50/50 ratio of phenylurazol (4/5) during electrolysis
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Figure 4 The e-Y-click protocol for BSA labeling. The formation Man-BSA, N3-BSA and Man-N3-BSA was successfully achieved using
e-Y-click as evidenced by mass spectrometry (SI) and western blot using silver staining, or a labelled mannose-binding lectin (Concanavalin
A = ConA). The N3 group could be further functionalized by the fluorescent FAM probe (detection with anti-FAM antibody) using the
commercially available DBCO-PEG4-5/6-FAM probe in a copper-free click-chemistry protocol.
and oxygen is reduced to hydrogen peroxide. Thus, GOx ki-
netics can be followed by hydrogen peroxide detection using
New bioconjugation techniques are being researched to de-
a well-established cascade with horseradish peroxidase
velop more effective antibody-drug conjugates (ADC) for
(HRP). The HRP uses H2O2 to oxidize the substrate o-dianis-
vectorized immunotherapy. ADC combines a specific mono-
idine (3, 3’-dimethoxybenzidine) into an oxidized yellow-or-
clonal antibody (mAb) acting as a “magic bullet” for the se-
ange product which is detected spectrophotometrically at λ=
436 nm (Figure 6A).35 GOx was electrochemically labeled
with 4 (Figure S9) but a strong matrix effect, probably due to
the presence of N-glycosylated GOx isoforms, prevented the
accurate determination of the average number of modified Y.
lective delivery of covalently linked cytotoxic compound(s)
in the tumoral environment. To assess the applicability of the
e-Y-click protocol for mAb functionalization, 4 was reacted
with epratuzumab, a humanized anti-CD22 mAb which has
previously shown promising clinical activity in patients with
non-Hodgkin’s lymphoma.33 Satisfyingly, a shift of the
epratuzumab molecular weight, corresponding to an average
grafting of 2.3 phenylurazol 4 molecules, was evidenced by
ESI-MS (Figure 5).
Finally, we evaluated the softness of the e-Y-Click, by check-
ing if an enzymatic activity could be retained after the elec-
trochemical coupling. Glucose oxidase enzyme (GOx) was se-
lected as a biomedically relevant target used for blood glucose
monitoring and in cancer diagnosis and treatment.34 GOx cat-
alyze the oxidation of D-glucose into D-glucono-1,5-lactone,
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of chemical oxidizers. Specific issues including lysine modi-
fications due to PTAD decomposition, double Y modifica-
tions and thiols oxidations were not observed. The e-Y-Click
protocol may therefore complement the current arsenal of
techniques for the traceless preparation of a wide range of
peptide and protein conjugates.
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ASSOCIATED CONTENT
Supporting Information. “This material is available free of
charge via the Internet at http://pubs.acs.org.”
Characterization details and general procedures for e-Y-click.
Figure 5 The e-Y-click protocol for antibody labeling.
Epratuzumab was efficiently labeled with 4 as evidenced by
the mass shift in deconvoluted mass spectra (native
epratuzumab in blue, epratuzumab-conjugated in red).
AUTHOR INFORMATION
Corresponding Authors
* dimitri.alvarez-dorta@univ-nantes.fr; moham-
med.boujtita@univ-nantes.fr; sebastien.gouin@univ-nantes.fr.
Importantly, the enzyme kinetic of the GOx was not affected
by the e-Y-click protocol and native GOx and 4-GOx showed
a virtually identical conversion rate of the glucose substrate
(Figure 6B). Thus, the electrochemical protocol can be ap-
plied to enzymes without damaging their functionality, even
if a redox active coenzyme (flavin adenine dinucleotide FAD
for GOx) is buried in the active center. This can be explained
by the low accessibility of the coenzyme and by the low coef-
ficient diffusion of proteins at the electrode surfaces.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was carried out with financial support from the the
Centre National de la Recherche Scientifique (CNRS) and the
Ministère de l’Enseignement Supérieur et de la Recherche in
France. We thank Isabelle Louvet for HPLC analysis and Alain
Faivre-Chauvet for providing epratuzumab.
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