Facile and stabile linkages through tyrosine: Bioconjugation strategies with the tyrosine-click reaction

Article abstract of DOI:10.1021/bc300665t

The scope, chemoselectivity, and utility of the click-like tyrosine labeling reaction with 4-phenyl-3H-1,2,4-triazoline-3,5(4H)-diones (PTADs) is reported. To study the utility and chemoselectivity of PTAD derivatives in peptide and protein chemistry, we synthesized PTAD derivatives possessing azide, alkyne, and ketone groups and studied their reactions with amino acid derivatives and peptides of increasing complexity. With proteins we studied the compatibility of the tyrosine click reaction with cysteine and lysine-targeted labeling approaches and demonstrate that chemoselective trifunctionalization of proteins is readily achieved. In particular cases, we noted that PTAD decomposition resulted in formation of a putative isocyanate byproduct that was promiscuous in labeling. This side reaction product, however, was readily scavenged by the addition of a small amount of 2-amino-2-hydroxymethyl-propane- 1,3-diol (Tris) to the reaction medium. To study the potential of the tyrosine click reaction to introduce poly(ethylene glycol) chains onto proteins (PEGylation), we demonstrate that this novel reagent provides for the selective PEGylation of chymotrypsinogen, whereas traditional succinimide-based PEGylation targeting lysine residues provided a more diverse range of PEGylated products. Finally, we applied the tyrosine click reaction to create a novel antibody-drug conjugate. For this purpose, we synthesized a PTAD derivative linked to the HIV entry inhibitor aplaviroc. Labeling of the antibody trastuzumab with this reagent provided a labeled antibody conjugate that demonstrated potent HIV-1 neutralization activity demonstrating the potential of this reaction in creating protein conjugates with small molecules. The tyrosine click linkage demonstrated stability to extremes of pH, temperature, and exposure to human blood plasma indicating that this linkage is significantly more robust than maleimide-type linkages that are commonly employed in bioconjugations. These studies support the broad utility of this reaction in the chemoselective modification of small molecules, peptides, and proteins under mild aqueous conditions over a broad pH range using a wide variety of biologically acceptable buffers such as phosphate buffered saline (PBS) and 2-amino-2-hydroxymethyl- propane-1,3-diol (Tris) buffers as well as others and mixed buffered compositions.

Full text of DOI:10.1021/bc300665t

Communication
pubs.acs.org/bc
Facile and Stabile Linkages through Tyrosine: Bioconjugation
Strategies with the Tyrosine-Click Reaction
Hitoshi Ban,‡ Masanobu Nagano,‡ Julia Gavrilyuk, Wataru Hakamata, Tsubasa Inokuma,
and Carlos F. Barbas, III*
The Skaggs Institute for Chemical Biology and the Departments of Chemistry and Molecular Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037, United States
S
* Supporting Information
ABSTRACT: The scope, chemoselectivity, and utility of the
click-like tyrosine labeling reaction with 4-phenyl-3H-1,2,4-
triazoline-3,5(4H)-diones (PTADs) is reported. To study the
utility and chemoselectivity of PTAD derivatives in peptide and
protein chemistry, we synthesized PTAD derivatives possessing
azide, alkyne, and ketone groups and studied their reactions with
amino acid derivatives and peptides of increasing complexity.
With proteins we studied the compatibility of the tyrosine click
reaction with cysteine and lysine-targeted labeling approaches and
demonstrate that chemoselective trifunctionalization of proteins is
readily achieved. In particular cases, we noted that PTAD
decomposition resulted in formation of a putative isocyanate
byproduct that was promiscuous in labeling. This side reaction
product, however, was readily scavenged by the addition of a small amount of 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris)
to the reaction medium. To study the potential of the tyrosine click reaction to introduce poly(ethylene glycol) chains onto
proteins (PEGylation), we demonstrate that this novel reagent provides for the selective PEGylation of chymotrypsinogen,
whereas traditional succinimide-based PEGylation targeting lysine residues provided a more diverse range of PEGylated
products. Finally, we applied the tyrosine click reaction to create a novel antibody−drug conjugate. For this purpose, we
synthesized a PTAD derivative linked to the HIV entry inhibitor aplaviroc. Labeling of the antibody trastuzumab with this
reagent provided a labeled antibody conjugate that demonstrated potent HIV-1 neutralization activity demonstrating the
potential of this reaction in creating protein conjugates with small molecules. The tyrosine click linkage demonstrated stability to
extremes of pH, temperature, and exposure to human blood plasma indicating that this linkage is significantly more robust than
maleimide-type linkages that are commonly employed in bioconjugations. These studies support the broad utility of this reaction
in the chemoselective modification of small molecules, peptides, and proteins under mild aqueous conditions over a broad pH
range using a wide variety of biologically acceptable buffers such as phosphate buffered saline (PBS) and 2-amino-2-
hydroxymethyl-propane-1,3-diol (Tris) buffers as well as others and mixed buffered compositions.
to be one-step (Figure 1b). Intrinsic approaches to
bioconjugation are, however, limited to the development of
chemistry compatible with the 20 naturally occurring amino
acids and are challenged by the fact that any given amino acid is
likely to occur more than once in a protein. Regardless of the
approach, bioconjugation reactions ideally proceed rapidly,
selectively, and in high yield under physiological conditions
while preserving the target protein’s biological activity.1−4,29−31
Much of the chemistry developed to tap intrinsic amino acid
reactivity is many decades old and is centered on the
modification of lysine and cysteine side chains. The abundance
of lysine residues on the protein surface, however, makes site-
specific modification difficult. In contrast, cysteine is rare and
INTRODUCTION
■
Bioconjugation reactions exploit either the intrinsic chemical
reactivity of the biomolecule or introduce extrinsic function-
alities that can then be subsequently reacted in a bio-orthogonal
fashion. In recent years a wide range of extrinsic bioorthogonal
reactions have been developed (Figure 1a).1−4 This two-step
approach relies on the introduction of functionalities such as
ketones, aldehydes, azides, alkenes, or alkynes into the target
protein. In a second step, the introduced extrinsic function-
alities are selectively reacted to form oximes/hydrazones,1−12
Staudinger ligation products,1−4,13 triazole-Click prod-
ucts,1−4,14−18 or Diels−Alder,1−4,19−23 among others.1−4,24 In
addition to chemical routes, extrinsic functional groups can be
introduced into biomolecules via enzymatic modifica-
tion1−5,11,12 19−23 or genetic encoding.1−4,25−33 Thus, extrinsic
,
Received: December 17, 2012
Revised: March 13, 2013
approaches involve a minimum of two modification steps of the
biomolecule. In contrast, intrinsic approaches have the potential
© XXXX American Chemical Society
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Figure 1. Bioconjugation reactions: (a) two-step extrinsic; (b) one-step intrinsic approach.
Scheme 1. Model Tyrosine Click Reaction
most often found in disulfide-linked pairs in native proteins.
For cysteine modification, pretreatment by reduction to cleave
disulfide bonds is typically required; this is followed by reaction
with a reagent like maleimide.34 Recently, novel mono- and
dibromomaleimides have expanded the potential of cysteine-
targeted labeling.35,36 Less developed are methods for the
selective modification of the aromatic amino acid side chains of
tryptophan37,38 and tyrosine,39−43 but a number of promising
approaches have been recently reported. Among nucleophilic
amino acids, tyrosine has unique reactivity due to the acidic
proton of the phenol ring.44 The alkyation or acylation reaction
of tyrosine under basic conditions proceeds at the oxygen.
Under acidic conditions, an ene-like reaction occurs at a carbon
atom on the aromatic ring. Bioconjugation at carbon in mild,
biocompatible, metal-free conditions was reported using
Mannich-type additions to imines or cerium(IV) ammonium
nitrate (CAN) as an oxidation reagent.39,43 However, in the
former, the modification of tyrosine with imines formed in situ
is limited by the requirement of an excess of highly reactive
formaldehyde. Tryptophan and amine containing side chains
and reduced disulfides form formaldehyde adducts under these
reaction conditions. The latter, oxidative coupling with
electron-rich aniline derivatives, results in the coupling of
both tyrosine and tryptophan. An alternative tyrosine
modification with cyclic imines solves this problem but often
reacts in an uncontrolled way with either one or two
equivalents of cyclic imine. Recently, we discovered that we
could exploit the reactivity of certain diazodicarboxylate-related
molecules and the intrinsic reactivity of tyrosine to create a
rapid aqueous ene-type reaction, the tyrosine-click reaction.45
As a background for the development of this reaction, it should
be noted that substituted phenols react with highly reactive
electrophiles such as diazodicarboxylates in organic solvents in
the presence of activating protic or Lewis acid additives.46−54
However, highly reactive diazodicarboxylate reagents decom-
pose rapidly in aqueous media and stabile diazodicarboxyamide
reagents do not react efficiently with phenols in aqueous media;
thus most diazodicarboxyamides are not suitable as bioconju-
gation reagents.55 Although cyclic diazodicarboxylate reagents
can be activated by interaction with cationic species such as
protons or metals, cyclic diazodicarboxyamides like 4-phenyl-
3H-1,2,4-triazoline-3,5(4H)-dione (PTAD) are not activated by
protic or Lewis acid additives.55 Our initial study involved a
survey of the reactivities and stabilities of diazodicarboxylate
and diazodicarboxyamide; these studies led us to develop the
tyrosine-click reaction of PTAD with the phenolic side chain of
tyrosine (Scheme 1).
The tyrosine click reaction is illustrated in Scheme 1
involving the reaction in this case of N-acyl tyrosine methyl
amide 1 with PTAD 2a in mixed organic solvent/aqueous
media. In sodium phosphate buffer (pH 7)/acetonitrile (1:1), 1
reacted rapidly (reaction was complete in less than 5 min) with
1.1 equiv of PTAD 2a to provide 3a in quantitative yield.
PTAD labeling was more efficient than 4-methyl-3H-1,2,4-
triazoline-3,5(4H)-dione (MTAD) labeling;56 reaction of
MTAD 2b with amide 1 gave 3b in 57% isolated yield as
electronic modulation of the triazolinedione system is key to
controlling this reaction.45 Here we present a comprehensive
study concerning the synthesis and characteristics of versatile
triazolinedione-based labeling reagents, studying of the scope,
stability, and chemoselectivity of these reagents, and examine
their applications in bioconjugation reactions with small
molecules, peptides, and proteins.
B
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solid (2 steps, 12%). This compound was purified by short
column chromatography (CHCl3/MeOH). 1H NMR (300
MHz, DMSO-d6): δ 10.4 (br, 2H), 7.31−7.27 (m, 2H), 7.02−
6.95 (m, 2H), 4.87 (s, 2H), 2.16 (s, 3H). 13C NMR (75 MHz,
DMSO-d6): δ 204.81, 157.96, 154.67, 128.57, 125.75, 115.57,
73.13, 27.16. HRMS: calcd for C11H12N3O4 (MH+) 250.0822,
found 250.0826. 4-(4-Azidophenyl)-1,2,4-triazolidine-3,5-dione
(8d). The title compound 8d was prepared from 4-azidoaniline
hydrochloride, and was obtained as white solid (2 steps, 35%).
1H NMR (300 MHz, DMSO-d6): δ 10.5 (br, 2H), 7.50 (d, J =
9.0 Hz, 2H), 7.23 (d, J = 9.0 Hz, 2H). 13C NMR (75 MHz,
DMSO-d6): δ 154.25, 139.59, 129.76, 128.53, 120.47. HRMS:
calcd for C8H7N6O2 (MH+) 219.0625, found 219.0617. General
procedure B: To a 0.5 M solution of compound 6 (1.0 equiv)
and Et3N (1.8 equiv) in THF (5 mL) was added 4-nitrophenyl
chloroformate (1.8 equiv) at 0 °C. The resulting solution was
stirred at room temperature overnight. Ethyl hydrazinecarbox-
ylate 4 (2.6 equiv) and Et3N (2.6 equiv) were added at room
temperature and stirred at 40 °C for 4 h. Then, EtOAc and
water were added. The organic layer was separated and washed
once with water. The resulting aqueous layer was combined and
extracted twice with EtOAc. The combined organic layer was
dried over MgSO4, and concentrated in vacuo. The obtained
crude solid was washed with EtOAc, dried, and dissolved in
MeOH (0.2 M solution) followed by addition of K2CO3 (3.0
equiv). The calculation was done based on the crude material.
The suspension was stirred under reflux for 3 h. The reaction
mixture was acidified with 12 N HCl to pH 2 and then
concentrated in vacuo. The generated white solids were washed
with water and Et2O to give 8. 4-(4-Ethynylphenyl)-1,2,4-
triazolidine-3,5-dione (8e). The title compound 8e was
prepared from 4-ethynylaniline hydrochloride, and was
EXPERIMENTAL PROCEDURES
■
General. 1H NMR and C NMR spectra44 were recorded on
Bruker DRX-600 (600 MHz) or Varian MER-300 (300 MHz)
spectrometers in the stated solvents using tetramethylsilane as
an internal standard. Chemical shifts were reported in parts per
million (ppm) on the δ scale from an internal standard (NMR
descriptions: s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; br, broad). Coupling constants, J, are reported in
Hertz. Mass spectroscopy was performed by The Scripps
Research Institute Mass Spectrometer Center. Analytical thin-
layer chromatography and flash column chromatography were
performed on Merck Kieselgel 60 F254 silica gel plates and
Silica Gel ZEOprep 60 ECO 40−63 Micron, respectively.
Visualization was accomplished with anisaldehyde or KMnO4.
High performance liquid chromatography (HPLC) was
performed on Shimadzu GC-8A using Vydac 218TP C18
GRACE Column (22 mm × 250 mm) for purification and
Hewlett-Packard series 1100 using MCI GEL C18 Mitubishi
Chemical Column (4.6 mm × 250 mm) for analysis. Unless
otherwise noted, all the materials were obtained from
commercial suppliers, and were used without further
purification. All solvents were the commercially available
grade. All reactions were carried out under argon atmosphere
unless otherwise mentioned. Amide starting materials, tyrosine,
histidine, tryptophan, serine, cysteine, lysine, and (Ile3)-
pressionoic acid, were commercially available compounds or
prepared according to published procedures.1 A peptide, H2N-
VWSQKRHFGY-CO2H, was custom-synthesized by Abgent,
Inc. Chymotrypsinogen A (MW 25 kDa) (ImmunO) was used
as a model protein for PEGylation.
Synthesis of 1,2,4-Triazolidine-3,5-diones 8. Method A:
To a 0.2 M solution of ethyl hydrazinecarboxylate 4 (1.0 equiv)
in THF was added 1,1′-carbonyldiimidazole (CDI, 1.0 equiv) at
room temperature. The resulting solution was stirred at room
temperature. After 2 h, aniline 5 (1.0 equiv) and Et3N (2.0
equiv) were added at room temperature and stirred overnight.
Then, EtOAc and 10% HCl were added. The organic layer was
separated and washed once with 10% HCl and water. The
resulting aqueous layer was extracted once with EtOAc. The
combined organic layer was dried over MgSO4, and
concentrated in vacuo. The obtained crude solid was washed
with EtOAc, dried, and dissolved in MeOH (0.2 M solution)
followed by addition of K2CO3 (3.0 equiv). The calculation was
done based on the crude material. The suspension was stirred
under reflux for 3 h. The reaction mixture was acidified with 12
N HCl to pH 2 and then concentrated in vacuo. The generated
white solids were washed with water and EtOAc to give 8. 4-(4-
Propargyloxyphenyl)-1,2,4-triazolidine-3,5-dione (8a). The title
1
obtained as white solid (2 steps, 38%). H NMR (300 MHz,
DMSO-d6): δ 10.6 (br, 2H), 7.60−7.57 (m, 2H), 7.54−7.50
(m, 2H), 4.27 (s, 1H). 13C NMR (75 MHz, DMSO-d6): δ
153.83, 133.36,1 33.07, 126.67, 121.59, 83.81, 82.41. HRMS:
calcd for C10H8N3O2 (MH+) 202.0611, found 202.0619. 4-(4-
Acetylphenyl)-1,2,4-triazolidine-3,5-dione (8f). The title com-
pound 8f was prepared from 4′-aminoacetophenone, and was
1
obtained as white solid (2 steps, 15%). H NMR (300 MHz,
DMSO-d6): δ 10.7 (br, 2H), 8.09−8.06 (m, 2H), 7.71−7.68
(m, 2H), 2.62 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ
198.18, 153.67, 137.09, 136.26, 129.74, 126.17, 27.76. HRMS:
calcd for C10H10N3O3 (MH+) 220.0717, found 220.0713.
Oxidation of 3H-1,2,4-Triazole-3,5(4H)-diones. To a
0.05 M solution of compound 8 (1.0 equiv) in CH2Cl2 was
added 1,3-dibromo-5,5-dimethylhydantoin 10 (1.0 equiv) at
room temperature. The resulting solution was stirred at room
temperature. After 2 h, silica sulfuric acid57 (SiO2−OSO3H, 4
times weight to starting material) was added at room
temperature and stirred at room temperature. After 30 min,
the silica sulfuric acid was removed by filtration. Then, volatile
materials were evaporated in vacuo to give 9. The obtained
material was relatively unstabile against light and humidity in
solution at room temperature. Therefore, it was used for next
reaction without additional purification after confirmation of
1
compound 8a was obtained as white solid (2 steps, 28%). H
NMR (300 MHz, DMSO-d6): δ 10.6 (br, 2H), 7.60−7.57 (m,
2H), 7.54−7.50 (m, 2H), 4.27 (s, 1H). 13C NMR (75 MHz,
DMSO-d6): δ 158.83, 133.36, 133.07, 126.67, 121.59, 83.81,
82.41. HRMS: calcd for C10H8N3O2 (MH+) 202.0611, found
202.0619. 4-(4-(2-Azidoethoxy)phenyl)-1,2,4-triazolidine-3,5-
dione (8b). The title compound 18b was obtained as white
1
1
solid (2 steps, 28%). H NMR (300 MHz, DMSO-d6): δ 10.4
purity by H NMR (see SI). 4-(4-(Propargyloxy)phenyl)-3H-
(br, 2H), 7.36−7.33 (m, 2H), 7.07−7.03 (m, 2H), 4.21 (t, J = 6
Hz, 2H), 3.66 (t, J = 6 Hz, 2H). 13C NMR (75 MHz, DMSO-
d6): δ 158.16, 154.63, 128.67, 125.85, 115.63, 68.03, 50.44.
HRMS: calcd for C10H11N6O3 (MH+) 263.0887, found
263.0889. 4-(4-(2-Oxopropoxy)phenyl)-1,2,4-triazolidine-3,5-
dione (8c). The title compound 8c was obtained as white
1,2,4-triazole-3,5(4H)-dione (9a). The title compound 9a was
prepared from 8a (50.0 mg, 0.216 mmol), and was obtained as
a deep red solid (42.0 mg, 85%). 1H NMR (300 MHz, CDCl3):
δ 7.41−7.37 (m, 2H), 7.15−7.12 (m, 2H), 4.75 (d, J = 3.0 Hz,
2H), 3.64 (t, J = 3.0 Hz, 1H). 4-(4-(2-Azidoethoxy)phenyl)-
3H-1,2,4-triazole-3,5(4H)-dione (9b). The title compound 9b
C
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was prepared from 8b (49.0 mg, 0.187 mmol), and was
obtained as deep red oil (39.6 mg, 81%). 1H NMR (300 MHz,
CDCl3): δ 7.40−7.35 (m, 2H), 7.10−7.06 (m, 2H), 4.20 (t, J =
3.0 Hz, 2H), 3.64 (t, J = 3.0 Hz, 2H). 4-(4-(2-Oxopropoxy)-
phenyl)-3H-1,2,4-triazole-3,5(4H)-dione (9c). The title com-
pound 9c was prepared from 8c (47.0 mg, 0.189 mmol), and
was obtained as deep purple solid (34.9 mg, 81%).1H NMR
(300 MHz, CDCl3): δ 7.42−7.38 (m, 2H), 7.05−7.02 (m, 2H),
4.61 (s, 2H), 2.31 (s, 3H). 4-(4-Azidophenyl)-3H-1,2,4-
triazole-3,5(4H)-dione (9d). The title compound 9d was
prepared from 8d (40 mg, 0.183 mmol), and was obtained as
deep red solid (34.1 mg, 86%). 1H NMR (300 MHz, CDCl3): δ
7.51−7.46 (m, 2H), 7.22−7.17 (m, 2H). 4-(4-Ethynylphenyl)-
3H-1,2,4-triazole-3,5(4H)-dione (9e). To a solution of
compound 9e (4.43 mg, 0.022 mmol) in MeCN (44 μL) was
added 1,3-dibromo-5,5-dimethylhydantoin (6.29 mg, 0.022
mmol) at room temperature. The resulting solution was stirred
at room temperature for 10 min. Completion of the reaction
was monitored by the change of reaction solution color to a
characteristic deep red color. The obtained material decom-
posed quickly at room temperature. Therefore, the 0.5 M
MeCN reaction solution was used for next step without
purification.
Synthesis of Aplaviroc-Urazole. Aplaviroc-alkyne (26):
To a solution of Azido-alkyne (See SI) (200 mg, 0.670 mmol)
in diethyl ether (2 mL) was added triphenylphosphine (264
mg, 1.00 mmol) at 0 °C and stirred at room temperature for 3
h. Then, deionized water 200 mL was added to reaction
mixture and stirred for 12 h. 10 % HCl aq. was added, followed
by wash with diethyl ether. The aqueous layer was basified to
pH 10 with 5 N NaOH and extracted with dichlorometha-
ne/iPrOH (4:1) 5 times. The organic layer was dried over
Na2SO4, and concentrated in vacuo. The crude amine 25 was
used to the next reaction without further purification. To a
solution of Aplaviroc 2458 prepared by the previously reported
method (300 mg, 0.513 mmol) in DMF (10 mL) was added
BOP (174 mg, 0.639 mmol), triethylamine (362 mL, 2.596
mmol), and amine-alkyne 25 (174 mg, 0.639 mmol) at room
temperature and stirred for 12 h. Then, dichloromethane were
added and was separated and washed sat. NaHCO3 aq. and
brine. The organic solution was dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel
chromatography (Dichloromethane/MeOH = 9/1) to give 26
(181 mg, 42%) as thin yellow oil. 1H NMR (400 MHz,
CDCl3): δ 7.83−7.79 (m, 2H), 7.34−7.28 (m, 2H), 7.02−6.96
(m, 5H), 6.47 (br t, 1H), 6.42 (br s, 1H), 4.01 (dd, J = 1.4, 5.7
Hz, 1H), 3.68−3.60 (m, 10H), 3.54 (t, J = 4.8 Hz, 2H), 3.46−
3.38 (m, 4H), 2.97−2.84 (m, 4H), 2.74−2.69 (m, 2H), 2.54−
2.47 (m, 2H), 2.41−2.27 (m, 2H). 2.20−2.07 (m, 4H), 2.02 (t,
J = 2.6, 1H), 1.98−1.89 (2H), 1.73−1.53 (m, 8H), 1.40−1.13
(m, 8H), 0.96−0.85 (t, J = 7.2, 4H). 13C NMR (125 MHz,
MeOD-d4): δ 173.02, 171.97, 168.41, 165.92, 160.57, 156.64,
132.35, 129.57, 119.81, 117.95, 82.69, 78.94, 70.50, 70.27,
70.23, 69.69, 69.64, 69.55, 61.42, 59.37, 56.98, 53.94, 49.77,
47.27, 42.87, 40.10, 39,98, 39.42, 36.09, 34.98, 32.17, 31.65,
31.54, 30.02, 29.63, 26.53, 26.02, 20.46, 14.75, 13.30. HRMS:
calcd for C46H65N5O9 (MH+) 832.4855, found 832.4854.
Aplaviroc-urazole (27): To a solution of 8b (20 mg, 0.763
mmol) and 27 (70 mg, 0.0839 mmol) in tert-BuOH/H2O (3
mL/1 mL) was added THPTA59 (458 mL, 0.0229 mmol, 50
mM solution in H2O), copper sulfate 5 hydrate (114 mL,
0.0229 mmol, 50 mg/mL solution in H2O), and sodium
ascorbate (91 mL, 0.0229 mmol, 50 mg/mL solution in H2O)
at room temperature and stirred for 30 min. Then, chloroform
was added and washed with sat. NaHCO3 aq. and brine.
Combined organic layer was dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel
chromatography (EtOAc/MeOH = 4/1) to give 27 (43 mg,
1
52%) as thin yellow solid. H NMR (400 MHz, MeOD-d4): δ
7.82−7.80 (m, 3H), 7.45−7.43 (m, 2H), 7.26−7.24 (m, 2H),
7.03−7.01 (m, 4H), 6.97−6.95 (m, 2H), 4.74 (m, 2H), 4.48
(m, 2H), 4.13 (m, 1H), 3.95 (m, 2H), 3.68−3.60 (m, 10H),
3.54 (t, J = 4.8 Hz, 2H), 3.46−3.38 (m, 4H), 2.97−2.84 (m,
2H), 2.74−2.69 (m, 2H), 2.54−2.47 (m, 2H), 2.41−2.27 (m,
2H). 2.20−2.07 (m, 4H), 2.02 (t, J = 2.6, 1H), 1.98−1.89 (2H),
1.73−1.53 (m, 8H), 1.40−1.13 (m, 8H), 0.96−0.85 (t, J = 7.2,
4H). 13C NMR (125 MHz, MeOD-d4): δ 173.51, 171.84,
168.40, 165.80, 160.35, 158.03, 157.15, 155.25, 154.88, 52.16,
46.76, 132.64, 129.53, 128.05, 123.27, 119.67, 118.08, 115.07,
79.13, 70.53, 70.27, 70.13, 69.54, 69.47, 66.93, 56.87, 49.92,
49.88, 40.1221, 39.96, 39.13, 35.30, 31.49, 26.49, 25.96, 21.46,
20.41, 13.15. HRMS: calcd for C56H75N11O12 (MH+)
1094.5669, found 1094.5660.
Synthesis of Model Compounds Used in Stability
Studies. 1-(2-Hydroxy-5-methylphenyl)-4-phenyl-1,2,4-triazo-
lidine-3,5-dione (29). To a solution of p-cresol (80 mg, 0.740
mmol) in THF (5 mL) was added NaH (35.5 mg, 0.885 mmol)
at 0 °C. After 20 min, PTAD (127 mg, 0.725 mmol) was added
at 0 °C and stirred at room temperature for 3 h. Then, EtOAc
and 10% HCl were added. The organic layer was separated and
washed once with brine. The resulting aqueous layer was
extracted once with EtOAc. The combined organic layer was
dried over MgSO4, and concentrated in vacuo. The residue was
purified by silica gel chromatography (CHCl3/MeOH) to give
1
29 (158 mg, 75%) as a white solid. H NMR (600 MHz,
DMSO-d6): δ 9.86 (br, 1H), 7.53−7.49 (m, 4H), 7.43−7.40
(m, 1H), 7.19 (d, J = 2.0 Hz, 1H), 7.10 (dd, J = 8.3, 2.0 Hz,
1H), 6.87 (d, J = 8.3 Hz, 1H), 2.24 (s, 3H). 13C NMR (150
MHz, DMSO-d6): δ 151.98, 151.64, 151.46, 131.86, 130.98,
129.56, 128.80, 127.91, 127.72, 126.03, 122.89, 116.57, 19.67.
HRMS: calcd for C15H14N3O3 (MH+) 284.1030, found
284.1028. Benzyl-3-(N-phenylsuccinimide)-thioether (30). To
a solution of N-phenylmaleimide (2 equiv, 279 mg, 1.610
mmol) in EtOH (2.85 mL)/pH7.0 HEPES buffer (2.85 mL)
was added benzylthiol (1 equiv, 100 uL, 0.805 mmol) in MeCN
(2.85 mL) at room temperature. After 12 h, EtOAc and H2O
were added. The organic layer was separated and the resulting
organic layer was extracted twice with EtOAc. The combined
organic layer was dried over Na2SO4, and concentrated in
vacuo. The residue was precipitated and washed by Et2O/
1
EtOAc to give 30 (140 mg, 58%) as white solids. H NMR
(400 MHz, CDCl3): δ 7.51−7.26 (m, 10H), 4.29 (d, J = 13.6
Hz, 1H), 3.90 (d, J = 13.6 Hz, 1H), 3.64 (dd, J = 3.7, 9.3 Hz,
1H), 3.16 (dd, J = 9.3, 18.9 Hz, 1H), 2.58 (dd, J = 3.7, 18.9 Hz,
1H).
Peptide Modification. Labeled peptide (11a). To a 2 mM
solution of custom-synthesized peptide 10 (1.82 mL, 3.82
μmol) in 100 mM pH 7.0 NaH2PO4/Na2HPO4 buffer, 100 mM
solution of PTAD 9a (114 μL, 11.5 μmol) in MeCN was added
(9.55 μL × 12 times, interval 1 min.) at room temperature. The
resulting solution was stirred at room temperature for 30 min.
The crude reaction was analyzed directly by ESI-LC/MS at 254
nm UV absorption and corresponding MS. The reaction
mixture was then diluted with MeCN (1.00 mL). The obtained
crude material was purified by reversed phase HPLC (mobile
phase; gradient of MeCN/0.1% TFA water, 30:70 to 50:50 over
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30 min, Rt; 14.5 min, detection; UV 254 nm) to give 11a (4.40
mg, 61%) as white amorphous solid. HRMS: calcd for
C73H93N21O17 (MH+) 1536.7131, found 1536.7125. Reversed
phase HPLC purity 95.1% (mobile phase; gradient of MeCN/
0.1% TFA water, 0:100 to 100:0 over 30 min, Rt; 15.8 min,
detection; UV 254 nm). Labeled peptide (11b). The
compound 11b was prepared from custom-synthesized peptide
10 (5 mg, 3.82 μmol) and 9b (2.99 mg, 11.5 μmol), and was
obtained as white amorphous solid (4.40 mg, 60%). Reverse
phase HPLC conditions for isolation: mobile phase: gradient of
MeCN/0.1% TFA water, 30:70 to 50:50 over 30 min, Rt 15.6
min, detection at UV 254 nm). HRMS: calcd for C72H94N24O17
(MH+) 1567.7301, found 1567.7236. Reversed phase HPLC
purity 91.3% (mobile phase: gradient of MeCN/0.1% TFA
water, 0:100 to 100:0 over 30 min, Rt 15.9 min, detection at
UV 254 nm). Labeled peptide (11c). The compound 11c was
prepared from custom-synthesized peptide 10 (5 mg, 3.82
μmol) and 9c (2.84 mg, 11.5 μmol), and was obtained as white
amorphous solid (4.60 mg, 63%). Reverse phase HPLC
conditions for isolation: mobile phase: gradient of MeCN/
0.1% TFA water, 30:70 to 50:50 over 30 min, Rt 14.8 min,
detection at UV 254 nm). HRMS: calcd for C73H95N21O18
(MH+) 1554.7236, found 1554.7220. Reversed phase HPLC
purity 93.6% (mobile phase: gradient of MeCN/0.1% TFA
water, 0:100 to 100:0 over 30 min, Rt 15.2 min, detection at
UV 254 nm).
and 16c; or BSA: 17a, 17b, and 17c) were recorded by a
Microplate spectrophotometer (Spectra Max Gemini; Molec-
ular Devices) using wavelength of excitation 485 nm and
emission 538 nm.
Pegylation of Chymotrypsinogen A. Synthesis of PEG-
urazole (22): In the 1.5 mL Eppendorf tube were mixed 5k
PEG-alkyne 21 (See SI) (15 μL of 48 mM solution in DMF,
0.72 μmol, 1 equiv) and 1,2,4-triazolidine-3,5-dione azide 2045
(30 μL of 24 mM solution in DMF, 0.72 μmol, 1 equiv)
followed by addition of a small piece of copper wire and copper
sulfate (0.72 μL, 100 mM solution in DI water). The reaction
mixture was vortexed gently and kept at 37 °C for 2 h with
intermittent vortexing. Copper wire was removed and copper
ions were scavenged from the reaction mixture using
“CupriSorb” resin (Seachem) overnight at room temperature.
The Cuprisorb resin was filtered and product polymer was
precipitated out with cold ether, centrifuged, and ether
decanted. The resulting white solid as PEG-urazole (22) was
washed with cold ether two times and dried. Isolated yield 4.0
mg, 95%. MALDI-TOF MWav = 5921. Pegylation of Chymo-
trypsinogen A with PTAD-PEG: To the 0.65 mL Eppendorf tube
containing 5k PEG-PTAD precursor 22 (50 μL, 10 mM
solution in DMF) was added 1,3-dibromo-5,5-dimethylimida-
zolidine-2,4-dione (0.49 μL, 100 mM solution in DMF). The
reaction mixture was vortexed gently and formation of the light
cranberry red color was observed, characteristic for the
presence of desired PTAD reagent. The reagent was kept on
ice and used for the protein modification immediately. To the
1.5 mL Eppendorf tube containing chymotrypsinogen A (MW
25 kDa purchased from ImmunO) (50 μL of 1 mg/mL
solution; 2 nM, 1 equiv) was added freshly prepared 5k PEG-
PTAD solution (2 μL of 10 mM solution in DMF, 20 nM, 10
equiv). The reaction was vortexed briefly and kept at room
temperature for 30 min. The product pegylated chymotrypsi-
nogen A 23 was purified by gel filtration using 7k MWCO Zeba
Spin Desalting column (Pierce) and characterized by MALDI-
TOF and gel electrophoresis.
Sequential Bioconjugation of Albumin Using Three
Orthogonal Chemistries. (Step 1) Reaction of albumin with
dansyl derivative. To a 1.5 mL centrifuge tube were added 300
μL of filtered albumin solution in H2O (pH 6.0) containing 1.5
mg albumin followed by addition of 10 μL of 140 mM 1-ethyl-
3-(3-dimethylaminopropyl) carbodiimide hydrochloride in
H2O (pH 6.0) and 5 μL of 136 mM 11-(dansylamino)
undecanoic acid in DMSO. Reaction tubes were gently shaken
at 250 rpm and incubated for 14.5 h at 37 °C. The reaction
mixtures were applied to Micro Bio-Spin Chromatography
Columns (Bio-Gel P6 Gel, Bio Rad) and exchanged into
̅
nanopure water. The purified albumin construct was charac-
terized by MALDI-TOF MS. The fluorescence signal of the
modified albumins 12 and 13 were recorded by a Microplate
spectrophotometer (Spectra Max Gemini; Molecular Devices)
using wavelength of excitation 320 nm and emission 555 nm.
(Step 2) Reaction of dansyl-albumins with PTAD derivative. The
dansyl-albumin solutions 150 μL were applied to Micro Bio-
Bioconjugation of Herceptin with Aplaviroc-PTAD. To
the 0.65 mL Eppendorf tube containing urazole-Aplaviroc 27
(10 μL, 6 mM solution in DMF) was added 1,3-dibromo-5,5-
dimethylimidazolidine-2,4-dione (10 μL, 6 mM solution in
DMF). The reaction mixture was vortexed gently and
formation of the light cranberry red color was observed,
characteristic for the presence of desired PTAD reagent. The
reagent was kept on ice and used for the protein modification
immediately. To the 0.65 mL Eppendorf tube containing 70 μL
of 1.0 mg/mL Trastuzumab (herceptin) in 100 mM pH 7.4 Na
phosphate buffer was added freshly prepared 3.92 μL of 3 mM
PTAD-Aplaviroc in DMF in 5 aliquots at 2 s intervals (* use
fresh pipet tip for each addition). After 15 min, the resulting
solution was purified by gel filtration using 7k MWC Zeba Spin
Desalting column (Pierce). The concentration of recovered
protein was 0.75 mg/mL (NanoDrop, IgG mode). The increase
of molecular weight of 1376 was observed by MALDI-TOF MS
analysis, that corresponds to an average 1.3 aplaviroc molecules
per molecule of herceptin. Herceptin−apraviroc conjugate (28)
was used for HIV neutralization assay without additional
purification.
Spin Chromatography Columns (Bio-Gel P6 Gel, Bio Rad) to
̅
exchange the buffer to 100 mM Na Phosphate buffer pH 7.4.
The resulting solutions were diluted to 30 μM concentration.
To a 1.5 mL centrifuge tube was added 99 μL of 30 μM of
dansyl-albumin solution followed by addition of 1 μL of 100
mM PTAD solution in MeCN. Reactions were gently shaken
and incubated at 25 °C for 15 min. The reaction mixtures were
applied to Micro Bio-Spin Chromatography Columns (Bio-Gel
P6 Gel, Bio Rad) to exchange the buffer to SSC buffer pH 7.0.
̅
The purified albumin constructs (HSA: 14a, 14b, and 14c; or
BSA: 15a, 15b, and 15c) were characterized by MALDI-TOF
MS. (Step 3) Reaction of dual labeled albumins with fluorescein-5-
maleimide. Labeling with fluorescein-5-maleimide (Thermo
Scientific, Catalog No. 46130) was performed according to
manufacturer’s procedure. The reaction mixtures were applied
HIV Neutralization Assay. Replication-incompetent HIV-1
enveloped pseudovirus was generated by cotransfection of
293T cells with JR-FL HIV-1 Env-expressing plasmid and
pSG3ΔEnv as previously described.60 Serial dilutions of
samples (50 μL) along with wt b12, 2D7, 2G12, and an
to Micro Bio-Spin Chromatography Columns (Bio-Gel P6 Gel,
̅
Bio Rad) to exchange buffer to H2O. The purified albumin
constructs was characterized by MALDI-TOF MS. The
fluorescence signal of the modified albumins (HSA: 16a, 16b,
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Scheme 2. Synthesis of PTAD Derivatives
isotype control antibody, DEN3, were added to TZM-bl target
cells (50 μL) and preincubated for 1 h at 37 °C. Following
incubation 250TCID50 of pseudovirus (100 μL) was added to
each well and incubated at 37 °C. Luciferase reporter gene
expression was evaluated 48 h post infection. The percentage of
virus neutralization at a given antibody concentration was
determined by calculating the reduction in luciferase expression
in the presence of antibody relative to virus-only wells. The
antibody dilution causing 50% reduction (50% inhibitory
concentration [IC50]) was calculated by regression analysis
using GraphPad Prism.
added 100 μL of 1 mg/mL 29 or 30, followed by the incubation
at 37 °C (final volume: 2 mL, final concentration: 50 mg/mL,
DMSO content: 5%). 100 μL aliquots were withdrawn at the
following time points: 3 min, 15 min, 30 min, 1 h, 4 h, 8 h, 24 h,
48 h, 120 h, and 168 h. Each 100 μL aliquot was precipitated by
addition of 600 μL of MeCN. Samples were centrifuged and
600 μL of supernatant were transferred into a new 1.5 mL
Eppendorf tube. The solution was centrifugally evaporated
followed by addition of 100 μL of 2 mg/mL methyl 4-
hydroxybenzoate as a internal standard (IS) for the subsequent
HPLC assay. 80 μL aliquots of supernatants were taken and 40
μL of each sample solution was injected and analyzed by
reverse phase HPLC under optimized conditions [(Analysis
time: 20 min, Flow rate: 1.0 mL/min, Column: Phenomenex
(4.6 × 250), Detection wavelength: 254 nm, Isostatic: 40−80%
MeCN in 1% TFA/H2O), TR = 14.70 min (30), TR = 6.23 min
(IS), TR = 7.40 min (29)].
Chemical Stability Study. Acidic or Basic Conditions.
The solution of compound 29 or 30 (0.0353 mmol) in 10%
HCl (0.5 mL) in MeOH (1.5 mL) and in 10% NaOH (0.5 mL)
in MeOH (1.5 mL) was stirred at room temperature for 12 h,
respectively. Then, EtOAc and water were added. In the case of
basic condition, EtOAc was added after acidification with 10%
HCl up to pH 3. The organic layer was separated and washed
once with water. The resulting aqueous layer was extracted
once with EtOAc. The combined organic layer was dried over
MgSO4, and concentrated in vacuo. The residue was purified by
silica gel chromatography (EtOAc) to recover compounds as
white solids. The recovery of 29 (8.9 mg, 89%) or 30 (9.0 mg,
86%) under acidic conditions and 29 (10.2 mg, quant.) under
basic conditions. 30 decomposed under basic hydrolysis
conditions within 15 min, as monitored by TLC. The
RESULTS AND DISCUSSION
■
In order to develop a versatile family of PTAD based
conjugation chemistries, we have focused on the design,
preparation, and utility of PTAD analogues possessing readily
derivatizable linker arms compatible with widely used
bioorthogonol coupling chemistries; click chemistry, Staudinger
ligation chemistry, and oxime/hydrazone chemistry. The
coupling reaction between ethyl hydrazinecarboxylate 4 and
anilines 5 or 6 was performed by Method A or B (Scheme 2)
depending on the nucleophilicity of aniline. The anilines used
were commercially available or were synthesized from
commercially available compounds by the methods detailed
in Scheme 2. Method A was used in the reaction of 4 with
aniline 5: After activation of 4 by treatment with CDI, aniline 5
was reacted with the activated ester in THF at room
temperature to afford coupling intermediate 15. The reaction
of the less nucleophilic aniline 6 was carried out via Method B.
First, 6 was converted to the corresponding activated ester
using 4-nitrophenyl chloroformate, then reacted with 4 to
1
decomposed compounds were purified and analyzed by H
NMR and LC-MS. Hydrolysis products constituted the yield of
85% (see the structures above).
Stability Study of a Modified p-Cresol in Thermal
Condition. Compound 29 (4.00 mg, 0.0141 mmol) or 30
(4.00 mg, 0.0135 mmol) was heated at 120 °C for 1 h
according to literature.56 The recovery amounts were both 4.00
mg (quant.). No decomposition has been detected by 1H
NMR. 3 Physiological stability in human blood plasma:61 To the 2
mL Eppendorf tube containing 1900 μL of fresh whole human
blood plasma (Normal Blood Donor Program at TSRI) was
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afford coupling intermediate 7 in THF at room temperature.
Obtained intermediate 7 was cyclized in the presence of K2CO3
in MeOH under reflux without isolation. Finally triazolidine 8
was converted to desired triazole 9 by oxidization of the N−N
single bond to an N−N double bond with 1,3-diboromo-5,5-
dimethylhydantoin 10 according to a literature procedure.57
Side products derived from 10 and unreacted starting material
were removed by scavenging with silica sulfuric acid (SiO2−
OS3H), because PTAD products 9 were unstabile during silica-
gel column chromatography. The oxidation reaction was readily
monitored by observing the change in the reaction mixture
from colorless to deep red. Reagents, 9a, 9b, 9c, and 9d were
obtained as solids or oils but solutions were relatively unstabile.
The products 9e and 9f were unstabile to isolation at room
reaction with electronically poor reagent 9f. The reagents
substituted with electronically rich moieties, 9a, 9b, and 9c,
efficiently modified 1. These results suggest that an electron
donating substitution on the phenyl ring provides stability in
water without compromising reactivity toward the phenolic side
chain of tyrosine.
In order to explore the potential of the tyrosine click reaction
using these reagents for labeling of more complicated targets,
we synthesized dodecapeptide 10, H2N-VWSQKRHFGY-
CO2H, which has a tyrosine at the C-terminus and contains
the potentially reactive amino acids Trp, Ser, Glu, Lys, Arg, and
His (Scheme 3). Because this peptide is designed to present
amino acids with potentially reactive and competing side-
chains, it serves as a stringent test of the chemoselectivity of this
reaction. The reaction was performed using 3.0 equiv of PTAD
or PTAD derivative in 6% MeCN/phosphate buffer (pH 7) at
room temperature. After purification using reversed-phase
HPLC, labeled peptides were obtained in approximately 60%
yield (Scheme 6). Significantly, a single Tyr modified
compound was observed in each reaction by LC-MS
(Supporting Information) and HRMS and MS/MS data
indicated tyrosine-selective modification. In MS/MS analyses,
products obtained using all tested PTAD analogues showed
similar fragmentation patterns and all daughter ions contained
the ions of modified tyrosine peptide (Supporting Informa-
tion). In our initial report of this reaction,45 we had shown that
both Trp and Lys can react, albeit inefficiently, with PTAD in
50% MeCN/phosphate buffer (pH 7) at room temperature
when studied in isolation. However, when N-acyl tyrosine
methyl amide 1 was mixed with the corresponding Trp and Lys
amino acid derivatives and reacted with PTAD under these
conditions, only Tyr modification was observed. Other
competitive labeling studies provided the same outcome:
selective Tyr labeling (see SI, ref 45). These studies, together
with the study of labeling of peptide 10 (which bears
potentially competing functional groups in the same molecule)
demonstrate here that the highly selective reaction of Tyr with
PTAD derivatives is significantly favored under aqueous
buffered conditions. As we initially reported, labeling with
PTAD derivatives is effective in PBS, 2-amino-2-hydroxymeth-
yl-propane-1,3-diol (Tris), 4-(2-hydroxyethyl)-1-piperazinee-
thanesulfonic acid (HEPES), and a variety of other buffers. In
some cases, for example, where tyrosines of the target
compound or protein are less accessible or reactive, we have
noted that an isocyanate decomposition product of the PTAD
may be formed that is promiscuous in its labeling and that the
products of this type of side reaction are observed. This is the
case for chymotrypsinogen labeling with PTADs (see SI). This
problem, however, is readily solved by using Tris buffer or by
simply adding a small quantity of Tris to form a mixed buffered
solution. The primary amine of the Tris buffer we believe then
acts to scavenge the isocyanate decomposition product of
PTADs to minimize production of the side-reaction product
(see Supporting Information).
1
temperature. Purity of products was confirmed by H NMR.
The reactivity of the PTAD analogues was evaluated in
reaction with N-acyl tyrosine methyl amide 1, and results are
shown in Table 1. All reactions were complete in less than 5
min based on color change; however, the reaction products
were characterized after a more extended reaction time of 30
min. Electronically neutral reagents 9d and 9e gave the same
results as PTAD with comparable percent conversions and
without generation of side products. In contrast, significant
amounts of uncharacterized side products were generated in the
Table 1. Reactivity of PTAD Derivatives with N-Acyl
Tyrosine Methyl Amide 1
In our earlier study, we established the efficiency of protein
labeling using a PTAD-rhodamine dye derivative and found
that bovine serum albumin (BSA) was efficiently labeled in
buffered solutions containing minimal organic cosolvent at pH
ranging from 2 to 10 with 37−98% labeling efficiency (see ref
45, SI). To explore the potential of the tyrosine click for protein
multifunctionalizations, here we studied trifunctionalization at
tyrosine, cysteine, and lysine residues of bovine serum albumin
(BSA) and human serum albumin (HSA). BSA contains 60
a
Reactions were performed in 100 mM NaH2PO4−Na2HPO4 (pH 7)/
b
MeCN (1:1) at room temperature. Percent conversion was
c
1
determined by H NMR. 0.5 M PTAD in MeCN mixture solution.
d
Reaction conditions were 100 mM NaH2PO4−Na2HPO4 (pH 7)/
MeCN (1:1.5).
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Scheme 3. Tyrosine Click Reaction of Designed Decapeptide with PTAD Derivatives to Test the Chemoselectivity and
Efficiency of the Reaction
Scheme 4. Triple-Labeling of Albumins at Lysine, Cysteine, and Tyrosine
lysines, 21 tyrosines, and 35 cysteines, whereas HSA has 55
lysines, 19 tyrosines, and 35 cysteines. Cysteine and lysine were
modified with a fluorescein maleimide and 11-(dansylamino)
undecanoic acid, respectively (Scheme 4). Labeling of albumins
at lysine was performed using 50 equiv of 11-(dansylamino)
undecanoic acid and 100 equiv of N-[3-(dimethylamino)-
propyl]-N′-ethylcarbodiimide hydrocholide (EDC HCl) in
water at 37 °C for 14.5 h. After the gel filtration, MALDI
TOF analysis revealed that the modified HSA (12) and
modified BSA (13) had 3.8 and 4.1 dansyl residues, respectively
(Table 2). Next, tyrosines were reacted in 1% MeCN/100 mM
phosphate buffer (pH 7.4) using 30 equiv of PTAD derivatives
9a, 9b, and 9c gave products with 4 to 8 modified residues
(Table 2). Final labeling at cysteine in SSC buffer (pH 7.0) was
achieved using 1 mM fluorescein-5-maleimide in DMSO at
room temperature for 2 h (Table 2). As noted in Table 2, each
of the desired labels could be efficiently installed onto the target
proteins. Fluorescence properties and molecular masses of the
modified BSA and HSA are given in the Supporting
Information. Thus, PTAD derived reagents bearing bioorthog-
onal alkyne, azide, and ketone groups were readily prepared and
efficiently modified small molecules, peptides, and proteins that
can subsequently be further functionalized using click
H
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precursor reagent 22 (Scheme 5, see details in Supporting
Information). We used this reagent to study the modification of
Chymotrypsinogen A, which contains four tyrosine and
fourteen lysine residues. Reactions were performed in buffer
(pH 7.4) with 10 equiv of freshly oxidized PEG-PTAD or
freshly dissolved PEG-NHS. Reactions were kept at room
temperature for one hour with intermittent mixing. Excess
reagents were removed using 7-kDa MWCO Zeba Spin
Desalting columns, and the products were characterized by
gel electrophoresis and MALDI-TOF MS (Supporting
Information). We observed formation of mono-, bis-, tri-, and
tetra-PEG addition products upon NHS conjugation and
predominant formation of mono-PEG addition products
upon PTAD conjugation. We have noted low reactivity of the
chymotrypsinogen tyrosine residues accounting for the limited
labeling of this protein (Supporting Information). Starting
material was not completely consumed in either reaction but
could be recovered and recycled.
Table 2. Triple-Labeling of BSA and HSA through Three
Different Bioconjugation Chemistries
a
estimated
number of
available
number of
residues
labeled by
amidation
number of
residues
labeled by
tyrosine-click
number of
residues labeled
by Michael
reaction
protein
HSA
AAs/total
Lys 30/55
Tyr 15/19
Cys 1/35
Lys 35/60
Tyr 15/21
Cys 20/35
4.1 (12)
6.7 (14b)
4.6 (14c)
3.8 (13)
7.4 (15b)
3.9 (15c)
6.5 (14a)
1.2 (16b)
1.6 (16c)
8.3 (15a)
2.3 (17b)
2.7 (17c)
1.8 (16a)
BSA
1.3 (17a)
a
Compounds numbers are shown in parentheses. Estimated numbers
of surface accessible amino acids (AAs) from ThermoScientific or
estimated by PyMOL v 1.5.
chemistries and other well established bioorthogonal reaction
chemistries.
Because of the significance of antibody−drug conjugates in
cancer and other therapeutic applications,64−66 we studied the
conjugation of the CCR5 antagonist aplaviroc (24) with a
monoclonal antibody. As a model monoclonal antibody, we
used the well-characterized antibody trastuzumab.67 Aplaviroc
was prepared as we previously reported.58 The carboxylic acid
moiety of aplaviroc was condensed with an alkyne linker (25)
to give aplaviroc having alkyne moiety (26), and then the click
reaction with azide-urazole (9c) gave the PTAD-aplaviroc
precursor (27). This precursor was oxidized to produce the
PTAD moiety, then immediately used for labeling of
trastuzumab in 0.1 M phosphate buffer (pH 7) at room
temperature. After the removal of the small molecule by gel
One of the most important protein conjugation reactions in
the pharmaceutical industry concerns the introduction of
poly(ethylene glycol) chains onto proteins (PEGylation) to
modify their pharmacokinetic properties.62,63 Protein PEGyla-
tion is most commonly employed using maleimide- or N-
hydroxysuccinimide-based PEGylation reagents. The high
abundance of the lysine moieties on protein surfaces often
results in generation of multiple PEG addition products and
necessitates complicated separation procedures. Conjugation at
cysteine residues typically requires the introduction of surface
accessible cysteine residues by mutation of the parental protein
sequence. To explore the potential of the tyrosine click reaction
for protein PEGylation we prepared a 5-kDa PEG-PTAD
Scheme 5. Protein PEGylation with a Linear PEG5000 PTAD Reagent
I
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Scheme 6. Tyrosine Click Bioconjugation of Trastuzumab Antibody with a Small Molecule HIV Entry Inhibitor
filtration, a product with a single aplaviroc was observed by
MALDI-TOF MS spectrum (Supporting Information).
conjugates and supports a chemical approach to multispecific
antibodies.68,69
In order to study the bioactivity of conjugated 28, HIV
neutralization activity of the aplaviroc−trastuzumab conjugate
(28) was tested in TZM-bl cells expressing CCR5 and clade B
pseudovirus JR-FL as shown in Figure 2. The IC50 value of
Key to many bioconjugation chemistries is the stability of the
designed linkage. To further stability of the tyrosine click
reaction in bioconjugation chemistry, we studied the stability of
C−N linkage installed using PTAD reagents in comparison
with the more commonly utilized C−S linkage provided by
maleimide coupling chemistry (Scheme 7). As noted in our
original communication on the PTAD adduct to p-cresol (29),
it is stabile to acidic or basic conditions at room temperature for
24 h or at 120 °C for 1 h. On the other hand, 30, containing an
S−C bond, was stabile in acidic conditions, but was hydrolyzed
within 5 min in basic condition, although the S−C bond was
not cleaved. 30 demonstrated a thermal stability similar to that
of 29 after heat treatment for 1 h. This study suggests that the
1,2,4-triazolidine-3,5-dione linkage is hydrolytically and ther-
mally stabile, whereas the maleimide linkage is unstabile in
basic conditions. Next, stability was evaluated in human blood
plasma in anticipation of the use of the tyrosine click reaction in
protein conjugates, specifically antibody−drug conjugates
(ADCs).70 For this purpose, we studied the stability of 29
and 30 by incubation in fresh human blood plasma at 37 °C for
one week (Figure 3). At various time points the reaction was
quenched by precipitation with MeCN and analyzed by
reversed-phase HPLC (Supporting Information). Compound
29 was found to be completely stabile over the course of the 7-
day experiment, while 30 decomposed after hours. This data
demonstrates that the Tyr click linkage is significantly more
Figure 2. JR-FL HIV-1 pseudovirus neutralization assay: aplaviroc 24
■
▲
(•), trastuzumab ( ) and aplaviroc−trastuzamab 28 ( ).
aplaviroc−trastuzumab conjugate 28 was 11.3 nM; trastuzamab
alone had no neutralization activity. Interestingly, this value was
very close to that of aplaviroc (24) alone (5.6 nM) indicating
that the tyrosine click conjugation chemistry did not negatively
impact the activity of the drug. No significant loss in
trastuzumab binding was noted as determined by ELISA.
This study and our earlier study45 concerning the bioconjuga-
tion of a peptide to trastuzumab indicates that the tyrosine click
reaction is applicable the preparation of antibody−drug
Scheme 7. Linkage Stability Study
J
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AUTHOR INFORMATION
Corresponding Author
*E-mail: carlos@scripps.edu.
■
Author Contributions
‡H.B. and M.N. contributed equally.
Funding
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported by National Institutes of Health
grants Pioneer Award DP1 CA174426, RO1 AI095038, and
The Skaggs Institute for Chemical Biology. We thank Prof.
Dennis Burton and Angelica Cuevas for HIV-1 assays (The
Scripps Research Institute). M.N. thanks The Uehara Memorial
Foundation for a postdoctoral fellowship for research abroad.
We thank Dr. Qi-Yang Hu for discussion.
Figure 3. Stability in human plasma at various time points. Peak Area
is from HPLC. IS: internal standard; Ethyl 4-hydroxy benzoate.
stabile in human blood plasma than the maleimide linkage and
is consistent with reports that protein conjugates prepared with
maleimide undergo maleimide exchange with reactive thiols in
albumin, free cysteine, or glutathione.71,72 Furthermore, thiol-
maleimide is prone to oxidation, and this facilitates the retro-
Michael reaction and subsequent decomposition.73 In view of
our studies here and reports concerning maleimide based
linkages, the tyrosine click reaction provides a more robust
linkage for bioconjugation than maleimide based connections.
ABBREVIATIONS
CCR5, CC chemokine receptor 5; PEG, poly(ethylene glycol)
■
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CONCLUSIONS AND IMPLICATIONS
■
The studies described herein indicate that the tyrosine click
reaction of PTAD derivatives is a highly efficient and
chemoselective strategy for small molecule, peptide, and
protein conjugation. The reactions of PTAD and designed
derivatives developed here were selective for the phenolic side
chain of tyrosine residues and proceeded in buffered aqueous
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ASSOCIATED CONTENT
* Supporting Information
Full experimental procedures and characterization data are
available for all new compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
■
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