Anaerobic conditions to reduce oxidation of proteins and to accelerate the copper-catalyzed "click" reaction with a water-soluble bis(triazole) ligand

Article abstract of DOI:10.1039/c0cc05376g

Oxidation of protein (bovine albumin serum) by air still occurred under the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction conditions even in the presence of a Cu(i)-stabilizing tris(triazole) ligand. Anaerobic conditions not only avoided th

Full text of DOI:10.1039/c0cc05376g

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2011, 47, 3186–3188
www.rsc.org/chemcomm
COMMUNICATION
Anaerobic conditions to reduce oxidation of proteins and to accelerate
the copper-catalyzed ‘‘Click’’ reaction with a water-soluble bis(triazole)
ligandw
Amit Kumar,a King Lib and Chengzhi Cai*a
Received 5th December 2010, Accepted 12th January 2011
DOI: 10.1039/c0cc05376g
Oxidation of protein (bovine albumin serum) by air still occurred
under the copper-catalyzed azide–alkyne cycloaddition (CuAAC)
reaction conditions even in the presence of a Cu(I)-stabilizing
tris(triazole) ligand. Anaerobic conditions not only avoided the
oxidation of the protein, but also greatly accelerated the CuAAC
reaction using a water-soluble bis(triazole) Cu(I) ligand.
to the risk of explosion. In this work, we investigated a series
of water-soluble ligands 1c and 4–6 containing 1–3 triazole
moieties. While ligand 1c was prepared in 3 steps (see ESIw),
the ligands 4–6 were readily prepared in one step from the
commercially available starting materials 7 and 8 in yields
ranging from 65–91% (see ESIw). As an example, the synthesis
of 4 is shown in Scheme 1.
A modified fluorogenic assay10 was used to evaluate the
ligands under various reaction conditions (Scheme 2). We
introduced an oligo(ethylene glycol) (OEG) chain in the
conjugated alkyne 9 to render it water soluble. Upon CuAAC
reaction of the non-fluorescent alkyne 9 with an azide, e.g. 8, a
highly fluorescent, conjugated triazole product, e.g. 10, was
formed, which allowed for convenient monitoring of the
reaction and estimating the yields by fluorescence measure-
ment (see ESIw). To facilitate the comparison of efficiency of
the ligands, we purposefully chose the slower reaction between
8 and 910 to form the fluorescent product 10, instead of the
rapid reaction between an alkyne and the highly reactive
conjugated azide that could be completed within 15 min.9
The copper-catalyzed azide–alkyne cycloaddition (CuAAC)
reaction1–4 and its copper-free, strained-alkyne variant5 is
one of the most widely used methods for bioconjugation.4
The former in general is faster, and does not require the
strained alkynes (some of them are commercially available
but costly). For the CuAAC reaction in air, sodium ascorbate
is commonly used to maintain the catalytically active
Cu(I) oxidation state.2 While this method is practical, the
Cu(I)/Cu(II)/ascorbate/O2 redox system generates reactive
oxy species and electrophiles that are harmful to many biological
systems.6 Finn and co-workers recently recommended general
conditions for using water-soluble tris-triazole derivatives as
co-catalysts that not only greatly accelerated the reaction but
also served as a sacrificial reductant to reduce the oxidative
damage.6 In this work, we found that these conditions still
could not eliminate the rapid oxidation of bovine serum
albumin (BSA) as a model protein. Therefore, for oxidatively
labile bio-systems, the reaction may require anaerobic conditions.
We show that a water-soluble bis(triazole) Cu(I) ligand is
among to date the most efficient co-catalysts for the CuAAC
reaction under anaerobic conditions.
A rapid reaction is highly desirable for bioconjugation.
Many Cu(I)-ligands have been reported to accelerate the
CuAAC reaction, in particular the tris-triazole-based ligands
1a,7 and its water-soluble analogues 1b6 and 2,7 and the
bathophenanthroline 3.8 The preparation of 2 in pure form
required four steps with a low overall yield,9 using a low
molecular weight azide that is not commercially available due
Scheme 1 One-step synthesis of the ligand 4.
a Department of Chemistry, University of Houston, Houston
TX 77204, USA. E-mail: cai@uh.edu; Fax: +1 713 743 2709;
Tel: +1 713 743 2710
b Department of Radiology, The Methodist Hospital Research
Institute, Houston, TX 77030, USA
w Electronic supplementary information (ESI) available: Experimental
procedures for synthesis and characterization. See DOI: 10.1039/
c0cc05376g
Scheme 2 Using the fluorogenic probes for optimization of the
CuAAC reaction conditions.
c
3186 Chem. Commun., 2011, 47, 3186–3188
This journal is The Royal Society of Chemistry 2011

View Article Online
Although the bis(triazole) and the mono(triazole) ligands 5
and 6 greatly accelerate the Cu(I)/Cu(II) redox cycle in the
presence of ascorbate and O2, Fokin and co-workers reported
that these ligands also speeded up the CuAAC reaction in
oxygen-free conditions.7 We compared the tris-, bis- and
mono(triazole) ligands (1c, 4, 5 and 6) as well as the water-
soluble bathophenanthroline 38 for promoting the CuAAC
reaction under oxygen-free conditions. The reaction was per-
formed with 8 (1 mM) and 9 (0.5 mM) in the presence of
sodium ascorbate (125 mM), CuSO4 (125 mM) and the ligand
with a Cu/ligand ratio of 1 : 2 in 50 mM HEPES buffer,
pH 7.2. To eliminate possible errors of quantification using
fluorescence, the yields of the reaction at various durations
were quantified by HPLC (see ESIw) and plotted in Fig. 1d.
The results showed that the rate of the reaction remained low
in the presence of the mono(triazole) ligand 6. Also, the
absence of O2 did not improve the efficiency of the TBTA
analogue 1c, attributable to its excellent stabilization of Cu(I)
against oxidation.7 However, the less hindered tris(triazole)
ligand 4 performed better under anaerobic conditions, and
was even more efficient than the TBTA analogue 1c at the
beginning of the reaction. This result indicates that the slight
decrease of efficiency of 4 than 1c in air was likely due to the
faster oxidation of the less hindered 4–Cu(I) complex than the
1c–Cu(I) complex.
Fig. 1 Yields vs. time profiles of the CuAAC reaction of 8 (1 mM)
and 9 (0.5 mM) with CuSO4 (125 mM), Cu/ligand ratio of 1 : 2 in
50 mM HEPES buffer, pH 7.2, in air in the presence of (a) ligand 1c,
(b) ligands 4, (c) ligand 5, and various ascorbic acid concentrations,
and (d) under anaerobic conditions with 1.25 mM ascorbate and
ligands 1c, 3, 4, 5, and 6.
The most efficient co-catalyst for the reaction performed in
an oxygen-free environment was the bis(triazole) ligand 5; the
reaction was completed in 40 min under the above conditions
(Fig. 1d). This ligand was also more efficient than the batho-
phenanthroline 3, previously reported to be the most efficient
co-catalyst for the CuAAC reaction in aqueous solution under
anaerobic conditions.8 Therefore, among the ligands 3–6, the
bis(triazole) ligand 5 has the best combination of electronic
and steric effects for binding the metal with the alkyne and
azide leading to triazole formation. We note that despite the
inconvenience, running the CuAAC reaction in oxygen-free
conditions does bring the following advantages: (1) elimi-
nating the formation of reactive by-products harmful to the
biological system (see below), (2) maintaining the catalytically
active Cu(I) state, and (3) greatly accelerating the reaction
using the most efficient (but oxygen-sensitive) catalyst, such as
Cu(I)/5.
When the reaction of 8 (1 mM) and 9 (0.5 mM) was
performed in air in the presence of CuSO4 (0.125 mM) and
the ligand 1c or 411 (0.25 mM), the rate was not affected by the
ascorbate concentration in the range of 1–5 mM (Fig. 1a and b),
indicating a good stabilization of the Cu(I) oxidation state by
these tris(triazole)methyl-amine ligands that bind Cu(I) in the
tetrahedral configuration. Indeed, Fokin and co-workers
showed that a similar ligand (2) largely increased the redox
potential of Cu(I)/Cu(II) by B300 mV and also decreased the
electron transfer kinetics.7 The most efficient ligand we tested
for the reaction in air was the OEG-modified TBTA (1c),
completing in 4 h in the presence of only 1.25 mM ascorbate.
The tris(triazole) ligand 4 without the benzyl substituent was
less efficient in air, but easily prepared by a one step reaction
(Scheme 1). On the other hand, the mono- and bis(triazole)
ligands 5 and 6 performed poorly in air, even with a large
excess of ascorbate (e.g. Fig. 1c). These ligands greatly pro-
moted the oxidation of Cu(I) to Cu(II) in air, as indicated by
the rapid color change of the solution from colorless to blue.
The high oxygen sensitivity of Cu(I) complexes with 5 and 6
may be attributed to the stronger electron-donating and less
steric hindrance of these ligands as compared to the tristriazole
ligand 1. The resultant Cu(II) species can be reduced back
to Cu(I) by ascorbate in the reaction system, but at the expense
of generating reactive oxy species (radicals, peroxides and
superoxides) and strong electrophiles that are harmful to the
biological systems and to the ligand itself.6 In addition to rapid
consumption of the excess of ascorbate, the oxidation of Cu(I)
by O2, which was greatly accelerated by the ligand 5 or 6, also
led to a decrease of the effective concentration of Cu(I),
depending on the rates of the redox reactions, thereby decreasing
the rate of the CuAAC reaction.
To evaluate the extent of oxidation in oxidatively labile
biological systems during the CuAAC reaction under various
conditions, we used an amino-rich protein, bovine albumin
serum (BSA), as a model. The formation of carbonyl groups in
proteins has been widely used as a marker of oxidative damage
of proteins, e.g. during oxidative stress in biological systems.12
We used an enzymatic assay kit (OxiSelectt protein carbonyl
ELISA, Cell Biolabs Inc.)13 to estimate the amount of carbonyl
groups generated on BSA after subjecting it to CuAAC
reaction conditions, as detailed in ESI.w Briefly, the BSA
samples were immobilized and treated with 2,4-dinitro-
phenyl-hydrazine (DNPH) that reacted with the active carbonyl
groups on the protein. The resultant hydrazone was quantified
by binding with the anti-DNP antibody followed by the horse-
radish peroxidase conjugated secondary antibody that catalyzed
a colorimetric reaction (see ESIw). A calibration curve correlating
the absorbance (generated by the colorimetric reaction)
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 3186–3188 3187

View Article Online
well as the Cu(I) stabilizing ligand 4 (condition b, Fig. 2), the
detectable carbonyl groups (excluding those reacted with AG)
on BSA were still increased by 30% within 10 min exposure to
air. Therefore, for oxidatively labile biosystems, it may be
necessary to perform the current CuAAC reaction under
oxygen-free conditions to eliminate oxidation.18 Indeed, much
less oxidation was found under our ‘‘oxygen-free’’ conditions
(condition c, Fig. 2) even in the presence of the bis(triazole)
ligand 5 that greatly accelerates the oxidation. The observed
low degree of oxidation (8% after 1 hour and 17% after 2
hours) was likely due to the presence of trace amounts of
oxygen in our anaerobic chamber, and perhaps also due to the
incomplete removal of copper species before exposing the
samples to air.
Fig. 2 The normalized amount of carbonyl generated on BSA after
being subjected to various CuAAC conditions (a–d) in an aerobic or
anaerobic environment for various durations. A fully reduced BSA
standard (RB, without carbonyl) and an oxidized BSA standard (OB,
with B0.5 carbonyl per BSA molecule) were used for the normalization.
RB (0.5 mg mLÀ1) was used for the following conditions: (a) 0.1 mM
CuSO4, 2.5 mM Na-ascorbate, 0.5 mM 4 in 0.1 M PBS buffer (pH 7.0);
(b) 0.1 mM CuSO4, 2.5 mM Na-ascorbate, 5 mM amino guanidine
(AG) and 0.5 mM 4 in 0.1 M PBS buffer/DMSO 95 : 5 (pH 7.0); (c)
0.125 mM CuSO4, 1.25 mM Na-ascorbate, 0.25 mM 5 in 50 mM
HEPES buffer (pH 7.2). Each data point was the mean of 3 replicates
and the error bar represented the standard deviation.
In conclusion, we show that a rapid oxidation of oxidatively
labile proteins may still occur even when a tris(triazole) ligand
is used to stabilize the Cu(I) oxidation state for the CuAAC
reaction performed in air. In such cases, the CuAAC reaction
and the subsequent removal of copper and ascorbate can be
performed under oxygen-free conditions. Although the operation
is not convenient, it brings the benefit of a large rate enhance-
ment by using the bis(triazole) ligand 5 and the significant
reduction of oxidative degradation of the biological and materials
systems highly sensitive to oxidation.
We thank the Welch Foundation (grant E-1498), National
Science Foundation (grant DMR-0706627), the Institute of
Biomedical Imaging Science and the National Institutes of
Health (grant EY018303) for support of this work.
with the amount of carbonyl on BSA was obtained using a
series of BSA standards (Cell Biolabs Inc.), including the
reduced BSA (RB, containing nearly no active carbonyl
groups), partially oxidized BSA, and the fully oxidized BSA
(OB, containing B7.5 nmol carbonyl per mg, corresponding
to B0.5 carbonyl per BSA). The calibration curve was linear
from 0–0.5 carbonyl per BSA (see ESIw). The reduced BSA
standard (RB) was then subjected to various CuAAC reaction
conditions. After rapid removal of the copper and other
reagents by centrifuge through size exclusion columns, the
samples were immediately subjected to the enzymatic assay.
The relative amount of carbonyl generated on the BSA was
represented by the normalized absorbance of the sample
against a reduced BSA (RB, without carbonyl, Cell Biolabs
Inc.) and an oxidized BSA (OB, with B0.5 carbonyl per BSA
molecule). The results are summarized in Fig. 2.
Notes and references
1 C. W. Tornoe, C. Christensen and M. Meldal, J. Org. Chem., 2002,
67, 3057.
2 V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B. Sharpless,
Angew. Chem., Int. Ed., 2002, 41, 2596.
3 (a) H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem.,
Int. Ed., 2001, 40, 2004; (b) J. E. Hein and V. V. Fokin, Chem. Soc.
Rev., 2010, 39, 1302.
4 M. A. Gauthier and H.-A. Klok, Chem. Commun., 2008, 2591.
5 J. C. Jewett and C. R. Bertozzi, Chem. Soc. Rev., 2010, 39, 1272.
6 V. Hong, A. K. Udit, R. A. Evans and M. G. Finn, ChemBioChem,
2008, 9, 1481.
7 T. R. Chan, R. Hilgraf, K. B. Sharpless and V. V. Fokin, Org.
Lett., 2004, 6, 2853.
Under similar conditions (condition a, Fig. 2) reported by
Hong et al.6 without the use of amino guanidine (AG) and
DMSO, over 30% increase of carbonyl groups on BSA
occurred in air within 1 minute. The same conditions led to
66% increase of carbonyl groups in 10 minutes. When AG and
DMSO were added (condition b, Fig. 2), the increase of
carbonyl groups was dropped to 30% after 10 minutes. In a
control experiment, treatment of the oxidized BSA standard
(OB) with 5 mM amino guanidine (AG) in 0.1 M PBS buffer
(pH 7.0) for 2 h resulted in a 40% decrease of the carbonyl
groups. This result can be attributed to the condensation of
AG with the reactive carbonyl groups on the oxidized
BSA.14,15 It suggests that AG can be used to reduce cross-
linking of the oxidized protein causing aggregation. In addition,
AG and DMSO might also function as a radical scavenger.16,17
However, even in the presence of 5 mM AG and 5% DMSO as
8 W. G. Lewis, F. G. Magallon, V. V. Fokin and M. G. Finn, J. Am.
Chem. Soc., 2004, 126, 9152.
9 V. Hong, S. I. Presolski, C. Ma and M. G. Finn, Angew. Chem.,
Int. Ed., 2009, 48, 9879.
10 M. Sawa, T. L. Hsu, T. Itoh, M. Sugiyama, S. R. Hanson,
P. K. Vogt and C. H. Wong, Proc. Natl. Acad. Sci. U. S. A.,
2006, 103, 12371.
11 A. Kumar, U. J. Erasquin, G. T. Qin, K. Li and C. Z. Cai, Chem.
Commun., 2010, 46, 5746.
12 E. R. Stadtman and R. L. Levine, Amino Acids, 2003, 25, 207.
13 H. Buss, T. P. Chan, K. B. Sluis, N. M. Domigan and
C. C. Winterbourn, Free Radical Biol. Med., 1997, 23, 361.
14 S. Picard, S. Parthasarathy, J. Fruebis and J. L. Witztum, Proc.
Natl. Acad. Sci. U. S. A., 1992, 89, 6876.
15 Y. AlAbed and R. Bucala, Chem. Res. Toxicol., 1997, 10, 875.
16 M. Kazachkov, K. Chen, S. Babiy and P. H. Yu, J. Pharmacol.
Exp. Ther., 2007, 322, 1201.
17 C. Courderot-Masuyer, F. Dalloz, V. Maupoil and L. Rochette,
Fundam. Clin. Pharmacol., 1999, 13, 535.
18 B. C. Bundy and J. R. Swartz, Bioconjugate Chem., 2010, 21, 255.
c
3188 Chem. Commun., 2011, 47, 3186–3188
This journal is The Royal Society of Chemistry 2011