A highly efficient oxidative condensation reaction for selective protein conjugation

Article abstract of DOI:10.1039/c4cc01551g

We hereby report a mild and efficient coupling reaction between alkyl aldehydes and aryl diamines. In the presence of a Cu²⁺ or a Zn ²⁺ ion, oxygen (O₂) in air is able to promote the oxidative condensation of the two readily preparable functional groups, forming stable benzimidazole linkages in neutral aqueous solution at room temperature (RT). We demonstrated that the reaction could be utilized to label a T4 lysozyme protein containing a chemically installed aryl diamine group with a fluorescent aldehyde dye molecule at 37 °C. the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc01551g

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
A highly efficient oxidative condensation reaction
for selective protein conjugation†
Cite this: Chem. Commun., 2014,
50, 7469
Ao Ji,‡ Wei Ren‡ and Hui-wang Ai*
Received 28th February 2014,
Accepted 22nd May 2014
DOI: 10.1039/c4cc01551g
www.rsc.org/chemcomm
We hereby report a mild and efficient coupling reaction between alkyl a hydrazide or a hydrazine functional group were utilized to
aldehydes and aryl diamines. In the presence of a Cu2+ or a Zn2+ ion, react with aldehyde.9a,14 However, these conjugation reactions often
oxygen (O2) in air is able to promote the oxidative condensation of the suffer from drawbacks such as the favoring of acidic conditions and
two readily preparable functional groups, forming stable benzimidazole incomplete conversion due to the reversible feature.15 To address the
linkages in neutral aqueous solution at room temperature (RT). We problems, anilines have been developed as nucleophilic catalysts to
demonstrated that the reaction could be utilized to label a T4 lysozyme assist bioconjugation at neutral pH,15b,16 but the general effective-
protein containing a chemically installed aryl diamine group with a ness of these catalysts remains to be investigated. Under some
fluorescent aldehyde dye molecule at 37 8C.
circumstances, aniline catalysts were reported to be neutral or even
detrimental to reaction kinetics and equilibrium.14a,17 Recently, a
In the past few decades, chemical coupling reactions that are modified Pictet–Spengler reaction has been developed:15a,18 small
compatible with common biomolecules have attracted intensive molecules were conjugated to aldehyde-containing proteins using
attention.1 Examples (Table S1, ESI†) include azide–alkyne cycloaddi- the modified reaction to afford stable irreversible linkages, but a
tion,2 thiol–ene coupling reactions,3 the Staudinger–Bertozzi ligation,4 slightly acidic pH range was still required. In this context, we set to
a photoclick tetrazole–alkene cycloaddition,5 the cyanobenzothiazole develop additional aldehyde coupling reactions that can be robustly
condensation,6 and a few other Diels–Alder cycloaddition reactions operated in neutral aqueous solution. Herein we describe our recent
including the ones between tetrazines and ring-strained alkenes or effort in developing an oxidative condensation reaction between
alkynes.7 These reactions have now been widely utilized to assemble alkyl aldehydes and aryl diamines, and employing this highly
organic molecules and molecular libraries,8 label biomolecules efficient and mild coupling reaction to site-specifically label a model
with fluorescent dyes and other biophysical probes,9 and con- protein at the physiological pH and 37 1C.
jugate biomolecules with polyethylene glycols (PEGs), radioactive
The direct condensation of aldehydes and aryl diamines is a
isotopes, toxins or drugs for medical applications.10 Despite the popular route for synthesizing benzimidazoles, a group of hetero-
progress, a further expanded set of bioconjugation reactions is cyclic aromatic compounds known for their antiviral, antimitotic
still in high demand to match diverse applications.1,11 Further- and anthelmintic properties.19 The aryl diamine functional group is
more, new expansion would provide additional, and sometimes unreactive to all natural functional groups in proteins, which is a
better, choices when multiple mutually compatible reactions are favorable property for applications in bioconjugation. However,
employed to modify one or several biomolecules.12
existing condensation methods often require conditions (e.g. organic
Aldehydes may interact with biological nucleophiles such as solvents, strong acids, elevated temperature, or microwave) that
amines and thiols, but the equilibrium in water favors the reverse are too harsh for biomolecular labeling.20 In one example, boric
hydrolytic reaction.13 Due to its small size and the availability of acid was reported to catalyze the condensation between
selective reactions, aldehyde has been utilized as a reactive o-phenylenediamine and aromatic aldehydes in water at RT.21
chemical handle to label biomolecules in vitro and in living cells. However, we failed to obtain the reported benzimidazole products.
In most existing examples, molecules containing an alkoxyamine, Recently, Jiao and coworkers described an elegant 12 hour catalyst-
free condensation of aryl diamines and cyclopropyl aldehydes in
toluene,19b a solvent inappropriate for bioconjugation. To identify
reaction conditions suitable for biomolecular labeling, we examined
Department of Chemistry, University of California Riverside, 501 Big Springs Road,
Riverside, CA 92521, USA. E-mail: huiwang.ai@ucr.edu
† Electronic supplementary information (ESI) available: General methods for
various combinations of aldehydes, diamines and common Lewis
organic synthesis and protein labeling, SDS-PAGE analysis and spectroscopic
acid catalysts in aqueous phosphate buffer (50 mM, pH 7.4). We
were fortunate to find that, in the presence of oxygen (O2) in air and
characterization. See DOI: 10.1039/c4cc01551g
‡ These two authors contributed equally to this work.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 7469--7472 | 7469

View Article Online
Communication
ChemComm
a catalytic amount of Cu2+ or Zn2+ metal ion, aryl diamines and alkyl 10 mM CuSO4 in open air at RT. In a 50 min time period,
aldehydes can react quickly to form corresponding benzimidazoles portions of the reaction mixture were removed and processed
in good yields at RT.
immediately for NMR analysis. A characteristic change in proton
We further explored the substrate scope of this condensation chemical shift of the methoxy group substituted in the aryl ring was
reaction. Alkyl aldehyde seemed to be required. The reaction observed (Fig. 1a). By integrating the areas of the corresponding
between benzaldehyde and o-phenylenediamine gave no benzimid- peaks, we determined the molar concentrations of the product and
azole product (entry 1, Table 1). In comparison, benzimidazoles the reactants at each time point. A second-order kinetics model was
could be synthesized from various aryl diamines and butyraldehyde utilized to fit the data (Fig. 1b), and the rate constant of the
(entries 2–5). In general, electron-donating groups (e.g. –OCH3 and coupling reaction was determined to be 1.61 MÀ1 sÀ1. The rate of
–CH3) substituted on the diamine aryl ring can accelerate the the reaction is comparable to many commonly used bioconjugation
reaction and enhance the yields, while electron-withdrawing groups reactions (Table S1, ESI†).12b,22
(e.g. –NO2 and –COOMe) disfavor the formation of benzimidazoles.
The two catalysts for the coupling reaction, Cu2+ and Zn2+, are
The reaction between 4-methoxybenzene-1,2-diamine and o-phenylene- essential elements for life and have reasonable compatibility
diamine completed nearly-quantitatively in 30 min (entry 4), while with biomolecules.1,23 We next explored the use of the oxidative
4-nitrobenzene-1,2-diamine under the same reaction condition resulted condensation reaction to site-specifically label proteins. We
in no significant amount of benzimidazole over 10 hours (entry 6). The acknowledge that a variety of elegant chemical, enzymatic and
reaction with cyclopropanecarboxaldehyde (entry 7) progressed equally genetic methods may possibly be adapted to introduce alkyl
well as the one with butyraldehyde (entry 3). It is also worth noting that, aldehyde or aryl diamine functional groups into proteins.9a,24
these condensation reactions (entries 2–5 and 7) can be performed in In our model experiment, we used a cysteine–maleimide Michael
phosphate buffer, as well as in neat water and organic solvents such as reaction to chemically install an aryl diamine functional group to
tetrahydrofuran (THF), as long as Cu2+ or Zn2+ is supplemented. Both a T4 lysozyme protein. Briefly, T4 lysozyme containing a single
metal ions and O2 in air are essential for the excellent reaction yields to cysteine at the surface residue 68 (T4L-N68C) was recombinantly
form benzimidazoles (entry 2).
expressed in E. coli. A bifunctional linker molecule containing an
We next investigated the kinetics of the metal ion-catalyzed aromatic diamine and a cysteine-reactive maleimide was chemically
reaction. A mixture of 4-methoxybenzene-1,2-diamine and butyr- synthesized (Scheme S1, ESI†) and utilized to react with T4L-N68C.
aldehyde was stirred in neutral phosphate buffer containing The successful conversion (Fig. 2a and b) was confirmed using
Table 1 Oxidative coupling of aldehydes and aryl diamines in the presence of a Cu2+ or a Zn2+ catalyst (1 mol%)
Molar ratioa
(diamine : aldehyde)
Isolated
Entry
1
R1
H
R3
Reaction time
5 h
Product
Catalyst
yieldb (%)
H
H
Ph
1 : 2
1 : 2
3a
CuSO4
ZnCl2
Air: 0
Air: 0
2
H
n-Propyl
1.5 h
3b
CuSO4
ZnCl2
No catalyst
CuSO4
Air: 84
Air: 66
Air: 15
N2: o5c
3
4
5
6
Me
H
H
n-Propyl
n-Propyl
n-Propyl
n-Propyl
Cyclopropyl
1 : 2
1 : 2
1 : 5
1 : 2
1 : 2
1 h
3c
3d
3e
3f
CuSO4
ZnCl2
Air: 84
Air: 63
Ome
COOMe
NO2
H
30 min
4 h
CuSO4
ZnCl2
Air: 92
Air: 94
H
CuSO4
ZnCl2
Air: 63
Air: 55
H
10 h
1 h
CuSO4
ZnCl2
Air: 0
Air: 0
7
Me
3g
CuSO4
ZnCl2
Air: 81
Air: 67
a
b
Additional volatile aldehydes were used to maximize diamine conversion. Isolated yields were calculated from the moles of the benzimidazole
c
products after column chromatography and the initial moles of aryl diamines. Residual benzimidazole product may form during workup
procedures performed in air.
7470 | Chem. Commun., 2014, 50, 7469--7472
This journal is ©The Royal Society of Chemistry 2014

View Article Online
ChemComm
Communication
Fig. 1 (a) 1H NMR time course showing the conversion of 4-methoxy-
benzene-1,2-diamine to its corresponding benzimidazole. (b) Reaction
kinetics of the formation of the benzimidazole product over time.
Fig. 2 Direct infusion ESI-TOF MS characterization of T4 lysozyme proteins
((a) T4L-N68C; (b) T4L containing an installed aryl diamine functional group;
(c) T4L labeled with an aldehyde Coumarin 343 dye in the presence of 2.5 mM
CuSO4).
electrospray ionization-time of flight mass spectrometry (ESI-
TOF MS). After obtaining the T4L protein with an installed aryl
diamine group, we tested its reaction with an aldehyde-
containing Coumarin 343 dye (Schemes S2 & S3, ESI†). The
protein (50 mM) was incubated with the dye (200 mM) in
phosphate buffer (50 mM, pH 7.4) supplemented with CuSO4
(2.5 mM). The mixture was left in open air at 37 1C. The reaction was
monitored with ESI-TOF MS in a direct infusion mode. It showed
that the protein was quantitatively converted to a fluorescently
labeled product in 2 hours (Fig. 2c). In the control experiment when
T4L-N68C and the aldehyde were directly mixed, no reaction
was observed (Fig. S1a, ESI†). The proteins were also analyzed on
a SDS-PAGE gel (Fig. S2a, ESI†). A fluorescent band was seen for the
Coumarin 343-labeled protein. After removing the excess dye, the
fluorescence of the protein was characterized (Fig. S2b, ESI†).
The excitation and emission spectra matched the cyan fluorescence
of Coumarin 343. Efficient labeling was also observed in the
presence of ZnCl2, but not in the absence of metal ions (Fig. S1
and S2, ESI†). All these results collectively support that the oxidative
condensation reaction is a potent and selective method that can be
utilized for site-specific protein conjugation.
In summary, we have identified a mild condition to couple
aryl diamines and alkyl aldehyde in neutral aqueous solution. The
resulting benzimidazole derivatives are a family of important
bioactive compounds, whose preparation is being actively pursued
by many researchers.19b,25 The one-step transformation reported
here is a very effective way to prepare these compounds. The
coupling reaction has favorable kinetics, excellent conversion and
good biocompatibility. We successfully used it to label a protein
containing a pre-installed aryl diamine functional group. Since the
two coupling functional groups are readily accessible and small in
structure, broad applications of this novel bioconjugation reaction
can be expected. We are currently developing strategies to geneti-
cally encode aldehydes and diamines in living cells, which are
expected to further ease the use of the condensation reaction for
selective protein modification. Because the metal ion concentration
used here is considerably lower than that in previous studies which
reduced Cu2+ for live-cell alkyne–azide click reactions,1,14b,26 an
integration of this condensation reaction with strategies to
introduce the functional groups into biomolecules may permit
site-specific labeling of biomolecules on the surface of or inside
living cells.
We gratefully acknowledge support from the University
of California Riverside. We thank the UCR NMR Facility and
the High Resolution Mass Spectrometry Facility for analysis of
small molecules and proteins.
Notes and references
1 E. M. Sletten and C. R. Bertozzi, Angew. Chem., Int. Ed., 2009, 48,
6974–6998.
2 (a) C. W. Tornoe, C. Christensen and M. Meldal, J. Org. Chem., 2002,
67, 3057–3064; (b) V. V. Rostovtsev, L. G. Green, V. V. Fokin and
K. B. Sharpless, Angew. Chem., Int. Ed., 2002, 41, 2596–2599; (c) J. M.
Baskin, J. A. Prescher, S. T. Laughlin, N. J. Agard, P. V. Chang,
I. A. Miller, A. Lo, J. A. Codelli and C. R. Bertozzi, Proc. Natl. Acad.
Sci. U. S. A., 2007, 104, 16793–16797.
3 C. E. Hoyle and C. N. Bowman, Angew. Chem., Int. Ed., 2010, 49,
1540–1573.
4 E. Saxon and C. R. Bertozzi, Science, 2000, 287, 2007–2010.
5 R. K. Lim and Q. Lin, Acc. Chem. Res., 2011, 44, 828–839.
6 H. Ren, F. Xiao, K. Zhan, Y. P. Kim, H. Xie, Z. Xia and J. Rao, Angew.
Chem., Int. Ed., 2009, 48, 9658–9662.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 7469--7472 | 7471

View Article Online
Communication
ChemComm
7 (a) N. K. Devaraj and R. Weissleder, Acc. Chem. Res., 2011, 44,
816–827; (b) K. Lang, L. Davis, J. Torres-Kolbus, C. Chou, A. Deiters
and J. W. Chin, Nat. Chem., 2012, 4, 298–304; (c) K. Lang, L. Davis,
S. Wallace, M. Mahesh, D. J. Cox, M. L. Blackman, J. M. Fox and
J. W. Chin, J. Am. Chem. Soc., 2012, 134, 10317–10320.
2013, 78, 1184–1189; (c) M. Rashidian, M. M. Mahmoodi, R. Shah,
J. K. Dozier, C. R. Wagner and M. D. Distefano, Bioconjugate Chem.,
2013, 24, 333–342; (d) P. Crisalli and E. T. Kool, Org. Lett., 2013, 15,
1646–1649; (e) Y. Zeng, T. N. Ramya, A. Dirksen, P. E. Dawson and
J. C. Paulson, Nat. Methods, 2009, 6, 207–209.
8 (a) H. C. Kolb and K. B. Sharpless, Drug Discovery Today, 2003, 8, 17 (a) Y. Taga, M. Kusubata, K. Ogawa-Goto and S. Hattori, Mol. Cell.
1128–1137; (b) S. L. Ng, P. Y. Yang, K. Y. Chen, R. Srinivasan and
S. Q. Yao, Org. Biomol. Chem., 2008, 6, 844–847; (c) V. T. Bhat,
A. M. Caniard, T. Luksch, R. Brenk, D. J. Campopiano and
M. F. Greaney, Nat. Chem., 2010, 2, 490–497.
Proteomics, 2012, 11, M111.010397; (b) J. E. Hudak, R. M. Barfield,
G. W. de Hart, P. Grob, E. Nogales, C. R. Bertozzi and D. Rabuka,
Angew. Chem., Int. Ed., 2012, 51, 4161–4165.
18 P. Agarwal, J. van der Weijden, E. M. Sletten, D. Rabuka and
C. R. Bertozzi, Proc. Natl. Acad. Sci. U. S. A., 2013, 110, 46–51.
9 (a) P. Wu, W. Shui, B. L. Carlson, N. Hu, D. Rabuka, J. Lee and
C. R. Bertozzi, Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 3000–3005; 19 (a) M. Alamgir, D. C. Black and N. Kumar, in Bioactive Heterocycles
(b) J. C. Jackson, J. T. Hammill and R. A. Mehl, J. Am. Chem. Soc.,
2007, 129, 1160–1166; (c) V. Muralidharan and T. W. Muir, Nat.
Methods, 2006, 3, 429–438; (d) S. T. Laughlin and C. R. Bertozzi, Nat.
Protocols, 2007, 2, 2930–2944; (e) H. W. Ai, Anal. Bioanal. Chem.,
2012, 403, 2089–2102.
III, ed. M. Khan, Springer, Berlin, Heidelberg, 2007, ch. 88, vol. 9,
pp. 87–118; (b) C. Zhang, L. Zhang and N. Jiao, Green Chem., 2012,
14, 3273–3276; (c) D. A. Horton, G. T. Bourne and M. L. Smythe,
Chem. Rev., 2003, 103, 893–930.
20 (a) L. M. Dudd, E. Venardou, E. Garcia-Verdugo, P. Licence,
A. J. Blake, C. Wilson and M. Poliakoff, Green Chem., 2003, 5,
187–192; (b) J.-P. Wan, S.-F. Gan, J.-M. Wu and Y. Pan, Green Chem.,
2009, 11, 1633–1637; (c) C. Mukhopadhyay and P. K. Tapaswi, Tetra-
hedron Lett., 2008, 49, 6237–6240; (d) K. Bahrami, M. M. Khodaei and
F. Naali, J. Org. Chem., 2008, 73, 6835–6837; (e) Y.-X. Chen, L.-F. Qian,
W. Zhang and B. Han, Angew. Chem., Int. Ed., 2008, 47, 9330–9333.
10 (a) H. Cho, T. Daniel, Y. J. Buechler, D. C. Litzinger, Z. Maio,
A. M. Putnam, V. S. Kraynov, B. C. Sim, S. Bussell, T. Javahishvili,
S. Kaphle, G. Viramontes, M. Ong, S. Chu, G. C. Becky, R. Lieu,
N. Knudsen, P. Castiglioni, T. C. Norman, D. W. Axelrod,
A. R. Hoffman, P. G. Schultz, R. D. DiMarchi and B. E. Kimmel, Proc.
Natl. Acad. Sci. U. S. A., 2011, 108, 9060–9065; (b) C. H. Kim, J. Y. Axup,
A. Dubrovska, S. A. Kazane, B. A. Hutchins, E. D. Wold, V. V. Smider 21 Z. Karimi-Jaberi and M. Amiri, E-J. Chem., 2012, 9, 167–170.
and P. G. Schultz, J. Am. Chem. Soc., 2012, 134, 9918–9921; (c) K. Nwe 22 S. I. Presolski, V. Hong, S.-H. Cho and M. G. Finn, J. Am. Chem. Soc.,
and M. W. Brechbiel, Cancer Biother. Radiopharm., 2009, 24, 289–302.
11 C. R. Bertozzi, Acc. Chem. Res., 2011, 44, 651–653.
12 (a) M. D. Best, Biochemistry, 2009, 48, 6571–6584; (b) R. K. Lim and
Q. Lin, Chem. Commun., 2010, 46, 1589–1600.
2010, 132, 14570–14576.
23 K. M. Dean, Y. Qin and A. E. Palmer, Biochim. Biophys. Acta, 2012,
1823, 1406–1415.
24 (a) M. Rashidian, J. K. Dozier and M. D. Distefano, Bioconjugate
Chem., 2013, 24, 1277–1294; (b) M. Rashidian, J. M. Song, R. E. Pricer
and M. D. Distefano, J. Am. Chem. Soc., 2012, 134, 8455–8467;
(c) M. Rashidian, J. K. Dozier, S. Lenevich and M. D. Distefano,
Chem. Commun., 2010, 46, 8998–9000.
13 W. P. Jencks, J. Am. Chem. Soc., 1959, 81, 475–481.
14 (a) X. Shi, Y. Jung, L. J. Lin, C. Liu, C. Wu, I. K. Cann and T. Ha, Nat.
Methods, 2012, 9, 499–503; (b) J. D. Cohen, P. Zou and A. Y. Ting,
ChemBioChem, 2012, 13, 888–894; (c) L. Yi, H. Sun, Y. W. Wu, G. Triola,
H. Waldmann and R. S. Goody, Angew. Chem., Int. Ed., 2010, 49, 9417–9421. 25 (a) S.-K. Xiang, W. Tan, D.-X. Zhang, X.-L. Tian, C. Feng, B.-Q. Wang,
15 (a) P. Agarwal, R. Kudirka, A. E. Albers, R. M. Barfield, G. W. de Hart,
P. M. Drake, L. C. Jones and D. Rabuka, Bioconjugate Chem., 2013,
24, 846–851; (b) A. Dirksen and P. E. Dawson, Bioconjugate Chem.,
2008, 19, 2543–2548.
16 (a) A. Dirksen, T. M. Hackeng and P. E. Dawson, Angew. Chem., Int.
Ed., 2006, 45, 7581–7584; (b) P. Crisalli and E. T. Kool, J. Org. Chem.,
K.-Q. Zhao, P. Hu and H. Yang, Org. Biomol. Chem., 2013, 11,
7271–7275; (b) T. Xiao, S. Xiong, Y. Xie, X. Dong and L. Zhou, RSC
Adv., 2013, 3, 15592–15595.
26 C. Besanceney-Webler, H. Jiang, T. Zheng, L. Feng, D. Soriano del
Amo, W. Wang, L. M. Klivansky, F. L. Marlow, Y. Liu and P. Wu,
Angew. Chem., Int. Ed., 2011, 50, 8051–8056.
7472 | Chem. Commun., 2014, 50, 7469--7472
This journal is ©The Royal Society of Chemistry 2014