Site-selective three-component reaction for dual-functionalization of peptides

Article abstract of DOI:10.1039/c3cc38673b

A site-selective dual-functionalization of peptides is presented, involving readily available maleimides as well as N-hydroxylamines. The modification proceeds through a three component 1,3-dipolar cycloaddition, forming a stable product. This was exemplified by the one-pot attachment of two molecular imaging moieties to a tumor binding cyclic peptide, and was extended to the conjugation of a DOTA chelator to a 12 kDa protein. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3cc38673b

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
Site-selective three-component reaction for
dual-functionalization of peptides†
Cite this: Chem. Commun., 2013,
49, 1936
a
ab
Henrik K. Munch,z Jakob E. Rasmussen,z Gina Popa,b Jørn B. Christensena and
Received 3rd December 2012,
Accepted 21st January 2013
Knud J. Jensen*a
DOI: 10.1039/c3cc38673b
www.rsc.org/chemcomm
A site-selective dual-functionalization of peptides is presented, attachment of moieties such as a metal ligand, nanoparticle,
involving readily available maleimides as well as N-hydroxyl- and fluorophore to a single multi-functional targeting molecule.
amines. The modification proceeds through a three component A facile ‘one-site multifunction’ chemical strategy would be
1,3-dipolar cycloaddition, forming a stable product. This was beneficial as it would reduce the number of modifications.
exemplified by the one-pot attachment of two molecular imaging
Here we present a one-pot, three-component peptide
moieties to a tumor binding cyclic peptide, and was extended to modification method based on a 1,3-dipolar cycloaddition
the conjugation of a DOTA chelator to a 12 kDa protein.
(DCA) of (1) a functionalized maleimide, (2) an N-hydroxyl-
amine, and (3) a peptide with an oxidizable serine residue at
Site-selective strategies for chemical modification of peptides the N-terminus or potentially other aldehyde moieties, and its
and proteins are essential tools for many areas of research and extension to proteins (Fig. 1). Maleimides are among the most
for the development of biopharmaceuticals, yet they remain a efficient dipolarophiles in DCA.10 Conveniently, the majority
chemical challenge due to the multiplicity of functional groups of labeling compounds commonly attached to proteins and
present and the requirement for mild, aqueous reaction con- peptides are commercially available as maleimides.
ditions. Several innovative methods have been developed to
Nitrones, in turn, are easily obtained from aldehydes and
address these challenges,1 either targeting specific low-abundance primary N-hydroxylamines under aqueous conditions, converting
surface accessible amino acids or through N-terminal modifi- the excellent nucleophile and electrophile into a new reactive
cations.2 Cycloaddition reactions in particular have proven dipole set-up for a subsequent cycloaddition. Nitrones have
versatile for site-selective modifications, due to their practical already been applied in bioorthogonal reactions and with rates
simplicity and general orthogonality to functional groups found comparable to those found for azide-alkyne DCA;5 however,
in most biomolecules, exemplified by their use in Huisgen strained alkynes requiring multi-step syntheses were used as
alkyne–azide3 and Diels–Alder based4 modification methods. dipolarophiles and only a single functionality was added
Other recently reported cycloadditions include the strain although the authors allude to the possibility of dual modifi-
promoted alkyne-nitrone 1,3-dipolar cycloaddition (SPANC),5 as cation. The peptide and protein aldehydes can be obtained by
well as alkyne-diazocarbonyl (SPADC) and -nitrile oxide (SPANOC) initial oxidation with NaIO4 of an N-terminal serine residue to
cycloadditions,6 providing important additional tools for chemo- form a 2-oxoacetamide.11 The chemoselective DCA between the
selective reactions.7
However, these methods aim at introducing a single
functionality at each reaction site. Modifying biomolecules with
multiple functionalities would thus normally require two or
more chemoselective reactions. Recent advances in multimodal
molecular imaging techniques,8 for instance, target a synergistic
combination of multiple imaging modes9 through covalent
a Department of Chemistry, Faculty of Science, University of Copenhagen,
Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark. E-mail: kjj@chem.ku.dk
b The Novo Nordisk Foundation Center for Protein Research, Faculty of Health
Sciences, Blegdamsvej 3b, DK-2200, Copenhagen N, Denmark
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
Fig. 1 An N-terminal serine provides an anchor for attaching multiple function-
alities via a three-component one-pot modification method.
c3cc38673b
‡ These authors contributed equally to this work.
c
1936 Chem. Commun., 2013, 49, 1936--1938
This journal is The Royal Society of Chemistry 2013

View Article Online
Communication
ChemComm
Table
1
Model study of N-terminal one-pot modification with varying
N-hydroxylamines and maleimides
Entry
pH
Peptide
R
R0
T (1C)
Conv. (%)
1a
2a
3b
4
4.5
4.5
4.5
7.0
7.0
4.5
4.5
4.5
4.5
4.5
4.5
1
1
1
1
1
2
2
2
2
2
1
CH3
CH3
CH3
CH3
CH3
Cy
Ph
Bn
CH3
17
18
CH3
CH3
MDCC
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
rt
86
84d
>98
89
50
50
rt
50
37
37
37
37
37
50
5
95d
97
6
7c
8
>98
92
>98
95
9
10
11
Fig. 2 HPLC monitoring of a model 3-component modification. Peptide 2 is
oxidized to 2-oxoacetamide 3, subsequently forming nitrone 4 by addition of
N-methylhydroxylamine. DCA with N-methylmaleimide yields the isoxazolidine
product 5, as well as the byproduct 6 formed by reaction of aniline with excess
maleimide.
>98
3-Component modification of model peptides according to procedure A
b
for 15 hours. a 10 eq. aniline. 5 eq. MDCC (N-(2-(1-maleimidyl)ethyl)-
c
7-(diethylamino)coumarin-3-carboxamide). Additional 6 eq. of phenyl-
d
hydroxyl-amine was added. 2 hours.
nitrone and a functionalized maleimide thus forms a stable giving the desired isoxazolidine product 5 in 58% isolated yield
isoxazolidine core which combines the three components over three steps. This represents a combined yield of two
(Fig. 2).
diastereoisomers formed during the cycloaddition. NMR
We initially studied the reaction on model peptide H-Ser- revealed a 48 : 52 ratio of the two diastereoisomers by integra-
Gln-Trp-Phe-NH2 (1) in acetate buffer at pH 4.5, near the tion of the doublets at 4.92 and 4.88 ppm originating from the
reported optimal pH range for nitrone formation.12 The rate two H5 signals in the formed isoxazolidine rings (ESI†).
limiting step under acidic conditions is the initial nucleophilic
attack forming the carbinolamine,7 suggesting the possibility signals indicates that both diastereoisomers are exo products
to employ nucleophilic catalysis. originating from the re- and si-face cycloaddition between the
The same 8.1 Hz coupling constant of the overlapping H3
A one-pot protocol would require quenching of the iodate maleimide and the nitrone. Varying the amino acid adjacent to
formed as a byproduct in the initial oxidation step, as iodate the N-terminal serine (Glu, Lys, Phe, and Gly) did not noticeably
has been shown to oxidize the hydroxylamine component, influence conversion rates for the condensation nor the DCA
forming undesirable nitroso compounds.5 We also observed (see Table S1, ESI†). However, the chiral environment on a
that the DCA was hampered without this quenching step (data protein may likely induce the preferred formation of one
not shown). This problem has been addressed by other groups isomer.14 Using the maleimide-functionalized fluorophore
by quenching with hydrophobic scavengers such as 4-methoxy- MDCC,15 full conversion to the desired isoxazolidine product
benzenethiol.5 In our hands, this led to the formation of large was obtained at 50 1C (entry 3, Table 1). Expanding our method
amounts of precipitates which necessitated removal by filtra- to incorporate a functionalized nitrone, we employed several
tion. To circumvent this problem, we found that excess iodate commercially available N-substituted hydroxylamines to give
could be successfully reduced by treatment with potassium the corresponding nitrones in good to excellent conversion.
iodide and sodium ascorbate. We observed no interference or A 1,5-hydroxylamino-AEDANS fluorescent reagent (17) was
side-reactions from this reductive work-up. This scavenging prepared from commercially available 1,5-IAEDANS16 by
strategy thus enabled the one-pot formation of the nitrone treatment with NH2OH (see ESI†). Subsequent reaction of the
from the serine peptide by subsequent treatment with resulting peptide-AEDANS nitrone with N-methylmaleimide
N-methylhydroxylamine, using aniline or p-anisidine as the afforded the fluorescent peptide in 95% conversion (entry 10,
nucleophilic catalyst.13 Subsequent DCA with N-methylmaleimide Table 1).
formed the isoxazolidine product in excellent conversion over
three steps (see Table 1).§
This strategy was furthermore applied17 to the peptide
ligation of two short peptide fragments in the N - N0 direction.
The reaction proceeded at room temperature overnight, but Nitrone condensation of a peptide hydroxylamine HO-GQWF-
could be significantly accelerated by gentle heating to 50 1C, NH2 (18) with the oxidized peptide 1 and subsequent DCA at
lowering reaction times to just 2 hours (entries 2 and 3, 50 1C with N-methylmaleimide provided the ligation product in
Table 1).
The reaction proceeded equally well in phosphate buffer at
98% yield (entry 11, Table 1).
The transmembrane glycoprotein integrin avb3 receptor is
pH 7 (entries 4 and 5, Table 1). A second peptide SGYGGFL (2) an attractive target for diagnostic cancer imaging as the protein
was modified according to procedure A (entry 9, Table 1), is overexpressed in several tumor forms such as melanomas,
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 1936--1938 1937

View Article Online
ChemComm
Communication
The wide selection of commercially available maleimides,
functionalized with metal ligands, PEGs, fatty acids, and fluoro-
phores is a distinctive advantage of this strategy. Peptide and
protein aldehydes were conveniently prepared by chemical
oxidation of precursors with an N-terminal Ser; however, they
could potentially also be accessed by a variety of other methods.
The three-component reaction can be used for introduction of
one or two functionalities in a peptide or for ligation of two
peptide chains. We also demonstrated its value for site-specific
protein modification. We envisage this one-pot protocol to have
wide applicability beyond the exemplifications reported here.
Fig. 3 Peptide 20 modified according to procedure A with 1,5-hydroxylamino-
AEDANS and 5 eq. DOTA-maleimide giving product 21, as a model for potential
modifications of targeting peptides.
Notes and references
ovarian and lung carcinoma, neuroblastomas, glioblastomas,
and breast cancer.18 The receptor specifically recognizes
Arg-Gly-Asp domains in peptides and several linear and cyclic
RGD targeting peptide analogs have been designed, such as
c(RGDfK), 19. As a model for a bimodal radio- and optical
imaging agent, we attached a fluorophore and the radio-isotope
chelator DOTA to a c(RGDfK) peptide in a one-pot reaction.
Boc-Ser(tBu)-OH was coupled to the Lys side-chain of 19 to
provide 20. Subsequent three-component modification with
N-hydroxylamine-AEDANS fluorophore (17) and DOTA-maleimide
then gave the desired bimodal compound 21 in 79% isolated yield
(Fig. 3).
§ Experimental: General modification procedure A/B: All components
are freshly prepared prior to each reaction (A: 200 mm NaOAc pH 4.5/
CH3CN 1 : 1; B: 100 mm phosphate buffer pH 7.0/CH3CN 1 : 1).
A 10 mm peptide solution is treated with NaIO4 (1.2 eq.). Oxidation
proceeds immediately and the formed iodate is quenched with sodium
ascorbate (4 eq.) and KI (6.6 eq.). After 15 min, aniline (3 eq., at pH 4.5)
or p-anisidine (3 eq., at pH 7.0) and the appropriate N-hydroxylamine
(3 eq. if not otherwise stated) are added, and the solution is monitored
until nitrone formation is complete. The appropriate maleimide
(25 eq.) is then added and the solution kept at rt or with gentle shaking
in a heater shaker for a specified amount of time.
1 S. D. Tilley, N. S. Joshi and M. B. Francis, Wiley Encycl. Chem. Biol,
John Wiley & Sons, Inc., 2009, vol. 4, pp. 158–174.
2 J. M. Gilmore, R. A. Scheck, A. P. Esser-Kahn, N. S. Joshi and
M. B. Francis, Angew. Chem., Int. Ed., 2006, 45, 5307–5311.
3 (a) C. W. Tornøe, 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, 1053–1057.
¨
¨
4 A. D. de Arau´jo, J. M. Palomo, J. Cramer, M. Kohn, H. Schroder,
R. Wacker, C. Niemeyer, K. Alexandrov and H. Waldmann, Angew.
Chem., Int. Ed., 2006, 45, 296–301.
5 (a) X. Ning, R. P. Temming, J. Dommerholt, J. Guo, D. B. Ania,
M. F. Debets, M. A. Wolfert, G.-J. Boons and F. L. van Delft, Angew.
Chem., 2010, 122, 3129–3132; (b) C. S. McKay, J. Moran and
J. P. Pezacki, Chem. Commun., 2010, 46, 931–933.
6 B. C. Sanders, F. Friscourt, P. A. Ledin, N. E. Mbua, S. Arumugam,
J. Guo, T. J. Boltje, V. V. Popik and G.-J. Boons, J. Am. Chem. Soc.,
2010, 133, 949–957.
Fig. 4 Site-selective modification of OXR1 at pH 7.5, forming the desired DOTA-
functionalized protein.
7 E. M. Sletten and C. R. Bertozzi, Angew. Chem., Int. Ed., 2009, 48,
6974–6998.
8 S. Faulkner and N. J. Long, Dalton Trans., 2011, 40, 6129–6143.
9 (a) S. Lee and X. Chen, Mol. Imaging, 2009, 8, 87–100;
(b) L. E. Jennings and N. J. Long, Chem. Commun., 2009, 3511–3524.
10 K. B. G. Torssell, Nitrile Oxides, Nitrones, and Nitronates in Organic
Synthesis, VCH Publishers, New York, 1988.
11 M. Villien, S. Deroo, E. Gicquel, E. Defrancq, C. Moucheron,
A. Kirsch-De Mesmaeker and P. Dumy, Tetrahedron, 2007, 63,
11299–11306.
In a first demonstration of this protocol for protein modi-
fication, we conjugated a DOTA-maleimide chelator to the
N-terminus of the 12 kDa LysM domain of human oxidation
resistance protein 1 (OXR1). OXR1 is highly conserved among
eukaryotes and is believed to be involved in the repair of
oxidative DNA damage.19,20 To avoid aggregation, the protein
concentration was lowered to 700 mM and the temperature kept 12 J. E. Reimann and W. P. Jencks, J. Am. Chem. Soc., 1966, 88,
3973–3982.
at 4 1C, which necessitated longer reaction times. Rewardingly,
after 4 days, the product was observed by ESI-LCMS in approxi-
13 A. Dirksen, T. M. Hackeng and P. E. Dawson, Angew. Chem., Int. Ed.,
2006, 45, 7581–7584.
mately 50% yield (see ESI†). Thus, our protocol proved effective 14 H. K. Munch, S. T. Heide, N. J. Christensen, T. Hoeg-Jensen,
P. W. Thulstrup and K. J. Jensen, Chem.–Eur. J., 2011, 17, 7198–7204.
15 J. Siegrist, T. Kazarian, C. Ensor, S. Joel, M. Madou, P. Wang and
for site-selective introduction of a ligand by a stable linkage
onto the protein OXR1. Likely, it can be further extended to
S. Daunert, Sens. Actuators, B, 2010, 149, 51–58.
introduce two ligands on some proteins (Fig. 4).
The presented site-selective three-component peptide and
protein modification method proceeds with excellent conver-
16 E. N. Hudson and G. Weber, Biochemistry, 1973, 12, 4154–4161.
17 For a recent example see: Z. Liu, Y. Yan, F. T. Chin, F. Wang and
X. Chen, J. Med. Chem., 2009, 52, 425–432.
18 S. Lee, J. Xie and X. Chen, Chem. Rev., 2010, 110, 3087–3111.
sions under mild aqueous conditions. The advantages over 19 P. L. Oliver, M. J. Finelli, B. Edwards, E. Bitoun, D. L. Butts, E. B.
E. Becker, M. T. Cheeseman, B. Davies and K. E. Davies, PLoS Genet.,
2011, 7, e1002338.
20 M. R. Volkert, N. A. Elliott and D. E. Housman, Proc. Natl. Acad. Sci.
U. S. A., 2000, 97, 14530.
oxime formation include that three components are linked
and that the resulting isoxazolidine products provide
a
chemically robust linkage between the conjugated components.
c
1938 Chem. Commun., 2013, 49, 1936--1938
This journal is The Royal Society of Chemistry 2013