Multivalent peptide and protein dendrimers using native chemical ligation

Article abstract of DOI:10.1002/anie.200500635

A wide variety of well-defined multivalent peptides and proteins can be made by conjugating synthetic peptides and recombinantly expressed proteins to cysteine-functionalized dendrimers using native chemical ligation (see picture). This modular approach provides access to dendrimers that are attractive both for understanding fundamental issues of multivalency in biological interactions as well as for biomedical applications. (Chemical Equation Presented)

Full text of DOI:10.1002/anie.200500635

Communications
Bioorganic Chemistry
DOI: 10.1002/anie.200500635
Multivalent Peptide and Protein Dendrimers
Using Native Chemical Ligation**
Ingrid van Baal, Hinke Malda, Silvia A. Synowsky,
Joost L. J. van Dongen, Tilman M. Hackeng,
Maarten Merkx,* and E. W. Meijer*
Multiple, simultaneous interactions are often used in biology
to enhance the affinity and specificity of binding, an effect
that is known as multivalency.[1] The principle of multivalency
has been recognized as an important strategy for the
development of (semi)synthetic ligands with high affinity
and specificity for biological targets.[1–6] Dendrimers are well-
defined, hyperbranched polymers with a high density of
functional groups and are therefore attractive scaffolds for
multivalent display of natural ligands.[7–9] Dendrimers have
been successfully applied in the design of multivalent sugar
ligands,[10–12] but much less work has been reported on
multivalent peptide and protein dendrimers. Solid-phase
peptide synthesis has been used to construct dendritic peptide
wedges by condensation of successive generations of lysine
residues followed by functionalization of the a and e amine
groups with peptides.[13,14] These so-called multiple antigen
peptides (MAPs)typically contain two to eight peptide chains
and have been found to be more immunogenic than single
[*] I. van Baal, H. Malda, J. L. J. van Dongen, Dr. M. Merkx,
Prof. E. W. Meijer
Laboratory of Macromolecular and Organic Chemistry
Eindhoven University of Technology
P.O. Box 513, 5600 MB, Eindhoven (The Netherlands)
Fax: (+31)40-245-1036
E-mail: m.merkx@tue.nl
e.w.meijer@tue.nl
S. A. Synowsky
Department of Biomolecular Mass Spectrometry
Bijvoet Center for Biomolecular Research and
Utrecht Institute for Pharmaceutical Sciences
Utrecht University
Sorbonnelaan 16, 3584 CA Utrecht (The Netherlands)
Dr. T. M. Hackeng
Department of Biochemistry
Cardiovascular Research Institute Maastricht
University Maastricht
P.O. Box 616, 6200 MD Maastricht (The Netherlands)
[**] I. van Baal and H. Malda contributed equally to this work. We
acknowledge grants from the department of Biomedical Engineer-
ing of the Eindhoven University of Technology and the NRSC
Catalysis. We thankRobert H. H. van den Heuvel and Albert J. R.
Heck(Utrecht University, The Netherlands) for useful discussions
and mass spectrometry equipment, Alex Faesen for preparation of
illustrations, and Marcel van Genderen and AnoukDirksen for
helpful discussions.
Supporting information (synthesis and characterization of the
dendrimers, including mass spectra for all the peptide and protein
dendrimers) for this article is available on the WWW under http://
www.angewandte.org or from the author.
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peptides. Dendrimers functionalized with cysteine-reactive
chloroacetyl[15] and maleimide[16] groups have been used to
conjugate cysteine-containing peptides, but this strategy
cannot be applied to peptides and proteins containing func-
tionally important native cysteine residues. Convergent syn-
thesis of peptide dendrimers has also been reported by
condensation of ketone-functionalized polyamidoamine
(PAMAM)dendrimers with aminooxy-functionalized pep-
tides, but this method is unsuitable for native proteins.[9] The
few reports of protein ligation to dendrimers typically involve
the coupling of a single protein (for example, an antibody)
using nonspecific conjugation chemistry that results in con-
jugation at multiple sites on the protein. Recently, the
preparation of multivalent hemoglobin dendrimers was
reported that involved specific conjugation of a fourth
generation PAMAM dendrimer to hemoglobin using a
hemoglobin-specific cross-linking agent.[17] However, no gen-
eral synthetic strategy is currently available that allows
conjugation of dendrimers with both oligopeptides and
recombinant proteins in a chemoselective manner.
large proteins (by multistep ligation of several peptide
fragments), the synthesis of proteins with synthetic moieties
such as fluorescent dyes and biotin tags, and the immobiliza-
tion of peptides and proteins on surfaces.[19–23] The application
of native chemical ligation was recently extended to recom-
binantly expressed proteins by the development of expression
systems based on self-cleavable intein domains that generate
proteins containing N-terminal cysteine or C-terminal thio-
ester groups.[24,25] Herein we report the application of native
chemical ligation as an attractive and general synthetic
strategy to conjugate both oligopeptides and recombinantly
expressed proteins to dendrimers, thus resulting in multi-
valent peptide and protein dendrimers.
Dendrimers can either be functionalized with thioester
groups or with cysteine residues to allow native chemical
ligation. We chose the latter strategy because the synthesis of
these cysteine dendrimers is more straightforward. Cysteine
dendrimers generations 1–3 (Scheme 1)were synthesized by
reaction of the amine end groups of poly(propyleneimine)
dendrimers with succinimide-activated and trityl-protected
cysteine residues (Scheme 1). Deprotection of the trityl-
protected amine and thiol groups using trifluoroacetic acid
(TFA)yielded the cysteine-functionalized dendrimers 1–3 in
good yields. Analysis of 1–3 by electrospray ionization mass
spectrometry (ESI-MS)showed that the cysteine residues
were reduced in the presence of the mild reductant b-
mercaptoethanol. Removal of the reductant and addition of
Native chemical ligation was first reported by Dawson
et al. as a unique method to ligate two unprotected peptide
fragments to form a native peptide bond.[18] This chemo-
selective reaction occurs spontaneously between a peptide
with a C-terminal thioester and a peptide with an N-terminal
cysteine residue under aqueous conditions at neutral pH.
Native chemical ligation has allowed the chemical synthesis of
Scheme 1. Top: synthesis of cysteine-functionalized poly(propyleneimine) dendrimers: activation of trityl-protected cysteine with disuccinimidylcar-
bonate in acetonitrile (1), coupling of activated cysteine to poly(propyleneimine) dendrimer using triethylamine in dichloromethane (2), and
deprotection of cysteine end groups using trifluoroacetic acid, 2.5% triisopropylsilane, and 2.5% water (3). Bottom: schematic structures of cys-
teine dendrimers 1, 2, and 3.
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Table 1: Overview of peptide and protein dendrimers obtained by native
chemical ligation.
hydrogen peroxide resulted in the immediate formation of
dendrimers with two, four, or eight intramolecular disulfide
bonds, respectively (see Supporting Information). To assess
the reactivity of the cysteine dendrimers in native chemical
ligation reactions we performed ligation reactions with
several peptides functionalized with an MPAL thioester at
their C-terminus.[19] Ligation of 1 with four equivalents of
LYRAG, a peptide previously used in studies on native
chemical ligation,[19] resulted in the formation of the peptide
tetramer 1-(LYRAG)4 (Figure 1a). Analysis by liquid chro-
matography/mass spectrometry (LC-MS)showed that the
reaction went to completion as no 1-(LYRAG)1–3 were
detected (not shown). Ligation reactions were also performed
between LYRAG and dendrimers 2 and 3 to test the
efficiency of the ligation reaction for higher generations of
Dendrimer
Observed mass [Da] Calculated average mass
[Da][a]
1-(LYRAG)4
2-(LYRAG)8
3139.7
6419.7
3139.8
6419.9
12307.6
3243.5
6627.1
28291
3-(LYRAG)16
1-(GRGDSGG)4
2-(GRGDSGG)8
1-(GFP)1
12262[b]
3243.2
6627.1
28288.6 (0.5)[c]
1-(GFP)1(GRGDSGG)3 30172.5 (0.7)[c]
30177
110976
1-(GFP)4
110971.1 (4.7)[c]
[a] Average masses were calculated assuming the complete reduction of
the cysteine groups. [b] In addition to the signal of 3-(LYRAG)16, the
MALDI-TOF spectrum showed signals consistent with 3-(LYRAG)12–15
[c] The number between brackets is the experimental error as determined
using the program MaxEnt 1.
.
cysteine dendrimers. Ligation of 2 with LYRAG
again went to completion and yielded the peptide
octamer 2-(LYRAG)8 exclusively (Figure 1b). The
MALDI-TOF spectrum obtained after ligation of 3
with LYRAG showed a signal consistent with the
formation of 3-(LYRAG)16, but also contained
signals attributable to 3-(LYRAG)12–15. Similar
results were obtained for other oligopeptides such
as GRGDSGG (Table 1). The RGD sequence
present in this peptide is known to bind to various
extracellular integrin receptors and can thus be
used to attach dendrimers to cell surfaces.
Green fluorescent protein (GFP)was chosen as
a model protein to study the ligation of folded
proteins to cysteine dendrimers. GFP was cloned
into the IMPACT vector pTXB1 to yield an E. coli
expression vector for a fusion protein of GFP with
an intein domain and a chitin-binding domain
(CBD). The cloning strategy used here meant that
the GFP contained eight additional residues at the
C-terminus of GFP, that is, NEFLEGSS. Overnight
incubation of the GFP-intein-CBD protein bound
to the chitin column with MESNA resulted in
cleavage of the peptide bond to the GFP and the
formation of GFP thioester. ESI-MS analysis
showed the presence of a single protein signal
with a mass of 27705 Da that is consistent with the
MESNA thioester of GFP (theoretical mass:
27703.9 Da). The GFP thioester was first ligated
to dendrimer 1 using a large excess of the den-
drimer. ESI-MS of the reaction mixture after
incubation for 20 h at room temperature showed
the clean conversion of GFP thioester to 1-(GFP)1,
with no indication of the presence of the initial
GFP thioester or dendrimer with more than one
GFP (Figure 2). The large difference in molecular
weight between 1-(GFP)1 and 1 allowed the easy
Figure 1. Native chemical ligation of the MPAL-thioester of LYRAG to first (a) and second
(b) generation cysteine dendrimers 1 and 2. Dendrimer was dissolved in 0.1m Tris, 6m gua-
nidine, 2% (v/v) thiophenol, and 2% (v/v) benzyl mercaptan pH 7.0–7.5 and treated with
four (1) or eight equivalents (2) of MPAL-peptide for 1 h at 378C. The mass spectra were
obtained by ESI-MS after removal of the buffer components by RP-HPLC.
removal of excess of 1 from the ligation mixture by
repeated concentration/dilution steps using a cen-
trifugal filter with a 10-kDa molecular-weight cut-
off. Subsequently, 1-(GFP)1 was treated with an
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Figure 2. a) Synthesis of a dendrimer containing both protein and peptide end groups. GFP thioester (75 mm) was treated with a large excess of
dendrimer 1 (2 mm) for 20 h at 208C to yield the monofunctionalized 1-(GFP)1 exclusively. After removal of remaining dendrimer 1, 1-(GFP)1 was
treated with excess GRGDSGG-MPAL peptide (375 mm) for 4 h at 208C to yield 1-(GFP)1(GRGDSGG)3 exclusively. b) Experimental (inset) and
deconvoluted ESI-MS of 1-(GFP)1. c) Experimental (inset) and deconvoluted ESI-MS of 1-(GFP)1(GRGDSGG)3.
excess of the MPAL thioester of GRGDSGG. ESI-MS
showed the presence of a single, high-molecular-weight
signal at 30173 Da, which is exactly the mass expected for
1-(GFP)1(GRGDSGG)3 assuming formation of two intra-
molecular disulfide bonds. This synthetic methodology should
be generally applicable to prepare dendrimers with (exactly)
one copy of any recombinant protein and multiple copies of
any peptide or other synthetic ligand with a thioester
functionality.
To test whether our synthetic strategy can be used to
obtain multivalent protein dendrimers, a 25 mm solution of
dendrimer 1 was incubated with four or eight equivalents of
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GFP thioester and the ligation reaction was monitored using
species that elutes from the column shows a limited number of
signals that can be deconvoluted to a single mass of
110971 Da. This mass corresponds almost exactly to the
average mass calculated for 1-(GFP)4 with two disulfide
bonds (110972 Da). The other detected ions originated from
GFP, 1-(GFP)1, 1-(GFP)2, and 1-(GFP)3 and clearly reveal
that size-exclusion liquid chromatography coupled to mass
spectrometry under native conditions is an ideal analytical
tool to separate and analyze these protein mixtures. More-
over, the high m/z values and the narrow charge distribution
indicate that the proteins may still be properly folded in 1-
(GFP)4.[28,29]
In summary, we have demonstrated that native chemical
ligation is an attractive ligation reaction to functionalize
dendrimers with both oligopeptides and recombinantly
expressed proteins. Furthermore, dendrimers can be derivat-
ized with exactly one copy of a recombinant protein, and the
remaining cysteine residues can then be further functional-
ized with oligopeptides. Our approach allows chemoselective
dendrimer conjugation to the C-terminus of a protein, which
is much less likely to interfere with protein function than
other less-specific protein-ligation strategies. The modular
approach presented here provides access to a wide variety of
well-defined multivalent peptides and proteins that are
attractive both for understanding the fundamental mecha-
sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE)(Figure 3a). Analysis of the reaction after 20 h
showed the presence of four distinct bands at 27, 55, about
120, and about 170 kDa that we attribute, respectively, to
GFP thioester and 1-(GFP)1, 1-(GFP)2, 1-(GFP)3, and 1-
(GFP)4. The bands of 1-(GFP)3 and 1-(GFP)4 appear at a
higher apparent molecular weight than expected, which is
probably a consequence of the branched structure of these
multivalent proteins. A similar effect of branching on the
apparent molecular weight has been reported for ubiquiti-
nated proteins, which are a natural form of branched
proteins.[26] The limited solubility of GFP under the con-
ditions suitable for native chemical ligation (100–200 mm GFP
thioester)probably makes the ligation of GFP slower than the
peptide-ligation reactions that are typically performed at 2–
10 mm.[27] A second factor that may prevent the full con-
version into 1-(GFP)4 is increased steric crowding that occurs
when attaching four 27-kDa proteins around a 0.7-kDa
dendritic core.
The ligation product was further purified using size-
exclusion chromatography and analyzed using electrospray
ionization time-of-flight mass spectrometry under native
conditions to provide definitive evidence for the formation
of the GFP tetramer (Figure 3b, c). The spectrum of the first
Figure 3. a) Native chemical ligation of dendrimer 1 (25 mm) with four (reaction 1) or eight equivalents (reaction 2) of GFP thioester in 0.1m
sodium phosphate, 2% (v/v) thiophenol, and 2% (v/v) benzyl mercaptan pH 7–7.5 for 20 h at 208C. b) Size-exclusion chromatogram of the liga-
tion reaction mixture on an analytical Superdex 200 PC 3.2/30 column showing the total ion current (blacktrace) and the selected ion current for
1-(GFP)4 (blue trace). c) Experimental (inset) and deconvoluted ESI-MS of 1-(GFP)4 (fraction that eluted between 25.7 and 27.6 min).
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[21] R. Y. P. Lue, G. Y. J. Chen, Y. Hu, Q. Zhu, S. Q. Yao, J. Am.
nisms of multivalency in biological interactions and for
biomedical applications in targeted drug delivery, molecular
imaging, and immunology.
Chem. Soc. 2004, 126, 1055.
[22] D. S. Y. Yeo, R. Srinivasan, G. Y. J. Chen, S. Q. Yao, Chem. Eur.
J. 2004, 10, 4664.
[23] J. A. Camarero, Y. Kwon, M. A. Coleman, J. Am. Chem. Soc.
2004, 126, 14730.
[24] T. W. Muir, D. Sondhi, P. A. Cole, Proc. Natl. Acad. Sci. USA
1998, 95, 6705.
Experimental Section
All peptide ligation experiments were performed in
a buffer
containing 0.1m tris(hydroxymethyl)aminomethane (tris), 6m guani-
dine, 2% (v/v)thiophenol, and 2% (v/v) a-toluenethiol (benzyl
mercaptan)pH 7.0–7.5. One equivalent of dendrimers 1, 2, or 3 was
treated with 4, 8, or 16 equivalents of peptide thioester
(ca. 10 mgmLÀ1), respectively, with stirring for 1 h at 378C. The
ligation products were analyzed by LC-MS. All protein-ligation
experiments were performed at 208C in a buffer containing 0.1m
sodium phosphate, 2% (v/v)thiophenol, and 2% (v/v)benzyl
mercaptan pH 7–7.5. Precipitates were removed after the reaction
by centrifugation. 1-(GFP)1 was obtained by ligation of 75 mm GFP
thioester with 2 mm dendrimer 1 for 20 h at 208C. Remaining
dendrimer was removed by repeated concentration and dilution of
the reaction mixture using an Amicon Ultra-4 centrifugal concen-
trator with a 10-kDa cut-off. 1-(GFP)1(GRGDSGG)3 was obtained by
subsequent ligation with 375 mm GRGDSGG-MPAL for 4 h at 208C,
followed by analysis using LC-MS.
[25] R. David, M. P. O. Richter, A. G. Beck-Sickinger, Eur. J.
Biochem. 2004, 271, 663.
[26] A. Borodovsky, H. Ovaa, N. Kolli, T. Gan-Erdene, K. D.
Wilkinson, H. L. Ploegh, B. M. Kessler, Chem. Biol. 2002, 9,
1149.
[27] We have observed similar noncomplete ligation reactions for
small, hydrophobic peptides with limited solubility.
[28] A. J. R. Heck, R. H. H. van den Heuvel, Mass Spectrom. Rev.
2004, 23, 368.
[29] R. H. H. van den Heuvel, A. J. R. Heck, Curr. Opin. Chem. Biol.
2004, 8, 519.
Received: February 21, 2005
Published online: July 11, 2005
Keywords: bioorganic chemistry · dendrimers · multivalency ·
.
peptides · proteins
[1] M. Mammen, S.-K. Choi, G. M. Whitesides, Angew. Chem. 1998,
110, 2908; Angew. Chem. Int. Ed. 1998, 37, 2754.
[2] L. L. Kiessling, L. E. Strong, J. E. Gestwicki, Curr. Opin. Chem.
Biol. 2000, 4, 696.
[3] L. L. Kiessling, L. E. Strong, J. E. Gestwicki, Annu. Rev. Med.
Chem. 2000, 35, 321.
[4] E. Fan, Z. Zhang, W. E. Minke, Z. Hou, C. L. M. J. Verlinde,
W. G. J. Hol, J. Am. Chem. Soc. 2000, 122, 2663.
[5] P. L. Kitov, J. M. Sadowska, G. Mulvey, G. D. Amstrong, H. Ling,
N. S. Pannu, R. J. Read, D. R. Bundle, Nature 2000, 403, 669.
[6] J. Rao, J. Lahiri, L. Isaacs, R. M. Weis, G. M. Whitesides, Science
1998, 280, 708.
[7] A. W. Bosman, H. M. Janssen, E. W. Meijer, Chem. Rev. 1999,
99, 1665.
[8] U. Boas, P. M. H. Heegard, Chem. Soc. Rev. 2004, 33, 43.
[9] J. P. Mitchell, K. D. Roberts, J. Langley, F. Koentgen, J. N.
Lambert, Bioorg. Med. Chem. Lett. 1999, 9, 2785.
[10] J. D. Reuter, A. Myc, M. M. Hayes, Z. Gan, R. Roy, D. Qin, R.
Yin, L. T. Piehler, R. Esfand, D. A. Tomalia, J. R. J. R. Baker,
Bioconjugate Chem. 1999, 10, 271.
[11] I. Vrasidas, N. J. de Mol, R. M. J. Liskamp, R. J. Pieters, Eur. J.
Org. Chem. 2001, 24, 4685.
[12] W. B. Turnbull, J. F. Stoddart, Rev. Mol. Biotechnol. 2002, 90,
231.
[13] C. Rao, J. P. Tam, J. Am. Chem. Soc. 1994, 116, 6975.
[14] K. Sadler, J. P. Tam, Rev. Mol. Biotechnol. 2002, 90, 195.
[15] M. Sakamoto, A. Ueno, H. Mihara, Chem. Commun. 2000, 2000,
1741.
[16] M. Zhou, D. Bentley, I. Ghosh, J. Am. Chem. Soc. 2004, 126, 734.
[17] R. Kluger, J. Zhang, J. Am. Chem. Soc. 2003, 125, 6070.
[18] P. E. Dawson, T. W. Muir, I. Clark-Lewis, S. B. Kent, Science
1994, 266, 776.
[19] T. M. Hackeng, J. H. Griffin, P. E. Dawson, Proc. Natl. Acad. Sci.
USA 1999, 96, 10068.
[20] T. J. Tolbert, C.-H. Wong, J. Am. Chem. Soc. 2000, 122, 5421.
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