Synthesis of n-terminally linked protein dimers and trimers by a combined native chemical ligation-CuAAC click chemistry strategy

Article abstract of DOI:10.1021/ol9016468

A novel method for the synthesis of N-terminally linked protein multimers is reported. Azide and alkyne thioesters were synthesized for the N-terminal modification of expressed proteins using native chemical ligation (NCL). Proteins modified by these moie

Full text of DOI:10.1021/ol9016468

ORGANIC
LETTERS
2009
Vol. 11, No. 18
4144-4147
Synthesis of N-Terminally Linked
Protein Dimers and Trimers by a
Combined Native Chemical
Ligation-CuAAC Click Chemistry
Strategy
Junpeng Xiao† and Thomas J. Tolbert*,†,‡
Interdisciplinary Biochemistry Graduate Program and Department of Chemistry,
Indiana UniVersity, Bloomington, Indiana 47405
tolbert@indiana.edu
Received July 18, 2009
ABSTRACT
A novel method for the synthesis of N-terminally linked protein multimers is reported. Azide and alkyne thioesters were synthesized for the
N-terminal modification of expressed proteins using native chemical ligation (NCL). Proteins modified by these moieties can be joined together
to form homodimers and homotrimers via Cu(I)-catalyzed azide-alkyne [3 + 2] cycloaddition (CuAAC) click chemistry. The orthogonal nature
of this reaction allows the production of protein heteromultimers, and this is demonstrated by synthesis of a protein heterodimer.
Multivalent interactions occur in many biological processes
such as signal transduction, immune responses, and viral
invasion.1 These multivalent interactions have been the
inspiration for the design of peptide and protein multimers
for use in biochemical and medicinal applications because
of the higher affinity and specificity of multimers compared
to monomers for cellular and viral targets. Multivalency has
been utilized to improve detection of antigen-specific T-cells
by peptide-MHC complexes,2-4 to produce and improve
antiviral peptides against HIV,5-8 and in vaccine design.9
Because of the many applications of protein and peptide
multimers there is significant interest in their synthesis.
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† Interdisciplinary Biochemistry Graduate Program.
‡ Department of Chemistry.
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10.1021/ol9016468 CCC: $40.75
Published on Web 08/25/2009
 2009 American Chemical Society

Several approaches have been taken to the synthesis of
multivalent peptides and proteins. The most common ap-
proach is to utilize amino acid side chains during or after
peptide synthesis to create branched structures. This was first
applied by Tam to produce multiple antigen peptides (MAPs)
by initiating the synthesis of dendrimeric peptides on-resin
utilizing both the NR and Nε amino groups of lysine.9 This
approach has been extended by Tam and others to use
cysteine and non-natural amino acids to form disulfide-,10,11
thioether-,5,6,12,13 thiazolidine-,7,14 triazole-,15,16 and oxime-
linked17 multivalent peptides. While these approaches work
well for producing multimeric synthetic peptides, the ap-
plication of these strategies to expressed proteins is com-
plicated by the increased number of amino acid residues in
proteins and the necessity of introducing non-natural chemi-
cal functionalities onto proteins. To address these issues, the
chemoselective reaction expressed protein ligation (EPL) has
been utilized to incorporate synthetic peptides and non-
natural functionalities onto the C-termini of expressed
proteins.18-21 This has allowed the production of C-
terminally linked multimers of expressed proteins.22,23
Because many proteins cannot be conjugated through their
C-termini without affecting biological activity and because
it is also desirable to produce protein heteromultimers, we
have explored other chemoselective strategies for the produc-
tion of protein multimers. Herein we report a novel strategy
for the synthesis of N-terminally linked homo- and hetero-
multimers of expressed proteins using a combination of
NCL24,25 with CuAAC click chemistry.26 This strategy takes
advantage of NCL to selectively modify the N-terminus of
expressed proteins with azide and alkyne moieties and the
bio-orthogonal nature of the CuAAC reaction to produce
homo- and heteromultimers.
the fusion protein with TEV protease produces a protein
containing a N-terminal cysteine.27 Next, azide/alkyne-
containing thioesters are specifically ligated to protein
N-terminal cysteines via NCL (Figure 1). Finally, the
Figure 1. Synthesis of azide/alkyne-functionalized proteins.
N-terminal azide- and alkyne-containing proteins are either
directly coupled together to form dimers or coupled onto
alkyne- and azide-containing linkers to form linker-conju-
gated multimers through CuAAC click chemistry. Using this
strategy, not only can N-terminally linked expressed protein
homodimers and homotrimers be produced, but synthetically
challenging N-terminally linked expressed protein het-
erodimers can also be produced. In addition to protein
multimer formation, expressed proteins that are N-terminally
modified by azide and alkyne moieties by the methods
described here can also be utilized in the myriad of
applications that have been developed for CuAAC click
chemistry, such as protein modification, immobilization, and
protein-polymer conjugation.28-36
In our strategy, the target protein is expressed as a fusion
protein which contains a tobacco etch virus NIa (TEV)
protease cleavable His-tag at the N-terminus. Treatment of
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Synthesis of an azide-containing thioester was accom-
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Org. Lett., Vol. 11, No. 18, 2009
4145

acid thioester 3. The tert-butyl-protected 3 was then treated
with 95% TFA to give water-soluble azidoacetic acid
thioester 4 with an overall 87% yield (Scheme 1A). The same
synthetic scheme was also used to synthesize the water-
soluble pentynoic acid thioester 7 with an overall 80% yield
(Scheme 1B).
Scheme 1
.
Synthesis of Azide- and Alkyne-Containing
Thioesters
To demonstrate the feasibility of this strategy, the C2
domain of protein G, an immunoglobulin-binding protein
found in group G streptococci,37 was chosen as a model
protein to work with. It was expressed as a fusion protein
with a TEV protease cleavable His-tag in E. coli.38 TEV
protease cleavage of the protein G fusion protein produced
protein G with an N-terminal cysteine (Figure S1A, Sup-
porting Information). The cleaved protein G was separated
from the His-tag by Ni2+-NTA affinity chromatography and
then dialyzed against 20 mM sodium phosphate buffer pH
7.5 at 4 °C for 12 h.
The azide-functionalized protein G 8 was obtained by
ligating azidoacetic acid thioester 4 to the N-terminal cysteine
of protein G using NCL. The ligation was initiated by adding
4 mM 4 and 30 mM sodium 2-mercaptoethanesulfonate into
the protein G solution (4 mg/mL). After 24 h incubation,
the reaction mixture was purified by reversed-phase HPLC
and lyophilized to give azide-protein G 8, as confirmed by
ESI-MS (Figure S1B, Supporting Information). The ligation
of pentynoic acid thioester 7 with cleaved protein G was
conducted using the same conditions that were used with 4.
The ligation of 7 proceeded well and produced alkyne-
functionalized protein G 9, as confirmed by ESI-MS
(alkyne-protein G 9, Figure S1C, Supporting Information).
A general procedure for the CuAAC reaction was utilized
for the synthesis of protein dimers and trimers. Proteins were
dissolved in 1 mL of 50 mM of sodium phosphate buffer
pH 7.5, and then CuSO4 and L-ascorbic acid were added to
final concentrations of 1 and 2 mM, respectively. Protein
multimerization reactions were incubated at room tempera-
ture for 12 h and then purified. The N-terminally linked
homodimer of protein G 10 was obtained by directly coupling
azide-protein G 8 (3 mg/mL, 0.44 mM) with alkyne-protein
G 9 (4 mg/mL, 0.59 mM) using the general CuAAC reaction
conditions described above (Figure 2A). Purification of the
homodimer 10 by reversed-phase HPLC was not possible
because 10 has the same retention time as 8 and 9. Because
Figure 2. (A) Synthesis of protein G homodimer 10; (B) ESI-MS
of 10, calcd 13637.6, found 13637.5.
of this, size-exclusion chromatography was used to separate
10 from unreacted 8 and 9. After purification and lyophiliza-
tion, 5.2 mg of the protein G homodimer 10 was obtained
and characterized by analytical HPLC and ESI-MS (Figure
S9A (Supporting Information), Figure 2B).
In addition to dimer formation by directly coupling azide
and alkyne modified proteins, a diazide linker 11 (the
synthesis of 11 is described in the Supporting Informa-
tion)39,40 and a trialkyne linker 12 were utilized to demon-
strate the application of our strategy for linker-conjugated
expressed protein homodimer and homotrimer formation.
The linker-conjugated protein G homodimer 13 was
synthesized by a one-pot approach (Figure 3A). The diazide
linker 11 (0.2 mM) was incubated with 3 equiv of
alkyne-protein G 9 (4 mg/mL, 0.59 mM) under CuAAC
reaction conditions. The reaction mixture was then purified
by size-exclusion chromatography and lyophilized to give
2.0 mg of the linker-conjugated protein G homodimer 13
(Figure S9B (Supporting Information), Figure 3B). The
linker-conjugated homotrimer 14 was also obtained by a one-
pot incubation of tripropargylamine 12 (0.15 mM) with
approximately 5 equiv of azide-protein G 8 (5 mg/mL, 0.73
mM) using the general CuAAC reaction conditions (Figure
3C). Size-exclusion chromatography purification of the
reaction mixture afforded 1.9 mg of the protein G homotrimer
14 (Figure S9C (Supporting Information), Figure 3D).
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4146
Org. Lett., Vol. 11, No. 18, 2009

Figure 3. (A) Synthesis of linker-conjugated protein G homodimer
13; (B) ESI-MS of 13, calcd 13834.6, found 13836.0; (C) synthesis
of linker-conjugated protein G homotrimer 14; (D) ESI-MS of 14,
calcd 20592.1, found 20594.5.
Figure 4. (A) Synthesis of protein G-C37H6 heterodimer 16; (B)
ESI-MS of 16, calcd 12528.3, found 12530.0.
An advantage of using this strategy to produce N-
terminally linked protein dimers is that the azide and alkyne
moieties can be attached to two different proteins and then
be used to produce heterodimers that are synthetically
challenging to obtain by other methods. We demonstrate this
using azide-protein G 8 and C37H6, an HIV entry inhibitor
peptide we have produced by bacterial expression (Figure
S2A, Supporting Information).41 Ligation of 7 to the N-
terminal cysteine of C37H6 produced alkyne-C37H6 15
(Figure S2B, Supporting Information), and then protein
G-C37H6 heterodimer 16 was obtained by coupling 8 (3
mg/mL, 0.44 mM) with excess 15 (3.5 mg/mL, 0.61 mM)
under the general CuAAC reaction conditions followed by
size-exclusion chromatography purification. This afforded 5.0
mg of the heterodimer 16 (Figure S9D (Supporting Informa-
tion), Figure 4).
In conclusion, a general strategy for the synthesis of
N-terminally linked multimeric expressed proteins was
developed by combining NCL with CuAAC click chemistry.
This strategy utilizes NCL to incorporate the azide and alkyne
moieties onto N-terminal cysteines of expressed proteins. The
azide- and alkyne-modified proteins can then be coupled
together through CuAAC to form N-terminally linked
expressed protein homodimers. In addition, linker-conjugated
expressed protein homodimers and homotrimers were also
obtained in a one-pot reaction between azide- or alkyne-
functionalized proteins and alkyne- or azide-containing
linkers. This strategy is general and could be extended to
other expressed protein multimers, and the linkers could be
adjusted by changing the linker length and rigidity. Further-
more, N-terminally linked expressed protein heterodimers,
which are difficult to produce selectively by existing
methods, can be prepared easily by coupling two different
proteins at their N-terminus using the orthogonal CuAAC
reaction. Thus, our strategy may be generally applied to
prepare multimeric bioactive proteins and peptides for drug
development and vaccine design. In addition, modification
of proteins with N-terminal azide and alkyne moieties can
enable the utilization of other CuAAC click chemistry
applications in protein chemistry, such as site-specific protein
modification, immobilization, and polymer conjugation.
Acknowledgment. This work was supported by Indiana
University.
Supporting Information Available: Experimental pro-
cedures and characterization data. This material is available
free of charge via the Internet at http://pubs.acs.org.
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