Highly-efficient and versatile fluorous-tagged Cu(i)-catalyzed azide-alkyne cycloaddition ligand for preparing bioconjugates

Article abstract of DOI:10.1039/c5cc06858d

A novel ligand (FBTTBE) for Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) has been developed, which demonstrates not only superior catalytic efficiency but also the ease of removing toxic copper species. FBTTBE has also been successfully applied in the synthesis of radiometal-labeled peptide and antibody without observable transchelation with the non-radioactive Cu(i) catalyst.

Full text of DOI:10.1039/c5cc06858d

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Chemical Communication
COMMUNICATION
Highly-Efficient and Versatile Fluorous-Tagged Cu(I)-Catalyzed
Azide-Alkyne Cycloaddition Ligand for Preparing Bioconjugates
Lingyi Sun, †a Yongkang Gai,†a Carolyn J. Andersona and Dexing Zeng*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
A
novel ligand (FBTTBE) for Cu(I)-Catalyzed Azide-Alkyne approach is highly desired.
Cycloaddition (CuAAC) has been developed, which demonstrates
Here we report the development of a novel fluorous tagged
not only superior catalytic efficiency but also the ease of removing tris(triazolylmethyl)amine-based Cu(I) stabilizing ligand
toxic copper species. FBTTBE has also been successfully applied in (FBTTBE; Figure 1). This ligand has great promise towards
the synthesis of radiometal-labeled peptide and antibody without facilitating the removal of toxic catalytic species while
observable transchelation with the non-radioactive Cu(I) catalyst.
maintaining high catalytic efficiency. The use of a fluorous tag
enables the easy separation of the toxic catalyst from the
product (non-fluorous species) via the Fluorous Solid-Phase
Extraction (F-SPE) approach16, whereby the separation is
accomplished by simply passing the reaction mixture through a
fluorous silica gel. The bis(tert-butyltriazolyl) methyl amine
based catalytic core shows significantly improved kinetics
compared with two commercially available Cu(I) ligands, TBTA
and THPTA (Figure 1).17 This new design of the catalytic ligand
integrates homogenous solution phase reaction conditions
with a phase-tag separation, while maintaining high reactivity
as well as strong capacity to fully complex the copper ions. It is
believed that the synergy of the fluorous-tag and the catalytic
core in the designed FBTTBE ligand will result in much broader
applications of CuAAC. The linker between the fluorous tag
and catalytic core provides the necessary distance to reduce
possible steric effects, and in the future it can be replaced by a
PEGylated linker to counter the loss of hydrophilic groups (i.e.,
the hydroxyl in THPTA) for improved aqueous solubility.
Click chemistry, particularly the copper(I)-catalyzed azide
alkyne cycloaddition (CuAAC)1, has found applications in a
wide range of modern chemistry-related areas, including
organic chemistry, drug discovery, drug delivery and chemical
biology.2-6 However, the toxicity of copper(I) from the
generation of reactive nitrogen and oxygen species7 limits its
application in living systems.8,9 For example, upon treatment
of 1 mM CuSO4, 1.5 mM sodium ascorbate, and 0.1 mM TBTA
(
Figure 1), Zebrafish embryos do not survive beyond 15 min.10
Therefore, the removal of copper species is typically required
in order to avoid cytotoxicity caused by residual copper ions in
biological applications, adding another layer of complexity to
the application of CuAAC in living systems. To overcome the
cumbersome copper removal problem, major efforts have
been made to minimize the risk caused by this metal catalyst.
New methodologies and techniques have been developed,
including copper-free variants of azide-alkyne click chemistry
(e.g., strain-promoted azide–alkyne cycloaddition (SPPAC) and
resin-supported catalyst systems).11-14 However, these
strategies cannot fulfill all the requirements due to their
inherent deficiencies, including relatively sluggish kinetics in
SPAAC and copper leaching problems observed in the resin-
supported catalyst systems.15 Therefore, a more efficient
Figure 1. FBTTB, TBTA and THPTA.
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In our study, a model FBTTBE ligand was synthesized via Upon formation of the triazole ring, strong fluorescence at 477
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DOI: 10.1039/C5CC06858D
multiple steps (Scheme 1). Alcohol
azide to generate azide . Subsequently,
3,3-diethoxy-1-propyne through copper catalyzed click CuAAC, followed by THPTA, with TBTA having the lowest
reaction to give the corresponding triazole , which was then reactivity (Figure 2). The reaction catalyzed by Cu(I)-FBTTBE
converted to the. triazolylcarbaldehyde via TFA
(trifluoroacetic acid) treatment. Facilitated by the reduction
reagent NaBH(OAc)3, intermediate was then prepared
through the reaction between
and propargyl amine.18
Intermediate was synthesized by treating the alcohol first
with thionyl chloride, followed by azidation using sodium
azide. In the final step, the FBTTBE ligand was obtained
through the click reaction between and
1 was treated with sodium nm can be quantitatively measured to determine the extent of
2
2 was reacted with the reaction. FBTTBE showed the greatest ability to accelerate
a
3
4
5
4
7
6
8
5
7.
As discussed above, the fluorous-tag containing FBTTBE
ligand features a rapid F-SPE removal capability. Utilizing
radioactive 64Cu2+, the trapping efficiency of the fluorous silica
gel was determined. In this experiment, 64Cu2+ (100 μCi) was
added to a non-radioactive Cu2+ solution, and the resulting
carrier-added 64Cu2+ (200 μM) was then mixed with 1.5 equiv.
of FBTTBE followed by 1.0 eq. of NaAA; the mixture was
passed through the fluorous silica gel after a 5 min incubation.
Over 99% of the radioactivity remained on silica gel,
demonstrating that FBTTBE-Cu(I) can be efficiently trapped.
Therefore, it is anticipated that the removal of toxic copper
species after CuAAC can be greatly simplified to a one-step
filtration using FBTTBE as the catalytic ligand.
In order to investigate its catalytic efficiency, the reactivity
of the synthesized FBTTBE ligand was then compared with two
widely used ligands TBTA and THPTA. Specifically, we
compared the relative reactivity of the canonical Cu(I) catalysts
in the form of TBTA–Cu(I), THPTA–Cu(I) and FBTTBE–Cu(I) via a
reported fluorogenic assay17 based on the reaction between
propargyl amine and 3-azido-7-hydroxycoumarin (Scheme S1).
Figure 2. Comparison of Cu(I) stabilizing ligands in the CuAAC
between propargyl amine and 3-azido-7-hydroxycoumarin
([azidocoumarin] = 10 µM; [propargyl amine] = 15 µM, ligand :
Cu(I) = 1.5 : 1).
completed in around 20 min at ambient temperature using 10
μM of Cu(I) and 1.5 eq. (15 μM) of FBTTBE. In contrast, no
more than 40% yield was achieved after 12 h with the TBTA
and THPTA ligands. This implies that FBTTBE renders a much
higher catalytic capacity in CuAAC compared with TBTA and
THPTA. The catalytic efficiency of the non-fluorous tagged
analogue BTTBE was found to be higher than that of FBTTBE
(
Figure S1), indicating the fluorous tag will affect the reactivity
of the catalytic core. This decreasing of catalytic efficiency
could be reversed by adding excess amount of FBTTBE which
could be conveniently removed by the fluorous silica gel after
the reaction.
The superior catalytic efficiency as well as the ease of
separating toxic copper species when using FBTTBE drove us to
further investigate its application in preparing agents that
could be used in living systems, such as a fluorescent dye
attached peptide for fluorescent microscopy staining.
Specifically, CuAAC between Cy3-azide and acetylene-AE105 (a
urokinase-type plasminogen activator receptor, uPAR-targeted
peptidic ligand) catalyzed by various Cu(I)-ligands was
compared (Figure S2). The Cu(I)-FBTTBE catalyzed CuAAC was
complete in 5 min, whereas neither Cu(I)-TBTA nor Cu(I)-
THPTA could achieve over 50% yield, even after incubating for
8 h. More importantly, both the Cu(I)-FBTTBE catalyst and
excess FBTTBE ligand were rapidly removed by F-SPE. The
resulting Cy3-AE105 conjugate was then used to stain human
U87MG glioblastoma cells that overexpress uPAR (Figure 3).
In addition to the rapid and complete removal of toxic
copper species and excellent catalytic efficiency, the FBTTBE
catalyst also demonstrated the capability of minimizing
transchelation between non-radioactive copper and
radiometals. In 2006, Marik and Sutcliffe reported the first use
of this method in preparing [18F]fluoropeptides,19 and since
then a large body of work employing the CuAAC has been
Scheme 1. Synthesis of the FBTTBE ligand. Reagents and
conditions: (a) NaN3, H2SO4: H2O = 1:1 (w/w); (b) 3,3-diethoxy-
1-propyne, NaHCO3, CuSO4, sodium ascorbate (NaAA), t-BuOH :
H2O = 1:1 (v/v); (c) TFA, DCM : H2O = 2:1 (v/v); (d) propargyl
amine, NaBH(OAc)₃ , Dichloroethane; (e) 1). SOCl2, DMF, 2).
NaN3, DMF: THF = 1:1 (v/v); (f) CuSO4, NaAA, t-BuOH : H2O = 1:1
(v/v).
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Figure 3. Microscopy staining of U87MG cells with A) Cy3-AE105;
and B) Cy3-AE105 with blocking (be more specific).
presented in the preparation of 18F-based PET radiotracers.20
However, its application in radiometal-based probes still
remains noticeably underdeveloped, possibly attributed to the
transchelation between non-radioactive copper ions and those
radiometals.21-25 Even with an extremely low concentration of
radiometal-containing reactants (e.g., 10-100 nM) and rapid
decay of these radionuclides, a relatively large amount of the
catalyst is required to ensure that the pmol-reactant reaction
can complete within a relatively short time. This leads to not
only difficulty in catalyst removal but also significant
radiometal transchelation, resulting in significant decreases in
specific activity of the corresponding radiopharmaceuticals.17
Here, a click reaction used for preparing the 64Cu-labeled
peptide was employed to evaluate the performance of FBTTBE
in addressing both removal of toxic copper and the radiometal
transchelation problems. Specifically, azide modified CB-
TE1K1P chelator (N3-CB-TE1K1P)26 was incubated with 64Cu for
5 min at 90 °C, and the resulting N3-(64Cu)CB-TE1K1P chelate
was then conjugated to acetylene-AE105 peptide via CuAAC
catalyzed by different ligands (Scheme S2). FBTTBE exhibited
much higher catalytic efficacy than either TBTA or THPTA
Figure 4. HPLC results: A) N3-CB-TE1K1P(64Cu) click reaction with
acetylene-AE105 catalyzed by 1.0 eq. of FBTTBE; B) Spectrum
after the fluorous-silica gel purification of the reaction mixture.
more sensitive to radiolabeling conditions such as pH,
temperature and incubation time than small molecules and
peptides. As a proof-of concept study, a monoclonal anti-EGFR
antibody (cetuximab) was functionalized with an acetylene
group using a slightly modified procedure from what was
previously reported.27 The resulting acetylene-cetuximab was
conjugated with N3-(64Cu)CB-TE1K1P via the Cu(I)-FBTTBE
catalyzed CuAAC under mild conditions (37 °C for 30 min) that
would not denature the protein. The results were encouraging
in that 50% of the (64Cu)CB-TE1K1P was attached to
cetuximab. The specific activity of the resulting 64Cu-labeled
cetuximab was 10 μCi/μg after purification by a Zeba desalting
column. Together, these data demonstrated the broad
applicability of the Cu(I)-FBTTBE catalyst for radiometal
labeling of peptides and antibodies.
(Figure S3). The rapid F-SPE removal capability was then
investigated by comparing the UV peaks in the two HPLC
traces of the above CuAAC mixtures before and after F-SPE
purification. The retention time of the FBTTBE/FBTTBE-Cu(I)
appeared to be 22.3 min according to the HPLC trace of the
CuAAC mixture before F-SPE purification (Figure 4A). After the
F-SPE purification, it was found that the UV peak representing
the FBTTBE/FBTTBE-Cu(I) completely disappeared (Figure 4B),
indicating that the catalyst was completely removed by the
fluorous silica gel. In addition, the reaction mixture was also
analyzed by the radio-HPLC to monitor potential
transchelation. It was found that the radio-purity of the final
product (retention time = 20 min, Figure 4A) was >98%, while
there was no radioactive peak at either 1.5 or 22.3 min for Cu
ions and FBTTBE-Cu(I), respectively. Therefore, FBTTBE can be
regarded as an excellent ligand for the preparation of
radiometal-based radiopharmaceuticals. The resulting
(64Cu)CB-TE1K1P-AE105 was subsequently used for PET/CT
imaging of nude mice bearing subcutaneous U87MG
xenografts that overexpress uPAR (Figure 5)
The FBTTBE ligand has also been successfully applied in the
rapid radiometal labeling of antibodies. Proteins are typically
.
Figure 5. PET/CT imaging of mice bearing U87MG xenografts: A) 1
h p.i; B) 4 h p.i.; c) with blockade, 4 h p.i.
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18.
D. Soriano del Amo, W. Wang, H. Jiang, C. Besanceney, A.
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C. Yan, M. Levy, Y. Liu, F. L. Marlow and P. Wu, J Am Chem
DOI: 10.1039/C5CC06858D
Soc, 2013, 2010, 132, 16893-16899.
J. Marik and J. L. Sutcliffe, Tetrahedron Lett, 2006, 47,
6681-6684.
D. Zeng, B. M. Zeglis, J. S. Lewis and C. J. Anderson, J Nucl
Med, 2013, 54, 829-832.
V. Kumar and D. K. Boddeti, Recent Res Cancer, 2013, 194,
189-219.
C. J. Anderson and R. Ferdani, Cancer Biother Radio, 2009,
24, 379-393.
I. Verel, G. W. Visser, R. Boellaard, M. Stigter-van Walsum,
G. B. Snow and G. A. van Dongen, J Nucl Med, 2003, 44,
1271-1281.
K. Willowson, N. Forwood, B. W. Jakoby, A. M. Smith and
D. L. Bailey, Med. Phys, 2012, 39, 7153-7159.
W. H. Bakker, R. Albert, C. Bruns, W. A. Breeman, L. J.
Hofland, P. Marbach, J. Pless, D. Pralet, B. Stolz, J. W.
Koper and et al., Life Sci, 1991, 49, 1583-1591.
D. Zeng, Q. Ouyang, Z. Cai, X.-Q. Xie and C. J. Anderson,
Chem Com, 2014, 50, 43-45.
In summary, a novel CuAAC ligand FBTTBE has been
successfully developed that not only demonstrated higher
catalytic efficiency than two commercial available ligands
(TBTA & THPTA), but also simplified the removal of toxic
copper species after reactions, rendering it an ideal ligand for
CuAAC. In addition, transchelation was avoided when applying
FBTTBE in preparing radiometal-based pharmaceuticals,
broadening the application of CuAAC beyond 18F-chemistry.
Although reactions presented here are primarily geared
toward PET and molecular imaging, FBTTBE can be applied in
almost any CuAAC where the high reaction rate as well as the
complete removal of copper species are desired.
We thank Dr. Sai Zhao for his assistance with the synthesis and
fluorogenic assay. This work was supported by the National
Institute of Biomedical Imaging and Bioengineering grant R21-
EB017317. Preclinical PET/CT imaging is supported in part by
P30CA047904 (UPCI CCSG).
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D. Zeng, Y. Guo, A. G. White, Z. Cai, J. Modi, R. Ferdani and
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