Copper(I)-Chelated Cross-Linked Cyclen Micelles as a Nanocatalyst for Azide-Alkyne Cycloaddition in Both Water and Cells

Article abstract of DOI:10.1002/adsc.201900773

Featuring the dendrimer-like properties, the cross-linked small-molecule micelles (cSMs) have been shown to be a good alternative to dendrimers in many applications. Following this trend, herein the copper(I)-chelated cross-linked cyclen micelles (Cu

Full text of DOI:10.1002/adsc.201900773

UPDATES
DOI: 10.1002/adsc.201900773
Copper(I)-Chelated Cross-Linked Cyclen Micelles as a
Nanocatalyst for Azide-Alkyne Cycloaddition in Both Water and
Cells
Fuqing Xiang,a Bing Li,a Pengxiang Zhao,c Jiangbing Tan,a Yunlong Yu,a and
Shiyong Zhanga, b,*
a
National Engineering Research Centre for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu 610064, China
Fax: +86-28-85411109
Phone: +86-28-85411109
E-mail: szhang@scu.edu.cn
b
College of Chemistry, Sichuan University, 29 Wangjiang Road, Chengdu 610064, China
Institute of Materials, China Academy of Engineering Physics, No. 9, Huafengxincun, Jiangyou, 621908, China General
c
methods
Manuscript received: June 24, 2019; Revised manuscript received: August 30, 2019;
Version of record online: ■■■, ■■■■
Supporting information for this article is available on the WWW under https://doi.org/10.1002/adsc.201900773
copper catalysts supported in organic nanomaterials
Abstract: Featuring the dendrimer-like properties,
the cross-linked small-molecule micelles (cSMs)
have recently attracted attention due to their superiority
in overcoming these problems.[2b,3] Among them,
have been shown to be a good alternative to
polymer nanoparticles and dendrimers are currently
dendrimers in many applications. Following this
two main supports for the loading of coppers.
trend, herein the copper(I)-chelated cross-linked
cyclen micelles (CuI@cCMs) were created as a
However, most of the polymer nanoparticle supported
copper catalysts are heterogeneous and face the short-
nanocatalyst for azide-alkyne cycloaddition. Both
coming of low activity due to the diffusion
alkynes and azides with diverse structures per-
limitations;[4] besides, polymer nanoparticles suffer
formed with excellent reactivity in water at the
also the stability issue due to their non-covalent bond
parts-per-million (ppm) catalyst usage. Recycle
synthesis. The dendrimers, benefited from their precise
experiments disclosed that the nanocatalyst had only
molecular weight, spherical nanostructure, functional-
slight decrease of catalytic efficiency after reusing
many times. Importantly, the CuI@cCMs could
ized exterior, and superstability, can take the advan-
tages of both heterogeneous and homogenous
easily enter cells and carry out the intracellular
catalysts,[2b,5] and seem to be a more superior catalyst
catalysis for lighting up and/or killing cancer cells.
carrier. In this regard, Astruc and coworkers did
seminal studies,[5b] and in some of them the copper
Keywords: Cross-linked small-molecule micelles;
Cyclen; Azide-alkyne cycloaddition; CuI; Intracellu-
dosage was even reduced to parts-per-million (ppm)
level and could be recycled many times.[3b,c] However,
lar catalysis
although these dendrimer supported metal catalysts are
promising, the dendrimer carriers themselves require
complex chemical synthesis. Moreover, the copper
ions were always protected in the dendrimer’s cavity
which was unfavorable to the contact between copper
Introduction
ions and substrates, thus resulting in the reaction
The copper catalyzed azide-alkyne cycloaddition kinetic drop.[6]
(CuAAC) has proven to be a pivotal advance in
The cross-linked small-molecule micelles (cSMs)
chemical ligation strategies, with applications ranging are covalently surface- or core-captured spherical
from synthetic organic chemistry to material chemistry, micelles assembled by the amphiphiles with typically
and to biomedicinal chemistry.[1] However, the classi- molecular weight below 1000.0 Da.[7] Featuring the
cal CuAAC reactions suffer from problems of high dendrimer-like properties (e.g., spherical shape, con-
catalyst dosage and absent recyclability.[2] In this case, trollable size, functionalized exterior, and good stabil-
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Scheme 1. Schematic illustration of the preparation of CuI@cCMs
ity), the cSMs have been shown to be a good micelle concentration (CMC, ~65.8 μM, Figure S1)
alternative to dendrimers in many applications.[7,8] In with a hydrodynamic diameter of ~36.0 nm (Fig-
organic synthesis, the cSMs supported rhodium ure 1a). The cross-linked cyclen micelles (cCMs) were
catalyst,[9] zinc complex,[10] gold(0) nanoclusters,[11] prepared by adding dithiothreitol (DTT) and 2,2-
and copper(0) nanoparticles[12] have successfully been dimethoxy-2-phenylacetophenone (DMPA, a photo-
developed to catalyze various organic reactions in initiator) to CMs solution under UV (254 nm) irradi-
1
aqueous phase with excellent catalytic efficiency, ation. The disappearance of alkenyl protons in H
selectivity, and recyclability.
NMR spectra demonstrated the successful cross-link-
In this study, we reported the copper(I)-chelated ing of the micelles (Figure S2). Notably, the average
cross-linked 1,4,7,10-tetraazacyclododecane (cyclen) diameter of cCMs was around 45.0 nm (Figure 1a), a
micelles (CuI@cCMs) as a nanocatalyst for azide- little bit larger than that of CMs. This is reasonable
alkyne cycloaddition in both water and cells. As shown considering that the cross-linking agents were intro-
in Scheme 1, the cyclen group can not only provide the duced into the micelle core after cross-linking.[7,8b]
hydrophilic part of the micelles, but also locate the
The preparation of the nanocatalyst CuI@cCMs
copper ions on the surface of micelles through involved addition of anhydrous CuSO4 to cCMs
coordination so as to facilitate the contact between
substrates and catalytic centers. Under the optimized
I
°
reaction condition of 20 ppm Cu @cCMs and 35 C for
24 h, the click reaction reached 82–99% isolated yields
for twenty substrates. The CuI@cCMs maintained a
high catalytic efficiency (>90%) even after five-time
recycles. Importantly, the CuI@cCMs could easily
enter cells and carry out the intracellular catalysis for
lighting up and/or killing cancer cells. The killing
efficiency was even twice higher than that of the
directly use original drug. With the help of cyclen on
the particle surface, the cCMs can also coordinate
other transition metal ions easily and therefore hold
great potential as a general platform for the synthesis
of various nano-catalysts according to need.
Results and Discussion
Compound 5 was prepared in two steps by the
Figure 1. Characterization of the cCMs and CuI@cCMs. a)
alkylation of di-allylamine with readily available 1,12-
Distribution of the hydrodynamic diameters of CMs, cCMs and
dibromododecane, followed by the alkylation of com-
CuI@cCMs. b) UV-vis spectroscopy of cCMs, free CuII, Cu
II@cCMs and CuI@cCMs. c) TEM micrograph of cCMs. d)
pound
4
with
1,4,7,10-tetraazacyclododecane
TEM micrograph of CuI@cCMs. All samples were stained with
an aqueous solution of 2% phosphotungstic acid.
(Scheme 1). The amphiphile 5 was spontaneously
assembled into cyclen micelles (CMs) above its critical
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solution and then in situ reduction of the precatalyst suggesting the stability of the CuI@cCMs. Then, to
CuII@cCMs by dropwise addition of sodium L(+)-as- verify the generality of CuI@cCMs, a series of cyclo-
corbate (NaAsc) under nitrogen atmosphere (Scheme 1 addition reactions between azides and alkynes under
and see the Supplementary Information for details). the optimal reaction condition were conducted (Ta-
The preparation process of CuI@cCMs was monitored ble 1). To our delight, the click reactions between the
by UV-vis spectroscopy, dynamic light scattering azides with either donating or withdrawing substituting
(DLS), and transmission electron microscopy (TEM). groups and terminal alkyne with different substituents
As shown in Figure 1b, in UV-vis spectroscopy, an both obtained good yields (entries 1–18). The func-
aqueous solution of CuSO4 had an absorption in the tional biomolecules with fluorogenic and medicinal
range of 650–800 nm, which is attributed to the CuII d- interests were synthesized by the click reaction in
d transition.[13] The CuII@cCMs showed also an aqueous media, and the separation yields of both were
absorption peak at 650–800 nm while the intensity above 90% (entries 19 and 20). In general, twelve
weakened, which indicated that the CuII was located on different azides and five different terminal alkynes
cyclen successfully (Figure 1b).[13–14] The CuI@cCMs underwent CuI@cCMs catalyzed click reaction, with
demonstrated no absorption at 650–800 nm, while a isolated yields ranging from 82% to 99% under the
new peak appeared between 300 and 400 nm (main optimal reaction condition, validating the quite sub-
absorption at 370 nm, Figure 1b) which was attributed strate adaptability of CuI@cCMs.
as a metal to ligand charge transfer (MLCT)
To test the recyclability of the CuI@cCMs, phenyl-
transition.[14] The DLS measurement revealed that the acetylene and benzyl-azide were employed as the
average diameter of CuI@cCMs was around 45.0 nm, model substrates. At the end of each cycle, the catalyst
consistent with the size of cCMs. The TEM character- was recovered by extraction-filtration process, and the
ization showed the CuI@cCMs have similar morphol- organic layer containing the click products was
1
ogy and size with the cCMs (Figure 1c, d). Interest- concentrated and analyzed by H NMR spectroscopy.
ingly, by dyeing with phosphotungstic acid, the Cu In this way, the catalyst still maintained its catalytic
I@cCMs was positively stained while the cCMs was efficiency above 90% after the fifth cycle (Figure 2,
negatively stained.[15] This phenomenon happened see the Supplementary Information for details). These
possibly because the coordination of CuI changed the results suggested that the CuI@cCMs would be served
combination way between phosphotungstic acid and as an economical nanocatalyst for organic synthesis.
cCMs, consequently leading to the change of staining
After confirming the excellent reaction efficiency
mode (Figure 1c, d). Since the cyclen has a strong of CuI@cCMs in aqueous phase, their potential to act
coordination ability toward a variety of cations, we as an intracellular catalyst was tested in living cells. As
used TiIII, PdII, FeIII to chelate with cCMs in aqueous
phase, respectively. It was found that they were all
successfully coordinated with cCMs according to the
characterization of UV-vis, Zeta potential and induc-
tively coupled plasma-atomic emission spectroscopy
(ICP-AES) (Figure S3–S6, see Supplementary Infor-
mation for details). This result suggests that the cCMs
are a general platform to form various metal nano-
catalysts.
The reaction between benzyl-azide and phenyl-
acetylene was set as the standard model to test the
catalytic efficiency of CuI@cCMs with various
I
°
amounts of Cu under nitrogen atmosphere at 35 C in
aqueous solution (Table S1). The studies showed that
the best condition was 20 ppm CuI@cCMs under
°
nitrogen atmosphere and 35 C for 24 h, which led to a
full conversion of starting substrates and 90% isolated
yield. It should be noted that the addition of 20 mol%
triethylamine did not improve the catalytic efficiency
(Table S1, entries 2 and 4), suggesting that the cyclen
served a dual role in this regard. At the end of the
reaction, the copper content of CuI@cCMs was
determined via ICP-AES, which showed that the
residual Cu was 19.3 ppm, very close to the copper
content of CuI@cCMs without catalysis (20.0 ppm)
(see the Supplementary Information for details),
Figure 2. Recyclability of the CuI@cCMs ([Cu]=100 ppm) in
click reaction in water. a) The click reaction between benzyl
azide (0.2 mmol) and phenylacetylene (0.201 mmol) under nitro-
°
gen at 35 C. b) The relationship between the number of cycles of
CuI@cCMs and the catalytic efficiency.
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a well-known coumarin derivative, 3-azido-7-coumarin
is often used in the alkyne-azide cycloaddition reaction
to determine whether the reaction takes place by
generating new “light up” coumarin derivatives.[12,16]
Thus, the potential for intracellular CuI@cCMs catal-
ysis of the click reaction between 3-azido-7-coumarin
(1k) and phenyl-acetylene (2a) were tested in living
cells using confocal microscopy (Figure 3a). Briefly,
Table 1. Click” reactions between various terminal alkynes and
azides using CuI@cCMs in water[a]
Figure 3. Investigation of the catalytic activity of CuI@cCMs
in living cells. a) The CuI@cCMs mediated cycloaddition of 3-
azido-7-coumarin (1k) and phenyl acetylene (2a). b) Confocal
laser-scanning microscopy (CLSM) and intensity comparison of
the intracellular catalysis of CuI@cCMs on the “click” reaction
between precursors 1k (50 μM) and 2a (50 μM). c) Model
study of the intracellular synthesis of anticancer agent 3t from
precursors 1l and 2e in HeLa cells, catalyzed by CuI@cCMs. d)
Viability of HeLa cells in precursors 1l, 2e and 3t.
human cervical cancer (HeLa) cells were preincubated
with CuI@cCMs ([Cu]=20 ppm in Dulbecco’s modi-
fied
Eagle medium, DMEM) for 1 h; the DMEM was
removed and the cells were washed three times by
phosphate buffered saline (PBS) to eliminate extrac-
ellular CuI@cCMs, and subsequently incubated with
°
1k (50 μM) and 2a (50 μM) substrates at 37 C for
1 h. As seen in Figure 3b, only weak fluorescence
could be observed in the absence of CuI@cCMs,
whereas cells were intensely fluorescent in the pres-
ence of the catalyst, suggesting the generation of
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fluorescent molecule 1a. High resolution mass spec- catalysts, which would play a role in wide organic
trometry (HR-MS) confirmed that compound 3s with a synthesis.
molecular weight of 306.0874 was synthesized in cells
([M+H]+, Figure S9). The experimental results
Experimental Section
showed that the CuI@cCMs is not only an excellent
solution catalyst, but also an ideal catalyst for intra- General Procedure for the CuI@cCMs Catalyzed
cellular click reaction.
Azide-Alkyne Cycloaddition in Water
To further investigate the application scope of Cu
I@cCMs, intracellular generation of active drugs for
cancer therapy was conducted in HeLa cells. Typically,
the triazole-containing anticancer agent 3t, which is
known to inhibit tubulin polymerization to interfere
with the mitotic spindle assembly during cell division,
A 20 ppm of catalyst CuI@cCMs, alkyne (0.105 mmol), organic
azide (0.1 mmol), and 1.0 mL water were added to a Schlenk
°
flask. The reaction mixture was then stirred at 35 C under
nitrogen atmosphere for 24 h. The progress of the reaction was
monitored by TLC. After completion of the reaction, the
reaction mixture was extracted with EtOAc (3×5 mL). The
was synthesized from two benign precursors 1l and 2e combined organic phase was washed with brine (10 mL) and
(Figure 3c).[12,16] As shown in Figure 3d, when HeLa
dried over anhydrous Na2SO4. The solvent was removed under
reduced pressure and the residue was purified by column
chromatography (petroleum/ EtOAc) to afford the acquire
product.
cells were incubated with different combinations of
precursor 1l, 2e, [1l+2e], and CuI@cCMs for 24 h,
there showed negligible cytotoxicity. By contrast, the
combination of [1l+2e+CuI@cCMs] showed re-
Catalytic Activity Study of CuI@cCMs in Living
markable cytotoxicity to HeLa cells. The HR-MS
confirmed the formation of compound 3t in the cells
with the molecular weight of 357.1563 ([M+H]+,
Figure S10). Interestingly, the cytotoxicity of [1l+2e
+CuI@cCMs] was even twice than that of anticancer
agent 1b (10.2% vs 20.5%), possibly owing to the high
uptake rate of precursors and subsequently in situ
generation of high local concentration of active drugs
realized by the CuI@cCMs near the nucleus.[17] Thus,
the CuI@cCMs catalyzed in situ generation of active
drugs seems to be an effective strategy to improve the
utilization rate of drugs.
Cells
HeLa cells were plated in DMEM in a 96-well plate grown for
°
24 h at 37 C with 5% CO2. The medium was removed and a
solution of CuI@cCMs ([Cu]=20 ppm) in DMEM was added
°
and incubated for 1 h at 37 C. Then the medium was removed,
and 3-azido-7-coumarin (1k, 50 μM) and phenylacetylene (2a,
50 μM) were added. The solution was incubated for 1 h at
°
37 C. As a control, the samples without any catalyst were
°
incubated for the same time at 37 C. After incubation, cells
were washed twice with PBS, and the fluorescence of
compound 3s was detected by confocal laser-scanning micro-
scopy. The catalytic activity of the nanocatalyst in living cells
was determined by the quantitative fluorescence signals as a
function of time.
The biocompatibility of CuI@cCMs was finally
evaluated (see the Supplementary Information for
details). As shown in Figure S7, the cCMs demon-
strated no cytotoxicity even at a concentration of
150 μg/mL. Due to the protection of cyclen of cCMs,
the CuI@cCMs showed little cytotoxicity as well, with
full cell viability at concentrations up to 100 ppm.
However, the conventional catalyst CuBr exhibited
significant concentration-dependent cytotoxicity with
the survival rate of treated cells less than 80% at a
concentration of 2.5 ppm CuI and less than 50% at
50 ppm CuI (Figure S8).
CuI@cCMs Catalyzed Intracellular Azide-Alkyne
Cycloaddition for in situ Generation of the Anti-
cancer Agent 3t
HeLa cells were plated in DMEM in a 96-well plate and grown
for 24 h at 37 C with 5% CO2. Then the medium was removed
°
and a solution of CuI@cCMs ([Cu]=20 ppm) in DMEM was
added and incubated for 1 h. Then the medium was removed,
and different combinations of precursor 1l (50 μM), 2e
(50 μM), [1l (50 μM) + 2e (50 μM)] and 3t (50 μM) were
added and incubated for 24 h. After the incubation, the culture
media was removed and fresh media (100 μL) containing CCK-
8 (10 μL) was added to each well. The plates were incubated at
Conclusions
In summary, we developed cross-linked cyclen mi-
celles for copper(I)-chelation (CuI@cCMs) to catalyze
°
37 C for another 2 h. The absorbances of each sample at
the azide-alkyne cycloaddition. Featuring the robust 450 nm were measured using a varioscan flash microplate
reader. The generation of anticancer agent 3t was determined
stability, water solubility, easy contact with substrates,
and biocompatibility, the CuI@cCMs exhibited re-
markable catalytic activity both in water and cells at
the parts-per-million (ppm) level. The nano-catalyst
could be recovered easily and had only slight decrease
of catalytic efficiency after reusing five times. Besides
copper, the cross-linked cyclen micelles could coor-
dinate other transition metals as to form various nano-
by a cell viability assay (vide infra).
Acknowledgements
This work was supported by National Natural Science
Foundation of China (No. 21975165, 51673130 and 51703145).
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We are grateful for the characterization examination test
provided by Center of Testing and Analysis, Sichuan University
amd “ceshigo” (www.ceshigo. com).
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Copper(I)-Chelated Cross-Linked Cyclen Micelles as a Nano-
catalyst for Azide-Alkyne Cycloaddition in Both Water and
Cells
Adv. Synth. Catal. 2019, 361, 1–7
F. Xiang, B. Li, P. Zhao, J. Tan, Y. Yu, S. Zhang*