Chemoselective sequential "click" ligation using unsymmetrical bisazides

Article abstract of DOI:10.1021/ol300899n

Unsymmetrical bisazides containing chelating and nonchelating azido groups undergo chemoselective three-component copper(I)-catalyzed azide-alkyne conjugation reactions with two different alkyne molecules. In conjunction with the reactivity gap between ar

Full text of DOI:10.1021/ol300899n

ORGANIC
LETTERS
2012
Vol. 14, No. 10
2590–2593
Chemoselective Sequential “Click”
Ligation Using Unsymmetrical Bisazides
Zhao Yuan, Gui-Chao Kuang, Ronald J. Clark, and Lei Zhu*
Department of Chemistry and Biochemistry, Florida State University, Tallahassee,
Florida 32306-4390, United States
lzhu@chem.fsu.edu
Received April 7, 2012
ABSTRACT
Unsymmetrical bisazides containing chelating and nonchelating azido groups undergo chemoselective three-component copper(I)-catalyzed
azideÀalkyne conjugation reactions with two different alkyne molecules. In conjunction with the reactivity gap between aromatic and aliphatic
alkynes, a bistriazole molecule can be generated with excellent regioselectivity by mixing two alkynes and a bisazide in a single reaction
container. This method is applicable in aqueous solutions at neutral pH, which may lend utilities in bioconjugation applications.
Chemoselective sequentialligation has beenemployed in
applications including multiple labeling1 and syntheses of
macromolecular assemblages.2 The two strategies in devis-
ing a sequential ligation are (1) successive coupling reac-
tions of the same type that require the deprotection of the
subsequent reactive site,1a,2d,e and (2) the utilization of two
or more completely different reactions.1b,c,2aÀc Recent
research in bifunctional molecular linkers highlights the
key challenge in developing chemoselective sequential
ligationprotocols, which istoachievecompleteregiochem-
ical control of the coupling reactions in a timely and cost-
effective manner, while providing broad functional group
tolerance.
Figure 1. Unsymmetrical bisalkyne linkers.
widely applied “click” reactions,5 appear to be the methods
of choice in developing bifunctional molecular linkers, due to
their enormous substrate scopes and rapid reaction kinetics.
Several examples of AAC-based bifunctional linkers are
listed in Figure 1, in which differentiating the reactivities of
alkyne substrates leads to chemoselectivity in sequential
ligation reactions. Aucagne and Leigh introduced a
double-click method, in which two CuAAC reactions of
compound 1 are spaced by a TMS-alkyne deprotection
step to gain chemoselectivity.6 In bisalkyne linker 2 by
Girard et al., an electron-deficient propiolamide, which
reacts with an azide thermally (i.e., without a copper
catalyst), pairs up with a propargylic group targeted for a
CuAAC reaction.7 The pairing of cyclooctyne and terminal
The copper(I)-catalyzed azideÀalkyne cycloaddition
(CuAAC)3 and its metal-free variants,4 which are the most
(1) (a) Gramlich, P. M. E.; Warncke, S.; Gierlich, J.; Carell, T.
Angew. Chem., Int. Ed. 2008, 47, 3442. (b) Sanders, B. C.; Friscourt,
F.; Ledin, P. A.; Mbua, N. E.; Arumugam, S.; Guo, J.; Boltje, T.; Popik,
V. V.; Boons, G.-J. J. Am. Chem. Soc. 2011, 133, 949. (c) Yi, L.; Sun, H.;
Itzen, A.; Triola, G.; Waldmann, H.; Goody, R. S.; Wu, Y.-W. Angew.
Chem., Int. Ed. 2011, 50, 8287.
(2) (a) Durmaz, H.; Dag, A.; Altintas, O.; Erdogan, T.; Hizal, G.;
Tunca, U. Macromolecules 2007, 40, 191. (b) Galibert, M.; Dumy, P.;
Boturyn, D. Angew. Chem., Int. Ed. 2009, 48, 2576. (c) Kempe, K.;
Hoogenboom, R.; Jaeger, M.; Schubert, U. S. Macromolecules 2011, 44,
ꢀ
€
6424. (d) Ehlers, I.; Maity, P.; Aube, J.; Konig, B. Eur. J. Org. Chem.
2011, 2474. (e) Valverde, I. E.; Lecaille, F.; Lalmanach, G.; Aucagne, V.;
Delmas, A. F. Angew. Chem., Int. Ed. 2012, 51, 718.
(3) Hein, J. E.; Fokin, V. V. Chem. Soc. Rev. 2010, 39, 1302.
(4) Sletten, E. M.; Bertozzi, C. R. Acc. Chem. Res. 2011, 44, 666.
(5) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
2001, 40, 2004.
(6) Aucagne, V.; Leigh, D. A. Org. Lett. 2006, 8, 4505.
(7) Elamari, H.; Meganem, F.; Herscovici, J.; Girard, C. Tetrahedron
Lett. 2011, 52, 658.
r
10.1021/ol300899n
Published on Web 05/04/2012
2012 American Chemical Society

alkyne is utilized in compound 3 by Beal et al., in which the
two triple bonds react via strain-promoted thermal reaction
and copper(I)-catalyzed means, respectively.8
groups with inherently different reactivities into one substrate,
such as 4À7 (Figure 2). These bisazides allow for sequential
CuAAC reactions with two distinct alkynes in a one-pot
procedure, without a protection/deprotection cycle. Moreover,
it was shown previously that different alkynes have various
reactivities to chelating azides,10b which makes it possible to
obtain a single product by simply mixing two alkynes and a
bisazide together in one experimental sequence.
Although effective to various degrees, these earlier
approaches suffer from a few limitations. The double-
click method requires a protection/deprotection sequence,
which adds to the workload. The thermal AAC reactions
using strained or electron-deficient alkynes are relatively
slow at rt, in addition to the lack of regioselectivity in
affording 1,4- or 1,5-disubstituted triazoles. Furthermore,
the propiolamide derivatives are prone to Michael ad-
dition with a nucleophile, thus limiting the scope of sub-
strates in sequential ligations. In another noteworthy
double-click method, amino-substituted organic azides
are employed in which a diazo transfer reaction is required
to activate the amino group to azido for the second
CuAAC reaction.9 Herein, wereporta double-conjugation
method in which two CuAAC reactions occur sequentially
in a single reaction mixture without an intervening depro-
tection step. This method affords excellent regioselectivity
while preserving the fast kinetics and large substrate scope
of the CuAAC reaction.
Figure 2. Unsymmetrical bisazides.
The syntheses of unsymmetrical bisazides 4À7 contain-
ing chelating and nonchelating azido groups are included
in the Supporting Information. Bisazides 4 and 7 contain a
2-(azidomethyl)pyridyl chelating component, and an ali-
phatic and a benzylic nonchelating azide, respectively. In
bisazide 5, the N3 nitrogen atom on the triazolyl ring and
the C4-azidomethyl group constitute a chelating compo-
nent, while a nonchelating aliphatic azido group is at-
tached via a 4-carbon linker. Compound 6 has a quinoline
core with a chelating 2-azidomethyl and a nonchelating
6-azidomethyl group. In 4À7, the chelating azido group
would react with an alkyne molecule first under the Cu-
(OAc)2-accelerated conditions,10b leaving the nonchelat-
ing azido group for the CuAAC reaction with the second
alkyne under more strongly reducing conditions.
Scheme 1. Azide Selectivity in CuAAC Reactions
Our group discovered that copper(II) acetate (Cu(OAc)2)
and chelating azides possess uniquely high reactivities in
CuAAC reactions.10 The reactivity difference between che-
lating and nonchelating azides is demonstrated in the two
CuAAC reactions starting with an equal molar mixture of
2-picolylazide and benzylazide (Scheme 1). In the presence of
only Cu(OAc)2, the added alkyne selectively reacts with the
chelating 2-picolylazide to afford triazoles A and C. The
subsequent addition of sodium ascorbate increases the con-
centration of the copper(I) catalyst, leading to the second tri-
azole formation (B and D) involving the nonchelating benzyl-
azide. The chemoselectivity in organic azides (chelating vs
nonchelating) opens up an opportunity to introduce two azido
Figure 3. ORTEP diagram of [Cu2(7)2Cl4] (30% ellipsoids).
Black, carbon and hydrogen; blue, nitrogen; green, chlorine;
orange, copper.
The single crystal structure of complex [Cu2(7)2Cl4]
(Figure 3) reveals the selective azido-copper interaction
in bisazide 7, which is the source of its chemoselectivity in
CuAAC reactions. The copper(II) center is square pyra-
midal, where the bidentate chelating 2-(azidomethyl)-
pyridyl moiety and two chloride ions constitute the square
plane. The nonchelating azido group is left unbound.
1-Ethynyl-4-nitrobenzene and propargyl alcohol were
employed as alkyne substrates in demonstrating the
(8) Beal, D. M.; Albrow, V. E.; Burslem, G.; Hitchen, L.; Fernandes,
C.; Lapthorn, C.; Roberts, L. R.; Selby, M. D.; Jones, L. H. Org. Biomol.
Chem. 2012, 10, 548.
(9) Guiard, J.; Fiege, B.; Kitov, P. I.; Peters, T.; Bundle, D. R.
Chem.;Eur. J. 2011, 17, 7438.
(10) (a) Brotherton, W. S.; Michaels, H. A.; Simmons, J. T.; Clark,
R. J.; Dalal, N. S.; Zhu, L. Org. Lett. 2009, 11, 4954. (b) Kuang, G.-C.;
Guha, P. M.; Brotherton, W. S.; Simmons, J. T.; Stankee, L. A.; Nguyen,
B. T.; Clark, R. J.; Zhu, L. J. Am. Chem. Soc. 2011, 133, 13984.
Org. Lett., Vol. 14, No. 10, 2012
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Table 1. Chemoselective Formation of Mono- and Bistriazolesa
a Azide (0.1 mmol) and alkyne (0.1 mmol) with 10 mol % of Cu(OAc)2 at rt; entries 1À8 in CH3OH/CH2Cl2 (1:1 v/v, 0.5 mL). b Isolated yield after
purification by column chromatography. c 2À3% impurity is contained in the isolated product, presumably the reverse regioisomer and/or
homobistriazole(s). d HEPES buffer (0.5 mL, 0.5 M, pH 7.0).
chemoselective double-click reaction involving bisazides
4À7 (eq 1, Table 1). The addition of the first alkyne to a
solution of a bisazide in a 1:1 molar ratio in the presence of
10 mol % Cu(OAc)2 resulted in the formation of mono-
triazoles T1ÀT8 (Table 1). The high isolated yields
(entries 1À8) indicated that the structures of bisazides
and alkynes have only a marginal influence on the effi-
ciency and chemoselectivity of the first CuAAC reaction.
Addition of the second alkyne with a catalytic amount of
Cu(OAc)2 (10 mol %) and sodium ascorbate (20 mol %) to
the CH2Cl2/CH3OH solution of the purified monotriazole
products resulted in the formation of bistriazoles
TT1ÀTT8 in good yields (yield of F). The results shown
in Table 1 from the two-step sequence demonstrate the
high reactivity of the chelating azide, which undergoes the
Cu(OAc)2-mediated cycloaddition with an alkyne without
affecting the nonchelating azide. The addition of sodium
ascorbate affords a high concentration of copper(I), which
is apparently needed for the nonchelating azido group to
ligate with the second alkyne. Thus, by using appropriate
reagents, sequential chemoseletive ligation of unsymme-
trical bisazides 4À7, eachof which contains a chelating and
nonchelating azido group, is achieved.
In addition to the stepwise syntheses of mono- and
bistriazoles, we carried out a “one-pot” double-click liga-
tion experiment without the isolation of the monotriazole
intermediate (eq 2, Table 1). The introduction of the first
alkyne into the CH2Cl2/CH3OH solution of a bisazide in
the presence of 10 mol % Cu(OAc)2 was followed 5 h later
by the addition of the second alkyne, which was accom-
panied by a sodium ascorbate solution to reduce Cu(OAc)2.
The CuAAC reaction between the nonchelating azide and
the second alkyne proceeded to afford single detectable
bistriazole products in excellent yields (Table 1, yield of G).
It is notable that the order of alkyne addition, not the
identity of the alkyne, directs the double-click reaction to
reach different bistriazole products. From comparison of
the overall isolated yields of bistriazoles from the stepwise
procedure (Table 1, yield of F), the higher yields of the one-
pot procedure (yield of G) suggest that not only the
chemoselectivity of the double-click reaction is maintained
but also efficient isolation of products with minimal
material loss is managed without separating the mono-
triazole intermediate.
One important benefit of the CuAAC reaction for
applications in chemical biology and material science is
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Org. Lett., Vol. 14, No. 10, 2012

the undiminished reactivity under physiological condi-
tions. Bisazide 7 was chosen to test the viability of chemo-
selective double ligation in an aqueous solution buffered
by HEPES at pH 7. The bistriazole products from both
stepwise (Table 1, entries 9 and 10) and one-pot procedures
were obtained in good isolated yields, which lends promise
in bioconjugation applications.11
Table 2. One-Pot, Three-Component Reactionsa
In our previous study, it was revealed that reactivities of
various alkynes differ under Cu(OAc)2-mediated conditions.10b
For example, enhanced acidity of an alkyne leads to the
increased rate of the CuAAC reaction, because alkyne depro-
tonation was shown to be kinetically significant.10b The differ-
ences in reactivities of both alkynes and azides make it possible
to perform chemoselective three-component (two alkynes with
different reactivities and a bisazide) reactions.
In the three-component reaction involving two different
alkynes and bisazide 7 (eq 3, Table 2), the alkyne with a higher
reactivity (e.g., 1-ethynyl-4-nitrobenzene and 4-ethynyltoluene)
shallundergoCu(OAc)2-mediated CuAAC reaction selectively
with the chelating azido group of 7. After the first reaction is
completed, the addition of sodium ascorbate to the reaction
mixture would lead to the second CuAAC reaction between
the nonchelating azido group and the less reactive alkyne
(e.g., propargyl alcohol and 1-hexyne).
a Bisazide 7 (0.1 mmol) and two alkynes (0.1 mmol) in CH3CN (0.5
mL) with addition of Cu(OAc)2 (10 mol %) followed by adding NaAsc
(20 mol %) after 5 h at rt. b Isolated yield after purification by column
chromatography. c 3% impurity is contained in the isolated product
presumably the reverse regioisomer and/or homobistriazole(s).
1-hexyne were consumed sequentially to afford bistriazole
TT10. The methyl groups of the two alkynes could con-
ceivably be replaced by other structural entities, thus
offering an attractive tool for the selective conjugation of
multiple components under mild conditions.
In conclusion, a method for the one-pot chemoselective
double-click reaction of three components is described.
This strategy employs unsymmetrical bisazides containing
chelating and nonchelating azido groups, which exhibit
different reactivities in CuAAC reactions. The easy pre-
paration and simple structures of bisazides 4À7 suggest
that they can be used as convenient conjugation linkers to
join two ethynyl-functionalized building blocks with high
regioselectivity under mild, including physiological condi-
tions. Exploring the utilities of the bisazide linkers in areas
of bioconjugation and material sciences is an ongoing
effort in our laboratory.
In agreement with the above expectation, the inclusion
of two alkynes (1-ethynyl-4-nitrobenzene and propargyl
alcohol) with bisazide 7 in CH3CN resulted in the first
cycloaddition selectively between the chelating azido
group and 1-ethynyl-4-nitrobenzene, and the second
CuAAC reaction between the nonchelating azido and
propargyl alcohol to afford the bistriazole in 70% isolated
yield (Table 2, entry 1). Compared to propargyl alcohol,
1-hexyne has a lower reactivity in CuAAC reaction. The
large disparity between the reactivities of 1-ethynyl-4-
nitrobenzene and 1-hexyne manifested in the one-pot
synthesis of bistriazole TT9, which was obtained in an
almost quantitative yield (Table 2, entry 2). The one-pot
reaction of entry 2 was monitored via 1H NMR spectro-
scopy, from which the exclusive ligation between the
chelating azido group and 1-ethynyl-4-nitrobenzene was
observed in CD3CN in the presence of the idle 1-hexyne
(Figures S1ÀS2).12
Acknowledgment. This work was supported by the U.S.
National Science Foundation (CHE-0809201).
4-Ethynyltoluene and 1-hexyne are also effective in
the one-pot double-click reaction involving bisazide 7, in
which the aromatic 4-ethynyltoluene and the aliphatic
Supporting Information Available. Experimental proce-
dures, characterization of new compounds, .cif file of
[Cu2(7)2Cl4], and additional figures. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
(11) Kalia, J.; Raines, R. T. Curr. Org. Chem. 2010, 14, 138.
(12) The second CuAAC reaction after the addition of sodium
ascorbate did not take place at the prescribed concentration levels in
the NMR tube, even at an elevated temperature.
The authors declare no competing financial interest.
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