Tailored ligand acceleration of the cu-catalyzed azide-alkyne cycloaddition reaction: Practical and mechanistic implications

Article abstract of DOI:10.1021/ja105743g

Tris(heterocyclemethyl)amines containing mixtures of 1,2,3-triazolyl, 2-benzimidazoyl, and 2-pyridyl components were prepared for ligand acceleration of the copper-catalyzed azide-alkyne cycloaddition reaction. Two classes of ligands were identified: thos

Full text of DOI:10.1021/ja105743g

Published on Web 09/23/2010
Tailored Ligand Acceleration of the Cu-Catalyzed
Azide-Alkyne Cycloaddition Reaction: Practical and
Mechanistic Implications
Stanislav I. Presolski, Vu Hong, So-Hye Cho, and M.G. Finn*
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research
Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
Received June 29, 2010; E-mail: mgfinn@scripps.edu
Abstract: Tris(heterocyclemethyl)amines containing mixtures of 1,2,3-triazolyl, 2-benzimidazoyl, and
2-pyridyl components were prepared for ligand acceleration of the copper-catalyzed azide-alkyne
cycloaddition reaction. Two classes of ligands were identified: those that give rise to high reaction rates in
coordinating solvents but inhibit the process when used in excess relative to copper and those that provide
for fast catalysis in water and are not inhibitory in excess. Several “mixed” ligands were identified that
performed well under both types of conditions. Kinetics measurements as a function of ligand:metal ratio
and catalyst concentration were found to be consistent with an active Cu2L formulation. Since strongly
bound chelating agents are not always the most effective, achieving optimal rates requires an assessment
of the potential donor molecules in the reaction mixture. Simple rules are provided to guide the user in the
choice of effective ligands and reaction conditions to suit most classes of substrates, solvents, and
concentrations.
Introduction
with a revealing exploration of the dependence of the reaction
on the nature of the solvent and the ligand:Cu ratio.
The CuI-catalyzed azide-alkyne cycloaddition1 (CuAAC)
click reaction has found use in a remarkably wide range of
settings and applications. Simple Cu(I) salts1a or the convenient
combination of a Cu(II) precursor and a reducing agent
(typically, CuSO4 and sodium ascorbate,1b top of Figure 1)
provide species that mediate azide-alkyne ligation at high
enough rates for many purposes, approximately 106 times faster
than without the metal.2 For more demanding applications, the
use of certain Cu-binding ligands has been found to accelerate
the CuAAC reaction even more, up to several thousand times
over the ligand-free process.3 We focused on C3V-symmetric
tris(heterocyclemethyl)amines, derivatives of the tris(triazolyl-
methyl) structure 1 (Figure 1) obtained by CuAAC reaction of
tripropargylamine and organic azides.3a Recently, we reported
benzimidazole-based ligands 2 to be superior under conditions
of low catalyst loading and high substrate concentration.4
Tris(pyridylmethyl)amines such as 3 are well-known ligands
for copper5 and provide some CuAAC rate acceleration.6 We
describe here the mixing of these binding motifs for the purposes
of catalyst optimization and mechanistic investigation, along
Results
We prepared the “hybrid” tris(heterocyclemethyl)amine de-
rivatives shown in Figure 1, mixing 1,2,3-triazolyl, 2-benzimid-
azolyl, and 2-pyridyl donors on the ligand “arms.” Catalysts
incorporating these ligands were compared by calorimetry on
the reaction of benzyl azide with phenylacetylene in 4:1 DMSO:
water, as we have done previously.4 Shown in Figure 2A are
the values of maximum heat output, representing the peak
activity of each catalyst at the start of the reaction. Instead of
a direct dependence of rate on heterocycle content, a nonlinear
pattern was observed for both triazole and pyridyl systems:
(6) (a) References to rates of different types of CuAAC catalysts are
included in the following: Meldal, M.; Tornøe, C. W. Chem. ReV.
2008, 108, 2952–3015. (b) Other recently reported CuAAC catalysts
of different types include the following. (i) D´ıez-Gonza´lez, S.; Nolan,
S. P. Angew. Chem., Int. Ed. 2008, 46, 9013–9016. (ii) Fabbrizzi, P.;
Cicchi, S.; Brandi, A.; Sperotto, E.; van Koten, G. Eur. J. Org. Chem.
2009, 5423–5430 (aminothiolate complexes useful in organic solvents).
(iii) Campbell-Verduyn, L. S.; Mirfeizi, L.; Dierckx, R. A.; Elsinga,
P. H.; Feringa, B. L. Chem. Commun. 2009, 2139–2141 (phosphora-
midite complexes in DMSO-water, rate constants not given). (iv)
Candelon, N.; Laste´coueres, D.; Diallo, A. K.; Aranzaes, J. R.; Astruc,
D.; Vincent, J.-M. Chem. Commun. 2008, 741–743 (Cu-tren complex
showing high turnover numbers at elevated temperature in organic
(1) (a) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002,
67, 3057–3062. (b) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.;
Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41, 2596–2599.
(2) Lewis, W. G.; Green, L. G.; Grynszpan, F.; Radic, Z.; Carlier, P. R.;
Taylor, P.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2002,
41, 1053–1057.
¨
solvents). (v) Ozcubukcu, S.; Ozkal, E.; Jimeno, C.; Perica´s, M. A.
Org. Lett. 2009, 11, 4680–4683 (tris(triazolyl)methyl complex, rate
constants not given). (vi) D´ıez-Gonza´lez, S.; Correa, A.; Cavallo, L.;
Nolan, S. P. Chem.sEur. J. 2006, 12, 7558–7564 (N-heterocyclic
carbene catalysts). (c) The fastest noncatalyzed cycloaddition reactions
of strained alkynes and organic azides appear to be photoinitiated cases
reported by Boons et al., approximately 100 times slower than the
CuAAC reactions described here: Poloukhtine, A. A.; Mbua, N. E.;
Wolfert, M. A.; Boons, G.-J.; Popik, V. V. J. Am. Chem. Soc. 2009,
131, 15769–15776.
(3) (a) Chan, T. R.; Hilgraf, R.; Sharpless, K. B.; Fokin, V. V. Org. Lett.
2004, 6, 2853–2855. (b) Lewis, W. G.; Magallon, F. G.; Fokin, V. V.;
Finn, M. G. J. Am. Chem. Soc. 2004, 126, 9152–9153.
(4) Rodionov, V. O.; Presolski, S.; Gardinier, S.; Lim, Y.-H.; Finn, M. G.
J. Am. Chem. Soc. 2007, 129, 12696–12704.
(5) Wei, N.; Murthy, N. N.; Chen, Q.; Zubieta, J.; Karlin, K. D. Inorg.
Chem. 1994, 33, 1953–1965.
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14570 J. AM. CHEM. SOC. 2010, 132, 14570–14576
10.1021/ja105743g  2010 American Chemical Society

Ligand Acceleration of CuAAC
A R T I C L E S
Figure 1. Ligand structures. (1-3) “Parent” ligands for CuAAC reactions. (4-10) “Hybrid” ligands based on 1,4-triazole (red), benzimidazole (black), and
pyridine (blue) groups. (11-13) Ligands previously shown to be strongly accelerating. (14, 15) Modified hybrid ligands. (16-22) Truncated ligands containing
fewer than three heterocyclic arms. Abbreviations below the compound numbers refer to the nature of the heterocyclic arms: Trz ) triazole, Bim )
benzimidazole, Py ) pyridine.
ligands containing a single benzimidazole and two triazoles or
pyridines were approximately as effective as the tris(benzimid-
azole) structure. This reprises an earlier observation that a
bis(benzimidazole)-mono(benzothiazole) ligand was less ef-
fective than its mono(benzimidazole) and tris(benzimidazole)
congeners.4
somewhat different picture emerged (Figure 2B). The benzimid-
azole-triazole series displayed the same sawtooth pattern of
rate vs heterocycle content as in Figure 2A, but the benzimid-
azole-pyridine series did not, with mono(benzimidazole)-bis
(pyridine) ligand 77 proving to be superior, even with respect
to optimized tris(benzimidazole) derivatives 11 and 12 reported
earlier.4 The “mixed ligand” approach therefore provides strong
improvement under these conditions, presumably reflecting
When the rates of more dilute reactions were measured by
aliquot quenching and determination of triazole formation,4 a
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A R T I C L E S
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Figure 2. Reactions in 4:1 DMSO:H2O. (A) Maximum heat output (mW) by calorimetry for two series of reactions catalyzed by Cu complexes of hybrid
ligands. Conditions: [BnN3] ) 360 mM, [PhCCH] ) 450 mM, [CuSO4] ) 2 mM, [Ligand] ) 2 or 4 mM, as indicated. (B) Observed second-order specific
activities for CuAAC reactions under more dilute conditions by aliquot quenching and LC-MS analysis: [BnN3] ) 1 mM, [PhCCH] ) 20 mM, [CuSO4] )
50 µM, [Ligand] ) 100 µM. (C) Observed second-order specific activities for CuAAC reactions accelerated by 7 as a function of ligand:copper ratio.
Conditions: [BnN3] ) 1 mM, [PhCCH] ) 20 mM, [CuSO4] ) 10 µM, [Ligand] ) varied. For these and all other kinetic data, see Supporting Information
for details of ascorbate concentration, pH, and temperature.
differences in the partitioning of coordination complexes of
copper among active and inactive forms.
various Cu-ligand combinations in mixtures of 10% DMSO
and 90% water or buffer.10 Most of the experiments were
performed with 0.1 mM azide and 0.2 mM alkyne to simulate
the conditions at which bioconjugation reactions are done in
our laboratory, with the results shown in Figure 3.
The relatively poor performance of catalysts containing 1, 3,
8, and 9 (Figure 2B) suggested that at least one benzimidazole
arm is necessary for high catalytic rates in DMSO-rich mixtures.
However, paring the ligand down to a single benzimidazoyl-
methylamine motif was ineffective, with 16 and 17 failing to
show ligand-accelerated catalysis. Adding a second benzimi-
dazole arm (18 and 20) proved to be inhibitory (rates lower
than the ligand-free process; data not shown).
We previously reported that tripodal ligands such as 2, 11,
and 12 strongly accelerate the CuAAC reaction even at high
ligand:Cu ratios.4,8 This is also true for the best hybrid ligand
(7) as illustrated in Figure 2C: at a very low Cu concentration
(10 µM), the reaction rate was maximal at [L]:[Cu] ) 2:1 but
slowed only modestly with 8 equivalents of ligand. It is
important to note that tris(benzimidazole) ligands such as 2,
11, and 12 do not reliably support catalytic turnover at Cu
concentrations less than 50 µM;8 ligand 7 is clearly superior in
this regard.
The pattern of relative reactivities for ligands 1-10 under
aqueous conditions was significantly different than in 80%
DMSO, when compared at their respective optimal ligand:Cu
ratios (Figure 3A vs 2B). While hybrid ligands 7 and 10
maintained high activity in both solvent systems, 8 and 9 (and,
to a lesser extent, 5 and 1) achieved excellent rates only in the
absence of large quantities of DMSO. A survey of reaction rate
as a function of ligand:metal ratio revealed three classes of
catalysts in water. Class I was comprised of four ligands that
maintained strong activity when used in a 2- or 4-fold molar
excess relative to Cu, in sharp contrast to their behavior in
DMSO (ligands 1, 9, 13, and 15; Figure 3B1 vs 2C and ref
8).11 Class II contains ligands that show a peak in activity at
ligand:Cu ratios of 0.5 or 1.0 but lose most of that activity when
used in greater amounts (ligands 7, 8, 10, 11, 14, and, to a lesser
extent, 4; Figure 3B2). Class III consists of relatively inactive
systems over all ligand:metal ratios, including tripodal ligands
2, 3, 5, and 6 (Figure 3B2). These behavioral classes track
closely with the relative affinities for CuI displayed by the
ligands and determined by the number and metal-binding powers
of the heterocyclic arms (triazoles , pyridines < benzimid-
azoles).8
Aqueous Reactions. The above experiments, as well as our
original kinetics measurements,4,8,9 were conducted in 80%
DMSO and 20% water in order to keep all components of the
reaction soluble, including both hydrophilic ascorbate and
hydrophobic reactants and products. Since the CuAAC reaction
has found frequent application in aqueous media, we tested
(7) (a) Pascaly, M.; Duda, M.; Schweppe, F.; Zurlinden, K.; Mu¨ller, F. K.;
Krebs, B. J. Chem. Soc., Dalton Trans. 2001, 828–837. (b) Yang,
X.-P.; Su, C.-Y.; Kang, B.-S.; Feng, X.-L.; Xiao, W.-L.; Liu, H.-Q.
J. Chem. Soc., Dalton Trans. 2000, 3253–3260.
(10) Potassium phosphate buffers are ideal as they cover a wide pH range
and do not contain chloride, which can be inhibitory at high
concentrations.
(11) Compound 15 was prepared in an attempt to make 9 more water
soluble with hydroxypropyl side chains, in the same way that 13 is a
derivative of 1.
(8) Rodionov, V. O.; Presolski, S.; D´ıaz, D. D.; Fokin, V. V.; Finn, M. G.
J. Am. Chem. Soc. 2007, 129, 12705–12712.
(9) Rodionov, V. O.; Fokin, V. V.; Finn, M. G. Angew. Chem., Int. Ed.
2005, 44, 2210–2215.
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Ligand Acceleration of CuAAC
A R T I C L E S
Figure 3. Reactions in 9:1 H2O:DMSO. (A) Observed second-order specific activities: [BnN3] ) 100 µM, [PhCCH] ) 200 µM, [CuSO4] ) 50 µM,
[Ligand] ) 25 µM. (B) Comparison of hybrid ligands at different ligand:copper ratios under the same conditions. The ligands are grouped by class and color
coded according to their heterocyclic content: black ) two or three benzimidazoles in the structure; dark blue ) two or three pyridines; red ) two or three
triazoles; light blue ) one of each heterocycle. (C) Comparison of ligands 7 and 1 under bioconjugation conditions.
Figure 4. Sequential ligand addition experiments. (A) CuAAC reactions in 10% aqueous DMSO were initiated with 50 µM CuSO4 and 100 µM of the first
indicated ligand. At 5 min, a concentrated solution of the second ligand was added to a final concentration of 100 µM (vertical dotted line). (B) As in part
A with reactions performed in 80% DMSO. For both sets of experiments, [BnN3] ) 100 µM, [PhCCH] ) 200 µM.
Binding Comparisons. We performed two experiments to test
the assumption that the class II mixed pyridine-benzimidazole
ligand 7 binds CuI more tightly than a class I tris(triazole) ligand
such as 1. First, a solution of 1, 7, and CuSO4 (200 µM each)
in the presence of 5 mM sodium ascorbate in 1:9 DMSO:H2O
was analyzed by electrospray mass spectrometry. In this mixture,
more than 90% of the CuI was bound by 7 (Supporting
Information). Second, the amount of triazole formed in the
presence of sequentially added ligands was determined by
aliquot quenching and LC-MS analysis, as shown in Figure 4.
In 10% aqueous DMSO containing relatively low concentrations
of azide and alkyne, two equivalents of ligand 1 per Cu atom
accelerated the CuAAC reaction whereas 7 was an inhibitor
(Figure 4A), the latter consistent with data shown in Figure 3B.
When 7 was added after 5 min to either a ligand-free reaction
or a reaction containing 1, a brief jump in activity was observed
before triazole formation ceased. These bursts of activity
reflected the transient formation of a ligand-accelerated catalyst
before an inhibitory equilibrium was reached. The same
experiments performed in 80% DMSO showed none of these
inhibitory features (Figure 4B), with the Cu(7)2 stoichiometry
forming a more active catalyst whether or not 1 was present.
Thus, in both inhibitory and acceleratory modes, in water or
DMSO, 7 bound CuI with higher affinity than its triazole
counterpart 1.
Solvent Effects. The loss of catalytic activity for a variety of
ligands when used in 2-fold excess relative to copper in 90%
water (Figure 3B) was quite striking, as this phenomenon had
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Presolski et al.
Figure 5. CuAAC rates with variations in solvent and ligand:metal ratio. Observed second-order specific activities for reactions accelerated by Cu ·7 in
20% H2O, 10% DMSO, and 70% polar organic cosolvent. [CuSO4] ) 50 µM, [7] ) variable, [BnN3] ) 100 µM, [PhCCH] ) 200 µM. The ligand:Cu ratio
in each solvent was varied from 0 to 2, as indicated. The donor abilities of the polar organic cosolvent are given by the basicity parameter ꢀ′ (representing
the free energy of potassium ion transport corrected for solvent polarity).12b Values for NMP were estimated from data reported in ref 12a.
not been observed in extensive studies in 4:1 DMSO:water
(Figure 2C).8 In order to further explore this solvent effect, we
performed the standard CuAAC reaction in mixtures containing
20% water, 10% DMSO, and 70% of a polar cosolvent (a
consistent formulation that kept all reactants and products
dissolved). As shown in Figure 5, DMSO, N-methylpyrrolidone
(NMP), and N,N-dimethylformamide (DMF) were the only
solvents in which reaction rates were not greatly diminished at
a 2:1 ligand:Cu ratio relative to the rates at 1:1 and 0.5:1 ratios.
These outcomes correlated better with the basicity parameter
ꢀ′ (representing the free energy of potassium ion transport
corrected for solvent polarity) than with the dielectric constant
of the solvents (Figure 5).12 Therefore, we suggest that DMSO,
Figure 6. Determination of rate order in [Cu ·7] in 4:1 and 1:9 mixtures
NMP, and DMF (as well as alkyne, see Supporting Information)
competitively bind CuI, preventing the strongly chelating ligands
from forming inhibitory complexes and keeping weakly chelat-
ing ligands from the metal. Acetonitrile, a more strongly
coordinating solvent due to its π-accepting ability, largely
inhibited the catalytic activity of both the Cu ·7 and the Cu ·1
systems under these conditions. Conversely, water was the only
solvent in which an excess of ligand (2:1 ligand:Cu ratio)
inhibited the CuAAC reaction relative to the ligand-free rate.
Kinetic Rate Orders. The rate order of the CuAAC reaction
in catalyst was determined with a constant [L]:[Cu] ratio. For
the most effective hybrid ligand, 7, the reaction was found to
be approximately third order in (Cu)(L) in 80% DMSO,
compared to first order in 90% water (Figure 6).13 Under the
latter conditions, the Cu ·7 system behaved the same regardless
of whether 0.5 or 1 equiv of ligand was employed, whereas the
rate order in Cu ·1 was sensitive to the [L]:[Cu] ratio (Figure
S10, Supporting Information).
of DMSO:H2O. [Cu] was varied from 10 to 200 µM, [7] was varied
according to the given ratio, [BnN3] ) 100 µM or 1 mM in 80% DMSO
and 100 µM in 90% H2O, [PhCCH] ) 2[BnN3]. In 80% DMSO, the
reactions containing <20 µM Cu stopped before completing many turnovers,
so initial rates are reported for these experiments. The correlations for
reactions in 80% DMSO should therefore be regarded as approximate
determinations of catalytic rate order.
On the basis of the above results, two general categories of
reaction media can be identified: those containing molecules
that have affinity for CuI metal centers, including solvents such
as DMSO, DMF, and NMP (“strong-donor conditions”), and
those poor in such molecules, such as solvents dominated by
water (“weak-donor conditions”). The best ligands for these two
conditions likewise are distinguished by their CuI binding ability.
For example, class I ligands such as 1, 8, and 9 contain no
benzimidazoles and are therefore relatively weak binders. As a
result, DMSO can compete with these ligands for Cu binding
sites, diminishing ligand-based acceleration (Figure 2B). How-
ever, under weak-donor conditions, these class I ligands form
the most productive Cu(I) complexes (Figure 3A). Class II
ligands such as 7 are stronger chelators, containing two or more
benzimidazole or pyridine groups, and therefore can maintain
productive Cu binding in the presence of competing solvent
donors such as DMSO (Figure 2B). In water, however, these
ligands block access to the metal center when used in 1:1 or
greater ligand:Cu ratios (Figure 3B2 and 3C). Class I ligands
are apparently incapable of forming such inhibitory complexes
and remain active at high [L]:[Cu] ratios (Figure 3B1).
Discussion
No single Cu(I) complex has been identified as an optimal
catalyst or precatalyst for CuAAC reactions under all conditions.
(12) (a) Marcus, Y. Pure Appl. Chem. 1983, 55, 977–1021. (b) Gajewski,
J. J. J. Am. Chem. Soc. 2001, 123, 10877–10883.
(13) In the ligand-free CuAAC reaction, terminal alkynes form binuclear
complexes which have been isolated as yellow precipitates: Buckley,
B. R.; Dann, S. E.; Heaney, H. Chem.sEur. J. 2010, 16, 6278–6284.
Such complexes were not observed in reactions accelerated by either
class I or class II ligands.
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A R T I C L E S
Figure 7. Proposed mono- and binuclear speciation in the CuAAC process. X ) anionic donor such as σ-acetylide, halide, hydroxide, or triazolide; S )
neutral intermolecular donor such as DMSO, other solvent, π-alkyne, or organic azide. Productive complexes are not formed when only one heterocyclic
donor arm (L) exists.
The behavior of the one- and two-armed ligands is also
instructive. The mono(benzimidazole-methyl) amines 16 and
17 did not affect CuAAC reactions in either aqueous or organic
media, showing that these molecules cannot bind copper in either
a productive or an inhibitory manner. Adding another benzimi-
dazole group produced chelates (18 and 20) that strongly
suppressed the CuAAC reaction at a 1:1 ligand:Cu ratio even
in 80% DMSO (data not shown). The bis(benzimidazole-
methyl)amine motif therefore must trap CuI in an inactive
form.4,14 Ligands 5 and 6, which contain this motif, are thereby
at a disadvantage at higher overall Cu-ligand concentrations
(Figure 2A). This putative coordination trap can be relieved in
a manner dependent on the conditions. In strong-donor solvents,
adding a benzimidazole arm (2) or replacing one of the
benzimidazole arms with triazole (4) or pyridine (7) provides
much more active catalysis at higher concentrations (Figure
2A).4,14 Under weak-donor conditions, replacing a benzimid-
azole with triazole or pyridine and using lower ligand:Cu ratios
gives the same result (Figure 3B2).
Proposed Mechanism. Consideration of the ligand-accelerated
CuAAC reaction mechanism must include the concepts that
inhibitory species can attenuate catalyst performance and that
solvent coordination can be important. Both points arise in part
from the fact that organic azide, a very weakly coordinating
ligand, needs access to the metal center. Figure 7 summarizes
our speculation concerning the types of Cu complexes that may
be present and the different possibilities for catalytic cycles that
are consistent with our experimental findings, although many
possibilities exist for the details of coordination and structure.
We suggest that the distributions among the various types of
complexes are determined by the ligand structure and the
donor-acceptor properties of the solvent or other additives, as
indicated by the blue notations, and that these distributions
determine many of the behavioral features of the CuAAC
reaction under different conditions.
Ligand-free CuI complexes are relatively inactive and in
equilibrium with mononuclear complexes such as 23, which are
not optimal because they lack the activating power of a second
Cu center.15 Binuclear species having a ligand:Cu ratio of 2:2
(or higher), such as 24, are inactive, since their coordinative
saturation makes it difficult for azide to gain access to a Cu
center. At a minimum, two heterocyclic arms and a center
nitrogen donor are necessary to bring two Cu atoms together
with open coordination sites for azide and alkyne reactants, and
indeed, dipodal ligands as 19 and 22 can be somewhat effective
(Figure 3B3). However, the versatility of these triazole-
containing ligands is limited, since they are easily displaced by
donor solvents; conversely, dipodal ligands containing strong-
donor arms form inhibitory complexes as discussed above.
More reliable access to two Cu centers is provided by a ligand
with three ligating arms, as in generalized structure 25 (Figure
7).13 Binding of alkyne and azide gives an active structure such
as 26, similar to previous proposals for triazole formation,8,15
leading to complex 27. We suggest that the fate of this species
determines the overall kinetic behavior with respect to rate order
in Cu and ligand. When competition for Cu binding is minimal
(weak-donor environment), the binuclear Cu2L species remains
intact, exhibiting a rate law of the form of eq 1, with apparent
first-order dependence on the concentration of Cu ·ligand com-
plex because no change in molecularity occurs through the
catalytic cycle (boxed in Figure 7). This corresponds to the “90%
H2O” plots in Figure 6.
(14) The attempted synthesis of 13 by triple CuAAC condensation of
3-azido-1-propanol with tripropargylamine is very difficult to push
past the bis(triazole) stage in water, presumably because the (triazole-
CH2)2N-propargyl intermediate is inhibitory. The details of this
phenomenon in the context of the preparation of 13 are described in
the Supporting Information of ref 16.
(15) Ahlquist, M.; Fokin, V. V. Organometallics 2007, 26, 4389–4391.
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rate ) k[Cu2 · Ligand]
(1)
The user of the CuAAC click reaction is therefore encouraged
to evaluate each case in terms of competing donor centers that
may be present in the solvent, substrates, or additives. The
matching of accelerating ligands to the conditions (stronger
ligands for “strong-donor” situations, weaker ligands for “weak-
donor” conditions) as well as the proper attention to ligand:
metal ratio should enable the selection of effective CuAAC
reactions in many demanding situations.
If, on the other hand, a strong-donor environment induces
complex 27 to break up during the catalytic cycle to give a
Cu-triazolyl 28 and the mononuclear complex 23, the reas-
sembly of one ligand and two Cu centers can be required for
each turnover, leading to rate eq 2, consistent with the third-
order dependence shown in Figure 6 for “80% DMSO”
reactions.
Conclusions
rate ) k[Ligand][Cu]2
(2)
The Cu(I) complex of the mono(benzimidazole)-bis(pyridine)
ligand 7 is a superior catalyst for CuAAC under synthetic
organic conditions. At ligand:Cu ratios greater than 1, ligand 7
can be inhibitory due to the blocking of coordination sites on
the metal, but such deactivating interactions are disrupted by
donor solvents such as DMSO and NMP or by high concentra-
tions of alkyne. Under aqueous conditions, the use of weaker
ligands is therefore recommended, minimizing the tendency to
form inhibitory copper chelates. In strong-donor environments,
however, strong ligands are needed to bring two copper centers
together to form catalytically active complexes.
The differing kinetic outcomes shown in Figure 6 therefore do
not necessarily suggest the existence of different triazole-forming
complexes under the two conditions but rather two different
pathways of catalyst regeneration.
Choosing the Best Catalyst. The studies described above of
CuAAC reaction rates under varying conditions and with
varying catalysts were spurred by anecdotal reports of occasional
failure of this otherwise supremely forgiving transformation and
of our own inability to generalize previous findings made in
DMSO-rich solvents to other reaction media. We now find it
helpful to consider two general types of reaction conditions when
selecting an appropriate form of the copper catalyst. “Prepara-
tive” reactions are those in which the concentrations of alkyne
are greater than 10 mM (often much greater), the concentration
of copper is approximately 50 µM, making for catalyst loadings
of 0.5% or less, and the reaction medium may contain large
amounts of DMSO or similar solvents. These are the “strong-
donor” conditions discussed above. “Weak-donor” conditions
are often used in bioconjugation reactions, involving substrate
concentrations less than 250 µM, catalyst loadings from 20%
to greater than 100% relative to alkyne, and solvent mixtures
dominated by water. [Note, however, that “strong-donor”
conditions may also occur with biomolecules or other substrates
in water, if those substrates contain large numbers or high local
concentrations of competing donor groups such as histidine
imidazoles.]
Ligands with stronger donor arms, such as benzimidazoles
and pyridines, are recommended for strong-donor conditions,
in which donor solvent and/or alkyne balance the coordination
equilibria to provide sufficient concentrations of active com-
plexes. The mixed benzimidazole-pyridine ligand 7 is more
active than the substituted tris(benzimidazoles) 11 and 12,
remains catalytically active at copper concentrations below 50
µM, and is considerably easier to prepare, so we now recom-
mend it for synthetic applications.
Experimental Section
Synthesis procedures and characterization data for the ligands
are given in the Supporting Information. All kinetic procedures and
analyses were performed as described in refs 4 and 9. Electrospray-
ionization mass spectrometry of Cu complexes was performed in
positive-ion mode on samples in 9:1 water:DMSO, injected directly
into the spray chamber of an Agilent 1100 (G1946D) instrument
being eluted with 9:1 MeCN:water containing 0.1% TFA. Changing
the carrier composition or the sample solvent system did not affect
the spectra in a meaningful way. Ascorbate stock was added 1 min
prior to analysis to allow reduction of the Cu(II) species. The
Cu(I)-ligand complexes were usually the major ions observed, but
their Cu(II) counterparts were always detected, possibly due to
reoxidation in the liquid handler and MS spray chamber. DMSO
dimers with a proton or a sodium ion were observed in all spectra.
Oxidation of the ligands (M + 16) was noticeable on several
occasions, a phenomenon that is discussed elsewhere.16 Addition
of phenylacetylene to mixtures containing ligand 7 did not affect
the mass spectral composition.
Acknowledgment. This work was supported by The Skaggs
Institute for Chemical Biology and the NIH (RR021886). We thank
Drs. Jane Kuzelka, Yeon-Hee Lim, and Valentin Rodionov for
assistance with ligand syntheses and Professors Valery Fokin and
K. Barry Sharpless for valuable discussions.
Supporting Information Available: Experimental details,
including ligand syntheses and characterization, numerical tables
for all plots, and mass spectra of Cu-ligand mixtures. This
material is available free of charge via the Internet at http://
pubs.acs.org.
For bioconjugation reactions, either a well-controlled amount
of 7, 11, or 14 (1:2 ratio to Cu) or an excess of triazole-rich
ligands such as 9, 13, or 15 are recommended. The widest pH
range can be accommodated with ligand 7 (see Supporting
Information) and, presumably, its analogues in class II. How-
ever, we have most often used the tris(triazolyl) compound 13
at neutral pH because of certain beneficial effects of excess
ligand with respect to oxidative byproducts produced in the
presence of ascorbate and oxygen, as described elsewhere.16
JA105743G
(16) Hong, V.; Presolski, S. I.; Ma, C.; Finn, M. G. Angew. Chem., Int.
Ed. 2009, 48, 9879–9883.
9
14576 J. AM. CHEM. SOC. VOL. 132, NO. 41, 2010