With the goal of identifying alkyne-like reagents for use in click chemistry, but without Cu catalysts, we used B3LYP density function theory (DFT) to investigate the trends in activation barriers for the 1,3-dipolar cycloadditions of azides with various cyclooctyne, dibenzocyclooctyne, and azacyclooctyne compounds. Based on these trends, we find monobenzocyclooctyne-based reagents that are predicted to have dramatically improved reactivity over currently employed reagents

Chenoweth, David

Chenoweth, Kimberly

Goddard, William A. III

English

Caltech Authors - Main

PAPER www.rsc.org/obc | Organic & Biomolecular Chemistry
Cyclooctyne-based reagents for uncatalyzed click chemistry:
A computational survey†
Kimberly Chenoweth,‡ David Chenoweth§ and William A. Goddard III*
Received 12th June 2009, Accepted 16th September 2009
First published as an Advance Article on the web 9th November 2009
DOI: 10.1039/b911482c
With the goal of identifying alkyne-like reagents for use in click chemistry, but without Cu catalysts, we
used B3LYP density function theory (DFT) to investigate the trends in activation barriers for the
1,3-dipolar cycloadditions of azides with various cyclooctyne, dibenzocyclooctyne, and azacyclooctyne
compounds. Based on these trends, we ﬁnd monobenzocyclooctyne-based reagents that are predicted to
have dramatically improved reactivity over currently employed reagents.
Introduction
An explosion of techniques has energized the bioorthogonal
ligation chemistry ﬁeld, with new methodologies involving oxime
and hydrazone formation, Staudinger ligation, and azide–alkyne
[3+2] cycloaddition.1 Although many areas of chemistry beneﬁt
from click chemistry, the toxicity of the Cu catalyst in the [3+2]
azide–alkyne cycloaddition has prohibited in vivo applications.1d,1e
Thus, room-temperature labeling reagents that avoid using copper
are highly desirable.
Similarly, click reactions provide a means for unique surface
functionalization and labelling strategies with application in
nanotechnology and surface science. For example, the Heath
group and others have developed strategies for functionalizing
Si and Au surfaces with azides, which could be attached to alkyne-
terminated labels or rotaxanes for sensors or nanomechanical
systems.2 Here it would be most useful to avoid the requirement of
copper catalysts while accelerating the reactions to be fast at room
temperature.
Recently, the Bertozzi group3 demonstrated a new strain-release
cyclooctyne labelling reagent (1, Scheme 1) that proceeds in the
absence of a Cu catalyst at physiological temperatures, although
with a ca. 52-fold reduction in reaction rate.1a The increased
reactivity of cyclooctyne toward [3+2] cycloaddition has been
attributed to strain release, and estimates of the cyclooctyne ring
strain range from 10–19 kcal/mol.4 The ground-state destabilizing
effect of the triple bond is thought to drive the bioorthogonal
ligation reactions (e.g. attachment of chemical reporter molecules)
in the absence of a catalyst; however, this has been challenged
with the concept of dipole distortion.4d,9 Bertozzi demonstrated
that gem-diﬂuoro substitution a to the alkyne leads to a reaction
rate 63 times faster than the simple cyclooctyne, which has
been attributed to the greater LUMO-lowering effect of the
Materials and Process Simulation Center, Division of Chemistry and Chem-
ical Engineering, California Institute of Technology, Pasadena, California,
91125, USA. E-mail: wag@wag.caltech.edu; Fax: +1 (626)585-0918;
Tel: +1 (626)395-2731
† Electronic supplementary information (ESI) available: Absolute energies
and cartesian coordinates. See DOI: 10.1039/b911482c
‡ Current address: Department of Chemistry, Smith College, Northamp-
ton, Massachusetts 01063, USA.
§ Current address: Department of Chemistry, Massachusetts Institute of
Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA
Scheme 1 Strain-release azide–alkyne cycloaddition reactions.
ﬂuoro substituent.3c In addition, dibenzocyclooctyne reagents
have allowed visualization of glycoconjugates in living cells using
confocal microscopy while a diﬂuorinated cyclooctyne derivative
has proven useful for investigating in vivo glycan trafﬁcking
dynamics.3c,5
First, we investigated the best current bioorthogonal
cyclooctyne-based labelling reagents to gain insight into reactivity
patterns associated with electronic, steric, and strain effects by
assessing trends in the activation energies, thus providing a basis
of comparison for potential monobenzocyclooctyne reagents.
In addition, we investigated the decomposition mechanism ob-
served for a monoﬂuorinated cyclooctyne reagent. Next, we
assessed dibenzocyclooctyne- and azacyclooctyne-based reagents
and compared the calculated barriers to the cyclooctynes. Finally,
we investigated new monobenzocyclooctynes and compared the
calculated barriers to that of cyclooctyne and dibenzocyclooctyne
reagents. To date, these monocyclooctynes have not been used in
bioorthogonal ligation chemistry but provide improved reactivity
compared to previously reported cyclooctyne-based reagents.
Computational details
All calculations were performed using the B3LYP hybrid DFT
functional6 with the 6-311G** basis set7 as implemented in the
Jaguar 6.5 software package.8 Vibrational frequencies have been
calculated at all stationary points to obtain zero-point energies
(ZPE) and free energies. All stationary points have been identiﬁed
as local minima (zero imaginary frequencies) or transition states
(TS) (one imaginary frequency). We should emphasize that the
interest here is in identifying the cyclooctyne reagent with the
lowest barrier for cycloaddition to an azide (click chemistry).
These DFT calculations may overestimate absolute reaction
energies by a few kcal/mol.4d
This journal is © The Royal Society of Chemistry 2009 Org. Biomol. Chem., 2009, 7, 5255–5258 | 5255
Table 1 Activation energy (in kcal/mol) for transition states TS1 and
TS2 in Fig. 1. Best is 1e, conﬁrmed experimentally3c
DE‡ DG‡
Reactant R1 R2 TS1 TS2 TS1 TS2
1a H H 12.8 — 24.9 —
1b Me H 13.7 13.6 26.1 26.1
1c Me F 13.7 11.3 26.1 24.7
1d H F 12.6 10.5 24.6 23.4
1e F F 12.1 9.3 24.6 22.1
Results and discussion
Cyclooctyne-based reagents
We ﬁrst studied a series of model ﬂuorinated cyclooctynes
analogous to the bioorthogonal reagents developed in the Bertozzi
group. To determine how various substituents affect the reactivity
of cyclooctyne with methyl azide, we calculated the activation
energies (Table 1) for formation of both triazole regioisomers for
a range of substituents (Fig. 1). We ﬁnd that methyl substitution
adjacent to the triple bond of cyclooctyne (1b), leads to a slight
increase in the activation barrier; however, ﬂuorination at the
a-position (1c, 1d, 1e) leads to a consistent decrease in the activa-
tion energy as the number of ﬂuorines increase. In addition, chang-
ing from the diﬂuorinated cyclooctyne (1e) to a mono-ﬂuorinated
cyclooctyne (1d) increases the azide–alkyne cycloaddition barrier
by DDG‡ =1.3 kcal/mol for TS2, which is consistent with the
factor of ~10 decrease in reaction rates observed experimentally.3c
The reaction of gem-diﬂuorocyclooctyne (1e) with methyl azide
leads to a transition state barrier for 1e-TS2 of 22.1 kcal/mol,
which is DDG‡ = 2.8 kcal/mol (DDE‡ = 3.5 kcal/mol) lower in
energy than reaction with cyclooctyne (1a), leading to a 106-fold
rate enhancement. This is consistent with the observed 63-fold
rate enhancement (from 1.2 ¥ 10-3 M-1 s-1 to 7.6 ¥ 10-2 M-1 s-1) for
similar substrates.3 [The slight disparity in rate and regioselectivity
(63 vs. 106) may be attributed to the solvation effects9a and the
difference between the benzyl azide used in experiments compared
to our use of methyl azide as a model system.] Our calculated
activation barriers are consistent with more recent theoretical
Fig. 1 Azide–cyclooctyne reaction pathways. R1 (pink) and R2 (orange)
are deﬁned in Table 1.
studies for reactionwithmethyl azide, and the trends in the barriers
are consistent with reaction with phenyl azide.4d,9
Decomposition of the product (2c) from cyclooctyne 1c
Interestingly, the Bertozzi group observed that when reacting
reagents analogous to 1c with azides one of the triazole regioi-
somers underwent decomposition where the other isomer was
stable.2b Experimental observations have indicated that ﬂuorina-
tion at a tertiary carbon position a to the alkyne in a cyclooctyne-
based reagent can enhance the elimination processes.2b To gain
insight into the experimentally observed regioisomeric decompo-
sition of the triazole products formed from cycloaddition of the
monoﬂuorinated cyclooctyne 1c, we propose a reaction interme-
diate to the decomposition reaction and a mechanism based on
steric A1,3 strain that precludes decomposition of product isomer
3c (Fig. 2). This suggests the possibility of a resonance-assisted
(hyperconjugation involving the NCH3 lone pair) steric-free
pathway, as a limiting case, that proceeds through a carbocation
intermediate (2c-cation), formed via ﬂuorine elimination, to the
decomposition product 2c-OH resulting from SN1-type attack by
water, with the actualmechanismprobably lying somewhere on the
continuum between the SN1 and SN2 extremes. We optimized the
structures for the carbocation intermediates in the decomposition
pathway (see Fig. 2), leading to a DFT energy for carbocation
3c-cation higher byDE= 12.7 kcal/mol than that of 2c-cation, due
primarily to A1,3 strain. This precludes full resonance stabilization
by the triazole ring, as shown by the resulting angles in Fig. 2. This
energetic difference in carbocations explains the decomposition of
2c versus the stability of 3c, providing insight into the importance
of the appropriate substitution a to the alkyne and A1,3 strain for
future reagent design.
Fig. 2 Decomposition pathway for a-monoﬂuorinated cyclooc-
tyne-based reagents.
5256 | Org. Biomol. Chem., 2009, 7, 5255–5258 This journal is © The Royal Society of Chemistry 2009
Azacyclooctyne-based reagent
Next, we evaluated the barriers for azide–alkyne cycloaddition
for a heterocyclic and heteroatom-substituted cyclooctyne-based
reagent (4, Fig. 3). Interest in this reagent lies in the experimental
observation that it improves sensitivity of azide detection due to
its superior polarity and water solubility due to the presence
of the methoxy groups.3e Experimentally, the azacyclooctyne,
6,7-dimethoxyazacyclooct-4yne (DIMAC), was found to provide
a rate enhancement of 2.5-fold over 1a for the 1,3¢-dipolar
cycloaddition reaction with benzyl azide (from 1.2 ¥ 10-3 M-1 s-1 to
3.0 ¥ 10-3 M-1 s-1).3e Our calculations ﬁnd a similar increase in rate
for a model DIMAC compound (4) over cyclooctyne (1a) with a
decrease in barrier height of DDG‡ = 0.3 kcal/mol, corresponding
to a rate enhancement of ~2.
Fig. 3 Azide–azacyclooctyne cycloaddition reaction pathway.
Dibenzocyclooctyne-based reagents
Another class of reagents investigated for use as bioorthogonal
reagents is the dibenzocyclooctynes. In order to compare these
systems to the nonbenzoid cyclooctyne-based reagents, we calcu-
lated the activation energies for cycloaddition with methyl azide
(Fig. 4). Structure 7fwithR4=R5=His comparable to the reagent
with R4 =OH andR5 =H.5 We ﬁnd that the cycloaddition barrier
for 7f is 12.3 kcal/mol (Table 2), which is 0.5 kcal/mol lower than
for cyclooctyne 1a (Table 1). It has been proposed that the biaryl
substitution would increase ring strain and provide conjugation
Fig. 4 Azide–dibenzocyclooctyne cycloaddition reaction pathways. R4 is
hydrogen, and R5 (pink) is deﬁned in Table 2.
Table 2 Activation energy (kcal/mol) for transition state TS3 in Fig. 2
Reactant R5 DE‡ DG‡
7a NH2 12.2 25.5
7b OMe 11.6 24.9
7c OEt 11.7 25.1
7d N(CH3)2 12.2 25.7
7e H 12.3 25.5
7f F 11.8 25.2
7g Cl 11.7 25.0
7h NO2 11.2 25.1
7i CN 11.3 24.7
with the alkyne, resulting in improved reactivity.5 However, we
ﬁnd from our calculations that the resulting steric interaction
between the methyl group on the azide and the hydrogen of the
phenyl group (A1,3 strain) competes directly with the activating
effects of the aryl ring, resulting in hampered reactivity compared
to Bertozzi’s ﬂuorinated cyclooctyne-based reagents. The rate
constants for dibenzocyclooctynol were reported5 to have a rate
acceleration of approximately three orders of magnitude over
that of cyclooctyn-ol. However, our calculations ﬁnd very similar
barriers between cyclooctyne and a dibenzocyclooctyne (e.g., with
cyclooctyne, leading to a factor of 2 enhancement). The origin
of this discrepancy might be related to solvent effects, reaction
conditions, the use of benzyl azide instead of methyl azide, or
differences in the substrates. Based on our calculations and results
in the literature,4d,9 the origin of this discrepancy might be related
to the speciﬁc experimental reaction conditions, and it would be
useful for the rates of the various cyclooctyne reagents to be
examined in a thorough experimental study. In addition, we ﬁnd
that para-substitution on the phenyl group has very little inductive
effect and does not signiﬁcantly reduce the activation barriers
(Table 2).
Benzocyclooctyne-based reagents
Based on these new insights into azide–alkyne cycloadditions,
we propose new cyclooctyne reagents bearing a single aryl
substituent (Fig. 5) under the hypothesis that decreasedA1,3 strain,
transannular hybridization-induced ring strain, and electron-
withdrawing substituents could yield a superior reagent with
improved reactivity. We ﬁnd that the position of the phenyl group
with respect to the alkyne has a signiﬁcant effect on the reactivity
toward cycloaddition. Thus fusing a benzene ring to carbon 3–4
of the cyclooctyne system 9-TS1 and 9-TS2 (Fig. 5) leads to
the largest barriers to cycloaddition, 25.3 and 24.9 kcal/mol
respectively. Moving the aryl fusion to the 4–5 position of the
cyclooctyne system decreases the barrier heights for 10-TS1 and
10-TS2 to 23.3 and 23.5 kcal/mol, respectively. This decrease
in activation energy and the energetic switch in regioisomeric
activation energies is presumably due to superposition of reduced
A1,3 interactions and decreased electronic effects from the aryl
group. Fusing the aryl group at the 5–6 carbon position of the
cyclooctyne ring 11-TS further decreases the barrier for cycload-
dition with methyl azide to 22.1 kcal/mol, making it competitive
with some of the best ﬂuorinated cyclooctyne systems (1e with
22.1 kcal/mol). In addition, we ﬁnd that a 6-membered aromatic
ring in this position provides increased angle compression, leading
to improved reactivity, whereas a 5-membered aromatic ring
This journal is © The Royal Society of Chemistry 2009 Org. Biomol. Chem., 2009, 7, 5255–5258 | 5257
Fig. 5 Azide–benzocyclooctyne (BC8y) cycloaddition transition-state
structures and activation barriers in kcal/mol. Bond distances are in A˚
and angles are in degrees. The best is gem-diﬂuorobenzocyclooctyne, 10.
results in less angle compression and decreased reactivity (see Fig.
S1†). These benzocyclooctynes have been reported to be isolable,
but they have not been utilized for copper-free click chemistry.10
Combining the positional effects of the aryl substituent and the
LUMO lowering electron-withdrawing effects of the gem-diﬂuoro
substituent, we ﬁnd signiﬁcantly decreased activation energies
to 19.7 kcal/mol for the 12-TS2 cycloaddition transition state
structure shown in Fig. 2 (and 21.7 kcal/mol for 12-TS1). This is
lower than the barrier for 1e (DDG‡ = 2.4 kcal/mol), making it
57 times faster, and suggesting a rate of 57 ¥ (7.6 ¥ 10-2 M-1 s-1) =
4.4M-1 s-1.11 Since 1e has been observed to be 52 times slower than
the copper-catalyzed system, the gem-diﬂuorobenzocyclooctyne
(12) should exhibit activity comparable to or faster than the
copper-catalyzed click reaction.3c We expect that this new class
of benzocyclooctyne reagents will provide a range of stabilities
and reactivities to enable room-temperature copper-free click
chemistry for applications in biology, nanotechnology, and surface
science. In addition, note that the simple benzocyclooctyne 11 has
the same barrier and rate as the best previous copper-free click
reagent, gem-diﬂuorocyclooctyne (1e), making it a potentially
useful reagent.
This class of benzocyclooctynes takes advantage of the optimal
placement of sp2 centers for the enhancement of ring strain and
the proper placement of electron-withdrawing substituents for
LUMO-lowering effects while avoiding the detrimental effects of
A1,3 interactions.
Conclusions
This exercise demonstrates how modern computational chemistry
provides a valuable tool for a priori design of optimal reagents
for novel applications in biological systems and materials science,
dramatically reducing the number of experiments required to
navigate structure-sequence space. In particular, we show that
combining transannular hybridization-induced ring strain with
proper placement of LUMO-lowering electron-withdrawing sub-
stituents, while avoiding deleterious effects of A1,3 interactions,
provides guiding principles for designing superior cyclooctyne-
based reagents with improved reactivity.
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5258 | Org. Biomol. Chem., 2009, 7, 5255–5258 This journal is © The Royal Society of Chemistry 2009


Cyclooctyne-based reagents for uncatalyzed click chemistry: A computational survey

109, 189. 11 Structures were rendered using UCSF Chimera, Resource for Biocomputing, Visualization, and Informatics,

2, 565; (d)

Structures were rendered using UCSF Chimera, Resource for Biocomputing, Visualization, and Informatics, University of California, San Francisco (supported by

http://authors.library.caltech.edu/17100/1/Chenoweth2009p6546Org_Biomol_Chem.pdf

Cyclooctyne-based reagents for uncatalyzed click chemistry: A computational survey

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