Reactivity of biarylazacyclooctynones in copper-free click chemistry.

Abstract

The 1,3-dipolar cycloaddition of cyclooctynes with azides, also called "copper-free click chemistry", is a bioorthogonal reaction with widespread applications in biological discovery. The kinetics of this reaction are of paramount importance for studies of dynamic processes, particularly in living subjects. Here we performed a systematic analysis of the effects of strain and electronics on the reactivity of cyclooctynes with azides through both experimental measurements and computational studies using a density functional theory (DFT) distortion/interaction transition state model. In particular, we focused on biarylazacyclooctynone (BARAC) because it reacts with azides faster than any other reported cyclooctyne and its modular synthesis facilitated rapid access to analogues. We found that substituents on BARAC's aryl rings can alter the calculated transition state interaction energy of the cycloaddition through electronic effects or the calculated distortion energy through steric effects. Experimental data confirmed that electronic perturbation of BARAC's aryl rings has a modest effect on reaction rate, whereas steric hindrance in the transition state can significantly retard the reaction. Drawing on these results, we analyzed the relationship between alkyne bond angles, which we determined using X-ray crystallography, and reactivity, quantified by experimental second-order rate constants, for a range of cyclooctynes. Our results suggest a correlation between decreased alkyne bond angle and increased cyclooctyne reactivity. Finally, we obtained structural and computational data that revealed the relationship between the conformation of BARAC's central lactam and compound reactivity. Collectively, these results indicate that the distortion/interaction model combined with bond angle analysis will enable predictions of cyclooctyne reactivity and the rational design of new reagents for copper-free click chemistry.

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Reactivity of Biarylazacyclooctynones in Copper-Free Click Chemistry
Chelsea G. Gordon,†Joel L. Mackey,⊥John C. Jewett,†,#Ellen M. Sletten,†K. N. Houk,*,⊥
and Carolyn R. Bertozzi*,†,‡,§
†Departments of Chemistry and‡Molecular and Cell Biology and§Howard Hughes Medical Institute, University of California -
Berkeley, Berkeley, California 94720, United States
⊥Department of Chemistry and Biochemistry, University of California - Los Angeles, Los Angeles, California 90095, United States
*
S Supporting Information
ABSTRACT: The 1,3-dipolar cycloaddition of cyclooctynes with
azides, also called “copper-free click chemistry”, is a bioorthogonal
reaction with widespread applications in biological discovery. The
kinetics of this reaction are of paramount importance for studies of
dynamic processes, particularly in living subjects. Here we performed a
systematic analysis of the effects of strain and electronics on the
reactivity of cyclooctynes with azides through both experimental
measurements and computational studies using a density functional
theory (DFT) distortion/interaction transition state model. In
particular, we focused on biarylazacyclooctynone (BARAC) because
it reacts with azides faster than any other reported cyclooctyne and its modular synthesis facilitated rapid access to analogues. We
found that substituents on BARAC’s aryl rings can alter the calculated transition state interaction energy of the cycloaddition
through electronic effects or the calculated distortion energy through steric effects. Experimental data confirmed that electronic
perturbation of BARAC’s aryl rings has a modest effect on reaction rate, whereas steric hindrance in the transition state can
significantly retard the reaction. Drawing on these results, we analyzed the relationship between alkyne bond angles, which we
determined using X-ray crystallography, and reactivity, quantified by experimental second-order rate constants, for a range of
cyclooctynes. Our results suggest a correlation between decreased alkyne bond angle and increased cyclooctyne reactivity.
Finally, we obtained structural and computational data that revealed the relationship between the conformation of BARAC’s
central lactam and compound reactivity. Collectively, these results indicate that the distortion/interaction model combined with
bond angle analysis will enable predictions of cyclooctyne reactivity and the rational design of new reagents for copper-free click
chemistry.
■INTRODUCTION
Since its initial introduction as a bioorthogonal reaction, the
strain-promoted 1,3-dipolar cycloaddition between cyclo-
octynes and azides (Figure 1a) has been utilized in a range of
biological studies.1The reaction was developed in response to
the dearth of tools available for the study of biomolecules in
their native environments, and was designed to proceed rapidly
and selectively in vivo without perturbing native biochemical
functionality. Due to the strain activation inherent to
cyclooctynes,2the reaction proceeds at a rate that is sufficient
for in vivo labeling while avoiding the use of the toxic copper(I)
catalysts traditionally employed in “click chemistry” with
terminal alkynes.3,4As a result, the reaction between cyclo-
octynes and azides is often referred to as “copper-free click
chemistry.”
In an effort to further enhance the utility of the strain-
promoted reaction, several groups have contributed to a series
of structurally varied cyclooctyne scaffolds that display
differential reactivities toward the azide. A selection of these
compounds is shown in b−d of Figure 1. All were designed on
the basis of assuming that one can alter the reactivity of a
cyclooctyne through modulation either of strain or electronics.
Cyclooctyne 1 (also called “Oct”, Figure 1b), the first
cyclooctyne developed specifically as a bioorthogonal reagent,
displays a second-order rate constant of 2.4 × 10−3M−1s−1for
the reaction with benzyl azide.5,6It was later shown that this
rate can be enhanced through installation of fluorine atoms at
the propargylic position. Monofluorinated cyclooctyne
(MOFO, 2) displays a second-order rate constant of 4.3 ×
10−3M−1s−1, whereas difluorinated cyclooctyne (DIFO, 3)
reacts with a rate constant of 7.6 × 10−2M−1s−1(1.8-fold and
32-fold faster than Oct, respectively).3a,6The rate-enhancing
effects of strain were subsequently demonstrated in the context
of dibenzocyclooctyne (DIBO, 4),7a,bdibenzoazacyclooctyne
(DIBAC, 5),7cand biarylazacyclooctynone (BARAC, 6),7d
which respectively react with azides 24-fold to 375-fold faster
than is observed for Oct (Figure 1d).
The intention implicit in the design of compounds 4−6 was
to increase strain by adding sp2centers to the cyclooctyne ring.
However, there may be more subtle consequences of these
compounds’ structural modifications that actually oppose the
Received:
Published: May 3, 2012
January 4, 2012
Article
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desired reactivity outcome. For example, while aryl ring fusion
may enhance strain, the “flagpole” hydrogen atoms ortho to the
aryl/cyclooctyne ring junction were predicted by Goddard and
co-workers to decrease reactivity by steric interference with the
azide in the transition state.8aThe experimentally observed
reactivity of biarylcyclooctynes likely reflects a balance of these
rate-enhancing and -diminishing effects. This example under-
scores the difficulty inherent to rational design of new
cyclooctynes with tailored kinetic properties. Many structural
perturbations affect more than one contributor to reactivity,
e.g., strain, sterics, and electronics. Without a better under-
standing of their relative influence on transition state activation
energies, these parameters cannot be readily optimized to
achieve a desired outcome. In principle, one might derive a set
of rules that links structure and reactivity solely on the basis of
empirical data. However, cyclooctynes are generally challenging
synthetic targets that do not lend themselves to extensive
analoging.
In previous work, density functional theory (DFT) models
have been employed to analyze reactions of cyclooctynes with
azides and have proven to be useful predictors of relative
transition state activation energies.8Here we performed a
systematic analysis of the effects of strain and electronics on the
reactivity of cyclooctynes with azides, using both empirical data
and a DFT-based distortion/interaction transition state
model.8b,cWe focused our analysis on a series of differentially
substituted BARAC analogues, comparing their second-order
rate constants in the cycloaddition reaction with benzyl azide to
those predicted by DFT calculations. We also investigated the
relationship between alkyne bond angles and reactivity and
determined the conformation of BARAC’s central lactam,
which has significant energetic consequences. The results
herein provide a framework for understanding and predicting
cycloaddition kinetics.
■DISTORTION/INTERACTION MODEL
The distortion/interaction model deconstructs the activation
energy of a reaction into two components: the distortion
energy, which is dependent on ground state strain, and the
interaction energy, which is governed by transition state
electronics.8b,cIn this model, the transition state energy
(ΔE⧧) of a reaction is defined as the sum of distortion energy
(ΔE⧧d), the energy required to distort the alkyne and azide into
their preferred transition state conformations, and interaction
energy (ΔE⧧i), the energy lowering upon favorable orbital
overlap between the azide and the alkyne (ΔE⧧= ΔE⧧d+
ΔE⧧i). Thus, the activation energy of a reaction can be altered
by changing either the distortion energy or the interaction
energy required to reach the transition state.
In the case of the 1,3-dipolar cycloaddition of 2-butyne with
methyl azide (Figure 2a), our calculations indicate that 29.9
kcal/mol energy is required to distort the ground state
substrates into their preferred transition state conformations.
Upon distortion, the alkyne and azide interact, lowering the
energy of the overall system by −9.0 kcal/mol through a
favorable orbital overlap that can only be achieved via the
geometry of the distorted state. Combining the effects of
distortion and interaction, we calculate an overall transition
state activation energy of 20.9 kcal/mol (ΔE⧧= ΔE⧧d+ ΔE⧧i).
In reality, distortion and interaction are not independent
processes but instead occur simultaneously to bring reactants
directly to their transition state geometries. However, this
model breaks up activation energy into two imaginary
distortion and interaction processes to allow a more detailed
analysis of reaction strain and electronics.
An example of how electronic perturbation can affect
interaction and distortion energies is illustrated by comparing
the above reaction coordinate to that for the reaction of
1,1,1,4,4,4-hexafluoro-2-butyne with methyl azide (Figure 2b).
In this case, the interaction energy is more negative than that
calculated for 2-butyne (ΔE⧧i,hexafluoroalkyne= −12.2 kcal/mol vs
ΔE⧧i,2‑butyne = −9.0 kcal/mol). This change results from
electronic perturbation of the alkyne LUMO energy via mixing
of the alkyne π-system with σ*C−F. Alabugin and co-workers
have shown that the observed acceleration results from
hyperconjugative stabilization of the cycloaddition transition
state via electron donation from the in-plane alkyne π-orbital to
the σ*C−F-orbital.9
As both 1,1,1,4,4,4-hexafluoro-2-butyne and 2-butyne are
relatively unstrained molecules, we would expect their
distortion energies to be similar for the reaction with methyl
azide. However, calculations indicate a larger total distortion
energy for the reaction of 2-butyne by 5.3 kcal/mol
(ΔE⧧d,total,2‑butyne= 29.9 kcal/mol and ΔE⧧d,total,hexafluoroalkyne=
24.6 kcal/mol). The data in a and b of Figure 2 indicate that
this difference results both from a change in alkyne transition
state distortion energy upon perfluorination as well as from a
decrease in azide distortion energy. That both the alkyne and
azide undergo shifts in distortion energy in the perfluorinated
case indicates that the transition state occurs at an earlier point
on the reaction coordinate. Calculated azide bond angles
Figure 1. Reagent development for copper-free click chemistry. (a)
The 1,3-dipolar cycloaddition between azides and cyclooctynes. (b)
Oct, the first cyclooctyne developed as a bioorthogonal reagent, and its
corresponding second-order rate constant for the reaction with benzyl
azide.5,6The reactivity of a cyclooctyne can be altered through (c)
electronic6,3aand (d) strain modulation.7All rate constants are second
order (M−1s−1) and were measured at room temperature in CD3CN
except for values noted with an asterisk (*) which were measured in
CD3OD.
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provide further evidence for an early transition state upon
perfluorination. Whereas the azide is bent to 137° in the
transition state of the 2-butyne reaction, it is only bent to 143°
in the transition state of the perfluorinated-alkyne reaction
(Figure 2). Thus, the effect of propargylic fluorination is 2-fold
when considering the distortion/interaction model; the fluorine
atoms enhance transition state interaction energies, which
facilitates an early transition state where less distortion is
required for both reactants.
The effects of distortion energy modulation in the context of
the cyclooctyne have previously been discussed8b,cand are
summarized in Figure 2c. The alkyne bond angles in
cyclooctyne are bent from linearity, and the molecule is
therefore destabilized relative to a linear isomer. Because it is
already distorted toward the transition state geometry, the
cyclooctyne requires less distortion energy to reach its
preferred transition state geometry than does a linear alkyne
(ΔE⧧d,2‑butyne= 10.1 kcal/mol and ΔE⧧d,cyclooctyne= 2.1 kcal/
mol). In addition, calculations show that the distortion energy
of methyl azide is lower for the reaction with cyclooctyne than
it is for the reaction with 2-butyne. We again attribute this
difference to the position of the transition state along the
reaction coordinate; here, ground state destabilization of the
strained cyclooctyne generates an early transition state, thereby
reducing the distortion required for the azide to reach its
transition state geometry. This shift in the position of the
transition state in response to the greater exothermicity of the
strained alkyne reaction is also consistent with the Hammond
Postulate. As a result of this significant reduction in distortion
energy, cyclooctyne displays a lower overall activation energy
for the cycloaddition reaction than does 2-butyne. In this way,
strain (i.e., distortion) promotes the cycloaddition.
Because this model is capable of distinguishing the effects of
strain and electronics on transition state energies, we chose to
apply it to the study of cyclooctyne reactivity. We began by
preparing a series of substituted BARAC analogues and
analyzing their reactivities experimentally and computationally.
■REACTIVITY OF BARAC AND ANALOGUES
The modular nature of BARAC’s synthesis7drendered this
scaffold amenable to derivatization with aryl ring substituents.
Previous analyses of the difluorinated cyclooctyne DIFO (3,
Figure 1c) suggested that addition of fluorine atoms at the
propargylic position enhances reaction rates by increasing
interaction energies and decreasing distortion energies in the
transition state.8b,cWe were curious as to whether comparable
interaction energy changes might be affected through
installation of fluorine atoms on BARAC’s aryl rings, and,
alternatively, whether electron-donating methoxy groups would
affect reaction kinetics in an opposite manner. As well, we
sought to analyze the steric effects of flagpole methyl
substituents on the rate of the reaction. Accordingly,
compounds 7−16 (Figure 3a) were selected as the targets for
our study. Compounds 7−10 possess a single fluorine atom
Figure 2. Distortion/interaction model. (a) Activation energy (ΔE⧧) for the reaction between 2-butyne and methyl azide is the sum of distortion
energy (ΔE⧧d) and interaction energy (ΔE⧧i). (b) Perfluorination of the alkyne reduces ΔE⧧of the reaction by increasing the magnitude of
stabilizing interactions in the transition state and decreasing distortion energy. (c) Constraining the alkyne into an eight-membered ring reduces ΔE⧧
by decreasing the distortion energy required to bend the starting materials into their preferred transition state conformations. For a−c, calculated
values are electronic energies, the potential energy of the molecule on a vibrationless potential energy surface. As all reactions are represented on
separate energy diagrams, the depictions are only intended to facilitate comparisons of ΔE⧧, ΔE⧧d, and ΔE⧧ivalues and not the overall energies of
starting materials or triazole products. Calculations were performed using B3LYP/6-31G(d). See Supporting Information for details.
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variously positioned around BARAC’s two aryl rings.
Compound 11 is doubly fluorinated. On the other end of the
spectrum, compounds 12 and 13 have single methoxy groups
on the aryl rings. Compound 14 is monofluorinated at the
flagpole position, potentially generating steric hindrance in the
transition state. Finally, compounds 15 and 16 possess methyl
substituents at the flagpole position. All analogues were
synthesized according to the route published by Jewett et
al.7dDetails are provided in the Supporting Information (SI).
As a platform for computational studies, we first analyzed the
bond angles and conformation of the parent compound
BARAC using X-ray crystallography and DFT calculations.
Figure 4a shows DFT geometry optimizations performed with
B3LYP and the 6-31G(d) basis set. The results indicate that the
trans conformation of the central amide bond is preferred to the
Figure 3. Bond angles and reactivities of BARAC analogues. (a) BARAC analogues targeted for our initial study of distortion/interaction
modulation. (b) Reactivity was probed empirically by measuring the second-order rate constant for the reaction of each analogue with benzyl azide in
CD3CN at rt by1H NMR spectroscopy. (c) Table shows both calculated and measured (X-ray crystallography data shown in parentheses) alkyne
bond angles for compounds 6−16 as well as measured second-order rate constants and activation free energies (ΔG⧧exp) for the model reaction with
benzyl azide. Also shown are calculated interaction (ΔE⧧i,calc) and total distortion energies (ΔE⧧d,calc= ΔE⧧d,calc,azide+ ΔE⧧d,calc,alkyne) as well as overall
electronic energies of activation (ΔE⧧calc) and free energies of activation (ΔG⧧calc) for the reaction of each analogue with methyl azide. All
computational data provided for compounds 6−16 are for the trans-BARAC isomer. *Free energies were calculated for the reaction in acetonitrile.
**The second-order rate constants shown for 15 and 16 were measured in CDCl3due to the limited solubility of 15 in CD3CN.
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cis conformation by ΔΔGsolv= 9.4 kcal/mol in acetonitrile. X-
ray data support this model, indicating that in crystal form,
BARAC preferentially occupies the trans conformation (Figure
4b).
Interestingly, DFT calculations show that the conformation
of the amide functionality dramatically influences BARAC’s
alkyne bond angles. As shown in Figure 4a, both alkyne angles
in trans-BARAC are 153°, whereas in cis-BARAC the angles are
compressed to 143° and 150°. These results suggest that cis-
BARAC is higher in energy than trans-BARAC due to increased
alkyne distortion and also imply that cis-BARAC would display
enhanced reactivity relative to that of trans-BARAC in the
cycloaddition reaction with azides. DFT calculations of free
energies of activation in acetonitrile (ΔG⧧solv) for this reaction
support our hypothesis. trans-BARAC has a ΔG⧧solvof 24.0
kcal/mol, whereas cis-BARAC has a ΔG⧧solvof 18.0 kcal/mol.
We performed energy optimization calculations on sub-
stituted BARAC analogues 7−16 as well, and the results also
indicated that the trans conformation is preferred to the cis
conformation by ΔΔGsolv= 9.1−9.7 kcal/mol (SI). We then
calculated ΔG⧧solvvalues for the reactions of compounds 7−16
with methyl azide, and results indicate that for compounds 7−
13, reaction of the trans-conformer is a lower-energy process by
3.0−4.5 kcal/mol than is isomerization to the cis-conformer
followed by reaction to form the triazole. These results suggest
that compounds 7−13 react predominantly as the trans
conformer. Thus, we focus the remainder of the calculations
in this section on trans-BARAC. As discussed later, ΔG⧧solv’s for
reactions of the cis- and trans-conformers are similar in energy
for compounds 14−16, suggesting a Curtin-Hammett con-
trolled reaction.
The table shown in Figure 3c shows the results of DFT
calculations of alkyne bond angles as well as empirical bond
angle measurements obtained by X-ray crystallography (shown
in parentheses) for select compounds in the trans conforma-
tion. We conclude from these data that aryl substitution does
not impose significant structural changes, as all compounds
exhibit alkyne bond angles close to 153°.
We next measured the second-order rate constants of the
reactions of 6−16 with benzyl azide (Figure 3b). Compounds
6−10 and 12−13 exhibited rate constants between 0.9 and 1.2
M−1s−1, with differences that do not lie outside the range of
experimental error.10,11Compound 11, however, showed a
roughly 75% increase in reactivity relative to that of 6 (k11= 1.6
± 0.1 M−1s−1versus k6= 0.9 ± 0.1 M−1s−1). Given the similar
reactivities of 6−10, 12, and 13, we expected these compounds
to display transition state distortion and interaction energies of
similar magnitude. DFT calculations were used to model the
reaction of each analogue with methyl azide, as calculations
show that methyl azide behaves similarly to benzyl azide for the
reaction with 6 (data provided in the SI). In discussing the
results of these calculations, we define triazole 17 (Figure 3b)
as the syn-regioisomer and triazole 18 (Figure 3b) as the anti-
regioisomer.
Computational data indicate that compounds 6−10, 12 and
13 do in fact exhibit similar energetics in their transition states,
with distortion energies for all seven compounds lying between
16.2 and 16.6 kcal/mol for the syn-regioisomer and between
16.5 and 16.8 kcal/mol for the anti-regioisomer. These data are
consistent with our observations that the compounds have
nearly identical alkyne bond angles (additional structural data
are provided in the SI). Similarly, calculated interaction
energies were alike for these compounds with values between
−8.1 and −8.6 kcal/mol for the syn-regioisomer and between
−8.7 and −9.0 kcal/mol for the anti-regioisomer. As a result,
overall energies of activation (ΔE⧧= ΔE⧧d+ ΔE⧧i) for these
compounds are quite comparable, falling within the range of
8.0−8.3 kcal/mol for the syn-regioisomer and between 7.7−8.0
kcal/mol for the anti-regioisomer.
Calculated free energies of activation in acetonitrile (ΔG⧧calc)
are also similar for compounds 6−10, 12 and 13. For both the
syn- and anti-regioisomers, ΔG⧧calclies between 23.7 and 24.1
kcal/mol. These calculations systematically overestimate the
Figure 4. Structural analysis of BARAC. (a) DFT calculations (B3LYP/6-31G(d)) of cis- and trans-BARAC. (b) Front and side view of BARAC
obtained via X-ray crystallography. Crystalline BARAC exists as the trans conformer. Thermal ellipsoid plots are shown at 50% probability.
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