Bioorthogonal Hydroamination of Push–Pull-Activated Linear Alkynes

Article abstract of DOI:10.1002/anie.202104863

A bioorthogonal reaction between N,N-dialkylhydroxylamines and push–pull-activated halogenated alkynes is described. We explore the use of rehybridization effects in activating alkynes, and we show that electronic effects, when competing stereoelectronic and inductive factors are properly balanced, sufficiently activate a linear alkyne in the uncatalyzed conjugative retro-Cope elimination reaction while adequately protecting it against cellular nucleophiles. This design preserves the low steric profile of an alkyne and pairs it with a comparably unobtrusive hydroxylamine. The kinetics are on par with those of the fastest strain-promoted azide-alkyne cycloaddition reactions, the products regioselectively formed, the components sufficiently stable and easily installed, and the reaction suitable for cellular labeling.

Full text of DOI:10.1002/anie.202104863

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Bioorthogonal Hydroamination of Push-Pull-Activated Linear
Alkynes
Authors: Dahye Kang, Sheldon T. Cheung, and Justin Kim
This manuscript has been accepted after peer review and appears as an
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202104863
Link to VoR: https://doi.org/10.1002/anie.202104863

10.1002/anie.202104863
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Bioorthogonal Hydroamination of Push-Pull-Activated Linear
Alkynes
Dahye Kang, Sheldon T. Cheung, and Justin Kim*
[*]
Dr. D. Kang, Dr. S. T. Cheung, Prof. Dr. J. Kim
Department of Cancer Biology
Dana-Farber Cancer Institute
Boston, MA 02215 (USA)
Department of Biological Chemistry and Molecular Pharmacology
Harvard Medical School
Boston, MA 02115 (USA)
E-mail: Justin_Kim@dfci.harvard.edu
Supporting information for this article is given via a link at the end of the document.
Abstract:
A
bioorthogonal
reaction
between
N,N-
dialkylhydroxylamines and push-pull-activated halogenated alkynes is
described. We explore the use of rehybridization effects in activating
alkynes, and we show that electronic effects, when competing
stereoelectronic and inductive factors are properly balanced,
sufficiently activate a linear alkyne in the uncatalyzed conjugative
retro-Cope elimination reaction while adequately protecting it against
cellular nucleophiles. This design preserves the low steric profile of an
alkyne and pairs it with a comparably unobtrusive hydroxylamine. The
kinetics are on par with those of the fastest strain-promoted azide-
alkyne cycloaddition reactions, the products regioselectively formed,
the components sufficiently stable and easily installed, and the
reaction suitable for cellular labeling.
Since its introduction two decades ago,[1] the copper-
catalyzed azide-alkyne cycloaddition (CuAAC) reaction has
proven indispensable in fields ranging from materials science to
chemical biology (Figure 1A).[2-3] It is a reaction with widespread
appeal for its rapid kinetics, ease of execution, and for the near
universal adaptability of its reaction components. The relatively
inert azide and terminal alkyne are two of the smallest functional
groups available, and either component can be incorporated with
ease into biological macromolecules, metabolites, and probes
without imposing significant perturbations on the system under
evaluation. The ability to incorporate these motifs into biological
systems through unnatural amino acids using orthogonal tRNA
systems and methionine auxotrophs,[4-6] through lipid and
nucleotide modifications,[7-10] or through metabolic engineering[7,
11-12] have been transformational.
Bertozzi and co-workers have since expanded the
application of the azide-alkyne cycloaddition to live cell and in vivo
systems by employing strained cyclooctynes in lieu of copper
catalysts.[13-14] Strain-promoted reactions are now a staple of the
bioorthogonal compendium with notable examples featuring
trans-cyclooctene,[15] norbornene,[16] quadricyclane,[17] and
cyclopropene[18-19] in inverse-electron demand Diels-Alder
cycloadditions, dipolar cycloadditions, and phosphine ligations.
Importantly, cyclooctyne has undergone substantial geometric[20-
24] and electronic[25-28] tuning in a bid to enhance reaction kinetics
with azides,[29] tetrazines,[15, 30] sydnones,[31] diazo[32] compounds,
and N,N-dialkylhydroxylamines (Figure 1B).[33]
Figure 1. Alkyne activation. (A) Metal-catalyzed azide-alkyne cycloaddition. (B)
Strain-promoted alkyne hydroamination. (C) Hydroamination of a push-pull-
activated linear alkyne.
uncatalyzed reaction kinetics comparable to that of strain-
promoted variants while preserving the size, regioselectivity, and
bioorthogonality of this classic reaction component (Figure 1C).
We first directed our attention to the alkyne’s electronics. Despite
the common presumption that polarized alkenes and alkynes lack
34-35]
chemoselectivity in biological contexts,[29,
we reasoned it
would be feasible to thread the narrow window of chemoselectivity
by deconstructing the electronic effects of the alkyne’s
substituents into their mesomeric and inductive components.
In this work, we revisited the terminal alkyne and explored
alternative modes of activation with the aim of achieving
1
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100
75
50
25
0
8
9
Scheme 1. Substituent effects on hydroamination.
10
11
12
13
14
15
Positing that cellular nucleophiles are particularly attuned to
π-electron density, often reacting by Michael-type addition into π-
deficient alkynes, we surmised that mild π-withdrawing
substituents might be tolerated if offset by compensatory π-
donation from a second substituent in a push-pull manner.
Moreover, such constraints on the mesomeric and
stereoelectronic effects would not preclude strong σ-withdrawing
effects by either or both substituents. The potential magnitude of
the influence of σ-withdrawing effects sans reinforcing π-effects
is apparent in the instability of 1-fluoroalkynes[36] as well as the
computed acceleration of the Bergman cycloaromatization
reaction per the rehybridization effect.[37] Strategic halogenation
at the terminal and propargylic positions of a linear alkyne
appeared promising, a search for an appropriate reaction partner
notwithstanding.
0
500
1000
1500
2000
2500
Reaction time (s)
We demonstrated recently that N,N-dialkylhydroxylamines
and cyclooctynes form a bioorthogonal reaction pair and their
union yields biologically compatible enamine N-oxide ligation
products (Figure 1B). With this precedent, we reasoned that the
retro-Cope elimination reaction would also prove suitable not just
in our bid to miniaturize the alkyne, but to achieve an uncatalyzed
bioorthogonal ligation reaction between a pair of exceptionally
small reaction components, hearkening back to the CuAAC
reaction. While promising in concept, few enamine N-oxide
products derived by intermolecular retro-Cope elimination had
been reported, and of these few, each save one was the product
of a reaction with a strong Michael acceptor.[38] Alkyne π-
activation appeared key in accessing these compounds and
circumventing competing Cope elimination and Meisenheimer
rearrangement-based degradation pathways.[39-41] Intriguingly, an
outlier, ethoxyethylene, indicated that both π-donors and
acceptors are actually prime for the task.[42] Agnosticism of the
reaction to the dearth or surplus of π-electron density boded well
for a push-pull system.
We first evaluated the impact of inductively withdrawing
halogen and chalcogen substituents at the propargylic and
terminal positions of the alkyne. Using reaction conditions
previously optimized for product stability,[43] we found the retro-
Cope elimination reaction between propargyl ether 1 and N,N-
diethylhydroxylamine (2) was completed in 18 h whereas
hydroamination of terminal alkyne 4 was incomplete even after 10
d. Terminal halogenation also had a similar accelerating effect as
the conversion of 6-chlorohex-5-yn-1-ol (6) was nearly complete
by 24 h. Most excitingly, the regioselectivities induced by the
propargyl and terminal alkyne substituents appeared to be
Figure 2. Effects of terminal and propargylic modification. (A) Reactivity screen
using alkynes 8–15. NMR conversion with trifluorotoluene as internal standard.
(B) Synthesis of alkynes 9–15. R=PMB. PMB=p-methoxybenzyl. (C) Second-
order rate constants for hydroamination of alkynes 11–15 in CD3CN at rt.
reinforcing. Hydroamination of unactivated alkyne 4 resulted in
the preferential formation of the Markovnikov adduct, but this
preference was overturned in favor of the anti-Markovnikov
products for both propargyl ether 1 and chloroalkyne 6. These
data suggested that synergy could be achieved if the propargylic
and terminal substituent effects were combined.
2
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100
50
0
13, PBS
13, GSH
14, PBS
14, GSH
15, PBS
15, GSH
23, PBS
23, GSH
23, Cell lysate
0
2
4
6
8
Time (h)
Figure 4. Stability of alkynes 13–15 in 50% CD3CN/PBS in the presence or
absence of glutathione (GSH, 2 mM). Stability of enamine N-oxide 23 in PBS in
the presence or absence of glutathione (2 mM) or HEK293T cell lysate.
monitored by 1H (alkyne 8) or 19F (alkynes 9–15) NMR (Figure 2A).
Robust reactivity toward N,N-dialkylhydroxylamines was
observed with halogenated alkynes 13–15 while alkynes 11 and
12 exhibited moderate reactivity. Difluoroalkyne 10 did undergo
partial conversion over 48 h; however, no conversion could be
observed for propargyl fluoride 9 and propargyl ether 8 over the
same period. Their conversion to the corresponding enamine N-
oxides could only be achieved upon heating to 60 ℃ (see
Supporting Information). The data indicated that the rate of
hydroamination correlates positively with the addition of
electronegative substituents to the propargylic position as well as
to alkyne termini, as anticipated.
Second-order rate constants were determined for
moderately reactive substrates 11 and 12 under pseudo first-
order conditions using excess hydroxylamine. Rate constants for
the most reactive substrates 13–15 were determined by reaction
with equimolar hydroxylamine (Figure 2C). Rate accelerations of
4.1, 63, and 240-fold were achieved over the parent difluoroether
11 by addition of an iodine, bromine, or chlorine atom,
respectively, to the alkyne terminus. Interestingly, removal of a
propargylic oxygen from chloroalkyne 14 produced chloroalkyne
15, which was still marginally faster than bromoalkyne 13
possessing the propargylic ether. With absolute rate constants on
the order of 0.1-1 M-1s-1, the rate of hydroamination of alkynes 13–
15 is comparable to the fastest bioorthogonal strain-promoted
azide-alkyne cycloaddition reactions.[23, 45]
32
24
16
8
34
39
44
49
s-character of C1 in C1–X (%)
Figure 3. Computational studies on the effects of alkyne halogenation. (A) s-
Characters (s-char) of alkyne sp-carbons were analyzed, and activation free
energies (ΔG‡) were computed for the reaction of alkynes with hydroxylamine
23. (B) Correlation of percent s-character of C1 in the C1-X bond and activation
free energy for hydroamination of the alkyne with N,N-dimethylhydroxylamine.
We next set out to investigate the role of the rehybridization
effect in driving the hydroamination of electronically modified
linear alkynes. Deviation of an alkyne’s atomic orbitals from
canonical hybridization schemes in accordance with Bent’s rule[46]
was expected to result in ground state destabilization of
haloalkynes – instability, which reaction might alleviate.[36-37, 47]
DFT calculations were performed at the M06-2X[48] level of
theory for reactions between N,N-dimethylhydroxylamine (22)
and model alkynes 20a–20o. We first confirmed that the
computed activation energies (ΔG‡) accurately recapitulated the
reactivity trends observed experimentally (Figure 3A). We then
performed natural bond orbital (NBO) analysis. In ground state
calculations of the alkyne component, electronegative atoms,
whether at the propargylic or terminal position, reduced the s-
character in the bonding orbital of the most proximal sp-hybridized
carbon consistent with Bent’s rule. Notably, the effect of terminal
halogenation was much more pronounced, and the s-character of
C1 in the C1-X bond deviated significantly (Cl, 40%; F, 35%) from
To examine whether these predictions would be borne out
experimentally, substrates 8–15 were synthesized (Figure 2A).
Grignard addition of ethynylmagnesium bromide into aldehyde 16
provided propargyl alcohol 17, which was readily converted to
either propargyl fluoride 9 using diethylaminosulfur trifluoride
(DAST) or to propargyl difluoride 10 by sequential oxidation and
difluorination with Dess-Martin periodinane and DAST,
respectively. Chloroalkyne 15 was obtained by halogenation of
the corresponding acetylide (Figure 2B). Difluoropropargyl ethers
11–14 were accessed by SN2' addition of a sodium alkoxide into
bromodifluoroallene
halogenation (Figure 2B).
18,[44]
desilylation,
and
acetylide
With the desired alkynes in hand, we screened their
reactivity toward N,N-diethylhydroxylamine (2). Alkynes 8–15
were each incubated with 5 equivalents of hydroxylamine 2 in
CD3CN at room temperature, and the reaction conversions were
3
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Figure 5. In vitro and live cell labeling by bioorthogonal hydroamination. (A) HaloTag protein was conjugated to chloroalkyne 24 and modified by TAMRA-
hydroxylamine 25 then visualized by in-gel fluorescence or fluorescence microscopy. (B) Structures of chloroalkyne 24 and TAMRA-hydroxylamine 25. (C) Time-
dependent in-gel fluorescence analysis of hydroamination between HaloTag protein-alkyne 24 conjugate (18 µM) and hydroxylamine 25 (200 μM) for 1-60 min at
rt. (D) Concentration-dependent in-gel fluorescence analysis of hydroamination between HaloTag protein-alkyne 24 conjugate (18 μM) and hydroxylamine 25 (25-
200 μM) upon incubation for 1 h at rt. (E) Cell surface HaloTag-GFP expressed on HEK293T cells was labeled with TAMRA by bioorthogonal hydroamination
between alkyne 24 and hydroxylamine 25. Merge is a composite of Hoechst 33342, GFP, and TAMRA channels. Scale bar = 50 µm. TAMRA = tetramethylrhodamine.
the canonical 50%. A plot of the reaction free energies against the
percent s-character of C1 in the C1-X bond of terminally
functionalized propynes or difluoropropargyl ethers revealed a
strong positive linear relationship consistent with other reactions
respectively, in the added presence of 2 mM glutathione. These
stabilities compare favorably with contemporary bioorthogonal
transformations.[49-50] Notably, bromoalkyne 13, which was less
reactive toward hydroamination than alkynes 14 and 15, proved
unacceptably sensitive to thiols, degrading with a half-life of <30
min under identical conditions. This observation is consistent with
the hypothesis that σ-withdrawing/π-donating alkyne substituents
such as halogens simultaneously promote hydroamination and
attenuate conjugate addition by cellular nucleophiles. Additionally,
enamine N-oxide 23 showed no observable degradation over 24
h at room temperature in aqueous solutions containing 2 mM
glutathione or HEK293T cell lysate.
We continued to evaluate the viability of the reaction both in
vitro and in cells (Figures 5A and 5B). After verifying that the
reaction between chloroalkyne 14 and N,N-diethylhydroxylamine
proceeds as expected under aqueous conditions and in the
presence of protein (Figure S2), purified recombinant HaloTag
protein[51-52] was incubated with HaloTag linker-conjugated
difluoropropargyl ether 24 for 10 min at room temperature in pH
7.0 phosphate buffer to provide alkyne-conjugated protein. This
protein was then treated with 200 µM TAMRA- hydroxylamine 25
and analyzed at various time points by in-gel fluorescence.
that exhibit rate enhancements deriving from substantial
47]
rehybridization effects (Figure 3B).[37,
NBO analysis also
revealed that propargylic modifications had a much more muted
effect on alkyne rehybridization despite its more significant impact
on reducing the activation free energy. In combination with the
divergent regioselectivities imparted by each modification, it is
plausible that the accelerating effects of propargylic halogen
substituents can be imputed more heavily on their
stereoelectronic rather than inductive effects, while the opposite
may be true for their terminal counterparts. Nonetheless, alkyne
halogenation at either site or in combination provides an effective
alternative to strain-activation in the context of the retro-Cope
elimination reaction.
We next evaluated the stability of alkynes 13–15 under
physiologically relevant conditions (Figure 4). Alkynes 14 and 15
showed no degradation by 19F NMR over 7 d in 50%
CD3CN/phosphate-buffered saline (PBS), pH 7.0 at room
temperature while displaying half-lives of 14 h and 43 h,
4
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experiment demonstrated expected time-dependent labeling over
1 h (Figure 5C). Labeling also proved concentration-dependent
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(Figure 5D). No labeling was observed in the absence of either
the alkyne or hydroxylamine. Formation of the desired protein-
fluorophore adduct upon hydroamination was verified by ESI-MS
(Figure S11).
Finally, we explored live cell labeling by hydroamination
(Figures 5E and S12). HEK293T cells were transiently transfected
with a cell surface HaloTag-GFP construct, treated with 10 μM
HaloTag linker-conjugated difluoropropargyl ether 24, washed,
and incubated with 50 μM TAMRA-conjugated hydroxylamine 25
for 1 h. The cells were then fixed and visualized by confocal
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with the GFP signal of transfected cells. Importantly, negative
controls lacking the alkyne and/or hydroxylamine did not exhibit
significant labeling. These experiments demonstrated the
specificity and efficacy of the reaction in cellular ligation
applications.
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We have described a bioorthogonal reaction between N,N-
dialkylhydroxylamines and halogenated alkynes. We explored the
use of rehybridization effects in activating alkynes, and we found
that electronic effects, when competing stereoelectronic and
inductive factors are properly balanced, sufficiently activate a
linear alkyne in the uncatalyzed conjugative retro-Cope
elimination reaction while adequately protecting it against cellular
nucleophiles. This design preserves the low steric profile of an
alkyne and pairs it with a comparably unobtrusive hydroxylamine.
The kinetics are on par with those of the fastest strain-promoted
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azide-alkyne
cycloaddition
reactions,
the
products
regioselectively formed, the components sufficiently stable and
easily installed, and the reaction suitable for cellular labeling.
Furthermore, this method introduces a unique enamine N-oxide
molecular linchpin, which has potential for further functional
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10.1002/anie.202104863
Angewandte Chemie International Edition
COMMUNICATION
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A rapid and regioselective click reaction between N,N-dialkylhydroxylamines and linear alkynes is described. This bioorthogonal
reaction between two small components is enabled by the rehybridization effect of terminally halogenated alkynes and the careful
balancing of alkyne π-electron density through a push-pull mechanism.
Institute and/or researcher Twitter usernames: @JKimLab
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