Light Harvesting for Rapid and Selective Reactions: Click Chemistry with Strain-Loadable Alkenes

Article abstract of DOI:10.1016/j.chempr.2017.11.007

Intramolecular strain is a powerful driving force for rapid and selective chemical reactions, and it is the cornerstone of strain-induced bioconjugation. However, the use of molecules with built-in strain is often complicated as a result of instability or selectivity issues. Here, we show that such strain, and subsequent cycloadditions, can be mediated by visible light via the harvesting of photochemical energy. Through theoretical investigations and molecular engineering of strain-loadable cycloalkenes, we demonstrate the rapid chemoselective cycloaddition of alkyl azides with unstrained cycloalkenes via the transiently (reversibly) formed trans-cycloalkene. We assess this system via the rapid bioconjugation of azide-functionalized insulin. An attractive feature of this process is the cleavable nature of the linker, which makes a catch-and-release strategy possible. In broader terms, we show that conversion of photochemical energy to intramolecular ring strain is a powerful strategy that can facilitate complex chemical transformations, even in biomolecular systems. Probing, isolating, and/or manipulating biologically relevant macromolecules is central to the study of their function in living systems. However, the synthetic tools available for performing the chemistry necessary for such studies are often difficult to use or limited in utility. In the approach presented here, light is converted to molecular strain energy, which can in turn be used for performing rapid and highly selective chemistry on macromolecular systems. Because it involves chemically stable and chemoselective reactions, this research not only opens up new possibilities for biomolecular functionalization and manipulation but also promises to make such experiments accessible to a broader class of researchers. The central concept of strain-loadable alkenes is general and provides a firm foundation for light-activated chemistry in complex environments. Strain-loadable alkenes are cycloalkenes that, when irradiated in the presence of a visible-light-absorbing photocatalyst, undergo double-bond isomerization. Because of engineered geometrical constraints, this isomerization results in significant molecular strain. Weaver and colleagues exploit this strain to dramatically accelerate the cycloaddition with azides, which are otherwise unreactive, in mixed molecular environments.

Full text of DOI:10.1016/j.chempr.2017.11.007

Article
Light Harvesting for Rapid and Selective
Reactions: Click Chemistry with Strain-
Loadable Alkenes
Kamaljeet Singh, Christopher J.
Fennell, Evangelos A. Coutsias,
Reza Latifi, Steve Hartson,
Jimmie D. Weaver
jimmie.weaver@okstate.edu
HIGHLIGHTS
Engineered alkenes for the
capture and conversion of
photochemical to strain energy
Strain-free reagents for strain-
induced conjugation allow easy
synthetic handling
Visible-light-mediated strain
loading provides ultimate
functional-group tolerance
Light-induced conjugation
provides spatial-temporal control
opportunity
Strain-loadable alkenes are cycloalkenes that, when irradiated in the presence of a
visible-light-absorbing photocatalyst, undergo double-bond isomerization.
Because of engineered geometrical constraints, this isomerization results in
significant molecular strain. Weaver and colleagues exploit this strain to
dramatically accelerate the cycloaddition with azides, which are otherwise
unreactive, in mixed molecular environments.
Singh et al., Chem 4, 1–14
January 11, 2018 ª 2017 Elsevier Inc.
https://doi.org/10.1016/j.chempr.2017.11.007

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Article
Light Harvesting for Rapid and Selective
Reactions: Click Chemistry
with Strain-Loadable Alkenes
1
1
2
1
3
Kamaljeet Singh, Christopher J. Fennell, Evangelos A. Coutsias, Reza Latifi, Steve Hartson,
and Jimmie D. Weaver1,4,
*
SUMMARY
The Bigger Picture
Intramolecular strain is a powerful driving force for rapid and selective chemical
reactions, and it is the cornerstone of strain-induced bioconjugation. However,
the use of molecules with built-in strain is often complicated as a result of insta-
bility or selectivity issues. Here, we show that such strain, and subsequent cyclo-
additions, can be mediated by visible light via the harvesting of photochemical
energy. Through theoretical investigations and molecular engineering of strain-
loadable cycloalkenes, we demonstrate the rapid chemoselective cycloaddition
of alkyl azides with unstrained cycloalkenes via the transiently (reversibly)
formed trans-cycloalkene. We assess this system via the rapid bioconjugation
of azide-functionalized insulin. An attractive feature of this process is the cleav-
able nature of the linker, which makes a catch-and-release strategy possible. In
broader terms, we show that conversion of photochemical energy to intramo-
lecular ring strain is a powerful strategy that can facilitate complex chemical
transformations, even in biomolecular systems.
Probing, isolating, and/or
manipulating biologically relevant
macromolecules is central to the
study of their function in living
systems. However, the synthetic
tools available for performing the
chemistry necessary for such
studies are often difficult to use or
limited in utility. In the approach
presented here, light is converted
to molecular strain energy, which
can in turn be used for performing
rapid and highly selective
chemistry on macromolecular
systems. Because it involves
chemically stable and
INTRODUCTION
chemoselective reactions, this
research not only opens up new
possibilities for biomolecular
functionalization and
The study of biomolecules in their native environments is facilitated by selective
1
–3
chemistry, empowering research into a wide array of areas.
To be useful, such re-
actions need to be extremely rapid and have minimal reactivity with native functional
groups, a balance that is highly desirable yet highly difficult to strike. Strain-induced
couplings have proven useful in bioconjugation (Figure 1A) but often require signif-
icant effort to ensure that the reactivity is used productively. The use of external stim-
uli, such as light, can offer an additional level of control not available in other
manipulation but also promises to
make such experiments accessible
to a broader class of researchers.
The central concept of strain-
loadable alkenes is general and
provides a firm foundation for
light-activated chemistry in
4
–7
methods.
The ability to use light to toggle chemistry on and off offers the conve-
nience of spatial and temporal control. Several examples of light-induced bio-
conjugation systems have been developed over the last decade (Figure 1B), and
most commonly, these make use of short-wavelength light. This light is typically
used to convert an otherwise unreactive molecule to a reactive molecule that un-
dergoes bond formation with a non-natural functional group. In this manner, upon
complex environments.
UV irradiation, tetrazoles can extrude N
2
to form a highly reactive nitrile imine
8
–11
dipole,
which undergoes cycloaddition with alkenes. This strategy has been
1
2
used by Lin to label proteins within Escherichia coli cells. In addition, Lin has
also shown that reactive nitrile ylides generated from the photolysis (302 nm) of
1
3
14
2
H-azirines can be used for the PEGylation of a lysozyme. Similarly, cycloprope-
1
5
nones have been used as a photolabile surrogate for strained cycloalkynes, which,
upon unmasking, undergo rapid reaction with azides. Although these works have
begun to demonstrate the utility of ‘‘photochemical click’’ reactions, the use of
Chem 4, 1–14, January 11, 2018 ª 2017 Elsevier Inc.
1

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Figure 1. Conjugation Strategies
11
07 Physical Science, Department of Chemistry,
Oklahoma State University, Stillwater, OK 74078,
USA
high-energy UV light is a significant drawback because of its often detrimental ef-
fects on living organisms. Two-photon processes, which utilize lower energy pho-
tons, could help alleviate the need for high-energy photons, but they can suffer
2
Department of Applied Mathematicise and
Statistics, Stony Brook University, New York, NY
1794, USA
1
3Department of Biochemistry and Molecular
Biology, Oklahoma State University, Stillwater,
OK 74078, USA
9
,16
from low quantum yields, and these strategies are still under development.
Tet-
razines have been a popular motif in conjugation chemistry because of their highly
reactive nature, which allows them to undergo rapid [4 + 2] cycloaddition reac-
4Lead Contact
1
7,18
tions.
The cycloaddition of tetrazines with trans-cyclooctenes provides the
*Correspondence: jimmie.weaver@okstate.edu
https://doi.org/10.1016/j.chempr.2017.11.007
3
6
ꢀ1 ꢀ1 19,20
greatest rate constants to date (from 2 3 10 to 1 3 10 M
s
),
partly as a
2
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result of the strained nature of the trans-cyclooctene, the most strained isolable
alkene known. However, the built-in reactivity of both partners can decrease the
1
7
practicality of this system via non-trivial syntheses and handling concerns. Very
2
1
recently, Fox and co-workers have shown that visible light and catalytic methylene
blue can be used to oxidize dihydrotetrazine in situ to give the highly reactive tetra-
zine, which undergoes rapid conjugation, circumventing some of the aforemen-
tioned issues. However, in all of these examples, the light stimulation serves to irre-
versibly unmask a reactive molecule, and little additional control is gained in
comparison with non-stimulated strain-induced reactions. In order for the field of
light-mediated bioconjugation to move forward, we need new strategies that pro-
vide enhanced control of reactivity.
In 2014, we showed that we could use catalytic amounts of fac-tris-[2-phenylpyridi-
2
nato-C ,N]iridium(III) [Ir(ppy)
3
] and blue light-emitting diodes (LEDs) to facilitate the
2
2
isomerization of substituted styrenes toward the less conjugated Z isomers. We
were curious to find out if we could find an alkene that, upon isomerization, would
capture some of the photochemical energy in the form of ring strain that could be
utilized for bimolecular couplings (Figure 1C). If successful, we would be able to
apply visible light as a trigger that would generate a reactive alkene potentially
capable of undergoing bioconjugation. It was expected that this might circumvent
some of the circuitous syntheses of popular strained molecules currently being
used for bioorthogonal coupling. Furthermore, unlike other photo-initiated pro-
cesses that utilize photochemical energy to irreversibly unmask the reactive group,
our system would simply capture photochemical energy, which would give rise to a
steady, catalytic concentration of a more reactive form of the same molecule, a
currently unexplored strategy.
RESULTS AND DISCUSSION
2
3
At the outset, we considered a variety of coupling partners, and azides stood out
as a good candidate. They are relatively inert toward biological functional groups,
result in minimal perturbation to the system, and are known to undergo reaction
2
4,25
26,27
with alkenes to form triazolines.
It is also known that electron-deficient
30
2
8,29
and strained
alkenes react faster with azides. Bach calculated the energy bar-
rier for the cycloaddition of methyl azide to cyclooctyne and E-cyclooctene, and
found that addition of methyl azide to cyclooctyne has a lower barrier than E-cyclo-
octene by 3.1 kcal/mol. In addition, Houk and co-workers performed a theoretical
study on the cycloaddition of strained alkenes and azides and concluded that only
trans-cyclooctene would be sufficiently rapid for use at room temperature, perhaps
2
8
somewhat diminishing interest in the alkene azide cycloaddition. However, in this
study, only isolable trans-cycloalkenes were considered, leaving the possibility for
potential discovery of a smaller alkene that would react more rapidly. Early photo-
chemical studies have shown that the trans-cycloalkenes could be formed via excita-
tion to the excited state with UV light, which suggest that formation could be
3
1–35
possible with visible light and a photocatalyst.
We initiated our study by using (E)-1-phenylcyclooct-1-ene (E-PC8; Scheme 1A). We
were pleased to see the isomerized alkene (Z)-1-phenylcyclooct-1-ene (Z-PC8) was
formed under these conditions. Although the photostationary state equilibrium
was disappointing from a synthetic view (19%–29%), it was encouraging from an en-
ergetic standpoint. Caldwell and co-workers have estimated the energy difference
3
6
for the E and Z isomers of phenyl cyclooctene at 13.3 kcal/mol, suggesting that
we were able to skew the product distribution away from the thermodynamic
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Scheme 1. The Development of the Molecular Mousetrap
(
(
(
(
(
A) First effort to capture photochemical energy as ring strain.
B) Attempts to chemically capture strained alkenes. Significant dimerization was observed for entries 3 and 4; see Supplemental Information for details.
C) Calculated torsion angle energy landscapes about the ring alkene bonds in (left) PC7 and (right) BC7.
D) Reaction results for BC6, BC7, and BC8 triazoline formation.
E) Two views of the crystal structure of the major product, showing the trans-ring fusion.
4
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equilibrium by nearly ten orders of magnitude using visible light. Because our inten-
tion was to generate the reactive alkenes in situ rather than isolate the unstable mol-
ecules, we were not dissuaded.
We next evaluated the ability to form triazolines as a function of alkene ring size
(
Scheme 1B). It was anticipated that the trans-cycloalkene would be continually
consumed by the azide, and the photocatalytic system would reestablish the equi-
3
7
librium. In contrast to their reactivity upon UV-light irradiation, alkyl azides are
generally stable to visible light, which is important to avoid competing types of
photochemistry. We were pleased to see that 7- and 8-membered trans-alkenes (en-
3
8
tries 1–4) afforded the desired triazoline product. Although the rates of the reac-
tion displayed some electronic dependence in the PC8 system, in general, it was
disappointingly slow (3–5 days). In contrast, despite no observation of the trans
1
isomer ( H nuclear magnetic resonance [NMR]) in the PC7 series, the reaction
required less than 0.67 days to reach completion. This suggested a strong correla-
tion between ring strain and reaction rate and provided evidence that a transient
alkene could result in faster coupling than the PC8 system. Unfortunately, significant
3
9
dimerization was also observed in the case of the PC7 system. Meanwhile, cyclo-
hexene, PC6, failed to give any conversion even after 5 days of irradiation (entry 5).
In order to better understand the observed reactivity and guide future efforts, we
performed a computational evaluation of the energy landscapes showing conver-
sion between the Z and E forms of PC6, PC7, and PC8. We generated both singlet
2
3
and triplet excitation landscapes with MP2/cc-pVTZ calculations in order to assess
both the strain energy loaded upon isomerization and gauge how features of the
excitation process could influence the reactive behavior of these systems. As ex-
pected, Z-PC7 (Scheme 1C; Figure S10) has over twice the strain energy of Z-PC8
(
Figure S12), supporting our observed reactivity. Although PC6 has a similar excita-
tion energy, the trans isomer, when accessible, is only transiently stable with a rever-
sion barrier to the cis isomer near room temperature thermal energy. The predicted
short-lived nature helps explain the inability of PC6 to undergo bimolecular triazo-
line formation, indicating that reactivity is most likely limited to intramolecular
processes.
Given that a transient amount of the trans-cycloheptene gave a substantial rate in-
crease in comparison with observable amounts of the PC8 system, we sought to
modify these systems to increase the concentration of the reactive conformer(s).
We envisioned that rigidification of the cycle could potentially result in increased
strain energy and increased barriers to interconversion. These changes, along with
potentially more efficient energy transfer due to the fusion of the aryl ring that rein-
forces conjugation with the double bond, might lead to increased concentration of
more reactive strained species. Thus, we fused the essential aryl group to the ring
system, giving benzocyclohexene, -heptene, and -octene (Z-BC6, Z-BC7, and
Z-BC8; Scheme 1D). We performed similar energy landscape calculations on these
compounds (Scheme 1C; Figures S7, S9, and S11), and they partly support these ex-
pectations. In the case of BC7, there is indeed a roughly 7 kcal/mol increase in strain
energy in relation to PC7 upon isomerization; however, the interconversion barrier is
essentially unchanged. The forced conjugation in BC7 lowers the triplet excitation
energy from the unstrained singlet conformer in relation to the PC7 excitation en-
ergy, which exists over a 30 kcal/mol band as a result of thermally accessible ground
state conformers. This lower excitation energy of BC7 will most likely result in an
increased excited-state population when under visible-light irradiation. Calculations
on BC6 and BC8 (Figures S7 and S11) predict poor triazoline formation reactivity for
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both. In BC6, a strained form is geometrically disallowed. In BC8, because of atomic
crowding in the octene ring, conjugation is severely broken and raises the excitation
energy well above the triplet energy of the photocatalyst used. We synthesized and
tested the photocatalytic reactivity of these three aryl fused-ring systems (Scheme
1
D), and the results are fully consistent with the calculations. Of these, BC7 certainly
shows the greatest promise given its predicted higher strain energy and greater po-
tential concentration of reactive species, both supporting an expected increase in
the rate of the subsequent bimolecular cycloadditions. Our attempts to trap the
transient E-BC7 with benzyl azide were successful. We were pleased to find that
the reaction went to completion in less than 1 day, with only two regioisomers as
2
3
the sole products. Both are single diastereomers with trans-ring junctions. An
X-ray structure of the major product (Scheme 1E) clearly shows the trans-ring fusion
of the triazoline, which is most readily explained by a concerted or nearly concerted
[
3 + 2] cycloaddition of the azide and E-BC7. Control reactions indicate the necessity
2
3
of the photocatalyst and indicate that there is no background reaction with Z-BC7.
Despite our initial excitement over these preliminary findings, there were a number
of issues that might preclude its utility for bioconjugation purposes that needed to
be addressed. These issues included slow kinetics, problematic dimerization of
alkene, use of excess azide, strict use of inert environment, use of pure MeCN, the
unknown toxicity of iridium and its potential to cause undesired redox chemistry,
and the instability of the triazoline motif. Fortuitously, the dimerization of the BC7
system proved to be much slower than that of the PC7 system, so slow that it was
no longer problematic. We then focused on increasing the rate of the reaction
and utilizing the azide as the limiting reagent. Although the reaction works with
either alkene or azide as the limiting component, we assumed that the azide was
the most valuable component of the reaction mixture. This seemed reasonable
given the overwhelming number of examples of incorporation of azides into biomol-
ecules, and functioning under azide limiting conditions would allow this chemistry to
function as a drop-in solution and require little to no alterations of existing biological
systems. Optimization included solvent, photocatalyst structure and loading, alkene
2
3
loading, photon dependency, substrate concentration, and temperature effects.
We found that use of dimethylformamide (DMF), commercially available fac-tris-
0
ꢁ
3 3
-ppy) (Cat. 3) at 1.2 mol %, 60 C, and 3 equiv of the alkene partner all
Ir(4 -CF
led to increases in the rate of the reaction. Under optimal reaction conditions,
2
3
both the photocatalyst and the alkene fell out of the rate expression, such that
no further rate increase was observed by increasing the concentration of these re-
agents. Under these conditions, a first-order rate constant was determined to be
ꢀ3
ꢀ1
23
k = 2.4 G 0.9 3 10
s
, with a half-life of only t1/2 = 4.8 min. Furthermore, we
expect that this is a lower estimate for the rate constant because it was performed
with an easily monitored substrate, which is known to react more slowly than others.
In direct competition experiments between the photoconjugation of several azides
with BC7 and Cu-catalyzed azide alkyne cycloaddition (CuAAC), the photoconjuga-
2
3
tion reaction was found to be up to 100-fold faster. CuAAC has been used for
bioconjugation, which suggests that the photoconjugation of BC7 takes place
with sufficient rates for bioconjugation applications.
In order to better understand the nature of the reaction, we began to investigate the
scope of the reaction by using these optimized conditions (Scheme 2). In bio-
conjugation, the surface of biomolecules can severely limit access to the azide sub-
strate, so we were pleased to see that the reaction works well across a range of ste-
rically hindered azides, even with sterically demanding 1-azidoadamantane (4i).
Often, the modest isolated yields are a reflection of the separation of the minor
6
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Scheme 2. Visible-Light-Mediated [3 + 2] Cycloaddition of Benzofused Cycloheptenes and Azides
regioisomer during purification, not a lack of reactivity. Despite the frequent use of
4
0–42
photocatalysts in promoting redox reactions,
the conditions proved to be
tolerant of a number of biologically relevant functional groups, such as amino acid
derivatives (4j and 4m), amines (4d, 4f, and 4l), amides (4c and 4m), alcohols (4g
and 4n), esters (4e), electron-rich arenes (4h), and purine bases or sugar molecules
(
4n). In addition, it tolerates a number of unnatural functional groups such as aryl-
bromides (4l) and -fluorides (4b), and alkynes (4k). This functional-group tolerance
is most likely a result of the mild conditions and the fast quenching of the excited-
state photocatalyst by the alkene. In other words, excess alkene serves to protect
sensitive functional groups by dissipating the photochemical energy in the form of
4
3
bond motion, much like the protective action of cinnamic acid-based sunscreens.
Smooth intermolecular coupling to give 4k demonstrates the orthogonal nature of
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these reaction conditions toward traditional alkyne and azide cycloadditions that
would be expected to give the fused triazole product. The doubly orthogonal nature
of the reaction makes it even more attractive, because alkynes could be used later for
4
4
further functionalization via traditional click chemistry.
Although the geometry of the ring is a prerequisite to reactivity, initial exploration
suggests that the aryl ring (4c and 4m) and the seven-membered ring (4l) can both
tolerate substitution. In the future, further evaluation will focus on the development
of probes and fine-tuning of the reactivity and improvement in the physicochemical
properties of the molecule. Acetamides (4c and 4m) were found to be well tolerated
in the reaction and allowed the facile coupling of a biotin-labeled alkene with a pro-
tected amino acid derivative (4m). This example highlights a particular strength of
this approach to strain-induced coupling: derivatization of the alkene is rapid and
trivial, and it provides biochemical tools that are indefinitely stable until subjected
to the reaction conditions. In the case of conjugation chemistry, it may be advanta-
geous if the stereochemical environment of the substrate does not affect the rate of
conjugation, in case it resulted in failed couplings. Fortunately, stereocenters on the
azide do not hamper the cycloaddition (4j, 4m, and 4n).
Finally, knowing that we want to use this chemistry on biological systems in which
proteins might interfere in undesired ways, we subjected BC7 and 4-fluorobenzyl
azide to normal reaction conditions but, instead of pure DMF, a 4:1 DMF:10%
aqueous BSA solution was used. Gratifyingly, under these conditions 79%
1
9
(
F NMR yield) of the desired product 4b was formed. Given the possibility of inter-
ference or unselective reaction on the protein surface, a high yield of the desired
4
5
product indicates that the BSA interfered with neither yield nor chemoselectivity.
Serendipitously, it also led us to discover that the reaction was further accelerated by
the presence of water, given that the reaction takes nearly 50 min to complete in dry
DMF but <15 min (upper limit) in the presence of the BSA solution. Under these
conditions, the reaction mixture was homogeneous, but at higher water loadings
the photocatalyst precipitates, which precludes using this specific set of reaction
components in a purely aqueous system. However, we believe the aqueous solubi-
lity can be increased via a number of strategies, and this is currently being pursued in
our laboratory. We were pleased to find that this acceleration was a general trend
(
Scheme 2, conditions B), with most substrates experiencing a roughly 3-fold
increase in rate, although we have made no attempts to directly quantify this
acceleration. The source of this acceleration could be a decrease in the transition
z
state volume (DV ), a case in which the use of water has been shown to lead to an
4
6,47
acceleration.
Alternatively, it is possible that the trans-alkene adopts a
zwitterionic form to relieve strain and is stabilized by a more polar solvent, leading
to a greater concentration of the reactive form.
Tetrazines are known to have better reactivity than azides and undergo cycloaddition
with strained alkenes. Thus, we briefly explored this possibility. We exposed E-PC8
ꢁ
to 3,6-diphenyl-1,2,4,5-tetrazine in the presence of Cat. 3c and MeCN at 45 C, and
2
3
no cycloaddition product was observed. Importantly, no strained Z-PC8 was detected
as is the case in the absence of the tetrazine) and both the reactants were recovered
(
unchanged after 18 hr of the irradiation. One explanation for this is that the fuchsia-
colored 3,6-diphenyl-1,2,4,5-tetrazine could itself absorb in the visible-light region
and might be responsible for the observed lack of reactivity.
In contrast to triazoles, triazolines are not aromatic and are quite susceptible to
further chemistry. Traditionally, the observed rate of triazoline decomposition was
8
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Table 1. Stability Studies of Triazoline Products
Entry
4a and 4b Conditions
Result
ꢁ
1
2
3
4
5
6
4a Cat-Ir(ppy)
3
, 60 C, Ar, DMF, blue LEDs
no decomposition, 24 hr
ꢁ
4a isolated compounds, 4 C, 2 years
4a-major, no decomposition; 4a-minor, decomposition
4a-minor, decomposition
4a-minor, some decomposition
no decomposition
4a DMF, exposed to air
4a silica chromatography
4a basic alumina chromatography
4b TFA, 5 min
rapid decomposition, 5b 38%
comparable with that of its formation, possibly deterring investigation of its use in
1
conjugation chemistry. However, the use of BC7 under photocatalytic conditions
effectively amplifies the rate of triazoline formation, such that appreciable
decomposition does not occur on the reaction timescale, allowing isolation of the
triazoline products. Because triazolines can be formed and isolated, the inherently
greater lability of triazolines might be a unique asset if decomposition could be
4
8
induced intentionally. In other words, rapid conjugation could then be followed
by controlled release of the amine product. In fact, Gamble and co-workers have
demonstrated that hydrolysis-susceptible triazolines formed via the 1,3-dipolar
cycloaddition of azide and trans-cyclooctene is a viable prodrug activation
4
9
strategy.
To investigate the stability, we subjected 4a to a number of conditions (Table 1).
Extended reaction time (24 hr) led to no detectable decomposition (entry 1). However,
ꢁ
upon isolation and storage at 4 C, the major isomer showed no decomposition for up to
2
years (and counting). The minor regioisomer is notably less stable and decomposes
both upon storage (entry 2) and in solution (ꢂ7 days) even in the absence of the photo-
catalyst (entry 3). Decomposition is further accelerated when chromatography on silica
is attempted (entry 4). Basic alumina was found to be an appropriate chromatography
medium; it did not lead to decomposition of the minor isomer (entry 5). However, upon
exposure of the products to acidic environments, we observed rapid decomposition
2 2
and gas evolution, presumably N and CO . The isolation of a-tetralone from decom-
5
0
posed mixtures was surprising but has precedent and presumably arises from ring
contraction, followed by hydrolysis, oxidation, and an oxidative decarboxylation.
Upon brief exposure of the crude reaction mixture containing 4a to trifluoroacetic
acid (TFA) (entry 6), a 38% yield of 5b was obtained, suggesting that this might be a
viable release strategy. Further studies aimed at increasing this yield via optimization
of the alkene structure and conditions are underway. As an aside, it could also provide
an alternative route to the Staudinger reaction, which produces hard-to-separate triphe-
nylphosphine oxide during the reduction of azides.
Given the frequent use of dyes as fluorophores for labeling cells in many
bioconjugation reactions, the effect of fluorophores on the reaction outcome was
evaluated. It was anticipated that the outcome of the reactions could potentially
Chem 4, 1–14, January 11, 2018
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Chem (2017), https://doi.org/10.1016/j.chempr.2017.11.007
N
N
N Bn
Cat 3 (1.2 mol%)
o
6
0 C, Ar
Bn N3
+
DMF/H O (4:1, 0.2 M)
2
4
a
blue LEDs
Dye (1 mol%)
1
.0 equiv 3.0 equiv
+ regioisomer
SO Na
3
O
OH
CF3
OH
N
HO
CF3
Ir
N
N
N
N
Sunset yellow
abs λmax 485
Alizarin
abs λmax 435
O
F3C
Cat 3
NMR yield = 45%
NMR yield = 57%
NaO S
3
O
SO Na
3
SO Na
3
O
O
O
O
O
Cl
N
Cl
O
N
Ph
HO
2
O
OH
OH
Orange G
abs λmax 500
',7'-dichlorofluorescein
O
Perylene-3,4,9,10-
abs λmax 520
tetracarboxylic dianhydride
abs λmax 520
NMR yield = 50%
NMR yield = 42% with
10 mol% dye
NMR yield = 41%
NMR yield = 53%
Scheme 3. Reaction Outcome in the Presence of Various Fluorophores
be effected partially, or even completely, in the presence of dyes as a result of
competitive absorption of photons or possibly undesired quenching of the excited
photocatalyst by the dye. Hence, experiments were performed in which various
dyes, absorbing at different wavelengths, were added to the reaction mixture.
The results are tabulated below (Scheme 3). The desired triazoline product 4a was
formed, albeit with lower efficiencies than the standard conditions in the absence
of any extra dye. Increasing the amount of dye (2,7-dichlorofluorescein) present in
the reaction to more than 8-fold that of the photocatalyst, resulted in a 42% NMR
yield of the desired triazoline product, 4a, within 6 hr of irradiation. With some effort,
it should be possible to design a system that selectively excites a photocatalyst even
2
3
in the presence of another dye, which is supported by the results of Scheme 3.
In order to showcase the applicability of this novel photoconjugation approach, we
applied the reaction conditions on a small tripeptide. A commercially available
tripeptide was modified with an azide and then conjugated with the BC7 under
standard conditions. Within 4 hr of the irradiation, complete conversion to the
desired conjugated tripeptide mass was observed by liquid chromatography-mass
2
3
spectrometry.
Although we had observed that the reaction could take place selectively in the pres-
ence of BSA, we also investigated the ability to perform the chemistry directly on a
biomolecule. This would demonstrate that the photoconjugation takes place at
sufficient rate to be useful even with large molecules, which have smaller diffusion
constants and are present at much lower concentrations, typical of proteins. The
N termini of insulin (bovine), a peptide hormone that plays a key role in the
10
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Chem (2017), https://doi.org/10.1016/j.chempr.2017.11.007
Figure 2. Demonstration of Biotin Conjugation to Insulin via Strain-Loadable Alkene Coupling
There are three modifiable sites on the insulin structure highlighted in red: the N termini on the A
and B strands and the lysine residue on the B strand. The matrix-assisted laser desorption/
ionization spectra on the right show modification and identification of two variants of the
monoconjugated protein.
metabolism of glucose and is made up of two peptide chains connected by disulfide
bridges, were unselectively modified with an azide-modified benzoylating reagent
(
A; Figure 2). The mixture of un-, mono-, di-, and tri-modified insulin (21.8 mM)
was next subjected to the photoconjugation reaction (Cat. 3, 0.1 mg), with biotin-
modified benzocycloheptene (BC7-B, 1.2 mM), which took place at room tempera-
ture and with no degassing. In less than 15 min, all of the modified insulin was
consumed. New masses consistent with the mono- and di-conjugated product
5
1
were detected. The conjugation of insulin was further supported by the SDS-PAGE
experiment and subsequent silver staining. New bands of slightly higher molecular
2
3
weight than the unmodified insulin and azide-modified insulin were detected.
When the alkene was left out of the irradiated reaction mixture (12 hr, not shown),
the modified insulin was broken into its constituent chains, highlighting the protec-
tive nature of the alkene and suggesting that even redox-sensitive biomolecules can
tolerate the reaction conditions, provided the alkene is present to quench the
photocatalyst. It is quite possible that the tertiary structure of insulin was lost as a
result of the solvent. The use of DMF is due to the low solubility of this particular
photocatalyst in water, and it is expected that modifications to the structure of the
photocatalyst can lead to increased aqueous solubility, which could eliminate this
problem. As a side note, it could also lead to diminished redox potentials, which
could make it even more compatible with redox-sensitive substrates. Furthermore,
photoconjugation is not necessarily limited to Ir-based photocatalysts. Recently,
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Gilmour and co-workers have shown that riboflavin was also a competent photoca-
talyst for photoisomerization of alkenes, indicating that it might be possible to use
the water-soluble vitamin with the appropriate modifications to accomplish the
5
2
photoconjugation.
In an attempt to move toward more biocompatible conditions (i.e., mostly
aqueous solvent), we performed a reaction with a more water-soluble catalyst
2
6ꢀ
4-F Ir(ppy) bpy] PF (1.0 mg of catalyst is completely miscible in 1.0 mL of 3:2
+
[
DMF/water) than Cat. 3 (less than 10 ppm in 3:2 DMF/water). However, the catalyst
has lower triplet energy (51.4 versus 56.4 kcal/mol), which was expected to
5
3
decrease the overall efficiency of the reaction. Indeed, when a more aqueous-
rich 3:2 DMF/water solvent system was used, the reaction produced the desired
triazoline product 4a, but required 9 hr of irradiation to reach completion. This result
suggests that designing a photocatalyst that is more water soluble and emits at a
higher energy will allow this reaction to take place in water.
Conclusions
In conclusion, we have taken the idea of energy harvesting from concept to practice
by providing conditions that allow kinetically and thermodynamically stable benzo-
fused cycloheptene, Z-BC7, to undergo fast and chemoselective cycloaddition with
substituted azides upon exposure to visible light. The reaction takes place under
mild conditions that can be used to work with biological molecules. The operative
mechanism involves energy transfer from a photocatalyst to Z-BC7—which results
in isomerization to an extremely strained and kinetically unstable alkene, E-BC7,
meaning that no background reaction occurs in the absence of photocatalyst or
light—and provides a strategy for temporal control that is simply not available to
strain-promoted reactions. The designed relief mechanism of BC7 is unique among
photo-initiated processes. In contrast to others, it provides a steady-state concen-
tration of highly reactive alkene throughout the duration of the reaction. The reac-
tion tolerates a broad range of azides that give triazoline products. A unique aspect
of this conjugation strategy is that the products can be conveniently and purpose-
fully decomposed by the addition of weak acid sources, releasing the amine that
corresponds to the azide. Finally, we have shown that the conjugation reaction
can be performed on modified proteins such as insulin (which are reasonably soluble
and stable in organic solvents) in short reaction times and at biologically relevant
concentrations and temperatures. From the initial synthetic concept to an applied
tool ready for further development, this work presents a powerful and flexible
strategy for complex chemical transformations via the conversion of photochemical
energy to intramolecular ring strain, facilitating transformations even on challenging
bimolecular systems.
EXPERIMENTAL PROCEDURES
An NMR tube was charged with a solution of azide (1.0 equiv), alkene S2 or 7 (3.0
equiv), photocatalyst 3 (1.2 mol %) in DMF (0.2 M) or DMF/water (4:1, 0.2 M), and
a sealed capillary containing deuterated benzene. The tube was capped with a
rubber septum, and the reaction mixture was degassed with argon bubbling for
1
0–15 min. The tubes were then sealed with Parafilm and placed in the light bath.
1
The progress of the reaction was monitored by H NMR. To minimize unintentional
reaction, the tube was quickly covered with aluminum foil after removal from the
light bath and kept under dark until being loaded into the NMR instrument. After
completion of the reaction, the mixture was diluted with EtOAc and washed with
4
water. The organic layer was dried over MgSO and concentrated to obtain the
12
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crude product, which was purified by normal phase chromatography. Normal phase
chromatography was performed with a Teledyne Isco automated chromatography
system with basic alumina as the stationary phase and either EtOAc/hexanes or
2 2
MeOH/CH Cl as the mobile phase unless otherwise noted.
DATA AND SOFTWARE AVAILABILITY
The structure of triazoline 4a in this article has been deposited in the Cambridge
Crystallographic Data Center under accession number CCDC: 1525656.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
1
13
1
8 schemes, 13 figures, 1 table, and H and C NMR data and can be found with
this article online at https://doi.org/10.1016/j.chempr.2017.11.007.
ACKNOWLEDGMENTS
The research results discussed in this publication were made possible in total or in
part by funding through the award for project number HR-14-072 from the
Oklahoma Center for the Advancement of Science and Technology. The computing
for this project was performed at the High Performance Computing Center at
Oklahoma State University and supported in part through National Science Founda-
tion (NSF) grant OCI–1126330. Mass spectrometry analyses were performed in the
Genomics and Proteomics Center at Oklahoma State University with resources
supported by the NSF MRI and EPSCoR programs (award DBI/0722494).
AUTHOR CONTRIBUTIONS
Conceptualization, J.D.W. and K.S.; Methodology, J.D.W. and K.S.; Investigation,
K.S.; Formal Analysis, C.J.F. and E.C., Writing – Original Draft, J.D.W.; Writing – Re-
view & Editing, J.D.W., K.S., and C.J.F.; Funding Acquisition, J.D.W.; Resources,
J.D.W., S.H., and R.L.; Supervision, J.D.W.
DECLARATION OF INTERESTS
A provisional patent concerning this technology has been filed.
Received: June 9, 2017
Revised: October 10, 2017
Accepted: November 16, 2017
Published: December 21, 2017
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