Phenanthridine-based nitrones as substrates for strain-promoted alkyne-nitrone cycloadditions

Article abstract of DOI:10.1139/cjc-2018-0253

Over the past decade, bioorthogonal chemistry that facilitates the efficient conjugation of biomolecules has expanded from the copper-catalyzed alkyne-azide cycloadditions to a multitude of diverse reactions, varying additives and reactional partners, and most often offering better alternatives with faster rates and lower toxicity of employed reactants. Among these, the copper-free strain-promoted cycloaddition reactions have been demonstrated to be more promising, offering a reaction without toxic metal catalysts and with faster inherent kinetic rate constants. The strain-promoted alkyne-nitrone cycloadditions are easily tunable from both the (strained) alkyne and nitrone perspective, both compounds giving the opportunity to modulate the rate of reaction by substituting various positions. Previously, acyclic nitrones have been evaluated in the strain-promoted alkyne-nitrone reactions; however, they were notably prone to hydrolysis. Some five-membered ring endocyclic nitrones developed concomitantly offered the advantage of relatively fast kinetics and better resistance to degradation in aqueous conditions and have been successfully used for labelling of biomolecules in living systems. Herein, we have prepared and studied nitrones inspired by the phenanthridine scaffold that efficiently undergo strain-promoted alkyne-nitrone reactions. Phenanthridine nitrones react fast with strained cyclooctynes with large bimolecular rate constants while maintaining bioorthogonality and resistance to hydrolysis.

Full text of DOI:10.1139/cjc-2018-0253

Page 1 of 21
Phenanthridine-based nitrones as substrates for strain-
promoted alkyne-nitrone cycloadditions
Miroslava Strmiskova, Didier A. Bilodeau, Mariya Chigrinova, John Paul Pezacki*
Department of Chemistry and Biomolecular Sciences, Centre for Chemical and Synthetic Biology, University of
Ottawa, 10 Marie-Curie private, Ottawa, ON, Canada, K1N 6N5
*
Corresponding author: John.Pezacki@uOttawa.ca; Tel. 613-562-5800 ext 3346
1

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Abstract
Over the past decade, bioorthogonal chemistry that facilitates the efficient conjugation of
biomolecules has expanded from the copper-catalyzed azide-alkyne cycloadditions to a multitude
of diverse reactions, varying additives and reactional partners, and most often offering better
alternatives with faster rates and lower toxicity of employed reactants. Among these, the copper-
free strain-promoted cycloaddition reactions have been demonstrated to be more promising,
offering a reaction without toxic metal catalysts, and with faster inherent kinetic rate constants.
The strain-promoted azide-nitrone cycloadditions are easily tunable from both the (strained)
alkyne and nitrone perspective, both compounds giving the opportunity to modulate the rate of
reaction by substituting various positions. Previously, acyclic nitrones have been evaluated in the
strain-promoted alkyne-nitrone reactions, however, they were notably prone to hydrolysis. Some
five-member ring endocyclic nitrones developed concomitantly offered the advantage of relatively
fast kinetics and better resistance to degradation in aqueous conditions, and have been successfully
used for labelling of biomolecules in living systems. Herein we have prepared and studied nitrones
inspired by the phenanthridine scaffold that efficiently undergo strain-promoted alkyne-nitrone
reactions. Phenanthridine nitrones react fast with strained cyclooctynes with large bimolecular rate
constants while maintaining bioorthogonality and resistance to hydrolysis.
Key words
Phenanthridine, bioorthogonal labelling, nitrone, SPANC
2

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Introduction
Studying biological processes in their natural environment has been made possible using
bioorthogonal reactions that have little impact on metabolism and homeostasis. The challenging
task of selectively targeting one family or one type of biomacromolecule has been tackled by
1
constantly developing the bioorthogonal reaction toolbox. The requirements for a reaction to be
considered a good candidate for bioorthogonal chemistry2-4 include, but are not limited to, fast
reactivity in aqueous biological environment at requisite temperatures, excellent selectivity
towards its target in presence of all available biological components as well as low toxicity or
perturbation to the biological system. As such, the copper-catalyzed alkyne-azide cycloaddition
(CuAAC) has proved itself particularly useful in labelling cell surface and intracellular
macromolecules, such as proteins, lipids,8-10 nucleic acids,11-13 and saccharides. However, the
use of toxic copper concentrations has been shown to be incompatible with labelling of living cells.
Several ways help to circumvent this, including the use of various ligands that help reduce copper
toxicity,5, 15 and more importantly, development of copper-free reactions using strained alkynes,
5
6-7
14
1
6
such as the strain-promoted alkyne-azide cycloaddition (SPAAC), or the rapid inverse-electron
demand Diels-Alder (IEDDA) tetrazine ligation.17-18 The tetrazine ligation was shown to be a
valuable tool for PET imaging19-22 and radioimaging probe construction, benefiting from fast
23
1
7
kinetics, and it is also a useful ligation reaction for chemical proteomic analysis. As an alternative
to azide 1,3-dipoles, nitrone dipoles24-26 have been used to enhance reactivity towards cyclooctynes
and their general reactivity has been recently reviewed in detail by Merion et al.27
Our group and others have applied the strain-promoted alkyne-nitrone cycloaddition (SPANC)
to a variety of living systems, exploring the nitrone-functionalized ligand,28 nitrone-derived
unnatural amino acid29-30, antibiotic and sugar incorporation and also nitrone-functionalized
29
31
3

Page 4 of 21
3
2
reporters. As such, both cyclic and acyclic nitrones were shown to be broadly useful for labelling
living bacteria30 via incorporation of KDO-nitrone or vancomycin-nitrone in bacterial
peptidoglycan, as well as for cellular labelling.29, 33
We have first investigated the reactivity of acyclic nitrones by varying substituents on α-C position
with the strained dibenzocyclooctyne (DIBO).24, 26, 33-34 We have shown that acyclic nitrones with
electron-withdrawing substituents on α-C position reacted faster than unsubstituted nitrones, or
2
4
those bearing electron-donating substituents on α-C. However, more reactive acyclic nitrones
3
3
suffered from poor stability in aqueous conditions (Scheme 1), which are necessary to carry out
labelling of biomolecules. Endocyclic nitrones, on the other hand, are excellent reactants for
2
8
SPANC due to their stability in aqueous media. We have therefore investigated the reactivity of
several endocyclic nitrones with strained alkyne biarylazacyclooctynone (BARAC, Scheme 2),
and we reported a series of nitrones that are highly reactive and resistant to hydrolysis in both
3
3
acidic and basic conditions, which we demonstrated by using several endocyclic nitrones for
cellular labelling and labelling of living bacteria.29, 33 Some nitrones undergo a rearrangement upon
SPANC reaction and their reactivity has been described.34 Similarly, a rearrangement of the
product can occur in the case of CMPO nitrone used with a terminal alkyne in the copper-catalyzed
3
2
reaction (CuANCR). Therefore, further reactions based on the nitrone group are warranted with
more stable and more reactive nitrone dipoles to be developed. We hypothesized that diaryl
nitrones based on the phenanthridine ring system, could lead to increased stability of the resulting
nitrone in aqueous conditions, while retaining reactivity in strain-promoted cycloaddition owing
to it’s similarity to other diaryl nitrones (Scheme 1).
Phenanthridines have been historically intensively studied biologically active compounds
and used in a wide range of applications as the gold-standard DNA- and RNA-intercalating agents.
4

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For a long time, the human medical applications of phenanthridines have been avoided due to their
potential carcinogenic and mutagenic properties, relevant especially in the case of ethidium
bromide derivatives. Recently, the scientific community noted an increased interest in
3
5
phenanthridines and their alkaloid analogues derivatives due to their antiparasitic properties, as
recently reviewed by Tumir et al. This could give rise to the possibility of using SPANC chemistry
to introduce biologically active compounds to bioconjugates or to construct novel carriers
containing the medicinal and biochemical effects of phenanthridines. It is also possible that nitrone
derivatives will not be as biologically active and serve as bioorthogonal groups.
Here, we report phenanthridine-nitrone derivatives and their kinetic characterization in a
SPANC reaction, and as promising scaffolds for new bioconjugation reactions. We show that these
phenanthridine-nitrones have low toxicity and potential broad applications in cellular labelling,
owing to their inherent tunable spectroscopic properties, their stability and compatibility with
aqueous conditions.
Results and Discussion
In order to explore the reactivity and stability of phenanthridine-derived nitrones for
bioorthogonal SPANC reactions, derivatives first needed to be synthesized. Phenanthridine-
derived nitrones 1 and 5 were prepared as previously reported in the literature.36 Briefly,
phenanthridine 5-oxide 1 was synthesized in one step from commercially available phenanthridine,
using freshly prepared phosphomolybdic acid as catalyst, affording 1 in 73% overall yield. 3-
methoxyphenanthridin-6(5H)-one, a precursor of 5, was obtained following the procedure reported
3
7
in the literature (Scheme S1). Then, the carbonyl of 3-methoxyphenanthridin-6(5H)-one was
transformed into (trifluoromethyl)sulfonyl- group using Tf O and subsequently removed with
2
5

Page 6 of 21
palladium acetate to afford 3-methoxyphenanthridine. Finally, 5 was prepared from 3-
methoxyphenanthridine in a similar manner as 1, using phosphomolybdic acid as catalyst, as
3
6
reported previously. All synthetic steps are detailed in the Supplemental information section.
Next, to evaluate the reactivity of phenanthridine-derived nitrones, kinetics studies were
performed in a SPANC reaction with a model strained alkyne BARAC, as described in the
Supplementary Material. A typical kinetic profile of nitrone 1 and BARAC under pseudo-first
order conditions is shown in Figure 1, along with a plot of kobs against nitrone concentration and
absorption spectra of reactants and products. As predicted, the reactivity of both phenanthridine
nitrones 1 and 5 (Table S1, S2, Figure S1, S2) is diminished in comparison with acyclic phenyl-
substituted nitrone 6 (Table 1). This is likely due to the loss of electron-donating effect of the
phenyl group of 6 that is now involved in the delocalized electron system of nitrones 1 and 5. With
3
3
a k constant approximately 3.5 times higher than the benzyl-substituted acyclic nitrone, the
2
strain offered by the three aromatic six-member rings gives a notable kinetic advantage to both 1
and 5. However, when compared to the five-member ring endocyclic nitrones, such as CMPO,33
the rate constants k of 1 and 5 were about 5 times lower, consistently with the fact that five-
2
member rings provide more strain than six-member rings. Furthermore, methoxy-substituted
phenanthridine-nitrone 5 is slightly more reactive than unsubstituted 1, probably owing to the
increased energy of the HOMO of 5. Finally, both nitrones 1 and 5 remain about 10 times more
reactive in the SPANC reaction than benzyl-azide in the SPAAC reaction. They are more resistant
to hydrolysis than their acyclic counterparts and retain the advantageous position for further
development of this scaffold. This establishes nitrones 1 and 5 as useful reactants for bioorthogonal
SPANC reactions
6

Page 7 of 21
In order to assess the bioorthogonality of phenanthridine-derived nitrones, toxicity profiles
were determined. Certain phenanthridine derivatives are notoriously toxic and even considered to
be mutagenic and carcinogenic due to their DNA-intercalating properties. Some phenanthridine
3
8
derivatives are also effective against human breast cancer cell proliferation. Here we assessed
the toxicity of nitrones 1 and 5 using MTT assays. Phenanthridine-nitrone derivatives showed no
toxic effect on human hepatoma Huh 7.5 cells (Figure 2) in the range of concentration commonly
used for cellular labelling, likely because of the lack of phenanthridine substituents necessary for
intercalation between nucleic acid base pairs.39-40 We compared their toxicity to a benchmark
compound, coumarin-nitrone 8 (Scheme 2, Figure 3), for which we determined an IC of 12.6
5
0
4
1
µM, owing likely to the intrinsic toxicity of coumarin-derived compounds. . The low toxicity in
the useful range of concentration for cellular labelling makes phenanthridine nitrones good
candidates for further development and use for labelling of biomolecules in living cells.
Because some phenanthridines display interesting fluorescence properties, we next sought to
study the fluorescence of phenanthridine-based nitrones. Unsubstituted phenanthridine scaffold
and a wide range of phenanthridine derivatives are fluorescent and are therefore used as potent
3
5
markers for DNA, owing to their DNA-intercalating properties. Given the high tunability of
phenanthridine spectroscopic properties, we measured both the excitation and emission spectra of
nitrones 1, 5 and 8 (Figure 3). While converting nitrogen of the initial phenanthridine to nitrone in
compound 1 did not cause any significant shift of excitation or emission maxima, nitrone 5
emission peak is largely red-shifted, similarly to other phenanthridine derivatives with substituents
3
9
on C3. As spectroscopic properties of phenanthridines are highly tunable, we expect the herein
presented nitrones to be first members of a larger groups of analogs with broad range of tunable
spectroscopic properties.
7

Page 8 of 21
We also briefly examined the potential of nitrone 1 and 5 to provide fluorogenic reactions.
Despite that neither of the two compounds is fluorogenic in reaction with strained alkynes (data
not shown), the emission spectrum of 5 is red-shifted enough to be a donor pair for Förster
resonance energy transfer (FRET) to a green fluorophore, such as the photostable cyanine-based
fluorescent dye CF488A (Figure 4A). In a SPANC reaction of nitrone 5 with the bicyclononyne
3
0
BCN-CF488A (Biotium, also previously used in SPANC reactions with nitrones ), a time-
dependent 1.3-fold increase of CF488A fluorescence at 515 nm upon irradiation of nitrone 5 at
3
80 nm is observed, as well as a similar decrease of nitrone fluorescence at 420 nm (Figure 4B,
S3). In absence of BCN, the fluorescence of nitrone 5 barely changes over the course of the
reaction (48 hours). Similarly, in absence of nitrone 5 and presence of phenanthridine control
(which lacks the reactive nitrone moiety), the emission at 515 nm did not change over the course
of the reaction, further suggesting that the observed fluorescence increase at 420 nm and decrease
at 515 nm is due to FRET of the product of SPANC reaction between nitrone 5 and BCN-CF488A
(Figure S3). Due to the length and small scale of the reaction, we have not isolated the formed
product which would allow us to further characterize its spectroscopic properties and confirm the
nature of the fluorescence change as FRET. However, we can conclude that irrespective of the
mechanism, the SPANC reaction with nitrone 5 and BCN-CF488A induces spectroscopic changes
in the SPANC product which allow us to easily follow the progress of the reaction (in contrast to
simple absorbance or fluorescence measurements which do not change substantially over time).
Additionally, the nitrone dipole can be further developed to offer a more efficient energy transfer,
or simply a higher brightness for direct use as fluorophore-nitrone electrophiles for SPANC
fluorescent labelling of strained alkyne-tagged biomolecules.
8

Page 9 of 21
We have also investigated whether the quantum yield of nitrones 1 and 5 changes upon
incubation with double-stranded DNA (dsDNA), and observed only a slight decrease of
fluorescence intensity (Figure S4, S5), which suggest, along with the fact that nitrones 1 and 5 lack
the necessary components to intercalate DNA, their spectroscopic properties remain the same,
whether in presence or in absence of dsDNA. We also observed a larger decrease of fluorescence
intensity after addition of BARAC, likely due to the fact that BARAC has a large absorption band
between 220-400 nm. Overall, we were able to conclude that addition of dsDNA does not affect
nitrone spectroscopic properties which is beneficial for broad applications of phenanthridine-
nitrones in cellular and intracellular labelling. Again, this is consistent with the nitrones 1 and 5
being bioorthogonal, rather than bioactive molecules.
Conclusion. We have designed and synthesized a series of cyclic nitrones based on the
phenanthridine scaffold. These were found to be stable to aqueous conditions and showed
-1
-1
bimolecular rate constants of k ~10 M s . Coumarin-fused nitrones were then synthesized and
2
studied in the context of their ability to impart fluorescence onto cyclooctyne reactants. Lack of
DNA-intercalating properties and related quantum yield change is in this case beneficial, as the
nitrone compounds are meant for SPANC labelling of intracellular biomolecules. Furthermore, we
demonstrated that phenanthridine-nitrone 5 can be used as a FRET donor with suitable
fluorophores and has an immense potential for fluorescent microscopy and general bioorthogonal
labelling compound.
Supplementary material
Supplementary data for this paper are available on the journal web site
(http://www.nrcresearchpress.com/journal/cjc)
9

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Acknowledgements
The authors thank Prof. Jeffrey Keillor (University of Ottawa) for the use of Cary-Eclipse
fluorimeter for FRET measurements. We also acknowledge the support of the Natural Sciences
and Engineering Research Council of Canada (NSERC), funding reference number 210719.
1
0

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Figure Captions:
Scheme 1. Reaction of acyclic diphenyl nitrone and cyclic phenanthridine nitrones with strained
alkynes in the strain-promoted alkyne-nitrone cycloaddition.
Figure 1. Kinetic plots for phenanthridine nitrone 1 reactions with BARAC. (A) Pseudo-
first order kinetics of BARAC reaction with 400 µM of 1. (B) Pseudo-first order rate constants
vs nitrone 1 concentration.
Figure 2. Toxicity of phenanthridine nitrones against Huh7.5 human hepatoma cells. Huh 7.5
cells were incubated with 0 – 100 µM of each compound for 24 hours, after which a 2.5 mg/mL
solution of MTT reagent in PBS was added and cells were incubated for further 3-4 hours. Cell
viability was determined by measuring absorbance at 562 nm of formed formazan.
Scheme 2. Reference molecules. Coumarin-nitrone 8 (left), based on acyclic phenyl-substituted
nitrone, was used as a control in this study. BARAC (right) was used as a model strained alkyne
for kinetics measurements.
Figure 3. Fluorescence spectrum of phenanthridine and nitrone compounds. Excitation and
emission spectra of 2 µM 1, 5 or phenanthridine control, and 1 µM 8 (stock in DMSO) have been
acquired in PBS at room temperature, using 5 nm increments, on a SpectraMax i3 plate reader
(Molecular Devices). Paired excitation and emission spectra have been normalized to the same
levels of fluorescence intensity. *Estimated value. Excitation maximum is likely below 280 nm.
Figure 4. Nitrone 5 as FRET pair with BCN-CF488A. (A). FRET pair design in SPANC
reaction. (B) FRET in SPANC reaction of BCN-CF488A (Biotium) and nitrone 5. 10 µM of
nitrone 5 was combined with 10 µM BCN-CF488A in PBS and SPANC reaction was followed by
FRET between the phenanthridine-nitrone 5 donor and the CF488A acceptor upon excitation at
3
80 nm (left). The reaction was performed under second order conditions to see a comparable
increase of acceptor and decrease of donor fluorescence. Fluorescence decrease at 420 nm and
increase at 515 nm overtime are shown (right). Fluorescence scans were acquired at 25 °C on a
Cary-Eclipse Fluorimeter (Varian) equipped with a temperature control unit, using 5 nm slit for
both excitation and emission.
1
4

Page 15 of 21
Table 1. Second order rate constants for addition of BARAC on nitrones 1, 5, 6, and 7.
-
1 -1
Nitrone
Structure
k (M s )
2
1
8.1
N
O
5
6
9.7
N
O
OMe
59a
N
O
7
0.96b
N3
a
33
b
42
see Mackenzie et al, 2014 , see Jewett et al, 2010
1

Page 16 of 21
N
O
N
O
diphenyl nitrone
SPANC
R1
R2
R1
phenanthridine
nitrones
N
O
R = H, OMe
2
N
O
R2
R1
Scheme 1
1

Page 17 of 21
N
N
O
O
O
O
N
O
N
OH
8
BARAC
Scheme 2
1

Page 18 of 21
A
B
0
1
2
3
4
5
6
7
8.00
-
6
.00
.00
.00
-
-
-
-
-
-
4
2
0.00
0.00 0.20 0.40 0.60 0.80 1.00
0
200
400
600
800
Time (s)
Concentration of nitrone 1 (mM)
Figure 1
1

Page 19 of 21
120
120
100
100
80
80
60
60
40
40
IC50 > 100 µM
IC50 > 100 µM
2
0
20
0
0
0
.01
0.1
1
10
100
0.01
0.1
1
10
100
nitrone 1 (µM)
nitrone 5 (µM)
125
100
75
50
IC50 = 12.6 µM
25
0
0
.01
0.1
1
10
100
nitrone 8 (µM)
Figure 2
1

Page 20 of 21
1
0000000
1
40000000
120000000
00000000
0000000
60000000
0000000
8
6
4
2
000000
000000
000000
000000
0
1
5
1
N
O
OMe
N
O
8
4
nitrone 1, Ex. max 320 nm
nitrone 1, Em. max 385 nm
nitrone 5, Ex. max 320 nm
nitrone 5, Em. max 425 nm
20000000
0
3
00 350 400 450 500 550 600 650 700 750
3
00 350 400 450 500 550 600 650 700 750
wavelength (nm)
wavelength (nm)
5
4
3
2
1
0000000
1
2000000
N
O
O
0000000
0000000
0000000
0000000
0
10000000
8000000
O
N
N
8
6
4
2
000000
000000
000000
0
nitrone 8, Ex. max 415 nm
nitrone 8, Em. max 490 nm
Emission blank
phenanthridine, Em. max 370 nm
phenanthridine, Ex. max 280 nm*
Excitation blank
3
00 350 400 450 500 550 600 650 700 750
300 350 400 450 500 550 600 650 700 750
wavelength (nm)
wavelength (nm)
Figure 3
1

Page 21 of 21
A
B
1
00
90
80
0
4
hours
hours
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
85
nitrone 5
CF488A
8 hours
1.2 x
1
1
2 hours
6 hours
75
20 hours
24 hours
1.3 x
70
2
8 hours
32 hours
6 hours
65
3
4
4
4
0 hours
4 hours
7 hours
60
5
5
5
4
50
50
0
4
0
10
20
30
40
00 425 450 475 500 525 550 575 600
wavelength (nm)
time (hours)
Figure 4
1