Study and application of noncatalyzed photoinduced conjugation of azides and cycloocta-1,2,3-selenadiazoles

Article abstract of DOI:10.1039/c6cc01789d

The non-catalyzed cycloaddition of eight structurally different azides with cyclooctyne generated in situ by the photolysis of cycloocta-1,2,3-selenadiazole gives 1,2,3-triazole derivatives as the main products. The application of this reaction was demonstrated by the photoconjugation reaction of cycloocta-1,2,3-selenadiazole with an avidin-modified biotin complex to introduce a new strategy in the non-catalyzed synthesis of bioconjugates.

Full text of DOI:10.1039/c6cc01789d

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DOI: 10.1039/C6CC01789D
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COMMUNICATION
Study and Application of Noncatalyzed Photoinduced Conjugation
of Azides and Cycloocta‐1,2,3‐selenadiazoles
P. Jedináková,a P. Šebej,b,† T. Slanina,b P. Klánb and J. Hlaváča,c*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
starting material.
The non‐catalyzed cycloaddition of eight structurally different
azides with cyclooctyne generated in‐situ by the photolysis of
cycloocta‐1,2,3‐selenadiazole gives 1,2,3‐triazole derivatives as
the main products. The application of this reaction was
demonstrated on photoconjugation reaction of cycloocta‐1,2,3‐
selenadiazole with an avidin‐modified biotin complex to introduce
a new strategy in the non‐catalyzed synthesis of bioconjugates.
A cyclooctyne system can also be generated from the
corresponding 1,2,3‐selenadiazole, which is easily prepared by
converting cyclooctanone to its semicarbazone, followed by its
subsequent reaction with selenium dioxide.14‐16 However, the
transformation of cycloocta‐1,2,3‐selenadiazole derivatives to
cyclooctynes requires harsh conditions, such as thermolysis at
temperatures over 115 °C,17‐19 thermolysis on copper
powder,20‐24 or a reaction mediated by n‐BuLi.14 Such
conditions limit the direct use of cycloocta‐1,2,3‐
selenadiazoles for the labeling of biomolecules and the
isolation of a generated cyclooctyne system must precede the
azide‐cyclooctyne conjugation.
Transformation of a 1,2,3‐selenadiazole derivative to system
bearing triple bond is published only by Krantz and co‐workers,
who described the formation of ethynylselenol by light‐
induced decomposition of 1,2,3‐selenadiazole25. The formation
of the product was indicated only by IR spectroscopy in an
argon or nitrogen matrix at 8 K. Other examples of the
Strain‐promoted alkyne‐azide cycloaddition (SPAAC) was first
described in 1961 by Krebs, who reported the reaction of cy‐
clooctyne with phenylazide affording 1,2,3‐triazole as a
product.1 This reaction has been extensively studied as a
copper‐free conjugation (click) reaction since 2004, when
Bertozzi and co‐workers used SPAAC as a biorthogonal
reaction with biotinylated cyclooctyne for labeling
biomolecules and living cells for the first time.2 Subsequently,
various cyclooctyne derivatives and their aza analogues have
been designed3,4 to improve the kinetic parameters of this
reaction and used in biological applications, such as cell
labeling,5,6 enzyme screening,7 fluorescent imaging8,9 or
radiolabeling.10,11
photochemical
transformation
of
1,2,3‐selenadiazole
derivatives have also been reported, but photoproducts other
than alkynes were formed. For example, the irradiation of 4,6‐
dihydrofuro[3,4‐d][1,2,3]selenadiazole gave selenirene and
selenoketone derivatives, benzo[d][1,2,3]selenadiazole was
converted to 6‐fulveneselone,26 simple 1,2,3‐selenadiazole
afforded etheneselenone,27 and its 4‐ethoxycarbonyl
derivative gave the corresponding 1,3‐diselenole derivative.28
In this work, we studied the photochemically‐induced noncata‐
lyzed conjugation reaction of cyclooctyne generated in situ
The complex multi‐step procedures needed for the
construction of a triple bond in substituted cyclooctyne
systems are the main drawbacks for their applications.12 On
the other hand, the photochemical production of cyclooctyne
ring
in
5,6‐didehydro‐11,12‐dihydrodibenzo[a,e]‐
from 6,7‐dihydro‐1H‐
cyclopropa[c][8]annulen‐1‐one
dibenzo[a,e]‐cyclopropa[c][8]annulen‐1‐one developed by
Popik and co‐workers13 and used for cell labeling, is an elegant
procedure, but it suffers from a rather difficult synthesis of the
from cycloocta‐1,2,3‐selenadiazole
azides 2a‐h at ambient temperature to form 1,2,3‐triazoles 3a‐
3h (Scheme 1).
1 with structurally different
The azides 2a‐h represent substrates possessing different
electronic and steric properties.
a. Institute of Molecular and Translation Medicine, Hnevotinska 5, 779 00 Olomouc,
Czech Republic.
Cycloocta‐1,2,3‐selenadiazol
1
was prepared from 2‐
cyclooctylidene‐hydrazinecarboxamide according to the
previously described procedure.29 Azides 2a‐c30 and 2d31 were
synthesized from the corresponding amines Benzyl azide 2g
was prepared from benzylchloride.32 5‐ and 5´‐azides derived
from 5‐methyluridine were synthesized according to our
recently published procedure.33 The analytical standards,
triazoles 3a‐h, were prepared by heating a mixture of
b. Department of Chemistry and RECETOX, Faculty of Science, Masaryk University,
Kamenice 5, 625 00 Brno, Czech Republic.
c. Department of Organic Chemstry, Faculty of Science, Palacký University, 17.
Listopadu 12, 771 46 Olomouc, Czech Republic
†Present address: Department of Chemistry, Columbia University, 3000 Broadway,
New York, New York 100 27, United States.
* hlavac@orgchem.upol.cz
Electronic Supplementary Information (ESI) available: Experimental procedures,
experimental data, UV/VIS spectra, copies of NMR spectra, LC/MS data, MALDI.
See DOI: 10.1039/x0xx00000x
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cycloocta‐1,2,3‐selenadiazole
2a‐h (see ESI).
1
and the corresponding azide of 20–35 ppm (Figure S47) were also attributed to cyclooctyne
View Article Online
in accordance with the published data.37DOI: 10.1039/C6CC01789D
Scheme 1: Noncatalyzed Conjugation Reactions of Cycloocta‐
1,2,3‐selenadiazole
Figure 1: A detail of the 1H NMR spectra of a mixture of 1,2,3‐
selenadiazole and triazole product 3e before irradiation
1.
1
(black line) and after 2 h of irradiation (blue line).
The irradiation wavelength had to be carefully chosen for each
pair of reactants. As the absorption spectrum of the principal
The cyclooctyne half‐life in the mixture with azide 2e was
estimated to be approximately 40 min (for details see ESI;
Figure S1). The quantum yield of triazole 3e formation upon
irradiation of a methanolic solution of the 1,2,3‐selenadiazole
chromophore cycloocta‐1,2,3‐selenadiazole
partially overlaps with those of the azides 2a‐2h (Figures
S5−S12) and the photoproducts 3a 3h (Figures S13‐S20), the
1 (Figure S4)
−
undesired light absorption by the azides should be avoided or
at least be largely suppressed. The absorption tail of the
1
(c = 1.9
mol dm–3) at
± 0.05 using valerophenone38,39 as an actinometer.
 
10–3 mol dm–3) in the presence of 2e (c = 6.1 10–3

irr = 313 nm was determined to be Φ (3e) = 0.10
starting material
1 allowed us to use irradiation wavelengths
up to 325 nm (Figure S4).
To determine the multiplicity of the excited state involved in
the cyclooctyne formation, the influence of oxygen as a triplet
quencher on the reaction rate was studied. Degassed, aerated
The azide 2e has been selected as a reagent for an initial
photochemical reaction study because it shows no absorption
above 300 nm (Figure S9). During the irradiation of a solution
of equimolar amounts of
1
and 2e in methanol (c = 3 × 10–3
and oxygenated solutions of
1 and 2e in methanol‐d4 were
mol dm–3) at 313 nm, a red solid precipitated. According to
UV/VIS spectrum, having absorption maximum at λabs of
approximately 500 nm, the solid was identified as selenium
nanoparticles with a diameter of approximately 180 nm34
(Figure S22), which may become an important internal optical
filter. Upon exhaustive irradiation, triazole 3e was found to be
the major product formed in a 28% yield, whereas 62% of
azide 2e remained unreacted in the solution according to
GC/MS analysis (Figure S23). Due to the thermal instability of
the 1,2,3‐selenadiazoles,35 GC/MS could not be used to
irradiated using a medium‐pressure Hg lamp in an NMR tube.
The changes in the concentration of
1 at different reaction
conversions were followed by 1H NMR (Table S1). The
presence of oxygen evidently but not significantly slowed the
reaction, thus the productive triplet‐excited state lifetime
must be relatively short and its reaction competes with a
diffusion‐limited quenching, or the singlet state is also involved
in the reaction.
In addition, sensitization of
was performed to find whether the same process occurs in the
triplet manifold. Irradiation of a degassed solution of 2e and
1 by triplet‐excited benzophenone
1
,
determine the concentration of the unreacted derivative
Therefore, NMR was used to monitor reaction progress.
1.
benzophenone in methanol resulted in the formation of 3e
(Table 1, Figure S49); the same product as that obtained by
direct irradiation) in substantially higher yields compared to
those observed when the same reaction mixture was purged
with oxygen. The irradiation wavelength was 366 ± 1 nm to
ensure that benzophenone is the only absorbing species
(Figure S4 and Figure S21). Table 1 also demonstrates that
oxygen quenched the sensitized production of 3e. Such a
result supports further our assumption that 3e is produced at
Upon irradiation of a solution of
1
(c ~ 0.03 mol dm–3) and 2e
(c ~ 0.03 mol dm–3) in methanol‐d4 in a NMR tube (Pyrex glass;
λ > 280 nm, Figure S3) by a medium‐pressure Hg lamp for 2 h,
the conversion reached 38%, and the triazole 3e was a major
product. The product formation was evident from appearance
of the new triplet signals at 2.81 and 2.94 ppm assigned to the
methylene group of the triazole derivative and a decreased
signal intensity for the methylene groups of 1,2,3‐
1
least in part from triplet‐excited 1.
selenadiazole
1
at 3.32 ppm (Figure 1). The H NMR spectrum
of the product obtained by irradiation corresponds to that of
the isolated triazole 3e prepared by thermolysis (Figure S33).
To detect cyclooctyne as a possible primary photoproduct,
Table 1. Sensitization of 1a
Yield of 3e/%a
Irradiation time (h)
b
c
irradiation of a solution of 1
in methanol‐d4 (c ~ 0.04 mol dm–3)
O2
4
N2
7
was performed for 1 h. The 1H NMR showed the signals at 1.64
and 2.10 ppm (Figure S46), what corresponds to the spectrum
of cyclooctyne reported in the literature.36 A new signal in the
13C NMR spectrum at 93.8 ppm and three signals in the range
2
16
24
41
a
1
(c ~ 0.03 mol dm–3), 2e (c ~ 0.03 mol dm–3) and
benzophenone (c ~ 0.3 mol dm–3) in methanol‐d4 irradiated at
366 nm; the yields were determined by 1H NMR. b Sample was
2 | J. Name., 2012, 00, 1‐3
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c
O
purged with oxygen for 10 min. Sample was purged with
nitrogen for 10 min.
H
N
View Article Online
DOI: 10.1039/C6CC01789D
O
O
HO
HN
O
Se
N
4
N
O
The photolysis of an equimolar mixture of
1 and the
corresponding azide 2a‐d
,
f‐h (c ~ 0.03 mol dm–3) was carried
O
O
N
CF3
out afterwards, and the reaction conversion was monitored by
NMR (see Table S2, Method I). The lowest triazole yield (6‐8%)
was observed for 3‐azidocoumarine 2d, which has been
reported to be photolabile.43 For the other azides 2a‐c and 2f‐
O
5a-without avidin
5b-with avidin
O
O
N3
O
HN
NH
h
, triazole yields were 11−39%. Further irradiation did not
S
increase the conversion, which may indicate a role of selenium
hv
O
O
N
as an internal filter (see above) and potential side
photoreactions of the azides known from literature.40‐42
.
O
O
S
N
H
O
N
CF3
To enhance the reactivity of cycloocta‐1,2,3‐selenadiazole
avoiding inadvertent photolysis of the azides , a solution of
1 by
O
O
NH
2
1
HN
in methanol‐d4 was initially irradiated for 1 h to form
cyclooctyne, and then an equimolar amount of the
N
O
HO
N
O
O
N
H
corresponding azide
2 was added. Subsequently, the mixture
O
6a-without avidin
6b-with avidin
was kept in the dark, and the reaction conversions were
monitored by NMR (Table S2, Method II). Surprisingly, the
triazole yields were similar or lower compared to those
previously determined. The only exception was the reaction of
azide 2d (the most photoactive azide43), where the post‐
irradiation addition of this azide prevented its
photodecomposition and the total yield of triazole increased.
Scheme 2: Conjugation reaction of modified cycloocta‐1,2,3‐
selenadiazole and appropriate azido derivatives.
In all cases, the starting cycloocta‐1,2,3‐selenadiazole
1 always
remained in the reaction mixture. Because of selenium internal
filter formation we were not able to further increase the yield
of the triazoles 3 under the conditions used.
To demonstrate the applicability of cycloocta‐1,2,3‐
selenadiazole photoconjugation reaction with azides, labeling
of biomolecules was studied. Selenium nanoparticles (Figure
S22), which are formed during the reaction, have already been
proven to be nontoxic44, what meets the basic requirements
for conjugation reactions applied in molecular biology.
First we prepared a modified derivative of cycloocta‐1,2,3‐
selenadiazole
4 having a PEG moiety for its better water
Figure 2: A details of the 1H NMR spectra of the mixture of modified
biotin 5a/5b and cycloocta‐1,2,3‐selenadiazole 4 before reaction
(red line), after 1h irradiation (green line) and subsequent 1h
irradiation (blue line).
solubility and the azidobiotine 5a (Scheme 2, the synthesis is
described in ESI) as model reaction partners and performed
the photoinduced conjugation.
The reaction mixture was irradiated with a medium‐pressure
mercury arc filtered through the Pyrex glass of an NMR tube
Further evidence for the reaction on avidin‐bound azide 5b
,
1
for 1 h. The H NMR analysis showed the formation of new
was brought by MALDI analyses of the modified protein,
where the corresponding appearance of higher‐mass
compounds was observed (See Figure S54). As the binding
constant of avidin‐biotin is very high at room temperature (KD
= 10−15 mol dm−3),45 we assume that the reaction of azide
immobilized by avidin proceeded on a bound substrate and
not on a substrate temporarily released from the protein.
signals at approximately 5.80 ppm and a decrease in the signal
intensity of azidomethylene group in the starting material at
approximately 3.42 ppm (Figure 2, S51). The MS spectrum of
the product of this reaction also provided evidence for the
formation of triazole 6a (see Figure S53).
The reactivity was then tested on modified protein 5b as a
biomolecule representative. Complex 5b was prepared by
mixing of avidin with modified biotin 5a (c ~ 0.013 mol dm–3).
The concentration of 5a was higher than corresponds to the
binding capacity of avidin to allow monitoring of the reaction
course by an NMR analysis of the non‐bound biotinylated
azide 5a present in reaction mixture in sufficient
Interestingly the conversion of 1,2,3‐selenadiazole
4 for both
reactions with substrate 5a (with and without presence of
avidine) was possible to enhance by the irradiation for
additional one hour. The intensity of the newly formed signals
described above significantly increased (about 2‐fold), whereas
the signals at approximately 3.17, 3.44 and 4.35 ppm nearly
disappeared (Figures 2, S51 and S52). We assume that the
conversion in this case was higher, because the selenium as
internal filter was formed in low amounts because of lower
concentration of selenadiazole.
concentration. After the addition of 1,2,3‐selenadiazole 4, the
reaction mixture was irradiated with a medium‐pressure
mercury arc filtered through the Pyrex glass of an NMR tube
for 1 h. In 1H NMR spectrum we observed the same changes as
in reaction without avidin.
According to these observations, the conjugation reaction be‐
tween the cyclooctyne analogue obtained from the
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biotinylated azide immobilized by avidin proceeds successfully. 16 I. Lalezari, A. Shafiee, J. Heterocycl. Che
D
m
OI
.
:
,
1
1
0
9
.1
7
0
2View Article Online
3
,
9/
9
C
,
6
1
C
4
C
1
01789D
1
.
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Research for this project was supported by the Ministry of
Education, Youth and Sport of the Czech Republic (project
IGA_PrF_2015_007), Technological Agency of Czech Republic
30 J. M. Altimari, B. Niranjan, G. P. Risbridger, S. S. Schweiker, A.
(project
TE01020028)
the European
Social
Fund
E. Lohning, L. C. Henderson, Bioorg. Med. Chem., 2014, 22
,
(CZ.1.07/2.3.00/30.0060 and CZ.1.07/2.3.00/20.0009) and the
Czech Science Foundation (GA13‐25775S). The infrastructure
of this project (Institute of Molecular and Translation
Medicine) was supported by the National Program of
sustainability (project LO1304). The RECETOX research
infrastructure was supported by the projects of the Czech
Ministry of Education (LO1214 and LM2015051). The authors
express their thanks to Lukáš Maier for his help with the NMR
measurements and Tomáš Ozdian for MALDI analyses.
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