Synthesis and physical chemistry of s-tetrazines: Which ones are fluorescent and why?

Article abstract of DOI:10.1002/ejoc.200900964

New fluorescent tetrazines have been prepared and their electrochemistry and fluorescence efficiency evaluated. The occurrence of fluorescence as well as the wavelength were found to be strongly dependent on the substituents, which have to be electronegative heteroatoms. This has been rationalized through a computational study that showed that the crucial factor is the nature of the HOMO, which determines the existence or not: of fluorescence. Wiley-VCH Verlag GmbH & Co. KGaA.

Full text of DOI:10.1002/ejoc.200900964

FULL PAPER
DOI: 10.1002/ejoc.200900964
Synthesis and Physical Chemistry of s-Tetrazines: Which Ones are Fluorescent
and Why?
Yong-Hua Gong,[a,b] Fabien Miomandre,[a] Rachel Méallet-Renault,[a] Sophie Badré,[a]
Laurent Galmiche,[a] Jie Tang,[b] Pierre Audebert,*[a] and Gilles Clavier*[a]
Keywords: Nitrogen heterocycles / Fluorescence / Electrochemistry / Ab initio calculations / Frontier orbitals
New fluorescent tetrazines have been prepared and their
electrochemistry and fluorescence efficiency evaluated. The
occurrence of fluorescence as well as the wavelength were
found to be strongly dependent on the substituents, which
have to be electronegative heteroatoms. This has been ra-
tionalized through a computational study that showed that
the crucial factor is the nature of the HOMO, which deter-
mines the existence or not of fluorescence.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
makes them especially attractive for sensing applications.
Our first studies showed indeed that 3-chloro-6-methoxy-s-
tetrazine was among the best of these compounds because
the combination of chlorine and an alkoxy substituent in a
s-tetrazine appeared to lead to a high fluorescence yield. On
the other hand, other s-tetrazines substituted by nitrogen or
sulfur atoms and the diaryl-s-tetrazines previously prepared
by us appeared to lack any fluorescence. Thus, we became
interested in understanding the origin of this behavior and
would like eventually to be able to predict it.
We describe herein the preparation of several families of
s-tetrazines, featuring not only the fluorescent OR- and Cl-
substituted tetrazines, but also alkylthio- and dialkylamino-
substituted tetrazines, tetrazines unsymmetrically substi-
tuted by various heteroatoms, and diaryl-s-tetrazines (see
Schemes 1 and 2 and Table 1). Many of these compounds
are original, and their absorption and fluorescence proper-
ties have been investigated. To rationalize the observed
properties, quantum calculations were performed on all the
s-tetrazines prepared and on a few additional examples. The
results show in particular that fluorescence occurs when the
HOMO orbital has a nonbonding n character, but is absent
if the HOMO is a π orbital. The HOMO and the closest
occupied HOMO-1 orbitals are very similar in energy in
most s-tetrazines and their frequent inversion has been
found to correlate with the occurrence or not of fluores-
cence, giving a valuable predictive tool for the fluorescence
of this class of compound.
Introduction
s-Tetrazines are relatively old molecules, the discovery of
which dates back to the end of the 19th century. The first
synthesis was reported by Pinner who performed the reac-
tion of equimolar quantities of hydrazine and benzonitrile
and prepared, upon further oxidation of the dihydrotetra-
zine intermediate, the deep-red 3,6-diphenyl-s-tetrazine.[1]
All s-tetrazines are highly colored, electroactive heterocy-
cles that display a very high electron affinity, which allows
them to be reduced at low-to-very-low potentials; they are
indeed the least electron-rich class of neutral CN heterocy-
cles.[2] They have a low-lying π* orbital that leads to a
nǞπ* transition in the visible light region, which is respon-
sible for their red color. s-Tetrazines are largely used as
dienes in inverse Diels–Alder cycloaddition reactions,[3] but
are also used in the development of new nitrogen-rich ener-
getic materials.[4] The chemistry of tetrazines has recently
been reviewed.[5]
We became interested in s-tetrazines to develop new ma-
terials having both special optical and electrochemical fea-
tures. In the course of these studies, we noticed that s-tetra-
zines substituted with heteroatoms can display fluorescence
properties.[6,7] Earlier reports of such behavior are
scarce.[8,9] Some of these compounds are even fluorescent
in the crystalline state, which certainly makes them the
smallest organic fluorophores ever prepared and therefore
[a] PPSM, ENS Cachan, CNRS, UniverSud,
61 av President Wilson, 94230 Cachan, France
Fax: +33-1-47402454
Results and Discussion
E-mail: audebert@ppsm.ens-cachan.fr
All the compounds described were prepared either by
starting from the easily accessible dichlorotetrazine by nu-
cleophilic substitution or by a modification of the classical
Pinner synthesis[10] (Scheme 3).
gclavier@ppsm.ens-cachan.fr
[b] Department of Chemistry, East China Normal University,
Shanghai, 200062, P.R. China
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.200900964.
Eur. J. Org. Chem. 2009, 6121–6128
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
6121

P. Audebert, G. Clavier et al.
FULL PAPER
Scheme 1. s-Tetrazines substituted by heteroatoms.
Scheme 2. s-Tetrazines substituted by aromatic groups (s-tetrazine and diphenyl-s-tetrazine have been included for comparison but were
not synthesized by us).
Most s-tetrazines substituted by O, N, or S heteroatoms
Table 1. Substituents in the generic s-tetrazines 1–14.
(Scheme 1 and Table 1) were prepared from the s-tetrazine
Substituent X
Substituent Y
1. This compound is easily prepared in five steps from gua-
nidine hydrochloride on a multigram scale. Our need for
this valuable starting material led us to optimize some of
the steps reported in the literature (see the Supporting In-
formation for details). Nucleophilic substitution of chlorine
by amines required the addition of 2 equiv. of reactant to
trap the hydrochloric acid formed. Alternatively, triethyl-
amine can be used. Monosubstitution was accomplished at
room temperature in high yields in diethyl ether or acetoni-
trile. Disubstitution was achieved by heating the reaction
for a few hours. Thiols proved to be very reactive with 1,
as previously reported.[11] Indeed, it was very difficult to
isolate the monosubstituted product of the reaction with
1
2
3
4
5
6
7
8
9
Cl
Cl
Cl
Cl
Cl
OMe
naphthalen-1-yloxy
pyrrolidin-1-yl
2,3-diphenylaziridin-1-yl
OMe
O(CH2)4OH
O(CH2)4OH
SMe
SMe
S-nBu
pyrrolidin-1-yl
Cl
OMe
OMe
O(CH2)4OH
OMe
10 SMe
11 S-nBu
12 pyrrolidin-1-yl
13 3,5-dimethyl-1H-pyrazol-1-yl 3,5-dimethyl-1H-pyrazol-1-yl
14 3,5-dimethyl-1H-pyrazol-1-yl OMe
6122
www.eurjoc.org
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 6121–6128

Synthesis and Physical Chemistry of s-Tetrazines
Scheme 3. Synthesis of s-tetrazines: a) nucleophilic substitution; b) modified Pinner’s synthesis.
the disubstituted product always predominating. In those played by the substituents on the position of the LUMO
cases in which we did obtain alkylthio(chloro)-s-tetrazines, level: Strong electron-withdrawing groups like chlorine or
the second chlorine could be replaced by oxygen by reaction 3,5-dimethylpyrazolyl shift the standard potential towards
with alcohols at reflux or under pressure. Compounds 17 more positive values, whereas electron-rich substituents like
and 18 were prepared in this way. In contrast, alcohols re- ferrocene or pyrrole shift it in the opposite direction. Sub-
acted with 1 with more difficulty. We tried various methods stitution of oxygen by sulfur, for example, replacing alkoxy
to accomplish monosubstitution in high yields. The use of with a methylthio group, has little influence on the redox
alcoholate proved not to be very satisfactory (20% yield on potential of the s-tetrazine. Very interesting is the compari-
average) as a considerable degradation took place. A low son between N-substituted s-tetrazines like pyrrolidine and
temperature (–78 °C) was required in this case. We at- pyrrole: In the former case, the reduction occurs at a very
tempted the reaction with a variety of inorganic bases but negative potential due to the π-donor effect of the lone pair
with little improvement. The use of pressure led to better on the nitrogen that destabilizes the anion radical, whereas
results (50% yields). Only when we used 2,4,6-collidine in in the latter, this effect is strongly alleviated by the aromatic
dichloromethane did we obtain monoalkoxy-s-tetrazine in character of the substituent.
high yields (90% or more) at room temperature. Addition
of a drying agent such as molecular sieves to keep moisture
off the reaction mixture allows a high yield to be main-
Table 2. Half-wave reduction potential of s-tetrazines in dichloro-
methane vs. ferrocene.
Substituent X
Substituent Y
E°red [V]
tained when the reaction time is prolonged (Ͼ1 h). The sec-
ond substitution was still a problem. Indeed, only alcohol-
ates at a low temperature (–78 °C) in THF gave acceptable
results with either s-tetrazine 1 (symmetrical product) or
chloro(alkoxy)-s-tetrazines (unsymmetrical product). Other
s-tetrazines can be used for nucleophilic substitution. Bis-
(methylthio)-s-tetrazine (10) was for a long time the inter-
mediate of choice in this type of chemistry[12] until the dis-
covery of an easy access to 1. Even this derivative was at
first prepared from 10.[13] The sole derivative prepared by
nucleophilic substitution from 10 in this work is 9. Disubsti-
tution in this case was sometimes possible. 3,5-Dimeth-
ylpyrazolyl can also act as a leaving group, but less ef-
ficiently than chlorine.[11] There are recent examples of sub-
stitution with amines.[14] We found that 13 reacted readily
with methanol under pressure to give both mono- and di-
substituted derivatives.
All the diaryl-s-tetrazines (Scheme 2) were prepared by
our modified version of the Pinner synthesis followed by
oxidation with isoamyl nitrite, which is a mild oxidant and
gives s-tetrazines cleanly and in high yields (Scheme 3).
The redox potentials for the first electrochemical re-
duction of s-tetrazines were measured in dichloromethane
(Table 2) and are correlated to the LUMO, with discrepanc-
ies related to solvation variations. However, it should be
pointed out that the s-tetrazine core in which the electron is
1
2
3
4
5
6
9
Cl
Cl
Cl
Cl
Cl
OMe
OMe
Cl
OMe
–0.68[a]
–0.99[a]
–0.97[a]
–1.35
naphthalen-1-yloxy
pyrrolidin-1-yl
2,3-diphenylaziridin-1-yl
OMe
SMe
SMe
pyrrolidin-1-yl
3,5-dimethyl-1H-pyrazol-1-yl
OMe
–1.04
–1.25[a]
–1.23
10 SMe
12 pyrrolidin-1-yl
13 3,5-dimethyl-1H-pyrazol-1-yl
14 3,5-dimethyl-1H-pyrazol-1-yl
15
16
19
20
–1.20
–1.73
–0.96[c,d]
–0.89
–0.99[a]
–0.90
–0.94[b]
–1.24
21
–1.21[b]
–0.97[c]
–1.07[c]
–1.31[c]
–1.24[c]
–1.25[e,f]
–1.28[d,g]
–1.32[d,g]
–1.16[h,i]
–1.21[h,i]
24 3,5-dimethyl-1H-pyrazol-1-yl
25 pyrrol-1-yl
26 pyrrol-2-yl
27 2-thienyl
28 2,2Ј-bithienyl-5-yl
29 ferrocenyl
30 4-ferrocenylphenyl
s-Tetrazine
pyrrol-1-yl
pyrrol-1-yl
pyrrol-2-yl
2-thienyl
2,2Ј-bithienyl-5-yl
ferrocenyl
4-ferrocenylphenyl
Diphenyl-s-tetrazine
[a] From ref.[3] [b] From ref.[5] [c] From ref.[14] [d] Not fully revers-
ible. [e] From ref.[15] [f] As a polymer. [g] From ref.[16] [h] From
ref.[15] [i] Recorded in acetonitrile.
These trends in the redox potentials are confirmed by the
located (Figure 1) undergoes only small structural changes. positions of the corresponding LUMO levels. In particular,
Besides, the first reduction is located on the s-tetrazine core, the differences between the reduction potentials in compari-
as evidenced by the fully reversible behavior observed for son with 1 (in V) are very similar to the corresponding dif-
most compounds. The redox potentials highlight the role ferences in the LUMO energy levels (in eV), as shown in
Eur. J. Org. Chem. 2009, 6121–6128
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6123

P. Audebert, G. Clavier et al.
FULL PAPER
the spectral features are very similar to their monomeric
equivalents (compare 15 and 19 with 2 or 17 and 9). For
differently substituted s-tetrazines, a more complicated situ-
ation is found, but in some cases two bands can be seen to
arise from the second transition (compounds 20 and 23).
The fluorescence spectra of all the compounds were re-
corded. Only the s-tetrazines substituted by heteroatoms
display fluorescence, and only in the case of oxygen and
chlorine substituents are relatively high fluorescence quan-
tum yields observed. Most s-tetrazines substituted by aro-
matic rings were found not to be fluorescent. Nevertheless
diphenyl-s-tetrazine is reported to be weakly fluorescent.[17]
The position of the emission band is also correlated to the
Figure 1. Spin density (isodensity 0.004 a.u.) from B3LYP calcula-
tions on the radical anions of 1 (left) and 13 (right).
Table 3 and Figure 2. These results confirm that the re-
duction of these compounds is centered on the four nitro-
gen atoms of the aromatic core with very little reorganiza-
tion in addition to the electron transfer.
Table 3. Values of the differences between standard potentials and
the differences between LUMO levels compared with tetrazine 1 as electron-donating strength of the substituents. The most ef-
the standard.
ficient derivatives are those containing one chlorine and one
alkoxy. This is true for monomer 2, but also, and very inter-
estingly, for the multichromophoric 15 and 19. In these
molecules, the fluorescence parameters (quantum yields
and lifetimes) are retained, which indicates that the s-tetra-
zines do not interact. The presence of one sulfur atom, as
in 16, induces a marked decrease in the fluorescence. This
loss is even more dramatic when sulfur is combined with
oxygen in 17. The presence of two sulfur atoms leads to a
nearly complete loss of fluorescence as demonstrated for 11.
Unfortunately, the multichromophoric systems with dif-
ferent s-tetrazines 18 and 20–23 exhibit the characteristics
of the less fluorescent subunit are retained.
Compound
Measured ∆E° [V]
Calculated ∆ELUMO [eV]
2
0.31
0.67
0.57
1.05
0.28
0.70
0.55
1.27
4
6
12
In this study, to highlight the high sensitivity of the emis-
sive properties of the s-tetrazine to the nature of the substit-
uent, one case is of particular importance. When one con-
siders compounds 4 and 5, it appears, very surprisingly at
first sight, that a very small change in the nature of the
amine (aziridine to pyrrolidine) can completely switch off
the fluorescence of the s-tetrazine. This result led us to look
at the electronic structure of these derivatives in more
depth; we looked at a possible relation between the struc-
ture of the s-tetrazines and their photophysical properties.
A survey of the literature attracted our attention to the spe-
Figure 2. Correlation between the differences in the LUMO ener-
gies of s-tetrazines 2, 4, 6, and 12 in comparison with 1 and the
differences in their standard potentials (R2 = 0.9904).
The absorption and fluorescence (when relevant) wave- cial crystal structure of an analogue of compound 4, bis(a-
lengths for the s-tetrazines are reported in Table 4. The ab- ziridin-1-yl)-s-tetrazine,[18] in which the rigidity of the azir-
sorption spectra exhibit two main bands (λ1 and λ2) and in idine ring forces it out of the plane of the s-tetrazine. As a
some cases a smaller one (λ3). The first transition is attrib- comparison, in piperazine, a larger cyclic amine, the nitro-
uted to the nǞπ* transition of the s-tetrazine. This forbid- gen atom attached to the tetrazine ring was found to be
den transition has a small molar extinction coefficient ε that conjugated with the aromatic ring.[14a] Our DFT calcula-
ranges from 400 to 2000 Lmol–1 cm–1. Furthermore, this tions were able to account for this structure and revealed
band is weakly sensitive to the substitution pattern on the one interesting consequence of this structural difference. We
ring, as expected for this type of transition.[16] The second found that the nature of the HOMO and HOMO-1 orbitals
band is attributed to a πǞπ* transition and is more intense in compounds 4 and 5 are reversed (Figure 3), which re-
(the ε values are usually higher by a factor of 10). Its posi- flects the difference in the donating ability of the nitrogen
tion is strongly correlated to the electronegativity of the atoms in the two cycles. For instance, studies on the influ-
substituents present on the ring. As expected electron-with- ence of the nature of the cyclic amine on the color of azo
drawing groups shift this band to higher energies (hypsoch- dyes have shown that the electron-donating power of the
romic shift). An intermediate situation is reached when two nitrogen atom decreases in the following order: Pyrrolidinyl
different heteroatoms are present when compared to their Ͼ piperidinyl Ͼ azetidinyl ϾϾ aziridinyl.[19]
symmetrical counterparts (for example, see 1, 2, and 6 or 1,
We then systematically performed calculations on all the
4, and 12). The nature of the alkyl chain has little influence, s-tetrazine derivatives that we had prepared and on some
as evidenced by the very similar behavior of 6, 7, and 8. others of potential interest and plotted the four most im-
Finally, note that in the case of multi-s-tetrazine derivatives, portant orbitals in relation to fluorescence processes, that
6124
www.eurjoc.org
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 6121–6128

Synthesis and Physical Chemistry of s-Tetrazines
Table 4. Absorption and fluorescence maxima recorded in dichloromethane.
Substituent X
Substituent Y
UV/Vis absorption
Fluorescence
λ1abs [nm] λ2abs [nm] λ3abs [nm]
λem[a] [nm]
φf[b]
τ [ns][c]
1
2
3
4
5
6
7
8
Cl
Cl
Cl
Cl
Cl
OMe
515
520
523
514
522
524
526
528
528
528
528
525
524
529
521
522
529
524
518
528
529
522
529
542
542
307
327
–
–
551; 567
567
567
–
576
575
572
575
–
0.14
0.38
0.004
–
0.045
0.11
0.10
0.09
–
58
269
281
–
259
275
–
160
120; 6
–
–
49
naphthalen-1-yloxy
pyrrolidin-1-yl
2,3-diphenylaziridin-1-yl
OMe
O(CH2)4OH
O(CH2)4OH
SMe
436
365
345
347
348
394
422
428
496
383
367
328
391
396
394
323
397; 349
403
330
398; 348
320
298
Cl
OMe
OMe
O(CH2)4OH
OMe
SMe
S-nBu
pyrrolidin-1-yl
59
–
–
–
–
–
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
SMe
S-nBu
287
290
274
278
–
270
273
258
263
269
257
288
269
259
252
–
–
590
–
–
0.0009
–
–
Ͻ1Ͼ[d]
–
pyrrolidin-1-yl
3,5-dimethyl-1H-pyrazol-1-yl
OMe
3,5-dimethyl-1H-pyrazol-1-yl
3,5-dimethyl-1H-pyrazol-1-yl
–
–
–
578
572
576
580
577
565
585
585
570
575
575
602
–
0.36
0.025
0.005
0.005
0.29
0.006
0.002
0.13
0.006
0.0006
–
144
13; 6
7; 2
Ͻ5Ͼ[d]
150
–
–
59
10
1.5
20
s-Tetrazine[e]
Diphenyl-s-tetrazine[f]
[a] λex = λ1abs. [b] φf Ϯ8%. [c] τϮ2%. [d] Average of a multiexponential decline. [e] From ref.[2] [f] From ref.[17]
Figure 3. Calculated HOMO and HOMO-1 orbitals of compounds
4 and 5.
is, the HOMO, the HOMO-1, the LUMO, and the
LUMO+1 (see Figure 4 and Supporting Information). It
appears that for substituents that are good electron donors
(in the case of amines, aromatics, etc.), the HOMO orbital
is of π type and the fluorescent character vanishes. This
inversion of orbitals has already been proposed in an earlier
report by Gleiter et al.,[20] who recorded and rationalized
the photoelectron spectra of s-tetrazines. Neugebauer and
Figure 4. Positions of the HOMO (o), HOMO-1 (᭡), and LUMO
(᭿) orbitals of 1, 2, 6, 9 10, and 3-chloro-6-methylthio-s-tetrazine.
Black denotes π-type orbitals and white n-type orbitals.
It was thus clear that the HOMO orbital plays a crucial
co-workers also studied the radical cations of s-tetrazines[21] role because of the mixing or not of the heteroatom lone
and observed that, depending on the substituents, the radi- pair or the π system of the aromatic substituent. It is also
cal was delocalized only on the central ring (n type s-tetra- clear that the LUMO and LUMO+1 orbitals are only
zines) or on the overall molecule (π type s-tetrazines). In weakly affected by the substituents, as could be expected,
contrast, even though a lot of calculations (semiempirical, especially in the case of the LUMO. This orbital corre-
HF, or DFT) have already been conducted on s-tetrazine[22] sponds to the π* orbital of the tetrazine ring and is located
and its derivatives, these trends have never been highlighted entirely on the four-nitrogen-atom system and its position
before.
depends mainly on the inductive effect of the substituents,
Eur. J. Org. Chem. 2009, 6121–6128
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6125

P. Audebert, G. Clavier et al.
FULL PAPER
as suspected on the basis of the electrochemistry and UV/ development of sensors as well as electrofluorescent
Vis absorption spectra.
switches is currently underway.
On the other hand, the HOMO and HOMO-1 orbitals
are strongly affected by the substituent. A very interesting
situation of orbital-crossing may happen depending on the Experimental Section
substituent. If the substituent is a weak mesomer donor, or
Synthesis: Solvents were dried by conventional methods, distilled
exerts only inductive effects without orbital-mixing (in the
case of chlorine and alkoxy substituents), insufficient or-
bital-mixing occurs and the tetrazine π system is the
HOMO-1 orbital, whereas the lone pairs of the tetrazine
from nitrogen, and deoxygenated prior to use. Starting compounds
were purchased and used without further purification. s-Tetrazines
1,[23] 2,[6] 3,[6] 4,[11] 6,[6] 7,[7] 8,[7] 9,[24] 10,[25] 11,[11] 13,[26] 15,[7] 19,[27]
20,[27] 21,[7] 22,[27] 23,[27] 24,[10] 25,[10] 26,[10] 27,[10] 28,[28] 29,[29] and
ring mix and form the HOMO. On the other hand, when 30[29] were synthesized according to reported procedures. The
NMR spectra were recorded with a Bruker AC 300 spectrometer
with a 5 mm probehead. The 1H and 13C NMR chemical shifts are
referenced relative to the residual proton signal and the central
peak of the carbon triplet of the deuteriated solvent (CDCl3),
respectively.
the substituents are good mesomer donors and mix suffi-
ciently with the accepting tetrazine π system (in the case of
all aromatics, amino, and alkylthio type substituents), this
latter orbital energy rises above the energy of the n orbital
(which is more or less constant) and forms the HOMO
(whereas the n system is the HOMO-1 orbital). The case of
9 is a special one in which both orbitals are almost at the
same level, which explains the very weak fluorescence ob-
served. In these limiting cases, a better representation of
the orbital spacing can be obtained by running a further
minimization in dichloromethane. For example, compound
4 has two orbitals that are very similar in energy (n:
–0.24529 hartree; π: –0.24588 hartree) in the gas phase,
whereas in dichloromethane they are reversed and more
widely spaced (π: –0.24329 hartree; n: –0.25496 hartree),
which reflects better its nonfluorescent nature. Note that
some s-tetrazine derivatives, for example, dimethyl- and di-
phenyl-s-tetrazines, previously reported as weakly fluores-
cent fit the observed trend. On the other hand, known com-
pounds like the bis(trifluoromethyl)-, dicyano-, or bis(x-
pyridyl)-s-tetrazine (x = 2, 3, or 4) have never been de-
scribed as fluorescent but will be tested in due course.
We can also understand from these results the behavior
of the multi-s-tetrazine compounds prepared. Indeed, the
relative positions of the orbitals of the simple s-tetrazines
shown in Figure 4 show that the HOMO of the least fluo-
rescent one is always of higher energy. Hence, the emissive
properties of compounds 15–23 are always governed by the
less emissive nucleus (thio oxygen for 20 or dithiol for 21).
Hopefully, calculations will provide a reliable predictive tool
for the design of future fluorescent multi-s-tetrazine com-
pounds.
3-Chloro-6-(2,3-diphenylaziridin-1-yl)-s-tetrazine (5): In an Erlen-
meyer flask, s-tetrazine 1 (0.1 g, 0.7 mmol) was dissolved in dry
diethyl ether (25 mL). 2,3-Diphenylaziridine[30] (0.3 g, 0.15 mmol)
was added dropwise at room temperature. The reaction was stirred
for 10 min and water (20 mL) was added. The organic layer was
separated and the aqueous phase was extracted with diethyl ether
(20 mL). The organic layers were combined, dried with MgSO4,
and concentrated under vacuo. Compound 5 was isolated by col-
umn chromatography (SiO2, CH2Cl2) in 66% yield (0.135 g). 1H
NMR (300 MHz, CDCl3, 25 °C): δ = 3.13 (s, 2 H), 7.32 (m, 10
H) ppm. 13C NMR (75 MHz, CDCl3, 25 °C): δ = 50.3, 127.1, 128.7,
128.83, 133.5, 164.0, 167.0 ppm. HRMS (ESI+): calcd. for
C16H1335ClN5 [M + H]+ 310.0854; found 310.0866.
3,6-Bis(pyrrolidin-1-yl)-s-tetrazine (12): In a 250 mL three-necked
round-bottomed flask, s-tetrazine 1 (2.02 g, 13.3 mmol) was dis-
solved in dry diethyl ether (130 mL). Pyrrolidine (4.40 mL,
52.7 mmol) was added dropwise at room temperature. The reaction
was heated at reflux for 2 h and quenched with water (100 mL).
The organic layer was separated and the aqueous phase was ex-
tracted with diethyl ether (100 mL). The organic layers were com-
bined, dried with MgSO4, and concentrated in vacuo. Compound
4 was isolated by column chromatography (SiO2, CH2Cl2) in the
first fraction and compound 12 was then recovered by using a 1:10
mixture of ethyl acetate/CH2Cl2; yield 29% (0.85 g). 1H NMR
(300 MHz, CDCl3, 25 °C): δ = 2.01 (m, 4 H), 3.62 (m, 4 H) ppm.
13C NMR (75 MHz, CDCl3, 25 °C): δ = 25.5, 46.5, 158.3 ppm.
HRMS (ESI+): calcd. for C10H16N6Na [M + Na]+ 243.1329; found
243.1326.
3-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-methoxy-s-tetrazine
(14):
s-Tetrazine 13 (1.0 g, 3.7 mmol), methanol (3 mL), and a small
amount of sodium sulfate were placed in a pressure-resistant reac-
tor. The reaction vessel was sealed and warmed at 100 °C for 2 h.
After cooling, the mixture was dissolved in CH2Cl2 and extracted
twice with water. The combined organic layers were dried with
MgSO4 and concentrated in vacuo. Compound 6 was isolated by
column chromatography (SiO2, CH2Cl2) in the first fraction and
compound 14 was then recovered by using a 1:10 mixture of ethyl
acetate/CH2Cl2; yield 67% (0.51 g). 1H NMR (300 MHz, CDCl3,
25 °C): δ = 2.31 (s, 3 H), 2.58 (s, 3 H), 4.28 (s, 3 H), 6.10 (s, 1
H) ppm. 13C NMR (75 MHz, CDCl3, 25 °C): δ = 13.7, 14.2, 56.9,
111.1, 143.0, 153.5, 159.4, 166.4 ppm. HRMS (ESI+): calcd. for
C8H10N6ONa [M + Na]+ 229.0808; found 229.0814.
Conclusions
From the results above it is clear that tetrazines, already
recognized for their applications in energetic materials and
coordination chemistry, also have good potential for the
elaboration of new performance molecular materials. Ow-
ing to the efficient synthesis of a fluorescent s-tetrazine
comprising one alkoxy substituent and a chlorine atom, we
now have access to a large variety of derivatives. The factors
determining whether fluorescence occurs or not has been
thoroughly investigated and now opens the way to the elab-
oration of more fluorescent molecules. The synthesis and
1,4-Bis(6-chloro-s-tetrazin-3-ylthio)butane (16): Butane-1,4-dithiol
(0.4 g, 3.3 mmol) and triethylamine (0.93 mL, 6.6 mmol) were
study of additional new s-tetrazines and their use in the mixed in acetonitrile (30 mL) in a dropping funnel and the mixture
6126
www.eurjoc.org
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 6121–6128

Synthesis and Physical Chemistry of s-Tetrazines
was added slowly to a solution of s-tetrazine 1 (1.0 g,6.6 mmol) in
acetonitrile (10 mL) at room temperature. After the addition the
mixture was stirred for another 2 h until TLC indicated the reaction
was over (1:5 ethyl acetate/petroleum ether). The solids were fil-
tered, washed with diethyl ether, and the filtrate evaporated. After
column chromatography (SiO2, 1:6 ethyl acetate/petroleum ether),
0.18 g (15%) of the product 16 was obtained. 1H NMR (300 MHz,
CDCl3, 25 °C): δ = 2.02 (m, 4 H), 3.37 (m, 4 H) ppm. 13C NMR
(75 MHz, CDCl3, 25 °C): δ = 27.5, 30.0, 165.6, 175.7 ppm. HRMS
(ESI+): calcd. for C8H835Cl2N8S2Na [M + Na]+ 372.9583; found
372.9587.
495 nm excitation) pumped by an argon ion laser. The Levenberg–
Marquardt algorithm was used for nonlinear least-squares fits.
Theoretical Modeling: All the geometry optimizations were per-
formed in vacuo with a Nec TX7 with 32 Itanium 2 processors at
ENS Cachan. The hybrid density functional B3LYP potential with
the 6-31(d)G* basis set was used as implemented in Gaussian 03.[31]
Harmonic vibrations were also calculated for all the obtained struc-
tures to verify that a true minimum was observed. In some cases
(see text) a solvent model was included (IEFPCM) to account for
the effects of solvation on the electronic properties. Orbitals and
spin densities were generated using the cubgen module of Gaussian
and visualized with GaussView 3.0 of Gaussian Inc.
1,4-Bis(6-methoxy-s-tetrazin-3-ylthio)butane (17): s-Tetrazine 16
(94 mg, 0.27 mmol) and MgSO4 (0.5 g) were added to methanol
(50 mL) and the mixture was heated at reflux under Ar for 20 h.
After filtration of the solids the filtrate was evaporated and the
residue was purified by column chromatography (SiO2, 3:10 ethyl
acetate/petroleum ether). After evaporation of the solvents 46 mg
Supporting Information (see also the footnote on the first page of
this article): Detailed preparations of tetrazine 1, UV/Vis and
fluorescence spectra of 5, 9, 11, 14 16, 17 and 18 and plots of the
energy levels of calculated orbitals for all compounds.
1
(50%) of 17 was obtained. H NMR (300 MHz, CDCl3, 25 °C): δ
[1] a) A. Pinner, Ber. Dtsch. Chem. Ges. 1897, 30, 1871; b) A.
Pinner, Justus Liebigs Ann. Chem. 1897, 297, 221–271.
[2] A. R. Katritzky in Handbook of Heterocyclic Chemistry; Perga-
mon Press, Oxford, 1986.
[3] a) J. Sauer, 1,2,4,5-Tetrazines in: Comprehensive Heterocyclic
Chemistry II (Eds.: A. R. Katritzky, C. W. Rees, E. F. V.
Scriven), Pergamon Press, Oxford, 1996, vol. 6, p. 901; b) D. L.
Boger, Tetrahedron 1983, 39, 2869–2939; c) D. L. Boger, Chem.
Rev. 1986, 86, 781–793; d) D. L. Boger, Chemtracts: Org. Chem.
1996, 9, 149–189.
[4] a) D. E. Chavez, M. A. Hiskey, R. D. Gilardi, Angew. Chem.
Int. Ed. 2000, 39, 1791–1793; b) M. H. V. Huynh, M. A. His-
key, D. E. Chavez, D. L. Naud, R. D. Gilardi, J. Am. Chem.
Soc. 2005, 127, 12537–12543; c) D. E. Chavez, M. A. Hiskey,
R. D. Gilardi, Org. Lett. 2004, 6, 2889–2891; d) H. Abou-Ra-
chid, Y. Song, A. Hu, S. Dudiy, S. V. Zybin, W. A. Goddard,
J. Phys. Chem. A 2008, 112, 11914–11920.
[5] N. Saracoglu, Tetrahedron 2007, 63, 4199–4236.
[6] P. Audebert, F. Miomandre, G. Clavier, M. C. Vernières, S.
Badré, R. Méallet-Renault, Chem. Eur. J. 2005, 11, 5667–5673.
[7] Y.-H. Gong, P. Audebert, J. Tang, F. Miomandre, G. Clavier,
S. Badré, R. Méallet-Renault, J. Marrot, J. Electroanal. Chem.
2006, 592, 147–152.
[8] a) M. Chowdhury, L. Goodman, J. Chem. Phys. 1962, 36, 548–
549; b) M. Chowdhury, L. Goodman, J. Chem. Phys. 1963, 38,
2979–2985.
[9] F. Gückel, A. H. Maki, F. A. Neugebauer, D. Schweitzer, H.
Vogler, Chem. Phys. 1992, 164, 217–227.
[10] P. Audebert, S. Sadki, F. Miomandre, G. Clavier, M. C. Verni-
eres, M. Saoud, P. Hapiot, New J. Chem. 2004, 28, 387–392.
[11] Z. Novak, B. Bostai, M. Csekei, K. Lorincz, A. Kotschy, Het-
erocycles 2003, 60, 2653–2668.
[12] For recent examples, see: a) S. C. Benson, L. Lee, L. Yang, J. K.
Snyder, Tetrahedron 2000, 56, 1165–1180; b) D. L. Boger, S. E.
Wolkenberg, J. Org. Chem. 2000, 65, 9120–9124.
= 2.03–1.96 (m, 4 H), 3.35–3.30 (m, 4 H), 4.24 (s, 6 H) ppm. 13C
NMR (75 MHz, CDCl3, 25 °C): δ = 28.0, 30.2, 56.5, 166.3,
171.4 ppm. HRMS (ESI+): calcd. for C10H14N8O2S2Na [M +
Na]+ 365.0573; found 365.0593.
4-{6-[4-(6-Chloro-s-tetrazin-3-ylthio)butylthio]-s-tetrazin-3-yloxy}-
butan-1-ol (18): s-Tetrazine 17 (143 mg, 0.41 mmol), butane-1,4-
diol (0.365 mL, 4.1 mmol), NaHCO3 (34 mg, 0.4 mmol), MgSO4
(0.3 g), and dichloromethane (6 mL) were added to a 20 mL pres-
sure tube. The mixture was stirred and heated at 120 °C for 2 h and
then cooled to room temperature. After column chromatography
(SiO2, ethyl acetate) 36 mg (22%) of the product 18 was obtained.
1H NMR (300 MHz, CDCl3, 25 °C): δ = 1.60 (br., 1 H), 1.80 (m,
4 H), 2.02 (m, 4 H), 3.32 (m, 4 H), 3.73 (t, J = 6.26 Hz, 2 H), 4.61
(t, J = 6.44 Hz, 2 H) ppm. 13C NMR (75 MHz, CDCl3, 25 °C): δ
= 25.2, 27.7, 28.0, 28.9, 30.2, 30.3, 62.3, 69.7, 165.8, 166.1, 171.2,
175.9 ppm. HRMS (ESI+): calcd. for C12H1735ClN8O2S2Na [M +
Na]+ 427.0497; found 427.0505.
Cyclic Voltammetry: The electrochemical studies were performed
using an EG&G PAR 273 potentiostat interfaced to a PC com-
puter. The reference electrode used was an Ag+/Ag electrode filled
with 0.01  AgNO3. This reference electrode was checked versus
ferrocene, as recommended by IUPAC. In our case, E°(Fc+/Fc) =
0.045 V in acetonitrile or dichloromethane with 0.1  tetrabutylam-
monium perchlorate (TBAP). TBAP was purchased from Fluka
(puriss). Acetonitrile (Aldrich, 99.8%), dichloromethane (SDS,
99.9%), and toluene (Aldrich, 99.5%) were used as received. All
solutions were deaerated by bubbling with argon for a few minutes
prior to electrochemical measurements.
Photophysical Studies
[13] U. Schirmer, B. Wuerzr, N. Meyer, F. A. Neugebauer, H. Fi-
scher, German Patent DE3508214-A1, 1986 [Chem. Abstr.
1986, 106, 45718p].
[14] a) C. Glidewell, P. Lightfoot, B. J. L. Royles, D. M. Smith, J.
Chem. Soc. Perkin Trans. 2 1997, 1167–1174; b) G. L. Rusinov,
N. I. Latosh, I. I. Ganebnykh, R. I. Ishmetova, N. K. Ignat-
enko, O. N. Chupakhin, Zh. Org. Khim. 2006, 42, 772–780;
Russ. J. Org. Chem. 2006, 42, 757–765.
[15] H. Fischer, T. Muller, I. Umminger, F. A. Neugebauer, H.
Chandra, M. C. R. Symons, J. Chem. Soc. Perkin Trans. 2
1988, 413–421.
[16] J. Waluk, J. Spanget-Larsen, E. W. Thulstrup, Chem. Phys.
1995, 200, 201–213.
Steady-State Spectroscopy: A UV/Vis Varian CARY 500 spectro-
photometer was used. Excitation and emission spectra were mea-
sured with a SPEX Fluorolog-3 (Jobin–Yvon) instrument. A right-
angled configuration was used. The optical densities of the samples
were checked to be less than 0.1 to avoid reabsorption artifacts.
The relative fluorescence quantum yields of the s-tetrazines were
measured relative to that of Rhodamine 6G in ethanol (φf = 0.95).
The solutions were of equal absorbance at the excitation wave-
length (λexc=529 nm).
Time-Resolved Spectroscopy: The fluorescence decay curves were
obtained by a time-correlated single-photon-counting method
using a titanium–sapphire laser (82 MHz, repetition rate lowered
to 4 or 0.8 MHz depending on the lifetime measured by a pulse-
peaker, 1 ps pulse width, a doubling crystal was used to reach
[17] a) UV/Vis: S. Makarov, E. Kudrik, K. Davydov, Russ. J. Gen.
Chem. 2006, 76, 1599–1603; b) fluorescence: S. Ghosh, M.
Chowdhury, Chem. Phys. Lett. 1982, 85, 233–238.
Eur. J. Org. Chem. 2009, 6121–6128
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6127

P. Audebert, G. Clavier et al.
FULL PAPER
[18] C. Krieger, H. Fischer, F. A. Neugebauer, F. Guckel, D.
Schweitzer, Acta Crystallogr., Sect. C 1987, 43, 1412–1415.
[19] G. Hallas, J.-H. Choi, Dyes Pigm. 1999, 40, 99–117.
[20] R. Gleiter, V. Schehlmann, J. Spanget-Larsen, H. Fischer, F. A.
Neugebauer, J. Org. Chem. 1988, 53, 5756–5762.
[21] a) H. Fischer, I. Umminger, F. A. Neugebauer, H. Chandra,
M. C. R. Symons, J. Chem. Soc., Chem. Commun. 1986, 837–
838; b) H. Fischer, T. Muller, I. Umminger, F. A. Neugebauer,
H. Chandra, M. C. R. Symons, J. Chem. Soc. Perkin Trans. 2
1988, 413–421.
[22] For recent examples (and for earlier ones, see cited references
within), see: a) C. Adamo, V. Barone, Chem. Phys. Lett. 2000,
330, 152–160; b) D. Xie, J. Zeng, J. Comput. Chem. 2004, 25,
1487–1494; c) M. Odelius, B. Kirchner, J. Hutter, J. Phys.
Chem. A 2004, 108, 2044–2052.
[23] M. D. Helm, A. Plant, J. P. A. Harrity, Org. Biomol. Chem.
2006, 4, 4278–4280 and the accompanying ESI.
[29]
[30]
I. Janowska, F. Miomandre, G. Clavier, P. Audebert, J.
Zakrzewski, K. H. Thi, I. Ledoux-Rak, J. Phys. Chem. A 2006,
110, 12971–12975.
D. Tanner, C. Birgersson, A. Gogoll, K. Luthman, Tetrahedron
1994, 50, 9797–9824.
[31] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria,
M. A. Robb, J. R. Cheeseman, J. A. Montgomery Jr., T.
Vreven, K. N. Kudin, J. C. Burant, J. M. Millam, S. S. Iyengar,
J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N.
Rega, G. A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K.
Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y.
Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J. E. Knox, H. P.
Hratchian, J. B. Cross, C. Adamo, J. Jaramillo, R. Gomperts,
R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pom-
elli, J. W. Ochterski, P. Y. Ayala, K. Morokuma, G. A. Voth, P.
Salvador, J. J. Dannenberg, V. G. Zakrzewski, S. Dapprich,
A. D. Daniels, M. C. Strain, O. Farkas, D. K. Malick, A. D.
Rabuck, K. Raghavachari, J. B. Foresman, J. V. Ortiz, Q. Cui,
A. G. Baboul, S. Clifford, J. Cioslowski, B. B. Stefanov, G. Liu,
A. Liashenko, P. Piskorz, I. Komaromi, R. L. Martin, D. J.
Fox, T. Keith, M. A. Al-Laham, C. Y. Peng, A. Nanayakkara,
M. Challacombe, P. M. W. Gill, B. Johnson, W. Chen, M. W.
Wong, C. Gonzalez, J. A. Pople, Gaussian 03, Revision C.02,
Gaussian, Inc., Wallingford, CT, 2004.
[24] S. M. Sakya, K. K. Groskopf, D. L. Boger, Tetrahedron Lett.
1997, 38, 3805–3808.
[25] D. L. Boger, S. M. Sakya, J. Org. Chem. 1988, 53, 1415–1423
(supplementary data).
[26] M. D. Coburn, G. A. Buntain, B. W. Harris, M. A. Hiskey,
K. Y. Lee, D. G. Ott, J. Heterocycl. Chem. 1991, 28, 2049–2050.
[27] Y.-H. Gong, P. Audebert, G. Clavier, F. Miomandre, J. Tang,
S. Badré, R. Méallet-Renault, E. Naidus, New J. Chem. 2008,
32, 1235–1242.
[28] P. Audebert, S. Sadki, F. Miomandre, G. Clavier, Electrochem.
Commun. 2004, 6, 144–147.
Received: August 24, 2009
Published Online: October 30, 2009
6128
www.eurjoc.org
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 6121–6128