Reactivity of 7-Azanorbornenes in Bioorthogonal Inverse Electron-Demand Diels–Alder Reactions

Article abstract of DOI:10.1002/ejoc.201700411

In the preparation of multicomponent compounds, the accumulation of components through sequential click reactions is an attractive strategy. In this work we examined the reactivity of various N-substituted 7-azanorbornenes in inverse electron-demand Diels–Alder (iEDDA) reactions with tetrazines to explore the potential of 7-azanorbornenes as clickable hub molecules. The iEDDA reaction of 7-azanorbornene is expected to proceed faster when the nitrogen atom at the 7-position is substituted with an electron-donating substituent. Contrary to this expectation, the electron-donating alkyl-bearing derivative reacted much more slowly than those bearing electron-withdrawing acyl groups. The results of DFT calculations indicate that the reaction rates correlate well with an increase in sp² character of the 7-nitrogen atoms: The ease of conversion of the more stable exo conformer into the more reactive endo conformer may lower the activation energy of the first rate-determining hetero-Diels–Alder step. Indeed, the reaction rates of N-acylated 7-azanorbornenes, which have a more planar nitrogen atom, were found superior to those of other derivatives and comparable to those of norbornenes. Finally, we successfully labeled a tetrazine on a protein surface by fluorophore-conjugated 7-azanorbornene in the presence of other proteins.

Full text of DOI:10.1002/ejoc.201700411

A Journal of
Accepted Article
Title: Reactivity of 7-Azanorbornenes in Bioorthogonal Inverse
Electron-Demand Diels-Alder Reactions
Authors: Fumika Karaki, Kenji Ohgane, Hirotaka Imai, Kennosuke Itoh,
and Hideaki Fujii
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using the Digital Object Identifier (DOI) given below. The VoR will be
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the VoR from the journal website shown below when it is published
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201700411
Link to VoR: http://dx.doi.org/10.1002/ejoc.201700411
Supported by

10.1002/ejoc.201700411
European Journal of Organic Chemistry
FULL PAPER
Reactivity of 7-Azanorbornenes in Bioorthogonal Inverse
Electron-Demand Diels–Alder Reactions
Fumika Karaki*[a,b], Kenji Ohgane[c], Hirotaka Imai[b,d], Kennosuke Itoh[a,b], Hideaki Fujii[a,b]
Abstract: In preparing multi-component compounds, accumulation
of the components through sequential click reactions is an attractive
strategy. Here, we examined the reactivity of variously N-substituted
7-azanorbornenes in inverse electron-demand Diels–Alder (iEDDA)
reactions with tetrazines to explore the potential of 7-
azanorbornenes as clickable hub molecules. An iEDDA reaction of
7-azanorbornene is expected to proceed faster when the 7-nitrogen
is substituted with an electron-donating substituent. Contrary to this
expectation, the electron-donating alkyl-bearing derivative reacted
much slower than electron-withdrawing acyl-bearing ones. DFT
calculation implied that the reaction rates correlated well with the
increase in sp2 character of the 7-nitrogen atoms: the ease of
conversion from the more stable exo conformers to the more
reactive endo conformers may lower the activation energy of the first
rate-determining hetero-Diels–Alder step. Indeed, the reaction rates
of N-acylated 7-azanorbornenes, which have the more planar
nitrogen, were superior to the other derivatives and comparable to
norbornenes. Finally, we successfully labeled a tetrazine on a
protein surface by fluorophore-conjugated 7-azanorbornene in the
presence of other proteins.
biological activity are often the result. Small-sized
polysubstituted clickable hub molecules should be particularly
useful to address these issues.
Highly efficient biocompatible cross-linking reactions are
referred to as click reactions, of which the copper-catalyzed 1,3-
dipolar cycloaddition reaction between an azide and a terminal
alkyne (CuAAC) is the most famous example.[8-10] Despite the
wide use of CuAAC, the toxicity of copper catalysts limits the
application in living systems. To overcome this issue, copper-
free click reactions have been extensively studied.[11] The
inverse electron demand Diels–Alder reaction (iEDDA)
represents one class of metal-catalyst-free click reactions, with
reaction rates that are faster than other copper-free click
reactions.[12] IEDDA reactions start with the Diels–Alder reaction
of tetrazine, an electron deficient diene, and strained alkenes
like transcyclooctene, norbornene and cyclopropene (Scheme 1,
Figure 1). The highly strained cycloadduct 1 expels molecular
nitrogen via
a
retro-Diels–Alder reaction, giving 4,5-
dihydropyridazine 2, and subsequent proton transfer leads to the
1,4-dihydropyridazine product 3.[13] Although norbornene has a
relatively lower reaction rate than other strained alkenes, it is
useful due to its synthetic accessibility and its small molecular
size.[14,15] Thus, multi-valent norbornenes should be feasible
clickable hub molecules as mentioned above.[16] Despite this
possibility, only studies on the reactivity of 5- or 5,6-substituted
Introduction
In recent years, there has been an urgent need to prepare multi-
component compounds in many scientific fields.[1-6] Since linear
synthesis of such complicated molecules is usually laborious,
convergent methods by click chemistry have been effective.[7] In
such a strategy of multiple conjugations, however, the linker
moieties often enlarge the entire molecular size of the designed
compounds. As the molecules become larger, poor water
solubility, poor membrane permeability, and diminished
norbornenes in the iEDDA reaction have thus far been
[17-19]
conducted
and reaction of 7-substituted norbornene has
rarely been reported, which may be attributed to synthetic
inaccessibility of this class of compounds.[20,21]
To address this issue, we set out to investigate the reactivity of
variously N-substituted 7-azanorbornenes (ANors), which would
R1
R1
R1
N
N
R1
R2
R3
R2
R3
R2
R3
–N2
R2
R3
N
N
N
N
N
N
N
+
N
HN
N
R1
[a]
Dr. F. Karaki, Dr. K. Itoh, Prof. Dr. H. Fujii.
Laboratory of Medicinal Chemistry
R1
R1
R1
strained alkene
1
2
3
School of Pharmacy, Kitasato University
5-9-1, Shirokane, Minato-ku, Tokyo 108-8641, Japan
E-mail: karakif@pharm.kitasato-u.ac.jp
Dr. F. Karaki, Prof. Dr. H. Imai, Dr. K. Itoh, Prof. Dr. H. Fujii
Medicinal Research Laboratories
School of Pharmacy, Kitasato University
5-9-1, Shirokane, Minato-ku, Tokyo 108-8641, Japan
Dr. K. Ohgane
Synthetic Organic Chemistry Laboratory
RIKEN
2-1, Hirosawa, Wako-shi, Saitama 351-0198, Japan
Prof. Dr. H. Imai
Scheme 1. iEDDA reaction between tetrazine and strained alkene.
[b]
[c]
[d]
7
4
3
5
2
1
6
cyclopropene
transcyclooctene
norbornene
Laboratory of Hygienic Chemistry
School of Pharmacy, Kitasato University
5-9-1, Shirokane, Minato-ku, Tokyo 108-8641, Japan
Figure 1. Structures of strained alkenes in iEDDA reactions.
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201700411
European Journal of Organic Chemistry
FULL PAPER
1) NBS, AgNO3
acetone
rt, 15 h
be easily established via the Diels–Alder reaction between
alkene and pyrrole. As various substituents can be introduced to
the 7-nitrogen atom after construction of the skeleton, ANors can
serve as a multi-valent scaffold to install multiple functionalities,
such as fluorophores and ligands for proteins. Moreover, thanks
to the newly introduced nitrogen atoms, ANors would have
enhanced aqueous solubility and would be suitable for use in
aqueous biological environments.[22] In this report, we
synthesized variously N-substituted ANors and investigated their
reactivity in iEDDA reactions. We also confirmed the
bioorthogonality of the reaction between tetrazine and ANors.
Boc
N
CO2Me
Boc
N
Boc
N
NaBH4
Br
+
2) N-Boc-pyrrole
90 ºC, 30 h
47% (2 steps)
CO2Me
DMSO-H2O
–15 ºC to –5 ºC
1 h, 80%
H
CO2Me
CO2Me
4
5
6
6’
endo : exo = 67 : 33
O
O
OtBu
N
OtBu
CH2OH
H
N
NaBH4, CaCl2
NOESY
or
6 or 6’
H
H
EtOH-THF
rt
NOESY
NOESY
H
HO
7 81%
7’ 68%
–
H2
N
X
X
Ph
CF3CO2
PhXCl
sat. NaHCO3 aq.
N
Ph
N
NaBH4, CaCl2
TFA
6
CH2Cl2
0 ºC, 1.5 h
CH2Cl2
rt
EtOH-THF
rt
CO2Me
CH2OH
CO2Me
8
9
X = CO 74% (2 steps)
11 X = CO 72%
12 X = SO2 60%
10 X = SO2 62% (2 steps)
S
S
Results and Discussion
N
Ph
NaBH4, CaCl2
N
Ph
Lawesson's reagent
9
THF
rt, 16 h
100%
EtOH-THF
rt, 6 h
95%
Synthesis of the ANor derivatives is summarized in Scheme 2.
The N-protected ANor 6 was constructed in 25% yield in just 3
steps. This synthetic sequence was as effective as other copper-
free click reagents.[11] The synthetic route started from a Diels–
Alder reaction between the alkyne and the pyrrole to afford 5.[23]
Although there is a report that the ethoxycarbonyl counterpart of
5 was successfully reduced with sodium borohydride in dimethyl
sulfoxide,[24] we noticed that the addition of water was
indispensable for effective conversion. This outcome may be
attributed to retarded hydroboration of the unsubstituted C=C
double bond of 5, 6, and 6' in the presence of water with the in
situ generated borane.[25] The 5-endo product 6 and 5-exo
product 6' were successfully separated by column
CO2Me
CH2OH
14
13
Scheme 2. Synthesis of ANor derivatives 11, 12 and 14.
Table 1. Half-lives of the 7-azanorbornenes in chloroform-d determined by 1H
NMR.
X
t1/2
X
R
+
CDCl3
R
pyrrole
alkene
R = -CO2Me
compound
R = -CH2OH
compound
energy gap (eV)[a]
energy gap (eV)[a]
t1/2
t1/2
X
X
+
+
stable[b,c]
7.33 d
stable[b,c]
stable[b]
stable[b]
stable[b]
8
9
0.271
0.050
0.044
0.033
16
11
0.437
0.215
0.210
0.198
NH2
NBz
NH2
chromatography. The stereochemistry of
6 and 6' were
NBz
determined by NOESY analysis after reduction of the 5-
methoxycarbonyl substituents to hydroxymethyl groups
6
NBoc 14.5 h
NH
< 1 h
7
NBoc
NH
15
17
(compounds
7
and 7'),[26] due to the instability of 5-
[a] The orbital energy gaps between HOMOpyrrole and LUMOalkene were
calculated at the B3LYP/6-311+G(d,p) level. [b] No retro-Diels–Alder product
was observed after seven days. [c] Trifluoroacetic acid salts were dissolved in
chloroform-d.
methoxycarbonyl-bearing ANor derivatives (see below). The N-
tert-butoxycarbonyl group of the intermediate 6 was deprotected
under acidic conditions and the resultant ammonium salt 8 was
acylated or sulfonylated to afford 9 or 10, respectively. The 5-
methoxycarbonyl moieties of 9 and 10 were then reduced to
provide 11 and 12. The carbonyl moiety of 9 was converted to
thiocarbonyl group to afford 13, and the 5-methoxycarbonyl
group of 13 was reduced to provide 14.
N-Boc-pyrrole
(A)
methyl acrylate
96 h
24 h
During the synthesis of ANors mentioned above, we noticed
that some ANors were susceptible to retro-Diels–Alder
decomposition. Thus, we determined the half-lives of the ANor
derivatives from the decay of 1H NMR signals (Table 1, Figure 2).
While ANors with 5-hydroxymethyl groups were stable
regardless of the N-substituents (compounds 7, 11, 16 and 17),
ANors with 5-methoxycarbonyl groups and with electron-
donating substituents on the 7-nitrogen atoms were prone to
degradation (compound 15 < 6 < 9). The instability of these
(B)
6 h
0 h
1
0.8
0.6
0.4
0.2
0
y
=
0.9475e-0.0461x
R² 0.9910
=
1,1,2,2-tetrachloroethane
bridgehead
0
10
20
30
40
50
time (h)
Figure 2. Typical 1H NMR time-course data and a decay curve of ANor 6. (A)
1H NMR time course data of 6. 1,1,2,2-Tetrachloroethane was used as an
internal standard. (B) Decay curve of 6 determined by comparing the relative
values of integration for the peaks corresponding to the bridgehead protons
and the peak at 5.96 ppm (1,1,2,2-tetrachloroethane).
compounds may result from the lowering of the HOMOpyrrole
LUMOalkene energy gaps caused by these substituents and the
lowered activation energies of the retro-Diels–Alder reactions
–
that released the strain. Calculation of the HOMOpyrrole
–
LUMOalkene energy gaps supported this possibility; both the 5-
methoxycarbonyl groups and the electron-donating substituents
on the 7-nitrogen atoms decreased these energy gaps (Table 1).
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10.1002/ejoc.201700411
European Journal of Organic Chemistry
FULL PAPER
O
Table 2. Second-order rate constants of 7-azanorbornenes in iEDDA reaction
with dipyridyltetrazine.
Boc
N
–
H2
N
CF3CO2
phenyl chloroformate
EtN(iPr)2
N
OPh
TFA
CH2Cl2
0 ºC, 1 h
CH2Cl2
–20 ºC to rt, 14 h
60% (2 steps)
CH2OH
CH2OH
CH2OH
7
16
18
N
N
R
R
O
N
N
k
N
N
N
N
N
N
NHPh
+
+
isomers
HN
PhNCO, K2CO3
H2O:MeOH = 95:5
CH2OH
or
CH3CN
CH2OH
N
acetone
0 ºC, 40 min
73% (2 steps)
N
CH2OH
19
[a]
[b]
k (H2O-MeOH, M-1s-1
1.14±0.05
)
k (CH3CN, M-1s-1
0.0967±0.0164
0.0384±0.0035
)
compound
R
N
Ph
11
18
-C(O)Ph
-C(O)OPh
0.901±0.054
BnBr, K2CO3
0.436±0.111[c]
0.0575±0.0172
0.0605±0.0061
CH2OH
14
19
-C(S)Ph
acetone
0 ºC, 1 h
89% (2 steps)
20
-C(O)NHPh
0.330±0.003
12
20
0.0360±0.0037
n.d.[d]
0.0031±0.0002
0.0239±0.0023
-SO2Ph
-CH2Ph
Scheme 3. Synthesis of ANor derivatives 18–20.
The measurements were repeated three times for each compound and the
data are shown as mean ± S.D. [a] Determined from the decay of the
absorption at 297 nm. [b] Determined from the decay of the absorption at 535
nm to avoid the wavelength absorbed by the cycloadducts. [c] Determined
from the decay of the absorption at 525 nm as the dienophile had strong
absorption at 297 nm. [d] n.d. = not determined. The reaction was too slow
and the rate could not be determined in this assay system.
Taking the instability of 5-methoxycarbonyl-bearing ANors into
consideration, we next synthesized several other ANor
derivatives with the 5-hydroxymethyl substituent (Scheme 3).
The tert-butoxycarbonyl group of 7 was deprotected under acidic
conditions, and the resultant ammonium salt 16 was converted
to 18–20.
Table 3. Energy gaps between π*tetrazine– π7-azanorbornene and π*7-azanorbornene–π
We evaluated the rate of the iEDDA reaction between ANor
derivatives and 3,6-di(4-pyridyl)-1,2,4,5-tetrazine by their
second-order rate constants in aqueous media (Table 2, left
column).[17,19] As this reaction is of the inverse electron-demand
type, electron-rich dienophiles with high HOMO energy were
expected to react faster. Indeed, the amide 11 reacted faster
.
tetrazine
O
O
O
Me
S
N
S Me
N
R
R
O
N
N
H
H
than the thioamide 14 with the nitrogen atom bearing a more
H
H
[27]
substantial positive charge
whereas compound 12, bearing
exo
rotamer 1
π*tet–πANor (eV)
rotamer 2
endo
the most electron-deficient sulfonamide moiety, gave a much
slower reaction rate compared to compounds 11, 14, 18 and 19.
On the other hand, the reactivities of the carbonyl series
(compounds 11, 18 and 19) were positively correlated with the
electron-withdrawing characters of the substituents. In addition,
compound 20, bearing an electron-donating alkyl group, reacted
sluggishly. The obtained outcomes may stem from the possibility
that protonation of the phenyl-substituted nitrogen of 19 or the
amine nitrogen of 20 in aqueous media diminished the electron
density of the olefin moieties in these derivatives. To investigate
this possibility, we determined the reaction rates in aprotic
acetonitrile (Table 2, right column). Compared with the rate
constants in aqueous solution, the rate constants decreased
dramatically in accordance with a previous report.[28] Indeed, the
urea 19 reacted faster than the carbamate 18, and the amine 20
reacted with tetrazine in acetonitrile at a measurable reaction
rate. Furthermore, the trifluoroacetic acid salt of 20 hardly
reacted with the tetrazine in acetonitrile, which further supported
our hypothesis (data not shown). However, 20 reacted still
slower than electron-deficient carbonylated or thiocarbonylated
compounds 11, 14, 18 and 19. Hence, we assumed the
existence of other factors that influenced the reactivity and
undertook a search for these factors.
π*ANor–πtet (eV)
compound
exo
exo
R
endo
endo
21
5.188
5.009
–
4.984
4.835
5.167
9.317 9.332
9.402 9.413
-C(O)Me
22 -C(O)OMe
23 -C(S)Me
–
9.113
24 -C(O)NHMe
4.836
4.992
4.806
4.901
4.992
4.434
9.466 9.467
8.931 9.227
9.335 9.148
9.629 9.793
25
-SO2Me (rotamer 1)
25 -SO2Me (rotamer 2) 4.645
26
4.567
-Me
The orbital energy gaps between the indicated 7-azanorbornenes (ANor) and
unsubstituted tetrazine (tet) are shown. Two assumed exo and endo
conformers defined as shown above were each calculated, except for the
thioamide 23, which converged to
a consistent conformation. For the
sulfonamide 25, existence of two rotamers was also considered. The lowest
energy gaps are shown by bold letters.
As the π* orbital of tetrazine and the π orbital of the strained
alkene interact in iEDDA reactions, these energy gaps are
important factors that determine the reactivity.[29] To investigate
the orbital energies of variously N-substituted ANors and
tetrazine, we performed DFT calculations at the B3LYP/6-
311+G(d,p) level (Table 3). Most of the endo conformers, which
seem to be less stable due to the steric repulsion between the R
groups and the 5,6-exo-protons, had lower energy gaps and
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10.1002/ejoc.201700411
European Journal of Organic Chemistry
FULL PAPER
Table 5. Energy differences and planarity of the 7-nitrogen atoms of
cycloadducts.
were expected to react much more efficiently (Table 3, left
column).[30] The increase in electronegativity of the R groups
resulted in the lower πANor orbital energies and the larger energy
gaps (e.g., 21 > 22 > 24 >> 26). However, the larger energy
gaps did not necessarily provide the decrease in the reactivity as
the amide 11 reacted faster than the other derivatives (Table 2).
This result may imply that the reaction proceeded in normal-
electron-demand Diels–Alder fashion.[31] However, calculated
energy gaps between π*ANor and πtetrazine (πtet) were much larger
than those of π*tet and πANor (Table 3, right column). Thus, these
outcomes confirmed that the reactions proceeded in iEDDA
fashion and that the energy gaps alone are insufficient to explain
R
R
N
N
N
N
N
N
N
H
H
H
N
N
H
N
H
H
H
H
exo
endo
exo
energy
endo
energy
(kcal/mol) θ (deg) (kcal/mol) θ (deg)
compound
R
–
–
–
–
–
–
–
–
–
–
–
–
21'
–
–
–
–
353.31
349.23
356.68
342.46
-C(O)Me
the reactivity.
22' -C(O)OMe
23' -C(S)Me
24' -C(O)NHMe
25'
25' -SO2Me (rotamer 2)
26'
As the exo and the endo conformers are expected to have
different reactivities, we next investigated the relative stability of
these conformers and the transition energies between them
(Table 4). We show the planarity of 7-nitrogen atoms depicted
-SO2Me (rotamer 1)
0.000 340.66
6.394 350.34
0.000 330.99
6.020 350.05
-Me
Structures of exo-cycloadducts were optimized as this type of addition is
estimated to be preferred over endo-addition in the case of norbornene.[17] For
21'–25', two initial exo/endo structures converged to
a
consistent
conformation.
by θ (the sum of the three valence angles around the nitrogen
atom),[32,33] as we expected a relation between the planarity and
the flipping energy. We also optimized structures of the hetero-
Diels–Alder cycloadducts (1, Scheme 1) as this addition would
be the rate-determining step of this sequential reaction (Table
5).[30] As expected, the endo starting materials with lower π*tet–
πANor energy gaps were less stable (Table 4). On the other hand,
cycloadducts 21'–25' exclusively existed in the endo-
conformation (two initial exo and endo structures converged to
the endo structures) and the endo structure was favored in the
case of 26' (Table 5). Hence, the major reaction pathway should
be that in which the more stable exo starting materials invert the
direction of the N-substituents to give the endo-conformers and
then cycloaddition would occur to give the endo-products. The
energy required to invert the substituents decreased (compound
26 > 25 > 24 > 22 > 21 > 23) as the θ of the either conformer
increased, i.e., as the sp2 characters of the nitrogen atoms
increased (Table 4). Remarkably, this correlated well with the
reaction rates of ANors except for the thioamide variant, which
had a significantly higher HOMO-LUMO gap than the other
derivatives (Figure 3). The lower the flipping energy from exo to
endo, which was brought about by the increased sp2 characters
of the 7-nitrogen atoms, may result in lower activation energy of
the cycloaddition and enhanced reaction rates of ANors with
planar nitrogen atoms.
Figure 3. Relationships between sp2 characters of the 7-nitrogen atoms and
the rates in iEDDA reaction. Reaction rates in aqueous solution (Table 2)
were plotted against θ of the most stable conformers/rotamers (Table 4). Data
points represent the mean with S.D. depicted by error bars (n =3).
Table 4. Energy differences and planarity of the 7-nitrogen atoms of 7-
azanorbornenes.
R
N
R
R
R
N
N
c
b
N
a
exo
θ = a+b+c
TS
TS
endo
exo
energy
(kcal/mol) θ (deg) (kcal/mol) θ (deg) (kcal/mol) θ (deg)
endo
energy
energy
compound
R
As the amide 11 had superior reactivity with respect to the
other derivatives (Table 2), we synthesized other acylated ANors
(Scheme 4) and set out to investigate the reaction rates. While
the ANors bearing 4-substituted benzoyl groups (compound 27
and 28) and the cyclohexane carbonyl group (compound 29)
were obtained by straightforward acylation of ammonium salt 16,
N-acetyl derivative 30 could not be isolated by this procedure
due to the highly hydrophilic nature of this compound. Thus, we
acetylated both the 7-nitrogen and the hydroxyl group of the
21
-C(O)Me
0.000 347.08
0.000 341.83
0.525 359.96
1.249 359.98
0.149 349.12
0.377 344.67
22
23
24
25
25
26
-C(O)OMe
–
–
–
–
-C(S)Me
-C(O)NHMe
-SO2Me (rotamer 1)
–
357.03
0.000 337.48
0.363 342.30
2.371 359.90
3.252 359.90
4.706 359.99
0.102 339.45
0.000 335.90
3.683 349.07
1.953 328.52
-SO2Me (rotamer 2) 0.040 331.13
0.000 326.72
11.55
359.96
-Me
For the thioamide 23, two initial exo/endo structures converged to a consistent
conformation. Values of the conformers with lower π*tetrazine–π7-azanorbornene
energy gaps (Table 3) are shown by bold letters.
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10.1002/ejoc.201700411
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FULL PAPER
O
N-acylated ANors are comparable to those of norbornenes (k =
H2
N
–
CF3CO2
CH2OH
Boc
acid chloride
2 M NaOH aq.
M–1s–1
in
the
case
of
5-endo-
N
N
R
1.76±0.013
TFA
hydroxymethylnorbornene),[17] apart from the one bearing a
strongly inductive substituent.
acetone, rt
CH2Cl2
rt, 1 h
CH2OH
CH2OH
7
16
27 = p-C6H4-OMe 88% (2 steps)
28 = p-C6H4-NO2 72% (2 steps)
29 = cyclohexyl 78% (2 steps)
Finally, we prepared fluorescein isothiocyanate (FITC)-
conjugated ANor 33 and biotin-conjugated tetrazine 39 to
confirm the bioorthogonality of the iEDDA reaction between the
ANors and the tetrazines (Scheme 5). After protection of the
hydroxyl group of the intermediate 7, the tert-butoxycarbonyl
group was cleaved, and the resultant ammonium salt was
condensed with the N-protected glycine. Then, the O-acetyl
group was cleaved under basic conditions to afford 32. The tert-
butoxycarbonyl group of 32 was cleaved under acidic conditions,
and the resultant ammonium salt was allowed to react with 5-
FITC to provide the triethylamine salt of the FITC-conjugated
ANor 33. Synthesis of the biotin-conjugated tetrazine 39 started
from condensation between biotin (34) and the linker moiety to
afford 35. A previously reported tetrazine 37 [34] was reacted with
carbonyldiimidazole to provide the activated urea 38.
Condensation between 38 and the N-deprotected biotin unit 36
afforded the desired biotin-conjugated tetrazine 39.
O
N
1) TFA, CH2Cl2
rt, 1 h
2) 2 M HCl-Et2O
3) Ac2O, pyridine
rt, 7 h
4) MeOK, MeOH
rt, 1 h, 83% (4 steps)
CH2OH
30
1) Ac2O, pyridine
rt, 13 h
2) TFA, CH2Cl2
rt, 1 h
O
N
CCl3
3) trichloroacetic anhydride
pyridine, rt, 9 h
4) MeOK, MeOH
rt, 20 min
CH2OH
31
28% (4 steps)
Scheme 4. Synthesis of the N-acylated ANor derivatives.
Table 6. Second-order rate constants of N-acylated 7-azanorbornenes.
We first examined the effect of buffer components and protein
on the iEDDA reaction. The iEDDA reaction between FITC-ANor
33 and tetrazine-biotin 39 was conducted in Tris-buffered saline
(TBS) or 1% bovine serum albumin (BSA) in TBS. The iEDDA
O
O
N
N
N
R
N
R
k
N
N
N
N
N
+
+
isomers
HN
H2O:MeOH = 95:5
product was trapped with
a streptavidin-coated plate and
CH2OH
CH2OH
N
N
detected by fluorescence from the FITC moiety (Figure 4A). As
shown in Figure 4B, the reaction of 33 and 39 smoothly
proceeded even in the presence of BSA under aqueous buffered
conditions. Next, we examined if a tetrazine on a protein surface
could be detected with FITC-conjugated 33. As a model, we
k (H2O-MeOH, M-1s-1
1.07±0.02
)
compound
R
30
29
-Me
cyclohexyl
1.08±0.02
immobilized the biotin-conjugated tetrazine 39 onto
a
27
28
31
4-methoxyphenyl
4-nitrophenyl
-CCl3
1.13±0.08[a]
0.749±0.013[b]
0.0316±0.0005
streptavidin-coated plate and performed the iEDDA reaction with
33 (Figure 5A). As shown in Figure 5B, the immobilized tetrazine
39 was successfully labeled with 33 in the presence of other
proteins (1% BSA in TBS or 10% fetal calf serum (FCS) in
Dulbecco's Modified Eagle Medium (DMEM)). Thus, the reaction
between tetrazine and ANor is expected to be applicable to the
conjugation under physiological conditions.
The reaction rates were determined from the decay of the absorption at 297
nm. The measurements were repeated three times for each compound and
the data are shown as mean ± S.D. [a] Determined from the decay of the
absorption at 307 nm as the dienophile had absorption at 297 nm. [b]
Determined from the decay of the absorption at 525 nm as the dienophile had
absorption at 297 nm.
1) Ac2O, DMAP
pyridine
rt, 40 min
2) TFA, CH2Cl2
rt, 1 h
OH
ammonium salt, and then cleaved the O-acetyl group with
anhydrous workup to obtain the desired compound 30. Before
the acetylation step, the counter anion was exchanged from
trifluoroacetate to chloride to prevent trifluoroacetylation of the 7-
nitrogen atom. In the synthesis of N-trichloroacetylated 31, the
hydroxyl group of compound 7 was acetylated prior to the
deprotection of the tert-butoxycarbonyl group and N-
trichloroacetylation.
O
H
N
H
N
O
1) TFA
CH2Cl2
rt, 1.5 h
Boc
NHBoc
N
N
N
S
O
2) 5-FITC, Et3
MeOH
rt, 24 h
N
3) N-Boc-glycine
O
iBuCOCl, Et3
THF, rt, 19 h
4) MeOK, MeOH
rt, 20 min
N
CH2OH
CH2OH
O
CH2OH
7
26% (2 steps)
32
OH
·1/3Et3
N
33
84% (4 steps)
O
S
O
O
S
-
CF3CO2
EDCI, DMAP
N-Boc-1,3-propanediamine
TFA
CH2Cl2
+
HN
NH
HN
H
NH
HN
NH
NHBoc
NH2
H
N
H
N
H
H
H
H
H
DMF
rt, 27 h
74%
CO2
H
rt, 30 min
S
O
O
34
36
35
N
N
The reaction rates of N-acylated ANor derivatives are shown in
Table 6. We expected that the steric effects on the reactivity
would be negligible as the N-acetylated 30 and the N-
cyclohexane-carbonylated 29 had similar rate constants.
Although the inductive effect of the trichloroacetyl group
(compound 31) had strong impact on the reactivity, the
mesomeric effect was quite small, as the presence of 4-
substituents on the benzoyl groups had little effect (compare
compound 11 (Table 2), 27 and 28). Most of the reaction rates of
N
N
1) TFA
CH2Cl2
rt, 1 h
N
N
N
N
N
36, Et3
N
O
N
N
H
N
H
N
N
N
H
N
2) CDI, Et3
CH2Cl2
rt, 20 h
N
N
DMF
rt, 15 h
41%
HN
NH
H
N
BocHN
H
H
O
O
37
73% (2 steps)
38
S
O
39
Scheme 5. Preparation of FITC-conjugated ANor 33 and biotin-conjugated
tetrazine 39.
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201700411
European Journal of Organic Chemistry
FULL PAPER
were reported as δ values (ppm) reference to tetramethylsilane. Mass
spectra (MS) were obtained on JMS-AX505HA, JMS-700 MStation, or
JMS-100LP instrument by applying an electrospray ionization (ESI)
method or fast atom bombardment (FAB) method. The progress of the
reaction was determined on Merck Silica Gel Art. 5715 (TLC). Silica gel
was purchased from Fuji Silysia (CHROMATOREX® PSQ 60B (60 µm),
CHROMATOREX® COOH MB 100-40/75 (60 µm) and SCAVENGER
SO3H SILICA).
(A)
(B)
10
9
8
7
6
5
4
3
2
1
0
FITC
N
N
N
N
N
N
+
N
HN
3% BSA-TBS
0.3% BSA-TBS
TBS
CH2OH
CH2OH
biotin
distilled water
without 29
without 39
0
0.2
0.4
0.6
0.8
1
streptavidin plate
dilution rate of the sample
Figure 4. IEDDA reaction between FITC-ANor 33 and tetrazine-biotin 39 in the
presence of proteins under buffered aqueous conditions. (A) Schematic
representation of the experiment. (B) FITC-ANor 33 and tetrazine-biotin 39
were allowed to react in the indicated solutions. The trapped conjugate was
detected by the fluorescence emitted by the FITC moiety. Data points
represent the mean with the range of two samples depicted by error bars (n =
2) except for the data obtained with distilled water (n = 3, S.D.).
7-(tert-Butyl) 2-methyl-3-bromo-7-azabicyclo[2.2.1]hepta-2,5-diene-
2,7-dicarboxylate (5): Compound 5 was synthesized according to the
literature procedure with some modifications.[23] To a solution of methyl
propiolate (4) (6.40 g, 76.1 mmol) in acetone (75 mL) were added
silver(I) nitrate (631 mg, 3.71 mmol) and N-bromosuccinimide (16.0 g,
90.1 mmol) and the mixture was stirred at ambient temperature. After 15
h, the mixture was poured into water (200 mL) and extracted with
dichloromethane (100 mL × 3). The combined organic layer was washed
with saturated aqueous sodium hydrogen carbonate (50 mL × 2) and
brine (50 mL), dried over sodium sulfate, and concentrated under
reduced pressure. To the residue was added tert-butyl 1-
pyrrolecarboxylate (60 mL, 360 mmol) and the mixture was stirred at 90
ºC for 30 h. After cooling to ambient temperature, the excess tert-butyl 1-
pyrrolecarboxylate was removed under reduced pressure (0.5 mm Hg, >
90 ºC). Column chromatography of the residue (silica-gel 124 g, n-
hexane/ethyl acetate = 100/1 to 15/1) gave the title compound (11.8 g,
35.8 mmol, 47.1% for 2 steps) as a yellow oil. The 1H NMR spectrum was
identical to that reported in the literature.[23]
(A)
(B)
3
2
1
0
FITC
N
2.5 pmol
1.25 pmol
0.313 pmol
N
N
N
N
CH2OH
biotin
BSA-TBS
DMEM
streptavidin plate
7-(tert-Butyl)
2-methyl-7-azabicyclo[2.2.1]hept-5-ene-2-endo-2,7-
dicarboxylate (6) and 7-(tert-butyl) 2-methyl-7-azabicyclo[2.2.1]hept-
5-ene-2-exo-2,7-dicarboxylate (6'): A solution of 5 (5.85 g, 17.7 mmol)
in dimethyl sulfoxide (45 mL) and water (9 mL) was cooled with an ice–
methanol bath (the bath temperature was –15 ºC at the start of the
reaction). To the cooled mixture was added sodium borohydride (1.34 g,
35.4 mmol) and the mixture was stirred for 1 h with the flask immersed in
the bath (after the reaction, the final temperature of the bath was –5 ºC).
Then, water (100 mL) was added and the mixture was extracted with
ethyl acetate (100 mL × 3). The combined organic layer was washed
extensively with water (50 mL × 6) and brine (50 mL), dried over sodium
sulfate, and concentrated under reduced pressure. Column
chromatography (silica-gel 122 g, n-hexane/ethyl acetate = 30/1 to 5/1)
gave the endo-isomer 6 (2.41 g, 9.50 mmol, 53.6%) as a yellow solid and
Figure 5. Detection of streptavidin-trapped biotin-tetrazine 39 with FITC-ANor
33. (A) Schematic representation of the experiment. (B) The indicated
amounts of biotin-tetrazine 39 were incubated in streptavidin-coated plates,
and the tetrazine 39 immobilized on the plate was detected with FITC-ANor 33
in 1% BSA-TBS or DMEM supplemented with 10% FCS. Data points
represent the mean with s.e.m. depicted by error bars (n = 6).
Conclusions
We synthesized variously N-substituted ANors, and determined
the iEDDA reaction rates with a tetrazine. The N-acylated ANors,
which reacted as fast as norbornenes, unexpectedly reacted
quickly compared to the ANors bearing other N-substituents.
The sp2 character of the nitrogen atoms was suggested to be a
major determinant of the reactivity of the N-substituted ANors.
We successfully showed the bioorthogonality of the reaction. As
the iEDDA reaction between norbornene and tetrazine is
orthogonal with respect to the CuAAC and strain-promoted
alkyne-azide cycloaddition (SPAAC), multiply substituted ANors
can be versatile clickable hub molecules applicable for
constructing multi-component compounds.
the exo-isomer 6' (1.18 g, 4.66 mmol, 26.3%) as
a yellow oil.
Stereochemistries of the isomers were determined after reduction of the
methoxycarbonyl groups (see compounds 7 and 7'). endo-isomer 6: 1H
NMR (CDCl3, 400 MHz): δ 1.42 (s, 9H), 1.51–1.57 (m, 1H), 2.14–2.22 (m,
1H), 3.13–3.28 (m, 1H), 3.32 (s, 3H), 4.68 (br s, 1H), 4.90 (br s, 1H),
6.17–6.22 (m, 1H), 6.36–6.45 (m, 1H). 13C NMR (CDCl3, 100 MHz): δ
28.2 (3C), 28.8, 42.5, 51.8, 60.4, 61.4, 80.3, 132.5, 137.1, 154.8, 172.8.
HR-MS (FAB): Calcd for C13H20NO4 [M+H]+: 254.1392. Found: 254.1388.
IR (KBr): 2981, 1728, 1696, 1368, 1314, 1281, 1256, 1213, 1098, 1029
cm–1. exo-isomer 6': 1H NMR (CDCl3, 400 MHz): δ 1.38–1.48 (m, 1H),
1.40 (s, 9H), 2.29–2.41 (m, 2H), 3.73 (s, 3H), 4.75 (br s, 1H), 4.94 (br s,
1H), 6.27–6.44 (m, 2H). 13C NMR (CDCl3, 100 MHz): Splits of some
peaks were observed due to the existence of conformers. δ 28.2 (3C),
28.5 (0.5C), 29.1 (0.5C), 43.4 (0.5C), 44.0 (0.5C), 52.1, 59.2, 60.0, 80.1,
134.8 (0.5C), 135.8 (0.5C), 136.8 (0.5C), 137.7 (0.5C), 155.2, 173.5. HR-
MS (FAB): Calcd for C13H20NO4 [M+H]+: 254.1392. Found: 254.1394. IR
Experimental Section
(neat): 2979, 1739, 1705, 1368, 1295, 1171, 1081, 1038, 952, 859 cm–1
.
General: All reagents and solvents were obtained from commercial
suppliers and were used without further purification other than N-
bromosuccinimide, which was recrystallized from boiling water. Infrared
tert-Butyl 5-endo-(hydroxymethyl)-7-azabicyclo[2.2.1]hept-2-ene-7-
carboxylate (7): To solution of (252 mg, 0.993 mmol) in
(IR) spectra were recorded on
a JASCO FT/IR-460Plus. Nuclear
a
6
magnetic resonance (NMR) spectra were recorded on an Agilent
Technologies VXR-400NMR for 1H NMR and 13C NMR. Chemical shifts
tetrahydrofuran (2 mL) and ethanol (3.5 mL) were added calcium chloride
(226 mg, 2.03 mmol) and sodium borohydride (153 mg, 4.04 mmol) at 0
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201700411
European Journal of Organic Chemistry
FULL PAPER
ºC and the mixture was stirred at ambient temperature for 10 h. Then,
saturated aqueous ammonium chloride (20 mL) was added and the
mixture was extracted with ethyl acetate (20 mL × 3). The combined
organic layer was washed with brine (10 mL), dried over sodium sulfate,
and concentrated under reduced pressure. Column chromatography
(silica-gel 6 g, n-hexane/ethyl acetate = 3/1 to 1/1) gave the title
compound (182 mg, 0.807 mmol, 81.3%) as a yellow oil. 1H NMR (CDCl3,
400 MHz): δ 0.57–0.70 (m, 1H), 1.42 (9H, s), 2.02–2.11 (m, 1H), 2.44–
2.55 (m, 1H), 3.20 (dd, J = 10.0, 10.0 Hz, 1H), 3.42–3.55 (m, 1H), 4.61
(br s, 1H), 4.75 (br s, 1H), 6.25 (br s, 1H), 6.36 (br s, 1H), a proton (OH)
was not observed. 13C NMR (CDCl3, 100 MHz): Splits of some peaks
were observed due to the existence of conformers. δ 27.6 (0.5C), 28.2
(3C), 28.3 (0.5C), 40.5 (0.5C), 41.0 (0.5C), 59.8 (0.5C), 60.4 (0.5C), 61.1
(0.5C), 61.7 (0.5C), 64.9, 80.0, 131.6 (0.5C), 132.7 (0.5C), 135.8 (0.5C),
136.9 (0.5C), 155.3. HR-MS (ESI): Calcd for C12H19NNaO3 [M+Na]+:
248.1263. Found: 248.1251. IR (neat): 3409, 2975, 1680, 1367, 1284,
[M+Na]+: 280.0950. Found: 280.0936. IR (neat): 2952, 1732, 1644, 1380,
1206, 1027, 849, 699, 671 cm–1
.
Methyl
carboxylate (10): To
7-(phenylsulfonyl)-7-azabicyclo[2.2.1]hept-5-ene-2-endo-
solution of (51.4 mg, 0.203 mmol) in
a
6
dichloromethane (1.0 mL) was added trifluoroacetic acid (0.2 mL) at 0 ºC
and the mixture was stirred at this temperature for 1.5 h. Then saturated
aqueous sodium hydrogen carbonate (4.0 mL) and dichloromethane (3.0
mL) were added at this temperature. To the mixture was added
benzenesulfonyl chloride (52 µL, 0.41 mmol) at 0 ºC and the mixture was
stirred at ambient temperature for 5 h. After separation of the two layers,
the aqueous layer was extracted with dichloromethane (10 mL × 3). The
combined organic layer was washed with saturated aqueous sodium
hydrogen carbonate (10 mL × 2) and brine (10 mL), dried over sodium
sulfate, and concentrated under reduced pressure. Column
chromatography (silica-gel 2 g, n-hexane/ethyl acetate = 3/1) gave the
title compound (36.9 mg, 0.126 mmol, 62.0% for 2 steps) as a yellow oil.
1H NMR (CDCl3, 400 MHz): δ 1.51 (dd, J = 11.5, 3.8 Hz, 1H), 2.33–2.41
(m, 1H), 3.34 (ddd, J = 9.1, 4.6, 4.0 Hz, 1H), 3.61 (s, 3H), 4.69–4.72 (m,
1H), 4.90–4.92 (m, 1H), 5.73 (dd, J = 5.8, 1.9 Hz, 1H), 5.92 (dd, J = 5.8,
2.1 Hz, 1H), 7.44–7.49 (m, 2H), 7.52–7.57 (m, 1H), 7.70–7.74 (m, 2H).
13C NMR (CDCl3, 100 MHz): δ 30.0, 43.4, 52.0, 62.8, 63.3, 128.1 (2C),
129.1 (2C), 130.7, 132.9, 134.8, 139.1, 172.0. HR-MS (ESI): Calcd for
C14H15NNaO4S [M+Na]+: 316.0620. Found: 316.0616. IR (neat): 3584,
1169, 1092, 1036 cm–1
.
tert-Butyl
carboxylate (7'): To
5-exo-(hydroxymethyl)-7-azabicyclo[2.2.1]hept-2-ene-7-
solution of 6' (50.2 mg, 0.198 mmol) in
a
tetrahydrofuran (0.4 mL) and ethanol (0.7 mL) were added calcium
chloride (49.8 mg, 0.449 mmol) and sodium borohydride (33.2 mg, 0.878
mmol) at 0 ºC and the mixture was stirred at ambient temperature for 24
h. Then, saturated aqueous ammonium chloride (10 mL) was added and
the mixture was extracted with ethyl acetate (10 mL × 3). The combined
organic layer was washed with brine (5 mL), dried over sodium sulfate,
and concentrated under reduced pressure. Column chromatography
(silica-gel 2 g, n-hexane/ethyl acetate = 3/1 to 1/1) gave the title
compound (30.2 mg, 0.134 mmol, 67.6%) as a colorless oil. 1H NMR
(CDCl3, 400 MHz): δ 1.28–1.34 (m, 1H), 1.36–1.46 (m, 1H), 1.41 (s, 9H),
1.73–1.80 (m, 1H), 3.57 (dd, J = 10.6, 9.1 Hz, 1H), 3.73 (dd, J = 10.6, 5.4
Hz, 1H), 4.64 (br s, 2H), 6.32 (br s, 2H), a proton (OH) was not observed.
13C NMR (CDCl3, 100 MHz): δ 28.3 (3C), 28.7, 41.2, 59.8, 61.3, 65.1,
80.1, 135.6 (2C), 155.7. HR-MS (ESI): Calcd for C12H19NNaO3 [M+Na]+:
248.1263. Found: 248.1274. IR (neat): 3411, 2976, 1682, 1367, 1297,
1735, 1447, 1343, 1207, 1163, 1101, 731, 689 cm–1
.
(5-endo-(Hydroxymethyl)-7-azabicyclo[2.2.1]hept-2-en-7-
yl)(phenyl)methanone (11): To a solution of 9 (77.6 mg, 0.302 mmol) in
tetrahydrofuran (0.9 mL) and ethanol (1.5 mL) were added calcium
chloride (69.3 mg, 0.624 mmol) and sodium borohydride (45.1 mg, 1.19
mmol) at 0 ºC and the mixture was stirred at ambient temperature. After 8
h, additional calcium chloride (37.3 mg, 0.336 mmol) and sodium
borohydride (28.1 mg, 0.743 mmol) were added and the mixture was
stirred for 15 h. Then, saturated aqueous ammonium chloride (20 mL)
was added and the mixture was extracted with ethyl acetate (20 mL × 3).
The organic layers were combined and washed with brine (10 mL), dried
over sodium sulfate, and concentrated under reduced pressure. Column
chromatography (silica-gel 2 g, n-hexane/ethyl acetate = 1/2) gave the
title compound (50.0 mg, 0.218 mmol, 72.3%) as a colorless oil. 1H NMR
(CDCl3, 400 MHz): The conformer ratio was 55:45 determined by
comparing the relative values of integration for the peaks observed at
5.29 ppm and 5.11 ppm (the bridgehead protons). δ 0.65–0.79 (m, 1H),
1.80–2.40 (m, 2H), 2.44–2.54 (m, 0.55H), 2.61–2.71 (m, 0.45H), 3.12 (dd,
J = 10.5, 10.5 Hz, 0.55H), 3.25 (dd, J = 10.5, 10.5 Hz, 0.45H), 3.52 (d, J
= 10.5 Hz, 0.55H), 3.53 (d, J = 10.5 Hz, 0.45H), 4.61 (br s, 0.45H), 4.75
(br s, 0.55H), 5.11 (br s, 0.55H), 5.29 (br s, 0.45H), 6.18–6.25 (m, 0.55H),
6.27–6.35 (m, 0.45H), 6.37–6.46 (m, 0.45H), 6.48–6.54 (m, 0.55H),
7.35–7.55 (m, 5H). 13C NMR (CDCl3, 100 MHz): δ 26.7 (0.55C), 29.4
(0.45C), 39.7 (0.45C), 41.9 (0.55C), 57.6 (0.55C), 59.0 (0.45C), 62.2
(0.45C), 63.4 (0.55C), 64.6 (0.55C), 64.7 (0.45C), 127.8 (0.45C), 127.9
(0.55C), 128.4 (2C), 130.7 (2C), 131.3 (0.55C), 133.4 (0.45C), 134.9
(1C), 135.2 (0.45C), 137.4 (0.55C), 167.7 (0.45C), 168.0 (0.55C). HR-MS
(ESI): Calcd for C14H15NNaO2 [M+Na]+: 252.1001. Found: 252.0989. IR
1256, 1159, 1089, 1045, 858 cm–1
.
Methyl 7-benzoyl-7-azabicyclo[2.2.1]hept-5-ene-2-endo-carboxylate
(9): To a solution of 6 (434 mg, 1.71 mmol) in dichloromethane (8.5 mL)
was added trifluoroacetic acid (1.7 mL) at 0 ºC and the mixture was
stirred at this temperature for 1.5 h. Then, saturated aqueous sodium
hydrogen carbonate (24 mL) and dichloromethane (26 mL) were added
at this temperature. To the mixture was added benzoyl chloride (395 µL,
3.40 mmol) at 0 ºC and the mixture was stirred at ambient temperature
for 14 h. After separation of the two layers, the aqueous layer was
extracted with dichloromethane (30 mL × 3). The combined organic layer
was washed with saturated aqueous sodium hydrogen carbonate (30 mL
× 3) and brine (20 mL), dried over sodium sulfate, and concentrated
under reduced pressure. Column chromatography (silica-gel 18 g, n-
hexane/ethyl acetate = 3/1) gave the title compound (327 mg, 1.27 mmol,
74.3% for 2 steps) as a yellow oil. 1H NMR (CD3OD, 400 MHz): The
conformer ratio was 55:45 determined by comparing the relative values
of integration for the peaks observed at 5.31 ppm and 5.12 ppm (the
bridgehead protons). δ 1.59 (br s, 0.45H), 1.62 (br s, 0.55H), 2.15–2.24
(m, 0.55H), 2.24–2.34 (m, 0.45H), 3.24–3.37 (m, 1H), 3.59 (s, 1.35H),
3.66 (s, 1.65H), 4.71 (br s, 0.55H), 4.87 (br s, 0.45H), 5.12 (br s, 0.45H),
5.31 (br s, 0.55H), 6.22 (br s, 0.45H), 6.37 (br s, 0.55H), 6.41 (br s,
0.55H), 6.57 (br s, 0.45H), 7.42–7.56 (m, 5H). 13C NMR (CD3OD, 100
MHz): δ 28.6 (0.45C), 30.9 (0.55C), 42.3 (0.55C), 44.6 (0.45C), 52.4 (1C),
59.3 (0.45C), 60.1 (0.55C), 63.9 (0.55C), 64.4 (0.45C), 128.8 (1C), 129.7
(2C), 132.4 (2C), 133.2 (0.45C), 134.4 (0.55C), 135.4 (0.55C), 135.5
(0.45C), 137.7 (0.55C), 139.1 (0.45C), 169.4 (0.45C), 169.6 (0.55C),
173.3 (0.45C), 173.7 (0.55C). HR-MS (ESI): Calcd for C15H15NNaO3
(neat): 3398, 2942, 1614, 1567, 1414, 1037, 923, 854, 700, 671 cm–1
.
(7-(Phenylsulfonyl)-7-azabicyclo[2.2.1]hept-5-en-2-endo-yl)methanol
(12): To a solution of 10 (45.9 mg, 0.157 mmol) in tetrahydrofuran (0.3
mL) and ethanol (0.5 mL) were added calcium chloride (50.8 mg, 0.458
mmol) and sodium borohydride (34.6 mg, 0.915 mmol) at 0 ºC and the
mixture was stirred at ambient temperature for 24 h. Then, saturated
aqueous ammonium chloride (10 mL) was added and the mixture was
extracted with ethyl acetate (10 mL × 3). The combined organic layer was
washed with brine (10 mL), dried over sodium sulfate, and concentrated
under reduced pressure. Column chromatography (silica-gel 1 g, n-
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10.1002/ejoc.201700411
European Journal of Organic Chemistry
FULL PAPER
hexane/ethyl acetate = 3/1 to 1/1) gave the title compound (24.7 mg,
0.0931 mmol, 59.5%) as a colorless oil. 1H NMR (CDCl3, 400 MHz): δ
0.57 (dd, J = 11.3, 4.1 Hz, 1H), 2.17–2.25 (m, 1H), 2.62–2.72 (m, 1H),
3.03 (dd, J = 10.6, 10.4 Hz, 1H), 3.46 (dd, J = 10.8, 5.9 Hz, 1H), 4.62–
4.66 (m, 1H), 4.77–4.81 (m, 1H), 5.74 (dd, J = 5.8, 1.6 Hz, 1H), 5.83 (dd,
J = 5.8, 1.8 Hz, 1H), 7.42–7.48 (m, 2H), 7.50–7.56 (m, 1H), 7.68–7.73 (m,
2H), a proton (OH) was not observed. 13C NMR (CDCl3, 100 MHz): δ
28.7, 41.4, 62.5, 63.7, 64.1, 128.0 (2C), 129.0 (2C), 129.9, 132.6, 133.7,
139.3. HR-MS (ESI): Calcd for C13H15NNaO3S [M+Na]+: 288.0670.
Found: 288.0662. IR (neat): 3521, 2943, 1447, 1332, 1161, 1086, 1029,
Phenyl
carboxylate (18): To
5-endo-(hydroxymethyl)-7-azabicyclo[2.2.1]hept-2-ene-7-
a solution of 7 (65.7 mg, 0.292 mmol) in
dichloromethane (1.5 mL) was added trifluoroacetic acid (0.3 mL) at 0 ºC
and the mixture was stirred at this temperature for 1 h. Then, the mixture
was concentrated under reduced pressure and the excess trifluoroacetic
acid was removed by repeated evaporation with toluene. To a solution of
the residue in dry dichloromethane (2.0 mL) was added N,N-
diisopropylethylamine (153 µL, 0.900 mmol) and the solution was cooled
with an ice–methanol bath (the bath temperature was –20 ºC at the start
of the reaction). To the cooled mixture was added phenyl chloroformate
(43 µL, 0.34 mmol) and the mixture was allowed to warm to ambient
temperature and stirred for 14 h. Then saturated aqueous sodium
hydrogen carbonate (20 mL) was added and the mixture was extracted
with dichloromethane (20 mL × 3). The combined organic layer was
washed with brine (10 mL), dried over sodium sulfate, and concentrated
under reduced pressure. Column chromatography (silica-gel 3 g, n-
hexane/ethyl acetate = 1/1) gave the title compound (47.5 mg, 0.194
mmol, 66.4% for 2 steps) as a brown oil. 1H NMR (CDCl3, 400 MHz):
Splits of some peaks were observed due to the existence of conformers.
δ 0.63–0.79 (m, 1H), 1.80–2.11 (m, 1H), 2.11–2.22 (m, 1H), 2.54–2.64
(m, 1H), 3.17 (dd, J = 10.5, 9.8 Hz, 1H), 3.49 (brs, 1H), 4.78 (br s, 0.5H),
4.88 (br s, 0.5H), 4.91 (br s, 0.5H), 5.01 (br s, 0.5H), 6.31 (br s, 1H), 6.42
731, 689 cm–1
.
Methyl 7-(phenylcarbonothioyl)-7-azabicyclo[2.2.1]hept-5-ene-2-
endo-carboxylate (13): To a solution of 9 (105 mg, 0.408 mmol) in
tetrahydrofuran (2.0 mL) was added Lawesson's reagent (181 mg, 0.449
mmol) and the mixture was stirred at ambient temperature. After 16 h,
the solvent was removed under reduced pressure. Column
chromatography (silica-gel 10 g, n-hexane/ethyl acetate = 5/1) gave the
title compound (111 mg, 0.406 mmol, 99.5%) as a yellow oil. 1H NMR
(CDCl3, 400 MHz): The conformer ratio was 54:46 determined by
comparing the relative values of integration for the peaks observed at
4.75 ppm and 4.96 ppm (the bridgehead protons). δ 1.68 (dd, J = 12.0,
4.0 Hz, 0.54H), 1.74 (dd, J = 11.9, 3.9 Hz, 0.46H), 2.14–2.21 (m, 0.54H),
2.50–2.58 (m, 0.46H), 3.09–3.14 (m, 0.46H), 3.49–3.55 (m, 0.54H), 3.62
(s, 1.38H), 3.71 (s, 1.62H), 4.74–4.77 (m, 0.54H), 4.94–4.97 (m, 0.46H),
5.74–5.77 (m, 0.46H), 5.93–5.96 (m, 0.54H), 6.28 (dd, J = 6.0, 2.2 Hz,
0.46H), 6.43–6.47 (m, 1.08H), 6.64 (dd, J = 6.0, 2.5 Hz, 0.46H), 7.32–
7.43 (m, 5H). 13C NMR (CDCl3, 100 MHz): δ 26.4 (0.46C), 30.3 (0.54C),
39.3 (0.46C), 43.3 (0.54C), 52.1 (0.46C), 52.2 (0.54C), 62.5 (0.54C),
63.1 (0.46C), 64.1 (0.54C), 64.3 (0.46C), 126.9 (0.92C), 127.0 (1.08C),
128.28 (1.08C), 128.33 (0.92C), 130.0 (1C), 131.4 (0.46C), 133.3
(0.54C), 135.4 (0.54C), 137.6 (0.46C), 141.04 (0.54C), 141.08 (0.46C),
171.3 (0.46C), 172.0 (0.54C), 193.0 (0.46C), 193.6 (0.56C). HR-MS
(ESI): Calcd for C15H16NO2S [M+H]+: 274.0902. Found: 274.0890. IR
(br s, 1H), 7.03–7.09 (m, 2H), 7.15–7.21 (m, 1H), 7.30–7.36 (m, 2H). 13
C
NMR (CDCl3, 100 MHz): Splits of some peaks were observed due to the
existence of conformers. δ 27.5 (0.5C), 28.6 (0.5C), 40.3 (0.5C), 41.3
(0.5C), 60.0 (0.5C), 60.4 (0.5C), 61.3 (0.5C), 61.6 (0.5C), 64.6, 121.6
(2C), 125.4, 129.3 (2C), 131.7 (0.5C), 133.0 (0.5C), 135.7 (0.5C), 137.0
(0.5C), 151.0, 153.2. HR-MS (ESI): Calcd for C14H15NNaO3 [M+Na]+:
268.0950. Found: 268.0930. IR (neat): 3423, 1726, 1493, 1368, 1277,
1203, 1041, 863, 741, 689 cm–1
.
(5-endo-(Hydroxymethyl)-N-phenyl-7-azabicyclo[2.2.1]hept-2-ene-7-
carboxamide (19): To a solution of 7 (65.8 mg, 0.292 mmol) in
dichloromethane (1.5 mL) was added trifluoroacetic acid (0.3 mL) at 0 ºC
and the mixture was stirred at this temperature for 1 h. Then the mixture
was concentrated under reduced pressure and the excess trifluoroacetic
(neat): 2951, 1738, 1434, 1327, 1206, 1027, 883, 758 cm–1
.
acid was removed by repeated evaporation with toluene. To
a
(5-endo-(Hydroxymethyl)-7-azabicyclo[2.2.1]hept-2-en-7-
suspension of the residue and potassium carbonate (170 mg, 1.23 mmol)
in dry acetone (0.6 mL) was added phenyl isocyanate (37.5 µL, 0.346
mmol) at 0 ºC and the mixture was stirred at this temperature for 40 min.
Then, water (10 mL) was added and the mixture was extracted with ethyl
acetate (20 mL × 3). The combined organic layer was washed with brine
(10 mL), dried over sodium sulfate, and concentrated under reduced
pressure. Column chromatography (silica-gel 2 g, n-hexane/ethyl acetate
= 1/1 to 1/3) gave the title compound (52.1 mmol, 0.213 mmol, 73.0% for
2 steps) as a colorless amorphous. 1H NMR (CDCl3, 400 MHz): δ 0.70
(dd, J = 11.6, 4.2 Hz, 1H), 1.51 (br s, 1H), 2.14–2.21 (m, 1H), 2.57–2.66
(m, 1H), 3.17–3.25 (m, 1H), 3.54–3.60 (m, 1H), 4.71–4.74 (m, 1H), 4.79–
4.82 (m, 1H), 6.30 (dd, J = 5.8, 2.1 Hz, 1H), 6.44 (dd, J = 5.8, 2.5 Hz, 1H),
6.44 (br s, 1H), 7.00–7.05 (m, 1H), 7.24–7.29 (m, 2H), 7.34–7.38 (m, 2H).
13C NMR (CDCl3, 100 MHz): δ 27.8, 40.8, 60.5, 62.0, 64.4, 120.0 (2C),
123.3, 128.8 (2C), 131.8, 136.5, 138.7, 155.8. HR-MS (ESI): Calcd for
C14H16N2NaO2 [M+Na]+: 267.1110. Found: 267.1097. IR (neat): 3310,
yl)(phenyl)methanetione (14): To a solution of 13 (84.0 mg, 0.307
mmol) in tetrahydrofuran (0.6 mL) and ethanol (1.1 mL) were added
calcium chloride (66.8 mg, 0.602 mmol) and sodium borohydride (46.8
mg, 1.24 mmol) at 0 ºC and the mixture was stirred at ambient
temperature for 6 h. Then saturated aqueous ammonium chloride (10
mL) was added and the mixture was extracted with ethyl acetate (20 mL
× 3). The combined organic layer was washed with brine (10 mL), dried
over sodium sulfate, and concentrated under reduced pressure.
Preparative TLC (n-hexane/ethyl acetate = 1/3) gave the title compound
(71.7 mg, 0.292 mmol, 95.1%) as a yellow oil. 1H NMR (CDCl3, 400
MHz): The conformer ratio was 54:46 determined by comparing the
relative values of integration for the peaks observed at 4.82 ppm and
4.67 ppm (the bridgehead protons). δ 0.79–0.86 (m, 1H), 1.47 (br s,
0.46H), 1.66 (br s, 0.54H), 2.06–2.14 (m, 0.46H), 2.34–2.42 (m, 0.54H),
2.45–2.54 (m, 0.54H), 2.76–2.85 (m, 0.46H), 3.12–3.20 (m, 0.54H),
3.30–3.38 (m, 0.46H), 3.52–3.62 (m, 1H), 4.65–4.69 (m, 0.46H), 4.81–
4.84 (m, 0.54H), 5.69–5.72 (m, 0.54H), 5.82–5.85 (m, 0.46H), 6.31 (dd, J
= 6.1, 2.3 Hz, 0.54H), 6.40 (dd, J = 6.1, 2.5 Hz, 0.46H), 6.49 (dd, J = 6.1,
2942, 1651, 1538, 1443, 1337, 1087, 1032, 911, 751 cm–1
.
2.4 Hz, 0.46H), 6.59 (dd, J = 6.1, 2.5 Hz, 0.54H), 7.31–7.41 (m, 5H). 13
C
(7-Benzyl-7-azabicyclo[2.2.1]hept-5-en-2-endo-yl)methanol (20): To a
solution of 7 (66.9 mg, 0.297 mmol) in dichloromethane (1.5 mL) was
added trifluoroacetic acid (0.3 mL) at 0 ºC and the mixture was stirred at
this temperature for 1 h. Then the mixture was concentrated under
reduced pressure and the excess trifluoroacetic acid was removed by
repeated evaporation with toluene. To a suspension of the residue and
potassium carbonate (166 mg, 1.20 mmol) in dry acetone (0.6 mL) was
added benzyl bromide (41 µL, 0.35 mmol) at 0 ºC and the mixture was
stirred at this temperature for 1 h. Then water (10 mL) was added and
NMR (CDCl3, 100 MHz): δ 25.2 (0.54C), 29.3 (0.46C), 37.8 (0.46C), 41.2
(0.54C), 62.3 (0.5C), 63.6 (0.5C), 63.8 (0.5C), 64.2 (0.5C), 64.4 (0.5C),
65.2 (0.5C), 126.8 (0.92C), 126.9 (1.08C), 128.2 (0.92C), 128.3 (1.08C),
129.7 (0.46C), 129.8 (0.54C), 130.7 (0.54C), 132.9 (0.46C), 134.4
(0.46C), 136.7 (0.54C), 141.27 (0.54C), 141.32 (0.46C), 191.3 (0.46C),
191.6 (0.54C). HR-MS (ESI): Calcd for C14H16NOS [M+H]+: 246.0953.
Found: 246.0957. IR (neat): 3389, 1477, 1320, 1259, 1031, 1000, 921,
757 cm–1
.
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201700411
European Journal of Organic Chemistry
FULL PAPER
the mixture was extracted with ethyl acetate (20 mL × 3). The organic
layer was washed with brine (10 mL), dried over sodium sulfate, and
concentrated under reduced pressure. Column chromatography (silica-
gel 2 g, chloroform/methanol = 100 /0 to 100/7) gave the title compound
(57.1 mg, 0.265 mmol, 89.3% for 2 steps) as a brown oil. 1H NMR (CDCl3,
400 MHz): δ 0.54 (dd, J = 11.1, 3.7 Hz, 1H), 2.01–2.09 (m, 1H), 2.26 (br
s, 1H), 2.43–2.52 (m, 1H), 3.16 (dd, J = 10.4, 10.1 Hz, 1H), 3.40 (dd, J =
10.4, 6.3 Hz, 1H), 3.47 (s, 2H), 3.76–3.78 (m, 1H), 3.88–3.91 (m, 1H),
6.03 (d, J = 5.5 Hz, 1H), 6.18 (dd, J = 5.5, 1.7 Hz, 1H), 7.19–7.31 (m, 5H).
13C NMR (CDCl3, 100 MHz): δ 28.1, 40.8, 51.5, 64.7, 65.1, 65.8, 126.9,
128.3 (2C), 128.8 (2C), 129.0, 133.8, 139.3. HR-MS (ESI): Calcd for
C14H18NO [M+H]+: 216.1388. Found: 216.1378. IR (neat): 3348, 2932,
2.62–2.72 (m, 0.44H), 3.13 (dd, J = 10.4, 9.3 Hz, 0.56H), 3.25 (dd, J =
10.1, 9.9 Hz, 0.44H), 3.52–3.61 (m, 1H), 4.52 (br s, 0.44H), 4.67 (br s,
0.56H), 5.15 (br s, 0.56H), 5.31 (br s, 0.44H), 6.22–6.27 (m, 0.56H),
6.31–6.38 (m, 0.44H), 6.42–6.47 (m, 0.44H), 6.52–6.58 (m, 0.56H),
7.64–7.71 (m, 2H), 8.24–8.29 (m, 2H). 13C NMR (CDCl3, 100 MHz): δ
26.5 (0.56C), 29.5 (0.44C), 39.5 (0.44C), 41.9 (0.56C), 57.8 (0.56C),
59.2 (0.44C), 62.0 (0.44C), 63.3 (0.56C), 64.4 (0.56C), 64.6 (0.44C),
123.8 (2C), 128.8 (0.88C), 128.9 (1.12C), 131.1 (0.56C), 133.6 (0.44C),
135.1 (0.44C), 137.6 (0.56C), 140.8, 149.1, 165.4. HR-MS (ESI): Calcd
for C14H14N2NaO4 [M+Na]+: 297.0851. Found: 297.0861. IR (neat): 3399,
1624, 1599, 1522, 1349, 1037, 822, 708 cm–1
.
1495, 1453, 1360, 1103, 1032, 720, 698 cm–1
.
Cyclohexyl(5-endo-(hydroxymethyl)-7-azabicyclo[2.2.1]hept-2-en-7-
yl)methanone (29): To a solution of 7 (55.6 mg, 0.247 mmol) in
dichloromethane (1.0 mL) was added trifluoroacetic acid (0.2 mL) at 0 ºC
and the mixture was stirred at ambient temperature for 1 h. Then, the
mixture was concentrated under reduced pressure and the excess
trifluoroacetic acid was removed by repeated evaporation with toluene.
The residue was dissolved in acetone (1.0 mL) and 2 M aqueous sodium
hydroxide (1.0 mL), and to this mixture was added cyclohexanecarbonyl
chloride (68 µL, 0.50 mmol) at ambient temperature. After stirring for 6 h,
the mixture was poured into saturated aqueous sodium hydrogen
carbonate (10 mL) and extracted with dichloromethane (10 mL × 3). The
combined organic layer was washed with brine (10 mL), dried over
sodium sulfate and concentrated under reduced pressure. Column
chromatography (silica-gel 3 g, n-hexane/ethyl acetate = 1/3 to 1/4) gave
the title compound (45.1 mg, 0.192 mmol, 77.7% for 2 steps) as a brown
oil. 1H NMR (CDCl3, 400 MHz): The conformer ratio was 56:44
determined by comparing the relative values of integration for the peaks
observed at 4.86 ppm and 4.69 ppm (the bridgehead protons). δ 0.60 (dd,
J= 11.2, 4.5 Hz, 0.56H), 0.78 (dd, J = 11.2, 4.5 Hz, 0.44H), 1.14–1.42 (m,
4H), 1.42–1.57 (m, 1H), 1.59–1.71 (m, 3H), 1.71–1.82 (m, 2H), 1.98–2.10
(m, 1H), 2.16–2.31 (m, 1H), 2.42–2.55 (m, 1H), 2.81 (br s, 0.56H), 2.97
(br s, 0.44H), 3.09–3.23 (m, 1H), 3.46–3.54 (m, 0.44H), 3.57 (dd, J =
10.6, 5.6 Hz, 0.56H), 4.69 (br s, 0.44H), 4.86 (br s, 0.56H), 5.01 (br s,
0.56H), 5.18 (br s, 0.44H), 6.28 (dd, J = 5.8, 1.8 Hz, 0.56H), 6.32–6.39
(m, 0.88H), 6.42 (dd, J = 5.8, 2.1 Hz, 0.56H). 13C NMR (CDCl3, 100
MHz): δ 25.64 (0.56C), 25.67 (0.44C), 25.72 (0.44C), 25.76, 25.79
(0.56C), 26.9 (0.56C), 28.8 (0.44C), 28.9 (0.56C), 29.0, 29.9 (0.44C),
40.0 (0.44C), 41.7 (0.56C), 41.8 (0.44C), 42.3 (0.56C), 56.7 (0.56C),
58.2 (0.44C), 59.3 (0.44C), 60.6 (0.56C), 64.6 (0.56C), 64.7 (0.44C),
131.4 (0.56C), 133.5 (0.44C), 135.3 (0.44C), 137.5 (0.56C), 171.7
(0.44C), 172.0 (0.56C). HR-MS (ESI): Calcd for C14H21NNaO2 [M+H]+:
258.1470. Found: 258.1476. IR (neat): 3398, 2930, 2855, 1620, 1450,
(5-endo-(Hydroxymethyl)-7-azabicyclo[2.2.1]hept-2-en-7-yl)(4-
methoxyphenyl)methanone (27): To a solution of 7 (33.0 mg, 0.147
mmol) in dichloromethane (0.5 mL) was added trifluoroacetic acid (0.1
mL) at 0 ºC and the mixture was stirred at ambient temperature for 1 h.
Then the mixture was concentrated under reduced pressure and the
excess trifluoroacetic acid was removed by repeated evaporation with
toluene. The residue was dissolved in acetone (0.5 mL) and 2 M
aqueous sodium hydroxide (0.5 mL), and to this mixture was added 4-
methoxybenzoyl chloride (56.0 mg, 0.328 mmol) at ambient temperature.
After stirring for 22 hours, the mixture was poured into saturated aqueous
sodium hydrogen carbonate (10 mL) and extracted with dichloromethane
(10 mL × 3). The combined organic layer was washed with brine (5 mL),
dried over sodium sulfate, and concentrated under reduced pressure.
Column chromatography (silica-gel 2 g, n-hexane/ethyl acetate = 1/3 to
1/4) gave the title compound (33.5 mg, 0.129 mmol, 88.2% for 2 steps)
as a colorless oil. 1H NMR (CDCl3, 400 MHz): The conformer ratio was
58:42 determined by comparing the relative values of integration for the
peaks observed at 4.80 ppm and 4.66 ppm (the bridgehead protons). δ
0.62–0.78 (m, 1H), 1.96–2.40 (m, 2H), 2.48 (br s, 0.58H), 2.65 (br s,
0.42H), 3.06–3.36 (m, 1H), 3.52 (dd, J = 10.7, 6.1 Hz, 1H), 3.83 (s, 3H),
4.66 (br s, 0.42H), 4.80 (br s, 0.58H), 5.06 (br s, 0.58H), 5.24 (br s,
0.42H), 6.21 (br s, 0.58H), 6.31 (br s, 0.42H), 6.41 (br s, 0.42H), 6.50 (br
s, 0.58H), 6.89 (d, J = 8.6 Hz, 2H), 7.50 (d, J = 8.6 Hz, 2H). 13C NMR
(CDCl3, 100 MHz): δ 26.6 (0.58C), 29.4 (0.42C), 39.6 (0.42C), 41.9
(0.58C), 55.4, 57.8 (0.58C), 59.1 (0.42C), 62.5 (0.42C), 63.8 (0.58C),
64.5, 113.6 (2C), 127.1, 129.9 (2C), 131.3 (0.58C), 133.5 (0.42C), 135.2
(0.42C), 137.4 (0.58C), 161.6, 167.9 (0.42C), 168.2 (0.58C). HR-MS
(ESI): Calcd for C15H17NNaO3 [M+Na]+: 282.1106. Found: 282.1097. IR
(neat): 3398, 2939, 1607, 1513, 1422, 1256, 1174, 1030, 845 cm–1
.
1214, 1048 cm–1
.
(5-endo-(Hydroxymethyl)-7-azabicyclo[2.2.1]hept-2-en-7-yl)(4-
nitrophenyl)methanone (28): To a solution of 7 (51.0 mg, 0.226 mmol)
in dichloromethane (1.0 mL) was added trifluoroacetic acid (0.2 mL) at 0
ºC and the mixture was stirred at ambient temperature for 1 h. Then the
mixture was concentrated under reduced pressure and the excess
trifluoroacetic acid was removed by repeated evaporation with toluene.
The residue was dissolved in acetone (1.0 mL) and 2 M aqueous sodium
hydroxide (1.0 mL), and to this mixture was added 4-nitrobenzoyl
chloride (202 mg, 1.09 mmol) at ambient temperature. After stirring for 2
days, the mixture was poured into saturated aqueous sodium hydrogen
carbonate (10 mL) and extracted with dichloromethane (10 mL × 3). The
combined organic layer was washed with brine (10 mL), dried over
sodium sulfate, and concentrated under reduced pressure. Column
chromatography (silica-gel 2 g, n-hexane/ethyl acetate = 1/3 to 1/4) gave
the title compound (44.8 mg, 0.163 mmol, 72.1% for 2 steps) as a brown
amorphous. 1H NMR (CDCl3, 400 MHz): The conformer ratio was 56:44
determined by comparing the relative values of integration for the peaks
observed at 4.67 ppm and 4.52 ppm (the bridgehead protons). δ 0.74 (dd,
J = 11.6, 4.1 Hz, 0.56H), 0.80 (dd, J = 11.7, 4.0 Hz, 0.44H), 1.96 (br s,
0.56H), 2.05–2.13 (m, 0.44H), 2.17–2.29 (m, 1H), 2.46–2.56 (m, 0.56H),
1-(5-endo-(Hydroxymethyl)-7-azabicyclo[2.2.1]hept-2-en-7-yl)ethan-
1-one (30): To a solution of 7 (227 mg, 1.01 mmol) in dichloromethane
(3.0 mL) was added trifluoroacetic acid (0.6 mL) at 0 ºC and the mixture
was stirred at ambient temperature for 1 h. Then the mixture was
concentrated under reduced pressure and the excess trifluoroacetic acid
was removed by repeated evaporation with toluene. To the residue was
added 2 M hydrogen chloride in diethyl ether (3 mL), and the mixture was
concentrated under reduced pressure. This process was repeated further
four times. To a solution of the residue in pyridine (6 mL) was added
acetic anhydride (284 µL, 3.00 mmol) at ambient temperature and the
mixture was stirred at this temperature for 7 h. Then the mixture was
concentrated under reduced pressure and the pyridine was removed by
repeated evaporation with toluene. To the residue were added methanol
(5 mL) and potassium methoxide (379 mg, 5.41 mmol), and the mixture
was stirred at ambient temperature for 1 h. Then SO3H silica gel (4.9 g)
was added and the mixture was filtered through a pad of Celite. The pad
was washed with methanol (40 mL) and the filtrate was concentrated
under reduced pressure. Column chromatography (silica-gel 17 g,
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201700411
European Journal of Organic Chemistry
FULL PAPER
chloroform/methanol = 100/0 to 100/4) gave the title compound (140 mg,
0.835 mmol, 82.9% for 4 steps) as a colorless oil. 1H NMR (CDCl3, 400
MHz): The conformer ratio was 55:45 determined by comparing the
relative values of integration for the peaks observed at 4.78 ppm and
4.60 ppm (the bridgehead protons). δ 0.62 (dd, J = 11.5, 4.2 Hz, 0.55H),
0.78 (dd, J = 11.6, 4.2 Hz, 0.45H), 1.93 (s, 1.35H), 1.95 (s, 1.65H), 1.97–
2.12 (m, 1H), 2.42–2.52 (m, 1H), 3.13 (dd, J = 10.5, 10.5 Hz, 0.55H),
3.18 (dd, J = 10.8, 10.8 Hz, 0.45H), 3.19 (br s, 1H), 3.50 (dd, J = 11.0,
6.2 Hz, 0.45H), 3.57 (dd, J = 10.7, 5.7 Hz, 0.55H), 4.59–4.62 (m, 0.45H),
4.76–4.79 (m, 0.55H), 4.98–5.01 (m, 0.55H), 5.15–5.18 (m, 0.45H), 6.29
(dd, J = 5.9, 2.2 Hz, 0.55H), 6.32–6.38 (m, 0.90H), 6.41 (dd, J = 5.9, 2.4
Hz, 0.55H). 13C NMR (CDCl3, 100 MHz): δ 20.9, 27.1 (0.55C), 29.5
(0.45C), 40.1 (0.45C), 42.1 (0.55C), 56.8 (0.55C), 58.3 (0.45C), 60.2
(0.45C), 61.5 (0.55C), 64.3 (0.55C), 64.4 (0.45C), 131.5 (0.55C), 133.2
(0.45C), 135.2 (0.45C), 136.9 (0.55C), 166.1 (0.45C), 166.4 (0.55C). HR-
MS (ESI): Calcd for C9H13NNaO2 [M+H]+: 190.0844. Found: 190.0851. IR
ambient temperature for 1 h. Then, the mixture was concentrated under
reduced pressure and the excess trifluoroacetic acid was removed by
repeated evaporation with toluene to provide an ammonium salt. In
another flask, to a mixture of N-(tert-butoxycarbonyl)glycine (528 mg,
3.01 mmol) and triethylamine (840 µL, 6.06 mmol) in tetrahydrofuran (60
mL) was added isobutyl chloroformate (338 µL, 2.60 mmol) at 0 ºC and
the mixture was stirred at this temperature for 10 minutes. Then, a
solution of the crude ammonium salt in tetrahydrofuran (30 mL) was
added at 0 ºC and the mixture was stirred at ambient temperature for 19
h. Then water (100 mL) was added and the mixture was extracted with
ethyl acetate (50 mL × 3). The combined organic layer was washed with
saturated aqueous sodium hydrogen carbonate (30 mL × 3) and brine
(30 mL), dried over sodium sulfate, and concentrated under reduced
pressure. To the residue were added methanol (10 mL) and potassium
methoxide (157 mg, 2.24 mmol), and the mixture was stirred at ambient
temperature for 20 min. Then the mixture was poured into saturated
aqueous ammonium chloride (30 mL) and extracted with ethyl acetate
(40 mL × 3). The combined organic layer was washed with brine (20 mL),
dried over sodium sulfate, and concentrated under reduced pressure.
Column chromatography (silica-gel 14 g, n-hexane/ethyl acetate = 1/3 to
1/5) gave the title compound (484 mg, 1.71 mmol, 84.4% for 4 steps) as
a pale brown amorphous. 1H NMR (CDCl3, 400 MHz): The conformer
ratio was 55:45 determined by comparing the relative values of
integration for the peaks observed at 4.80 ppm and 4.64 ppm (the
bridgehead protons). δ 0.66 (dd, J = 11.6, 4.2 Hz, 0.55H), 0.79 (dd, J =
11.7, 4.2 Hz, 0.45H), 1.44 (s, 9H), 2.00–2.13 (m, 1H), 2.42–2.56 (m, 1H),
3.15 (dd, J = 10.7, 10.5 Hz, 0.55H), 3.21 (dd, J = 10.5, 10.2 Hz, 0.45H),
3.47–3.64 (m, 1H), 3.64–3.80 (m, 1H), 3.80–3.98 (m, 1H), 4.64 (br s,
0.45H), 4.80 (br s, 0.55H), 5.04 (br s, 0.55H), 5.19 (br s, 0.45H), 5.32 (br
s, 1H), 6.27 (dd, J = 5.9, 1.9 Hz, 0.5H), 6.30–6.40 (m, 1H), 6.43 (dd, J =
5.9, 2.0 Hz, 0.5H), a proton (OH) was not observed. 13C NMR (CDCl3,
100 MHz): δ 27.1 (0.55C), 28.4 (3C), 29.4 (0.45C), 40.0 (0.55C), 42.1
(0.45C), 42.2, 57.5 (0.55C), 58.6 (0.45C), 58.8 (0.45C), 59.9 (0.55C),
64.5 (0.55C), 64.7 (0.45C), 79.8, 131.4 (0.55C), 133.2 (0.45C), 135.4
(0.55C), 137.2 (0.45C), 155.9, 164.3 (0.45C), 164.4 (0.55C). HR-MS
(ESI): Calcd for C14H22N2NaO4 [M+Na]+: 305.1477. Found: 305.1476. IR
(neat): 3389, 2944, 1633, 1454, 1145, 1037, 995, 919, 849, 793 cm–1
.
2,2,2-Trichloro-1-(5-endo-(hydroxymethyl)-7-azabicyclo[2.2.1]hept-2-
en-7-yl)ethan-1-one (31): A solution of 7 (92.7 mg, 0.412 mmol) and
acetic anhydride (57 µL, 0.60 mmol) in pyridine (2 mL) was stirred at
ambient temperature for 13 h. Then the mixture was concentrated under
reduced pressure and the residue was taken up in dichloromethane (30
mL). The organic layer was washed with 1 M hydrochloric acid (10 mL ×
4) and brine (10 mL), dried over sodium sulfate, and concentrated under
reduced pressure. To a solution of the residue in dichloromethane (1.5
mL) was added trifluoroacetic acid (0.3 mL) at 0 ºC and the mixture was
stirred at ambient temperature for
1 h. Then the mixture was
concentrated under reduced pressure and the excess trifluoroacetic acid
was removed by repeated evaporation with toluene. To a solution of the
residue in pyridine (2 mL) was added trichloroacetic anhydride (85 µL,
0.47 mmol) at ambient temperature and the mixture was stirred at this
temperature for 9 h. Then, the mixture was concentrated under reduced
pressure and the pyridine was removed by repeated evaporation with
toluene. To the residue was added methanol (2 mL) and potassium
methoxide (141 mg, 2.01 mmol) and the mixture was stirred at ambient
temperature for 20 min. Then SO3H silica gel (1.5 g) was added and the
mixture was filtered through a pad of Celite. The pad was washed with
methanol (20 mL) and the filtrate was concentrated under reduced
pressure. Column chromatography (silica-gel 6 g, n-hexane/ethyl acetate
= 2/1 to 1/1) gave the title compound (30.9 mg, 0.114 mmol, 27.8% for 4
steps) as a pale brown oil. 1H NMR (CDCl3, 400 MHz): The conformer
ratio was ca. 1:1, which was difficult to be determined from the NMR due
to the broadening of the signals. δ 0.69–0.82 (m, 1H), 1.71 (br s, 1H),
2.16 (br s, 0.5H), 2.31 (br s, 0.5H), 2.59 (br s, 0.5H), 2.76 (br s, 0.5H),
3.17–3.31 (m, 1H), 3.54–3.64 (m, 1H), 5.08 (br s, 0.5H), 5.22 (br s, 0.5H),
5.29 (br s, 0.5H), 5.43 (br s, 0.5H), 6.30–6.54 (m, 2H). 13C NMR (CDCl3,
100 MHz): δ 26.9 (0.5C), 28.5 (0.5C), 39.6 (0.5C), 40.7 (0.5C), 59.9
(0.5C), 61.2 (0.5C), 62.1 (0.5C), 63.3 (0.5C), 64.3, 92.9, 131.3 (0.5C),
132.5 (0.5C), 135.1 (0.5C), 136.6 (0.5C), 154.97 (0.5C), 155.04 (0.5C).
HR-MS (ESI): Calcd for C9H1035Cl237ClNNaO2 [M+Na]+: 293.9645. Found:
293.9652. IR (neat): 3434, 2946, 1681, 1423, 1036, 862, 836, 809, 714
(neat): 3399, 2977, 1705, 1646, 1454, 1367, 1251, 1168, 1050, 865 cm–1
.
1-(3',6'-Dihydroxy-3-oxo-1H-spiro[isobenzofuran-1,9'-xanthen]-5-
endo-yl)-3-(2-(5-(hydroxymethyl)-7-azabicyclo[2.2.1]hept-2-en-7-yl)-
2-oxoethyl)thiourea (33): To a solution of 32 (228 mg, 0.809 mmol) in
dichloromethane (5 mL) was added trifluoroacetic acid (1 mL) at 0 ºC and
the mixture was stirred at ambient temperature for 1.5 h. Then, the
mixture was concentrated under reduced pressure and the excess
trifluoroacetic acid was removed by repeated evaporation with toluene.
To a solution of the residue and triethylamine (333 µL, 2.40 mmol) in
methanol (5 mL) was added fluorescein 5-isothiocyanate (315 mg, 0.809
mmol) at ambient temperature and the mixture was stirred at this
temperature for 24 h with protection from light. Then, the mixture was
concentrated under reduced pressure. Column chromatography (CO2H
silica-gel 15 g, chloroform/methanol = 100/3 to 10/1) gave the title
compound (1/3 triethylammonium salt, 126 mg, 0.208 mmol, 25.7% for 2
steps) as an orange solid. 1H NMR (dimethyl sulfoxide-d6, 400 MHz): The
conformer ratio was 51:49 determined by comparing the relative values
of integration for the peaks observed at 0.59 ppm and 0.65 ppm. δ 0.59
(dd, J = 11.5, 4.1 Hz, 0.51H), 0.65 (dd, J = 11.6, 4.1 Hz, 0.49H), 0.97 (t, J
= 7.1 Hz, 3H, triethylamine), 1.83–1.92 (m, 0.5H) 2.04–2.14 (m, 0.5H),
2.22–2.32 (m, 0.5H), 2.40–2.58 (m, 0.5H), 2.53 (q, J = 7.1 Hz, 2H,
triethylamine), 2.88–3.01 (m, 1H), 3.20–3.50 (m, 1H), 4.13–4.23 (m, 1H),
4.26–4.41 (m, 1H), 4.63 (br s, 0.5H), 4.67 (br s, 0.5H), 4.86 (br s, 1.5H),
4.93 (br s, 0.5H), 6.27–6.34 (m, 1H), 6.45 (dd, J = 5.9, 2.5 Hz, 1H), 6.53–
6.65 (m, 4H), 6.67 (d, J = 2.3 Hz, 2H), 7.19 (d, J = 8.3 Hz, 1H), 7.76 (d, J
= 8.4 Hz, 1H), 8.08 (br s, 1H), 8.35 (s, 1H), 10.1 (br s, 2H), 10.3 (br s,
1H). 13C NMR (dimethyl sulfoxide-d6, 100 MHz): δ 18.6 (triethylamine),
cm–1
.
tert-Butyl (2-(5-endo-(hydroxymethyl)-7-azabicyclo[2.2.1]hept-2-en-
7-yl)-2-oxoethyl)carbamate (32): A solution of 7 (457 mg, 2.03 mmol),
acetic anhydride (284 µL, 3.00 mmol) and N,N-dimethyl-4-aminopyridine
(2.2 mg, 18 µmol) in pyridine (5 mL) was stirred at ambient temperature
for 40 min. Then, the mixture was concentrated under reduced pressure
and the residue was taken up in dichloromethane (50 mL). The organic
layer was washed with 0.5 M hydrochloric acid (20 mL × 5) and brine (20
mL), dried over sodium sulfate, and concentrated under reduced
pressure. To a solution of the residue in dichloromethane (10 mL) was
added trifluoroacetic acid (2 mL) at 0 ºC and the mixture was stirred at
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201700411
European Journal of Organic Chemistry
FULL PAPER
27.1 (0.5C), 28.9 (0.5C), 41.8, 45.7, 56.0 (triethylamine), 57.3 (0.5C),
58.3 (0.5C), 58.7 (0.5C), 59.7 (0.5C), 63.0, 102.2 (2C), 109.7 (2C), 112.6
(2C), 116.4, 124.2, 126.7, 129.1 (2C), 129.3, 132.0 (0.5C), 132.2 (0.5C),
136.0 (0.5C), 136.3 (0.5C), 141.2, 147.2, 151.9 (2C), 159.6 (2C), 164.0,
168.5 (2C), 180.3 (0.5C), 180.4 (0.5C). HR-MS (ESI): Calcd for
C30H24N3O7S [M]–: 570.1335. Found: 570.1362. IR (KBr): 3855, 3753,
dimethylformamide (1 mL) was stirred at ambient temperature for 15 h.
Then, the mixture was concentrated under reduced pressure and N,N-
dimethylformamide was removed by repeated evaporation with toluene.
Recrystallization from boiling methanol gave the title compound (11.3 mg,
21.4 µmol, 40.8%) as a pink solid. 1H NMR (dimethyl sulfoxide-d6, 400
MHz): δ 1.21–1.39 (m, 2H), 1.40–1.69 (m, 6H), 2.06 (t, J = 7.4 Hz, 2H),
2.57 (d, J = 12.4 Hz, 1H), 2.81 (dd, J = 12.4, 5.1 Hz, 1H), 2.99 (s, 3H),
2.98–3.13 (m, 5H), 4.10–4.15 (m, 1H), 4.27–4.35 (m, 1H), 4.33 (d, J =
6.0 Hz, 2H), 6.05 (t, J = 5.7 Hz, 1H), 6.34 (s, 1H), 6.41 (s, 1H), 6.54 (t, J
= 6.1 Hz, 1H), 7.52 (d, J = 8.4 Hz, 2H), 7.76 (t, J = 5.6 Hz, 1H), 8.42 (d, J
= 8.4 Hz, 2H). 13C NMR (dimethyl sulfoxide-d6, 100 MHz): δ 20.8, 25.3,
28.0, 28.2, 30.2, 35.2, 36.1, 37.1, 39.8, 42.7, 55.4, 59.2, 61.0, 127.4 (2C),
127.8 (2C), 130.1, 146.0, 158.1, 162.7, 163.2, 167.0, 172.0. HR-MS
(ESI): Calcd for C24H33N9NaO3S [M+Na]+: 550.2325. Found: 550.2305.
IR (KBr): 3292, 2929, 1703, 1631, 1573, 1405, 1364, 1266, 890, 796,
3651, 3247, 1736, 1609, 1459, 1176, 1110, 848 cm–1
.
tert-Butyl (3-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-
d]imidazol-4-yl)pentanamido)propyl)carbamate (35): To a mixture of
biotin (34) (217 mg, 0.887 mmol), tert-butyl N-(3-aminopropyl)carbamate
(154 mg, 0.886 mmol) and N,N-dimethyl-4-aminopyridine (13.1 mg,
0.107 mmol) in N,N-dimethylformamide (8 mL) was added 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (215 mg, 1.12
mmol) at ambient temperature and the mixture was stirred at this
temperature for 27 h. Then, the mixture was concentrated under reduced
pressure and N,N-dimethylformamide was removed by repeated
evaporation with toluene. Column chromatography (silica-gel 20 g,
chloroform/methanol = 20/1 to 12/1) gave the title compound (261 mg,
561 cm–1
.
Determination of the half-lives of 7-azabicyclo[2.2.1]heptene
derivatives: The compound (ca. 3 mg) and 1,1,2,2-tetrachloroethane (as
an internal standard, ca. 5 mg) were dissolved in chloroform-d (pre-
passed through a column of alumina), and immediately after that, the 1H
NMR spectrum was recorded. 1H NMR spectra were repeatedly recorded
over the interval of several hours or days and the ratio of the remaining
starting material was determined by comparing the relative values of
integration for the peaks corresponding to the bridgehead protons and
the peak at 5.96 ppm (1,1,2,2-tetrachloroethane). The half-lives were
determined from the following equation: t1/2 = 0.693/λ where λ is the
decay constant. For free amines, the trifluoroacetic acid salt, 1,1,2,2-
tetrachloroethane, and a large excess amount of triethylamine were
dissolved and the half-lives were determined as for the others.
1
0.652 mmol, 73.6%) as a colorless solid. H NMR (CD3OD, 400 MHz): δ
1.39–1.49 (m, 2H), 1.43 (s, 9H), 1.54–1.79 (m, 6H), 2.20 (t, J = 7.4 Hz,
2H), 2.70 (d, J = 12.7 Hz, 1H), 2.92 (dd, J = 12.8, 5.0 Hz, 1H), 3.06 (t, J =
6.8 Hz, 2H), 3.17–3.24 (m, 1H), 3.19 (t, J = 6.9 Hz, 2H), 4.30 (dd, J = 7.9,
4.5 Hz, 1H), 4.49 (dd, J = 7.8, 4.3 Hz, 1H), four protons (NH×4) were not
observed. 13C NMR (CD3OD, 100 MHz): δ 26.8, 28.8 (3C), 29.5, 29.8,
30.7, 36.8, 37.7, 38.7, 41.0, 57.0, 61.6, 63.4, 80.0, 158.5, 166.1, 176.1.
HR-MS (ESI): Calcd for C18H32N4NaO4S [M+Na]+: 423.2042. Found:
423.2038. IR (KBr): 3303, 2930, 1703, 1646, 1527, 1460, 1366, 1251,
1172, 727 cm–1
.
N-(4-(6-Methyl-1,2,4,5-tetrazin-3-yl)benzyl)-1H-imidazole-1-
carboxamide (38): Tetrazine 37 was prepared according to the literature
Determination of the rate constants: The rate constants k were
procedure.[34] To
a
solution of 37 (152 mg, 0.505 mmol) in
measured under pseudo first-order conditions using
concentration of 3,6-di(4-pyridyl)-1,2,4,5-tetrazine and an excess amount
of 7-azanorbornene (ANor) derivatives. mL solution of ANor
a constant
dichloromethane (5 mL) was added trifluoroacetic acid (1 mL) at 0 ºC and
the mixture was stirred at ambient temperature for 1 h. Then, the mixture
was concentrated under reduced pressure and the excess trifluoroacetic
acid was removed by repeated evaporation with toluene. To a mixture of
the residue and triethylamine (208 µL, 1.50 mmol) in dichloromethane (5
mL) was added carbonyldiimidazole (105 mg, 0.646 mmol) at 0 ºC and
the mixture was stirred at ambient temperature for 20 h. Then the mixture
was diluted with dichloromethane (60 mL). The mixture was washed with
saturated aqueous sodium hydrogen carbonate (20 mL) and brine (20
mL), dried over sodium sulfate, and concentrated under reduced
pressure. Column chromatography (silica-gel 5 g, n-hexane/ethyl acetate
= 1/3 to 1/5) gave the title compound (108 mg, 0.367 mmol, 72.6% for 2
steps) as a pink solid. 1H NMR (CD3OD, 400 MHz): δ 3.03 (s, 3H), 4.67
(s, 2H), 7.05 (s, 1H), 7.08 (dd, J = 1.6, 1.0 Hz, 1H), 7.63 (d, J = 8.6 Hz,
2H), 7.67 (dd, J = 1.6, 1.4 Hz, 1H), 8.31 (dd, J = 1.4, 1.0 Hz, 1H), 8.55 (d,
J = 8.6 Hz, 2H). 13C NMR (CD3OD, 100 MHz): δ 20.8, 43.3, 116.6, 127.6
(2C), 128.2 (2C), 129.7, 130.8, 136.0, 143.2, 149.1, 163.1, 167.1. HR-MS
(ESI): Calcd for C14H13N7NaO [M+Na]+: 318.1079. Found: 318.1073. IR
(KBr): 3222, 1720, 1542, 1406, 1362, 1282, 1246, 1039, 890, 653, 566
A
1
derivatives (0.313, 0.625, 1.25, 2.5 or 5 mM in water/methanol = 95/5, or
1.25, 2.5, 5, 10 or 20 mM in acetonitrile) and a 1 mL solution of 3,6-di(4-
pyridyl)-1,2,4,5-tetrazine (0.05 mM in water/methanol = 95/5, or 0.1 mM
in acetonitrile) were mixed in an UV-cuvette and immediately inserted in
HITACHI U-2800 spectrophotometer. The decay in the absorption of the
tetrazine at the indicated wavelengths were observed over 60 min at 23
ºC. The data were fitted to a single exponential equation. The observed
rate constants k' were plotted against the concentration of the ANors, and
the second-order constants k were obtained from the slope of the plots.
Each measurement was repeated three times and the mean and the
standard deviation of the rate constant k were determined. Typical time-
course decrease of the absorbance and the plots of the rate constants k'
against the concentration of the ANors are shown in Figure S1. The
formation of the 1,4-dihydropyridazine products was verified by HR-MS
analysis of the reaction mixtures, and this is shown in Table S1.
Conjugation of FITC-ANor 33 and tetrazine-biotin 39 in protein-
containing buffers: Streptavidin and Immulon® 2HB Flat Bottom 96-well
Microtiter Plates were purchased from Wako Pure Chemical Industries
Ltd. (Cat. No. 192-17864) and Thermo Scientific (Cat. No. 3455),
respectively. A 10 µg/mL solution of streptavidin in coating buffer (15 mM
sodium carbonate, 35 mM sodium hydrogen carbonate and 3 mM sodium
azide) was prepared and this was placed in the wells of the plates. The
plates were allowed to stand overnight at 4 ºC. After washing with 0.1%
Tween-20 in TBS (TBST), the plates were blocked with 3% BSA-TBS.
FITC-ANor 33 (250 µM) and tetrazine-biotin 39 (25 µM) were dissolved in
the indicated solutions (2% DMSO was used as co-solvent) and reacted
for 20 h at ambient temperature. Serial dilutions of the conjugate were
prepared and the diluted conjugate solutions were placed on the plates.
cm–1
.
N-(3-(3-(4-(6-Methyl-1,2,4,5-tetrazin-3-yl)benzyl)ureido)propyl)-5-
((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-
yl)pentanamide (39): To a solution of 35 (32.2 mg, 80.4 µmol) in
dichloromethane (0.5 mL) was added trifluoroacetic acid (0.1 mL) at 0 ºC
and the mixture was stirred at ambient temperature for 30 min. Then, the
mixture was concentrated under reduced pressure and the excess
trifluoroacetic acid was removed by repeated evaporation with toluene.
The residual ammonium salt 36 was used for the next reaction without
further purification. A mixture of 38 (15.5 mg, 52.5 µmol), triethylamine
(31 µL, 224 µmmol), and the ammonium salt 36 in N,N-
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10.1002/ejoc.201700411
European Journal of Organic Chemistry
FULL PAPER
After incubation for 2 h, the plates were washed with TBST, and TBS
was placed to the wells. Fluorescence intensity was measured with
FlexStation 3 (Molecular Devices, ex. 492 nm, em. 515 nm). Data points
represent the mean with the range of two samples depicted by error bars
(n = 2) except for distilled water (n = 3, S.D.).
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FITC-ANor 33: A 100 nM solution of tetrazine-biotin 39 in 1% BSA-TBS
was prepared (0.004% DMSO was used as co-solvent) and this was
placed in the wells of streptavidin-conjugated plates prepared as above.
After 2 h, the plates were washed with TBST. A 200 µM solution of FITC-
ANor 33 in 1% BSA-TBS was prepared (0.8% DMSO was used as a co-
solvent) and this was placed in the wells of the plates. After 2 h, the
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as above.
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Ground and transition states were confirmed by the number of imaginary
frequencies. For transition states, the related vibration motion of the
imaginary frequency was also observed to ensure that it corresponds to
N-R bending. Zero-point energy corrections were applied with the
appropriate scaling. The optimized structures and the frontier orbitals are
shown in the Supporting Information.
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Acknowledgements
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We are grateful to Dr. Kenichiro Nagai and Ms. Noriko Sato (the
Institute of Instrumental Analysis of Kitasato University) for help
with structure determination of newly synthesized compounds.
We thank Dr. Yotaro Matsumoto (Graduate School of
Pharmaceutical Sciences, Tohoku University) for helpful advices
in calculations and Dr. Takeshi Kumagai (School of
Pharmaceutical Sciences, Kitasato University) for helpful
discussion in biological experiments. This work was supported
by Grant-in-Aid for Young Scientists (B) (JPSP KAKENHI Grant
Number 16K18912) and Kitasato University Research Grant for
Young Researchers.
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This article is protected by copyright. All rights reserved.

10.1002/ejoc.201700411
European Journal of Organic Chemistry
FULL PAPER
FULL PAPER
Reactivity of 7-Azanorbornene
in Tetrazine Click Reaction
Key Topic Click chemistry, inverse
electron-demand Diels–Alder reaction
The reactivities of variously N-substituted
7-azanorbornenes in inverse electron-
demand Diels–Alder reactions with
tetrazines were explored. The rates of N-
acylated derivatives were superior to other
derivatives with stronger electron-donating
substituents. This outcome may be
attributed to the sp2 characters of the
nitrogen atoms and lower flipping energy of
the N-substituents. The reaction was
bioorthogonal, and 7-azanorbornenes
would be suitable for conjugation under
physiological conditions.
R
N
R'
R'
N
N
N
N
HN
N
N
R
+
Dr. Fumika Karaki*, Dr. Kenji Ohgane,
Prof. Dr. Hirotaka Imai, Dr. Kennosuke
Itoh, Prof. Dr. Hideaki Fujii
N
R
R'
R'
O
S
O
N
O
O
R
R
NHR
>
H2
C
Alkyl
N
N
N
≈
>
>
Page No. – Page No.
R
R
R
R
R
Title
Fast
sp2
Slow
sp3
Reactivity of 7-Azanorbornenes in
Bioorthogonal Inverse Electron-Demand
Diels–Alder Reactions
This article is protected by copyright. All rights reserved.