Synthesis of macrocyclic tetrazoles for rapid photoinduced bioorthogonal 1,3-dipolar cycloaddition reactions

Article abstract of DOI:10.1002/chem.201002360

Quick as a flash! A series of conformationally constrained, macrocyclic tetrazoles were expediently prepared by double alkylation of a bis(o-phenolyl)tetrazole precursor. Several of them showed excellent reactivity toward norbornene in a photoinduced tetrazole-alkene cycloaddition reaction in organic solvent, as well as toward a norbornene-modified protein in phosphate-buffered saline buffer (see scheme). Copyright

Full text of DOI:10.1002/chem.201002360

COMMUNICATION
DOI: 10.1002/chem.201002360
Synthesis of Macrocyclic Tetrazoles for Rapid Photoinduced Bioorthogonal
1,3-Dipolar Cycloaddition Reactions
Zhipeng Yu, Reyna K. V. Lim, and Qing Lin*[a]
There is increasing interest to develop robust bioorthogo-
nal reactions for site-specific modification of biomolecules
within living systems.[1] A prominent example is the copper-
catalyzed azide–alkyne cycloaddition reaction (CuAAC).[2]
Since copper is toxic to cells, there have been substantial ef-
forts directed toward developing copper-free alternatives for
applications in living cells. One strategy involves activation
of dipolarophiles by constraining them in macrocyclic rings.
Several cyclooctynes have been developed for rapid 1,3-di-
polar cycloadditions with azide.[3] However, the use of ring
structures containing strained dipoles for rapid bioorthogo-
nal 1,3-dipolar cycloaddition reactions is extremely rare,[4]
despite the fact that many cyclic dipoles have been used suc-
Scheme 1. Representative cyclic 1,3-dipoles.
cessfully in organic synthesis, for example, cyclic azomethine
imine,[5] cyclic carbonyl ylide,[6] cyclic azomethine ylide,[7]
and cyclic nitrones[8] (Scheme 1). To the best of our knowl-
edge, cyclic nitrile imines—a class of 1,3-dipoles[9] important
for the preparation of pyrazolines—have not been reported
in the literature.
tion, generate a cyclic nitrile imine with an overall reduced
rotational freedom. Herein we report the synthesis of a
series of conformationally constrained macrocyclic tetra-
zoles and the characterization of their reactivity toward
both a terminal alkene and a strained alkene in organic sol-
vents as well as a norbornene-modified lysozyme in phos-
phate buffered saline (PBS).
To create the attachment sites for a chemical “bridge”, we
synthesized two linear tetrazoles, 2,5-bis(o-phenolyl)tetra-
zole (1) and 2-o-phenolyl-5-o-anilinyltetrazole (7) using the
Kakehi method.[14] For macrocyclization by bis-alkylation,
we treated tetrazole 1 with dibromoalkanes (1.1 equiv) with
variable lengths of the carbon chain and screened four alkali
metal bases, LiOH, Na2CO3, K2CO3, and Cs2CO3, anticipat-
ing that alkali metal ions with the right size may facilitate
ring closure by bringing the two phenol O atoms to the N1
side of tetrazole 1 together through chelation (Table 1).
Gratifyingly, we found that bases with larger alkali metals,
such as Cs+ and K+, in general afforded higher yields of the
desired macrocyclic products (Table 1, entries 7, 8, 11, 12,
15, 16, 19, and 20); tetrazole 2 (Table 1, entries 3 and 4) had
the lowest yields, presumably due to the small ring size (12-
membered ring). Li+ and Na+ bases generally produced
only trace amounts of the desired macrocyclic products to-
We recently reported the use of diaryltetrazoles as photo-
activatable precursors to the reactive nitrile imine dipoles
for photoinduced, bioorthogonal cycloaddition reactions
with both electron-deficient alkenes[10] and unactivated ter-
minal alkenes.[11] In a photocrystallographic study, we found
that a bent nitrile imine was generated in situ upon photoir-
radiation of a diaryltetrazole.[12] The bent geometry is postu-
lated to exhibit reduced distortion energy and thus higher
reactivity based on recent computational studies.[13] To rein-
force the nitrile imine geometry in this reactive conforma-
tion, we hypothesized that inserting a short bridge between
the ortho positions of the two flanking phenyl rings to form
a macrocyclic diphenyltetrazole should, upon photoirradia-
[a] Dr. Z. Yu, R. K. V. Lim, Prof. Dr. Q. Lin
Department of Chemistry
State University of New York at Buffalo
Buffalo, NY 14260 (USA)
Fax : (+1)716-645-6963
E-mail: qinglin@buffalo.edu
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201002360.
Chem. Eur. J. 2010, 16, 13325 – 13329
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Table 1. Synthesis of the macrocyclic tetrazoles.[a]
razole 1 than with the smaller ions. We then designed
tetrazole 9 (Table 1), suspecting that the amido-H may
form an internal hydrogen bond with tetrazole-N1,
which would reinforce the bent nitrile imine geometry.
Thus, tetrazole 9 was readily prepared through amida-
tion of tetrazole 7 with 4-chlorobutanoyl chloride fol-
lowed by an intramolecular O-alkylation. Notably,
Na2CO3 was found to be the optimal base for the ring-
closure reaction (compare entries 21–24 in Table 1).
The structures of macrocyclic tetrazoles 6 and 9 were con-
firmed by X-ray crystallography (Figure 1). To our surprise,
the amide bond in 9 was engaged in a pair of intermolecular
À
hydrogen bonds between N5 H and N1 of the neighboring
tetrazole instead of internal interactions. The three conjoin-
ing aromatic rings are slightly twisted out of the tetrazole
plane with the torsional angles N1-N2-C2-C3 and N1-C1-
C8-C13 of 45.12 and À44.388, respectively (for tetrazole 6),
Entry
Base
Product
Yield [%][b]
1
2
3
4
LiOH·H2O
Na2CO3
K2CO3
2
2
2
2
trace
<5[c]
<30[c]
38
Cs2CO3
5
6
7
8
LiOH·H2O
Na2CO3
K2CO3
3
3
3
3
trace
trace
51
Cs2CO3
78
9
10
11
12
LiOH·H2O
Na2CO3
K2CO3
4
4
4
4
trace
trace
<35[c]
45
Cs2CO3
13
14
15
16
LiOH·H2O
Na2CO3
K2CO3
5
5
5
5
trace
trace
<60[c]
67
Cs2CO3
17
18
19
20
LiOH·H2O
Na2CO3
K2CO3
6
6
6
6
trace
trace
<50[c]
61
Cs2CO3
21
22
23
24
LiOH·H2O
Na2CO3
K2CO3
9
9
9
9
trace
70
<20[c]
trace
Cs2CO3
[a] For bis-O-alkylation, reactions were carried out by heating tetrazole 1
at reflux with dibromide (1.1 equiv) and base (3 equiv) in acetone over-
night. For intramolecular O-alkylation, tetrazole 8 was heated at reflux in
acetonitrile with base (3 equiv). [b] Isolated yields were reported unless
noted otherwise. [c] Estimated yields based on TLC.
gether with large amounts of intractable oligomers. This
metal ion size dependency suggests that the larger ions, Cs+
and K+, may form more stable, tridentate chelates with tet-
Figure 1. ORTEP diagrams of macrocyclic tetrazole 6 (top) and the
dimer of macrocyclic tetrazole 9 (bottom) shown at 50% probability.
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Cycloaddition Reactions of Macrocyclic Tetrazoles
COMMUNICATION
Table 2. Photoinduced 1,3-dipolar cycloaddition reactions of macrocyclic and acylic tetrazoles with alkenes.[a]
and À62.46 and 37.178, respec-
tively (for tetrazole 9). These
distortions might be due to
lone-pair repulsions between
N1 and O1 (distance: 2.815 ꢁ
for 6, 2.848 ꢁ for 9) and be-
tween N1 and O2 (distance:
2.857 ꢁ for 6, 3.249 ꢁ for 9).
To assess the reactivity of
the macrocyclic tetrazoles, we
carried out photoinduced cy-
cloaddition reactions with
either 4-penten-1-ol, an ana-
logue of
a
bioorthogonal
alkene reporter homoallylgly-
cine,[15] or a strained alkene re-
porter norbornene[16] in ethyl
acetate (Table 2). Because the
reaction proceeds through two
distinct steps, a ring-opening
step to generate the cyclic ni-
trile imine in situ and the sub-
sequent [3+2] cycloaddition
reaction,[10] we monitored the
reactions by thin-layer chro-
matography (TLC) and discon-
tinued the reactions once the
tetrazole starting materials
were consumed. The macrocy-
clic tetrazoles gave, in general,
higher yields of the pyrazoline
cycloadducts (with the excep-
tions of entries 5, 6, and 11 in
Entry
Alkene
Tetrazole
Pyrazoline
Time[b] [min]
Yield[c] [%]
1
2
3
4
5
6
7
a
a
a
a
a
a
a
2
3
4
5
6
9
10
2a
3a
4a
5a
6a
9a
10a
120
75
84
180
120
300
120
59
71
60
70
46[d]
43
58
8
9
b
b
b
b
b
b
b
2
3
4
5
6
9
10
2b
3b
4b
5b
6b
9b
10b
108
54
120
240
90
95
91
81
60
84
84
76
10
11
12
13
14
120
60
[a] Tetrazole (0.1 mmol) and alkene dipolarophile (10 mmol) in EtOAc (250 mL) were irradiated with a
302 nm UV lamp in quartz flask. [b] Time was determined by tracing the disappearance of the starting materi-
als on TLC. [c] Isolated yields. [d] The pyrazoline adduct was unstable upon standing.
Table 2) compared to their acyclic counterparts (Table 2, en-
tries 7 and 14). As expected, norbornene serves as a more
reactive dipolarophile than 4-penten-1-ol, affording the cor-
responding pyrazolines in higher yields (compare entries 8–
14 with 1–7, Table 2). Among the macrocyclic tetrazoles, the
three- and four-carbon-linked tetrazoles 2 and 3 showed
markedly higher reactivity with shorter reaction times and
higher yields (Table 2, entries 2, 8, and 9), presumably due
to the smaller ring sizes and thus reduced conformational
flexibility. Interestingly, the O-xylylene-linked tetrazole 6
gave a moderate yield with 4-penten-1-ol (Table 2, entry 5),
but a good yield with norbornene (Table 2, entry 12), which
can be attributed to a compact exo transition state (TS) sta-
À
bilized by the attractive edge-to-face C H···p interaction be-
À
tween the norbornene bridge C H and the xylyl p elec-
trons.[17] Gratifyingly, the X-ray structure of pyrazoline 3b
was obtained, showing that the two phenyl rings are almost
coplanar with the pyrazoline ring with torsional angles N1-
N2-C15-C16 and N1-C1-C9-C14 of 30.8 and À26.68, respec-
tively (Figure 2).
Figure 2. ORTEP diagram of the macrocyclic pyrazoline 3b shown at
50% probability.
The excellent reactivity of the macrocyclic tetrazoles
toward norbornene prompted us to measure the photophysi-
cal properties of the resulting macrocyclic pyrazolines
(Table 3). Four macrocyclic pyrazolines showed bathochro-
mic shifts in labs (Table 3, entries 1, 2, 5, and 6) relative to
the pyrazoline control 10b (Table 3, entry 7), while two pyr-
azolines showed the hypsochromic shifts (Table 3, entries 3
Chem. Eur. J. 2010, 16, 13325 – 13329
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Q. Lin et al.
Table 3. Photophysical properties of macrocyclic pyrazolines.[a]
of photoactivatable tetrazole reagents for the bioorthogonal
tetrazole–alkene cycloaddition reaction in living systems.
[c]
Entry
Pyrazoline
labs [nm]
e [mÀ1 cmÀ1
]
lem [nm][b]
FF
1
2
3
4
5
6
7
2b
3b
358
364
348
348
358
376
350
20356
10190
1702
456
466
468
464
451
477
502
0.30
0.35
0.35
0.17
0.32
0.25
0.15
4b[d]
5b[d]
6b
Experimental Section
830
13852
19440
12534
Photoinduced cycloaddition of lyso-norbornene with macrocyclic tetra-
zoles: Various tetrazoles (1 mL; 4 mm in DMSO, to a final concentration
of 200 mm) were added to a 20 mL solution of either lysozyme (lyso) or
lyso-norbornene (10 mm) in PBS buffer, pH 7.4, in a 96-well microtiter
plate. The mixtures were irradiated with a handheld 302 nm UV lamp for
1 min before quenching by addition of 6ꢂSDS (5 mL) sample buffer,
boiled for 5 min at 958C, loaded onto a NuPAGE 12% Bis-Tris gel (Invi-
trogen), and then subjected to protein electrophoresis. The lyso-pyr cy-
cloadducts in the gel were visualized by illuminating the gel with a hand-
held 365 nm UV lamp and the resulting image was captured by a digital
camera. After image acquisition, the same gel was stained with Coomas-
sie blue to confirm the size and equal loading of proteins.
9b
10b
[a] Compounds were dissolved in PBS/CH3CN (1:1) at concentrations of
5 mm unless noted otherwise. [b] lex =348 nm. [c] Quantum yields were
determined by using 4’,6-diamino-2-phenylindole (DAPI) as the standard
(FF =0.58 in DMSO). [d] 20 mm concentrations were used.
and 4), presumably due to their twisted macrocyclic geome-
tries. Compared with 10b, the macrocyclic pyrazolines ex-
hibited invariable hypsochromic shifts in the maximum
emission wavelength (lem) along with increases in the fluo-
rescence quantum yield (FF).
To examine whether the macrocyclic tetrazoles can be
used to label a norbornene-containing protein, we incubated
tetrazoles 2–6 (200 mm) with a norbornene-modified lyso-
zyme (10 mm) in PBS buffer. The mixtures were photoirradi-
ated with a handheld 302 nm UV lamp for 1 min prior to
quenching with sodium dodecyl sulfate (SDS) sample buffer.
In-gel fluorescence analysis (Figure 3) indicated specific pyr-
CCDC-783273 (6), 783275 (9), and 783274 (3b) contains the supplemen-
tary crystallographic data for this paper. These data can be obtained free
of charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
Acknowledgements
We gratefully acknowledge the National Institutes of Health (GM 85092)
for financial support. We thank William Brennessel at University of Ro-
chester for X-ray structural determination.
Keywords: alkenes
·
bioorthogonal
chemistry
·
biotechnology · cycloaddition · tetrazoles
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Cycloaddition Reactions of Macrocyclic Tetrazoles
COMMUNICATION
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Received: August 17, 2010
Published online: October 28, 2010
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