The Excited-State Triple Proton Transfer Reaction of 2,6-Diazaindoles and 2,6-Diazatryptophan in Aqueous Solution

Article abstract of DOI:10.1021/jacs.7b01672

3-Me-2,6-diazaindole ((2,6-aza)Ind) was strategically designed and synthesized to probe water molecule catalyzed excited-state proton transfer in aqueous solution. Upon electronic excitation (λ_max ～ 300 nm), (2,6-aza)Ind undergoes N(1)-H to N(6) long-distance proton transfer in neutral H₂O, resulting in normal (340 nm) and proton-transfer tautomer (480 nm) emissions with an overall quantum yield of 0.25. The rate of the water-catalyzed proton transfer shows a prominent H/D kinetic isotope effect, which is determined to be 8.3 × 10⁸ s^-1 and 4.7 × 10⁸ s^-1 in H₂O and D₂O, respectively. Proton inventory experiments indicate the involvement of two water molecules and three protons, which undergo a relay type of excited-state triple proton transfer (ESTPT) in a concerted, asynchronous manner. The results demonstrate for the first time the fundamental of triple proton transfer in pure water for azaindoles as well as pave a new avenue for 2,6-diazatryptophan, an analogue of tryptophan exhibiting a similar ESTPT property with (2,6-aza)Ind, to probe biowaters in proteins.

Full text of DOI:10.1021/jacs.7b01672

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)
Article
The Excited-State Triple Proton Transfer Reaction of 2,6-
Diazaindoles and 2,6-Diazatryptophan in Aqueous Solution
Kun-you Chung, Yi-Han Chen, Yi-Ting Chen, Yen-Hao Hsu,
Jiun-Yi Shen, Chi-Lin Chen, Yi-An Chen, and Pi-Tai Chou
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 20 Apr 2017
Downloaded from http://pubs.acs.org on April 20, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of the American Chemical Society is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 9
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
Kun-You Chung, Yi-Han Chen, Yi-Ting Chen, Yen-Hao Hsu, Jiun-Yi Shen, Chi-Lin Chen, Yi-An Chen and Pi-
Tai Chou*
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Department of Chemistry and Center for Emerging Material and Advanced Devices, National Taiwan University, Taipei 10617,
Taiwan, R.O.C.
KEYWORDS Excited state proton transfer, water-catalyzed triple proton transfer, proton pump
ABSTRACT: 3-Me-2,6-diazaindole ((2,6-aza)Ind) was strategically designed and synthesized to probe water molecule catalyzed excited-
state proton transfer in aqueous solution. Upon electronic excitation (λmax ~ 300 nm), (2,6-aza)Ind undergoes N(1)-H to N(6) long-distance
proton transfer in neutral H2O, resulting in normal (340 nm) and proton-transfer tautomer (480 nm) emissions with an overall quantum
yield of 0.25. The rate of the water-catalyzed proton transfer shows a prominent H/D kinetic isotope effect, which is determined to be 8.3 
108 s-1 and 4.5  108 s-1 in H2O and D2O, respectively. Proton inventory experiments indicate the involvement of two water molecules and
three protons, which undergo a relay type of excited-state triple proton transfer (ESTPT) in a concerted, asynchronous manner. The results
demonstrate for the first time the fundamental of triple proton transfer in pure water for azaindoles as well as pave a new avenue for 2,6-
diazatryptophan, an analogue of tryptophan exhibiting a similar ESTPT property with (2,6-aza)Ind, to probe bio-waters in proteins.
1. Introduction
state proton transfer such as keto-enol isomerization takes place,
leading to equilibrium between normal and tautomer species in
Excited-state proton transfer (ESPT) reactions occur in a wide
variety of chemical systems. One old and famous class is the
photoacids, of which phenol and - or - naphthol with the –OH
group serving as the proton donor are representative.1 For these
photoacids, pKa of the –OH group is drastically reduced in the
excited state, resulting in the deprotonation, i.e., release of protons,
in neutral aqueous solution, giving rise to anion emission. In this
case, the bulk water molecules serve as the proton acceptor. In non-
aqueous organic solvents, especially aprotic solvents, the solvent
molecule is not basic enough to accept the proton, so most
photoacids do not undergo ESPT. Alternatively, with the addition
of a proton acceptor such as carbonyl oxygen or pyridinyl nitrogen
that forms a hydrogen bond (H-bond) with the proton donor (e.g.
–OH or -NH) excited-state intramolecular proton transfer (ESIPT)
may take place, resulting in a tautomer species.2-4
water, which complicates the spectroscopic and dynamic studies15-
17
In our viewpoint, an ideal probe for proton transfer reaction in
water should not avoid water interference but rather fully utilize the
water molecules, which can catalyze the proton transfer reaction.
Also, such a probe should display great bio-compatibility and high
emission quantum yield (Q.Y.). A case18 in point is the recently
developed 2,7-diazaindole and its tryptophan analogue (2,7-
aza)Trp (see Scheme 1). Upon UV (e.g., 310 nm) excitation, the
N(1)-H isomer for 2,7-diazaindole and (2,7-aza)Trp undergoes
water catalyzed double proton transfer reaction, resulting in an
N(7)-H tautomer that exhibits green emission (see Scheme 1).
(2,7-aza)Trp has been successfully applied to replace tryptophan in
proteins and probe the water microsolvation in the proximity of
tryptophan in Thromboxane18 and Ribonuclease T1.19 The
importance of these studies stems from the structural resemblance
between tryptophan and (2,7-aza)Trp. Therefore, under minimum
structural perturbation, the results provide valuable information for
the protein-water correlation and further exploration of the protein
functionality.
In the above two cases, the anion species (for photoacids) and
proton-transfer tautomer (ESIPT molecules) being generated
exhibit emission that is red-shifted dramatically as compared to the
normal Franck-Condon absorption. This unique property has been
intensively studied to shed light on their associated proton-transfer
mechanisms, as well as widely applied in various optoelectronic
applications.3,5-8 In terms of bio-applications, however, the
photoacids are also sensitive to the pH environment. In numerous
cases, the existence of anions in the ground state is non-negligible
in the physiological condition.9-11 On the other hand, the ESIPT
properties are subject to environmental perturbation, such as
solvent polarity and/or external H-bonding formation. Polar and
protic solvents perturb ESIPT significantly by rupturing an
intramolecular H-bond, altering the ESIPT dynamics and/or
quenching of tautomer emission.10,12-14 In certain cases, the ground-
Despite the prominence in its excited-state proton transfer
(ESPT) reaction catalyzed by water, (2,7-aza)Trp unfortunately
has a relatively low emission quantum yield of ~0.03 (normal plus
tautomer emission), which may be interfered with by unwanted
bioluminescence. In addition, (2,7-aza)Trp requires the formation
of 1:1 (water: (2,7-aza)Trp in molar ratio) H-bonded complex to
execute ESPT, which is effective only when water molecules are in
proximity. As for sensing the water relay or water clusters in protein,
if the proton donor and acceptor of the tryptophan analogue are
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 9
Scheme 2. The synthetic route of (2,6-aza)Ind
widely separated, for which ESPT requires the formation of an H-
1
2
3
4
5
6
bonded complex with a relay of n (n  2) water molecules, the
result on the one hand may provide complementary information
that cannot be accessed by (2,7-aza)Trp. Consequently, the
combination of different probes should make water sensing more
effective, broadening the horizon for probing bio-water in proteins.
Scheme 1. Water-catalyzed ESPT property of 2,7-diazaindole and (2,7-aza)Trp
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For (2,6-aza)Ind, we obtained the single crystal X-ray structure
shown in Figure 1a, in which (2,6-aza)Ind was found to form a
tetramer via the hydrogen bond between N(1)-H (hydrogen bond
donor) in one molecule and N(6) (hydrogen bond acceptor) in the
other molecule alternatively. The intermolecular hydrogen bond
was determined to range from 1.769 Å to 1.890 Å. The packing
view in Figure 1b shows that the H-bonded tetramer of (2,6-
aza)Ind is nearly planar, and the inter-layer distance is 3.554 Å. The
unit cell is constructed by the π-π interaction between different
layers of (2,6-aza)Ind molecules and the H-bond interaction
between (2,6-aza)Ind N(2) and solvent H2O.
Up to current stage only few groups reported the hypothetical
formation of cyclic H-bonded complex involving two water
molecules. Prototypical examples are 7-azaindole in gas phase20,21
and 2-(3’-pyridyl)indole in alcohol/water systems.22,23 Yet, no
proton-transfer tautomer emission was observed for these systems
in water. Instead, the intense 2-(3’-pyridyl)indole emission in
nonpolar solvent was strongly quenched by addition of protic
solvents due to the formation of 1:2 2-(3’-pyridyl)indole:solvent
H-bonded complex, which enhances the single bond rotation and
hence the depopulation of the excited state.24
Herein we proposed that the analogue of 2,7-diazaindole,
namely 2,6-diazaindole and its bio-derivative 2,6-diazatryptophan,
may be an ideal case in point for probing multiple proton transfer
catalyzed by water molecules. In this strategic design, N(2) must be
kept to increase the acidity of N(1)-H proton18 to facilitate ESPT
via long range relay of water molecules (see Figure 1). Structurally,
the long distance between N(1)-H and N(6), together with a C(7)-
H steric interference, makes ESPT possible only under the catalysis
of n  2 water molecules. While there are no reports so far of
photophysical properties of 2,6-diazaindole, let alone 2,6-
diazatryptophan, it would be of fundamental and practical
importance to initiate this seminal research. In this study, we
mainly focus on the photophysical properties of compound 3-Me-
2,6-diazaindole ((2,6-aza)Ind), which is also the precursor for the
synthesis of 2,6-diazatryptophan ((2,6-aza)Trp). As elaborated in
the following sections, comprehensive spectroscopic and dynamic
studies have led us to establish a mechanism incorporating water-
catalyzed triple proton transfer for (2,6-aza)Ind in the excited state.
We also prove that (2,6-aza)Trp possesses a photophysical
property similar to that of (2,6-aza)Ind, brightening the prospect of
sensing bio-water in proteins.
Figure 1. (a) Molecular structure of (2,6-aza)Ind; thermal ellipsoids drawn at
50% probability level. (b) Packing view of (2,6-aza)Ind taken along the c axis.
Carbon, nitrogen, and hydrogen atoms are grey, blue, and green, respectively.
Proton-transfer tautomerism of (2,6-aza)Ind
Manifested in neutral water (pH = 7.4), (2,6-aza)Ind reveals two
remarkable fluorescent bands, one with the peak wavelength at 340
nm and the other, being much red shifted, maximized at 480 nm
(Figure 2). First of all, according to the absorption titration (Figure
S1), the pKa of (2,6-aza)Ind and protonated (2,6-aza)Ind were
determined to be 12.1 and 5.34, respectively. Therefore, at pH =
7.4, the presence of anionic and cationic forms is negligible. The
corresponding excitation spectra monitored at the 340 and 480 nm
bands, respectively, are identical (see Figure S2); they are also the
same as the absorption spectrum, indicating that the two emission
bands share the same ground-state origin. As a result, the possibility
of two species or trace impurities contributing to the two emission
bands can be eliminated. In aprotic solvents such as toluene, ethyl
ether and CH3CN, (2,6-aza)Ind exhibits only the normal emission
band, which is located at ~330 nm regardless of significant changes
in solvent polarity from toluene to acetonitrile (see Figure S3).
2. Results and Discussion
Synthesis and structural characterization
To surpass the limitations of the traditional tryptophan (Trp)
analogues for probing bio-waters, we strategically designed and
synthesized the compound (2,6-aza)Ind and its derivative, a new
type of unnatural tryptophan, 2,6-diazatryptophan ((2,6-aza)Trp).
For clarity, we mainly focused on (2,6-aza)Ind, while the results
and prospect of (2,6-aza)Trp in bio-applications will be discussed
in the last section. Starting from 3-amino-4methylpyridine 1, (2,6-
aza)Ind was prepared via a relevant synthetic route depicted in
Scheme 2.25 Detail of the corresponding syntheses and
characterization is elaborated in the experimental section.
ACS Paragon Plus Environment

Page 3 of 9
Journal of the American Chemical Society
(e.g., 510 nm, see Figure 3). The tautomer emission then
1
2
3
4
5
6
7
8
undergoes population decay with a lifetime of 5.07 ns. Assuming
that the decay of the normal emission is dominated by ESPT, the
kobs for the normal emissions, measured to be 8.3108 s-1 and
4.5108 s-1 in H2O and D2O, respectively, manifest a kinetic isotope
effect with k(H)/k(D) of 1.84. The significant kinetic isotope
effect implies that proton transfer should be directly involved in the
rate-determined step.
9
10
11
12
Figure 2. Absorption (solid line) and emission spectrum (dashed line) of (2,6-
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aza)Ind in neutral H2O (red) and D2O (blue) and N(1)Me-(2,6-aza)Ind (green)
in H2O. The peak at 340 nm represents the normal form emission, and the one
at 480 nm is the tautomer emission (λex = 300 nm).
From the chemistry point of view, we also synthesized the
methyl derivatives of (2,6-aza)Ind, namely N(1)Me-(2,6-aza)Ind
and N(6)Me-(2,6-aza)Ind (see experimental section), which
represent the normal and proton-transfer tautomer forms and
exhibit single emission band maximized at 360 nm (Figure 2 and
Figure S4) and 485 nm (Figure S5), respectively, in water. It can
thus be unambiguously concluded that (2,6-aza)Ind undergoes
water-catalyzed proton transfer reaction that competes with the
normal emission (340 nm), giving rise to a 480 nm proton-transfer
tautomer emission. Remarkably, the quantum yield of the dual
emission was measured to be 0.25, which is ~8.3 folds higher than
that (Q.Y.~0.03) of the overall emission of 2,7-diazaindole (normal
plus tautomer emission).18 From the bio-imaging point of view, this
tautomer fluorescence brings a new dimension and further expands
the dynamic range of sensing. What is more, as elaborated in the
following sections, ESPT of (2,6-aza)Ind, incorporating triple
proton transfer in water, is unprecedented and is of prime
fundamental importance.
Figure 3. Relaxation dynamics of (2,6-aza)Ind in H2O (black) and D2O (blue).
Sample was monitored at (a) 340 nm and (b) 510 nm respectively (λex = 290
nm).
We then performed a temperature dependent study to analyze the
reaction kinetic isotope parameters. In this approach we utilized
the decay rate of N(1)Me-(2,6-aza)Ind emission to simulate the
overall non-proton transfer decay rate of (2,6-aza)Ind. Accordingly,
the proton transfer rate kESPT could be extracted by
ESPT Kinetics and isotope effect
Further affirmation of the excited-state proton transfer for (2,6-
aza)Ind is given by the correlation of relaxation dynamics among
emission bands (Figure 3). When monitored at the short
wavelength band of 340 nm, (2,6-aza)Ind exhibits a single decay
component of 1.20 ns in water. On the other hand, at the red edge
of the 510 nm emission, the relaxation dynamics consist of a finite
rise component of 1.18 ns (Figure 3b) and a decay component of
3.61 ns. The 1.18 ns rise time of the tautomer emission, within
experimental uncertainty (± 0.05 ns), correlates well with the 1.20
ns decay time. These data clearly support the water-catalyzed ESPT
from N(1) to N(6), showing the precursor (340 nm, the N(1)-H
isomer) and successor (480 nm, the N(6)-H isomer) type of
kinetic relationship assisted by the water molecules (see Scheme 3).
Accordingly, we assigned the 3.61 ns decay as the population decay
of the tautomer.
kESPT  kN* for (2,6-aza)Ind kN* for N(1)Me-(2,6-aza)Ind
Upon monitoring the decay of the normal emission for (2,6-
aza)Ind and N(1)Me-(2,6-aza)Ind from 313 K (40oC) to 277 K
(4oC) in H2O and D2O, the temperature dependent rate constant
of the proton transfer reaction kESPT(T) can thus be obtained.
Figure 4 shows a plot for the logarithm of kESPT(T) versus the
reciprocal of temperature in H2O and D2O according to the
Arrhenius equation (1).
lnkESPT (T)  ln A Ea / RT
(1)
where Ea is the reaction activation energy and A is the frequency
factor. As a result, for (2,6-aza)Ind, Ea of water catalyzed ESPT are
calculated to be 2.64 ± 0.03 kcal mol-1 in H2O and 2.66 ± 0.05 kcal
mol-1 in D2O, which are considered to be identical within
experimental error. The extrapolation value A represents the
maximum rate of kESPT for the normal emissions in H2O or D2O,
which differ greatly, being 5.271010 s-1 in H2O and 2.721010 s-1 in
D2O; AH/AD is determined to be 1.94, consistent with that deduced
at room temperature (vide supra).
Figure 2 also shows the steady-state absorption and emission
spectrum of (2,6-aza)Ind in D2O. In comparison to that in H2O,
despite the same dual emission band with the same corresponding
peak wavelengths, the intensity ratio for the tautomer versus
normal emissions diminishes significantly in D2O, implying that the
ESPT rate may be slower in D2O. This viewpoint is supported by
the rather long decay time constant of 2.15 ns upon monitoring at
the normal emission band (e.g., 340 nm) in D2O, which correlates
well with the rise time of 2.21 ns for the tautomer emission band
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 9
Table 1 Photophysical properties of (2,6-aza)Ind and its derivatives in H2O and D2O.
1
2
3
4
cpds
λabs
λema (nm)
Q.Y.
Ac (s-1)
Eac (kcal mol-1)
obsb (ns)
3.511010
2.300.09
(2,6-aza)Ind in H2O
300
340 (N*), 480 (T*)
0.16 (N*); 0.09 (T*)
1.27 (N*); 1.18(-) 3.61 (T*)
5
6
7
8
1.981010
2.250.04
(2,6-aza)Ind in D2O
300
313
313
370
300
340 (N*), 480 (T*)
0.23 (N*); 0.05 (T*)
2.15 (N*); 2.27(-) 5.07 (T*)
N(1)Me-((2,6-aza)Ind) in H2O
N(1)Me-((2,6-aza)Ind) in D2O
N(6)Me-((2,6-aza)Ind) in H2O
(2,6-aza)Trp in H2O
360
10.93
360
12.55
9
485
3.51
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
330 (N*),465 (T*)
0.77(N*); 0.74(-) 5.40 (T*)
aThe samples were excited at the lowest transition band. N* = normal emission; T* = tautomer emission bLifetime data were measured using a TCSPC system coupled with a hydrogen-
filled flash lamp as the light source. cA and Ea were determined by the Arrhenius plot using lnkESPT versus 1/T.
less than that of N* decay for (2,6-aza)Ind. Accordingly, the time
dependent [N*]t for (2,6-aza)Ind in water, derived from Scheme 3,
is expressed as28-30
k1kpt
t
[N* ]t =[N* ]0ek1 kpt
(2)
Based on eq. (2), two simplified cases are discussed. If kpt is >> k-1,
the overall ESPT rate constant k1kpt/(k-1 + kpt) in eq. (2) can be
approximately equal to k1. This result implies that the rate-
determining step is solvent reorientation. In this case the energy
required for breaking and reformation of hydrogen bonds should
be H/D isotope dependent. This interpretation is inconsistent with
the experimental results of the H/D independent Ea value.
Alternatively, if k-1 >> kpt, the rate constant in eq. (2) is then
simplified to kpt(k1/k-1), which is essentially equivalent to kpt×Keq,
where Keq is the equilibrium constant between I* and N*.
Thermodynamically, Keq = e-G/RT, whereG specifies the free
energy difference between I* and N* (G(I*)G(N*)). Therefore,
Figure 4. The Arrhenius plot of (2,6-aza)Ind in H2O (black) and D2O (red)
monitored at 330 nm (λex = 300 nm), and the linear fit of lnkESPT versus 1/T.
To rationalize the above interesting kinetic isotope effect for
ESPT, we proposed the existence of an intermediate I*. I* is
associated with the formation of a specific (2,6-aza)Ind/H2O
hydrogen bonded (H-bonded) complex, in which the
configuration of water molecules versus (2,6-aza)Ind is ready for
the proton transfer.23,26-30 Due to the great separation between
N(1)-H and N(6), it is reasonable to propose that I* is composed
of (2,6-aza)Ind and water in a stoichiometric ratio of 1:2, in which
the two water molecules are H-bonded at N(1)-H and N(6) sites
of (2,6-aza)Ind, respectively, forming a cyclic H-bonded complex
(see Scheme 3 below).
the overall rate constant of this water assisted ESPT) reaction, kESPT
is equal to
kESPT  kpteG/RT (3)
,
Scheme 3. The proposed water-catalyzed ESPT property of (2,6-aza)Ind and
(2,6-aza)Trp
According to Scheme 3 several remarks can be made: 1. In the
ground state, such an ordered cyclic complex (I) should have
higher free energy than all other randomly water-solvated (2,6-
aza)Ind, denoted as N. Therefore, the population of I should be
negligible. 2. Upon excitation of the randomly water solvated from
N to N*, N* I* water reorientation takes place with a rate of k1. 3.
Once I* forms, it either quickly breaks down to the randomly
water-solvated N* with a rate of k-1 or undergoes triple proton
transfer reaction, forming the N(6)-H tautomer (see Scheme 3)
with a rate of kpt. Since k-1 < k1 or kpt, from the classical kinetic
approach, a steady state is established for I* such that d[I*]/dt ~ 0.
Furthermore, it is reasonable to assume pre-equilibrium between
N* and I* because of the relatively small knr and kr for N*. This
viewpoint is supported by the N(1) methylated (2,6-aza)Ind,
namely N(1)Me-(2,6-aza)Ind, for which ESPT is prohibited due to
the lack of the N(1)-H proton. Figure S4 shows steady-state
absorption and emission spectra, as well as fluorescence decay of
N(1)Me-(2,6-aza)Ind. Its emission decay rates kobs (= kr + knr )
were measured to be as small as 9.1 × 107s-1 and 8.0 × 107s-1 in H2O
and D2O, respectively, at room temperature, which is ~5-8 folds
The principle of eq. (3) is similar to that derived from the transition
state theory except that G in (3) is the true equilibriums not
activation free energy so there was no kBT/h factor deduced from the
transition state theory. In theory, G should be identical between
(2,6-aza)Ind in H2O and D2O and is virtually H/D isotope
independent, while kpt involves proton tunneling and thus is H/D
isotope dependent. The latter also echoes the report that tunneling
of hydrogen significantly contributes to enzyme kinetics in
biological systems.31 This interpretation is also reminiscent of water
molecule assisted excited state proton transfer in 7-azaindole and
its derivatives,27,32 except the much different structure proposed in
the intermediate, where a relay of two water molecules17 undergoes
ACS Paragon Plus Environment

Page 5 of 9
Journal of the American Chemical Society
triple proton transfer in the electronically excited (2,6-aza)Ind,
bringing the proton from N(1) to N(6) (see Scheme 3).
Mechanism of excited state triple proton transfer
After careful optimization of the eight kxxx values (see SI for
detail), kH and kD as a function of XD are well fitted by eqs. (4) and
(5), respectively. The results clearly support the assumption that I*
of (2,6-aza)Ind incorporates two water molecules, forming a cyclic
water relay to connect N(1) and N(6) via hydrogen bonds, where
the triple proton transfer takes place. In theory, the concerted,
synchronous triple-proton-transfer requires that all the bond
breakages and formations take place simultaneously in a single step
and all proceed in the same extent. Therefore, the primary isotope
1
2
3
4
5
6
7
8
The next key interest is to study whether the excited-state proton
transfer for (2,6-aza)Ind is concerted or non-concerted. The
former should fit the above proposed mechanism involving a relay
type of proton transfer from the 1:2 (2,6-aza)Ind:H2O cyclic H-
bonded intermediate. For this case, it is of fundamental importance
to probe whether ESPT is a concerted, synchronous or a concerted,
asynchronous reaction. The latter, by widely accepted definition,
does not require a strict simultaneity or symmetry, but it only
implies that the motions of the protons associated in the reaction
are correlated.26,33-35 To gain deep insight into the associated
mechanism, we then performed a proton inventory experiment, in
which the molar fraction of deuterium oxide in water was varied to
analyze the kinetic results statistically.26,33
effect must be equal to the secondary isotope effect; that is, (kHHH
/
9
1/3
kDHH) = (kDHH/ kHDD) = (kHHH/ kDDD
)
must be satisfied, which is
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
the core of rule of the geometric mean.33,36 Conversely, the reaction
is considered to be concerted, asynchronous with a tunneling effect
involved if the rule of the geometric mean does not hold. As shown
in Table S1, the quadratic fitting yields values of (kHHH/ kDHH),
1/3
(kDHH/ kHDD), and (kHHH/ kDDD
)
of 1.54, 0.75, and 1.26,
respectively (Table S1). The result does not satisfy the rule of the
geometric mean. The larger kHHH/ kDHH implies that the proton
transfer from the N(1)-H site, is much more driven by the
tunneling effect. In brief, based on the proton inventory experiment,
we thus conclude the operation of excited-state triple proton
transfer (ESTPT), which can be ascribed to a concerted,
asynchronous reaction rather than a concerted, synchronous
reaction or a separated two-step reaction.
We have concluded the overall rate of ESPT, kESPT, to be kpt×Keq,
where only the I*  T* proton transfer process kpt accounts for the
kinetic H/D isotope effect (vide supra). Because Keq is independent
of the isotope effect, upon variation of the molar fraction of H2O
versus D2O, the observed decay of N* should be proportional to kpt
and can thus be used as a reference to monitor the intrinsic kpt for I*
 T* in various H2O/D2O mixtures. This makes the following
proton inventory experiment simple. Excited-state triple proton
transfer in a mixed solution of H2O and D2O should at least
consider eight different types of decay: H-H-H, H-D-H, H-H-D,
and H-D-D, and D-H-H, D-D-H, D-H-D, and D-D-D. We then
used N(1)H and N(1)D to specify the non-deuterated and
deuterated I* species. The time-dependent N(1)H and N(1)D
concentration in the H2O/D2O mixture can thus be written as
follows17 (see SI for detailed derivation):
d[N(1)H]
 [(kHHH  kHDD  kHDH  kHHD ) XD2 [(kHDH  kHHD  2kHHH ) XD  kHHH ][N(1)H]
dt
 kH [N(1)H]
(4)
 [(kDHH  kDDD  kDDH  kDHD ) XD2 [(kDDH  kDHD  2kDHH ) XD  kDHH ][N(1)D]
 kD[N(1)D]
d[N(1)D]
dt
(5)
where XD is the molar ratio of D2O. kXXX (X = D or H) specifies the
rate constant of different deuterated species. The superscript X on
the left specifies the N(1)-H(or D) followed by H(or D) of two
water molecules in the complex. For a given relaxation dynamics of
(2,6-aza)Ind in a mixed solvent of H2O and D2O, we can describe
the decay as a double-exponential decay,
Figure 5. The (a)kH and (b) kD of (2,6-aza)Ind in water versus the molar
fraction of deuterium oxide XD plot. Solid line is the best quadratic fitting of the
data. From the best fitting result, we can obtain that kHHH, kDHH, kHDD, and kDDD
are 6.97108 s-1, 4.52108 s-1, 6.02108 s-1, and 3.52108 s-1, respectively.
F(t)  (1 XD )exp(kH t)  XD exp(kD t)
(6)
Excited-state triple proton transfer in (2,6-aza)Trp
Upon a change in the fraction XD, the resulting decay of N* can
be fitted by the double-exponential decay function F(t) (6) with
known XD to obtain kH and kD. As a result, the plot of kH and kD as a
function of XD in Figure 5 reveals a quadratic relationship between
kH (or kD) and XD, consistent with the quadratic relationship
expressed in eqs. (4) and (5). If the mechanism is a two separated
single steps such as the deprotonation at N(1)-H followed by an
uptake of a proton at N(6), N(6) would become a much stronger
base due to the re-localization of the negative charge. One thus
predicts the rate of the N(6) protonation in water to be very fast.
Given this circumstance the mechanism can be seemed as a single
deprotonation process at N(1)-H which is inconsistent with the
observed quadratic relation of rate constant versus deuterium
solvent concentration in the proton inventory experiment.
A brightening prospect of water-catalyzed ESTPT for bio-
applications lies in the derivative of (2,6-aza)Ind, namely (2,6-
aza)Trp (see Scheme 4), which is deemed as the analogue of the
natural amino acid tryptophan. The synthesis of (2,6-aza)Trp was
nontrivial, for it took further four-step synthesis from (2,6-aza)Ind
and produced a final yield of 15% (see Scheme 4 and experimental
section). As the two compounds share identical core
chromophores, the photophysical property of (2,6-aza)Trp is
expected to be similar to that of (2,6-aza)Ind. Based on the
absorption spectrum shown in Figure 6, (2,6-aza)Trp in neutral
H2O (pH = 7.4) reveals exclusively the normal N(1)-H isomer.
Upon electronic excitation (e.g., 300 nm), dual emission bands
maximized at 330 nm and 465 nm were observed, in which the
lifetime of 0.77 ns of the 330 nm emission correlated well with a
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 9
0.74 ns rise time of the 465 nm emission (see Table 1 and Figure microsolvation in proteins. Comprehensive studies of (2,6-aza)Trp
1
2
3
4
5
6
7
8
S6), indicating the occurrence of excited-state proton transfer in
(2,6-aza)Trp. The proton-transfer tautomer emission (the 465 nm
band) then undergoes a population decay of 5.40 ns to the ground
state. Also revealed in Figure 6 is the emission spectrum of (2,6-
aza)Trp in D2O, for which the different ratiometric emission from
that in H2O clearly manifests the H/D isotope effect similar to
those elaborated in (2,6-aza)Ind. In light of the combination of
pronounced water catalyzed ESTPT and biocompatibility, (2,6-
aza)Trp must be a good candidate to replace tryptophan in probing
the microsolvaton of water in proteins to provide complementary
information on protein structure and dynamics.18,19
incorporated in proteins are currently in progress.
4. Experimental Section
Syntheses and Structural Characterization
All solvents were distilled from appropriate drying agents prior to
use. Commercially available reagents were used without further
purification unless otherwise stated. All reactions were monitored
by TLC. Column chromatography was carried out using silica gel
1
from Merck (230-400 mesh). H and 13C NMR spectra were
9
recorded on a Varian Unity 400 spectrometer at 400 MHz and 100
MHz, respectively. Chemical shifts (δ) are recorded in parts per
million (ppm) and coupling constants (J) are reported in Hertz
(Hz). Mass spectra were obtained using a Gas Chromatograph-
Mass Spectrometer (Finnigan MAT TSQ-46C GC/MS/MS/DS).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 4. The synthetic route of (2,6-aza)Trp
Synthesis of (2,6-aza)Ind 25
Starting from 3-amino-4methylpyridine 1 (see Scheme 2),
sodium hexamethyldislazane and Boc anhydride were used to
protect the amino group, which gave compound 2. The methyl
group in the fourth position of compound 2 was homologated to an
ethyl group by iodomethane with treatment of t-BuLi to get
compound 3. The protecting group Boc was eliminated by the
treatment of TFA, and subsequent the cyclization of the
1
intermediate 4 produced the (2,6-aza)Ind. H NMR (400MHz,
CDCl3); δ 8.98 (s, 1H), 8.32 (d, J = 5.6 Hz, 1H), 7.58 (d, J = 5.6 Hz,
13
1H), 2.61 (s, 3H). C NMR (100 MHz, CDCl3): δ 142.8, 138.1,
137.9, 134.5, 126.6, 114.5, 12.0.
Synthesis of compound 7 (Scheme 4)
Starting from (2,6-aza)Ind, Boc anhydride was used to protect
the N(1)-H, which gave 525. The protected compound then reacted
with NBS and BPO to obtain compound 6. Next, NaH (60%, 0.054
g, 1.3 mmol) was added to a solution of diethylacetamidomalonate
(0.29 g, 1.3 mmol) dissolved in THF (8 mL) at 0C under N2.
After 1 h, 6 (0.28 g, 0.9 mmol) in THF (8 mL) was added to the
mixture and the solution was stirred at RT for 2 h, followed by
further reflux at 55-60°C overnight. After cooling, the mixture was
quenched with ice and extracted with CH2Cl2. The organic layer
was dried over MgSO4, filtered, and evaporated. The crude product
was purified by silica gel column chromatography with
Hexane/EtOAc mixture as eluent to afford 7 (0.088 g, 22%) as
Figure 6. Absorption (dotted line) and emission (solid line) spectra of (2,6-
aza)Trp in H2O (black) and D2O (blue).
3. Conclusion
In summary, the synthesis, characterization, and photophysical
properties of (2,6-aza)Ind and (2,6-aza)Trp have been studied.
Remarkably, (2,6-aza)Ind in water undergoes the excited-state
triple proton transfer reaction in a concerted, asynchronous
manner that is proved via the proton inventory experiments. The
overall rate of excited-state triple proton transfer is governed by the
pre-equilibrium between water randomly solvated (2,6-aza)Ind
and cyclic 2:1 (water:(2,6-aza)Ind) hydrogen bonded complex,
followed by the intrinsic proton transfer. The latter undergoes
proton tunneling that is sensitive to the H/D effect, yielding a
tautomer with 480 nm emission. The results provide an ideal
paradigm to probe the fundamentals of water catalyzed triple
proton transfer in pure water. It is conceivable that such an H-
bonded relay may exist in the gas phase, particularly in the cold gas
generated from a molecular beam, such that the in-depth
fundamentals of ESTPT can be probed by high-resolution
spectroscopy, free from perturbation by external media. From the
application point of view, the water catalyzed ESTPT, together
with its good emission yield, should make (2,6-aza)Trp an ideal
unnatural tryptophan analogue for probing the water
1
white solids. H NMR (400 MHz, CDCl3): δ 9.49 (s, 1H), 8.47 (d,
J = 5.16 Hz, 1H), 8.37 (d, J = 6.4, 1H), 4.49 (t, J = 6, 1H), 3.84-3.82
(m, 2H).
Synthesis of (2,6-aza)Trp
Compound 7 (0.89 g, 2.0 mmol) in 12 M HCl was heated to
reflux for 8 h. The reaction mixture was concentrated under
reduced pressure to afford (2,6-aza)Trp as a white solid in the HCl
1
salt form. H NMR (400 MHz, D2O): δ 9.44 (s, 1H), 8.42 (d, J =
6.8 Hz, 1H), 7.52 (d, J = 5.48, 1H), 6.64 (s, 1H), 4.32-4.27 (m, 4H),
4.08 (s, 2H), 1.88 (s, 3H), 1.69 (s, 9H), 1.31 (t, 6H).
Synthesis of N(1)Me-(2,6-aza)Ind
NaOH (36 mg, 0.9 mmol) was added to a solution of (2,6-
aza)Ind (100 mg, 0.75 mmol) dissolved in THF (30 mL) at RT
under N2. After 15 mins, methyl iodide (0.056 mL, 0.9 mmol) was
added to the mixture and the solution was stirred at RT for 4 hours;
then the mixture was extracted with EtOAc. The organic layer was
dried over MgSO4, filtered and evaporated. The crude product was
purified by silica gel column chromatography with EtOAc as eluent
ACS Paragon Plus Environment

Page 7 of 9
Journal of the American Chemical Society
1
(8)Zhang, Z.; Chen, Y.-A.; Hung, W.-Y.; Tang, W.-F.; Hsu, Y.-H.; Chen,
to afford N(1)Me-(2,6-aza)Ind (99 mg, 90%) as white solids. H
C.-L.; Meng, F.-Y.; Chou, P.-T. Chem. Mater. 2016, 28, 8815.
(9)Stoner-Ma, D.; Jaye, A. A.; Matousek, P.; Towrie, M.; Meech, S. R.;
Tonge, P. J. J. Am. Chem. Soc. 2005, 127, 2864.
(10)Mandal, A.; Misra, R. J. Lumin. 2014, 150, 25.
(11)Laptenok, S. P.; Conyard, J.; Page, P. C. B.; Chan, Y.; You, M.; Jaffrey,
S. R.; Meech, S. R. Chem. Sci. 2016, 7, 5747.
(12)McMorrow, D.; Kasha, M. J. Am. Chem. Soc. 1983, 105, 5133.
(13)Van Benthem, M. H.; Gillispie, G. D. J. Phys. Chem. 1984, 88, 2954.
(14)Strandjord, A. J. G.; Barbara, P. F. J. Phys. Chem. 1985, 89, 2355.
(15)Lee, S.-I.; Jang, D.-J. J. Phys. Chem. 1995, 99, 7537.
(16)Park, S.-Y.; Kim, B.; Lee, Y.-S.; Kwon, O.-H.; Jang, D.-J. Photochem.
Photobiol. Sci. 2009, 8, 1611.
(17)Kwon, O.-H.; Mohammed, O. F. Phys. Chem. Chem. Phys. 2012, 14,
8974.
(18)Shen, J.-Y.; Chao, W.-C.; Liu, C.; Pan, H.-A.; Yang, H.-C.; Chen, C.-L.;
Lan, Y.-K.; Lin, L.-J.; Wang, J.-S.; Lu, J.-F.; Chun-Wei Chou, S.; Tang, K.-C.;
Chou, P.-T. Nat. Commun 2013, 4, 2611.
(19)Chao, W.-C.; Shen, J.-Y.; Lu, J.-F.; Wang, J.-S.; Yang, H.-C.; Wee, K.;
Lin, L.-J.; Kuo, Y.-C.; Yang, C.-H.; Weng, S.-H.; Huang, H.-C.; Chen, Y.-H.;
Chou, P.-T. J. Phys. Chem. B 2015, 119, 2157.
(20)Sakota, K.; Komoto, Y.; Nakagaki, M.; Ishikawa, W.; Sekiya, H. Chem.
Phys. Lett. 2007, 435, 1.
(21)Sakota, K.; Inoue, N.; Komoto, Y.; Sekiya, H. J. Phys. Chem. A 2007,
111, 4596.
(22)Kyrychenko, A.; Herbich, J.; Wu, F.; Thummel, R. P.; Waluk, J. J. Am.
Chem. Soc. 2000, 122, 2818.
(23)Waluk, J. Acc. Chem. Res. 2003, 36, 832.
(24)Kyrychenko, A.; Wu, F.; Thummel, R. P.; Waluk, J.; Ladokhin, A. S. J.
Phys. Chem. B 2010, 114, 13574.
(25)Tucker, T. J.; Sisko, J. T.; Tynebor, R. M.; Williams, T. M.; Felock, P. J.;
Flynn, J. A.; Lai, M.-T.; Liang, Y.; McGaughey, G.; Liu, M.; Miller, M.;
Moyer, G.; Munshi, V.; Perlow-Poehnelt, R.; Prasad, S.; Reid, J. C.;
Sanchez, R.; Torrent, M.; Vacca, J. P.; Wan, B.-L.; Yan, Y. J. Med. Chem.
2008, 51, 6503.
1
2
3
4
5
6
7
8
NMR (400 MHz, CDCl3): δ 8.86 (s, 1H), 8.25 (d, J = 5.6 Hz, 1H),
7.51 (d, J = 5.6 Hz, 1H), 4.07 (s, 1H), 2.54 (s, 1H). 13C NMR (100
MHz, CDCl3) : δ 141.1, 137.9, 137.4, 133.4, 126.8, 113.9, 35.6,
11.7. HRMS calcd. for C8H9N3 (M+H)+: 147.0796; found:
147.0788.
Synthesis of N(6)Me-(2,6-aza)Ind
CH3I (8 mL) was added to a solution of (2,6-aza)Ind (180
mg,1.35 mmol) dissolved in toluene (20 mL), and the solution was
heated at reflux under N2 atmosphere for 6 h. After 6 h, the
precipitated material was filtered off. The white precipitate was
dissolved in 35% ammonia in water (100 mL) and extracted with
CH2Cl2. The organic layer was dried over MgSO4, filtered, and
evaporated to dryness to afford N(6)Me-(2,6-aza)Ind (125 mg,
63%). 1H NMR (400 MHz, CDCl3): δ 8.74 (s, 1H), 7.63 (d, J = 6.8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
Hz, 1H), 7.22 (d, J = 6.8 Hz, 1H), 4.19 (s, 1H), 2.70 (s, 1H). C
NMR (100 MHz, CDCl3) : δ 145.9, 142.1, 134.8, 125.1, 123.8,
116.0, 46.5, 11.8. HRMS calcd. for C8H9N3 (M+H)+: 147.0875;
found: 147.0869.
Spectroscopic Measurements
The steady-state absorption and emission spectra were
measured by a Hitachi U-3310 Spectrophotometer and an
Edinburgh FS920 Fluorimeter respectively, both of which had been
calibrated. In brief, nanosecond time-resolved experiments were
performed by using an Edinburgh FLS920 time-correlated single
photon-counting (TCSPC) system with a pulsed hydrogen-filled
lamp as the excitation source. Data were fitted with the sum of
exponential functions using a nonlinear least-squares procedure in
combination with a convolution method.
(26)Kwon, O.-H.; Lee, Y.-S.; Yoo, B. K.; Jang, D.-J. Angew. Chem. Int. Ed.
2006, 45, 415.
Supporting Information. Details of synthetic procedures,
characterization, pH titration, proton inventory and photophysical
measurements. This material is available free of charge via the Internet
at http://pubs.acs.org.” F
(27)Chapman, C. F.; Maroncelli, M. J. Phys. Chem. 1992, 96, 8430.
(28)Moog, R. S.; Maroncelli, M. J. Phys. Chem. 1991, 95, 10359.
(29)Mente, S.; Maroncelli, M. J. Phys. Chem. A 1998, 102, 3860.
(30)Hsieh, C.-C.; Chen, K.-Y.; Hsieh, W.-T.; Lai, C.-H.; Shen, J.-Y.; Jiang,
C.-M.; Duan, H.-S.; Chou, P.-T. ChemPhysChem 2008, 9, 2221.
(31)Kohen, A.; Klinman, J. P. Acc. Chem. Res. 1998, 31, 397.
(32)Hsieh, W.-T.; Hsieh, C.-C.; Lai, C.-H.; Cheng, Y.-M.; Ho, M.-L.;
Wang, K. K.; Lee, G.-H.; Chou, P.-T. ChemPhysChem 2008, 9, 293.
(33)Chen, Y.; Gai, F.; Petrich, J. W. J. Am. Chem. Soc. 1993, 115, 10158.
(34)Kwon, O.-H.; Zewail, A. H. Proc. Natl. Acad. Sci. U.S.A. 2008, 105,
E79.
Email: chop@ntu.edu.tw (P.-T. Chou.)
The authors declare no competing financial interests.
(35)Kwon, O.-H.; Lee, Y.-S.; Park, H. J.; Kim, Y.; Jang, D.-J. Angew. Chem.
Int. Ed. 2004, 43, 5792.
(36)Irie, M. J. Am. Chem. Soc. 1983, 105, 2078.
P.-T. Chou thanks the Ministry of Science and Technology, Taiwan for
the financial support.
(1)Tolbert, L. M.; Solntsev, K. M. Acc. Chem. Res. 2002, 35, 19.
(2)Hsieh, C.-C.; Jiang, C.-M.; Chou, P.-T. Acc. Chem. Res. 2010, 43, 1364.
(3)Tang, K.-C.; Chang, M.-J.; Lin, T.-Y.; Pan, H.-A.; Fang, T.-C.; Chen, K.-
Y.; Hung, W.-Y.; Hsu, Y.-H.; Chou, P.-T. J. Am. Chem. Soc. 2011, 133,
17738.
(4)Demchenko, A. P.; Tang, K.-C.; Chou, P.-T. Chem. Soc. Rev. 2013, 42,
1379.
(5)Park, S.; Kwon, O.-H.; Kim, S.; Park, S.; Choi, M.-G.; Cha, M.; Park, S.
Y.; Jang, D.-J. J. Am. Chem. Soc. 2005, 127, 10070.
(6)Kwon, J. E.; Park, S. Y. Adv. Mater. 2011, 23, 3615.
(7)Padalkar, V. S.; Seki, S. Chem. Soc. Rev. 2016, 45, 169.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 9
1
2
3
Table of Contents artwork
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 9 of 9
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The Excited-State Triple Proton Transfer Reaction
65x27mm (300 x 300 DPI)
ACS Paragon Plus Environment