Synthesis and properties of the p -Sulfonamide analogue of the green fluorescent protein (GFP) Chromophore: The mimic of GFP chromophore with very strong N-H photoacid strength

Article abstract of DOI:10.1021/acs.orglett.8b00257

The para-sulfonamide analogue (p-TsABDI) of a green fluorescent protein (GFP) chromophore was synthesized to mimic the GFP chromophore. Its S₁ excited-state pK_a^? value in dimethylsulfoxide (DMSO) is -1.5, which is strong enough to partially protonate dipolar aprotic solvents and causes excited-state proton transfer (ESPT), so it can partially mimic the GFP chromophore to further study the ESPT-related photophysics and the blinking phenomenon of GFP. In comparison with 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) (pK_a = 7.4, pK_a^? = 1.3 in water), p-TsABDI (pK_a = 6.7, pK_a^? = -1.5 in DMSO) is a better photoacid for pH-jump studies.

Full text of DOI:10.1021/acs.orglett.8b00257

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Synthesis and Properties of the p‑Sulfonamide Analogue of the
Green Fluorescent Protein (GFP) Chromophore: The Mimic of GFP
Chromophore with Very Strong N−H Photoacid Strength
Yi-Hui Chen, Robert Sung,† and Kuangsen Sung*
Department of Chemistry, National Cheng Kung University, Tainan, Taiwan
S
* Supporting Information
ABSTRACT: The para-sulfonamide analogue (p-TsABDI) of a green fluorescent
protein (GFP) chromophore was synthesized to mimic the GFP chromophore. Its
S1 excited-state pK*a value in dimethylsulfoxide (DMSO) is −1.5, which is strong
enough to partially protonate dipolar aprotic solvents and causes excited-state
proton transfer (ESPT), so it can partially mimic the GFP chromophore to further
study the ESPT-related photophysics and the blinking phenomenon of GFP. In
comparison with 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) (pKa = 7.4, pK*a = 1.3 in water), p-TsABDI (pKa = 6.7, pK*a = −1.5
in DMSO) is a better photoacid for pH-jump studies.
roton transfer is a cornerstone of acid/base-catalyzed
excitation at 395 nm, the neutral p-HBDI chromophore
Pchemical reactions, where the acid/base strength of undergoes a rapid ESPT, emitting the dominant GFP
catalysts plays an important role on the proton transfer.1
Among acid catalysts, N−H acids are not as common as
oxoacids, because N−H acids are much less acidic, such as NH3
(pKa = 41)2a vs H2O (pKa = 31.4)2b in dimethylsulfoxide
(DMSO), CH3C(O)NH2 (pKa = 25.5)2c vs CH3CO2H (pKa =
fluorescence at 508 nm with 80% fluorescence quantum
yield.7 It has been proposed that the hydrogen-bonding
network, which is comprised of the neutral p-HBDI
chromophore, an internal water molecule, Ser205, and
Glu222, promotes rapid ESPT from p-HBDI.8a,b This results
in an ESPT phenomenon in GFP where the fluorescence
wavelength is increased. This is indirectly proven in the non-
ESPT-related blue fluorescent emission of the double mutant
S205 V/T203 V of wtGFP.8c Unfortunately, there remains
some ambiguity surrounding the ESPT-related photophysics of
the wtGFP chromophore, such as charge transfer; thus, further
understanding of the GFP chromophore and its analogues is an
important issue.
12.6)2c in DMSO, and NH4 (pKa = 10.5 in DMSO, 9.2 in
+
H2O) vs H3O+ (pKa = −1.7 in H2O).2d
Since the pioneering work of Forster3a and Weller,3b it has
been found that the acid strength of some oxoacids can be
significantly boosted by photoexcitation. The first singlet (S1)
excited-state acid strength (pK*a ) of some oxoacids can be
several orders of magnitude (ΔpK*a ) stronger than that of its
ground state (pKa),4a such as phenol (pKa = 9.82, pKa* = 4 in
water),4b 7-cyano-2-naphthol (pKa = 8.75, pKa* = −1.3 in
water),4c and 8-hydroxypyrene-1,3,6-trisulfonate, HPTS (pKa =
7.4, pK*a = 1.3 in water).4d In addition, few N−H acids have
been found to be boosted by photoexcitation,4e such as
tryptophan (pKa = 9.30, pKa* = 7.8 in water).4f These
photoacids can be initiated by light to generate protons for
proton-transfer reactions at a specified instant in time, so a pH-
jump study has been one of important applications for these
photoacids, and HPTS is one of the best, highly used
photoacids for pH-jump studies.4g,h
One of the issues the ESPT-related photophysics of GFP
may help to solve is the GFP blinking phenomenon. It was
attributed to the temporary conversion to a nonfluorescent
form, which has been variously suggested to be triplet
formation,9a,b proton transfer,9c,d or Z/E isomerization.9e−g
The identity of the nonfluorescent form and the photophysics
of GFP blinking phenomenon remain unsolved.9h The GFP
blinking phenomenon occurs right after the ESPT of GFP.
How is the ESPT-related photophysics of GFP linked to the
nonfluorescent form where the GFP blinking phenomenon
happens? Uncovering the ESPT-related photophysics of GFP
may help us to understand the GFP blinking phenomenon,
because it is likely due to the ESPT-related photophysics.
Green fluorescent protein (GFP) and its mutants have
attracted interest as fluorescent biological labels in the last two
decades.5 The GFP chromophore is also a photoacid that
involves excited-state proton transfer (ESPT).4a The wild-type
GFP (wtGFP) is a globular protein of 238 amino acids that are
folded into an 11-stranded β-barrel with the p-hydroxybenzy-
lideneimidazolinone (p-HBDI) chromophore located at the
center of the β-barrel.6 The major electronic absorption (395
nm) of the wtGFP comes from the neutral form of the p-HBDI
chromophore. Its minor electronic absorption (475 nm) comes
from the electronic absorption of the p-HBDI anion.7 Upon
Received: January 24, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b00257
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
To study ESPT-related photophysics of GFP, p-HBDI was
attempted to reproduce the ESPT phenomenon of wtGFP, but
it failed to display ESPT, because the p-HBDI excited state
undergoes much faster radiationless transitions.10a In this paper,
we report that the p-sulfonamide analogue (p-TsABDI) of the
GFP chromophore, which displays ESPT, may qualify to be a
model for further study of ESPT-related photophysics of the
wtGFP choromphore. In addition, we also report that the pKa
of p-TsABDI can be significantly decreased by 8.2 through
photoexcitation at a specified instant in time, and that makes it
better than HPTS in pH-jump studies.4g,h
For the fluorescent emission spectrum of p-TsABDI, it
displays dual fluorescence in polar aprotic solvents (see Table 1
and Figure 1) The major peak is located at 425 nm (ΦF = 1.1 ×
For the synthesis of p-TsABDI (Scheme 1), reduction of p-1
to p-ABDI with SnCl2 was attempted,10b but it was not so
Figure 1. Normalized electronic absorption (dotted line) and
fluorescent emission spectra (solid line) of p-TSABDI in water
(cyan), DMSO (purple), acetonitrile (dark blue), or THF (red).
Scheme 1. Preparation of p-TsABDI
10−3) in DMSO, 437 nm (ΦF = 7 × 10−4) in acetonitrile, and
441 nm (ΦF = 1.1 × 10−3) in tetrahydrofuran (THF), and the
other is a weak shoulder fluorescence at 520 nm (ΦF = 6.0 ×
10−5) in DMSO, 512 nm (ΦF = 4.0 × 10−5) in acetonitrile, and
510 nm (ΦF = 1.0 × 10−5) in THF. This is quite unusual.
To check if the weak shoulder fluorescence of p-TsABDI is
caused by ESPT, it was compared with the fluorescence of the
p-TsABDI anion. The p-TsABDI anion was prepared by
titration of p-TsABDI in acetonitrile or DMSO with NaOH(aq)
with 3 μL of water added (see Figure 2 and Table 1). Its
successful in our hands. It is better to reduce p-1 to p-ABDI
with Pd-catalyzed hydrogenation. Then, p-TsABDI was
prepared by treating p-ABDI with 1.2 equiv of tosyl chloride
in the presence of NEt3.
Because p-TsABDI includes both the acidic sulfonamide and
the basic amidine moieties, it may exist as a neutral or
zwitterionic structure in an aprotic or protic solvent. In order to
identify the solution structures, ESPT phenomena, and
photoacid strength of p-TsABDI in an aprotic or protic solvent,
electronic absorption and fluorescent emission spectra of p-
TsABDI and its cation and anion were measured and compared
in a protic solvent (H2O), aprotic solvents, or mixed
CH3CN(aprotic)−H2O(protic) solvents (see Table 1).
Table 1. Photophysical Properties of Neutral, Zwitterionic,
Anionic, and Cationic p-TsABDI
Figure 2. Change in the electronic absorption (top) and in the
fluorescent emission spectra (bottom) for the titration of p-TsABDI
(1.4 × 10−5 M) in CH3CN with NaOH(aq) (0−9 × 10−4 M) with 3 μL
of water added.
lowest-energy electronic absorption is significantly red-shifted
to 450 nm (ε = 1.2 × 104 M−1 cm−1) in DMSO and 441 nm (ε
= 1.3 × 104 M−1 cm−1) in acetonitrile. It is consistent with
another similar experimental result that the electronic
absorption of the p-HBDI anion is red-shifted by ∼75 nm, in
comparison with that of p-HBDI.10d This result indicates that
the sulfonamide N−H acid of p-TsABDI is not dissociated in
polar aprotic solvents. The fluorescent emission of the p-
TsABDI anion is also significantly red-shifted to 501 nm (ΦF =
8.0 × 10−4) in DMSO and 507 nm (ΦF = 9.0 × 10−4) in
acetonitrile, which looks like the weak shoulder fluorescence of
p-TsABDI. However, according to the electronic absorption
spectra of p-TsABDI, p-TsABDI in dipolar aprotic solvents
does not have any trace of the p-TsABDI anion (see Figure 3).
Hence, we suggest that the weak shoulder fluorescence of p-
TsABDI in DMSO, acetonitrile, and THF is important
evidence for the ESPT of p-TsABDI (see Figure 3). We
suggest that the weak shoulder fluorescence of p-TsABDI
comes from its dissociated S1 excited state while the other
solvent
λabs (nm)
ε (M−1 cm−1
)
λf (nm)
ϕF (× 10−3
)
Neutral Compounds
1.1 × 104
a
DMSO
378
378
372
372
375
375
425
520
437
512
441
510
1.1
a
DMSO
1.1 × 104
0.06
0.7
a
CH3CN
CH3CN
8.6 × 103
a
8.6 × 103
0.04
1.1
a
THF
7.4 × 103
a
THF
7.4 × 103
0.01
Zwitterionic Compounds
9.1 × 103
H2O
408
502
0.1
Anionic Compounds
9.1 × 103
H2O
408
450
441
502
501
507
0.1
0.8
0.9
DMSO
CH3CN
1.2 × 104
1.3 × 104
Cationic Compounds
9.0 × 103
CH3CN
384
437
0.9
a
The solvent was dried.
B
DOI: 10.1021/acs.orglett.8b00257
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Figure 4. Change in the electronic absorption (top) and in the
fluorescent emission spectra (bottom) for the titration of p-TsABDI
(1.7 × 10−5 M) in CH3CN with HCl(aq) (0−1.2 × 10−4 M) with 3 μL
of water added.
Figure 3. Evidence of the ESPT of p-TsABDI. The electronic
absorptions (top) and the fluorescent emission spectra (bottom) of
the neutral p-TsABDI (solid line) and the p-TsABDI anion (dotted
line) in CH3CN (dark blue) or DMSO (red).
fluorescent emission spectra of p-TsABDI were compared with
those of the p-TsABDI anion in various ratios of mixed
CH3CN−H2O solvents (see Figure 5). Upon increasing the
major fluorescence of p-TsABDI is from its undissociated S1
excited state (see Scheme 2).
Scheme 2. Proposed Photoexcitation, Fluorescence, ESPT
and Proton Recombination of p-TsABDI
Figure 5. Electronic absorptions (top) and the fluorescent emission
(bottom) spectra of the neutral p-TsABDI (solid line) and the p-
TsABDI anion (dotted line) in mixed CH3CN−H2O solvents (dark
blue for acetonitrile, red for 2:1 CH3CN−H2O, lime green for 1:2
CH3CN−H2O, and purple for water).
To check if p-TsABDI exists as a neutral or zwitterionic
structure in an aprotic solvent, its electronic absorption was
compared with that of the p-TsABDI cation. The p-TsABDI
cation was prepared by titration of p-TsABDI in acetonitrile
with HCl(aq) with 3 μL of water added (see Figure 4 and Table
1). Its lowest-energy electronic absorption is slightly red-shifted
to 384 nm (ε = 9.0 × 103 M−1 cm−1) and its fluorescent
emission is also ∼437 nm (ΦF = 9.0 × 10−4). It is consistent
with another experimental result that the electronic absorption
of the p-HBDI cation is red-shifted by ∼20 nm, in comparison
with that of p-HBDI.10d This result confirms that p-TsABDI in
CH3CN exists in the neutral structure, instead of the
zwitterionic structure. If p-TsABDI in CH3CN exists in the
zwitterionic structure, protonation of p-TsABDI would make its
electronic absorption blue-shifted. The p-TsABDI cation is
supposed to have a proton added on the amidine nitrogen,
according to the experimental result that the pKa value for the
amidinium N−H proton of the p-HBDI cation is 1.4 in
water.10e
water percentage of mixed CH3CN−H2O solvents, the
electronic absorption at 372 nm decreases with a simultaneous
increase of the electronic absorption at 408 nm for p-TsABDI,
while the lowest-energy electronic absorption of the p-TsABDI
anion is blue-shifted with gradual migration from 441 nm to
408 nm. It means that p-TsABDI in mixed CH3CN−H2O
solvents exists only in two structures: the undissociated neutral
structure for the electronic absorption at 372 nm and the
zwitterion structure for the electronic absorption at 408 nm.
However, p-TsABDI in CH3CN and DMSO exists only in the
undissociated neutral structure, as shown in Figure 3. However,
the p-TsABDI anion always exists in the anionic structure,
whereas the ground state of the p-TsABDI anion is highly
stabilized by various percentages of water through hydrogen
bonding, causing blue-shifted migration of the electronic
absorption. When the water percentage of mixed CH3CN-
H2O solvents nears 100%, the lowest-energy electronic
absorptions of p-TsABDI and the p-TsABDI anion are both
located at 408 nm, indicating that p-TsABDI in H2O exists only
To check if p-TsABDI exists as a neutral or zwitterionic
structure in a protic solvent, the electronic absorption and
C
DOI: 10.1021/acs.orglett.8b00257
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
in the zwitterion structure and the electronic absorption spectra
of the zwitterionic p-TsABDI and the p-TsABDI anion are
almost the same.
TsABDI exists in the zwitterionic structure, whose electronic
absorption and fluorescent emission spectra are almost the
same as those of the p-TsABDI anion. In comparison with
HPTS (pKa = 7.4, pKa* = 1.3 in water),4d which is one of the
best, highly used photoacids for pH-jump studies, p-TsABDI
(pKa = 6.7, pK*a = −1.5 in DMSO) is a better photoacid than
HPTS for pH-jump studies. Unlike p-HBDI, p-TsABDI
displays ESPT so it qualifies to be a model for further study
of the ESPT-related photophysics of GFP. Hopefully, it could
be used to uncover the photophysics of the GFP blinking
phenomenon as well.
Upon increasing the water percentage of mixed CH3CN−
H2O solvents, both the major and the weak shoulder
fluorescent emissions of p-TsABDI at 437 and 512 nm and
the fluorescent emission of the p-TsABDI anion at 507 nm
decreases significantly. It is likely because hydrogen bonding
with water increases radiationless relaxation of their S1 excited
states.11 When the water percentage of mixed CH3CN−H2O
solvents reaches 100%, the fluorescent emissions of p-TsABDI
and the p-TsABDI anion are both located at 502 nm, indicating
that the emission spectra of the zwitterionic p-TsABDI and the
p-TsABDI anion are also almost the same.
ASSOCIATED CONTENT
■
S
* Supporting Information
To understand the ground-state acid strength of p-TsABDI,
Shi’s method12a was used, and the ground-state pKa values of
N−H acids of formamide and p-TsABDI in DMSO were
calculated using the combined methods of B3PW91/6-311+
+G(3df,2p)//B3LYP/6-31+G(d)//HF//CPCM/UA0. The
calculated ground-state pKa value of formamide in DMSO is
23.1, which is very close to the experimental value of 23.5.2d
The calculated ground-state pKa value of p-TsABDI in DMSO
is 6.7. This is consistent with the experimental result that p-
TsABDI, which exists as a neutral structure in CH3CN and
DMSO, turns into a zwitterionic structure in water.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.8b00257.
Acidity, synthesis, characterization, H and 13C NMR
spectra (PDF)
1
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: kssung@mail.ncku.edu.tw.
ORCID
According to the thermodynamics of the Forster cycle,4a an
excited state is a stronger acid than its ground state if the
absorption or emission spectrum of the conjugated base is
characterized by a red shift, relative to that of the conjugated
acid.4a Hence, the S1 excited state of p-TsABDI is a stronger
acid than its ground state. The Forster equation is
Kuangsen Sung: 0000-0003-3920-6674
Present Address
†Faculty of Family Medicine, Northern Ontario School of
Medicine, Ontario, Canada.
Notes
(hυ1 − hυ2)
*
The authors declare no competing financial interest.
pKa = pKa − N
A
2.3RT
ACKNOWLEDGMENTS
We thank the National Science Council of Taiwan for financial
support (No. NSC104-2119-M-006-013).
■
where frequency (υ) is suggested to be the average of
absorption frequency (υa) and fluorescence frequency (υf) for
better accuracy.12b In the case of p-TsABDI in DMSO,
c
c
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