Design and concise synthesis of a novel type of green fluorescent protein chromophore analogue

Article abstract of DOI:10.1021/ol301901e

A small molecular model compound for the green fluorescent protein chromophore was readily synthesized by a novel condensation reaction of (thio)imidate with imino-ester via an aziridine intermediate. This compound showed fluorescence in the solid and fro

Full text of DOI:10.1021/ol301901e

ORGANIC
LETTERS
2012
Vol. 14, No. 17
4406–4409
Design and Concise Synthesis of a Novel
Type of Green Fluorescent Protein
Chromophore Analogue
Masahiro Ikejiri,† Moe Tsuchino,† Yoshiko Chihara,† Takao Yamaguchi,‡
Takeshi Imanishi,‡ Satoshi Obika,‡ and Kazuyuki Miyashita*,†
Faculty of Pharmacy, Osaka Ohtani University, Nishikiori-Kita 3-11-1, Tondabayashi,
Osaka 584-8540, Japan, and Graduate School of Pharmaceutical Sciences,
Osaka University, Yamadaoka 1-6, Suita, Osaka 565-0871, Japan
miyasik@osaka-ohtani.ac.jp
Received July 10, 2012
ABSTRACT
A small molecular model compound for the green fluorescent protein chromophore was readily synthesized by a novel condensation reaction of
(thio)imidate with imino-ester via an aziridine intermediate. This compound showed fluorescence in the solid and frozen solution states but not in
the solution state. Its fluorescent property was successfully applied in the detection of dsDNA.
A number of functional fluorescent molecules have been
developed as useful imaging tools thus far, which, for
instance, can help visualize various biological events through
the highly sensitive detection of biologically important
molecules.1 The development of a green fluorescent protein
(GFP) is one of the most remarkable milestones in this
research field.2 Despite the enormous contribution of the
GFP to biological research, only a limited number of small
molecular model compounds have been reported for a
GFP chromophore.3À8 This is probably because the GFP
chromophore does not exhibit fluorescence when it is
outside the barrel structure of the GFP. It is well-known that
the conformation of the GFP chromophore is strictly re-
stricted to a Z-form in the barrel structure (Figure 1), which
enables the GFP to exhibit fluorescence.9 However, without
the barrel structure, fluorescent quenching of the GFP
chromophore is induced by molecular motion such as the
double bond isomerization of the 4-hydroxyphenylmethylene
† Osaka Ohtani University.
‡ Osaka University.
(1) For a recent review, see: Kobayashi, H.; Ogawa, M.; Alford, R.;
Choyke, P. L.; Urano, Y. Chem. Rev. 2010, 110, 2620–2640.
(2) GFP review:Zimmer, M. Chem. Rev. 2002, 102, 759–781.
(3) (a) He, X.; Bell, A. F.; Tonge, P. J. Org. Lett. 2002, 4, 1523–1526.
(5) Notable phenomena have been reported, in which the encapsula-
tion of a modified GFP chromophore induced fluorescence: (a)
Baldridge, A.; Samanta, S. R.; Jayaraj, N.; Ramamurthy, V.; Tolbert,
L. M. J. Am. Chem. Soc. 2010, 132, 1498–1499. (b) Cacciarini, M.;
Nielsen, M. B.; de Castro, E. M.; Marinescu, L.; Bols, M. Tetrahedron
Lett 2012, 53, 973–976.
˚
(b) Petersen, M. A.; Riber, P.; Andersen, L. H.; Nielsen, M. B. Synthesis
2007, 3635–3638. (c) Lincke, K.; Sølling, T.; Andersen, L. H.; Klærke,
˚
B.; Rahbek, D. B.; Rajput, J.; Oehlenschlæger, C. B.; Petersen, M. A.;
(6) Wu, L.; Burgess, K. J. Am. Chem. Soc. 2008, 130, 4089–4096.
(7) Kojima, S.; Ohkawa, H.; Hirano, T.; Maki, S.; Niwa, H.; Ohashi,
M.; Inouye, S.; Tsuji, F. I. Tetrahedron Lett. 1998, 39, 5239–5242.
(8) Recently, Jaffrey and co-workers reported fluorescent RNA
aptamers that bind modified GFP chromophores:Paige, J. S.; Wu,
K. Y.; Jaffrey, S. R. Science 2011, 333, 642–646.
(9) Niwa, H.; Inouye, S.; Hirano, T.; Matsuno, T.; Kojima, S.;
Kubota, M.; Ohashi, M.; Tsuji, F. I. Proc. Natl. Acad. Sci. U.S.A.
1996, 93, 13617–13622.
Nielsen, M. B. Chem. Commun. 2010, 46, 734–736. (d) Kartritzky, A. R.;
Yoshioka-Tarver, M.; El-Gendy, B. M.; Hall, C. Tetrahedron Lett. 2011,
52, 2224–2227.
(4) Tolbert’s group reported a series of GFP chromophore ana-
logues: (a) Tolbert, L. M.; Baldridge, A.; Kowalik, J.; Solntsev, K. M.
Acc. Chem. Res. 2012, 45, 171–181and references therein. (b) Baranov,
M. A.; Lukyanov, K. A.; Borissova, A. O.; Shamir, J.; Kosenkov, D.;
Slipchenko, L. V.; Tolbert, L. M.; Yampolsky, I. V.; Solntsev, K. M.
J. Am. Chem. Soc. 2012, 134, 6025–6032.
r
10.1021/ol301901e
Published on Web 08/16/2012
2012 American Chemical Society

Table 1. Reaction of Thioimidate 3a with Imino-Ester 2
run
temp (°C)
70
additive (equiv)
time (days)
yield (%)
Figure 1. Structures of the GFP chromophore and our model
compound 1.
1
2
3
4
5
6
7
none
7.0
4.0
7.0
3.0
2.5
1.0
1.0
55
80
none
60
reflux
80
none
tracea
58
BuSH (1.0)
EtOH (5.0)
AcOH (2.0)
MsOH (2.0)
moiety,9,10 and the resultant E-isomer exhibits extremely
weak fluorescence.11
80
43
reflux
rt to reflux
78
0b
We realized that this characteristic feature of the GFP
chromophore could lead to the development of a new
fluorescence switch with an on/off switching mechanism.5
On this basis, we designed a diphenylmethylene derivative
1 as a novel type of GFP chromophore model compound.
This compound can be regarded as a combination of the
Z- and E-forms of the benzylidene moiety, and accord-
ingly, it can be expected to not exhibit the fluorescent
quenching caused by the E-form isomer. Since certain
types of bridged diarylmethylene groups are known to
function as a shaft of light-driven molecular motors that
can be rotated using UV irradiation,12 the diphenylmethy-
lene moiety of 1 is also expected to rotate upon exposure to
UV irradiation to consume the excitation energy. We
suppose that this rotation could cause fluorescence to
switch off, and if it is controlled, it could turn fluorescence
on. To the best of our knowledge, there has been no report
on GFP chromophore model compounds that have such a
diarylmethylene moiety.
a Almost no reaction. b Complex mixture.
and 3a in hot toluene, although a long reaction time was
required (Table 1, runs 1 and 2). However, contrary to
expectations, the product was not obtained under refluxing
toluene (run 3). As this result appeared to suggest an
important role of the volatile methanethiol and/or ethanol
produced in the reaction pathway, we observed the reac-
tion in the presence of a protic additive (runs 4À7). It was
found that protic additives shortened the reaction time
and that acetic acid in refluxing toluene produced the best
result (run 6). The use of a stronger acid (methanesulfonic
acid) was found ineffective (run 7).
Table 2. Synthesis of Cyclic Imidazolinones 1bÀe
For the synthesis of compound 1, we initially attempted
to apply previously reported methods that were mainly
used for the syntheses of monoarylmethylene deriva-
tives,6,13 considering that no effective methods are known
to have been reported for synthesizing diarylmethylene
derivatives. However, we could not obtain 1a. Thus, we
endeavored to develop a novel synthetic method for
obtaining diarylmethylene derivatives and eventually iden-
tified a suitable concise condensation reaction with imino-
ester 2 and thioimidate 3a, with the understanding that a
highly reactive aziridine intermediate could transform into
the desired (diphenylmethylene)imidazolinone 1a, as
shown by the reaction given in Table 1. The product, 1a,
was obtained in 55À60% yield, as expected, by mixing 2
run
product 1
X
n
time (days)
yield (%)
ꢀ
(10) Voliani, V.; Bizzarri, R.; Nifosi, R.; Abbruzzetti, S.; Grandi, E.;
1
2
3
4
5
6
b
c
S
S
S
S
S
O
2.0
2.0
2.0
2.0
4.0
2.0
1.0
1.5
1.5
1.5
1.0
1.0
43
68
77
42
75
85
Viappiani, C.; Beltram, F. J. Phys. Chem. B 2008, 112, 10714–10722.
(11) Dong, J.; Abulwerdi, F.; Baldridge, A.; Kowalik, J.; Solntsev,
K. M.; Tolbert, L. M. J. Am. Chem. Soc. 2008, 130, 14096–14098and
references therein.
(12) Vicario, J.; Walko, M.; Meetsma, A.; Feringa, B. L. J. Am.
Chem. Soc. 2006, 128, 5127–5135.
d
e
e
d
(13) Baldridge, A.; Kowalik, J.; Tolbert, L. M. Synthesis 2010, 2424–
2436.
Org. Lett., Vol. 14, No. 17, 2012
4407

Under the conditions of run 6, listed in Table 1, cyclic
thioimidates 3bÀe (X = S) also reacted with 2 to give
bicyclic imidazolinones 1bÀe in moderate to good yields
(Table 2, runs 1À5). The reaction with imidate 3d (X = O)
was found to proceed similarly (run 6). The structure of the
reaction product 1d was confirmed by X-ray crystallo-
graphic analysis, as shown in Figure 2.
Figure 4. Photographs of fluorescence for 1aÀe. [A] Pictures of
solid 1aÀe under visible light (top panel) and 365 nm UV light
(bottom panel). [B] Pictures of 1.0 Â 10À3 M DMSO solutions of
1aÀe under 365 nm UV light (top) and those of the frozen state
(bottom). [C] Pictures of frozen solutions of 1c and d in benzene
(1.0 Â 10À3 M), biphenyl (1.0 Â 10À3 mol/kg), and 4-t-butylcy-
clohexanone (1.0 Â 10À3 mol/kg) under 365 nm UV light. The
DMSO and benzene solutions were prepared at rt and then
frozen at À20 °C. The biphenyl and 4-t-butylcyclohexanone
solutions were prepared at 90 °C and then solidified at rt.
Figure 2. X-ray-based ORTEP drawings of 1d. Ellipsoids are set
at 50% probability.
Regarding the reaction mechanism (Figure 3), the attack
of the imine nitrogen, and not the R-carbanion, of 2 on the
thioimidate 3a appears to be the first step, which forms the
iminium intermediate 4. The intramolecular aziridine for-
mation of 5 and concomitant aziridine ring cleavage of 6 is
expected to give product 1a, through which the diphenyl-
methylene group can migrate from nitrogen to R-carbon via
the aziridine intermediate 6. Although we could not isolate
the aziridine intermediate 6, Ayyangar and co-workers
reported an aziridine synthesis from the imino-ester 2.14
became fluorescent when frozen (Figure 4B). Benzylidene-
type model compounds are also known to exhibit fluores-
cence in a frozen solution state at substantially low
temperatures (ethanol at 77 K);7,9 however, 1bÀe showed
fluorescence even at higher temperatures from around À20 °C
(e.g., in DMSO and benzene) to room temperature (e.g., in
biphenyl and 4-tert-butylcyclohexanone) (Figure 4C). From
the present results, it seems that a fused six-membered ring
with the imidazolinone ring is essential, and a fused benzene
ring in conjugation with the imidazolinone ring expectedly
increases the fluorescent intensity. A table summarizing the
data of the fluorescent spectra for 1aÀe is given in the
Supporting Information (Table S1).
The fluorescent profiles of 1aÀe suggest, as expected,
that molecular motion, including the isomerization of the
double bond, rather than the aggregation of the molecules,
is related to the fluorescent switching phenomenon. The
molecular motion involving double bond isomerization is
not restricted in the solution state, which leads to effective
and nonfluorescent energy consumption. In contrast, in a
conformationally rigid system, such as in a crystalline state
and a frozen solution state, the rotation of the double bond
bearing large substituents such as phenyl groups is steri-
cally prohibited, resulting in fluorescence.
An X-ray crystallographic analysis of 1d (Figure 2)
revealed that the relationship between the phenyl ring at
the Z-position (Z-Ph) and the imidazolinone ring (IMN) is
flatter than that between the phenyl ring at the E-position
(E-Ph) and IMN. The dihedral angle between Z-Ph and
IMN is 31.4°, whereas that between E-Ph and IMN is
71.4°. This steric relation between Z-Ph and IMN of 1d is
not as flat as that of the GFP chromophore in the barrel
structure (the dihedral angle between the 4-hydroxyphenyl
ring and the IMN of the GFP chromophore is3.7°).16 Even
Figure 3. Possible reaction mechanism for 1a.
We next investigated the fluorescent properties of the
compounds 1 and found that 1bÀe exhibited fluorescence
under specific conditions (Figure 4). For 1bÀe, although
their solution state in various solvents did not produce
fluorescence, their crystalline state inducedfluorescence, as
shown in Figure 4A.15 In addition, although a solution of
1bÀe in DMSO was almost completely nonfluorescent, it
(14) Rao, M. N.; Holkar, A. G.; Ayyangar, N. R. Tetrahedron Lett.
1989, 30, 4717–4720.
(15) Several GFP chromophore analogues are known to show fluo-
rescence in the solid state: (a) Dong, J.; Solntsev, K. M.; Tolbert, L. M.
J. Am. Chem. Soc. 2009, 131, 662–670. (b) Chuang, W. T.; Hsieh, C. C.;
Lai, C. H.; Lai, C. H.; Shih, C. W.; Chen, K. Y.; Hung, W. Y.; Hsu,
Y. H.; Chou, P. T. J. Org. Chem. 2011, 76, 8189–8202.
(16) Quillin, M. L.; Anstrom, D. M.; Shu, X.; O’Leary, S.; Kallio, K.;
Chudakov, D. M.; Remington, S. J. Biochemistry 2005, 44, 5774–5787.
4408
Org. Lett., Vol. 14, No. 17, 2012

then, thisrelationbetween Z-Ph and IMN of 1dapparently
reveals the existence of some conjugation between them.
Encouraged by the results, we next investigated the
application of our compound 1 to the detection of double-
stranded DNA (dsDNA), based on the following consid-
erations: (1) the relatively flat structure of Z-Ph and IMN
could intercalate into dsDNA or bind at a minor groove of
dsDNA; and (2) such a phenomenon would restrict the
rotation of the diphenylmethylene group, acting as a switch
that would turn ON fluorescence as a consequence. On the
basis of this concept, we designed and synthesized 1f, which
had a hydrophilic and interactive unit with DNA, as shown
in Scheme 1. To prepare an imidate part, lactam 717 was
converted into 8 by methylation. The imidazolinone struc-
ture was constructed with imino-ester 2 and imidate 8 in the
same manner, and the hydroxyl group of the product 9 was
connected with the hydrophilic unit by a Mitsunobu reac-
tion after a conversion into 10. Finally, the dimethylamino
group of 11 was transformed into an ammonium salt to
afford 1f.
of 1f in the presence and absence of DNA (S/N g 500) is
significantly higher than, for example, that of ethidium
bromide (S/N = ∼10),18 which is generally employed in
dsDNA detection.
Figure 5. Fluorescent spectra and a picture of 1f in the presence
of dsDNA (0.01% of 1f (2.1 Â 10À4 M) and 0.025% of DNA in
water, λex = 327 nm). The same concentration of ethidium
bromide (EtzBr) was used as a standard for the relative intensity
(2.1 Â 10À4 M, λex = 330 nm, λem = 594 nm, 24 h).
Scheme 1
In summary, we designed and synthesized fluorescent
switchable GFP chromophore analogues 1 by employing a
novel condensation reaction. Interestingly, this reaction
accompanies an unprecedented migration of a diphenyl-
methylene group from nitrogen to R-carbon via an
aziridine intermediate. Several compounds in 1 showed
remarkable fluorescence in the crystalline and frozen
solution states, whereas they did not do so in the solution
state. This characteristic fluorescent behavior was success-
fully applied in the detection of dsDNA by employing 1f.
In the fluorescence of 1bÀe, dynamic double bond isomer-
ization appears to function as an effective switch for
fluorescence, which makes these compounds potentially
applicable to the detection of not only dsDNA but also a
variety of chemical and biological events.8
The fluorescent profile of 1f was examined in the
absence and presence of fish dsDNA, and the results are
shown in Figure 5. Similar to the other compounds, 1f did
not exhibit fluorescence in a solution state. However, in the
presence of dsDNA, 1f exhibited fluorescence even in a
solution state. The intensity of the fluorescence gradually
Supporting Information Available. Detailed experimen-
tal procedures, spectral data, and a CIF of 1d. This
material is available free of charge via the Internet at
http://pubs.acs.org.
increased and reached near maximum after 24 h (λem
:
551 nm). A noteworthy point is that, because 1f without
DNA is almost completely nonfluorescent, the S/N value
(18) Olmsted, J., III; Kearns, D. R. Biochemistry 1977, 16, 3647–
3654.
(17) Occhiato, E. G.; Ferrali, A.; Menchi, G.; Guarna, A.; Danza, G.;
Comerci, A.; Mancina, R.; Serio, M.; Garotta, G.; Cavalli, A.; De Vivo,
M.; Recanatini, M. J. Med. Chem. 2004, 47, 3546–3560.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 17, 2012
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