An efficient and concise method to synthesize locked GFP chromophore analogues

Article abstract of DOI:10.1016/j.tetlet.2016.10.021

A series of GFP analogues, which are fluorescent in the solid state at room temperature, but weakly fluorescent in solution, have been synthesized via an oxazolone formation process that involves a condensation reaction in the presence of a Lewis acid following a Knoevenagel condensation. A ring opened intermediate is formed which cyclizes readily upon heating to produce the imidazolinone. This method is faster, simpler and produces higher yields than alternative methods. A few analogues represent locked GFP derivatives where the exocyclic single bond rotation has been stopped. Weak fluorescence, even after stopping single bond rotation, indicates that restriction of conformation is not effectively controlled and that the double bond rotation is solely responsible for the major non-radiative pathway.

Full text of DOI:10.1016/j.tetlet.2016.10.021

Accepted Manuscript
An efficient and concise method to synthesize locked GFP chromophore ana-
logues
Soumit Chatterjee, Peter Karuso
PII:
S0040-4039(16)31324-7
DOI:
http://dx.doi.org/10.1016/j.tetlet.2016.10.021
TETL 48191
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
24 September 2016
4 October 2016
7 October 2016
Please cite this article as: Chatterjee, S., Karuso, P., An efficient and concise method to synthesize locked GFP
chromophore analogues, Tetrahedron Letters (2016), doi: http://dx.doi.org/10.1016/j.tetlet.2016.10.021
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Tetrahedron Letters
An efficient and concise method to synthesize locked GFP chromophore
analogues
*
Soumit Chatterjee and Peter Karuso
Department of Chemistry and Biomolecular Sciences. Macquarie University, Sydney, NSW 2109, Australia
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A series of GFP analogues, which are fluorescent in the solid state at room temperature, but
weakly fluorescent in solution, have been synthesized via an oxazolone formation process that
involves a condensation reaction in the presence of a Lewis acid following a Knoevenagel
condensation. A ring opened intermediate is formed which cyclizes readily upon heating to
produce the imidazolinone. This method is faster, simpler and produces higher yields than
alternative methods. A few analogues represent locked GFP derivatives where the exocyclic
single bond rotation has been stopped. Weak fluorescence, even after stopping single bond
rotation, indicates that restriction of conformation is not effectively controlled and that the
double bond rotation is solely responsible for the major non-radiative pathway.
Available online
Keywords:
Green Fluorescent Protein
HBDI
fluorescence
Knovenagel condensation
oxazolinone
2
009 Elsevier Ltd. All rights reserved.
2
1-23
The Green Fluorescent Protein (GFP) family is extensively
used in cell biology as a genetically encoded, fluorescent
phosphorous oxychloride, were tried but to no avail.
An
alternate route was explored via Knovenagel condensation of N-
1-6
24,25
marker.
In wild-type GFP, the chromophore is (Z)-5-(4-
(p-
HBDI). However, the isolated chromophore is non-fluorescent
acylglycines.
4
This TiCl mediated aldol condensation, was
26
successfully implemented by Tepe and co-workers and
subsequently by Chou and co-workers to condense indanones
with 2,3-dimethylimidazolin-4-one, for example going directly
hydroxybenzylidene)–3,5-dihydro-4H-imidazol-4-one
7
–
4
8-10
(
f
< 10 ) in contrast to the protein (
f
= 0.8).
This has been
27
found to be due to non-radiative pathways ( and  rotation in the
excited state) for the free fluorophore that is not available to the
chromophore inside the constrained GFP -barrel.
from compound 1f to 4f.
11-14
Although
numerous structural analogues of the GFP chromophore have
been prepared, only a few synthetic routes are utilized. In
general, these routes start with Erlenmeyer azalactone synthesis,
in which the reaction of N-acylglycines with arylaldehydes under
the influence of sodium acetate in acetic anhydride results in the
formation of 4-arylidene-5-oxazolinones (Scheme 1). Direct
condensation of the oxazolinones with primary amines in the
presence of a base is the default approach for synthesizing the
9,15-17
final 4-arylidene-5-imidazolinones (Scheme 1, Route A).
An
indirect approach proceeding via a cinnamide (3a), followed by
1
8-
dehydration is generally higher yielding (Scheme 1, Route B).
20
In our hands, route B was more time efficient with more than
double the yield obtained when starting from p-
hydroxybenzaldehyde despite the extra synthetic step. The reason
for this was the quantitative conversion of 2a to 3a, which
required no purification (other than filtration) and the second step
simply requires column chromatography. However, the
Erlenmeyer method (the first step in Scheme 1) fails with
arylketones due to their poor reactivity compared to aldehydes. A
number of variations, including catalytic lead acetate, bases and
Scheme 1. Synthesis of phenyl substituted p-HBDI
—
——
*
Corresponding author. Tel.: +61-2-9850-8290; fax: +61-2-9850-8313; e-mail: peter.karuso@mq.edu.au

2
1
9,20
However, 2,3-dimethylimidazolin-4-one is difficult to
synthesize compared to 2-phenyloxazolinone. The former
requires three steps starting from N-methylacetamide and
literature procedures failed.
Fortuitously, it was discovered
that drying a TLC plate with a hot-air gun turned the colorless
acrylamide spot yellow. Elution of this spot separated it into two
yellow bands that corresponded to 4b and 2b (ESI-MS).
Optimization of this reaction revealed that heating the solid
acrylamides (3) in a furnace at 350 °C for 1 min (Scheme 2) led
to formation of the desired products in good to high yield (Table
S4; ESI) and recovery of 2, which was recycled, led to a
cumulative yield of over 90% for all compounds. Some amount
of 2 was always formed regardless of how carefully water was
excluded. NMR spectroscopy indicated that only the Z-isomer
was formed for all imidazolinones (4) except 4b, where both
were formed. For example an NOE was observed between H8’
and H2” in 4d. This can only arise from the Z-isomer. The steric
hindrance associated with the 5- or 6-membered ring, as well as
restricted rotation of the exocyclic double bond play a key role in
formation of a single isomer.
2
4
chloroacetyl chloride compared to the latter which can be
synthesized on a gram scale from N-acylglycines (e.g. hippuric
acid), in one step without purification, simply by heating in acetic
28
anhydride for 30 minutes then pouring into ice/water.
With this background, we set about to design a better
synthesis of GFP chromophores that would be especially useful
for generating conformationally restricted HBDI analogues via
29
oxazolinone formation.
A series of oxazolinones (2) were synthesized by the
condensation of arylketones (1a-h) with phenyloxazolone in
good yield (Scheme 2). NMR analysis showed that for a few
oxazolinones (2), both Z- and E- isomers were formed (ESI).
Keeping in mind that conversion to the final imidazolinone could
lead to isomerization and that any isomers could be separated at
the end, these were not separated. To our dismay, refluxing
oxazolinones (2) in ethanol with aqueous methylamine did not
Steady state absorption and emission spectra were then
recorded for each compound (Table 1). Compound 4c was non-
fluorescent but 4f was 30 times more fluorescent than p-HBDI
and had the highest quantum yield of all the compounds
synthesized.
8
form the corresponding imidazolinones (4) but rather, a white
precipitate which was shown to be the ring opened N-alkyl-2-
acylamino-3-arylacrylamides (3b-h). Formation of this type of
compound has previously been reported by both Chien and co-
Table 1. Steady state absorption and emission maxima and
quantum yields for imidazolinones (4) in different solvents
19,20
workers and Diederichsen and co-workers.
opened products (3) was optimized by stirring the oxazolinones
2) in dichloromethane at room temperature and adding
The yield of ring-
max
max
Compound
Solvent
abs
em
f
-
4
(
(nm)
390
398
347
357
436
436
379
379
391
403
381
381
377
400
400
(nm)
480
470
496
492
-
× 10
acetonitrile saturated with dry methylamine (2 equiv.). This
minimized formation of the corresponding carboxylic acids and
the reactions were generally complete within 15 minutes except
for 2e and 2h, which required one hour due to concomitant
deprotection of the phenol. The yields were generally 98-100%
and 80-90% for 3e and 3h.
4a
4b
4c
H O
1.9
4.2
2.2
1.6
-
2
tert-BuOH
2
H O
tert-BuOH
2
H O
tert-BuOH
-
-
4d
2
H O
497
460
552
530
481
461
463
488
489
14
19
8.7
13
30
25
5.4
6.6
4.4
tert-BuOH
4e
2
H O
tert-BuOH
4f
2
H O
tert-BuOH
acetonitrile
4g
4h
2
H O
tert-BuOH
All of the conformationally restricted analogues (4d-h) had
increased quantum yields compared to 4a-c (Table 1).
Femtosecond fluorescent transients for 4d, 4e, 4f and 4h in water
and tert-butanol (Fig. 1B and Fig. S8; ESI) indicated a very fast
relaxation process (< 5 ps) that was relatively solvent
independent. This is in agreement with the observed low
quantum yield (< 1%) of all the compounds (Table 1). The faster
component of < 1 ps predominated for all the compounds in both
solvents (Table S6; ESI).
To understand this rapid relaxation we conducted a series of
DFT calculations. Scanning the angle (Fig. 2) revealed that the
Z-isomer of 4e is more stable by ~2.5 kcal/mol than the E-isomer,
but more importantly, that the activation barrier to rotation is ~34
kcal/mol indicating that there is not isomerism in the ground
state. However, in the first excited state the potential energy
surface (PES) is relatively flat with a conical intersection with the
ground state at a  angle of ~ 100° (Fig. 2). The dip in energy of
1
the S state when the imidazolone and tetrahydronaphthalene
rings are perpendicular is associated with concomitant flexing of
2
3
the cyclohexene (R /R bridge; Scheme 2). This would allow a
facile return to the ground state (internal conversion) without
emission of a photon and could explain the low quantum yields
observed (Table 1) and the very fast decay recorded in
upconversion experiment (Fig. 1 and S8). This indicates that
restricting the  angle is not sufficient for a high quantum yield
Scheme 2. Efficient synthesis of restrained p-HBDI analogues
Dehydrative cyclization of acrylamide (3b) to imidazolinone
(4b) proved to be more difficult than anticipated and standard

and that the  angle also needs to be physically restrained to
fluorescent in the solid state but poorly fluorescent in solution,
indicating either the restriction of conformation () is not
sufficient or that other non-radiative pathways are involved.
However, these studies provide valuable information on the
design and synthesis of more fluorescent analogues and these
will be reported in due course.
generate room temperature HBDI analogues.
A
Acknowledgment
This research is supported by the Australian Research Council
(DP0877999) to P.K. We gratefully acknowledge the Australian
Research Council for the funding support of S.C. We thank Prof.
Anindya Datta for permitting us to use absorption
spectrophotometer, fluorimeter and fluorescence upconversion
instrument in his laboratory.
References and notes
3
00 350 400 450 500 550 600 650 700

1
.
Chudakov, D. M.; Matz, M. V.; Lukyanov, S.; Lukyanov, K. A.
Physiol. Rev. 2010, 90, 1103-1163.
/ nm
2
3
.
.
Tsien, R. Y. Annu. Rev. Biochem. 1998, 67, 509-544.
Green Fluorescent Proteins; Academic Press: San Diego, 1999;
Vol. 58.
B
4
5
.
.
Paige, J. S.; Wu, K. Y.; Jaffrey, S. R. Science 2011, 333, 642-646.
Addison, K.; Heisler, I. A.; Conyard, J.; Dixon, T.; Bulman Page,
P. C.; Meech, S. R. Faraday Discuss. 2013, 163, 277-296.
Cheng, C.-W.; Huang, G.-J.; Hsu, H.-Y.; Prabhakar, C.; Lee, Y.-
P.; Diau, E. W.-G.; Yang, J.-S. J. Phys. Chem. B 2013, 117, 2705-
6
.
2
716.
7
8
9
1
1
.
.
.
Cody, C. W.; Prasher, D. C.; Westler, W. M.; Prendergast, F. G.;
Ward, W. W. Biochemistry 1993, 32, 1212-1218.
Kojima, S.; Ohkawa, H.; Hirano, T.; Maki, S.; Niwa, H.; Ohashi,
M.; Inouye, S.; Tsuji, F. I. Tetrahedron Lett. 1998, 39, 5239-5242.
Chen, K.-Y.; Cheng, Y.-M.; Lai, C.-H.; Hsu, C.-C.; Ho, M.-L.;
Lee, G.-H.; Chou, P.-T. J. Am. Chem. Soc. 2007, 129, 4534-4535.
0. Hsieh, C.-C.; Chou, P.-T.; Shih, C.-W.; Chuang, W.-T.; Chung,
M.-W.; Lee, J.; Joo, T. J. Am. Chem. Soc. 2011, 133, 2932-2943.
1. Weber, W.; Helms, V.; McCammon, J. A.; Langhoff, P. W. Proc.
Natl. Acad. Sci. U.S.A. 1999, 96, 6177-6182.
0
1
2
3
4
5
Delay/ps
Figure 1. A) Normalized absorption (black) and emission
spectra (red) and B) femtosecond fluorescence upconversion
decay trace of 4h in water. ex = 400 nm; em = 490 nm
12. Mandal, D.; Tahara, T.; Webber, N. M.; Meech, S. R. Chem. Phys.
Lett. 2002, 358, 495-501.
1
1
1
1
1
1
1
3. Litvinenko, K. L.; Webber, N. M.; Meech, S. R. J. Phys. Chem. A
003, 107, 2616-2623.
4. Mandal, D.; Tahara, T.; Meech, S. R. J. Phys. Chem. B 2004, 108,
102-1108.
5. Dong, J.; Solntsev, K. M.; Poizat, O.; Tolbert, L. M. J. Am. Chem.
Soc. 2007, 129, 10084-11085.
2
1
6. Dong, J.; Solntsev, K. M.; Tolbert, L. M. J. Am. Chem. Soc. 2006,
1
28, 12038-12039.
7. Song, W.; Strack, R. L.; Svensen, N.; Jaffrey, S. R. J. Am. Chem.
Soc. 2014, 136, 1198-1201.
8. Baranov, M. S.; Lukyanov, K. A.; Ivashkin, P. E.; Yampolsky, I.
V. Synth. Commun. 2013, 43, 2337-2342.
9. Lee, C.-Y.; Chen, Y.-C.; Lin, H.-C.; Jhong, Y.; Chang, C.-W.;
Tsai, C.-H.; Kao, C.-L.; Chien, T.-C. Tetrahedron 2012, 68, 5898-
5
907.
2
2
0. Stafforst, T.; Diederichsen, U. Eur. J. Org. Chem. 2007, 899-911.
1. Cativiela, C.; Diaz De Villegas, M. D.; Meléndez, E. J.
Heterocycl. Chem. 1985, 22, 1655-1657.
2
2
2. ꢀꢁꢂa
ꢃ
sꢄ ꢅꢆꢇ ꢈꢉꢊeꢋ, M. P.; Cativiela, C. Tetrahedron 2004, 60, 885-
8
91.
3. Balakrishna, M. S.; Kaboudin, B. Tetrahedron. Lett. 2001, 42,
1127-1129.
Figure 2. DFT//bp86/SV(P) calculated energies for E-/Z-
2
2
4. Wu, L.; Burgess, K. J. Am. Chem. Soc. 2008, 130, 4089-4096.
5. Baldridge, A.; Kowalik, J.; Tolbert, L. M. Synthesis 2010, 14,
isomerization () for 4e, showing the ground state (S
0
; triangles),
first excited state (S ; squares) and the ground state energy at the
1
2
424-2436.
6. Sharma, V.; Lansdell, T. A.; Jin, G.; Tepe, J. J. J. Med. Chem.
004, 47, 3700-3703.
first excited state geometry (circles)
2
2
For the phenols (4a, 4e and 4h) pH variation studies of
27. Hsu, Y.-H.; Chen, Y.-A.; Tseng, H.-W.; Zhang, Z.; Shen, J.-Y.;
Chuang, W.-T.; Lin, T.-C.; Lee, C.-S.; Hung, W.-Y.; Hong, B.-C.;
Liu, S.-H.; Chou, P.-T. J. Am. Chem. Soc. 2014, 136, 11805-
absorption spectra were used to calculate the ground state pK
the respective compounds (Fig. S4-7, Table S5; ESI). The pK
a
of
of
a
1
1812.
8. Conway, P. A.; Devine, K.; Paradisi, F. Tetrahedron 2009, 65,
935-2938.
29. Kang, J.; Zhao, G.; Xu, J.; Yang, W. Chem. Commun. 2010, 46,
868-2870.
4
e (8.52) was an order of magnitude higher than that of the other
2
two, suggesting that the conjugate base of 4e is relatively
unstable, perhaps due to the presence of a flexible six membered
ring that can undergo facile conformational changes (chair-boat-
chair), even in the ground state.
2
2
In conclusion, we have developed a new, simple, facile and
high yielding route to GFP-chromophore analogues via
oxazolinone formation. The locked analogues are brightly

4
Supplementary Material
Synthesis of all the compounds, 1D NMR spectra, steady state
absorption and emission spectra and femtosecond fluorescence
decay
traces.

Highlights



Synthesis of a series of conformationally
locked GFP fluorophores was achieved
A new faster, simpler and higher yielding
synthesis of GFP fluorophores is reported
Double bond rotation is the major non-
radiative pathway in these analogues

6
Graphical Abstract
To create your abstract, type over the instructions in the
template box below.
An Efficient and Concise Method to
Synthesize Locked GFP Chromophore
Analogues
Leave this area blank for abstract info.
Soumit Chatterjee, and Peter Karuso
Fonts or abstract dimensions should not be changed or altered.