PCl3-mediated synthesis of green/cyan fluorescent protein chromophores using amino acids

Article abstract of DOI:10.1039/c5nj03144c

Convenient synthesis involving aromatic amino acids, acetyl chlorides, and anilines in the presence of phosphorous trichloride has been established for the rapid synthesis of functionalized imidazolinones. With all the starting materials which are commercially available, low cost and stable, the construction of the target products has been accomplished via tandem transformations involving a key C-N coupling process, leading to the formation of two C(sp²)-N, one C=N, and one C=C bonds.

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PCl3-Mediated Synthesis of Green/Cyan
Fluorescent Proteins Chromophore using
Amino Acids
Cite this: DOI: 10.1039/x0xx00000x
Received 00th January 2012,
Accepted 00th January 2012
Thokchom Prasanta Singh and Raja Shunmugam*
DOI: 10.1039/x0xx00000x
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Figure 1. Imidazolinones containing flourescent protein in nature.
Convenient synthesis involving aromatic amino acids, acetyl
chlorides, and anilines in presence of phosphorous trichloride
has been established for the rapid synthesis of functionalized
imidazolinones. With all the starting materials which are
commercially available, low cost and stable, the construction of
the target products has been accomplished via tandem
transformations involving a key C−N coupling, leading to the
formation of two C(sp2)−N, one C=N, and one C=C bonds.
While each of these known protocols for imidazolinone synthesis
has specific features and/or advantages, the design of new tandem
reactions, using simpler starting materials to expand the structural
diversity of the products, is still highly desirable and remains a
challenge. In continuation of our interest in nitrogen containing
heterocycles,14a-b herein we report a new phosphorous trichloride-
Nitrogen containing heterocycles are of special interest in catalyzed tandem approach for the synthesis of 4-arylidene-5-
synthetic organic chemistry,1 since they occur in a wide variety of imidazolinones involving the assembly of N-chloroacetyl-amino
natural products. Among them, imidazolinone is a fundamental non- acids (1a’/b’) and anilines (3), wherein (1a’/b’) are generated in situ
aromatic naturally-occurring heterocycle that has been intensively by the direct use of the corresponding aromatic amino acids (1) and
used in the synthesis of functional materials and pharmaceuticals.2 acetyl chlorides (2) in one pot (Scheme 1).
The imidazolinone substructures are found to act as the
chromophores of the fluorescent proteins (FPs) in nature, for
example, green fluorescent protein (GFP), cyan fluorescent protein
(CFP), blue fluorescent protein (BFP) and for red kaede fluorescent
protein (RFP) (Figure 1).3a-c Imidazolinones show various biological
and pharmaceutical activities such as, angiotensin II receptor,4 dual
p38αMAPK and ERK1/2 inhibitors,5 and monoamine oxidase
(MAO) inhibitory.6 They are also used as herbicides,7 anticonvulsant
agents,8 anti-parkisonian,9 and anti-inflammatory agents.10 The
omnipresence of 4-arylidene-5-imidazolinones and their intrinsic
Scheme 1. Synthesis of 4-arylidene-5-imidazolinones in one-pot
system
photochemical phenomena have made it intriguing synthetic targets
and useful chemical models for investigating the mechanism of the
fluorescence proteins.11a-d Till date numerous structural analogues of
Originally, the reaction of L-tyrosine (1a), chloroacetyl
chloride (2), and aniline (3a) were employed to probe the expected
transformation. We then envisioned that cyclization of N-
chloroacetyl-L-tyrosine 1a’ with aniline 3a would give
imidazolinone 4a’ in presence of an appropriate catalyst. But to our
surprise, the final product was identified to be 4-arylidene-5-
12a-k
FPs have been reported,
however a literature perusal revealed
that only few synthetic routes have been utilized for the synthesis of
highly functionalized imidazolinone that allow substitutions at 2-
position upon a ring-closing condensation of α-amidoamides.13a-b
imidazolinone 4a,
a well known Green fluorescent protein
chromophore using PCl3 in acetonitrile and the expected
imidazolinone 4a’ was not obtained (Scheme 1).
Then, we decided to investigate the solvent, catalysts loading and
temperature effects on the efficiency of cyclization which could
contribute eventually to higher conversion of imidazolinone. As
shown in Table 1, comparison of different typical catalysts such as
PCl3, POCl3 and SOCl2 revealed that PCl3 was the optimal catalyst
(Table 1, entries 1−5). Increasing the amount of catalyst (2 mmol)
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did not improve the yield (entry 1), whereas decreasing the amount
of catalyst (0.5 mmol) decreased the yield (entry 2). The subsequent
examination of reaction media of different polarity, including
toluene, DMF, DCM, THF and ethanol demonstrated that a polar
aprotic solvent was better favoured by the model reaction, and
acetonitrile was among the best ones followed by THF (Table 1,
entries 1,4−8). Further, variation of the reaction temperature proved
that 50 °C was the favoured temperature (Table 1, entries 3 and 9).
The reaction did not occur in the absence of mentioned catalysts
even after 48 h stirring (entry 12). Finally, the optimized reaction
conditions were obtained in the presence of 1 equiv of PCl3 in
MeCN at 50 °C for 3 h under ambient air (entry 1). It is noteworthy
that only one regioselective imidazolinone is obtained by following
this protocol.
Table 1. Optimization of 4-arylidene-5-imidazolinones synthesisa
entry
solvent
catalyst
(mmol)
PCl3[2]
PCl3[0.5]
PCl3[1]
POCl3[1]
SOCl2[1]
PCl3[1]
PCl3[1]
PCl3[1]
PCl3[1]
PCl3[1]
PCl3[1]
No catalyst
temp
(°C)
50
50
50
50
50
110
130
40
yield(%)b
MeCN
MeCN
MeCN
MeCN
MeCN
Toluene
DMF
DCM
THF
EtOH
MeCN
MeCN
75
70
75
25
50
25
-
-
70
-
Optimal reaction conditions: 1a (2.0 mmol), 2 (2.4 mmol) and 3
(2.0 mmol) using PCl3 (1 mmol) in MeCN (2.5 mL) in 3 h.
Table 3. Synthesis of Cyan FP chromophores using L-tryptophan
80
40
70
50
75
-
aReaction conditions: 1a’ (2 mmol), 2 (2 mmol) and solvent (2.5
mL) stirred at given temperature for 2-10 h (TLC). bIsolated
yields.
Under the optimized reaction conditions, we began to explore the
scope of this annulation by employing various anilines (3a−i) to get
different GFP chromophores (Table 2). Thus, a small library of GFP
chromophores was established, in which anilines bearing electron-
donating groups such as −Me and −OMe on the phenyl ring
successfully provided the corresponding imidazolinone in good
yields (4b-e), whereas halogen-substituted anilines gave moderate
yields (4f-i). However, anilines containing electron withdrawing
group like -NO2, -COOH and –OH did not proceed to give the
products even after reacting for 24 h. Simultaneously, we also
focused our attention to the scope of another aromatic amino acid viz
L-tryptophan to prove the general applicability of this protocol for
the synthesis of imidazolinone. Thus, the reaction of L-tryptophan
and chloroacetyl chloride yielded N-chloroacetyl-L-tryptophan (1b’),
which was allowed to react with various anilines (3a-d) to afford the
corresponding imidazolinones 5a−d, which were again Cyan
fluorescent protein chromophores (Table 3). This assured the
broader scope of application of the present protocol toward
imidazolinone synthesis. Meanwhile, investigations on this protocol
for preparation of Blue FP chromophores using histamine are still
going on in our laboratory.
Optimal reaction conditions: 1b (2.0 mmol), 2 (0.2 mmol) and
3(2.0 mmol) using PCl3 (1.0 mmol) in MeCN (2.5 mL) in 3 h.
It is noteworthy that no imidazolinone was produced in the reaction
involving an alkylamine, such as methyl amine, benzylamine or 4-
butylaniline (Scheme 2).
Scheme 2. Attempted synthesis with an aliphatic amine
Further, we intended to functionalize 2-methyl substituted
imidazolinone 4 to get synthetic analogues of red Kaede FP
chromophore 7 by the formation of the π-conjugated product on
reaction with p-nitrobenzaldehyde. Following the reported
literature,[11d, 13b-c] we tested for different Lewis acids such as AlCl3,
FeCl3 and ZnCl2 as catalyst in different reaction media. However, the
best yields for our system was found using AlCl3 (5 mol %) in THF
Table 2. Synthesis of Green FP chromophores using L-tyrosine
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as solvent in excellent yields. Thus, 4a, 4f, and 4h were selected and
successfully converted to the corresponding synthetic analogues of
red Kaede FP chomophores 7a, 7b, and 7c on condensation with p-
nitrobenzaldehyde (1:1 ratio) in the presence of THF at 80 oC (Table
4).
Table 4. Synthesis of Red FP chromophores analogues
Scheme 3. Proposed Mechanism of Imidazolinone synthesis
In summary, we have described the application of L-tyrosine and L-
tryptophan to synthesis 4-arylidene-5-imidazolinones in a one-pot
reaction. In these reactions, at least four different active sites were
involved; one ring, one C=N bond, one C=C bond and two C-N
bonds were constructed with all reactants efficiently utilized in the
chemical transformation. To the best of our knowledge, no report on
the use of L-tyrosine and L-tryptophan as starting materials for the
synthesis of Green/Cyan FPs chromophore is known. Further,
functionalization of methyl from G/C FPs chromophore with p-
nitrobenzaldehde was successfully carried out giving synthetic
analogues of red Kaede FP chromophores. Undoubtedly, this
cycloannulation opens a new convenient and an effective way to
construct the 4-arylidene-5-imidazolinones from readily available
starting materials.
Reaction conditions: 2 (1.0 mmol), 6 (1.0 mmol) and AlCl3 (5
mol %) in 2.0 ml of THF at 80 oC for 4 h.
1
In the H NMR spectrum of 4a, a singlet at δ 7.01 was observed,
which could be assigned to the >C=C-H proton and singlet at 2.19
ppm for CH3 linked to C-2 correlating well with other 4-arylidene-5-
imidazolinones. In the 13C NMR spectrum, the signals at δ 16.08 and
160.46 were due to –CH3 linked to C-2 and tertiary C-2 linked to
two N atoms, respectively, and the characteristic peak at δ 115.8 was
assigned for allynic
C linked to imidazolone. The relative
configuration of the product was determined based on single-crystal
X-ray diffraction of 4a, figure 2. In compound 7a, there was no
1
methyl peak in H NMR and 13C NMR spectra, all the protons were
1
in aromatic region in H NMR. Analytical and spectral data further
supported the structures of all the newly synthesized compounds.
Experimental Section
Preparation of 4a:
In a 25 mL round bottom flask, L-Tyrosine 1a (2 mmol) was
dissolved in diethyl ether/water (1:1 v/v) mixture (2 mL) in an ice
bath (4 oC), and 2 mL of 4 M NaOH solution was added to mixture.
Then, chloroacetyl chloride (2 mmol) dissolved in 1.0 mL of diethyl
ether was added dropwise to the amino acid solution simultaneously
with aqueous 4 M NaOH solution 2 mL over a 20-minute period
under vigorous stirring. The aqueous layer was acidified to pH 1
with aqueous 4 M HCl and extracted with ethyl acetate. The
combined organic layers were washed with saturated NaCl solution,
dried with anhydrous Na2SO4, and concentrated under vacuum. To
this solid product, aniline 3a (2 mmol) in CH3CN (2.5 mL) were
taken. PCl3 (1.0 mmol) was added and refluxed for 2 hour, where the
reaction mixture turns yellow in colour as indicated by TLC. The
reaction mixture was poured into ice-cold water and extracted with
ethyl acetate (20 mL). The combined organic layer was washed with
water for three times, dried over anhydrous Na2SO4 and organic
layer was evaporated. The crude products were columned using
Figure 2. X-ray crystal structure of 4a at 50 % thermal ellipsoids.
A possible mechanism is proposed in Scheme 3. The reaction of 1a
and chloroacetyl chloride 2 leading to the formation of N-
chloroacetyl-L-tyrosine 1a’ in a basic solution, which undergoes
amination with aniline 3a initiated by PCl3 to generate intermediate
I. Intramolecular cyclization of the aniline NH with a nucleophilic
attack at the carbonyl group followed by elimination of H2O leads to
the intermediate II. Then, tertiary H of C-4 participate in double
bond extension leading to the elimination HCl to give intermediate
III, which further rearrange to give more stable product 4a.
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hexane/ethyl acetate (80:20) and were recrystallized from EtOH to
afford the pure product 4a in 75 % yield.
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E-mail: sraja@iiserkol.ac.in
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TOC Content:
PCl3-Mediated Synthesis of Green/Cyan Fluorescent Proteins Chromophore using
Amino Acids
Thokchom Prasanta Singh and Raja Shunmugam*
An efficient synthesis of green and cyan fluorescent proteins chromophore from L-tyrosine and L-
tryptophan using PCl3 has been successfully developed.
This journal is © The Royal Society of Chemistry 2013
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