Simple and stereocontrolled preparation of benzoylated phenylalanine using friedel - Crafts reaction in trifluoromethanesulfonic acid for photoaffinity labeling

Article abstract of DOI:10.3987/COM-13-12815

Simple and stereocontrolled preparation of benzoylated phenylalanine derivatives from optically pure phenylalanine using Friedel-Crafts reaction in trifluoromethanesulfonic acid (TfOH) is reported; these derivatives are useful for photoaffinity labeling. Protected or unprotected phenylalanine derivatives were converted to benzoyl derivatives in TfOH at room temperature in a short time without loss of optical purity. The reaction condition was applied to synthesize novel photoreactive phenylalanine derivative, which has two photophores (benzophenone and diazirine). The detail analysis of photo-irradiation for two different photophores contained phenylalanine derivative was also investigated. The Japan Institute of Heterocyclic Chemistry.

Full text of DOI:10.3987/COM-13-12815

HETEROCYCLES, Vol. 87, No. 10, 2013
2119
HETEROCYCLES, Vol. 87, No. 10, 2013, pp. 2119 - 2126. © The Japan Institute of Heterocyclic Chemistry
Received, 21st August, 2013, Accepted, 28th August, 2013, Published online, 12th September, 2013
DOI: 10.3987/COM-13-12815
SIMPLE
AND
STEREOCONTROLLED
PREPARATION
OF
BENZOYLATED PHENYLALANINE USING FRIEDEL ꢀ CRAFTS
REACTION IN TRIFLUOROMETHANESULFONIC ACID FOR
PHOTOAFFINITY LABELING
Yuta Murai,1,2 Lei Wang,1 Yasuyuki Muto,1 Yasuko Sakihama,1 Yasuyuki
Hashidoko,1 Yasumaru Hatanaka,3 and Makoto Hashimoto1,*
1Graduate School of Agriculture of Hokkaido University, Kita 9, Nishi 9, Kita-ku,
2
Sapporo 060-8589, Japan. Faculty of Advanced Life Science, Frontier Research
Center for Post-Genome Science and Technology, Hokkaido University, Kita 21,
3
Nishi 11, Kita-ku, Sapporo 001-0021, Japan. Graduate School of Medicine and
Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194,
Japan; E-mail: hasimoto@abs.agr.hokudai.ac.jp
Abstract –Simple and stereocontrolled preparation of benzoylated phenylalanine
derivatives from optically pure phenylalanine using FriedelꢀCrafts reaction in
trifluoromethanesulfonic acid (TfOH) is reported; these derivatives are useful for
photoaffinity labeling. Protected or unprotected phenylalanine derivatives were
converted to benzoyl derivatives in TfOH at room temperature in a short time
without loss of optical purity. The reaction condition was applied to synthesize
novel photoreactive phenylalanine derivative, which has two photophores
(benzophenone and diazirine). The detail analysis of photo-irradiation for two
different photophores contained phenylalanine derivative was also investigated.
Photoaffinity labeling is a valuable chemical method for studying the interactions of biologically active
molecules with their target proteins. In photoaffinity labeling, a covalent bond is formed between ligand
and target proteins upon irradiation with UV light.1 For instance, to study biologically active peptide
interactions for a target protein, a photoreactive !-amino acid will be required and is a powerful probe for
photoaffinity labeling. It has been reported for the preparation of benzophenone contained !-amino acid

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derivatives, which were synthesized from halomethyl derivatives of benzophenone and !-amino-
malonate equivalents using racemic2 or asymmetric3 methods (Figure 1: A). But there are no reports on
direct construction of benzophenone moiety on phenylalanine with FriedelꢀCrafts reaction due to low
solubility of phenylalanine derivatives in appropriate solvent for the reaction. Here, we report a useful
strategy for direct construction of benzophenone moiety on optically pure phenylalanine using a Friedelꢀ
Crafts reaction in trifluoromethanesulfonic acid (TfOH) with stereocontrolled manner (Figure 1: B).
Figure 1. Synthesis of benzoylated phenylalanine derivatives: (A) previous report, (B) this report.
We have recently reported that TfOH can effectively dissolve !-amino acid derivatives and catalyze
Friedel-Crafts reaction.4 We attempted direct construction of benzoylated phenylalanine by using TfOH
(Scheme 1). Optically pure unprotected phenylalanine (L-1 or D-1) was dissolved in TfOH at 0 °C and
stirred for 1 h, followed by the addition of benzoyl chloride (5, 8 eq). The reaction mixture was stirred at
room temperature. Although the starting material was consumed completely in 12 h, the reaction became
a complex mixture. The isolated 4-benzoylphenylalanine (6) was less than 30 % yield without loss of
optical purity (Table 1, Entries 1-2). No improvement was observed, when the reaction mixture was
heated (Table 1, Entries 3-4). Next, we considered to prevent the acid-base reaction between amino group
o
and TfOH. Optically pure N-Ac-L-Phe (L-2) was subjected to benzoylation at room temperature, 60 C
o
and 80 C. Although chemical yield was improved using L-2, the product (7) was loss of its optical
activity (Table 1, Entries 5-7). Optically pure N-Ac-L-Phe-OMe (L-3) was subject to Friedel-Crafts
reaction at room temperature. Consumption of L-3 and construction of benzophenone were observed
within 1 h. But the !-amino acid skeleton was converted to oxazole derivative (9) between acetamide and
methyl ester (Table 1, Entry 8). And we observed that L-3 did not form an oxazole derivative 9 without
benzoyl chloride 5 under the same conditions. These results indicated that oxazole formation was
promoted by in situ formation of mixed anhydride from benzoyl chloride and TfOH then the mixed
anhydride reacted with 3. It is consistent with the formation of oxazoles from N-acylamino acid methyl
esters in the presence of triflic anhydride.5 To prevent oxazole formation, we changed the protecting

HETEROCYCLES, Vol. 87, No. 10, 2013
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group for amino moiety from acetamide to trifluoroacetamide: the trifluoromethyl group is highly
electron-withdrawing and sterically-bulky to prevent imino-ester formation. N-TFA-L-Phe-OMe (L-4)
was treated at room temperature and completely consumed for 10 h. The yield of desired product L-8
improved by using compound L-4 without loss of optical purity (Table 1, Entry 9). The same treatment of
D-4 also afforded D-8 without loss of optical purity (Table 1, Entry 10).
Scheme 1. Simple and stereocontrolled preparation of benzoylated phenylalanine derivatives using
Friedel–Crafts reaction in trifluoromethanesulfonic acid.
Yield
Entry
Material
Condition
Product
ee (%)
(%)
28
29
26
18
44
58
59
63
45
41
1
2
L-1
D-1
L-1
L-1
L-2
L-2
L-2
L-3
L-4
D-4
rt, 12 h
rt, 12 h
L-6
D-6
L-6
L-6
L-7
L-7
L-7
9
98a
98a
98a
98a
74b
79b
9b
3
60 °C, 1 h
80 °C, 1 h
rt, 48 h
4
5
6
60 °C, 2 h
80 °C, 1 h
rt, 1 h
7
8
-
9
rt, 10 h
L-8
D-8
98c
98c
10
rt, 10 h
Table 1. The conditions and results of FriedelꢀCrafts reaction; Determination of enantiomeric excess:
a
b
Chirobiotic T (Astec), condition; MeOH/water = 1/9, as N-Ac-Phe(benzoyl)-OMe after
methylation with thionyl chloride in MeOH at room temperature for 24 h, CHIRALPAK AD
(Daicel), condition; hexane/iso-propanol = 7/3, Compound 8 converted to compound 6 with 1
M NaOH, condition the same of a.
c
(Trifluoromethyl)phenyldiazirine (TPD) is one of the important photophores in photoaffinity labeling.6
Although TPD is expensive and sensitive to high temperatureꢀunder strong acid condition,7 we have

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reported that TPD can be applied to the FriedelꢀCrafts reaction in TfOH at room temperature to
synthesize TPD contained (bis) homophenylalanine derivatives.8 Although both benzophenone and TPD
are excited at 350 nm, the reactive species generated from TPD was faster than that of benzophenone.9
But it has not been reported the construction of two photophores in one molecule and comprehensive
analysis of photoreactive properties. We tried to construct two kinds of photophore (benzophenone and
diazirine) on phenylalanine using our development synthesis. N-TFA-L-Phe-OMe (L-4) was treated with
benzoyl chloride derivative (11)10 in TfOH at room temperature until L-4 was consumed completely. The
desired product (L-12) was obtained in high yield without decomposition of diazirine moiety.
Deprotection of L-12 proceeded with 1 M NaOH to yield compound L-13 (Scheme 2). The same
treatment of N-TFA-D-Phe-OMe (D-4) afforded optically active D-13 as above without loss of optical
purity.
Scheme 2. Synthesis of compound (12) using Friedel-Crafts reaction in TfOH. Determination of
enantiomeric excess: Chirobiotic T (Astec), condition; methanol/water = 1/9.
We also confirmed the photoreactive properties of 13. We have already demonstrated that the
concentration of the diazirinyl compound had to be set to less than 1 mM to minimize isomerization to the
diazo compound.11 Methanolic solution of L-13 was irradiated with black light (100 W) for 10 min. The
maximum absorption at 350 nm for diazirine moiety of L-13 decreased with time-dependent manner
(Figure 2: A). The half-life of L-13 was determined to be 35 seconds. The photolyzed mixture was
subjected to UPLC-TOF-MS. The major product afforded a molecular weight of m/z 382, which was
consisted with methanol insertion to diazirine moiety and benzophenone is still remained (L-14a, Figure
2: B, I). One of the minor product afforded a molecular weight of m/z 414, which arose from the reaction
of both photophores with methanol (L-15a, Figure 2: C, I). It was slightly difficult to predict detail
structure of the reaction product in methanol from mass spectrometric analysis. Deuterated methanol
(CD3OD) was subjected to photolysis. For major product, which was activated only TPD, 4 mass number
increased, m/z 386 in CD3OD (Figure 2: B, II). The result indicated “D” and “OCD3” were incorporated to

HETEROCYCLES, Vol. 87, No. 10, 2013
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the diazirine moiety (L-14b). On the other hand, minor product, which was activated both TPD and
benzophenone, afforded 6 mass number increased peak m/z 420 (L-15b).
A)
B) major product
C) minor product
Figure 2. Photolysis of 1 mM of compound L-13 in methanol or deuterated methanol with 100 W black
light in 10 minutes. A) UV-vis spectral change of photolyzed mixture. B) Mass spectrum of
the major product photolyzed L-13 in methanol or deuterated methanol. C) Mass spectrum of
the minor prouct photolyzed L-13 in methanol or deuterated methanol.
These results indicated that benzophenone part increased two mass units. The photoirradiated structure
was considered to be generated by the bond formation between carbonyl carbon of benzophenone and
carbon of methanol (Figure 2C, II). Additionally we confirmed that methanolic or deuterated methanolic

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solution of benzophenone derivatives 6 did not decompose by photo-irradiation within 10 min. These
results indicated that diazirine photophore substitutions at p-position of benzophenone promoted
activation of benzophenone faster than unsubstituted one.
In summary, FriedelꢀCrafts reaction with TfOH as solvent and catalyst enabled us to employ simple and
stereocontrolled benzoylation of optically active phenylalanine to prepare photoreactive phenylalanine
derivatives. Additionally, this synthetic method was applicable to construction of two kinds of
photophore (benzophenone and diazirine) in phenylalanine derivative. And photolysis study of 13
revealed that we could control the reactivity between TPD and benzophenone in the same molecule by
photo-irradiation times. These simple preparations will be acceptable for all biochemist and contribute to
understanding peptide-receptor interactions with photoaffinity labeling.
EXPERIMENTAL
1H, 13C and 19F NMR spectra were measured by JEOL ECA 500 and EX 270 spectrometers for structural
determinations. ESI-TOF-MS spectra were measured with a Waters LCT Premier XE spectrometer.
General procedure for Friedel-Crafts reaction
TfOH (0.5 mL) was added to phenylalanine derivatives (0.1 mmol) at 0 °C in a tube with screw cap and
PTFE-faced rubber liner. After solution became homogeneous, benzoyl chloride (3~8 equivalents) was
added at 0 °C. The reaction mixture was stirred at indicated temperature until the stating material was
consumed completely, then poured into cold water and AcOEt. The organic layer was washed with
saturated aqueous NaCl, dried over MgSO4, and then filtrated. The filtrate was concentrated, and the
residue was subjected to silica column chromatography (AcOEt/MeOH/H2O = 8/1/0.5 for unprotected
Phe) to purify product.
4-(4-Benzoylbenzy)-5-methoxy-2-methyloxazole (9)
1H NMR (CDCl3) ! 7.77 (2H, d, J = 6.9 Hz), 7.73 (2H, d, J = 8.0 Hz), 7.57 (1H, t, J = 7.2 Hz), 7.46 (2H, t,
J = 7.7 Hz), 7.36 (2H, d, J = 8.6 Hz), 3.88 (3H, s), 3.78 (2H, s), 2.33 (3H, s); 13C NMR (CDCl3) ! 196.50,
155.03, 152.49, 144.52, 137.79, 135.56, 132.24, 130.48, 129.99, 128.44, 128.20, 61.15, 30.87, 14.30;
ESI-TOF-MS, M+H+ Calcd for C19H18NO3 308.1281, Found 308.1257.
(S)-Methyl
2-(2,2,2-trifluoroacetamido)-3-(4-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoyl)-
phenyl)propanoate (L-12)
TfOH (1 mL) was added to N-L-TFA-Phe-OMe (27.2 mg, 0.1 mmol) and 4-(3-(trifluoromethyl)-3H-
diazirin-3-yl)benzoyl chloride (98.3 mg, 0.396 mmol) at 0 °C. The reaction mixture was warmed to room
temperature and stirred until the stating material was consumed completely, then poured into cold water
and AcOEt. The organic layer was washed with saturated aqueous NaCl, dried over MgSO4, and then
evaporated. The crude product was purified by silica column chromatography (AcOEt/hexane = 1/8 to

HETEROCYCLES, Vol. 87, No. 10, 2013
2125
1
1/3) to yield L-12 (37.5 mg, 77%) as colorless solid. H NMR (CDCl3) ! 7.80 (2H, d, J = 8.6 Hz), 7.74
(2H, d, J = 7.9 Hz), 7.30 (2H, d, J = 8.6 Hz), 7.22 (2H, d, J = 7.9 Hz), 6.87 (1H, brs, NH), 4.96 (1H, dd, J
13
= 5.6, 5.9 Hz), 3.82 (3H, s), 3.37 (1H, dd, J = 14.2, 5.9 Hz), 3.24 (1H, dd, J = 14.2, 5.6 Hz); C NMR
2
(CDCl3) ! 194.94, 170.10, 156.59 (q, JCF = 38.2 Hz), 140.09, 138.31, 136.13, 133.22, 130.50, 130.13,
1
1
129.35, 126.35, 121.86 (q, JCF = 275.4 Hz), 115.48 (q, JCF = 289.4 Hz), 53.31, 53.07, 37.22, 28.40 (q,
2JCF = 39.7 Hz); F NMR (CDCl3) ! -66.19; ESI-TOF-MS, M+H+ Calcd for C21H16F6N3O4 488.1032,
19
Found 488.1022; [!]D +82.0 (c 1.0 CHCl3, 25 °C).
The same treatment of N-TFA-D-Phe-OMe (26.9 mg, 0.1 mmol) as described above gave D-12 (38 mg,
79%) as a colorless solid. [!]D -82.0 (c 1.0 CHCl3, 25 °C).
(S)-2-Amino-3-(4-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoyl)phenyl)propanoic acid (L-13)
Compound L-12 (29.4 mg, 0.06 mmol) in MeCN (2.0 mL), MeOH (1.0 mL) and 1 M NaOH (1.0 mL) was
stirred for 2 h at room temperature. The solution was evaporated, then the residue was purified by silica
column chromatography (AcOEt/MeOH/H2O = 4/1/1) to yield L-13 (21.0 mg, 92%) as colorless solid. 1H
NMR (CD3OD) ! 7.86 (2H, d, J = 8.6 Hz), 7.77 (2H, d, J = 7.9 Hz), 7.48 (2H, d, J = 7.9 Hz), 7.42 (2H, d,
J = 8.6 Hz), 4.23 (1H, t, J = 6.9 Hz), 3.41 (1H, dd, J = 14.7, 6.9 Hz), 3.23 (1H, dd, J = 14.2, 6.9 Hz); 13C
NMR (CD3OD) ! 196.66, 170.93, 141.30, 139.96, 137.61, 134.12, 131.67, 131.46, 130.86, 127.63,
1
2
19
123.41 (q, JCF = 274.3 Hz), 54.76, 37.17, 29.51 (q, JCF = 40.8 Hz); F NMR (CD3OD) ! -66.78;
ESI-TOF-MS, M+H+ Calcd for C18H15F3N3O3 378.1066, Found 378.1052; [!]D -6.0 (c 0.2 MeOH, 25 °C).
The same treatment of compound D-12 (30.3 mg, 0.06 mmol) as described above gave D-13 (22.1 mg,
94%) as a colorless solid. [!]D +6.0 (c 0.2 MeOH, 25 °C).
Photolysis of compound 13 in methanol
A 1 mM solution of compound 13 in MeOH or CD3OD (1 mL) was placed in a quartz cuvette. After the
sample atmosphere was replaced with nitrogen, photolysis was carried out with 100 W blacklight at a
distance 1 cm from the surface of light source. Spectra were measured after each minute, and then the
half-life was calculated from the decrease of the absorbance around 350 nm.
Determination of enantiomeric excess
Compound 6 and 8
1 mM MeOH solution of 6 was performed with Chirobiotic T (Astec), 4.6ꢁ250 mm, eluted with 10 %
MeOH/H2O; flow rate, 1 mL/min.; UV detection at 210 nm. L-6; tR = 25.4 min., D-6; tR = 27.1 min.
For 8; After deprotection of 8 with 1 M NaOH, which was performed in the same manner as compound 6.
Compound 7
After methylation of 7 with thionyl chloride in MeOH at room temperature for 12 h, 1 mM MeOH
solution of the compound was performed with CHIRALPAK AD (Daicel), eluted with

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hexane/iso-propanol 7/3; flow rate, 1 mL/min.; UV detection at 210 nm. L-7; tR = 10.8 min., D-7; tR = 7.8
min.
ACKNOWLEDGEMENTS
MH thanks the Fugaku Foundation for financial support. Part of this work was performed under the
Cooperative Research Program of “Network Joint Research Center for Materials and Devices”
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