Facile preparation of N-Tosyl-L-Phenylalanine chloromethyl ketone

Article abstract of DOI:10.2174/157017809790443005

A method for the facile preparation of N-tosyl-L- phenylalanine chloromethyl ketone was developed. L- Phenylalanine was tosylated and converted to the activated ester of p-nitrophenol. Subsequent reaction with dimethylsulfoxonium methylide and treatment with lithium chloride and methanesulfonic acid afforded the α-chloroketone.This practical procedure avoided the use of toxic and explosive diazomethane.

Full text of DOI:10.2174/157017809790443005

648
Letters in Organic Chemistry, 2009, 6, 648-649
Facile Preparation of N-Tosyl-L-Phenylalanine Chloromethyl Ketone
Bing-Lei Gao, Shu-Tao Ma and Zhao-Peng Liu*
Department of Organic Chemistry, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, P. R. China
Received May 11, 2009: Revised October 09, 2009: Accepted October 14, 2009
Abstract: A method for the facile preparation of N-tosyl-L-phenylalanine chloromethyl ketone was developed. L-
Phenylalanine was tosylated and converted to the activated ester of p-nitrophenol. Subsequent reaction with
dimethylsulfoxonium methylide and treatment with lithium chloride and methanesulfonic acid afforded the ꢀ-
chloroketone. This practical procedure avoided the use of toxic and explosive diazomethane.
Keywords: Serine protease inhibitors, ꢀ-chloroketone, N-tosyl-L-phenylalanine chloromethyl ketone, ylides, sulfur.
N-Tosyl-L-phenylalanine chloromethyl ketone (TPCK) is
an irreversible serine protease inhibitor. It inhibits
chymotrypsin and chymotrypsin-like proteases and it is not
active against trypsin [1]. TPCK also inactivates some
cysteine proteases such as bromelain, clostripain and papain
by alkylating the cysteine residue of the active site rather
than on the imidazole group of a histidyl residue, as in the
case of chymotrypsin [2]. TPCK is mostly used in mixtures
with other protease inhibitors throughout the protein
isolation to prevent protein degradation. It also exhibits wide
range of biochemical effects on cellular functions including
chemotaxis [3], apoptosis [4], gene expression [5], and cell
degranulation [6]. Despite the increasing interests on the
the steady-state amount of CH2N2 in a reaction vessel and
make these hazards manageable [8]. However, the
disadvantages and inconvenience with the use of the
dangerous and toxic diazomethane make this approach
problematic for large-scale manufacture of N-protected ꢀ’-
amino-ꢀ-chloroketones. Thus, a safe and convenient method
is of value for the preparation of TPCK. We report here a
new synthesis of TPCK applying dimethylsulfoxonium
methylide one-carbon homologation methodology as an
alternative to the use of diazomethane (Scheme 1).
L-Phenylalanine was reacted with p-toluenesulfonyl
chloride in aqueous NaOH solution to give N-tosyl-L-
phenylalanine in 85% yield [9]. Given the fact that the
NHTs
NH2
COOH
NHTs
COOH
p-Nitrophenol
TsCl
O
NaOH
DCC, DMAP, DMF
O
NO2
1
2
NHTs
O
NHTs
Me3S(O)I
tert-BuOK, THF
LiCl, MeSO3H
THF
O
S
Cl
TPCK
Me
Me
O
3
Scheme 1. Synthetic route to TPCK.
biological activities of TPCK, the chemical synthesis of
TPCK can not be easily achieved. The only method reported
in literature utilized the one-carbon homologation strategy
using the uniquely useful reagent diazomethane. The
tosylated L-phenylalanine was converted to its acid chloride,
which was then treated with diazomethane and finally with
hydrogen chloride [7]. Diazomethane is highly toxic, shock-
sensitive and explosive gas. It is usually used as an ethereal
solution generated in situ. For small and intermediate-scale
preparation, engineering solutions can be applied which limit
reaction of Boc-protected L-phenylalanine methyl ester with
Corey’s sulfur ylide generated from trimethylsulfoxonium
iodide afforded the chain-extended ꢁ-keto dimethyl-
sulfoxonium ylide in moderate yield and accompanied with
complete racemization [10], an activated ester of N-tosyl-L-
phenylalanine would be required for the efficient
transformation without epimerization of the protected amino
acid. Therefore, N-tosyl-L-phenylalanine was next converted
into its 4-nitrophenyl (Np) ester. Catalytic amount of DMAP
was necessary for this transformation when DCC was used
as the condensing reagent, and the activated 4-nitrophenyl
ester (Ts-Phe-ONp) 2 was obtained in 75% yield.
*Address correspondence to the author at the Department of Organic
Chemistry, School of Pharmaceutical Sciences, Shandong University, Jinan
250012, P. R. China; Tel: +86-531-88382006; Fax: +86-531-88382548;
E-mail: liuzhaop@sdu.edu.cn
Dimethylsulfoxonium methylide was generated by
treatment of trimethylsulfoxonium iodide with potassium
tert-butoxide in refluxing anhydrous THF. The reaction of
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© 2009 Bentham Science Publishers Ltd.

Facile Preparation of N-Tosyl-L-Phenylalanine Chloromethyl Ketone
Letters in Organic Chemistry, 2009, Vol. 6, No. 8
649
Ts-Phe-ONp 2 with 3 equiv of the resulting dimethylsulfo-
xonium methylide solution at 0°C for 1 h led to the ꢀ-keto
sulfur ylide smoothly. Extractive workup afforded
compound 3, which was used for the next transformation
without purification.
ethyl acetate. The combined extracts were washed with water
and brine. The solution was dried over anhydrous sodium
sulfate, filtered and concentrated by rotary evaporation to
afford the crude sulfur ylide 3 as a light yellow solid (4.50 g,
95%), which was carried on to the next step without further
purification.
Treating a THF solution of the ꢀ-keto sulfur ylide with
anhydrous hydrogen chloride resulted in the conversion of
the ꢀ-keto sulfur ylide to the ꢁ-chloroketone. As a practical
source of HCl, the combination of lithium chloride and
methanesulfonic acid proved to be effective, and the ꢁ-
chloroketone (TPCK) was obtained by recrystallization in
good yield and high optical purity (above 99.2% ee) [11]. Its
physical data (mp, optical rotation) are identical with an
authentic sample from Sigma-aldrich [12].
Crude 3 (4.50 g, 11.40 mmol) was dissolved in
anhydrous THF (60 mL) and the solution was cooled to 0 °C
under N2. Lithium chloride (0.60 g, 13.5 mmol) and
methanesulfonic acid (0.90 mL, 13.2 mmol) were added. The
temperature was slowly raised to 70 °C and stirring was
continued for 2 h. After cooling, the reaction was quenched
by addition of water (60 mL). The organic layer was
separated and ethyl acetate (30 mL) was added. The organic
solution was washed with saturated NaHCO3, water, brine
and dried (Na2SO4). Removal of solvent afforded the crude
chloroketone which was recrystallized with 95% EtOH to
give pure TPCK (2.25 g, 56%) as a white powder: mp
In summary, we have developed a new method for the
preparation of N-tosyl-L-phenylalanine chloromethyl ketone
(TPCK) in four steps with an overall yield of 36%. In all the
processes, no chromatographic purification is needed, and
what is more, the use of the dangerous diazomethane is
avoided. Thus, this provided a safe and practical way for the
facile preparation of TPCK.
20
106ꢂ108 °C; [ꢁ]D ꢂ86.5 (c 0.04, EtOH); IR (KBr): 3239,
1740, 1597, 1159, 1085 cm-1; 1H NMR (600 MHz, CDCl3) ꢀ
2.42 (s, 3H); 2.89—2.93 (m, 1H); 2.96—2.99 (m, 1H);
4.01—4.02 (m, 2 H); 4.30 (m, 1H); 5.03 (d, J = 7.8 Hz, 1H);
6.98 (dd, J = 7.2, 1.8 Hz, 2H); 7.21—7.25 (m, 5H); 7.55 (d,
J = 8.4 Hz, 2H); Anal. Calcd for C17H18ClNO3S: C, 58.03;
H, 5.16; N, 3.98. Found: C, 58.14; H, 5.10; N, 3.91.
EXPERIMENTAL SECTION
(S)-4-Nitrophenyl-2-(4-methylphenylsulfonamido)-3-
phenylpropanoate (Ts-Phe-ONp) 2: Ts-Phe-OH (6.39 g, 20
mmol) was dissolved in 20 mL DMF, p-nitrophenol (4.17 g,
30 mmol), DCC (4.95 g, 24 mmol) and DMAP (0.15 g, 1.2
mmol) were added. The reaction mixture was stirred at room
temperature overnight. The solid was removed by filtration
and the filtrate was added to 1 L water. Standing for 4 h, the
solid was collected by filtration and recrystallized from
CH2Cl2/cyclohexane to give 2 (6.60 g, 75%) as a yellow
ACKNOWLEDGEMENT
This work was supported by the Natural Science
Foundation of China (30873134).
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20
powder: mp 152-154 °C; [ꢁ]D -57.4 (c 0.10, CHCl3); IR
1
(KBr): 3227, 1768, 1592, 1513, 1208, 1090 cm-1; H NMR
(600 MHz, CDCl3) ꢀ 2.42 (s, 3H); 3.11-3.15 (m, 1H); 3.20-
3.25 (m, 1 H); 4.42-4.46 (m, 1H); 5.11 (d, J = 9.6 Hz, 1H);
6.82 (d, J = 9.0 Hz, 2H); 7.14-7.15 (m, 2H); 7.27 (d, J = 8.4
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(d, J = 9.6 Hz, 2H); Anal. Calcd for C22H20N2O6S: C, 59.99;
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HPLC: Shimadzu LC-10AT VP; Detector: SPD-10A VP; Column:
Chiralpak® IA (No. 1A00CE-MD018); Fluent: hexane:i-PrOH =
90:10; Flow rate: 1.0 mL/min; TPCK showed a single peak at 15.2
min, its optical purity was above 99.2%.
N-Tosyl-L-phenylalanine chloromethyl ketone (TPCK):
A solution of 1.0 M potassium tert-butoxide in anhydrous
THF (48 mL, 48 mmol) was added at room temperature to a
suspension of trimethylsulfoxonium iodide (8.01 g, 36
mmol) in anhydrous THF (45 mL). The mixture was heated
at reflux for 2 h under N2 atmosphere, and was then cooled
to 0 °C. A solution of 2 (5.28 g, 12 mmol) in anhydrous THF
(60 mL) was added dropwise and the resultant solution was
stirred for 1 h. The reaction was quenched with water (60
mL). The mixture was filtered through a short pad of celite
and the solvent was removed by rotary evaporation. The
concentrated mixture was rinsed into a separatory funnel
with 90 mL of water and extracted with three portions of
[10]
[11]
[12]
Physical data for an authentic sample of TPCK: Mp 106ꢂ109 °C;
[ꢁ]D20 ꢂ86.0 (c 0.04, EtOH).