Facile synthesis of new L-arginine derivatives as substrates for trypsin-like proteases

Article abstract of DOI:10.1081/SCC-200030965

Facile and efficient synthetic routes for fluorogenic anilides of L-arginine have been developed. The amide bond formation uses phosphorous oxychloride as a condensing agent. The synthesis is simple, rapid, and affords excellent yields.

Full text of DOI:10.1081/SCC-200030965

SYNTHETIC COMMUNICATIONSw
Vol. 34, No. 19, pp. 3465–3471, 2004
Facile Synthesis of New L-Arginine
Derivatives as Substrates for
Trypsin-Like Proteases
*
Yongjun Gao and Yuan C. Lee
Department of Biology, Johns Hopkins University,
Baltimore, Maryland, USA
ABSTRACT
Facile and efficient synthetic routes for fluorogenic anilides of L-arginine
have been developed. The amide bond formation uses phosphorous oxy-
chloride as a condensing agent. The synthesis is simple, rapid, and affords
excellent yields.
Key Words: 2-Aminobenzoic acid; Fluorogenic substrates; Trypsin;
Synthesis.
*
Correspondence: Yuan C. Lee, Department of Biology, Johns Hopkins University,
144 Mudd Hall, 3400 North Charles Street, Baltimore, MD 21218, USA; E-mail:
yclee@jhu.edu.
3465
DOI: 10.1081/SCC-200030965
0039-7911 (Print); 1532-2432 (Online)
www.dekker.com
Copyright # 2004 by Marcel Dekker, Inc.

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Gao and Lee
INTRODUCTION
Proteolytic enzymes are involved in a diverse range of biological processes.
Their convenient and sensitive determination is of great interest and importance,
since they serve as indicators for the evaluation of several diseases.[1] Because
of their sensitivity, fluorogenic substrates have been conveniently used to probe
enzyme structures and mechanisms. Many synthetic chromogenic or fluoro-
genic substrates composed of peptidyl amides of aromatic amines are widely
used to detect and quantify protease and to define their amino acid specifici-
ties.[2] 2-Aminobenzoic acid (Abz), also known as anthranilic acid (AA), has
been frequently used as a convenient fluorescent group in internally quenched
fluorescent peptides as substrates for several proteolytic enzymes,[3] in which
the AA is usually at the C-terminal of peptides. Upon hydrolysis, AA can be
easily and sensitively measured by fluorescence. Recently, Panigraphi reported
the synergistic fluorescence enhancement of anthranilic acid with Tb.[4] These
studies prompted us to design and synthesize new N-acyl L-argininyl 2-amino-
benzoic acids as substrates for trypsin.
The synthesis of anilides of arginine has always been a difficult task
because of the poor nucleophilicity of the anilino group, making it difficult
to couple with the carboxyl group of arginine. The routine coupling
methods used in peptide synthesis, including the DCC method[5] and the
mixed anhydride method,[6] did not give satisfactory results. To remedy this
problem, two methods were developed in which phosphorus trichloride (the
phosphorazo-method)[7] or phosphorus pentaoxide in diethyl phosphite was
used.[8] Even with these methods, the yields varied between 30–44%. In
1991, Riijkers et al.[9] described the synthesis of p-nitroanilides of several
amino acids in excellent yields by using phosphorus oxychloride as a conden-
sing reagent. We adopted phosphorus oxychloride as the coupling reagent for
syntheses of a variety of anilides of L-arginine as substrates for trypsin.
RESULTS AND DISCUSSION
First, N-benzyloxycarbonyl-L-arginine-2-aminobenzoic acid (ZAAA)
was synthesized. The choice of this new fluorogenic substrate was based on
the fact that benzyloxycarbonyl (Cbz)-protected Arg-anilides showed high sus-
ceptibility to tryptic hydrolysis,[10] and their syntheses were efficient and devoid
of of racemerization.[9] As shown in Sch. 1, N-benzyloxycarbonyl-L-arginine
was coupled with ethyl 2-aminobenzoate by using phosphorus oxychloride in
pyridine as a coupling reagent. The reaction proceeded very smoothly and
rapidly to give 1 in high yield (92%). N-Benzyloxycarbonyl-L-arginine-2-ami-
nobenzoic acid 2 was obtained after deesterification of 1 with the yield of 82%.

L-Arginine Derivatives as Substrates for Trypsin-Like Proteases
3467
Scheme 1. Facile synthetic route for Z-L-arginine-2-aminobenzoic acid.
p-Nitroanilides of N-benzoyl-L-arginine and N-acetyl-L-arginine are often
used as substrates for proteolytic enzymes, but their synthesis from
N-benzoyl-L-arginine and N-acetyl-L-arginine is more difficult and trouble-
some because of multistep operation (including protection and deprotection
of guanidine group), racemization, and low yield.[10] In fact, we initially
tried to use conventional coupling reagents such as DCC, EDAC, and
EEDQ for anilide formation of N-benzoyl-L-arginine and N-acetyl-L-arginine
with ethyl 2-aminobenzoate, but failed to obtain useful results. Even with the
powerful condensing agent, phosphorus oxychloride, the reaction still pro-
ceeded rather slowly and the products were complex. Encouraged by the
facile preparation of compound 1, decided to use 1 as a starting material for
preparation of N-benzoyl-L-arginine-2-aminobenzoic acid, and N-acetyl-L-
arginine-2-aminobenzoic acid as new substrates for trypsin.
The schemes used to synthesize 6 and 7 are presented in Sch. 2. Ethyl
N-benzyloxycarbonyl-L-arginine 2-aminobenzoate 1 was successfully de-N-
protected with 3 M HBr in acetic acid to give the intermediate 3 in quantitative
yield. The product 3, without purification, was then benzoylated with benzoyl
chloride in the sodium carbonate solution to give compound 4, which was
deesterified with 0.2 M sodium hydroxide solution at room temperature for
5 h to give the product 6. In a similar manner, compound 7 was prepared by
acetylation of intermediate 3 with acetic anhydride, followed by hydrolysis
with 0.2 M sodium hydroxide solution.
EXPERIMENTAL
Material and General Procedure
N2-carbobenzyloxy-L-arginine, ethyl 2-aminobenzoate, and phosphorus
oxychloride were purchased from Aldrich (Milwaukee, WI). Melting points

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Gao and Lee
Scheme 2. Efficient synthetic routes for benzoyl-L-arginine-2-aminobenzoic acid and
acetyl-L-arginine-2-aminobenzoic acid.
were determined with a Fisher-Johns apparatus and were not corrected.
1H-NMR spectra were recorded with a Varian-400 NMR spectrometer at
resonance frequencies of 400 MHz. The chemical shifts (d) were in parts
per million (ppm) relative to Me4Si (0 ppm, 1H) as internal standards.
FABHRMS data were obtained at the mass spectrometry facility at Michigan
State University. Thin-layer chromatography (TLC) was performed on alumi-
num sheets coated with silica gel 60 F254 (thickness, 0.25 mm; E. Merck) and
spots were visualized under ultraviolet (UV) light. The ratios of solvents used
for TLC are expressed in vol/vol.
Ethyl N-Benzyloxycarbonyl-L-arginine 2-aminobenzoate (1)
N2-carbobenzyloxy-L-arginine (3.08 g, 10 mmol) was suspended in dry
pyridine (35 mL), mixed with ethyl 2-aminobenzoate (1.68 mL, 11.3 mmol),
and the mixture was cooled to 158C. After phosphorus oxychloride
(1.13 mL, 12 mmol) in pyridine (5 mL) was added dropwise to the mixture,
it was warmed to room temperature and stirred for 3 h until completion of
the reaction (monitored by TLC; BuOH/HOAc/H20 4 : 1 : 5). The reaction
mixture was then quenched with crushed ice and water (50 mL). The
product was extracted with EtOAc (3 ꢀ 100 mL) and the combined EtOAc
solutions were washed successively with saturated NaHCO3 solution
(100 mL) and NaCl solution (100 mL). The EtOAc solution was evaporated
in vacuo, and the residue was coevaporated with toluene, EtOAc, followed
by MeOH to remove residual pyridine. The crude product was suspended in

L-Arginine Derivatives as Substrates for Trypsin-Like Proteases
3469
diethyl ether, filtered, and washed with diethyl ether (3 ꢀ 50 mL) to give a
white powder (4.18 g) in a yield of 92%. This compound was characterized
1
by H NMR and HRMS.
1H NMR (400 MHz, DMSO-d6) d (ppm): 1.59–1.93 (m, 4 H, NHCH2-
CH2CH2), 2.09 (m, 3 H, CH3), 3.13 (m, 2 H, NHCH2CH2CH2), 3.85 (t, 2 H,
OCH2CH3), 4.08 (s, 1 H, a-H), 5.10 (s, 2 H, OCH2Ph), 7.21 (m, 2 H, NH),
7.32–7.44 (m, 5 H, ArH), 7.64 (br, 1 H, ArH), 7.87 (br, 1 H, NH), 7.99 (br,
1
1 H, ArH), 8.09 (d, J ¼ 7 Hz, ArH), 8.49 (d, J ¼ 8.4 Hz, H, NH).
HRMS (FAB þ ) calcd for C23H30N5O5 [M þ H]þ 456.2247, found:
456.2248.
N-Benzyloxycarbonyl-L-arginine-2-aminobenzoic acid (2)
Compound 1 (980 mg, 2.15 mmol) was dissolved in a mixture of 15 mL of
0.2 M NaOH and 15 mL of EtOH. The mixture was stirred at room tempera-
ture for 7 h, when TLC (EtOAc : HOAc : H2O 3 : 2 : 1) showed that the starting
material disappeared completely. The solvent was concentrated to 5 mL and
mixed with 50 mL EtOH. After adjusting the pH to 6.5 with 1 M HCl, the solu-
tion was evaporated to give a powder, which was shaken with EtOH (50 mL).
The NaCl precipitate formed was filtered off, and the filtrate was concentrated
to give a white solid. Further purification was performed by gel filtration with
a LH-20 column (185 ꢀ 5 cm) in 95% ethanol. The fractions containing the
product were pooled and concentrated to give a white powder (835 mg).
1
M.p.: 154–1558C. Yield: 82%. This compound was characterized by H
NMR and HRMS.
1H NMR (400 MHz, DMSO-d6) d (ppm): 1.59–1.82 (m, 4 H, NHCH2-
CH2CH2), 3.08 (m, 2 H, NHCH2CH2CH2), 4.03 (s, 1 H, a-H), 5.03 (s, 2 H,
OCH2Ph), 6.97 (br, 1 H, NH), 7.24–7.34 (m, 6 H, ArH), 7.81 (s, 1 H, ArH),
7.99 (d, J ¼ 7 Hz, 1 H, ArH), 8.45 (br, 1 H, NH).
HRMS (FAB þ ) calcd for C21H26N5O5 [M þ H]þ 428.1934, found:
428.1935.
N-Benzoyl-L-arginine-2-aminobenzoic acid (6)
Ethyl N-benzyloxycarbonyl-L-arginine-2-aminobenzoate (1.60 g,
3.51 mmol) was dissolved in 25 mL of 3 M HBr in acetic acid. The mixture
was stirred at room temperature for 4 h, when TLC (EtOAc : HOAc : H2O
3 : 2 : 1) showed a total disappearance of the starting material. The reaction
mixture was evaporated and coevaporated with 95% EtOH (2 ꢀ 50 mL) to
give a pale yellow solid (1.5 g). It was used in the next step without further
purification. (TLC : EtOAc : HOAc : H2O 3 : 2 : 1 showed one spot.)

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Gao and Lee
The above solid (1.50 g) was dissolved in water (45 mL) and then cooled
to 08C. After the addition of benzoyl chloride (0.4 mL) and ether (45 mL) to
the solution, an aqueous solution of sodium carbonate (518 mg in 5.3 mL)
was added dropwise into it with vigorous stirring over a period of 15 min.
The reaction mixture was kept for 1 h at 08C and then for 5 h at room tempera-
ture. TLC (EtOAc : HOAc : H2O 3 : 2 : 1) showed complete disappearance of
the starting material and formation of a new product. The ether was
removed by evaporation. The solution was adjusted to pH 7 with 2 M HCl
and was evaporated to dryness, and mixed with ethanol (100 mL). After the
NaCl precipitate was filtered off, the ethanol solution was evaporated to
give a pale yellow solid. It was used in the next step without further purifi-
cation. TLC (EtOAc : HOAc : H2O 3 : 2 : 1) showed one spot.
The residue was dissolved in a mixture of 0.2 M NaOH (80 mL) and
EtOH (80 mL), and after stirring at room temperature for 5 h, TLC
(EtOAc : HOAc : H2O 3 : 2 : 1) showed that a main product was formed. The
volume was then reduced to 20 mL, and EtOH (100 mL) was added. After
adjusting the pH to 6.5 with 1 M HCl, the solution was evaporated to give
a powder, which was mixed with EtOH (50 mL). After the NaCl preci-
pitate was filtered off, the ethanol solution was concentrated to give a
white powder. Further purification was achieved by LH-20 gel filtration
(185 ꢀ 5 cm in 95% EtOH). Final product was obtained as a white
solid (992 mg). The overall yield after the three-step operation was
1
65%. M.p.: 182–1838C. This compound was characterized by H NMR and
HRMS.
1H NMR (400 MHz, DMSO-d6) d (ppm); 1.57–1.96 (m, 4 H, NHCH2-
CH2CH2), 3.11 (m, 2 H, NHCH2CH2CH2), 4.51 (s, 1 H, a-H), 6.97 (br, 1 H,
NH), 7.28–7.52 (m, 6 H, ArH), 7.90–7.98 (br, 3 H, ArH), 8.46 (d, J ¼ 8.4 Hz,
1 H, ArH), 8.75 (br, 1 H, NH).
HRMS (FAB þ ) calcd for C20H24N5O4 [M þ H]þ 398.1828, found:
398.1829.
N-Acetyl-L-arginine-2-aminobenzoic acid (7)
The procedure for the synthesis of 7 was similar to that for 6, except for
using 1.64 mmol of 3, 0.18 mL of acetic anhydride, and an aqueous sodium
carbonate solution (280 mg in 2.8 mL H2O). After deesterification with
0.2 M NaOH, product 7 was obtained. It should be noted that product 7 was
difficult to dissolve in 95% EtOH. Further purification was performed by
gel filtration with a Sephadex G-25 column (185 ꢀ 5 cm, 0.1 M HOAc as
eluent). Final product was obtained as a white solid (390 mg). M.p.: 195–
1968C. The overall yield was 60%.

L-Arginine Derivatives as Substrates for Trypsin-Like Proteases
3471
Characterization data by 1H NMR and HRMS are as follows.
1H NMR (400 MHz, DMSO-d6) d (ppm): 1.59–1.83 (m, 4 H, NHCH2-
CH2CH2), 1.93 (s, 3 H, CH3CO), 3.16 (m, 2 H, NHCH2CH2CH2), 4.23
(s, 1 H, a-H), 6.97 (m, 1 H, NH), 7.29 (m, 1 H, ArH), 7.78–8.01 (m, 3 H,
ArH, NH), 7.99 (br, 1 H, ArH), 8.40–8.50 (br., 2 H, NH), 8.72 (br., 1 H, NH).
HRMS (FABþ) calcd for C15H22N5O4 [M þ H]þ 336.1672, found:
336.1671.
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Received in the USA June 2, 2004