Anisylazoformylarginine: A superior assay substrate for carboxypeptidase B type enzymes

Article abstract of DOI:10.1016/S0960-894X(02)00128-2

Anisylazoformylarginine (CH₃OC₆H₄-N=N-CO-Arg-OH) is rapidly hydrolyzed at the acyl-arginine linkage by the zinc-enzyme porcine carboxypeptidase B. The catalytic reaction is readily monitored spectrophotometrically by disappearance of the intense absorption (349 nm, ε 19,100) of the azo chromophore, which chemically fragments after substrate cleavage.

Full text of DOI:10.1016/S0960-894X(02)00128-2

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1193–1194
Anisylazoformylarginine: A Superior Assay Substrate for
Carboxypeptidase B Type Enzymes
William L. Mock* and Daniel J. Stanford
Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607-7061, USA
Received 13 December 2001; accepted 5 February 2002
Abstract—Anisylazoformylarginine (CH OC H –N¼N–CO–Arg–OH) is rapidly hydrolyzed at the acyl-arginine linkage by the
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zinc-enzyme porcine carboxypeptidase B. The catalytic reaction is readily monitored spectrophotometrically by disappearance of
the intense absorption (349 nm, e 19,100) of the azo chromophore, which chemically fragments after substrate cleavage. # 2002
Elsevier Science Ltd. All rights reserved.
Assay for catalytic activity of metalloproteases is much
simplified by employment of substrates incorporating
the anisylazoformyl residue (Fig. 1). As previously
attenuation of fibrinolysis.7,8 An internal-quench fluor-
escent-type of substrate for an enzyme having this spe-
cificity may be difficult to secure, due to absence of an
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exemplified for thermolysin and carboxypeptidases A
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excitation acceptor or donor in the P half of the sub-
strate. A preparation of substrate Aaf-Lys–OH was
and B, enzymic removal of the CH OC H –N=N–
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CO– moiety (acronym: Aaf) from a substrate is fol-
lowed by a spontaneous fragmentation of the released
acyl segment. The intense light absorption of the Aaf
previously reported. However, that procedure was
laborious in that it entailed phthaloyl-group protection
of the lysine side chain, and a two-step deprotection. We
now report an expedient synthesis of Aaf-Arg–OH that
avoids this difficulty. Catalytic activity of the new sub-
strate with carboxypeptidase B is superior to that of
Aaf-Lys–OH.
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chromophore (lmax ꢀ350 nm, e ꢀ19,000 M cm ) is
thereby abolished, allowing a convenient and sensitive
spectrophotometric detection of enzymic catalysis.
This assay tool is especially valuable for exopeptidases
that remove cationic amino acids (Lys–OH or Arg–OH)
from the C-terminus of oligopeptides, as during meta-
Due to the greater basicity of the guanidine function-
ality, it may be protected by protonation, while the a-
amino group in arginine is acylated during substrate
preparation (Fig. 2). The proto-chromophore reactant
CH OC H NHNHCOOPh is secured from commer-
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bolism of a number of hormones,
as well as in the
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cially available anisylhydrazine and diphenyl carbon-
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ate. The initial hydrazo acylation product is oxidized
directly to the azo substrate, which forms a crystalline
hydrochloride suitable for storage.
Figure 1. Structure of substrates and chemical transformation cata-
lyzed by zinc peptidases (Aaf-Phe–OH cleaved by carboxypeptidase A;
Aaf-Lys–OH and Aaf-Arg–OH cleaved by carboxypeptidase B).
*Corresponding author. Fax: +1-312-996-0431; e-mail: wlmock@uic.
edu
.
Figure 2. Preparation of Aaf-Arg–OH HCl.
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960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00128-2

1194
W. L. Mock, D. J. Stanford / Bioorg. Med. Chem. Lett. 12 (2002) 1193–1194
The new substrate was directly compared with Aaf-Lys–
OH with regard to cleavage by porcine carboxy-
peptidase B. Michaelis–Menten kinetics was observed as
previously in a continuous spectrophotometric analysis,
by following a decrease in substrate absorption at 350
mL of 1.23 N methanolic hydrochloric acid solution, to
a pH of ꢀ3 by pH paper, yielding a dark brown solu-
tion near the end of the titration. Solvent was removed
by rotary evaporation under reduced pressure. The
residue was redissolved in methanol and again rotary
evaporated (2ꢁ) to remove any excess hydrochloric
acid. The residue was taken up in a minimum amount of
hot methanol and was diluted to the cloud point with
ethyl ether. Orange rosettes formed; the material was
recrystallized by repeat precipitation from methanol by
ether, with drying by evacuation. This yielded 0.222 g
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nm: For Aaf-Arg–OH a value for the specificity con-
ꢂ
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À1
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stant kcat/K of 1.0ꢁ10 s
M
was obtained at 25 C,
m
pH 7.9 (0.05 M Tris buffer), which exceeds that for Aaf-
Lys–OH by a factor of 9.9-fold. The kinetic difference
between the two substrates largely resides in the value for
K (44ꢃ2 mM for Aaf-Arg–OH; k 44ꢃ1 s ).
À1
m
cat
(
53% from powder) of orange needles of p-anisylazo-
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formyl-l-arginine hydrochloride: mp 121–123 C (dec);
IR (KBr) 1653, 1708 cm ; UV–vis (H O) 349 nm, e
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Preparation
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19,100 M
cm
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H NMR (400 MHz, CD OD) d
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Nitrogen-purged dimethylformamide (4 mL) was added
to a stirred solution of l-arginine (1.32 g, 7.62 mmol) in
nitrogen-purged water (6 mL) maintained at 70 C.
1.73–1.80 (m, 2H), 1.87–1.97 (m, 1H), 2.07–2.16 (m,
1H), 3.24–3.28 (m, 2H), 3.92 (s, 3H), 4.54–4.58 (m, 1H),
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7.11 (d, 2H, J=9 Hz), 7.98 (d, 2H, J=9 Hz); C NMR
(100 MHz, CD OD) d 26.6, 30.0, 42.1, 54.6, 56.6, 115.9,
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Phenyl p-anisylhydrazoformate (2.93 g, 11.3 mmol) in
nitrogen-purged dimethylformamide (6 mL) was added.
The brown homogeneous solution was stirred under a
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127.5, 147.2, 158.8, 164.3, 166.4, 174.5. Anal. calcd for
C H N O Cl (372.8): C, 45.10; H, 5.68; N, 22.54.
Found: C, 44.96; H, 5.49; N, 22.22.
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nitrogen atmosphere for 1.5 h at 60 C. After cooling,
the solution was diluted with water (50 mL) and then
was extracted with ethyl acetate (3ꢁ50 mL). The aqu-
eous layer was submitted to rotary evaporation under
reduced pressure, and the residue was redissolved in
methanol and again concentrated, yielding 3.9 g of a
yellow residue of crude p-anisylhydrazoformyl-l-argi-
nine. This material was immediately taken up in
methanol (50 mL) and was stirred with excess manga-
nese dioxide (3.31 g, 38.1 mmol) at room temperature
for 2 h. The mixture was then passed through a pad of
diatomaceous filter agent to remove the Mn remainder.
The resulting clear orange solution was concentrated by
rotary evaporation under reduced pressure. The residue
was taken up in methanol (20 mL) and was diluted with
ethanol (20 mL), and the homogeneous solution was
again evaporated to dryness, yielding 2.24 g (87% from
l-arginine) of an orange, glassy powder. A portion of
this (0.424 g, >90% by NMR, ꢀ1.13 mmol) was dis-
solved in 5.0 mL of methanol and was titrated with 0.8
References and Notes
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8. Wang, W.; Boffa, M. B.; Bajzar, L.; Walker, J. B.;
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