psi(SO2NH) transition state isosteres of peptides. Synthesis and bioactivity of sulfonamido pseudopeptides related to carnosine.

Article abstract of DOI:10.1016/S0014-827X(99)00079-8

This paper reports the synthesis of tauryl dipeptides related to carnosine. In particular H-Tau-His-OH (5), H-Tau-His(pi-Me)-OH (6) and H-Tau-His(tau-Me)-OH (9) are described. The enzyme carnosinase has been isolated from pig kidney and after purification

Full text of DOI:10.1016/S0014-827X(99)00079-8

www.elsevier.nl/locate/farmac
Il Farmaco 54 (1999) 673–677
C(SO2NH) transition state isosteres of peptides. Synthesis and
bioactivity of sulfonamido pseudopeptides related to carnosine
c
Anna Calcagni a, Pier Giuseppe Ciattini a, Antonio Di Stefano b, Silvestro Dupre` ,
Grazia Luisi b, Francesco Pinnen b,*, Domenico Rossi a, Alessandra Spirito c
a Dipartimento di Studi Farmaceutici e Centro di Studio per la Chimica del Farmaco del CNR, Uni6ersita` ‘La Sapienza’, P. le Aldo Moro 5,
I-00185 Rome, Italy
b Istituto di Scienze del Farmaco, Uni6ersita` ‘G. D’Annunzio’, Via dei Vestini, I-66100 Chieti, Italy
c Dipartimento di Scienze Biochimiche, Uni6ersita` ‘La Sapienza’, Rome, Italy
Received 5 January 1999; accepted 30 June 1999
Abstract
This paper reports the synthesis of tauryl dipeptides related to carnosine. In particular H-Tau-His-OH (5), H-Tau-His(p-Me)-
OH (6) and H-Tau-His(t-Me)-OH (9) are described. The enzyme carnosinase has been isolated from pig kidney and after
purification has been used to test the stability and the inhibitory activity of the three new analogues. H-Tau-His-OH (5) and
H-Tau-His(t-Me)-OH (9) were found to possess weak inhibitory properties towards carnosinase, while H-Tau-His(p-Me)-OH (6)
proved to be devoid of any significant activity. All the three sulfonamido pseudopeptides 5, 6 and 9 show stability to carnosinase
activity. © 1999 Elsevier Science S.A. All rights reserved.
Keywords: Carnosinase inhibitors; Carnosine; Sulfonamido peptides; Taurine; Transition state
b-alanyl-containing dipeptides, i.e. b-alanyl-histidine
(carnosine) and b-alanyl-p-methyl-histidine (anserine)
(Fig. 1). These peptides occur at surprisingly high
concentration in muscle and brain, the two tissues
with the most active oxidative metabolism, where they
have been postulated to play a role as natural antiox-
idants, free-radical scavengers and wound healing
agents [17–20]. In particular it has been recently
shown that carnosine inhibits protein alteration medi-
ated by oxygen free-radicals and related toxic species
such as hypochlorite anions and malondialdehyde
[21]. However, the half-life in the body and thus the
beneficial effects of these compounds are significantly
limited due to the activity of the enzyme carnosinase
and related peptidases which cleave the b-Ala-His [or
His(Me)] peptide bond.
1. Introduction
In recent years there has been an increasing interest
in the structural modification of biologically active
peptides in order to optimize potency, selectivity and
metabolic stability [1–8].
We reported previously studies on peptides contain-
ing the residue of taurine (2-aminoethanesulfonic acid;
Tau) at the N-terminal or internal position [9–13].
These compounds, which are characterized by the
presence of a sulfonamido junction, present several
interesting aspects which are connected with the bio-
logical relevance of the taurine residue as well as the
stability toward enzymatic hydrolysis, the high polar
character and the sulfur tetrahedral structure making
it suitable for the design of tight binding enzyme in-
hibitors (transition state analogue protease inhibitors)
[9–16]. To carry out our work in this field, we fo-
cused attention on a family of biologically relevant
This report describes the synthesis and properties
of the three novel pseudodipeptides tauryl-histidine
(5), tauryl-p-methyl-histidine (6) and tauryl-t-methyl-
histidine (9), structurally related to carnosine, anserine
and isoanserine [22], respectively. In contrast with the
majority of previously reported sulfonamido pseudo-
* Corresponding author. Tel.: +39-0871-355-5337; fax: +39-
0871-355-5322.
0014-827X/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(99)00079-8

674
A. Calcagni et al. / Il Farmaco 54 (1999) 673–677
2. Chemistry
Synthesis of the new carnosine analogue 5 was per-
formed as described in Scheme 1; the same procedure
has been adopted for the preparation of the anserine
and isoanserine analogues 6 and 9. As in the case of
the previously studied tauryl peptides [9–13], the use
of N-benzyloxycarbonyl chloride (Z-Tau-Cl) as acy-
lating agent seemed a convenient synthetic route to
obtain the N-protected tauryl dipeptide methyl esters
1, 2 and 7. In the case of the synthesis of Z-Tau-His-
OMe (1), coupling of Z-Tau-Cl with equimolar
amounts of H-His-OMe gave unsatisfactory results
owing to the concomitant acylation at the imidazole
ring N1-position of the histidine residue. Good yields
of the expected compound 1 were, however, obtained
by using an excess (two equivalents) of the amino
component (see Section 5). The structures of the new
sulfonamido analogues 5, 6 and 9 are in accordance
with their spectroscopic properties; a correlation of
Fig. 1. b-Alanyl-containing dipeptides (numbering of the imidazole
ring follows the IUPAC-IUB recommendations.
1
the H and 13C NMR data with those of carnosine is
reported in Table 1. In particular, the 13C NMR
spectrum (D2O) of the three analogues 5, 6 and 9
reveals that, in agreement with the different chemical
environment, the Tau Ca signal appears shifted
downfield (l 52.44, 52.60 and 52.75, respectively)
[10,11,13] as compared with the corresponding b-Ala
Ca resonance in carnosine [24].
peptides, these compounds are characterized by the
simple SO2NH replacement, retaining all the other
structural features of the corresponding bioactive par-
ent. Thus, compounds 5, 6 and 9 are very suitable
models to investigate the potential of the SO2NH
junction to mimic the transition state for enzymatic
amide hydrolysis [23]; in addition, they may represent
therapeutically useful agents which maintain the an-
tioxidant properties of carnosine and related dipep-
tides being at the same time not cleaved by serum
dipeptidases [21].
3. Biological results
The three analogues under study have been tested
as substrates of the enzyme carnosinase and as in-
hibitors of the enzymatic hydrolysis of carnosine.
Scheme 1. Reagents and conditions: (i) TEA, CHCl3, 0°C, 1 h, then r.t. 18 h; (ii) 1 N NaOH, (2:1) acetone–H2O, r.t. 5 h; (iii) H2, 10% Pd–C,
(2:1) MeOH–H2O, 3 h, r.t. Compounds 6 and 9 have been prepared by following the same procedure reported for compound 5.

A. Calcagni et al. / Il Farmaco 54 (1999) 673–677
675
Table 1
1H (300 MHz) and 13C (75.43 MHz) NMR dataa for carnosine and its sulfonamido analogues 5, 6 and 9
Residue
H-b-Ala-His-OH (carnosine) H-Tau-His-OH (5)
H-Tau-His(p-Me)-OH (6)
lH lC
H-Tau-His(t-Me)-OH (9)
lH
lC
lH
lC
lH
lC
b-Ala or Tau
Ca
2.6 m
3.2 m
35.09
38.62
3.05 m
3.15 m
52.44 3.2 m
36.84 3.35 m
52.60
37.04
3.2 m
3.35 m
52.75
37.10
Cb
CO
174.37
His or His(Me)
Ca
Cb
C2
4.5 m
58.08
3.85 m
61.28 4.0 m
61.50
3.9 m
2.95 (5.0; 8.4; 15.4)
7.65 s
60.57
30.28
140.82
131.88
127.79
34.48
3.1 (4.9; 8.5; 14.6) 31.83
7.7 s
2.8 (4.6; 9.1; 15.1) 32.87 2.9 (4.5; 9.0; 14.7) 33.97
138.47
136.11
120.23
7.6 s
6.8 s
138.13 7.6 s
135.31
120.42 6.95 s
3.65 s
140.45
138.51
122.34
36.23
C4
C5
Me
CO
6.95 s
6.8 s
3.55 s
180.71
180.40
180.58
179.49
a In D2O at 21°C. 13C-chemical shifts are relative to internal dioxane. Coupling constants (in parentheses, Hz) for mutually coupled protons are
given only once, at their first occurrence in the Table. The assignments for proton-bearing carbons were confirmed by APT experiments.
The sulfonamido bond turned out to be stable
against hydrolytic activity of carnosinase; after 1 h
incubation in the presence of 20 mM sulfonamido
analogues 5, 6 and 9, the amount of histidine detected
was less than 2% when compared with the reaction in
the presence of carnosine.
H-Tau-His-OH (5), at a concentration of 5 mM,
inhibits the hydrolysis of carnosine of about 15%.
Preincubation of the enzyme for 1 h with the inhibitor
does not improve the inhibition effect. H-Tau-His(t-
Me)-OH (9) shows a very similar behavior with about
15% inhibition under the same conditions. H-Tau-
His(p-Me)-OH (6), however, does not inhibit the
carnosinase reaction under these conditions. A detailed
study of the inhibition course with H-Tau-His-OH (5)
(data not shown) indicates the presence of a competi-
tive inhibition pattern, with an approximate Ki value of
0.25 mM.
5, 6 and 9 represent interesting stable mimics of physio-
logical components whose protective effects have been
extensively reported [21].
On the other hand, data concerning the carnosinase
inhibitory activity of the new products are rather unex-
pected and seem to confirm a recent theoretical investi-
gation concerning the capability of phosphonamidates
and sulfonamides to behave as protease transition state
isosteres [23]. Two major properties of the high-energy
intermediate for amide hydrolysis have been considered
by the authors: the tetrahedral geometry and the elec-
trostatic potential. The first property ensures that the
inhibitor will fit into the binding site, while the second
ensures that the inhibitor will interact with the enzyme
in a fashion similar to the transition state [23]. Our
results clearly show that the inhibition exerted by the
sulfonamido carnosine analogue 5 on the carnosinase
activity is not very efficient and the calculated Ki value
(0.25 mM) indicates that the inhibitor has an affinity
for the enzyme similar to the substrate. By considering
the tetrahedral hybridization of the sulfur atom of the
sulfonamides and its geometrical analogy with the
amide hydrolysis transition state, it seems probable that
the scarce inhibition activity shown by 5 and 9 should
be attributed to an unfavorable electrostatic potential
of sulfonamides as recently suggested by Houk and
co-workers [23].
4. Discussion
The specificity of carnosinase involves the presence of
the
with this enzyme [25]. The new sulfonamido pseu-
dopeptides described here, containing the -histidine
L-histidine residue in many of the substrates tested
L
and related amino acids, have been found to be stable
towards the activity of the serum enzyme carnosinase.
Although this result would be expected by considering
the high chemical stability of the SO2NH function, it
should be noted that experiments concerning the enzy-
matic stability of tauryl peptides towards b-alanyl spe-
cific peptidases are, at the present, not available; in fact
all substrates of carnosinase referred to in the literature
possess an amide or peptide bond prone to hydrolysis.
Thus, he results reported here show that the analogues
Regarding the complete inactivity shown by 6, it
should be noted that the substrate specificity studies
with mouse kidney carnosinase [26] showed that anser-
ine is a poor substrate for the enzyme as compared with
carnosine. The p-methyl group apparently gives steric
hindrance impairing the access to the bond to be hy-
drolyzed; H-Tau-His(p-Me)-OH (6) is the modified
analogue of anserine and, as expected, shows only a
weak inhibition.

676
A. Calcagni et al. / Il Farmaco 54 (1999) 673–677
5. Experimental
5.1. Chemistry
wmax (CHCl3): 3265, 3180, 3110, 1720, 1690, 1150 cm−1
.
1H NMR (DMSO-d6): l (ppm) 2.85 [2H, m, His(t-Me)
b-CH2], 2.9 (2H, m, Tau a-CH2), 3.25 (2H, m, Tau
b-CH2), 3.55 (3H, s, NtMe), 3.65 (3H, s, OMe), 4.2
[1H, m, His(t-Me) a-CH)], 5.05 (2H, s, CH2O), 6.9 [1H,
s, His(t-Me) C5H)], 7.35 (6H, m, ArH and Tau NH),
7.45 [1H, s, His(t-Me) C2H], 7.85 [1H, d, J=8.2 Hz,
His(t-Me) NH]. Anal. (C, H, N, S) for C18H24N4O6S.
Melting points are uncorrected. Optical rotations
were taken at 20°C with a Schmidt–Haensch Polar-
tronic D polarimeter. IR spectra were recorded employ-
ing a Perkin–Elmer 983 spectrophotometer. 1H (300
MHz) and 13C (75.43 MHz) NMR spectra were deter-
mined on a Varian XL-300 instrument. 13C NMR
chemical shifts were measured relative to internal diox-
ane (67.40 ppm) for compounds 5, 6 and 9 and relative
to internal tetramethylsilane for the other compounds.
Elemental analyses were performed in the laboratories
of the Servizio Microanalisi del CNR, Area della
Ricerca di Roma, Montelibretti, Italy and were within
90.4% of theoretical values.
5.1.2. Hydrolysis of Z-Tau-Xaa-OMe: general
procedure
To a solution of the above reported methyl esters (1.3
mmol) in a mixture of (2:1) acetone–H2O (10 ml) 1 N
NaOH (2.0 ml) was added at room temperature. After
5 h at room temperature the solution was evaporated
under reduced pressure and the residue taken up in
H2O. The aqueous alkaline solution was washed with
CH2Cl2 and the pH adjusted to 5.0 with 1 N HCl. The
residue obtained after evaporation was extracted with a
mixture of (95:5) CHCl3–MeOH to give the corre-
sponding acids as vitreous oils.
5.1.1. Preparation of Z-Tau-Xaa-OMe: general
procedure
To a stirred ice-cold suspension of the corresponding
2HCl.Xaa-OMe (6.0 mmol), triethylamine (TEA) (2.4 g,
24.0 mmol) in CHCl3 (5 ml) was added. After 10 min,
solutions of N-benzyloxycarbonyltauryl chloride (3.0
mmol for compound 1, 8.0 mmol for compounds 2 and
7) in CHCl3 (3 ml) were added dropwise at 0°C over a
period of 20 min. Stirring was continued for 1 h at 0°C
and 18 h at room temperature. The mixture was then
filtered and the resulting solution diluted with CHCl3
and washed with saturated aqueous NaHCO3 and H2O.
The residue obtained after drying and evaporation was
chromatographed on silica gel using CHCl3–MeOH
(9:1 for compound 1; 95:5 for compounds 2 and 7) as
eluant to give the corresponding methyl esters.
Z-Tau-His-OMe (1). Yield 0.65 g (53%). M.p. 131–
132°C (EtOAc). [h]D= −16.0° (c=2, EtOH). IR wmax
(CHCl3): 3330, 3300, 3050, 1730, 1680, 1535, 1150
Z-Tau-His-OH (3). Yield 0.5 g (96%). [h]D= +11.0°
(c=2, EtOH). IR wmax (KBr): 3400, 3150, 1705, 1600,
1150 cm−1 1H NMR (DMSO-d6) l (ppm) 2.9–3.0
.
(4H, m, His b-CH2 and Tau a-CH2), 3.3 (2H, m, Tau
b-CH2), 4.1 (1H, m, His a-CH), 5.0 (2H, s, CH2O), 6.9
(1H, d, His C5H), 7.3 (7H, m, ArH, Tau NH and His
NH), 7.75 (1H, s, His C2H). Anal. (C, H, N, S) for
C16H20N4O6S.
Z-Tau-His(p-Me)-OH (4). Yield 0.5 g (95%). [h]D=
+7.5° (c=1, H2O). IR wmax (KBr): 3410, 3160, 1735,
1
1585, 1150 cm−1. H NMR (DMSO-d6): l (ppm) 2.9–
3.2 [4H, m, His(p-Me) b-CH2 and Tau a-CH2], 3.35
(2H, m, Tau b-CH2), 3.6 (3H, s, NpMe) 4.05 [1H, m,
His(p-Me) a-CH)], 5.05 (2H, s, CH2O), 6.9 [1H, s,
His(p-Me) C5H)], 7.35 (5H, m, ArH), 7.9 [1H, s, His(p-
Me) C2H]. Anal. (C, H, N, S) for C17H22N4O6S.
1
cm−1. H NMR (DMSO-d6): l (ppm) 2.85 (2H, m, His
Z-Tau-His(t-Me)-OH (8). Yield 0.5 g (95%). [h]D=
b-CH2), 2.95 (2H, m, Tau a-CH2), 3.3 (2H, m, Tau
b-CH2), 3.6 (3H, s, OMe), 4.2 (1H, m, His a-CH), 5.0
(2H, s, CH2O), 6.8 (1H, d, His C5H), 7.3 (6H, m, ArH
and Tau NH), 7.55 (1H, s, His C2H), 7.9 (1H, d, J=6.5
Hz, His NH), 11.8 (1H, broad, His NtH). Anal. (C, H,
N, S) for C17H22N4O6S.
+12.0° (c=2, EtOH). IR wmax (KBr): 3415, 3145, 1710,
1
1605, 1145 cm−1. H NMR (CDCl3) l (ppm) 2.85 [2H,
m, His(t-Me) b-CH2], 2.9 (2H, m, Tau a-CH2), 3.25
(2H, m, Tau b-CH2), 4.1 [1H, m, His(t-Me) a-CH)], 5.1
(2H, s, CH2O), 6.9 [1H, s, His(t-Me) C5H)], 7.4 (5H, m,
ArH), 7.55 [1H, s, His(t-Me) C2H]. Anal. (C, H, N, S)
for C17H22N4O6S.
Z-Tau-His(p-Me)-OMe (2). Yield 0.7 g (27%); oil.
[h]D= +18.0° (c=2, CHCl3). IR wmax (CHCl3): 3445,
3350, 1745, 1715, 1510, 1150 cm−1. H NMR (DMSO-
5.1.3. Preparation of carnosine-, anserine- and
1
d6): l (ppm) 3.0 [2H, m, His(p-Me) b-CH2], 3.35 (2H,
m, Tau a-CH2), 3.45 (2H, m, Tau b-CH2), 3.6 (3H, s,
NpMe), 3.7 (3H, s, OMe), 4.1 [1H, m, His(p-Me) a-
CH)], 5.05 (2H, s, CH2O), 6.8 [1H, s, His(p-Me) C5H)],
7.3 (6H, m, ArH and Tau NH), 7.55 [1H, s, His(p-Me)
C2H], 8.05 [1H, d, J=8.5 Hz, His(p-Me) NH]. Anal.
(C, H, N, S) for C18H24N4O6S.
isoanserine-analogues 5, 6 and 9: general procedure
The above described N-protected peptide acids (1.1
mmol) were hydrogenated in a mixture of (2:1)
MeOH–H2O (8 ml) in the presence of 10% Pd on
activated charcoal (0.25 g). The mixture had been vig-
orously stirred for 1 h at room temperature and a
further 0.1 g of catalyst was added. After additional 2 h
the catalyst was filtered off and the filtrate evaporated
under reduced pressure to afford the corresponding
Z-Tau-His(t-Me)-OMe (7). Yield 1.4 g (54%). M.p.
144–145°C (EtOAc). [h]D= +5.0° (c=2, CHCl3). IR

A. Calcagni et al. / Il Farmaco 54 (1999) 673–677
677
TLC pure title compounds, further purified by
crystallization.
was measured using the method reported by Lenney
and co-authors [30].
Carnosine-analogue (5). Yield 0.25 g (87%). M.p.
240–242°C (EtOH–H2O). [h]D= −16.0° (c=2, H2O).
IR wmax (KBr): 3370, 3130, 1650, 1595, 1565, 1150
cm−1. Anal. (C, H, N, S) for C8H14N4O4S.
Anserine-analogue (6). Yield 0.29 g (95%). M.p.
233–234°C (dec., EtOH–H2O). [h]D= −10.0° (c=2,
References
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Anal. (C, H, N, S) for C9H16N4O4S.
.
Isoanserine-analogue (9). Yield 0.27 g (91%). M.p.
221–222°C (iPrOH–H2O). [h]D= −11.0° (c=2, H2O).
IR wmax (KBr): 3200, 1655, 1610, 1140 cm−1. Anal. (C,
H, N, S) for C9H16N4O4S.
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Carnosine and dithiothreitol (DTT) were purchased
from Sigma. DEAE cellulose was a Bio-Rad product.
Histidine was a Merck product. o-Phtaldialdehyde, Tris
and trichloroacetic acid were Fluka products. The other
reagents were analytical reagent grade.
The enzyme carnosinase (aminoacyl-L-hystidine hy-
drolase EC 3.4.13.3) [27] was obtained from pig kidney,
further purified by ion-exchange chromatography on a
DEAE cellulose column in phosphate buffer 0.01 M,
pH 7.6. After washing, the enzyme was eluted with the
same buffer containing 0.4 M NaCl. The enzyme prepa-
ration used throughout this work shows a specific
activity, with 10 mM carnosine as substrate, of 0.02
mM histidine min−1 mg−1 protein without activation
and of 0.05 mM min−1 mg−1 protein when the enzyme
was activated with 0.01 M MnCl2 (60 min preincuba-
tion in 50 mM Tris–HCl buffer, pH 8 at 25°C). The
protein concentration was 6.9 mg ml−1, determined by
Lowry’s method [28] with bovine serum albumin as
standard. With carnosine as substrate, the enzyme
preparation shows a Km value of 0.5 mM which corre-
sponds to the literature value for carnosinase of mouse
[26] and hog kidney peak 1 [29]. Carnosinase activity
was followed by determining the amount of histidine
produced after 30 min incubation at 30°C in 50 mM
Tris–HCl buffer, pH 8. The incubation mixture con-
tained: carnosine 20 mM, enzyme 2.1 mg, 50 mM
Tris–HCl buffer, pH 8, 1 mM DTT, inhibitor 0.5–5
mM, water to a final volume of 2 ml. The determina-
tion of the amount of histidine produced by the enzyme
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