Transglutaminase surface recognition by peptidocalix[4]arene diversomers

Article abstract of DOI:10.1016/j.tetlet.2005.01.078

A series of N-linked tetrakis(tetrapeptido)calix[4]arene diversomers, 3A-P, has been synthesized by coupling of a cone calix[4]arene tetracarboxylic acid chloride with tetrapeptides 1A-P obtained in a parallel fashion. The inhibition activity of 3A-P towards tissue and microbial transglutaminase was evaluated by in vitro assays with a labeled substrate. Kinetic analysis using one of the most active derivatives (3A) showed a noncompetitive inhibition with respect to the amino acceptor substrate and an uncompetitive inhibition with respect to amino donor substrate. Experimental results are in accordance with an inhibition due to a protein specific surface recognition on a region noncomprising the enzyme active site.

Full text of DOI:10.1016/j.tetlet.2005.01.078

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 1611–1615
Transglutaminase surface recognition by peptidocalix[4]arene
diversomers
Simona Francese,a Anna Cozzolino,b Ivana Caputo,c Carla Esposito,a,* Marco Martino,a
Carmine Gaeta,a Francesco Troisia and Placido Neria,*
a
`
Dipartimento di Chimica, Universita di Salerno, Via S. Allende 43, I-84081 Baronissi (Salerno), Italy
b
`
Dipartimento di Pediatria, Universita di Napoli ‘Federico II’, Via Pansini 5, I-80131 Napoli, Italy
` `
Dipartimento di Scienze degli Alimenti, Universita di Napoli ‘Federico II’, Via Universita 100, I-80055 Portici (Napoli), Italy
c
Received 16 December 2004; revised 17 January 2005; accepted 18 January 2005
Abstract—A series of N-linked tetrakis(tetrapeptido)calix[4]arene diversomers, 3A–P, has been synthesized by coupling of a cone
calix[4]arene tetracarboxylic acid chloride with tetrapeptides 1A–P obtained in a parallel fashion. The inhibition activity of 3A–P
towards tissue and microbial transglutaminase was evaluated by in vitro assays with a labeled substrate. Kinetic analysis using
one of the most active derivatives (3A) showed a noncompetitive inhibition with respect to the amino acceptor substrate and an
uncompetitive inhibition with respect to amino donor substrate. Experimental results are in accordance with an inhibition due to
a protein specific surface recognition on a region noncomprising the enzyme active site.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Transglutaminases (TGs; EC 2.3.2.13) form a large fam-
ily of enzymes that catalyze an acyl transfer reaction
between the c-carboxamide group of the protein-bound
glutamine residue and the primary amino group of the
protein-bound lysine residue or biogenic polyamines.1
Due to the TG-catalyzed post-translational modification
of selected protein substrates, these enzymes play impor-
tant roles in several biological functions.2 Recent find-
ings suggest that some TGs, normally expressed at low
levels in many tissue types, are activated and/or over-
expressed in a variety of disorders, which include celiac
sprue and Huntington disease.3 Consequently, there is a
strong interest in the search of TG inhibitors as poten-
tial therapeutic agents.4
somers6 and their evaluation as inhibitors of two TG
isoforms.
In the design of peptidocalixarenes, we first decided to
use a calix[4]arene scaffold blocked in the cone confor-
mation and bearing tetrapeptide chains N-linked at the
upper rim.7 In this way, a convergent presentation to
the protein surface is favoured by the fixed conforma-
tion of the scaffold, while the intramolecular interchain
interaction, observed for some C-linked derivatives,
should be disfavoured.8 In addition, glycine was chosen
as the first amino acid of the peptide chain because of
the minimal steric bulkiness of the a-substituent, which
would allow its appropriate folding over the protein
surface.
In this regard, a potential alternative to conventional
small-molecule inhibitor approach, could be the exploi-
tation of the principles of protein surface recognition,
successfully applied, in the last few years, by Hamilton
and co-workers.5 Following these lines, here we report
the synthesis of a series of peptidocalix[4]arene diver-
In order to have a more general unbiased approach, we
decided to initially test peptidocalix[4]arenes bearing an
apolar, anionic or cationic ending residue. Thus, tetra-
peptides 1A–C ending with tyrosine, aspartic acid and
lysine, respectively, were synthesized by using the solu-
tion phase methodology reported by Carpino and co-
workers,9 which relies on the successive coupling of
Fmoc-protected amino acid chlorides (Scheme 1). The
tetrapeptides were then coupled with the known tetra-
propoxycalix[4]arene tetracarboxylic acid chloride 2,7
blocked in the cone conformation, in the presence of
Keywords: Calixarenes; Peptidocalixarenes; Transglutaminase; Surface
protein recognition; Enzyme inhibitors.
*
Corresponding authors. Tel.: +39 089 965262; fax: +39 089
965296; e-mail addresses: cesposito@unisa.it; neri@unisa.it
0040-4039/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.01.078

1612
S. Francese et al. / Tetrahedron Letters 46 (2005) 1611–1615
1. (c)
O
X4
O
O
O
O
H
N
H
N
H
N
H
N
(a)
Fmoc
Fmoc
OR
Fmoc
Fmoc
H2N
PS
PS
PS
O
+
N
H
OH
+
OR
Cl
HO
O
X3
X4
X3
X4
X4
1. (d)
2. (e)
1. (b) 2. (a)
X2
O
X4
O
X3
O
O
1. (d)
X3
H
H
N
H
H
N
N
OR
Fmoc
N
N
H
N
H
PS
O
Fmoc
Fmoc
N
O
N
H
H
O
2. (e)
X3
X4
O
O
X2
X4
O
1. (d) 2. (e)
1. (b) 2. (a)
O
X2
O
X4
O
X3
O
X1
H
N
H
N
H
H
N
OR
N
Fmoc
N
H
N
H
O
Fmoc
PS
N
H
N
H
O
X4
O
X1
X3
O
O
X2
1. (e)
X4
2. (f) or (g)
(b)
O
OR
H2N AA1 AA2 AA3 AA4
X2
O
O
H
N
RO
A
B
C
D
E
F
Gly Phe Gly Tyr
Asp
OR
H2N
OR
OR
RO
N
H
N
AA4
Gly Gly Phe
Gly Lys
H
AA4
O
X1
X3
AA4
AA3
O
AA4
AA3
Gly Leu
AA3
AA2
1A-P
AA3
Gly Val Phe Tyr
Gly Phe Val Tyr
Gly Trp Ala Tyr
Gly Ala Leu Tyr
Gly Trp Ala Tyr
Gly Phe Gly Phe
Gly Phe Leu Phe
Gly Val Leu Phe
Gly Leu Val Phe
N(CH2CH3)3
CH2Cl2
AA2
AA2
AA2
AA1
+
AA1
AA1
HN
z
AA1
z
z
z
NH
O
NH
O
HN
O
O
G
O
O
O
H
I
O
O
O
O
O
O
O
O
J
K
Pr
Pr
Pr
Pr
Pr
Pr
Pr
Pr
L
2 Z = Cl
2a Z = OH
M
N
O
P
Gly Phe Val Phe
Gly Leu Phe Phe
Gly Val Phe Phe
Gly Leu Gly Phe
3A-P
R = Me (fully protected)
R = H (fully deprotected)
R = H (deprotected under basic conditions)
R = Me (deprotected under acid conditions)
3Aa-Pa
3Ab-Pb
3Ac-Pc
Scheme 1. Reagents and conditions: (a) NaHCO3, 5%, Fmoc-AA-Cl, CH2Cl2; (b) TAEA, CH2Cl2; (c) CH2Cl2/DMF (150/3), DIC, 0 ꢁC, DMAP; (d)
20% piperidine, DMF, 30 min; (e) HBTU, Fmoc-AA-OH, DIPEA, DMF; (f) MeOH, N(Et)3, DMF, 2 d; (g) (1) CF3COOH, (Et)3SiH, CH2Cl2, 1 h;
(2) (trimethylsilyl)diazomethane. Side chain of Tyr and Asp amino acids were protected by tert-butyl group; e-amino group of Lys was protected by
Boc group. Left- and right-side routes were used to prepare tetrapeptides 1A–C or 1D–P, respectively.
NEt3, to give tetrakis(tetrapeptido)calix[4]arenes 3A–C,
as methyl ester derivatives. These compounds, as well
as all the other diversomers successively described, were
structurally characterized by NMR, ESI/MS and ele-
mental analysis. In all instances no significant racemiza-
tion was evidenced by NMR and [a]D measurements.
ated casein (DMC).10 As shown in panels A (Figs. 1and
2) the tetrakis(tetrapeptido)calix[4]arene 3A, ending
with tyrosine was found to be more effective than 3B
and 3C towards both TG isoforms.
Successive deprotection treatments of 3A–C led to deriv-
atives 3Aa–Ca, fully deprotected at both the side chain
and the terminal residue. In addition, partially deprotec-
ted compounds 3Ab–Cb and 3Ac–Cc were also obtained
by a single treatment under basic or acid conditions,
respectively (Scheme 1).11 However, all these derivatives
showed a lower inhibition activity with respect to the
parent compounds 3A–C (Figs. 1and 2 , panels A).
The synthesized tetrapeptidocalix[4]arenes were in vitro
screened (by using a concentration ranging between 0.01
and 1mM) for their inhibition activity towards tissue
TG (tTG) (Fig. 1) and microbial TG (mTG) (Fig. 2).
The assays were carried out by measuring the incorpora-
3
tion of H-labeled spermidine (Spd) into N,N-dimethyl-
Figure 1. Tissue transglutaminase (tTG) activity in the presence of the synthesized tetrapeptidocalix[4]arene diversomers (see Scheme 1). Inhibitors
were used in a concentration ranging between 0.01and 1mM (only data for the highest concentration are shown). Columns represent an average of
three independent sets of experiments with two replicates and the bars represent the standard deviations. Comparison of inhibition activity of
protected, fully and partially deprotected derivatives is shown only for 3A,D and 3I and their corresponding free tetrapeptides (1A,D and 1I), whereas
only the datum for the most active form is shown in the other instances.

S. Francese et al. / Tetrahedron Letters 46 (2005) 1611–1615
1613
Figure 2. Microbial transglutaminase (mTG) activity in the presence of the synthesized tetrapeptidocalix[4]arene diversomers (see Scheme 1).
Inhibitors were used in a concentration ranging between 0.01and 1mM (only data for the highest concentration are shown). Columns represent an
average of three independent sets of experiments with two replicates and the bars represent the standard deviations. Comparison of inhibition activity
of protected, fully and partially deprotected derivatives is shown only for 3A,D and 3I and their corresponding free tetrapeptides (1A,D and 1I),
whereas only the datum for the most active form is shown in the other instances.
Interestingly, no significant inhibition was found for free
tetrapeptides 1A–C and tetracarboxylic acid 2a, suggest-
ing a specific, multipoint surface binding interaction of
tetrapeptidocalix[4]arenes 3 in enzyme inhibition.
Compounds 3D–H were subsequently deprotected11 to
give 3Da–Ha, 3Db–Hb and 3Dc–Hc derivatives. Among
them 3Da was found to exhibit the best inhibitory effect
towards tTG (34% activity reduction) and mTG (35%
activity reduction) (Figs. 1and 2 , panels B, respectively).
On the basis of the above results, we directed our atten-
tion to the synthesis of further apolar peptidocalixarenes
ending with tyrosine residue and having a combination
of different residues at intermediate positions of the pep-
tide chain. The synthesis of such tetrapeptides, namely
1D–H, was performed in a parallel fashion, in the solid
phase by using an automated synthesis workstation
(Chemspeed ASW1000 synthesizer).12 A polystyrene
Wang resin was used as solid support on which Fmoc-
protected amino acid were then coupled with HBTU
as condensing agent (Scheme 1).
The solid phase synthesis was then extended to tetrapep-
tides 1I–P,11–13 with phenylalanine ending residue, to
give tetrapeptidocalix[4]arenes 3I–P (Scheme 1). These
compounds generally showed a low level of inhibition
activity, whilst the corresponding deprotected com-
pounds, 3Ia–Pa, were more effective (Figs. 1and 2 , pan-
els C, respectively). In particular, 3Ia was found to be
the best inhibitor for tTG (ꢀ40% activity reduction),
while 3Na and 3Pa were more effective on mTG
(ꢀ58% and 62% activity reduction, respectively).
The tetrapeptides were detached from the solid sup-
port13 and then coupled with 2 to give tetrapeptidoca-
lix[4]arenes 3D–H that were tested for their inhibition
activity towards tTG and mTG (Figs. 1and 2 , panels
B, respectively). These compounds exhibited a very
low inhibition activity towards both the TG isoforms,
thus evidencing the relevance of the internal sequence
on inhibition activity.
The proposed TG inhibition by tetrapeptidocalix[4]-
arenes 3 through a specific complex formation was
confirmed by nondenaturing gel electrophoresis on
acrylamide/agarose gel (Fig. 3, panel A).14 In fact, the
effective formation of a tTG/3A complex, which
migrated towards the cathode faster than the uncom-
plexed enzyme, was observed in the lane corresponding
to their mixture.
Figure 3. Study on tTG inhibition by 3A. Panel A: nondenaturing gel electrophoresis on acrylamide/agarose gel of free tTG (left lane) and its mixture
with 3A (right lane). Panel B: kinetic analysis of tTG inhibition by 3A. Lineweaver–Burk plots were made by using N,N-dimethylated casein (DMC)
(B1) and spermidine (Spd) (B2) as variable substrates. tTG from guinea pig liver was incubated in the absence (rhombuses) or in the presence of 3A
(0.1mM, squares; 1mM, triangles).

1614
S. Francese et al. / Tetrahedron Letters 46 (2005) 1611–1615
3. Kim, S.-Y.; Jeitner, T. M.; Steinert, P. M. Neurochem. Int.
2002, 40, 85.
Further information on the type of inhibition (competi-
tive, noncompetitive, and uncompetitive) of 3A on tTG
activity were obtained through a kinetic study.15 As
shown in Figure 3, 3A exhibited a noncompetitive inhi-
bition with respect to the amino acceptor substrate
DMC (Fig. 3, panel B1) and uncompetitive with respect
to amino donor substrate Spd (Fig. 3, panel B2). These
results lend support to a tTG/3A complex formation
promoted by a specific surface recognition on a region
noncomprising the enzyme active site (hot spot).5c Con-
sequently, TG inhibition could be due to a conforma-
tional rearrangement of the active form produced by
the enzyme/inhibitor recognition interaction.
4. (a) Kogen, H.; Kiho, T.; Tago, K.; Miyamoto, S.; Fujioka,
T.; Otsuka, N.; Suzuki-Konagai, K.; Ogita, T. J. Am.
Chem. Soc. 2000, 122, 1842; (b) Hausch, F.; Halttunen, T.;
Ma¨ki, M.; Khosla, C. Chem. Biol. 2003, 10, 225.
5. (a) Park, H. S.; Lin, Q.; Hamilton, A. D. J. Am. Chem.
Soc. 1999, 121, 8; (b) Blaskovich, M. A.; Lin, Q.; Delarue,
F. L.; Sun, J.; Park, H. S.; Coppola, D.; Hamilton, A. D.;
Sebti, S. M. Nat. Biotechnol. 2000, 18, 1065; (c) Peczuh,
M. W.; Hamilton, A. D. Chem. Rev. 2000, 100, 2479; (d)
Jain, R. K.; Hamilton, A. D. Org. Lett. 2000, 2, 1721; (e)
Park, H. S.; Lin, Q.; Hamilton, A. D. Proc. Natl. Acad.
Sci. U.S.A. 2002, 99, 5105; (f) Gradl, S. N.; Felix, J. P.;
Isacoff, E. Y.; Garcia, M. L.; Trauner, D. J. Am. Chem.
Soc. 2003, 125, 12668; (g) Mecca, T.; Consoli, G. M. L.;
Geraci, C.; Cunsolo, F. Bioorg. Med. Chem. 2004, 12,
5057.
6. For an account on peptidocalixarenes, see: (a) Casnati, A.;
Sansone, F.; Ungaro, R. Acc. Chem. Res. 2003, 36, 246;
(b) For a general review on calixarenes, see: Calixarenes
2001; Asfari, Z., Bo¨hmer, V., Harrowfield, J., Vicens, J.,
Eds.; Kluwer, 2001; (c) For examples of on-bead peptido-
calixarene libraries, see: Hioki, H.; Ohnishi, Y.; Kubo, M.;
Nashimoto, E.; Kinoshita, Y.; Samejima, M.; Kodama,
M. Tetrahedron Lett. 2004, 45, 561, and refs therein.
7. Sansone, F.; Barboso, S.; Casnati, A.; Fabbi, M.; Pochini,
A.; Ugozzoli, F.; Ungaro, R. Eur. J. Org. Chem. 1998,
897.
On the basis of the above results, we concluded that the
sequences Gly-Phe-Gly-Tyr (3A) and Gly-Phe-Gly-Phe
(3Ia) are the most effective tTG inhibitors in the frame
of our tetrapeptidocalix[4]arene library. Interestingly,
both compounds have the same internal sequence (Phe-
Gly), which appears to be important for a specific sur-
face recognition of tTG. Furthermore, the presence of
an apolar aromatic moiety in the side chain of the termi-
nal amino acid appears to be also necessary. In fact, a
comparable activity was observed for 3Ia and 3A ending
with phenylalanine and But-O-protected tyrosine,
respectively, whereas the corresponding O-deprotected
derivative 3Aa showed a lower efficiency (Fig. 1). On
the other hand, the best inhibitory effect on mTG was
observed with derivatives 3Na and 3Pa (Fig. 2), having
a very similar sequence (Gly-Leu-Phe-Phe and Gly-Leu-
Gly-Phe, respectively), again supporting the specificity
of surface recognition by different diversomers on the
two TG isoforms.
8. Lazzarotto, M.; Sansone, F.; Baldini, L.; Casnati, A.;
Cozzini, P.; Ungaro, R. Eur. J. Org. Chem. 2001, 595.
9. (a) Beyermann, M.; Bienert, M.; Niedrich, H.; Carpino, L.
A.; Sadat-Aalaee, D. J. Org. Chem. 1990, 55, 721; (b)
Carpino, L. A.; Sadat-Aalaee, D.; Beyermann, M. J. Org.
Chem. 1990, 55, 1673.
10. TGs inhibition assay: TGs activity was assayed according
to a modified reported procedure (Achyuthan, K. E.;
Greenberg, C. S. J. Biol. Chem. 1987, 262, 1901) Briefly,
mTG or tTG (1.28 · 10À7 M) were incubated at 37 ꢁC for
1h in 125 mM Tris–HCl, pH 7.5, 70 nM [ 3H]Spd, 10 mM
dithiothreitol, 2.5 mM CaCl2 (for tTG only) and 0.2 mg
DMC (0.1mL of final volume), in the absence or in the
presence of each chemical compound (0.01, 0.1 or
1 · 10À3 M). Data have been reported as the mean the
standard error (SE) obtained from three separate deter-
minations in which each point was performed in duplicate.
Statistical analysis was performed with StudentÕs t-test.
11. The complete deprotection of compounds 3A–P to give
3Aa–Pa, was obtained by consecutive treatments, when
required, to remove the OMe protection at C-terminus
carboxylic group (LiOH, EtOH, 12 h, rt), the But-O-
protection at the side chain of Tyr and Asp (3 N HCl,
MeOH, 16 h, rt), or the Boc protection of e-amino group
of Lys (TFA, CH2Cl2, 2 h, rt). Partially deprotected
derivatives 3Ab–Pb and 3Ac–Pc, were obtained, when
applicable, by a single treatment under basic or acid
conditions, respectively.
In conclusion, this study demonstrates that TGs can be
inhibited by peptidocalix[4]arene diversomers by means
of unconventional protein surface recognition, but
further efforts are required to improve their limited
efficiency. The extension of this strategy to the elabora-
tion of a second generation of TGs inhibitors may include
the less symmetrical attachment of diverse peptide chains
on the same scaffold or the use of a calix[4]arene skeleton
bearing both NH2 and COOH groups to give peptidomi-
metic N,C-linked derivatives.16 Both approaches are cur-
rently under study in our laboratory.
Acknowledgements
Financial supports from the Italian MIUR (COFIN-
2003, Supramolecular Devices Project and Prj. No.
200306302_004) are gratefully acknowledged. Thanks
are due to Dr. A. DÕAmato (Department of Chemistry,
University of Salerno) for ESI-MS measurements.
12. The compounds 1D–P were synthesized by general Fmoc
solid phase synthesis, see: Chan, W.; White, P. D. Fmoc
Solid Phase Peptide Synthesis: A Practical Approach;
Oxford University Press: New York, 2000. The computer-
controlled automated synthesizer ASW100 was manufac-
tured by CHEMSPEED Ltd–Rheinstrasse 32, CH 4302
Augst, Switzerland.
References and notes
13. Tetrapeptides 1D–H were detached form the solid support
by treatment with MeOH, N(Et)3, in DMF for 2 d
[conditions (f) in Scheme 1], while for derivatives 1I–P a
treatment with CF3COOH, (Et)3SiH in CH2Cl2, for 1h,
was used [conditions (g), Scheme 1].
1. Folk, J. E.; Finlayson, J. S. Adv. Protein Chem. 1977,
31, 1 .
2. (a) Lorand, L.; Graham, M. Nat. Rev. Mol. Cell Biol.
2003, 4, 140; (b) Esposito, C.; Caputo, I. FEBS J. 2005,
272, 615.

S. Francese et al. / Tetrahedron Letters 46 (2005) 1611–1615
1615
14. Gel electrophoresis: tTG (5.12 · 10À7 M) and 3A (5 mM)
were incubated for 1h at 37 ꢁC in 125 mM Tris–HCl,
pH 7.5 (10 lL of final volume), mixed with the buffer
sample and loaded onto a nondenaturing acrylamide/
agarose (2.5%/0.4%) gel.
15. Kinetic assay: tTG activity was measured using DMC or
Spd as variable substrates in the absence or presence of 3A
(0.1and 1mM). The reaction mixture contained different
concentrations of DMC (0.5, 1, 2, 4 lM) with a fixed
concentration of Spd (0.6 lM) or different concentrations
of Spd (0.1, 0.2, 0.4, 0.6 lM) with a fixed concentration of
DMC (4 lM).
16. Similar N,C-linked peptidocalix[4]arene derivatives were
very recently reported by Ungaro group: Sansone, F.;
Baldini, L.; Casnati, A.; Chierici, E.; Faimani, G.; Ugozz-
oli, F.; Ungaro, R. J. Am. Chem. Soc. 2004, 126, 6204.