Synthesis of a conformationally constrained tyrosine-glycine dipeptide mimetic: Design of a potential substrate of Syk kinase

Article abstract of DOI:10.1016/S0040-4039(02)00635-4

The synthesis of a conformationally restricted tyrosine-glycine dipeptide mimetic and its insertion into an octapeptide is described.

Full text of DOI:10.1016/S0040-4039(02)00635-4

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 3769–3771
Synthesis of a conformationally constrained tyrosine–glycine
dipeptide mimetic: design of a potential substrate of Syk kinase
Paolo Ruzza,* Andrea Calderan, Alessio Osler, Stefano Elardo and Gianfranco Borin
CNR-Biopolymer Research Center, via F. Marzolo, 1-35131 Padova, Italy
Received 15 February 2002; accepted 14 March 2002
Abstract—The synthesis of a conformationally restricted tyrosine–glycine dipeptide mimetic and its insertion into an octapeptide
is described. © 2002 Elsevier Science Ltd. All rights reserved.
as cell cycle, growth and differentiation.2 In particular,
protein tyrosine kinases (PTKs) are enzymes involved,
by the phosphorylation of specific tyrosine residues, in
the transmission of mitogenic signals and in the regula-
tion of cell cycle.3 During the course of a program to
develop useful competitive inhibitors of these enzymes,
we found that the replacement of the phosphorylatable
tyrosine with its Htc (1,2,3,4-tetrahydro-7-hydroxyiso-
quinoline-3-carboxilic acid) constrained analog in some
of their peptide-substrates, strongly inhibits the phos-
phorylation by Lyn and c-Fgr, two kinases of Src-fam-
ily. On the contrary, Syk can phosphorylate
Htc-analog, albeit with a lower efficiency with respect
to the native sequence. Htc analog has only two
allowed low-energy side chain orientations about the
CaꢀCb bond: the gauche (−) or the gauche (+).
A critical area for the design of biological active pep-
tides is the three dimensional structure of the side chain
moieties. As illustrated in Fig. 1, each side chain x1
torsional angle can assume three low energy staggered
conformations: gauche (−), gauche (+), and trans. Yet
the orientation of the side chain group of an L-amino
acid residue with respect to the peptide backbone will
be dramatically different: for gauche (−), the side chain
points towards the N-terminus of the peptide chain; for
trans, the side chain points towards the C-terminus, and
for gauche (+) the side chain points on the peptide
backbone.1 Clearly, in short peptides, different disposi-
tions of side chain groups in terms of x1 space can
provide a remarkable diversity of chemical surfaces,
even considering a single peptide backbone template
(a-helix, b-turn, …).
Phosphorylation is one of the major post-traslational
modifications that regulate many cellular processes such
In order to correlate the side chain orientation of the
phosphorylatable tyrosine residue, present into short
peptide sequences, with the efficiency and the selectivity
of the analogs as substrates for Src and Syk/Zap70
PTK families we fixed the side chain of Tyr into the
trans conformation by using an aminobenzazepine-type
structure. Indeed 4-amino-2-benzazepin-3-ones have
proven very useful for studying the biological active
conformations of peptides.4 In this structure, the side
chain of tyrosine is fixed in the gauche (+) or the trans
conformer by cyclizing the aromatic ring onto the
nitrogen of the succeeding amino acid.
Figure 1. Newman projections of the three staggered x1
In this paper, we report the synthesis of a conforma-
tionally constrained tyrosine–glycine dipeptide mimetic
(Hba-Gly, 5) and its insertion into a peptide corre-
sponding to a modified sequence of a phosphorylation
domain of the haematopoietic lineage-specific protein
HS1. The key point of this synthetic route is the
rotamers in L-amino acids.
Keywords: peptide mimetics; protein tyrosine kinases; Syk PTK;
constrained Tyr analogs.
* Corresponding author. Tel.: +39.049.8275258; fax: 39.049.8275239;
e-mail: paolo.ruzza@unipd.it
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)00635-4

3770
P. Ruzza et al. / Tetrahedron Letters 43 (2002) 3769–3771
conversion of the oxazolidinone 3 into the benzazepine
derivative 4. In our procedure (Scheme 1) the phenolic
moiety of the tyrosine residue was protected as benzyl
derivative and the Hba-Gly analog was precisely per-
formed using TFMSA, adapting the method described
by Flynn and de Laszlo for the corresponding phenyl-
TFMSA. This is very advantageous for our purpose,
since the obtained compound may be used in Fmoc/
Boc peptide synthesis. Dephthaloylation of 4 by treat-
ment with hydrazine hydrate in ethanol afforded the
dipeptide mimetic in 70% yield. The corresponding
Fmoc derivative 5 was obtained by treatment with
Fmoc-OSu in water–dioxane solution (75% yield).
alanine–glycine dipeptide.5,6 In
a previous work,
Casimir et al.7 reported that in the same conditions this
TFMSA treatment led to a mixture of by-products.
Therefore, they preferred to use SnCl4 as Lewis acid
catalyst, and the 2,6-dichloro benzyl group for the
tyrosine hydroxy protection, to obtain the correspond-
ing acid stable 2,6-dichloro benzyl benzazepine
derivative.
The utility of dipeptide mimetics as tools to probe the
binding requirements of peptides of interest is depen-
dent on their compatibility with standard peptide cou-
pling and deprotection methods. The Hba-Gly mimetic
replaced the phosphorylatable tyrosine residue into the
synthetic peptide Glu-Asp-Asp-Glu-Tyr-Glu-Glu-Val, a
modified sequence of the phosphorylation domain of
the HS1 protein.10 The Fmoc derivative 6 was incorpo-
rated into the sequence using the classical solid Fast-
Fmoc/HBTU protocol,11 and the corresponding peptide
was obtained in good yield after HPLC purification.12
The obtained peptide possessed a phosphorylation
The synthetic pathway is in succession shortly
described. Condensation of Pht-Tyr(OBzl)-OH 1 with
glycine tert-butyl ester utilizing the DCCI/HOSu
reagent provided the corresponding dipeptide in 93%
yield. The Pht-Tyr(Bzl)-Gly-OH dipeptide
2 was
obtained in quantitative yield by treatment of com-
pound 1 with 50% TFA in DCM. Starting from this
dipeptide, the oxazolidinone 3 was formed by conden-
sation of 2 with paraformaldehyde, in presence of p-
toluensulfonic acid, in toluene refluxed in a Dean–Stark
apparatus (84% yield). The preparation of the oxazo-
lidinone 3 starting from the dipeptide Pht-Tyr-Gly-OH,
unprotected to the phenolic moiety, does not succeed
due to a polymerization reaction to a phenol–formalde-
hyde polymer.8 On the other hand, when the phenolic
hydroxyl group of tyrosine was protected as its benzyl-
oxycarbonyl ester or 2,6-dichloro benzyl ether, treat-
ment of 3 with TFMSA gave only recovered starting
material for more than 80% (our experiments). To our
satisfaction, the treatment of oxazolidinone 3, under
acidic conditions of Pictet–Spengler reaction (17%
TFMSA in CH2Cl2),9 provided the desired benzazepine
structure 4 in 70–75% overall yield. In this condition
the benzyl group was removed by the treatment with
efficiency comparable to the L-Htc containing peptide.
In addition, the NMR data of the Hba-Gly containing
octapeptide (3Jab and NH-CHbCHb%, CHa-CHbCHb%
NOE) are consistent with a CaHCbH2 moiety in a trans
conformation, for the tyrosine analog.13
In conclusion, dipeptide mimetic 5 is a versatile confor-
mationally constrained intermediate which can be
incorporated into peptides to help to define the impor-
tant determinants for selective phosphorylation by
protein tyrosine kinases.
Acknowledgements
The authors are most grateful to Mr. U. Anselmi for
skilful technical assistance and for performing amino
acid analyses. This work was supported by CNR (Pro-
Scheme 1. Reagents and conditions: (1) H-Gly-OtBu (1 equiv.), DCCI (1 equiv.), HOSu (1 equiv.), TEA (1 equiv.), CH2Cl2, 93%;
(2) 50% TFA in CH2Cl2, 99%; (3) (CH2O)n (3×10 equiv.), pTos-OH (0.1 equiv.), toluene, reflux, 7 h, 84%; (4) 17% TFMSA in
CH2Cl2, rt, overnight in N2 atm, 70–75%; (5) NH2–NH2·H2O, EtOH, reflux, 2 h, 70%; (6) Fmoc-OSu (2 equiv.), Na2CO3 (1
equiv.), water–dioxane 1:1, rt, overnight, 75%.

P. Ruzza et al. / Tetrahedron Letters 43 (2002) 3769–3771
3771
getto Strategico ‘Controlli post-trascrizionali dell’e-
spressione genica’ and Progetto Strategico ‘Biotecnolo-
gie’).
sizer User’s Manual, Applied Biosystems Inc., Foster
City, CA, 1989.
12. ESI-MS m/z=1095 [MH]+; tr (min) 25.6 min. (Elution
conditions: eluent A, 0.05% TFA in water; eluent B,
0.05% TFA in 9:1 v/v acetonitrile–water; column Jupiter
C18, 10 m, 250×4.6 mm; flow rate 1 ml/min; detection at
216 and 275 nm; isocratic elution at 5% B for 3 min and
then linear gradient from 5 to 30% B in 40 min). Amino
acid ratios in acidic hydrolyzate: Asp 1.92 (2), Glu 4.07
(4), Val 0.98 (1), Hba-Gly 0.95 (1).
References
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13. Characteristic data for the key compounds. Compound 2:
1H NMR (DMSO-d6): l (ppm) 7.81–7.55 (m, arom. Pht);
7.35 (m, arom. Bzl); 7.2–6.95 (d, arom. Tyr); 5.06 (s, CH2
Bzl); 4.65 (m, CaH); 3.85 (CaH Gly); 3.18–2.8 (dd, CbH).
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5.55–5.30 (d, CH2); 5.15 (m, CaH Tyr); 4.98 (m, CH2
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