Design and synthesis of conformationally constrained, extended and reverse turn pseudopeptides as Grb2-SH2 domain antagonists

Article abstract of DOI:10.1016/S0040-4039(03)00013-3

A series of conformationally constrained and flexible pseudopeptide derivatives of the tripeptide pYVN were prepared as potential antagonists of interactions of phosphotyrosine peptides with the Grb2-SH2 domain. The conformationally constrained compounds

Full text of DOI:10.1016/S0040-4039(03)00013-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 1571–1574
Design and synthesis of conformationally constrained, extended
and reverse turn pseudopeptides as Grb2-SH2 domain
antagonists
Hilary R. Plake, Thomas B. Sundberg, Angela R. Woodward and Stephen F. Martin*
Department of Chemistry and Biochemistry and Institute of Cellular and Molecular Biology, The University of Texas,
Austin, TX 78712, USA
Received 5 December 2002; revised 20 December 2002; accepted 30 December 2002
Abstract—A series of conformationally constrained and flexible pseudopeptide derivatives of the tripeptide pYVN were prepared
as potential antagonists of interactions of phosphotyrosine peptides with the Grb2-SH2 domain. The conformationally con-
strained compounds contained trans- and cis-cyclopropanes as replacements to enforce locally extended and reverse turn peptide
conformations, respectively. © 2003 Elsevier Science Ltd. All rights reserved.
Small peptides bind to proteins in well-defined confor-
mations, so there has been considerable interest in
developing rigid replacements of peptide secondary
structural elements for incorporation into non-peptidic
analogues.1 The rationale for introducing conforma-
tional constraints into flexible molecules owes its origin
to the widely held belief that preorganizing such ligands
into their biologically active conformations will provide
analogues that have higher binding affinities as a conse-
quence of a more favorable entropy of binding. Of
course, there is the obvious caveat that such constraints
must not introduce any bad contacts and that all the
attractive interactions are maintained in the complex.2
that the DGbinding for flexible and constrained analogues
of this tetrapeptide were approximately the same
because the favorable entropic effect of restricting inter-
nal rotors was offset by unfavorable enthalpic effects
that could not be attributed to specific ligand–protein
interactions or lack thereof. Thus, introducing confor-
mational constraints does not necessarily lead to higher
affinity ligands, especially when entropy–enthalpy com-
pensation intervenes.5
In order to explore the generality of entropy–enthalpy
compensation in pseudopeptide–protein interactions,
we were attracted to studying the binding of Ac-pTyr-
Val-Asn-NH2 (pYVN, 1) and pseudopeptide derivatives
thereof to the SH2 domain of the mammalian growth
receptor bound protein 2 (Grb2). While the phosphoty-
rosine residue, pY, of pYVN binds to the Grb2-SH2
domain in an extended orientation, the ligand adopts
an unusual type b-turn at the pY+1 position;6 this
structural feature has been imitated in the design of
novel analogues.7
We have explored the use of 1,2,3-trisubstituted cyclo-
propanes as rigid peptide replacements in designing
novel enkephalin analogues as well as potent inhibitors
of renin, HIV-1 protease, matrix metalloproteinases,
and Ras farnesyltransferase.3 The cyclopropane
replacements in these compounds were designed to
constrain the backbones and position the amino acid
side chains in a predetermined orientations correspond-
ing to the putative biologically active conformations of
the related peptides. We recently conducted a detailed
thermodynamic and structural study of the complexes
of several Ac-pTyr-Glu-Glu-Ile derivatives bound to
the Src-SH2 domain.4 Somewhat surprisingly, we found
Preliminary modeling studies suggested that the cyclo-
propane rings in 2 and 3 should mimic the salient
three-dimensional structural features of 1 when bound
to the Grb2-SH2 domain. Compounds 4 and 5 would
then serve as flexible controls of 2 and 3, respectively.
The cyclopropane in 2, which enforces a local extended
conformation on the peptide backbone of the pY
residue, is derived by replacing the amide nitrogen atom
of the native tripeptide 1 with a carbon atom and
connecting this atom to C(b) of the tyrosine side chain
Keywords: peptide mimics; conformational constraints; cyclo-
propanes; SH2 domain.
* Corresponding author. Tel.: +1-512-471-3915; fax: +1-512-471-4180;
e-mail: sfmartin@mail.utexas.edu
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(03)00013-3

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H. R. Plake et al. / Tetrahedron Letters 44 (2003) 1571–1574
(Scheme 1, mode a). Examination of X-ray structures
of complexes of the Grb2-SH2 domain and peptide-
derived ligands reveals that the N-terminal amide of 1
is not likely involved in interactions with the SH2
domain.6,7 Nevertheless, we wanted to preserve the
N-terminal functional character of 1 in our ligand
design, so a N-methyl carboxamide group was placed
on the cyclopropane ring of 2.
(1)
(2)
The cyclopropane in 3, which is proposed to favor a
reverse turn in the backbone at the pY+1 position,
arises from replacing the carbonyl carbon atom on 1
with a sp3-carbon and linking this atom to C(b) of the
valine side chain (Scheme 1, mode b). Based upon
previous work in our labs,3g,i we were cognizant of the
potential liabilities of replacing an amide bond with an
amino linkage when the amide group itself is in some
way involved in binding. However, because neither the
CO of Val nor the NH of Asn in 1 interact directly with
the Grb-SH2 domain, it seemed reasonable to assess the
impact of an aminoethyl substitution in this system. If
a basic nitrogen atom were highly detrimental to bind-
ing, second generation ligands could be designed in
which an ether function would link the Asn and Val
residues. The geminal dimethyl groups on the cyclo-
propane ring of 3 would not occupy precisely the same
positions as the methyl groups in 1, but initial modeling
suggested they should not incur unfavorable interac-
tions with the SH2 domain. We now report the synthe-
ses and preliminary biological evaluation of 2–5 as
analogues of 1.
Synthesis of the constrained pseudopeptide 3 com-
menced with the hydrazinolysis of the known lactone
811 followed by a Curtius reaction of the resultant
hydrazide that led to the cyclic carbamate 9 (Scheme 2).
Base-induced hydrolysis of the carbamate moiety in 9
furnished an amino alcohol that was N-alkylated with
the triflate 10, which was prepared in situ from (R)-
dimethyl malate, to give 11 as the only isolated
stereoisomer in 49% overall yield.12 Reaction of 11 with
NH3 in MeOH in the presence of a catalytic amount of
NaCN delivered a diamide that was transformed into
12 via N-protection and oxidation of the primary alco-
hol function. One-carbon degradation of the carboxyl
group in 12 via a modified Curtius reaction led to the
protected diaminocyclopropane 13.13 The N-Boc pro-
Compounds 2 and 4 were prepared as single isomers by
coupling the known acids 6 and 74,8 with the dipeptide
H-Val-Asn-NH2 in the presence of HATU, followed by
global removal of the benzyl protecting groups by
catalytic hydrogenolysis (Eqs. (1) and (2)).9,10
Scheme 2. Reagents and conditions: (a) H2NNH2·H2O,
MeOH; (b) HONO, Et2O/H2O, 0°C, then toluene, 80°C; (c)
Ba(OH)2, aq. dioxane; (d) 10, 2,6-lutidine, i-Pr2NEt, CH2Cl2,
0°C to rt; (e) NH3, NaCN (cat.), MeOH, 50°C; (f) Cbz-Cl,
i-Pr2NEt, THF, CH3CN, D; (g) RuCl3, NaIO4, NaHCO3,
CH3CN/CCl4/H2O; (h) EtOCOCl, Et3N, 0°C, NaN3; (i) t-
BuOH, D; (j) CF3CO2H; (k) HATU, Ac-pY*-OH, 2,6-
lutidine, DMF, −10°C to rt; (l) H2, Pd/C, EtOH.
Scheme 1.

H. R. Plake et al. / Tetrahedron Letters 44 (2003) 1571–1574
1573
tecting group was removed using CF3CO2H, and the
resultant unstable amine was immediately coupled with
1, have significantly lower binding affinities. Comparing
the binding of 4 and 5 suggests that introducing an
amino linkage between Asn and Val is somewhat detri-
mental. However, the nature of the substitution on
cyclopropane ring in 3 also has a negative impact on
binding, and further variations of the cyclopropane-
derived replacement in analogues of 1 must be evalu-
ated in order to ascertain whether a cyclopropane ring
may stabilize a turned structure.
N-acetyl-L
-Tyr(PO3Bn2)-OH (Ac-pY*-OH)14 using
HATU to give the protected tripeptide 14 as a single
isomer in 60% overall yield. Global deprotection by
hydrogenolysis gave 3 in virtually quantitative yield.15
The synthesis of the flexible analogue 5 began with
converting Boc-valine into the thioester 15 with DCC
and EtSH in 89% yield (Scheme 3). The thioester
moiety of 15 was reduced using the Fukuyama
protocol16 to give an aldehyde that was then coupled
A more detailed analysis of the energetic parameters
DH and DS associated with the binding of these and
other related ligands to the Grb2-SH2 domain and
X-ray crystallographic studies of selected complexes are
the subjects of ongoing studies. The results of these
investigations will be reported in due course.
with
L-aspartic acid dimethyl ester hydrochloride by
reductive amination using NaBH(OAc)3 to produce 16
as a single diastereomer.17 The amine 16 thus obtained
was isolated as a single diastereomer in 58% yield over
the two steps. The ester groups in 16 were transformed
into their respective amides using NH3 in the presence
of NaCN as before to provide 17. Acid-catalyzed
removal of the N-Boc protecting group and coupling
the resultant amine with Ac-pY*-OH in the presence of
HATU afforded the protected tripeptide 18 as a single
isomer in 30% overall yield. Complete removal of the
benzyl protecting groups by catalytic hydrogenolysis in
the presence of HCl then delivered 5.18
Acknowledgements
We thank the National Institutes of Health, The
Robert A. Welch Foundation, the Texas Advanced
Research Program, Pfizer, Inc. and Merck Research
Laboratories for their generous support of our
research. T.B.S. and A.R.W. are also grateful to Pfizer,
Inc. for support by Summer Undergraduate Research
Fellowships. We are also thankful to Dr. James P.
Davidson for helpful discussions.
We have determined the binding constants, Ka (M−1) of
1–5 with the Grb2-SH2 domain in a series of prelimi-
nary studies as follows: 1, 4.9( 0.4)×105; 2, 5.2( 0.7)×
105; 3, <1×103; 4, 2.9( 0.7)×105; and 5, 2.0( 0.5)×104.19
Thus, analogues of 1 with conformationally constrained
and flexible replacements of a phosphotyrosine residue
(e.g. 2 and 4) exhibit similar binding affinities. Based
upon these data, it seems likely that the orientations of
the substituents on the cyclopropane ring in 2 mimics
the bound conformation of the corresponding groups in
1 and 4. On the other hand, 3 and 5, which contain
rigid and flexible replacements of the valine residue in
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15. 1H NMR spectrum of 3 (500 MHz, D2O): l 7.22 (d,
J=8.5 Hz, 2 H), 7.19 (d, J=8.5 Hz, 2 H), 4.52–4.49 (m,
1 H), 3.52–3.49 (m, 1 H), 3.14–2.89 (comp m, 2 H), 2.62
(dd, J=15.0, 5.7, 1 H), 2.53 (dd, J=15.0, 8.0 Hz, 1 H),
2.30 (d, J=6.0 Hz, 1 H), 2.00 (d, J=6.0 Hz, 1 H), 1.95 (s,
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9. The structure assigned to each compound is in complete
accord with its spectral (1H and 13C NMR, IR, mass)
characteristics; molecular compositions of all new com-
pounds were determined by high resolution mass mea-
surements of purified materials. All yields are based on
isolated, purified materials judged >95% pure by 1H
NMR spectroscopy.
17. Based upon 1H NMR evidence, no epimerization
occurred at the carbon atom bearing the isopropyl group
during the conversion of 15 into 16. The reaction
sequence was performed with racemic 15 to establish that
1
the two epimers could be distinguished by H NMR.
18. 1H NMR spectrum of 5 (500 MHz, D2O): l 7.27 (d,
J=8.2 Hz, 2 H), 7.17 (d, J=8.2 Hz, 2 H), 4.57 (dd,
J=8.4, 7.0 Hz, 1 H), 4.26 (dd, J=7.7, 4.9 Hz, 1 H),
3.98–3.94 (m, 1 H), 3.21 (dd, J=12.9, 4.3 Hz, 1 H), 3.13
(dd, J=13.9, 7.0 Hz, 1 H), 3.05–2.97 (m, 2 H), 2.95–2.87
(m, 2 H), 1.96 (s, 3 H), 1.90–1.84 (m, 1 H), 0.88 (dd,
J=16.9, 6.8 Hz, 6 H).
10. 1H NMR spectrum of 2 (500 MHz, D2O): l 7.27 (d,
J=8.7 Hz, 2 H), 7.12 (d, J=7.4 Hz, 2 H), 4.73–4.70 (m,
1 H), 4.13 (d, J=6.9 Hz, 1 H), 2.96–2.94 (m, 1 H),
2.87–2.84 (m, 2 H), 2.76–2.74 (m, 1 H), 2.60 (s, 3 H), 2.54
(dd, J=10.0, 4.8 Hz, 1 H), 2.17–2.08 (m, 1 H), 0.99 (d,
J=8.1 Hz, 3 H), 0.97 (d, J=8.1 Hz, 3 H). 1H NMR
spectrum of 4 (500 MHz, D2O): l 7.17–7.09 (comp m, 4
H), 4.57 (dd, J=7.7, 6.1 Hz, 1 H), 3.96 (d, J=7.8 Hz, 1
H), 3.13–3.08 (m, 1 H), 2.81–2.77 (comp m, 3 H), 2.68–
2.65, (m, 3 H), 2.64 (s, 3 H), 2.53–2.41 (comp m, 2 H),
1.93 (app hep, J=6.8 Hz, 1 H), 0.85 (d, J=6.8 Hz, 6 H).
19. Binding constants were determined using isothermal titra-
tion calorimetry following the procedure outlined in:
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