Stabilisation of β-hairpin conformations in a protein surface mimetic using a bicyclic template derived from (2S,3A,4R)-diaminoproline

Article abstract of DOI:10.1039/a804412k

A trifunctional template, derived by formal coupling of (S)-aspartic acid and (2S,3R,4R)-diaminoproline (available from vitamin C) as a diketopiperazine, was incorporated by solid-phase peptide synthesis into a protein loop mimetic containing the sequence

Full text of DOI:10.1039/a804412k

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Stabilisation of b-hairpin conformations in a protein surface mimetic using a
bicyclic template derived from (2S,3R,4R)-diaminoproline
Marc E. Pfeifer and John A. Robinson*†
Institute of Organic Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
A trifunctional template, derived by formal coupling of
(S)-aspartic acid and (2S,3R,4R)-diaminoproline (available
from vitamin C) as a diketopiperazine, was incorporated by
solid-phase peptide synthesis into a protein loop mimetic
containing the sequence -Ala-Asn-Pro-Asn-Ala-Ala-; this
was shown by NMR analysis to adopt a stable b-hairpin
conformation in DMSO.
mented already on a multi-kilogram scale in a commercial
synthesis of b-lactamase inhibitors.7 As shown in Scheme 1, 5
CONH2
O
Asn4
Pro3
N
O
N
H
Asn2
Ala5
O
N
H
Many proteins exert their biological activity through inter-
actions involving relatively small regions of their exposed
surfaces. Small synthetic molecules that mimic surface features
of proteins are therefore of potential interest in the design of
novel drug candidates. Unlike folded proteins, however, short
linear peptides are inherently flexible molecules. To overcome
this problem, much attention has been focused recently on the
design of templates to constrain peptide chains into biologically
relevant secondary and tertiary structures.1,2 We report here a
novel bicyclic template, comprising a diketopiperazine derived
from aspartic acid and (2S,3R,4R)-diaminoproline, which was
designed to stabilize b-hairpin conformations, as typically
found in protein loops connecting adjacent antiparallel
b-strands.
H2NOC
CO2Allyl
O
Me
N
H
O
O
H
N
Me
O
H
Ala6
N
Ala1
N
H
Me
HN
O
NHFmoc
O
H
O
N
N
H
H
N
HO2C
O
OH
1
2
O
O
Pro3
CONH2
Asn4
N
H
N
In previous work,3,4 we described the synthesis of template 1,
and its incorporation into the loop mimetic 2, containing the
NPNA (Asn.Pro.Asn.Ala) motif found in a tandemly repeated
form in the circumsporozoite protein of Plasmodium falci-
parum.5 Based on NMR and MD studies, we could show that 2
adopts a stable b-turn conformation within the NPNA motif,
while the 4-amido N-atom in the 4-aminoproline moiety of the
template prefers a pseudo-equatorial position,3,6 as depicted in
Fig. 1. Here, we set out to introduce an additional amino group
in an axial position at the 3-position of the pyrrolidine ring, as
in 3, which could then be used as an anchoring group to more
accurately position a peptide loop in a b-hairpin geometry, as in
4.
Asn2
H2NOC
O
O
H
N
N
N
H
Ala5
CO2H
O
O
O
Me
Me
Ala1
N
H
Me
H
H
H
N
N
Ala6
NHBz
O
HN
O
H
O
O
H
NHFmoc
NHBz
N
N
H
3
O
H
4
H
A convenient gram-scale synthesis of (2S,3R,4R)-diamino-
proline was established by exploiting a known route to the
bicyclic b-lactam 5 from vitamin C, which has been imple-
BzHN
HO
H
H
DMB
Boc
N
N
Vitamin C
CO2H
i–v
N
N
O
O
H
H
Z
Z
6
5
vi, vii
O
BzHN
NHBoc
N
H
H
NHBz
N
CO2Me
viii–xii
N
Z
H
O
NHFmoc
7
3
Scheme 1 Reagents and conditions: i, PhthH, Ph3P, THF, DEAD (61%); ii,
MeNHNH2, DMF, 80 °C; iii, Bz2O, Et3N, CH2Cl2 (66% over 2 steps); iv,
K2S2O8, Na2HPO4, aq. MeCN, 78 °C (77%); v, Boc2O, Et3N, DMAP,
CH2Cl2 (60%); vi, Na2CO3, aq. THF; vii, CH2N2, Et2O (98% over 2 steps);
viii, H2, Pd/C, DMF (96%); ix, Z-Asp(OBut)-OH, HATU, HOAt, Pri2EtN,
CH2Cl2 (80%); x, H2, Pd/C, DMF (100%); xi, TFA, CH2Cl2 (89%); xii,
Fmoc-OSucc, Pri2EtN, CH2Cl2 (60%)
Fig. 1 Average solution structures of (a) 2 and (b) 4 deduced by SA (see
text). The side-chains of Ala and Asn, and all hydrogen atoms apart from
peptide NHs, are omitted for clarity. N, O and amide H atoms = white, C
atoms = grey.
Chem. Commun., 1998
1977

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Table 1 Temperature coefficientsa (2Dd/T), H/D exchange ratesb (t1/2) for amide protons, and 3J coupling constantsc measured for 4
3J/Hz
Residue
2(Dd/T)/ppb K21
t1/2/min
3Ja,NH
3Ja,b
3Jb,g
3Jg,d
Ala1
6.0
3.2
1.4
0.0
5.1
1.9d 4.2e
5.1
33
280
350
260
7.5
7.6
9.4
7.0
9.1
8.9d 7.0e
< 2.0
6.9
—
—
—
—
—
4.3
—
—
—
—
—
—
10.1, 9.6
—
Asn2
Asn4
Ala5
5.0, 4.1
10.3, 3.9
6.8
6.9
3.5
Ala6
dApro7
Asp8
23
> 104 d 240e
4.5
3.1, 4.1
a The temperature coefficients for the peptide amides are given; dApro7 refers to the diaminoproline and Asp8 to the aspartate moieties, respectively, of the
template. Measurements were made in d6-DMSO in the range 295–320 K. b The half-lifes (t1/2) of amide resonances were determined by fitting residual peak
intensities after dissolution in d6-DMSO + 10% d4-methanol to an exponential function. The exchange rates may be classified as: fast [Asp8 NH and Asn2/
Asn4 side chain NHs (data not shown)]; medium (Ala1, Ala6); slow [Asn2, Asn4, Ala5, dApro7 C(g)-NH]; and very slow [dApro7 C(b)-NH]. c Measured using
1D and/or E.COSY spectra. d For C(b)-NH. e For C(g)-NH
was converted into 6 by a Mitsunobu reaction8 and exchange of
protecting groups, and the b-lactam ring was then opened to
yield after esterification the orthogonally protected (2S,3R,4R)-
diaminoproline derivative 7. Thereafter, coupling to
Z-Asp(OtBu)-OH, cyclisation to afford the diketopiperazine,
and further manipulation of the protecting groups gave 3 in
good overall yield.
The template 3 could be incorporated into the cyclic peptide
4 using standard solid-phase methods and Fmoc chemistry.9 For
example, 3 was coupled to Tentagel S-AC resin, and the peptide
chain was then elaborated to afford H-Ala-Asn(Mtt)-Pro-
Asn(Mtt)-Ala-Ala-Template-Resin. After cleavage from the
resin with 1% TFA in CH2Cl2, the linear precursor was cyclized
using HATU/HOAt‡ in DMF, and all side-chain protecting
groups were then removed with TFA in CH2Cl2 (35:60) and
TIPS (5% v/v). After purification by HPLC, the cyclic peptide
4 was obtained from 3 in 11% yield.
protons in the template, in particular within the diaminoproline
moiety, show values consistent with the geometry found in the
SA structures, with the C(b)-NH axial, and the C(g)-NH
equatorial. To a first approximation, therefore, the experimental
data are interlocking and consistent with the derived SA
structures, which indicate a significantly populated b-hairpin
conformation in the backbone of 4. The molecule should not be
viewed as rigid, however, and MD simulations may provide a
more detailed description of allowed conformational dynamics
on the MD time-scale in this system.
Studies are now underway to determine how general this
approach is to the construction of conformationally defined
b-hairpin loop mimetics of diverse size and sequence. The
amino functionality at C(g) in the template may be useful in this
context to allow its attachment to a solid-support for solid-phase
syntheses, as well as for coupling to other carrier molecules.
The authors thank the Swiss National Science Foundation for
financial support, and Dr Pflieger, F. Hoffmann-La Roche,
Basel, for a generous gift of compound 5.
The preferred conformation of 4 was studied in d6-DMSO (4
has low solubility in water at pH 5) at 305 K, a temperature at
1
which the amide NH protons are optimally resolved in 1D H
NMR spectra. A relatively stable b-hairpin conformation in the
peptide backbone was indicated in NOESY spectra of 4 by
NOEs connecting Ala1 H(a) as well as Asn2 NH with Ala5 NH,
which were not observed in earlier studies3,6 of 2. A b-turn in
the NPNA motif was also indicated, in particular, by a relatively
strong Asn4 to Ala5 dNN NOE, as well as NOEs between Asn2
H(b)s and Ala5 NH, as observed in earlier studies3,4,6 of 2.
Average solution structures were determined by dynamic
simulated annealing§ (SA) using distance restraints derived
from NOE build-up curves in a series of NOESY spectra with
increasing mixing times. The SA structures showed no major
distance restraint violations (e.g. > 0.2 Å) and revealed a well
defined b-hairpin backbone conformation, including a bI turn in
the NPNA motif, as in the representative structure 4 shown in
Fig. 1.
A critical test of the accuracy of the SA structures is to
examine how well they also account for other experimental
data, in particular, 3J coupling constants, relative H/D exchange
rates of amide protons, and amide proton chemical shift
temperature coefficients. Structure 4 predicts intramolecular
hydrogen-bonding across the hairpin, involving the C(b)-NH
with O(d) of the template, as well as Asn2 NH with Ala5 CO
(indicated by dotted lines in 4 and in Fig. 1). We observe very
low amide proton temperature coefficients for these two amide
NH groups, as well as relatively slow amide NH exchange rates,
measured in d6-DMSO with 10% v/v d4-methanol (Table 1),
data which indicate the involvement of these NH groups in
intramolecular hydrogen-bonding. In addition, the 3J values for
Notes and References
† E-mail: robinson@oci.unizh.ch
‡ Abbreviations: HATU
methyluronium hexafluorophosphate; HOAt
triazole; TIPS = triisopropylsilane.
§ The method used for SA calculations has been described in detail
elsewhere (refs. 3 and 6).
= O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetra-
= 1-hydroxy-7-azabenzo-
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29.
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Tetrahedron, 1997, 53, 12 789.
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Pluschke and J. A. Robinson, J. Am. Chem. Soc., 1998, 120, 7439.
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I. Heinze, C. Hubschwerlen, EP 0 508 234 A2, 1991.
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Practical Approach, IRL Press, Oxford, 1989.
Received in Cambridge, UK, 10th June 1998; 8/04412K
1978
Chem. Commun., 1998