Accessing the disallowed conformations of peptides employing amide-to-imidate modification

Article abstract of DOI:10.1039/c1cc13515e

Selective modification of the C-terminal amide in peptides to dihydrooxazine (a novel stable imidate isostere) by intramolecular nucleophilic cyclo-O-alkylation of the corresponding N-(3-bromopropyl)amides results in constraining of the C-terminal residue in natively disallowed conformations both in crystals and in solution.

Full text of DOI:10.1039/c1cc13515e

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COMMUNICATION
Accessing the disallowed conformations of peptides employing
amide-to-imidate modificationw
Damodara N. Reddy,a Ravula Thirupathib and Erode N. Prabhakaran*a
Received 14th June 2011, Accepted 5th July 2011
DOI: 10.1039/c1cc13515e
Selective modification of the C-terminal amide in peptides to
dihydrooxazine (a novel stable imidate isostere) by intra-
molecular nucleophilic cyclo-O-alkylation of the corresponding
N-(3-bromopropyl)amides results in constraining of the C-terminal
residue in natively disallowed conformations both in crystals and in
solution.
The range of disallowed dihedral angles (f, c) for residues in
Scheme 1 General protocol for the conversion of C-terminal amide-
peptides is governed by their local steric and electrostatic
clashes.1 Rare tolerances of violations in these angles2 are
attributed to distortions in both local and global bond
characteristics of the peptides.2,3 Discerning the origins of
such disallowed angles and the consequent distortions in the
body of the peptides is essential for a complete understanding
of the protein fold;4 to improve the crystal database for
validation of rare but acceptable residue conformations;1d,5
and for validation and improvement of theoretical models6
that evaluate the interactions that define the Ramachandran
space. Unlike for the ordered secondary structures such as
b-sheets,7 a-helices8 and b-turns9 currently there are no models
for residues constrained in disallowed folds.10 We reasoned
that residues may be stabilized in disallowed folds in peptides
if a neighbouring group and hence its local unfavorable clashes
can be selectively modified to a motif that favors such space.
Steric clashes of the type HÁ Á ÁXiÆn involving the backbone
amide hydrogen1a,b,11 (H) contribute to B60% of disallowed
f, c space.12 Conversion of a backbone amide to an imidate
(A - I) will remove the corresponding H and hence the steric
clashes related to it in peptides (Scheme 1). Importantly, this
will introduce an H-bond acceptor N (of imidate) in place of
an H-bond donor NH (of amide), which will allow formation
of unusual H-bonding interactions between the imidate N and
the neighbouring Hs and hence constrain residues in otherwise
inaccessible dihedral angles.
to-imidate (A - I) in peptides.
We address all these three concerns by the selective conversion
of a backbone amide in peptides to the relatively stable cyclic
imidate isostere, 5,6-dihydro-4H-1,3-oxazine (dihydrooxazine),
by base-mediated intra-molecular nucleophilic cyclo-O-alkylation
reaction (Scheme 1). In the design of such a model, it is important
that the conformation of the peptide body is influenced mainly
by the ‘‘unusual’’ backbone dihedral angles of the modified
residue rather than by the structure of the modified motif
(dihydrooxazine) itself. This is optimally achieved when the
dihydrooxazine replaces a C-terminal amide bond, where it is
as far isolated from the peptide body as possible, than a
central or an N-terminal amide bond.
Here we replace the C-terminal amide with a dihydrooxazine
by reacting the C-terminal N-(3-bromopropyl)amide derivatives
of peptides with NaH in THF (Scheme 1). Thus pseudopeptides,
containing aliphatic (2–7) and polar (8–9) side chains on the
C-terminal residue, oligopeptides (10–11) and b-amino acid
derivatives (12) were synthesized and isolated as the exclusive
products by simply filtering off the inorganic NaBr salt
without the need for any workup or column chromatographic
purification. This is a distinct advantage of this method over
the others13 for its application in longer peptides. It is notable
that the Ca of the C-terminal residue is not epimerized under
these conditions.12
The conversion of A - I is challenging owing to difficulties in
selective synthesis, stability and purification of the imidate motif.
Peptides containing the stereochemically constrained dipeptide
segment Pro-Aib (Aib is a-aminoisobutyric acid) are good
models14 to probe the effects of structural modifications on
their rigid turn conformations. The Pro-Aib containing dipeptide
derivative 1a folds in a stable Type II b-turn conformation in
solution15 and in crystals16 and contains a strong 4 - 1
intramolecular H-bond involving the C-terminal H. This turn
conformation is conserved in 1c, the precursor to the A - I
analogue 2, as revealed from the conformational analysis of 1c
a Department of Organic Chemistry, Indian Institute of Science,
CV Raman Avenue, Bangalore, India 560012.
E-mail: erodeprabhakaran@gmail.com
b Solid State and Structural Chemistry Unit, Indian Institute of
Science, CV Raman Avenue, Bangalore, India 560012
w Electronic supplementary information (ESI) available: Syntheses,
crystal data, 1H, 13C, 2D NMR, solution structure analyses. CCDC
[CCDC NUMBER(S)]. For ESI and crystallographic data in CIF or
other electronic format see DOI: 10.1039/c1cc13515e
c
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Fig. 2 The relevant portions of (a) 1H NMR and (b) FTIR spectra of
peptides 2 and 13 in chloroform (10 mM).
disallowed in the presence of the other known imidate isosteres
namely the oxazolines, whose geometry is inappropriate for
effecting the H-bond in the C5i structure and the esters14d,25
where the oxygen replacing the amide N is not a H-bond
acceptor.26 Either isostere constrains Aib only in allowed
(non-C5i) conformations unless forced by resonance27 or by cyclic
constraints.28 This highlights the significant energy contribution
of the H-bond in the C5i structure of the A - I analogue 2.
To gain insight into the persistence of the C5i structure in
solution, we examined 2 by FT-IR and NMR spectroscopy in
chloroform (10 mM). The major IR absorption bands for Aib
Fig. 1 (a) Syntheses and crystal structures of the dihydrooxazine
containing peptidomimic and the hydrolyzed derivative 1d;
2
(b) Ramachandran plot showing the allowed (blue) and disallowed
(yellow) dihedral angles in peptides and the backbone dihedral angles
in 2 (blue arrow); (c) the depth cue rendition of the ‘‘top’’ view of 2
showing the staggered orientation of OPro and OOx between the two Cb
atoms of Aib.
and its chemically stable variant17 1b in CHCl3 (1H NMR) and
in MeOH (CD).12
The model peptidomimic 2 crystallized from a mixture of
dichloromethane and hexane (4 : 1). In the crystal structure
(Fig. 1a) the Aib residue is in a nonhelical conformation
(fAib = À177.11, cAib = 5.11; cAib is defined as the angle
NH and Piv CO appear at 3335 cmÀ and 1602 cmÀ
1
1
respectively (Fig. 2). This is attributed to the presence of g-turn
conformation (C7) at Pro in 2, based on similar values for the
model compound 1329 which contains the g-turn. However,
the band at 3456 cmÀ1 corresponding to the non-H-bonded
NH of 13 is absent in 2. Additionally, the 1H NMR signal for
Aib H is significantly down field shifted in 2 (d 7.75 ppm) than
in 13 (d 6.67 ppm), indicating that it is further H-bonded30
with the dihydrooxazine N, in the C5i structure as in the crystals.
Thus 2 exists in a 3-centered H-bonded state containing the C5i
structure in solution (Fig. 2).
N
Aib–CaAib–CAib–NOx) which is in a space (f = 180 Æ 101,
c = 0 Æ 101) that is strictly disallowed1b,2,14d for residues in
natural peptides (Fig. 1b) due to soft steric clashes (HiÀ1Á Á ÁH
and NiÀ1Á Á ÁH)1a,c,d,14d,18 involving their C-terminal H and
hard sphere eclipsing repulsions (CbÁ Á ÁO).1b,19 This violation
is allowed for Aib in 2 in the presence of (i) two Cb atoms in it
and (ii) the new-found flexibility in the peptide due to removal
of the C-terminal H involved in the 4 - 1 H-bond. This is not
only because the A - I conversion removes both the HiÀ1Á Á ÁH
and NiÀ1Á Á ÁH soft clashes, but it also introduces the dihydro-
oxazine nitrogen (N), a good H-bond acceptor,20 which
interacts with the Aib H in a planar geometry12 that is
optimum for H-bonding21 in a 5-membered ring H-bonded
structure (C5i) (Fig. 1a).
This natively disallowed C5i structure in 2 influences the
conformation of Pro which is in a relatively high energy fold16
(f Pro = À71.71, cPro = À19.21) as seen for the i + 1th residue
in Types I and III b-turns.16,22 The hydrogen bond in the C5i
structure, between the dihydrooxazine N and the central
peptide N–H in the turn, is essential for stabilizing this fold.
Usually a side chain functional group (His, Ser) or a solvent
molecule acts as the H-bond acceptor to stabilize such folds in
proteins.23 The energy gained through this intramolecular
N–HÁ Á ÁN interaction allows the staggered orientation of the
Pro oxygen (OPro) between the two Cb groups of Aib (Fig. 1c),
which is again strictly disallowed in natural peptides14d due to
strong CbÁ Á ÁOiÀ1 hard sphere clashes.
It is remarkable that an H-bond in non-optimal geometry in
C5i enables the accommodation of three hard sphere repulsive
interactions1b in 2. Such a frustrated24 conformational space is
c
9418 Chem. Commun., 2011, 47, 9417–9419
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The carbamylamide NH signals (1H NMR) for 3, 4 and 5
(d = 5.23, 5.39 and 5.95 ppm respectively) are downfield
shifted compared to those for the corresponding esters,
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Boc-Gly-OMe, Boc-Ala-OMe and Boc-Aib-OMe (d
=
5.00 ppm, 5.05 ppm and 5.01 ppm respectively),31 due to
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The dihydrooxazine is stable in water. However, it can be
hydrolyzed under basic conditions (1 eq. K2CO3) and peptide
2 is cleanly converted to the alcohol 1d (Fig. 1a), whose crystal
structure is similar to that of 1a (RMSD = 0.025 A) (f Pro, =
À57.81, cPro = 139.21, fAib = 61.41, cAib = 25.11).12 Hence
the A - I conversion is chemically and conformationally
reversible (I - A), a desirable feature for probing the
conformations of peptides containing either the amide or the
dihydrooxazine under similar non-denaturing aqueous conditions.
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c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 9417–9419 9419