β-Turn structure in glycinylphenylalanine dipeptide based N-amidothioureas

Article abstract of DOI:10.1039/c3cc44336a

Transforming the C-terminal amide of a glycinylphenylalanine dipeptide into N-amidothiourea affords a β-turn structure in the formed dipeptide based N-amidothioureas, which can be readily identified by an induced CD signal from the achiral phenylthiourea chromophore.

Full text of DOI:10.1039/c3cc44336a

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b-Turn structure in glycinylphenylalanine dipeptide
based N-amidothioureas†
Cite this: Chem. Commun., 2013,
49, 8943
Xiao-Sheng Yan, Kun Wu, Yuan Yuan, Ying Zhan, Jin-He Wang, Zhao Li and
Yun-Bao Jiang*
Received 9th June 2013,
Accepted 5th August 2013
DOI: 10.1039/c3cc44336a
www.rsc.org/chemcomm
Transforming the C-terminal amide of a glycinylphenylalanine
dipeptide into N-amidothiourea affords a b-turn structure in the
formed dipeptide based N-amidothioureas, which can be readily
identified by an induced CD signal from the achiral phenylthiourea
chromophore.
Scheme 1 Structures of peptides, N-amidoureas and N-amidothioureas.
The b-turn is one of the important structural motifs for maintaining
the secondary structures of proteins1 and some of the peptides or
proteins bearing b-turn structure have found significant pharma-
ceutical and biomedical applications.2 Although it has been now
shown that some of the amino acid residues, for example proline,
would facilitate the formation of b-turn structure in peptides con-
taining those residues,1b it is not straightforward to predict which
sequence would afford this structure and the use of such unmodi-
fied peptides as therapeutics is limited due to their poor pharmaco-
kinetic properties. Azapeptides represent a family of modified
peptides in which the b-turn structure is promoted, since the
replacement by a nitrogen atom of the a-carbon of one or more
amino acid residues in the peptide backbone leads to a more rigid
Na–C(O) motif and electronic repulsion between the lone pairs of the
two adjacent nitrogen atoms, resulting in constrained j and c
dihedral angles (Scheme 1).3 The b-turn structure involves four
amino acid residues, i.e. the i to i + 3 residues, and is stabilized by
a ten-membered ring hydrogen bond. The four amino acid residues
would therefore be included in a chiral environment if at least one of
the amino acid residues, in particular the one within the ‘‘ring’’, i + 1
or i + 2 amino acid residue, is chiral. This specific steric effect may
allow the chirality to be transferred across a long distance and the
induced chiral information such as the circular dichroism (CD)
signal from the involved achiral chromophore may serve to identify
the turn structure. We recently showed that N-amidothioureas
exhibited much stronger hydrogen bonding ability towards oxoanions
than the urea counterparts (the azaGly containing peptides,4
Scheme 1), partly because of the higher acidity of the thioureido –NH
protons.5 Given the structural similarity of thioamide to oxoamide,6
we envisaged that within the peptide-based N-amidothioureas the
b-turn structure might be created if the thioureido –NH is designed
to act as a hydrogen bonding donor. Here we report our first attempt
by capping the amino acid based N-amidothioureas (1, Scheme 2),7
with a simple achiral glycine residue at the N-terminal of the amino
acid residue in 1. Thus created glycinylphenylalanine dipeptide
based N-amidothioureas (L-2 and D-2, Scheme 2) were shown to bear
a b-turn structure in both solid state and solution phase. Despite the
more stable b-turn structure in 2 than that in the urea counterpart
Department of Chemistry, College of Chemistry and Chemical Engineering,
MOE Key Laboratory of Analytical Sciences, and Collaborative Innovation Center of
Chemistry for Energy Materials (iChEM), Xiamen University, Xiamen 361005,
China. E-mail: ybjiang@xmu.edu.cn; Fax: +86 592 218 5662;
Tel: +86 592 218 8372
† Electronic supplementary information (ESI) available: Synthesis (Scheme S1)
and characterization of 2, L-3, L-4a and L-4b and spectral titration traces and data
(Tables S1–S3, Schemes S1 and S2, Fig. S1–S15). CCDC 914552 and 926794. For
ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/
c3cc44336a
Scheme 2 Molecular structures of 1, 2, L-3, L-4a and L-4b. Chiral carbons in all
molecules are indicated by ‘‘*’’.
c
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in the achiral glycine residue since they are included in a five-
membered ring next to the chiral carbon center of the phenyl-
alanine residue, which means that such a hydrogen bond exists at
least in the tested three deuterated solvents of increasing polarity
and hydrogen bonding capacity, CDCl3, CD3CN and DMSO-d6
(Fig. S2, ESI†). The ten-membered ring hydrogen bond involves
four amino acid residues and is stabilized by COÁÁÁHN hydrogen
bond linking residues i and i + 3, termed b-turn in peptides and
proteins.1 This b-turn structure is of type II according to the
Ramachandran j and c dihedral angles (Table S1, ESI†). The b-turn
structure causes the chiral phenylalanine residue and the achiral
phenylthiourea moiety to come into close proximity and brings the
achiral phenylthiourea moiety into a chiral environment, explaining
the observed exciton-coupled CD signal of 2 (Fig. 1b). DFT calcula-
tions agree with the crystal structure of L-2 (Fig. S3, ESI†).
Fig. 1 Absorption (a) and CD (b) spectra of 2 in CH3CN. [2] = 40 mM.
1D and 2D NMR data both show that the b-turn structure of 2
exists in solution phase as well. 1H NMR spectra of 2 in DMSO-d6–
CD3CN binary solvents of varying composition reveal that the
signals of NHh and NHk protons are insensitive to variation in
solvent composition, suggesting their involvement in the intra-
molecular hydrogen bonds, whereas those of NHi and NHj are
sensitive which means that these two protons are solvent accessible
(Fig. S4a, ESI†), as shown by the crystal structure of L-2 (Fig. 2).
2D NMR exhibits NOE signals for coupling of Ha/Hf and of Hb/Hf,
providing direct evidence for the b-turn structure in the solution
phase (Fig. S5, ESI†). It is therefore concluded that it is the b-turn
structure in 2 that leads to the intramolecular chirality transfer
from the phenylalanine residue to the phenylthiourea moiety.
Because of the intramolecular hydrogen bonding nature, thus
stabilized b-turn structure could exist in organic aqueous solutions
with a limited extent of water. Our NMR titrations and CD spectral
data in H2O–CH3CN solutions suggest that the b-turn structure
may remain in up to 10–15% by volume of water (Fig. S4b and S6,
ESI†). The observed temperature independent CD spectrum of L-2
in CH3CN over 25–45 1C indicates that the b-turn structure is stable
within the tested temperature range (Fig. S7, ESI†).
it is nevertheless easier to be broken upon anion binding, again
because of the higher hydrogen bonding ability of thiourea.
N-Amidothioureas L-2 and D-2 were readily prepared from
(N,N-dimethylglycinyl)phenylalanine following a procedure out-
lined in Scheme S1 of ESI.† Fig. 1 presents the absorption and CD
spectra of 2 in acetonitrile. Mirrored CD spectra of L-2 and D-2 are
observed (Fig. 1b), confirming that the chirality originates from
the phenylalanine residue. By comparison with the absorption
spectrum, the CD signal at 235 nm is assigned to the chiral
phenylalanine residue,8 while that at 270 nm arises from the
achiral phenylthiourea chromophore. This observation is signifi-
cant since no CD signal at 270 nm was observed from 1 suggesting
that its phenylthiourea chromophore remains achiral (Fig. S1,
ESI†).7 The thiourea moiety in 2 is thus turned chiral by simply
capping 1 with an achiral glycine residue (Scheme 2), which
demonstrates the key role that the glycine residue in 2 plays in
facilitating the intramolecular chirality transfer, despite its achiral
nature and its distance from the thiourea moiety.
The crystal of L-2 suitable for X-ray crystallography was success-
fully grown by slow evaporation of its solution in CH2Cl2. Crystal
structure of L-2 (Fig. 2) shows two intramolecular five-membered
ring hydrogen bonds (N2–HhÁÁÁN1, 2.298 Å; N5–HkÁÁÁN3, 2.260 Å)
and one ten-membered ring hydrogen bond (N5–HkÁÁÁO1, 2.401 Å).
The first five-membered ring hydrogen bond was also confirmed by
the splitting of NMR signals of geminal CH2 protons (Ha and Hb)
As one of the thioureido –NH protons is involved in the b-turn,
it was expected that the b-turn structure may collapse upon addition
of an anion since the anion will bind to the thiourea moiety,
a well-known anion binding site.5,9 Fig. 3 shows absorption and
Fig. 2 X-ray crystal structure of L-2. Dashed pink lines highlight the intra-
Fig. 3 Absorption (a, L-2) and CD (b, L-2; c, D-2) spectra of 2 in CH3CN in the
presence of the AcOÀ anion. [2] = 40 mM. AcOÀ exists as the (n-Bu)4N+ salt.
molecular hydrogen bonds.
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and CD spectral response toward AcOÀ in CH3CN, since they are
similar to those of L-2 (Fig. S14 and S15, ESI†).
In summary, we report that, despite a minor difference by only an
achiral glycine residue, the dipeptide based N-amidothiourea 2
differs dramatically from the amino acid based 1 in the conforma-
tion and chirality transfer, which affords a new entry to the b-turn
structural motif by transforming the C-terminal amide of a dipeptide
glycinylphenylalanine into an N-amidothiourea moiety. Thus created
b-turn structure can be readily identified by a CD signal resulting
from the achiral phenylthiourea chromophore. The b-turn structure
in 2 is not only more stable than that in azapeptides but it is also
Scheme 3 Proposed hydrogen bonding network in the 2–AcOÀ complex.
CD spectra of 2 in acetonitrile in the presence of the acetate anion. more labile to anion binding. These observations, together with
With increasing AcOÀ concentration, the absorption of the phenyl- the interesting chirality transfer in both the dipeptide based
thiourea chromophore at 270 nm shifts to 266 nm, while a new N-amidothiourea and its anion binding complex, may help
band develops at 296 nm, with an isosbestic point at 244 nm guide the design of peptidomimetic pharmaceuticals.11 They
(Fig. 3a). This means a clean interaction of AcOÀ with 2. The also establish a new entry to chiral thioureas of intensive
absorption at 296 nm can be analogously assigned to the charge current interest in organocatalytic asymmetric syntheses.12
transfer transition of the anion binding complex.5 Interestingly, the Work is now underway to validate the generality of the present
CD signal at 270 nm of the phenylthiourea origin reverses gradually, strategy of creating a b-turn structure, despite being validated
along with the development of a new CD band at 304 nm. The in our preliminary assays in a small library, and to evaluate
resultant opposite and coupled Cotton effects at 304 nm and chiral communication along the peptide backbone.
270 nm suggest that the chirality of the phenylthiourea moiety in
This work was supported by MOST (2011CB910403) and NSF
2 has been overturned and extended to the anion–thiourea binding of China (91127019, 21275121 and J1030415). We thank Professor
block.7 The mirror-imaged CD spectra of AcOÀ–L-2 and AcOÀ–D-2 Xin Lu for his support in DFT calculations.
solutions (Fig. 3) again confirm that the chirality originates from the
Notes and references
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Two structures for the AcOÀ–2 complex are possible (Scheme S2,
ESI†), of which model I that contains a g-turn is more stable than
model II, in view of the steric effect (Scheme 3).
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