β,γ-Diamino acid: An original building block for hybrid α/γ-peptide synthesis with extra hydrogen bond donating group

Article abstract of DOI:10.1007/s00726-014-1831-y

Using a β,γ-diamino acid, several small hybrid α/γ peptides have been synthesized and their conformations investigated through extensive NMR studies and molecular dynamics. A tripeptide and a tetrapeptide have thus shown several hydrogen bonds in solution, including a 13-membered ring involving the β-nitrogen.

Full text of DOI:10.1007/s00726-014-1831-y

Amino Acids
DOI 10.1007/s00726-014-1831-y
ORIGINAL ARTICLE
β,γ‑Diamino acid: an original building block for hybrid
α/γ‑peptide synthesis with extra hydrogen bond donating group
Andrii Stanovych · Régis Guillot · Cyrille Kouklovsky ·
Emeric Miclet · Valérie Alezra
Received: 24 July 2014 / Accepted: 21 August 2014
©
Springer-Verlag Wien 2014
Abstract Using a β,γ-diamino acid, several small hybrid
α/γ peptides have been synthesized and their conformations
investigated through extensive NMR studies and molecular
dynamics. A tripeptide and a tetrapeptide have thus shown
several hydrogen bonds in solution, including a 13-mem-
bered ring involving the β-nitrogen.
hybrid β/γ-peptide (Guo et al. 2010), β-turns (Chatterjee
et al. 2009), or original structures like 9-helices (Sharma et al.
2006b), 12-helices (Dinesh et al. 2013; Jadhav et al. 2013),
or 12/10-helices (Giuliano et al. 2013). The only limitation
so far for peptides containing γ-amino acid residues lies in
the synthesis of original and stereo-controlled γ-amino acids.
4
Several diversely substituted γ -amino acids have been syn-
Keywords Peptide · γ-amino acid · Foldamer · NMR ·
Molecular dynamics · Structure
thesized and incorporated in peptides but few of them contain
heteroatom substituent (Machetti et al. 2000; Farrera-Sinfreu
et al. 2004; Edwards et al. 2006; Sharma et al. 2006a, 2009;
3,4
Mathieu et al. 2013) and only one of them is γ -disubstituted
(without any substituent in 2-position) but no defined struc-
ture has been determined for it (Brenner and Seebach 2001).
For a few years we have developed a synthesis of
orthogonally protected β,γ-diamino acids, ready to use in
peptide synthesis (Hoang et al. 2007, 2009; Bouillère et al.
2011a, 2012). These types of compounds can be incor-
porated in peptides by the nitrogen in β-position or in
γ-position, the second one being source of hydrosolubility
(if deprotected), functionalization, or source of supplemen-
tary hydrogen bonding. We have already shown that tripep-
tides containing one β,γ-diamino acid issued from l-valine
Introduction
For several years, the field of peptidic foldamers has pro-
duced a huge amount of interesting secondary structures,
going from the α-helix mimetics to the β-turn analogues
or more original new structures (Guichard and Huc 2011;
Roy et al. 2011; Martinek and Fülöp 2012). In this area,
the β-peptides have contributed to the major extent but the
γ-peptides or hybrid peptides tend to increase their part (Vas-
udev et al. 2010; Bouillère et al. 2011b). Several secondary
structures have thus been described such as 13-helix, with
were able to present a C hydrogen bonded turn around
9
the β,γ-diamino acid (Thétiot-Laurent et al. 2012). In this
paper, we describe the structure adopted by a tripeptide and
a tetrapeptide containing a β,γ-diamino acid issued from
l-leucine, with a different relative configuration (Fig. 1).
The goal was to determine the influence of the relative con-
figuration of the β-substituent.
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-014-1831-y) contains supplementary
material, which is available to authorized users.
A. Stanovych · R. Guillot · C. Kouklovsky · V. Alezra (*)
Laboratoire de Méthodologie, Synthèse et Molécules
Thérapeutiques, ICMMO UMR 8182, Univ Paris-Sud, CNRS,
Bât 410, 91405 Orsay, France
e-mail: valerie.alezra@u-psud.fr
Results and discussion
E. Miclet (*)
Laboratoire des BioMolécules, UMR 7203 CNRS-UPMC-ENS,
UPMC Univ Paris 06, 4 Place Jussieu, 75005 Paris, France
e-mail: emeric.miclet@upmc.fr
The synthesis of the β,γ-diamino acid was performed
according to our developed strategy, starting from
1
3

A. Stanovych et al.
Fig. 1 Structures of former tripeptide (Thétiot-Laurent et al. 2012)
and tetrapeptide of this work (color figure online)
Scheme 2 Synthesis of peptides. a L-Val, EDCI, HOBt, DIPEA,
DMF; b H , Pd/C MeOH; c L-Leu, EDCI, HOBt, DIPEA, DMF; d
2
L-Ala, EDCI, HOBt, DIPEA, DMF
Fig. 2 Partial view of the packing of dipeptide 8 (color figure online)
It can be performed either using our previously developed
conditions (Zn activated by 1,2-dibromoethane, tert-butyl-
bromoacetate, yield = 56 %) or activated by ultrasound
(Lee et al. 1997). This latter procedure led to a better yield
(
yield = 66 %) and also to a methyl ester which is orthogo-
nal to the Boc protecting group. The resulting enaminoester
2
or 3 (present as a single stereoisomer) was then reduced
Scheme 1 Synthesis of β,γ-diamino acid
into a mixture of diastereomers (dr close to 1:1 in both
cases). In the case of the tert-butyl ester, the separation
of the diastereomers was performed at this stage and the
relative stereochemistry of compound 6a was established
thanks to its crystallographic structure. For the methyl
ester, the diastereomer separation was efficient after protec-
tion. Orthogonally protected deoxyaminostatine 5a (or 7a
via compound 6a) was thus synthesized in 5 (6) steps in
28 % (16 %) yield instead of 11 steps in 5 % yield.
l-leucine, the two key-steps being a Blaise reaction and
the subsequent reduction of the double bond (Scheme 1).
The synthesis was nevertheless slightly modified in few
steps: the formation of the nitrile was achieved in quantita-
tive yield using trifluoroacetic anhydride instead of phos-
phorus oxychloride and the Blaise reaction was conducted
successfully directly on the monoprotected aminonitrile 1.
1
3

β,γ-Diamino acid
the NHβ in the β,γ-diamino acid residue and the l-leucine
C=O (Fig. 3). We have never observed so far the implica-
tion of the NHβ in the β,γ-diamino acid residue and this
conformation was very different from the one observed
with the previous tripeptide [in which the β,γ-diamino acid
is issued from l-Val (Thétiot-Laurent et al. 2012)]. We then
decided to synthesize a tetrapeptide and added an l-alanine
at the N-terminus.
O
iBu
H
H
N
Ph
O
N
CO Me
2
N
H
O
O
iBu
BocHN
9
O
Me
O
iBu
H
H
N
N
CO Me
The tetrapeptide 10 formed a gel in cyclohexane
2
tBuO
N
N
−1
H
H
(c = 37 mmol L ) but was highly soluble in chloroform. At
O
iBu
O
BocHN
5 mM, it showed downfield chemical shifts for some NH,
in particular NH valine (δ = 7.8 ppm at 300 K) and also
the NHβ in the β,γ-diamino acid residue (δ = 5.85 ppm),
suggesting the presence of intramolecular hydrogen bonds.
Extensive NMR experiments (COSY, TOCSY, NOESY,
HMBC, HSQC) were recorded on a 5-mM solution in
10
Fig. 3 Hydrogen bonds obtained from the simulated annealing proto-
col (pink C , purple C , green C , blue C , and orange C ) of trip-
eptide 9 and tetrapeptide 10 (color figure online)
13
12
10
9
7
CDCl and complete proton and carbon resonance assign-
3
ment was performed. Solvent titration studies showed that
very low Δδ were observed for the same NH (NH valine
and NHβ in the β,γ-diamino acid residue). NOESY spec-
trum revealed the presence of numerous cross peaks, which
were converted into distance restraints. Among the inter-
residue correlations, some long-distance correlations were
significant of hydrogen bonding and proximity of the Boc
group and the β,γ-diamino acid. For instance, the tBu of
Boc shows cross peaks with the NHγ and the NHβ in the
β,γ-diamino acid.
Following the same protocol, vicinal coupling con-
stants and NOE distance restraints were introduced in the
simulated annealing protocol. Superimposition of the 10
lowest energy structures showed the presence of 2 three-
centered hydrogen bonds (Figs. 3, 4). In the first one, the
NH valine was located between the CO of leucine (form-
Compound 5a could be quantitatively converted to
compound 7a by saponification. Compound 7a was next
engaged in peptide synthesis. We chose the same α-amino
acids as before to have insights into the influence of the
relative stereochemistry. The peptide synthesis was per-
formed under classical conditions (Scheme 2). The dipep-
tide 8 was crystalline and the X-ray structure revealed the
formation of a hydrogen-bonded ladder structure (Fig. 2).
Every nitrogen atom and carbonyl group (except for the
ester) are involved in a hydrogen bond. Successive dipep-
tides are parallel and the three N···O distances are around
2
.97 Å and the N–H···O angles between 156° and 170°,
which are significant of usual hydrogen bonding. The
molecules are thus linked into infinite chain via hydrogen
bond, the chains forming a parallel β-sheet like arrange-
ment. The tripeptide 9 was then synthesized but no crys-
tal structure could be obtained. We thus performed NMR
studies, including solvent titration with DMSO. This latter
showed that the NH-Val and the NHβ in the β,γ-diamino
acid residue had very low Δδ, which indicates that both
could be involved in intramolecular hydrogen bond. This
is supported by the NHβ chemical shift (δ = 6.43 ppm),
significantly higher than those observed for free carba-
mate NH. NMR NOESY experiment showed among other
a weak long-distance correlation between the NH-Val and
the Cbz methylene. The NOESY correlations were con-
verted into distance restraints thanks to the vicinal distance
between Hα and Hβ of the valine residue (2.1 Å). Starting
from extended folds, 200 structures have been calculated
using a simulated annealing protocol and both vicinal cou-
pling constants and NOE distance restraints. Superimpo-
sition of the 10 lowest energy structures showed the pres-
ence of two intramolecular hydrogen bonds. The first one
forms a C12 turn and involves the NH-Val and the Cbz car-
ing a C turn around the β,γ-diamino acid) and the CO of
9
alanine (forming a C12 turn as found for the tripeptide 9,
d(NH···O) = 2.4 Å). The second three-centered hydro-
gen bond recalled the architecture observed in oligoureas
foldamers where one hydrogen bond acceptor simultane-
ously interacts with two hydrogen bond donors (Guichard
et al. 2008). In tetrapeptide 10, the CO of the terminal Boc
group was hydrogen bonded to both the NHγ and the NHβ
of the β,γ-diamino acid, leading to C10 turn and C13 turns,
respectively. Thus, the N-terminal elongation of peptide 9
had two consequences. First, the presence of the supple-
mentary CO (of the terminal Boc group) made it possible
to adopt the C /C double H-bonds fold. Secondly, since
1
0
13
the C13 was not compatible with the C pseudocycle, we
7
observed the disruption of the latter and the formation of
a hydrogen bond between the released CO (leucine) and
the NH of the l-valine. This formed a C turn around the
9
β,γ-diamino acid, as reported before in tripeptide with a
different relative configuration (Fig. 1) (Thétiot-Laurent
et al. 2012). In this longer tetrapeptide, the presence of
bonyl group, and the second forms a C turn and involves
7
1
3

A. Stanovych et al.
(a)
(
a)
1
2-membered ring
…
d(NH O) = 2.06 Å
(b)
7
-membered ring
…
d(NH O) = 1.87Å
1
0-membered ring
…
d (NH O) = 2.41 Å
(
b)
1
3-membered ring
(c)
…
d (NH O) = 1.84Å
(d)
Fig. 4 Overlay of the 10 lowest energy structures of a tripeptide 9
and b tetrapeptide 10 (hydrogen-bonds are shown in black lines; for
clarity, only the backbone is shown) (color figure online)
(e)
a supplementary CO (of the terminal Boc group) should
favor longer hydrogen bonds, such as this C13 pseudo
cycle, which reminds the hydrogen bonds present in the
α helix. These structures, involving the β nitrogen, show
the importance of having a supplementary nitrogen in the
β-position.
Fig. 5 Trajectories obtained for peptide 9 and 10 in the time course
of the MD simulation (25 ns). NH···O=C distances revealing the
formation for 9 of a 7-membered ring hydrogen bond (a) and a
We then wanted to assess the stability of the NMR struc-
tures at 300 K and performed molecular dynamics calcula-
1
2-membered ring hydrogen bond (b) and for 10 of a 13-membered
ring hydrogen bond (c), a 10-membered ring hydrogen bond (d) and a
2-membered ring hydrogen bond (e)
tions in CHCl solvent boxes. For peptide 9, the 12- and
3
7
-membered rings were conserved during the whole time
1
course of the simulation, as attested by the short d(NH–O)
distances (Fig. 5a, b). Similar calculations were carried out
on peptide 10 which highlighted the stability of the C10 and
C13 pseudo cycles involving the β,γ-diamino acid (Fig. 5c,
d).
In contrast, it showed that the H-bond between the NH
valine and CO Leucine was quickly disrupted, allowing the
amide proton to specifically interact with the CO of the ala-
nine residue (C12 turn, Fig. 5e). This hydrogen bond was
stable in both peptides 9 and 10 and is probably favored by
the (3S,4S) stereochemistry of the central β,γ-diamino acid,
Conclusion
To conclude, we have reported the synthesis of new hybrid
α/γ-peptides incorporating a β,γ-diamino acid residue.
NMR data, structure calculations and molecular dynamics
have shown that this latter was involved in the formation of
C10 and C13 turns leading to a stable three-centered hydro-
gen bonds arrangement. Such a backbone conformation
required both a four-residues long peptide and a (3S,4S)
configuration of the β,γ-diamino acid residue. In particu-
lar, a different backbone conformation was obtained for
whereas the (3R,4S) configuration preferred the C pseudo-
9
cycle (Fig. 1) (Thétiot-Laurent et al. 2012).
1
3

β,γ-Diamino acid
tripeptide 9 which lacks an N-terminal CO group to estab-
lish a long C13 pseudocycle, as observed in the α-helices.
The presence of a second amino group could then be seen
as a tool for long-range peptide structuration. Further inves-
tigations with longer peptides are under study in our group.
selective NMR at 13C natural abundance. Magn Reson Chem
6:918–924
Guo L, Almeida AM, Zhang W et al (2010) Helix formation in pre-
organized β/γ-peptide foldamers: hydrogen-bond analogy to
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132:7868–7869
Hoang CT, Alezra V, Guillot R, Kouklovsky C (2007) A stereoselec-
tive entry into functionalized 1,2-diamines by zinc-mediated
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4
Acknowledgments This research was supported by the M.E.S.R.
(doctoral Grant to A.S.) and by ANR Grants (no ANR-08-JCJC0099
and ANR-12-JS08-0005-01).
Conflict of interest The authors declare that they have no conflict
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