Aza-β3-cyclotetrapeptides

Article abstract of DOI:10.1021/jo8013963

(Figure Presented) The cyclization of aza-β³-tetrapeptides gives access to new CTP (cyclotetrapeptide) analogues. These stereocontrolled templates are assembled without any asymmetric synthesis. X-ray crystallographic structure and NMR analysis show that the macrocyclic scaffold is characterized by a fully cooperative intramolecular H-bond network, in sharp contrast with the nanotubular assemblies observed for β³-cyclotetrapeptides. This folding property reduces considerably the polarity of aza-β³- tetrapeptides and should be useful in addressing intracellular targets.

Full text of DOI:10.1021/jo8013963

Aza-ꢀ3-cyclotetrapeptides
important roles as recognition and binding sites in numerous
biological events.5 ꢀ-Turns are also implied in the receptor
affinity of Somatostatin,6 Bradykinin,7 and other biologically
important peptides.8,9 Natural CTPs also display interesting
biological effects.10 In particular, Apicidin shows antiprotozoal
activities with possible development as antimalarial agents.11
Yet, all these attractive features are counterbalanced by the fact
that the CTP scaffold, with four planar amide groups constrained
in a 12-membered ring, remains hardly accessible syntheti-
cally.12 Several groups, both academic and pharmaceutical, have
developed new synthetic strategies to obtain easily accessible
natural CTP analogues, with high conformational homogeneity.
The incorporation of a single ꢀ3-amino acid, which induces a
pseudo γ-turn, gives efficient synthesis of rigid 13-membered
ring CTP mimetics,13 also used to develop RGD mimetics.14
The introduction of a reduced peptide bond proved to be a
valuable approach toward the synthesis of Apicidin analogues.15
Cyclic ꢀ-tetrapeptoids also appear as a novel promising scaf-
fold.16
Arnaud Salau¨n,† Cle´mence Mocquet,† Romain Perochon,†
Aure´lien Lecorgne,† Barbara Le Grel,† Michel Potel,‡ and
Philippe Le Grel*,†
ICMV and CSM, UMR CNRS 6226, UniVersite´ de Rennes I,
263 aVenue du Ge´ne´ral Leclerc, 35042 Rennes, Cedex, France
philippe.legrel@uniV-rennes1.fr
ReceiVed June 26, 2008
Aza-ꢀ3-peptides,17 built up with NR-substituted hydrazinoace-
tic acids18 (aza-ꢀ3-amino acids) (Figure 1, top), are pseudopep-
tides which differ mainly from regular peptides by the replace-
ment of amide bonds by hydrazide bonds and the location of
the chirality on the NR nitrogen centers (Figure 1, middle). These
pseudopeptidic foldamers are characterized by an intramolecular
H-bond network (Figure 1, bottom) based on a repeated
bifurcated C8 pseudocycle (hydrazinoturn) and a phenomenon
of chiral scanning along the backbone resulting from nitrogen
The cyclization of aza-ꢀ3-tetrapeptides gives access to new
CTP (cyclotetrapeptide) analogues. These stereocontrolled
templates are assembled without any asymmetric synthesis.
X-ray crystallographic structure and NMR analysis show
that the macrocyclic scaffold is characterized by a fully
cooperative intramolecular H-bond network, in sharp
contrast with the nanotubular assemblies observed for ꢀ3-
cyclotetrapeptides. This folding property reduces consider-
ably the polarity of aza-ꢀ3-tetrapeptides and should be useful
in addressing intracellular targets.
(5) (a) Lewis, P. N.; Nomany, F. A.; Sheraga, H. A. Proc. Natl. Acad. Sci.
U.S.A. 1971, 68, 2293. (b) Wilmot, C. M.; Thornton, J. M. J. Mol. Biol. 1988,
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Candelore, M. R.; Donaldson, G.; Maechler, L. J. Am. Chem. Soc. 1992, 114,
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Cyclization is a common tool to introduce steric and angular
constraints in small peptidic segments, and to probe the
conformational requirements for biological activity.1 Cyclotet-
rapeptides (CTPs) seem attractive for drug design, due to a
molecular weight ranging in the drug-like gap, to an intrinsic
resistance to exopeptidases but also to endopeptidases which
are mostly active against ꢀ-strand conformation,2 and to the
potential side chain diversity that can be introduced by using
both proteogenic and nonproteogenic amino acids.3 Their cyclic
nature makes them more rigid than the linear precursors, what
is an entropic advantage upon binding, although they can show
residual conformational heterogeneity.4 CTPs represent mini-
malist ꢀ-turns mimetics, the most common reverse turn,
comprising four amino acid residues making almost a complete
180° turn in the direction of the peptide chain. ꢀ-Turns, generally
located on the solvent-exposed surface of proteins, play
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J. L.; Schreiber, S. L. J. Am. Chem. Soc. 1996, 118, 10412. (c) Kawai, M.;
Pottorf, R. S.; Rich, D. H. J. Med. Chem. 1986, 29, 2409. (d) Templeton, G. E.
Microb. Toxins 1972, 8, 160. (e) Pringle, R. B. Plant Physiol. 1970, 46, 45.
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Crumley, T. M.; Alloco, J. J.; Cannova, C.; Meinke, P. T.; Colleti, S. L.;
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A. B.; Keavey, K. N.; Jaxa-Chamiec, A.; Seale, P. W.; Stead, P.; Upton, R. J.;
Croft, S. L.; Clegg, W.; Elsegood, M. R. Bioorg. Med. Chem. Lett. 2001, 11,
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2008, 10, 921.
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Floc’h, M.; Le Grel, P. J. Org. Chem. 2001, 66, 4923.
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Synlett 2000, 3, 331.
† ICMV.
‡ CSM.
(1) Rizo, J.; Gierash, L. M. Annu. ReV. Biochem. 1992, 61, 387–418.
(2) Tyndall, J. D. A.; Nall, T.; Fairlie, D. P. Chem. ReV. 2005, 105, 973.
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Raynal, I.; Yiotakis, A.; Dive, V. J. Am. Chem. Soc. 1997, 119, 5239. (c) Loiseau,
N.; Gomis, J. M.; Santolini, J.; Delaforge, M.; Andre´, F. Biopolymers 2003, 69,
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10.1021/jo8013963 CCC: $40.75
Published on Web 10/08/2008
2008 American Chemical Society
J. Org. Chem. 2008, 73, 8579–8582 8579

FIGURE 2. C6-conformation possibly disturbing the blue NH to
participate to the hydrazinoturn network.
FIGURE 1. Comparative structures between aza-ꢀ3- and ꢀ3-amino acid
(top) and between peptides and aza-ꢀ3-peptides (middle). The hydrazi-
noturn network (bottom).
TABLE 1. Yields of Macrocyclization
FIGURE 3. Comparative X-ray structures of compounds 12 and 13
showing the divergences between internal-bond networks.
compd
nos.
yields
(%)
R1
R2
R3
R4
the nitrogen atom inside the aromatic ring (compounds 8, 9
versus 10, 11). We assume that this phenomenon is related to
the possible H-bonding of the 2-pyridyl ring nitrogen atom with
the adjacent hydrazidic NH, closing a six-membered pseudocy-
cle (C6-conformation). This interaction could disturb the hy-
drazinoturn network (C8-conformation) of the precursor in such
a way that the head-to-tail folding should be energetically
disfavored (Figure 2).
1
2
3
4
5
6
7
8
Bn
Bn
3-HO-Bn
3-HO-Bn
Bn
H
Bn
Bn
Bn
Bn
Bn
76
4-HO2C-Bn 50
3-MeO-Bn 3-HO-Bn
3-MeO-Bn
78
71
74
40a
35
65
59
15
27
Me
iBu
iBu
Bn
Bn
Bn
H
iBu
iBu
iBu
H
H
Bn
3-pyridyl methyl Bn
3-pyridyl methyl iBu
2-pyridyl methyl iBu
3-pyridyl methyl Bn
3-pyridyl methyl iBu
2-pyridyl methyl iBu
2-pyridyl methyl iBu
9
10
11
3-HO-Bn
Me
Compounds 6 and 7, containing glycine analogues, are also
obtained with lower yields. Compound 6 can nevertheless be
quantitatively prepared from compound 5 by debenzylation in
the presence of Pd/C and a catalytic amount of acetic acid.
Compounds containing glycine analogues are synthetically
useful as they can be functionalized by reaction with electro-
philes.21 The presence of the unsubstituted NRΗ group of the
hydrazinoglycine is a competitive reactive site during the
coupling steps and thus a possible yield-limiting factor. This
point has been previously discussed.22 The NRΗ group could
also be involved in alternative conformations. In fact, during
the study of small molecular models related to aza-ꢀ3-peptides,
we observed interesting divergences between the solid state
structures of compounds 12 and 13 (Figure 3). While compound
12 is organized in the classical hydrazinoturn (left), compound
13 adopts an extended conformation, the NRΗ being H-bonded
to both the amide NH and the N-terminus carbonyle (“pseu-
dospiranic” or 2 × C5-conformation, right). The two conforma-
tions differ by a 180° rotation around the N-N bond, but both
respect the orthogonal disposition of the adjacent nitrogen lone
pairs, which is energetically favorable.23
a 95% starting from 5.
pyramidal inversion.19 These conformational properties are
revealed to be very favorable for macrocyclization and allowed
us to obtain a series of aza-ꢀ3-cyclohexapeptides in high yields.20
These macrocycles, as well as their acylated derivatives,21 are
characterized by an internal hydrazinoturn network that gives
them a highly ordered conformation and controls the relative
configuration of the NR nitrogen atoms. Thus, it was interesting
to check the possibility of obtaining CTPs analogues showing
similar properties. We present here a preliminary study bound
to show the potential of aza-ꢀ3-CTPs as readily accessible CTP
analogues. The structural parameters that govern the efficiency
of cyclization are discussed. The conformation of the macrocycle
is analyzed on the basis of NMR experiments and X-ray
structures.
Tetrameric precursors were assembled following our previous
papers.17,19a Cyclizations were performed in DCM containing
an excess of EDCI/HOBT, by adding slowly a solution of the
fully deprotected tetramers to obtain a final concentration of
around 1 mM.20 The yields of macrocyclization are gathered
in Table 1. Compounds bearing pyridyl methyl side chains were
obtained in very different yields depending on the position of
The differences in the solid state also found expression in
solution. Actually, the ∆δ value between the two amide NHs
of compound 13 is 1.60 ppm (see the Supporting Information,
S23), significantly lower than the canonical ∆δ value (around
2.60 ppm) associated with the hydrazinoturn like in 12.19b The
significant decrease of the ∆δ value is very presumably related
to the possible competition between the two conformations in
(19) (a) Salau¨n, A.; Potel, M.; Roisnel, T.; Gall, P.; Le Grel, P. J. Org. Chem.
2005, 70, 6499. (b) Salau¨n, A.; Favre, A.; Le Grel, B.; Potel, M.; Le Grel, P. J.
Org. Chem. 2006, 71, 150.
(20) Le Grel, P.; Salau¨n, A.; Potel, M.; Le Grel, B.; Lassagne, F. J. Org.
Chem. 2006, 71, 5638.
(21) Le Grel, P.; Salau¨n, A.; Mocquet, C.; Le Grel, B.; Roisnel, T.; Potel,
M. J. Org. Chem. 2008, 73, 1306.
(22) Viret, J.; Collet, A.; Pichon-Pesme, V.; Aubry, A. New J. Chem. 1988,
12, 253.
(23) Dewar, M. J. S.; Jennings, W. B. J. Am. Chem. Soc. 1973, 95, 1562.
8580 J. Org. Chem. Vol. 73, No. 21, 2008

FIGURE 5. Reversal of a hydrazinoturn.
rapeptoid,16 giving the macrocycle dimensions which approach
those of CTPs in spite of the difference in ring sizes (16-
membered versus 12-membered). The conformation of aza-ꢀ3-
CTPs diverges also radically from that of the closely related
isosteric ꢀ3-CTPs (RSRS isomer). The latter shows an all-trans
configuration, with the same relative orientation of the NH and
CO groups as in our case, but the vectors point almost
perpendicularly to the mean plane of the ring, leading to a
nanotubular assembly mediated by intermolecular H-bonding
both in the solid state and in solution.24 The intermolecular/
intramolecular switch of the H-bond network, elicited by the
CꢀH/NR exchange, thus translates into very different solubilities.
While aza-ꢀ3-CTPs dissolve in most common organic solvents
(except compound 3), ꢀ3-CTPs are highly insoluble materials.
The coalescence, observed at high temperature in C2D2Cl4
reflects the fast interconversion between the two mirror images
at the NMR time scale (see the Supporting Information, S25).
The measured Tc for compound 4 gives an inversion rate
between 1 and 0.1 s-1. Despite the less stable conformation the
macrocycle must go through during the interconversion process
being inherently undetectable, it should be assumed that the
reversal of chirality at the level of a hydrazinoturn (Figure 5)
requires H-bonds breaking, together with pyramidal inversion
at the NR nitrogen atom (blue arrow), and π/2 rotation around
the N-N and N-CH2 bonds (red arrows). The energy cost
associated with the breaking of the cooperative hydrazinoturn
network certainly contributes significantly to the short life of
the intermediate species.
In summary, during this preliminary work we have performed
the synthesis of a series of new CTPs analogues, bearing
appreciable side chain diversity on chiral nitrogen atoms. These
compounds present a conformation that differ from that of the
closely related cyclic ꢀ3-tetrapeptides and cyclic ꢀ-tetrapeptoids.
The cyclization drives to the diastereoselection of the syndio-
tactic chiral sequence (RSRS/SRSR) in an all-trans form stabi-
lized by an internal H-bond network concerning all the hydrazide
groups. The macrocyclic backbone undergo a “flip-flop” phe-
nomenon through undetectable transient species. As for the
immunosupressive undecapeptide Cyclosporine A, which binds
to the cytoplasmic protein cyclophilin,25 it has been shown
recently that internal H-bonding in cyclic peptides enhances their
ability to cross cell-membrane models and thus to possibly
address intracellular targets.26 This property should give inter-
esting biological application to aza-ꢀ3-CTPs and aza-ꢀ3-cyclo-
hexapeptides. The abiotic character of these compounds is also
of potential pharmacological benefits, as it can confer protection
from biological degradation.27 Compounds 4 and 11 bear side
chains which are related to the substitution pattern of the
FIGURE 4. Cristal structure of compound 8. Top and side views. For
clarity, original side chains are replaced by methyl groups, and all
hydrogen atoms except NHs have been removed. H-bonds appear as
dotted lines.
solution in the case of compound 13. Combined together, the
experimental observations and the discussion concerning the
lower yields observed for compounds 6, 7, 10, and 11 seem to
indicate that the integrity of the hydrazinoturn network is
important to achieve efficient cyclization. Besides these less
favorable cases, all other macrocycles were obtained in ap-
preciable yields, including precursors bearing unprotected
functional groups.
The structural elements that modulate the shape of CTPs are
also present in aza-ꢀ3-CTPs. This includes cis/trans isomerism
at the level of the hydrazidic bond, and up/down orientation of
the hydrazide carbonyle with regard to the mean plane of the
macrocycle. The nitrogen pyramidal inversion is an additionnal
1
source of conformational flexibility in our case. Yet, the H
NMR spectra invariably show a single set of sharp signals. No
modification occurs by lowering the temperature of the sample
to 213 K (see the Supporting Information). Hydrazide NHs,
around 9 ppm, almost insensitive to the concentration or addition
of DMSO (see the Supporting Information, S26), clearly take
part in intramolecular hydrogen bonds. The geminal protons of
methylenic groups adjacent to chiral nitrogen centers give rise
to AB spin systems, which coalesce by increasing the temper-
ature (around 398 K for compound 4), while the NHs signals
remain unmodified. The all-trans form can be unambiguously
assigned referring to the carbonyle chemical shifts which range
roughly between 167 and 170 ppm. Actually, the cis and trans
geometry of the hydrazidic bond can be correlated to the
carbonyl chemical shift, with values ranging respectively around
174-175 ppm in the cis-isomer, and not exceeding 170 ppm
for the trans-isomer.20
By analogy with the cyclohexamer series,20 we assume that
the major conformation of aza-ꢀ3-cyclotetrapeptides is an all-
trans form in which the hydrazide NHs are implied in hydrazi-
noturn, enforcing the chiral sequence to be syndiotactic. These
conclusions are supported by X-ray structures. All crystals
examined are racemates (except the meso compound 1), with
syndiotactic chiral sequence. Only intramolecular H-bonding
is observed, each NH participating in a hydrazinoturn. Viewed
from the top, the backbone resembles a square, with the chiral
nitrogen atoms at the corners, with the atoms between two
consecutive chiral centers being almost coplanar (Figure 4). The
side view reveals a wavy form that defines a saddle horse shape.
The NH and CO vectors point alternatively upside and downside
relative to a mean plane that crosses the four Nꢀ atoms, making
angles of 45° with this plane. The side chains are oriented
alternatively upside and downside in pseudoequatorial positions.
The molecular contraction that results from internal H-bonding
differentiates strongly this structure from the recently published
all-cis structure of the alternative 16-membered cyclic ꢀ-tet-
(24) Seebach, D.; Matthews, J. L. HelV. Chim. Acta 1997, 80, 173.
(25) Schreiber, S. L.; Crabtree, G. R. Immunol. Today 1992, 13, 136.
(26) (a) Rezai, T.; Yu, B.; Millhauser, G. L.; Jacobson, M. P.; Lokey, R. S.
J. Am. Chem. Soc. 2006, 128, 2510. (b) Rezai, T.; Bock, J. E.; Zhou, M. V.;
Kalyanaraman, C.; Lokey, R. S.; Jacobson, M. P. J. Am. Chem. Soc. 2006, 128,
14073.
(27) Lelais, G.; Seebach, D. HelV. Chim. Acta 2003, 86, 4152.
J. Org. Chem. Vol. 73, No. 21, 2008 8581

powder that was purified by flash chromatography (DCM/ EtOAc
50/50) and crystallized in EtOAc.
Purification of compounds 6, 7, 10, and 11 was performed on
silica gel (DCM, then AcOEt/EtOH 80/20). Monocrystals for 11
were obtained in AcOEt.
neuropeptide Leu-Enkephaline, showing that the extension of
the synthesis to biologically relevant targets is conceivable.
Experimental Section
Synthesis of 6 starting from 5: Macrocycle 5 (1 g) was
dissolved in 20 mL of methanol containing two drops of acetic
acid. Pd/C (10%, 50 mg) was added, and the mixture was stirred
for 12 h under hydrogen atmosphere. After filtration on celite,
methanol was evaporated. The residue was further coevaporated
with toluene (2 × 10 mL) then with DCM (2 × 10 mL) to afford
6 quantitatively as a white powder.
General Macrocyclization Procedure for Compounds 1-11.
Typically, a Boc-aza-ꢀ3-OH tetramer (1 mmol) was treated with a
mixture of DCM/TFA (6 mL/4 mL) during 12 h. The excess of
TFA was then coevaporated under reduced pressure with toluene
(3 × 20 mL) then ether (3 × 20 mL) until a white foam appeared.
The crude residue was dissolved in 20 mL of DCM and 10 mmol
of triethylamine was added. This solution was poured drop by drop
into a solution of EDCI (8 mmol) and HOBT (8 mmol) in 1.5 L of
DCM. The reaction was stirred vigorously for 72 h (unoptimized).
The volume was then reduced to around 100 mL. The addition of
20 mL of 1 N HCl under stirring gave rise to the apparition of a
white solid (HOBT, HCl), which was filtrated by succion. The
solution was then washed successively with 20 mL of 1 N HCl,
twice with 20 mL of water, and twice with 20 mL of 1 N NaHCO3,
dried on Na2SO4, and evaporated. In the cases of compounds 1-5,
8, and 9, the crude reaction product was obtained as an off-white
Acknowledgment. We are grateful for the support provided
by ANR (National Research Agency).
Supporting Information Available: Experimental details,
NMR spectra of compounds 1-13, and X-ray crystallography
data for compounds 4, 8, 11, and 13. This material is available
free of charge via the Internet at http://pubs.acs.org.
JO8013963
8582 J. Org. Chem. Vol. 73, No. 21, 2008