Postsynthetic modification of C3-symmetric aza- β3-cyclohexapeptides

Article abstract of DOI:10.1021/jo7021394

(Chemical Equation Presented) We have synthesized a series of C ₃-symmetric aza-β³-cyclohexapeptides with functionally diverse side chains carrying a good functional diversity. The very simple chemical sequence that we used (debenzylation/acylation) makes it certain that the series synthesized could be easily expanded, leading to a wide family of C₃-symmetric cyclohexapeptides analogues. The macrocyclic backbone of the azaβ³-cyclohexapeptides shows a highly ordered conformation that is sustained by a dense intramolecular H-bond network where all endocyclic NHs are hydrogen bonded, the side chains being projected in equatorial position around the macrocycle. The resulting internal secondary structure relies on the cooperative alternation of two slightly different C ₈-bifidic pseudocycles, which differ mainly by the hybridization of the N^α nitrogen atom (N-N_sp²-turn and N-N_sp²-turn). In both cases, the nitrogen lone pair participates to stabilize the pseudocycle. This has been established by NMR experiments and X-ray diffraction analysis. As in the precursors, the nitrogen stereocenters are characterized by a strikingly slow rate of pyramidal inversion, considering the size of the macrocycle.

Full text of DOI:10.1021/jo7021394

Postsynthetic Modification of C3-Symmetric
Aza-â3-Cyclohexapeptides
Philippe Le Grel,*,† Arnaud Salau¨n,† Cle´mence Mocquet,† Barbara Le Grel,†
Thierry Roisnel,‡ and Michel Potel‡
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 October 2, 2007
We have synthesized a series of C3-symmetric aza-â3-cyclohexapeptides with functionally diverse side
chains carrying a good functional diversity. The very simple chemical sequence that we used
(debenzylation/acylation) makes it certain that the series synthesized could be easily expanded, leading
to a wide family of C3-symmetric cyclohexapeptides analogues. The macrocyclic backbone of the aza-
â3-cyclohexapeptides shows a highly ordered conformation that is sustained by a dense intramolecular
H-bond network where all endocyclic NHs are hydrogen bonded, the side chains being projected in
equatorial position around the macrocycle. The resulting internal secondary structure relies on the
cooperative alternation of two slightly different C8-bifidic pseudocycles, which differ mainly by the
R
3
2
hybridization of the N nitrogen atom (N-Nsp -turn and N-Nsp -turn). In both cases, the nitrogen lone
pair participates to stabilize the pseudocycle. This has been established by NMR experiments and X-ray
diffraction analysis. As in the precursors, the nitrogen stereocenters are characterized by a strikingly
slow rate of pyramidal inversion, considering the size of the macrocycle.
Introduction
This has been experimentally confirmed by several examples
and recently further extended to pseudopeptides.3d,g,6 In a
previous work, we showed that aza-â3-hexapeptides, where the
C3-symmetric compounds are attractive molecules, both
aesthetically and functionally. They have been used to perform
asymmetric catalysis and molecular recognition and to develop
nanoarchitecture.1 Among them, C3-symmetric peptidic or
pseudopeptidic macrocycles were recently used for the molecular
recognition of carbohydrates,2 as selective receptors for cations
or anions,3 and to mimic trimeric CD40L, an important ligand
of the tumor necrosis factor receptor (TNFR superfamily).4 De
Santis and co-workers were the first to postulate5 that the
synthesis of cyclic hexapeptides should be easier for precursors
with syndiotactic chiral sequence (alternation of (L)- and (D)-
amino acids), due to a favorable pre-organized conformation.
(3) (a) Kubik, S.; Goddard, R. J. Org. Chem. 1999, 64, 9475-9486. (b)
Kubik, S. J. Am. Chem. Soc. 1999, 121, 5846-5855. (c) Kubik, S.; Goddard,
R. Eur. J. Org. Chem. 2001, 311-322. (d) Yang, D.; Qu, J.; Li, W.; Zhang,
Y. H.; Ren, Y.; Wang, D. P.; Wu, Y. D. J. Am. Chem. Soc. 2002, 124,
12410-12411. (e) Kubik, S.; Kirchner, R.; Nolting, D.; Seidel, J. J. Am.
Chem. Soc. 2002, 124, 12752-12760. (f) Otto, S.; Kubik, S. J. Am. Chem.
Soc. 2003, 125, 7804-7805. (g) Yang, D.; Li, X.; Sha, Y.; Wu, Y. D. Chem.
Eur. J. 2005, 11, 3005-3009. (h) Heinrichs, G.; Kubik, S.; Lacour, J.; Vial,
L. J. Org. Chem. 2005, 70, 4498-4501. (i) Rodriguez-Docampo, Z.; Pascu,
S. I.; Kubik, S.; Otto, S. J. Am. Chem. Soc. 2006, 128, 11206-11210.
(4) (a) Fournel, S.; Wieckowski, S.; Sun, W.; Trouche, N.; Dumortier,
H.; Bianco, A.; Chaloin, O.; Habib, M.; Peter, J. C.; Schneider, P.; Vray,
B.; Toes, R. E.; Offringa, R.; Melief, C. J. M.; Hoebeke, J.; Guichard, G.
Nat. Chem. Biol. 2005, 1, 377-382. (b) Hymowitz, S. G.; Ashkenazi, A.
Nat. Chem. Biol. 2005, 1, 353-354. (c) Chaloin, O.; Hoebeke, J.; Fournel,
S.; Guichard, G. J. Am. Chem. Soc. 2007, 129, 13480-13492.
(5) De Santis, P.; Morosetti, S.; Rizzo, R. Macromolecules 1974, 7, 52-
58.
† ICMV.
‡ CSM.
(1) Gibson, S. E.; Castaldi, M. P. Chem. Commun. 2006, 3045-3062.
(2) Bitta, J.; Kubik, S. Org. Lett. 2001, 3, 2637-2640.
10.1021/jo7021394 CCC: $40.75 © 2008 American Chemical Society
Published on Web 01/16/2008
1306
J. Org. Chem. 2008, 73, 1306-1310

Synthesis of C3-Symmetric Aza-â3-cyclohexapeptides
FIGURE 3. Hydrogenolysis of aza-â3-cyclohexapeptide 1: synthesis
of 2 (aza-â3-Gly-aza-â3-Leu)3.
FIGURE 1. Structural relationship between â3-peptides and aza-â3-
peptides.
FIGURE 4. Acylation of cyclohexamer 2 (aza-â3-Gly-aza-â3-Leu)3.
Compounds 3a-d: R ) -C6H5, -CH2CH2Br, -CH2CHdCH2, and
-CH2CO2Et, respectively.
striking analogy appears between the macrocycle reversal and
the interconversion of the two chair forms of cyclohexane.
The potential of C3-symmetric molecules further improves
when they carry functional side chains, likely to take part in
host-guest interactions or catalytic processes. Aza-â3-cyclo-
hexapeptides are built from NR-substituted hydrazino acetic
acids, whose synthesis use aldehydes or ketones,9 allowing the
introduction of a good variety of side chains. This upstream
functionalization strategy is currently being developed by our
team. We report herewith a very convenient downstream
approach, which relies on the linkage of functional arms on a
preformed C3-symmetric macrocycle. The conformation of the
new macrocycles is compared with those of the precursors,
based on NMR spectroscopic data and X-ray crystallographic
structure.
FIGURE 2. (a) Hydrazinoturn or N-N turn, a bifidic hydrogen-bonded
C8 pseudocycle. (b) Equilibrium between the two “chair forms” of aza-
â3-cyclohexapeptides.
CHâ units of â3-peptides are replaced by nitrogen atoms (Figure
1), are very suitable precursors of macrocycles.7 In such
compounds, the syndiotactic arrangement favorable to cycliza-
tion is spontaneously achieved during the chiral tuning that
results from the pyramidal inversion at the sp3 nitrogen
stereocenters.
In contrast with the conformational versatility of their
precursors, aza-â3-cyclohexapeptides have a well-defined sec-
ondary structure that has been deduced from both solid state
and NMR analysis. The ring backbone is organized by an
uninterrupted network of reverse-turn, referred to as an hy-
drazinoturn or N-N-turn,8 which is a bifidic eight-membered
pseudocycle (Figure 2a).
Results and Discussion
Among the side chains that can be regioselectively introduced
on aza-â3-cyclopeptides, the benzylic group is particularly useful
as it can easily be removed by hydrogenolysis. This character-
istic offers the opportunity to introduce new functionalities by
subsequent reactions with electrophiles.
We started the synthesis of modified macrocycles with C3-
symmetric aza-â3-cyclohexamer 1 (aza-â3-Phe-aza-â3-Leu)3
which carried alternating benzyl and isobutyl side chains. The
hydrophobic aza-â3-leucine residue was introduced to retain a
good solubility in organic solvents for all new compounds. This
was particularly important in order to perform NMR spectra in
common solvent (CDCl3). A first attempt to remove the benzyl
groups of macrocycle 1 was made using 10% Pd/C in methanol.
The cleavage was slow and a mixture of partially deprotected
oligomers was obtained. In contrast, the addition of p-toluene-
sulfonic acid induced a complete deprotection with a very low
level of side products within 12 h at room temperature (Figure
3).10
These turns are linked together to confer a C3-symmetric
conformation to the macrocycle. Consequently, the chiral
sequence of the nitrogen centers keeps alternating. The mac-
rocycle oscillates slowly (around one time per second based on
variable-temperature experiments) between two mirror images
(Figure 2b). This represents an exceptionally slow pyramidal
inversion considering the size of the ring. By joining the chiral
nitrogen centers by virtual bonds (green lines in Figure 2b), a
(6) (a) Khazanovich, N.; Granja, J. R.; McRee, D. E.; Milligan, R. A.;
Ghadiri, M. R. J. Am. Chem. Soc. 1994, 116, 6011-6012. (b) Hartgerink,
J. N.; Granja, J. R.; Milligan, R. A.; Ghadiri, M. R. J. Am. Chem. Soc.
1996, 118, 43-50. (c) Clark, T. D.; Buehler, L. K.; Ghadiri, M. R. J. Am.
Chem. Soc. 1998, 120, 651-656. (d) Clark, T. D.; Buriak, J. M.; Kobayashi,
K.; Isler, M. P.; McRee, D. E.; Ghadiri, M. R. J. Am. Chem. Soc. 1998,
120, 8949-8962. (e) Bong, D. T.; Clark, T. D.; Granja, J. R.; Ghadiri, M.
R. Angew. Chem., Int. Ed. 2001, 40, 988-1011. (f) Rosenthal-Aizman, K.;
Svensson, G.; Unde´n, A. J. Am. Chem. Soc. 2004, 126, 3372-3373. (g)
Ashkenasy, N.; Horne, W. S.; Ghadiri, M. R. Small 2006, 1, 99-102.
(7) Le Grel, P.; Salau¨n, A.; Potel, M.; Le Grel, B.; Lassagne, F. J. Org.
Chem. 2006, 71, 5638-5645.
The next step was to introduce new side chains by reacting
HNR nucleophilic sites of macrocycle 2 with electrophilic
isocyanates RNdCdO (Figure 4). Compounds 3a-d were
obtained in quantitative yields starting from crude 2 and the
(9) Cheguillaume, A.; Doubli-Bounoua, I.; Baudy-Floc’h, M.; Le Grel,
P. Synlett 2000, 3, 331-334.
(10) Achiwa, K.; Yamada, S. I. Tetrahedron Lett. 1975, 31, 2701-2704.
(8) Salau¨n, A.; Favre, A.; Le Grel, B.; Potel, M.; Le Grel, P. J. Org.
Chem. 2006, 71, 150-158.
J. Org. Chem, Vol. 73, No. 4, 2008 1307

Le Grel et al.
FIGURE 5. 1H NMR (500 MHz) spectra of compounds 1 and 3b (aza-â3-CONHCH2CH2Br-aza-â3-Leu)3 (CDCl3, 10 mM, 298 K).
appropriate isocyanate. Our results clearly indicate that this
reaction can be easily performed and allows the introduction
of a wide variety of functional groups on the aza-â3-cyclo-
hexapeptidic backbone.
in eight-membered hydrogen-bonded pseudocycles. In contrast,
the signal of the urea NHu is strongly affected by the presence
of DMSO, indicating that the exocyclic NHs do not interfere
significantly with the intramolecular H-bonding pattern. The
downfield chemical shift values observed for NHh′s (around 11.0
ppm) compared with the NHhs values in an hydrazinoturn
(around 10.2 ppm) could reflect the higher acidity of NHh′
induced by the acylation of the NR nitrogen atom. On the other
hand, the chemical shift of a hydrazidic NHh in 3a-d (around
9.7 ppm) which are shielded from around 0.5 ppm relative to 1
could be rationalized by the lack of the sp3NR-HN contact.
2D-NOESY spectra of macrocycles 3a-d were recorded. For
all endocyclic NHs, strong NOEs between NHi and CRHi but
medium NOEs between NHi and CRHi-1 were observed (Figure
7), which are fully consistent with the previous interpretation.
As in compound 1, NR-methylene protons of 3a-d are
nonequivalent. AX- (∆δ ≈ 1.50 ppm) and AB-systems are
observed for (aza-â3-CONHR) and (aza-â3-Leu) units, respec-
tively. In C2D2Cl4, these signals coalesce around 378 K, close
to the value of 388 K observed for 1.7 Clearly, the strong
slowing of nitrogen pyramidal inversion that characterizes aza-
â3-cyclohexapeptides still occurs in modified compounds 3a-
d.
Analysis of the Conformation of Cyclohexamers 3a-d
(aza-â3-CONHR-aza-â3-Leu)3 in CDCl3. Theoretically, the
acylation of 2 turns the hybridization of NR nitrogen atoms from
sp3 to sp2. This last point raised some interesting questions: How
does the alternation of NRsp3/NRsp2 nitrogen atoms influence
the network of hydrazinoturns? To what extent does this
structural modification have an effect upon the conformation
of new macrocycle? Is the C3-symmetrical conformation
retained? Interestingly, the following data show that the
conformations of macrocycles are very similar, before and after
acylation.
1
The complete assignment of H and 13C NMR spectra was
made possible using classical 2D-COSY, HMBC, and HMQC
sequences. The spectral characteristics of cyclohexamers (aza-
â3-CONHR-aza-â3-Leu)3 present recurrent features. As for
1
precursors 1 and 2, H NMR spectra of 3a-d show only two
sets of signals, as illustrated in Figure 5 for compounds 1 and
3b. Consequently, macrocycles 3a-d still adopt a C3-sym-
metrical conformation in solution.
The backbones of compounds 3a-d combine two types of
CO-NH bonds: on the one hand, the CO-NH-Nsp3 hy-
drazidic linkage of (aza-â3-Leu) units, and on the other hand,
the CO-NH-Nsp2 acylated fragment of (aza-â3-CONHR)
groups. To discriminate both endocyclic NHs, the former were
noted NHh and the latter NHh′ (the exocyclic NH is noted NHu
for urea NH) (Figure 5). The downfield signals of NHs inside
the ring, combined with their insensitivity to dilution or addition
of increasing amounts of DMSO-d6 (Figure 6),11 are charac-
teristic of intramolecular H-bonding. These results can be
reasonably interpreted by assuming that the NHh′s participate
to hydrazinoturns and that consequently the NHhs are involved
After numerous attempts, we succeeded in getting suitable
crystals for X-ray diffraction with compound 3c (dichlo-
romethane/toluene). The high resolution of the structure revealed
the presence of disordered solvent molecules but allowed us to
resolve the conformation of the macrocycle. The molecule shows
a C3-symmetry axis. Inside the macrocycle, all NH protons are
intramolecularly H-bonded. Exocyclic NHs close five-membered
pseudocycles whereas endocyclic NHs are alternatively engaged
into two different bifurcated eight-membered rings. Bifidic
hydrogen-bonded C8 pseudocycles involving the lone pair of
the sp3 NR nitrogen atoms of (aza-â3-Leu) groups are typically
hydrazinoturns. Corresponding torsional angles ω, φ, θ, and ψ,
close to average values observed for aza-â3-cyclohexapeptides7
(ω ) 180°, φ ) -115°, θ ) +90°, and ψ ) (10° for an (S)-
(11) Kopple, K. D.; Ohnishi, M.; Go, A. Biochemistry 1969, 8, 4087.
1308 J. Org. Chem., Vol. 73, No. 4, 2008

Synthesis of C3-Symmetric Aza-â3-cyclohexapeptides
1
FIGURE 6. Superposition of H NMR (200 MHz) spectra in the presence of increasing amounts of DMSO-d6 (0-9%, from bottom to top) in a
10 mM solution of compound 3b (1 mL in CDCl3 at room temperature).
TABLE 2. Nr Bond Angles (deg) of N-Nsp -Turn and
3
2
N-Nsp -Turn in Cyclohexapeptide 3c
sp3 NR bond angles
Nâ-NR-CR Nâ-NR-CH2 CH2-NR-CR
sum of sp3 NR
bond angles
335.43
3
N-Nsp -turn
110.42
110.01
115.0
sp2 NR bond angles
Nâ-NR-CR Nâ-NR-CO CO-NR-CR
sum of sp2 NR
bond angles
2
N-Nsp -turn
116.12 115.93 117.38
349.43
FIGURE 7. Summary of NHs NOEs observed for cyclic backbone of
hexamers 3a-d (10 mM in CDCl3) at 288 K. s, strong NOE; m,
medium NOE.
TABLE 3. Distances (Å) and Angles (deg) of Bifurcated
3
2
Intramolecular H-Bond of N-Nsp -Turn and N-Nsp -Turn in
3
TABLE 1. Torsional Angles (deg) of (S)-N-Nsp -Turn and
Cyclohexapeptide 3c
2
N-Nsp -Turn in Cyclohexapeptide 3c
intra-
molecular
H bonds
distances (Å)
angles (deg)
torsional angles
Oi-HNi+2 NRi+1-HNi+2 Oi-H-Ni+2 NRi+1-H-Ni+2
ω
φ
θ
ψ
3
N-Nsp -turn
2.19
2.08
2.15
2.32
131.02
141.13
111.91
109.0
3
(S)-N-Nsp -turn
+178.82
-179.21
-113.04
+100.53
+89.83
-97.82
+7.17
+5.21
2
N-Nsp -turn
2
N-Nsp -turn
pairs of electrons of the two adjacent sp2 nitrogen atoms, which
minimizes electronic repulsions and steric hindrance. A slight
pyramidalization of the sp2 NR nitrogen atom toward hydrazidic
HN is observed which contributes to reinforce the H bond
closing the five-membered pseudocycle. The NR bond angles
hydrazinoturn), are listed in Table 1. Unexpectedly, the second
intramolecular C8 pattern also relies on a bifurcated H-bond
system where hydrazidic NHhi+2 interacts with both the carbonyl
group of the residue i and the Pz lone pair of the sp2 NR nitrogen
atom of residue i + 1 (aza-â3-CONHR). As illustrated in Table
1, the two sets of dihedral angles are very similar.12Therefore,
we decided to refer to this new local folded structure as a
3
2
of N-Nsp -turn and N-Nsp -turn are given in Table 2. Distances
and angles associated with the two bifurcated intramolecular
3
2
H-bond systems are listed in Table 3. N-Nsp -turn and N-Nsp
-
2
N-Nsp -turn (de facto, the foremost “classical hydrazinoturn”
turn conformations are depicted in Figure 8 (right).
3
will be noted N-Nsp -turn). The value of the torsional angle φ
The exocyclic NHs are intramolecularly H-bonded with the
Pz lone pair of the NHh′ sp2 nitrogen atom (Nâ-HN ) 2.32 Å).
The top view (Figure 8, left) indicates that the 24-membered
macrocycle exhibits an hexagonal shape with side chains in
radial positions. The side view in Figure 9 shows the wavy
conformation of the cyclic covalent backbone. The ring is also
) +100° corresponds to an orthogonal arrangement of the lone
(12) As in syndiotactic combination, two sets of dihedral angles with
opposite signs are observed.
3
(13) The N-Nsp -turn is represented in the R configuration in order to
2
facilitate the comparison with the N-Nsp -turn.
J. Org. Chem, Vol. 73, No. 4, 2008 1309

Le Grel et al.
3
2
FIGURE 8. (Left) Solid state conformation of cyclohexapeptide 3c: top view. (Right, top and bottom) (R)-N-Nsp -turn and N-Nsp -turn conformations
with corresponding torsional angles.13 Hydrogen bonds are indicated by dotted lines.
Experimental Section
General Hydrogenolysis Procedure.
Macrocycle 1 (800 mg, 0,92 mmol) dissolved in 40 mL of
methanol, was debenzylated quantitatively under hydrogen
atmospheric pressure in the presence of 80 mg of 10% Pd/C
with 1 equiv of para-toluenesulfonic acid monohydrate. After
12 h of stirring, the solution was filtrated on Celite and
evaporated. The crude residue was dissolved in 100 mL of CH2-
FIGURE 9. Solid state conformation of cyclohexapeptide 3c: side
Cl2 and the solution was washed vigorously with 5 mL of 0.5
view. Side chains are partially represented for clarity. Hydrogen bonds
N NaHCO3. After two extractions (CH2Cl2 and EtOAc), drying
are indicated by dotted lines.
of the organic phase on Na2SO4 and evaporation afforded
macrocycle 2 as a white powder (415 mg, 0.69 mmol, 75%).
This compound was carried on without further purification.
characterized by a median plane containing all endocyclic NH
nitrogen atoms. The six CO-NH bonds of the backbone are
nearly perpendicular to the cycle mean plane and are alterna-
General Acylation Procedure.
Compounds 3a-d (0.69 mmol) were obtained from crude 2
(0.69 mmol) and an excess (six equivalents) of the appropriate
isocyanate. The reaction is completed at room temperature in
dichloromethane in a few hours. Excess of isocyanate was
neutralized with ethanol (5 mL). After evaporation, the crude
residues gave rise to the apparition of white solids corresponding
to the expected macrocycles. The solids were then washed in
ether and isolated by filtration in almost quantitative yields.
tively orientated in opposite directions. Finally, the secondary
structure associated with the alternation of both types of
hydrazinoturns induces a very compact conformation superim-
posable with those of corresponding aza-â3-cyclohexapeptides
precursors.
Conclusions
The preliminary results presented give us confidence in our
ability to introduce a wide diversity of functional groups on
the aza-â3-cyclohexapeptidic backbone, leading to numerous
structural modulations and applications in the future. The
conformational homogeneity of the ring-backbone, which relies
on intramolecular H-bond network and exceptionally slow
nitrogen pyramidal inversion, is kept in all the series of
macrocycles. Most interesting is the ability to achieve a well-
defined scaffold without any asymmetric synthesis. Combined
with the highly efficient synthesis of the macrocyclic precursors,
the robust chemical sequence described here to perform their
refunctionalization makes it a very attractive route to a large
family of aza-â3-pseudopeptidic macrocycles. Actually, work
in progress by our team shows that the strategy depicted here
is limited to neither cyclohexamers nor C3-symmetric macro-
cycles.
Acknowledgment. We are grateful for the support provided
by ANR (National Research Agency).
Supporting Information Available: Characterization data for
1
compounds 1, 2, and 3a-d; copies of H NMR and 13C NMR
spectroscopic data; graphics showing the variation of the chemical
shifts of NHs as a function of the concentration of DMSO-d6 in
CDCl3 (0-9%) for compound 3b; superposition of 1H NMR spectra
at different temperatures in C2D2Cl4 for compounds 3a; portions
of the 2D-NOESY spectrum and the summary of NOEs observed
for compound 3c in CDCl3 at 298 K (s, strong NOE; w, weak
NOE); and crystallographic data in CIF format for compounds 2
and 3c. This material is available free of charge via the Internet at
http://pubs.acs.org.
JO7021394
1310 J. Org. Chem., Vol. 73, No. 4, 2008