Synthesis and structural study of highly constrained hybrid cyclobutane-proline γ,γ-peptides

Article abstract of DOI:10.1007/s00726-011-0912-4

Two diastereomeric series of hybrid γ,γ-peptides derived from conveniently protected derivatives of (1R,2S)- and (1S,2R)-3-amino-2,2- dimethylcyclobutane-1-carboxylic acid and cis-4-amino-l-proline joined in alternation have efficiently been prepared thro

Full text of DOI:10.1007/s00726-011-0912-4

Amino Acids (2011) 41:673–686
DOI 10.1007/s00726-011-0912-4
ORIGINAL ARTICLE
Synthesis and structural study of highly constrained hybrid
cyclobutane-proline c,c-peptides
•
Raquel Gutierrez-Abad Daniel Carbajo
•
´
Pau Nolis Carles Acosta-Silva Juan A. Cobos
•
•
•
•
•
Ona Illa Miriam Royo Rosa M. Ortun˜o
Received: 14 January 2011 / Accepted: 2 April 2011 / Published online: 4 May 2011
Ó Springer-Verlag 2011
Abstract Two diastereomeric series of hybrid c,c-pep-
tides derived from conveniently protected derivatives of
(1R,2S)- and (1S,2R)-3-amino-2,2-dimethylcyclobutane-1-
carboxylic acid and cis-4-amino-L-proline joined in alter-
nation have efficiently been prepared through convergent
synthesis. High-resolution NMR experiments show that
these compounds present defined conformations in solution
affording very compact structures as the result of intra and
inter residue hydrogen-bonded ring formation. (R,S)-
cyclobutane containing peptides adopt more twisted con-
formations than (S,R) diastereomers. In addition, all these
c-peptides have high tendency to aggregation providing
vesicles of nanometric size, which were stable when
allowed to stand for several days, as verified by transmis-
sion electron microscopy.
Abbreviations
Boc
t-Butoxycarbonyl
t-Butyl
tBu
tBuOH
t-Butanol
Circular dichroism
Benzyl carbamate
CD
Cbz
DIPEA
DMAP
EDAC
N,N-Diisopropylethylamine
4-Dimethylaminopyridine
1-Ethyl-3-(3-dimethylaminopropyl)
carbodiimide
GABA
HMBC
HSQC
Hz
c-Aminobutyric acid
Heteronuclear multiple bond correlation
Heteronuclear single quantum correlation
Hertz
Me
Methyl
MeOH
NMR
NOE
Methanol
Keywords Hybrid c,c-peptides ꢀ Cyclobutane ꢀ
cis-4-amino-L-proline ꢀ Hydrogen bonds ꢀ
Nuclear magnetic resonance
Nuclear overhauser effect
Secondary structures ꢀ Self-assembly ꢀ Vesicles
NOESY Nuclear overhauser effect spectroscopy
ppm Parts per million
PyBOP Benzotriazol-1-yl-
oxytripyrrolidinophosphonium
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-011-0912-4) contains supplementary
material, which is available to authorized users.
hexafluorophosphate
ROESY Rotational nuclear overhauser effect
spectroscopy
´
R. Gutierrez-Abad ꢀ C. Acosta-Silva ꢀ J. A. Cobos ꢀ O. Illa ꢀ
R. M. Ortun˜o (&)
Departament de Quımica, Universitat Autonoma de Barcelona,
TEM
TFA
THF
Transmission electron microscopy
Trifluoroacetic acid
´
Bellaterra, 08193 Barcelona, Spain
e-mail: rosa.ortuno@uab.cat
`
Tetrahydrofuran
TOCSY Total correlation spectroscopy
D. Carbajo ꢀ M. Royo
Combinatorial Chemistry Unit, Barcelona Science Park,
University of Barcelona, Baldiri Reixac, 10,
08028 Barcelona, Spain
Introduction
P. Nolis
Servei de RMN, Universitat Autonoma de Barcelona, Bellaterra,
08193 Barcelona, Spain
Foldamers with heterogeneous backbones (hybrid peptides)
`
consisting, in most cases, of a,b-, a,c-, or b,c-amino acids
123

´
R. Gutierrez-Abad et al.
674
(DePol et al. 2004; Guo et al. 2009, 2010; Hecht and Huc
2007; Horne and Gellman 2008) have been prepared with
the aim to enhance the ability of peptide oligomers to fold
in defined manners. Also, combinations of different b,b-
(Cheng et al. 2001; Izquierdo et al. 2004) and, in less
extent, c,c-amino acids (Brenner and Seebach 2001; See-
bach et al. 2002; Aguilera et al. 2008) have been used for
these purposes. Moreover, replacement of linear residues
by alicyclic ones has resulted, in some instances, in the
formation of strong secondary structures and in changing
their biological properties (Koglin et al. 2003; Lang et al.
2006).
construction of c-oligomers that can be modified by intro-
duction of alkyl chains or other functional groups by
manipulation of the pyrrolidine nitrogen. The ability to fold
into defined secondary structures of homopeptides
containing some of these compounds has been investigated
(Farrera-Sinfreu et al. 2004). To mention some applications,
4-aminoprolines and derivatives have been used for the syn-
thesis of distamycin analogues with DNA-binding affinity
(Woods et al. 2002), for the preparation of cell-penetrating
peptides (Farrera-Sinfreu et al. 2005), and for the synthesis of
helical dendronized polymers (Zhang and Schlu¨ter 2007;
´
Zhang et al. 2008; Rodrıguez-Ropero et al. 2009).
We have previously synthesized b- and c-cyclobutane
amino acids and incorporated them into different oligo-
mers. For b-peptides constituted by cyclobutane b-amino
acids, we have observed secondary structures derived from
the formation of six-membered hydrogen-bonded rings due
to COꢀꢀꢀHN intra-residual interactions in the case of all-cis-
cyclobutane derivatives, from a dimer up to an octamer
(Torres et al. 2010). In contrast, for b-dipeptides containing
two trans-cyclobutane amino acids or one cis and another
trans, we have found the formation of eight-membered
rings as the consequence of inter-residual CO(i)ꢀꢀꢀHN(i?1)
hydrogen bonds (Torres et al. 2009). In the case of hybrid
b-peptides consisting of cis-cyclobutane amino acids and
b-alanine joint in alternation, a 14-helix resulted as the
preferred folding showing thus the ability of the cyclobu-
tane ring to induce secondary structures (Izquierdo et al.
2004). Some of these peptides presented activity as
We decided to combine cis-4-amino-L-proline derivative
3 (Fig. 1) (Torino et al. 2009) and enantiomeric amino
acids 1 and 2 to afford diastereomeric hybrid c,c-peptides
with the purpose to study the influence of both cyclic
residues on the structural features of the resultant c-pep-
tides. In this article, we report the synthesis of several
oligomers, dimers, tetramers and hexamers, for each dia-
stereomeric series and their structural study mainly based
in high resolution NMR techniques. The ability of some of
these compounds to aggregate has been also investigated
by TEM and results are reported herein.
Materials and methods
General
´
metallocarboxypeptidase inhibitors (Fernandez et al. 2009).
The chemicals and reagents (Sigma–Aldrich, Fluka,
Madrid, Spain) were of analytical grade and were used
without further purification. Anhydrous dichloromethane
was freshly distilled when needed under a nitrogen atmo-
sphere from calcium chloride. Anhydrous methanol was
freshly distilled from calcium hydride. TLC on silica gel-
coated aluminium plates was performed in the systems
indicated for each product in the following description. The
compounds were visualized by exposure to UV light at
254 nm, dipping in a basic potassium permanganate solu-
tion or in an acid vanillin solution. Flash chromatography
purifications were carried out on silica gel (200–400 mesh).
Melting points were recorded on a Reicher Klofler block
and values are uncorrected. 1H NMR and 13C NMR spectra
were measured in a Bruker Avance 600 apparatus (1H at
600.13 MHz, 13C at 150.9 MHz) in CDCl3 solution at
298 or 273 K. The 2D-H,H-COSY, 2D-H,C-HSQC and
In addition, some of these oligomers self-assemble hier-
archically producing stable gels or fibres in common
´
organic solvents (Rua et al. 2007; Gorrea et al. 2011).
We have also prepared fully protected cyclobutane
enantiomeric c-amino acids 1 and 2 (Fig. 1), which have
been used for the preparation of short c-peptides consisting
of all cyclobutane amino acids, with the same or different
configuration, and in hybrid c-peptides obtained by
sequential alternation with GABA residues. Preliminary
results show the tendency of these oligomers to adopt
extended conformations or to form b-sheets (Aguilera et al.
2008).
4-Aminoprolines when included in oligomers, can behave
both as an a- and as a c-amino acid with an additional amino
function. The key lies on which amino group is involved
in the peptide bond. They are useful scaffolds for the
Fig. 1 Monomers used in this
work for the synthesis of hybrid
c-peptides
123

Synthesis and structural study of highly constrained hybrid
675
Fig. 2 1H-NMR spectrum of 3
acquired in a 600 MHz
spectrometer at 273 K. Trans
isomer is marked T and cis
isomer is marked C
1
2D-H,C-HMBC spectra were recorded and used for the
structural assignment of proton and carbon signals. IR
spectra were obtained from samples in neat form with an
Attenuated Total Reflectance (ATR) accessory. High res-
olution mass spectra were recorded using a direct inlet
system (ESI) in electrospray ionization mode.
Trans-conformer H NMR (600 MHz; CDCl3): 1.46 (s,
9H, H9), 1.96 (d, J = 14.9 Hz, 1H, H7 proR), 2.48 (m, 1H,
H7 proS), 3.49 (d, J = 11.4 Hz, 1H, H5 proS), 3.61 (m, 1H,
H5 proR), 3.77 (s, 3H, H1), 4.34 (dd, J = 9.8 Hz,
J0 = 2.3 Hz, 1H, H3), 4.42 (m, 1H, H6), 5.10 (m, 2H,
H11 CH2), 5.93 (d, J = 9.2 Hz, 1H, H10), 7.33–7.37 (m, 5H,
H11 Ar). 13C NMR (150 MHz; CDCl3): 28.3 (C9), 35.8 or
36.8 (C7), 50.7 (C6), 52.9 (C1), 53.7 (C5), 57.6 (C3), 66.8
(C11 CH2), 80.7 (C9q), 128.2, 128.3 and 128.6 (C11 Ar),
136.3 (C11q), 154.2 (C8), 155.8 (C11 CO), 174.8 (C2).
Triprotected Proline (3)
Previously described product (Torino et al. 2009). Detailed
NMR of conformers at 273 K in CDCl3 (see Fig. 2 for
numbering).
c-Dipeptide (9)
1
Cis-Conformer H NMR (600 MHz; CDCl3): 1.41 (s,
9H, H9), 1.98 (d, J = 14.9 Hz, 1H, H7 proR), 2.48 (m, 1H,
H7 proS), 3.58 (d, J = 11.4 Hz, 1H, H5 proS), 3.61 (m, 1H,
H5 proR), 3.75 (s, 3H, H1), 4.26 (dd, J = 9.8 Hz,
J0 = 2.5 Hz, 1H, H3), 4.42 (m, 1H, H6), 5.10 (m, 2H,
H11 CH2), 5.86 (d, J = 9.2 Hz, 1H, H10), 7.33–7.37 (m, 5H,
H11 Ar). 13C NMR (150 MHz; CDCl3): 28.3 (C9), 35.8 or
36.8 (C7), 49.7 (C6), 52.6 (C1), 53.1 (C5), 57.7 (C3), 66.8
(C11 CH2), 80.7 (C9q), 128.2, 128.3 and 128.6 (C11 Ar),
136.3 (C11q), 153.5 (C8), 155.7 (C11 CO), 174.7 (C2).
Acid (4) (292 mg, 0.58 mmol), DIPEA (0.30 mL,
1.57 mmol) and PyBOP (420 mg, 0.78 mmol) were dis-
solved in anhydrous dichloromethane (15 mL). The mix-
ture was stirred for 5 min under nitrogen atmosphere, and
then a solution of amine 8 (92 mg, 0.58 mmol) in anhy-
drous dichloromethane (10 mL) was added via cannula.
After stirring at room temperature for 2 h the reaction
crude was washed with a saturated aqueous sodium
bicarbonate solution. The organic phase was dried over
123

´
R. Gutierrez-Abad et al.
676
magnesium sulfate and the solvents were removed under
vacuum. The reaction crude was purified by column
chromatography on neutral silica gel (ethyl acetate/hexane
1:1) to afford pure dipeptide (9). Yield 292 g (quantita-
(C8), 29.3 (C25), 31.1 (C15), 31.9 (C32), 43.0 (C3), 44.3
(C20), 45.8 (C23), 46.2 (C6), 49.1 (C14), 50.0 and 50.1 (2C,
C5, C22), 50.7 (C31), 51.4 (C1), 55.2 (C30), 55.5 (C13), 59.2
(C11), 59.3 (C28), 66.4 (C36 CH2), 80.9 and 81.1 (2C, C17q
,
C34q), 127.9, 128.0 and 128.4 (C36 Ar), 136.7 (C36q), 156.1
and 156.3 (3C, C16, C33, C36 CO), 171.2 (C19), 172.2 (C27),
172.4 (C10), 172.7 (C2). IR (ATR, cmax cm-1) 3,355, 2,926,
2,854, 1,700, 1,671, 1,542, 1,459, 1,398. HRMS (ESI)
calculated for C43H64N6NaO11 [M ? Na]? 863.4525;
found: 863.4534.
20
tive). White solid. mp 52–54°C (from Et2O). ½aꢁD -13.8 (c
0.99, CH2Cl2) (see Fig. 6 for numbering). 1H NMR
(600 MHz; CDCl3): 0.87 (s, 3H, H7), 1.27 (s, 3H, H8), 1.40
(s, 9H, H17), 2.12–2.21 (m, 2H, H4 proR, H15 proS), 2.25 (m,
1H, H4 proS), 2.40 (d, J = 13.7 Hz, 1H, H15 proR), 2.62 (dd,
J = 10.0 Hz, J0 = 8.0 Hz, 1H, H3), 3.47 (d, J = 11.6 Hz,
1H, H13 proS), 3.55 (m, 1H, H13 proR), 3.70 (s, 3H, H1), 4.08
(dd, J = 17.8 Hz, J0 = 8.6 Hz, 1H, H5), 4.30 (m, 1H, H14),
4.43 (d, J = 8.7 Hz, 1H, H11), 5.10 (m, 2H, H19 CH2), 6.85
(d, J = 6.2 Hz, 1H, H18), 7.32–7.37 (m, 5H, H19 Ar), 7.72
(d, J = 8.0 Hz, 1H, H9). 13C NMR (150 MHz; CDCl3):
17.0 (C7), 26.2 (C4), 28.3 (C17), 29.0 (C8), 31.5 (C15), 43.1
(C3), 46.3 (C6), 50.1 (C5), 50.7 (C14), 51.4 (C1), 55.1 (C13),
59.21 (C11), 66.4 (C19 CH2), 81.2 (C17q), 128.0 and 128.4
(C19 Ar), 136.7 (C19q), 156.0 (C19 CO), 156.2 (C16), 172.3
(C10), 172.8 (C2). IR (ATR, cmax cm-1) 3,312, 2,956,
2,876, 1,677, 1,521, 1,393. HRMS (ESI) calculated for
C26H37N3O7 [M ? Na]? 526.2524; found: 526.2506.
c-Hexapeptide (13)
Acid (12a) (140 mg, 0.17 mmol), DIPEA (0.12 mL,
0.60 mmol) and PyBOP (161 mg, 0.30 mmol) were dis-
solved in anhydrous dichloromethane (10 mL), the mixture
was stirred for 5 min under nitrogen atmosphere, and then
a solution of amine (11) (70 mg, 0.19 mmol) in anhydrous
dichloromethane (5 mL) was added via cannula. After
stirring at room temperature for 2 h the reaction crude was
washed with a saturated aqueous sodium bicarbonate
solution. The organic phase was dried over magnesium
sulfate and the solvents were removed under vacuum. The
reaction crude was purified by column chromatography on
neutral silica gel (ethyl acetate to ethyl acetate/methanol
19:1) to afford pure hexapeptide (13). Yield 180 mg (81%).
c-Tetrapeptide (12)
Acid (10) (90 mg, 0.19 mmol), DIPEA (0.12 mL,
0.60 mmol) and PyBOP (161 mg, 0.29 mmol) were dis-
solved in anhydrous dichloromethane (10 mL). The mix-
ture was stirred for 5 min under nitrogen atmosphere, and
then a solution of amine (11) (65 mg, 0.8 mmol) in
anhydrous dichloromethane (5 mL) was added via cannula.
After stirring at room temperature for 2 h, the reaction
crude was washed with a saturated aqueous sodium
bicarbonate solution. The organic phase was dried over
magnesium sulfate and the solvents were removed under
vacuum. The reaction crude was purified by column
chromatography on neutral silica gel (ethyl acetate) to
afford pure tetrapeptide (12). Yield 145 mg (96%). White
20
White solid. mp 128–131°C (from AcCN/H2O). ½aꢁD 18.5
(c 0.1, CH2Cl2). (See Supplementary Material for num-
1
bering) H NMR (600 MHz; CDCl3): 0.76–0.81 (m, 9H,
H7, H24, H24 and H41), 1.12–1.17 (m, 9H, H8, H25 and H42),
1.35–1.38 (m, 27H, H17, H34 and H51), 2.03–2.26 (m, 12H,
H4, H15, H21, H32, H38 and H49), 2.41–2.44 (m, 2H, H20 and
H37), 2.54 (m, 1H, H3), 3.26–3.44 (m, 6H, H13, H30 and
H47), 3.61 (s, 3H, H1), 3.96–3.97 (m, 3H, H5, H22 and H39),
4.21 (m, 1H, H48), 4.31 (m, 1H, H45), 4.34 (m, 1H, H28),
4.36–4.38 (m, 3H, H11, H14 and H31), 5.01 (m, 2H, H53
CH2), 6.93 (d, J = 6.8 Hz, 1H, H52), 7.23–7.28 (m, 5H,
H53 Ar), 7.56 (d, J = 8.1 Hz, 1H, H43), 7.60 (d, J =
8.9 Hz, 1H, H26), 7.66 (d, J = 9.0 Hz, 1H, H9), 7.94 (d,
J = 7.1 Hz, 1H, H18), 8.03 (d, J = 7.2 Hz, 1H, H35). IR
(ATR, cmax cm-1) 3,298, 2,957, 1,673, 1,520, 1,392, 1,367.
HRMS (ESI) calculated for C60H91N9O15 [M ? Na]?
1,200.6527; found: 1,200.6521.
20
solid. mp 79–81°C (from H2O). ½aꢁD -310.8 (c 0.06,
1
CH2Cl2) (see Fig. 7 for numbering). H NMR (600 MHz;
CDCl3): 0.85 and 0.86 (s, 6H, H7 and H24), 1.22 (s, 3H,
H8), 1.26 (s, 3H, H25), 1.45 (s, 18H, H17 and H34), 2.10–
2.35 (m, 8H, H4 proS, H4 proR, H15 proS, H15 proR, H21 proS
,
H21 proR, H32 proS, H32 proR), 2.48 (m, 1H, H20), 2.60 (m,
1H, H3), 3.34 (m, 1H, H30 proS), 3.44 (m, 1H, H13 proR),
3.52 (m, 1H, H30 proR), 3.68 (s, 3H, H1), 4.00–4.08 (m, 2H,
H5 and H22), 4.30 (m, 1H, H31), 4.38–4.47 (m, 3H, H11, H14
and H28), 5.09 (m, 2H, H36 CH2), 6.93 (d, J = 6.1 Hz, 1H,
H35), 7.30–7.36 (m, 5H, H36 Ar), 7.56 (d, J = 7.9 Hz, 1H,
H26), 7.70 (d, J = 8.4 Hz, 1H, H9), 7.81 (d, J = 6.4 Hz,
1H, H18). 13C NMR (150 MHz; CDCl3): 16.5 (C24), 17.0
(C7), 26.0 (C21), 26.2 (C4), 28.3 (6C, C17 and C34), 29.0
c-Dipeptide (14)
Acid (4) (210 mg, 0.57 mmol), DIPEA (0.34 mL,
1.71 mmol) and PyBOP (480 mg, 0.86 mmol) were dis-
solved in anhydrous dichloromethane (15 mL). The mixture
was stirred for 5 min under nitrogen atmosphere, and then a
solution of amine ent-8 (90 mg, 0.57 mmol) in anhydrous
dichloromethane (5 mL) was added via cannula. After
123

Synthesis and structural study of highly constrained hybrid
677
stirring at room temperature for 2 h the reaction crude was
washed with a saturated aqueous sodium bicarbonate
solution. The organic phase was dried over magnesium
sulfate and the solvents were removed under vacuum. The
reaction crude was purified by column chromatography on
neutral silica gel (ethyl acetate/hexane 1:1) to afford pure
dipeptide (14). Yield 276 mg (95%). White solid. mp 59–
H
11 and H28), 4.48 (m, 1H, H14), 5.04 (d, J = 12.3 Hz, 1H,
H36 CH2), 5.12 (d, J = 12.3 Hz, 1H, H36 CH2), 6.80 (d,
J = 7.1 Hz, 1H, H35), 7.32–7.38 (m, 5H, H36 Ar), 7.63–7.72
(m, 3H, H9, H18 and H26). 13C NMR (150 MHz; CDCl3):
17.0 (C25), 17.3 (C8), 25.8 (C4), 26.3 (C21), 28.4 (6C, C17,
C34), 28.8 (C24), 29.0 (C7), 31.9 (C15), 32.1 (C32), 43.0 (C3),
44.7 (C20), 45.6 and 45.9 (2C, C25, C6), 49.0 (C1), 49.9 (2C,
C5, C22), 50.7 (C31), 51.7 (C14), 55.2 (2C, C13, C30), 59.2 (2C,
C11, C28), 66.6 (C36 CH2), 81.2 and 81.3 (2C, C17q, C34q),
128.1, 128.3 and 128.5 (C36 Ar), 136.6 (C36q), 156.1 and
156.2 (3C, C16, C33, C36 CO), 171.1 (C19), 172.1 (C27), 172.5
(C10), 173.0 (C2). IR (ATR, cmax cm-1) 3,640, 3,571, 3,292,
2,956, 1,665, 1,524, 1,391, 1,368. HRMS (ESI) calculated
for C43H65N6O11 [M ? H]? 841.4706; found: 841.4698.
62°C (from Et2O). ½aꢁ20 -109.4 (c 0.39, CH2Cl2) (see Fig. 6
D
1
for numbering). H NMR (600 MHz; CDCl3): 0.95 (s, 3H,
H7), 1.28 (s, 3H, H8), 1.47 (s, 9H, H17), 2.11 (m, 1H,
H4 proS), 2.16 (m, 1H, H15 proS), 2.32 (m, 1H, H4 proR), 2.38
(m, 1H, H15 proS), 2.61 (dd, J = J0 = 8.9 Hz, 1H, H3), 3.45
(dd, J = J0 = 10.8 Hz, 1H, H13 proS), 3.54 (dd,
J = 10.8 Hz, J0 = 4.8 Hz, 1H, H13 proR), 3.69 (s, 3H, H1),
4.06 (dd, J = 17.2 Hz, J0 = 8.5 Hz, 1H, H5), 4.30 (m, 1H,
H14), 4.42 (d, J = 8.8 Hz, 1H, H11), 5.06 (d, J = 12.3 Hz,
1H, H19 CH2), 5.13 (d, J = 12.3 Hz, 1H, H19 CH2), 6.68
(d, J = 6.4 Hz, 1H, H18), 7.32–7.37 (m, 5H, H19 Ar), 7.67
(d, J = 7.6 Hz, 1H, H9). 13C NMR (150 MHz; CDCl3):
17.2 (C8), 26.3 (C4), 28.3 (3C, C17), 28.8 (C7), 31.8 (C15),
43.1 (C3), 46.0 (C6), 50.0 (C5), 50.7 (C14), 51.5 (C1), 55.1
(C13), 59.2 (C11), 66.5 (C19 CH2), 81.2 (C17q), 128.0, 128.2
and 128.5 (C19 Ar), 136.7 (C19q), 156.0 (C19 CO), 156.3
(C16), 172.1 (C2), 172.9 (C10). IR (ATR, cmax cm-1) 3,305,
2,958, 1,679, 1,671, 1,541, 1,406. HRMS (ESI) calculated
for C26H37N3O7 [M ? Na]? 526.2524; found: 526.2504.
c-Hexapeptide (16)
Acid (15a) (90 mg, 0.10 mmol), DIPEA (0.06 mL,
0.30 mmol) and PyBOP (80 mg, 0.15 mmol) were dis-
solved in anhydrous dichloromethane (10 mL). The mix-
ture was stirred for 5 min under nitrogen atmosphere, and
then a solution of amine (14b) (60 mg, 0.10 mmol) in
anhydrous dichloromethane (5 mL) was added via cannula.
After stirring at room temperature for 2 h the reaction
crude was washed with a saturated aqueous sodium
bicarbonate solution. The organic phase was dried over
magnesium sulfate and the solvents were removed under
vacuum. The reaction crude was purified by column
chromatography on neutral silica gel (ethyl acetate to ethyl
acetate/methanol 19:1) to afford pure hexapeptide (16).
Yield 100 mg (84%). White solid. mp 144–146°C (from
c-Tetrapeptide (15)
Acid (14a) (160 mg, 0.33 mmol), DIPEA (0.2 mL,
1.00 mmol) and PyBOP (280 mg, 0.50 mmol) were dis-
solved in anhydrous dichloromethane (15 mL). The mixture
was stirred for 5 min under nitrogen atmosphere, and then a
solution of amine (14b) (120 mg, 0.33 mmol) in anhydrous
dichloromethane (5 mL) was added via cannula. After stir-
ring at room temperature for 2 h the reaction crude was
washed with a saturated aqueous sodium bicarbonate solu-
tion. The organic phase was dried over magnesium sulfate
and the solvents were removed under vacuum. The reaction
crude was purified by column chromatography on neutral
silica gel (ethyl acetate) to afford pure tetrapeptide (15) Yield
255 mg (92%). White solid. mp 129–131°C (from Et2O).
20
AcCN/H2O). ½aꢁD -131 (c 0.13, CH2Cl2) (see supple-
mentary material for numbering). 1H NMR (600 MHz;
CDCl3): 0.93–0.97 (m, 9H, H8, H25 and H42), 1.21–1.31
(m, 9H, H7, H24 and H41), 1.44–1.46 (m, 27H, H17, H34 and
H51), 2.10–2.44 (m, 14H, H4, H15, H21, H32, H38, H49, H20
and H37), 2.64 (m, 1H, H3), 3.37–3.53 (m, 6H, H13, H30 and
H47), 3.69 (s, 3H, H1), 4.02–4.07 (m, 3H, H5, H22 and H39),
4.29 (m, 1H, H48), 4.37–4.43 (m, 3H, H11, H28 and H45),
4.47–4.51 (m, 2H, H14 and H31), 5.04 (d, J = 12.3 Hz, 1H,
H53 CH2), 5.12 (d, J = 12.3 Hz, 1H, H53 CH2), 6.81 (d,
J = 7.2 Hz, 1H, H52), 7.33–7.37 (m, 5H, H53 Ar), 7.62 (d,
J = 7.9 Hz, 1H, H26), 7.67–7.75 (m, 4 H, H9, H18, H35 and
H43). IR (ATR, cmax cm-1) 3,292, 2,957, 1,666, 1,534,
1,392, 1,368. HRMS (ESI) calculated for C60H91N9O15
[M ? Na]? 1,200.6527; found: 1,200.6509.
20
½aꢁD -16.4 (c 0.57, CH2-Cl2) (see supplementary material
1
for numbering). H NMR (600 MHz; CDCl3): 0.93 (s, 3H,
H25), 0.95 (s, 3H, H8), 1.27 (s, 3H, H7), 1.31 (s, 3H, H24), 1.46
(s, 18H, H17 and H34), 2.10–2.20 (m, 4H, H4 proS, H15 proS
,
H21 proS and H32 proS), 2.24–2.27 (m, 1H, H15 proR), 2.32–2.45
(m, 4H, H4 proR, H20 proR, H21 proR and H32 proR), 2.63 (dd,
J = 10.1 Hz, J0 = 7.8 Hz, 1H, H3), 3.38 (d, J = 11.6 Hz,
1H, H13 proS), 3.45 (d, J = 11.5 Hz, 1H, H30 proS), 3.50–3.55
(m, 2H, H13 proR and H30 proR), 3.70 (s, 3H, H1), 4.02–4.07
(m, 2H, H5 and H22), 4.29 (m, 1H, H31), 4.45–4.50 (m, 2H,
Results and discussion
Scheme 1 shows the synthesis of both enantiomers 1 and 2
from (-)-verbenone as a common chiral precursor.
123

´
R. Gutierrez-Abad et al.
678
This compound is commercially available and the dou-
the supplementary material. We started studying fully
protected 4-aminoproline 3 itself because no detailed data
were provided in the literature on this compound. 600 MHz
1H-NMR spectrum of 3 acquired at 273 K clearly showed
split resonances in most of the protons. It is widely known
that dynamic rotation of the conjugated NC bond in Boc
group is very slow within the NMR time scale giving rise
to cis/trans conformers. The key point for the unambiguous
assignment of both conformers was the NOE contacts
observed in tert-Bu group. While trans isomer correlates
tert-Bu with H5 protons, cis isomers does it with Me1 and
H3 proton. Both conformers are almost equally populated
(Fig. 2).
1D selective TOCSY experiments irradiating at H10
protons allowed us to obtain cis/trans proline protons in
separate subspectra thus facilitating the cis/trans chemical
shift assignment (see the supplementary material).
13C-NMR spectrum acquired at 273 K also exhibited split
resonances for most of the signals due to cis/trans isomers.
It is noticeable that CO8 signal difference between cis and
trans isomer was about 113 Hz, while the difference found
between cis/trans isomers in CO2 in the methyl ester and
CO11 in the Cbz group was much lower (16 and 9 Hz,
respectively), therefore confirming that conformational
rotational barrier is due to NC bond in the Boc group.
2D-NOESY spectrum acquired at 273 K was the key point
to assign unequivocally cis and trans isomers. The
expanded region showing tert-Bu cross peaks indicated
that the slightly major component has cross peaks with
H3 and Me1 protons and, therefore, can be attributed to cis
conformer. On the other hand, minor component corre-
lates with H5 protons, consequently assigned to trans
conformer.
ble bond was oxidatively cleaved to afford quantitatively
(-)-cis-pinononic acid without epimerization (Moglioni
et al. 2000). Activation of the carboxyl group by formation
of a mixed anhydride through reaction with ethyl chloro-
formate and subsequent reaction with sodium azide affor-
ded an acyl azide which was submitted to Curtius
rearrangement by heating in toluene in the presence of
benzyl alcohol to afford compound 5. Lieben degradation
of the methyl ketone and esterification of the resulting
carboxylic acid produced orthogonally protected amino
acid 2. Alternatively, (-)-cis-pinononic acid was protected
as a tert-butyl ester and the ketone was submitted to Lieben
degradation to give carboxylic acid 6, which was trans-
formed into benzyl carbamate 7 following the same
transformations as in the case of 2. Removal of the tert-
butyl ester in 7 and subsequent methylation provided
enantiomer 1 (Aguilera et al. 2008).
Benzyl carbamate in this compound was reductively
cleaved by reaction with ammonium formate in the pres-
ence of 10% Pd/C in refluxing methanol affording free
amine 8 (Scheme 2). This compound was reacted with
partially protected 4-amino proline 4 (Fisher et al. 2006)
under usual coupling conditions by using PyBOP and
DIPEA in anhydrous dichloromethane to provide hybrid
c-dipeptide 9 in quantitative yield.
Tetrapeptide 12 was prepared through a convergent
synthesis by coupling acid 10, obtained from 9 by mild
saponification with 1 M LiOH, and the amine resulting
from removal of the benzyl carbamate in a second mole-
cule of 9. In this way, 12 was obtained in 96% yield.
Finally, deprotection of the carboxyl group in 12 followed
by coupling with amine 11 afforded hexamer 13 in 81%
yield.
Interestingly, while inverting H10 proton in 1D selective
NOE experiment, independently of cis or trans isomer,
NOE effects were observed at pro-S H5 (H5S) and pro-R H7
(H7R) protons, suggesting that NH10 proton in both con-
formers is pointing to the carbonyl oxygen of the methyl
Similarly, the diastereomeric series of hybrid c-peptides
14–16 (Fig. 3) was synthesized.
Next, we decided to investigate the conformational bias
of these products in solution. Full details are provided in
Scheme 1 Reagents,
conditions, yields: (a) RuCl3,
NaIO4, CH2Cl2/CH3CN/H2O,
rt, quantitative; (b) ClCO2Et;
(c) NaN3 (90% for 4, 98% for 6,
two steps); (d) BnOH, toluene,
reflux (92%); (e) NaOBr,
dioxane/H2O (80% for 2, 93%
for 5); (f) CH3I, Cs2CO3, DMF
t
(95%); (g) BuOH, DMAP,
EDAC, Et3N, anhydrous THF,
0°C (60%); (h) TFA, Et3SiH,
CH2Cl2 (94%)
123

Synthesis and structural study of highly constrained hybrid
679
Scheme 2 Reagents,
conditions, yields:
(a) (NH4)HCO2, 10% Pd/C,
MeOH, reflux (quantitative);
(b) PyBOP, DIPEA, anhydrous
CH2Cl2 (quantitative for 9, 96%
for 12, 81% for 13); (c) 1 M
LiOH, 2:10 THF/H2O
(quantitative)
ester group probably due to the presence of a hydrogen
bond. Also, conformational exchange signals were detected
in that experiment between cis and trans isomers (see the
supplementary material). Furthermore, two small signals
resonating *0.5 ppm downfield with respect to the major
signals were observed and were assigned to minor con-
formations without such hydrogen bonding. According to
all NMR experiments performed, the conformational
equilibrium of modified proline 3 can be depicted as shown
in Fig. 4.
The next step was to investigate if the presence of the
cyclobutane residues in the hybrid oligomers has any
influence on this equilibrium and if preferred conforma-
tions change. Therefore, the two diastereomeric dimers 9
and 14 were studied. Differently to 4-aminoproline 3, the
respective 1H-NMR spectra of both 9 and 14 clearly
showed a single major conformation. Strongly deshielded
position of H9 suggested a hydrogen bond between NH9
and CO16 building a seven-membered ring stacked to the
five-membered proline ring fixing, therefore, Boc rotamer
to trans position (see Fig. 6 for atom numeration). NOE
experiments confirmed such hypothesis as described
below. 1D selective TOCSY experiment irradiating at the
NH protons allows the separation of proline and cyclobu-
tane subspectra, which affords a clear visualization of the
correct proton assignment of the molecule.
1
Finally, variable H-NMR temperature experiments led
to visualize the coalescence temperature between major
cis/trans Boc isomers (see the supplementary material). As
conformer populations are almost equal, Eyring’s equation
was used to approximately determine a rotational barrier of
18.1 kcal/mol for the cis/trans equilibrium.
123

´
R. Gutierrez-Abad et al.
680
Fig. 3 Structures of hybrid
c-peptides 14, 15, 16
Considering dimer 14, J coupling values together with
1D selective NOE experiments (Fig. 5) over NH protons
give an excellent visualization of the conformational
structure of the molecule. Selective inversion of H9 gives a
strong NOE effect with H11. This fact, together with the
highly deshielded position found for that proton, clearly
Furthermore, exchange peaks are observed in selective
NOE experiment indicating the presence of minor confor-
mations during the NMR experiment time scale (500 ms).
1
Those conformers can be seen in the H-NMR spectrum
background but close to noise level, making their study
difficult. However, the broadness and the chemical shift
position of the exchange peaks observed in NOE experi-
ments give an idea of the nature of the different con-
formers. For instance, NH9 exchanges with a broad peak
(*40 Hz at half height) that resonates at 6.2, 1.5 ppm far
away from its initial 7.7 ppm position. The large dis-
placement and the broadness of the line suggest that finding
H9 in this position is due to the loss of the hydrogen bond.
On the other hand, NH18 behavior is more difficult to
explain because several exchange lines are observed. Two
signals (6.4 and 6.5 ppm), which are very close to the
initial peak position (6.7 ppm) are observed. Those signals
are equally broad at half height of the inverted peak
(*20 Hz). Both slight chemical shift displacement and
very similar signal broadness suggest that peaks come from
a conformational exchange without hydrogen bond break-
ing, thus from another part of the molecule. This is prob-
ably due to Cbz rotation from a trans to a cis position and
the above mentioned H9 hydrogen bond loss. Also,
although small, a broad peak appears at 5.9 ppm (*40 Hz
at half height), which is attributed to hydrogen bond
breaking. Low intensity is explained because once the bond
is broken water can have access to that proton and
exchange with it, which is in fact seen in the spectrum.
indicates a strong hydrogen bond formation with Boc
3
carbonyl group. Furthermore, coupling constant JH9H5
=
7.8 Hz (dihedral angle *150°) and NOE effects observed
with H5, H4S and Me8 indicate the spatial disposition of the
cyclobutane ring.
Similarly to that observed in 4-aminoproline 3, NH18
proton in Cbz group has an unusual deshielded position
suggesting the formation of a hydrogen bond with CO10. A
similar hydrogen bond was previously reported for a
c-dipeptide consisting of two residues of 4-aminoproline
protected in a different manner than compound 3 (Farrera-
Sinfreu et al. 2004). In our case, this is corroborated by
NOE effects observed to H15R and H13S protons. However,
there are slight differences in NOE intensities compared
with 3. In the dimer 14, comparatively, more NOE signal is
observed in H15R with respect to H13S proton indicating a
shift toward this proton. This can be explained due to the
conformational restriction of CO10 that belongs to the new
seven-membered ring, which consequently shifts a bit the
hydrogen bond CO10–NH18. The slight change is also
noticeable in 3JH14H18 coupling value, which changed from
8.8 Hz (dihedral angle *160°) in triprotected proline to
6.5 Hz in the dimer (dihedral angle *140°).
123

Synthesis and structural study of highly constrained hybrid
681
Fig. 4 Expanded region of
1H-NMR spectrum at 273 K
with the detailed integration
values for each conformer
123

´
R. Gutierrez-Abad et al.
682
Fig. 5 1D selective NOESY
experiments irradiating
NH protons in 14. Mixing time
1
was set to 500 ms. H-NMR
spectrum is added for
comparison. Besides NOE
peaks, exchange peaks are also
observed
1
suggesting a stronger hydrogen bond. Variable H-NMR
A pictorial conformational structure of the major con-
former is presented in Fig. 6a.
temperature experiments corroborate such hypothesis (see
the supplementary material).
A similar study was carried out on dipeptide 9 leading to
the same conclusions. The conformation for 9 deduced
from NOEs and J coupling values is depicted in Fig. 6b.
In order to qualitatively compare hydrogen bonding
strength of NH9 and NH18 in these dipeptides, 50 lL of
deuterated methanol were added into the NMR tube. The
tube was then hand-shaken and left to equilibrate for
10 min. The spectrum clearly shows that, while approxi-
mately half of the signal of NH9 prevails, NH18 has com-
pletely disappeared indicating a total deuterium exchange,
therefore being experimentally demonstrated that NH9
hydrogen bond is less accessible than NH18 one, thus
To ascertain if this conformation was preserved in tet-
ramers, c-tetrapeptides 12 and 15 were investigated. The
respective H-NMR spectra clearly showed a single major
1
conformation although several minor conformers are visi-
ble in the spectrum and as exchange peaks in 2D ROESY
spectra. All 1H and 13C resonances have been assigned with
the help of standard 2D NMR experiments which are
reported in the supplementary material (TOCSY, ROESY,
HSQC and HMBC). The result of these studies was the
confirmation for the same preferred conformation found for
c-dipeptides 9 and 14. These conformers are depicted in
Fig. 6 Conformation of
a dipeptide 14 and b dipeptide 9
deduced from NOEs and
J coupling values. NOE effects
are indicated with arrows and
hydrogen bonds indicated with
lines
123

Synthesis and structural study of highly constrained hybrid
683
Fig. 7 in which the inter residue strongest ROE contacts are
indicated by bold arrows.
fact accounts for the higher strength of the former as
1
deduced from H NMR experiments. The geometries of
1
c-Hexapeptides 13 and 16 were also examined by H
the resultant structures were optimized at B3LYP/6-
31G(d) level of theory in chloroform solution for tetra-
peptides 12 and 15 (see the supplementary material) and in
gas phase for hexapeptides 13 and 16 (Fig. 9). Structural
trends are similar for tetra- and hexapeptides in each series.
Comparing the calculated structures for 13 and 16 some
features are remarkable. The first one is that, in 13, each
cyclobutane NH proton is involved, in average, in a
bifurcated hydrogen bond, that means an inter residual
NH(i)ꢀꢀꢀOC(i-1) hydrogen bond with the carbamate of the
sequentially preceding proline moiety and a second one
with the amide carbonyl of the same residue, NH(i)ꢀꢀꢀOC(i).
This last interaction is not observed in the terminal
cyclobutane residue. In contrast, for hexapeptide 16, only
inter residual hydrogen bonding is predicted and the mol-
ecule is more twisted than 13. The presence of the intra and
inter residue hydrogen bonds in 13 originates a differenti-
ated chemical environment for each NH proton, which in
turn explains the splitted pattern in the NH region of the 1H
NMR spectra. In contrast, the preferred conformation for
16 presents a similar environment for all NH protons,
thus resulting in an overlapping of the corresponding
NH signals.
NMR. A similar preferential conformation as that for
dipeptides and tetrapeptides was concluded. Nevertheless,
other minor conformers were also observed.
1
Compared analysis of the amide NH region in the H
NMR spectra of these c-tetra- and c-hexapeptides showed
that 12 and 13 in one diastereomeric series, and 15 and 16
in the other one presented different peak splitting. Thus,
while all NH signals presented differentiated chemical
shifts for 12 and 13, overlapped NH signals were observed
for 15 and 16. On the basis of these results, we could
suggest different molecular arrangements for the two
c-peptide series in good agreement with the observed sig-
natures in the CD spectra (see below).
Figure 8 shows the CD spectra of all the synthesized
c-peptides as 0.01 M solutions in MeOH. This concentra-
tion is low enough to avoid self-aggregation. We can
observe a different pattern and also a different sign of the
maximum absorption peaks in each series thus reflecting
the opposite chirality of the cyclobutane residues in 9, 12
and 13 with respect to diastereomers 14–16. Well-defined
Cotton effects were observed for tetra- and hexapeptides 12
and 13, respectively.
Theoretical calculations were done to understand the
differences observed for both diastereomeric series (see the
supplementary material for details). Molecular Dynamics
were performed on the significant structures obtained from
the conformational search. It is remarkable that the inter
residue hydrogen bond is always present during the
dynamics while the proline intra residue one is not. This
Once the conformational bias in solution was verified,
we explored the ability of some of these compounds to self-
assemble. Thus, 2 mM solutions of the hybrid c-peptides in
MeOH were prepared and incubated for 24 h. TEM
micrographs showed the formation of nicely defined sphere
vesicles of nanometric size, as shown in Fig. 10 for com-
pounds 9, 12 and 14. Incubation of samples for a week did
Fig. 7 Main conformation for
a tetrapeptide 12 and
b tetrapeptide 15 deduced from
3
ROE connections and JNHCH
coupling values. Hydrogen
bonds are indicated with lines.
ROE contacts are indicated with
arrows. The bold ones
correspond to the strongest
contacts in each peptide
123

´
R. Gutierrez-Abad et al.
684
Fig. 8 CD spectra of
a c-peptides 9, 12 and 13,
and b 14–16 as 0.01 mM
solutions in MeOH
Fig. 9 Preferred conformations
for hexapeptides 13 (a) and 16
(b) as obtained at B3LYP/
6-31G(d) level of theory in gas
˚
phase. Distances are in A. All
hydrogen atoms except
NH have been omitted for
clarity
123

Synthesis and structural study of highly constrained hybrid
685
Fig. 10 TEM images of the vesicles formed by a dipeptide 9,
b tetrapeptide 12, c hexapeptide 13, d dipeptide 14, e tetrapeptide 15,
f hexapeptide 16 from 2 mM solutions in MeOH (1 day incubation
for a, b, d, e and 4 days incubation for c, f placed onto a carbon-film
coated copper grid
not alter the shape of these assemblies. Hexamers 13 and
16 required longer incubation times to form defined
aggregates.
allowed to stand for several days. Possible applications of
these and other related hybrid peptides are under active
investigation.
Acknowledgments Authors thank financial support from Spanish
´
Ministerio de Ciencia e Innovacion (grants CTQ2007-61704/BQU,
CTQ2008-00177/BQU, and CTQ2010-15408/BQU) and Generalitat
de Catalunya (grant 2009SGR-733). Time allocated in the Servei de
Conclusions
`
`
`
´
Two diastereomeric series of hybrid c,c-peptides have been
synthesized from enantiomeric cyclobutane amino acids 1
or 2 and 4-aminoproline 3 joined in alternation. The
presence of the cyclobutane moiety in these compounds
leads to very defined conformations in solution affording
compact structures as the result of two kinds of hydrogen-
bonded ring formation. The intra residue interaction
between the Cbz–NH and the amide CO, equivalent to that
already observed in the monomer, was retained. Further-
more, a new and strong hydrogen bond was formed by inter
residue interaction between the Boc CO of a proline moiety
and the NH of the cyclobutane residue giving a second
seven-membered ring. Moreover, in the (S,R)-cyclobutane
containing series (peptides 12 and 13), this NH proton is
also involved in an intra residue hydrogen bond with the
amide CO producing, in average, a third seven-membered
ring. As a result, (S,R)-cyclobutane peptides are more rigid
than the (R,S) diastereomers 15 and 16. The latter peptides
Ressonancia Magnetica Nuclear and Servei de Microscopia Electro-
nica (UAB) is gratefully acknowledged. R G-A thanks Ministry of
Education for a predoctoral fellowship.
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