Synthesis and conformational analysis of heterogeneous cyclic oligomers of 6-amino-6-deoxygalactonic acid and phenylalanine

Article abstract of DOI:10.1002/ejoc.201200463

A series of heterogeneous oligomers based on the sugar Iμ-amino acid 6-amino-6-deoxygalactonic acid and D-or L-phenylalanine have been synthesised and cyclised. The resulting cyclic heterogeneous oligomers have been analysed by molecular dynamics and NMR spectroscopy to describe their conformational and topological features. These initial studies have provided valuable insight into the structural properties of these cyclopeptides.

Full text of DOI:10.1002/ejoc.201200463

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FULL PAPER
DOI: 10.1002/ejoc.201200463
Synthesis and Conformational Analysis of Heterogeneous Cyclic Oligomers of
6-Amino-6-deoxygalactonic Acid and Phenylalanine
Raquel G. Soengas,*[a,b] Marco Fontanella,[c] Jose I. Santos,[c] George W. J. Fleet,[b] and
Jesús Jiménez-Barbero*[c]
Keywords: Peptidomimetics / Cyclopeptides / Amino acids / Carbohydrates / Conformation analysis
A series of heterogeneous oligomers based on the sugar ε-
amino acid 6-amino-6-deoxygalactonic acid and - or
by molecular dynamics and NMR spectroscopy to describe
their conformational and topological features. These initial
D
L-
phenylalanine have been synthesised and cyclised. The re- studies have provided valuable insight into the structural
sulting cyclic heterogeneous oligomers have been analysed
properties of these cyclopeptides.
used in tissue engineering of cartilage[11] and have provided
a set of novel integrin inhibitors.[12]
Introduction
Cyclic peptides are widely known as antibiotics and for
other chemotherapeutic uses.[1] Accordingly, there has been
a great deal of interest in their chemical[2] and enzymatic[3]
synthesis. The two most widely used cyclic peptides, grami-
cidin and cyclosporine, are 30- and 33-membered rings,
respectively, and significant activity was also found in a 42-
ring analogue of gramicidin.[4] In the past few years, it has
become clear that cyclic peptides also have potential appli-
cations in nanotube technology.[5] For example, cyclopept-
ides can adopt the required flat ring-shaped conformations
with self-complimentary recognition surfaces, thereby con-
structing hydrogen-bonded and open-ended hollow peptide
nanotubes, which can act as artificial trans-membrane ion
channels.[6] Bioassays have shown that these cyclic peptides
increase membrane permeability because they disrupt the
trans-membrane ion potential, cause rapid cell death and
act preferentially on Gram positive and negative bacterial
membranes relative to mammalian cells. The potential bio-
logical interest of this type of derivative as antibiotics has
led to extensive current studies into cyclopeptide nanotubes.
Sugar amino acids (SAAs) have been reported to be im-
portant components for peptidomimetics[7] and, moreover,
linear oligomers of conformationally locked[8] SAAs pro-
vide many examples in which there is a predisposition
towards secondary structures in relatively small mo-
lecules.[9] Cyclic peptides that contain SAAs[10] have been
In the course of our investigations on cyclic peptides con-
taining SAAs, we have synthesised several cyclic homo-
oligomers of 6-amino-6-deoxy-d-galactonic acid with dif-
ferent ring sizes.[13] There is a strong predisposition for the
macrocyclisation of linear pentafluorophenyl esters of 6-
amino-6-deoxy-d-galactonic acid to homooligomers in
good yield. Herein, we report the preparation and the con-
formational study of a series of heterogeneous cyclic oligo-
mers with different ring sizes. The compounds contain 6-
amino-6-deoxygalactonic acid and phenylalanine; an α-
amino acid with a hydrophobic side chain. It was envisaged
that these cyclic peptides could have biological and/or struc-
tural properties of interest.
Results and Discussion
Standard peptide coupling methods were employed to
couple the galactose-derived azido ester 1[14] to l-phenylal-
anine to give dipeptide 2. Hydrolysis of the methyl ester,
followed by reaction of the resulting acid 3 with penta-
fluorophenol and 1-[3-(dimethylamino)propyl]-3-ethyl-
carbodiimide hydrochloride (EDCI·HCl) in dioxane, af-
forded the pentafluorophenyl ester. Catalytic hydrogenation
of the azido group to an amine led to subsequent cycli-
sation to the corresponding 10-membered cyclic peptide 4
(Scheme 1).
Catalytic hydrogenation of dimeric azido ester 2 afforded
amino ester 5, which was coupled to dimeric acid 3. Thus,
coupling of the two units of dipeptide afforded tetramer
6, which was then hydrolysed to the corresponding acid 7.
Compound 7 was then transformed into the cyclic tetramer
8 via the pentafluorophenyl ester.
[a] Departament of Chemistry & QOPNA, University of Aveiro,
3810-193 Aveiro, Portugal
[b] Chemistry Research Laboratory, Department of Chemistry,
University of Oxford,
Mansfield Road, Oxford, OX1 3TA, UK
[c] Centro de Investigaciones Biológicas, CSIC,
Ramiro de Maeztu 9, 28040 Madrid, Spain
Homepage: http://www.cib.csic.es/en/grupo.php?idgrupo=71
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201200463.
Linear hexapeptide 9 was synthesised by coupling amine
5 and acid 7. Hydrolysis of the methyl ester was followed
Eur. J. Org. Chem. 0000, 0–0
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Scheme 1. Reagents and conditions: (i) 1. Ba(OH)2,THF/H2O. 2. l-Phe-OMe, O-(benzotriazol-1-yl)-N,N,NЈ,NЈ-tetramethyluronium tetra-
fluoroborate (TBTU), Et3N, DMF, 78% (two steps). (ii) Ba(OH)2, THF/H2O, quant. (iii) 1. Pentafluorophenol, EDCI·HCl, dioxane. 2.
H2, Pd black, dioxane, 35% (two steps). (iv) H2, Pd black, dioxane, quant. (v) 3, TBTU, Et3N, DMF, 52%. (vi) 1. Pentafluorophenol,
EDCI·HCl, dioxane. 2. H2, Pd black, dioxane, 22% (two steps). (vii) 7, TBTU, Et3N, DMF, 38%. (viii) 1. Ba(OH)2, THF/H2O, quant.
2. Pentafluorophenol, EDCI·HCl, dioxane. 3. H2, Pd black, dioxane, 14% (two steps).
by formation of the pentafluorophenyl ester and catalytic Conformational Analysis of Cyclopeptides
hydrogenation of the azido group afforded cyclic hexamer
10.
The 3D structures of model compounds 4, 8, 10, 14 and
The preparation of cyclic peptides derived from dimer 19 were investigated by NMR spectroscopy and molecular
11, which is the epimer of 2 obtained by coupling galactose dynamics (MD) simulations. NMR spectroscopic analysis
azido ester 1 and d-Phe, was investigated in a similar way was carried out by using standard techniques at 400 and
(Scheme 2). Interestingly, the synthesis of cyclic dimer 13 500 MHz in CDCl3.
was unsuccessful; catalytic hydrogenation of the penta-
For each peptide, the 1H NMR spectrum contained a
fluorophenyl ester of 12 gave rise to cyclic tetramer 14. The single set of resonances (Figure 1), indicating that the pos-
structure of 14 was unequivocally determined by X-ray sible conformational equilibria were fast on the NMR
crystallographic analysis (see part f of Figure 2).[15] Linear chemical shift timescale.[16] COSY analysis allowed unam-
hexamer 18 gave rise to the corresponding cyclic hexamer biguous assignment of the resonances. The molecular back-
19.
bone conformations were then investigated by analysing the
2
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Cyclic Peptide Mimics
Scheme 2. Reagents and conditions: (i) 1. Ba(OH)2,THF/H2O. 2. d-Phe-OMe, TBTU, Et3N, DMF, 75% (two steps). (ii) Ba(OH)2,THF/
H2O, quant. (iii) 1. Pentafluorophenol, EDCI·HCl, dioxane. 2. H2, Pd black, dioxane, 38% (two steps); (iv) H2, Pd black, dioxane, quant.
(v) 12, TBTU, Et3N, DMF, 55%. (vi) 12, TBTU, Et3N, DMF, 30%. (vii) 1. Pentafluorophenol, EDCI·HCl, dioxane; 2. H2, Pd black,
dioxane, 20% (two steps).
1
Figure 1. H NMR spectra of cyclic peptides.
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Figure 2. Representative lowest-energy structures of 4 (a), 8 (b), 14 (c), 10 (d) and 19 (e) calculated by ROESY-restrained MD with the
fewest violations of ROESY data, and X-ray analysis of cyclopeptide 14 (f).
2
2D NOESY and ROESY spectra with the aid of molecular moiety (φ: 67.6°; ψ: –55.8°). In this structure, Saa NH is
modelling protocols. In particular, a set of structures consis- involved in an explicit hydrogen bond with 2Saa C=O
tent with the NMR spectroscopic analyses were generated (2.02 Å) and this stabilises the structure. This situation is
by employing restrained MD simulations followed by en- also consistent with the chemical shifts of the NH proton.
1
2
ergy minimisation with the AMBER force field. The con- Thus, while Phe NH resonates at δ = 6.35 ppm, Saa NH
formations with the lowest internal energy and the lowest (involved in hydrogen bonding) appears downfield at δ =
number of violations were selected from the experimental 7.46 ppm (Figure 1).
data and analysed (see Figure 2 and Supporting Infor-
mation).
As far as tetrapeptide 8 is concerned, the conformational
analysis protocol predicted the existence of an equilibrium
The representative structure of dipeptide 4 calculated by between the three conformers A, B and C (20:20:60). The
MD is the single conformer represented in Figure 2 (a). The major conformer is C (Figure 2, b), which has a highly sym-
calculated coupling constants and NOEs for 4 are in good metrical structure that is stabilised by hydrogen bonding be-
agreement with the experimental data (Table 1 and the Sup- tween all the NH moieties. In particular, 2Saa NH and 4Saa
4
2
porting Information). The structure of this cyclic dipeptide NH are hydrogen-bonded with Saa C=O and Saa C=O
4 is compatible with an inverse γ-turn centred on the Phe with distances of 1.89 and 1.91 Å, respectively, whereas
4
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Cyclic Peptide Mimics
Table 1. Experimental and theoretical coupling constants for cyclic peptides 4, 8, 10, 14 and 19.
Protons
Residue
4
Exp.
8
10
Exp.
14
Theor.
Theor.
Exp.
Theor.
Exp.
Theor.
Theor.
Exp.
Hα,Hβ
2Saa
4Saa
6Saa
2Saa
4Saa
6Saa
2Saa
4Saa
6Saa
2Saa
4Saa
6Saa
2Saa
4Saa
6Saa
6.6
–
–
2.1
–
–
8.3
–
–
8.9
–
–
–
–
–
7.8
–
–
15.4
–
6.5
–
–
2.8
–
–
7.9
–
–
9.3
–
–
–
–
–
3.1
–
–
11.4
–
6.7
6.7
–
4.1
4.1
–
8.2
8.2
–
4.9
4.9
–
9.5
9.5
–
6.7
6.7
–
8.1
8.1
–
6.2
6.2
–
4.2
3.3
–
9.3
9.3
–
3.8
3.7
–
10.6
10.4
–
5.2
5.2
–
7.2
7.4
–
6.5
6.5
6.5
3.5
3.5
3.5
7.9
7.9
7.9
5.5
5.5
5.5
7.9
7.9
7.9
7.5
7.5
7.5
–
7.2
7.2
7.2
3.8
3.4
3.9
8.6
8.8
8.6
5.4
5.0
5.2
7.9
7.5
7.8
7.5
7.7
7.7
–
9.5
9.5
–
2.4
2.4
–
10.1
10.1
–
3.3
3.3
–
9.3
9.3
–
11.3
11.3
–
3.1
3.1
–
10.3
10.3
–
2.1
1.6
–
9.8
9.7
–
1.9
4.1
–
10.2
10.4
–
11.8
11.8
–
3.1
3.0
–
7.5
7.5
7.5
7.2
7.2
7.2
10.0
10.0
10.0
2.2
2.2
2.2
8.2
8.2
8.2
11.5
11.5
11.5
–
6.4
9.4
9.9
2.0
6.5
9.4
9.7
9.9
8.3
2.5
4.2
2.8
9.6
11.0
10.9
11.8
7.1
10.0
2.9
1.8
3.7
Hβ,Hγ
Hγ,Hδ
Hδ,Hε
Hδ,HЈε
Hα,CHPh 1Phe
3Phe
5Phe
Hα,CHЈPh 1Phe
3Phe
–
–
–
–
–
–
5Phe
–
–
3
1Phe NH and Phe NH are hydrogen-bonded to the corre- the existence of hydrogen-bonded species exclusively on
sponding O atom of the SAA (2.11 and 2.10 Å, respec- chemical shifts and to confirm our hypothesis, we per-
tively). The structures of the minor conformers are similar formed H/D exchange experiments. However, the NH ex-
and analogous hydrogen bonds are formed. Indeed, the change behaviour was indeed similar for all of the cyclic
only difference between these conformers is that they have peptides and no clear-cut conclusions could be addressed.
less symmetrical structures than the major conformer.
It is probable that there is certain population of a hydrogen-
The MD-derived conformational equilibrium of tetra- bonded species for 14, but its lifetime is short and cannot
peptide 14, which contains d-Phe instead of the parent l- be experimentally detected.
amino acid residue, shows the presence of two conformers,
The hexamer 10 has three slightly different conformers,
A and B (60:40). In this case, hydrogen bonds are not ob- A, B and C (90:5:5), in equilibrium. Only one hydrogen
served. The peptide bonds are fairly extended. Short intra- bond is observed in the major isomer (Figure 2, d) and
molecular CH/Ar distances were found between the iso- there are no salient structural features. Folding is again
propylidene protecting group and the aromatic carbon of present in the sugar residue, while the peptide bonds are
one phenylalanine residue (2.58 Å). One additional NH/π extended.
2
contact was also found between Saa NH and the aromatic
The representative structure of hexamer 19, as calculated
ring of Phe (2.7 Å). Thus, the molecule adopts a compact by MD, involves three conformations in equilibrium, A, B
structure that is stabilised by non-polar contacts, without and C (20:20:60). Two hydrogen bonds are observed in the
any intramolecular hydrogen bonds (Figure 2, c). It is note- major isomer (Figure 2, e) and, once again, folding is evi-
worthy that the crystal structure of peptide 14 is different dent in the sugar residue, while the peptide bonds are ex-
(Figure 2, f) to that observed in solution. In the solid state, tended.
this molecule has a pseudo-chair-like structure stabilised by
hydrogen bonding between NH and C=O groups on oppo-
site sides of the ring (1Phe NH–3Phe C=O 1.91 Å; 3Phe
Conclusions
NH19–1Phe C=O 1.93 Å). However, the coupling constants
calculated for the crystal structure are very different to
We have prepared a variety of cyclopeptides based on
those observed experimentally; thus confirming the signifi- alternate units of d- or l-Phe and a galactose-derived ε-
cant differences between the structure of the peptide in amino acid. The conformational properties of these cyclo-
solution and that in the solid state.
peptides have been analysed and it was found that they dis-
According to MD calculations for the structure of 14 in played distinct topological features. Cyclodipeptide 4 is a γ-
solution, the tetrapeptide forms no hydrogen bonds in solu- turn mimetic, which could be useful as a template in the
tion. However, in the NMR spectrum of 14, the signals of preparation of molecular entities with different applica-
the amide protons are shifted downfield; this is a typical tions. The l-cyclotetrapeptide has a highly symmetrical
sign of the formation of hydrogen bonds and would mean structure stabilised by internal hydrogen bonding. In con-
that the structure in solution would be comparable to that trast, the d-cyclotetrapeptide has a 3D structure that is
found by crystal analysis. Because it is very difficult to base largely dominated by the tendency of the sugar residue to
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110.8, 112.1 [2ϫ C(CH3)2], 127.7, 129.1, 129.6, 135.5 (C6H5), 170.9
(CONH), 171.9 (CO2CH3) ppm. MS (ESI+): m/z (%) = 463.44 (23)
[M + H]+. HRMS: calcd. for C22H31N4O7 [M + H]+ 463.2193;
found 463.2190.
fold, while the amide bonds adopt extended conformations
close to antiparallel β sheets. The overall structure thus re-
sembles a β hairpin.[17] Finally, the cyclohexamers also dis-
play similar behaviour, with the sugar residue folded back,
while the peptide bonds are fairly extended. These different
(6-Azido-6-deoxy-2,3:4,5-di-O-isopropylidene-D-galactonyl)-L-phen-
topologies may be employed as templates for further deco- ylalanine (3): Barium hydroxide (1.21 g, 7.09 mmol) was added to
a stirred solution of 2 (1.09 g, 2.36 mmol) in THF (37 mL) and
water (74 mL). The reaction mixture was stirred for 3 h at room
temp. TLC analysis (ethyl acetate/cyclohexane, 1:1) indicated com-
plete conversion of starting material (Rf = 0.4) to one product (Rf
= 0.0–0.1). The reaction mixture was acidified by addition of
DOWEX 50W (H+), which was then removed by filtration, and the
filtrate was concentrated in vacuo to give 3 (1.02 g, quant.), which
was used in the next step without any further purification.
ration with interacting groups for applications in molecular
recognition studies.
Experimental Section
General: TLC was performed on aluminium sheets coated with 60
F254 silica, visualised by using a spray of 0.2% w/v cerium(IV)
sulfate and 5% ammonium molybdate in 2 m sulfuric acid or 0.5%
ninhydrin in methanol. Purification by flash column chromatog-
raphy was performed on Sorbsil C60 40/60 silica. NMR spectra
were recorded on Bruker DPX 400 or DQX 400 spectrometers in
the deuterated solvent stated. IR spectra were recorded on a Per-
kin–Elmer 1750 FTIR spectrophotometer and on a Bruker Tensor
27 FTIR spectrophotometer as thin films on NaCl. Optical rota-
tions were measured on a Perkin–Elmer 241 polarimeter with a
path length of 1.0 dm. Concentrations are quoted in g per 100 mL.
Low-resolution mass spectra were recorded by using electrospray
ionisation (ES) measured on Micromass BioQ II-ZS, Micromass
500 OAT, Micromass TofSpec 2E, Micromass GCT or Micromass
Platform 1 mass spectrometers. High-resolution mass spectra were
measured on a Waters 2790-Micromass LCT by ES.
Cyclo[(6-amino-6-deoxy-2,3:4,5-di-O-isopropylidene-
D-galactonyl)-
L-phenylalanine] (4): The crude acid 3 (1.02 g, 2.36 mmol) was dis-
solved in 1,4-dioxane (8 mL). Pentafluorophenol (870 mg,
2.83 mmol) and EDCI·HCl (543 mg, 4.72 mmol) were added and
the mixture was stirred at room temp. under an atmosphere of ar-
gon. After 16 h, TLC analysis (ethyl acetate/cyclohexane, 1:2) indi-
cated complete conversion of the starting material (Rf = 0.0) to a
major UV-active product (Rf = 0.50). The solvent was removed in
vacuo and the residue was dissolved in dichloromethane (50 mL).
The solution was washed with an aqueous solution of sodium hy-
drogen carbonate (5% w/v, 20 mL) and an aqueous solution of
citric acid (5% w/v, 20 mL). The organic layer was dried (MgSO4),
filtered and concentrated in vacuo. The residue was purified by
flash column chromatography (ethyl acetate/cyclohexane, 1:4) to
yield (6-azido-6-deoxy-2,3:4,5-di-O-isopropylidene-d-galactonyl)-
l-phenylalanine pentafluorophenyl ester (1.14 g, 79 %) as a un-
stable oil. This pentafluorophenyl ester (0.65 g, 1.06 mmol) was dis-
solved in 1,4-dioxane (60 mL), palladium black (10 mg) was added
and the flask was flushed with argon. The reaction mixture was
flushed with hydrogen and stirred under a hydrogen atmosphere
for 15 h. TLC analysis (ethyl acetate/cyclohexane, 1:1) indicated
complete conversion of the starting material (Rf = 0.90 UV) into a
major product (Rf = 0.24). The solvent was removed in vacuo and
the oil residue was purified by flash column chromatography (ethyl
acetate/cyclohexane, 3:2) to yield cyclic dimer 4 (148 mg, 35%) as
Syntheses of the Cyclic Peptides
(6-Azido-6-deoxy-2,3:4,5-di-O-isopropylidene-D-galactonyl)-L-phen-
ylalanine Methyl Ester (2): Barium hydroxide (1.98 g, 11.56 mmol)
was added to a stirred solution of 1 (1.21 g, 3.84 mmol) in THF
(37 mL) and water (74 mL). The reaction mixture was stirred for
3 h at room temp. TLC analysis (ethyl acetate/cyclohexane, 1:1)
indicated complete conversion of starting material (Rf = 0.4) into
one product (Rf = 0.0–0.1). The reaction mixture was acidified by
addition of DOWEX 50W (H+), which was then removed by fil-
tration, and the filtrate was concentrated in vacuo to give 6-azido-
6-deoxy-2,3:4,5-di-O-isopropylidene-d-galactonic acid. The crude
acid was added to a stirred solution of l-phenylalanine hydrochlo-
ride methyl ester (1.11 g, 3.84 mmol, quant.) in DMF (35 mL).
TBTU (1.85 g, 5.76 mmol) was added and the solution was stirred
for 5 min. Triethylamine (1.1 mL, 7.68 mmol) was added and the
reaction mixture was stirred at room temp. for 20 h under an atmo-
sphere of argon. TLC analysis (ethyl acetate/cyclohexane, 1:2) indi-
cated complete conversion of the acid starting material (Rf = 0.0)
to a major product (Rf = 0.29). The solvent was removed in vacuo
(co-evaporation with toluene). The residue was purified by flash
column chromatography (ethyl acetate/cyclohexane, 1:2), yielding 2
(1.38 g, 78%) as a colourless oil. Rf = 0.29, ethyl acetate/cyclohex-
an off-white solid. Rf = 0.28, ethyl acetate/cyclohexane, 3:2. [α]2D1
+19.9 (c = 2.6 in CHCl ). ν = (NaCl): 3383 (N-H, br.), 1668
=
˜
3
max
1
(C=O, amide) cm–1. H NMR (CDCl3): δ = 1.32, 1.36, 1.40, 1.47
[4ϫ s, 12 H, 4ϫ C(CH3)2], 2.90 (d, J = 15.4 Hz, 2 H, CH2Ph),
2.97–3.04, 3.74–3.77 (2ϫ m, 2 H, Saa Hε, Saa HЈε), 3.80–3.82 (m,
1 H, Saa Hδ), 3.81 (dd, JHα,Hβ = 6.6 Hz, JHβ,Hγ = 2.1 Hz, 1 H, Saa
Hβ), 3.96 (dd, JHγ,Hδ = 8.3 Hz, JHβ,Hγ = 2.1 Hz, 1 H, Saa Hγ), 4.36
(d, JHα,Hβ = 6.6 Hz, 1 H, Saa Hα), 4.42–4.49 (m, 1 H, Phe Hα),
6.35 (d, J = 6.4 Hz, 1 H, Phe NH), 7.23–7.36 (m, 5 H, 5ϫ ArH),
7.46 (d, J = 7.5 Hz, 1 H, Saa NH) ppm. 13C NMR (CDCl3): δ =
26.1, 26.5, 26.7, 26.9 [2ϫ C(CH3)2], 39.8, 40.3 (CH2Ph, Saa CHδ),
55.1 (Saa CHε), 73.8, 74.2, 79.2, 79.9 (Saa CHα, Phe CHα, Saa
CHγ, Saa Hβ), 110.8, 112.1 [2ϫ C(CH3)2], 127.1, 128.6, 129.2,
136.3 (C6H5), 169.7, 172.0 (2ϫ CONH) ppm. MS (ESI+): m/z (%)
= 427.18 (29) [M + Na]+. HRMS: calcd. for C21H28N2NaO6 [M +
Na]+ 427.1845; found 427.1851.
ane, 1:2. [α]2D1 = +64.9 (c = , 0.5 in CHCl ). ν
= (NaCl): 3412
˜
3
max
(N-H, br.), 2104 (-N3), 1746 (C=O, CO2Me), 1684 (C=O, amide)
cm–1. 1H NMR (CDCl3): δ = 1.31, 1.40, 1.44, 1.48 [4ϫ s, 12 H, 2ϫ
C(CH3)2], 3.16 (ABq, J = 5.8 Hz, 2 H, CH2Ph), 3.31 (dd, JHε,HЈε
13.3, JHδ,Hε = 4.7 Hz, 1 H, Saa Hε), 3.65 (dd, JHε,HЈε = 13.3,
JHδ,HЈε = 3.3 Hz, 1 H, Saa HЈε), 3.47 (s, 3 H, CO2CH3), 4.06–4.10 (6-Amino-6-deoxy-2,3:4,5-di-O-isopropylidene-
=
D
-galactonyl)-L-phen-
(m, 1 H, Saa Hδ), 4.24–4.27 (m, 2 H, Saa Hβ, Saa Hγ), 4.48 (d, ylalanine Methyl Ester (5): Compound 2 (1.21 g, 2.62 mmol) was
JHα,Hβ = 5.8 Hz, 1 H, Saa Hα), 4.86–4.91 (m, 1 H, Phe Hα), 7.05 dissolved in 1,4-dioxane (60 mL), palladium black (1.21 g) was
(d, J = 8.2 Hz, 1 H, NH), 7.12 (ABq, J = 6.8 Hz, 2 H, 2ϫ ArH), added and the flask was flushed with argon. The reaction mixture
7.25–7.34 (m, 3 H, 3ϫ ArH) ppm. 13C NMR (CDCl3): δ = 26.1, was flushed with hydrogen and stirred under a hydrogen atmo-
27.3, 27.4, 27.4 [2 ϫ C(CH3)2], 38.2 (CHPh2), 52.1 (C-9), 52.8
sphere for 15 h. TLC analysis (ethyl acetate/cyclohexane, 1:1) indi-
(-CH-), 53.0 (CO2CH3), 77.3 (C-5), 78.0 (2ϫ -CH), 79.4 (-CH-), cated complete conversion of the starting material. The solvent was
6
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Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O20463
/KAP1
Date: 22-08-12 16:34:22
Pages: 12
Cyclic Peptide Mimics
removed in vacuo to afford 5 (1.10 g, 2.62 mmol), which was used
without any further purification.
nyl)-l-phenylalanine–(6-amino-6-deoxy-2,3:4,5-di-O-isopropylid-
ene-d-galactonyl)-l-phenylalanine pentafluorophenyl ester, which
was used without any further purification. This pentafluorophenyl
ester was dissolved in 1,4-dioxane (4 mL), palladium black (43 mg)
was added and the flask was flushed with argon. The reaction mix-
ture was flushed with hydrogen and stirred under a hydrogen atmo-
sphere for 15 h. TLC analysis (ethyl acetate/cyclohexane, 2:3) indi-
cated complete conversion of the starting material (Rf = 0.31 UV)
into a major product (Rf = 0.42). The solvent was removed in vacuo
and the oil residue was purified by flash column chromatography
(ethyl acetate/cyclohexane, 2:1) to yield cyclic tetramer 8 (21.8 mg,
22%) as an off-white solid. Rf = 0.30, ethyl acetate/cyclohexane,
(6-Azido-6-deoxy-2,3:4,5-di-O-isopropylidene-
ylalanine–(6-amino-6-deoxy-2,3:4,5-di-O-isopropylidene-
onyl)- -phenylalanine Methyl Ester (6): Compound 3 (0.95 g,
D
-galactonyl)-
L-phen-
D
-galact-
L
2.26 mmol) was added to a stirred solution of 5 (0.91 g, 2.26 mmol)
in DMF (18 mL). TBTU (1.02 g, 3.14 mmol) was added and the
solution was stirred for 5 min. Triethylamine (0.36 mL, 2.26 mmol)
was added and the reaction mixture was stirred at room temp. for
20 h under an atmosphere of argon. TLC analysis (ethyl acetate/
cyclohexane, 1:1) indicated complete conversion of the acid starting
material (Rf = 0.0) into a major product (Rf = 0.29). The solvent
was removed in vacuo. The residue purified by flash column
chromatography (ethyl acetate/cyclohexane, 1:1) to yield 6 (1.17 g,
52%) as a colourless oil. Rf = 0.29, ethyl acetate/cyclohexane, 1:1.
1
2:1. H NMR (CDCl3): δ = 1.32, 1.36, 1.40, 1.47 [4ϫ s, 24 H, 8ϫ
C(CH3)2], 3.06–3.12 (dd, 2 H, J = 8.1 Hz, J = 6.7 Hz, 2ϫ CHPh),
3.20–3.25 (dd, J = 8.1 Hz, J = 6.7 Hz, 4 H, 2ϫ CHPh), 3.42–3.49
(dd, 2 H, JHε,Hδ = 4.9 Hz, 1Saa Hε, 2Saa Hε), 3.59–3.65 (dd, JHЈε,Hδ
= 9.5 Hz, 2 H, 1Saa HЈε, 2Saa HЈε), 3.87 (dd, 2 H, JHγ,Hδ = 8.2 Hz,
[α]2D1 = +22.9 (c = 1.3 in CHCl ). ν
= (NaCl): 3321 (N-H, br.),
˜
3
max
2103 (-N3), 1746 (C=O, CO2Me), 1672 (C=O, amide) cm–1 1H
.
1
2
JHβ,Hγ = 4.1 Hz, 2 H, Saa Hγ, Saa Hγ), 4.05 (dd, JHα,Hβ = 6.7,
NMR (CDCl3): δ = 1.30, 1.31, 1.32, 1.35, 1.44, 1.46, 1.49 [7ϫ s,
24 H, 8ϫ C(CH3)2], 3.05–3.09 (m, 2 H, CH2Ph), 3.16 (ABq, J =
5.6 Hz, 2 H, CH2Ph), 3.32 (dd, J = 13.6, J = 4.8 Hz, 1 H, 2Saa
Hδ), 3.37–3.49 (m, 2 H, 1Saa Hε, 1Saa HЈε), 3.62–3.67 (m, 2 H,
1Saa Hγ, 2Saa Hγ), 3.74 (s, 3 H, CO2CH3), 3.96–4.01 (m, 1 H, 1Saa
Hδ), 4.07 (dd, JHε,HЈε = 8.0, JHδ,HЈε = 5.6 Hz, 1 H, 2Saa HЈε), 4.17–
4.28 (m, 3 H, 1Saa Hβ, 2Saa Hβ, 2Saa Hε), 4.41 (d, JHα,Hβ = 6.0 Hz,
JHβ,Hγ = 4.1 Hz, 1Saa Hβ, 2Saa Hβ), 4.12 (ddd, 2 H, JHε,Hδ
4.9 Hz, JHЈε,Hδ = 9.5 Hz, JHγ,Hδ = 8.2 Hz, Saa Hδ, Saa Hδ), 4.42
(d, JHα,Hβ = 6.7 Hz, 2 H, Saa Hα, Saa Hα), 4.73 (dd, J = 6.7, J
=
1
2
1
2
= 8.1 Hz, 2 H, 1Phe Hα, Phe Hα), 7.06 (br. s, 2 H, 1Saa NH, Saa
NH), 7.20 (br. s, 2 H, 1Phe NH, 2Phe NH), 7.23–7.36 (m, 10 H,
10ϫ ArH) ppm. 13C NMR (CDCl3): δ = 26.1, 26.5, 26.7, 26.9 [4ϫ
C(CH3)2], 39.8, 40.3 (2ϫ CH2Ph, 1Saa CHε, 2Saa CHε), 55.1 (1Saa
CHδ, 2Saa CHδ), 73.8, 74.2, 79.2, 80.0 (1Saa CHβ, 2Saa CHβ, 1Saa
CHγ, 2Saa CHγ, 1Saa CHα, 2Saa CHα, 1Phe CHα, 2Phe CHα),
110.8, 112.1 [4ϫ C(CH3)2], 127.1, 128.6, 129.2, 136.3 (2ϫ C6H5),
169.7, 172.0 (2ϫ CONH) ppm. MS (ESI+): m/z (%) = 831.38 (100)
[M + Na]+. HRMS: calcd. for C42H56N4NaO12 [M + Na]+:
831.3795; found 831.3788.
2
2
1
2
1 H, Saa Hα), 4.46 (d, JHα,Hβ = 6.0 Hz, 1 H, Saa Hα), 4.56–4.62
2
1
(m, 1 H, Phe Hα), 4.84–4.89 (m, 1 H, Phe Hα), 6.07–6.09 (m, 1
1
1
H, Saa NH), 7.07 (d, J = 8.4 Hz, 1 H, Phe NH), 7.11–7.33 (m,
11 H, 10ϫ ArH, 2Phe NH) ppm. 13C NMR (CDCl3): δ = 26.1,
26.3, 27.1, 27.4, 27.5 (8ϫ CH3), 38.2, 39.2, 41.2, 52.1 (2ϫ CH2Ph,
1Saa CHε, Saa CHε), 52.8, 53.1, 54.8, 76.7, 76.9, 77.4, 77.7, 78.9,
2
79.1, 79.5 (10ϫ -CH-), 110.1, 110.8, 112.1 [4ϫ C(CH3)2], 127.6,
127.7, 129.1, 129.2, 129.6, 135.9, 136.7 (2ϫ C6H5), 170.6, 170.8,
171.1, 171.8 (3ϫ CONH, CO2CH3) ppm. MS (ESI+): m/z (%) =
865.74 (100) [M – H]+, 866.74 (50) [M]+. HRMS: calcd. for
C43H57N6O14 [M – H]+: 865.3984; found 865.3994.
(6-Azido-6-deoxy-2,3:4,5-di-O-isopropylidene-
ylalanine–(6-amino-6-deoxy-2,3:4,5-di-O-isopropylidene-
onyl)- -phenylalanine–(6-aAmino-6-deoxy-2,3:4,5-di-O-isopropylid-
ene- -galactonyl)- -phenylalanine Methyl Ester (9): Compound 7
D
-galactonyl)-L-phen-
D
-galact-
L
D
L
(724 mg, 0.86 mmol) was added to a stirred solution of 5 (388 mg,
0.86 mmol) in DMF (12 mL). TBTU (334 mg, 1.03 mmol) was
added and the solution was stirred for 5 min. Triethylamine
(0.12 mL, 0.86 mmol) was added and the reaction mixture was
stirred at room temp. for 20 h under an atmosphere of argon. TLC
(ethyl acetate/cyclohexane, 2:1) indicated complete conversion of
the acid starting material (Rf = 0.0) to a major product (Rf = 0.29).
The solvent was removed in vacuo (co-evaporation with toluene).
The residue was pre-adsorbed into silica and purified by flash col-
umn chromatography (ethyl acetate/cyclohexane, 2:1) to yield 9
(415 mg, 38%) as a colourless oil. Rf = 0.27, ethyl acetate/cyclohex-
(6-Azido-6-deoxy-2,3:4,5-di-O-isopropylidene-
ylalanine–(6-amino-6-deoxy-2,3:4,5-di-O-isopropylidene-
onyl)- -phenylalanine Pentafluorophenyl Ester (7): Barium hydrox-
D
-galactonyl)-
L-phen-
D
-galact-
L
ide (59 mg, 0.35 mmol) was added to a stirred solution of 6 (0.10 g,
0.12 mmol) in THF (5 mL) and water (10 mL). The reaction mix-
ture was stirred for 3 h at room temp. TLC analysis (ethyl acetate/
cyclohexane, 1:1) indicated complete conversion of starting mate-
rial (Rf = 0.29) into one product (Rf = 0.0–0.1). The reaction mix-
ture was acidified by addition of DOWEX 50W (H+), which was
then removed by filtration, and the filtrate was concentrated in
vacuo to give 7 (0.10 g, quant.), which was used without any fur-
ther purification.
ane, 2:1. [α]2D3 = +10.7 (c = 0.5 in CHCl ). νmax = (NaCl): 3317 (N-
˜
3
H, br.), 2103 (-N3), 1744 (C=O, CO2Me), 1664 (C=O, amide) cm–1.
1H NMR (CDCl3): δ = 1.34, 1.36, 1.37, 1.39, 1.40, 1.40, 1.49, 1.51,
1.53 [9ϫ s, 36 H, 12ϫ C(CH3)2], 3.08–3.15 (m, 4 H, 2ϫ CH2Ph),
Cyclo[(6-azido-6-deoxy-2,3:4,5-di-O-isopropylidene-
phenylalanine–(6-amino-6-deoxy-2,3:4,5-di-O-isopropylidene-
lactonyl)- -phenylalanine] (8): The crude acid 7 (101 mg, 0.12 mmol)
D-galactonyl)-
L-
D
-ga-
1
L
3.20 (ABq, J = 5.5 Hz, 2 H, CH2Ph), 3.34–3.66 (m, 6 H, Saa Hε,
3
1
2
3
was dissolved in 1,4-dioxane (2 mL). Pentafluorophenol (43 mg,
0.32 mmol) and EDCI·HCl (27 mg, 0.14 mmol) were added and
the mixture was stirred at room temp. under an atmosphere of ar-
gon. After 16 h, TLC analysis (ethyl acetate/cyclohexane, 1:1) indi-
cated complete conversion of the starting material (Rf = 0.0) into
a major UV-active product (Rf = 0.40). The solvent was removed
in vacuo and the residue dissolved in dichloromethane (10 mL).
The solution was washed with an aqueous solution of sodium hy-
2Saa Hε, Saa Hε, Saa HЈε, Saa HЈε, Saa HЈε), 3.67–3.71 (m, 2
1
2
H, Saa Hγ, Saa Hγ), 3.77 (s, 3 H, COCH3), 4.01–4.06 (m, 2 H,
2
3
1Saa Hδ, Saa Hδ), 4.10 (dd, J = 8.0, J = 5.6 Hz, 1 H, Saa Hγ),
1
2
3
3
4.20–4.31 (m, 4 H, Saa Hβ, Saa Hβ, Saa Hβ, Saa Hδ), 4.43 (d,
JHα,Hβ = 6.0 Hz, 1 H, 2Saa Hα), 4.45 (d, JHα,Hβ = 6.0 Hz, 1 H,
1Saa Hα), 4.50 (d, JHα,Hβ = 5.8 Hz, 1 H, Saa Hα), 4.61–4.68 (m,
3
2
3
1
2 H, Phe Hα, Phe Hα), 4.89–4.93 (m, 1 H, Phe Hα), 6.16–6.19
(m, 2 H, 1Saa NH, Saa NH), 7.10 (d, J = 6.8 Hz, 1 H, Phe NH),
2
1
drogen carbonate (5% w/v, 5 mL) and an aqueous solution of citric 7.25–7.36 (m, 2Phe NH, 3Phe NH, 15 ϫ ArH) ppm. 13C NMR
acid (5% w/v, 5 mL). The organic layer was dried (MgSO4), filtered,
and concentrated in vacuo. The residue was filtered through silica
to yield (6-azido-6-deoxy-2,3:4,5-di-O-isopropylidene-d-galacto-
(CDCl3): δ = 25.6, 25.8, 26.7, 26.87, 27.02 (12ϫ CH3), 37.7, 38.8,
40.7, 40.7, 51.7 (3ϫ CH2Ph, 1Saa CHε, 2Saa CHε, 3Saa CHε),
51.7, 52.3, 52.6, 54.3, 54.4, 76.4, 76.7, 77.0, 77.2, 77.6, 78.3, 78.5,
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
7

Job/Unit: O20463
/KAP1
Date: 22-08-12 16:34:22
Pages: 12
R. G. Soengas, J. Jiménez-Barbero et al.
FULL PAPER
78.6, 78.7, 80.0 (15 ϫ -CH-), 109.6, 109.7, 110.3, 111.6 [6 ϫ
C(CH3)2], 127.1, 127.2, 128.6, 128.7, 129.2, 135.4, 136.2 (3 ϫ
C6H5), 170.0, 170.3, 170.4, 170.6, 171.3 (5ϫ CONH, CO2CH3)
ppm. MS (ESI+): m/z (%) = 1270.38 (100) [M – H]+.
into one product (Rf 0.0–0.1). The reaction mixture was acidified
by addition of DOWEX 50W (H+), which was then removed by
filtration, and the filtrate was concentrated in vacuo to give 6-
azido-6-deoxy-2,3:4,5-di-O-isopropylidene-d-galactonic acid. The
crude acid was added to a stirred solution of d-phenylalanine hy-
drochloride methyl ester (0.74 g, 3.42 mmol, quant.) in DMF
(30 mL). TBTU (1.33 g, 4.11 mmol) was added and the solution
was stirred for 5 min. Triethylamine (1.0 mL, 6.85 mmol) was
added and the reaction mixture was stirred at room temp. for 20 h
under an atmosphere of argon. TLC analysis (ethyl acetate/cyclo-
hexane, 1:2) indicated complete conversion of the acid starting ma-
terial (Rf = 0.0) into a major product (Rf = 0.29). The solvent was
removed in vacuo (co-evaporation with toluene). The residue was
pre-adsorbed into silica and purified by flash column chromatog-
raphy (ethyl acetate/cyclohexane, 1:2) to yield 11 (1.18 g, 75%) as
Cyclo[(6-Amino-6-deoxy-2,3:4,5-di-O-isopropylidene-
D-galactonyl)-
L
-phenylalanine–(6-amino-6-deoxy-2,3:4,5-di-O-isopropylidene-
D-
galactonyl)-L-phenylalanine–(6-amino-6-deoxy-2,3:4,5-di-O-isoprop-
ylidene-D-galactonyl)-L-phenylalanine] (10): Barium hydroxide
(77 mg, 0.45 mmol) was added to a stirred solution of 9 (190 mg,
0.15 mmol) in THF (4 mL) and water (8 mL). The reaction mixture
was stirred for 3 h at room temp. TLC analysis (ethyl acetate/cyclo-
hexane, 1:1) indicated complete conversion of the starting material
into one product. The reaction mixture was acidified by addition
of DOWEX 50W (H+), which was then removed by filtration, and
the filtrate was concentrated in vacuo to give (6-azido-6-deoxy-
2,3:4,5-di-O-isopropylidene-d-galactonyl)-l-phenylalanine–(6-
amino-6-deoxy-2,3:4,5-di-O-isopropylidene-d-galactonyl)-l-phen-
ylalanine–(6-amino-6-deoxy-2,3:4,5-di-O-isopropylidene-d-galact-
onyl)-l-phenylalanine. The crude acid was dissolved in 1,4-dioxane
(2 mL). Pentafluorophenol (55 mg, 0.30 mmol) and EDCI·HCl
(34 mg, 0.18 mmol) were added and the mixture was stirred at
room temp. under an atmosphere of argon. After 16 h, TLC analy-
sis (ethyl acetate/cyclohexane, 1:1) indicated complete conversion
of the starting material (Rf = 0.0) into a major UV-active product
(Rf = 0.3). The solvent was removed in vacuo and the residue was
dissolved in dichloromethane (10 mL). The solution was washed
with an aqueous solution of sodium hydrogen carbonate (5% w/v,
5 mL) and an aqueous solution of citric acid (5% w/v, 5 mL). The
organic layer was dried (MgSO4), filtered and concentrated in
vacuo. The residue was purified by flash column chromatography
(ethyl acetate/cyclohexane, 1:1) to yield (6-azido-6-deoxy-2,3:4,5-
di-O-isopropylidene-d-galactonyl)-l-phenylalanine–(6-amino-6-de-
oxy-2,3:4,5-di-O-isopropylidene-d-galactonyl)-l-phenylalanine–(6-
amino-6-deoxy-2,3:4,5-di-O-isopropylidene-d-galactonyl)-l-phen-
ylalanine pentafluorophenyl ester. This pentafluorophenyl ester
was dissolved in 1,4-dioxane (6 mL). Palladium black (57 mg) was
added and the flask was flushed with argon. The reaction mixture
was flushed with hydrogen and stirred under a hydrogen atmo-
sphere for 15 h. TLC analysis (ethyl acetate/cyclohexane, 1:1) indi-
cated complete conversion of the starting material (Rf = 0.30 UV)
into a major product (Rf = 0.1). The solvent was removed in vacuo
and the oil residue was purified by flash column chromatography
(ethyl acetate/cyclohexane, 3:1) to yield cyclic tetramer 10 (25 mg,
14%) as an off-white solid. Rf = 0.28, ethyl acetate/cyclohexane,
a colourless oil. Rf = 0.29, ethyl acetate/cyclohexane, 1:2. [α]2D2
=
+3.0 (c = 1.2 in CHCl ). ν
= (NaCl): 3412 (N-H, br.), 2104
˜
3
max
(-N3), 1744 (C=O, CO2Me), 1679 (C=O, amide) cm–1. 1H NMR
(CDCl3): δ = 1.17, 1.40, 1.43, 1.46 [4ϫ s, 12 H, 2ϫ C(CH3)2], 3.01–
3.33 (m, 2 H, CH2Ph), 3.63 (dd, JHε,HЈε = 13.3, JHδ,Hε = 3.3 Hz, 1
1
1
H, Saa Hε), 3.65 (dd, JHε,HЈε = 13.3, JHδ,HЈε = 3.3 Hz, 1 H, Saa
HЈε), 3.76 (s, 3 H, CO2CH3), 4.04–4.31 (m, 3 H, 1Saa Hγ, 1Saa
1
1
Hδ, Saa Hβ), 4.48 (d, JHα,Hβ = 9.0 Hz, 1 H, Saa Hα), 4.83–4.94
(m, 1 H, 2-H), 6.94 (d, J = 8.0 Hz, 1 H, NH), 7.10–7.32 (m, 5 H,
5ϫ ArH) ppm. 13C NMR (CDCl3): δ = 26.3, 27.3, 27.3, 27.4 [2ϫ
C(CH3)2], 38.2 (CHPh2), 52.0 (1Saa CHε), 52.9 (-CH-), 53.0
(CO2CH3), 77.0, 77.6, 78.0, 79.4 (4 ϫ -CH-), 110.7, 111.1 [2ϫ
C(CH3)2], 127.6, 129.1, 129.6, 136.1 (C6H5), 170.7 (CONH), 172.0
(CO2CH3) ppm. MS (ESI+): m/z (%) = 463.44 (25) [M + H]+.
HRMS: calcd. for C22H31N4O7 [M + H]+ 463.2195; found
463.2190.
(6-Azido-6-deoxy-2,3:4,5-di-O-isopropylidene-D-galactonyl)-D-phen-
ylalanine Acid (12): Barium hydroxide (0.74 g, 4.31 mmol) was
added to a stirred solution of 18 (0.66 g, 1.44 mmol) in THF
(22 mL) and water (44 mL). The reaction mixture was stirred for
3 h at room temp. TLC analysis (ethyl acetate/cyclohexane, 1:1)
indicated complete conversion of the starting material (Rf 0.4) into
one product (Rf = 0.0–0.1). The reaction mixture was acidified by
addition of DOWEX 50W (H+), which was then removed by fil-
tration, and the filtrate was concentrated in vacuo to give (6-azido-
6-deoxy-2,3:4,5-di-O-isopropylidene-d-galactonyl)-d-phenylalanine
(0.62 g, quant.), which was used without any further purification.
Cyclo[(6-Azido-6-deoxy-2,3:4,5-di-O-isopropylidene-
D
D
-galactonyl)-
-phenylalanine–(6-amino-6-deoxy-2,3:4,5-di-O-isopropylidene- -ga-
-phenylalanine] (14): The crude acid (0.62 g, 1.44 mmol)
D
1
3:2. [α]2D3 = +1.2 (c = 0.2 in CHCl3). H NMR (CDCl3): δ = 1.41–
lactonyl)-
D
1.52 [m, 36 H, 12ϫ C(CH3)2], 3.12 (s, 6 H, 3ϫ CH2Ph), 3.39–3.45
was dissolved in 1,4-dioxane (8 mL). Pentafluorophenol (529 mg,
2.87 mmol) and EDCI·HCl (330 mg, 1.72 mmol) were added and
the mixture was stirred at room temp. under an atmosphere of ar-
gon. After 16 h, TLC analysis (ethyl acetate/cyclohexane, 1:2) indi-
cated complete conversion of the starting material (Rf = 0.0) into
a major UV-active product (Rf 0.50). The solvent was removed in
vacuo and the residue dissolved in dichloromethane (50 mL). The
solution was washed with an aqueous solution of sodium hydrogen
carbonate (5% w/v, 20 mL) and an aqueous solution of citric acid
(5% w/v, 20 mL). The organic layer was dried (MgSO4), filtered
and concentrated in vacuo. The residue was purified by flash col-
umn chromatography (ethyl acetate/cyclohexane, 1:4) to yield (6-
1
2
3
1
(m, 3 H, Saa HЈε, Saa HЈε, Saa HЈε), 3.48–3.54 (m, 3 H, Saa
2
3
Hε, Saa Hε, Saa Hε), 3.94 (dd, 3 H, JHβ,Hγ = 3.5 Hz, JHγ,Hδ
=
7.9 Hz, 1Saa Hγ, 2Saa Hγ, 3Saa Hγ), 4.05 (ddd, JHγ,Hδ = 7.9, JHδ,Hε
= 5.5, JHδ,HЈε = 7.9 Hz, 3 H, 1Saa Hδ, 2Saa Hδ, 3Saa Hδ), 4.19
1
2
3
(dd, JHα,Hβ = 6.5, JHβ,Hγ = 3.5 Hz, 3 H, Saa Hβ, Saa Hβ, Saa
Hβ), 4.32 (d, JHα,Hβ = 6.5 Hz, 3 H, Saa Hα, Saa Hα, Saa Hα),
1
2
3
1
2
3
4.61 (d, J = 7.5 Hz, 2 H, Phe Hα, Phe Hα, Phe Hα), 6.50 (br. s,
3 H, 1Phe NH, 2Phe NH, 3Phe NH), 7.23–7.36 (m, 10 H, 10ϫ
ArH), 7.38 (br. s, 3 H, 1Saa NH, 2Saa NH, 3Saa NH) ppm. MS
(ESI+): m/z (%) = 1235.57 (23) [M + Na]+. HRMS: calcd. for
C63H84N6NaO18 1235.5740; found 1235.5745.
(6-Azido-6-deoxy-2,3:4,5-di-O-isopropylidene-D-galactonyl)-D-phen- azido-6-deoxy-2,3:4,5-di-O-isopropylidene-d-galactonyl)-d-phenyl-
ylalanine Methyl Ester (11): Barium hydroxide (1.98 g, 11.56 mmol)
was added to a stirred solution of 1 (1.08 g, 3.42 mmol) in THF
(30 mL) and water (60 mL). The reaction mixture was stirred for
3 h at room temp. TLC analysis (ethyl acetate/cyclohexane, 1:1)
indicated complete conversion of the starting material (Rf = 0.4)
alanine pentafluorophenyl ester (0.67 g, 78 %) as a unstable oil,
which was directly dissolved in 1,4-dioxane (60 mL). Palladium
black (67 mg) was added and the flask was flushed with argon. The
reaction mixture was flushed with hydrogen and stirred under a
hydrogen atmosphere for 15 h. TLC analysis (ethyl acetate/cyclo-
8
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© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O20463
/KAP1
Date: 22-08-12 16:34:22
Pages: 12
Cyclic Peptide Mimics
hexane, 1:1) indicated complete conversion of the starting material
(Rf = 0.90 UV) into a major product (Rf = 0.24). The solvent was
removed in vacuo and the oil residue was purified by flash column
chromatography (ethyl acetate/cyclohexane, 3:2) to yield cyclic tet-
ramer 14 (161 mg, 38%) as an off-white solid. Rf = 0.30, ethyl acet-
ate/cyclohexane, 2:1. [α]2D3 = +2.8 (c = 0.4 in CHCl3). 1H NMR
onyl)-D-phenylalanine Acid) (17): Barium hydroxide (318 mg,
1.83 mmol) was added to a stirred solution of 16 (523 mg,
0.61 mmol) in THF (5 mL) and water (10 mL). The reaction mix-
ture was stirred for 3 h at room temp. TLC analysis indicated com-
plete conversion of starting material into one product. The reaction
mixture was acidified by addition of DOWEX 50W (H+), which
(CDCl3): δ = 1.37, 1.45, 1.46, 1.49 [4ϫ s, 24 H, 4ϫ C(CH3)2], 3.13– was then removed by filtration and the filtrate was concentrated in
1
3.17 (m, 4 H, Saa Hε, 2Saa Hε, 2ϫ CHPh), 3.37 (dd, J = 8.2 Hz,
vacuo to give 17 (517 mg, 0.61 mmol). The crude acid was used
without any further purification.
J = 11.3 Hz, 2 H, CHPh), 3.44 (d, JHγ,Hδ = 10.1 Hz, 2 H, 1Saa Hγ,
2Saa Hγ), 4.02–4.06 (m, 2 H, Saa HЈε, Saa HЈε), 4.09 (d, JHα,Hβ
1
2
(6-Azido-6-deoxy-2,3:4,5-di-O-isopropylidene-
ylalanine–(6-amino-6-deoxy-2,3:4,5-di-O-isopropylidene-
onyl)- -phenylalanine–(6-amino-6-deoxy-2,3:4,5-di-O-isopropylidene-
-galactonyl)- -phenylalanine Methyl Ester (18): Compound 17
D
-galactonyl)-
D-phen-
1
2
= 9.5 Hz, 2 H, Saa Hβ, Saa Hβ), 4.46 (d, JHγ,Hδ = 10.1 Hz, 2 H,
D
-galact-
1Saa Hδ, Saa Hδ), 4.54 (d, JHα,Hβ = 9.5 Hz, 2 H, Saa Hα, Saa
2
1
2
D
1
2
Hα), 4.72 (dd, J = 7.2, J = 11.3 Hz, 2 H, Phe Hα, Phe Hα), 6.82
(d, J = 8.2 Hz, 2 H, 1Saa NH, 2Saa NH), 7.24–7.34 (m, 10 H, 10ϫ
ArH), 8.01 (d, J = 7.2 Hz, 2 H, 1Phe NH, 2Phe NH) ppm. 13C
NMR (CDCl3): δ = 25.8, 26.1, 26.7, 26.8 [4ϫ C(CH3)2], 30.3, 39.8
D
D
(517 mg, 0.61 mmol) was added to a stirred solution of (6-amino-
6-deoxy-2,3:4,5-di-O-isopropylidene-d-galactonyl)-d-phenylalanine
(265 mg, 0.61 mmol) in DMF (10 mL). TBTU (238 mg, 0.73 mmol)
was added and the solution was stirred for 5 min. Triethylamine
(0.09 mL, 0.61 mmol) was added and the reaction mixture was
stirred at room temp. for 20 h under an atmosphere of argon. TLC
analysis (ethyl acetate/cyclohexane, 2:1) indicated complete conver-
sion of the acid starting material (Rf = 0.0) into a major product
(Rf = 0.29). The solvent was removed in vacuo (co-evaporation
with toluene). The residue purified by flash column chromatog-
raphy to yield 18 (202 mg, 30%) as a colourless oil. Rf = 0.27, ethyl
1
2
2
(2ϫ CH2Ph, Saa CHε, Saa CHε), 54.6 (1Saa CHδ, Saa CHδ),
73.5, 75.2, 76.6, 78.4 (1Phe CHα, 2Phe CHα, 1Saa CHα, 2Saa CHα,
1Saa CHβ, 2Saa CHβ, 1Saa CHγ, 2Saa CHγ), 108.8, 111.9 [4ϫ
C(CH3)2], 127.0, 128.7, 129.2, 136.3 (2ϫ C6H5), 169.9, 171.9 (4ϫ
CONH) ppm. MS (ESI+): m/z (%) = 831.38 (100) [M + Na]+.
(6-Amino-6-deoxy-2,3:4,5-di-O-isopropylidene-D-galactonyl)-D-phen-
ylalanine Methyl Ester (15): Compound 11 (1.63 g, 3.52 mmol) was
dissolved in 1,4-dioxane (80 mL). Palladium black (0.20 g) was
added and the flask was flushed with argon. The reaction mixture
was flushed with hydrogen and left stirring under a hydrogen atmo-
sphere for 15 h. TLC (ethyl acetate/cyclohexane, 1:1) indicated
complete conversion of the starting material. The solvent was re-
moved in vacuo to afford 15 (1.51 g, quant.), which was used with-
out any further purification.
acetate/cyclohexane, 2:1. [α]2D3 = +5.7 (c = 2.2 in CHCl ); ν
=
˜
3
max
(NaCl): 3324 (N-H, br.), 2104 (-N3), 1744 (C=O, CO2Me), 1665
1
(C=O, amide) cm–1. H NMR (CDCl3): δ = 1.17, 1.19, 1.20, 1.30,
1.32, 1.33, 1.34, 1.40, 1.45 [9ϫ s, 36 H, 12ϫ C(CH3)2], 3.03–3.10
(m, 4 H, 2ϫ CH2Ph), 3.18–2.28 (m, 2 H, CH2Ph), 3.36–3.60 3.74–
3.80 (m, 6 H, 1Saa Hε, 2Saa Hε, 3Saa Hε, 1Saa HЈε, 2Saa HЈε, 3Saa
HЈε), 3.74–3.80 (m, 2 H, 1Saa Hγ, 2Saa Hγ), 3.73 (s, 3 H, COCH3),
1
3.96–4.26 (m, 7 H, Saa Hδ, 2Saa Hδ, 3Saa Hδ, 3Saa Hγ, 1Saa Hβ,
(6-Azido-6-deoxy-2,3:4,5-di-O-isopropylidene-
ylalanine–(6-amino-6-deoxy-2,3:4,5-di-O-isopropylidene-
onyl)- -phenylalanine Methyl Ester (16): Compound 12 (1.55 g,
D
-galactonyl)-
D-phen-
2Saa Hβ, 3Saa Hβ), 4.30–4.43 (m, 3 H, 1Saa Hα, 2Saa Hα, 3Saa
D
-galact-
2
Hα), 4.60–4.68 (m, 2 H, Phe Hα, 3Phe Hα), 4.85 (dd, J = 7.2, J =
D
3.2 Hz, 1 H, 1Phe Hα), 6.35–6.39 (m, 2 H, 1Saa NH, 2Saa NH),
3.52 mmol) was added to a stirred solution of 15 (1.51 g,
3.52 mmol) in DMF (20 mL). TBTU (1.37 g, 4.23 mmol) was
added and the solution was stirred for 5 min. Triethylamine
(0.50 mL, 3.53 mmol) was added and the reaction mixture was left
stirring at room temp. for 20 h under an atmosphere of argon. TLC
analysis (ethyl acetate/cyclohexane, 1:1) indicated complete conver-
sion of the acid starting material (Rf = 0.0) into a major product
(Rf = 0.28). The solvent was removed in vacuo. The residue was
purified by flash column chromatography to afford 16 (1.68 g,
55%) as a colourless oil. Rf = 0.28, ethyl acetate/cyclohexane, 1:1.
2
7.00 (d, J = 7.2 Hz, 1 H, 1Phe NH), 7.10–7.30 (m, 17 H, Phe NH,
3Phe NH, 15ϫ ArH) ppm. 13C NMR (CDCl3): δ = 25.7, 26.6, 26.7,
26.9, 27.0 (12ϫ CH3), 37.7, 38.9, 40.4, 40.6, 51.6 (3ϫ CH2Ph, 1Saa
CHε, 2Saa CHε, 3Saa CHε), 52.4, 52.6, 53.9, 54.0, 75.9, 76.1, 76.6,
77.2, 77.5, 77.6, 77.8, 78.0, 78.4, 78.5, 78.9 (15 ϫ -CH-), 109.4,
109.5, 110.2, 111.2, 111.3, 111.4 [6ϫ C(CH3)2], 127.0, 127.1, 127.2,
128.6, 128.7, 129.1, 135.5, 136.2 (3ϫ C6H5), 170.3, 170.4, 170.7,
171.5 (5ϫ CONH, CO2CH3) ppm. MS (ESI+): m/z (%) = 1270.38
(100) [M – H]+.
[α]2D1 = +22.9 (c = 1.3 in CHCl ); ν
= (NaCl): 3331 (N-H, br.),
˜
3
max
Cyclo[(6-Amino-6-deoxy-2,3:4,5-di-O-isopropylidene-
D-galactonyl)-
2103 (-N3), 1744 (C=O, CO2Me), 1667 (C=O, amide) cm–1 1H
.
D
-phenylalanine–(6-amino-6-deoxy-2,3:4,5-di-O-isopropylidene-
D-
NMR (CDCl3): δ = 1.16, 1.17, 1.31, 1.39, 1.43 [5ϫ s, 24 H, 8ϫ
galactonyl)-D-phenylalanine–(6-amino-6-deoxy-2,3:4,5-di-O-isoprop-
2
C(CH3)2], 2.99–3.28 (m, 5 H, 2ϫ CH2Ph, Saa Hδ), 3.37–3.48 (m,
ylidene-D-galactonyl)-D-phenylalanine] (19): Barium hydroxide
2 H, 1Saa Hε, 2Saa Hε), 3.54–3.80 (m, 5 H, 1Saa Hγ, 2Saa Hγ,
(47 mg, 0.27 mmol) was added to a stirred solution of 18 (117 mg,
0.09 mmol) in THF (2 mL) and water (4 mL). The reaction mixture
was stirred for 3 h at room temp. TLC analysis (ethyl acetate/cyclo-
hexane, 1:1) indicated complete conversion of the starting material
into one product. The reaction mixture was acidified by addition
of DOWEX 50W (H+), which was then removed by filtration, and
the filtrate was concentrated in vacuo to give (6-azido-6-deoxy-
2,3:4,5-di-O-isopropylidene-d-galactonyl)-d-phenylalanine–(6-
amino-6-deoxy-2,3:4,5-di-O-isopropylidene-d-galactonyl)-d-phen-
ylalanine–(6-amino-6-deoxy-2,3:4,5-di-O-isopropylidene-d-galact-
onyl)-d-phenylalanine. The crude acid was dissolved in 1,4-dioxane
(1 mL). Pentafluorophenol (34 mg, 0.18 mmol) and EDCI·HCl
(21 mg, 0.11 mmol) were added and the mixture was stirred at
1
1
2
1
CO2CH3), 4.00–4.24 (m, 5 H, Saa Hδ, Saa HЈε, Saa HЈε, Saa
Hβ, Saa Hβ), 4.37–4.44 (m, 2 H, 5-H, 15-H), 4.66 (dd, J = 1.8, J
= 3.7 Hz, 1 H, Phe Hα), 4.84 (dd, J = 1.8, J = 3.4 Hz, 1 H, Phe
2
2
1
1
1
Hα), 6.33–6.39 (m, 1 H, Phe NH), 6.99 (d, J = 2.0 Hz, 1 H, Saa
NH), 7.08–7.32 (m, 10 H, 10ϫ ArH) ppm. 13C NMR (CDCl3): δ
= 25.6, 26.5, 26.0, 26.8, 26.9 (8ϫ CH3), 37.6, 38.0, 40.3, 51.5 (2ϫ
CH2Ph, 1Saa CHε, 2Saa CHε), 52.3, 52.5, 53.8, 75.8, 76.0, 76.5,
77.1, 77.4, 78.4, 78.7 (10ϫ -CH-), 109.4, 110.1, 111.2 [4ϫ C(CH3)
2], 127.0, 127.1, 128.5, 128.9, 129.0, 135.4, 136.1 (2ϫ C6H5), 170.3,
171.4, 171.8 (3ϫ CONH, CO2CH3) ppm. MS (ESI+): m/z (%) =
865.75 (49) [M + H]+. HRMS: calcd. for C43H57N6O14 (M – H)+:
865.3984; found 865.3992.
(6-Azido-6-deoxy-2,3:4,5-di-O-isopropylidene-
ylalanine–(6-amino-6-deoxy-2,3:4,5-di-O-isopropylidene-
D
-galactonyl)-
D
-phen- room temp. under an atmosphere of argon. After 16 h, TLC analy-
sis (ethyl acetate/cyclohexane, 1:1) indicated complete conversion
D
-galact-
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
9

Job/Unit: O20463
/KAP1
Date: 22-08-12 16:34:22
Pages: 12
R. G. Soengas, J. Jiménez-Barbero et al.
FULL PAPER
of the starting material (Rf = 0.0) into a major UV-active product and also compared to those estimated from ROESY experiments
(Rf = 0.3). The solvent was removed in vacuo and the residue dis-
solved in dichloromethane (10 mL). The solution was washed with
an aqueous solution of sodium hydrogen carbonate (5% w/v, 5 mL)
and an aqueous solution of citric acid (5% w/v, 5 mL). The organic
layer was dried (MgSO4), filtered and concentrated in vacuo. The
residue was purified by flash column chromatography (ethyl acet-
ate/cyclohexane, 1:1) to yield (6-azido-6-deoxy-2,3:4,5-di-O-iso-
propylidene-d-galactonyl)-d-phenylalanine–(6-amino-6-deoxy-
2,3:4,5-di-O-isopropylidene-d-galactonyl)-d-phenylalanine–(6-
amino-6-deoxy-2,3:4,5-di-O-isopropylidene-d-galactonyl)-d-phen-
ylalanine pentafluorophenyl ester (74.5 mg), which was dissolved
in 1,4-dioxane (7 mL). Palladium black (74 mg) was added and the
flask was flushed with argon. The reaction mixture was flushed
with hydrogen and stirred under a hydrogen atmosphere for 15 h.
TLC analysis (ethyl acetate/cyclohexane, 1:1) indicated complete
conversion of the starting material (Rf = 0.30 UV) into a major
product (Rf = 0.1). The solvent was removed in vacuo and the oil
residue was purified by flash column chromatography (ethyl acet-
ate/cyclohexane, 3:1) to yield cyclic tetramer 19 (22 mg, 20%) as an
as described in ref.[22]
Supporting Information (see footnote on the first page of this arti-
1
cle): Copies of the H and 13C NMR spectra for cyclopeptides 4,
8, 10, 14, and 19 and results from the MD simulations.
Acknowledgments
Financial support to R. S. provided through the European Com-
munity’s Human Potential Programme under contract HPRN-CT-
2002-00173 is gratefully acknowledged.
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off-white solid. Rf = 0.28, ethyl acetate/cyclohexane, 3:2. [α]2D3
=
1
+0.8 (c = 0.1 in CHCl3). H NMR (CDCl3): δ = 1.06, 1.27, 1.31,
1.33 [4ϫ s, 36 H, 12ϫ C(CH3)2], 2.99–3.07 (m, 9 H, 1Saa Hε, 2Saa
Hε, Saa Hε, 3ϫ CH2Ph), 3.80–3.86 (m, 3 H, Saa HЈε, 2Saa HЈε,
3
1
3Saa HЈε), 3.90–3.94 (m, 6 H, 1Saa Hβ, 2Saa Hβ, 3Saa Hβ, 1Saa
2
3
1
Hδ, Saa Hδ, Saa Hδ), 4.04 (d, JHγ,Hδ = 10.0 Hz, 3 H, Saa Hγ,
2Saa Hγ, Saa Hγ), 4.46 (d, JHα,Hβ = 7.5 Hz, 3 H, Saa Hα, Saa
3
1
2
3
1
2
Hα, Saa Hα), 4.56 (dd, J = 8.5, J = 11.5 Hz, 3 H, Phe Hα, Phe
3
1
2
3
Hα, Phe Hα), 6.45 (d, J = 8.4 Hz, 2 H, Saa NH, Saa NH, Saa
NH), 7.24–7.34 (m, 10 H, 10ϫ ArH), 7.55 (d, J = 8.5 Hz, 3 H,
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230–253.
1Phe NH, Phe NH, 3Phe NH) ppm. 13C NMR (CDCl3): δ = 25.6,
2
1
2
26.5, 26.6, 26.9 [6ϫ C(CH3)2], 37.7, 41.2 (CH2Ph, Saa CHε, Saa
CHε, 3Saa CHε), 54.2 (1Saa CHδ, 2Saa CHδ, 3Saa CHδ), 75.3,
75.7, 78.1, 78.3 (1Phe CHα, 2Phe CHα, 3Phe CHα, 1Saa CHα, 2Saa
CHα, 3Saa CHα, 1Saa CHγ, 2Saa CHγ, 3Saa CHγ, 1Saa CHβ, 2Saa
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3
CHβ, Saa CHβ), 109.3, 111.6 [6ϫ C(CH3)2], 127.2, 128.7, 129.1,
136.1 (3ϫ C6H5), 170.5, 171.4 (6ϫ CONH) ppm. MS (ESI+): m/z
(%) = 1235.57 (21) [M + Na]+. HRMS: calcd. for C63H84N6NaO18
1235.5740; found 1235.5749.
Conformational Analysis. NMR Spectroscopy: 1H NMR spectro-
scopic assignments were performed by using standard 1D, 2D-
COSY, NOESY, ROESY and HSQC experiments on a Bruker Av-
ance 500 MHz spectrometer at 300 K. The coupling constants were
obtained from analysis of the 1H NMR spectra by using Mes-
treNova software. Proton–proton interatomic distances were esti-
mated from the enhancements measured by 2D ROESY experi-
ments, using mixing times of 300 and 400 ms.
Molecular Modelling: Molecular mechanics, MD and Monte Carlo
(MC) studies were conducted with the MACROMODEL program
as implemented in the Maestro package.[18] The AMBER* force
field was used.[19] The energies were minimised by using the Polak–
Ribière PR conjugate gradient method. The generalised Born sur-
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lations. The starting coordinates for dynamics calculations were
those obtained after energy minimisations. Simulations were car-
ried out over 2 ns at 300 K. MC studies were conducted by using
default parameters implemented in MACROMODEL; 300 trial
structures were generated for each molecule. Coupling constants
were calculated for the obtained structures by using the empirical
Karplus equation proposed by Haasnoot et al.[21] and compared
to those experimentally available. Interatomic H–H distances and
estimated NOEs were calculated by using the Mestrelab program
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10
www.eurjoc.org
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O20463
/KAP1
Date: 22-08-12 16:34:22
Pages: 12
Cyclic Peptide Mimics
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Received: April 11, 2012
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Job/Unit: O20463
/KAP1
Date: 22-08-12 16:34:22
Pages: 12
R. G. Soengas, J. Jiménez-Barbero et al.
FULL PAPER
Peptide Mimetics
R. G. Soengas,* M. Fontanella,
J. I. Santos, G. W. J. Fleet,
J. Jiménez-Barbero* ........................ 1–12
Synthesis and Conformational Analysis of
Heterogeneous Cyclic Oligomers of 6-
Amino-6-deoxygalactonic Acid and Phen-
ylalanine
The preparation of a series of cyclopep-
tides, formed by cyclisation of linear het-
erogeneous oligomers of 6-amino-6-deoxy-
galactonic acid and d- or l-phenylalanine,
is reported. Molecular dynamics and NMR
spectroscopy studies have given valuable in-
sight into their properties, indicating that
one of these structures, l-cyclodipeptide, is
a peptide mimetic of potential interest in
medicinal chemistry.
Keywords: Peptidomimetics / Cyclopept-
ides / Amino acids / Carbohydrates / Con-
formation analysis
12
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