Fructose-based proline analogues: Exploring the prolyl trans/cis-amide rotamer population in model peptides

Article abstract of DOI:10.1002/ejoc.201000983

A D-fructose moiety in which a D-proline ring has been engineered with a spiranic junction in the glycidic scaffold has been used as a proline mimetic. The bicyclic structure, which possesses high structural rigidity, was then included in model peptides t

Full text of DOI:10.1002/ejoc.201000983

FULL PAPER
DOI: 10.1002/ejoc.201000983
Fructose-Based Proline Analogues: Exploring the Prolyl trans/cis-Amide
Rotamer Population in Model Peptides
Laura Cipolla,*[a] Cristina Airoldi,[a][‡‡] Davide Bini,[a][‡‡] Maria Gregori,[a][‡][‡‡]
Filipa Marcelo,[b] Jesús Jiménez-Barbero,[a,b] and Francesco Nicotra[a]
Keywords: Peptides / Peptidomimetics / Carbohydrates / Molecular modeling / Molecular dynamics
A
D
-fructose moiety in which a
D
-proline ring has been engi-
peptides to explore their prolyl cis/trans amide rotamer pop-
ulations. Model peptide conformations were studied by mo-
lecular dynamics and NMR experiments.
neered with a spiranic junction in the glycidic scaffold has
been used as a proline mimetic. The bicyclic structure, which
possesses high structural rigidity, was then included in model
Introduction
Proline (Pro), being the only cyclic proteinogenic amino
acid, plays an important and unique role in determining the
structural properties of peptides and proteins.[1] At the same
time, the cis/trans isomerisation of the aminoacyl–proline
bonds plays an important role in the folding and stabilisa-
tion of protein structures. Proline is the sole amino acid to
have its side-chain fused into the peptide backbone. This
unique feature limits its rotation about amide bond, thereby
reducing the energy gap between the prolyl amide cis and
trans isomers and making them almost isoenergetic. As a
consequence, proline has a much greater tendency to form
cis amide bonds than other peptidic bonds, which usually
exist almost exclusively in the trans form (Figure 1).
Figure 1. cis/trans isomerisation of amides in peptidic bonds in ge-
Hence, proline is crucial for inducing reverse-turn struc-
tures such as β-turns and β-hairpins. Depending upon the
cis/trans stereochemistry of the Xaa-Pro amide bond, pro-
line is often involved in either a type I(IЈ)–II(IIЈ) or type VI
β-turn. In particular, the cis geometry induces the peptide
backbone to fold into a type VI β-turn in which proline
occupies the i + 2 position whereas type I(IЈ) and type
II(IIЈ) β-turns assume structures in which proline is in posi-
tion i + 1 and generates a trans amide bond with the preced-
ing amino acid in position i.
neral (A) and of the aminoacyl–proline bonds in particular (B).
Nature frequently modulates the thermodynamic sta-
bility of peptide and protein structures controlling the pro-
lyl cis/trans isomerisation, which can actually be considered
a conformational switch.[2] The cis/trans interconversion is
intrinsically slow and is catalysed in vivo by the evolution-
arily conserved group of peptidyl prolyl cis/trans isomerase
enzymes. A number of regulatory mechanisms of cellular
processes induced by the intrinsic conformational switch af-
forded to peptides by prolyl cis/trans isomerisation has re-
cently being elucidated.[3] Another powerful tool influenc-
ing the cis/trans population, also employed by nature, is the
incorporation of conformationally constrained amino acids
into a peptide chain thus reducing its intrinsic flexibility.
This goal can be achieved, for example, by the introduction
of suitable functionalities into the proline cycle: of the sev-
eral modified prolines,[4] the most common found in nature
are (2S,4R)-hydroxyproline (Hyp) and (2S,3S)-Hyp, gener-
[a] Department of Biotechnology and Biosciences,
University of Milano-Bicocca,
P.za della Scienza 2, 20126 Milano, Italy
Fax: +39-0264483565
E-mail: laura.cipolla@unimib.it
[b] Centro de Investigaciones Biológicas, CSIC,
Ramiro de Maeztu 9, 28040 Madrid, Spain
[‡] Present Address: Department of Experimental Medicine, Uni-
versity of Milano-Bicocca, Via Cadore 48, 20052 Monza, Italy.
[‡‡] These authors contributed equally to this work.
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201000983.
128
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Fructose-Based Proline Analogues
ated in post-translational processes exclusively in the Y and logue 1 on prolyl cis/trans isomerisation, simple N-acetyl-
X positions of collagen (Xaa-Yaa-Gly) repeats, respectively, proline analogues (Scheme 1) and N-acetylprolyl-glycine
with the enzymatic 4R hydroxylation being by far the domi- peptides (Scheme 2) were synthesised. Modifications of the
nant modification.[5]
glycine-proline peptide motif are of particular interest as it
Owing to the importance of cis/trans isomerism, proline is present in collagen[23] as well as in small peptides found
derivatives and analogues that allow the strict control of the to have neuroprotective properties[24] and the ability to in-
cis/trans population to modulate the thermodynamic sta- hibit HIV replication.[25] Furthermore, the Gly-Pro-Gly
bility of peptide and protein structures are being intensively peptide sequence is known to induce reverse turns.[26]
sought.[6] Conformationally constrained peptide analogues
are useful in deciphering the receptor-bound conformation
of biologically active peptides and in the design of novel
peptides with important biomedical properties.[7]
Over the years, several proline analogues have been sug-
gested to control prolyl cis/trans isomerism, for example,
through steric or stereoelectronic influences, designing Cβ-,
Cγ- and Cδ-substituted prolines,[8] including fluoropro-
lines,[9] 4-mercaptoproline,[10] azaprolines,[11] pseudopro-
lines,[12] bicyclic proline analogues[13] or other constrained
mimetics[14] and even glycosylated[15] or acylated Hyp.[16]
To nucleate β-turns, a different approach relies on the use
of the d-Pro residues.[17] For this moiety, the chemical na-
ture of the pyrrolidine ring restricts the φ value to 160Ϯ20°.
An additional and attractive concept for the design of novel
conformationally constrained proline analogues is based on
sugar amino acids (SAAs), which are defined by an α-
amino acid moiety [CH(NH2)CO2H] engineered on a
carbohydrate scaffold (furan or pyran).[18] The relative ri-
gidity of the furan or pyran ring combined with the poly-
functional nature of the carbohydrate scaffold has inspired
the design of unusual and conformationally constrained
Scheme 1. Synthesis of model peptide Ac-DProSpiro-NHMe from
compound 1. Reagents and conditions: a) MeNH2, HBTU, DI-
amino acids and novel peptidomimetics. Only a few proline-
PEA, MeCN (54%); b) DEA, CH3CN; c) Ac2O, Py, DMAP, DCM
based SAAs currently exist.[19] Proline-based SAAs are par-
(83% over two steps); d) HCO2NH4, Pd/C (200% in weight), re-
flux; 2 d, quant.yield.
ticularly interesting because the presence of the sugar cycle
in addition to the pyrrolidine ring confers increased rigidity,
which may permit better control of the cis/trans isomerism.
We recently synthesised a d-fructose scaffold possessing
high structural rigidity on which a d-proline moiety has
been engineered with a spiranic junction.[20] This scaffold
Synthesis
The peptide Ac-DProSpiro-NHMe (5) was synthesised in
was found to be highly versatile and we proposed its use in solution as outlined in Scheme 1. Amino acid 1 was coupled
the synthesis of conformationally constrained GABA ana- directly to methylamine by using HBTU [2-(1H-benzotria-
logues[21] and 1,4-benzodiazepine-2,5-dione chimeric deriv- zol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate]
atives.[22] To further explore the usefulness of this sugar as coupling reagent in MeCN to produce amide 2 in 54%
scaffold, we envisaged the possibility of exploring the prolyl yield. Deprotection of the amine (to give 3) followed by
cis/trans-amide rotamer population induced by our proline acylation produced diamide 4 in 83% yield over two steps.
mimetic in model peptides.
The last step in the synthesis was the deprotection of the
hydroxy groups; this reaction proved to be very trouble-
some. In fact many different conditions and reagents were
tried [a) H2, Pd/C, AcOH; b) H2, Pd(OH)2/C, AcOH;
c) FeCl3, DCM; d) NaI, BF3·OEt2, MeCN; e) Me3SiI,
Results and Discussion
We supposed that the d-proline analogue 1 (dProSpiro, see CCl4/DCM (1:1)] before finding an effective procedure[27]
Schemes 1 and 2), characterised by a spiro bicyclic structure (60 equiv. of HCO2NH4, Pd/C, reflux, 2 d, quant. yield) af-
derived from d-fructose, should provide conformational fording title compound 5.
constraints on the proline ring as a result of the spiro-like
Peptide Ac-DProSpiro-Gly-NHMe (13) was prepared by
junction combined with the relative rigidity of the fructose solid-phase peptide synthesis (SPPS), as illustrated in
furan ring and the pyrrolidine ring. Hence we decided to Scheme 2. The peptide was synthesised on Novagel®
include analogue 1 in model peptides and to study their HMPA high-loading resin (Novabiochem). Fmoc-protected
conformation by molecular dynamics and NMR experi- glycine 6 was coupled to the resin by using 3 equiv. of the
ments. To assess the influence of the bicyclic proline ana- Fmoc derivative activated with 1-(mesitylsulfonyl)-3-nitro-
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129

L. Cipolla et al.
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Scheme 2. Synthesis of model peptide Ac-DProSpiro-Gly-NHMe from compound 1. Reagents and conditions: a) 1. MSNT, MeIm, dry
DCM; 2. 10% Ac2O in dry DMF; b) 1. Pip (20% in dry DMF). 2. 1, HBTU, NEM, dry DMF; c) 20% Pip in dry DMF; d) 10% Ac2O
in dry DMF; e) TFA/TIS/H2O (95:2.5:2.5); f) MeNH2, HBTU, DIPEA, MeCN; g) HCO2NH4, Pd/C (200% in weight), reflux, 2 d, quant.
yield.
1,2,4-triazole (MSNT) (3 equiv.) in the presence of 1-
methylimidazole (MeIm, 2.5 equiv.) in DCM. The proline
scaffold 1 (reaction cycle of 1 h, 3.0 equiv.) was coupled to
the growing peptide chain upon preactivation with HBTU
and N-ethylmorpholine (NEM). Fmoc groups were re-
moved by treatment with 20% piperidine in DMF; com-
pound 9 was then acetylated with 10% acetic anhydride in
DMF.
The peptide 11 was cleaved from the resin with a solution
of TFA/triisopropylsilane (TIS)/H2O (9.5:0.25:0.25). Ad-
dition of Et2O caused the precipitation of side-products;
filtration and evaporation of the suspension afforded pure
11 (24% overall yield). Coupling of 11 to methylamine
using HBTU as coupling reagent in MeCN afforded com-
pound 12. Removal of the benzyl ether protecting groups
as described previously provided the deprotected compound
13 (Scheme 2).
To best characterise the behaviour of the rigid proline
scaffold as turn-inducer, molecular dynamic and NMR ex-
periments were performed.
NMR Spectroscopic Studies
1
The H NMR resonances of compounds 4, 5, 12 and 13
were assigned by performing COSY and HSQC experi-
ments. The major isomer was assigned as the trans amide
isomer by 2D NOESY experiments which showed interpro-
Figure 2. cis and trans isomer resonances assigned on the basis of
ton effects from the N-acetyl methyl group to the prolyl δ
NOE contacts of the N-acetyl methyl group as depicted in the
scheme. The 2D NOESY spectrum of compound 5 in D2O is re-
protons (Figure 2). In the minor cis isomer a NOE contact
was observed between the N-acetyl methyl group and the ported as an example.
prolyl α-proton (Figure 2).
Due to the different solubilities of these compounds, vents: the O-benzylated derivatives 4 and 12 were dissolved
NMR characterisations were performed in different sol- in CDCl3 and compounds 5 and 13 were dissolved in D2O.
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Fructose-Based Proline Analogues
To compare their behaviour, we also analysed their proper- Amide Proton Temperature Dependence
ties in [D6]DMSO.
To verify the propensity of compounds 4, 5, 12 and 13
to induce turns, we investigated their ability to form intra-
molecular hydrogen bonds between the oxygen of the N-
acetyl group and the NH-CH3 or the NH-Gly proton. In
general, strongly hydrogen-bonded amide protons show a
low temperature coefficient [Δδ(NH)/ΔT; –2.6 ppbK–1 or
smaller in absolute terms] and a high chemical shift
(δϾ7.0 ppm) in comparison with the corresponding pro-
tons in reference compounds and non-hydrogen-bonded
Measurement of Kcis/trans
The ratio of cis/trans isomers was determined by integrat-
ing as many well-resolved peaks as possible for each isomer
and calculating the average value for all the peaks of each
isomer at 298 K (Table 1).[28] Different behaviour was ob-
served depending on the solvent polarity. In CDCl3 com-
pounds 4 and 12 showed only the trans conformer whereas
in [D6]DMSO the cis conformer represented 33 and 25% of
the total, respectively. On the other hand, compounds 5 and
13 displayed very similar behaviour when dissolved in D2O
and [D6]DMSO; the trans conformer was present as 77 and
73% of the total, respectively, for molecule 5 and as 83%
for 13 in both solvents.
amide protons have
a
low-temperature coefficient
low chemical shift
(–2.6 ppbK–1 or smaller) and
a
(δϽ7.0 ppm). In contrast, amide protons in equilibrium be-
tween hydrogen-bonded and non-hydrogen-bonded states
show
a large temperature dependence (larger than
–2.6 ppbK–1 in absolute terms).[14a] The Δδ(NH)/ΔT values
for our molecules were calculated from the results of
DNMR (dynamic NMR) experiments over the temperature
range 298–324 K in CDCl3 and 298–368 K in [D6]DMSO.
The NH chemical shifts at 298 K and Δδ(NH)/ΔT values
for molecules 4, 5, 12 and 13 are reported in Table 1. Com-
pounds 4 (NH-CH3) and 12 (NH-Gly) in CDCl3 presented
very low Δδ(NH)/ΔT and chemical shift values, which me-
ans they are non-hydrogen-bonded. Conversely, the NH
protons showed high Δδ(NH)/ΔT and chemical shift values
both in [D6]DMSO (for all compounds) and D2O (for 5
and 13), which suggests the existence of an equilibrium be-
tween the hydrogen-bonded and non-hydrogen-bonded
states in these solvents.
Thermodynamics
The effect of temperature on the Kcis/trans values for com-
pounds 4, 5, 12 and 13 was explored in [D6]DMSO to allow
comparison between compounds; the resulting van’t Hoff
plots are shown in Figure 3. The ΔH°, ΔS° and ΔG°298 K
values were calculated from lnKct = (–ΔH°/R)(1/T) + ΔS°/
R and ΔG° = ΔH° – TΔS. The data are reported in Table 1.
DNMR experiments allowed us also to determine the
coalescence temperature, about 388 K, which was essen-
tially the same for all the molecules reported in Table 1 (see
also Figures 1S–6S in the Supporting Information).
Kinetics
The rates of cis/trans amide isomerisation [Eq. (1)] were
calculated for compound 13 by using 2D EXSY (Exchange
SpectroscopY)[29] NMR in [D6]DMSO at different tempera-
tures (303, 310, 313, 318, 323 and 328 K). For each tem-
perature two EXSY spectra were acquired with mixing
Figure 3. VanЈt Hoff plots for compounds 4, 5, 12 and 13.
Table 1. Thermodynamic and kinetic parameters and amide temperature dependence for compounds 4, 5, 12 and 13 in different deuterated
solvents.
[b]
Solvent
cis/trans at
298 K
ΔH°[a]
ΔS°[a]
ΔG°298 K
Tc [K] δ(NHGly) at
Δδ(NHGly)/
δ(NHCH3) at Δδ(NHCH3)/
298 K [ppm] ΔT [ppbK–1
[kJmol–1
]
[JK–1mol–1
]
[kJmol–1
]
298 K [ppm]
ΔT [ppbK–1
]
]
4
5
CDCl3
[D6]DMSO
Ͻ0.05
0.50
6.96 (trans)
8.18 (cis)
7.88 (trans)
–1.7
–4.7
–5.2
–5.562
–13.01
–1.684
388
D2O
[D6]DMSO
0.30
0.37
–1.928
–5.005
–3.404
1.80
–7.82
0.60
–2.465
–2.676
–3.582
388
8.19 (cis)
–4.9
–5.0
–8.0
–5.1
–3.6
7.88 (trans)
7.37 (trans)
7.90 (cis)
12 CDCl3
[D6]DMSO
Ͻ0.05
0.33
6.45 (trans)
8.50 (cis)
8.54 (trans)
–0.1
–5.7
–5.8
388
388
13 D2O
[D6]DMSO
0.20
0.20
7.72 (trans)
7.91 (cis)
7.74 (trans)
8.52 (cis)
8.59 (trans)
–5.7
–6.4
–5.5
–3.9
[a] Calculated by fitting of the van’t Hoff plots to the equation lnKct = (–ΔH°/R)(1/T) + ΔS°/R. [b] Calculated from ΔG° = ΔH°–TΔS°.
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FULL PAPER
times of 0.0 and 0.7 s, the first being the reference experi- were performed by using the GB/SA solvent model for
ment. The volumes of the cross and diagonal peaks corre- water and DMSO. The results were completely parallel for
sponding to the prolyl α proton of both cis and trans iso- both solvents. AMBER provided the worst agreement with
mers were collected and the magnetisation exchange rate experimental data because it estimated the cis isomer to be
constants k1 and k–1 were calculated by using the program more stable in all cases, whereas OPLS and MM3* pre-
EXSYCalc.[30] The results of the 2D EXSY experiments are dicted the trans analogue to be the major isomer, in agree-
summarised in Table 2.
ment with the NMR results (see Tables 1S–4S in the Sup-
porting Information). In any case, given the small values of
the relative energies, all the results of the molecular me-
chanics calculations may be considered as fairly satisfac-
tory. In any case, from the geometry point of view, and
when the structures were analysed, it became evident that
the presence of the spiro moiety at the γ carbon atom of
the proline ring causes the substituents at the different posi-
tions of the pyrrolidine moiety to adopt well-defined orien-
tations. When the proline amide and the substituent at Cα
are considered, the adopted orientations do not allow them
to form stable intramolecular hydrogen bonds as they have
different spatial orientations.
MM3*-based molecular dynamics calculations were also
performed to verify the conformational stability of the cal-
culated conformers and to predict the access to a larger
conformational space and the possible occurrence of intra-
molecular hydrogen bonds. No attempts to search for larger
conformational transitions were performed. As deduced
from the simultaneous presence of cis and trans isomers in
the NMR spectra, the timescale for interconversion be-
tween the rotamers is on the millisecond timescale, still very
far from the possibility of MD simulations. The MM3*-
calculated geometries were found to be stable during the
MD simulations with certain minor fluctuations around the
starting geometry and the occurrence of hydrogen bonds
was very minor (see Figures 4 and S7, the latter in the Sup-
porting Information).
(1)
Table 2. k1, k–1 and the ratios of cis/trans isomers calculated on the
basis of 2D EXSY experiments recorded at different temperatures.
T [K]
k1 [s–1]
k–1 [s–1]
cis/trans
303
310
313
318
323
328
0.021
0.113
0.209
0.392
0.586
0.728
0.005
0.021
0.048
0.087
0.146
0.183
0.153
0.157
0.158
0.160
0.165
0.170
All the NMR spectroscopic data support the existence of
an equilibrium of the proline amide bond with significant
percentages of the cis isomer in polar solvents such as water
and DMSO. This is not the case in less polar solvents such
as chloroform in which only the trans isomer is present.
From an analysis of the thermodynamic parameters, the
trans isomer is always more stable from the enthalpy view-
point with the entropy term being either negative (for the
benzylated analogues) or positive (for the free hydroxy com-
pounds). This indicates that the interplay between the sol-
vent and the substituents at the sugar scaffold may play a
key role in modulating the conformational equilibrium.
From the spatial arrangement perspective and thinking of
the possibility of this scaffold acting as a turn-inducer, the
NOE and variable-temperature data suggest that no stable
intramolecular hydrogen bonds form and that these mole-
cules do not adopt stable turn-like structures. At this point
molecular mechanics and dynamics calculations were per-
formed to try to understand this behaviour.
Computational Studies
The major aim of the molecular mechanics calculations
was to provide 3D models to support the experimental
NMR results, which suggested that no turn-like structures
were present in solution in a significant proportion. There-
fore the calculations were performed by using three dif-
ferent force fields, AMBER, OPLS and MM3*, as im-
plemented in the Maestro package, built with either a cis or
trans orientation at the proline amide bond for compounds
4, 5, 12 and 13. In all cases very small relative steric energy
values were found between the trans and cis conformers,
with the largest relative difference of 4.2 kJmol–1 (AM-
BER) found for compound 13. In most of the cases the
difference was below 2.0 kJmol–1, in semi-quantitative
Figure 4. Structures obtained by MD simulations for compounds
5 (A, trans conformer; B, cis conformer) and 13 (C: trans con-
former; D: cis conformer) in water. In the dynamics, both conform-
ers, cis and trans, are conformationally stable. This is more evident
for 5. The hydrogen atoms have been removed from the figures for
agreement with the experimental observations. Calculations the sake of clarity.
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Fructose-Based Proline Analogues
and diethylamine (0.1 mL) and stirred at room temperature under
Ar. After 1 h, the solvents were evaporated and crude 3 was used
directly in the next reaction.
Conclusions
A variety of molecules containing a d-proline moiety
spiro-fused to a d-fructose ring at the Cγ carbon of the Pro
ring have been prepared as putative structural scaffolds. It
has been shown that the nature of the remote substituents
of the fructose moiety modulates the conformational equi-
librium at the proline amide bond, which also depends
upon the polar/apolar features of the solvent. An enthalpy/
entropy compensation phenomenon is observed in the equi-
librium, which is always enthalpy-driven towards the trans
isomer, but the entropy term for the cis form may substan-
tially compensate the enthalpy term. For these molecules
no persistent intramolecular hydrogen bond is formed that
may drive the geometry of a peptide chain to adopt a stable
turn-like structure. In fact, the presence of the fused spiro
moiety at Cγ strongly rigidifies the pyrrolidine ring and
causes the amide substituent and the Cα substituent to
point in rather divergent orientations, which could be em-
ployed in other types of design. These observations support
the idea that when molecular recognition processes are in-
volved, the existence of a certain flexibility is essential to
allow the counterparts to adopt well-defined intra- and
intermolecular arrangements.
AcNH-DProSpiro(OBn)-NHMe (4): Acetic anhydride (0.020 mL,
0.055 mmol), pyridine (0.010 mL, 0.055 mmol) and a catalytic
amount of 4-(dimethylamino)pyridine (DMAP) were added to a
solution of crude compound 3 dissolved in dry DCM (1 mL) and
the reaction was stirred at room temperature for 1 h. MeOH was
added and the mixture extracted with DCM washing with aq. 5%
HCl. The organic phase was then dried (Na2SO4), filtered and con-
centrated. The crude residue was purified by flash chromatography
(EtOAc/EtOH, 8:2) to afford 4 (17 mg) as a white solid (83% over
two steps). 1H NMR: δ = 7.299–7.163 (m, 15 H, Ph), 6.963 (s, NH),
4.473 (m, 7 H, OCH2Ph, α-H), 4.085 (d, J = 2.4 Hz, 1 H, 1-H),
4.052 (ddd, J = 9.4, 5.4, 4.1 Hz, 1 H, 3-H), 3.944 (dd, J = 2.4,
4.1 Hz, 1 H, 2-H), 3.709 (d, J = 11.7 Hz, 1 H, δa-H), 3.563 (d, J =
11.7 Hz, 1 H, δb-H), 3.444 (m, 2 H, 4a-H, 4b-H), 2.690 (d, J =
4.8 Hz, 3 H, NHCH3), 2.625 (dd, J = 6.5, 13.7 Hz, 1 H, βa-H),
2.176 (dd,
J = 8.6, 13.7 Hz, 1 H, βb-H), 1.938 (s, 3 H,
CH3CO) ppm. 13C NMR: δ = 171.614, 171.151 (s, CO), 138.133,
137.845, 137.675 (s, Cquat arom.), 128.940–127.577 (d, CH arom.),
90.267 (s, C-γ), 85.524 (d, C-1), 83.648 (d, C-2), 81.655 (d, C-3),
73.558, 72.231, 72.103, 70.508 (t, 3 OCH2Ph, C-4), 59.157 (d, C-
α), 54.588 (t, C-δ), 37.030 (t, C-β), 26.594 (q, NHCH3), 22.958 (q,
CH3CO) ppm.
AcNH-DProSpiro-NHMe (5): Pd/C (0.006 g) and ammonium for-
mate (0.020 g, 0.322 mmol) were added to a solution of compound
4 (0.006 g, 0.0107 mmol) in dry MeOH and the solution was stirred
at reflux for 24 h. The same quantities of Pd/C and ammonium
formate were added again and the solution was allowed to react
for an additional 24 h. Palladium was then removed by filtration
through a Celite pad, washing with DCM and MeOH. Evaporation
of the solvents afforded pure 5 as a yellowish oil (quant. yield). 1H
NMR (D2O): δ = 4.355 (dd, J = 7.9, 9.7 Hz, 1 H, α-H), 4.087 (d,
J = 4.9 Hz, 1 H, 1-H), 3.987 (dd, J = 4.9, 6.1 Hz, 1 H, 2-H), 3.867
(m, 2 H, δa-H, 3-H), 3.704 (dd, J = 3.4, 12.4 Hz, 1 H, 4a-H), 3.664
(d, J = 12.6 Hz, 1 H, δb-H), 3.626 (dd, J = 5.1, 12.4 Hz, 1 H, 4b-
H), 2.681 (s, 3 H, NHCH3), 2.475 (dd, J = 7.9, 13.8 Hz, 1 H, βa-
H), 2.053 (s, 3 H, COCH3), 2.023 (dd, J = 9.7, 13.8 Hz, 1 H, βb-
H) ppm. 13C NMR (D2O): δ = 171.988, 170.762 (s, CON), 91.336
(s, C-γ), 82.630 (d, C-3), 79.310 (d, C-1), 76.488 (d, C-2), 61.549 (t,
C-4), 56.901 (d, C-α), 55.075 (t, C-δ), 39.426 (t, C-β), 26.525 (q,
NHCH3), 22.375 (q, CH3CO) ppm.
Experimental Section
General Methods: All solvents were dried with molecular sieves for
at least 24 h prior to use. When dry conditions were required, the
reaction was performed under Ar. Thin-layer chromatography
(TLC) was performed on silica gel 60F254-coated glass plates
(Merck) with UV detection when possible or charring with a concd.
H2SO4/EtOH/H2O solution (5:45:45, v/v/v). Flash column
chromatography was performed on silica gel 230–400 mesh
(Merck). Routine 1H and 13C NMR spectra were recorded using
CDCl3 as solvent unless otherwise stated. Chemical shifts are re-
ported in ppm downfield from TMS as internal standard (aromatic
signals have been omitted); J values are given in Hz.
Fmoc-NH-DProSpiro(OBn)-NHMe (2): Compound
1 (0.050 g,
0.067 mmol) was dissolved in dry MeCN (2.5 mL). Addition of
diisopropyl(ethyl)amine (0.046 mL, 0.27 mmol) was followed by
addition of HBTU (0.051 g, 0.135 mmol) and methylamine (40%
in water, 0.011 mL, 0.135 mmol). The reaction mixture was stirred
at room temperature for 30 min, then diluted with water (6 mL)
and extracted with DCM, dried (Na2SO4) and then concentrated
under reduced pressure. Compound 2 was obtained as a yellowish
oil (0.027 mg, 54% yield). 1H NMR: δ = 7.778–6.989 (m, 23 H,
Ph), 6.499 (br. s, 1 H, NHcis), 5.816 (br. s, 1 H, NHtrans), 4.267 (m,
11 H, 3 OCH2Ph, CH2Fmoc, CHFmoc, α-H, 3-H), 3.915 (br. s, 1
H, 1-H), 3.779 (br. s, 1 H, 2-H), 3.446 (m, 2 H, 4a-H, 4b-H), 2.914
(m, 5 H, NHCH3trans, δa-H, δb-H), 2.718 (d, J = 10.2 Hz, 3 H,
NHCH3cis), 2.365 (m, 2 H, βa-H, βb-H) ppm. 13C NMR: δ =
157.915, 153.642 (s, CO), 144.164, 143.907, 141.503, 140.033,
138.271, 137.875, 137.861 (s, CquatFmoc and arom.), 131.297–
120.205 (CH arom.), 92.690 (s, C-γ), 85.774, 84.001, 82.143 (d, C-
1, C-2, C-3), 73.569, 71.969, 70.763, 67.782, 67.982 (t, OCH2Ph, C-
4, OCH2Fmoc), 60.400 (d, C-α), 53.592 (t, C-δ), 42.878 (d,
CHFmoc), 38.829 (t, C-β), 27.095 (q, NHCH3) ppm.
N-Acetyl-DProSpiro(OBn)-Gly-OH (11): Compound 11 was synthe-
sised manually on the solid phase by using a disposable plastic
syringe fitted with a Teflon filter (pore size 70 μm) and connected
to a vacuum waste bottle by a two-way Teflon valve. The synthesis
was performed on Novagel® (250 mg, 0.16 mmol) derivatised with
an HMPA linker. The completeness of all reactions was monitored
by using the Bromophenol Blue test. FmocHN-glycine-COOH
(3 equiv.) was coupled to the resin by activation with 1-(mesitylsul-
fonyl)-3-nitro-1,2,4-triazole (MSNT, 3 equiv.) in the presence of 1-
methylimidazole (MeIm, 2.5 equiv.) in dry DCM (2ϫ45 min). The
resin was treated with 10% Ac2O in dry DMF (2ϫ10 min); then,
the Fmoc group was removed with 20% piperidine. The resin was
washed with dry DMF between each coupling and deprotection
step. The glycosyl amino acid building block 1 (3.0 equiv.) was cou-
pled (1h) after preactivation for 5 min with HBTU (2.8 equiv.) and
NEM (4.5 equiv.) in dry DMF. The N-terminal Fmoc protecting
group was cleaved as usual and then the resin was treated (10 min.)
with 10% acetic anhydride in dry DMF. After washing with DMF,
AcOH and DCM, compound 10 was cleaved from the solid phase
H2N-DProSpiro(OBn)-NHMe
0.027 mmol) was dissolved in a 9:1 mixture of dry MeCN (0.9 mL)
(3):
Compound
2
(0.020 g,
Eur. J. Org. Chem. 2011, 128–136
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
133

L. Cipolla et al.
FULL PAPER
by incubation with a 9.5:0.25:0.25 mixture of TFA/triisopropylsi-
lane/H2O for 2h. The resin was thoroughly washed with this mix-
ture and the liquids were collected and concentrated in vacuo. Ad-
dition of Et2O caused the precipitation of side-products; filtration
and evaporation of the ethereal solution afforded pure 11 (20 mg,
streLab Research s.r.l.) and kinetic parameters were calculated with
EXSYCalc.[30] For 1D NMR experiments, compounds 4, 5, 12 and
13 were dissolved in CDCl3, [D6]DMSO or D2O in a concentration
range of between 0.05 and 20 mm to verify the absence of intermo-
lecular interactions. DNMR experiments were then performed on
1
24% yield). 1H NMR (CD3OD): δ = 7.913–6.797 (m, 15 H, Ph), 2 mm solutions of each compound. The H NMR spectra were re-
4.527 (m, 7 H, OCH2Ph, α-H), 4.135 (br. s, 1 H, 3-H), 4.070–3.637
(m, 6 H, δa-H, δb-H, 1-H, 2-H, a-αGlyH, b-αGly-H), 3.530 (m, 2 H,
corded at different temperatures and then processed with Mes-
treNova. Solvent chemical shifts were calculated according to
4a-H, 4b-H), 2.519 (dd, J = 8.4, 13.5 Hz, 1 H, βa-H), 2.126 (dd, J Gottlieb et al.[31]
= 8.8, 13.5 Hz, 1 H, βb-H), 1.978 (s, 3 H, CH3CO) ppm. 13C NMR
Molecular Mechanics and Dynamics: Molecular mechanics and dy-
(CD3OD): δ = 177.365, 176.027, 175.448 (s, CO), 142.166, 142.013,
141.875 (s, Cquat arom.), 132.399–131.417 (d, CHarom.), 94.445 (s,
C-γ), 89.416, 86.926, 86.058 (d, C-1, C-2, C-3), 77.033, 75.851,
75.611 (t, OCH2Ph), 74.171 (t, C-4), 63.574 (d, C-α), 58.678 (t, C-
δ), 42.772 (t, C-αGly), 33.507 (t, C-β), 25.099 (q, CH3CO) ppm.
namics studies were conducted with Macromodel 9.6[32] as im-
plemented in version 8.5.110 of the Maestro suite[33] using distinct
force fields (AMBER*,[34] MM3*[35] and OPLS*[36]). The starting
coordinates for dynamics calculations (MM3* as the force field)
were those obtained after energy minimisation of both the cis and
trans conformations. In particular, a systematic variation of the tor-
sional degrees of freedom of the molecules permitted different
starting structures to be constructed that were further minimised
to provide the corresponding local minima. For each (cis or trans)
geometry and force field, the conformer with the lowest energy was
considered (also for the MD simulations). The existence of the
spiro-junction precluded further motion around the two five-mem-
bered rings. Simulations were carried out over 2 ns at 400 K with
a 0.05 fs time step and a 5 ps equilibration step; 200 structures were
sampled and minimised for further analysis. The continuum GB/
SA solvent model[37] was employed and the general PRCG (Polak–
Ribiere Conjugate Gradient) method for energy minimisation was
used. An extended cut-off was applied and the SHAKE procedure
for bonds was not selected.
AcHN-DProSpiro(OBn)-Gly-NHMe (12): Compound 11 (0.020 g,
0.033 mmol) was dissolved in dry MeCN (1.2 mL) and diiso-
propyl(ethyl)amine (0.023 mL, 0.132 mmol), HBTU (0.025 g,
0.066 mmol) and methylamine (40% in water, 0.006 mL,
0.066 mmol) were added. The reaction mixture was stirred at room
temperature for 1 h, then diluted with water (3 mL), extracted with
DCM, dried (Na2SO4) and then concentrated under reduced pres-
sure. Pure 12 was provided as a yellowish oil (0.010 mg, 49% yield).
1H NMR (DMSO): δ = 8.475 (t, J = 5.9 Hz, 1 H, NHGly), 7.651
(d, J = 4.5 Hz, 1 H, NHCH3), 7.349–7.286 (m, 15 H, Ph), 4.525
(m, 6 H, OCH2Ph), 4.245 (dd, J = 8.2, 8.8 Hz, 1 H, α-H), 4.128
(br. s, 1 H, 3-H), 4.074 (br. s, 1 H, 2-H), 4.008 (br. s, 1 H, 1-H),
3.844 (d, J = 11.4 Hz, 1 H, δa-H), 3.689 (m, 1 H, a-αGly-H), 3.528
(m, 4 H, δb-H, b-αGly-H, 4a-H, 4b-H), 2.564 (d, J = 4.5 Hz, 3 H,
NHCH3), 2.315 (dd, J = 7.5, 12.9 Hz, 1 H, βa-H), 1.962 (dd, J =
9.9, 12.9 Hz, 1 H, βb-H), 1.933 (s, 3 H, CH3CO) ppm. 13C NMR:
δ = 172.029, 171.024, 170.984 (s, CON), 137.629, 137.355, 136.211
(s, Cquat arom.), 128.867–127.781 (d, CHarom.), 90.547 (s, C-γ),
85.745, 83.144, 82.192 (d, C-1, C-2, C-3), 73.589, 72.184, 72.107,
70.468 (t, OCH2Ph, C-4), 60.801 (d, C-α), 55.332 (t, C-δ), 43.606
(t, NHCH2), 38.260 (t, C-β), 26.527 (q, NHCH3), 22.870 (q,
CH3CO) ppm.
Supporting Information (see also the footnote on the first page of
this article): plots of NH chemical shifts versus temperature for
compounds 4, 5, 12 and 13 (Figures S1–S6), energy values calcu-
lated by MM for the cis and trans isomers of compounds 4, 5, 12
and 13 in different solvents and employing the OPLS, MM3* and
AMBER* force fields and the structures obtained by MD for com-
pounds 4, 5, 12 and 13.
AcHN-DProSpiro-Gly-NHMe (13): Compound 12 (0.010 g,
0.0162 mmol) was debenzylated as previously described for peptide
5 (0.010 g Pd/C, 0.030 g, 0.486 mmol ammonium formate). Com-
Acknowledgments
We gratefully acknowledge financial support by Regione Lom-
bardia (Programma Operativo Regione Lombardia Ob. 3 Fondo
Sociale Europeo 2000–2006), Sovvenzione Globale Progetto In-
genio A0001127/2007, Consorzio Interuniversitario Nazionale
“Metodologie e Processi Innovativi di Sintesi” (C.I.N.M.P.I.S.) and
the Fondo di Ateneo per la Ricerca FAR 2008. The Fundação para
Ciência e Tecnologia (FCT) Portugal is also thanked for post-doc-
toral research (grant SFRH/BDP/65462/2009).
1
pound 13 was obtained as a yellowish oil (quant. yield). H NMR
(D2O): δ = 4.42 (dd, J = 7.8, 9.5 Hz, 1 H, α-H), 4.09 (d, J = 4.9 Hz,
1 H, 1-H), 3.98 (dd, J = 4.9, 5.9 Hz, 1 H, 2-H), 3.89 (m, 3 H, δa-
H, 3-H, a-αGly-H), 3.79 (m, 1 H, b-αGly-H), 3.68 (m, 3 H, 4a-H,
4b-H, δb-H), 2.68 (d, J = 4.9 Hz, 3 H, NHCH3), 2.52 (dd, J = 7.8,
13.8 Hz, 1 H, βa-H), 2.08 (m, 1 H, βb-H), 1.96 (s, 3 H,
CH3CO) ppm. 13C NMR (DMSO): δ = 178.757, 177.224, 174.665
(s, CO), 94.035 (s, C-γ), 88.388, 84.534, 81.531 (d, C-1, C-2, C-3),
66.547 (t, C-4), 64.820 (d, C-α), 60.094 (t, C-δ), 47.681 (t, NHCH2),
44.304 (t, C-β), 30.976 (q, NHCH3), 27.986 (q, CH3CO) ppm.
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Received: July 12, 2010
Published Online: November 24, 2010
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