Arabinose-derived bicyclic amino acids: Synthesis, conformational analysis and construction of an αvβ3-selective RGD peptide

Article abstract of DOI:10.1039/b110453e

The synthesis, NMR structure determination, and molecular modelling of the conformationally restricted diastereomeric sugar azido acids 1 and 2 are presented. The bicyclic structures of these compounds are obtained through a iodocyclization reaction on th

Full text of DOI:10.1039/b110453e

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Arabinose-derived bicyclic amino acids: synthesis, conformational
analysis and construction of an ꢀvꢁ3-selective RGD peptide
Francesco Peri,*a Roberta Bassetti,a Enrico Caneva,b Luca de Gioia,a Barbara La Ferla,a
Marco Presta,c Elena Tanghettic and Francesco Nicotra*a
1
a Department of Biotechnology and Biosciences, University of Milano-Bicocca, I-20126 Milano,
Italy. E-mail: francesco.peri@unimib.it, fancesco.nicotra@unimib.it; Fax: ϩ39.02.64483565
b Department of Organic and Industrial Chemistry, Via Venezian 21, I-20133, Milano, Italy
c Unit of General Pathology and Immunology, Department of Biomedical Sciences and
Biotechnology, School of Medicine, University of Brescia, I-25123, Brescia, Italy
Received (in Cambridge, UK) 16th November 2001, Accepted 18th January 2002
First published as an Advance Article on the web 7th February 2002
The synthesis, NMR structure determination, and molecular modelling of the conformationally restricted
diastereomeric sugar azido acids 1 and 2 are presented. The bicyclic structures of these compounds are obtained
through a iodocyclization reaction on the C-allyl glycoside of the -arabinofuranose. Cyclic tetrapeptide 11
containing the amino acid derived from 1 linked to the RGD sequence has been synthesized; this compound
was found to be a selective antagonist of αvβ3 integrins expressed on GM 7373 cells.
bicyclic compounds derived from natural products as dipeptide
Introduction
analogues to stabilize the peptide chain in a reverse-turn con-
formation has been the object of extensive studies.13 Among
these, 5,5-14 and 6,5-bicyclic15 compounds have been reported
as β-turn mimetics.
An increasing interest has recently been devoted to sugar-
derived amino acids (SAAs) that have been used as conform-
ationally restricted non-peptide isosteres and have been intro-
duced into the peptide backbone in order to achieve desirable
secondary structures. In this context, the pyran ring of SAAs
derived from α--glucopyranose was used as a rigid or semi-
rigid template to induce linear, β-turn and γ-turn conform-
ations in peptides.1 The same sugar amino acids have been used
to induce the bioactive conformation in small RGD (Alg-Gly-
Aso) cyclic peptides, obtaining selective antagonists for the αvβ3
integrin.2 In a slightly different conceptual approach, α--
glucopyranose was employed as a peptidomimetic scaffold
devoid of an amide backbone to accommodate the side-chain
functionalities responsible for the biological activity of the
peptide hormone somatostatin.3 In this context, RGD mimics4
and thrombin inhibitors5 were prepared by assembling amino
acid side chain functionalities on -glucose. The same sugar
was used to replace the ester-amide backbone of the cyclic depsi-
peptide hapalosin.6 Recently, this approach has been extended
to a model study in solution for the preparation of solid-phase
libraries of compounds based on the α--glucose scaffold.7
Finally, sugar-derived amino acid monomers were assembled
to form oligomers capable of adopting defined secondary struc-
tures in solution. Oligomers of pyranose sugar amino acids8
have been synthesized, and furanosidic sugar amino acids have
been used as rigid platforms in the design and synthesis of
carbopeptoids.9 Homo-oligomers of tetrahydrofuran amino
acids were shown to adopt a novel repeating β-turn-type
secondary structure in tetramers10 and a left-handed helical
conformation in octamers11 (the generic name of foldamers has
been proposed for this type of structure).12
Results and discussion
We reported in a note16 the synthesis and the structural charac-
terization of the bicyclic sugar azido acids 1 and 2 (Fig. 1) and
Fig. 1 Structures of the azido acid 1 and 2 (epimers on C-2).
Numbering shown is non-systematic.
the incorporation of 1 into a cyclic RGD peptide. In this paper
we report the synthetic procedure with full experimental details,
the conformational analysis of azido acids 1 and 2, and the
biological activity of the peptide.
Compounds 1 and 2 can be directly condensed to N-
protected amino acids in the solid phase by using a coupling
method based on simultaneous in situ activation of the
carboxylic group and reduction of the azide functionality.17 The
bicyclic structures of 1 and 2 were obtained according to
the synthetic strategy shown in Scheme 1.
The commercially available 2,3,5-tri-O-benzyl--arabino-
furanose was converted into the anomeric acetate 3 (mixture
of anomers), which was allylated by treatment with allyl-
trimethylsilane (ATMS)† in the presence of the Lewis acid
SAAs possess a cyclic pyranose or furanose core that reduces
the overall conformational mobility of the molecule and allows
it to maintain a defined mutual disposition of the amino and
carboxylic groups. This is an important prerequisite to the
induction of peptide sequences in which SAAs are inserted to
fold into regular secondary structures. In this paper we present
the design and the synthesis of SAAs with a bicyclic core: the
conformational freedom of such molecules is further reduced
by the presence of two fused five-membered rings. The use of
† Abbreviations: ATMS, allyltrimethylsilane; DIC, N,N Ј-diisopropyl-
carbodiimide; DIPEA, diisopropylamine; DMAP, 4-(dimethylamino)-
pyridine; FCS, fetal calf serum; HBTU, 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate; HOBt, N-hydroxy-
benzotriazole; NIS, N-iodosuccinimide; Pmc, 2,2,5,7,8-pentamethyl-
chroman-6-ylsulfonyl; SASRIN, Super Acid Sensitive ResIN; TFA, tri-
fluoroacetic acid; TIS, triisopropylsilane; TMS tetramethylsilane.
638
J. Chem. Soc., Perkin Trans. 1, 2002, 638–644
This journal is © The Royal Society of Chemistry 2002
DOI: 10.1039/b110453e

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Scheme 1 Reagents and conditions (and yields): i) ATMS, BF3ؒEt2O, dry CH3CN, rt, (75%); ii) NIS, dry THF, rt (80% on the β-anomer);
iii) Bu4NN3, toluene, 60 ЊC (60%, separation of diastereomers by column chromatography); iv) Ac2O–TFA, rt (95%); v) MeONa, MeOH, rt (75%);
vi) CrO3–H2SO4, acetone–water, rt (72%).
promoter BF3ؒEt2O. The reaction, effected at rt in dry aceto-
nitrile, afforded 418 as a 1 : 1 mixture of α and β diastereomers
(75% yield). Iodocyclization (Scheme 1) was then carried out
with NIS in dry THF at rt affording bicyclic iodo ether 5 as a
mixture of diastereomers (80% yield based on the β-anomer),
Fig. 2 Observed NOEs in 1.
the major isomer having the 2R configuration. This reaction is
the crucial step for the synthesis of the bicycle and its mechan-
ism is based on the opening of the intermediate iodonium ion
by attack of the γ-benzyloxy groups in the 5-exo-mode with
formation of a cyclic iodo ether with concomitant debenzyl-
ation.19 Only the β-anomer of 4 reacted and this allowed its
easy separation from the α-anomer. Pure α-anomer of 4 was
then isolated and characterized. The reaction of the iodo
derivative 5 with tetrabutylammonium azide in toluene gave
azido derivative 6 (60 ЊC, 60% yield). Compounds (R)-6 and
(S)-6 were separated by flash chromatography and their relative
configurations at C-2 assigned by NOESY analysis: an intense
correlation peak was observed between H-2 and H-3a protons
for the diastereomer (S)-6, that was absent for (R)-6. The
subsequent reactions were carried out separately on the two
diastereomers with similar yields. Compound 6 was regioselec-
tively debenzylated at the primary hydroxy group by acetolysis
(Ac2O, CF3COOH, rt, 95%), followed by saponification of the
acetate on C-2Ј (75%) and Jones oxidation (72%). Anomers 1
and 2 (S or R configuration at C-2), were obtained with similar
yields over the last three synthetic steps.
1 was then performed by executing five 2D NOESY experi-
ments with different mixing-time-values,20 between 0.6 and 1.4
seconds, to cover the full range of molecular relaxation times in
the two-spin approximation. Since the presence of conform-
ational equilibria may introduce errors in quantitative NMR-
derived data, molecular dynamics simulations were performed
for compound 1. The structures obtained after the simulated
annealing MD procedure were finally optimized by MM.21
Final structures were clustered to obtain distinct conformer
families and ranked by potential-energy values. Crucial struc-
tural properties of the low-energy conformers were compared
with the analogous data inferred from NOE experiments.
Some interproton distances relevant for the definition of the
conformation of 1 derived from NOE and from MD-MM are
compared in Table 1.
As exemplified from the obtained data, in compound 1 the
distances calculated by MM/MD are in excellent agreement
with those derived from NOE experiments. The only discrep-
ancy between the two sets of measures are H-3a/H-4 and H-3a/
H-2 distances that are underestimated by NMR calculations.
This is due to the total superimposition between the H-3a and
the benzyl methylene signals in the 1H-NMR spectrum, leading
to an increment of NOEs volume and a consequent interproton
distance reduction. It is, however, noteworthy that all the high-
energy conformers of 1 obtained by MM/MD are characterized
by molecular geometries presenting interproton distances that
differ consistently from those derived from NOE experiments.
In a first qualitative investigation of the conformation of 1 by
1H-NMR spectroscopy in CDCl3, intense NOEs were observed
for H-6a/H-1b, H-2/H-1b, H-2/H-3a and H-5/H-6a couples (Fig.
2). These NOEs indicate that the hydrogen couples H-2/H-3a
and H-5/H-6a are in close spatial proximity; this situation is
compatible with a conformation of the bicycle in which the two
tetrahydrofuran rings are disposed with both the oxygen atoms
pointing upward. A quantitative conformational analysis of
J. Chem. Soc., Perkin Trans. 1, 2002, 638–644
639

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Table 1 Reference geminal interproton distances in Å
resin (1% TFA in CH2Cl2 with immediate neutralization of
the effluents with pyridine) maintaining all protecting groups,
purified by flash chromatography on silica gel, cyclized in a
dilute solution of DMF (peptide concn. 0.5 mM) in the
presence of HBTU, HOBt and DIPEA and finally totally
deprotected with TFA–TIS–water (95 : 2.5 : 2.5), affording 11
in 35% yield over the last two steps. This compound was finally
tested for its capacity to inhibit selectively the αvβ3-mediated
endothelial cell adhesion. Fetal bovine aortic endothelial GM
7373 cells,26 that express α5β1 and αvβ3 integrins,27 were used
for the inhibition tests. Experiments performed with specific,
neutralizing anti-integrin antibodies had demonstrated that
αvβ3 integrin mediates adhesion and spreading of GM 7373
cells on immobilized vitronectin (VN) whereas α5β1 integrin is
involved in the interaction of these cells with immobilized
fibronectin (FN).27 Control cRADfV peptide is instead ineffec-
tive. On this basis, we evaluated the capacity of 11 to affect
the adhesion of GM 7373 cells to immobilized VN and FN.
Non-tissue culture plastic 96-well plates were incubated with
carbonate buffer containing 20 µg mlϪ1 VN (Fig. 4, open
Protons
MM/MD (1)
MM/MD (2)
NOE (1)
1Јa–1Јb*
5–6a
3a–5
3a–6a
2–3a
3a–4
6a–1b
6a–1a
2–1b
1Јa–1a
1Јb–1b
1.76
2.71
2.86
2.46
2.82
2.93
2.39
2.77
2.33
1.76
2.84
3.12
2.46
3.80
2.98
2.37
2.77
3.05
1.80
2.61
2.70
2.21
2.49
2.47
2.28
2.68
2.30
2.61
2.65
The coherence between the NMR and MD/MM data in com-
pound 1 indicates that the MMϩ forcefield is suitable to carry
out a reliable conformational analysis for these structures:
starting from this assumption the conformers corresponding to
the energy minima for the azido acid 2 were obtained by MD/
MM (Table 1). A conformer of 2 corresponding to an absolute
energy minimum was unambiguously found, presenting a
spatial disposition of the atoms constituting the bicycle dif-
ferent to that of 1. In Fig. 3 the minimum-energy conformers
found for compounds 1 and 2 are shown.
Fig. 4 Inhibition of integrin-dependent endothelial cell adhesion.
Fig. 3 Minimum-energy conformers of 1 and 2.
symbols) or FN (closed symbols). Then, GM 7373 cells were
seeded onto coated plates in the absence or in the presence of
30 µM cRGDfV or cRADfV (Fig. 4a) or of increasing concen-
trations of 11 (Fig. 4b). Cells were allowed to adhere for 2 h
at 37 ЊC. Then, adherent cells were fixed, stained, and plates
were read with a microplate reader at 595 nm. Data are the
mean S.D. of three determinations and are expressed as % of
cell adhesion observed in the absence of antagonist. In agree-
ment with its higher affinity for αvβ3 integrin, the cyclic
cRGDfV peptide inhibits more efficiently GM 7373 cell
adhesion to VN-coated plastic in respect to FN-coated sub-
stratum (Fig. 4a). Control cRADfV peptide is instead ineffec-
tive. As shown in Fig. 4b, 11 inhibits endothelial cell adhesion
to VN, exerting only a limited effect on FN-mediated cell
adhesion.
We envisaged the use of conformationally constrained SAA 1
in the construction of a cyclic peptide containing the Arg-Gly-
Asp sequence, with the aim of obtaining new inhibitors of the
adhesive interaction between the integrins and their receptor.22
Cyclic peptide 11 was synthesized on a solid phase (Scheme 2)
using the Fmoc–tBu strategy.
The dipeptide Arg(Pmc)-Gly was assembled on SASRIN®
resin with an average loading of about 0.5 mmol gϪ1, then
compound 1 was coupled in the presence of HBTU, HOBt
and DIPEA. The solid-phase reduction of the azide proved
to be problematic. Using PPh3 in THF–water mixture,23 the
phosphazene intermediate obtained was difficult to transform
quantitatively into the azide by treatment with acids or bases;
SnCl2 and PhSH in presence of Et3N24 led to cleavage of the
peptide from resin; dithiothreitol (DTT) in presence of diaza-
bicycloundecene (DBU) gave an extremely low reduction yield
even after several reiterations of the reaction; finally, trimethyl-
silyl chloride in dry CH2Cl2 cleaved the peptide from the resin.
We then explored the possibility of reducing the N-terminal
azide and simultaneously couple the Fmoc-Asp(tBu), in a one-
pot reaction, by using DIC, Bu3P, and HOBt in dry DMF–
toluene (2 : 1) at rt for 24 h.25 This reaction was found to be very
efficient and high yielding (about 95%, as assessed from the
loading value calculated after Fmoc removal from the aspartic
acid residue), in sharp contrast with the inefficiency of other
well-established methods we employed to reduce solid-phase-
bound azide. The reduction could be monitored conveniently
by the disappearance of the N3 stretch (2100 nm) in the IR
spectrum obtained from a few resin beads. After N-terminal
Fmoc deprotection, the tetrapeptide 9 was cleaved from the
Conclusions
Sugar azido acids 1 and 2 were synthetically derived from the
common precursor -arabinofuranose. Through molecular
dynamics and molecular modelling calculations a minimum-
energy conformation was unambiguously found for 1, that
showed interproton distances very similar to those derived from
NOE experiments in solution. The minimum-energy confor-
mation of 2 was also calculated; both 1 and 2 present a very
limited conformational mobility due to the bicyclic structure
that makes the corresponding amino acids interesting building
blocks for the construction of conformationally restricted
peptides. The amino acid derived from 1 was inserted in the
cyclic RGD peptide 11. We demonstrated in vitro that, like
cyclic cRGDfV peptide, 11 acts as selective antagonist of αvβ3
integrins expressed on GM 7373 cells.
640
J. Chem. Soc., Perkin Trans. 1, 2002, 638–644

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Scheme 2 Reagents and conditions (and yields): i) HBTU, HOBt, DIPEA, DMF, rt, 4 h; ii) Fmoc-Asp(tBu), DIC, Bu3P, HOBt in dry DMF–toluene
(2 : 1), rt (95%); iii) 20% piperidine in DMF; iv) 1% TFA in CH2Cl2, neutralization of the effluents with pyridine; v) cyclization: 0.5 mM peptide in
DMF, HBTU, HOBt, DIPEA, 72 h, rt (47%); vi) 95% TFA, 2.5% TIS, 2.5% water, 10 h, rt (75%).
tions are prepared as follows. Solution A: a 10% solution of
diisopropylethylamine (DIPEA) in dry DMF; solution B: a
Experimental
General procedures
1% solution of TNBS in dry DMF. A small portion of the resin,
previously washed with DMF, is suspended in a mixture of
equal amounts of the two solutions. After a few minutes the
presence of solid-phase-bound amino groups is detected by
the appearance of an intense red color on the beads.
Purification of peptides was performed by semi-preparative
reversed-phase HPLC on a Waters HPLC system (515 pumps)
equipped with a Merck LiChrosper 100 RP18 column (250 ×
10 mm; 10 µm), and elution with a flow rate of 7 ml min with
linear gradients of eluent A (0.1% TFA in water) and eluent B
(0.1% TFA in acetonitrile). Analytical HPLC purity checks
were performed using a Merck LiChrosper 100 RP18 column
(250 × 4 mm; 5 µm), elution at a flow rate of 1 ml minϪ1, using
gradients of A and B eluents. Detection at 214 and 280 nm
with an dual-wavelength absorbance detector Waters 2487.
Petroleum ether refers to the fraction with distillation range
40–60 ЊC.
All solvents were dried over molecular sieves (Fluka), for at
least 24 h prior to use. Thin-layer chromatography (TLC) was
performed on Silica Gel 60 F254 plates (Merck) with detection
with UV light when possible, or charring with a solution con-
taining conc. H2SO4–MeOH–H2O in the proportions 5 : 45 : 45.
Flash column chromatography was performed on silica gel
230–400 mesh (Merck). N α-Fmoc-protected amino acids were
purchased from Calbiochem-Novabiochem AG (Laufelfingen,
CH) with the following protecting groups for their side chains:
pentamethylchroman-6-ylsulfonyl (Pmc) for arginine; tert-butyl
(tBu) for aspartic acid. SASRIN® resin was purchased from
Bachem (CH). Solid-phase peptide synthesis was carried out
manually in plastic syringe with septum and Teflon stopcock on
the bottom. Usual work-up refers to dilution with an organic
solvent, washing with water to neutrality (pH test paper),
drying with Na2SO4, filtration, and evaporation under reduced
1
pressure. H and 13C NMR spectra were recorded on a Varian
Mercury 400 MHz spectrometer, using TMS as internal stand-
ard. All chemical shifts (δ) are quoted in ppm and coupling
constants (J) in Hz. The numbering used for the difuran NMR
assignments is as shown in structure 5 Scheme 1.
1-(2Ј,3Ј,5Ј-Tri-O-benzyl-D-arabinofuranosyl)prop-2-ene (4)
To
a stirred solution of 1-O-acetyl-2,3,5-tri-O-benzyl--
arabinofuranose (10.0 g, 21.6 mmol) in dry CH3CN (75 ml)
were added allyltrimethylsilane (6.9 ml, 43.2 mmol) and
BF3ؒEt2O (2.7 ml, 21.6 mmol) at rt under argon atmosphere.
After 1 h 30 min, solid Na2CO3 (5 g) was added to the solution
to neutralize the acid, the solvent was partly evaporated, and
the residue was diluted with CH2Cl2 (70 ml). The organic layer
was submitted to usual work-up and purified by column flash-
chromatography (9.5 : 0.5, petroleum ether–EtOAc) to afford
4 (7.2 g, 75%) as a colorless oil (mixture of α and β anomers;
α : β = 1 : 1, as determined from the integration ratio of the
1H-NMR signals). Anomers were not separable by chroma-
tography. MS(MALDI-TOF): m/z 468.7 [M ϩ H ϩ Na]ϩ, 484.5
IR spectra were recorded on a Biorad FT-IR spectrometer
FTS-40A coupled to an IR microscope Biorad UMA-40A.
[α]D-Values were measured at 20 ЊC on a Perkin-Elmer 241
digital polarimeter and are given in units of 10Ϫ1 deg cm2 gϪ1
.
Mass spectra were recorded on a MALDI 2 Kompakt Kratos
instrument, using gentisic acid (2,5-dihydroxybenzoic acid,
DHB) or α-cyano-4-hydroxycinnamic acid (CHCA) as
matrices. Elemental analyses were performed with a Perkin-
Elmer Series II Analyzer 2400.
Colorimetric assays for solid-phase peptide synthesis. TNBS
(2,4,6-trinitrobenzenesulfonic acid) test:28 two standard solu-
J. Chem. Soc., Perkin Trans. 1, 2002, 638–644
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[M ϩ H ϩ K]ϩ; elemental analysis Calc. (%) for C29H32O4: C,
(2R,4aS,5R,6R,6aS)- and (2S,4aS,5R,6R,6aR)-2-Azidomethyl-
78.35; H, 7.26. Found: C, 77.95; H, 6.48.
6-benzyloxy-5-(acetoxymethyl)hexahydrofuro[3,2-b]furan
[(R)-7] and [(S)-7]
(2R, 4aS, 5R, 6R, 6aR) and (2S, 4aS, 5R, 6R, 6aR)-2-Iodo-
methyl-6-benzyloxy-5-(benzyloxymethyl)-hexahydrofuro[3,2-b]-
furan (5)
A solution of (R)-6 or (S)-6 (480 mg, 1.2 mmol) in 4 : 1 Ac2O–
TFA (10 ml) was stirred at room temperature for 45 min. After
this time, the reaction was quenched by addition of a mixture
of ice and water and the product was extracted with AcOEt.
After usual work-up and column chromatography (8 : 2, petrol-
eum ether–AcOEt) pure compound (R)-7 or (S)-7 was obtained
as colorless oil (395 mg, 95% for both compounds).
To a solution of 4 (mixture of anomers, 0.436 g, 0.98 mmol)
in dry THF (25 ml) was added NIS (0.663 g, 2.95 mmol) at rt
under argon atmosphere. The reaction mixture was stirred at
this temperature for 6 days. After this time, TLC analysis of
the crude product (eluent 9.5 : 0.5, petroleum ether–AcOEt)
showed that a part of 4 did not react (α-anomer) and that two
distereomeric iodo derivatives were formed (two very close
TLC spots with Rf = 0.3). The crude reaction product was then
diluted with CH2Cl2 (25 ml), and the organic layer was sub-
jected to usual work-up. The oily residue was purified by
column chromatography (9.5 : 0.5, petroleum ether–AcOEt) to
give 5 (mixture of diastereomers) as a pale yellow oil (0.185 g,
80% calculated on the β-anomer of 4). The α-anomer of 4 was
recovered unchanged.
(R)-7. δH (400 MHz; CDCl3; 35 ЊC) 7.4–7.2 (5 H, m, Harom),
4.79 (1 H, br t, J6A,1 4.3, H-6a), 4.65 (1 H, m, H-3a), 4.60 (2 H,
ABq, CH2Ph), 4.29 (1 H, m, H-2), 4.25 (1 H, dd, J2ЈA,2ЈB 11.9,
J2ЈA,5 3.8, H-2Јa), 4.12 (1 H, dd, J2ЈB,2ЈA 11.9, J2ЈB,5 6.4, H-2Јb),
4.02 (1 H, m, H-5), 3.80 (1 H, br d, J4,3A 5.7, H-4), 3.52 (1 H, dd,
J1ЈA,1ЈB 13.1, J1ЈA,2 3.4, H-1Јa), 3.22 (1 H, dd, J1ЈB,1ЈA 13.1, J1ЈB,2
5.1, H-1Јb), 2.17 (1 H, m, H-1a), 2.11 (1 H, s, CH3), 1.78 (1 H,
m, H-1b); δC (300 MHz, CDCl3) 170.8, 88.38, 85.26, 83.64,
82.08, 77.81, 72.22, 64.18, 53.55, 35.68, 20.78; [α]2D0 ϩ28.0 (c 1,
CHCl3); MS (MALDI-TOF): m/z 371.3 [M ϩ H ϩ Na]ϩ, 387.6
[M ϩ H ϩ Kϩ]ϩ; C17H21N3O5 requires C, 58.78; H, 6.09; N,
12.10. Found: C, 59.11; H, 5.91; N, 11.87%.
(2R,4aS,5R,6R,6aR)- and (2S,4aS,5R,6R,6aR)-2-Azidomethyl-
6-benzyloxy-5-(benzyloxymethyl)hexahydrofuro[3,2-b]furan
[(R)-6)] and [(S)-6]
To a solution of 5 (mixture of diastereomers, 1.5 g, 3.15 mmol)
was added tetrabutylammonium azide‡ (1.8 g, 6.3 mmol) under
argon atmosphere and the mixture was stirred at 70 ЊC for 24 h.
The solvent was then evaporated in vacuo, the residue was
diluted with AcOEt, and after usual work-up and column
chromatography (8 : 2, petroleum ether–AcOEt) the two
diastereomers (R)-6 and (S)-6 were separated and isolated as
colorless oils [500 mg of (R)-6, 40%; 250 mg of (S)-6, 20%].
(S)-7. δH (400 MHz; CDCl3; 35 ЊC) 7.4–7.2 (5 H, m, Harom),
4.73 (1 H, br t, J6A,1 4.9, H-6a), 4.60–4.50 (3 H, m, H-3a,
CH2Ph), 4.25 (3 H, m, H-2, H-2Јa, H-2Јb), 4.04 (1 H, m, H-5),
3.91 (1 H, br d, J 5.6, H-4), 3.48 (1 H, dd, J1ЈA,1ЈB 12.8, J1ЈA,2 8.0,
H-1Јa), 3.23 (1 H, dd, J1ЈB,1ЈA 12.8, J1ЈB,2 4.0, H-1Јb), 2.22 (1 H m,
H-1a), 2.09 (1 H, s, CH3), 1.91 (1 H, m, H-1b); δC (300 MHz,
CDCl3) 185.5, 89.67, 85.00, 83.90, 83.57, 80.72, 72.69, 64.15,
55.13, 35.73, 20.84; [α]2D0 ϩ48.9 (c 1, CHCl3); MS (MALDI-
TOF): m/z 371.6 [M ϩ H ϩ Na]ϩ, 387.8 [M ϩ H ϩ K]ϩ;
C17H21N3O5 requires C, 58.78; H, 6.09; N, 12.10. Found: C,
58.51; H, 6.31; N, 11.77%.
(R)-6. δH (400 MHz; CDCl3; 35 ЊC) 7.4–7.2 (10 H, m, Harom),
4.79 (1 H, br t, J6A,1 5.2, H-6a), 4.71 (1 H, m, H-3a), 4.7–4.5
(4 H, 2ABq, CH2Ph), 4.29 (1 H, m, H-2), 4.03 (1 H, ddd, J 6.2,
J 6.2, J 3.8, H-5), 3.86 (1 H, dd, J 6.2, J 1.2, H-4), 3.61 (1 H, dd,
J 10.5, J 3.8, H-2Јa), 3.59 (1 H, dd, J2ЈB,2ЈA 10.5, J2ЈB,5 6.2, H-2Јb),
3.52 (1 H, dd, J1ЈA,1ЈB 13.0, J1ЈA,2 3.5, H-1Јa), 3.24 (1 H, dd, J1ЈB,1ЈA
13.0, J1ЈB,2 3.5, H-1Јb), 2.19 (1 H, dd, J1A,1B 13.4, J1A,6A 5.2,
H-1a), 1.78 (1 H, ddd, J1B,1A 13.4, J1B,2 10.2, J1B,6A 6.3, H-1b);
δC (300 MHz, CDCl3) 88.72, 85.60, 83.53, 83.35, 77.70, 73.41,
72.10, 70.18, 53.60, 35.82; [α]2D0 ϩ21.4 (c 1, CHCl3); MS
(MALDI-TOF): m/z 419.2 [M ϩ H ϩ Na]ϩ, 435.0 [M ϩ H ϩ
K]ϩ; C22H25N3O4 requires C, 66.82; H, 6.37; N, 10.63. Found:
C, 67.53; H, 7.01; N, 11.21%.
(2R,4aS,5R,6R,6aS)- and (2S,4aS,5R,6R,6aR)-2-Azidomethyl-
6-benzyloxy-5-(hydroxymethyl)hexahydrofuro[3,2-b]furan
[(R)-8] and [(S)-8]
To a solution of (R)-7 or (S)-7 (1 g, 2.88 mmol) in dry MeOH
(50 ml) was added sodium metal in catalytic amount under
argon atmosphere. The solution was stirred at room tem-
perature for 30 min. After this time Amberlite IRA-120 (Hϩ)
resin was added and the mixture was vigorously stirred for
10 min. Then resin was removed by filtration and the solvent
was evaporated. After usual work-up and chromatography
(1 : 1, petroleum ether–AcOEt), pure compound (R)-8 or
(S)-8 was recovered as colorless oil (660 mg, 75% for both
compounds).
(S)-6. δH (400 MHz; CDCl3; 35 ЊC) 7.4–7.2 (10 H, m, ArH ),
4.72 (1 H, br t, J6A,1 4.4, H-6a), 4.65–4.45 (5 H, m, 2 × CH2Ph
and H-3a), 4.24 (1 H, m, H-2), 4.03 (1 H, ddd, J5,4 6.0, J5,6 3.7,
H-5), 3.94 (1 H, br d, J4,5 6.0, H-4), 3.65 (1 H, dd, J2ЈA,2ЈB 10.3,
J2ЈA,5 3.0, H-2Јa), 3.58 (1 H, dd, J2ЈB,2ЈA 10.3, J2ЈB,5 5.9, H-2Јb),
3.48 (1 H, dd, J1ЈA,1ЈB 12.5, J1ЈA,2 8.1, H-1Јa), 3.16 (1 H, dd, J1ЈB,1ЈA
12.5, J1ЈB,2 4.0, H-1Јb), 2.20 (1 H, ddd, J1A,1B 14.1, J1A,2 8.4, J1A,6A
5.6, H-1a), 1.91 (1 H, m, H-1b); δC (300 MHz, CDCl3) 90.00,
85.40, 85.03, 83.63, 80.23, 73.64, 72.57, 70.00, 55.28, 35.80;
[α]2D0 ϩ29.3 (c 1, CHCl3); MS (MALDI-TOF): m/z 396.4 [M ϩ
H]ϩ, 419.6 [M ϩ H ϩ Na]ϩ, 435.3 [M ϩ H ϩ K]ϩ; C22H25N3O4
requires C, 66.82; H, 6.37; N, 10.63. Found: C, 66.51; H, 6.81;
N, 11.01%.
(R)-8. δH (400 MHz; CDCl3; 35 ЊC) 7.4–7.2 (5H, m, Harom),
4.78 (1 H, br t, J6A,1 4.3, H-6a), 4.65 (1 H, dd, J3A,4 4.3, H-3a),
4.62 (2 H, ABq, CH2Ph), 4.29 (1 H, m, H-2), 3.90 (2 H, m, H-4,
H-5), 3.81 (1 H, dd, J2ЈA,2ЈB 11.4, J2ЈA,5 2.3, H-2Јa), 3.63 (1 H, dd,
J2ЈB,2ЈA 11.4, J2ЈB,5 3.4, H-2Јb), 3.51 (1 H, dd, J1ЈA,1ЈB 13.2, J1ЈA,2
3.7, H-1Јa), 3.22 (1 H, dd, J1ЈB,1ЈA 13.2, J1ЈB,2 5.0, H-1Јb), 2.14
(1 H, dd, J1A,1B 13.6, J1A,2 5.5, H-1a), 1.94 (1 H, m, H-1b);
δC (300 MHz, CDCl3) 88.84, 84.62, 84.58, 83.35, 77.80, 72.27,
62.67, 53.61, 35.76; [α]2D0 ϩ21.9 (c 1, CHCl3); MS (MALDI-
TOF): m/z 328.2 [M ϩ Na]ϩ, 344.1 [M ϩ K]ϩ; C15H19N3O4
requires C, 59.01; H, 6.27; N, 13.76. Found: C, 57.11; H, 6.79;
N, 13.23%.
‡ Tetrabutylammonium azide was prepared from sodium azide accord-
ing to the following procedure: to a stirred solution azide (228 mg,
3.5 mmol) in water (2 ml) was added Bu4NOH (9.2 ml of a 20%
aqueous solution) and the solution was stirred at rt for 30 min. After
this time, the aqueous solution was extracted with CH2Cl2 (10 ml × 3),
the organic layer was dried (Na2SO4), and the solvent removed in vacuo
by adding toluene, until a solid powder is obtained. CAUTION:
Bu4NN3 became explosive when solution was evaporated to dryness
and the solid residue was heated.
(S)-8. δH (400 MHz; CDCl3; 35 ЊC) 7.4–7.2 (5 H, m, Harom),
4.72 (1 H, bt, J6A,1 4.2, H-6a), 4.60 (2 H, ABq, CH2Bn), 4.48
(1 H, d, J3A,4 4.2, H-3a), 4.22 (1 H, m, H-2), 3.98 (2 H, m, H-4,
H-5), 3.82 (1 H, dd, J2ЈA,2ЈB 11.8, J2ЈA,5 2.7, H-2Јa), 3.68 (1 H, dd,
642
J. Chem. Soc., Perkin Trans. 1, 2002, 638–644

View Article Online
J2ЈB,2ЈA 11.8, J2ЈB,5 4.4, H-2Јb), 3.46 (1 H, dd, J1ЈA,1ЈB 12.9, J1ЈA,2
6.7, H-1Јa), 3.33 (1 H, dd, J1ЈB,1ЈA 12.9, J1ЈB,2 4.1, H-1Јb), 2.25
(1 H, ddd, J1A,1B 14.2, J 8.2, J 6.0, H-1a), 1.94 (1 H, m, H-1b);
δC (300 MHz, CDCl3) 89.83, 86.67, 84.40, 83.73, 79.91, 72.63,
62.95, 54.97, 35.61; [α]2D0 ϩ47.03 (c 1, CHCl3); MS (MALDI-
TOF): m/z 328.6 [M ϩ Na]ϩ, 344.4 [M ϩ K]ϩ. C15H19N3O4
requires: C, 59.01%; H, 6.27%; N, 13.76%. Found: C, 58.45%;
H, 5.99%; N, 14.01%.
stirred 10 min at this temperature. Then a solution of tributyl-
phosphine in dry toluene (1.7 ml of a 0.6 mM solution) and the
resin (0.5.g) were added and the suspension was allowed to
warm to rt. and shaken under argon atmosphere for 24 h. The
resin was rinsed with DMF and CH2Cl2 and, after N-terminal
Fmoc removal, treated with 1% TFA in CH2Cl2 (3 × 10 min).
Effluents containing peptide were neutralized at 0 ЊC with
2% pyridine in CH2Cl2. Solvents were evaporated in vacuo, the
residue was diluted with CH2Cl2 (10 ml), and the organic
layer was washed with water to partly remove pyridinium salts,
dried (Na2SO4), and solvent was evaporated. TLC analysis of
the crude revealed the presence of protected peptide (eluent
CHCl3–EtOH–AcOH 7 : 3 : 0.2, Rf = 3.5) which was purified by
column chromatography (CHCl3–EtOH–AcOH 7 : 3 : 0.2) and
obtained as a colorless oil (120 mg, 0.13 mmol, 56% based
on the loading of the resin). Peptide 9: MS (MALDI-TOF):
m/z 945.8 [M ϩ H]ϩ, 968.2 [M ϩ Na]ϩ, 983.7 [M ϩ K]ϩ.
A solution of peptide 9 (100 mg, 0.11 mmol), HBTU (58 mg,
0.15 mmol), HOBt (21 mg, 0.16 mmol) and DIPEA (36 µl,
0.21 mmol) in dry DMF (212 ml, concn. peptide = 0.5 mM) was
stirred for 72 h at rt. Solvent was evaporated in vacuo and the
crude residue was purified by RP-HPLC. After lyophilization
peptide 10 was recovered as a colorless oil (48 mg, 47%).
Peptide 10: MS (MALDI-TOF): m/z 927.8 [M ϩ H]ϩ, 950.2
[M ϩ Na]ϩ, 965.7 [M ϩ K]ϩ.
(2R,4aS,5S,6S,6aR)- and (2S,4aS,5S,6S,6aR)-2-Azidomethyl-
6-benzyloxy-5-carboxyhexahydrofuro[3,2-b]furan (2) and (1)
To a solution of (R)-8 or (S)-8 (320 mg, 1.1 mmol) in acetone
(20 ml) was added dropwise a solution of CrO3 (550 mg,
5.5 mmol) in 3 M H2SO4 (13 ml) over a period of 10 min at 0 ЊC,
the reaction mixture was then allowed to warm at room tem-
perature and stirred for 3 h. The acetone was then evaporated
in vacuo from the mixture and the product was extracted with
AcOEt. The organic layer was washed twice with 0.1 M HCl,
and after usual work-up and chromatography (9.5 : 0.45 : 0.05,
CHCl3–EtOH–AcOH), pure compound 1 or 2 was obtained as
a colorless oil.
2. δH (400 MHz; CDCl3; 35 ЊC) 7.4–7.2 (5 H, m, Harom), 4.93
(1 H, m, H-6a), 4.69 (2 H, ABq, CH2Ph), 4.66 (1 H, m, H-5),
4.47 (1 H, br d, J 2.0, H-4), 4.39 (1 H, d, J3A,4 3.3, H-3a), 4.18
(2 H, m, H-2), 3.58 (1 H, dd, J1ЈA,1ЈB 12.8, J1ЈA,2 7.4, H-1Јa), 3.33
(1 H, dd, J1ЈB,1ЈA 12.8, J1ЈB,2 3.7, H-1Јb), 2.35 (1 H, m, H-1a), 2.12
(1 H, m, H-1b); δC (300 MHz, CDCl3) 174.34, 87.39, 85.88,
85.14, 83.24, 79.42, 72.20, 54.41, 36.04; [α]2D0 Ϫ8.90 (c 1, CHCl3);
MS (MALDI-TOF): m/z 342.4 [M ϩ Na]ϩ, 358.4 [M ϩ K]ϩ;
C15H17N3O5 requires C, 56.42; H, 5.37; N, 13.16. Found: C,
57.02; H, 5.67; N, 13.70%.
Peptide 10 (30 mg, 0.03 mmol) was dissolved in TFA–TIS–
water (95 : 2.5 : 2.5 by vol; 1 ml) and the solution was stirred for
10 h at rt. Solvent was evaporated in vacuo and the crude reidue
was purified by RP-HPLC. After lyophilization, peptide 11
was recovered as a white powder (13 mg, 75%). Peptide 11:
m/z 604.8 [M]ϩ, 627.2 [M ϩ Na]ϩ, 643.7 [M ϩ K]ϩ.
NMR Spectroscopy
1H and 13C NMR spectra were recorded with TMS as internal
standard. The signals of the aromatic carbons in the 13C NMR
spectra are not reported. Diluted and degassed d-chloroform
solutions of compounds (concentrations around 30–40 mM)
were examined either on a Bruker AC-300 or an Avance-400
spectrometer. In order to select the best NMR method to
analyze the conformation in solution of bicycle 1, preliminary
measurements were performed on a similar bicyclic compound.
1. δH (400 MHz; CDCl3; 35 ЊC) 7.4–7.2 (5 H, m, Harom), 4.97
(1 H, m, H-6a), 4.67 (2 H, ABq, CH2Ph), 4.61 (1 H, m, H-5),
4.47 (1 H, br d, J 2.9, H-4), 4.42 (1 H, m, H-2), 4.39 (1 H, m,
H-3a), 3.51 (1 H, dd, J1ЈA,1ЈB 13.2, J1ЈA,2 2.4, H-1Јa), 3.33 (1 H,
dd, J1ЈB,1ЈA 13.2, J1ЈB,2 3.9, H-1Јb), 2.38 (1 H, m, H-1a), 1.96 (1 H,
m, H-1b); δC (300 MHz, CDCl3) 175.21, 87.95, 85.43, 84.78,
83.32, 79.91, 71.65, 53.81, 34.64; [α]2D0 Ϫ1.76 (c 1, CHCl3);
MS (MALDI-TOF): m/z 342.4 [M ϩ Na]ϩ, 358.4 [M ϩ K]ϩ;
C15H17N3O5 requires C, 56.42; H, 5.37; N, 13.16. Found: C,
56.02; H, 5.91; N, 12.78%.
A
first set of interproton distances was derived from
NOE-difference-1D experiments,29 repeated at different values
of NOE generation time (distances were evaluated in the two-
spin approximation30 or, when possible, with the NOE ratio
method).31 A second set of measurement was obtained by a
series (4 or 5) of 2D-NOESY experiments with different
mixing-time-values,32 between 0.4 and 1.3 seconds, to cover the
full range of molecular relaxation times in the two-spin
approximation. Examination of curves of cross-peaks volumes
vs. NOESY mixing times allowed us to match maximum NOE
value for each nucleus and to calculate distances with the well-
Solid-phase peptide synthesis
SASRIN® resin (0.5 g) was swollen in CH2Cl2 (4 ml) for 30
min, then treated with a solution of FmocGlyOH (420 mg,
0.7 mmol), DIC (216 µl, 0.7 mmol), and HOBt (189 mg,
0.7 mmol), and DMAP (11 mg, 0.05 mmol) in dry DMF
(2.5 ml) for 12 h at rt. After this time, the resin was washed
several times with DMF (5 ml) and CH2Cl2 (5 ml), and capped
with Ac2O–pyridine–DMF (2 : 4 : 94 by vol) in the presence
of catalytic DMAP during 2 h. The resin was then rinsed with
DMF and CH2Cl2 and treated with piperidine–DMF (1 : 4 by
vol; 10, 10 and 5 min). Loading (0.47 mmol gϪ1) was calculated
by detecting UV absorbance of the effluents at 290 nm. The
resin was then treated with a solution of FmocArg(Pmc)OH
(311 mg, 0.47 mmol), HBTU (170 mg, 0.45 mmol), and DIPEA
(160 µl, 0.94 mmol) in DMF (3 ml) for 2 h at, rt. The coupling
was repeated until the TNBS test was negative. After washing
and Fmoc deprotection, the resin was treated with azido acid
1 (220 mg, 0.7 mmol), HBTU (246 mg, 0.65 mmol), HOBt
(95 mg, 0.7 mmol), and DIPEA (240 µl, 1.4 mmol) in DMF–
CH2Cl2 (2 : 1 by vol; 3 ml) for 4 h at, rt. The resin was washed
with DMF and CH2Cl2 and submitted to one-pot azide reduc-
tion and aspartic acid coupling reaction. To a solution of
FmocAsp(tBu)OH (189 mg, 0.46 mmol) and HOBt (62 mg,
0.46 mmol) in dry DMF (3 ml) was added DIC (72 µl, 0.46
mmol) at 0 ЊC under argon atmosphere and the solution was
known relationship between NOE enhancement and rϪ6 33
.
Finally, an ROESY experiment34 was used, when necessary, to
confirm uncertainties in NOE attributions. The geminal con-
stant interproton distance (1.8 Å) between non-equivalent
nuclei was taken as reference31 in distance calculations either
for 1D or for 2D experiments. Results derived from NOESY
experiments were more regular, numerous and reliable in fitting
with our MD simulation and so, representing the best reference,
have been used to analyze conformations of 1 (five different
experiments with mixing times between 0.6 and 1.4 s).
Molecular dynamics
The potential-energy surface has been modelled employing
the MMϩ forcefield,21 according to the following strategy.
Molecular geometries were optimized by MM and then sub-
jected to up to 10 cycles of simulated annealing MD. In particu-
lar, the temperature was raised from 50 up to 700 K in 10 ps and
J. Chem. Soc., Perkin Trans. 1, 2002, 638–644
643

View Article Online
7 R. Hirschmann, L. Ducry and A. B. Smith, J. Org. Chem., 2000, 65,
8307.
8 (a) E. F. Fuchs and J. Lehmann, Chem. Ber., 1975, 108, 2254;
(b) E. F. Fuchs and J. Lehmann, Carbohydr. Res., 1975, 45,
135; (c) E. F. Fuchs and J. Lehmann, Carbohydr. Res., 1976, 49,
267; (d ) K. C. Nicolaou, H. M. Florke, G. Egan, T. Barth and
V. A. Estevez, Tetrahedron Lett., 1995, 36, 1775.
9 (a) T. K. Chakraborty, S. Ghosh, S. Jayaprakash, J. A. R. P. Sarna,
V. Ravikanth, P. V. Diwan, R. Nagaraj and A. C. Kunwar, J. Org.
Chem., 2000, 65, 7710; (b) T. K. Chakraborty, S. Ghosh, M. H. V. R.
Rao, A. C. Kunwar, H. Cho and A. K. Ghosh, Tetrahedron Lett.,
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then lowered to 20 K in 20 ps. The temperature was raised to
high values (700 K) to allow possible conformational transi-
tions in the sterically constrained molecules 1 and 2.
Cell cultures
Fetal bovine aortic endothelial GM 7373 cells were obtained
from the NIGMS Human Genetic Mutant Cell Repository
(Institute for Medical Research, Camden, NJ). They corre-
spond to the BFA-1c multilayered transformed clone described
by Grinspan et al.26 GM 7373 cells were grown in Eagle’s
minimal essential medium (MEM) containing 10% fetal calf
serum (FCS), vitamins, and essential and non-essential amino
acids.
Cell-adhesion assay
100 µl aliquots of 100 mM NaHCO3, pH 9.6 (carbonate buffer),
containing 20 µg mlϪ1 of the adhesive molecule under test, were
added to polystyrene non-tissue-culture microtiter plates. After
16 h of incubation at 4 ЊC the solution was removed and wells
were washed three times with cold phosphate-buffered saline
(PBS). For the cell-adhesion assay, confluent cultures of GM
7373 cells were trypsinized, washed, and resuspended with the
appropriate medium. 50,000 GM 7373 cells were resuspended
in 200 µl of medium/1% FCS and were immediately seeded
onto coated wells. Routinely, cell adhesion was allowed to occur
for 2 h at 37 ЊC. Then, wells were washed with 2 mM EDTA
in PBS. Adherent cells were fixed in 3.7% paraformaldehyde–
0.1 M sucrose in PBS, washed with PBS, and stained with
Methylene Blue–Azur II (1 : 1 by vol). Plates were read with a
microplate reader at 595 nm.
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Acknowledgements
This work was supported in part by CNR (Progetto Finalizzato
Biotecnologie), MURST (Cofin 2000), Associazione Italiana
per la Ricerca sul Cancro, and Istituto Superiore di Sanità
(AIDS Project) to M. P.
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