Synthesis and conformational studies of a hybrid cyclic peptide based on cis-β-furanoid sugar amino acid (FSAA) and ornithine

Article abstract of DOI:10.1002/hlca.200890138

The conformational control of a 14-helix nucleating template, cis-β-furanoid sugar amino acid (FSAA), over a flexible δ-amino acid, ornithine is studied in a FSAA-ornithine cyclic tetrapeptide. Extensive NMR and MD studies reveal that the cyclic peptide adopts a three-dimentional bowl-shape cavity, which promotes six- and seven-membered intra- and inter-residue H-bonding, in polar and nonpolar solvents, respectively.

Full text of DOI:10.1002/hlca.200890138

Helvetica Chimica Acta – Vol. 91 (2008)
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Synthesis and Conformational Studies of a Hybrid Cyclic Peptide Based on
cis-b-Furanoid Sugar Amino Acid (FSAA) and Ornithine
by Srivari Chandrasekhar*a), Birudaraju Sarithaa), Police Nareshb), Marelli Udayakiranb),
Chada Raji Reddya), and Bharatam Jagadeesh*b)
a) Organic Division-I, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India
b) Centre for Nuclear Magnetic Resonance, Indian Institute of Chemical Technology, Tarnaka,
Hyderabad 500007, India (fax: (þ)91-40-27160512; e-mail: srivaric@iict.res.in and bj@iict.res.in)
The conformational control of a 14-helix nucleating template, cis-b-furanoid sugar amino acid
(FSAA), over a flexible d-amino acid, ornithine is studied in a FSAA-ornithine cyclic tetrapeptide.
Extensive NMR and MD studies reveal that the cyclic peptide adopts a three-dimentional bowl-shape
cavity, which promotes six- and seven-membered intra- and inter-residue H-bonding, in polar and non-
polar solvents, respectively.
Introduction. – Amongst the unnatural oligopeptides, b-peptides that exhibit well-
defined secondary folding (ꢀfoldamersꢁ) [1] have gained immense scientific interest in
recent years. The interests are mainly due to their ability to form stable secondary
structural scaffolds with a relatively small number of b-amino acid residues compared
to the natural a-peptides, and due to their potential applications in drug delivery. In this
regard, sugar amino acids (SAA) have a special appeal as they serve as bridge between
carbohydrates and proteins [2]. Recently, we have reported that homooligomers of a
novel peptide building block, cis-b-furanoid sugar amino acid (FSAA) [3], adopt
robust right handed 14-helices. Subsequently, we have shown that the conformational
space (represented by dihedral angles) of the FSAA can be expanded to a more flexible
residue b-Ala in linear [4] and cyclic [5] heterooligomers consisting of FSAA and b-Ala
residues at alternate positions. These linear and cyclic heteropeptides also exhibited
robust 14-helical folding [4] and C2-symmetric b-sheet-like peptide rings, which self-
assembled to form tubular structures [5], respectively, suggesting that FSAA exerts
conformational control of the peptide backbone [6] and diverse secondary structures
can be derived.
As a part of our ongoing research activity on SAA based molecular design, we are
interested in developing cyclic hybrid peptides that can accommodate various
combinations of functional side chains. In fact, the cyclization of linear peptides is a
widely accepted strategy to constrain the conformational freedom of the backbone and
to gain improved accessibility for molecular recognition, a process essential for
biological activity [7]. The present work aims in this direction and reports the synthesis
and conformation of a cyclic hybrid tetrapeptide consisting of an alternating FSAA and
ornithine (equivalent of d-amino acid), 1. This example is also expected to provide
insight into the conformational control exerted by the rigid FSAA over a more flexible
and longer spacer ornithine, compared to b-Ala in a heterocyclic peptide [5]. To the
ꢂ 2008 Verlag Helvetica Chimica Acta AG, Zürich

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best of our knowledge, this is the first synthesis of a C2-symmetric cyclic peptide
with alternating SAA and d-amino acid residues. Detailed NMR, ESI-MS, FT-IR,
and restrained MD simulations techniques were employed to characterize the peptide
1.
Results and Discussion. – The monomer of FSAA, 3, was synthesized from the
known azido sugar derivative 2 [8], which was used to prepare the dimer 5 by coupling
with Na-Boc-Nd-Z-l-ornithine using the standard solution-phase coupling method
using EDCI and HOBt as coupling agents in dry CH2Cl2. Initially, the free amine of
FSAA 3 was coupled with the carboxylic acid group of the protected ornithine 4, which
afforded the protected dimer 5 in 85% yield (Scheme 1). The ester group of compound
2 was hydrolyzed with LiOH in THF/H2O to afford the acid 6. The benzyloxycarbonyl
group of 5 was deprotected under hydrogenation reaction conditions to obtain the
amine 7, which was coupled with the dimer acid 6 under EDCI/HOBt coupling
conditions to give tetramer 8 in 81% yield. After hydrolysis of the Me ester group in
compound 8, the obtained acid was esterified with pentafluoro phenol to afford 9 in
76% yield. Compound 9 was cyclized under standard hydrogenation conditions to get
the desired 20-membered cyclic peptide 1 in 71% yield (Scheme 2).
Scheme 1. Synthesis of 5
a) Pd/C, H2, AcOEt, r.t., 1 h, 95%. b) EDCI, HOBt, CH2Cl2; 08 to r.t., 24 h, 85%.
Preliminary molecular mechanics calculations carried out on the 20-membered
backbone of 1 by using the conformational space search method [9] showed that FSAA
maintains gauche-conformation [3] around CaÀCb, similar to that observed for 14-
helical folds. Information on the preferred conformation of 1 (4 mm) was obtained in
both non-polar and polar solvents (CDCl3, (D6)DMSO) by carrying out 1D- and 2D-

Helvetica Chimica Acta – Vol. 91 (2008)
Scheme 2. Synthesis of 1
1269
a) LiOH, THF/H2O 3 :1, 08 to r.t., 2 h. b) Pd/C, H2, AcOEt, r.t. c) EDCI, HOBt, CH2Cl2, 08 to r.t., 24 h,
81%. d) C6F5OH, EDCI, CH2Cl2, 08 to r.t., 18 h, 76%.
(gDQCOSY, TOCSY, and ROESY) NMR studies. The 1D spectra in both solvents
revealed a single set of resonances, at low (253 – 278 K, CDCl3) and high (300 – 323 K)
temperatures, and were consistent with the C2 symmetry of the molecule. The complete
resonance assignments were accomplished by using a combination of 1D-NMR (1H),
COSY, TOCSY, and ROESY data. The ROESY spectra were recorded at different
mixing times (tmix ¼ 180 to 400 ms). The data was found to be consistent over the range,
and the analysis of the data for tmix ¼ 300 ms is discussed here. The chemical shifts of
amide H-atoms were found to be constant as a function of the sample dilution from
10 mm to ca. 0.5 mm, thereby confirming the absence of aggregation. ESI-MS data
([1 þ H]þ, [1 þ Na]þ), also support this possibility with the values 799 and 821 Da,
respectively, corresponding to the monomeric cyclic tetramer 1, and signals corre-
sponding to aggregats were absent.
3
Conformational Analysis of Compound 1 in CDCl3. The value of J(HCa,HCb) of
FSAA is observed to be ca. 5.0 Hz, which is a clear sign for a gauche conformation
around the CaÀCb bond [3]. The other 3J couplings along the backbone J(HCb,NH) ¼
6.0 and J(HCb,CaH) ¼ 5.0 Hz were obtained by selective homonuclear decoupling
(Table 1).
The observed coupling J(NH,CbH) ¼ 6 Hz, and the strong NOEs NH/CaH and NH/
CbH of FSAA suggest a predominantly gauche conformation around NHÀCbH of
FSAA as well (Figs. 1 and 2). Absence of an NHOrn/NHFSAA NOE and the presence of
medium range NOEs, NHOrn/CaHFSAA and NHOrn/CdHFSAA further indicate that the two
NH groups (Orn and FSAA) are anti (phasing in opposite direction). These findings
collectively infer a cyclic backbone conformation of 1 in CDCl3, with the CO groups of
Orn pointing towards the inner cavity, while the CO groups of FSAA being in opposite

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Table 1. 1H-NMR Chemical Shifts (d, in ppm) and Coupling Constants (J, in Hz) of 1 in CDCl3
Residue
Sugar
Ornithine
NH
CaH
CbH
Cb1H
CgH
Cg1H
CdH
Cd1H
BocNH
8.03 (d, J ¼ 6.0)
7.09 (t, J ¼ 6.1)
4.67( m)
1.7 m)
1.26 (m)
1.61 (m)
1.36 (m)
3.88 (m)
2.99 (m)
5.22 (d, J ¼ 8.1)
4.81 (d, J ¼ 5.0)
4.06 (dd, J ¼ 5.0, 6.0)a)
2 (
–
5.03 (d, J ¼ 3.1)
–
6.31 (d, J ¼ 3.1)
–
–
a) The coupling constants were obtained by selective homonuclear decoupling.
Fig. 1. Schematic representation of observed
NOEs of 1 in CDCl3. The solid and dotted
arrows represent strong and weak NOEs, resp.
Fig. 2. Selected NOESY regions for 1 in CDCl3. a) Intra-residue NOEs in FSAA involving the NHFSAA
H-atom. b) Inter-residue NOEs involving the CbH and CaH of FSAA, and NHOrn H-atoms. c) In contrast
to the data in DMSO, inter-residue NH/NH NOEs are not observed.
direction. This conformation brings the NHFSAA to a proximity to COBoc, an
arrangement that can favor seven-membered inter-residue H-bonding. The downfield

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shift of amide H-atoms of FSAA (8.03 ppm) supports the possibility of its participation
in H-bonding [3].
In order to assess the involvement of amide H-atoms in H-bonding, DMSO titration
studies were carried out by sequentially adding DMSO (up to 33% v/v) to the CDCl3
solution. The variations of NH chemical shifts over the range of titration are observed
to be À 0.4, 1.0, and 1.6 ppm for NHFSAA, NHOrn, and NHBoc, respectively, which imply
that NHFSAA is involved in H-bonding (possibly inter-residue H-bond), while the NHOrn
and NHBoc seem to be solvent-exposed.
Conformational Analysis of Compound 1 in DMSO. The CbH of FSAA shows a
3
resolved doublet of doublet with J(CbH,NH) ¼ 7.5, J(CbH,CaH) ¼ 4.5 Hz (Table 2),
which suggests that the gauche conformation is retained around the CaÀCb bond of
FSAA, while the NHÀCbH bond strongly deviates from gauche conformation, contrary
to the observation in CDCl3.
Table 2. 1H-NMR Chemical Shifts (d, in ppm) and Coupling Constants (J, in Hz) of Compound 1 in
(D6)DMSO
Residue
Sugar
Ornithine
NH
CaH
CbH
Cb1H
CgH
Cg1H
CdH
Cd1H
BocNH
7.32 (d, J ¼ 7.5)
8.11 (t, J ¼ 5.4)
3.78 (m)
1.44 (m)
1.44 (m)
1.44 (m)
1.44 (m)
3.04 (m)
3.04 (m)
7.08 (d, J ¼ 6.3)
4.61 (d, J ¼ 4.5)
4.25 (dd, J ¼ 4.5, 7.5)
–
4.43 (d, J ¼ 3.9)
–
5.88 (d, J ¼ 3.9)
–
–
Strong NOEs, (NH/CgH)FSAA, (NH/CdH)FSAA, and a weak NOE, (NH/CbH)FSAA
imply that NHFSAA and CbHFSAA are in trans conformation. On the other hand, a strong
NOE between NHOrn and CaHFSAA indicates that these two H-atoms are syn to each
other. This flip in the orientation of NHOrn and NHFSAA is reflected in their
corresponding dihedral angles (F). The temperature dependence of the NHFSAA
chemical shifts showed a small coefficient (Dd/DT) of À 4.0 ppb/K compared to that
of NHOrn and NHBoc (À6.5 ppb/K, and À 10.5 ppb/K, resp.). This temperature
coefficient for NHFSAA and the corresponding NOEs (Figs. 3 and 4) suggest a
possibility of six-membered (NHÀCO)FSAA H-bonding (intra-residue).
The involvement of NH groups in H-bonding is also further corroborated by FT-IR
studies, which show a characteristic broad NH-stretching band around 3297cm À1 and
C¼O stretching peaks around 1670 cmÀ1 [10].
Restrained Molecular Dynamics. The distance restraints used in molecular dynamics
were derived from the ROESY cross-peak intensities, which are volume-integrated and
normalized with respect to the CH2 H-atoms (1.8 Š) or FSAA-g- and -d-H-atoms
(2.43 Š) (Table 3).
The restrained MD calculations were carried out following a simulated annealing
protocol [11]. However, ambiguity in ornithine residue assignments has limited us to
use only eight NOEs for restrained molecular dynamics. Dielectric constants of 4.7and

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Fig. 3. Schematic representation of observed
NOEs of 1 in DMSO. The solid and dotted
arrows represent strong and weak NOEs,
resp.
Fig. 4. Selected NOESY regions for 1 in DMSO. a) Intra-residue NOE in FSAA involving the NHFSAA H-
atom. b) Inter-residue NOEs involving the CbH and CaH of FSAA, and NHOrn H-atoms. c) Inter-residue
NH/NH NOEs.
47.0 were used for CDCl and DMSO, respectively. Initially, the molecule was heated up
3
to 500 K and gradually cooled down to 300 K in two steps, for a 1 ns time period. At
300 K, a prolonged dynamics (5 ns) was run. By collecting a snapshot for every 50000
history files, 100 structures were obtained, which were energy minimized by using the
conjugate method. Among these structures, the lowest energy structures were
subjected to restrained (distances and torsion angles) MD studies.
During the MD trajectory over a period of 500000 fs, 100 structures were collected
by taking a snapshot for every 5000 fs. Superposition of these structures showed a good
convergence (Fig. 5), where at least the backbone is well-defined, suggesting a
predominant single conformation for 1 in both solvents during the timescale of
observation.
The individual structures were further subjected to energy minimization using the
conjugate method (Fig. 6). No violations larger than 0.15 Š occurred for any distance
restraints except for the NHFSAA/CaHOrn (À0.25 Š) in DMSO. Analysis of the snapshots
of MD of 1 showed that the backbone dihedral angles, F, q, and Y, of FSAA were
À 70.25, À 26.55, and 138.43 in CDCl3, and 167.64, À 37.16, and À 91.87in DMSO,

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Table 3. Distance Restraints Used in Molecular Dynamics for 1
Residue
Atom
Residue
Atom
Distance range [Š]
CDCl3 as the solvent medium
FSAA
FSAA
FSAA
Ornithine
Ornithine
FSAA
FSAA
Ornithine
NH
NH
NH
NH
Ornithine
Ornithine
FSAA
FSAA
FSAA
FSAA
FSAA
Ornithine
H(a)
H(b)
H(a)
H(d)
H(a)
H(a)
H(a)
H(d)
3.56 – 2.91
3.63 – 2.97
4.07– 3.33
4.36 – 3.56
3.32 – 2.72
4.26 – 3.48
3.48 – 2.84
2.64 – 2.16
NH
H(g)
H(d)
H(a)
DMSO as the solvent medium
FSAA
FSAA
FSAA
Ornithine
Ornithine
Ornithine
FSAA
FSAA
Ornithine
NH
NH
NH
NH
NH
NH
H(b)
H(d)
H(a)
FSAA
FSAA
Ornithine
FSAA
FSAA
FSAA
Ornithine
FSAA
H(d)
H(b)
H(a)
NH
H(a)
H(b)
H(a)
H(a)
H(d)
3.89 – 3.18
3.25 – 2.66
3.20 – 2.6
4.19 – 3.80
2.77 – 2.27
4.08 – 3.3
4.48 – 3.67
4.08 – 3.34
3.20 – 2.6
Ornithine
Fig. 5. Superposition of 40 snapshots (MD) for 1 in CDCl3 (a) and DMSO (b). H-Atoms and Boc groups
are omitted for the sake of clarity.
respectively. While the rigid FSAA motif continued to maintain a gauche conformation
around CaÀCb (q), the change in the solvent polarity caused a flip in the orientation of
the C¼O and NH groups of FSAA and Orn, which is clearly reflected in their F
and Y values. The resultant conformations favor a seven-membered inter-residue
(NHFSAAÀCOBoc) H-bond (2.16 Š) in CDCl3, and a relatively weak 6-membered intra-
residue (FSAA) H-bond (2.63 Š) in DMSO. Nevertheless, FSAA seems to have
significant control over the backbone folding, which is stabilized by either six- or seven-
membered rings by H-bonding, and the cyclization enhanced the rigidity of the overall
peptide macrocycle in both solvents (Fig. 6). These findings were consistent with the
analysis of the NMR data.

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Helvetica Chimica Acta – Vol. 91 (2008)
Fig. 6. Superposition of the 40 minimum energy structures (MD simulated) of 1 in CDCl3 (a) and DMSO
(b). H-Atoms and Boc groups are omitted for the sake of clarity.
To explore the extent of the influence of rigid FSAA scaffold over a more flexible
ornithine, we have also studied the linear analogue of the cyclic peptide 1, 8. The data
exhibited gauche conformation for q (À408). The observed 3J(NH,CbH) ¼ ca. 9 Hz for
the FSAA residues corresponds to an antiperiplanar arrangement between these H-
atoms, similar to the earlier observations in FSAA-based 14-helix oligomers [3].
However, no clearly determined secondary structure could be derived from the CD
spectrum for the linear analog of 1. Furthermore, except the third residue NH-5 (Boc-
NH of ornithine) all the NH H-atoms of the oligomer seem to be participating only in
weak H-bonds, as suggested by their temperature coefficients (Dd/DT) of ca. 5.5 ppb/
K. One can infer that the FSAA can still modulate the conformational space of the
adjacent flexible spacer to some extent. Nevertheless, a detailed structural study on this
linear oligomer along with the CgÀCd constrained ornithine residues would be
necessary for gaining insight into the conformational propensities.
Conclusions. – In summary, the present work describes the synthesis and
conformational analysis of a C2-symmetric cyclic tetrapeptide 1, with alternating b-
and d-amino acids, which adopts a 3D-bowl shape structure in both polar and non-polar
solvents. The study could also reveal that in a linear analogue of 1, FSAA can nominally
modulate the conformational space of the flexible ornithine. Further investigations on
the linear and cyclic peptide with constrained ornithine (by introducing side chains at
selected positions) may yield more information on various folding propensities.
Experimental Part
General. Commercial reagents were used without further purification. All solvents were purified by
standard techniques. Column chromatography (CC) was carried out on silica gel (SiO2, 60 – 120 mesh).
Optical rotations: Jasco Dip 360 digital polarimeter. CD Spectra: Jasco J-75 spectrometer at 258 in
MeOH, using a 1 mm path-length CD cell. IR Spectra: Perkin-Elmer 683 spectrometer. NMR Spectra: in
CDCl3 or DMSO on a Varian Unity Inova-500 MHz or an Avance-II 600 MHz spectrometer. Chemical
shifts (d) in ppm, referenced to the tetramethylsilane (TMS) as internal standard; coupling constants (J)
in Hz. Two dimensional (2D), total correlation spectroscopy (TOCSY), and rotating frame nuclear
Overhauser effect spectroscopy (ROESY) experiments were carried out. All the experiments were
carried out in phase-sensitive mode. MS: Finnigan MAT 1020B or Micromass VG 70 – 70 H spectrometer
at 70 eV with a direct inlet system. Model building and molecular dynamics simulations were carried out

Helvetica Chimica Acta – Vol. 91 (2008)
1275
using Insight II (97.0)/Discover1 program on a SiliconGraphics Octane and Fuel workstations using
IRIX64 (6.5) operating system.
Na-Boc-Nd-Cbz-Orn-fSAA-OMe (¼ Methyl 3-{[N2-(tert-Butoxycarbonyl)-N5-(phenoxycarbonyl)-
l-ornithyl]amino}-3-deoxy-1,2-O-(1-methylethylidene)-a-d-xylofuranoate; 5). To a soln. of methyl 3-
azido-3-deoxy-1,2-O-(1-methylethylidene)-a-d-xylofuranoate (2; 1 g, 4.6 mmol) in AcOEt (20 ml) was
added 10% Pd/C (0.05 g). After hydrogenation of the mixture for 1 h at atm. pressure, the catalyst was
filtered off, and the filtrate was concentrated in vacuo to give methyl 3-amino-3-deoxy-1,2-O-(1-
methylethylidene)-a-d-xylofuranoate (3; 0.92 g). The compound N2-(tert-butoxycarbonyl)-N5-(phenoxy-
carbonyl)-d-ornithine (4; 1.39 g, 3.8 mmol) was dissolved in CH2Cl2 and treated with 1-hydroxybenzo-
triazole (HOBt; 0.52 g, 4.2 mmol) and N-[3-(dimethylamino)propyl]-N’-ethylcarbodiimide (EDCI;
0.97g, 5 mmol) at 0 8. The mixture was stirred at r.t. for 30 min, and the free amine 3 (0.92 g, 4.2 mmol)
was added. The mixture was stirred at r.t. for further 24 h and diluted with aq. HCl (1m, 50 ml), and
extracted with CH2Cl2 (3 ꢀ 50 ml). The combined org. layers were extracted with aq. NaHCO3 (50 ml)
and brine (50 ml), dried, concentrated in vacuo, and purified by CC (SiO2, AcOEt/hexanes 2 :3) to yield
5 (2.03 g, 85%) as a white solid. M.p. 153 – 1558. [a]2D5 ¼ À7.5 (c ¼ 1, CHCl3). IR (neat): 3348, 2980, 1701,
1660, 1521, 1246, 1029, 754. 1H-NMR (CDCl3, 500 MHz): 7.45 – 7.30 (m, 5 H); 7 .17 d(, J ¼ 8.7, 1 H); 5.99 –
5.90 (m, 1 H); 5.25 – 5.20 (m, 1 H); 5.10 – 5.00 (m, 3 H); 4.95 – 4.90 (m, 1 H); 4.82 – 4.80 (m, 1 H); 4.52 –
4.50 (m, 1 H); 4.20 – 4.18 (m, 1 H); 3.7 3 (s, 3 H); 3.45 – 3.30 (m, 1 H); 3.21 – 3.18 (m, 1 H); 2.14 – 1.92 (m,
3 H); 1.81 – 1.79 (m, 1 H); 1.50 (s, 3 H); 1.40 (s, 9 H); 1.26 (s, 3 H). 13C-NMR (75 MHz, CDCl3): 172.0;
168.4; 156.6; 155.5; 136.4; 128.4; 128.07; 128.0; 112.7; 104.8; 83.8; 76.3; 66.6; 56.4; 52.4; 40.1; 29.9; 28.2;
26.6; 26.1. HR-MS: 588.2532 ([M þ Na]þ, C27H39N3NaO1þ0 ; calc. 588.2533).
Nd-Cbz(Na-Boc-Orn-fSAA)2-OMe (¼ Methyl (3aR,5S,6R,6aR)-6-{[(2S)-2-[(tert-Butoxycarbonyl)-
amino]-5-({[(3aR,5S,6R,6aR)-6-({(2S)-2-[(tert-butoxycarbonyl)amino]-5-[(phenoxycarbonyl)amino]-
pentanoyl}amino)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl]carbonyl}amino)pentanoyl]ami-
no}-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxole-5-carboxylate; 8). Compound 5 (1 g, 1.7mmol) was
dissolved in 12 ml of THF/H2O (3 :1), followed by the addition of LiOH (0.22 g, 5.3 mmol). After stirring
at r.t. for 2 h, the mixture was acidified to pH 2 with NaHSO4 and extracted with AcOEt (2 ꢀ 50 ml).
Then, the combined org. layers were dried over anh. Na2SO4 and concentrated under vaccum to give 3-
{[N2-(tert-butoxycarbonyl)-N5-(phenoxycarbonyl)-l-ornithyl]amino}-3-deoxy-1,2-O-(1-methylethylidene)-
a-d-xylofuranoic acid (6; 0.87g), which was used without further purification for the next reaction.
To a soln. of 5 (1.2 g, 2.1 mmol) in AcOEt (10 ml) 10% Pd/C (0.05 g) was added. After
hydrogenation of the mixture for 12 h at atm. pressure, the catalyst was filtered off, and the filtrate was
concentrated in vacuo to give methyl 3-{[N2-(tert-butoxycarbonyl)-l-ornithyl]amino}-3-deoxy-1,2-O-(1-
methylethylidene)-a-d-xylofuranoate (7; 0.85 g). Compound 6 (0.83 g, 1.5 mmol) was dissolved in CH2Cl2
and treated with HOBt (0.24 g, 1.7mmol) and EDCI (0.4 g, 2 mmol) at 0 8. This mixture was stirred at r.t.
for 30 min, and the free amine 7 (0.75 g, 1.7 mmol) was added. The mixture was stirred at r.t. for 24 h and
diluted with aq. HCl (1m, 50 ml) and extracted with CH2Cl2 (3 ꢀ 50 ml). The combined org. layers were
extracted with aq. NaHCO3 (50 ml) and brine (50 ml), dried, concentrated in vacuo, and purified by CC
(SiO2, AcOEt/hexane 4 :1) to yield compound 8 (1.36 g, 81%) as a white solid. M.p. 183 – 1858. [a]D25
¼
À14.0 (c ¼ 0.5, CHCl3). IR (neat): 3326, 2981, 1660, 1530, 1255, 1165, 1029, 753. 1H-NMR (500 ml
CDCl3 þ 20 ml DMSO, 600 MHz): 8.2 (d, J ¼ 9.64, 1 H); 7.83 (d, J ¼ 8.77, 1 H); 7.82 (t, J ¼ 6.07, 1 H); 7.35
(m, 4 H); 7 .30 (m, 1 H); 7 .10 (t, J ¼ 5.5, 1 H); 6.60 (d, J ¼ 7.97, 1 H); 6.53 (d, J ¼ 7.84, 1 H); 6.0 (d, J ¼ 3.15,
1 H); 5.94 (d, J ¼ 3.20, 1 H); 5.03 (s, 2 H); 4.7 8 (d, J ¼ 4, 1 H); 4.72 (dd, J ¼ 4, 9.6, 1 H); 4.64 (d, J ¼ 3.9,
1 H); 4.53 (dd, J ¼ 3.95, 8.77, 1 H); 4.46 (d, J ¼ 3.32, 1 H); 4.45 (d, J ¼ 3.27, 1 H); 3.93 (m, 2 H); 3.66 (s,
3 H); 3.24 (m, 1 H); 3.04 (m, 3 H); 1.58 (m, 2 H); 1.50 (m, 4 H); 1.47 (m, 8 H); 1.41 (s, 9 H); 1.39 (s, 9 H);
1.29 (s, 6 H). 13C-NMR (75 MHz, CDCl3): 172.6; 172.1; 168.4; 167.0; 156.9; 155.5; 136.5; 128.4; 127.8;
112.3; 104.9; 84.0; 79.4; 66.4; 56.6; 52.3; 30.5; 28.5; 26.6; 26.2. HR-MS: 987.4509 ([M þ Na]þ;
C45H68N6NaOþ17 ; calc. 987.4538).
Di(tert-butyl) [(3aR,4aR,10S,12aR,12bR,15aR,16aS,22S,24aR,24bR)-Tetracosahydro-2,2,14,14-tet-
ramethyl-5,11,17,23-tetraoxobis[1,3]dioxolo[4,5]furo[3,2-b:3’,2’-l][1,5,11,15]tetraazacycloicosine-10,22-
diyl]biscarbamate (1; [12]). Compound 8 (0.317g, 0.3 mmol) was dissolved in 12 ml THF/H 2O (3 :1),
followed by the addition of LiOH (0.041 g, 0.9 mmol). After stirring at r.t. for 2 h, the mixture was
acidified to pH 2.0 with NaHSO4 and extracted with AcOEt (2 ꢀ 50 ml). Then, the combined org. layers

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Helvetica Chimica Acta – Vol. 91 (2008)
were dried over Na2SO4 and concentrated under vaccum to give the acid, which was dissolved in CH2Cl2
and treated with EDCI (0.05 g, 0.3 mmol) and pentafluorophenol (0.05 g, 0.3 mmol) at 08. The mixture
was stirred at r.t. for 18 h and diluted with H2O (20 ml), and extracted with CH2Cl2 (2 ꢀ 50 ml). The
combined org. layers were extracted with brine (50 ml), dried, and concentrated in vacuo to give
pentafluorophenyl (3aR,5S,6R,6aR)-6-{[(2S)-2-[(tert-butoxycarbonyl)amino]-5-({[(3aR,5S,6R,6aR)-6-
({[(tert-butoxycarbonyl)amino]propyl}amino]acetyl}amino)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]di-
oxol-5-yl]carbonyl}amino)pentanoyl]amino}-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxole-5-carboxy-
late (9). To the soln. of 9 (0.317g, 0.2 mmol) in AcOEt was added 10% Pd/C (0.050 g). After
hydrogenation of the mixture for 24 h at atm. pressure, the catalyst was filtered off, and the filtrate was
concentrated in vacuo and purified by CC (SiO2, CHCl3/MeOH 25 :1) to yield 1 (0.16 g, 71%) as a white
solid. M.p. 230 – 232. [a]2D5 ¼ À43.3 (c ¼ 0.015, CHCl3). IR (neat): 3297, 2919, 2850, 1690, 1677, 1532, 1451,
1368, 1217, 1165, 102, 861.1H-NMR(CDCl3, 500 MHz): 8.0 (d, J ¼ 5.0, 1 H); 7.09 (t, J ¼ 6.08, 1 H); 6.31 (d,
J ¼ 3.1, 1 H); 5.22 (d, J ¼ 8.0, 1 H); 5.03 (d, J ¼ 3.14, 1 H); 4.81 (d, J ¼ 5.05, 1 H); 4.67( m, 1 H); 4.06 (t,
J ¼ 5.0, 1 H); 3.88 (m, 1 H); 2.99 (m, 1 H); 1.7 2 (m, 1 H); 1.62 (m, 1 H); 1.51 (s, 3 H); 1.43 (s, 9 H); 1.36
(m, 1 H); 1.33 (s, 3 H); 1.26 (m, 1 H). 13C-NMR (75 MHz, CDCl3): 173.5; 170.0; 156.0; 111.5; 106.3; 84.5;
81.5; 79.7; 59.1; 50.6; 37.3; 33.0; 28.2; 26.7; 26.0; 25.7. HR-MS: 821.3931 ([M þ Na]þ, C36H58N6NaO1þ4 ; calc.
821.3908).
B. S., P. N. , and M. U. are thankful to CSIR, New Delhi, for fellowship.
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Received February 6, 2008