Conformational studies of 3,4-dideoxy furanoid sugar amino acid containing analogs of the receptor binding inhibitor of vasoactive intestinal peptide

Article abstract of DOI:10.1016/j.tet.2004.07.032

Conformational analysis of vasoactive intestinal peptide (VIP) receptor binding inhibitor Leu¹-Met²-Tyr³-Pro ⁴-Thr⁵-Tyr⁶-Leu⁷-Lys⁸ 1 by various NMR techniques and constrained molecular dynamics (MD) simulation studies revealed that the molecule had a turn structure involving its Tyr ³-Pro⁴-Thr⁵-Tyr⁶ moiety with intramolecular hydrogen bond between Tyr⁶NH→Tyr³CO. In order to mimic the structure of 1, peptidomimetic analogs 2-4 were synthesized using conformationally constrained scaffolds of 3,4-dideoxy furanoid sugar amino acids (2S,5R)-ddSaa1 5 and its enantiomer (2R,5S)-ddSaa2 6. All these analogs displayed well defined three-dimensional structures akin to that found in 1. Peptides 2 and 3, which differed only in the sugar amino acid stereochemistry, show propensity of structures with identical intramolecular hydrogen bonds between ThrNH→MetCO. A similar structure with a hydrogen bond between TyrNH→MetCO was observed in 4. Graphical Abstract.

Full text of DOI:10.1016/j.tet.2004.07.032

Tetrahedron 60 (2004) 8329–8339
Conformational studies of 3,4-dideoxy furanoid sugar amino acid
containing analogs of the receptor binding inhibitor of vasoactive
intestinal peptide
a
a
a
Tushar K. Chakraborty,a, V. Ramakrishna Reddy, G. Sudhakar, S. Uday Kumar,
*
T. Jagadeshwar Reddy,a S. Kiran Kumar,a Ajit C. Kunwar,a, Archna Mathur, Rajan Sharma,
b
b
*
Neena Guptab and Sudhanand Prasadb,
*
aBioorganic Laboratory, Organic Division-III, Indian Institute of Chemical Technology, Tarnaka, Uppal Road, Hyderabad 500 007, India
bDabur Research Foundation, 22 Site IV, Sahibabad, Ghaziabad 201 010, India
Received 14 May 2004; revised 25 June 2004; accepted 15 July 2004
Available online 4 August 2004
Abstract—Conformational analysis of vasoactive intestinal peptide (VIP) receptor binding inhibitor Leu1-Met2-Tyr3-Pro4-Thr5-Tyr6-Leu7-
Lys8 1 by various NMR techniques and constrained molecular dynamics (MD) simulation studies revealed that the molecule had a turn
structure involving its Tyr3-Pro4-Thr5-Tyr6 moiety with intramolecular hydrogen bond between Tyr6NH/Tyr3CO. In order to mimic the
structure of 1, peptidomimetic analogs 2–4 were synthesized using conformationally constrained scaffolds of 3,4-dideoxy furanoid sugar
amino acids (2S,5R)-ddSaa1 5 and its enantiomer (2R,5S)-ddSaa2 6. All these analogs displayed well defined three-dimensional structures
akin to that found in 1. Peptides 2 and 3, which differed only in the sugar amino acid stereochemistry, show propensity of structures with
identical intramolecular hydrogen bonds between ThrNH/MetCO. A similar structure with a hydrogen bond between TyrNH/MetCO
was observed in 4.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
lipoconjugates have been synthesized and tested for their
anti cancer activities.7 This prompted us to undertake the
development of new peptidomimetic analogs of 1 based on
conformationally constrained nonproteinogenic scaffolds in
order to increase their physiological stabilities.
Vasoactive intestinal peptide (VIP) is a widely distributed
naturally occurring neuropeptide containing 28 amino acids
with wide ranging biological activities.1 It has been found
that VIP receptors are over-expressed on a variety of
malignant tumor cells that are also associated with the
synthesis and secretion of detectable levels of the VIP by the
malignant cells themselves.2 The in vitro studies suggest
that VIP acts as a growth factor and plays a dominant role in
the sustained or indefinite proliferation of cancer cells.
Focus is on the development of VIP receptor binding
inhibitors that will have the potential to arrest the growth of
malignant cells.3 The peptide sequence Leu1-Met2-Tyr3-
Pro4-Thr5-Tyr6-Leu7-Lys8 1 is known to be one such VIP
receptor binding inhibitor.4 The role of this octapeptide as a
VIP receptor binding inhibitor5 and its anti cancer activities
in combination with other neuropeptide analogs6 ha1ve
been well established. Several novel analogs of this peptide
containing a,a-dialkylated amino acids and its
It was envisaged that the design of the peptidomimetic
analogs of 1 could be greatly facilitated by the knowledge of
its three-dimensional structure. This prompted us to
undertake first the structural studies of the octapeptide 1.
In this paper, we describe the detailed conformational
analysis of 1 using various NMR techniques that established
a well-defined turn structure involving Tyr3-Pro4-Thr5-Tyr6
residues. Based on these structural studies, novel peptido-
mimetic analogs 2–4 were subsequently developed using
3,4-dideoxy furanoid sugar amino acids 5 (ddSaa1) and 6
(ddSaa2) as building blocks. In recent years, sugar amino
acids have emerged as a class of versatile templates that
have been used extensively as conformationally constrained
scaffolds in many peptidomimetic studies.8 Insertion of
sugar amino acids (2S,5R)-ddSaa1 5 and its enantiomer
(2R,5S)-ddSaa2 6 as dipeptide isosteres in place of the Tyr3-
Pro4 segment of 1 led to the formation of analogs 2 and 3,
respectively.
Keywords: 3,4-Dideoxy furanoid sugar amino acids; VIP receptor;
Conformation; NMR.
* Corresponding authors. Tel.: C91-40-27193154; fax: C91-40-
27193108; e-mail: chakraborty@iict.res.in
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.07.032

8330
T. K. Chakraborty et al. / Tetrahedron 60 (2004) 8329–8339
dideoxy furanoid sugar amino acids ddSaa1 and ddSaa2.9
The starting material for the synthesis of Fmoc-ddSaa1 was
(5S)-5-(hydroxymethyl)dihydrofuran-2(3H)-one 7, which
was prepared from L-glutamic acid in two steps using
known methods.10 Protection of the primary hydroxyl of 7
as the trityl ether, and subsequent reduction of the lactone
using DIBAL-H furnished the lactol 8 as a mixture of
isomers. Acylation of the lactol hydroxyl group led to the
formation of a glycosyl acetate intermediate that was treated
with trimethylsilyl cyanide in the presence of BF3–Et2O to
get a diastereomeric mixture of the glycosyl cyanides 9.11
Reduction of the cyanide group of 9 with LiAlH4 gave a
primary amine intermediate that was protected in situ using
FmocOSu to furnish the intermediate 10. The faster moving
spot on the TLC was the desired (2S,5R)-isomer that could
be separated easily at this stage using standard silica gel
column chromatography. The relative stereochemistries of
Compounds 2 and 3 displayed nucleation of well-defined
turn structures, similar to the one found in 1, with an
intramolecular hydrogen bond between ThrNH/MetCO.
Further truncation of 1, replacing three of its amino acids
Tyr3-Pro4-Thr5 with ddSaa1 and deleting one residue each
from both the termini, still retained the essential 10-
membered b-turn like structure in the resulting analog 4
with a TyrNH/MetCO hydrogen bond, underlying the
turn-inducing effect of 2,5-syn furanoid sugar amino acids.
The details of the synthesis and structural studies of peptide
1 and its analogs 2–4 are described here.
2. Results and discussions
2.1. Synthesis of sugar amino acids 5 and 6 with
Fmoc-protection
1
the separated isomers were further confirmed by H NOE
difference spectroscopic studies. Irradiation of the C2–H
Scheme 1 describes the synthesis of Fmoc-protected 3,4-

T. K. Chakraborty et al. / Tetrahedron 60 (2004) 8329–8339
8331
Scheme 1. Reagents and conditions: (a) pyridine, TrCl, DMAP (cat.), CH2Cl2, 0 8C to rt, 12 h; (b) DIBAL-H, CH2Cl2, K78 8C, 10 min; (c) Et3N, Ac2O,
DMAP (cat.), CH2Cl2, 0 8C to rt, 30 min; (d) TMSCN, BF3–Et2O, CH3CN, rt, 4 h; (e) LiAlH4, Et2O, 0 8C, 5 min, reflux, 3 h; (f) FmocOsu, CH2Cl2, 0 8C to rt,
1 h; (g) Jones reagent, acetone, 0 8C to rt, 3 h; (h) H2, 10% Pd(OH)2–C, MeOH, rt, 8 h; (i) TBDPSCl, Et3N, DMAP (cat.), DMF, 0 8C to rt, 3 h; (j) Ph3P,
imidazole, I2, toluene, reflux, 3 h; (k) H2, 10% Pd(OH)2–C, MeOH, rt, 1 h; (l) TBAF, THF, 0 8C to rt, 3 h; (m) NaIO4, RuCl3$3H2O (cat.), CH3CN:CCl4:H2O
(1:1:1.5), 0 8C, 2 h; (n) TFA, CH2Cl2, 0 8C, 2 h; (o) FmocOSu, 10% aqueous Na2CO3, dioxane, 0 8C to rt, 12 h.
signal of the (2S,5R)-isomer 10 enhanced the peak of C5–H
confirming their syn-relationship. Oxidation of the primary
hydroxyl of 10 to carboxylic acid using Jones’ reagent
furnished the target molecule Fmoc-ddSaa1 11.
cleaved from the resin by treatment with trifluoroacetic
acid, crystalline phenol, ethanedithiol, thioanisole and
deionized water for 2–3 h at room temperature. The crude
peptides obtained by precipitation with cold dry ether were
purified by preparative HPLC and used for conformational
analysis by NMR.
Synthesis of the other enantiomer of 11, Fmoc-ddSaa2, was
started with the known compound 12, which was prepared
by us earlier from D-glucose.12 Debenzylation of 12 was
followed by selective protection of the primary hydroxyl
group as the TBDPS ether to get the intermediate diol 13.
Next, the diol 13 was transformed into the 3,4-dideoxy
intermediate 14 in three steps—reductive elimination of the
3,4-diol moiety using Ph3P/I2/imidazole,13 hydrogenation of
the resulting 3,4-olefin function and finally, desilylation of
the C1-hydroxyl group using TBAF. Compound 14 was
transformed into the required Fmoc-protected product 15
following a three-step process—oxidation of the primary
hydroxyl to get the carboxylic acid, Boc-deprotection using
trifluoroacetic acid in dichloromethane, followed by Fmoc
protection using FmocOSu to furnish the required product
15.
2.3. Conformational analysis. NMR studies
NMR studies of 1–4 were carried out in DMSO-d6. The
spectra were well resolved and most of the spectral
parameters could be obtained easily and are reported in
the Tables 1–4. While the assignments were carried out with
the help of total correlation spectroscopy (TOCSY),16
rotating frame nuclear Overhauser effect spectroscopy
(ROESY)17 experiments provided the information on the
proximity of protons, the details of which are provided in
Section 4. Variable temperature studies were carried out to
measure the temperature coefficients of the amide proton
chemical shifts (Dd(D(), which provided information about
their involvements in intramolecular hydrogen bonds.18 The
cross-peak intensities in the ROESY spectra, shown
schematically in Figures 1–4, were used for obtaining the
restraints in the simulated molecular dynamics (MD)
calculations,19 the detailed protocol of which is included
in Section 4.
2.2. Synthesis of peptides 2–4
Peptides 2–4 were synthesized by solid phase method on
Wang resin using the Fmoc strategy.14 Substitution levels
for automated synthesis were preferably between 0.9 and
1.2 mmol amino acid per gram resin. Preferably, DIPC/
HOBt or HBTU/DIPEA and PyBOP/DIPEA were used as
activating reagents in the coupling reactions.15 The coupling
reactions were carried out in DMF, CH2Cl2 or NMP15 or a
mixture of these solvents. The Fmoc group was cleaved by
using 20% piperidine in DMF for 30 min. Usually,
3–4 equiv of activating agents and 3–4 equiv of Fmoc
protected amino acid per resin nitrogen equivalent were
used per coupling step. The side chain protecting groups
were deprotected and the peptide was simultaneously
2.4. Conformational analysis of 1
Proline, the unique cyclic natural amino acid, can undergo
cis–trans rotamerisation with preceding amino acid,
because of the presence of imide bond. Invariably the
trans rotamer predominates the cis rotamer in solution.
Peptide 1 shows two sets of resonances with 6:1 ratio,
because of the existence of cis/trans isomers about Tyr3-
Pro4 amide linkage. The observation of strong rOe cross-
peaks, as shown in
A
in Figure 1, between

8332
T. K. Chakraborty et al. / Tetrahedron 60 (2004) 8329–8339
Table 1. 1H chemical shifts (d in ppm), coupling constants (J in Hz) and temperature coefficients (Dd/DT in ppb/deg K) of 1 in DMSO-d6 at 500 MHz
Amino acid
Leu1
NH
—
CaH
3.71 (m)
CbH
CgH
1.59 (m)
CdH
Others
Dd/DT
1.45–1.49 (m)
0.86 (d, JZ6.7 Hz),
0.84 (d, JZ6.8 Hz)
Met2
Tyr3
8.49 (d)
(JZ8.5 Hz)
8.20 (d)
4.39 (m)
1.86, 1.74 (m)
2.41 (m)
2.02 (SCH3)
K5.5
K7.8
4.55 (ddd)
(JZ4.5, 8.2 Hz)
2.86 (dd, JZ4.5,
14.3), 2.67 (dd,
JZ8.9, 14.3 Hz)
1.96, 1.88 (m)
3.96 (m)
7.04, 6.61 (Ph),
9.15 (OH)
(JZ7.8)
Pro4
Thr5
—
7.78 (d)
(JZ8.1 Hz)
7.68 (d)
4.43 (m)
1.82 (m), 1.81 (m)
1.01 (d, JZ6.4 Hz)
3.59 (m), 3.43 (m)
4.18 (dd)
(JZ4.2 Hz)
4.44 (m)
4.91 (OH)
K6.0
K3.6
Tyr6
2.93 (dd, JZ4.4,
14.3 Hz), 2.71 (dd,
JZ9.1, 14.3 Hz)
1.45–1.47 (m)
6.99, 6.60 (Ph)
(JZ8.1 Hz)
Leu7
Lys8
7.94 (d)
(JZ8.2 Hz)
7.99 (d)
4.30 (q)
(JZ8.2 Hz)
4.13 (dt)
1.58 (m)
0.87, 0.83
1.36 (m)
K6.9
K6.9
1.72 (m)
1.60 (m), 1.54 (m)
2.76
(JZ7.9 Hz)
(JZ5.0 Hz)
Table 2. 1H chemical shifts (d in ppm), coupling constants (J in Hz) and temperature coefficients (Dd/DT in ppb/deg K) of 2 in DMSO-d6 at 500 MHz
Amino acid
NH
8.08 (bs)
CaH
3.80 (m)
CbH
CgH
CdH
Others
Dd/DT
Leu
Met
ddSaa1
Thr
Tyr
1.52–1.61 (m)
0.87 (d), 0.88 (d)
2.03 (SCH3)
3.34, 3.16
4.94 (OH)
6.98 (d, JZ8.4 Hz), K4.7
6.59 (d, JZ8.4 Hz)
8.61 (d) (JZ8.1 Hz) 4.41 (dt) (JZ5.2 Hz) 1.91 (m), 1.80 (m) 2.48 (m), 2.43 (m)
8.33 (d) (JZ5.8 Hz) 4.30 (m) 1.87 (m), 2.11 (m) 1.46 (m)
7.60 (d) (JZ8.4 Hz) 4.19 (dd) (JZ4.6 Hz) 3.98 (m)
7.87 (d) (JZ8.2 Hz) 4.49 (dt) (JZ4.4 Hz) 2.90 (dd, JZ4.5,
14.2 Hz), 2.67 (dd,
K5.5
K7.3
K2.8
3.96
0.99 (m)
JZ8.7, 14.2 Hz)
1.45–1.47
8.08 (d) (JZ7.7 Hz) 4.15 (dt) (JZ5.0 Hz) 1.72
Leu
Lys
7.96 (d) (JZ8.1 Hz) 4.31 (m)
1.60 (m) 0.87 (d), 0.84 (d)
1.60 (m), 1.54 (m) 1.36 (m)
K6.5
K7.8
2.76
Table 3. 1H chemical shifts (d in ppm), coupling constants (J in Hz) and temperature coefficients (Dd/DT in ppb/deg K) of 3 in DMSO-d6 at 500 MHz
Amino acid
Leu
NH
8.07 (bs)
CaH
3.80 (m)
CbH
1.50 (m)
CgH
1.59 (m)
CdH
Others
Dd/DT
0.87 (d, JZ6.5 Hz),
0.87 (d, JZ6.5 Hz)
—
Met
8.61 (d) (JZ8.1 Hz) 4.39 (ddd) (JZ5.0,
1.90 (m), 1.80 (m) 2.47 (m), 2.42 (m)
2.03 (SCH3)
K5.0
K6.7
K2.2
8.4 Hz)
8.25 (d) (JZ5.8 Hz) 4.28 (dd) (JZ5.2,
8.3 Hz)
ddSaa2
Thr
1.88 (m)
1.51 (m)
3.97 (m)
3.24 (m), 3.12 (m)
7.53 (d) (JZ8.2 Hz) 4.17 (d) (JZ4.6 Hz) 3.95 (m)
0.95 (d, JZ6.3 Hz)
5.00 (d, JZ5.5 Hz,
OH)
Tyr
7.93 (d) (JZ8.3 Hz) 4.46 (dt) (JZ4.3 Hz) 2.92 (dd, JZ4.4,
14.1 Hz), 2.67 (dd,
6.98 (d, JZ8.5 Hz), K4.9
6.60 (d, JZ8.5 Hz)
JZ8.9, 14.1 Hz)
7.94 (d) (JZ8.1 Hz) 4.31 (dt) (JZ6.1, 8. 1.45–1.47 (m)
1 Hz)
Leu
Lys
1.60 (m)
0.87 (d, JZ6.5 Hz),
0.83 (d, JZ6.5 Hz)
K6.1
8.07 (d) (JZ7.5 Hz) 4.14 (m)
1.72 (m)
1.60 (m), 1.54 (m) 1.35
2.75 (m), 7.65 (bs)
K7.4
Table 4. 1H chemical shifts (d in ppm), coupling constants (J in Hz) and temperature coefficients (Dd/DT in ppb/deg K) of 4 in DMSO-d6 at 500 MHz
Amino acid
NH
8.16 (bs)
CaH
3.84 (m)
CbH
CgH
CdH
Others
Dd/DT
Met
ddSaa1
2.48 (m), 1.98 (m) 3.30 (m), 3.30 (m)
1.29 (m), 1.84 (m) 1.66 (m), 2.05 (m) 3.94 (m)
2.05 (s, SCH3)
3.12 (m), 3.22 (m)
—
K4.7
8.69 (t) (JZ5.8 Hz) 4.18 (dd) (JZ5.4,
8.0 Hz)
7.56 (d) (JZ8.8 Hz) 4.58 (ddd) (JZ4.1,
9.3 Hz)
Tyr
Leu
2.96 (dd, JZ4.4,
14.0 Hz), 2.67 (dd,
JZ9.8, 14.0 Hz)
6.63 (d, JZ8.5 Hz), K2.8
7.07 (d, JZ8.5 Hz),
9.15 (OH)
8.38 (d) (JZ8.0 Hz) 4.23 (ddd) (JZ6.0,
8.5 Hz)
1.63–1.52 (m)
0.90 (d, JZ6.7 Hz),
0.84 (d, JZ6.7 Hz)
K6.9

T. K. Chakraborty et al. / Tetrahedron 60 (2004) 8329–8339
8333
Figure 1. (A) Schematic representation of the proposed b-turn involving Tyr3-Pro4-Thr5-Tyr6 residues in 1 with some of the prominent long-range rOes seen in
its ROESY spectrum. (B) Stereo view of the 25 superimposed energy-minimized structures of 1 sampled during 50 cycles of the 300 ps constrained MD
simulations following the simulated annealing protocol: H-bonded region (left), full structure (right).
Tyr3CaH4Pro4CdH and Tyr3CaH4Pro4Cd0H in the
ROESY spectrum show that the major isomer has a trans
imide bond preceding Pro4. Moderate magnitude of Dd/
DTZK3.6 ppb/deg K for Tyr6NH indicates the propensity
of a structure, with its participation in intramolecular
hydrogen bonding. Observation of rOe cross-peaks between
Thr5NH4Tyr6NH and Tyr6NH4Leu7NH as well as the
participation of Tyr6NH in hydrogen bonding shows the
existence of a 10-membered b-turn around Pro4-Thr5
residues. The presence of similar intensities of rOe cross-
peaks of Thr5NH4Pro4CaH and Thr5NH4Thr5CaH
imply that the observed turn is a type-II b-turn.
The molecular dynamics calculations on 1 clearly show
structures with a type-II b-turn about Pro4-Thr5 residues.
Figure 1 depicts the assembly B of the backbone super-
imposed turn structures of the 25 samples collected during
300 ps simulated annealing protocol (detailed protocol has
been given in the Supporting Information). It clearly shows
a type-II b-turn about Pro4-Thr5 residues, where as the other
Figure 2. (A) Schematic representation of the proposed b-turn like structures involving Met-ddSaa1-Thr residues in 2 with some of the prominent long-range
rOes seen in its ROESY spectrum. (B) Stereo view of the 25 superimposed energy-minimized structures of 2 sampled during 50 cycles of the 300 ps
constrained MD simulations following the simulated annealing protocol: H-bonded region (left), full structure (right).

8334
T. K. Chakraborty et al. / Tetrahedron 60 (2004) 8329–8339
Figure 3. (A) Schematic representation of the proposed b-turn like structures involving Met-ddSaa2-Thr residues in 3 with some of the prominent long-range
rOes seen in its ROESY spectrum. (B) Stereo view of the 25 superimposed energy-minimized structures of 3 sampled during 50 cycles of the 300 ps
constrained MD simulations following the simulated annealing protocol: H-bonded region (left), full structure (right).
part of the peptide backbone shows an irregular (extended)
conformation. The average pair wise backbone RMSD for
five-membered sugar ring. Temperature coefficient, Dd/
DTZK2.8 ppb/deg K, for Thr5NH showed that it partici-
pates in intramolecular hydrogen bonding. Appearances
of the sequential NHi4NHiC1 rOe cross-peaks
(MetNH4ddSaa1NH, ddSaa1NH4ThrNH, ThrNH4
TyrNH, TyrNH4LeuNH and LeuNH4LysNH) in the
ROESY spectrum show that there is a propensity towards a
helical structure. The presence of rOes between
˚
the structures is 0.47G0.16 A.
2.5. Conformational analysis of 2
As compared to 1, in peptide 2, the dipeptide isostere
(2S,5R)-ddSaa1 5, has been inserted in place of the Tyr3-
Pro4 residues. The relative configuration of the ddSaa1 at
the C2 and C5 carbons was confirmed by the observation
of the rOe cross-peak between ddSaa1C2–H4C5–H,
which imply that these protons are on the same side of the
ddSaa1NH4ThrNH,
ThrNH4TyrNH,
ThrNH4
0
ddSaa1C6–H2(a,a ) (A in Fig. 2) coupled with the intramo-
lecular hydrogen bonding of ThrNH imply that the molecule
has a b-turn like structure around Met-ddSaa1-Thr residues,
Figure 4. (A) Schematic representation of the proposed b-turn like structures involving Met-ddSaa1-Tyr residues in 4 with some of the prominent long-range
rOes seen in its ROESY spectrum. (B) Stereo view of the 25 superimposed energy-minimized structures of 4 sampled during 50 cycles of the 300 ps
constrained MD simulations following the simulated annealing protocol: H-bonded region (left), full structure (right).

T. K. Chakraborty et al. / Tetrahedron 60 (2004) 8329–8339
8335
which is stabilized by the ThrNH/MetCO H-bond. The
3. Conclusion
observed b-turn is similar to that observed earlier by us20 and
others21 for oligomers containing furanoid sugar amino acids.
The MD calculations on 2 show the existence of a 10-
memberedhydrogenbondedturnstructurebetweenThrNH/
MetCO, which mimics a regular b-turn structure, where as the
rest of the peptide backbone seem to take an extended
conformation. Figure 2 shows an ensemble B of 25
conformations superimposed at the turn structure during 50
cycles of 300 ps simulated annealing MD run. The average
3,4-Dideoxy furanoid sugar amino acids 5 and 6, having a
syn relationship between C2–H and C5–H, belong to the
growing family of very useful molecular building blocks of
sugar amino acids and display remarkable propensities to
induce well defined turn structures in small peptides. The
b-turn structure found in VIP anatagonist 1 was successfully
reproduced by introducing the nonproteinogenic dipeptide
isostere 5 and 6 in the molecule, resulting in the
development of novel peptidomimetic analogs 2–4.
Although both ddSaa1 5 and ddSaa2 6 gave rise to the
similar 10-membered hydrogen bonded structures, the turn
induced by the latter with (2R,5S) stereochemistry was more
pronounced than those derived from the former having the
(2S,5R) stereochemistry. These studies may help in design-
ing mimics of the bioactive peptide conformations of small
peptides, where proline residue participates in well-defined
b-turn structures.
˚
pair wise backbone RMSD is 0.87G0.39 A.
2.6. Conformational analysis of 3
In compound 3, containing the dipeptide isostere (2R,5S)-
ddSaa2 6, the observed conformation of the peptide is
similar to that of 2, which had the isomeric sugar amino
acid. The small magnitude of Dd/DTZK2.2 ppb/deg K for
Thr5NH confirms its participation in hydrogen bonding
probably with Met2C]O. Similar to 2, all the sequential
NHi4NHiC1 rOe cross-peaks (MetNH4ddSaa2NH,
ddSaa2NH4ThrNH, ThrNH4TyrNH, TyrNH4LeuNH
and LeuNH4LysNH) in the ROESY spectrum were
observed. It supports the presence of an incipient helix.
Participation of the ThrNH in hydrogen bonding, as well as
the observation of rOe cross-peaks between
ddSaa2NH4ThrNH, ThrNH4TyrNH and ThrNH4
4. Experimental
4.1. General experimental procedures
All reactions were carried out in oven or flame-dried
glassware with magnetic stirring under a nitrogen atmo-
sphere using dry, freshly distilled solvents, unless otherwise
noted. Reactions were monitored by thin layer chromato-
graphy (TLC) carried out on 0.25 mm silica gel plates with
UV light, I2, 7% ethanolic phosphomolybdic acid-heat or
2.5% ethanolic anisaldehyde (with 1% AcOH and 3.3%
conc. H2SO4)—heat as developing agents. Silica gel finer
than 200 mesh was used for flash column chromatography.
Yields refer to chromatographically and spectroscopically
homogeneous materials unless otherwise stated. Melting
points are uncorrected. IR spectra were recorded as neat
liquids or KBr pellets on FT-IR Nicolet-740. Mass spectra
were obtained on Micromass Autospec and Quattro
spectrometers under liquid secondary ion mass spectro-
metric (LSIMS) and electron spray ionisation (ESI)
techniques, respectively. Optical rotations were measured
with a Jasco Dip-370 and Horiba Sepa-300 digital
polarimeters.
0
ddSaa2C6–H2(a,a ) (A in Fig. 3) indicate that the molecule
has a b-turn like structure involving Met-ddSaa2-Thr
residues. The observed b-turn is similar to that found in 2
and in various oligomers containing furanoid sugar amino
acids.20,21
Molecular mechanics calculations clearly show the exist-
ence of a 10-membered H-bond between ThrNH/MetCO.
Figure 3 shows an ensemble B of 25 conformations
superimposed at the turn structure during 50 cycles of
300 ps simulated annealing MD run. The average pair wise
˚
backbone RMSD is 0.41G0.23 A.
2.7. Conformational analysis of 4
In compound 4, TyrNH shows small magnitude of Dd/DT
(K2.8 ppb/deg K), indicating its participation in intra-
molecular hydrogen bonding. The presence of ROESY
cross-peaks between TyrNH4ddSaa1C2–H and
TyrNH4ddSaa1C6–H coupled with the participation of
TyrNH in hydrogen bonding suggests that a 10-membered
b-turn like structure, similar to the one observed earlier by
us and others,20,21 is stabilized by a hydrogen bond between
TyrNH/MetCO. Appearance of a rOe cross-peak between
the ddSaa1C2–H4ddSaa1C5–H indicates that the two
protons are on the same side of the five-membered ring. The
3JNH–CaH of 8.8 and 8.0 Hz for Tyr and Leu residues,
respectively, indicate that there is propensity of structures
with backbone f angles in the b-region of the Ramachan-
dran plot. The cross-peak intensities in the ROESY
spectrum of 4 (A in Fig. 4) were used for obtaining the
restraints in the MD calculations. The molecular dynamics
calculations showed the existence of a b-turn like structure
as shown in B in Figure 4 having a 10-membered
intramolecular hydrogen bond. The average pair wise
4.2. NMR spectroscopy
NMR spectra of the peptides 1–4 were recorded on Varian
Unity-Inova 500 MHz spectrometer at 30 8C with 2–10 mM
solutions in appropriate solvents using TMS as internal
standard or the solvent signals as secondary standards and
the chemical shifts are shown in d scales. Multiplicities of
NMR signals are designated as s (singlet), d (doublet),
t (triplet), q (quartet), br (broad), m (multiplet, for
unresolved lines), etc. 13C NMR spectra were recorded on
Bruker Avance-300 spectrometer at 75 MHz with complete
proton decoupling. The chemical shift assignments were
carried out with the help of two-dimensional total
correlation spectroscopy (TOCSY)16 and rotating frame
nuclear Overhauser effect spectroscopy (ROESY) exper-
iments,17 the later also provided the information on the
proximity of protons. All the experiments were carried out
in the phase sensitive mode.22 The spectra were acquired
˚
backbone RMSD is 1.20G0.33 A.

8336
T. K. Chakraborty et al. / Tetrahedron 60 (2004) 8329–8339
with 2!256 or 2!192 free induction decays (FID)
containing 8–16 transients with relaxation delays of 1.0–
1.5 s. The ROESY experiments were performed with
mixing time of 0.3 s. For ROESY experiments a spin-
locking field of about 2 kHz was used. The TOCSY
experiments were performed with the spin locking fields
of about 10 kHz and a mixing time of 0.08 s. The two-
dimensional data were processed with Gaussian apodization
in both the dimensions. To obtain the temperature
coefficients of NH-chemical shifts, the spectra were
recorded between 30 and 70 8C (at 30, 40, 50, 60, and
70 8C) in DMSO-d6. The temperature coefficients Dd/DT
were determined from the slopes of the linear regression
lines obtained from the chemical shift versus temperature
plots (see Supplementary Information).18
room temperature for 12 h. The reaction was then quenched
with saturated NH4Cl solution, the organic layer was
separated and the aqueous layer was extracted with
EtOAc. The combined organic extracts were washed
with water, brine, dried (Na2SO4) and concentrated in
vacuo. Purification by column chromatography afforded
(5S)-5-[(triphenylmethoxy)methyl]dihydrofuran-2(3H)-
one (110.6 g) in 77% yield.
The product (110 g, 307 mmol) was dissolved in CH2Cl2
(600 mL) and the solution was cooled to K78 8C. DIBAL-H
(1.2 M in toluene, 281 mL, 337.7 mmol) was added
dropwise and stirred for 15 min at this temperature. The
reaction mixture was then quenched with MeOH followed
by saturated sodium potassium tartrate solution and stirred
for 1 h. The organic layer was separated and the aqueous
layer was extracted with EtOAc. The combined organic
extracts were washed with water, brine, dried (Na2SO4) and
concentrated in vacuo. Purification by column chromato-
graphy afforded the title compound 8 (95 g, 86%) as a
mixture of diastereomers at the anomeric position. Data for
8: RfZ0.35 (silica gel, 40% EtOAc in petroleum ether); IR
4.3. Molecular dynamics
Molecular mechanics/dynamics calculations were carried
out using the Sybyl 6.8 program on a Silicon Graphics O2
workstation. The Tripos force field, with default parameters,
was used throughout the simulations.
1
(neat) nmax 3448, 3350, 1479, 1433, 1219, 1075 cmK1; H
NMR (CDCl3, 200 MHz, mixture of isomers 1:1) d 7.48–
7.22 (m, 15H, ArH), 5.62 and 5.48 (two m, 1H, anomeric
H), 4.45 (dq, JZ7.5, 5.3 Hz) and 4.27 (m) (total 1H), 3.30
and 3.22 (two dd, JZ9.8, 3.7, 9.8, 4.5 Hz) and 3.10 (d, JZ
5.3 Hz) (total 2H), 2.83 (d, JZ6.0 Hz) and 2.50 (m) (total
1H), 2.08–1.82 (m, 3H), 1.70 (m, 1H); 13C NMR (CDCl3,
75 MHz, mixture of isomers) d 144.00, 143.78, 128.74,
128.68, 127.77, 127.71, 127.02, 126.89, 98.91, 98.68, 79.31,
77.57, 66.84, 66.10, 34.03, 32.74, 25.93, 25.25; MS (ESI):
m/z (%): 383 (100) [MCNa]C, 399 (55) [MCK]C; HRMS
(ESI): Calcd for C24H24O3Na [MCNa]C 383.1623, found
383.1606.
A dielectric constant of 47 Debye was used in all
minimizations as well as in MD runs. Minimizations were
done first with steepest decent, followed by conjugate
gradient methods for a maximum of 2000 iterations each or
RMS deviation of 0.005 kcal/mol, whichever was earlier.
The energy-minimized structures were then subjected to
MD studies. A number of inter atomic distance constraints
(more than three bond away) were used in the MD studies
that were derived from the rOe cross-peaks (see Sup-
plementary Information) on the basis of two-spin approxi-
˚
mation by taking the TyrCbH protons distance (1.8 A) as an
internal standard. For all the amide bonds, the torsional
angle 1808 was used as a constraint. No H-bonding
constraint was used. For distance constraints, a force
4.3.2. (5S)-Tetrahydro-5-(hydroxymethyl)-2-furancar-
bonitrile (9). The lactol 8 (95 g, 264 mmol) was dissolved
in CH2Cl2 (500 mL) and the solution was cooled to 0 8C.
Et3N (55.2 mL, 396 mmol) was added drop wise. After
10 min, Ac2O (30 mL, 317 mmol) was added followed by
DMAP (6.48 g, 53 mmol) and stirred for 30 min. The
reaction was then quenched with saturated NH4Cl solution,
the organic layer was separated and the aqueous layer was
extracted with EtOAc. The combined organic extracts were
washed with water, brine, dried (Na2SO4) and concentrated
in vacuo. Purification by column chromatography afforded
the expected acetate intermediate (102 g) in 96% yield as a
mixture of diastereomers at the anomeric position.
˚
constant of 15 kcal/A was applied in the form of flat bottom
˚
potential well and a force constant of 5 kcal/A was
employed for the dihedral angle constraints.19 The energy-
minimized structures were subjected to constrained MD
simulations for duration of 300 ps using 50 cycles, each of
6 ps period, of the Simulated Annealing protocol. The
atomic velocities were applied following Boltzmann
distribution about the center of mass, to obtain a starting
temperature of 700 deg K.23
After simulating for 1 ps at high temperature, the system
temperature was reduced exponentially over a 5 ps period to
reach a final temperature of 300 8K. Structures were
sampled after every two cycle, leading to an ensemble of
total 25 structures. The sampled structures were energy-
minimized using the above-mentioned protocol and the
superimposed structures obtained by backbone alignment
are shown in Figures 1–4. To determine the backbone and
the average pair-wise heavy atom RMSD, the structures
were analyzed using the MOLMOL program.24
The acetate (100 g, 249 mmol) was dissolved in acetonitrile
(500 mL) and to it trimethylsilyl cyanide (50 mL,
375 mmol) was added at room temperature. This was
followed by the addition of BF3.Et2O (25.2 mL, 199 mmol)
and the reaction mixture was stirred for 4 h. The solution
was then concentrated and purified by column chromato-
graphy to afford the title compound 9 (20.2 g) as a mixture
of diastereomers in 64% yield. Data for 9: RfZ0.39 (silica
gel, 70% EtOAc in petroleum ether); IR (neat) nmax 3410
(br), 2896, 1752, 1704, 1432, 1204, 1166, 1120, 1040,
784 cmK1; 1H NMR (CDCl3, 500 MHz, mixture of isomers
1:1) d 4.79 and 4.71 (two dd, JZ4.2, 7.8, 3.62, 7.8 Hz, total
1H), 4.33–4.28 and 4.21–4.15 (two m, total 1H), 3.78 (m,
4.3.1. (5S)-Tetrahydro-5-[(triphenylmethoxy)methyl]-2-
furanol (8). To a solution of 7 (46 g, 396 mmol) in dry
CH2Cl2 (800 mL), Et3N (82.8 mL, 594 mmol), trityl
chloride (121.4 g, 435.6 mmol) and DMAP (9.68 g,
79.2 mmol) were sequentially added at 0 8C and stirred at

T. K. Chakraborty et al. / Tetrahedron 60 (2004) 8329–8339
8337
1H), 3.66 and 3.56 (two dd, JZ6.03, 12.0, 4.83, 12.0 Hz,
2H), 2.38–2.25 and 2.13–2.00 (two m, 2H), 2.21–2.14 and
1.94–1.86 (two m, 2H); 13C NMR (CDCl3, 75 MHz, mixture
of isomers) d 119.40, 119.00, 82.19, 80.82, 66.72, 66.40,
64.27, 63.90, 31.99, 31.61, 26.52, 26.03; MS (ESI): m/z (%):
150 (12) [MCNa]C; HRMS (ESI): Calcd for C6H9NO2Na
[MCNa]C 150.0531, found 150.0531.
(DMSO-d6, 75 MHz) d 174.97, 156.53, 144.00, 140.86,
127.79, 127.23, 125.37, 120.25, 79.22, 76.51, 65.66, 46.84,
44.34, 29.98, 27.80; MS (ESI): m/z (%): 390 (100) [MC
Na]C, 406 (10) [MCK]C; HRMS (ESI): Calcd for
C21H21NO5Na [MCNa]C 390.1317, found 390.1310.
4.3.5. N-(9-Fluorenylmethoxycarbonyl)-6-amino-2,5-
anhydro-1-O-(tert-butyldiphenyl)silyl-6-deoxy-D-glucitol
(13). To a solution of compound 12 (9.56 g, 21.55 mmol) in
MeOH was added 10% Pd(OH)2 on C (1.08 g). It was
hydrogenated for 8 h under atmospheric pressure using a H2
balloon. The reaction mixture was then filtered through a
short pad of Celite and the filter cake was washed with
MeOH. The filtrate and washings were combined and
concentrated in vacuo. The residue was azeotroped with dry
toluene and used directly in the next step without further
purification.
4.3.3. (2S,5R)-5-[N-(9-Fluorenylmethoxycarbonyl)-ami-
nomethyl]tetrahydro-2-furanmethanol (10). To the sol-
ution of 9 (20 g, 157.48 mmol) in dry ether (350 mL) at
0 8C, LiAlH4 (14.94 g, 393.7 mmol) was added portion
wise. After the addition was over the solution was refluxed
for 3 h. It was then cooled to 0 8C and quenched by the
sequential addition of water (15 mL), 3 M NaOH (15 mL)
and water (45 mL). Stirring was continued till free flowing
solids were formed. Then the mixture was filtered through a
sintered funnel, and washed thoroughly with EtOAc. The
combined organic filtrate and washings were concentrated
in vacuo to dryness. The resulting crude amine was
dissolved in CH2Cl2 (250 mL), FmocOSu (58.4 g,
173.23 mmol) was added at 0 8C and stirred for 1 h at rt.
Then the solution was concentrated in vacuo and directly
subjected to purification by column chromatography to
separate the isomers furnishing the title compound 10
(15.1 g, 57% yield) as colorless oil. Data for 10: RfZ0.33
(silica gel, 70% EtOAc in petroleum ether); [a]26DZK4.47
(c 1.9, CHCl3); IR (neat) nmax 3426 (br), 2924, 2850, 2337,
The above-prepared intermediate triol was dissolved in dry
DMF (65 mL) and treated at 0 8C under a nitrogen
atmosphere with Et3N (4.5 mL, 32.33 mmol). After
10 min, TBDPSCl (6.08 mL, 23.71 mmol) followed by
DMAP (264 mg, 2.16 mmol) were added. After stirring for
3 h at room temperature, the reaction mixture was diluted
with EtOAc, washed with saturated NH4Cl and brine, dried
(Na2SO4) and concentrated in vacuo. Purification by column
chromatography (SiO2, 30–35% EtOAc in petroleum ether
eluant) afforded the title compound 13 (9.84 g, 91% in two
steps). Data for 13: RfZ0.6 (silica gel 60% EtOAc in
petroleum ether); [a]2D6ZK22.83 (c 2.37, CHCl3); IR (neat)
nmax 3418 (br), 3214, 2970, 2932, 2360, 1695, 1513, 1428,
1391, 1366, 1252, 1221, 1167, 1111, 824, 770, 704, 613,
1
1704, 1538, 1436, 1220, 1085, 772, 656 cmK1; H NMR
(CDCl3, 500 MHz) d 7.76 (d, JZ7.84 Hz, 2H, ArH), 7.60
(d, JZ7.24 Hz, 2H, ArH), 7.39 (t, JZ7.24 Hz, 2H, ArH),
7.31 (t, JZ7.24, Hz, 2H, ArH), 5.22 (bs, 1H, NH), 4.45–
4.39 (m, 2H, OCH2 of Fmoc), 4.22 (t, JZ6.64 Hz, 1H,
OCH2CH of Fmoc), 4.08–4.01 (m, 2H), 3.75 (dd, JZ11.5,
1.8 Hz, 1H), 3.48 (dd, JZ11.5, 4.83 Hz, 1H), 3.36–3.22 (m,
2H), 2.45 (bs, 1H, OH), 2.02–1.95 (m, 1H), 1.93–1.86 (m,
1H), 1.84–1.78 (m, 1H), 1.68–1.55 (m, 1H); 13C NMR
(CDCl3, 75 MHz) d 157.07, 144.53, 143.90, 141.25, 128.48,
127.97, 127.61, 127.00, 125.03, 119.89, 80.30, 78.88, 66.58,
64.37, 47.16, 45.29, 28.44, 26.47, 22.65; MS (ESI): m/z (%):
376 (95) [MCNa]C; HRMS (ESI): Calcd for
C21H23NO4Na [MCNa]C 376.1525, found 376.1491.
505 cmK1 1H NMR (CDCl3, 500 MHz) d 7.71 (d, JZ
;
7.7 Hz, 2H, ArH), 7.65 (d, JZ7.7 Hz, 2H, ArH), 7.44–7.37
(m, 6H, ArH), 4.89 (br s, 1H, NH), 4.24 (m, 1H), 4.01 (dd,
JZ3.6, 8.3 Hz, 2H), 3.99 (dd, JZ4.7, 8.9 Hz, 1H), 3.94 (dd,
JZ4.7, 8.3 Hz, 1H), 3.73 (q, JZ4.7 Hz, 1H), 3.63 (br s, 1H,
OH), 3.41 (m, 1H), 3.37 (m, 1H), 2.58 (br s, 1H, OH), 1.43
(s, 9H, Boc), 1.06 (s, 9H, SitBu); 13C NMR (CDCl3,
75 MHz) d 156.58, 135.58, 135.49, 132.51, 132.30, 129.94,
127.80, 83.01, 79.63, 79.43, 79.08, 63.35, 42.41, 28.33,
26.73, 18.98; MS (LSIMS): m/z (%): 402 (95) [MCH–
C5H8O2]C; HRMS (LSIMS): Calcd for C22H32NO4Si [MC
H–C5H8O2]C 402.2101, found 402.2108.
4.3.4. (2S,5R)-5-[N-(9-Fluorenylmethoxycarbonyl)-ami-
nomethyl]tetrahydro-2-furancarboxylic acid (11). To a
solution of 10 (14 g, 39.62 mmol) in acetone (100 mL) at
0 8C, was added freshly prepared Jones reagent carefully
and drop by drop till the orange color persisted. The reaction
mixture was stirred for 0.5 h at 0 8C, then warmed to rt, and
stirred for an additional 3 h. Then the reaction mixture was
quenched with isopropanol (80 mL) and diluted with
EtOAc, washed with water, brine, dried (Na2SO4) and
concentrated in vacuo. Purification by colomun chromato-
graphy afforded the title compound 11 (9.72 g) in 67%
yield. Data for 11: RfZ0.4 (10% MeOH in CHCl3);
[a]26DZK6.4 (c 0.74, MeOH); IR (neat) nmax 3418 (br),
2898, 2841, 2363, 1628, 1436, 1372, 1256, 1134, 1070, 762,
4.3.6.
(2R,5S)-5-[N-(tert-Butoxycarbonyl)-amino-
methyl]tetrahydro-2-furanmethanol (14). To a solution
of compound 13 (9.84 g, 19.61 mmol), Ph3P (20.58 g,
78.45 mmol) and imidazole (5.34 g, 78.45 mmol) at reflux
in toluene (200 mL), I2 (14.93 g, 58.84 mmol) was added in
small portions with stirring. The white finely dispersed
complex initially formed was transformed into a clear
yellow solution that darkened as iodine was liberated at the
bottom of the reaction vessel, a dark tarry complex was
formed from which the product was gradually dissolved.
After 3 h, the reaction mixture was cooled and iodine
(4.98 g, 19.6 mmol) was added, followed by aqueous
sodium hydroxide (6.28 g, 156.88 mmol, 120 mL water).
The mixture was stirred until virtually all of the tarry red
deposits were dissolved. The mixture was transferred into a
separating funnel. The aqueous layer was separated and the
organic layer was washed successively with water, saturated
aqueous sodium thiosulphate, saturated aqueous NaHCO3
1
651 cmK1; H NMR (DMSO-d6, 500 MHz) d 7.87 (d, JZ
7.81 Hz, 2H, ArH), 7.68 (d, JZ7.21 Hz, 2H, ArH), 7.64 (br
s, 1H, NH), 7.40 (t, JZ7.2 Hz, 2H, ArH), 7.32 (t, JZ7.2 Hz,
2H, ArH), 4.34–4.19 (m, 4H), 4.01 (t, JZ6.01 Hz, 1H),
3.22–3.10 (m, 2H), 2.16 (td, JZ8.4, 20.4 Hz, 1H), 1.95 (m,
1H), 1.88 (m, 1H), 1.58 (td, JZ8.4, 19.8 Hz, 1H); 13C NMR

8338
T. K. Chakraborty et al. / Tetrahedron 60 (2004) 8329–8339
and brine. The organic layer was dried (Na2SO4), filtered
and concentrated in vacuo. Purification by column chroma-
tography (SiO2, 15–18% EtOAc in petroleum ether eluant)
afforded the olefin intermediate (7.79 g, 85%).
with EtOAc, washed with water, brine, dried (Na2SO4) and
concentrated in vacuo. Purification by column chromato-
graphy (SiO2, 12–15% MeOH in chloroform eluant)
afforded the title compound 15 (2.17 g, 85% yield). Data
for 15: RfZ0.4 (10% MeOH in CHCl3); [a]2D6ZC8.13 (c
1.01, MeOH); IR (neat) nmax 3304 (br), 3019, 1698, 1622,
1449, 1331, 1216, 1099, 977, 943, 753, 696 cmK1; 1H NMR
(DMSO-d6, 500 MHz) d 8.25 (br s, 1H, NH), 7.87 (d, JZ
7.2 Hz, 2H, ArH), 7.71 (d, JZ7.2 Hz, 2H, ArH), 7.40 (t, JZ
7.2 Hz, 2H, ArH), 7.31 (t, JZ7.2 Hz, 2H, ArH), 4.34–4.19
(m, 4H), 4.05 (t, JZ6.01 Hz, 1H), 3.25–3.15 (m, 2H), 2.08
(m, 1H), 1.94 (m, 1H), 1.80 (m, 1H), 1.61 (td, JZ8.4,
19.8 Hz, 1H); 13C NMR (DMSO-d6, 75 MHz) d 176.24,
156.64, 143.93, 140.69, 127.62, 127.12, 125.38, 120.10,
78.55, 78.08, 65.57, 46.70, 43.97, 30.28, 27.28; MS
(LSIMS): m/z (%): 390 (65) [MCNa]C; HRMS (LSIMS):
Calcd for C21H21NO5Na [MCNa]C 390.1317, found
390.1304.
To a solution of the olefin intermediate (6.52 g,
13.94 mmol) in MeOH (100 mL) was added 10% Pd(OH)2
on C (700 mg). It was hydrogenated for 1 h under
atmospheric pressure using a H2 balloon. The reaction
mixture was then filtered through a short pad of Celite and
the filter cake was washed with MeOH. The filtrate and
washings were combined and concentrated in vacuo. The
residue was azeotroped with dry toluene to afford the 3,4-
dideoxy intermediate, which was used directly in the next
step without further purification.
A solution of the above-prepared dideoxy compound in
THF (42 mL) was treated at 0 8C with TBAF (1 M in THF,
15.33 mL, 15.33 mmol). The reaction mixture was stirred at
room temperature for 3H, quenched with saturated NH4Cl
solution, and extracted with EtOAc. The combined organic
extracts were washed with brine, dried (Na2SO4) and
concentrated in vacuo. Purification by column chromato-
graphy (SiO2, 65–70% EtOAc in petroleum ether eluant)
afforded the title compound 14 (2.09 g, 65% in two steps).
Data for 14: RfZ0.4 (silica gel, 70% EtOAc in petroleum
ether); [a]2D6Z(55.59 (c 1.6, CHCl3); IR (neat) nmax 3322
(br), 3014, 2976, 1692, 1516, 1393, 1366, 1251, 1218, 1169,
1
4.3.8. Data for 1. H NMR (DMSO-d6, 500 MHz): see
Table 1; quant. HPLC profile: RP-18 column, 50!300 mm,
mobile phase: AZwater (0.1% TFA), BZacetonitrile
(0.1% TFA), gradient: 20–40% B in 20 min, detection at
220 nm, retention timeZ7.71 min; MS (LSIMS): m/z (%):
1029 (4.6) [MCH]C.
1
4.3.9. Data for 2. H NMR (DMSO-d6, 500 MHz): see
1
1080, 978, 941, 771, 697, 665 cmK1; H NMR (CDCl3,
Table 2; quant. HPLC profile: RP-18 column, 50!300 mm,
mobile phase: AZwater (0.1% TFA), BZacetonitrile
(0.1% TFA), gradient: 20–40% B in 20 min, detection at
220 nm, retention timeZ6.96 min; MS (LSIMS): m/z (%):
895 (70) [MCH]C, 917 (20) [MCNa]C; HRMS (LSIMS):
Calcd for C42H71N8O11S [MCH]C 895.4963, found
895.4970.
500 MHz) d 4.89 (t, JZ5.6 Hz, 1H, NH), 4.05 and 4.01 (two
m, 2H), 3.74 (dd, JZ2.7, 11.7 Hz, 1H), 3.49 (dd, JZ4.9,
11.7 Hz, 1H), 3.24 (ddd, JZ3.7, 5.5, 13.5 Hz, 1H), 3.18 (dd,
JZ6.7, 13.5 Hz, 1H), 2.66 (br s, 1H), 1.99 (m, 1H), 1.88 (m,
1H), 1.69 (m, 1H), 1.64 (m, 1H), 1.45 (s, 9H, Boc); 13C
NMR (CDCl3, 75 MHz) d 156.58, 80.35, 79.36, 78.99,
64.33, 44.94, 28.47, 28.31, 26.41; MS (LSIMS): m/z (%):
132 (95) [MCH–C5H8O2]C, 232 (35) [MCH]C; HRMS
(ESI): Calcd for C11H21NO4Na [MCNa]C 254.1368, found
254.1369.
1
4.3.10. Data for 3. H NMR (DMSO-d6, 500 MHz): see
Table 3; quant. HPLC profile: RP-18 column, 50!300 mm,
mobile phase: AZwater (0.1% TFA), BZacetonitrile
(0.1% TFA), gradient: 20–40% B in 20 min, detection at
220 nm, retention timeZ6.60 min; MS (LSIMS): m/z (%):
895 (100) [MCH]C, 917 (11) [MCNa]C; HRMS
(LSIMS): Calcd for C42H71N8O11S [MCH]C 895.4963,
found 895.4982.
4.3.7. (2R,5S)-5-[N-(9-Fluorenylmethoxycarbonyl)-ami-
nomethyl]tetrahydro-2-furancarboxylic acid (15). A
mixture of NaIO4 (5.58 g, 26.07 mmol) and RuCl3$3H2O
(22.5 mg, 0.087 mmol) in CH3CN/CCl4/H2O (1:1:1.5,
53 mL) was stirred at rt for 45 min and then added to a
solution of the alcohol (2.01 g, 8.69 mmol) in CH3CN
(25 mL) at 0 8C. After stirring for 5 min, an additional
amount of NaIO4 (1.86 g, 8.69 mmol) was added to the
reaction mixture. After 2 h, it was diluted with EtOAc,
washed with saturated aqueous NH4Cl, brine, dried
(Na2SO4) and concentrated in vacuo. Purification by column
chromatography afforded the acid (1.71 g, 80%).
1
4.3.11. Data for 4. H NMR (DMSO-d6, 500 MHz): see
Table 4; quant. HPLC profile: RP-18 column, 50!300 mm,
mobile phase: AZwater (0.1% TFA), BZacetonitrile
(0.1% TFA), gradient: 20–40% B in 20 min, detection at
220 nm, retention timeZ7.33 min; MS (LSIMS): m/z (%):
553 (98) [MCH]C, 575 (9) [MCNa]C; HRMS (LSIMS):
Calcd for C26H41N4O7S [MCH]C 553.2695, found
553.2686.
To a solution of the acid in dry CH2Cl2 (16 mL) at 0 8C,
trifluoroacetic acid (5 mL) was added and stirred for 2 h at
rt. The excess trifluoroacetic acid was then evaporated off on
rotary evaporator and dried thoroughly under high vacuum.
The resulting TFA salt was dissolved in dioxane and cooled
to 0 8C. Then 10% aqueous Na2CO3 (17.4 mL) followed by
FmocOSu (2.35 g, 6.97 mmol) were added and stirred for
12 h at rt. The dioxane was then evaporated in vacuo and the
residual aqueous layer was washed with EtOAc. The
aqueous layer was acidified with 1 M HCl pHz2, extracted
Acknowledgements
We thank CSIR, New Delhi for research fellowships
(V. R. R., G. S., S. U. K. and S. K. K.) and DST, New
Delhi for financial support.

T. K. Chakraborty et al. / Tetrahedron 60 (2004) 8329–8339
8339
H.; Koert, U.; Herrschaft, B.; Harms, K. Eur. J. Org. Chem.
1999, 2977–2990.
Supplementary data
Supplementary data associated with this article can be found
at doi:10.1016/j.tet.2004.07.032.
10. (a) Gringore, O. H.; Rouessac, F. P. Org. Synth. 1985, 63,
121–126. (b) Hanessian, S.; Murray, P. J. Tetrahedron 1987,
43, 5055–5072.
11. Koert, U.; Stein, M.; Harms, K. Tetrahedron Lett. 1993, 34,
2299–2302.
12. Chakraborty, T. K.; Ghosh, S. J. Ind. Inst. Sci. 2001, 81,
117–123.
References and notes
13. (a) Garegg, P. J.; Samuelsson, B. Synthesis 1979, 469–470. (b)
Garegg, P. J.; Samuelsson, B. Synthesis 1979, 813–814.
14. DCCZ1,3-dicyclohexylcarbodiimide; DIPCZ1,3-diisopro-
pylcarbodiimide; HOBtZ1-hydroxybenzotriazole; HBTUZ
O-benzotriazol-1-yl-N,N,N0,N0-tetramethyluronium hexafluoro-
phosphate; DIPEAZN,N-diisopropylethylamine; PyBOPZ
(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluoro-
phosphate; NMPZN-methyl-2-pyrrolidinone
15. (a) Bodanszky, M.; Bodanszky, A. The Practices of Peptide
Synthesis; Springer: New York, 1984. (b) Grant, G. A.
Synthetic Peptides: A User’s Guide; W.H. Freeman: New
York, 1992. (c) Bodanszky, M. Peptide Chemistry: A Practical
Textbook; Springer: Berlin, 1993.
1. (a) Said, S. I.; Mutt, V. Eur. J. Biochem. 1972, 28, 199–204.
(b) Dey, R. D.; Shannon, W. A.; Said, S. I. Cell Tissue Res.
1981, 220, 231–238. (c) Coles, S. J.; Said, S. I.; Reid, L. M. Am.
Rev. Respir. Dis. 1981, 124, 531–536. (d) Polak, J. M.; Bloom,
S. R. Exp. Lung Res. 1982, 3, 313–328. (e) Cameron, A. R.;
Hohnson, C. F.; Kirkpatrick, C. T.; Kirkpatrik, M. C. Q. J. Exp.
Physiol. 1983, 68, 413–426. (f) Saga, T.; Said, S. I. Trans. Assoc.
Am. Physicians 1984, 97, 304–310. (g) Laitinen, A.; Partanen,
M.; Hervonen, A.; Pelto-Huikko, M.; Laitinen, L. A. Histochem-
istry 1985, 82, 213–219.
2. (a) Jiang, S.; Kopras, E.; McMichael, M.; Bell, R. H.; Ulrich,
C. D. Cancer Res. 1997, 57, 1475–1480. (b) Ogasawara, M.;
Murata, J.; Ayukawa, K.; Saimi, I. Cancer Lett. 1997, 116,
111–116. (c) Shaffer, M. M.; Carney, D. N.; Korman, L. Y.;
Lebovic, G. S.; Moody, T. W. Peptides 1987, 8, 1101–1106.
3. (a) Virgolini, I.; Yang, Q.; Li, S.; Angelberger, P.; Neuhold,
N.; Niederle, B.; Scheithauer, W.; Valent, P. Cancer Res.
1994, 54, 690–700. (b) Maruno, K.; Absood, A.; Said, S. I. Proc.
Natl. Acad. Sci. U.S.A. 1998, 95, 14373–14378.
16. (a) Cavanagh, J.; Fairbrother, W. J.; Palmer, A. G.; Skelton,
N. J. Protein NMR Spectroscopy; Academic: San Diego, 1996.
(b) Wu¨thrich, K. NMR of Proteins and Nucleic Acids; Wiley:
New York, 1986.
17. Hwang, T. L.; Shaka, A. J. J. Am. Chem. Soc. 1992, 114,
3157–3159.
18. (a) Adams, P. D.; Chen, Y.; Ma, K.; Zagorski, M. G.;
¨
Sonnichsen, F. D.; McLaughlin, M. L.; Barkley, M. D. J. Am.
4. Singh, H.; Kumar, A.; Courtney, M.; Townshed, J. R.; Samad,
Z.; Singh, P. Ann. N.Y. Acad. Sci. 1988, 527, 679–681.
5. Gozes, I.; Brenneman, D. E.; Fridkin, M. M.; Moody; T. US
Patent 5 217 953, 1993, Chem Abstr. No. 116:249474x, 1992.
6. Mukherjee, R.; Jaggi, M. US Patent 6 156 725, 1996; use the
patent number in http://patft.uspto.gov/netahtml/srchnum.htm
to get the details.
Chem. Soc. 2002, 124, 9278–9286. (b) Kessler, H.; Bats,
J. W.; Griesinger, C.; Koll, S.; Will, M.; Wagner, K.
J. Am. Chem. Soc. 1988, 110, 1033–1049. (c) Kessler, H.
Angew. Chem., Int. Ed. Engl. 1982, 21, 512–523.
19. Kessler, H.; Griesinger, C.; Lautz, J.; Mu¨ller, A.; F. van
Gunsteren, W.; Berendsen, H. J. C. J. Am. Chem. Soc. 1988,
110, 3393–3396.
7. Burman, A. C.; Prasad, S.; Mukherjee, R.; Singh, A. T.;
Mathur, A.; Gupta, N. US Patent 6 489 297, 2002, Chem Abstr.
No. 137:385116t, 2002.
20. Chakraborty, T. K.; Jayaprakash, S.; Srinivasu, P.; Madhaven-
dra, S. S.; Ravi Sankar, A.; Kunwar, A. C. Tetrahedron 2002,
58, 2853–2859.
8. For recent reviews see: (a) Gruner, S. A. W.; Locardi, E.;
Lohof, E.; Kessler, H. Chem. Rev. 2002, 102, 491–514. (b)
Chakraborty, T. K.; Ghosh, S.; Jayaprakash, S. Curr. Med.
Chem. 2002, 9, 421–435. (c) Chakraborty, T. K.; Jayaprakash,
S.; Ghosh, S. Comb. Chem. High Throughput Screening 2002,
5, 373–387. (d) Schweizer, F. Angew. Chem., Int. Ed. 2002, 41,
230–253. (e) Peri, F.; Cipolla, L.; Forni, E.; La Ferla, B.;
Nicotra, F. Chemtracts Org. Chem. 2001, 14, 481–499.
9. For some earlier references of 3,4-dideoxy furanoid sugar
amino acids see: (a) Chakraborty, T. K.; Ghosh, S.;
Jayaprakash, S.; Sarma, J. A. R. P.; Ravikanth, V.; Diwan,
P. V.; Nagaraj, R.; Kunwar, A. C. J. Org. Chem. 2000, 65,
6441–6457. (b) Schrey, A.; Vescovi, A.; Knoll, A.; Rickert, C.;
Koert, U. Angew. Chem., Int. Ed. 2000, 39, 900–902. (c)
Schrey, A.; Osterkamp, F.; Straudi, A.; Rickert, C.; Wagner,
21. Hungerford, N. L.; Claridge, T. D. W.; Watterson, M. P.;
Aplin, R. T.; Moreno, A.; Fleet, G. W. J. J. Chem. Soc., Perkin
Trans. 1 2000, 3666–3679.
22. States, D. J.; Haberkorn, R. A.; Ruben, D. J. J. Magn. Reson.
1982, 48, 286–292.
23. For details see: (a) Sybyl Force Field Manual, version 6.6,
1999, pp 217–229 (http://www.tripos.com/custResources/
applicationNotes/triad/index.html). (b) Evans, J. N. S.
Biomolecular NMR Spectroscopy; Oxford University Press:
Oxford, 1995.
24. Etezady-Esfarjani, T.; Hilty, C.; Wu¨thrich, K.; Rueping, M.;
Schreiber, J.; Seebach, D. Helv. Chim. Acta 2002, 85,
1197–1209.