The role of the disulfide bond in amyloid-like fibrillogenesis in a model peptide system

Article abstract of DOI:10.1039/b509083k

Three terminally protected short peptides Bis[Boc-D-Leu(1)-Cys(2)-OMe] 1, Bis[Boc-Leu(1)-Cys(2)-OMe] 2 and Bis[Boc-Val(1)-Cys(2)-OMe] 3 exhibit amyloid-like fibrillar morphology. Single crystal X-ray diffraction analysis of peptide 1 clearly demonstrates that it adopts an overall extended backbone molecular conformation that self-assembles to form an intermolecular hydrogen-bonded antiparallel supramolecular β-sheet structure in crystals. Scanning electron microscopic (SEM) images, transmission electron microscopic (TEM) images and Congo red binding studies vividly demonstrate the amyloid-like fibril formation of peptides 1, 2 and 3. However, after reduction of the disulfide bridge of peptides 1, 2 and 3, three newly generated peptides Boc-D-Leu(1)-Cys(2)-OMe 4, Boc-Leu(1)-Cys(2)-OMe 5 and Boc-Val(1)-Cys(2)-OMe 6 are formed and all of them failed to form any kind of fibril under the same conditions, indicating the important role of the disulfide bond in amyloid-like fibrillogenesis in a peptide model system. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b509083k

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A R T I C L E
The role of the disulfide bond in amyloid-like fibrillogenesis
in a model peptide system
Apurba Kumar Das,a Michael G. B. Drew,b Debasish Haldara and Arindam Banerjee*a
a Department of Biological Chemistry, Indian Association for the Cultivation of Science,
Jadavpur, Kolkata 700 032, India. E-mail: bcab@mahendra.iacs.res.in;
Fax: +91-33-2473-2805
b School of Chemistry, The University of Reading, Whiteknights, Reading, UK RG6 6AD
Received 28th June 2005, Accepted 20th July 2005
First published as an Advance Article on the web 11th August 2005
Three terminally protected short peptides Bis[Boc–D-Leu(1)–Cys(2)–OMe] 1, Bis[Boc–Leu(1)–Cys(2)–OMe] 2 and
Bis[Boc–Val(1)–Cys(2)–OMe] 3 exhibit amyloid-like fibrillar morphology. Single crystal X-ray diffraction analysis of
peptide 1 clearly demonstrates that it adopts an overall extended backbone molecular conformation that
self-assembles to form an intermolecular hydrogen-bonded antiparallel supramolecular b-sheet structure in crystals.
Scanning electron microscopic (SEM) images, transmission electron microscopic (TEM) images and Congo red
binding studies vividly demonstrate the amyloid-like fibril formation of peptides 1, 2 and 3. However, after reduction
of the disulfide bridge of peptides 1, 2 and 3, three newly generated peptides Boc–D-Leu(1)–Cys(2)–OMe 4,
Boc–Leu(1)–Cys(2)–OMe 5 and Boc–Val(1)–Cys(2)–OMe 6 are formed and all of them failed to form any kind of
fibril under the same conditions, indicating the important role of the disulfide bond in amyloid-like fibrillogenesis
in a peptide model system.
Introduction
Self-assembly of short model peptides containing suitable molec-
ular conformations to form supramolecular b-sheet architecture
is a highly emerging field of current research.1,2 Recent reports
have demonstrated that self-assembling b-sheet peptide scaffolds
can act as biomaterials for neurite outgrowth and they are useful
for tissue repair and tissue engineering.3 Accumulating evidence
indicates that many fatal neurodegenerative diseases including
Alzheimer’s disease,4 Huntington’s disease,5 Parkinson’s disease6
and prion related encephalopathies7 occur due to the formation
and deposition of amyloid fibrils as plaques in specific regions
of the brain. Other examples of amyloid diseases include
type II diabetes where misfolded islet amyloid polypeptides
(IAPP) form amyloid fibrils in the pancreas8 and fatal familial
cardiomyopathy amyloid deposition occurs in cardiac tissues.9
Patients undergoing long term haemo-dialysis generally suffer
from b2-microglobulin (b2M) amyloidosis.10 Recently, Goto and
coworkers have established that the disulfide bond plays an es-
sential role in the amyloid fibril formation of b2-microglobulin.10
A recent report also suggests that in transthyretin amyloidosis,
Cys10 mixed disulfides of TTR mutants are more amyloidogenic
than the wild type transthyretin under mildly acidic conditions.11
Austen and his coworkers have demonstrated that a disulfide
bond is required for the formation of b-sheet structure and
amyloid fibrils in Familial British Dementia (FBD), a rare
autosomal dominant neurodegenerative disorder that shares
features of Alzheimer’s disease.12
Fig. 1 The schematic presentation of peptides 1–6.
Previously, Atkins and Lyon have shown that the oxidized
disulfide form of glutathione self-assembles into an extended
network of intermolecular, antiparallel sheets in appropriate
organic solvents to form gels.13 We are involved in developing
model peptides that form supramolecular parallel or antiparallel
b-sheets in crystals and amyloid-like fibrils in the solid state14
and we are interested in investigating the role of the disulfide
bond in amyloid-like fibril formation in our model peptide
system. To this end we have synthesized three terminally pro-
tected short model peptides Bis[Boc–D-Leu(1)–Cys(2)–OMe]
1, Bis[Boc–Leu(1)–Cys(2)–OMe] 2 and Bis[Boc–Val(1)–Cys(2)–
OMe] 3 (Fig. 1). Compounds Boc–D-Leu(1)–Cys(2)–OMe 4,
Boc–Leu(1)–Cys(2)–OMe 5 and Boc–Val(1)–Cys(2)–OMe 6
(Fig. 1) were obtained from the reduction of the disulfide
bond of peptides 1, 2 and 3. In this paper we address the
question of whether the presence of the disulfide bridge is
necessary to form amyloid-like fibrils or not. We present here the
detailed structural analysis of peptide 1 using single crystal X-
ray diffraction study and the fibrillation study of this peptide and
other structure related peptides (peptides 2 and 3) in the solid
state. Congo red binding studies of the fibrils generated from
peptides 1, 2 and 3 were performed in order to probe whether
these fibrils are amyloid-like or not. Whether the peptides with
reduced disulfide bonds (peptides 4, 5 and 6) can form fibrils is
also addressed here.
3 5 0 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 5 0 2 – 3 5 0 7
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
©

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molecules. Each b-sheet column is then stacked via van der
Waals interactions to form a highly ordered supramolecular
structure along the axis parallel to the crystallographic c
direction (Fig. 4). Fig. 5 presents a schematic illustration of
the stepwise self-assembly of peptide 1 into the supramolecular
b-sheet architecture.
Results and discussion
Single crystal X-ray diffraction study
Colorless monoclinic crystals of peptide 1, suitable for an X-ray
diffraction study were obtained from a methanol–water solution
by slow evaporation. The molecule shown in Fig. 2 contains a
crystallographic C2 axis though the S–S bond. There are no
intramolecular hydrogen bonds. The dihedral angle values φ1
(122.2(6)◦) and w1 (−111.2(5)◦) fall within the parallel b-sheet
region whereas φ2 (50.1(6)◦) and w2 (40.6(6)◦) fall within the
right handed a-helical region of the Ramachandran map.15 Here,
the adoption of positive φ, w values of the D-leucine residue is
a common feature. However, the molecular conformation of
this peptide looks like an overall extended backbone b-strand
structure (Fig. 2). There is an intramolecular disulfide bond that
connects the two similar parts of the molecule (Fig. 1). The
dihedral angle around the S–S bond is 87.9(2)◦. Self-assembly
of the individual monomers leads to the formation of a b-sheet
ribbon along the crystallographic b axis (Fig. 3). The b-sheet
ribbon structure is stabilized by four intermolecular hydrogen
bonds (two from each part of the molecule) N3–H3 · · · O4 and
N6–H6 · · · O7 (Table 1) that connect the individual peptide
Fig. 4 Higher order packing of peptide 1 along the crystallographic c
axis forming quaternary b-sheet structures. Hydrogen bonds are shown
as dotted lines.
Fig. 5 Schematic illustration of the stepwise self-assembly of disulfide
bridge peptide 1 into the supramolecular b-sheet architecture.
FT-IR studies
The FT-IR studies of all reported peptides indicate that NH-
stretching vibrations fall within the range from 3315–3341 cm−1
corresponding to an intermolecular H-bonded aggregated struc-
ture and the CO-stretching vibrations are within the range
from 1653–1689 cm−1 (Table 2) which are suggestive of inter-
molecularly H-bonded aggregated sheet-like structures for these
peptides in the solid state.16
Fig. 2 The ORTEP diagram of peptide 1 with atomic numbering
scheme. Ellipsoids are at a level of 30% probability.
Morphological study
To examine the role of the disulfide bond in fibril formation,
morphological studies of peptides 1, 2 and 3 were carried out
using a scanning electron microscope (SEM) and a transmission
electron microscope (TEM). The SEM images of peptides 1,
2 and 3 (Fig. 6) of the dried fibrous material grown from
methanol–water clearly demonstrate that the aggregates in
the solid state have amyloid-like fibrillar morphology.17 The
SEM image of peptide 1 shows the entangled fibrillar network
structure while the SEM image of peptide 2 exhibits the
filamentous fibrillar structure. Fig. 6(c) shows the SEM picture
Fig. 3 Packing of peptide 1, showing the self-assembly of indi-
vidual monomers, leads to the formation of an intermolecular hy-
drogen-bonded supramolecular b-sheet ribbon structure along the
crystallographic b axis. Hydrogen bonds are shown as dotted lines.
Table 2 Infrared (IR) absorption frequencies (cm−1) for all reported
peptides in the solid state (on a KBr pellet)
Peptide
CO stretch
NH bend
NH stretch
Table 1 Intermolecular hydrogen bonding parameters of peptide 1 in
the crystal state
1
2
3
4
5
6
1655 (s)
1664 (s)
1654 (s)
1662 (s)
1679 (s)
1689 (s), 1653 (m)
1532 (m)
1540 (m)
1532 (m)
1527 (m)
1540 (m)
1531 (m)
3331 (s)
3315 (s)
3330 (s)
3341 (s)
3337 (s)
3330 (s)
˚
˚
D–H · · · A
H · · · A/A
D · · · A/A
D–H · · · A/◦
N3–H3 · · · O4a
2.18
2.13
3.04
2.98
178
169
N6–H6 · · · O7b
a Symmetry element x, 1 + y, z. b Symmetry element x, −1 + y, z.
s = strong, m = medium
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 5 0 2 – 3 5 0 7
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Fig. 6 SEM images of (a) peptide 1 showing fibrillar morphology, (b) peptide 2 showing filamentous fibrillar morphology and (c) peptide 3 showing
fibrillar morphology in the solid state.
of peptide 3 suggesting a fibrillar network structure. The TEM
image of peptide 1 also shows remarkable amyloid-like fibrillar
morphology (Fig. 7). The representative TEM image of peptide
1 reveals that the peptide exists as a bunch of long unbranched
filaments. However, peptides 4, 5 and 6 have failed to form any
kind of fibril under the same conditions. The SEM image of
peptide 6 does not show any kind of fibrillar structure in the
solid state (Fig. 8).
Fig. 9 Optical microscopic images of (a) peptide 2 fibrils stained with
a Congo red dye showing a green-gold birefringence, a characteristic
feature of amyloid fibrils and (b) peptide 5 stained with a Congo red
dye does not show any birefringence observed at 100 × magnification
between crossed polarizers.
From the FT-IR studies of peptides 1–6, it is clear that all
these peptides form intermolecularly hydrogen-bonded sheet-
like aggregated structures. The single crystal X-ray structure
of peptide 1 clearly established that it has self-assembled to
form an intermolecularly hydrogen-bonded supramolecular b-
Fig. 7 TEM image of peptide 1 shows amyloid-like fibrillar morphol-
ogy in the solid state.
sheet structure and it forms amyloid-like fibrils in the solid
state upon further aggregation. Similar fibrillation has been
observed for peptides 2 and 3 containing disulfide bonds. All
these peptide fibrils bind with a physiological Congo red dye and
exhibit typical birefringence under a cross polarizer suggesting
the similarity of these fibrils to amyloid fibrils. However, peptides
in which disulfide bonds are reduced (peptides 4, 5 and 6) are
unable to form any kind of fibrillation. This clearly indicates
that disulfide bonds must play a definite role in amyloid-like fibril
formation in our model peptide system. It has also been reported
that the disulfide linkage plays a crucial role in amyloid fibril
formation from various non-related amyloidogenic proteins.10–12
The linkage between the disulfide bond and fibril formation is
closely related. Our model peptide system vividly demonstrates
the role of the disulfide bridge in the formation of amyloid-like
fibrils.
Fig. 8 SEM image of peptide 6 does not show any kind of fibrillar
morphology in the solid state.
The morphological resemblance of peptide 1, 2 and 3 fibrils
with amyloid plaque was also studied by Congo red staining.
It has been reported that Congo red binds with amyloid
fibrils responsible for various neurodegenerative diseases like
Alzheimer’s disease, Parkinson’s disease and shows distinct
birefringence under polarized light.17 To determine the similarity
of these aggregated fibrils with Alzheimer’s b-amyloid fibrils,
fibrils obtained from peptides 1, 2 and 3 have been stained
with Congo red and observed through cross polarizers. All
these peptide fibrils bind with Congo red and exhibit typical
birefringence under a polarized microscope. Fig. 9(a) shows
a typical green-gold birefringence of Congo red bound fibrils
of peptide 2 viewed through a cross polarizer. These results
are consistent with Congo red binding to an amyloid cross-b-
sheet fibrillar structure.18 Peptides 4, 5 and 6 do not exhibit
typical birefringence when they are stained with Congo red and
viewed through cross polarizers. Fig. 9(b) does not show any
birefringence when peptide 5 is stained with Congo red and
viewed under a polarized microscope.
Conclusions
This paper clearly demonstrates that peptide 1 containing
a disulfide bond self-assembles to form an intermolecularly
hydrogen-bonded antiparallel supramolecular b-sheet structure,
as it is apparent from its single crystal X-ray diffraction study.
The subunit of the supramolecular b-sheet of peptide 1 is a
new motif, a non-intramolecularly hydrogen bonded structure
with a buried interchain disulfide bond. This peptide and other
structurally related peptides 2 and 3 form fibrils upon further
aggregation. FT-IR data indicate that these other peptides (2
and 3) containing disulfide bonds also form intermolecularly
hydrogen-bonded b-sheet like structures. These fibrils show a
close resemblance to amyloid fibrils. However, peptides 4, 5 and
6 without any disulfide bonds have failed to form amyloid-like
fibrils under the same conditions. This establishes the role of the
disulfide bond in amyloid-like fibril formation in our model
peptide studies. So, this study of amyloid-like fibril forming
3 5 0 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 5 0 2 – 3 5 0 7

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model peptides with antiparallel b-sheet structures containing a
disulfide bond at atomic resolution, may assist in understanding
the self-assembly mechanism of amyloid fibril formation and
the role of the disulfide bond in this type of fibrillation. It is an
important issue to find out the specific cause for the loss of fibril
formation after reducing the disulfide bridge. It might be the
increased molecular weight (i. e. the larger hydrogen bonding
capacity per unit) and/or the presence of the disulfide bond
itself. Studies regarding this direction are yet to be explored and
it is going on in our laboratory.
corresponding methyl ester hydrochloride by neutralization and
subsequent extraction with ethyl acetate and the ethyl acetate
extract was concentrated to 8 mL. It was then added to the
reaction mixture, followed immediately by 1.75 g (8.5 mmol)
DCC and 1.15 g (8.5 mmol) of HOBt. The reaction mixture
was stirred for three days. The residue was taken up in ethyl
acetate (40 mL) and the DCU was filtered off. The organic layer
was washed with 2 M HCl (3 × 40 mL), brine (2 × 50 mL),
1 M sodium carbonate (3 × 40 mL), brine (2 × 40 mL), dried
over anhydrous sodium sulfate and evaporated in vacuo to yield
2 as a white solid. Purification was done by silica gel column
(100–200 mesh) using chloroform–methanol as eluent. Yield =
2.57 g (3.7 mmol, 87.1%). (Found: C, 51.85; H, 7.68; N, 8.11%.
C30H54N4O10S2 requires: C, 51.87; H, 7.78; N, 8.07%); IR(KBr):
Experimental
Synthesis of peptides
3315, 1749, 1664, 1540 cm−1; [a]D = +47.4 (c 0.21, CHCl3);
20
The dipeptide subunits 1, 2 and 3 employed in this report
were synthesized by conventional solution phase methodology.19
The Boc group was used for N-terminal protection and the
C-terminus was protected as a methyl ester. Couplings were
mediated by dicyclohexylcarbodiimide–1-hydroxybenzotriazole
(DCC–HOBt). The final compounds were fully characterized by
IR spectroscopy, 1H NMR spectroscopy and mass spectrometry.
1H NMR (300 MHz, CDCl3) d 7.6 (Cys(2) NH, 2H, d, J =
6.8 Hz), 5.24 (Leu(1) NH, 2H, d, J = 8.3 Hz), 4.89 (CaH of
Cys(2), 2H, m), 4.30 (CaH of Leu(1), 2H, m), 3.75 (–OCH3,
6H, s), 3.12 (CbHs Cys(2), 4H, m), 1.65 (CbHs & CcH of Leu(1),
6H, m), 1.43 (Boc–CH3s, 18H, s), 0.93 (CdHs of Leu(1), 12H,
m). MALDI-MASS (M + Na)+ = 717.1, Mcalcd = 694.
Bis[Boc–Val(1)–Cys(2)–OMe] 3
Bis[Boc–D-Leu(1)–Cys(2)–OMe] 1
Boc–Val(1)–OH 9. A solution of valine (1.17 g, 10 mmol)
in a mixture of dioxan (20 mL), water (10 mL) and 1 M NaOH
(10 mL) was stirred and cooled in an ice–water bath. Di-tert-
butylpyrocarbonate (2.4 g, 11 mmol) was added and stirring
was continued at room temperature for 6 h. Then the solution
was concentrated in vacuo to about 40–60 mL, cooled in an ice–
water bath, covered with a layer of ethyl acetate (about 50 mL)
and acidified with a dilute solution of KHSO4 to pH 2–3 (Congo
red). The aqueous phase was extracted with ethyl acetate and this
operation was done repeatedly. The ethyl acetate extracts were
pooled, washed with water and dried over anhydrous Na2SO4
and evaporated in vacuo. Pure material was obtained as a white
solid. Yield = 1.91 g (8.8 mmol, 88%).
Boc–D-Leu(1)–OH 7. A solution of D-leucine (1.31 g,
10 mmol) in a mixture of dioxan (20 mL), water (10 mL) and 1 M
NaOH (10 mL) was stirred and cooled in an ice–water bath. Di-
tert-butylpyrocarbonate (2.4 g, 11 mmol) was added and stirring
was continued at room temperature for 6 h. Then the solution
was concentrated in vacuo to about 40–60 mL, cooled in an ice–
water bath, covered with a layer of ethyl acetate (about 50 mL)
and acidified with a dilute solution of KHSO4 to pH 2–3 (Congo
red). The aqueous phase was extracted with ethyl acetate and this
operation was done repeatedly. The ethyl acetate extracts were
pooled, washed with water and dried over anhydrous Na2SO4
and evaporated in vacuo. Pure material was obtained as a white
solid.
Yield = 2.01 g (8.7 mmol, 87%).
Bis[Boc–Val(1)–Cys(2)–OMe] 3. 1.84 g (8.5 mmol) of Boc–
Val(1)–OH 9 in 15 mL of DMF were cooled in an ice–water bath
and H–cystine–OMe was isolated from 2.9 g (8.5 mmol) of the
corresponding methyl ester hydrochloride by neutralization and
subsequent extraction with ethyl acetate and the ethyl acetate
extract was concentrated to 8 mL. It was then added to the
reaction mixture, followed immediately by 1.75 g (8.5 mmol)
DCC and 1.15 g (8.5 mmol) of HOBt. The reaction mixture
was stirred for three days. The residue was taken up in ethyl
acetate (40 mL) and the DCU was filtered off. The organic
layer was washed with 2 M HCl (3 × 40 mL), brine (2 ×
50 mL), 1 M sodium carbonate (3 × 40 mL), brine (2 × 40 mL),
dried over anhydrous sodium sulfate and evaporated in vacuo
to yield 3 as a white solid. Purification was done by silica gel
column (100–200 mesh) using chloroform–methanol as eluent.
Yield = 2.4 g (3.6 mmol, 85%). (Found: C, 50.49; H, 7.49; N,
8.35%. C28H50N4O10S2 requires: C, 50.45; H, 7.51; N, 8.41%);
Bis[Boc–D-Leu(1)–Cys(2)–OMe] 1. 1.96 g (8.5 mmol) of
Boc–D-Leu(1)–OH 7 in 15 mL of DMF were cooled in an
ice–water bath and H–cystine–OMe was isolated from 2.9 g
(8.5 mmol) of the corresponding methyl ester hydrochloride by
neutralization and subsequent extraction with ethyl acetate and
the ethyl acetate extract was concentrated to 8 mL. It was then
added to the reaction mixture, followed immediately by 1.75 g
(8.5 mmol) DCC and 1.15 g (8.5 mmol) of HOBt. The reaction
mixture was stirred for three days. The residue was taken up
in ethyl acetate (40 mL) and the DCU was filtered off. The
organic layer was washed with 2 M HCl (3 × 40 mL), brine (2 ×
50 mL), 1 M sodium carbonate (3 × 40 mL), brine (2 × 40 mL),
dried over anhydrous sodium sulfate and evaporated in vacuo
to yield 1 as a white solid. Purification was done by silica gel
column (100–200 mesh) using chloroform–methanol as eluent.
Yield = 2.5 g (3.6 mmol, 84.7%). (Found: C, 51.89; H, 7.82;
N, 8.1%. C30H54N4O10S2 requires: C, 51.87; H, 7.78; N, 8.07%);
20
IR(KBr): 3330, 1748, 1691, 1654, 1532 cm−1; [a]D = +69.9
1
(c 0.21, CHCl3); H NMR (300 MHz, CDCl3) d 7.46 (Cys(2)
20
IR(KBr): 3331, 1730, 1692, 1655, 1532 cm−1; [a]D = +74.7
NH, 2H, d, J = 6.6 Hz), 5.36 (Val(1) NH, 2H, d, J = 8.6 Hz),
4.91 (CaH of Cys(2), 2H, m), 4.08–4.13 (CaH of Val(1), 2H, m),
3.75 (OCH3, 6H, s), 3.09 (CbHs Cys(2), 4H, m), 2.07 (CbH of
Val(1), 2H, m), 1.43 (Boc–CH3s, 18H, s), 0.99 (CcHs of Val(1),
12H, m). ESI-MS (M + Na)+ = 689.3, Mcalcd = 666.
(c 0.2, CHCl3); 1H NMR (300 MHz, CDCl3) d 7.09 (Cys(2) NH,
2H, d, J = 9 Hz), 4.99 (D-Leu(1) NH, 2H, d, J = 11.2 Hz), 4.79
(CaH of Cys(2), 2H, m), 4.19 (CaH of D-Leu(1), 2H, m), 3.76
(–OCH3, 6H, s), 3.19 (CbHs Cys(2), 4H, m), 1.66 (CbHs & CcH
of D-Leu(1), 6H, m), 1.45 (Boc–CH3s, 18H, s), 0.92 (CdHs of
D-Leu(1), 12H, m). ESI–MS (M + Na)+ = 717.2, Mcalcd = 694.
The disulfide bonds of peptides 1, 2 and 3 were reduced by
refluxing with indium and NH4Cl at 60 ◦C in dry ethanol for
2 hours to obtain the peptides Boc–D-Leu(1)–Cys(2)–OMe 4,
Boc–Leu(1)–Cys(2)–OMe 5 and Boc–Val(1)–Cys(2)–OMe 6.21
Bis[Boc–Leu(1)–Cys(2)–OMe] 2
Boc–Leu(1)–OH 8. See ref. 20.
Boc–D-Leu(1)–Cys(2)–OMe 4. (Found: C, 51.75; H, 8.07;
Bis[Boc–Leu(1)–Cys(2)–OMe] 2. 1.96 g (8.5 mmol) of Boc–
Leu(1)–OH 8 in 15 mL of DMF were cooled in an ice–water bath
and H–cystine–OMe was isolated from 2.9 g (8.5 mmol) of the
N, 8.1%. C15H28N2O5S requires: C, 51.72; H, 8.05; N, 8.05%);
20
IR(KBr): 3341, 2383, 1751, 1686, 1662, 1527cm−1; [a]D = +70.5
(c 1.12, CHCl3); 1H NMR (300 MHz, CDCl3) d 7.10 (Cys(2) NH,
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 5 0 2 – 3 5 0 7
3 5 0 5

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1H, d, J = 6 Hz), 5.00 (D-Leu(1) NH, 1H, d, J = 9 Hz), 4.78
(CaH of Cys(2), 1H, m), 4.18 (CaH of D-Leu(1), 1H, m), 3.78
(OCH3, 3H, s), 3.19 (CbHs Cys(2), 2H, m), 1.64 (CbHs & CcH
of D-Leu(1), 3H, m), 1.45 (Boc–CH3s, 9H, s), 0.94 (CdHs of
D-Leu(1), 6H, m). ESI-MS (M + Na)+ = 371.2, Mcalcd = 348.
with MoKa radiation using the MARresearch Image Plate Sys-
tem. The crystal was positioned at 70 mm from the Image Plate.
100 frames were measured at 2◦ intervals with a counting time
of 5 min to give 2802 independent reflections. Data analysis was
carried out with the XDS program.22 The structure was solved
using direct methods with the Shelx86 program.23 The non-
hydrogen atoms were refined with anisotropic thermal parame-
ters. The hydrogen atoms were included in geometric positions
and given thermal parameters equivalent to 1.2 times those of
the atom to which they were attached. The structure was refined
on F2 using Shelxl.24 The final R values were R1 0.0807 and wR2
0.1785 for 2488 data with I > 2r(I). The largest peak and hole
Boc–Leu(1)–Cys(2)–OMe 5. (Found: C, 51.7; H, 8.02; N,
8.09%. C15H28N2O5S requires: C, 51.72; H, 8.05; N, 8.05%);
IR(KBr): 3337, 2566, 1715, 1679, 1540 cm−1; [a]D = +23.8
20
1
(c 1.1, CHCl3); H NMR (300 MHz, CDCl3) d 7.63 (Cys(2)
NH, 1H, d, J = 6.3 Hz), 5.31 (Leu(1) NH, 1H, d, J = 7.8 Hz),
4.86 (CaH of Cys(2), 1H, m), 4.30 (CaH of Leu(1), 1H, m), 3.74
(OCH3, 3H, s), 3.08 (CbHs Cys(2), 2H, m), 1.68 (CbHs & CcH of
Leu(1), 3H, m), 1.42 (Boc-CH3s, 9H, s), 0.93 (CdHs of Leu(1),
6H, m). ESI-MS (M + Na)+ = 371.4, Mcalcd = 348.
−3
˚
in the final difference Fourier were 0.43 and −0.26 e A .†
Acknowledgements
Boc–Val(1)–Cys(2)–OMe 6. (Found: C, 50.35; H, 7.75; N,
We thank the EPSRC and the University of Reading, UK
for funds for the Image Plate System. This research was also
supported by a grant from the Department of Science and
Technology (DST), India (project no. SR/S5/OC-29/2003).
A. K. Das wishes to acknowledge the CSIR, New Delhi, India
for financial assistance.
8.43%. C14H26N2O5S requires: C, 50.3; H, 7.78; N, 8.38%);
IR(KBr): 3330, 2354, 1747, 1689, 1653, 1531 cm−1; [a]D
=
20
1
+49.4 (c 1.02, CHCl3); H NMR (300 MHz, CDCl3) d 7.45
(Cys(2) NH, 1H, d, J = 5.7 Hz), 5.37 (Val(1) NH, 1H, d, J =
8.6 Hz), 4.90 (CaH of Cys(2), 1H, m), 4.13 (CaH of Val(1), 1H,
m), 3.75 (OCH3, 3H, s), 3.11 (CbHs Cys(2), 2H, m), 2.11 (CbH
of Val(1), 1H, m), 1.43 (Boc–CH3s, 9H, s), 0.96 (CcHs of Val(1),
6H, m). ESI-MS (M + Na)+ = 357.4, Mcalcd = 334.
References
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Crystal data for peptide 1
C30H54N4O10S2, Mw = 694.91, monoclinic, space group C2, a =
◦
˚
25.41(3), b = 5.071(7), c = 15.318(17) A, b = 106.77(10) , U =
† CCDC reference number 276179. See http://dx.doi.org/10.1039/
b509083k for crystallographic data in CIF or other electronic format.
3
1890 A , Z = 2, Dcalc = 1.221 g cm−3. Intensity data were collected
˚
3 5 0 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 5 0 2 – 3 5 0 7

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O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 5 0 2 – 3 5 0 7
3 5 0 7