Synthesis, redox properties, and conformational analysis of vicinal disulfide ring mimics

Article abstract of DOI:10.1016/j.tet.2008.11.085

A vicinal disulfide ring (VDR) results from disulfide-bond formation between two adjacent cysteine residues. This eight-membered ring is a rare motif in protein structures and is functionally important to those few proteins that posses it. This article focuses on the construction of strained and unstrained VDR mimics, discernment of the preferred conformation of these mimics, and the determination of their respective disulfide redox potentials.

Full text of DOI:10.1016/j.tet.2008.11.085

Tetrahedron 65 (2009) 1257–1267
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis, redox properties, and conformational analysis of vicinal
disulfide ring mimics
,
Erik L. Ruggles a, P. Bruce Deker b, Robert J. Hondal a
*
a Department of Biochemistry, University of Vermont, Burlington, VT 05405, USA
b Department of Chemistry, University of Vermont, Burlington, VT 05405, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 October 2008
Received in revised form 21 November 2008
Accepted 25 November 2008
Available online 30 November 2008
A vicinal disulfide ring (VDR) results from disulfide-bond formation between two adjacent cysteine
residues. This eight-membered ring is a rare motif in protein structures and is functionally important to
those few proteins that posses it. This article focuses on the construction of strained and unstrained VDR
mimics, discernment of the preferred conformation of these mimics, and the determination of their
respective disulfide redox potentials.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Cyclocystine
Dithiocine
Dithiazacanones
Eight-membered ring
Vicinal cysteines
Thiol–disulfide redox
1. Introduction
important role for this ring system is to act as a conformational
switch and it is able to do so in two ways. First it may act as
a conformational switch by lowering the trans/cis isomerization
barrier of the peptide backbone. Several NMR studies of model
1.1. VDR biological function
A vicinal disulfide ring (VDR, 1) is an eight-membered ring
structure that occurs as a result of disulfide-bond formation be-
tween vicinal cysteines and is a very rare occurrence in protein
structures (Fig. 1). In the most current search for this structure in
the Brookhaven Protein Data Bank (PDB), Perczel and co-workers
found 31 occurrences out of ca. w28,000 deposited protein struc-
tures.1 Given its rare occurrence in the PDB, this ring system must
have a unique structural/functional assignment where it is found.
In the Janus-faced atracotoxins’ mutation of this motif to a vicinal
serine pair resulted in no change in tertiary geometry of the pro-
tein, however, the mutant was devoid of toxicity.2 In the case of
methanol dehydrogenase, reduction of the VDR is accompanied by
complete loss of enzymatic activity. Here it has been proposed that
the ring aids in either electron transfer or conformational rigidity of
the protein during substrate binding.3,4 We believe one very
peptides containing this ring have demonstrated that an eight-
membered ring formed by a vicinal disulfide is in dynamic equi-
librium between trans and cis conformations about the amide bond
of the ring and that conversion from the trans form to the cis form
would dramatically alter protein topology.5,6 Such a process is
proposed to occur in the nicotinic acetylcholine receptor’s (nAChR)
binding site. Upon binding with acetylcholine, or other agonists, the
ring undergoes a conformational change that makes it two orders
of magnitude more resistant to reduction.7 This type of confor-
mational regulation has also been proposed for the vicinal disulfide
ring of cytochrome C from Utricularia.8 Second, it may act as a redox
conformational switch. Examples of this type of switch have been
shown in human RNase H1,9 and the hSH3N domain of the adhesion
and degranulating promoting adapter protein (ADAP).10 The
mechanism by which the switch acts is through the change in
peptide backbone conformation that is observed on going from the
reduced state to the oxidized state. Upon oxidation of vicinal cys-
teines, formation of the eight-membered ring has the effect of
* Corresponding author. Department of Biochemistry, B413 Given Building, Uni-
versity of Vermont, 89 Beaumont Avenue, Burlington, VT 05405, USA. Tel.: þ1 802
656 8282; fax: þ1 802 656 8220.
constraining backbone torsion angles,
4, j, and u, similar to the
effect that proline (a five-membered ring) has on the backbone.11,12
This change in backbone conformation is rather dramatic as has
E-mail address: robert.hondal@uvm.edu (R.J. Hondal).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.11.085

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E.L. Ruggles et al. / Tetrahedron 65 (2009) 1257–1267
that all of the ring structures were in a strained, distorted trans
conformation with average values of
lying between 161ꢁ and
u
ꢀ172ꢁ with deviations from 180ꢁ as large as 44ꢁ being observed.1
This discrepancy may be explained by the dynamic nature of the
VDR, not properly being accounted for in X-ray protein structures in
particular. In an NMR study of the bass hepcidin structure it was
noted that the most flexible region of the protein was the area
containing the VDR.22 Peptide bonds prefer a trans conformation,
with a torsion angle of 180ꢁ, so that the nitrogen lone pair can have
maximal delocalization into the
p-system. This also minimizes
steric repulsions from large peptidyl side chains. However, small
rings that have a central bond with restricted rotation will prefer to
adopt a cis conformation to minimize ring strain. These disparate
facts raise the question of what the preferred amide geometry of
a VDR will be. This point was first raised by North, whose inspection
of molecular models of smaller amide rings (less than eight atoms)
closed by a disulfide was found to have exclusively cis-amide geo-
metry (analogous to cycloalkenes), while larger rings (more than
eight atoms) had trans-amide geometry.23 The transition point
between amide-disulfide ring systems with all cis geometry and all
trans geometry was eight atoms. These eight-membered ring sys-
tems (VDRs) seem to be a special case as they can adopt either
geometry and can oscillate between the two types in solution. This
ability to easily switch between cis- and trans-amide geometries
may make a VDR a good ‘molecular switch’,24 regulating the con-
formation of proteins as has been proposed for the nAChR.8,25
Multiple amide (trans/cis) and disulfide (þ/ꢀ90ꢁ) conformations
are energetically feasible in the unique case of a VDR. An all carbon
analogue of a VDR that could be used as a point of comparison is
cyclooctene. Both are eight-membered ring systems with a central
bond that has some type of unsaturation; the element of unsatu-
ration in cyclooctene is a carbon–carbon double bond, while a VDR
contains an amide bond with only 40% double bond character.
While both a VDR and cyclooctene are similar in having restricted
rotation about a central bond and ring size, there are significant
conformational differences. For cyclooctene, the cis isomer is more
energetically stable than the trans as a result of the ring strain re-
quired to incorporate a trans double bond. This ring strain is
Figure 1. VDR mimics.
been observed in the case of the hSH3N domain of ADAP. In both
RNase H1 and the hSH3N domain of ADAP, it has been proposed that
this motif acts as a cellular redox state sensor, as oxidation of the
vicinal cysteine pair in RNase H1 leads to inactivation of the en-
zyme.9 The redox potential of the vicinal cysteine pair for the
hSH3N domain has been determined to be ꢀ228 mV.10 This value is
within the range of disulfide redox potentials found in proteins, but
is near the oxidizing end of the scale. We posit that the relative
instability of this particular disulfide bond is by design and is due to
the strained nature of the ring. The strain in the ring is due to the
distorted trans conformation that the ring adopts as determined by
NMR spectroscopy. This next point is addressed in the following
section.
1.2. VDR conformation
When the field of peptide structure was relatively new, it was
predicted by Chandrasekaran and Balasubramanian that a VDR
dipeptide should have a nonplanar cis geometry with a dihedral
angle (
L
u
) of ꢀ12ꢁ.13 The dihedral angle for the cyclic disulfide
-cysteinyl-L-cysteine was later experimentally determined to be
ꢀ7ꢁ by X-ray crystallography.14,15 Similar cis
u
-dihedral angles were
demonstrated by the higher DHH2 of trans-cyclooctene (34.4 kcal/
also observed in the bicyclic diketopiperazine ring system of cyclo-
mol) compared to the cis isomer (23.0 kcal/mol).26 By contrast and
as noted above, a VDR has a fluxional nature that permits both cis
and trans conformers due to: (i) the longer carbon–sulfur and
sulfur–sulfur bonds, which makes this eight-membered ring larger
and more flexible than its all carbon analogue and (ii) the amide
bond having only partial double bond character allows for rotation
about the C–N axis.
L
-cystine.16,17 An NMR study of VDR 6 in which R2 is replaced with
OtBu and the acetyl group replaced with Boc (Fig. 1) found evidence
for two conformers in chloroform, both with cis-amide geometry, in
agreement with the earlier crystallographic and NMR studies.18
However, if the amino terminus in 6 is replaced with gem-dimethyl
substituents, two conformations are observed both with trans-
amide geometries by NMR and X-ray crystallography.19 Another
solid state and solution conformational study revealed that a trans
Since a VDR is a potential regulatory conformational switch in
proteins, it would be useful to understand the factors that poten-
tiate amide geometry in this system. Studies by North and co-
workers found that cyclo-[(R)-cysteinyl-(R)-penicillamine] adopts
a cis conformation, while a trans conformation is observed upon
changing the penicillamine stereochemistry from R to S.23,27 We
conformer was observed if the stereochemistry of the carbon
the ring amide nitrogen is inverted, as is in the case of phenylacetyl-
-cysteinyl-
-penicillamine.20 When VDR 6 was placed in the con-
a to
L
D
text of a heptamer peptide, the conformer population that was cis
was found to be 70ꢂ5% as determined by NMR spectroscopy.21
However, when VDR 6 was part of the pentameric peptide TCCPD,
found in the nAChR, both cis and trans conformers of the VDR were
found to exist in solution and these conformers were found to in-
terconvert, lending support for the idea that the ring motif could act
as a protein conformational regulatory switch.7 An NMR study of
the isolated dipeptide 6 done in aqueous solution revealed the
presence of two trans conformers (designated as Tꢀ and T0ꢀ) and
two cis conformers (designated as Cþ and Cꢀ), with trans and cis
isomers being in equilibrium with a ratio of w60:40, with trans
being slightly favored.5
have confirmed this observation by constructing cyclo-[
Cys]. Similar conformational studies showed that switching the
stereochemistry of the C on the N-terminal Cys results in
L-Cys-D-
a
a strained trans conformation of the molecule with no cis con-
formers present (data not shown). Thus both stereochemistry and
substituents about the ring effect amide geometry of a VDR.
As we were interested in VDRs in the context of a redox regu-
latory switch, we wanted to determine the redox potential of a cis
VDR and a trans VDR. We wanted to mimic the situation in proteins,
thus we avoided changing the stereochemistry of the a-carbon and
focused instead on changing the properties of the amide bond by
N-methylation. It has been previously established that N-methyl-
ation of the amide bond increases the population of the cis con-
former in peptides.28 Construction of cis and trans VDRs would then
In contrast to the model studies of small peptides above,
a complete analysis of the PDB performed by Perczel and co-
workers of the 31 deposited structures containing a VDR showed

E.L. Ruggles et al. / Tetrahedron 65 (2009) 1257–1267
1259
allow us to assess how VDR conformation affects the redox po-
tential of the disulfide bond, and explore our VDR hypothesis that
a VDR with a cisoid peptide bond will be a weaker oxidant than
a VDR with a transoid peptide bond. Correspondingly, this means
that the dithiol with a cisoid peptide bond will be the stronger
reductant.
predominating. The redox data (Table 1) support our hypothesis by
displaying a monomeric redox potential for cis-olefin 2 (ꢀ318 mV)
and a dimeric redox potential for the trans-olefin 3 (ꢀ329 mV).31
The inability to observe a monomeric potential for the trans mimic
and its propensity to dimerize suggest a strong oxidative potential.
The ring strain necessary to incorporate the trans-olefin is repre-
sented by a fragile disulfide bond and results in an inability to form
the cyclic monomer. Thus the cis-dithiocine is more stable than
trans, making 3 a much stronger oxidant in comparison to 2. The
dithiol of 2 is thus a very strong reductant. These initial results
supported our VDR hypothesis.
2. Results and discussion
2.1. Determination of redox potentials
In order to test our hypothesis, variants of the VDR are needed
with different elements of rigidity (2–7, Fig. 1). Fully cis- or trans-
dithiocines 2 and 3 mimic opposite ends of the amide rotational
spectrum. Dithiazacanones 4 and 5 are not as rigid as compared to 2
and 3 due to the replacement of the central alkene with an amide
bond. North has previously reported that amide 4 exists exclusively
in a trans conformation.29 While N-Me amide 5 is unknown in the
literature it is predisposed for a cis conformation as a result of steric
interactions.28 Dithiazacanones 4 and 5 are the ‘parent’ compounds
for VDR mimics as they contain no appendages that would affect
the conformation of the ring. Disulfides 6 and 7, however, are di-
peptides that have substituents on Ca and represent a minimalist
VDR. Compound 6 was constructed by Leo and co-workers and
displayed four major conformations, two being cis and two trans,
with a trans/cis ratio of 60:40.5 Compound 7 has not been reported
in the literature, but N-methylation should favor a cis-amide ori-
entation as is the case in 5.28 The redox potentials are determined
by equilibrium thiol–disulfide exchange, with the varying concen-
trations of reduced and oxidized forms monitored by 1H NMR
(Fig. 2).30,31 Employment of oxidized or reduced butane dithiol
(BDTox and BDTred, respectively), a species of known redox potential
E0(BDT), allows for the redox potential of the VDR mimics to be
determined by Eqs. 1 and 2.
ꢀ1
K
¼ Kred ¼ ½8ꢃ½BDToxꢃ=½2ꢃ½BDTredꢃ
(1)
(2)
ox
E0 ¼ E0ðBDTÞ ꢀ 0:03 logðKox
Þ
Scheme 1. Reagents and conditions for dithiocine synthesis: (a) m-CPBA, K2HPO4,
DCM/H2O; (b) 10% H2SO4, THF; (c) NaIO4, THF; (d) NaBH4, H2O, THF; (e) DEAD, Ph3P,
AcSH, Et2O; (f) K2CO3, MeOH; (g) CsF/Celite, air, ACN 30 mM; (h) H2SO4, MeOH (97%),
(i) LAH, Et2O.
2.2. Synthesis, conformation, and redox potential
of dithiocines 2 and 3
Our earlier communication involves the synthesis and redox
properties of dithiocines 2 and 3.31 The dithiocine sulfhydryl pre-
cursors were readily available from diol intermediates 11 (Scheme
1) and 15 upon Mitsunubo thioesterification and hydrolysis
(Scheme 1).32,33 Dithiocine 2 was isolated in good yield upon CsF/
Celite mediated oxidation.34 However, dithiocine 3 was never iso-
lated even under dilute conditions with the cyclic dimer 18
2.3. Synthesis, conformation, and redox potential
of dithiazacanones 4 and 5
Construction of dithiazacanones 4 and 5 begins with sulfhydryl
trityl protection of both acid 19 and amine 20 (Scheme 2).29,35
Figure 2. 1H NMR thiol–disulfide equilibrium redox experiment for cis-dithiocine 2.

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E.L. Ruggles et al. / Tetrahedron 65 (2009) 1257–1267
Table 1
VDR redox potentials
Cisoid
Transoid
(4)
(1)
ꢀ318 mV
ꢀ320 mV
ꢀ329 mV
Figure 3. Dithiazacanone VDR conformations.
(2)
(3)
(5)
(6)
ꢀ278 mV
geometry is demonstrated by this species flexibility at room tem-
perature, reminiscent of dithiocine 2.31 This flexibility was dem-
onstrated by 1H NMR coalescence experiments (Supplementary
data). The observed redox potentials (Table 1), determined by Eqs. 1
and 2, for both 4 (ꢀ278 mV) and 5 (ꢀ320 mV) are in support of the
VDR hypothesis. Amide 4 exists in a trans conformation and as
a result of the ring strain needed to accommodate this geometry, it
is the stronger oxidant when compared to 5. The trans geometry of
the precursor dithiol of 4 disfavors disulfide-bond formation since
the sulfur atoms are distant, e.g., not close to each other in space.
Amide 5 is the weaker oxidant as a result of the cis-amide bond
relieving ring strain and thus stabilizing the disulfide. In this case,
the precursor dithiol of 5 most likely has a significant population of
the cis conformer, which brings the sulfur atoms closer in space and
favors intramolecular disulfide-bond formation. This fact makes
the dithiol form of 5 a good reductant. These redox data for the
dithiazacanone VDR mimics support our VDR hypothesis since
the more strained system is the stronger oxidant.
The redox potential of disulfide 4 (ꢀ278 mV) is particularly in-
teresting in light of the determined redox potential of the single
vicinal disulfide bond of the hSH3N domain in ADAP being
ꢀ228 mV.10 Disulfide 4 has the highest redox potential (strongest
oxidant) of all the mimics in Table 1. Since both North and ourselves
have demonstrated that the amide bond geometry is in an all trans
configuration, it is expected to be the most strained of the amide-
containing disulfides that are in this study. While it is not a perfect
mimic of the strained disulfide bond within the hSH3N domain of
ADAP, the results in Table 1 clearly demonstrate how straining the
amide bond within a VDR affects the redox potential of the disulfide
bond. Clearly a disulfide bond can be more strained in the context of
a folded domain such as that found in the hSH3N domain of ADAP
compared to when the disulfide bond is isolated and brought out of
its protein context, as is disulfide 4. This is the first study that we
are aware of that quantitates redox potentials of vicinal disulfide
bonds and makes a comparison to strain in the peptide torsion
angle.
ꢀ363 mV
ꢀ311 mV
Standard amide coupling provides common intermediate 23.29,36
Dithiol deprotection,37 followed by oxidation provides 4 in low
yield along with two unidentified oligomeric products.34 Amide N-
methylation of 23 generates 24 in mediocre yield,38 after which
analogous sulfhydryl deprotection and oxidation afford 5.34,37 De-
spite the fact that the dithiazacanones are similar in structure they
differ in conformation (Fig. 3). Prior to this report, North observed
that disulfide 4 existed in one trans conformation.29 However, here
we have observed that VDR 4 populates two trans conformations
with a ratio of 1.7:1.0. In support of our result, after determining the
separate spin systems in VDR 4 by COSY NMR, ROESY experiments
showed magnetization transfer between the two species specifi-
cally between the conformers CaN protons and CbC]O protons. This
result supports the existence of two conformers (Supplementary
data). The trans-amide bond geometry was determined at 5 ꢁC by
ROESY signals between the amide proton and CaC]O, CbC]O, and Cb
N
protons. As a result of the strong ROESY signals between the amide
proton and axial CaC]O and CbN protons, a cis-amide conformation
can be ruled out. Disulfide 5 exists in one predominating confor-
mation, however, the amide geometry is now cis. Here, TOCSY NMR
determined the different spin systems within VDR 5 and ROESY
signals showed a strong through-space interaction between the Ca
N
and CbC]O protons as well as a weaker CaN and CaC]O contact. These
through-space interactions would not be possible if the amide
geometry was trans. A much weaker contact between the N-Me and
Cb protons was also observed. Additional support of a cis-amide
N
2.4. Synthesis, conformation, and redox potential
of dipeptides 6 and 7
Construction of the VDR dipeptides begins with fashioning 6.5
Amide coupling of 27 and 28 provides 29 in good yield (Scheme
3).36 Sulfhydryl deprotection and oxidation afford 6 in modest
isolated yield.34,37 Construction of 7 was much more labor in-
tensive. Amine methylation proved difficult under a variety of
conditions. Fortunately, N-methylation was achieved via oxazoli-
dinone intermediate 32.39,40 The oxazolidinone ring opens upon
treatment with TES/TFA to provide N-Me cysteine 33, whose sulf-
hydryl is StBu protected.41 Methyl esterification and Fmoc depro-
tection ensues,42,43 followed by amide coupling with acid 27
generate dipeptide 35.44 The StBu group is removed,45 and after
treatment with DTNP cyclizes spontaneously under acidic condi-
tions to produce N-Me VDR 7.46 While the true pathway for this
Scheme 2. Reagents and conditions for dithiazacanone synthesis: (a) 4-DMAP, TrtCl,
Et3N, DMF; (b) 21, 22, DCC, HOBt, Et3N, DCM/DMF; (c) TES, TFA, DCM; (d) CsF/Celite, air,
ACN, 1 mM; (e) NaH, MeI, DMF.

E.L. Ruggles et al. / Tetrahedron 65 (2009) 1257–1267
1261
Scheme 3. Reagents and conditions for dipeptide synthesis: (a) DCC, HOBt, Et3N, DCM/DMF; (b) TES, TFA, DCM; (c) CsF/Celite, air, ACN, 1 mM; (d) CSA, (CH2O)n, PhH, reflux; (e) TFA,
TES, DCM; (f) K2CO3, MeI, DMF; (g) Et2NH, DCM; (h) 27, HATU, Hu¨ nigs, DCM; (i) DTT, NMM, DMF; (j) (1) DTNP, (2) TFA, (3) TES, DCM; (k) DTT, NMM, DCM.
cyclization is currently under investigation, a possible mechanism
employs an addition–elimination sequence (Scheme 4). First the
trityl-protected sulfur atom is deprotected, which generates a free
sulfhydryl. The unmasked thiol now attacks the vicinal cysteinyl
sulfur atom, whose electrophilicity is enhanced by the Npys group,
expelling pyridinyl-thione. The attacking sulfhydryl’s proton is
subsequently scavenged. Problematic to this step was also the
production of diNpys-protected dipeptide 37, resultant from DTNP
protection of both sulfur atoms. Compound 37 can also be trans-
formed into VDR 7 upon treatment with dithiothreitol in similar
yield.45 As with the dithiazacanones, VDR 6 and 7 are similar in
structure but not in conformation (Fig. 4). VDR 6 exists as a mixture
of four detectable interconverting cisoid (Cþ/Cꢀ 36:5) and transoid
(Tꢀ/T0ꢀ 42:17) amide conformers with transoid being preferred in
accordance with what was observed prior by Leo and co-workers
(Fig. 4, trans/cis 59:41).5 VDR 7 exists in one major conformation
that is cisoid in nature (Fig. 4). These conformations were de-
termined in a similar manner as dithiazacanone VDRs 4 and 5. The
observed redox potentials, determined by Eqs. 1 and 2, for both 6
(ꢀ311 mV) and 7 (ꢀ363 mV) are in support of our hypothesis (Table
1). While both VDR dipeptides are flexible N-Me VDR 7 exists
preferentially in a cisoid confirmation while 6 is primarily transoid.
In accord with prior results VDR 6 is a stronger oxidant, when
compared to 7, as a result of a weak disulfide bond that is a conse-
quence of incorporating a strained transoid amide geometry within
the eight-membered ring. Conversely, VDR 7 is a weaker oxidant,
when compared to 6, due to a stronger disulfide bond that is a re-
sult of a less strained cisoid ring system. These redox data for the
dipeptide VDR mimics support our hypothesis that dithiols that
form cisoid eight-membered rings will be stronger reductants in
comparison to dithiols that form transoid eight-membered rings.
This means that a transoid VDR will be much more reactive toward
thiol–disulfide exchange reactions and this enhanced reactivity
could be harnessed by proteins for either catalytic or regulatory
mechanisms. As noted above, all of the vicinal disulfide bonds in
proteins so far have been found to be in such a strained state.
3. Summary
It is interesting to compare trans-dithiol 17 to that of a VDR
found in proteins. When the central torsional angle is constrained
to 180ꢁ, as is the case for 17, intramolecular disulfide-bond for-
mation is impossible. A disulfide bond can form between the
nearest neighbors in peptidyl systems because the peptide bond is
not as rigid as an olefin. This allows the central peptide bond of the
VDR to adopt a strained trans geometry with the lone pair of
electrons on the nitrogen atom out of phase with the
p system to
varying degrees depending on the strain in the system. This amide
bond strain allows disulfide-bond formation to occur. Here we have
demonstrated a correlation between amide strain and redox po-
tential in a VDR. We also have demonstrated that this strain can be
relieved, by forcing the VDR to adopt the cis conformation via N-
methylation. A VDR with cis geometry is less strained as evidenced
by the lower redox potentials of all of the VDR mimics in this study.
While there are currently no known examples of a vicinal disulfide
bond with cis-amide geometry in the PDB, several redox enzymes
contain a vicinal disulfide bond as part of their redox cycle and
could use cis-amide geometry to influence the redox potential of
the VDR. Of particular interest to us are eukaryotic thioredoxin
reductases that contain a C-terminal vicinal disulfide bond as part
of their active site. The redox potential of the disulfide bond of the
protein substrate (thioredoxin) is ꢀ270 mV.47 Thus for efficient
Scheme 4. Proposed mechanism for the formation of VDR 7.

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E.L. Ruggles et al. / Tetrahedron 65 (2009) 1257–1267
Figure 4. Dipeptide VDR conformations.
catalysis to occur, the redox potential of the vicinal disulfide bond
of thioredoxin reductase should be more negative (more reducing)
than this value. One way to achieve a lower redox potential would
be to have the VDR of thioredoxin reductase to adopt a cis-amide
geometry as shown here. A problem of having the VDR of a protein
adopt a cis conformation is that it forces the peptide main chain to
make a sharp turn, greatly altering protein topology and affecting
protein function. This is most likely the cause of enzyme in-
activation in RNase H1 upon oxidation of the vicinal cysteine pair.
This problem is largely avoided in the cases of VDRs found in thio-
redoxin reductase and mercuric ion reductase since the vicinal
cysteine pair occurs at the C-terminus of these enzymes, which
would minimize the effect of the conformation on the rest of
peptide chain. The conformation of the VDR of thioredoxin re-
ductase is an area of intense research in our laboratory.
4.2. Synthesis of dithiocine 2
4.2.1. 7-Oxa-bicyclo[4.1.0]hept-3-ene (10)
In an air-dried flask, 10.0 mL (106 mmol) 1,4-cyclohexadiene
was added quickly to a stirring room temperature biphasic solution
of 24.38 g (108 mmol) m-CPBA and 18.8 g (108 mmol) K2HPO4 in
762 mL DCM/H2O (151:1). The reaction mixture was stirred at room
temperature for 18 h. Reaction was quenched with 200 mL aqueous
NaHCO3(satd) and layers were separated. DCM layer was washed
once with 150 mL 5% aqueous Na2SO3 and once with 150 mL
aqueous NaHCO3(satd)
. The combined aqueous washes were
extracted two sequential times with 100 mL DCM. The DCM ex-
tractions were combined, dried over MgSO4, filtered, and concen-
trated to afford 9.56 g (94%) of 10 as a clear oil. Rf¼0.63 (1:2
hexanes/EtOAc); 1H NMR (500 MHz, CDCl3)
d 5.42 (s, 2H), 3.22 (s,
2H), 2.56 (d, J¼14.0 Hz, 2H), 2.43 (d, J¼13.5 Hz, 2H); 13C NMR
(125 MHz, CDCl3)
d 121.5 (CH), 50.8 (CH), 24.9 (CH2); IR (neat)
4. Experimental details
(neat) 1736 (m), 1422 (m), 1214 (m), 1021 (m) cmꢀ1; LRMS (EI) m/z
96.0 [(Mþ), calcd for C6H8O: 96.0].49
4.1. Materials and methods
4.2.2. Hex-3-ene-1,6-diol (11)
Reactions employed oven-dried glassware under argon unless
otherwise noted. Argon was passed through a column of anhydrous
CaSO4 before use. Amino acids were purchased from Synpep Corp
(Dublin, CA). All other chemicals were purchased from either
Sigma–Aldrich (Milwaukee, WI) or Fisher Scientific (Pittsburgh, PA)
and were used as-received or purified by standard procedures.48
Reactions were monitored by thin layer chromatography (TLC)
using glass 0.25 mm silica gel plates with UV indicator. Flash
chromatography was performed using columns packed with 230–
400 mesh silica gel as a slurry in the elution solvent, unless oth-
erwise noted. Gradient flash chromatography was conducted by
adsorption of product mixtures on silica gel, packing onto fresh
silica bed as a slurry in minimal hexanes, and eluting with a con-
tinuous gradient as noted in parentheses. Reverse-phase high
performance liquid chromatography (RP-HPLC) was executed using
In an air-dried flask, 12 mL H2SO4 was added to a stirring room
temperature solution of 6.17 g (64.2 mmol) epoxide 10 in 200 mL
THF/H2O (1:1). Upon complete addition, the reaction mixture was
refluxed (oil bath¼85 ꢁC) for 1 h and then cooled to 0 ꢁC. Over
30 min 14.4 g (67.4 mmol) NaIO4 was added in three portions after
which the reaction mixture was stirred for 1.5 h. A solution of 3.66 g
(96.7 mmol) NaBH4 in 15 mL H2O was then added dropwise via
addition funnel to the cold reaction mixture. Upon complete ad-
dition, the reaction mixture was allowed to warm to room tem-
perature and subsequently stirred for 30 min. After quenching with
100 mL aqueous NH4Cl(satd), the layers were separated and the
aqueous layer was extracted three consecutive times with 100 mL
DCM. The DCM extractions were combined, dried over MgSO4, fil-
tered, and concentrated. The diol was purified after impregnation
onto flash silica gel via gradient flash chromatography (2:1 hex-
anes/EtOAc to 1:2 to EtOAc) to provide 3.55 g (48%) of 11 as a light
yellow oil. Rf¼0.28 (1:2 hexanes/EtOAc); 1H NMR (500 MHz, CDCl3)
a Shimadzu VP system with a SymmetryÓ C18-5
Waters (4.6ꢄ150 mm) for analytical analysis and
tryPrepÔ C18-7
m
m column from
Symme-
m column from Waters (19ꢄ150 mm) for pre-
a
m
d
5.50 (m, 2H), 3.59 (t, J¼6.0 Hz, 4H), 3.52 (br s, 2H), 2.29 (q,
paratory separation. Melting points were determined on a Meltemp
apparatus and are uncorrected. Proton and carbon NMR data were
obtained with a Varian or Bruker ARX 500 spectrometer at 20 ꢁC
unless otherwise noted. Chemical shifts for 1H NMR and 13C NMR
are reported in parts per million (ppm) relative to tetramethylsilane
J¼6.2 Hz, 4H); 13C NMR (125 MHz, CDCl3)
d 128.7 (CH), 61.5 (CH2),
30.4 (CH2); IR (neat) 3376 (m), 3336 (m), 1740 (m), 1462 (m), 1350
(m), 1259 (m), 1042 (m) cmꢀ1; LRMS (EI) m/z 116.0 [(Mþ), calcd for
C6H12O2: 116.0].50
(d
¼7.24 ppm for 1H NMR) or chloroform-d (
d
¼77.0 ppm for 13C
4.2.3. Thioacetic acid S-(6-acetylsulfanyl-(Z)-hex-3-enyl) ester (12)
To a chilled (0 ꢁC) stirring solution of 13.5 g (51.4 mmol) Ph3P in
500 mL anhydrous Et2O, 8.11 mL (51.4 mmol) DEAD was added.
After 1 h, a solution of 3.80 mL (53.4 mmol) AcSH and 2.30 g
(19.8 mmol) diol 11 in 165 mL anhydrous Et2O was added dropwise
via addition funnel over 30 min. Upon complete addition, the re-
action mixture was kept at 0 ꢁC for 1 h and then warmed naturally
to room temperature and then stirred for 10 h. The reaction
NMR), respectively. Infrared spectra were recorded with a Perkin–
Elmer 2000 FT-IR spectrophotometer. Low resolution mass spectra
were obtained with a Hewlett Packard 5988 GCMS. High resolution
mass spectra were performed by the University of South Carolina
Mass Spectrometry Laboratory. A Voyager-DEÔ PRO Workstation
(Applied Biosystems) was used for mass spectral analysis of peptide
samples.

E.L. Ruggles et al. / Tetrahedron 65 (2009) 1257–1267
1263
mixture was filtered and triturated with cold anhydrous Et2O. The
filtrate was concentrated. The oil was purified via flash chroma-
tography using 10:1 hexanes/EtOAc as eluent to provide 3.49 g
(76%) of 12 as a clear oil. Rf¼0.46 (4:1 hexanes/EtOAc); 1H NMR
H2O. After filtration, the filtrate layers were separated. The aqueous
layer was extracted two consecutive times with 50 mL Et2O. The
ethereal extractions were combined, dried over MgSO4, filtered,
and concentrated to afford 1.23 g (88%) of 15 as a clear oil. Rf¼0.17
(500 MHz, CDCl3)
d
5.37 (t, J¼4.8 Hz, 2H), 2.82 (t, J¼6.8 Hz, 4H), 2.26
(1:2 hexanes/EtOAc); 1H NMR (500 MHz, CDCl3)
d
5.49 (t, J¼1.5 Hz,
(s, 6H), 2.24 (m, 4H); 13C NMR (125 MHz, CDCl3)
d
195.1 (C), 128.8
2H), 3.59 (t, J¼6.0 Hz, 4H), 3.57 (br s, 2H), 2.24 (q, J¼2.0 Hz, 4H); 13C
(CH), 30.3 (CH3), 28.5 (CH2), 27.0 (CH2); IR (neat) 3038 (w), 1682 (s),
1355 (m), 1130 (s), 1100 (s) cmꢀ1; HRMS (EI) m/z 233.0672 [(Mþ),
calcd for C10H16O2S2: 233.0670].
NMR (125 MHz, CDCl3) d 129.2 (CH), 61.4 (CH2), 35.7 (CH2); IR
(neat) 3386 (s), 3336 (s), 1741 (m), 1435 (m), 1365 (m), 1051
(s) cmꢀ1; LRMS (EI) m/z 116.0 [(Mþ) calcd for C6H12O2: 116.0].51
4.2.4. Hex-3-ene-1,6-dithiol (8)
4.3.3. Thioacetic acid S-(6-acetylsulfanyl-(E)-hex-3-enyl) ester (16)
Procedure and workup analogous to 12 using 2.32 g (19.9 mmol)
15. After workup and concentration, the oil obtained was purified
via flash chromatography using 10:1 hexanes/EtOAc as eluent to
provide 3.61 g (78%) of 16 as a clear oil. Rf¼0.42 (4:1 hexanes/
In an air-dried flask, 0.872 g (6.31 mmol) K2CO3 was added to
a stirring solution of 1.00 g (4.30 mmol) 12 in MeOH at room
temperature. The reaction mixture was filtered, concentrated, and
taken up in 100 mL DCM/H2O (1:1). After separation, the DCM layer
was washed once with 50 mL aqueous NH4Cl(aq) and two sequential
times with 50 mL H2O. The aqueous layers were combined and
extracted two consecutive times with 50 mL DCM. The DCM ex-
tractions were combined, dried over MgSO4, filtered, and concen-
trated to afford 0.59 g (94%) of 8 as a foul-smelling oil. Rf¼0.11 (4:1
EtOAc); 1H NMR (500 MHz, CDCl3)
d
5.44 (m, 2H), 2.88 (t, J¼7.3 Hz,
195.6
4H), 2.30 (s, 6H), 2.23 (m, 4H); 13C NMR (125 MHz, CDCl3)
d
(C), 129.7 (CH), 32.3 (CH2), 30.5 (CH3), 28.7 (CH2); IR (neat) 3015
(w), 1686 (s), 1429 (m), 1353 (ms), 1131 (s) cmꢀ1; HRMS (EI) m/z
233.0662 [(MþH), calcd for C10H16O2S2: 233.0670].
hexanes/EtOAc); 1H NMR (500 MHz, CDCl3)
d
5.49 (t, J¼4.7 Hz, 2H),
2.72 (t, J¼7.0 Hz, 4H), 2.47 (q, J¼7.0 Hz, 4H), 1.25 (br s, 2H); 13C NMR
4.3.4. (E)-Hex-3-ene-1,6-dithiol (17)
(125 MHz, CDCl3)
d
128.9 (CH), 38.4 (CH2), 27.2 (CH2); IR (neat) 3010
Procedure and workup analogous to 8 using 0.718 g (3.09 mmol)
16 as substrate. After workup and concentration, the oil obtained
was purified without delay, to avoid oligomer formation, after
impregnation onto flash silica gel via gradient flash chromatogra-
phy (hexanes to 10:1 to 4:1 to 1:1 to EtOAc) to provide 0.421 g (92%)
of 17 as a clear foul-smelling oil. Rf¼0.55 (4:1 hexanes/EtOAc); 1H
(vw), 1690 (m), 1640 (s), 1415 (m), 1360 (m), 1235 (w), 1199 (s), 1100
(w) cmꢀ1; HRMS (EI) m/z 148.0382 [(Mþ) calcd for C6H12S2:
148.0380].
4.2.5. 3,4,7,8-Tetrahydro-[1,2]dithiocine (2)
In an air-dried flask, 0.853 g (4.02 mmol) CsF/Celite was added
to a stirring solution of 0.390 g (2.66 mmol) 8 in 89 mL ACN
(30 mM) at room temperature. The reaction mixture was vigor-
ously stirred at room temperature for 5 days. After filtration, the
solid was triturated with ACN and the filtrate concentrated to
provide an oil, which was purified via alumina (basic, activity 1)
chromatography using hexanes as eluent to provide 0.249 g (65%)
of 2 as a clear oil with a distinct disulfide odor. Rf¼0.87 (4:1 hex-
NMR (500 MHz, CDCl3)
d
5.47 (m, 2H), 2.57 (q, J¼7.7 Hz, 4H), 2.33
(m, 4H), 1.42 (t, J¼7.7 Hz, 2H); 13C NMR (125 MHz, CDCl3)
d 129.6
(CH), 38.7 (CH2), 25.6 (CH2); IR (neat) 3015 (w), 1739 (ms), 1429 (s),
1278 (ms), 1227 (ms) cmꢀ1; HRMS (EI) m/z 148.0379 [(Mþ) calcd for
C6H12S2: 148.0380].
4.3.5. 1,2,9,10-Tetrathia-cyclohexadeca-5,13-diene (18)
Procedure and workup analogous to 2 using 1.85 g (12.7 mmol)
17 in ACN (30.0 mM). After workup and concentration, the oil
anes/EtOAc); 1H NMR (500 MHz, CDCl3)
d
5.67 (m, 2H), 2.87 (m,
2H), 2.64 (m, 4H), 2.50 (2H); 13C NMR (125 MHz, CDCl3)
d
129.7
obtained was purified via gradient alumina (basic, activity 1)
chromatography (hexanes to 50:1 to 25:1 to 10:1 to 4:1 to EtOAc) to
provide 0.192 g (21%) of 18 as a distinctly disulfide-smelling oil.
(CH), 39.4 (CH2), 27.2 (CH2); IR (neat) 3010 (w), 1645 (w), 1448 (m),
1407 (m), 1279 (m),1249 (w), 966 (w), 886 (w), 726 (s) cmꢀ1; HRMS
(EI) m/z 146.0225 [(Mþ), calcd for C6H10S2: 146.0224].
Rf¼0.65 (4:1 hexanes/EtOAc); 1H NMR (500 MHz, CDCl3)
d 5.62 (m,
2H), 2.79 (t, J¼7.1 Hz, 4H), 2.40 (m, 4H); 13C NMR (125 MHz, CDCl3)
4.3. Synthesis of dithiocine dimer 18
d 129.8 (CH), 39.7 (CH2), 31.8 (CH2); IR (neat) 3010 (w), 1450 (m),
1410 (m), 1268 (m) cmꢀ1; HRMS (EI) m/z 292.0446 [(Mþ), calcd for
4.3.1. (E)-Hex-3-enedioic acid dimethyl ester (14)
C12H20S4: 292.0448].
In an air-dried flask, one drop H2SO4 was added to a stirring
solution of 2.01 g (13.9 mmol) trans-mucionic acid in 50 mL MeOH
at room temperature. The reaction mixture was heated to reflux
(oil-bath¼90 ꢁC) and stirred for 16 hr. Cooled to room temperature
and partitioned with 40 mL DCM/H2O (1:1). After separation, the
aqueous layer was extracted two consecutive times with 20 mL
DCM. The DCM extractions were combined, dried over MgSO4, fil-
tered, and concentrated to afford 2.32 g (97%) of 14 as a sweet-
smelling oil. Rf¼0.41 (2:1 hexanes/EtOAc); 1H NMR (500 MHz,
4.4. Synthesis of dithiazacanone 4
4.4.1. 3-Tritylsulfanyl-propionic acid (21)
In an air-dried flask, 10.5 g (37.6 mmol) TrtCl was added to
a stirring solution of 3.30 mL (37.6 mmol) 19 in 754 mL (0.05 M)
DCM at room temperature. The reaction mixture was stirred at
room temperature for 14 h and then concentrated. The solid was
purified upon recrystallization from MeOH/H2O to provide 10.7 g
(82%) 21 as a white solid.39 Rf¼0.60 (4:1 hexanes/EtOAc); mp¼202–
CDCl3)
d
5.61 (m, 2H), 3.59 (s, 6H), 3.01 (d, J¼1.5 Hz, 4H); 13C NMR
(125 MHz, CDCl3)
d
171.5 (C), 125.7 (CH), 51.5 (CH2), 37.3 (CH3); IR
204 ꢁC; 1H NMR (500 MHz, DMSO-d6)
d 12.2 (br s, 1H), 7.33 (s, 12H),
(neat) 1729 (s), 1453 (m), 1360 (w), 1251 (m), 1154 (s) cmꢀ1; LRMS
7.24 (m, 3H), 2.29 (t, J¼7.3 Hz, 2H), 2.17 (t, J¼7.3 Hz, 2H); 13C NMR
(EI) m/z 172.0 [(Mþ) calcd for C8H12O4: 172.0].51
(125 MHz, CDCl3) d 178.6 (C), 144.5 (C), 129.4 (CH), 127.7 (CH), 126.4
(CH), 66.6 (C), 33.3 (CH2), 26.7 (CH2); IR (neat) 3463 (w), 3010 (s),
4.3.2. (E)-Hex-3-ene-1,6-diol (15)
2748 (m), 2650 (m), 2569 (m), 1700 (vs), 1429 (s), 1231 (s) cmꢀ1
;
To a stirring solution of 0.919 g (24.2 mmol) LAH in 16 mL
anhydrous THF previously cooled to 0 ꢁC, a solution of 2.08 g
(12.1 mmol) 14 in 4 mL anhydrous THF was added dropwise via
syringe. Upon compete addition, the reaction mixture was stirred at
0 ꢁC for 10 min then heated to reflux (oil bath¼40 ꢁC) and stirred
for 4 h. Cooled to room temperature and quenched cautiously with
HRMS (EI) m/z 347.1110 [(MꢀH), calcd for C22H20O2S: 347.1106].29,35
4.4.2. 2-Tritylsulfanyl-ethylamine (22)
In an air-dried flask, 4.90 g (17.6 mmol) TrtCl was added to
a stirring solution of 2.00 g (17.6 mmol) 20 in TFA at room tem-
perature where upon dissolution resulted in a deep red color. The

1264
E.L. Ruggles et al. / Tetrahedron 65 (2009) 1257–1267
reaction mixture was set-aside for 1 h and subsequently poured
into 200 mL water. The solution was made basic by the addition of
concentrated KOHaq and vacuum-filtered. The solid was dried over
CaSO4 and high-vacuum. The mono/di-protected mixture was dis-
solved in 1:1 HCl/ACN and poured into ether (10:1 ether/(HCl/
ACN)). The solution was filtered and the solid triturated with ether.
Concentrated KOHaq was added to the ethereal solution (1:1) and
stirred vigorously mixed for 45 min. After partitioning, the ethereal
was dried over MgSO4, filtered, and concentrated to afford a 5.62 g
(100%) of 22 as a white solid. Rf¼0.17 (4:1 hexanes/EtOAc);
mp¼146–148 ꢁC; 1H NMR (500 MHz, CDCl3) 7.41 (m, 6H), 7.27 (m,
6H), 7.20 (m, 3H), 2.68 (br s, 2H), 2.52 (t, J¼6.4 Hz, 2H), 2.36 (t,
EtOAc); mp¼178–181 ꢁC; 1H NMR (500 MHz, DMSO-d6)
d major
conformer: 8.15 (m, 1H), 3.32 (m, 2H), 2.90 (m, 2H), 2.79 (m, 2H),
2.47 (m, 2H); minor conformer: 8.22 (m, 1H), 3.31 (m, 2H), 2.89 (m,
2H), 2.84 (m, 2H), 2.52 (m, 2H); 13C NMR (125 MHz, DMSO-d6)
d
major conformer: 170.1 (C), 37.6 (CH2), 37.0 (CH2), 34.4 (CH2), 33.9
(CH2); minor conformer: 173.3 (C), 39.0 (CH2), 38.9 (CH2), 37.5 (CH2),
34.9 (CH2); IR (neat) 3287 (m), 3068 (w), 2925 (w), 1633 (s), 1542
(s), 1416 (m), 1358 (m), 1258 (m), 1201 (m), 710 (w) cmꢀ1; HRMS
(EI) m/z 163.0120 [(Mþ), calcd for C5H9NOS2: 163.0126].29
4.5. Synthesis of N-Me dithiazacanone 5
J¼6.4 Hz, 2H); 13C NMR (125 MHz, CDCl3)
d
144.6 (C), 129.5 (CH),
4.5.1. N-Methyl-3-tritylsulfanyl-N-(2-tritylsulfanyl-ethyl)-
propionamide (24)
127.9 (CH), 126.7 (CH), 66.6 (C), 40.4 (CH2), 34.7 (CH2); IR (neat)
3379 (br m), 3057 (w), 2932 (w), 1680 (m), 1484 (m), 1439 (m), 1206
(m), 1130 (m), 698 (s) cmꢀ1; HRMS (EI) m/z 320.1489 [(MþH), calcd
for C21H21NS: 320.1473].29,35
A solution of 1.55 g (2.38 mmol) 23 in 5 mL anhydrous DMF was
added dropwise to a solution of 0.22 g (5.72 mmol) NaH in 20 mL
anhydrous DMF previously chilled to 0 ꢁC. After complete addition,
the reaction mixture was stirred at 0 ꢁC for 10 min, after which
1.18 mL (19.0 mmol) MeI was added rapidly dropwise. The reaction
mixture was stirred at 0 ꢁC for 30 min and then warmed naturally
to room temperature. The reaction mixture was stirred at room
temperature for 14 h and then quenched with excess phosphate
buffer (pH¼7). The solution was then poured into 100 mL EtOAc
and the biphasic solution separated. The EtOAc layer was washed
once with 100 mL brine, dried over MgSO4, filtered, and concen-
trated. After impregnation onto flash silica, the N-methyl amide
was purified via gradient flash chromatography (hexanes to 10:1 to
4:1 to 1:1 to EtOAc to DCM) to provide 1.37 g (87%) 24 in two in-
distinguishable conformations (w1.0:1.0) as a white solid. Rf¼0.45
(4:1 hexanes/EtOAc); mp¼146–148; 1H NMR (500 MHz, CDCl3)
4.4.3. 3-Tritylsulfanyl-N-(2-tritylsulfanyl-ethyl)-propionamide (23)
To a stirring solution of 1.39 g (3.99 mmol) acid 21 and 1.26 g
(3.95 mmol) amine 22 in 40 mL DCM was added 0.69 mL
(5.01 mmol) Et3N followed by 1.06 g (7.84 mmol) HOBt. For com-
plete dissolution, 2 mL DMF was added. The reaction mixture was
stirred at room temperature for 18 h and then vacuum-filtered. The
filtrate was partitioned with the addition of 50 mL 10% aq NaHCO3.
After separation, the DCM layer was washed once with 50 mL 10%
aq NaHCO3. The DCM layer was dried over MgSO4, filtered, and
concentrated. After impregnation onto flash silica, the acid was
purified via gradient flash chromatography (hexanes to 10:1 to 4:1
to 1:1 to EtOAc) to afford 1.72 g (67%) 23 as a white solid. Rf¼0.07
(4:1 hexanes/EtOAc); mp¼138–141 ꢁC; 1H NMR (500 MHz, CDCl3)
d
conformer mixture: 7.45 (dd, J¼16.1, 7.3 Hz, 24H), 7.28 (m, 24H),
d
7.39 (m, 12H), 7.25 (m, 12H), 7.19 (m, 6H), 5.29 (t, J¼6.0 Hz, 1H),
7.22 (m, 12H), 3.06 (t, J¼7.3 Hz, 2H), 2.80 (t, J¼7.3 Hz, 2H), 2.56 (s,
3H), 2.49 (m, 4H), 2.47 (s, 3H), 2.34 (t, J¼7.3 Hz, 2H), 2.29 (t,
J¼7.3 Hz, 2H), 2.02 (t, J¼7.3 Hz, 2H), 1.93 (t, J¼7.3 Hz, 2H); 13C NMR
2.99 (q, J¼6.2 Hz, 2H), 2.44 (t, J¼7.3 Hz, 2H), 2.34 (t, J¼6.2 Hz, 2H),
1.92 (t, J¼7.5 Hz, 2H); 13C NMR (125 MHz, CDCl3)
d 170.5 (C), 144.6
(C), 144.5 (C), 129.5 (CH), 129.4 (CH), 127.9 (CH), 127.8 (CH), 129.7
(CH),126.6 (CH), 66.8 (C), 66.7 (C), 38.0 (CH2), 35.5 (CH2), 31.8 (CH2),
27.6 (CH2); IR (neat) 3469 (m), 3058 (m), 2933 (m), 1703 (s), 1488
(125 MHz, CDCl3) d conformer mixture: 170.6 (C), 170.5 (C), 144.8 (C),
144.7 (C), 144.6 (C), 144.4 (C), 129.6 (CH), 129.5 (CH), 129.5 (CH),
129.4 (CH), 127.9 (CH), 127.8 (CH), 127.7 (CH), 127.7 (CH), 126.8 (CH),
126.6 (CH), 126.5 (CH), 126.5 (CH), 67.2 (C), 66.7 (C), 66.7 (C), 66.6
(C), 48.8 (CH2), 47.2 (CH2), 35.6 (CH3), 33.1 (CH3), 32.7 (CH2), 32.1
(CH2), 29.8 (CH2), 29.5 (CH2), 27.2 (CH2), 27.0 (CH2); IR (neat) 3464
(m), 3062 (m), 2924 (m), 1694 (m), 1489 (m), 1444 (s), 1148 (s), 1009
(s), 755 (s), 695 (s) cmꢀ1; HRMS (EI) m/z 664.2714 [(MþK), calcd for
C44H41NOS2: 664.2708].
(s), 1444 (s), 1252 (m), 1155 (m), 1074 (m), 1009 (m), 695 (s) cmꢀ1
;
HRMS (EI) m/z 688.2094 [(MþK), calcd for C43H39NOS2:
688.2110].29
4.4.4. 3-Mercapto-N-(2-mercapto-ethyl)-propionamide (25)
In an air-dried flask, 256 mL TFA was added to a stirring solution
of 5.00 g (7.69 mmol) 23 in 512 mL (15 mM) DCM at room tem-
perature. The yellow color produced was then quenched by the
addition of 4.96 mL (30.7 mmol) TES. Upon complete addition, the
reaction mixture was stirred at room temperature for 1 h and then
concentrated. The oil was redissolved in 500 mL DCM and con-
centrated. This was repeated two additional times. After impreg-
nation onto flash silica, the dithiol was purified via flash
chromatography (4:1hexanes/EtOAc) to provide 1.16 g (92%) 25 as
4.5.2. 3-Mercapto-N-(2-mercapto-ethyl)-N-methyl-
propionamide (26)
Procedure and workup analogous to 25 using 1.37 g (2.06 mmol)
24 as substrate. After impregnation onto flash silica, the disulfide
was purified via gradient flash chromatography (hexanes to 10:1 to
4:1 to EtOAc to DCM) to provide 0.33 g (92%) 25 in two in-
distinguishable conformations (w1.0:1.0) as a foul-smelling oil.
a
foul-smelling oil. Rf¼0.72 (1:1 hexanes/EtOAc); 1H NMR
Rf¼0.32 (1:2 hexanes/EtOAc); 1H NMR (500 MHz, CDCl3)
d con-
(500 MHz, CDCl3)
d
5.98 (br s, 1H), 3.46 (q, J¼6.3 Hz, 2H), 2.82 (q,
former mixture: 3.65 (br m, 2H), 3.56 (br m,1H), 3.36 (br m,1H), 3.07
(br m, 4H), 2.95 (br s, 6H), 2.86 (br m, 4H), 2.76 (br m, 3H), 2.62 (br
m, 1H), 1.72 (br m, 2H), 1.62 (br m, 2H); 13C NMR (125 MHz, CDCl3)
J¼8.7 Hz, 2H), 2.68 (q, J¼8.7 Hz, 2H), 2.52 (t, J¼6.3 Hz, 2H), 1.63 (t,
J¼8.7 Hz, 1H), 1.39 (t, J¼8.7 Hz, 1H); 13C NMR (125 MHz, CDCl3)
d
170.7 (C), 42.3 (CH2), 40.3 (CH2), 24.6 (CH2), 20.4 (CH2); IR (neat)
d conformer mixture: 176.0 (C), 175.6 (C), 49.8 (CH2), 49.5 (CH2), 33.5
3283 (m), 3076 (w), 2933 (w), 1640 (s), 1539 (s), 1420 (m), 1355 (m),
258 (m), 1198 (m) cmꢀ1; HRMS (EI) m/z 243.4738 [(Mþ2K), calcd
for C5H11NOS2: 243.4735].
(CH3), 33.4 (CH3), 29.7 (CH2), 29.6 (CH2), 25.4 (CH2), 25.3 (CH2), 24.7
(CH2), 24.7 (CH2); IR (neat) 3301 (w), 2927 (m), 1671 (s), 1628 (s),
1548 (m),1414 (m),1135 (s),1046 (m), 704 (m) cmꢀ1; HRMS (EI) m/z
179.0437 [(Mþ), calcd for C6H13NOS2: 179.0439].
4.4.5. [1,2,5]-Dithiazocan-6-one (4)
Procedure and workup analogous to 2 using 0.39 g (2.35 mmol)
25 as substrate in ACN (1 mM). After workup, the oil was triturated
with hexanes overnight. The white solid obtained after filtration
was triturated with excess DCM to provide 70.2 mg (18%) 4 in two
conformations (1.7:1.0) as a white solid. Rf¼0.65 (1:1 hexanes/
4.5.3. 5-Methyl-[1,2,5]dithiazocan-6-one (5)
Procedure and workup analogous to
2 using 96.1 mg
(0.53 mmol) 26 in ACN (1 mM). After impregnation onto flash silica,
the disulfide was purified via gradient flash chromatography
(hexanes to 10:1 to 4:1 to 2:1 to EtOAc to DCM to 10%MeOH/DCM)

E.L. Ruggles et al. / Tetrahedron 65 (2009) 1257–1267
1265
to provide 33.8 mg (36%) 5 in indistinguishable interconverting
conformations as a water-white oil. Rf¼0.09 (4:1 hexanes/EtOAc);
5.10 (m, 1H), 4.98 (m, 1H), 3.53 (m, 1H), 3.05 (m, 1H), 3.02 (m, 1H),
2.74 (m, 1H), 2.05 (s, 3H); Cþ conformer: 8.29 (s, 1H), 7.58 (s, 1H),
7.42 (s, 1H), 7.37 (s, 1H), 5.23 (m, 1H), 5.11 (m, 1H), 3.41 (m, 1H), 3.20
(m, 1H), 3.15 (m, 1H), 2.74 (m, 1H), 2.02 (s, 3H); 13C NMR (125 MHz,
1H NMR (500 MHz, CDCl3)
d conformer mixture: 4.25 (m, 2H), 3.51
(m, 2H), 3.16 (m, 2H), 2.93 (m, 8H), 2.88 (m, 6H), 2.76 (m, 2H); 13C
NMR at ꢀ18 ꢁC (125 MHz, CDCl3)
d
major: 174.2 (C), 54.3 (CH2), 37.8
CDCl3)
d
Tꢀ conformer: 180.3 (C), 176.4 (C), 175.3 (C), 59.1 (CH), 56.7
(CH2), 36.8 (CH2), 34.1 (CH3), 30.7 (CH2); minor: 176.4 (C), 47.2
(CH2), 37.9 (CH2), 37.3 (CH2), 36.6 (CH2), 32.2 (CH3); IR (neat) 3464
(m), 2921 (m), 1632 (s), 1462 (m), 1397 (m), 1284 (w), 1172
(w) cmꢀ1; LRMS (EI) m/z 2921 (m), 1632 (s), 1462 (m), 1397 (m),
1284 (w), 1172 (w); HRMS (EI) m/z 177.0283 [(Mþ), calcd for
C6H11NOS2: 177.0282].
(CH), 49.8 (CH2), 48.6 (CH2), 24.4 (CH3); T0ꢀ conformer: 176.1 (C),
175.6 (C), 174.9 (C), 57.4 (CH), 54.4 (CH), 50.9 (CH2), 43.3 (CH2), 24.4
(CH3); Cꢀ conformer: 176.5 (C), 174.0 (C), 173.8 (C), 55.6 (CH), 55.2
(CH), 45.0 (CH2), 43.3 (CH2), 24.4 (CH3); Cþ conformer: 176.1 (C),
174.7 (C), 173.6 (C), 61.7 (CH), 52.2 (CH), 44.5 (CH2), 41.2 (CH2), 24.3
(CH3); IR (neat) 3265 (br s), 1642 (s), 1516 (m), 1403 (m), 1179
(w) cmꢀ1; HRMS (EI) m/z 264.0466 [(Mþ), calcd for C8H13N3O3S2:
264.0476].5
4.6. Synthesis of dipeptide 6
4.6.1. 2R-(2R-Acetylamino-3-tritylsulfanyl-propionylamino)-
3-tritylsulfanyl-propionamide (29)
4.7. Synthesis of N-Me dipeptide 7
Peptide coupling of 0.50 g (1.23 mmol) 27 and 0.45 g
(1.25 mmol) 28 was analogous to the production of compound 23 in
DCM/DMF (0.1 M DCM). After impregnation onto flash silica, the
protected dipeptide was purified via gradient flash chromatogra-
phy (10:1 hexanes/EtOAc to 4:1 to 1:1 to EtOAc to DCM to
4.7.1. 4R-tert-Butyldisulfanylmethyl-5-oxo-oxazolidine-3-
carboxylic acid 9H-fluoren-9-ylmethyl ester (32)
To a room temperature solution of 10.0 g (23.1 mmol) Fmoc-
Cys(StBu)-OH in 230 mL PhH under natural atmosphere was added
3.59 g (155.1 g:1 mmol) p-formaldehyde and 0.37 g (7 mol %)
camphorsulfonic acid. The reaction mixture was heated to reflux
(oil bath¼100 ꢁC) in a Dean–Stark apparatus and stirred for 14 h.
The reaction mixture was concentrated to provide a clear oil. After
impregnation onto flash silica, the oxazolidinone was purified via
gradient flash chromatography (hexanes to 10:1 to 4:1 to 1:1 to
2.5%MeOH/DCM to 10%MeOH/DCM) to provide 0.72 g (79%) 29 as
20
a white solid. Rf¼0.56 (5%MeOH/DCM); [
a
]
ꢀ22.2 (c 0.09, CHCl3);
D
1H NMR (500 MHz, CDCl3)
d
7.47 (m, 12H), 7.32 (m, 12H), 7.26 (m,
6H), 6.45 (d, J¼7.8 Hz, 2H), 6.16 (d, J¼6.8 Hz, 1H), 5.67 (s, 1H), 4.21
(dd, J¼12.6, 6.8 Hz, 1H), 4.09 (dd, J¼13.1, 6.3 Hz, 1H), 2.90 (dd,
J¼12.6, 6.3 Hz, 1H), 2.73 (dd, J¼13.1, 6.3 Hz, 1H), 2.66 (dd, J¼13.1,
6.3 Hz, 1H), 2.56 (dd, J¼13.1, 5.3 Hz, 1H), 1.92 (s, 3H); 13C NMR
EtOAc) to provide 9.63 g (94%) 32 as a white viscous gum. Rf¼0.71
20
(125 MHz, CDCl3)
d
171.6 (C), 170.3 (C), 169.7 (C), 144.2 (CH), 144.0
(EtOAc); [
d
a
]
ꢀ13.6 (c 0.23, CHCl3); 1H NMR (500 MHz, CDCl3)
D
(CH), 129.4 (CH), 129.2 (CH), 128.0 (CH), 127.9 (CH), 129.9 (C), 126.8
(C), 67.1 (C), 67.0 (C), 52.2 (CH), 51.8 (CH), 33.2 (CH2), 32.9 (CH2),
22.8 (CH3); IR (neat) 3255 (m), 3056 (m), 2930 (w), 1744 (w), 1650
(s), 1488 (s), 1442 (s), 1032 (w), 697 (s) cmꢀ1; HRMS (EI) m/z
772.2637 [(MþK), calcd for C46H43N3O3S2: 772.2644].
7.76 (d, J¼7.6 Hz, 2H), 7.55 (d, J¼7.6 Hz, 2H), 7.40 (t, J¼7.6 Hz, 2H),
7.31 (m, 2H), 5.5–5.2 (br m’s, 2H), 4.75–4.35 (br m’s, 2H), 4.24 (br s,
1H), 4.01 (br s, 1H), 3.53 (br s, 0.5H), 3.20 (br s, 0.5H), 2.97 (br s,
0.5H), 2.66 (br s, 0.5H), 1.26 (s, 9H); 13C NMR (125 MHz, CDCl3)
d
170.8 (C),152.2 (C),143.4 (C),141.4 (C),127.9 (CH),127.2 (CH),124.6
(CH), 120.0 (CH), 78.4 (CH2), 73.9 (CH2), 67.6 (CH), 55.3 (C), 48.2
(CH), 47.2 (CH2), 29.6 (CH3); IR (neat) 1800 (s), 1714 (s), 1417 (s),
1288 (m),1129 (m),1052 (m), 910 (w), 737 (m) cmꢀ1; HRMS (EI) m/z
444.1308 [(Mþ), calcd for C23H25NO4S2: 444.1303].39
4.6.2. 2R-(2R-Acetylamino-3-mercapto-propionylamino)-
3-mercapto-propionamide (30)
Procedure and workup analogous to 25 using 0.20 g
(0.26 mmol) 29 as substrate in DCM (2 mM). After impregnation
onto flash silica, the dithiol was purified via gradient flash chro-
4.7.2. 3-tert-Butyldisulfanyl-2R-[(9H-fluoren-9-ylmethoxy
matography (hexanes to DCM to EtOAc) to provide 64.5 mg (91%) of
carbonyl)-methyl-amino]-propionic acid (33)
20
30 as a foul-smelling oil. Rf¼0.1 (10%MeOH/DCM); [
a
]
ꢀ13.6 (c
To a room temperature stirring solution of 9.03 g (20.3 mmol)
32 in 101 mL CHCl3 under natural atmosphere was added 32.8 mL
(203 mmol) TES followed by the rapid addition of 50 mL TFA. The
reaction mixture was stirred at room temperature for 16 h and then
concentrated. The oil was dissolved in 150 mL DCM and concen-
trated. This was repeated three consecutive times. After impreg-
nation onto flash silica, MeCys 33 was purified via gradient flash
chromatography (hexanes to 10:1 to 4:1 to 2:1; 1:1 to EtOAc) to
D
0.22, CHCl3); 1H NMR (500 MHz, CDCl3)
d
6.99 (br m, 2H), 6.39 (br
m, 1H), 5.59 (br m, 1H) 4.60 (dd, J¼9.6, 6.4 Hz, 1H), 4.51 (dd, J¼13.3,
6.2 Hz,1H), 3.08 (dd, J¼7.9, 6.2 Hz,1H), 3.00 (dd, J¼13.3, 6.6 Hz, 1H),
2.90 (dd, J¼12.4, 7.3 Hz, 1H), 2.81 (dd, J¼13.1, 6.6 Hz, 1H), 2.08 (s,
3H), 1.70 (m, 1H), 1.60 (m, 1H); 13C NMR (125 MHz, CDCl3)
d 178.2
(C), 175.4 (C), 173.9 (C), 67.1 (CH), 65.0 (CH), 32.3 (CH2), 31.9 (CH2),
23.7 (CH3); IR (neat) 3282 (m), 3021 (m),1741 (m),1633 (s),1535 (s),
1444 (s), 1211 (m), 1171 (s), 699 (s) cmꢀ1; HRMS (EI) m/z 304.0186
[(MþK), calcd for C8H15N3O3S2: 304.0192].
provide 8.05 g (89%) 33 as a white solid in a 1.5:1.0 conformer ratio.
20
Rf¼0.32 (EtOAc); [
a
]
ꢀ30.4 (c 0.33, CHCl3); 1H NMR (500 MHz,
D
CDCl3)
d
major conformer: 10.36 (br s,1H), 7.82 (m, 2H), 7.69 (m, 2H),
4.6.3. 7R-Acetylamino-6-oxo-[1,2,5]dithiazocane-4R-carboxylic
acid amide (6)
7.46 (m, 2H), 7.39 (m, 2H), 4.91 (dd, J¼10.5, 4.3 Hz,1H), 4.52 (m, 2H),
4.36 (t, J¼7.1 Hz, 1H), 3.47 (dd, J¼13.9, 4.3 Hz, 1H), 3.25 (dd, J¼13.9,
10.7 Hz, 1H), 3.13 (s, 3H), 1.43 (s, 9H); minor conformer: 10.36 (br s,
1H), 7.81 (m, 2H), 7.68 (m, 2H), 7.46 (m, 2H), 7.39 (m, 2H), 4.86 (m,
1H), 4.63 (dd, J¼10.5, 6.4 Hz,1H), 4.57 (m,1H), 4.32 (t, J¼6.0 Hz,1H),
3.33 (dd, J¼12.2, 5.5 Hz, 0.5H), 3.23 (dd, J¼13.9, 4.7 Hz, 1H), 3.06 (s,
3H), 2.84 (dd, J¼13.7,10.5 Hz, 0.5H),1.41 (s, 9H); 13C NMR (125 MHz,
Procedure and workup analogous to
2 using 70.9 mg
(0.26 mmol) 30 as substrate in ACN (1 mM). After impregnation
onto flash silica, the disulfide was purified via gradient flash
chromatography (DCM to 2.5%MeOH/DCM to 5%MeOH/DCM to
20
10%MeOH/DCM). Rf¼0.27 (10%MeOH/DCM); [
a
]
D
ꢀ24.4 (c 0.08,
D2O); 1H NMR (500 MHz, DMSO-d6/D2O)
d
Tꢀ conformer: 8.09 (s,
CDCl3) d major conformer: 174.4 (C), 156.5 (C), 143.5 (C), 141.0 (C),
1H), 7.73 (s, 1H), 7.39 (s, 1H), 7.28 (s, 1H), 4.96 (m, 1H), 4.29 (m, 1H),
3.68 (m, 1H), 3.42 (m, 1H), 3.39 (m, 1H), 3.28 (m, 1H), 2.11 (s, 3H);
T0ꢀ conformer: 8.62 (d, J¼5.0 Hz, 1H), 8.03 (d, J¼5.1 Hz, 1H), 7.53
(m, 1H), 7.09 (s, 1H), 4.95 (m, 1H), 3.98 (m, 1H), 3.63 (m, 1H), 3.59
(m, 1H), 3.44 (m, 1H), 3.43 (m, 1H), 2.07 (s, 3H); Cꢀ conformer: 8.54
(m, 1H), 8.22 (d, J¼7.2 Hz, 1H), 8.15 (d, J¼7.2 Hz, 1H), 6.92 (m, 1H),
127.4 (CH), 126.8 (CH), 124.8 (CH), 124.6 (CH), 67.9 (CH2), 59.5 (CH),
48.0 (C), 46.8 (CH), 38.8 (CH2), 33.4 (CH3), 29.7 (CH3); minor con-
former: 174.3 (C), 156.1 (C), 143.6 (C), 141.0 (C), 127.2 (CH), 126.9
(CH), 124.9 (CH), 124.7 (CH), 67.8 (CH2), 58.5 (CH), 47.9 (C), 46.7
(CH), 39.0 (CH2), 32.7 (CH3), 29.7 (CH3); IR (neat) 3353 (w), 1702 (s),
1449 (s), 1399 (m), 1317 (s), 1165 (s), 1129 (m), 976 (w), 738

1266
E.L. Ruggles et al. / Tetrahedron 65 (2009) 1257–1267
(s) cmꢀ1; HRMS (EI) m/z 446.1457 [(Mþ), calcd for C23H27NO4S2:
4.7.5. 2R-[(2R-Acetylamino-3-tritylsulfanyl-propionyl)-methyl-
amino]-3-mercapto-propionic acid methyl ester (36)
446.1460].39
To a room temperature solution of 32.8 mg (52.4
mmol) 35 in
4.7.3. 3-tert-Butyldisulfanyl-2R-[(9H-fluoren-9-ylmethoxy
0.5 mL DMF were added 80.9 g (0.52 mmol) dithiothreitol and 5 mL
carbonyl)-methyl-amino]-propionic acid methyl ester (34)
(52.4 mol) N-methyl morpholine, stirred for 24 h, and then con-
centrated. Reaction mixture was transferred into 50 mL EtOAc and
the EtOAc layer was washed two consecutive times with 50 mL H2O
and once with 50 mL brine. The EtOAc layer was dried over MgSO4,
filtered, and concentrated. The solid was purified via reverse-phase
In an air-dried flask, 0.08 g (0.59 mmol) K2CO3 was added to
a stirring solution of 0.26 g (0.54 mmol) 33 in 0.5 mL DMF pre-
viously chilled to 0 ꢁC. Upon complete addition, 0.06 mL
(1.08 mmol) MeI was added and the reaction mixture was stirred at
0 ꢁC for 30 min. The ice bath was removed and the reaction mixture
was stirred for 1 h. The reaction mixture was then filtered and
concentrated. After impregnation onto flash silica, the N-Me amino
ester was purified via gradient flash chromatography (hexanes to
HPLC to provide 23.6 mg (84%) 36 as a white solid in a 1.0:1.0
20
conformer ratio. Rf¼0.38 (EtOAc); [
a
]
D
ꢀ64.0 (c 0.07, CHCl3); 1H
NMR (500 MHz, CDCl3)
d
conformer mixture: 7.37 (m, 12H), 7.29 (m,
12H), 7.22 (m, 6H), 6.59 (m, 2H), 4.86 (m, 3H), 4.64 (m, 1H), 3.67 (s,
3H), 3.66 (s, 3H), 3.07 (m, 2H), 2.91 (s, 3H), 2.83 (s, 3H), 2.75 (m, 1H),
2.70 (m, 1H), 2.56 (m, 2H), 2.54 (m, 2H), 2.03 (s, 3H), 2.00 (s, 3H),
1.59 (t, J¼8.3 Hz, 1H), 1.51 (t, J¼8.7 Hz, 1H); 13C NMR (125 MHz,
10:1 to 4:1 to 1:1 to EtOAc to DCM) to provide (95%) 34 as a water-
20
white oil in a 2.0:1.0 conformer ratio. Rf¼0.86 (EtOAc); [
a
]
ꢀ268.5
D
(c 0.35, CHCl3); 1H NMR (500 MHz, CDCl3)
d
major conformer: 7.76
(d, J¼7.5 Hz, 2H), 7.60 (d, J¼7.5 Hz, 2H), 7.40 (t, J¼7.5 Hz, 2H), 7.31
(dt, J¼7.5, 7.3 Hz, 2H), 4.79 (dd, J¼10.3, 4.5 Hz, 1H), 4.41 (dd, J¼13.9,
7.0 Hz, 2H), 4.30 (t, J¼7.0 Hz, 1H), 3.74 (s, 3H), 3.33 (dd, J¼13.9,
4.7 Hz, 1H), 3.11 (dd, J¼13.9, 10.5 Hz, 1H), 3.01 (s, 3H), 1.34 (s, 9H);
minor conformer: 7.76 (d, J¼7.5 Hz, 2H), 7.58 (d, J¼7.3 Hz, 2H), 7.40
(t, J¼7.5 Hz, 2H), 7.31 (dt, J¼7.5, 7.3 Hz, 2H), 4.72 (dd, J¼9.0, 4.5 Hz,
1H), 4.52 (dd, J¼10.1, 5.5 Hz, 1H), 4.46 (dd, J¼9.6, 5.5 Hz, 1H), 4.2.5
(t, J¼5.5 Hz, 1H), 3.61 (s, 3H), 3.11 (dd, J¼13.9, 10.5 Hz, 1H), 2.93 (s,
3H), 2.73 (dd, J¼13.9, 9.8 Hz, 1H), 1.32 (s, 9H); 13C NMR (125 MHz,
CDCl3) d conformer mixture: 173.3 (C), 172.3 (C), 171.4 (C),170.7
(C),170.2 (C), 169.7 (C), 144.5 (C), 144.4 (C), 129.5 (CH), 129.5 (CH),
127.9 (CH), 127.9 (CH), 126.8 (CH), 126.7 (CH), 67.1 (CH), 67.0 (CH),
59.4 (C), 58.9 (C), 54.0 (CH), 53.2 (CH), 52.6 (CH3), 52.5 (CH3), 34.4
(CH2), 34.1 (CH2), 33.8 (CH2), 31.7 (CH2), 30.0 (CH3), 29.6 (CH3), 23.1
(CH3), 23.0 (CH3); IR (neat) 3288 (w), 3058 (w), 1740 (s), 1637 (s),
1487 (s), 1443 (s), 1241 (s), 1154 (s), 1097 (m), 1033 (m), 742 (s), 699
(s) cmꢀ1; HRMS (EI) m/z 575.1443 [(MþK), calcd for C29H32N2O4S2:
575.1441].
CDCl3)
d major conformer: 169.5 (C), 150.5 (C), 143.8 (C), 141.3 (C),
127.6 (CH), 127.0 (CH), 125.1 (CH), 124.8 (CH), 73.1 (CH2), 59.6 (CH),
48.1 (CH3), 47.1 (CH2), 42.7 (C), 39.6 (CH2), 33.2 (CH3), 29.9 (CH3);
minor conformer: 170.6 (C), 153.2 (C), 144.5 (C), 139.9 (C), 127.4 (CH),
126.8 (CH), 125.0 (CH), 124.8 (CH), 67.8 (CH2), 62.0 (CH), 52.5 (CH3),
46.5 (CH2), 42.8 (C),39.4 (CH2), 33.1 (CH3), 29.8 (CH3); IR (neat) 3010
(w), 1743 (s), 1700 (s), 1449 (m), 1312 (m), 1164 (s), 1000 (m), 737
(s) cmꢀ1; HRMS (EI) m/z 459.1539 [(Mþ), calcd for C24H29NO4S2:
459.1538].
4.7.6. 7R-Acetylamino-5-methyl-6-oxo-[1,2,5]dithiazocane-4R-
carboxylic acid methyl ester (7)
In an air-dried flask, 21.0 mg (67.9
pyridine) was added to a stirring solution of 36.1 mg (67.2
m
mol) 2,20-dithiobis(5-nitro-
mol) 36
m
in 25 mL DCM at room temperature producing a yellow color over
time. After 14 h, 10 mL TFA was added and an even brighter yellow
color being produced over time. After 2 h, the reaction was scav-
enged by the addition of 0.05 mL (0.33 mmol) TES creating a water-
white reaction. Upon complete addition, the reaction mixture was
stirred at room temperature for 8 h, once again with slow evolution
of yellow color, and concentrated. The yellow solid was purified via
reverse-phase HPLC to provide 7.0 mg (36%) 7 as a white solid in
4.7.4. 2R-[(2R-Acetylamino-3-tritylsulfanyl-propionyl)-methyl-
amino]-3-tert-butyldisulfanyl-propionic acid methyl ester (35)
To a room temperature solution of 0.89 g (1.95 mmol) 34 in
19.5 mL DCM was added 2.04 mL (19.5 mmol) Et2NH, stirred for
1.5 h, and then concentrated. After dissolution in 21 mL DMF, 0.86 g
(2.14 mmol) 27 and 0.81 g (2.14 mmol) HATU were added. Upon
complete dissolution, 0.48 mL (2.92 mmol) Hu¨nigs base was added
and the reaction mixture was stirred at room temperature for 3 h.
Reaction mixture was poured into 100 mL EtOAc. The EtOAc layer
was washed two consecutive times with 100 mL 10% aqueous
NaHCO3, once with 100 mL H2O and once with 100 mL brine. The
EtOAc layer was dried over MgSO4, filtered, and concentrated. After
impregnation onto flash silica, the dipeptide was purified via gra-
dient flash chromatography (hexanes to 10:1 to 5:1 to 2:1 to 1:1 to
a single conformation and 12.5 mg (31%) 37 as a white solid in
20
a single conformation. Rf¼0.14 (EtOAc); [
a
]
D
ꢀ19.2 (c 0.02, CHCl3);
1H NMR (500 MHz, CDCl3)
d
6.85 (s, 1H), 5.31 (dd, J¼11.9, 3.6 Hz,
1H), 5.13 (t, J¼7.6 Hz, 1H), 3.81 (s, 3H), 3.38 (dd, J¼13.7, 3.9 Hz, 1H),
3.04 (d, J¼14.0 Hz, 1H), 2.96 (d, J¼13.7 Hz, 1H), 2.89 (dd, J¼11.3,
2.4 Hz, 1H), 2.86 (s, 3H), 2.01 (s, 3H); 13C NMR (125 MHz, CDCl3)
d
171.7 (C), 169.3 (C), 169.0 (C), 57.5 (CH), 52.1 (CH), 53.1 (CH3), 42.4
(CH2), 38.9 (CH2), 30.3 (CH3), 22.7 (CH3); IR (neat) 3332 (m), 1741
(s), 1637 (s), 1436 (w), 1407 (w), 1240 (w) cmꢀ1; HRMS (EI) m/z
293.0623 [(Mþ), calcd for C10H16N2O4S2: 293.0630].
EtOAc to 10%MeOH/DCM) to provide (68%) 35 as a white solid in
4.7.7. 2R-{[2R-Acetylamino-3-(5-nitro-pyridin-2-yldisulfanyl)-
propionyl]-methyl-amino}-3-(5-nitro-pyridin-2-yldisulfanyl)-
propionic acid methyl ester (37)
20
a 1.0:1.0 conformer ratio. Rf¼0.45 (EtOAc); [
a
]
ꢀ131.6 (c 0.06,
D
CHCl3); 1H NMR (500 MHz, CDCl3)
d
conformer mixture: 7.39 (m,
12H), 7.25 (m, 12H), 7.18 (m, 6H), 6.46 (br t, J¼7.8 Hz, 2H), 4.93–4.82
(series of m’s, 3H), 4.64 (dd, J¼9.7, 4.8 Hz, 1H), 3.61 (s, 3H), 3.60 (s,
3H), 3.29 (t, J¼4.3 Hz, 1H), 3.26 (t, J¼4.3 Hz, 1H), 3.05 (dd, J¼13.6,
10.2 Hz, 1H), 2.93 (dd, J¼14.1, 10.2 Hz, 1H), 2.87 (s, 3H), 2.79 (s, 3H),
2.51 (m, 4H), 1.91 (s, 3H), 1.90 (s, 3H), 1.29 (s, 9H), 1.28 (s, 9H); 13C
Compound 37 was obtained as a single conformer during the 36
20
to 7 transformation (see Section 4.7.6). Rf¼0.34 (EtOAc); [
a
]
ꢀ26.2
D
(c 0.06, CHCl3); 1H NMR (500 MHz, CDCl3)
d
9.31 (s, 2H), 8.43 (d,
J¼8.7 Hz, 2H), 7.82 (t, J¼8.7 Hz, 2H), 6.65 (d, J¼8.7 Hz, 1H), 5.39 (m,
1H), 5.05 (m, 1H), 3.75 (s, 3H), 3.56 (dd, J¼14.1, 5.3 Hz, 1H), 3.36 (dd,
J¼13.6, 4.8 Hz, 1H), 3.26 (dd, J¼13.6, 9.7 Hz, 1H), 3.18 (s, 3H), 3.12
(dd, J¼13.6, 7.8 Hz, 1H), 2.09 (s, 3H); 13C NMR (125 MHz, CDCl3)
NMR (125 MHz, CDCl3)
d conformer mixture: 175.1 (C), 174.6 (C),
171.2 (C), 170.9 (C), 169.8 (C), 169.7 (C), 144.4 (C), 144.4 (C), 129.5
(CH), 129.5 (CH), 127.9 (CH), 127.8 (CH), 126.7 (CH), 126.7 (CH), 66.9
(CH), 66.9 (CH), 61.0 (C), 59.4 (C), 58.0 (CH), 57.8 (CH), 52.4 (CH3),
52.4 (CH3), 48.4 (C), 48.1 (C), 38.7 (CH2), 38.4 (CH2), 34.4 (CH2), 34.3
(CH2), 29.9 (CH3), 29.8 (CH3), 29.7 (CH3), 29.7 (CH3), 23.1 (CH3), 23.1
(CH3); IR (neat) 3327 (m), 2926 (s), 1734 (m), 1623 (m), 1567 (m),
d
191.2 (C), 191.0 (C), 165.9 (C), 165.3 (C), 164.3 (C), 145.7 (CH), 145.2
(CH), 141.2 (C), 139.8 (C), 131.9 (CH), 131.7 (CH), 119.9 (CH), 119.9
(CH), 63.8 (CH), 52.9 (CH), 48.4 (CH3), 37.6 (CH2), 29.8 (CH2), 23.4
(CH3), 19.6 (CH3); IR (neat) 3291 (br w), 3060 (w), 2925 (w), 1739
(m), 1645 (s), 1587 (s), 1563 (s), 1514 (s), 1430 (vs), 1096 (s), 1007
(w) cmꢀ1; HRMS (EI) m/z 603.0459 [(Mþ), calcd for C20H22N6O8S4:
603.0460].
1242 (m) cmꢀ1
C33H40N2O4S3: 624.2150].
;
HRMS (EI) m/z 624.2140 [(MþH), calcd for

E.L. Ruggles et al. / Tetrahedron 65 (2009) 1257–1267
1267
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