Synthesis and properties of disulfide-bond containing eight-membered rings

Article abstract of DOI:10.1016/j.tetlet.2006.04.023

The cyclocystine ring structure (CRS, 3), which results from a disulfide-bond between adjacent cysteine residues, is a rare motif in protein structures and is functionally important to those few proteins that posses it. This letter will focus on the const

Full text of DOI:10.1016/j.tetlet.2006.04.023

Tetrahedron Letters 47 (2006) 4281–4284
Synthesis and properties of disulfide-bond containing
eight-membered rings
Erik L. Ruggles and Robert J. Hondal*
Department of Biochemistry, 89 Beaumont Avenue, Given Building, University of Vermont, Burlington, VT 05405, USA
Received 22 February 2006; revised 31 March 2006; accepted 5 April 2006
Available online 5 May 2006
Abstract—The cyclocystine ring structure (CRS, 3), which results from a disulfide-bond between adjacent cysteine residues, is a rare
motif in protein structures and is functionally important to those few proteins that posses it. This letter will focus on the construc-
tion of CRS mimics and the determination of their respective redox potentials.
Ó 2006 Elsevier Ltd. All rights reserved.
A disulfide-bond between adjacent cysteine residues (3,
Fig. 1) is a very rare occurrence in protein structures.
Currently, 32 out of ca. ꢀ28,000 proteins structurally
angle of 180° is not allowed for a CRS due to its inabil-
ity to form the disulfide-bond. Hence, the nitrogen lone-
pair must come slightly out of phase with the p-system
to allow disulfide-bond formation.
identified in the Brookhaven Protein Data Bank
1
(
PDB) carry this unique motif. In every case the amide
bond of the CRS is reported to be in a strained trans
geometry with an average x value of 171°. Peptide
A reasonable model for a CRS is cyclooctene. Energeti-
cally, the cis isomer is more stable than the trans as a
result of the ring strain required to incorporate a trans
double bond. This ring strain is demonstrated by the
higher DHHydrogenation of trans-cyclooctene (34.4 kcal/
1
bonds prefer a trans conformation with a torsion angle
of 180° so that the nitrogen lone-pair can have maximal
delocalization into the p-system, while minimizing steric
repulsions from peptidyl side-chains. However, the small
ring nature of the eight-membered CRS allows for mul-
tiple amide conformations to be energetically feasible.
The amide bond could adopt a cis conformation, which
still allows for delocalization of the nitrogen lone-pair,
but would cause the main peptidyl-chain to have a kink
in it. A ‘strained’ trans conformation allows the main
chain to remain relatively unaltered, but still allows
for partial delocalization of the nitrogen lone-pair into
the p-system. Our model studies show that a torsion
2
mol) compared to the cis isomer (23.0 kcal/mol). If
the cyclooctene analogy is applied to the eight-mem-
bered CRS, one might expect cis amide geometry to pre-
dominate. This is not what has been observed
experimentally in the PDB. Investigations into proteins
that carry the CRS reveal that this motif is important
3
for activity. The central focus of this study is to assess
how CRS conformation affects the redox potential of
the disulfide-bond. Our central hypothesis is that a
CRS with a cis peptide bond should be much more
reducing (low redox potential) than a CRS with a trans
peptide bond.
In order to test this hypothesis, cis and trans substrates
were constructed in both oxidized (1 and 2) and reduced
forms (10 and 13). Both systems have a central bond
(
cis/trans-olefin) with restricted geometry that mimics
the 0° and 180° amide conformations of a cis and trans
CRS. The synthesis of these compounds has not been
reported previously, though the theoretical value for
Figure 1. Small molecule mimics of CRS.
4
the redox potential of 1 has been calculated.
Keywords: Cyclocystine; Dithiocine; Vicinal disulfide-bond; Thiol–
disulfide redox.
*
Corresponding author. Tel.: +1 802 656 8282; fax: +1 802 656
220; e-mail: Robert.Hondal@uvm.edu
Retrosynthetically, dithiocines 1 and 2 are available
upon intramolecular oxidation of the appropriate
8
0040-4039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.04.023

4282
E. L. Ruggles, R. J. Hondal / Tetrahedron Letters 47 (2006) 4281–4284
redox potential (E0(BDT)), allows for the redox potential
of the CRS mimics to be determined by Eqs. 1 and 2.
ꢁ
1
ox
K
¼ Kred ¼ ½CRSredꢂ½BDToxꢂ=½CRSoxꢂ½BDTred
ꢂ
ð1Þ
ð2Þ
E
0
¼ E0ðBDTÞ ꢁ 0:03 logðKoxÞ
The synthesis of cis-dithiocine 1 begins with the epoxi-
dation of 1,4-cyclohexadiene to generate 8 almost quan-
8
titatively (Scheme 1). A subsequent one-pot protocol
entails diol formation, followed by oxidative ring cleav-
age and reduction of the intermediate acyclic dial to pro-
Figure 2. Retrosynthesis of dithiocines.
dithiol precursor. Originally the dithiol substrates were
to be constructed via dithioureic salt 6. However, while
the synthesis of 6 was non-problematic, the harsh condi-
tions necessary for the generation of the dithiol led to
5
significant by-product formation. This led to the use
of dithioester 7 as the intermediary target, which could
6
undergo saponification easily (Fig. 2).
The redox properties were determined by thiol–disulfide
exchange, with the varying concentrations of reduced
1
7
and oxidized forms monitored by H NMR (Fig. 3).
Employment of oxidized or reduced butane dithiol
BDTox and BDTred, respectively), a species of known
(
Scheme 1. Synthesis of cis-dithiocine (1).
Figure 3. Proton NMR equilibrium redox experiment for cis- and trans-dithiols.

E. L. Ruggles, R. J. Hondal / Tetrahedron Letters 47 (2006) 4281–4284
4283
Scheme 2. Attempted synthesis of trans-dithiocine (2).
1
9
Figure 4. H NMR temperature dependent coalescence experiment for
cis-dithiocine (1).
duce 9 in good overall yield. The formation of dithio-
1
0
ester cis-7 occurs under Mitsunobu conditions, after
which mild saponification affords dithiol 10. Air-oxida-
6
tion mediated by CsF impregnated celite produced the
1
1
desired cis-dithiocine 1. Disulfide formation does
occur without the CsF–celite additive; however, reaction
times are significantly longer.
when compared to the trans isomer. As a result, cis-
dithiocine 1 is much more stable than trans-dithiocine
2. The stability and absence of ring-strain in 1, when
compared to 2, is also demonstrated via temperature
1
The synthesis of trans-dithiocine precursor 13 begins
with trans-mucionic acid (Scheme 2). Methyl esterifica-
dependent H NMR coalescence experiments (Fig. 4).
1
2
tion and reduction generates trans-diol 12. Analogous
to the construction of 1, Mitsunobu thioesterification
and mild saponification affords trans-dithiol 13 in good
overall yield. While the formation of dithiol 13 was not
troublesome, oxidative intramolecular construction of 2
was never realized even under dilute conditions. The
only disulfide-bond containing compound isolated was
that of cyclic-dimer 14.
It is interesting to compare trans-dithiol 13 to that of a
CRS found in proteins. A disulfide-bond can form be-
tween nearest neighbors because the peptide bond is
not as rigid as an olefin. This allows the central peptide
bond to ‘twist’ slightly allowing disulfide-bond forma-
tion to occur with a strained transoid geometry. When
the central torsional angle is constrained to 180°, as is
the case for 13, disulfide-bond formation is impossible.
Redox enzymes, such as mammalian thioredoxin reduc-
tase, may take advantage of the low redox potential of
adjacent cysteine residues in a cis configuration, which
The redox potentials were then determined by equilib-
rium thiol–disulfide exchange. These redox experiments
reached equilibrium in approximately 5 days in DMSO-
d6. Integration of olefinic signals, as well as others,
allowed for the respective redox potentials to be deter-
mined (Fig. 3).13 This resulted in a derived cyclomono-
meric redox potential of ꢁ0.318 eV for dithiol 10
1
7
cycle between reduced and oxidized states. This is
especially true for this enzyme since the redox pair
occurs at the C-terminus, which would minimize the
effect of a cisoid peptide bond on the main chain.
1
4
(
Fig. 3a), which is in close agreement with the
4
predicted redox potential for this compound. The cyc-
Acknowledgments
lodimeric redox potential of trans-dithiol 13 was deter-
1
5
mined to be ꢁ0.329 eV (Fig. 3b). The inability to
determine the cyclomonomeric redox potential for the
disulfide-bond of 2 alludes to its highly oxidative
character.
This study was supported by NIH Grant GM070742 to
R.J.H. E.L.R. was supported by NIH Grant PHS T32
HL07594.
The ease of monomeric disulfide formation for the pro-
References and notes
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ˇ
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(125 MHz, CDCl
HRMS (EI) m/z 148.0379 [(M ) calcd for C H S :
148.0380]. Compound 14: H NMR (500 MHz, CDCl )
3 2 2
) d 128.9 (CH), 38.4 (CH ), 27.2 (CH );
+
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6 12 2
1
3
1
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6
d 5.62 (m, 4H), 2.79 (t, J = 7.1 Hz, 8H), 2.40 (m, 8H);
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20 4
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d c j g
, H , H and H H NMR signals (Fig. 3a),
which led to a ratio of 1.00:1.64:3.60:3.60 (1:BDTred
:
10:BDTox). Equilibrium concentrations were then deter-
mined by comparison of the equilibrium ratio to initial
concentrations of 1 and BDTred. Insertion into Eq. 2
produces
3. Redox experiments were conducted under dry Ar, with
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followed by sonication (10 min) at aspirator pressure.
Standard Redox Procedure: Dithiocine 1 (1.0 equiv) and
E
0
¼ ꢁ0:345–0:03 log½ð1:30Þð3:08Þ=ð4:69Þð6:76Þꢂ
ꢁ0:318 eV:
¼
15. The redox potential of 13 was determined by the integration
1
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6
, after
of H , H , H and H H NMR signals (Fig. 3b), which
d
c
i
g
which a 10 mM NaOD/D O (5 mol% NaOD) was added.
led to a ratio of 1.00:2.00:1.11:1.93 (14:BDTred:13:BDTox).
Equilibrium concentrations were then determined by
comparison of the equilibrium ratio to initial concentra-
2
Residual O was removed by Ar-sparging for 30 min. The
2
1
redox experiment was monitored by H NMR and reached
equilibrium in 5 days. Compound 10:
1
H
NMR
tions of 13 and BDT . Use of a cyclodimeric derived form
ox
(
500 MHz, CDCl ) d 5.49 (t, J = 4.7 Hz, 2H), 2.72 (t,
of Eq. 2 produces
3
J = 7.0 Hz, 4H), 2.47 (q, J = 7.0 Hz, 4H), 1.25 (br s, 2H);
1
3
2
2
2
C NMR (125 MHz, CDCl
3
) d 128.9 (CH), 38.4 (CH
2
),
E ¼ ꢁ0:345–0:03 log½ð2:84Þð5:30Þ =ð3:14Þ ð5:12Þ ꢂ
0
+
2
7.2 (CH ); HRMS (EI) m/z 148.0382 [(M ) calcd for
2
1
¼ ꢁ0:329 eV:
C H S : 148.0380]. Compound 1: H NMR (500 MHz,
6
12
2
CDCl
3
) d 5.67 (m, 2H), 2.87 (br s, 2H), 2.64 (br s, 4H),
1
3
2.50 (br s, 2H); C NMR (125 MHz, CDCl
3
) d 129.7
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(
CH), 39.4 (CH ), 27.2 (CH ); HRMS (EI) m/z 146.0225
2
2
+
1
[(M ) calcd for C H S : 146.0224]. 13: H NMR
6
10 2
(
4
500 MHz, CDCl
H), 2.33 (m, 4H), 1.42 (t, J = 7.7 Hz, 2H); C NMR
3
) d 5.47 (m, 2H), 2.57 (q, J = 7.7 Hz,
13