Double-helical cyclic peptides: Design, synthesis, and crystal structure of figure-eight mirror-image conformers of adamantane-constrained cystine- containing cyclic peptide cyclo (Adm-Cyst)3

Article abstract of DOI:10.1021/jo0003807

A large number of macrocycles containing alternating repeats of cystine diOMe(-NH-CH-(CO₂Me)-CH₂-S-)₂ and either a conformationally rigid aromatic/alicyclic moiety or a flexible polymethylene unit (X) in the cyclic backbone with ring size varying from 13- to 78-membered have been examined by spectral (¹H NMR, FT-IR, CD) and X-ray crystallography studies for unusual conformational preferences. While ¹H NMR measurements indicated a turnlike conformation for all macrocycles, stabilized by intramolecular NH···CO hydrogen bonding, as also supported by FT-IR spectra in chloroform, convincing proof for β-turn structures was provided by circular dichroism studies. Single-crystal X-ray studies on 39-membered cyclo (Adm-L-Cyst)₃ revealed a double-helical fold (figure-eight motif for the macrocycle. Only a right-handed double helix was seen in the macrocycle constructed from L- cystine. The mirror-image macrocycle made up of D-cystine units exhibited a double helix with exactly the opposite screw sense, as expected. The enantiomeric figure-eights were stabilized by two intramolecular NH···CO hydrogen bonds and exhibited identical ¹H NMR and FT-IR spectra. The CD spectra of both isomers had a mirror-image relationship. The present results have clearly brought out the importance of cystine residues in inducing turn conformation that may be an important deciding factor for the adoption of topologically important structures by macrocycles containing multiple S-S linkages.

Full text of DOI:10.1021/jo0003807

J . Org. Chem. 2000, 65, 4415-4422
4415
Dou ble-Helica l Cyclic P ep tid es: Design , Syn th esis, a n d Cr ysta l
Str u ctu r e of F igu r e-Eigh t Mir r or -Im a ge Con for m er s of
Ad a m a n ta n e-Con str a in ed Cystin e-Con ta in in g Cyclic P ep tid e Cyclo
(Ad m -Cyst)3
Darshan Ranganathan,*,† V. Haridas,† R. Nagaraj,‡ and Isabella L. Karle*,§
Discovery Laboratory, Indian Institute of Chemical Technology, Hyderabad-500 007, India,
Centre for Cellular and Molecular Biology, Hyderabad-500 007, India, and Laboratory for the
Structure of Matter, Naval Research Laboratory, Washington, DC 20375-5341
ranganathan@iict.ap.nic.in
Received March 15, 2000
A large number of macrocycles containing alternating repeats of cystine diOMe(-NH-CH-
(CO2Me)-CH2-S-)2 and either a conformationally rigid aromatic/alicyclic moiety or a flexible
polymethylene unit (X) in the cyclic backbone with ring size varying from 13- to 78-membered
have been examined by spectral (1H NMR, FT-IR, CD) and X-ray crystallography studies for unusual
1
conformational preferences. While H NMR measurements indicated a turnlike conformation for
all macrocycles, stabilized by intramolecular NH‚‚‚CO hydrogen bonding, as also supported by
FT-IR spectra in chloroform, convincing proof for â-turn structures was provided by circular
dichroism studies. Single-crystal X-ray studies on 39-membered cyclo (Adm-L-Cyst)3 revealed a
double-helical fold (figure-eight motif) for the macrocycle. Only a right-handed double helix was
seen in the macrocycle constructed from L-cystine. The mirror-image macrocycle made up of D-cystine
units exhibited a double helix with exactly the opposite screw sense, as expected. The enantiomeric
figure-eights were stabilized by two intramolecular NH‚‚‚CO hydrogen bonds and exhibited identical
1 H NMR and FT-IR spectra. The CD spectra of both isomers had a mirror-image relationship. The
present results have clearly brought out the importance of cystine residues in inducing turn
conformation that may be an important deciding factor for the adoption of topologically important
structures by macrocycles containing multiple S-S linkages.
In tr od u ction
and sheets are quite well understood, the reasons for
adopting a double-helical fold still remain to be identified.
Several simple models of a figure-eight motif from
macrocyclic ligands7-14 have been reported. While in
metal-assisted figure eights9,13 it is the metal ion and its
preference for a particular coordination geometry that
dictates the formation of a double-helical fold, in simple
macrocycles, apart from the ring size, the major control-
ling factors appear to be the stabilizations due to
hydrogen bond formation and or aromatic π-π interac-
tions.
The figure-eight motif representing the simplest form
of a double-helical arrangement in a ring has recently
attracted much attention not only for its unique su-
pramolecular architecture but also for its topological
implications in biological systems.1-6 Although large
cyclic peptides are known to have a natural tendency to
fold their backbone, assisted by either intracyclic hydro-
gen bonding or aromatic π-π stacking, into stabilized
conformations of various secondary structural elements,
for example, â-turns, â-sheets, and helical motifs, and
while the structural factors responsible for inducing turns
Interestingly, while molecules with a figure-eight
topology are quite well-known in the field of helicines,15
* To whom correspondence should be addressed. (D.R.) Honorary
Faculty Member J awaharlal Nehru Centre for Advanced Scientific
Research. Fax: (040)7173387. (I.L.K.) Code 6030, Naval Research
Laboratory, Washington, DC. Fax: 202-767-6874.
(7) Mascal, M.; Moody, C. J .; Morrel, A. I.; Slawin, A. M. Z.; Williams,
D. J . J . Am. Chem. Soc. 1993, 115, 813.
(8) Mascal, M.; Wood, I. G.; Begley, M. J .; Batsanov, A. S.; Wals-
grove, R.; Slawin, A. M. Z.; Williams, D. J .; Drake, A. F.; Siligardi, G.
J . Chem. Soc., Perkin Trans. 1 1996, 2427.
(9) Comba, P.; Fath, A.; Hambley, T. W.; Richens, T. D. Angew.
Chem., Int. Ed. Engl. 1995, 34, 1883.
(10) Sessler, J . L,; Weghorn, S. J .; Lynch, V.; J ohnson M. R. Angew.
Chem., Int. Ed. Engl. 1994, 33, 1509.
(11) Vogel, E.; Broring, M.; Fink, J .; Rosen, D.; Schmickler, H.; Lex,
J .; Chan, K. W. K.; Wu, Y.-D.; Plattner, D. A.; Nendel, M.; Houk, K.
N. Angew. Chem., Int. Ed. Engl. 1995, 34, 2511.
(12) Broring, M.; J endrny, J .; Zander, L.; Schmickler, H.; Lex, J .;
Wu, Y.-D.; Nendel, M.; Chen, J .; Plattner, D. A.; Houk, K. N.; Vogel,
E. Angew. Chem., Int. Ed. Engl. 1995, 34, 2515.
(13) Koerner, R.; Olmstread, M. M.; Ozarowski, A.; Phillips, S. L.;
Van Calcar, P. M.;.Winkler, K.; Balch, A. L. J . Am. Chem. Soc. 1998,
120, 1274.
(14) Karle, I. L.; Ranganathan, D.; Haridas, V., J . Am. Chem. Soc.
1996, 118, 10916.
† Indian Institute of Chemical Technology.
‡Centre for Cellular and Molecular Biology.
§ Naval Research Laboratory.
(1) Sumners, D. W., The Role of Knot Theory in DNA Research. In
Geometry and Topology; McCrory, C., Schifrin, T., Eds.; Marcel
Dekker: New York, 1987; pp 297-318.
(2) Dietrich-Buchecker, C. O.; Sauvage, J .-P. Interlocked and Knot-
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(3) Bates, A. D.; Maxwell, A. DNA Topology; Oxford University
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(4) Mansfield, M. L. Nat. Struct. Biol. 1994, 1, 213.
(5) Liang, C.; Mislow, K. J . Am. Chem. Soc. 1994, 116, 3588.
(6) The only topological link known to date that is composed entirely
of amino acid residues is the catenated structure of â-subunit of human
chorionic gonadotropin (see ref 6 in Liang, C.; Mislow, K. J . Am. Chem.
Soc. 1994, 116, 11189).
10.1021/jo0003807 CCC: $19.00 © 2000 American Chemical Society
Published on Web 06/16/2000

4416 J . Org. Chem., Vol. 65, No. 14, 2000
Ranganathan et al.
F igu r e 1. Synthesis of X-constrained cystine-based macrocycles.
cyclophanes,16 and polypyrroles,10-12 to our knowledge,
this motif is rarely observed among cyclic peptides.
Patellamide-D, a 26-membered cytotoxic cyclic peptide
of marine origin containing an unusual combination of
thiazole and oxazoline amino acids in the ring, provides
a unique example of a figure-eight conformation.17,18
Some features of figure-eight structure were also noticed
in a related cyclic peptide alkaloid lissoclinamide-7, a 21-
membered macrocycle containing two thiazole rings in
addition to an oxazoline heterocycle.19
important insights into the nature of factors responsible
for driving the conformation of a macrocyclic peptide into
a double-helical fold. While intramolecular hydrogen
bonding may play only a minor role, the presence of a
type-II â-turn conformation appears to be the deciding
factor in the choice between an open and a double-helical
structure for a cyclic peptide. Among the conformational
constraints that can be used to induce type-II â turns,
incorporation of cystine amino acid was shown by us20
to be particularly reliable. In the present work, a large
variety of cystine-containing cyclic peptides with the
general structure cyclo (X-Cyst-)n wherein cystine (Cyst
) (HN-CH(CO2Me)-CH2-S-)2) residue alternates with a
conformationally rigid alicyclic (1,3-adamantanedicarbo-
nyl or 2,3-trans-norbornenedicarbonyl) or aromatic (1,3-
benzene- or 2,6-pyridinedicarbonyl) or flexible (1,3-
propanedicarbonyl) unit X in a ring with adjustable size
(13- to 78-membered; n ) 1, 2, 3...6) were prepared and
examined for unusual folds and conformational prefer-
ences (Figure 1).
Among the non-natural cyclic peptides, a 22-membered
tryptophan-derived macrocycle was demonstrated to fold
into a left-handed double helix stabilized by transannular
hydrogen-bonding.7 The corresponding decarboxy ana-
logue, however, did not show any double helical character
presumably because of its much decreased tendency
toward intramolecular hydrogen bonding.8
Our recent work14 reporting on the design of adaman-
tane-constrained cystine-based cyclic peptides provided
(15) Thulin, B.; Wennerstrom, O. Acta Chem. Scand. Ser. B 1976,
30, 688.
(16) Shenk, R.; Mullen, K.; Wennerstrom, O. Tetrahedron Lett. 1990,
31, 7367.
(17) Schmitz, F. J .; Ksebati, M. B.; Chang, J . S.; Wang, J . L.;
Hossain, M. B.; v.d. Helm, D.; Engel, M. H.; Serban, A.; Silfer, J . A. J .
Org. Chem. 1989, 54, 3463.
(18) Ishida, T.; In, Y.; Shinozaki, F.; Doi, M.; Yamamoto, D.;
Hamada, Y.; Shiori, T.; Kamigauchi, M.; Sugiura, M. J . Org. Chem.
1995, 60, 3944.
Resu lts a n d Discu ssion
The common synthetic strategy used for the prepara-
tion of cystine-based macrocycles was one-step condensa-
tion of cystine-diOMe (generated in situ from dihydro-
chloride and triethylamine) with the corresponding
(19) Wipf, P.; Fritch, P. C.; Geib, S. J .; Selfer, A. M. J . Am. Chem.
Soc. 1998, 120, 4105.
(20) Ranganathan, D.; Haridas, V.; Karle, I. L. J . Am. Chem. Soc,
1998, 120, 2695.

Double-Helical Cyclic Peptides
J . Org. Chem., Vol. 65, No. 14, 2000 4417
Ta ble 1. Yield Distr ibu tion of P r od u cts in th e Con d en sa tion of Cystin e DiOMe w ith Va r iou s Tem p la tes (X)
dicarbonyl dichloride X (COCl)2 in the presence of tri-
ethylamine in dry CH2Cl2 under high dilution conditions.
The products were separated by careful chromatography
on silica gel columns using gradient elution with a
mixture of chloroform and methanol and characterized
by FAB MS or ES MS for oligomeric nature.
and 13%, respectively (not very different from cystine
case), the non S-S diamines gave poor yields of cycliza-
tion products. Thus, while L-lysine in a two-step reaction
afforded a 24-membered macrocycle (11) in a total yield
of ∼20%, the simple 1,ω-diamino hexane on direct
condensation with 1,3-adamantane dicarbonyl dichloride
led to two cyclic products 8 and 9 (arising from 2 + 2
and 4 + 4 cyclization, respectively) in abysmally poor
total yield of ∼10%, with the 26-membered 2 + 2
macrocycle as the major component (8%) (Figure 2). The
above results support the notion that within a narrow
window of reaction conditions the disulfide linkage
manifests itself in increased macrocyclization yield.
In their 1H NMR spectra, regardless of the ring size
and the nature of “X”, all cystine-based macrocycles
exhibited a single set of resonances for Cyst and X units
indicating the presence of high symmetry in the rings.
The presence of strong Cyst NH-CâH2 cross-peaks in
their ROESY NMR spectra (Supporting Information)
supported â-turn features. The other prominent cross-
peaks were between Cyst NH and X protons indicating
trans-orientation of the X-carbonyls. FT-IR studies (Sup-
porting Information) in chloroform solution (at 298 K)
indicated considerable population of intramolecularly
hydrogen-bonded conformations as shown by the pres-
ence of a concentration-independent broad band at 3310-
3330 cm-1 in the NH stretch region of most cystine-based
cyclic peptides attributed to hydrogen-bonded NH groups.
1H NMR VT studies (conducted in DMSO-d6 between 303
and 343 K) corroborated FT-IR results. Most cystine-
based macrocycles exhibited relatively low values of
temperature coefficients (dδ/dT varying from ∼-1 to -5
ppb/K) indicating intramolecularly hydrogen-bonded NH
groups (Supporting Information).
Interestingly, none of the condensation reactions (with
the sole exception of 1c) resulted in a single macrocycle.
Thus, 1,3-adamantane dicarbonyl dichloride (1a ) yielded
a mixture of four macrocycles identified as 2 + 2 (3a ), 3
+ 3 (4a ), 4 + 4 (5a ), and 5 + 5 (6a ) condensation products,
cystine diOMe and 1,3-pyridinedicarbonyl dichloride (1b)
afforded three macrocyclic products identified as 2 + 2
(3b), 3 + 3 (4b), and 4 + 4 (5b), and only one macrocycle
characterized as 2 + 2 product (3c) was obtained in the
case of 1,3-benzenedicarbonyl dichloride (1c). With nor-
bornene trans-2,3 dicarbonyl dichloride (1d ), the conden-
sation of cystine diOMe led to the isolation of three
products identified as arising from 1 + 1 (2d ), 2 + 2 (3d ),
and 3 + 3 (4d ) cyclization. As anticipated, the use of
flexible 1,3-propanedicarbonyl dichloride (1e) afforded six
macrocycles (2e-7e) starting from the minimum size 13-
membered to maximum 78-membered. Table 1 presents
the yield distribution of products in the condensation of
cystine diOMe with various templates (X).
In another series of experiments, in order to evaluate
the role of S-S linkage in facilitating macrocyclization,
it was envisaged to replace cystine with several 1,ω-
diamines of approximately equal chain length, for ex-
ample, cystamine (achiral, contains S-S), lysine (chiral,
lacks S-S), and simple 1,6-diaminohexane (achiral, lacks
S-S linkage). Condensation reaction of thus selected 1,ω-
diamines with 1,3-adamantanedicarbonyl dichloride un-
der identical conditions afforded cyclic products varying
in total yield of 10-59% (Figure 2). For example, while
cystamine produced two macrocycles arising from 2 + 2
(12) and 3 + 3 (13) cyclization reaction in yields of 46
Convincing support for â-turn-type structures was
provided by circular dichroism (CD) spectra.21 CD studies
were carried out in trifluoroethanol (TFE) solution at 298

4418 J . Org. Chem., Vol. 65, No. 14, 2000
Ranganathan et al.
F igu r e 2. Synthesis of adamantane-containing noncystine-based macrocycles.
K at a concentration of ∼0.5 µM. The presence of a
prominent positive band at ∼213 nm in cystine-contain-
ing macrocycles with X ) Adm, at 218 nm with X ) Pyr,
at 229 nm with X ) Ph, and at 217 nm with X )
(-CH2-)3 strongly suggested type-II â-turn conformation.
A comparison of CD spectra in pyridine-containing cys-
tine-based 3 + 3 and 4 + 4 macrocycles of 39- and 52-
membered rings, respectively, is presented in Figure 3.
The adamantane-containing macrocycles of cystine in 26-,
39-, 52-, and 65-membered rings showed CD bands as
presented in Figure 4. The same trend was shown with
cystine macrocycles containing 1,3-propanedicarbonyl
unit in 26-, 39-, and 52-membered rings (Figure 5). The
turn structure became highly pronounced in norbornene
containing cystine macrocycles as shown by the presence
of a sharp CD maximum at 208 nm in 12-membered cyclo
(NBE-Cyst) and at 222 nm in 24-membered cyclo (NBE-
Cyst)2 (Supporting Information).
F igu r e 3. CD spectra of cyclo (Pyr-Cyst)3 (a) and cyclo (Pyr-
Cyst)4 (b).
Suitable crystals could be obtained only with adaman-
tane-containing 39-membered cystine cyclic peptide 4a .
The crystal structure revealed a figure-eight conforma-
tion with a double cavity. Interestingly, despite the 3-fold
symmetry in its formula (Figure 7a), the cyclo (Adm-L-
Cyst)3 molecule crystallizes with a 2-fold rotation axis
that passes through one adamantyl group and through
(21) The CD studies for all cyclopeptides reported in this paper were
carried out in the wavelength region of 200-250 nm. The S-S
chromophore of cystine unit is known to absorb at ∼260 nm (Woody,
R. W.; Dunker, A. K. In Circular Dichroism and the Conformational
Analysis of Biomolecules; Fasman, G. D., Ed.; Plenum Press: New
York, 1966; pp 109-157) and does not interfere in this region. The
CdC chromophore of norbornene unit also does not absorb in this
region as shown by us recently (J . Am. Chem. Soc. 1998, 120, 8448) in
the comparison of olefinic and the dihydro analogues of norborneno
peptides, wherein it was found that both exhibit a prominent positive
CD band at ∼211 nm. A large number of cystine-containing cyclic
peptides have been studied by us (Angew. Chem., Int. Ed. Engl. 1996,
35, 1105; J . Am. Chem. Soc. 1996, 118, 10916; J . Am. Chem. Soc. 1998,
120, 2695) for CD spectra and are found to absorb in the range of 210-
230 nm, the peptide chromophore region. We believe therefore that
the CD bands exhibited by cystine-containing cyclic peptides described
in the present work are largely due to normal peptide chromophores.
F igu r e 4. CD spectra of cyclo (Adm-L-Cyst)2 (a), cyclo (Adm-
L-Cyst)3 (b), cyclo (Adm-L-Cyst)4 (c), and cyclo (Adm-L-Cyst)5
(d).
the center of the opposing S-S bond (Figure 7b). As seen
in Figure 7b, while the two symmetry equivalent ada-
mantane units have their carbonyl units anti to each
other (see O6 and O7), the carbonyl groups in the central

Double-Helical Cyclic Peptides
J . Org. Chem., Vol. 65, No. 14, 2000 4419
at CR atom predetermines the helical sense, would the
D-Cyst containing macrocycle, cyclo (Adm-D-Cyst)3, adopt
the mirror image form (conformation b in Figure 9) and
furthermore would two forms “a” and “b” be equally
present in an achiral, cystamine-based macrocycle? While
cystamine-based adamantane-constrained macrocycles
12 and 13, prepared in an analogous manner by the
reaction of 1,3-adamantanedicarbonyl dichloride with
cystamine under high dilution conditions did not yield
suitable crystals for X-ray studies, the 39-membered
D-cystine-based macrocycle cyclo (Adm-D-Cyst)3 crystal-
lized in colorless prisms. The presence of the dimer cyclo
(Adm-D-Cyst)2 and the tetramer cyclo (Adm-D-Cyst)4 was
confirmed by FAB-MS spectra. As in the L-Cyst case, the
yield distribution was ∼50:20:5 in the D-Cyst-based
macrocycles of dimer, trimer, and tetramer structure.
Interestingly, in both L- and D-Cyst series, it was only
the 3 + 3 cyclic trimer that showed any tendency to
crystallize.
F igu r e 5. CD spectra of cyclo (Glut-Cyst)2 (a), cyclo (Glut-
Cyst)3 (b), and cyclo (Glut-Cyst)4 (c).
The crystal structure of enantiomeric cyclo (Adm-D-
Cyst)3 was shown (Figure 7c) to be the exact mirror image
of the L-Cyst-based macrocycle, including the disorder at
S3-S3a. The result had been expected, although there
are rare exceptions in which, under the same conditions,
enantiomers of peptides have crystallized in different
space groups and exhibited different orientations in the
side chains (Balaram and Karle, to be published). A
comparison of the two structures (Figure 7b,c) shows that
the only difference between the two enantiomers is the
sign of the handedness at the interstrand crossing. While
in L-Cyst macrocycle it is the right-handed helix on the
top, in the D-isomer the top helix assumes the left-handed
conformation. The space-filling diagrams of the two
enantiomers (L and D) are compared in Figure 10, looking
down the 2-fold axes of the molecules. The D-Cyst
enantiomer also shows two internal hydrogen bonds
(Figure 8b).
F igu r e 6. Comparison of CD spectra of cyclo (Adm-L-Cyst)3
(a) and cyclo (Adm-D-Cyst)3 (b). All spectra were taken in TFE
solution at 298 K with 0.5 µM concentration.
adamantane unit (see O1 and O7) adopt a syn conforma-
tion that is in a favorable orientation to form intramo-
lecular NH‚‚‚OC bonds (N(3)‚‚‚O(1a) 3.027 Å; H‚‚‚O )
2.21 Å) with the amide NHs of the middle cystine unit
(Figure 8a) that stabilizes the figure-8-fold in cyclo (Adm-
L-cyst)3.
The D-cyst enantiomer showed almost identical 1H
NMR spectra. The appearance of only a single set of
resonances for cystine and adamantane protons coupled
with the observation that, like in L-Cyst, these remain
unaffected in the temperature range of 20-90 °C indi-
cated that the molecule has a single conformation in
solution. The anti arrangement for the 1,3-adamantane
carbonyls seen for the two symmetry equivalent ada-
mantane units in the crystal structure was also suggested
by the enhanced ROE between the NH and the adaman-
tane methylene protons in the ROESY spectrum of both
L- and L-enantiomers of cyclo(Adm-Cyst)3 (Supporting
Information). FT-IR and 1H NMR VT studies indicate
only modest amount of intramolecular hydrogen bonding
(Supporting Information).
In principle, the figure-eight (double-helical arrange-
ment) motif could have a left or a right disposition of the
intercrossing helices (Figure 9, parts a and b, respec-
tively). The fact that only one arrangement (Figure 9a)
is seen in cyclo (Adm-L-Cyst)3 suggests control by the
L-chirality of the cystine unit in the macrocycle formation.
The choice of “a” over the alternate form “b” (Figure 9)
is analogous to the choice of right- or left-handed helices
in linear peptides where the backbone atoms form a right-
handed helix when the side chains have the L-configu-
ration at the CR atoms and a left-handed helix when the
side chains have the D-configuration at the CR atoms. In
cases where the CR atoms are rendered achiral by the
presence of two CH3 groups (as in Aib residue), both
right- and left-handed helices occur as shown, for ex-
ample, in the crystal structure analyses of X-(Aib)n-Y
peptides.22 In the present case, there are six chiral CR
atoms, two for each L-cystine residue, that predispose the
figure-eight backbone to assume the conformation “a” in
Figure 9, rather than conformation “b”. Conformation “b”
is not favored, if not impossible, because the six COOCH3
moieties that extend outward (Figure 7b) would have to
turn inward for “b” and cause considerable steric inter-
ference. The question would arise then that if chirality
The circular dichroism spectrum of the D-isomer con-
firmed the enantiomeric nature of the molecule. Com-
parison of the CD spectra of L- and D-isomers shows the
exact mirror-image relationship (Figure 6). The type-II
â-turn structure in both enantiomers was indicated by
the presence of an intense CD band at ∼213 nm. Thus,
1
collectively H NMR, FT-IR, and CD studies supported
the enantiomeric Figure-8-fold for the L- and D-macro-
cycles in solution.
Although the adoption of the figure-eight motif has
been demonstrated only in the 39-membered cyclo (Adm-
Cyst) trimer, the possibility of presence of extended
double-helical structures in higher (52- and 65-membered
cyclicoligomers) cannot be ruled out. The 26-membered
(22) Toniolo, C.; Crisma, M.; Bonora, G. M.; Benedetti, E.; di Blasio,
B.; Pavone, V.; Pedone, C.; Santini, A. Biopolymers 1991, 31, 129.

4420 J . Org. Chem., Vol. 65, No. 14, 2000
Ranganathan et al.
F igu r e 7. (a) Chemical structure of cyclo (Adm-Cyst)3. (b) X-ray structure of cyclo (Adm-L-Cyst)3. A 2-fold rotation axis passes
through C(1) and C(6) and halfway between S(3) and S(3a). The view is offset somewhat from being down the x axis in order to
illustrate better the Figure 8. Several interatomic distances that indicate the size of the cavity are as follows: C(1)-S(3), 5.8 Å;
N(2)-C(4), 4.36 Å; N(2)-S(3a), 6.20 Å; N(1)-C(12), 3.94 Å; C(18)-C(3a), 4.77 Å; O(6)-C(5), 3.85 Å. (c) X-ray structure of cyclo
(Adm-D-Cyst)3.
F igu r e 8. (a) View of cyclo (Adm-L-Cyst)3 down the z axis. Only the most prevalent of the disordered sites for S(3)-S(3a) and the
bonded CH2’s (at the bottom of the macrocycle) are shown in all the diagrams. The dotted lines indicate the only two intramolecular
hydrogen bonds, N(3)‚‚‚O(1a) and N(3a)‚‚‚O(1) with an N‚‚‚O distance of 3.03 Å. (b) View of cyclo (Adm-D-Cyst)3 down the z axis.
multiple disulfide (S-S) bonds in protein and polypep-
tides with topologically important features. A rotaxane-
like motif of three cystine residues present in various
growth factors and related proteins has been referred to
as a knot.24-26
Con clu sion
F igu r e 9. Presence of L-substituents on chiral CR atoms
(designated by O) results in the preference of (a) for the figure-
The cystine-containing macrocycles reported here rep-
resent a novel class of cyclic peptides capable of adopting
eight conformation of the backbone of (Adm-L-Cyst)3 rather
1
topologically important conformations. H NMR, FT-IR,
than (b). The heavy lines are above the plane of the paper. If
and CD studies have shown these cystine-based macro-
cycles to adopt â-turn-like features in their structures
which are largely stabilized by intramolecular hydrogen
bonds. The 39-membered cyclo (Adm-L-Cyst)3 and its
enantiomer cyclo(Adm-D-Cyst)3 are shown by their X-ray
crystal structure to adopt figure-eight conformations with
exact mirror image relationship, and the spectroscopic
measurements on these molecules confirm that each
enantiomer has a single conformation in solution. The
increasing number of cystine residues in these macro-
cycles are likely to be of consequence in designing
macrocycles with extended double helical structures that
there were no chiral atoms in the macrocycle, then conforma-
tion (a) could be twisted into (b).
lower homologue, however ,appears to be less likely to
have a figure-eight motif, since the crystal structure of
related 26-membered 2 + 2 macrocycle containing alter-
nating repeats of m-phenylene and cystine units was
shown to adopt a collapsed open-ring structure.20 Thus,
apart from the ring size, the factors that may control the
figure-eight formation in macrocyclic peptides appear to
be the presence of turn-inducing units. In the present
example, the presence of three cystine residues promotes
the attainment of a double-helical fold. Interestingly, a
survey23 has shown the significance of the presence of
(24) McDonald, N. Q.; Hendrickson, W. A. Cell 1993, 73, 421.
(25) Schlunegger, M. P.; Grutter, M. G. J . Mol. Biol. 1993, 231, 445.
(26) Murray-Rust, P.; McDonald, N. W.; Blundell, T. L.; Hosang,
M.; Oefner, C.;. Winkler, F.; Bradshaw, R. A. Structure 1993, 1, 153.
(23) Liang, C.; Mislow, K. J . Am. Chem. Soc. 1989, 111, 6132.

Double-Helical Cyclic Peptides
J . Org. Chem., Vol. 65, No. 14, 2000 4421
F igu r e 10. Space-filling diagrams of (a) cyclo (Adm-L-Cyst)3 and (b) cyclo (Adm-D-Cyst)3.
1
Cyclo(Ad m -cysta )3 (13): yield 13%; mp 142-143 °C;
H
may have relevance in providing new insights into
protein folding mechanism and in the design of topologi-
cally important macrocycles.
NMR (200 MHz, CDCl3 + DMSO-d6) δ 1.60-2.28 (m, 42H),
2.85 (m, 12H), 3.50 (m, 12H), 7.32 (brd, 6H); FAB MS m/z 1022
(62) [M + H]+. Anal. Calcd for C48H72N6O6S6 (mol wt 1020):
C, 56.47; H, 7.05; N, 8.23. Found: C, 56.19; H, 6.98; N, 8.19.
Exp er im en ta l Section
1
Cyclo(Ad m -D-Cyst )2: yield 50%; mp 118-120 °C;
H
NMR (300 MHz, CDCl3) δ 1.57-2.32 (m, 28H), 3.13 (m, 4H),
3.28 (m, 4H), 3.78 (s, 12H), 4.81 (m, 4H), 6.61 (d, J ) 6.71 Hz,
4H); FAB MS (m/z) 913 (100) [M + H]+. Anal. Calcd for
All amino acids used were of the L-configuration [except for
(Adm-D-Cyst)n]. Melting points are uncorrected. 1H NMR
ROESY experiments were performed using 0.2 and 0.3 s
mixing time with pulse spin locking with 30 pulses and 2 kHz
spin locking field. FAB MS were obtained using m-nitrobenzyl
alcohol as a matrix. The circular dichroism (CD) spectra were
recorded in quartz cells of 1 mm path length at 25 °C.
Reactions were monitored wherever possible by TLC. Silica
gel (Merck) was used for TLC, and column chromatography
was done on silica gel (100-200 mesh) columns, which were
generally made from a slurry in hexane or a mixture of hexane
and ethyl acetate. Products were eluted with either a mixture
of ethyl acetate/hexane or chloroform/methanol.
C
40H56N4O12S4 (mol wt 912): C, 52.63; H, 6.14; N, 6.14.
Found: C, 52.70; H, 6.23; N, 6.09.
Cyclo(Ad m -D-Cyst)3: yield 15%; mp 173-175 °C; 1 H NMR
(400 MHz, CDCl3) δ 1.67-2.28 (m, 42H), 3.18 (m, 12H), 3.78
(s, 18H), 4.80 (m, 6H), 6.72 (d, J ) 7.18 Hz, 6H); FAB MS m/z
1369 (63) [M + H]+. Anal. Calcd for C60H84N6O18S6 (mol wt
1368): C, 52.63; H, 6.14; N, 6.14. Found: C, 52.90; H, 6.23;
N, 6.21.
Rea ction of 1, 6-Dia m in oh exa n e w ith 1,3-Ad a m a n -
ta n ed ica r bon yl Dich lor id e. (a) Synthesis of Macrocycles 8
and 9. To an ice-cooled and stirred solution of 1,6-diamino-
hexane (1 mmol) in dry CH2Cl2 (150 mL) containing triethyl-
amine (2.5 mmol) was added 1,3-adamantanedicarbonyl dichlo-
ride (1 mmol in 50 mL dry CH2Cl2) over a period of 0.5 h and
the mixture stirred for 12 h (TLC). The reaction mixture was
worked up by washing, sequentially, with ice-cold 2 N H2SO4,
H2O, and 5% NaHCO3 (∼20 mL each), drying the organic layer
with anhyd MgSO4, and evaporating in vacuo. The residue was
chromatographed on a column of silica gel, and elution with a
mixture of chloroform/methanol yielded two products which
were identified as macrocycles 8 and 9.
Gen er a l P r oced u r e for th e P r ep a r a tion of Cyclo [(X-
Cyst)n ]. A solution of freshly prepared X (COCl)2 (X ) 1,3-
adamantane, 1,3-benzene, 2,6-pyridine, 2,3-trans-norbornene,
and 1,3-propane unit) (1 mmol) in dry CH2Cl2 (50 mL) was
added dropwise over 0.5 h to a well-stirred solution of L- or
D-cystine dimethyl ester hydrochloride (1 mmol) and triethyl-
amine (4.5 mmol) in dry CH2Cl2 (∼150 mL) at 0 °C. The reac-
tion mixture was stirred at room temperature for 4-6 h and
monitored by TLC. The reaction mixture was washed sequen-
tially with ice-cold 2 N H2SO4, H2O, and 5% NaHCO3 (∼20
mL each). The organic layer was dried over anhydrous MgSO4
and concentrated in vacuo. The residue was chromatographed
on a column of silica gel using either a mixture of ethyl acetate/
hexane or chloroform/methanol as eluent to afford the X-
constrained cystine macrocycles. A similar procedure was
followed for the preparation of cystamine-based macrocycles.
Selected Da ta . Detailed spectral and analytical data have
been reported for cyclo[(Adm-L-Cyst)n; n ) 2, 3, 4, 5], cyclo-
[Ar-L-Cyst)n; n ) 2, 3, 4; Ar ) Pyr or Ph], cyclo[(Glut-L-Cyst)n;
n ) 1, 2...6; Glut ) -CO-CH2-CH2-CH2-CO-], and cyclo-
[(NBE-L-Cyst-)n; n ) 1, 2, 3; NBE ) 2,3-trans norbornene
dicarbonyl unit] in refs 20 and 27-29, respectively. Selected
data for all new compounds reported in the present paper are
given below.
8: yield 8%; mp 360 °C dec; IR (KBr) 3386, 3314, 1644, 1552,
1526 cm-1 1H NMR (300 MHz, DMSO-d6) δ 1.00-2.12 (m,
;
44H), 2.98 (m, 8H), 7.35 (brs, 4H); FAB MS (m/z) 609 (100)
(MH)+. Anal. Calcd for C36H56N4O4 (mol wt 608): C, 71.05; H,
9.21; N, 9.21. Found: C, 70.98; H, 9.09; N, 9.33.
9: yield 2%; IR (KBr) 3352, 1644, 1552, 1533 cm-1 1 H NMR
;
(300 MHz, DMSO-d6) δ 0.09-2.09 (m, 88H), 3.00 (m, 16H),
7.35 (brs, 8H); FAB MS (m/z) 1217 (72) (MH)+. Anal. Calcd
for C72H112N8O8 (mol wt 1216): C, 71.05; H, 9.21; N, 9.21.
Found: C, 71.13; H, 9.09; N, 9.30.
Syn th esis of Lysin e-Ba sed Ma cr ocycle (11). (a ) Syn -
th esis of NR-Z-Lys-Ad m (10). A solution of freshly prepared
1,3-adamantanedicarbonyl dichloride (1 mmol) in dry CH2Cl2
(50 mL) was added dropwise to a well-stirred solution of
NRZ-LysOMe‚HCl (2 mmol) in dry CH2Cl2 (75 mL) containing
triethylamine (4 mmol) at 0 °C over 0.5 h and mixture stirred
at room temperature for 12 h (TLC). The reaction mixture was
worked up by washing, sequentially, with ice-cold 2 N H2SO4,
H2O, and 5% NaHCO3 (∼20 mL each), drying the organic layer
with anhyd MgSO4, and evaporating in vacuo. The residue was
Cyclo(Ad m -cysta )2 (12): yield 46%; mp 297-298 °C; 1H
NMR (200 MHz, DMSO-d6) δ 1.60-2.32 (m, 28H), 2.95 (m,
8H), 3.50 (m, 8H), 7.85 (brd, 4H); FAB-MS m/z 681 (64) [M +
H]+. Anal. Calcd for C32H48N4S4O4 (mol wt 680): C, 56.47; H,
7.05; N, 8.23. Found: C, 56.63; H, 7.13; N, 8.30.
(27) Ranganathan, D.; Haridas, V.; Madusudanan, P.; Roy, R.;
Nagaraj, R.; J ohn, G. B.; Sukhaswami, M. B. Angew. Chem., Int. Ed.
Engl. 1996, 35, 1105 and ref 14.
(28) Ranganathan, D.; Haridas, V.; Sivakama Sundari, C.; Bala-
subramanian, D.; Madhusudanan, K. P.; Roy R.; Karle, I. L. J . Org.
Chem. 1999, 64, 9230.
(29) Ranganathan, D.; Haridas, V.; Kurur, S.; Nagaraj, R.; Bik-
shapathy, E.; Kunwar, A. C.; Sarma, A.V. S.; Vairamani, M. J . Org.
Chem. 2000, 65, 365.

4422 J . Org. Chem., Vol. 65, No. 14, 2000
Ranganathan et al.
chromatographed on silica gel with a mixture of ethyl acetate/
hexane as eluents.
16.945(1) Å, c ) 25.882(1) Å, V ) 7274.6 Å3, dc ) 1.331 g/cm3,
Z ) 4, Cu radiation, λ ) 1.5418 Å. Crystals were obtained from
chloroform and ethyl acetate. The molecule lies on a 2-fold axis.
The structure was solved by direct phase determination and
refined by full-matrix anisotropic least squares. Distance
restraints were applied to the disordered ethyl acetate mol-
ecule that lies on a 2-fold axis, to one methyl ester group with
high mobility and one C-S-S-C group highly disordered
around a crystallographic 2-fold axis. R ) 9.1 for 433 param-
eters and 2130 independent data (>3σ(F)). Absolute configu-
ration determined by the anomalous scattering of the S atoms.
(b) (Adm-D-Cyst)3, the same as above except a ) 16.536(2) Å,
b ) 16.946(1) Å. c ) 26.080(2) Å; R ) 9.7 for 417 parameters
and 2793 independent data (>3σ(F)).
Selected d a ta : yield 57%; oily; [R]26D ) -4.36 (c 4.3, CHCl3);
IR (KBr) 3379 (br), 1749, 1730, 1710, 1639, 1560, 1540, 1532
1
cm-1
;
H NMR (90 MHz, CDCl3) δ 1.10-2.32 (m, 26H), 3.24
(m, 4H), 3.72 (m, 6H), 4.30 (m, 2H), 5.10 (s, 4H), 5.75 (m, 4H),
7.35 (brs, 10H).
(b) Syn th esis of Cyclo(Lys-Ad m ) (11). A solution of
freshly prepared 1,3-adamantanedicarbonyl dichloride (2 mmol)
in dry CH2Cl2 (50 mL) was added dropwise to a well-stirred
solution of 2 mmol of N-deprotected (H2NR-Lys)2Adm (2
mmol) in dry CH2Cl2 (100 mL) containing triethylamine (4
mmol) at 0 °C over 0.5 h and mixture stirred at room
temperature for 24 h (TLC). The reaction mixture was worked
up by washing, sequentially, with ice-cold 2 N H2SO4, H2O,
and 5% NaHCO3 (∼20 mL each), drying the organic layer with
anhyd MgSO4, and evaporating in vacuo. The residue was
chromatographed on silica gel using a mixture of ethyl acetate/
hexane as eluents.
Su p p or tin g In for m a tion Ava ila ble: X-ray diffraction
analyses: tables of atomic coordinates, bond lengths and bond
angles, anisotropic thermal parameters, hydrogen atom coor-
dinates, and torsion angles for (Adm-L-Cyst)3 and (Adm-D-
Cyst)3. Solution data: 1H NMR, ROESY NMR, VT NMR, FAB
MS, and CD spectra for (X-Cyst)n where n ) 2-5. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Selected d a ta : yield 20%; mp 272 °C; 1 H NMR (300 MHz,
CDCl3) δ 1.30-2.29 (m, 40H), 3.09 (m, 2H), 3.43 (m, 2H), 3.73
(s, 6H), 4.53 (m, 2H), 6.11 (brs, 2H), 6.46 (d, J ) 9.0 Ηz, 2Η);
FAB MS m/z 697 [M + H]+.
Cr yst a l St r u ct u r e An a lysis. (a) (Adm-L-Cyst)3, C60H84
-
N6O18S6. C4H8O2, space group C2221, a ) 16.587(1) Å, b )
J O0003807