Serine-based cyclodepsipeptides on an adamantane building block: Design, synthesis, and characterization of a novel family of macrocyclic membrane ion-transporting depsipeptides

Article abstract of DOI:10.1021/ja971517m

A simple two-step synthetic strategy provides a straightforward entry to a large variety of adamantane-containing serine-based cyclodepsipeptides. The design is flexible with respect to the choice of an amino acid, the ring size, and the nature of the tem

Full text of DOI:10.1021/ja971517m

11578
J. Am. Chem. Soc. 1997, 119, 11578-11584
Serine-Based Cyclodepsipeptides on an Adamantane Building
Block: Design, Synthesis, and Characterization of a Novel
Family of Macrocyclic Membrane Ion-Transporting
Depsipeptides
Darshan Ranganathan,*,† V. Haridas,† K. P. Madhusudanan,‡ Raja Roy,‡
R. Nagaraj,§ and G. B. John§
Contribution from the Biomolecular Research Unit, Regional Research Laboratory (CSIR),
TriVandrum 695019, India, Central Drug Research Institute, Lucknow, India, and Centre for
Cellular and Molecular Biology, Hyderabad, India
ReceiVed May 12, 1997X
Abstract: A simple two-step synthetic strategy provides a straightforward entry to a large variety of adamantane-
containing serine-based cyclodepsipeptides. The design is flexible with respect to the choice of an amino acid, the
ring size, and the nature of the template as illustrated here with the preparation of a large variety of serine-based
macrocycles, for example, 18-membered simple cyclo(Adm-Ser-)2 (4), 21-membered, S-S bridged, cyclo(Adm-
Ser-Cyst-Ser-) (10), 24-membered cyclo(Adm-Ser-Val-)2 (5a), cyclo(Adm-Ser-Leu-)2 (5b), cyclo(Adm-Ser (Leu)-
Val)2 (6), pyridine-containing cyclo(Adm-Ser-Val-Py-Val-Ser-) (13), 26-membered cyclo(Adm-Ser-Ser-)2 (8) and
crown ether hybrid cyclo(Adm-Ser-TEG-Ser-) (12), and 36-membered cyclo(Adm-Ser-)4 (7) and provides built-in
handles (in the form of protected NH2 and COOH groups) for attachment of suitable pendants leading to attractive
models that may have multiple uses as membrane ionophores, scaffolds, or templates in the design of artificial
proteins and for studying the structure-function relationship in biological receptors. This novel class of macrocyclic
peptides are demonstrated to adopt â-turn type conformation and possess high efficiency in transporting Na+, Ca2+
,
and Mg2+ ions across model membranes. Amongst the cyclodepsipeptides reported here, the 24-membered macrocycle
6, containing two leucine residues symmetrically placed on the exterior of the ring, was found to be the most efficient
ion-transporter in lipid bilayer membranes. Interestingly, no appreciable ion-transport was noticed by 18-membered
cyclodepsipeptide (4) and by macrocycles 10, 12, and 13 possessing only one adamantane unit in their cyclic
framework. These results show that a minimum of two adamantane units in a 24-membered ring size appears to be
the optimum requirement for efficient membrane ion transport.
Introduction
hydrophilic interior capable of binding an ion. Valinomycin
meets all these requirements by having a balled hydrophobic
exterior provided by a large number of isopropyl groups of
valine and hydroxyvaleric acid (HyV) residues and at the same
time showing conformational flexibility3 by striking a balance
of more polar amide carbonyl and less polar ester carbonyls to
complex with less charge dense potassium ions.
An interesting feature of most naturally occuring cyclopep-
tides or cyclodepsipeptides is the presence of an N-methylamino
acid or a D-amino acid or a Proline residue in their cyclic
framework.1 This must be Nature’s strategy to create bends in
the peptide backbone in order to facilitate cyclization.4 In
valinomycin, Nature has taken particular care to arrange
alternating sequence of D and L amino acids introducing
repeating bends and resulting in a tennis-ball-seam arrangement
without imposing much rigidity on the structuresa feature
particularly important to any successful ionophore because not
only must a metal ion be bound and transported but also it must
be released after transport if the ionophore is to be useful.5
In recent years, there has been a virtual explosion in the
number of papers that have appeared in literaure on the design
of nonpeptidic macrocyclic ionophores, such as crown ethers,
cryptands, spherands, calixarenes, and cyclophanes, etc., incor-
Cyclodepsipeptides belong to a special class of compounds
well-known for their biological functions as antibiotics and
regulators of ion-transport in membranes.1 This special property
may be attributed to their capability to complex and release the
metal ions at a rate most desirable for good transporters.
Amongst the naturally occurring cyclodepsipeptides, valino-
mycinsa cyclododecadepsipeptide containing three cyclic re-
peats of a four residue (D-Val-L-Lac-L-Val-D-HyV) segment with
alternating amide (CONH) and ester (COO) bondssoccupies a
central place because of its special property of selectively
transporting potassium ions across biological membranes.2
In order to be an efficient ion-transporter, a molecule must
not only be able to bind a metal ion selectively and tightly but
also must be flexible enough to release the ion fairly rapidly.
Additionally, to be compatible with a biomembrane, the
ionophore must have a hydrophobic exterior while having a
* To whom correspondence should be addressed. FAX: +91-0471-
490186/491712. E-mail: darshan@csrrltrd.ren.nic.in.
† Biomolecular Research Unit.
‡ Central Drug Rsearch Institute.
§ Centre for Cellular and Molecular Biology.
X Abstract published in AdVance ACS Abstracts, November 1, 1997.
(1) Ovchinnikov, Y. A.; Ivanov, V. T.; Shkrob, A. M. Membrane ActiVe
Complexones; Elsevier: Amsterdam, 1974. Ovchinnikov, Y. A.; Ivanov,
V. T. Tetrahedron Report No.1; Pergamon Press: New York, 1976.
(2) Izatt, R. M.; Bradshaw, J. S.; Nielson, S. A.; Lamb, J. D.; Christensen,
J. J.; Chem. ReV. 1985, 85, 271-339. Marrone, T. J.; Merz, K. M., Jr., J.
Am. Chem. Soc. 1995, 117, 779.
(3) Smith, G. D.; Daux, W. L.; Langs, D. A.; DeTitta, G. T.; Edmonds,
J. W.; Rohrer, D. C.; Weeks, C. M. J. Am. Chem. Soc. 1975, 97, 7242.
Karle, I. L.; Flippen-Anderson, J. L. J. Am. Chem. Soc. 1988, 110, 3253.
Karle, I. L. J. Am. Chem. Soc. 1975, 97, 4379. Patel, D. J.; Tonelli, A. E.
Biochemistry 1973, 12, 486. Tabeta, R.; Saito, H. Biochemistry 1985, 24,
7696.
S0002-7863(97)01517-5 CCC: $14.00 © 1997 American Chemical Society

Synthesis of Macrocyclic Membrane Ion Carriers
J. Am. Chem. Soc., Vol. 119, No. 48, 1997 11579
Scheme 1
porating aromatic, non aromatic, carbohydrate, or steroid units
in their cyclic frame work, and even pendant attachments have
been done in order to increase their potential for forming three-
dimensional complexes and as efficient ion-transporters in
membranes.6
Although a large number of simple structural analogs of
valinomycin and some related cyclodepsipeptide antibiotics of
natural origin have been prepared,5 to our knowledge, the reports
on the de noVo design of membrane ion-transporting macrocyclic
peptides or depsipeptides are still scarce.7
-CH2OH8 is used for ester bond formation and rigid, low
molecular weight, lipophilic adamantane building blocks provide
the desired membrane permeability and conformational con-
straint for efficient ion transport in membranes. The present
strategy9 is flexible with respect to the ring size, the choice of
an amino acid, and the choice of a building block as illustrated
here with the preparation of 18-membered (4), 21-membered
(10), 24-membered (5a,b, 6, and 13), 26-membered (8 and 12),
and 36-membered (7) serine-based macrocycles. An additional
advantage is provided by the built-in handles in 4-13 (in the
form of protected NH2 and COOH groups) that can be ligated
via peptide chemistry to a variety of subunits, for example, a
long alkyl amine, a polypeptide chain, a polysaccharide unit,
or even a peptide dendron, providing attractive models for novel
artificial protein design10 and membrane ion-transport. The
design also permits the incorporation of ionophoric pendants
such as crown ethers either as part of the cyclic backbone as
illustrated here with the preparation of macrocycle 12 or attached
to the amino and carboxy side arms.
In this paper, we report the design, synthesis, and character-
ization of a new class of cyclodepsipeptides wherein a serine
(4) Proline has been established as a frequent cause of chain reversal in
globular proteins (Crawford, J. L; Lipscomb, W. N; Schollman, C. G. Proc.
Natl. Acad. Sci. U.S.A. 1973, 70, 538. Chou, P. Y.; Fasman, G. D.
Biochemistry 1974, 13, 222). This property arises in part from the rigidity
of the pyrrolidine ring, which restricts the possible values of the angle of
rotation φ around the CRN bond to between 105 and 125°, thus diminishing
the allowed space on the conformational maps (Venkatachalam, C. M.
Biopolymers 1969, 6, 1425). While proline is a proteinous amino acid,
D-amino acids are not known to occur in natural proteins (Stryer, L.
Biochemistry, 2nd ed.; Freeman: New York, 1981; p 774). Their frequent
occurrence in microbial linear or cyclopeptides is truly remarkable and may
be necessary to facilitate cyclization. The D-amino acids by virtue of their
opposite chirality and hence opposite geometry are likely sites for bends
stabilizing the cyclic backbone (Ramakrishnan,C.; Sarathy, K. P. Int. J.
Pep. Protein Res. 1969, 1, 63).
Results and Discussion
The two-step synthesis envisaged here (Scheme 1) involves
first the condensation of an N,C-protected serine amino acid or
its peptide (2) with 1,3-adamantane dicarbonyl dichloride (1)
to give the bis-Ser derivative 3a-d, which after N-deprotection
and recoupling with 1 affords the desired cyclodepsipeptides
(5) Marrone, T. J.; Merz, K. M., Jr. J. Am. Chem. Soc. 1992, 114, 7542.
Also see: Fyles, T. M. in Bioorganic Chemistry Frontiers; Springer Verlag:
New York, 1991, Vol. 1, pp 71-113. Gokel, G. W.; Eschegoyen, L. Ibid.
pp 115-141. Shinkai, S. Ibid. pp 161-201.
(6) For recent updates on these topics see: ComprehensiVe Supramo-
lecular Chemistry, Vol.1; Molecular Recognition: Receptors For Cationic
Guests; Gokel, G. W., Guest Ed.; Pergamon, Elsevier Science Ltd.: Oxford,
1996. ComprehensiVe Supramolecular Chemistry, Vol. 2, Molecular
Recognition: Receptors for Molecular Guests; Vogtle, F., Guest Ed.;
Pergamon, Elsevier Science Ltd.: Oxford, 1996. Gokel, G. W.; Murillo,
O. Acc. Chem. Res. 1996, 29, 425-432.
(7) See ref 1 and: Roeske, R. W.; Kennedy, S. J. In Chemistry and
Biochemistry of Amino Acids, Peptides and Proteins; Weinstein, B., Ed.;
Marcel Dekker, Inc.: New York, 1983; Vol. 7, p 205-256. Garcia-
Echeverria, C.; Albericio, F.; Giralt, E.; Pons, M. J. Am. Chem. Soc. 1993,
115, 11663. Ghadiri, M. R.; Granja, J. R.; Buchler, L. N. Nature 1994,
369, 301. Khazanovich, N.; Granja, J. R.; Mc Ree, D. I.; Milligan, R. A.;
Ghadiri, M. R. J. Am. Chem. Soc. 1994, 116, 6011. Ranganathan, D.;
Haridas, V.; Madhusudanan, K. P.; Roy, R.; Nagaraj, R.; John, G. B.;
Sukhaswami, M. B. Angew. Chem., Int. Ed. Engl. 1996, 35, 1105. Karle, I.
L; Ranganathan, D.; Haridas, V. J. Am. Chem. Soc. 1996, 118, 10916.
(8) Interestingly, while hydroxy acids like lactic acid and hydroxyvaleric
acids are commonly employed for depsibond formation in cyclodepsipep-
tides, hydroxy amino acids are rarely used, and there are hardly any
examples where proteinous aminoacids like serine and tyrosine are exploited
either by nature or in the laboratory for the construction of cyclodepsipep-
tides (see refs 1 and 14).
(9) The first illustration of the template-based strategy for the synthesis
of a large number of adamantane-containing cystine cyclic peptides was
recently reported by us (Ranganathan, D.; Haridas, V.; Madhusudanan, K.
P.; Roy, R.; Nagaraj, R.; John, G. B.; Sukhaswami, M. B. Angew. Chem.,
Int. Ed. Engl. 1996, 35, 1105. Karle, I. L; Ranganathan, D.; Haridas, V. J.
Am. Chem. Soc. 1996, 118, 10916).
(10) Carey, R. T.; Mutter, M. In Molecular conformation and Biological
Interactions; Balaram, P., Ramaseshan, S., Eds.; Indian Academy of
Sciences: Bangalore, 1991; pp 457-468. Grove, A.; Mutter, M.; Rivier, J.
E.; Montal, M. J. Am. Chem. Soc. 1993, 115, 5919. Zhang, L.; Tam, J. P.
J. Am. Chem. Soc. 1997, 119, 2363.

11580 J. Am. Chem. Soc., Vol. 119, No. 48, 1997
Ranganathan et al.
Figure 1.
4-7 in good yields (Scheme1). Thus, the 18-membered serine
cyclodepsipeptide (4), the simplest member of the family, was
obtained in an overall yield of 59% from Z-Ser-OMe (2, R )
The versatility of the present strategy was further demon-
strated by the preparation of novel macrocyclic cyclodepsipep-
tides, for example, with an S-S bridge (10), a crown ether
hybrid (12), and containing a pyridyl unit in the cyclic
framework (13). This group of macrocycles (Figure 2) provided
another set of attractive molecules for ion-transport studies.
The N,N′ bis ser (NR-Boc) cystine diOMe (9)sprepared from
cystine diOMe and Boc-SerOH using DCC/NOH succinimide
couplingswas treated with adamantane dicarbonyl dichloride
(1) in dry CH3CN at 0 °C in the presence of DMAP (Scheme
2). The 21-membered cystine containing cyclodepsipeptide 10,
obtained in moderate yields (30%), was fully characterized. The
Boc-protection on the amino groups can easily be removed
(TFA/CH2Cl2) without touching the S-S linkage, which is not
the case with carbobenzoxy deprotection.
For the preparation of cyclodepsipeptide-crown hybrid (12),
tetraethylene glycol (TEG) was treated with NR-Boc-Ser-(CH2-
OBzl)OH in the presence of DMAP under DCC/NOH succin-
imide coupling conditions. The N,O-protected bis-Ser derivative
(11) was selectively O-deprotected (Pd/C/H2) and directly treated
with 1 and DMAP under high dilution conditions to give the
crown depsipeptide 12 in 25% yield (Scheme 2). The macro-
cycle 13 with a pyridyl unit in the backbone was constructed
from bis-Ser intermediate 3b by NR-deprotection (Pd/C/H2)
followed by treatment with 2,6-pyridinedicarbonyl dichloride
(Scheme 2).
OMe, R′ ) Z) and 1 via the bis-ser intermediate 3a.
A
noteworthy feature of this reaction was the formation of a small
amount (∼2%) of 2 + 2 cyclization product 7 (Figure 1)
containing four cyclic repeats of adamantane and serine units
connected with alternating pairs of ester and amide bonds in a
36-membered cyclic framework. In terms of ring size, the
macrocycle 7 can be considered as equivalent to valinomycinsa
36-membered cyclododecadepsipeptide. Interestingly, the FAB-
MS spectrum (Supporting Information) showed the presence
of (M + H)+, (M/2 + H)+, and (M/4 + H)+ peaks, which
supported the 4-fold symmetry. The presence of only a single
set of proton resonances for adamantane and serine units in the
1H NMR of 7 also indicated the highly symmetrical nature of
the macrocycle.
For the preparation of 24-membered macrocycles 5a and 5b,
the intermediate bis-depsipeptides 3b and 3csobtained by the
direct condensation of serine peptides Z-Val-Ser-OMe and
Z-Leu-Ser-OMe, respectively, with the adamantane template
1swere N-deprotected (Pd/C, 5%, H2) and recoupled with 1.
The cyclodepsipeptide 6 containing two leucine residues on the
exterior of the macrocycle 5a was crafted with the aim of
increasing the lipophilicity, most desirable for efficient mem-
brane penetration. The macrocycle 6 was prepared from Z-Val-
Ser-Leu-OMe using essentially the same procedure as for 5a
and 5b (Scheme 1). The 26-membered all-Ser cyclodepsipep-
tide 8 could be obtained either by a single-step condensation
of Z-Ser-Ser-OMe with 1 or by an alternate route with a two-
step sequence (Scheme 1) involving first the conversion of 3a
into 3e (by N-deprotection followed by coupling with Z-SerOH)
and the subsequent depsicoupling of 3e with adamantane
template 1 (Scheme 1). The cyclodepsipeptides from both the
routes were found to be identical in all respects; the yield,11
however, was much better (53%) in the two-step procedure.
The bis-Z macrocycle 8 could easily be N-deprotected (Pd/C,
5%, H2) to give a novel bis-amino derivative of 8, potentially
useful for ion-complexation studies.
Interestingly, while the 18-membered macrocycle 4 did not
1
show any significant features in its H NMR and CD spectra,
the ROESY NMR of 24-membered macrocycles 5a,b and 6
indicated development of secondary structural features.
A
particularly diagnostic feature that signifies the formation of a
â-turn-type structure in peptides was the presence of strong Ser
NH-Val/Leu CRH cross-peaks in the ROESY spectra (supporting
(11) The one-step condensation of Z-Ser-Ser-OMe with 1 gave a mixture
of products, of which macrocycle 8 formed only a small portion (∼10%).
The major compound (∼30%) of the reaction was tentatively identified
(FAB-MS) as the 3 + 3 macrocyclesa 39-membered cyclodepsipeptide
containing three cyclic repeats of alternating 1,3-adamantanedicarbonyl and
Z-Ser-Ser-OMe units.

Synthesis of Macrocyclic Membrane Ion Carriers
J. Am. Chem. Soc., Vol. 119, No. 48, 1997 11581
Figure 2.
Scheme 2
information) of 5a,b and 6. A very enhanced ROE between
Val/Leu CRH and Ser CâH2 protons further supported the â-turn-
type conformation. The syn orientation of amide carbonyls at
the Val/Leu end was indicated by the presence of only a weak
ROE effect between the Val/Leu NH and the adamantane
methylene protons. Variable-temperature (VT) studies con-
ducted in CDCl3 with 5a,b and 6 over the temperature range
298-328 K showed relatively low temperature coefficients for
ring Val/Leu NH (dδ/dT in ppb/K ) -2.8 (5a), -1.5 (5b) and
-3 (6)), indicating possible involvement of these protons in
stabilizing turn conformation via 10-membered hydrogen-
bonded rings.
minima at ∼200 and 210 nmsattributed to a distorted â-turn
structuresthe compound 5a exhibited a broad minimum at ∼215
nm with a shoulder at 202 nm, a feature typical of a type I
â-turn in cyclic peptides.12 The macrocycles maintain their
â-turn conformation even in aqueous solvents as was shown
by their CD spectra in 15% aqueous methanol (Supporting
Information).
With a variety of cyclodepsipeptides in hand, we set out to
measure the capability of these molecules to transport ions in
model membranes.
Using the fluorescent dye method13 with valinomycin as a
standard reference,9 it was found that while the 18-membered
cyclodepsipeptide (4) showed near absence of any ion transport
Further support for the turn structure in 24-membered
macrocycles 5a,b and 6 was provided by their CD spectra
(supporting Information). Thus, in trifluroethanol (TFE) solu-
tion, while 5b showed a minimum at 220 nm and 6 showed
(12) Woody, R. W. In Peptides: Analysis, Synthesis, Biology; Uden-
friend, S., Meienhofer, J., Eds.; Academic Press: NY, 1985; Vol. 7, pp
16-115.

11582 J. Am. Chem. Soc., Vol. 119, No. 48, 1997
Ranganathan et al.
conformation and the presence of built-in handles for ligating
lipophilic chains or secondary structural elements on their
exterior would make them attractive models for studying ion-
transport in membranes, structure-function relationship in
receptors, and protein-folding mechanisms and as conforma-
tionally well-defined scaffolds or templates for use in the
synthesis of artificial proteins. The novel architecture and the
demonstrated high efficiency of these molecules for ion-transport
in model membranes combined with their extremely simple and
straightforward synthesis from commercially available starting
materials is expected to provide additional incentives for the
design of future serine-based cyclodepsipeptides on an ada-
mantane building block.
Experimental Section
All amino acids used were of L-configuration. Melting points were
recorded on a Fisher-Johns melting point apparatus and are uncorrected.
Optical rotations were measured with an automatic JASCO polarimeter;
concentrations are given in grams/100 mL. Infrared spectra were
recorded on a Perkin-Elmer/1600-FT spectrometer either in chloroform
solutions, as neat liquids, or as KBr pellets, and prominent peaks are
expressed in cm-1
.
1 H NMR spectra were recorded on Varian UNITY-
400, Bruker WM-300, Hitachi R-600, and JEOL 90 MHz instruments.
The chemical shifts are reported in δ (ppm) with TMS at 0.00 as an
internal reference. ROESY experiments were performed using 0.2 s
mixing time with pulsed spin locking with 30° pulses and 2 kHz spin-
locking field. FAB-MS were obtained on a JEOL SX-120/DA-6000
instrument using m-nitrobenzyl alcohol as the matrix. Reactions were
monitored wherever possible by TLC. Silica gel G (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.
The circular dichroism (CD) spectra were recorded on JASCO J- 715
instrument in quartz cells of 1 mm path length at 25 °C. For ion-
transport study, small unilamellar vesicles of palmitoyloleoylphos-
phatidylcholine were prepared by sonication of a suspension of
multilamellar vesicles with a Branson sonifier having a microtip. The
vesicles were loaded with K+ (300 mM). The diffusion potential was
set up by diluting the concentrated stock of the lipid vesicles 100-fold
into buffer (Hepes, 10 mM, pH 7.4) containing either 300 mM NaCl,
250 mM CaCl2, or 200 mM MgCl2. Fluorescent dye 3,3′-bis(ethylthio)-
dicarbocyanine iodide [dis-C2-(5)] was then added followed by vali-
nomycin (0.3 µg/mL; lipid: Vm ratio 300:1). Fluorescence spectra were
recorded in a Hitachi 4010 spectrofluorimeter at 25 °C. The ion-
transport capability of cyclodepsipeptides (5a-8) was evaluated by
monitoring the dissipation of a valinomycin-mediated K+ diffusion
potential, creating a diffusion potential-like valinomycin (ref 13) and
by the chlorotetracycline-Ca2+ assay (ref 15).
Figure 3. Effect of the addition of cyclodepsipeptide 6 (5.6 µM) on
the diffusion potential set up by valinomycin (at a lipid: Vm ratio of
300:1) for Na+ (a), Ca2+ (b), and Mg2+ (c) as monitored by the
fluorescence of cyanine dye dis-C2 (5). Maximum efficiency was seen
at a lipid:peptide ratio of 300:1. The points Vm and c denote the points
of addition of valinomycin and cyclodepsipeptide 6, respectively.
capability,14 the larger macrocycles 5a,b and 6-8 were all found
to be very effective in translocating Na+, Mg2+, and Ca2+ ions
across model membranes (small unilamellar vesicles). Amongst
these macrocycles, the cyclodepsipeptide 6, with two leucine
residues on the exterior of the ring, showed maximum efficiency
and was demonstrated (Figure 3) to be almost as efficient as
valinomycin (with respect to the lipid:ionophore ratio) in
transporting Na+, Mg2+, and Ca2+ ions. None of these
macrocycles caused the release of entrapped carboxyfluorescein,
indicating that the movement of ions across the lipid bilayer
was not due to the formation of large pores or detergent-like
action. Although the transport mechanism is yet to be estab-
lished, the nonselective nature of ion-influx suggests encapsula-
tion of the ions by macrocyclic peptides. Interestingly, the
cyclodepsipeptides 10, 12, and 13, all containing only one
adanantane unit, and despite having strong metal-complexing
ligands in their cyclic framework, were found to to be totally
devoid of any membrane ion-transport capability, thus support-
ing our earlier notion9 that a minimum number of two
adamantane units in the appropriate ring size are necessary for
membrane penetration.
General Procedure for the Preparation of 1, 3-Adamantane Bis-
Ser Depsipeptides 3a-d Listed in Scheme 1. A solution of 1,3-
adamantanedicarbonyl dichloride (1, 1 mmol, freshly prepared from
dicarboxylic acid by refluxing with 3 M excess of SOCl2 for 3 h,
followed by drying in vacuo) in dry CH2Cl2 (10 mL) was added
dropwise over a period of 0.5 h to a well stirred and ice-cooled solution
of NR-Z-Ser-OMe or its di- or tripeptide (2 mmol) in dry CH3CN (30
mL) containing N,N′-dimethyl-4-aminopyridine (DMAP, 2 mmol) and
the mixture stirred at room temperature for 12 h. Solvents were
evaporated in Vacuo, the residue was triturated with ethyl acetate (∼100
mL), the extract was washed sequentially with 20 mL each of ice cold
2 N H2SO4, H2O, and 5% aqueous NaHCO3, and the organic layer
was dried over anhydrous MgSO4 and evaporated in Vacuo. The residue
was purified on a short column of silica gel using a mixture of ethyl
acetate and hexane as eluent to afford the adamantane-supported bis-
Ser peptides 3a-d in nearly quntitative yields.
In summary, the adamantane-constrained, serine-based cy-
clodepsipeptides described here represent a new class of peptide
ionophores. The intrinsic propensity of these macrocycles (as
exemplified with 5a,b, 6, and 8) to adopt a â-turn-type
(13) Loew, L. M.; Rosenberg, I.; Bridge, M.; Gitter, C. Biochemistry
1983, 22, 837. Loew, L. M.; Benson, L.; Lazarovici, P.; Rosenberg, I.
Biochemistry 1985, 24, 2101. Shai, Y.; Bach, D.; Yanovsky, A. J. Biol.
Chem. 1990, 265, 20202. Glaser, S. M.; Cumsky, M. G. J. Biol. Chem.
1990, 265, 8808. Roise, D. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 608.
Epand, R. M.; Shai,Y.; Segrest, J. P.; Anantharamaiah, G. M. Biopolymers
1995, 37, 319. Minn, A. J.; Velez, P.; Schendel, S. L.; Liang, H.; Muchmore,
S. W.; Fesik. S. W.; Fill, M.; Thompson, C. B. Nature 1997, 385, 353.
(14) Cyclodepsipeptides of ring size smaller than eight residues do not
normally form complexes with metal ions; their carbonyls are oriented
outward. Roeske, R. W.; Kennedy, S. J. In Chemistry and Biochemistry of
Amino Acids, Peptides and Proteins; Weinstein, B., Ed.; Marcel Dekker,
Inc.: New York, 1983, Vol. 7, pp 205-256.
3a: yield 99%; syrup; [R]25 ) +32.82 (c 6.35, CHCl3); IR (KBr)
D
3380, 2958, 2920, 2871, 1744, 1712, 1645, 1522, 1454, 1391, 1349,
1
1212 cm-1; H NMR (90 MHz, CDCl3) δ 1.53-2.20 (m, 14H), 3.70
(15) Nagaraj, R.; Mathew, M. K.; Balaram, P. FEBS Lett. 1980, 121,
365.

Synthesis of Macrocyclic Membrane Ion Carriers
J. Am. Chem. Soc., Vol. 119, No. 48, 1997 11583
(s, 6H), 4.39 (m, 4H), 4.65 (m, 2H), 5.10 (s, 4H), 5.97 (d, J ) 7.5 Hz,
2H), 7.31 (s, 10H).
mixture of Z-Ser (2 mmol), DCC, and N-hydroxysuccinimide (2 mmol
each) in dry dichloromethane (30 mL) was added, in one lot, the NR-
Z-deprotected (Pd/C, 5%, H2) solution of 3a (1 mmol) in dry ethyl
acetate (30 mL) and the reaction mixture stirred at room temperature
for 24 h. Usual peptide workup and purification of the crude product
(silica gel column with CHCl3/MeOH eluents) afforded the title
compound in 79% yield: thick syrup; IR (KBr) 3385, 2944, 1738, 1687,
1675, 1569, 1535, 1456, 1246, 1231 cm-1; 1H NMR (200 MHz, CDCl3)
δ 1.56-2.22 (m, 14H), 3.78 (s + m, 8H), 3.99 (m, 2H), 4.38 (m, 6H),
4.83 (m, 2H), 5.11 (s, 4H), 6.03 (d, J ) 6.31 Hz, 2H), 7.33 (s, 10H),
7.57 (d, J ) 6.31 Hz, 2H).
3b: yield 90%; mp 128-130 °C; [R]25D ) +53.24 (c 2.05, CHCl3);
IR (KBr) 3315, 3075, 2965, 2870, 1746, 1695, 1661, 1558, 1536, 1513,
1458, 1395 cm-1; 1H NMR (90 MHz, CDCl3) δ 0.93 (m, 12H), 1.53-
2.24 (m, 16H), 3.81 (s, 6H), 4.42 (m, 6H), 4.92 (m, 2H), 5.09 (brs,
4H), 5.65 (d, J ) 7.5 Hz, 2H), 7.55 (s + m, 12H).
3c: yield 93%; mp 118-119 °C; IR (KBr) 3391, 3298, 2964, 2871,
1
1736, 1718, 1666, 1538, 1457 cm-1; H NMR (300 MHz, CDCl3) δ
0.89 (brs, 12H), 1.40-2.16 (m, 20H), 3.80 (s, 6H), 4.27 (brd, 2H),
4.51 (m, 4H), 4.92 (brs, 2H), 5.04 (brs, 2H), 5.11 (brs, 2H), 5.54 (brs,
2H), 7.32 (brs, 10H), 7.47 (brs, 2H).
3d: yield 82%; mp 148-150 °C; IR (KBr) 3312, 3078, 2965, 1747,
1697, 1660, 1544, 1532, 1461, 1378, 1249, 1211 cm-1; 1H NMR (300
MHz, CDCl3) δ 0.92 (brs, 24H), 1.62-2.18 (m, 22H), 3.71 (s, 6H),
4.15 (m, 2H), 4.26 (m, 2H), 4.46 (m, 2H), 4.96 (m, 4H), 5.15 (m, 2H),
6.03 (brs, 2H), 7.30 (s + m, 12H), 7.61 (brs, 2H).
Preparation of Cyclodepsipeptides 4-7. General Procedure: (a)
N-Deprotection of Adamantane-Supported Bis-Nr-Z-Ser-Depsipep-
tides 3a-d. A solution of bis NR-Z-peptide 3a-d (1 mmol) in dry
ethyl acetate (∼10 mL) was admixed with Pd/C, 5% (peptide/catalyst
1:0.5 w/w) and hydrogenolyzed using Parr hydrogenation apparatus.
The reaction mixture after complete NR-deprotection (TLC) was filtered
through a sintered funnel, the residue was washed with dry ethyl acetate
(20 mL), and the filtrate was directly used for the coupling reaction in
the next step.
(b) Coupling of Adm-Supported Bis-Ser-Ser Peptide 3e with 1,3-
adamantanedicarbonyl dichloride. A freshly prepared solution of
1,3-adamantanedicarbonyl dichloride (1, 1 mmol) in dry dichlo-
romethane (50 mL) was added dropwise over a period of 0.5 h to a
well-stirred and ice-cooled solution of 3e (1 mmol) in dry CH3CN (100
mL) containing DMAP (2 mmol). After 12 h of stirring at room
temperature, the reaction mixture was worked up by evaporating the
solvents in Vacuo, triturating the residue with ethyl acetate (100 mL),
and washing the extract, sequentially, with 2 N H2SO4, H2O, and 5%
NaHCO3 solution (20 mL each), drying the organic layer with
anhydrous MgSO4, and evaporating in Vacuo. The residue was
chromatographed on a small column of silica gel and eluted with a
mixture of CHCl3/MeOH to afford the title compound 8 in 53% yield:
mp 200-203 °C; [R]25 ) +63.69 (c 1.6, CHCl3); IR (KBr) 3392,
D
3278, 3042, 2943, 2865, 1742, 1718 (sh), 1679, 1533, 1502 (sh), 1452
1
cm-1; H NMR (300 MHz, CDCl3) δ 1.50-2.25 (m, 28H), 3.88 (m,
(b) Condensation of Nr-Deprotected 1,3-Adamantane Bis-Ser
Depsipeptides 3a-d with 1,3-Adamantanedicarbonyl Dichloride (1).
To a well-stirred and ice-cooled solution of NR-deprotected bis-
Serdepsipeptide (1 mmol in ∼100 mL of dry EtOAc) containing 2 mmol
of triethylamine was added dropwise a solution of freshly prepared
1,3-adamantanedicarbonyl dichloride (1 mmol in 50 mL of dry CH2-
Cl2) over a period of 0.5 h and the mixture stirred at room temperature
for 12 h. The solvents were removed in Vacuo, and the residue was
taken up in CH2Cl2 (∼100 mL) and washed, sequentially, with 20 mL
each of the ice-cold 2 N H2SO4, H2O, and 5% aqueous NaHCO3. The
organic layer was dried over anhydrous MgSO4 and evaporated in Vacuo
and the residue purified on a column of silica gel using either a mixture
of EtOAc/hexane or CHCl3/MeOH as eluent to afford adamantane-
constrained Ser cyclodepsipeptides 4-7 in good yields.
2H), 4.00 (s, 6H), 4.39 (d, J ) 11.2 Hz, 2H), 4.53 (m, 2H), 4.73 (d, J
) 11.2 Hz, 2H), 4.85 (brs, 2H), 4.95 (brs, 2H), 5.10 (s, 4H), 5.45 (d,
J ) 8.6 Hz, 2H), 7.35 (brs, 10H), 7.88 (d, J ) 6.25 Hz, 2H); FAB MS
m/z 1057 (50) (M + H)+.
Synthesis of Monoadamantyl Ser-Based Cyclodepsipeptides 10,
12, and 13. Preparation of Cyclo(Adm-Ser-Cyst-Ser-) (10). (a) Nr-
Boc-Ser-Cyst-Ser Nr-Boc-bis-depsipeptide (9). To a well-stirred and
ice-cooled solution of NR-Boc-Ser-OH (10 mmol) in dry CH2Cl2 (∼50
mL) admixed with N-hydroxysuccinimide (10 mmol) and dicyclohexy-
lcarbodiimide (DCC, 10 mmol) was added a freshly prepared solution
of cystine diOMe (generated in situ at 0 °C from 5 mmol of cystine
dimethyl ester dihydrochloride and 10 mmol of dry triethylamine in
dry CH2Cl2). After 2 days of stirring at room temperature, the reaction
mixture was filtered, the residue washed with CH2Cl2, the filtrate
washed, sequentially, with 1 N H2SO4, H2O, and 5% aqueous NaHCO3
(20 mL each), dried (anhydrous Mg SO4), and evaporated in Vacuo,
and the residue was cleaned up on a small column of silica gel using
CHCl3/MeOH as eluents to give the title bis-Ser peptide in 75% yield:
4: yield 59%; mp 205-206 °C; [R]25 ) +72.82 (c 3.85, CHCl3);
D
IR (KBr) 3426, 2922, 2859, 1749, 1715, 1670, 1556 (sh), 1512, 1457,
1385, 1356 cm-1; 1H NMR (300 MHz, CDCl3) δ 1.65-2.23 (m, 28H),
3.78 (s, 6H), 4.44 (m, 4H), 4.84 (m, 2H), 6.34 (d, J ) 7.5 Hz, 2H);
FAB MS m/z 615 (100) (M + H)+.
syrup; IR (KBr) 3402, 2988, 1750, 1730, 1679, 1548, 1533, 1500 cm-1
;
5a: yield 60%; mp 149-151 °C; [R]25 ) -2.47 (c 2.2, CHCl3);
D
1H NMR (90 MHz, CDCl3) δ 1.46 (s, 18H), 3.17 (d, J ) 6.3 Hz, 4H),
3.78 (s, 6H), 4.0-4.49 (m, 6H), 4.87 (m, 2H), 5.88 (d, J ) 7.6 Hz,
2H), 7.63 (d, J ) 7.6 Hz, 2H); FAB MS m/z 643 (14) (MH)+, 543
(42) (M - Boc + H)+, 443 (100) (M - 2 × Boc + H)+.
IR (KBr) 3324, 2917, 2864, 2807, 1743, 1653 (br), 1558, 1523, 1505,
1459, 1377 cm-1; 1H NMR (400 MHz, CDCl3) δ 0.93, 0.95 (d, d, J )
6.9 Hz, 6.7 Hz, 6H, 6H), 1.67-2.24 (m, 30H), 3.78 (s, 6H), 4.24 (dd,
J ) 3.4, 7.7 Hz, 2H), 4.29 (m, 2H), 4.49 (dd, J ) 3.0, 8.5 Hz, 2H),
4.89 (m, 2H), 6.15 (d, J ) 8.3 Hz, 2H), 6.94 (d, J ) 8.1 Hz, 2H); FAB
MS m/z 813 (46) (M + H)+.
(b) Condensation of the Bis-Ser peptide 9 with 1,3-Adaman-
tanedicarbonyl dichloride. A dilute solution of freshly prepared 1,3
adamantanedicarbonyl dichloride (1, 1 mmol in 50 mL of dry CH2Cl2)
was added dropwise over a period of 0.5 h to a well-stirred and ice-
cooled solution of the bis-Ser peptide 9 (1 mmol in 100 mL of dry
CH3CN) containing DMAP (2 mmol) and the reaction mixture stirred
for 12 h at room temperature. Workup as for 8 and purification of the
residue on a short column of silica gel using CHCl3/MeOH as eluent
afforded the title cyclodepsipeptide 10 in 30% yield: thick syrup; IR
(KBr) 3387, 2940, 2866, 1740, 1688, 1536, 1514, 1458, 1371, 1251
5b: yield 55%; mp 104-105 °C; IR (KBr) 3374, 2938, 2865, 1744,
1
1678, 1647,1529, 1456 cm-1; H NMR (400 MHz, CDCl3) δ 0.89,
0.95 (d, d, J ) 6.4 Hz, 6.4 Hz, 6H, 6H), 1.55-2.24 (m, 34H), 3.78 (s,
6H), 4.32 (dd, J ) 4.5, 6.8 Hz, 2H), 4.40 (dd, J ) 2.2, 8.6 Hz, 2H),
4.49 (m, 2H), 4.93 (m, 2H), 6.07 (d, J ) 7.7 Hz, 2H), 7.30 (d, J ) 8.6
Hz, 2H); FAB MS m/z 841 (100) (M + H)+.
6: yield 50%; mp 151-153 °C; [R]25 ) -32.81 (c 1.3, CHCl3);
D
IR (KBr) 3318, 3072, 2942 (br), 2867, 1747, 1700, 1678, 1650 (br),
1544, 1514, 1474 cm-1; 1H NMR (400 MHz, CDCl3) δ 0.89 (m, 24H),
1.50-2.23 (m, 36H), 3.68 (s, 6H), 4.16 (t, J ) 7.1 Hz, 2H), 4.31 (d,
J ) 5.3 Hz, 4H), 4.52 (m, 2H), 4.73 (m, 2H), 6.08 (d, J ) 7.5 Hz,
2H), 6.69 (d, J ) 8.3 Hz, 2H), 6.97 (d, J ) 8.1 Hz, 2H); FAB MS m/z
1039 (100) (M + H)+.
1
cm-1; H NMR (300 MHz, CDCl3) δ 1.49 (s, 18H), 1.67-2.17 (m,
14H), 3.14 (brd, 2H), 3.35 (brd, 2H), 3.78 (s, 6H), 4.17 (m, 2H), 4.45
(brd, 2H), 4.62 (brd, 2H), 4.86 (m, 2H), 5.43 (d, J ) 6.51 Hz, 2H),
7.19 (brd, 2H); FAB MS m/z 831 (10) (MH)+, 731 (42) (M - Boc +
H)+, 631 (100) (M - 2 × Boc + H)+.
7: yield 2%; syrup; [R]25D ) +29.49 (c 1.1, CHCl3); IR (KBr) 3416,
2927, 2865, 1742, 1671, 1648, 1557, 1524, 1459, 1353 cm-1; 1H NMR
(200 MHz, CDCl3) δ 1.60-2.32 (m, 56H), 3.79 (s, 12H), 4.43 (m,
8H), 4.82 (m, 4H), 6.53 (d, J ) 7.6 Hz, 4H); FAB MS m/z 1229 (65)
(M + H)+, 615 (23) (M/2 + H)+, 308 (26) (M/4 + H)+.
Preparation of Crown Ether-Containing, Adamantane-Con-
strained Ser Cyclodepsipeptide (12). (a) Preparation of the Precur-
sor Bis-Nr-Boc-Ser-(CH2-OBzl)-Tetraethylene Glycol Ester (11). To
a well-stirred and ice-cooled mixture of Boc-Ser(CH2-OBzl)-OH (2
mmol), DCC (2 mmol), and N-hydroxysuccinimide (2 mmol) in dry
CH2Cl2 (20 mL) was added a solution of tetraethylene glycol (1 mmol)
in CH2Cl2 (∼20 mL) containing DMAP (2 mmol). After 24 h of stirring
Preparation of Cyclo(Adm-Ser-Ser-)2 (8). (a) 1,3-Adamantane
Bis-Ser-Ser Depsipeptide (3e). To a well-stirred and ice-cooled

11584 J. Am. Chem. Soc., Vol. 119, No. 48, 1997
Ranganathan et al.
at room temperature, the reaction mixture was workedup as for 9 and
the residue chromatographed over a short column of silica gel with a
mixture of ethyl acetate/hexane as eluent to afford the title compound
in 60% yield: syrup; IR (KBr) 3440 (br), 2985, 2940, 2879, 1757,
1722, 1633, 1579, 1503, 1458, 1370, 1350, 1291, 1252, 1169, 1115
were removed in Vacuo, and the residue was dissolved in CH2Cl2 (100
mL), washed with 5% NaHCO3 (20 mL), dried (anhydrous MgSO4),
and evaporated in Vacuo. The residue on purification on a small column
of silica gel and elution with CHCl3/MeOH gave the title compound
13 in 42% yield: mp 136-137 °C; IR (KBr) 3315, 2965, 2941, 1742,
1670, 1544, 1447 cm-1; 1H NMR (200 MHz, CDCl3) δ 1.02 (m, 12H),
1.27-1.83 (m, 14H), 2.53 (m, 2H), 3.77 (s, 6H), 4.26 (m, 2H), 4.46
(m, 4H), 4.80 (m, 2H), 7.35 (d, J ) 7.1 Hz, 2H), 8.03 (1H, t, J ) 7.1
Hz), 8.32 (d, J ) 7.1 Hz, 2H), 8.67 (d, J ) 8.6 Hz, 2H); FAB MS m/z
756 (71) (M + H)+.
1
cm-1; H NMR (300 MHz, CDCl3) δ 1.44 (s, 18H), 3.57-3.87 (m,
16H), 4.28 (brs, 4H), 4.48 (m, 6H), 5.43 (brd, 2H), 7.28 (brs, 10H);
FAB MS m/z 755 (20) (M + Li+), 649 (31) (M - Boc + H)+, 549
(14) (M - 2 × Boc + H)+.
(b) Condensation of O-Deprotected Bis-Ser-Tetraethylene Glycol
Ester 11 with 1,3-Adamantanedicarbonyl Dichloride (1). A solution
of 1,3-adamantanedicarbonyl dichloride (1 mmol) in dry CH2Cl2 (50
mL) was added dropwise to a well-stirred and ice-cooled solution of 1
mmol of O-deprotected (Pd/C, 5%, H2) 11 in dry ethyl acetate (∼100
mL) containing DMAP (2 mmol). The reaction mixture was stirred at
room temperature for 12 h and worked up as described in the general
procedure for 4-7. The residue was chromatographed on a small
column of silica gel and eluted with a mixture of ethyl acetate/hexane
to afford the crown hybrid 12 in 25% yield: thick syrup; IR (KBr)
3356 (br), 2932, 2863, 1733, 1718, 1641, 1576, 1555, 1523, 1501, 1466,
Acknowledgment. This work was financially supported by
the Department of Science and Technology, New Delhi. We
are most grateful to Professor S. Ranganathan (RRL, Trivan-
drum) and Professor D. Balasubramanian (CCMB, Hyderabad)
for helpful advice and comments. We thank Mr. N. Sridharsa
summer trainee under the Research Fellowship Programme of
Jawaharlal Nehru Centre For Advanced Scientific Researchsfor
some experimental help.
1
1372 cm-1; H NMR (300 MHz, CDCl3) δ 1.46 (s, 18H), 1.67-2.06
Supporting Information Available: 1H NMR of 5a,b and
6, ROESY NMR of 5a,b and 6, FAB-MS of 4, 5a,b, 6-8, 10,
12, and 13, CD spectra of 5a and 6 in TFE, CD spectra of 5a
and 6 in aqueous MeOH, and ion-transport diagrams of 7 and
8 (22 pages). See any current masthead page for ordering and
Internet access instructions.
(m, 14H), 3.64 (m, 12H), 4.59 (m, 8H), 4.61 (m, 2H), 5.45 (br, 2H);
FAB MS m/z 779 (100) (M + Na+).
Preparation of Cyclo(Adm-Ser-Val-Pyr-Val-Ser-) (13). A solu-
tion of 2,6-pyridinedicarbonyl dichloride (1 mmol) in dry CH2Cl2 (50
mL) was added dropwise to a well-stirred and ice-cooled solution of
N-deprotected (Pd/C, 5%, H2) 1,3-adamantane bis Ser-Val depsipeptide
3b (1 mmol) in dry ethyl acetate (100 mL) containing 2 mmol of
triethylamine and the reaction mixture stirred for 12 h. The solvents
JA971517M