Proc. Natl. Acad. Sci.

USA
Vol. 85, pp. 4118-4122, June 1988
Chemistry

Crystal and molecular structure of didemnin B, an antiviral and

cytotoxic depsipeptide
(x-ray structure/conformation/immunosuppressive agent)
M. B. HosSAIN*, D. VAN DER HELM*, J. ANTELt, G. M. SHELDRICKt, S. K. SANDUJAf,
AND A. J. WEINHEIMER*
*Department of Chemistry, The University of Oklahoma, Norman, OK 73019; tInstitute of Inorganic Chemistry, University of Gottingen, Gottingen, Federal
Republic of Germany; and tDepartment of Medicinal Chemistry, University of Houston, Houston, TX 77004
Communicated by Isabella L. Karle, February 23, 1988 (received for review August 28, 1987)

ABSTRACT Didemnin B, a highly active depsipeptide iso-Sto Hp

0 0 0 0
isolated from a Caribbean tunicate, crystallizes from chloro- it

form/benzene in the orthorhombic space group C2221, with CmNHCHCHOHCH2CmOCHCCH(CHs)C

cell parameters a = 14.990 0.003 A, b = 22.574 0.004 6
t!H-CH2CH3 6H(CH3)2
A, c = 41.112 : 0.009 A, V = 13911.7 A3 at 138 K and a 11 I CH3
calculated density of 1.143 g/cm3 based on C,7H89N7O1l. R-N (CH3)CHC-NHCH
1.5C6H6^H20 and eight formula units per cell. The overall (CH3)ICHCH2 CH2-<ZOCH CHICHIC2
agreement factor R = 0.052 for 7699 reflections, 20..] = 1500, CuCHOCCHN(CH3)C-C _CCHN
Cu Kia radiation. The structure determination revealed that
didemnin B contains an isostatine residue instead of a statine CH3 0 0
residue. The conformation of the 23-membered depsipeptide Thr Melyr Pro Lou

ring is stabilized by one transannular hydrogen bond. The ring DIDEMNIN A, R=H
does not show the antiparallel fl-pleated-sheet structure but,
instead, has a fold in the shape of a bent figure-eight. The linear DIDEMNIN B, R=CH3CHOHC-N4 C_
peptide moiety, containing N-methylleucine and lactylproline, 0

forms a B(1)-bend and is folded back toward the cyclic
backbone, giving the overall molecule a globular character.
Comparison with the structure of cyclosporin A shows distinct
stereochemical differences between the two molecules. It is
suggested that didemnin B and cyclosporin A are unlikely to
have a common receptor binding site.
Didemnins, a class of depsipeptides, were originally isolated
from a Caribbean tunicate of the family Didemnidae (Tridi-
demnum genus) and were structurally characterized by
spectroscopic and degradative studies (1). The didemnins
consist of an unusual depsipeptide ring structure that con-
tains one unit of 2-(2-hydroxyisovaleryl)propionate (Hip) and
one unit of an isomer of the amino acid statine. The total
synthesis of didemnins, A, B, and C has been reported (2). All
didemnins contain the same cyclic depsipeptide backbone
but differ from one another in the substituent attached to
N-methylleucine in the linear peptide side chain (Fig. 1).
The didemnins have been reported to be highly active
against P388 leukemia and B16 melanoma (3) and L1210
leukemia and CHO cells in vitro (4, 5). In addition, didemnins
effectively inhibit the replication of several DNA and RNA
viruses in vitro (4) and protect female mice from genital herpes
simplex virus 2 infection (3). Didemnin B in general is many
times more active and potent than the parent compound,
didemnin A, in all these tests. In the case of the L1210 mouse
leukemia cell line (5), the concentration inhibiting cell growth
by 50% (IC50) is 1-2 ng/ml (0.9-1.8 nM) for didemnin B,
exemplifying the antiproliferative potency of this compound.
Didemnin B has been reported to have significant activity
against a variety of human tumors, including carcinoma of the
breast, ovary, kidney and lung, mesothelioma, sarcoma, and
hairy-cell leukemia, in in vitro stem-cell assays (3, 6).
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. 1734 solely to indicate this fact.
DIDEMNIN C, R= CH3CHOHC-
FIG. 1. Chemical structures of didemnins A, B, and C. iso-Sta,
isostatine.
Preliminary studies on the mechanism of action of these
antibiotics showed that didemnin B is an inhibitor of protein
synthesis and that it does not bind DNA (7). It was suggested
that its biological activity is mediated primarily through its
inhibition of protein synthesis and to a lesser extent by its
inhibition of DNA synthesis (8). Unlike other polypeptide
antibiotics, such as valinomycin and gramicidin, didemnin B
(and also A) does not function as an ionophore (8).
It has been reported that didemnin B has potent immuno-
suppressive activity both in vitro and in vivo (9). The
structural interest in didemnin B is heightened by the fact that
the most commonly used immunosuppressive agent, cyclo-
sporin A (CsA), another cyclic polypeptide, bears some
structural resemblance to didemnin B. The potency of di-
demnin B as an inhibitor of lymphocyte proliferation in
response to B- and T-cell mitogens and alloantigens is 100-
1000 times that of CsA (9).
Because of its cytotoxicity and its activity against tumors in
animals, didemnin B is being developed as an antitumor agent.
Didemnin B is undergoing phase II trials by the National
Cancer Institute. We believe that the solid-state molecular
conformation ofthis highly potent drug will provide a basis for
further studies into the mechanism of its activity.
EXPERIMENTAL PROCEDURE
Extraction and Purification. An aqueous isopropanol ex-
tract of Didemnidum species, probably D. solidum, was
concentrated to remove the isopropanol and then extracted
with chloroform. The residue from the chloroform extract
was partitioned in the usual manner (10) and chromato-
graphed on Sephadex LH-20 (Pharmacia) in methanol. The
Abbreviations: Hip, 2-(2-hydroxyisovaleryl)propionate; CsA, cyclo-
sporin A.
 Chemistry: Hossain et al. Proc. Natl. Acad. Sci. USA 85 (1988) 4119
Table 1. Fractional atomic coordinates (x, y, z) and temperature factors (Ueq) for didemnin B
Atom X X 104 y X 104 z X 105 Ueqt X 103 Atom x x 10l y X 104 z X i05 Ueqt X 103
C(1) 6898 (3) 2313 (2) 6509 (9) 27 (1) C(39) 5475 (3) 1284 (2) 14887 (9) 29 (1)
C(2) 6661 (3) 2833 (2) 4226 (9) 33 (1) N(5) 4684 (3) 1336 (1) 16879 (8) 36 (1)
C(3) 7083 (3) 2750 (2) 857 (9) 30 (1) C(40) 4101 (3) 888 (2) 17024 (9) 31 (1)
C(4) 7925 (3) 2870 (2) 119 (10) 22 (1) 0(10) 4244 (2) 408 (1) 15694 (6) 32 (1)
C(5) 8285 (3) 2746 (2) -2931 (9) 31 (1) C(41) 3204 (3) 1011 (2) 18681 (10) 35 (1)
C(6) 7740 (3) 2495 (2) -5287 (9) 34 (1) C(42) 2773 (4) 443 (2) 19966 (1) 55 (2)
0(1) 8012 (2) 2358 (1) -83% (7) 46 (1) C(43) 1818 (4) 535 (2) 21239 (15) 65 (2)
C(7) 9822 (3) 2472 (2) -9250 (12) 51 (2) C(44) 1191 (5) 758 (4) 18731 (21) 95 (3)
C(8) 6853 (3) 2374 (2) -4607 (10) 36 (1) C(45) 1490 (5) -54 (3) 22684 (22) 104 (3)
C(9) 6511 (3) 2503 (2) -1579 (10) 33 (1) N(6) 3282 (2) 1465 (1) 21181 (7) 32 (1)
N(1) 6826 (2) 1722 (1) 5017 (7) 26 (1) C(46) 3764 (3) 1315 (2) 24156 (11) 47 (2)
C(10) 7633 (3) 1464 (2) 3653 (10) 34 (1) C(47) 2988 (4) 2017 (2) 20493 (11) 47 (2)
C(11) 5998 (2) 1501 (2) 4529 (8) 24 (1) C(48) 3063 (3) 2499 (2) 23087 (11) 44 (1)
0(2) 5332 (2) 1770 (1) 5402 (6) 28 (1)
C(12) 5911 (3) 893 (2) 2831 (9) 27 (1) 0(11)t 2820 (4) 2157 (2) 17551 (12) 44 (2)
C(13) 5934 (3) 942 (2) -888 (9) 35 (1) 0(11)A 2355 (4) 2097 (3) 18433 (15) 26 (2)
C(14) 4989 (3) 1122 (2) -1747 (10) 39 (1)
C(15) 4401 (3) 808 (2) 749 (9) 32 (1) C(49)t 2144 (5) 2800 (4) 23443 (23) 54 (3)
N(2) 5020 (2) 658 (1) 3422 (7) 25 (1) C(49)A 2160 (8) 2559 (4) 25384 (25) 37 (3)
C(16) 4820 (3) 429 (1) 6378 (9) 24 (1)
0(3) 5394 (2) 375 (1) 8497 (6) 27 (1) C(S0)t 2365 (3) 3459 (3) 24149 (23) 50 (3)
C(17) 3859 (3) 251 (2) 6979 (9) 27 (1) C(50)A 2350 (8) 3220 (5) 26482 (27) 41 (4)
C(18) 3299 (3) 797 (2) 7945 (10) 37 (1)
C(19) 2301 (4) 678 (3) 8174 (20) 82 (3) C(51)t 3101 (5) 3633 (3) 22011 (19) 44 (2)
C(20) 1934 (5) 561 (6) 4756 (33) 168 (6) C(51)A 2788 (7) 34% (5) 23323 (27) 36 (3)
C(21) 1839 (5) 1212 (4) 9799 (20) 97 (3)
N(3) 3813 (2) - 182 (1) 9611 (7) 26 (1) N(7)t 3618 (4) 3063 (2) 21600 (15) 32 (3)
C(22) 3431 (2) -716 (2) 9298 (9) 25 (1) N(7)A 3279 (5) 2999 (3) 22000 (17) 21 (4)
0(4) 3190 (2) -928 (1) 6686 (7) 37 (1)
C(23) 3271 (3) - 1051 (2) 12486 (9) 28 (1) C(52)t 4458 (5) 3007 (3) 20804 (16) 32 (2)
C(24) 2280 (3) - 1023 (2) 13358 (11) 39 (1) C(52)A 3971 (6) 2999 (4) 19856 (21) 20 (2)
C(25) 3583 (3) - 1689 (2) 12081 (9) 28 (1)
0(5) 3079 (2) -2195 (1) 12314 (8) 42 (1) 0(12)t 4816 (3) 2510 (2) Z0703 (12) 39 (2)
C(26) 4566 (3) - 1784 (2) 11247 (9) 28 (1) 0(12)A 4341 (4) 2529 (3) 19190 (16) 27 (2)
C(27) 4947 (3) - 2404 (2) 11816 (12) 39 (1)
C(28) 4563 (3) -2853 (2) 9394 (14) 54 (2) C(53)t 5087 (5) 3599 (3) 19902 (15) 71 (2)
C(29) 4862 (4) -2608 (2) 15307 (14) 57 (2) C(53)A 4333 (7) 3571 (4) 18388 (25) 29 (3)
0(6) 5052 (2) -1353 (1) 13149 (6) 27 (1)
C(30) 5763 (2) - 1092 (2) 11715 (9) 24 (1) 0(13)t 5921 (4) 3415 (2) 20403 (13) 45 (2)
0(7) 6009 (2) - 1202 (1) 8991 (6) 31 (1) 0(13)A 3837 (6) 4079 (3) 18772 (25) 43 (2)
C(31) 6158 (3) -640 (2) 13970 (9) 27 (1)
C(32) 7078 (3) -428 (2) 12967 (10) 30 (1) C(54)t 4843 (6) 3725 (4) 16407 (21) 50 (3)
0(8) 7741 (2) -854 (1) 13728 (8) 44 (1) C(54)A 5087 (5) 3599 (3) 19902 (15) 71 (2)
C(33) 7337 (3) 145 (2) 14728 (10) 33 (1)
C(34) 8289 (3) 357 (2) 13887 (14) 50 (2) C(55) 5910 (3) 18% (2) 14516 (9) 30 (1)
C(35) 8369 (4) 529 (2) 10303 (16) 63 (2) C(56) 6543 (4) 2077 (2) 17222 (11) 51 (2)
C(36) 8588 (4) 878 (2) 15975 (18) 71 (2) 0(14) 6412 (2) 1874 (1) 11474 (6) 27 (1)
C(37) 8565 (5) 784 (3) 19547 (20) 96(3) C(57) 6359 (3) 2367 (2) 9635 (9) 25 (1)
N(4) 6677 (2) 600 (1) 14024 (7) 29 (1) 0(15) 5965 (2) 2808 (1) 10411 (6) 29 (1)
C(38) 6127 (3) 828 (2) 16263 (9) 31 (1) W 2114 (2) 3027 (7) 13934 (7) 36 (1)
0(9) 6154 (2) 711 (1) 19180 (7) 46 (1)
Estimated standard deviation for last digit is given in parentheses.
teq = 3 IIUj Oaj*a(a..aj.)
ij aj .

tDisordered atoms. Atoms marked A have 40%o occupancy.

Water molecule.
major fraction, didemnin B, crystallized readily from
chloroform/hexane.
X-Ray Diffraction. A crystal of dimensions 0.81 x 0.72 x
0.32 mm was used for all x-ray measurements. Cell param-
eters were obtained by a least-squares fit to the 20 values
of 48 (at 138 K) reflections measured with Cu Ka1 radiation.
Intensities of all unique reflections with 20max < 1500 were
measured at 138 2 K with Cu Ka radiation on an Enraf-
Nonius CAD-4 diffractometer. The 0-20 scan technique was
employed with a variable scan width of (0.7 + 0.20 tan o)r and
a variable horizontal aperture of (3.5 + 0.86 tan 6) mm. Three
standard reflections were monitored every 2 hr of x-ray
exposure, and they showed a maximum fluctuation of less
than 2%. Of 7699 unique reflections that were measured, 7127
reflections were considered observed [I 2 2o-(I), where I is
the intensity and ais the standard deviation]. Intensities were
corrected for Lorentz and polarization factors.
Structure Determination and Refinement. The structure
proved resistant to all attempts to solve it by conventional
direct methods. It was eventually solved by using the
program SHELXS-86 (11) with the following strategy. Mul-
tiple random-start tangent refinement was performed for a
4120 Chemistry: Hossain et al.
FIG. 2. Atom numbering scheme (nonconventional).
subset of 60 strong E values chosen from the centrosymmet-
ric 0 k I projection. The most consistent phase sets (judged by
the 183 triplets involving subset reflections only) were then
expanded to 485 general reflections by using all 9752 triplets.
The strongest 1000 negative quartets were employed actively
in this phase refinement, in which the subset reflections were
given an initial weight of 1, and the rest a weight of 0.1. One
solution out of the 5215 in the final refinement gave convinc-
ing figures of merit and the resulting E-Fourier map yielded
79 correct atoms.
The lactylproline fragment of the linear chain part of the
molecule was found to be disordered. The 10 disordered
atoms were refined with (60/40)% occupancy as determined
from difference Fourier syntheses. The solvent in the crystal
consists of one water molecule and two molecules of ben-
zene, one of which occupies a special position (2-fold). Of the
total of 100 hydrogen atoms in the structure, 68 were located
from a difference Fourier map and were refined isotropically.
Another 12 hydrogen atoms were placed on their calculated
positions and were kept fixed. Hydrogen atoms belonging to
the disordered part of the molecule and those belonging to the
benzene solvates were not included in the calculations. The
final refinement of the structure was carried out by using a
block-diagonal least-squares routine (12) that included ani-
sotropic thermal parameters for the non-hydrogen atoms.
The refinement was carried to convergence, producing an
agreement factor R (= SIMF1/EIF0d of 0.045 for 7127 reflec-
tions included in the least-squares calculations and an R of
0.052 for all 7699 reflections.
RESULTS AND DISCUSSION
The final atomic parameters for the non-hydrogen atoms of
didemnin B are given in Table 1. (Supplementary material
consisting of observed and calculated structure factors,
FIG. 3. Stereoview of a single molecule of didemnin B. For the disordered fragment, only those atom sites that have 60%o occupancy are
shown here and in all subsequent figures.
Proc. Natl. Acad. Sci. USA 85 (1988)
FIG. 4. A view of the peptide chain (thick line) in didemnin B.
Intramolecular hydrogen bonds are shown by broken lines.
anisotropic thermal parameters, hydrogen-atom parameters,
bond distances, and bond angles is available from D.v.d.H.)
The atom numbering scheme is shown in Fig. 2. A stereoview
of a single molecule of didemnin B is displayed in Fig. 3. The
crystal structure determination of the molecule demonstrates
that didemnin B contains an isostatine residue rather than a
statine residue as reported earlier (1, 3-7). The absolute
configuration has not been determined but is based on the
original assignment (1) of L amino acids for six of the seven
amino acid residues in the compound. It is apparent that only
the N-methylleucine residue has the D configuration. The
absolute configuration of the asymmetric centers of the Hip
unit is (2S, 45), which confirms the results of synthetic
studies (1, 13). The configuration of the asymmetric centers
of the isostatine residue is (35, 4R, 5S), in agreement with the
results obtained in the total synthesis of the didemnins (2).
The 23-membered depsipeptide macrocycle assumes a
highly irregular shape but displays very little of the antipar-
allel a-pleated structure often seen in cyclic peptides. In-
stead, the cyclic part of the structure can be described as a
bent figure-eight (Fig. 4). The linear peptide moiety contain-
ing the N-methylleucine and lactylproline is folded back
toward the cyclic backbone, giving the overall molecule a
highly asymmetric and globular character in contrast to many
other large cyclic peptide structures, which have relatively
more flat and symmetric geometry. This conformation of the
didemnin B molecule is stabilized by three intramolecular
 Chemistry: Hossain et al.
N-H ..O hydrogen bonds (Fig. 4). Of the three hydrogen
bonds, only one [N(4) ..0(3), 3.020 A] is within the cyclic
backbone, and it forms a bridge across the middle of the
macrocyclic ring by linking the isostatine amide group with
the leucine carbonyl oxygen atom. The second hydrogen
bond [N(5)-- 0(12), 3.087 A] is within the linear part of the
peptide chain, and the third [N(3)' * O(10), 2.906 Al, by far the
strongest, binds the linear chain to the cyclic backbone. The
second of these three hydrogen bonds is involved in a
conventional /3(II)-turn of the peptide side chain, involving
L-proline and N-methyl-D-leucine at the corners. The (4, 4i)
torsion-angle values of (-65, 1250) and (1030, -29) com-
pare well with those expected for a /3(II)-turn (14). There are
three more turns in the depsipeptide chain. One turn is
formed by the Hip residue. The other two are formed by
L-threonine and isostatine residues and by L-proline and
N,O-dimethyl-L-tyrosine residues, respectively. They can be
described as /3-turns on the basis of their geometry, but they
are unconventional because there is no possibility for hydro-
gen bonding. The relevant torsion angles in these two turns,
which correspond to (4), qi) angles of a regular /3-turn, are
(-690, 1560) and (1130, - 580) for the threonine-isostatine turn
and ( -75, 1630) and (510, 400) for the proline-dimethyltyro-
sine turn (see also Table 2). They result in van der Waals
contacts of 3.785 A and 3.903 A between O(10) and C(13) and
between 0(3) and 0(14), respectively. These observations
indicate that hydrogen-bond formation, commonly associ-
ated with a /-turn, may not be the primary cause for the
folding.
The conformation of the threonine residue seems to play a
critical role in shaping the overall molecular conformation, as
the threonine C" forms the junction of the cyclic and linear
peptide chains. It is interesting that in several antibiotic
peptides, including virginiamycin S, vernamycin B, patricin
A, stendomycin, and telomycin (15), a threonine residue
occupies a similar position as it does in the didemnins. In all
these cases, an ester bond is formed between the threonine
hydroxyl and the C-terminal carboxyl in the cyclic part of the
molecule, and the threonine amino group is acylated to form
a linear peptide side chain. In the solution conformation of
patricin A (16), a hydrogen bond has been proposed between
the amide hydrogen of the succeeding residue [N(4)H] and
the ester oxygen atom [0(14)] of the threonine. However, in
didemnin B the observed geometry does not favor this
description [N(4)H-.. 0(14), 2.68 Al.
Selected torsion angles of the didemnin B molecule are
listed in Table 2. The peptide torsion angles (w) show that the
Table 2. Selected torsion angles
Angle value,*
Bonds degrees
N(1)-C(11)-C(12)-N(2) 163.0
C(11)-C(12)-N(2)-C(16) -75.4
C(12)-N(2)-C(16)-C(17) 173.6
N(2)-C(16)-C(17)-N(3) 158.0
C(16)-C(17)-N(3)-C(22) -124.1
C(17)-N(3)-C(22)-C(23) -167.4
N(3)-C(22)-C(23)-C(25) -133.6
C(22)-C(23)-C(25)-C(26) 60.0
C(23)-C(25)-C(26)-0(6) 39.4
C(25)-C(26)-0(6)-C(30) -141.8
C(26)-0(6)-C(30)-C(31) 177.4
0(6)-C(30)-C(31)-C(32) 166.1
C(30)-C(31)-C(32)-C(33) 163.9
C(31)-C(32)-C(33)-N(4) -58.2
C(32)-C(33)-N(4)-C(38) 113.2
C(33)-N(4)-C(38)-C(39) -178.1
N(4)-C(38)-C(39)-C(55) -81.0
*Standard deviations range between 0.50 and 0.7.
Proc. Natl. Acad. Sci. USA 85 (1988) 4121
amide and ester bonds are all trans and do not deviate from
planarity by exceptional amounts (o values range from - 167
to + 1740). The bond distances and bond angles, except for
the disordered part, are quite normal and fall within the range
of those observed in other depsipeptide structures. The
conformations of all the alkyl side chains are generally
staggered. The two proline rings in the molecule have
opposite conformations: the one in the cyclic part of the
molecule has an endo conformation in which the y ring
carbon is puckered on the same side as the proline carbonyl
group, whereas the proline ring in the linear chain has an exo
conformation. The methyl group of the tyrosine side chain
lies in the plane of the aromatic ring. The disorder in the
lactylproline moiety does not affect the hydrogen bonding.
The most significant change is a rotation of the terminal
hydroxyl group.
Fig. 5 gives a view of the didemnin B molecule that shows
the cup-like fold of the cyclic backbone. This conformation
of the cyclic moiety resembles the cavity formation of many
large polypeptides that form metal complexes. However, a
closer look at the oxygen atoms in the present structure
shows that most of them are pointed away from the interior
of the cavity. This may explain why didemnin B does not
complex alkali cations and does not act as an ionophore (8).
Fig. 5 also gives indications of possible active site(s) for the
didemnin B molecule. The way in which the two nearly planar
sections, the tyrosine side chain (I) and the lactylproline
moiety (II), extend out of the main body of the molecule
suggests roles for them in interactions with receptors. An-
other possible interacting group is the hydroxyl group of the
isostatine residue (III in Fig. 5), which also sticks outward.
The three possible interacting groups of didemnin B form a
roughly trigonal array. The lone water molecule in the
structure, in symmetry-related positions, forms strong hy-
drogen bonds with all three of these groups. The water
molecule (W) acts as a donor to the oxygen atom of the
tyrosine side chain [W ...O)(1), 2.784 0.004 A] and to the
proline carbonyl in the linear chain [W ...O(11), 2.681 0.006
A] and as an acceptor for the isostatine hydroxyl proton
[0(8).. W, 2.697 0.004 Ai.
Didemnin B shows significant immunosuppressive activity
and has been reported to be 100-1000 times more active than
CsA (9), and a recent study showed that didemnin B is much
more potent than CsA in its ability to inhibit binding of
prolactin to human lymphocytes (17). The cyclosporin bind-
ing site does not have significant affinity for didemnin B (18).
It is therefore important to compare the two structures. The
Angle value,*
Bonds degrees
C(38)-C(39)-C(55)-0(14) 81.2
C(39)-C(55)-0(14)-C(57) 139.0
C(55)-0(14)-C(57)-C(1) 180.0
0(14)-C(57)-C(1)-N(1) 40.0
C(57)-C(1)-N(1)-C(11) 51.3
C(1)-N(1)-C(11)-C(12) 177.4
N(4)-C(38)-C(39)-N(5) 156.0
C(38)-C(39)-N(5)-C(40) -68.6
C(39)-N(5)-C(40)-C(41) -169.5
N(5)-C(40)-C(41)-N(6) -29.0
C(40)-C(41)-N(6)-C(47) 103.0
C(41)-N(6)-C(47)-C(49) 180.0
N(6)-C(47)-C(48)-N(7) 124.6
C(47)-C(48)-N(7)-C(52) -63.5
C(25)-C(26)-C(27)-C(28) 69.6
N(4)-C(33)-C(34)-C(35) -58.6
C(33)-C(34)-C(36)-C(37) 56.6
4122 Chemistry: Hossain et al.
I that can bind receptors. The role of the linear peptide chain,
11
FIG. 5. A view of the didemnin B molecule showing the fold of
the cyclic peptide ring and the disposition of possible interacting
groups: I, tyrosine side chain; II, lactylproline moiety; and III, the
hydroxyl group in isostatine. The water molecule (W) is shown in
three symmetry-related positions. Broken lines indicate hydrogen
bonds.
comparison shows that despite some common features (ali-
phatic side chains and a D amino acid), there are major
structural differences in the two molecules. CsA is a regular
cyclic polypeptide with 11 amino acid residues and has no
linear peptide side chain or ester bond, whereas didemnin B
is an irregular depsipeptide that has both a cyclic and a linear
peptide segment. In the CsA crystal structure, the larger
segment of the macrocyclic peptide ring has antiparallel
pleated-sheet structure; in didemnin B, the cyclic backbone
is severely folded and shows very little pleated-sheet struc-
ture. In addition, the oxygen atoms and the amide groups are
more effectively shielded by the lipophilic groups in CsA than
in didemnin B, due to a larger number of aliphatic side chains
(eight compared to four in didemnin B) and of methylated
amide groups (seven compared to 2 in didemnin B). In the
proposed solution structure of CsA (19), which is also
presumed to be the active conformer, the long alkyl chain of
the (4R)-4-[(E)-2-butenyl]-4,N-dimethyl-L-threonine residue
extends outward from the cyclic peptide backbone and
presumably interacts with receptors. The equivalent segment
in didemnin B is the lactylproline moiety, which extends out
of the cyclic peptide backbone. It is unlikely that the binding
site of an unsaturated alkyl chain in CsA would also bind the
more polar lactylproline group of didemnin B. The structural
difference between the two molecules is also reflected in their
solvent interactions. Didemnin B forms three strong hydro-
gen bonds with a solvent water molecule, whereas CsA does
not interact with solvent. From all these considerations, we
suggest that the two molecules do not have comparable
binding sites.
Proc. Natl. Acad. Sci. USA 85 (1988)
Concluding Remarks. Didemnin B has diverse biological
activities and is unique in its structural features among the
known cyclic polypeptides. The results of the present work
suggest that didemnin B has more than one reacting group
particularly the lactylproline moiety, is probably the most
significant, considering the dramatic change in bioactivity
effected by the modification of didemnin A to didemnin B.
The high activity of didemnin B indicates specificity for its
interactions, while the variety of its activities, suggests that
different interaction sites are involved in its various biological
functions.
This research was supported by the National Institutes of Health
under Grants GM21822 and CA17562 (D.v.d.H.).
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