Isolation and structure determination of Cryptophycins 38, 326, and 327 from the terrestrial Cyanobacterium Nostoc sp. GSV 224

Article abstract of DOI:10.1021/np0499665

Cryptophycin-38 (2), -326 (3), and -327 (4) are three new trace constituents of the terrestrial cyanobacterium Nostoc sp. GSV 224. Cryptophycin-38 is a stereoisomer of cryptophycin-1 (1) and to date is the only naturally occurring analogue that possesses

Full text of DOI:10.1021/np0499665

J . Nat. Prod. 2004, 67, 1403-1406
1403
Isola tion a n d Str u ctu r e Deter m in a tion of Cr yp top h ycin s 38, 326, a n d 327 fr om
th e Ter r estr ia l Cya n oba cter iu m Nostoc sp . GSV 224†
Sreedhara Chaganty, Trimurtulu Golakoti, Carl Heltzel, Richard E. Moore,* and Wesley Y. Yoshida
Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii 96822
Received J anuary 12, 2004
Cryptophycin-38 (2), -326 (3), and -327 (4) are three new trace constituents of the terrestrial cyanobac-
terium Nostoc sp. GSV 224. Cryptophycin-38 is a stereoisomer of cryptophycin-1 (1) and to date is the
only naturally occurring analogue that possesses a S,S epoxide group in unit A. Cryptophycin-327 is a
geometric isomer that differs from 1 in having a cis ∆2-double bond in unit A. Cryptophycin-326 is related
to cryptophycin-21, but has two chlorines ortho to the methoxy group in unit B. The relative and absolute
stereochemistries of 2 have been related to known cryptophycins by semisynthesis and/or spectral analysis.
Cryptophycins are potent antitumor and antifungal
peptolides associated with the terrestrial cyanobacteria
Nostoc sp. GSV 224 and ATCC53789 (Nostocaceae).1-5 The
most abundant member of this class of cyclic depsipeptides
is cryptophycin-1 (1). In addition to 1, 24 other analogues
have been isolated from the GSV 224 cyanobacterium.4,5
Interestingly HPLC analysis shows that each cyanobac-
terium exhibits the same array of cryptophycins.5,6 Nev-
ertheless, the two cyanobacteria are significantly different
genetically and biochemically. The 16S rDNAs differ by
2.8% in homology (GenBank Accession AF062637 for
GSV224 and AF062638 for ATCC53789), and the other
secondary metabolites that are found in GSV224 appear
to be different from the ones found in ATCC53789. For
example, GSV224 produces a class of peptolides, called
nostopeptolides, that are totally absent in the ATCC53789
cyanobacterium.6 Furthermore, the genes that encode for
the biosynthesis of the nostopeptolides in GSV224 (Gen-
Bank Accession AF204805) are not found in ATCC53789.7
Conversely the ATCC53789 species produces an unusual
class of cyclic peptides (nostocyclopeptides) that are not
present in GSV224.8 Similarly, the genes assigned to the
biosynthesis of the nostocyclopeptides in ATCC53789 (Gen-
Bank Accession AY167420) are absent in GSV224.9
In this paper we report the isolation and identification
of three more trace cryptophycins (2-4) from GSV 224.
Lyophilized GSV 224 was extracted with 4:1 CH3CN/CH2-
logues appear to be present in the ATCC53789 Nostoc by
Cl2, and the concentrated extract was fractionated by
HPLC analysis.10
reversed-phase flash column chromatography with mix-
Cryptophycin-38 (2) has the molecular formula C35H43
-
tures of H2O/CH3CN as previously described.2 All of the
cryptophycins were eluted with 35% H2O/CH3CN, and this
mixture was further separated by reversed-phase HPLC
into a number of fractions. One of the fractions, a peak
having a relative retention time of 1.16 compared with 1.00
for 1, contained the three new analogues, viz., cryptophy-
cin-38 (2), cryptophycin-326 (3), and cryptophycin-327 (4).
Further fractionation led to pure 3 and two subfractions.
Repeated chromatography of the two subfractions by
normal-phase and reversed-phase HPLC led to pure 2 and
4. The total structures of 2-4 were established in a
straightforward manner using a combination of spectral
and chemical (semisynthesis) techniques. The same ana-
ClN2O8 based on its HREIMS. The fragmentation pattern
in the EIMS of 2 is essentially identical with that of 1.
Diagnostic fragment ion peaks at m/z 412/414 (ion a), 280/
282 (ion b), 227 (ion c), and 195/197 (ion d)11 strongly
suggested that the elemental compositions, but not neces-
sarily the gross structures, of the four amino/hydroxy acid
units of 2 and 1 are the same. Although the 1H NMR
spectra of 2 (Table 1) and 12 were comparable, the chemical
shifts of some of the proton signals were significantly
different, particularly those for H-7 and H-8 in unit A.
These NMR differences suggested that the epoxide group
in unit A of 2 has a different stereochemistry. The proposed
structure, including the absolute stereochemistry, was
rigorously established by comparing the physical (optical
1
† Dedicated to the late Dr. D. J ohn Faulkner (Scripps) and the late
Dr. Paul J . Scheuer (Hawaii) for their pioneering work on bioactive marine
natural products.
* To whom correspondence should be addressed. Tel: (808) 956-7232.
Fax: (808) 956-5908. E-mail: moore@gold.chem.hawaii.edu.
rotations, HPLC retention times) and spectral (EIMS, H
NMR, and 13C NMR) data for natural 2 with the data for
the semisynthetic 7S,8S (2), 7R,8S (cryptophycin-39, 5),
and 7S,8R (cryptophycin-77, 6) epoxides. The data fit
10.1021/np0499665 CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/24/2004

1404 J ournal of Natural Products, 2004, Vol. 67, No. 8
Notes
Ta ble 1. 1H NMR Data for Cryptophycin-38, -326, and -327 in CDCl3
δH (mult.; J in Hz
cryptophycin-326
unit
A
position
cryptophycin-38
5.80 (d; 15.4)
6.68 (ddd; 15.4, 9.9, 5.4)
2.65 (dt; -14.5, 9.9-11.2)
2.55 (brdd; -14.5, 5.4)
5.12 (ddd; 11.2, 5.0, 1.6)
1.76 (pd; 7-7.8, 5.0)
1.03 (d; 7.0)
cryptophycin-327
5.81 (d; 11.3)
6.00 (ddd; 11.3, 9.6, 5.1
3.88 (ddd; -15.5, 9.6, 5.1)
2.50 (dt; -15.5, 5.1)
5.05 (dt; 8.1, 5.1)
1.93 (dp; 8.1, 7.4)
1.15 (d; 7.4)
2
3
5.75 (dd; 15.3, 1.1)
6.69 (ddd; 15.3, 10.4, 5.0)
2.45 (ddd; -14.0, 11.3, 10.4)
2.58 (dddd; -14.0, 5.0, 1.7, 1.1)
5.18 (ddd; 11.3, 5.0, 1.7)
1.81 (dqd; 7.5, 6.9, 5.0)
1.15 (d; 6.9)
4 (proS)
4 (proR)
5
6
6-Me
7
8
2.88 (dd; 7.8, 2.0)
3.58 (d; 2.0)
2.93 (dd; 7.5, 1.9)
3.70 (d; 1.9)
2.92 (dd; 7.4, 1.9)
3.71 (d; 1.9)
10/14
7.22 (m)
7.25 (brd; 8.4)
7.25 (m)
11/13
12
2
7.28-7.36 (m)
7.36 (m)
7.37 (m)
4.76 (ddd; 8.7, 7.6, 6.1)
5.80 (d; 8.7)
7.34 (m)
7.34 (m)
4.68 (ddd; 8.3, 7.3, 6.5)
5.72 (d; 8.3)
7.28-7.36 (m)
B
4.81 (m)
5.70 (d; 8.7)
2-NH
3proR
3proS
5
3.03 (dd; -14.4, 7.4)
3.13 (dd; -14.4, 5.5)
7.22 (d; 2.1)
2.93 (dd; -14.4, 7.6)
3.15 (dd; -14.4, 6.1)
7.16 (s)
2.94 (dd; -14.4, 7.3)
3.18 (dd; -14.4, 6.5)
7.24 (d; 2.0)
7-OMe
3.86 (s)
3.86 (s)
3.87 (s)
8
6.83 (d; 8.4)
6.84 (d; 8.6)
9
2
2′
7.07 (dd; 8.4, 2.1)
2.71 (pd; 6.7-7.3, 4.0)
7.16 (s)
2.60 (m)
2.55 (m)
7.10 (dd; 8.6, 2.0)
2.78 (dqd; 9.4, 6.9, 4.5)
C
D
2-Me
3proS
3proR
3-NH
2
3
1.22 (d; 7.3)
1.17 (d; 6.9)
3.29 (dt; -13.5, 6.7)
3.49 (dt; -13.5, 4-5)
6.96 (brt; 5-6.7)
4.90 (dd; 10.0, 3.3)
1.72 (m)
3.35 (dddd; -13.9, 5.5, 5.0, 4.2)
3.63 (dddd; -13.9, 8.9, 5.0, 4.0)
6.86 (t; 5.0)
4.88 (dd; 10.2, 3.3)
1.68 (m)
3.11 (ddd; -13.4, 9.4, 5.6)
3.60 (ddd; -13.4, 6.9, 4.5)
6.99 (dd; 6.9, 5.6)
4.88 (dd; 10.5, 3.2)
1.56 (m)
3′
4
1.49 (m)
1.69 (m)
1.31 (m)
1.68 (m)
1.21 (m)
1.60 (m)
5
0.86 (d; 6.7)
0.85 (d; 6.3)
0.75 (d; 6.5)
5′
0.89 (d; 6.5)
0.84 (d; 6.3)
0.76 (d; 6.5)
perfectly with the data for semisynthetic 2. It was clear
from the vicinal 1H-1H coupling constants that 2 was a
trans-epoxide (J 7,8 ) 2.0 Hz for 2 and 2.0 Hz for 1), not a
cis-epoxide (J 7,8 ) 3.7 Hz for 5 and 4.4 Hz for 6); moreover,
chemical shifts also indicated that 2 was a trans-epoxide
two chlorines have to be in a symmetrical 3,5-dichloro-4-
methoxyphenyl group since the 13C chemical shifts for unit
B of 3 (Table 2) are essentially identical with those found
for unit B of cryptophycin-31 (7).2 The remaining 13C
chemical shifts are the same as those assigned to units A,
C, and D of cryptophycin-21 (8).4 The optical rotation of 3
([R]D +9° in CDCl3) is dextrorotatory, as are 1, 7, and 8
([R]D +34°, +51°, and +40° in MeOH, respectively).
(δH7 ) 2.88 and δH8 ) 3.58 for 2 vs δH7 ) 2.92 and δH8
)
3.69 for 1), not a cis-epoxide (δH7 ) 3.18 and δH8 ) 4.15 for
5 and δH7 ) 3.12 and δH8 ) 4.07 for 6).
Cryptophycin-327 (4) has the molecular composition
C
35H43ClN2O8 based on HRFABMS. Even though many of
the 1H chemical shifts were significantly different, analysis
of the COSYspectrum led us to conclude that units A-D
in 4 and 1 were similar. The major differences in the 1H
NMR spectra of the two compounds were the chemical shift
of C-3 and the magnitude of the vicinal coupling between
H-2 and H-3 in unit A. In 4 the ∆2-double bond was clearly
cis (11.3 Hz), whereas in 1 it was trans (15.5 Hz). Most of
the remaining coupling constants were similar, except for
the ones between H2-4 and H-5 in unit A (11.1 and 1.9 Hz
for 1, but both 5.1 Hz for 4) and between H-2 and H2-3 in
unit C (6.3 and 3.8 Hz for 1, but 9.4 and 4.5 Hz for 4).
An explanation of these coupling constant differences
was obtained by a molecular modeling study of 1 and 4.
At the top of Figure 1 is shown the molecular model (CS
Chem3D Pro, version 4.0) of the preferred conformation of
1 based on X-ray crystallographic and NMR studies.2 The
molecular model of 4 was generated in the following
manner: First the trans NH-CO-CHdCH-CH2 segment
overlapping units A and B was highlighted and then
deleted from the molecular model of 1. Protons automati-
cally appeared where the amide N and methylene C had
previously been. Next a cis NH2-CO-CHdCH-CH3 mol-
ecule was constructed, energy minimized, and moved to the
place where the trans segment had originally been located
in the molecular model of 1. Care was taken to position
Cryptophycin-326 (3) has the molecular formula C34H40
Cl2N2O8 based on HRFABMS. In the EIMS of 3 charac-
teristic fragment ions are observed at m/z 446/448/450 (ion
a), 314/316 (ion b), 229/231 (ion d), and 227 (ion c),
indicating that another chlorine was located in unit B. The
-

Notes
J ournal of Natural Products, 2004, Vol. 67, No. 8 1405
Ta ble 2. 13C NMR Data for Cryptophycin-38, -326, and -327
in CDCl3
δC
unit carbon cryptophycin-38 cryptophycin-326 cryptophycin-327
A
1
2
3
4
165.5
125.3
141.4
36.7
165.7
125.4
141.1
36.7
165.8
125.4
138.0
29.2
5
76.9
76.0
76.4
6
41.0
13.4
40.6
13.4
38.99
13.4
6-Me
7
63.2
62.9
63.9
8
56.1
58.9
59.4
9
137.1
125.4
128.6
128.6
171.0
53.6
136.7
125.3
128.7
128.5
170.9
53.5
136.8
125.9
128.7
128.52
170.9
53.6
10/14
11/13
12
1
B
2
3
35.1
35.0
34.9
4
5
6
7
129.9
131.0
122.5
154.0
56.3
134.6
129.6
129.3
151.1
60.7
129.7
131.1
119.8
154.1
56.1
7-OMe
8
9
1
2
112.3
128.4
175.6
38.3
129.3
129.6
172.3
32.3
112.2
128.50
174.6
39.4
C
D
2-Me
14.1
41.1
170.8
71.5
39.3
24.7
21.4
23.1
13.8
42.3
170.7
70.8
39.02
24.5
3
1
2
3
4
5
5′
34.6
170.2
71.1
39.5
24.4
21.2
22.8
F igu r e 1. Molecular models depicting the preferred conformations of
cryptophycin-1 (1) and cryptophycin-327 (4). The absolute stereochem-
istries of the two molecules are assumed to be identical. In the model
for 1 H-5 in unit A is anti to H-4proR and gauche to H-4proS and in
unit C H-2 is gauche to both protons on C-3. In the model for 4 H-5 in
unit A is gauche to both protons on C-4 and H-2 in unit C is anti to
H-3proS and gauche to H-3proR.
22.8
21.1
(i) Cryptophycin-38 (2) is weaker because the configuration
of the epoxide has to be R,R for maximum activity.
Changing it to S,S as it is in 2 results in a several hundred-
fold loss in cytotoxicity. Cryptophycin-38 (2) shows IC50
values averaging 54.7 nM compared with 0.0092 nM for
1.2 Similarly the semisynthetic S,R (5) and R,S (6) ana-
logues show IC50 values of 72 and 654 nM, respectively.
(ii) Cryptophycin-326 (3) is weaker (IC50 1160 nM) than 1
because addition of a second chlorine to the aromatic ring
in unit B results in at least a 10-fold to 100-fold loss of
cytotoxicity.2 (iii) An E ∆2-double bond in unit A of 1
appears to be important for optimum activity. Changing
it to Z as it is in cryptophycin 327 (4) results in virtual
loss of cytotoxicity (IC50 14 100 nM)
the amide nitrogen and methylene carbon so that they were
coincidental with the positions of the two protons that had
appeared on C-2 of unit B and C-5 of unit A, respectively,
after deletion of the trans NH-CO-CHdCH-CH2 seg-
ment. MM2 energy minimization led to a molecular model
(not shown) in which the conformation of the cis NH-CO-
CHdCH-CH2 segment in unit A was essentially identical
with the one shown in the bottom drawing of Figure 1. The
5.1 Hz coupling constants between H2-4 and H-5 were
entirely consistent with the model, as H-5 was gauche to
both of the protons on C-4. ROESY data also supported
the structure and conformation. The H-5 signal (δ 5.05)
showed NOE cross-peaks to the signals at δ 1.15 (Me on
C-6, 2.7 Å), 1.93 (H-6, 2.3-3.1 Å), 2.50 (H-4proR, 2.5 Å),
2.92 (H-7, 2.6 Å), and 3.88 (H-4proS, 2.4 Å). Moreover, the
H-4proR signal showed additional NOE cross-peaks to the
signals at δ 1.15 (Me on C-6, 2.7 Å) and 6.00 (H-3, 2.5 Å).
The chemical shift for H-4proS was much further downfield
compared with the chemical shifts of H-4proR in 4 and H2-4
in 1 due to deshielding by the C-1 carbonyl of unit A.
In the preferred conformation of 1, H-2 in unit C has to
be gauche to both protons on C-3 to explain the medium to
small sized couplings (6.3 and 3.8 Hz) that are observed.
In the preferred conformation of 4, however, H-2 has to be
anti to one of the protons on C-3 (9.4 Hz) and gauche to
the other one (4.5 Hz). Using the small procedure described
above, the CH2-CH-CH3 segment in unit C (type shown
in preferred conformation of 1) was deleted and replaced
with the segment in which the methine proton is anti to
one of the methylene protons. MM2 energy minimization
led to the conformation for 4 shown at the bottom of Fig-
ure 1.
Exp er im en ta l Section
NMR An a lysis. NMR spectra were determined on an
1
11.75-T instrument operating at 500 MHz for H and 125 MHz
for 13C. 1H chemical shifts are referenced in CDCl3 to residual
CHCl3 (7.26 ppm); 13C chemical shifts are referenced to the
solvent (CDCl3, 77.0 ppm).
Biologica l Ma ter ia l. The cyanobacterium was grown in
mass culture as previously described.2
Extr a ction a n d Isola tion . Lyophilized Nostoc sp. GSV 224
(660 g) was extracted with 4:1 MeCN/CH2Cl2, and the extract
(15.0 g) was subjected to reversed-phase flash column chro-
matography to give 6.0 g of material that contained only
cryptophycins. Reversed-phase HPLC of the mixture of cryp-
tophycins on a 250 × 10 mm Econosil C18 column (10 µm)
with 30:70 H2O/MeCN (flow rate 6.0 mL/min, detection at 254
nm) led to a fraction (tR 44 min, 105 mg) that consisted of three
new cryptophycins (by comparison cryptophycin-1 (1) eluted
at tR 38 min on this column). Normal-phase HPLC of this
fraction on an Econosil silica column (250 × 10 mm, 5 µm)
with 1:1 EtOAc/hexanes (4.0 mL/min) gave cryptophycin-326
(3) (tR 11.5 min, 0.8 mg) and two subfractions eluting at 7 min
The three new analogues are weaker cytotoxins than 1
against the human tumor cell line KB (nasopharyngeal).

1406 J ournal of Natural Products, 2004, Vol. 67, No. 8
Notes
(A) and 20 min (B). HPLC of subfraction A (2 mg) on a smaller
Econosil C18 column (250 × 4.6 mm, 5 µm, 35:65 H2O/MeCN,
1.5 mL/min) yielded cryptophycin-327 (4) (tR 13 min, 0.5 mg).
HPLC of subfraction B on a 250 × 22 mm column (Econosil
C18, 10 µm) with 32.5:67.5 H2O/MeCN (6.0 mL/min) produced
cryptophycin-38 (2) (tR 57 min, 0.6 mg).
Ack n ow led gm en t. This research was supported by Grant
No. CA12623 from the National Cancer Institute, Department
of Health and Human Services. General cytotoxicity assays
were carried out by M. Lieberman, Department of Chemistry,
University of Hawaii.
Cr yptoph ycin -38 (2): [R]D -15° (CHCl3, c 0.4); UV (MeOH)
Su p p or tin g In for m a tion Ava ila ble: Experimental Section for
semisynthesis of 5 and 6. This material is available free of charge via
the Internet at http://pubs.acs.org.
λmax (ꢀ) 202 (131 250), 218 (71 417), 280 (6192) nm; IR (film)
1
νmax 3409, 2959, 1746, 1666, 1537, 1257 cm-1; H NMR data,
Table 1; 13C NMR data, Table 2; EIMS m/z (rel int) 654/656
(11/4), 412/414 (29/7, ion a), 280/282 (14/5, ion b), 253 (6), 227
(43, ion c), 195/197 (68/27, ion d), 184 (25), 155 (51), 141 (35),
91 (100); HREIMS m/z 654.2725 (calcd for C35H4335ClN2O8, m/z
654.2708).
Refer en ces a n d Notes
(1) Trimurtulu, G.; Ohtani, I.; Patterson, G. M. L.; Moore, R. E.; Corbett,
T. H.; Valeriote, F. A.; Demchik, L. J . Am. Chem. Soc. 1994, 116,
4729-4737.
(2) Golakoti, T.; Ogino, J .; Heltzel, C. E.; Husebo, T. L.; J ensen, C. M.;
Larsen, L. K.; Patterson, G. M. L.; Moore, R. E.; Mooberry, S. L.;
Corbett, T. H.; Valeriote, F. A. J . Am. Chem. Soc. 1995, 117, 12030-
12049.
Cr yp top h ycin -326 (3): [R]D +9° (CHCl3, c 0.4); UV (MeOH)
λmax (ꢀ) 208 (39 659), 218 (25 255), 280 (1368) nm; IR (film)
ν
max 3401, 3280, 2958, 2932, 2872, 1738, 1667, 1537, 1268, 1174
(3) Moore, R. E.; Corbett, T. H.; Patterson, G. M. L.; Valeriote, F. A. Curr.
Pharm. Des. 1996, 2, 317-330.
cm-1; 1H NMR data, Table 1; 13C NMR data, Table 2; FABMS
m/z (rel int) 675/677/679 (16/11/3, MH+); HRFABMS m/z
675.2250 (calcd for C34H4135Cl2N2O8, m/z 675.2240).
(4) Barrow, R. A.; Hemscheidt, T.; Liang, J .; Paik, S.; Moore, R. E.; Tius,
M. A. J . Am. Chem. Soc. 1995, 117, 2479-2490.
(5) Subbaaraju, G. V.; Golakoti, T.; Patterson, G. M. L.; Moore, R. E. J .
Nat. Prod. 1997, 60, 302-305.
Cr yp top h ycin -327 (4): [R]D +18° (CHCl3, c 0.3); UV
(MeOH) λmax (ꢀ) 208 (29 091), 218 (18 323), 280 (1815) nm; IR
(film) νmax 3409, 2960, 1726, 1659, 1503, 1259, 1196 cm -1 ; 1H
NMR data, Table 1; 13C NMR data, Table 2; FABMS m/z (rel
int) 655/657 (5/3, MH+); HRFABMS m/z 655.2805 (calcd for
(6) Golakoti, T.; Yoshida, W. Y.; Chaganty, S.; Moore, R. E. Tetrahedron
2000, 56, 9093-9102.
(7) Golakoti, T.; Yoshida, W. Y.; Chaganty, S.; Moore, R. E. J . Nat. Prod.
2001, 64, 54-59.
(8) Hoffmann, D.; Hevel, J . M.; Moore, R. E.; Moore, B. S. Gene 2003,
311, 169-178.
C
35H4435ClN2O8, m/z 655.2786).
(9) Becker, J . E.; Moore R. E.; Moore, B. S. Gene 2004, 325, 35-42.
(10) In the HPLC profile of the mixture of cryptophycins on a 25 cm × 22
mm column of Econosil C18 (10 µm) with 65:35 MeCN/H2O at a flow
rate of 6 mL/min (UV detection at 254 nm), the peak containing 2, 3,
and 4 appears as a shoulder at 62 min (relative tR ) 62/53 ) 1.17
where 53 is the tR for 1 in min.
Sem isyn th esis of 2. A solution of cryptophycin-3 (3 mg)
and m-chloroperbenzoic acid (3.5 mg) in 1 mL of dry CH2Cl2
was stirred at 60 °C for 2 h, then washed successively with
aqueous 5% sodium sulfite (1 mL) and 5% sodium carbonate.
The organic layer was evaporated to obtain the reaction
product, which was separated by reversed-phase HPLC (Econo-
sil C18, 250 × 10 mm, 10 µm) using 30% H2O/CH3CN to give
1 (1.2 mg) and 2 (0.7 mg).
(11) For an explanation of ions a, b, c, and d, refer to Scheme 1 in our
first publication.1
NP0499665