Structure elucidation at the nanomole scale. 1. Trisoxazole macrolides and thiazole-containing cyclic peptides from the nudibranch Hexabranchus sanguineus

Article abstract of DOI:10.1021/np8007649

A single specimen of Hexabranchus sanguineus, a nudibranch from the Indo-Pacific that is known to sequester kabiramides B and C and other trisoxazole macrolides, yielded new kabiramide analogues, 9-desmethylkbiramide B and 33-methyltetrahydrohalichondrami

Full text of DOI:10.1021/np8007649

732
J. Nat. Prod. 2009, 72, 732–738
Structure Elucidation at the Nanomole Scale. 1. Trisoxazole Macrolides and
Thiazole-Containing Cyclic Peptides from the Nudibranch Hexabranchus sanguineus
Doralyn S. Dalisay,† Evan W. Rogers,† Arthur S. Edison,‡ and Tadeusz F. Molinski*,†,§
Department of Chemistry and Biochemistry and Skaggs School of Pharmacy and Pharmaceutical Sciences, MC0358, 9500 Gilman DriVe, La
Jolla, California 92093-0358, and National High Magnetic Field Laboratory and Department of Biochemistry & Molecular Biology, UniVersity
of Florida, GainesVille, Florida 32610
ReceiVed December 1, 2008
A single specimen of Hexabranchus sanguineus, a nudibranch from the Indo-Pacific that is known to sequester kabiramides
B and C and other trisoxazole macrolides, yielded new kabiramide analogues, 9-desmethylkbiramide B and
33-methyltetrahydrohalichondramide, and two new unexpected thiazole-containing cyclic peptides in submicromolar
amounts. The structures of these cyclic peptides were determined by analyses of 1D and 2D NMR spectra recorded
1
with a state-of-the-art 1 mm H NMR high-temperature superconducting microcryoprobe, together with mass spectra.
In addition to two proline residues, each peptide contains a thiazole- or oxazole-modified amino acid residue, together
with conventional amino acid residues. All of the amino acid residues were L, as determined by Marfey’s analysis of
the acid hydrolysates of the peptides. This is the first report of cyclic thiazole peptides from H. sanguineus. Since
thiazole-oxazole-modified peptides are typically associated with cyanobacteria and tunicates, the finding may imply a
dietary component of the H. sanguineus that was previously overlooked.
The Indo-Pacific nudibranch Hexabranchus sanguineus (a shell-
less opistobranch mollusk) and its egg masses contain extraordinary
bioactive polyketide macrolide natural products known as “trisox-
azole” macrolides. The three contiguous 2,4-disubstituted oxazole
rings, which constitute the outstanding structural feature of trisox-
azole macrolides, are integrated within a larger macrolide ring that
is further appended by an aliphatic chain terminated by an N-methyl-
N-vinylformamide. The first examples of trisoxazoles, ulapualides
A and B1 and kabiramide C (1), were reported in 1986;2 however,
a subsequent report described the related analogue halichondramide
from the sponge Halichondria sp. collected in Palau.3 At about
the same time, dihydrohalichondramide and tetrahydrohalichon-
dramide were found in specimens of H. sanguineus,4 collected in
Kwajalein atoll, and also in egg masses laid by live individuals in
captivity. Like many dorid nudibranchs, H. sanguineus is a specialist
predator with a spongiverous diet; in an aquarium, the nudibranch
was found to consume only Halichondria containing trisoxazole
macrolides and not sponges of other species or genera.5 These
observations provided strong evidence that H. sanguineus acquires
its suite of trisoxazole compounds from a selective sponge diet and
the first confirmatory evidence of the passage of trisoxazoles
macrolides from sponge to nudibranch to progeny.
Trisoxazole macrolides exhibit extremely potent antifungal and
cytotoxic activities, properties that appear to correlate with their
tight binding to G-actin, depolymerization of F-actin, and disruption
of actin filament formation and organization.6 Finally, trisoxazoles
are concentrated in tissue of Hexabranchus at levels higher than
those found in the sponge and provide the animal with a potent
chemical defense against crustaceans and fish. For example, the
common Pacific wrasse Thalasomma lunare was deterred from
consuming krill pellets that were treated with trisoxazoles
at concentrations as low as 10 ppm.5
sanguineus collected in Fiji uncovered 12 and 27 as the major
antifungal metabolites; however at that time NMR instrumentation
was insufficient to allow investigation of the minor constituents
and the extract fractions were archived (-20 °C) for almost two
decades. In 2007-2008, we gained access to two 600 MHz NMR
spectrometers with highly sensitive probes, a custom-built 1 mm
high-temperature superconducting (HTS) NMR cryoprobe12 (Na-
tional High Magnetic Field Laboratory) and a commercial 1.7 mm
cryoprobe, which allowed full characterization of the very minor
components of H. sanguineus. The HTS cryoprobe was constructed
with four Helmholtz pairs (1H, 2H, 13C, and 15N) of planar sapphire
supporting plates that were coated with a thin film of YBCO
(yttrium-barium-copper oxide). The HTS coils and conventional
cryoprobe preamplifier components are cooled to 20 K using a
commercial cryogenic cooling system.12 The combination of high
intrinsic coil sensitivity, low noise, and very small fill volume
(∼5-10 µL) give 1H NMR spectra with exceptional mass sensitivity
and opens investigations of new natural products with complex
structures from rare sources where only as little as a few nanomoles
may be available.13 In this report, we describe our application of
the microcryoprobes with unmatched sensitivity to elucidate the
structures of the very minor natural products from a single specimen
of H. sanguineus: two new trisoxazole analogues, 9-O-desmethyl
kabiramide B (3) and 33-methyltetrahydrohalichondramide (5),
together with two unexpected thiazole-containing peptides, which
we named sanguinamides A (7) and B (8). These complex structures
were fully characterized from submicromole samples by 1D and
2D 1H and 13C NMR data together with MS and degradative
correlation.
Results and Discussion
Since 1986, over two dozen analogues, including kabiramide B
(2),7 have been described from H. sanguineus8 and other sponges
of the genera Jaspis,9 Mycale,10 and Pachastrissa.11 Our own
investigations of the extract from a single specimen of H.
A single specimen of H. sanguineus was collected by hand in
Fiji (1987) in the Yasawa Island chain. The deep-red acetone extract
of H. sanguineus was partitioned between ethyl acetate and water.
The ethyl acetate-soluble portion was fractionated by silica chro-
matography followed by C18 reversed-phase chromatography to give
the new compounds 9-O-desmethylkabiramide B (3, 0.04% w/w
dry weight), 33-methyltetrahydrohalichondramide (5, 0.07%), and
sanguinamides A (7, 0.023%) and B (8, 0.011%) in addition to the
known compounds 1 (2.2%),2 27 (0.12%), kabiramide D7 (0.24%),
and 33-methyldihydrohalichondramide7 (0.28%).
* To whom correspondence should be addressed. Tel: +1 (858) 551-
1662. Fax: +1 (858) 822-0386. E-mail: tmolinski@ucsd.edu.
† Department of Chemistry and Biochemistry, UC San Diego.
§ Skaggs School of Pharmacy and Pharmaceutical Sciences, UC San
Diego.
‡ University of Florida.
10.1021/np8007649 CCC: $40.75
2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/02/2009

Trisoxazoles and Thiazole Peptides from H. sangineus.
Journal of Natural Products, 2009, Vol. 72, No. 4 733
J coupling. The absolute configurations of several of the trisoxazoles
have been assigned by separate groups using different approaches.
Panek and co-workers assigned mycalolide A by degradation,
synthesis of degradation products, and, ultimately, the natural
product.14 None of the trisoxazole macrolides have delivered
crystals suitable for X-ray analysis; however the excellent work of
Rayment and co-workers provided unambiguous absolute configu-
rations for trisoxazoles by separate single-crystal X-ray analyses6
of G-actin bound with 1, jaspisamide A (6),9 halichondramide,4,5
and ulapualide.15b Not only did this reveal the relationship of the
latter natural products to the marine-derived macrolides reidispon-
giolides and sphinxolides,14 but this milestone work served to
remove stereochemical ambiguities and corrected15b an anomalous
assignment16 of at least one trisoxazole macrolide based on the
“facts and fantasies”17 of untested metal-chelation hypotheses.18
The C-9 configuration changes in different trisoxazoles6 and the
C-9-OMe substitutent may be R or ꢀ; however the configurations
of other methyl-branched stereocenters in trisoxazole families appear
to be invariant. For reasons of optimum deuterium-lock strength,
1
the H NMR spectra of 3 were recorded in CD3CN (600 MHz, 1
mm HTS probe); however NMR data for other trisoxazoles8 have
been mostly reported in CDCl3. Comparisons based on chemical
shift alone may compromise arguments for the C-9 relative
configuration in 3. For example, the polar H-bond acceptor solvent
CD3CN may coordinate the free OH groups and influence the local
conformation and electronic environment around the 1,3-diol moiety
and chemical shifts with respect to 2. In order to resolve this
problem, we examined the Rayment X-ray structures6 of jaspisa-
mide A (6) and kabiramide C (1), which share similar C-6-C-9
segments but have epimeric C-9 configurations. Although both
X-ray structures were determined for G-actin-bound trisoxazoles,
the macrolide ring segments show minimal contacts to protein
residues and their conformations are likely to be similar to the
solution structures. The ring conformations of 1 and 6 (Figure 1)
are remarkably similar; the three contiguous oxazole rings form a
planar wall, tilted approximately at an angle of ∼22° to the averaged
macrolide ring plane defined by the ester CdO group and the
extended (zigzag) C-2-C-9 chain. In both molecules, the C-7-O
bond of the oxygen substituent (OH in 1 and CdO in 6) points
endo to the macrolide ring, and the C-8-Me bond is perpendicular
to the average macrolide plane. The major difference between the
two molecules is the orientation of the C-9 OMe group. In
jaspisamide A (6), the ꢀ-OMe group lies pseudoequatorial, almost
in-plane with the macrolide ring, but in kabiramide C (1), C-9 has
the epimeric configuration with a pseudoaxial R-OMe substitutent
almost anti to the C-8 methyl group. The corresponding dihedral
angles H-8-C-8-C-9-H-9 for 1 and 6 are -78° and +68°,
respectively, and give rise to an H-9 1H NMR signal that is a broad
singlet (δ 5.19, bs) for 1 and a doublet for 6 (δ 4.98, d, J ) 4.4
Hz).9 Since the vicinal coupling constant between H-8 and H-9 in
3 is very small (J ≈ 0 Hz), this compound must have the same
C-9 R-OMe configuration found in 1. Using similar arguments, the
J values and chemical shifts of 4 and 5 were found to be similar to
those of halichondramide,3,4 and we tentatively assigned the C-9
ꢀ-OMe configuration to 5, as depicted here.
Compound 3 was shown to be the homologue of 2 with the
formula C46H67N5O14Na established by HRESIMS (m/z 936.4574,
[M + Na]+) and most likely arises from loss of a C-methyl or an
O-methyl group. As with all trisoxazoles, the NMR spectra were
observed as slowly interconverting rotamers (∼2:1) due to restricted
rotation about the N-methyl-formamide bond. The 1H NMR
spectrum of 3 was similar to that of 2 except only two OMe singlets
(δ 3.28, s; 3.29, s) were present instead of three. The COSY,
HMBC, and HSQC data indicated that 3 had the same carbon
1
framework as 2 including the characteristic H NMR singlets for
the three oxazole rings (δ 7.72, s, H-11; 8.32, s H-14; 8.27, s, H-17).
Since 2 has OMe groups at C-9, C-26, and C-32, one of these must
be replaced by an OH group in 3. The HMBC and HSQC data for
3 showed that the position of the new OH group was at C-9 because
the two OMe signals (δ 3.28, s; 3.29, s) were located unambiguously
at C-26 (δ 56.8, q) and C-32 (δ 60.3, q) through long-range
correlations. The remaining 1H NMR signals of 3 were essentially
identical to those of 2.
Compound 5 has a molecular formula of C45H67N4O12 as
determined by HRESIMS (m/z 855.4738 [M + H]+) with one CH2
unit more than tetrahydrohalichondramide (4).4 The 1H NMR data
of 5 revealed five C-methyl signals (δ 1.02, d, J ) 6.8 Hz; 0.87, d,
J ) 7.2 Hz; 0.84, d, J ) 7.2 Hz; 0.92, d, J ) 6.8 Hz and 1.16, d,
J ) 6.8 Hz) corresponding to C-8-Me, C-23-Me, C-27-Me, C-31-
Further examination of the minor components of H. sanguineus
gave, unexpectedly, two new peptides, 7 and 8. Sanguinamide A
(7) was isolated as a minor component from a fraction containing
the trisoxazole compounds. HRESIMS gave the formula
C37H52N7O6S (m/z 722.3683 [M + H]+) with 16 degrees of
1
Me, and C-33-Me, respectively. The H NMR signal due to H-33
in 5 appeared as a multiplet (δ 2.38, m) and corresponds to
replacement by CH for the two diastereotopic CH2 signals in 4 (δ
2.50, m, 1H; 2.17, m, 1H). Careful analysis of COSY and TOCSY
spectra revealed correlations of the C-33-Me signal (δ 1.16, d, J )
6.8 Hz) to H-32, H-33, H-34, and H-35 and located the additional
C-methyl group at C-33. Thus, 5 has the structure 33-methyltet-
rahydrohalichondramide.
The absolute configurations of 3 and 5 were assigned by
comparisons to the trisoxazoles kabiramide C (1) and jaspisamide
A (6) and deductions based on conformational analysis and 1H-1H
1
unsaturation. The H NMR data of 7 revealed signals typical of a
peptide: four amide NH proton signals (Table 1, δ 9.19, d, J ) 7.2
Hz; 8.78, d, J ) 7.2 Hz; 8.11, d, J ) 7.8 Hz; 6.11, bs) and six
R-CH signals (δ 3.38 d, J ) 7.6 Hz, H-13; 4.22, ddd, J ) 10.1,
5.8, 1.7 Hz, H-18; 4.26, dd, J ) 10.0, 7.2 Hz, H-7; 4.67, d, J )
7.9 Hz, H-2; 4.69, qd, J ) 7.8, 6.4 Hz, H-27; 5.09, t, J ) 7.2 Hz,
H-33). HSQC showed a downfield broad 1H singlet (δ 8.01, s, H-31)

734 Journal of Natural Products, 2009, Vol. 72, No. 4
Dalisay et al.
Table 1. NMR Data of Sanguinamide A,7 (600 MHz, CDCl3)
#
δCa, mult.
δH, mult. (J in Hz)
HMBC
L-Pro-1
1
2
3
170.6, qC
60.8, CH
30.5, CH2
4.67, d (7.9)
1.81, m
1, 6
1, 4, 5
2.68, dd (12.0, 5.2)
1.90, 2.02, m
3.67, 4.00, m
4
5
25.0, CH2
48.1, CH2
2
4
L-Ile-1
6
7
8
9
10
11
NH
174.5, qC
56.3, CH
35.5, CH
25.3, CH2
15.1, CH3
10.3, CH3
4.26, dd (10.0, 7.2)
2.30, m
1.24, 1.70, m
1.02, d (6.8)
0.91, t (7.6)
9.19, d (7.2)
6, 8, 9, 12
8, 10
7, 8, 9
8, 9
12
L-Pro-2
12
13
14
171.50, qC
60.9, CH
30.4, CH2
3.38, d (7.6)
0.93, m
12, 17
12, 16
2.10, dd (12.2, 6.1)
1.70, 1.50, m
3.50, 3.53, m
15
16
22.0, CH2
46.3, CH2
13
15
L-Phe
17
18
19
169.2, qC
54.5, CH
37.9, CH2
4.22, ddd (10.1, 5.8, 1.7)
2.98, dd (11.9, 5.8)
19, 26
17
3.08, dd (11.9, 11.9)
20
21
22
23
NH
134.3, qC
129.3, CH
127.8, CH
129.1, CH
7.16, d (7.10)
7.24, m
7.28, m
20, 22, 23
Figure 1. X-ray structures of trisoxazoles bound to G-actin (data
adapted from Rayment et al., ref 6): (a) jaspisamide A (6), torsional
angles C-8(Me)-C-8-C-9-C-10 ) -57.4°, H-8-C-8-C-9-H-9
) +67.9°; (b) kabiramide C (1), torsional angles C-8(Me)-C-8-C-
9-C-10 ) -67.3°, H-8-C-8-C-9-H-9 ) +75.8°. Side chains
of each compound (upper left) and protein residues have been
removed for clarity.
6.11, br s
L-Ala
26
27
28
171.0, qC
49.1, CH
17.9, CH3
4.69, qd (7.8, 6.4)
1.48, d (6.4)
26, 28, 29
26, 27
NH
8.11, d (7.8)
26, 29
coupled to a 13C signal (δ 123.1, d, C-31) characteristic of C-4 in
a 2-substituted thiazole-4-carboxamide. This was verified by HMBC
data, which showed correlations from H-31 to other thiazole ring
carbon signals at δ 148.5, 160.3, and 168.1.
Thz
29
30
31
32
160.3, qC
148.5, qC
123.1, CH
168.1, qC
8.01, s
29, 30, 32
The identities of six remaining amino acid residuesstwo Pro,
two Ile, Ala, and Pheswere revealed by analysis of COSY,
TOCSY, HSQC, and HMBC data (Table 1). Therefore, sanguina-
mide A (7) is a modified heptapeptide. The amino acid sequence
of 7 was defined from the HMBC correlations between the R-CH
and amide-NH proton signals with the adjacent CdO group. An
HMBC correlation was observed from the R-CH of Pro-1 (H-2)
and the CdO of Ile-1 (δ 174.5, s, C-6). The amide-NH proton of
Ile-1 (δ 9.19) showed a correlation to CdO of Pro-2 (δ 171.5, s,
C-12). In turn, the R-proton of Pro-2 (H-13) showed a cross-peak
to the CdO of Phe (δ 169.2, s, C-17), and the R-CH signal of Phe
(H-18) showed connectivity to the CdO group of Ala (δ 171.0, s,
C-26).
The above HMBC correlations provided the partial structure
Pro1-Ile1-Pro2-Phe-Ala, which was connected to the thiazole-
modified amino acid as follows. The H-31 singlet showed an HMBC
correlation to the Thz CdO (δ 160.3, s, C-29), which in turn was
correlated to 1H NMR signals of the Ala R-CH (H-27) and NH (δ
8.11). The last residue, Ile-2, identified through COSY correlations,
constitutes the thiazole-modified amino acid residue. Mutual
correlations (Figure 2a) were observed from the Ile-2 NH (δ 8.78),
R-CH (H-33), and H-31 signals to the thiazole carbon C-32 (δ
168.1, s). Finally, an HMBC correlation between the amide proton
at δ 8.78 and R-CH signal (H-33) of Ile2-thiazole fragment and
CdO of Pro-1 (δ 170.6, s, C-1) returns to the first residue, closes
the ring, and completes the structure of cyclic peptide 7.
Sanguinamide B (8), C33H43N8O6S2 (HRESIMS m/z 711.2723
[M + H]+), is a larger peptide than 7 with 17 degrees of
L-Ile-2
33
34
35
36
37
NH
56.3, CH
40.9, CH
25.9, CH2
15.0, CH3
10.3, CH3
5.09, dd (7.2, 7.2)
1.80, m
1.17, 1.50, m
0.77, d (6.7)
0.91, t (7.6)
8.78, d (7.2)
1, 32, 34, 35
35
34, 36
33, 34, 35
34, 35
1, 32
a δ’s measured by indirect detection, HSQC, and HMBC (1JCH ) 6
b
Hz). 1Hf13C.
unsaturation. The 1H NMR features of 8 are indicative of differences
in amino acid composition from 7, in particular the presence of
three azole-modified amino acids instead of one. The backbone 1H
NMR signals included three amide-NH protons, confirmed by MS
deuterium exchange (δ 9.60, d, J ) 7.2 Hz; 9.26, d, J ) 6.6 Hz;
8.42, d, J ) 6.6 Hz), and five R-CH proton resonances (δ 4.39, d,
J ) 6.6 Hz, H-7; 4.61, d, J ) 6.0 Hz, H-2; 5.29, dd, J ) 16.8, 7.8
Hz, H-23; 5.56, dq, J ) 7.2, 6.6, Hz, H-32). Evidence for the
presence of three five-membered ring heterocycles include three
1
downfield H NMR singlets (δ 7.95, s, H-16; 8.12, s, H-30; 8.27,
s, H-13), which were coupled (HSQC) to their respective 13C nuclei
(δ 121.8, d, C-16; 124.1 d, C-30; 141.5 d, C-13), accounting for
two thiazoles and one 2-substituted oxazole 4-carboxamide. Long-
range H-H couplings from the two broad singlets H-16 and H-30
to those 13C signals in the two thiazole rings (Table 2) were
identified as before with 7; however the third singlet, H-13, showed
long-range couplings to carbon signals that were significantly shifted

Trisoxazoles and Thiazole Peptides from H. sangineus.
Journal of Natural Products, 2009, Vol. 72, No. 4 735
experiments, and the sequence was solved as before using HMBC
correlations of the R-CH and -NH resonances to the adjacent CdO
13C signals. The -NH of Val (δ 9.26) was correlated to the CdO
of Pro-1 (δ 170.4, s, C-6), and the R-CH of Pro-1 (H-7) was
correlated to the CdO of the oxazole-4-carboxamide (δ 169.2, s,
C-11). The H-16 signal showed an HMBC correlation to hetero-
cyclic 13C nuclei (δ 157.7, C-14; 142.3, C-15; 172.9, C-17),
indicating that the oxazole and thiazole rings in 8 were connected
to each other as shown (Figure 2b). Extending the sequence, the
R-CH of Pro-2 (H-18) was correlated with C-17 (δ 172.9, s),
showing that four of the five cyclic amino acid residues were
contiguous.
Completion of the sequence was done by correlating the R-CH
of Pro-2 (H-18) to the CdO of Leu and the R-CH of Leu to the
CdO of the Thz-2 ring. The thiazole-modified amino acid Ala-
Thz (Thz-1) was established by identifying correlations from R-CH
(H-32), NH, and ꢀ-Me (δ 1.68, d, J ) 6.4 Hz) to 13C resonances
within the heterocyclic ring and C-31 (δ 170.6, Figure 2c), as with
7. Finally, the macrolactam ring of the octapeptide 8 was completed
by connecting the Ala R-CH (H-32) and the amide proton (δ 8.42,
d) to the CdO signal of Val (δ 159.1, s, C-1).
Rotation about tertiary proline amide bonds is restricted, and, in
peptides, the Pro residue may adopt either s-trans or s-cis
conformations. The syn ꢀ-CH2 group is shielded by the CdO group
in s-cis Pro residues with respect to the γ-CH2 group; therefore the
difference in 13C chemical shifts between the Cꢀ and Cγ signals in
Pro amino acid residues (∆δꢀγ ) δꢀ - δγ) in peptides is diagnostic
of s-cis and s-trans conformations of the Pro amide bond; s-cis
Pro residues have a larger difference than s-trans Pro residues.19
The ∆δꢀγ values measured for Pro-1 and Pro-2 in the heptapeptide
7 (5.5 and 8.4 ppm, respectively) indicate the s-cis conformation
for both residues. In contrast, the more flexible octapeptide 8 has
smaller ∆δꢀγ’s for Pro-1 (4.2 ppm) and Pro-2 (2.4 ppm), which is
consequently assigned the s-trans configuration at each residue.
Figure 2. Subunits of sanguinamide A (7, a) and sanguinamide B
1
(8, b,c), showing H-1H COSY and HMBC correlations.
Table 2. 1H and 13C NMR Data of Sanguinamide B, 8 (600
MHz, CDCl3)
#
δCa, mult.
δH, mult. (J in Hz)
L-Val
HMBCb
1
2
3
4
5
159.1, qC
58.7, CH
30.6, CH
20.4, CH3
20.2, CH3
4.61, d (6.0)
2.93, m
0.87, d (6.6)
1.15, d (6.6)
9.26, d (6.6)
1
-NH
2, 3, 6
L-Pro-1
6
7
8
9
170.4, qC
60.9, CH
29.4, CH2
25.2, CH2
45.4, CH2
4.39, d (6.6)
1.74, 2.80, m
2.22, 1.94, m
3.75, 3.34, m
6, 11
6
10
Oxz
11
12
13
14
169.2, qC
136.7, qC
141.5, CH
157.7, qC
The absolute configuration of both 7 and 8 was determined by
total hydrolysis of the peptides followed Marfey’s analysis.20 Given
the known propensity for racemization at R-CH centers of thiazole-
modified peptides, samples of 7 and 8 were split into two pools;
one of which was first subjected to ozonolysis prior to total
hydrolysis (6 M HCl, 110 °C, 16 h) to degrade the heterocyclic
rings. The limited supply of 7 and 8 precluded the standard Marfey’s
method20 and required optimized derivatization followed by sub-
nanomole detection of the each amino acid derivative using
ultrahigh-pressure liquid chromatography-mass spectrometry (UPLC-
MS) and single-ion monitoring (SIM). These criteria were fulfilled,
and complete configurational analysis was achieved on ∼30-50
µg of each peptide; all the amino acid residues in 7 and 8 were
found to have the L-configuration.
8.27, s
12, 14
Thz-1
7.95, s
15
16
17
142.3, qC
121.8, CH
172.9, qC
14, 15, 17
L-Pro-2
18
19
20
21
58.7, CH
31.1, CH2
28.7, CH2
48.0, CH2
4.57, d (6.0)
2.94, 2.07, m
2.10, 1.81, m
3.81, 3.97, m
17, 22
17
L-Leu
22
23
24
25
26
27
-NH
170.8, qC
48.4, CH
41.4, CH
25.6, CH2
23.2, CH3
22.0, CH3
5.29, dd (16.8, 7.8)
2.02, m
1.63, m
0.99, d (6.6)
1.03, d (6.7)
9.60, d (7.2)
22, 28
Some comment is in order with respect to the absolute mass
sensitivity of a 600 MHz spectrometer equipped with a 1 mm HTS
cryoprobe (measurement of compounds 3 and 5) or a commercial
1.7 mm cryoprobe (compounds 7 and 8). Both probes have the
significant advantage of cryocooled (∼20 K) preamplifier and probe
components and fill volumes far smaller than conventional 5 mm
1H inverse-detect cryoprobes (∼600 µL). Mass sensitivity is
inversely proportional to the diameter of the coil, but this increased
mass sensitivity comes at a cost of reduced volume. The smaller
diameter of the HTS 1 mm probe, with a fill volume of ∼5-7 µL,
provides high mass sensitivity but also requires a relatively high
sample concentration. The mass sensitivity of the two probes can
be compared from S/N values of 0.1% ethylbenzene/CDCl3: 292:1
and 1120:1 for the 1 and 1.7 mm probes, respectively. When
normalizing for the total volume of the sample, the 1 mm HTS
probe has a slightly higher mass sensitivity than the 1.7 mm
Thz-2
28
29
30
31
160.1, qC
149.2, qC
124.1, CH
170.6, qC
8.12, s
28, 29, 31
L-Ala
5.56, qd (7.2 6.6)
1.68, d (6.6)
32
33
47.8, CH
24.8, CH3
1, 31
31
-NH
8.42, d (7.2)
1, 31
a δ’s measured by indirect detection, HSQC, and HMBC (1JCH ) 6
b
Hz). 1Hf13C.
downfield (δ 136.7, C-12; 157.7, C-14), indicative of oxazole. Spin
systems for five additional amino acid residuesstwo Pro, Val, Leu,
and Alaswere identified by 1H NMR, COSY, and TOCSY
cryoprobe: 292/7 µL ≈ 42 µL-1 versus 1120/30 µL ≈ 37 µL-1
.
Thus, there is a slight mass sensitivity advantage for the 1 mm
probe, but the larger volume of the 1.7 mm probe provides greater

736 Journal of Natural Products, 2009, Vol. 72, No. 4
Dalisay et al.
under reduced pressure to give a dark orange oil. The second batch of
acetone extract was separated by silica chromatography using a shallow
gradient (1% MeOH/CH2Cl2, 2% MeOH/CH2Cl2, 4% MeOH/CH2Cl2
and 50% MeOH/CH2Cl2), giving six fractions. Fraction 90.001.6 was
purified twice by reversed-phase HPLC (Dynamax, 5 µm, C18 column,
10 × 250 mm, 80:20 MeOH/H2O, 3.5 mL/min, and (Dynamax, 5 µm,
C18 column, 4.6 × 250 mm, 80:20 MeOH/H2O, 1.5 mL/min), yielding
620 µg of pure 2 (tR ) 12.10 min). Fraction 90.001.5 was separated
under reversed-phase HPLC (Dynamax semiprep, 25 × 1 cm; 83:17
MeOH/H2O, 3.5 mL/min) to obtain three fractions. Fraction 90.001.5A
was purified by reversed-phase HPLC (Zorbax Agilent, 5 µm, C18
column, 4.6 × 250 mm, 50:50 CH3CN/H2O), yielding two major
fractions of 1 (16.64 mg) and crude 4. Final purification by reversed-
phase HPLC (Phenylhexyl Luna, 4.6 × 250 mm, 50:50 CH3CN/H2O,
1 mL/min, detection at 230 nm) yielded 3 (620 µg, tR ) 15.2 min),
Table 3. Minimum Inhibitory Activity, MIC (µg/mL), of
Trisoxazole Macrolides against Pathogenic Candida and
Cryptococcus neoformans Strains (positive control, amphotericin
B, AMB)
Candida albicans
C. neoformans
ATCC 14503
C. glabrata
var. grubii
1
0.016
0.50
2.00
0.250
0.016
0.25
0.50
1.00
0.125
0.25
0.125
1.00
2.00
0.50
0.016
2
3
5
AMB
overall sensitivity and can be used to study less concentrated
samples. In practice both probes gave exceptionally high mass
sensitiVity for compounds of relatively high MW and a large number
of resonances. Perhaps more significant is the fact that complete
NMR data sets of submicromolar amounts of molecules 3, 5, 7,
and 8 (C37-C45)sgreater in complexity than the majority of
submicromolar natural products measured with capillary or
microprobes21,13swere easily obtained in time frames similar to
those encountered in conventional NMR. Clearly, this portends
success for natural products discovery that previously was unat-
tainable, particularly applications in characterization of novel drug
leads from rare marine organisms.22
The antifungal mechanism of action of trisoxazole macrolides
is likely related to tight binding to G-actin, depolymerization of
F-actin, and disruption of actin filament formation and organization.6
Compounds 1-3 and 5 and the clinical antifungal agent amphot-
ericin B (AMB) were assayed for antifungal activity against
Candida sp. and Cryptococcus neoformans (Table 3). Compound
1 showed the most potency against all fungal strains tested with
an MIC nearly equal to the AMB. Compound 3 was least inhibitory
against the strains of Candida and Cryptococcus.
additional 1 (5.64 mg, tR ) 16.40 min), and pure 4 (1.30 mg, tR
)
21.04 min). Fraction 90.001.5B was further purified by reversed-phase
HPLC (Phenylhexyl Luna, 4.6 × 250 mm, 50:50 CH3CN/H2O, 1.5 mL/
min) to provide 8 (190 µg, tR ) 11.0 min), 7 (390 µg, tR ) 12.30
min), and 5 (1.3 mg, tR ) 18.0 min).
9-O-Desmethyl kabiramide B (3): colorless film; UV (MeOH) λmax
1
(log ε) 243 nm (4.38); IR (ATR) ν 3200-3600 br, 1618 cm-1; H
NMR (600 MHz, CD3CN, 1 mm HTS probe) δ 8.36 (0.7H, s, H-42),
8.32 (1H, s, H-14), 8.27 (1H, s, H-17), 8.04 (0.3H, s, H-42), 7.72 (1H,
s, H-11), 7.25 (1H, ddd, J ) 14.0, 8.5, 5.8 Hz, H-20), 7.07 (0.3H, d,
J ) 14.0 Hz, H-35), 6.39 (1H, d, J ) 14.0 Hz, H-19), 6.62 (0.7H, d,
J ) 14.0 Hz, H-35), 5.19 (1H, br s, H-9), 5.15 (1H, m, H-3), 5.16
(0.3H, d, J ) 14.0 Hz, H-34), 5.08 (0.7H, d, J ) 14.0 Hz, H-34), 5.07
(1H, dd, J ) 10.7, 7.0 Hz, H-24), 3.99 (1H, m, H-22), 3.92 (1H, br s,
H-7), 3.84 (1H, br s, C-9-OH), 3.50 (1H, br s, C-7-OH), 3.36 (1H, d,
J ) 6.0 Hz, C-22-OH), 3.29 (3H, s, C-32-MeO), 3.28 (3H, s, C-26-
OMe), 3.27 (1H, m, H-32), 3.09 (3H, m, C-35-NMe), 3.06 (1H, m,
H-26), 2.74 (1H, dd, J ) 7.2, 6.5 Hz, H-31), 2.58 (0.3H, m, H-33),
2.52 (1H, m, H-29′), 2.51 (1H, m, H-21′), 2.50 (1H, m, H-29), 2.50
(1H, m, H-2′), 2.45 (0.7H, m, H-33), 2.31 (1H, m, H-21), 2.30 (1H, m,
H-2), 2.15 (1H, dd J ) 8.0, 7.0 Hz, H-8), 1.81 (1H, m, H-5), 1.81
(1H, m, H-6), 1.81 (1H, m, H-6′), 1.72 (1H, m, H-27), 1.71 (1H, m,
H-28), 1.65 (1H, m, H-23), 1.64 (1H, m, H-25′), 1.58 (1H, m, H-4′),
1.50 (1H, m, H-25), 1.31 (1H, m, H-4), 1.22 (1H, m, H-28′), 1.36 (3H,
d, J ) 7.0 Hz, H-41), 0.95 (3H, d, J ) 7.0 Hz, H-36), 0.91 (3H, d, J
) 7.0 Hz, H-40), 0.90 (3H, d, J ) 7.0 Hz, H-38), 0.87 (3H, d, J ) 8.0
Hz, H-37), 0.82 (3H, d, J ) 7.0 Hz, H-39); 13C NMR (indirect detection,
HSQC, and HMBC (JCH ) 6 Hz), 600 MHz) δ 214.1 (C, C-30), 175.8
(C, C-1), 163.5 (NHCHO), 145.4 (CH, C-20), 138.3 (CH, C-17), 137.9
(CH, C-14), 136.9 (C, C-10), 135.9 (CH, C-11), 131.4 (C, C-13), 130.7
(C, C-16), 111.4 (113.7*) (CH, C-34), 88.4 (89.6*) (CH, C-32), 82.02
(CH, C-26), 74.5 (CH, C-24), 74.0 (CH, C-9), 69.5 (CH, C-22), 68.7
(CH, C-7), 67.6 (CH, C-3), 60.3 (C-32-OMe), 56.8 (C-26-OMe), 49.7
(CH, C-31), 45.1 (CH2, C-4), 43.1 (CH2, C-6), 42.9 (CH, C-23), 41.3
(CH2, C-29), 41.2 (CH2, C-2), 38.3 (CH, C-8), 38.1 (38.0*) (CH, C-33),
36.1 (CH2, C-21), 35.0 (CH, C-27), 33.7 (CH2, C-25), 27.7 (NMe),
26.5 (CH, C-5), 25.6 (CH2, C-28), 18.3 (19.3*) (CH3, C-41), 18.1 (CH3,
C-36), 15.7 (CH3, C-40), 15.5 (CH3, C-39), 9.5 (CH3, C-38) (*minor
isomer); HREIMS m/z 936.4574 [M + Na]+ (calcd for C46H67N5O14Na,
936.4582).
Two new antifungal trisoxazole macrolides, 3 and 5, were
isolated from H. sanguineus in addition to two unexpected cyclic
peptides, 7 and 8. The structures of all four compounds were solved
by MS and NMR, aided by data collected on extremely mass-
sensitive NMR cryo-microprobes with limited materials at the
nanomole scale. Cyclic peptides 7 and 8 have not been found in
sponges consumed by H. sanguineus, suggesting either a different
as-yet unidentified dietary source or de noVo biosynthesis.
Experimental Section
General Experimental Procedures. All solvents for HPLC purifica-
tion were HPLC grade. CD spectra were recorded on a Jasco J810
spectropolarimeter in 0.2 cm quartz cells at 23 °C unless otherwise
stated. UV-vis spectra were recorded in a dual-beam Jasco V630
spectrometer in 1 cm quartz cells. 1H and 13C NMR spectra were
recorded in CDCl3 using either a Varian Mercury-400 (400 MHz),
Varian Unity-500 (500 MHz), Bruker DMX-600 (600 MHz) equipped
with a 1.7 mm {13C}1H CPTCI probe, or a custom built high-
temperature superconducting probe (1 mm {13C,15N}1H HTS, 600 MHz;
design and performance features of this probe are described else-
where).12 NMR spectra were measured in CDCl3 and referenced to
residual solvent signals (1H, δ 7.26 ppm; 13C, δ 77.16 ppm). HRMS
measurements were measured at The Scripps Research Institute (TOF-
MS) or University of California, San Diego (EI-MS) mass spectrometry
facilities. IR spectra were recorded on thin films using a Jasco 4100
FTIR and attenuated total reflectance (ATR, 3 mm ZnSe) plate. LCMS
was carried out on a ThermoFisher Accela UPLC coupled to an MSQ
single quadrupole mass spectrometer operating in positive ion mode,
unless otherwise stated. Semipreparative HPLC was carried out on a
Varian SD200 system equipped with a dual-pump and UV-1 UV
detector under specified conditions.
33-Methyl tetrahydrohalichondramide (5): colorless film; UV
(MeOH) λmax (log ε) 233 nm (4.53); IR (ATR) ν 3200-3600 br, 1584
cm-1; 1H NMR 600 MHz (CDCl3) δ 8.28 (0.7H, s, H-41), 8.10 (1H, s,
H-17), 8.07 (0.3H, s, H-41), 8.05 (1H, m, H-14), 7.63 (1H, s, H-11),
7.13 (0.3H, d, J ) 14.0 Hz, H-35), 6.99 (1H, dt, J ) 16.0, 8.0 Hz,
H-20), 6.46 (1H, d, J ) 0.7 Hz, H-35), 6.43 (1H, d, J ) 16.0 Hz,
H-19), 5.20 (1H, m, H-24), 5.09 (1H, m, H-34), 4.39 (1H, d, J ) 3.0
Hz, H-9), 4.22 (1H, m, H-3), 3.93 (1H, m, H-7), 3.30 (1H, m, H-32),
3.50 (3H, s, C-9-OMe), 3.35 (3H, s, C-26-OMe), 3.34 (3H, s, C-32-
OMe), 3.04 (3H, s, NMe), 2.97 (1H, br d, J ) 10.0 Hz, H-26), 2.64
(1H, dq, J ) 9.0, 7.0 Hz, H-31), 2.69 (1H, m, H-6), 2.67 (1H, m, H-6′),
2.53 (1H, m, H-2), 2.48 (1H, m, H-21), 2.48 (2H, m, H-29), 2.43 (1H,
br qd, J ) 7.0, 4.5 Hz, H-8), 2.38 (1H, m, H-33), 2.26 (1H, m, H-21′),
1.85 (2H, m, H-5), 1.79 (1H, m, H-28′), 1.77 (2H, m, H-23), 1.71 (2H,
m, H-27), 1.71 (1H, m, H-4), 1.71 (1H, m, H-22′), 1.61 (1H, m, H-4′),
1.56 (1H, m, H-25), 1.34 (1H, m, H-22′), 1.16 (3H, d, J ) 7.0 Hz,
H-40), 1.02 (3H, d, J ) 7.0 Hz, H-36), 0.92 (3H, d, J ) 7.0 Hz, H-39),
0.87 (3H, d, J ) 7.0 Hz, H-37), 0.84 (3H, d, J ) 7.0 Hz, H-38); 13C
NMR (indirect detection, HSQC, and HMBC (JCH ) 8 Hz), 600 MHz)
δ 213.6 (C, C-30), 173.2 (C, C-1), 162.1(160.8*) (NHCHO), 162.0
Extraction and Isolation. The nudibranch Hexabranchus sanguineus
was collected in the Yasawa Islands, Fiji, in 1987. The sample (N )
1) was immediately frozen and stored at -20 °C until extraction (∼12
months). Frozen tissue was thawed and extracted with acetone (2 times),
and each acetone extract was filtered and concentrated before partition-
ing between EtOAc and water. The EtOAc layers were concentrated

Trisoxazoles and Thiazole Peptides from H. sangineus.
Journal of Natural Products, 2009, Vol. 72, No. 4 737
(C, C-18), 156.3 (C, C-15), 155.0 (C, C-12), 143.2 (CH, C-20), 141.3
(C, C-10), 137.7 (CH, C-17), 137.4 (CH, C-14), 136.7 (CH, C-11),
130.9 (C, C-13), 129.9 (124.6*) (CH, C-35), 129.7 (C, C-16), 116.3
(CH, C-19), 111.0 (113.0*) (CH, C-34), 82.4 (CH, C-32), 82.4 (CH,
C-9), 81.9 (CH, C-26), 73.9 (CH, C-24), 71.0 (CH, C-7), 68.7 (CH,
C-3), 61.2 (C-9-OMe), 58.9 (C-26-OMe), 57.9 (C-32-OMe), 48.9 (CH,
C-31), 42.5 (CH2, C-2), 42.1 (CH2, C-29), 38.8 (CH, C-8), 37.6 (CH,
C-33), 36.7 (CH2, C-4), 35.7 (CH, C-23), 34.7 (CH, C-27), 33.3 (CH2,
C-6), 32.1 (CH2, C-25), 31.5 (CH2, C-22), 28.8 (CH2, C-21), 27.6
(33.4*) (NMe), 25.3 (CH2, C-28), 22.4 (CH2, C-5), 19.4 (CH3, C-40),
15.6 (CH3, C-38), 13.7 (CH3, C-37), 13.3 (CH3, C-39), 9.1 (CH3, C-36)
(*minor isomer); HREIMS m/z 855.4738 [M + H]+ (calcd for
C45H67N4O12, 855.4750).
neoformans var. gattii. The fungi were grown and maintained in
Sabouraud dextrose agar, SDA plates (BBL, 211584), and incubated
at 30 °C for 24 h (Candida sp.) or 48 h (C. neoformans). The in
Vitro susceptibility of each compound was determined by the broth
microdilution method according to the guidelines of the National
Committee for Clinical Laboratory Standards (NCCLS).23 Briefly,
2-fold serial dilutions of compounds were prepared in 96-well
microtiter plates (Corning Incorporated, 3595) from stock solutions
in an RPMI-1640 broth medium (Sigma) buffered to a final pH of
7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS; Sigma)
to a final volume of 100 µL. A stock solution was prepared in
dimethyl sulfoxide (DMSO, Sigma) for the various compounds and
for amphotericin B, AMB (Sigma), which was used as positive
control. The final drug concentrations tested were from 0.062 to 64
µg/mL and from 0.0078 to 8 µg/mL for amphotericin B. All fungal
strains were tested in replicate in each run of the experiments. Cell
growth was determined by the OD at 600 nm using a Spectramax
Plus 384 microplate reader (Molecular Devices, CA). The MIC end
point was defined as the lowest concentration with complete (g90%)
growth inhibition.
Sanguinamide A (7): colorless film; IR (ATR) ν 2922, 2852, 1733,
1
1646, 1541 cm-1; CD (MeOH) λmax (∆ε) 224 nm (-14.5); H (600
MHz, 1.7 mm probe) and 13C NMR, see Table 1; HREIMS m/z
722.3683 [M + H]+ (calcd for C37H52N7O6S, 722.3694)
Sanguinamide B (8): colorless film; UV (MeOH) λmax (log ε) 250
nm (4.7); IR (ATR) ν 2980, 2357, 1589 cm-1; CD (MeOH) λmax (∆ε)
1
235 nm (-4.30); H (600 MHz, 1.7 mm probe) and 13C NMR, see
Table 2; HREIMS m/z 711.2723 [M + H]+ (calcd for C33H43N8O6S2,
711.2723).
Acknowledgment. We are grateful to T. M. Zabriskie (Oregon
State University) for collection of H. sanguineus, C. M. Ireland
(University of Utah) for expedition logistics, T. Ku¨hn (Bruker,
Switzerland) for some of the 1.7 mm cryoprobe NMR data, and
Brandon I. Morinaka for helpful discussions. We thank J. Pawlik
(University of North Carolina, Wilmington) for the image of H.
sanguineus (graphic abstract). Mass spectra were provided by Scripps
Research Institute MS Facility and Y. Su (UCSD MS Facility). This
research was supported by grants from the NIH (RO1 AI 039987,
CA122256 to T.F.M.) and National High Magnetic Field Laboratory
support (to A.S.E.). The 1 mm HTS instrument time was provided
by the NSF-funded NHMFL external user program.
Determination of Absolute Configuration of 7 and 8. (a) Ozo-
nolysis and Hydrolysis of Peptides. Samples of peptide were split
into two pools, A and B. The first pool (A) was ozonolyzed prior to
hydrolysis as follows: 7 (50 µg) and 8 (30 µg) were individually
suspended in ozone-saturated MeOH (150 µL) at -78 °C for 5 min
before concentration of the solvent under a N2 stream at -78 °C to
remove ozone, then to dryness at 0 °C. Pool B samples, 7 (50 µg),
8 (30 µg), and ozonolyzed samples from pool A were each
individually resuspended in degassed, freshly distilled constant-
boiling 6 N HCl (200 µL) and heated in a flame-sealed glass tube
at 110 °C for 15 h. The mixtures were concentrated to dryness under
a stream of N2 and redissolved in 100 µL of H2O.
1
20
Supporting Information Available: H NMR, 2D NMR, and CD
(b) Marfey’s Derivatization with L-FDLA. Each of the above
spectra of 3, 5, 7, and 8. This material is available free of charge via
the Internet at http://pubs.acs.org.
hydrolysates was treated with a solution of 2,4-dinitrophenyl-5-fluoro-
L-leucinamide (100 µL, 1% w/v in acetone), followed by 1.0 M
NaHCO3 (25 µL), then heated in a sealed tube at 80 °C for 10 min.
The mixture was cooled and quenched with 1.0 M HCl (25 µL).
Authentic L- and D-amino acid standards were treated under identical
conditions.
References and Notes
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(c) LC-MS Analysis. The Marfey’s derivatives (see above) were
analyzed by LC-MS using a ThermoElectron Accela series ultrahigh-
pressure liquid chromatograph (UPLC) with a Thermo Hypersil Gold
C-18 column (50 mm × 2.1 mm, 1.9 µm) or Agilent Zorbax SB-
Aq C18 column (250 mm × 4.6 mm, 5 µm) connected to a PDA
and ThermoFinnigan MSQ quadrapole mass spectrometer. LC
parameters were as follows: Hypersil: flow rate 0.5 mL/min, initial
80% solvent A (H2O + 0.1% formic acid), 20% solvent B
(acetonitrile), hold 0.3 min, at 9 min 40% A, at 10 min 100% B
hold for 3 min, at 14 min 80% A hold for 2 min; Zorbax SB-Aq:
flow rate 1.0 mL/min, initial 95% solvent A (H2O + 0.1% formic
acid) 5% solvent B (acetonitrile), hold 2 min, at 65 min 40% A, at
67 min 100% B hold for 5 min, at 73 min 95% A hold for 5 min.
Injection volume was 3 µL. PDA parameters were as follows:
channel A 340 nm, channel B 254 nm. MSQ parameters were as
follows: ESI-MS, selected ion monitoring at m/z 384.15, 410.17,
412.18, 426.20, 460.18 [M + H]+, span 1.5 amu, dwell 0.6 s, cone
75 V, probe temperature 450 °C. Retention times for the amino acids
on the Hypersil in min tR) (L/D) were as follows: alanine (4.28/
5.16), proline (4.32/4.99), valine (5.07/6.61), phenylalanine (5.85/
7.12), allo-isoleucine (5.64/7.26), isoleucine (5.67/7.36), leucine
(5.75/7.39). Because of overlap of peaks of the allo-isoleucine and
isoleucine FDLA derivatives on the C18 Hypersil column, these
substituted amino acids were analyzed on the Zorbax SB-Aq column,
which gave the following retention times: allo-isoleucine (55.79/
62.38), isoleucine (56.17/62.42). Co-injections of L-allo-isoleucine
and L-isoleucine FDLA derivatives with the corresponding deriva-
tives from 7 and 8 were used to verify the assignments.
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measurements suggests that trisoxazole macrolides do not chelate
divalent metal ions and, with the exception of Hg2+, ligate poorly with
In Vitro Antifungal Testing. The fungal isolates used in this study
were strains of Candida albicans (2 clinical isolates that are
fluconazole-resistant, strain UCDFR1 and 96-489, and a reference
strain, ATCC 14503) and clinical isolates of Candida glabrata,
Candida krusei, Cryptococcus neoformans var. grubii, and C.

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NP8007649