Stereochemical elucidation of new sagittamides CF from a didemnid ascidian

Article abstract of DOI:10.1071/CH10059

Four new minor congeners, sagittamides CF, were isolated from an unidentified Didemnid tunicate that previously afforded sagittamides A and B. The structures were determined by interpretation of spectroscopic data, degradation to amino acids, and comparisons with sagittamide A. An unexpected change in relative configuration of the hexacetoxy C5C10 stereoelement is present in sagittamides D and F. A tentative assignment of configuration was possible through a systematic deduction based on analysis of ¹³C NMR data and symmetry considerations.

Full text of DOI:10.1071/CH10059

RESEARCH FRONT
Full Paper
CSIRO PUBLISHING
www.publish.csiro.au/journals/ajc
Aust. J. Chem. 2010, 63, 935–941
Stereochemical Elucidation of New Sagittamides C–F
from a Didemnid Ascidian
Sarah C. Lievens,A Brandon I. Morinaka,B and Tadeusz F. MolinskiB,C,D
ADepartment of Chemistry, University of California, Davis, 1 Shields Avenue, Davis, CA 95616, USA.
BDepartment of Chemistry and Biochemistry, University of California, San Diego,
9500 Gilman Drive, La Jolla, CA 92093-0358, USA.
CSkaggs School of Pharmacy and Pharmaceutical Sciences, University of California,
San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358, USA.
DCorresponding author. Email: tmolinski@ucsd.edu
Four new minor congeners, sagittamides C–F, were isolated from an unidentified Didemnid tunicate that previously
afforded sagittamides A and B. The structures were determined by interpretation of spectroscopic data, degradation to
amino acids, and comparisons with sagittamide A. An unexpected change in relative configuration of the hexacetoxy
C5–C10 stereoelement is present in sagittamides D and F. A tentative assignment of configuration was possible through
a systematic deduction based on analysis of 13C NMR data and symmetry considerations.
Manuscript received: 29 January 2010.
Manuscript accepted: 31 May 2010.
Introduction
least-squares fit to the predicted values of RDCs.[5] The outcome
of these experiments corrected earlier reported values of 2,3JCH
and unambiguously confirmed the Kishi assignment.
Previously, we reported the isolation and structure elucidation
of long-chain polyacetoxylated α,ω-diamides sagittamidesA (1)
(Fig. 1) and B (2)[1,2] from a didemnid tunicate collected in
Micronesia. The structures of these natural products embody a
C5–C10 stereohexad of six contiguous acetoxyl groups, an oxi-
dation motif not commonly found in other lipid natural products.
The structures are suggestive of polyketide (PK) biosynthe-
sis with incorporation of several hydroxymalonate extender
units.[1] Elucidation of the relative stereochemistry of the C5–
C10 segment in 1[2] was a difficult, non-trivial problem. The
first proposed configuration of anti-anti-anti-syn-anti was based
on progressive stereochemical analysis and comparison with a
series of 16 hexaol peracetates model compounds derived from
d-xylose and d-ribose; however, this assignment differed from
that of Kishi and coworkers who proposed an anti-anti-syn-anti-
syn isomer.[3] Kishi’s independent assignment of configuration
of the stereohexad is based on application of iterative 3JHH vic-
inal coupling constant analysis and total synthesis of 1 and
a diastereomer in which both terminal l-amino acid groups
were replaced with d-lysine and d-ornithine respectively. The
1H NMR chemical shifts of the C5–C10 hexad were identical
to those of the natural product and the absolute configurations
(directionality of C5–C10 stereocentres) were determined by
matching the correct synthetic diastereomer with the natural
Further investigations of the tunicate extract have now
resulted in the isolation of four additional minor constituent
sagittamides C (3), D (4), E (5), and F (6), comprising two
homologues and two stereoisomers of 1. Here we report the
structures 3–6 by analysis of spectroscopic data in compari-
son with those of 1 and degradation and identification of the
constituent amino acids. A tentative stereochemical assignment
of the configuration of the C5–C10 stereohexad in the new
stereoisomers is provided from 13C NMR data and symmetry
considerations, along with an appraisal of 1H NMR JHH analy-
sis in the context of second-order coupling of near-symmetrical
polyol stereoelements.
Results and Discussion
High resolution-fast atom bombardment mass spectrometry
(HR-FABMS) of sagittamide C (3) showed pseudomolecular
ions at m/z 1016.5901 ([M + H]+) and m/z 1038.6 ([M + Na]+)
corresponding to a molecular formula of C50H85N3O18 and it
is therefore a higher homologue of 1 (C48H81N3O18) differing
by two CH2 units. Inspection of the 1H NMR spectrum of 3 in
CD3OD showed very few differences from 1, and the 13C NMR
data of 3 were almost identical with 1 except for the presence of
two additional peaks in the midchain CH2 cluster at δ 30.5 ppm.
Thus, 3 has a longer C28 α,ω-dicarboxyl chain compared with
C26 in 1. COSY and HMBC analysis of 3 showed the same cor-
relations linking the proximal acetoxylated C5 through to the
chain terminus C1 as seen in 1; consequently, the additional two
CH2 groups were placed in the long-chain segment between C11
and C28.
1
product. The 500-MHz H NMR spectrum of co-mixtures of
natural 1 with each of the two synthetic diastereomers (ratio of
2:1) showed only one discrete set of six OAc singlets for the
synthetic l-Lys, l-Orn diastereomer, whereas the mismatched
d-Lys, d-Orn diastereomer showed more than six singlets due to
slight diastereomeric differences. We re-examined the relative
2
configuration of natural 1 by careful remeasurements of JCH
3
1
and JCH using the HSQC-HECADE[4] measurement of H–
Sagittamide D (4), C48H81N3O18 (m/z 988.5605, [M + H]+),
13C residual dipolar couplings (RDCs) in aligned media and a
was isomeric with 1. 1H NMR analysis of 4 confirmed the
© CSIRO 2010
10.1071/CH10059
0004-9425/10/060935

RESEARCH FRONT
936
S. C. Lievens, B. I. Morinaka, and T. F. Molinski
OAc OAc OAc
confirmed the common formula C49H83N3O18. The mass of
compound 5 was greater than 1 by one CH2 unit, and 1H NMR
analysis showed both 1 and 5 had identical signals for the aceto-
xylated C5–C10 hexad. Consequently, the configuration of 5 is
the same as that of 1. The 1H NMR chemical shifts and vicinal J
values of the hexaacetate C5–C10 segment in compound 6 were
the same as those of sagittamide D (4). Therefore, 5 and 6 differ
in the configuration of one or more stereocentres in C5–C10 but
each shares the configuration of their respective homologues 1
and 4.
H
N
5
7
1
2ꢂ COOH
9
8
10
6
OR OR OAc
O
O
COOH
NH2
26
2ꢁ
N
m
n
(28) H
1 R ꢀ Ac, m ꢀ 1, n ꢀ 1 Sagittamide A
2 R ꢀ H, m ꢀ 1, n ꢀ 1
3 R ꢀ Ac, m ꢀ 2, n ꢀ 1
5 R ꢀ Ac, m ꢀ 1, n ꢀ 2
B
C
E
Given that 5 is a homologue of 1 and no methyl branches
or N- or O-methyl groups were present, it appeared the extra
CH2 unit in 5 was associated with a difference in lipid chain
length or one of the amino-acid residues. 2D NMR analysis of
5 shows the same signals associated with a Val end group, but
careful assessment of the HSQC spectrum revealed a new cross-
peak corresponding to an extra CH2 (δC 21.1; δH 1.3, m) that
is consistent with the γ-CH2 of lysine.[6] Exhaustive hydrolysis
of 5 (6 M HCl, 110◦C, 12 h) followed by treatment of the dried
hydrolysate with 5-fluoro-2,4-dinitrophenyl-l-alaninamide
(l-FDAA, l-Marfey’s reagent, 80◦C, 10 min) followed by HPLC
analysis gave peaks that co-eluted with standard l-FDAA-l-
Val and a new peak with a retention time identical with that of
standard l-FDAA-l-Lys adduct,[7] proving that 5 and 6 contain
l-Lys instead of l-Orn in the distal amide group.
OAc OAc OAc
OAc OAc OAc
H
N
COOH
O
O
COOH
NH2
26
N
n
H
Sagittamide D
F
4
6
n ꢀ
n ꢀ
1
2
OAc OAc OAc
5
OMe
10
We addressed the configurational relationship between
stereoisomeric sagittamides A (1) and D (4) in the following
manner. From biosynthetic considerations, it is likely that the
change in configuration in 4 with respect to 1 involves inversion
of a minimal number of stereocentres, possibly only one. The
salient feature of the 1H and 13C NMR spectra of 4 and 6, which
is lacking in 1, is the high degree of symmetry for both 1H and
13C NMR spectra, which reduces the C5–C10 spin systems to
three sets of almost-degenerate chemical shifts. The chemical
shifts of C5, C6, and C7 of 4 (Table 1) differ from each other
significantly, but are virtually identical with their counterparts at
C10, C9, and C8, in that order. This suggests that the change in
configuration produces local symmetry in 4 resulting in either
a local pseudo-C2 axis or a mirror plane σ (pseudo-meso), in
contrast to the lack of symmetry (C1) present in 1. Fig. 2 depicts
the structures of all possible isomers of 1 (Fig. 2a), bearing
symmetrical C5–C10 stereo-hexads, grouped as pseudo-C2 and
pseudo-meso isomers (Fig. 2bꢀ–hꢀ). An analysis of the configu-
ration of 4, assuming parsimonious changes from 1, excludes
most of these permutations and all of their respective mirror
images because they require inversions of three of more centres.
This leaves two possibilities for 4 as an epimer of 1 at either C5
or C10 (Fig. 2b and c respectively).
We favour a C10 epimer of 1 as the configuration for 4
from symmetry considerations and by comparison of 4 with
16 stereoisomeric model compounds used in structure elucida-
tion of sagittamide A (1),[2] including 7, two pseudo-C2 models
8, 9, and two pseudo-meso isomers 10, 11.[8] The 13C NMR
signals of 4 and pseudo-symmetrical isomers of 8–10 were,
as expected, almost identical within each matched pair of sig-
nals C5/C10, C6/C9, and C7/C8 (ꢀδ < 0.1 ppm), but differed
betweenthepairs.Althoughall13CNMRchemicalshiftsforC5–
C10 (seeAccessory Publication, Table S1) occurred in the range
δ 67.2–71.7 ppm, the range differences for C6/C7 and C8/C9
pairs were consistently larger (ꢀδ = 1.9–2.8 ppm) in pseudo-C2
isomers than in pseudo-meso isomers (ꢀδ = 0.1 ppm). Because
the chemical shifts differences for C6/C7 and C8/C9 in sagit-
tamide D (4) are within the larger range (ꢀδ = 2.5 ppm), we may
OAc OAc OAc
O
7
(a)
OAc OAc OAc
5
Rꢂ
R
10
OAc OAc OAc
anti-anti-anti-syn-anti
(5S,6S,7S,8R,9S,10S)
Original assignment
(b)
OAc OAc OAc
5
Rꢂ
R
10
OR OR OAc
anti-anti-syn-anti-syn
(5S,6S,7S,8S,9S,10R)
Kishi assignment (correct)
Fig. 1. The absolute stereostructure of sagittamide A (1). (a) Original
assignment (ref. [2]) and (b) revised assignment (ref. [3]).
presence of the hexad of the contiguous AcO–CH signals and
terminal ornithine and valine residues; however, the stereochem-
istry of the C5–C10 segment was different as indicated by
significant changes in the 1H and 13C NMR chemical shifts. The
1H NMR chemical shifts (600 MHz, CD3OD) of the four inter-
nal OCH proton multiplets at δ 5.42, 5.15, 5.13, and 5.11 ppm in
1 were shifted to δ 5.32, 5.30, 5.15, and 5.13 ppm in 4; similarly,
the 13C NMR signals (CD3OD) for the C5–C10 acetoxylated
CH signals in 1 (δ 73.4, 71.4, 69.2, 68.4, 70.4, 71.9 ppm respec-
tively) now appeared as a symmetrical pattern (δ 73.4, 69.03,
71.0, 71.0, 68.99, 73.6 ppm). The same high symmetry was also
observed when the 1H and 13C NMR spectra of 4 were recorded
in [D6]DMSO (Table 1).
Sagittamides E (5) and F (6) were isomeric with each
other; pseudomolecular ions observed in HR-FABMS at m/z
1002.5764 ([M + H]+) and 1002.5731 ([M + H]+) respectively,

RESEARCH FRONT
Sagittamides C–F
937
Table 1. Partial 1H and 13C NMR data for 1 and 4 (CD3OD)
See Experimental section for a complete listing of values for 4
Carbon no.
1 (ref. [1])
4
A
B,C
D
δH
δC
δH
δC
4a
4b
5
6
7
8
9
10
11
1.59 (m, 1H)
1.63 (m, 1H)
4.92 (m, 1H)
5.11 (dd, 8.3, 4.0, 1H)
5.15 (dd, 8.3, 1.5, 1H)
5.42 (dd, 9.9, 1.5, 1H)
5.13 (dd, 9.9, 1.8, 1H)
4.82 (td, 6.8, 1.8, 1H)
1.42 (m, 2H)
29.1
1.66 (m, 2H)
28.6
73.4
71.4
69.2
68.4
70.4
71.9
31.8
4.82 (m, 1H)E
73.4
69.03G
71.0
71.0
68.99G
73.6
5.15 (dd, 8.9, 2.8, 1H)F
5.33 (dd, 8.9, 1.9, 1H)H
5.32 (dd, 8.9, 1.9, 1H)H
5.15 (dd, 8.9, 2.8, 1H)F
4.80 (dt, 10.7, 2.8, 1H)
1.59 (m, 2H)
28.7
A400 MHz.
B800 MHz.
CJ values from the 800-MHz 1H NMR spectrum. H4 and H11 assignments confirmed from DQFCOSY (500 MHz).
D125 MHz.
EObscured by solvent.
F,G,HInterchangeable pairs.
assign 4 as a pseudo-C2 isomer. The isomers depicted in Fig. 2d
and e were eliminated because they each would require inversion
of three stereocentres of 1 to obtain 4;[9] this narrows the choice
for 4 to a C5 or C10 epimer of 1 (Fig. 2b or c respectively).
Unfortunately, the required models corresponding to Fig. 2b
and c were not available for direct comparison. Nevertheless,
partial comparison of the C8–C10 stereotriad in the model com-
pounds with those of 1 and 4 suggested a reasonably confident
assignment of the stereochemistry. The C7–C10 anti-anti-syn
relative configuration of 7 matches well for 1 (Fig. 3b) but poorly
for the C9–C10 chemical shifts of 4 (Fig. 3a). Therefore, by
elimination, it is likely that C10 in sagittamide D (4) is inverted
with respect to 1, and 4 has the configuration corresponding to
Fig. 2c. The anti-anti-syn-anti-anti relative configuration for 4,
corresponding to Fig. 2c, is also supported by the closer match
of C8–C10 chemical shifts with an anti-anti stereoelement (see
4 and model 11, Fig. 4d).
allowed validation of the assignments by the Kishi method;[3]
specifically, that configuration of stereotriads may be assigned
confidently if the differences of J profiles between the database
and 4 exceed the tolerance ꢁ|ꢀHz| ≥ 3.3 Hz. Four sequential
overlapped stereotriads were matched to predict the relative
stereochemistry of 1;[3a] however, because of the symmetry in
4, only a match with the first of two overlapped stereotriads
for C5–C6–C7–C8 was required; both remaining stereochemi-
cal possibilities (Fig. 2b and c) have anti-syn-anti in the second
stereotriad, C6–C7–C8–C9. Pleasingly, contiguous JHH analysis
of 4 also narrows down to the same two configurations obtained
from 13C NMR analysis; unfortunately, the sequential JHH cou-
plings fall within the tolerance of analysis of C5–C6–C7–C8 for
both anti-anti-syn (Fig. 2c, ꢁ|ꢀHz| = 2.5 Hz) and syn-anti-syn
(Fig. 2b, ꢁ|ꢀHz| = 1.6 Hz).[11] Consequently, interpretation of
contiguous JHH values in 4 is equivocal and, in this case, the pre-
ceding argument for configuration by 13C NMR chemical shift
and symmetry arguments is stronger.
An alternative approach draws on Kishi’s vicinal coupling
constant profile analysis by matching JHH values of contigu-
ous overlapping stereotriads (syn-anti relative configurations)
in acyclic peracetoxy compounds to databases of averaged JHH
ranges obtained from multiple literature sources.[3] At lower
field strength (500 MHz, [D6]DMSO, or CD3OD, Fig. 5), the 1H
NMR chemical shifts of the pairs of signals H7/H8 and H6/H9 in
4 were not resolved. In fact, because of overlapping spin states,
corresponding to the frequencies of the upfield and downfield
multiplet components in coupled H7 and H8, the H6/H9 multi-
plets exhibited characteristics of second-order (virtual) coupling
and it was not possible to obtain reliable δ and J values under
these conditions. It is often overlooked that second-order cou-
pling in 1H NMR spectra of all pseudo-symmetrical stereoseg-
ments will limit not only the Kishi-type JHH analysis, but also
integrated Murata-type J-based analyses.[10] Symmetry places
critical demands for NMR signal dispersion that can sometimes
be achieved at higher fields. The second-order effects in the
1H NMR spectrum of 4 were removed at 800 MHz (CD3OD,
Fig. 5) and partial separation of all four multiplets observed.
Careful measurements of the vicinal coupling constants in 4 for
proton signals H5/H6 (J = 2.8 Hz), H6/H7 (J = 8.9 Hz), H7/H8
(J = 1.9 Hz), H8/H9 (J = 8.9 Hz), and H9/H10 (J = 2.8 Hz)
As noted above, the relative configurations of sagittamides
D (4) and F (6) are identical in the C4–C10 region by NMR;
therefore, we assign both compounds as (5S,6S,7S,8S,9S,10S).
Note this is not a fully rigorous proof because lack of the model
compound with exactly the predicted configuration (cf. Fig. 2c)
prevents direct comparison with 4, and the foregoing stereo-
chemical assignments of 4 and 6 must be considered, at best,
tentative.
Long-chain lipids that are functionalized at both α,ω-
termini are relatively rare in nature. Examples among
marine natural products include the ‘two-headed’ sphingolipids
derived from sponges rhizochalin[12] and isorhizochalin[13]
from Rhizochalina incrustata, oceanapiside from Oceanapia
philipensis,[14] calyxoside from Calyx sp.,[15] and leucettamols
from Leucetta microrhaphis.[16] A variation on the latter theme is
found in rhapsamine, an unusual linear C28 polyene, terminally
substituted by 1,3-diamino-2-propanol groups, from theAntarc-
tic sponge L. leptorhapsis.[17] Long-chain dicarboxylic acid
derivativesareevenlesscommonbutdooccurincertainbacteria.
For example, very-long-chain dimethyl branched C29–C32 α,ω-
dicarboxylic acids comprise some 40% of the fatty acyl mem-
brane lipids of the anaerobic bacterium Thermoanaerobacter

RESEARCH FRONT
938
S. C. Lievens, B. I. Morinaka, and T. F. Molinski
(a)
(aꢂ)
3
2
OAc OAc OAc
OAc OAc OAc
C5
C6
C7
C8
C9
C10
(a)
(b)
C1
5
7
9
5
R
R
8
10
6
10
Rꢂ
Rꢂ
OAc OAc OAc
OAc OAc OAc
1
(5S,6S,7S,8S,9S,10R) 1
(5R,6R,7R,8R,9R,10S) ent-1
0
C2
(b)
(bꢂ)
OAc OAc OAc
OAc OAc OAc
ꢃ1
ꢃ2
ꢃ3
5
5
R
R
C5
C6
C7
C8
C9
C10
10
10
Rꢂ
Rꢂ
Rꢂ
OAc OAc OAc
OAc OAc OAc
3
4
5
6
7
8
9 10 11
3 4 5 6 7 8 9 10 11
(c)
(cꢂ)
OAc OAc OAc
OAc OAc OAc
Carbon number
5
5
C10
R
R
C5
C6
C7
C8
C9
10
10
Rꢂ
Rꢂ
Rꢂ
Fig. 3. Comparison of 13C NMR ꢀδ values for 1, 4, and 7 (125 MHz,
[D6]DMSO, 298 K). (a) ꢀδ = δ(4) − δ(7). (b) ꢀδ = δ(1) − δ(7).
OAc OAc OAc
OAc OAc OAc
(d)
(dꢂ)
OAc OAc OAc
OAc OAc OAc
the PK zwittermicin A,[20] and other examples have since been
described.[21]
5
5
C5
C6
C9
R
R
C7
10
10
Rꢂ
Rꢂ
C8
C10
OAc OAc OAc
OAc OAc OAc
Two explanations may be advanced to explain the epimeric
configurations in the minor congeners 4 and 6. Both substrate
control and enzyme stereospecificity of PKS ketosynthase (KS)
and ketoreductase KR modules determine 1,3-tacticity and 1,2-
relative stereochemistry respectively, in the nascent PK chain
(Fig. 6). Generally, the configuration of the β-carbon in PKS
chain extension is determined by intrinsic properties of the oper-
ative KR domain. If sequential additions of hydroxymalonyl
CoA (Fig. 6i) are syndiotactic (alternating configurations at
the HO-bearing C2 carbon, Fig. 6ii), as they appear to be in
the major natural product sagittamide A (1), stereoregularity
in C9–C10 may be disrupted by possible ‘slippage’ through
enolization and partial epimerization at the α-carbon before
reduction of the intermediate α-hydroxy-β-ketothioester in the
growing chain. Such lack of stereofidelity is perhaps not unex-
pected as hydroxymalonyl thioesters, with lower pKas for the
α-CH than malonyl- and α-methylmalonyl thioesters, are more
prone to spontaneous epimerization. Alternatively, the tacticity
of the first of the three hydroxy-ketide additions to the growing
long-chain acyl thioester may be lax compared with the last two
where preset stereocentres impose order. Other explanations for
the origin of the (AcO–CH)6 hexad (e.g. cytochrome P450 oxi-
dations of a regular malonate-extended PK) seem less appealing
because they do not explain the regular, tight clustering of oxi-
dation states at C5–C10. Further investigations are required to
elaborate the details of the intriguing biosynthesis of 1–6.
(e)
(eꢂ)
OAc OAc OAc
OAc OAc OAc
5
5
C5
C7
C8
R
R
C6
10
10
C9
C10
OAc OAc OAc
OAc OAc OAc
meso
(f)
(fꢂ)
OAc OAc OAc
OAc OAc OAc
5
5
R
R
C6
C7
C5
10
10
Rꢂ
Rꢂ
Rꢂ
Rꢂ
Rꢂ
Rꢂ
C8
OAc OAc OAc
OAc OAc OAc
C9
C10
(g)
(gꢂ)
OAc OAc OAc
OAc OAc OAc
5
5
R
R
C5
C7
C9
10
10
C6
OAc OAc OAc
OAc OAc OAc
C8
C10
(h)
(hꢂ)
OAc OAc OAc
OAc OAc OAc
C6
C8
C5
5
5
7
9
7
9
R
R
8
10
8
6
10
6
Rꢂ
Rꢂ
C6
C8
OAc OAc OAc
OAc OAc OAc
C10
(i)
R
(iꢂ)
OAc OAc OAc
OAc OAc OAc
C7
C8
C5
5
7
5
9
7
9
R
8
10
6
10
8
6
C6
C7
OAc OAc OAc
OAc OAc OAc
C10
Fig. 2. C5–C10 stereohexads of (a) sagittamideA (1) and all stereoisomers
possessing pseudo-symmetrical properties. (b)–(e) Local two-fold rotation
axis (pseudo-C2), or (f)–(h) σ, local mirror plane (pseudo-meso). (aꢀ)–(hꢀ) are
mirror images of (a)–(h) respectively. Locants next to each structure are the
stereocentres in 1 that must be inverted to achieve the depicted configuration.
Conclusion
In conclusion, we have characterized sagittamides C–F (2–6),
four new relatives of sagittamide A (1) comprising a pair of
higher homologues extended by (CH2)2 in the long chain and a
second pair of homologues in which the l-Orn in 1 is replaced
with l-Lys. Sagittamides A–C (1–3) and E (5) share the same
relative configuration at C5–C10, whereas sagittamides D (4)
and F (6) are tentatively assigned as epimeric at C10 with respect
to 1.
ethanolicus 39E, and appear to be biosynthesized by a
unique transmembrane ω-oxidation-coupling mechanism.[18]
The biosynthesis of sagittamides 1–6 is unknown; however, the
contiguous stereoregular hexad of acetoxyls between C5 and
C10 in the structure of 1 and its congeners may have a different
origin to post-translationally modified fatty acids. An alterna-
tive hypothesis invokes polyketide synthase (PKS) biosynthesis
starting with malonyl CoA or acetyl CoA and ketosynthase-
catalyzed chain extension with three hydroxymalonyl CoA units,
with partial reduction of the keto groups, with the remainder of
the chain assembled by malonyl CoA units in the conventional
manner.[19]After our disclosure of the structure of 1,[1] the first
experimental evidence for PK chain extension by a hydroxy-
malonate extender was reported by Thomas and coworkers in
Experimental
General Procedure
Solvents used for purification of 3–6 were of HPLC grade.
Fourier-transform (FT)-IR spectra were measured on a Mattson
Galaxy FTIR spectrometer and optical rotations were measured
on a Jasco DIP-370 polarimeter. NMR spectra were recorded
on various instruments: Varian Inova 400 MHz, Bruker DRX

RESEARCH FRONT
Sagittamides C–F
939
Chain growth
3
3
2
(a)
2
(c)
OAc OAc OAc
5
R
1
0
1
10
Rꢁ
0
OAc OAc OAc
ꢃ1
ꢃ2
ꢃ3
ꢃ4
ꢃ1
ꢃ2
ꢃ3
ꢃ4
1
OH OH OH
R
XꢂS
4
5
6
7
8
9
10 11
4
5
6
7
8
9 10 11
O
O
O
O
ii
C#
3
2
3
2
(b)
(d)
OH
HO
SCoA RꢂS
R
1
1
3X
O
O
O
i
0
0
ꢃ1
ꢃ2
ꢃ3
ꢃ4
ꢃ1
ꢃ2
ꢃ3
ꢃ4
Fig. 6. Hypothetical assembly of sagittamide A (1) from polyketide syn-
thase (PKS)-mediated sequential chain extensions of hydroxymalonyl CoA
to the growing ketide chain R(C=O)SRꢀ. Direction of chain growth is chosen
arbitrarily. Intermediate ii does not imply ordering of reduction steps.
4
5
6
7
8
9
10 11
4
5
6
7
8
9 10 11
OAc OAc OAc
OAc OAc OAc
δ = 49.00 ppm; [D6]DMSO, δ = 40.45 ppm). LR-ESIMS and
MS-MS were obtained by direct infusion into aThermoFinnigan
MSQ quadrupole spectrometer. Reversed-phase HPLC purifica-
tions were carried out on Phenomenex C18 Luna (10 × 250 mm
or 4.6 × 150 mm) columns with flow rates of 3–4 mL min−1 and
coupled to an evaporative light scattering detector or a refrac-
tive index detector. HR-MS measurements were obtained from
University California, Riverside, Mass Spectrometry Facility.
Rꢂ
Rꢂ
R
R
R
OAc OAc OAc
OAc OAc OAc
8
10
OAc OAc OAc
OAc OAc OAc
9
5
Rꢂ
7
Rꢂ
8
6
10
R
OAc OAc OAc
OAc OAc OAc
9
11
R ꢀ n-C3H7, Rꢂ ꢀ (CH2)3COOMe
Animal Material
Fig. 4. Comparison of 13C NMR (125 MHz, [D6]DMSO, 298 K) ꢀδ
values for 4 and models (a) ꢀδ = δ(4) − δ(8). (b) ꢀδ = δ(4) − δ(9).
(c) ꢀδ = δ(4) − δ(10). (d) ꢀδ = δ(4) − δ(11).
An unidentified colonial ascidian (01–17–133) was collected
in 2001 by hand from shallow water (∼1 m) at Arrow Island,
Micronesia, in 2001 and kept frozen until required. The ani-
mal had a typical appearance of other colonial Didemnum
species consisting of thin sheets of dark-brown pigmented tunic
with lighter, orange-tan zooids that were less apparent in the
frozen material. A crepe-paper like texture was apparent in the
frozen material. A preserved type sample (ethanol) is archived
at University of California, San Diego.
(a)
(b)
Extraction and Isolation
The tunicate (261.6 g, frozen) was macerated and suspended
in 1:1 MeOH/CH2Cl2 and stirred overnight before filtering. The
remaining marc was then ground and re-extracted twice more
with 1:1 MeOH/CH2Cl2 and the extracts combined and concen-
trated to a brown solution that was partitioned against hexanes.
The H2O content of the MeOH layer was adjusted to 40% and
the mixture extracted twice with CHCl3. The aqueous methanol
layer was concentrated to remove all organic solvent and the
residual aqueous extract partitioned with n-butanol.
5.34 5.33 5.32 5.31 5.30 5.29
5.16 5.15 5.14 5.13 5.12
d
The CHCl3 layer was dried, then separated using silica chro-
matography eluting with a gradient of 5% MeOH/CH2Cl2 to
100% MeOH. The most polar fraction contained nearly pure
sagittamides. Concurrently, the n-butanol fraction was dried,
then separated by size-exclusion chromatography (LH-20), elut-
ing with MeOH.The sagittamide-containing fractions from both
silica and LH-20 were finally purified by reversed-phase HPLC
(1:1 CH3CN:H2O, 0.5% TFA, 10 × 250 mm C18 column) to
yield sagittamide C (3) (5.9 mg, 0.0023% wet weight), sagit-
tamide D (4) (18.1 mg, 0.0069% wet weight) as a mixture with 1,
Fig. 5. Partial 1H NMR spectra (CD3OD, 23◦C) of sagittamide D (4) at
500 MHz (top) and 800 MHz (bottom). (a) H7/H8; (b) H6/H9. See Table 1
for J values.
600 MHz with 5-mm cryoprobe (UC Davis), orVarian Xsens 500
and Varian 800 spectrometers (both cryoprobes, UC San Diego)
and referenced to internal residual solvent (1H NMR CD2HOD,
δ = 3.30 ppm; [D6]DMSO, δ = 2.50 ppm; 13C NMR CD3OD,

RESEARCH FRONT
940
S. C. Lievens, B. I. Morinaka, and T. F. Molinski
sagittamideE (5)(3.0 mg, 0.0011%wetweight), andsagittamide
F (6)(1.3 mg, 0.00050%wetweight). PuresagittamideD (4)was
obtained by repurification on an analytical C18 reversed-phase
HPLC column.
2.25 (m, H2, H2, H25, H25), 2.15 (m, H3ꢀ), 2.08 (s, OAc), 2.07
(s, OAc), 2.03 (s, OAc), 2.020 (s, OAc), 2.008 (s, OAc), 2.007
(s, OAc), 1.97 (m, H3βꢀ), 1.73 (m, H3β, H3αꢀꢀ, H5ꢀꢀ, H5ꢀꢀ), 1.62
(m, H4β, H24, H24), 1.51 (m, H3α), 1.44 (m, H11, H11), 1.38
(m, H12, H12), 1.28 (br s, H13 through H23, H4ꢀꢀ, H4ꢀꢀ), 0.97
(d, 6.6, H5ꢀ), 0.96 (d, 6.6, H4ꢀ). δC (150 MHz, CD3OD) 176.5
(C26), 175.8 (C1), 174.9 (C1ꢀ), 174.8 (C1ꢀꢀ), 172.5 (OAc), 172.3
(OAc), 171.9 (OAc), 171.6 (OAc), 171.5 (OAc), 171.4 (OAc),
73.2 (C5), 71.7 (C10), 71.2 (C6), 70.3 (C9), 69.0 (C7), 68.2 (C8),
59.0 (C2ꢀ), 59.0 (C2ꢀꢀ), 40.2 (C6ꢀꢀ), 36.9 (C25), 36.1 (C2), 31.9
(C11), 31.6 (C3ꢀ), 30.8–30.4 (C13 through C23), 29.7 (C3ꢀꢀ),
29.0 (C4), 26.9 (C24), 26.2 (C12), 25.2 (C5ꢀꢀ), 22.9 (C3), 21.1
(C4ꢀꢀ), 21.0 (OAc), 20.9 (OAc), 20.88 (OAc), 20.87 (OAc), 20.83
(OAc), 20.76 (OAc), 19.7 (C5ꢀ), 18.4 (C4ꢀ).+m/z (HR-FABMS)
1002.5764 [M + H]+; Calc. for C49H84N3O18, 1002.5744.
Sagittamide F ( 6): δH (600 MHz, CD3OD) 5.32 (dd, 8.7, 1.8,
H7 or H8), 5.30 (dd, 8.7, 1.8, H7 or H8), 5.15 (t, 3.0, H6 or H9),
5.13 (t, 3.0, H6 or H9), 4.83 (m, H5 or H10), 4.80 (tt, 10.5, 2.4,
H5 or H10), 4.43 (dd, 8.4, 5.4, H2ꢀꢀ), 4.32 (dd, 5.4, H2ꢀ), 2.95
(t, 6.3, H5ꢀꢀ, H5ꢀꢀ), 2.25 (m, H2, H2, H25, H25), 2.16 (m, H3ꢀ),
2.067 (s, OAc), 2.063 (s, OAc), 2.029 (s, 2 OAc), 1.99 (OAc),
1.98 (OAc), 1.73 (m, 5H), 1.65 (m, 2H), 1.62 (m, 2H), 1.52 (m,
4H), 1.28 (br s, H13 through H23, H4ꢀꢀ, H4ꢀꢀ), 0.97 (d, 7.2, H4ꢀ),
0.96 (d, 7.2, H5ꢀ). m/z (HR-FABMS) 1002.5731 [M + H]+; Calc.
for C49H84N3O+18, 1002.5744.
Sagittamide C ( 3): colourless glass. δH (600 MHz, CD3OD)
5.42 (dd, 9.9, 1.5, H8), 5.15 (dd, 8.4, 1.8, H7), 5.13 (dd, 9.9, 2.1,
H9), 5.11 (dd, 8.4, 3.6, H6), 4.88 (m, H5), 4.84 (m, H10), 4.42
(dd, 8.1, 5.1, H2ꢀꢀ), 4.32 (d, 6.0, H2ꢀ), 2.95 (br t, 6.6, H5ꢀꢀ), 2.25
(m, H2, H2ꢀ, H27, H27ꢀ), 2.16 (hep, 6.0, H3ꢀ), 2.08 (s, OAc),
2.07 (s, OAc), 2.024 (s, OAc), 2.020 (s, OAc), 2.008 (s, OAc),
2.006 (s, OAc), 1.98 (m, H4βꢀ), 1.73 (m, H3β, H3αꢀ, H3βꢀ, H4αꢀ),
1.60 (m, H4α, H4β, H26α, H26β), 1.52 (m, H3α), 1.43 (m, H11α,
H11β), 1.37 (m, H12α, H12β), 1.27(br s, 24H), 0.97(d, 7.2, H4ꢀ),
0.96 (d, 7.2, H5ꢀ). δC (150 MHz, CD3OD) 176.5 (C28), 175.8
(C1), 174.9 (C1ꢀ), 174.8 (C1ꢀꢀ), 172.4 (OAc), 172.3 (OAc), 171.9
(OAc), 171.6 (OAc), 171.5 (OAc), 171.3 (OAc), 73.2 (C5), 71.7
(C10), 71.2 (C6), 70.3 (C9), 69.0 (C7), 68.2 (C8), 59.0 (C2ꢀ),
52.7 (C2ꢀꢀ), 40.2 (C5ꢀꢀ), 36.8 (C27), 36.1 (C2), 31.9 (C11 or C3ꢀ),
31.6 (C11 or C3ꢀ), 30.8–30.4 (C13 through C26), 29.7 (C3ꢀ),
29.0 (C4), 27.0 (C26), 26.2 (C12), 22.9 (C3), 21.1 (OAc), 20.94
(OAc), 20.90 (OAc), 20.86 (OAc), 20.82 (OAc), 20.75 (OAc),
19.7 (C5ꢀ), 18.4 (C4ꢀ). m/z (HR-FABMS) 1016.5901 [M + H]+;
Calc. for C50H86N3O+18, 1016.5901.
Sagittamide D ( 4): colourless glass. [α]2D4 −31.0 (c 0.92,
MeOH). νmax (ATR)/cm−1 2926, 2854, 1748, 1681, 1539, 1434,
1371, 1214, 1139, 1033 cm−1. δH (600 MHz, [D6]DMSO) 8.02
(d, 7.8, NHꢀꢀ), 7.93 (d, 8.4, NHꢀ), 5.25 (dd, 9.0, 1.2, H7 or H8),
5.23 (dd, 8.4, 1.2, H7 or H8), 5.04 (m, H6 or H9), 5.01 (m, H6
or H9), 4.79 (m, H5 or H10), 4.76 (m, H5 or H10), 4.14 (m,
H), 4.12 (m, H), 2.77 (t, 6.9, H5ꢀꢀ), 2.12 (m, H2, H2, H25, H25,
H3ꢀ), 2.03 (s, 2 OAc), 2.01 (s, OAc), 2.00 (s, OAc), 1.98 (s, OAc),
1.96 (s, OAc), 1.75 (m, H3ꢀꢀ), 1.57 (m, H3, H3, H24, H24, H3ꢀꢀ,
H4ꢀꢀ, H4ꢀꢀ), 1.47 (m, H4, H4, H11), 1.34 (m, H11), 1.23 (br s,
H13 through H23), 0.86 (d, 6.6, H4ꢀ and H5ꢀ). δC (150 MHz,
[D6]DMSO) 174.5 (C1ꢀ), 174.2 (C1ꢀꢀ), 173.2 (C26), 172.8 (C1),
170.9 (OAc), 170.6 (OAc), 170.5 (OAc), 170.4 (OAc), 170.3
(OAc), 170.1 (OAc), 72.3 (C5 and C10), 70.5 (C6 or C9), 70.3
(C6 or C9), 68.0 (C7 and C8), 58.0 (C2ꢀ), 52.3 (C2ꢀꢀ), 39.4 (C5ꢀꢀ),
36.1 (C25), 35.5 (C2), 31.2 (C11), 30.7 (C3ꢀ), 30.0–29.6 (C13
through C23), 29.4 (C4), 29.2 (C3ꢀꢀ), 26.2 (C24), 25.6 (C4ꢀꢀ),
24.8 (C12), 21.7 (C3), 21.55–21.47 (6 OAc), 20.1 (C5ꢀ), 19.0
(C4ꢀ). δH (500 MHz, CD3OD) 5.31 (m, H7 and H8), 5.14 (m,
H6 and H9), 4.81 (m, H10), 4.79 (m, H5), 4.43 (dd, 8.0, 5.2,
H2ꢀꢀ), 4.32 (d, 5.8, H2ꢀ), 2.96 (t, 6.9, H5ꢀꢀ), 2.25 (m, H2, H25),
2.16 (m, H3ꢀ), 2.070 (s, OAc), 2.066 (s, OAc), 2.03 (s, 2 OAc),
2.00 (s, OAc), 1.98 (s, OAc), 1.96 (m, H3ꢀ), 1.73 (m, H3ꢀ, H4ꢀ),
1.66 (m, H4, H3), 1.61 (m, H24), 1.59 (m, H11), 1.52 (m, H4),
1.32 (m, H12, H12), 1.28 (br s, H13 through H23), 0.97 (d, 6.9,
Marfey’s Amino Acid Analysis of Sagittamide E (5)
Sagittamide E (5) was hydrolyzed (6 M HCl, 110◦C, overnight)
and the dried hydrolysate derivatized with Marfey’s reagent[7]
according to the previously described protocol.[1] The l-FDAA
derivatives co-eluted with those of l-lysine and l-valine.
Accessory Publication
1HNMR, COSY, HSQC, andHMBCspectraof 3–6areavailable
on the Journal’s website.
Acknowledgements
We are grateful the Federated States of Micronesia for permission to collect
in territorial waters and Stam for assistance with collections. We thank J.
DeRopp (UC Davis), A. Mrse, and X. Huang (UCSD) for assistance with
NMR measurements andY. X. Su (UCSD) for high-resolution MS measure-
ments. The 500-MHz NMR spectrometers were purchased with funds from
US National Science Foundation (CRIF, CHE0741968). This work was sup-
ported by grants from the National Institutes of Health, US Public Health
Service (CA85602 and CA122256).
References
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OL050717X
1
H5ꢀ), 0.96 (t, 6.9, H4ꢀ). For H NMR (800-MHz partial data
and accurate J values of H5–H10), see Table 1. δC (125 MHz,
CD3OD) 176.5 (C26), 175.8 (C1), 174.8 (C1ꢀ), 174.7 (C1ꢀꢀ),
172.35 (OAc), 172.33 (OAc), 171.7 (OAc), 171.6 (OAc), 171.49
(OAc), 171.46 (OAc), 73.6 (C10), 73.4 (C5), 71.0 (C6 and C9),
69.03 (C7 or C8), 68.99 (C7 or C8), 59.0 (C2ꢀ), 52.7 (C2ꢀꢀ),
40.2 (C5ꢀꢀ), 36.8 (C25), 36.1 (C2), 31.6 (C3ꢀ), 30.8–30.1 (C13
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Sagittamide E ( 5): colourless glass. δH (600 MHz, CD3OD)
5.42 (dd, 9.9, 1.5, H8), 5.15 (dd, 8.4, 1.8, H7), 5.13 (dd, 10.2, 1.8,
H9), 5.11 (dd, 8.4, 3.6, H6), 4.90 (m, H5), 4.84 (m, H10), 4.42
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RESEARCH FRONT
Sagittamides C–F
941
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