Thiazoline peptides and a tris-phenethyl urea from Didemnum molle with Anti-HIV activity

Article abstract of DOI:10.1021/np300270p

As part of our screening for anti-HIV agents from marine invertebrates, the MeOH extract of Didemnum molle was tested and showed moderate in vitro anti-HIV activity. Bioassay-guided fractionation of a large-scale extract allowed the identification of two new cyclopeptides, mollamides E and F (1 and 2), and one new tris-phenethyl urea, molleurea A (3). The absolute configurations were established using the advanced Marfey's method. The three compounds were evaluated for anti-HIV activity in both an HIV integrase inhibition assay and a cytoprotective cell-based assay. Compound 2 was active in both assays with IC₅₀ values of 39 and 78 μM, respectively. Compound 3 was active only in the cytoprotective cell-based assay, with an IC₅₀ value of 60 μM.

Full text of DOI:10.1021/np300270p

Article
pubs.acs.org/jnp
Thiazoline Peptides and a Tris-Phenethyl Urea from Didemnum molle
with Anti-HIV Activity
Zhenyu Lu,† Mary Kay Harper,† Christopher D. Pond,‡ Louis R. Barrows,‡ Chris M. Ireland,†
and Ryan M. Van Wagoner*,†
‡
†Department of Medicinal Chemistry and Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah
84112, United States
S
* Supporting Information
ABSTRACT: As part of our screening for anti-HIV agents
from marine invertebrates, the MeOH extract of Didemnum
molle was tested and showed moderate in vitro anti-HIV
activity. Bioassay-guided fractionation of a large-scale extract
allowed the identification of two new cyclopeptides,
mollamides E and F (1 and 2), and one new tris-phenethyl
urea, molleurea A (3). The absolute configurations were
established using the advanced Marfey’s method. The three
compounds were evaluated for anti-HIV activity in both an HIV integrase inhibition assay and a cytoprotective cell-based assay.
Compound 2 was active in both assays with IC50 values of 39 and 78 μM, respectively. Compound 3 was active only in the
cytoprotective cell-based assay, with an IC50 value of 60 μM.
uman immunodeficiency virus (HIV) was first estab-
peptides, and they used this information to engineer the
Hlished as the causative agent of acquired immunodefi- production of novel related peptides in E. coli.6
ciency syndrome (AIDS) over 20 years ago.1,2 In the past
In the process of bioassay-guided fractionation of the extract
of D. molle, we identified three compounds. Interestingly, two
of the compounds (1 and 2) corresponded to peptides that
were initially identified by genome sequencing of Prochloron
spp. and their predicted products detected by LC-MS analysis
of a single ascidian colony (1)7a or of E. coli heterologously
expressing the corresponding biosynthetic pathway (2).7b
However, this report marks the first time these compounds
have been fully characterized as isolated natural products.
These peptides were shown to be ribosomally produced with all
residues presumably having the natural L-configuration. Using
the advanced Marfey’s method we found, however, that the
thiazoline and phenylalanine residues in both 1 and 2 were
present predominantly in a D-configuration. Herein we report
the isolation and structure elucidation of compounds 1−3, as
well as their HIV-1 inhibitory activity.
decade, clinical treatments of HIV-infected patients with
inhibitors of reverse transcriptase and protease have led to
significant improvements in reducing the viral load and the
progression of AIDS. However, these treatments can become
ineffective due to rapid mutations of the virus. HIV-1 integrase
is an enzyme that is critical for integration of the HIV genome
into the host genome.3 This process is unique to the virus and
is absent in the host and, therefore, presents an attractive target
for the development of single and/or combination anti-HIV
therapy.
As part of an International Cooperative Biodiversity Group
(ICBG) involving a collaboration of institutions in Papua New
Guinea and the United States, we have established a natural
product-based antiviral drug discovery program specifically
targeting HIV. Organisms of interest have included plants,
marine invertebrates, and endophytic fungi. During an initial
screen, a MeOH extract prepared from the ascidian Didemnum
molle collected from Papua New Guinea was shown to inhibit
HIV-1 replication at 100 μg/mL, while being devoid of
cytoxicity at this concentration. D. molle is known for producing
cyclic hexa-, hepta-, and octapeptides characterized by an
alternating sequence of thiazole, thiazoline, or oxazoline
heterocycles and hydrophobic amino acids, many of which
show remarkably high levels of cytotoxicity.4 Researchers have
speculated that these peptides might be biosynthesized by
symbiotic prochlorophytes. Recently, Schmidt et al. localized
the genes responsible for the biosynthesis of patellamide to the
symbiont Prochloron didemni.5 They also found that Prochloron
spp. generate a diverse library of patellamides and related cyclic
RESULTS AND DISCUSSION
■
The molecular formula of C35H50N6O7S for compound 1 was
provided by HRESIMS and supported by NMR data (Table 1).
1
The H and 13C NMR spectra indicated peptidic metabolites
due to the presence of four NH doublets (δH 6.62−9.28), six
Hα multiplets (δH 3.97−5.40), and six putative amide carbonyl
13C signals (δC 168−175).
Detailed analysis of the 1D and 2D NMR data of 1
established six amino acid residues: phenylalanine, proline,
serine, leucine, threonine, and cysteine present as a thiazoline
Received: April 12, 2012
Published: July 30, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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Table 1. NMR Data for 1 in DMSO-d6 (1H 500 MHz, 13C
125 MHz)
position
δC
δH (J in Hz)
Pro
HMBC
ROESY
1
2
169.6, C
60.0, CH
4.67, d (7.0)
1, 3, 4, 5,
1Ser
3, 2Ser, 3Phe
2, 4, 5
3
4
5
29.0, CH2 1.60, m
2.17, m
1, 2, 4, 5
20.7, CH2 0.48, m
1.49, m
2, 3
3, 5
3, 4
(Tzn). In addition, a reversed-isoprene functionality attached to
the Thr residue was apparent (Table 1).
45.7, CH2 2.93, br t (10.0) 4, 3, 1Ser
The amino acid sequence of 1 was assigned from a
combination of inter-residue ROESY and HMBC correlations
(Table 1). The Hα of proline (δH 4.67) showed an HMBC
correlation to the serine carbonyl carbon (δC 168.6) and
ROESY correlations to both Hα of serine (δH 4.08) and Hβ of
phenylalanine (δH 3.57). Hα (δH 4.08) and NH (δH 9.07) of
serine both showed HMBC correlations to the leucine carbonyl
carbon (δC 172.7). NH (δH 6.62) of leucine showed ROESY
correlations to NH (δH 7.37) of threonine. Hα (δH 3.97) of
threonine showed an HMBC correlation to the thiazoline
carbonyl carbon (δC 170.3). Hα (δH 5.40) of thiazoline showed
an HMBC correlation to the phenylalanine thioimide carbon
(δC 175.1). Analysis of the MS-MS fragmentation of 1
identified several fragments consistent with the sequence as
deduced by NMR (Figure S17; Table S1). The sequence of
residues for this peptide is consistent with the peptide
mollamide E as predicted by genetic sequencing and confirmed
by mass spectrometry.7a Hence we have used that name for 1.
The absolute configurations of the amino acid residues in 1
were established by acid hydrolysis (under argon) and
derivatization with Marfey’s reagent8,9 followed by comparative
LC-MS analysis with derivatized standard D- and L-amino acids.
It should be noted that the acid conditions used for hydrolysis
simultaneously promote the cleavage of the reverse-prenylated
ether group in 1. The analysis established the L-configuration
for Cα of proline, serine, leucine, and threonine. To determine
the configuration of Cβ of prenyl-Thr, derivatization of standard
L-Thr and L-allo-Thr was carried out, and the resulting elution
profiles were compared against the corresponding residues
from the peptide hydrolysate. Co-injection analysis with the
two standards showed the peptide to contain exclusively L-Thr.
Two peaks were observed for the phenylalanine residue
corresponding to L-Phe and D-Phe at a ratio of 3:7, respectively
(Figure S16). It has been reported that the thiazoline-based
amino acid in cyclic peptides may undergo racemization during
hydrolysis.10,11 Given the 40% enantiomeric excess observered
for D-Phe in the hydrolysate and absence of signs of
stereoisomerism in the NMR spectra of the intact peptide,
the natural product most likely contains exclusively D-Phe at
this position. In order to determine the configuration of Cα of
the thiazoline moiety, 1 was first hydrolyzed under argon. The
hydrolysis product was derivatized with cystamine and then
derivatized with Marfey’s reagent. D- and L-Cysteine standards
were processed in the same manner. The LC-MS analysis
revealed that the product derived from 1 had the same
retention time as the D-cysteine standard (Figure S15). Finally,
the configuration of the Ser−Pro peptide bond was examined.
A large difference in chemical shift between Cβ and Cγ of Pro
(ΔδC = +8.3 ppm) and the presence of ROESY correlations
between Hα of Ser and Hα of Pro are both consistent with a cis
geometry for the Ser−Pro peptide bond.12
3.22, m
Ser
1
168.6, C
2
55.2, CH
4.08, m
1, 3, 1Leu
1, 2
3, 2Pro, NH
2, NH
3
60.3, CH2 3.58, m
9.07, br s
NH
3, 1Leu
2, 3, 2Leu
Leu
1
2
172.7, C
50.3, CH
4.34, dd (7.5)
1, 3, 4,
1Thr‑ether
3, 5, 6, NH,
NHSer
3
42.8, CH2 1.44, m
1, 2, 5, 6
2, 3, 5, 6
3, 4, 6
2, 4, 5, 6
3, 5, 6
4
23.8, CH
1.72, m
5
22.1, CH3 0.96, d (6.5)
22.7, CH3 0.92, d (6.5)
6.62, d (7.5)
2, 3, 4
6
3, 4, 5
2, 3, 4
NH
1, 2,
1Thr‑ether
2, NHThr‑ether
Thr-ether
169.5, C
1
2
60.7, CH
3.97, dd (8.5,
2.5)
1, 3, 4, 1Tzn 4, NH
3
66.7, CH
4.05, m
1′
2, 3
2′, 3′, 4, NH
2, 3
4
20.9, CH3 1.17, d (6.0)
75.5, C
1′
2′
3′
4′
26.2, CH3 1.23, s
26.2, CH3 1.23, s
1′, 3′, 4′
1′, 2′, 4′
3, 4′, 5′, NH
3, 4′, 5′, NH
2′, 3′, 5′
143.7, CH
5.85, dd (18.0, 1′, 2′, 3′
11.0)
5′
113.6, CH2 5.05, d (11.0)
5.17, d (18.0)
1′, 4′
2′, 3′ 4′
NH
7.37, d (8.5)
2, 3, 1Tzn
2, 3, 2′, 3′, 2Tzn,
NHLeu
Tzn
1
2
170.3, C
77.5, CH
5.40, dd (11.0, 1, 1Phe
2.0)
3, NHThr‑ether
2
3
32.9, CH2 3.45, dd (11.0, 1, 2, 1Phe
11.0)
3.72, dd (11.0,
2.0)
Phe
1
2
3
175.1, C
51.6, CH
4.97, m
1, 3
3, 5/5′, NH
36.8, CH2 3.17, m
3.57, m
1, 2, 4, 5/5′ 2, 5/5′, NH, 2Pro
4
138.2, C
5/5′
6/6′
7
128.6, CH
128.0, CH
126.0, CH
7.17, m
3, 5′/5, 7
4, 6′/6
5/5′
2, 3, NH
7.28, m
7.19, m
NH
9.28, d (9.0)
2, 3, 1Pro
2, 3, 5/5′
The HRESIMS spectrum of compound 2 suggested a
molecular formula of C33H46N6O5S, which was supported by
NMR data (Table 2). The H and 13C NMR spectra of 2
1
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Table 2. NMR Data for 2 in DMSO-d6 (1H 500 MHz, 13C
125 MHz)
residues: one phenylalanine, two prolines, one valine, one
isoleucine, and one cysteine present as a thiazoline. As with
compound 1, the amino acid sequence of 2 was determined
from a combination of inter-residue ROESY and HMBC
correlations (Table 2) in comparison with MS-MS fragmenta-
tion (Figure S18; Table S2), which was Phe, Pro, Pro, Val, Ile,
and Tzn. Similarly to 1, the absolute configurations for the
amino acids were determined as D-Phe, L-Pro, L-Pro, L-Val, L-Ile,
and D-Tzn. Here again there was an enantiomeric excess of 20%
favoring D-Phe over L-Phe (Figure S16) but no evidence of
multiple configurational isomers for 2 by NMR. Similar to 1,
the Pro2−Pro1 peptide bond was determined to adopt a cis
geometry on the basis of the large chemical shift difference
between Cβ and Cγ (ΔδC = +8.2 ppm) for Pro1 and the
presence of ROESY correlations between Hα of Pro2 and Hα of
Pro1. A trans peptide geometry was determined for Val−Pro2
based on the small chemical shift difference between Cβ and Cγ
(ΔδC = +3.4 ppm) for Pro2 and the presence of ROESY
position
δC
δH (J in Hz)
Pro1
HMBC
ROESY
1
2
169.5, C
59.8, CH
4.56, d (7.0)
1, 3, 4, 5,
1Pro2
3, 4, 2Pro2, NHPhe
2, 4, 5
3
4
5
29.0, CH2 1.59, m
2.23, m
1, 2, 4, 5
20.8, CH2 0.48, m
1.46, m
2, 3
2, 3, 5
3, 4
45.3, CH2 2.72, br t (10.0) 3, 4, 1Pro2
3.11, m
Pro2
1
2
169.4, C
58.4, CH
4.18, dd (7.3,
7.3)
1, 3, 4, 5,
1Val
3, 4, 2Pro1
2, 4, 5
3
4
5
28.1, CH2 1.60, m
2.27, m
1, 2, 4, 5
12
correlations between Hα of Val and H2 of Pro2. Compound
2 was named mollamide F.
δ
24.7, CH2 1.79, m
1.99, m
2, 3, 5
2, 3, 5
The HRESIMS spectrum of compound 3 suggested a
1
molecular formula of C26H29N3O2. The H and 13C NMR
47.3, CH2 3.56, m
3.77, m
2, 3, 4
3, 4, 2Val
spectra exhibited signals corresponding to six methylene groups
and three aromatic rings (Table 3). Examination of the 1D and
Val
1
2
170.4, C
Table 3. NMR Data for 3 in C6D6 (1H 500 MHz, 13C 125
MHz)
54.6, CH
4.35, dd (8.5,
1, 3, 4, 1Ile
3, 4, 5, NH, 5Pro2
8.5)
3
30.5, CH
2.00, m
1, 2, 4, 5
2, 3, 5
2, 4, 5
position
δC
δH (J in Hz)
3.39, m
HMBC
2, 3, 15
4
18.2, CH3 0.93, d (6.5)
18.4, CH3 1.01, d (6.5)
7.49, d (9.0)
2, 3, NH, 5Pro2
2, 3, NH, 5Pro2
5
2, 3, 4
1
41.1, CH2
35.7, CH2
139.1, C
NH
1, 2, 1Ile
2, 3, 4, 2Ile, 3Ile,
NHIle
2
2.63, m
1, 3, 4/4′
3
Ile
4/4′
5/5′
6
128.8, CH
128.6, CH
126.5, CH
42.5, CH2
33.6, CH2
138.5, C
7.05, m
7.13, m
7.06, m
3.52, m
2.73, m
2, 6, 4′/4
3, 5′/5
4/4′
1
2
170.6, C
59.6, CH
4.01, dd (9.5,
9.5)
1, 3, 4, 5,
1Tzn
4, 5, 6, NH
7
8, 9, 16
3
34.4, CH
2.05, m
1
4, 5, 6, NH,
NHVal
8
7, 9, 10, 14,
9
4
5
15.1, CH3 0.87, m
24.9, CH2 1.14, m
1.52, m
2, 3, 5
2, 3, 5
10
11
12
13
14
15
16
17
18
19
20/20′
21/21′
22
137.1, C
2, 3, 4, 6
2, 3, 4, NH
126.6, CH
125.9, CH
129.9, CH
130.8, CH
170.1, C
6.96, m
9, 13, 15
10, 14
9
6.86, dd (7.2, 7.2)
7.03, m
6
10.2, CH3 0.85, m
3, 5
2, 3
NH
7.77, d (9.0)
2, 3, 1Tzn
2, 3, 5, 2Tzn
NHVal
,
6.97, m
10, 12
Tzn
1
2
170.0, C
77.3, CH
158.1, C
5.30, dd (10.5, 1, 1Phe
2.5)
3, NHIle
2
41.9, CH2
36.8, CH2
140.0, C
3.34, m
2.60, m
16, 18, 19
17, 19, 20, 20′
3
33.1, CH2 3.40, m
3.71, dd (10.5,
4.0)
Phe
1, 2, 1Phe
129.0, CH
128.4, CH
126.0, CH
7.02, m
7.11, m
7.03, m
18, 22, 20′/20
19, 21′/21
20/20′
1
2
3
173.3, C
52.0, CH
4.88, m
1, 3
3, 5/5′, NH
36.5, CH2 3.38, m
3.51, m
1, 2, 4, 5/5′ 2, 5/5′, NH
2D NMR data of 3 indicated unambiguously the presence of
three phenethylamine fragments (A, B, and C). Connections
between these fragments were established through analysis of
the HMBC spectrum. An HMBC correlation from the low-field
methylene residue of B (H-7, δH 3.52) to a carbonyl carbon (C-
16, δC 158.1) and correlation from the low-field methylene
residue of C (H-17, δH 3.34) to the same carbonyl carbon
indicated B and C were connected through a urea group. The
methylene of A (H-1, δH 3.39) showed an HMBC correlation
to a carbonyl carbon (C-15, δC 170.1), to which an aromatic
proton of B (H-11, δH 6.96) was also correlated, suggesting A
4
138.8, C
5/5′
6/6′
7
129.2, CH
128.0, CH
126.1, CH
7.19, m
3, 7, 5′/5
4, 6′/6
5/5′
2, 3, NH, 5Pro1
5Pro1
7.25, m
7.17, m
NH
9.17, d (8.5)
2, 3, 1Pro1
2, 3, 5/5′, 2Pro1
(Table 2) were very similar to those of 1 with minor differences
such as the absence of the isoprene signal. Detailed analysis of
the 1D and 2D NMR data of 2 established six amino acid
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of the extract (2.0 g) was separated on HP20SS resin using a gradient
of H2O to IPA in 25% steps and a final wash of 100% MeOH to yield
five fractions. The HIV active F2 (50/50 H2O/IPA) was further
fractionated by a flash C18 column eluted with a gradient of MeOH/
H2O to give five fractions (Fr2.1−2.5). The active Fr2.2 was
chromatographed by HPLC using a Phenomenex Luna C18 column
(250 × 10 mm) employing 60% CH3CN/40% H2O at 4 mL/min to
yield compound 2 (1.4 mg, tR = 6.4 min) and compound 3 (1.0 mg, tR
= 8.0 min). The active Fr2.3 was chromatographed by HPLC using a
Phenomenex Luna C18 column (250 × 10 mm) employing 60%
CH3CN/40% H2O at 4 mL/min to yield compound 1 (4.0 mg, tR =
9.2 min).
and B to be connected through a benzene-attached amide
group. Thus the structure of 3, named molleurea A, was
established. Compound 3 is similar to N,N′-diphenethylurea,
which is a common metabolite in tunicates and has been
reported to act as an antidepressant and to promote adipocyte
differentiation.13,14
Compounds 1−3 were evaluated for anti-HIV activity in
both an HIV integrase inhibition assay and a cytoprotective
cell-based assay. In the cytoprotective assay, 2 and 3 showed
HIV inhibition with IC50 values of 78 and 60 μM, respectively,
whereas 1 did not show any inhibition at 78 μM. The cyclic
peptides (1 and 2) are structurally similar to dolastatin 3, which
was reported to have HIV integrase inhibition activity.15 We
found 2 inhibited HIV-1 integrase with an IC50 value of 39 μM,
whereas 1 and 3 showed no activity at 100 μg/mL. On the basis
of our results, residues from the southern region of 2 as
depicted appear to play a critical role in binding to HIV
integrase.
There are only two other reports of S-(D-) thiazoline
occurring in cyanobactin-like peptides.16,17 The occurrence of
epimerization in 1 and 2 was unexpected given the earlier study
by Schmidt that detected 2 by sequencing and MS7 that used
animals from the same collection site as this study. The absence
of any apparent epimerase enzymes in the sequenced pathways
suggests that epimerization of Phe and Tzn in the peptides
reported here could result from thermodynamic relaxation
under the constrained peptide geometry as allowed by the
stereochemical lability of sites adjacent to the thioimide
functionality.18 To test for formation of other stereoisomers
under conditions favoring epimerization, intact 1 was incubated
in 20% piperidine in DMF for 24 h at room temperature. The
single ion recording for [M + H]+ of the reaction mixture
showed no changes (Figure S19), suggesting that no chromato-
graphically distinguishable stereoisomers formed during the
incubation. This result is consistent with the isolated material
being at thermodynamic equilibrium with respect to the
configuration of its most labile stereocenters.
Mollamide E (1): colorless, amorphous powder; [α]20D −21 (c 0.1,
MeOH); UV (MeOH) λmax (log ε) 208 (3.90), 254 (3.36) nm; IR
(film) νmax 3390, 3273, 2952, 2921, 2360, 2341, 1683, 1651, 1540,
1507, 1375, 1339, 1271, 1147, 1116, 1074, 1011, 954, 923, 865, 755,
700, 667 cm−1; 1H and 13C NMR, Table 1; HRESIMS m/z 699.35307
[M + H]+ (calcd for C35H51N6O7S, 699.35345; Δ −0.5 ppm).
Mollamide F (2): colorless, amorphous powder; [α]20D −24 (c 0.1,
MeOH); UV (MeOH) λmax (log ε) 208 (3.89), 256 (3.29) nm; IR
(film) νmax 3384, 3264, 2965, 2932, 2877, 1652, 1624, 1539, 1507,
1455, 1387, 1313, 1275, 1209, 1106, 1031, 999, 921, 876, 751, 700,
1
667 cm−1; H and 13C NMR, Table 2; HRESIMS m/z 639.33145 [M
+ H]+ (calcd for C33H47N6O5S, 639.33232; Δ −1.4 ppm).
Molleurea A (3): colorless, amorphous powder; UV (MeOH) λmax
(log ε) 210 (3.74), 266 (2.68) nm; IR (film) νmax 3310, 3061, 3025,
2926, 2859, 1634, 1558, 1455, 1363, 1315, 1266, 1195, 828, 805, 748,
1
699 cm−1; H and 13C NMR, Table 3; HRESIMS m/z 416.23299 [M
+ H]+ (calcd for C26H30N3O2, 416.23326; Δ −0.6 ppm).
Acid Hydrolysis of Peptides. Compounds 1 and 2, 100 μg each,
were separately dissolved in degassed 6 N HCl (600 μL) and heated in
sealed glass vials (under argon) at 110 °C for 17 h. The solvent was
removed in vacuo.
LC-MS Analysis of D/L-FDLA Derivatives (refs 8, 9). The acid
hydrolysates of 1 and 2 were dissolved in H2O (100 μL) separately,
and 1 N NaHCO3 (20 μL) and 1% 1-fluoro-2,4-dinitrophenyl-5-L-
leucinamide (L-FDLA solution in acetone, 100 μL) were added. The
mixtures were then heated to 40 °C for 50 min. The solutions were
cooled to room temperature, neutralized with 1 N HCl (20 μL), and
then dried in vacuo. The residues were dissolved in 1:1 CH3CN/H2O
and then analyzed by LC-MS. Amino acid standards were derivatized
with L-FDLA in a similar manner. Analysis of the L-FDLA derivatives
was performed on a Supelcosil LC-18 column (150 × 4.6 mm, 5 μm)
employing a linear gradient of 25% CH3CN/75% 0.01 M formic acid
to 70% CH3CN/30% 0.01 M formic acid at 0.5 mL/min over 45 min.
The LC-MS analysis of 1 established the presence of D-
phenylalanine (tR = 37.11 min, 70%) [L-phenylalanine (tR = 31.60
min), 30%], L-proline (tR = 24.29 min) [D-proline (tR = 27.60 min)], L-
serine (tR = 20.38 min) [D-serine (tR = 21.16 min)], L-leucine (tR =
31.10 min) [D-leucine (tR = 39.15 min)], and L-threonine (tR = 20.10
min) [D-threonine (tR = 24.88 min)].
Because L-Thr and L-allo-Thr could not be resolved clearly using
this method, they were separated using a Luna C5 column (250 × 4.6
mm, 5 μm) with a mobile phase of 40 mM ammonium acetate (A) and
70% CH3CN and 30% MeOH (B), from 5% to 40% B over 70 min at
1 mL/min. The derivative of 1 was co-injected with L-Thr and L-allo-
Thr standards, which clearly revealed that the residue from 1 is L-Thr
[L-Thr (tR = 56.00 min), L-allo-Thr (tR = 56.99 min)].
The LC-MS analysis of 2 established the presence of D-
phenylalanine (tR = 37.11 min, 60%) [L-phenylalanine (tR = 31.60
min), 40%], L-proline (tR = 24.29 min) [D-proline (tR = 27.60 min)], L-
valine (tR = 28.37 min) [D-valine (tR = 35.33 min)], and L-isoleucine
(tR = 30.72 min) [D-isoleucine (tR = 38.71 min)].
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a Jasco DIP-370 polarimeter. UV spectra were acquired
in spectroscopy grade MeOH using a Hewlett-Packard 8452A diode
array spectrophotometer. IR spectra were recorded on a JASCO FT/
IR-420 spectrophotometer. NMR data were collected using a Varian
INOVA 500 (1H 500 MHz, 13C 125 MHz) NMR spectrometer with a
3 mm Nalorac MDBG probe with a z-axis gradient and utilized
residual solvent signals for referencing (δH 2.50, δC 39.52 for DMSO-
d6 and δH 7.16, δC 128.06 for C6D6). High-resolution ESIMS analyses
were performed on a Bruker (Billerica, MA, USA) APEXII FTICR
mass spectrometer equipped with an actively shielded 9.4 T
superconducting magnet (Magnex Scientific Ltd.) and an external
Bruker APOLLO nanospray ESI source. LC-MS analyses were carried
out using a Waters Micromass Q-TOF Micro integrated LC-MS
system employing negative ion ESI mode with an ion source
temperature of 100 °C, a desolvation temperature of 300 °C, and
desolvation with nitrogen gas at a flow rate of 400 L/h. Analytical and
semipreparative HPLC were accomplished utilizing a Beckman System
Gold 126 solvent module equipped with a 168 PDA detector. All
reagents were purchased and used without additional purification.
Biological Material. The Didemnum molle ascidian was collected
by hand using scuba from New Britain, Papua New Guinea (S
5°17.382′, E 150°6.089′). A voucher specimen is maintained at
University of Utah under accession number PNG07-2-050.
Because L-Ile and L-allo-Ile could not be resolved clearly using this
method, they were separated using an Agilent Eclipse Plus C18 column
(150 × 4.6 mm, 3.5 μm) with a binary mobile phase of 40 mM
ammonium acetate (A) and CH3CN (B), with a gradient of 20−30%
B over 30 min at 1 mL/min. The derivative of 2 was co-injected with
L-Ile and L-allo-Ile standards, which clearly revealed that the residue
from 2 is L-Ile [L-Ile (tR = 20.10 min), L-allo-Ile (tR = 19.78 min)].
Extraction and Isolation. The frozen ascidian (480 g wet wt) was
exhaustively extracted with MeOH to yield 8.4 g of extract. A portion
1439
dx.doi.org/10.1021/np300270p | J. Nat. Prod. 2012, 75, 1436−1440

Journal of Natural Products
Article
The absolute configuration at the α-carbon of the thiazoline amino
acids of 1 and 2 were determined to be S by adding cystamine after
acid hydrolysis (under argon). 2-Amino-3-((2-aminoethyl)disulfanyl)-
propanoic acid was detected by LC-MS. The mixtures were then
derivatized with L-FDLA. L/D-cysteine standards were treated in a
manner similar to the above. The analysis of the L-FDLA derivatives
was performed on a Supelcosil LC-18 column (150 × 4.6 mm, 5 μm)
employing a linear gradient of 25% CH3CN/75% 0.01 M formic acid
to 70% CH3CN/30% 0.01 M formic acid at 0.5 mL/min over 45 min:
D-2-amino-3-((2-aminoethyl)disulfanyl)propanoic acid (tR = 34.49
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ASSOCIATED CONTENT
■
S
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* Supporting Information
̈
1H NMR, 13C NMR, HMBC, and ROESY spectra for
compounds 1−3. Ion chromatograms of the co-injection of L-
FDLA derivatives of the hydrolysis product of 1 with L-Thr and
L-allo-Thr; the co-injection of L-FDLA derivatives of the
hydrolysis product of 2 with L-Ile and L-allo-Ile; selected ion
chromatograms for L-FDLA derivatives of L- and D-Phe and the
corresponding residues from 1 and 2; comparison between the
hydrolysis product of 1 and 2 with L/D-cysteine standards in
negative ion mode; MS-MS spectra and fragment assignments
for 1 and 2; LC-MS chromatogram for reaction of 1 with 20%
piperidine in DMF. This material is available free of charge via
the Internet at http://pubs.acs.org.
Prod. 2008, 71, 1193−1196.
(18) Schmidt, E. W. Department of Medicinal Chemistry, University
of Utah, Salt Lake City, UT. Personal communication, March 2012.
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AUTHOR INFORMATION
■
Corresponding Author
*Tel: (801) 581-4932. Fax: (801) 585-6208. E-mail: r.m.
vanwagoner@pharm.utah.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by NIH support through the Fogarty
International Center, ICBG 5U01T006671. Funding for the
Varian INOVA 500 MHz NMR spectrometer was provided
through NIH grant RR06262.
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dx.doi.org/10.1021/np300270p | J. Nat. Prod. 2012, 75, 1436−1440