Cyclic hexapeptide dimers, antatollamides A and B, from the ascidian Didemnum molle. A tryptophan-derived auxiliary for L- and D-amino acid assignments

Article abstract of DOI:10.1021/acs.joc.7b01659

Two dimerized cyclic hexapeptides, antatollamides A 1 and B (2), were isolated from the colonial ascidian Didemnum molle collected in Pohnpei. The amino acid compositions and sequences were determined by interpretation of MS and 1D and 2D NMR data. Raney Ni reduction of antatollamide A cleaved the dimer to the corresponding monomeric cyclic hexapeptide with replacement of Cys by Ala. The amino acid configuration of 1 was established, after total hydrolysis, by derivatization with a new chiral reagent, (5-fluoro- 2,4-dinitrophenyl)-N^α-L-tryptophanamide (FDTA), prepared from L-Trp, followed by LCMS analysis; all amino acids were found to be L-configured except for D-Ala.

Full text of DOI:10.1021/acs.joc.7b01659

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Article
Cyclic Hexapeptide Dimers, Antatollamides A and B,
from the Ascidian, Didemnum molle. A Tryptophan-
Derived Auxiliary for L- and D-Amino Acid Assignments
Mariam N Salib, and Tadeusz F Molinski
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b01659 • Publication Date (Web): 28 Aug 2017
Downloaded from http://pubs.acs.org on August 29, 2017
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Cyclic Hexapeptide Dimers, Antatollamides A and B, from
the Ascidian, Didemnum molle. A Tryptophan-Derived
Auxiliary for L- and D-Amino Acid Assignments
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Mariam N. Salib† and Tadeusz F. Molinski*,†,
§
†Department of Chemistry and Biochemistry and §Skaggs School of Pharmacy and
Pharmaceutical Sciences, University of California, San Diego,
9500 Gilman Dr. MC-0358, La Jolla, California 92093-0358.
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ABSTRACT: Two dimerized cyclic hexapeptides, antatollamides A (1) and B (2) were
isolated from the colonial ascidian Didemnum molle collected in Pohnpei. The amino acid
compositions and sequences were determined by interpretation of MS and 1D and 2D NMR data.
Raney Ni reduction of antatollamide A cleaved the dimer to the corresponding monomeric cyclic
hexapeptide with replacement of Cys by Ala. The amino acid configuration of 1 was established,
after total hydrolysis, by derivatization with a new chiral reagent, (5-fluoro-2,4-dinitrophenyl)-Nα-
L-tryptophanamide (FDTA), prepared from L-Trp, followed by LCMS analysis; all amino acids
were found to be L-configured except for D-Ala.
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Ascidians are well-known sources of diverse peptides, peptolides and
depsipeptides,1 many with profound biological activities. For example, the antiproliferative
aplidine (plitidepsin), discovered from a temperate Atlantic species of Aplidium, is now in phase II
and III trials for treatment of multiple myeloma and T-cell lymphoma.2 Various species of
Lissoclinum and Didemnum are the sources of a unique family of ribosomal RNA-derived
peptides, including thiazoline-, thiazole-, oxazoline-, and oxazole-containing peptides
lissolinamides, patellamides, and ulithiacyclamide: all are now collectively called cyanobactins.3
Didemnum molle, a common Indo-Pacific colonial tunicate known for its symbiotic relationship
with the cyanobacterium, Prochloron, is the source of prenylated azole-containing peptides
mollamides,4,5 and oligomeric phenethylamine/N-phenethylureas.5
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In our search for antiproliferative and antiinfective natural products from marine
invertebrates, we examined D. molle, collected from Pohnpei, Federated States of Micronesia and
discovered the antatollamides A (1) and B (2). Peptides 1 and 2, which are closely related to
mollamide F (3),5 are isomeric dimers of cyclic hexapeptides linked through a Cys-Cys disulfide
bond and differ only in configuration at the restricted amide bonds of the Pro residues. In the
course of these discoveries, we developed a chiral auxiliary; a variant of the Marfey’s reagent that
resolved hitherto difficult or impossible stereoassignments of amino acid residues and was used to
determine that the Cys residues of 1 are of the D-configuration while the remaining residues are L-.
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RESULTS AND DISCUSSION
Separation of the MeOH-soluble extract of lyophilized D. molle by solvent partitioning and
reversed phase HPLC gave two new peptides, 1 and 2. HRMS analysis of 1 showed two prominent
sodium adduct molecular ions: [M+Na]+ at m/z 1333.6433, and the reduced monomeric form at
+
m/z 679.3250 [M/2+H+Na]2 , corresponding to formulas C66H94N12O12S2 and C33H48N6O6S,
respectively. Analysis of 1D and 2D NMR data of 1 - including the presence of four amide proton
signals (δ 7.04 (d, 9), 8.43 (d, 6.6), 7.69 (d, 7.8) and 8.15 (d, 7.8), six α protons (δ 4.24 (t, 7.5),
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4.50 (m), 4.39 (t, 8.4), 3.84 (t, 6.6), 4.46 (m) and 4.53 (m)), and six expected amide carbonyl C
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NMR signals (δ 170.6, 170.4, 170.3, 170.2, 169.71 and 169.67) – supported a peptide structure.
Exhaustive analysis of HSQC, HMBC, COSY, TOCSY and NOESY data identified six spin
systems assigned to the amino acid residues as Cys, two Pro, Ile, Phe, and Val. Due to congestion
of the 13C NMR carbonyl signals, which hampered analysis of long-range heteronuclear
correlations, the amino acid sequence was established from the NOESY data; specifically,
observation of nOe crosspeaks (Figure 1) from the amide NH signals of each residue, i, to the
adjacent CHα proton signals of the adjacent residue, i–1.
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A second dimeric cyclohexapeptide 2, isomeric with 1, was separated from the latter by
reversed phase HPLC. Analysis of the 1D and 2D NMR data for 2 (Table 1) revealed that both 1
and 2 shared the same amino acid sequence, but exhibited significant differences in the 1H and 13C
NMR chemical shifts of the Pro residues. Pro residues, in particular those involved in oligo-Pro
sequences in conformationally restricted cyclic peptides, are known to exist in stable s-cis and s-
trans configurations which interchange on slow time scales. The Pro amide bond configuration is
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empirically assigned from differences in the C NMR chemical shifts (Δδ) of their respective β-
and γ-carbons.6 Small and large chemical shift differences in the Pro-1 and Pro-2 residues of 1,
respectively (Δδβγ = 3.3 and 9.2 ppm), confirmed a trans-cis Pro-1–Pro-2 configuration.
Conversely, 2 adopts the cis-trans Pro-1–Pro-2 configuration (Δδβγ = 9.5 and 4.2 ppm). The two
diastereomers were stable; no interconversion between 1 and 2 or other isomers was observed over
the time frame of these measurements (weeks).
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Table 1. NMR data for 1 and 2 (600 MHz, DMSO-d6)
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1
2
a
b
a
b
Position
δC
δH , mult (J, Hz)
δC
169.9
58.7
31.5
δH , mult (J, Hz)
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C=O
α
β
170.3
58.7
28.3
Pro-1
CH
4.24, t (7.5)
CH2 1.62, m
2.29, m
CH2 1.83, m
1.98, m
CH2 3.54, q (8.4)
3.79, m
CH
CH2
4.27, m
1.30, m
1.89, m
1.53, m
1.59, m
3.20, m
3.27, m
γ
25.0
47.5
22.0
46.7
CH2
CH2
δ
Pro-2 C=O
170.6
60.5
30.6
C
170.2
58.7
28.9
C
CH
CH2
α
β
CH
CH2 1.82, m
2.12, dd (12, 6.0)
4.50, m
4.16. dd (8.1, 5.1)
1.66, m
2.19, m
1.82, m
1.90, m
γ
21.4
45.9
CH2 0.94, m
1.59, m
CH2 2.90, t (9.9)
3.20, m
24.7
47.6
CH2
CH2
δ
3.49, m
3.76, m
Val
Ile
C=O
169.67 C
171.2
56.0
29.7
19.0
19.5
C
α
β
γ
54.8
30.3
18.0
19.2
CH
CH
4.39, t (8.4)
2.02, m
CH3 0.86, d (6.6)
CH3 0.94, d (6.6)
7.04, d (9.0)
CH
CH
CH3
CH3
4.28, m
1.91, m
0.81, m
0.83, m
NH
C=O
α
7.08, d (9.6)
170.2
59.7
35.2
15.6
25.0
C
CH
CH
169.8
60.9
35.4
15.9
25.7
C
3.84, t (6.6)
1.85, m
CH3 0.89, d (7.2)
CH2 1.25, m
1.47, m
CH
CH
CH3
CH2
3.70, t (6.3)
1.76, m
0.88, d (7.2)
1.23, m
1.49, m
β
β-Me
δ
δ
11.3
CH3 0.85, t (7.2)
8.43, d (6.6)
11.6
CH3
0.84, m
8.22, d (5.4)
NH
C=O
α
Cys
Phe
169.71 C
170.9
54.6
26.2
C
CH
CH2
54.8
24.8
CH
4.46, m
4.57, m
2.69, m
2.82, m
7.26, m
CH2 2.48, mc
β
NH
C=O
α
7.69, d (7.8)
170.4
55.0
38.0
C
CH
171.6
55.0
37.8
C
CH
CH2
4.53, m
CH2 3.00, dd (9.9, 3.6)
3.12, dd (13.2, 6.0)
C
4.42, m
2.89 dd (13.8, 10.8)
3.22, m
β
γ
137.6
138.1
C
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δ
ε
129.2 CH
128.3 CH
7.15, d (7.2)
7.25, m
129.6 CH
128.6 CH
126.8
7.18, m
7.27, m
ζ
NH
126.4
C
7.19, m
8.15, d (7.8)
C
7.19, m
8.46, d (8.4)
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a. 125 MHz Multiplicity determined from HSQC spectra. b, 600 MHz. c, 2H integration
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(b)
(a)
H
N
Bn
α
HN
O
N
O
H
s-trans-Pro-1
O
α'
O
N
HN
Pro-1
Pro-2
H
O
N
i-Pr
α''
O
O
S
s-cis-Pro-2
NH
N
O
O
N
H
N
H
2
Figure 1. Antatollamide A (a) Summary of NOESY correlations observed for 1 (DMSO-
d6, 600 MHz, tm = 500 mS) (b) 3D approximation of the s-trans-Pro-1–s-cis-Pro-2 element (amide
bonds highlighted in color).
The conformational changes introduced by s-cis/s-trans isomerism in 1 and 2 dramatically
influenced their ECD spectra. The ECD spectrum of 1 (Figure 2) is dominated by three Cotton
effects – one positive and two negative (λ 196 nm (Δε +18.7), 204 (–28.4), 221 (–24)) – that arise
largely from π-π* exciton transitions of the amide carbonyl groups. In contrast, the signature
features in the ECD spectrum of 2 are two considerably weaker positive Cotton effects (λ 196 nm
(Δε +13.8), 204 (–5.5) 213 (+7.7)). We have noted earlier that similarly constrained medium ring
peptide macrocycles, with embedded Pro residues – for example, syllisamides G and H from the
Caribbean sponge, Stylissa caribica,7 and sanguinamides A and B from the Spanish dancer
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nudibranch, Hexabranchus sanguineus8 – display ECD spectra that are exquisitely sensitive to s-
cis/s-trans isomerism.
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In theory, dimerism and Pro s-cis/s-trans isomerism in 1 and 2, may generate up to 16
diastereomers, however, no evidence of isomers other than 1 and 2 were detected in the extracts of
D. molle. From this we surmise that the 1 and 2 are not stereorandom isomers, but the consequence
of a thermodynamically favorable "well" resulting from specific non-bonded constraints in the
rigid hexapeptide motif.
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Figure 2. ECD Spectra of antatollamides A (1) and B (2) (CH3CN, 23 ˚C)
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The absolute configurations of all the individual amino acids of 1 were secured by
Marfey’s analysis9 using a new variant of his eponymous reagent. Dimeric 1 was cleaved to the
monomeric cyclic peptide 4 by desulfurization of the Cys-S-S-Cys unit to two D-Ala residues
(Raney Ni, H2O-MeOH, 65 ˚C). Inspection of the 1H NMR spectrum of 4 revealed essentially the
same signals as 1 with the appearance of a new methyl signal (δ 1.03, d, J = 6.6 Hz, Hβ, Ala).
After hydrolysis of 4 (6 M HCl, 110 ˚C), the amino acid residues were derivatized with a versatile,
new L-tryptophanamide variant of Marfey’s reagent (see below) and their configurations assigned
by comparative analysis by LCMS (reversed phase HPLC, H2O (0.1% HCOOH)-CH3CN gradient,
see Experimental for details) along with standards. The following residues were identified: L-Pro,
L-Val, L-Ile, D-Ala and L-Phe, and confirmed by coinjection with standards. It is notable that the
latter conditions failed to separate Ile and L-allo-Ile DTA derivatives, however successful
resolution (tR) was obtained with salt buffered mobile phase (Zorbax SB-Aq, H2O (NH4OAc)-
CH3CN gradient: L-Ile (34.41), and L-allo-Ile (33.38). See Experimental for details). The
configurations of the amino acid residues in 2 are presumed to be the same as those of 1; too little
sample remained available to analyze 2 independently.
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α
The 5-fluoro-2,4-dinitrophenyl-N -L-tryptophanamide reagent, FDTA (8), was developed
as an alternate reagent to the conventional Marfey’s reagent, FDAA,9 for the determination of the
absolute configuration of amino acids. Compound 8 was synthesized using a variant of the
literature method (Scheme 1).9 L-Trp was converted to the corresponding methyl ester 6 that, in
turn, was ammonialysed to the primary amide, L-Trp-NH2 (7, NH4OH, MeOH, quantitative). SNAr
coupling of 7 (1 equiv) with 1,5-difluoro-2,4-dinitrobenzene delivered FDTA (8, m.p. 220 – 222
˚C). Reagent 8 was stable at room temperature in the dark with no detectable signs of
decomposition after four months. The extinction coefficient of the long-wavelength UV-vis
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transition of 8 (λmax 339 nm, ε log 4.12) is comparable to Marfey's reagent (λmax 338 nm, ε log
4.18),9 but the indole chromophore confers π-π* transitions to 8 of comparable intensity at shorter
wavelength (e.g. λmax 252, ε log 4.04) that may find utility in detection.
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Scheme 1. Preparation of L-FDTA (8) and Application in Configurational Analysis of 1.
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While Marfey’s analysis using the conventional DNP-alaninamide provides good
resolution for differentiation of L- and D-enantiomers of non-polar amino acids, it notably
performs poorly with some polar amino acids and fails entirely to discriminate certain N-Me-
amino acids (e.g. L- and D-N-Me-Ala, see Table 2),10,11 but the corresponding L-DTA derivatives
are well-resolved.
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Other methods to overcome resolution problems in Marfey's analysis include variants of
the original reagent synthesized by replacements of the L-Ala-NH2 group in FDAA with more
a,12
a
11
non-polar amino acid amides (e.g. L-Val-NH2
and L-Leu-NH2),11 which increase the
hydrophobic properties of the analytes, or conventional analysis using a C3 reversed phase column
under combined "2D and 3D" HPLC conditions.13 As mentioned above, derivatives of L-Ile and L-
allo-Ile prepared from 8 are resolved with a difference in tR of more than 60 seconds.
Table 2. Comparative UHPLC Retention times (tR, min) and Orders of Elution of L-DTA and L-
DAA (Marfey’s) Derivatives of Selected L- and D- Amino Acids (aa)
tR, min •DAA
tR, min •DTA
αc
Δtb
Δtb
αc
aa
L-
D-
L-
D-
L→D
L→D
Ala
Ser
13.08
10.43
9.73
15.52
10.93
9.73
11.31
19.95
10.51
17.04
22.06
22.01
–
2.44 1.19
0.50 1.05
0.00 1.00
–0.38 1.03
0.55 1.03
–1.16 1.11
0.37 1.02
3.66 1.20
3.69 1.20
5.20
3.02
2.72
4.15
8.39
3.49
3.82
12.67
7.38 2.18
3.02 0.00
2.72 0.00
4.15 0.00
8.39 0.00
4.29 0.80
3.82 0.00
16.18 3.51
1.42
1.00
1.00
1.00
1.00
1.23
1.00
1.28
L→D
L→D
d
d
Asn
Cite
D→L
L→D
D→L
L→D
L→D
L→D
L→D
L→D
D→L
10.93
19.40
11.67
16.67
18.40
18.32
34.41
33.38
d
N-Me-Ala
N-Me-Asp
iso-Serf
Ile
D→L
d
L→D
allo-Ile
Ileg
allo-Ileg
–
–
–
–
–
a RP C18 column, 50 x 2.1 mm (1.9 µm) and a flow rate = 0.50 mL.min–1. Elution was completed with a
linear gradient of 15% – 45% CH3CN/H2O–0.1% HCOOH for 25 minutes, followed by 100% CH3CN for 2
minutes and re-equilibration with 15% CH3CN-H2O-0.1% HCOOH for 3 minutes before the next
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measurement; void time, tVº= 0.53 min. b ∆t = tR (D-aa) – tR (L-aa). c α = [tR(B) – tVº)]/[tR(A) – tVº], where A,
B are the faster and slower eluting components, respectively. d Coelution. eCitrulline f 3-Amino-2-
hydroxypropanoic acid. g Agilent Zorbax SB-Aq column, 4.6 x 250 mm (5 µm), stepped gradient, initial
conditions 30%-40% CH3CN-20 mM NH4OAc-0.1% TFA, 40 min; 40% – 50% CH3CN-20 mM NH4OAc-
0.1% TFA, flow rate = 0.7 mL.min–1.
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Among the cyanobactins, peptide 1 is most similar to mollamide F (3) reported by Ireland
b
and coworkers from a D. molle collected from New Britain, Papua New Guinea.5 Both are cyclic
peptides with two differences: 1 contains L-Phe and free D-Cys dimerically linked through a
disulfide bridge (D-cystine) while mollamide F is monomeric, containing D-Phe and an (S)-
thiazoline residue (the latter presumably arises from D-Cys by cyclodehydration). The mechanism
and gene clusters responsible for formation of thiazoline and oxazoline peptide residues in
ascidians are now well known,14 and it is clear that Prochloron in D. molle harbors the
biosynthetic capacity for cyanobactin production. Mollamide F (3) appears to be formally related
to 1 through heterocyclodehydration of D-Cys to the (S)-thiazoline residue and the epimerization
of L-Phe to D-Phe. The unexpected D-thiazoline configuration of 3 – and by analogy, D-Cys in 1 –
has drawn comment because the responsible biosynthetic gene cluster in Prochloron3 from D.
molle reveals, “absence of any apparent epimerase enzymes in the sequenced pathways”.5
Disulfide-linked peptides are among the more cytotoxic cyanobactins. Each of the
structures of ulithiacyclamide (11)15 and ulithiacyclamide B (12) – highly cytotoxic cyanobactins
from Lissoclinum patella (IC50 (KB cells) 35 and 17 and ng.mL–1, respectively16) – has two Cys
residues linked by an intramolecular disulfide bond.17 To the best of our knowledge, 1 and 2 are
the only cyanobactins that have intermolecular dimerization through Cys. In order to compare the
antiproliferative properties of 1 and 11, both were assayed against a chronic lymphocytic leukemia
(CLL) primary cell line. While compound 11 was modestly potent (IC50 ~300 nM, or 400 ng.mL–
1), the open-disulfide dimeric analog 1 lacked substantial cell cytotoxicity (IC50 >104 nM).
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CONCLUSION
In conclusion, two new dimeric hexapeptides, antatollamide A (1) and B (2) were isolated
from the ascidian, Didemnum molle collected in Pohnpei, and characterized by NMR, chemical
degradation to constituent amino acids, and derivatization with L-FDTA – a novel
tryptophanamide variant of Marfey’s reagent to give DTA-amino acid derivatives. Under UHPLC
analysis, DTA-derivatives of critical amino acids show superior resolution compared to
conventional Marfey’s derivatives. NMR studies of the antatollamides revealed two configurations
for the Pro-Pro dipeptide segment with the predominant natural product, 1, bearing the trans-cis
amide bond configuration.
EXPERIMENTAL SECTION
General Experimental Procedures. UV-Vis spectra were measured on a JASCO V-630
double beam spectrometer. ECD spectra were recorded on a JASCO J-810 spectropolarimeter and
optical rotations were measured on a JASCO P-2000 at the D-double emission line of Na. FTIR
spectra were collected on thin film samples using a JASCO FTIR-4100 fitted with an ATR
accessory (ZnSe plate). Direct-detected 1H and inverse-detected 2D NMR spectra were measured
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on a Jeol ECA (500 MHz) spectrometer, equipped with a 5 mm 1H{13C} 5 mm probe, or a Bruker
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Avance III (600 MHz) NMR spectrometer fitted with a 1.7 mm H{13C} microcryoprobe. 13C
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NMR spectra were collected on a Varian NMR spectrometer (125 MHz) equipped with a 5 mm
Xsens 13C{1H} cryoprobe. NMR spectra were referenced to solvent signals (δH 2.50 ppm, residual
CHD2SOCD3; δC 39.52 ppm, (CD3)2SO-d6). High-resolution ESITOF analyses were carried out on
an Agilent 1200 HPLC coupled to an Agilent 6350 TOFMS. Low-resolution MS measurements
were made on a Thermoelectron Surveyor UHPLC coupled to an MSD single-quadrupole detector.
HPLC was performed on an Agilent 1100 HPLC system.
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Biological Material. Didemnum molle (01-10-039) was collected in 2001 from a shallow
reef near Ant Atoll, Pohnpei, Federated States of Micronesia (6˚ 53.963' N, 158˚ 20.525' E), using
snorkel, at a depth of approximately –2 m. Voucher samples are archived in the Department of
Chemistry and Biochemistry.
Extraction and Isolation. The D. molle tunicate was lyophilized (dry wt. 35.5 g) and
extracted twice by homogenization with CH2Cl2/MeOH (1:1, 2 x 400 mL) and the filtered extracts
were combined and concentrated under reduced pressure to yield the crude extract which was
dissolved in MeOH-H2O (9:1) and separated starting with progressive solvent partitioning as
follows. The aqueous MeOH solution (400 mL) layer was repeatedly extracted with hexane (2 x
400 mL) to provide the hexane-soluble ‘A’ layer (0.6493 g). Water was added to the MeOH-H2O
layer (6:4 final ratio of MeOH-H2O) and the mixture twice partitioned against CH2Cl2 (2 x 400
mL). The combined lower layers were concentrated to deliver the CH2Cl2-MeOH soluble ‘B-layer’
(1.50 g).
The B-layer was separated by flash chromatography (SiO2, step gradient of MeOH in
CH2Cl2), and the fraction eluted with 1:9 MeOH-CH2Cl2 (0.2163 g) was further partitioned by
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size-exclusion chromatography (Sephadex LH-20, MeOH) into nine fractions according to TLC
(UV-activity, p-anisaldehyde staining). The third fraction (73.8 mg) containing 1 and 2 was
separated by preparative reversed-phase HPLC (Phenomenex, Kinetex C18 column, 5 µ, 150 x 21.2
mm, linear gradient, initial conditions 90:10 H2O-0.1% TFA-CH3CN for three minutes to CH3CN
over 18 minutes for a total of 30 minutes, flow rate = 11 mL·min–1) to yield 22 fractions which
included the known compounds N,N-diphenylurea and mollurea.5 The eighth fraction (6.2 mg) was
purified by reversed-phase analytical HPLC (Luna Phenyl Hexyl column, 250 x 4.60 mm, step
gradients, initial conditions 90:10 H2O-0.1% TFA-CH3CN for 3 minutes, 50: 50 for 15 min, 30:70
for 15 min to 100% CH3CN for the last 5 min; flow rate = 0.700 mL·min–1) to antatollamide A (1,
2.2 mg, tR = 24.80 min) and antatollamide B (2, 0.1 mg, tR = 23.32 min).
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Antatollamide A (1): colorless solid; [α]23.5 –97 ± 1.6 (c 0.22, CH3CN); UV (CH3CN)
D
λ 220 nm (ε log10 4.19). ECD (CH3CN, c 8.39 x 10–5 M, 23 ˚C) λ 196 nm (Δε +18.7), 204 (–28.4),
221 (–24); FTIR (ATR, ZnSe plate) ν 3484, 3300, 2965, 2877, 1653, 1520, 1456, 1318, 1205,
1139, 1027, 840, 802, 723 cm–1; HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for C66H94N12O12S2Na
1333.6454; Found 1333.6433. [M/2+H+Na]+ Calcd for C33H48N6O6SNa 679.3248; Found
679.3250; 1H NMR and 13C NMR. See Table 1.
Antatollamide B (2): colorless solid; [α]23.5 –22 ± 3 (c 0.080, CH3CN); UV (CH3CN)
D
λ 219 nm (ε log10 4.09). ECD (CH3CN, c = 3.05 x 10–5 M, 23 ˚C) λ 196 nm (Δε +13.8), 204 (–
5.5), 215 (+7.7); FTIR (ATR, ZnSe plate) ν 3445, 2968, 1678, 1633, 1523, 1440, 1331, 1204,
1138, 1028, 839, 802, 723 cm–1; HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for 1333.6454; Found
1
1333.6448. [M/2+H+Na]+ Calcd for C33H48N6O6SNa 679.3248; Found 679.3248; H NMR and
13C NMR. See Table 1.
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Desulfurization of Antatollamide A (1) – Desthio-antatollamide A (4). A suspension of
Raney Ni (Raney® 2800 approx. 50 µL, 50% w/v H2O) was added to a solution of 1 (0.5 mg, 0.4
µmol) in MeOH (1.5 mL). The solution was purged with N2 (5 min), then stirred at 65 ºC for 3 h
while monitoring the disappearance of 1 (LC-MS). The mixture was cooled to room temperature,
and passed onto an equilibrated SPE cartridge (C18) and eluted with MeOH to deliver desthio-
antatollamide A (4, 0.4 mg, 84%) as colorless solid that was used for acid hydrolysis (see below).
An analytical sample of 4 was obtained by purification of an aliquot of crude compound (100 µg)
by HPLC, using the same conditions for purification of 1 and 2, and quantitated by QSCS (40
µg).18. Colorless solid; 1H NMR (600 MHz, DMSO-d6) δ (ppm) 8.24 (d, J = 7.2 Hz, 1H), 8.22 (d,
J = 8.4 Hz, 1H), 7.55 (d, J = 7.2 Hz, 1H), 7.25 (m, 2H), 7.19 (m, 1H), 7.13 (m, 2H), 4.50 (m, 2H),
4.42 (t, J = 7.2 Hz, 1H), 4.37 (t, J = 8.4 Hz, 1H), 4.20 (m, 2H), 3.81 (m, 1H), 3.79 (t, J = 6.3 Hz,
1H), 3.53 (q, J = 8.4 Hz, 1H), 3.20 (m, 1H), 3.10 (dd, J = 13.8, 6.0 Hz, 1H), 2.96 (dd, J = 13.2, 9.6
Hz, 1H), 2.89 (t, J = 9.6 Hz, 1H), 2.25 (m, 1H), 2.10 (m, 1H), 2.03 (m, 1H), 1.96 (m, 1H), 1.81 (m,
3H, 1.62 (m, 1H), 1.56 (m, 1H), 1.46 (m, 1H), 1.24 (m 2x 1H), 1.03 (d, J = 6.6 Hz, 3H), 0.93 (d, J
= 7.2 Hz, 3H), 0.88 (d, J = 7.2 Hz, 3H), 0.86 (m, 6H).; HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for
C33H48N6O6Na 647.3528; Found 647.3528.
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Acid Hydrolysis of Desthio-antatollamide A (4). Compound 4 (0.4 mg, 0.640 µmol) was
dissolved in 6 M HCl (500 µL) and heated at 110 ºC with stirring for 12 h. The solution was
cooled to 23 ˚C and extracted with EtOAc (3 X 500 µL). The combined organic layers were dried
α
under a stream of N2 and derivatized with (5-fluoro-2,4-dinitrophenyl)-N -L-tryptophanamide
(FDTA, 8) to yield the DTA-amino acid derivatives for LCMS analysis.
α
Synthesis of 2-((5-fluoro-2,4-dinitrophenyl)-N -tryptophanamide (FDTA, 8). Thionyl chloride
(3.6 mL, 49.7 mmol) was slowly added to a suspension of L-tryptophan (1.00 g, 4.90 mmol) in
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anhydrous MeOH (50 mL) at 0 °C. The solution was heated at reflux for 20 h, cooled, and the
solvent removed under reduced pressure. The crude residue was dissolved in EtOAc, and the
solution washed with saturated Na2CO3 followed by brine. The organic layer was dried over
anhydrous Na2SO4, filtered and concentrated to yield methyl ester 6 (1.41 g, 81 %) as a pale
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1
yellow solid, identical by H NMR and ESI-MS with literature values.19 Concentrated aqueous
ammonia (15 mL, 0.222 mol) was added dropwise to a solution of 6 (1.41 g, 6.47 mmol) in
anhydrous MeOH (2 mL) at 0 °C, then the mixture warmed to room temperature with stirring until
no 6 remained (TLC, ninhydrin). Removal of the solvent gave the primary amide 7 as an off-white
solid (quantitative), identical by 1H NMR and ESI-MS with literature values.20
Compound 8 was prepared according to a variation of the original protocol reported by
Marfey.9 Anhydrous MgSO4 (1.25 g) was added to a stirred solution of 7 dissolved in NaOH (0.5
mL, 0.5 mmol) and acetone (10 mL). The solution was filtered, 1,5-difluoro-2,4-dinitrobenzene
(0.10 g, 0.51 mmol) was added and the solution stirred for 0.5 h at room temperature. Standard
workup gave 8 (0.122 g, 63 %); recrystallization from aqueous acetone delivered pure 8 as bright
orange rosettes, m.p. 220 – 222 ˚C ˚C; [α]D –230 (c 1.0, acetone); UV (MeOH) λmax 220 nm (ε
log10 4.57), 252 (4.04), 266 (4.07), 339 (4.12); FTIR (ATR, ZnSe plate) ν 3416, 3330, 3196, 3114,
2925, 2253, 1682, 1630, 1579, 1542, 1520, 1457, 1421, 1367, 1329, 1287, 1126, 1099, 1050, 922,
834, 742, 707 cm–1. 1H NMR (600 MHz, DMSO-d6, JHF = 1H-19F coupling): δ 10.93 (bs, 1H), 8.94
(d, J = 7.8 Hz, 1H), 8.79 (d, JHF = 8.4 Hz, 1H), 7.85 (bs, 1H), 7.57 (d, J = 7.8 Hz, 1H), 7.53 (bs,
1H), 7.30 (d, J = 8.4 Hz, 1H), 7.18 (s, 1H), 7.04 (t, J = 7.5 Hz, 1H), 6.94 (t, J = 7.5 Hz, 1H), 6.69
(d, JHF = 14.4 Hz, 1H), 4.67 (m, 1H), 3.38 (dd, J = 14.4, 4.8 Hz, 1H), 3.27 (dd, J = 14.4, 7.2 Hz,
1H); 13C NMR (125 MHz, DMSO-d6, JCF = C-19F coupling): δ 171.5 Cq, 158.7, Cq (JCF = 265
13
Hz), 148.3 Cq (JCF = 14.0 Hz), 136.2 Cq, 127.3 CH, 127.2 Cq, 127.1 Cq (br), 125.0 Cq (JCF = 9.5
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Hz), 124.6 CH, 121.2 CH, 118.6 CH, 118.4 CH, 111.4 CH, 108.4 Cq, 102.1 CH (JCF = 27.4 Hz),
56.8 CH, 28.4 CH2; HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for C17H14N5O5FNa 410.0871; Found
410.0878.
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Absolute Configuration of the Amino Acids of Desthio-antatollamide A (4) and
Antatollamide A (1). The acid hydrolysate obtained from 4 (see above) was treated with FDTA
(8, 2 mg, 5 µmol) in acetone (200 µL) and 1M NaHCO3 (50 µL, 50 µmol), and the mixture heated
at 85 ºC, with stirring, for 30 minutes. After cooling, the mixture was neutralized with 1M HCl (50
µL, 50 µmol) and concentrated under a stream of N2. The residue was dissolved in H2O (100 µL)
and centrifuged to remove insoluble material before analysis by LCMS (Hypersil Gold, C18
column, 50 x 2.1 mm, 1.9 µm, linear gradient, initial conditions 85:15 H2O-0.1% HCOOH-CH3CN
to 55:45 over 25 minutes, flow rate = 0.50 mL·min–1). The retention times (tR, min) for the
standard amino acids DTA-amino acid derivatives were as follows: L-Ala (13.00), D-Ala (15.52),
L-Pro (13.51), D-Pro (15.21), L-Val (16.24), D-Val (19.76), L-Ile and L-allo-Ile (co-elution,
18.30), D-Ile and D-allo-Ile (co-elution, 21.98), L-Phe (18.72), and D-Phe (21.53). LCMS analysis
of DTA derivatives obtained from 4 showed the presence of L-Pro (13.57), D-Ala (15.47), L-Ile or
L-allo-Ile (18.26), L-Phe (18.77), and L-Val (16.22). By correlation, 1 has the same absolute
configuration.
As L-Ile and L-allo-Ile DTA derivatives co-eluted under the above conditions, they were resolved
under a different combination of solid phase, solvent gradient and buffer (Agilent Zorbax SB-Aq
column, 4.6 x 250 mm, 5 µm, step gradient, initial conditions 30%-40% CH3CN-20 mM NH4OAc-
0.1% TFA, 40 min; 40%-50% CH3CN-20 mM NH4OAc-0.1% TFA, 5 min. Flow rate = 0.70
mL·min–1). The retention times for the standard amino acid DTA derivatives were as follows: L-
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Ile (tR = 34.41 min), and L-allo-Ile (tR = 33.38 min). The HPLC analysis of the DTA derivatives
obtained from 4, co-injected with L-Ile and L-allo-Ile, established the presence of L-Ile.
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Cultured CLL Cytotoxicity Assay. Primary chronic lymphocytic leukemia (CLL) cells were
seeded in RPMI 1640 with 10% of FBS (Fetal Bovine Serum) in 96-well plates (106 cells/well/100
µL) and treated with compounds at determined concentrations (0-104 nM). Cells were harvested
after 48 h, washed and stained with PI (Propidium Iodide) and DioC6 (30 min, 37 ºC). After the
incubation time, the cells were washed and run in a flow cytometer (FACS Calibur) with detection
of fluorescence in channels 1 (DioC6) and 3 (PI). Cells that were positive by DioC6 were
considered alive while cells that were positive by PI were considered dead.
ACKNOWLEDGEMENTS.
We thank J. MacMillan for assistance with collection of D. molle, Y. Su for HRMS data,
A. Mrse and B. Duggan for assistance with NMR measurements, and J. Castro and J. Vélez Luján
(Moores Cancer Center) for cytotoxicity data. We are grateful to the Federated States of
Micronesia for permission to collect in territorial waters. The 500 MHz NMR and the HPLC
TOFMS spectrometers were purchased with funding from the NSF (Chemical Research
Instrument Fund, CHE0741968) and the NIH Shared Instrument Grant (S10RR025636)
programs, respectively. This research was supported by the NIH (AI100776).
ASSOCIATED CONTENT.
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Supporting Information. The Supporting Information is available free of charge on the ACS
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Publications website at DOI: xxxx. 1D and 2D NMR spectra of 1 and 2, H NMR spectrum of 4
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and 1H and 13C NMR spectra of 8.
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AUTHOR INFORMATION: Tel: +1 (858) 534-7115. Fax: +1 (858) 822-0386. Email:
tmolinski@ucsd.edu
Notes.
The authors declare no competing financial interest
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