Symplocin A, a linear peptide from the bahamian cyanobacterium symploca sp. configurational analysis of N, N -dimethylamino acids by chiral-phase HPLC of naphthacyl esters

Article abstract of DOI:10.1021/np200861n

The absolute stereostructures of the components of symplocin A (3), a new N,N-dimethyl-terminated peptide from the Bahamian cyanobacterium Symploca sp., were assigned from spectroscopic analysis, including MS, 2D NMR, and Marfey's analysis. The complete a

Full text of DOI:10.1021/np200861n

Article
pubs.acs.org/jnp
Symplocin A, a Linear Peptide from the Bahamian Cyanobacterium
Symploca sp. Configurational Analysis of N,N-Dimethylamino Acids
by Chiral-Phase HPLC of Naphthacyl Esters
Tadeusz F. Molinski,*,‡,§ Kirk A. Reynolds,‡ and Brandon I. Morinaka‡
‡Department of Chemistry and Biochemistry and §Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California,
San Diego, La Jolla, California 92093-0358, United States
S
* Supporting Information
ABSTRACT: The absolute stereostructures of the components
of symplocin A (3), a new N,N-dimethyl-terminated peptide from
the Bahamian cyanobacterium Symploca sp., were assigned from
spectroscopic analysis, including MS, 2D NMR, and Marfey’s an-
alysis. The complete absolute configuration of symplocin A,
including the unexpected D-configurations of the terminal N,N-
dimethylisoleucine and valic acid residues, was assigned by
chiral-phase HPLC of the corresponding 2-naphthacyl esters,
a highly sensitive, complementary strategy for assignment of
N-blocked peptide residues where Marfey’s method is in-
effectual or other methods fall short. Symplocin A exhibited
potent activity as an inhibitor of cathepsin E (IC50 300 pM).
inear peptides terminated with N,N-dimethyl amino acid
residues (DMAAs) are a minority family of natural prod-
to DMAA and 2-hydroxy acids, and may find application to
other N-terminal blocked peptides.
L
ucts among the larger array of cyclic peptides reported from
cyanobacteria.1 For example, the exceedingly potent anticancer
compounds dolastatin-152 and -10 (1)3which has completed
phase I clinical trials4were initially found in the sea hare
Dolabella auricularia from the Indian Ocean and, later, in the
cyanobacterium Symploca sp.1b collected in Palau. Grassys-
tatins A and B (2a,b),1c two DMAA-terminated peptides from
Lyngbya cf. confervoides, and grassystatin C (2c) exhibit selective
inhibition of the protease cathepsin E. Gallinamide A,1a from a
Panamanian Schizothrix sp., is moderately active against the
malarial parasite, Plasmodium falciparum, and the cytotoxic
belamide A,1d from a Panamanian Symploca sp., shows tubulin-
destabilizing activity.
While analyses of many highly modified α-amino acids have
been achieved5 conveniently by application of Marfey’s an-
alysis,3 the configuration of DMAA-containing peptides is com-
plicated by lack of reactivity: the hindered N-terminal amino
acid is inert in standard Marfey’s analysis and Edman degrada-
tion. A similar problem is encountered with analysis of
2-hydroxy acids that occasionally replace α-amino acid residues in
DMAA-modified peptides or other cyclic peptides arising from
nonribosomal polypeptide synthetase (NRPS) pathways.6 Here,
we report a new peptide, symplocin A (3), from a mixed cyano-
bacterial assemblage containing Symploca sp. collected in the
Bahamas and its complete stereoassignment aided by a new pro-
tocol for DMAA and other nonstandard residues by analysis of
their corresponding 2-naphthacyl esters. The sensitivity of the
latter protocol is comparable to Marfey’s method and well-suited
RESULTS AND DISCUSSION
■
The molecular formula for 3, C56H86N8O14, was established
from HRESIMS data (m/z 1094.6257, [M + H]+). The high N-
content of 3 together with the presence of several CO
groups (HMBC) was suggestive of a peptide; this was con-
firmed by identification of peptide residue 1H and 13C spin sys-
tems through analysis of COSY, TOCSY, HSQC, and HMBC
spectra. Amino acid (aa) analysis of the hydrolysate of 3 (6 M
HCl, 110°) revealed the presence of the residues glycine (Gly),
proline (Pro), serine (Ser), tyrosine (Tyr), and valine (Val).
The 1H NMR spectrum of 3 (CD3CN, Table 1) showed broad
N−H doublets coupled to α-CH protons of amino acid re-
sidues, the partial sequence of which was established by HMBC
(Figure 1). A downfield AA′BB′ system (δ 6.73, d, J = 8.3 Hz;
7.09, d, J = 8.3 Hz) was assigned to the Tyr residue and sup-
ported by HMBC C−H correlations (Table 1). The remaining
aromatic proton signals were suggestive of a phenylalanine
(Phe) residue; however, Phe was not detected in standard an-
alysis for standard proteinogenic amino acids. An HSQC spec-
trum showed the presence of eight C-substituted methyl groups,
indicating hydrophobic amino acid residues. In addition, three
upfield N-Me signals (δ 2.92, s; 2.83, s, 6H, two overlapped Me)
appeared in the 1H NMR spectrum of 3. The HMBC spectrum
Special Issue: Special Issue in Honor of Gordon M. Cragg
Received: October 25, 2011
Published: February 23, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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1
(3S,4R)-diasteromer 5b was obtained, along with 5a (2:3, H
NMR), when borohydride reduction of the pyrrolidindione was
carried out after removal of the N-Boc protecting group (TFA-
CH2Cl2), followed by acid hydrolysis (6 M HCl, 110 °C,
2.5 h).10,12 The diastereomeric ratio of the mixture of 5a:5b
was the same as that of the 4-hydroxypyrrolidinone from which
it derived, suggesting that no epimerization had taken place at
C-4.13 HPLC-Marfey’s analysis of 3, as described above, re-
vealed symplocin A contains the (3R,4S) diastereomer of
statine.
Assignment of absolute configurations of the N,N-dimethy-
lisoleucine and valic acid residues presented a larger obstacle:
both are insufficiently nucleophilic to react with Marfey’s re-
agents (FDAA7 or FDLA14). As an alternative, α-bromoaceto-
phenone has been used extensively for derivatization of carbo-
xylic acids to their corresponding phenacyl esters.15 We anti-
cipated that free N,N-Ile and valic acid, present in the peptide
hydrolysate of 3, would undergo O-alkylation exclusively at
their carboxy groups at pH = 6, conditions under which the OH
group in α-hydroxy acids and α-N,N-dimethylamino groups
should be protonated and unreactive. Subsequent assignment
of the configuration of the ester products should be feasible
under chiral-phase HPLC conditions.
The four stereoisomers of N,N-dimethylisoleucine (6a−d)
were prepared by separate reductive alkylation (CH2O, H2,
Pd−C)16 of stereoisomers of Ile. For reasons of sensitivity, we
chose α-bromo-2-acetonaphthone (7) to prepare naphthacyl
esters. The latter possesses two advantages over phenacyl esters:
a stronger chromophore (λmax 248 nm, log10 ε 3.9413) that is
inherently fluorescent. Compounds 6a−d were separately con-
verted (Scheme 2) into the corresponding 2-naphthacyl esters
8a−d, which were readily visualized and collected from pre-
parative TLC plates under long-wavelength UV irradiation.
Optimized conditions for chiral-phase HPLC separation of the
four esters (Chiralcel OD-H, 2:98 i-PrOH + 0.1% TFA−hexane,
0.5 mL/min) gave baseline separations of 8a−d (tR = 28.60,
29.91, 27.42, 25.22, respectively). Derivatization of the acid
hydrolysate of 3 and HPLC under identical conditions gave a
compound that co-eluted with 8b; therefore, 3 contains N,N-
Me2-D-Ile.
The absolute configuration of valic acid was determined in a
similar manner. Samples of L- and D-valic acid (9a,b), prepared
by diazotization−hydrolysis17 of L- and D-Val (Scheme 2), were
converted to the corresponding 2-naphthacyl esters 10a,b
under conditions similar to those described above. The two en-
antiomers were separated with baseline resolution on chiral-
phase HPLC (tR = 30.20; 18.16, respectively). The EtOAc-
soluble material, obtained by two-phase extraction of the acid
hydrolysate of 3, was derivatized in the same manner. Chiral-
phase HPLC of this material showed only the presence of O-
(2-naphthacyl) ester of valic acid (D-10b) that co-eluted with
an authentic standard. Therefore, D-valic acid is present in 3,
and the complete configuration of the peptide is revealed.
Peptide 3 is most similar to grassystatins A and B (2a,b);
however, important differences in the structures of 3 and 2a,b
are apparent. Compounds 2a,b contain two units of S valic acid,
whereas 3 has an R valic acid residue. Hydrolysis of statine-like
residues is sometimes accompanied by epimerization at C-3.
We find that under conditions of acid hydrolysis of 3 the statine
residue undergoes only partial epimerization at C-3, unlike that
of grassypeptin A (2a), or the homologated-Phe γ-amino acid resi-
due in stictamides A−C,18 or the isostatine residue in didemnin B.19
In contrast, the C-4 stereocenter (pyrrole numbering) of 4a
showed a correlation of the methyl group (δ 2.92, s) to a spin
system consistent with N-MePhe.
A TOCSY experiment readily traced the α-CH proton signal
(δ 3.94, d) to a spin network belong to isoleucine (Ile). Integra-
tion of the δ 2.83 singlet in the 1H NMR spectrum showed the
presence of two equivalent methyl groups; thus 3 is a linear
peptide terminated with an N,N-dimethylisoleucine residue. An
O-CH3 group (δH 3.65 s) was linked to the terminal proline
methyl ester (O-MePro) by HMBC correlations. The COSY
and HMBC correlations from the downfield shifted CH (C-45
δ 81.2) were consistent with a 3-methyl-2-hydroxybutanoyl re-
sidue (2-valic acid). Finally, the remaining COSY and HMBC
correlations of 3 revealed the presence of a γ-amino acid, statine
(4-amino-3-hydroxy-6-methylheptanoic acid).
The configurations of the proteinogenic amino acid residues
were determined, uneventfully, after hydrolysis of 3 (6 M HCl,
110 °C) and derivatization−HPLC analysis using Marfey’s method.7
The α-amino acid residues Tyr, Ser, Val, and O-MePro were found
to be of the L-configuration, and the remaining residues were as-
signed either by Marfey’s method (N-MePhe, statine) or an alter-
native O-derivatization (2-naphthacyl esters; see below) after pre-
paration of amino acid standards as follows.
N-Me-L-Phe was prepared (Scheme 1) by methylation of N-
Boc-L-phenylalanine ethyl ester (freshly prepared Ag2O, CH3I)
to obtain N-Boc-N-methyl-L-phenylalanine ethyl ester followed
by acid deprotection (3 M HCl, 80 °C).8 Comparison of the L-
Marfey’s derivative of the peptide hydrolysate of 3 with that of
standard N-MePhe (both L- and D-FDAA reagents; tR = 33.35 and
44.65 min, respectively) revealed the presence of N-Me-D-Phe.
Two diastereomers of statine were prepared by modifica-
tions of published procedures from Schmidt9 and Galeotti10
(Scheme 1). (S)-N-Boc-Leu underwent intermolecular cou-
pling−cyclization with Meldrum’s acid (DCC, DMAP) to pro-
duce the corresponding pyrrolidin-2,4-dione,11 which was reduced
(NaBH4) diastereoselectively to provide the 4-hydroxypyrrolidi-
none (4S,5S)-4a (pyrrole numbering) as a single diastereomer.
Ring-opening of 4a (NaOMe, MeOH) followed by depro-
tection (TFA) gave pure (3S,4S)-statine (5a). Alternatively, the
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1
Table 1. H (600 MHz) and 13C NMR (125 MHz) for 3 (CD3CN)
a
b
amino acid
no.
δC, mult.
δH, mult (J in Hz)
COSY
3a, 3b
HMBC
1, 3, 4
ROESY
O-Me-Pro
1
2
173.4, C
59.1, CH
29.5, CH2
4.26, dd (2.3, 5.9)
1.77, m
3a, 3b
3a
2, 3b
1, 2, 4, 5
1, 4, 5
2, 3, 5
2, 5
2, 3b, 5
2, 3a
3b
2.12, m
2, 3a
4a
25.7, CH2
1.85, m
3b, 5
4b
1.78, m
3a, 3b, 5
4a, 4b
3a, 3b, 5
3a
5
47.4, CH2
52.5, CH2
168.7, C
3.31, m
3, 4
6
3.65, s
1
N-MePhe
7
8
57.5, CH
35.3, CH2
5.42, t (7.1)
2.75, m
9a, 9b
8, 9b
8, 9a
7, 9, 10, 16
8, 10, 11
5, 11
9a
7, 8, 9b, 11
8, 9b, 11
9b
3.18, m
7, 8, 10, 11
10
138.7, C
11/15
12/14
13
130.2, CH
129.0, CH
127.2, CH
30.6, CH3
169.5, C
7.23, m
7.23, m
7.18, m
2.92, s
9, 10, 13
10, 13
12/14
8, 17
8, 9a, 9b, 16
8, 9a, 9b, 16
16
Gly
Val
17
18a
18b
NH
19
41.5, CH2
3.94, m
4.02, m
7.45, m
NH
NH
17, 19
17, 19
16
16
18b
172.5, C
c
20
59.9, CH
30.9, CH
18.4, CH3
22.56, CH3
4.12
NH, 21
20, 23
21
21, 22, 23
20, 22, 23
20, 21
21, 22, 25, 28a
21
2.08, m
0.91, s
0.86, s
7.36, m
20, 23
21
22
23
21
20, 21
21
NH
24
20
21, 25, 30
Sta
172.7, C
25a
25b
26
41.1, CH2
2.36, m
2.42, m
3.92, m
3.83, m
1.29, m
1.49, m
1.57, m
0.88, s
26
26
24, 26, 27
26,28, NH(Val), NH
25
24, 26, 27
c
71.4, CH
52.5, CH
39.7, CH2
c
c
c
27
28a
28b
29
27, 28b
28a
25.4, CH
23.6, CH3
22.4, CH3
30
29
29
28, 30, 31
31
30
31
0.85, s
b
NH
32
7.04
27
Ser
171.8, C
56.9, CH
62.6, CH2
172.5, C
56.1, CH
36.9, CH2
33
4.31, m
3.77, m
34, NH
33
34
32
34, NH
34
Tyr
35
36
4.58, m
2.81, m
3.14, m
37a, 37b, NH
36, 37b
37, 38, 32
35, 39/43
37a,b, NH
37b
37a
37b
38
36, 37a
35,36, 39/43
36, 37a, 39/43
129.0, C
39/43
40/42
41
131.0, CH
115.9, CH
156.6, C
7.09, d (8.3)
6.72, d (8.4)
40/42
39/43
37,38, 40/42, 41
38, 41, 40/42
37a,b, 36
NH
44
7.94, m
44
36, 37a, 46, 39/43
Valic acid
45
81.2, CH
31.1, CH
16.8, CH3
18.9, CH3
168.0, C
4.87, d (4.1)
2.01, m
46
46, 47, 48
45, 47, 48
45, 46, 48
45, 46, 47
46, 47, 48, NH (Tyr)
46
45, 47, 48
45, NH (Tyr)
46, 45
47
0.67, d (6.8)
46
46
d
48
0.84
46
N,N-Me2-Ile
49
50
71.8, CH3
34.1, CH
14.0, CH3
27.5, CH2
3.94, d(4.5)
2.15, m
0.95, s
51
51, 52, 53
51, 52, 53
52
51
50, 52
51
49, 50, 52, 53, 54
50, 51, 53
52
53a
53b
1.32, m
1.42, m
50, 51, 52, 54
50, 51
53a
50, 53a
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Table 1. continued
a
b
amino acid
no.
54
55/56
δC, mult.
δH, mult (J in Hz)
COSY
53a,b
50
HMBC
51, 52
ROESY
12.0, CH3
43.0, CH3
0.96, s
2.83, s
a
b
c
d
n
Determined from edited HSQC. JCH = 8 Hz. Significant overlap with isoleucine. Overlapped
CH3CN(aq)). The 2-naphthacyl ester approach provides an
independent, highly sensitive tool for configurational analysis of
DMAAs. Our finding that the α-CH carbon in 1 in the terminal
N,N-Me2-Ile has the D-configuration refutes an earlier as-
sumption, “the precedent that all marine natural products con-
taining an N,N-dimethyl terminal amino acid residue possess
the L configuration at this center”,1a and offers a more satisfying
experimental solution for independent verification of DMAA
configuration.
Figure 1. HMBC correlations in compound 3.
Symplocin A (3) lacked significant cytotoxicity against
cultured tumor cells (HCT-116) but exhibited potent activity
as an inhibitor of the protease enzyme cathepsin E22
(IC50 300 pM), comparable to that of pepstatin.23 This is
consistent with the findings by Luesch and co-workers, who
reported inhibition of cathepsin E by grassystatins A and B
(IC50 886 and 354 pM, respectively).1c
Scheme 1. Synthesis of Diastereomeric Statine Standards,
(3S,4S)-5a and (3R,4S)-5b
a
In conclusion, a new peptide, symplocin A (3), from
Symploca sp. has been characterized and shown to contain the
N,N-Me2-D-Ile terminal amino acid residue and an acetate-
homologated γ-amino acid, (3S,4S)-statine. A new method to
assign the absolute configurations of N,N-dimethylamino acids
and 2-hydroxy acids was deployed, along with Marfey’s analysis,
to secure the complete stereostructure of 3.
a
EXPERIMENTAL SECTION
See refs 9 and 10.
■
General Experimental Procedures. General procedures are
described elsewhere.24
Scheme 2. Synthesis of 2-Naphthacyl Esters of N,N-
Dimethylisoleucine Stereoisomers and (S)- and (R)-Valic
Acid
Microbial Material. A mixed cyanobacterial assemblage (10−10−
039) was collected at San Salvador Island, Bahamas, in July 2010, at a
depth of 25 m, and frozen until required. The following characteristics
of the major filamentous components (>70%) were observed by
comparative microscopy: presence of sheath, cell dimensions: width
∼7.5 μm, length ∼5 μm, shallow constrictions with a single trichome,
lack of calyptra, which are characteristic of Symploca sp.25 On the basis
of the general appearance of the type sample slide, approximately 75−
80% of the type sample appears to be Symploca, and the remaining
sample is made up of diatoms, other cyanobacteria resembling the
genus Lyngbya spp. A voucher specimen is archived at UC San Diego,
Department of Chemistry and Biochemistry.
Extraction and Isolation. A sample of a cyanobacterial assemblage
(116 g wet wt) was extracted with MeOH (2 × 900 mL over 8 h). The
concentrated extract was partitioned between EtOAc (3 × 700 mL)
and H2O (300 mL), and the organic layer concentrated under reduced
pressure to give a green solid (130.0 mg). The EtOAc extract (121.5 mg)
was subjected to Sephadex LH-20 chromatography eluting with 100%
MeOH to give 45 fractions. Fractions 12−14 (22.9 mg) were com-
bined, dried under reduced pressure, and subjected to semipreparative
reversed-phase HPLC (C18, 2 mL/min, gradient, 40:60 to 100:0
(CH3CN + 0.1% TFA(aq))−(H2O + 0.1% TFA(aq)) over 40 min) to
give 3 (3.1 mg).
3: colorless, amorphous solid; [α]22.5D +16.0 (c 2.18, MeOH); FTIR
(Scheme 1) does not epimerize under similar conditions. Con-
sequently, the observed ratio of 5a:5b in the hydrolysate of 3
suggests the major diastereomer retains the configuration
(3S,4S) of the intact peptide.
The configurational analysis of DMAAs has, in the past,
relied on total synthesis (e.g., the need for total synthesis of
1 and its diastereomers and systematic spectroscopic com-
parisons20) or methods of lower sensitivity21 based on chiral-
phase HPLC (penicillamine-bonded phase, elution with Cu2+,
1
(ATR) ν 3311, 2972, 1745, 1671, 1518, 1447, 1204, 1138 cm−1; H
and 13C NMR, see Table 1; HRESIMS m/z 1095.6330 [M + H]+
(calcd for C56H86N8O14, 1095.6336).
N,N-Dimethyl-L-isoleucine (ref 26). A mixture of L-isoleucine (0.020
g in 0.8 mL water), formaldehyde (0.049 mL, 37% aq), and 10% Pd−C
(20 mg, 0.188 mmol) was stirred vigorously overnight under 1 atm of
H2. After venting, the mixture was heated to reflux and filtered hot (×2),
and the filtrate concentrated under reduced pressure to afford N,N-
dimethyl-L-isoleucine26 as a colorless solid: 1H NMR (300 MHz, D2O)
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δ 3.39 (1H, d, J = 10.2 Hz), 3.30 (1H, s), 2.87 (6H, s), 2.01 (1H, m),
1.41 (1H, m), 1.03 (3H, d, J = 7.3 Hz), 0.89 (3H, t, J = 7.4 Hz).
N-Boc-L-Phenylalanine Ethyl Ester. To a solution of L-phenyl-
alanine ethyl ester (1.0 g in 10 mL CH2Cl2) was added di-tert-butyl
dicarbonate (1.31 g, 5.57 mmol) followed by dry, distilled diiso-
propylethylamine (10 mL, 7.42 mmol). The mixture was stirred at
room temperature (rt; 0.5 h), extracted (3 × 25 mL H2O), dried over
MgSO4, and concentrated under reduced pressure to yield 1.0 g of N-
Boc-L-phenylalanine ethyl ester: 1H NMR (300 MHz, CDCl3) δ 7.12−
7.33 (m, 5H), 4.97 (1H, m), 4.56 (1H, m), 4.15 (3H, q, J = 7.1 Hz),
3.07 (1H, br s), 1.52 (3H, s), 1.41 (9H s), 1.22 (3H, t, J = 7.2 Hz).
N-Methyl-L-Phenylalanine Hydrochloride. A mixture of N-Boc-
phenylalanine ethyl ester (0.4 g, 1.4 mmol), freshly prepared Ag2O
(1.28 g, 5.5 mmol), and CH3I (1.54 mL, 11.0 mmol) in DMF (15 mL)
was stirred overnight at rt. The mixture was diluted with H2O
(300 mL) and extracted (200 mL of CHCl3, 3×), and the combined
organic extracts were dried over MgSO4 and concentrated. The
product was purified by flash chromatography (silica, 1:10 acetone−
hexane, flow rate: 25 mL/min) to obtain N-Boc-N-methyl-L-phenyl-
alanine ethyl ester27 (0.236 g, 56%). A solution of N-Boc-N-methyl-L-
phenylalanine ethyl ester (1 mg) in MeOH (0.25 mL) was treated
with HCl (3 M HCl(aq), 0.25 mL). The reaction mixture was stirred
for 2 h at rt and extracted with EtOAc (3 × 1 mL). The combined
organic extracts were concentrated under a stream of N2 to give pure
N-methyl-L-phenylalanine hydrochloride. 1H NMR data were in
agreement with literature values.27
pure (3S,4S)-N-Boc statine9 (21.4 mg, 38%) with data consistent with
literature values. H NMR (300 MHz, CDCl3) δ 4.02 (1H, m), 3.61
1
(1H, m), 2.55 (2H, d, J = 3.0 Hz), 1.43 (9H, s), 0.91 (6H, m). A
sample of the latter compound (1.0 mg, 0.36 μmole) was stirred in
CH2Cl2−TFA (1:1, 0.5 mL) at rt for 2 h to yield, after removal of
1
volatiles, (3S,4S)-statine TFA salt (5a, quant): H NMR (400 MHz,
D2O) δ 4.10 (1H, m), 3.31 (1H, m), 2.66 (2H, m), 1.68 (1H, m), 1.50
(2H, m), 0.91 (6H, m).9
(3R,4S)-Statine TFA Salt (5b). S-N-Boc pyrrolidin-2,4-one was
subject to deprotection (TFA), followed by NaBH4 reduction10 and
hydrolysis (6 M HCl, 110 °C, 2.5 h) to give a mixture of diastereomers
5a:5b (62:38). LRMS m/z 176.4 [M + H]+ (calcd 176.2).
(S)-Valic Acid. To an ice-cold solution of L-valine (100 mg,
0.854 mmol) in 2 M H2SO4 (0.97 mL) was added NaNO2 (116 mg)
in H2O (0.155 mL) over 4 h, and the mixture allowed to warm to rt
with stirring over 6 h. The reaction mixture was saturated with NaCl
and extracted with EtOAc (10 × 20 mL), and the combined organics
were dried over MgSO4. Removal of the volatiles under reduced pres-
1
sure afforded (S)-valic acid (9a) as a viscous oil (0.053 g, 53%): H
NMR (300 MHz, CDCl3) δ 4.16 (1H, d, J = 3.4 Hz), 2.16 (1H, m),
1.07 (3H, d, J = 6.9), 0.93 (3H, d, J = 6.9 Hz).28
(S)-Valic Acid 2-Naphthacyl Ester (10a). To a solution of (S)-
valic acid (9a, 20 mg, 0.17 mmol in 0.50 mL of EtOAc) was added α-
bromo-2-acetonaphthone (0.042 g, 0.17 mmol) in EtOAc (2.5 mL)
followed by Et3N (23 μL). The solution was stirred at rt for 3 h followed
by extraction with H2O (3 × 2 mL). The combined organic layers were
washed, sequentially, with 10% citric acid (1 mL), 7% NaHCO3 (1 mL),
and brine (1 mL), and the volatiles were removed under reduced pres-
sure. The residue was dissolved in EtOAc and separated by preparative
TLC (1:1 hexane−EtOAc), and the middle-eluting product band was
extracted from silica gel (2 mL of EtOAc) to yield L-valic acid
(4S,5S)-4-Hydroxypyrrolidin-2-one (4a). A solution of N-Boc-L-
leucine (0.5 g, 2.16 mmol) in CH2Cl2 (5 mL) was cooled to 0 °C and
treated with Meldrum’s acid (0.809 g, 2.16 mmol), DMAP (0.369 g,
3.2 mmol), and freshly triturated DCC (0.5 g, 2.51 mmol). The
solution was warmed to rt, left to stir for 3 h, and poured into cold
EtOAc (50 mL). The solution was then filtered, and the filtrate was
washed with cold, aqueous NaHSO4 (8 mL, 5% w/v), followed by
brine (8 mL). After concentration under reduced pressure, the
resulting solid was redissolved in EtOAc (20 mL) and heated at
reflux (0.5 h). After removal of volatiles, the residue was purified by
flash chromatography (silica, gradient: 1:9 to 1:1 EtOAc−hexane).
Fractions identified by TLC were then combined and concentrated to
yield the known pyrrolidine-2,4-dione9 (0.42 g, 42%), which was used
2-naphthacyl ester (10a, 5.0 mg): colorless solid; [α]26 +5.0 (c 1.01,
D
EtOAc); UV (MeOH), λmax (log ε) 248 (4.34), 283 (3.65), 338 (2.95)
nm; FTIR (ATR): ν 3349, 2921, 2851, 1741, 1695 cm−1; 1H NMR (400
MHz, CDCl3) δ 8.42 (1H, s), 7.94 (5H, m), 7.63 (2H, m), 5.69 (1H, d,
J = 21.6 Hz), 5.52 (1H, d, J = 21.6 Hz, 5.18 (1H, d, J = 5.8 Hz), 2.53
(1H, m), 1.20 (6H, m); 13C NMR (100 MHz, CDCl3) δ 191 (C), 168
(C), 136 (C), 133 (C), 131 (C), 129.9 (CH), 129.8 (CH), 129.3 (CH),
129.2 (CH), 128 (CH), 127 (CH), 123 (CH), 84.1 (CH), 67.0 (CH2),
30.0 (CH), 19.0 (CH3), 17.4 (CH3).; HRESIMS m/z 309.1098 [M + Na]+
(calcd for C17H18O4Na, 309.1097).
1
directly in the next step. H NMR (300 MHz, CDCl3): δ 4.40 (1H,
m), 3.28 (1H, d, J = 18.9 Hz), 3.16 (1H, d, J = 18.9 Hz), 1.87−2.01
(1H, m), 1.56 (9H, s), 1.1−1.4 (1H, m), 0.95 (6H, d, J = 6 Hz), in
agreement with literature values.9
( )-D,L-10a,b was prepared from ( )-valic acid in a similar manner.
See below for chiral-phase HPLC retention times.
A solution of the above-described pyrrolidine-2,4-dione (0.186 g,
0.71 mmol) in CH2Cl2−AcOH (3.5 mL, 9:1) was cooled to 0 °C and
treated with NaBH4 (0.055 g, 1.4 mmol) in two portions. The mixture
was stirred at 0 °C for 30 min, concentrated, and redissolved in EtOAc
(7 mL). The organic solution was washed with 5% aqueous NaHCO3
(3 × 3 mL). The crude product was then purified by flash chrom-
atography (silica, gradient 3:1 EtOAc−hexane to EtOAc) to yield
N,N-Dimethyl-L-isoleucine 2-Naphthacyl Ester (8a). To a
solution of N,N-dimethyl-L-isoleucine (6a, 1.18 mg, 11.4 μmol) in
EtOH (200 μL) was added NaHCO3 (3 mg, 23 μmol) followed by α-
bromo-2-acetonaphthone (7, 4 mg, 11.4 μmol). The mixture was stirred
at rt overnight, dried down, and separated by preparative TLC (9:1
hexane−EtOAc). The band corresponding to product was extracted with
CH2Cl2 to yield N,N-dimethyl-L-isoleucine naphthacyl ester (8a, 0.8 mg,
9
1
known 4-hydroxypyrrolidinone 4a (55.5 mg, 30% yield): H NMR
(400 MHz, DMSO) δ 5.29 (1H, d, 9.66), 4.30 (1H, m), 4.00 (1H, m),
2.45 (2H, dq, J = 16.8, 6.9), 1.72 (2H, m), 1.44 (9H, s), 0.90 (6H, m),
in agreement with literature values.9
40%): [α]23 +12.2 (c 0.35, CH3CN); UV (CH3CN) λmax 248 (log ε
D
4.45), 283 (3.73), 338 (3.00) nm; FTIR (ATR) ν 2920, 2851, 2346,
1
1680, 1466 cm−1; H NMR (400 MHz, CDCl3) δ 8.45 (1H, s), 7.93
(4H, m), 7.60 (2H, m), 5.53 (2H, s), 3.06 (2H, d, J = 10.1), 2.41 (6H, s),
1.93 (1H, m), 1.72 (1H, m), 1.21 (2H, m), 0.99 (3H, d, J = 6.6), 0.93
(3H, t, J = 7.5 Hz); 13C NMR (100 MHz, CDCl3) δ 192 (C), 171 (C),
136 (C), 132 (C), 131 (C), 130 (CH), 129 (CH), 128 (CH), 127 (CH),
123 (CH), 72.5 (CH), 65.6 (CH2), 41.6 (CH), 33.6 (CH), 25.3 (CH2),
15.8 (CH3), 10.7 (CH3); HRESIMS m/z 328.1909 [M + H]+ (calcd for
C20H26NO3, 328.1907).
Chiral-Phase HPLC Analysis of N,N-Dimethyl-L-isoleucine 2-
Naphthacyl Esters 8a−d. Stereoisomerically pure and mixed N,N-
Me2 isoleucines [allo-L-Ile, 6c, 1:1 mixture of L-Ile, 6a, and allo-D-
Ile (6d, Sigma), and a 1:1:1:1 mixture of 6a−d (Sigma)] were con-
verted to the corresponding naphthacyl esters 8a−d, as described
above for pure S-6a, and analyzed by chiral-phase HPLC (Chiralcel
OD-H, 250 × 4.60, 0.5 mL.min−1, 2:98 i-PrOH + 0.1% TFA−hexane)
and gave the following retention times: tR = 28.60, 29.91, 27.42, 25.23 min,
respectively.
(4R,5S)-4-Hydroxypyrrolidin-2-one (4b). To a suspension of
pyrrolidine-2,4-dione (0.128 g, 0.78 mmol) in CH2Cl2 (1 mL) was
added TFA (1 mL), and the the mixture was stirred for 20 min. The
solution was concentrated under a stream of N2 to provide crude
product (0.121 g), which was dissolved in CH2Cl2−AcOH (9:1,
4 mL). NaBH4 (60 mg, 1.56 mmol) was added at 0 °C in two port-
ions, and the mixture was allowed to warm to rt over 45 min with
stirring. The organic solution was washed with 5% NaHCO3(aq) (3 ×
5 mL), and the organic phase dried over MgSO4, concentrated, and
purified by flash chromatography (silica, 9:1 CH2Cl2−MeOH) to yield
a mixture of 4a:4b (62:38, 38.0 mg, 30%).
(3S,4S)-Statine TFA Salt (5a). A mixture of 4a (55.5 mg in 1 mL
MeOH) and NaOMe (0.5 mL, 0.44 M) was stirred for 1 h, con-
centrated under reduced pressure, and taken up in EtOAc (10 mL).
The EtOAc solution was then washed with H2O (5 mL) and brine
(5 mL). Acidification of the aqueous layer (1 M HCl, 0.5 mL) yielded
429
dx.doi.org/10.1021/np200861n | J. Nat. Prod. 2012, 75, 425−431

Journal of Natural Products
Article
Hydrolysis of Symplocin A. Symplocin A (3, 200 μg) was sub-
jected to acid hydrolysis (6 N HCl, 120 °C, 20 h), the mixture was
cooled, and a portion was extracted with EtOAc. The organic layer was
dried and used in the preparation of 10a (see below), and the re-
mainder of the aqueous hydrolysate was dried under a stream of nitro-
gen and high vacuum before conversion to Marfey’s derivatives or
2-naphthacyl esters as described below.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +1 (858) 534-7115. Fax: +1 (858) 822-0386. E-mail:
tmolinski@ucsd.edu.
Notes
The authors declare no competing financial interest.
Preparation of 2-Naphthacyl Esters from the Peptide Hydro-
lysate of 3. An aliquot of the peptide hydrolysate of 3 (∼100 μg in
200 μL of EtOAc) of the ethyl acetate-soluble fraction was treated
with α-bromo-2-acetonaphthone (200 μg) followed by triethylamine
(∼10 μL). The reaction was stirred overnight at rt followed by isola-
tion by preparative TLC (1:1 EtOAc−hexane). The extracted band was
identified as product by LRMS (m/z 287.5 [M + H]+; calcd 287.1) as
valic acid 2-naphthacyl ester (10). The product was concentrated under
a stream of N2 and redissolved (1 mg/mL, 1:1 hexane−i-PrOH) and
analyzed by chiral-phase HPLC (see below).
ACKNOWLEDGMENTS
■
We thank the UC Davis Molecular Structure Facility for amino
acid composition analysis and Y. Su of the UC San Diego Small
Molecular Mass Spectrometry facility for MS fragmentation
data. The 500 MHz NMR spectrometers were purchased with a
grant from the NSF (CRIF, CHE0741968). This work was
generously supported by the NIH (AI039987).
An aliquot of the total hydrolysate (2.0 mg) was dissolved in EtOH
(250 μL) and adjusted to pH = 6 (LiOH(aq)). To the reaction
mixture was added α-bromo-2-acetonaphthone (4.2 mg). The reaction
mixture was left to stir at rt overnight. The reaction mixture was then
dried under a stream of N2, redissolved in CH2Cl2 (0.5 mL), and sepa-
rated by preparative TLC (1:9 EtOAc−hexane). The product band
containing 8 (LRMS, m/z 328.52 [M + H]+; calcd 328.18) was con-
centrated under a stream of N2, reconstituted (1 mg/mL, 1:1 hexane−
i-PrOH), and analyzed by chiral-phase HPLC (Chiracel OD-H, 250 ×
4.6 mm, 0.5 mL·min−1, 2:98 i-PrOH + 0.1% TFA−hexane) along with
standard naphthacyl esters 8a−d prepared as described above. The
natural product-derived N,N-dimethylisoleucine 2-naphthacyl ester
was identified as (2R,3R)-8b (tR = 28.75) and confirmed by co-
injection with standard 8b.
Chiral-Phase HPLC Analysis of 2-Naphthacyl Valic Acid Esters. An
aliquot of the EtOAc-soluble hydrolysate of peptide 3 (∼100 μg) was
derivatized with α-bromo-2-acetonaphthone (see above) and com-
pared with the standard 2-naphthacyl esters (10a,b) (prepared from
( )-valic acid 9a,b, see above) by chiral-phase HPLC (Chiracel
OD-H, 250 × 4.60, 0.5 mL·min−1, 25:75 i-PrOH−hexane), giving the
following respective retention times: tR = 18.17, 30.14, and 18.16 min.
The identity of the natural product-derived S-10b and the synthetic
standard was confirmed by co-injection.
Marfey’s Analysis of 3. Aliquots (∼100 μg) of hydrolyzed symplo-
cin A (3) were dissolved in 1 M NaHCO3(aq) (400 μL) and treated,
separately, with L-FDAA or D-FDAA (50 μL, 2 mg/mL in acetone).
The mixture was stirred at 80 °C for 30 min followed by the addition
of HCl (2 M, 200 μL) and dilution with CH3CN (100 μL). The
freshly prepared Marfey’s derivatives of the peptide hydrolysate and
standard amino acids were analyzed under two sets of conditions.
Condition A (RP HPLC, Agilent Eclipse XDB-C18, 1.0 mL/min, gra-
dient elution with 1:4 to 1:1 CH3CN−H2O + 0.1% HCOOH over
45 min, monitoring at 340 nm) was used to assign Ser, Tyr, and N-
MePhe. Condition B (RP HPLC, Agilent Eclipse XDB-C18, 1.0 mL/min,
gradient elution with 1:9 to 100% CH3CN−H2O + 0.1% formic acid over
45 min, monitoring at 340 nm) was used to assign Pro and Val. The
retention times (min) of authentic L-FDAA derivatives were L-Ser
(22.39), D-Ser (23.6), L-Pro (31.25), D-Pro (33.0), L-Tyr (26.08),
D-Tyr (30.79), L-Val (33.42), D-Val (37.84), L-N-MePhe (33.35), and
D-N-MePhe (44.46). The Marfey’s derivatives of hydrolyzed 3 gave
retention times of 22.00, 31.92, 27.37, 33.21, and 43.71 min, corres-
ponding to L-Ser, L-Pro, L-Try, L-Val, and D-N-MePhe, respectively,
which were confirmed by co-injections.
DEDICATION
■
Dedicated to Dr. Gordon M. Cragg, formerly Chief, Natural
Products Branch, National Cancer Institute, Frederick, Mary-
land, for his pioneering work on the development of natural
product anticancer agents.
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