Insights into lomaiviticin biosynthesis. isolation and structure elucidation of (-)-homoseongomycin

Article abstract of DOI:10.1021/np400355h

The dimeric diazofluorenes known as the lomaiviticins are produced by the marine bacterium Salinispora pacifica DPJ-0019. Investigation of the fermentation broth of DPJ-0019 has yielded the first monomeric benzo[b]fluorene isolated from this species, (-)-

Full text of DOI:10.1021/np400355h

Communication
pubs.acs.org/jnp
Insights into Lomaiviticin Biosynthesis. Isolation and Structure
Elucidation of (−)-Homoseongomycin
Christina M. Woo, Shivajirao L. Gholap, and Seth B. Herzon*
Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States
S
* Supporting Information
ABSTRACT: The dimeric diazofluorenes known as the lomaiviticins are produced by the
marine bacterium Salinispora pacifica DPJ-0019. Investigation of the fermentation broth of DPJ-
0019 has yielded the first monomeric benzo[b]fluorene isolated from this species,
(−)-homoseongomycin (13). (−)-Homoseongomycin (13) is related to the known natural
product seongomycin (10), which is co-produced with the monomeric diazofluorenes known as
the kinamycins. We describe the synthesis of the isotopically labeled derivative
homoseongomycin-d5 (14), via the intermediacy of the diazofluorene “prelomaiviticin-d5”
(12). Our studies establish that (−)-homoseongomycin (13) may be derived from
prelomaiviticin (11) and suggest that 13 and 10 are shunt or detoxification metabolites in lomaiviticin and kinamycin
biosynthesis, respectively.
Herein, we report the isolation and structure determination
of (−)-homoseongomycin (13), which constitutes the first
monomeric metabolite related to the lomaiviticins that has been
isolated from DPJ-0019. We also describe an efficient synthesis
of an isotopically labeled derivative of “prelomaiviticin”
(prelomaiviticin-d5, 12) and demonstrate that this intermediate
can be efficiently converted to (−)-homoseongomycin-d5 (14).
This latter result suggests that both seongomycin (10) and
(−)-homoseongomycin (13) are derived from the correspond-
ing diazofluorenes 9 and 11 by reduction and addition of N-
acetyl-L-cysteine. This work provides the isotopically labeled
derivative prelomaiviticin-d5 (12) for feeding studies.
(−)-Homoseongomycin (13) was isolated by UV−vis-
guided fractionation (310 nm) of the fermentation broth of
DPJ-0019. Detailed fermentation conditions have been
described.4b Briefly, the bacteria were grown in SPYESS
media in the presence of HP-20 beads for 9 d at 28 °C. The
beads were isolated by filtration and collected, and the collected
beads were washed with MeOH. Concentration of the MeOH
extracts and purification by reversed-phase chromatography
afforded a dark green solid mass (500 mg/L), which was further
purified by reversed-phase HPLC (CH3CN−H2O gradient
containing 0.1% TFA).12 (−)-Homoseongomycin (13) was
obtained as a dark purple solid, in yields of 13−20 mg/L over
several fermentations. Purified (−)-homoseongomycin (13) is
soluble in DMF and DMSO but has limited solubility in
MeOH, CH2Cl2, and THF.
acteria of the genus Salinispora, first discovered by Fenical
1
Band co-workers in 2003,
have proven to be a rich
reservoir of structurally diverse bioactive natural products
including the salinosporamides,2 the lymphostins,3 and the
lomaiviticins.4 (−)-Lomaiviticins A and B (1, 2, respectively)
are cytotoxic, dimeric, C2-symmetric bacterial metabolites that
were first isolated from Salinispora pacifica 37L366 in 2001
(Figure 1A).4a The carbon skeleton of (−)-lomaiviticins A and
B (1, 2) comprises two diazotetrahydrobenzo[b]fluorene
(diazofluorene) functional groups linked by a single carbon−
carbon bond.5 We recently reported the isolation of
(−)-lomaiviticins C−E (3−5, respectively),4b which contain a
hydroxyfulvene in place of one of the diazo groups, from
Salinispora pacifica DPJ-0019.6
Two studies toward elucidating the biosynthesis of the
lomaiviticins have been reported. The lomaiviticin biosynthetic
gene cluster was recently characterized in Salinispora tropica
CNB-440.7 In addition, the biosynthesis of the aminosugar
residue of the lomaiviticins has been studied in E. coli.8 By
comparison, the biosynthesis of the related monomeric
diazofluorenes known as the kinamycins (see 6−8)9 has been
extensively studied.5a In the latter pathway, the metabolite
prekinamycin (9, Figure 1B)10 was suggested to serve as a
precursor to the kinamycins by a series of D-ring oxidations and
acyl transfer steps (Figure 1B).5a The related metabolite
seongomycin (10) is co-produced with the kinamycins.11 No
evidence for incorporation of seongomycin (10) in kinamycin
biosynthesis was presented. Given the structural homology
between the lomaiviticins and the kinamycins, it seems
plausible that the lomaiviticins are synthesized by dimerization
of two monomeric intermediates, and a putative dimerase
enzyme has been identified in S. tropica CNB-440.7 At present,
the timing of dimerization remains undefined, and no
monomeric precursors have been identified in the fermentation
broth of the producing organisms.
HRMS analysis of 13 indicated a molecular formula of
C24H21NO7S. The structure of 13 was elucidated by 1D- and
2D-NMR analysis and IR spectroscopy. NMR data are
presented in Table 1. C−H correlations were established by
HMQC and HMBC experiments. An exchangeable proton at δ
Received: May 1, 2013
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
dx.doi.org/10.1021/np400355h | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Communication
1
Table 1. H NMR (500 MHz) and 13C NMR (125 MHz)
a
Data for (−)-Homoseongomycin (13)
δC, type
(125 MHz)
δH, mult. (J in Hz, 500
position
MHz)
HMBC (H→C)
1
114.4, CH
143.9, C
114.3, CH
149.9, C
121.2, C
114.8, C
152.5, C
134.7, C
116.1, CH
135.7, CH
119.7, CH
162.6, C
7.09, s
6.63, s
12, 11, 4a, 4, 3
2
3
12, 4a, 4, 1
4
4a
4b
5
5a
6
7.36, br d (7.5)
7.52, dd (8.0, 8.0)
6.95, br d (8.0)
9a, 8, 7, 5
9, 6, 5a
7
8
9, 9a, 6
9
9-OH
9a
10
10a
11
11a
12
13
14
13.54, s
9, 8, 6
116.1, C
184.3, C
128.3, C
141.3, C
140.6, C
28.4, CH2
15.6, CH3
34.9, CH2
2.58, q (8.0)
13, 3, 2, 1
12, 2
1.19, t (7.5)
3.80, dd (13.5, 4.5)
3.45, dd (11.0, 7.5)
4.37, dt (8.5, 4.5)
16, 15, 11
15
16
17
18
19
52.4, CH
171.8, C
18, 16, 14
19, 18, 15
18
8.27, d (8.0)
169.3, C
22.2, CH3
1.73, s
a
NMR spectra were recorded in DMSO-d6 at 24 °C. Carbons 4b, 10,
10a, and 11a were assigned by analogy to seongomycin (10).
to carbons at δ 114.3 (C-3), 28.4 (C-12), and 121.2 (C-4a),
1
established this as a 3-ethyl-5,6-disubstituted phenol. H and
13C NMR data designated an N-acetyl cysteine subunit, which
was connected to the core off the molecule at C-11 (δ 141.3)
by HMBC. Collectively, these data suggested a structure similar
to seongomycin (10). The remaining carbon−carbon con-
nectivity was assigned by HMBC experiments and by analogy
to seongomycin (10). The absolute configuration of the
cysteine side chain was not rigorously established and was
assumed to be the same as that of seongomycin (10).
Figure 1. (A) Structures of lomaiviticins A−E (1−5) and kinamycins
A−C (6−8). (B) Structures of prekinamycin (9), seongomycin (10),
prelomaiviticin (11), (−)-homoseongomycin (13), and isotopically
labeled derivatives.
Chemical synthesis of (−)-homoseongomycin-d5 (14)
confirmed these assignments (Scheme 1).13 Beginning with
cyclohex-2-en-1-one (15), copper-catalyzed 1,4-addition of
ethyl-d5-magnesium iodide followed by oxidation of the
resulting ketone (not shown) formed 5-(ethyl-d5)-cyclohex-2-
en-1-one (16, 56%, two steps). Copper-catalyzed 1,4-addition
of trimethylsilylmethylmagnesium chloride, trapping of the
resulting enolate with chlorotrimethylsilane, and palladium-
mediated oxidation provided the β-(trimethylsilylmethyl)-α,β-
unsaturated ketone 17 (66%, two steps). Fluoride-mediated
13.54 [confirmed by solvent exchange (CD3OD) and HMQC
experiments] and a three-proton spin system in the range of δ
7.52−6.95 (COSY) were diagnostic of a juglone residue. A
quartet at δ 2.58, integrating for 2 H and coupled to a triplet at
δ 1.19 (3H, J = 7.5 Hz), suggested the presence of an arylethyl
substructure. HMBC correlations between a signal at δ 6.63
(H-3) and carbon resonances at δ 28.4 (C-12), 149.9 (C-4),
and 114.4 (C-1), as well as a singlet at δ 7.09 (H-1) correlated
B
dx.doi.org/10.1021/np400355h | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Communication
a
Scheme 1. Synthesis of Prelomaiviticin-d5 (12) and Homoseongomycin-d5 (14)
a
Conditions: (1) D5C2MgI, CuI, Et2O, −40 °C, 92%. (2) Et3N, TMSOTf, CH2Cl2, 0 °C; then Pd(OAc)2, CH3CN, 24 °C, 61%. (3) TMSCH2MgCl,
CuI, HMPA, Et3N, TMSCl, THF, −30 → −60 → −78 °C. (4) Pd(OAc)2, CH3CN, 24 °C, 66%, two steps. (5) TASF(Et), CH2Cl2, −78 °C, 72%.
(6) Pd(OAc)2, polymer-supported PPh3, Ag2CO3, toluene, 80 °C, 54%. (7) TfN3, DIPEA, CH3CN, 24 °C, 98%. (8) TMSOTf, Et3N, CH2Cl2, 0 °C;
then DDQ, CH2Cl2t-BuOH pH 7 buffer, 24 °C, 89%. (9) TFA, CH2Cl2, 24 °C, 91%. (10) N-Ac-L-cysteine, K2CO3, DMF, 24 °C, 55%.
coupling of the β-(trimethylsilylmethyl)-α,β-unsaturated ketone
17 with O-(methoxymethyl)-2-bromo-3-methoxyjuglone
(18)13 generated a γ-quinonylated enone (not shown) that
was cyclized by heating with palladium acetate in the presence
of polymer-supported triphenylphosphine (PS-PPh3), to form
the hydroxyfulvene 19 (39%, two steps). Diazo transfer to the
hydroxyfulvene 19 (trifluoromethanesulfonyl azide) provided
the diazofluorene 20 (98%). Enoxysilane generation (trimethyl-
silyl trifluoromethanesulfonate, triethylamine) followed by
oxidation (1,2-dichloro-5,6-dicyanobenzoquinone, DDQ)
formed the phenol 21 (89%).
8.0, 8.0 Hz, H-7), 7.35 (1H, d, J = 7.5 Hz, H-6), 7.15 (1H, s, H-
1), 6.87 (1H, d, J = 8.0 Hz, H-8), 6.64 (1H, s, H-3), 4.57 (1H,
dd, J = 8.5, 4.0 Hz, H-15), 4.01 (1H, dd, J = 13.5, 4.5 Hz, H-
14), 3.55 (1H, dd, J = 14.0, 9.0 Hz, H-14), 2.65−2.58 (1H, m,
H-12), 1.77 (3H, s, H-19), 1.26 (3H, t, J = 7.5 Hz, H-13); 13
C
NMR (125 MHz, DMF-d7) δ 185.7 (C), 172.9 (C), 170.6 (C),
164.4 (C), 152.1 (C), 151.1 (C), 145.8 (C), 144.3 (C), 143.3
(C), 136.7 (CH), 135.7 (C), 130.1 (C), 122.2 (C), 120.7
(CH), 117.4 (CH), 117.2 (CH), 116.6 (C), 116.2 (CH), 115.7
(C), 53.9 (CH), 36.6 (CH2), 29.8 (CH2), 22.8 (CH3), 16.3
(CH3); HRMSESI m/z 468.1132 [M + H]+ (calcd for
C24H22NO7S, 468.1117).
Acid-catalyzed deprotection of the methoxymethyl protecting
group provided prelomaiviticin-d5 (12, 91%). Treatment of
prelomaiviticin-d5 (12) with N-acetyl-L-cysteine and potassium
carbonate generated (−)-homoseongomycin-d5 (14, 55%).
NMR spectroscopic data for (−)-homoseongomycin-d5 (14)
closely matched those of (−)-homoseongomycin (13), and
equimolar mixtures of the natural and synthetic material
exhibited similar chromatographic and spectroscopic properties
(HPLC, 1H, 13C NMR analysis). The mild conversion of
prelomaiviticin-d5 (12) to (−)-homoseongomycin-d5 (14)
observed here suggests that prekinamycin (9) and prelomaivi-
ticin (11) are converted to the seongomycins 10 and 13 by
thiol addition intracellularly or in the fermentation media.
In summary, we have described the isolation and structure
elucidation of (−)-homoseongomycin (13), a monomeric
metabolite related to the lomaiviticins. We have provided
evidence that the seongomycins are readily formed from
diazofluorenes, suggesting they are shunt or detoxification
metabolites in the biosynthesis of the kinamycins and
lomaiviticins. The synthesis of prelomaiviticin-d5 (12)
described herein enables feeding studies to probe lomaiviticin
biosynthesis.
ASSOCIATED CONTENT
* Supporting Information
Experimental procedures and detailed characterization data for
all new compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Tel: 1-203-436-8571. Fax: 1-203-432-6144. E-mail: seth.
herzon@yale.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support from the American Cancer Society, the
National Science Foundation (Graduate Research Fellowship
to C.M.W.), the Searle Scholars Program, and Yale University is
gratefully acknowledged. S.B.H. is a fellow of the David and
Lucile Packard and the Alfred P. Sloan Foundations, is a
Camille Dreyfus Teacher−Scholar, and is a Cottrell Scholar of
the Research Corporation for Science Advancement.
(−)-Homoseongomycin (13): amorphous, purple solid;
[α]2D0 −525 (c 0.001, CH3OH); UV (0.1% TFA in CH3CN−
H2O, diode array detector) λmax (log ε) 225, 229, 234, 282 nm;
IR (ATR-FTIR) νmax 3333 (br), 1621 (s), 1585 (s), 1451 (m)
REFERENCES
■
1
cm−1; H and 13C NMR data (500 and 125 MHz, DMSO-d)
(1) Fenical, W.; Jensen, P. R.; Palladino, M. A.; Lam, K. S.; Lloyd, G.
K.; Potts, B. C. Bioorg. Med. Chem. 2009, 17, 2175−2180.
see Table 1; 1H NMR (400 MHz, DMF-d7) δ 13.66 (1H, s, C-
9-OH), 8.34 (1H, d, J = 8.0 Hz, H-17), 7.56 (1H, dd, J = 8.0,
8.0 Hz, H-7), 7.42 (1H, br s, H-6), 7.24 (1H, br s, H-1), 6.97
(1H, br s, H-8), 6.81 (1H, br s, H-3), 4.69 (1H, ddd, J = 8.0,
4.8, 4.8 Hz, H-15), 3.98 (1H, br s, H-14), 3.72 (1H, br s, H-14),
2.64 (2H, br s, H-12), 1.83 (3H, s, H-19), 1.24 (3H, t, J = 7.6
Hz, H-13); 1H NMR (500 MHz, CD3OD) δ 7.42 (1H, dd, J =
(2) (a) Feling, R. H.; Buchanan, G. O.; Mincer, T. J.; Kauffman, C.
A.; Jensen, P. R.; Fenical, W. Angew. Chem., Int. Ed. 2003, 42, 355−
357. (b) Macherla, V. R.; Mitchell, S. S.; Manam, R. R.; Reed, K. A.;
Chao, T. H.; Nicholson, B.; Deyanat-Yazdi, G.; Mai, B.; Jensen, P. R.;
Fenical, W. F.; Neuteboom, S. T. C.; Lam, K. S.; Palladino, M. A.;
Potts, B. C. M. J. Med. Chem. 2005, 48, 3684−3687. (c) Williams, P.
G.; Buchanan, G. O.; Feling, R. H.; Kauffman, C. A.; Jensen, P. R.;
C
dx.doi.org/10.1021/np400355h | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Communication
Fenical, W. J. Org. Chem. 2005, 70, 6196−6203. (d) Reed, K. A.;
Manam, R. R.; Mitchell, S. S.; Xu, J.; Teisan, S.; Chao, T.-H.; Deyanat-
Yazdi, G.; Neuteboom, S. T. C.; Lam, K. S.; Potts, B. C. M. J. Nat.
Prod. 2007, 70, 269−276.
(3) Miyanaga, A.; Janso, J. E.; McDonald, L.; He, M.; Liu, H.;
́
Barbieri, L.; Eustaquio, A. S.; Fielding, E. N.; Carter, G. T.; Jensen, P.
R.; Feng, X.; Leighton, M.; Koehn, F. E.; Moore, B. S. J. Am. Chem.
Soc. 2011, 133, 13311−13313.
(4) (a) He, H.; Ding, W. D.; Bernan, V. S.; Richardson, A. D.;
Ireland, C. M.; Greenstein, M.; Ellestad, G. A.; Carter, G. T. J. Am.
Chem. Soc. 2001, 123, 5362−5363. (b) Woo, C. M.; Beizer, N. E.;
Janso, J. E.; Herzon, S. B. J. Am. Chem. Soc. 2012, 134, 15285−15288.
(5) For reviews of diazofluorene-containing natural products and
diazofluorene-based cytotoxic agents, see: (a) Gould, S. J. Chem. Rev.
1997, 97, 2499−2510. (b) Marco-Contelles, J.; Molina, M. T. Curr.
Org. Chem. 2003, 7, 1433−1442. (c) Arya, D. P. Top. Heterocycl. Chem.
2006, 2, 129−152. (d) Nawrat, C. C.; Moody, C. J. Nat. Prod. Rep.
2011, 28, 1426−1444. (e) Herzon, S. B. The Kinamycins. In Total
Synthesis of Natural Products. At the Frontiers of Organic Chemistry; Li, J.
J., Corey, E. J., Eds.; Springer-Verlag: Berlin, 2012. (f) Herzon, S. B.;
Woo, C. M. Nat. Prod. Rep. 2012, 29, 87−118.
(6) DPJ-0019 was recently identified as Salinispora pacifica by
genotypic and phenotypic analysis, see: Ahmed, L.; Jensen, P.; Freel,
K.; Brown, R.; Jones, A.; Kim, B.-Y.; Goodfellow, M. Antonie van
Leeuwenhoek 2013, 103, 1069−1078.
(7) Kersten, R. D.; Lane, A. L.; Nett, M.; Richter, T. K. S.; Duggan, B.
M.; Dorrestein, P. C.; Moore, B. S. ChemBioChem 2013, 14, 955−962.
(8) Zhang, H. Metabolic Engineering for the Heterologous
Biosynthesis of Erythromycin A and Associated Polyketide Products
in Escherichia coli. Ph.D. Thesis, Tufts University, Medford, MA, 2011;
pp 71−72.
(9) For isolation of the kinamycins, see: (a) Ito, S.; Matsuya, T.;
Omura, S.; Otani, M.; Nakagawa, A. J. Antibiot. 1970, 23, 315−317.
̅
(b) Hata, T.; Omura, S.; Iwai, Y.; Nakagawa, A.; Otani, M. J. Antibiot.
̅
1971, 24, 353−359. (c) Omura, S.; Nakagawa, A.; Yamada, H.; Hata,
̅
T.; Furusaki, A.; Watanabe, T. Chem. Pharm. Bull. 1971, 19, 2428−
2430. (d) Furusaki, A.; Matsui, M.; Watanabe, T.; Omura, S.;
̅
Nakagawa, A.; Hata, T. Isr. J. Chem. 1972, 10, 173−187. (e) Omura, S.;
̅
Nakagawa, A.; Yamada, H.; Hata, T.; Furusaki, A. Chem. Pharm. Bull.
1973, 21, 931−940. (f) Cone, M. C.; Seaton, P. J.; Halley, K. A.;
Gould, S. J. J. Antibiot. 1989, 42, 179−188. (g) Seaton, P. J.; Gould, S.
J. J. Antibiot. 1989, 42, 189−197.
(10) Gould, S. J.; Chen, J.; Cone, M. C.; Gore, M. P.; Melville, C. R.;
Tamayo, N. J. Org. Chem. 1996, 61, 5720−5721.
(11) (a) Carney, J. R.; Hong, S.-T.; Gould, S. J. Tetrahedron Lett.
1997, 38, 3139−3142. (b) Gould, S. J.; Hong, S. T.; Carney, J. R. J.
Antibiot. 1998, 51, 50−57. The optical rotation of seongomycin (10)
was not determined.
(12) Purification with 0.1% TFA accompanies a visual shift in the
solution from green to purple.
(13) Woo, C. M.; Lu, L.; Gholap, S. L.; Smith, D. R.; Herzon, S. B. J.
Am. Chem. Soc. 2010, 132, 2540−2541.
D
dx.doi.org/10.1021/np400355h | J. Nat. Prod. XXXX, XXX, XXX−XXX