Cytosine-type nucleosides from marine-derived Streptomyces rochei 06CM016

Article abstract of DOI:10.1038/ja.2015.72

Rocheicoside A (3), a nucleoside analog possessing a novel 5-(hydroxymethyl)-5-methylimidazolidin-4-one substructure, was isolated from marine-derived actinomycete Streptomyces rochei 06CM016, together with a new (4) and three known compounds. Structures of the new metabolites were elucidated by one-dimensional (¹H and ¹³C) and 2D NMR (COSY, HMQC and HMBC) and HR-TOF-MS analyses. All the metabolites exhibited significant antimicrobial activity. A plausible mechanism was proposed for compound 3's formation from amicetin.

Full text of DOI:10.1038/ja.2015.72

The Journal of Antibiotics (2015), 1–6
2015 Japan Antibiotics Research Association All rights reserved 0021-8820/15
www.nature.com/ja
&
ORIGINAL ARTICLE
Cytosine-type nucleosides from marine-derived
Streptomyces rochei 06CM016
Semiha Çetinel Aksoy1, Ataç Uzel1 and Erdal Bedir2
Rocheicoside A (3), a nucleoside analog possessing a novel 5-(hydroxymethyl)-5-methylimidazolidin-4-one substructure, was
isolated from marine-derived actinomycete Streptomyces rochei 06CM016, together with a new (4) and three known compounds.
Structures of the new metabolites were elucidated by one-dimensional (1H and 13C) and 2D NMR (COSY, HMQC and HMBC)
and HR-TOF-MS analyses. All the metabolites exhibited significant antimicrobial activity. A plausible mechanism was proposed
for compound 3’s formation from amicetin.
The Journal of Antibiotics advance online publication, 1 July 2015; doi:10.1038/ja.2015.72
INTRODUCTION
RESULTS AND DISCUSSION
Antibiotics are natural compounds produced by microorganisms The isolation process performed using the sediment sample resulted in
acquiring a bioactive mesophilic actinomycete 06CM016. The isolate
showed antibacterial activity against Escherichia coli (17 mm) and
MRSA (22 mm), and antifungal activity against Candida albicans
(37 mm). Sequencing using primers 27F and 1492R revealed that
the isolate was a member of Streptomyces genus. When the molecular
identification evaluated with the phenotypical results (data not
shown), the isolate was identified as Streptomyces rochei. After large-
scale fermentation, 1.92 g of EtOAc extract was acquired. Bioassay-
guided fractionation using the extract led to the isolation of five
compounds (1–5), of which 1 (plicacetin), 2 (norplicacetin) and 5
were identified as cytosine-type nucleoside derivatives, by comparison
of their spectral data with those previously reported (Figure 1).14–17
The high resolution time of flight mass spectrum (HR-TOF-MS) of
as secondary metabolites to kill or inhibit other microorganisms.
As their first discovery in the middle of the twentieth century,
they had an important role in the treatment of infectious diseases.
Up to now, pharmaceutical industry has primarily targeted
drugs from soil organisms, and of the antibiotics in clinical use,
most are of bacterial or fungal origin. Among the bacteria,
Actinobacteria are particularly noteworthy, as they produce
antibiotics such as streptomycin, neomycin, chloramphenicol,
chlortetracycline and erithyromycin.1,2 Members of Actinomycetes,
especially Streptomyces, are fruitful organisms, producing ~ 80% of the
antibiotics.3,4
The acquired resistance of pathogen microorganisms due to their
high mutation rates, toxic effects of some known antibiotics on
human health and the existence of naturally resistant bacteria
require the discovery of novel antibiotics.5,6 As antibiotic discovery
efforts result in identified secondary metabolite frameworks with
known mode of actions, recently, the researchers have started to
focus on microorganisms living in extreme conditions, namely
ocean and ocean bottoms, to discover novel chemical entities.2,7,8
It is thought that these novel compounds in general are produced
as response to stress conditions.9 This research line led to the
discovery of many new microbial species producing bioactive
compounds.10,11 Recent studies have revealed that unusual marine
Actinomycetes are good candidates for the discovery of new natural
products.12
25
3 ½½aꢀD ¼ 5:7 (c 0.0035, MeOH)] gave molecular ions of m/z 631.3123
[(+) mode] and 629.2387 [(− ) mode], attributed to [M+H]+ and
[M− H]− , respectively. Together with the 13C NMR data, accurate
mass data supported a molecular formula of C30H42N6O9. Initial
inspection of the one-dimensional NMR spectra (1H, 13C and DEPT)
also suggested a cytosine derivative, similar with compounds 1 and 2.
1H NMR spectrum of 3 exhibited a tertiary and two secondary
up-field methyl groups (δ 1.10, 1.16 and 1.27, respectively), a tertiary
lower field methyl group (δ2.38, 6H) attributable to a N(CH3)2 unit, a
para-disubstituted aromatic ring (δ 7.79, 2H, d, J = 8.8 Hz; δ 8.07, 2H,
d, J = 8.8 Hz) and two vinyl protons (δ 7.34, 1H, d, J = 7.6 Hz; δ 8.16,
1H, d, J = 7.6 Hz; Table 1). The chemical shifts of the latter protons
are typical of the CH(5) and CH(6) resonances of a cytosine unit.18–20
On the basis of the HMQC connectivity, this assumption was
substantiated, which indicated characteristic C-5 and C-6 signals at
97.1 and 145.7 p.p.m., respectively.
As part of our ongoing studies,13 the aim of this study was to
discover new antimicrobial molecules from marine Actinomycetes
living in unexplored areas of Mediterranean Sea.
1Ege University, Department of Biology, Basic and Industrial Microbiology Section, Izmir, Turkey and 2Ege University, Department of Bioengineering, Izmir, Turkey
Correspondence: Dr SÇ Aksoy, Ege University, Department of Biology, Basic and Industrial Microbiology Section, Bornova, Izmir 35100, Turkey.
E-mail: semiha.cetinel@hotmail.com
or Professor Dr E Bedir, Ege University, Department of Bioengineering, Bornova, Izmir 35100, Turkey.
E-mail: erdalbedir@gmail.com
Received 12 February 2015; revised 27 April 2015; accepted 7 May 2015

Cytosine-type nucleosides from S. rochei
SÇ Aksoy et al
2
Figure 1 Structures of 1–5.
1
In addition, H NMR spectrum of 3 displayed typical signals for electron donating amino group of p-amino benzoic acid, possibly an
two sugar units, namely amicetose and amosamine as in 1 and 2.16–20
amide functionality.
After exclusion of the above-mentioned parts, the remaining side
chain of the molecule had the formula C5H9N2O2. On the basis of the
comprehensive examination of one-dimensional and 2D NMR data of
3, and the comparison of these data with those of related metabolites,
well-known cytosine-type antibiotics, namely amicetin, bamicetin
and oxamicetin,16,19,20–22 it was inferred that a framework derived
from α-methylserine was connected to the benzoyl unit via an amide
bond. Four of the left over carbons at δ 176.9, 66.2, 65.6, 19.3 (C-16
(s), C-19 (t), C-17 (s), and C-20 (q), respectively) in the 13C NMR
spectrum, a singlet methyl at δ 1.10 (H3-20) and two methylene
protons at δ 3.32 and 3.62 (each d, J = 5.6 Hz, H2-19) in the 1H NMR
spectrum were evident for the integration of the aforementioned
amino-acid moiety. Furthermore, in the HMBC spectrum, long-range
correlations between C-16/C-17 and H3-20/H2-19 substantiated our
assumption. The remaining NMR signals indicated an isolated
methylene group (H2-21: δ 4.73, 4.85, each d, J = 7.6 Hz; C-21:
δ 63.1, t), secured by the COSY and HMQC spectra. One of the two
unsaturation numbers calculated for the side chain was readily
accounted for the amide carbonyl at δ 176.9, suggestive of a
cyclization. Thus, incorporation of the remaining carbon should
afford a ring system. In this case, there are two possibilities, either –
N–CH2–N– or –N–CH2–O– bridges. In case of the latter (oxazolidine
ring), C-21 was supposed to be resonated around 80–85 p.p.m. in the
13C NMR spectrum.23–25 However, the observed carbon data (δ 63.1)
was entirely compatible with –N–CH2–N– connection,26 referring a
5-(hydroxymethyl)-5-methylimidazolidin-4-one skeleton. The stereo-
chemistry of C-17 was assigned by a combination of 2D NOESY data
and molecular modeling studies performed on 3. In regard to the
absolute configuration of C-17, the minimum energy conformations
of R and S stereoisomers were calculated with a three-dimensional
computer-generated model. In the case of R-configuration at C-17
The two major spin systems of the disaccharidic part were deduced
from the COSY spectrum starting from the well resolved anomeric
protons (Figure 2). The H-1′ resonance of amicetose (δ 5.73) showed
cross peaks with two methylene protons (H-2′: δ 1.73 and 2.07),
which, in turn, coupled with H2-3′ (δ 1.50 and 2.29). H2-3′ exhibited
correlation with H-4′ (δ 3.36), whereas the latter proton showed cross
peaks with H-5′ (δ 3.65), which coupled with an up-field methyl
signal at δ 1.16 (H3-6′). In a similar manner, the spin system of
amosamine moiety could be traced from the low-field H-1″ (δ 4.81) to
H3-6″ (1.27; Table 1). The use of HMQC spectrum of 3 allowed the
assignment of the carbon signals ascribable to the saccharides, which
also verified the presence of inner amicetose and terminal amosamine
moieties. The absolute configurations of these deoxy sugars were
suggested to be D based on the same biosynthetic origin as in 1, 2 and
5. This assumption was substantiated by carrying out acidic hydrolysis
on compound 3.
The interfragment relationship deduced from the HMBC spectrum
helped us to locate the O- and N-glycosidic linkages, shown in
Figure 2. Thus, the anomeric proton of the amosamine unit (δ 4.81)
displayed a long-range correlation to C-4′ of amicetose (δ 73.6),
whereas the anomeric carbon of the latter sugar showed a 3J C–H
heteronuclear correlation with H-6 of cytosine moiety (δ 8.16).
Moreover, the methyl signal observed at δ 2.38 (6H) was readily
assigned to C-4″-[N(CH3)2] on the basis of a long-range connectivity
to C-4″ (δ 70.5).
The combined use of COSY, HMQC and HMBC spectra also
permitted the complete assignment of the aromatic ring (Table 1,
Figure 2) as p-substituted benzoyl extending from cytosine moiety.
The proton chemical shift of H-11(13) was notably deshielded
compared with those in compound 1 (δ 7.79 and 6.57, respectively),
implying an electron-withdrawing group at p-position instead of an (D-serine derivative), CH3-20 is required to show NOE interactions
The Journal of Antibiotics

Cytosine-type nucleosides from S. rochei
SÇ Aksoy et al
3
Table 1 13C and 1H NMR data of 3 and 4 recorded in DMSO-d6
(100 and 400 MHz, respectively)
identity of the gene cluster. Total of 21 genes, including 3 for
regulation and transportation, 10 for disaccharide biosynthesis and 8
for the formation of the amicetin skeleton by the linkage of cytosine,
p-amino benzoic acid and the terminal α-methylserine moieties, were
described putatively in amicetin biosynthesis by in silico sequence
analysis. In the case of 3, after biosynthesis of amicetin, an additional
enzymatic step is required for a carbon transfer followed by a ring
formation between N-15 and N-18 atoms. A comprehensive search
pointed to N5,N10-methylene tetrahydrofolate (THF), an important
metabolite for the synthesis of purines, thymidine, methionine,
choline and lipids as the major source of C1 units in biosynthesis.
Conversion of THF to 5,10-methylene-THF is one of the well-known
enzymatic processes: the serine hydroxymethyltransferase, a pyridoxal
5′-phosphate-dependent enzyme, converts serine to glycine, transfer-
ring a methyl group to THF, thus producing 5,10-methylene-THF
(Figure 3).26–28 With respect to the additional carbon’s incorporation
(C-21), a plausible mechanism is proposed (Figure 4), which involves
a serine hydroxymethyltransferase homolog in generation of the
imidazolidine ring.27–29
δC (3)
δH (3) (p.p.m.),
δC (4)
δH (4) (p.p.m.),
C\H
(p.p.m.)
J (Hz)
(p.p.m.)
J (Hz)
1
—
—
—
—
2
154.3
—
—
151.5
—
—
3
—
—
—
4
163.6
97.1
145.7
—
151.3
84.7
165.4
—
—
6.06 s
—
5
7.34 d (7.6)
8.16 d (7.6)
—
6
7
—
8
167.2
128.3
130.1
117.6
142.4
117.6
130.1
—
—
166.5
119.2
130.5
113.1
153.8
113.1
130.5
—
—
9
—
—
10
11
12
13
14
15
16
17
18
19
8.07 d (8.8)
7.79 d (8.8)
—
7.80 d (8.4)
6.59 d (8.4)
—
7.79 d (8.8)
8.07 d (8.8)
6.59 d (8.4)
7.80 d (8.4)
6.00 br s
To the best of our knowledge, such a methylene insertion between
the p-amino of p-amino benzoic acid moiety and the nitrogen atom of
D-serine residue is encountered for the first time in secondary
metabolites.
176.9
65.6
—
—
—
66.2
3.32 d, 3.62 d
(5.6)
In addition to 3, another new metabolite (4) with a simple
structural core was isolated. The HR-TOF-MS of 4 provided
[M− H]− and [M+H]+ at m/z 245.0593 and 247.0864, respectively,
20
21
19.3
63.1
1.10 s
4.73, 4.85, each d
(7.6)
1
indicating the molecular formula C11H10N4O3. Inspection of H and
13C NMR spectra of 4 suggested the presence of a 6-monosubstituted
uracil residue instead of the cytosine moiety established in compounds
1–3 and 5.28–30 The substituted part was deduced to be p-amino
1′
82.6
5.73 dd (1.6,
10.4)
2′
3′
29.2
27.0
1.73 m, 2.07m
1.50 m, 2.29 d
(10.8)
1
benzamide as in the other isolates. The assignment of the H and 13C
NMR signals of 4 was secured by COSY, HMQC and HMBC
experiments (Table 1). On the basis of the spectral evidence, the
structure of 4 was established as shown in Figure 1.
4′
5′
6′
1″
2″
3″
4″
73.6
76.9
19.6
95.2
73.4
68.7
70.5
42.6
66.2
18.9
3.36 m
3.65 m
Amicetin-type nucleoside analogs are not common in nature, and
about 10 derivatives have been identified so far, namely bamicetin,14
plicacetin,14 norplicacetin,15 oxamicetin,21 SF2457,31 oxyplicacetin32
and cytosaminomycins A to D.33 Amicetin and its analogs have been
described as potent antibiotics against microorganisms, including
archaea, bacteria and eukarya. These compounds exhibit their activity
via peptidyl transferase inhibition blocking protein biosynthesis.34
Compounds 1–5 showed stronger activity than the positive controls
against the test organisms with MIC values ranging from 4 to
16 μg ml− 1 (Table 2), substantiating antibiotic property of cytosine-
type nucleosides towards microorganisms from different origins.
1.16 d (6.0)
4.81 d (3.6)
3.27 dd (3.6, 9.6)
3.77 t (10.0)
2.00 m
4″-N(CH3)2
5″
6″
2.38 s
3.63 m
1.27 d (6.0)
with CH2-21 due to their close proximity (2.64 Å) above the rigid
imidazolidin-4-one ring system, whereas the S-configuration (L-serine)
is predicted to provide a correlation between CH2-19 and CH2-21
(2.58 Å) (calculated CH3-20/CH2-21 distance = 4.07 Å). On the basis
of the correlation between CH3-20 and CH2-21 observed in the 2D
NOESY spectrum, the R-configuration of C-17 was concluded (see
Supplementary Information).
A detailed literature search revealed that compound 3 was the first
nucleoside derivative possessing imidazolidin-4-one framework. This
prompted us to engage in the biosynthetic means of this exceptional
ring system. Although the structure of amicetin, the originator of 3,
was established in 1962, characterization of its biosynthesis was
reported in 2012 by Zhang et al.16 In that study, first, the amicetin
biosynthesis gene cluster from S. vinaceusdrappus NRRL 2363 was
cloned, followed by heterologous expression in S. lividans TK64, which
MATERIALS AND METHODS
Sampling station and isolation
A marine sediment sample, collected from Kaş, Turkey at a depth of 35 m by
Scuba diving, was used for isolation of marine actinomycetes. The isolation
procedure on the sediment was performed according to the method of
Maldonado et al.35 M1 medium (10 g soluble starch, 4 g yeast extract, 2 g
peptone and 15 g agar, 1000 ml seawater) was used for marine actinomycete
isolation.36
Antimicrobial activity
Before the molecular characterization, a preliminary fermentation was per-
formed with broth version of the same medium at 28 °C for 14 days at
150 r.p.m. to confirm antimicrobial activity. Sequential 25-ml aliquots of the
fermentation broth were removed from the culture every 4 days from day 2 to
14, and activities of the ethyl acetate extracts were monitored using disc
resulted in the production of amicetin and its analogs, confirming the diffusion method on Mueller-Hinton agar against four different bacteria
The Journal of Antibiotics

Cytosine-type nucleosides from S. rochei
SÇ Aksoy et al
4
Figure 2 Spin systems and key long-range correlations of 3.
28 °C for 3 days on a rotary shaker at 150 r.p.m. Five milliliter of the culture
medium was transferred into 200 ml of M1 liquid medium in 1-l flasks, and
further incubation was carried out at 28 °C for 10 days. In total, 56 l of
fermentation liquid was obtained, and after removal of the cells by centrifuga-
tion, the broth was gradually extracted with EtOAc. After evaporation at 40 °C
in vacuo, EtOAc extract was acquired (1.92 g; 0.034 g l− 1 yield).
Thin-layer chromatography
To compare chemical compositions of fractions exhibiting activity and to
determine the required solvent systems for chromatographical separations,
silica gel (aluminum foil, prepared plate, Kieselgel 60 F254, 0.2 mm, Merck,
Art.5554, Merck KGaA, Darmstadt, Germany) TLC analyses were performed.
Solvent systems consisting of CHCl3:MeOH (9:1), CHCl3:MeOH:H2O
(85:15:0.5, 80:20:2, 70:30:3 and 61:32:7), EtOAc:MeOH:H2O (100:17.5:13.5)
and Hexane:EtOAc:MeOH (10:10:2 and 10:10:3) were used.
Figure 3 SHMT catalyzed 5,10-methylene-THF formation. PLP, pyridoxal
5′-phosphate; SHMT, serine hydroxymethyltransferase; THF, tetrahydrofolate.
Bioassay-guided fractionation
(Enteroccocus faecium DSM 13590: vancomycin resistant; E. coli O157:H7 RSKK
234: streptomycin, sulfisoxazole and tetracycline resistant; Staphylococcus
aureus: methicillin resistant; Pseudomonas aureginosa ATCC 27853) and a yeast
species (C. albicans DSM 5817).
Silica gel (Kieselgel 60, 70–230 mesh, Merck), reversed-phase silica gel
(RP-C18, Sepralyte 40 μm) and Sephadex LH-20 as stationary phases on
preparative thin layer, open column and high-performance flash chromato-
graphy (Biotage, Uppsala, Sweden) systems (see isolation scheme in the
Supplementary Material) were used for purification studies.
Molecular identification
DNA isolation was carried out by a modified method of Liu et al.37 The isolate
was identified using sequencing of the 16S ribosomal DNA region.38
Primers for amplification of the 16S ribosomal RNA region were 27F
(5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′-TAC GGC
TAC CTT GTT ACG ACT T-3′). The amplification reaction was performed
with Corbett Cool Gradient Palm Cycler CGI-96 (Concorde, Sydney, NSW,
Australia) under the following conditions as described elsewhere.13 Sequence
analysis of 16S ribosomal RNA region was carried out on an ABI 3130XL
automated sequencer (Applied Biosystems, Foster City, CA, USA). The
sequence (JX912315.1) obtained was compared pairwise using a BLASTN
search and aligned with the sequences of related species retrieved from
GenBank, NCBI.
Acidic hydrolysis of compound 1
Compound 3 (3.1 mg) was refluxed in 1 M HCl for 4 h. After removal of the
solvent, the residue was partitioned between CHCl3 and H2O. The H2O
solution was neutralized with 5% NaOH, and desalted on Sephadex LH-20
column by passing MeOH. The sugar fraction was concentrated in vacuo and
purified on preparative TLC using CHCl3:Me2CO (1:1) to afford two
saccharides, providing positive color reaction to anisaldehyde-H2SO4
(3a: 0.3 mg; 3b: 0.4 mg). Positive optical rotation data were observed for both
25
sugar moieties (D-amicetose: ½aꢀD ¼ þ1:6 (c 0.0023, Me2CO); D-amosamine
25
½aꢀD ¼ þ1:3 (c 0.0024, Me2CO)).
Broth microdilution method
Large-scale fermentation
MICs of the purified compounds were performed by the microdilution method
For large-scale fermentation, a loopful culture of S. rochei 06CM016 was
inoculated in a flask containing 50 ml of M1 medium, and it was incubated at described by CLSI.39
The Journal of Antibiotics

Cytosine-type nucleosides from S. rochei
SÇ Aksoy et al
5
Figure 4 A plausible mechanism for the transformation of amicetin into compound 3. PLP, pyridoxal 5′-phosphate.
ACKNOWLEDGEMENTS
This work was supported by the Scientific and Technological Research Council
Table 2 MIC values of compounds 1–5 and positive controls against
the test organisms
of Turkey (TUBITAK, Project No: 109S361) and the Scientific Research
Foundation of Ege University (10-FEN-012).
Author contributions: The manuscript was written through contributions of
all authors. All authors have given approval to the final version of the
manuscript.
MIC (μg ml− 1
)
E. coli O157:H7
MRSA DSM
11729
C. albicans DSM
Compounds
RSKK 234
5817
1
8
4
8
8
4
4
8
8
1
2
Bèrdy, J. Bioactive microbial metabolites. J. Antibiot. (Tokyo) 58, 1–26 (2005).
Lam, K. S. Discovery of novel metabolites from marine actinomycetes. Curr. Opin.
Microbiol. 9, 245–251 (2006).
2
16
16
16
16
64
4
3
8
4
16
16
—
32
—
32
—
4
3
4
Sathiyaseelan, K. & Stella, D. Isolation, identification and antimicrobial activity of
marine actinomycetes isolated from Parangipettai. Recent Res. Sci. Technol. 3,
74–77 (2011).
Thenmozhi, M. & Krishnan, K. Anti‐Aspergillus activity of Streptmyces sp. VITSTK7
isolated from bay of Bengal coast of Puducherry, India. J. Nat. Environ. Sci. 2,
1–8 (2011).
5
Lincomycin
Nalidixicasid
Azithromycin
Cefaclor
16
8
5
6
Koehn, F. E. & Carter, G. T. The evolving role of natural products in drug discovery. Nat.
Rev. Drug Discov. 4, 206–220 (2005).
Talbot, G. H. et al. Bad bugs need drugs: an update on the development pipeline from
the Antimicrobial Availability Task Force of the Infectious Diseases Society of America.
Clin. Infect. Dis. 42, 657–668 (2006).
Ceftriaxone
Clindamycin
Tetracycline
Ampicillin sulbactam
Amphotericin B
Fluconazole
—
16
8
4
7
8
Fenical, W. et al. in From Monsoons to Microbes: Understanding the Ocean’s Role in
Human Health (ed Fenical W.) 71–86 (National Academies Press, Washington, 1999).
Alvarez-Mico, X., Jensen, P. R., Fenical, W. & Hughes, C. C. Chlorizidine, a cytotoxic
5H-pyrrolo[2,1-a]isoindol-5-one-containing alkaloid from a marine Streptomyces sp.
Org. Lett. 15, 988–991 (2013).
32
0.5
8
The Journal of Antibiotics

Cytosine-type nucleosides from S. rochei
SÇ Aksoy et al
6
9
Nam, S. J., Kauffman, C. A., Paul, L. A., Jensen, P. R. & Fenical, W. Actinoranone,
Structural evidence for the existence of the anomeric effect. Tetrahedron 58,
4439–4444 (2002).
a
cytotoxic meroterpenoid of unprecedented structure from
a marine adapted
Streptomyces sp. Org. Lett. 15, 5400–5403 (2013).
10 Donia, M. & Hamann, M. T. Marine natural products and their potential applications as
anti-infective agents. Lancet Infect. Dis. 3, 338–348 (2003).
25 Kleyer, D. L. & Koch, T. H. A photochemical ring contraction of an imino lactam. J. Org.
Chem. 47, 3145–3148 (1982).
26 Matthews, R. G.
& Drummond, J. T. Providing one-carbon units for biological
11 Hughes, C. C., Prieto-Davo, A., Jensen, P. R. & Fenical, W. The marinopyrroles,
antibiotics of an unprecedented structure class from a marine Streptomyces sp. Org.
Lett. 10, 629–631 (2008).
12 Blunt, J. W., Copp, B. R., Munro, M. H. G., Northcote, P. T. & Prinsep, M. R. Marine
natural products. Nat. Prod. Rep. 21, 1–49 (2004).
13 Özcan, K. et al. Diversity and antibiotic-producing potential of cultivable marine-derived
actinomycetes from coastal sediments of Turkey. J. Soil. Sediment. 13, 1493–1501
(2013).
14 Haskell, T. H. et al. The isolation and characterization of three crystalline antibiotics
from Streptomyces plicatus. J. Am. Chem. Soc. 80, 743–747 (1958).
15 Evans, J. R. & Weare, G. Norplicacetin, a new antibiotic from Streptomyces plicatus.
J. Antibiot. 30, 604–606 (1977).
methylations. Chem. Rev. 90, 1275–1290 (1990).
27 Schirch, V. & Szebenyi, D. M. E. Serine hydroxymethyltransferase revisited. Curr. Opin.
Chem. Biol. 9, 482–487 (2005).
28 Bardagí, J. I. & Rossi, R. A. A novel approach to the synthesis of 6-substituted uracils in
three-step, one-pot reactions. J. Org. Chem. 73, 4491–4495 (2008).
29 Angelaccio, S. Extremophilic SHMTs: From structure to biotechnology. BioMed Res. Int.
2013, 1–10 (2013).
30 Al-Turkistani, A. A., Al-Deeb, O. A., El-Brollosy, N. R. & El-Emam, A. A. Synthesis and
antimicrobial activity of some novel 5-alkyl-6-substituted uracils and related derivatives.
Molecules 16, 4764–4774 (2011).
31 Itoh, J. & Miyadoh, S. SF2457, a new antibiotic related to amicetin. J. Antibiot. (Tokyo)
45, 846–853 (1992).
16 Zhang, G. et al. Characterization of the amicetin biosynthesis gene cluster from
Streptomyces vinaceusdrappus NRRL 2363 implicates two alternative strategies for
amide bond formation. Appl. Environ. Microbiol. 78, 2393–2401 (2012).
17 Latosińska, J. N., Seliger, J., Grechishkin, V. S. & Spychala, J. Studies of the electronic
structure of 4-N-cytosine derivatives by NMR-NQR double resonance spectroscopy.
Magn. Reson. Chem. 37, 881–884 (1999).
32 Chen, Y., Zeeck, A., Chen, Z. & Zahner, H. Studies on metabolites produced by
Streptomyces ramuloses Tue-34. II. The structural elucidation of oxyplicacetin, a new
member of amicetin group. Kangshengsu 10, 285–295 (1985).
33 Haneda, K. et al. Cytosaminomycins, new anticoccidial agents produced by
Streptomyces sp KO-8119. 1. Taxonomy, production, isolation and physicochemical
and biological properties. J. Antibiot. (Tokyo) 47, 774–781 (1994).
34 Kirillov, S., Porse, B. T., Vester, B., Woolley, P. & Garrett, R. A. Movement of the 3'-end
of tRNA through the peptidyl transferase centre and its inhibition by antibiotics. FEBS
Lett. 406, 223–233 (1997).
18 Sugimura, H.
& Watanabe, K. Formal synthesis of cytosamine—A component of
nucleoside antibiotics, the amicetin family. Synth. Commun. 31, 2313–2321 (2001).
19 Flynn, E. H., Hinman, J. W., Caron, E. L. & Woolf, D. O. The chemistry of Amicetin, a
new antibiotic. J. Am. Chem. Soc. 75, 5867–5871 (1953).
20 Stevens, C. L., Nagarajan, K. & Haskell, T. H. The structure of amicetin. J. Org. Chem.
2, 2991–3005 (1962).
35 Maldonado, L. A. et al. Diversity of cultivable actinobacteria in geographically
widespread marine sediments. Antonie van Leeuwenhoek 87, 11–18
(2005).
21 Konishi, M., Naruishi, M., Tsuno, T., Tsukiura, H. & Kawaguchi, H. Oxamicetin, a new
antibiotic of bacterial origin. II. Structure of oxamicetin. J. Antibiot. (Tokyo) 26,
757–764 (1973).
36 Mincer, T. J., Jensen, P. R., Kauffman, C. A. & Fenical, W. Widespread and persistent
populations of a major new marine actinomycete taxon in ocean sediments. Appl.
Environ. Microbiol. 68, 5005–5011 (2002).
22 Glaeske, K. W. & West, F. G. Chirality transfer from carbon to nitrogen to carbon via
37 Liu, D., Coloe, S., Baird, R. & Pedersen, J. Rapid mini-preparation of fungal DNA
cyclic ammonium ylides. Org. Lett. 1, 31–34 (1999).
for PCR. J. Clin. Microbiol. 38, 473 (2000).
23 Katarzyńska, J. et al. 4-Methylpseudoproline derived from α-methylserine—synthesis
and conformational studies. Org. Biomol. Chem. 10, 6705–6716 (2012).
24 Selambarom, J., Carre, F., Fruchier, A., Roque, J. P. & Pavia, A. A. Stereochemical
study of 1, 3-N,X-heterocycles derived from alpha-aminoacids and formaldehyde.
38 Hall, V., Evans, T. L. & Duerden, B. I. Identification of actinomyces, propionibacteria,
lactobacilli and bifidobacteria by amplified 16S rDNA restriction analysis. Anaerobe 7,
55–57 (2001).
39 Clinical and Laboratory Standards Institute CLSI. document M100-S17 (2007).
Supplementary Information accompanies the paper on The Journal of Antibiotics website (http://www.nature.com/ja)
The Journal of Antibiotics