Kocurin, the true structure of PM181104, an anti-methicillin-resistant Staphylococcus aureus (MRSA) thiazolyl peptide from the marine-derived bacterium Kocuria palustris

Article abstract of DOI:10.3390/md11020387

A new thiazolyl peptide, kocurin (1), was isolated from culture broths of a marine-derived Kocuria palustris. Its structural elucidation was accomplished using a combination of spectroscopic and chemical methods, including HRMS, extensive 1D and 2D NMR analysis, MS/MS fragmentation, and chemical degradation and Marfey's analysis of the resulting amino acid residues. The structure herein reported corrects that previously assigned to PM181104 (3). Kocurin displayed activity against methicillin-resistant Staphylococcus aureus (MRSA), with MIC values in the submicromolar range.

Full text of DOI:10.3390/md11020387

Mar. Drugs 2013, 11, 387-398; doi:10.3390/md11020387
OPEN ACCESS
Marine Drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Kocurin, the True Structure of PM181104, an
Anti-Methicillin-Resistant Staphylococcus aureus (MRSA)
Thiazolyl Peptide from the Marine-Derived Bacterium
Kocuria palustris
1
1
1
1
1
Jesús Martín , Thiciana da S. Sousa , Gloria Crespo , Sara Palomo , Ignacio González ,
1
1
2,†
2
José R. Tormo , Mercedes de la Cruz , Matthew Anderson , Russell T. Hill ,
1
1
1,
Francisca Vicente , Olga Genilloud and Fernando Reyes *
1
Fundación MEDINA, Centro de Excelencia en Investigación de Medicamentos Innovadores en
Andalucía, Avda. del Conocimiento 3, Granada 18016, Spain;
E-Mails: jesus.martin@medinaandalucia.es (J.M.); thicy85@yahoo.com.br (T.S.S.);
gloria.crespo@medinaandalucia.es (G.C.); sara.palomo@medinaandalucia.es (S.P.);
ignacio.gonzalez@medinaandalucia.es (I.G.); jose.tormo@medinaandalucia.es (J.R.T.);
mercedes.delacruz@medinaandalucia.es (M.C.); francisca.vicente@medinaandalucia.es (F.V.);
olga.genilloud@medinaandalucia.es (O.G.)
2
Institute of Marine and Environmental Technology, University of Maryland Center for
Environmental Science, 701 E Pratt Street, Baltimore, MD 21202, USA;
E-Mails: andersomenator@gmail.com (M.A.); hill@umces.edu (R.T.H.)
†
Current address: Algenol Biofuels, 16121 Lee Rd Suite 110, Fort Myers, FL 33912, USA.
*
Author to whom correspondence should be addressed; E-Mail: fernando.reyes@medinaandalucia.es;
Tel.: +34-958993965; Fax: +34-958846710.
Received: 11 December 2012; in revised form: 9 January 2013 / Accepted: 21 January 2013 /
Published: 4 February 2013
Abstract: A new thiazolyl peptide, kocurin (1), was isolated from culture broths of a
marine-derived Kocuria palustris. Its structural elucidation was accomplished using a
combination of spectroscopic and chemical methods, including HRMS, extensive 1D and
2
D NMR analysis, MS/MS fragmentation, and chemical degradation and Marfey’s analysis
of the resulting amino acid residues. The structure herein reported corrects that previously
assigned to PM181104 (3). Kocurin displayed activity against methicillin-resistant
Staphylococcus aureus (MRSA), with MIC values in the submicromolar range.

Mar. Drugs 2013, 11
388
Keywords: marine-derived bacteria; Kocuria palustris; thiazolyl peptides; structure
elucidation; antibacterial activity
1
. Introduction
The increasing resistance to known antibiotics developed by bacterial pathogens is one of the greatest
threats to human health worldwide. Methicillin-resistant Staphylococcus aureus (MRSA) infections
have received much attention and are still a major cause of death, killing more Americans every year
(~19,000) than emphysema, HIV/AIDS, Parkinson’s disease, and homicide combined [1]. Therefore,
there remains an urgent need to find new anti-MRSA antibiotics with novel modes of action.
Thiazolyl peptides constitute a class of naturally occurring compounds with potent in vitro activity
against Gram-positive bacteria. The structure of these polycyclic peptides contains a central
pyridine/tetrahydropyridine ring, with up to three thiazolyl substituents at the 2-, 3-, and 6-positions of
the central heterocycle. This structural class includes close to 100 molecules [2], with the first member,
micrococcin, isolated in 1948 [3], and some new members of the thiazomycin family reported as
recently as 2009 [4]. Despite its interesting in vitro submicromolar antibacterial activity against MRSA,
the development of this class of compounds as clinical agents has been hampered due to extremely poor
physicochemical properties, most notably low aqueous solubility and unfavorable pharmacokinetics.
As part of our continuing program for the search of new bioactive molecules, 44 bacterial strains of
the family Micrococcaceae isolated from marine sponges collected in the Florida Keys (USA) were
surveyed for the presence of genes encoding non ribosomal peptide synthetase (NRPS) and polyketide
synthase (PKS) enzymes and their fermentation extracts were tested for antibiotic activity against
clinically relevant strains: Gram-positive bacteria (Bacillus subtilis and methicillin-resistant
Staphylococcus aureus), Gram-negative bacteria (Acinetobacter baumannii) and yeast (Candida albicans).
Growth inhibition of MRSA was observed in acetone extracts of three of these strains [5]. This
antibacterial activity was reproduced in new fermentations and the active component, kocurin (1)
(Figure 1), was isolated from one of these strains belonging to the genus Kocuria by reversed phase C18
chromatography and semipreparative HPLC, and was identified as a new member of the thiazolyl
peptide family of antibiotics. The structural elucidation of kocurin was accomplished using a
combination of spectroscopic and chemical methods, including HRMS, extensive 1D and 2D NMR
analysis, and chemical degradation and Marfey’s analysis of the resulting amino acid residues.
2
. Results and Discussion
The producing bacterium, K. palustris, was fermented for 1 day in R358 medium. A 7 L fermentation
was centrifuged and the cell pellet was extracted with methanol (3 × 50 mL). This extract was
chromatographed on a reversed phase C18 column using a gradient H O/MeOH and the bioactive
2
fractions were finally purified by repeated semipreparative and preparative HPLC to yield 1.4 mg of 1 as
a white solid.
Kocurin (1) was assigned a molecular formula of C H N O S by ESI-TOF mass spectrometry
69
66 18 13 5
+
(
m/z 1515.3739, calc. for [M + H] 1515.3733). Two absorption maxima in the UV spectrum at 218,

Mar. Drugs 2013, 11
389
3
07, and a shoulder at 349 nm together with the presence of five sulphur atoms in the molecular formula
1
of 1 identified the compound as a member of the thiazolyl peptide class. The H NMR spectrum (Table 1)
displayed downfield singlets due to the presence of four thiazoles (7.45, 7.86, 8.01 and 8.09 ppm) and
two doublets characteristic of a 2,3,6-trisubstituted pyridine ring (8.24 and 8.36 ppm). Other low field
proton signals were attributable to protons of the aromatic rings of phenylalanine (7.17, 7.23 and 7.18 ppm)
and tyrosine units (6.45 and 6.60 ppm). Three pairs of methylene groups resonating at 5.52 and 6.69 ppm,
5
1
.48 and 6.54 ppm, and 5.38 and 6.58 ppm correlated in the HSQC spectrum with carbons at 102.8,
03.6 and 103.5, respectively, accounting for three dehydroalanine residues. Correlations observed in
the HMBC spectrum from these three pairs of proton signals to carbons at 133.9 and 163.1, 134.4 and
1
62.0, and 132.9 and 165.3 corroborated the identity of these three structural units.
Figure 1. Structures of kocurin (1), GE 37468 A (2) and PM181104 (3).

Mar. Drugs 2013, 11
390
1
13
Table 1. H and C NMR data (500 and 125 MHz, CDCl ) for Kocurin (1).
3
No.
1
δ (C)
150.8
138.6
118.6
122.5
151.4
155.8
129.9
154.2
12.1
δ (H), Multiplicity, J
δ (C)
117.6
131.1
170.5
61.7
δ (C), Multiplicity, J
6.45, d, 8.2
39
40
41
42
43
44
45
46
47
48
49
50
2
8.36, d, 8.2
8.24, d, 8.2
6.60, d, 8.2
3
4
5.33, t, 7.5
2.58, m; 2.07, m
2.37, m; 2.17, m
3.99, m, 2H
5
34.2
6
25.7
7
48.2
8
173.5
148.6
114.0
159.7
154.2
9
2.88, s, 3H
1
1
1
0
1
2
163.1
7.45, s
8.01, s
8.65, d, 9.7
4.90, m
47.7
37.6
2
.16, m
13
51
122.5
0
.50, dd, 17.4, 3.0
1
1
1
1
1
1
2
4
5
6
7
8
9
0
172.3
171.9
149.0
122.4
160.3
52
53
54
55
56
57
58
167.9
151.4
127.0
159.4
8.29, s
10.05, s
7.86, s
8.35, d, 6.4
5.13, m
133.9
102.8
53.5
39.0
6.69, br s; 5.52, br s
3
.58, m
2
1
59
163.1
3
.36, dd, 14.0, 3.1
2
2
2
2
2
2
2
2
3
3
3
2
3
4
5
6
7
8
9
0
1
2
135.7
129.9
128.4
127.2
128.4
129.9
172.9
78.3
60
61
62
63
64
65
66
67
68
69
70
7.44, d, 7.4
4.90, m
7.23, d, 7.8
7.28, t, 7.8
7.17, t, 7.8
7.28, t, 7.8
7.23, d, 7.8
47.6
18.4
172.3
61.1
27.8
25.1
47.3
169.6
1.55, d, 6.8, 3H
4.71, dd, 7.8, 3.3
2.38, m; 2.09, m
2.17, m; 2.09, m
3.71, m, 2H
4.83, ddd, 13.3, 8.7, 2.6
3.76, m; 3.58, m
35.9
169.1
8.89, s
7.40, d, 9.3
5.21, m
134.4
103.6
6.54, d, 1.9
3
3
3
4
51.4
36.4
71
72
5.48, br s
3
2
.14, dd, 14.1, 3.0
.94, dd, 14.1, 4.5
162.0
3
3
5
6
125.6
131.1
73
74
8.86, s
6.60, d, 8.2
6.45, d, 8.2
132.9
103.5
165.3
6
.58, d, 1.4
3
3
7
8
117.6
155.6
75
76
5
.38, br s

Mar. Drugs 2013, 11
391
1
Other building blocks identified in the H NMR spectrum and confirmed via COSY, HSQC and
HMBC correlations were an asparagine unit (8.65, 4.90, 2.56 and 0.50 ppm), an alanine (7.44, 4.90 and
1
.55 ppm), two prolines and a thiazoline ring (4.83, 3.76, and 3.58 ppm). The unusual low field chemical
shift of the methine carbon of the latter residue (78.3 ppm) was due to the presence of a cyclic secondary
ketimine bond in addition to the downfield effect of the adjacent carbonyl group. Finally, one
methyloxazole residue was also present in the structure of 1 as revealed by the presence of a singlet
methyl group at δ 2.88 ppm that correlated in the HMBC spectrum to 3 carbons at δ 129.9 (C-7),
H
C
1
55.8 (C-8) and 163.1 (C-10, weak, 4-bond correlation).
Once all the subunits present in the structure of the new peptide were identified, the sequence of
amino acids was established by analysis of key correlations observed in the HMBC spectrum (Figure 2)
and MS/MS analysis. HMBC correlations confirmed that the central pyridine nucleus was surrounded
by two of the thiazolyl and the methyloxazole units and allowed the planar structure of the compound to
be established as depicted in Figure 2. Additional support for the sequence determined by HMBC came
from the analysis of key fragments observed in the MS/MS spectrum of the molecule. Thus, the
sequence Dha-Dha-Pro-Ala for the C-terminal amino acid side chain was determined from fragments at
m/z 1498, 1429, 1360, 1263, and 1192 (Figure 2). The planar structure of kocurin was found to be similar
to that of the antibiotic peptide GE 37468 A (2) [6,7], the major structural differences found between
both compounds being the replacement of the methylhydroxyproline unit in the cycle of 2 by proline in 1
and the presence of additional amino acid units in the side chain of the latter compound.
Figure 2. Key HMBC correlations and MS/MS fragments observed in the spectra of
kocurin (1).

Mar. Drugs 2013, 11
392
The absolute configurations of the amino acid residues in kocurin were determined by application of
the Marfey’s methodology [8]. A hydrolysis of the sample with HCl 6 N (110 °C, overnight) and
derivatization of the hydrolysate with 1-fluoro-2,4-dinitrophenyl-5-L-valinamide (L-FDVA) revealed
the presence of L-Tyr, L-Pro (×1) and L-Ala on the basis of comparison of their retention times with those
of derivatized standards. In order to liberate those amino acids adjacent to thiazole rings to determine
their absolute configurations, it was necessary to cleave these by ozonolysis prior to hydrolysis.
Treatment of a solution of 1 in CHCl /MeOH with ozone for 10 min followed by acid hydrolysis with
3
6
N HCl (110 °C, overnight) and derivatization with L-FDVA identified L-Asp, L-Phe, L-Cya, L-Pro (×2)
and L-Ala as components of the hydrolyzate by LC/MS. Tyr was not found in this analysis, probably due
to its decomposition during the ozonization of the peptide. The ozonolysis of the sample prior to the acid
hydrolysis had the additional advantage of reducing the epimerization of the thiazoline present in the
molecule, which has been described to readily occur in the presence of mild acid or base. A 1.6:1 ratio of
L- and D-Cya was obtained in the LC/MS analysis, confirming L-Cys as the residue present in the
1
13
structure of kocurin. The L-Cys assignment was also supported by comparative H and C NMR data of
the thiazoline in 1 to those reported for other molecules containing the same residue.
Once the complete structure of the compound had been determined, a literature search revealed the
existence of two patents describing compounds having the same molecular formula as kocurin, the
thyazolyl peptide PM181104 (3), isolated from a marine-derived Kocuria sp. (ZMA B-1/MTCC 5269) [9]
and a molecule with the same planar structure as kocurin obtained from Kocuria sp. Strain
MI-67-EC3-038, isolated from a marine sample collected in the Southeast coast of Spain [10]. Although
no explanation is given in the first patent about the rationale followed in the structural elucidation of 3,
we believe that kocurin and PM181104 are actually the same molecule. Although the same residues can
be identified by NMR in both structures, on the basis of biogenetic grounds we firmly believe that the
structure proposed for 3 is not correct, probably due to a misinterpretation of some HMBC correlations
observed for carbons resonating very closely, and that the actual compound isolated in the work
described in the patent has the structure herein proposed for kocurin. It is well known that thiazolyl
peptides arise from a cascade of post-translational modifications on 50 to 60 residue pre-peptide
precursors that trim away the N-terminal leader sequences (40 residues) while at the C-terminal,
1
4 to 18 residues are converted into the mature scaffold [11]. In this sense, kocurin would be the result
of post-translational modifications of a linear C-terminal peptide containing 17 amino acids
Ser-Thr-Asn-Cys-Phe-Cys-Tyr-Pro-Cys-Cys-Ser-Cys-Ser-Ala-Pro-Ser-Ser) whereas the biosynthesis
of PM181104 would require the participation of two independent peptide units of
3 (Cys-Phe-Cys-Tyr-Pro-Cys-Asp/Asn-Cys-Ser-Thr-Pro-Ser-Ser) and 4 (Ser-Cys-Ser-Ala) amino acid
(
1
residues (Figure 3). A second inconsistency in the structure proposed for compound 3 is the presence of
two C-terminal amino acids. Due to this, the MS/MS fragmentation pattern generated by this compound
should contain additional ions to those observed for 1, originated by losses of amino acids from both side
chains. Among others, ions at m/z 1427 and 1358 accounting for the elimination of the terminal alanine
and alanine-DHA residues in PM181104, not observed in the spectrum of kocurin, should appear in the
MS/MS spectrum of the molecule. Unfortunately, no MS/MS fragmentation of PM181104 is reported in
the patent and only the molecular ion of the compound is included as part of the structural information.
1
Finally, a comparison of the H NMR in DMSO-d of kocurin with that reported in the patent for
6
PM181104 definitely confirms that both molecules are identical.

Mar. Drugs 2013, 11
393
The biological activity of kocurin was tested against a panel of bacterial and fungal pathogens,
including wild-type MRSA and S. aureus strains resistant to thiazomycin, other Gram-positive
(Bacillus subtilis and Enterococcus faecium) and Gram-negative (Escherichia coli, Acinetobacter
baumannii and Pseudomonas aeruginosa) bacteria and one fungus (Candida albicans). Kocurin
strongly inhibited the growth of MRSA MB5393 with a MIC value of 0.25 μg/mL. In addition, the
compound also displayed antibacterial activity against B. subtilis and E. faecium in a solid agar test, with
zones of inhibition (ZOI) of 9 and 10 mm when tested at a concentration of 8 μg/mL. On the other hand,
the compound displayed ZOI of 5 mm when a 10 μL aliquot of a 2 μg/mL solution was spotted on plates
containing cultures of both, wild-type and thiazomycin resistant S. aureus strains, revealing that the
mechanism of action of the compound is different from that of thiazomycin. Finally, kocurin did not
inhibit the growth of Gram-negative bacteria or C. albicans when tested at a concentration of 16 μg/mL.
Figure 3. Structural units required for the biosynthesis of kocurin (1) and PM181104 (3).
3
. Experimental Section
3
.1. General Experimental Procedures
Optical rotations were determined using a JASCO P-2000 polarimeter. UV spectra were obtained
with an Agilent 1200 DAD. NMR spectra were recorded on a Varian “INOVA 500” spectrometer at
1
13
1
5
7
00/125 MHz ( H/ C). Chemical shifts were reported in ppm using residual CDCl (δ 7.26 for H and
3
1
3
3
7.0 for C) as internal reference. HMBC experiments were optimized for a J
of 8 Hz.
CH
(
+)-ESI-TOFMS was performed on a Bruker maXis spectrometer.
.2. Producing Microorganism
The producing strain F-276,345 was obtained from a sponge sample collected in Florida Keys, USA [12].
3
Upon phylogenetic analysis based on partial 16S rRNA gene sequences of related type species, the strain
F-276,345 was identified as a new member of the species Kocuria lacustris [5].

Mar. Drugs 2013, 11
.3. Fermentation of the Producing Microorganism
A 7.2 liter fermentation of Kocuria palustris F-276,345 was obtained as follows: a first seed culture
394
3
of the strain F-276,345 was prepared by inoculating three 50 mL tubes containing 12 mL each of MY
seed medium (D-(+)-Glucose 10 g/L, Bacto Yeast Extract 3 g/L, Proteose-peptone 5 g/L, Malt Extract
3
g/L, adjusted to pH 7.0), with 0.5 mL of a frozen inoculum stock of the producing strain and incubating
the tube at 28 °C with shaking at 220 rpm for about 24 h. A second seed culture was prepared by
inoculating eight 250 mL flasks containing 50 mL each of MY seed medium with 2.5 mL of the first
seed. A 5% aliquot of the second seed culture was transferred to each of the forty-eight 500 mL flasks
containing 150 mL of the production medium R358, which is a modified version of the one used by
Jensen et al. [13], which consists of starch from potato 10 g/L, Bacto Yeast Extract 4 g/L, Bacto Peptone
2
g/L, FeSO .7H O (5 mL of a 8 g/L stock solution), and KBr (5 mL of a 20 g/L stock solution) at
4 2
pH 7.0. The flasks were incubated at 28 °C for 1 day in a rotary shaker at 220 rpm and 70% humidity
before harvesting.
3
.4. Extraction and Isolation
The culture broth was centrifuged for 15 min at 8500 rpm and the supernatant was discarded. The cell
pellet was extracted with MeOH (3 × 50 mL), filtered and the solvent was evaporated to dryness. The
dried extract was subjected to reversed phase flash chromatography (15.5 g of RediSep C18, gradient
from 20% MeOH to 100% MeOH in 12.5 min + 100% MeOH for 10 min, 10 mL/min, UV detection at
2
10 and 305 nm) yielding a fraction eluting between 12.5 and 14.4 min that was subjected to
semipreparative HPLC (X-Bridge Phenyl, 10 × 15 mm, 5 μm, gradient H O–CH CN from 25% to 80%
2
3
CH CN in 35 min, 3.6 mL/min, UV detection) to yield 1.4 mg of kocurin (1) as a white amorphous solid.
3
2
5
Data for kocurin (1): White amorphous solid; [α] +27.0 (c 0.11, CHCl /MeOH 1:1); UV (DAD)
D
3
−
1
1
13
λmax 218, 307, 349 (sh) nm; IR (ATR) νmax 3342, 2922, 2853, 1649, 1512, 1427 cm ; H and C NMR:
+
see Table 1; (+)-ESI-TOFMS m/z 1515.3739 [M + H] (calc. for C H N O S , 1515.3733, Δ 0.3 ppm).
69
66 18 13 5
3
.5. Ozonolysis, Acid Hydrolysis, and Marfey’s Analysis of Kocurin (1)
A sample of kocurin (0.1 mg, 50 mM) was treated with 6 N HCl in a sealed vial at 110 °C for 24 h.
The solution was concentrated to dryness in vacuo. The hydrolysate was reconstituted in H O (50 μL)
2
and treated with a solution of 1-fluoro-2,4-dinitrophenyl-5-L-valine-amide (L-FDVA, 150 μL, 1% in
acetone) and a 1 M solution of NaHCO (20 μL) in a sealed vial at 40 °C for 1 h. The reaction mixture
3
was neutralized with 1 N HCl (20 μL) and an aliquot (10 μL) was diluted with CH CN (40 μL). The
3
resulting solution was analyzed by LC-MS employing a Waters X-Bridge C18 column (4.6 × 150 mm,
5
μm) and a gradient elution profile of 10% B (90% CH CN, 10% H O, 1.3 mM TFA, 1.3 mM
3 2
ammonium formiate)/90% A (10% CH CN, 90% H O, 1.3 mM TFA, 1.3 mM ammonium formiate) to
3
2
5
5% B/45% A over 35 min at flow of 1 mL/min. The hydrolysate of kocurin contained L-Ala (13.7 min),
L-Pro (14.9 min) and L-Tyr (30.5 min). The retention time of the L-FDVA derivatives of the authentic
amino acids were as follows: L-Ala (13.8 min), D-Ala (18.3 min), L-Pro (14.9 min), D-Pro (18.1 min),
L-Tyr (30.6 min) and D-Tyr (34.8 min).

Mar. Drugs 2013, 11
395
A second sample (0.2 mg) of the peptide was dissolved in 2 mL of CHCl /CH OH, and ozone was
3
3
bubbled through the solution for 10 min. This solution was evaporated to dryness on a rotary evaporator.
Subsequent hydrolysis and Marfey’s analysis of the resulting residue was performed as described above.
The resulting solution was analyzed by LC-MS employing a Waters X-Bridge C18 column (4.6 × 150 mm,
5
μm) and a gradient elution profile of 10% B/90% A to 35% B/65% A over 35 min and then to 100% B
in 1 min and held at 100% B for 4 min using a flow of 1 mL/min. The hydrolysate of the ozonolysis
product gave L-Ala (19.0 min), L-Asp (14.1 min), L-Phe (35.2 min) and L-Pro (21.4 min). The retention
time of the L-FDVA derivatives of the authentic amino acids were as follows: L-Ala (19.0 min), D-Ala
(28.5 min), L-Asp (14.1 min), D-Asp (16.4 min), L-Phe (34.6 min), D-Phe (37.8 min), L-Pro (20.8 min)
and D-Pro (27.3 min). A second chromatographic method using the same solvent system as the previous
methods (Waters X-Bridge C18 column (4.6 × 150 mm, 5 μm) and isocratic elution 3% B/97% A over
3
5 min at flow of 1 mL/min) was used for the identification of the L-Cya residue (17.4 min)
Cya L:D, 1.5:1). The retention times of the L-FDVA derivatives of cysteic acid were: L-Cya (17.4 min),
D-Cya (18.5 min).
(
3
.6. Antifungal Assays
Frozen stocks of C. albicans were used to inoculate Sabouraud Dextrose Agar (SDA) plates for
confluent growth. Plates were incubated for 24 h, at 35 °C. The grown colonies were harvested from the
SDA plates and suspended in RPMI-1640 modified medium. Modified RPMI-1640 medium was
prepared as follows: 20.8 g of RPMI powder (Sigma) were poured into a 2 L flask, together with 13.4 g
of YNB, 1.8 L of milliQ water, 80 mL of 1 M HEPES and 72 mL of glucose 50%. The volume was
adjusted to 2 L and filtered. The OD660 was adjusted to 0.25 using RPMI-1640 modified as diluent and
blank. This inoculum was diluted 1:10 and kept on ice until used to inoculate 96-well microtiter plates.
For the assay, 90 μL/well of the 1:10 diluted inoculum were mixed with 1.6 μL/well of compound
solution in DMSO and 8.4 μL/well of RPMI-1640 modified medium. Amphotericin B and Penicillin G
were used as internal positive and negative controls respectively. After dispensing the inocula, the
samples and the controls, the assay plates were read in a Tecan Ultraevolution spectrophotometer at
6
12 nm for T (zero time). Then, the plates were statically incubated at 37 °C for 20 h. After incubation,
0
the plates were shaken in a DPC Micromix-5 and read again for T (final time). Percentage of growth
f
inhibition was calculated using the following equation:
%
Inhibition = 100 × {1 − [(Tf Sample − T0 Sample) − (Tf Blank − T0 Blank)]/[(Tf Growth − T0 Growth) −
(1)
(Tf Blank − T0 Blank)]}
Compound 1 was serially diluted in DMSO with a dilution factor of 2 to provide 10 concentrations
starting at 160 μg/mL. The MIC was defined as the lowest concentration of an antimicrobial or
antifungal compound that inhibited ≥95% of the growth of a microorganism after overnight incubation.
The data were analysed using the Genedata Screener program (Genedata AG, Switzerland). In all
experiments performed in this work the RZ’ factor obtained was between 0.85 and 0.95.

Mar. Drugs 2013, 11
.7. Antibacterial Assays
For the liquid media antibacterial tests, thawed stock inocula suspensions from cryovials of each
396
3
microorganism (MRSA, A. baumannii, E. coli and P. aeruginosa) were streaked onto Luria-Bertani agar
plates (LBA, 40 g/L) and incubated at 37 °C overnight to obtain isolated colonies. Single colonies of
each microorganism were inoculated into 10 mL of Luria-Bertani broth medium (LB, 25 g/L in 250 mL
Erlenmeyer flasks) and incubated overnight at 37 °C with shaking at 220 rpm and then diluted in order to
6
5
obtain assay inocula of approximately 1.1 × 10 CFU/mL (MRSA) or 5–6 × 10 CFU/mL (A. baumannii,
E. coli and P. aeruginosa).
For the assay 90 μL/well of the diluted inoculum were mixed with 1.6 μL/well of each compound
dissolved in DMSO and 8.4 μL/well of LB medium. Kanamycin and amphotericin B (MRSA),
rifampicin and amphotericin B (A. baumannii), novobiocin and amphotericin B (E. coli), and
ciprofloxacin and amphotericin B (P. aeruginosa) were included as internal plate controls. Absorbance
at 612 nm was measured with a Tecan UltraEvolution spectrophotometer (Tecan, Durham, USA) at
T0 (zero time) and immediately after that, plates were statically incubated at 37 °C for 20 h. After this
period, the assay plates were shaken using the DPC Micromix-5 and once more the absorbance at OD
6
12 nm was measured at T (final time). Percentage inhibition of growth was calculated using the same
f
equation previously described for C. albicans.
Assays in solid agar plates were performed in order to determine the antimicrobial susceptibility of
kocurin against B. subtilis MB964, E. faecium MB5571 resistant to vancomycin and β-lactam antibiotics
and thiazomycin-resistant S. aureus strains. The B. subtilis MB964 assay plates were prepared by adding
8
1
mL (1.5 × 10 CFU/mL) of a spore suspension (Difco) directly to 1 liter of cooled nutrient agar yeast
extract medium (NAYE, nutrient agar 23 g/L, yeast extract 2 g/L). The E. faecium strain was grown in a
shaken (220 rpm) culture of brain heart infusion broth (BHI, 37 g/L) at 37 °C. The assay plates were
prepared by inoculating the same cooled media plus agar (15 g/L) with 3.3% of the inoculum adjusted to
an optical density (OD) range of 0.22–0.35 at 660 nm. The wild-type S. aureus Smith MB2865 and
thiazomycin-resistant S. aureus MB5832 strains were grown in 10 mL of LB medium overnight.
Overnight growth was adjusted to an optical density of 0.2 at 600 nm. The suspension was then added to
molten LB agar in the proportion of 25 mL/L. The activity was determined by measuring the differences
of the zone of clearances observed between the wild type versus the resistant plate. Compound 1 was
serially diluted in DMSO 20% with a dilution factor of 2 to provide 10 compound concentrations starting
at 128 μg/mL for all the agar assays. Ten microliters of each compound concentration were applied to
the agar plate containing the pathogenic microorganism.
4
. Conclusions
A thiazolyl pepetide bioactive against MRSA, kocurin (1), has been isolated from cultures of the
marine sponge-derived bacterium Kocuria palustris. Its challenging structural elucidation was
completed using a combination of spectroscopic and chemical methods, including HRMS, extensive 1D
and 2D NMR analysis, MS/MS fragmentation, and chemical degradation and Marfey’s analysis of the
resulting amino acid residues. The structure herein proposed for the compound corrects that previously
reported for PM181104 in a US patent. The identification of a new bioactive compound in a member of

Mar. Drugs 2013, 11
397
the Microccocaceae suggests that this family of actinomycetes represents an alternative source for the
discovery of new medically relevant small molecules. The relative abundance in marine environments,
intra-familiar diversity, and ease of growth of these bacteria makes them an attractive target for the
discovery of new antibiotics.
Acknowledgments
The authors thank the assistance of Catalina Moreno in the preparation of the extracts and Dr. Ignacio
Pérez-Victoria for recording the NMR experiments. The polarimeter, IR and NMR equipment used
in this work were purchased via two grants for scientific and technological infrastructures
from the Ministerio de Ciencia e Innovación [Grants No. PCT-010000-2010-4 (NMR) and
INP-2011-0016-PCT-010000-ACT6 (Polarimeter and IR)]. This is contribution No. 13104 from the
Institute of Marine and Environmental Technology and contribution No. 4728 from the University of
Maryland Center for Environmental Science.
References
1
.
Klevens, R.M.; Morrison, M.A.; Nadle, J.; Petit, S.; Gershman, K.; Ray, S.; Harrison, L.H.;
Lynfield, R.; Dumyati, G.; Townes, J.M.; et al. Invasive methicillin-resistant Staphylococcus aureus
infections in the United States. JAMA 2007, 298, 1763–1771.
2
3
4
.
.
.
Bagley, M.C.; Dale, J.W.; Merritt, E.A.; Xiong, X. Thiopeptide antibiotics. Chem. Rev. 2005, 105,
6
85–714.
Su, T.L. Micrococcin, an antibacterial substance formed by a strain of Micrococcus. Br. J. Exp.
Pathol. 1948, 29, 473–481.
Zhang, C.; Herath, K.; Jayasuriya, H.; Ondeyka, J.G.; Zink, D.L.; Occi, J.; Birdsall, G.;
Venugopal, J.; Ushio, M.; Burgess, B.; et al. Thiazomycins, thiazolyl peptide antibiotics from
Amycolatopsis fastidiosa. J. Nat. Prod. 2009, 72, 841–847.
5
.
Palomo, S.; González, I.; de la Cruz, M.; Martín, J.; Tormo, J.R.; Anderson, M.; Hill, R.T.; Vicente, F.;
Reyes, F.; Genilloud, O. Sponge-derived Kocuria and Micrococcus spp. as new sources of thiazolyl
peptide antibiotics. Mar. Drugs 2013, submitted.
6
7
.
.
Ferrari, P.; Colombo, L.; Stella, S.; Selva, E.; Zerilli, L.F. Antibiotic GE37468 A: A novel inhibitor
of bacterial protein synthesis. II. Structure elucidation. J. Antibiot. 1995, 48, 1304–1311.
Stella, S.; Montanini, N.; Le Monnier, F.; Ferrari, P.; Colombo, L.; Marinelli, F.; Landini, P.;
Ciciliato, I.; Goldstein, B.P.; Selva, E.; et al. Antibiotic GE37468 A: A new inhibitor of bacterial
protein synthesis. I. Isolation and characterization. J. Antibiot. 1995, 48, 780–786.
Marfey, P. Determination of D-amino acids. II. Use of a bifunctional reagent,
8
9
.
.
1
,5-difluoro-2,4-dinitrobenzene. Carlsberg Res. Commun. 1984, 49, 591–596.
Mahajan, G.B.; George, S.D.; Ranadive, P.V.; Mishra, P.D.S.; Eyyammadichiyil, S.S.;
Panshikar, R.M.; Sawant, S.N.; Krishna, S.; Sivakumar, M.; Pari, K.; et al. PM181104 and Related
Antibacterial Compounds, Production, Pharmaceutical Compositions, and Therapeutic Use. PCT
Int. Appl. WO 2007119201A2, 25 October 2007.

Mar. Drugs 2013, 11
398
1
1
0. Cañedo Hernández, L.M.; Romero Millán, F.; Fernández Medarde, A.; Fernández Chimeno, R.I.;
Hidalgo Villar, J.C. Peptides as bioactive compounds. PCT Int. Appl. WO 2012062906A1,
8 May 2012.
1
1. Walsh, C.T.; Acker, M.G.; Bowers, A.A. Thiazolyl peptide antibiotic biosynthesis: A cascade of
post-translational modifications on ribosomal nascent proteins. J. Biol. Chem. 2010, 285,
2
7525–27531.
1
1
2. Montalvo, N.; Mohamed, N.; Enticknap, J.; Hill, R. Novel actinobacteria from marine sponges.
Antonie Van Leeuwenhoek 2005, 87, 29–36.
3. Jensen, P.R.; Williams, P.G.; Oh, D.-C.; Zeigler, L.; Fenical, W. Species-specific secondary
metabolite production in marine actinomycetes of the Genus Salinispora. Appl. Environ. Microbiol.
2
007, 73, 1146–1152.
Samples Availability: Not available.
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2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
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