Journal of Molecular Structure 1297 (2024) 137008

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Journal of Molecular Structure

journal homepage: www.elsevier.com/locate/molstr

Marihysin B, a new cyclic lipopeptide from culture broth of Staphylococcus

sp. - antimicrobial and glucoamylase inhibitory activities
Amgad I.M. Khedr a, *, Gamal A. Mohamed b, Sabrin R.M. Ibrahim c, d, *,
Reda F.A. Abdelhameed e, f, Tagyedeen H. Shoaib g, Abdulrahim A. Alzain g, Koji Yamada h,
Mohamed S. Refaey i
a
Department of Pharmacognosy, Faculty of Pharmacy, Port Said University, Port Said 42526, Egypt
b
Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia
c
Department of Chemistry, Batterjee Medical College, Jeddah 21442, Saudi Arabia
d
Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt
e
Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt
f
Department of Pharmacognosy, Faculty of Pharmacy, Galala University, New Galala 43713, Egypt
g
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Gezira, Wad Madani 21111, Sudan
h
Garden for Medicinal Plants, Graduate School of Biomedical Sciences, Nagasaki University, Bunkyo-machi 1-14, Nagasaki 852-8521, Japan
i
Department of Pharmacognosy, Faculty of Pharmacy, University of Sadat City, Sadat City, Menoufia 32897, Egypt

A R T I C L E I N F O A B S T R A C T

Keywords: A new cyclic lipopeptide, marihysin B (1) and the well-known marihysin A (2) were isolated from Staphylococcus
Staphylococcus sp. sp. culture broth obtained from Corallina officinalis L. Extensive 1D (1H and 13C) and 2D (1H–1H COSY, HSQC,
Marihysin B HMBC, and NOESY) NMR spectral investigations were used to determine the structures of isolated compounds.
Cyclic lipopeptide
With inhibition zone widths of 11 to 25 mm and MICs of 25 µg/mL, compounds 1 and 2 showed marked anti­
Antimicrobial
Marine environment
fungal activity against Aspergillus niger, Penicillum crustosum, Trichophyton concentricum, and Schizophyllum
Life below water commune compared to nystatin (inhibition zone diameters 23–31 mm). Additionally, computational study was
Molecular docking done to determine the possible mechanism underlying the antifungal activity of the two cyclic lipopeptides
Drug discovery against Schizophyllum commune glucoamylase. These substances showed similar binding affinities to the target’s
Glucoamylase reference compound.

1. Introduction creation of new pharmaceuticals. [10,11] Peptides and modified pep­

Diverse habitats in marine environments are found in a variety of
hydrostatic pressure, temperature, and salinity conditions. [1] Marine
microorganisms have evolved diverse adaption techniques, such as the
creation of certain biometabolites, to flourish in such conditions.[1–6]
Marine microorganisms have gained popularity as a possible source of
several biologically active substances, stimulated the interest of
biomedical researchers for the creation of various drugs and have
attracted a large number of these compounds. [5,7-9] Cultured marine
bacteria have become a rich source of secondary metabolites with novel
structural characteristics and biological activity, which may help in the
tides produced by marine microorganisms are of particular interest.[7]
amongst them, Cyclic lipopeptides demonstrated a variety of bio­
activities, including antifungal, anticancer, and anti-inflammatory
properties.[12–14] Our previous research on Staphylococcus sp. led to
isolation of various metabolites belonging to different classes as dike­
topiperazines,[15] cyclic tetrapeptide, [16] and cyclic depsipeptide.
[17] As part of our continuous investigation for new and/or bioactive
metabolites from marine bacteria, Staphylococcus sp. (No. P-100,
826–4–6) was further chemically investigated, resulting in the isolation
of a new cyclic lipopeptide termed marihysin B (1), together with the
known compound; marihysin A (Fig. 1).

All authors have read and agreed to the published version of the manuscript.
* Corresponding authors.
E-mail addresses: a_mansour7799@yahoo.com (A.I.M. Khedr), gahussein@kau.edu.sa (G.A. Mohamed), sabrin.ibrahim@bmc.edu.sa (S.R.M. Ibrahim),
abdelhameed@pharm.suez.edu.eg (R.F.A. Abdelhameed), Shoaibth37@hotmail.com (T.H. Shoaib), abdulrahim.altoam@gmail.com (A.A. Alzain), kyamada@
nagasaki-u.ac.jp (K. Yamada), mohamed.said@fop.usc.edu.eg (M.S. Refaey).

https://doi.org/10.1016/j.molstruc.2023.137008
Received 6 September 2023; Received in revised form 25 October 2023; Accepted 3 November 2023
Available online 4 November 2023
0022-2860/© 2023 Elsevier B.V. All rights reserved.
A.I.M. Khedr et al.
Here, we discussed how separated metabolites were isolated, their
structural elucidation, and their antibacterial bioassay. The outcomes of
our antimicrobial evaluations also showed that the evaluated extracts
lacked antibacterial activity. However, a wide spectrum of antifungal
activity was demonstrated by these extracts. In light of these results, our
research uses computational methods to explore the probable binding
processes of these candidate compounds. To accomplish this, glucoa­
mylase was chosen as a target enzyme. This selection is significant as
glucoamylase plays a pivotal role as a major allergen in Schizophyllum
commune fungal infections, underscoring the importance of under­
standing its interaction with the compounds in question.
2. Materials and methods
2.1. General experimental procedures
IR spectra were obtained with JASCO FT/IR-410 spectrophotome­
ters. With the use of a JASCO DIP-370 digital polarimeter, optical ro­
tations were measured. A Unity plus 500 spectrometer (Varian Inc., U.S.
A.) was used to record NMR spectra. Coupling constants are expressed in
Hz, while 1H NMR chemical shifts are expressed in δ values relating to
the solvent peaks at δH 7.19, 7.55, and 8.71 for C5D5N. Chemical shifts
for C5D5N solvent peaks at δC 123.5, 135.5, and 149.9 are used in 13C
NMR chemical shift expressions. Column chromatography was per­
formed using Sephadex LH-20 (Pharmacia Fine Chemical Co.Ltd, Lin­
cang, China), Wakogel C-300 (Wako Pure Chemical Industries, Ltd,
Osaka, Japan, 45 75 m), and Cosmosil 5C18–140 PREP (Nakarai Tesque,
No.379–34). TLC analysis was carried out using RP-18 F254s plates and
precoated silica gel plates (Kieselgel 60 F254, 0.25 mm, Merck, Darm­
stadt, Germany). JMS DX-303 spectrometer (JEOL Ltd., Japan) was used
to record HRFABMS spectra. On a Develosil C-30-UG-5 (250×4.6 mm i.
d., Nomura Chemical Co., Aichi, Japan) with a TOSOH RI-8020 detector
and a JASCO BIP-I HPLC pump, preparative HPLC was carried out at a
flow rate of 1.5 mL/min. We bought standard amino acids from the
Tokyo, Japan-based Sigma-Aldrich Chemical Co.
2.2. Bacterial material and fermentation
Corallina officinalis L. (Corallinaceae) was isolated from the marine
Staphylococcus sp. (strain number P-100,826–4–6), which was discov­
ered in 2010 on the Nagasaki Shitsu coast of Japan. The Garden for
Medicinal Plants, Graduated School of Biomedical Sciences, Nagasaki
University is home to the bacterial subcultures. The bacterium was
cultured using rotary-shaking at 120 rpm for 28 days at 25 ºC in a
Fig. 1. Structures of isolated compounds (1 and 2).
2
Journal of Molecular Structure 1297 (2024) 137008
seawater-based medium (D-glucose 1 %/polypeptone 0.5 %/yeast
extract 0.3 %/KH2PO4 0.3 %/MgSO4 0.1 %/pH 7.5). The 32 L of culture
broth was sonicated and then filtered. By utilizing EtOAc, the filtrated
broth (32 L) was divided into an EtOAc layer and an aqueous layer.
2.3. Extraction and isolation
Under reduced pressure, the entire EtOAc layer (30 L) was condensed
to yield 5.2 g of extract. Under reduced pressure, the aqueous layer was
concentrated to produce 5 liters of aqueous suspensions which were then
chromatographed on diaion HP-20 column with H2O, 60 % aq. MeOH,
100 % MeOH, and acetone, respectively to give 60 % aq. MeOH (28.3 g),
100 % MeOH (8.0 g), and acetone (3.9 g) fractions. The MeOH fraction
(8.0 g) was dissolved in a 1:1:0.2, v/v/v mixture of CHCl3, MeOH, and
H2O to yield 4.6 g of soluble part and 3.3 g of insoluble part. On
Sephadex LH-20, the soluble portion (4.6 g) was chromatographed with
(CHCl3:MeOH:H2O; 5:5:1 v/v/v) to produce seven fractions (A-G). ODS
column chromatography (MeOH:H2O; 2:8~10:0 v/v) was used to
separate fraction C (750 mg) into seven fractions (C1-C7). Using a
CHCl3:MeOH gradient elution, the C6 fraction (295 mg) was processed
to silica gel column chromatography to produce 3 subfractions (C6a -
C6c). The reversed phase Develosil C-30-UG-5 (250 × 4.6 mm i.d, Aichi,
Japan) which eluted by 75 % MeOH/H2O (in isocratic manner) was used
to chromatograph the C6b subfraction (115 mg), using a TOSOH RI-8020
detector and the rate of flow was adjusted at 1.5 mL/min, which pro­
duced the compound 1 (13 mg, white amorphous powder) and 2 (32 mg,
white amorphous powder).
2.4. Spectral data
Marihysin B (1): White amorphous powder, [α]30 D − 57.8 (c 0.0575,
pyridine); IR νmax (dry film) 3312, 2967, 2860, 1730, 1686 cm− 1; NMR
data (see Table 1); FABMS m/z: 1043.64 [M + H]+; HRFABMS m/z:
1065.5688 [M+Na]+ (calcd for C49H78N12O13Na, 1065.5709., Δ–2.1
mmu).
Marihysin A (2): White amorphous powder, [α]30 D − 69.5 (c 0.0575,
pyridine); IR νmax (dry film) 3297, 2947, 2794, 1727, 1673 cm− 1; NMR
data (see Table 2); FABMS m/z: 1043.6 [M + H]+.
2.5. Determination of amino acids configuration
A sample of 1 or 2 (1.5 mg) was hydrolysed in 6 N HCl (1 mL) at
110 ◦ C for 16 h. After concentration to dryness, the residue was dis­
solved in 50 μL of H2O and 40 μL of 1 M aq. NaHCO3 and 100 μL of 1 % of
A.I.M. Khedr et al. Journal of Molecular Structure 1297 (2024) 137008

Table 1
1
H-, 13C NMR, 1H–1H-COSY, HMBC and NOESY data of compound 1 (recorded in C5D5N-d5; in ppm, J in Hz).
13 1 1
Position C H NMR H, 1H-COSY HMBC NOESY
NMR

Pro 1 176.6 –
2 62.23 4.50, m H-15, H-19, H-22, H-35
3 29.4 2.13, m H–C(4) H-4, H-5, H-7, H-36, H-37
4 25.3 1.75, m, 2.05, H–C(5) C3 H-5, H-7, H-35, H-36, H-37, H-38, H-39
m
5 48.1 4.04, m H–C(4) H-3, H-4, H-7, H-8, H-35, H-36, H-37
Gln 6 173.3 –
7 51.1 5.17, m H–C(8) H-3, H-4, H-5, H-8, H-13, H-18, H-23, H-32, H-35, H-36,
H-37
8 27.2 2.55, m, 2.71, H–C(7) H-2, H-5, H-7, H-9, H-18, H-12, H-16, H-35
m
9 31.4 2.69, m, 2.74, C8, C10 H-2, H-4, H-7, H-8, H-19, H-36
m
10 176.6
10–NH2 7.15 (s), 8.40
(s)
7-NH 7.60, s
A2Pr 11 173.5 –
12 51.1 5.17, m H-C(13) C13
13 36.1 3.34, m, 3.42, H–C(12) C11, C12 H-7, H-15, H-18, H-22, H-31
m
13–NH2 7.20 (s), 8.60
(s)
12-NH 8.12, br.s
Ser 14 172.0 –
15 57.2 5.01, m H-C(16) H-13, H-18, H-23, H-31, H-32
16 62.2 4.50, m, 4.56, H–C(15)
m
16-OH 5.45, m
15-NH 8.20, br.s
1
Asn 17 172.3 –
18 52.2 5.44, m H-C(19) H-5, H-8, H-9, H-13, H-16, H-38
19 37.4 3.26, m H–C(18) C18, C20 H4, H-8, H-9, H-15 H-16, H-18, H-22, H-36
20 173.5
20-NH2 7.55 (s), 8.12 H-36
(s)
18–NH 9.0, br.s
Tyr 21 174.6 – – –
22 57.1 4.90, m H-C(23) H-2, H-16
23 36.7 3.34, m, 3.42, H–C(21) C21, C25/C29 H-13, H-15, H-22
m
24 128.2 –
25, 29 130.8 7.39, dd, J = C23, C25/C29 H-13, H-15, H-23
8.5
26, 28 116.2 7.20, dd, J = C26/C28 H-25, H-29
8.5
27 156.7 C25/C29, C26/
C28
27–OH 9.45, s
22-NH 9.70, s
2
Asn 30 174.6 –
31 52.1 5.35, m H-C(32) H-7, H-8, H-9, H-13
32 36.7 3.34, m, 3.42, H–C(31), C31, C33 H-13, H-15, H-22
m
33 172.2
33–NH2 7.40 (s), 8.60
(s)
31-NH 8.70, br.s
Amino-methylpentadecanoic 34 174.5 – – – –
acid
35 42.8 2.71, m, 3.10, H-C(36) C34, C36 H-5, H7, H-8
m
36 47.5 4.56, m H–C(35), H–C H-4, H-5, H-7, H-9, H-19, H-38, H-39
(37)
37 35.6 1.88, m, 1.90, H–C(36) C36 H-3, H-5, H-7, H-8, H-9, H-35, H-36
m
38 26.2 1.40, m H-4, H-36
39 27.5 1.18, m
40 28.0 1.45, m
41 29.6 2.13, m
42 29.8 2.13, m
43 30.0 1.18, m
44 30.1 1.18, m C45
(continued on next page)

3
A.I.M. Khedr et al.
Table 1 (continued )
13 1 1
Position C H NMR
NMR
45 39.1 1.10, m
46 34.4 1.26, m
47 22.7 0.95, m, 1.0, m H–C(46)
48 11.5 0.89, m
49 19.3 0.86 (t, J = 8) H-C(46)
36–NH 8.10, s
Nα-(5-fluoro-2,4-dinitrophenyl)-l-leucinamide (FDLA) in acetone were
added. FDLA contains a reactive fluorine atom which can be used for the
reaction with a mixture of l- and d-amino acids and resulting di­
astereomers can be separated and estimated by HPLC. The mixture was
heated at 37 ◦ C for 1 h and 20 μL of 1 N HCl was added. The solution was
evaporated to dryness to furnish a yellow residue. The residue was
dissolved in H2O/CH3CN (500 μL) and analysed by reversed-phase HPLC
(Mightysil RP-18 (250 × 4.6 mm i.d., Kanto Chemical Co., Inc., Tokyo,
Japan), mobile phase; (MeCN:H2O; 4:6, v/v) with flow rate; 1 mL/min,
detection; UV 340 nm) and comparing with standard l- and d- amino
acids.
2.6. Antimicrobial activity
Antimicrobial activities of compounds at concentration 125 μg/disc
were tested by agar disk diffusion method [18] against Gram +ve (Ba­
cillus subtilis subsp. subtilis, Staphylococcus aureus subsp. aureus) and
Gram –ve(Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens
subsp. marcescens) bacteria and fungi (Aspergillus niger, Penicillium
crustosum, Schizophyllum commune, and Trichophyton concentrieum). The
tube-dilution method was used to determine the MIC for compounds 1
and 2. [19,20] Ciprofloxacin (50 µg/disc) and nystatin (100 µg/disc)
were used as standard antibacterial and antifungal agents, respectively.
2.7. Computational studies
2.7.1. Ligands preparation
The macrocycle conformational sampling (MCS) tool of Prime
module was used for the preparation of the marihysin A and marihysin
B. [21] For each compound, the settings were maintained with the
default entries and the simulation cycles were set to be 1000, and the
lowest energy conformer was then selected.
2.7.2. Protein preparation
The crystal structure of starch-binding domain from Rhizopus oryzae
glucoamylase -protein that is complexed with isomaltotriose (Fig. 2) was
obtained from protein data bank with an identification code of (4BFO).
[22]
The protein was then prepared for the docking procedure using the
Protein Preparation Wizard within the Schrödinger suite.[23] This tool
encompassed several steps, including the addition of missed hydrogen
atoms, addition of missing residues and loops, resolution of atomic
overlaps, determination of missing bond orders, assessment of ligand
protonation states, and enhancement of the hydrogen-bonding network.
Additionally, water molecules found in the crystal structure were
eliminated, and the orientations of water molecules within a 3-Å radius
of the co-crystallized ligand were optimized and retained.[24] Subse­
quently, the entire protein structure undergoes optimization and energy
minimization using the OPLS4 (Optimized Potentials for Liquid Simu­
lations) force field. Following this optimization step, a comprehensive
assessment of the protein’s reliability is conducted through a protein
reliability report, confirming its suitability for more processing.
2.7.3. Receptor grid generation and glide molecular docking
In order to set the target for the docking procedure, an essential
initial step involved generating a receptor grid. This receptor grid
Journal of Molecular Structure 1297 (2024) 137008
H, 1H-COSY HMBC NOESY
C49
C49
C48
C45, C48
generation step played a critical role in identifying the precise location
where the ligand would bind, thus laying the groundwork for the sub­
sequent docking process. The grid was constructed using the coordinates
of a co-crystallized ligand with glucoamylase. Following the receptor
grid generation, the next phase encompassed the execution of a molec­
ular docking procedure. This intricate process was carried out utilizing
the Glide module, a component of the comprehensive Schrödinger suite
of molecular modelling tools, which enabled the ligand to interact with
the target protein in a highly accurate and precise manner. [25–27]
3. Results and discussion
3.1. Purification of compounds 1 and 2
The fermented broth of the marine bacterium Staphylococcus sp. was
extracted with ethyl acetate. Successive fractionation of the combined
extracts was done using Diaion HP-20/Sephadex LH-20/ODS/silica gel
column chromatography. Final purification on a C18-reversed-phase-
HPLC column to afford 1 (13 mg) and 2 (32 mg) (Fig. 1).
3.2. Structural characterization of compounds 1 and 2
Compound 1 (13 mg) was obtained as a white amorphous powder. Its
molecular formula, C49H78N12O13, was determined based on HR-FAB-
MS ion peak (Fig. S7) at m/z 1065.5688 [M+Na]+ (calcd for
C49H78N12NaO13, 1065.5709). The 1H and 13C NMR spectra of 1
(Table 1, Figs S1 and S2) showed typical signals for a lipopeptide. [28]
The intense absorptions between 1600‒1700 cm− 1 and between 3100‒
3400 cm− 1 in the IR spectrum suggested the presence of the amide C = O
and NH groups, respectively. Compound 1 was negative to ninhydrin
reagent but positive after hydrolysis with 6 M HCl. [29] The 13C NMR
spectrum of 1 revealed signal corresponding to C = O carbon atoms of
ester and amide at δC 176.6 to 172.0. The 1H NMR spectrum together
with H1-H1-COSY and HSQC spectra of 1 revealed the presence of eight
methine atoms at δH (5.44, 5.35, 5.17 (2H), 5.01, 4.90, 4.65, and 4.50)
and eleven NH/NH2 groups ranging from δH 7.15 to 9.70. The 1H NMR
signals at δH 7.39 (dd, J = 8.5 Hz, 2H) and 7.20 (dd, J = 8.5 Hz, 2H) and
the 13C NMR signals at δC 156.7, 130.8, 128.2, and 116.2 are charac­
teristic for a p-disubstituted aromatic ring, which was identified as
tyrosine by HSQC, 1H–1H COSY, and HMBC data.
Likewise, combining the NMR data of 1 (Table 1) with 1H–1H-COSY,
HSQC, HMBC, (Figs 3 and S3–S5), compound 1 was determined as a
lipopeptide comprised of diamino propionic acid, one tyrosine, two
asparagine, one proline, one glutamine, and one serine residues, and a
long-chain fatty acid and this was in accordance with those reported in
literature.[30] The H atom signals at δH 1.18–1.45 (m, 16 H) 0.89 (m,
3H), and 0.86 (t, J = 8, 3H), and the carbon atom signals at δC
(22.7–39.1), 11.5, and 19.3 indicated the existence of branched-type
long-chain amino fatty acid in 1.[31] It was reported that heptapep­
tide belonged to iturin-like family, having heptapeptide attached to a
β-amino fatty acid chain of variable length (C14–C17) [32]. Accord­
ingly, the fatty acid residue in 1 was assigned as anteisomethyl-branched
fatty acid called β-aminomethyl pentadecanoic acid (AMPDA) residue
that was confirmed by the analysis of HMBC and 1H–1H-COSY spectra
(Figs 3, S3 and S5).[20] Based on HMBC spectra (Table 1 and Fig. S13)
and those reported in literature,[33] the carboxyl terminal (from Pro)
4
A.I.M. Khedr et al. Journal of Molecular Structure 1297 (2024) 137008

Table 2
1
H-, 13C NMR, 1H–1H-COSY, HMBC and NOESY data of compound 2 (recorded in C5D5N-d5; in ppm, J in Hz).
13 1 1
Position C NMR H NMR H, 1H-COSY HMBC NOESY

Pro 1 176.9 –
2 62.23 4.25, m H-C(3) C1 H-16, H-37
3 29.4 2.2, m, 2.3, m H–C(2) C1, C2, C5
4 25.4 1.90, m, 2.10, m H-C(5) C2, C3
5 48.3 4.10, m H–C(4) C3, C4 H-9, H-17, H-19, H-38, H-39, H-46
Gln 6 173.3 –
7 51.0 5.20, m H–C(8), 7-NH C6, C8, C10 H-17, H-37
8 27.1 2.50, m, 2.70, m H–C(7), H–C(9) C7, C9, C10 H-5, H-9, H-19
9 31.4 2.60, m, 2.70, m C7, C8, C10 H-5, H-8, H-19, H-36
10 177.0
10–NH2 7.70 (s), 8.40 (s) C10
7-NH 7.65, s H-C(7)
Asn1 11 173.7 –
12 51.2 5.22, m H–C(13), 12-NH C11, C13, C14
13 36.1 3.38, m, 3.40, m H-C(12) C11, C12, C14` H-16, H-19, H-23
14 174.4
14–NH2 7.60 (s), 8.50 (s) C14
12-NH 9.45, br.s H-C(12) H-10
Ser 15 171.7 –
16 57.0 4.90, m H–C(17), 16-NH C15, C17 H-13, H-33
17 62.25 4.50, m, 4.54, m H–C(16) C15
17-OH 5.45, m
16-NH 8.10, br.s H-C(16)
Asn2 18 172.3 – – – –
19 52.0 5.40, m H–C(20), 19-NH C18, C20 H-5, H-8, H-9, H-13, H-17, H-39
20 37.4 3.10, m H–C(19) C19, C21
21 173.7
21-NH2 7.70 (s), 8.30 (s) C21 H-37
19–NH 9.0, br.s H-C(19)
Tyr 22 174.1 – – –
23 57.1 4.80, m C22, C26/C30 H-13
24 36.7 3.20, m C22, C25, C26/C30
25 128.2 –
26, 30 130.8 7.40, dd (J = 8.5) C25, C27, C28, C29
27, 29 116.2 7.20, dd (J = 8.5) C25, C26, C28
28 156.7
28–OH 9.58, S
23-NH 9.40, br.s H-C(23)
3
Asn 31 174.6 – – – –
32 51.7 5.40, m H–C(33), 32-NH C31, C33 –
33 36.7 3.35, m, 3.40, m H-C(32), C32, C34 H-13, H-16, H-19, H-23
34 172.1
34–NH2 7.75 (s), 8.60 (s)
32-NH 8.60, s H-C(32)
β-Amino-tetradecanoic acid 35 174.2 – – – –
36 42.8 2.70, m, 3.0, m H-C(37), 37-NH C35 H-17, H38, H-46
37 47.5 4.60, m H–C(36), H–C(38) C35, C39 H-2, H-7, H-13
38 35.5 1.54, m, 1.58, m H–C(37), H–C(39) C40 H-5, H-17, H-39
39 29.4 1.20, m H–C(38) C41 H-5, H-17, H-19, H-38
40–44 29.7 1.20, m C42, C43
45 31.9 1.20, m C44
46 26.2 1.48, m, 1.50, m H-17, H-36, H-38, 39
47 22.7 1.25, m H–C(48) C45, C48
48 14.2 0.86 (t, J = 8) H-C(47) C47
37–NH 7.55, s H-C(37) C1 H-21

and the amino terminal (from Asn) of the peptide chain (Pro-Gl­
n-A2Pr-Ser-Asn-Tyr-Asn) were linked to the amino group and the
carboxyl group of β-amino fatty acid, respectively, which is a typical
characteristic of iturin lipopeptides.[33] The relative configuration of 1
was established by NOESY correlations (Table 1 and Figs 4 and S6). The
NOESY correlations between NH protons and adjacent amino acids
methine protons clearly identified the following amide bonds:
Gln-CO/A2Pr-NH (δH 5.17/8.12), A2Pr-CO/Ser-NH (δH 5.17/8.20),
Ser-CO/Asn1-NH (δH 5.01/9.0), Asn1-Co/Tyr-NH (δH 5.44/9.70), and
Asn2-CO/AMPDA-NH (δH 5.35/8.10), in addition to the correlations
between Tyr-CO/Tyr-NH (δH 4.90/9.70) and AMPDA-CO/Asn2 (δH
4.56/8.7). The connectivity between AMPDA and Pro-was also estab­
lished by NOESY correlation between α-methine proton of AMPDA and
methine proton of Pro (δH 4.56/5.40). These data, together with HMBC
correlations as shown in (Fig. 3), finally enabled to set up the structure of
1 as cyclo(Pro-Gln-2,3α diaminopropoinic acid-­
Ser-Asn1-Tyr-Asn2-β-aminomethyl-pentadecanoic acid). The amino
acids absolute configuration of 1 was recognized by the reaction of
Marfey`s reagent [34] with the crude hydrolysate followed by
co-injection of standard amino acids using HPLC analysis. The hydro­
lysate was identified to possess l-Asn, l-Pro, l-Gln, l-Ser, and l-Tyr. The
configuration of A2Pr and C-3 of AMPDA was proposed by careful
analysis of NOESY correlations (Table 1 and Fig. S6). The NOESY cor­
relations between β-proton (δH 4.56) of AMPDA/α-proton (δH 5.35) of
Asn[2] and β-proton (δH 4.56) of AMPDA/α-proton (δH 4.50) of
Pro-revealed that the configuration at C-3 of AMPDA have the same
configuration of l-Asn[2] and l-Pro. Moreover, the configuration of A2pr
has the same configuration of methine proton of l-Ser-and

5
A.I.M. Khedr et al.
Fig. 2. 3D structure of Glucoamylase (PDB ID: 4BFO) with the co-crystalized
reference ligand.
l-Gln-Therefore, 1 was lastly characterized as cyclo(L Pro-L Gln-L 2,3α
diaminopropoinic acid -L Ser-Lasn[1] -LTyr-Lasn[2] - β-amino­
methyl-pentadecanoic acid) which was named staphylolipopeptides A.
Compound 2 (32 mg) was obtained as a white amorphous powder. It
gave [M + H]+ and [M+Na]+ peaks in the positive FAB-MS at m/z
1043.6 and 1065.6 (Fig. S15), respectively, which were consistent with
molecular formula C48H74N12O14 (18◦ of unsaturation). The 1H and 13C
NMR spectra of 2 (Table 2, Fig.s S8 and S9) together with DEPT
(Fig. S10), 1H–1H-COSY, HSQC, and HMBC, (Figs 3 and S11–S13)
spectra revealed that compound 2 is nearly similar to compound 1 in the
presence of lipopeptide nucleus, consisting of one tyrosine, three
asparagine, one proline, one glutamine, and one serine residues, and a
long-chain fatty acid. [30] The H-atom signals at δH 1.20–1.50 (m, 16 H)
and 0.86 (t, J = 8), and the carbon atom signals at δC (22.7–31.9), and
14.2 indicated that the amino fatty acid it was straight long-chain type.
[29] This fatty acid was identified as β-aminotetradecanoic acid residue.
[31]
Fig. 3. Key HMBC ( ) and 1H–1H-COSY (
6
Journal of Molecular Structure 1297 (2024) 137008
In the HMBC (Table 2 and Fig. S13), the carboxyl terminal (from Pro)
and the amino terminal (from Asn) of the peptide chain (Pro-Gln-Asn-
Ser-Asn-Tyr-Asn) were linked to the amino group and the carboxyl
group of β-amino fatty acid, respectively, which is a typical character­
istic of iturin lipopeptides. [32] The relative configuration of compound
one was established from NOESY correlations presented at (Table 2 and
Figs 4 and S14). The amino acids absolute configuration of 2 was
assigned as 3 L-Asn, 1 L-Pol,1 L Gln, 1 Ser-and 1 L-Tyr-using Marfey`s
method. The NOESY correlations of ATDA β-proton (δH 4.6) /Asn3α-­
proton (δH 5.4) and ATDA β-proton (δH 4.6)/Pro α-proton (δH 4.25)
revealed that the C-3 configuration of ATDA was the same configuration
as that of l-Asn[3] and l-Pro (Table 2 and Fig. S14). Therefore,
2 was identified as cyclo(L-Pro-l-Gln-l-Asn1-l-Ser-LAsn2-l-Tyr-l-Asn3-β-
aminotetradecanoic acid) which is known as marihysin A.
It is noteworthy that 2 was previously isolated from the marine
B. marinus B-9987 fermentation broth, which was obtained from tissues
of the rhizophere of Suaeda salsa in the intertidal zone of the Bohai Bay,
China. [34] This compound demonstrated broad-spectrum efficacy
against plant pathogens (MICs 100–200 g/mL).[35]
The antimicrobial activities (Table 3) of compounds 1 and 2 (con­
centration of 100 µg/disk) were examined for their growth inhibition of
9 microorganisms including Gram-negative, Gram-positive bacteria and
fungi using agar disk diffusion method with replication (n = 3). As a
result, these metabolites showed no antibacterial activity against
B. subtilis, S. aureus, P. aeruginosa, S. marcescens, and E. coli. Whilst they
exhibited potent antifungal activity against A. niger, P. crustosum, T.
concentricum, and S. commune (Table 3) with MIC value of 25 µg/mL.
3.3. Molecular docking analysis
After the preparation of the of the ligands and the receptor grid of
glucoamylase, molecular docking was implemented using glide module.
The enzyme glucoamylase, our focal point, holds immense importance
as it plays a pivotal role in the breakdown of starch into β-d-glucose
through the process of hydrolysis. In terms of its structure, glucoamylase
comprises two distinct carbohydrate binding sites. Site I is formed by
three remarkably conserved aromatic residues, namely Trp47, Tyr83,
and Tyr94, while site II is constituted by Tyr32 and Phe58. Notably, site
II serves as the primary recognition binding site for carbohydrates, while
site I complements site II by facilitating the binding of ligands. [22]
In previous research, it has been proposed that Tyr32, situated at site
II, could potentially serve as an essential residue in recognizing long-
) correlations of compounds 1 and 2.
A.I.M. Khedr et al. Journal of Molecular Structure 1297 (2024) 137008

Fig. 4. Key elements of NOESY correlations of compound 1 (A: general correlations; B: characteristic correlations) and compound 2.

chain polysaccharides. Moreover, experiments involving ITC-binding involve hydrogen bonding (both direct and water mediated) and provide
affinities revealed that amongst the residues in starch binding do­ crucial details about the binding modes of the compounds. In the case of
mains, Tyr32 at site II holds the utmost significance for binding soluble the reference ligand (Fig. 5C), it is noticed that it establishes direct
polysaccharide ligands.[36,37] hydrogen bonds with Tyr32 (at 2.22 Å) and Lys34 (at 2.11 Å). Addi­
In table 4, it can be seen that the docked molecules gave comparable tionally, it forms a water-bridged hydrogen bond with Tyr32 (at 3.48 Å),
docking scores. Specifically, the reference ligand attained a score of Ser33 (at 3.63 Å), Lys34 (at 4.55 Å), and Phe58 (at 3.77 Å and 3.79 Å).
− 6.081 kcal/mol, marihysin A scored − 5.508 kcal/mol, and marihysin Furthermore, it engages in several hydrogen bonds with various water
B yielded a docking score of − 4.326 kcal/mol. molecules located within the active site. These interactions highlight the
Examining the docked poses of the of the studied compounds pro­ ligand’s strong affinity for the active site, which is likely essential for its
vided insightful information into the specific interactions occurring biological activity.
between the ligands and the active site of the protein. These interactions In contrast, as in Fig. 5A, the compound marihysin A demonstrates a
different pattern of hydrogen bonds primarily with water molecules
within the active site. This suggests a distinct binding mechanism
Table 3 compared to the reference ligand.
Antimicrobial activity of compounds 1 and 2.
In Fig. 5B, the candidate compound marihysin B is observed to
Inhibition zone (mm, 125 µg/disc) establish water-bridged hydrogen bonds with Tyr32 (at 4.13 Å) and
Tested strains 1a 2a Ciprof.b Nyst.c Phe58 (at 4.48 Å). Notably, these interactions could reflect a potential
binding affinity, as they resemble the interactions of the reference
S. aureus NA NA 23±0.17 –
B. subtillis NA NA 31±0.13 – ligand.
E. coli NA NA 24±0.12 – Further analysis of the 2D and 3D graphs (Figs 5 and 6) interactions
P. aeruginosa NA NA 29±0.08 –
S. marcescens NA NA 19±0.09 –
S. commune 19±0.11 20±0.09 – 23±0.15 Table 4
T. concentricum 11±0.10 13±0.13 – 31±0.13 The docking scores of the three docked compounds in glucoamylase active site.
A. niger 24±0.12 25±0.10 – 24±0.18
P. crustosum 22±0.12 22±0.12 – 29±0.0.23 Compound Docking score Glide g score
a
= 125 µg/disc Co-crystallized reference − 6.081 − 6.081
b Marihysin A − 5.508 − 5.508
Ciprofloxacin as antibacterial standard (50 µg/disc)
c Marihysin B − 4.326 − 4.326
Nystatin antifungal standard (100 µg/disc); NA= Not Active.

7
A.I.M. Khedr et al.
Fig. 5. 2D interactions of the two compounds and the reference complexed with the Glucoamylase binding site (PDB ID: 4BFO) docking poses using Glide. A.
Marihysin A B. Marihysin B C. Reference.
provided highlights of additional types of interactions. These in­
teractions primarily involved hydrophobic and polar contacts with
specific amino acid residues of the protein.
Specifically, the reference ligand presented hydrophobic interactions
with Tyr32, Phe58, and Trp70, as well as polar interactions with Asn29,
Lys34, Ser57, and Glu68.
In comparison, the known compound marihysin A also engages in
interactions with the protein. It forms hydrophobic contacts with Tyr32
and Phe58, along with polar interactions with Asn29, Ser57, and Glu68.
Similarly, the candidate compound marihysin B demonstrates in­
teractions with the protein. These interactions include hydrophobic
contacts with Tyr32, Phe58, and Trp70, as well as polar interactions
with Asn29, Lys34, Ser57, and Glu68. Remarkably, there is a significant
overlap in the nature of interactions observed between these compounds
and the amino acids of the protein. This implies a level of consistency in
how these ligands bind, hinting at shared binding preferences or motifs.
These shared interactions may indicate the presence of common binding
regions on the protein’s surface that are particularly favourable for these
compounds.
To gain a comprehensive understanding of the importance of these
interactions and their implications for the compounds’ binding strengths
and potential biological functions, more detailed structural and
biochemical analyses are warranted. Moreover, conducting comparative
studies like these can offer valuable insights for the development of
novel therapeutic agents by identifying recurring interaction patterns
that can be leveraged in drug design efforts.
Fig. 6. 3D interactions of the two compounds and the reference complexed with the Glucoamylase binding site (PDB ID: 4BFO) docking poses using Glide. A.
Marihysin A B. marihysin B C. Reference.
8
Journal of Molecular Structure 1297 (2024) 137008
4. Conclusion
A new cyclic lipopeptide namely marihysin B (1) and marihysin A (2)
were isolated and characterized from Staphylococcus sp. culture broth
using various chromatographic and spectral analyses, as well as chem­
ical method. These compounds demonstrated marked antifungal po­
tential in the antimicrobial assay. Also, they had notable binding
affinities to Schizophyllum commune glucoamylase in the computational
study. This molecular docking analysis furnishes valuable insights into
how these compounds interact with the active site of the protein. It
emphasizes the critical role of comprehending these interactions in the
field of drug discovery and design, as they are pivotal for crafting
compounds with desired therapeutic properties. Further exploration
through structural and functional studies is essential to fully grasp the
significance of these interactions within the context of biological activity
and selectivity.
Funding source
This work was not supported by any funding source.
Ethical approval
Not required.
A.I.M. Khedr et al.
Data availability
The original contributions presented in the study are included in the
article. Further inquiries can be directed to the corresponding author.
Supplementary data
Fig. S1: 1H NMR spectra of compound 1 (C5D5N-d5, 500 MHz);
Fig. S2: 13C NMR spectra of compound 1 (C5D5N-d5, 125 MHz); Fig. S3:
1 1
H, H-COSY spectra of compound 1; Fig. S4: HSQC spectra of com­
pound 1; Fig. S5: HMBC spectra of compound 1; Fig. S6: NOESY spectra
of compound 1; Fig. S7: HR-FAB-MS spectra of compound 1; Fig. S8: 1H
NMR spectra of compound 2 (C5D5N-d5, 500 MHz); Fig. S9: 13C NMR
spectra of compound 2 (C5D5N-d5, 125 MHz); Fig. S10: DEPT spectra of
compound 2 (C5D5N-d5, 125 MHz); Fig. S11: 1H,1H-COSY spectra of
compound 2; Fig. S12: HSQC spectra of compound 2; Fig. S13: HMBC
spectra of compound 2; Fig. S14: NOESY spectra of compound 2;
Fig. S15: Positive-FAB-MS spectra of compound 2.
Declaration of Competing Interest
The authors declare that the research was conducted in the absence
of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.molstruc.2023.137008.
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