K. Sivonen et al.
and actively shielded z-axis (xyz-axes) gradient systems (Varian) in
death. They might also contribute to a better understanding of
the functional purpose of the production by cyanobacteria of
nostocyclopeptides like Ncp-M1.
[D6]DMSO at RT. 13C and 15N HSQC experiments were optimised for
1
1JCH =150 Hz and JNH =95 Hz, and 13C and 15N HMBC experiments
n
were optimised for JCH =7 Hz and nJNH =4 Hz.
IR spectra (500 to 4000 cmꢀ1) were recorded on a Bruker Vertex 70
FTIR spectrometer (Bruker Optics) equipped with a microplate HTS-
XT accessory unit. Dissolved peptide was placed on a silicon plate
and evaporated, and the transmission spectrum was measured.
Experimental Section
Cyanobacteria strains and extraction for activity screening: The
cyanobacterial strains studied were planktonic Portuguese freshwa-
ter samples (IZ samples) as well as planktonic and benthic Baltic
Sea samples (M samples). The strain (XSPORK 13A) used to purify
the novel Ncp-M1 antitoxin was isolated from a gastropod collect-
ed from shallow seawater at the Cape of Porkkala, Finland, and
was classified as a Nostoc sp. based on morphology and 16S se-
quencing. Sampling, isolation of strains from the extraction solu-
tion, strain purification, mass cultivation, freeze-drying and cultur-
ing were as previously described.[19,28,29]
Partial amino acid hydrolysis of Ncp-M1 was performed by dissolv-
ing Ncp-M1 (100 mg) in HCl (300 mL, 1.2m) and allowing the solu-
tion to hydrolyse for 70 min at 1058C. The acid was evaporated,
and the residue dissolved in water. Peptide fragments were ana-
lysed by LC/MS.
1
Nostocyclopeptide M1: H, 13C and 15N NMR data are presented in
Table 1; IR (solid on a silicon plate): nmax =3329, 2961, 2920, 2851,
1640, 1539, 1517, 1449, 1244, 828 cmꢀ1; UV (MeOH): lmax =224,
276; HR-TOFMS m/z 882.4391 [M+H]+ (calcd for C47H60N7O10 ꢀ0.4
mmu error), 900.4467 [M+H2O+H]+, 922.4223 [M+H2O+Na]+,
DBE=22.
Extracts for screening of antiapoptotic activity were made by sus-
pending freeze-dried cyanobacteria (10 mg) in CH3OH/CH2Cl2 (1:1
v/v, 1 mL). After homogenisation, the samples were extracted on
ice overnight in the dark. The samples were separated in a centri-
fuge (10 min, 10000g), the supernatant was collected, and the
pellet was washed twice with ice-cold CH3OH/CH2Cl. The superna-
tants from the wash were combined with the extract, and evapo-
rated to dryness. The residues were resuspended in DMSO (25 mL,
Sigma–Aldrich) for cell-toxicity and enzyme assays.
Derivatisation of nostocyclopeptide M1
Acetylation: Ncp-M1 (30 mg) was dissolved in ammonium bicarbon-
ate solution (400 mL, 50 mm), and acetylation reagent (1 mL;
1.2 mL MeOH and 400 mL acetic anhydride) was added. The solu-
tion was incubated for 1.5 h at room temperature, evaporated and
dissolved in 0.1% HCOOH/MeCN (1:1) prior to mass spectrometric
flow injection analysis.
Purification of Ncp-M1: Freeze-dried biomass (16.4 g) was extract-
ed three times for 45 min with MeOH (400 mL). The extract was
evaporated to dryness, and the residue was redissolved in MeOH
(100 mL) and filtered before being passed through two solid-phase
extraction cartridges (StrataX Polymeric Sorbent, 30 mg, Phenom-
enex, Torrance, CA). The volume of the MeOH effluent was reduced
to 15 mL before being extracted with heptane (3ꢁ15 mL), evapo-
rated to dryness and dissolved in 10% aqueous MeOH (3.5 mL).
HPLC purification was performed with a HP 1100 Series modular
chromatograph (Agilent Technologies). The sample was injected
into the Luna C18 column (150ꢁ4.6 mm, 5 mm, Phenomenex) in
batches (0.3–0.5 mL). Nostocyclopeptide was eluted isocratically
with 75% water (HPLC grade, Rathburn)/25% MeCN (LiChrosolv,
Merck) at 408C, and the column was washed with 95% aqueous
MeCN between the injections (Figure S1A). The fractions contain-
ing Ncp-M1 were evaporated to dryness under a jet of N2. The
purity of Ncp-M1 was verified by HPLC (Figure S1B).
DNPH derivative: Ncp-M1 (20 mg) was dissolved in 0.5% trifluoro-
acetic acid (TFA; 50 mL), and a saturated solution of 2,4-dinitro-
phenyl hydrazine in MeCN/water (1:1) containing 0.5% TFA (50 mL)
was added. The reaction mixture was incubated for 30 min at
378C, and after adding MeCN (25 mL), the sample was analysed by
LC/MS.
Acetylated Marfey derivative (FDAA): Ncp-M1 (50 mg) was dissolved
in water (50 mL), and NaHCO3 (20 mL, 1m) and 1% FDAA (Marfey re-
agent, Pierce) in acetone (100 mL) was added. After incubation for
1 h at 378C, the reaction was terminated with 1n HCl (20 mL). The
Ncp-M1–FDAA derivative was purified by adding water (800 mL) to
the reaction solution (200 mL, 50% MeCN in water) and then pass-
ing the solution through a solid-phase extraction cartridge (StrataX
Polymeric Sorbent, 30 mg; particle size, 33 mm; pore size, 85 ꢂ;
Phenomenex) that was equilibrated with 85% MeCN (1 mL). The
cartridge was washed (2ꢁ200 mL of 30% MeCN and 200 mL of 60%
MeCN). The FDAA derivative was eluted with MeOH (2ꢁ200 mL).
After evaporation, the residue was acetylated as described above
by using 10% acetylation reagent.
Chemical analyses: An HP 1100 Series modular HPLC containing
diode array detector (Agilent Technologies, Palo Alto, USA) inter-
faced with an Esquire3000 plus ESI ion-trap mass spectrometer
(Bruker Daltonics) was used for the mass spectrometric analyses.
Samples were separated on a Luna C18 column (150ꢁ4.6 mm,
5 mm, Phenomenex) eluted with various water or 0.1% HCOOH
and MeCN or MeOH gradients at 408C. Flow-injection analysis was
used for the purified Ncp-M1 to obtain MS and MSn spectra.
Enantiomeric analysis of amino acid: Ncp-M1 (0.1 mg) was added to
6n HCl (0.3 mL) and allowed to hydrolyse at 1108C for 22 h before
the solvent was evaporated. The residue was dissolved in water
(50 mL) and analysed by chiral HPLC on a Phenomenex Chirex d-
penicillamine column (150ꢁ2 mm), eluting with 5% MeCN in
CuSO4 (1.9 mm) at 0.15 mLminꢀ1 and 308C with UV detection at
245 nm. An aliquot of the residue dissolved in water (50 mL) was
treated as described for Marfey (FDAA) derivatisation. The sample
was then diluted with MeCN (200 mL) and analysed by LC/MS. Ref-
erence Marfey derivatives of l- and d-amino acids were prepared
similarly with amino acid solutions (50 mL, 50 mm). Reference com-
pound (2S,4S)-4-MePro was obtained by hydrolysing spumigins A,
B1, B2 and C purified from Nodularia spumigena AV1[30] at 1128C
for 24 h.
Accurate mass measurements of Ncp-M1 dissolved in 1% formic
acid/MeCN (1:1) were made on a ESI-MS Micromass Q-tof Ultima
tandem mass spectrometer (Micromass, Manchester, UK). Ncp-M1
solution was injected into the mass spectrometer at a flow rate of
0.5 mL minꢀ1. The mass spectrometer was operated in positive ion
electrospray mode with a source temperature of 808C and a po-
tential of 3.5 kV applied to the nano-LC probe. In MS/MS mode, a
collision energy of 30 eV was used.
NMR spectra were recorded on Varian Inova 600 (and 800) spec-
1
trometers equipped with H/13C/15N triple resonance probe heads
1598
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 1594 – 1599
Jokela, Jouni
