Allelopathic Activity of the Iron Chelator Anachelin?–?A Molecular Hybrid with a Dual Mode of Action

Received Date: 30-Apr-2016

Accepted Date: 11-Jun-2016

Article Type: Full Paper

Allelopathic Activity of the Iron Chelator Anachelin – A

Molecular Hybrid with a Dual Mode of Action

a

b

c

b

Suman De Sarkar , Judith F. Blom , Yann Bethuel , Friedrich Jüttner , and

a, c, d, e

Karl Gademann*

a

Department of Chemistry, University of Basel, St. Johanns-Ring 19, CH-4056 Basel

b

Limnological Station, Institute for Plant and Microbial Biology, University of Zurich,

Seestrasse 187, CH-8802 Kilchberg

c

Laboratorium für Organische Chemie, Swiss Federal Institute of Technology (ETH)

Zürich, Vladimir-Prelog-Weg 1 – 5/10, CH-8093 Zürich

d

Chemical Synthesis Laboratory (SB-ISIC-LSYNC), Swiss Federal Institute of

Technology (EPFL), CH-1015 Lausanne

Department of Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057

Zürich, e-mail: karl.gademann@uzh.ch

e

This article has been accepted for publication and undergone full peer review but has

not been through the copyediting, typesetting, pagination and proofreading process, which

may lead to differences between this version and the Version of Record. Please cite this

article as doi: 10.1002/.201600123

One of the compounds suggested to be responsible for the cyanobacterial

dominance over competing green algae is identified. Evidence is provided on the

molecular, chemical level that the iron chelator anachelin from the cyanobacterium

Anabaena cylindrica promotes both the growth of cyanobacteria and reduces the

growth of competing chlorophytes. These results illustrate a molecular strategy of

addressing two challenges (nutrient availability and algal competition) by one

molecule. Such strategies could be implied in harmful algal blooms in marine and

freshwater environments.

Keywords: Natural products, Organic synthesis, Siderophores, Chemical

ecology, Mechanism of action.

Introduction

The growth of phytoplankton has direct implications to fields such as global

carbon budgeting, climate regulation and for fisheries through its central role in both

[

1]

marine and freshwater ecosystems. The growth of prokaryotic cyanobacteria and

eukaryotic algae is determined by resource limitations including light, inorganic and

organic nutrients, the attack of grazers and pathogens, and interspecific inhibition

[

2]

through allelochemicals. The importance of phosphorous and nitrogen has been

well documented, and recently also the impact of iron on phytoplankton growth has

[

3 – 5]

found new interest.

Whereas iron is one of the most abundant elements on

earth, its bioavailability is very low due to the prevalence of insoluble iron oxide

[

6][7]

hydrate species.

Cyanobacteria have met this challenge by producing small

[8 – 10]

molecule iron chelators called siderophores.

Over 60 years ago, Fogg and

Westlake suggested that cyanobacteria secrete siderophores that ‘may have

[

11]

important effects on the growth of organisms in the water’. This hypothesis was

expanded by Tassigny and Lefèvre, who raised the possibility of allelochemical

[

12]

action of such secreted compounds in cyanobacterial and algal blooms.

[

13]

[14]

Murphy and co-workers, and more recently Matz and co-workers,

provided further experimental support and concluded that the excretion of

siderophores by species of the cyanobacterium Anabaena enables other

cyanobacteria to grow, to outcompete, and thus to dominate eukaryotic algae. So far

[

11 – 14]

only supernatants of cultures or crude extracts were studied.

In particular, the

presence of additional unrelated allelopathic compounds was never ruled out and the

responsible compounds were never characterized on the molecular level.

Anachelin (1) was isolated from the cyanobacterium Anabaena cylindrica and

features an interesting structural diversity combining polyketide, peptide and alkaloid

[

15]

[16]

fragments (Fig. 1). We have studied its conformation, suggested a biogenetic

[

17]

[18]

hypothesis and established the configuration by total synthesis. However, up to

now, no biological studies of this metabolite have been reported. In particular, while

the catecholate unit (green in 1) is proposed to facilitate iron uptake and can be used

to functionalize surfaces, the role of the polyketide fragment (red in 1) remained

unknown.

Herein, we provide evidence through chemical synthesis combined with

[

15]

growth experiments that the cyanobacterial siderophore anachelin (1) enhances

growth of the cyanobacterium Microcystis aeruginosa concomitant with allelopathic

activity against the competing green alga Kirchneriella contorta. We devised a new

synthetic route based on several organocatalytic transformations to the polyketide

fragment of anachelin, and through the synthesis of additional derivatives, the

structural elements in anachelin (1) that are responsible for this dual mode of action

have been identified. Anachelin (1) thus presents a fully elucidated example of a

cyanobacterial siderophore with allelopathic properties against competing

photoautotrophic microorganisms (dual mode of action).

Results and Discussion

Synthesis

In order to study the effects of the constitution and configuration of anachelin

(1) on biological activity, anachelin (1), three truncated derivatives 2 – 4 and two

epimers 5 and 6 (Fig. 1) were prepared by chemical synthesis. As a representative

of the alkaloid fragment, Boc-protected anachelin chromophore 2 was prepared in

eight steps starting from L-DOPA using an oxidative aza-annulation as the key

[

17][19]

step.

The peptide fragment 3 was synthesized by debenzylation of a protected

[

18]

analog previously reported by us. In order to block the otherwise free terminal

amino group and mask unwanted reactivity, the chemically inert butyloxycarbonyl

(Boc) group was introduced in fragments 2 and 3. The preparation of

diastereoisomers 5 and 6 was carried out using previously reported building

[

18]

blocks.

The polyketide fragment 4 was addressed next, and although we have

[

18][19]

developed routes to this unit,

we needed an optimized process to secure

amounts for biological testing. Therefore, a concise approach featuring

organocatalytic key steps was developed for the synthesis of the challenging

polyketide fragment 4. The synthesis commenced with the addition of

[

20]

allylmagnesium chloride to Garner’s aldehyde (7) (Scheme 1). Unfortunately, the

chiral substrate was unable to induce good selectivity in the addition step and 3:1

mixture was obtained in favor of desired diastereoisomer. Thus, we utilized the

reliable Duthaler–Hafner method that results excellent yield and selectivity for the

[

21]

allyl alcohol 8 (87%, dr >20:1). The chiral allyl alcohol 8 was then protected, and

silylated 9 was subjected to cross metathesis with crotonaldehyde to the α,β-

unsaturated aldehyde 10 in good yield (93% over two steps). We then envisioned the

use of an organocatalytic approach for the installation of the β-OH group and

[

22]

oxidation of the aldehyde to the ester. In the first step, a catalyst controlled

asymmetric epoxidation was performed with the proline-derived secondary amine

[

23]

catalysts B and C. Sterically demanding catalyst B resulted in good selectivity with

moderate yield, whereas slightly diminished selectivity but good yield was obtained

with biphenyl-substituted catalyst C. The chiral epoxy aldehyde was then directly

subjected to the N-heterocyclic carbene (NHC) catalyzed internal redox process in

[

24]

the same pot. In situ generated NHC by deprotonation of corresponding azolium

ion D facilitates the epoxide opening followed by esterification to yield ester 11 in

good yield and selectivity.

We then proceeded with the synthesis and silyl protection of the free OH

group, which delivered the fully protected ester 12 (Scheme 2). The next task was

the introduction of the amide functionality after selective deprotection of Boc-group.

After some unsuccessful attempts using Lewis acids (AlCl ) or stronger Brønsted

3

acids (HCl), it was found that both the acetonide and Boc group can be cleaved

using a mixture of CH Cl and CF CO H at 0 °C for one hour. Undesired, desilylated

2

3

2

product formation was observed by carrying out the reaction at higher temperature

and upon longer reaction time. The amino alcohol was then subjected to amide

.

coupling employing EDC HCl and HOBt as coupling reagents to give 13. Finally,

removal of the protecting groups by sequential hydrogenation and acid mediated

desilylation completed the synthesis of target compound 4 in nine steps and 40%

overall yield starting from Garner’s aldehyde. This route therefore allowed for the

generation of sufficient material for subsequent biological testing, which was carried

out next.

Effect of Anachelin on Growth of Microcystis aeruginosa

We first wanted to evaluate the effect of the siderophore anachelin (1) on the

growth of other cyanobacteria. Therefore, we investigated the growth inducing

[

15]

properties of anachelin (1), the siderophore of Anabaena cylindrica, on a bloom-

forming cyanobacterium (Microcystis aeruginosa). We prepared synthetic

[

18]

anachelin, which was shown to be spectroscopically identical to a natural sample

and, in particular, free of any cyanobacterial toxins potentially associated with natural

samples. The addition of anachelin (1) promoted growth of the toxic cyanobacterium

M. aeruginosa in a dose-dependant manner (Fig. 2). Small amounts of anachelin

(starting at 800 nM concentration) were sufficient to lead to a significant higher

growth compared to the control.

Having established a positive impact of anachelin (1) on M. aeruginosa, we

wanted to investigate a variety of concentrations of both siderophore and Fe (Fig. 3).

This beneficial effect on cyanobacterial growth was further substantiated at two

different Fe concentrations in the medium (0.1 μM and 10 μM), but the observed

effects decreased with increasing iron concentration (Fig. 3,a and 3,a), likely due to

higher growth rates of M. aeruginosa. Whereas at lower iron concentration an almost

fivefold increase in growth compared to control could be observed, the increase at

higher iron concentrations in the medium was only twofold. The reason for this

growth enhancing property is likely to reside in the binding of iron by the siderophore

resulting thus in higher bioavailability of iron to the cyanobacterium. M. aeruginosa

[

25]

itself is known to produce hydroxamate-type siderophores. However, the

phenomenon that microbes can uptake siderophores they do not produce by

[

26]

themselves is well known and was described as ‘siderophore piracy’. In addition, it

could be demonstrated that uncultured bacteria were enabled to grow via the

[

27]

secretion of siderophores of a distantly related and previously cultured bacterium.

Additionally, it was shown that within the genome of some bacteria the number of

[

28]

siderophore uptake genes is much higher than the number of synthesis genes.

From all these findings it can be concluded that the inability to produce siderophores

and thus the dependence on other organisms might allow microbial growth to

[

27]

establish a community only in an environment with favored neighbours. Although

only known from heterotrophic bacteria so far, a similar ‘siderophore piracy’ might

also take place in cyanobacteria. Still it is unknown whether the iron-anachelin

complex delivers iron directly or through a specific transporter into the Microcystis

cell. However, it seems that M. aeruginosa is a strong competitor under iron

[

25]

limitation.

In a first conclusion, these experiments demonstrate that anachelin (1) is not

[

15]

only able to enhance growth of its producer, but also to enhance growth of other

cyanobacteria such as M. aeruginosa, likely via facilitated Fe uptake by chelation.

Effect of Anachelin (1) on Growth of Kirchneriella contorta

We then investigated the action of anachelin on a competing planktonic green

algae (chlorophyte, Kirchneriella contorta). Anachelin (1) inhibited the growth of this

species in a dose-dependant manner, starting at nanomolar concentrations; a

significant impact could be observed at low micromolar concentrations (Fig. 4). This

effect was not due to iron sequestering by anachelin, as this allelopathic effect was

also found to be present at a large excess of iron: at 10 μM total Fe(III) concentration,

nanomolar concentrations of anachelin were sufficient to negatively impact the

growth of the green algae (Fig. 4). A growth reduction of 15% was already observed

at 900 nM concentrations; at 60 μM a growth reduction of 33% was found. From

these experimental data, it can be concluded that this allelopathy is most likely

independent of iron concentration, thus questioning the ‘iron monopoly’

[

13]

hypothesis, and adding support to the hypothesis of additional inhibitory activity

unrelated to iron chelation. Matz and co-workers could show that siderophores

derived from Anabaena flos-aquae inhibited the growth of the green alga

Chlamydomonas reinhardtii via uncharacterized toxicity in iron-limited medium but

[

14]

not due to competition of iron. Additionally, we observed a significant growth

inhibitory effect of anachelin against Chlamydomonas which reflects its general

allelopathic activity (see Supporting Information).

Taken together, these results provide evidence that anachelin (1) is able to

promote the growth of the cyanobacterium M. aeruginosa, and that this natural

product inhibits the growth of two competing green algae. Such a dual mode of

action for an iron chelator is surprising. We then wanted to evaluate the structural

requirements for these effects, and in particular, find out if certain fragments of

anachelin (1) were responsible for this divergent activity.

Effect of Fragments on Growth of Kirchneriella contorta

In order to investigate the chemical mode of action of this allelopathic activity,

and to identify the structural requirements, derivatives 2 – 4 of anachelin (1) were

tested for the growth inhibition of K. contorta (Fig. 5). First we investigated the role of

the quaternary ammonium functionality, which is known for manifold biological

[

29]

activities including potent antibacterial and antifungal properties. Fragment 2 was

in principle able to inhibit the growth of the green algae but not to the same extent as

the anachelin itself. At a very high concentration of 60 μM fragment 2 reduced the

growth of the green algae of about 17% (Fig. 5,a). Virtually the same inhibition effect

was observed with anachelin (1) already at high nanomolar concentrations that is

two orders of magnitude lower. Several quaternary ammonium compounds have

been found to negatively effect the growth of green algae such as Scenedesmus

[

30]

intermedius, Dictyosphaerium chlorelloides, and Pseudokirchneriella subcapitata.

Thus, it is possible that the quaternary ammonium group of fragment 2 supported the

total activity of anachelin, but to a minor extent. In contrast, fragment 3 did not have

any negative impact on the growth of K. contorta, even at very high concentrations

(Fig. 5,b). At two concentrations, a significant positive effect was observed, but the

overall tendency was not different from the control. Comparing the fragments 2 and 3

one can speculate, that the impact of the quaternary ammonium, if bound to the

tripeptide as in fragment 3 or in anachelin (1), is negligible for the overall growth

inhibitory effect of anachelin.

Cyanobacteria are well recognized for the production of polyketides with

[

31]

broad biological activities. In a next experiment, the polyketide fragment 4 of

anachelin as prepared through the organocatalyic route above, was tested for the

growth inhibitory effect on K. contorta. The polyketide inhibited the growth of the

green algae in a dose-dependant manner, starting at nanomolar concentrations (Fig.

5

,c). A significant impact could be observed at low micromolar concentrations. At 60

μM a growth reduction of 38% was observed. Thus, the impact of fragment 4 on the

growth of the green algae resembled the one of anachelin (1). It can be concluded

that virtually the complete antialgal activity was retained in the polyketide fragment 4

(Fig. 5).

Effect of Diastereoisomers on Growth of Kirchneriella contorta

We next investigated whether this inhibitory and allelopathic activity is

unrelated to iron binding. Therefore, two diastereoisomers of anachelin, i.e.,

compounds 5 and 6 were evaluated, that are still able to bind iron, but differ in the

configuration of the polyketide fragment. Interestingly, both the 3,5-epi-anachelin (5)

and the 3,5,6-epi-anachelin (6) were hardly affecting the growth of K. contorta (Fig.

6

). Only the 3,5-epi-anachelin showed a growth reduction of 12% at high (60 μM)

concentrations. Additionally, using 3,5,6-epi-anachelin, the molecule in which the

configuration of all three OH groups of the polyketide was inverted, no significant

growth inhibition could be observed. This demonstrates that the relative configuration

in the polyketide fragment of anachelin is crucial for allelopathic activity and that the

observed growth inhibitory activity is unrelated to iron binding and siderophore

activity.

The Dual Mode of Action

All these results provide strong evidence on a molecular level that the

siderophore anachelin possesses a dual mode of action both promoting the growth

of cyanobacteria and concomitantly inhibiting the growth of competing

phytoplankton. The two bioactivities are promoted by different fragments of the same

molecule: whereas the tetrahydroquinolinium fragment (shown in green in 1) binds

[

18]

iron and is thus mainly considered responsible for growth promotion, the

polyketide fragment (shown in red in 1) is responsible for the allelopathic growth

inhibition. It is interesting to note that the different bioactivities reside in fragments

that might have different biosynthetic origins: The iron binding in the alkaloid part and

the allelopathic activity in the polyketide fragment. As anachelin can be considered a

product of mixed polyketide synthase and non-ribosomal peptide synthetase

pathways, it can be speculated that these fragments, and thus the corresponding

activities, have biosynthetically merged over time in one single compound. These

experimental findings for a cyanobacterium are complemented by dual mode of

action hybrids (sideromycins) isolated from bacteria, where both iron chelating and

[

32]1

antibiotic fragments are complementary present.

In addition, anachelin (1)

provides an interesting example of natural product with two different fragments

[

34]

merged. While many natural products can be structurally simplified, in this case,

one biological property would be lost. Based on structural considerations, such

combined fragments having different functions could be operative in other natural

2

products from cyanobacteria, such as the cyanopeptolins or the aeruginosins.

1

2

[33]

For a review on natural product hybrids, see

.

[35]

For our work in this area, see

.

Possible Role of Anachelin in the Environment

In the ecological context, it should be pointed out that the producer of

anachelin, Anabaena cylindrica, is a filamentous freshwater cyanobacterium that can

form biofilms and can occur also as planktonic organism in ponds and small lakes. In

such densely populated environments, where cooperative behavior may be

successful, secreted iron chelators are likely to be taken up by other cells of the

filament or by other filaments thus increasing the overall amount of bioavailable iron.

At the same time, the anachelin that is lost by diffusion can be predicted to display a

secondary benefit by inhibiting the growth of competing organisms like eukaryotic

algae. By using one secondary metabolite, Anabaena cylindrica can address two

main challenges related to phytoplankton growth, i.e. nutrient limitation and inhibition

of competitors.

Conclusions

In conclusion, we have shown that the iron chelator anachelin (1) stimulates

growth of Microcystis aeruginosa while at the same time reducing the growth of two

competing green algae, i.e. Kirchneriella and Chlamydomonas. Chemical synthesis

of the derivatives such as 2 – 4 and diastereoisomers such as 5 and 6 allowed for

the identification of the crucial fragment for this activity, where the polyketide moiety

was shown to be responsible for allelopathy. Anachelin (1) can thus be considered a

molecular hybrid with a dual mode of action. These experiments described herein

provide therefore detailed experimental evidence for theories documented over the

[

11][14]

last 50 years regarding the role of iron chelators related to algal blooms.

The data presented in this study provide a strong rationale on a molecular

level for the hypothesis that the excretion of iron chelators such as anachelin

constitutes a strategy of cyanobacteria to dominate competing species.

Supplementary Material

Supporting information for this article is available on the WWW under

http://dx.doi.org/10.1002/hlca.201600123.

Author Contribution Statement

J. F. B. and K. G. conceived this work; J. F. B. and K. G. designed the

experiments; S. D. S., J. F. B., and Y. B. conducted the experiments; S. D. S., J. F.

B., Y. B., F. J., and K. G. discussed the results and commented on the manuscript,

S. D. S., J. F. B., and K. G. wrote the manuscript.

Experimental Part

Abbreviations

Boc = tert-butoxycarbonyl; CDI = carbonyldiimidazole; DMAP = N, N-

dimethylaminopyridine; EDC = ethyl-(N, N-dimethylaminopropyl)car-bodiimide; ether

refers to diethylether; FC = flash chromatography; HOBt = 1-hydroxybenzotriazole;

NMM = N-methyl-morpholine; RT = room temperature; TBS = tert-butyl-dimethylsilyl;

TFA = trifluoroacetic acid; THF = tetrahydrofuran; THQ = 3-amino-6,7-dihydroxy-1,1-

dimethyl-1,2,3,4-tetrahydro-quinolinium; Z = benzyloxycarbonyl.

General

Unless otherwise stated, chemicals were purchased from Sigma–Aldrich,

ABCR, Acros, or Lancaster and used without further purification. Solvents for work-

up and chromatography were distilled from technical quality. Solvents used for

chemical transformations were either puriss quality or dried by filtration through

activated aluminium oxide under Ar or N (H O content < 10 ppm, Karl–Fischer

2

titration). Reactions involving air or moisture sensitive reagents or intermediates

were performed under Ar or N in glassware which had been oven dried or dried by a

2

heat gun under high vacuum. Concentration under reduced pressure was performed

by rotary evaporation at 40 °C (unless otherwise specified). Yields refer to purified,

dried and spectroscopically pure compounds. Analytical thin layer chromatography

(TLC) was performed on Merck silica gel 60 F254 plates (0.25 mm thickness) pre-

coated with fluorescent indicator. The developed plates were examined under UV

light and stained with ceric ammonium molybdate followed by heating. Flash

chromatography was performed using silica gel 60 (230 – 240 mesh) from Fluka

1

using a forced flow eluant at 0.3 – 0.5 bar pressure. H-NMR spectra were recorded

on Varian Gemini Bruker DPX 400 MHz or Bruker DRX 600 MHz spectrometers at

2

98 or 348 K in the indicated deuterated solvent, unless otherwise stated. Data are

reported as follow: chemical shift (δ, ppm), multiplicity (s, singulet; d, doublet; t,

triplet; q, quartet; m, multiplet or not resolved signal; br., broad signal; p, pseudo

signal), coupling constant (J,), integration. All signals were referenced to the internal

solvent signal as standard (CDCl , δ(H) 7.26) or ((D )DMSO, δ(H) 2.50) or

3

6

13

1

(

(D )methanol, δ(H) 3.31). C-NMR spectra were recorded with H-decoupling on

4

Varian Gemini 100 MHz or Bruker DPX 125 MHz spectrometers at 298 K or 348 K in

the indicated deuterated solvent, unless otherwise stated. All signals were

referenced to the internal solvent signal as standard (CDCl , δ(C) 77.0) or

3

(

(D )DMSO, δ(C) 39.52) or ((D )methanol, δ(C) 49.00). IR Spectra were recorded

6

4

–1

using a Varian 800 FT-IR ATR spectrometer. The absorptions are reported in cm .

T

Optical rotations [α]_Dwere measured at the sodium D line using a 1 ml cell with a 1

dm path length on a Jasco P-2000 digital polarimeter. The concentration c is given in

g/100 ml and the used solvent is CHCl . All mass spectra (HR-ESI-MS) were

3

recorded by the Mass spectrometric Service of University of Bern on Sciex QSTAR

Pulsar mass spectrometer using electrospray ionization. Compounds 1 and 2 were

prepared according to literature procedure [18][19].

N-(tert-Butoxycarbonyl)-L-threonyl-D-seryl-N-[(3S)-1,2,3,4-tetrahydro-6,7-

dihydroxy-1,1-dimethylquinolinium-3-yl]-L-serinamide (3). Boc-L-Thr(OBn)-D-

[

20]

Ser(OBn)-L-Ser(OBn)-(3S)-THQ(OBn) chloride (15 mg, 14.5 μmol) was dissolved

in MeOH (150 μl) and glacial AcOH (30 μl) was added. The reaction vessel was

flushed three times with Ar and Pd/C (10 %) was added. The reaction vessel was

flushed with H three times and the mixture stirred at r.t. for 4 h. The mixture was

2

then filtered over Celite, and the solvent evaporated under reduced pressure. The

residual acid was coevaporated with CHCl to give target compound 3 (quant.).

3

24

1

[

α]_D= –310.8 (c = 0.48 in H O). FT-IR (neat): 3282, 2976, 2484, 1657, 1525. H-

2

NMR (D O, 300 MHz): 1.17-1.24 (m, 3 H); 1.35 (s, 6 H); 1.41 (s, 3 H); 1.95 (s,

2

residual AcOH); 2.9-3.07 (m, 1 H); 2.07-3.2 (m, 1 H); 3.57 (m, 7 H); 3.89 (m, 4 H);

4

.04-4.24 (m, 2 H); 4.38-4.52 (m, 2 H); 4.56-4.70 (m, 1 H); 6.8 (s, 1 H); 7.23 (s, 1 H).

+

HR-MS: 584.2916 (M , C H N O ; calc. 584.2926).

26

42

5

10

Preparation of Compound 4

tert-Butyl (4S)-4-[(1S)-1-hydroxybut-3-en-1-yl)-2,2-dimethyl-1,3-oxazolidine-3-

carboxylate (8) was prepared by the addition of Garner aldehyde [20] (7) into (R,R)-

Duthaler–Hafner reagent and allyl magnesium chloride complex following the

[

21a]

literature procedure.

The resulting alcohol was protected to afford the known tert-

butyl (4S)-4-[(1S)-1-{[tert-butyl(dimethyl)silyl]oxy}but-3-en-1-yl]-2,2-dimethyl-1,3-

[

21b]

oxazolidine-3-carboxylate (9) and the spectra matched those reported.

tert-Butyl (4S)-4-[(1S,3E)-1-{[tert-Butyl(dimethyl)silyl]oxy}-5-oxopent-3-

en-1-yl]-2,2-dimethyl-1,3-oxazolidine-3-carboxylate (10). Crotonaldehyde (0.64

ml, 7.86 mmol, 6.0 equiv.) was added to a stirred soln. of 9 (505 mg, 1.31 mmol, 1.0

equiv.) and Grubbs–Hoveyda 2nd generation catalyst (16.4 mg, 26.2 μmol, 0.02

equiv.) in CH Cl (15 ml). The resulting mixture was heated to reflux for 18 h and

2

then cooled to r.t. The solvent was removed in vacuo and the residue was purified by

flash chromatography (SiO ; 15:1 pentane/AcOEt) to yield 10 as a colorless oil (511

2

26

mg, 1.24 mmol, 94%). R (pentane/AcOEt 15:1) 0.3. [α]

= –39.5 (c = 1.0 in CHCl₃).

f

D

1

FT-IR (neat): 2931, 1695, 1366, 1253, 1170, 1074, 836, 776. H-NMR (600 MHz,

(

D )DMSO, 348 K): 9.60 (d, J = 7.7, 1 H); 7.04 (ddd, J = 15.4, 8.0, 6.6, 1 H); 6.21

6

(dd, J = 15.6, 7.7, 1 H); 4.42 – 4.38 (m, 1 H); 4.18 – 4.14 (m, 1 H); 4.06 – 4.02 (m, 2

H); 2.64 – 2.59 (m, 1 H); 2.53 – 2.46 (m, 1 H); 1.63 (s, 3 H); 1.56 (s, 9 H); 1.52 (s, 3

13

H); 0.96 (s, 9 H); 0.18 (s, 3 H); 0.14 (s, 3 H). C-NMR (151 MHz, (D )DMSO, 348 K):

6

1

4

92.8; 155.3; 151.4; 134.0; 93.7; 79.2; 69.5; 62.0; 59.6; 34.0; 27.7; 25.7; 25.1; 22.5;

+

7.1; –5.0; –5.2. HR-ESI-MS (pos.): 414.2666 ([M + H] , C H NO Si ; calc.

21

40

5

14.2670).

tert-Butyl (4S)-4-[(1S,3R)-1-{[tert-Butyl(dimethyl)silyl]oxy}-3-hydroxy-5-

methoxy-5-oxopentyl]-2,2-dimethyl-1,3-oxazolidine-3-carboxylate (11). H O₂

2

(

35% aq., 78 ml, 0.96 mmol, 1.2 equiv.) was added to stirred soln. of 10 (331 mg,

.80 mmol, 1.0 equiv.) and (S)-(–)-α,α-diphenyl-2-pyrrolidinemethanol trimethylsilyl

ether (27.4 mg, 0.08 mmol, 0.1 equiv.) in CH Cl (4 ml) at r.t. under Ar atmosphere.

0

2

Stirring was continued at r.t. for 16 h and then the solvent was removed. The crude

mixture was further dried under high vacuum (2 h, r.t.), the diastereoselectivity of the

1

crude epoxy aldehyde was measured by H-NMR, and the residue was then

dissolved in CH Cl (4 ml). To the mixture were then sequentially added 6,7-dihydro-

2

-(pentafluorophenyl)-5H-pyrrolo[2,1-c][1,2,4]triazol-2-ium tetrafluoroborate (14.4

mg, 0.04 mmol, 0.05 equiv.), MeOH (162 μl, 4.0 mmol, 5.0 equiv.) and NEt (68 μl,

3

0

.4 mmol, 0.5 equiv.). Stirring was continued for 12 h at r.t. The solvent was

removed in vacuo and the residue was purified by flash chromatography (SiO , 6:1

2

pentane/AcOEt) to yield the target compound 11 (inseparable mixture of

diastereomers) as colorless oil (298 mg, 0.64 mmol, 81%). R (pentane/AcOEt 6:1)

f

1

0

4

3

.3. For mixture of diastereomers and rotamers: H-NMR (400 MHz, CDCl , 298 K):

3

.43 – 4.26 (m, 1 H); 4.23 – 4.06 (m, 2 H); 4.00 – 3.80 (m, 2 H); 3.78 – 3.54 (m, 3 H);

.43 – 3.17 (m, 1 H); 2.59 – 2.35 (m, 2 H); 1.71 – 1.62 (m, 2 H); 1.59 – 1.36 (m, 15

13

H); 0.95 – 0.79 (m, 9 H); 0.23 – 0.03 (m, 6 H). C-NMR (101 MHz, CDCl , 298 K):

3

1

6

2

72.3; 152.7 and 152.1; 95.0 and 94.3; 80.3; 70.6 and 70.1; 67.5; 63.1 and 62.8;

0.4 and 59.7; 51.6; 41.6 and 41.3; 37.0; 28.6 and 28.4; 26.5 and 25.9; 25.7 and

5.7; 23.9 and 22.4; 17.9 and 17.7; –3.8 and –4.1 and –4.9 and –5.3. HR-ESI-MS

+

(

pos.): 462.2876 ([M + H] , C H NO Si ; calc. 462.2882).

22

44

7

tert-Butyl (4S)-4-[(5S,7R)-7-(2-Methoxy-2-oxoethyl)-2,2,3,3,9,9,10,10-

octamethyl-4,8-dioxa-3,9-disilaundecan-5-yl]-2,2-dimethyl-1,3-oxazolidine-3-

carboxylate (12). Lutidine (104 μl, 0.88 mmol, 2.0 equiv.) and TBSOTf (152 μl, 0.66

mmol, 1.5 equiv.) were successively added to a soln. of 11 (203 mg, 0.44 mmol, 1.0

equiv.) in CH Cl (2 ml) at –20 °C. The mixture was stirred at –20 °C for 1 h and then

2

slowly warm to r.t. over 1 h. Then CH Cl was added and the org. phase was

2

washed 3 × with citric acid (10%) and 2 × with H O, dried over Na SO , and filtered.

2

4

The solvent was removed in vacuo and the residue was purified by flash

chromatography (SiO , 50:1 to 30:1 pentane/AcOEt) to yield 12 as colorless oil (12

2

as major diastereomer 209 mg and 12′ as minor diastereomer 35 mg, 0.42 mmol

26

overall, 96% overall yield). Data of 12: R (pentane/AcOEt 30:1) 0.30. [α]

= –55.3

f

D

(

c = 1.0 in CHCl ). FT-IR (neat): 2955, 2931, 1743, 1705, 1473, 1384, 1255, 1170,

3

1

8

37, 777. H-NMR (600 MHz, (D )DMSO, 348 K): 4.31 – 4.24 (m, 1 H); 4.19 – 4.07

6

(

m, 1 H); 4.02 – 3.96 (m, 1 H); 3.95 – 3.91 (m, 1 H); 3.91 – 3.86 (m, 1 H); 3.59 (s, 3

H); 2.50 – 2.46 (m, 1 H); 2.25 (dd, J = 14.5, 8.5, 1 H); 1.70 – 1.60 (m, 2 H); 1.52 (s, 3

H); 1.45 (s, 9 H); 1.41 (s, 3 H); 0.89 (s, 9 H); 0.85 (s, 9 H); 0.11 (s, 6 H); 0.05 (s, 3

13

H); 0.01 (s, 3 H). C-NMR (151 MHz, (D )DMSO, 348 K): 170.6; 151.3; 93.6; 79.2;

6

–

7.1; 66.6; 62.0; 59.3; 50.5; 40.9; 38.1; 27.7; 25.5; 25.2; 25.2; 22.6; 17.1; 17.1; –4.5;

+

4.7; –5.3; –5.4. HR-ESI-MS (pos.): 576.3741 ([M + H] , C H NO Si ; 576.3746).

28

58

7

2

26

Data of minor diastereomer 12′: R (pentane/AcOEt 30:1) 0.33. [α]_D= –15.9

f

(

c = 0.5 in CHCl ). FT-IR (neat): 2955, 2931, 1743, 1705, 1473, 1384, 1255, 1170,

3

1

8

37, 777. H-NMR (600 MHz, (D )DMSO, 348 K): 4.29 – 4.22 (m, 1 H); 4.21 – 4.15

6

(

m, 1 H); 3.98 – 3.93 (m, 1 H); 3.93 – 3.87 (m, 2 H); 3.59 (s, 3 H); 2.58 (dd, J = 14.9,

.6, 1 H); 2.45 (dd, J = 14.9, 7.3, 1 H); 1.70 (ddd, J = 14.3, 5.9, 2.1, 1 H); 1.59 – 1.54

4

(

m, 1 H); 1.53 (s, 3 H); 1.45 (s, 9 H); 1.41 (s, 3 H); 0.88 (s, 9 H); 0.84 (s, 9 H); 0.12

13

(

s, 6 H); 0.06 (s, 3 H); 0.03 (s, 3 H). C-NMR (151 MHz, (D )DMSO, 348 K): 170.4;

6

1

2

51.5; 93.5; 79.1; 68.3; 67.6; 61.8; 59.7; 50.4; 43.4; 38.6; 27.7; 25.7; 25.2; 25.2;

+

2.4; 17.1; 17.1; –4.4; –4.6; –4.8; –5.1. HR-ESI-MS (pos.): 576.3749 ([M + H] ,

+

C H NO Si ; 576.3746).

28

58

7

2

Methyl (3R,5S,6S)-6-{[2-(Benzyloxy)benzoyl]amino}-3,5-bis{[tert-

butyl(dimethyl)silyl]oxy}-7-hydroxyheptanoate (13). TFA (0.4 ml) was added

drop-wise to a soln. of 12 (118 mg, 0.20 mmol, 1.0 equiv.) in CH Cl (2.0 ml) at 0 °C.

2

The mixture was stirred at 0 °C for 1 h, and then, the solvent was removed under

reduced pressure keeping the temp. below 5 °C. Excess TFA was removed using

high vacuum (3 h) at r.t. The crude material 13′ was dissolved in dry CH Cl (1.0 ml)

2

and used for next step without further purification.

.

EDC HCl (47 mg, 0.24 mmol, 1.2 equiv.), HOBt (33 mg, 0.24 mmol, 1.2

equiv.), and NMM (68 μl, 0.60 mmol, 3.0 equiv.) were successively added to a soln.

of O-Bn-salicylic acid (60 mg, 0.24 mmol, 1.2 equiv.) in CH Cl (1.0 ml) at r.t. The

2

mixture was stirred at r.t. for 30 min, and then a soln. of 13′ in CH Cl (1.0 ml) was

2

added. Stirring was continued for 20 h. The solvent was removed in vacuo and the

residue was purified by flash chromatography (SiO , 4:1 pentane/AcOEt) to yield 13

2

26

as colorless oil (99 mg, 0.15 mmol, 75%). R (pentane/AcOEt 4:1) 0.2. [α]

= +9.9

f

D

(

c = 1.0 in CHCl ). FT-IR (neat): 2954, 2930, 1729, 1601, 1487, 1451, 1295, 1237,

3

1

7

086, 887. H-NMR (400 MHz, CDCl , 298 K): 8.21 (d, J = 8.1, 1 H); 8.18 (dd, J =

3

.8, 1.8, 1 H); 7.45 – 7.29 (m, 6 H); 7.05 (p t, J = J = 7.5, 1 H); 6.96 (d, J = 8.3, 1

1

2

H); 5.28 (s, 2 H); 4.39 – 4.28 (m, 1 H); 4.21 – 4.07 (m, 2 H); 3.76 – 3.62 (m, 2 H);

.49 (s, 3 H); 2.54 (br. s, 1 H); 2.40 (d, J = 6.0, 2 H); 1.78 – 1.61 (m, 2 H); 0.88 (s, 9

3

13

H); 0.83 (s, 9 H); 0.11 (s, 3 H); 0.09 (s, 3 H); 0.08 (s, 3 H); 0.07 (s, 3 H). C-NMR

(

101 MHz, CDCl , 298 K): 171.2; 166.4; 156.6; 135.8; 132.8; 132.4; 128.8; 128.4;

3

1

27.2; 121.8; 121.4; 113.1; 70.9; 68.9; 66.2; 64.5; 54.0; 51.3; 43.1; 41.5; 25.8; 25.7;

+

7.8; 17.8; –4.4; –4.5; –4.6; –4.8. HR-ESI-MS (pos.): 646.3580 ([M + H] ,

+

C H NO Si ; calc. 646.3590).

34

56

7

2

Methyl (3R,5S,6S)-3,5-Bis{[tert-butyl(dimethyl)silyl]oxy}-7-hydroxy-6-[(2-

hydroxybenzoyl)amino]heptanoate (4′). Compound 13 (34.5 mg, 53.4 μmol, 1.0

equiv.) was dissolved in dry CH OH (2.0 ml), and glacial AcOH (200 μl) was added.

3

The reaction flask was flushed three times with Ar, and then 10% Pd/C (11.4 mg,

1

0.7 μmol, 0.2 equiv.) was added. The reaction flask was flushed three times with

H , and the mixture was stirred 2.5 h at r.t. The mixture was then filtered over Celite

2

and solvent was removed in vacuo to yield 4′ as a colorless oil (29.5 mg, 53.1 μmol,

2

5

9

3

9%). R (pentane/AcOEt 4:1) 0.5. [α]_D= –14.4 (c = 1.0 in CHCl ). FT-IR (neat):

f

3

1

343, 2930, 2360, 1740, 1636, 1536, 1362, 1254, 1097, 1038, 835, 776. H-NMR

(

400 MHz, CDCl , 298 K): 7.43 – 7.37 (m, 1 H); 7.31 (d, J = 7.9, 1 H); 6.98 (d, J =

3

8

4

3

.3, 1 H); 6.93 (d, J = 8.2, 1 H); 6.89 – 6.81 (m, 1 H); 4.28 (dd, J = 13.5, 6.5, 1 H);

.21 (dd, J = 8.1, 4.9, 1 H); 4.17 – 4.09 (m, 1 H); 3.80 (dd, J = 10.5, 6.6, 1 H); 3.74 –

.64 (m, 1 H); 3.53 (s, 3 H); 2.50 (dd, J = 14.7, 5.0, 1 H); 2.43 (dd, J = 14.7, 7.3, 1

H); 2.09 (br. s, 1 H); 1.82 – 1.68 (m, 2 H); 0.94 (s, 9 H); 0.88 (s, 9 H); 0.15 (s, 3 H);

1

3

0

1

4

+

.13 (s, 3 H); 0.11 (s, 3 H); 0.08 (s, 3 H). C-NMR (101 MHz, CDCl , 298 K): 171.1;

3

70.5; 161.6; 134.4; 125.3; 118.8; 118.7; 114.0; 68.2; 66.1; 63.6; 52.9; 51.5; 43.0;

2.1; 25.9; 25.7; 17.9; 17.8; –4.2; –4.5; –4.6; –4.9. HR-ESI-MS (pos.): 556.3111 ([M

+

H] , C H NO Si ; calc. 556.3120).

27

50

7

2

Methyl (3R,5S,6S)-3,5,7-Trihydroxy-6-[(2-

hydroxybenzoyl)amino]heptanoate (4). A soln. of HCl (4M) in dioxane (91 μl) was

added drop-wise to a soln. of 4′ (28.9 mg, 52.0 μmol, 1.0 equiv.) in dry MeOH (2.1

ml) at 0 °C. The mixture was stirred at 0 °C for 1.5 h. All volatiles were removed

under reduced pressure while keeping the soln. temp. below 5 °C. The residue was

further dried using high vacuum (1 h) at r.t. to provide the desired product as

26

colorless oil (16.9 mg, 51.6 μmol, 99%). R (CH Cl /MeOH 10:1) 0.3. [α]

= –23.7

f

2

D

(c = 1.0 in MeOH). FT-IR (neat): 3350, 2955, 2363, 1721, 1636, 1546, 1443, 1307,

1

7

3

256, 757. H-NMR (400 MHz, MeOD, 298 K): 7.87 (dd, J = 8.1, 1.4, 1 H); 7.41 –

.33 (m, 1 H); 6.95 – 6.88 (m, 2 H); 4.32 – 4.23 (m, 1 H); 4.23 – 4.12 (m, 2 H); 3.79 –

.68 (m, 2 H); 3.62 (s, 3 H); 2.52 (dd, J = 15.1, 4.8, 1 H); 2.44 (dd, J = 15.1, 8.0, 1

13

H); 1.78 – 1.63 (m, 2 H). C-NMR (101 MHz, MeOD): 173.7; 170.3; 160.1; 134.6;

30.0; 120.3; 118.1; 117.9; 69.1; 67.7; 62.8; 55.8; 52.0; 43.2; 41.7. HR-ESI-MS

1

+

(

pos.): ■329.1456■Authors: you gave C H NO ■ ([M + H] , C H NO ; calc.

15

23

7

15 22

7

3

28.1391).

Preparation of 3,5-epi-Anachelin (5)

Boc-L-Thr(OBn)-D-Ser(OBn)-L-Ser(OBn)-(3S)-THQ(OBn) [18][20] (25 mg, 14.9

μmol) was dissolved in dioxane (0.4 ml) and HCl·dioxane (4M, 90 μmol) was added

at 0 °C. It was stirred 1 h at this temp. and 3 h at r.t. The solvent was removed under

reduced pressure and the product dried under high vacuum 2 h. It was used without

further purification.

To a soln. of (3S,5R,6S)-6-{[2-(benzyloxy)benzoyl]amino}-3,5,7-tris{[tert-

butyl(dimethyl)silyl]oxy}heptanoic acid [18][20] (11 mg, 14.9 μmol) in DMF (0.4 ml) at

i

–

18 °C was added BuOCOCl (1.9 μl, 14.9 μmol) and NMM (5.4 μl, 49.2 μmol). The

mixture was stirred at this temp. for 20 min. Then, H-L-Thr(OBn)-D-Ser(OBn)-L-

Ser(OBn)-(3S)-THQ(OBn) (15 mg, 14.9 μmol) dissolved in DMF (0.4 ml) and NMM

3.3 μl, 29.8 μmol) was added dropwise at –18 °C and it was allowed to warm over 4

(

h. The solvent was evaporated under reduced pressure and the residue taken up in

AcOEt. It was washed 3 x with H O, and the resulting H O was extracted 3 x with

2

AcOEt. The combined org. phases were dried over Na SO , filtered, and evaporated

2

4

under reduced pressure. FC (CH Cl /MeOH, 10:1) gave (3S,5R,6S)-6-{[2-

2

(benzyloxy)benzoyl]amino}-3,5,7-tris{[tert-butyl(dimethyl)silyl]oxy}heptanoic acid-L-

Thr(OBn)-D-Ser(OBn)-L-Ser(OBn)-(3S)-THQ(OBn) (9 mg, 5.4 μmol, 36%) as a

colorless oil.

(3S,5R,6S)-6-{[2-(benzyloxy)benzoyl]amino}-3,5,7-tris{[tert-

butyl(dimethyl)silyl]oxy}heptanoic acid-L-Thr(OBn)-D-Ser(OBn)-L-Ser(OBn)-(3S)-

THQ(OBn) (9 mg, 5.4 μmol) was dissolved in MeOH (350 μl), and glacial AcOH (30

drops) was added. The reaction vessel was flushed 3 × with Ar, and then Pd/C (10

%

, 7 mg) was added. The reaction vessel was three times flushed with H , and the

2

mixture was stirred 2.5 h at r.t. The mixture was then filtered over Celite and the

solvent removed under reduced pressure. The residue was dissolved in cold MeOH

containing 1% HCl (1ml) and the soln. stirred for 4 h at 0°C. Then the solvent was

evaporated under reduced pressure and the residual acid was coevaporated three

times with CHCl . It was dissolved in D O and lyophilized to give 3,5-epi-anachelin

3

2

(5; N-{(3S,5R,6S)-3,5,7-trihydroxy-6-[(2-hydroxybenzoyl)amino]heptanoyl}-L-

threonyl-D-seryl-N-[(3S)-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-

tetrahydroquinolinium-3-yl]-L-serinamide; 2.3 mg, 2.9 μmol, 55%) as light yellow

2

5

fluffy powder. [α]_D= –60.0 (c = 0.1 in H O). FT-IR (neat): 3679, 1643, 1609, 1571,

2

1

+

1

537. H-NMR data: see Supporting Information. HR-ESI-MS: 779.3448 (M ,

+

C H N O ; calc. 779.3458).

35

51

6

14

Preparation of 3,5,6-epi-Anachelin (6)

Boc-L-Thr(OBn)-D-Ser(OBn)-L-Ser(OBn)-(3S)-THQ(OBn)

[

18][20]

(38 mg, 35.4

μmol) was dissolved in dioxane (0.6 ml) and HCl·dioxane (4M, 100 μmol) was added

at 0 °C. It was stirred 1 h at this temp. and 3 h at r.t. The solvent was removed under

reduced pressure and the product dried under high vacuum 2 h. It was used without

further purification. To a soln. of (6R)-6-(2-benzyloxy-benzoylamino)-(3S,5R)-3,5,7-

[

18][20]

tris-(tert-butyl-dimethyl-silanyloxy)-heptanoic acid

(24 mg, 32.2 μmol) in DMF

(0.6 ml) at –18 °C was added iBuOCOCl (4.2 μl, 32.2 μmol) and NMM (13 μl, 113

μmol). The mixture was stirred at this temp. for 20 min. Then H-L-Thr(OBn)-D-

Ser(OBn)-L-Ser(OBn)-(3S)-THQ(OBn) (36 mg, 35.4 μmol) dissolved in DMF (0.5 ml)

and NMM (7 μl, 38 μmol) was added dropwise at –18 °C and it was allowed to warm

over 4 h. The solvent was evaporated under reduced pressure and the residue taken

up in AcOEt. It was washed three times with H O, and the resulting water extracted

2

three times with AcOEt. The combined org. phases were dried over Na SO , filtered,

2

4

and evaporated under reduced pressure. FC (CH Cl /MeOH 10:1) gave (3S,5R,6R)-

2

6

-[benzoyl(benzyloxy)amino]-3,5,7-tris{[tert-butyl(dimethyl)silyl]oxy}heptanoic acid-L-

Thr(OBn)-D-Ser(OBn)-L-Ser(OBn)-(3S)-THQ(OBn) (27 mg, 16 μmol, 50%) as

colorless oil.

This compound (9 mg, 5.3 μmol) was dissolved in MeOH (350 μl) and glacial

AcOH (30 drops) was added. The reaction vessel was flushed three times with Ar,

and then Pd/C (10 %, 7 mg) was added. The reaction vessel was three times flushed

with H and the mixture was stirred 2.5 h at r.t. The mixture was then filtered over

2

Celite, and the solvent removed under reduced pressure. The residue was dissolved

in cold MeOH containing 1% HCl (1 ml) and the soln. stirred for 4 h at 0 °C. Then,

the solvent was evaporated under reduced pressure and the residual acid was co-

evaporated three times with CHCl . It was dissolved in D O and lyophilized to give

3

2

3

,5,6-epi-anachelin (6; N-{(3S,5R,6R)-3,5,7-trihydroxy-6-[(2-

hydroxybenzoyl)amino]heptanoyl}-L-threonyl-D-seryl-N-[(3S)-6,7-dihydroxy-1,1-

dimethyl-1,2,3,4-tetrahydroquinolinium-3-yl]-L-serinamide; 2.1 mg, 2.6 μmol,

2

5

3

0%) as light yellow fluffy powder. [α]_D= –26.7 (c = 0.12 in H O). FT-IR (neat):

2

1

+

679, 1638, 1530. H-NMR: see Supporting Information. HR-ESI-MS: 779.3447 (M ,

+

C H N O ; calc. 779.3458).

35

51

6

14

Biological Assays

The blue-green-colored unicellular cyanobacterium Microcystis aeruginosa

PCC 7806 was provided by R. Rippka (Pasteur Culture Collection, Paris, France);

the unicellular green alga Kirchneriella contorta SAG 11.81 was provided by the

Culture Collection of Algae at the University of Göttingen, Germany. Both strains

were grown in Erlenmeyer flasks on a cyanobacterial medium as described by

[

36]

Jüttner and co-workers on 10 μM Fe-citrate at 20 °C under continuous light from

fluorescent tubes (7 μmol m-2 s-1). For the inhibition growth assays, the

microorganisms were transferred into commercial 96-well microtiter plates endowed

with 180 μl cyanobacterial medium or 180 μl Fe-limited cyanobacterial medium (0.1

μM Fe-citrate). 20 μl cell suspension were added to each well having then an OD675

nm of approximately 0.1 at the beginning of the experiment. The plates were

-2

-1

incubated at 20 °C under continuous light from fluorescent tubes (7 μmol m s ).

Measurements of OD₆₇₅were performed daily on an Absorbance Microplate Reader

(Spectra Max 190, Molecular Devices Corporation, Sunnyvale, CA, USA). Anachelin,

the synthesised fragments, and the diastereoisomers were added on the 5th day in

2

0 μl dissolved in H O. Each compound was tested in five to six concentrations,

2

ranging from 8 nM to 90 μM (end concentrations in the wells). The OD₆₇₅was chosen

for the determination of the in vivo absorption of chlorophyll a. To test the validity of

this method, dilutions were prepared ranging from an OD₆₇₅of 0.01 to 1.2 (M.

2

aeruginosa) and of 0.01 to 1.8 (K. contorta). The R values of the linear regression

curves were 0.998 and 0.999, resp.

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1]

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004, 82, 436–442.

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15] a) H. Beiderbeck, K. Taraz, H. Budzikiewicz, A. E. Walsby, ‘Anachelin, the

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Received April 30, 2016

Accepted June 11, 2016

Article Doi

Allelopathic Activity of the Iron Chelator Anachelin?–?A Molecular Hybrid with a Dual Mode of Action

DOI: 10.1002/hlca.201600123

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