Altering the sex pheromone cyclo(L-pro-l-pro) of the diatom seminavis robusta towards a chemical probe

Article abstract of DOI:10.3390/ijms22031037

As a major group of algae, diatoms are responsible for a substantial part of the primary production on the planet. Pennate diatoms have a predominantly benthic lifestyle and are the most species-rich diatom group, with members of the raphid clades being motile and generally having heterothallic sexual reproduction. It was recently shown that the model species Seminavis robusta uses multiple sexual cues during mating, including cyclo(L-Pro-L-Pro) as an attraction pheromone. Elaboration of the pheromone-detection system is a key aspect in elucidating pennate diatom life-cycle regulation that could yield novel fundamental insights into diatom speciation. This study reports the synthesis and bio-evaluation of seven novel pheromone analogs containing small structural alterations to the cyclo(L-Pro-L-Pro) pheromone. Toxicity, attraction, and interference assays were applied to assess their potential activity as a pheromone. Most of our analogs show a moderate-to-good bioactivity and low-to-no phytotoxicity. The pheromone activity of azide-and diazirine-containing analogs was unaffected and induced a similar mating behavior as the natural pheromone. These results demonstrate that the introduction of confined structural modifications can be used to develop a chemical probe based on the diazirine-and/or azide-containing analogs to study the pheromone-detection system of S. robusta.

Full text of DOI:10.3390/ijms22031037

International Journal of
Molecular Sciences
Article
Altering the Sex Pheromone Cyclo(L-Pro-L-Pro) of the Diatom
Seminavis robusta towards a Chemical Probe
Eli Bonneure 1 , Amber De Baets , Sam De Decker , Koen Van den Berge , Lieven Clement , Wim Vyverman
and Sven Mangelinckx *
1
2
3
3
2
1
,
1
Department of Green Chemistry and Technology—SynBioC, Faculty of Bioscience Engineering,
Ghent University, Coupure Links 653, 9000 Ghent, Belgium; eli.bonneure@ugent.be (E.B.);
amber.debaets@gmail.com (A.D.B.)
2
3
Department of Biology—Protistology and Aquatic Ecology, Faculty of Sciences, Ghent University,
Krijgslaan 281/S8, 9000 Ghent, Belgium; samddecker@gmail.com (S.D.D.); wim.vyverman@ugent.be (W.V.)
Department of Applied Mathematics, Computer Science and Statistics, Faculty of Sciences, Ghent University,
Krijgslaan 281/S9, 9000 Ghent, Belgium; koen.vandenberge@ugent.be (K.V.d.B.);
lieven.clement@ugent.be (L.C.)
*
Correspondence: sven.mangelinckx@ugent.be; Tel.: +32-(0)9-264-5951
Abstract: As a major group of algae, diatoms are responsible for a substantial part of the primary
production on the planet. Pennate diatoms have a predominantly benthic lifestyle and are the most
species-rich diatom group, with members of the raphid clades being motile and generally having
heterothallic sexual reproduction. It was recently shown that the model species Seminavis robusta
uses multiple sexual cues during mating, including cyclo(L-Pro-L-Pro) as an attraction pheromone.
Elaboration of the pheromone-detection system is a key aspect in elucidating pennate diatom life-cycle
regulation that could yield novel fundamental insights into diatom speciation. This study reports
the synthesis and bio-evaluation of seven novel pheromone analogs containing small structural
alterations to the cyclo(L-Pro-L-Pro) pheromone. Toxicity, attraction, and interference assays were
applied to assess their potential activity as a pheromone. Most of our analogs show a moderate-
to-good bioactivity and low-to-no phytotoxicity. The pheromone activity of azide- and diazirine-
containing analogs was unaffected and induced a similar mating behavior as the natural pheromone.
These results demonstrate that the introduction of confined structural modifications can be used
to develop a chemical probe based on the diazirine- and/or azide-containing analogs to study the
pheromone-detection system of S. robusta.
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Citation: Bonneure, E.; De Baets, A.;
De Decker, S.; Van den Berge, K.;
Clement, L.; Vyverman, W.;
Mangelinckx, S. Altering the Sex
Pheromone Cyclo(L-Pro-L-Pro) of the
Diatom Seminavis robusta towards a
Chemical Probe. Int. J. Mol. Sci. 2021,
2
2, 1037. https://doi.org/10.3390/
ijms22031037
Keywords: diatom; pheromone; diketopiperazine; chemical probe; diazirine
Received: 14 December 2020
Accepted: 19 January 2021
Published: 21 January 2021
1
. Introduction
Diatoms play a key role in the biosphere. As an important group of phytoplankton,
they account for about one-fifth of the photosynthesis on earth and form the basis of
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with regard to jurisdictional claims in
published maps and institutional affil-
iations.
many of the aquatic ecosystems that exist on our planet [
opportunities for biotechnological applications [3–6].
1,2]. In addition, they present
The benthic diatom Seminavis robusta is an experimental model organism for diatom
research [ ]. S. robusta has a typical diatom life cycle: the cell size declines during mitotic
division and is restored after sexual reproduction, using a heterothallic mating system
7,8
Copyright: © 2021 by the authors. (Figure 1) [
9
]. Once the cell size reaches the sexual size threshold, both mating types
+
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
start producing a sex inducing pheromone (SIP). The presence of SIP induces mating
type – (MT ) to produce an attraction pheromone: the diketopiperazine cyclo(L-Pro-L-Pro)
−
(1) (Figure 2) [10].
4
.0/).
Int. J. Mol. Sci. 2021, 22, 1037. https://doi.org/10.3390/ijms22031037
https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 1037
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Figure 1. Lifecycle of Seminavis robusta. (
diatom’s cell size. (
both mating types (MT and MT ) start producing a sexual inducing pheromone (SIP). (
A
) Vegetative cell division leads to a diminution of the
B
) Once the cell size shrinks below the sexual size threshold of about 52
µ
m,
+
−
−
C
) MT
+
starts producing attraction pheromone cyclo(L-Pro-L-Pro) (
1
) when it detects SIP . After sensing
−
+
−
SIP , MT becomes motile and is attracted to the pheromone producing MT . The physiological
concentration of the pheromone is 100 nM. ( ) Cell size restitution: mating is followed by pairing
D
of gametangial cells, gamete formation and fusion, followed by expansion of the zygotes into two
auxospores which subsequently germinate to produce a large vegetative cell each [7,9–11].
Figure 2.
right) Trans-4-hydroxy-L-proline
Modified compounds were then coupled with proline to create analogs of 1.
(left) Sex pheromone cyclo(L-Pro-L-Pro) (1) of S. robusta [10], a symmetrical cyclic dipeptide.
(
2
was used to synthesize proline analogs with a C-4 modification.
The diketopiperazine scaffold is present in a plethora of metabolites [12–14]. Regardless of
the broad occurrence of these cyclopeptides, their receptors are not extensively character-
+
ized. Bilcke et al. [11] found that in S. robusta MT genes encoding for transmembrane
−
proteins were upregulated in response to SIP , suggesting that a G protein-coupled recep-
tor (GPCR) is responsible for the perception of 1.
We evaluated the possibility of modifying the pheromone to a chemical probe, in order
to identify the putative receptor. However, certain structural restrictions should be taken
into account. Lembke et al. [15] described that the diketopiperazine structure of
modified while retaining its activity, but only if both of the flanking ring structures were
present. The authors also tested a hydroxyl-substituted analog of (cyclo(4-OH-Pro-4-OH-
1 can be
1
Pro) that proved to be inactive. This observation demonstrates the confined chemical space
in which the pheromone is positioned.
Photoaffinity labeling has, since it was first reported in 1962 by Westheimer et al. [16],
provided the scientific community with a broad array of tools to study ligand-protein inter-
actions [17
diketopiperazine
a lower cross-linking efficiency in comparison to other photoreactive groups, diazirines
–19] and has proven to be useful for GPCR characterization [20
–23]. In the case of
1
, the (alkyl) diazirine is an attractive photoreactive group. While having

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have a small footprint. Therefore, we deemed that the inclusion of an alkyl diazirine would
be the best option to render a bioactive probe.
Photoaffinity labeling (PAL) probes are more useful if means are provided to conjugate
a reporter group to the cross-linked target receptor. An elegant approach is the use of
click chemistry, which has been widely adopted since its introduction by Sharpless et al.
in 2001 [24
could allow for an easy detection of the target.
A chemical probe derived from could also be applied in other disciplines. The dike-
–27]. The inclusion of a click-chemistry compatible moiety, such as an azide,
1
topiperazine was reported to be a constituent of pheromonal secretions in two other species,
namely in the femoral gland of the Sceloporus virgatus lizard [28] and the mating plug of
the Bombus terrestris bumblebee [29], and was also identified in the Aspergillus fumigatus
fungus, the Lucilia sericata blowfly and the Antarctic Pseudoalteromonas haloplanktis bac-
terium [28]. Recently, 1 was also shown to be an effective induced resistance stimulus for
rice plants [30], triggering a systemically enhanced defense against the root-knot nematode
Meloidogyne graminicola.
2. Results
New pheromone analogs were synthesized according to literature protocols. We inte-
grated chemical moieties that could be used for photo-cross-linking and click chemistry
and evaluated the modifiability of the pheromone scaffold at the C-4 position of one of
the two proline rings (Figure 2). Trans-4-hydroxy-L-proline (2, Figure 2) is an inexpen-
sive commercial analog of L-proline and allows a straightforward modification at the
C-4 position.
After synthesis and purification, the phytotoxicity of the analogs was examined
and comparative studies were performed to assess the equivalence of the analogs and
the natural pheromone. The comparative studies were based on an interference assay,
an attraction assay and a short evaluation of the conformation of the diketopiperazines.
2.1. Diketopiperazine Synthesis
Seven diketopiperazines (3 to 9, Figure 3) were synthesized in close resemblance to 1.
These pheromone analogs contain the same cyclo(L-Pro-L-Pro) backbone with an additional
functional group attached onto the C-4 of one of the L-proline rings, consisting of a diazirine
(3), an azide (4 and 5), a hydroxyl (6), a methoxy (7), and an acetal (8). In analog (9), one of
the proline rings was substituted with its four-membered azetidine equivalent.
Figure 3. A library of pheromone analogs, which was used in this study. They were synthesized and
evaluated for their pheromonal potency and phytotoxicity. The diazirine ( ) was introduced as a
photo-cross-linker and the azide ( and ) as a useful handle that is compatible with click chemistry.
Analogs allowed us to investigate the chemical space surrounding the hydrophobic ring structure.
3
4
5
6–9

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Prior to diketopiperazine synthesis, a small library of six L-proline analogs was
synthesized, starting from commercial trans-4-hydroxy-L-proline
A. A Boc group was introduced [31] and oxoproline 11 and diazirinylproline 12 were
subsequently synthesized according to Van der Meijden et al. [32]. Azidoprolines 16 and
were synthesized using a method adopted from Marusawa et al. [33]. Methyl ester 13
2 as shown in Scheme
1
18
was synthesized using the method of Chalker et al. [34].
A
B
Scheme 1. A library of six proline analogs was synthesized and coupled with proline to their corresponding diketopiperazine.
) Starting from L-Hyp-OH ( ) several proline derivatives were synthesized. Compounds surrounded by a bracket were
used for diketopiperazine synthesis. Compounds 16 18 underwent a N-Boc-deprotection step prior to the coupling reaction.
Reagents and conditions: (a) Boc O, NaOH, tBuOH/H O, 16 h, 20 C, 89%; (b) Jones reagent, acetone, 3 h, 0 C to 20 C,
(
A
2
–
◦
◦
◦
2
2
◦
0%; (c) H NOSO H, NH , 6.5 h, ∆; (d) I , Et N, MeOH, 0 C, 31%; (e) HCl, MeOH, 16 h, ∆, 90%; (f) Boc O, Et N, THF, 16
2 3 3 2 3 2 3
5
◦
◦
◦
◦
h, 20 C, 79%; (g) MsCl, Et N, THF, 16 h, 0 C to 20 C, 42%; (h) NaN , DMF, 16 h, 70 C, 94%; (i) BzONa, DMSO, 17 h,
3
3
◦
◦
9
0 C, 91%; (j) K CO , MeOH, 1 h, 20 C, 68% (see Supplementary Material S7 [31
–
41]). (
was adopted from Campbell et al. [35]. The same procedure was applied for the synthesis of diketopiperazines
45]). Reagents and
HCl, OxymaPure, DIPEA, DMF, 16 h, 20 C; (l) HCl, MeOH, 16 h, 0 C to 20 C; (m) piperidine, DMF,
B) Synthesis of diketopiperazine
2
3
1
(
3–9
see Supplementary Material S7.13 for an overview and Supplementary Material S8 for NMR data [42–
◦
◦
◦
conditions: (k) EDC
h, 20 C, 29 to 70%.
·
◦
1
Diketopiperazines
modified from Campbell et al. [35], of which a general overview is given in Scheme
B. OxymaPure [46 47] was used as additive and DMF was used as solvent during the
coupling step. Diketopiperazines and were synthesized by coupling H-Pro-OMe HCl
Apollo Scientific) with respectively Boc-Pro-OH [31] and Boc-Aze-OH (ChemPur). Pro-
line analogs 11 and 12 were coupled with H-Pro-OMe HCl to their corresponding dike-
topiperazines and , with analog 11 undergoing acetal formation during the acidic
Boc-removal in methanol. Proline analogs 16 and 18 were coupled with Boc-Pro-OH
after being treated with TFA in DCM or HCl in methanol, yielding respectively and
H-Hyp-OMe·HCl 13) was coupled with Boc-Pro-OH and methylated using MeI to-
wards 36]. Proline analog 17 was coupled, after Boc-removal with hydrochloric acid,
with Boc-Pro-OH, yielding 6.
1 and 3–9, presented in Figure 3, were synthesized with a method
1
,
1
9
·
(
·
8
3
5
4.
(
7
[
2.2. Diketopiperazine Phytotoxicity
Diketopiperazines are a large class of compounds with a broad range of activities [12].
Recently, some diketopiperazines were reported to be algicidal [48,49]. For this reason,
it was not implausible that small changes to the pheromone scaffold could induce algicidal
effects and thereby could affect the physiology of S. robusta.
We determined the short-term phytotoxicity of the newly synthesized analogs, as the
exposure time to the pheromone analogs during the bioassays is limited. Pulse Ampli-
tude Modulation (PAM) fluorometry was used to measure the chlorophyll fluorescence

Int. J. Mol. Sci. 2021, 22, 1037
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of photosystem II (PSII), a parameter that is related to the overall fitness of algae [50].
The technique has been successfully exploited to assess toxic effects of herbicides [51,52],
heavy metals [53], volatile organic compounds [54], pharmaceuticals [55], and quorum
sensing related compounds [56] towards microalgae.
Cultures of S. robusta were treated with 10
a PSII inhibitor [57 58]. The quantum yield of PSII (Y ) was measured for 30 min and the
resulting data of the analogs was interpolated between
µM of 1, 3–9 and with 1 µM of DCMU,
,
II
1
(set to 0% inhibition) and DCMU
(
100% inhibition) (Figure 4, see Supplementary Material S1 for original Y data).
II
Figure 4. One of the tested diketopiperazines (7) shows a significant phytotoxic effect at 10
µM, with an average inhibition
of 10.2%. (left) The represented time-series are an interpolation between the two control treatments (see Supplementary
Material S1 for original data) and are the average over five replicates. The slopes of the inhibition curves of compounds
3,
are positive (< 0.02% min 1, first 10 min not included) but not significant (p > 0.22). (right) The quantum yield
using a two sample t-test. The p-value is shown for
every pheromone analog, the gray line represents a p-value of 0.05. Only one analog, methoxy-substituted analog , shows
a significant difference with 1. The data of analog 7 are significant (p < 0.05) for all time points, except the first (p = 0.058).
−
5
,
and
9
of PSII (Y ) of the pheromone analogs was compared with those of
1
II
7
The inhibition of Y was significant for methoxy-substituted analog
7
, with an average
inhibition of 10%. The other pheromone analogs can be categorized in two groups. The first
group comprising of compounds 5, and shows little phytotoxicity, with an average
inhibition of 6, 3.9, and 4.8% respectively at 10 M (p > 0.13, Figure 4). The second group,
consisting of compounds 4, 6, and 8, shows an average inhibition of less than 1%.
II
3,
9
µ
2.3. Interference Assay
The pheromone potency of the analogs was evaluated according to an interference
+
assay, in which the attraction of MT towards a point source of the natural pheromone is
+
disturbed by the presence of a pheromone analog [10
,
15]. The MT cells were conditioned
−
−
with spent medium from a MT culture, that was evaluated for the presence of the SIP
conditioning factor [10].
Diketopiperazines
in three independent experiments. Treatments with compound
experiment, allowing us to compare the outcome of the pheromone analogs interexperi-
3
to
9
were tested in concentrations of 10 nM, 100 nM, and 10 µM
1
were included in every
mentally. Oasis HLB beads, that were coated with a load of 2 nmol of
were used as a pheromone point source.
1 per mg of beads,
Each experiment included two control treatments. In the first control treatment
positive control), no pheromone (analog) was added. The attraction towards the coated
(
beads was not disturbed and maximal. For a second control treatment, blank beads were
added (negative control) to measure coincidental contact of the algae with the beads.
The total amount of treatments per experiment was 11 or 14, equaling to respectively
three or four analogs at three concentrations and two control treatments. In the case of
experiments A and B, two replicates were provided per multi-well plate, occupying 22 of
the available 24 wells. In experiment C, every multi-well plate was provided with only one

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replicate (14 out of 24 wells). The activity screening of the pheromone analogs was spread
over three independent experiments according to their availability.
The interference caused by adding
1 to the culture medium (Figure 5) was significant
for all three of the tested concentrations, except in experiment C. A normal dose-response
relationship, where a low concentration yields a limited interference and a high concen-
tration yields an increased interference, was expected. However, it was noted that the
dose-response trend of 1 was variable.
Figure 5. Interference assays performed on S. robusta with seven asymmetrical pheromone analogs. Numbers on top
represent the different experiments that were carried out. The presented values are the estimated average fraction of
attractive beads over 6 (experiments
model (details can be found in the Supplementary Material S5.1). Error bars represent the 95% confidence interval of the
estimates. Significance is expressed compared to the positive control. Significance levels: (p < 0.1), * (p < 0.05), ** (p < 0.01),
** (p < 0.001).
A and B) or 4 (experiment C) replicates, based on a Binomial generalized linear
·
*
The pheromone analogs were also compared to
tration of the pheromone (Figure 6). The analogs’ estimated average fraction of attractive
beads was interpolated between the positive control (no interference) and at 100 nM
set as 100% interference). The interpolation makes it possible to compare the pheromonal
1 at 100 nM, the physiological concen-
1
(
potency of the analogs relative to the natural pheromone and irrespective of the experiment
in which they were measured (see Supplementary Material S5.2).
Figure 6. Chemical modifications alter the pheromonal affinity. The interference values of the
analogs at 100 nM were interpolated between the positive control and the interference of
1 at 100 nM.
The calculated 95% confidence intervals of the interpolated values can be found in Supplementary
Material S5.3. The actual values of analogs 3 and 4 are 113% and 108%.
2.4. Attraction Assay
Diketopiperazines 3, 4, and 5 were also evaluated using an attraction assay, where the
attraction towards pheromone-analog-coated beads was assessed (Figure 7). Beads were

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coated in the same manner as was previously done with diketopiperazine
1
(with a load of
−
1
2
nmol mg ). As demonstrated by Figure 7, the beads coated with compounds 3–5 gave
similar results as the positive control.
+
Figure 7. Asymmetrical pheromone analogs 3, 4, and 5 attract S. robusta MT in a similar fashion
as the natural pheromone
experiments (n = 6 for diketopiperazine
1
. The diketopiperazines were tested in three separate and independent
and n = 4 for diketopiperazines and ). The beads were
coated with a load of 2 nmol mg , · = outlier.
3
4
5
−
1
3
. Discussion
The data from the PAM fluorometry measurements (Figure 4) showed that diketopiper-
azine caused a significant 10% reduction of Y . Although it is unclear how compound
could affect the physiology of S. robusta, the results from the interference assay might
be impaired due to the slightly toxic nature of . The other analogs showed a small but
7
II
7
7
non-significant inhibition of Y . We therefore assumed that the use of these compounds
II
would not alter the physiological behavior of S. robusta.
The impact of adding a diazirine (
scaffold was rather limited. In case of the diazirine
of (100 nM). This suggests that the small, though polar, symmetrical increase in molecular
3
) or an azide (4 and 5) to the cyclo(L-Pro-L-Pro)
3, the interference at 100 nM equals that
1
3
volume of 3% (or 5.9 Å ) at position C-4 does not affect its activity (see Supplementary
Material S6 for molecular volumes and molecule models [59]). However, when the volume
is further incremented, the activity of the compound decreases as demonstrated by acetal
The two methoxy substituents increase the molecular volume by 52.7 Å , yielding an
8
.
3
overall volume increase of 30% compared to 1.
In the case of the azides
observed. Our interference data suggests that a substitution of the azide on the pseudo-
axial position of the L-proline ring (compound ), causes less steric hindrance between
the ligand and its putative receptor than on the pseudo-equatorial position (compound ).
4 and 5, a slight difference between both diastereomers was
4
5
The significance of this difference could not be determined because the compounds were
evaluated in two independent experiments. Moreover, an interaction between the treatment
and experiment is to be expected due to differences in diatom cell size and density.
The limited impact of the diazirine and azide moieties is also reflected by the outcome
of the attraction assay. Beads coated with compounds
was comparable to diketopiperazine . The results of both assays demonstrate that these
compounds could interact with the putative GPCR in the same manner as 1.
The interference caused by hydroxy analog was less pronounced. Lembke et al.
3, 4, or 5 had an attractiveness that
1
6
reported that a symmetrical hydroxy analog did not show any activity [15]. Here we
observed a significant interference, albeit only at the highest concentration tested. The in-
troduction of a polar hydroxy group clearly decreases the pheromonal activity, but the
pseudo-equatorial positioning of the substituent could be a contributing factor. In com-

Int. J. Mol. Sci. 2021, 22, 1037
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parison: the pseudo-axial positioning of the methoxy group (7) seems to have less impact
on the pheromonal activity. Yet, it is not clear how much of the interference effect of
caused by the toxic nature of this compound.
7 is
The remaining azetidine analog
9
, with a two-carbon side-chain, showed a reduced
M. This result validates the structural
activity compared to , though still significant at 10
1
µ
restrictions defined by Lembke et al. and suggests a preference of a three-carbon side-chain
over a two-carbon side-chain [15].
While Figure 6 shows that the negative impact of some of the substitutions can be
quite pronounced, all analogs (3–9) cause significant interference at 10 µM, which suggests
that the modifications can still be tolerated. We also tried to evaluate whether the substi-
tutions on the flanking ring structures impacted the conformation of the analogs’ central
diketopiperazine ring, possibly affecting the interaction with the receptor.
Siemion and coworkers found an empirical relationship between the torsion of the
1
3
C -C
α
-C’=O fragment (
θ
) of the proline moiety and the difference between the C chemi-
| + 2.47) [60].
β
cal shifts of Cβ and C in proline-containing cyclic peptides (∆δβγ = 0.081 |
γ
θ
The authors were also able to use the established relationship to determine the equilibrium
between the boat and planar conformation of several proline-containing diketopiper-
azines [61].
◦
The calculated torsion values of the pheromone analogs are smaller or equal to 30 and
within the ∆δβγ prediction interval for the postulated torsion angle of diketopiperazine
1
(4.91 ppm
there are no indications that the adopted conformation of the central diketopiperazine ring
of the diketopiperazines deviates from the preferred boat conformation of . Additionally,
there was no significant correlation found between the torsion angles and the bioactivity
data presented in Figure 6) of the analogs (Pearson correlation coefficient = 0.41, p = 0.3).
±
1.48, see Supplementary Material S6) [60]. Based on these calculations,
1
(
These observations suggest that the introduced chemical moieties could have a minor to
no effect on the diketopiperazine conformation.
Aside from the molecular dimensions and conformation, the substitutions also impact
the lipophilic character of the pheromone analogs. The LogP values of the analogs were
calculated (see Supplementary Material S6) and a positive correlation between these LogP
values and the bioactivity (data presented in Figure 6) was found, however the correlation
was not significant (Pearson correlation coefficient = 0.57, p = 0.14).
In conclusion, we have shown that it is possible to modify the pheromonal scaffold in
an asymmetrical fashion, while retaining its pheromonal activity. Although structural re-
strictions demand hydrophobic ring flanking structures, nitrogen-containing substitutions,
such as diazirines or azides, are tolerated as presented in the structure activity relation-
ship map (Figure 8). The substitution with a hydroxy group seems to be less favored.
The available space in the binding pocket of the putative pheromone receptor is limited,
as demonstrated earlier by Lembke et al. [15], where a symmetrical variant of diketopiper-
azine
molecular volume and the less favored hydroxy substitution, the volumetric restriction
reflects in our findings; the introduction of the acetal in diketopiperazine largely reduces
the bioactivity of the pheromone scaffold. The results of diazirine and azide analogs
to demonstrate that these substituents can be incorporated into the cyclo(L-Pro-L-Pro)
6 was found to be inactive. Though this could be a combined effect of the increased
8
3
5
scaffold to probe the putative diatom pheromone receptor and to possibly characterize the
action of 1 on other organisms as well.

Int. J. Mol. Sci. 2021, 22, 1037
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Figure 8. A structure activity relationship map for cyclo(L-Pro-L-Pro) analogs. This study reflects on the functionality and
stereochemistry of both R groups and on the effect of the inclusion of a 4-membered flanking ring structure. The general
requirement of the flanking ring structures was described by Lembke et al. The diketopiperazine stereochemistry was
studied by Gillard et al. and Lembke et al. [10,15].
4. Methods
4
.1. Strain and Culture Conditions
+
−
Seminavis robusta DCG 0105 (85A, MT ) and DCG 0107 (85B, MT ) were used in
this study. Both are available in the diatom culture collection of the Belgian Coordi-
nated Collection of Microorganisms (BCCM/DCG, bccm.belspo.be/about-us/bccm-dcg)
−
1
at Ghent University. Cultures were grown in artificial sea water: 34.5 g L of Tropic Marin
−
Wartenberg, Germany), 0.08 g L NaHCO (Sigma-Aldrich, St. Louis, Missouri, USA)
3
1
(
enriched with Guillard’s F/2 solution (Sigma-Aldrich, St. Louis, Missouri, USA) at 20 mL
−
1
L
inside a 250 mL cell culture flask. Light was provided in a 12 h:12 h light/dark cycle,
using cool white fluorescent lamps (Philips, Amsterdam, Netherlands) with an intensity of
−
2
−1 ◦
and the temperature was held at 18 C. Cultures were kept axenically
2
0 to 30
µmol m
s
−
1
−1
by treating them regularly with antibiotics (0.5 g L Penicillin, 0.5 g L Ampicillin, 0.1 g
Streptomycin and 0.05 g L Gentamycin, Sigma-Aldrich, St. Louis, Missouri, USA).
Stock cultures were kept at 4 C with a light intensity of 2 to 5
−
1
−1
L
◦
−2 −1
s . All cultures
µmol m
used in this study had a mean cell length between 20 and 35 µm.
4
.2. Interference Assay
The interference assay was adopted from Lembke et al. [15] and a diagram of the
assay is provided in the Supplementary Material S4. Oasis HLB beads were coated with
−
1
cyclo(L-Pro-L-Pro) as described by Gillard et al., with a load of 2 nmol mg
[10]. In short,
a 10 mg SPE cartridge was washed with methanol (1 mL) and Milli-Q water (2 mL). A 2 mL
solution of cyclo(L-Pro-L-Pro) (20 nM) was eluted over the cartridge. The SPE material was
◦
suspended in 2 mL Milli-Q water and was stored as a stock solution at
aliquots. Before usage, a dilution was made by adding 20
−
20 C in 50 µL
µ
L of the stock suspension to
980 µL of Milli-Q water.
Axenic S. robusta 85A cultures were inoculated in a 24-well culture plate with a total of
1
mL medium per well and dark-synchronized for 40 h by wrapping the plate in aluminum
foil. After synchronization, the cultures were conditioned by adding 1 mL 85B filtered
medium (see Supplementary Material S2). Cyclo(L-Pro-L-Pro)–coated beads (50 L of the
µ
diluted suspension) were added after 6 to 7 h and images were taken of the cell cultures
with the BioTek Cytation3 Cell Imaging Multi-Mode Reader. For this study, pheromone
(
5
analogs) were applied in concentrations of 10 nM, 100 nM and 10
mM stock solution. After addition of the pheromone (analogs), the cell medium was
mixed gently by pipetting the cell medium up and down.
µM, starting from a

Int. J. Mol. Sci. 2021, 22, 1037
10 of 14
In every interference experiment a positive and negative control is included. The pos-
itive control is the response of conditioned 85A cells towards cyclo(L-Pro-L-Pro)–coated
beads, whereas the negative control assesses the attractiveness of non-coated beads.
Beads were assigned attractive or not attractive depending on a defined threshold,
being a number of cells attached to a bead. In an effort to maximize the response difference
between the positive control and the highest cyclo(L-Pro-L-Pro)
1 concentration tested
(
10 M), an interference assay was set-up with 4 different cell densities. Aliquots of an
µ
exponentially growing stock culture were inoculated in a 24-well plate containing 2 mL
of f/2 medium and incubated for 48 h. The cell medium was renewed with 1 mL of f/2
medium, cell densities were determined and the cultures were dark-adapted for 40 h.
The interference test was carried out and the response towards the beads was assessed.
The number of cells attached to every bead were evaluated after manual image capturing
and categorized with the number attached cells varying from 1 to 9. The differences
between both treatments, the positive control and 10
density and threshold were calculated (see Supplementary Material S3).
The highest difference in response can be found at a density of 7
a threshold of 1 cell per bead. The choice of a low threshold gives a higher difference
between both treatments, but the downside is that the variance on the data is also higher
µ
M of cyclo(L-Pro-L-Pro)
1
, for every
7
−2
×
10 cells m and
(see Supplementary Material S3). This could explain the high variability in the interference
test that was discussed in this study (Figure 5).
4.3. Attraction Assay
The attraction assay was adopted from Gillard et al. [10]. The procedure is sim-
ilar to the interference assay described above, without the addition of (a) pheromone
analog) into the culture medium. Oasis HLB beads loaded with synthetic pheromone
analogues were made using the same procedure as for cyclo(L-Pro-L-Pro)–coated beads.
Binding efficiencies were not determined.
(
3–5
4.4. Microscopy and Image Processing
4.4.1. Automatic Image Capturing
Images were captured using a Cell Imaging Multi-Mode Reader (Cytation3, BioTek,
Winooski, Vermont, USA), mounted with a 10x objective and controlled using Gen5 soft-
ware (3.03.14, BioTek, Winooski, Vermont, USA). For the interference assays, image mon-
tages (7 by 7 images) were captured using three image channels: bright field, Cyanine 5
(
CY5), and Green Fluorescent Protein (GFP). The CY5-channel was used to image the cell
chlorophyll while the GFP-channel was used to image the beads. The bright field was
used to autofocus the image and as reference. An algorithm was written in Python 3.6
(
Python Software Foundation, https://www.python.org/) to assess the attractiveness of
the beads (visualized in Figure 9). In short: both CY5 and GFP images were converted to a
binary image after applying a threshold (Otsu’s method [62]). Next, beads were identified
in the GFP image and if there was overlap between a bead and a cell, the bead was assigned
as attractive. This method was used by default, unless mentioned otherwise.
4.4.2. Manual Image Capturing
Images were captured on a Zeiss Observer A1 inverted microscope (Zeiss, Jena,
Germany) equipped with a Zeiss EC plan-NEOFLUAR 10x or 4x lens (Zeiss, Jena, Germany)
and coupled to a Nikon DS-Fi2 U3 camera (Nikon, Tokyo, Japan), operated by the NIS-
elements software package (Nikon, Tokyo, Japan). Beads were classified attractive after
manual evaluation.
4.5. Data Analysis
The statistical analysis of the interference assays was done in R 3.5 [63]. The number of
attractive beads (summarized per well) were modeled as a Poisson generalized linear model
in function of treatment (positive control, negative control and different compound effects)

Int. J. Mol. Sci. 2021, 22, 1037
11 of 14
and a fixed plate effect that captures the variability between plates (replicates). The different
treatment concentrations were modeled as separate treatment effects, effectively allowing
for an interaction between the treatment and concentration. The total number of beads
were added as offset to the model, thereby effectively modeling the fraction of attractive
beads. In every experiment, one replicate per treatment was present per 24-well plate in a
randomized fashion; four to six plates were used per experiment. Statistical inference was
based on Wald tests using the asymptotic Chi-squared null distribution.
Figure 9. Assignment of (non-)attractive beads. (
during a typical interference assay (bar = 100 m). (
Protein (GFP, green) and Cyanine 5 (CY5, red) image channels. Some of the red areas are shifted due to motile nature of the
algae (bar = 100 m). ( ) The bead is represented by a green disc on the GFP channel. In this particular case, the green area
A) One bright field image of the 7 by 7 image montage that was recorded
µ
B
) An overlay of the bright field image and both Green Fluoreschent
µ
C
overlaps with two red areas and the bead is classified as attractive (bar = 25 µm). (D) The bead does not overlap with an
alga and is classified as not attractive (bar = 25 µm).
4.6. Phytotoxic Evaluation
The short-term phytotoxicity of the synthesized pheromone analogs was estimated
using Pulse-Amplitude-Modulation fluorometry. Axenic S. robusta 85A cultures were
inoculated in a 48-well plate (Greiner Bio-One, Kremsmünster, Austria). Diketopiperazine
1
and the seven analogs were added to a concentration of 10
µM. A treatment with PSII
blocker DCMU (1 M) was added as a control [57 58]. After addition of the compounds, the
µ
,
plate was incubated for 10 min at room temperature and Y was monitored with a MAXI
II
Imaging PAM M-series fluorometer (Walz Mess- und Regeltechnik, Effeltrich, Germany) for
3
m
0 min, with a saturation pulse every 2 min and actinic light turned on (15
µ
mol photons
−1
s ); sequential data were averaged over five replicates. The results are reported
−
2
as relative inhibition of Y , after interpolating the Y values of the pheromone analogs
II
II
between those of
1 and DCMU (DCMU is 100% inhibition). The statistical analysis was
done in R 3.5.2 using the lm() function.
Supplementary Materials: Supplementary materials can be found at https://www.mdpi.com/1422
-
0067/22/3/1037/s1.
Author Contributions: Conceptualization, E.B., S.D.D., K.V.d.B., W.V. and S.M.; validation, E.B.
and S.D.D.; formal analysis, E.B., K.V.d.B. and L.C.; investigation, A.D.B. and E.B.; resources, S.M.
and W.V.; writing—original draft preparation, E.B.; writing—review and editing, W.V. and S.M.;
visualization, E.B.; supervision, S.M. and W.V.; funding acquisition, S.M. and W.V. All authors have
read and agreed to the published version of the manuscript.
Funding: This research was funded by Ghent University via a BOF-GOA grant (BOF-GOA No. 01G01715).
Part of the research was carried out with infrastructure funded by EMBRC Belgium—FWO project
GOH3817N. The computational resources and services used in this work were provided by the VSC
(
Flemish Supercomputer Center), funded by the Research Foundation—Flanders (FWO) and the
Flemish Government—department EWI.
Institutional Review Board Statement: Not applicable.

Int. J. Mol. Sci. 2021, 22, 1037
12 of 14
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in Supplementary Material.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
CY5
GFP
Cyanine 5
Green Fluorescent Protein
GPCR G protein-coupled receptor
MT
PAL
PAM
PSII
SIP
Mating type
Photoaffinity labeling
Pulse amplitude modulation
Photosystem II
Sex inducing pheromone
Quantum yield of PSII
YII
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