Versatile synthesis of the signaling peptide glorin

Article abstract of DOI:10.3762/bjoc.13.27

We present a versatile synthesis of the eukaryotic signaling peptide glorin as well as glorinamide, a synthetic analog. The ability of these compounds to activate glorin-induced genes in the social amoeba Polysphondylium pallidum was evaluated by quantita

Full text of DOI:10.3762/bjoc.13.27

Versatile synthesis of the signaling peptide glorin
Robert Barnett1, Daniel Raszkowski2, Thomas Winckler2 and Pierre Stallforth*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2017, 13, 247–250.
1Leibniz Institute for Natural Product Research and Infection Biology,
Hans Knöll Institute – HKI, Junior Research Group Chemistry of
Microbial Communication, Beutenbergstr. 11, D-07745 Jena,
Germany and 2Faculty of Biology and Pharmacy, Institute of
Pharmacy, Department of Pharmaceutical Biology, University of Jena,
Semmelweisstrasse 10, D-07743 Jena, Germany
doi:10.3762/bjoc.13.27
Received: 29 September 2016
Accepted: 27 January 2017
Published: 08 February 2017
This article is part of the Thematic Series "Chemical biology".
Guest Editor: H. B. Bode
Email:
Pierre Stallforth* - pierre.stallforth@leibniz-hki.de
* Corresponding author
© 2017 Barnett et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
Dictyostelium; glorin; multicellularity; Polysphondylium; signaling
molecules; social amoebae
Abstract
We present a versatile synthesis of the eukaryotic signaling peptide glorin as well as glorinamide, a synthetic analog. The ability of
these compounds to activate glorin-induced genes in the social amoeba Polysphondylium pallidum was evaluated by quantitative
reverse transcription PCR, whereby both compounds showed bioactivity comparable to a glorin standard. This synthetic route will
be useful in conducting detailed structure–activity relationship studies as well as in the design of chemical probes to dissect glorin-
mediated signaling pathways.
Introduction
The emergence of multicellularity from unicellular ancestors is gate to form a multicellular organism. Eventually, they culmi-
considered a major evolutionary transition [1]. This transition nate in fruiting bodies to spread some of the population as
has occurred not only once, in fact more than 25 independent dormant spores into the environment. Secondary metabolites
instances of this event are known. The resulting increase in bio- often constitute the key signaling molecules in these develop-
logical complexity requires fine-tuned differentiation and mental processes [3,4]. For instance, aggregation of the
cell–cell communication mechanisms. The social amoebae are amoebae is initiated by pulses of chemoattractive, low-molecu-
exquisite organisms to study the emergence of multicellularity lar weight signaling molecules – so-called acrasins [5]. Addi-
since they can exist in both a unicellular and a multicellular tionally, it has been shown that natural products are also
stage with a well-orchestrated developmental cycle linking the involved in interspecies interactions of social amoebae and
two [2]. The unicellular amoebae feed on bacteria and divide by bacteria [6-10]. A detailed investigation of both inter- and
binary fission. Upon depletion of their food source, they aggre- intraspecies interactions will give insight into the fundamentals
247

Beilstein J. Org. Chem. 2017, 13, 247–250.
of cell signaling and access to a rich source of novel natural more stable than glorin (1) [14]. The molecules were shown
products.
to be bioactive, effectively mediating the induction
of gene expression during early development, as
We describe a practical synthesis of the modified dipeptide determined by quantitative reverse-transcription PCR (RT-
glorin (1, Figure 1), the assumed acrasin for many of the early- qPCR).
diverged species of social amoebae. While numerous species of
Results and Discussion
social amoebae such as Polysphondylium pallidum,
Dictyostelium fasciculatum [11], and D. caveatum [12], Synthesis of glorin and glorinamide
amongst others respond chemotactically to glorin (1), the Two glorin syntheses have been published [15,16], one of
acrasin has only been isolated from P. violaceum [13]. Despite which lacked sufficient data to be reproducible and the other
its crucial role in the initiation of multicellularity, little is one displayed limited versatility. Therefore, we focused on
known about glorin’s biosynthesis, signaling pathways, or deg- designing a robust synthesis that would allow for facile access
radation. To facilitate further studies, our chemical route allows to glorin derivatives required for structure–activity relationship
for a facile synthesis of glorin derivatives and glorin-based studies. Eventually, these studies can lead to the construction of
chemical probes.
various chemical probes to identify the unknown glorin recep-
tor.
Here, we report the synthesis of glorin (1), as well as the novel
synthetic analog glorinamide 2: a compound with comparable Syntheses of glorin and glorinamide (Scheme 1) started from
bioactivity, that is hydrolytically – and thus metabolically – commercially available L-ornithine (3) and benzyloxycarbonyl-
Figure 1: Glorin (1) and glorinamide 2.
Scheme 1: Synthesis of glorin (1) and glorinamide 2. Reagents and conditions: a) TMSCl, MeOH, rt, 12 h; b) NaOEt, EtOH, 0 °C to rt, 30 min, 97%
over two steps; c) H2CO, p-TsOH, toluene, Dean–Stark conditions, 3 h, 76%; d) for 7a: NaOEt, EtOH, 0 °C to rt, 30 min, 76%, for 7b: H2NEt, THF, rt,
16 h, 75%; e) for 8a: isobutyl chloroformate, NMM, 4, DMF, −15 °C to rt, 2 h, 69%, for 8b: HBTU, Et3N, 4, DMSO, rt, 3 h, 69%; f) Pd/C, H2, MeOH, rt,
1 h, 74% 9a, 97% 9b; g) iPr2EtN, DMAP, propionic anhydride, DCM, rt, 2 h, 92% 1, 97% 2.
248

Beilstein J. Org. Chem. 2017, 13, 247–250.
protected L-glutamic acid 5. In an improvement on previous
two-step syntheses, L-ornithine δ-lactam 4 was synthesized
from L-ornithine in a one-pot procedure, whereby the latter was
converted into the corresponding methyl ester using trimethyl-
silyl chloride in methanol [17] and cyclization was achieved
under basic conditions using sodium ethoxide; the lactam was
prone to racemization under strongly basic conditions, this was
avoided by short reaction times with sodium ethoxide, and by
avoiding strongly basic reaction conditions in subsequent steps.
A key challenge in the syntheses of glorin and analogs is the
differentiation between the α- and the γ-carboxylic acid groups
of L-glutamic acid for selective esterification or amidation.
α-Selective functionalization was achieved via synthesis of
oxazolidinone 6 from Cbz-L-glutamic acid (5) with paraform-
aldehyde and p-toluenesulfonic acid under dehydrating condi-
tions [18]. Opening of the oxazolidinone with sodium ethoxide
as nucleophile thus yielded ester 7a, while addition of ethyl-
amine yielded amide 7b. Subsequent amide bond formation
with lactam 4 using isobutyl chloroformate or HBTU as cou-
pling reagents furnished the protected dipeptides 8a and 8b, re-
spectively. Hydrogenolysis with hydrogen gas and palladium on
charcoal gave free amines 9a and 9b. Glorin (1) and glori-
namide 2 were then obtained by treating amines 9a and
9b, respectively, with propionic anhydride. The main
advantage of our synthesis over previous syntheses is that
ours allows for late-stage functionalization of the
α-amino group of the glutamic acid moiety. This is
particularly useful for the rapid generation of different α-amide
analogues.
Biological activity of glorin and glorinamide
The bioactivities of synthetic glorin (1) and glorinamide 2 were
assayed by their ability to elevate the expression of the glorin-
induced gene PPL_09347 in the social amoeba P. pallidum, as
previously determined [11]. We chose the gene PPL_09347,
which encodes the Dictyostelium discoideum ortholog of
profilin I, an actin binding protein required for the actin
cytoskeleton organization, as a model gene. Upon glorin (1)
exposure, this gene was found to be up-regulated by about
50-fold [11]. To this end, the compounds were dissolved in
DMSO/water and added in two 1 μM portions (30 min apart) to
Figure 2: Gene induction in P. pallidum (model gene PPL_09347)
without test compounds (negative control), commercial glorin (positive
control), synthetic glorin (1) and glorinamide 2. Top: Gene expression
is presented as ‘fold change’ of gene expression in stimulated vs
vegetatively-growing cells. Data are means of three biological repli-
cates ± S.D. **: p < 0.01; ***: p < 0.001 vs vegetative cells (Student’s
t-test). Bottom: Gene expression presented as ‘fold change’ of glorin-
stimulated vs unstimulated cells. Data are means of five biological
replicates ± S.D. and GraphPad Prism 6 was used for visualization of
the results.
a suspension of starving P. pallidum. After 1 h, the cells were Conclusion
harvested and total RNA was extracted. The differential regula- In summary, we have devised a versatile and robust synthetic
tion of PPL_09347 was determined by RT-qPCR. Synthetic route to glorin that allows for a wide range of derivatizations.
glorin (1) and glorinamide 2 led to similar expression of Glorin, as well as the hydrolytically more stable derivative
PPL_09347, comparable to commercial glorin as positive glorinamide, were shown to display comparable glorin-induced
control (Figure 2). While a small baseline induction of gene expression in Polysphondylium pallidum. In future this
PPL_09347 without test compounds is always observed synthesis will facilitate the construction of a library of glorin
(Figure 2, top), induction by glorin and glorinamide led to a sig- derivatives for a detailed structure–activity relationship study.
nificant increase in expression level above baseline (Figure 2, Ultimately, we wish to synthesize chemical probes of glorin for
bottom).
the identification of the unknown glorin receptor.
249

Beilstein J. Org. Chem. 2017, 13, 247–250.
6. Klapper, M.; Götze, S.; Barnett, R.; Willing, K.; Stallforth, P.
Experimental
Angew. Chem., Int. Ed. 2016, 55, 8944–8947.
doi:10.1002/anie.201603312
Quantitative reverse transcription PCR: P. pallidum
PN500 cells were cultured in association with Escherichia coli
K12 cells. Cells were harvested before first signs of aggrega-
tion became visible. Cells were washed three times in 17 mM
phosphate buffer (pH 6.2) to remove bacteria and suspended at
2 × 107 cells/mL in 17 mM phosphate buffer (pH 6.2) with
shaking at 150 rpm. After 1 h of starvation, 1 µM glorin
(Phoenix Pharmaceuticals, Burlingame CA, USA), synthetic
glorin (1), or glorinamide 2 (100 µM stock solutions with 3%
DMSO) or water were added to the cells every 30 min for 1 h.
Cells were centrifuged for 30 min after the last addition and
stored in pellets of 2 × 107 cells at −80 °C.
7. Stallforth, P.; Brock, D. A.; Cantley, A. M.; Tian, X.; Queller, D. C.;
Strassmann, J. E.; Clardy, J. Proc. Natl. Acad. Sci. U. S. A. 2013, 110,
14528–14533. doi:10.1073/pnas.1308199110
8. Jousset, A. Environ. Microbiol. 2012, 14, 1830–1843.
doi:10.1111/j.1462-2920.2011.02627.x
9. Ballestriero, F.; Daim, M.; Penesyan, A.; Nappi, J.; Schleheck, D.;
Bazzicalupo, P.; Di Schiavi, E.; Egan, S. PLoS One 2014, 9, e109201.
doi:10.1371/journal.pone.0109201
10.Mazzola, M.; de Bruijn, I.; Cohen, M. F.; Raaijmakers, J. M.
Appl. Environ. Microbiol. 2009, 75, 6804–6811.
doi:10.1128/AEM.01272-09
11.Asghar, A.; Groth, M.; Siol, O.; Gaube, F.; Enzensperger, C.;
Glöckner, G.; Winckler, T. Protist 2012, 163, 25–37.
doi:10.1016/j.protis.2010.12.002
Total RNA was prepared using the QIAGEN RNeasy kit and
cDNA was synthesized using the QIAGEN Omniscript kit and
an oligo(dT) primer. Expression of the glorin-induced model
gene PPL_09347 was determined by RT-qPCR as described
[11,19]. Expression of PPL_09347 in different cDNA samples
was standardized to the reference gene glyceraldehyde-3-phos-
phate dehydrogenase (gpdA, SACGB accession number
PPL_12017; http://sacgb.fli-leibniz.de/cgi/index.pl).
12.Waddell, D. R. Nature 1982, 298, 464–466. doi:10.1038/298464a0
13.Shimomura, O.; Suthers, H. L.; Bonner, J. T.
Proc. Natl. Acad. Sci. U. S. A. 1982, 79, 7376–7379.
doi:10.1073/pnas.79.23.7376
14.Nogrady, T.; Weaver, D. F. Medicinal chemistry: a molecular and
biochemical approach, 3rd ed.; Oxford University Press: New York,
2005.
15.Lee, Y. S.; Lee, S. W.; Park, W. J. Bull. Korean Chem. Soc. 1987, 8,
58–59.
16.Ball, J. B.; Craik, D. J.; Alewood, P. F.; Morrison, S.; Andrews, P. R.;
Nicholls, I. A. Aust. J. Chem. 1989, 42, 2171–2180.
doi:10.1071/CH9892171
Supporting Information
17.Li, J.; Sha, Y. Molecules 2008, 13, 1111–1119.
doi:10.3390/molecules13051111
Supporting Information File 1
18.Silva, L. L.; Joussef, A. C. J. Nat. Prod. 2011, 74, 1531–1534.
doi:10.1021/np200234e
Detailed experimental procedures, compound
characterization data, and copies of NMR spectra.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-13-27-S1.pdf]
19.Lucas, J.; Bilzer, A.; Moll, L.; Zündorf, I.; Dingermann, T.; Eichinger, L.;
Siol, O.; Winckler, T. PLoS One 2009, 4, e5012.
doi:10.1371/journal.pone.0005012
Acknowledgements
License and Terms
We thank A. Perner and H. Heinecke (Hans Knöll Institute,
Jena) for MS and NMR measurements. We are grateful for
financial support from the Leibniz Association, and the Fonds
der Chemischen Industrie (VCI) (P.S.). This work was also sup-
ported by the DFG-funded graduate school of excellence Jena
School for Microbial Communication (R.B. and D.R.).
This is an Open Access article under the terms of the
Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0), which
permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
References
The license is subject to the Beilstein Journal of Organic
Chemistry terms and conditions:
1. Grosberg, R. K.; Strathmann, R. R. Annu. Rev. Ecol. Evol. Syst. 2007,
38, 621–654. doi:10.1146/annurev.ecolsys.36.102403.114735
2. Chisholm, R. L.; Firtel, R. A. Nat. Rev. Mol. Cell Biol. 2004, 5, 531–541.
doi:10.1038/nrm1427
(http://www.beilstein-journals.org/bjoc)
3. Morris, H. R.; Taylor, G. W.; Masento, M. S.; Jermyn, K. A.; Kay, R. R.
Nature 1987, 328, 811–814. doi:10.1038/328811a0
4. Weijer, C. J. Curr. Opin. Genet. Dev. 2004, 14, 392–398.
doi:10.1016/j.gde.2004.06.006
The definitive version of this article is the electronic one
which can be found at:
doi:10.3762/bjoc.13.27
5. Konijn, T. M.; van de Meene, J. G. C.; Bonner, J. T.; Barkley, D. S.
Proc. Natl. Acad. Sci. U. S. A. 1967, 58, 1152–1154.
doi:10.1073/pnas.58.3.1152
250