Extending the Salinilactone Family

Article abstract of DOI:10.1002/cbic.201900764

Five new members of the salinilactone family, salinilactones D–H, are reported. These bicyclic lactones are produced by Salinispora bacteria and display extended or shortened alkyl side chains relative to the recently reported salinilactones A–C. They were identified by GC/MS, gas chromatographic retention index, and comparison with synthetic samples. We further investigated the occurrence of salinilactones across six newly proposed Salinispora species to gain insight into how compound production varies among taxa. The growth-inhibiting effect of this compound family on multiple biological systems including non-Salinispora actinomycetes was analyzed. Additionally, we found strong evidence for significant cytotoxicity of the title compounds.

Full text of DOI:10.1002/cbic.201900764

Accepted Article
Title: Extending the Salinilactone Family
Authors: Christian Schlawis, Tim Harig, Stephanie Ehlers, Dulce G
Guillen-Matus, K. E. Creamer, Paul Jensen, and Stefan
Schulz
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.201900764
Link to VoR: http://dx.doi.org/10.1002/cbic.201900764
A Journal of
www.chembiochem.org

ChemBioChem
10.1002/cbic.201900764
COMMUNICATION
Extending the Salinilactone Family
[
a]
[a]
[a]
[b]
[b]
[b]
C. Schlawis, T. Harig, S. Ehlers, D. G. Guillen-Matus, K. E. Creamer, P. R. Jensen, S.
[
a]
Schulz
Abstract: Five new members of the salinilactone family,
salinilactones D-H, are reported. These bicyclic lactones are
produced by Salinispora bacteria and display extended or shortened
alkyl side chains compared to the recently reported salinilactones A-
C. They were identified by GC/MS, gas chromatographic retention
index and comparison with synthetic samples. Wefurther investigated
the occurrence of salinilactones across six newly proposed
Salinispora species to gain insight into how compound production
varies among taxa. The growth inhibiting effect of this compound
family on multiple biological systems including non-Salinispora
actinomycetes were analyzed. Additionally, we found strong evidence
for significant cytotoxicity of the title compounds.
headspace using closed-loop-stripping-analysis (CLSA)[9] and
GC/MS.
Marine Salinispora bacteria are known for the production of a wide
variety of natural products,[ often with promising biological
activities, e. g. salinipostins[2] or salinosporamide A.[3] Besides
these metabolites, they also produce a wide variety of volatile
compounds such as alcohols, ketones, esters, amides, ureas,
imides, and sulfinamides.[4] Recently, we discovered a new group
of bicyclic lactones in the headspace of Salinispora cultures using
a combination of GC/MS, coupled gas chromatography/direct
1]
deposition infrared spectroscopy (GC/DD-FTIR)
, spectra
calculations and synthesis.[5] These compounds, called
salinilactones A-C (Figure 1, 1a-c), are structurally related to the
A-factor family of g-butyrolactone (GBL) autoregulators[6,7] and
also might have a signaling function. They display a unique
bicyclo[3.1.0]-lactone ring system not known from other natural
products and are volatile in contrast to known GBL autoregulators.
Salinilactone biosynthesis has been linked to the spt gene cluster,
with a knock-out mutant of the asfA homolog spt9 in Salinispora
arenicola CNS-205 failing to produce both salinilactones and the
related salinipostins.[5,8]
Figure 1. Structures of salinilactones A-H, 1a-1h.
Five compounds with mass spectra similar to those of
salinilactones A-C were detected and tentatively termed
salinilactones D-H (Figure 1, 1d-h). Their structures were
proposed based on their molecular mass, gas chromatographic
retention indices I (Table 1) and mass spectra (Supporting
information, Figure S1).
Employing GC/MS analysis and synthesis, we were able to
identify five additional salinilactones produced by Salinispora
bacteria. We report here on the identification, synthesis and
biological evaluation of these compounds.
Eleven Salinispora strains representing nine different species
were analyzed for the presence of salinilactone derivatives in their
[
[
a]
b]
Prof. Dr. Stefan Schulz, Christian Schlawis, Tim Harig, Stephanie
Ehlers,
Institut für Organische Chemie, TU Braunschweig,
Hagenring 30, 38106 Braunschweig (Germany),
Email: Stefan.Schulz@tu-bs.de
Prof. Dr. P. Jensen, D. G. Guillen-Matus, K. E. Creamer,
Scripps Institution of Oceanography, UC San Diego, 9500 Gilman
Drive, La Jolla, CA 92093-0204 (USA)
Supporting information for this article is given via a link at the end of
the document.
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201900764
COMMUNICATION
Table 1. Known salinilactones with gas chromatographic retention index I
and molecular mass.
Compound
I: nat./syn.
alkyl chain
R =)[a]
Molecular
mass
(
Salinilactone A (1a)
Salinilactone B (1b)
Salinilactone C (1c)
Salinilactone D (1d)
Salinilactone E (1e)
Salinilactone F (1f)
Salinilactone G (1g)
Salinilactone H (1h)
1413 / 1410
1472 / 1468
1528 / 1527
1287 / 1285
1315 / 1315
1369 / 1368
1566 / 1566
1634 / 1635
/ 1629
2-methylpropyl
n-butyl
182
182
196
154
168
168
196
210
210
3-methylbutyl
ethyl
isopropyl
n-propyl
n-pentyl
3-methylpentyl
4-methylpentyl
1i
[a]Referring to the general structure shown in Figure 2A.
In GC, an additional methylene group in an alkyl chain results in
an increase of I of about 100 units. When a lower increase is
observed, a methyl-branched chain is present. This general
pattern led to the proposal of the side chain length and branching
of the newly discovered bicyclic lactones. The characteristic ions
at m/z 140 and 122 in the mass spectra, already known from
salinilactones A-C,[5] arise from McLafferty-rearrangement and
subsequent loss of water and facilitated the identification of
salinilactones F to H (Figure 2).
Figure 3. Mass spectra of synthetic salinilactones: A) 1d (salinilactone D); B)
1e (salinilactone E); C) 1f (salinilactone F); D) 1g (salinilactone G); E) 1h
(salinilactone H).
A)
B)
125
O
C)
1
+H
40
O
O
O
O
O
R'
The mass spectra of the two salinilactones D and E do not display
the ions m/z 140 and 122 because the alkyl chain is too short to
undergo McLafferty-rearrangement. Instead, an ion at m/z 125 is
observed, resulting from ketone a-cleavage (Figure 2). Also, both
compounds display prominent fragments from water loss at m/z
136 and 150, respectively. Because salinilactone E has the same
mass as salinilactone F, the only other possible side chain is
isopropyl, fitting the shorter I = 1315. Finally, salinilactone D with
the lowest I = 1287 likely had an ethyl side chain, evident by its
R
R
O
O
O
1a-1f
1d-e
loss of H O
1a-1c, 1f-h
subsequent
2
loss of H O
2
[
M-18]+
m/z 122
Figure 2. A) General structure of salinilactones 1a-h; B) mass spectrometric
fragmentation of short chain salinilactones; C) fragmentation of long chain
salinilactones.
+
M ion at m/z 154.
To support these structural proposals, compounds 1d-1i, were
synthesized following the already established route utilizing an
intramolecular palladium-catalyzed cyclization of enyne esters
(
Scheme 1).[5,10] The syntheses of the shorter side chain lactones
Salinilactone F showed an I = 1369, 100 units shorter than the
value for salinilactone B with a butyl side chain, and was therefore
proposed to be the n-propyl analog. Similarly, I = 1566 indicated
a n-pentyl side chain in salinilactone G. Salinilactone H did not fit
this pattern with I = 1634. Therefore, a methyl branched side chain
was proposed, either 3- or 4-methylbutyl, because 2-methylbutyl
would likely show a different mass spectrum.
started from the commercially available alkynes or alkynoic acids.
The longer homologues 1h and 1i were synthesized from the
corresponding alcohol or bromide from which the required
alkynes 3h and 3i were produced by substitution with lithium
acetylide. All these synthetic routes finally converged into the
enyne ester synthesis. No asymmetric synthesis was undertaken,
but so far all investigated salinilactones 1a-1c have an (1R,5S)-
configuration.[5]
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201900764
COMMUNICATION
and Amycolatopsis mediterranei, all of which are known to
[7,12]
produce GBLs.
None of these strains produced salinilactones (Table 2). The
genus Micromonospora is close relative to the genus
a
[13]
Salinispora. The absence of salinilactone production in M. nigra
and M. echinospora suggests it is not a common feature outside
of the genus Salinispora.
The structural relation of the salinilactones to the GBL
autoregulators points to a specific function in the ecology of
Salinispora. Therefore, we performed several assays to probe the
biological activity of the salinilactones. Inhibition assays showed
inhibitory activity of salinilactone
B against Actinoplanes
teichomyceticus DSM 43866 and Micromonospora echinospora
DSM43816 at higher concentrations matching our previous
results[5] for Salinispora arenicola CNH-996A and Streptomyces
violaceoruber A3(2). Surprisingly, Micromonospora nigra
DSM43818 was impacted quite heavily by salinilactone B, while
the remaining other actinomycetes S. griseus, S. lavendulae and
Amycolatopsis mediterranei were not affected (Figure S3, Table
Scheme 1. Synthesis of bicyclic lactones 1d-1i. The starting points of the
synthetic routes differ depending on the commercial availability of precursors.
2).
Table 2. Actinomycetes tested for salinilactone production and inhibitory
activity in agar diffusion assays. Minimum loading of salinilactone B needed
to observe growth inhibition.
Mass spectra as well as I values of the synthetic samples 1d-1g
matched those of the naturally occurring salinilactones D-G
(Figure S1), confirming their identity.
Compounds 1h and 1i were synthesized as candidate structures
of salinilactone H. The position of the methyl branch had to be
assigned by comparison of I of the two synthetic compounds with
the naturally occurring lactone because the mass spectra of both
compounds were similar and the natural compound spectra were
of poor quality due to the miniscule amounts in the samples
Actinomycete
salinilactone
production
Inhibition / Loading
Yes / 50 µg[5]
No
Salinispora arenicola
CNH-996A
Yes
(~500 µg/l 1b)[5]
Salinispora arenicola
CNS-205
Yes
No
No
No
No
(Figures S2, S3). Lactone 1h thus proved to be identical with
salinilactone H, exhibiting an anteiso-methyl group.
Yes / 50 µg[5]
No
Streptomyces
violaceoruber A3(2)
We recently proposed the designation of six new Salinispora
species in addition to the three that have already been
[11]
described. Consequently, we tracked salinilactone production
in members of all nine species as represented by 11 different
strains. For this purpose, CLSA of liquid cultures and agar plates
was performed (Table S2). Salinilactone production was
widespread in the Salinispora genus. Nine out of 11 strains
produced salinilactones, although in variable amounts. The two
strains that did not produce any compounds both lacked the spt
gene cluster (Figure 4). These strains are dispersed in the genus
suggesting two independent loss events of the gene cluster. The
amounts of natural 1i were so small that it was only detected after
prolongation of the extraction time from 24 h to 48 h in the high
producing strain S. arenicola CNS-205. Hence it is not included in
Table S2 or Figure 4.
Streptomyces griseus
DSM40236
Micromonospora nigra
DSM43818
Yes / 30 µg
Yes / 100 µg
Micromonospora
echinospora
DSM43816
Streptomyces
lavendulae
DSM40069
No
No
No
No
Amycolatopsis
mediterranei
DSM43304
No
The bouquet of compounds is usually very similar among strains,
although the amount detected varied (Figure 4). Salinilactones A,
B, and F often were the most abundant compounds, with both
strains of S. pacifica and S. arenicola producing the highest yields
and greatest diversity of salinilactones.
Actinoplanes
teichomyceticus
DSM43866
Yes / 100 µg
The gene cluster is highly conserved across all nine strains in
which it was detected although observed in different genomic
environments that include varied numbers and types of regulatory
elements that could play a role in the amounts and types of
salinilactones produced. Differences in production of agar and
liquid cultures were usually small (Table S2).
Hence, the inhibitory activities of salinilactone
considerably both among strains of the same genus and, in the
case of S. arenicola, between strains of the same species.
B varies
We also analyzed liquid and agar plate CLSA-extracts of a range
of actinomycetes including Streptomyces griseus, S. lavendulae,
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201900764
COMMUNICATION
Figure 4. Species phylogeny in relation to salinilactone production and BGC organization. Salinispora species tree generated from 77 concatenated, single-copy genes using autoMLST;[16] with 1000 bootstraps and Micromonospora
nigra DSM 43818 as an outgroup. Salinilactone A-G production is indicated by black circles of various size corresponding to Table S2. When the salinipostin BGC is present, 20kb up and downstream are shown. Genes are
colored based on COG function. Black asterisks indicate regulatory genes: transcriptional regulators include tetR (pink), arsR (purple), gntR (grey), fadR (maroon), luxR (light blue), ompR (light purple), and XRE (dark blue)
families.
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201900764
COMMUNICATION
To get some insight into the chemical ecology of the marine
sediment affiliated genus Salinispora, a toxicity assay with brine
shrimps was performed with the racemic synthetic products to
evaluate the protective value of these compounds. Interestingly,
salinilactones A-C and G exhibited significant activity in the
assays, which are commonly used for evaluation of cytotoxicity of
bioactive compounds (Table 3).[14]
acknowledges funding from the NIH under award R01
GM085770.
Keywords: microbial volatiles • A-factor • toxic compounds •
GC/MS • biosynthesis
[
1]
a) P. R. Jensen, P. G. Williams, D.-C. Oh, L. Zeigler, W. Fenical, Appl.
Environ. Microbiol. 2007, 73, 1146; b) M. Nett, H. Ikeda, B. S. Moore,
Nat. Prod. Rep. 2009, 26, 1362.
Table 3. LC50 values of salinilactones in standard brine shrimp (Artemia
salina) cytotoxicity assay ± SD, n = 3.
[
2]
3]
C. J. Schulze, G. Navarro, D. Ebert, J. DeRisi, R. G. Linington, J. Org.
Chem. 2015, 80, 1312.
Compound
LC50 [µM]
[
a) T. A. M. Gulder, B. S. Moore, Angew. Chem. Int. Ed. 2010, 49, 9346;
b) R. H. Feling, G. O. Buchanan, T. J. Mincer, C. A. Kauffman, P. R.
Jensen, W. Fenical, Angew. Chem. Int. Ed. Engl. 2003, 42, 355.
a) U. Groenhagen, A. L. L. de Oliveira, E. Fielding, B. S. Moore, S.
Schulz, ChemBioChem 2016, 17, 1978; b) T. Harig, C. Schlawis, L.
Ziesche, M. Pohlner, B. Engelen, S. Schulz, J. Nat. Prod. 2017, 80, 3289.
C. Schlawis, S. Kern, Y. Kudo, J. Grunenberg, B. S. Moore, S. Schulz,
Angew. Chem. Int. Ed. 2018, 57, 14921.
Salinilactone A (1a)
Salinilactone B (1b)
Salinilactone C (1c)
Salinilactone F (1f)
Salinilactone G (1g)
Berberine chloride[14]
117±51
91±26
[
4]
85±24
[
[
5]
6]
576±140
87±33
a) A. S. Khokhlov, L. N. Anisova, I. I. Tovarova, E. M. Kleiner, I. V.
Kovalenko, O. I. Krasilnikova, E. Y. Kornitskaya, S. A. Pliner, Z. Allg.
Mikrobiol. 1973, 13, 647; b) S. Horinouchi, T. Beppu, Mol. Microbiol.
25.2±2.9
1994, 12, 859.
[
[
7]
8]
J. M. Willey, A. A. Gaskell, Chem. Rev. 2011, 111, 174.
G. C. A. Amos, T. Awakawa, R. N. Tuttle, A.-C. Letzel, M. C. Kim, Y.
Kudo, W. Fenical, B. S. Moore, P. R. Jensen, Proc. Natl. Acad. Sci. U.
S. A. 2017, 114, E11121-E11130.
The brine shrimp activity is similar to that of berberine chloride, a
compound well-known for its cytotoxic effects.[15] This Indicatesa
a potential defensive function of the salinilactones against
grazers. The lower activities of salinilactone A and F might be the
result of the compounds volatility. As the activity of salinilactones
B, C and G is very similar, the non-polar side chain seems to have
negligible effect compared to the bicyclic core. A reaction of the
highly strained three membered ring with a nucleophile is
conceivable as this kind of compound is known for reactions of
[
9]
a) K. Grob, F. Zürcher, J. Chromatogr. A 1976, 117, 285; b) C. A. Citron,
J. Gleitzmann, G. Laurenzano, R. Pukall, J. S. Dickschat, ChemBioChem
2
012, 13, 202; c) S. Schulz, J. Fuhlendorff, H. Reichenbach, Tetrahedron
004, 60, 3863.
2
[10] L. L. Welbes, T. W. Lyons, K. A. Cychosz, M. S. Sanford, J. Am. Chem.
Soc. 2007, 129, 5836.
[
11] N. Millán-Aguiñaga, K. L. Chavarria, J. A. Ugalde, A.-C. Letzel, G. W.
Rouse, P. R. Jensen, Sci. Rep. 2017, 7, 3564.
[17]
this type at elevated temperatures employing alcohols or thiols.
[
12] a) O. V. Efremenkova, Russ J Bioorg Chem 2016, 42, 457; b) S. Schulz,
S. Hötling, Nat. Prod. Rep. 2015, 32, 1042; c) E. Takano, T. Nihira, Y.
Hara, J. J. Jones, C. J. L. Gershater, Y. Yamada, M. Bibb, J. Biol. Chem.
In conclusion, we identified five new members of the salinilactone
family, salinilactones D-H, by GC/MS studies and synthesis of
candidate structures. The production of salinilactones in a wide
variety of Salinispora strains was confirmed. The absence of
salinilactone production in strains lacking AfsA the salinipostin
biosynthetic gene cluster supports our previously postulated
biosynthetic pathway. Additionally, salinilactone B inhibits a range
of actinomycetes including salinilactone and other GBL
producers. Finally, salinilactones exhibit significant cytotoxicity in
brine shrimp assays demonstrating the ability of salinilactones to
affect higher organisms, even though the specific biological
function of the salinilactones remains unclear.
2000, 275, 11010.
[
13] a) P. R. Jensen, C. Mafnas, Environ. Microbiol. 2006, 8, 1881; b) L. A.
Maldonado, W. Fenical, P. R. Jensen, C. A. Kauffman, T. J. Mincer, A.
C. Ward, A. T. Bull, M. Goodfellow, Int. J. Syst. Evol. Microbiol. 2005, 55,
1759.
[
[
[
14] P. N. Solis, C. W. Wright, M. M. Anderson, M. P. Gupta, J. D. Phillipson,
Planta Med. 1993, 59, 250.
15] S. Jantová, L. Cipák, M. Cernáková, D. Kostálová, J. Pharm. Pharmacol.
2003, 55, 1143.
16] M. Alanjary, K. Steinke, N. Ziemert, Nucleic Acids Res. 2019, 47, W276-
W282.
[17] a) P. S. Skerry, N. A. Swain, D. C. Harrowven, Donald Smyth, Gordon
Bruton, R. C. D. Brown, Chem. Commun. 2004, 1772; b) N. A. Swain, R.
C. D. Brown, Gordon Bruton, Chem. Commun. 2002, 2042.
Acknowledgements
We thank Jessica Grube for the cultivation of several
actinomycetes, their extraction and inhibition testing. PRJ
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201900764
COMMUNICATION
Entry for the Table of Contents (Please choose one layout)
Layout 1:
COMMUNICATION
Five new salinilactones have been
identified by GC/MS and synthesis
extending the family of salinilactones
to eight compounds. Inhibition assays
with actinomycetes, tracking of
salinilactone production in Salinispora
bacteria and brine shrimp assays
provided insight into the occurrence
and biological purpose of
Author(s), Corresponding Author(s)*
Page No. – Page No.
Title
salinilactones.
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.