Viriditins from Byssochlamys spectabilis, their stereochemistry and biosynthesis

Article abstract of DOI:10.1016/j.tetlet.2019.151446

Byssochlamys spectabilis (anamorph Paecilomyces variotii) strain 10536 was isolated as an endophyte from grapevine and investigated for its secondary metabolite production. Cultures of B. spectabilis yielded the known compound viriditin A (1) and its new derivative viriditin B (2), which showed pronounced cis–trans-amide isomerism. The previously unknown absolute configuration of C–2 and C–13 in 1 were assigned by Mosher's method. Marfey's method confirmed 2S stereochemistry after Jones oxidation and hydrolysis. A series of feeding experiments with [1-¹³C], [2-¹³C] and [1, 2-¹³C₂]-acetate as well as [methyl-¹³C]-methionine indicated a polyketide biosynthetic pathway. Compound 1 showed weak cytotoxicity against the cell line KB3.1 with an IC₅₀ = 30 μg/ml.

Full text of DOI:10.1016/j.tetlet.2019.151446

Journal Pre-proofs
Viriditins from Byssochlamys spectabilis, their stereochemistry and biosynthe-
sis
Sebastiàn Lòpez-Fernàndez, Andrea Campisano, Barbara J. Schulz, Michael
Steinert, Marc Stadler, Frank Surup
PII:
S0040-4039(19)31244-4
DOI:
https://doi.org/10.1016/j.tetlet.2019.151446
TETL 151446
Reference:
Tetrahedron Letters
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Accepted Date:
22 August 2019
5 November 2019
20 November 2019
Please cite this article as: Lòpez-Fernàndez, S., Campisano, A., Schulz, B.J., Steinert, M., Stadler, M., Surup, F.,
Viriditins from Byssochlamys spectabilis, their stereochemistry and biosynthesis, Tetrahedron Letters (2019), doi:
https://doi.org/10.1016/j.tetlet.2019.151446
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Graphical Abstract
Viriditins from Byssochlamys spectabilis,
Leave this area blank for abstract info.
their stereochemistry and biosynthesis
Sebastiàn Lòpez-Fernàndez, Andrea Campisano, Barbara J. Schulz, Michael Steinert, Marc Stadler and Frank
Surup
OH
N
HO
HO
O
O
1
OH
O
O
N
N
2
O
trans-isomer
cis-isomer

1
Tetrahedron Letters
journal homepage: www.elsevier.com
Viriditins from Byssochlamys spectabilis, their stereochemistry and biosynthesis
Sebastiàn Lòpez-Fernàndeza,b,c,¥, Andrea Campisanoc, Barbara J. Schulzb, Michael Steinertb, Marc Stadlera,d
and Frank Surupa,d
a Department Microbial Drugs, Helmholtz Zentrum für Infektionsforschung, Inhoffenstraße 7, 38124 Braunschweig, Germany
b Institute of Microbiology. Technische Universität Braunschweig, Spielmannstraße 7, 38106 Braunschweig, Germany
c Research and Innovation Centre, Fondazione Edmund Mach (FEM), S. Michele all’Adige (TN), Italy
d German Centre for Infection Research Association (DZIF), partner site Hannover-Braunschweig, Inhoffenstraße 7, 38124 Braunschweig, Germany
———
ARTICLE INFO
ABSTRACT
 Corresponding author. Tel.: +49-531-6181-4256; fax: +49-531-6181-9499; e-mail: frank.surup@hemlholtz-hzi.de
¥ current address: Institut Pasteur, Unit Biodiversity and epidemiology of bacterial pathogens, Rue du Dr. Roux 25, 75015 Paris, France
Article history:
Received
Received in revised form
Accepted
Available online
Byssochlamys spectabilis (anamorph Paecilomyces variotii) strain 10536 was isolated as an
endophyte from grapevine and investigated for its secondary metabolite production. Cultures of
B. spectabilis yielded the known compound viriditin A (1) and its new derivative viriditin B (2),
which showed pronounced cis-trans-amid isomerism. The so far unknown absolute
configuration of C–2 and C–13 in 1 were assigned by Mosher’s method. Marfey’s method
confirmed 2S stereochemistry after Jones oxidation and hydrolysis. A series of feeding
experiments with [1-13C], [2-13C] and [1, 2-13C2]-acetate as well as [methyl-13C]-methionine
indicated a polyketide biosynthetic pathway. Compound 1 showed weak cytotoxicity against the
cell line KB3.1 with an IC50 = 30µg/ml.
Keywords:
Structure elucidation
Natural Products
Secondary metabolites
Endophytes
2009 Elsevier Ltd. All rights reserved.
Grapevine (Vitis vinifera L) is a native European plant that has
been used for wine production since ancient times.1 Like many
other plants, grapevine can be asymptomatically colonized by
microorganisms, which are called endophytes.2 It has been
established that endophytes interact with grapevine metabolism
when colonizing their roots and can both activate and block
pathways for plant defense, which suggests that in planta, a
chemical exchange between endophytes and the host takes place.
Additionally, endophytes must co-habit with many organisms
that are competitors for space and nutrients. This species co-
occurrence and resource overlap forces interactions that can
result in the production of secondary metabolites.3,4
with solutions at 200 and 500 ppm of chlorine dioxide and citric
acid-activated sodium chlorite).10
Also, species of the genus have been found in a remarkable
number of marines sources, including the white mangrove
Laguncularia racemosa and marine algae showing their ability of
adaptation to extreme environments probably explained by their
rather strong spore adaptation.11
Since endophytes have great potential for natural product
synthesis,12 we investigated the secondary metabolite production
of the endophytic fungus Byssochlamys spectabilis strain 10536
(DSM 109444) after its isolation from grapevine (Fig. S1 and
S2). In the crude extracts from cultures of strain 10536, we
observed three main peaks (Fig. S3).
Endophytic fungi also live closely associated with wild and
cultivated grapevine. For example, in V. vinifera fungal
endophytic diversity varies according to host cultivar type
showing the tight association of fungal endophytes with their
plant host.5
The genus Byssochlamys (Thermoascaceae: Eurotiales)
consists of fungal species closely related to the genera
Penicillium and Aspergillus.6 It forms a well-defined clade in the
Eurotiales.7
Members of Byssochlamys are typically isolated from the soil,
and thus fruit near the plant can be contaminated with conidia
from the fungus.8 Some species of Byssochlamys produce highly
resistant ascospores that are both heat and low oxygen tension
resistant,9 which pose a great challenge for disinfection protocols
based on pasteurization. However treatment with chemical agents
shows little recovery of either dormant and heat- activated spores
in the anamorphic phase of B. spectabilis (including disinfection

2
Tetrahedron
In our experiments, viriditin (1) co-eluted with a slightly more
5
18
OH
lipophilic fraction that contained the two peaks 2t and 2c (Fig.
S3), which interconverted into each other after isolation by
preparative HPLC. Both 2t and 2c had the molecular formula
C18H27NO3, as determined by HRESIMS, indicating the formal
loss of 2H compared to viriditin A (1). The proton and carbon
spectra of 2t/c were similar to 1, with the key differences being
the substitution of methine C–13 by a ketone. Consequently, 2
was assigned as the 13-keto derivative of 1 and named viriditin
B. However, two sets of resonances were observed in the 1H and
13C spectra for the CH2–1 to CH–12 part with a ratio of 4:6,
indicating indicated spontaneous inter-conversion between cis-
and trans-isomers. The coalescence temperature is at
approcimately 358K (Fig. 3). A ROESY correlation between 7–H
and 5–H2 indicated a trans-amide configuration of the main
isomer, whereas a ROESY correlation was observed between 7–
H and 2–H for the minor cis–isomer. Although the cis/trans
isomerization of proline moieties plays a key role in protein
folding,16 it has only rarely been observed for secondary
metabolites.17 A minor isomer can also be observed in the case of
1. However, less than 5% of 1 exists in the cis-amide
configuration.
17
6
N
2
13
10
1
HO
HO
O
1
2
O
N
trans-isomer
O
O
OH
O
N
cis-isomer
Figure 1. Structure of viriditin A (1) and its new derivative viriditin
B (2), which showed a pronounced cis-trans-amide isomerism.
Viriditin (1) was isolated by preparative HPLC as a pale-green
oil, with the molecular formula C18H29NO3 determined by
HRESIMS and indicative of five units of unsaturation. The
proton and 1H,13C HSQC spectra showed signals of two methyls,
five olefinic and two aliphatic methines (one of them an
oxymethine) along with seven methylenes (one of them an
oxymethylene). In addition, the carbon spectrum revealed the
presence of one conjugated ester and one quaternary olefinic
carbon. Detailed analysis of the COSY, TOCSY and HMBC data
confirmed its planar structure and confirmed its identity as
viriditin (Fig. 1).13 Its structure, although partially elucidated, had
not been completely resolved in terms of stereochemistry. Since
we have provided evidence of a new derivative of 1, we propose
to rename this compound as viriditin A. Also because no
stereochemistry had been assigned to the C–2 and C–13
stereocenters of 1, the absolute configuration of C–13 was
assigned by Mosher´s method.14 Because SR values of -
methoxy--(trifluoro-methyl-) phenylacetic acid (MTPA) esters
were positive for 14–H2 – 17–H3 and negative for 1–H2 – 12–H
(Fig. 2), a 13R configuration was deduced. Furthermore, the
signal patterns and chemical shift differences of the germinal
protons 1–Ha and 1–Hb ( +0.25 ppm for the (S)-MPTA ester,
 +0.20 ppm for the (R)-MPTA ester) indicated a 2S
configuration. To confirm the stereochemistry of C–2, 1 was
oxidized and hydrolyzed to yield free proline, which was
converted into its Marfey’s derivative by treatment with 1-fluoro-
2,4-dinitrophenyl-5-L—alanine amide (FDAA).15 HPLC analysis
of the FDAA derivative of the viriditin hydrolysate and those of
authentic S- and R-proline disclosed the presence of S-proline in
the hydrolysate. Thus, the absolute configuration of 1 was
determined as 2S,13R.
Figure 3. Section of the 1H NMR spectra (700 MHz, DMSO-d6) of 2
measured at 298 – 378 K showing the coalescence of the 2–H signal
for cis and trans-amid-isomers.
Analysis of the bioactivity revealed that 1 possesses very
weak activity against B. subtilis (MIC = 300 µg/mL), no activity
against any other bacterium of our compilation of test
organisms.18 Additionally, no activity was observed against
endophytic gram positive and gram negative bacteria isolated
from V. vinifera (Table S1). Derivative 2 had no biological
activity against the bacterial strains tested. Cytotoxicity was
determined against cervical cancer cell line KB-3-1 and mouse
fibroblast cell line L929. Only 1 was toxic to KB3.1 with an IC50
= 30µg/ml, but no activity was detected against the cell line L929
(Table S2).
To determine the biosynthetic pathway of 1 and 2, a series of
feeding experiments were carried out with stable isotope-labeled
precursors. [1-13C], [2-13C] and [1,2-13C2]-acetate as well as
[methyl-13C]-methionine. The isotopes were fed to growing
cultures of B. spectabilis 10536. The incorporation of non-
labeled control substances, including L-proline, into the growth
medium, did not affect growth of the fungus (Fig. S5A); however
it did affect biosynthesis of 1 and 2. Acetate increased the
production of both molecules, but addition of 0.1 mg of L-proline
reduced the production of 1 and 2 by half, suggesting a possible
anabolic repression of its synthesis. Addition of L-methionine
also exerted a negative effect on the biosynthesis of 2. Analysis
of B. spectabilis fermentation broths through HPLC-MS
demonstrated accumulation of both 1 and 2 (Fig. S5B);
production of 1 and 2 reached a concentration peak at 96 h for 1
and 290 h for 2 (Fig. S5C). Both compounds were best produced
in a glucose-rich medium compared to other media, even in
biomimetic conditions where yeast and even grapevine leaves
were added (Fig. S5D ANOVA P value = 0.00004).
Figure 2. SR values for MPTA esters of 1 diagnostic for 2S,13R
+0.16/
+0.16
-0.01
+0.07/
+0.09
CH3
OMTPA
+0.11
+0.04
CH3
-0.09
-0.02
-0.04
+0.03/
N
+0.09
-0.06
0
+0.06/
+0.04
-0.04
+0.07
-0.19
O
OMTPA
0/
-0.05
configuration.

3
13C enrichments of
1
were analyzed by 13C NMR
spectroscopy.19 High incorporation rates at positions C–7, C–9,
C–11, C–13, C–15 and C–17 of the carbon skeleton upon feeding
with [1-13C]-acetate as well as at positions C–6, C–8, C–10, C–
12, C–14 and C–16 upon feeding with [2-13C]-acetate clearly
indicated a polyketide biosynthetic pathway (Fig. 4, Table 1).
Furthermore, a feeding experiment with [1,2-13C2]-acetate proved
intact incorporation of six acetate units by strong spin-spin
couplings and gave evidence for the direction of the polyketide
chain. A feeding experiment with [methyl-13C]-methionine
showed strong signal enrichment of C–18, demonstrating an S-
adenosylmethionin (SAM) mediated methylation. The C–1 – C–5
moiety most likely derives from proline, which has yet to be
proved experimentally. The chemical structure, together with the
feeding experiments, provides a biosynthesis model for the
viriditins. Presumably, biosynthesis of the viriditins starts with
the assembly of six acetate units by a polyketide synthase; the
fourth unit is methylated with SAM by a methyltransferase.
Table 1. 13C NMR data from feeding experiments on the
biosynthetic pathway of 1
Atom
#
δc [ppm]
[1-13C-]
[2-13C-]
[1,2-13C2-]
[methyl-13C-]
Figure 4. Hypothetical biosynthetic pathway of viriditins. ACP-S=
acyl moiety bound to the Acyl Carrier Protein through a sulfur bond.
NRPS = Non ribosomal peptide synthase; PKS = Polyketide
synthase. Proline = incorporation remains to be experimentally
confirmed.
acetate a
acetate a
acetate b
methionine a
1
67.7
0
0.0
n.o.
n.o.
n.o.
34
0.0
2
61.5
-0.10
-0.06
-0.06
0.3
0.2
0.0
3
28.3
-0.01
0.9
0.0
Byssochlamys spectabilis, or rather its anamorph
Paecilomyces variotii, has been shown producing a great
versatility of secondary metabolites with varied bioactivities.23
These include the antifungals varioxepine A and the pentacyclic
4
24.4
0.1
5
48.1
-0.7
-0.2
7.8
34
0.05
n.o.
0.1
6
triterpenoid
betulin,24,25
the
lipopeptide
antibiotics
167.6
120.9
143.4
126.1
144.4
134.8
138.9
68.6
1.1
67
leucionostatins,26 anthraquinones paeciloquinones,27 the pyridone
alkaloid paecilomide28 and paecilaminol29. Cornexistin for
example, has a selective herbicide activity against invading
mono- a dycotiledoneous plants with no effect in corn, and was
proposed as a plant protection strategy30 whereas variotin31,
isolated from the anamorph P. variotii has a strong antimycotic
activity against foot skin infections caused by Tricophyton,
Dermatophyton, Microsporia and Dermatomyces species.
Viriditin A (1), also an antimycotic, was first isolated from
cultures of by Apergillus viridi-nutans, together with variotin.13
7
n.o.
0.8
67
8
-0.4
6.5
56
0.06
n.o.
0.1
9
n.o.
1.4
56
10
11
12
13
14
15
16
17
18
-0.3
7.4
54
n.o.
1.8
54
n.o.
0.7
1.6
48
n.o.
0.5
8.4
48
0.02
0.1
37.2
-0.3
5.6
35
Recently the role of B. spectabilis viriditoxin in the
antagonism against the plant pathogens Fusarium moliniforme,
Biscogniauxia mediterranea and Phytophtora cinnamomi was
demonstrated.32 These are produced by different strains of
Bysssochlamys although not necessarily by endophytic forms.
27.5
-0.1
0.7
35
0.01
0.1
22.6
-0.7
6.0
35
14.0
-0.03
-0.1
35
0.2
12.8
-0.6
n.o.
19.7
With all these metabolites being produced with little
nutritional requirements and the fact that B. spectabilis can both
contaminate man made products and be persistent in them, an
immense challenge in terms of sterilization of the good for
human consumption is being faced. Strict guidelines show that
the Eurotiales are a major concern for good manufacturing
practices and the metabolites associated with the fungus have to
be taken into account when preparing quality control schemes a
critical point analysis.
aValues of percentage of incorporation (%I) were normalized to the unlabeled
C1 in the proline moiety.
b Coupling constants (in Hz).
n.o. = not observed
Taking into account the common NRPS-PKS hybrid
biosynthesis, a proline building block is most likely condensed to
a PKS-chain by a non-ribosomal peptide synthase and cleaved
off reductively (Fig. 4). Our results point to a textbook style
hybrid PKS-NRPS biosynthesis for the viriditins, which
resembles the biosynthesis proposed for variotin based on
labeling experiments with 14C-labeled precursors20 The same
arrangement, the loading of a PKS chain onto proline building
block catalyzed by a NRPS module, most likely also occurs for
the closely related structures of scalusamides A–C and
formosusins A–C.21,22
Acknowledgments
We are grateful to Christel Kakoschke, Sabrina Karwehl,
Cäcilia Bergmann, Vanessa Stiller and Wera Collisi for recording
spectra and expert technical assistance, respectively. We thank
Christiane Baschien for identification of B. spectabilis and
Daniele Prodorutti and Claudia Longa for assistance in isolation

4
Tetrahedron
of the fungus. This work was funded by Provincia Autonoma di
Trento, progetto PAT - Call 2 Team 2009 - Incoming –
Mecagrafic, and SLF was supported in the form of
17. Seebach D, Ko SY, Kessler H, Köck M, Reggelin M,
Schmieder P, Walkinshaw MD, Bölsterli JJ. Helv. Chim.
Acta 1991, 74, 1953–1990.
a
18. Sandargo B, Michehl M, Praditya D, Steinmann E, Stadler
M, Surup F. Org. Lett. 2019, 21, 3286–3289.
19. Scott AI, Townsend CA, Okada K, Kajiwara M, Cushley
RJ, Whitman PJ. J Am Chem Soc. 1974, 96, 8069-8080.
20. Tanaka N. In Biosynthesis; D. Gottlieb, and P. D. Shaw,
Eds.; Springer Berlin Heidelberg: Berlin, Heidelberg, 1967;
pp 216–221.
21. Tsuda M, Sasaki M, Mugishima T, Komatsu K, Sone T,
Tanaka M, Mikami Y, Kobayashi J. J. Nat. Prod. 2005, 68,
273–276.
"Stipendium" from Helmholtz Zentrum für Infektiosnforschung
for the year 2017.
References and notes
1. Ramos-Madrigal J, Runge AKW, Bouby L, Lacombe T,
Samaniego Castruita JA, Adam-Blondon A-F, Figueiral I,
Hallavant C, Martínez-Zapater JM, Schaal C, Töpfer R,
Petersen B, Sicheritz-Pontén T, This P, Bacilieri R, Gilbert
MTP, Wales N. Nat. Plants 2019, 5, 595–603.
2. Lòpez-Fernàndez S, Compant S, Vrhovsek U, Bianchedi
PL, Sessitsch A, Pertot I, Campisano A. Plant Soil. 2016,
405, 155–175.
22. Mizushina Y, Suzuki-Fukudome H, Takeuchi T, Takemoto
K, Kuriyama I, Yoshida H, Kamisuki S, Sugawara F.
Bioorg. Med. Chem. 2014, 22, 1070–1076.
23. Mioso R, Toledo Marante FJ, Herrera Bravo de Laguna I.
Appl. Biochem. Biotechnol. 2015, 177, 781–791.
24. Zhang P, Mándi A, Li X-M, Du F-Y, Wang J-N, Li X,
Kurtán T, Wang B-G. Org. Lett. 2014, 16, 4834–4837.
25. Barakat K, Saleh M. J. Appl. Pharm.Sci. 2016, 6, 034–040.
26. Wang G, Liu Z, Lin R, Li E, Mao Z, Ling J, Yang Y, Yin
W-B, Xie B. PLOS Pathogens. 2016, 12: e1005685.
27. Petersen F, Fredenhagen A, Mett H, Lydon NB, Delmendo
R, Jenny H-B, Peter HH. J. Antibiot. 1995, 48, 191–198.
28. Teles APC, Takahashi JA. Microbiol. Res. 2013, 168, 204–
210.
3. Schulz BJ, Rabsch L, Junker C. In Seed Endophytes; S. K.
Verma, and J. F. White, Jr, Eds.; Springer International
Publishing: Cham, 2019; pp 171–189.
4. Freilich S, Zarecki R, Eilam O, Segal ES, Henry CS, Kupiec
M, Gophna U, Sharan R, Ruppin E. Nat. Commun. 2011, 2,
589.
5. Pancher M, Ceol M, Corneo PE, Longa CMO, Yousaf S,
Pertot I, Campisano A. Appl. Environ. Microbiol. 2012; 78,
4308–4317.
6. Yilmaz N, Visagie CM, Houbraken J, Frisvad JC, Samson
RA. Stud. Mycol. 2014, 78, 175–341.
29. Ui H, Shiomi K, Suzuki H, Hatano H, Morimoto H,
Yamaguchi Y, Masuma R, Sakamoto K, Kita K, Miyoshi H,
Tomoda H, Tanaka H, Ōmura S. J. Antibiot. 2006, 59, 591–
596.
30. Amagasa T, Paul RN, Heitholt JJ, Duke SO. Pestic.
Biochem. Physiol. 1994, 49, 37–52.
31. Kobori T, Kato H, Miyazaki J. J Antibiot. 1960; 13, 211–
215.
32. Rodrigo S, Santamaria O, Halecker S, Lledó S, Stadler M.
Ann. Appl. Biol. 2017, 171, 464–476.
7. Houbraken J, Varga J, Rico-Munoz E, Johnson S, Samson
RA. Appl. Environ. Microbiol. 2008, 74, 1613–1619.
8. Houbraken J, Samson RA, Frisvad JC. In Advances in Food
Mycology; A. D. Hocking, , J. I. Pitt, , R. A. Samson, and U.
Thrane, Eds.; Springer US: Boston, MA, 2006; Vol. 571, pp
211–224.
9. Samson RA, Houbraken J, Varga J, Frisvad JC. Persoonia
2009, 22, 14–27.
10. Dijksterhuis J, Meijer M, van Doorn T, Samson R, Rico-
Munoz E. Int. J. Food Microbiol. 2018, 285, 27–33.
11. Zhang P, Li X-M, Wang J-N, Li X, Wang B-G. Chin. Chem.
Lett. 2015, 26, 313–316.
12. Brader G, Compant S, Mitter B, Trognitz F, Sessitsch A.
Curr. Opin. Biotechnol. 2014, 27, 30–37.
Supplementary Material
13. Omolo JO, Anke H, Chhabra S, Sterner O. J. Nat. Prod.
2000, 63, 975–977.
14. Hoye TR, Jeffrey CS, Shao F. Nat. Protoc. 2007, 2, 2451–
2458.
15. Fujii K, Ikai Y, Oka H, Suzuki M, Harada K. Anal. Chem.
1997, 69, 5146–5151.
16. Wedemeyer WJ, Welker E, Scheraga HA. Biochemistry
2002, 41, 14637–14644.
Supplementary data (Experimental procedures, 1D and 2D
NMR, HPLCESIMS spectra for 1 and 2) associated with this
article can be found, in the online version, at
http://dx.doi.org/xyz.
Click here to remove instruction text...

5
Highlights
-
-
-
-
Isolation of viriditin and its derivative viriditin B
from Byssochlamys spectabilis
Variable temperature NMR indicates cis-trans-
amid isomerism of viriditin B
Elucidation of absolute configuration by
Mosher’s and Marfey’s methods
Feeding of 13C-labelled precursors reveals the
biogenesis