Total synthesis and structural revision of (+)-cristatumin C

Article abstract of DOI:10.1021/np400969u

Naturally occurring (+)-cristatumin C, a bis-pyrrolidinoindoline diketopiperazine alkaloid isolated from Eurotium cristatum EN-220, is the 2R,3R,11S,15R,2′R,3′R,11′S,15′S enantiomer, as confirmed by total synthesis.

Full text of DOI:10.1021/np400969u

Note
pubs.acs.org/jnp
Total Synthesis and Structural Revision of (+)-Cristatumin C
́
́
Paula Lorenzo, Rosana Alvarez, and Angel R. de Lera*
́
Departamento de Química Organica, Universidade de Vigo, Lagoas-Marcosende, 36310 Vigo, Spain
S
* Supporting Information
ABSTRACT: Naturally occurring (+)-cristatumin C, a bis-pyrrolidinoindoline
diketopiperazine alkaloid isolated from Eurotium cristatum EN-220, is the
2R,3R,11S,15R,2′R,3′R,11′S,15′S enantiomer, as confirmed by total synthesis.
ungi are rich sources of secondary metabolites derived
F
from tryptophan.1,2 Several members of this alkaloid class
have been recently isolated from the culture extract of Eurotium
cristatum EN-220, an endophytic fungus of the marine alga
Sargassum thunbergii.3 The heterodimeric (+)-cristatumin C (6)
stands out as the most complex of the isolates since it is
composed of two subunits of a diketopiperazine-fused
hexahydropyrrolo[2,3-b]indole connected to each other
through the two equally configured contiguous quaternary
stereogenic centers C3 and C3′.
Although these compounds are derived in Nature from the
condensation of tryptophan with other amino acids,4,5 the
biosynthetic machinery combines these simple building blocks
using a variety of connection patterns, C3−C3′ being the most
common, within either homodimeric or heterodimeric
structures. Stereochemical diversity is also achieved through
the presence of either antipode of the amino acid components.
Although the C11 and C15 chiral centers of the diketopiper-
azine can be found in the cis or trans relative configuration, with
the exception of (−)-ditryptophenaline (3)6 all other members
of this family of homodimeric and heterodimeric bis-
pyrrolidinoindoline diketopiperazine alkaloids have opposite
configuration at the C2/C3 hexahydropyrroloindole junction
relative to C11 (cf. 1, 2, 4, 5 in Figure 1).4,5 After having
corrected the proposed structure7 of (+)-asperdimin to 5,8 we
were surprised to find yet another cis relative configuration at
C3′−C11′−C15′ in the purported structure of (+)-cristatumin
C (6) (one of the three different drawings used in the article to
depict the same natural product).3 On the basis of our previous
experience with this family of alkaloids,8−10 we surmised by
analogy that the natural isolate had the relative and absolute
configuration shown in 7 and carried out the first total synthesis
of this compound to test this hypothesis. As anticipated, the
proposed structure of the natural product (+)-cristatumin C
(6) needs to be revised to the stereoisomer 7.3
Figure 1. Representative dimeric pyrrolidinoindoline diketopiperazine
alkaloids, with the numbering system indicated in structure 1.
deprotection of the Fmoc groups in the tetrapeptide 8, itself
obtained by peptide bond formation in homodimeric
hexahydropyrroloindole 9 (Scheme 1). Compound 9 is a
Our synthesis of bis-pyrrolidinoindoline diketopiperazines is
based on the final assembly of the diketopiperazine rings after
Received: November 20, 2013
Published: January 17, 2014
© 2014 American Chemical Society and
American Society of Pharmacognosy
421
dx.doi.org/10.1021/np400969u | J. Nat. Prod. 2014, 77, 421−423

Journal of Natural Products
Note
Scheme 1. Retrosynthetic Analysis of (+)-Cristatumin C
Scheme 2. Synthesis of (+)-Cristatumin C (7)
powerful synthetic platform11 for the straightforward con-
struction of both C3−C3′ symmetric8,9,12 and nonsymmetric
heterodimers.8,9 The latter are acquired by the sequential
peptide coupling of the tetraamine in 98 with two different
amino acids. In the case of (+)-cristatumin C (6/7) L-alanine
and D-valine are chosen since their absolute configurations were
secured by application of Marfey’s method13 based on chiral
HPLC analysis of derivatives of the acid hydrolysate of natural
6.3
The peptide coupling reactions of compound 9 were
sequentially carried out with N-Fmoc-D-valine (10) (1.0
equiv, −15 °C) and N-Fmoc-L-alanine (11) (1.2 equiv, 25
°C) in the presence of 1-[bis(dimethylamino)methylene]-1H-
1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate
(HATU), Et3N, and DMF14 to give tetrapeptide 8 (Scheme
2). This was not fully characterized due to the presence of
rotamers (also of a secondary product, vide infra), which
complicated the analysis of the spectra. Hence, Fmoc-
deprotection under basic conditions (Et2NH in MeOH at 25
°C) and spontaneous cyclization afforded heterodimeric
diketopiperazine 7 in 50% combined yield. Also isolated in
19% yield was the homodimer 12, the result of the 2-fold
condensation of 9 and protected D-valine 10. Its formation
could not be suppressed even at the low temperature of the first
coupling (−15 °C). This result indicates that the steric bulk of
the amino acid used in the first reaction greatly influences the
selectivity of the coupling.8,9 As additional support of this
assumption, if the order of amino acids is reversed, coupling 9
first with N-Fmoc-L-alanine (11) at −15 °C then with
protected D-valine 10, heterodimeric compound 7 was obtained
in 43% yield and homodimer 13 in 41% yield (Scheme 2).15
As anticipated, the spectroscopic data (including optical
rotation, [α]20 +347.6 (c 0.62, MeOH); lit.3 [α]20 +315.7 (c
D
D
1.21, MeOH)) of 7 matched those of the natural product3 (see
the Supporting Information). The relative configuration of
synthetic (+)-cristatumin C (7) is consistent with the analysis
of one- and two-dimensional NMR experiments. Particularly
revealing were the NOESY correlations of H11′ and H15′ and
the lack of correlation between H11 and H15, which confirmed
the relative configuration at these positions.
To summarize, the relative and absolute configuration of the
purported structure of the natural product (+)-cristatumin C3
has been corrected by total synthesis of a rationally guided
stereoisomer. We have shown elsewhere that the assignment
based merely on the spectroscopic data of this family of natural
products can be misleading.10 Although the configuration of the
C15 stereocenter is always confirmed through Marfey’s
degradative amino acid analysis, the (sometimes weak) NOE
correlation of H15 with its neighboring atom is insufficient
evidence to establish the relative configuration at C11. In
addition, the configuration of the C2−C3 pyrrolidinoindoline
fusion atoms cannot be determined in the absence of X-ray
diffraction data or comparison of DFT-computed and
experimental CD curves.16 Chemical synthesis remains in
those cases as the ultimate structural proof.17
EXPERIMENTAL SECTION
■
General Experimental Procedures. Solvents were dried
according to published methods and distilled before use. All reagents
were commercial compounds of the highest purity available. Reactions
were carried out under an argon atmosphere, unless indicated
otherwise. Analytical TLC was performed on aluminum plates with
422
dx.doi.org/10.1021/np400969u | J. Nat. Prod. 2014, 77, 421−423

Journal of Natural Products
Note
Merck Kieselgel 60F254 and visualized by UV irradiation (254 nm) or
by staining with a ethanolic solution of phosphomolybdic acid. Flash-
column chromatography was carried out using Merck Kieselgel 60
(230−400 mesh) under pressure. IR spectra were obtained on a
JASCO IR 4200 spectrophotometer from a thin film deposited onto
NaCl glass. Specific rotations were obtained on a JASCO P-1020
polarimeter. Mass spectra and HRMS (ESI+) were taken on an Apex
III FT ICR MS (Bruker Daltonics) apparatus. H NMR spectra were
recorded in CD3OD or DMSO-d6 at ambient temperature on a Bruker
AMX-400 or AMX-600 spectrometer at 400 or 600 MHz, respectively,
ASSOCIATED CONTENT
* Supporting Information
Comparison of the NMR data of natural and synthetic
■
S
cristatumin C, spectroscopic data for homodimers 12 and 13,
1
and copies of H NMR and 13C NMR spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
1
AUTHOR INFORMATION
Corresponding Author
■
*Tel: +34-986-812316. Fax: +34-986-818622. E-mail: qolera@
with residual protic solvent as the internal reference (CD3OD, δH
=
́
uvigo.es (A. de Lera).
3.31 ppm or DMSO-d6, δH = 2.50 ppm). Chemical shifts (δ) are given
in parts per million (ppm), and coupling constants (J) are given in
hertz (Hz). The proton spectra are reported as follows: multiplicity,
coupling constant J, number of protons, assignment. 13C NMR spectra
were recorded in CD3OD or DMSO-d6 at ambient temperature on a
Bruker AMX-400 at 100 MHz, with the central peak of CD3OD (δC =
49.15 ppm) or DMSO-d6 (δC = 39.51 ppm) as the internal reference.
DEPT135 and two-dimensional (COSY, HSQCed, HMBC, and
NOESY) sequences were used where appropriate to aid in the
assignment of signals.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by funds from the Spanish MINECO
(SAF2010-17935-FEDER), Xunta de Galicia (Grant
08CSA052383PR from DXI+D+i; Consolidacion 2006/15
from DXPCTSUG; INBIOMED-FEDER “Unha maneira de
facer Europa”).
■
́
Synthesis of Cristatumin C (7). To a cooled (−15 °C) solution of
the tetraamine 98 (60 mg, 0.14 mmol) and N-Fmoc-D-valine (10) (47
mg, 0.14 mmol) in DMF (2.3 mL) were added HATU (53 mg, 0.14
mmol) and Et3N (38 μL, 0.28 mmol, 2.0 equiv). The reaction mixture
was stirred at −15 °C for 5.5 h and allowed to reach 0 °C, and then N-
Fmoc-L-alanine (11) (56 mg, 0.18 mmol), HATU (68 mg, 0.18
mmol), and Et3N (50 μL, 0.47 mmol, 2.6 equiv) were added. The
cooling bath was removed, and the mixture was further stirred for 19 h
at 25 °C. The reaction was quenched by the addition of H2O (10 mL)
and extracted with EtOAc (3 × 20 mL). The combined organic layers
were washed with H2O (25 mL) and dried over Na2SO4, and the
solvents were removed under reduced pressure. The residue was
purified by flash chromatography on silica gel (98:2 CH2Cl2/MeOH)
to give the corresponding tetrapeptide 8, which was used directly in
the next step.
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