Stereochemical determination of the leupyrrins and total synthesis of leupyrrin A1

Article abstract of DOI:10.1021/jacs.5b01894

The stereochemical determination of the potent antifungal agents leupyrrin A₁ and B₁ and the total synthesis of leupyrrin A₁ are reported. The relative and absolute configuration was determined by a combination of high field NMR studies, molecular modeling, and chemical derivatization. The expedient total synthesis involves a one-pot sequential Zr-mediated oxidative diyne-cyclization/regioselective opening sequence for preparation of the unique dihydrofuran ring, a highly stereoselective one-pot approach to the butyrolactone, a challenging sp²-sp³ Suzuki coupling and a high-yielding Shiina macrolactonization.

Full text of DOI:10.1021/jacs.5b01894

Communication
pubs.acs.org/JACS
Stereochemical Determination of the Leupyrrins and Total Synthesis
of Leupyrrin A1
Daniel Herkommer,† Sebastian Thiede,† Paul R. Wosniok,† Sandra Dreisigacker,†,§ Maoqun Tian,†,||
Thomas Debnar,†,⊥ Herbert Irschik,‡ and Dirk Menche*,†
†Kekule-
́
Institut fur Organische Chemie und Biochemie, Universitat Bonn, Gerhard-Domagk-Str. 1, 53121 Bonn, Germany
̈
̈
‡Microbial Drugs, Helmholtz Centre for Infection Research (HZI), Inhoffenstr. 7, D-38124 Braunschweig, Germany
S
* Supporting Information
butyrolactone and the dihydrofuran moieties.3 They are
ABSTRACT: The stereochemical determination of the
potent antifungal agents leupyrrin A1 and B1 and the total
synthesis of leupyrrin A1 are reported. The relative and
absolute configuration was determined by a combination
of high field NMR studies, molecular modeling, and
chemical derivatization. The expedient total synthesis
involves a one-pot sequential Zr-mediated oxidative
diyne-cyclization/regioselective opening sequence for
preparation of the unique dihydrofuran ring, a highly
stereoselective one-pot approach to the butyrolactone, a
challenging sp2−sp3 Suzuki coupling and a high-yielding
Shiina macrolactonization.
distinguished by an 18-membered nonsymmetric macrodiolide
core incorporating an unusually substituted γ-butyrolactone ring
together with a pyrrole and an oxazoline ring in combination with
a side chain containing a unique dihydrofuran with two exocyclic
alkylidenes.4 The important biological properties and their
natural scarcity, coupled with their intriguing molecular
architectures and most unusual biosyntheses, render the
leupyrrins highly attractive targets for further development.
Herein we report determination of the full stereochemistry of
the leupyrrins and the first total synthesis of leupyrrin A1 (1).
Configurational assignment relied on application of high-field
NMR studies in combination with molecular modeling and
derivatization. The total synthesis was accomplished by an
expedient strategy, involving innovative, highly stereoselective
sequential approaches to the butyrolactone and the furan
moieties, a challenging sp2-sp3 coupling of advanced inter-
mediates and a high yielding Shiina macrolactonization.
he leupyrrins (Figure 1) are structurally unique macro-
T
diolides from the myxobacterium Sorangium cellulosum.1
The planar constitutions of the leupyrrins (Figure 1) were
elucidated by Bode and Hofle on the basis of NMR data (1H and
̈
13C NMR, COSY, HMQC, and HMBC).1 They contain up to
seven stereogenic centers. Only one of them (C-3′) has been
rigorously assigned by degradation. Furthermore, a trans-
orientation of H-13 to H-14 in the oxazoline ring as well as
trans-configurations of H-3 to both H-4 and C-23 in the
butyrolactone moiety have been proposed for leupyrrin A1 (1)
on the basis of conformational NMR studies.
For full stereochemical determination, we focused our studies
on leupyrrin B1, due to optimal signal resolution and low degrees
of conformational flexibility.5 Optimum 1H signal dispersion was
obtained in CD3OD and CDCl3 at the highest available field
strength (600 MHz) allowing for complete assignment of all
resonances. Notably, the coupling constants and NOESY
correlations suggest the macrocyclic core to be relatively rigid,
while a certain degree of flexibility has to be considered for the C-
6 to C-8 and the C-25 region.
Figure 1. Planar structures of leupyrrins A1 (1) and B1 (2): potent
antifungal agents from the myxobacterium Sorangium cellulosum.
They display impressive biological properties, including
antiproliferative and anti-HIV activities2 and highly potent
antifungal effects in nanomolar concentrations.1 On a molecular
level they efficiently inhibit DNA, RNA, and protein syntheses.
In cellular systems, no interaction with the respiratory chain,
membrane disruption, or conductivity changes were observed,
which suggest that an unusual and so far undefined molecular
target may be involved. As shown for the most prominent
representatives leupyrrins A1 (1) and B1 (2) their unique
architectures are derived by a most diverse biosynthesis reported
for myxobacteria so far, that includes a polypropionate (C-1 to C-
7), a nonribosomal peptide (C-8 to C-15), a dicarboxylic acid (C-
1′ to C-8′), and a polyketide segment (C-16 to C-21) and also
includes highly unusual biosynthetic constructions of the γ-
Within the C-1 to C-6 subunit, a trans-, trans-configuration
between H-3 and H-4 and between H-3 and Me-23 was readily
confirmed,6 as shown in Figure 3 for the macrocyclic core 4, in
full agreement with the proposal of Bode and Hofle for leupyrrin
̈
A1 and a partial synthesis from our laboratory.7 As depicted in
Figure 2 for the C-12 to C-18 fragment, a homonuclear coupling
Received: February 20, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.5b01894
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
calculated data was obtained. Furthermore, the calculated
conformation of 4 with pyrrole and oxazoline nitrogen atoms
adopting an N,N-syn orientation accounted most accurately for
several key long-range ROESY correlations (i.e., from H6 to H9
and from H2′ to H14).
Finally, for assigning the absolute configuration of leupyrrin
B1, selective cleavage of the dicarboxylic acid was effectuated by
basic hydrolysis using K2CO3/MeOH to give a corresponding
triol (see SI). Mosher ester analysis then allowed designation of
the C3-configuration, as shown (Scheme 1). Structure 6
Figure 2. Conformations determined for the C12 to C18 subunit 3 of
leupyrrin B1 (2); coupling constants, 3JH,H (Hz) in parentheses.
constant of 4.0 Hz between H-13 and H-148 and a large coupling
between H-14 and H-15 (10.1 Hz) indicated an antiperiplanar
relationship between these protons. In combination with strong
NOE correlations from H-13 to H-15 and from H-14 to CH2-25,
a relative trans-relationship between H-13 and H-14 was
deduced, as shown for 3a, again in full agreement with previous
proposals.1,7
Scheme 1. Stereostructures for Leupyrrins A1 and B1 and
Retrosynthetic Analysis of Leupyrrin A1
Stereochemical correlation of the chiral center at C-26 to the
oxazoline moiety was based on a characteristic NOE-signal from
H-26 to H-14. In contrast, an analogous correlation between H-
13 and H-26 was much less pronounced, which defines the
dihedral angle between the oxazoline and the furan moiety, as
shown in Figure 3b.9 Together with a large coupling between H-
14 and H-15, the rigidity of the alkylidenesystem, and the
absence of NOE correlations of CH2-27 to either H-14 or H-25,
these data suggest a relative syn-configuration between H-14 and
H-26. Notably, the characteristic NOE signal from H-14 to H-26
had also been observed independently for a structurally related
synthetic fragment, which further corroborates this assignment.7
For determination of the relationship between the C1 to C6
and the C12 to C18 stereoclusters, molecular modeling was
applied on the two possible stereochemical permutations of a
truncated analogue (4) of leupyrrin B1 (Figure 3). Using
summarizes the assignment of the full relative and absolute
configuration for leupyrrin B1. On the basis of a common
biogenesis and close similarity of the spectral data, the
stereochemistry of leupyrrin A1 was assigned as 5, and the
structure of the other leupyrrins can be assigned accordingly.13
Efforts were then directed to a validation of our assignment by
a total synthesis of leupyrrin A1. As outlined retrosynthetically in
Scheme 1, our synthetic approach relies on the assembly of four
building blocks, i.e., 7, 8, 9, and 10. The oxazoline moiety was
planned to arise from a cyclodehydration of 8 with the vicinal
aminoalcohol derived from 10, while a cross-coupling of
boronate 7 with benzylic bromide 8 should deliver the sp2−sp3
fusion between C-6 and C-7. In principle, either methodology
could be employed to close the macrocyclic ring, as an alternative
to more conventional macrolactonizations with either end of 9,
thus offering considerable flexibility in the synthesis.
Figure 3. Lowest energy conformation (OPLS2005; GB/SA water
solvent model) of the truncated analogue 4 of leupyrrin B1.
Macromodel (Version 8.5), 20,000 step Monte Carlo searches
were carried out with the OPLS2005 force field and the
generalized Born/surface area (GB/SA) water solvent model and
with implementation of a conformational restraint of the
antiperiplanar orientation of H-4 and C-24 based on the NOE
data (see above).10 As starting geometries for each of the two
stereochemical permutations, both N,N-syn and N,N-anti
orientations of the nitrogen atoms of the pyrrole and the
oxazoline heterocycles (cf. C11−C12 dihedral angle) were
employed, resulting in four input structures.11 The calculations
revealed a series of discrete families of low energy conformations
for the stereoisomers within 20.00 kcal mol−1 of the global
minimum.12 The calculated dihedral angles for the lowest energy
conformation of 4 (see Figure 3) to the corresponding series of
coupling constants, as determined by NMR, resulted in a close
match, while for the other three stereochemical permutations, a
much lower degree of resemblance between spectral and
For a synthesis of the butyrolactone 7, an innovative 3-step
one-pot process was designed. As shown in Scheme 2, this
involves a hemiacetal opening of readily available lactol 11,14
nucleophilic addition to the resulting aldehyde 12 with iso-
Scheme 2. One-Pot Synthesis of the Butyrolactone Fragment
B
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Journal of the American Chemical Society
Communication
propenylmagnesium bromide and an intramolecular trans-
esterification to 14. This process proceeded with excellent
selectivity giving the desired hydroxy ester 14 as a single
diastereomer.15 Notably, this route is highly concise and
compares favorably to a previous preparation of the
butyrolactone from our group.7 After installment of suitable
protective groups, the derived terminal alkene 15 was elaborated
to vinylic boronic ester 7 by a cross metathesis with sterically
demanding methyl-propenylboronic acid pinacol ester in the
presence of Hoveyda−Grubbs II catalyst, which proceeded in
good yields and E/Z-selectivities (9:1).16,17
Scheme 4. Completion of the Total Synthesis of Leupyrrin A1
For synthesis of bis-alkylidene-substituted dihydrofuran 10, a
further advancement and application of our Zr-mediated
cyclization strategy, as previously developed in or group, was
evaluated.7,18 In detail, the elaboration toward a densely
functionalized diyne, i.e., 21 was envisioned (Scheme 3). The
Scheme 3. Tandem Synthesis of the Dihydrofuran Moiety
could be accomplished in good yields using catalytic amounts of
(tBu3P)2Pd and cesium carbonate in a THF/water mixture. To
the best of our knowledge this presents the first example of a
Suzuki-reaction at the benzylic position of a pyrrole.27
Simultaneous removal of the Boc- and tert-butyl ester group
was achieved under Lewis acidic conditions to give 24.28
For esterification of 9 with 10, the Shiina protocol proceeded
smoothly and in high yields.29 Mild reaction conditions (TASF)
then had to be applied for the liberation of the vicinal
aminoalcohol 25 to minimize partial migration of the ester
group to C-13. For coupling of 24 with 25, a protocol of Wipf and
co-workers30 was applied, involving an HATU mediated amide
formation and cyclodehydration with DAST, giving the desired
oxazoline 26 in good yields and with inversion of the hydroxyl-
bearing stereogenic center at C-13, as expected.30
required diyne was obtained from readily available propargylic
alcohol 1719 involving an etherification with 16,7,18 an HWE
homologation of a derived aldehyde 18, a Sharpless epoxidation,
subsequent regioselective opening of the epoxide 20 with
ammonia at the propargylic center,20 and introduction of suitable
protective groups.21,22 Following our previously developed
procedure, the crucial 3-step sequential process was initiated
by a zirconocene-mediated cyclization. After regioselective
opening of the derived zirconacyclopentadiene 22 with NBS in
situ, final protonation of 22 gave rise to the desired furan 23. This
process proceeded with high yields, considering the complexity
of the substrate, and excellent selectivities (>20:1), even on gram
scale. This outcome is presumably based on a remote
coordination of the metal center to the terminal OMe-ether, in
agreement with our mechanistic and protective group concept.18
To the best of our knowledge, this presents the most advanced
application of Zr-mediated oxidative cyclizations in complex
target synthesis. Finally, the required methyl group was installed
using slightly modified Negishi coupling conditions, and the
terminal hydroxyl was liberated with in situ generated HCl in high
yields.23,24
After liberation of seco-acid 27 by cleavage of the tert-butyl
ester with TMSOTf and mild saponification of the acetate using
potassium carbonate in a methanol/THF/water mixture, the
desired macrolactonization to 28 could be effectuated using the
Shiina protocol under high dilution conditions (0.001 M) in
excellent yield (92%). Ultimately, removal of the remaining
primary TBS-group proceeded smoothly with TASF in
acetonitrile to reveal the natural product 5 in 76% yield. The
spectroscopic data (1H NMR, 13C NMR) and specific rotation
20
20
(synthetic, [α]D = +11.0, c = 0.31, MeOH; natural, [α]D
=
+12.0, c = 4.06, MeOH) of our synthetic material were in
agreement with those published for an isolated sample of
leupyrrin A1, thus allowing confident assignment of the relative
and absolute configuration of leupyrrin A1 and validating our
earlier proposal.1
In summary, a full stereochemical assignment for the leupyrrin
class of natural products was suggested based on the results of
high field NMR studies, molecular modeling, and chemical
derivatization. This assignment was ultimately validated by a
stereocontrolled total synthesis of leupyrrin A1 (5), which
proceeds in 21 steps (longest linear sequence, starting from
known compounds 16 and 17) with a 6.3% overall yield.31 This
represents the first synthesis of a member of this family. Salient
features of our strategy were an advantageous sp2−sp3 Suzuki
In a rationale to allow for high degrees of convergence, our
strategy for fragment union relied on first building up the
Western and Eastern fragments, i.e., 7 with 825 and 926 with 10,
respectively (Scheme 4). After extensive evaluation of reaction
parameters (catalyst, base, and solvent), it was found that the
challenging sp2−sp3 Suzuki cross-coupling between 7 and 8
C
DOI: 10.1021/jacs.5b01894
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
(10) Conformational searches without constraints resulted in
structures with an anti-periplanar orientation of Me-24 to C-3, which
can be excluded due strong NOE correlation from H-3 to H-24 and the
absence of a correlation between H-3 and H-6.
(11) The chemical shift of the pyrrole NH proton in CDCl3 (9.0 ppm)
indicates an N,N-syn-orientation between the pyrrole and the oxazoline:
Afonin, A. V.; Ushakov, I. A.; Pavlov, D. V.; Ivanov, A. V.; Mikhaleva, A.
I. Magn. Reson. Chem. 2010, 48, 685.
(12) For analysis of the conformers, all output structures were
superimposed and identical conformations of the macrocycle were
clustered, resulting in two to five families of macrocyclic conformations.
(13) This assignment is in agreement with biosynthetic considerations
suggesting C-14 to be (R)-configured based on the origin from
threonine.
cross-coupling for C6/C7 bond formation and a Zr-mediated
diyne-cyclization of a highly functionalized substrate for the
regioselective dihydrofuran synthesis. This route will be readily
amenable for preparing significant amounts of leupyrrin A1 as
well as designed analogues to explore the biological potential and
highly unusual biosynthesis of this unique class of metabolites.
ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed experimental procedures and full characterization
(PDF). This material is available free of charge via the Internet
at http://pubs.acs.org.
(14) Lactol 11 was obtained in four steps from commercial malonate
ester involving a yeast mediated reduction: Fronza, G.; Fuganti, C.;
Grasselli, P.; Malpezzi, L.; Mele, A. J. Org. Chem. 1994, 59, 3487.
(15) The high degrees of asymmetric induction may possibly be
derived via a cyclic Cram or a Felkin-Ahn transition state.
(16) Wang, Z. J.; Jackson, W. R.; Robinson, A. J. Org. Lett. 2013, 15,
3006.
(17) The minor isomer could be removed by column chromatography;
little or no conversion was observed with vinylboronic or 1-
propenylboronic acid pinacol ester under a variety of conditions.
(18) Debnar, T.; Dreisigacker, S.; Menche, D. Chem. Commun. 2013,
49, 725.
AUTHOR INFORMATION
■
Corresponding Author
*dirk.menche@uni-bonn.de
Present Addresses
§Pharmaceutical Industry, Germany.
||Scripps Research Institute, La Jolla, California 92037, United
States.
⊥Department of Chemistry, University of Basel, CH-4056 Basel,
Switzerland.
Notes
(19) Compound 17 was readily available by asymmetric alkyne
addition as described in: Katukojvala, S.; Barlett, K. N.; Lotesta, S. D.;
Williams, L. J. J. Am. Chem. Soc. 2004, 126, 15348.
The authors declare no competing financial interest.
(20) (a) Olofsson, B.; Somfai, P. J. Org. Chem. 2002, 67, 8574.
ACKNOWLEDGMENTS
■
(b) Lindstrom, U. M.; Olofsson, B.; Somfai, P. Tetrahedron Lett. 1999,
̈
We thank the DFG (ME 2756/7-1; GK 850: scholarship to S.D.)
for generous funding, Andreas J. Schneider for HPLC-support,
and Dr. Frank Rominger (Univ. Heidelberg) for X-ray analysis.
40, 9273.
(21) A TEOC protective group was chosen to enable high selectivities
in the zirconacyclopentadiene opening, based on our model.18
(22) The absolute configuration was confirmed by Mosher ester
analysis of an epoxide-reduction product of 20: see SI. The relative
conformation was confirmed by NOE experiments on 21.
(23) This method proved more reliable as compared to a previously
evaluated procedure using BuLi and Me2SO4, see refs. 7 and 18.
(24) Shi, J.-C.; Zeng, X.; Negishi, E.-I. Org. Lett. 2003, 5, 1825.
(25) Bromide 8 was obtained from 5-methylpyrrole-2-carboxylic acid
by introduction of the tert-butyl-ester, Boc-protection, and bromination
with NBS (66%, 3 steps; see SI), see: (a) Olson, S.; Slossberg, L. H.
Tetrahedron Lett. 2003, 44, 61. (b) Ashkenazi, T.; Pinkert, D.;
Nudelman, A.; Widberg, A.; Wexler, B.; Wittenbach, V.; Flint, D. Pest.
Manag. Sci. 2007, 63, 974.
(26) Dicarboxylic acid 9 was synthesized in 3 steps (57%) following a
reported procedure involving an Evans-type asymmetric alkylation:
Hoekstra, M. S.; Sobieray, D. M.; Schwindt, M. A.; Mulhern, T. A.;
Grote, T. M.; Huckabee, B. K.; Hendrickson, V. S.; Franklin, L. C.;
Granger, E. J.; Karrick, G. L. Org. Process Res. Dev. 1997, 1, 26.
(27) For selected cross couplings at benzylic positions of other 5-ring
heterocycles, see (a) isoxazoles: McDaniel, S. W.; Keyari, C. M.; Rider,
K. C.; Natale, N. R.; Diaz, P. Tetrahedron Lett. 2011, 52, 5656. (b)
furans: Kuriyama, M.; Shinozawa, M.; Hamaguchi, N.; Matsuo, S.;
Onomura, O. J. Org. Chem. 2014, 79, 5921. (c) thiophenes: Shimizu,
M.; Tomioka, Y.; Nagao, I.; Hiyama, T. Synlett 2009, 3147.
(28) (a) Jones, A. B.; Villalobos, A.; Linde, R. G., II; Danishefsky, S. J. J.
Org. Chem. 1990, 55, 2786. (b) Trzeciak, A.; Bannwarth, W. Synthesis
1996, 1433.
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̈
̈
̈
(4) Four more leupyrrins have been reported. Leupyrrins A2 and B2
bear an additional double bond between C-19 and C-20. Leupyrrins C
and D are formal derivatives of leupyrrins A1 and B1 with a terminal OH-
group in the side chain instead of the OMe-ether.
(5) Higher degrees of signal overlap were observed for the other
leupyrrins. In addition, the double bond in the dicarboxylic part makes
this compound less flexible, which was critical for correlation of remote
stereogenic centers. The observed coupling constants and NOE data in
CD3OD and CDCl3 were very similar for the leupyrrins, which suggest
that they adopt similar conformations in these solvents.
(6) In detail, characteristic NOE correlations were observed from H-3
to H-22 and H-24 as well as from H-4 to H-6 and H-23. In combination
with an antiperiplanar relationship of H-3 and H-4, as deduced from a
large vicinal coupling constant between these protons (9.2 Hz), these
data suggest an anti-, anti-configuration between H-2 and H-3 and
between H-3 and Me-23: see SI for details.
(7) Debnar, T.; Wang, T.; Menche, D. Org. Lett. 2013, 15, 2774.
(8) These values are in full agreement with usual data, see e.g.:
(a) Morris, L. A.; Jantina Kettenes van den Bosch, J.; Versluis, K.;
Thompson, G. S.; Jaspars, M. Tetrahedron 2000, 56, 8345. (b) Toske, S.
G.; Fenical, W. Tetrahedron Lett. 1995, 36, 8355.
(29) Shiina, I.; Kubota, M.; Oshiumi, H.; Hashizume, M. J. Org. Chem.
2004, 69, 1822.
(30) Phillips, A. J.; Uto, Y.; Wipf, P.; Reno, M. J.; Williams, D. R. Org.
Lett. 2000, 2, 1165.
(31) The stereochemical assignment for leupyrrin B1 was independ-
ently proven by X-ray crystallography (see SI for details).
(9) Furthermore, H-15 shows a strong NOE contact both to H-13 and
to Me-32, which confirms the configuration of the exocyclic double
bonds between C-15 and C-16, as well as between C-17 and C-18.
D
DOI: 10.1021/jacs.5b01894
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX