Total Synthesis and Structural Revision of Aeruginosin KT608A

Article abstract of DOI:10.1021/acs.orglett.7b01822

The synthesis of the presumed structure of aeruginosin KT608A was accomplished for the first time. The unusual d-diepi-Choi core was prepared from tyrosine via C-H activation and heterogeneous hydrogenation. Due to differences in the spectral data of synthetic and natural samples, a revised structure featuring l-diepi-Choi was proposed, which was synthesized and confirmed to be identical. On the basis of these findings, revised structures for six additional aeruginosins (KT608B, KT650, GH553, DA495A, DA511, and KB676) are presented.

Full text of DOI:10.1021/acs.orglett.7b01822

Letter
pubs.acs.org/OrgLett
Total Synthesis and Structural Revision of Aeruginosin KT608A
Manuel Scherer† and Karl Gademann*
Department of Chemistry, University of Zurich, 8057 Zurich, Switzerland
S
* Supporting Information
ABSTRACT: The synthesis of the presumed structure of
aeruginosin KT608A was accomplished for the first time. The
unusual D-diepi-Choi core was prepared from tyrosine via C−
H activation and heterogeneous hydrogenation. Due to
differences in the spectral data of synthetic and natural
samples, a revised structure featuring L-diepi-Choi was
proposed, which was synthesized and confirmed to be
identical. On the basis of these findings, revised structures
for six additional aeruginosins (KT608B, KT650, GH553,
DA495A, DA511, and KB676) are presented.
the (S) configuration in L-Choi.4 Consequently, this Choi
eruginosins constitute a group of linear modified
Atetrapeptides isolated form cyanobacteria and marine isomer is referred to as L-diepi-Choi. Aeruginosins DA495A,
DA511, and KB676 have been assigned an inverted
configuration at the 6-position, thus resulting in L-6-epi-Choi
(Figure 1).5
sponges. These compounds are characterized by a central 6-
hydroxyoctahydroindole-2-carboxylic acid (Choi) motif which
is decorated with an arginine mimicking residue on the C-
terminus. The N-terminus often features a hydrophobic amino
acid on the second position and a phenyllactic acid derivative or
a fatty acid on the first position. Since the first isolation of an
aeruginosin in 1994 by Murakami and co-workers,1 this class of
natural products gained attention as protease inhibitors2 and,
more recently, as potent biotoxins.3 Today, around 60 different
aeruginosins are known, with many of them possessing
modifications such as additional halogen atoms, sulfate, and/
or glycosyl groups.2 The vast majority (>50) of aeruginosins
consist of a Choi moiety with (2S,3aS,6R,7aS) configuration,
referred to as L-Choi (Figure 1). Recently, structures with
variations of the Choi residue have been assigned to various
congeners. Aeruginosin EI461 displays the inverted (R)
configuration both at the 3a and 7a positions, compared to
Aeruginosin KT608A (1) was isolated along with aerugino-
sins KT608B, KT650, and GH553 from Microcystis aeruginosa
bloom material from Lake Kinneret, Israel.6 These aeruginosins
have been assigned a D configuration of the Choi motif
(inverted at the 2 position), which is unique compared to other
aeruginosins. Besides the D configuration, the Choi unit of
KT608A (1) was suggested to differ additionally from the
common L-Choi by epimerization at the 3a and 7a positions (D-
diepi-Choi). Further structural features of aeruginosin KT608A
(1) are an agmatine (Agma) residue on the C-terminus and a
D-phenylalanine and hydroxyphenyllactic acid moiety on the N-
terminus (Figure 2).6 In the past, large efforts have been made
Figure 2. Originally assigned (1) and revised (2) structures of
aeruginosin KT608A.
Received: June 15, 2017
Figure 1. Structures of the Choi nucleus in different aeruginosins.
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.7b01822
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
aiming at the synthetic preparation of L-Choi containing
aeruginosins,7 whereas the synthesis of D-diepi-Choi congeners
has rarely been explored. In addition, to the best of our
knowledge, the presence of the D-diepi-Choi in aeruginosins has
not yet been confirmed by total synthesis.
In this study, we report the first total synthesis of the putative
structure of aeruginosin KT608A (1), and after disclosing
evidence for the incorrect assignment of the configuration, an
alternative structure (2) was proposed and verified by total
synthesis (Figure 2). In addition, we disclose a new strategy
toward the Choi unit based on C−H activation and
stereoselective hydrogenation reactions.
before reaching full conversion to enhance yields. Subsequent
removal of the trifluoroacetyl group using NaBH4 furnished D-
diepi-Choi derivative 7 (Scheme 1).
For the introduction of the phenyllactic acid (Hpla) and the
phenylalanine (Phe) residues on the N-terminus of aeruginosin
KT608A (1), we envisioned the incorporation of these two
moieties as one building block. The required Hpla-Phe-OH
fragment 10 was prepared over four synthetic steps from
known phenyllactic acid derivative 812 (Scheme 2). Peptide
Scheme 2. Synthesis of Hpla-Phe-OH Fragment 10
The synthesis of aeruginosin KT608A (1) commenced with
the attachment of pyridine-2-sulfonyl chloride8 to TBS-
protected tyrosine methyl ester 39 (Scheme 1). 2-Pyridine-
Scheme 1. Synthesis of D-diepi-Choi Building Block 7
coupling of acid 8 with D-phenylalanine methyl ester followed
by selective saponification of the acetyl ester afforded Hpla-
Phe-OMe dipeptide 9 in 65% yield over two steps. Subsequent
TBS protection of the obtained alcohol followed by hydrolysis
of the methyl ester yielded Hpla-Phe-OH building block 10.
The assembly of the different building blocks was initiated by
coupling D-diepi-Choi derivative 7 with Hpla-Phe-OH fragment
10 (Scheme 3). For the peptide bond formation DMTMM was
used as coupling reagent to give tripeptide 11 in good yield
with no observable isomerization.13 Hydrolysis of the methyl
ester and subsequent coupling of the acid with agmatine side
chain 1214 provided tetrapeptide 13 in moderate yield. Global
deprotection commenced with catalytic hydrogenation to
sulfonamides have been successfully introduced as directing
groups for intramolecular aminations of arylethylamines by Yu
and co-workers.10 Since the intramolecular amination of 3 to
the corresponding indoline only proceeded in moderate yields
around 50%, the TBS protecting group was replaced by the
more electron-withdrawing acetyl group. To this goal,
derivative 3 was treated with TBAF solution followed by
acetylation of the obtained alcohol using AcCl and DMAP to
give acetyl ester 4. Pleasingly, the intramolecular amination of 4
could be achieved at elevated temperature with Pd(OAc)2 as
catalyst and PhI(OAc)2 as oxidant to give indoline 5 in a very
good yield of 80%. Treatment of indoline 5 with methanolic
Mg0 solution led to the cleavage of the sulfonamide directing
group and acetyl ester. All attempts to perform the reduction of
the indoline to the octahydroindole at this stage were not
rewarded with any success. The low reactivity of the indoline
toward hydrogenation was assigned to the presence of the free
amino and hydroxy groups. Thus, the amine was trifluoroacety-
lated and the hydroxyl group equipped with a MOM protecting
group to give fully protected indoline 6. Fortunately, indoline 6
could be readily transformed to the octahydroindole by
heterogeneous hydrogenation using Rh/C as catalyst at 15
bar H2 pressure.11 Thus, the carboxyl group of 6 led to a
substrate-controlled all-syn addition of the introduced hydrogen
atoms and thus to the formation of only one diastereoisomer.
Due to the modest stability of the trifluoroacetyl amide under
the applied hydrogenation conditions, the reaction was stopped
Scheme 3. Synthesis of the Presumed Structure of
Aeruginosin KT608A (1)
B
DOI: 10.1021/acs.orglett.7b01822
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
remove the Bn group. Subsequent removal of the Boc and
MOM groups was achieved by treatment with diluted TFA
solution. Surprisingly, the TBS group proved to be stable under
the applied acidic conditions. Thus, additional treatment with
diluted HF solution was required to complete the cleavage of
the TBS group. To hydrolyze partially formed trifluoroacetyl
ester, formed during the second deprotection step, the reaction
mixture was treated with aqueous Na2CO3 solution to give pure
aeruginosin KT608A (1) in excellent yield.
15 with NaBH4 followed by protection of the resulting alcohol
with a MOM group afforded octahydroindole 16. Removal of
the Cbz group gave L-diepi-Choi derivative 17 (Scheme 4). The
Scheme 4. Synthesis of the Revised Structure of Aeruginosin
KT608A (2)
However, comparing the NMR spectral data of the
synthesized sample to the values reported for the isolated
aeruginosin KT608A revealed large deviations. The most
apparent difference in the NMR spectra arises from the ratio
of the cis and trans rotamer characteristics for aeruginosins. The
NMR spectra of isolated aeruginosin KT608A appear in a 1:1
ratio at 300 K in DMSO-d6, whereas spectra of the synthesized
sample revealed a ratio of 3:1 favoring the trans rotamer. In
addition, the observed chemical shifts were different from the
reported values as well: while Choi H-2, H-3α, H-3β, H-7(eq),
and H-7(ax) resonate at 4.24, 1.81, 1.96, 1.60, and 1.90 ppm in
the synthetic sample of aeruginosin, the signals of their
equivalents in the isolated sample have been reported to appear
at 4.82, 2.38, 1.70, 2.37, and 0.83 ppm.15 Reviewing the
literature data for several compounds revealed distinct
similarities of the shifts belonging to the Choi unit of natural
aeruginosin KT608A and the L-diepi-Choi signals of aeruginosin
EI461.4
Due to the endo configuration of the D-diepi-Choi, the
carbonyl group is in close proximity to H-7(ax) leading to a
drastic deshielding thereof (m, 1.90 ppm). In the exo
configuration of the L-diepi-Choi such an effect is missing,
resulting in an upfield-shifted signal of H-7(ax) (q, 0.83 ppm).
In addition, the H-2 signal is strongly influenced by the
configuration at C-2. In D-diepi-Choi, the dihedral angles of
H2−C2−C3−H3α and H2−C2−C3−H3β correspond to 20
and 140°, respectively, resulting in a pseudotriplet multiplicity
1
of the H-2 signal in the H NMR spectrum (t, 4.24 ppm). On
the other hand, the equivalent dihedral angles in the L-diepi-
Choi amount to 20 and 100°, respectively. As a result, the Choi
H-2 signal in the L-diepi-Choi appears as a doublet (Figure
3).4,16 To support the hypothesis that the structure of
aeruginosin KT608A contains an L-diepi-Choi motif, the revised
structure 2 of aeruginosin KT608A was prepared. To this goal,
L-diepi-Choi derivative 17 needed to be accessed. Following the
procedures of Wipf and co-workers,17 ketone 15 was prepared
over five steps from tyrosine derivative 14. Reduction of ketone
assembly of the building blocks was performed analogously to
the synthesis of 1. Coupling of L-diepi-Choi derivative 17 to the
Hpla-Phe-OH fragment 10 provided tripeptide 18. Hydrolysis
of the L-diepi-Choi methyl ester followed by linkage of the acid
to agmatine side chain 12 yielded tetrapeptide 19. Global
deprotection was carried out as mentioned above and afforded
aeruginosin 2 consisting of an L-diepi-Choi core. The NMR
spectral data of the revised structure 2 were in full agreement
with values reported for the natural aeruginosin KT608A. This
evidence strongly corroborates the hypothesis that aeruginosin
KT608A contains a Choi motif with an L-diepi rather than a D-
diepi configuration.
After revealing the revised structure of aeruginosin KT608A
by total synthesis, we reviewed the NMR spectral data of
aeruginosins KT608B, KT650, GH553, DA495A, DA511, and
KB676 as published in the literature.5,6 The NMR spectra of all
these aeruginosins displayed characteristic signals assigned to
the L-diepi-Choi rather than the D-diepi-Choi (for KT608B,
KT650, and GH553) or the L-6-epi-Choi (for DA495A, DA511,
and KB676), respectively. This strongly supports the
hypothesis that the structures of these aeruginosins have also
been assigned incorrectly and that an L-diepi-Choi configuration
is present in all of these secondary metabolites.18 Consequently,
the existence of the D-diepi-Choi and the L-6-epi-Choi
configuration in natural aeruginosins has to be questioned at
Figure 3. Differences in the chemical shifts of the Choi H-2 and H-7 in
the D-diepi- and L-diepi-Choi.
C
DOI: 10.1021/acs.orglett.7b01822
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Hall, A.; Johnstone, S.; Tremblay, M.; Parlanti, L. J. Am. Chem. Soc.
2002, 124, 13342−13343. (f) Diethelm, S.; Schindler, C. S.; Carreira,
E. M. Org. Lett. 2010, 12, 3950−3953. (g) Diethelm, S.; Schindler, C.
S.; Carreira, E. M. Chem. - Eur. J. 2014, 20, 6071−6080. (h) Trost, B.
M.; Kaneko, T.; Andersen, N. G.; Tappertzhofen, C.; Fahr, B. J. Am.
Chem. Soc. 2012, 134, 18944−18947. (i) Dailler, D.; Danoun, G.;
Ourri, B.; Baudoin, O. Chem. - Eur. J. 2015, 21, 9370−9378. (j) Dailler,
D.; Danoun, G.; Baudoin, O. Angew. Chem., Int. Ed. 2015, 54, 4919−
4922.
present on the basis of the synthetic work and structural
analysis reported in this study.
In conclusion, the total synthesis of the proposed structure 1
of aeruginosin KT608A is presented. The unique D-diepi-Choi
core was prepared via C−H activation followed by heteroge-
neous hydrogenation. Differences in the NMR spectral data of
synthesized and isolated aeruginosin KT608A led to a structural
revision of the structure. The proposed revised structure 2
featuring an L-diepi-Choi core configuration was established by
total synthesis. Furthermore, on the basis of these synthetic
studies combined with structural analysis by spectroscopic
means, revised structures for six additional aeruginosins are
proposed.
(8) Yang, D.; Mao, S.; Gao, Y.-R.; Guo, D.-D.; Guo, S.-H.; Li, B.;
Wang, Y.-Q. RSC Adv. 2015, 5, 23727−23736.
(9) Cao, B.; Xiao, D.; Joullie, M. M. Org. Lett. 1999, 1, 1799−1801.
(10) Mei, T. S.; Leow, D.; Xiao, H.; Laforteza, B. N.; Yu, J. Q. Org.
Lett. 2013, 15, 3058−3061.
(11) Mezei, T.; Porcs-Makkay, M.; Simig, G. US Patent 198988A1,
2004.
ASSOCIATED CONTENT
* Supporting Information
■
(12) Valls, N.; Vallribera, M.; Lop
Chem. 2002, 67, 4945−4950.
́
ez-Canet, M.; Bonjoch, J. J. Org.
S
(13) Falchi, A.; Giacomelli, G.; Porcheddu, A.; Taddei, M. Synlett
2000, 2, 275−277.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.7b01822.
(14) (a) Han, S.; Moore, R. A.; Viola, R. E. Bioorg. Chem. 2002, 30,
81−94. (b) Sarabia, F.; San
́
chez-Ruiz, A.; Chammaa, S. Bioorg. Med.
Detailed experimental procedures, full characterization,
and all spectra (PDF)
Chem. 2005, 13, 1691−1705.
(15) For a more detailed comparison of the H and 13C spectra of
natural and synthetic aeruginosin KT608A, see the Supporting
Information.
1
AUTHOR INFORMATION
■
́
(16) Valls, N.; Lopez-Canet, M.; Vallribera, M.; Bonjoch, J. Chem. -
Corresponding Author
*E-mail: karl.gademann@uzh.ch.
ORCID
Eur. J. 2001, 7, 3446−3460.
(17) (a) Wipf, P.; Methot, J. L. Org. Lett. 2000, 2, 4213−4216.
(b) Pierce, J. G.; Kasi, D.; Fushimi, M.; Cuzzupe, A.; Wipf, P. J. Org.
Chem. 2008, 73, 7807−7810.
Karl Gademann: 0000-0003-3053-0689
Present Address
†(M.S.) Department of Chemistry, University of British
Columbia, Vancouver, BC V6T 1Z1, Canada.
(18) For the proposed revised structures of aeruginosins KT608B,
KT650, GH553, DA495A, DA511, and KB676, see the Supporting
Information.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Simon Jurt (University of Zurich) for support with
NMR spectroscopy measurements. Cedric Luthi, Angela
Amsler, and Agron Ilazi (all University of Zurich) are gratefully
acknowledged for skillful technical assistance.
■
̈
REFERENCES
■
(1) Murakami, M.; Okita, Y.; Matsuda, H.; Okino, T.; Yamaguchi, K.
Tetrahedron Lett. 1994, 35, 3129−3132.
(2) For a review on aeruginosins and their biological activity, see:
Ersmark, K.; Del Valle, J. R.; Hanessian, S. Angew. Chem., Int. Ed. 2008,
47, 1202−1223.
(3) (a) Kohler, E.; Grundler, V.; Haussinger, D.; Kurmayer, R.;
̈
Gademann, K.; Pernthaler, J.; Blom, J. F. Harmful Algae 2014, 39,
154−160. (b) Scherer, M.; Bezold, D.; Gademann, K. Angew. Chem.,
Int. Ed. 2016, 55, 9427−9431.
(4) (a) Ploutno, A.; Shoshan, M.; Carmeli, S. J. Nat. Prod. 2002, 65,
973−978. (b) Valls, N.; Vallribera, M.; Carmeli, S.; Bonjoch, J. Org.
Lett. 2003, 5, 447−450.
(5) (a) Adiv, S.; Carmeli, S. J. Nat. Prod. 2013, 76, 2307−2315.
(b) Elkobi-Peer, S.; Carmeli, S. Mar. Drugs 2015, 13, 2347−2375.
(6) Lifshits, M.; Carmeli, S. J. Nat. Prod. 2012, 75, 209−219.
(7) For syntheses, see: (a) Valls, N.; Vallribera, M.; Font-Bardía, M.;
Solans, X.; Bonjoch, J. Tetrahedron: Asymmetry 2003, 14, 1241−1244.
(b) Valls, N.; Lopez-Canet, M.; Vallribera, M.; Bonjoch, J. J. Am.
Chem. Soc. 2000, 122, 11248−11249. (c) Hanessian, S.; Tremblay, M.;
Petersen, J. F. W. J. Am. Chem. Soc. 2004, 126, 6064−6071.
(d) Hanessian, S.; Del Valle, J. R.; Xue, Y. F.; Blomberg, N. J. Am.
Chem. Soc. 2006, 128, 10491−10495. (e) Hanessian, S.; Margarita, R.;
D
DOI: 10.1021/acs.orglett.7b01822
Org. Lett. XXXX, XXX, XXX−XXX