Synthesis of the peptaibol framework of the anticancer agent culicinin D: Stereochemical assignment of the AHMOD moiety

Article abstract of DOI:10.1021/ol302852q

The postulated structure of the potent anticancer peptaibol culicinin D has been synthesized using Fmoc-based solid-phase peptide synthesis (SPPS). Comparison of the ¹H NMR data for the reported structure of culicinin D with the data obtained for the two synthetic polypeptides epimeric at C-6 in the AHMOD unit established the C-6 stereochemistry of the AHMOD residue in the natural product to be (R).

Full text of DOI:10.1021/ol302852q

ORGANIC
LETTERS
XXXX
Vol. XX, No. XX
000–000
Synthesis of the Peptaibol Framework
of the Anticancer Agent Culicinin D:
Stereochemical Assignment of the
AHMOD Moiety
Kuo-yuan Hung, Paul W. R. Harris, and Margaret A. Brimble*
School of Chemical Sciences and The Maurice Wilkins Centre for Molecular
Biodiscovery, The University of Auckland, 23 Symonds Street, Auckland, New Zealand
m.brimble@auckland.ac.nz
Received October 17, 2012
ABSTRACT
The postulated structure of the potent anticancer peptaibol culicinin D has been synthesized using Fmoc-based solid-phase peptide synthesis
(SPPS). Comparison of the 1H NMR data for the reported structure of culicinin D with the data obtained for the two synthetic polypeptides epimeric
at C-6 in the AHMOD unit established the C-6 stereochemistry of the AHMOD residue in the natural product to be (R).
Culicinin D (1) (Figure 1) is a member of the linear
peptaibol family of peptides isolated from the cultures of
the fungus Culicinomyces clavisporus.1 Not only has culi-
cinin D (1) been found to suppress proliferation of PTEN-
negative MDA468 breast tumor cells with an impres-
sive IC50 value of <6 nM,1 this intriguing compound also
contains 2-amino-6-hydroxy-4-methyl-8-oxodecanoic acid
(AHMOD) 2 which is an unnatural amino acid found in
many members of the peptaibol family.2 In addition to the
unique AHMOD 2 unit, structural analysis of culicinin D (1)
revealed the presence of 2-amino-4-methyldecanoic acid
(AMD) 3 and 2-(2-aminopropylamino)ethanol (APAE) 4.
While experimental evidence established that both the
leucine and proline residues in natural culicinin D (1) were
L-amino acids1a and that the absolute stereochemistry of
the AMD 3 unit was (2S,4R),3 the chiral centers in the
AHMOD 2 and APAE 4 building blocks were only
assumed to exhibit the (S)-configuration.1a,4 With the
overarching aim of developing a synthetic program to
enable future investigations of the biological activity of
culicinin D and related members of the peptaibol family,
we herein report the first synthesis of the postulated struc-
ture of culicinin D (1). Importantly, the synthesis estab-
lished the absolute stereochemistry of C-6 in the AHMOD
moiety of the natural product to be (R).
Lengthy syntheses of fully protected AHMOD 2, AMD 3,
and APAE 4 residues with Boc-protected N-termini have
been reported.4,5 Moreover, the one reported synthesis of
(1) (a) He, H.; Janso, J. E.; Yang, H. Y.; Bernan, V. S.; Lin, S. L.; Yu,
K. J. Nat. Prod. 2006, 69, 736–741. (b) Baker, D. D.; Chu, M.; Oza, U.;
Rajgarhia, V. Nat. Prod. Rep. 2007, 24, 1225–1244.
(2) See, for example: (a) Fukushima, K.; Arai, T.; Mori, Y.; Tsuboi,
M.; Suzuki, M. J. Antibiot. 1983, 36, 1613–1630. (b) Grafe, U.; Ihn, W.;
Ritzau, M.; Schade, W.; Stengel, C.; Schlegel, B.; Fleck, W. F.; Kunkel,
W.; Hartl, A.; Gutsche, W. J. Antibiot. 1995, 48, 126–133. (c) Degenkolb,
T.; Heinze, S.; Schlegel, B.; Dornberger, K.; Mollmann, U.; Dahse,
H. M.; Grafe, U. J. Antibiot. 2000, 53, 184–190. (d) Pruksakorn, P.; Arai,
M.; Kotoku, N.; Vilcheze, C.; Baughn, A. D.; Moodley, P.; Jacobs,
W. R., Jr.; Kobayashi, M. Bioorg. Med. Chem. Lett. 2010, 20, 3658–
3663.
(3) Zhang, W.; Ding, N.; Li, Y. J. Pept. Sci. 2011, 17, 576–580.
(4) (a) Hadrami, M. E.; Lavergne, J. -P.; Viallefont, P.; Itto, M. Y. A.;
Hasnaoui, A. Tetrahedron Lett. 1991, 32, 3985–3988. (b) Degenkolb, T.;
Berg, A.; Gams, W.; Schlegel, B.; Grafe, U. J. Pept. Sci. 2003, 9, 666–
678.
(5) (a) Zhang, W.; Sun, T. -T.; Li, Y. -X. J. Pept. Sci. 2009, 15, 366–
368. (b) Zhang, W.; Li, X.; Ding, N.; Li, Y. J. Pept. Sci. 2012, 18, 163–
169.
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10.1021/ol302852q
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enabling the assignment of the absolute chirality at C-6 in
the AHMOD residue of the natural product.
Scheme 1. Retrosynthesis of (2S,4S,6S)-AHMOD 5a,
(2S,4S,6R)-AHMOD 5b, and (2S,4R)-AMD 6
Figure 1. Postulated structure of culcinin D (1) showing the
three unnatural building blocks abbreviated as (2S,4S,6S)-
AHMOD 2, (2S,4R)-AMD 3, and (S)-APAE 4.
AHMOD has been identified as difficult to reproduce on a
large scale.6 In order to adopt Fmoc solid-phase peptide
synthesis (SPPS) to form the linear peptide backbone of
culicinin D (1), the three key building blocks were prepared
using alternative synthetic routes that afford the more
desirable Fmoc-protected amino acids. It was decided to
use 2-chlorotrityl resin as the solid support for SPPS, thus
enabling successful anchoring of the hydroxyl group7 in
the C-terminal APAE residue and also allowing facile
cleavage of the polypeptide using 1% TFA in order to
minimize degradation of the sensitive β-hydroxy ketone
motif in AHMOD 2.
Focusing initially on the synthesis of Fmoc-protected
AHMOD 5a and Fmoc-protected AMD 6, it was realized
that both building blocks were accessible from the com-
mon aldehyde 7 that in turn is derived from olefin 8
(Scheme 1). Olefin 8 is available from asymmetric alkyla-
tion of Belokon complex 98 with iodide 10.9 Subsequent
asymmetric aldol reaction of aldehyde 7 with butan-2-one
11 should allow access to Fmoc-protected AHMOD 5.
Bis-boc protection of aldehyde 7 was required to pre-
vent intramolecular cyclization of the amino group onto
the incumbent aldehyde group.10 Importantly, the new
route proposed for the synthesis of AHMOD 5 and
AMD 6 enables access to both (2S,4S,6S)-AHMOD 5a
and (2S,4S,6R)-AHMOD 5b that can be readily incorpo-
rated into the culicinin D polypeptide skeleton thereby
The synthesis of both AHMOD 5a and 5b commenced
with asymmetric alkylation of Belokon complex 9 with
iodide 10 affording olefin 8 after Boc protection and
methyl ester formation (Scheme 2). Iodide 10 in turn was
readily prepared by asymmetric alkylation of N-propionyl
pseudoephedrine with allyl iodide.9 Exhaustive Boc pro-
tection of olefin 8 by refluxing with (Boc)2O, Et3N and
DMAP in CH2Cl2 for 3 days, delivered olefin 12 in 97%
yield that was converted into aldehyde 7 in 89% yield via
dihydroxylation followed by oxidative cleavage using
OsO4, 2,6-lutidine and NaIO4.11 Treatment of butan-2-
one 11 with (þ)-Ipc2BCl and Et3N followed by addition of
aldehyde 7 effected the desired aldol reaction in 72% yield
forming an inseparable mixture of AHMOD 13a and 13b
(dr 2.6:1) with the facial selectivity favoring formation of
AHMOD 13a.12
After removal of both Boc groups in AHMOD 13a and
13b using 10% TFA in CH2Cl2, Fmoc protection of the
resultant amines afforded a chromatographically separ-
able mixture of Fmoc-protected AHMOD 14a (48%) and
14b (18%). The absolute configuration of C-6 in the major
epimer AHMOD 14a was confirmed to be (S) by Mosher
ester analysis13 as predicted from the use of (þ)-Ipc2BCl to
effect the key asymmetric aldol reaction.12 Treatment of
both AHMOD 14a and 14b with powdered NaOH in 0.8
M CaCl2 solution (i-PrOH/H2O v/v 7:3)14 allowed hydro-
lysis of the methyl ester in the presence of the base-labile
(6) Private communication from ALMAC Sciences.
(7) (a) Frechet, J. M. J.; Nuyens, L. J. Can. J. Chem. 1976, 54, 926–
934. (b) Wenschuh, H.; Beyermann, M.; Haber, H.; Seydel, J. K.;
Krause, E.; Bienert, M.; Carpino, L. A.; El-Faham, A.; Albericio, F.
J. Org. Chem. 1995, 60, 405–410.
(11) Yu, W.; Mei, Y.; Kang, Y.; Hua, Z.; Jin, Z. Org. Lett. 2004, 6,
(8) Belokon’, Y. N.; Zeltzer, I. E.; Bakhmutov, V. I.; Saporovskaya,
M. B.; Ryzhov, M. G.; Yanovsky, A. I.; Struchkov, Y. T.; Belikov, V. M.
J. Am. Chem. Soc. 1983, 105, 2010–2017.
3217–3219.
(12) Goodman, J. M.; Paterson, I. Tetrahedron Lett. 1992, 33, 7223–
7226.
(13) (a) Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973, 95, 512–
519. (b) Hoye, T. R.; Jeffrey, C. S.; Shao, F. Nat. Protoc. 2007, 2, 2451–
2458.
(9) Hung, K. -y.; Harris, P. W. R.; Brimble, M. A. J. Org. Chem.
2010, 75, 8728–8731.
(10) (a) Kokotos, G.; Padron, J. M.; Martı
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B
Org. Lett., Vol. XX, No. XX, XXXX

Scheme 2. Synthesis of (2S,4S,6S)-AHMOD 5a and (2S,4S,6R)-
AHMOD 5b
Scheme 3. Synthesis of (2S,4R)-AMD 6
the synthesis led to clean formation of the desired peptide
via in situ generation of the Fmoc-Aib acid fluoride.15
Fmoc group thus affording Fmoc-protected AHMOD 5a
and 5b in 88% and 92% yield, respectively, ready for
incorporation into SPPS.
For the synthesis of AMD 6, the hydrophobic side chain
was introduced by Wittig reaction of the ylide generated
from phosphonium salt 15 with aldehyde 7 in toluene
at ꢀ78 °C, affording cis-olefin 16 (cis/trans > 95%) in
86% yield (Scheme 3). Hydrogenation of the olefin over
10% Pd/C in MeOH at 20 °C and 30 psi gave Boc-
protected methyl ester 17 in 88% yield. Both Boc groups
in methyl ester 17 were removed by stirring in 50% TFA in
CH2Cl2 and treatment of the crude product with Fmoc-
OSu and 10% Na2CO3 in 1,4-dioxane furnished Fmoc-
protected methyl ester 18in90% yield over 2 steps. Finally,
saponification of methyl ester 18 with powdered NaOH in
0.8 M CaCl2 solution (i-PrOH/H2O v/v 7:3) delivered the
desired building block AMD 6 in 84% yield after purifica-
tion by flash chromatography.
Prior to the synthesis of culicinin D (1) itself, optimal
reaction conditions for SPPS were investigated by synthe-
sis of an analogue of culicinin D 19 in which the valuable
AHMOD 5 and AMD 6 building blocks were substituted
by L-leucine (Figure 2). Using HATU as the coupling
reagent with DIPEA in DMF, the synthesis of analogue
19 proceeded smoothly until the point where peptide bond
formation between two Aib residues proved problematic
affording an unknown byproduct in 50% yield. Gratify-
ingly, replacement of HATU with tetramethylfluorofor-
mamidiniumhexafluorophosphate(TFFH) atthispoint in
Figure 2. Analogue of culicinin D 19 where AHMOD 5 and
AMD 6 are both substituted by L-leucine.
SPPS of culicinin D (1) itself began with attachment
of Fmoc-protected (S)-APAE 20 (see the Supporting
Information) onto 2-chlorotrityl-functionalized amino-
methyl polystyrene resin16 (Scheme 4). Although APAE-
loaded resin 21 was only obtained with a loading yield of
23%,17 SPPS of culicinin D (1) from resin 21 proceeded
uneventfully initially using HATU/DIPEA to effect pep-
tide bond formation between the appropriate building
blocks including AHMOD 5a and AMD 6. TFFH/
DIPEA was used to effect the difficult coupling between
peptidyl resin 22 and sterically hindered Fmoc-Aib-OH
forming peptidyl resin 23. SPPS was continued using
HATU/DIPEA to effect coupling of subsequent amino
acids. The final L-proline residue was precoupled to butyric
acid before incorporation into the solid-phase synthesis of
culicinin D (1).18
(16) (a) Quibell, M.; Packman, L. C.; Johnson, T. J. Am. Chem. Soc.
1995, 117, 11656–11668. (b) Harris, P. W. R.; Yang, S. H.; Brimble,
M. A. Tetrahedron Lett. 2011, 52, 6024–6026.
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L.; Subra, G.; Marinez, J.; Amblard, M. Angew. Chem., Int. Ed. 2010, 49,
117–120. (b) Rizzi, L.; Cendic, K.; Vaiana, N.; Romeo, S. Tetrahedron
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5401–5402. (b) Carpino, L. A.; Beyermann, M.; Wenschuh, H.; Bienert,
M. Acc. Chem. Res. 1996, 29, 268–274.
(18) Xiang, D. F.; Patskovsky, Y.; Xu, C.; Fedorov, A. A.; Fedorov,
E. V.; Sisco, A. A.; Sauder, J. M.; Burley, S. K.; Almo, S. C.; Raushel,
F. M. Biochemistry 2010, 49, 6791–6803.
Org. Lett., Vol. XX, No. XX, XXXX
C

Scheme 4. SPPS of the Postulated Structure of Culicinin D (1) and the Revised Structure of Culicinin D 24
The epimer of culicinin D (1), namely culicinin D 24, was
also synthesized in a similar manner incorporating AHMOD
5binto the SPPS toenableabsolute stereochemistry assign-
ment at C-6 of the AHMOD moiety in the natural product.
The formation of both benzyl-protected culicinin D 25
(using AHMOD 5a) and 26 (using AHMOD 5b) was
confirmed by MS analysis of a sample obtained after resin
cleavage thus confirming that the β-hydroxyketone motif
in the AHMOD unit was intact at this stage.
Pleasingly, brief exposure ofbothpeptaibols to1% TFA
in CH2Cl2 during the resin cleavage step only caused
negligible degradation of the sensitive β-hydroxy ketone
motif in the AHMOD residues. Finally, the benzyl group
in the APAE residues of 25 and 26 was removed by
hydrogenolysis over 10% Pd/C in MeOH to successfully
afford culicinin D peptaibols 1 and 24 in >95% purity
after purification by semipreparative HPLC. Comparison
of the 1H NMR data of the synthetic postulated structure
of culicinin D (1) and its epimer 24 with the data reported
for the natural product (see the Supporting Information)
confirmed that the absolute chirality at C-6 in the AHMOD
moiety of the natural product agreed with the epimeric
culicinin D structure 24 in which the stereochemistry at C-6
in the AHMOD residue was in fact (R).19
In summary, both Fmoc-protected AHMOD 5a and 5b
and Fmoc-protected AMD 6 were efficiently synthesized
from a common intermediate. The syntheses of these
building blocks together with APAE 20 enabled the first
SPPS of the postulated structure of the potent anticancer
peptaibol culicinin D (1) and the polypeptide 24 epimeric
at C-6 in the AHMOD unit. Comparison of the 1H NMR
data for the synthetic postulated structure of culicinin D
(1) and its epimeric peptaibol 24 with the data reported for
the natural product established that the C-6 stereochem-
istry in the AHMOD moiety in the natural product is in
fact (R). The synthetic work reported herein establishes
that the structure of culicinin D should be revised to
structure 24 with (6R) stereochemistry in the AHMOD
residue.
Acknowledgment. We acknowledge financial support
from the University of Auckland (K.-y.H.) and the Maur-
ice Wilkins Centre for Molecular Biodiscovery.
Supporting Information Available. Experimental pro-
cedures and spectroscopic data for all compounds pre-
pared. This material is available free of charge via the
Internet at http://pubs.acs.org.
(19) An authentic sample of the natural product could not be
obtained for comparison.
The authors declare no competing financial interest.
D
Org. Lett., Vol. XX, No. XX, XXXX