Total synthesis of chloptosin: A dimeric cyclohexapeptide

Article abstract of DOI:10.1002/chem.201003216

Here we describe in full our investigations into the synthesis of the dimeric cyclohexapeptide chloptosin in 17 linear steps. Particularly, this work features an organocatalytic tandem process for the synthesis of the embedded piperazic acids, in which a differentially protected azodicarboxylate is used together with pyrrolidinyl tetrazole as the catalyst. The central biaryl bond is being formed by Stille coupling of two sterically demanding ortho-chloropyrroloindole fragments. The inherent flexibility of the synthetic strategy proved beneficial as the route could be adjusted smoothly during the progression of the synthesis programme. Copyright

Full text of DOI:10.1002/chem.201003216

FULL PAPER
DOI: 10.1002/chem.201003216
Total Synthesis of Chloptosin: A Dimeric Cyclohexapeptide
Alexander J. Oelke,[a] Francesca Antonietti,[a] Leonardo Bertone,[a]
Philippa B. Cranwell,[a] David J. France,[a] Rebecca J. M. Goss,[b] Tatjana Hofmann,[a]
Stephan Knauer,[a] Steven J. Moss,[c] Paul C. Skelton,[a] Richard M. Turner,[a]
Georg Wuitschik,[a] and Steven V. Ley*[a]
Abstract: Here we describe in full our
investigations into the synthesis of the
dimeric cyclohexapeptide chloptosin in
17 linear steps. Particularly, this work
features an organocatalytic tandem
process for the synthesis of the embed-
ded piperazic acids, in which a differen-
tially protected azodicarboxylate is
used together with pyrrolidinyl tetra-
zole as the catalyst. The central biaryl
bond is being formed by Stille coupling
of two sterically demanding ortho-
chloropyrroloindole fragments. The in-
herent flexibility of the synthetic strat-
egy proved beneficial as the route
could be adjusted smoothly during the
progression of the synthesis pro-
gramme.
Keywords: cyclopeptide
· natural
products · organocatalysis · piperaz-
ic acid · total synthesis
Introduction
Chloptosin (1; Figure 1) is a dimeric cyclohexapeptide that
was isolated from the culture broth of the Streptomyces
strain MK498-98F14,[1] which also produces polyoxypeptins
A and B.[2] It is reported to exhibit apoptotic activity in the
apoptosis-resistant human pancreatic adenocarcinoma cell
line AsPC-1 (with an EC50 of 1.6 mm after 24 h) as well as in
several apoptosis-sensitive cancer cell lines. Apoptosis is the
process of programmed cell death (PCD) that occurs in mul-
ticellular organisms and is often associated with characteris-
tic morphological and biochemical changes. In this regard,
chloptosin was found to induce nuclear fragmentation after
24 h and internucleosomal DNA fragmentation after 18 h at
2–6 mm in AsPC-1 cells, thereby indicating the induction of
apoptosis. In addition, chloptosin exhibits strong antimicro-
bial activity against Gram-positive bacteria, such as Staphy-
lococcus, Micrococcus, and Bacillus strains (MIC range 130–
Figure 1. Structure of chloptosin (1).
510 nm) including methicillin-resistant Staphylococcus
aureus (MRSA), but its LD50 value was relatively high (50–
100 mgkgÀ1) to the four-week-old ICR male mice.[1] As de-
fective apoptotic processes often play an essential role in
many diseases, including cancer, and since solid tumours
cells are often resistant to apoptosis, chloptosin has become
an interesting target for drug development. Furthermore,
chloptosin may be an excellent molecular tool for apoptosis
mechanism studies.
In terms of structure, chloptosin (1) is dimeric with a
biaryl linkage connecting two identical cyclic hexapeptide
subunits.[3] These cyclic hexapeptides contain, in alternating
enantiomeric forms, exclusively non-proteinogenic amino
acids including the unusual (R)- and (S)-piperazic acids as
well as a 6-chloropyrroloindole residue.
Piperazic acids such as 6 (Scheme 1) are cyclic amino
acids that contain a hydrazine unit within the six-membered
ring. The first examples of these molecules were isolated by
Hassal from the monamycin family of natural products in
the late 1960s.[4] More recently, a variety of piperazic acid-
containing natural products have been prepared by total
synthesis, namely sanglifehrin by Nicolaou and Paquette,[5]
members of the luzopeptin family by Boger and Ciufolini,[6]
several members of the azinothrycin family and precursors
for monamycin by Hale,[7] piperazimycin by Ma[8] and hi-
[a] Dr. A. J. Oelke, Dr. F. Antonietti, L. Bertone, P. B. Cranwell,
Dr. D. J. France, Dr. T. Hofmann, Dr. S. Knauer, P. C. Skelton,
Dr. R. M. Turner, Dr. G. Wuitschik, Prof. Dr. S. V. Ley
Department of Chemistry
University of Cambridge
Lensfield Road, Cambridge, CB2 1EW (UK)
Fax : (+44)1223-336-442
E-mail: svl1000@cam.ac.uk
[b] Dr. R. J. M. Goss
Department of Chemistry, University of East Anglia
Norwich, NR4 7TJ (UK)
[c] Dr. S. J. Moss
Biotica Technology Ltd.
Chesterford Research Park
Cambridge CB10 1XL (UK)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201003216.
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Scheme 1. Synthetic strategy (Boc=tert-butoxycarbonyl; TBS=tert-butyldimethylsilyl; Troc=2,2,2-trichloroethyloxycarbonyl).
mastatin by Danishefsky.[3] Although himastatin is structur-
ally related to chloptosin and possesses a central dimeric
pyrroloindole core, the surrounding cyclic peptide back-
bones exist as depsipeptides and contain hydroxyisovaleric
acid instead of one of the piperazic acid units. In his synthe-
sis, Danishefsky chose to assemble the molecule by elabora-
tion of a dimeric pyrroloindole core.
tion conditions of metal-catalysed C C bond forming pro-
cesses developed in more recent years would allow this
transformation to proceed on a fully functionalised chlopto-
sin monomer. Should this route fail, we anticipated that
using an extension of Danishefskyꢁs work on himastatin,
early dimerisation of a 6-chloropyrroloindole core fragment
followed by bi-directional peptide assembly would offer an
alternative approach.[3,11] Further disconnection of the mo-
nomer reveals a pyrroloindole unit 2 and non-proteinogenic
amino acid derivatives 3, 4, 5, 6, and ent-6. The pyrroloin-
dole core could be prepared by a selenocyclisation of a 6-
chlorotryptophan derivative,[12] which could, in turn, be ob-
tained using a Chen annulation.[13] Annulation precursors 8
and 9 are readily obtained from commercially available ma-
terials.[14] Although d-valine 3, d-threonine 4 and O-methyl
l-serine 5 are non-proteinogenic, they are nonetheless avail-
able commercially. The piperazic acids 6 and ent-6, however,
represent a significant synthetic challenge and our primary
interest lay in their selective synthesis. Although several
methods for their preparation have been reported in the lit-
erature,[15,16] only a few use catalytic enantioselective meth-
ods to impart the stereochemical information. In addition, it
would be desirable if the piperazic acids were obtained in
an orthogonally protected fashion such that they could be
used directly in the synthesis programme. This was anticipat-
ed to proceed using organocatalytic methods, which have
been developed previously in our group.[17] In contrast to
our previous work that utilised symmetric azodicarboxylates,
this new strategy necessitates a regioselective functionalisa-
tion of a differentially substituted azodicarboxylate 11 with
aldehyde 12 promoted by the tetrazole catalyst 10.[18]
À
Since the disclosure of the structure in 2000[1] chloptosin
has received significant attention from the synthesis com-
munity and several synthetic approaches have been pub-
lished.[9] However, it was not before 2010 that the first syn-
theses of this interesting molecule were finally completed.[10]
In early 2010, the Yao group reported a total synthesis of
chloptosin that was based on their previous work towards
the central dimeric bis-pyrroloindole amino acid present in
the molecule.[9c] In this work, the central biaryl bond was
formed at the earliest possible stage by a benzidine rear-
rangement following reduction of m-chloronitrobenzene. N-
Alkylation using a serine derivative installed the stereocen-
tre and a sequence of Heck reaction and oxidative cyclisa-
tion furnished the initial bis-pyrroloindole core fragment.
Here we describe fully the outcome of a number of strat-
egies for the preparation of chloptosin (1), ultimately lead-
ing to a successful completion of the synthesis.
Synthetic strategy: Our initial approach to chloptosin (1)
utilises the inherent C2 symmetry along the central biaryl
linkage, as this would allow late-stage dimerisation by cross-
coupling reaction between an aryl iodide fragment and an
organometal derivative (Scheme 1). It was hoped that the
well-known high functional group tolerance and mild reac-
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Total Synthesis of Chloptosin
FULL PAPER
Our initial coupling approach then focussed on the con-
nection between the pyrroloindole N-terminus and the pen-
tapeptide C-terminus, and subsequent macrolactamisation
between the pyrroloindole C-terminus and the remaining
valine N-terminus (steps 1 and 2, boxed in Scheme 1). Con-
version of the aryl iodide to a corresponding organostan-
nane (step 3), followed by Stille coupling (step 4), and
global deprotection (step 5) would then complete the syn-
thesis of chloptosin. Although it was reasoned that this ap-
proach would be the most attractive way to the natural
product, it was noticed that, with appropriate peptide frag-
ments available, macrocyclisation could be carried out be-
tween all other amino acid residues, apart from the two dif-
ficult-to-form N-terminal piperazic acid bonds marked by
red boxes, thus potentially greatly extending the synthesis
programme (cf. Scheme 1).
was reproducible up to a scale of about 1 g. Unfortunately,
on further scale-up, all attempts to achieve similar yields
and selective product formation remained unsuccessful and
a complex, inseparable mixture of starting material together
with reduction products of both esters to the aldehyde and
alcohol oxidation levels was obtained.
The annulation reaction proceeded with reasonable yield
using conditions reported by Zhu[13b] via initial imine forma-
tion between aniline 8 and aldehyde 9, tautomerisation to
the enamine and intramolecular Heck reaction. Tryptophan
15 was then converted into its N,N’-di-Boc-protected conge-
ner 16 by a simple deprotection-reprotection sequence. This
reaction also suffered from low yields on scale-up (more
than 500 mg 15), predominantly resulting from unselective
Boc removal. At this stage, it was recognised that the multi-
gram quantities of tryptophan 16 that were likely to be re-
quired for the total synthesis would not be accessible by this
route. Because of the problems encountered in the selective
reduction of 14 to 9, and conversion of 15 to 16, an alterna-
tive strategy was sought to provide 16 in fewer steps and
higher yield.
The second approach for the preparation of 6-chlorotryp-
tophan derivative 16 employed 6-chloroindole 17 and l-
serine as commercially available starting materials, and uti-
lised an enantioselective, pyridoxal phosphate dependent
biotransformation. For this transformation, a lysate was pre-
pared from commercially available Escherichia coli pre-
transformed with pSTB7, a high copy number plasmid ex-
pressing tryptophan synthase from Salmonella enterica.[20]
The biotransformation reaction itself proceeded well on
scale, furnishing desired 6-chloroptyptophan (18) in moder-
ate, but scalable 35% yield (Scheme 3). In the final scale-up
Results and Discussion
Preparation of the pyrroloindole core: The first subgoal on
the pyrroloindole synthesis was the preparation of an appro-
priately protected 6-chlorotryptophan derivative, which
would serve as precursor for subsequent selenocyclisation
(16, Scheme 2).[19] Thus, aniline 8 was prepared from com-
Scheme 2. First route to 6-chlorotryptophan 16. a) NaNO2, H2SO4,
H3PO4, 08C to RT, then KI, RT, 90%; b) Fe powder, EtOH/AcOH 1:1,
1008C, 85%; c) TMSCl, MeOH, RT; d) Boc2O, Et3N, MeOH, 08C; e)
Boc2O, DMAP, MeCN, RT, 86% over 3 steps; f) DIBAL-H, Et2O,
À788C, variable yield, up to 91%; g) DABCO, Pd
ACHTUNGRTEN(NGNU OAc)2, molecular
sieves 4 ꢂ, DMF, 858C, 66%; h) HCl (1.0m in Et2O), Et2O, RT; i) Boc2O,
NaOH, nBu4N·HSO4, CH2Cl2, RT, 75% over 2 steps (DABCO=1,4-
Scheme 3. Second route to 6-chlorotryptophan 16. a) Cell lysate contain-
ing tryptophan synthase, l-serine, pyridoxal phosphate, KH2PO4, H2O,
diazabicycloACHTUNGTRENNUNG[2.2.2]octane; DIBAL-H=diisobutylaluminium hydride;
DMAP=N,N-dimethylaminopyridine; DMF=N,N-dimethylformamide;
TMS=trimethylsilyl).
RT, 35%; b) SOCl2, MeOH,
Bu4N·HSO4, CH2Cl2, RT, quant.
0 to 658C, 99%; c) Boc2O, NaOH,
mercially available 4-chloro-2-nitroaniline (13) in two
steps.[14c] First, the amine group was converted into the aryl
iodide via a standard Sandmeyer protocol. This was fol-
lowed by nitro group reduction using iron in acetic acid, af-
fording the desired aniline 8 in high yield and on syntheti-
cally useful multigram scale. For the synthesis of the re-
quired aldehyde annulation partner 9, a procedure initially
described by Martin et al. was used.[14a] Following methyl
esterification and double Boc protection of the amine func-
tionality of l-glutamic acid 14, the side-chain methyl ester in
the corresponding intermediate was selectively reduced to
aldehyde 9 using DIBAL-H. This reaction worked well and
phase, a biotransformation was carried out on 660 mmol
scale in a 30 L fermenter, which provided 40 g of 18 in a
single reaction. The protocol allowed simple isolation of the
indole starting material by extraction with diethyl ether and
the recovered indole could then be resubjected without any
further purification to another biotransformation reaction.
After three cycles, the combined yield then reached synthet-
ically useful 75% and provided access to multigram quanti-
ties of 18. Unprotected amino acid 18 was then progressed
to selenocyclisation precursor 16 in 99% yield via methyl
esterification and double Boc protection. This short se-
quence then intercepted with the previous route, and again
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S. V. Ley et al.
worked well on multigram scale and reduced the total
number of steps from nine to three.
to the 3,6-dihydropyridazine products by addition of sodium
hydride and vinyltriphenylphosphonium bromide.[21] Mecha-
nistically, base-promoted addition of the secondary carba-
mate of 24 to vinyl phosphonium bromide 23 provided tran-
sient phosphorane 25, which immediately underwent an in-
tramolecular Wittig ring closure to give the desired 3,6-dihy-
dropyridazine 26. In order to permit N-selective functionali-
sation of the piperazic acids, we chose to employ
orthogonally protected azodicarboxylate 11 in the a-amina-
tion–cyclisation protocol (Scheme 5). The Boc and Troc
groups were deliberately chosen to provide both a general
route to piperazic acids and, more importantly, the chlopto-
sin total synthesis. It was thought that by use of these pro-
tecting groups, the electronic environment on the nitrogen
atoms would be different enough to achieve regioselective
attack of the enamine in the organocatalytic a-amination
The next aim was the formation of the pyrroloindole
framework using a selenocyclisation reaction. Based on orig-
inal findings by Danishefsky and co-workers[12a] and previ-
ous work within our group on the synthesis of the okarami-
nes,[12b,c] it was envisaged that the new tryptophan substrates
such as the 6-chloro substituted 16 would undergo diastereo-
selective syn–cis pyrroloindole formation upon treatment
with N-phenylseleno phthalimide (N-PSP). This proved to
be the case and phenylselenopyrroloindole 19 was obtained
in high yield as the only diastereoisomer (Scheme 4). The
Scheme 4. Preparation of pyrroloindole 21. a) N-PSP, PPTS, Na2SO4,
CH2Cl2, RT, 89%; b) m-CPBA, wet CH2Cl2, 08C to RT, 80%; c) TESCl,
DBU, DMF, 508C, 95%; d) ICl, 2,6-di-tert-butylpyridine, CH2Cl2, 558C,
sealed tube, 88% (m-CPBA=meta-chloroperbenzoic acid; DBU=1,8-
diazabicycloACHTUNGTRENNUNG[5.4.0]undec-7-ene; PPTS=pyridinium para-toluene sulfo-
nate; N-PSP=N-phenylseleno phthalimide; TES=triethylsilyl).
syn–cis substitution pattern on the pyrroloindole ring in 19
was assigned based on a similar selenocyclisation reaction of
a 6-chlorotryptophan derivative as reported by Yao.[9c] How-
ever, it was noted that the reaction between 16 and N-PSP
was considerably slower than with its more electron-rich,
unsubstituted tryptophan congener and optimum results
were obtained after a reaction time of six days.
It was then necessary to convert the phenylseleno group
on pyrroloindole 19 into the hydroxyl group that is required
for the natural product. This was achieved by oxidation of
the selenide to the corresponding selenone using m-chloro-
perbenzoic acid in wet dichloromethane as solvent. The hy-
droxyl group was then protected as its TES ether 20 upon
treatment with an excess of TES chloride and DBU. Finally,
the aryl iodide was installed by the action of iodine mono-
chloride.[3c] The low reactivity of 20 was overcome by heat-
ing 30 equivalents of both iodine monochloride and 2,6-di-
tert-butylpyridine together with 20 in a sealed tube for 48 h.
A new organocatalytic route to differentially protected pi-
perazic acids: Recently, we have developed an organocata-
lytic one-pot procedure for the enantioselective synthesis of
3,6-dihydropyridazines from achiral aldehydes and ketones
using the tetrazole catalyst 10.[17a,b] In this sequence, an or-
ganocatalytic a-amination reaction between an aldehyde
(such as 12) and an azodicarboxylate (such as 11) furnished
hydrazino aldehyde 24. This intermediate was then cyclised
Scheme 5. Organocatalytic preparation of piperazic acid building blocks.
a) TrocCl, NMM, THF, 08C to RT; b) NBS, pyridine, toluene, RT, quant.
(2 steps); c) 10, CH2Cl2, À58C, then 23, NaH, THF, À58C to RT, 90%,
e.r. 92:8; d) PtO2, H2 (10 bar), THF, RT, 98%; e) HCl (4m in dioxane),
CH2Cl2, THF, 08C to RT; f) NaIO4, RuCl3·nH2O, H2O, acetone, MeCN,
RT, 94% (2 steps); g) TESOTf, 2,6-lutidine, CH2Cl2, 08C to RT, 94%; h)
Zn, NaH2PO4, THF, H2O, RT, 99%; i) (PhSeO)2O, THF, RT, 85%
(NBS=N-bromosuccinimide; NMM=N-methylmorpholine; Tf=tri-
fluoromethanesulfonyl; THF=tetrahydrofuran).
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Total Synthesis of Chloptosin
FULL PAPER
step. The starting material was prepared in quantitative
yield in two steps from tert-butyl carbazate by Troc protec-
tion and oxidation. More importantly, when 11 was subject-
ed to the a-amination conditions, hydrazino aldehyde 24
was formed in high yield and e.r. as the sole product from
the reaction mixture by regioselective attack on the Boc-
protected nitrogen atom. ent-26 was obtained in similar
yield and e.r. by using corresponding tetrazole catalyst ent-
10.
Hydrogenation of the double bond present in 26 using
Crabtreeꢁs catalyst[22] gave key intermediate 27 in high yield.
The functional groups present in 27 could be selectively un-
masked as required for further conversion to piperazic acid
6. Treatment of 27 with hydrochloric acid led exclusively to
cleavage of the silyl protecting group in the presence of the
tert-butyl carbamate, and, after oxidation using catalytic
ruthenium in the presence of sodium periodate, provided 6,
the required precursor for C-terminal peptide couplings. Re-
crystallisation of protected piperazic acids 6 and ent-6 fur-
ther improved the enantiomeric purity to >200:1.
Our initial route to prepare piperazic acid 6 involved a
number of shortcomings that limited throughput on multi-
gram scales. Among these were the cost of Crabtreeꢁs hy-
drogenation catalyst (step d in Scheme 5), and the low over-
all yield as a result of the final recrystallisation of the piper-
azic acid 6. A screening of heterogenous hydrogenation cat-
alysts revealed that platinum oxide under 10 bar of hydro-
gen pressure would also effect the desired transformation
Scheme 6. First route to tripeptide 37 and formation of carboxy anhy-
dride 38. a) Boc2O, NaHCO3, H2O, MeOH, RT, quant.; b) allyl bromide,
K2CO3, DMF, 08C to RT, 93%; c) TFA, toluene, 08C, quant.; d) HATU,
iPr2NEt, DMF, 08C to RT, 99%; e) Ghosezꢁ reagent 40, benzene, 58C to
RT; f) TFA, toluene, 08C to RT, 91%; g) AgCN, benzene, 808C, yield
variable, up to 35% (70% brsm); h) Ghosezꢁ reagent 40, CH2Cl2, 58C to
RT; i) MeOH, RT, 88% (HATU=2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate; TFA=trifluoroacetic acid).
À
while leaving the Troc carbamate and the N N bond un-
touched. The yield of the final recrystallisation of 6 was im-
proved by conducting the crystallisation over a 12 h time
period with slow temperature gradient using a Web-cam
monitoring device.[23]
Preparation of the corresponding N-terminal deprotected
precursor 28 could also be accomplished using a mixture of
TES triflate and 2,6-lutidine,[24] to effect selective removal of
the Boc group in the presence of the TBS ether. Although
not en route to chloptosin (1), it was also possible to cleave
the Troc group of 27 using zinc in buffered aqueous tetrahy-
drofuran,[25] providing naturally occurring piperazinic acid
derivative 29 after C=N oxidation with benzene seleninic
anhydride.[26] Each manipulation of hydrogenated intermedi-
ate 27 proceeded in excellent yield with no loss of enantio-
meric purity and without affecting the remaining protecting
groups present. All reactions could provide multigram quan-
tities of the requisite piperazic acid derivatives required for
the projected total synthesis.
to di-protected derivative 32 via standard conditions, namely
Boc protection and formation of the allyl ester by treatment
with allyl bromide and potassium carbonate. Coupling of 33
with acid ent-6 then furnished dipeptide 34 in nearly quanti-
tative yield as a single diastereoisomer. Having dipeptide 34
À
in hand, the stage was set for the crucial piperazic acid
piperazic acid bond-forming event. In the initial screening
of conditions, amine 36 was reacted together with acid 6 and
a range of standard peptide coupling reagents, such as
HATU, PyBrOP, TOTU or DPPA.[27] Unfortunately, no con-
version of the amine could be detected under these condi-
tions. Activation of 6 by formation of its acid fluoride also
did not lead to peptide coupling. Eventually, conditions sim-
ilar to those reported by Hale were applied,[7c] and indeed,
heating amine 36 in a mixture with acid chloride 35 and
silver cyanide afforded tripeptide 37 in variable low to mod-
erate yield. When this reaction was carried out on a scale
greater than 0.1 mmol, the yield was generally between 10
and 25% and all attempts to improve the outcome were un-
successful. Since amine 36 could be recovered from the reac-
tion mixture, the low yield was attributed to decomposition
Towards the pentapeptide fragment: Since the route devel-
oped previously for the synthesis of piperazic acid deriva-
tives (see above) gave access to enantiomerically pure C-ter-
minally unmasked piperazic acids 6 and ent-6 by recrystalli-
sation, our approach was to employ both enantiomers 6 and
ent-6 for the assembly of the pentapeptide fragment
(Scheme 6).
In order to prepare the required protected serine deriva-
tive 33, commercial l-serine methyl ether 30 was progressed
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S. V. Ley et al.
of acid chloride 35 during the reaction. When acid chloride
35 (prepared in situ) was stirred at ambient temperature for
one hour before being quenched by addition of methanol,
formation of amine 39 was detected. In order to explain this
surprising outcome of the reaction, a reaction pathway pro-
ceeding via N-carboxyanhydride 38 was proposed and later
verified by isolation of 38 through evaporation of the vola-
tiles without addition of methanol.
Given the fact that formation of 38 could not easily be
prevented under the necessary reaction conditions, it was
clear that access to larger quantities of tripeptide 37 would
require a different approach. Nevertheless, the material
present at this stage could be used to progress further in the
proposed synthesis of pentapeptide fragment 49. Peptide
chain 37 was further elongated after Boc removal by cou-
pling with Troc-d-valine using the optimised acid chloride/
silver cyanide conditions reported previously and tetrapep-
tide 44 was obtained in 62% yield (Scheme 7). The Troc
protecting group on the valine residue was selected to facili-
tate later global amine deprotection and also to prevent for-
mation of an N-carboxy anhydride analogous to 38. Finally,
acid 45 was liberated upon treatment of 44 with palladi-
um(0) and morpholine, and the resulting tetrapeptide 45
then subjected to HATU mediated coupling with d-
In summary, pentapeptide 49 was successfully prepared
using enantiomerically pure recrystallised piperazic acid in
both enantiomeric forms and no epimerisation was detected
at any stage along the synthetic pathway. However, due to
the low-yielding peptide coupling step between the two pi-
perazic acids, other possible routes towards pentapeptide 49
were investigated and finally, one based on initial piperazic
À
acid piperazic acid coupling was selected as the optimum
procedure.
Alternative preparation of the pentapeptide fragment: This
alternative strategy based on initial union of the two piper-
azic acids present in the pentapeptide fragment 49 forges
the difficult-to-install bond at an early stage in the synthesis
and makes use of the higher reactivity of aminoalcohol ent-
28 as N-terminal coupling partner (Scheme 8).[16b] While
threonineACHTUNGTRENNUNG(TBS) allyl ester 48 to afford pentapeptide frag-
ment 49.
Scheme 8. Second route to tripeptide 37. a) Ghosezꢁ reagent 40, CH2Cl2,
08C; b) pyridine, CH2Cl2, 08C to RT, 70%; c) HF/pyridine, pyridine, 08C
to RT; d) RuCl3, NaIO4, acetone, H2O, RT, 76% (2 steps); e) H-l-
Ser(Me)-Oallyl, HATU, iPr2NEt, DMF, 08C to RT, 74%.
acid 6 was used in enantiomerically pure form, the coupling
partner amine ent-28 was only available in 92:8 e.r. Howev-
er, based on Horeauꢁs principle,[28] resulting dipeptide 50
was expected to be enantiomerically pure and consequently
this strategy only necessitated the efficient isolation of de-
sired diastereoisomer 50. Further functionalisation of 50 via
the described deprotection-oxidation sequence (cf.
Scheme 5) and peptide coupling with an appropriate serine
derivative would then intercept with the previous route and
deliver tripeptide 37.
While early studies with standard peptide coupling re-
agents were rather disappointing, initial results using piper-
azic acid chloride 34 in conjunction with a base were encour-
aging, and eventually, the optimised conditions depicted in
Scheme 8 were developed. Keeping the temperature of the
reaction below ambient effectively suppressed formation of
N-carboxy anhydride 38, and gave the desired dipeptide 50
in 71% yield. Moreover, due to a substantial difference in
polarity between 50 and its undesired (S,S)-diastereoisomer,
Scheme 7. Preparation of pentapeptide fragment 49. a) Ghosezꢁ reagent
40, CH2Cl2, 08C; b) TFA, toluene, 08C to RT, 81%; c) AgCN, benzene,
808C, 62%; d) [PdACHTUNGTRENNUNG(PPh3)4], morpholine, RT, 85%; e) Boc2O, NaOH,
H2O, tBuOH, RT; f) TBSOTf, Et3N, CH2Cl2, 08C to RT, 75% (2 steps);
g) allyl bromide, K2CO3, DMF, 08C to RT, 78%; h) TFA, toluene, 08C,
quant.; i) HATU, iPr2NEt, DMF, 08C to RT, 70%.
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Total Synthesis of Chloptosin
FULL PAPER
dipeptide 50 was obtained cleanly by standard flash column
chromatography. This reaction was also scalable to deliver
multigram quantities of dipeptide 50 with just a minor de-
crease in yield (50% on 10 mmol scale). Dipeptide 50 was
then converted into tripeptide 37 using the conditions devel-
oped earlier, namely TBS removal, oxidation of the result-
ing alcohol to the carboxylic acid and peptide coupling with
serine derivative 33 to afford 37 in 25–36% yield from pi-
perazic acids 6 and ent-28 and thus intercepted with the pre-
vious route.
would then provide further insight into the undesired side
reaction.
Accordingly, Fmoc-protected valine 55 was subjected to
the peptide coupling conditions employed earlier, and
Fmoc-protected tetrapeptide 57 was obtained in 63% yield
(Scheme 10). This intermediate was then further advanced
Assembly of chloptosin monomer I: With both the pyrro-
loindole fragment 21 and pentapeptide 49 in hand, it was
the time to investigate fragment union, macrocyclisation and
ultimately the envisaged dimerisation of the fragments. In-
vestigations therefore began with liberation of the C-termi-
nus of pentapeptide fragment 49, which proceeded smoothly
upon treatment with palladium(0) and morpholine, and re-
moval of the Boc protecting groups from pyrroloindole 21
(Scheme 9). Fragment union between acid 51 and amine 52
Scheme 10. Preparation of pentapeptides 58 and 59 and fragment union.
a) Ghosezꢁ reagent 40, benzene 58C to RT; b) AgCN, benzene, 808C,
63%; c) [Pd
ACHTUNGTRENNUNG
G
PhSiH3, CH2Cl2, 08C to RT, 54%; f) 52, HATU, HOAt, collidine,
CH2Cl2, À108C to RT, 55%.
via removal of the allyl ester, coupling with threonine deriv-
ative 48 and again, allyl ester removal. As a result of an
Fmoc protecting group being part of the assembled mole-
cule, conditions for allyl ester removal had to be adjusted,
such that morpholine was replaced by phenyl silane. Some-
what surprisingly, this in turn led to partial loss of the threo-
nine TBS protecting group and ultimately an inseparable
mixture of 58 and 59 was obtained. The mixture was none-
theless used for the fragment union with pyrroloindole 52,
affording hexapeptides 60 and 61 ready for Fmoc deprotec-
tion. However, after treatment with piperidine, the desired
free valine N-terminus was not isolated but instead, analysis
of the reaction mixture by high resolution mass spectrome-
try (HRMS) showed loss of trichloroethanol, possibly result-
ing from attack of the free primary valine amine formed in
the deprotection step into the Troc group.
Scheme 9. Fragment union. a) [PdACHTNUGRTNEUNG(PPh3)4], morpholine, RT, 82%; b)
TFA, 08C, 98%; c) HATU, HOAt, collidine, CH2Cl2, À108C to RT, 60%
(HOAt=1-hydroxy-7-azabenzotriazole).
was achieved by the action of HATU as coupling agent, af-
fording the expected linear hexapeptide 53 in an inseparable
mixture with its des-TBS-congener 54. Treatment with lead–
cadmium couple[29] then effected simultaneous removal of
all three Troc protecting groups, which was required for the
desired cyclisation to take place. Unfortunately, the corre-
sponding free amine was found to oxidise spontaneously
upon exposure to air.[5d,16b] In view of this result, it was de-
cided to introduce an orthogonally Fmoc-protected valine
derivative into the pentapeptide. Selective removal of either
the valine or the piperazic acid carbamate protecting groups
It was reasoned that this side reaction could only be sur-
mounted by cleavage of the Troc protecting groups on both
piperazic acid residues. Although 62 could be converted
cleanly to the corresponding des-Troc fragment 63
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4189

S. V. Ley et al.
action between threonine and serine would benefit from
greater reactivity of the coupling partners.
Assembly of chloptosin monomer III: Starting from tetra-
peptide fragment 57 and pyrroloindole fragment 21, two
routes to hexapeptide 70 were feasible: following saponifica-
tion of methyl ester 21, peptide coupling with tetrapeptide
69 would furnish a pentapeptide intermediate, which would
then be advanced to 70 via Boc removal and coupling with
Boc-ThrACHTNUTRGNE(NUG TBS)-OH. Alternatively, a pyrroloindole-threonine
Scheme 11. Protecting group manipulations on pentapeptide fragment 62.
a) PbCd couple, NH4OAc, THF, H2O, RT; b) 10% piperidine in DMF
(degassed), RT, quant. (2 steps).
dipeptide could be converted into 70 by saponification of
the methyl ester and coupling with tetrapeptide 69, and
studies along both these pathways were investigated
(Scheme 13).
(Scheme 11), the ensuing Fmoc removal once again led, ini-
tially, to a difficult to characterise oxidised product as de-
tected by HRMS. Although oxidation could be suppressed
by degassing both solvent and reagent prior to their use and
giving crude 64 by removal of the volatiles in vacuo, the pro-
pensity of the substrate for oxidation led us to consider an
initial coupling between the pyrroloindole C-terminus and
valine N-terminus, and ring closure was then expected to
occur between the pyrroloindole core and threonine (cf.
Synthetic Strategy, Scheme 1, steps 1 and 2 in reversed
order).
With regard to the first route, saponification of 21 pro-
ceeded as expected and coupling of the resulting acid with
tetrapeptide 69 using PyBrOP afforded the linear pentapep-
tide intermediate in 91% yield. Cleavage of the Boc pro-
Assembly of chloptosin monomer II: The desired fragment
union between pyrroloindole 65 and pentapeptide 64 pro-
ceeded well with activation by PyBrOP and afforded hexa-
peptide 66 and its des-Boc-derivative 67 as a mixture
(Scheme 12). The allyl ester was then removed from 66 and
67 in the presence of the aryl halide functionalities using a
palladium(0)/morpholine mixture, and the remaining Boc
groups were removed upon treatment with TFA.
All attempts to effect the cyclisation reaction of 68 under
a variety of conditions were unsuccessful, presumably due to
the relatively low reactivity of the secondary nitrogen atom
of the pyrroloindole and competing degradation. In order to
circumvent this problem, we reasoned that a cyclisation re-
Scheme 13. Third fragment union approach and successful macrocyclisa-
tion. a) PbCd couple, NH4OAc, THF, H2O, RT; b) 10% piperidine in
DMF (degassed), RT, quant. (2 steps); c) LiOH, THF, MeOH, H2O, RT,
97%; d) 69, PyBrOP, iPrN2Et, DMF, 08C to RT, 91%; e) TFA, toluene,
08C to RT; f) Boc-Thr
ACHTUNGTRENNUNG
RT, 52% (2 steps); g) TFA, 08C; h) Boc-ThrACHTUNGTRENNNUG
collidine, 08C to RT, 85% (2 steps); i) LiOH, H2O, THF, MeOH, RT; j)
69, PyAOP, HOAt, iPrN2Et, CH2Cl2, 08C to RT, 54% (2 steps); k)
Scheme 12. Alternative fragment union approach. a) LiOH, THF,
MeOH, H2O, RT, 92 %; b) 64, PyBrOP, iPr2NEt, CH2Cl2, 08C to RT,
[PdACHTGNUETRN(NUG PPh3)4], morpholine, MeCN, 08C; l) TFA, toluene, 08C to RT; m)
95%; c) [Pd
(PPh3)4], morpholine, MeCN, RT; d) TFA, toluene, 08C to
HATU, HOAt, iPrN2Et, DMF, 08C to RT, 79% (3 steps); n) HF/pyri-
dine, pyridine, 08C to RT, 44% (PyAOP=(7-azabenzotriazol-1-yl-oxy)-
tripyrrolidinophosphonium hexafluorophosphate).
RT, quant. (2 steps) (PyBrOP=bromotripyrrolidinophosphonium hexa-
fluorophosphate).
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Total Synthesis of Chloptosin
FULL PAPER
tecting groups occurred without incident and coupling with
Boc-ThrACHTUNGTRENNUNG(TBS)-OH then furnished the desired linear mono-
ether and aryl chloride functionality present in 71
(Scheme 15). However, when the important dimerisation re-
action was attempted using the conditions developed for the
pyrroloindole core, we were never able to isolate a dimerisa-
tion product. Reaction monitoring by HRMS suggested that
oxidation of the reaction partners might have occurred
during the reaction conditions although it was not possible
to characterise precisely where this arose in the molecule.
mer 70 in moderate yield. The second route also successfully
led to hexapeptide 70 in similar 46% overall yield. On a
larger scale, quenching and separation of PyBrOP proved to
be difficult, and traces of reagent led to further elimination
of water from 70 presumably via generation of hydrobromic
acid, and even after purification by column chromatography
no improvement was noted. This problem was solved by
using PyAOP as an alternative reagent, which unfortunately
decreased the yield for the coupling step from 67 to 54%.
Linear hexapeptide 70 was converted into the required
cyclisation precursor by a short sequence involving removal
of the allyl ester and Boc group (Scheme 13). The crude ma-
terial obtained was subjected to macrolactamisation condi-
tions, and by using HATU activation, affording cyclic mono-
mer 71 in high yield. Cleavage of the silyl ether then fur-
nished chloptosin monomer 72, which pleasingly showed
many spectral similarities to the natural product as found by
1H and 13C NMR analysis.
Scheme 15. Preparation of stannane 75 and dimerisation attempt. a)
[PdACHTNUGTRNEUNG(PPh3)4], Me6Sn2, toluene, 608C, 80%; b) various cross-coupling con-
ditions, see text.
Dimerisation studies on the pyrroloindole fragment: Having
À
successfully prepared chloptosin monomer 72, the key aryl
aryl bond formation was investigated. Because quantities of
the chloptosin monomer 72 were limited, pyrroloindole 21
was chosen as the test substrate for model studies.
It was thought that oxidation might have happened within
the peptide ring, most probably within the two piperazic
acids. In order to gain further insight into this undesired
side-reaction, tetrapeptide 57 was subjected to the Stille re-
action conditions, where in fact, a double oxidation was de-
tected by mass spectrometric analysis. The source of the oxi-
dation reaction was identified when copper(I) iodide was
added to 57 and the mixture heated in DMF to 1008C for
30 min.[30] Heating of 57 with the other components of the
Stille reagent mix did not result in oxidation. A short
screening of copper sources and reaction conditions was car-
ried out, leading to the conclusion that any copper source
with sufficient heating would lead to oxidation of the tetra-
peptide fragment 57 (Table 1). With the evidence that cop-
per(I) was the cause of the problematic oxidation during the
Stille coupling of monomers 71 and 75, a variety of alterna-
tive Stille coupling conditions were applied, including those
reported by Fu[31] and Fꢃrstner.[32] Unfortunately, none of
those led to any detectable dimerisation: the iodide starting
material was still detected at the end of the reaction period,
while the tin derivative was consumed by conversion into a
variety of palladium-containing intermediates and by hydro-
destannylation.
The Stille reaction was selected as a starting point for
these studies and consequently, organostannane derivative
73 was prepared in moderate yield from pyrroloindole
iodide 21 using tetrakis(triphenylphosphine) palladium(0)
catalyst and hexamethylditin (Scheme 14). Dimerisation by
reaction of iodide 21 with organostannane 73 smoothly af-
forded bis-pyrroloindole 74 in high yield as a mixture of two
atropisomers along the central biaryl bond. The use of mi-
crowave heating for this transformation reduced the reac-
tion time significantly from 12 h to 30 min.
Scheme 14. Dimerisation studies via Stille reaction. a) Me6Sn2,
[PdACHTUNGTRENNUNG(PPh3)4], toluene, 608C, 57%; b) 21, [Pd2dba3], Ph3As, CuI, DMF, mw,
1108C, 56% (dba=dibenzylideneacetone).
Owing to these difficulties in the attempted Stille dimeri-
sation of the chloptosin monomer, formation of the central
biaryl linkage using a Suzuki coupling was explored as an al-
ternative strategy on a variety of pyrroloindole systems
bearing different protecting groups. Attempts to convert
aryl iodide 76 to the corresponding pinacol boronate ester
77 resulted in partial cleavage of the TES ether
(Scheme 16). Attempting to couple pinacol boronate ester
77 with aryl iodide 76 using NaOH as the base allowed cou-
pling at elevated temperature, although yields were relative-
ly low.
Dimerisation attempts on chloptosin monomer: Having
found reliable conditions for the cross-coupling reaction of
6-chloropyrroloindole systems on core fragment 21, work
was then centred on the application of these conditions to
chloptosin monomer 71.
Pleasingly, a transition metal-catalysed reaction trans-
formed iodo-monomer 71 into its trimethyltin analogue 75
despite the plethora of amide, basic amine, alcohol, aminal,
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4191

S. V. Ley et al.
Table 1. Observed oxidation of tetrapeptide fragment 57.
which would then be subjected to the peptide coupling con-
ditions developed on the monomer synthesis to arrive after
cyclisation directly at the natural product (Figure 2).
Entry[a] Cu source
T [8C] tteaction [min] Results
1
2
3
4
5
6
7
CuI
CuI
RT
100
30
30
30
20
30
30
30
no oxidation
double oxidation
double oxidation
no oxidation
mixture
Figure 2. Bi-directional fragment coupling.
CuI, mixture degassed 100
CuI
CuCN
CuTC
CuTC
50
In order to examine this route, Boc-protected dimer 74
was treated with TFA to effect liberation of the amine func-
tionalities. Unfortunately, we were never able to remove all
four Boc groups without substantial degradation. Therefore,
Boc-pyrroloindole 21 was converted into Cbz-congener 76
via amine deprotection and Cbz reprotection (cf.
Scheme 16). Conversion of iodide 76 into stannane 80 and
subsequent Stille reaction furnished Cbz-protected dimer 79
in similar yield, without the need for further optimisation
(Scheme 17). Finally, hydrogenolysis of the Cbz groups
using palladium on charcoal smoothly afforded dimeric pyr-
roloindole 81.
100
RT
100
no oxidation
double oxidation
[a]To a solution of tetrapeptide 57 in DMF in a microwave vial was
added the copper source at ambient temperature. The vial was closed
and stirred at the appropriate temperature for the time stated. A sample
was removed from the reaction mixture and analysed by nanospray high
resolution mass spectrometry (TC=thiophene-2-carboxylate).
Scheme 17. Preparation of dimeric pyrroloindole 81. a) [PdACHTUNGTRENNUNG(PPh3)4],
Me6Sn2, toluene, 558C, 84%; b) 76, [Pd2dba3], Ph3As, CuI, DMF, 1008C
(mw), 94%; c) Pd/C, H2, EtOAc, RT, quant. (Cbz=benzyloxycarbonyl).
Scheme 16. Dimerisation studies via Suzuki reaction. a) TFA, 08C to RT;
b) CbzCl, K2CO3, THF, H2O, RT, 81% (2 steps); c) bis(pinacolato)dibor-
on, [PdCl2dppf], KOAc, DMSO, 808C, 37% 77, 32% 78; d) [PdACHTNUTRGNEUNG(PPh3)4],
NaOH, toluene, H2O, 50 8C, 36% (DMSO=dimethylsulfoxide; dppf=
1,1’-bis(diphenylphosphino)ferrocene).
Dimeric amine 81 was then successfully coupled to threo-
nine derivative 82 by the action of HATU (Scheme 18).
Saponification then formed the free di-acid 84 with simulta-
neous removal of the pyrroloindole TES ether. This material
was converted into linear chloptosin 85 by coupling with tet-
rapeptide 69. In contrast to the corresponding coupling be-
tween the monomer and 69 where PyAOP was used as cou-
pling reagent, we found that in this bi-directional coupling
the best yields were obtained by using BEP activation.[10a]
Removal of the allyl esters was achieved using palladium(0)
and dimethylamine as a volatile nucleophile that could be
used in excess and then removed by evaporation. Following
this protocol allowed us to use the crude material obtained
directly in the subsequent Boc cleavage and macrocyclisa-
tion steps. Finally, a desilylation reaction using buffered
TBAF concluded our synthesis and furnished the natural
product. The synthetic chloptosin obtained via this sequence
was identical in all respects to an authentic sample.[35]
Numerous examples of one-pot dimerisations via in situ
generation of aryl boronate esters, followed by Suzuki cou-
pling may be found in the literature.[33]
Unfortunately, attempts to dimerise chloptosin monomer
71 under a variety of conditions were unsuccessful. Like-
wise, attempts to isolate the intermediate pinacol boranate
ester were unsuccessful. Although literature precedent again
exists for Suzuki couplings in the presence of basic
amines,[34] it may be possible that these functional groups
are responsible for the difficulty encountered in this chal-
lenging cross-coupling reaction.
Bidirectional fragment union: In light of these results from
the dimerisation attempts of chloptosin monomer 71 it was
decided to change the strategy once more and to approach
the natural product from dimerised pyrroloindole core
74.[3,10a] Boc removal should give a dimeric pyrroloindole,
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Total Synthesis of Chloptosin
FULL PAPER
biological testing can be realised that could provide valuable
new information concerning the mechanism of action of this
fascinating molecule.
Experimental Section
A full account on experimental procedures, characterisation data and
spectra for all compounds reported in this manuscript can be found in
the Supporting Information.
Acknowledgements
The authors gratefully acknowledge financial support from the Alexand-
er von Humboldt Foundation (to S.K. and G.W.), the Swiss National Sci-
ence Foundation (to T.H.) and the B.P. Endowment and the EPSRC (to
S.V.L.). The authors would like to thank Dr. J. E. Davies, Cambridge, for
providing a crystal structure, and the Cambridge University Chemical
Laboratory NMR services for their cooperation with the analytical equip-
ment.
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Scheme 18. Completion of the synthesis. a) HATU, HOAt, collidine,
À108C to RT, 94%; b) LiOH, H2O, THF, RT; c) 69, BEP, HOAt,
iPr2NEt, CH2Cl2, DMF, À108C to RT, 56% (2 steps); d) [Pd
ACHTUNGTRNE(NUNG PPh3)4],
Me2NH, THF, 08C to RT; e) TFA, toluene, 08C to RT; f) PyBOP, HOAt,
iPr2NEt, CH2Cl2, DMF, 08C to RT, 43% (3 steps); g) TBAF (1m in
THF), HOAc, THF, 08C to RT, 65% (BEP=2-bromo-1-ethylpyridinium
tetrafluoroborate, PyBOP=(benzotriazol-1-yl-oxy)tripyrrolidinophospho-
nium hexafluorophosphate; TBAF=tetrabutylammonium fluoride).
Conclusion
A total synthesis of chloptosin (1) has been achieved in a
total of 30 steps (17 steps in the longest linear sequence)
and in 4% overall yield from azodicarboxylate 11.
We have used this molecule as a platform to demonstrate
and further improve a range of methods, including a seleno-
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catalytic tandem process for the enantioselective prepara-
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blocks. However, along the way to this synthetic success, we
have been challenged by unexpected fast diketopiperazine
formations, by unwanted oxidation reactions and highly sen-
sitive protecting groups, all of which contrived to force a re-
planning of the synthesis route.
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Received: November 8, 2010
Published online: March 15, 2011
4194
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