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Total synthesis of cyclomarins A, C and D, marine

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Cite this: Org. Biomol. Chem., 2016,

cyclic peptides with interesting anti-tuberculosis
14, 6036 and anti-malaria activities†
Philipp Barbie and Uli Kazmaier*

Received 14th April 2016,
Accepted 19th May 2016
DOI: 10.1039/c6ob00800c
www.rsc.org/obc
Cyclomarins are cyclic heptapeptides containing four unusual amino acids. New synthetic protocols
toward their synthesis have been developed, leading to the synthesis and biological evaluation of three
natural occurring cyclomarins. Interestingly, cyclomarins address two completely different targets: Clp C1,
a subunit of the caseinolytic protease of Mycobacterium tuberculosis (MTB), as well as PfAp3Ase of Plas-
modium falciparum. Therefore, cyclomarins are interesting lead structures for the development of drugs
against tuberculosis and malaria.

Introduction lethal. Therefore, the development of new drugs, especially

with a new mode of action, is highly desired.
Many people in industrial nations think that diseases such as In 1999, Clardy et al. reported the isolation and structure
tuberculosis (TB) are more or less wiped out now, but in elucidation of a family of new cyclic peptides from a marine
reality, almost 30% of humanity is infected by Mycobacterium streptomycete CNB-982, called cyclomarins (Fig. 1).8 Cyclo-
tuberculosis (MTB).1 Luckily, the disease, which mainly affects marin A is the major metabolite, while cyclomarin B and C are
the lungs, progresses to the active disease in only 10% of only formed in 2–3%.
infected people, and only the active infection can lead to Later on, a fourth derivative cyclomarin D, in principle
further infections. Although, a range of drugs is available to N-desmethylcyclomarin C, was isolated from another bacter-
cure the disease,2 around 9 million people become newly ium Salinispora arenicola CNS-205.9 The crude extract as well
infected each year, while 1.5 million die from the disease. as isolated cyclomarin A show only moderate activity toward
Therefore, TB takes up position two in the list of lethal infec- tumour cell lines (low μM) and some antiviral activity.10 Also
tions behind HIV/AIDS.3 an interesting activity towards Mycobacterium tuberculosis was
A major issue in the treatment of TB is caused by the high observed, while the mode of action was unclear at the begin-
tendency of the bacteria to develop resistance towards the drug ning.11 Cyclomarin A was found to be bacteriocidal against
used.4 The bacteria are clasped around by the macrophages,
but they are not killed, and they can even grow and replicate in
them.5 In addition, it is also desired, that antituberculosis
drugs not only kill replicating bacteria (bacteriocide), but also
have a sterilizing effect on nonreplicating persistent cells
(bacteriostatic effect). These non growing bacteria are more or
less drug-resistant, and therefore long lasting treatments are
required to cure the disease.6 Multidrug-resistant tuberculosis
(MDR-TB) is caused by bacteria which cannot be combated by
first-line medications, using isoniazid and rifampicin.7 In the
meanwhile extensively drug resistant strains (XDR-TB) are even
resistant toward second-line drugs, e.g. amikacin, kanamycin,
or capreomycin. In general, infections with these strains are

Institute of Organic Chemistry, Saarland University, P.O. Box 151150,

66041 Saarbrücken, Germany. E-mail: u.kazmaier@mx.uni-saarland.de
† Electronic supplementary information (ESI) available: Copies of 1H and 13
C
NMR spectra. See DOI: 10.1039/c6ob00800c Fig. 1 Cyclomarins from marine streptomycetes.

6036 | Org. Biomol. Chem., 2016, 14, 6036–6054 This journal is © The Royal Society of Chemistry 2016
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bacteria replicating in culture broth as well as in human-
derived macrophages and a series of multidrug resistant clini-
cal isolates. Detailed studies at Novartis indicate, that ClpC1, a
subunit of a caseinolytic protease, is the actual target of
CycA.12 Very recently, the same group at Novartis reported also
a strong activity against Plasmodium falciparum, inhibiting the
PfAp3Aase of the protozoan parasite, in a nanomolar range
without affecting the human homolog hFHIT.13 Therefore, not
only from a synthetic view but also from a pharmaceutic/
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have powerful tools in hand to get access to the different
stereoisomers. While anti-configured amino acids can easily
be obtained via allylic alkylations,27 the corresponding syn-
products are accessible via chelate enolate Claisen rearrange-
ment.30 Using this approach, the two stereogenic centres can
easily be introduced by using a chiral allyl alcohol. Unfortu-
nately, the dimethylated unsaturated side chain cannot be
introduced directly in a stereoselective fashion, since the
corresponding tertiary alcohol would be achiral. To circumvent

medicinal point of view a synthetic protocol toward the cyclo- this problem we decided to generate a monomethyl-substi-
marins is highly desired. tuted amino acid and to introduce the dimethyl group via oxi-
The cyclomarins are cyclic heptapeptides containing two dative cleavage and subsequent olefination, as reported
proteingenic amino acids [(S)-Ala, (S)-Val)], an N-methylated previously be Yao et al.28 (Scheme 1).
(S)-Leu, as well as four unusual (S)-amino acids. Two of them We started the synthesis with an enzymatic kinetic resolu-
are also found in other natural products. δ-Hydroxyleucine tion of secondary alcohol 1, using novozyme 435. The reaction
(HyLeu) is a building block in depsipeptides isolated from was monitored by GC. Since the remaining (desired)
Paecilomyces lilacinus and Bipolaris zeicola,14 while β-methoxy- (S)-alcohol 1 and the (R)-acetate are relatively volatile com-
phenylalanine (MePhe) is also found in the discokiodines.15 pounds, which cannot be separated by distillation, we decided
The two remaining amino acids 2-amino-3,5-dimethylhex-4- to couple the remaining allyl alcohol directly with Boc-glycine
enoic acid (ADHA) and N-(1,1-dimethyl-2,5-epoxypropyl)- and to separate and purify the desired allyl ester (S)-2
β-hydroxytryptophan (DEHT) [or N-(1,1-dimethyl-2-propenyl)- (Scheme 2), which was obtained in enantiopure form. Ester 2
β-hydroxytryptophan (DPHT), respectively] are unique and only was subsequently subjected to a chelate enolate Claisen
found in the cyclomarins, although similar N-prenylated rearrangement giving rise to the γ,δ-unsaturated amino acid
tryptophans have also been observed in the ilamycins.16 It with perfect chirality transfer and excellent diastereo-
should be mentioned, that a few years ago a closely related selectivity.30 The free acid was directly converted into the
cyclic peptide M 10709 was isolated from a clinical streptomy- methyl ester 3. According to Yao28 we subjected 3 to an ozono-
cete, where the ADHA is replaced by a Val.17 lysis using a reductive workup, and the aldehyde formed was
So far, all SAR studies have been carried out with cyclo- subjected to a Wittig reaction. This sequence was found not to
marin A, obtained by fermentation, and semisynthetic deriva-
tives obtained from it.11a Interestingly, the epoxide
functionality in CycA is not responsible for the biological
activity, at least not against MTB, while the OH group of
HyLeu is. Nothing is known so far about the importance of the
β-OH group of the tryptophan.
With respect to the interesting biological activities of the
Scheme 1 Retrosynthesis of protected ADHA.
cyclomarins it is not surprising that a series of synthetic
studies have been carried out towards the unusual building
blocks,18 especially those found also in other natural
products.19–22 Our group is also involved in the development of
new synthetic methods towards such unusual amino acids23
using chelated glycine ester enolates as nucleophiles,24 which
can be used in a wide range of reactions, such as aldol reac-
tions,25 Michael additions26 or allylic alkylations.27 So far, only
one synthesis of the minor metabolite CycC has been reported,28
and our group communicated very recently the first total syn-
thesis of CycA.29 Herein, we report detailed synthesis of the
cyclomarins A, C and D, details of the synthesis of the building
blocks as well as biological data of these three natural products.

Results and discussion

Synthesis of the protected amino acid building blocks
Boc-protected (2S,3R)-2-amino-3,5-dimethylhex-4-enoic acid
(ADHA) methyl ester. We started our investigations with the
synthesis of the γ,δ-unsaturated amino acid ADHA, since we Scheme 2 Synthesis of protected ADHA.

This journal is © The Royal Society of Chemistry 2016 Org. Biomol. Chem., 2016, 14, 6036–6054 | 6037

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be trivial. The aldehyde formed in the ozonolysis was configur-
ationally labile and epimerized almost completely during puri-
fication via flash chromatography. Therefore, it had to be used
as the crude product in the Wittig reaction. In this step a high
excess of the Wittig reagent had to be used, and even with 5
equiv. only 27% of the desired product (S)-4 could be isolated,
as a 1 : 1 diastereomeric mixture. Obviously, the large excess of
the rather basic Wittig ylide caused complete epimerization of
the α-chiral aldehyde. Attempts to reduce the amount of Wittig
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reagent, e.g. by removing the acidic proton from the Boc-NH by
double Boc-protection, failed.
Therefore, we decided to switch to other olefination
methods. By far, the best results were obtained using a special
version of the Julia–Kocienski olefination, a protocol which is
normally used to generate (E)-double bonds.31 The E/Z-selecti-
vity is not relevant in our case, but the interesting point was
the relatively low basicity of the olefination reagent. If sulfone
A 32 was deprotonated with LHMDS before the fresh prepared
aldehyde (crude product) was added, the reaction was finished
after only 5–10 min at −78 °C. The desired product 4 was
obtained almost epimerization-free and with high diastereo-
selectivity. The best results were obtained with 2.3 equiv. of
the deprotonated sulfone. During the synthesis of the linear
heptapeptide we recognised, that the Boc protecting group
could not be removed in the presence of the β-hydroxytrypto-
phan without decomposition, because of its acid sensibility.
Therefore, we finally replaced the Boc group of 4 by an Alloc
protecting group (5).
Protected N-methyl-(2S,4R)-5-hydroxyleucin (N-Me-HyLeu).
Our first approach for the synthesis of this unusual amino
acid was also based on the chelate Claisen rearrangement as a
key step to provide a methyl-substituted γ,δ-unsaturated amino
acid. Kinetic resolution using acylase and regio- and stereo-
selective hydroboration/oxidation should give access to the
desired amino acid (Scheme 3).
Since enzymatic deacetylation of amino acid derivatives is a
suitable protocol for the synthesis of enantiomerically pure
amino acids,33 we decided to subject acetylated allyl ester 6 to
a chelate Claisen rearrangement under the same conditions as
before. Although the rearrangement proceeded cleanly, in our
first attempts only around 50% of the desired product could
be obtained after flash chromatography, because of the high
polarity of the acetylated amino acid. Therefore, we decided to
change the workup procedure. By simply washing the crude
rearrangement product 7 with chloroform/pentane (3 : 1) and
afterwards with pentane, 7 could be obtained in pure form in
high yield. Subsequent enzymatic amide cleavage provided a
mixture of (R)-7 and the desired free (S)-amino acid. To avoid
Scheme 3 Retrosynthesis of protected HyLeu.
6038 | Org. Biomol. Chem., 2016, 14, 6036–6054
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Scheme 4 Synthesis of protected dehydroleucines.
loss during workup, we decided to protect the free amino acid
directly from the mixture (Scheme 4).
After esterification, the two different N-protected amino
acid esters 9 and 10 could be separated by flash chromato-
graphy. Both enantiomers could be obtained in high overall
yield (over 3 steps) and with good ee.
With the unsaturated amino acid ester 10 in hand, we inves-
tigated the next crucial step, the stereoselective introduction of
the 5-OH-group via hydroboration (Table 1). In our first
attempt, the hydroboration using BH3–THF, a mixture of the
two regioisomeric alcohols was obtained after oxidative
workup as an almost 1 : 1 mixture. No reaction was observed
using disiamylborane, while thexylborane gave rise to the
desired primary alcohol 12, albeit in only low yield and
diastereoselectivity. By far the best results were obtained with
9-BBN providing (S)-12 as a single regioisomer as an acceptable
3 : 1 diastereomeric mixture.
To determine the relative configuration at the newly formed
stereogenic centre, we saponified methyl ester 12 and sub-
jected the hydroxy acid to an acid-catalysed lactonisation. The
major stereoisomer was obtained in enantiomerically pure
form after chromatography. The six-membered lactone 13 was
well suited to determine the configuration of the methyl group
via NOESY (Scheme 5). A strong NOE was observed between
the α-H and the methyl group at the γ-position, clearly indicat-
ing that the N-Boc-group and the methyl group have an anti-
orientation in the lactone ring, corresponding to the (2S,4S)-
configuration, unfortunately the wrong diastereomer.
Therefore, we had to develop an alternative route towards
the desired (2S,4R)-isomer. We decided to use an asymmetric
catalytic hydrogenation approach to introduce the stereogenic
centre at the α-position and to adopt the configuration at the
Table 1 Hydroboration/oxidation of dehydroleucine 10
Entry Borane Equiv. 11 [%] 12 [%] dr (12)
1 BH3·THF 0.3 20 25 1:1
2 Disiamylborane 1.1 — —
3 Thexylborane 1.1 — 36 1.5 : 1
4 9-BBN 1.0 — 60 3:1
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Organic & Biomolecular Chemistry
Scheme 5 Determination of configuration of (S)-12.
γ-position from commercially available Roche ester 14
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(Scheme 6). After silylation of the primary OH-functionality
(15)34 a Dibal-H reduction of the ester provided the corres-
ponding aldehyde. Also this aldehyde was extremely sensitive
and configurationally labile, and was therefore directly
coupled with phosphonoglycinates 16 according to Schmidt
et al.35 Since the protecting group of the dehydroamino acid
has a strong influence on the selectivity of the subsequent
asymmetric hydrogenation, and good results are generally
obtained with N-acetyl derivatives, we used this protecting
group also in the phosphonoglycinate (16a). But in our case
the olefination with this reagent was rather sluggish, and an
epimerization of the α-chiral aldehyde could not be sup-
pressed. In addition, the yield of 17a was rather low. Neverthe-
less, we subjected this dehydroamino acid to a catalytic
hydrogenation using (R)-monophos as a chiral ligand.36 At 20
bar hydrogen pressure, the hydrogenation was complete after
3 h and the desired amino acid 18a was obtained in good yield
and high ee, but as a 3 : 1 diastereomeric mixture (4R : 4S) (as
the result of the aldehyde epimerisation).
Therefore, we investigated olefinations using other pro-
tected phosphonoglycinates. With the Cbz-protected phos-
phonate 16b the condensation with the fresh prepared
aldehyde proceeded cleanly at −78 °C, and the desired dehy-
droamino acid 17b could be obtained in almost enantiomeri-
cally pure form in good overall yield. Luckily, the ee-value in
the hydrogenation step was comparable to the acetyl derivative
and only 5% of the wrong diastereomer (2S,4S) was observed.
To finalize the synthesis of the required building block 20, 18b
Scheme 6 Synthesis of protected N-Me-HyLeu 20.
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was saponified to the free acid 19, which was subsequently
N-methylated at −5 °C in excellent overall yield.
Protected (2S,3R)-3-methoxyphenylalanine (MePhe). In prin-
ciple, β-oxygenated amino acids can be obtained by aldol reac-
tion of chelated enolates,25 but in this case, the anti-products
are formed preferentially, while MePhe, found in the cyclo-
marins, the syn-orientation of the stereogenic centres requires
an alternative strategy. We decided to investigate chelate-
controlled additions of arylorganometallics towards a chiral
serine-derived aldehyde 21.37 This aldehyde can also be
obtained via Dibal-H reduction of protected serine ester, and
should be directly used without purification to avoid racemiza-
tion (Scheme 7). First, we used phenylmagnesium bromide
(3 equiv.) as nucleophile, giving rise to the desired aldol
product in acceptable yield (60%) but only moderate diastereo-
selectivity (dr 7 : 3). Much better results were obtained after
transmetallation to titanium. Although a large excess of the
titanium reagent, prepared in situ, was required for good yield,
the desired product 22 was obtained as a single diastereomer.
Its configuration was determined by deprotonating with NaH
and formation of oxazolidinone 23. Coupling constants of J =
4.9 Hz for the two ring protons are typical for trans-configured
oxazolidinones.38 The formation of the syn-configured
addition product 22 can be explained via the chelate Cram-
model.39 The methylation of the β-OH group in 22 was by far
not as trivial as it looks like at first sight. Typical reaction con-
ditions such as Ag2O/MeI or pyridine/MeOTf did not lead to
significant amounts of methylated product 24, even not after
3 d of reaction time. But if 22 was deprotonated with an excess
of LHMDS in DMF, the methylation with MeI was complete
after only 10 min at −10 °C. Subsequently, the silyl ether was
cleaved and the primary alcohol was oxidised to acid 25 in
quantitative yield.
Protected β-hydroxytryptophans. Most efforts had to be dedi-
cated to the synthesis of the tryptophan building blocks, not
only because of the high acid-sensitivity of the β-OH group,
but also of the terminal double bond of the reverse prenyl
group. When we started our investigations, only one protocol
was known for the introduction of the tert-prenyl group,
Scheme 7 Synthesis of protected MePhe 25.
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Table 2 Optimization of the carbonyl addition

Entry React. cond. 1 React. cond. 2 Yield [%] dr

1 THF, 0 °C, 90 min −30 °C, 2 h 70a n.d.

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2 THF, −20 °C, 15 min −30 °C, 2 h 19 85 : 15

Scheme 8 Retrosynthesis of protected β-hydroxytryptophan.
requiring relatively high amounts of a Pd-catalyst (40 mol%)
and silver and copper salts in excess.40 Therefore, we planned
to introduce this functionality in a late state of the synthesis,
preferentially more or less at the end. Since the relative con-
figuration of the two stereogenic centres (syn) is the same as in
the MePhe building block, we decided to use the same type of
carbonyl addition also for their introduction. Our retro-
synthetic plan is shown in Scheme 8.
After the carbonyl addition and protection of the β-OH
group by a suitable orthogonal protecting group (PG′) the
indole protecting group (PG″) had to be removed to allow the
introduction of the tert-prenyl group. Finally, after selective
cleavage of the primary OH-protecting group, the desired
building block should be accessible by oxidation.
We decided to start our synthesis with N-Boc-protected
iodoindole 26. The Boc-protecting group was chosen because
it can be removed from an indole nitrogen under basic con-
ditions41 without affecting aliphatic N-Boc-protecting groups.
This would allow to use the same building block 21 as in the
last synthesis. 26 can easily be obtained from indole in two
steps,42 but the synthesis is not trivial, since 3-iodoindoles are
rather unstable, especially with free NH, decomposing at temp-
eratures above 0 °C rapidly. Therefore, the reactions had to be
carried out fast and 26 should be used directly in the next syn-
thetic steps. Since 26 was not converted into an organometallic
reagent so far, we had to find suitable conditions for the
desired halogen–metal exchange (Table 2). The metallated
intermediate formed was directly reacted with fresh prepared
21 as described before. In our first attempt, we used sec-
BuMgCl for metalation at 0 °C and the aldehyde was added at
−30 °C. Complete conversion was observed after two hours,
but we were not able to obtain the desired alcohol in pure
form and to determine the diastereomeric ratio from the NMR
spectra (entry 1). Transmetallation at −20 °C for only 15 min
gave rise to 27 with good diastereoselectivity, but only moder-
ate yield (entry 2). As a major side product, the direct addition
product of the Grignard reagents was found, clearly indicating,
that the halogen–metal exchange requires more time. The best
results were obtained after prolonged metalation time and if
the aldehyde was reacted at −20 °C (entry 3). The diastereo-
selectivity was slightly worse, but under these conditions, the
major diastereomer could be obtained in pure form by flash
chromatography.
3 THF, −10 °C, 45 min −20 °C, 2 h 65b 80 : 20
−20 °C to rt
4 (1) THF, −10 °C, 45 min −78 °C to rt, 61 60 : 40
18 h
(2) CH2Cl2, TiCl(OiPr)3,
−78 °C
a
Impure compound. b Isolated yield of the pure major diastereomer.
Interestingly, transmetallation to titanium, like in the syn-
thesis of 22, brought no improvement (entry 4). The reaction
was clean and the yield acceptable, but the diastereoselectivity
dropped to 6 : 4. It should be mentioned, that in case of
Ti(OiPr)4 as titanium source only traces of coupling product
could be obtained.
In the first instance, the β-OH functionality of 27 was pro-
tected as the acetate (28), a protecting group orthogonal to the
others (Scheme 9). Also here, the diastereomers could be sep-
arated by flash chromatography and pure syn-28 was subjected
to silyl deprotection. But unfortunately, under all cleaving con-
ditions used, a 1,3 acyl-migration of the acetate group was
observed. Therefore, we decided to switch to the MOM protect-
ing group (29), which allowed a clean cleavage of the silyl group
without protecting group migration. Oxidation of the primary
alcohol 30, as described before, and subsequent esterification
gave rise to double Boc-protected hydroxytryptophan 31.
To replace the indole Boc-group by the desired tert-prenyl
group we subjected 31 to a series of cleavage conditions,42 but
all of them failed. In all cases only the elimination product
was obtained. Therefore, the Boc-protecting group on the
indole nucleus is definitely not the best choice. It should be
mentioned, that a benzyl group at this position is more suit-
Scheme 9 Synthesis of protected tert-prenylated β-hydroxytryptophan
31.

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able,43 but we observed, that also the α-Boc-protecting group
cannot be removed without decomposition of the very sensitive
β-hydroxytryptophan.
Recently, Stanley et al. reported a new protocol for the syn-
thesis of N-tert-prenylated indoles, requiring only small
amounts of a Pd-catalyst (4 mol%) and Xantphos (4 mol%) as
ligand.44 Under these conditions, electron-poor indoles can be
prenylated with good regioselectivity. With this method in
hand, we decided to introduce the prenyl group just at the
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beginning of the synthesis to avoid problems during N-depro-
tections. In this context the α-Boc-protecting group was
replaced by an Alloc-protecting group, which can be cleaved
under much milder conditions. Since the prenylation only
works with electron-deficient indoles, we used an indole-3-
carboxylate as starting material. By using tert-prenyl-tert-butyl-
carbonate an excellent yield of 32 was obtained with only
1 mol% of [allylPdCl]2 as catalyst, although the reaction took
two days to completion. During that time we kept the reaction
temperature at 0 °C to avoid the formation of the n-prenylated
product. In the next step the “activating” ester functionality
had to be replaced by iodine. Attempts to saponify the ester
and to decarboxylate the salt directly by heating or under
microwave irradiation failed. But after acidic workup and
heating the neat carboxylic acid to 180 °C decarboxylation took
place, providing the desired indole 33 in almost quantitative
yield. It should be mentioned, that before decarboxylation the
acid has to be washed to neutral, since traces of mineral acids
caused side reactions, resulting in a significant drop of the
yield. Indole 33 was iodinated also in excellent yield. The
whole sequence could be carried out on 10 gram scale provid-
ing 34 in an overall 91% yield (Scheme 10).
In analogy to the synthesis of MePhe (Scheme 7) Alloc-pro-
tected (D)-serine 35 45 was subjected to Dibal-H reduction
under the same conditions as described before (Scheme 11).
For the metalation of 34 we chose a procedure described by
Knochel et al. using a combination of ZnCl2, LiCl and Mg.46
Under these conditions probably an indole-zinc–magnesium
complex is formed, which we hoped will give better selectiv-
ities than the magnesium derivative used before. The fresh
prepared aldehyde (35′) was added at −78 °C and the mixture
was allowed to warm to room temperature overnight. And
indeed, the desired product 36 could be obtained in good yield
and selectivity. We decided to introduce also a TBDMS protect-
ing group on the secondary OH-group, since it is possible to
Scheme 10 Synthesis of tert-prenylated iodoindole 34.
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Scheme 11 Synthesis of protected tert-prenylated β-hydroxytrypto-
phan 40.
cleave primary alcohols selectively in the presence of secondary
ones.47 Surprisingly, no O-protection could be obtained under
standard conditions using TBDMSCl/imidazole, only
decomposition of 36 was observed, illustrating the moderate
stability of this compound. The combination of TBDMSOTf/
lutidine was found to be the reagent of choice, while the best
yield was obtained at −35 °C. After only 15 min the desired
product 37 could be obtained in 87% yield. The primary
O-TBDMS group was removed using NH4F in methanol.48 In
contrast to the generally used Bu4NF here not a “naked” fluor-
ide is the cleaving reagent, but a highly solvated one. Of
course, this solvated F− is less reactive, but therefore also more
selective. Attempts to oxidize the primary alcohol 38 in
analogy to the Boc-protected derivative 30 failed, and we had
to investigate alternative strategies. The best results were
obtained using a Parikh–Doering protocol.49 The aldehyde
formed is extremely sensitive and undergoes rapid decompo-
sition. Therefore, it should be directly subjected to further oxi-
dation. Since the free acid is also not very stable, we decided to
oxidize the aldehyde directly to the corresponding methyl ester
39 using N-iodosuccinimide in methanol.50 If the reaction
temperature was kept at 0 °C during all steps and workups, 39
could be obtained in 90% yield. The free acid 40 could be
obtained by careful saponification of 39 in acceptable yield and
should be directly used in the peptide coupling step. As a side
product, elimination of the β-O-functionality was observed.
While 40 is a useful building block for the synthesis of
cyclomarins C and D, for cyclomarin A the corresponding
epoxidised tryptophan is required. To use an analogous proto-
col we decided to synthesize epoxy iodoindole 41 from indole
34 according to the literature via Sharpless dihydroxylation/
tosylation/epoxidation (Scheme 12).22b Unfortunately, the
selectivity in the dihydroxylation step was only moderate. The
best results were obtained using (DHQD)2Pyr as ligand, but
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Scheme 12 Synthesis of protected epoxidised β-hydroxytryptophan
45.
also here the ee does not exceed 80%.22b To avoid compli-
cations in the metalation step, caused by the epoxide, we
decided to use another procedure to generate the indolylzinc
intermediate. 41 was lithiated at −78 °C with n-BuLi and sub-
sequently transmetallated at the same temperature by addition
of ZnBr2.46 The zinc reagent formed was directly reacted with
fresh prepared aldehyde 35′, giving rise to the desired product
42. According to NMR and HPLC only two diastereomers could
Scheme 13 Synthesis of cyclomarins C and D.
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Organic & Biomolecular Chemistry
be detected in a ratio of 9 : 1. The secondary alcohol was silyl-
protected as described before, but in this case, surprisingly no
complete conversion could be obtained, and the silyl ether 43
was isolated in 75% as a single stereoisomer. Obviously, the
minor stereoisomer of 42 did not react under these reaction
conditions. The cleavage of the primary silyl group was inter-
rupted after 48 h, when the formation of side products was
observed. The primary alcohol 44 could easily be separated
from unreacted 43 (17% recovered). The final oxidation steps
proceeded without complications as described before. Ester 45
was saponified directly before the peptide coupling and was
used without purification.
With these building blocks in hand, we next focused on the
synthesis of the linear peptides required for the cyclization.
Since we decided to close the ring between the N-Me-Leu and
the unsaturated amino acid ADHA, we could use the same
tetrapeptide for the synthesis of all three cyclomarins, because
they differ only in the fifth and sixth amino acids. Starting
from N-methylated (S)-methyl leucinate, coupling with Cbz-
protected (S)-valine gave rise to dipeptide 46 (Scheme 13). BEP
(2-bromo-1-ethyl-pyridiniumtetrafluoroborate)51 was used as
coupling reagent for the connection of these two sterically hin-
dered amino acids. To avoid a formation of the corresponding
dioxopiperazine during the subsequent hydrogenation, this
reaction was carried out in the presence of HCl, giving rise to
the corresponding hydrochloride. For the next coupling step
25 was activated as a mixed anhydride, which was directly
coupled with the dipeptide salt. The Boc-protecting group was
removed under standard conditions and the same protocol
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Organic & Biomolecular Chemistry Paper

was also used for the next coupling step. For the synthesis of
cyclomarin C the tetrapeptide was reacted with N-methylated
amino acid 20, while 19 was used for cyclomarin
D. Hydrogenolytic removal of the Cbz-protecting groups from
the heptapeptides proceeded smoothly and tert-prenylated
tryptophan 40 was coupled using again BEP in case of the
N-methyl derivative and EDC/HOBT for the non methylated
derivative. The N-terminal Alloc-protecting group was removed
using Pd-catalysis, using TPPTS [tri-(sodium-metasulfonato-
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phenyl)-phosphane] as a ligand, and the final amino acid 5

was coupled using EDC/HOBT. The yields obtained for the two
heptapeptides 51 were almost the same. At this stage of the
synthesis we had to decide if we would cleave the silyl-protect-
ing groups already in the heptapeptide and to undertake the
cyclization with the unprotected OH-functionalities, or later on
in the cyclic peptide. Attempts to remove both TBDMS-protect-
ing groups of 51a with TBAF provided the corresponding diol
in a slow reaction in only 48%, while several side products
were formed.
The next step, the saponification of the C-terminal methyl
ester caused severe problems, resulting in a decomposition
and fragmentation ( probably via retro aldol of the β-hydroxy-
tryptophan) of the molecule. Therefore, we decided to saponify
esters 51 first and to remove the silyl protecting groups later
on from the cyclic products. Cleavage of the Alloc protecting
group was performed under the previous conditions. Without
purification, the free heptapeptide was cyclized using PyBOP/
iPr2NEt by slow addition via syringe pump over a period of Scheme 14 Synthesis of cyclomarin A.
12 h, giving a final concentration of 1 mM. Under these con-
ditions, 52a was obtained in a very good yield of 76% (over
three steps). HPLC analysis showed the formation of around
5% of a second stereoisomer, probably caused by a partial epi- An interesting observation was made by comparison of the
1
merization of the C-terminal Leu on activation. But this minor H NMR spectra of the synthetic cyclomarins with the isolated
isomer could easily be removed by chromatography. In the ones8 (see ESI†). On the first view, the spectra looked almost
case of the desmethyl derivative 52b the yield was somewhat identical, but by having a closer look it was obvious, that an
lower, which can probably be explained by a less suitable con- NH signal at 6.8 ppm was shifted to higher field and that the
formation for cyclization of the linear precursors. Finally, the signals of the free OH groups were not found at all. Since their
primary O-silyl group was removed selectively using the NH4F/ shifts are dependent on the solvent and concentration, this
MeOH protocol, a reaction which was rather sluggish at room fact is not so surprising, but also two shifts of an alkyl multi-
temperature. It took 5 d for complete cleavage of the protecting plet (1.8 ppm) and a methyl group (0.5–0.6 ppm) did not corre-
group and during this rather long time, a range of side reac- late. All these signals belong to the hydroxyleucine. Since the
tions could occur, resulting in a moderate yield of only 40%. spectrum of the isolated natural product contains not only a
Better results were obtained by warming the reaction mixture huge “water peak” at 1.56 ppm, but also a strong signal of
to 40 °C overnight, which caused complete cleavage. Sub- CDCl3, one can assume that the isolated sample was measured
sequently, the β-OH tryptophan could be deprotected with under significantly higher dilution, while we took our spec-
exactly 1 equiv. of Bu4NF at 0 °C in only 30 min. Under these trum also under “dry conditions”. To figure out if these facts
optimized conditions, the two cyclomarins C and D could be might result in a different conformation of our sample, we
obtained in a yield around 65% (over two deprotection steps). diluted it and added 5 μl water. And indeed, under these con-
For the synthesis of cyclomarin A, pentapeptide 49a was ditions, the spectra of the isolated and the synthetic cyclo-
N-deprotected by catalytic hydrogenation (Scheme 14). In par- marin C matched perfectly. The same effect was also observed
allel, tryptophan ester 45 was saponified, and the crude acid with the other members of the family.
was coupled with the deprotected peptide using BEP. The All three cyclomarins have been tested on their antituber-
further steps were carried out in analogy to the synthesis of culotic activity.52 The MIC of the synthetic cyclomarin A (0.5 µM)
the cyclomarins C and D. The yields obtained were comparable matches perfectly the previously reported value. Interestingly,
to the previous examples, only the final deprotection of the the synthetic cyclomarin C showed exactly the same activity.
silyl groups gave a slightly lower yield. Obviously, the epoxide moiety in the tryptophan side chain is

This journal is © The Royal Society of Chemistry 2016 Org. Biomol. Chem., 2016, 14, 6036–6054 | 6043

Paper
not essential for the activity towards Mycobacterium tuberculosis.
This is in good agreement with observations made by the
Novartis group investigating derivatives of cyclomarin A result-
ing from epoxide opening reactions.11a Cyclomarin D proved
to be less active, showing a MIC in the range of 8–32 µM.
Conclusions
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In conclusion, we have shown that the unusual amino acids of
the cyclomarins can be synthesized in enantiomerically pure
form in high yields. With these building blocks in hand, the
cyclomarins A, C and D could be synthesised, using a finely
adjusted protecting group strategy. All the cyclomarins show
activity towards Mycobacterium tuberculosis in the low μM
range, while cyclomarine A and C are the most potent ones.
Experimental
General experimental details
All air- or moisture-sensitive reactions were carried out in
dried glassware (>100 °C) under an atmosphere of nitrogen or
argon. Dried solvents were distilled before use. The products
were purified by flash chromatography on silica gel columns
(Macherey-Nagel 60, 0.063–0.2 mm) and with a flash chromato-
graphy system [Reveleris (Grace), RediSep-columns 4 g, 12 g,
24 g and 40 g from Axel Semrau], using mixtures of ethyl
acetate and petroleum ether as eluents. Alternatively, a C-18
silica column was applied, using acetonitrile/water as eluent.
Analytical TLC was performed on pre-coated silica gel plates
(Macherey-Nagel, Polygram® SIL G/UV254). Visualization was
accomplished with UV-light, KMnO4 or a ceric ammonium
molybdate chamber. Melting points were determined with a
melting point apparatus MelTEMP II by Laboratory devices and
are uncorrected. 1H, and 13C spectra were recorded with a
Bruker AV II 400 [400 MHz (1H), 100 MHz (13C)], or a Bruker AV
500 [500 MHz, (1H), 125 MHz (13C)] spectrometer in CDCl3
unless otherwise specified. NMR spectra were evaluated using
Mestrec. Chemical shifts are reported in ppm relative to
Si(CH3)4 and CHCl3 was used as the internal standard [δ(1H) =
7.26 ppm, δ(13C) = 77.0 ppm]. High resolution mass spectra
were recorded with a Finnigan MAT 95 spectrometer using the
CI technique (M < 600 g mol−1) or with an Bruker maXis 4G
UHR-TOF-spectrometer using the ESI technique (M >
600 g mol−1). Optical rotations were measured with a Perkin-
Elmer polarimeter (model 341) in a thermostated (20 °C ±
1 °C) cuvette. The radiation source used was a sodium vapor
lamp (λ = 589 nm). The concentrations are given in g per
100 ml. HPLC/MS analysis was performed with a Shimadzu
system (LC: 10A-series with Autosampler, MS: LCMS-2020).
Elemental analyses were performed at the Saarland University.
Preparation and analytical data of cyclomarin A and its syn-
thetic intermediates war reported previously.29
N-Acetyl-glycine-(2-methallyl) ester (6). To a suspension of
N-acetyl glycine (10.5 g, 89.8 mmol) and 2-methyl-allyl alcohol
6044 | Org. Biomol. Chem., 2016, 14, 6036–6054
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Organic & Biomolecular Chemistry
(6.30 mL, 74.8 mmol) in THF (500 mL) DMAP (9.16 g,
75.0 mmol) and DCC (18.6 g, 90.0 mmol) were added at room
temperature. The reaction mixture was heated to reflux for
18 h. Half of the solvent was removed in vacuo and the solution
was filtrated after cooling to room temperature. The filtrate
was completely evaporated and the residue was dissolved in di-
chloromethane (20 mL). The solution was washed with 1 N
KHSO4 and dried over Na2SO4. The resulting ester (11.2 g,
48.9 mmol, 65%) was obtained as a white crystalline solid,
melting point: 95–98 °C. Rf (6): 0.23 (ethyl acetate). 1H NMR
(400 MHz, MeOD): δ = 1.76 (s, 3 H), 2.00 (s, 3 H), 3.97 (s, 2 H),
4.57 (s, 2 H), 4.93 (m, 1 H), 4.98 (m, 1 H) ppm. 13C NMR
(100 MHz, MeOD): δ = 19.5, 22.3, 42.1, 69.3, 113.5, 141.3,
171.1, 173.9 ppm.
rac-N-Acetyl-4-dehydroleucine (rac-7). In a Schlenk flask, di-
isopropyl amine (7.00 mL, 49.8 mmol) was dissolved in dry
THF (40 mL) and cooled down to −40 °C. To the resulting solu-
tion, n-BuLi (1.6 M in hexanes, 31.1 mL, 49.8 mmol) was
added dropwise. After stirring for 5 min at this temperature,
the cooling bath was removed and the mixture was allowed to
warm up to room temperature.
In a second Schlenk flask, ZnCl2 (3.26 g, 23.9 mmol) was
heated with a heat gun in vacuo and dissolved in dry THF
(40 mL) after cooling. To this solution 6 (2.85 g, 16.6 mmol)
was added, dissolved in dry THF (40 mL).
Both solutions were cooled down to −78 °C and the LDA-
solution was added slowly to the substrate solution via transfer
cannula. The resulting mixture was allowed to warm up to
room temperature overnight. After dilution with diethyl ether
(100 mL) 1 N KHSO4-solution was added (50 mL). The aqueous
layer was saturated with NaCl and extracted with diethyl ether
(3 × 100 mL). The combined organic layers were dried over
Na2SO4 and the solvent was evaporated in vacuo. The crude
residue was washed with a chloroform–pentane mixture (3 : 1,
50 mL) and with pentane (2 × 50 mL). The resulting acid
(2.51 g, 14.7 mmol, 89%) was obtained as a white crystalline
solid. Melting point: 124–128 °C. 1H NMR (400 MHz, MeOD):
δ = 1.76 (s, 3 H), 1.96 (s, 3 H), 2.38 (dd, J = 14.3 Hz, 10.0 Hz,
1 H), 2.58 (dd, J = 14.3 Hz, 4.9 Hz, 1 H), 4.58 (dd, J = 10.0 Hz,
4.9 Hz, 1 H), 4.78 (s, 1 H), 4.83 (s, 1 H) ppm. 13C NMR
(100 MHz, MeOD): δ = 22.0, 22.3, 40.9, 52.1, 114.1, 142.4,
173.3, 175.3 ppm. HRMS (CI): calculated: for C8H13NO3 [M]+
171.0895, found 171.0900.
(R)-N-Acetyl-4-dehydroleucine methyl ester [(R)-9], (S)-N-Boc-
4-dehydroleucine methyl ester [(S)-10]. Racemic N-acetyl de-
hydroleucine, rac-(7) (2.41 g, 14.6 mmol) was suspended in
water (100 mL) and the pH was set to 6.8–7.0 by the means of
an automated titration apparatus filled with 0.1 M NaOH. To
this mixture Acylase I (73 mg, isolated aminoacylase EC
3.5.1.14. from porcine kidney) was added. The mixture was
stirred for 48 h at room temperature while the pH was main-
tained between 6.5 and 7.0. To this mixture NaHCO3 (2.10 g,
25.0 mmol) and a solution of di-tert-butyl dicarbonate (4.37 g,
20.0 mmol) in dioxane (50 mL) was added. The mixture was
stirred overnight and acidified carefully with 1 N aq. KHSO4
(50 mL), before it was extracted with ethyl acetate (3 × 100 mL).
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Organic & Biomolecular Chemistry
The combined organic layers were dried over Na2SO4 and the
solvent was removed in vacuo.
The residue was dissolved in dry DMF (20 mL) and was
cooled to 0 °C. To this solution K2CO3 (2.52 g, 18.3 mmol) and
methyl iodide (2.74 mL, 60.2 mmol) were added. The mixture
was stirred overnight at room temperature and diluted with
ethyl acetate (100 mL), after TLC showed complete consump-
tion of the starting material. The mixture was washed succes-
sively with 1 N aq. KHSO4, sat. aq. NaHCO3, 5% aq. NaS2O3,
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water and brine. After drying (Na2SO4), the solvent was
removed in vacuo and column chromatography of the crude
residue (silica, 1. petroleum ether/ethyl acetate 8 : 2; 2. ethyl
acetate) gave rise to (R)-9 (1.21 g, 6.28 mmol, 45%, 97% ee) as
a colourless resin and (S)-10 (1.53 g, 6.28 mmol, 43%, 98% ee)
as a colourless oil.
D = −22.6 (c = 1.0,
(R)-9: Rf [(R)-9]. 0.37 (ethyl acetate). [α]20
CHCl3). 1H NMR (400 MHz, CDCl3): δ = 1.73 (s, 3 H), 2.00 (s,
3 H), 2.38 (dd, J = 13.9 Hz, 8.2 Hz, 1 H), 2.54 (dd, J = 13.9 Hz,
5.5 Hz, 1 H), 3.73 (s, 3 H), 4.40 (ddd, J = 8.2 Hz, 5.5 Hz, 5.5 Hz,
1 H), 4.74 (m, 1 H), 4.85 (m, 1 H), 5.90 (d, J = 5.5 Hz, 1 H) ppm.
13
C NMR (100 MHz, CDCl3): δ = 21.7, 22.8, 40.4, 50.5, 52.2,
114.3, 140.4, 169.8, 172.8 ppm. HRMS (CI): calculated for
C9H16NO3 [M + H]+ 186.1125, found 186.1129.
(S)-10: Rf [(S)-10]. 0.65 (ethyl acetate). [α]20
D = +9.6 (c = 1.0,
CHCl3). Mixture of rotamers. Major rotamer: 1H NMR
(400 MHz, CDCl3): δ = 1.43 (s, 9 H), 1.74 (s, 3 H), 2.36 (dd, J =
13.8 Hz, 8.4 Hz, 1 H), 2.51 (dd, J = 13.8 Hz, 5.5 Hz, 1 H), 3.73
(s, 3 H), 4.40 (m, 1 H), 4.75 (s, 1 H), 4.85 (s, 1 H), 4.93 (d, J =
6.8 Hz, 1 H) ppm. 13C NMR (100 MHz, CDCl3): δ = δ = 21.8,
28.2, 40.7, 51.8, 52.1, 79.8, 114.4, 140.5, 155.2, 173.0 ppm.
Minor rotamer (selected signals) 1H NMR (400 MHz, CDCl3):
δ = 4.21 (bs, 1 H), 4.62 (bs, 1 H) ppm. HRMS (CI): calculated
C12H22NO3 [M + H]+ 244.1543, found 244.1547.
(2S,4R/S)-N-Boc-5-hydroxyleucine methyl ester (12). To a
solution of (S)-10 (500 mg, 2.06 mmol) in THF (10 mL) a
9-BBN-solution (0.5 M in THF, 412 µL, 206 µmol) was added
dropwise. After TLC showed complete conversion, 30% H2O2
(2 mL) and phosphate buffer ( pH 7, 2 mL) were added. The
mixture was stirred overnight, diluted with sat. aq. Na2S2O3
solution and extracted with ethyl acetate (3 × 20 mL). The com-
bined organic layers were dried (Na2SO4) and the solvent was
evaporated in vacuo. Column chromatography (silica,
petroleum ether, ethyl acetate 1 : 1) gave rise to alcohol 12
(283 mg, 1.08 mmol, 53%) as a colourless resin and a mixture
of diastereomers [dr = 3 : 1 (NMR)]. Rf [(2S,4S)-12]: 0.28;
Rf [(2S,4R)-12]: 0.29 ( petroleum ether/ethyl acetate 1 : 1). [α]20
D =
+15.7 (c = 1.0, CHCl3). [(2S,4S)-12]: 1H NMR (400 MHz, CDCl3):
δ = 0.97 (d, J = 6.7 Hz, 3 H), 1.43 (s, 9 H), 1.59 (m, 1 H), 1.75
(m, 2 H), 1.97 (bs, 1 H), 3.43 (dd, J = 10.6 Hz, 6.2 Hz, 1 H), 3.57
(dd, J = 10.6 Hz, 4.9 Hz, 1 H), 3.73 (s, 3 H), 4.33 (m, 1 H), 5.13
(d, J = 7.0 Hz, 1 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 17.1,
20.6, 28.3, 36.3, 49.9, 51.8, 71.9, 79.9, 155.4, 173.0 ppm.
[(2S,4S)-12] (selected signals): 1H NMR (400 MHz, CDCl3): δ =
1.89 (m, 2 H), 4.45 (m, 1 H), 5.25 (d, J = 6.0 Hz, 1 H) ppm.
HRMS (CI): calculated for C12H25NO5 [M + H]+ 262.1649, found
262.1637.
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(3S,5S)-3-Boc-amino-5-methyltetrahydro-2H-pyran-2-one (13).
To a solution of the alcohol 12 (94.0 mg, 360 µmol, dr 3 : 1) in
MeOH (3 mL) aq. NaOH (1 M, 380 mL, 380 µmol) was added at
0 °C. The mixture was allowed to warm to room temperature
overnight and the solvent was evaporated in vacuo. 1 N KHSO4
(3 mL) and ethyl acetate (10 mL) were added to the residue.
The layers were separated and the organic layer was washed
with water. The solvent was removed in vacuo after drying over
Na2SO4. The residue was taken up in toluene (3 mL) and
p-TsOH (6.0 mg, 36 µmol) was added. The mixture was heated
to reflux until TLC showed complete conversion. The mixture
was diluted with ethyl acetate (10 mL), and washed succes-
sively with 1 N KHSO4, sat. NaHCO3 and brine. The organic
layer was dried over Na2SO4 and the solvent was removed
in vacuo. Column chromatography (silica, petroleum ether/
ethyl acetate 1 : 1) of the residue yielded pure major diastereo-
mer (2S,4S)-13 (17.0 mg, 74 µmol, 21%) together with a mixed
fraction [15.0 mg, 66 µmol, 18%, ratio (2S,4S):(2S,4R) 3 : 2] as
colourless oils. Rf [(2S,4S)-13]: 0.38; Rf [(2S,4R)-13]: 0.37
( petroleum ether/ethyl acetate 1 : 1). [(2S,4S)-13]: 1H NMR
(400 MHz, CDCl3): δ = 1.04 (d, J = 6.9 Hz, 3 H), 1.33 (m, 1 H),
1.44 (s, 9 H), 2.27 (m, 1 H), 2.60 (m, 1 H), 3.98 (dd, J = 11.4 Hz,
6.9 Hz, 1 H), 4.25 (m, 1 H), 4.37 (ddd, J = 11.4 Hz, 4.8 Hz,
1.1 Hz, 1 H), 5.32 (bs, 1 H) ppm. 13C NMR (100 MHz, CDCl3):
δ = 18.3, 27.7, 28.3, 35.1, 49.9, 74.1, 80.2, 155.4, 171.8 ppm.
HRMS (CI): calculated for C11H20NO4 [M + H]+ 230.1387, found
230.1393.
(2S,4R)-N-Cbz-4-methyl-5-[(tert-butyldimethylsilyl)oxy]-leucine
(19). To a solution of the methyl ester 18 29 (110 mg, 270 µmol)
in MeOH (3 mL) 1 M NaOH (300 µL, 300 µmol) was added at
0 °C. The mixture was allowed to warm to room temperature
over night and the solvent was removed in vacuo. The residue
was dissolved in water and was washed with dichloromethane
(5 mL). The aqueous layer was acidified using 1 N KHSO4 and
extracted with dichloromethane (3 × 10 mL). The resulting
organic layers were combined, dried over NaSO4 and concen-
trated in vacuo. The acid 19 (106 mg, 270 µmol) was obtained
as a colourless oil and was used without further purification.
Rf (19): 0.27 ( petroleum ether/ethyl acetate 7 : 3). [α]20
D = +2.5
(c = 1.0, CHCl3). Mixture of rotamers. Major rotamer: 1H NMR
(400 MHz, CDCl3): δ = 0.04 (s, 3 H), 0.05 (s, 3 H), 0.88 (s, 9 H),
0.93 (d, J = 6.7 Hz, 3 H), 1.57 (m, 1 H), 1.81 (m, 1 H), 1.93 (m,
1 H), 3.40 (dd, J = 9.9 Hz, 6.5 Hz, 1 H), 3.58 (dd, J = 9.9 Hz,
4.8 Hz, 1 H), 4.39 (m, 1 H), 5.09 (d, J = 12.3 Hz, 1 H), 5.13 (d,
J = 12.3 Hz, 1 H), 5.82 (d, J = 7.6 Hz, 1 H), 7.27–7.38 (m, 5 H)
ppm. 13C NMR (100 MHz, CDCl3): δ = −5.5, −5.5, 17.5, 18.3,
25.9, 32.4, 35.9, 52.3, 67.0, 67.4, 128.0, 128.1, 128.5, 136.2,
156.2, 177.1 ppm. Minor rotamer (selected signals): 1H NMR
(400 MHz, CDCl3): δ = 0.91 (d, J = 5.0 Hz, 3 H), 3.64 (m, 1 H),
3.74 (m, 1 H), 4.33 (bs, 1 H), 5.82 (bs, 1 H) ppm. HRMS (CI):
calculated for C20H34NO5Si [M + H]+ 396.2201, found
396.2199.
(4R,5R)-4-[(tert-Butyldimethylsilyloxy)methyl]-5-phenyloxazoli-
din-2-one (23). To a solution of alcohol 22 29 (80.0 mg,
210 µmol) in dry THF (2 mL) NaH (60% dispersion in mineral
oil, 16.5 mg, 410 µmol) was added at 0 °C. The mixture was
Org. Biomol. Chem., 2016, 14, 6036–6054 | 6045

Paper
allowed to warm to room temperature overnight and was
quenched with 1 N KHSO4. The mixture was extracted with
diethyl ether (3 × 10 mL) and the combined organic layers
were washed with sat. aq. NaHCO3 and brine. The solvent was
removed in vacuo after drying over Na2SO4. Column chromato-
graphy (silica, petroleum ether/ethyl acetate 1 : 1) gave rise to
the oxazolidione 23 (44.0 mg, 143 µmol, 68%) as a colourless
resin. Rf (23): 0.29 (petroleum ether/ethyl acetate 8 : 2). [α]20D =
+27.3 (c = 1.0, CHCl3). 1H NMR (400 MHz, CDCl3): δ = 0.09 (s,
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3 H), 0.1 (s, 3 H), 0.91 (s, 9 H), 3.74 (m, 2 H), 3.79 (m, 1 H), 5.34
(d, J = 4.9 Hz, 1 H), 6.53 (bs, 1 H), 7.34–7.41 (m, 5 H) ppm. 13C
NMR (100 MHz, CDCl3): δ = −5.5, 18.1, 25.7, 61.1, 64.4, 80.0,
125.5, 128.6, 128.8, 139.0, 159.4 ppm. HRMS (CI): calculated for
C16H26NO3Si [M + H]+ 308.1676, found 308.1674.
(2R,3R)-N,N′-(Bis-Boc)-1-O-(tert-butyldimethylsilyl)-3-hydroxy-
tryptophanol (27). To a solution of D-N-Boc-O-(tert-butyldi-
methylsilyl)-serine methyl ester29 (334 mg, 1.00 mmol) in dry
toluene (10 mL) DIBAL-H (1 M in hexanes, 1.4 mL, 1.4 mmol)
was added at −78 °C over a period of 30 min. After TLC
showed complete consumption of the starting material, di-
chloromethane (10 mL) was added, followed by 1 M HCl. The
mixture was allowed to warm to 0 °C and was stirred at that
temperature until the solution became clear. After extraction
of the aq. layer with dichloromethane (3 × 10 mL), the com-
bined organic layers were washed with sat NaHCO3 dried over
Na2SO4 and the solvent was removed in vacuo. The crude alde-
hyde 21 was immediately used in the next step.
The iodinated Boc-indole 26 53 (1.03 g, 3.00 mmol) was dis-
solved in dry THF (10 mL) and cooled down to −10 °C. To this
solution, diisopropyl magnesium chloride (2 M in diethyl
ether, 1.50 mL, 3.00 mmol) was added dropwise. The mixture
was stirred for 45 min at that temperature and cooled down to
−20 °C.
The crude aldehyde 21 was dissolved in dry THF (5 mL) and
added to the solution of the indole at −20 °C. After stirring for
2 h at −20 °C, the mixture was allowed to warm up to 0 °C and
was quenched with 1 N aq. KHSO4. After extraction with ethyl
acetate (3 × 20 mL), the combined organic layers were washed
with sat. NaHCO3 and brine. After drying (Na2SO4), the solvent
was removed in vacuo and the residue was subjected to column
chromatography (silica, petroleum ether/ethyl acetate 9 : 1).
The pure major diastereomer 27 (339 mg, 651 µmol, 65%) was
obtained as a colourless resin. Rf (27): 0.31 ( petroleum ether/
ethyl acetate 8 : 2). (2S,3R)-27: [α]20 D = −19.1 (c = 1.0,
CHCl3).1H NMR (400 MHz, CDCl3): δ = 0.09 (s, 6 H), 0.94 (s,
9 H), 1.41 (s, 9 H), 1.65 (s, 9 H), 3.65 (s, 1 H), 3.82 (m, 1 H),
3.89 (dd, J = 10.1 Hz, 3.9 Hz, 1 H), 4.02 (m, 1 H), 5.24–5.30 (m,
2 H), 7.22 (dd, J = 8.0 Hz, 8.0 Hz, 1 H), 7.31 (dd, J = 8.0 Hz, 8.0
Hz, 1 H), 7.49 (s, 1 H), 7.64 (d, J = 8.0 Hz, 1 H), 8.16 (d, J = 8.0
Hz, 1 H) ppm. 13C NMR (100 MHz, CDCl3): δ = −5.5, 18.2, 25.9,
28.2, 28.4, 55.0, 64.9, 69.3, 79.7, 83.6, 115.3, 119.6, 121.9,
122.6, 123.3, 124.5, 128.6, 135.8, 149.5, 155.8 ppm. (2S,3S)-27:
1
H NMR (selected signals, 400 MHz, CDCl3): δ = (s, 3 H), 0.07
(s, 3 H), 0.92 (s, 9 H), 1.47 (s, 9 H), 4.07 (m, 1 H), 5.45 (d, J =
9.1 Hz, 1 H) ppm. 13C NMR (selected signals, 100 MHz,
CDCl3): 53.6, 63.6, 71.3, 124.6, 128.1, 136.0, 149.6, 156.3 ppm.
6046 | Org. Biomol. Chem., 2016, 14, 6036–6054
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HRMS (CI): calculated for C27H44N2O6Si [M]+ 520.2963, found
520.2991.
(2R,3R)-N,N′-(Bis-Boc)-1-O-(tert-butyldimethylsilyl)-3-acetoxy-
tryptophanol (28). To a solution of alcohol 27 (305 mg,
587 µmol) in dichloromethane (6 mL) triethylamine (90.0 µL,
645 µmol), acetic anhydride (61.0 µL, 645 mmol) and DMAP
(7.3 mg, 0.06 mmol) were added at 0 °C. The mixture was
allowed to warm to 0 °C overnight, diluted with dichloro-
methane (20 mL) and washed with 1 N KHSO4, sat. NaHCO3
and brine. The solvent was removed in vacuo after drying
(Na2SO4) and the residue was purified by column chromato-
graphy (silica, petroleum ether/ethyl acetate 95 : 5). Acetate 28
(276 mg, 491 µmol, 84%) was obtained as a yellow resin.
Rf (28): 0.62 ( petroleum ether/ethyl acetate 9 : 1). [α]20D = −61.4
(c = 1.0, CHCl3). Mixture of rotamers. Major rotamer: 1H NMR
(400 MHz, CDCl3): δ = −0.02 (s, 6 H), 0.01 (s, 6 H), 0.91 (s,
9 H), 1.46 (s, 9 H), 1.65 (s, 9 H), 2.06 (s, 3 H), 3.46 (dd, J = 10.2
Hz, 2.1 Hz, 1 H), 3.58 (dd, J = 10.2 Hz, 4.1 Hz, 1 H), 4.33 (m,
1 H), 5.03 (d, J = 9.8 Hz, 1 H), 6.23 (d, J = 8.8 Hz, 1 H), 7.24
(ddd, J = 8.0 Hz, 8.0 Hz, 1.0 Hz, 1 H), 7.33 (m, 1 H), 7.61 (s,
1 H), 7.81 (d, J = 8.0 Hz, 1 H), 8.17 (d, J = 8.0 Hz, 1 H) ppm.
13
C NMR (100 MHz, CDCl3): δ = −5.6, −5.6, 18.2, 21.1, 25.9,
28.2, 28.4, 54.4, 62.5, 69.1, 79.5, 83.9, 115.3, 117.1, 120.3,
122.9, 124.8, 128.3, 135.7, 149.4, 155.7, 170.5 ppm. Minor
rotamer: 1H NMR (selected signals, 400 MHz, CDCl3): δ = 1.36
(s, 9 H), 4.12 (m, 1 H), 4.72 (m, 1 H) ppm. HRMS (CI): calcu-
lated for C29H64N2O7Si [M]+ 562.3069, found 562.3073.
(2R,3R)-N,N′-(Bis-Boc)-1-O-(tert-butyldimethylsilyl)-3-(methoxy-
methylenoxy)-tryptophanol (29). To a solution of the secondary
alcohol 27 29 (1.12 g, 2.15 mmol) in dry dichloromethane
(10 mL) DIPEA (2.07 mL, 11.8 mmol) and MOMCl (820 µL,
10.8 mmol) were added at 0 °C. The mixture was warmed to
room temperature overnight and was diluted with ethyl acetate
(50 mL) after TLC showed full conversion. The resulting
mixture was washed with 1 N KHSO4, sat NaHCO3 and brine.
The solvent was evaporated after drying over Na2SO4. Column
chromatography (silica, petroleum ether/ethyl acetate 9 : 1) of
the crude residue gave rise to the protected alcohol 29
(971 mg, 1.72 mmol, 80%) as a colourless resin. Rf (29): 0.43
( petroleum ether/ethyl acetate 8 : 2). [α]20 D = −73.8 (c = 1.0,
CHCl3). 1H NMR (400 MHz, CDCl3): δ = 0.03 (s, 6 H), 0.04 (s,
6 H), 0.92 (s, 9 H), 1.40 (s, 9 H), 1.65 (s, 9 H), 3.39 (s, 3 H), 3.82
(dd, J = 9.8 Hz, 3.2 Hz, 1 H), 3.89 (dd, J = 9.8 Hz, 6.2 Hz, 1 H),
4.07 (m, 1 H), 4.56 (d, J = 6.7 Hz, 1 H), 4.63 (d, J = 6.7 Hz, 1 H),
5.05 (d, J = 9.3 Hz, 1 H), 5.11 (d, J = 5.6 Hz, 1 H), 7.21 (dd, J =
7.4 Hz, 7.4 Hz, 1 H), 7.30 (dd, J = 7.4 Hz, 7.4 Hz, 1 H), 7.55 (s,
1 H), 7.72 (d, J = 7.4 Hz, 1 H), 8.14 (bs, 1 H) ppm. 13C NMR
(100 MHz, CDCl3): δ = −5.5, −5.4, 18.2, 25.8, 28.1, 28.3, 55.6,
55.7, 62.3, 70.4, 79.1, 83.6, 94.3, 115.2, 118.4, 120.3, 122.6,
124.5, 128.9, 135.8, 149.5, 155.7 ppm. HRMS (CI): calculated
for C29H48N2O7Si [M]+ 564.3225, found 564.3225.
(2R,3R)-N,N′-(Bis-Boc)-3-(methoxymethylenoxy)-tryptophanol
(30). To a solution of the protected tryptophanol 29 (864 mg,
1.50 mmol) in THF (10 mL) TBAF trihydrate (473 mg,
1.50 mmol) was added at 0 °C. The mixture was warmed to
room temperature and the solvent was removed in vacuo after
This journal is © The Royal Society of Chemistry 2016

Organic & Biomolecular Chemistry
TLC showed complete conversion. Column chromatography
(silica, petroleum ether/ethyl acetate 1 : 1) of the residue gave
rise to the primary alcohol 30 (643 mg, 95%) as a colourless
resin. Rf (30): 0.31 ( petroleum ether/ethyl acetate 1 : 1). [α]20
D =
−85.3 (c = 1.0, CHCl3). 1H NMR (400 MHz, CDCl3): δ = 1.35 (s,
9 H), 1.66 (s, 9 H), 2.77 (bs, 1 H), 3.41 (s, 3 H), 3.82 (dd, J =
11.1 Hz, 5.2 Hz, 1 H), 3.89 (m, 1 H), 4.04 (m, 1 H), 4.56 (d, J =
6.7 Hz, 1 H), 4.63 (d, J = 6.7 Hz, 1 H), 5.12 (d, J = 5.1 Hz, 1 H),
5.20 (bs, 1 H), 7.22 (m, 1 H), 7.31 (m, 1 H), 7.57 (s, 1 H), 7.67
Published on 31 May 2016. Downloaded by National Taiwan University on 7/10/2020 2:17:27 PM.
(d, J = 7.7 Hz, 1 H), 8.13 (d, J = 7.8 Hz, 1 H) ppm. 13C NMR
(100 MHz, CDCl3): δ = 28.1, 55.9, 56.2, 63.1, 71.3, 79.6, 83.9,
94.3, 115.3, 117.8, 120.0, 122.7, 124.6, 124.7, 128.8, 135.7,
149.5, 156.1 ppm. HRMS (CI): calculated for C23H34N2O7 [M]+
450.2361, found 450.2370.
(2S,3R)-N,N′-(Bis-Boc)-3-(methoxymethylenoxy)-tryptophan
methyl ester (31). To a solution of the primary alcohol 30
(612 mg, 1.36 mmol) in acetonitrile (1.5 mL) and phosphate
buffer ( pH 6.4, 1.5 mL) TEMPO (42.0 mg, 272 µmol) and PhI-
(OAc)2 (BAIB, 44.0 mg, 136 µmol) were added at room tempera-
ture. The resulting mixture was cooled to 0 °C before NaClO2
(80%, 507 mg, 4.49 mmol) was added. After stirring for 15 min
at this temperature, 1 N KHSO4 was added and the layers were
separated. The aqueous layer was extracted with dichloro-
methane (3 × 5 mL) and the combined organic layers were
washed with 5% Na2S2O3. After drying over Na2SO4, the
solvent was evaporated and the residue was taken up in diethyl
ether (10 mL), before diazomethane was added. The solvent
was removed in vacuo and column chromatography (silica, pet-
roleum ether/ethyl acetate 85 : 15) gave rise to ester 31
(540 mg, 1.13 mmol, 83%) as a colourless resin. Rf (31): 0.43
( petroleum ether/ethyl acetate 8 : 2). [α]20 D = −59.8 (c = 1.0,
CHCl3). 1H NMR (400 MHz, CDCl3): δ = 1.28 (s, 9 H), 1.66 (s,
9 H), 3.32 (s, 3 H), 3.80 (s, 3 H), 4.53 (d, J = 6.9 Hz, 1 H), 4.62
(d, J = 6.9 Hz, 1 H), 4.66 (dd, J = 9.7 Hz, 2.5 Hz, 1 H), 5.46–5.50
(m, 2 H), 7.23 (m, 1 H), 7.30 (m, 1 H), 7.60 (s, 1 H), 7.65 (d, J =
7.8 Hz, 1 H), 8.11 (d, J = 7.7 Hz, 1 H) ppm. 13C NMR (100 MHz,
CDCl3): δ = 28.1, 28.1, 52.4, 55.7, 58.3, 71.5, 79.7, 83.9, 94.0,
115.3, 116.9, 119.7, 122.8, 124.5, 124.6, 128.8, 135.5, 149.4,
155.3, 170.9 ppm. HRMS (CI): calculated for C24H35N2O8
[M + H]+ 479.2388, found 479.2389.
(2R,3R)-N-Alloc-N′-(tert-prenyl)-1-O-(tert-butyldimethylsilyl)-3-
hydroxytryptophanol (36)
DIBAL-H-reduction. To a solution of (D)-N-Alloc-O-(tert-
butyldimethylsilyl)-serine methyl ester 35 29 (1.27 g,
4.00 mmol) in dry toluene (40 mL) DIBAL-H (1 M in hexanes,
4.00 mL, 4.00 mmol) was added at −78 °C over a period of
30 min. The mixture was stirred for 2–3 h at this temperature
and after TLC showed full conversion, dichloromethane
(20 mL) followed by 1 M HCl (10 mL) were added. The result-
ing mixture was allowed to warm to 0 °C and was stirred at this
temperature until both layers became clear. The layers were
separated and the aqueous layer was extracted with dichloro-
methane (3 × 30 mL). The combined organic layers were
washed with sat. NaHCO3 and dried over NaSO4. The solvent
was removed in vacuo and the crude aldehyde was used
immediately without further purification.
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Zinc-reagent. In a Schlenk flask, ZnCl2 (1.80 g, 13.2 mmol)
and LiCl (736 mg, 18.0 mmol) were dried with a heat gun
in vacuo and dissolved in dry THF (15 mL) after cooling down
to room temperature. Then, Mg-turnings (729 mg, 30.0 mmol)
and a solution of the iodoindole 34 (3.73 g, 12.0 mmol) in dry
THF (15 mL) were added subsequently. To the resulting
mixture dibromoethane (70 µL) was added, what caused an
exothermic reaction. After heat development had ceased, stir-
ring was continued for 2 h at room temperature and the excess
Mg-turnings were filtrated under inert gas atmosphere.
Addition reaction. The solution of the zinc-reagent was
cooled to −78 °C and a solution of the crude aldehyde in dry
THF (15 mL) was added dropwise. The reaction mixture was
allowed to warm up to room temperature, diluted with diethyl
ether (40 mL) and quenched with sat. aq. NH4Cl. The aqueous
layer was extracted with diethyl ether (3 × 50 mL) and the com-
bined organic layers were washed subsequently with sat. aq.
NaHCO3, 5% aq. Na2S2O3 and brine. After drying over Na2SO4,
the solvent was removed in vacuo and column chromatography
(silica, petroleum ether/ethyl acetate 9 : 1 to 8 : 2) gave rise to
the secondary alcohol 36 (1.41 g, 2.98 mmol, 75%, >90% ds).
The product proved to be extremely sensitive and had to be
used immediately in the next step or stored under inert atmo-
sphere below −20 °C. Rf (36): 0.20 ( petroleum ether/ethyl
D = −34.7 (c = 1.0, CHCl3). (2R,3R)-36: H NMR
acetate 8 : 2). [α]20 1
(400 MHz, CDCl3): δ = 0.08 (s, 6 H), 0.94 (s, 9 H), 1.74 (s, 6 H),
3.28 (bs, 1 H), 3.80 (m, 1 H), 3.87 (dd, J = 10.1 Hz, 4.1 Hz, 1 H),
4.14 (m, 1 H, 14 H), 4–58 (m, 1 H), 5.15 (d, J = 17.5 Hz, 1 H),
5.21 (d, J = 10.7 Hz, 1 H), 5.18–5.32 (m, 2 H), 5.36 (m, 1 H),
5.52 (d, J = 8.5 Hz, 1 H), 5.91 (m, 1 H), 6.13 (dd, J = 17.5 Hz,
10.7 Hz, 1 H), 7.08 (ddd, J = 7.1 Hz, 7.1 Hz, 1.2 Hz, 1 H), 7.12
(ddd, J = 6.9 Hz, 6.9 Hz, 1.4 Hz, 1 H), 7.33 (s, 1 H), 7.50 (d, J =
7.6 Hz, 1 H), 7.71 (d, J = 7.1 Hz, 1 H) ppm. 13C NMR (100 MHz,
CDCl3): δ = −5.6, 18.2, 25.8, 27.9, 28.0, 55.8, 59.0, 64.7, 65.5,
69.1, 113.5, 113.9, 117.5, 119.1, 119.3, 120.9, 123.1, 127.6,
132.9, 135.8, 156.6 ppm. (2R,3S)-36: (selected signals,
400 MHz, CDCl3): δ = 0.07 (s, 6 H), 0.93 (s, 9 H), 1.75 (s, 6 H),
3.14 (bs, 1 H), 3.66 (m, 2 H), 4.06 (m, 1 H), 4.63 (m, 2 H), 5.63
(d, J = 8.7 Hz, 1 H), 7.21 (m, 1 H), 7.38 (s, 1 H), 7.67 (d, J = 8.2
Hz, 1 H) ppm. 13C NMR (selected signals, 100 MHz, CDCl3):
δ = −5.7, 25.7, 28.0, 59.1, 63.4, 118.0, 119.2, 119.7, 127.2,
132.5 ppm. HRMS (CI): calculated for C26H40N2O4Si [M]+
472.2752, found 472.2753.
(2R,3R)-N-Alloc-N′-(tert-prenyl)-1-O-(tert-butyldimethylsilyl)-
3-[(tert-butyldimethylsilyl)oxy]-tryptophanol (37). A solution of
the secondary alcohol 36 (340 mg, 719 µmol) in dry dichloro-
methane (7 mL) was cooled down to −35 °C and a solution of
TBDMSOTf (180 µL, 791 µmol) and 2,6-lutidine (170 µL,
1.45 mmol) in dry dichloromethane (2 mL) was added drop-
wise. After addition was complete, the mixture was stirred for
5 min at this temperature and quenched with sat. aq. NaHCO3.
After warming up to room temperature, the mixture was
diluted with dichloromethane (20 mL) and the organic layer
was washed with subsequently with 1 N aq. KHSO4 and water.
After drying over Na2SO4 the solvent was removed in vacuo.
Column chromatography (silica, petroleum ether/ethyl acetate
Org. Biomol. Chem., 2016, 14, 6036–6054 | 6047

Paper
8 : 2) gave rise to the diastereomerically pure, protected alcohol
37 (369 mg, 629 µmol, 87%) as a colourless resin. Rf (37): 0.34
( petroleum ether/ethyl acetate 9 : 1). [α]20 D = −54.3 (c = 1.0,
CHCl3). Mixture of rotamers. Major rotamer: 1H NMR
(400 MHz, CDCl3): δ = −0.18 (s, 3 H), 0.06 (s, 3 H), 0.10 (s,
6 H), 0.89 (s, 9 H), 0.97 (s, 9 H), 1.72 (s, 3 H), 1.73 (s, 3 H), 3.63
(d, J = 6.2 Hz, 2 H), 3.92 (m, 1 H), 4.52 (d, J = 5.4 Hz, 2 H),
4.98–5.35 (m, 5 H), 5.38 (d, J = 3.7 Hz, 1 H), 5.91 (m, 1 H), 6.13
(dd, J = 17.4 Hz, 10.7 Hz, 1 H), 7.02–7.14 (m, 2 H), 7.21 (s, 1 H),
Published on 31 May 2016. Downloaded by National Taiwan University on 7/10/2020 2:17:27 PM.
7.47 (d, J = 8.1 Hz, 1 H), 7.65 (d, J = 7.4 Hz, 1 H) ppm. 13C NMR
(100 MHz, CDCl3): δ = −5.4, −5.3, −4.6, 18.2, 18.2, 25.8, 25.9,
28.0, 57.9, 58.9, 61.8, 65.3, 66.6, 113.4, 113.7, 115.3, 117.5,
118.8, 119.5, 120.6, 123.3, 127.6, 133.2, 135.6, 144.1,
156.0 ppm. Minor rotamer: 1H NMR (selected signals,
400 MHz, CDCl3): δ = −0.14 (s, 3 H), 0.05 (s, 3 H), 0.09 (s, 6 H),
1.76 (s, 3 H), 1.81 (s, 3 H), 3.85 (m, 1 H), 4.39 (m, 2 H), 5.42
(m, 1 H), 5.65 (m, 1 H), 6.13 (dd, J = 17.4 Hz, 10.7 Hz, 1 H),
7.24 (s, 1 H), 7.59 (d, J = 6.8 Hz, 1 H) ppm. 13C NMR (selected
signals, 100 MHz, CDCl3): δ = −5.3, 28.1, 58.2, 62.2, 117.0,
118.9, 120.6, 123.7, 127.3 ppm. HRMS (CI): calculated for
C28H45N2O4Si2 [M − C4H9]+ 529.2912, found 529.2922.
(2R,3R)-N-Alloc-N′-(tert-prenyl)-3-[(tert-butyldimethylsilyl)oxy]-
tryptophanol (38). To a solution of the fully protected diol 37
(1.05 g, 1.84 mmol) in methanol (20 mL) NH4F (683 mg,
18.5 mmol) was added at 0 °C. The mixture was allowed to
warm up to room temperature and was stirred for 48 h in total.
Ethyl acetate was added (60 mL) and the resulting mixture was
washed subsequently with water, sat. NaHCO3 and brine. After
drying over Na2SO4, the solvent was removed in vacuo and
column chromatography (silica, petroleum ether/ethyl acetate
8 : 2) of the residue yielded the primary alcohol 37 (722 mg,
1.53 mmol, 83%) as a colourless resin. Rf (37): 0.28 ( petroleum
D = −34.5 (c = 1.0, CHCl3). H NMR
ether/ethyl acetate 7 : 3). [α]20
1
(400 MHz, CDCl3): δ = −0.12 (s, 3 H), 0.06 (s, 3 H), 0.89 (s,
9 H), 1.72 (s, 3 H), 1.72 (s, 3 H), 3.75 (m, 2 H), 3.99 (m, 1 H),
4.53 (m, 2 H), 5.10 (d, J = 17.4 Hz, 1 H), 5.21 (d, J = 10.7 Hz,
1 H), 5.15–5.31 (m, 4 H), 5.88 (m, 1 H), 6.12 (dd, J = 17.4 Hz,
10.7 Hz, 1 H), 7.07 (m, 1 H), 7.11 (m, 1 H), 7.25 (s, 1 H),
7.48 (d, J = 7.8 Hz, 1 H), 7.62 (d, J = 7.4 Hz, 1 H) ppm. 13C NMR
(100 MHz, CDCl3): δ = −5.4, −5.7, 18.1, 25.8, 28.0, 58.1,
59.1, 63.2, 65.6, 68.6, 113.6, 113.9, 114.2, 117.6, 119.1, 119.3,
120.9, 123.6, 127.5, 132.8, 135.6, 143.9, 156.7 ppm. HRMS (CI):
calculated for C26H39N2O3Si [M − OH]+ 455.2724, found
455.2730.
(2S,3R)-N-Alloc-N′-(tert-prenyl)-3-[(tert-butyldimethylsilyl)oxy]-
tryptophan methyl ester (39). The primary alcohol 38 (300 mg,
643 µmol) was dissolved in a mixture of dry dichloromethane
(5 mL) and dry DMSO (4 mL). Triethylamine (450 µl,
3.17 mmol) was added and the mixture was cooled to 0 °C.
A solution of pyridine·SO3 (403 mg, 2.54 mmol) in dry DMSO
(2.5 mL) was added over a period of 15 min. The mixture was
allowed to warm up slowly to room temperature and after TLC
showed full conversion, ethyl acetate (30 mL) was added and
the resulting mixture was washed subsequently with 1 N
KHSO4, sat. NaHCO3, 5% Na2S2O3 and brine. After drying over
Na2SO4, the solvent was removed in vacuo at 0 °C and the
6048 | Org. Biomol. Chem., 2016, 14, 6036–6054
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Organic & Biomolecular Chemistry
resulting very sensitive crude aldehyde was immediately used
in the next step.
K2CO3 (262 mg, 1.90 mmol) was added to a solution of the
aldehyde in methanol (8 mL) at 0 °C. After addition of
2-methyl-2-butene (1 mL), N-iodosuccinimide (428 mg,
1.90 mmol) was added under light exclusion. After TLC
showed full conversion, the reaction was quenched with 5%
Na2S2O3 and extracted with dichloromethane (3 × 15 mL). The
combined organic layers were dried over Na2SO4 and the
solvent was removed in vacuo. Column chromatography (silica,
petroleum ether/ethyl acetate 9 : 1) gave rise to the methyl ester
39 (286 mg, 572 µmol, 90%) as a yellow oil. Rf (39): 0.34 ( pet-
D = −63.8 (c = 1.0, CHCl3).
roleum ether/ethyl acetate 8 : 2). [α]20
Mixture of rotamers. Major rotamer: 1H NMR (400 MHz,
CDCl3): δ = −0.15 (s, 3 H), 0.00 (s, 3 H), 0.88 (s, 9 H), 1.72 (s,
3 H), 1.73 (s, 3 H), 3.78 (s, 3 H), 4.45 (m, 2 H), 4.56 (dd, J =
9.4 Hz, 2.3 Hz, 1 H), 5.08 (d, J = 17.4 Hz, 1 H), 5.13–5.27 (m,
3 H), 5.61 (d, J = 9.4 Hz, 1 H), 5.66 (d, J = 2.3 Hz, 1 H), 5.85 (m,
1 H), 6.12 (dd, J = 17.4 Hz, 10.7 Hz), 7.06–7.14 (m, 2 H), 7.24 (s,
1 H), 7.48 (m, 1 H), 7.63 (m, 1 H) ppm. 13C NMR (100 MHz,
CDCl3): δ = −5.6, −4.7, 18.1, 25.6, 28.0, 28.1, 52.2, 59.0, 60.3,
65.6, 69.6, 113.5, 113.8, 113.9, 117.6, 118.8, 119.1, 120.8, 123.,
127.1, 132.8, 135.5, 143.9, 156.0, 171.2 ppm. Minor rotamer:
1
H NMR (selected signals, 400 MHz, CDCl3): δ = −0.12 (s, 3 H),
0.06 (s, 3 H), 0.90 (s, 9 H), 1.76 (s, 3 H), 1.80 (s, 3 H), 3.81 (s,
3 H), 4.02 (dd, J = 13.4 Hz, 5.4 Hz, 1 H), 4.30 (dd, J = 13.4 Hz,
5.1 Hz, 1 H), 4.53 (m, 1 H), 4.84 (d, J = 17.2 Hz, 1 H), 4.90 (d,
J = 10.3 Hz, 1 H), 5.36 (m, 1 H), 5.41 (d, J = 9.9 Hz, 1 H) ppm.
13
C NMR (selected signals, 100 MHz, CDCl3): δ = −5.4, −4.7,
25.7, 27.8, 51.8, 60.4, 65.4, 69.7, 113.6, 116.9, 118.3, 124.1,
126.8, 132.1, 155.6, 171.1 ppm. HRMS (CI): calculated for
C21H25N2O4 [M − TBDMSO]+ 369.1809, found 369.1813.
N-Cbz-(2S,4R)-[5-(tert-butyldimethylsilyl)oxy-leucyl]-(S)-alanyl-
(2S,3R)-(3-methoxyphenylalanyl)-(S)-valyl-(S)-N-methylleucine
methyl ester (49b). To the tetrapeptide 48 29 (118 mg,
194 µmol) HCl (4 M in dioxane, 500 µL, 2.0 mmol) was added
at room temperature. After TLC showed full conversion, the
solvent was removed in vacuo. The obtained hydrochloride was
dissolved in dry dichloromethane (3 mL), the acid 19 (70.0 mg,
177 µmol) was added and the mixture was cooled down to
0 °C. To the resulting solution, EDC·HCl (37.1 mg, 194), NMM
(22 µL, 0.19 mmol) and HOBt (3.4 mg, 22 µmol) were added
successively. The mixture was allowed to warm up to room
temperature, diluted with ethyl acetate (20 mL) and washed
with 1 N KHSO4, sat. NaHCO3 and brine. The solvent was
removed in vacuo after drying over Na2SO4. Flash chromato-
graphy (4 g C-18-silica, H2O/MeCN gradient) gave rise to the
pentapeptide 49b (123 mg, 139 µmol, 79%) as a colourless
resin. Rf (49b): 0.34 ( petroleum ether/ethyl acetate 1 : 1). [α]20
D =
−52.3 (c = 1.0, CHCl3). 1H NMR (500 MHz, CDCl3): δ = 0.04 (s,
3 H), 0.05 (s, 3 H), 0.87 (s, 9 H), 0.89 (d, J = 6.8 Hz, 3 H), 0.91
(d, J = 6.4 Hz, 3 H), 0.94 (d, J = 6.7 Hz, 3 H), 0.95 (d, J = 6.6 Hz,
3 H), 1.00 (d, J = 6.8 Hz, 3 H), 1.23 (d, J = 7.0 Hz, 3 H), 1.49 (m,
1 H), 1.64–1.85 (m, 5 H), 2.14 (m, 1 H), 2.99 (s, 3 H), 3.32 (s,
3 H), 3.40 (dd, J = 10.1 Hz, 6.4 Hz, 1 H), 3.53 (dd, J = 10.1 Hz,
4.7 Hz, 1 H), 3.69 (s, 3 H), 4.33 (m, 1 H), 4.34 (m, 1 H), 4.69
This journal is © The Royal Society of Chemistry 2016

Organic & Biomolecular Chemistry
(dd, J = 7.7 Hz, 3.6 Hz, 1 H), 4.80–4.84 (m, 2 H), 5.11 (s, 1 H),
5.35 (dd, J = 10.5 Hz, 5.3 Hz, 1 H), 5.95 (d, J = 6.7 Hz, 1 H), 6.67
(d, J = 5.9 Hz, 1 Hd), 6.78 (d, J = 7.7 Hz, 1 H), 7.19–7.36 (m,
11 H) ppm. 13C NMR (125 MHz, CDCl3): δ = −5.5, −5.4, 17.3,
17.7, 18.2, 18.4, 19.5, 21.4, 23.3, 24.8, 25.9, 31.2, 31.4, 32.2,
36.1, 36.9, 49.4, 52.1, 53.4, 54.1, 54.5, 57.5, 57.8, 67.0, 67.7,
81.3, 126.8, 128.1, 128.1, 128.2, 128.3, 128.5, 136.2, 136.8,
156.5, 168.4, 171.5, 172.0, 172.0, 172.1 ppm. HRMS (ESI): calcu-
lated for C46H74N5O10Si [M + H]+ 884.5199, found 884.5206.
Published on 31 May 2016. Downloaded by National Taiwan University on 7/10/2020 2:17:27 PM.
N-Alloc-(2S,3R)-{N′-(tert-prenyl)-3-[(tert-butyldimethylsilyl)oxy]-
tryptophanyl}-N-methyl-(2S,4R)-[5-(tert-butyldimethylsilyl)oxy-
leucyl]-(S)-alanyl-(2S,3R)-(3-methoxyphenylalanyl)-(S)-valyl-(S)-
N-methyl-leucine methyl ester (50a). To a solution of the
methyl ester 39 (34.2 mg, 68.0 µmol) in methanol (700 µL) aq.
NaOH (1 M, 125 µL, 125 µmol) was added. The mixture was
allowed to warm to room temperature an stirring was contin-
ued for 2 d. After TLC showed complete conversion, pH-buffer
(1 mM phosphate buffer, pH 6.4) was added to the solution
and the solvent was evaporated in vacuo.
The pentapeptide 49a (67 mg, 75.1 µmol) was dissolved in
methanol (1 mL) and 10% Pd/C (7 mg) was added under nitro-
gen atmosphere. The nitrogen was replaced by hydrogen and
the mixture was stirred for 2–3 h at 1 atm until TLC showed
complete conversion. The solution was filtered over celite and
the solvent was removed in vacuo. The crude amine was dis-
solved in dry dichloromethane (1 mL) together with the crude
acid. The mixture was cooled down to −20 °C and DIPEA
(12 µL, 71 µmol) followed by BEP (21.0 mg, 76.7 µmol) were
added. The mixture was warmed up to room temperature over-
night and diluted with ethyl acetate (10 mL), after LCMS-analy-
sis showed full conversion. The mixture was washed with 1 N
KHSO4, sat. NaHCO3 and brine. The solvent was removed
in vacuo after drying over Na2SO4. Flash chromatography (4 g
C-18-silica, H2O/MeCN gradient) gave rise to the hexapeptide
50a (46.1 mg, 37.4 µmol, 55%) as a colourless resin. If the reac-
tion is conducted with the purified acid 40, the peptide is
obtained in 69% yield (from 40). Rf (50a): 0.77 (dichloro-
methane/diethyl ether 1 : 1). [α]20 D = −62.8 (c = 1.0, CHCl3).
Mixture of rotamers. Major rotamer: 1H NMR (500 MHz,
CDCl3): δ = −0.26 (s, 3 H), 0.00 (s, 3 H), 0.01 (s, 3 H), 0.10 (s,
3 H), 0.73 (d, J = 6.6 Hz, 3 H), 0.86 (s, 9 H), 0.91 (s, 9 H),
0.86–0.96 (m, 12 H), 1.19 (m, 1 H), 1.21 (d, J = 7.1 Hz, 3 H),
1.35 (m, 1 H), 1.48 (m, 1 H), 1.68 (s, 3 H), 1.69 (s, 3 H), 1.72
(m, 2 H), 1.91 (m, 1 H), 2.10 (m, 1 H), 2.60 (s, 3 H), 2.96 (s,
3 H), 3.30 (s, 3 H), 3.31 (m, 1 H), 3.37 (dd, J = 9.9 Hz, 4.9 Hz,
1 H), 3.68 (s, 3 H), 4.26 (m, 1 H), 4.45–4.57 (m, 3 H), 4.65 (dd,
J = 7.6 Hz, 3.6 Hz, 1 H), 4.74–4.83 (m, 3 H), 5.03–5.38 (m, 6 H),
5.65 (d, J = 5.3 Hz, 1 H), 5.89 (m, 1 H), 6.08 (dd, J = 17.4 Hz,
10.5 Hz, 1 H), 6.84 (d, J = 7.6 Hz, 1 H), 7.00–7.09 (m, 3 H),
7.15–7.29 (m, 5 H), 7.39 (d, J = 8.7 Hz, 1 H), 7.44 (d, J = 8.3 Hz,
1 H), 7.70 (d, J = 7.2 Hz, 1 H), 7.93 (d, J = 6.5 Hz, 1 H) ppm.
13
C NMR (125 MHz, CDCl3): δ = −5.4, −5.4, −5.3, 17.2, 17.2,
17.3, 18.2, 18.3, 19.5, 21.4, 23.3, 24.8, 25.8, 25.9, 28.0, 28.2,
29.1, 31.2, 31.4, 32.2, 36.9, 49.7, 52.0, 54.1, 54.5, 56.9, 57.4,
57.5, 57.8, 59.1, 66.1, 67.4, 71.8, 81.0, 113.5, 113.6, 113.9,
117.8, 119.4, 121.3, 121.5, 123.2, 126.8, 127.5, 128.2, 128.2,
This journal is © The Royal Society of Chemistry 2016
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Paper
132.3, 135.9, 137.0, 143.7, 156.7, 168.5, 168.6, 168.6, 170.4,
170.6, 171.6, 171.6, 171.6, 171.9, 172.1, 172.1 ppm. Minor
rotamer: 1H NMR (selected signals, 500 MHz, CDCl3): δ =
−0.97 (m, 1 H), −0.34 (s, 3 H), −0.05 (s, 3 H), −0.04 (s, 3 H),
0.32 (d, J = 6.6 Hz, 3 H), 0.79 (s, 9 H), 0.83 (s, 9 H), 0.98 (d, J =
6.8 Hz, 3 H), 1.05 (d, J = 7.1 Hz, 3 H), 1.61–1.66 (m, 4 H), 2.19
(m, 1 H), 2.49 (m, 1 H), 2.73 (s, 3 H), 2.86 (dd, J = 7.3 Hz,
3.5 Hz, 1 H), 3.01 (s, 3 H), 3.27 (s, 3 H), 3.68 (s, 3 H), 3.87 (m,
1 H), 4.59 (dd, J = 7.2 Hz, 3.5 Hz, 1 H), 5.04 (m, 1 H), 6.00–6.07
(m, 2 H), 6.72 (d, J = 7.4 Hz, 1 H), 7.35 (d, J = 8.8 Hz, 1 H), 7.85
(d, J = 7.1 Hz, 1 H) ppm. 13C NMR (selected signals, 125 MHz,
CDCl3): δ = −4.9, −4.7, 14.9, 17.3, 18.0, 18.2, 19.4, 21.3, 23.3,
24.7, 25.6, 25.9, 27.9, 28.2, 29.7, 31.2, 31.8, 50.0, 54.2, 54.5,
57.2, 59.0, 65.6, 68.3, 69.3, 81.4, 112.3, 114.0, 117.4, 119.6,
121.1, 124.2, 126.9, 127.7, 132.8, 135.3, 136.9, 143.8,
156.4 ppm. LC-MS: Luna 3μ C18, 50 × 4.6 mm, 0.9 ml min−1,
MeCN/H2O gradient from 10% MeCN to 100% MeCN in
5 min, 100% MeCN for 15 min, tR (50a) = 14.19 min, m/z =
1255 ([M + Na]+). HRMS (ESI): calculated for C65H106N7O12Si2
[M + H]+ 1232.7433, found 1232.7359.
N-Alloc-(2S,3R)-{N′-(tert-prenyl)-3-[(tert-butyldimethylsilyl)oxy]-
tryptophanyl}-(2S,4R)-[5-(tert-butyldimethylsilyl)oxy-leucyl]-(S)-
alanyl-(2S,3R)-(3-methoxyphenylalanyl)-(S)-valyl-(S)-N-methyl-
leucine methyl ester (50b). In analogy to the synthesis of 50a,
the methyl ester 39 (65.0 mg, 130 µL) was saponified with
150 µL 1 N aq. NaOH and worked up using a buffer ( pH 6.4).
The pentapeptide 49b (117 mg, 132 µmol) was dissolved in
methanol (1.5 mL) and 10% Pd/C (7.0 mg) was added under
nitrogen. The nitrogen was replaced by a hydrogen atmosphere
and the mixture was stirred until TLC showed complete con-
version. After filtering over celite, the solvent was removed
in vacuo. The crude amine was dissolved in dry dichloro-
methane (1.5 mL) together with the buffer salt of the crude
acid 39. The mixture was cooled to −20 °C and EDC·HCl
(25.0 mg, 132 µmol) and HOBt (3.5 mg, 24 µmol) were added.
The mixture was allowed to warm up to room temperature and
diluted with ethyl acetate (10 mL) after LC-MS-analysis showed
complete conversion. The solution was washed with 1 N aq.
KHSO4, sat. aq. NaHCO3 and brine. After drying over Na2SO4,
the solvent was removed in vacuo. Flash chromatography (4 g
C-18-silica, H2O/MeCN gradient) gave rise to the hexapeptide
50b (85.2 mg, 69.9 µmol, 54%) as a colourless resin. Rf (50b):
0.42 (ethyl acetate/petroleum ether 1 : 1). [α]20 D = −62.8 (c = 1.0,
CHCl3). 1H NMR (500 MHz, CDCl3): δ = −0.07 (s, 3 H), 0.05 (s,
6 H), 0.07 (s, 3 H), 0.86–0.98 (m, 12 H), 0.89 (s, 9 H), 0.92 (s,
9 H), 1.00 (d, J = 6.8 Hz, 3 H), 1.17 (d, J = 7.1 Hz, 3 H),
1.46–1.53 (m, 2 H), 1.66 (s, 3 H), 1.68 (s, 3 H), 1.68–1.79 (m,
3 H), 1.95 (m, 1 H), 2.17 (m, 1 H), 3.01 (s, 3 H), 3.35 (s, 3 H),
3.45 (dd, J = 10.0 Hz, 5.6 Hz, 1 H), 3.50 (dd, J = 10.0 Hz, 5.5 Hz,
1 H), 3.69 (s, 3 H), 4.23 (m, 1 H), 4.40 (m, 1 H), 4.51–4.54 (m,
3 H), 4.69 (dd, J = 7.7 Hz, 3.5 Hz, 1 H), 4.81 (dd, J = 8.8 Hz,
6.3 Hz, 1 H), 4.89 (d, J = 3.5 Hz, 1 H), 5.08 (d, J = 17.4 Hz, 1 H),
5.19 (d, J = 10.7 Hz, 1 H), 5.20 (m, 1 H), 5.27 (m, 1 H), 5.37 (dd,
J = 10.5 Hz, 5.3 Hz, 1 H), 5.58 (d, J = 6.3 Hz, 1 H), 5.70 (d, J =
2.7 Hz, 1 H), 5.88 (m, 1 H), 6.10 (dd, J = 17.4 Hz, 10.7 Hz, 1 H),
6.71 (d, J = 6.7 Hz, 1 H), 6.78 (d, J = 7.6 Hz, 1 H), 7.02 (dd, J =
Org. Biomol. Chem., 2016, 14, 6036–6054 | 6049

Paper
7.3 Hz, 7.3 Hz, 1 H), 7.08 (dd, J = 7.4 Hz, 7.3 Hz, 1 H),
7.20–7.30 (m, 6 H), 7.34 (s, 1 H), 7.36 (d, J = 8.8 Hz, 1 H), 7.46
(d, J = 7.3 Hz, 1 H), 7.56 (d, J = 7.3 Hz, 1 H) ppm. 13C NMR
(125 MHz, CDCl3): δ = −5.3, −5.4, −4.8, 17.1, 17.4, 17.8, 18.2,
18.3, 19.5, 21.4, 23.3, 24.8, 25.7, 26.0, 28.0, 31.2, 31.3, 32.2,
36.1, 37.0, 49.5, 51.9, 52.1, 54.3, 54.5, 57.5, 57.8, 59.2, 61.0,
66.0, 67.6, 68.6, 81.2, 113.1, 113.5, 113.9, 118.0, 119.0, 119.2,
120.9, 124.0, 126.8, 126.9, 128.1, 128.3, 132.5, 135.5, 137.1,
143.9, 156.5, 168.6, 168.9, 171.4, 171.7, 172.0, 172.2 ppm.
Published on 31 May 2016. Downloaded by National Taiwan University on 7/10/2020 2:17:27 PM.
LC-MS: Luna 3μ C18, 50 × 4.6 mm, 0.9 ml min−1, MeCN/H2O
gradient from 10% MeCN to 100% MeCN in 5 min, 100%
MeCN for 15 min, tR (50b) = 13.76 min, m/z = 1241 ([M + Na]+).
HRMS (ESI): calculated for C64H104N7O12Si2 [M + H]+
1218.7276, found 1218.7254.
N-Alloc-(2S,3R)-(2-amino-3,5-dimethylhex-4-enoyl)-(2S,3R)-
(2-amino-3,5-dimethylhex-4-enoyl)-(2S,3R)-{N′-(tert-prenyl)-3-
[(tert-butyldimethylsilyl)oxy]-tryptophanyl}-N-methyl-(2S,4R)-
[5-(tert-butyldimethylsilyl)oxy-leucyl]-(S)-alanyl-(2S,3R)-(3-methoxy-
phenylalanyl)-(S)-valyl-(S)-N-methyl-leucine methyl ester (51a). To
a solution of the hexapeptide 50a (101 mg, 81.9 µmol) in a
mixture of acetonitrile (500 µL) and water (400 µL) TPPTS
(1.9 mg, 3.2 µmol), Pd(OAc)2 (20 mM in MeCN, 80 µL,
1.6 µmol) and diethylamine (42 µL, 410 µmol) were sub-
sequently added. After stirring for 90 min at room temperature
(LC-MS-analysis), the solvent was removed in vacuo and the
residue was dissolved in dry dichloromethane (1.2 mL). To the
resulting solution the acid (2S,3R)-5 (21.7 mg, 90.0 µmol) was
added, followed by HOBt (11.7 mg, 34.0 µmol) and EDC·HCl
(18.9 mg, 98.6 µmol) at 0 °C. The mixture was allowed to warm
to room temperature and was diluted with ethyl acetate (5 mL)
after LC-MS-analysis showed full conversion. The resulting
solution was washed with water, sat aq. NaHCO3 and brine.
The organic layer was dried over Na2SO4 and the solvent was
removed in vacuo. Flash chromatography (4 g C-18-silica, H2O/
MeCN gradient) gave rise to the heptapeptide 51a (100 mg,
72.9 µmol, 89%) as a colourless resin. Rf (51a): 0.60 (dichloro-
methane/diethyl ether 1 : 1). [α]20 D = −47.0 (c = 1.0, CHCl3).
Mixture of rotamers. Major rotamer: 1H NMR (500 MHz,
CDCl3): δ = −0.26 (s, 3 H), −0.05 (s, 3 H), −0.03 (s, 3 H), 0.10 (s,
3 H), 0.83 (s, 9 H), 0.93 (s, 9 H), 0.77–0.99 (m, 15 H), 1.07 (d,
J = 6.3 Hz, 3 H), 1.19 (m, 1 H), 1.23 (d, J = 7.1 Hz, 3 H), 1.40 (m,
1 H), 1.48 (m, 1 H), 1.56 (s, 3 H), 1.65 (s, 3 H), 1.68 (s, 3 H),
1.68 (s, 3 H), 1.73 (m, 2 H), 1.96 (m, 1 H), 2.12 (m, 1 H), 2.57
(s, 3 H), 2.86 (m, 1 H), 2.97 (s, 3 H), 3.27 (s, 3 H), 3.31 (m, 2 H),
3.68 (s, 3 H), 4.16 (m, 1 H), 4.26 (m, 1 H), 4.51–4.68 (m, 4 H),
4.73–4.82 (m, 3 H), 4.94 (d, J = 11.0 Hz, 1 H), 5.05–5.40 (m,
6 H), 5.53 (d, J = 10.0 Hz, 1 H), 5.92 (m, 1 H), 6.07 (m, 1 H),
6.68 (bs, 1 H), 6.76 (d, J = 7.5 Hz, 1 H), 7.00–7.09 (m, 3 H),
7.13–7.27 (m, 5 H), 7.35 (d, J = 8.6 Hz, 1 H), 7.45 (d, J = 8.2 Hz,
1 H), 7.82 (d, J = 7.6 Hz, 1 H), 8.34 (d, J = 5.9 Hz, 1 H) ppm.
13
C NMR (125 MHz, CDCl3): δ = −5.4, −5.4, −5.3, −5.2, 17.2,
17.2, 17.3, 17.4, 18.0, 18.0, 18.2, 19.5, 21.4, 23.3, 24.7, 25.7,
25.8, 25.9, 28.0, 28.2, 29.2, 21.2, 31.2, 31.4, 32.2, 35.8, 37.0,
49.9, 52.1, 54.1, 54.4, 56.5, 57.4, 57.5, 57.8, 59.0, 59.1, 65.9,
67.1, 72.0, 81.1, 112.2, 113.7, 114.0, 117.9, 119.4, 121.1, 121.4,
123.2, 124.5, 126.8, 127.4, 128.2, 128.2, 132.7, 134.6, 135.9,
6050 | Org. Biomol. Chem., 2016, 14, 6036–6054
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Organic & Biomolecular Chemistry
137.0, 143.7, 155.6, 168.3, 168.5, 168.9, 169.7, 170.1, 170.2,
170.4, 171.1, 171.4, 171.5, 171.6, 171.9, 172.0, 172.1 ppm. (Car-
bonyl signals not distinguishable from the rotameric signals).
Minor rotamer 1H NMR (selected signals, 500 MHz, CDCl3):
δ = −1.05 (m, 1 H), −0.34 (s, 3 H), −0.02 (s, 3 H), 0.00 (s, 3 H),
0.01 (s, 3 H), 0.32 (d, J = 6.6 Hz, 3 H), 0.80 (s, 9 H), 0.86 (s,
9 H), 1.12 (d, J = 7.1 Hz, 3 H), 1.60 (m, 1 H), 2.44 (m, 1 H), 2.78
(s, 3 H), 2.86 (m, 1 H), 2.99 (s, 3 H), 3.32 (s, 3 H), 3.69 (s, 3 H),
3.87 (m, 1 H), 4.02 (m, 1 H), 4.59 (dd, J = 7.2 Hz, 3.5 Hz, 1 H),
4.97 (d, J = 9.4 Hz, 1 H), 7.32 (d, J = 8.1 Hz, 1 H), 7.44 (d, J = 8.5
Hz, 1 H), 7.68 (d, J = 8.5 Hz, 1 H) ppm. 13C NMR (selected
signals, 125 MHz, CDCl3): δ = −4.9, −4.8, 15.0, 19.5, 24.8, 25.7,
28.2, 31.8, 49.4, 52.1, 54.0, 54.5, 55.6, 65.6, 68.3, 69.2, 81.4,
113.6, 114.0, 117.9, 119.4, 119.5, 124.2, 124.9, 127.0, 127.6,
128.1, 128.3, 132.8, 133.8, 135.4, 136.8, 143.8, 155.8 ppm.
LC-MS: Luna 3μ C18, 50 × 4.6 mm, 0.9 ml min−1, MeCN/H2O
gradient from 10% MeCN to 100% MeCN in 5 min, 100%
MeCN for 15 min, tR (51a) = 15.57 min, m/z = 1394 ([M + Na]+).
HRMS (ESI): calculated for C73H118N8O13Si2Na [M + Na]+
1393.8249, found 1393.8240.
N-Alloc-(2S,3R)-(2-amino-3,5-dimethylhex-4-enoyl)-(2S,3R)-
(2-amino-3,5-dimethylhex-4-enoyl)-(2S,3R)-{N′-(tert-prenyl)-3-
[(tert-butyldimethylsilyl)oxy]-tryptophanyl}-(2S,4R)-[5-(tert-
butyldimethylsilyl)oxy-leucyl]-(S)-alanyl-(2S,3R)-(3-methoxyphenyl-
alanyl)-(S)-valyl-(S)-N-methyl-leucine methyl ester (51b). In
analogy to the synthesis of 51a, the hexapeptide 50b (75.0 mg,
61.6 µmol) was dissolved in a mixture of acetonitrile (500 µL)
and water (300 µL) and was deprotected and worked up using
TPPTS (2.3 mg, 4 µmol), Pd(OAc)2 (20 mM in MeCN, 100 µL,
2 µmol) and diethylamine (32 µL, 310 µmol). The obtained
residue was dissolved in dry dichloromethane (800 µL). To this
solution the acid (2S,3R)-5 (16.4 mg, 68.2 µmol), HOBt
(8.4 mg, 62.2 µmol) and EDC·HCl (13.1 mg, 68.2 µmol) were
added at 0 °C. The mixture was allowed to warm to room temp-
erature and was diluted with ethyl acetate (5 mL), after LC-MS-
analysis showed full conversion. The mixture was washed with
water, sat. NaHCO3 and brine. The solvent was removed
in vacuo after drying over Na2SO4. Flash chromatography (4 g
C-18-silica, H2O/MeCN gradient) gave rise to the heptapeptide
51b (65.1 mg, 47.9 µmol, 78%) as a colourless resin. Rf (51b):
0.56 (ethyl acetate/petroleum ether 1 : 1).[α]20 D = −73.5 (c = 1.0,
CHCl3). 1H NMR (500 MHz, CDCl3): δ = −0.09 (s, 3 H), 0.03 (s,
3 H), 0.04 (s, 3 H), 0.06 (s, 3 H), 0.56 (d, J = 6.8 Hz, 1 H),
0.86–0.94 (m, 6 H), 0.88 (s, 9 H), 0.92 (s, 9 H), 0.98 (d, J = 6.4
Hz, 3 H), 1.01 (d, J = 6.8 Hz, 3 H), 1.06 (d, J = 6.6 Hz, 3 H), 1.19
(d, J = 7.2 Hz, 3 H), 1.48 (s, 3 H), 1.49–1.58 (m, 2 H), 1.59 (s, 3
H), 1.68 (s, 3 H), 1.70 (s, 3 H), 1.74 (m, 2 H), 1.90–1.99 (m, 2
H), 2.30 (m, 1 H), 2.77 (m, 1 H), 3.03 (s, 3 H), 3.35 (s, 3 H), 3.36
(m, 1 H), 3.60 (dd, J = 4.2 Hz, 9.8 Hz, 1 H), 3.68 (s, 3 H), 3.86
(m, 1 H), 4.15 (m, 1 H), 4.40 (m, 1 H), 4.54 (dd, J = 13.0 Hz, 5.9
Hz, 1 H), 4.62 (dd, J = 6.1 Hz, 1.6 Hz, 1 H), 4.66–4.70 (m, 2 H),
4.79 (dd, J = 8.8 Hz, 7.5 Hz, 1 H), 4.82 (d, J = 9.3 Hz, 1 H), 5.03
(d, J = 2.2 Hz, 1 H), 5.09 (d, J = 17.3 Hz, 1 H), 5.11 (bs, 1 H),
5.19 (d, J = 10.7 Hz, 1 H), 5.30–5.40 (m, 3 H), 5.82 (d, J = 1.6
Hz, 1 H), 5.96 (m, 1 H), 6.06 (dd, J = 17.3 Hz, 10.7 Hz, 1 H),
6.99 (m, 1 H), 7.05–7.12 (m, 4 H), 7.18–7.28 (m, 5 H), 7.36 (s,
This journal is © The Royal Society of Chemistry 2016

Organic & Biomolecular Chemistry
1 H), 7.43–7.46 (m, 3 H), 7.70 (d, J = 7.8 Hz, 1 H) ppm. 13C
NMR (125 MHz, CDCl3): δ = −5.4, −5.4, −5.0, 15.7, 17.0, 17.5,
17.8, 18.0, 18.1, 18.3, 19.5, 21.5, 23.3, 24.7, 25.8, 25.9, 26.0,
27.9, 28.0, 30.7, 31.3, 32.1, 33.3, 36.1, 37.2, 49.5, 52.0, 52.7,
54.5, 54.7, 57.5, 58.2, 59.1, 61.0, 61.2, 66.7, 67.3, 68.5, 81.3,
113.7, 114.0, 114.2, 119.0, 119.2, 119.4, 121.2, 122.8, 123.9,
127.0, 127.0, 127.7, 128.2, 131.9, 135.3, 135.8, 138.0, 143.7,
157.3, 169.4, 170.2, 171.6, 171.9, 172.1, 172.4, 172.6 ppm.
LC-MS: Luna 3μ C18, 50 × 4.6 mm, 0.9 ml min−1, MeCN/H2O
Published on 31 May 2016. Downloaded by National Taiwan University on 7/10/2020 2:17:27 PM.
gradient from 10% MeCN to 100% MeCN in 5 min, 100%
MeCN for 15 min, tR (51b) = 15.42 min, m/z = 1383 ([M + Na]+).
HRMS (ESI): calculated for C72H117N8O13Si2 [M + H]+
1357.8273, found 1357.8279.
O-O′-Bis-(tert-butyldimethylsilyl)-cyclomarin C (52a). The
linear heptapeptide 51a (97.0 mg, 70.7 µmol) was dissolved in
methanol (1 mL) and aq. NaOH (1 M, 80 µL, 80 µmol) was
added. The mixture was stirred for 2 d at room temperature.
The solvent was removed in vacuo and taken up in acetonitrile
(400 µL) and 1 mM phosphate buffer ( pH 6.4, 350 µL). To the
resulting solution TPPTS (1.6 mg, 2.8 µmol), Pd(OAc)2 (20 mM
in MeCN, 70 µL, 1.4 µmol) and diethylamine (37 µL,
360 µmol) were added at room temperature. After stirring for
2 h at room temperature (LC-MS-analysis), the solvent was
removed via lyophilisation. The obtained residue was dissolved
in dry dichloromethane (25 mL).
PyBOP (73.9 mg, 140 µmol) and DIPEA (24 µL, 140 µmol)
were dissolved in dry dichloromethane (55 mL) in a three-
necked-flask with pressure equalization. The solution of the
peptide was added with a syringe pump to the coupling
reagent over a period of 12 h. After the addition was complete,
stirring was continued for additional 6 h. The solvent was
removed in vacuo and flash chromatography (4 g C-18-silica,
H2O/MeCN gradient) gave rise to the cyclic heptapeptide 52a
(67.2 mg, 53.5 µmol, 76% from 51a) as a colourless resin. Rf
(52a): 0.60 (dichloromethane/diethyl ether 3 : 2). [α]20 D = −70.1
(c = 1.0, CHCl3). 1H NMR (500 MHz, CDCl3): δ = −1.02 (ddd, J =
13.0 Hz, 10.1 Hz, 2.8 Hz, 1 H), −0.30 (s, 3 H), −0.07 (s, 3 H),
−0.05 (s, 3 H), 0.13 (s, 3 H), 0.16 (d, J = 6.6 Hz, 3 H), 0.67 (d, J =
6.5 Hz, 3 H), 0.81 (s, 9 H), 0.92 (s, 9 H), 0.96 (d, J = 6.7 Hz,
3 H), 0.98 (d, J = 5.7 Hz, 3 H), 0.99 (d, J = 5.8 Hz, 3 H), 1.01 (m,
1 H), 1.08 (d, J = 6.6 Hz, 3 H), 1.19 (m, 1 H), 1.26 (d, J = 7.5 Hz,
3 H), 1.28 (s, 3 H), 1.54 (ddd, J = 13.0 Hz, 13.0 Hz, 4.5 Hz, 1 H),
1.62 (s, 3 H), 1.63–1.73 (m, 3 H), 1.65 (s, 3 H), 1.74 (s, 3 H),
2.26 (m, 1 H), 2.41 (ddd, J = 13.1 Hz, 11.7 Hz, 4.3 Hz, 1 H), 2.47
(m, 1 H), 2.49 (s, 3 H), 2.84 (m, 1 H), 2.87 (s, 3 H), 3.36 (s, 3 H),
4.06 (dd, J = 10.6 Hz, 9.5 Hz, 1 H), 4.34 (dd, J = 9.1 Hz, 9.1 Hz,
1 H), 4.40 (dd, J = 9.1 Hz, 1.7 Hz, 1 H), 4.52 (dd, J = 11.9 Hz,
2.6 Hz, 1 H), 4.69–4.77 (m, 2 H), 4.81 (dd, J = 4.8 Hz, 4.8 Hz,
1 H), 5.01–5.08 (m, 2 H), 5.09 (d, J = 4.8 Hz, 1 H), 5.18 (d, J =
10.7 Hz, 1 H), 5.21 (d, J = 9.1 Hz, 1 H), 6.03 (dd, J = 17.4 Hz,
10.7 Hz, 1 H), 6.45 (d, J = 1.7 Hz, 1 H), 6.98–7.02 (m, 2 H), 7.06
(m, 1 H), 7.17–7.19 (m, 3 H), 7.21–7.27 (m, 3 H), 7.43 (d, J = 8.4
Hz, 1 H), 7.71 (d, J = 7.8 Hz, 1 H), 7.87 (d, J = 9.1 Hz, 1 H), 7.98
(d, J = 9.4 Hz, 1 H), 8.18 (d, J = 10.6 Hz, 1 H) ppm. 13C NMR
(125 MHz, CDCl3): δ = −5.4, −5.4, −5.3, −4.9, 15.3, 18.1, 18.3,
18.4, 18.8, 19.5, 20.0, 20.5, 22.1, 23.6, 25.4, 25.8, 25.9, 28.0,
This journal is © The Royal Society of Chemistry 2016
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28.2, 29.2, 29.4, 30.9, 31., 32.0, 35.1, 39.0, 50.1, 55.7, 55.9, 56.5,
57.7, 57.8, 58.6, 58.7, 59.0, 68.2, 72.1, 79.8, 112.0, 113.7, 114.1,
118.1, 119.3, 121.3, 123.0, 124.8, 127.4, 127.8, 128.2, 128.6,
134.6, 135.2, 135.9, 143.6, 168.0, 168.2, 169.1, 169.5, 170.6,
171.4, 172.4 ppm. LC-MS: Luna 3μ C18, 50 × 4.6 mm, 0.9 ml
min−1, 100% MeCN, tR (52a) = 14.50 min, m/z = 1278
([M + Na]+). HRMS (ESI): calculated for C68H111N8O10Si2
[M + H]+ 1255.7956, found 1255.7938.
O-O′-Bis-(tert-butyldimethylsilyl)-cyclomarin D (52b). In
analogy to the synthesis of 52a, the linear heptapeptide 51b
(58.0 mg, 42.7 µmol) was dissolved in methanol (500 µL) and
saponified using 1 M NaOH (50 µl, 50 µmol). The reaction was
worked up as described, after TLC showed full conversion
(48 h). The N-terminal deprotection was also conducted as
described, using 20 mM Pd(OAc)2 (50µL, 1 µmol), TPPTS
(1.2 mg, 2 µmol) and diethylamine (22 µL, 214 µmol) in a
mixture of acetonitrile (300 µL) and phosphate buffer ( pH 6.4,
200 µL). The residue was dissolved in dry dichloromethane
(10 mL) and added to a solution of PyBOP (47.0 mg,
90.3 µmol) and DIPEA (16 µL, 92 µmol) in dry dichloro-
methane (30 mL) via a syringe pump over a period of 12 h.
After stirring for 18 h in total, the solvent was removed
in vacuo and flash chromatography (4 g C-18-silica, H2O/MeCN
gradient) gave rise to the cyclic heptapeptide 52b (24.3 mg,
19.6 µmol, 46%) as a colourless resin. Rf (52b): 0.71 (ethyl
D = −83.2 (c = 1.0, CHCl3). H NMR (500 MHz,
acetate). [α]20 1
CDCl3): δ = −0.30 (s, 3 H), −0.16 (s, 3 H), 0.02 (s, 3 H), 0.03 (s,
3 H), 0.79–1.00 (m, 18 H), 0.88 (s, 9 H), 0.90 (s, 9 H), 1.24–1.34
(m, 9 H), 1.37 (d, J = 7.2 Hz, 3 H), 1.59 (s, 3 H), 1.67 (m, 1 H),
1.76 (s, 3 H), 1.81 (s, 3 H), 1.82–1.93 (m, 3 H), 2.03 (m, 1 H),
3.29 (s, 3 H), 3.42 (dd, J = 10.0 Hz, 5.1 Hz, 1 H), 3.48 (dd, J =
10.0 Hz, 4.9 Hz, 1 H), 3.86 (m, 1 H), 3.94 (m, 1 H), 4.34 (dd, J =
2.4 Hz, 2.4 Hz, 1 H), 4.36 (m, 1 H), 4.51–4.72 (m, 2 H), 4.77 (d,
J = 9.6 Hz, 1 H), 4.96 (d, J = 4.8 Hz, 1 H), 4.98 (m, 1 H), 5.21 (d,
J = Hz, 1 H), 5.25 (d, J = 10.7 Hz, 1 H), 5.66 (d, J = 2.4 Hz, 1 H),
5.90 (bs, 1 H), 6.17 (dd, J = 17.6 H, 10.7 Hz, 1 H), 7.07 (m, 1 H),
7.12 (m, 1 H), 7.17–7.49 (m, 11 H), 7.60 (d, J = 7.9 Hz, 1 H),
7.78 (bs, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = −5.5, −5.4,
−4.9, 16.8, 17.2, 18.2, 18.3, 18.9, 18.9, 21.6, 23.4, 24.6, 25.8,
25.9, 25.9, 27.7, 27.9, 29.7, 30.2, 32.5, 33.2, 35.8, 36.2, 50.4,
52.7, 54.9, 55.8, 57.5, 59.0, 59.2, 62.3, 67.9, 68.7, 80.5, 113.6,
113.7, 113.8, 119.2, 119.4, 121.4, 123.7, 125.8, 126.5, 127.9,
128.3, 133.9, 136.0, 136.8, 143.9, 168.8, 169.0, 170.6, 171.3,
171.5, 171.9, 172.6 ppm. LC-MS: Luna 3μ C18, 50 × 4.6 mm,
0.9 ml min−1, 100% MeCN, tR (52b) = 9.87 min, m/z = 1264
([M + Na]+). HRMS (ESI): calculated for C67H109N8O10Si2
[M + H]+ 1241.7800, found 1241.7739.
Cyclomarin C
To a solution of the protected, cyclic heptapeptide 52a
(50.0 mg, 39.8 µmol) in methanol (400 µL) NH4F (14.8 mg,
400 µmol) was added at 0 °C. The mixture was warmed to
room temperature overnight and carefully heated to 40 °C
(bath temperature) for an additional 18 h. The solvent was
removed in vacuo after LC-MS-analysis showed complete con-
version. The residue was dissolved in THF (400 µL) at 0 °C and
Org. Biomol. Chem., 2016, 14, 6036–6054 | 6051

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TBAF (1 M in THF, 44 µL, 44 µmol) was added. After stirring
for 30 min at this temperature, the solvent was removed at
0 °C and the residue was purified by preparative HPLC (Luna,
100 mm/10 mm/5 µ, 5 mL min−1, H2O/MeCN 45 : 55). Cyclo-
marin C (27.2 mg, 26.5 µmol, 67%) was obtained as a white
solid. Rf (cyclomarin C): 0.71 (ethyl acetate). [α]20D = −30.4 (c =
1.0, CHCl3). 1H NMR (500 MHz, CDCl3): δ = 0.56 (ddd, J = 13.8
Hz, 6.4 Hz, 3.1 Hz, 1 H), 0.64 (d, J = 6.5 Hz, 3 H), 0.70 (d, J =
6.8 Hz, 3 H), 0.84 (d, J = 6.6 Hz, 3 H), 0.89 (d, J = 6.6 Hz, 3 H),
Published on 31 May 2016. Downloaded by National Taiwan University on 7/10/2020 2:17:27 PM.
0.94 (d, J = 6.7 Hz, 3 H), 1.04 (m, 1 H), 1.06 (d, J = 6.7 Hz, 3 H),
1.25 (s, 3 H), 1.29 (d, J = 7.3 Hz, 3 H), 1.38–1.44 (m, 2 H), 1.68
(m, 1 H), 1.70 (s, 6 H), 1.72 (s, 3 H), 2.18–2.32 (m, 4 H), 2.70 (s,
3 H), 2.82 (s, 3 H), 2.47 (dd, J = 11.1 Hz, 5.6 Hz, 1 H), 2.84 (dd,
J = 11.1 Hz, 4.3 Hz, 1 H), 3.36 (s, 3 H), 4.10 (dd, J = 10.5 Hz, 9.8
Hz, 1 H), 4.38 (dd, J = 8.7 Hz, 8.7 Hz, 1 H), 4.57 (dd, J = 4.9 Hz,
4.9 Hz, 1 H), 4.74–4.79 (m, 2 H), 4.80–4.87 (m, 1 H), 4.89 (dd,
J = 4.8 Hz, 4.8 Hz, 1 H), 5.07 (d, J = 5.4 Hz, 1 H), 5.16 (d, J =
17.4 Hz, 1 H), 5.21 (d, J = 10.7 Hz, 1 H), 5.30 (d, J = 4.9 Hz,
1 H), 6.06 (dd, J = 17.4 Hz, 10.7 Hz, 1 H), 6.85 (d, J = 4.9 Hz,
1 H), 7.04 (ddd, J = 7.2 Hz, 0.8 Hz, 1 H), 7.08–7.13 (m, 2 H),
7.19 (m, 2 H), 7.22–7.26 (m, 3 H), 7.29 (s, 1 H), 7.49 (d, J = 7.2
Hz, 1 H), 7.56 (d, J = 7.9 Hz, 1 H), 7.94 (d, J = 8.7 Hz, 1 H), 8.06
(d, J = 9.5 Hz, 1 H), 8.18 (d, J = 10.5 Hz, 1 H) ppm. 13C NMR
(125 MHz, CDCl3): δ = 17.6, 18.5, 18.9, 19.3, 20.0, 20.8, 22.4,
23.4, 25.0, 25.7, 27.8, 27.9, 29.3, 29.6, 30.8, 33.0, 33.3, 35.5,
38.9, 50.6, 53.0, 55.3, 55.9, 57.7, 58.1, 58.6, 59.2, 59.2, 66.3,
68.6, 80.0, 111.3, 113.8, 114.3, 118.9, 119.5, 121.5, 123.1, 124.8,
126.8, 127.8, 128.3, 128.7, 134.4, 135.1, 135.8, 143.7, 168.4,
168.9, 169.6, 170.6, 170.9, 171.6, 172.5 ppm. LC-MS: Luna 3μ
C18, 50 × 4.6 mm, 0.9 ml min−1, MeCN/H2O 1 : 1, tR (cyclo-
marin C) = 7.12 min, m/z = 1050 ([M + Na]+), 1010 ([M − OH]+).
HRMS (ESI): calculated for C56H83N8O10 [M + H]+ 1027.6227,
found 1027.6242.
Cyclomarin D
The cyclic heptapeptide 52b (21.0 mg, 16.9 µmol) was de-
protected and worked up as described for cyclomarin C using
NH4F (6.6 mg, 170 µmol) in methanol (200 µL) at 40 °C. The
solvent was removed after LC-MS-analysis showed full conver-
sion. The residue was dissolved in THF (200 µL) at 0 °C and
TBAF (1 M in THF, 20 µL, 20 µmol) was added. The mixture
was stirred for 45 min and the solvent was evaporated at 0 °C.
Preparative HPLC (Luna, 100 mm/10 mm/5 µ, 5 mL min−1,
H2O/MeCN 45 : 55) gave rise to cyclomarin D (11.0 mg,
10.9 µmol, 64%) as a colourless solid. [α]20 D = −24.4 (c = 1.0,
CHCl3). 1H NMR (500 MHz, CDCl3): δ = 0.66 (d, J = 6.6 Hz,
3 H), 0.86–0.90 (m, 6 H), 0.91 (d, J = 6.6 Hz, 3 H), 0.94 (d, J =
6.5 Hz, 3 H), 1.01 (d, J = 6.6 Hz, 3 H), 1.26 (s, 3 H), 1.35 (m,
1 H), 1.39 (d, J = 7.2 Hz, 3 H), 1.50 (m, 1 H), 1.55 (s, 3 H), 1.62
(m, 1 H), 1.76 (s, 3 H), 1.78 (m, 3 H), 1.79–1.84 (m, 4 H), 2.08
(m, 1 H), 2.39 (s, 3 H), 2.69 (m, 1 H), 3.28 (s, 3 H), 3.34 (m,
1 H), 3.54 (m, 1 H), 3.91 (m, 1 H), 4.12 (m, 1 H), 4.19–4.25 (m,
2 H), 4.40 (bs, 1 H), 4.52–4.61 (m, 2 H), 4.74 (d, J = 9.3 Hz,
1 H), 4.93 (d, J = 4.0 Hz, 1 H), 5.00 (m, 1 H), 5.20–5.23 (m, 2 H),
5.61 (bs, 1 H), 6.15 (dd, J = 17.6 Hz, 10.7 Hz, 1 H), 6.32 (bs,
1 H), 7.03–7.15 (m, 3 H), 7.24–7.39 (m, 5 H), 7.44–7.50
6052 | Org. Biomol. Chem., 2016, 14, 6036–6054
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Organic & Biomolecular Chemistry
(m, 4 H), 7.66 (d, J = 7.7 Hz, 1 H), 7.79 (d, J = 7.4 Hz, 1 H) ppm.
13
C NMR (125 MHz, CDCl3): δ = 17.0, 17.9, 18.0, 18.6, 19.2,
21.4, 23.3, 25.8, 25.8, 27.7, 27.8, 30.3, 30.5, 32.3, 33.0, 36.0,
36.4, 36.5, 51.5, 52.2, 55.3, 55.8, 57.4, 59.2, 67.3, 68.2, 81.0,
112.6, 113.5, 114.0, 119.1, 119.3, 121.3, 123.3, 125.4, 127.0,
127.6, 128.4, 128.4, 133.9, 136.0, 136.6, 144.1, 169.7, 170.3,
171.1, 172.2, 172.6, 172.8, 173 ppm. LC-MS: Luna 3μ C18, 50 ×
4.6 mm, 0.9 ml min−1, MeCN/H2O 1 : 9 to 100% MeCN in
5 min, 100% MeCN, tR (cyclomarin D) = 7.75 min, m/z = 1026
([M + Na]+), 996 ([M − OH]+). HRMS (ESI): calculated for
C55H81N8O10 [M + H]+ 1013.6070, found 1013.6022.
Acknowledgements
Financial support from the Deutsche Forschungsgemeinschaft
was gratefully acknowledged. We are especially thankful to
Marie Schneefeld from Hannover Medical School for investi-
gating the biological activities of the cyclomarins.
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