The Total Synthesis of the Bioactive Natural Product Plantazolicin A and Its Biosynthetic Precursor Plantazolicin B

Article abstract of DOI:10.1002/chem.201603157

Herein, we describe our full investigations into the synthesis of the peptide-derived natural product plantazolicin A, a compound that demonstrates promising selective activity against the causative agent of anthrax toxicity, and its biosynthetic precurso

Full text of DOI:10.1002/chem.201603157

DOI: 10.1002/chem.201603157
Full Paper
&
Natural Product Synthesis
The Total Synthesis of the Bioactive Natural Product
Plantazolicin A and Its Biosynthetic Precursor Plantazolicin B
Sabine Fenner,[a, b] Zoe E. Wilson,[a] and Steven V. Ley*[a]
Abstract: Herein, we describe our full investigations into the
synthesis of the peptide-derived natural product plantazolici-
n A, a compound that demonstrates promising selective ac-
tivity against the causative agent of anthrax toxicity, and its
biosynthetic precursor plantazolicin B. This report particularly
focuses on the challenging preparation of the arginine con-
taining thiazole fragment, including the development of
a robust, high yielding procedure that avoids the use of sul-
furating agents. Extensive studies on the design of a coher-
ent protecting group strategy and the establishment of
a
step-efficient dicyclization/oxidation approach allowed
high levels of convergence for the construction of the oxa-
zole fragments. This has led to a unified, highly convergent
synthesis for both plantazolicin A and B.
Introduction
hand side of 1 have shown respectable activity against B. an-
thracis, they also exhibit reduced selectivity, with their activity
thought to occur by a different mechanism.[7a,9]
The need for new antibiotics, especially those that work by
novel mechanisms of action, is undisputed.[1] The rise of antibi-
otic-resistant bacteria is thought, in part, to have been due to
the use of broad spectrum antibiotics and therefore narrow
spectrum antibacterial compounds are garnering increased at-
tention.[1] Natural products have proven to be a significant
source of both chemically and mechanistically diverse antibiot-
ics.[2] The isolation of the bioactive molecule plantazolicin A (1)
and its biosynthetic precursor[3] plantazolicin B (2) was first re-
ported in 2011 from the Bacillus amyloliquifaciens FZB42,[4]
a bacterium that has been recently reclassified as Bacillus vele-
zensis FZB42.[5] These molecules both consist of an unusual
linear 14 amino acid sequence that is highly modified to give
two polyazole subunits (Figure 1), and plantazolicin A has been
predicted to sit in a dynamic hairpin-like conformation.[6]
The promising bioactivity of plantazolicin A, along with its
novel and challenging structure, has led to significant interest
in the synthesis of these molecules. To date, our recently com-
municated synthesis of plantazolicin A (1)[10] is the second of
three reported total syntheses of this natural product and the
only report of the synthesis and full characterisation of biologi-
cal precursor plantazolicin B (2). Sꢀssmuth et al. reported the
synthesis of plantazolicin A (1) in 2013.[11] Moody et al. have re-
cently reported an elegant total synthesis of plantazolicin A (1)
based on rhodium-catalysed carbene N-H insertion.[6] The syn-
thesis and biological evaluation of shortened plantazolicin ana-
logues, and their use to further elucidate the biosynthesis of
the natural products, has also been described by both the
Mitchell[3c,9] and Sꢀssmuth[3b] groups. Herein, we fully disclose
our ultimately successful endeavours towards these molecules.
It has been observed that the bioactivity of these molecules
is dependent on the N-terminus dimethylation, with plantazoli-
cin A (1) showing selective and potent activity against Bacillus
anthracis, the causative agent of anthrax toxicity,[7] whereas
biosynthetic precursor plantazolicin B (2) exhibits reduced bio-
logical activity.[8] It is thought that plantazolicin A acts by caus-
ing membrane depolarisation and lysis of B. anthracis selective-
ly by taking advantage of a locally weakened cell membrane.[7a]
Additionally, whereas some truncated analogues of the left-
Synthetic strategy
While there are many potential approaches to the synthesis of
these molecules, in terms of convenience and efficiency, care-
ful planning is important to allow the exploitation of afforda-
ble and readily available building blocks. Accordingly, our syn-
thetic endeavours were focused on the use of natural amino
acids as building blocks where possible. The initial synthetic
strategy for the synthesis of plantazolicin A (1) and B (2) was
designed to be as convergent as possible, with the natural
products being split into two pentacycles by disconnecting at
the hinge point between the two isoleucine residues. It was
imagined that it would be necessary to form the sensitive oxa-
zolidine ring after the final coupling to avoid its hydrolysis. As
the mechanism of the dehydrative cyclisation proceeds with
inversion, this necessitates the incorporation of an l-allo-threo-
nine residue at this position. For the left-hand side it was
[a] Dr. S. Fenner, Dr. Z. E. Wilson, Prof. S. V. Ley
Department of Chemistry, University of Cambridge
Lensfield road, CB21EW, Cambridge (UK)
E-mail: svl1000@cam.ac.uk
[b] Dr. S. Fenner
Now employed by:
GlaxoSmithKline, Gunnels Wood Rd, SG12NY, Stevenage (UK)
Supporting information and the ORCID identification number(s) for the au-
thor(s) of this article can be found under http://dx.doi.org/10.1002/
chem.201603157.
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Figure 1. Plantazolicin A (1) and B (2) showing numbering of residues.
Results and Discussion
hoped that the arginine containing fragment, which differenti-
ates between plantazolicin A (1) and B (2) could be installed at
an advanced stage to ensure maximum convergence between
the two syntheses. Our protecting group strategy was based
on having protecting groups on the guanidine moiety of the
arginine residue and the C-terminus, which could be simulta-
neously removed (carboxyl benzyl (Cbz) and benzyl (Bn)),
whereas the N-terminus was orthogonally protected to allow
synthesis of plantazolicin A (1) and B (2) from a common
route. It was envisaged that the two thiazole rings could be
built by using modified Hantzsch thiazole syntheses, and that
oxazole rings could be assembled through iterative amino acid
couplings followed by cyclisation and oxidation by using the
methodology of Wipf and Williams[12] (Scheme 1).
Synthesis of LHS fragments
Synthesis of arginine thiazole fragments: One of the most
challenging sections of plantazolicin proved to be the synthe-
sis of the two coupling partners, which contained arginine de-
rived thiazoles 6 and 7. Originally we had decided to simply
employ a modified Hantzsch thiazole synthesis[13] for the for-
mation of the desired heterocycle. Differentially protected argi-
nine 12 was chosen as the starting material because it is com-
mercially available, but should allow for the selective deprotec-
tion of the alpha nitrogen to allow N-dimethylation to give
access to 6, the required coupling partner for plantazolicin A,
or left with the Boc-protected alpha nitrogen 7 for plantazoli-
cin B (Scheme 2).
Scheme 1. Initial retrosynthetic strategy for the synthesis of plantazolicin A (1) and B (2).
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The condensation of cysteine ethyl ester (22) with an amino
acid derived aldehyde[17] allows the formation of thiazolidines
from amino acid derived precursors without requiring the use
of unpleasant sulfurating reagents. It was envisaged that if ar-
ginine derived aldehyde 23 could be accessed, this would pro-
vide a rapid access to thiazole 7 from readily available starting
materials (Scheme 4).
Scheme 2. Initial retrosynthesis for arginine derived thiazoles 6 and 7.
The route began with the amidation of Boc-Arg(Cbz)2-OH 12
with the crude amide being directly converted into the thioa-
mide by using the sulfurating agent Belleau’s reagent[14] in an
acceptable 77% yield. The key modified Hantzch thiazole for-
mation[13] was then carried out to afford thiazole 7 in a modest
33% yield. Pleasingly, it was found that the alpha nitrogen of
the arginine residue could be deprotected by using trifluoro-
acetic acid (TFA) and although N-methylation was at first un-
successful when attempted using the Eschweiler–Clarke reac-
tion,[15] it was found that reductive amination could be carried
out by using sodium cyanoborohydride[16] to afford dimethyl
arginine thiazole 6 in 54% yield (Scheme 3). However, the
overall yield of only 25% for thiazole 7 was limiting, and addi-
tionally it was found that the yield of the thiazole formation
tended to be variable when it was carried out on scale, neces-
sitating an alternative approach for thiazole 7.
Scheme 4. Alternative retrosynthesis for thiazole 7.
Straightforward coupling of differentially protected arginine
12 with N,O-dimethylhydroxylamine 25 afforded amide 26,
which could then be reduced to the required amino acid de-
rived aldehyde quantitatively. The crude aldehyde was directly
coupled with cysteine ethyl ester hydrochloride 22 in biphasic
solution with substoichiometric potassium bicarbonate,[17a] to
afford thiazolidine 24. This could then be oxidised by using
manganese dioxide, to reproducibly afford thiazole 7 in four
steps (three purifications) and an overall yield of 52%
(Scheme 5).
As the synthesis progressed, it was found that hydrolysis of
the ethyl ester of 6 and 7 to afford the free acid was problem-
atic, with significant amounts of degradation being seen under
a range of conditions for the hydrolysis. To avoid this problem,
cysteine methyl ester hydrochloride 27 was used as the alde-
hyde coupling partner in the established route to afford
methyl ester protected thiazoles 28 and 29 (Scheme 6).
Additionally, as the protecting group strategy developed, it
became necessary to have the arginine guanidine nitrogen
atoms protected by using a tert-butyloxycarbonyl protecting
group. Initially this was accomplished by removing the nitro-
gen protecting groups from the four synthesised thiazoles, 6,
7, 28 and 29, and directly reprotecting them by using di-tert-
butyl dicarbonate and diisopropyethylamine to afford the di-
(31 and 32) or tri- (33 and 34) Boc protected thiazoles in eight
or six steps, respectively (Scheme 7).
Scheme 3. Modified Hantzsch synthesis of arginine thiazoles 6 and 7. Re-
agents and conditions: a) i. isobutylchloroformate, NMM, THF, À208C,
10 min; ii. NH4OH (35% in H2O), 08C, 5.5 h; b) Belleau’s reagent, THF 08C–rt,
2.5 h, 77%; c) ethyl bromopyruvate, KHCO3, DME, À158C, 5 min then tri-
fluoroacetic anhydride, 2,6-lutidine, DME, À158C, 4 h, 33%; d) i. TFA, CH2Cl2,
rt, 3.5 h, ii. 37% aq. CH2O, CH3OH, rt, 40 min then NaBH3CN, rt, 14.5 h, 54%.
NMM=N-methylmorpholine.
A more step-efficient method for the preparation of the gua-
nidine Boc protected thiazoles was next attempted, starting
from carboxybenzyl arginine 35. The free acid was converted
into the methyl ester and the crude ester Boc protected as pre-
viously established. This afforded a separable 4.7:1 ratio of the
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Nd,Nw’ (31a) and Nw,Nw’ (31b) protected isomers in 57%
overall yield. Both isomers could then be progressed through
to the thiazole. It was found that esters 36 could be reduced
to the required aldehyde without over-reduction to the corre-
sponding alcohol, then directly used in the thiazolidine forma-
tion as previously employed. However, it was found that re-
moval of the Cbz group from Na with retention of the Boc
protection on the guanidine functionality was challenging.
When hydrogenation of thiazole 37a was attempted by using
palladium(II) chloride, buffered with potassium carbonate to
ensure retention of the Boc protection, only a small amount of
the desired Cbz deprotected product was formed, which could
then be N-dimethylated as previously to afford only 18% of
31a, with 42% of starting material 37a also being recovered.
This indicated that the Cbz deprotection was, in fact, mediated
by the acid produced when palladium(II) chloride was reduced
to palladium(0), rather than solely by hydrogenation.
Scheme 5. Alternative synthesis of thiazole 7. Reagents and conditions:
a) CH3ONHCH3·HCl 25, NiPr2Et, HOBt, EDCI, CH2Cl2, rt, 16 h, 96%; b) i. DIBAL-
H, CH2Cl2, À788C, 40 min; ii. Cys-OEt·HCl 22, KHCO3, MeOH/toluene/H2O, rt,
15 h, 81%; c) MnO2, toluene, 808C, 24 h, 67%. DIBAL-H=diisobutylalumini-
um hydride.
When palladium(II) chloride was used unbuffered, although
the Na was fully deprotected, the Boc groups were also par-
tially deprotected. While pleasingly it was seen that controlling
the pH of the reaction mixture allowed for the selective N-
methylation at Na, this did necessitate an additional Boc repro-
tection step to afford the two differently protected isomers of
thiazole 31 (Scheme 8).
Boc-N-Arg(Boc2)-OH 39 is commercially available, which
meant that both the methyl (33) and ethyl (34) esters of the
tri-boc thiazole could be synthesised in just four steps (three
purifications) by using the established route. The N-dimethyl
thiazoles could then be accessed through Boc deprotection, re-
ductive amination at the Na position and Boc reprotection.
Global Boc deprotection was first attempted by using trifluoro-
acetic acid, which afforded an 11% yield of the desired dimeth-
yl thiazole 31a after reductive amination and reprotection.
Pleasingly, however, it was found that deprotection by using
anhydrous hydrochloric acid followed by reductive amination
and reprotection afforded 52% yield of thiazole 31 as a separa-
ble 35:17 (a/b) mixture of the two protected isomers
(Scheme 9).
Scheme 6. Synthesis of arginine thiazole methyl esters 28 and 29. Reagents
and conditions: a) i. DIBAL-H, CH2Cl2, À788C, 50 min; ii. Cys-OMe·HCl 27,
KHCO3, MeOH/toluene/H2O, rt, 15.5 h, 75%; b) MnO2, toluene, 808C, 19.5 h,
57%; c) i. TFA, CH2Cl2, rt, 5 h; ii. 37% aq. CHO, CH3OH, rt, 1 h then NaBH3CN,
rt, 18 h, 52%.
In an effort to develop a more step-efficient approach to the
N-dimethyl thiazoles, the preparation of protected N-dimethyl
arginine 43 was next attempted. Reductive amination then
protection of the free base of arginine methyl ester hydrochlo-
ride 44 resulted in a low 5% yield of the desired product 43a.
Pleasingly however, arginine 45 could be Na-dimethylated by
following a reported procedure,[18] the acid esterified and Boc
protection carried out to afford a 62% yield of 43 as a separa-
ble 2.1:1 (a/b) mixture of the two regioisomers. It was found
that 43a could be reduced, coupled with cysteine methyl ester
hydrochloride 27 and oxidised to afford thiazole 31a in 14%
yield (Scheme 10). Although this route required one less step
than that of Scheme 9, the overall yield was significantly lower
(6% vs. 13% 31a and 6% 31b) so it was decided to use the
route from Boc-N-Arg(Boc2)-OH 39 for the synthesis of both
the target thiazoles.
Scheme 7. Swapping protection on arginine thiazoles. Reagents and condi-
tions: a) i. PdCl2, H2, MeOH, 45 min (31 and 34)/50 min (32)/30 min (33);
ii. Boc2O, NiPr2Et, CH2Cl2, rt, 4 days (31, 32 and 34)/ 6 days (33), 31=86%,
32=24%, 33=87%, 34=87%.
Synthesis of threonine thiazole fragment: Known threo-
nine derived thiazole 11[19] was obtained from Boc-threonine
13 by the same approach as was applied to the synthesis of
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Scheme 8. Alternative protecting group strategy for arginine thiazoles. Reagents and conditions: a) i. Si(CH3)3Cl, MeOH, rt, 18.5 h; ii. Boc2O, NiPr2Et, MeOH,
CH2Cl2, rt, 6 days, 47% (36a) and 10% (36b); b) i. DIBAL-H, CH2Cl2, À788C, 3 h 10 min (38a)/3 h (38b); ii. Cys-OMe·HCl 27, KHCO3, toluene/H2O/MeOH (36b
only), rt, 21 h (38a)/22 h (38b), 38a=64%, 38b=54%; c) MnO2, toluene, 808C, 24 h (37a)/20 h (37b), 37a=54%, 37b=17%; d) i. K2CO3, PdCl2, H2, MeOH,
rt, 3 h; ii. 37% aq. CHO, MeOH, rt, 1 h then NaBH3CN, rt, 20.5 h, 18% (31a) and 42% (37a); e) i. PdCl2, H2, MeOH, rt, 1 h; ii. 37% aq. CH2O, MeOH, rt, 1 h then
NaBH3CN, rt, 20.5 h; iii. Boc2O, NiPr2Et, MeOH, CH2Cl2, rt, 4 days, 36% (31a) and 23% (31b).
Scheme 10. Direct synthesis of dimethyl arginine thiazole 31a. Reagents
and conditions: a) i. Ambersepꢂ 900-OH, MeOH, rt, 5 min; ii. CH2O, MeOH, rt,
1 h 40 min then NaBH3CN, rt, 18 h; iii. Boc2O, NiPr2Et, MeOH, CH2Cl2, rt, 7
Scheme 9. Synthesis of arginine thiazoles from tri-Boc arginine. Reagents
and conditions: a) CH3ONHCH3·HCl 25, NiPr2Et, HOBt, EDCI, CH2Cl2, rt, 16 h,
99%; b) i. DIBAL-H, CH2Cl2, À788C, 1 h; ii. Cys-OMe·HCl 27 (41) or Cys-
OEt·HCl 22 (42), KHCO3, MeOH/toluene/H2O, rt, 41.5 h (41)/19 h (42),
41=78%, 42=73%; c) MnO2, toluene, 808C, 15 h (33)/21 h (34), 33=48%,
34=59%; d) i. TFA, CH2Cl2, rt, 1 h; ii. 37% aq. CH2O, CH3OH, rt, 1.5 h then
NaBH3CN, rt, 20 h; iii. Boc2O, NiPr2Et, rt, 4 days, 11% (31a); e) i. HCl, 1,4-diox-
ane, rt, 1 h; ii. 37% aq. CH2O, CH3OH, rt, 1 h then NaBH3CN, rt, 15.5 h; iii. Bo-
c2O, NiPr2Et, CH2Cl2, rt, 4 days, 35% (31a) and 17% (31b). HOBt=1-hydro-
benzotriazole, EDCI=N-(3-dimethylaminopropyl)-N’’-ethyl-carbodiimide hy-
drochloride.
days, 5% (43a); b) i. 37% aq. CHO, NaBH3CN, NaOAc, H2O, rt, 16.5 h; ii.
Si(CH3)3Cl, MeOH, rt, 18 h; iii. Boc2O, NiPr2Et, MeOH, CH2Cl2, rt, 13 days, 42%
(43a) and 20% (43b); c) i. DIBAL-H, CH2Cl2, À788C, 1 h; ii. Cys-OMe·HCl 27,
KHCO3, toluene/H2O, rt, 18 h; iii. MnO2, toluene, 808C, 23 h, 14%.
azabicyclo[5.4.0]undec-7-ene (DBU) according to a one-pot
method of Wipf and Williams[12] to afford dicycle 49 in 43%
yield. The synthesis of di-cycle 49 in 54% overall yield from 48
by using flow chemistry has been previously reported by this
group.[20] Di-cycle 49 was then coupled with Boc deprotected
threonine-isoleucine dipeptide 50 before cyclisation and oxida-
tion to afford a modest 15% yield of tri-azole 8 (Scheme 12).
The final oxidation was also attempted using manganese diox-
ide, however this only resulted in partial conversion into the
desired product, even with large excesses of oxidant.
the arginine thiazoles to afford 11 in 42% overall yield
(Scheme 11).
Synthesis of tri-azole: The route to tri-azole 8 started with
the hydrolysis of the ethyl ester of threonine thiazole 11 using
lithium hydroxide before it was coupled with threonine methyl
ester hydrochloride 15 to afford 48 in 57% yield. Cyclisation
was effected by using the fluorinating reagent Deoxo-Fluorꢂ
and oxidation with bromotrichloromethane (BrCCl3) and 1,8-di-
To facilitate a more convergent approach to tri-azole 8, it
was next decided to determine whether the two cyclisation
and oxidation steps could be carried out concurrently. Tripep-
tide 52 was synthesised in 80% yield by straightforward pep-
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Scheme 11. Synthesis of threonine thiazole 11. Reagents and conditions:
a) i. 2,2-dimethoxypropane, PPTS, THF, reflux, 15 h; ii. CH3ONHCH3·HCl 25,
NiPr2Et, HOBt, EDCI, CH2Cl2, rt, 21 h, 49%; b) i. CH3ONHCH3·HCl 25, NiPr2Et,
HOBt, EDCI, CH2Cl2, rt, 22 h; ii. 2,2-dimethoxypropane, PPTS, THF, reflux, 18 h,
86%; c) i. DIBAL-H, CH2Cl2, À788C, 30 min; ii. Cys-OEt·HCl 22, KHCO3, tolu-
ene/H2O/MeOH, rt, 15 h, 83%; d) MnO2, toluene, 808C, 24 h, 59%.
PPTS=para-toluene sulfonate.
Scheme 13. Convergent synthesis of tri-azole 8. Reagents and conditions:
a) LiOH·H2O, MeOH/H2O, rt, 3 h; b) anhydrous HCl, 1,4-dioxane, rt, 30 min;
c) NiPr2Et, HOBt, EDCI, CH2Cl2, rt, 17.5 h, 71%; d) Deoxo-Fluorꢂ, CH2Cl2,
À208C, 2.5 h then DBU, BrCCl3 (portionwise), À208C to 08C, 110 h, 64%.
BrCCl3 and DBU used, the dicyclisation and oxidation could be
affected in 64% yield (Scheme 13).
Completion of LHS fragments: Attention then turned to
the completion of the two left-hand side fragments 54 and 55.
Whereas the N-Boc deprotections were routinely effected by
using anhydrous hydrochloric acid in dioxane, it was found
that the addition of water to the reaction mixture allowed the
simultaneous removal of the Boc protection and the acetal
from 8; in contrast, when anhydrous HCl was used, only the
Boc protection was lost. It was found that the methyl esters of
31 and 33 could be carefully hydrolysed at low temperature
and the deprotected molecules coupled by using 1-[bis(dime-
thylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-
oxide hexafluorophosphate (HATU) and diisopropylethylamine
to afford cyclisation precursors 56 and 57. The established cyc-
lisation/oxidation conditions were then employed to afford
pentacycles 54 and 55 with overall yields of 8 and 12%, re-
spectively, for the longest linear pathways (Scheme 14).
Synthesis of the RHS fragment
Synthesis of tetraoxazole fragment: The preparation of right-
hand side pentacycle 5 began with the preparation of tetracy-
cle 9. This synthesis started with the coupling of Boc-protected
isoleucine 16 with serine methyl ester hydrochloride 17 fol-
lowed by cyclisation and oxidation to install the first ring, with
the remaining three rings being installed iteratively by the
same process, affording tetracycle 9 in eleven steps and 10%
overall yield (Scheme 15).
Scheme 12. Linear synthesis of tri-azole 8. Reagents and conditions:
a) i. LiOH·H2O, THF/H2O, 08C—rt, 13 h; ii. Thr-OMe·HCl 15, NiPr2Et, HOBt,
EDCI, CH2Cl2, rt, 17 h, 57%; b) Deoxo-Fluorꢂ, CH2Cl2, À208C, 2 h then DBU,
BrCCl3, À208C to 08C, 60 h, 43%; c) Ile-OMe·HCl 14, NiPr2Et, HOBt, EDCI,
CH2Cl2, rt, 17 h, 99%; d) LiOH·H2O, THF/H2O, 08C to rt, 18 h; e) anhydrous
HCl, 1,4-dioxane, rt, 45 min; f) NiPr2Et, HOBt, EDCI, CH2Cl2, rt, 19 h, 31% (3
steps); g) Deoxo-Fluorꢂ, CH2Cl2, À208C, 2 h then DBU, BrCCl3, À208C to 08C,
48 h, 15%.
With the synthesis of tetraoxazole 9 achieved, attention
turned to whether this route could be improved by the use of
multiple cyclisations in one step. However, it was found that
the synthesis of the cyclisation precursors limited the number
of rings we could attempt to form at once. Whereas tripeptide
65 and diserine 66 could be obtained by using standard solu-
tion-phase coupling conditions, all attempts at gaining access
to tri- or tetraserine containing molecules were unsuccessful,
which could in part be due to the high polarity of these com-
pounds and resulting solubility problems in organic solvents.
tide couplings using EDCI and 1-hydroxybenzotriazole (HOBt)
(see the Supporting Information). This was coupled with threo-
nine thiazole 11 to give cyclisation precursor 53. It was found
that by increasing the reaction time and the equivalents of
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Scheme 14. Completion of left-hand fragments 54 and 55. Reagents and conditions: a) LiOH·H2O, MeOH/H2O, 08C, 1.5 h; b) HCl (1,4-dioxane/H2O), 1,4-diox-
ane, rt, 1 h; c) HATU, NiPr2Et, CH2Cl2/DMF, 08C to rt, 22 h, 56=61%, 57=66%; d) Deoxo-Fluorꢂ, CH2Cl2, À208C, 2 h then DBU, BrCCl3, À208C to 08C, 20 h
(54)/ 15 h (55), 54=69%, 55=92%. DMF=N,N-dimethylformamide.
Scheme 15. Linear synthesis of tetraoxazole 9. Reagents and conditions: a) Ser-OMe·HCl 17, NiPr2Et, HOBt, EDCI, CH2Cl2, 20 h, 91%; b) Deoxo-Fluorꢂ, CH2Cl2,
À208C, 30 min then BrCCl3, DBU, À208C to 38C, 8 h, 81%; c) i. LiOH·H2O, THF/MeOH/H2O, 08C to rt, 18 h; ii. Ser-OMe·HCl 17, NiPr2Et, HOBt, EDCI, CH2Cl2, 20 h,
84%; d) Deoxo-Fluorꢂ, CH2Cl2, À208C, 30 min then BrCCl3, DBU, À208C to 38C, 8 h, 78%; e) i. LiOH·H2O, THF/MeOH/H2O, 08C, 3 h; ii. Ser-OMe·HCl 17, NiPr2Et,
HOBt, EDCI, CH2Cl2, 20 h, 79%; f) Deoxo-Fluorꢂ, CH2Cl2, À208C, 30 min then BrCCl3, DBU, À208C to 38C, 8 h, 73%; g) i. LiOH·H2O, THF/MeOH/H2O, 08C to rt,
8 h; ii. Ser-OMe·HCl 17, NiPr2Et, HOBt, EDCI, CH2Cl2, 20 h, 71%; h) Deoxo-Fluorꢂ, CH2Cl2, À208C, 30 min then BrCCl3, DBU, À208C to 38C, 6.5 h, 53%.
When tripeptide 65 was submitted to the cyclisation/oxidation
conditions it was found that under optimised conditions only
23% dioxazole product 61 was formed, with the major product
(61%) being the dicyclised but only mono-oxidised product
67, which meant that the most efficient synthesis of dioxazole
61 would be the linear approach of Scheme 15.
The second dicyclisation precursor 68 was synthesised by
either coupling 61 and diserine 66, or by the less convergent
coupling of dioxazole serine 64 with Ser-OMe·HCl 17 (see the
Supporting Information). Pleasingly, dicyclisation/oxidation of
68 proceeded in 77% yield to afford tetraoxazole 9 in 47%
overall yield from 61, which is a marked improvement on the
linear conversion of 61 into 9, which had an overall yield of
22%, so this approach was incorporated into the final route
(Scheme 16).
It was found that tetraoxazole 9 was very poorly soluble,
which proved problematic when the hydrolysis of the ester of
this compound was attempted to enable progression, which
was also observed by Sꢀssmuth et al. for a similar intermediate
in their work towards plantazolicin A (1).[11] To try to avoid this
problem, tetraoxazoles with ethyl ester 70 and benzyl ester 71
protection were obtained from trioxazole 63 (Scheme 17).
Deprotection of the three tetraoxazoles was then attempted.
Through use of high temperatures and mixed solvent systems
it was found that both methyl ester 9 and ethyl ester 70 could
be deprotected by using lithium hydroxide, with methyl ester
9 being slightly higher yielding in initial tests, and was there-
fore carried forward (Scheme 18). Multiple attempts at remov-
ing the benzyl ester of 71, both by hydrolysis and hydrogena-
tion were unsuccessful, so this route was abandoned.
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Scheme 16. Convergent synthesis of tetraoxazole 9. Reagents and conditions: a) i. LiOH·H2O, THF/MeOH/H2O, 08C, 2 h 45 min; ii. Ser-OMe·HCl 17, NiPr2Et,
HOBt, EDCI, CH2Cl2, 20 h, 74%; b) Deoxo-Fluorꢂ, CH2Cl2, À208C, 1 h 45 min then BrCCl3, DBU, À208C to À108C, 30 min, À108C to 08C, 6 h, 23% (61) and 61%
(67); c) Ser-OMe·HCl 17, NiPr2Et, HOBt, EDCI, CH2Cl2, 20 h, 88%; d) LiOH·H2O, THF/MeOH/H2O, 08C to rt, 2 h; e) anhydrous HCl, 1.4-dioxane, 08C to rt, 3.5 h;
f) NiPr2Et, HOBt, EDCI, CH2Cl2, 20 h, 61%; g) Deoxo-Fluorꢂ, CH2Cl2, À208C, 30 min then BrCCl3, DBU, À208C to 08C, 8 h, 77%.
benzyl (10) esters of the required l-allo-threonine-phenylala-
nine dipeptide were synthesised to help establish what pro-
tecting group was most appropriate for the C-terminus
(Scheme 19).
These dipeptides were then coupled by using HATU to the
tetraoxazole 9 to give three potential right-hand side coupling
partners, 5, 83 and 84. Benzyl ester 5 was then dehydratively
cyclised to oxazoline 85 by using diethylaminosulfur trifluoride
(DAST) in a modest 39% yield. It was seen that the key cou-
pling constant of the doublet of 5-MeOxl13-C4H (4.32 ppm, J=
7.6 Hz) correlated well to this coupling in the natural product
(4.23 ppm, J=7.6 Hz),[4a] which was a reassuring preliminary in-
Scheme 17. Synthesis of alternatively protected tetraoxazoles 70 and 71. Re-
agents and conditions: a) i. LiOH·H2O, THF/MeOH/H2O, 08C to rt, 26 h; ii. Ser-
OEt·HCl 74 (72)/Ser-OBn·HCl 75 (73), NiPr2Et, HOBt, EDCI, CH2Cl2, 20 h,
72=90%, 73=84%; b) Deoxo-Fluorꢂ, CH2Cl2, À208C, 30 min (70)/45 min
(71) then BrCCl3, DBU, À208C to 08C, 20 h, 70=48%, 71=83%.
Scheme 18. Deprotection of tetraoxazoles 9 and 70. Reagents and condi-
tions: a) 9: LiOH·H2O, THF/MeOH/H2O, 08C to rt, 22 h, 62%*; 70: LiOH·H2O,
CCl3H/MeOH/H2O, 508C to 558C, 19 h, 56%*. *crude yields.
Scheme 19. Synthesis of C-terminus dipeptides. Reagents and conditions:
a) i. NaHCO3, H2O, rt, 15 min then Boc2O, MeOH, rt, 13.5 h (77)/13 h (10);
ii. Phe-OMe·HCl 82 (77)/Phe-OBn·HCl 19 (10), NiPr2Et, HOBt, EDCI, CH2Cl2,
20 h, 77=79%, 10=88%; b) HOCH2CH2Si(CH3)3, EDCI, DMAP, CH2Cl2, 08C to
rt, 18 h, 74%; c) anhydrous HCl, 1,4-dioxane, rt, 2 h; d) NaHCO3, H2O, rt,
15 min then Boc2O, MeOH, rt, 15.5 h; e) HATU, NiPr2Et, CH2Cl2, 08C to rt,
16 h, 81%. DMAP=4-dimethylaminopyridine.
Given the uncertainty over our overall protecting group
strategies, methoxy- (77), trimethylsilyl- (TMSE À 78) and
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Scheme 20. Synthesis of right-hand side coupling partners, showing key H NMR coupling constant. Reagents and conditions: a) LiOH·H2O, MeOH/CHCl3/H2O,
558C to 658C, 7 h to 2 days; b) anhydrous HCl, 1,4-dioxane, 08C to rt, 30 min; c) NiPr2Et, HOBt, EDCI, CH2Cl2, DMF (83 only) 20 h, 83=9%, 5=37%; d) HATU,
NiPr2Et, CH2Cl2/DMF, 08C to rt, 20 h (5)/18 h (84), 5=60%, 84=77%; e) DAST, CH2Cl2, À788C, 1 h then K2CO3, À788C, 15 min then rt, 30 min, 39% (85).
dication that the cyclodehydration was proceeding with inver-
sion, consistent with an SN2-like ring-closing mechanism
(Scheme 20). This was supported by the fact that when 86 was
synthesised by using an analogous route from natural l-threo-
nine, the coupling constant was considerably larger (4.78 ppm,
J=10.0 Hz) (see the Supporting Information).
the coupled product 87 in a modest 38% yield. The threonine
residue of coupled model 87 was then dehydratively cyclised
by using Deoxo-Fluorꢂ, because this had been reported to be
more effective at cyclising threonine residues than DAST,[12] af-
fording oxazoline 88 in 74% yield. Attention then turned to
the deprotection of the C- and N-termini where it was found
that it was possible to remove the benzyl protecting group by
hydrogenation with palladium(II) chloride, before trifluoroace-
tic acid (TFA) was used to remove the Boc protecting group
and acetal to afford the deprotected product 89 in a crude
73% yield (Scheme 21). Given that the benzyl protecting
group required an extra deprotection step, and the methyl
ester removal conditions were unlikely to be compatible with
the oxazoline ring, it was decided to progress the synthesis by
using TMSE protected right-hand side 84.
Coupling and completion
Model coupling and deprotections: With the synthesis of the
two left-hand side fragments 54 and 55 and the right-hand
side with three different forms of C-terminus protection 5, 83
and 84 completed, it was then desirable to establish suitable
conditions for the end game of the route using a model,
before attempting it on the actual substrates.
Coupling and completion of plantazolicin A and B: The
coupling and cyclisation conditions established by using the
Tri-azole 8 and dipeptide 10 were deprotected by using the
established conditions before coupling with HATU to afford
Scheme 21. Model system to investigate end game. Reagents and conditions: a) LiOH, MeOH/H2O, 08C to rt, 19 h; b) anhydrous HCl, 1,4-dioxane, 08C to rt,
30 min; c) HATU, NiPr2Et, CH2Cl2/DMF, 08C to rt, 19 h, 38%; d) Deoxo-Fluorꢂ, CH2Cl2, À208C, 1 h, 74%; e) i. PdCl2, H2, MeOH, 2 h; ii. TFA, CH2Cl2, 6 h, 73% (crude
yield).
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Scheme 22. Synthesis of plantazolicin A (1) and B (2). Reagents and conditions: a) LiOH, THF/H2O, 08C, 2 h 25 min; b) anhydrous HCl, 1,4-dioxane, 08C to rt,
30 min; c) HATU, NiPr2Et, CH2Cl2/DMF, 08C to rt, 16 h; d) Deoxo-Fluorꢂ, CH2Cl2, À208C, 24 h (90)/17 h (91), 90=43%, 91=35%; e) TFA, rt, 2 h (1)/ 1 h (2),
1=59%, 2=64%.
ing Information. Additional data related to this publication is avail-
able at the University of Cambridge Institutional Data Repository
model were then applied to the prepared plantazolicin cou-
pling partners 54, 55 and 84 to afford the protected natural
products 90 and 91. Pleasingly, it was found that treating
these with neat TFA led to the removal of all protecting
groups, as long as care was taken to exclude water to avoid
hydrolysis of the oxazoline ring (Scheme 22). Purification was
carried out by using HPLC and full comparison of synthetic
and natural plantazolicin A is included in our communication
of this work[10] and Moody’s synthesis of plantazolicin A[6] in-
cluded a HPLC comparison of their synthetic 1 to material syn-
thesised by this route. To our knowledge the characterisation
of biosynthetic precursor plantazolicin B (2) has not been car-
ried out elsewhere.
(http://dx.doi.org/10.17863/CAM.486).
Acknowledgements
We gratefully acknowledge generous funding from the
German Academic Exchange Service DAAD (S.F.), The Royal So-
ciety (Newton International Fellowship to Z.E.W.), and the Engi-
neering and Physical Sciences Research Council (grants EP/
K009494/1 and EP/K039520/1).
Keywords: antibiotics · heterocycles · natural products
polyazole · total synthesis
·
Conclusions
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Received: July 1, 2016
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FULL PAPER
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Natural Product Synthesis
S. Fenner, Z. E. Wilson, S. V. Ley*
&& – &&
The Total Synthesis of the Bioactive
Natural Product Plantazolicin A and Its
Biosynthetic Precursor Plantazolicin B
The full story: Our full investigations
into the synthesis of the linear polythia-
zole/oxazole plantazolicin A (see figure),
which exhibits desirable selective bioac-
tivity against Bacillus anthracis, and its
biosynthetic precursor plantazolicin B is
reported. The convergent route devel-
oped utilised amino acids as the build-
ing blocks and allowed access to both
molecules from a common route.
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