Total synthesis of Microcin B17 via a fragment condensation approach

Article abstract of DOI:10.1021/ol102916b

The total synthesis of the 43 amino acid antibacterial peptide Microcin B17 (MccB17) is described. The natural product was synthesized via a convergent approach from a heterocycle-derived peptide and peptide thioester fragments prepared via Fmoc-strategy solid phase peptide synthesis (SPPS). Final assembly was achieved in an efficient manner using two Ag(I)-assisted peptide ligation reactions to afford MccB17 in excellent overall yield.

Full text of DOI:10.1021/ol102916b

ORGANIC
LETTERS
2
011
Vol. 13, No. 4
80–683
Total Synthesis of Microcin B17 via a
Fragment Condensation Approach
6
Robert E. Thompson, Katrina A. Jolliffe,* and Richard J. Payne*
School of Chemistry, The University of Sydney, NSW 2006, Australia
kate.jolliffe@sydney.edu.au; richard.payne@sydney.edu.au
Received December 2, 2010
ABSTRACT
The total synthesis of the 43 amino acid antibacterial peptide Microcin B17 (MccB17) is described. The natural product was synthesized via a
convergent approach from a heterocycle-derived peptide and peptide thioester fragments prepared via Fmoc-strategy solid phase peptide
synthesis (SPPS). Final assembly was achieved in an efficient manner using two Ag(I)-assisted peptide ligation reactions to afford MccB17
in excellent overall yield.
Microcin B17 (MccB17, 1) is a 43 amino acid antibacterial
peptide secreted from various strains of the bacterium
thiazole and oxazole heterocycles throughout the native
3
peptide backbone.
To date there has been only one total synthesis of this
complex biomolecule. This work, reported by Jung and co-
workers, utilized a linear solid-phase peptide synthesis
1
Escherichia coli. MccB17 targets DNA gyrase, a member of
the type II topoisomerase family of enzymes, essential for
2
DNA replication in prokaryotic organisms. MccB17 displays
(
SPPS) approach, incorporating preformed oxazole and
extensive post-translational modification, whereby specific
serine and cysteine residues have undergone enzymatic dehy-
drative cyclization and dehydrogenation, affording several
4
thiazolecontainingaminoacidsintothesequence. Despite
the successful assembly of the natural product, the practical
length limitation of SPPS, coupled with the difficulties
5
(
1) (a) Asensio, C.; P ꢀe rez-Dı
´
az, J. C.; Martı
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associated with preparing glycine rich sequences, makes the
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1
0.1021/ol102916b r 2011 American Chemical Society
Published on Web 01/14/2011

Scheme 1. Retrosynthesis of Microcin B17 (MccB17,1)
this strategy. In addition to this synthetic approach, MccB17
and several analogues have been prepared via biosynthetic
Fmoc-strategy SPPS. We envisaged the assembly via two
9
cysteine-free direct aminolysis ligation reactions.
6
means. While this represents a powerful approach for
Protecting groups on the N-termini of peptide thioesters
3 and 4, specifically N-trifluoroacetamide and N-fluore-
nylmethylcarbamate (Fmoc) groups respectively, were in-
corporated to prevent polymerization of fragments during
the ligation reactions.
accessing these post-translationally modified peptides, it
cannot provide access to a comprehensive suite of analogues
for detailed structure-activity studies. Given the limitations
associated with these prior approaches, we sought to develop
a more convergent route that would also be amenable to the
synthesis of a library of MccB17 variants for biological
testing in the future.
Ligation-based approaches for the synthesis of large
polypeptides and proteins are of widespread interest as they
allow rapid access to biologically and pharmaceutically
7
relevant biomolecules. We reasoned that a convergent liga-
tion-based assembly of MccB17 would circumvent difficul-
ties associated with the Fmoc-strategy SPPS of large peptides
and, in addition, would provide convenient access to ana-
6
,8
logues unobtainable by biosynthetic means.
Our
synthetic approach toward MccB17 involved disconnec-
tion at two junctions, namely Gly19-Gly20 and Gly34-
Gly35 (Scheme 1). This provided target peptide 2 and
peptide thioesters 3 and 4 that could be obtained through
Figure 1. Fmoc-protected heterocyclic amino acids.
Preformed Fmoc-protected building blocks (5-8,
Figure 1) were obtained using established procedures for
1
0
oxazole and thiazole synthesis and were incorporated
directly into the Fmoc-strategy SPPS of fragments 2-4
(
5) Mousavi, A.; Hotta, Y. Appl. Biochem. Biotechnol. 2005, 120,
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6) (a) Roy, R. S.; Kelleher, N. L.; Milne, J. C.; Walsh, C. T. Chem.
1
(see Supporting Information for experimental details).
The preparation of C-terminal fragment 2 began from
(
Biol. 1999, 6, 305–318. (b) Zamble, D. B.; Miller, D. A.; Heddle, J. G.;
Maxwell, A.; Walsh, C. T.; Hollfelder, F. Proc. Natl. Acad. Sci. U.S.A.
Fmoc-Ile-preloaded Wang resin (9, Scheme 2). Removal
of the N-terminal Fmoc group using 20% piperidine in
DMF was followed by coupling to Fmoc-His(Trt)-OH
using benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium
2001, 98, 7712–7717.
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7) (a) Kent, S. B. H. Chem. Soc. Rev. 2009, 38, 338–351. (b) Kan, C.;
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Walsh, C. T. Chem. Biol. 1998, 5, 373–384. (c) Roy, R. S.; Belshaw, P. J.;
Walsh, C. T. Biochemistry 1998, 37, 4125–4136. (d) Kelleher, N. L.;
Hendrickson, C. L.; Walsh, C. T. Biochemistry 1999, 38, 15623–15630.
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Q.; Tan, Z. P.; Kan, C.; Hua, Z. H.; Ranganathan, K.; Danishefsky, S. J.
Angew. Chem., Int. Ed. 2007, 46, 7383–7387. (c) Payne, R. J.; Ficht, S.;
Greenberg, W. A.; Wong, C.-H. Angew. Chem., Int. Ed. 2008, 47, 4411–
4415.
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Nat. Prod. Rep. 1999, 16, 249–263.
Org. Lett., Vol. 13, No. 4, 2011
681

Scheme 2. Synthesis of Peptide Fragment 2 via Fmoc-SPPS
Scheme 3. Synthesis of Peptide Thioester 3 via Fmoc-SPPS
hexafluorophosphate (PyBOP) and N-methylmorpholine
NMM) in DMF. Assembly of the remaining peptide
(
sequence was achieved using iterative Fmoc-strategy SPPS.
Notably, only 1.5 equiv of Fmoc-protected oxazole building
block 5 were coupled to the growing resin-bound peptide
[
using 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethylur-
onium hexafluorophosphate (HATU) and NMM in DMF]
to prevent excess waste of this precious building block. This
coupling strategy was also applied to the incorporation of
heterocyclic building blocks 6, 7, and 8 (see below). Acid-
olytic cleavage of the fully assembled peptide 10 from the
N-diisopropylethylamine. Acidolytic cleavage from the
resin then afforded the crude peptide thioester 3. Poor
solubility of this fragment hampered purification attempts
by HPLC; however the highly efficient synthesis permitted
isolation through precipitation in diethyl ether, to provide
peptide thioester 3 in 87% yield and greater than
resin using TFA/triisopropylsilane (TIS)/H O (95/2.5/2.5, v/
2
v/v) afforded MccB17(35-43) 2 in 78% yield after reversed-
phase HPLC purification (see Supporting Information).
Synthesis of peptide thioester fragment 3 was achieved
9
0% purity and, importantly, without epimerization of the
penultimate asparagine residue (see Supporting Informa-
tion).
1
1
using the side chain anchoring strategy. This method has
been shown to be an efficient route to peptide thioesters
using Fmoc-strategy SPPS and, importantly, proceeds
without epimerization of the resin bound amino acid. To
this end, Rink amide resin was deprotected using 20%
piperidine in DMF and Fmoc-Asp-OAll was subsequently
immobilizedusing PyBOPand NMM inDMF toafford11
Synthesis of the final fragment, N-terminal peptide thio-
ester 4, was performed on 2-chlorotrityl chloride resin
(Scheme 4). Loading of Fmoc-Gly-OH was conducted in
dichloromethane using N,N-diisopropylethylamine to fur-
nish resin bound 14. The desired sequence was subse-
quently assembled on the resin via Fmoc-strategy SPPS.
1
2
Two 2,4-dimethoxybenzyl groups were incorporated
into the amide backbone to suppress the aggregating
effects of the extended polyglycine sequences which we
had encountered in our initial synthetic endeavors. Cleav-
age of the fully assembled protected resin-bound peptide
(
Scheme 3). Iterative Fmoc-strategy SPPS was then con-
ducted as described for 2 to give resin bound 12. Introduc-
tion of the C-terminal thioester moiety was initiated by
treatment of 12 with Pd(PPh ) and phenylsilane to liber-
3
4
ate the free C-terminal acid and was followed by coupling
of HCl H-Gly-S(CH ) CO Et (13) using HATU and N,
1
5 was achieved using hexafluoro-2-propanol/CH Cl
2 2
3
2 2
2
(4:1, v/v). The crude peptide was immediately subjected
to the thioesterification procedure reported by Kajihara
and co-workers, using PyBOP, ethyl 3-mercaptopropio-
nate, and N,N-diisopropylethylamine in NMP. Global
deprotection and reversed-phase HPLC purification af-
forded the N-Fmoc protected peptide thioester 4 in 18%
overall yield and 28 linear steps.
(
10) (a) Videnov, G.; Kaiser, D.; Kempter, C.; Jung, G. Angew.
1
3
Chem., Int. Ed. 1996, 35, 1503–1506. (b) Moody, C. J.; Bagley, M. C.
J. Chem. Soc., Perkin Trans. 1 1998, 601–607. (c) Phillips, A. J.; Uto, Y.;
Wipf, P.; Reno, M. J.; Williams, D. R. Org. Lett. 2000, 2, 1165–1168. (d)
Deeley, J.; Bertram, A.; Pattenden, G. Org. Biomol. Chem. 2008, 6,
1994–2010. (e) Aguilar, E.; Meyers, A. I. Tetrahedron Lett. 1994, 35,
2473–2476. (f) Bredenkampf, M. W.; Holzapfel, C. W.; van Zyl, W. J.
Synth. Commun. 1990, 20, 2235–2249.
11) (a) Wang, P.; Miranda, L. P. Int. J. Pept. Res. Ther. 2005, 11,
17–123. (b) Ficht, S.; Payne, R. J.; Guy, R. T.; Wong, C.-H. Chem.;
Eur. J. 2008, 14, 3620–3629. (c) Yang, Y.-Y.; Ficht, S.; Brik, A.; Wong,
C.-H. J. Am. Chem. Soc. 2007, 129, 7690–7701. (d) Ajish Kumar, K. S.;
Harpaz, Z.; Haj-Yahya, M.; Brik, A. Bioorg. Med. Chem. Lett. 2009, 19,
(
(12) Cardona, V.; Eberle, I.; Barthelemy, S.; Beythien, J.; Doerner,
B.; Schneeberger, P.; Keyte, J.; White, P. D. Int. J. Pept. Res. Ther. 2008,
14, 285–292.
(13) (a) Kajihara, Y.; Yoshihara, A.; Hirano, K.; Yamamoto, N.
Carbohydr. Res. 2006, 341, 1333–1340. (b) Hirano, K.; Kajihara, Y.
J. Carbohydr. Chem. 2010, 29, 84–91.
1
3870–3874.
6
82
Org. Lett., Vol. 13, No. 4, 2011

Scheme 4. Synthesis of Peptide Thioester 4 via Fmoc-SPPS
Scheme 5. Ligation-Based Assembly of MccB17 (1)
and N,N-diisopropylethylamine in DMSO (Scheme 5).
After 24 h the reaction had proceeded to completion as
assessed by LC-MS analysis. At this point, in situ deprotec-
tion of the N-terminal Fmoc-carbamate moiety afforded
MccB17 (1) in 45% yield over two steps after reversed-phase
HPLC purification. Pleasingly, the spectroscopic data ob-
tained for synthetic MccB17 were consistent with those
With the peptide and peptide thioester fragments (2-4)
now in hand, attention turned to the ligation-based assem-
bly of the natural product. The ligation between peptide 2
and peptide thioester 3 was first investigated using the
direct aminolysis ligation reaction recently reported by
15
4
reported for both isolated and previously synthesized
MccB17 (see Supporting Information). Specifically,
9
c
1
Wong and co-workers. To this end, 2 and 3 were reacted
in 4:1 v/v NMP/6 M Gn HCl with 1 M HEPES at pH
H NMR, HSQC, HMBC, and mass spectral analysis of
synthetic 1 correlated fully.
3
7
.5 with the addition of thiophenol to facilitate thioester
In summary, we have completed an efficient total synthesis
of the 43 amino acid post-translationally modified antibac-
terial peptide MccB17. Key features in our approach include
the use of Fmoc-SPPS to rapidly assemble three key peptide
and peptide thioester fragments which, under Ag(I)-
mediated ligation conditions, were effectively assembled to
the target molecule. Given the efficient and convergent
nature of the synthetic route described, it is anticipated that
it will prove amenable to the preparation of libraries of
MccB17 analogues that can be evaluated for antibacterial
activity. Moreover, it may also find utility in the total
synthesis of related peptide-based natural products and
associated analogues, work toward which is currently under-
way in our laboratories and will be reported in due course.
exchange to the more reactive thiophenylester (Scheme 5).
After 7 days the ligation reaction was treated with aqueous
hydrazine to facilitate deprotection of the N-terminal
trifluoroacetamide moiety, furnishing MccB17(20-43) 16 in
38% yield over the two steps. Owing to the modest yield
obtained, we chose to investigate an alternative coupling
strategy. The presence of a nonepimerizable C-terminal
glycine residue permitted the use of a Ag(I)-promoted
9
a,b,14
ligation reaction.
As such, coupling of 2 and 3 in the
presence of AgNO , 3-hydroxy-1,2,3-benzotriazin-4(3H)-
3
one (HOOBt), and N,N-diisopropylethylamine in DMSO
was performed (Scheme 5). Gratifyingly after 18 h, a near-
quantitative conversion to the desired product was achieved,
as assessed by LC-MS analysis. At this stage, in situ hydra-
zinolysis of the N-terminal trifluoroacetamide moiety and
subsequent HPLC purification provided MccB17(20-43)
Acknowledgment. We would like to acknowledge
Dr. Ian Luck, School of Chemistry, The University of
Sydney, for assistance with NMR spectroscopy. We also
acknowledge an Australian Postgraduate Award for PhD
funding (R.E.T.).
16 in 70% yield over two steps. Given the success of the silver
ion mediated formation of 16, we chose to employ the same
methodology for the crucial final ligation. To this end, pep-
tide 16 and thioester 4 were treated with AgNO , HOOBt,
3
Supporting Information Available. Experimental pro-
1
cedures and characterization for all compounds. H and
C NMR spectra and HPLC and mass spectral data for
novel compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
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M.; Shinohara, N.; Tanabe, S.; Takahashi, T.; Okura, K.; Itoh, H.;
Mizoguchi, Y.; Iida, M.; Lee, N.; Matsuoka, S. Nat. Chem. 2010, 2, 280–
1
3
285.
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15) Bayer, A.; Freund, S.; Jung, G. Eur. J. Biochem. 1995, 234, 414–426.
Org. Lett., Vol. 13, No. 4, 2011
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