Convergent and Modular Synthesis of Candidate Precolibactins. Structural Revision of Precolibactin A

Article abstract of DOI:10.1021/jacs.6b02276

The colibactins are hybrid polyketide-nonribosomal peptide natural products produced by certain strains of commensal and extraintestinal pathogenic Escherichia coli. The metabolites are encoded by the clb gene cluster as prodrugs termed precolibactins. clb⁺ E. coli induce DNA double-strand breaks in mammalian cells in vitro and in vivo and are found in 55-67% of colorectal cancer patients, suggesting that mature colibactins could initiate tumorigenesis. However, elucidation of their structures has been an arduous task as the metabolites are obtained in vanishingly small quantities (μg/L) from bacterial cultures and are believed to be unstable. Herein we describe a flexible and convergent synthetic route to prepare advanced precolibactins and derivatives. The synthesis proceeds by late-stage union of two complex precursors (e.g., 28 + 17 → 29a, 90%) followed by a base-induced double dehydrative cascade reaction to form two rings of the targets (e.g., 29a → 30a, 79%). The sequence has provided quantities of advanced candidate precolibactins that exceed those obtained by fermentation, and is envisioned to be readily scaled. These studies have guided a structural revision of the predicted metabolite precolibactin A (from 5a or 5b to 7) and have confirmed the structures of the isolated metabolites precolibactins B (3) and C (6). Synthetic precolibactin C (6) was converted to N-myristoyl-d-asparagine and its corresponding colibactin by colibactin peptidase ClbP. The synthetic strategy outlined herein will facilitate mechanism of action and structure-function studies of these fascinating metabolites, and is envisioned to accommodate the synthesis of additional (pre)colibactins as they are isolated.

Full text of DOI:10.1021/jacs.6b02276

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Article
A convergent and modular synthesis of candidate
precolibactins. Structural revision of precolibactin A.
Alan R. Healy, Maria I. Vizcaino, Jason M. Crawford, and Seth B Herzon
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 30 Mar 2016
Downloaded from http://pubs.acs.org on March 30, 2016
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Page 1 of 16
Journal of the American Chemical Society
Title: A convergent and modular synthesis of candidate precolibactins. Structural revision of precolibactin A.
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a,b
a,b
a,b,c
,a,d
Authors: Alan R. Healy, Maria I. Vizcaino, Jason M. Crawford,
and Seth B. Herzon*
Affiliations:
a
Department of Chemistry, Yale University, New Haven, Connecticut 06520, USA
Chemical Biology Institute, Yale University, West Haven, Connecticut, 06516, USA
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c
Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, Connecticut, 06536, USA
d
Department of Pharmacology, Yale School of Medicine, New Haven, Connecticut, 06520, USA
Email: seth.herzon@yale.edu
Abstract.
The colibactins are hybrid polyketide-nonribosomal peptide natural products produced by certain strains of
commensal and extraintestinal pathogenic E. coli. The metabolites are encoded by the clb gene cluster as
+
pro-drugs termed precolibactins. clb E. coli induce DNA double-strand breaks (DSBs) in mammalian cells in
vitro and in vivo and are found in 55–67% of colorectal cancer patients, suggesting that mature colibactins
could initiate tumorigenesis. However, elucidation of their structures has been an arduous task as the
metabolites are obtained in vanishingly small quantities (µg/L) from bacterial cultures and are believed to be
unstable. Herein we describe a flexible and convergent synthetic route to prepare advanced precolibactins
and derivatives. The synthesis proceeds by late-stage union of two complex precursors (e.g., 28 + 17 → 29a,
9
0%) followed by a base-induced double dehydrative cascade reaction to form two rings of the targets (e.g.,
2
9a → 30a, 79%). The sequence has provided quantities of advanced candidate precolibactins that exceed
those obtained by fermentation, and is envisioned to be readily-scaled. These studies have guided a structural
revision of the predicted metabolite precolibactin A (from 5a or 5b to 7) and have confirmed the structures of
the isolated metabolites precolibactins B (3) and C (6). Synthetic precolibactin C (6) was converted to N-
myristoyl-D-Asn and its corresponding colibactin by colibactin peptidase ClbP. The synthetic strategy outlined
herein will facilitate mechanism of action and structure–function studies of these fascinating metabolites, and is
envisioned to accommodate the synthesis of additional (pre)colibactins as they are isolated.
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Page 2 of 16
Introduction.
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Bacteria residing in and on humans (the human microbiota) play an integral role in regulating
1
physiology and disease. The intestinal tract has been estimated to contain 500–1000 species of bacteria
2
constituting ~1.5 kg of biomass. Certain strains of gut commensal and extraintestinal pathogenic E. coli
3
harbour a gene cluster (clb or “pks”) that encodes a group of molecules termed precolibactins. Precolibactins
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are substrates for colibactin peptidase ClpP, a protease encoded within the clb gene cluster. ClbP is anchored
4
within the inner periplasmic membrane of the bacteria and removes an N-acyl-D-asparagine side chain from
the precolibactins. This cleavage step converts precolibactins to cytotoxic colibactins and likely constitutes a
5
+
prodrug resistance mechanism in the bacteria. clb E. coli induce DNA double-strand breaks (DSBs) in
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a
6
7
mammalian cells in vitro and in vivo. Host inflammation promotes proliferation of E. coli and expression of
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clb, the clb pathway promotes colorectal cancer in colitis-susceptible mice treated with azoxymethane, and
+
7,9
two studies revealed the presence of clb E. coli in 55–67% of colorectal cancer patients. Collectively, these
data suggest that colibactins initiate tumorigenesis by a mechanism involving induction of DNA DSBs.
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P = predicted structure
I = isolated structure
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O
O
H2N
H2N
O
O
O
CH3
O
H
H
N
N
n-C13H27
N
n-C13H27
N
CH3
H
H
O
CH3
O
CH3
NH
O
1
Vizcaino et al. 2014 (I)
2 Vizcaino and Crawford 2015 (I)
Brotherton and Balskus 2015 (I)
Bian et al. 2015 (I, 35–55 µg/L)
O
OH
10
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O
O
H2N
S
N
N
O
O
O
HN
H
N
H2N
O
n-C13H27
N
H
H
N
O
CH3
NH
n-C13H27
N
H
O
O
CH3
NH
O
precolibactin B (3)
4 Li et al. 2015 (I, 30 µg/L)
Li et al. 2015 (I, 2.5 µg/L)
O
O
O
H2N
O
N
N
R =
5a
OH
O
H
N
NH
S
R
S
n-C13H27
N
H
O
CH3
O
NH
O
N
N
5
b
OH
precolibactin A (5a or 5b) Vizcaino and Crawford 2015 (P)
either 5a or 5b possible based on prior data
S
S
O
OH
S
OH
O
N
O
NH
SH
S
N
N
O
O
H2N
O
S
N
O
O
H2N
O
H
N
N
n-C13H27
N
H
H
N
O
CH3
NH
n-C13H27
N
H
O
O
CH3
NH
precolibactin C (6)
Li et al. 2015 (P)
O
revised structure of precolibactin A (7)
Zha et al. 2016 (I, 10 µg/L)
Figure 1. Structures of isolated, predicted, and synthesized candidate precolibactins. Fermentation yields
(micrograms of product per liter of fermentation broth) are shown in parentheses, where available.
Fully elaborated (pre)colibactins have been difficult to isolate in homogenous form, and the definitive
structures of the most active metabolite(s) are not known. This has been attributed to the low levels of natural
production of the metabolites, their instability under fermentation conditions, and the inflammation-dependant
5
c
10
up-regulation of the native clb gene cluster. The metabolites 1, 2, 3 (referred to hereafter as “precolibactin
1
0
10
B”), and 4 were obtained in vanishingly small quantities (2.5–55 µg/L for 2–4) from the fermentation broth of
+
genetically-engineered clb E. coli and implicated as shunt metabolites and/or degradation products in the
colibactin biosynthetic pathway (Figure 1). Using the isolation of 2, as well as HRMS analysis, isotope
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labelling, and bioinformatics based on established biosynthetic logic, the structure of precolibactin A was
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10a
predicted as 5a or 5b.
Key elements within the proposed structures include a hydrophobic N-terminal
fragment, a spirocyclic aminocyclopropane, and (read from left to right) a thiazoline–thiazole chain. As the
1
0a
presence of the thiazoline–thiazole fragment was inferred by bioinformatic analysis, 5a and 5b could not be
unequivocally distinguished at that time, and the absolute stereochemistry of the putative thiazoline ring was
not determined. A compound with an exact mass corresponding to 5a was observed in unpurified extracts, but
0
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0
10a
all efforts to isolate this structure were hampered by its low levels of production and instability. The pyridone
structure 6 (referred to hereafter as “precolibactin C”) was recently proposed as a candidate precolibactin
1
1
based on biosynthetic considerations, isolation of precolibactin B (3), and HRMS analysis, and during the
preparation of this manuscript, Balskus and co-workers reported the isolation of precolibactin C (6) from a
1
2
mutant strain (0.5 mg of 6 was obtained from an optimized 48-L fermentation). Although one can envision
cyclodehydration of 5a or 5b to form pyridones resembling 6, the biosynthetic relationship between these
10a
structures had not been established. 2 was shown to weakly cross-link DNA in vitro, suggesting that the
13
colibactins may damage DNA by induction of replication-dependant DSBs. Detailed structure–function
analyses of the colibactins have been impossible to conduct owing to their low yields of natural production and
the absence of a synthetic route to the targets. However, the aminocyclopropane fragments within 2–6 are
reminiscent of yatakemycin, CC-1065, and the duocarmycins, which have been shown to alkylate DNA via
1
4
15
nucleophilic ring-opening, and the biheterocyclic fragment may serve as a DNA intercalation motif.
In light of the immense difficulties associated with isolating natural precolibactins, chemical synthesis
provides an attractive avenue to resolve the ambiguities surrounding the composition of the active
metabolite(s) and to enable mechanism of action and structure–function studies. Studies indicate the
1
2,16
presence of an aminomalonyl unit in the biosynthetic pathway,
suggesting additional colibactins are
formed, but no evidence relevant to the structures of these metabolites exists, to our knowledge.
Consequently, we initially focused on the synthesis of the predicted structures of precolibactin A (5a and 5b)
and precolibactin C (6), as these represent the most advanced precolibactins for which structural data had
been presented. Herein we report a convergent and high-yielding synthesis of structures 5a and 5b by
cyclization of a fully linear precursor, establish that these materials are distinct from natural precolibactin A,
propose and validate by synthesis a revised structure for precolibactin A (as 7), demonstrate that acyclic
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Journal of the American Chemical Society
precolibactins undergo cyclodehydration to form the pyridone residues found in precolibactin B (3), 4, and
precolibactin C (6) under mild conditions, and confirm the structures of precolibactins B (3) and C (6) by total
synthesis. We anticipate that this route will be amenable to synthesis of more advanced structures, including
those containing aminomalonyl units, as they are proposed.
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Results.
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As shown in Figure 1, at the time we began our studies, the structure of precolibactin A had been
predicted as 5a or 5b. Consequently, we designed our synthetic route to accommodate either heterocyclic
sequence and to provide access to the bithiazole found in precolibactin C (6). The synthesis of the common
left-hand fragment began with N
α
-(tert-butoxycarbonyl)-D-asparagine, which was coupled with (S)-hex-5-en-2-
1
7
amine (8, prepared in three steps, 96% yield, and 88% ee from pent-4-en-al)
using N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC•HCl). Cleavage of the tert-butoxycarbonyl
protective group (hydrochloride acid) provided the amine hydrochloride 9 (84%, two steps). Acylation of the
amine 9 with myristoyl chloride, followed by oxidative cleavage of the alkene (ruthenium chloride, sodium
periodate) generated the carboxylic acid 10 (78%, two steps).
a. EDC•HCl, HOBT
O
ClH3N
O
H2N
H2N
CH3
8
H
N
OH
b. HCl
BocNH
ClH3N
O
O
CH3
8
4% (two steps)
N-Boc-D-Asn
9
O
H2N
O
c. myristoyl chloride
O
Et N
H
N
3
n-C13H27
N
H
OH
d. RuCl3, NaIO4
O
CH3
78% (two steps)
1
0
Scheme 1. Synthesis of the carboxylic acid 10. Reagents and conditions: a. (S)-hex-5-en-2-amine hydrogen
chloride (8), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC•HCl), hydroxybenzotriazole (HOBT), N,N-
diisopropylethylamine (DIPEA), DMF, 23 °C, 84%; b. HCl, CH
2
Cl
2
–1,4-dioxane (7:1), 23 °C, >99%; c. Myristoyl
O–EtOAc–CH CN (3:2:2), 50 °C, 95%.
chloride, triethylamine (Et N), DMF, 23 °C, 82%; d. RuCl , NaIO
3
3
4
, H
2
3
The isomeric thiazoline–thiazole and the related bithiazole fragments were prepared by the sequences
1
8
shown in Scheme 2. Deuterated cysteine labelling experiments supported preservation of the L-amino acid
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so we selected L-cysteine as the building block for the thiazoline ring.
Treatment of N-(tert-butoxycarbonyl)aminoacetonitrile (11) with L-cysteine ethyl ester provided the thiazoline
2 (85%, Scheme 2A). Aminolysis of the ester, followed by heating with Lawesson’s reagent, generated the
1
0a
configuration in precolibactin A
1
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5
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9
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1
1
1
1
1
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2
2
2
2
2
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5
5
5
5
5
5
5
6
1
thioamide 13 (>99%, two steps). Exposure of 13 to bromopyruvic acid in the presence of triethylamine formed
the thiazoline–thiazole 14 (71%). The thiazoline–thiazole and subsequent intermediates were found to be
exceedingly unstable toward hydrolytic ring-opening and, to a lesser and variable extent, oxidation to a
bithiazole. Accordingly, the identification of conditions to isolate and purify these intermediates without
exposure to water was essential to the success of the route. Cleavage of the tert-butoxycarbonyl protective
group (hydrochloric acid, >99%) generated the amine 15. Coupling (silver trifluoroacetate, triethylamine) of the
amine 15 with the β-ketothioester 16 (prepared in one step and 56% yield from N-(tert-butoxycarbonyl)-1-
0
1
2
3
4
5
6
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8
9
0
1
2
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8
9
0
1
2
3
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7
8
9
0
1
2
3
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5
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7
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9
0
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2
3
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5
6
7
8
9
0
1
7
aminocyclopropane-1-carboxylate) followed by carbamate cleavage (hydrochloric acid, >99%) furnished the
thiazoline–thiazole fragment 17.
The isomeric thiazole–thiazoline fragment was prepared by a modified sequence (Scheme 2B).
Treatment of N-(tert-butoxycarbonyl)-2-aminoethanthioamide (18) with ethyl bromopyruvate formed the
thiazole 19 (74%). Aminolysis of 19 followed by dehydration of the resulting primary amide (trifluoroacetic
anhydride, triethylamine) generated the nitrile 20 (84%, two steps). Coupling of 20 with L-cysteine formed the
thiazole–thiazoline 21 (97%). In contrast to the isomeric intermediate 14, 21 was found to be stable toward
aqueous workup and atmospheric oxygen. A three-step sequence analogous to that described above provided
the thiazole–thiazoline 23 (69% overall).
The bithiazole fragment was prepared by the sequence shown in Scheme 2C. Aminolysis of the ester
1
9 followed by heating with Lawesson’s reagent formed the thioamide 24 (>99%, two steps). Treatment of the
thioamide 24 with bromopyruvic acid in the presence of calcium carbonate formed the bithiazole 25 (58%).
1
9
Prior efforts to prepare and isolate 25 were impeded by its instability; we found that rigorous exclusion of
water during work-up and purification facilitated the isolation of 25 and subsequent intermediates in
homogenous form. A three-step sequence analogous to that used to prepare 17 and 23 then generated the
bithiazole fragment 27 (72% overall).
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A.
a.
L
-cys-OEt
BocHN
N
O
b. NH3
BocHN
N
S
d. BrCH C(O)CO H
N
N
S
CO2H
1
2
3
N
2
2
BocHN
BocHN
S
S
S
Et N
OEt
c. Lawesson's
reagent
NH2
Et N
3
3
11
12
13
14
e. HCl, >99%
8
5%
71%
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
>
99% (two steps)
O
O
O
O
CO H
CO H
f. AgOTFA, Et N, 63%
3
2
2
HCl•H N
N
N
thiazoline–thiazole
sequence
N
2
H N
N
2
BocHN
+
N
H
St-Bu
g. HCl, >99%
S
S
S
S
1
7
16
15
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
B.
S
h. BrCH C(O)CO Et
N
O
i. NH3
BocHN
N
k.
L
-cys
CO2H
2
2
BocHN
BocHN
N
N
N
BocHN
S
S
S
NH2
j. TFAA, Et3N
84% (two steps)
CaCO3
OEt
Et N
S
3
1
8
19
20
21
l. HCl, >99%
7
4%
97%
O
O
O
O
CO H
CO H
m. AgOTFA, Et N, 69%
3
2
2
HCl•H N
N
N
thiazole–thiazoline
sequence
N
S
2
H N
N
S
2
BocHN
+
N
H
St-Bu
n. HCl, >99%
S
S
2
3
16
22
C.
N
O
o. NH3
BocHN
N
S
q. BrCH2C(O)CO2H
CaCO3
N
N
CO2H
BocHN
BocHN
S
S
S
OEt
p. Lawesson's
NH2
S
reagent
1
9
24
25
r. HCl, >99%
58%
>
99% (two steps)
O
O
O
O
CO H
2
CO H
s. AgOTFA, Et N, 72%
3
2
HCl•H N
N
N
bithiazole
sequence
N
S
2
H N
N
N
2
BocHN
+
St-Bu
t. HCl, >99%
S
H
S
S
2
7
16
26
Scheme 2. A. Synthesis of the thiazoline–thiazole 17. B. Synthesis of the thiazole–thiazoline 23. C. Synthesis
of the bithiazole 27. Reagents and conditions: a. L-(+)-cysteine ethyl ester hydrochloride, Et N, CH OH, 23 °C,
85%; b. NH , CH OH–H O (2:1), 23 °C, >99%; c. Lawesson’s reagent, CH Cl , 23 °C, >99%; d. bromopyruvic
acid, Et N, CH OH, reflux, 71%; e. HCl, CH Cl –1,4-dioxane (4:1), 23 °C, >99%; f. silver trifluoroacetate
AgOTFA), Et N, DMF, 0 °C, 63%; g. HCl, CH Cl –1,4-dioxane (4:1), 23 °C, >99%; h. ethyl bromopyruvate,
, EtOH, 23 °C, 74%; i. NH , CH OH–H O (2:1), 23 °C, >99%; j. trifluoroacetic anhydride (TFAA), Et N,
, 0→23 °C, 84%; k. L-(+)-cysteine, Et N, CH OH, reflux, 97%; l. HCl, CH Cl –1,4-dioxane (8:1), 23 °C,
>99%; m. AgOTFA, Et N, DMF, 0 °C, 69%; n. HCl, CH Cl –1,4-dioxane (4:1), 23 °C, >99%; o. NH , CH OH–
O (2:1), 23 °C, >99%; p. Lawesson’s reagent, CH Cl , 23 °C, >99%; q. bromopyruvic acid, CaCO , EtOH, 23
C, 58%; r. HCl, CH Cl –1,4-dioxane (4:1), 23 °C, >99%; s. AgOTFA, Et N, DMF, 0 °C, 72%; t. HCl, CH
,4-dioxane (4:1), 23 °C, >99%.
3
3
3
3
2
2
2
3
3
2
2
(
3
2
2
CaCO
3
3
3
2
3
CH
2
Cl
2
3
3
2
2
3
2
2
3
3
H
2
2
2
3
°
2
2
3
2
Cl
2
–
1
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
To complete the synthesis of the precolibactin A skeleton, the carboxylic acid 10 was first converted to
the β-ketothioester 28 by activation with carbonyl diimidazole followed by the addition of 3-(tert-butylthio)-3-
2
0
oxopropanoic acid and magnesium ethoxide (Scheme 3). Silver-mediated coupling of 28 with the heterocyclic
fragments 17, 23, or 27 then formed the penultimate intermediates 29a–c (90%, 87%, and 86% for 29a, 29b,
and 29c, respectively). The stabilities of the fully linear precursors 29a–c paralleled those of 17, 23, or 27; the
thiazole–thiazoline 29b was stable toward aqueous work-up, while the thiazoline–thiazole 29a and the
bithiazole 29c were unstable toward aqueous conditions.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a.
O
O
O
O
H N
O
H N
O
2
2
b. 17, 23, or 27
HO
St-Bu
O
O
O
O
10
H
N
H
N
H
N
CDI, Mg(OEt)2
AgOTFA, Et N
3
R
n-C13H27
N
H
St-Bu
n-C13H27
N
H
N
H
O
CH3
O
CH3
O
O
9
5%
2
8
2
9a–c
S
S
S
S
S
S
R =
N
N
N
N
N
N
CO H
CO H
CO H
2
2
2
2
9a (90%)
29b (87%)
29c (86%)
Scheme 3. Synthesis of the acyclic advanced precursors 29a–c. Reagents and conditions: a. Carbonyl
diimidazole (CDI), 4 Å molecular sieves, DMF, then malonic acid half-thioester, magnesium ethoxide
(
Mg(OEt)
2
), 23 °C, 95%; b. 17, 23, or 27, AgOTFA, Et N, DMF, 0 °C, 90% (29a); 87% (29b); 86% (29c).
3
Given the higher stability of the thiazole–thiazoline 29b, this compound was used to develop conditions
to effect the key cyclization reaction (to 5b). Surprisingly, we found that in preparative experiments treatment
of 29b with potassium carbonate in methanol at 0 °C resulted in formation of the pyridone 30b (80%, Scheme
4
A). Similar outcomes were obtained on exposure of 29b to ammonium carbonate in ethanol or aqueous
sodium hydroxide. Under these conditions, accumulation of the putative monocyclized intermediate 5b was
not observed (LC/MS analysis). The pyridone 30b was fully characterized and spectroscopic data for this
compound were in good agreement with the isolated metabolite precolibactin B (3; Table 1). In particular, H-2
and H-4 of 30b resonated at 6.16 and 5.59/5.50 ppm, respectively; these values are nearly identical to those
11
recorded for natural precolibactin B (3; 6.16 and 5.61/5.48 ppm, respectively). A plausible mechanism for the
formation of the pyridone 30b involves cyclodehydration to 5b, 1,2-addition of the primary amide to the
8
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Journal of the American Chemical Society
adjacent carbonyl, and aromatization. The facile formation of 30b from 29b provides evidence that the putative
and isolated colibactin metabolites 3, 4, and 6 may derive from related acyclic precursors, although the timing
of cyclizations in the modular biosynthetic pathway remains unknown. The cyclization of the thiazoline–
thiazole derivative 29a and the bithiazole derivative 29c proceeded in a strictly analogous manner to provide
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
7
the fully cyclized derivatives 30a or precolibactin C (6), respectively. The mass spectroscopic fragmentation
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
data and H NMR data for synthetic precolibactin C (6), as well as LC/MS co-injection with metabolite extracts,
10,12
matched those of natural material (Figure S1, Tables S1 and S5).
In addition, synthetic precolibactin C (6)
was converted to N-myristoyl-D-Asn and its corresponding colibactin in a ClbP-dependent manner, indicating
that precolibactin C (6) represents a suitable substrate for ClbP (Figure S3). This sequence provided
multimilligram quantities of 30a, 30b, and precolibactin C (6), and is envisioned to be easily scalable.
A.
O
B.
24 h
30b
H2N
O
O
O
H
N
2
H
3
N
R
n-C13H27
N
H
N
H
1
4
O
CH3
O
O
2
9a–c
a. K2CO3, CH3OH, 0 °C or
b. K2CO3, DMSO, 24 °C
7.5 h
30b
O
5b
O
H2N
O
2
4
O
H
N
N
H
3
R
n-C13H27
N
H
O
CH3
NH
3.5 h
1
O
29b
5a–c
time 5b
30b
(
not detected under conditions a,
detected under conditions b)
cyclodehydration
O
H2N
O
R
O
29b
0 h
4
2
N
H
N
n-C13H27
N
H
O
CH3
NH
1
O
3
0a (15 mg), 30b (8 mg), or precolibactin C (6, 12 mg)
S
S
S
S
S
S
R =
N
N
N
N
N
N
CO2H
CO2H
CO2H
2
9a, 5a, and 30a
29b, 5b, and 30b
29c, 5c, and 6
Scheme 4. A. Cyclodehydration of the linear precursors 29a–c. Reagents and conditions: a. K
2 3 3
CO , CH OH,
0
°C, 79% (30a); 80% (30b); 83% (30c) or b. K CO , dimethyl sulfoxide, 24 °C (for 29b). B. UV trace (254 nm)
2
3
of the cyclization of 29b using potassium carbonate in dimethyl sulfoxide at 24 °C.
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Page 10 of 16
We ultimately found that treatment of 29b with potassium carbonate (~3.0 equiv) in dimethyl sulfoxide
at 24 °C proceeded more slowly and allowed for detection of 5b in the reaction mixture (LC/MS analysis,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
Scheme 4B). By conducting the reaction in dimethyl sulfoxide-d , signals consistent with the monocyclized
1
intermediate 5b could also be observed by H NMR analysis (Table 1).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
Table 1. Selected H chemical shifts and LC/MS data for 29b, 5b, 30b,
a
and precolibactin B (3).
Position
1
2
3
4
r
t (min)
2
5
3
9b
b
0b
8.83
8.78
8.46
8.44
3.60
—
6.16
6.16
8.94
8.65
4.56
—
3.52
3.48
3.55
b
b
5.59, 5.50
5.61, 5.48
6
(500 MHz, 23 °C).
1
0
precolibactin B (3)
H spectroscopic data recorded in DMSO-d
Complete H NMR spectra (Figure S2) and LC/MS conditions are
presented in the Supporting Information. Not resolved.
a1
1
b
Mass-selective LC/HRMS-QTOF analysis was conducted to determine if either 5a or 5b corresponded
+
to the structure of natural precolibactin A. As shown in Figure 2, the concentrated ethyl acetate extracts of clb
10a
+
E. coli ΔclbP
displayed a single prominent peak of m/z = 816.3788, which corresponds to [M+H] for the
proposed structure of precolibactin A. However, the retention times of synthetic 5a and 5b were distinct and
the signals did not coalesce upon co-injection with the natural sample (Figures 2A and 2B, respectively). The
retention times of 5a and 5b were nearly identical (t
r
= 15.70 and 15.80 min, respectively), as expected, and
= 16.56 min) suggested a significant discrepancy in
their differences with respect to natural precolibactin A (t
structure.
r
1
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Journal of the American Chemical Society
3
3
2
1
A.
B.
natural precolibactin A
natural precolibactin A
1
2
3
4
5
6
7
8
9
2
1
0
6
0
3
5
a
5b
2
1
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
0
6
co-injection
co-injection
3
2
1
0
3
0
15
15.5
16
16.5
17
15
15.5
16
16.5
17
Time (min)
Time (min)
+
Figure 2. A. Mass-selective (m/z = 816.3788) LC/HRMS-QTOF analysis of the ethyl acetate extracts of clb E.
coli ΔclbP (top), synthetic 5a (middle), and co-injection (bottom). B. Mass-selective (m/z = 816.3788)
+
LC/HRMS-QTOF analysis of the ethyl acetate extracts of clb E. coli ΔclbP (top), synthetic 5b (middle), and co-
4
injection (bottom). Y axis corresponds to relative ion intensity (×10 ).
In light of the facile cyclization of our synthetic intermediates to the pyridone residues found in
precolibactins B (3) and C (6), we reasoned that precolibactin A may also incorporate this substructure. The
1
0a
original isotope labeling, HRMS, and MSMS data for natural precolibactin A
could not exclude this
assignment. In addition, careful inspection of the initial report revealed that precolibactin A production was
optimized by increasing the concentration of L-cysteine in the media 5-fold (to 1 g/L). Walsh and co-workers
2
1
have previously reported the formation of cysteine derailment products in the biosynthesis of yersiniabactin.
Accordingly, we hypothesized that the structure of natural precolibactin A may comprise the pyridone found in
precolibactins B (3) and C (6) and a terminal cysteine residue appended to a single thiazole ring (Scheme 5A).
This structure (7) possesses an exact mass that is identical to the originally predicted structures 5a or 5b and
would similarly match the reported amino acid isotope labeling studies. Such a change in the terminal
1
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Page 12 of 16
heterocyclic fragment would also be consistent with the large differences in retention times between 5a or 5b
and natural precolibactin A. The synthesis of the revised precolibactin A structure 7 was readily-accomplished
using our synthetic strategy (Scheme 5B). Treatment of N-(tert-butoxycarbonyl)-2-aminoethanthioamide (18)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
2
with bromopyruvic acid followed by removal of the tert-butoxycarbonyl protective group generated the
thiazole 31 (74%, two steps). Silver trifluoroacetate-mediated coupling of 31 and the thioester 16, followed by
carbamate cleavage, formed the amine 32 (55%, two steps). Coupling of the amine 32 with the thioester 28
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
Scheme 3; silver trifluoroacetate, triethylamine), followed by double-cyclization (potassium carbonate,
methanol) generated precolibactin B (3; 67% over two steps, 36 mg). NMR spectroscopic data for synthetic
precolibactin B (3) and LC/MS co-injection with metabolite extracts matched those of natural material (Figure
1
1,12
17
S1, Tables S3, S4),
thereby confirming the structure of the natural product.
Finally, coupling of
precolibactin B (3) with L-cysteine mediated by N-hydroxysuccinimide (NHS) and EDC•HCl generated 7 (89%).
+
Mass-selective LC/HRMS-QTOF analysis against the concentrated ethyl acetate extracts of clb E. coli
10a
ΔclbP revealed that 7 corresponded exactly to natural material (Figure 3). In addition, both synthetic 7 and
natural precolibactin A displayed identical mass spectral fragmentation patterns, providing further confirmation
1
0a
of structure (Table S2). As natural precolibactin A is not isolable in amounts sufficient for NMR analysis,
a
2
3
direct comparison of the NMR spectra of synthetic and natural precolibactin A is not possible at this time.
OH
A.
M+H] = C39H58N O S2+, 816.3783
+
[
O
7
8
O
O
NH
O
SH
H N
O
2
O
O
S
N
N
H
N
NH
O
N
O
N
OH
H N
n-C13H27
N
H
2
S
O
O
CH3
S
NH
H
N
O
n-C13H27
N
H
O
CH3
NH
O
originally proposed structure of precolibactin A (5a)
revised structure of precolibactin A (7)
B.
O
O
S
a. BrCH C(O)CO H, CaCO
HCl•H N
N
O
c. 16, AgOTFA, Et
d. HCl
N
3
2
2
3
2
O
H N
N
2
BocHN
N
H
S
NH2
b. HCl
OH
S
OH
1
8
31
32
7
4% (two steps)
55% (two steps)
OH
e. 28, AgOTFA, Et N
3
O
g.
L
-cys
O
S
O
EDC•HCl
NHS
OH
N
S
N
H N
O
O
2
O
f. K
2
CO
3
O
O
N
precolibactin A (7)
H N
O
2
H
N
H
N
DMF
CH
OH
3
H
N
n-C13H27
N
H
N
H
8
9%
n-C13H27
N
H
67%
two steps)
O
CH3
O
O
(
O
CH3
NH
O
precolibactin B (3, 36 mg)
33
1
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Scheme 5. A. The originally predicted (5a) and revised (7) structures of precolibactin A. B. Synthesis of the
revised structure of precolibactin A (7). Reagents and conditions: a. bromopyruvic acid, CaCO , EtOH, 23 °C,
4%; b. HCl, CH Cl –dioxane (3:1), 23 °C, >99%; c. AgOTFA, Et N, DMF, 0 °C, 55%; d. HCl, CH Cl –1,4-
dioxane (3:1), 23 °C, >99%; e. AgOTFA, Et N, DMF, 0 °C; f. K CO , CH OH, 0 °C, 67% (two steps); g. L-
cysteine, N-hydroxysuccinimide (NHS), EDC•HCl, Et N, DMF, 0→23 °C, 89%.
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
7
2
2
3
2
2
3
2
3
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
natural precolibactin A
20
1
0
0
20
7
1
0
0
3
co-injection
0
0
0
2
1
0
16
16.5
Time (min)
17
+
Figure 3. Mass-selective (m/z = 816.3788) LC/HRMS-QTOF analysis of the ethyl acetate extracts of clb E.
coli ΔclbP (top), synthetic 7 (middle), and co-injection (bottom).
Discussion and Conclusion.
The colibactins are a fascinating family of natural products that are produced by certain strains of
commensal and extraintestinal E. coli, and the pathway has been implicated in the progression of colorectal
7,8
cancer.
Despite over a decade of intensive research, their complete structures and mechanism of action
1
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Page 14 of 16
have remained unresolved. As highlighted in Figure 1, many of these compounds have been isolated in
astoundingly low yields (µg/L) by painstaking fermentation experiments. By bringing the power of modern
bioinformatics, enzymology, and mass spectrometry to bear on this problem, the structures of additional
precolibactins, which are recalcitrant to isolation, have been predicted.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
At the time we began our work, 3–6 represented the most complex precolibactin structures in the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0
10a
11
literature. Precolibactin B (3) and 4 were fully characterized by isolation, while 5a and precolibactin C (6)
1
2
were predicted. While this manuscript was in preparation, Balskus and co-workers reported the isolation of
precolibactin C (6; 0.5 mg from a 48 L fermentation) from a mutant strain. Biosynthetic studies now suggest
12,16
that additional precolibactins incorporating an aminomalonyl unit exist,
has been presented, to our knowledge.
but no evidence for their structures
We have developed a high-yielding and modular synthetic route to the most advanced known
precolibactin structures. The left hand fragment 10, which is common to all of the precolibactins, is prepared in
six steps and 63% overall yield from pent-4-en-al (Scheme 1). We have executed the synthesis of four distinct
heterocyclic side chain fragments, in 4–7 steps and 37–41% overall yield (Schemes 2 and 5). Finally, these
intermediates are elaborated to advanced precolibactins in three steps and ~50% overall yield (Schemes 3, 4,
and 5). We have confirmed the structures of the isolated precolibactins B (3) and C (6) by chemical synthesis,
and revised the structure of precolibactin A, from 5a or 5b to 7. This structural revision also supports an
unexpected biosynthetic route to colibactin bithiazole formation, in which biosynthesis of the first thiazole ring
may precede heterocyclization and oxidation of the C-terminal L-cysteine moiety. This is in contrast to
2
4
bioinformatic proposals for bleomycin bithiazole biosynthesis. Our synthetic studies also provide insights into
the reactivities of these structures, and the facile cyclization of the linear precursors 29a–c to pyridones
suggests this element as a common substructure. We envision that the synthetic strategy we have presented
will be amenable to the synthesis of precolibactins incorporating an aminomalonyl substituent or other
modifications, as their structures are proposed.
The synthetic route outlined herein provides a means to procure sufficient quantities of material to study
the cellular responses to isolated colibactins and elucidate their mechanism of action for the first time. As
+
noted in the introduction, mammalian cells were shown to accumulate DNA DSBs when co-cultured with pks
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E. coli cells, but no essential follow-up studies employing single metabolites derived from the clb pathway
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have been reported, to our knowledge. We expect that our modular synthetic strategy will finally open the door
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to examining these types of questions regarding colibactin’s mode of action with molecular-level resolution.
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Acknowledgement. Financial support from the National Institutes of Health (R01GM110506 to S.B.H. and
DP2-CA186575 to J.M.C.) is gratefully acknowledged. We thank Mr. Herman Nikolayevskiy and Mr. Steven
1
Swick for assistance with DFT calculations.
Supporting Information Available.
Supplementary Figures S1–S6, Supplementary Tables S1–S5,
Supplementary Scheme S1, general experimental procedures, and detailed experimental procedures and
characterization data for all new compounds. This data is available free of charge at www.pubs.acs.org.
References.
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. For a review, see: Donia, M. S.; Fischbach, M. A. Science 2015, 349, 395.
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. Dubois, D.; Baron, O.; Cougnoux, A.; Delmas, J.; Pradel, N.; Boury, M.; Bouchon, B.; Bringer, M. A.;
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5. Wu, W.; Vanderwall, D. E.; Turner, C. J.; Hoehn, S.; Chen, J.; Kozarich, J. W.; Stubbe, J. Nucleic Acids
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216.
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3. Synthetic precolibactin A (7) was found to readily undergo disulfide bond formation, which may explain its
instability in complex cellular organic extracts.
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Nat. Prod. 2011, 74, 526.
5. Our data suggest that prior proposals of the colibactin mechanism of action may require revision. See the
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Supporting Information for a discussion.
TOC Graphic.
OH
O
O
• precolibactin A (7)
R =
R
•
revised structure
HN
SH
S
N
N
O
O
•
precolibactin B (3)
H2N
O
R =
R =
OH
H
N
• confirmed by synthesis
n-C13H27
N
H
S
N
• precolibactin C (6)
O
CH3
NH
O
•
confirmed by synthesis
CO2H
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