Gene expression enabling synthetic diversification of natural products: Chemogenetic generation of pacidamycin analogs

Article abstract of DOI:10.1021/ja1060406

Introduction of prnA, the halogenase gene from pyrrolnitrin biosynthesis, into Streptomyces coeruleorubidus resulted in efficient in situ chlorination of the uridyl peptide antibotic pacidamycin. The installed chlorine provided a selectably functionalizable handle enabling synthetic modification of the natural product using mild cross-coupling conditions in crude aqueous extracts of the culture broth.

Full text of DOI:10.1021/ja1060406

Published on Web 08/16/2010
Gene Expression Enabling Synthetic Diversification of Natural Products:
Chemogenetic Generation of Pacidamycin Analogs
Abhijeet Deb Roy, Sabine Gru¨schow, Nickiwe Cairns, and Rebecca J. M. Goss*
School of Chemistry, UniVersity of East Anglia, Norwich NR4 7TJ, U.K.
Received July 8, 2010; E-mail: r.goss@uea.ac.uk
municated. As the test bed for these experiments, we have chosen
Abstract: Introduction of prnA, the halogenase gene from pyr-
the uridyl peptide antibiotic (UPA) pacidamycin, a compound with
rolnitrin biosynthesis, into Streptomyces coeruleorubidus resulted
an unusual structure and with good activity against the Gram-
in efficient in situ chlorination of the uridyl peptide antibotic
negative pathogen Pseudomonas aeruginosa.8 No natural haloge-
pacidamycin. The installed chlorine provided a selectably func-
nated UPA analogs exist. At the time of carrying out these
tionalizable handle enabling synthetic modification of the natural
experiments the pacidamycin producer, Streptomyces coeruleoru-
product using mild cross-coupling conditions in crude aqueous
bidus AB11383F-64, was genetically uncharacterized. Although
extracts of the culture broth.
little is known about the biosynthesis of these antibiotics, the
enzymes involved in the assembly of pacidamycin have been shown
to be highly flexible and able to incorporate a series of unnatural
substrate analogues.9,10
Natural products represent a treasure trove of medicinally
relevant compounds: over the past 3 decades over 70% of
To enable the realization of the concept two challenges needed
antimicrobials and over 60% of antitumor agents entering clinical
to be addressed: first the introduction and expression of a gene that
trials have been based on natural products.1 Generation of natural
would produce an active protein capable of acting in concert with
product analogs is an important area enabling improvement of
the natural biosynthetic machinery to introduce a halogen; second
activity and overcoming poor physicochemical properties.
application of sufficiently mild and aqueous derivatization condi-
Conventional methods of analog generation include total syn-
tions to enable selective cross-coupling chemistry at the halogen
thesis, semisynthesis, and mutasynthesis.2
without decomposition of the thermally unstable natural product.
Here we describe a new paradigm in natural product analog
As we had previously demonstrated the striking promiscuity of the
generation, the introduction of a gene to act in concert with an
pacidamycin biosynthetic enzymes toward 7-halotryptophans,9 we
existing biosynthetic pathway so as to install a chemical handle
enabling selective functionalization of the natural product (Scheme
undertook to explore the introduction of prnA, the tryptophan
7-halogenase gene from pyrrolnitrin biosynthesis, to complement
1). The biosynthetic introduction of a halogen into a compound
the pacidamycin biosynthetic genes.3 The gene was cloned into
affords the, until now unrealized, potential to enable selective
the integrative plasmid pIJ1025711 in which prnA was placed under
postbiosynthetic derivatization. Over the past 5 years the structures
of a series of halogenases have been reported.3-6 Four examples
the control of the constitutive promoter ermE*. The expression
construct was introduced into the pacidamycin producer S. coeru-
leorubidus through conjugation from Escherichia coli to give strain
RG-5059. The resulting strain produced chloropacidamycin 1
(Scheme 1) alongside nonhalogenated pacidamycins. The isolated
yields of chloropacidamycin 1 were approximately 1 mg per liter
culture. This titer is comparable to 1 obtained from precursor-
directed biosynthesis.9 Typically, the ratio of 1 to its parent
pacidamycin was 1:5 but could go as high as 4:1 in favor of the
of the heterologous expression of halogenases have been described
so far. These reports involve the introduction of a halogenase into
closely structurally and biosynthetically related systems and the
generation of additional analogs of known halometabolites.7 We
report the first introduction of a halogenase to complement a
genetically undefined and biosynthetically unrelated pathway giving
access to a new series of halometabolites. The postbiosynthetic
diversification of these new unnatural products is also com-
Scheme 1. Gene Expression Enabling Synthetic Diversificationa
a The introduction of prnA, a gene encoding a halogenase, into the pacidamycin producer results in the generation of chlorinated pacidamycins. The
chlorine may be used as a selectably functionalizable handle enabling further synthetic diversification.
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J. AM. CHEM. SOC. 2010, 132, 12243–12245 12243

C O M M U N I C A T I O N S
chlorinated pacidamycin. The factors governing the observed
variability are currently not fully understood.
We then turned our attention to the chemical functionalization
of the halogen handle and the possibility of derivatizing the natural
product as a component of a crude aqueous extract. We envisaged
that the introduction of the chlorine to the pacidamycin skeleton
would make it a suitable substrate for Pd-mediated Suzuki-Miyaura
and related cross-coupling reactions. Pacidamycin is a particularly
demanding, and therefore useful, model for synthetic derivatization;
it contains a number of potentially reactive functional groups that
render selective synthetic modifications difficult, it is thermally
unstable,8 and due to its highly polar nature it is insoluble within
organic solvents. So far, few examples have been reported of the
palladium-catalyzed cross-coupling reactions of hydrophilic aryl
chlorides with aryl boronic acids under aqueous conditions. The
few reported examples employ high temperatures and long reaction
times, making these conditions incompatible with thermally unstable
natural products.12 Therefore, we sought to develop milder reaction
conditions to enable the Suzuki-Miyaura coupling of chlorinated
natural products.
Initially, we explored the cross-coupling of unprotected 7-chlo-
rotryptophan as the model compound. With our previously devel-
oped conditions using tris(3-sulfonatophenyl)phosphine trisodium
salt (TPPTS) as ligand13 we were unable to effect the cross-coupling
of this relatively unreactive species; however, by applying extended
heating in a microwave up to 10% conversion could be observed.
In order to improve the reaction we explored a range of water-
soluble catalysts and ligands (Supporting Information (SI)) and
found the sterically more demanding and electron-rich SPhos ligand,
designed by Buchwald, to work best.14 While direct application of
the reported Buchwald conditions to 7-chlorotryptophan led to no
detectable product, by investigating different palladium salts and
adding a small amount of acetonitrile to solvate SPhos we were
able to determine conditions that enabled rapid reaction with
7-chlorotryptophan (see SI).
Having optimized conditions that would facilitate the cross-
coupling of the 7-chlorotryptophan, we investigated their application
to the modification of chloropacidamycin 1. Cross-coupling using
boronic acid, Na2Cl4Pd-SPhos, and K2CO3 in water/acetonitrile
under microwave heating at 80 °C for 1 h allowed the modification
of not only purified chloropacidamycins but also crude extracts of
1. The Suzuki-Miyaura cross-coupling was explored with four
boronic acids. Reaction with phenyl boronic acid or 4-methoxy-
benzene boronic acid resulted in quantitative conversion of 1 to
give 2 and 3, respectively (Figure 1). Unsurprisingly, lower
conversions were observed with the more electron-deficient 4-ethoxy-
carbonyl phenylboronic acid. During this reaction the ethyl ester
was cleaved resulting in the corresponding carboxylic acid deriva-
tive 4 being formed as the major product. Remarkably, the cross-
coupling was equally effective with the less reactive furan-2-boronic
acid to give 5. Reaction products were analyzed by LC-MS/MS.
Three fragments proved to be diagnostic for the presence of
additional substituents on the tryptophan residue (Figure 1).
Economical access to natural product analogs is an essential
component of drug discovery. We have demonstrated that a gene
can be expressed in complement to a genetically undefined
biosynthetic pathway installing a handle that enables further
selective synthetic diversification. The derivatization of the resulting
natural product has been achieved through the application of
exceptionally mild cross-coupling conditions. This has enabled the
modification of the natural product within aqueous extracts of
the fermentation broth, and in its semipurified and purified states.
The applicability of this approach to a genetically undefined system,
Figure 1. MS/MS analysis of pacidamycin analogs. Diagnostic fragment ions
are shown. Percent conversion based on consumption of 1 is listed next to the
corresponding partial spectra. [a] denotes isolated yield; [b] the reaction was
carried out on 1 as a component of a crude aqueous extract.
and a highly functionalized and thermally unstable natural product
implies great potential for this technology.
Acknowledgment. We are grateful to Prof. K.-H. van Pe´e (TU
Dresden) and Prof. M. J. Bibb (John Innes Centre) for provision
of plasmids pCIB7805 and pIJ10257, respectively. We thank Dr.
L. M. Hill (JIC) and the National Mass Spectrometry Service
(Swansea) for MS analysis. We are grateful to the Royal Society
for an RS Dorothy Hodgkin Fellowship for R.J.M.G. and to The
Leverhulme Trust for their support of this project F/00204/AO,
F/00204/AF.
Supporting Information Available: Full experimental details and
analytical data. This material is available free of charge via the Internet
at http://pubs.acs.org.
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