Chemoenzymatic acylation of aminoglycoside antibiotics

Article abstract of DOI:10.1039/b802248h

The chemoenzymatic installation of the clinically valuable (S)-4-amino-2-hydroxybutyryl side chain onto a number of 2-deoxystreptamine- containing aminoglycosides is described using the purified Bacillus circulans biosynthetic enzymes BtrH and BtrG in combination with a synthetic acyl-SNAC surrogate substrate. The Royal Society of Chemistry.

Full text of DOI:10.1039/b802248h

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Chemoenzymatic acylation of aminoglycoside antibioticsw
Nicholas M. Llewellyn* and Jonathan B. Spencerz
Received (in Cambridge, UK) 8th February 2008, Accepted 15th May 2008
First published as an Advance Article on the web 20th June 2008
DOI: 10.1039/b802248h
The chemoenzymatic installation of the clinically valuable (S)-4-
amino-2-hydroxybutyryl side chain onto a number of 2-deoxy-
streptamine-containing aminoglycosides is described using the
purified Bacillus circulans biosynthetic enzymes BtrH and BtrG
in combination with a synthetic acyl-SNAC surrogate substrate.
has been demonstrated to proceed with the unusual protection
of a potentially labile ACP-bound g-aminobutyryl intermedi-
ate as the N-g-L-glutamyl amide prior to installation of the
stereogenic C-2 hydroxyl. The g-L-glutamylated side chain is
then transferred onto the aminoglycoside substrate ribosta-
mycin by the acyltransferase BtrH, and the resulting
acyl-aminoglycoside species is cleaved by the g-L-glutamyl
cyclotransferase BtrG to reveal butirosin B (Scheme 1A).
Further examination of this biosynthetic machinery re-
vealed BtrH to possess modest tolerance for close structural
relatives of ribostamycin, including neamine, paromomycin,
and neomycin, as non-native side chain acceptors;
The aminoglycosides are a diverse class of bactericidal anti-
biotic natural products of clinical importance.1 Most of the
significant aminoglycoside families, including the neomycin-,
kanamycin-, and gentamicin-class aminoglycosides, consist of
the core aminocyclitol 2-deoxystreptamine (2-DOS) decorated
with various aminosugar substituents.2 This family of antibio-
tics binds the A-site stem loop feature of the bacterial 16S
rRNA,3,4 thereby inhibiting protein synthesis via interference
with codon fidelity,5 translocation,6 tmRNA aminoacylation,7
and ribosome recycling.8 Additionally, aminoglycosides and
their derivatives exhibit a number of interesting activities
toward other nucleic acid targets that may lead to the discovery
of novel antiviral agents and genetic disorder therapies.9–14
The continuing proliferation of bacterial resistance to anti-
biotics has spurred the investigation of diverse and intriguing
strategies to counter important resistance mechanisms.15–17
One of the most successful strategies is the rational modifica-
tion of existing drugs to improve their activity against resistant
organisms. Aminoglycoside derivatives bearing the (S)-4-
amino-2-hydroxybutyryl (AHBA) side chain are of particular
importance for their improved activity against aminoglyco-
side-resistant organisms, including methicillin-resistant
Staphylococcus aureus (MRSA).18 The AHBA pharmaco-
phore, originally observed in the natural product butirosin,19
is semisynthetically incorporated into the aminoglycoside
antibiotics kanamycin A and dibekacin to afford amikacin
and arbekacin, respectively.20,21 The regiospecific synthetic
acylation of densely functionalized and structurally diverse
aminoglycosides commonly requires multi-step, substrate-
specific protection/deprotection schemes.22–24 The obvious
appeal of a general biocatalytic route to regiospecifically
acylated aminoglycosides has, therefore, led our group to
investigate the biosynthetic origins of the AHBA moiety in
butirosin produced by Bacillus circulans NRRL B3312.25,26
The ACP-mediated biosynthesis of AHBA from L-glutamate
Department of Chemistry, University of Cambridge, Lensfield Road,
Cambridge, United Kingdom CB2 1EW. E-mail: nml30@cam.ac.uk;
Fax: +44 (0)1223 336362; Tel: +44 (0)1223 336710
w Electronic supplementary information (ESI) available: Experimental
details, synthetic methods, and LC-ESI-MS/MS spectra. See DOI:
10.1039/b802248h
Scheme 1 (A) Biosynthetic incorporation of the AHBA side chain in
butirosin B. (B) Synthetic N-acetylcysteamine thioester used as a
surrogate acyl donor for chemoenzymatic acylation.
z Deceased.
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Fig. 1 (A) Chemoenzymatic conversion of neomycin to neokacin. Incubation of neomycin (left) with BtrH and 2 results in essentially complete
conversion to g-L-Glu-AHBA-neomycin (centre, no peak corresponding to neomycin observed). This intermediate is then quantitatively cleaved by
incubation with BtrG to reveal neokacin (right, no peak corresponding to g-L-Glu-AHBA-neomycin observed). (B) Chemoenzymatic conversion
of kanamycin to amikacin. Incubation of kanamycin (left) with BtrH and 2 results in partial conversion (B15%) to g-L-Glu-AHBA-kanamycin
(centre). This intermediate is then quantitatively cleaved by incubation with BtrG to reveal amikacin (right, no peak corresponding to g-L-Glu-
AHBA-kanamycin observed, overlapping peak corresponding to unconverted kanamycin omitted). LC-ESI-MS traces with selective ion
monitoring (SIM) for each relevant species are given below the corresponding structure. See ESIw for mass spectra and MS/MS fragmentation
data for each species.
furthermore, BtrG exhibits good activity against unnatural
g-L-Glu-AHBA-aminoglycosides.26 This finding encouraged
us to investigate the application of this system to the produc-
tion of a greater diversity of AHBA-aminoglycoside species.
For this purpose, it was desirable to circumvent the ACP
(BtrI) as the acyl donor, as reconstitution of the complete
multi-enzyme biosynthetic pathway in vitro is inconvenient.
Further, while the native acyl donor g-L-Glu-AHBA-S-BtrI (1)
may be prepared by loading synthetic g-L-Glu-AHBA-CoA26
onto apo-BtrI with the promiscuous phosphopantetheinyl
transferase Sfp,27 the spent holo-BtrI cannot be reloaded with
another acyl group, and the small quantity of 1 that can
reasonably be prepared limits the overall theoretical produc-
tivity of this non-catalytic approach. It is well established that
synthetic N-acetylcysteamine thioesters are suitable surrogates
for carrier protein-bound substrates;28,29 therefore, we elected
to pursue a chemoenzymatic strategy in which a synthetic N-
acetylcysteamine thioester (SNAC) is substituted for 1 as the
side chain donor. g-L-Glu-AHBA-SNAC (2, Scheme 1B) was
synthesized by reported strategies,26,29 and the competency of
this substrate as an acyl donor was tested by incubation with
purified recombinant BtrH and ribostamycin. Satisfyingly,
quantitative acylation of ribostamycin was observed by LC-
ESI-MS/MS analysis of the reaction mixture. Further,
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incubation of 2 with BtrH and a variety of non-native ami-
noglycoside acceptors led to acylation of all substrates tested,
with varying levels of conversion. Paromamine, neamine,
paromomycin, neomycin, and apramycin are all well tolerated
as substrates for BtrH with essentially complete conversion
observed (Fig. 1A and ESIw). 2-DOS is also accepted with
moderate conversion (B70%) to the acylated species within
the assay period (ESIw). Kanamycin and gentamicin C are
acylated with modest conversion (B15% and B10%, respec-
tively) (Fig. 1B and ESIw). Notably, a number of these
aminoglycoside substrates (2-DOS, paromamine, apramycin,
kanamycin, and neomycin) had previously been tested with the
native acyl donor 1 without any detectable side chain incor-
poration;26 these new results demonstrate that BtrH possesses
broader substrate tolerance than previously thought. Investi-
gation into the reason for the improved substrate scope when
using 2 as the acyl donor is ongoing. The effect may be due
exclusively to the higher concentration of 2 that is reasonably
achieved relative to the less abundantly available 1; alterna-
tively, possible interactions between BtrH and BtrI might
impose a discriminatory conformational state on BtrH. All
of the acyl-aminoglycosides produced by the action of BtrH
are quantitatively and rapidly deglutamylated by incubation
with BtrG, revealing the expected AHBA-aminoglycoside
species (Fig. 1 and ESIw). However, 2 was also found to be
a good substrate for BtrG; therefore, the in vitro construction
of AHBA-aminoglycosides by this method must be accom-
plished by stepwise incubation, first with BtrH and 2, then
followed by BtrG. In order to test the scalability of this
chemoenzymatic strategy for the preparation of AHBA-ami-
noglycosides and to confirm the expected regiochemistry of the
acylation step, 3.2 mg of paromamine was incubated in a 5 mL
reaction first with 2 and BtrH, then with BtrG. Following
purification, 3.5 mg of AHBA-paromamine was isolated (82%
yield), and NMR analysis of the product unambiguously
confirmed that the AHBA side chain is indeed attached to
the C-1 amine (ESIw).
Notes and references
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In summary, the in vitro chemoenzymatic acylation of a
wide range of 2-DOS-containing aminoglycoside substrates
with the valuable AHBA pharmacophore has been demon-
strated up to the milligram scale. The described method
affords convenient access to a number of unnatural AHBA-
aminoglycosides. Additional work is underway to further scale
up the productive capacity of this chemoenzymatic system and
to more thoroughly investigate the substrate promiscuity of
these enzymes. It is anticipated that this method will provide
an attractive alternative to synthetic approaches to interesting
acyl-aminoglycoside species.
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