Biomimetic synthesis and structural refinement of the macrocyclic dimer aminoglycoside 66-40C - The remarkably selective self-condensation of a putative aldehyde intermediate in the submerged culture medium producing sisomicin

Article abstract of DOI:10.1039/b925668g

Aminoglycoside 66-40C, an unprecedented 16-membered bis-azadiene macrocyclic natural product isolated from the Micromonospora producer of the antibiotic sisomicin, was synthesized following a biomimetic strategy which definitively established its origin as arising from a remarkably selective non-enzymatic macro-dimerization. The Royal Society of Chemistry.

Full text of DOI:10.1039/b925668g

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Biomimetic synthesis and structural refinement of the macrocyclic dimer
aminoglycoside 66-40C—the remarkably selective self-condensation
of a putative aldehyde intermediate in the submerged culture medium
producing sisomicinw
Stephen Hanessian* and Juan Pablo Maianti
Received (in Cambridge, UK) 8th December 2009, Accepted 2nd February 2010
First published as an Advance Article on the web 15th February 2010
DOI: 10.1039/b925668g
Aminoglycoside 66-40C, an unprecedented 16-membered
bis-azadiene macrocyclic natural product isolated from the
Micromonospora producer of the antibiotic sisomicin, was
synthesized following a biomimetic strategy which definitively
established its origin as arising from a remarkably selective
non-enzymatic macro-dimerization.
pseudotrisaccharide which was designated as aminoglycoside
66-40C.3 Degradative studies by Mallams and co-workers,
then at the Schering-Plough Corporation, established the
structure and stereochemistry of aminoglycoside 66-40C as
the dimer shown as expression 2 (Fig. 1).3 Critical experiments
included careful osmometric determination of the molecular
weight of aminoglycoside 66-40C compared to the parent
sisomicin and its degradation to the monomer aldehyde 3
under strongly acidic conditions.3 However, the authors made
no allusion to the possibility of self-condensation of this
monomeric aldehyde to regenerate aminoglycoside 66-40C.
The dimer exhibited no antibacterial activity, instead it was
used to access several 60N-substituted sisomicins by reductive
amination from the 60-aldehyde (3).4
The submerged fermentation of Micromonospora inyoensis
gives the broad-spectrum aminoglycoside antibiotic sisomicin
(1) as the principal product (Fig. 1).1,2 Among the minor
components produced by the same medium is a 40,50-unsaturated
The dimeric 16-membered C2-symmetrical bis-Schiff’s base
structure of aminoglycoside 66-40C is unprecedented among
members of the aminoglycoside antibiotic family. Moreover, it
is the only minor fermentation product which appears to be
biosynthetically derived through additional reactions on the
parent antibiotic (Fig. 1).2,3,5
The unique features of aminoglycoside 66-40C make it an
intriguing synthetic target. Detailed structural consideration
suggests its formation from a non-enzymatic intermolecular
double condensation of a putative 60-aldehyde intermediate 3
onto the 3-amino group of a congener.
We report herein the validation of this hypothesis through
the first concise biomimetic synthesis, and structural refinements
of aminoglycoside 66-40C, starting with the readily available
sisomicin sulfate (Scheme 1).1 Conversion of the 300 0N-Cbz
tetra-azido derivative 5 to the 60-aldehyde (6) was achieved in
excellent yield, following our recently reported oxidation of
dihydropyran allylic azides with SeO2.6
Treatment with trimethylorthoformate and TFA led to the
dimethyl acetal intermediate 7, as a key latent form of the
60-aldehyde. Cleavage of the N-Cbz group with concomitant
reduction of the azide groups under Birch conditions afforded
masked sisomicin 60-acetal 8. Treatment with dilute sulfuric
acid immediately liberated the monomer aldehyde 3, which
upon neutralization with barium hydroxide underwent
smooth self-condensation. The dimerization event could be
followed by sequential H-NMR owing to the individual
signals of the functionalities involved (Fig. 2). Filtration of
the barium sulfate precipitate followed by lyophilization gave
essentially pure aminoglycoside 66-40C, which was purified
from residual salts by bench-top ammoniacal silica-gel column
Fig. 1 Sisomicin (1), aminoglycoside 66-40C (2) and their hypothetical
biosynthetic relationship through a putative 60-aldehyde monomer (3).
Department of Chemistry, Universite´ de Montre´al, C. P. 6128,
´
Succ. Centre-Ville, Montreal, QC, Canada H3C 3J7.
E-mail: stephen.hanessian@umontreal.ca; Fax: +1-514-343-5728;
Tel: +1-514-343-6738
w Electronic supplementary information (ESI) available: Experimental
details, spectroscopic data and a model of aminoglycoside 66-40C in
MOL format. See DOI: 10.1039/b925668g
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Scheme 1 Synthesis and biomimetic dimerization of aminoglycoside 66-40C (2), refined structure and key nOe (blue) and HMBC correlations
(red).
chromatography. The physical, spectroscopic and chemical
characteristics of the product were identical to those reported.3
A quasi first order proton spectrum of aminoglycoside
66-40C in D2O is indicative of the prevalence of a preferred
rigid macrocycle conformer. As originally presumed, homo-
and heteronuclear correlation experiments attest to the imine
location on N-3 of the deoxystreptamine ring B.3 In addition,
nuclear Overhauser effect (nOe) spectroscopy revealed spatial
proximity between the 60-imine proton and both the vinylic
40-H and the axial proton 3-H of ring B refining the originally
proposed structure with an in-line arrangement for the
trans, trans-bis-azadiene linkers bridging rings A and B
(Scheme 1 and Fig. 3).
Fig. 3 Model of aminoglycoside 66-40C (2, green), overlaid on the
skeleton of paromomycin (yellow),10 RMS error 0.23 A.9
Intrigued by this unique macrocyclic dimerization event, we
envisioned the analogous process for a nominal motif, namely
the 60-aldehyde of sisamine, a pseudodisaccharide which could
be derived by periodate degradation of the vicinal amino
alcohol in sisomicin derivative 4 (Scheme 2).7
The reliable transformations we developed were applied
unexceptionally to tetra-azido sisamine 9, to access a sisamine
core aldehyde 11 which behaved essentially like 2, self-condensing
to yield sisamine dimer 12.
Likewise, we observed that the self-condensation is slow for
acidic to neutral salt solutions of the monomeric aldehydes
(3 and 11), but occurs readily in mild alkaline solutions which
warrant the presence of the 3-amino group in its nucleophilic
form (approx. pKa E 7 to 8).2,8 These results, the similarity of
nOe, and all other spectroscopic characteristics of dimers 2
and 12, suggest that the sisamine core imparts the remarkable
selectivity over the vast space of mono-, di- and oligomeric
imine variations available.
In order to further explore the solution properties of
aminoglycoside 66-40C, we studied various pH conditions to
determine if it exists in dynamic equilibria with partially open
or monomeric species. We performed crossover experiments
using sisamine dimer 12, as a labeling entity which permitted
monitoring the behavior of the mixtures by LCMS (Scheme 3).
Testament to the stability of aminoglycoside 66-40C, negligible
amounts of crossover heterodimer product (13) were detected
when dimers were incubated as their free-base or neutral
Fig. 2 Sequential H-NMR spectra in D2O revealing the conversions
from acetal 8 to aldehyde 3, and finally self-condensation to dimer 2.
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conformation in solution (Fig. 3).12 In fact, the innate con-
formation of aminoglycosides might be the determining factor
for the spontaneous self-selection of the putative 60-aldehyde
intermediate (3), responsible for the prevalence of this unusual
macrocyclic motif in a complex biological medium, where a
plethora of other potential nucleophiles are also present.
In conclusion, our synthetic efforts have uncovered the
remarkable tendency of the putative sisomicin 60-aldehyde 3,
and related congeners such as 11 to undergo non-enzymatic self-
and/or cross-dimerization leading to exquisitely C2-symmetrical
macrocyclic Schiff’s bases such as aminoglycoside 66-40C. As a
result, the structure and solution conformation of the latter were
refined over the original proposal.3 Although the biological
function of aminoglycoside 66-40C remains speculative, such a
metabolite is conceivable as a pathway feedback regulator or an
extracellular signal in M. inyoensis.
We thank Fonds de Recherche en Sante du Quebec (FRSQ)
´ ´
for a fellowship, and continued support from the Natural
Sciences and Engineering Research Council of Canada
(NSERC).
Scheme 2 Degradation of tetra-azido sisomicin ring C and synthesis
of the minimal dimer motif (12). Key nOe and HMBC correlations.
Notes and references
1 M. J. Weinstein, J. A. Marquez, R. T. Testa, G. H. Wagman,
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1976, 9, 537.
2 D. P. Arya, Aminoglycoside Antibiotics: From Chemical Biology to
Drug Discovery, Wiley, Hoboken, New Jersey, 2007; C. Walsh,
Antibiotics: Actions, Origins, Resistance, ASM Press, Washington,
DC, 2003.
3 D. H. Davies, A. K. Mallams, J. McGlotten, J. B. Morton and
R. W. Tkach, J. Chem. Soc., Perkin Trans. 1, 1977, 1407;
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E. L. Moss Jr. and J. A. Waitz, J. Med. Chem., 1978, 21, 189.
5 R. T. Testa and B. C. Tilley, J. Antibiot., 1976, 29, 140;
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K. J. Rinehart Jr. and W. T. Shier, J. Antibiot., 1970, 23, 464;
G. H. Wagman and J. Weinstein, Journal of Chromatography
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Amsterdam, 1978, vol. 15.
6 S. Hanessian, J. Szychowski and J. P. Maianti, Org. Lett., 2009, 11,
429. DHP = 3,4-dihydro-[2H]-pyran.
7 H. Paulsen, R. Jansen and P. Stadler, US pat., 4 195 173, 1980;
H. Paulsen and R. Jansen, Carbohydr. Res., 1981, 92, 305.
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9 Hyperchem 8 suite, geometry optimizations and molecular dynamics
calculations run using amber99 parameter set. Pair fitting
RMS error for all 16 sp3 matching atoms of rings A and B of
paromomycin. Fig. 3 image produced in PyMol.
10 Q. Vicens and E. Westhof, Structure (London), 2001, 9, 647.
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Scheme 3 Crossover experiments between aminoglycoside 66-40C (2)
and its truncated sisamine analog 12, monitored by LCMS.
acetate salt forms, in aqueous solutions over periods of >72 h.
Under slight excess of acetic acid we observed slow hydrolysis
to aldehydes 3 and 11 (approx. 25% over 72 h, H-NMR),
nonetheless, scrambling between dimers was incomplete.
These results indicate that aminoglycoside 66-40C is a resilient
dimer, and does not readily undergo dynamic equilibration
even under acid catalysis. On the other hand, the crossover
heterodimer (13) could be readily generated by neutralizing a
mixture of aldehydes 3 and 11 obtained by sulfuric acid
treatment of the corresponding homodimers (Scheme 3).
Models of the structure of dimers 2 and 12,9 respecting the
azadiene conformation indicated from nOe’s, suggest that the
conformations of rings A and B are unperturbed compared
to aminoglycosides co-crystallized within A-site RNA oligo-
nucleotide models,10,11 also believed to be the lowest energy
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Chem. Commun., 2010, 46, 2013–2015 | 2015