Ramoplanin A2 and Ramoplanose Aglycon
A R T I C L E S
In our hands, and unlike the conformational heterogeneity
alluded to by McCafferty,15 the ramoplanin aglycon 48 adopts
a single, rigid conformation in solution indistinguishable from
that of the natural product (1H NMR) although this was not
explored with 2D techniques. Similarly, we have not observed
stability differences between the natural product and the aglycon.
We do, like McCafferty, observe losses in material upon reverse
phase chromatography of 2 or 48. This poor recovery (ca. 50%),
in our assessment, is not derived from degradation (no other
products detected), but rather the poor physical properties of
the compounds including their complete and irretrievable
adherence to plastic and related materials. Consequently, we
do not view the role of the disaccharide subunit of ramoplanin
A2 as one that conveys stability or conformational rigidity to
the molecule,15 but rather one that, among other roles, may alter
and potentially improve solubility or physical properties. Thus,
the final, single step HF deprotection of 47 to provide the
aglycon 48 was most effectively conducted with a trituration
purification (EtOAc) of the final product avoiding a reverse
phase chromatography purification and the simple recovery
losses. Notably, cyclic depsipeptide intermediates bearing the
Fmoc, Trt, and SES protecting groups (e.g., 38) do not suffer
these same recovery properties and could be purified by standard
versus reverse phase chromatography, and 47 bearing the Asn1
acyl side chain and Trt/SES protecting groups could be purified
by reverse phase chromatography without analogous apparent
recovery losses.
Figure 4.
material loss that accompanies HPLC purification. Thus, the
use of anhydrous HF for deglycosidation of ramoplanin A1-
A3 is exceptionally effective providing the corresponding
aglycons in conversions as high as 92% and offers a superb
improvement over existing methods.
Reaction of 48 with SESCl (Et3N, DMF, -20 °C) provided
a mixture of 49 and 50 with the Orn4 amine reacting only
sluggishly under a wide range of reaction conditions, Scheme
10. Importantly, authentic 49 prepared in this manner proved
identical (1H NMR, HPLC, UV, MS) with the synthetic material
described above. Notably, 49 and 50 have proven to be key
derivatives of 2 useful in the definition of the site of action of
ramoplanin and the details of these and related studies will be
disclosed in due time.
Antimicrobial Activity of Key Derivatives. The results of
an antimicrobial evaluation of several of the key derivatives of
ramoplanin A2 that were prepared herein are summarized in
Figure 4 using Staphylococcus aureus (ATCC 25923) which is
among the least sensitive wild-type bacteria. Consistent with
the original36 and more recent subsequent observations,15 the
antimicrobial potency of ramoplanin A2 (2) and its aglycon 48
were not distinguishable with the latter typically displaying
slightly greater potency in our single assay. Consequently,
glycosidation does not contribute to intrinsic in vitro antimi-
crobial potency. Similarly, acycloramoplanin A2 and its aglycon,
in which the macrocyclic lactone was hydrolyzed, were inactive
(MIC > 128 µg/mL) and found to be >250- to 500-fold less
potent. Previous studies detailed an approximate 2000-fold loss
in antimicrobial activity upon hydrolysis of the macrocyclic
lactone of ramoplanin A215,18 and we see analogous drops in
the activity of not only this derivative, but its aglycon as well.
Similarly, 49, in which both the Orn4 and Orn10 δ-amino groups
are SES protected, was found to be inactive (MIC >128 µg/
mL, >500-fold loss in activity). Analogous observations were
recently disclosed by McCafferty15 with Orn4, Orn10-diacetyl
ramoplanin A2 which was found to be 500-fold less potent than
ramoplanin A2 highlighting the importance of these two basic
amines which are conserved among all members of this class
of antimicrobial natural products. Much more interestingly, the
mono SES derivative 50 (MIC ) 4 µg/mL), in which only the
Orn10 δ-amino group is protected and the Orn4 amine is not,
was 16-fold less potent than the free aglycon 48, but >32-fold
more potent than the diSES derivative 49. Clearly, both the Orn4
and Orn10 amines contribute to the antimicrobial activity and
the latter result suggests, but does not require, that the Orn4
free amine is more important than the Orn10 amine. Recent work
of Walker’s6 enlisting alanine derivatives of the Orn4 and Orn10
δ-amines, which maintain but move or extend the position of
the free amines, found that the Orn4-Ala was an active
antimicrobial agent (MIC ) 0.8 µg/mL) and bound peptidogly-
Deglycosidation of Ramoplanin and Functionalization of
the Ramoplanin A2 Aglycon. In an effort to secure comparison
samples of authentic 48 and preceding synthetic intermediates,
we examined methods for the deglycosidation of ramoplanin
and the subsequent functionalization of the ramoplanin A2
aglycon. Past protocols for the deglycosidation of ramoplanin
have relied on the treatment of the ramoplanin complex with
either: (1) trimethylsilyl iodide or trimethylsilyl chloride in the
presence of sodium iodide followed by hydrolysis, or (2) a
strong mineral acid (HCl) in the presence of a lower alcohol
(e.g., BuOH) under anhydrous conditions (entry 1).36 Typically,
pure ramoplanin A2 aglycon is obtained in 20-30% yield using
optimized conditions for these methods. In addition to low
conversions, the isolation of pure ramoplanin aglycon requires
a tedious HPLC purification that results in some loss of material
and contributes to the low conversions. We have found that the
use of anhydrous HF cleanly cleaves the dimannose sugar of
ramoplanin, without affecting the sensitive ester linkage or acyl
side chains.37 Thus, treatment of the ramoplanin complex with
anhydrous HF cleanly provided the A1-A3 aglycons contami-
nated only with mannose-derived reaction byproducts, Scheme
10. Reverse phase HPLC purification of the mixture which
serves to separate the A1-A3 aglycons (20-50% CH3CN-
HCOONH4 (aq, 0.05 M)) provided pure A1 (3%), A2 (46%),
A3 (3%), albeit with a recovery loss due to the physical
properties of the natural product aglycons. Enlisting pure
ramoplanin A2 (2), the HF deglycosidation followed by a simple
EtOAc trituration to remove the deglycosidation byproducts
provided the pure A2 aglycon (48) in 92% yield avoiding the
(36) Ciabatti, R.; Cavalleri, B. (BioSearch Italia S.p. A.) US patent 5491128;
Chem. Abstr. 1990, 112, 179 893.
(37) Wanner, J.; Tang, D.; McComas, C. C.; Crowley, B. M.; Jiang, W.; Moss,
J.; Boger, D. L. Bioorg. Med. Chem. Lett., in press. See also: Boger, D.
L.; Menezes, R. F.; Yang, W. Bioorg. Med. Chem. Lett. 1992, 2, 959.
9
J. AM. CHEM. SOC. VOL. 125, NO. 7, 2003 1885