C O M M U N I C A T I O N S
Scheme 3
to linear aglycones as well as cyclic analogues. This property
expands the opportunities for glycodiversification of secondary
metabolites. More importantly, this study implicates that the
substitution patterns nearby the glycosylation site, and not the ring
conformation, are the major recognition determinants for glyco-
syltransferases.16 This finding sets the stage for a future investigation
to establish the minimal recognition elements for this class of
glycosyltransferases. However, the fact that 8 and 4 are better
substrates (than 20 and 9, respectively) indicates that the specificity
of DesVII is also sensitive to the substituents distant from the
glycosylation site. It has been shown that 8 can be cyclized to 4 by
pik-TE in vitro.12 It remains to be tested if the thioesterase is also
capable of cyclizing the glycosylated linear polyketides.
as the N-acetylcysteamine (NAC) thioester via esterification of 19
with N-acetylcysteamine in 82% yield. Selective oxidation of 20
afforded the desired product 8.12
Acknowledgment. This work was supported by the National
Institutes of Health Grant GM54346.
Supporting Information Available: The conditions for the enzy-
matic glycosylations as well as characterization of glycosylated products
(PDF). This material is available free of charge via the Internet at http://
pubs.acs.org.
Incubation of 8 with TDP-D-desosamine (7) in the presence of
DesVII and DesVIII resulted in the formation of a more polar
compound.13 This compound was purified by HPLC, and the high-
resolution CI-MS mass (calcd for C29H53N2O7S [M + H]+
573.3573, found 573.3566) is consistent with the anticipated
glycosylated product 21 (Scheme 3). The 7-OH compound 20 was
also tested for chemical competence as a substrate for DesVII/
DesVIII. Two new compounds were isolated from this incubation
mixture. The major product is singly glycosylated as indicated by
the high-resolution CI-MS mass (calcd for C29H55N2O7S [M + H]+
575.3730, found 575.3729). The predicted structure of this product
is 22. The minor and less polar product has a molecular mass
corresponding to the dehydrated 22 (CI-MS calcd for C29H53N2O6S
[M + H]+ 557.3624, found 557.3618). The dehydration is unlikely
enzyme-catalyzed but an artifact of the incubation (pH 8) or
isolation conditions. HPLC analysis of the incubation mixtures
enabled the estimation of a 12% yield for 21 from 8 and a 9%
yield for 22 and the dehydrated product from 20. The low yields
of the products precluded a thorough characterization of the
structures, but a single product formation in all cases suggests that
these compounds are glycosylated at the 3-OH group.14
For comparison, glycosylation of the cyclic aglycone 4 and the
C-7 reduced form 9 were also examined under the same conditions.
Again, a single glycosylated product was isolated in each case.
NMR analysis confirmed the structure of the product resulting from
9 to be 23 (obtained in 45% yield), with the correct MS mass (calcd
for C25H46NO6 [M + H]+ 456.3325, found 456.3327). As expected,
compound 5 was obtained from the incubation with 4 in nearly
quantitative yield (CI-MS calcd for C25H44NO6 [M + H]+ 454.3169,
found 454.3173).15 Clearly, the cyclic forms are much better
substrates: the yield of glycosylation is 5-fold higher for 9 versus
20 and at least 8-fold more for 4 versus 8. The analysis also shows
that DesVII prefers the C-7 keto group over the C-7 OH group (8
versus 20, and 4 versus 9).
References
(1) For reviews, see: (a) Rawlings, B. J. Nat. Prod. Rep. 2001, 18, 190-
227. (b) Rawlings, B. J. ibid. 231-281. (c) Staunton, J.; Weissman, K. J.
Nat. Prod. Rep. 2001, 18, 380-416. (d) Walsh, C. Antibiotics: Actions,
Origins, Resistance; ASM Press: Washington, D.C., 2003. (e) Weissman,
K. J. Philos. Trans. R. Soc. London, Ser. A 2004, 362, 2671-2690.
(2) Xue, Y.; Zhao, L.; Liu, H.-w.; Sherman, D. H. Proc. Natl. Acad. Sci.
U.S.A. 1998, 95, 12111-12116.
(3) Aglycone 4 is produced under the growth conditions where the ketosyn-
thase (KS6) domain of module 6 is expressed in a truncated form. Under
conditions when a full length KS6 is expressed, the released cyclic product
is the 14-membered ring narbonolide, which is the precursor for
narbomycin and pikromycin. [(a) Xue, Y.; Sherman, D. H. Nature 2000,
403, 571-575. (b) Beck, B. J.; Yoon, Y. J.; Reynolds, K. A.; Sherman,
D. H. Chem. Biol. 2002, 9, 575-583.]
(4) For reviews, see: (a) Strohl, W. R. Metab. Eng. 2001, 3, 4-14. (b)
Rodriguez, E.; McDaniel, R. Curr. Opin. Microbiol. 2001, 4, 526-534.
(c) Walsh, C. T. ChemBioChem 2002, 3, 125-134.
(5) Borisova, S. A.; Zhao, L.; Melanc¸on, C. E., III; Kao, C.-L.; Liu, H.-w. J.
Am. Chem. Soc. 2004, 126, 6534-6535.
(6) A similar requirement has since been observed for other glycosyltrans-
ferases involved in the biosynthesis of macrolides [(a) Melancon, C. E.,
III; Takahashi, H.; Liu, H.-w. J. Am. Chem. Soc. 2004, 126, 16726-
16727. (b) Yuan, Y.; Chung, H. S.; Leimkuhler, C.; Walsh, C. T.; Kahne,
D.; Walker, S. J. Am. Chem. Soc. 2005, 127, 14128-14129] and
anthracyclines [(c) Lu, W.; Leimkuhler, C.; Gatto, G. J. J.; Kruger, R.
G.; Oberthur, M.; Kahne, D.; Walsh, C. T. Chem. Biol. 2005, 12, 527-
534]. Preliminary data suggested that DesVIII and its homologues do not
directly participate in the coupling reaction but may serve as chaperones
to activate the respective glycosyltransferase [see ref 6b and (d) Borisova,
S. A.; Zhang, C.; Takahashi, H.; Zhang, H.; Wong, A. W.; Thorson, J.
S.; Liu, H.-w. Angew. Chem., Int. Ed., 2006, published on the Web].
(7) Borisova, S. A.; Zhao, L.; Sherman, D. H.; Liu, H.-w. Org. Lett. 1999, 1,
133-136.
(8) Attempts to cleave the internal double bond using OsO4/NaIO4 were
unsuccessful, possibly due to the migration of the acetyl group from the
C-7 hydroxyl to the C-8 hydroxyl of the diol intermediate. Ozonolysis
yielded the acyclic dialdehyde 11 without complications.
(9) Paquette, L. A.; Chang, S.-K. Org. Lett. 2005, 7, 3111-3114.
(10) Blakemore, P. R.; Cole, W. J.; Kocienski, P. J.; Morley, A. Synlett 1998,
26-28.
(11) Such a transformation could occur by a â-fragmentation facilitated by
the oxyanion generated after the addition of 12 to 11.
(12) A more efficient synthesis of 8 based on direct hydrolysis to open the
macrolactone ring of 4 was recently reported [(a) Aldrich, C. C.; Beck,
B. J.; Fecik, R. A.; Sherman, D. H. J. Am. Chem. Soc. 2005, 127, 8441-
8452. (b) Aldrich, C. C.; Venkatraman, L.; Sherman, D. H.; Fecik, R. A.
J. Am. Chem. Soc. 2005, 127, 8910-8911]. However, the hydrolysis was
slow, and two C-2 stereoisomers were generated during hydrolytic ring
opening, which had to be separated by HPLC.
In summary, this study demonstrated for the first time that a
macrolide glycosyltransferase can also recognize and process the
linear precursor of its macrolactone substrate with reduced but
measurable activity. The reaction is regioselective because only one
singly glycosylated product is generated in each case despite the
presence of multiple potential glycosylation sites. This finding is
significant for three reasons. First, a similar capability is possible
for other macrolide glycosyltransferases, and the glycosylation of
the linear polyketide may be a minor glycosylation pathway during
the biosynthesis of macrolide antibiotics. Second, the substrate
flexibility of macrolide glycosyltransferases is apparently extended
(13) See Supporting Information for details.
(14) However, glycosylation at 11-OH remains a possibility.
(15) Since neither 9 nor 23 possess a chromophore, an HPLC instrument
equipped with the universal Corona charged aerosol detector (CAD) was
used to show the conversion of 9 to 23 as the only product.
(16) This is in agreement with our previous observation of aglycone specificity
for DesVII (see ref 6d).
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