Mutasynthesis of Glycopeptide Antibiotics: Variations of Vancomycin's AB-Ring Amino Acid 3,5-Dihydroxyphenylglycine

Article abstract of DOI:10.1021/ja0499389

In the mutasynthetic approach, the ΔdpgA mutant of the vancomycin-type glycopeptide antibiotic producer Amycolatopsis balhimycina, which is deficient in the synthesis of 3,5-dihydroxyphenylglycine (DPg), was supplemented with synthetic DPg analogues to obtain the corresponding modified glycopeptides. Sterically more demanding 3,5-disubstituted methoxy derivatives as well as monosubstituted DPg analogues were accepted as substrates. These facts indicate that steric and electronic requirements suffice in several cases for the oxidative closure of the AB ring, thus leading to the generation of novel antibiotically active glycopeptide derivatives. The results represent a further step in evaluating the potential of mutasynthesis for peptidic secondary metabolites. Copyright

Full text of DOI:10.1021/ja0499389

Published on Web 04/27/2004
Mutasynthesis of Glycopeptide Antibiotics: Variations of Vancomycin’s
AB-Ring Amino Acid 3,5-Dihydroxyphenylglycine
Stefan Weist,† Claudia Kittel,‡ Daniel Bischoff,† Bojan Bister,† Volker Pfeifer,‡ Graeme J. Nicholson,†
Wolfgang Wohlleben,‡ and Roderich D. Su¨ssmuth*,†
Institute of Organic Chemistry, Auf der Morgenstelle 18, and Institute of Microbiology/Biotechnology,
Auf der Morgenstelle 28, UniVersita¨t Tu¨bingen, D-72076 Tu¨bingen, Germany
Received January 5, 2004; E-mail: roderich.suessmuth@uni-tuebingen.de
Vancomycin (Figure 1a) is an antibiotic of last resort for
treatment of life-threatening infections with methicillin-resistant
Staphylococcus aureus (MRSA).1 Emerging enterococcal and
staphylococcal resistances against vancomycin require the evalu-
ation of approaches to novel vancomycin-related glycopeptides.
However, due to their structural complexity, generation of molecular
diversity in glycopeptides, in particular in the D-Ala-D-Ala peptide-
binding region of the heptapeptide backbone (aglycon), via total
synthesis poses inherent problems.
Alternative biotechnological approaches would constitute attrac-
tive options, and considerable success has been achieved in the
enzymatic attachment of various carbohydrate residues to glyco-
peptide aglycons.2 Recently, Boger and co-workers demonstrated
Figure 1. Structure formulas of glycopeptide antibiotics vancomycin (a)
and balhimycin (b). The highlighted amino acid is (S)-3,5-dihydroxyphe-
that permethylation of vancomycin-type aglycons resulted in
improved activity against VanB-resistant bacteria.3 These results
indicate that aglycon modifications still are a promising target for
the development of novel glycopeptide compounds. Here we report
on the generation of a series of modified glycopeptide antibiotics
by mutasynthesis in which the C-terminal amino acid (S)-3,5-
dihydroxyphenylglycine (DPg) has been substituted, employing a
number of phenylglycines or their mandelic and phenylacetic acid
analogues, respectively.
nylglycine (DPg, AA-7); ovcn ) 4-oxovancosamine.
cine (DMeOPg), as well as the corresponding mandelic acid and
phenylacetic acid analogues. After supplementation of A. balhi-
mycina ∆dpgA cultures, the presence of glycopeptides in the culture
filtrates was proved by HPLC-ESI-MS (Table 1) with the charac-
teristic chlorine isotope pattern as a marker as well as via plate
diffusion tests, the latter indicating restored antibiotic activity which
was comparable to that of balhimycin. The balhimycin derivatives
from the 3-MeOPg supplementation were characterized by high-
resolution ESI-FTICR-MS (Figure 2 and Supporting Information).
Feeding of 4-hydroxy-substituted (R)- and (S)-HPg, the latter
also being the product of the HPg biosynthesis pathway, did not
compensate for the lack of the DPg moiety, and no glycopeptides
were detected with HPLC-MS. Supplementation with the synthetic
amino acids HMeOPg and DMeOPg resulted in tricyclic glyco-
peptides, the majority of which were glucosylated at 4HPg. In
contrast to the products obtained by supplementation with the
natural substrate DPg, no glycosylation with 4-oxovancosamine
(ovcn) was detected (Table 1). Employment of the monosubstituted
3-HPg and its methoxy analogue 3-MeOPg as substrates also led
to antibiotically active tricyclic derivatives. Glycosylation patterns
comprised modifications with glucose, 4-oxovancosamine, and
ureido-vancosamine (urvcn) in analogy to wild-type metabolites.4
However, using 3-HPg and 3-MeOPg, bicyclic glycopeptides
lacking the AB-ring biaryl were also detected. The absence of the
AB-ring in the bicyclic glycopeptides was confirmed by LC-MS/
MS experiments.
Balhimycin (Figure 1b) is a vancomycin-type glycopeptide
antibiotic produced by the actinomycete Amycolatopsis balhimy-
cina.4 Recent advances in the investigation of balhimycin biosyn-
thesis by means of gene inactivation and analysis of the metabolites
have provided an understanding of the central steps of the assembly
of the aglycon from a linear peptide precursor.5 Combined with
the knowledge of the biosynthesis of the aromatic amino acid
â-hydroxytyrosine,6 4-hydroxyphenylglycine (HPg),7 and 3,5-
dihydroxyphenylglycine (DPg),8-10 these findings constitute the
basis for the further biotechnological generation of altered glyco-
peptide aglycons. Thus, in previous work, structural variations at
the 3-chloro-â-hydroxytyrosine (Cht) moieties by means of muta-
tional biosynthesis led to the first fluorinated vancomycin-type
glycopeptide antibiotic.11 Subsequently, we directed our attention
5
toward the atropisomeric AB macrocycle formed between HPg
and 7DPg, which is of crucial importance for the antibiotic activity
of this compound family.12 Inactivation of the dpgA gene, which
is involved in the first step of DPg biosynthesis, rendered a
glycopeptide-deficient ∆dpgA mutant, the antibiotic activity of
which could be restored by supplementation of the culture medium
with DPg.8 To elucidate structural and steric requirements for the
mutasynthetic assembly of DPg analogues and their putative
precursors into glycopeptides, a variety of substrates were synthe-
sized, including 3-hydroxy- (3-HPg), 3-methoxy- (3-MeOPg),
3-hydroxy-5-methoxy- (HMeOPg), and 3,5-dimethoxyphenylgly-
In a second set of feeding experiments, mandelic acid (MA) and
phenylacetic acid (PA) analogues of HMeOPg, DMeOPg, 3-HPg,
and 3-MeOPg were tested, since 3,5-dihydroxyphenylacetic acid
(DPA)9,10 and 3,5-dihydroxymandelic acid (DMA)10 derivatives
have both been postulated, albeit controversially, as precursors in
the biosynthesis of DPg. Except for DPA, none of the phenylacetic
acid analogues succeeded in being accepted as a substrate. Of the
mandelic acid derivatives, both DMA and 3-HMA yielded balhi-
† Institute of Organic Chemistry.
‡ Institute of Microbiology/Biotechnology.
9
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J. AM. CHEM. SOC. 2004, 126, 5942-5943
10.1021/ja0499389 CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Structures of Mutasynthetically Generated Balhimycins
from Supplementation of ∆dpgA Mutant with Substituted
Phenylglycines and the Influence of This Substitution Pattern upon
AB-Ring Closurea
strate recognition by the peptide synthetases to form the heptapep-
tide are met by the monosubstituted substrates 3-HPg and 3-MeOPg.
The additional detection of bicyclic glycopeptides with 3-HPg/3-
MeOPg suggests that narrow constraints on the orientation of the
ring of this amino acid are posed by the oxygenase OxyC12 in order
to achieve AB-ring formation and thus the production of antibi-
otically active products. Consequently, the AB-ring bridge is
expected to be formed exclusively in the position ortho to the
electron-donating hydroxy or methoxy substituent. Interestingly,
substrate specifity of the ∆dpgA mutant is more discriminating
toward mandelic and phenylacetic acids in comparison to their
amino acid analogues. This increased discrimination could be
associated with their intracellular uptake and/or degradation, or
preferably with an additional number of biosynthetic steps required
before their loading onto the peptide synthetase.
In summary, we have shown that the mutasynthesis approach
can be extended to the AB macrocycle, allowing the generation of
a variety of antibiotically active vancomycin-type derivatives
selectively modified at the DPg residue. Access to such compounds
by synthetic routes is extremely laborious, and our approach is a
first step toward a biotechnological generation of modified glyco-
peptide antibiotics.
[M + H]+
m/z
AB
ring
b
1
2
3
4
5
6
c
AA
R
R
R
R
R /R
1291.3*
1305.3*
3-HPg
3-HPg
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H/Me
Me/Me
H/Me
Me/Me
H/Me
Me/Me
-
-
-
-
+
+
+
+
+
+
+
-
-
+
+
+
H
H
H
H
H
H
H
H
H
H
H
H
1129.2*,d 3-HPg
Hd
Hd
1143.2*,d 3-HPg
1430.3
1303.2
1628.5
1487.4
1430.2
1289.1
1303.1
1291.2*
1305.2*
1305.1
1319.1*
1319.2*
3-HPg
3-HPg
ovcne
H
3-MeOPg OMe
3-MeOPg OMe
3-MeOPg OMe
3-MeOPg OMe
3-MeOPg OMe
3-MeOPg OMe
3-MeOPg OMe
HMeOPg OH
HMeOPg OH
urvcnf ovcne H/Me
urvcnf
ovcne
H
H
H
H
H
H
H
H
H
H
H/Me
H/H
H/H
H/Me
H/H
H/Me
H/H
Acknowledgment. This work was supported by an Emmy-
Noether fellowship of the Deutsche Forschungsgemeinschaft (SU
239/1-2). We thank Prof. G. Jung for generous support.
H
H
H
H
H
H
Supporting Information Available: General experimental proce-
dures and characterization data (PDF). This material is available free
of charge via the Internet at http://pubs.acs.org.
OMe
OMe
H/Me
H/H
DMeOPg OMe OMe
References
a According to LC-ESI-MS experiments. Major metabolites are indicated
with an asterisk. The most probable orientation of substituents at the DPg
moiety in the metabolites is shown. b Supplemented amino acid. c Indicates
whether the AB ring (AA 5-7) is closed (+) or open (-). d Non-
glucosylated (aglycon). e ovcn ) 4-oxovancosamine (Figure 1). f urvcn )
ureido-vancosamine (Figure 2).
(1) Reviews: (a) Nicolaou, K. C.; Boddy, C. N C.; Bra¨se, S.; Winssinger, N.
Angew. Chem., Int. Ed. 1999, 38, 2096-2152. (b) Hubbard, B. K.; Walsh,
C. T. Angew. Chem., Int. Ed. 2003, 42, 730-765.
(2) Losey, H. C.; Jiang, J.; Biggins, J. B.; Oberthur, M.; Ye, X.-Y.; Dong, S.
D.; Kahne, D.; Thorson, J. S.; Walsh, C. T. Chem. Biol. 2002, 9, 1305-
1314.
(3) McComas, C. C.; Crowley, B. M.; Hwang, I.; Boger, D. L. Bioorg. Med.
Chem. Lett. 2003, 13, 2933-2936.
(4) Chatterjee, S.; Vijayakumar, E. K. S.; Nadkarni, S. R.; Patel, M. V.;
Blumbach, J.; Ganguli, B. N.; Fehlhaber, H.-W.; Kogler, H.; Vertesy, L.
J. Org. Chem. 1994, 59, 3480-3484.
(5) (a) Su¨ssmuth, R. D.; Pelzer, S.; Walk, T.; Wohlleben, W.; Jung, G. Angew.
Chem., Int. Ed. 1999, 38, 1976-1979. (b) Pelzer, S.; Su¨ssmuth, R.;
Heckmann, D.; Recktenwald, J.; Huber, P.; Jung, G.; Wohlleben, W.
Antimicrob. Agents Chemother. 1999, 1565-1573.
(6) Puk, O.; Huber, P.; Bischoff, D.; Recktenwald, J.; Jung, G.; Su¨ssmuth,
R. D.; van Pe´e, K.-H.; Wohlleben, W.; Pelzer, S. Chem. Biol. 2002, 9,
225-235.
(7) Hubbard, B. K.; Thomas, M. G.; Walsh, C. T. Chem. Biol. 2000, 7, 931-
942.
(8) Pfeifer, V.; Nicholson, G. J.; Ries, J.; Recktenwald, J.; Schefer, A. B.;
Shawky, R. M.; Schro¨der, J.; Wohlleben, W.; Pelzer, S. J. Biol. Chem.
2001, 42, 38370-38377.
Figure 2. ESI-FTICR-MS (internal calibration) of two-fold chlorinated
ureido-7[3-MeOPg]-balhimycin; urvcn ) ureido-vancosamine.
(9) (a) Chen, H.; Tseng, C. C.; Hubbard, B. K.; Walsh, C. T. Proc. Natl.
Acad. Sci. U.S.A. 2001, 98, 14901-14906. (b) Tseng, C. C.; McLoughlin,
S. M.; Kelleher, N. L.; Walsh, C. T. Biochemistry 2004, 43, 979-980.
(10) (a) Sandercock, A. M.; Charles, E. H.; Scaife, W.; Kirkpatrick, P. N.;
O’Brien, S. W.; Papageorgiou, E. A.; Spencer, J. B.; Williams, D. H.
Chem. Commun. 2001, 1252-1253. (b) Li, T.-L.; Choroba, O. W.; Hong,
H.; Williams, D. H.; Spencer, J. B. Chem. Commun. 2001, 2156-2157.
(11) Weist, S.; Bister, B.; Puk, O.; Bischoff, D.; Pelzer, S.; Nicholson, G. J.;
Wohlleben, W.; Jung, G.; Su¨ssmuth, R. D. Angew. Chem., Int. Ed. 2002,
41, 3383-3385.
mycin derivatives. The acceptance of both mandelic and phenyl-
acetic acids would favor the proposed DPA biosynthesis path-
ways.9,10
According to our results, the presence of at least one hydroxy
or methoxy substituent in the 3-position at the phenylglycine is of
crucial importance for its acceptance as a substrate for the formation
of bicyclic and tricyclic aglycons. A 3,5-disubstitution pattern with
either hydroxy or methoxy groups as well as a “mixed” substitution
pattern (HMeOPg) is tolerated and leads to completely cyclized
aglycons. Apparently, steric and electronic requirements for sub-
(12) (a) Bischoff, D.; Pelzer, S.; Bister, B.; Nicholson, G. J.; Stockert, S.;
Schirle, M.; Wohlleben, W.; Jung, G.; Su¨ssmuth, R. D. Angew. Chem.,
Int. Ed. 2001, 40, 4688-4691. (b) Bischoff, D.; Pelzer, S.; Nicholson, G.
J.; Stockert, S.; Wohlleben, W.; Jung, G.; Su¨ssmuth, R. D. Angew. Chem.,
Int. Ed. 2001, 40, 1693-1696.
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