Catalytic site-selective synthesis and evaluation of a series of erythromycin analogs

Article abstract of DOI:10.1016/j.bmcl.2008.09.019

The generation of a series of analogs of erythromycin A (EryA, 2) is described. In this study, we compared two peptide-based catalysts-one originally identified from a catalyst screen (5) and its enantiomer (ent-5)-for the selective functionalization of EryA. The semi-synthetic analogs were subjected to MIC evaluation with two bacterial strains and compared to unfunctionalized EryA.

Full text of DOI:10.1016/j.bmcl.2008.09.019

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6007–6011
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Catalytic site-selective synthesis and evaluation of a series of
erythromycin analogs
Chad A. Lewis a, Janie Merkel b, Scott J. Miller a,
*
a Department of Chemistry, Yale University, 225 Prospect Street, New Haven, CT 06520-8107, USA
b Center for Chemical Genomics, Yale University, 219 Prospect Street, New Haven, CT 06520, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The generation of a series of analogs of erythromycin A (EryA, 2) is described. In this study, we compared
two peptide-based catalysts—one originally identified from a catalyst screen (5) and its enantiomer (ent-
5)—for the selective functionalization of EryA. The semi-synthetic analogs were subjected to MIC evalu-
ation with two bacterial strains and compared to unfunctionalized EryA.
Received 25 August 2008
Accepted 3 September 2008
Available online 9 September 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Catalysis
Natural product
Erythromycin
Peptide
Antibiotic
The use of venerable natural product scaffolds as lead structures
for new drugs and drug candidates has generally been a successful
endeavor.1 In this spirit, molecules such as penicillin (1) and eryth-
romycin (2), among others, have been used for decades as tem-
plates in the continuing battle against the numerous antibiotic
resistant bacterial strains such as methicillin-resistant Staphylococ-
cus aureus2–4 (Fig. 1).
The generation of analogs of natural products is challenging, in
part due to the complexity of the interesting structures. If one were
to desire analog compounds that are derived from natural products
readily available from fermentation, then the direct, selective func-
tionalization of the natural product could provide rapid access to
site-specifically modified natural compounds without recourse to
de novo synthesis or biosynthesis of each analog. In the case of pol-
yol natural products, the specter of differentiating the same func-
tional groups (RꢀOH, R0ꢀOH, etc.) within the molecule provides
a daunting challenge for catalysis. Polyketides present a particu-
larly interesting class for this challenge, due to the complexity of
structure and the typical presence of an abundance of hydroxyl
groups available for synthetic derivatization. Differential function-
alization of these polyol arrays has been met with limited success.
Semi-synthetic techniques typically employ enzymatic catalysts.5
Natural product modification using small molecule catalysts is also
possible.6,7
been previously employed to produce analogs that were then
tested for activity against S. aureus and Klebsiella pneumoniae.8 It
was found that the 11-hydroxyl is the least reactive of the second-
ary hydroxyl groups; access to 11-acylated materials required pro-
tecting group strategies to block the 20-position and 400-position.
The 20-position is subject to autocatalytic acylation, owing to the
vicinal dimethylamine. We focused our attention on overturning
this reactivity pattern by employing peptide-based catalysts to
produce 20,11-diacylerythromycin analogs (3) directly, rather than
via the 20,400-diacylerythromycin analogs 4, followed by an addi-
tional step involving potentially complicated ester hydrolyses.
After careful screening of peptide-based catalysts, a lead (5,
Fig. 2) was identified by examination of product ratios achieved
at a common level of reaction conversion.6 (1H NMR proved to be
an effective technique for the initial determination of product ra-
tios.) Acetic anhydride was utilized initially as the acyl donor with
O
Me
HO
Me
Me
Me
OH
Me
HO
O
11
OH
Me
H
N
2'
NMe2
Me
R
S
Me
Me
O
O
O
N
The inherent reactivity of erythromycin A (EryA, 2) toward cat-
alytic functionalization with achiral catalysts is known and had
O
O
O
OMe
Me
COOH
Me
4"
OH
O
1
2
Me
* Corresponding author.
E-mail address: scott.miller@yale.edu (S.J. Miller).
Figure 1. Penicillin G and erythromycin A (EryA).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.019

6008
C. A. Lewis et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6007–6011
Table 2
O
O
CHCl3, Et3N
5-10 mol % Catalyst
20,11-Functionalized analogs of EryA
R
O
R'
O
Ery A
O
OH
R
O
R
O
O
Me
Me
OH
OH
Me
HO
Me
OH
O
O
Me
O
O
Me
OH
R
R
O
Me
O
O
Me
O
O
Me
Me
O
Me
Me
Me
OH
Me
NMe2
NMe2
R
R
O
R'
Me
O
O
Me
Me
O
Me
Ery A
NMe2
O
O
Me
Me
O
CHCl3, Et3N
10 mol % cat.
O
O
Me
O
O
O
OMe
Me
O
O
OMe
Me
O
O
OMe
Me
Me
Me
O
O
Me
O
OH
Me
R
Me
O
OH
2',4"-Product
(3)
2',11-Product
(4)
Me
O
Entry
Compound
R
Cat. 5 yield (%)
Cat. ent-5 yield (%)
1
2
4
6
Me
Et
37
28
44
50
Catalytic NMI:
Catalytic 5:
O
Me
Me
Me
N
N
H
3
4
5
7
8
9
56
53
58
52
68
71
N
N
Me
N
O
H
HN
O
NHBoc
Me
Ratio:
N
O
NHBoc
Catalyst 5
NBoc
3 to 4:
NH
O
5
2 to >10 : 1
O
Ratio: 3 to 4:
Me
product of interest. In addition, they may be employed as mecha-
nistic probes of so-called ‘non-linear effects’.9 In the present con-
text, however, enantiomeric catalysts offer a special opportunity
to probe robust, alternative (i.e., enantiomeric) catalyst architec-
tures in the common spatial context of a stereochemically com-
plex, single-enantiomer natural product substrate.10 Notably, when
the enantiomer of peptide catalyst 5 (ent-5) was evaluated, it
proved to be more selective than 5. Intriguingly, catalyst ent-5 also
leads to preferential formation of 4 over 3. As shown in Table 1,
catalyst ent-5 actually leads to an enhancement of selectivity for
the production of compound 4 in all cases we examined, with reac-
tions run to a common level of conversion. Most notably, with sim-
ple anhydrides, product ratios were enhanced from modest levels
to more useful levels when catalyst 5 was exchanged for catalyst
ent-5 (acetate: 1:4.5–1:7, entry 1; propionate: 1:3.5–1:>10, entry
2). Presumably the molecular recognition issues leading to analo-
gous sense of selectivity are different for the two enantiomeric cat-
1: 3.5 to >10
Figure 2. Catalyst-dependent functionalization of EryA.
triethylamine employed as base. Catalyst loadings of 5–10 mol %
were found to be satisfactory. Notably, catalyst 5 was effective at
reversing the inherent selectivity observed with N-methylimidaz-
ole (NMI) as a catalyst. Product distributions with catalytic NMI
typically produce 3–4 ratios ranging from 2:1 to >10:1, depending
on the anhydride employed. Catalyst 5, on the other hand, exhibits
reversals of this selectivity, with ratios of 3–4 ranging from 1:3.5 to
1:>10, depending on the nature of the anhydride.
While various analogs of catalyst 5 were studied in the initial
screen, we also seized the opportunity to perform an intriguing,
targeted experiment. We wished to compare the performance of
5 to its enantiomer (ent-5), to see how its performance might dif-
fer. Typically, in studies of enantioselective catalysis, enantiomeric
catalysts are employed to produce the opposite enantiomer of a
Table 3
Synthesis of tris-functionalized EryA
Table 1
Comparative selectivities obtained with catalysts 5 and ent-5
O
R
OH
O
O
Me
Me
Me
OH
Me
N
O
O
P
OPh
N
H
Me
N
R'
NMe2
O
H
Me
O
O
OPh
Me
Me
O
Me
HN
Cl
O
2',11
Erythromycin
O
Me
O
DMAP, NEt3
CHCl3
N
O
NHBoc
NBoc
NH
O
O
O
OMe
Me
O
Me
Catalyst ent-5
O
O
P
OPh
OPh
Me
Me
O
Entry Compound
R
Cat. 5 Ratio 3 : 4 Cat. ent-5 Ratio 3 : 4
Entry
Compound
R
R0
Yield (%)
82
1
2
4
6
Me
Et
1:4.5
1:3.5
1:7
1:>10
1
2
10
11
Et
Et
3
7
1:5
1:8
52
69
58
4
8
1:>10
1:>10
3
4
12
13
Me
NHBoc
Me
NHBoc
Me
5
9
1:9
1:>10
Me
5
5
5

C. A. Lewis et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6007–6011
6009
Table 4
MIC testing of erythromycin analogs
O
Me
HO
Me
Me
Me
OH
Me
R'O
O
OR
Me
NMe2
Me
O
O
O
O
OMe
Me
Me
OR"
O
Me
Entry
Compound
R
R0
R00
MIC (lg mL)
S. aureus 29213
E. faecalis 29212
O
O
O
O
O
1
2
3
4
5
6
7
H
32
32
Me
Me
O
O
O
H
32
>32
>32
>32
>32
>32
>32
>32
8
H
NHBoc
NHBoc
9
H
Me
6
Me
6
O
O
10
P(O)(OPh)2
Me
Me
O
O
O
O
O
O
6
7
11
12
13
14
15
16
17
18
19
2
P(O)(OPh)2
P(O)(OPh)2
P(O)(OPh)2
>32
>32
>32
32
>32
>32
>32
>32
>32
32
NHBoc
Me
8
Me
Me
6
6
O
O
O
9
H
NHBoc
Me
O
10
11
12
13
14
15
H
H
H
H
H
H
>32
16
Me
Me
6
6
O
O
O
O
H
H
H
H
H
Me
16
32
32
>32
32
NHBoc
32
Me
6
H
1
2
alysts, a situation that is mirrored in biological ligand–receptor
interactions.11
the catalysts for the acetylation, it was found that the yield
increased from 37% to 44% (entry 1). A more dramatic example
of the effect of ent-5 was observed for the reaction of erythromycin
A and propionic anhydride, with an increase in yield from 28% to
Operationally, catalyst ent-5 also leads to increased overall
yield of the 20,11-functionalized products (Table 2). Comparing

6010
C. A. Lewis et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6007–6011
O
OH
O
R
R
O
O
Me
12
Me
Me
Me
OH
Me
HO
Me
OH
9
9
O
O
R
R
NMe2
Me
O
O
Me
O
O
O
Me
NMe2
Me
Me
O
Me
6
12
6
O
Me
O
Me
O
O
O
O
OMe
Me
O
O
OMe
Me
Me
Me
O
OH
O
OH
Me
Me
Macrolide Form A
Macrolide Form B
11
H
O
Me
HO
NMe2
Me
O
9
6
OH
2'
O
O
Me
HO
12
Me
O
O
O
Me
MeO
Me
Me
Me
OH
4"
Me
Figure 3. Ketalization of 20,11-functionalized EryA.
50% (entry 2). Yield increases were also observed for the b-alanyl
(Boc) derivative (53–68%, entry 4) and the octanoyl derivative
(58–71%, entry 5). Improved yields of natural product analogs in
derivatization studies are no small matter, as isolation of pure
materials is often difficult from complex mixtures of products,
with each component present in minute and comparable
quantities.
strains. Two analogs that retained some of the activity of EryA
against S. aureus were the propionate analog 16 (entry 11, 16 g/
mL) and the 4-pentenoate derivative 17 (entry 12, 16 g/mL).
l
l
The presence of esters at the 20-position has been previously re-
ported to require hydrolysis for activity.15 Consistent with these
findings, the analogs with free 20-hydroxyl did exhibit some in-
crease in effectiveness (e.g., entry 1 vs entry 11; entry 2 vs entry
12).
Upon isolation of the 20,11-functionalized substrates (i.e., com-
pounds like 4), the corresponding 1H and 13C NMR spectra reveal a
subtle molecular rearrangement involving the hemiketalization of
the C9 ketone. As shown in Figure 3, functionalization of the C11-
OH leads to the loss of a hydrogen bond between the 11-hydroxyl
to the C9 ketone, and this event has been suggested as the basis for
the rearrangement. The hemiketalization has furthermore been
suggested to involve engagement of the C12-OH group,12 leading
to the formation of structures like 4 (macrolide form B). The alter-
native possibility for hemiketalization, involving the C6-hydroxyl,
has also been discussed. However, the 1H NMR data collected for
the analogs in this study is most consistent with that reported by
Everett et al. for related compounds,10 suggesting preferential for-
mation of the 9,12-hemiketal.
In summary, we have used two peptide-based catalysts to
reorder the inherent reactivity offered by the natural product
scaffold provided by EryA. These catalysts have allowed the
straightforward access to useful quantities of a number of natu-
ral product analogs, which were then evaluated against two
strains of bacteria. The ability to use chiral catalysts for selective
modification of complex molecules could prove to be a useful
tool in the direct modification of natural products quite broadly.
Such experiments also raise fascinating issues in the area of
asymmetric catalysis, which are aggressively under study in
our laboratory at the present time.
Acknowledgment
Utilizing peptide catalysts 5 and ent-5 for derivatization of EryA
with other anhydrides allowed straightforward access to a number
of analogs directly and differentially functionalized at the 20- and
11-positions (Table 2). Synthesis of these derivatives in sufficient
quantities (at least 8.0 mg, each) permitted biological testing of
the first generation analogs. In addition, each provides a valuable
precursor for further functionalization reactions. The isolated
20,11-diacylated products could be either hydrolyzed to the 11-
functionalized products, or the 400-position could be phosphory-
lated to provide tris-functionalized erythromycins. Resistance to
EryA has been associated with the phosphorylation of the natural
product by a number of bacteria.13 Phosphorylation of the 400-posi-
tion was easily achieved with the chemical methods reported in
this study. A representative list of the trifunctionalized analogs
we produced is shown in Table 3.
We are grateful to the NIH/NIGMS (GM-068649) for generous
support.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.09.019.
References and notes
1. Wilson, R. M.; Danishefsky, S. J. J. Org. Chem. 2006, 71, 8329.
2. Walsh, C. T.; Wright, G. D. Chem. Rev. 2005, 105, 391.
3. Fisher, J. F.; Meroueh, S. O.; Mobashery, S. Chem. Rev. 2005, 105, 395.
4. McDaniel, R.; Welch, M.; Hutchinson, C. R. Chem. Rev. 2005, 105, 543.
5. (a) Abel, U.; Koch, C.; Speitling, M.; Hansske, F. G. Curr. Opin. Chem. Biol. 2002, 6,
453; (b) Gu, J.; Ruppen, M. E.; Cai, P. Org. Lett. 2005, 7, 3945.
6. Lewis, C. A.; Miller, S. J. Angew. Chem. Int. Ed. 2006, 45, 5616.
7. (a) Chen, M. S.; White, M. C. Science 2007, 318, 783; (b) Peddibhotla, S.; Dang,
Y.; Liu, J. O.; Romo, D. J. Am. Chem. Soc. 2007, 129, 12222.
8. (a) Jones, P. H.; Perun, T. J.; Rowley, E. K.; Baker, E. J. J. Med. Chem. 1972, 15, 631;
(b) Martin, Y. C.; Jones, P. H.; Perun, T. J.; Grundy, W. E.; Bell, S.; Bower, R. R.;
Shipkowitz, N. L. J. Med. Chem. 1972, 15, 635.
These analogs, along with those noted above, were evaluated
for their activity against S. aureus and Enterococcus faecalis. Utiliz-
ing the MIC method (minimum inhibitory concentration),14 the
performance of analogs is summarized in Table 4.
In comparison to EryA (entry 15), the analogs generally did not
exhibit the impressive levels of activity against the bacterial
9. Blackmond, D. G. Acc. Chem. Res. 2000, 33, 402.

C. A. Lewis et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6007–6011
6011
10. The issue is related to that of double diastereoselectivity. See: Masamune, S.;
Choy, W.; Petersen, J. S.; Sita, L. R.. Angew. Chem. Int. Ed. 1985, 97, 1.
11. Feng, S.; Schreiber, S. L. J. Am. Chem. Soc. 1997, 119, 10873.
12. Everett, J. R.; Hunt, E.; Tyler, J. W. J. Chem. Soc., Perkin Trans. 1991, 2,
1481.
13. Katz, L.; Ashley, G. W. Chem. Rev. 2005, 105, 499. references therein.
14. (a) Bou, G. Methods Mol. Biol. 2007, 391, 29; (b) Lorian, V. Antibiotics in
Laboratory Medicine, 5th ed.; Lippincott Williams and Wilkins, 2005.
15. Tardrew, P. L.; Mao, J. C.-H.; Kenney, D. Appl. Microbiol. 1969, 18,
159.