Site- and Stereoselective Chemical Editing of Thiostrepton by Rh-Catalyzed Conjugate Arylation: New Analogues and Collateral Enantioselective Synthesis of Amino Acids

Article abstract of DOI:10.1021/jacs.7b08775

The synthesis of complex, biologically active molecules by catalyst-controlled, selective functionalization of complex molecules is an emerging capability. We describe the application of Rh-catalyzed conjugate arylation to the modification of thiostrepton, a complex molecule with potent antibacterial properties for which few analogues are known. By this approach, we achieve the site- and stereoselective functionalization of one subterminal dehydroalanine residue (Dha16) present in thiostrepton. The broad scope of this method enabled the preparation and isolation of 24 new analogues of thiostrepton, the biological testing of which revealed that the antimicrobial activity of thiostrepton tolerates the alteration of Dha16 to a range of amino acids. Further analysis of this Rh-catalyzed process revealed that use of sodium or potassium salts was crucial for achieving high stereoselectivity. The catalyst system was studied further by application to the synthesis of amino esters and amides from dehydroalanine monomers, a process which was found to occur with up to 93:7 er under conditions milder than those previously reported for analogous reactions. Furthermore, the addition of the same sodium and potassium salts as applied in the case of thiostrepton leads to a nearly full reversal of the enantioselectivity of the reaction. As such, this study of site-selective catalysis in a complex molecular setting also delivered synergistic insights in the arena of enantioselective catalysis. In addition, these studies greatly expand the number of known thiostrepton analogues obtained by any method and reveal a high level of functional group tolerance for metal-catalyzed, site-selective modifications of highly complex natural products.

Full text of DOI:10.1021/jacs.7b08775

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Article
Site- and Stereoselective Chemical Editing of Thiostrepton
by Rh-Catalyzed Conjugate Arylation: New Analogs and
Collateral Enantioselective Synthesis of Amino Acids
Hanna M. Key, and Scott J. Miller
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b08775 • Publication Date (Web): 04 Oct 2017
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Journal of the American Chemical Society
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Site- and Stereoselective Chemical Editing of Thiostrepton by Rh-
Catalyzed Conjugate Arylation: New Analogs and Collateral Enanti-
oselective Synthesis of Amino Acids
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Hanna M. Key† and Scott J. Miller†*
† Department of Chemistry, Yale University, New Haven, Connecticut 06520-8107, United States
ABSTRACT: The synthesis of complex, biologically active molecules by catalyst-controlled, selective functionalization of complex molecules is an
emerging capability. We describe the application of Rh-catalyzed conjugate arylation to the modification of thiostrepton, a complex molecule with
potent antibacterial properties for which few analogs are known. By this approach, we achieve the site- and stereoselective functionalization of one
subterminal dehydroalanine residue (Dha16) present in thiostrepton. The broad scope of this method enabled the preparation and isolation of 24 new
analogs of thiostrepton, the biological testing of which revealed that the antimicrobial activity of thiostrepton tolerates the alternation of Dha16 to a
range of amino acids. Further analysis of this Rh-catalyzed process revealed that use of sodium or potassium salts was crucial for achieving high stere-
oselectivity. The catalyst system was studied further by application to the synthesis of amino esters and amides from dehydroalanine monomers, a
process which was found to occur with up to 93 : 7 er under conditions milder than those previously reported for analogous reactions. Furthermore,
the addition of the same sodium and potassium salts as applied in the case of thiostrepton lead to a nearly full reversal of the enantioselectivity of the
reaction. As such, this study of site-selective catalysis in a complex molecular setting also delivered synergistic insights in the arena of enantioselective
catalysis. In addition, these studies greatly expand the number of known thiostrepton analogs obtained by any method and reveal a high level of func-
tional group tolerance for metal-catalyzed, site-selective modifications of highly complex natural products.
Introduction
O
Dha16
N
Catalyst-controlled, selective functionalization of highly complex natu-
ral products is an emerging approach to generate new compounds with
potential applications as pharmaceuticals or tools to interrogate the bio-
logical function of the parent molecule.1 Although efficient in principle,
this approach necessarily confronts a series of challenges, including (a)
the compatibility of the reagents and reaction conditions with a highly
functionalized substrate and (b) the site- and stereoselectivity with
which reactions occur.1 Nonetheless, a growing number of reports have
demonstrated the potential to achieve catalyst-controlled, site- and,
when applicable, stereoselective functionalizations of several functional
groups present in biologically relevant compounds.2
H
NH2
O
N
Dha17
O
H
S
N
Dha3
N
O
H
N
O
S
O
H
N
N
N
H
O
N
HN
O
NH
O
S
O
HO
O
H
OH
NH
O
N
HN
NH
N
N
H
N
S
S
OH
O
HO
OH
Thiostrepton 1
SH
NHR’
Thiostrepton (1, Fig. 1) is a canonical, complex, and highly potent anti-
biotic containing multiple, potentially reactive functional groups. De-
RHN
3
NHR’
O
RHN
Phenylalanine
spite being readily available by fermentation, thiostrepton has seen lim-
ited use as a pharmaceutical due to poor pharmacological properties.
O
R
4
5
Since its initial discovery in 1955, the biosynthesis and chemical syn-
ArB(OH)2
NHR’
NHR’
6-8
RHN
RHN
RHN
thesis of thiostrepton have been described, but only a few derivatives
Phenylanaline
Analogs
O
O
O
9-11
have been reported.
The complexity of 1, exemplified in part by its
X
three dehydroalanines, one dehydrothreonine, four thiazoles, one thia-
zoline, three isolated hydroxyl groups, a diol, an amine, an imine, and a
highly-substituted pyridine, may contribute to the paucity of synthetic,
semisynthetic, or biosynthetic derivatives that have been prepared and
studied. Consequently, thiostrepton is a scientifically heuristic platform
for the investigation of late-stage, site-, and stereoselective functionali-
zation.
OH
Other Unnatural
Amino Acids
NHR’
NHR’
RHN
O
Figure 1. The structure of thiostrepton 1 (top) and the suitability of
dehydroalanine (Dha) for late stage chemical editing by Rh-cata-
lyzed conjugate addition (bottom). The Dha residues in thiostrep-
ton are shown in blue and are numbered in accordance with the pep-
tide precursor. Strategies to incorporate Dha from amino acids are
also shown.
The three dehydroalanine motifs within 1 are intriguing sites for selec-
tive functionalization (Fig. 1). Dehydroalanines are found in numerous
medicinally important compounds including lantibiotics, thiopeptides,
12,13
and other highly modified peptides of microbial origin.
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Moreover, in addition to their natural occurrence, dehydroalanines can
Using either KOH or Et3N as base, the major product observed is com-
1
2
3
4
5
6
7
8
pound 3 (37–43% conversion by UPLC, Fig. 2b, entries 1–2). Smaller
and roughly equal amounts of 2A and 2B also form, each as a mixture of
two diastereomers. The site isomers 2A/2A’ and 2B/2B’ were assigned
by 1D and 2D NMR analysis, which included a consideration of the
chemical shifts of the signals from the carbonyl carbons in the remaining
be incorporated into complex molecules and proteins by b-elimination
14
of serine-OH or cysteine-SH functional groups. Unlike the canonical
twenty amino acids, dehydroalanine is electrophilic, making it suitable
for selective functionalization even within the complex environment of
15,16
a natural product, peptide, or protein.
As such, a strategy for cata-
1
Dha residue of the product, as well as the correlations observed in H–
lytic, nucleophilic addition to dehydroalanines in a complex environ-
ment would enable post-synthetic, catalyst-controlled chemical editing
of many naturally-occurring compounds (Fig. 1).
13C HMBC spectra (see section III of the Supporting Information for a
detailed explanation). Due to the significant amount of base-mediated
cleavage that occurred under the initial reaction conditions, we investi-
gated alternative bases that were either sterically hindered amines or
non-nucleophilic inorganic bases. We found that K2CO3 delivers both
higher conversion to 2A/2A’ and 2B/2B’ (16% total) and lower con-
version to product 3 (25%) than were observed in the analogous reac-
tions containing either KOH or Et3N as base (Fig. 2b, entry 3). Moreo-
ver, these catalytic conditions lead to 3 : 1 site-selectivity favoring func-
tionalization of the subterminal Dha16 over that of terminal Dha17.
This selectivity is alternate to that reported for un-catalyzed conjugate
additions of thiols to thiostrepton, which are reported to occur at the
terminal alkene.10 To our knowledge, there are no previously methods
known to functionalize selectively the subterminal Dha (Dha16) of thi-
ostrepton. When the reaction in the presence of K2CO3 is conducted at
room temperature, the ratio of conjugate addition products to cleavage
products further increases to approximately 1 : 1.5 (Fig. 2b, entry 4), alt-
hough the overall conversion decreases slightly. The catalyst derived
from the enantiomerically pure ligand S,S-BINAP affords more of the
conjugate addition products than are produced by the racemic ligand,
but overall, the yields of all reactions conducted using BINAP were low
(Fig. 2b, entry 5).
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With this objective, we aimed to apply Rh-catalyzed conjugate addition
to functionalize selectively the dehydroalanines present in thiostrepton.
While more commonly employed to create a b-stereocenter from an un-
saturated carbonyl, in a few cases stereoselective, Rh-catalyzed conju-
gate addition to a dehydroalanine motif has been achieved, resulting in
the formation of an a-stereocenter by asymmetric protonation of a Rh-
17-23
enolate.
Although reported catalytic methods require high reaction
temperatures and specific substrates and reagents to achieve high yields
and enantioselectivities, we hypothesized that further development of
this reaction could make it suitable for both site- and diastereoselective
editing of complex molecules containing dehydroalanines. Herein, we
report a rhodium catalyst that operates under mild conditions and that
controls both the site and the diastereoselectivity of the conjugate aryla-
tion of diverse boronic acids to thiostrepton. Furthermore, the applica-
tion of these same reaction conditions to the asymmetric synthesis of
protected amino acids from dehydroalanine monomers also led to the
collateral discovery of a rare example of enantio-divergence owing to an
achiral additive.
To identify a more active and selective catalyst, we evaluated ~20 chiral
and achiral phosphine and diene ligands in place of BINAP (representa-
tive examples; Fig. 2b, entries 6–9; see section VII of the Supporting In-
formation for additional results). Catalysts derived from most of the lig-
ands that were evaluated afford little to no conjugate addition products.
Of note, while the aforementioned BINAP-based ligands form the con-
jugate addition product 2A with up to 3:1 site-selectivity, the analogous
reaction conducted with DPPB as the ligand enables a reversal of site-
selectivity; Product 2B forms as the major product (1 : 5, 2A : 2B), albeit
with only 4% conversion (Fig. 2b, entry 6). In sharp contrast to reac-
tions conducted with any other ligand, reactions
Results and Discussion
Site-Selective Arylation of Thiostrepton. To develop a Rh-catalyzed
conjugate addition of boronic acids to thiostrepton, we began with reac-
tions conditions typical for Rh-catalyzed conjugate addition reactions.
We found that the reaction between 4-methoxyphenyl boronic acid and
thiostrepton 1 at 50 °C using [Rh(nbd)2]BF4/rac-BINAP as catalyst led
to the formation of two classes of products: products 2A/2A’ and
2B/2B’, resulting from conjugate additions to either Dha16 or Dha17,
respectively, and products 3 and 3’, arising from a documented, base-
mediated cleavage of Dha16 and/or Dha17 from thiostrepton (Fig. 2a).9
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OMe
O
O
H
O
Dha16
N
B(OH)2
1
H
N
N
NH2
+
NH2
a.
S
O
O
O
H
H
N
O
2
3
2 equiv.
0.003 mmol
N
MeO
N
H
N
NH2
O
Thiostrepton
Dha17
S
2B, 2B’
H
S
N
O
N
OMe
[Rh(nbd)2]BF4/ Ligand (50 mol %)
Base (2 equiv)
2A, 2A’
Thiostrepton
4
5
6
O
N
O
N
Dioxane : H2O (7 : 1)
X oC, 14 h
H
N
NH2
O
O
S
N
H
S
O
H
N
+
N
H
+
N
NH2
O
O
N
HN
S
O
3
3’
N
NH
O
7
8
Thiostrepton
S
O
(Product due to base
mediated cleavage)
(Product due to base
mediated cleavage)
HO
O
H
OH
Thiostrepton
NH
O
N
HN
NH
9
N
Temp
Base
Conv. to A + B
3(%, A : B)3
Entry
Ligand
b.
3Conv. to 3 (%)3
N
H
(oC)
S
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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N
S
OH
2.5 mg (0.0015 mmol)
O
50
6%
8%
16%
8%
12%
4%
3trace3
45%
67%
3(2 : 3)3
(2 : 1)
(3 : 1)
(3 : 1)
(3 : 1)
(1 : 5)
—
313
2
3
4
5
6
7
8
9
3rac-BINAP3
rac-BINAP
rac-BINAP
rac-BINAP
S,S-BINAP
DPPB
S,S-DIOP
S,S-chiraphos K2CO3
S,S-chiraphos K2CO3
KOH
Et3N
43%
37%
25%
13%
8%
HO
OH
50
50
rt
rt
rt
rt
rt
3503
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
c.
8%
10%
12%
7%
(8 : 1)
(10 : 1)
CF3
d.
OMe
F
Cl
O
O
O
O
O
O
O
O
H
N
H
N
H
N
H
N
N
H
NH2
N
H
NH2
N
H
NH2
N
H
NH2
S
S
S
S
N
O
N
O
N
O
N
O
2
71% conversion
(1.5 equiv. boronic acid,
50 oC, 14 hr)
10 : 1 site selectivity
4
76% conversion
5
92% conversion
6
Thiostrepton
Thiostrepton
Thiostrepton
Thiostrepton
63% conversion
(1.25 equiv. boronic acid,
rt, 14 hr)
(1.5 equiv. boronic acid,
rt, 14 hr)
10 : 1 site selectivity
(1.5 equiv. boronic acid,
rt, 14 hr)
>10 : 1 site selectivity
8 : 1 site selectivity
O
tBu
Me
OCF3
N
O
O
O
O
O
O
O
O
H
N
H
N
H
N
H
N
N
H
NH2
N
H
NH2
N
H
NH2
N
H
NH2
S
S
S
S
N
O
N
O
N
O
N
O
9
42% conversion
(2.0 equiv. boronic acid,
50 oC, 14 hr)
6 : 1 site selectivity
7
65% conversion
(2 equiv. boronic acid,
50 oC, 14 hr)
8 : 1 site selectivity
8
10
74% conversion
(1.5 equiv. boronic acid,
50 oC, 14 hr)
9 : 1 site selectivity
Thiostrepton
Thiostrepton
Thiostrepton
Thiostrepton
67% conversion
(2 equiv. boronic acid,
50 oC, 14 hr)
5 : 1 site selectivity
O
S
N
NH
O
O
NH
O
O
O
O
O
O
O
O
O
H
N
H
N
H
N
H
N
N
H
NH2
N
H
NH2
N
H
NH2
N
H
NH2
S
S
S
S
N
O
N
O
N
O
N
O
11
13
59% conversion
(4 equiv. boronic acid,
65 oC, 14 hr)
Site selectivity: ND*
12
14
64% conversion
(1.5 equiv. boronic acid,
rt, 14 hr)
Site selectivity: ND*
Thiostrepton
Thiostrepton
Thiostrepton
Thiostrepton
49% conversion
(4 equiv. boronic acid,
65 oC, 14 hr)
81% conversion
(2 equiv. boronic acid,
rt, 14 hr)
5 : 1 site selectivity
4 : 1 site selectivity
Figure 2. Conjugate additions of boronic acids to thiostrepton. a. Scheme depicting the products 2A, 2B, and 3 observed in the reaction
using 4-OMe-phenyl boronic acid. Products 2A/2A’ and 2B/2B’ are pairs of diastereomers. Products 3 and 3’ arise from base mediated
cleavage of the Dha residues. b. Tabulated results of the reaction depicted in Fig. 2a under varied conditions. Reaction temperatures at or
above 80 oC resulted in significant decomposition of the starting material without product formation. Conversions to particular products were
determined by UPLC using uncorrected areas. Conversion to 2A and 2B are the sum of the areas due to the two diastereomers of each product.
The two diastereomers of 2A can be separated by UPLC. The two diastereomers of 2B co-elute as a single peak. See Fig. 2c and Supporting
Information for site assignment. c. Representative UPLC trace of the crude reaction mixture formed under the conditions described in Entry
9. d. Thiostrepton analogs prepared by Rh-catalyzed conjugate arylation. Reaction conditions are as described in Fig. 2a. Tabulated conver-
sions describe the sum of the conversion to the monoarylation products with the site selectivity as listed (see sections III-IV of the Supporting
Information for details). *Products arising from reaction at Dha16 and Dha17 could not be separated completely. e. Representative UPLC
traces demonstrating the purity of the products obtained by preparative HPLC and submitted for biological testing (see Table 1).
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1
2
3
4
5
6
7
8
9
conducted with S,S-Chiraphos (Fig. 2b, entries 8) occur with moder-
Stereoselective Arylation of Thiostrepton. Having achieved cata-
ate conversion to the conjugate addition products (45%), 8 : 1 site-
selectivity for subterminal Dha16 over Dha17 and 4 : 1 chemoselectiv-
ity for conjugate addition over tail cleavage in reactions conducted at
room temperature. At 50 °C (Fig. 2b, entry 9), the conjugate addition
reaction proceeds to 67% conversion without loss of site or chemose-
lectivity. In a single purification, the four monoarylation products of
the reaction under these conditions were isolated in 61% combined
yield, revealing a high correlation between the conversion determined
by UPLC (67%) and the isolated yield (61%). The four individual
monoarylation products were purified by reverse phase preparative
HPLC (See section III of the Supporting Information).
lyst-controlled site-selectivity, we turned our attention to modifying
the reaction conditions to achieve catalyst-controlled stereoselectiv-
ity. This goal confronts at least two significant challenges. First, alt-
hough the targeted transformation is diastereoselective, the reaction
occurs on a flexible region of the molecule that lacks proximal stereo-
centers. Thus, substrate-controlled diastereoselectivity is unlikely,
and indeed under the conditions employed in Fig. 3, all products
formed in ~1:1 dr. The second issue concerns catalyst control. Re-
lated precedents involving enantioselective conjugate addition of bo-
ronic acids to simple dehydroalanine substrates identify asymmetric
10
11
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13
14
15
16
17
18
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protonation of an intermediate Rh-enolate as the enantio-determining
17,19,24
step.
Solutions to the general problem of enantioselective pro-
tonation of Rh-enolates rely on stoichiometric, acidic additives (such
With these reaction conditions in hand, we evaluated the scope of the
reaction (Fig 2d). A wide tolerance for boronic acids was observed,
with the site-selectivity for Dha16 maintained in all cases (see section
IV of the Supporting Information for details). Electron-neutral and
electron-poor boronic acids were generally found to be more reactive
than was 4-OMe phenyl boronic acid. The reaction of 3-CF3-
phenylboronic acid forms the conjugate addition products 5 in 92%
conversion at room temperature with only 1.5 equiv. of boronic acid.
The combined monoarylation products of this reaction were obtained
in 70% isolated yield. Ortho-, meta-, and para-substituted arylboronic
acids were tolerated in the reaction, which was also suitable for the in-
corporation of heteroarenes into the thiostrepton scaffold. Finally, the
dansylamide fluorophore can be incorporated into thiostrepton using
this conjugate addition strategy, forming compound 14. Although in-
itial experiments were performed at high catalyst loading, we evaluated
the reaction of electron neutral (4-F) and electron poor (3-CF3) phe-
nylboronic acids at lower catalyst loading and found that with these
substrates, the reactions proceed to high conversion (75% and 78%,
as substituted phenols or phthalamides) in place of protic solvents to
17
achieve high enantioselectivities. These approaches have been ap-
plied specifically to the synthesis of protected amino acids from dehy-
droalanines, and high enantioselectivities and yields have been
achieved. However, in addition to the use of stoichiometric organic
additives, specialized ligands (difluorophos or bulky bidentate phos-
phites), and rarer aryl trifluoroborates, the reactions require high tem-
°
peratures (>100 C). This in particular implied that a direct imple-
mentation of literature conditions was not possible for the complex
structure of 1.
We sought to define reaction conditions that would lead to stereose-
lectivity through evaluation of the reaction between thiostrepton and
4-F-phenylboronic acid in the absence of water and in the presence of
various alternative proton sources (Fig. 3a). We found that only trace
product forms in reactions containing either 2-methoxyphenol or
phthalamide as acids (Fig. 3b, entries 1-4). In contrast, using isopro-
panol (10 vol %) in place of water, the conjugate addition products
form with 6:1 site-selectivity (4A: 4B), with the major site isomer 4A
forming in 4:1 dr (Fig. 3b, entry 5). However, only 11% conversion to
conjugate addition products was observed, and further efforts to im-
prove the conversion by modifying other reaction parameters were not
successful. Using methanol in place of isopropanol (Fig. 3b, entries
7), we found that the conversion improves to 62% with a modest de-
crease in diastereoselectivity (3 : 1 dr) compared to that observed
when using isopropanol (4 : 1 dr).
°
respectively) at 50 C using only 6.25 mol% catalyst. Together, these
results demonstrate the possibility to generate a wide range of deriva-
tives of a complex molecule by Rh-catalyzed conjugate addition.
However, little to no diastereoselectivity was observed, with all prod-
ucts reported in Fig. 2 forming as an approximately equal mixture of
two diastereomers. Intrinsically, there remains value in this type of un-
selective outcome, because the individual diastereomers of the prod-
ucts formed from 11 of the 12 boronic acids shown in Fig. 2d were sep-
arable, enabling testing of their biological properties (vide infra).
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F
B(OH)2
a.
1
O
O
O
O
O
O
H
N
H
N
H
N
2
3
4
5
2 equiv.
F
N
H
NH2
N
H
NH2
N
H
NH2
S
+
S
S
[Rh(nbd)2]BF4/ S,S-chiraphos (50 mol %)
N
O
N
O
N
O
Additive(s)
Dioxane
65 oC, 14 h
Thiostrepton
Thiostrepton
Thiostrepton
F
4A
4B
6
7
Conv. to A + B
(%, A : B)3
Condition
Proton Source
b.
Base
d.r. (A)3
c.
Dioxane/ H2O
K2CO3
4A
8
9
4A’
1
2
None
2-Methoxyphenol
Phthalamide
IPA
None
3<5%
—
—
—
K2CO3
K2CO3
None
<5%
<5%
<5%
11%
22%
62%
85%
84%
30%
43%
—
3
3
Double Addition Products
—
—
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4B
4A
4B’
3’/1
4
—
—
5
IPA
K2CO3
None
36 : 13
10 : 1
5 : 1
10 : 1
9 : 1
8 : 1
8 : 1
4 : 1
1 : 1
3 : 1
1 : 1
4 : 1
3 : 1
3 : 1
6
MeOH
Dioxane/ MeOH
Na2CO3
7
MeOH
K2CO3
Et3N
8
MeOH
4A’
4B’
9
MeOH
Na2CO3
KCl
3
4B
Double Addition Products
3’/1
10
11
MeOH
MeOH
NaCl
CF3
F
OMe
d.
O
O
H
O
O
H
O
O
H
N
N
N
H
NH2
N
H
NH2
S
N
N
S
N
H
NH2
O
N
O
S
4A
N
O
Thiostrepton
Thiostrepton
2A
S,S-chiraphos
84% conversion, 4 : 1 dr
R,R-catASium D
5A
S,S-chiraphos
89% conversion, 4 : 1 dr
R,R-catASium D
S,S-chiraphos
Thiostrepton
52% conversion, 4 : 1 dr
R,R-catASium D
59% conversion, 1 : 3 dr
70% conversion, 1 : 2 dr
67% conversion, 1 : 2 dr
O
O
O
O
O
S
H
N
N
N
H
NH2
N
Thiostrepton
HN
S
Ph2P
PPh2
N
O
NH
O
Ph2P
12A
S,S-chiraphos
72% conversion, 3 : 1 dr
R,R-catASium D
S,S-Chiraphos
11A
O
Thiostrepton
PPh2
S,S-chiraphos
NH2
20% conversion, 4 : 1 dr
R,R-catASium D
trace product
R,R-catASium D
32% conversion, 1 : 3 dr
Figure 3. Reaction conditions for diastereoselective conjugate addition of boronic acids to thiostrepton. a. Model reaction used to
develop a diastereoselective process. b. Outcome of the model reaction conducted under varied reaction conditions. c. HPLC traces
of crude reactions conducted under aqueous conditions (described in Fig. 2) and methanolic conditions (Fig. 3b, entry 9). d. Outcome
of diastereoselective reactions using varied boronic acid substrates. Reactions were conducted under conditions shown in Fig. 3b,
entry 9. UPLC traces of crude reactions are shown to exemplify the change in diastereoselectivity in the presence of each ligand.
Conversions are based on integration of the UPLC traces using uncorrected areas. Stereochemistry is depicted based on analogy to
the stereochemical outcome of reactions of Dha monomers conducted under comparable conditions (vide infra).
Given this improvement in conversion when using MeOH, we re-eval-
uated the effect of the base on the diastereoselectivity of the reaction
under methanolic conditions (Fig. 3b, entries 6-10), and we found
that only sodium and potassium bases afford diastereoselectivity,
while reactions containing amine, cesium, and rubidium bases form
the product with ~1 : 1 dr. Sodium carbonate was identified as the
most suitable base for the reaction (Fig. 3b, entry 9); in the presence
of 0.4 equiv. of Na2CO3, the reaction occurs with 84% conversion, 9:1
site selectivity, and 4: 1 diastereoselectivity for the formation of the
major site isomer (Fig. 3b-c). Although the assignment of the absolute
stereochemistry of 4A and 4A’ (and the other analogs that were pre-
pared) is challenging and remains an ongoing effort in our laboratory,
the stereochemistry depicted in Figure 3 is tentatively assigned on the
basis of preliminary studies that established the absolute stereochem-
ical outcome of reactions of dehydroalanine monomers conducted un-
der the same catalytic conditions (vide infra).
(Fig. 3d and section IV of the Supporting Information). These studies
revealed that electron-rich and electron-poor, ortho-, meta-, and para-
substituted, and heteroaromatic boronic acids all react with compara-
ble levels of diastereoselectivity. In contrast, reactions catalyzed by the
opposite enantiomer of the catalyst (R,R-chiraphos) occur with con-
versions that are 2-3 times lower and with negligible diastereoselectiv-
ity, suggesting a mismatched effect between this enantiomer of the cat-
alyst and the chiral structure of thiostrepton. However, we found that
reactions containing the pseudoenantiomeric ligand R,R-catASium D
proceeded in higher conversions and with higher selectivities than the
analogous reactions containing R,R-Chiraphos; reactions containing
R,R-catASium D formed the opposite diastereomer of the products
compared to those formed in reactions containing S,S-chiraphos (Fig.
3d). The least reactive heteroaryl boronic acids did not react in the
presence of catalysts formed from R,R-catASium D. The UPLC traces
of crude reactions shown in Figure 3d exemplify catalyst-controlled se-
lection for the different thiostrepton diastereomers formed during
conjugate arylation.
Under the same conditions, we evaluated the selectivity of conjugate
additions reactions between thiostrepton and other boronic acids
ACS Paragon Plus Environment

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Page 6 of 9
1
a. Enantiodivergent Conjugate Addition of Boronic Acids
2
3
4
[Rh(nbd)2]BF4 (5 mol %)
S,S-chiraphos (5 mol %)
MeOH (8 equiv.)
[Rh(nbd)2]BF4 (5 mol %)
S,S-chiraphos (5 mol %)
MeOH (8 equiv.)
MeO2C
NHAc
MeO2C
NHAc
MeO2C
NHAc
5
6
+
Na2CO3 (1.0 equiv.)
Dioxane, 65 oC, 9 hr
No additive
Dioxane, 65 oC, 9 hr
B(OH)2
F
F
7
8
15A
98% conversion
91: 9 er
15B
Condition A
Condition B
F
2 equiv.
100% conversion
84 : 16 er
9
b.
d. Synthesis of Unnatural Amino Esters and Amino Amides
[Rh(nbd)2]BF4 (5 mol %)
Ligand (5 mol %)
Dioxane : MeOH (50 : 1)
MeO2C
NHAc
+
MeO2C
NHAc
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
B(OH)2
2 equiv.
MeO2C
NHAc
Additive
65o C, 9 hr
F
MeO2C
NHAc
MeO2C
NHAc
MeO2C
NHAc
F
CF3
Conversion
e.r.
Condition
Ligand
Additive
O
Cl
F
313
2
3
4
5
6
7
8
3S,S-chiraphos2
S,S-chiraphos
S,S-chiraphos
S,S-chiraphos
S,S-chiraphos
S,S-chiraphos
R,R-chiraphos
R,R-chiraphos
3K2CO3 (1 equiv.)3
K2CO3 (0.5 equiv.)
Na2CO3 (1.0 equiv.)
KCl (1.0 equiv.)
NaCl (1.0 equiv.)
None
310 : 903
10 : 90
9 : 91
15 : 85
21 : 79
83 : 17
87 : 13
27 : 73
395%
94%
98%
18%
42%
>99%
>99%
>99%
19
15
91% yield
93 : 7 er
20
92% yield
91 : 9 er
21
82% yield
86 : 14 er
90% yield
90 : 10 er
(Condition A)
Na2CO3 (1.0 equiv.)
None
(Condition A)
(Condition A)
(Condition B)
O
HO
c. Generality of Reversal of Enantioselectivity
O
NHBoc
MeO2C
AcHN
NHAc
OMe
O
O
O
N
H
AcHN
BocHN
AcHN
OMe
OBn
OMe
O
F
N
H
F
22
72% yield
89 : 11 er
24
67% yield
86 : 14 er
23
MeO
F
47% yield
89 : 11 er
(Condition B)
18
17
16
(Condition B)
(Condition A)
No additive: 85 : 15 er
Na2CO3: 23 : 77 er
No additive: 78 : 21 er
Na2CO3: 17 : 83 er
No additive: 74 : 26 er
Na2CO3: 20 : 80 er
Figure 4. Outcome of conjugate addition reactions between protected Dha monomers and aryl boronic acids. Absolute configura-
tions are assigned by comparison to literature data or by analogy (See Supporting Information for details) a. Observation of reversal
of enantioselectivity in reactions conducted with and without additive. b. Analysis of the influence of varied cation sources on the
outcome of the conjugate addition reaction. c. Demonstration of reversal of enantioselectivity in the presence of varied substrates.
d. Synthesis of protected amino esters and amino amides using the Rh-catalyzed conjugate addition methods shown in Fig. 4a.
Reactions were conducted with [Rh(cod)2]BF4 in place of [Rh(nbd)2]BF4.
To probe the effect of the base on the diastereoselectivity, we con-
ducted the reaction to form thiostrepton analog 4 in the presence of
either NaCl or KCl salts in places of their analogous carbonate bases,
and we found that these salts effected the reaction with comparable
levels of diastereoselectivity to that achieved with the carbonate bases,
albeit with somewhat lower yields (Fig. 3b, entries 10-11). This sug-
gests that the sodium or potassium cations may play a role in organiz-
ing the substrate and/or the catalyst to favor the formation of one dia-
stereomer.
This surprising reversal of enantioselectivity sheds light on the chal-
lenge to develop reactions relying on asymmetric protonation of Rh-
enolates and complements prior work that found potassium aryl-tri-
fluoroborates, rather than boronic acids, to be required for high enan-
19,25
tioselectivity in analogous reactions.
Ongoing studies in our labor-
atory seek to understand further these observations.
In addition to offering insight into the diastereoselectivity of reactions
of thiostrepton, we were intrigued by the high level of enantioselectiv-
ity achieved in this reaction, given the limited number of catalyst sys-
tems known for enantioselective conjugate addition of boronic acids.
A limited optimization of the reaction conditions revealed that
[Rh(cod)2]BF4 afforded slightly higher selectivity than
[Rh(nbd)2]BF4 (93 : 7 versus 91 : 9 er) in the model reaction between
4-F phenylboronic acid and Ac-Dha-OMe. Using these conditions, we
prepared a series of additional amino ester and amino amide com-
pounds (Fig. 4d).
Implications for Enantioselective Catalysis. Having observed this
effect in the case of a diastereoselective reaction of a complex mole-
cule, we hypothesized that it could also be influential in enantioselec-
tive reactions of simpler substrates. We thus explored analogous con-
jugate addition reactions using protected Dha monomers in place of
thiostrepton. The effect of the salt was found to be substantial (Fig.
4a). The omission of the alkali bases or salts from the reaction lead to
nearly a full reversal of the enantioselectivity of the reaction. The ad-
dition of 1 equiv. of Na2CO3 to the reaction lead to the formation of
the product with 98% conversion and 91:9 er in favor of the (S)-enan-
tiomer (Fig 4b, entry 3). The reaction conducted in the absence of
any additive occurred with 83:17 er in favor of the (R)-enantiomer
(Fig 4b, entry 6). The reversal of selectivity was found to be conserved
across several boronic acid and dehydroalanine substrates (Fig. 4c.)
Biological Activity of Thiostrepton Analogs. The aggregate of
these studies enabled preparative semi-synthesis of 24 thiostrepton
derivatives, including the first derivatives that are selectively function-
alized at subterminal Dha16. This set of thiostrepton analogs signifi-
cantly expands the number of known analogs of this highly complex
ACS Paragon Plus Environment

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Journal of the American Chemical Society
natural product. Nineteen of the analogs were evaluated as antimicro-
magnitude of the difference in activity between the two diastereomers
1
2
3
4
5
6
7
8
bial agents against nine resistant and non-resistant organisms (see
Supporting Information section XIII for complete data set). The ma-
jority of the analogs that were tested retained levels of activity that
were within an order of magnitude of that of thiostrepton (Table 1).
Many analogs retained activity that was within a factor of two of the
level observed for thiostrepton (bolded entries, Table 1), with 2A’
having the highest activity. The data also show that the stereochemis-
try of the newly formed amino acids does affect the activity, with the
typically found to be 2-4 fold. The least active analogs were found to
be those for which the Dha was modified by a very bulky or hydropho-
bic amino acid (such as 8A/A’ and 14A/A’). Together, these results
show that conjugate arylation of Dha 16 may be a suitable strategy for
modification of thiostrepton in order to improve the pharmacological
properties of the molecule without inherent loss of activity.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 1. Biological Activity of Analogs of Thiostrepton (See Supporting Information for details and additional data)
MIC (ug/mL) for a range of organisms (VSE = Vancomycin susceptible, VRE = vancomycin resistant)
Compound
S. aureus
E. faecalis
E. faecalis
E. faecium
S. pneumoniae
S. pyogenes
(Ar substituent)
Thiostrepton 1
Vancomycin
MRSA
VSE
VRE
VRE
PSSP
0.25
1
0.5
2
0.25
>32
0.12
>32
0.002
0.25
0.002
0.5
2A (4-OMe)
2A’ (4-OMe)
4A (4-F)
1
0.5
1
1
1
1
2
1
1
1
2
1
2
4
1
2
1
2
2
2
4
4
1
0.5
1
1
0.25
0.5
1
0.008
0.002
0.008
0.015
0.008
0.008
0.015
0.008
0.004
0.008
0.03
0.004
0.002
0.008
0.008
0.004
0.004
0.004
0.004
0.002
0.008
0.015
0.008
0.015
0.004
0.004
0.015
0.008
0.03
4A’ (4-F)
2
1
5A (3-CF3)
2
1
1
5A’ (3-CF3)
2
1
0.5
1
6A (4-Cl)
2
1
6A’ (4-Cl)
2
1
1
7A (4-Me)
8A (4-tBu)
8A’ (4-tBu)
2
0.5
2
0.5
2
4
8
2
2
9A (4-OCF3)
10A (4-Morph.)
10A’ (4-Morph.)
11A (Benzofuran)
11A’ (Benzofuran)
12A (Dibenzofuran)
14A (Dansyl)
14A’ (Dansyl)
2
2
1
0.008
0.015
0.008
0.008
0.015
0.008
0.03
4
2
1
2
1
0.5
1
2
1
4
4
2
2
2
1
32
64
8
2
4
2
0.15
0.15
products. Yet, many challenges remain. These include extending our
findings to the comprehensive functionalization of alternate Dha sites,
the dehyrothreonine site, if not all other loci of unsaturation in the
structure.
Conclusions
We have developed a high yielding, and site- and diastereoselective
conjugate addition reaction suitable for functionalization of the sub-
terminal dehydroalanine residue of thiostrepton. In doing so, we have
demonstrated the utility of this reaction to enable post-synthetic con-
version of a dehydroalanine residue to a wide range of unnatural amino
acids in a highly complex molecule; from the single, fully unprotected
thiostrepton substrate, twenty-four new derivatives of this antibiotic
were prepared by this approach. Moreover, we found that the simple
reaction conditions developed for thiostrepton were also suitable for
enantioselective conjugate addition to dehydroalanine monomers,
providing access to a range of unnatural amino acid derivatives in high
enantioselectivity. Thus, these synergistic studies of site-selective ca-
talysis in a complex molecular setting also delivered new fundamental
insights in the arena of enantioselective catalysis. In addition to
providing access to the new compounds described herein, this method
should enable the preparation of analogs of many other biologically
relevant molecules containing one or more dehydroalanine motifs.
They also exemplify the suitability of metal catalyzed asymmetric cat-
alysts to address problems in the tailoring of high complexity natural
ASSOCIATED CONTENT
The Supporting Information is available free of charge on the ACS
Publications website. Experimental methods, characterization of
all compounds, and copies of spectra can be found in the Support-
ing Information.
AUTHOR INFORMATION
Corresponding Author: scott.miller@yale.edu
Present Addresses
Hanna M. Key
Department of Chemistry
Grinnell College
Grinnell, IA 50112
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Nicolaou, K. C.; Zak, M.; Safina, B. S.; Lee, S. H.;
Estrada, A. A. Angew. Chem. Int. Ed. 2004, 43, 5092–
5097.
Jonker, H. R. A.; Baumann, S.; Wolf, A.; Schoof, S.;
Hiller, F.; Schulte, K. W.; Kirschner, K. N.; Schwalbe,
H.; Arndt, H. D. Angew. Chem. Int. Ed. 2011, 50,
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Myers, C. L.; Hang, P. C.; Ng, G.; Yuen, J.; Honek, J.
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Gruppi, F.; Xu, X.; Zhang, B.; Tang, J. A.; Jerschow,
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Funding Sources
We are grateful to the National Institute of General Medical Sciences
of the NIH (GM-068649) for support.
1
2
3
4
5
6
7
8
ACKNOWLEDGMENT
We are grateful to the U.S. National Institutes of Health for support of
this research.
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Insert Table of Contents artwork here
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R
O
O
H
NH2
H
O
N
NH2
O
N
N
O
N
H
O
S
H
N
S
N
B(OH)2
R
N
O
N
O
H
N
O
S
H
N
O
S
9
O
H
N
N
N
O
[Rh]/S,S-chiraphos
Dioxane/MeOH
H
N
N
O
HN
N
N
H
O
O
H
N
O
HN
NH
O
S
O
NH
HO
HN
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
H
S
OH
HO
HN
NH
O
H
O
OH
50-89% conversion
4 : 1 dr
> 5 : 1 site selectivity
N
NH
O
N
O
N
O
N
NH
NH
N
N
H
N
S
H
N
————————————————
24 new derivatives isolated
One Step, No Protecting Groups
S
S
OH
S
O
OH
O
HO
OH
HO
Thiostrepton
OH
9
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