Total synthesis of [ψ[C(=S)NH]Tpg4]vancomycin aglycon, [ψ[C(=NH)NH]Tpg4]vancomycin aglycon, and related key compounds: Reengineering vancomycin for dual D-Ala-D-Ala and D-Ala-D-Lac binding

Article abstract of DOI:10.1021/ja209937s

The total synthesis of [ψ[C(=S)NH]Tpg⁴]vancomycin aglycon (8) and its unique AgOAc-promoted single-step conversion to [ψ[C(=NH)NH] Tpg⁴]vancomycin aglycon (7), conducted on a fully deprotected substrate, are disclosed. The synthetic approach not only permits access to 7, but it also allows late-stage access to related residue 4 derivatives, alternative access to [ψ[CH₂NH]Tpg⁴]vancomycin aglycon (6) from a common late-stage intermediate, and provides authentic residue 4 thioamide and amidine derivatives of the vancomycin aglycon that will facilitate ongoing efforts on their semisynthetic preparation. In addition to early stage residue 4 thioamide introduction, allowing differentiation of one of seven amide bonds central to the vancomycin core structure, the approach relied on two aromatic nucleophilic substitution reactions for formation of the 16-membered diaryl ethers in the CD/DE ring systems, an effective macrolactamization for closure of the 12-membered biaryl AB ring system, and the defined order of CD, AB, and DE ring closures. This order of ring closures follows their increasing ease of thermal atropisomer equilibration, permitting the recycling of any newly generated unnatural atropisomer under progressively milder thermal conditions where the atropoisomer stereochemistry already set is not impacted. Full details of the evaluation of 7 and 8 along with several related key synthetic compounds containing the core residue 4 amidine and thioamide modifications are reported. The binding affinity of compounds containing the residue 4 amidine with the model d-Ala-d-Ala ligand 2 was found to be only 2-3 times less than the vancomycin aglycon (5), and this binding affinity is maintained with the model d-Ala-d-Lac ligand 4, representing a nearly 600-fold increase in affinity relative to the vancomycin aglycon. Importantly, the amidines display effective dual, balanced binding affinity for both ligands (K_a2/4 = 0.9-1.05), and they exhibit potent antimicrobial activity against VanA resistant bacteria (E. faecalis, VanA VRE) at a level accurately reflecting these binding characteristics (MIC = 0.3-0.6 μg/mL), charting a rational approach forward in the development of antibiotics for the treatment of vancomycin-resistant bacterial infections. In sharp contrast, 8 and related residue 4 thioamides failed to bind either 2 or 4 to any appreciable extent, do not exhibit antimicrobial activity, and serve to further underscore the remarkable behavior of the residue 4 amidines.

Full text of DOI:10.1021/ja209937s

Article
pubs.acs.org/JACS
Total Synthesis of [Ψ[C(S)NH]Tpg4]Vancomycin Aglycon, [Ψ[C(
NH)NH]Tpg4]Vancomycin Aglycon, and Related Key Compounds:
Reengineering Vancomycin for Dual D-Ala-D-Ala and D-Ala-D-Lac
Binding
Jian Xie, Akinori Okano, Joshua G. Pierce, Robert C. James, Simon Stamm, Christine M. Crane,
and Dale L. Boger*
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines
Road, La Jolla, California 92037, United States
S
* Supporting Information
ABSTRACT: The total synthesis of [Ψ[C(S)NH]Tpg4]vancomycin
aglycon (8) and its unique AgOAc-promoted single-step conversion to
[Ψ[C(NH)NH]Tpg4]vancomycin aglycon (7), conducted on a fully
deprotected substrate, are disclosed. The synthetic approach not only
permits access to 7, but it also allows late-stage access to related residue 4
derivatives, alternative access to [Ψ[CH2NH]Tpg4]vancomycin aglycon
(6) from a common late-stage intermediate, and provides authentic residue
4 thioamide and amidine derivatives of the vancomycin aglycon that will
facilitate ongoing efforts on their semisynthetic preparation. In addition to
early stage residue 4 thioamide introduction, allowing differentiation of one
of seven amide bonds central to the vancomycin core structure, the approach relied on two aromatic nucleophilic substitution
reactions for formation of the 16-membered diaryl ethers in the CD/DE ring systems, an effective macrolactamization for closure
of the 12-membered biaryl AB ring system, and the defined order of CD, AB, and DE ring closures. This order of ring closures
follows their increasing ease of thermal atropisomer equilibration, permitting the recycling of any newly generated unnatural
atropisomer under progressively milder thermal conditions where the atropoisomer stereochemistry already set is not impacted.
Full details of the evaluation of 7 and 8 along with several related key synthetic compounds containing the core residue 4 amidine
and thioamide modifications are reported. The binding affinity of compounds containing the residue 4 amidine with the model D-
Ala-D-Ala ligand 2 was found to be only 2−3 times less than the vancomycin aglycon (5), and this binding affinity is maintained
with the model D-Ala-D-Lac ligand 4, representing a nearly 600-fold increase in affinity relative to the vancomycin aglycon.
Importantly, the amidines display effective dual, balanced binding affinity for both ligands (Ka 2/4 = 0.9−1.05), and they exhibit
potent antimicrobial activity against VanA resistant bacteria (E. faecalis, VanA VRE) at a level accurately reflecting these binding
characteristics (MIC = 0.3−0.6 μg/mL), charting a rational approach forward in the development of antibiotics for the treatment
of vancomycin-resistant bacterial infections. In sharp contrast, 8 and related residue 4 thioamides failed to bind either 2 or 4 to
any appreciable extent, do not exhibit antimicrobial activity, and serve to further underscore the remarkable behavior of the
residue 4 amidines.
to a community-acquired infection have intensified the need to
treat such resistant bacterial infections. It is estimated that
MRSA is responsible for 19 000 deaths and invasively afflicts
94 000 annually in the U.S. (2005) at a cost of $3−4 billion per
year.5c,d In addition, vancomycin-resistant strains of other
bacteria are also on the rise with U.S. ICU clinical isolates of
vancomycin-resistant Enterococcus faecalis (VRE) approaching
30% (2003),5 albeit in strains presently sensitive to other
antibiotics. Even more significant and feared by all is the recent
emergence of MRSA strains now resistant or insensitive to
vancomycin (VRSA and VISA). This poses a major health
INTRODUCTION
■
Vancomycin (1) is the most widely recognized member of a
large family of glycopeptide antibiotics that are used for the
treatment of resistant bacterial infections. Vancomycin was first
disclosed over 50 years ago (1955) by Eli Lilly,1 and its
structure was determined 25−30 years later (1983),2 long after
its clinical introduction in 1958. Clinical uses of vancomycin
include the treatment of patients on dialysis, patients allergic to
β-lactam antibiotics, and patients undergoing cancer chemo-
therapy.3 However, the most widespread clinical use of
vancomycin is the treatment of methicillin-resistant Staph-
ylococcus aureus (MRSA) infections, for which it is regarded as
the drug of last resort.4 The prevalence of MRSA in intensive
care units (ICU, 60% of SA infections in the U.S. are MRSA,
2003)5 and the movement of MRSA from a hospital-acquired
Received: October 21, 2011
Published: December 20, 2011
© 2011 American Chemical Society
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problem and has stimulated efforts to develop vancomycin
analogues6 or alternative antibiotics to combat this resistance.7
Vancomycin inhibits bacterial cell wall synthesis, binding to
the peptidoglycan peptide terminus N-acyl-D-Ala-D-Ala found in
cell wall precursors,8 by sequestering the substrate from
transpeptidase and inhibiting cell wall cross-linking. Shortly
after the disclosure of the structure of vancomycin, Williams
provided the NMR structure of a N-acyl-D-Ala-D-Ala complex
with the antibiotic that was found to be stabilized by an
extensive array of hydrophobic van der Waals contacts within
the vancomycin binding pocket and five key H-bonds lining the
pocket, and this has since been confirmed in X-ray studies of
such model complexes (Figure 1).9 In the most commonly
1). We provided an experimental estimation of the magnitude
of these two effects by examining the model ligands 2−4 that
indicated it is the repulsive lone pair interaction (100-fold), not
the H-bond loss (10-fold), that is responsible for the largest
share of the reduced binding affinity (1000-fold).12 Not only
were the estimates consistent with intuitive expectations, but
their combined magnitude matched expectations resulting from
the stabilizing binding energy of an amide H-bond (0.0−1.5
kcal/mol) and a destabilizing lone pair/lone pair interaction
(1.6−2.7 kcal/mol).13 These observations have significant
ramifications on the reengineering of vancomycin to bind D-
Ala-D-Lac, suggesting that the design could focus principally on
removing the destabilizing lone pair interaction rather than
reintroduction of a H-bond and that this may be sufficient to
compensate for most of the binding affinity lost with D-Ala-D-
Lac.
The resurgence of interest in vancomycin and the
identification of the molecular origin of the bacterial resistance
emerged as the first total syntheses of its family members were
completed.14 In our efforts complementary to those reported
by Evans15 and Nicolaou,16 an initial total synthesis of the
vancomycin aglycon17 was extended to the more complex
tetracyclic teicoplanin18 and ristocetin19 aglycons, as well as the
structurally related chloropeptins,20 in which the synthetic
approach was continuously refined.21 In the midst of these
studies and concurrent with efforts probing modifications to
vancomycin itself,22 we initiated efforts on the redesign of
vancomycin to bind D-Ala-D-Lac, completing the total synthesis
and evaluation of [Ψ[CH2NH]Tpg4]vancomycin aglycon (6)23
in which the residue 4 amide carbonyl was replaced with a
methylene group. Confirming the conclusions drawn from the
binding studies summarized in Figure 2 and relative to
Figure 1. Structure of vancomycin (1), schematic representation of
interaction with model ligands 2−4, and measured binding data.
encountered vancomycin-resistant bacterial strains (VanA and
VanB), a vancomycin challenge is sensed10 and subsequently
induces a remodeling of the precursor peptidoglycan terminus
from D-Ala-D-Ala to D-Ala-D-Lac.11 Normal D-Ala-D-Ala
production continues despite the presence of vancomycin,
but now a late-stage remodeling to D-Ala-D-Lac ensues to avoid
the action of the antibiotic. The substitution of a linking ester
for an amide with the exchange of a single atom (NH→O)
reduces the binding to vancomycin 1000-fold and accounts for
the 1000-fold higher MICs seen against vancomycin-resistant
bacteria.11 A subtle but important feature that emerged from
this understanding is the recognition that efforts to rationally
redesign vancomycin to directly treat such resistant bacteria,
which arise from this single atom change in a bacterial cell wall
precursor, should strive to devise compounds that not only
bind D-Ala-D-Lac, but that also maintain binding to D-Ala-D-Ala.
The altered complex of vancomycin with N-acyl-D-Ala-D-Lac
lacks the central H-bond of the D-Ala-D-Ala complex and suffers
a repulsive lone pair interaction between the vancomycin
residue 4 carbonyl and the D-Ala-D-Lac ester oxygens (Figure
Figure 2. Dual, balanced binding properties of [Ψ[CH2NH]Tpg4]-
vancomycin aglycon (6).
vancomycin aglycon (5), 6 exhibited a 40-fold increase in
affinity for D-Ala-D-Lac (4, Ka = 5.2 × 103 M−1) and a 35-fold
reduction in affinity for D-Ala-D-Ala (2, Ka = 4.8 × 103 M−1),
providing the first modified glycopeptide with dual, balanced
binding properties. Accurately reflecting these binding proper-
ties, 6 exhibited improved, but modest, antimicrobial activity
against vancomycin-resistant bacteria (VanA VRE MIC = 31
μg/mL). Thus, this removal of a single atom from the antibiotic
countered bacterial resistance that is derived from the single
atom change in the cell wall peptidoglycan precursor, provided
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the first reengineered vancomycin rationally designed to exhibit
dual D-Ala-D-Ala and D-Ala-D-Lac binding, and established the
successful foundation on which our continuing studies are
based.
Having first addressed the vancomycin analogue 6 for which
we were most confident in the projected binding characteristics,
we turned our attention to an additional key member in the
series [Ψ[C(NH)NH]Tpg4]vancomycin aglycon (7),23
replacing the residue 4 amide with the corresponding amidine
(Figure 3). Although the methylene derivative 6 exhibited the
would be valuable even if the properties of 7 had proved
disappointing. Herein, we report the total synthesis of [Ψ[C(
S)NH]Tpg4]vancomycin aglycon (8), its direct single-step
conversion to [Ψ[C(NH)NH]Tpg4]vancomycin aglycon
(7) by use of a unique AgOAc-promoted reaction, the
preparation of a series of related key compounds that also
contain the core residue 4 amidine and thioamide modifica-
tions, and details of their evaluation alongside the initial results
we communicated for 7.27
RESULTS AND DISCUSSION
■
Thionation Survey. Although not yet comprehensively
examined, efforts to selectively convert the amide linking
residues 4 and 5 to a thioamide in a vancomycin aglycon
derivative have not yet been successful in our hands.
Consequently, we surveyed progressively simpler partial
structures available from our vancomycin aglycon synthesis.
At the initiation of our efforts, we found that the residue 4
amide could be selectively converted to the corresponding
thioamide using Lawesson’s reagent within the isolated CD ring
system where it is the sterically more accessible of only two
possible amides, and that this could be further improved by
conducting the reaction on the t-butyldimethylsilyl (TBS) ether
protected phenol, providing a remarkably selective and high
yielding stage for thionation (Figure 4). At this early stage, the
Figure 3. Structures of [Ψ[C(NH)NH]Tpg4]vancomycin aglycon
(7), [Ψ[C(S)NH]Tpg4]vancomycin aglycon (8), and the potential
dual binding behavior of the amidine in 7.
desired dual binding and the expected enhancement in D-Ala-D-
Lac affinity, it also displayed an expected reduced D-Ala-D-Ala
affinity. The key feature to be addressed with [Ψ[C(
NH)NH]Tpg4]vancomycin aglycon (7) was whether the
incorporation of the residue 4 amidine could accommodate
or further enhance D-Ala-D-Lac binding by removing the
destabilizing lone pair repulsion and perhaps by serving as a H-
bond donor, while simultaneously maintaining the required
vancomycin affinity for D-Ala-D-Ala by virtue of serving as an
alternative H-bond acceptor (Figure 3). Such binding
characteristics of 7 were not easy to anticipate as it is not
clear whether the ester oxygen of D-Ala-D-Lac could actually
serve as a H-bond acceptor,24 or whether an amidine, which is
likely protonated, might remain a good H-bond acceptor for D-
Ala-D-Ala. Because the projected behavior of 7 was uncertain
and because the use of amidines as isosteres of amides in cyclic
peptides has been largely unexplored,25,26 the preparation of 7
was slated to follow that of 6 in our efforts. Key to the approach
to 7 pursued herein is the use of the residue 4 thioamide 8 for
amidine introduction in the last step, conducted on a fully
functionalized and fully deprotected vancomycin aglycon. This
strategy not only permits access to 7, but it also allows late-
stage access to a series of related key analogues including 8
itself, alternative access to our methylene derivative 6 from a
common late-stage intermediate (8), and provides authentic
samples of residue 4 thioamide and amidine derivatives of
vancomycin aglycon that will facilitate ongoing efforts on the
semisynthetic preparation of either 6 or 7. These latter two
features of the work ensured that our investment in the efforts
Figure 4. Thionation survey using Lawesson’s reagent in toluene.
CD ring system thioamide emerged as our key precursor from
which 7 and 8 were to be prepared. No efforts were made to
extensively profile the less successful reactions, and it is likely
that each would benefit from a detailed study.
With a thionation stage established and following a strategy
first introduced in our vancomycin aglycon synthesis, the
overall approach to 7 and 8 was anticipated to rely on two key
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aromatic nucleophilic substitution reactions for formation of
the 16-membered diaryl ethers in the sequential CD and DE
ring formations, a macrolactamization for closure of the 12-
membered biaryl AB ring system, and the defined order of CD,
AB, and DE ring closures to permit selective thermal
atropoisomerization of the newly formed ring systems or
their immediate precursors. Thus, in addition to any kinetic
atroposelectivity that may be achieved in each macrocyclization,
the order of ring closures follows their increasing ease of
thermal atropisomer equilibration (Ea for CD, AB, and DE ring
systems = 30.4, 25.1, and 18.7 kcal/mol, respectively),21
permitting the recycling of any newly generated unnatural
atropisomer under progressively milder thermal conditions
where any atropisomer stereochemistry already in place is not
impacted. This additional indirect control of the atropisomer
stereochemistry allows all synthetic material to be funneled into
the single atropdiastereomer found in the natural product.
CD Ring System Synthesis. As a result of these studies, we
were presented the opportunity to reexamine the CD
macrocyclization reaction enroute to the vancomycin aglycon
5, potentially improving our original synthesis and benefiting
from the experience gained with such aromatic nucleophilic
substitution reactions since its disclosure. The acyclic tripeptide
9 was prepared in three steps (72% overall) from the
constituent amino acid precursors as previously detailed
(Scheme 1).17 Treatment of 9 with K2CO3/CaCO3 (5 equiv
liberated fluoride as insoluble CaF2 preventing competitive silyl
ether deprotection and subsequent base-catalyzed retro aldol
cleavage of the newly formed CD ring system, slightly
improved the conversions (65%). Consequently, a reexamina-
tion of the impact of base (Li2CO3, Na2CO3, K2CO3, Cs2CO3,
Rb2CO3), additive (CaCO3), and solvent (THF, DMF,
DMSO) on the reaction was conducted, enlisting progressively
larger amounts of reagents (Supporting Information Table S1).
From these studies, K2CO3/CaCO3 reemerged as the most
effective combination and with the increased reagent amounts
(20 equiv each) now provided 10 as an approximate 1:1
mixture of atropisomers in yields as high as 68%. The most
significant improvements were observed by exploring the
impact of solvent while enlisting the increased reagent amounts.
As anticipated, the facility of the cyclization increased
substantially with DMSO > DMF > THF such that effective
rates of ring closure were observed at 25 °C (DMSO), 45 °C
(DMF), or 75 °C (THF). With the increased amounts of
reagents and especially with the larger amounts of CaCO3, the
reaction in anhydrous DMSO (0.005 M, 6−8 h, 25 °C) was
now unusually clean, affording 10 as a separable 1:1.2 mixture
of atropisomers in excellent yield (75−85%) free of apparent
silyl ether deprotection, retro aldol cleavage, or inadvertent
epimerization. Although the scale-up of this optimized reaction
intermittently suffered progressively lower conversions with the
reemergence of competitive desilylation and ester hydrolysis,
these were effectively addressed using either finely milled nano-
CaCO3 (avg size = 0.34 μm vs 26 μm diameter) or carefully
flame-dried reagents (20 equiv of K2CO3, 20 equiv of CaCO3, 3
wt equiv of 4 Å MS, 0.005 M DMSO, 10 h, 25 °C). The
isolated unnatural atropisomer (M)-10 was thermally equili-
brated, regenerating a 1:1 mixture of M:P atropisomers from
which additional amounts of the natural (P)-atropisomer were
isolated, and this was conducted as previously disclosed (o-
Cl2C6H4, 150 °C, 48 h, 85% recovery)17 or under improved
microwave conditions (o-Cl2C6H4, 210 °C, 12 min, 89%
recovery). Thus, the isolation and re-equilibration of the
unnatural atropisomer was used to funnel all material into the
synthetic sequence.
Scheme 1
The cyclized material (P)-10 was converted to the aryl
chloride 11 (82−89%) by reduction of the nitro group,
diazotization of the resulting aniline, and Sandmeyer
substitution following procedures previously disclosed.17
Although 11 could be converted to the corresponding
thioamide upon treatment with Lawesson’s reagent (1 equiv,
toluene, 80 °C, 8 h, 54−63%), protection of the phenol as its
TBS ether 12 (quant.), preventing its competitive reaction with
the reagent, and subsequent thionation with Lawesson’s reagent
(1 equiv, toluene, 55−60 °C, 2−3 h) provided 13 in superb
conversions (85−92%) with complete selectivity for the residue
4 amide. One of the challenges in characterizing 13 in early
studies was the presence of carbamate rotamers (1:1 to 1:8
depending on the solvent) that complicated assessment of the
reaction selectivity. Although these rotamers did not readily
coalesce with variable temperature NMR at modest temper-
atures (<60 °C) in a variety of solvents, N-Boc removal from
13 (HCO2H, CHCl3, 0 °C) provided the corresponding free
amine 14 as a single compound. Additionally and without
optimization, reduction of the thioamide 13 (H2, Ra−Ni,
MeOH, −20 to 0 °C, 3 h, 60%) in the presence of formamidine
acetate to prevent competitive aryl dechlorination and
subsequent selective phenol TBS ether deprotection of 15 (1
equiv of Bu4NF, 5 equiv of HOAc, THF, 25 °C, 0.5 h, 96%)
each, 4 Å MS, 0.005 M DMF, 45 °C, 12 h) provided 10 in good
yield (50−60%) as a separable 1:1 mixture of atropisomers,
mirroring the results originally disclosed.17 Increasing the
amount of CaCO3 (10 vs 5 equiv), which serves to scavenge
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provided 16 (eq 1).23 Alternatively, treatment of 15 with a large
excess of dimethyl dicarbonate (4 N aq NaOH−THF (1:2), 25
°C, 30 min, 91%) prior to subsequent selective phenol TBS
ether deprotection of 17 (2 equiv of Bu4NF, 5 equiv of HOAc,
THF, 25 °C, 1 h, 95%) provided 18, identical in all respects to
authentic material prepared in our earlier efforts.23 Not only
does this provide an alternative synthesis of the key
intermediate 18 employed in our synthesis of [Ψ[CH2NH]-
Tpg4]vancomycin aglycon23 (6) and provide precedent for a
late-stage conversion of 8 to 6, but the conversion of 13 to 18
also served to confirm the thionation site and macrocycle
stereochemistry.
Scheme 2
ABCD Ring System Synthesis. Completion of the ABCD
ring system required introduction of the 12-membered AB ring
system and was expected to follow protocols we first
introduced with vancomycin itself.17 Although direct Suzuki
coupling of 13 with the A-ring boronic acid 19 proceeded to
give the biaryl product 22 in good yield (69−79%) in early
efforts, the reaction proved difficult to dependably reproduce
(Scheme 2). Initially, sulfur byproducts from the thionation
reaction were suspected to shut down this reaction, but even
careful purification of 13 did not seem to alter this reaction
irreproducibility. Rather, it appeared to be directly related to
the presence of the substrate thioamide itself. Converting
thioamide 13 to the corresponding methyl thioimidate 20 (2
equiv of MeI, 4 equiv of K2CO3, acetone, 25 °C, 3 h, 92%),
protecting the coordinating thioamide as a less nucleophilic
thioimidate, provided a substrate that cleanly and reproducibly
participated in the Suzuki coupling reaction. Thus, coupling of
20 with the boronic acid 19 (2.5 equiv) catalyzed by Pd2(dba)3
(0.3 equiv) in the presence of the ligand (o-tol)3P (1.5 equiv)
in toluene/MeOH/1 M aq NaHCO3 (3/1/0.5; 0.1 M in
substrate) proceeds in 15−30 min at 80 °C, providing 21 as a
mixture of atropisomers (typically 1:1.2) in good yield (65−
80%). Like the substrate bearing a residue 4 amide for which we
first developed these reaction conditions,17 the Suzuki coupling
of 20 proceeds at remarkable rates and conversions considering
the hindered nature of the boronic acid and the electron-rich
character of the hindered aryl bromide. Liberation of the
thioamide (H2S, 2 equiv of collidine, MeOH, 25 °C, 2 h, 95%)
provided (P)-22 and its separable unnatural atropisomer. The
unnatural atropisomer (M)-22 was thermally equilibrated
under conditions that do not affect the CD atropisomer
stereochemistry (o-Cl2C6H4, 120 °C, 10−24 h or microwave at
160 °C, 30 min, 2.6−1.8:1 P:M 22 with 83−99% recovery) to
provide the thermodynamically preferred (P)-22, funneling all
material into the synthetic sequence. The use of Na2CO3 (vs
NaHCO3) in the Suzuki coupling led to lower conversions and
detectable epimerization, the inclusion of collidine in the
thioamide liberation minimized trace racemization, attempts to
equilibrate the separable thioimidate atropisomers were not
effective and led to the generation of a detectable new
diastereomer, and the overall conversions were higher if the
intermediate thioimidate atropisomers were not separated prior
to thioamide liberation where the preparative separation was
simpler and the product stability greater.
Ionic Pd-catalyzed benzyloxycarbonyl (Cbz) removal on (P)-
22 (0.3−0.5 of equiv Pd(OAc)2, 10 equiv of Et3SiH, 1−1.5
equiv of NMM, CH2Cl2, 25 °C, 24 h, 87%),28,29 silyl ether
deprotection of 23 (7 equiv of Bu4NF, 8 equiv of HOAc, THF,
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25 °C, 3 h, 95%), hydrolysis of the resulting methyl ester 24 (2
equiv of LiOH, 2:1 t-BuOH−H2O, 0 °C, 2 h, 87−100%), and
macrocyclization of the amino acid 25 (2−5 equiv of DEPBT,30
2−5 equiv of NMM, 0.002 M THF, 0−25 °C, 12−17 h, 70−
79%) provided 26, the fully functionalized ABCD ring system
bearing the core residue 4 thioamide. The use of ionic
hydrogenolysis conditions for the Cbz deprotection, which
provided 23 in yields as high as 97% (80−97%), avoided
competitive thioamide reduction observed to be problematic
with conventional reagents. Additionally, it provides an initial
and unusually stable triethylsilyl (TES) carbamate29 that
required subsequent deliberate deprotection to fully release
the free amine (saturated aq NH4Cl, 2 h). The silyl ether
removal with Bu4NF−HOAc (vs Bu4NF) prior to methyl ester
hydrolysis, rendering the methyl ester sterically more accessible,
as well as careful optimization of the hydrolysis reaction
conditions prevented competitive ester epimerization. Revers-
ing the order of ester and carbamate deprotections led to
competitive resilylation of the required free alcohol and phenol
under the ionic hydrogenolysis reaction conditions. Addition-
ally, a number of reagents were examined to effect the
macrolactamization of the AB ring system, including EDCI−
HOBt (42−50% and dr 2.5−3:1), FDPP (40−66% and dr 5:1),
and PyBop (20% and dr 1:2), which suffered comparatively
lower conversions and more extensive racemization than did 3-
(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4-one (DEPBT),30
which was superb at minimizing racemization (71% and dr
7:1).31 More subtly, conducting the macrocyclization with the
A-ring C-terminus MEM ether (vs methyl ester) accelerates the
rate of macrocyclization, diminishing competitive epimerization
of the activated carboxylic acid that accompanies ring closure,
and improves the overall conversions.17 However, it neces-
sitates a late-stage alcohol deprotection and subsequent
oxidation to provide the C-terminus carboxylic acid, a
conversion viewed as potentially problematic in the presence
of a residue 4 thioamide. Finally, the structure and
conformation of 26 initially were established by NMR,
exhibiting diagnostic NOE’s analogous to those observed with
vancomycin itself, confirming the atropisomer stereochemistry,
the cis amide between residues 5 and 6 central to the AB ring
Figure 5. X-ray structure of 2632 and its overlay with an X-ray
structure of vancomycin (1) taken from a bound complex with a
model D-Ala-D-Ala ligand (PDB code 1FVM, 1.8 Å resolution).33
Superimposition performed with the molecular modeling program
MOLOC34 and graphics generated using Chimera.35
challenging than it might appear with nearly all reagents
providing substantial amounts of racemization (e.g., EDCI−
HOAt, 0 °C, dr = 2:1, 60% 29). In earlier efforts on teicoplanin
where competitive racemization is even more problematic,18 we
found that DEPBT30 was the only reagent capable of
promoting the analogous coupling without significant race-
mization, and DEPBT (2 equiv) was also found to work well
for promoting the coupling (4 equiv of NaHCO3, THF, 0 °C,
16 h) of 27 with 28 (68%, dr > 4.5:1, 56% 29). A continued
survey of newer or alternative reagents also revealed that
commercially available propylphosphonic anhydride (T3P, 2.4
equiv)36 performed equally well (4 equiv of i-Pr2NEt, THF, 0
°C, 0.5−1.5 h), providing 29 in essentially identical conversions
(68%) and dr (4.5:1, 56% 29). In addition, the reaction
promoted by T3P proceeded at a much faster rate (1 vs 16 h)
and proved much easier to conduct because of the workup
water-soluble byproducts, suggesting that it represents an
attractive alternative reagent with coupling characteristics
similar to those of DEPBT. The final macrocyclization, an
aromatic nucleophilic substitution reaction for formation of the
diaryl ether and closure of the 16-membered DE ring system,
was effected by treatment of 29 with Cs2CO3 (3−4 equiv, 0.005
M DMSO, 9−12 h) at room temperature to provide 30 and its
DE atropisomer in superb yield (74−78%) as a separable 5−8:1
mixture of atropisomers favoring the natural (P)-isomer (58−
66%). A range of reagents were found to be effective for
promoting this cyclization (10 equiv of CsF, 50 equiv of
K2CO3−CaCO3, or 50 equiv of K2CO3) under remarkably mild
room-temperature reaction conditions when conducted in
DMSO, approaching or matching the conversions and
atropodiastereoselectivity (4−7:1) observed with Cs2CO3 (5−
5
5
system, and the location of the thioamide [e.g., C4a −H/C26−
5
6
5
6
6
H, C4a −H/C2 −H, C2 −H/C2 −H (cis amide), C5a −H/C3 −
6
6
H and C5a −H/C2 −H (atropisomer stereochemistry); vanco-
mycin numbering]. These conformational properties of 26
were not surprising because earlier studies, including those
describing the more flexible methylene analogue 6, showed that
the AB ring system and its rigid conformation bearing a cis
amide central to even its isolated structure define the
conformation characteristic of the vancomycin ABCD ring
system.17,23 The structure, stereochemistry, and conformation
of 26 were unambiguously established with a single-crystal X-
ray structure determination32 conducted on crystals grown
from acetone−hexane that overlays nearly precisely with the
ABCD ring system of vancomycin with the exception that the
CS bond length (1.66 Å) of the thioamide 26 exceeds the
CO bond length (1.23 Å) of the vancomycin amide (Figure
5).33
Introduction of the DE Ring System and Synthesis of
[Ψ[C(S)NH]Tpg4]Vancomycin Aglycon (8). This set the
stage for the introduction of the DE ring system. N-Boc
deprotection of 26 (85−98%) and subsequent coupling of the
amine 27 with 2817 provided substrate 29 for the final
cyclization (Scheme 3). This coupling reaction is more
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of reagent needed to promote the ring closure that simplified
workup of the reaction mixture. The minor unnatural DE
atropisomer was collected and, when accumulated in sufficient
quantities, thermally equilibrated (1:1, o-Cl2C6H4, microwave at
170 °C, 30 min) without impacting the AB and CD
atropisomer stereochemistry, funneling all material into the
synthetic sequence. Conversion of the aryl nitro substituent to
the corresponding chloride 32 (55−60%) proceeded smoothly
under conditions that avoid use of excess oxidant (t-BuONO)
and the resulting competitive thioamide oxidation. Compound
32 served as our storage point for the synthetic efforts from
which material was carried forward to 7 and 8 only as needed,
or diverted to allow the preparation of related key analogues
(e.g., 42−45). Protection of the secondary alcohols as their
TBS ethers (95%) and subsequent MEM ether deprotection
using B-bromocatecholborane (BCB) followed by N-Boc
reprotection provided the C-terminus primary alcohol 34
(75%), poised for oxidation to the corresponding carboxylic
acid 35 (Scheme 3).
Scheme 3
We approached this conversion with considerable concern,
recognizing that most reagents previously employed in the
vancomycin field would likely oxidize the residue 4 thioamide,
promoting its conversion to an amide. As a result, a series of
reagents were examined first with the primary alcohol derived
from 26 in hopes of identifying one capable of finessing this
conversion. These studies were largely unsuccessful, leading to
competitive or preferential thioamide oxidation. With the
benefit of results derived from concurrent efforts on the
conversion of 34 to the corresponding amidine, we came to
recognize that the thioamide of 34 is deeply embedded in the
core structure, often sterically protected from reaction. As a
result, we found that Jones oxidation37 (4 equiv of CrO3, aq
H2SO4−acetone, 12−24 h) under controlled reaction con-
ditions selectively and directly oxidizes the primary alcohol 34
to the corresponding carboxylic acid 35 (63−69%) in a single
step with a manageable amount of competitive oxidative
conversion of the thioamide to the amide (≥10:1). Somewhat
larger amounts of the amide (5−7:1) were observed with use of
less H2SO4 as an additive, but were further minimized with the
use of a larger amount of the additive. Significantly, the amide
byproduct produced proved identical in all respects to authentic
material,17 confirming the structure and stereochemistry of the
precursor and product thioamides 34 and 35. More popular
oxidizing reagents and two-step procedures typically deployed
on such occasions including Dess−Martin periodinane, Swern
oxidation, SO3−Pyr, Bu4NRuO4 (TPAP), pyridinium dichro-
mate (PDC),38 pyridinium chlorochromate (PCC),38 and 2-
iodoxybenzoic acid (IBX) provided little or no desired reaction
or complex mixtures of products even when employed in the
presence of thiourea as a diversion additive, and 2,2,6,6-
tetramethypiperidine-N-oxyl/(bisacetoxyiodo)benzene
(TEMPO−BAIB) selectively provided what appeared to be the
isolable thioamide S-oxide. The subsequent penultimate
conversion of the nitrile found in 35 to the corresponding
primary carboxamide 36 was not successful when we enlisted
the mild conditions (H2O2, K2CO3, DMSO−H2O, 25 °C)
developed for vancomycin itself due to competitive thioamide
oxidation.17 Recognizing the robust stability of vancomycin to
acidic conditions, the nitrile hydrolysis to the carboxamide was
39
accomplished by exposure to TFA−H2SO4 (25 °C) or more
8:1). The distinguishing feature of Cs2CO3 that led to its use in
our efforts going forward was its ease of implementation
(nonhygroscopic), as well as the reduced number of equivalents
simply trifluoroacetic acid39 (TFA, 25 °C, 12 h, 95%), which
also served to remove the N-terminus Boc group. Finally,
treatment of 36 with AlBr3 (250 equiv) in EtSH (25 °C, 12
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h)17 served to globally remove the four aryl methyl ethers and
two TBS ethers, providing [Ψ[C(S)NH]Tpg4]vancomycin
aglycon (8) in good conversions (63%). The physical
properties and chromatographic behavior of 8, like that of the
intermediate thioamides, proved similar to vancomycin aglycon
or the corresponding residue 4 amides, albeit characteristically
being slightly less polar in their behavior.
routes to 7, the conversions of the thioamides in 13, 26, 32, and
34 to the corresponding amidines were examined. Although
these proved instructive and provided additional key derivatives
for comparative examination (e.g., 43 and 45), establishing the
experience needed to conduct this transformation within the
chemical and structural framework of a fully functionalized and
fully deprotected vancomycin aglycon, they were not especially
predictive of what approach might be successful when applied
to 8 itself. In retrospect, we now understand that many of the
subtleties of these observations reflect the protecting group
status of the earlier stage intermediates rather than the
reactions themselves, in particular the residue 3 nitrile that
can also react with NH3. Nonetheless, the efforts with 26
proved most predictive and, without optimization, several direct
conversions of its thioamide to the corresponding amidine were
found to be effective of which the AgOAc (vs HgCl2 or
Hg(OAc)2)41 promoted reaction (10 equiv, NH3−MeOH, 25
°C) proved to be the cleanest, most direct, and most effective
(Scheme 4). The Hg(II)-promoted reactions, while often clean
Synthesis of [Ψ[C(NH)NH]Tpg4]Vancomycin Agly-
con (7). Key to the work and central to our objectives was the
conversion of a thioamide to an amidine. Optimally, we felt this
should be conducted on 8 itself requiring only one additional
step for the preparation of 7, establishing it as a late-stage
penultimate intermediate from which additional deep-seated
and site specific changes in this key region of the vancomycin
core structure could be conducted. Thus, single-step treatment
of the fully deprotected vancomycin aglycon thioamide 8 with
AgOAc40 (10 equiv) in saturated NH3−MeOH at 25 °C (12 h)
provided the amidine 7, [Ψ[C(NH)NH]Tpg4]vancomycin
aglycon (>50%), cleanly as a colorless solid that proved to be
stable to extensive handling and purification (eq 2). Although
there is no direct precedent that we are aware of detailing the
use of AgOAc for promoting direct amidine generation upon
reaction of ammonia with a thioamide, it was examined on the
basis of its well-recognized thiophilicity.40 AgOAc proved more
effective than alternative, commonly employed reagents (e.g.,
HgCl2 or Hg(OAc)2)41 whose use in protic solvents with
ammonia was found to be precluded by extensive salt
precipitation. Beyond the homogeneous reaction conditions
and central to the success with AgOAc is the ability to use a
relatively poor nucleophile (NH3) in a protic solvent (MeOH)
where the substrate is readily soluble, in a reaction conducted
on the fully functionalized and deprotected vancomycin
aglycon without the detection of reagent-derived competitive
reactions. The product amidine 7 is considerably more polar
than 5 or 8, most likely reflecting amidine protonation, is
readily soluble in H2O or H2O−MeOH but insoluble in
MeCN, and required addition of TFA to the sample before
reverse-phase high performance liquid chromatography
(HPLC) purification (5 μm C18, 0.46 × 15 cm, 30%
CH3OH/H2O−0.07% TFA, 3 mL/min, Rt = 17.9 min or 5−
20% MeCN/H2O−0.07% TFA, 3 mL/min, Rt = 21.6 min).
Although our experience in handling 7 is still limited, the
conversion of 8 to 7 is clean and complete (>85% by LCMS),
although it is conservatively reported herein as 50%. We have
occasionally experienced losses of material during HPLC
purification that may be related to additional unappreciated
features of its physical properties that are under continued
examination.
Scheme 4
and more rapid, always suffered from poor recovery due in part
to the precipitated salts that form in the reaction mixtures with
NH3. Efforts to conduct the conversion in a stepwise fashion
with NH3 displacement on the intermediate methyl thioimidate
38 were surprisingly ineffective, although more powerful
nucleophiles including hydroxylamine cleanly react with 38.
Reduction (H2, Ra−Ni, or TiCl3) of the resulting hydrox-
yamidine (amidoxime) 39, also available directly through a
41
AgOAc or HgCl2 promoted reaction of hydroxylamine with
the thioamide, provided the corresponding amidine 40, but
required controlled conditions for reduction with Ra−Ni to
avoid competitive aryl dechlorination. Significantly, analogous
thioamide functionalizations are planned with 8 itself, and the
results of such studies will be disclosed in due course.
Additional Residue 4 Thioamide and Amidine
Derivatives. Previous studies have shown that the C-terminus
carboxylic acid of vancomycin does not have an impact on D-
Ala-D-Ala binding, and it has been explored extensively as a
productive derivatization site, tolerating conversion to ester,
The simplicity of this transformation does not do justice to
the efforts that went into its adoption. Throughout the course
of our studies and many times in pursuit of establishing the
viability of earlier stage amidine introductions in alternative
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amide, and alcohol derivatives. Similarly, phenol methylation is
well tolerated, often is beneficial for imparting antimicrobial
activity against vancomycin-resistant bacteria,22,42 and does not
significantly impact model D-Ala-D-Ala or D-Ala-D-Lac binding.
Consequently, earlier stage tricyclic thioamide and amidine
derivatives were also prepared bearing a C-terminus hydroxy-
methyl group with and without the corresponding phenol
methyl ether protection and made available for examination
(Scheme 5). These earlier stage intermediates not only allowed
to provide 43 (63%), which proved surprisingly more
challenging to work with43 than either 7 or 45 (below).
However, global deprotection of the four methyl ethers in
the thioamide 42 (250 equiv of AlBr3, EtSH, 25 °C, 4 h) cleanly
provided the free phenol 44 (61−69% from 32) without
impacting the thioamide. Single-step, direct conversion of 44 to
the corresponding amidine with AgOAc (10 equiv) in saturated
NH3−MeOH (25 °C, 24 h) provided the well-behaved and
stable amidine 45 (85%), which differs from the structure of 7
only at the C-terminus (hydroxymethyl group vs carboxylic
acid). Interestingly, reverse-phase HPLC purification of 45 (5−
20% MeCN/H2O−0.7%TFA) revealed that the compound is
actually slightly more polar than the corresponding carboxylic
acid 7. Finally and in the course of these efforts, we also
established that treatment of the corresponding residue 4 amide
47 with an even larger excess of AgOAc (50 equiv) in NH3−
MeOH for a more extended reaction time (48 h, 25 °C)
provided only recovered starting material, indicating no side
reactions at sites other than the thioamide might be expected
upon exposure to the reaction conditions.
Scheme 5
Binding to Model D-Ala-D-Ala and D-Ala-D-Lac Ligands.
The binding assays were conducted at pH 5.1 in a citrate buffer
as originally disclosed by Perkins.8,44 At this pH and regardless
of any structural perturbation in a protonated amidine pKa
(12.5), the residue 4 amidine would be expected to be fully
protonated in the absence of a ligand, even more so than a
simple amine (pKa = 11.5). Similarly, the presence of the excess
citrate in the buffer insures nonspecific binding of a ligand
carboxylic acid is not observed in the vancomycin binding
pocket. Thus, although the incorporation of the amidine in the
potentially hydrophobic vancomycin binding pocket might be
expected to lower the protonated amidine pKa, and one might
be concerned with whether the residue 4 amidine may
nonspecifically bind a ligand carboxylic acid, the binding
assay conditions mitigate such concerns, providing conditions
under which to critically evaluate only the specific interaction of
ligands with the candidate analogues.
Although [Ψ[C(S)NH]Tpg4]vancomycin aglycon (8) was
prepared as the key immediate precursor to 7, it proved
especially revealing to examine its binding characteristics with
the model D-Ala-D-Ala and D-Ala-D-Lac ligands 2 and 4,
respectively. Because thioamides are regarded as weaker H-
bond acceptors than the corresponding amides, the affinity of 8
for 2 was anticipated to be reduced, whereas its potential
behavior toward 4 was more difficult to predict. As reported
earlier,27 its behavior and that of the related thioamides 42 and
44 proved stunning (Figure 6). All three thioamides failed to
bind either the model D-Ala-D-Ala or D-Ala-D-Lac ligands 2 and
4 to any appreciable extent, and all three failed to exhibit
appreciable antimicrobial activity against sensitive or vancomy-
cin-resistant bacteria. The 1000-fold loss in affinity for the D-
Ala-D-Ala ligand 2 relative to the vancomycin aglycon (5) is
both general (8, 42, and 44) and remarkable, indicating that
this simple exchange in a single atom (O→S) is sufficient to
completely disrupt binding. Although the weaker H-bonding
capability of a thioamide thiocarbonyl may contribute to this
lowered affinity, the magnitude of the loss indicates something
more fundamental is responsible. As reported earlier,27 we
suggest that both the increased bond length of the thiocarbonyl
(1.66 vs 1.23 Å, see Figure 5) and the larger van der Waals radii
of sulfur (1.80 vs 1.52 Å) are sufficient to sterically displace and
completely disrupt the intricate binding of D-Ala-D-Ala. This
behavior of all three residue 4 thioamides, which initially
the development of refined conditions for the amidine
introduction, but their examination served to establish the
generality of the amidine and thioamide biological properties.
Concurrent C-terminus MEM ether and N-terminus Boc
deprotection of 32 by treatment with 4 N HCl−dioxane (25
°C, 8 h) and subsequent residue 3 nitrile hydrolysis of 41,
enlisting TFA treatment (25 °C, 12 h) followed by a workup
with MeOH (25 °C, 1 h) to convert the intermediate
trifluoroacetoxy imidate to the primary carboxamide, provided
thioamide 42 (66%, 2 steps). We later found that omitting the
HCl-catalyzed deprotections and simply treating 32 with TFA
(25 °C, 16 h) followed by workup with MeOH (25 °C, 18 h)
provided 42 directly (69%), effecting the MEM ether and Boc
deprotections along with the nitrile conversion to the primary
carboxamide. Single-step, direct conversion of 42 to the
corresponding amidine was effected by treatment with
AgOAc (10 equiv) in saturated NH3−MeOH (25 °C, 24 h)
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ligand 2 was only approximately 2-fold less than that of the
residue 4 amides including vancomycin aglycon itself and 15-
fold higher than the corresponding methylene derivative 6,
suggesting that the residue 4 amidine functions well as a H-
bond acceptor for the amide NH in the model ligand.
Moreover, this binding affinity of 7 and the closely related
amidine 45 was maintained with the model D-Ala-D-Lac ligand
4, representing a nearly 600-fold increase in affinity relative to
the residue 4 amides including the vancomycin aglycon (5) and
a more than 10-fold increase in affinity relative to the
methylene derivative 6. Significantly, 7 and the related residue
4 amidines display effective dual, balanced binding affinity for
both model ligands (Ka 2/4 = 0.9−1.05) at a level that is within
2−3 fold that exhibited by vancomycin for D-Ala-D-Ala.
Although the behavior of 7 and the related residue 4
amidines toward the model D-Ala-D-Ala ligand 2 may not be too
surprising on the surface, it requires a presumably ligand-
induced unprotonated (vs protonated) amidine to function
effectively as a H-bond acceptor for the ligand amide NH. The
behavior of the amidines toward the D-Ala-D-Lac ligand 4 is
even more remarkable. There is no precedent on which to
suggest that the residue 4 amidine could function as such a
good H-bond donor to the ester oxygen of the D-Ala-D-Lac
ligand to achieve this level of increased affinity. Rather, we have
suggested that this is largely the result of a now stabilizing
electrostatic interaction between the protonated amidine and
the ester oxygen lone pairs27 (Figure 7). Thus, removing the
Figure 7. Proposed dual binding behavior of amidine derivatives
toward D-Ala-D-Lac and D-Ala-D-Ala.
vancomycin carbonyl oxygen atom and its destabilizing lone
pair repulsion with the D-Ala-D-Lac ester oxygen atom and
replacing it with a protonated amidine nitrogen and its now
complementary stabilizing electrostatic interaction and H-bond
donor capability reinstates essentially full binding affinity to the
altered ligand. Although our studies do not experimentally
distinguish whether the amidine is protonated or unprotonated
when binding D-Ala-D-Lac and either is feasible, an unproto-
nated amidine would offer only the stabilization of a
questionable H-bond and less or little in the way of additional
electrostatic stabilization. As a result and because the amidine
(pKa = 12.5) would be expected to be fully protonated in the
absence of a ligand under the conditions of the assay (pH =
5.1), it is unnecessary to invoke and unlikely to involve binding
of the unprotonated amidine. Beautifully, this represents a
complementary single atom exchange in the antibiotic (O→
NH) to counter a corresponding single atom exchange in the
cell wall precursors of resistant bacteria (NH→O).
Figure 6. Comparison of residue 4 amide, thioamide, and amidine
derivatives.
tempered our expectations of 7, serves as a sharp contrast to the
remarkable behavior of the residue 4 amidines detailed below.
The behavior of the residue 4 amidine derivatives proved
truly remarkable (Figure 6). Both the bond length of an
amidine (1.30 vs 1.23 Å) and the van der Waals radii of a
nitrogen atom (1.55 vs 1.52 Å) closely approximate those of an
amide carbonyl and oxygen atom, suggesting they may serve as
effective amide isosteres in peptides. The binding affinity44 of 7
and the closely related amidine 45 with the model D-Ala-D-Ala
To further support the unique binding behavior of the
residue 4 amidines and that it is directly related to their
proposed interaction with the linking heteroatoms of the D-Ala-
D-Ala and D-Ala-D-Lac ligands 2 and 4, the binding of the
amidines 7 and 45 was also examined with the ketone ligand
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3,12 lacking a linking heteroatom. The amidines were found to
bind the ketone ligand 3 with affinities of 7.4 × 103 M−1 (7)
and 8.8 × 103 M−1 (45), representing binding approximately
10-fold less effective than their binding to either 2 or 4 and
consistent with both qualitative and quantitative expectations of
the behavior of an unprotonated amidine (Figure 7).
The binding of the amidine 45 with a series of additional
ligands was also examined, and its comparison with vancomycin
aglycon (5) demonstrates that they display identical trends
(Figure 8). The ligands 48 and 49, which have been previously
Figure 8. Additional comparison ligand binding affinities of amidine
45 versus amide 5.
examined with vancomycin and related compounds,44 represent
models of an alternative, less effective vancomycin binding site
in the bacterial cell wall (48)44c and a modified peptidoglycan
precursor found in a less prevalent form of vancomycin-
resistant (VanC) bacteria (49).44e For both ligands and
precisely in line with the differences observed with the D-Ala-
D-Ala ligand 2 (2−3 fold), the amidine 45 binding was 2−3-fold
lower than that of the vancomycin aglycon. Relative to the
respective binding to D-Ala-D-Ala (2) and like 5, amidine 45
binding to the D-Ala-Gly ligand 48, lacking only the methyl
group on the terminal amino acid, is reduced 5.5-fold (vs 5.5-
fold for 5), and that of D-Ala-D-Ser 49, bearing the additional
hydroxyl group, is lowered 23-fold (vs 36-fold for 5).
Importantly and also like 5,44c no binding of amidine 45 with
the L-Ala-L-Ala ligand 50 was observed, ruling out a nonspecific
interaction with the examined ligands. This study further
establishes that the amidines (1) bind such ligands in a manner
identical to the residue 4 amides, (2) are subject to the same
structural recognition features that dominate the vancomycin
interaction with D-Ala-D-Ala and related ligands, and (3)
eliminate the possibility that the amidines may be interacting
with the ligands in a different or unique manner.
Additionally, the binding titration assay final mixture of 45
with the D-Ala-D-Lac ligand 4, containing the final 10-fold
excess of ligand 4, was monitored for >2 months (room
temperature) for evidence of either N-acylation of 45 by 4 or
hydrolysis of 4 catalyzed by 45 that conceivably could
contribute to the properties of the residue 4 amidines.
However, both 45 and ligand 4 were unchanged under these
conditions (pH 5.1), and no evidence of substrate acylation or
ligand hydrolysis was detected (MS).
Figure 9. (Top) Ball-and-stick representation of an energy-minimized
structure (MOLOC34) of 7 (C-atoms: light blue) and (Ac)2-L-Lys-D-
Ala-D-Ala (C-atoms: salmon). (Bottom) Ball-and-stick representation
of an energy-minimized structure of 7 (C-atoms: light blue) and
(Ac)2-L-Lys-D-Ala-D-Lac (C-atoms: salmon). Hydrogen atoms of all
amides are depicted in white, H-bonds are depicted as green-dotted
lines, and H-bonding distances are indicated. The corresponding H-
bond distances for vancomycin binding to (Ac)2-L-Lys-D-Ala-D-Ala
(see Figure 5) in the X-ray structure are 1.9, 1.9, 1.8, 2.0, and 1.7 Å,
respectively. Structures obtained by modification of a cocrystal
structure of vancomycin aglycon and (Ac)2-L-Lys-D-Ala-D-Ala (PDB
code: 1FVM).
indistinguishable from those observed in the X-ray structure
of vancomycin bound to the model (Ac)2-L-Lys-D-Ala-D-Ala
ligand (2) illustrated in Figure 5. Analogous efforts to model
the binding of the protonated amidine with the model (Ac)2-L-
Lys-D-Ala-D-Ala ligand (2) did not provide as productive a
complex that maintains the intricate interactions characterizing
the natural product X-ray and NMR structures.
Antimicrobial Activity. Accurately reflecting their binding
properties, each of the residue 4 amidines exhibited potent and
comparable antimicrobial activity (MIC = 0.3−0.6 μg/mL)
against VanA resistant bacteria (E. faecalis, VanA VRE), the
most stringent of vancomycin-resistant bacteria, being roughly
1000-fold more potent than vancomycin (1) and vancomycin
aglycon (5), Figure 7, and equipotent to the activity they
display against sensitive bacterial strains (MIC = 0.3−2 μg/
mL). By contrast, but also consistent with their binding
properties, the residue 4 thioamides were found to be inactive
against VanA VRE (Figure 7) as well as sensitive bacteria. The
impressive antimicrobial activity of 7 and its generality observed
with the related amidines 43 and 45 suggest that the clinical
impact of such redesigned residue 4 amidine glycopeptide
antibiotics is likely to be important, charting a rational approach
Models of Amidine Binding. Energy-minimized models of
the free base amidine binding to (Ac)2-L-Lys-D-Ala-D-Ala (2)
and the protonated amidine binding to (Ac)2-L-Lys-D-Ala-D-Lac
(4) are presented in Figure 9 along with the modeled H-bond
distances. Most notably, those of the latter are nearly
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forward in the development of antibiotics for the treatment of
vancomycin-resistant bacterial infections.
the atropisomer stereochemistry, in addition to any kinetic
atroposelectivity achieved in the cyclizations, allowed all
synthetic material to be funneled into the single atropdiaster-
eomer characterizing the natural product.
CONCLUSIONS
■
The binding affinity of 7 and the closely related residue 4
amidine 45 with the model D-Ala-D-Ala ligand 2 was found to
be only 2-fold less than that of the vancomycin aglycon (5),
indicating that the amidine functions well as a H-bond acceptor
for the amide NH in the model ligand. More remarkable, this
binding affinity was maintained with the model D-Ala-D-Lac
ligand 4, representing a nearly 600-fold increase relative to the
vancomycin aglycon (5). Thus, removing the vancomycin
carbonyl oxygen atom and its destabilizing electrostatic
interaction with the D-Ala-D-Lac ester oxygen atom and
replacing it with a protonated amidine nitrogen and its
complementary stabilizing electrostatic interaction and H-
bond donor capability reinstates essentially full binding affinity
to the altered ligand. Significantly, 7 and the related amidine 45
display dual, balanced binding affinity for both ligands (Ka 2/4
= 0.9−1.05) that is within 2−3 fold that of vancomycin for D-
Ala-D-Ala. Accurately reflecting these binding properties, each
of the residue 4 amidines exhibited potent antimicrobial activity
(MIC = 0.3−0.6 μg/mL) against VanA resistant bacteria (E.
faecalis, VanA VRE), being 1000-fold more potent than
vancomycin (1) and vancomycin aglycon (5) and equipotent
to the activity they display against sensitive bacterial strains
(MIC = 0.3−2 μg/mL). Beautifully, this represents a single
atom exchange in the antibiotic (O→NH) to counter a
corresponding single atom exchange in the cell wall precursors
of resistant bacteria (NH→O), charting a rational path forward
in the development of antibiotics for the treatment of
vancomycin-resistant bacterial infections. Further residue 4
amide modifications building off these synthetic accomplish-
ments and the remarkable behavior of 7 enlisting 8 as a key
intermediate are under investigation, efforts directed at the
development of alternative approaches to the preparation of 7
or 8 are in progress, the impact of peripheral modification of
the key amidines for further enhancing their potency are being
examined, and the results of such studies will be disclosed in
due time.46
The induced remodeling of the dipeptide terminus of
peptidoglycan cell wall precursors from D-Ala-D-Ala to D-Ala-
D-Lac in resistant bacteria with the exchange of a single atom
reduces vancomycin binding affinity for its biological target by
1000-fold, leading to a loss in biological activity. Herein, we
reported the total synthesis45 of a key vancomycin analogue
designed to exhibit dual, balanced binding to both D-Ala-D-Ala
to D-Ala-D-Lac, incorporating a complementary single atom
exchange in the residue 4 amide, [Ψ[C(NH)NH]Tpg4]-
vancomycin aglycon (7). The approach, enlisting a unique and
optimal AgOAc-promoted late-stage single-step conversion
from [Ψ[C(S)NH]Tpg4]vancomycin aglycon (8), not only
permitted access to 7 and a series of earlier stage amidines, but
it now also allows late-stage access to related analogues and
alternative access to [Ψ[CH2NH]Tpg4]vancomycin aglycon
(6) from a common late-stage intermediate. The approach
relied on two aromatic nucleophilic substitution reactions of o-
nitrofluoroaromatics for formation of the 16-membered CD/
DE diaryl ether ring systems, an effective macrolactamization
for closure of the 12-membered biaryl AB ring system, and the
defined order of CD, AB, and DE ring closures (Figure 10).
ASSOCIATED CONTENT
■
S
* Supporting Information
Full experimental details and full refs 5c and 26a. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
boger@scripps.edu
ACKNOWLEDGMENTS
■
We are especially grateful for the financial support of the
National Institutes of Health (CA041101) and the Skaggs
Institute for Chemical Biology. We wish to thank Dr. D. B.
Horne for initial improvements in the macrocyclization of 9
and the initial successful selective thionation studies on 11, Dr.
Y. Zhang for extending these studies to the thionation of 12,
Dr. J. Elsner for substantial supplies of early intermediates
prepared at mid stage of the efforts, and Dr. Raj Chadha for the
X-ray crystal structure of 26. We would like to thank Dr. H.
Gukasyan (Pfizer) for the generation and characterization of
the nanosuspension of CaCO3 in DMSO. J.G.P. is the recipient
Figure 10. Summary of synthetic strategy.
This order of ring closures follows their increasing ease of
thermal atropisomer equilibration, permitting the recycling of
any newly generated unnatural atropisomer under progressively
milder thermal conditions where the atropisomer stereo-
chemistry already set is not impacted. This indirect control of
1295
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Richardson, T. I.; Trotter, B. W.; Katz, J. L. Angew. Chem., Int. Ed.
1998, 37, 2704. See also: (c) Evans, D. A.; Barrow, J. C.; Watson, P.
S.; Ratz, A. M.; Dinsmore, C. J.; Evrard, D. A.; DeVries, K. M.; Ellman,
J. A.; Rychnovsky, S. D.; Lacour, J. J. Am. Chem. Soc. 1997, 119, 3419.
(d) Evans, D. A.; Dinsmore, C. J.; Ratz, A. M.; Evrard, D. A.; Barrow, J.
C. J. Am. Chem. Soc. 1997, 119, 3417.
of a NIH postdoctoral fellowship (CA144333, 2009−2011),
A.O. is the recipient of a JSPS fellowship (2010−2011), R.C.J.
is a Skaggs Fellow (2006−2011), and S.S. was the recipient of a
Swiss NSF postdoctoral fellowship (SNSF PBZH2-115560,
2006−2007).
(16) (a) Nicolaou, K. C.; Takayanagi, M.; Jain, N. F.; Natarajan, S.;
Koumbis, A. E.; Bando, T.; Ramanjulu, J. M. Angew. Chem., Int. Ed.
1998, 37, 2717. (b) Nicolaou, K. C.; Natarajan, S.; Li, H.; Jain, N. F.;
Hughes, R.; Solomon, M. E.; Ramanjulu, J. M.; Boddy, C. N. C.;
Takayanagi, M. Angew. Chem., Int. Ed. 1998, 37, 2708. (c) Nicolaou, K.
C.; Jain, N. F.; Natarajan, S.; Hughes, R.; Solomon, M. E.; Li, H.;
Ramanjulu, J. M.; Takayanagi, M.; Koumbis, A. E.; Bando, T. Angew.
Chem., Int. Ed. 1998, 37, 2714. (d) Nicolaou, K. C.; Mitchell, H. J.;
Jain, N. F.; Winssinger, N.; Hughes, R.; Bando, T. Angew. Chem., Int.
Ed. 1999, 38, 240.
(17) (a) Boger, D. L.; Miyazaki, S.; Kim, S. H.; Wu, J. H.; Loiseleur,
O.; Castle, S. L. J. Am. Chem. Soc. 1999, 121, 3226. (b) Boger, D. L.;
Miyazaki, S.; Kim, S. H.; Wu, J. H.; Castle, S. L.; Loiseleur, O.; Jin, Q.
J. Am. Chem. Soc. 1999, 121, 10004.
(18) (a) Boger, D. L.; Kim, S. H.; Miyazaki, S.; Strittmatter, H.;
Weng, J.-H.; Mori, Y.; Rogel, O.; Castle, S. L.; McAtee, J. J. J. Am.
Chem. Soc. 2000, 122, 7416. (b) Boger, D. L.; Kim, S. H.; Mori, Y.;
Weng, J.-H.; Rogel, O.; Castle, S. L.; McAtee, J. J. J. Am. Chem. Soc.
2001, 123, 1862.
REFERENCES
■
(1) McCormick, M. H.; Stark, W. M.; Pittenger, G. E.; Pittenger, R.
C.; McGuire, J. M. Antibiot. Annu. 1955−1956, 606.
(2) (a) Harris, C. M.; Kopecka, H.; Harris, T. M. J. Am. Chem. Soc.
1983, 105, 6915. See also: (b) Harris, C. M.; Harris, T. M. J. Am.
Chem. Soc. 1982, 104, 4293. (c) Williamson, M. P.; Williams, D. H. J.
Am. Chem. Soc. 1981, 103, 6580. (d) Sheldrick, G. M.; Jones, P. G.;
Kennard, O.; Williams, D. H.; Smith, G. A. Nature 1978, 271, 223.
(e) Williams, D. H.; Kalman, J. R. J. Am. Chem. Soc. 1977, 99, 2768.
(3) Glycopeptide Antibiotics; Nagarajan, R., Ed.; Marcel Dekker: New
York, 1994.
(4) Reviews: (a) Kahne, D.; Leimkuhler, C.; Lu, W.; Walsh, C. T.
Chem. Rev. 2005, 105, 425. (b) Hubbard, B. K.; Walsh, C. T. Angew.
Chem., Int. Ed. 2003, 42, 730.
(5) (a) CDC. National Nosocomial Infections Surveillance (NNIS)
System Report, Data Summary from January 1992 Through June
2004, Issued October 2004. Am. J. Infect. Control 2004, 32, 470.
(b) Laxminarayan, R. Antibiotic Resistance: The Unfolding Crisis. In
Extending the Cure, Policy Responses to the Growing Treat of Antibiotic
Resistance; Laxminarayan, R., Malani, A., Howard, D., Smith, D. L.,
Eds.; Resources for the Future: 2007; Chapter 1, pp 25−37.
(c) Klevens, R. M.; et al. J. Am. Med. Assoc. 2007, 298, 1763.
(d) Walsh, C. T.; Fischbach, M. A. Sci. Am. 2009, 301, 44.
(6) Reviews: (a) Malabarba, A.; Nicas, T. I.; Thompson, R. C. Med.
Res. Rev. 1997, 17, 69. (b) Malabarba, A.; Ciabatti, R. Curr. Med. Chem.
2001, 8, 1759. (c) Van Bambeke, F. V.; Laethem, Y. V.; Courvalin, P.;
Tulkens, P. M. Drugs 2004, 64, 913.
(19) Crowley, B. M.; Mori, Y.; McComas, C. C.; Tang, D.; Boger, D.
L. J. Am. Chem. Soc. 2004, 126, 4310.
(20) (a) Garfunkle, J.; Kimball, F. S.; Trzupek, J. D.; Takazawa, S.;
Shimamura, H.; Tomishima, M.; Boger, D. L. J. Am. Chem. Soc. 2009,
131, 16036. (b) Shimamura, H.; Breazzano, S. P.; Garfunkle, J.;
Kimball, F. S.; Trzupek, J. D.; Boger, D. L. J. Am. Chem. Soc. 2010, 132,
7776. (c) Breazzano, S. P.; Boger, D. L. J. Am. Chem. Soc. 2011, 133,
18495.
(21) (a) Boger, D. L.; Weng, J.-H.; Miyazaki, S.; McAtee, J. J.; Castle,
S. L.; Kim, S. H.; Mori, Y.; Rogel, O.; Jin, Q. J. Am. Chem. Soc. 2000,
122, 10047. (b) Boger, D. L.; Castle, S. L.; Miyazaki, S.; Wu, J. H.;
Beresis, R. T.; Loiseleur, O. J. Org. Chem. 1999, 64, 70. (c) Boger, D.
L.; Miyazaki, S.; Loiseleur, O.; Beresis, R. T.; Castle, S. L.; Wu, J. H.;
Jin, Q. J. Am. Chem. Soc. 1998, 120, 8920. (d) Boger, D. L.; Beresis, R.
T.; Loiseleur, O.; Wu, J. H.; Castle, S. L. Bioorg. Med. Chem. Lett. 1998,
8, 721. (e) Boger, D. L.; Borzilleri, R. M.; Nukui, S.; Beresis, R. T. J.
Org. Chem. 1997, 62, 4721. (f) Boger, D. L.; Loiseleur, O.; Castle, S.
L.; Beresis, R. T.; Wu, J. H. Bioorg. Med. Chem. Lett. 1997, 7, 3199.
(g) Boger, D. L.; Zhou, J.; Borzilleri, R. M.; Nukui, S.; Castle, S. L. J.
Org. Chem. 1997, 62, 2054. (h) Boger, D. L.; Borzilleri, R. M.; Nukui,
S. J. Org. Chem. 1996, 61, 3561. (i) Boger, D. L.; Borzilleri, R. M.;
Nukui, S. Bioorg. Med. Chem. Lett. 1995, 5, 3091. (j) Boger, D. L.;
Borzilleri, R. M. Bioorg. Med. Chem. Lett. 1995, 5, 1187. (k) Boger, D.
L.; Nomoto, Y.; Teegarden, B. R. J. Org. Chem. 1993, 58, 1425.
(l) Boger, D. L.; Yohannes, D. J. Org. Chem. 1990, 55, 6000.
(22) (a) Crane, C. M.; Pierce, J. G.; Leung, S. S. F.; Tirado-Rives, J.;
Jorgensen, W. L.; Boger, D. L. J. Med. Chem. 2010, 53, 7229.
(b) Crane, C. M.; Boger, D. L. J. Med. Chem. 2009, 52, 1471.
(c) McComas, C. C.; Crowley, B. M.; Hwang, I.; Boger, D. L. Bioorg.
Med. Chem. Lett. 2003, 13, 2933. (d) McAtee, J. J.; Castle, S. L.; Jin,
Q.; Boger, D. L. Bioorg. Med. Chem. Lett. 2002, 12, 1319.
(23) Crowley, B. M.; Boger, D. L. J. Am. Chem. Soc. 2006, 128, 2885.
(24) Molcanov, K.; Kojic-Prodic, B.; Raos, N. Acta Crystallogr. 2004,
B60, 424.
(7) (a) Sussmuth, R. D. ChemBioChem 2002, 3, 295. (b) Gao, Y. Nat.
Prod. Rep. 2002, 19, 100. (c) Walker, S.; Chen, L.; Helm, J.; Hu, Y.;
Rew, Y.; Shin, D.; Boger, D. L. Chem. Rev. 2005, 105, 449. (d) von
̈
Nussbaum, F.; Brands, M.; Hinzen, B.; Weigand, S.; Habich, D. Angew.
̈
Chem., Int. Ed. 2006, 45, 5072.
(8) (a) Nieto, M.; Perkins, H. R. Biochem. J. 1971, 123, 789. Review:
(b) Perkins, H. R. Pharmacol. Ther. 1982, 16, 181.
(9) (a) Williams, D. H.; Williamson, M. P.; Butcher, D. W.;
Hammond, S. J. J. Am. Chem. Soc. 1983, 105, 1332. Review:
(b) Williams, D. H.; Bardsley, B. Angew. Chem., Int. Ed. 1999, 38, 1172.
X-ray: (c) Schaefer, M.; Schneider, T. R.; Sheldrick, G. M. Structure
1996, 4, 1509.
(10) For lead references, see: (a) Koteva, K.; Hong, H.-J.; Wang, X.
D.; Nazi, I.; Hughes, D.; Naldrett, M. J.; Buttner, M. J.; Wright, G. D.
Nat. Chem. Biol. 2010, 6, 327. (b) Hong, H.-J.; Hutchings, M. I.;
Buttner, M. J. Adv. Exp. Med. Biol. 2008, 631, 200.
(11) (a) Bugg, T. D. H.; Wright, G. D.; Dutka-Malen, S.; Arthur, M.;
Courvalin, P.; Walsh, C. T. Biochemistry 1991, 30, 10408. Reviews:
(b) Walsh, C. T. Science 1993, 261, 308. (c) Walsh, C. T.; Fisher, S. L.;
Park, I.-S.; Prahalad, M.; Wu, Z. Chem. Biol. 1996, 3, 21. (d) Lessard, I.
A.; Walsh, C. T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11028.
(e) Healy, V. L.; Lessard, I. A.; Roper, D. I.; Knox, J. R.; Walsh, C. T.
Chem. Biol. 2000, 7, R109.
(12) McComas, C. C.; Crowley, B. M.; Boger, D. L. J. Am. Chem. Soc.
2003, 125, 9314.
(25) (a) Moser, H.; Fliri, A.; Steiger, A.; Costello, G.; Schreiber, J.;
Eschenmoser, A. Helv. Chim. Acta 1986, 69, 1224. (b) Jones, R. C. F.;
Ward, G. J. Tetrahedron Lett. 1988, 29, 3853.
(13) Morgan, B. P.; Scholtz, J. M.; Ballinger, M. D.; Zipkin, I. D.;
Bartlett, P. A. J. Am. Chem. Soc. 1991, 113, 297. References cited
therein.
(26) (a) Kamenecka, T. M.; et al. Bioorg. Med. Chem. Lett. 2004, 14,
2323. (b) Inokuchi, E.; Oishi, S.; Kubo, T.; Ohno, H.; Shimura, K.;
Matsuoka, M.; Fujii, N. ACS Med. Chem. Lett. 2011, 2, 477.
(c) Inokuchi, E.; Yamada, A.; Hozumi, K.; Tomita, K.; Oishi, S.;
Ohno, H.; Nomizu, M.; Fujii, N. Org. Biomol. Chem. 2011, 9, 3421.
(27) Xie, J.; Pierce, J. G.; James, R. C.; Okano, A.; Boger, D. L. J. Am.
Chem. Soc. 2011, 133, 13946.
(14) Reviews: (a) Evans, D. A.; DeVries, K. M. Drugs Pharm. Sci.
1994, 63, 63. (b) Nicolaou, K. C.; Boddy, C. N. C.; Brase, S.;
Winssinger, N. Angew. Chem., Int. Ed. 1999, 38, 2096. (c) Boger, D. L.
Med. Res. Rev. 2001, 21, 356.
(15) (a) Evans, D. A.; Wood, M. R.; Trotter, B. W.; Richardson, T. I.;
Barrow, J. C.; Katz, J. L. Angew. Chem., Int. Ed. 1998, 37, 2700.
(b) Evans, D. A.; Dinsmore, C. J.; Watson, P. S.; Wood, M. R.;
1296
dx.doi.org/10.1021/ja209937s | J. Am. Chem.Soc. 2012, 134, 1284−1297

Journal of the American Chemical Society
Article
(28) Coleman, R. S.; Shah, J. A. Synthesis 1999, 1399.
(29) Sakaitani, M.; Ohfune, Y. J. Org. Chem. 1990, 55, 870.
(30) (a) Fan, C.-X.; Hao, X.-L.; Ye, Y.-H. Synth. Commun. 1996, 26,
1455. (b) Li, H.; Jiang, X.; Ye, Y.-H.; Fan, C.; Romoff, T.; Goodman,
M. Org. Lett. 1999, 1, 91.
(31) (a) Jiang, W.; Wanner, J.; Lee, R. J.; Bounaud, P.-Y.; Boger, D. L.
J. Am. Chem. Soc. 2002, 124, 5288. (b) Jiang, W.; Wanner, J.; Lee, R. J.;
Bounaud, P.-Y.; Boger, D. L. J. Am. Chem. Soc. 2003, 125, 1877.
(c) Shin, D.; Rew, Y.; Boger, D. L. Proc. Natl. Acad. Sci. U.S.A. 2004,
101, 11977. (d) Nam, J.; Shin, D.; Rew, Y.; Boger, D. L. J. Am. Chem.
Soc. 2007, 129, 8747. (e) Boger, D. L.; Colletti, S. L.; Honda, T.;
Menezes, R. F. J. Am. Chem. Soc. 1994, 116, 5607.
zotriazole; HOBt, 1-hydroxybenzotriazole; IBX, 2-iodoxybenzoic acid;
MS, molecular sieves; NMM, N-methylmorpholine; NMP, N-
methylpyrrolidinone; PyBop, (benzotriazol-1-yl)tripyrrolidinophos-
phonium hexafluorophosphate; Pyr, pyridine; Ra−Ni, Raney nickel;
T3P, propylphosphonic anhydride; TBS, t-butyldimethylsilyl;
TEMPO, 2,2,6,6-tetramethypiperidine-1-oxyl; TES, triethylsilyl; TFA,
trifluoroacetic acid; THF, tetrahydrofuran.
(32) The structure, stereochemistry, and conformation of 26 were
established with a single-crystal X-ray structure determination (CCDC
837960).
(33) Nitanai, Y.; Kikuchi, T.; Kakoi, K.; Hanamaki, S.; Fujisawa, I.;
Aoki, K. J. Mol. Biol. 2009, 5, 1422.
(34) Gerber, P. R.; Mueller, K. J. Comput.-Aided Mol. Des. 1995, 9,
251.
(35) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.;
Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. J. Comput. Chem. 2004,
25, 160.
(36) (a) Wissmann, H.; Kleiner, H.-J. Angew. Chem., Int. Ed. Engl.
1980, 19, 133. (b) Dunetz, J. A.; Xiang, Y.; Baldwin, A.; Ringling, J.
Org. Lett. 2011, 13, 5048.
(37) (a) Bowden, K.; Heilbron, I. M.; Jones, E. R. H.; Weedon, B. C.
L. J. Chem. Soc. 1946, 39. (b) Bowers, A.; Halsall, T. G.; Jones, E. R.
H.; Lemin, A. J. J. Chem. Soc. 1953, 2548.
(38) (a) Corey, E. J.; Suggs, J. W. Tetrahedron Lett. 1975, 2647.
(b) Corey, E. J.; Boger, D. L. Tetrahedron Lett. 1978, 2461. (c) Corey,
E. J.; Schmidt, G. Tetrahedron Lett. 1979, 399.
(39) Moorthy, J. N.; Singhal, N. J. Org. Chem. 2005, 70, 1926.
(40) (a) Shibuya, I.; Taguchi, Y.; Tsuchiya, T.; Oishi, A.; Katoh, E.
Bull. Chem. Soc. Jpn. 1994, 67, 3048. (b) Cacchi, S.; La Torre, F.;
Misiti, D. Chem. Ind. 1978, 669. (c) Corey, E. J.; Boger, D. L.
Tetrahedron Lett. 1978, 5.
(41) (a) Marchand-Brynaert, J.; Moya-Portuguez, M.; Huber, I.;
Ghosez, L. J. Chem. Soc., Chem. Commun. 1983, 818. (b) Sauve, G.;
Rao, V. S.; Lajoie, G.; Belleau, B. Can. J. Chem. 1985, 63, 3089.
(c) Bock, M. G.; DiPardo, R. M.; Evans, B. E.; Rittle, K. E.; Freidinger,
R. M.; Chang, R. S. L.; Lotti, V. J. J. Med. Chem. 1988, 31, 264.
(42) (a) Leung, S. S. F.; Tirado-Rives, J.; Jorgensen, W. L. Bioorg.
Med. Chem. 2009, 17, 5874. (b) Leung, S. S. F.; Tirado-Rives, J.;
Jorgensen, W. L. Bioorg. Med. Chem. Lett. 2009, 19, 1236.
(43) Samples of 43, even after repeated purification, can be isolated
pure, but re-equilibrate (<1 h) to three easily separable (HPLC)
interconverting components (2:1:1), each of which display the same
molecular weight: ESI-TOF HRMS m/z 1184.39 (M+ + H,
C57H63Cl2N9O15 requires 1184.39); see the Supporting Information.
Binding studies conducted with this equilibrating constitutional,
configurational, or conformational isomer mixture exhibited Ka’s of
3.9 × 104 M−1 (2) and 4.1 × 104 M−1 (4).
(44) UV-difference titration assays were run as previously described:
(a) Nieto, M.; Perkins, H. R. Biochem. J. 1971, 124, 845. (b) Nieto, M.;
Perkins, H. R. Biochem. J. 1971, 123, 773. (c) For D-Ala-Gly and L-Ala-
L-Ala, see: Nieto, M.; Perkins, H. R. Biochem. J. 1971, 123, 789.
(d) Perkins, H. R. Biochem. J. 1969, 111, 195. (e) For D-Ala-D-Ser, see:
van Wageningen, A. M. A.; Staroske, T.; Williams, D. H. Chem.
Commun. 1998, 1171.
(45) Szpilman, A. M.; Carreira, E. Angew. Chem., Int. Ed. 2010, 49,
9592.
(46) Abbreviations: BCB, B-bromocatecholborane; Boc, t-butylox-
ycarbonyl; Cbz, benzyloxycarbonyl or carboxybenzyl; dba, dibenzyli-
deneacetone; DEPBT, 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-
4-one; DMAP, 4-(dimethylamino)pyridine; DMF, N,N-dimethylfor-
mamide; DMSO, dimethylsulfoxide; EDCI, 1-[3-(dimethylamino)
propyl]-3-ethylcarbodiimide hydrochloride; FDPP, pentafluorophenyl
diphenylphosphinate; HATU, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium hexafluorophosphate; HOAt, 1-hydroxy-7-azaben-
1297
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