Total Syntheses and Initial Evaluation of [Ψ[C(=S)NH]Tpg4]vancomycin, [Ψ[=NH)NH]Tpg4]vancomycin, [Ψ[CH2NH]Tpg4]vancomycin, and Their (4-Chlorobiphenyl)methyl Derivatives: Synergistic Binding Pocket and Peripheral Modifications for the Glycopeptide Antibiotics

Article abstract of DOI:10.1021/jacs.5b01008

Full details of studies are disclosed on the total syntheses of binding pocket analogues of vancomycin bearing the peripheral l-vancosaminyl-1,2-d-glucosyl disaccharide that contain changes to a key single atom in the residue-4 amide (residue-4 carbonyl O → S, NH, H₂) designed to directly address the underlying molecular basis of resistance to vancomycin. Also disclosed are studies piloting the late-stage transformations conducted on the synthetically more accessible C-terminus hydroxymethyl aglycon derivatives and full details of the peripheral chlorobiphenyl functionalization of all of the binding-pocket-modified vancomycin analogues designed for dual d-Ala-d-Ala/d-Ala-d-Lac binding. Their collective assessment indicates that combined binding pocket and chlorobiphenyl peripherally modified analogues exhibit a remarkable spectrum of antimicrobial activity (VSSA, MRSA, and VanA and VanB VRE) and impressive potencies against both vancomycin-sensitive and vancomycin-resistant bacteria (MICs = 0.06-0.005 and 0.5-0.06 μg/mL for the amidine and methylene analogues, respectively) and likely benefit from two independent and synergistic mechanisms of action, only one of which is dependent on d-Ala-d-Ala/d-Ala-d-Lac binding. Such analogues are likely to display especially durable antibiotic activity that is not prone to rapidly acquired clinical resistance. (Chemical Equation Presented).

Full text of DOI:10.1021/jacs.5b01008

Article
pubs.acs.org/JACS
Total Syntheses and Initial Evaluation of
[Ψ[C(S)NH]Tpg4]vancomycin, [Ψ[C(NH)NH]Tpg4]vancomycin,
[Ψ[CH2NH]Tpg4]vancomycin, and Their (4-Chlorobiphenyl)methyl
Derivatives: Synergistic Binding Pocket and Peripheral Modifications
for the Glycopeptide Antibiotics
Akinori Okano, Atsushi Nakayama, Kejia Wu, Erick A. Lindsey, Alex W. Schammel, Yiqing Feng,
Karen C. Collins, 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: Full details of studies are disclosed on the total syntheses of
binding pocket analogues of vancomycin bearing the peripheral L-vancosaminyl-
1,2-D-glucosyl disaccharide that contain changes to a key single atom in the
residue-4 amide (residue-4 carbonyl O → S, NH, H2) designed to directly
address the underlying molecular basis of resistance to vancomycin. Also
disclosed are studies piloting the late-stage transformations conducted on the
synthetically more accessible C-terminus hydroxymethyl aglycon derivatives and
full details of the peripheral chlorobiphenyl functionalization of all of the
binding-pocket-modified vancomycin analogues designed for dual D-Ala-D-Ala/D-
Ala-D-Lac binding. Their collective assessment indicates that combined binding
pocket and chlorobiphenyl peripherally modified analogues exhibit a remarkable
spectrum of antimicrobial activity (VSSA, MRSA, and VanA and VanB VRE) and
impressive potencies against both vancomycin-sensitive and vancomycin-resistant
bacteria (MICs = 0.06−0.005 and 0.5−0.06 μg/mL for the amidine and
methylene analogues, respectively) and likely benefit from two independent and synergistic mechanisms of action, only one of
which is dependent on D-Ala-D-Ala/D-Ala-D-Lac binding. Such analogues are likely to display especially durable antibiotic activity
that is not prone to rapidly acquired clinical resistance.
antibiotic classes, although a time may come when this will
no longer be the case. More pressing is the emergence of
VRSA, which has already acquired resistance to other common
classes of antibiotics. Treatment options in such cases are
expected to be limited, and outside the new-generation
glycopeptide antibiotics, these presently include antibiotics
known to easily evoke resistance (linezolide, daptomycin).10,11
As a result, these latter antibiotics have been designated or
recommended for use as “reserve antibiotics”, ones that should
be employed sparingly to preserve their effectiveness as drugs
of last resort against intractable infections. This has intensified
interest in the development of alternative treatments for
resistant pathogens that display the remarkable clinical
durability of vancomycin.1,12,13
INTRODUCTION
■
The glycopeptide antibiotics are among the most important
class of drugs used in the treatment of resistant bacterial
infections.1 Vancomycin,2 teicoplanin,3 and a set of recently
approved semisynthetic derivatives, including oritavancin
(August 2014),4 dalbavancin (May 2014),5 and telavancin
(September 2009),6 are widely or increasingly used to treat
clinically refractory and resistant bacterial infections. The
parent member of this group, vancomycin (1), was disclosed
in 19562 and introduced into the clinic in 1958, although its
structure was only established 25−30 years later (Figure 1).7
After more than 50 years of clinical use and even with the
additional widespread use of glycopeptide antibiotics for
agricultural livestock (avoparcin), worldwide observation of
vancomycin-resistant pathogens has only slowly emerged. This
was first restricted to vancomycin-resistant enterococci (VRE),
initially detected in 1987 after 30 years of clinical use,8 but
recently includes the more feared emergence of vancomycin-
resistant Staphylococcus aureus (VRSA). which was first detected
in 2002.9 In spite of the increasing prevalence of VRE, such
infections presently remain sensitive to other common
The clinical durability can be attributed to several
complementary features of vancomycin that result in inhibition
of bacterial cell wall biosynthesis and integrity.14 Foremost of
the features responsible for this durability is its primary
Received: January 29, 2015
© XXXX American Chemical Society
A
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that are not yet recognized), it is most revealing that the
primary mechanism of clinical resistance to the glycopeptide
antibiotics (VanA and VanB) was transferred to pathogenic
bacteria from nonpathogenic vancomycin-producing organisms
that use this inducible mechanism to protect themselves during
vancomycin production.16 This underscores the fact that
pathogenic bacteria have not yet independently evolved
effective resistance mechanisms to the glycopeptide antibiotics
even after more than 50 years of widespread use,17 suggesting
that fundamental solutions to VanA and VanB resistance may
provide durable antibiotics with clinical lifetimes lasting 50
more years.
Because of their structural complexity, essentially all
analogues of the glycopeptide antibiotics consist of semi-
synthetic derivatives of the natural products obtained by
chemical, enzymatic, or mutasynthesis approaches.1,12 The
most significant of the modifications introduce peripheral
hydrophobic groups, and these are found in each of the
clinically approved semisynthetic derivatives (Figure 2). For
both dalbavancin and telavancin, the long hydrophobic alkyl
chains are thought to provide selective membrane anchoring
properties and to promote antibiotic dimerization without
impacting the binding affinity to the primary biological target D-
Ala-D-Ala.18 It is possible that such semisynthetic changes to the
glycopeptide antibiotics also avoid bacterial sensing of the
antibiotic challenge, and this may account for their VanB VRE
activity first observed with teicoplanin.19 Additionally,
telavancin has been shown to function not only through the
traditional mechanism of inhibition of cell wall synthesis by
binding of D-Ala-D-Ala but also through disruption of bacterial
membrane integrity, a mechanism typically not observed for the
glycopeptide antibiotics.20
One of the most widely recognized modifications is the 4-
chlorobiphenyl (4-CBP) substitution of a peripheral carbohy-
drate. This has been examined at range of positions in a variety
of glycopeptide antibiotics, most notably in oritavancin,4 the N-
(4-chlorobiphenyl)methyl derivative of chloroeremomycin, and
with vancomycin itself (CBP-vancomycin).21 In addition to
perhaps promoting antibiotic dimerization, membrane anchor-
ing, disruption of bacterial membrane integrity, and potentially
avoiding bacterial sensing of the antibiotic challenge, the unique
placement of the 4-chlorobiphenyl substituent introduces or
potentiates a second mechanism of action. The direct inhibition
of transglycosylases mediated by the modified carbohydrate has
been identified as a second, now effective, mechanism by which
oritavancin exhibits antimicrobial activity.22,23 Regardless of the
origin of the effects, such derivatives often increase the
antibiotic potency as much as 100-fold. While increasing
bacterial sensitivity to the antibiotics, VanA vancomycin-
resistant bacterial strains (minimum inhibitory concentration
(MIC) = ca. 10 μg/mL) remain 1000-fold less sensitive than
susceptible strains (MIC = ca. 0.01 μg/mL). This suggested to
us that combining such peripheral hydrophobic substitutions
with vancomycin binding pocket modifications that maintain D-
Ala-D-Ala binding and reinstate binding to D-Ala-D-Lac would
further increase their antimicrobial activity against not only
vancomycin-sensitive but also vancomycin-resistant bacteria to
truly remarkable levels.
Figure 1. Structures of vancomycin (1) and teicoplanin.
biological target (binding to D-Ala-D-Ala). This target is not
only unique to bacteria but also a structural component of the
bacterial cell wall and a substrate for an enzymatic reaction. It is
not a protein or nucleic acid target, and as a consequence, it is
not subject to alteration by genetic mutation. Moreover, the
ramifications of additional candidate binding sites within the
bacterial cell wall (not only D-Ala-D-Ala but also D-Ala-Gly and
Gly-Gly) have yet to be defined. Vancomycin’s primary
mechanism of action involves sequestration of the substrate
(D-Ala-D-Ala) for an essential late-stage enzyme (trans-
peptidase)-catalyzed reaction needed for peptidoglycan cross-
linking and bacterial cell wall maturation. However, it is
thought also to inhibit transglycosylase-catalyzed incorporation
of lipid intermediate II into the repeating polysaccharide
backbone of the bacterial cell wall. With this second mechanism
of action for vancomycin, it is not yet clear whether this
involves direct binding of the appended disaccharide to the
enzyme active site or requires binding to cell wall sites (e.g., D-
Ala-D-Ala, D-Ala-Gly, or Gly-Gly) that contribute to its
localization and subsequent indirect enzyme inhibition. Since
there may be two or more mechanisms of action that contribute
to the inhibition of bacterial cell wall maturation by
vancomycin, full bacterial resistance may require statistically
unlikely simultaneous changes to each to overcome all of the
contributing mechanisms. Just as importantly, the site of action
is at the bacterial cell wall surface and not at an intracellular
target. As a result, no bacterial cell wall penetration or import
mechanism is needed, and this allows vancomycin to avoid the
common resistance mechanisms mediated by efflux pumps,
blocked transport, and deactivation by cytosolic metabolic
enzymes.15 Regardless of the origin (and it is likely there are
additional features contributing to the durability of vancomycin
In an extension of work first directed at the total syntheses of
the naturally occurring glycopeptide antibiotics,24−27 we
recently described studies of the binding pocket redesign of
vancomycin14 that were the first to directly address the
underlying molecular basis of clinical resistance to vancomy-
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resistance (VanA and VanB) acquired from nonpathogenic
vancomycin-producing organisms16 also highlighted that such
modifications of the vancomycin binding pocket must target
compounds that not only establish binding to D-Ala-D-Lac but
also maintain D-Ala-D-Ala binding. This not only ensures that
they would display antimicrobial activity against vancomycin-
resistant bacteria (VanA and VanB) but additionally assures
that they would remain active against vancomycin-sensitive
bacteria.
Our initial studies provided [Ψ[CH2NH]Tpg4]vancomycin
aglycon (5),31 which displayed such dual binding properties by
virtue of removal of the lone-pair repulsion between the
vancomycin residue-4 carbonyl and D-Ala-D-Lac ester oxygens.
This change reinstated commensurate activity against VanA
VRE, validated the opportunities of the approach, and entailed
removal of a single atom from the vancomycin binding pocket
(Figure 3). These efforts were followed by the total synthesis of
Figure 2. Clinically approved semisynthetic glycopeptide antibiotics.
cin.28,29 In clinically resistant organisms (VanA and VanB),
synthesis of the bacterial cell wall precursors lipid intermediate
I and II continue complete with installation of their pendant N-
terminus D-Ala-D-Ala, but resistant bacteria sense the antibiotic
challenge.19 Through the use of a two-component cell surface
receptor sensing and subsequent intracellular signaling
system,19 they initiate a late-stage remodeling of their
peptidoglycan termini from D-Ala-D-Ala to D-Ala-D-Lac28 to
avoid the action of the antibiotic. The vancomycin binding
affinity for this altered ligand is reduced 1000-fold,28 resulting
in a corresponding 1000-fold loss in antimicrobial activity. The
destabilized binding to D-Ala-D-Lac is due to a combination of
the loss of a hydrogen bond central to binding of the ligand to
the antibiotic (10-fold) and an even more significant
destabilizing lone-pair repulsion between the vancomycin
residue-4 carbonyl and D-Ala-D-Lac ester oxygens (100-
fold).30 The elucidation of this inducible mechanism of
Figure 3. Vancomycin aglycon residue-4 modifications and dual
binding behavior of amidine 4.
[Ψ[C(NH)NH]Tpg4]vancomycin aglycon (4),32 providing
a modified antibiotic that not only maintains vancomycin’s
ability to bind the unaltered peptidoglycan D-Ala-D-Ala but also
binds the altered ligand D-Ala-D-Lac just as effectively by virtue
of its ability to serve as either a hydrogen-bond donor (for D-
Ala-D-Lac) or a hydrogen-bond acceptor (for D-Ala-D-Ala).
Whereas the former entails binding of the protonated amidine
with D-Ala-D-Lac and replaces the destabilizing carbonyl lone-
pair interaction with the ester oxygen lone pair with a
stabilizing electrostatic interaction and perhaps a reversed
hydrogen bond, the latter entails binding of D-Ala-D-Ala with
the unprotonated amidine serving as a hydrogen-bond acceptor
(Figure 3).33 Not only did amidine 4 display balanced binding
affinities for both target ligands within 2-fold of that exhibited
C
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by vancomycin aglycon with D-Ala-D-Ala, but it also exhibited
effective antimicrobial activity against VanA VRE, being
equipotent to the activity that vancomycin displays against
sensitive bacterial strains. These latter studies represented the
replacement of a single atom in the binding pocket of the
antibiotic aglycon (O → NH) to counter a complementary
exchange in the cell wall precursors of resistant bacteria (NH →
O). Just as remarkable, we established that [Ψ[C(S)NH]-
Tpg4]vancomycin aglycon (3), which served as the penultimate
precursor to 4, fails to bind D-Ala-D-Ala or D-Ala-D-Lac to any
appreciable extent and is inactive against both vancomycin-
sensitive and vancomycin-resistant bacteria (Figure 3).32 The
expectedly benign conversion of the residue-4 amide to a
thioamide with the exchange of a single atom in the binding
pocket (O → S) proved sufficient to completely disrupt ligand
binding. We attributed this loss in affinity largely to the
increased thiocarbonyl bond length and size of the sulfur atom,
which are sufficient to sterically displace the ligand out of the
binding pocket and completely disrupt the intricate binding of
D-Ala-D-Ala. Significantly, the comparison of 3 with 4
highlighted just how remarkable the behavior of amidine 4 is.
Herein we report full details of the recently disclosed34 total
syntheses of [Ψ[C(NH)NH]Tpg4]vancomycin, [Ψ[C(
S)NH]Tpg4]vancomycin, and their (4-chlorobiphenyl)methyl
derivatives and previously unreported studies first developed
with their corresponding synthetic C-terminus hydroxymethyl
precursors. We also report new efforts on the extension of these
studies to the total syntheses of [Ψ[CH2NH]Tpg4]vancomycin
and its corresponding (4-chlorobiphenyl)methyl derivative.
The latter new and previously undisclosed studies complete
an initial series of totally synthetic vancomycin analogues
bearing the peripheral L-vancosaminyl-1,2-D-glucosyl disacchar-
ide as well as their (4-chlorobiphenyl)methyl derivatives.
Collectively the compounds represent a key set of analogues
of vancomycin and its (4-chlorobiphenyl)methyl derivative
containing changes to a single atom in the binding pocket.
Their assessment indicates that combined pocket and
chlorobiphenyl peripherally modified analogues exhibit a
remarkable spectrum of antimicrobial activity (vancomycin-
sensitive S. aureus (VSSA), methicillin-resistant S. aureus
(MRSA), and VanA and VanB VRE) and impressive potencies
against both vancomycin-sensitive and vancomycin-resistant
bacteria and likely benefit from two independent and
synergistic mechanisms of action. Like vancomycin, such
analogues are likely to display especially durable antibiotic
activity not prone to rapidly acquired clinical resistance.
Figure 4. Notable improvements in the total syntheses of 3 and 5.
solvent adjustment, these minor side reactions were avoided
and, following protection of the secondary alcohols, cleanly
provided thioamide 9 free of amide contaminant or reduced
byproduct. These improvements were subsequently applied in
the conversion of 10 to 11 (1.1 vs 1.2 equiv of HBF4/t-
BuONO), which was conducted at lower reaction temperatures
(−15 °C for diazonium salt formation and −35 to 25 °C for
chloride substitution) and with the aryl nitro reduction
mediated by zinc nanoparticles (40 equiv, saturated aq.
NH4Cl-acetone, 25 °C, 30 min, quant.) versus the reported
H2/Pd−C to provide 11 with improved conversions. Finally,
the final stage of the synthesis of the fully deprotected
thioamide aglycon 3 was also improved. Reduction in the
amount of reagent used in the Jones oxidation35 (3 vs 4 equiv
of CrO3, aq. H2SO4/acetone, 24 h) under controlled reaction
conditions directly oxidized the primary alcohol to the
corresponding carboxylic acid (67−75%) in a single step with
little competitive oxidative conversion of the thioamide to the
RESULTS AND DISCUSSION
■
Resynthesis of [Ψ[C(S)NH]Tpg4]vancomycin Agly-
con and [Ψ[CH2NH]Tpg4]vancomycin Aglycon. The total
syntheses of the aglycons 3 and 5 were conducted following the
approaches previously disclosed,31,32 presenting the opportu-
nity to further improve on the routes. The first of the reactions
that was improved was the E-ring chloride introduction by
Sandmeyer substitution of an intermediate diazonium salt
(Figure 4). For 8, it was found that excess reagents (1.1 vs 1.0
equiv of HBF4/t-BuONO) can lead to subsequent reaction of
the thioamide, resulting in generation of minor amounts of the
corresponding amide. Additionally, it was found that if the
Sandmeyer substitution reaction of the intermediate diazonium
salt was conducted in 1:1 (vs 2−1.4:1) CH3CN/H2O, the small
amount of reduced (H vs Cl) byproduct could be eliminated.
With the use of stoichiometric amounts of reagents and this
D
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amide (>10:1). As detailed earlier, more popular oxidizing
reagents and two-step procedures typically deployed on such
occasions, including Dess−Martin periodinane, Swern oxida-
tion, SO3−Pyr, Bu4NRuO4 (TPAP), pyridinium dichromate
(PDC),36 pyridinium chlorochromate (PCC),37 2-iodoxyben-
zoic acid (IBX), and 2,2,6,6-tetramethypiperidine-N-oxyl−
(bis(acetoxy)iodo)benzene (TEMPO−BAIB), provided little
or no desired reaction or complex mixtures of products, even
when employed in the presence of thiourea as a diversion
additive, and on occasion provided the isolable thioamide S-
oxide. After trifluoroacetic acid (TFA)-mediated conversion of
the residue-3 nitrile to a primary carboxamide,38 which was
accompanied by N-Boc removal, global deprotection with the
use of increased amounts of reagent, a longer reaction time, and
better control of the reaction concentration (0.1 M, AlBr3,
EtSH, 25 °C, 72 h) cleanly effected the removal of the four aryl
methyl ethers as well as the slower silyl ether deprotections,
providing [Ψ[C(S)NH]Tpg4]vancomycin aglycon (3).
Notably, when this final global deprotection was conducted at
a more dilute reaction concentration (0.01 vs 0.1 M) under
otherwise identical conditions, the major product still contained
the residue-2 tert-butyldimethylsilyl (TBS) ether. These
modified conditions were also adopted for the global
deprotection leading to 5 (0.1 M, 100 equiv of AlBr3, EtSH,
25 °C, 78 h), effecting the removal of four aryl methyl ethers,
two TBS ethers, the N-Boc group, and the methoxycarbonyl
carbamate.39
optimization, conversion of the residue-3 nitrile 13 to the
corresponding primary carboxamide 14 (60 equiv of H2O2, 40
equiv of K2CO3, DMSO/H2O, 25 °C, 2 h, 81%) followed by
global deprotection enlisting AlBr3 (100 equiv, EtSH, 0.1 M, 25
°C, 8 h, 65%) provided 18 with removal of the four aryl methyl
ethers, the N-Boc group, the C-terminus (2-methoxyethoxy)-
methyl (MEM) ether, and the methoxycarbonyl carbamate. Its
preparation not only provided a sample of 18 for a pilot
examination of the initial glycosylation reaction and assured
that conditions for the final steps in the preparation of 5 were
further optimized but also presented the opportunity to
complete the comparison of a key series of aglycon residue-4
analogues, each bearing the C-terminus hydroxymethyl group.
Consistent with expectations and in line with the behavior of
5, the methylene derivative 18 exhibited antimicrobial activity
against VanA VRE (BM4166), reinstating the activity by virtue
of its projected dual D-Ala-D-Ala and D-Ala-D-Lac binding (see
Figure 3), albeit at a reduced level relative to the corresponding
amidine (Figure 5). Thus, like 5, 18 proved to be less active
Initial Studies Conducted with Synthetic Aglycon C-
Terminus Hydroxymethyl Derivatives. Because of their
greater accessibility, studies examining and optimizing the
sequential enzymatic glycosylation of the unprotected aglycons
and the subsequent (4-chlorobiphenyl)methyl introduction
were first conducted with the C-terminus hydroxymethyl
derivatives. Alongside the residue-4 amide, the studies were
first conducted with the residue-4 thioamide and amidine
derivatives, both of which were prepared as previously
disclosed.32 Subsequently, the most recent studies were
conducted with the residue-4 methylene derivative 18, which
was prepared from 1331 as shown in Scheme 1. Without
Scheme 1
Figure 5. Aglycon residue-4 analogues bearing a C-terminus
hydroxymethyl group.
than the amidine 17, but it displayed activity precisely in line
with expectations based on the ligand binding affinity measured
with 5.
For vancomycin, the carbohydrate introduction has been
approached by using either chemical40−42 or enzymatic43−46
glycosylations for sequential introduction of the glucose and
vancosamine sugars located on the central residue of the
aglycon or pseudoaglycon, respectively. Of these and as noted
elsewhere,40,43,44 the enzymatic glycosylations avoid the
protection and corresponding deprotection of aglycon
precursors required of chemical procedures, providing the
fully glycosylated products in two steps from the fully
deprotected aglycons. As a consequence, the sequential
glycosylations of the modified aglycon derivatives were
examined alongside the vancomycin aglycon and its C-terminus
hydroxymethyl derivative using the enzymatic approach.47 The
recombinant glycosyltranferases GtfE and GtfD from a
E
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vancomycin-producing strain of Amycolatopsis orientalis
(ATCC19795) were expressed in Escherichia coli from the
corresponding constructs43a and were purified to homogeneity
(His6 tag). Notably, although the endogenous glycosyl donors
for both enzymes are the TDP-sugars,43 UDP-sugars have been
shown to be equally effective cosubstrates for both enzymes.
Since the requisite NDP-sugar precursor UMP morpholidate is
commercially available48 and the corresponding activated TMP
is not, we prepared, explored, and adopted UDP-vancosamine
for use with GtfD.47 The UDP-vancosamine possessing the
required β-anomer stereochemistry was prepared using a
procedure described by Kahne49 to access TDP-vancosamine
with modifications to the synthetic route that incorporate
uridine versus thymidine.47 With the use of the purified
enzymes and the synthetic glycosyl donors UDP-glucose50 (for
GtfE) and UDP-vancosamine47 (for GtfD), conditions were
optimized for the two sequential glycosylations of vancomycin
aglycon (2) as well as its C-terminus hydroxymethyl derivative
15.47 Of the two glycosylation reactions, the initial GtfE-
catalyzed incorporation of glucose using UDP-glucose exhibited
the greatest aglycon substrate sensitivity, and those bearing a C-
terminus hydroxymethyl group were established to be much
less effective than the corresponding carboxylic acids. Our
previously reported optimization efforts focused on this
glycosylation reaction and examined simultaneously both 2
and 15 (37 °C).47 In the case of hydroxymethyl substrate 15,
whose reaction proceeded at a slow rate (Figure 6), preparative
The second glycosylation reaction catalyzed by GtfD using
synthetic UDP-vancosamine proceeded to completion rapidly
(<3 h) independent of the substrate, displaying no impact of
either the C-terminus hydroxymethyl group or the nature of
residue 4 (amide, thioamide, or methylene), and the reaction
conditions required little optimization (Scheme 2). Aside from
Scheme 2
Figure 6. Comparison of the relative rates and efficiencies of the GtfE-
catalyzed reactions of 15−18 (0.5 mM) vs vancomycin aglycon (2)
under the optimized conditions: Tricine (pH 9, 75 mM), TCEP (2
mM), UDP-glucose (2 mM for 2; 4 mM for 15−18), GtfE (5 μM),
MgCl2 (1 mM), glycerol (5% v/v), 37 °C.
amounts of product 19 (55%, 48 h)47 were obtained by
increasing the amount of enzyme used (20 vs 5 μM). The
residue-4 thioamide and the residue-4 methylene derivative 18
were capable of glycosylation using GtfE and UDP-glucose to
provide the pseudoaglycons 20 (35%; 65% based on recovered
16, 25 μM GtfE) and 22 (HPLC-scale, 22% with 5 μM GtfE),
while glycosylation of the residue-4 amidine 17 was not
sufficient to provide isolatable amounts of product (Figure 6).
Whereas the studies with amide 15 and thioamide 16 were
conducted on preparative scales, the studies with amidine 17
and the more recent methylene derivative 18 were conducted
only on an analytical scale as a prelude to studies with the
corresponding and more effective C-terminus carboxylic acids.
incorporating glycerol (10% v/v) and reducing the amount of
added bovine serum albumin (0.2 vs 1 mg/mL), the conditions
used are essentially those originally disclosed43 for the use of
this enzyme and provided both 23 (79%)47 and 24 (84%) in
excellent yields.
Direct conversion of thioamide 24 to the corresponding
amidine (10 equiv of AgOAc,51 sat. NH3/MeOH, 25 °C, 6 h,
50%, unoptimized)33 provided 25, the C-terminus hydrox-
ymethyl analogue of vancomycin containing the residue-4
amidine modification. Importantly, this latter reaction was
implemented without competitive deglycosylation, and the
entire three-step sequence (conversion of 16 to 25) could be
conducted without protecting groups. Most significantly, the
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approach defined an effective route to the key residue-4
amidine analogues despite their inability to directly participate
effectively in the initial enzymatic glycosylation reaction.
Subsequent introduction of the chlorobiphenyl group into 23
and 24 by selective reductive amination was conducted best
with preformation of the imine (1.3−1.5 equiv of 4-(4-
chlorophenyl)benzaldehyde, 5 equiv of i-Pr2NEt, DMF, 30 °C,
9−12 h) followed by subsequent imine reduction (100 equiv of
NaBH(OAc)3, 30 °C, 2 h) and provided 26 (67−74%) and 27
(74%) using conditions modified (NaBH(OAc)3 vs
NaCNBH3) from those disclosed by Kahne for (4-
chlorobiphenyl)methyl vancomycin itself (Scheme 2).52 Of
most significance, the reaction of the latter compound occurs
without observation of competitive reactions of either the
residue-4 thioamide (reduction) or the N-terminal free amine
(reductive amination). A final AgOAc-promoted (10 equiv, sat.
NH3/MeOH, 25 °C, 6 h) conversion of thioamide 27 to the
amidine provided 28 (48%, unoptimized),53 the 4-chlorobi-
phenyl derivative of 25. By design, the final reaction
introducing the amidine as well as the sequential glycosylation
reactions and the reductive amination could be conducted
effectively on fully functionalized substrates lacking protecting
groups and incorporating the vancomycin disaccharide.
Scheme 3
Total Syntheses of Vancomycin, [Ψ[C(S)NH]Tpg4]-
vancomycin, [Ψ[C(NH)NH]Tpg4]vancomycin, [Ψ-
[ C H 2 N H ] T p g 4 ] v a n c o m y c i n , a n d T h e i r ( 4 -
Chlorobiphenyl)methyl Derivatives. The studies piloted
with the C-terminus hydroxymethyl derivatives as well as
vancomycin aglycon itself defined the approach and provided
the experience needed to address the fully functionalized
residue-4-modified aglycons. The two sequential glycosylations
of vancomycin aglycon 2,47 the freshly prepared synthetic
thioamide 3,34 amidine 4,32 and the more recently reprepared
methylene analogue 531 were conducted with the recombinant
glycosyltransferases43 and the synthetic glycosyl donors (UDP-
glucose50/GtfE and UDP-vancosamine47/GtfD) to provide the
intermediate pseudoaglycons 29 (92%), 30 (75%, HPLC
conversion 86−92%), and 32 (72%, HPLC conversion 85−
95%) and subsequently vancomycin (1) (87%) and the fully
functionalized vancomycin analogues bearing single-atom
changes in the binding pocket, [Ψ[C(S)NH]Tpg4]-
vancomycin (33) (87%, HPLC conversion >95%) and
[Ψ[CH2NH]Tpg4]vancomycin (35) (76%, HPLC conversion
>95%) (Scheme 3). Notably, in combination with our earlier
efforts that provided a total synthesis of 2,24a the studies on the
conversion of 2 to 1 also served to complete a total synthesis of
vancomycin itself.
A comparison of the relative efficiencies of the initial
glycosylation reactions with 3 and 5 conducted on an analytical
scale alongside that of 2 is shown in Figure 7. Unlike the
significant impact of the C-terminal hydroxymethyl group but
like the well-tolerated N-terminus substitutions,43d,47 modifica-
tions to the vancomycin binding pocket itself had a minimal
impact on both the rate and overall efficiency of the initial
GtfE-catalyzed reaction. However, like the observations made
with amidine 17, amidine aglycon 4 failed to undergo successful
GtfE-catalyzed glycosylation. Although small amounts of
product could be detected by HPLC, the aglycon itself
underwent competitive conversion to several byproducts
under the basic conditions (pH 9) required for the reaction.
The second glycosylation reaction catalyzed by GtfD using the
cosubstrate UDP-vancosamine proceeded rapidly (<1.5 h)
regardless of the aglycon substrate, displaying no significant
impact of the nature of residue 4 (amide, thioamide, or
methylene), and the conditions required no further optimiza-
tion (Scheme 3). For [Ψ[C(NH)NH]Tpg4]vancomycin
(34), direct conversion of thioamide 33 to the corresponding
amidine (10 equiv of AgOAc, sat. NH3/MeOH, 25 °C, 6 h)
provided 34.53
Subsequent introduction of the chlorobiphenyl group with
[Ψ[C(S)NH]Tpg4]vancomycin (33) and [Ψ[CH2NH]-
Tpg4]vancomycin (35) by reductive amination (1.5 equiv of
4-(4-chlorophenyl)benzaldehyde, 5 equiv of i-Pr2NEt, DMF,
50−70 °C, 2 h; 100 equiv of NaCNBH3, 70 °C, 5 h) provided
37 (57%)53 and 39 (41%), respectively,53 on the unprotected
vancomycin analogues without optimization using conditions
piloted with CBP-vancomycin (36) itself (61−74%).52 Direct
AgOAc-promoted (10 equiv, sat. NH3/MeOH, 25 °C, 6 h)
conversion of thioamide 37 to the amidine provided 38
(45%),53 the chlorobiphenyl derivative of 34. Significantly, the
reductive amination was conducted without competing reaction
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Figure 7. Comparison of the relative rates and efficiencies of the GtfE-
catalyzed reactions of 2−5 (0.5 mM). Conditions: Tricine (pH 9, 75
mM), TCEP (2 mM), UDP-glucose (2 mM for 2; 4 mM for 3−5),
GtfE (5 μM), MgCl2 (1 mM), glycerol (5% v/v), 37 °C.
of either the thioamide or the N-terminus and residue-4
secondary amines, the entire three or four step sequence could
be conducted without protecting groups, and the amidine
introduction was implemented without competitive deglycosy-
lation. Finally, it is worth noting that the enzymatic
glycosylations were conducted on ca. 1−3 mg of substrate
with 1 mol % enzyme and 4 equiv of UDP-glucose or UDP-
vancosamine, reflecting our piloted laboratory scale. However,
the expression and purification of the enzymes and the
chemical synthesis of UDP-vancosamine,47 along with the
commercial availability of UDP-glucose,50 were conducted on
scales that would easily support laboratory preparations on
much larger scales (ca. 100-fold) than exemplified herein and
could easily be scaled beyond even this level.
Antimicrobial Activity. The pocket-modified vancomycin
analogues containing the C-terminus hydroxymethyl group
(23−25) and their chlorobiphenyl derivatives (26−28), as well
as the fully functionalized vancomycin analogues [Ψ[C(
S)NH]Tpg4]vancomycin, [Ψ[C(NH)NH]Tpg4]-
vancomycin, and [Ψ[CH2NH]Tpg4]vancomycin (33−35)
and their (4-chlorobiphenyl)methyl derivatives (37−39),
were examined alongside the corresponding vancomycin
(residue-4 amide) derivatives. As shown in Figure 8, the
antimicrobial activities of the compounds were evaluated
against a panel of Gram-positive bacteria that included VSSA,
MRSA, and both VanA (Enterococcus faecalis and Enterococcus
faecium) and VanB (E. faecalis) VRE, of which VanA is the most
stringent of the resistant organisms.54 Notably, one VanA VRE
tested (E. faecium ATCC BAA-2317) represents an emerging
challenging multidrug-resistant VanA VRE that is resistant not
only to vancomycin and teicoplanin but also ampicillin,
benzylpenicillin, ciprofloxacin, erythromycin, levofloxacin,
nitrofurantoin, and tetracycline. It is also insensitive to linezolid
but remains sensitive to tigecycline and dalfopristine.54 The
activities of the C-terminus hydroxymethyl derivatives paral-
leled those observed with the corresponding C-terminus
carboxylic acids and will not be discussed separately below.
However, they displayed the same trends and near identical
absolute MIC values, reinforcing the generality of the
observations and significance of the conclusions.
Figure 8. In vitro antimicrobial activity.
which they are based (see Figure 3). Although it is well-
established that the attached unmodified carbohydrate does not
alter the in vitro antimicrobial activity (potency) or influence
the target D-Ala-D-Ala or D-Ala-D-Lac binding, the disaccharide
impacts the in vivo activity, increasing the water solubility,
influencing the pharmacokinetic and distribution properties,
First and as expected, the activities of the pocket-modified
vancomycin analogues 33−35 matched the in vitro antimicro-
bial activities of the corresponding aglycon analogues 3−5 on
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and contributing a potential second mechanism of action. An
analogous impact on the vancomycin analogues 33−35 might
be expected since each represents the change of a single atom
in the binding pocket (residue-4 carbonyl O → S, NH, H2),
and they would be the preferred compounds (vs 3−5) with
which to probe in vivo activity. Within this series, vancomycin
displayed potent activity against VSSA and MRSA (MIC = 0.5
μg/mL) but was ineffective against VanA VRE (MIC = 250 μg/
mL) and only modestly active against VanB VRE (MIC = 8 μg/
mL) under the assay conditions employed. Consistent with its
lack of binding to either D-Ala-D-Ala or D-Ala-D-Lac, thioamide
33 proved to be inactive as an antimicrobial agent against both
sensitive and resistant bacteria (MICs >32 μg/mL). Both
amidine 3434 and the methylene analogue 35 reinstated the
activity against VanA VRE with MICs of 0.5 and 31 μg/mL,
respectively, precisely in line with expectations based on the
relative dual D-Ala-D-Ala and D-Ala-D-Lac binding affinities of
their aglycons and matching the activities observed with the
corresponding aglycons 4 and 5 (see Figure 3). Of most
significance, amidine 34 displayed a potency against VanA VRE
that matched the activity vancomycin displays against sensitive
bacteria (VSSA and MRSA, MICs = 0.5 μg/mL).
Given the distinct origins of their impacts on the
antimicrobial activity of vancomycin, we expected that
incorporation of the peripheral chlorobiphenyl modification
into the structure of the binding-pocket-modified vancomycin
analogues would further increase their antimicrobial activity
against not only vancomycin-sensitive but also vancomycin-
resistant bacteria to truly remarkable levels. Although this
conceivably could have been demonstrated by substitution of
the synthetic aglycons 2−5, we expected that the most
definitive assessment of the dual impact would be a direct
comparison of CBP-vancomycin (36) with 37−39 wherein a
series of key changes in a single atom in the binding pocket
were introduced, despite the synthetic challenges this posed.
This choice of both the site of modification and the use of the
chlorobiphenyl modification proved key to understanding the
behavior of such analogues and revealed unique insights into
the origin of the effects.
In line with reports of its impact, introduction of the (4-
chlorobiphenyl)methyl group into vancomycin (36 vs 1)
resulted in 100-fold improvements in the activity against
VanA and VanB VRE (MIC = 2.5 vs 250 μg/mL) and 20-fold
improvements against VSSA and MRSA (MIC = 0.03 vs 0.5
μg/mL) in the strains examined. In spite of the increases in
potency, it remained 100-fold less effective against VanA VRE
than against sensitive bacterial strains. Both amidine 38 and the
methylene analogue 39 displayed the same 100-fold increases in
activity against VanA VRE, exhibiting remarkable MICs of
0.005 and 0.06−0.13 μg/mL, respectively. Just as significantly,
introduction of the chlorobiphenyl group into either the
vancomycin amidine (to give 38) or the vancomycin methylene
analogue (to give 39) resulted in compounds with remarkable
spectra of activity at these impressive potencies. Both were
equally effective against both vancomycin-sensitive bacteria
(VSSA and MRSA) and vancomycin-resistant bacteria (VanA
and VanB VRE), of which VanA VRE proved especially
sensitive to the analogues. Both analogues exhibited MICs
below 1 μg/mL across the bacterial panel, and amidine 38 was
found to be on average 15-fold more potent than the methylene
analogue 39, precisely in line with their relative dual ligand
binding affinities. Moreover, amidine 38 not only matched the
activity that CBP-vancomycin (36) displays against vancomy-
cin-sensitive bacteria (VSSA and MRSA) but also exhibited this
extraordinary potency against VanA and VanB vancomycin-
resistant bacteria. In fact, the activity of 38 against the most
stringent of the resistant bacteria, VanA VRE, was nearly 10-
fold better than the potency it displayed against the sensitive
bacteria, representing a 500-fold increase in activity relative to
36 and a 50000-fold increase in activity relative to vancomycin
(1) itself. Thus, the introduction of chlorobiphenyl into the
pocket-modified vancomycin analogues 38 (MICs = 0.005−
0.06 μg/mL) and 39 (MICs = 0.06−0.5 μg/mL) synergistically
increased their potency against both vancomycin-sensitive and
vancomycin-resistant bacteria. Insights into this behavior came
from an examination of 37, the chlorobiphenyl derivative of
vancomycin thioamide (33). Introduction of the (4-
chlorobiphenyl)methyl group into 33 to give 37 reinstates
impressive and equally potent activity (MIC = 2−4 μg/mL)
against all vancomycin-sensitive and vancomycin-resistant
strains despite its inability to bind the primary cell wall target
D-Ala-D-Ala/D-Ala-D-Lac. It is unlikely that such effective
activity can be achieved simply by the effects of antibiotic
membrane anchoring, antibiotic dimerization, or disruption of
bacterial membrane integrity. Rather, it likely reflects potent
antimicrobial activity derived from a second mechanism of
action impacting cell wall synthesis unrelated to D-Ala-D-Ala/D-
Ala-D-Lac binding. In line with observations made with CBP-
vancomycin and analogues containing damaged binding
pockets, this most likely involves potent transglycosylase
inhibition mediated by direct binding to the enyzme.22,23
Because of the insights derived from the comparative
examination of the thioamides 33 and 37, the behavior of the
CBP-vancomycin amidine 38 and CBP-vancomycin methylene
analogue 39 likely represents a spectrum of activity and potency
derived from inhibition of bacterial cell wall synthesis through
two synergistic mechanisms, one involving inhibition of
transpeptidase-catalyzed cell wall cross-linking through dual
substrate (D-Ala-D-Ala and D-Ala-D-Lac) binding and the second
involving direct inhibition of transglycosylase independent of
such ligand binding. If this is the case, it suggests that resistance
is even more unlikely to emerge against such analogues since it
would entail simultaneous bacterial changes to two distinct
targets of the antibiotics, one of which is not subject to direct
genetic alterations. Therefore, both 38 and 39 are superb
candidates for preclinical development. Their preliminary
assessments not only indicate that they address the present-
day emerging vancomycin resistance and exhibit remarkable
spectra of activity and superb antimicrobial potency but also
that they are endowed with a unique combination of
characteristics that may allow them to display the 50-year
clinical durability of vancomycin.
Although at this stage still speculative, the four chlorobi-
phenyl derivatives 36−39 are also uniquely poised to help
unravel the subtleties of the mechanisms of action of such
modified glycopeptide antibiotics. Because of its inability to
bind either D-Ala-D-Ala or D-Ala-D-Lac, CBP-[Ψ[C(S)NH]-
Tpg4]vancomycin (thioamide 37) derives its antimicrobial
activity (MIC = 2−4 μg/mL) exclusively through a distinct
second mechanism of action that does not involve ligand
binding and likely involves direct inhibition of transglycosy-
lase.22,23 By virtue of its inability to bind D-Ala-D-Lac, CBP-
vancomycin (36) also likely derives its similarly potent activity
against vancomycin-resistant organisms (VanA VRE, MIC = 2.5
μg/mL) by this same mechanism potentially involving only the
direct inhibition of transglycosylase, whereas its more potent
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activity against vancomycin-sensitive organisms (VSSA and
MRSA, MIC = 0.03 μg/mL) is derived from the equally potent
and synergistic inhibition of both transpeptidase (via D-Ala-D-
Ala binding and substrate sequestration) and transglycosylase
(direct enzyme inhibition). As a result of the binding pocket
redesign and ability to exhibit fully effective dual D-Ala-D-Ala
and D-Ala-D-Lac binding combined with the peripheral
chlorobiphenyl-mediated potential direct inhibition of trans-
glycosylase, CBP-[Ψ[C(NH)NH]Tpg4]vancomycin (38)
picks up the ability to effectively inhibit transpeptidase in
vancomycin-resistant bacteria (VanA VRE) via D-Ala-D-Lac
binding, maintains the ability to inhibit transpeptidase in
vancomycin-sensitive bacteria (VSSA and MRSA) via D-Ala-D-
Ala binding, allows the potential indirect transglycosylase
inhibition through ligand binding, and benefits potentially from
an equally potent and synergistic direct inhibition of trans-
glycosylase independent of D-Ala-D-Ala/D-Ala-D-Lac binding.
The net result is an antibiotic that benefits from two equally
potent, independent, and synergistic mechanisms of action and
that displays remarkable antimicrobial potencies (MIC = 0.06−
0.005 μg/mL) against both vancomycin-sensitive and vanco-
mycin-resistant bacteria. In contrast but similarly interestingly,
the potency of CBP-[Ψ[CH2NH]Tpg4]vancomycin (39)
(MIC = 0.5−0.06 μg/mL) suggests that the principal
mechanism by which it acts is through the potential
chlorobiphenyl-mediated direct inhibition of transglycosylase,
but now with a second less-potent contribution derived from its
balanced, albeit reduced, dual ligand binding affinities for
inhibition of transpeptidase in either vancomycin-sensitive and
vancomycin-resistant bacteria. It is remarkable that the series
appears to display the combined activity of two independent
mechanisms, which act synergistically with one another, to
provide predictable potency trends derived independently from
the binding pocket modifications and the peripheral carbohy-
drate substitution. Kahne has shown that although the potency
of most lipid-linked glycopeptides or their aglycons lose activity
against VanA strains when their binding pocket is chemically
damaged,52 indicating that ligand binding is important to their
activity, a small subset (including CBP-vancomycin) retain
good antimicrobial activity even when their binding pocket is
chemically damaged.22 Moreover, it is such derivatives that
were shown by Kahne and Walker to effectively inhibit
transglycosylase without substrate or ligand binding, suggesting
that they directly bind and inhibit the enzyme. CBP-[Ψ[C(
S)NH]Tpg4]vancomycin embodies these same characteristics,
displays the same VanA VRE potency, and likely will display the
same behavior toward transglycosylases. As pointed out by
Kahne, is likely that this activity against VanA strains requires
specific positioning of the hydrophobic substituent attached to
the vancomycin disaccharide. As a consequence, it is especially
notable that our studies were conducted with single-atom
changes to the binding pocket of vancomycin and CBP-
vancomycin and not conducted on simpler, more accessible
aglycon derivatives.
acquired to a community-acquired infection in the last 10 years
has increased the number and intensified the need to treat such
resistant bacterial infections. In addition, vancomycin-resistant
bacterial strains are also on the rise, with U.S. ICU clinical
isolates of vancomycin-resistant Enterococcus faecalis approach-
ing 30%,55 albeit in strains presently sensitive to other
antibiotics. Most feared is the recent emergence of MRSA
strains now resistant or insensitive to vancomycin (VRSA and
VISA). This poses a major health problem and has intensified
efforts to develop antibiotics that not only combat this
resistance but also display the durability of vancomycin.7
Herein we have provided full details of the completion of the
total syntheses and evaluation of [Ψ[C(S)NH]Tpg4]-
vancomycin (33), [Ψ[C(NH)NH]Tpg4]vancomycin (34),
and [Ψ[CH2NH]Tpg4]vancomycin (35), fully adorned ana-
logues of vancomycin that contain single-atom changes or
exchanges in the binding pocket (residue-4 carbonyl O → S,
NH, H2) designed to probe and directly address the molecular
basis of vancomycin resistance; their corresponding C-terminus
hydroxymethyl analogues 23−25, on which the chemistry was
piloted; and their corresponding chlorobiphenyl derivatives
(26−28 and 36−39). The latter derivatives constitute
combined pocket and peripherally modified vancomycin
analogues anticipated to exhibit remarkable properties.
By design, the sequential enzyme-catalyzed glycosylation
reactions of the modified aglycons, the final amidine
introductions, and the intermediate reductive aminations used
for the chlorobiphenyl substitutions were conducted without
the need of protecting groups, establishing the foundation and
providing the methodology for potential semisynthetic or
biosynthetic preparations of such glycopeptide analogues. As
expected, the activities of the pocket-modified vancomycin
analogues 33−35 matched the in vitro antimicrobial activities
of the corresponding aglycon analogues 3−5 on which they are
based. In line with expectations based on the behavior of the
corresponding aglycons and in stark contrast to one another,
the vancomycin amidine and, to a lesser extent, the vancomycin
methylene analogue reestablish potent antimicrobial activity
against VanA VRE, whereas vancomycin thioamide is inactive
even against vancomycin-sensitive bacteria; results that parallel
the dual D-Ala-D-Ala and D-Ala-D-Lac binding affinities of the
corresponding aglycons. The introduction of a peripheral
chlorobiphenyl modification converts vancomycin thioamide
into an effective antimicrobial agent that is active against
vancomycin-sensitive and vancomycin-resistant bacteria (MIC
= 2−4 μg/mL), even though it is not capable of D-Ala-D-Ala/D-
Ala-D-Lac binding. It also converts the vancomycin amidine and
vancomycin methylene analogues into compounds with
remarkable spectra of activity (VSSA, MRSA, and VanA and
VanB VRE) and truly impressive potencies (MIC = 0.06−0.005
and 0.06−0.5 μg/mL, respectively) that are likely derived from
inhibition of cell wall biosynthesis through at least two distinct
mechanisms, only one of which is dependent on D-Ala-D-Ala/D-
Ala-D-Lac binding. In addition to indicating that the described
peripheral and productive pocket modifications are synergistic,
such analogues are likely to be even less prone to acquired
clinical resistance than vancomycin and to display durable
antibiotic activity.
CONCLUSIONS
■
Clinical uses of vancomycin include treatment of patients on
dialysis, allergic to β-lactam antibiotics, or undergoing cancer
chemotherapy.1 However, the most widely recognized use of
vancomycin is the treatment of methicillin-resistant Staph-
ylococcus aureus (MRSA) infections.1 The prevalence of MRSA
in intensive care units (ICUs) (60% of SA infections in the
United States are MRSA)55 and its movement from a hospital-
ASSOCIATED CONTENT
* Supporting Information
Full experimental details. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
S
J
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approaches, see: (e) Fowler, B. S.; Laemmerhold, K. M.; Miller, S. J. J.
Am. Chem. Soc. 2012, 134, 9755. (f) Griffith, B. R.; Krepel, C.; Fu, X.;
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Soc. 2007, 129, 8150. (g) Jain, R. K.; Trias, J.; Ellman, J. A. J. Am.
Chem. Soc. 2003, 125, 8740. (h) Weist, S.; Kittel, C.; Bischoff, D.;
AUTHOR INFORMATION
Corresponding Author
*boger@scripps.edu
Notes
■
Bister, B.; Pfeifer, V.; Nicholson, G. J.; Wohlleben, W.; Sussmuth, R.
̈
The authors declare no competing financial interest.
D. J. Am. Chem. Soc. 2004, 126, 5942.
(13) (a) Sussmuth, R. D. ChemBioChem 2002, 3, 295. (b) von
̈
ACKNOWLEDGMENTS
■
Nussbaum, F.; Brands, M.; Hinzen, B.; Weigand, S.; Habich, D. Angew.
̈
We gratefully acknowledge the financial support of the National
Institutes of Health (CA041101 to D.L.B.). We especially thank
Professor C. T. Walsh for the gift of the plasmid constructs
pET22b-gtfD-his6 and pET22b-gtfE-his6 for GtfD and GtfE
expression, Professor K. D. Janda (TSRI) for providing the
facilities for expressing and purifying the enzymes, and Dr. J. C.
Collins for initial exploration of the enzymatic glycosylations.
We thank Dr. M. Weiss for advanced intermediates en route to
additional 3 and 4 in early stages of the work, D. Ogasawara
and Dr. T. Michaels for quantities of the amino acid subunits
and early-stage intermediates in the synthesis of additional 3
and 4, and Dr. T. Mukaiyama for early-stage intermediates en
route to 5.
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