Next-Generation Total Synthesis of Vancomycin

Article abstract of DOI:10.1021/jacs.0c07433

A next-generation total synthesis of vancomycin aglycon is detailed that was achieved in 17 steps (longest linear sequence, LLS) from the constituent amino acid subunits with kinetically controlled diastereoselective introduction of all three elements of atropisomerism. In addition to new syntheses of three of the seven amino acid subunits, highlights of the approach include a ligand-controlled atroposelective one-pot Miyaura borylation-Suzuki coupling sequence for introduction of the AB biaryl axis of chirality (>20:1 dr), an essentially instantaneous and scalable macrolactamization of the AB ring system nearly free of competitive epimerization (>30:1 dr), and two room-temperature atroposelective intramolecular SNAr cyclizations for sequential CD (8:1 dr) and DE ring closures (14:1 dr) that benefit from both preorganization by the preformed AB ring system and subtle substituent effects. Combined with a protecting group free two-step enzymatic glycosylation of vancomycin aglycon, this provides a 19-step total synthesis of vancomycin. The approach paves the way for large-scale synthetic preparation of pocket-modified vancomycin analogues that directly address the underlying mechanism of resistance to vancomycin.

Full text of DOI:10.1021/jacs.0c07433

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Next-Generation Total Synthesis of Vancomycin
Maxwell J. Moore, Shiwei Qu, Ceheng Tan, Yu Cai, Yuzo Mogi, D. Jamin Keith, and Dale L. Boger*
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ABSTRACT: A next-generation total synthesis of vancomycin aglycon is detailed
that was achieved in 17 steps (longest linear sequence, LLS) from the constituent
amino acid subunits with kinetically controlled diastereoselective introduction of all
three elements of atropisomerism. In addition to new syntheses of three of the seven
amino acid subunits, highlights of the approach include a ligand-controlled
atroposelective one-pot Miyaura borylation−Suzuki coupling sequence for
introduction of the AB biaryl axis of chirality (>20:1 dr), an essentially instantaneous
and scalable macrolactamization of the AB ring system nearly free of competitive
epimerization (>30:1 dr), and two room-temperature atroposelective intramolecular
SNAr cyclizations for sequential CD (8:1 dr) and DE ring closures (14:1 dr) that
benefit from both preorganization by the preformed AB ring system and subtle
substituent effects. Combined with a protecting group free two-step enzymatic
glycosylation of vancomycin aglycon, this provides a 19-step total synthesis of
vancomycin. The approach paves the way for large-scale synthetic preparation of pocket-modified vancomycin analogues that
directly address the underlying mechanism of resistance to vancomycin.
the N-terminus of peptidoglycan precursors from D-Ala-D-Ala
to D-Ala-D-Lac.32 This single-atom exchange in the cell wall
precursors reduces vancomycin binding (1000-fold) and its
derived antimicrobial activity (1000-fold).33
INTRODUCTION
■
Vancomycin (1), first disclosed1 and introduced into the clinic
over 60 years ago, remains one of the clinically most effective
and important antibiotics for treatment of life-threatening
Gram-positive bacterial infections,2 including methicillin-
resistant S. aureus (MRSA). Its complex structure,3 the three
strained macrocyclic ring systems interwoven into the highly
functionalized and rigid tricyclic heptapeptide core, the
unusual centers of axial or planar chirality (atropisomers),
and its glycosylation present formidable synthetic challenges
(Figure 1). Three total syntheses of vancomycin aglycon4−6
and two total syntheses of vancomycin7,8 have been disclosed
to date. Related efforts have detailed total syntheses of
orienticin C,9 teicoplanin,10,11 and ristocetin A12 aglycons, and
a near endless number of methodology studies have been
conducted that address synthetic challenges posed by their
structures. The comprehensive reviews of Perkins,13 Wil-
liams,14,15 Nicolaou,16 Courvalin,17,18 Walsh,19,20 Kahne,2
ourselves,21,22 and others23−25 provide summaries of the rich
history on the isolation, structure elucidation, biosynthesis,
semisynthetic and synthetic studies, mechanism of action, and
mechanisms of resistance of the glycopeptide antibiotics.
The only clinically significant resistance to vancomycin even
after >60 years of use first emerged in enterococci (VanA and
VanB VRE, 1987)26 and more recently in S. aureus (VRSA,
2002).18 It was co-opted from nonpathogenic source
organisms that use an intricate mechanism to protect
themselves while producing vancomycin.27 It is induced
upon detection28−31 of a glycopeptide challenge that initiates
an orchestrated response, resulting in late-stage remodeling of
An extension of our efforts on the total synthesis of the
glycopeptide antibiotics has targeted vancomycin analogues,
containing deep-seated compensatory single-atom exchanges in
the binding pocket,34 that exhibit dual D-Ala-D-Ala/D-Ala-D-Lac
binding and antimicrobial activity against both vancomycin-
sensitive and -resistant organisms (235 and 336). These efforts,
along with peripheral modifications that introduce additional
independent mechanisms of action,37,38 have provided
extraordinarily potent analogues that display especially durable
antimicrobial activity (Figure 1). We have shown that
incorporation of two simple peripheral modifications (CBP
and C1) independently (e.g., 4−6) or simultaneously (8) into
the more accessible of our pocket-modified analogues at the
time, [ψ[CH2NH]Tpg4]vancomycin (2), provided potent
antimicrobial agents with up to three synergistic mechanisms
of action, each of which is individually effective against both
vancomycin-resistant and vancomycin-sensitive bacteria and
two of which are independent of D-Ala-D-Ala-D-Lac binding.37
The exceptional potency (MIC 0.01−0.005 μg/mL, VRE) and
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recycling), lengthy syntheses of several of the unnatural amino
acid subunits, and lack of kinetic atroposelectivity in the
construction of the AB and CD ring systems. While the latter
issue was originally addressed in our efforts by a defined order
of macrocyclizations that permitted thermal equilibration40
and recycling of the unnatural atropisomers, it required two
atropisomer separations that limit material throughput. There-
fore, we embarked on and herein detail a streamlined 17-step
total synthesis of vancomycin aglycon (10) that achieves high
kinetic diastereoselectivity for formation of each macrocyclic
ring and improves the overall yield of 10 more than 20-fold.
RESULTS AND DISCUSSION
■
Improved Synthesis of the A, C, and D Ring Amino
Acid Subunits. Our synthesis of vancomycin aglycon begins
with seven unnatural amino acid subunits (11−17), four of
which are either commercially available or readily prepared.
The remaining three were each prepared in a modest overall
yield (ca. 20%) in our previous efforts (Figure 2). The A ring6
Figure 1. (Top) Structure of vancomycin and key analogues with H-
bonding interactions between vancomycin and model ligands
indicated by dashed lines. (Middle) Added peripheral modifications
that synergistically improve activity by inducing membrane perme-
abilization (C1) or directly inhibiting transglycosylase (CBP), both
independent of D-Ala-D-Ala-D-Lac binding. (Bottom) VanA VRE
antimicrobial activity.
remarkable durability of the prototypical members, CBP-
[ψ[C(NH)NH]Tpg4]vancomycin (6) and C1,CBP-[ψ-
[CH2NH]Tpg4]vancomycin (8), has inspired our continued
examination of new analogues, including 9, and their expanded
preclinical examination.
Figure 2. Key retrosynthetic disconnections and initial starting
subunits.
In order to facilitate these studies, we have now developed a
scalable total synthesis that could provide sufficient amounts of
each analogue within the confines of an academic lab for initial
preclinical assessment of in vivo efficacy and safety. In contrast
to what many might think would be the most challenging
feature, we earlier reported a scalable enzymatic conversion of
the aglycon to the fully decorated natural product8 (2 steps) or
its pocket-modified analogues39 that is conducted without
protecting groups. However, the total synthesis of vancomycin
aglycon (10) and its residue 4 modified analogues required
redesign to overcome practical issues, including a long step
count (25), low overall yield (0.2% without atropisomer
(14) and D ring41 (12) subunits were accessed in 5 and 9 total
steps, respectively, with the requisite absolute stereochemistry
introduced by Sharpless asymmetric aminohydroxylations,42,43
while the C ring subunit12 (11) was prepared by a modestly
12,44
̈
diastereoselective (5:1 syn:anti) Schollkopf aldol reaction
with full control of the α-amino acid stereochemistry.
Substantially improved syntheses of these three subunits
were developed during the course of this work. With these
improvements, five of the subunits are now derived from
inexpensive chiral pool starting materials and only two require
B
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Scheme 1. Improved Synthesis of Starting Subunits
asymmetric synthesis. All but one of the subunits are now
available in >50% overall yield.
synthesis detailed herein. The successful implementation of
this route hinged on the use of 3,4,7,8-tetramethyl-1,10-
phenanthroline (tmphen) as the ligand in the C−H
borylation,47,48 the inclusion of pyridine in the Chan−Lam
methoxylation (Supporting Information Figure S1),49 and the
development of an efficient Miyaura borylation of the hindered
aryl bromide 22 with bis(ethyleneglycolato)diboron (B2eg2),
whereas typical Miyaura borylation conditions [B2pin2/
PdCl2(dppf)]50 delivered only trace product.
An asymmetric aminohydroxylation (AA) reaction reported
by Sharpless42 was used to prepare the A ring amino acid in
our prior efforts,6 proceeded in 68% yield (90% ee) on 2 mmol
(328 mg) scale, and provided regioisomeric products (3:1 rr).
However, the yield of this reaction diminished on larger scales
(>10 g, 33−41%) in our hands, and the product was invariably
contaminated with an additional inseparable ring-chlorinated
byproduct. An alternative, chiral pool route with complete
control of the absolute stereochemistry was developed based
on C−H activation and meta functionalization of the
commercially available inexpensive ($0.40/g) phenylglycine-
derived Evans auxiliary 18 (Scheme 1). A one-pot sequential
Ir-mediated bis-C−H borylation45/Chan−Lam methoxyla-
tion46 furnished 19 (50% overall) in a reaction that was easily
scaled to >20 g. Bromination of 19 followed by recrystalliza-
tion provided 20 (95%). To date, this three-step sequence has
provided more than 300 g of intermediate 20. Subsequent
oxazolidinone hydrolysis and N-trifluoroacetamide protection
of 21 provided 22 quantitatively. The lithium−halogen
exchange/B(OMe)3-trapping procedure previously used6 to
prepare the boronic acid 14 (59% yield) was superseded by a
modified Miyaura borylation, providing high yields (85%) of
cyclic boronate 23. In addition and importantly, the crude
reaction mixture containing boroxane 23 could be telescoped
directly into a subsequent atroposelective Suzuki coupling (see
below). Hence, the new route to the A ring subunit provides
the cyclic boronate 23 in 40% yield (5 steps), a 2-fold
improvement over the asymmetric aminohydroxylation route
(21%) with complete (chiral pool) control of the absolute
stereochemistry. Further, its implementation provides direct
access to 23, which is functionalized (trifluoroacetamide vs
NHCbz) for more effective use in the final streamlined total
The D ring phenylglycine 12 was previously the most
cumbersome subunit to prepare on scale, requiring 9 steps and
relying on an asymmetric aminohydroxylation42,43 (AA) to
install the needed chiral center (69%, 96% ee, 7:1
regioselectivity).41 We developed a new chiral pool 5-step
route to the D ring subunit starting from inexpensive ($0.24/
g) D-4-hydroxyphenylglycine (24), which was first converted
to dichlorinated methyl ester 25 in 62% yield (59% for 3 steps
on 100 g scale, Scheme 1) by modification of a reported
procedure.51 Although 25 proved sensitive to racemization
(Et3N, 23 °C), net hydroxylation of both chlorides in 25 could
be accomplished in a one-pot Miyaura borylation/oxidation
sequence (94%) with negligible loss of optical purity (99% ee
for 26). Saponification (LiOH, 96%, 98% ee) provided the
required 3,5-dihydroxy-4-methoxyphenylglycine 12 now avail-
able in a much shorter (5 vs 9 steps) and easily scalable
sequence with a 3-fold improvement in yield (56% yield for 5
steps) over our previous route (18% for 9 steps) and with
complete control (chiral pool) of the absolute stereochemistry.
Notably, the use of dichloride 25 as a substrate for the key
borylation−oxidation reaction was essential, whereas use of the
corresponding dibromide or diiodide substrates resulted in
considerable ring halogenation upon exposure to H2O2.
Preparation of the C ring subunit 11 previously was achieved
44
̈
by zirconium-mediated addition of a Schollkopf reagent
C
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(commercially available for >$100/g, or in 4 steps52 from D-
valine), proceeding in 5:1 dr (syn:anti) and 50% yield (syn
isomer). In order to avoid the needed separation of the alcohol
diastereomers and lengthy preparation or costly reagent
purchase, we turned to a Ti-mediated aldol reaction of the
known oxazolidinethione 27,53 which proceeds with near
perfect diastereoselection and enlists a more readily prepared
(2 steps, 68%) and recyclable chiral auxiliary (27) (Scheme 1).
Addition of 27 to 4-fluoro-3-nitrobenzaldehyde (28) under
conditions designed and disclosed by Crimmins54 to provide
the syn aldol product, followed by in situ methanolysis, cleanly
provided methyl ester 29 (90%, >99:1 dr, 27 g scale). In
addition, the recovered auxiliary 30 was recycled in a single
step to regenerate 27, without prior separation of 29 from 30.
Subsequent TIPS protection of alcohol 29 and Staudinger
reduction of 31 provided 11 in high yield (92% for 2 steps,
>99% ee, 56% overall for 5 steps) virtually free of the anti-
diastereomer (>99% de). To date, >320 g of 11 has been
prepared by this route.
Formal Total Synthesis of Vancomycin Aglycon:
Kinetically Controlled Diastereoselective Introduction
of All Three Elements of Atropisomerism. Concurrent
with the above efforts, we undertook studies on the
atroposelective construction of the AB biaryl axis of chirality
as well as diastereoselective formation of the CD and DE
macrocyclic diaryl ethers. Efficient elements of the synthesis of
the vancomycin core structure developed in our prior efforts
were maintained, enlisting a macrolactamization6 for closure of
the 12-membered biaryl AB ring system and two aromatic
nucleophilic substitution reactions for macrocyclization of the
16-membered diaryl ethers in the CD42/DE6 ring systems but
conducted in an altered order. The availability now of robust
methods for atroposelective Suzuki biaryl coupling reactions
suggested that we set the AB biaryl stereochemistry first. Then
following macrolactamization and using the preformed AB
macrocycle as an element of preorganization, we hoped to
achieve high kinetic atroposelectivity in the closure of the CD
ring system under substrate control, although precedent
suggested this might be improbable.9,55 If successful and
based on our previous work,6 we could anticipate that the
subsequent DE ring closure would proceed with excellent
substrate-controlled atroposelectivity (see Figure 2).
Figure 3. Key observations in the development of a catalyst-
controlled diastereoselective synthesis of AB axis of chirality.
catalyst [(R)-BINAP(O)-Pd0] from 1:1 (R)-BINAP/Pd-
(OAc)2. Our initial screen of available chiral ligands for the
coupling of 32 with 14 revealed that the (R)-BINAP/
Pd(OAc)2 combination was among the most effective of
those examined (Supporting Information Figure S2). However,
these initially favorable results from the PdII/BINAP system
proved difficult to maintain as we scaled the reaction, which we
found was due to precipitation of the active (R)-BINAP(O)−
Pd0 catalyst as a cherry-red solid prior to substrate addition.
Fortunately, highly reproducible results were obtained by first
heating a mixture of Pd(OAc)2, (R)-BINAP, and aryl bromide
in the presence of aqueous NaHCO3, thereby trapping the
active catalyst as the stable and soluble oxidative addition
complex, followed by slow addition of the boronic acid. The
superiority of this latter procedure was confirmed in
subsequent studies of the coupling of 34 with 23 that
consistently provided high yields of 35 (82−93%) on scales up
to 25 g. Finally, the oxidative addition complex 36 was isolated
in excellent yield (91%) from the reaction of (R)-BINAP,
Pd(OAc)2, and 32 in the absence of boronic acid, and 36 was
demonstrated to be catalytically competent in the coupling of
32 with 14 (TON = 60, Supporting Information Figure S4). In
comparison, the oxidative addition to 32 employing Pd2dba3
and (R)-BINAP(O) furnished 36 in a more modest yield
(24%), although it is worth highlighting the simplicity and
effectiveness of the “dump-and-stir” Pd2dba3/BINAP(O)
method, which may be preferable for small-scale experiments.
Biaryl 33 was converted to the macrolactamization precursor
42 as shown in Figure 4. The A ring Cbz protecting group was
exchanged for Alloc (H2, Pd/C; AllocCl, 83% for 2 steps) to
allow eventual amine deprotection under conditions that avoid
reduction of the C ring nitro group. Subsequent saponification
(LiOH, 99%), coupling of the carboxylic acid with 11
(DMTMM, 91%), Alloc deprotection (PhSiH3, Pd(PPh3)4,
91%), and methyl ester saponification (LiOH, 99%) provided
Although not incorporated into their total synthesis of
vancomycin, Nicolaou and co-workers later reported56 an
atroposelective Suzuki biaryl coupling conducted on model
substrates similar to our own that was mediated by (R)-BINAP
and Pd(OAc)2 (3:1 ligand:Pd). We suspected that the active
catalyst was actually the palladium-ligated bisphosphine mono-
oxide of (R)-BINAP [(R)-BINAP(O)−Pd0] based on the
frequently underappreciated role of such ligand complexes57,58
that are most often unknowingly generated in situ. Consistent
with this proposal, the coupling of 32 with 14 that employed a
59
2:1 combination of (R)-BINAP(O):Pd2dba3 (1:1 ligand:Pd)
afforded biaryl 33 with a consistently high yield and
atroposelectivity (>20:1 dr, up to 89% yield on 8 g scale)
(Figure 3). In contrast, the combination of (R)-BINAP and
Pd2dba3 was ineffective, confirming that mono-oxidation of the
BINAP ligand was essential to catalytic activity. While the (R)-
BINAP(O):Pd2dba3 system provided more than sufficient
material for initial studies, the use of a noncommercial ligand
as well as the variable quality of commercial Pd2dba3 (Pd-black
contamination)60 introduced practical challenges that we
hoped to circumvent by direct in situ generation of the
D
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provided the highest yields, particularly under conditions that
promote fast cyclization (Base: iPr2NEt > NMM. Solvent:
NMP (N-methylpyrrolidone) > DMF > MeCN.). These
studies culminated in a rapid macrolactamization reaction
that is complete on addition, conducted at a practical
concentration (0.1 M), and capable of furnishing 42 in 61%
yield on 10 g scale with negligible (≤2%) epimerization.
Subsequent Boc deprotection (HCO2H) of 42 and coupling of
the free amine with the D ring subunit carboxylic acid 12
(DEPBT, 72% for 2 steps) set the stage for studies on the key
CD ring closure.
The proposal to use the AB macrocycle as an element of
preorganization for the CD ring closure was first validated by
an improvement in the CD cyclization dr from 1:142 in the
absence of the AB macrocycle to 3:1 in its presence with
preferential formation of natural atropisomer 46b when the C
ring alcohol was protected as a TBS ether. The macro-
cyclization of substrate 45b was also unusually facile,
proceeding readily at 23 °C in DMF (Scheme 2).
Unfortunately, thorough screening of solvent, base, temper-
ature, and additives did not improve the reaction diaster-
eoselectivity beyond 3:1. Moreover, the TBS ether proved
sensitive to desilylation as noted in our previous work.
Conducting the cyclization on the unprotected alcohol 45a
reduced the diastereoselectivity to 2:1, suggesting that the
sterically bulky TBS ether improved the SNAr atroposelectivity.
Indeed, a progressive increase in the size of the silyl ether
protecting group improved the diastereoselectivity (TBS, 3:1
dr; TBDPS, 5:1 dr; TIPS, 7:1 dr). As an added benefit, TIPS-
protected 45d was considerably less prone to desilylation than
either 45b or 45c, although strict anhydrous conditions were
still required for optimal results. The challenge of installation
and removal of even larger silyl ether protecting groups (e.g.,
BIBS66 and supersilyl), as well as their potential to further
decelerate the macrolactamization, led us to select the TIPS
ether as the preferred alcohol protecting group.
A similar study of the CD macrocyclization of substrates
47b−d prior to macrolactamization of the AB ring system
displayed the same trends, where increasing the size of the silyl
ether protecting group improved the selectivity, although
diastereoselections were lower than those obtained for
substrates 45a−d that contain the intact AB ring system
(Scheme 2). In addition, the cyclizations of 47b−d proceeded
2−3 times more slowly than 45b−d bearing the intact AB ring
system under identical conditions (Cs2CO3, DMF, 23 °C, 10 h
vs 3−5 h, respectively). Collectively, these studies illustrate
that the rate and diastereoselectivity of the CD ring closures of
45a−d benefit from a combined preorganization provided by
the AB ring system, which preferentially adopts an inherent cis
amide conformation6 poised for cyclization and a subtle
tunable substituent effect that sterically further directs the nitro
group distal to the large silyl ether. An analogous study of the
SNAr cyclization of the isolated DE ring system (substrates
49a−d) demonstrated that an increase in the size of the silyl
ether protecting group on the E ring hydroxyl also
progressively favored the natural DE atropisomers 50a−d.
However, the diastereoselectivities achieved were modest, and
only the TIPS-protected substrate 49d favored formation of
the natural atropisomer (1.1:1 dr), whereas all other substrates
examined slightly favored formation of the unnatural DE
atropisomer (1:1.7−1:1.3 dr). Nonetheless, the consistent
correlation between the size of the silyl ether protecting group
and the reaction diastereoselectivity was ultimately key to
Figure 4. (Top) Synthesis of the AB ring system. (Bottom)
Optimization of the macrolactamization reaction.
41. Macrolactamization of 41 under previously optimized
conditions36 [3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-
4(3H)-one (DEPBT),61 N-methylmorpholine (NMM), 1
mM in THF, 3:1 dr (inseparable), 48% combined yield]
proved disappointing. Prior iterations on this route with
smaller C ring alcohol silyl ether protecting groups (TBS,
TBDPS) did not suffer from low yields or competitive
epimerization (60%, no epimerization and 60%, 13:1 dr,
respectively), demonstrating that the increased steric bulk of
the TIPS protecting group impeded productive cyclization.
However, TIPS protection proved essential to the success of a
subsequent diastereoselective CD ring closure, requiring an
improved method for the macrolactamization of 41 that avoids
epimerization of the C ring α-stereocenter.
Extensive reagent screening identified DMTMM62 (43) and
its PF6− salt63 (DMTMMH) (44), PyOxim,64 and COMU65 as
the only reagents capable of suppressing epimerization (>95:5
dr) in the macrolactamization of 41, consistent with their
capabilities for promoting the couplings of hindered carboxylic
acids (Figure 4). Further optimization of solvent, base, and
reagent under simulated high-dilution conditions (i.e., slow
addition of substrate to reagents) revealed that DMTMMH
E
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Scheme 2. CD and DE Ring Macrocyclization Studies
realizing the highly diastereoselective construction of the CD
and DE biaryl ethers.
Scheme 3. Completion of Formal Total Synthesis
Completion of the formal synthesis required introduction of
the DE ring system (Scheme 3). The high diastereoselection
(8:1) obtained for the room-temperature DE ring closure in
our previous work6 with substrates bearing the intact ABCD
ring system was observed with the E ring free alcohol substrate
(7:1) herein and was further improved to 11:1 by the subtle
impact of TIPS protection of the E ring hydroxyl group,
mirroring the results obtained above. Simultaneous C/E ring
TIPS deprotection (Bu4NF/HOAc, 98%), nitro group
reduction (Zn/NH4Cl), and double-Sandmeyer chlorination
(tBuONO, HBF4; CuCl/CuCl2, 54% for 2 steps) provided
known intermediate 56, thereby completing a formal total
synthesis of vancomycin aglycon 10 in 26 projected steps6
from the individual amino acid subunits and 0.7% overall yield
(major diastereomer only) without thermal atropisomer
equilibrations. This result compares favorably with our
previous synthesis,6 which proceeded in 25 steps but with a
more modest 0.2% overall yield when material recovered by
thermal atropisomer equilibrations is excluded.
Concurrent with the above efforts, we undertook further
optimization of the Sandmeyer chlorination to minimize
protodediazoniation, which in practice often leads to copolar
deschloro reaction byproducts. In previous efforts, we
mitigated this issue by employing a large excess of CuCl/
CuCl2 in a mixed CH3CN/H2O solvent system at low
temperature.39,42 In these prior studies, we also observed
that Sandmeyer chlorination of the C ring substituent was
especially challenging, whereas the less hindered D ring
substitution was more straightforward. In this work,
Sandmeyer substitution of model substrate 57 for the
challenging C ring substitution was problematic at 0 °C (3:1
58:59, 65%) (Figure 5). Further lowering the reaction
temperature (−35 °C) suppressed protodediazoniation to
acceptable levels (18:1), although the more difficult model
F
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Figure 5. Model studies of the Sandmeyer chlorination reaction.
t
General conditions: HBF4 (1.3 equiv), BuONO (1.3 equiv), solvent
1, 0 °C for 30 min, cooled to the specified temperature, treated with
CuCl (50 equiv) and CuCl2 (60 equiv) in solvent 2, and warmed to
room temperature over 1 h. Product ratios were determined by LC/
MS analysis of the crude reaction mixture. Isolated yields are given in
parentheses.
substrate 60 bearing the fully functionalized ABCD ring system
yielded a less satisfactory but good 9:1 ratio of 61:62 under
identical conditions. Disappointingly, inclusion of CCl4 as an
additional chlorine atom donor increased the amount of
reduction byproduct by >2-fold, contrary to earlier claims by
Zhu.67 Fortunately, the product ratio improved >3-fold (to
>32:1) in CD3CN at −35 °C, indicating that reduction
primarily occurs by solvent acetonitrile C−H abstraction. A full
study of factors influencing the reaction is summarized in
Supporting Information Figure S5. These optimal conditions
(CD3CN, −35 °C) translated smoothly to the simultaneous
double-Sandmeyer substitution of substrate 72 bearing the
fully functionalized ABCDE ring system in the further
streamlined total synthesis below, providing the dichlorinated
product 73 in 63% yield. Importantly, CD3CN is inexpensive
and available in bulk quantities, such that its use in the
Sandmeyer chlorination of 1 g of a late-stage intermediate such
as 72 would cost only $20.
Next-Generation Total Synthesis of Vancomycin.
With high kinetic diastereoselectivity achieved for all three
atropisomer elements as well as access to large quantities of the
amino acid subunits, we proceeded to streamline the approach
in anticipation of preparing significant quantities of pocket-
modified analogues by total synthesis. Foremost among our
concerns with the redesigned formal synthesis above were the
step count (26 steps LLS), low overall yield (<1%),
unnecessary A ring amine protecting group exchange, and
late-stage conversion of the C-terminus MEM ether to the
carboxylic acid. Although the latter feature was used to
preclude C-terminus epimerization of a precursor methyl ester
in prior studies, it introduced an inefficient deprotection−
oxidation sequence (53%, 4 steps)6 conducted toward the end
of the synthesis that is even more challenging for substrates
Figure 6. (Top) Substituent impact on macrolactamization of the AB
ring system. (Bottom) X-ray crystal structure of 66.
containing the oxidation-prone residue 4 thioamide36 used to
access pocket-modified analogues.
We incorporated the A ring subunit as the unprotected
alcohol 23 also now bearing an orthogonal trifluoroacetamide
protecting group (Scheme 4). The use of 22 in a direct, one-
pot borylation/atroposelective Suzuki coupling with BC
dipeptide 34, prepared by coupling 11 with 15 (DMTMM,
88%), provided 35 in superb yield and outstanding
diastereoselectivity (93%, unnatural atropisomer not detected),
the development of which is detailed in Figure 3. To date, this
reaction has been scaled to 25 g without loss of efficiency and
avoids isolation and handling of the sensitive boronic acid 23.
Subsequent primary alcohol oxidation and esterification
(TEMPO, PhI(OAc)2;68 tert-butyl trichloroacetimidate,69
82% for 2 steps, 26 g scale) secured the C-terminus in the
required oxidation state in high yield and at an early stage prior
to introduction of the D ring subunit (residue 4) that will bear
a key thioamide in future syntheses of pocket-modified
analogues. Alternative efforts to incorporate the C-terminus
tert-butyl ester into the A ring subunit prior to the biaryl
coupling resulted in racemization in studies conducted to date.
Through judicious choice of the amine protecting group,
simultaneous single-step deprotection of the C ring methyl
ester and A ring trifluoroacetamide of 64 (LiOH, 82%)
provided 65 without competitive tert-butyl ester hydrolysis and
G
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Scheme 4. Next-Generation Vancomycin Total Synthesis
set the stage for closure of the AB ring system. Gratifyingly, the
macrolactamization conditions developed herein (see Figure
6) proved most effective for 65 bearing the C-terminal tert-
butyl ester, despite the more electron-deficient nature of the
reacting amine. Not only did the macrocyclization of 65
(DMTMMH, iPr2NEt, NMP) proceed essentially instanta-
neously to provide 66 in higher yield (up to 85% yield, on
scales of up to 16.4 g) than the C-terminal MEM ether (61%
for 42) but alternative substrates that bear a C-terminal free
alcohol or TBS-protected alcohol also cyclized in inferior yields
(33% and 12%, respectively), revealing that the substrate
containing the least nucleophilic amine (65) cyclized most
effectively (Figure 6). Although this trend may appear
counterintuitive, adducts of the less effective starting amines
with the coupling reagent DMTMMH were invariably
observed, indicating that an amine triazinylation side reaction
was competitive with macrocyclizations of substrates bearing
more nucleophilic amines. Finally, the structure, relative
stereochemistry, and cis amide linking residues 5 and 6
(vancomycin numbering) were confirmed in a single-crystal X-
ray structure determination of 66 (Figure 6).70 In addition, the
suggested distal disposition of the large TIPS ether and aryl
nitro substituent poised for diastereoselective CD ring closure
was observed in the X-ray crystal structure of 66. In total, the
AB macrocycle was prepared in 53% yield (5 steps),
representing a significant improvement over the formal
synthesis above (37%/8 steps) with the added advantage of
early-stage oxidation of the C-terminus that avoids 4 additional
late-stage steps in the overall synthesis.
Completion of our next-generation total synthesis of
vancomycin aglycon 10 is outlined in Scheme 4. Selective N-
Boc deprotection of 66 under conditions that may permit
reversible tert-butyl ester deprotection/reprotection69 (H2SO4,
tBuOAc, 86% yield, 98% brsm, 6.9 g scale) followed by
coupling of amine 67 with the D ring subunit 12 provided 68
(88%) poised for CD ring closure. Further refinement of
conditions for the room-temperature macrocyclization of this
substrate provided the ABCD ring system 69 in superb yield
and excellent diastereoselectivity (84%, 8:1 dr) without
detectable epimerization of the C-terminal tert-butyl ester.
Attentive reaction monitoring permitted N-Boc deprotection
of 69 without significant tert-butyl ester deprotection (5%
TFA-CH2Cl2, 87%). Coupling of the amine 70 with the E ring
tripeptide 51 promoted by DMTMM provided 71 in excellent
yield with minimal epimerization of the β-cyanoalanine residue
(6:1 dr, 68% major diastereomer) that is ordinarily problem-
atic for this segment coupling.36,10 Room-temperature macro-
cyclization of the DE ring system proceeded with near
exclusive formation of the desired DE atropisomer (14:1 dr)
in high yield (82%), where the rate and substrate-controlled
diastereoselectivity benefit from both the preorganization
provided by the ABCD ring system observed in prior studies6
as well as the now added influence of the E ring TIPS alcohol
protecting group discovered in this work. Dual nitro group
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reduction followed by Sandmeyer chlorination under the
conditions developed herein (tBuONO/HBF4; CuCl/CuCl2,
CD3CN:H2O, −35 °C) yielded dichloride 73 (63% for 2
steps), and this proved more effective than Sandmeyer
substitution following removal of the two TIPS protecting
groups. Without optimization, final conversion to vancomycin
aglycon 10 was conducted in a now simplified three-step, one-
pot sequence consisting of desilylation (Bu4NF/HOAc, THF,
23 °C), nitrile hydration with concomitant Boc and tert-butyl
ester deprotections (neat TFA, 23 °C), and global O-
demethylation (AlBr3, EtSH, 23 °C; 53−58% for 3 steps
with removal of 9 protecting groups). This next-generation
total synthesis of vancomycin aglycon was completed in 17
steps and in 5% overall yield from the amino acid subunits with
excellent kinetic diastereoselectivity achieved for introduction
of each atropisomer element.
teria. It is possible that such durable analogues that act by up
to three MOAs might provide extraordinarily potent anti-
biotics, which display clinical lifetimes measured not in
decades, or even the half-century of vancomycin, but perhaps
in centuries. The total synthesis of vancomycin detailed herein
provides the opportunity to prepare and assess such
analogues.76,77
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c07433.
1
Full experimental details and copies of H, 13C, and 2D
NMR spectra (PDF)
Just as importantly, we previously implemented a practical
solution to what many may think would be the most
challenging feature of developing synthetic glycopeptide
antibiotics, the late-stage steps used for scalable conversion
of vancomycin aglycon to the fully decorated natural product
or its analogues. This entailed enzymatic glycosylation of the
aglycon(s) with the biosynthetic enzymes (glycosyltransferases
GtfE and GtfD)71−73 and commercial (UDP-glucose)74 or
readily accessible (UDP-vancosamine)75 glycosyl donors for
introduction of the disaccharide.8 The two enzymes have been
stably overexpressed in E. coli by Walsh, making them readily
available as reagents for use on any scale, like the UDP-glycosyl
donors.71−73 Moreover, this utilizes the fully functionalized
aglycon without introduction or removal of protecting groups
and was found to work effectively on the vancomycin aglycon
residue 4 thioamide that serves as our immediate precursor to
binding pocket analogues.8,38 Combined, this provides a 19-
step total synthesis of vancomycin 1 that proceeds in 3.7%
overall yield, substantially improving on prior approaches
(Figure 7).
AUTHOR INFORMATION
■
Corresponding Author
Dale L. Boger − Department of Chemistry and Skaggs Institute
for Chemical Biology, The Scripps Research Institute, La Jolla,
California 92037, United States; orcid.org/0000-0002-
3966-3317; Email: dale.boger@outlook.com
Authors
Maxwell J. Moore − Department of Chemistry and Skaggs
Institute for Chemical Biology, The Scripps Research Institute,
La Jolla, California 92037, United States; orcid.org/0000-
0003-4001-5330
Shiwei Qu − Department of Chemistry and Skaggs Institute for
Chemical Biology, The Scripps Research Institute, La Jolla,
California 92037, United States
Ceheng Tan − Department of Chemistry and Skaggs Institute
for Chemical Biology, The Scripps Research Institute, La Jolla,
California 92037, United States
Yu Cai − Department of Chemistry and Skaggs Institute for
Chemical Biology, The Scripps Research Institute, La Jolla,
California 92037, United States; orcid.org/0000-0001-
9140-4235
Yuzo Mogi − Department of Chemistry and Skaggs Institute for
Chemical Biology, The Scripps Research Institute, La Jolla,
California 92037, United States
D. Jamin Keith − Department of Chemistry and Skaggs Institute
for Chemical Biology, The Scripps Research Institute, La Jolla,
California 92037, United States; orcid.org/0000-0003-
4611-927X
Figure 7. Comparison of the total syntheses of vancomycin (1),
highlighting the late-stage enzymatic glycosylation. Yield and LLS
start from individual amino acid subunits.
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c07433
CONCLUSION
■
Notes
Despite the exceptional antimicrobial potency and durability of
the pocket-modified vancomycin analogues discovered by our
group, their preclinical evaluation has been slowed by the
challenges presented by their total synthesis. Herein, we
address this issue through the development of a scalable
atroposelective total synthesis of vancomycin that considerably
reduced the step count and increased the overall yield. With
this new approach in hand, our efforts are focused on the
preparation of new durable vancomycin analogues (e.g., 9) and
preclinical evaluation of the most promising candidates,
including those that express three synergistic mechanisms of
action (MOAs), two of which are independent of D-Ala-D-Ala/
D-Lac binding and each of which is independently effective
against vancomycin-resistant and vancomycin-sensitive bac-
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge financial support from the NIH
(CA041101, D.L.B.), postdoctoral support from Nippon
Chemiphar (Y.M.), and graduate fellowship support from the
Kellogg family (M.J.M.). We especially acknowledge and thank
Dr. Shouliang Yang for contributions to the synthetic studies
early in development, Dr. Jason Chen, Brittany Sanchez, and
Emily Sturgell for assistance with HRMS, LC, and chiral SFC
analysis, Dr. Thomas Razler (Bristol Myers Squibb) for helpful
discussion and for generously providing ligands, Dr. Dee
Huang and Dr. Laura Pasternack for NMR assistance, and
I
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determination of 66.
(23) Binda, E.; Marinelli, F.; Marcone, G. L. Old and new
glycopeptide antibiotics: Action and resistance. Antibiotics 2014, 3,
572−594.
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of key 1H−1H ROESY NMR spectra displaying the data are
presented.
L
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