Structure diversification of vancomycin through peptide-catalyzed, site-selective lipidation: A catalysis-based approach to combat glycopeptide-resistant pathogens

Article abstract of DOI:10.1021/jm501872s

The emergence of antibiotic-resistant infections highlights the need for novel antibiotic leads, perhaps with a broader spectrum of activity. Herein, we disclose a semisynthetic, catalytic approach for structure diversification of vancomycin. We have identified three unique peptide catalysts that exhibit site-selectivity for the lipidation of the aliphatic hydroxyls on vancomycin, generating three new derivatives 9a, 9b, and 9c. Incorporation of lipid chains into the vancomycin scaffold provides promising improvement of its bioactivity against vancomycin-resistant enterococci (Van A and Van B phenotypes of VRE). The MICs for 9a, 9b, and 9c against MRSA and VRE (Van B phenotype) range from 0.12 to 0.25 μg/mL. We have also performed a structure-activity relationship (SAR) study to investigate the effect of lipid chain length at the newly accessible G₄-OH derivatization site.

Full text of DOI:10.1021/jm501872s

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Article
Structure diversification of vancomycin through peptide-
catalyzed, site-selective lipidation: A catalysis-based
approach to combat glycopeptide-resistant pathogens
Sabesan Yoganathan, and Scott J. Miller
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501872s • Publication Date (Web): 11 Feb 2015
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Structure diversification of vancomycin through peptide-catalyzed, site-selective
lipidation: A catalysis-based approach to combat glycopeptide-resistant pathogens
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Sabesan Yoganathana,b and Scott J. Miller*a
aDepartment of Chemistry, Yale University, P.O. Box 208107, New Haven, CT 06520-
8107
b Current address: Department of Pharmaceutical Sciences, St. John’s University, 8000
Utopia Parkway, Queens, NY 11439
*To whom correspondence should be addressed. e-mail: scott.miller@yale.edu
KEY WORDS: vancomycin, glycopeptides, infectious diseases, antibiotics, peptide
catalysis, site-selective chemistry, vancomycin-resistant bacteria
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ABSTRACT
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Emergence of antibiotic-resistant infections highlights the need for novel
antibiotic leads, perhaps with a broader spectrum of activity. Herein, we disclose a
semisynthetic, catalytic approach for structure diversification of vancomycin. We have
identified three unique peptide catalysts that exhibit site-selectivity for the lipidation of
the aliphatic hydroxyls on vancomycin, generating three new derivatives 9a, 9b and 9c.
Incorporation of lipid chains into vancomycin scaffold provides promising improvement
of its bioactivity against vancomycin-resistant enterococci (Van A and Van B phenotypes
of VRE). The MICs for 9a, 9b, and 9c against MRSA and VRE (Van B phenotype) range
from 0.12 to 0.25 µg/mL. We have also performed a structure activity relationship (SAR)
study to investigate the effect of lipid chain length at the newly accessible G4-OH
derivatization site.
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INTRODUCTION
Natural products and their structural derivatives continue to be the lead
therapeutics for the treatment of various diseases.1-3 In particular, numerous antibiotics
approved for clinical applications have been either natural products or their derivatives.4
Emerging problem of drug resistance highlights the need for new drug leads to combat
infectious diseases, perhaps via development of novel synthetic analogues of existing
bioactive molecules.4-6 Synthetic endeavors to modify natural products can be a
challenging task due to their structural complexity and the presence of a large array of
reactive functional groups.2,7 As such, efficient and highly selective approaches that
employ minimal protecting group manipulations are desirable. In recent years, catalyst
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dependent diversification of complex drug molecules has gained considerable interest.8-11
Many important approaches, including biocatalysis, may be brought to bear on the
problem.12
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Our research group (Figure 1)13-18 and others19,20 have demonstrated that synthetic
oligopeptide-based catalysis enables a wide range of chemical transformations to
derivatize polyol natural products of medicinal interest.10,21,22 For example (Figure 1a),
we have developed peptide-based catalyst wherein the critical catalytic residue
p(methyl)-histidine (Pmh) is central to the selective transfer of acyl groups,13 phosphoryl
groups,23 sulfur-based electrophiles24,25 to individual sites in structures containing
multiple hydroxyl groups. As an example in the context of complex glycopeptides like
vancomycin (1a), we also showed that catalytic thiocarbonylation – as a prelude to site-
selective deoxygenation – could be enabled with the Pmh-based catalyst construct,
allowing excision of either the G6-oxygen atom, or the Z6-oxygen atom, in a catalyst-
dependent manner (Figure 1b).15
Please Insert Figure 1
Figure 1. (a) A schematic outline of various group transfer reactions catalyzed by Pmh-
based peptides for polyol modifications. (b) A representative set of catalyst-dependent
maneuvers leading to two unique deoxygenation vancomycin analogues.15
Catalytic, site-selective acyl transfer reactions of long-chain fatty acid
functionality (i.e., lipidation) present a particularly intriguing avenue for the synthesis of
positional isomers of lipidated analogues of glycopeptide-based polyols. Lipid groups in
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lipopeptide and lipoglycopeptide antibacterial natural products have been implicated in
their potency26-29 and such modifications enable medicinal chemists to tune the bioactivity
of antibacterial natural products.28,30,31 We describe in this paper several advances in the
use of Pmh-based catalysts for the preparation of site-selectively lipidated vancomycins,
along with data on their biological activity.
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The glycopeptide vancomycin and the teicoplanins, lipoglyco-peptides (Figure 2,
1a and 2) are potent antibiotics for the treatment of several drug resistant bacterial
infections, including methicillin-resistant Staphylococcus aureus (MRSA).26,32,33
Glycopeptides and lipoglycopeptides bind to the ‘Lys-DAla-DAla’ peptide region of
peptidoglycan monomer, which leads to inhibition of bacterial cell wall biosynthesis, and
cell death. Due to its unique mode of action and potency, vancomycin has been an
antibiotic of interest for synthetic modifications in order to target drug-resistant
pathogens.7,32,34-40 In particular, several analogues containing lipid motifs at the
chemically most accessible positions on vancomycin (V3-NH2, X1-NHMe, or G6-OH,
Figure 2) have been reported, which exhibit improved antibacterial activity against
glycopeptide-resistant bacteria.34,35,37
Please Insert Figure 2
Figure 2. Structures of vancomycin (1a), a G6-modified vancomycin (1b), and
teicoplanin A2-2 (2)
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Differentiating the reactivity of six aliphatic hydroxyls, the phenolic OH-group
and the resorcinol-like OH-groups on vancomycin is a challenging task. This has
restricted comprehensive structural modifications on the peripheral hydroxyls of
vancomycin to date. Moreover, limited knowledge is available regarding the importance
of these functional groups on structure and bioactivity. Our recent study of the catalytic
synthesis of two deoxy-vancomycin analogues noted above revealed biological
consequences.15 Z6-Deoxy-vancomycin exhibits conformational heterogeneity, and
modified potency in MIC assays. We speculate that additional perturbations to the
hydroxyl group array of vancomycin would also unveil additional modulation of
biological activity. Related observations followed our discovery of catalysts for the site-
selective phosphorylation of hydroxyls within the teicoplanin A2-2 structure.18
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Moreover, based on the heightened bioactivity of alkyl- and acyl-vancomycin
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analogues and teicoplanins,
we hypothesized that site-selective lipidation of
vancomycin would yield structurally diverse and biologically promising analogues. In
this vein, previous studies suggest that hydrophobic groups on vancomycin greatly
improve its potency via at least two distinct proposed mechanisms: (i) enhancement of
proximity induced binding of vancomycin analogues to peptidoglycan monomer via
bacterial cell membrane association31,43,44; (ii) binding of lipidated vancomycin
derivatives to transglycosylase and inhibition of cross-linking of disaccharides during
peptidoglycan biosynthesis.45-47 The research described below culminates in the discovery
of three unique peptide-based catalysts for site-selective incorporation of lipid motifs at
the G6, G4, and Z6 positions on vancomycin. Each is portable for the transfer of acyl side
chains of varying length, leading to a library of previously unknown vancomycin
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analogues. Their structural characterization and the antibacterial activity are also
disclosed.
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RESULTS AND DISCUSSION
Chemical catalysis
Based on previous studies, we began with the synthesis of a partially allyl-
protected vancomycin (3) in order to gain desirable solubility in organic solvents, and
prevent undesired reactions with amines, phenols or resorcinol-like OH-groups of 1a.48
Since vancomycin is readily available from commercial sources, we synthesized 3 on
multi-gram scale (15 g) via a sequential formation of alloc-carbamates, ally-ester and
allyl-ethers.15,48,49 From our initial screening, we found that symmetric anhydrides worked
well for the acyl-transfer reactions; while acyl chlorides showed unselective reactivity
and N-hydroxysuccinimide active esters were not reactive under the reaction conditions.
Under optimized reaction conditions, 3 exhibited no reactivity towards an
anhydride in the absence of a nucleophilic catalyst, and negligible reactivity in the
presence of N-methylimidazole (NMI) as an achiral control catalyst. Once we established
a sense of the background reactivity, we investigated a library of peptide-based catalysts
for the acyl-transfer reaction. All of these peptides contain the catalytic amino acid Pmh,
which assists in the acyl transfer reaction. For this study, we investigated two parallel
approaches to identify catalysts to achieve site-selective lipidation: (i) screening of
catalysts from legacy peptide-based catalysts in our laboratory. (These catalysts and their
analogues have been previously explored for regioselective or enatioselective
reactions.50,51), and (ii) Screening of catalyst structures designed by placing the Pmh
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residue within the ‘Lys-DAla-DAla’ scaffold. Analysis of a X-ray crystal structure of
vancomycin-ligand complex (Figure 3)52 and our previous findings15 suggest that
placement of the Pmh residue proximal to disaccharides on vancomycin often yields
selective modification of the hydroxyls based on this binding model.15-18
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Please Insert Figure 3
Figure 3. (a) a co-crystal structure of the ligand, N-Ac-Lys(Ac)-DAla-DAla (yellow)
bound to vancomycin and (b) a drawing of a segment of a designed catalyst over the
ligand (PDB 1FVM).52
Since numerous lipopeptides and lipoglycopeptides (i.e. daptomycin,
teicoplanins) generally contain a decanoyl-group as the lipid motif, we began the
lipidation chemistry using decanoic anhydride as the longest lipid chain. We first
screened a collection of 25 catalysts from the legacy peptide library. Starting material 3
was reacted with decanoic anhydride (2 equiv), 1,2,2,6,6-pentamethylpiperidine (PEMP,
2 equiv), and catalyst (20 mol%) in 3:1 CH2Cl2/THF (0.007 M) for 24 h (Figure 4 and
Scheme 1). Intriguingly, the peptide catalysts exhibited superior reactivity compared to
NMI, and provided reasonable conversion to the decanoyl-vancomycin derivatives (See
Supporting Information for details). In fact, we identified several catalysts that yielded a
mixture of G6 and G4-products in very high conversion, including catalyst 5. Each
product was characterized using a combination of LC-MS/MS fragmentation analysis,
and 2D-NMR spectroscopy to elucidate the site of modification (see Supporting
Information).
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Please Insert Scheme 1
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Scheme 1. Exploration of catalysts for site-selective modification of 3.
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Please Insert Figure 4
Figure 4. RP-HPLC traces for uncatalyzed, NMI (0.2 equiv) catalyzed, and peptide 5
catalyzed reactions of 3 using decanoyl anhydride.
Within the first set of legacy library, we were pleased to identify catalysts 6 and 7
for selective modification of the G4 (4a:4b:4c = 1:5:0, 87% conv. of 3) and Z6 (4a:4b:4c
= 0:1:17, 94% conv. of 3) hydroxyls respectively.53 It is worth noting that 6 and 7 were
historically relevant to us, having been previously identified as hit catalysts for the kinetic
resolution of formamides,54 and for the asymmetric phosphorylation of myo-inositol,23
respectively. Moreover, 7 was also a key catalyst for the Z6-selective thiocarbonylation of
vancomycin.15 Interestingly, no catalyst from the legacy peptide library delivered G6-
selectivity. Thus, we turned our attention to the second approach, where rationally
designed ‘Xaa-DAla-DPmh’ type catalysts were evaluated. As this approach had been
successful previously, allowing observation of high selectivity for G6-position for
thiocarbonylation, we hoped its selectivity would carry over to the transfer of a different
reagent. Strikingly, catalyst 8, which was a hit catalyst for the G6-selective
thiocarbonylation15 also yielded good selectivity for the acylation of the G6-position
(4a:4b:4c = 6:1:0, >99% conv. of 3).53 It is gratifying that we rediscovered new reactivity
and selectivity for 6, 7 and 8, which suggests that these ‘legacy catalysts’ are very
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versatile for various group transfer reactions. This state of affairs is observed in some
cases as different types of electrophiles, but not all, as we have observed previously.15
Nevertheless, a set of catalysts selective for three distinct hydroxyl groups within
vancyomycin (G6-, Z6- and G4-, with G4- being previously unknown in our experience)
represents an expansion of catalytic capabilities for derivatization of this particular
structure.
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Please Insert Figure 5
Figure 5. Structures of catalysts identified for selective lipidation.
Please Insert Figure 6
Figure 6. RP-HPLC traces for catalyst dependent reactivity of 3 with decanoyl
anhydride. (a) G4-selective catalysis with 6, (b) Z6-selective catalysis with 7, and (c) G6-
selective catalysis with 8.
Although the catalyst dependent acyl-transfer reaction enables modification of the
various hydroxyls, we wanted to confirm that the selectivity observed is not due to an
internal, intramolecular acyl group migration among the hydroxyls within a glucose ring.
More specifically, we wanted to ensure that conversion of 4a into 4b is not occurring
under the reaction condition. To investigate this, we prepared pure samples of 4a and 4b
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and subjected them to the reaction condition (Scheme 2). Since 2 equiv of PEMP was
used for the optimized acylation reaction to modify vancomycin, we subjected the
products (4a and 4b) to the same reaction condition to evaluate the acyl migration
pathway. We were pleased to find that 4a remained stable in the presence of up to 2 equiv
of PEMP for 20 h, suggesting that the G6-acyl group (4a) is not migrating to G4-position
(4b). When 4b was treated with 2 equiv of PEMP, it was stable for up to 5 h; however, at
20 h, 4b progressively equilibrated to 4a (4a: 4b = 1:1, see supporting materials),
indicating under strong basic conditions, the acyl group at G4-position migrated to the G6-
position. Yet, when only 0.5 equiv of PEMP was used, there was no equilibration of 4b to
4a for up to 20 h. These findings suggest that the migration is not occurring under mild
basic condition (with 0.5 equiv PEMP). In addition, when acylation reactions (Scheme 1)
were monitored over 24 h, 30 h and 40 h intervals, no change in selectivity was observed,
perhaps indicative of a buffering effect wherein the RCO2H byproduct of the anhydride
inhibits migration. Taken together, we believe that both 4a and 4b are formed in a
catalyst-dependent manner under kinetic control under our catalytic reaction conditions,
and once these products are formed, they remain stable under the reaction condition.
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Please Insert Scheme 2
Scheme 2. Investigation of acyl-migration within a glucose ring.
We also performed a brief study to understand better the nature of any catalyst-
substrate complex suggestive of the possible mode of binding of 6, 7, and 8 to the
vancomycin-based substrate. From earlier work,15 we have compelling evidence that 8
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binds to the ligand binding pocket of vancomycin through a series of hydrogen bonds
with the backbone amides (Figure 3b).55 However, it is unclear if the same sets of amide
bonds are also playing a role in recruiting 6 and 7. To probe this, we performed the
acylation reactions with each catalyst in the presence of Boc-Leu-DAla-DAla-OH(1 equiv)
as a competing ligand for the binding pocket (see Supporting Information). RP-HPLC
analysis confirmed that 8 considerably lost its selectivity (4a:4b, from 6:1 to 1.6:1) in the
presence of the ligand. Moreover, 6 also lost its selectivity to a noticeable extent (4a:4b,
from 1:5 to 1:1.6). On the other hand, 7 had insignificant effect in the presence of the
competing ligand. These results further support the hypothesis that ‘Xxa-DAla-DPmh’
type catalysts interact with the substrate in the canonical ligand binding pocket, and these
interactions contribute to high selectivity. As for the catalysts from the legacy library (6
and 7), it is difficult to propose a binding model for the observed selectivity, as we have
limited knowledge from these experiments.
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With the discovery of catalysts 6, 7, and 8, desired mono-acylated products were
synthesized in sufficient amounts (100 mg scale reaction) for structural characterization
and biological studies. Since starting material 3 is available in multi-gram scale, the
lipidation reactions can be done in gram-scale quantity to access these derivatives if
necessary (See Supporting Information for details). Following selective acylation, the
compounds were purified via RP-MPLC, then the allyl/alloc groups were removed
reliably under standard conditions (Pd(PPh3)2Cl2 and PhSiH3; Scheme 3). A final RP-
HPLC purification yielded the decanoyl-vancomycin analogues 9a (17 mg, 38% over two
steps), 9b (32 mg, 24% over two steps) and 9c (11 mg, 24% over two steps) in greater
than 95% purity based on RP-HPLC analysis (Please see Supporting Information).
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Please Insert Scheme 3
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Scheme 3. Site-selective incorporation of decanoyl motifs into vancomycin scaffold.
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The structure of the acyl-vancomycin derivatives 9a, 9b, and 9c were all
characterized by 2D-NMR spectroscopy. Figure 7 shows an overlaid edited-HSQC
spectrum of native vancomycin (green spots), and 9b (red and blue spots). All peaks have
been assigned using a combination of HSQC, COSY, TOCSY and HMBC experiments
and verified with literature reports.15,56 There are clear changes in the chemical shifts of
glucose ring protons relative to that of native vancomycin. The G4 proton has shifted
about 1.5 ppm downfield. The G3 and G5 protons also showed noticeable downfield shifts.
The G1 and G2 protons, on the other hand, exhibit a very small downfield shift, while the
G6 protons reflects a very small up-field shift (Figure 7). We also observed a minute
perturbation of some of the amino acid α-protons, perhaps due to subtle conformational
changes introduced by the decanoyl moiety. However, almost all of the remaining
protons’ chemical shifts overlap well with that of native vancomycin. Similarly, 9a and
9c were also characterized, where the chemical shift of G6 proton on 9a and the Z6 proton
on 9c had significant downfield shift, confirming the site of modification (see Supporting
Information).
Please Insert Figure 7
Figure 7. An overlay of HSQC-DEPT spectra of vancomycin analogue 9b (methyls and
methines shown in red, and methylenes shown in blue), and native vancomycin (green).
DEPT: distortionless enhancement by polarization transfer.
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We then used the newly identified G4-selective catalyst to facilitate the synthesis
of a library of acyl-vancomycin derivatives to perform a SAR study, wherein the acyl
side chain was varied in length and composition at this newly modifiable site. Butanoyl,
pentanoyl, hexanoyl, octanoyl, pentenoyl and methyl-adipoyl moieties were selectively
incorporated at the G4 position (10 – 15, Figure 8) using catalyst 6. The different aliphatic
chains enable an interrogation of the role of chain length for antibacterial activity. We
envisioned that incorporation of a terminal alkene (14), or a carboxylate-handle (15)
could provide a useful chemical handle for further derivatization of vancomycin scaffold
as a biomarker or imaging probe.57 Catalyst 6 was highly faithful in delivering the
anticipated selectivity for G4-OH site. Each new compound was synthesized using the
established protocol with catalyst 6 (20 mol%), and isolated in pure form by RP-HPLC.
The site of modification was confirmed by LC-MS/MS analysis and multidimensional
NMR spectroscopy (see Supporting Information).
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Please Insert Figure 8
Figure 8. Structures of G4-modified vancomycin derivatives.
Antibacterial evaluation
We evaluated the set of nine new lipidated-vancomycin derivatives against a
panel of both vancomycin sensitive and vancomycin resistant strains, including
methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant
Enterococcus faecalis (VRE). The activity is reported as minimum inhibitory
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concentration (MIC) where complete killing effect was observed. Vancomycin,
teicoplanin A2-2, and linezolid were used as control compounds to compare the MIC
values. All of the biologically tested compounds are greater than 95% pure as ascertained
by RP-HPLC, as exhibited in the SI (9a, page S-8; 9b, page S-11; 9c, page S-14;
10, page S-16; 11, page S-16; 12, page S-17; 13, page S-17; 14, page S-18;
15, page S-18). All decanoyl-vancomycin derivatives (9a, 9b, and 9c) exhibited
remarkable potency against all strains tested, even against a VRE (Van B phenotype)
strain. The G4-modified analogue 9b is 10 times more active against vancomycin-
sensitive strains and 64 times more active against a VRE strain (Van B phenotype). In
fact, the values are comparable or marginally enhanced in some cases compared to
teicoplanin A2-2. Remarkably, derivatives 9a, 9b, and 9c exhibit a promising activity of
16 µg/mL against Van A phenotype VRE strain as well. As a comparison, vancomycin
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and teicoplanin A2-2 exhibit an MIC value of 512 µg/mL and 128 µg/mL respectively
against Van A phenotype VRE strain based on literature data.37
We also evaluated the bioactivity of the derivatives containing shorter acyl chains
or functionalized acyl-moieties. The results indicate that analogues containing lipid chain
that is smaller than an octanoyl moiety exhibited reduced bioactivity against VRE strains.
A clear trend of loss of activity with reduced length of lipid chain is observed. However,
the bioactivity is not completely lost compared to native vancomycin. This trend may
support the previously proposed cell membrane binding model by which lipidated-
vancomycin analogues exhibit potency against VRE strains.34,35,37 Compounds 14 and 15
retained respectable bioactivity against vancomycin-sensitive strains, suggesting that
these sites are potential places for the incorporation of chemical probes for studying
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vancomycin and its biological fate.57
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Table 1. Antibacterial activity data for lipidated-vancomycin derivatives against
vancomycin-sensitive and vancomycin-resistant bacteria.
CONCLUSION
With the increased emergence of vancomycin resistance among clinically relevant
pathogens, significant effort is being directed towards identifying new vancomycin
analogues with improved bioactivity. Herein we report our discovery of three unique
peptide-catalysts that allow us to incorporate lipid moieties into vancomycin scaffold in a
highly site-selective manner. This semi-synthetic approach enables us to modify three
different positions (G4, G6, and Z6) on vancomycin. Since enhancing the bioactivity of
vancomycin via incorporating lipid motifs is of high interest in literature37,43,45,46, our
approach provides new opportunities to efficiently modify three distinct positions on
vancomycin. We also prepared a series of lipidated vancomycins derivatized at the site
for which the newest catalyst capability was achieved, the G4-OH group. The
antibacterial evaluation revealed that incorporation of a decanoyl group considerably
improves vancomycin’s activity against MRSA and VRE (Van A and Van B
phenotypes). That said, it seems that the added lipophilicity of the presently reported
vancomycin derivatives might render them more teicoplanin-like. Since teicoplanin is
known to be vulnerable to the development of resistance,58 it seems much further study
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would be required to for the presently reported compounds to contribute to useful
therapies.
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Ultimately, the site-selective catalysis approach may allow one not only to
identify biologically interesting analogues, but perhaps also to study the function of the
peripheral hydroxyl groups of vancomycin in a position-specific manner.57
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EXPERIMENTAL PROCEDURES
Materials and instrumentation. All commercially available reagents were
purchased and used without further purification, unless otherwise stated. Air and
moisture sensitive reactions were done under nitrogen atmosphere employing flame-dried
glassware. Commercially available solvents (> 99.0% purity) were used for column
chromatography without any further purification. Tetrahydrofuran (THF),
dichloromethane (CH2Cl2), and toluene were purified by a Seca Solvent Purification
System from Glass Contour (Nashua, NH). Proton and carbon NMR spectra were
recorded on a 600 MHz or 500 MHz spectrometer. Proton chemical shifts are reported in
ppm (δ) relative to residual CHCl3 (δ 7.26 ppm) or DMSO-d6 (2.50 ppm). Data are
reported as follows: chemical shift (multiplicity [singlet (s), doublet (d), doublet of
doublets (dd), doublet of doublets of doublets (ddd), triplet (t), quartet (q), quintet (p),
sextet (h), multiplet (m), apparent singlet (app. s), apparent doublet (app. d)], coupling
constants [Hz], integration). Carbon chemical shifts are reported in ppm with the
respective solvent resonance as the internal standard (CDCl3, δ 77.1 ppm, DMSO-d6, δ
40.0). Unless otherwise noted, all NMR spectra were acquired at ambient temperature.
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Infrared spectra (thin film) were recorded on a FT-IR, νmax (cm-1) and are partially
reported. Analytical thin-layer chromatography (TLC) was performed using Silica Gel 60
Å F254 precoated plates (0.25 mm thickness). The following visualization methods were
used for monitoring reactions and column chromatography: UV absorption by
fluorescence quenching; Ninhydrin spray (Ninhydrin: acetic acid: n-butanol/ 0.6 g:6
mL:200 mL) or PMA stain (phosphomolybdic acid, H2SO4). Flash column
chromatography was performed using Flash Silica Gel (32-63 micron). Mass
spectrometry data were collected using a Ultra high performance LC/MS, which was was
performed on a UPLC/MS instrument equipped with a reverse-phase C18 column (1.7
μm particle size, 2.1 x 50 mm), dual atmospheric pressure chemical ionization
(API)/electrospray (ESI) mass spectrometry detector, and photodiode array detector.
Minimum inhibitory concentrations (MICs) were determined by Micromyx, LLC
(Kalamazoo, MI) in accordance with CLSI guidelines.
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HR-LC/MS analyses were done on a Waters Acquity UPLC® instrument.
Method 1: Column = Waters Acquity UPLC® BEH C18 1.7 μm, 2.1 Å~ 100 mm;
Temperature = 25 °C; Solvent A = H2O (0.1% formic acid); Solvent B = MeCN (0.1%
formic acid); Flow Rate = 0.8 mL/min; Gradient: Start at 20% B, ramped to 100% B over
3 min, held at 100% B for 1 min. Method 2: Column = Waters Acquity UPLC® BEH C8
1.7 μm, 2.1 Å~ 100 mm; Temperature = 25 °C; Solvent A = H2O (0.1% formic acid);
Solvent B = MeCN (0.1% formic acid); Flow Rate = 0.3 mL/min; Gradient: Held at 5%
B for 1 min, ramped to 95% B over 5 min and held for 1.5 min.
RP-HPLC analysis and purification were done using these methods. Method 3:
Column = Waters SymmetryPrep C8 7 μm, 300 Å, 7.8 x 300 mm; Temperature = 25 °C;
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Solvent A = H2O (0.1% formic acid); Solvent B = MeCN (0.1% formic acid); Flow Rate
= 4.0 mL/min; Gradient: Held at 5% B for 3 min, ramped to 57% B over 17 min,
maintained at 57% B for 5 min, ramped to 99% B over 25 min, held at 99% B for 10 min,
ramped to 5% B over 2 min and held for 5 min. Method 4: Column = Waters
SymmetryPrep C8 7 μm, 300 Å, 19 x 300 mm; Temperature = 23 °C; Solvent A = H2O
(0.1% formic acid); Solvent B = MeCN (0.1% formic acid); Flow Rate = 24 mL/min;
Gradient: Held at 5% B for 3 min, ramped to 57% B over 17 min, held at 57% B for 3
min, ramped to 90% B over 20 min, held at 90% B for 3 min, ramped to 5% B over 2 min
and held for 2 min. Method 5: Column = Waters SymmetryPrep C8 7 μm, 300 Å, 19 x
300 mm; Temperature = 23 °C; Solvent A = H2O (0.1% formic acid); Solvent B = MeCN
(0.1% formic acid); Flow Rate = 24 mL/min; Gradient: Held at 2% B for 7 min, ramped
to 60% B over 20 min, ramped to 95% B over 2 min, held at 95% B for 2 min, ramped to
2% B over 2 min and held for 2 min.
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RP-MPLC purification was done on a Biotage® instrument. Method 6: Column =
Biotage SNAP C18, 120 g column with 12-g samplet; Temperature = 23 °C; Solvent A =
H2O; Solvent B = MeOH; Flow Rate = 50 mL/min; Gradient: Held at 75% B for 1
column volume (CV), ramped to 80% B over 4 CV, held at 80% B for 6 CV, ramped to
90% B over 1 CV, held at 90% B for 2 CV. Method 7: Column = Biotage SNAP C18 60
g column with 12-g samplet; Temperature = 23 °C; Solvent A = H2O ; Solvent B =
MeCN; Flow Rate = 50 mL/min; Gradient: Held at 5% B for 4 column volume (CV),
ramped to 40% B over 4 CV, ramped to 95% B over 18 CV, held at 95% B for 5 CV.
General procedure 1: Screening of peptide catalysts. In a flame-dried small
glass vial containing a stir bar, 3 (5 mg, 2.81 µmol, 1 equiv) and peptide catalyst (0.2
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equiv) were dissolved in THF (100 µL) to yield a clear, homogeneous solution. Then,
CH2Cl2 (300 µL) and PEMP (1.1 µL, 6.07 µmol, 2 equiv) were added to the vial.
Decanoic anhydride (2.1 µL, 5.62 µmol, 2 equiv) was added subsequently and the
reaction mixture was allowed to stir at rt for 24 h. An aliquot (20 µL) was taken and
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quenched with 2-propanol (500 µL), and analyzed by HR-LC/MS (Method 2) and RP-
HPLC (Method 3). These data were used for preliminary determination of conversion,
product selectivity, and site of reaction (See Supporting Information for HPLC data).
General procedure 2: Synthesis of decanoyl-vancomycin derivatives 9a-9c. In
a flame-dried 10-mL round bottom flask containing a stir bar, 3 (50 mg, 28.1 µmol, 1
equiv) and the peptide catalyst (5.69 µmol, 0.2 equiv) were dissolved in THF (1 mL) to
yield a clear, homogeneous solution. Then, CH2Cl2 (2 mL) and PEMP (56.2 µmol, 2
equiv) were added sequentially to the flask, and stirred under N2 atmosphere. A solution
of decanoic anhydride (56.2 µmol, 2 equiv) in CH2Cl2 (1 mL) was added over 10 h using
a syringe pump, and the reaction was allowed to stir for an additional 14 h. An aliquot
(20 µL) was taken and quenched with 2-propanol (500 µL), and analyzed by HR-LC/MS
(Method 2) and RP-HPLC (Method 3) to determine if the reaction was complete. At this
time, the reaction was quenched with 2-propanol (2 mL). The solution was then
concentrated in vacuo to a volume of ~1 mL and diluted with another 2 mL of 2-
propanol. The solution was loaded onto a C18 Biotage samplet and purified using RP-
MPLC (Method 7). Fractions containing the desired mono-decanoyl vancomycin
derivatives were combined and concentrated to yield the mixture of products as a white
residue.
The protected decanoyl vancomycin derivative (15.0 µmol, 1 equiv) was
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dissolved in degassed 1,4-dioxane (2 mL) in a 10-mL round bottom flask. The flask was
evacuated, and refilled with argon gas four times, and kept under argon atmosphere. To
the flask, Pd(PPh3)2Cl2 (10.6 mg, 15.1 µmol, 1 equiv), and AcOH (0.5 mL) were added
quickly, and the flask was sealed with a septum. The flask was again evacuated and
refilled with argon gas four times, and kept under argon atmosphere. Then, PhSiH3 (84
µL, 0.68 mmol, 45 equiv) was added drop wise over 30 min. The reaction was stirred for
another 2 h under argon atmosphere at rt. The reaction mixture turned from a cloudy
yellow mixture into a darker brown mixture over the course of the reaction. After 2 h of
stirring, the reaction mixture was poured into a separatory funnel containing 0.01 M
aqueous HCl (100 mL), and washed with hexanes (3 x 100 mL). The aqueous layer was
passed through a pad of celite. The filtrate was concentrated to ~20 mL, and purified
using preparative RP-HPLC (Method 5). The fractions containing the desired product
were concentrated in vacuo to remove the organic solvent, and the water was removed
via freeze-drying to yield 9a-9c a white residue in greater than 95% purity based on RP-
HPLC (Method 3), and 24 – 38% yield over 2 steps.
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Synthesis of G6-decanoyl vancomycin derivative 9a using general procedure
2. Protected G6-decanoyl vancomycin derivative was isolated as a white residue (29 mg
yield; 28.1 µmol scale reaction), which was carried forward for the next reaction.
Analytical RP-HPLC retention time of protected G6-decanoyl vancomycin derivative
(Method 3): 35.5 min; LC-MS (ESI+) for C96H117Cl2N9O29K [M+K]+: Calc’d = 1968.697;
found = 1968.696.
The deprotected G6-decanoyl vancomycin derivative 9a was isolated as a white
residue (17 mg, 38% over two steps). Analytical RP-HPLC retention time of 9a (Method
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3): 13.4 min; LC-MS (ESI+) for C76H94Cl2N9O25 [M+H]+: Calc’d = 1602.573; found =
1602.579. (See Supporting Information for HPLC & NMR data)
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Synthesis of G4-decanoyl vancomycin derivative 9b using general procedure
2. Protected G4-decanoyl vancomycin derivative was isolated as a white residue (47 mg
yield; 56.2 µmol scale reaction), which was carried forward for the next reaction.
Analytical RP-HPLC retention time of protected G4-decanoyl vancomycin derivative
(Method 3): 38.0 min; LC-MS (ESI+) for C96H117Cl2N9O29K [M+K]+: Calc’d = 1968.696;
found = 1968.695.
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The deprotected G4-decanoyl vancomycin derivative 9b was isolated as a white
residue (32 mg, 24% over two steps). Analytical RP-HPLC retention time of 9b (Method
3): 14.2 min; LC-MS (ESI+) for C76H94Cl2N9O25 [M+H]+: Calc’d = 1602.573; found =
1602.582. (See Supporting Information for HPLC & NMR data)
Synthesis of Z6-decanoyl vancomycin derivative 9c using general procedure
2. Protected Z6-decanoyl vancomycin derivative was isolated as a white residue (29 mg
yield; 28.1 µmol scale reaction), which was carried forward for the next reaction.
Analytical RP-HPLC retention time of protected Z6-decanoyl vancomycin derivative
(Method 3): 39.6 min; LC-MS (ESI+) for C96H118Cl2N9O29 [M+H]+: Calc’d = 1930.741;
found = 1930.742.
The deprotected Z6-decanoyl vancomycin derivative 9c was isolated as a white
residue (11 mg, 24% over two steps). Analytical RP-HPLC retention time of 9c (Method
3): 12.3 min; LC-MS (ESI+) for C76H94Cl2N9O25 [M+H]+: Calc’d = 1602.573; found =
1602.588. (See Supporting Information for HPLC & NMR data)
General procedure 3: Synthesis of G4-acyl vancomycin derivatives 10 – 15. In
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a flame-dried 10-mL round bottom flask containing a stir bar, 3 (100 mg, 56.2 mmol, 1
equiv) and catalyst 6 (8 mg, 11.6 µmol, 0.2 equiv) were dissolved in THF (2 mL) to yield
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a clear, homogeneous solution. Then, CH2Cl2 (5 mL) and PEMP (20.3 µL, 112.4 µmol, 2
equiv) were added sequentially to the flask, and stirred under N2 atmosphere. A solution
of anhydride (112.6 µmol, 2 equiv) in CH2Cl2 (1 mL) was added over 10 h using a
syringe pump, and the reaction was allowed to stir for an additional 14 h. An aliquot (20
µL) was taken and quenched with 2-propanol (500 µL), and analyzed by HR-LC/MS
(Method 2) and RP-HPLC (Method 3) to determine if the reaction was complete. At this
time, the reaction was quenched with 2-propanol (2 mL). The solution was then
concentrated in vacuo to a volume of ~1 mL and diluted with another 2 mL of 2-
propanol. The solution was loaded onto a C18 Biotage samplet and purified using RP-
MPLC (Method 7). Fractions containing the desired mono-decanoyl vancomycin
derivatives were combined and concentrated to yield the mixture of products as a white
residue (40 – 50% yield), which was carried forward for the next reaction.
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The protected G4-acyl vancomycin derivative (28 µmol, 1 equiv) was dissolved in
degassed 1,4-dioxane (2 mL) in a 10-mL round bottom flask. The flask was evacuated,
and refilled with argon gas four times, and kept under argon atmosphere. To the flask,
Pd(PPh3)2Cl2 (19.7 mg, 28 µmol, 1 equiv), and AcOH (0.5 mL) were added sequentially,
and the flask was sealed with a septum. The flask was again evacuated and refilled with
argon gas four times, and kept under argon atmosphere. Then, PhSiH3 (157 µL, 1.27
mmol, 45 equiv) was added drop wise over 30 min. The reaction was stirred for another 2
h under argon atmosphere at rt. The reaction mixture turned from a cloudy yellow
mixture into a darker brown mixture over the course of the reaction. After 2 h of stirring,
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the reaction mixture was poured into a separatory funnel containing 0.01 M aqueous HCl
(200 mL), and washed with hexanes (3 x 100 mL). The aqueous layer was passed through
a pad of celite. The filtrate was concentrated to ~20 mL, and purified using preparative
RP-HPLC (Method 5). The fractions containing the desired product were concentrated in
vacuo to remove the organic solvent, and the water was removed via freeze-drying to
yield a white residue (25 – 32% yield over two steps) in greater than 95% purity based on
RP-HPLC (Method 3). (See Supporting Information for HPLC & NMR data)
G4-butyryl-vancomycin derivative (10): Analogue 10 was synthesized using the
general procedure 3 and isolated as a white solid (14.5 mg, 17% over two steps).
Analytical RP-HPLC retention time (Method 3): 9.7 min; LC-MS (ESI+) for
C71H84Cl2N9O25 [M+H]+: Calc’d = 1518.479; found = 1518.469.
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G4-pentanoyl-vancomycin derivative (11): Analogue 11 was synthesized using
the general procedure 3 and isolated as a white solid (10 mg, 23% over two steps).
Analytical RP-HPLC retention time (Method 3): 9.8 min; LC-MS (ESI+) for
C71H84Cl2N9O25 [M+H]+: Calc’d = 1532.495; found = 1532.526.
G4-hexanoyl-vancomycin derivative (12): Analogue 12 was synthesized using
the general procedure 3 and isolated as a white solid (11 mg, 25% over two steps).
Analytical RP-HPLC retention time (Method 3): 11.2 min; LC-MS (ESI+) for
C72H86Cl2N9O25 [M+H]+: Calc’d = 1546.511; found = 1546.510.
G4-octanoyl-vancomycin derivative (13): Analogue 13 was synthesized using
the general procedure 3 and isolated as a white solid (14 mg, 32% over two steps).
Analytical RP-HPLC retention time (Method 3): 13.2 min; LC-MS (ESI+) for
C74H90Cl2N9O25 [M+H]+: Calc’d = 1574.542; found = 1574.540.
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G4-pentenoyl-vancomycin derivative (14): Analogue 14 was synthesized using
the general procedure 3 and isolated as a white solid (18 mg, 21% over two steps).
Analytical RP-HPLC retention time (Method 3): 10.2 min; LC-MS (ESI+) for
C71H82Cl2N9O25 [M+H]+: Calc’d = 1530.479; found =1530.476.
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G4-methyladipoyl-vancomycin derivative (15): Analogue 15 was synthesized
using the general procedure 3 and isolated as a white solid. Analytical RP-HPLC
retention time (Method 3): 10.1 min; LC-MS (ESI+) for C73H86Cl2N9O27 [M+H]+: Calc’d =
1590.500; found = 1590.529.
General procedure 4: Competition studies using Boc-Leu-DAla-DAla-OH, a
tripeptide ligand for vancomycin. In a flame-dried small glass vial containing a stir bar,
3 (5 mg, 2.81 µmol, 1 equiv), Boc-Leu-DAla-DAla-OH (1.1 mg, 2.94 µmol, 1 equiv) and
peptide catalyst (0.9 µmol, 0.3 equiv) were dissolved in THF (100 µL) to yield a clear,
homogeneous solution. Then, CH2Cl2 (300 µL) and PEMP (1.1 µL, 6.07 µmol, 2 equiv)
were added to the vial. Decanoic anhydride (2.1 µL, 5.62 µmol, 2 equiv) was added
subsequently and the reaction mixture was allowed to stir for 24 h at rt. An aliquot (20
µL) was taken and quenched with 2-propanol (500 µL), and analyzed by HR-LC/MS
(Method 2) and RP-HPLC (Method 3). These data were used for preliminary
determination of the conversion, product selectivity, and site of reaction (See Supporting
Information for HPLC data).
ACKNOWLEDGMENT
We are grateful to the National Institute of General Medical Sciences of the
United States National Institutes of Health (GM-068649) for support. We also wish to
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thank Dr. Brandon S. Fowler for providing peptide catalysts for the initial screening.
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ASSOCIATED CONENT
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Supporting Information Availability:
Figures, schemes and characterization (RP-HPLC data and NMR data) of the compounds
are provided. This material is available free of charge via the Internet at:
http://pubs.acs.org
AUTHOR INFORMATION
Corresponding Author
*Phone: 203-432-9885. Fax: 204-436-4900. E-mail: scott.miller@yale.edu
ABBREVIATIONS USED
NMI – N-methylimidazole; PEMP - 1,2,2,6,6-pentamethylpiperidine; pmh - p(methyl)-
histidine; DEPT: distortionless enhancement by polarization transfer; MSSA –
methicillin-sensitive Staphylococcus aureus; methicillin-resistant Staphylococcus aureus;
VRE – vancomycin-resistant Enterococcus faecalis.
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