Antibacterial sulfonimidamide-based oligopeptides as type I signal peptidase inhibitors: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2021.113699

Oligopeptide boronates with a lipophilic tail are known to inhibit the type I signal peptidase in E. coli, which is a promising drug target for developing novel antibiotics. Antibacterial activity depends on these oligopeptides having a cationic modification to increase their permeation. Unfortunately, this modification is associated with cytotoxicity, motivating the need for novel approaches. The sulfonimidamide functionality has recently gained much interest in drug design and discovery, as a means of introducing chirality and an imine-handle, thus allowing for the incorporation of additional substituents. This in turn can tune the chemical and biological properties, which are here explored. We show that introducing the sulfonimidamide between the lipophilic tail and the peptide in a series of signal peptidase inhibitors resulted in antibacterial activity, while the sulfonamide isostere and previously known non-cationic analogs were inactive. Additionally, we show that replacing the sulfonamide with a sulfonimidamide resulted in decreased cytotoxicity, and similar results were seen by adding a cationic sidechain to the sulfonimidamide motif. This is the first report of incorporation of the sulfonimidamide functional group into bioactive peptides, more specifically into antibacterial oligopeptides, and evaluation of its biological effects.

Full text of DOI:10.1016/j.ejmech.2021.113699

European Journal of Medicinal Chemistry 224 (2021) 113699
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Antibacterial sulfonimidamide-based oligopeptides as type I signal
peptidase inhibitors: Synthesis and biological evaluation
a
b
c
a
a
ꢀ
Andrea Benediktsdottir , Lu Lu , Sha Cao , Edouard Zamaratski , Anders Karlen ,
Sherry L. Mowbray b , Diarmaid Hughes , Anja Sandstrom
,
d
c
a, *
€
a Department of Medicinal Chemistry, BMC, Uppsala University, Box 574, SE-75123, Uppsala, Sweden
b Department of Cell and Molecular Biology, BMC, Uppsala University, Box 596, SE-75123, Uppsala, Sweden
c Department of Medical Biochemistry and Microbiology, BMC, Box 582, SE-75123, Uppsala, Sweden
d Uppsala University, Science for Life Laboratory, Department of Cell and Molecular Biology, Box 596, SE-751 24, Uppsala, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
Oligopeptide boronates with a lipophilic tail are known to inhibit the type I signal peptidase in E. coli,
which is a promising drug target for developing novel antibiotics. Antibacterial activity depends on these
oligopeptides having a cationic modification to increase their permeation. Unfortunately, this modifi-
cation is associated with cytotoxicity, motivating the need for novel approaches. The sulfonimidamide
functionality has recently gained much interest in drug design and discovery, as a means of introducing
chirality and an imine-handle, thus allowing for the incorporation of additional substituents. This in turn
can tune the chemical and biological properties, which are here explored. We show that introducing the
sulfonimidamide between the lipophilic tail and the peptide in a series of signal peptidase inhibitors
resulted in antibacterial activity, while the sulfonamide isostere and previously known non-cationic
analogs were inactive. Additionally, we show that replacing the sulfonamide with a sulfonimidamide
resulted in decreased cytotoxicity, and similar results were seen by adding a cationic sidechain to the
sulfonimidamide motif. This is the first report of incorporation of the sulfonimidamide functional group
into bioactive peptides, more specifically into antibacterial oligopeptides, and evaluation of its biological
effects.
Received 8 March 2021
Received in revised form
18 June 2021
Accepted 10 July 2021
Available online 13 July 2021
Keywords:
Sulfonimidamide
Antibacterial
Bacterial type I Signal peptidase
LepB
Oligopeptides
Serine-lysine protease
Bioisosteres
© 2021 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY
license (http://creativecommons.org/licenses/by/4.0/).
1. Introduction
chemical properties and new possibilities in drug design. The extra
nitrogen in the SIA scaffold acts as a weakly basic imine, providing a
The lack of new antibiotics, alongside the increase in resistance
to existing antibiotics, is a current alarming problem that re-
searchers all over the world are fighting to address. To discover new
antibiotics, novel lead structures that are potent and specific, and
possess minimal toxic effects, are needed. In medicinal chemistry,
optimization of validated hits is a fundamental task involving
strategies such as bioisosteric replacements of atoms or groups [1].
The sulfonamide (SA) functional group, one of the first examples of
a carboxamide isostere in drug design [2], has played a major role in
various important biologically-active compounds such as diuretics,
antivirals, and antibiotics [3]. Replacing one of the SA oxygens with
a nitrogen gives the isostere sulfonimidamide (SIA), which has
gained much momentum over the past ten years due to its unique
reactive handle for incorporating chemical modifications. This
substitution furthermore introduces chirality into the molecule, in
contrast to the achiral SA or carboxylic acids [4]. Previous reports
have shown that properties such as metabolic stability and solu-
bility are often improved with the SIA compared to the corre-
sponding SAs in small molecules [4,5]. Until now, the major studies
regarding SIAs have focused on the development and optimization
of novel synthetic routes to small molecule SIAs, whereas the
evaluation of SIA-based compounds in medicinal chemistry has
been less explored. Examples of small molecule SIA-based analogs
of known bioactive compounds are seen in Fig. 1, including onco-
lytic sulfonylureas [6], an SIA analog of the Alzheimer's drug
Begacestat [7], antimicrobial trifluoromethylated SIAs [8], and an
analog of the cancer drug Tasisulam [9].
A review by Arvidsson's group in 2016 [4] focusing on SIAs in
medicinal chemistry also highlighted the fact that most emphasis
* Corresponding author.
E-mail address: anja.sandstrom@ilk.uu.se (A. Sandstrom).
€
https://doi.org/10.1016/j.ejmech.2021.113699
0223-5234/© 2021 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

A. Benediktsdottir, L. Lu, S. Cao et al.
European Journal of Medicinal Chemistry 224 (2021) 113699
Fig. 2. Outline of this work, in which replacing the amide linker in the previous oli-
gopeptide boronate hit (A) with the sulfonamide and sulfonimidamide moiety (B) is
explored.
Fig. 1. Examples of sulfonimidamide-based analogs of small molecule bioactive
compounds.
macrocycles [27]. These compounds were also potent inhibitors of
the enzyme, but unfortunately did not solve the existing problems;
when the toxic effect was avoided, the antibacterial activity was
reduced. Investigation of the structure-activity relationships of the
substrate-based oligopeptides has identified some key structural
features important for antibacterial activity. A bulky lipophilic tail
at the N-terminus, an electrophilic and highly-reactive warhead at
the C-terminus, and a positive charge around the P5 position are all
important for antimicrobial activity (Fig. 2). In particular, the lipo-
philic group is crucial for enzymatic inhibition. This tail seems to be
needed for anchoring to the membrane where the LepB target is
situated; it thus helps the peptide part of the inhibitor to reach the
binding site. However, previous studies indicated that the lipophilic
tail might also be correlated with cytotoxicity and hemolytic side
effects [26,27]. To develop sufficiently potent lead molecules with
an acceptable toxicity profile, further exploration of the lipophilic
tails is of interest. Additionally, designs for improved antibacterial
activity of the oligopeptides in previous studies relied on either the
addition of a cationic side chain as seen in Fig. 2A [25] or replace-
ment of the proline with the cationic amino acid ornithine [27]. The
aim of the present work was to investigate the effect of the SIA
functionality, introduced between the lipophilic tail and P5, on
antibacterial potency via modulating permeability or efflux prop-
erties. Effects on toxicity and hemolysis of SIA-containing oligo-
peptides were also evaluated. Furthermore, the SIA offers an
additional hydrogen bond donor or acceptor, allows for an addi-
tional substituent, introduces chirality, and alters the geometry
between the peptide and the lipophilic chain compared to the
amide-linked oligopeptide. We wanted to take advantage of these
features and explore the differences of the SA versus SIA func-
tionalities in the oligopeptides. We furthermore saw an opportu-
nity to apply and challenge our previously reported synthetic
protocols [28,29] for introducing the SIA group into peptides in a
medicinal chemistry context.
has been placed on studies of the drug-related and physicochemical
properties of SIAs, in contrast to the paucity of studies on biological
evaluation. To the best of our knowledge, no evaluation of SIA in
bioactive peptides has yet been presented.
Multidrug-resistant Gram-negative bacteria have become one of
the primary concerns in the fight against antibiotic resistance, as
there are currently few effective drugs available to treat such in-
fections. The double cell membrane and special efflux mechanisms
make it very challenging to develop new effective Gram-negative
antibiotics [10e12]. Type I signal peptidase (SPase I), commonly
referred to as LepB in Escherichia coli [13], is a promising drug target
as it is limited to bacteria and vital for their survival and virulence.
SPase is a highly specific enzyme designed to cleave pre-proteins to
release a mature protein from the bacterial cytoplasmic membrane.
The active site is located at the outer surface of the cytoplasmic
membrane, making it accessible to any inhibitor capable of crossing
the outer membrane of Gram-negative bacteria. The peptidase has
a unique Ser-Lys catalytic dyad that differs from typical eukaryotic
serine proteases, which have a Ser-His-Asp catalytic triad archi-
tecture [14e16]. Targeting the Ser-Lys dyad and thus blocking the
SPase activity leads to the accumulation of the pre-proteins in the
membrane, eventually killing the bacteria. Despite the five classes
of molecules, including arylomycins [17e19], krisynomycin [20],
5S-penems [21,22], beta-aminoketone [23], and substrate-based
oligopeptides [24e27], that have been identified as inhibitors of
SPase I, there is to our knowledge still no candidate drug in
advanced stages of development. In our laboratory, De Rosa et al.,
previously optimized substrate-based oligopeptides by replacing
an aldehyde warhead with a boronic ester (Fig. 2A), which
improved the EcLepB IC50 from low micromolar to low nanomolar,
and at the same time imparted whole-cell antibacterial activity on
wild-type strains [26]. Although promising starting points, these
potent oligopeptide boronates were shown to be toxic to human
liver cells (HepG2) and to have hemolytic effects. Therefore, im-
provements are necessary to reduce the toxic effects while main-
taining the antibacterial activity of these compounds. Further
optimization of the linear boronic ester inhibitors was recently
attempted in our lab, resulting in novel boronic ester-linked
We report herein the design, synthesis, and biological evalua-
tion of novel SA- and SIA-linked oligopeptides targeting the E. coli
type I signal peptidase.
2

A. Benediktsdottir, L. Lu, S. Cao et al.
European Journal of Medicinal Chemistry 224 (2021) 113699
2. Results and discussion
of a bromobenzenesulfonyl chloride 1 with a proline methyl ester,
followed by a Suzuki-Miyaura cross-coupling of a (4-hexylphenyl)
boronic acid, then basic hydrolysis of the methyl ester to yield the
free carboxylic acid at the proline. Likewise, compound 4 was
prepared by substitution of an N-Boc-etylene diamine with
bromobenzenesulfonyl-chloride, followed by Suzuki-Miyaura
cross-coupling of the (4-hexylphenyl) boronic acid. The prepara-
tion of compound 7 started by protecting a bromobenzenesulfo-
namide 5 with a TBDPS group, which was followed with Suzuki-
Miyaura cross-coupling of the (4-hexylphenyl) boronic acid to
obtain the hexyl-biphenyl tail. The tripeptide 10 and tetrapeptide
11 were synthesized by manual SPPS on 2-CTC resin using standard
protocols for N-terminal 9-fluorenylmethyloxycarbonyl-protected
amino acids (Fmoc-AA-OH), using N,N-diisopropyl-ethylamine
(DIPEA) and 3-[Bis(dimethylamino)methyliumyl]-3H-benzotriazol-
1-oxide hexafluorophosphate (HBTU) and 20% piperidine in DMF
for Fmoc removal (Scheme 2). First, the Fmoc-Asn-(Trt)-OH 8 was
attached to a 2-CTC resin to yield 9, which was further reacted with
the remaining Fmoc-AAs, DIPEA, and HBTU and finally the Fmoc
group was removed by treating the resin with 20% piperidine so-
lution in DMF to yield 10 and 11. To further react the peptides with
the SA-based building blocks, the commercially-available sulfonyl
chloride 1a was coupled to the tetramer 11 (Scheme 3-c) and the
building block 3 was attached directly to the trimer 10 (Scheme 3-
a). Both peptides were then cleaved off the solid phase, resulting in
the SA-linked oligopeptides 12e13 (Scheme 3-d).
The syntheses of the SIA-linked oligopeptides started with
converting the SA building blocks 4 and 7 to a sulfonimidoyl-
chlorides (SIC) in solution, with the chlorinating agent triphenyl-
phosphine dichloride (Scheme 3-b). The SIC solution was then
added directly to the tetramer 11 on solid-phase as seen in Scheme
3-c. Any excess of unreacted building blocks was then simply
washed away from the oligopeptide-bound resins, after which the
resins were treated with 20% HFIP in DCM to selectively release the
oligopeptides as free C-terminal acids 14e16 (Scheme 3-d), with
the sidechain protecting groups still intact. Thanks to the TBDPS
group, diastereomers 14 and 15 were easily separated by prepara-
tive HPLC, in contrast to the diastereomers of the Boc-protected
aminoethyl compound 16 which were inseparable under these
conditions. After purification of the acids by preparative reversed-
phase HPLC, the final step included the attachment of the reac-
tive boronic ester warhead via peptide coupling of the
commercially-available (R)-boroAla-(þ)-pinanediol, in solution,
resulting in the final compounds 17e21 (Scheme 4). It is however
well known that activation of C-terminal carboxylic acids of pep-
tides can cause racemization via oxazolone formation [30]. To avoid
this as much as possible, the activation was carried out at low
temperature for 3 min before the addition of the amino boronate.
However, as previously observed by De Rosa et al., racemization
was unavoidable, resulting in partial loss of chiral integrity at the
asparagine chiral side chain, and yielding mixtures of epimers in
ratios ranging from 90:10 to 50:50. The epimers could not be
separated in the preparative RP-HPLC, and thus the final com-
pounds were isolated and tested as mixtures of epimers in analogy
with the corresponding inhibitors evaluated by De Rosa et al. [26].
2.1. Design and chemistry
The oligopeptide boronates with lipophilic tails designed and
synthesized in this study were based on a Pro-Thr-Ala-Asn peptide
sequence with an amino boronic ester as warhead at the N-ter-
minus, a rigid biphenyl tail, and with SA or SIA as a linker func-
tionality. Furthermore, for comparison, a SA-linked analog was
designed with a flexible dodecane tail to investigate if there were
differences from the more rigid tail analog. The SIA-linked oligo-
peptides were designed with the same sequence as the SA-linked
analog for direct comparison of the linkers. As previously
mentioned, the SIA group introduces chirality, therefore it was
important to be able to separate the two diastereomers to inves-
tigate them for possible differences. To facilitate simple separation
of the diastereomers, the SIA building blocks were designed with
the tert-butyldiphenylsilyl (TBDPS)-protected SA. This bulky group
has been shown to enhance differences between isomers; the fact
that it is highly UV-active also makes the separation much easier
[28]. We have previously shown that the TBDPS group is compat-
ible with solid-phase peptide chemistry (SPPS), and is stable when
cleaving the peptide with 10e20% hexafluoroisopropanol (HFIP) in
DCM from a 2-chlorotritylchloride (2-CTC) polymer resin, whereas
it was removed when using standard cleavage conditions such as
TFA [29]. As highlighted in the initial study on the oligo-boronates
[26], introducing a primary amine as a substituent on the proline in
P5 was important for antibacterial activity. With this in mind, we
designed an analog with an additional cationic sidechain (amino-
ethyl), utilizing the SIA moiety. The overall synthetic approach is
described below where the SA building blocks were first prepared
in solution (Scheme 1), the desired peptide sequences on solid
phase (Scheme 2), then the building blocks were attached to the
peptide sequence (Scheme 3), and finally the boronic ester warhead
was coupled to the free C-terminus (Scheme 4).
The syntheses of the SA-based building blocks 3, 4 and 7 are
outlined in Scheme 1. Compound 3 was prepared by a substitution
2.2. In vitro biology
The final compounds 17e21 were evaluated for in vitro inhibi-
tory potency against the E. coli SPase in a FRET-based functional
assay, reported in Table 1 as IC50 values (mM) [26]. The compounds
were also evaluated for their minimal inhibitory concentration
(MIC), against a panel of seven Gram-negative bacterial strains and
one Gram-positive organism, S. aureus [26]. The S. aureus spsB gene
encodes a catalytically active and essential Type I signal peptidase
Scheme 1. Syntheses of the sulfonamide-based building blocks 3, 4 and 7. Reagents
and conditions: a) (S)-methyl pyrrolidin-1-ium-2-carboxylate chloride, Et3N, DCM,
overnight b) tert-butyl N-(2-aminoethyl) carbamate, Et3N, DCM, 16h c) tert-butyl-
chlorodiphenylsilane, Et3N, anhydrous THF, overnight, 50ᵒC d) (4-hexylphenyl)
boronic acid, Pd(PPh3)4, K2CO3, Tol/EtOH (1:1), 4h e) LiOH, THF/MeOH/H2O (3:2:1),
12h.
3

A. Benediktsdottir, L. Lu, S. Cao et al.
European Journal of Medicinal Chemistry 224 (2021) 113699
Scheme 2. Solid-phase synthesis of peptides 10 and 11. Reagents and conditions: a) 2-CTC, DIPEA, DCM, r. t., 2 h, then MeOH, r. t., 0.5 h; b) 20% piperidine in DMF, twice,
5 min þ 20 min, r. t.; c) Fmoc-AA-OH, HBTU, DIPEA, DMF, r. t., 5 h; d) 20% piperidine in DMF (5 þ 20 min), DMF wash.
Scheme 3. Synthetic route of the SA- and SIA linked oligopeptides 12e16. Reagents and conditions: a) HBTU, DIPEA, DMF, 4h b) PPh3Cl2, DIPEA, anhydrous DCM c) DIPEA, N2, 0ᵒ d)
HFIP in DCM (1:4), 1h at rt.
Scheme 4. Final step in the synthesis of SA- and SIA-linked oligopeptide boronic acids 17e21 by peptide coupling of the boronic ester warhead to the linear oligopeptide carboxylic
acids 12e16. Reagents and conditions: a) (R)-boroAla-(þ)-pinanediol hydrochloride, HATU, DIPEA, DCM, 0 ꢀC, 1h; b) TFA/DCM 1:1, 2h. *Relative stereochemistry. The epimeric ratio
(er) and the diastereomeric ratio (dr) of the final compounds are seen in parentheses.
enzyme with similar conserved sequence motifs to LepB in E. coli
[31]. Further, the oligopeptides were evaluated on the human liver
cell line HepG2 for cytotoxicity, and in a hemolysis assay where the
heparinized human blood, values greater than 1% in the hemolysis
assay are regarded as a red flag [26].
All of the new oligopeptides (17e21) were able to inhibit the
compounds (100
mM) were tested, using red blood cells from
LepB protease, with IC50s ranging from 0.056 to 1.11 mM.
4

A. Benediktsdottir, L. Lu, S. Cao et al.
European Journal of Medicinal Chemistry 224 (2021) 113699
Table 1
Structures and in vitro testing results for compounds 17e23.
.
Cmpd. nr. R1
17
R2
H
EcLepB IC50
(
m
M) MIC (
E. c.a E. c.b E. c.c P. a.d P. a.e K. p.f A. b.f S. a.h
>64 16 >64 32
>64 >64 ꢁ1
m
g/mL)
Cytotoxicity IC50
1.20
(
m
M)i Hemolysis (%)
0.10 0.01
0.056
8
18
19
20
21
H
H
H
H
0.071
1.07
>64 >64 >64 >64 >64 >64 >64 >64 0.58
14.5 8.8
2.15 0.24
0.35 0.31
5.32 0.8
>64
>64
64
4
8
>64 >64 >64 >64
>64 >64 >64 >64
>64 >64 >64 64
4
2.95
2.99
5.20
1.11
4
32
8
4
0.133
16
ꢁ1
22j
23j
H
0.012
>64 64
16
1
>64 >64 >64 >64
4
4.7
1.1 0.2
5.7 0.3
CH2NHþ3 0.018
32
4
64
8
16
32
ꢁ1
18.6
aE. coli ATCC 25922 WT; bE. coli tolC (CH3130, efflux-defective mutant isogenic to ATCC 25922); cE. coli D22 (CGSG 5163, lps mutant, drug-hypersensitive); dP. aeruginosa
PAO1 WT; eP. aeruginosa efflux defective PAO750 (isogenic to PAO1); fK. pneumoniae ATCC 13883 WT; gA. baumanii ATCC 19606 WT; hS. aureus ATCC 29213 (Gram-positive
j
WT reference); iCytotoxicity of HepG2 cells; Reference compounds from De Rosa et al. [25].
As seen in Table 1, the SA-linked peptides 17 and 18 were more
potent inhibitors of the enzyme (0.056e0.071 M) compared to the
SIA-linked peptides 19e21 (0.133e1.11 M), but not as potent as the
previously reported amide-linked peptides 22 and 23
(0.012e0.018 M) [26]. The difference between 22 and 23 is that
the latter has a cationic sidechain in P5, which makes the com-
pound active on bacteria, able to inhibit growth at 4 g/mL on the
efflux-defective E. coli strain and at 32 g/mL on wild-type. Com-
vs. 5.20 mM). However, it should be noted that the values are rough
m
and should be analyzed with caution since the compounds are a
mixture of epimers/diastereomers. Thus, it might be that the in-
hibition potency, as well as cytotoxicity, for individual stereoiso-
mers could be higher. Since all compounds in Table 1 (17e23) are
mixed with certain amounts of D-Asn epimer, which could not be
separated, the exact influence of this epimer remains unknown.
The initial set of compounds were all designed with the
biphenyl lipophilic tail however, to evaluate whether the flexibility
of the tail in these types of compounds would have any impact on
activity or toxicity, we prepared 17, a direct analog of 18 but with a
flexible tail. Notably, the more flexible tail had an impact on the
antibacterial activity, resulting in activity on the efflux-defective
and hypersensitive E. coli mutants, and on S. aureus, unlike epi-
mers 18, which were inactive on all strains. The compounds
showing MIC activity against S. aureus (Table 1) are believed to do
so by targeting the SpsB enzyme but at present we have no direct
evidence to support this. The alternative possibility is that the ac-
tivity on S. aureus reflects general cytotoxicity, however, there is no
correlation between MIC on S. aureus and cytotoxicity measured by
hemolysis or IC50 on HepG2 cells (Table 1). The flexibility of the tail
did not result in any difference in cytotoxicity, however, the more
flexible analog had negligible hemolytic effects (0.1%) compared to
the more rigid one, where 14.5% hemolysis was observed. The same
effect was seen by De Rosa et al. [26], i.e. the more flexible tail was
moderately less hemolytic.
m
m
m
m
pound 22 and its new SA analog 18 do not have a cationic side chain
and lack antibacterial activity even on the efflux-defective strain
(>64
mg/mL). However, the SIA analogs 19e20 both inhibit bacterial
growth on the efflux-defective strain at 4
mg/mL, equally potent to
compound 23, which relies on the cationic side chain for this ac-
tivity. The drawback of the cationic sidechain in 23 was strong
(5.7%) hemolysis, whereas the hemolytic effects of the SIA-analogs
19e20 were lower, at 0.35e2.2%. The SIA analogs 19e21 were also
active on the hypersensitive E. coli strain at 8e32 mg/mL, however,
no activity was observed on wild-type strains, possibly because
they are substrates for efflux pump activity. The cytotoxic effects of
the SIA-linked peptides were 5-fold less than the SA analogs
(~2.97
mM vs. 0.58 mM), however without reaching safe levels,
defined as at least ten times lower than enzyme potency. Adding
the cationic aminoethyl group directly on the SIA handle (di-
astereomers 21) resulted in enhanced inhibition of the LepB pro-
tease as compared to the unsubstituted SIA peptides (19 and 20)
and 9-fold suppression of cytotoxicity compared to the SA (0.58 mM
5

A. Benediktsdottir, L. Lu, S. Cao et al.
European Journal of Medicinal Chemistry 224 (2021) 113699
3. Conclusions
overnight. The mixture was diluted with 1 M aq. HCl (35 mL) and
DCM (5 mL) and the two phases were separated in a funnel. The
organic phase was washed twice with 1 M HCl (30 mL). The com-
bined organic layers were washed twice with brine (25 mL), dried
over Na2SO4, filtered and the solvent was removed under reduced
pressure. The title compound 2a was obtained as white solid
(4.30 g, 97% yield) and used in the next step without further pu-
In exploring the effects of novel bioisosteric replacements, a
number of sulfonamide and sulfonimidamide-based oligopeptides
were designed and synthesized. The goal was to study whether
such small changes in large bioactive peptides would result in
marked differences in their properties. The biological evaluation of
the sulfonimidamide-based oligopeptides indicated that the bio-
isosteric replacement was tolerated, but did not result in optimized
inhibition compared to the previously-reported amide-based oli-
gopeptides. However, in the case of the sulfonimidamide 20, he-
molysis was decreased to basal levels, yet the compound was active
on an efflux-defective E. coli strain. Further, replacing the sulfon-
amide with sulfonimidamide decreased the cytotoxicity and led to
improved antibacterial properties. Additionally, utilizing the sul-
fonimidamide motif for adding a basic aminoethyl side chain to the
“N” handle resulted in reduced cytotoxicity compared to the sul-
fonamide. We foresee that the synthetic approaches herein
demonstrated and the awareness of SIA's ability to affect various
properties of bioactive peptides will inspire and facilitate explora-
tion of this functionality in peptide-based drug discovery.
rification. 1H NMR (400 MHz, Chloroform-d)
d
7.75 (AA0 of an
AA’XX’, 2H), 7.66 (XX0 of an AA’XX’, 2H), 4.35 (dd, J ¼ 8.2, 3.6 Hz,
1H), 3.71 (s, 3H), 3.45 (m, 1H), 3.35 (m, 1H), 2.09 (m, 1H), 2.06e1.91
(m, 2H), 1.83 (m,1H). 13C NMR (101 MHz, CDCl3)
d 172.2,137.3, 132.1,
128.8, 127.6, 60.2, 52.3, 48.1, 30.7, 24.5. HRMS-ESI (m/z): [MþH]þ
calcd for C12H15NO4SBr 347.9905; found, 347.9901.
4.1.2. Synthesis of tert-butyl (2-((4-bromophenyl)sulfonamido)
ethyl) carbamate (2b)
Commercially available 4-bromo-benzenesulfonate chloride
(2.16 mmol, 553 mg) was dissolved in 6 mL DCM, N-Boc-ethyl-
enediamine (1.95 mmol, 308 mL) and TEA (6.49 mmol, 905 mL) was
added dropwise and the mixture stirred overnight. After the
completion of the reaction, excess TEA was boiled away and then
the reaction mixture was diluted with 50 mL of water and extracted
two times with DCM, the organic phases collected and dried over
Na2SO4. The solvent was removed under reduced pressure and the
crude product then purified by silica gel chromatography (iso-
hexane/ethyl acetate 1:1) and a white solid 2b (797 mg, 96% yield)
4. Experimental section
4.1. Chemical synthesis
All chemicals and solvents were purchased from Sigma Aldrich,
Fisher Scientific, FluoroChem, Enamine and Ark Pharm chemicals
and were used without further purification. Analytical thin-layer
chromatography (TLC) was performed using Merck aluminum
sheets precoated with silica gel 60 F254. Column chromatography
was obtained. 1H NMR (400 MHz, DMSO‑d6)
d
7.81 (AA0 of an
AA’XX’, 2H), 7.71 (XX0 of an AA’XX’, 2H), 7.33 (bs, 1H), 6.76 (t,
J ¼ 5.8 Hz, 1H), 2.94 (m, 2H), 2.76 (m, 2H), 1.34 (s, 9H). 13C NMR
(101 MHz, DMSO‑d6)
d 155.9, 140.2, 132.8, 129.0, 126.7, 78.3, 42.7,
was performed on Merck silica gel 60 (40e63
m
m) 1H and 13C NMR
40.6, 28.6. C13H19BrN2O4S, MS (ESI): m/z 758.9 [2 M þ H]þ. HRMS-
ESI (m/z): [M þ CH3CN þ Na]þ calcd for C15H22BrN3O4SNa
442.0412; found, 442.0405.
spectra were recorded on Varian Mercury Plus instruments; 1H at
ꢀ
399.9 MHz and 13C at 100.6 MHz at 25 C. Chemical shifts (
d) are
reported in ppm and coupling constants (J) are reported in hertz
(Hz). Exact molecular masses were determined on Micromass Q-Tof
2 mass spectrometer equipped with an electrospray ion source.
Analytical RP-HPLC-MS was performed on a Gilson RP-HPLC system
with a Finnigan AQA quadrupole low-resolution mass spectrometer
in positive or negative ESI mode using an Onyx Monolithic C18
4.1.3. Synthesis of ((40-hexyl-[1,10-biphenyl]-4-yl)sulfonyl)-
L-proline
(3)
A 30 mL vial was charged with sulfonamide 2a (1.44 mmol,
500 mg) and hexylphenyl-boronic acid (2.87 mmol, 592 mg) fol-
lowed by addition of toluene/EtOH (1:1, 11 mL), 4.0 M potassium
carbonate (5.74 mmol, 1.44 mL) and at last Pd(PPh3)4 (0.0717 mmol,
82.9 mg). The vial was sealed and flushed with N2 gas and heated to
90 ꢀC and left stirring for 3 h when it was stopped after TLC and LC-
MS results confirmed that the starting materials were consumed.
The reaction mixture was washed with 1 M HCl (2 ꢂ 15 mL) and
DCM (2 ꢂ 15 mL) the organic phase was dried over Na2SO4 and
concentrated under reduced pressure. To the reaction mixture,
LiOH (4.18 mmol, 90.6 mg) was added along with 6 mL of THF/
MeOH/H2O (3:2:1) and left stirring overnight. The reaction mixture
was washed again with 1 M HCl (2 ꢂ 10 mL) and DCM (2 ꢂ 10 mL)
the organic phase was dried over Na2SO4 and concentrated under
reduced pressure. The crude mixture was then purified by flash
column chromatography (EtOAc/iso-hexane, 4:6). The pure frac-
tions with the product were collected and concentrated affording 3
as a white solid (432 mg, 80% yield). C23H29NO4S, MS (ESI): m/z
3 ꢂ 50 mm, 2.6
mm particle size,100 Å pore size (Phenomenex) with
gradients of MeCN in 0.05% aqueous HCOOH as mobile phase at a
flow rate of 2 mL/min. Preparative RP-HPLC was performed on a
system equipped with a Nucleodur C18 HTec 5 m column
m
(150 21.2 mm) or Phenomenex C8 5 mm column
ꢂ
a
(150 ꢂ 21.2 mm), using a H2O/MeCN gradient with 0.1% TFA or H2O/
MeCN gradient, flow rate 14 mL/min with 20 mM triethylammo-
nium acetate (TEAA), in both cases using UV detection at 220 nm
and 254 nm. High-resolution molecular masses (HRMS) were
determined on a Waters acuity UPLC mass spectrometer with an
ESI source, 7-T hybrid linear ion trap (LTQ), flow rate 0.25 mL/min
(MeCN/H2O 1:1). Compounds 1, 1a, 5, and 8 were purchased, and
compounds 2a and 2b are commercially available but synthesized
in this work. The rest of the compounds are new entities and have
been characterized by 1H NMR and 13C NMR and ESI (MS). All final
compounds were ꢃ95% pure as determined by analytical HPLC and
NMR.
416.1 [MþH]þ. 1H NMR (400 MHz, Chloroform-d)
d 7.93 (AA’ of an
AA'XX’, 2H), 7.74 (XX’ of an AA'XX’, 2H), 7.54 (AA’ of an AA'XX’, 2H),
7.29 (XX’ of an AA'XX’, 2H), 4.33 (m, 1H), 3.56 (m, 1H), 3.33 (m, 1H),
2.66 (t, J ¼ 7.8, 7.4 Hz, 2H), 2.15 (m, 1H), 2.07e1.92 (m, 2H),
1.69e1.62 (m, 2H), 1.34e1.31 (m, 2H), 1.27e1.25 (m, 6H), 0.89
(distorted triplet, w ¼ 13.9 Hz, 3H). 13C NMR (101 MHz, Chloroform-
4.1.1. Synthesis of methyl ((4-bromophenyl)sulfonyl)-L-prolinate
(2a)
To
a solution of (S)-2-(methoxycarbonyl)pyrrolidin-1-ium
chloride (12.7 mmol, 2.10 g) in dry DCM (35 mL) in a round bot-
tom flask, commercially available 4-bromo-benzenesulfonyl chlo-
ride (19.0 mmol, 4.86 g) and triethylamine (38.0 mmol, 5.30 mL)
d)
d 176.6, 146.5, 144.2, 136.8, 135.9, 129.6, 128.5, 128.0, 127.6, 60.9,
49.3, 36.1, 32.2, 31.8, 31.1, 29.4, 25.1, 23.1, 14.6. HRMS-ESI (m/z):
ᵒ
was carefully added at 0 C and left stirring for 30 min and then at rt
[MþH]þ calcd for C23H30NO4S, 416.1817; found, 416.1882.
6

A. Benediktsdottir, L. Lu, S. Cao et al.
European Journal of Medicinal Chemistry 224 (2021) 113699
4.1.4. Synthesis of tert-butyl (2-((40-hexyl-[1,10-biphenyl])-4-
sulfonamido)ethyl)carbamate (4)
manual solid-phase peptide synthesis (SPPS) from 2-
Chlorotritylchloride (2-CTC) polymer resin (1.63 mmol/g) in a
12 mL disposable syringe fitted with porous polyethylene filter. For
the Fmoc-amino acids the following sidechain protecting groups
were used: Thr-(t-Bu), Asn-(Trt). Standard Fmoc conditions were
used and the Fmoc protecting group was removed by treatment
with 20% piperidine in DMF (2 ꢂ 3 mL, 5 þ 20 min) and the polymer
was washed with DMF (5 ꢂ 3 mL, 5 ꢂ 2 min). Coupling of the first
amino acid Fmoc-Asn-(Trt)-OH (0.8 equiv.) to the 2-CTC resin (1
equiv.) was performed in anhydrous DCM (7.5 mL) in the presence
of DIPEA (3 equiv.) under agitation for 2 h at rt, after which MeOH
(1.2 mL) was added to cap the unreacted sites of the resin (15 min
agitation). The MeOH was removed by filtration and the resin
washed with DCM, DMF and again DCM and dried under vacuum
overnight. 1.46 g of Fmoc-Asn-(Trt)-CTC resin were obtained, with a
new loading of 0.68 mmol/g. Next, coupling of the appropriate
amino acid, Fmoc-AA-OH (4 equiv.) was performed in DMF (3 mL)
A 30 mL vial was loaded with sulfonamide 2b (1.23 mmol,
466 mg) and hexylphenyl-boronic acid (2.46 mmol, 506 mg) fol-
lowed by addition of 4.0 M K2CO3 (4.91 mmol, 1.23 mL), 10 mL
Toluene/EtOH (1:1) and triphenyl-phosphine)palladium (0)
(0.0614 mmol, 71.0 mg) was added. The vial was sealed, and the
reaction mixture was stirred at 100 ꢀC for 4h. Upon completion, the
reaction mixture was cooled down to rt and diluted with DCM
(30 mL) and brine solution (30 mL) were added, and the two phases
separated. The organic phases were collected and concentrated in
vacuo and purified by column chromatography (1.5:1 iso-hexane/
ethyl acetate) yielding 515 mg of 4 as white solid (91% yield). 1H
NMR (400 MHz, DMSO‑d6) d 7.91 (m, 1H), 7.85 (AA'XX’ of an AA'XX’,
4H), 7.67 (XX’ of an AA'XX’, 2H), 7.32 (AA’ of an AA'XX’, 2H), 6.79 (t,
J ¼ 5.8 Hz, 1H), 2.97 (m, 2H), 2.78 (m, 2H), 2.62 (t, J ¼ 8.05, 2H), 1.59
(m, 2H), 1.34 (s, 9H), 1.31e1.24 (m, 6H), 0.86 (distorted triplet,
w ¼ 14.1 Hz, 3H). 13C NMR (101 MHz, DMSO‑d6)
d
155.9, 144.3,
using
N,N,N0,N0-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium
143.4, 139.3, 136.3, 134.6, 129.5, 127.60, 127.58, 127.4, 78.3, 42.8,
35.2, 31.6, 31.3, 28.8, 28.6, 22.5, 14.4. HRMS-ESI (m/z):
[M þ MeCN þ Na] for C27H39N3O4NaS Calcd: 524.2559, Found:
524.2557 (M þ MeCN þ Na).
hexafluorophosphate (HBTU, 4 equiv.), in the presence of N,N-dii-
sopropylethylamine (DIPEA, 8 equiv.), shaking at rt for 4 h. In the
case of a-branched amino acids (e.g. Fmoc-Thr (t-Bu)-OH) or sec-
ondary amine (Fmoc-Pro-OH), the coupling was carried out over-
night. After each coupling, the resin was washed with DMF
(5 ꢂ 3 mL, 5 ꢂ 2 min) and subsequently de-protected and washed
as described above. After completion of the coupling cycle the resin
was washed with several portions of DMF, MeOH and DCM before it
was dried under high vacuum overnight. The final peptides were
cleaved from the resin by treatment with a solution of 1,1,1,3,3,3-
hexafluoro-2-propanol (HFIP)/DCM 1:4 (5 mL), followed by agita-
tion for 1h at rt. The resin was filtered off and washed several times
with DCM. The filtrate was collected in a centrifuge tube and
concentrated in a stream of nitrogen and the residue was directly
purified. The crude peptides were dissolved in H2O/MeCN and
4.1.5. Synthesis of 4-bromo-N-(tert-butyldiphenylsilyl)benzene-
sulfonamide (6)
In
a
solution
of
commercially
available
4-
bromobenzenesulfonamide (4.24 mmol, 1.00 g) in dry THF
(25 mL), tert-butyl-chloro-diphenylsilane (8.46 mmol, 2.20 mL) and
triethylamine (16.9 mmol, 2.36 mL) was added and left stirring at
50 ꢀC overnight. After the completion of the reaction the solvent
was removed in vacuo and the product recrystallized from pentane/
MeOH resulting in 1.70 g of compound 6 as grey crystal (85% yield).
1H NMR (400 MHz, DMSO‑d6)
d
8.33 (s, 1H), 7.61 (AA0 of an AA’XX’,
2H), 7.57 (BB0 of an BB’CC’D, 4H), 7.47 (D of an BB’CC’D, 2H), 7.37 (CC0
filtered through a 0.45 mm nylon membrane and purified in 6e8
runs by RP-HPLC. Selected fractions were analyzed by RP-HPLC-MS,
and those containing pure product were combined and lyophilized.
of an BB’CC’D, 4H), 7.31 (XX0 of an AA’XX’, 2H), 0.96 (s, 9H). 13C NMR
(101 MHz, DMSO‑d6)
d 143.5, 136.0, 132.1, 132.0, 130.3, 128.01,
127.95, 125.8, 27.2, 18.6. HRMS-ESI (m/z): [MþH]þ calcd for
C
22H23BrNO2SSi, 472.0402; found, 472.0410.
4.1.8. Synthesis of N2eO-(tert-butyl)-N-((dodecylsulfonyl)-L-
prolyl)-L-allothreonyl-L-alanyl-N4-trityl-
-asparagine (12)
L
4.1.6. Synthesis of N-(tert-butyldiphenylsilyl)-40-hexyl-[1,10-
biphenyl]-4-sulfonamide (7)
In a 12 mL disposable syringe, fitted with porous polyethylene
filter, the resin bound tetramer 11 was added (0.135 mmol, 300 mg,
loading 0.45 mmol/g) and swelled in DCM for 5 min. Then DCM was
filtered away and solution of 20% piperidine in DMF added and
reacted on rotating wheel. Into a falcon tube, commercially-
available dodecane sulfonyl chloride 1a was added (0.552 mmol,
149 mg) with TEA (1.11 mmol, 0.154 mL) dissolved in 12 mL
anhydrous DCM. This solution was then added to the syringe con-
taining the resin-bound peptide and left rotating on wheel for 5h.
The leftover reaction mixture was then washed away and the resin
washed with DCM (3 ꢂ 4 mL), DMF (3 ꢂ 4 mL), THF (2 ꢂ 4 mL) and
DCM (2 ꢂ 4 mL). The peptide was then cleaved from the resin and
the filtrate and two DCM washes were collected and the solvent
removed in vacuo. The crude peptide was purified by preparative
RP-HPLC (0.05% HCOOH in MeCN/H2O, 90e100% gradient, 20 min,
product eluted after 10 min) and the fractions containing the
desired compound were combined and lyophilized to get 88.1 mg
(70% yield) of desired protected peptide as white solid. 1H NMR
A 30 mL vial was loaded with TBDPS protected sulfonamide 6
(0.887 mmol, 421 mg) and hexylphenyl-boronic acid (1.77 mmol,
366 mg) followed by addition of 4.0 M K2CO3 (3.55 mmol,
0.887 mL), 10 mL Toluene/EtOH (1:1) and lastly triphenyl-
phosphine)palladium (0) (0.0444 mmol, 51.3 mg) was added. The
vial was sealed, and the reaction mixture stirred at 90 ꢀC for 3h.
Upon completion, the reaction mixture was cooled down to rt and
diluted with DCM (30 mL) and brine solution (30 mL) added, and
the two phases separated. The organic phases were collected and
concentrated in vacuo and purified by column chromatography
(0.5:3; ethyl acetate/iso-hexane) yielding 380 mg of 7 as white solid
(77% yield). 1H NMR (400 MHz, DMSO‑d6) 8.23 (s, 1H), 7.66 (AA0 of
d
an AA’XX’, 2H), 7.63 (AA0 of an AA’XX’, 2H), 7.60 (BB0 of an BB’CC’D,
4H), 7.48 (XX of an AA’XX’, 2H), 7.45 (D of an BB’CC’D, 2H), 7.37 (CC0
of an BB’CC’D, 4H),7.33 (XX of an AA’XX’, 2H), 2.63 (t, J ¼ 7.6 Hz, 2H),
1.60 (m, 2H), 1.34e1.27 (m, 6H), 0.96 (s, 9H), 0.87 (distorted triplet,
w ¼ 13.8 Hz, 3H). 13C NMR (101 MHz, DMSO‑d6)
d
143.6, 143.2,
(400 MHz, DMSO‑d6)
d
12.65 (s, 1H), 8.76 (s, 1H), 8.20 (d, J ¼ 8.1 Hz,
136.5, 136.1, 132.4, 130.3, 129.5, 127.9, 127.3, 126.9, 126.7, 115.4, 35.2,
31.6, 31.3, 28.8, 27.2, 22.6, 18.7, 14.4. (ESI): m/z 556.8 [MþH]þ.
HRMS-ESI (m/z): [M ꢄ H]þ calcd for C34H40NO2SSi, 554.2549;
found, 554.2531.
1H), 7.77 (d, J ¼ 8.1 Hz, 1H), 7.73 (d, J ¼ 7.2 Hz, 1H), 7.27e7.23 (m,
6H), 7.19e7.15 (m, 9H), 4.46 (m, 1H), 4.39 (m, 1H), 4.36 (m, 1H), 4.21
(dd, J ¼ 8.1, 3.2 Hz, 1H), 4.02 (m, 1H), 3.46e3.33 (m, 2H), 3.19e3.06
(m, 2H), 2.74 (dd, J ¼ 15.1, 7.2 Hz, 1H), 2.57 (m, 1H), 2.14 (m, 1H), 1.93
(m, 1H), 1.89e1.79 (m, 2H), 1.73e1.61 (m, 2H), 1.34 (m, 2H),
1.30e1.27 (m, 3H), 1.25e1.22 (m, 16H), 1.14 (s, 9H), 1.00 (d, J ¼
6.2 Hz, 3H), 0.84 (distorted triplet, w ¼ 13.6 Hz, 3H). 13C NMR
4.1.7. General synthesis of the peptides 12e16
The peptides 10 and 11 were synthesized on a 1.0 mmol scale by
7

A. Benediktsdottir, L. Lu, S. Cao et al.
European Journal of Medicinal Chemistry 224 (2021) 113699
(101 MHz, DMSO‑d6)
d
173.2, 171.95, 171.89, 169.2, 168.9, 145.2,
143.7, 143.2, 140.4, 136.3, 136.0, 135.6, 135.5, 129.5, 129.5, 129.0,
128.2, 127.9, 127.8, 127.8, 127.3, 127.0, 126.8, 74.5, 69.8, 67.8, 66.9,
62.5, 57.4, 49.9, 49.0, 48.4, 48.2, 40.5, 35.2, 31.6, 31.5, 31.3, 28.8, 28.5,
28.3, 27.5, 24.6, 22.5, 20.8, 19.62, 19.57, 18.7, 14.4. HRMS-ESI (m/z):
[MþH]þ calcd for C73H87N6O8SSi, 1235.6075; found, 1235.6100.
129.0, 127.9, 126.8, 74.3, 69.9, 67.0, 61.4, 57.9, 49.1, 48.9, 48.3, 31.8,
31.4, 29.4, 29.2 (2C), 29.0, 28.5, 28.2, 25.0, 23.0, 22.6, 19.5, 14.4. MS
(ESI): m/z 932.3 [MþH]þ. HRMS-ESI (m/z): [MþH]þ calcd for
C
51H73N5O9NaS, 954.5027; found, 954.5022.
4.1.9. Synthesis of N2eO-(tert-butyl)-N-(((40-hexyl-[1,10-biphenyl]-
4.1.11. Synthesis of N2eO-(tert-butyl)-N-(((R)-N-(tert-
4-yl)sulfonyl)-L-prolyl)-L-allothreonyl-L-alanyl-N4-trityl-
L
-
butyldiphenylsilyl)-40-hexyl-[1,10-biphenyl]-4-sulfonimidoyl)-L-
asparagine (13)
prolyl)-L-allothreonyl-L-alanyl-N4-trityl-
L
-asparagine (15)
To a stirred suspension of PPh3Cl2 in dry DCM (1 mL) under a N2
gas DIPEA (0.306 mmol, 53.4 L) was added and the reaction
mixture stirred for 30 min at rt then cooled to 0 ꢀC. A solution of the
TBDPS-protected sulfonamide building block (0.123 mmol,
The analog was prepared according to the general peptide
synthesis using the trimer 10 (the Fmoc group was removed prior
coupling by the general method of deprotection) (0.754 mmol,
877 mg, loading: 0.53 mmol/g), the sulfonamide building block 3
(1.04 mmol, 433 mg) in anhydrous DMF (7 mL), and adding HBTU
(1.04 mmol, 394 mg) and DIPEA (2.26 mmol, 0.394 mL). The peptide
was then cleaved from the resin and was directly purified by pre-
parative RP-HPLC (0.05% HCOOH in MeCN/H2O, 90e100% gradient,
20 min, product eluted after 8 min) and the fractions containing the
desired compound were combined and lyophilized to get 528 mg
(70% yield) of desired protected peptide as white soft solid. MS
m
7
80.0 mg) in dry DCM (4 mL) was added. The reaction mixture was
stirred for 30 min then added to the DMF swelled tetramer bound
resin 11 (0.341 mmol, 832 mg, loading: 0.41 mmol/g), in a filter
containing syringe and left rotating for 2h. The peptide was then
washed with 3 ꢂ 5 mL of DCM and DMF and then cleaved from the
resin. The crude was directly purified by preparative RP-HPLC
(0.05% HCOOH in MeCN, 30 min, product eluted after 14 min) and
the fractions containing the desired compound were combined and
lyophilized to get 54.9 mg (63.7% yield) of desired protected pep-
(ESI): m/z 1000.4.1H NMR (400 MHz, DMSO‑d6)
d 12.65 (s, 1H), 8.70
(s, 1H), 8.25 (d, J ¼ 8.0 Hz, 1H), 7.97 (m, 1H) 7.95 (AA0 of an AA’XX’,
2H), 7.89 (XX of an AA’XX’, 2H), 7.77 (d, J ¼ 7.2 Hz, 1H) 7.67 (AA0 of
an AA’XX’, 2H), 7.33 (XX of an AA’XX’, 2H), 7.27e7.22 (m, 6H),
7.20e7.15 (m, 9H) 5.16 (m, 1H), 4.49 (m, 1H) 4.40 (m, 1H), 4.36 (m,
1H), 4.25 (m, 1H), 4.05 (m, 1H), 3.42 (m, 1H), 3.22 (m, 1H) 2.77 (m,
1H) 2.62 (m, 3H), 1.86 (m,1H), 1.76 (m, 1H) 1.73 (m, 1H),1.61 (m, 2H)
1.55 (m, 1H), 1.28 (m, 10H), 1.16 (s, 9H), 1.05 (d, J ¼ 6.3 Hz, 3H) 0.86
(distorted triplet, w ¼ 14.0 Hz, 3H). 13C NMR (101 MHz, DMSO‑d6)
tide as white soft solid. 1H NMR (600 MHz, DMSO‑d6)
d 12.56 (broad
s, 1H), 9.13 (broad s, 1H), 8.20 (d, J ¼ 7.9 Hz, 1H), 7.94 (AA0 of an
AA’XX, 2H), 7.83 (d, J ¼ 8.0 Hz, 1H), 7.81 (d, J ¼ 7.2 Hz, 1H) 7.78 (AA0
of an AA’XX, 2H) 7.72 (XX0 of an AA’XX, 2H) 7.70 (BB0 of an BB’CC’D,
4H) 7.63 (D of an BB’CC’D, 2H), 7.34 (CC0 of an BB’CC’D, 4H), 7.29 (XX0
of an AA’XX’, 2H), 7.24 (m, 6H), 7.17 (m, 9H), 4.47 (m, 1H), 4.42 (m,
1H), 4.25 (dd, J ¼ 8.0, 3.0 Hz, 1H), 4.15 (dd, J ¼ 8.5, 3.5 Hz, 1H), 4.06
(m, 1H), 3.29 (m, 1H), 3.25 (m, 1H), 2.76 (dd, J ¼ 15.2, 6.0 Hz, 1H),
2.61 (m, 2H), 2.56 (m, 1H), 1.83 (m, 1H), 1.67 (m, 1H), 1.58 (m, 3H),
1.50 (m, 1H), 1.27e1.24 (m, 6H), 1.11 (m, 12H), 1.02 (m, 12H), 0.85
d
173.2, 172.0, 171.5, 169.2, 168.9, 162.8, 145.2, 145.0, 143.6, 136.1,
135.7, 129.6, 129.0, 128.7, 127.9 127.6, 127.4, 126.8, 74.4, 69.9, 67.0,
61.7, 58.0, 49.7, 48.3, 38.4, 36.2, 35.2, 31.6, 31.3, 31.1, 28.8, 28.5, 24.7,
22.5, 19.6, 19.5, 14.4. [MþH]þ. HRMS-ESI (m/z): [MþH]þ calcd for
(distorted t, w ¼ 14.1 Hz, 3H). 13C NMR (151 MHz, DMSO‑d6)
d 173.2,
C
57H70N5O9S, 1000.4894; found, 1000.4902.
172.0, 171.9, 169.2, 169.0, 145.2, 143.9, 143.3, 139.0, 136.3, 136.1,
135.5, 129.5, 129.0, 128.4, 127.9, 127.3, 127.2, 126.8, 74.3, 69.8, 66.8,
61.8, 58.0, 50.6, 49.7, 48.3, 40.5, 38.5, 35.2, 31.6, 31.3, 31.1, 28.8, 28.7,
28.4, 27.5, 24.7, 22.5, 19.7, 19.52, 19.46, 14.4. HRMS-ESI (m/z):
[MþH]þ calcd for C73H87N6O8SSi, 1235.6075; found, 1235.6102.
4.1.10. Synthesis of N2eO-(tert-butyl)-N-(((S)eN-(tert-
butyldiphenyl-silyl)-40-hexyl-[1,10-biphenyl]-4-sulfonimidoyl)-L-
prolyl)-L allothreonyl-L-alanyl-N4-trityl-
L
-asparagine (14)
To a stirred suspension of PPh3Cl2 (0.147 mmol, 49.2 mg) in dry
DCM (1.1 mL) under N2 atmosphere was added DIPEA
(0.306 mmol, 53.4 L). The reaction mixture was stirred for 20 min
a
4.1.12. Synthesis of N2eN-((N-(2-((tert-butoxycarbonyl)amino)
m
ethyl)-40-hexyl-[1,10-biphenyl]-4-sulfonimidoyl)-L-prolyl)-O-(tert-
at rt and thereafter cooled to 0 ꢀC. A solution of the TBDPS-
protected sulfonamide building block 7 (0.123 mmol, 80.0 mg) in
dry DCM (4 mL) was added, formation of a light brown-yellow
solution was observed immediately. The reaction mixture was
stirred for 30 min at 0 ꢀC then added to the DMF swelled tetramer-
bound resin 11 (0.341 mmol, 832 mg, loading: 0.41 mmol/g), in a
syringe for 2h on rotating wheel. The peptide was then washed
with 3 ꢂ 5 mL of DCM and DMF and then cleaved from the resin and
directly purified by preparative RP-HPLC (0.05% HCOOH in MeCN,
30 min, product eluted after 16 min) and the fractions containing
the desired compound were combined and lyophilized to get
59.5 mg (65.7% yield) of desired protected peptide as white soft
butyl)-L-allothreonyl-L-alanyl-N4-trityl-
L
-asparagine (16)
To a stirred suspension of PPh3Cl2 in dry DCM (1 mL) under a N2
atmosphere was added DIPEA (1.32 mmol, 230 L). The reaction
m
mixture was stirred for 30 min at rt then cooled to 0 ꢀC. A solution
of the Boc-protected sulfonamide building block 4 (0.430 mmol,
198 mg) in dry DCM (4 mL) was added, formation of a clear light
brown yellow solution was observed immediately. The reaction
mixture was stirred for 30 min at 0 ꢀC then added to the DMF
swelled tetramer bound resin 11 (0.341 mmol, 832 mg, loading:
0.41 mmol/g), in a filter containing syringe and left on a rotating
wheel for 2h. The peptide was then washed with 3 ꢂ 5 mL of DCM
and DMF and then cleaved from the resin and directly purified by
preparative RP-HPLC (0.05% HCOOH in MeCN for 30 min. The
product eluted after 16 min) and the fractions containing the
desired compound were combined and lyophilized to get 54.9 mg
(63.7% yield) of desired protected peptide as white fluffy solid. 1H
solid. 1H NMR (600 MHz, DMSO‑d6)
d 12.55 (broad s, 1H), 8.78
(broad s, 1H), 8.31 (d, J ¼ 7.9 Hz, 1H), 8.21 (d, J ¼ 7.3 Hz, 1H) 8.18 (d,
J ¼ 7.7 Hz, 1H) 7.83 (AA0 of an AA’XX, 2H), 7.83 (d, J ¼ 8.0 Hz, 1H),
7.81 (d, J ¼ 7.2 Hz, 1H) 7.78 (AA0 of an AA’XX, 2H) 7.72 (XX0 of an
AA’XX, 2H) 7.70 (BB0 of an BB’CC’D, 4H) 7.63 (D of an BB’CC’D, 2H),
7.34 (CC0 of an BB’CC’D, 4H), 7.30 (XX of an AA’XX’, 2H), 7.24 (m, 6H),
7.17 (m, 9H), 4.46 (m, 1H), 4.40 (m, 2H), 4.18 (dd, J ¼ 8.0, 3.0 Hz, 1H),
3.91 (m, 1H), 3.20 (m, 1H), 3.25 (m, 1H), 2.69 (dd, J ¼ 15.2, 6.0 Hz,
1H), 2.61 (m, 2H), 2.56 (m, 1H), 1.75 (m, 1H), 1.67 (m, 1H), 1.58 (m,
2H), 1.50 (m, 1H), 1.27e1.24 (m, 6H), 1.11e1.09 (m, 10H), 0.99 (m,
9H), 0.90 (m, 3H) 0.85 (distorted t, w ¼ 13.7 Hz, 3H). 13C NMR
NMR (400 MHz, DMSO‑d6)
d
9.07 (broad s, 1H), 8.32 (d, J ¼ 8.1 Hz,
1H), 8.16 (d, J ¼ 7.7 Hz,1H), 7.98 (d, J ¼ 8.3 Hz,1H), 7.88 (d, J ¼ 7.2 Hz,
1H), 7.84 (m, 1H), 7.65 (m, 1H), 7.32 (d, J ¼ 8.0 Hz, 1H), 7.27 (m, 3H),
7.24 (m, 6H), 7.17 (m,12H), 4.45e4.38 (m, 2H), 4.36 (m,1H), 4.21 (m,
1H), 3.99 (m, 1H), 3.80 (m, 1H), 2.99 (m, 1H), 2.87 (m, 1H), 2.70 (m,
2H), 2.63 (m, 1H), 2.54 (s, 9H), 2.06 (m, 2H), 1.75 (m, 1H), 1.67 (m,
2H), 1.60 (m, 1H), 1.37 (m, 3H), 1.26 (m, 7H), 1.16 (m, 2H), 1.13 (s,
10H), 1.05 (m, 1H), 0.99 (d, J ¼ 6.3 Hz, 3H), 0.86 (distorted triplet,
(151 MHz, DMSO‑d6)
d 173.2, 172.0, 171.8, 169.3, 168.6, 145.3, 145.3,
8

A. Benediktsdottir, L. Lu, S. Cao et al.
European Journal of Medicinal Chemistry 224 (2021) 113699
w ¼ 13.6 Hz, 3H). 13C NMR (126 MHz, DMSO‑d6)
d
172.8,171.3,168.9,
4.1.15. Synthesis of (2S)-2-((2S)-2-((3R)-2-((S)-1-((40-hexyl-[1,10-
biphenyl]-4-yl)sulfonyl)pyrrolidine-2-carboxamido)-3-hydroxy
butanamido)propanamido)-N1-((R)-1-((3aS,4S,6S,7aR)-3a,5,5-
trimethylhexahydro-4,6-methanobenzo[d] [1e3]dioxaborol-2-yl)
ethyl)succinamide (18)
Final compound 18 was prepared according to the general
procedure using the peptide 13 (0.114 mmol, 114 mg), and the
warhead (R)-BoroAla-(þ)-Pinanediol hydrochloride (0.181 mmol,
46.9 mg) in anhydrous DCM and in the presence of HATU
168.7, 168.6, 144.8, 129.1, 128.6, 127.4, 126.3, 77.4, 73.8, 69.3, 66.8,
61.7, 59.8, 57.6, 57.3, 49.4, 47.9, 46.4, 42.4, 42.2, 42.0, 40.4, 34.8, 31.1,
30.8, 30.3, 28.4, 28.0, 25.2, 22.1, 19.1, 18.9, 14.0. C64H84N7O10S MS
(ESI): m/z 1142.4 [MþH]þ. HRMS-ESI (m/z): [MþH]þ calcd for
C
64H84N7O10S, 1142.5995; found, 1142.6000.
4.1.13. General method for coupling the amino-boronic ester
warhead to peptides 12e16 and deprotection of sidechain
protecting groups
(0.228 mmol, 86.6 mg) and DIPEA (1.14 mmol, 198 mL). DCM was
evaporated and de-protection of the side chains was performed
according to the general method (TFA/DCM, 10:9, 3 mL). The crude
yellow oil was purified by preparative RP-HPLC (using TEAA as the
buffer, to avoid hydrolysis of the boronate to boronic acid),
affording 8.1 mg (9.2% yield) of the desired oligopeptide 18 as white
soft solid and as a 50:50 mixture of epimers. Characterization for
To a solution of the peptide (1 equiv.) in anhydrous DCM
(2e4 mL), HATU (2 equiv.) and DIPEA (3e10 equiv.) were added
under N2 flow and the mixture was stirred at 0 ꢀC for 5 min, after
which the warhead (2 equiv.) was added and the reaction mixture
was stirred for 30 min and then removed from the ice bath and
stirred for another 30 min at rt till completion (monitoring via LC-
MS). After completion, the solvent was removed under N2 stream
and the crude dissolved in minimum amount of DMSO for purifi-
cation by RP-HPLC, the pure product was then used for the final
step, deprotection of the sidechain protecting groups. The crude or
pure protected peptides (1 equiv.) were dissolved in a solution of
95% TFA in H2O or in a solution of TFA/DCM/TES (10:9:1) and stirred
at rt till completion (1 h). The solvents were removed under
reduced pressure and the residues were purified by preparative RP-
HPLC with TEAA/H2O/MeCN mobile system.
the major epimer: 1H NMR (400 MHz, DMSO‑d6)
d
8.37 (d, J ¼
8.0 Hz, 1H), 8.26 (m, 2H), 8.00 (m, 1H), 7.93 (AA0 of an AA’XX’, 4H),
7.68 (XX0 of an AA’XX’, 2H), 7.50 (m, 1H), 7.34 (XX0 of an AA’XX’, 2H),
7.11 (m, 2H), 5.02 (m, 1H), 4.81 (m, 1H), 4.31 (m, 1H), 4.27 (m, 1H),
4.09 (m, 2H), 3.92 (m, 1H), 3.65 (m, 2H), 3.62 (m, 1H), 3.45 (m, 1H),
3.21 (m, 1H), 3.14 (m, 3H), 2.64 (m, 3H), 1.84 (m, 1H), 1.76 (m, 1H),
1.70 (m, 1H), 1.60 (m, 2H), 1.55 (m, 1H), 1.29 (m, 2H), 1.26 (m, 6H),
1.24 (m, 8H), 1.09 (m, 1H), 0.96 (m, 2H), 0.86 (distorted triplet,
w ¼ 11.9 Hz, 3H), 0.82 (s, 6H). 13C NMR (126 MHz, DMSO)
d 177.3,
172.4, 171.3, 170.0, 169.8, 144.6, 143.1, 135.6, 135.0, 129.1, 128.3, 127.1,
126.3, 118.1, 97.2, 76.4, 66.7, 65.7, 61.4, 59.9, 57.7, 54.9, 49.2, 45.8,
35.9, 34.8, 31.1, 30.8, 30.7, 29.0, 28.3, 26.2, 24.2, 22.1, 19.7, 18.1, 17.6,
16.7, 14.9, 14.0, 12.5, 8.6. C46H67BN6O10S MS (ESI): m/z 907.47
[MþH]þ. RP-HPLC purity: C8 column >95%. HRMS-ESI (m/z):
[MþH]þ calcd for C46H68N6O10SB, 907.4811; found, 907.4818.
4.1.14. Synthesis of (2S)-2-((2S)-2-((3R)-2-((S)-1-(dodecylsulfonyl)
pyrrolidine-2-carboxamido)-3-hydroxybutan-amido)
propanamido)-N1-((R)-1-((3aS,4S,6S,7aR)-3a,5,5-trimethyl-
hexahydro-4,6-methanobenzo[d] [1e3]dioxaborol-2-yl)ethyl)
succinamide (17)
The final compound 17 was prepared according to the general
procedure using peptide 12 (0.0429 mmol, 40.0 mg), and the
commercially available warhead (R)-BoroAla-(þ)-Pinanediol hy-
drochloride (0.0858 mmol, 22.3 mg) in anhydrous DCM (1.2 mL)
and in the presence of HATU (0.0858 mmol, 32.6 mg) and DIPEA
4.1.16. Synthesis of (S)-2-((S)-2-((2S,3S)-2-((S)-1-((R)-40-hexyl-
[1,10-biphenyl]-4-sulfonimidoyl)pyrrolidine-2-carboxamido)-3-
hydroxy butanamido)propanamido)-N1-((R)-1-((3aS,4S,6S,7aR)-
3a,5,5-trimethylhexahydro-4,6-methanobenzo[d] [1e3]dioxaborol-
2-yl)ethyl)succinamide (19)
(0.172 mmol, 29.9
mixture dissolved in 900
m
L). The solvent was removed, and the crude
The final compound 19 was prepared according to the general
procedure using the peptide 14 (0.049 mmol, 51.8 mg), and the
warhead (R)-BoroAla-(þ)-Pinanediol hydrochloride (0.0837 mmol,
31.8 mg) in anhydrous DCM (1 mL) and in the presence of HATU
mL DMSO and purified by multiple runs
into the preparative RP-HPLC (20 mM TEAA MeCN/H2O, 90e100%
gradient for 20 min, product eluted at 9 min). The fractions with the
desired product were collected at 214 nm and lyophilized yielding
40.1 mg of pure white compound (84% yield) (RP-HPLC purity: C8
column 100%, MS (ESI): m/z 1137.9) with sidechains still protected.
Next, the deprotection of the side chains was performed according
to the general method (TFA/DCM/TES, 10:9:1, 3 mL) for 1h at rt. The
solvent was removed and the oily crude purified by preparative RP-
HPLC (20 mM TEAA MeCN/H2O, 55e100% 28 min gradient; product
eluted after 8 min) affording 11.5 mg (39% yield) of desired oligo-
peptide boronate 17 (epimeric ratio 90:10). Major epimer: 1H NMR
(0.0837 mmol, 31.8 mg) and DIPEA (0.209 mmol, 36.5 mL). DCM was
evaporated and de-protection of the side chains was performed
according to the general method (TFA/DCM, 10:9, 3 mL). The crude
yellow oil was purified by preparative RP-HPLC (using TEAA as the
buffer, to avoid hydrolysis of the boronate to boronic acid),
affording the total yield of the epimers 15 mg (25% yield) of the
desired oligopeptide 19 as white solid and as a 53:47 mixture of
epimers. Characterization for the major epimer: 1H NMR (600 MHz,
DMSO‑d6)
d
8.63 (m, 1H), 8.39 (d, J ¼ 6.9 Hz, 1H), 8.27 (d, J ¼ 8.2 Hz,
(400 MHz, DMSO‑d6)
d
8.65 (broad s, 1H), 8.08 (d, J ¼ 7.7 Hz, 1H),
1H), 8.06 (d, J ¼ 7.9 Hz, 1H), 7.97 (AA0 of an AA’XX’, 2H), 7.90 (AA0 of
an AA’XX’, 2H), 7.80 (d, J ¼ 8.4 Hz, 1H), 7.68 (XX0 of an AA’XX’, 2H),
7.34 (XX0 of an AA’XX’, 2H), 6.97 (m, 1H), 5.12 (m, 1H), 4.66 (s, 1H),
4.62 (d, J ¼ 7.0 Hz, 1H), 4.32 (m, 1H), 4.27 (m, 1H), 4.17 (m, 1H), 4.07
(m, 1H), 3.92 (m, 1H), 3.51e3.43 (m, 1H), 3.13 (m, 1H), 2.62 (m, 3H),
2.45 (m, 1H), 2.38 (m, 1H), 2.19 (m, 1H), 2.01 (m, 1H), 1.84 (m, 1H),
1.79 (m, 2H), 1.72e1.69 (m, 3H), 1.65 (m, 1H), 1.62e1.57 (m, 2H), 1.51
(m, 1H), 1.27 (m, 6H), 1.27e1.22 (m, 8H), 1.09e1.06 (m, 3H), 1.00 (m,
7.95 (d, J ¼ 6.8 Hz, 1H), 7.71 (d, J ¼ 8.5 Hz, 1H), 7.35 (s, 1H), 6.96 (s,
1H), 5.02 (d, J ¼ 5.2 Hz, 1H), 4.59 (q, J ¼ 6.9 Hz, 1H), 4.36 (dd, J ¼ 8.6,
3.6 Hz, 1H), 4.25 (m, 1H), 4.21 (m, 1H), 4.09 (m, 1H), 4.07 (m, 1H),
3.46e3.35 (m, 2H), 3.20e3.07 (m, 2H), 2.52 (m, 2H), 2.49e2.45 (m,
2H), 2.21 (m, 1H), 2.13 (m, 1H), 2.01 (m, 2H), 1.93 (m, 1H), 1.88e1.82
(m, 2H), 1.78 (m, 1H), 1.70e1.63 (m, 2H), 1.62 (m, 1H), 1.40e1.33 (m,
4H), 1.28e1.17 (m, 21H), 1.03 (d, J ¼ 6.3 Hz, 3H), 0.99 (m, 2H), 0.84
(distorted triplet, w ¼ 13.5 Hz, 3H), 0.80 (s, 6H). 13C NMR (101 MHz,
2H), 0.86 (distorted triplet, w ¼ 13.8 Hz, 3H), 0.80 (broad s, 6H). 13
C
DMSO‑d6)
d
174.1, 172.2, 171.2, 170.2, 83.3, 76.1, 67.0, 61.5, 57.9, 52.2,
NMR (126 MHz, DMSO‑d6) d 173.6, 172.3, 172.2, 170.8, 170.7, 143.9,
49.1, 49.0, 48.5, 47.1, 38.1, 31.8, 29.5, 29.42, 29.38, 29.3, 29.2, 29.0,
28.2, 27.6, 25.0, 24.4, 23.0, 22.6, 20.1, 18.2, 16.9, 14.4. RP-HPLC pu-
rity: C8 column >95%. C40H71BN6O10S MS (ESI): m/z 839.5 [MþH]þ.
HRMS-ESI (m/z): [M þ Na þ H]þ calcd for C40H72BN6O10S þ Na,
861.4938; found, 861.4949.
142.9, 135.7, 135.4, 129.1, 128.6, 127.9, 126.9, 82.9, 75.7, 66.7, 62.5,
57.4, 51.7, 49.9, 48.9, 48.7, 47.6, 45.7, 37.6, 36.6, 36.2, 34.7, 31.1, 30.8,
28.9, 28.3, 27.1, 26.0, 24.3, 23.9, 22.1, 20.1, 17.7, 17.5, 16.4, 14.0.
C
46H68BN7O9S, MS (ESI): m/z 906.47 [MþH]þ. RP-HPLC purity: C8
column >95%. HRMS-ESI (m/z): [MþH]þ calcd for C46H69N7O9SB,
9

A. Benediktsdottir, L. Lu, S. Cao et al.
European Journal of Medicinal Chemistry 224 (2021) 113699
906.4971; found, 906.5002.
127.0, 75.7, 51.7, 47.5, 45.7, 41.1, 40.0, 39.9, 39.7, 39.5, 39.4, 39.2, 39.0,
37.7, 36.2, 34.8, 31.1, 30.8, 28.9, 28.3, 28.1, 27.1, 26.0, 24.0, 23.9, 22.1,
22.0 (2C), 20.0, 16.4, 14.0, 8.6. RP-HPLC purity: C8 column >95%.
HRMS-ESI (m/z): [MþH]þ calcd for C48H73BN8O9S, 949.5393; found,
949.5410.
4.1.17. Synthesis of (2S)-2-((2S)-2-((3R)-2-((S)-1-((S)-40-hexyl-
[1,10-biphenyl]-4-sulfonimidoyl)pyrrolidine-2-carbox-amido)-3-
hydroxy butan-amido)propanamido)-N1-((R)-1-((3aS,4S,6S,7aR)-
3a,5,5-trimethyl-hexa-hydro-4,6-methanobenzo[d] [1e3]
dioxaborol-2-yl) ethyl)succinamide (20)
4.2. Bioassays
The final compound 20 was prepared according to the general
procedure using the peptide 15 (0.0404 mmol, 50.0 mg), and the
warhead (R)-BoroAla-(þ)-Pinanediol hydrochloride (0.0808 mmol,
21.0 mg) in anhydrous DCM (1.5 mL) in the presence of HATU
The E. coli signal peptidase activity and inhibition experiments
were conducted in a FRET assay with the two fluorophore labeled
substrates; dabcyl-VEVGGTATAYGAFSRPGLE-(EDANS)) and dabcyl-
KLTFGTVKPVQAIAYGYEILE-(EDANS)-OH (arrows indicate the ex-
pected cleavage sites). The linear increase in fluorescence, corre-
sponding to substrate cleavage, was monitored at 22 ꢀC for 2 h. For
the inhibition tests, EcLepB was pre-incubated with the compound
for 10 min at 22 ꢀC, then the reaction was followed at a substrate
(0.0808 mmol, 30.7 mg) and DIPEA (0.202 mmol, 35.2 mL). DCM was
evaporated and de-protection of the side chains was performed
according to the general method (TFA/DCM, 10:9, 2 mL). The crude
yellow oil was purified by preparative RP-HPLC (using TEAA as the
buffer, to avoid hydrolysis of the boronate to boronic acid),
affording the total yield of the epimers 7.0 mg (12% yield) of the
desired oligopeptide 20 as white solid and as a 84:16 mixture of
epimers. Characterization for the major epimer: 1H NMR (600 MHz,
(dabcyl-VGGTATAGAFSRPGLE (EDANS)-OH) concentration of 8 mM;
final EcLepB concentration was estimated to be 50 nM. Reaction
rates were plotted as a function of inhibitor concentration, and half
maximal inhibitory concentration (IC50) values were determined by
a non-linear regression analysis of the sigmoidal doseeresponse
curves in GraphPad Prism (GraphPad software Inc., CA, USA). The
cloning, expression and purification of the E. coli LepB protein is
fully described in the supporting information by De Rosa et al. [26].
For assessing the minimal inhibitory concentration (MIC), the
compounds were prepared in Mueller-Hinton II medium and
dispensed into a 96-well round-bottomed plate to give final assay
DMSO‑d6)
d
8.63 (m, 1H), 8.06 (d, J ¼ 7.9 Hz, 1H), 7.99 (m, 1H), 7.97
(AA0 of an AA’XX’, 2H), 7.90 (AA0 of an AA’XX’, 2H), 7.80 (d,
J ¼ 8.4 Hz, 1H), 7.68 (XX0 ozf an AA’XX’, 2H), 7.36 (s, 1H), 7.34 (XX0 of
an AA’XX’, 2H), 6.97 (m, 1H), 5.12 (m, 1H), 4.76 (s, 1H), 4.62 (q,
J ¼ 7.0 Hz, 1H), 4.27 (m, 1H), 4.23 (m, 1H), 4.17 (m, 1H), 4.09 (m, 1H),
4.06 (m, 1H), 3.41 (m, 1H), 3.14 (m, 1H), 2.62 (m, 3H), 2.38 (m, 1H),
2.19 (m, 1H), 2.01 (m, 1H), 1.84 (m, 1H), 1.79 (m, 1H), 1.72e1.69 (m,
3H), 1.65 (m, 1H), 1.62e1.57 (m, 2H), 1.51 (m, 1H), 1.27 (m, 6H), 1.24
(m, 6H), 1.21 (m, 3H), 1.08 (m, 3H), 1.00 (m, 2H), 0.86 (distorted
triplet, w ¼ 13.8 Hz, 3H), 0.80 (s, 6H). 13C NMR (126 MHz, DMSO‑d6)
concentrations from 64
mg/mL down to 0.25 mg/mL (two-fold
dilution series in 10 wells, with two control wells: medium control
with no bacteria or compound, and growth control with bacteria
added but no compound). Plates were covered and incubated
without shaking for 16e20 h at 35 ꢀC 2 ꢀC. MIC was read visually,
as complete inhibition of growth by the unaided eye, using the
medium-only wells as the control [26].
The compounds were evaluated for hemolytic activity using red
blood cells from heparinized human blood. Red blood cells (RBC)
were washed three times in Tyrode buffer (130 mM NaCl, 4 mM KCl,
2.8 mM Na acetate, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose,
1 mM CaCl2, adjusted to pH 7.4) and resuspended in the same
d
173.6, 172.3, 172.2, 170.8, 170.7, 143.9, 142.9, 135.7, 135.4, 129.1,
128.6, 127.9, 126.9, 82.9, 75.7, 66.7, 62.5, 57.4, 51.7, 49.9, 48.9, 48.7,
47.6, 45.7, 37.6, 36.6, 36.2, 34.7, 31.1, 30.8, 28.9, 28.3, 27.1, 26.0, 24.3,
23.9, 22.1, 20.1, 17.7, 17.5, 16.4, 14.0. C46H68BN7O9S, MS (ESI): m/z
906.47 [MþH]þ. RP-HPLC purity: C8 column >95%. HRMS-ESI (m/z):
[MþH]þ calcd for C46H69N7O9SB, 906.4971; found, 906.5002.
4.1.18. Synthesis of 2-((((S)-2-(((2S,3S)-1-(((S)-1-(((S)-4-amino-
1,4-dioxo-1-(((R)-1-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-
4,6-methanobenzo[d] [1e3]dioxaborol-2-yl)ethyl)amino)butan-2-
yl) amino)-1-oxopropan-2-yl)amino)-3-hydroxy-1-oxobutan-2-yl)
carbamoyl)pyrrolidin-1-yl) (40-hexyl-[1,10-biphenyl]-4-yl) (oxo)-l6-
sulfanylidene)amino)ethan-1-aminium (21)
buffer. Final concentrations in the hemolysis assay were 100
mM
compound, 1% DMSO, and 50% RBC, assayed in a 200 L vol. in a
m
microtiter plate. The mixture was incubated at 37 ꢀC for 45 min
with shaking (250 rpm). After incubation, RBCs were removed by
centrifugation and clear plasma was transferred to a fresh plate,
and the amount of hemoglobin measured using a spectrophotom-
eter at 540 nm. The complete lysis control contained 2% Triton X-
100 (in Tyrode buffer) instead of compound; the negative control
contained Tyrode buffer but no compound. Percent hemolysis was
calculated as: [Abs compound] e [Abs negative control]/[Abs
complete lysis control] e [Abs negative control] x 100. Values
The final compound 21 was prepared according to the general
procedure using the peptide 16 (0.0446 mmol, 50.9 mg), and the
warhead (R)-BoroAla-(þ)-Pinanediol hydrochloride (0.0891 mmol,
23.1 mg) in anhydrous DCM (900
mL) in the presence of HATU
(0.0891 mmol, 33.9 mg) and DIPEA (0.178 mmol, 31.0
mL). DCM was
evaporated and de-protection of the side chains was performed
according to the general method (TFA/DCM, 10:9, 2 mL). The crude
yellow oil was purified by preparative RP-HPLC (using TEAA as the
buffer, to avoid hydrolysis of the boronate to boronic acid),
affording the total yield of the epimers 8.1 mg (14% yield) of the
desired oligopeptide 21 as white solid and as a 90:10 mixture of
epimers. Characterization for the major epimer: 1H NMR (500 MHz,
greater than 1% hemolysis at 100
[26].
mM were regarded as a red flag
In vitro cytotoxicity was determined by a fluorometric micro-
culture cytotoxicity assay using the HepG2 cell line from ATCC, and
cultured in Eagle's Minimum Essential Medium (ATCC) supple-
mented with 10% fetal bovine serum, penicillin/streptomycin (100
DMSO‑d6)
d
9.06 (broad s, 2H), 8.71 (m, 2H), 8.31 (d, J ¼ 8.1 Hz, 1H),
8.23 (d, J ¼ 7.0 Hz,1H), 8.17 (d, J ¼ 8.2 Hz,1H), 8.09 (d, J ¼ 8.3 Hz,1H)
8.05 (AA0 of an AA’XX, 2H), 7.89 (XX0 of an AA’XX, 2H) 7.78 (m, 1H),
7.69 (AA0 of an AA’XX, 2H), 7.34 (XX of an AA’XX’, 2H), 4.61 (m, 1H),
4.37 (m, 2H), 4.28 (m, 2H), 4.23 (m, 1H), 4.06 (m, 1H), 3.64 (m, 1H),
3.38 (m, 1H), 3.27 (m, 1H), 2.90 (m, 1H), 2.63 (m, 2H), 2.19 (m, 2H),
2.02 (m, 2H), 1.88 (m, 2H), 1.76 (m, 1H), 1.59 (m, 1H), 1.31e1.27 (m,
8H), 1.24 (m, 3H), 1.22 (m, 2H), 1.20 (m, 1H), 1.09 (m, 3H), 1.04 (m,
3H), 1.00 (m, 3H), 0.94 (d, J ¼ 6.5 Hz, 3H), 0.86 (distorted t,
U/100 mg/mL) and L-glutamine 2 mM. HepG2 cells were passaged 2
times/week and used maximally for 20 passages. Cells were finally
seeded in Nunc 384-well assay plates at a density of 1000 cells/well.
Cytotoxicity was assessed after 72 h with cell survival presented as
survival index (SI, %) defined as fluorescence in test wells in percent
of control cultures with blank values subtracted. Criteria for a
successful assay included a signal-to-noise ratio in control cultures
>10, CV < 30% and a positive control (Bortezomib) SI of <5%. The
half maximal inhibitory concentration (IC50) was determined from
w ¼ 13.9 Hz, 3H), 0.80 (s, 6H). 13C NMR (126 MHz, DMSO)
d 173.7,
172.1, 171.9, 170.6, 170.1, 158.0, 157.7, 143.1, 135.7, 129.1, 128.5, 127.5,
10

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Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships which have or could be
perceived to have influenced the work reported in this article.
Acknowledgements
This work was supported by the Swedish Research Council (521-
2014-6711 and 2017e03953). We thank Lena Lenhammar and Rolf
Larsson at the Department of Medical Sciences for contributing to
the cytotoxicity studies.
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Author contributions
[20] A.G. Therien, J.L. Huber, K.E. Wilson, P. Beaulieu, A. Caron, D. Claveau,
K. Deschamps, R.G.K. Donald, A.M. Galgoci, M. Gallant, X. Gu, N.J. Kevin,
J. Lafleur, P.S. Leavitt, C. Lebeau-Jacob, S.S. Lee, M.M. Lin, A.A. Michels,
A.M. Ogawa, R.E. Painter, C.A. Parish, Y.-W. Park, L. Benton-Perdomo, M. Petcu,
J.W. Phillips, M.A. Powles, K.I. Skorey, J. Tam, C.M. Tan, K. Young, S. Wong,
AB carried out the synthesis, data analysis, and wrote the
manuscript. AS, AK, and EZ were involved in the project planning,
supervision, data analysis, and in manuscript editing. The enzy-
matic studies and data analysis were performed by LL. The micro-
biology and hemolysis assays were performed by SC. SLM and DH
were involved in the supervision, data analysis, and in manuscript
editing. All authors listed contributed to the manuscript.
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