Design, synthesis and in vitro biological evaluation of oligopeptides targeting E. coli type I signal peptidase (LepB)

Article abstract of DOI:10.1016/j.bmc.2016.12.003

Type I signal peptidases are potential targets for the development of new antibacterial agents. Here we report finding potent inhibitors of E. coli type I signal peptidase (LepB), by optimizing a previously reported hit compound, decanoyl-PTANA-CHO, through modifications at the N- and C-termini. Good improvements of inhibitory potency were obtained, with IC₅₀s in the low nanomolar range. The best inhibitors also showed good antimicrobial activity, with MICs in the low μg/mL range for several bacterial species. The selection of resistant mutants provided strong support for LepB as the target of these compounds. The cytotoxicity and hemolytic profiles of these compounds are not optimal but the finding that minor structural changes cause the large effects on these properties suggests that there is potential for optimization in future studies.

Full text of DOI:10.1016/j.bmc.2016.12.003

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and in vitro biological evaluation of oligopeptides
targeting E. coli type I signal peptidase (LepB)
Maria De Rosa a,g, Lu Lu , Edouard Zamaratski , Natalia Szałaj , Sha Cao , Henrik Wadensten ,
b,g
a
a
c
d
e
a
f
c
e
d
Lena Lenhammar , Johan Gising , Annette K. Roos , Douglas L. Huseby , Rolf Larsson , Per E. Andrén ,
Diarmaid Hughes , Peter Brandt , Sherry L. Mowbray
Uppsala University, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, BMC, Box 574, SE-751 23 Uppsala, Sweden
Uppsala University, Department of Cell and Molecular Biology, BMC, Box 596, SE-751 24 Uppsala, Sweden
Uppsala University, Department of Medical Biochemistry and Microbiology, BMC, Box 582, SE-751 23 Uppsala, Sweden
Uppsala University, Department of Pharmaceutical Biosciences, BMC, Box 591, SE-751 24 Uppsala, Sweden
Uppsala University Hospital, Department of Medical Sciences, Uppsala, Sweden
c
a
b,f,
⇑
a,
⇑
, Anders Karlén
a
b
c
d
e
f
Uppsala University, Science for Life Laboratory, Department of Cell and Molecular Biology, BMC, 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:
Type I signal peptidases are potential targets for the development of new antibacterial agents. Here we
report finding potent inhibitors of E. coli type I signal peptidase (LepB), by optimizing a previously
reported hit compound, decanoyl-PTANA-CHO, through modifications at the N- and C-termini. Good
improvements of inhibitory potency were obtained, with IC50s in the low nanomolar range. The best inhi-
Received 10 June 2016
Revised 14 October 2016
Accepted 3 December 2016
Available online xxxx
bitors also showed good antimicrobial activity, with MICs in the low
lg/mL range for several bacterial
species. The selection of resistant mutants provided strong support for LepB as the target of these com-
pounds. The cytotoxicity and hemolytic profiles of these compounds are not optimal but the finding that
minor structural changes cause the large effects on these properties suggests that there is potential for
optimization in future studies.
Keywords:
Antibacterials
Escherichia coli
Oligopeptides
Ó 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Solid-phase peptide synthesis
Type I signal peptidase
1
1
. Introduction
against drug-resistant strains. Of particular interest from the clin-
ical perspective are the ESKAPE pathogens (Enterococcus faecium,
The frequency of antimicrobial resistance has increased signifi-
Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter bau-
mannii, Pseudomonas aeruginosa, and Enterobacter species) that
cause the majority of healthcare-associated infections worldwide.3
In recent decades, type I signal peptidases (SPase I) have been
extensively explored as potential targets for new antibacterial
agents. Signal peptidases play a crucial role in the bacterial Sec
and Tat secretion pathways, catalyzing the cleavage of a largely
hydrophobic N-terminal sequence (the signal or ‘‘leader” peptide)
from their natural substrates (which include most pre-secretory
cantly in recent decades.1 There is an urgent need to discover and
,2
develop new drugs with novel modes of action that are effective
Abbreviations: dabcyl, 4-((4-(dimethylamino)phenyl)azo)benzoic acid; DCM,
dichloromethane; DIEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide;
E. coli, Escherichia coli; EDANS, 5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid;
EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; Fmoc, 9-fluorenylmethoxy-
0
0
carbonyl; HBTU, N,N,N ,N -tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluo-
rophosphate; HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]
4
proteins). Inhibition of SPase leads to an accumulation of pre-pro-
pyridinium 3-oxide hexafluorophosphate; HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol;
IC50, half maximal inhibitory concentration; MIC, minimal inhibitory concentration;
5,6
teins at the phospholipid bilayer and cell death. Bacterial SPases
6,7
are essential enzymes that differ significantly from their eukary-
SPPS, solid-phase peptide synthesis; TES, triethylsilane; SPase I, type
peptidase; TFA, trifluoroacetic acid.
I signal
8
otic counterparts, making these enzymes attractive targets for
⇑
Corresponding authors at: Uppsala University, Department of Medicinal
drug development.
Chemistry, Organic Pharmaceutical Chemistry, BMC, Box 574, SE-751 23 Uppsala,
Sweden (A. Karlén) and Department of Cell and Molecular Biology, BMC, Box 596,
SE-751 24 Uppsala, Sweden (S.L. Mowbray).
SPases I (EC 3.4.21.89) are endopeptidases that belong to the
serine protease family. They are ubiquitous and well conserved
among bacterial species. The biochemical properties of SPases I
E-mail addresses: Sherry.Mowbray@icm.uu.se (S.L. Mowbray), Anders.Karlen@
orgfarm.uu.se (A. Karlén).
9
from Gram-positive bacteria (B. subtilis, S. pneumoniae, and S.
g
10
Maria De Rosa and Lu Lu contributed equally to this work.
aureus ) as well as from Gram-negative bacteria have been
http://dx.doi.org/10.1016/j.bmc.2016.12.003
0
968-0896/Ó 2016 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: De Rosa M., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.12.003

2
M. De Rosa et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
studied in detail. SPase I of E. coli, commonly known as LepB, and
herein designated EcLepB, is the best characterized.11 SPase I
enzymes operate via a Ser/Lys catalytic dyad, which differs from
the classical Ser-His-Asp triad seen in most serine proteases. The
active site (including S91 and K146 in EcLepB)12 is located at the
outer surface of the cytoplasmic membrane, making it accessible
to any inhibitor able to cross the outer membrane of Gram-nega-
11,13
tives, or the cell wall of Gram-positives.
SPases have evolved
recognition criteria consistent with their need to cleave many,
but not all, peptide sequences. The canonical signal peptide starts
with a short positively-charged region, followed by a hydrophobic
segment of sufficient length to span the membrane in question in a
a) 5S-Penems
b) Arylomycins
R1
R1
O
HO
O
O
OR2
O
O
R3
HH
Me
Me
S
Me
S
O
OH
N
N
O
O
Me
O
HN
H
N
H
N
H
N
R4
O
O
O
N
N
H
N
Me
O
O
O
R2
O
O
O
O
Me
OH
1
. Allyl (5S,6S)-6-[(R)-acetoxyethyl]-penem-3-carboxylate
5. Tricyclic penem
Arylomycin A:
R1 = R = R3 = H; R4 = iso-C11,12,14, n-C12, anteiso-C13
1
= Acetyl, R2 = allyl
2
R
2
. Allyl (5S,6S)-6-[(R)-hydroxyethyl]-penem derivative
6. Arylomycin A : R
= iso-C
12
4
2
1
= H, R2 = p-nitrobenzyl
4
R
7. ArylomycinA-C16: R = n-C15
Carbamate derivatized penem
Arylomycin B:
. R = -NHEt, R2 = p-nitrobenzyl
1
R1 = NO ; R = R = H; R4 = iso-C11,12,14, n-C12, anteiso-C13,15
2
3
3
2
. R = -NHiPr, R2 = p-nitrobenzyl
1
4
ArylomycinC (lipoglycopeptides):
: R1 = H; R = deoxy-α-mannose;R = H or OH; R4 = iso- or n-C13,15
2
3
8
c) Oligopeptides
O
Me
O
Me
H
N
H
H
N
N
N
H
N
H
Me
O
O
O
O
O
Me
OH
2
H N
9
. Decanoyl-PTANA-CHO
Fig. 1. Classes of SPase I inhibitors.
Fig. 2. New PTANA oligopeptide analogues.
Please cite this article in press as: De Rosa M., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.12.003

M. De Rosa et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
3
presumably
a
-helical conformation, then the actual recognition
or more enzymes from different species are used to guide chem-
istry efforts, generating compounds that are then tested against a
number of different pathogens. In this way, the benefits of organic
synthesis efforts are maximized. In the current study, we aimed at
optimizing the structure of the known oligopeptide decanoyl-
sequence characterized by small, neutral residues in the ꢀ3 (P3)
and ꢀ1 (P1) positions relative to the point of cleavage, the so-called
Ala-X-Ala sequence. The fact that the substrates are pre-oriented,
by virtue of the placement of their hydrophobic segment in the
lipid bilayer, is thought to be a factor of importance for activity.
To date, the search for effective drugs has uncovered three classes
1
2
PTANA-CHO 9 (Fig. 1, EcLepB IC50 = 13.4 lM, E. coli MIC > 500 lg/
1
8
mL), in order to determine whether we could improve potency
inhibitors, namely penems (b-lactams),1
and oligopeptides (Fig. 1). However, useful thera-
4,15
ary-
against both the enzyme and bacterial cells.
of SPase
lomycins
I
1
6,17
18
In particular, we wished to investigate the influence of
modifications at the N- and C-termini, while keeping the
Pro-Thr-Ala-Asn-Ala (PTANA) peptide sequence unchanged. This
type of inhibitor was first reported by Bruton et al., and was based
on the consensus sequence of pre-proteins in S. aureus.19 In our
studies, the aldehyde warhead at the C-terminus was first replaced
pies targeting these enzymes have not yet been developed.
Our group is actively involved in the search for new starting
points for antibiotic development, and targeting the LepB enzyme
has been one of our areas of interest. A core premise in our work
is the STOP (Same-Target-Other-Pathogen) strategy, in which one
Cl
O
Cl
FmocHN
O
a
OH
O
+
FmocHN
Cl
O
O
NHTrt
NHTrt
10: Fmoc-Asn-OH
11: 2-CTC
12
O
Me
O
O
Me
O
H
H
H
N
H H
N
H
N
b, d
N
N
Fmoc
N
H
O
O
N
N
H
O
O
O
O
O
O
O
Me
OtBu
R
Me
OtBu
NHTrt
NHTrt
13
17a-c
O
Fmoc-aa-COOH
FmocHN
Me
O
O
Me
O
O
O
b, c
H
b, d
H
Fmoc
N
N
N
O
O
Me
N
H
O
O
H
O
O
NHTrt
1
2
1
4
NHTrt
18
NHTrt
BocHN
O
Me
O
O
Me
O
H
H
H
N
H H
H
N
N
N
Fmoc
N
H
O
O
b, c
N
N
H
O
16
O
O
O
O
Me
OtBu
O
Me
OtBu
NHTrt
NHTrt
15
19
C
6 13
H
O
Me
O
H
N
H
H
N
e
e
1
3
N
Fmoc
N
OH
O
H
O
O
Me
OtBu
2
0
NHTrt
(
a)
Me
O
Me
O
H H
H
1
7a-c
N
N
(b)
R =
( )6 Me
N
O
N
OH
O
H
O
R
O
Me
OtBu
1a-c
(c)
6 13
C H
2
NHTrt
O
Me
O
e
H
1
8
9
N
Me
N
H
OH
O
O
2
2
NHTrt
BocHN
O
Me
O
H H
N
H
N
e
1
N
H
OH
O
N
O
O
Me
OtBu
O
NHTrt
23
C
6
H
13
Scheme 1. Solid-Phase Synthesis of Peptides 20, 21a–c, 22, 23. Reagents and conditions: a) DIEA, DMF, rt, 2 h, then MeOH, rt, 15 min; b) 20% piperidine in DMF, 2 min + 10 min,
rt; c) Fmoc-AA-OH, HBTU, DIEA, DMF, rt, 2 h or 2 h + 2 h or overnight; d) acetic anhydride or decanoic anhydride, DIEA, DMF, rt, 20 h; e) HFIP:DCM 1:4, rt, 1 h, 60–88%
Please cite this article in press as: De Rosa M., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.12.003

4
M. De Rosa et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
with chemically more stable but equally reactive bioisosters such
extensively washed with DMF, methanol and dichloromethane,
then dried under high vacuum.
as
a
-keto-amides, boronic acid and ester (Fig. 2). We also intro-
0
duced a proline at P1 , which has been shown to convert serine
Coupling of the tail at the N-terminus, using acetic anhydride,
20,21
0
protease substrates into inhibitors.
Finally, an analogue simply
decanoic anhydride and 4 -hexyl-biphenyl-carboxylic acid, respec-
carrying the unmodified alanine was prepared to determine
whether the electrophile moiety at the C-terminus is really
required for antibacterial activity. At the N-terminus, the decanoyl
tail was replaced with a bulkier 4-(4-hexylphenyl)benzoyl group. A
few examples of derivatives without the fatty acid tail, but capped
at the N-terminus with an acetyl group, were also prepared, to
evaluate the effects on the activity profile. In addition, a positive
charge was introduced at the P5 residue in an attempt to increase
cell wall permeability and to improve the potency of the com-
pound inside the bacterial cell. Herein, we describe the design, syn-
thesis and in vitro evaluation of these new oligopeptides against
EcLepB. Other aspects, such as activity towards a panel of Gram-
negative bacteria, cytotoxicity and hemolysis, are also explored.
tively, afforded intermediates 17a–c, 18. In the case of the modified
proline 16, the fatty acid tail was directly coupled to the commer-
cially available 4-hydroxyproline 26 (Scheme 2).
Next, cleavage of resin-bound peptides 13, 17a–c, 18 and 19 by
treatment with a solution of 1,1,1,3,3,3-hexafluoro-2-propanol:
2
3
dichloromethane (1:4), afforded peptides 20, 21a–c, 22 and 23.
Scheme 2 depicts the synthesis of the modified proline 16. Starting
from 4-hydroxyproline methyl ester hydrochloric salt 26, selective
2
2
0
coupling of 4 -hexyl-biphenyl carboxylic acid 24 at the nitrogen
was achieved in high yield, via generation of the corresponding
chloride 25. The hydroxyl group was converted into a better leav-
ing group via tosylation and subsequent cyanation of intermediate
28 gave compound 29 as a mixture of two diastereomers in 55%
yield. Reduction of the nitrile, using a mixture of platinum oxide
and palladium on carbon in acidic medium at room temperature
under hydrogen atmosphere, afforded the chloride salt 30, which
was protected in high yield using di-tert-butyl dicarbonate in the
presence of triethylamine. Finally, treatment of the salt precursor
2
. Results and discussion
2.1. Chemistry
3
1 with lithium hydroxide in a mixture of methanol-THF-water
The peptides 20, 21a–c, 22, 23 were synthesized by manual
(
1:1:1) afforded the desired modified proline 16. The synthesis of
solid-phase peptide synthesis (SPPS) from 2-chlorotritylchloride
polymer resin 11 (Scheme 1, see Supplementary data for details).
Standard 9-fluorenylmethoxycarbonyl amino-protected amino
acids (Fmoc-aa-OH) were used, and the protecting group was
removed by treatment with 20% piperidine in N,N-dimethylfor-
mamide (DMF). Coupling of the appropriate amino acid was
the modified alanine carrying the warheads is shown in Scheme 3.
First, the Weinreb amide 34 was prepared by coupling the com-
mercially available N-tert-butoxycarbonyl-l-alanine 32 with N,O-
dimethylhydroxyamine 33, and then reduced to the correspond-
ing aldehyde 35 by treatment with lithium aluminum hydride at
ꢀ15 °C (Scheme 3-I). Reaction of the aldehyde 35 with the appro-
priate isonitrile 38a–b in the presence of acetic acid (functioning
both as the catalyst and as the protecting group of the newly
2
4
0
0
performed in DMF using N,N,N ,N -tetramethyl-O-(1H-benzotria-
zol-1-yl)uronium hexafluorophosphate (HBTU) as the coupling
reagent, in the presence of N,N-diisopropylethylamine (DIEA) as
the base, and shaking at room temperature for 2 h. When
formed secondary alcohol) at ꢀ78 °C, afforded the
a-protected
alcohols 39a–b in 40–84% yields. Isonitrile 38a was prepared by
microwave heating of a mixture of naphthylmethylamine 36 and
ethylformate 37 at 130 °C for 30 min. The obtained formamide
was dissolved in anhydrous THF and phosphorous oxychloride
was slowly added at ꢀ78 °C, stirring at room temperature over-
a
-branched amino acids (e.g. Fmoc-O-tert-butyl-l-threonine) or
secondary amines (Fmoc-l-proline) were involved, longer reaction
times were preferred (14 h) or a double coupling was performed
2 h + 2 h, treating the resin with a fresh solution of reagents). After
(
each round of coupling and deprotection steps, the resin was
6 13
C H
Cl
C
6
H
13
C
6
H
13
OH
H
2
N
O
a
b
c
O
O
O
N
O
OH
Cl
OMe
26
MeO
24
25
27
OH
C
6
H
13
C
6
H
13
6 13
C H
O
d
O
e
O
f
O
N
O
N
O
N
MeO
MeO
MeO
NH
3 Cl
OTos
CN
28
29
30
C
6
H
13
6 13
C H
O
g
O
O
N
O
N
MeO
HO
16
NHBoc
NHBoc
31
Scheme 2. Synthesis of the modified cationic proline 16. Reagents and conditions: a) SOCl
pyridine, anhy. DCM, 50 °C, 16 h, 90%; d) NaCN, DMSO, 50 °C, 16 h, 55%; e) Pd/C, PtO , H , EtOAc:MeOH (1:1), HCl, 1.5 h; f) Boc
g) LiOH, MeOH:THF:H O (1:1:1), rt, 20 h, 97%.
2
, 80 °C, 2 h, quantitative; b) NaHCO O:THF (1:2), rt, 18 h, 85%; c) p-TolSO
3
, H
2
2
Cl,
2
2
2
O, Et N, anhy. DCM, rt, 18 h, 70% over 2 steps;
3
2
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M. De Rosa et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
5
I) Synthesis of α-keto-amide warheads (41a-b)
NH
2
O
O
Me
36
37
g
Me
O
Me
CN
Me
NHMe
CN
38a
Me or
O
O
O
H
N
33
H
N
H
N
b
38b
OMe
Boc
OH
Boc
N
Boc
a
c
Me
Me Me
Me
32
34
35
Me
O
O
O
H
H
H
N
H
O
d, e
f
H
N
N
N
N
Boc
R1
Boc
R1
2
HCl H N
R1
Me
O
Me
40a-b
O
Me
41a-b
a: R1 = tBu
b: R1 = 1-naphthylmethyl
O
3
9a-b
1
a: R1 = tBu
b: R1 = 1-naphthylmethyl
a: R = tBu
b: R1 = 1-naphthylmethyl
II) Synthesis of a P1' proline analogue (45)
O
N
O
O
NHMe
NHMe
H
N
a
O
h
O
NHMe
H
N
+
N
H
2
H N
Fmoc
OH
Fmoc
N
O
Me
Me
Me
42
43
44
45
III) Acetylation of warheads (41b and 47)
O
H
i
O
H
N
H
N
HClH
2
N
N
Me
Me
O
O
Me
O
41b
46
Me
H
Me
H
Me
O
Me
Me
O
Me
i
H
N
HCl H
2
N
B
Me
O
B
O
O
Me
4
Me
48
7
0
Scheme 3. Synthesis of modified alanine carrying the warheads. I) Synthesis of a-keto-amides warheads 41a–b; II) Synthesis of a P1 proline analogue 45; III) Acetylation of
warheads 41b and 47. Reagents and conditions: a) CDI, DIEA, anhy. THF:anhy. DMF (2.5:1), rt, 18 h, 65–90%; b) LiAlH
4
, anhy. THF, ꢀ15 °C, 45 min, 87%; c) AcOH, ethyl acetate,
ꢀ
78 °C to rt, 18 h, 40–84%; d) K CO , H O:MeOH (1:2), rt, 1 h, e) EDCI, dichloroacetic acid, anhy. DCM:anhy. DMSO (7:1), 0 °C to rt, 1 h, 25–63%, over two steps; f) HCl(g), 1,4-
2
3
2
dioxane, rt, 5 min, quantitative; g) MW, 130 °C, 30 min then DIEA, POCl , anhy. THF, ꢀ78 °C to rt, 18 h; h) 30% piperidine in ethanol, rt, 15 min, 73%; i) acetic anhydride, DIEA,
3
anhy. DMF, rt, 3 h–overnight, 10–71%.
night. Deprotection of the obtained secondary alcohols 39a–b by
treatment with potassium carbonate and subsequent Pfitzner–
Moffatt oxidation gave precursors 40a–b in 25–63% yields.
Removal of the tert-butoxycarbonyl protecting group in acidic
The obtained peptides 20, 21a–c, 22 and 23 were coupled at the
C-terminus with the modified alanine carrying the warheads 41a–
b, 45, 47 and 49. Finally, deprotection of the side chains by using
aq. 95% trifluoroacetic acid or a mixture of dichloromethane:triflu-
oroacetic acid:triethylsilane (10:9:1) afforded the final oligopep-
tides 50a–p (Scheme 4).
medium afforded the final
a
-keto-amide hydrochloric salts 41a–b
0
in quantitative yields. The P1 proline analogue 45 was prepared
as described in Scheme 3-II. Coupling of commercially available
N-methylpyrrolidine-2-carboxamide 43 with Fmoc-l-alanine 42
gave the dipeptide 44, which was treated with 30% piperidine in
ethanol to remove the Fmoc protecting group, yielding the desired
2.2. Biology and SAR
2.2.1. Characterization of expressed protein
product 45. Further, the
a
-naphthylmethyl keto-amide 41b and
EcLepB activity was investigated on two substrates. The first,
dabcyl-VGGTATAGAFSRPGLE-(EDANS)-OH, was based on the
Mycobacterium tuberculosis antigen 85A sequence. This
commercial (R)-boroAla-(+)-pinanediol hydrochloride 47 were
capped at the nitrogen with an acetyl group by treatment with
acetic anhydride, yielding compounds 46 and 48, respectively
ꢀ
1
ꢀ5
substrate gave a kcat of 135 s and a K
m
of 2 ꢁ 10 M, resulting
6
ꢀ1 ꢀ1
(Scheme 3-III).
in
a
k
cat/K
m
value of 6.7 ꢁ 10 M
s
.
The second,
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6
M. De Rosa et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
R3
R3
O
Me
O
O
Me
O
Me
R2
H
N
H
H
N
H H
N
H
N
N
N
H
OH
O
N
N
H
N
H
O
R2
Me
a, b or a, c, b
O
R1
O
O
(HCl) NH
2
R1
O
O
Me
OtBu
O
Me
OH
NHTrt
NH
2
20, 21a-c, 22, 23
41a-b, 45, 47
2
50a-p
4
9 R : COOH
O
O
H
N
H Me
N
Me
O
O
Me
R1 = H, acetyl, 4'-hexyl-biphenyl, nonyl, Fmoc
R3 = H or Boc NH
R2
H
N
Me
=
O
Me
O
O
Me
OH
O
Me
B
N
B
OH
OH
O
H
Scheme 4. Coupling at C-terminus between peptides 20, 21a–c, 22 and 23 and modified alanine carrying warheads 41a–b, 45, 47 and 49. Reagents and conditions: a) HBTU or
HATU, DIEA, anhy. DMF or DCM, rt, 2 h; b) 95% TFA in H O or TFA:DCM:TES (10:9:1), rt, 2 h, 5–65% (over two steps); c) 30% piperidine in ethanol, rt, 3 h, 41–73%.
2
dabcyl-KLTFGTVKPVQAIAGYEILE-(EDANS)-OH, was based on the
produced rather inactive compounds. Next, based on recent find-
25
29,30
sequence of Streptococcus pyogenes streptokinase. This substrate
ings in the patent literature,
we introduced a boronic ester
ꢀ
1
ꢀ5
gave a kcat of 90 s and a K
of 1.5 ꢁ 10 M
m
of 6 ꢁ 10 M, for a kcat/K
m
value
and/or acid warhead at the C-terminus. Gratifyingly, a very good
inhibitor 50j was obtained, 25-fold more potent than the reference
inhibitor 9 (IC50 = 48 vs 1200 nM, see Table 2), that could serve as a
lead for further SAR studies. To start, the alkyl tail at the N-terminus
6
ꢀ1 ꢀ1
s ; these values compare favorably with the
reported activity of the S. pneumoniae SPase I on the same substrate
ꢀ
1
ꢀ4 25
(
k
cat 0.032 s , K
m
1.18 ꢁ 10 ). The values for both substrates
12
0
are in good agreement with earlier results for EcLepB. For further
assay work, we decided to use the antigen 85A-based sequence.
As documented in the Supplementary data, cleavage of labeled
and unlabeled substrates was investigated using mass spectrome-
try. In each case, intact substrate could be detected, and was shown
to decrease with time. Products were identified corresponding to
the C-terminal fragments predicted by SignalP,26 but not the N-ter-
minal fragments (perhaps reflecting additional degradation of the
latter).
was replaced with a bulkier aromatic fatty acid group; the 4 -hexyl-
biphenyl moiety was chosen, as it has been shown to improve
antimicrobial activity of the arylomycin series against a panel of
31
microorganisms. The resulting inhibitor 50k (IC50 = 12 nM) was
4-fold more potent than the parent 50j. Surprisingly,2
9,30
the
hydrolyzed analogue 50l having a boronic acid warhead showed
lower potency (IC50 = 230 nM) than the corresponding boronic
ester derivative 50k. From these data, it was clear that modifica-
tions of the warhead and tail both contributed to the significant
increase in potency of the new inhibitors compared to 9. According
3
2,33
2.2.2. Evaluation of EcLepB IC50 and MIC
to the literature,
the fatty acid tail in the arylomycin series is
All newly synthesized oligopeptides 50a–p were evaluated for
positioned close to the enzyme-membrane association surface,
and the backbone is tightly bound via a hydrogen-bonding net-
work. Consistent with this, replacement of the aromatic fatty acid
tail with an acetyl moiety (50o) or removal of the entire fatty acid
tail (50p) resulted in a large decrease in inhibitory activity
(IC50 = 26,000 nM). When the warheads alone (in the N-acylated
form, 46 and 48) were tested in the same assay, inhibition was also
very poor (Table 1). The active lipopeptides were then evaluated
against a panel of Gram-negative bacteria, as well as one Gram-
positive organism, S. aureus (Table 2). Compound 9 lacked antibac-
terial activity completely. Despite their low IC50 values, inhibitors
50j–l did not show useful activity against the Gram-negatives
but some activity could be seen against S. aureus. We decided to
introduce a positive charge at the P5 proline residue of the lipopep-
tides (compounds 50m and 50n), hoping to increase uptake and
thereby the antimicrobial potency. Our docking study of inhibitor
50n into EcLepB (Fig. 3), indicated that this group should fit
nicely into the enzyme active site. For the modeling, the fatty acid
tail of 50n was truncated to acetyl, as the enzyme lacks strong
determinants for orienting the tail. As seen in Fig. 3, the P2, P4,
and P5 side chains are exposed to bulk solvent and should tolerate
structural variations without loss of target affinity. A positively-
charged side chain at P5 could potentially interact with either
E83 or D143.
their inhibitory activity against the EcLepB enzyme, and antibacte-
rial activity against a panel of strains; the results are reported in
Tables 1 and 2. Table 1 shows the percentage inhibition for the ref-
erence compound 9, and for the new compounds with less than
5
0% inhibition when tested at 100
values (and MIC) for compounds that had more than 50% inhibition
of enzyme activity at 100 M (% inhibition data not shown).
lM. Table 2 presents the IC50
l
As seen in Table 1, our first compounds (50a–i) were very poor
inhibitors of the enzyme. As expected, the analogue 50a, missing
the warhead at the C-terminus, was almost completely inactive
at a concentration of 100 lM (EcLepB percentage of inhibi-
tion < 50%), indicating that a reactive electrophilic moiety at this
position promotes inhibition. Our initial idea for optimization of
the PTANA hit was to replace the aldehyde warhead of inhibitor
9
at the C-terminus with a chemically more stable but equally
3
4
reactive group, while keeping the decanoyl tail at the N-terminus
unchanged. We hypothesized that, similar to the 5S-penem type
of inhibitors, an electrophilic and highly reactive warhead could
covalently bind S91 in the active site of LepB.27 Following the clas-
sical approach of inhibiting serine protease enzymes with
a
-keto-
amides, two moieties were chosen, namely tert-butyl– (50b)
and -naphthylmethyl-keto-amides (50c). Surprisingly, the inhibi-
2
8
a
tory activity was severely compromised (compare 50b–c to inhibi-
0
tor 9). We introduced a proline in the P1 position, which has
The two new inhibitors, 50m–n, blocked the growth of most of
the bacteria tested, and were much more potent in this regard than
the reference inhibitor 9. It should be noted that these two inhibi-
tors have both a bulky aromatic fatty acid tail, a key element for
previously produced effective inhibitors,20 but this was likewise
not a viable approach (50d). Removal of the fatty acid tail (50e–
g) and/or truncation of the peptide PTANA sequence (50h–i) also
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M. De Rosa et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
7
Table 1
Compounds showing less than 50% inhibition of EcLepB, when tested at a concentration of 100
lM. Compound 9 was
included as a reference. Measurements were made as triplicates.
Cmpds
Structure
Me
% Inhibition
100
9a
O
O
Me
Me
Me
Me
Me
Me
H
N
H
H
N
N
N
H
OH
N
H
O
O
O
O
O
Me
NH
2
5
0a
O
Me
O
Me
38.0 ± 2.5
31.5 ± 4.5
14.4 ± 0.6
31.3 ± 2.6
22.5 ± 0.9
32.3 ± 2.0
35.8 ± 0.7
13.3 ± 0.2
26.6 ± 0.3
26.0 ± 8.1
31.0 ± 0.2
18.0 ± 1.1
H
N
H
H
N
OH
N
N
H
OH
N
H
O
O
O
O
O
Me
NH
2
5
0b
O
Me
O
Me
O
Me
Me
Me
H
N
H
H
N
N
N
H
OH
N
N
H
H
O
O
O
O
O
Me
NH
O
2
5
0c
O
Me
Me
O
H H
N
H
N
N
N
H
N
H
O
N
H
O
O
O
O
Me
OH
NH
2
5
0d
O
Me
O
Me
H H
H
N
N
N
N
N
H
N
H
O
Me
O
O
O
N
O
Me
O
OH
O
H
NH
2
5
0e
O
Me
Me
O
Me
Me
Me
H H
N
H
N
N
N
H
N
H
N
H
Me
Me
O
O
O
O
O
O
Me
OH
NH
2
5
0f
O
Me
O
Me
H H
N
H
N
N
N
H
OH
N
H
N
H
O
O
O
O
O
Me
NH
2
5
0g
O
Me
O
Me
H
N
H
H
N
N
N
N
H
N
H
Me
Me
Me
O
O
O
O
N
H
O
Me
OH
O
NH
Me
2
5
0h
O
O
Me
O
Me
O
H
Me
Me
N
N
H
N
H
N
H
O
O
O
NH
2
5
0i
Me
O
Me
O
H
N
Me
N
H
N
H
N
H
O
O
O
NH
2
5
0o
O
Me
O
Me
H H
N
H
N
O
Me
N
N
N
B
O
Me
Me
H
H
O
Me
Me
O
O
O
Me
OH
H
NH
2
4
6
8
O
O
Me
O
N
H
N
H
O
4
Me
O
Me
Me
N
B
O
Me
Me
H
H
a
Compound 9 is unstable, see footnote to Table 2.
Please cite this article in press as: De Rosa M., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.12.003

Table 2
Further characterization of compounds where in vitro inhibition was at least 50% in tests at 100 lM.
Cmpd
Structure
IC50 (nM) (95%
confidence interval)
MIC (
l
g/mL)
Cytoxicity on HepG2 Hemolysis
i
E. c.b
E. c.c
P. a.e
cells IC50
(lM)
(%)
E. c.
wt
P. a.
K. p.
wt
A. b.
wt
S. a.
wt
a
d
f
g
h
wt
O
Me
O
Me
Me
H
N
H
H
N
9j
N
N
H
N
1200 (800–1800)
48 (30–76)
>128 >128 >128 >128
128
>64
>64
>128 >128
>128
32
Ndk
Ndk
H
O
O
O
O
O
Me
O
OH
NH
2
Me
O
Me
Me
H H
H
N
N
O
Me
N
N
H
N
H
B
O
H
5
0j
Me
Me
>64
>64
>64
64
>64
16
>64
>64
>64
>64
>64
>64
7.8
4.7
0.4 ± 0.1
1.1 ± 0.2
O
O
O
O
Me
OH
NH
2
O
Me
O
Me
H H
N
H
N
O
Me
N
N
H
N
B
5
0k
Me
Me
Me
12 (8.9–18)
4
H
O
O
O
O
O
Me
OH
H
NH
2
O
Me
O
Me
H H
N
H
N
OH
N
N
H
N
H
B
OH
5
0l
Me
230 (150–360)
18 (15–20)
>64
32
>64
>64
>64
64
>64
>64
16
>64
32
4
>64
1.1 ± 0.2
5.7 ± 0.3
O
O
O
O
Me
OH
NH
2
NH
3
O
Me
O
Me
5
0m
H H
N
H
N
4
1
8
0.5
18.6
O
Me
N
N
H
N
H
O
B
O
Me
Me
Me
O
Me
O
O
OH
H
NH
2
NH
3
O
Me
O
Me
5
0n
0p
H H
N
H
N
6 (4.8–8.4)
16
2
0.5
64
4
8
8
0.25
>64
15.6
>64
2.5 ± 0.1
OH
N
N
H
N
H
O
B
Me
O
O
OH
O
Me
OH
NH
2
O
Me
O
Me
H H
N
H
N
O
Me
N
H
26,000 (17,000–
N
H
N
H
B
O
Ndk
5
Me
Me
>64
>64
>64
>64
>64
>64
>64
40,000)
O
O
O
Me
OH
H
NH
2

M. De Rosa et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
9
good affinity, and a cationic group added to the proline at P5,
which is apparently required for permeability. It was comforting
to see that introduction of a positive charge at the proline did
not compromise the inhibitory activity. In fact, compounds 50m–
n were among the most potent EcLepB inhibitors we obtained, with
IC50 values of 18 and 6 nM, respectively. The minor difference in
the IC50 values of the two compounds could potentially indicate
hydrolysis of the ester into the corresponding acid, although in
the active site of EcLepB, there is space between residues S91
and K146 to accommodate a bulky ester (see Fig. 3). Indeed,
hydrolysis could potentially be catalyzed by the enzyme in a sim-
ilar manner to that for the natural substrates. However, we note
that the observed reaction rates were linear over the 2 h time
course of the kinetics experiment, and so if such hydrolysis is
occurring, it must be rapid (during the 10-min pre-incubation
step). In this case, the boronic ester derivative appeared to be more
potent than the boronic acid, in contrast to the 50k–l result
described above.
2
.2.3. Evaluation of cytotoxicity and hemolysis
Inhibitors 50j–p were also evaluated on the HepG2 cell line36
for cytotoxicity (for details see Supplementary data), and most
were evaluated in a hemolysis assay; the results are reported in
Table 2. It was clear that there were two pairs of potent enzyme
inhibitors: 50j/50k and 50m/50n. Inhibitors 50j and 50k only
differ in the fatty-acid tail, and have similar EcLepB IC50s (48
and 12 nM, respectively). Both are cytotoxic, with HepG2 IC50
s
values of 7.8 and 4.7 M, respectively, although each performed
l
fairly well in the hemolysis assay. These compounds, in principle,
lack activity against Gram-negative organisms, although results
with S. aureus were more promising. Inhibitors 50m and 50n only
differ in the warhead, having either a boronic ester or boronic
acid group. They also have good EcLepB IC50s (18 and 6 nM,
respectively), although they gave poor results in both cytotoxicity
and hemolysis assays. The introduction of a positive charge at P5
of these two compounds appears to be correlated with the fact
that they are active on most of the bacterial strains evaluated.
However, these compounds effects on other properties do not
follow any clear pattern. In the case of the 50k/50m pair, the
introduction of the positively charged group is associated with
less cytotoxicity, but more hemolysis; inhibition of the enzyme
is not changed significantly. In the case of the 50l/50n pair,
inhibition of the enzyme is better, while cytotoxicity and
hemolysis are increased.
2.2.4. Profiling of LepB inhibitor from the patent literature
Finally, we synthesized one of the inhibitors from the WO
015/023898 patent application to include it in our work as a ref-
2
erence compound. We selected compound 50q, one of the more
potent compounds based on the reported EcLepB IC50 and E. coli
MIC values, and which, furthermore, could be easily synthesized.
Compound 50q is similar to our inhibitors (presented in Table 2)
in structure and activity profile. The synthesis was performed
according to the methods described in the patent, and the charac-
terization of compound 50q is detailed in the Supplementary data.
The most similar compound in our series 50m is 6-fold more
potent (EcLepB IC50 18 nM) than the reference compound 50q
(
EcLepB IC50 110 nM). However, 50q has a slightly better MIC on
wild-type strains of E. coli and K. pneumonia (Table 2). Compound
0q bears two positive charges as compared to one positive charge
5
in the analogue 50m, which may explain its higher hemolytic
activity.
2.2.5. Selection of resistant mutants
The spontaneous frequency of resistance, measured by plating
ꢀ8
efflux-deficient
D
tolC E. coli at 8 ꢁ MIC, was <5 ꢁ 10 for 50m,
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1
0
M. De Rosa et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Me
O
N
O
O
Me
O
Me
H
H
N
HN
OH
3
H N
N
H
N
H
B
OH
O
Me
OH
O
NH2
P5
P4
P3
P2
P1
Fig. 3. Molecular modeling of a covalently bound N-acetyl analogue of 50n (both diastereomers) into EcLepB (PDB code: 3IIQ),35 illustrating the direction of the hydrophobic
tail towards the membrane association surface. Boronic esters fit between S91 and K146, and the amine at P5 is in contact with E83 on the surface of the enzyme (the
numbering system for the protein has been adjusted to reflect the corrected sequence reported by Paetzel et al.).12 Mutations in amino acids in resistant strains from 50m are
numbered in red (vide infra). SiteMap-calculated volumes shown in yellow highlight the two hydrophobic regions contributing to the recognition of the Ala-X-Ala motif.
ꢀ
9
and <5 ꢁ 10
sequencing were raised against both 50m and 50q by serial
passage of E. coli MG1655 tolC at successively increasing concen-
for 50q. Resistant mutants for whole genome
9. A set of 16 oligopeptides 50a–p structurally related to the decan-
oyl-PTANA-CHO inhibitor 9 were designed, synthesized and evalu-
ated. In general, good improvement of inhibitory potency was
obtained. Four inhibitors (50j, 50k, 50m and 50n) were found to
be very active, showing IC50s in the low nanomolar range. In partic-
ular, inhibitor 50n was the most potent of this new series of
oligopeptides (IC50 = 6 nM). Two of the best inhibitors (50m–n)
also showed promising antimicrobial activity, with MICs in the
D
trations of each compound (Supplemental material). In each case
the great majority of the mutants (8/10 for 50m, 11/11 for 50q)
had acquired mutations in lepB causing amino acid substitutions
(
Tables S3 and S4). The clustered location of most of the mutations
in the structure of LepB (Glu83, Phe85, Ile87, Pro88, and Ile145)
supports the hypothesis that these inhibitors bind in the active site
of the enzyme (Fig. 3). The different mutations in LepB had similar
low lg/mL range for several strains. The results obtained showed
that a bulky fatty acid tail at the N-terminus, an electrophilic and
highly-reactive warhead at the C-terminus, and a positively
charged proline in the P5 position, are key features for obtaining
good inhibitors that can be used as potential starting points for
antibiotic drug discovery. Raising mutants based on 50m and
50q and performing whole genome sequencing of these enabled
us to confirm the mode of action of the synthesized inhibitors.
Out of 21 mutants, 19 carried mutations in LepB (12 had no addi-
tional mutations in any other genes), which lends strong support to
the premise that LepB is the target for our inhibitors. Although it is
also clear that more optimization will be required to obtain inhibi-
tors with favorable cytotoxicity and hemolytic profiles, we showed
that minor changes in the oligopeptides often produce large effects
in their biological profile and this information can be exploited in
future studies.
effects on susceptibility, increasing MIC from 1–2
lg/mL up to 8–
16 l
g/mL (Tables S3, S4). The precise mechanism by which the
LepB mutations cause resistance has not been investigated but
the nature of these binding-pocket mutations is consistent with
them acting to reduce compound affinity for LepB, and so supports
inhibition of LepB function as the whole cell mode of action of
compounds 50m and 50q. Several mutants carried a second muta-
tion in LepB at either Ala6 (50m) or Asp113 (50q). The significance
of these second mutations is not clear but the increase in MIC asso-
ciated with mutants carrying only single amino acid substitutions
shows that a single substitution in the binding pocket is sufficient
to fully account for the increase in MIC (Fig. 3, Tables S3, S4). Sev-
eral mutants also carried 1 or 2 mutations in additional genes
(
Tables S3, S4). The significance of these additional mutations is
unclear, but as 12 of 19 mutants carried only LepB mutations, we
can be confident that mutation in LepB is sufficient to confer the
resistant phenotype.
4. Experimental section
4.1. General methods
3
. Conclusions
Analytical thin layer chromatography (TLC) was performed
The rapid development of antimicrobial resistance represents
using Merck aluminum sheets precoated with silica gel 60 F254
Column chromatography was performed on Merck silica gel 60
(40–63 m). The microwave reactions were performed in a Biotage
Initiator producing controlled irradiation at 2450 MHz with a
.
one of the major challenges in the search for new antibiotics. Type
I signal peptidases (SPases I) are for a number of reasons attractive
targets for the development of antibacterial agents with a novel
mode of action. In this study, we explored the possibility of finding
potent EcLepB inhibitors by optimizing the existing hit compound
l
1
13
power of 0–300 W. H and C NMR spectra were recorded on Var-
1
13
ian Mercury Plus instruments; H at 399.9 MHz and
C at
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M. De Rosa et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
11
1
00.6 MHz at 25 °C. Exact molecular masses were determined on
41a (0.046 mmol, 0.008 g) in anhydrous DMF (2 mL), in the pres-
ence of HBTU (0.046 mmol, 0.017 g) and DIEA (0.115 mmol,
0.015 g). The crude material was purified by preparative RP-HPLC,
to get 9.5 mg (41%, MS (ESI) m/z 1008.8 [M+H] ) of desired pro-
tected peptide as a white solid. Deprotection of the side chains
was carried out according to the general procedure (treating the
Micromass Q-Tof2 mass spectrometer equipped with an electro-
spray ion source. Analytical RP-HPLC-MS was performed on a Gil-
son RP-HPLC system with a Finnigan AQA quadrupole low-
resolution mass spectrometer in positive or negative ESI mode
+
using an Onyx Monolithic C18 4.6 ꢁ 50 mm 5
lm (Phenomenex)
with MeCN in 0.05% aqueous HCOOH as mobile phase at a flow rate
2
crude material with 95% TFA in H O, 2 mL) after which the solvents
of 4 mL/min. Preparative RP-HPLC was performed on a system
were evaporated and the residue (yellow oil) was purified by
equipped with
a
Nucleodur C18 HTec
Phenomenex C8
O/CH
CN gradient with 20 mM TEAA, in both cases using UV
detection at 220 nm and 254 nm. The purity of the peptides was
determined by RP-HPLC using the columns: ACE C18
50 ꢁ 4.6 mm) and Nucleodur C18 HTec 5 m (4.6 ꢁ 50 mm),
O/CH CN gradient with 0.1% TFA or H O/CH CN gradient with
0 mM TEAA and UV detection at 220 nm and 254 nm. All peptides
showed purity above 95%.
5
5
l
m
column
column
preparative RP-HPLC, affording 0.4 mg (6%) of desired oligopeptide
+
(
(
H
150 ꢁ 21.2 mm) or
150 ꢁ 21.2 mm), using a H
O/CH
a
l
m
50b as white solid. C34
H
59
N
7
O
9
, MS (ESI): m/z 710.3 [M+H] . RP-
2
3
CN gradient with 0.1% TFA or
HPLC purity: C18 column > 95%. HRMS for C34
710.4453, Found 710.4446.
60 7 9
H N O Calcd:
2
3
5
4.2.5. (S)-2-((S)-2-((2S,3S)-2-((S)-1-Decanoylpyrrolidine-2-carbox-
1
(
l
3
amido)-3-hydroxybutanamido)propanamido)-N -((S)-4-((naphthalen-
H
2
3
2
1-ylmethyl)amino)-3,4-dioxobutan-2-yl)succinamide (50c)
The analogue was prepared according to the general procedure
using the peptide 21b (0.024 mmol, 0.021 g), the modified alanine
2
4
1b (0.048 mmol, 0.012 g) in anhydrous DMF (2 mL) in the pres-
ence of HBTU (0.048 mmol, 0.018 g) and DIEA (0.12 mmol,
.015 g). The crude material was purified by preparative RP-HPLC
4.2. Synthetic procedures
0
+
4.2.1. General method for the coupling between the peptides (20, 21a–
affording 14.7 mg (56%, MS (ESI) m/z 1092.4 [M+H] ) of desired
protected peptide as a white solid. The deprotection of the side
chains was performed according to the general procedure and
the crude peptide was purified by preparative RP-HPLC, affording
c, 22, 23) and the warheads (41a–b, 45 and 47)
To a solution of the peptide (1 eq.) in anhydrous DMF (2 or
5
2
mL) or anhydrous DCM (3 mL) HBTU or HATU or EDCI (1.2 or
eq.) and DIEA (3 or 6 eq.) were added under N flow and the mix-
2
6 mg (58%) of desired oligopeptide 50c as white solid.
+
ture was stirred at room temperature or 0 °C for 10 min, after
which the warhead (2 or 3 eq.) was added and the reaction mixture
was stirred at room temperature till completion (monitoring via
C
41
59
H N
7
O
9
, MS (ESI): m/z 794.3 [M+H] . RP-HPLC purity: C18 col-
umn > 95%. HRMS for
794.4427.
C
41
60
H N
7
O
9
Calcd: 794.4453, Found:
2
RPLC-MS). The solvent was evaporated under N stream and the
residue was used for the next step (deprotection or oxidation)
without or after purification by RP-HPLC.
4.2.6. (S)-2-((S)-2-((2S,3S)-2-((S)-1-Decanoylpyrrolidine-2-carbox-
amido)-3-hydroxybutanamido)propanamido)-N -((S)-1-((S)-1-(methyl-
1
carbamoyl)pyrrolidin-2-yl)-1-oxopropan-2-yl)succinamide (50d)
The analogue was prepared according to the general procedure
using the peptide 21b (0.031 mmol, 0.026 g), and the modified pro-
line 45 (0.037 mmol, 0.007 g) in anhydrous DCM (3 mL), in the
presence of HBTU (0.037 mmol, 0.014 g) and DIEA (0.074 mmol,
0.009 g). Deprotection of the side chains was carried out according
4
.2.2. General method for the side chain deprotection
The crude or pure protected peptides were dissolved in a solu-
tion of 95% TFA in H
and stirred at room temperature till completion (generally 1 or
h). The solvents were removed under reduced pressure and the
residues were purified by preparative RP-HPLC.
2
O or in a solution of TFA:DCM:TES (10:9:1)
2
2
to the general procedure (95% TFA in H O, 2 mL) after which the
solvents were evaporated and the residue (yellow oil) was purified
by preparative RP-HPLC, affording 0.014 g (62%, over two steps) of
desired oligopeptide 50d as a white solid. H NMR (400 MHz,
4
.2.3. Decanoyl-PTANA-OH, Decanoyl-l-prolyl-l-allothreonyl-l-alanyl-
1
l-asparaginyl-l-alanine (50a)
The analogue was prepared according to the general procedure
using peptide 21b (0.022 mmol, 0.019 g), alanine (l-Ala-OH) 49
6
DMSO-d , mixture of rotamers), d 8.11 (d, J = 8.1 Hz, 0.4H), 8.02–
7.94 (m, 1.8H rotamer), 7.83 (d, J = 7.2 Hz, 0.6H), 7.73–7.66 (m,
3.5H), 7.30 (s, 1H), 6.88 (s, 1H), 4.58–4.42 (m, 3.5H), 4.38 (dd,
J = 8.1, 3.1 Hz, 1H), 4.32–4.22 (m, 5H), 4.11–3.99 (m, 1.3H), 3.61–
3.27 (m, 3H), 2.56–2.54 (m, 3H), 2.35 (dd, J = 15.5, 7.3 Hz, 3H),
2.26 (t, J = 7.4 Hz, 2.5H), 2.22–2.10 (m, 1H) 2.06–1.69 (m, 15H),
(
0.044 mmol, 0.004 g) in anhydrous DMF (2 mL), in the presence
of HBTU (0.044 mmol, 0.016 g) and DIEA (0.11 mmol, 0.014 g).
The crude material was used directly for the deprotection step,
after which preparative RP-HPLC purification was performed.
3
.5 mg (25%, over 2 steps) of desired oligopeptide 50a as a white
1.49–1.44 (m, 5H), 1.28–1.18 (m, 35H), 1.04 (d, J = 6.4 Hz, 5H),
1
+
solid were obtained. H NMR (400 MHz, DMSO-d
s, 1H), 8.07 (d, J = 9.1 Hz, 1H), 7.89 (d, J = 6.3 Hz, 1H), 7.80 (d,
J = 7.7 Hz, 1H), 7.70 (d, J = 8.3 Hz, 1H), 7.30–7.22 (m, 1H), 6.89
6
, 90 °C), d 12.56
0.92–0.81 (m, 5H). C35
H
60
N
8
9
O , MS (ESI): m/z 737.3 [M+H] . RP-
(
HPLC purity: C18 column > 95%. HRMS C35
759.4381, Found: 759.4382.
60 8 9
H N O Na, Calcd:
(
(
3
2
bs, 1H), 6.53 (bs, 1H), 4.88–4.84 (m, 1H), 4.54–4.50 (m, 1H), 4.38
dd, J = 7.8, 2.4 Hz, 1H), 4.30–4.08 (m, 2H), 4.06–4.00 (m, 1H),
.55–3.47 (m, 2H), 3.03–2.99 (m, 2H), 2.67–2.66 (m, 1H), 2.34–
.32 (m, 1H), 2.26 (t, J = 8.0 Hz, 2H), 2.20–2.12 (m, 1H), 1.91–1.87
4.2.7. (S)-2-((S)-2-((2S,3S)-2-((S)-1-Acetylpyrrolidine-2-carboxamido)-
3-hydroxybutanamido)propanamido)-N -((S)-4-(tert-butylamino)-
3,4-dioxobutan-2-yl)succinamide (50e)
1
(
1
m, 1H), 1.75–1.71 (m, 2H), 1.49–1.44 (m, 2H), 1.40–1.12 (m,
6H), 1.05 (d, J = 6.8 Hz, 3H), 0.85 (t, J = 8.0 Hz, 3H). C29
The analogue was prepared according to the general procedure
using the peptide 21a (0.033 mmol, 0.025 g), and the warhead 41a
(0.101 mmol, 0.021 g) in anhydrous DMF (3 mL) in the presence of
HBTU (0.101 mmol, 0.038 g) and DIEA (0.198 mmol, 0.025 g). The
crude material was purified by RP-HPLC obtaining 8 mg (26%) of
50 6 9
H N O ,
+
MS (ESI): m/z 627.3 [M+H] . RP-HPLC purity: C18 column > 95%.
HRMS for C29H N O Calcd: 627.3718, Found: 627.3710.
51 6 9
1
4
(
.2.4. (S)-N -((S)-4-(tert-Butylamino)-3,4-dioxobutan-2-yl)-2-((S)-2-
pure protected peptide as a fluffy white solid. C41
H
65
N
7
O
9
, MS
(ESI): m/z 896.4 [M+H] . Deprotection of the side chains was car-
ried out according to the general procedure (95% TFA in H O,
+
(2S,3S)-2-((S)-1-decanoylpyrrolidine-2-carboxamido)-3-hydroxybu-
tanamido)propanamido)succinamide (50b)
2
The analogue was prepared according to the general procedure
using the peptide 21b (0.023 mmol, 0.020 g), the modified alanine
2 mL) after which the solvents were evaporated and the residue
(yellow oil) was purified by preparative RP-HPLC, affording
Please cite this article in press as: De Rosa M., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.12.003

1
2
M. De Rosa et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
3
.5 mg (65%) of desired final oligopeptide 50e as white solid. 1
H
30 5 6
RP-HPLC purity: C18 column > 95%. HRMS for C17H N O Calcd:
NMR (400 MHz, CDCl
3
+ 2 drops of DMSO-d
6
), d 4.53–4.51 (m,
400.2196, Found: 400.2203.
1
2
H), 4.04–4.01 (m, 1H), 3.80–3.67 (m, 4H), 3.21–2.90 (m, 2H),
.05–1.96 (m, 5H), 1.53–1.36 (m, 2H), 0.78–0.55 (m, 15H), 0.24
1
4.2.11. (S)-2-((S)-2-Acetamidopropanamido)-N -((S)-4-((naphthalen-
1-ylmethyl)amino)-3,4-dioxobutan-2-yl)succinamide (50i)
+
(
t, J = 8.1 Hz, 3H). C26
H
43
N
7
O
9
, MS (ESI): m/z 598.1 [M+H] . RP-
Calcd:
HPLC purity: C18 column > 95%. HRMS for C26
5
H
42
N
7
O
9
The analogue was prepared according to the general procedure
using the peptide 22 (0.051 mmol, 0.025 g), and the warhead 41b
96.2041, Found: 596.2061.
(
0.153 mmol, 0.045 g) in anhydrous DMF (3 mL) in the presence
4
.2.8. (S)-2-((S)-2-((2S,3S)-2-((S)-1-Acetylpyrrolidine-2-carboxamido)-
of HBTU (0.153 mmol, 0.058 g) and DIEA (0.306 mmol, 0.039 g).
The crude material was purified by RP-HPLC affording 0.018 g
1
3-hydroxybutanamido)propanamido)-N -((S)-4-((naphthalen-1-ylm-
ethyl)amino)-3,4-dioxobutan-2-yl)succinamide (50f)
The analogue was prepared according to the general procedure
using the peptide 21a (0.033 mmol, 0.025 g), and the warhead
(49%) of pure compound as a fluffy white solid. C43
H
43
N
5
O
6
, MS
(ESI): m/z 726.1 [M+H] . Deprotection of the side chains was car-
ried out according to the general procedure (95% TFA in H O,
+
2
4
1b (0.101 mmol, 0.030 g) in anhydrous DMF (3 mL) in the pres-
2 mL) after which the solvents were evaporated and the residue
ence of HBTU (0.101 mmol, 0.038 g) and DIEA (0.198 mmol,
(yellow oil) was purified by preparative RP-HPLC, affording 5 mg
1
0
.025 g). The crude material was purified by RP-HPLC affording
.018 g (54%) of pure compound as fluffy white solid.
, MS (ESI): m/z 980.5 [M+H] . Deprotection of the side
chains was carried out according to the general procedure (95%
TFA in H O, 2 mL) after which the solvents were evaporated
(43%) of desired oligopeptide 50i as white solid.
(400 MHz, CDCl + 2 drops of DMSO-d
H NMR
), d 7.80–7.63 (m, 1H),
0
C
a
3
6
+
56
H
65
N
7
O
9
7.62–7.32 (m, 2H), 7.13–7.07 (m, 3H), 6.88–6.80 (m, 1H), 5.95–
5.94 (m, 1H), 4.83–4.78 (m, 1H), 4.55 (s, 2H) 4.35–4.24 (m, 2H),
2
3.96–3.88 (m, 1H), 2.21 (s, 3H), 1.60 (d, J = 8.3 Hz, 3H), 0.87 (m,
+
and the residue (yellow oil) was purified by preparative RP-HPLC,
3H). C24
H
29
N
5
O
6
, MS (ESI): m/z 484.2 [M+H] . RP-HPLC purity:
affording 5 mg (43%) of desired oligopeptide 50f as a white solid.
H NMR (400 MHz, CDCl + 2 drops of DMSO-d ), d 7.35–7.32 (m,
3 6
C18 column > 95%. HRMS for C24
482.2021.
28 5 6
H N O Calcd: 482.2040, Found:
1
1
6
H), 7.24–7.20 (m, 1H), 7.15–7.12 (m, 1H), 6.80–6.68 (m, 4H),
.50–6.43 (m, 1H), 5.70–5.67 (m, 1H), 4.44–4.33 (m, 1H), 4.14
4.2.12. (S)-2-((S)-2-((2S,3S)-2-((S)-1-Decanoylpyrrolidine-2-carbox-
amido)-3-hydroxybutanamido)propanamido)-N -((R)-1-((3aS,4S,6S,
1
(
s, 2H), 4.01–3.87 (m, 1H), 3.68–3.53 (m, 4H), 2.96–2.80 (m,
2
C
H), 1.96–1.84 (m, 5H), 1.41–1.30 (m, 2H), 0.66–0.41 (m, 9H).
7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-
2-yl)ethyl)succinamide (50j)
+
33
H
43
N
7
O
9
, MS (ESI): m/z 682.0 [M+H] . RP-HPLC purity: C18 col-
Calcd: 680.3044, Found:
umn > 95%. HRMS for
C
33
H
42
N
7
O
9
The analogue was prepared according to the general procedure
using the peptide 21b (0.064 mmol, 0.055 g), and the warhead (R)-
BoroAla-(+)-Pinanediol hydrochloride 47 (0.128 mmol, 0.033 g) in
anhydrous DCM (3 mL) in the presence of EDCI (0.128 mmol,
0.020 g) and DIEA (0.192 mmol, 0.025 g). DCM was evaporated
and deprotection of the side chains was performed according to
the general method (TFA:DCM:TES, 10:9:1, 3 mL). The crude yel-
low oil was purified by preparative RP-HPLC (using TEAA, triethy-
lammonium acetate, as the buffer, to avoid hydrolysis of the
boronic ester to boronic acid), affording 0.010 g (20%, over two
6
80.3031.
4.2.9. (S)-2-((S)-2-((2S,3S)-2-((S)-1-Acetylpyrrolidine-2-carboxamido)-
1
3
-hydroxybutanamido)propanamido)-N -((S)-1-((S)-1-(methylcar-
bamoyl)pyrrolidin-2-yl)-1-oxopropan-2-yl)succinamide (50g)
The analogue was prepared according to the general procedure
using the peptide 21a (0.027 mmol, 0.020 g), and the modified
proline 45 (0.027 mmol, 0.005 g) in anhydrous DCM (3 mL) in
the presence of HBTU (0.027 mmol, 0.011 g) and DIEA
1
(
0.054 mmol, 0.007 g). Deprotection of the side chains was carried
steps) of desired oligopeptide 50j as a white solid. The H NMR
1
out according to the general procedure (95% TFA in H O, 2 mL)
2
showed a mixture 4.5:1 of two rotamers. H NMR (400 MHz,
after which the solvents were evaporated and the residue (yellow
oil) was purified by preparative RP-HPLC, affording 3 mg (18%,
over two steps) of desired oligopeptide 50g as a white solid.
6
DMSO-d , 90 °C), d 8.77–8.64 (m, 0.5H rotamer), 8.60 (d,
J = 2.9 Hz, 1H), 8.19 (dd, J = 17.6, 7.9 Hz, 0.5H rotamer), 8.13–8.10
(m, 0.5H rotamer), 8.07–8.04 (m, 1H), 8.03–7.93 (m, 1H), 7.72 (d,
J = 8.3 Hz, 1H), 7.37–7.29 (m, 1H), 6.93–6.94 (m, 1H), 5.02–4.99
(m, 0.5H rotamer), 4.70–4.53 (m, 1H), 4.39 (dd, J = 8.0, 3.1 Hz,
1H), 4.31 (dd, J = 8.6, 3.8 Hz, 0.5H rotamer), 4.27–4.15 (m, 2H),
44 8 9
RP-HPLC purity: C18 column > 95%. C27H N O , MS (ESI): m/z
+
6
25.3 [M+H] . HRMS for C27
H
44
N
8
O
9
Na Calcd: 647.3153, Found:
6
47.3143.
4
.11 (dd, J = 8.0, 4.1 Hz, 0.5H rotamer), 4.07–4.04 (m, 2H), 3.55–
1
4
.2.10. (S)-2-((S)-2-Acetamidopropanamido)-N -((S)-4-(tert-butyla-
3.45 (m, 2H), 2.44–2.39 (m, 1H), 2.26 (t, J = 8.6 Hz, 2H), 2.22–2.14
(m, 1H + 1H rotamer), 2.04–1.76 (m, 9H), 1.65–1.61 (m, 1H),
1.49–1.42 (m, 3H), 1.33–1.16 (m, 21H + 8H rotamer), 1.06–1.04
(m, 3H + 0.5H rotamer), 1.02–0.98 (m, 3H), 0,92 (t, J = 8.2 Hz,
mino)-3,4-dioxobutan-2-yl)succinamide (50h)
The analogue was prepared according to the general procedure
using the peptide 22 (0.051 mmol, 0.025 g), and the warhead 41a
(
0.153 mmol, 0.032 g) in anhydrous DMF (3 mL) in the presence
0.5H rotamer), 0.87–0.84 (m, 3H + 1H rotamer), 0.80 (s, 3H).
+
of HBTU (0.153 mmol, 0.058 g) and DIEA (0.306 mmol, 0.039 g).
The crude material was purified by RP-HPLC affording 0.013 g
(
38
C H65BN
6
O
9
, MS (ESI): m/z 761.3 [M+H] . RP-HPLC purity: C8
column > 95%. HRMS for C38
761.4974.
H66BN
O
6 9
Calcd: 761.4986, Found:
40%) of pure protected oligopeptide as a fluffy white solid.
+
C
36
H
43
N
5
O
6
, MS (ESI): m/z 642.1 [M+H] . Deprotection of the side
chains was carried out according to the general procedure (95%
TFA in H O, 2 mL) after which the solvents were evaporated and
0
0
4.2.13. (S)-2-((S)-2-((2S,3S)-2-((S)-1-(4 -Hexyl-[1,1 -biphenyl]-4-
carbonyl)pyrrolidine-2-carboxamido)-3-hydroxybutanamido)propa-
2
1
the residue (yellow oil) was purified by preparative RP-HPLC,
namido)-N -((R)-1-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-
affording 5 mg (62%) of desired oligopeptide 50h as a white solid.
H NMR (400 MHz, CDCl + 2 drops of DMSO-d , mixture of rota-
3 6
mers), d 7.66–7.64 (m, 1H), 7.51–7.46 (m, 1H), 7.34 (d, J = 6.4 Hz,
methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)succinamide (50k)
The analogue was prepared according to the general procedure
using the peptide 21c (0.026 mmol, 0.025 g), and the warhead
1
1
1
2
C
H), 6.86 (bs, 1H), 6.64 (bs, 1H), 5.95–5.94 (m, 1H), 4.83–4.78 (m,
H), 4.35–4.24 (m, 2H), 3.96–3.88 (m, 1H), 3.33–3.30 (m, 1H),
.21 (s, 3H), 1.60 (d, J = 8.3 Hz, 3H), 1.03–0.87 (m, 12H).
(R)-BoroAla-(+)-Pinanediol
hydrochloride
47
(0.052 mmol,
0.013 g) in anhydrous DCM (3 mL) in the presence of EDCI
(0.052 mmol, 0.008 g) and DIEA (0.126 mmol, 0.020 g). DCM was
evaporated and deprotection of the side chains was performed
+
+
17
29 5 6
H N O , MS (ESI): m/z 400.2 [M+H] , 799.2 [2 M+HM+H] .
Please cite this article in press as: De Rosa M., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.12.003

M. De Rosa et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
13
according to the general method (TFA:DCM:TES, 10:9:1, 3 mL).
The crude yellow oil was purified by preparative RP-HPLC (using
TEAA as the buffer), affording 6 mg (27%, over two steps) of
desired oligopeptide ester 50k. A small fraction (2.4 mg, 13%) of
boronic acid 50l was also isolated. Ester. 1H NMR (400 MHz,
4.2.17. (S)-2-((S)-2-((2S,3R)-2-((S)-1-acetylpyrrolidine-2-carboxamido)-
3-hydroxybutanamido)propanamido)-N -((R)-1-((3aS,4S,6S,7aR)-
3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-
yl)ethyl)succinamide (50o)
The analogue was prepared according to the general procedure
using the peptide 21a (0.054 mmol, 0.040 g), and the warhead (R)-
BoroAla-(+)-Pinanediol hydrochloride 47 (0.108 mmol, 0.028 g) in
anhydrous DCM (3 mL) in the presence of HATU (0.108 mmol,
0.041 g) and DIEA (0.162 mmol, 0.021 g). DCM was evaporated
and deprotection of the side chains was performed according to
the general method (TFA:DCM:TES, 10:9:1, 2 mL). The crude yel-
low oil was purified by preparative RP-HPLC (using TEAA as the
1
DMSO-d
6
, 90 °C), d 8.02–7.99 (bs, 1H), 7.89 (d, J = 3.9 Hz, 1H),
7
4
2
2
2
1
.77 (t, J = 4.5 Hz, 1H), 7.67 (d, J = 7.7 Hz, 2H), 7.60–7.52 (m,
H), 7.30 (d, J = 7.8 Hz, 2H), 4.61–4.59 (m, 2H), 4.29–4.20 (m,
H), 4.16 (d, J = 8.5 Hz, 1H), 4.08–4.02 (m, 1H), 3.62–3.59 (m,
H), 3.05–2.95 (m, 5H), 2.65 (t, J = 7.6 Hz, 2H), 2.28–2.20 (m,
H), 2.10–2.00 (m, 2H), 1.92–1.83 (m, 4H), 1.64–1.60 (m, 2H),
.34–1.24 (m, 18H), 1.05 (dd, J = 7.3, 3.4 Hz, 3H), 0.89 (t,
J = 8.2 Hz, 3H), 0.83 (s, 3H). C47
[
H
67BN
M+H] . RP-HPLC purity: C8 column > 95%. HRMS for C47
Calcd: 871.5141, Found: 871.5136.
6
O
9
, MS (ESI): m/z 871.5
buffer), affording 5 mg (14%, over two steps) of desired oligopep-
+
+
H68BN
6
O
9
tide ester 50o. C30
H49BN
6
O
9
, MS (ESI): m/z 649.1 [M+H] . RP-HPLC
purity: C8 column > 95%.
4
.2.18. (S)-2-((S)-2-((2S,3R)-3-hydroxy-2-((S)-pyrrolidine-2-carbox-
0
4
[
.2.14. ((3S,6S,9S,12R)-9-(2-Amino-2-oxoethyl)-1-((S)-1-(4 -hexyl-
1
amido)butanamido)propanamido)-N -((R)-1-((3aS,4S,6S,7aR)-3a,5,5-
trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)-
succinamide (50p)
The analogue was prepared according to the general procedure
using the peptide 20 (0.119 mmol, 0.110 g), and the commercial
0
1,1 -biphenyl]-4-carbonyl)pyrrolidin-2-yl)-3-((S)-1-hydroxyethyl)-6-
methyl-1,4,7,10-tetraoxo-2,5,8,11-tetraazatridecan-12-yl)boronic
acid (50l)
Compound 50l was obtained (2 mg, 10.5%) as a by-product of
the reaction between peptide 21c and (R)-BoroAla-(+)-Pinanediol
(
R)-BoroAla-(+)-Pinanediol
.062 g) in anhydrous DCM (10 mL) in the presence of HATU
0.238 mmol, 0.091 g) and DIEA (0.357 mmol, 0.046 g). After
preparative RP-HPLC purification (TEAA buffer) 0.075 g (55%) of
pure peptide as white solid were obtained. C66 10, MS
hydrochloride
47
(0.238 mmol,
+
hydrochloride 47. C37
7
C
H53BN
6
O
9
, MS (ESI): m/z 701.2 [M+Hꢀ2H
2
O] ,
0
(
+
19.2 [M+HꢀH
2
O] . RP-HPLC purity: C8 column > 95%. HRMS for
37 6 9
H53BN O
Na Calcd: 759.3865, Found: 759.3861.
6
H79BN O
+
0
(ESI): m/z 1127.6 [M+H] . 0.050 g of protected compound were
used for the Fmoc protecting group removal (30% piperidine in
ethanol, room temperature, 3 h) and the subsequent deprotection
of the side chains (performed according to the general method in
TFA:DCM:TES, 10:9:1, 3 mL). The crude yellow oil was purified
by preparative RP-HPLC (using TEAA) affording 0.010 g (41%, over
4
[
.2.15. (2S)-2-((2S)-2-((2S,3S)-2-((2S)-4-(aminomethyl)-1-(4 -hexyl-
1,1 -biphenyl]-4-carbonyl)pyrrolidine-2-carboxamido)-3-hydroxybu-
0
1
tanamido)propanamido)-N -((R)-1-((3aS,4S,6S,7aR)-3a,5,5-trimethyl-
hexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)succi-
namide (50m)
The analogue was prepared according to the general procedure
using the peptide 23 (0.050 mmol, 0.056 g), and the warhead (R)-
BoroAla-(+)-Pinanediol hydrochloride 47 (0.100 mmol, 0.026 g) in
anhydrous DCM (5 mL), in the presence of HATU (0.100 mmol,
two steps) of desired acetate salt oligopeptide 50p as a white solid.
1
6
H NMR (400 MHz, DMSO-d , 90 °C), d 8.27 (t, J = 6.8 Hz, 1H), 8.02
(
bs, 1H), 7.93 (d, J = 8.2 Hz, 1H), 7.81 (m, 1H), 4.67–4.48 (m, 1H),
4
3
3
2
1
1
4
.36–4.20 (m, 3H), 4.16–4.13 (m, 1H), 4.10–3.96 (m, 1H), 3.66–
.54 (m, 1H), 3.52 (s, 3H), 3.32–3.26 (m, 1H), 3.23–3.17 (m, 1H),
.10 (q, J = 7.3 Hz, 6H), 2.38–2.24 (m, 1H), 2.28–2.14 (m, 2H),
.07–2.01 (m, 1H), 1.94–1.87 (m, 5H), 1.83–1.81 (m, 1H), 1.77–
.74 (m, 1H), 1.70–1.69 (m, 1H), 1.67–1.65 (m, 1H) 1.28 (s, 3H),
.24–1.18 (m, 15H), 1.11 (d, J = 6.3 Hz, 3H), 1.05 (dd, J = 7.4,
0
.038 g) and DIEA (0.15 mmol, 0.020 g). DCM was evaporated and
deprotection of the side chains was performed according to the
general method (TFA:DCM:TES, 10:9:1, 3 mL). The crude yellow
oil was purified by preparative RP-HPLC (using TEAA, as the buffer),
affording 6 mg (21%, over two steps) of desired oligopeptide ester
5
0m. A small fraction (2.2 mg, 6.3%) of boronic acid 50n was also
6
.6 Hz, 3H), 0.81 (s, 3H). C30H51BN O10, MS (ESI): m/z 607.1 [M
isolated, along with a second fraction of the de-boronation by-pro-
duct 50o. Ester. C48H70BN O , MS (ESI): m/z 900.5 [M+H] . RP-HPLC
7 9
purity: C8 column > 95%. HRMS for C48
Found: 900.5397.
+
+
+H] . RP-HPLC purity: C8 column > 95%. HRMS for C30
Calcd: 607.3627, Found: 607.3647.
6 10
H52BN O
7 9
H71BN O Calcd: 900.5406,
4.3. Computational details
4
.2.16. ((3S,6S,9S,12R)-9-(2-amino-2-oxoethyl)-1-((2S)-4-(aminom-
All calculations were performed using the Schrödinger Small
Molecule Drug Discovery Suite (Schrödinger Release 2015-4) using
the OPLS-2005 force field. The E. coli crystal structure 3IIQ was
0
0
ethyl)-1-(4 -hexyl-[1,1 -biphenyl]-4-carbonyl)pyrrolidin-2-yl)-3-((S)-
3
7
35
1
1
-hydroxyethyl)-6-methyl-1,4,7,10-tetraoxo-2,5,8,11-tetraazatridecan-
2-yl)boronic acid (50n)
downloaded from the PDB and prepared using Protein Preparation
3
8
Compound 50n was obtained (2.2 mg, 6%) as a by-product of
Wizard. This implied the addition of hydrogens, the assignment
of protonation states and optimization of the hydrogen bond net-
work followed by a restrained minimization. To model the binding
of both diastereomers of the N-acetyl analogue of 50n, these
ligands were built manually into the active site of LepB, using
the protein bound conformation of arylomycin as a template. A
(R)-boroalanine was added to the C-terminal and the boron was
attached covalently to Ser91. The resulting complexes were further
refined by 1000 steps of conformational sampling by MCMM using
the reaction between peptide 23 and (R)-BoroAla-(+)-Pinanediol
hydrochloride 47. The compound was isolated as TFA salt after
preparative RPHPLC (TFA buffer). 1H NMR (400 MHz, DMSO-d
) d
.06 (bs, 1H), 7.98 (bs, 1H), 7.86 (bs, 1H), 7.76–7.70 (m, 3H),
6
8
7
2
4
.63–7.58 (m, 3H), 7.53 (bs, 1H), 7.42 (bs, 1H), 7.31 (d, J = 7.6 Hz,
H), 7.16 (s, 1H), 6.98 (bs, 1H), 6.53 (s, 1H), 4.97–4.94 (m, 1H),
.67 (t, J = 8.9 Hz, 1H), 4.27–4.25 (m, 2H), 4.09–4.07 (m, 1H), 3.74
(
2
(
t, J = 9.7 Hz, 1H), 3.45–3.39 (m, 2H), 2.95–2.90 (m, 2H), 2.67–
.60 (m, 4H), 2.33–2.31 (m, 2H), 1.61–1.58 (m, 1H), 1.33–1.17
m, 11H), 1.10 (d, J = 6.0 Hz, 3H), 1.00 (d, J = 6.2 Hz, 3H), 0.87 (t,
3
9
the GB/SA continuum solvation model for water in the optimiza-
tion step. Torsion angles sampled were those of the ligand and pro-
tein side chains in close contact with the ligand. The backbone of
the protein as well as amino acids more than 5 Å away from the
+
J = 7.8 Hz, 3H). C38
RP-HPLC purity: C8 column > 95%. HRMS for C38
66.4311 Found: 766.4310.
H56BN
7
O
9
, MS (ESI): m/z 748.2 [M+HꢀH
2
O] .
H
7 9
57BN O
Calcd:
4
0
7
ligand were kept fixed. SiteMap was used to identify and visual-
Please cite this article in press as: De Rosa M., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.12.003

1
4
M. De Rosa et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
ize the hydrophobic patches responsible for the Ala-X-Ala
recognition.
drug-hypersensitive lpxC mutant), P. aeruginosa (PAO1, wild-type;
PAO750, an efflux-defective mutant isogenic to PAO1), K. pneumo-
nia (ATCC 13833, wild-type), A. baumannii (ATCC 19606, wild-
type), and the Gram-positive S. aureus (ATCC 29213, wild-type).
In vitro antimicrobial activity of each compound was determined
by measuring the MIC using the broth micro-dilution technique
in cation-adjusted Mueller-Hinton II medium (Becton-Dickinson,
212322) according to EUCAST and CLSI guidelines (for details, see
4.4. Production of EcLepB
Details of EcLepB cloning, expression and purification are pro-
vided in the Supplementary data. Briefly, a full-length construct
was designed that included a His tag between the two transmem-
brane helices predicted at the N-terminal end, replacing residues
known to represent self-cleavage site in the wild type
6
4
1
the Supplementary data, 2.2).
a
3
4
sequence. The resulting protein was expressed at high levels in
E. coli, extracted from the membrane fraction with 1% reduced Tri-
ton X-100, then purified by metal-chelation chromatography with
good yield in the presence of 0.5% detergent.
4.8. Evaluation of cytotoxicity
Compounds were evaluated in a fluorometric microculture
cytotoxicity assay (FMCA) and the HepG2 cell line was used for
3
6
the study (see Supplementary data for more details). Cytotoxicity
was assessed after 72 h with cell survival presented as survival
index (SI, %) defined as fluorescence in test wells in percent of con-
trol cultures with blank values subtracted. The IC50 was deter-
mined from log concentration-effect curves in Graph Pad Prism
using a non-linear regression analysis.
4
.5. Assay
EcLepB activity and inhibition were determined using
a
FRET-pair assay, as detailed in the Supplementary data. Dabcyl-
VGGTATA;GAFSRPGLE(EDANS)-OH, and dabcyl-KLTFGTVKPVQAIA;
GYEILE(EDANS)-OH were used as substrates, where dabcyl was
the fluorescence acceptor and EDANS was the fluorescence donor;
the expected cleavage sites are indicated with arrows. The linear
increase in fluorescence, corresponding to substrate cleavage,
was monitored at 22 °C for 2 h. For inhibition tests, EcLepB was
pre-incubated with the compound for 10 min at 22 °C, then the
reaction was followed at a substrate (dabcyl-VGGTATAGAFSRP-
4.9. Hemolysis studies
Compounds were evaluated for hemolytic activity using red
blood cells from heparinized human blood (for details, see the Sup-
plementary data, 2.3).
GLE(EDANS)-OH) concentration of 8
lM; final EcLepB concentra-
4.10. Selection and sequence identification of resistant E. coli mutants
tion 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 dose–response curves in
GraphPad Prism (GraphPad software Inc., CA, USA).
Mutants of E. coli
D
tolC resistant to compounds 50m and 50q29
were isolated by serial passage with selection for growth at
increasing concentrations of compound. Mutations were identified
by whole genome sequencing of independently selected mutants
with increased MIC values (for details, see the Supplementary data,
2.4).
4.6. Substrate cleavage sites
Cleavage sites were investigated for two substrates (one unla-
Acknowledgements
beled peptide VGGTATAGAFSRPGL, as well as the dabcyl-VGGTA-
TAGAFSRPGLE(EDANS)-OH substrate) by mass spectrometry (MS),
as described in the Supplementary data. Briefly, a 5 fmol sample
of each intact substrate was loaded to the LC-MS for initial charac-
This work was supported by the Swedish Research Council
(521-2014-6711, 521-2013-2904, 521-2013-3105, 621-2014-
6215), the Swedish Foundation for Strategic Research (no. RIF14-
0078), a Swedish Institute scholarship (NS), Science for Life
Laboratory (SciLifeLab pilot infrastructure grant) and Uppsala
University. M.D.R. would like to thank Dr. Gunnar Lindberg for
his help with solid-phase synthesis.
terization. Subsequently, 10
lL of 5
lM substrate was incubated
with 2.3 M EcLepB. Samples (1
l
l
L) were taken at 1, 2, 3, 4 and
5
h, as well as overnight; each sample was diluted 1000 times with
0
.25% acetic acid, then loaded to the LC-MS to identify the prod-
ucts, both qualitatively and quantitatively. The samples from the
incubation were analyzed using nanoscale liquid chromatography
Supplementary data
(
nanoAdvance, Bruker Daltonics, Bremen, Germany) coupled to
an electrospray ionization quadrupole time-of-flight mass spec-
trometer (Maxis Impact, Bruker Daltonics). They were injected
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.12.003.
and desalted on a pre-column (100
Map100, 5 m, 100 Å) at a flow-rate of 10
fused silica needle emitter (15 cm, 75 m i.d., 375
New Objective, Woburn, MA, packed with Reprosil-Pur C18-AQ,
m resin) was used as spray emitter and analytical column.
The mobile phases were 0.1% formic acid in water (buffer A) and
.1% formic acid in acetonitrile (buffer B). Each sample was ana-
lyzed during a 20 min gradient (3–50% buffer B) at a flow rate of
00 nL/min at 50 °C. Mass spectra were collected in full MS scan
mode (m/z 50–2000).
l
m i.d. ꢁ 20 mm, C18 Pep-
L/min for 4 min. A
m o.d., Picotip,
l
l
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