Phosphinate, sulfonate, and sulfonamidate dipeptides as potential inhibitors of Escherichia coli aminopeptidase N

Article abstract of DOI:10.1016/j.bmcl.2005.08.055

In an effort to prepare novel inhibitors of bacterial aminopeptidase N (PepN), the phosphinate, propenylphosphinate, decylphosphinate, sulfonate, and sulfonamidate analogs of Ala-Ala were synthesized and tested as inhibitors. Phosphinate 1 was shown to inhibit PepN with a K_i of 10 μM, and propenylphosphinate 2 and decylphosphinate 3 inhibited PepN with a K_i of ca. 1 μM. Sulfonate and sulfonamidate analogs did not inhibit PepN.

Full text of DOI:10.1016/j.bmcl.2005.08.055

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5150–5153
Phosphinate, sulfonate, and sulfonamidate dipeptides
as potential inhibitors of Escherichia coli aminopeptidase N
Ke-Wu Yang, Frank C. Golich, Tara K. Sigdel and Michael W. Crowder*
Department of Chemistry and Biochemistry, 160 Hughes Hall, Miami University, Oxford, OH 45056, USA
Received 25 July 2005; revised 19 August 2005; accepted 22 August 2005
Available online 15 September 2005
Abstract—In an effort to prepare novel inhibitors of bacterial aminopeptidase N (PepN), the phosphinate, propenylphosphinate,
decylphosphinate, sulfonate, and sulfonamidate analogs of Ala–Ala were synthesized and tested as inhibitors. Phosphinate 1 was
shown to inhibit PepN with a Ki of 10 lM, and propenylphosphinate 2 and decylphosphinate 3 inhibited PepN with a Ki of
ca. 1 lM. Sulfonate and sulfonamidate analogs did not inhibit PepN.
Ó 2005 Elsevier Ltd. All rights reserved.
Aminopeptidases are ubiquitous hydrolases that are in-
volved with protein maturation, activation, and degra-
dation.1,2 These enzymes can hydrolyze the N-terminal
amino acid(s) from a peptide by using active site serine
or cysteine residues or by using metal ion catalysis.
The most common metal ion found at the active site
of aminopeptidases is Zn(II); however, there are known
aminopeptidases that utilize Fe(II), Mn(II), or Co(II).1
The aminopeptidases have also been grouped according
to their substrate specificities, physical and mechanistic
properties, and amino acid sequences.1,3
In bacteria, PepN is cytosolic,1 and the enzymes from a
number of Lactococcus and Lactobacillus strains have
been studied in detail because of the roles of PepN in
the making of cheese and other dairy products.1,11 In
Escherichia coli, PepN has been identified as the sole ala-
nyl aminopeptidase and is the major aminopeptidase in-
volved in ATP-independent processing during cytosolic
O
O
OH
OH
P
P
H N
2
H N
2
OH
OH
O
O
One particularly interesting enzyme is aminopeptidase
N (PepN). In mammals, PepN is membrane-associated
and has been identified as a receptor for several viruses
and in the activation mechanism of collagenolysis that
allows for tumor cell invasion.4–6 Since PepN apparently
is involved in a number of biomedically important pro-
cesses, a large number of inhibitors of mammalian PepN
have been reported. For example, several natural prod-
uct dipeptides, bestatin, phebestin, probestin, and
amastatin, have been shown to be potential inhibitors
of PepN, leucine aminopeptidase, methionine amino-
peptidase, and aminopeptidase A.7–9 Bartlett and co-
workers synthesized and tested phosphinate analogs of
bestatin as inhibitors of leucine aminopeptidase.10
Propylphosphinate, 1
Propenylphosphinate, 2
O
O
O
OH
OH
S
H N
2
P
H N
2
O
OH
O
O
Sulfonate, 4
O
H
N
OH
S
H N
2
O
O
Decylphosphinate, 3
Sulfonamidate, 5
O
O
OH
P
H N
2
OH
O
Keywords: Aminopeptidase N; Phosphinate; Inhibitor.
*
Propylphosphonate
Corresponding author. Tel.: +1 51 35 29 72 74; fax: +1 51 35 29 57
1; e-mail: crowdemw@muohio.edu
Figure 1. Structures of compounds described in this study.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.08.055

K.-W. Yang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5150–5153
5151
protein degradation.12 Given these roles, PepN and
other bacterial proteases have been recently identified
as potential antibiotic targets.13
Propylphosphinate 1 was previously reported to be an
inhibitor of the aminopeptidase VanX18,19 and was syn-
thesized as previously described.20 This compound was
shown to be a competitive inhibitor of PepN from E. coli
with a Ki value of 10 2 lM (Table 1). The comparable
phosphonate analog (Fig. 1) of 119 did not appreciably
inhibit PepN at concentrations up to 200 lM (data not
shown). We reasoned that the P–X–C bond angle (ideally
109.5° in 1 and 104° in the phosphonate analog) may play
a role in the relative binding strengths of these com-
pounds. Therefore, a compound with a large P–X–C bond
angle was tested as an inhibitor. (1-Aminoethyl)(2-car-
boxy-1-propenyl) phosphinic acid 2 was synthesized as
shown in Scheme 1 and as described in supplementary
materials. The overall yield of this synthetic strategy
was 27%. Propenylphosphinate 2 was a competitive inhib-
itor with a Ki of 1.0 0.2 lM (Table 1). As we hypothe-
sized, the lengthening of the P–X–C bond angle resulted
in tighter binding of the inhibitor.
In this work, we have tested a number of phosphinate,
sulfonate, and sulfonamidate analogs of alanyl–alanine
as inhibitors of PepN from E. coli (Fig. 1).
Previous studies have demonstrated that similar analogs
are transition state analog inhibitors of peptidases,14–17
and these inhibitors are typically tight binding and
specific.
Table 1. Inhibition constants for compounds 1–5a
Compound
Ki (lM)
10
Mode of inhibition
1
2
3
4
5
2
Competitive
Competitive
Competitive
1.0 0.2
1.1 0.1
>300
>300
In an effort to improve the binding affinity of these phos-
phinate dipeptide analogs to PepN, a hydrophobic sub-
stituent (C8H17) was attached to the b-carbon of 1 to
yield decylphosphinate 3. We reasoned that 3 would
a Ki values and modes of inhibition were determined as previously
described and using L-ala-p-nitroanilide as substrate and PepN from
E. coli as the enzyme in 50 mM Hepes, pH 7.0, at 25 °C.20
H
CO CH
2
3
Br
H N
2
P
OH
O
CH(CO C H )
2 5 2
CH
3
2
6
EtONa/EtOH
Br
Cbz-Cl
2
Na CO /NaOH
2
3
CH(CO C H )
/NaHCO
2
2
5 2
3
11
Br
CO CH
2
3
O
i. KOH/MeOH
ii.HCl/H O
2
H
H
CH
3
P
O
N
H
Br
H
CO H
2
OH
O
9
7
CO CH
2
3
CH N
MeONa
MeOH
2
2
12
13
Et O/CH Cl
2
2
2
H CO, Et NH
2
2
O
H
CO CH
2
3
H
P
O
N
CO CH
2
Br
3
CH
3
H
OCH
O
3
8
10
MeONa/MeOH
MeONa/MeOH
O
O
O
O
OCH
P
OCH
P
3
O
N
O
N
OCH
3
H
H
O
OCH
O
3
3
15
+
14
i. HBr/HOAc, ii. H
iii. Propylene oxide
+
i. HBr/HOAc, ii. H
iii. Propylene oxide
O
O
OH
P
OH
H N
2
O
OH
P
H N
2
OH
O
3
2
Scheme 1. Synthesis of propenylphosphinate 2 and decylphosphinate 3.

5152
K.-W. Yang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5150–5153
O
CH3CHO
+
NaHSO3
OH
O
CbzCl
OH
O
i. NH4OH
ii. HCl
H N
2
S
O
N
H
S
NaOH/NaHCO3/Na2CO
O
O
3
17
16
HnX
CO CH
2
3
O
O
SOCl2
CHCl3
Cl
O
X
CO CH
2 3
O
N
H
S
O
N
S
Et3N/CHCl3
X= O, n=1
X= N, n=2
H
O
O
O
18
19 X = O
20 X = NH
O
O
H
Pd/C, H2
O
N
OH
S
H N
2
OH
S
+
H N
2
MeOH/H2O
O
O
O
O
4
5
Scheme 2. Synthesis of sulfonate 4 and sulfonamide 5.
be a bifunctional inhibitor in which the substituent
would provide additional points of attachment to PepN.
(1-Aminoethyl)(2-carboxy-1-decyl) phosphinic acid 3
was synthesized in 34% overall yield as shown in Scheme
1 and as described in supplementary materials. Dec-
ylphosphinate 3 was shown to be a competitive inhibitor
of PepN with a Ki of 1.1 0.1 lM (Table 1). The inclu-
sion of the hydrophobic substituent on phosphinate 1
did result in improved binding affinity. Unfortunately,
efforts to synthesize a decylphosphinate analog of pro-
penylphosphinate 2 have been unsuccessful.
a double bond and a hydrophobic substituent are
included in the phosphinate dipeptide. These studies
also show that the use of sulfonate/sulfonamide analogs
to replace phosphinates is not an effective strategy to im-
prove the binding strength of the compounds.
Acknowledgments
This work was supported by the National Institutes of
Health (GM67928 to M.W.C.)
Previously, phosphinate analogs of peptides have been
reported to be very tight binding inhibitors of several
peptidases with Ki values reported as low as
10À15 M.16 The relative weaker binding to PepN sug-
gested that these compounds may not be good transition
state analogs. Nonetheless, we attempted to improve the
binding of the dipeptide analogs by preparing sulfonate
and sulfonamide analogs of Ala–Ala. We reasoned that
the sulfonate and sulfonamide analogs would require
less desolvation than the phosphinate analogs, and the
reduced desolvation energy would result in relatively
tighter binding.
Supplementary data
Supplementary data associated with this article can be
found in the online version at doi:10.1016/
j.bmcl.2005.08.055
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