Individual stereoisomers of phosphinic dipeptide inhibitor of leucine aminopeptidase

Article abstract of DOI:10.1016/j.bmcl.2008.01.107

Individual stereoisomers of the phosphinic pseudodipeptide hPheψ[P(O)(OH)CH₂]Phe were obtained by stereoselective liquid chromatographic separation as N- and C-terminally protected derivative on quinidine carbamate modified silica stationary ph

Full text of DOI:10.1016/j.bmcl.2008.01.107

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1550–1554
Individual stereoisomers of phosphinic dipeptide inhibitor
of leucine aminopeptidase
Artur Mucha,a,* Michael La¨mmerhofer,b Wolfgang Lindner,b
Małgorzata Pawełczakc and Paweł Kafarskia,c
aDepartment of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology,
Wybrzez_e Wyspian´skiego 27, 50-370 Wrocław, Poland
bChristian Doppler Laboratory for Molecular Recognition Materials, Department of Analytical Chemistry & Food Chemistry,
University of Vienna, Wa¨hringer Strasse 38, A-1090 Vienna, Austria
cInstitute of Chemistry, University of Opole, Oleska 48, 45-052 Opole, Poland
Received 10 January 2008; revised 29 January 2008; accepted 29 January 2008
Available online 2 February 2008
Abstract—Individual stereoisomers of the phosphinic pseudodipeptide hPhew[P(O)(OH)CH2]Phe were obtained by stereoselective
liquid chromatographic separation as N- and C-terminally protected derivative on quinidine carbamate modified silica stationary
phase. The stereoisomeric purity, exceeding 95% for each fraction, was determined before and after deprotection using two indepen-
dent methods. The absolute configuration was rationally assigned by application of enantiomerically pure phosphinic acid sub-
strates in the synthetic procedure and correlation with biological activity of the products. Substantial differences in inhibition of
leucine aminopeptidase by the individual isomers revealed novel insights into potency of the recently developed and remarkably
effective compound.
ꢀ 2008 Elsevier Ltd. All rights reserved.
The design and construction of targeted bioactive mole-
cules requires to take into consideration their strictly
stereoselective mode of action. Chirality is, in the most
cases, the key factor in the safety and efficacy of many
drug products.1,2 Usually only one enantiomer is
responsible for the desired activity, whereas its counter-
part could be inactive, possess some activity of interest,
be an antagonist of the active enantiomer or have a sep-
arate activity that could be either desirable or undesir-
able. Only in a few cases specific compositions of a
racemate or an enantiomeric pair demonstrated a syner-
gistic effect.1,2 Similarly, diastereoisomers of unique
three dimensional structure, basically determined by
the configuration of chiral elements such as stereogenic
centres present in the molecule, are able to approach
the receptors’ binding site and interact efficiently,
whereas the other forms are discriminated. Apparently,
this is particularly valid for peptides and their various
chemically modified analogues. Among the latter, phos-
phinic acid pseudopeptides represent isosters with a
scissile bond replaced by non-hydrolysable –PO2HCH2–
moiety. Tetrahedral phosphinates resemble the high-en-
ergy transition state of the peptide bond hydrolysis3–5
and consequently, they have been extensively applied
for effective and selective regulation of activity of vari-
ous proteases, particularly metallo-dependent ones.6–10
For these purposes phosphinic peptides were frequently
assayed in the form of their pure diastereoisomers, but
rarely was it the result of achievements in development
of stereoselective strategies for their preparation.11 Ele-
gant asymmetric hydrogenation of the appropriate
phosphinic dehydrodipeptide to obtain D-Ala-D-Ala
analogue, antibacterial agent acting as D-Ala-D-Ala li-
gase inhibitor, is one such example.12 The extended
pseudopeptides containing only one modified amide
bond are usually obtained by elongation of a suitably
protected dipeptide building block with chiral amino
acids using standard peptide synthesis methods. Then,
individual diastereoisomers are readily separated upon
conventional purification of a final product on reverse
phase HPLC (for example, see Ref. 13).
Keywords: Phosphinic dipeptides; Stereoselective separation; Leucine
aminopeptidase; Inhibitors.
*
The situation is more complicated in the case of phos-
phinic acid dipeptides. Possessing two non-defined
Corresponding author. Tel.: +48 71 320 3354; fax: +48 71 328
4064; e-mail: artur.mucha@pwr.wroc.pl
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.107

A. Mucha et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1550–1554
1551
asymmetric carbon atoms, obtained in non-stereoselec-
tive manner and lacking a chiral auxiliary, they repre-
sent mixture of two enantiomeric pairs which cannot
be separated using non-chiral techniques. Recently, we
have reported rationally designed phosphinic dipeptide
analogues of such type as effective inhibitors of bizinc
cytosolic leucine aminopeptidase (LAP, EC 3.4.11.1).14
Broad interests in LAP are directed towards detailed
evaluation of its mechanism of hydrolytic action, biolog-
ical significance and potential medical importance (for
recent references on mechanistic, physiological and
pathological implications of LAP activity see Refs. 15
and 16). All these accounts stimulate continuous interest
in agents regulating the protease activity.17 Phosphinic
dipeptides, although tested as stereoisomeric mixtures,
were ranked amongst the most potent low-molecular
weight inhibitors of LAP reported so far. For example,
homophenylalanyl-phenylalanine (compound 1, hPhew
[PO2HCH2]Phe, Fig. 1) homophenylalanyl-tyrosine
and leucyl-leucine analogues exhibited Ki’s in the low
nanomolar range.14 Furthermore, selected dipeptides
were even more successfully employed to inhibit recom-
binant leucyl aminopeptidase of Plasmodium falcipa-
rum,18 a potential target protease for the development
of new antimalarials.19 Excellent kinetic parameters of
suppression of the enzyme activity and the growth of
the pathogen, both in vitro as well as in vivo, were evi-
denced.18 As the result, the study provided new interest-
ing lead compounds for malaria treatment.
O
P
O
P
H
N
H
H
OH
N
O
Cbz
Cbz
OH
O
a-c
d
2
3
O
P
OH
O
P
H
N
*
*
OH
O
H N
2
*
*
Cbz
OH
O
O
e-g
RR-3
RS-3
SR-3
SS-3
RR-1
RS-1
SR-1
SS-1
Optical purity > 95%
Chiral stationary phase:
R R
Si
S
3R
silica
4S
H
N
H
N
O 9S
8R
O
O
4
N
Scheme 1. Synthesis and separation of the stereoisomers of
hPhew[PO2HCH2]Phe phosphinic dipeptide analogue. Reagents and
conditions: (a) hexamethyldisilazane, 100–110 ꢁC, 3 h; (b) H2C@
CH(CH2Ph)COOMe, 80–90 ꢁC, 3 h; (c) MeOH; (d) stereoselective
HPLC on a prototype of the commercial CHIRALPAK QD-AX
(10 lm) (250 · 20 mm id) (Chiral Technologies Europe) with stationary
phase 4, mobile phase acetonitrile/0.25 M formic acid (80:20, v/v) (pH of
the mixture adjusted to 4.0 with ammonia), ambient temperature, flow
rate 12 mL/min, elution order: R,R-3 < R,S-3 < S,R-3 < S,S-3; (e)
bromotrimethylsilane, 10 equiv, 3 d, rt; (f) H2O, then concd HCl; (g)
HPLC purification, gradient 20:80 (00) ! 40:60 (200) (v/v, acetonitrile/
water, both containing 0.1% of trifluoroacetic acid), tR = 17.8 min.
In this context, the challenge to synthesize/separate all
four pure stereoisomers and to specify their potency
seems to be fully justified. Here, we report the results
of our studies on resolution of the four isomers of
hPhew[PO2HCH2]Phe by combination of synthetic pro-
tocols and stereoselective liquid chromatographic meth-
ods. We also compare their activity towards leucine
aminopeptidase depending on the absolute configura-
tion. To the best of our knowledge, this represents very
first example of such studies on phosphinic acid
pseudodipeptides.
based on a quinidine derivative (4, Scheme 1) in HPLC
and CEC (capillary electrochromatography) separation
and purity analysis of the individual phosphinic dipep-
tide stereoisomers.20
The overall procedure to obtain individual stereoiso-
mers of phosphinic acid analogues of homophenylala-
nyl-phenylalanine (1) is outlined in Scheme 1.
Partially, it was also described in our previous paper
dealing with the application of a chiral stationary phase
The N-benzyloxycarbonyl (Cbz) protected phosphinic
pseudodipeptide methyl ester (3) was obtained in a
non-stereoselective manner by addition of the appropri-
ate acrylate to the suitably blocked phosphinic acid (2)
as described previously.14 The final material showed
two 31P NMR resonance signals of not equal integra-
tion, varying in the range 3:2–2:1 depending on the
batch. It was the result of more significant loss of the
up-field shifted enantiomeric pair in the purification pro-
cess (liquid chromatography/crystallization). Normally
unwanted (although straightforward), this was fortunate
in our case and greatly facilitated matching enantio-
meric and diastereomeric relationships (see below for
the details). The individual stereoisomers of the pro-
tected peptide were obtained by preparative stereoselec-
tive chromatography on a prototype of the commercial
CHIRALPAK QD-AX column (Chiral Technologies
Europe, Illkirch, France).20 The extensive optimization
O OH
P
OH
NH2
O
1, hPheψ[PO2HCH2]Phe
Ki = 66 nM (porcine kidney LAP) [14]
Ki = 13.2 nM (rec. P. falciparum LAP) [18]
(activity of the mixture of four stereoisomers)
Figure 1. The structure of the phosphinic acid analogue of homophe-
nylalanyl-phenylalanine and inhibition constants towards porcine
kidney and
aminopeptidase.
recombinant
Plasmodium
falciparum
leucine

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A. Mucha et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1550–1554
of the elution conditions did finally allow collection of
the four isomer fractions with sufficient purity. Newly
elaborated, superior CEC system using the same chiral
selector gave confirmation of their satisfactory stereoiso-
meric purity, exceeding 95% for each of the four frac-
tions corresponding to the individual stereoisomers.20
the stereoisomeric composition of compounds bearing
more than one asymmetric centre.
To define the absolute configurations of the pseudodi-
peptide samples additional experiments needed to be
performed. First of all, this was accomplished via the
synthesis of the substrate – Cbz-protected phosphinic
acid (2) in the optically pure forms. Usually, it proceeds
very efficiently by crystallization of diastereomeric salts
of 2 with enantiomeric a-methylbenzylamines as de-
scribed by Baylis et al.22 Surprisingly, although various
crystallization conditions were tested, we were not able
to achieve higher levels of enrichment than 70% of enan-
tiomeric purity. Thus, we returned to stereoselective li-
The obtained materials were treated with an excess of
bromotrimethylsilane (10-fold excess) in anhydrous
CH2Cl2 to remove both protecting groups in one step
without using alkaline conditions. Free pseudodipep-
tides were obtained after evaporation of volatile compo-
nents and subsequent hydrolysis of the residue with
water and concd HCl. The crude products were finally
purified by means of standard reverse phase HPLC.
After this step and before enzymatic assays their stereo-
isomeric purity was additionally verified. A method
developed for analysis of the enantiomeric excess of a-
aminophosphonates (phosphorus analogues of amino
acids) was used for this purpose. The method relies on
31P NMR analysis of diastereomeric inclusion com-
plexes formed between analytes bearing hydrophobic
side chains and cyclodextrins.21 In our case, treatment
of the hPhew[PO2HCH2]Phe stereoisomeric mixture (1)
with 10-fold excess of a-cyclodextrin gave satisfactory
separation for all four 31P NMR resonance signals
allowing their reliable integration (Fig. 2, bottom trace).
On the basis of the integrated signals it may be con-
cluded that the isomers with signals at 45.08 and
44.90 ppm as well as at 44.48 and 44.37 ppm represent
pairs of enantiomers. Furthermore, the analysis of indi-
vidual stereoisomers by 31P NMR confirmed their high
stereoisomeric purity of over 95%, which did not change
during deprotection (Fig. 2, upper traces). This success-
ful application of using cyclodextrins and a spectro-
scopic technique that is simple and straightforward to
interpret definitely opens new perspectives to determine
quid chromatography. The chiral acid
2
was
preparatively resolved into its antipodes using condi-
tions that had been developed in one of our previous
studies.23 As the result, R-2 (equivalent to the relative
configuration L) that was eluted first from the quinidine
carbamate column was isolated with enantiomeric pur-
ity of 99%, while S-2 (equivalent to the relative configu-
ration D), which eluted as second peak, had an
enantiomeric purity of 95%. Both were separately con-
verted to the target compound 1 by the same procedure
as described above for the racemic mixture. Two batches
of the final phosphinic dipeptide were obtained, each
containing two diastereoisomers with the defined config-
uration of the N-terminal amino acid part (Scheme 2).
The R,R/R,S-1 pair exhibited two signals at 44.90 and
44.48 ppm, whereas the S,R/S,S-1 pair at 45.08 and
44.37 ppm in 31P NMR experiments with the use of
a-cyclodextrin.
Significantly, the R,R/R,S-1 pair appeared much more
potent when tested for inhibition of leucine aminopepti-
dase. This should not be surprising as the protease is
responsible for cleavage of the N-terminal amino acids
of natural configuration L from polypeptide chains.24
Similar dependence is observed for the enzyme inhibi-
tors being much more effective in their relative configu-
ration L.17 Going into details, the fraction
corresponding to the 31P NMR signal at 44.90 ppm ap-
peared clearly to be of superior activity to regulate LAP
with Ki equal to 45 nM (Table 1). Thus, its configuration
can be finally specified as R,S (equivalent to the relative
one L,L) with high level of certainty. The R,R (L,D)-1 iso-
mer (44.48 ppm) exhibited six times lower potency. Con-
sequently, the enantiomer of the aforementioned
(45.08 ppm, S,R (D,D)-1) was far the weakest inhibitor
of LAP with Ki in micromolar range, while the enantio-
mer of the last mentioned (44.37 ppm, S,S (D,L)-1) was
again placed somewhere between the extremes.
S,R
R,S
R,R
S,S
Differences in activity of the studied stereoisomers to-
wards leucine aminopeptidase thus proved to be signifi-
cant indicating strong spatial preference of the enzyme
active site, particularly concerning the N-terminal frag-
ment. The closest literature examples to be compared
with are represented by phosphinic tripeptide analogues,
extended by an additional L-amino acid residue on the
45.2
45.0
44.8
44.6
44.4
44.2
δ [ppm]
Figure 2. 31P NMR spectra of the stereoisomeric mixture of
hPhew[PO2HCH2]Phe (1) (bottom trace) and separated individual
isomers with the absolute configuration defined (four upper traces).
Conditions of the experiments: 10-fold excess of a-cyclodextrin, D2O/
NaOD, pD 12.
C-terminus.
Gly-Trp-NH2 was described as very effective inhibitor
Cbz-Phew[PO2HCH2]-(3-phenylpropyl)
of stromelysin 3,4 while Phew[PO2HCH2]Leu-Phe

A. Mucha et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1550–1554
1553
O
P
lute configuration in the P1 position was less significant,
however, as generally the R (L) was slightly more pre-
ferred. We believe the recent correlation study on the
dipeptide analogue represents an interesting contribu-
tion to chemistry and biological activity relationship of
phosphinic pseudopeptides.
H
H
OH
N
Cbz
2
a
a
O
P
O
P
H
N
H
N
H
OH
H
OH
Cbz
Cbz
Acknowledgment
This work was supported by Komitet Badan
Naukowych.
S
-2
R
-2
b-f
b-f
References and notes
O
P
OH
O
P
OH
*
*
OH
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RR-1 (σ 44.48 ppm)
RS-1 (σ 44.90 ppm)
SR-1 (σ 45.08 ppm)
SS-1 (σ 44.37 ppm)
Scheme 2. Synthesis and separation of diastereoisomeric pairs with
fixed configuration on the N-terminal asymmetric carbon atom.
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the commercial CHIRALPAK QD-AX (15 lm) (250 · 16 mm id)
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mobile phase methanol/50 mM phosphate buffer (80:20; v/v), pH 5.6,
flow rate 4.5 mL/min, elution order: R-2 < S-2; (b) hexamethyldisilaz-
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Table 1. Inhibition of leucine aminopeptidase by individual stereoiso-
mers of hPhew[PO2HCH2]Phe phosphinic pseudodipeptide (for the
experimental details of the assay see Ref. 14)
Stereoisomer
of the hPhew[PO2HCH2]
Phe inhibitor
31P NMR: d
Ki [nM]
[D2O, ppm]a
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R,S-1 (L,L)
S,R-1 (D,D)
S,S-1 (D,L)
44.48
44.90
45.08
44.37
271
45
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8800
988
a In the presence of 10-fold excess of a-cyclodextrin.
appeared to be a dual neutral endopeptidase (neprily-
sin)/aminopeptidase N inhibitor.25 In the first case, the
potency of its three out of four stereoisomers is practi-
cally indistinguishable, with Ki varying in the range 5–
9 nM. Flexible phenylpropyl side chain in the P10 and
L-tryptophan in the P20 position seem responsible for
such privileged binding. Only the S,R,S (D,D,L) form is
significantly discriminated (Ki = 50 nM) and thus less
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factor. Diastereoisomers bearing the L leucine isoster
were found significantly more efficient than those with
D arrangement (roughly two orders of magnitude) in
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