Phosphinotripeptidic inhibitors of leucylaminopeptidases

Article abstract of DOI:10.3390/ijms22105090

Phosphinate pseudopeptide are analogs of peptides containing phosphinate moiety in a place of the amide bond. Due to this, the organophosphorus fragment resembles the tetrahedral transition state of the amide bond hydrolysis. Additionally, it is also capable of coordinating metal ions, for example, zinc or magnesium ions. These two properties of phosphinate pseudopeptides make them an ideal candidate for metal-related protease inhibitors. This research investigates the influence of additional residue in the P2 position on the inhibitory properties of phosphinopeptides. The synthetic strategy is proposed, based on retrosynthetic analysis. The N-C-P bond formation in the desired compounds is conveniently available from the three-component condensation of appropriate amino components, aldehydes, and hypophosphorous acid. One of the crucial synthetic steps is the careful selection of the protecting groups for all the functionals. Determination of the inhibitor activity of the obtained compounds has been done using UV-Vis spectroscopy and standard substrate L-Leu-p-nitroanilide toward the enzymes isolated from the porcine kidney (SsLAP, Sus scrofa Leucine aminopeptidase) and barley seeds (HvLAP, Hordeum vulgare Leucine aminopeptidase). An efficient procedure for the preparation of phosphinotripeptides has been performed. Activity test shown that introduction of additional residue into P2 position obtains the micromolar range inhibitors of SsLAP and HvLAP. Moreover, careful selection of the residue in the P2 position should improve its selectivity toward mammalian and plant leucyl aminopeptidases.

Full text of DOI:10.3390/ijms22105090

International Journal of
Molecular Sciences
Article
Phosphinotripeptidic Inhibitors of Leucylaminopeptidases
Michał Jewgin´ski 1,
*
, Kinga Haremza 1, Jesús M. de los Santos 2 , Zouhair Es Sbai 2, Bartosz Oszywa 3,
Małgorzata Pawełczak 3 , Francisco Palacios 2 and Rafał Latajka 1
1
˙
Department of Bioorganic Chemistry, Wrocław University of Science and Technology, Wybrzeze
Wyspian´skiego 27, 50-370 Wrocław, Poland; kinga.haremza@gmail.com (K.H.); rafal.latajka@pwr.edu.pl (R.L.)
Department of Organic Chemistry I, Faculty of Pharmacy, Lascaray Research Center, University of the Basque
Country (UPV/EHU), Paseo de la Universidad, 7, 01006 Vitoria, Spain; jesus.delossantos@ehu.es (J.M.d.l.S.);
zouhair.essbai@ehu.eus (Z.E.S.); francisco.palacios@ehu.es (F.P.)
2
3
Institute of Chemistry, University of Opole, Oleska 48, 45-052 Opole, Poland; bartoszoszywa@interia.pl (B.O.);
pamal@uni.opole.pl (M.P.)
*
Correspondence: michal.jewginski@pwr.edu.pl; Tel.: +48-71-320-24-61
Abstract: Phosphinate pseudopeptide are analogs of peptides containing phosphinate moiety in a
place of the amide bond. Due to this, the organophosphorus fragment resembles the tetrahedral
transition state of the amide bond hydrolysis. Additionally, it is also capable of coordinating metal
ions, for example, zinc or magnesium ions. These two properties of phosphinate pseudopeptides
make them an ideal candidate for metal-related protease inhibitors. This research investigates the
influence of additional residue in the P2 position on the inhibitory properties of phosphinopeptides.
The synthetic strategy is proposed, based on retrosynthetic analysis. The N-C-P bond formation in the
desired compounds is conveniently available from the three-component condensation of appropriate
amino components, aldehydes, and hypophosphorous acid. One of the crucial synthetic steps is
the careful selection of the protecting groups for all the functionals. Determination of the inhibitor
activity of the obtained compounds has been done using UV-Vis spectroscopy and standard substrate
L-Leu-p-nitroanilide toward the enzymes isolated from the porcine kidney (SsLAP, Sus scrofa Leucine
aminopeptidase) and barley seeds (HvLAP, Hordeum vulgare Leucine aminopeptidase). An efficient
procedure for the preparation of phosphinotripeptides has been performed. Activity test shown
that introduction of additional residue into P2 position obtains the micromolar range inhibitors of
SsLAP and HvLAP. Moreover, careful selection of the residue in the P2 position should improve its
selectivity toward mammalian and plant leucyl aminopeptidases.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Jewgin´ski, M.; Haremza, K.;
de los Santos, J.M.; Es Sbai, Z.;
Oszywa, B.; Pawełczak, M.; Palacios,
F.; Latajka, R. Phosphinotripeptidic
Inhibitors of Leucylaminopeptidases.
Int. J. Mol. Sci. 2021, 22, 5090.
https://doi.org/10.3390/ijms22105090
Academic Editor: Atsushi Matsuzawa
Keywords: molecular modeling; LAP inhibitors; barley aminopeptidase inhibitor; phosphinate
pseudopeptide; ligand-enzyme interaction; organophosphorus compound
Received: 13 April 2021
Accepted: 6 May 2021
Published: 11 May 2021
1. Introduction
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
The tetrahedral geometry of the phosphinate group introduced into the peptide chain
makes these analogs one of the most potent, reversible, and competitive inhibitors of
metalloproteases [1–3]. This phosphinate moiety mimics the structural and electronic
features of the tetrahedral transition state intermediate occurring during the enzymatic
hydrolysis of the amide bond. Additionally, this group is also capable of coordinating the
metal ion in the active site, which could also block the catalytic activity.
Leucyl aminopeptidases (E.C. 3.4.11.1) are the enzymes from the metallo-dependent
hydrolase family, responsible for the cleavage of N-terminal amino acids from the polypep-
tide substrates—due to this, they play an important role in the physiological processing
and degradation of protein and peptide. In mammals, the activity of LAP is involved in
the physiological metabolism of regulatory and bioactive peptides, as well as in antigen
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
presentation [4,5]. The disturbed activity of LAP has been connected with various patho-
logical disorders. The therapeutic application of the enzyme is connected with its role
during metastasis and tumorigenesis [4]. It has also been shown the LAP is a key enzyme
Int. J. Mol. Sci. 2021, 22, 5090. https://doi.org/10.3390/ijms22105090
https://www.mdpi.com/journal/ijms

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in the process of glutathione turnover in the liver [
6]. This enzyme is found in human
pathogens, e.g., Staphylococcus aureus or Plasmodium falciparum, and is required for virulence
or digestive proteolysis and nutrition delivery [ ]. The elevated level of LAP has also been
connected with certain pathological stress states, including inflammation, cataracts, cancer,
or HIV infection [ 12]. Potential therapeutic significance, resulting from the biological role
7
8–
of LAP, constantly stimulates research in the area of the methods of regulating its activity.
Although bacterial, parasitic, and animal aminopeptidase are well investigated, their
plant counterparts still required further research. The determination of their substrate
specificity, as well as the design of selective inhibitors, requires continuous studies. The
presence of aminopeptidases has been observed in plant parts characterized by intensive
processes of synthesis, conversion, or degradation of the protein [5,13–17].
Within this publication, the application of the phosphinic analogs of tripeptide (see
Figure 1) as inhibitors of leucine aminopeptidase and plant aminopeptidase will be dis-
cussed. It is known that the selectivity of such kinds of molecules is primarily dependent
on the structural feature of residues in positions Pn and Pn’ (see Figure 1). The structure
and character of these residues are crucial for the matching process of the inhibitor to the
enzyme binding pockets. The optimization of the character of residues in positions P1 and
P1’ in phosphinic dipeptides obtain the first most potent unnatural inhibitors of LAP [18].
The further investigation, focused on modification of side chains in the positions P1 and
P10, lead to numerous phosphinate and phosphinic dipeptidic inhibitors of LAP [19–24].
Figure 1. Schematic structure of phosphinic peptide with marked position P2-P2’.
Because both porcine LAP and barley aminopeptidase belong to exopeptidases, which
relieve N-terminal residue, structural modification of inhibitors was usually limited to
residues in positions P1 and/or P10. Grembecka et al. showed that the application of
the molecular modeling allowed to optimize the character of the residues side chains in
positions P1 and P10 in results giving the most potent inhibitors of LAP: hPheP[CH2]Phe
and hPheP[CH2]Tyr. However, within this study, we have investigated the influence
of residues in the P2 on inhibitory activity. It would be worth checking whether the
introduction of additional residue in the P2 position keeps at least moderate inhibitor
activity and changes the selectivity toward mammalian and plant aminopeptidase.
2. Results and Discussions
2.1. Chemistry
The synthetic targets of this work were phosphinic pseudotripeptide analogs 1–9 of
the structure, shown in Figure 2. The designed compounds possess several functional
groups of various characters, and thus, their synthesis is not trivial. To confront this
challenge, a retrosynthetic analysis has been performed for these phosphinopeptides
inhibitors (Scheme 1). The N-C-P bond formation in α-amino-H-phosphinic substrates 10
is conveniently available from the three-component condensation of appropriate amino
components, aldehydes, and hypophosphorous acid (Scheme 1, pathway c). With the
suitable protected phosphinic amino acid analogs 10 in hand, the Michael addition of these
phosphorus nucleophiles to appropriate carbon electrophiles (typically acrylic esters) is the
most commonly applied method for the phosphinate pseudopeptidic bond formation (P-C
bond formation) (Scheme 1, pathway b). The availability of the corresponding C-terminal

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ester 11 would allow multidirectional diversification of the P1 position at the final step
of the synthesis through coupling reaction at the N-terminus with different N-protected
α-amino acids (C-N bond formation) (pathway a, Scheme 1).
Figure 2. Putative phosphinic pseudotripeptides derived from leucine and homophenylalanine
containing modified P2 substituents as inhibitors of leucylaminopeptidase.

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OPG2
OH
H
O
H
O
Multicomponent
Kabachnik-Fields reaction
H
N
P
O
N
P
R
PG1
PG1
Coupling
reaction
Phospha-Michael
O
addition
R
11
a
b
Ar, R
H2N
10
OH
P
O
H
C-N
disconnection
P-C
disconnection
+
N
O
O
+
Ar, R
O
O
R
O
BocHN
CO2H
O
+
OH
O
c
NH2
+
P
PG
H
H
N-C-P
disconnection
R
Scheme 1. Retrosynthetic analysis of phosphinic pseudotripeptides derived from leucine and homophenylalanine.
Scheme 2 outlined the synthesis of the designed synthons 16 and 17 to prepare the
target phosphinic pseudotripeptides . Careful choice of the protecting groups for
1–9
three functions present in the molecules of phosphinopeptides precursors is crucial, since
their selective removal at the desired synthetic step is an absolute requirement. Different
approaches to prepare P-H substrates have been reported in the literature. Early work
by Schmidt [25] demonstrated the addition of hypophosphorous acid to imines to give
N-substituted
phorous acid, and an amine as the nitrogen source can also be used to prepare these
derivatives [ ]. In our case, we began with the use of the three-component reaction. Even
α-aminoalkylphosphinic acids. The condensation of an aldehyde, hypophos-
2
though the preparative yields are not high, the main advantages of this approach are
low cost and simplicity. Thus, the starting aminophosphinic acid analogs derived from
homophenylalanine 12 (R = Bn) and leucine 13 (R = iPr) (Scheme 2), were synthesized in
the reaction of an appropriate aldehyde (RCH2CHO), diphenylmethylamine hydrochloride
(Ph2CNH2
component Kabachnik-Fields reaction. The diphenylmethyl group can be hydrolyzed from
adducts under acidic conditions to obtain free -aminophosphoric acids. The free amino
group in the former -aminophosphoric acids can subsequently be protected by the benzy-
·
HCl), and hypophosphorous acid (H2P(O)OH), using a variant of the multi-
α
α
loxycarbonyl group (Cbz) in a water solution [26] to give the starting aminophosphinic
acid analogs 12 and 13 (Scheme 2). Phosphinic pseudodipeptides 14 and 15, containing a
homophenylalanyl or leucyl aminophosphinic acid, respectively, were synthesized using
an approach based on a phospha-Michael addition, which relies on a well-established
procedure developed previously [27,28]. Hence, N-benzyloxycarbonylaminophosphinic
acids 12 and 13 were activated to the more nucleophilic tervalent ester form by heating
with hexamethyldisilazane, followed by adding methyl acrylate as the Michael acceptor.
After work-up and purification, the protected pseudopeptides 14 and 15 were obtained
in moderate yields (Scheme 2). After a thorough overview of the literature, numerous
contradictory reports concerning the protection of the hydroxyphosphinyl moiety have
been found. We envisaged that proper protection of the hydroxyphosphinyl moiety is
essential for the efficient preparation of phosphinopeptides. Some researchers claim that
protection of the hydroxyphosphinyl function is not necessary during peptide coupling
reactions, either on the solid-phase [29] or in solution [30
size the serious limitations of coupling methods when phosphinopeptides are not properly
protected at the phosphorus center [33 35]. Such last evidence is strongly supported by
–32], while others strongly empha-
–
this work, since all attempts to use deprotection products coming from 14 and 15 in the
peptide coupling reaction were unsuccessful. This also proves the potential reactivity of
the phosphinic acid during the decoupling reaction. The adamantyl group was chosen
as a protecting group [
1,20,28,36,37], since it can be removed easily under the classical
deprotection conditions required for Boc-protecting amino acids coupled in the last step of

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the synthesis. The introduction of the adamantyl group was easily achieved by refluxing
phosphinic acids 14 and 15 with adamantly bromide (1-AdBr) and silver oxide in chlo-
roform. Fully protected pseudodipeptidic blocks derived from homophenylalanine 16
(R = Bn) and leucine 17 (R = iPr) are obtained in excellent yields (Scheme 2) and used as
building blocks for incorporation into peptides in solution.
O
P
O
CbzHN
CbzHN
P
O
a,b
H
OH
R
OH
R
O
12: R = Bn
13: R = iPr
14: R = Bn
15: R = iPr
O
CbzHN
P
O
c
OAd
R
O
16: R = Bn
17: R = iPr
Scheme 2. Synthesis of the key building blocks 16 and 17 bearing the side chains of pseudo-HPhe
and -Leu, respectively, and required for preparing the target phosphinopeptides
conditions: (a) HMDS, 110 C; (b) H2C=CHCO2Me, 90 C, 66–74%; (c) 1-AdBr, Ag2O, CHCl3, 65 C,
87–90%.
1
–
9
. Reagents and
◦
◦
◦
Phosphinic pseudodipeptides blocks 16 and 17 were subjected to coupling reaction
at the N-terminus with different N-protected -amino acids, leading to various P2 di-
L
versified phosphinic tripeptides 1–9. Prior to to the peptide coupling reaction, the ben-
zyloxycarbonyl group might be removed by hydrogenolysis or by the action of HBr in
AcOH. When deblocking the Cbz group of 16 and 17 using the method described by
Anwer and Spatola [38], which utilizes ammonium formate as a hydrogen donor, in the
presence of palladium/carbon catalyst, we found the formation of the pseudodiketopiper-
azines 18 and 19 as the main product of the reaction (Scheme 3), similarly as described
in the literature [28]. Satisfactory results for the decarbobenzyloxylation of compounds
16 and 17 were obtained by means of catalytic hydrogenation using H2 in the presence
of Pd/C catalyst. The N-terminus elongation approach was adopted to prepare the fi-
nal phosphinic pseudotripeptides, and in all instances, coupling reactions were achieved
with N-Boc-methionine (Boc-L-Met-OH) or TBDMS-protected N-Boc-homoserine (Boc-L-
Hse(TBDMS)-OH—was prepared, starting from -Hse-OH through protection first the
L
hydroxyl group as OTBDMS [39] followed by protection of the N-terminus as N-Boc pro-
tecting group [40]) mediated by peptide coupling agents EDC-HOBT in DMF [41,42]. This
procedure appeared to be a simple and satisfactory method for the N-terminus elongation
of the individual phosphinic dipeptides in solution since, after standard work-up and
purification on flash-column chromatography, tripeptide analogs 20
satisfactory yields (51–70%). Finally, deprotection of both N-terminus and hydroxyphos-
phinyl [43] groups in 20 21 under acidic conditions enabled us to obtain the desired fully
deprotected phosphinotripeptides . Formation of compound entails O-TBDMS depro-
–22 were obtained in
–
1
–2
3
tection of pseudotripeptide 22 by using standard conditions [44], previously to the removal
of the N-Boc and P-Ad protecting groups as before (Scheme 3).

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Scheme 3. Synthesis of phosphinic pseudotripeptide analogs 1–3 and their free acids 4 and 5. Reagents and conditions:
(a) HCO2NH4/Pd-C, MeOH, reflux or rt; (b) H2/Pd-C, MeOH; (c) Boc-L-Met-OH (R1 = CH2SMe) or Boc-L-Hse(TBDMS)-
◦
OH (R1 = CH2OTBDMS), HOBt, EDC, TEA, DMF, 51–70%; (d) TBAF, THF, 0 C to rt, 70%; (e) TFA/CH2Cl2 (1:1), 85–99%;
(f) NaOH/MeOH (0.4 M) them 0.5 M HCl, pH 1, 98–99%.
Phosphinotripeptides methyl esters 20 and 21 were subjected to hydrolysis by the
use of NaOH in MeOH followed by careful adjustment of pH by dilute hydrochloric acid.
The saponification of the methyl ester 20 and 21, in spite of the presence of the adamantyl
ester, is completely selective and allows for preparing phosphinotripeptides 24 and 25
,
respectively [28]. Consecutive removal of N-Boc and P-adamantyl protecting groups in
acidic conditions gave the target compounds as free acids
practically quantitatively (with yields exceeding 98%).
4
and
5
. Both steps proceeded
were synthesized
When a similar methodology was used, tripeptide analogs 6–9
(Scheme 4). Debenzylation of the of phosphinic pseudodipeptides blocks 16 and 17 at the
N-terminus, followed by the assembly with the suitable N-Boc protected derivative of
0
Bip (N-Boc-L-Bip-OH, N-Boc-L-4,4 -biphenylalanine) and Bpa (N-Boc-L-Bpa-OH, N-Boc-p-
benzoyl-L-phenylalanine) by using peptide coupling agents EDC-HOBT in DMF [41
,42],
afforded pseudotripeptide derivatives 26 29 in satisfactory yields (50–76%). Removal
–
of the N-Boc and P-adamantyl protecting groups was performed in acidic conditions
(TFA/CH2Cl2, 1:1), providing inhibitors 6–9.

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Scheme 4. Synthesis of phosphinic pseudotripeptide analogs 6–9. Reagents and conditions: (a) H2/Pd-C, MeOH;
(b) Boc-L-Bip-OH (R1 = Ph) or Boc-L-Bpa-OH (R1 = COPh), HOBt, EDC, TEA, DMF, 50–76%; (c) TFA/CH2Cl2 (1:1), 78–99%.
2.2. Enzyme Inhibition
The obtained phosphinopeptides were tested as inhibitors of mammalian leucine
aminopeptidase and plant aminopeptidase. As the model of mammalian enzyme, the
porcine leucine aminopeptidase (SsLAP) has been selected, while as the plant enzyme
model, the barley aminopeptidase has been chosen. LAP (leucine aminopeptidase) is
the multifunctional broad-band specific zinc-dependent aminopeptidase. The structure,
mechanism of the molecular action, as well as therapeutic significance of these enzymes,
are well known [5,45]. Barley aminopeptidase HvLAP is an example of plant aminopep-
tidase, catalyzing the removal of the N-terminal amino acids. As shown in our previous
studies, HvLAP preferably removes hydrophobic residue in the P1 position [46]. How-
ever, Kawaguchi and Doi showed that HvLAP has also got endopeptidase properties [47].
HvLAP is the metalloprotease which activity is modulated by several metal ions. As shown
in our previous studies, HvLAP activity is increased by Ca2+, Mg2+, and Mn2+ ions, and is
decreased by Co2+, Cd2+, Zn2+, and Fe2+ ions.
The inhibition type, mechanism, and inhibition constants (Ki) were determined using
graphical methods (see Figures 3 and 4 and Tables S1 and S2). The inhibition type was
determined by the Lineweaver-Burk plot based on the results of the inhibitory effect of the
compounds on the hydrolytic activity of L-Leu-p-nitroanilide. The synthesized molecules,
as it is shown in Table 1, exhibited moderate micromolar inhibitory activity toward SsLAP
and HvLAP and appeared to be competitive inhibitors. It should be remembered that
inhibitor activity was appointed for the racemic mixtures. All investigated compound
1
–
9
contains only S isomer in the P2 position, whereas in the P1 position, both R and S
isomers were possible. Comparing the obtained activities of the investigated compounds
toward mammalian and plant aminopeptidases allows us to make some general
1–9

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observations. First of all, although there are no published results that suggested to not
elongate the phosphonic inhibitors chain into the N-terminus site, the obtained compounds
have presented the biological activity. Additionally, SsLAP has better tolerance for the
residue in the P1 position, whereas HvLAP prefers hPhe residue in this position. The
compounds containing hPhe residue have activity at least two times higher than analogs
that possess Leu residue. It is in good agreement with the previous research, which showed
that hPhePO3H2 is more than two times more active than LeuPO2H3 [48].
Figure 3. The designation of type inhibition and the inhibition constant KI by Dixon method for compound 3 in the presence
of SsLAP.
Figure 4. The designation of type inhibition and the inhibition constant KI by Dixon method for compound 1 in the presence
of HvLAP.

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Table 1. The activity of pseudotripeptides as inhibitors of porcine LAP and barley LAP.
KI [µM]
Entry
Structure
SsLAP
HvLAP
1
Met-hPheP[CH2]Gly-OMe
532 ± 46
38 ± 9
2
3
Met-LeuP[CH2]Gly-OMe
Ser-LeuP[CH2]Gly-OMe
1090 ± 73
649 ± 43
121 ± 19
14 ± 1
4
5
Met-hPheP[CH2]Gly
Met-LeuP[CH2]Gly
229 ± 40
228 ± 10
S
N
O
H
N
P
OH
3618 ± 433
1035 ± 102
H2
OH
O
O
6
Bip-hPheP[CH2]Gly-OMe
175 ± 3
878 ± 70
7
Bip-LeuP[CH2]Gly-OMe
130 ± 14
1303 ± 84
8
9
Bpa-hPheP[CH2]Gly-OMe
95 ± 9
159 ± 25
Bpa-LeuP[CH2]Gly-OMe
507 ± 31
797 ± 76

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The best biological activity has been found for compound
compound toward HvLAP. It should be noticed that
of HvLAP than . What is interesting, compound has got relatively high activity despite
3
toward SsLAP and for
1
3 is only 2.5 times weaker inhibitor
1
3
that it contains Leu residue at the P1 position and not preferable hPhe. It can be suggested
that the hydrophilic side chain of Ser residue in the P2 position is responsible for some
additional intermolecular interactions, which improve the inhibitor activity. Additionally,
a comparison of the inhibitory activity of compound
1 and phosphonic acid analog of
hPhe toward barley aminopeptidase [48] shown that introduction of additional residues in
position P10 and P2 to a phosphonic acid analog of hPhe decreases the activity less than
two times. Additionally, sequence
1 provides greater selectivity of HvLAP inhibition over
SsLAP compared to the phosphine analog of homophenylleucine.
The introduction of the bulky residues, like biphenylalanine (Bip) or p-benzoyl
L-phenylalanine (Bpa) in the position P2 instead of L-Met or L-Ser, have been showing that
mammalian aminopeptidase has a much higher tolerance for the bigger side chain in this
position than plant aminopeptidase. What is also interesting, it seems that these bulky
residues improve the inhibitor activity towards SsLAP in comparison to the activity of
inhibitors with L-Met in the P2 position. Comparing the activity of this group of inhibitors
allows selecting compound
the most selective is inhibitor
8
as the most potent inhibitor toward both enzymes. Whereas,
, which inhibits SsLAP almost 10 times stronger than HvLAP.
7
This result is in full agreement with the structural preferences of HvLAP, which stronger
interacts with the inhibitors containing hPhe than Leu in the P1 position [48].
2.3. Molecular Docking
Rational design and development of biologically active compounds require under-
standing the nature of inhibitor-enzyme interactions. Simulation of the docking process is
one of the methods which might contribute to understanding the physical nature of those
interactions, and thus, to understand the binding mode of the investigated compounds to
the structure of selected aminopeptidases. Because there are no known crystal structures
of porcine LAP and barley LAP, for simulation of docking process, the bovine lens LAP
(BtLAP, Bos Taurus Lecine aminopeptidase) and tomato AP (SlLAP, Solanum lycopersicum
Leucine aminopeptidase) have been selected, respectively, as a model of mammalian and
plant enzymes. Taylor et al. showed that bovine LAP and porcine LAP share over 90%
amino acid sequence homology [49
crystal structure of bovine lens LAP can be applicable to the design of new inhibitors of
LAP from other organism tissues [ ]. Furthermore, Wanat et al., as well as Janiszewska
et al. showed that the known structure of tomato LAP is a good model of the barley seeds
LAP [22 24]. All the above-mentioned observations make the use of enzymes from two
different organisms for biological testing and molecular modeling highly justified. Because
for the biological test, racemic mixture SR and SS isomers of have been used, also
,50]. Moreover, Grembecka et al. showed that the
4
,
1–9
during the simulations of the docking process, both types of isomers have been considered.
Optimized structures of the investigated phosphinopeptides were obtained by using the
Gaussian09 software at the B3LYP/6-311g(d,p) level with the PCM solvent model and the
water selected as a solvent. Subsequently, obtained structures were docked to the active
pocket of bovine lens leucyl aminopeptidase and tomato aminopeptidase available from
Protein Data Bank (1LCP, 4KSI [51,52]), by using the Gold v2020.1 algorithm [53]. The
structure of the enzymes was protonated at experimental pH using an H++ web server [54].
As the results of the docking simulation, for each inhibitor, three possible orientations of
the docked molecule were obtained.
To validate chosen docking methodology redocking of LeuPO3H2 to the BtLAP has
been performed. As shown with the obtained results, the used methodology led to the
arrangement of the inhibitor in the enzyme active site similar to those from the X-ray
structure (PDB: 1LCP) (see Figure S26). Applied molecular docking protocol allows to
obtain the complexes of all investigated inhibitors 1–9 with both considered enzymes, and

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the best alignment of all investigated inhibitors in the active site of BtLAP and SlLAP are
shown on the figures in the Supporting Information (see Figures S27–S30 and Table S3).
To understand the influence of the P1 residue onto inhibitory activity toward mam-
malian leucyl aminopeptidase and plant aminopeptidase, the alignment of inhibitors
1
and in the active sites of BtLAP and SlLAP have been compared. The importance of
2
the inhibitory P1 residue is due to the interaction of its side chain with a cavity on the
enzyme surface (called S1 pocket) located adjacent to the catalytic residues. As it is shown
in Figure 5 in the case of the plant’s enzyme, if the designed inhibitor contains in the P1
position hPhe residue, the S1 pocket of SlLAP is completely occupied by its side chain.
Whereas, if in the P1 position of the inhibitor is located Leu residue, the S1 pocket is not
fulfilled completely, and bonded molecule still has some flexibility, which could result in
lower affinity. In the case of alignment of
1 and 2 in the active site of BtLAP, no significant
difference in the fulfilling of the S1 binding pocket could be observed. Such observation let
to explains the higher tolerance of mammalian leucyl aminopeptidase for variation of the
size of the side chain in the P1 position.
Figure 5. Alignment of (
-SR isomer in the active site of BtLAP (PDB: 1LCP). Magnesium and zinc ions are shown, respectively, as the green and
gray spheres. The surface of the active site is colored according to hydrophobicity.
a) 1-SR isomer and (b) 2-SR isomer in the active site of SlLAP (PDB: 4KSI), and (c) 1-SR isomer and
(d) 2
The results of the molecular modeling help to understand the phenomena of relatively
high activity of toward HvLAP. As it has already been noticed, the biological activity of
the investigated compounds , strongly depend on the type of the amino acid residue in
position P1, the higher affinity has been found for compounds containing aromatic residue
in this position— . However, inhibitor 3, although it contains in this critical position
leucine residue, inhibits the HvLAP only three times weaker than . We believe that this
3
1–9
1, 4, 6, 8
1
activity is the results of the introduction of L-Ser into position P2. Presence of the additional
hydroxylic group in the serine side chain is probably responsible for the generation of
additional intermolecular hydrogen bond, which could increase intermolecular interaction
and stabilize the alignment of the inhibitor in enzyme active site. In fact, performed
docking simulation, showed the presence of the additional hydrogen bond for the complex
of
in Figure 6, except for hydrogen bonds between: Side chain amine group of Lys354 and
phosphine group of ; guanidine moiety of Arg431 and C-terminal carboxylic group of
and interaction between phosphinic moiety and magnesium ions, which could also be
3 with plant aminopeptidase SlLAP comparison to complex of 2-SlLAP. As it is shown
3
3
found in SlLAP-2 complex, the arrangement of 3 in the SlLAP active site is stabilized by
one additional hydrogen bond between a hydroxylic group of serine and carboxylic group
of Ser359 or carboxylic group of Arg358 for SR-isomer and SS-isomer, respectively.

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Figure 6. Alignment of (a) 2-SR isomer, (b) 2-SS isomer, (c) 3-SR isomer, (d) 3-SS isomer in the active site of SlAP (PDB:
4KSI). Magnesium ions are shown as a green sphere. Green lines represent intermolecular hydrogen bonds; blue lines
represent ligand–metal interactions. The surface of the active site is colored according to hydrophobicity.
Molecular modeling of the most active phosphinic compound
3 with di-zinc mam-
malian leucine aminopeptidase BtLAP revealed a typical mode of binding. The phosphinate
group is involved in coordination with the catalytic metal ions (see Figure 7). C-terminal
carboxylate is involved in an intermolecular hydrogen bond with the guanidine moiety
of Arg431. The position of
3 is further stabilized by a salt bridge between the N-terminal
amine group of and the side chain carboxylic moiety of Asp273, as well as hydrogen
3
bond between the side chain hydroxyl group of serine residue of
of Ala451.
3 and carboxylic group
Figure 7. Modeled binding mode of 3-SS isomer in the active center of bovine leucyl aminopeptidase (PDB: 1LCP). Amino
acid residues of the inhibitor and enzyme are shown as sticks, while the zinc ions are shown as a gray sphere. Hydrogen
bonds are shown as thin green lines, metal ion—phosphinic moiety interaction is shown as a thin blue line, while the surface
of the active site is colored according to hydrophobicity.
In the case of inhibitors with aliphatic amino acids in position P2 (1–5), the typical
binding of inhibitors in the active site of the enzyme was observed—coordination of
catalytic metal ions by phosphinate and binding of the C-terminus of the inhibitor by
hydrogen bonding to the guanidine moiety of Arg431 for SlLAP or Arg336 for BtLAP
and interaction with the enzyme’s S1 cavity. For inhibitors 6–9, containing large aromatic
substituents in the P2 position, the situation is a bit more complicated. The conduced
molecular modeling for compounds containing both types of isomer in position P1 show
the different interaction of these inhibitors with the enzymatic active site, depending on
the stereochemistry of P1 residues. This situation is presented in detail for
7 and BtLAP
enzyme. The best pose found for the -SS isomer showed almost typical binding mode for
7
di-zinc leucine aminopeptidase (see Figure 8b), which means that zinc ions are coordinated
by phosphinate. One of the oxygen atoms of the phosphinate moiety is involved in ionic
+
interaction with NH3 of Lys250 (2.45 Å). However, due to the presence of bulky biphenyl

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side chain at the P2 position, the inhibitor is shifted in the active site into S10 direction. It
results in the interaction of Leu residue from the P1 position with the S10 binding pocket.
The N-terminal amine group forms a salt bridge with Asp273 (2.42 Å) and a hydrogen bond
with CO of Thr359 (2.43 Å). Moreover, the position of
7-SS in the active pocket is stabilized
by the interaction of -electrons of biphenyl moiety with the aliphatic and sulfuric groups
π
of Ala451 and Met454, respectively.
Figure 8. Modeled binding mode of (a) 7-SR isomer and (b) 7-SS isomer in the active center of
bovine leucyl aminopeptidase (PDB: 1LCP). Amino acid residues of the inhibitor and enzyme are
shown as sticks, while the zinc ions are shown as a gray sphere. Hydrogen bonds are shown as
thin green lines, metal ion—coordinating moieties interaction shown as a thin blue line, aromatic
π-electrons—aliphatic group, and sulfur atom shown as dashed lines. The surface of the active site is
colored according to hydrophobicity.
While the
7-SS isomer shows an almost canonical binding to the BtLAP active site,
the
7-SR isomer shows an unusual way of binding (see Figure 8a). The most important
difference is the coordination of the zinc ion by the amide CO of Bip residue instead of
phosphinate. The orientation of this group is additionally stabilized by the hydrogen
+
bonding to
ε
NH3 of Lys262 (2.60 Å). Moreover, in this case, the inhibitor molecule is
shifted into S10 direction, and leucine residue from the P1 position interacts with the
enzyme’s binding pocket S10. Inhibitor alignment in the active site is further stabilized by
the series of π-alkyl interactions involving the biphenyl moiety of Bip and aliphatic side
chains of Met270, Ala451, and Met454.
3. Materials and Methods
3.1. Chemistry
General information: Et3N was distilled and then dried over molecular sieves 70 Å.
Hydrocynamaldehyde, isovaleraldehyde, and methyl acrylate were freshly distilled before
use. EtOH, MeOH, CH2Cl2, CHCl3, THF, and DMF were freshly distilled and dried over
molecular sieves 70 Å. All other solvents and reagents were obtained from commercial
sources (Sigma-Aldrich, Madrid, Spain) and used without further purification. All reactions
were performed under an atmosphere of dry nitrogen. Melting points were obtained using
a Büchi Melting Point B-540 apparatus and are uncorrected. High-resolution mass spectra
(HRMS) were measured on a Agilent 6530 Accurate-Mass QTOF LC/MS (Santa Clara, CA,
USA) by the CI method using a mass spectrometer Q-TOF. 1H (400 MHz), 13C (100 MHz),
and 31P NMR (160 MHz) spectra were recorded on a Bruker Avance 400 (Bruker BioSpin
GmbH, Rheinstetten, Germany) spectrometer (see Figures S1–S25), respectively, in CDCl3,
DMSO-d6, CD3OD, or D2O. Chemical shifts (δH) are reported in parts per million (ppm).

Int. J. Mol. Sci. 2021, 22, 5090
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All coupling constants (J) values are given in Hz. Chemical shifts (δC) are reported in parts
per million (ppm) in a broadband decoupled mode. Flash-column chromatography was
performed using commercial grades of silica gel finer than 230 mesh. Analytical thin layer
chromatography was performed on precoated Merck silica gel 60 F254 plates, and spot
visualization was accomplished by UV light (254 nm) or KMnO4 solution.
3.1.1. [1-(N-Benzyloxycarbonylamino)-3-phenylpropyl]phosphinic Acid (12)
The phosphinic analog of homophenylalanine was synthesized in the reaction of
hydrocynnamaldehyde with diphenylmethylamine and hypophosphorous acid, followed
by hydrolysis of the adduct and N-terminal protection with benzyl chloroformate, similarly
as described in the literature [
2
,
55]. Global yield from three steps: 43%; mp 137–141 ◦C
◦
1
2
(lit. mp 142–143 C [55]); H NMR (400 MHz, DMSO-d6)
δ
7.72 (d, JPH = 8.8 Hz, 1H,
OH), 7.39–7.16 (m, 10H), 6.77 (d, 1JPH = 531.6 Hz, 1H, PH), 5.07 (s, 2H, PhCH2O), 3.56–3.51
(m, 1H, CH-P), 2.76–2.53 (m, 2H, CH2CH2Ph), 1.92–1.77 (m, 2H, CH2CH2Ph); 13C NMR
(
100 MHz, DMSO-d6)
δ
156.2 (d, 3JPC = 3.2 Hz), 141.1, 137.0, 128.4, 128.4, 128.3, 127.9, 127.7,
125.9, 65.6, 49.9 (d, 1JPC = 105.1 Hz), 31.4 (d, 2JPC = 12.4 Hz), 28.3 (d, 3JPC = 3.9 Hz); 31
P
NMR (160 MHz, DMSO-d6) δ
28.4; ESI-HRMS m/z calcd. for C17H20NNaO4P ([M+Na]+)
356.1028, found 356.1030.
3.1.2. [1-(N-Benzyloxycarbonylamino)-3-methylbutyl]phosphinic Acid (13)
The phosphinic analog of leucine was synthesized in the reaction of isovaleraldehyde
with diphenylmethylamine and hypophosphorous acid, followed by hydrolysis of the
adduct and N-terminal protection with benzyl chloroformate, similarly as described in the
literature [55]. Global yield from three steps: 46%; mp 149–151 ◦C (lit. mp 150–152 ◦C [2])
;
1H NMR (400 MHz, DMSO-d6)
δ
7.58 (d, 2JPH = 9.0 Hz, 1H, OH), 7.39–7.15 (m, 5H), 6.74
(d, 1JPH = 529.1 Hz, 1H, PH), 5.04 (s, 2H, PhCH2O), 3.69–3.60 (m, 1H, CH-P), 1.69–1.61 (m,
1H, CH2CH of iBu group), 1.56–1.32 (m, 2H, CH2CH of iBu group), 0.89 (d, 3JHH = 6.7 Hz
,
3H), 0.83 (d, 3JHH = 6.6 Hz, 3H); 13C NMR (100 MHz, DMSO-d6)
δ
156.2 (d, 3JPC = 3.4 Hz),
1
2
137.0, 128.3, 127.8, 127.6, 65.6, 48.7 (d, JPC = 105.5 Hz), 34.9 (d, JPC = 3.6 Hz), 23.9 (d,
3JPC = 12.2 Hz), 23.1, 21.0; 31P NMR (160 MHz, DMSO-d6)
δ 29.1; ESI-HRMS m/z calcd. for
C13H20NNaO4P ([M+Na]+) 308.1028, found 308.1025.
3.1.3. General Procedure for the Synthesis of 14 and 15
To a refluxing solution of compound 14 or 15 and 1-adamantyl bromide (1.5 equiv) in
CHCl3 (10 mL/mmol) was added silver(I) oxide (1.2 equiv) portionwise over 1 h. After
the solution was refluxed for 3 h, the solvent was removed in vacuo, and the residue
was treated with Et2O (5 mL/mmol). The resulting mixture was filtered through a sin-
tered glass vacuum filtration funnel with Celite, and the filtrates were evaporated. The
residue was purified by column chromatography (SiO2, AcOEt/hexanes 1:1) to yield the
desired compounds.
Methyl 3-[1-N-(benzyloxycarbonylamino)-3-phenylpropyl]hydroxyphosphinyl
propanoate (14)
(9.0 g, 74%) obtained as a white solid from phosphinic acid 12 (9.66 g, 29 mmol),
hexamethyldisilazane (30.85 mL, 145 mmol) and methyl acrylate (3.96 mL, 43.5 mmol)
as described in the general procedure. The crude product was worked up with Et2O
(120 mL/mmol) and left overnight for crystallization to give the title compound. mp:
163–166 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ
7.67 (d, 2JPH = 9.4 Hz, 1H, OH), 7.58–7.14
(m, 10H), 5.08 (AB system, J = 12.6 Hz, 2H, PhCH2O), 4.08 (bs, 1H, NH), 3.74–3.54 (m, 1H, CH-
P), 3.59 (s, 3H), 2.76–2.36 (m, 4H, PCH CH , CH CH Ph), 2.01–1.60 (m, 4H, PCH CH , CH CH Ph)
100 MHz, DMSO-d6)
172.6 (d, 3JPC = 15.8 Hz), 156.3 (d, 3JPC = 3.6 Hz), 141.2, 137.2, 128.4,
;
13C NMR
2
2
2
2
2
2
2
2
(
δ
128.4, 128.3, 127.8, 127.6, 125.9, 65.6, 51.6, 49.4 (d, 1JPC = 106.2 Hz), 31.6 (d, 2JPC = 11.8 Hz),
29.1, 26.1, 21.7 (d, 1JPC = 89.5 Hz); 31P NMR (160 MHz, DMSO-d6)
δ 46.2; ESI-HRMS m/z
calcd. for C21H27NO6P ([M+H]+) 420.1576, found 420.1579.

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Methyl 3-[1-N-(benzyloxycarbonylamino)-3-methylbutyl]hydroxyphosphinyl
propanoate (15)
The phosphinic dipeptide was obtained according to the procedures previously de-
scribed in the literature [18], and similar as described in the general procedure above. Yield:
66%; mp 150–155 ◦C (lit. mp 146–153 ◦C [18]); 1H NMR (400 MHz, DMSO-d6)
δ 7.54 (d,
2JPH = 9.4 Hz, 1H), 7.37–7.29 (m, 5H), 5.05 (AB system, J = 12.7 Hz, 2H, PhCH2O), 3.77–3.70
(m, 1H, CH-P), 3.59 (s, 3H), 2.56–2.40 (m, 2H, PCH2CH2), 1.85–1.78 (m, 2H, PCH2CH2),
1.63–1.38 (m, 3H, CHCH2 of iBu group), 0.89 (d, 3JHH = 6.5 Hz, 3H), 0.81 (d, 3JHH = 6.4 Hz,
3H); 13C NMR (100 MHz, DMSO-d6)
δ
172.6 (d, 3JPC = 15.7 Hz), 156.3 (d, 3JPC = 3.8 Hz),
137.2, 128.3, 127.8, 127.5, 65.5, 51.6, 48.2 (d, 1JPC = 106.9 Hz), 35.6, 26.2 (d, 2JPC = 2.3 Hz),
24.0 (d, 3JPC = 11.3 Hz), 23.3, 21.5 (d, 1JPC = 89.5 Hz), 20.8; 31P NMR (160 MHz, DMSO-d6)
δ 46.6; ESI-HRMS m/z calcd. for C17H27NO6P ([M+H]+) 372.1576, found 372.1585.
3.1.4. General Procedure for the Protection of Hydroxyphosphinyl Group in 14 and 15
To a refluxing solution of compound 14 or 15 and 1-adamantyl bromide (1.5 equiv) in
CHCl3 (10 mL/mmol) was added silver(I) oxide (1.2 equiv) portionwise over 1 h. After
the solution was refluxed for 3 h, the solvent was removed in vacuo, and the residue
was treated with Et2O (5 mL/mmol). The resulting mixture was filtered through a sin-
tered glass vacuum filtration funnel with Celite, and the filtrates were evaporated. The
residue was purified by column chromatography (SiO2, AcOEt/hexanes 1:1) to yield the
desired compounds.
Methyl
3-[(1-N-(benzyloxycarbonylamino)-3-phenylpropyl)adamantyloxyphoshinyl]propanoate (16
)
(9.60 g, 87%) obtained as a white solid from compound 14 (8.38 g, 20 mmol), 1-
adamantyl bromide (6.52 g, 30 mmol) and silver(I) oxide (5.56 g, 24 mmol) as described in
the general procedure. The residue was purified by column chromatography eluting from
(SiO2, hexanes) to (SiO2, AcOEt/hexanes 1:1) to afford the title compound. mp: 114–116 ◦C
;
1H NMR (400 MHz, CDCl3) 7.36–7.07 (m, 10H), 5.45 (d, 3JPH = 10.3 Hz, 1H, NH)major, 5.10
δ
(AB system, J = 12.3 Hz, 2H, PhCH2O), 4.96 (d, 3JPH = 10.3 Hz, 1H, NH)minor, 3.95–3.90 (m,
1H, CH-P), 3.64 (s, 3H), 2.83–2.45 (m, 4H, PCH2CH2, CH2CH2Ph), 2.18–1.88 (m, 13H, CCH2
and CHCH2 of Ad group, CH2CH2Ph, PCH2CH2), 1.63–1.57 (m, 6H, CHCH2CH of Ad
group); 13C NMR (100 MHz, CDCl3) 172.9 (d, 3JPC = 16.9 Hz), 172.8 (d, 3JPC = 17.7 Hz),
δ
156.4 (d, 3JPC = 5.6 Hz)major, 156.1 (d, 3JPC = 4.8 Hz)minor, 141.1major, 140.9minor, 136.4major
,
136.1minor, 129.3, 128.5, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.8, 126.2, 126.0, 125.9, 83.3
(d, 2JPC = 9.9 Hz)major, 83.0 (d, 2JPC = 10.0 Hz)minor, 67.2minor, 67.0major, 51.9minor, 51.8major
49.5 (d, 1JPC = 110.8 Hz), 44.4 (d, 3JPC = 3.3 Hz)minor, 44.3 (d, 3JPC = 3.3 Hz)major, 35.6, 32.1 (d,
,
2JPC = 11.2 Hz), 31.1, 29.6, 27.2, 23.6 (d, 1JPC = 90.0 Hz)minor, 23.5 (d, 1JPC = 89.9 Hz)major 31P
;
NMR (160 MHz, CDCl3) δ 48.4minor, 47.7major; ESI-HRMS m/z calcd. for C31H40NNaO6P
([M+Na]+) 576.2491, found 576.2496.
Methyl
3-[(1-N-(benzyloxycarbonylamino)-3-methylbutyl)adamantyloxyphoshinyl]propanoate (17
)
(8.19 g, 90%) obtained as a white solid from compound 15 (6.68 g, 18 mmol), 1-
adamantyl bromide (5.87 g, 27 mmol) and silver(I) oxide (5.01 g, 21.6 mmol) as described
in the general procedure. The residue was purified by column chromatography eluting
from (SiO2, hexanes) to (SiO2, AcOEt/hexanes 1:1) to afford the title compound. mp:
111–115 ◦C; 1H NMR (400 MHz, CDCl3)
δ
7.35–7.26 (m, 5H), 5.29 (bs, 1H, NH), 5.08 (AB
system, J = 12.4 Hz, 2H, PhCH2O)major, 5.08 (AB system, J = 12.1 Hz, 2H, PhCH2O)minor
,
4.84 (bs, 1H, NH), 3.99–3.90 (m, 1H, CH-P), 3.66 (s, 3H)minor, 3.64 (s, 3H)major, 2.70–2.44
(m, 2H, PCH2CH2), 2.14–1.93 (m, 9H, CCH2 and CHCH2 of Ad group), 1.73–1.37 (m, 11H,
CHCH2CH of Ad group, CHCH2 of iBu group, PCH2CH2), 0.91 (d, 3JHH = 6.4 Hz, 3H),
0.89 (d, 3JHH = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3)
172.8 (d, JPC = 17.8 Hz)minor, 156.2 (d, JPC = 5.1 Hz)major, 156.0 (d, JPC = 4.5 Hz)minor
δ
172.9 (d, 3JPC = 16.8 Hz)major
,
,
3
3
3

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136.4major, 136.2minor, 128.5major, 128.4minor, 128.2minor, 128.1major, 128.0minor, 127.9major, 83.0
(d, 2JPC = 10.0 Hz major, 82.7 (d, 2JPC = 10.0 Hz)minor, 67.1minor, 67.0major, 51.9minor, 51.8major
) ,
48.1 (d, 1JPC = 110.9 Hz)major, 48.0 (d, 1JPC = 105.6 Hz)minor, 44.5 (d, 3JPC = 3.3 Hz)minor, 44.3
(d, 3JPC = 3.2 Hz)major, 37.1minor, 36.7major, 35.6major, 35.6minor, 31.1, 27.2 (d, 2JPC = 1.1 Hz),
24.5 (d, 3JPC = 11.7 Hz)minor, 24.4 (d, 3JPC = 10.9 Hz)major, 23.7 (d, 1JPC = 89.5 Hz)minor, 23.5,
23.4, 23.4 (d, 1JPC = 89.9 Hz)major, 21.1; 31P NMR (160 MHz, CDCl3)
m/z calcd. for C27H40NNaO6P ([M+Na]+) 528.2491, found 528.2497.
δ 49.1, 48.5; ESI-HRMS
3.1.5. General Procedure for the Coupling Reaction of Building Blocks 16 and 17 Involving
HOBt and EDC
The N-(benzyloxycarbonyl) compounds 16 or 17 were N-deprotected by dissolving
in MeOH (15 mL/mmol) and stirring with 10% Pd/C catalyst (100 mg/mmol) under an
atmosphere of hydrogen 20 psi overnight at room temperature. The mixture was filtered
through a sintered glass vacuum filtration funnel with Celite, washed several times with
MeOH, and filtrate concentrated under reduced pressure. The residue and the correspond-
ing N-Boc-protected amino acid (1 equiv) were dissolved in DMF (5 mL/mmol) and stirred
at room temperature for 20 min with N-hydroxybenzotriazole (HOBt) (1.1 equiv) and
N-(dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC HCl) (1.1 equiv).
·
After adding Et3N (1.5 equiv), the suspension was stirred for 20 h at room temperature.
Then 10% aqueous solution of citric acid (50 mL/mmol) was added, and the mixture was
extracted with AcOEt (3
10% aqueous solution of citric acid (2
and saturated brine (1 20 mL/mmol). The organic layer was dried over MgSO4, filtrated,
×
20 mL/mmol). The combined organic layer was washed with
×
20 mL/mmol), 0.5 M NaHCO3 (2 × 20 mL/mmol),
×
and concentrated to dryness in a vacuum.
Methyl 3-[(adamantan-1-yloxy)-(1-((S)-2-(tert-butoxycarbonylamino)-4-
(methylthio)butan-amido)-3-phenylpropyl)phosphinyl]propanoate (20)
(2.41 g, 60%) obtained as a foamy white solid from N-benzyloxycarbonyl compound 16
(3.42 g, 6.18 mmol) and 10% Pd/C catalyst (618 mg) for the deprotection step, followed by
coupling reaction using N-Boc-methionine (1.54 g, 6.18 mmol), as described in the general
procedure. The crude product was purified by flash-chromatography eluting from (SiO2,
1
AcOEt/hexanes 1:6) to (SiO2, AcOEt/hexanes 1:1) to afford phosphinotripeptide 20. H
NMR (400 MHz, CDCl3) δ 7.50–7.44 (m, 1H, NH), 7.24–7.08 (m, 5H), 5.73 and 5.40 (bs, 1H,
NH), 4.31–4.19 (m, 2H, NHCHCO, CH-P), 3.64 (s, 3H)major, 3.63 (s, 3H)minor, 2.77–2.32 (m,
6H, PCH2CH2, CH2CH2Ph, CH2CH2S), 2.12–1.81 (m, 18H, CCH2 and CHCH2 of Ad group,
CH2CH2Ph, PCH2CH2, CH2CH2SCH3), 1.67–1.51 (m, 6H, CHCH2CH of Ad group), 1.37
(s, 9H)major, 1.36 (s, 9H)minor
;
13C NMR (100 MHz, CDCl3)
δ
172.7 (d, 3JPC = 16.5 Hz
)
,
,
,
,
minor
172.6 (d, 3JPC = 17.3 Hz)major, 172.3 (d, 3JPC = 11.2 Hz)minor, 172.3 (d, 3JPC = 11.7 Hz)major
155.4minor, 155.3major, 141.0minor, 140.8major, 128.4, 128.4, 128.3, 128.3, 126.0major, 125.9minor
83.5 (d, 2JPC = 10.2 Hz)minor, 83.4 (d, 2JPC = 10.3 Hz)major, 79.8, 54.4minor, 54.0major, 51.9major
51.8minor, 47.1 (d, 1JPC = 111.1 Hz)minor, 47.0 (d, 1JPC = 111.2 Hz
)
major, 44.3 (d, 3JPC = 3.5 Hz),
2
1
35.5, 32.2, 32.0 (d, JPC = 11.4 Hz), 31.1, 30.1, 29.4, 28.2, 27.3, 23.6 (d, JPC = 90.4 Hz),
15.3minor, 15.2major; δ 48.5, 48.4; ESI-HRMS m/z calcd. for
31P NMR (160 MHz, CDCl3)
C33H51N2NaO7PS ([M+Na]+) 673.3052, found 673.3045.
Methyl 3-[(adamantan-1-yloxy)(1-((S)-2-(tert-butoxycarbonylamino)-4-
(methylthio)butan-amido)-3-methylbutyl)phosphinyl]propanoate (21)
(907 mg, 70%) obtained as a foamy white solid from N-benzyloxycarbonyl compound
17 (1.09 g, 2.15 mmol) and 10% Pd/C catalyst (215 mg) for the deprotection step, followed
by coupling reaction using N-Boc-methionine (0.54 g, 2.15 mmol), as described in the
general procedure. The crude product was purified by flash-chromatography eluting from
(SiO2, AcOEt/hexanes 1:5) to (SiO2, AcOEt/hexanes 4:1) to afford phosphinotripeptide 21
.

Int. J. Mol. Sci. 2021, 22, 5090
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1H NMR (400 MHz, CDCl3)
δ
4.30–4.25 (m, 2H, CH-P, NHCHCO), 3.65 (s, 3H)major, 3.64 (s,
3H)minor, 2.66–2.46 (m, 4H, PCH2CH2, CH2CH2S), 2.15–1.98 (m, 14H, CCH2 and CHCH2
of Ad group, CH2CH2SCH3), 1.87–1.84 (m, 2H, PCH2CH2), 1.60–1.40 (m, 9H, CHCH2CH
i
3
of Ad group, CHCH2 of Bu group), 1.38 (s, 9H), 0.90 (d, JHH = 6.1 Hz, 3H), 0.84 (d,
3JHH = 6.2 Hz, 3H); 13C NMR (100 MHz, CDCl3)
δ
172.7 (d, 3JPC = 17.4 Hz), 171.9, 155.3,
83.3 (d, 2JPC = 10.1 Hz), 79.8, 53.9, 51.9, 45.6 (d, 1JPC = 111.5 Hz), 44.3, (d, 3JPC = 3.6 Hz),
36.3, 35.6, 32.1, 31.1, 30.1, 28.2, 27.3, (d, 2JPC = 4.1 Hz), 24.4 (d, 3JPC = 10.9 Hz), 23.4, 23.3 (d,
1JPC = 90.5 Hz), 21.1, 15.3; 31P NMR (160 MHz, CDCl3)
δ 49.3minor, 49.0major; ESI-HRMS
m/z calcd. for C29H51N2NaO7PS ([M+Na]+) 625.3052, found 625.3045.
Methyl 3-[(adamantan-1-yloxy)(1-((S)-2-(tert-butoxycarbonylamino)-4-((tert-butyldime-
thylsilyl)-oxy)butanamido)-3-methylbutyl)phosphinyl]propanoate (22)
(1.53 g, 51%) obtained as a white solid from N-benzyloxycarbonyl compound 17
(
2.21 g, 4.37 mmol) and 10% Pd/C catalyst (437 mg) for the deprotection step, followed
by coupling reaction using O-TBDMS-protected N-Boc-homoserine (Boc-L-Hse(TBDMS)-
OH was prepared starting from L-Hse-OH through protection first the hydroxyl group as
OTBDMS [39] followed by protection of the N-terminus as N-Boc protecting group [40])
(1.46 g, 4.37 mmol), as described in the general procedure. The crude product was puri-
fied by flash-chromatography eluti◦ng from 1:4 AcOEt/hexanes to AcOEt to afford phos-
1
phinotripeptide 22. mp: 156–158 C; H NMR (400 MHz, CDCl3)
δ 4.43–4.18 (m, 2H,
CH-P, NHCHCO), 3.75–3.68 (m, 2H, CH2-OSi), 3.64 (s, 3H)major, 3.63 (s, 3H)minor, 2.72–
2.49 (m, 2H, PCH2CH2), 2.18–1.91 (m, 13H, CCH2 and CHCH2 of Ad group, PCH2CH2,
i
t
CH2CH2OSi), 1.63–1.21 (m, 18H, CHCH2CH of Ad group, CHCH2 of Bu group, Bu
of Boc protecting group), 0.92–0.82 (m, 6H, CH2CH(CH3)2), 0.86 (s, 9H, SitBu), 0.03 (s,
6H, Si(CH3)2)major, 0.02 (s, 6H, Si(CH3)2)minor; δ 173.0 (d,
13C NMR (100 MHz, CDCl3)
3JPC = 16.6 Hz), 172.2minor, 172.0major, 155.8major, 155.4minor, 83.0 (d, 2JPC = 9.9 Hz)minor, 82.9
(d, 2JPC = 9.8 Hz)major, 80.5minor, 79.9major, 65.7, 55.2minor, 54.8major, 51.9major, 50.1minor, 45.6
(d, 1JPC = 109.9 Hz), 44.3major, 44.3minor, 41.6minor, 41.5major, 36.2major, 36.1minor, 35.7, 31.1,
30.8, 28.2major, 28.1minor, 27.2minor, 27.1major, 25.8minor, 25.8major, 24.4 (d, 3JPC = 10.9 Hz
)
,
,
major
3
1
24.1 (d, JPC = 10.9 Hz)minor, 23.5, 23.3 (d, JPC = 90.1 Hz), 21.0major, 20.9minor, –5.6minor
–5.7major;31P NMR (160 MHz, CDCl3)
δ 49.0major, 48.8minor; ESI-HRMS m/z calcd. for
C34H64N2O8PSi ([M+H]+) 687.4170, found 687.4164.
Methyl 3-[(adamantan-1-yloxy)(1-((S)-2-(tert-butoxycarbonylamino)-4-(hydroxybutan-
amido)-3-methylbutyl)phosphinyl]propanoate (23)
◦
To a stirred 0 C solution of phosphinotripeptide 22 (651 mg, 0.95 mmol) in THF
(
10 mL) was added a 1 M solution of tetrabutylammonium fluoride in THF (1.90 mL,
◦
1.90 mmol). The solution was stirred for 5 min at 0 C and then was allowed to warm to rt
and was stirred for 20 h. The mixture was diluted with water (4 mL) and extracted with
CH2Cl2 (3
×
4 mL). The combined organic layers were dried over MgSO4, and concentrated.
The residue was worked up with hexanes/Et2O 1:1 and left overnight for crystallization
to yield 384 mg (71%) of the title compound as a white solid. Due to the instability of this
compound, it was subjected to deprotection of Boc and adamantyl groups without further
purification steps. 1H NMR (400 MHz, CDCl3)
δ 4.43–4.18 (m, 2H, CH-P, NHCHCO),
3.72–3.63 (m, 2H, CH2-OH), 3.66 (s, 3H)minor, 3.65 (s, 3H)major, 2.73–2.43 (m, 2H, PCH2CH2),
2.14–1.92 (m, 13H, CCH2 and CHCH2 of Ad group, PCH2CH2, CH2CH2OH), 1.67–1.32 (m,
18H, CHCH2CH of Ad group, CHCH2 of iBu group, tBu), 0.92–0.82 (m, 6H, CH2CH(CH3)2);
31P NMR (160 MHz, CDCl3)
573.3305, found 573.3297.
δ
50.0, 49.9; ESI-HRMS m/z calcd. for C28H50N2O8P ([M+H]+)

Int. J. Mol. Sci. 2021, 22, 5090
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Methyl 3-[(adamantan-10-yloxy)-((1-((S)-3-([1,10-biphenyl]-4-yl)-2-(tert-butoxycarbonyl-
amino)propanamido)-3-phenylpropyl)phosphinyl]propanoate (26)
(1.05 g, 76%) obtained as a foamy white solid from N-benzyloxycarbonyl compound
16 (1.03 g, 1.87 mmol) and 10% Pd/C catalyst (187 mg) for the deprotection step, followed
0
by coupling reaction using N-Boc-L-4,4 -biphenylalanine (660 mg, 1.87 mmol), as described
in the general procedure. The crude product was purified by flash-chromatography
eluting from (SiO2, AcOEt/hexanes 1:6) to (SiO2, AcOEt) to afford phosphinotripep-
tide 26
. δ 7.55–7.05 (m, 14H), 4.52–4.21 (m, 2H, CH-P,
1H NMR (400 MHz, CDCl3)
NHCHCO), 3.65 (s, 3H)major, 3.61 (s, 3H)minor, 3.21–2.99 (m, 2H, CH2-biphenyl), 2.71–
2.40 (m, 4H, PCH2CH2, CH2CH2Ph), 2.11–1.73 (m, 13H, CCH2 and CHCH2 of Ad group,
CH2CH2Ph, PCH2CH2), 1.62–1.46 (m, 6H, CHCH2CH of Ad group), 1.39 (s, 9H)minor
,
3
1.36 (s, 9H)major
;
13C NMR (100 MHz, CDCl3)
δ
172.8 (d, JPC = 17.0 Hz)minor, 172.7
3
3
3
(d, JPC = 16.8 Hz
)
major, 171.0 (d, JPC = 11.2 Hz)minor, 170.9 (d, JPC = 11.4 Hz)major
,
155.5minor, 155.3major, 141.1, 140.9, 140.7, 140.6, 140.4, 140.4, 139.8, 139.8, 135.6, 135.6,
135.5, 135.4, 129.7, 129.3, 128.7, 128.7, 128.6, 128.5, 128.5, 128.4, 128.4, 128.3, 128.3, 128.2,
127.4, 127.3, 127.3, 127.3, 127.2, 127.1, 126.9, 126.9, 126.8, 126.8, 126.0, 125.9, 83.5 (d,
2JPC = 10.1 Hz)major, 83.0 (d, 2JPC = 10.1 Hz
)
minor, 80.4minor, 80.2major, 56.0major, 55.7minor
,
1
1
52.0minor, 51.9major, 47.3 (d, JPC = 110.0 Hz)minor, 47.2 (d, JPC = 110.0 Hz)major, 44.4 (d,
3JPC = 4.1 Hz)minor, 44.3 (d, 3JPC = 3.2 Hz
(d, 2JPC = 11.6 Hz major, 32.0 (d, 2JPC = 11.5 Hz
)major, 37.7minor, 37.3major, 35.5major, 35.4minor, 32.2
)
)
minor, 31.1major, 31.0minor, 29.4, 28.2minor,
1
1
28.1major, 23.6 (d, JPC = 90.7 Hz
)minor, 23.4 (d, JPC = 90.0 Hz)major;
31P NMR (160 MHz,
CDCl3)
δ
48.0major, 47.9minor; ESI-HRMS m/z calcd. for C43H56N2O7P ([M+H]+) 743.3825,
found 743.3824.
Methyl 3-[(adamantan-1-yloxy)-((1-((S)-3-([1,10-biphenyl]-4-yl)-2-(tert-butoxycarbonyl-
amino)propanamido)-3-methylbutyl)phosphinyl]propanoate (27)
(826 mg, 50%) obtained as a foamy white solid from N-benzyloxycarbonyl compound
17 (1.20 g, 2.38 mmol) and 10% Pd/C catalyst (238 mg) for the deprotection step, followed
0
by coupling reaction using N-Boc-L-4,4 -biphenylalanine (812 mg, 2.38 mmol), as described
in the general procedure. The crude product was purified by flash-chromatography eluting
from (SiO2, AcOEt/hexanes 1:4) to (SiO2, AcOEt/hexanes 3:1) to afford phosphinotripep-
1
tide 27. H NMR (400 MHz, CDCl3)
δ
7.57–7.25 (m, 9H), 4.46–4.21 (m, 2H, CH-P, NHCHCO),
3.66 (s, 3H)major, 3.61 (s, 3H)minor, 3.18–3.00 (m, 2H, CH2-biphenyl), 2.67–2.44 (m, 2H,
PCH2CH2), 2.14–1.90 (m, 11H, CCH2 and CHCH2 of Ad group, PCH2CH2), 1.63–1.43 (m,
i
9H, CHCH2CH of Ad group, CHCH2 of Bu group), 1.40 (s, 9H)minor, 1.38 (s, 9H)major
,
0.88 (d, JHH = 6.3 Hz, 3H), 0.81 (d, JHH = 6.2 Hz, 3H); 13C NMR (100 MHz, CDCl3)
172.8 (d, JPC = 18.5 Hz)minor, 172.8 (d, JPC = 17.0 Hz)major, 170.8major, 170.7minor, 155.5,
155.2, 140.7, 140.7, 140.5, 140.4, 139.7, 139.6, 135.6, 135.6, 135.5, 129.7, 129.7, 129.6, 128.7,
128.7, 128.7, 127.3, 127.2, 127.1, 126.9, 126.9, 126.8, 126.8, 83.2 (d, 2JPC = 10.2 Hz)major, 82.8
δ
3
3
3
3
(d, 2JPC = 10.1 Hz
)
minor, 80.1major, 79.9minor, 55.8major, 55.6minor, 51.9major, 51.8minor, 45.7 (d,
1JPC = 110.7 Hz)minor, 45.7 (d, 1JPC = 110.7 Hz)major, 44.4minor, 44.3major, 37.6minor, 37.3major
36.2minor, 35.9major, 35.6major, 35.5minor, 31.1major, 31.0minor, 28.2major, 28.1minor, 27.1 (d,
,
2JPC = 30.7 Hz)major, 27.0 (d, 2JPC = 28.2 Hz)minor, 24.3, 24.1, 23.4 (d, 1JPC = 89.6 Hz
)
,
,
minor
23.4major, 23.2 (d, 1JPC = 89.1 Hz)major, 21.1minor 31P NMR (160 MHz, CDCl3)
; δ 48.6minor
48.5major; ESI-HRMS m/z calcd. for C39H56N2O7P ([M+H]+) 695.3825, found 695.3809.
Methyl 3-[(adamantan-1-yloxy)-((1-((S)-3-(4-benzoylphenyl)-2-(tert-butoxycarbonyl-
amino)-propanamido)-3-phenylpropyl)phosphinyl]propanoate (28)
(1.26 g, 66%) obtained as a foamy white solid from N-benzyloxycarbonyl compound
16 (1.37 g, 2.47 mmol) and 10% Pd/C catalyst (247 mg) for the deprotection step, followed
by coupling reaction using N-Boc-p-benzoyl-L-phenylalanine (911 mg, 2.47 mmol), as de-

Int. J. Mol. Sci. 2021, 22, 5090
19 of 27
scribed in the general procedure. The crude product was purified by flash-chromatography
eluting from (SiO2, AcOEt/hexanes 1:3) to (SiO2, AcOEt/hexanes 4:1) to afford phos-
1
phinotripeptide 28. H NMR (400 MHz, CDCl3)
δ 7.75–7.08 (m, 14H), 4.44–4.22 (m, 2H,
CH-P, NHCHCO), 3.65 (s, 3H), 3.63 (s, 3H), 3.31–3.07 (m, 2H, CH2-4-benzoylphenyl), 2.64–
2.44 (m, 4H, PCH2CH2, CH2CH2Ph), 2.11–1.85 (m, 13H, CCH2 and CHCH2 of Ad group,
CH2CH2Ph, PCH2CH2), 1.62–1.57 (m, 6H, CHCH2CH of Ad group), 1.38 (s, 9H), 1.35 (s,
3
9H); 13C NMR (100 MHz, CDCl3)
δ
196.2minor, 196.1major, 173.0 (d, JPC = 14.1 Hz)minor
,
172.7 (d, 3JPC = 16.6 Hz)major, 171.4major, 170.7minor, 155.4minor, 155.3major, 141.9, 141.8, 141.7,
141.6, 141.0, 140.9, 140.7, 140.6, 137.5, 137.4, 136.2, 136.2, 136.1, 136.1, 132.4, 132.3, 132.2,
130.5, 130.5, 130.4, 130.4, 129.9, 129.9, 129.8, 129.3, 129.3, 129.2, 128.5, 128.4, 128.4, 128.4,
128.3, 128.2, 128.2, 126.1, 126.0, 83.5 (d, 2JPC = 93.8 Hz), 83.4 (d, 2JPC = 10.4 Hz), 80.3major
,
1
80.2minor, 55.6major, 55.5minor, 52.0minor, 52.0major, 47.5 (d, JPC = 104.4 Hz)minor, 47.4 (d,
1JPC = 109.8 Hz
)
major, 44.4 (d, 3JPC = 3.0 Hz)minor, 44.3 (d, 3JPC = 3.0 Hz)major, 37.9, 37.6, 35.5,
35.5, 32.0 (d, 2JPC = 11.6 Hz), 31.1minor, 31.1major, 29.5, 28.2minor, 28.1major, 27.1major, 26.8minor
,
23.6 (d, 1JPC = 90.8 Hz)minor, 23.4 (d, 1JPC = 90.7 Hz)major 31P NMR (160 MHz, CDCl3)
; δ
47.7, 47.6; ESI-HRMS m/z calcd. for C44H56N2O8P ([M+H]+) 771.3774, found 771.3786.
Methyl 3-[(adamantan-1-yloxy)-((1-((S)-3-(4-benzoylphenyl)-2-(tert-butoxycarbony)-
amino)-propanamido)-30-methylbutyl)phosphinyl]propanoate (29)
(923 mg, 51%) obtained as a foamy white solid from N-benzyloxycarbonyl compound
17 (1.26 g, 2.49 mmol) and 10% Pd/C catalyst (249 mg) for the deprotection step, followed
by coupling reaction using N-Boc-p-benzoyl-L-phenylalanine (919 mg, 2.49 mmol), as de-
scribed in the general procedure. The crude product was purified by flash-chromatography
eluting from (SiO2, AcOEt/hexanes 1:4) to (SiO2, AcOEt/hexanes 3:1) to afford phos-
phinotripeptide 29. δ 7.77–7.31 (m, 9H), 4.44–4.24 (m, 2H,
1H NMR (400 MHz, CDCl3)
CH-P, NHCHCO), 3.65 (s, 3H), 3.64 (s, 3H), 3.24–3.01 (m, 2H, CH2-4-benzoylphenyl), 2.64–
2.51 (m, 2H, PCH2CH2), 2.14–1.88 (m, 11H, CCH2 and CHCH2 of Ad group, PCH2CH2),
1.62–1.41 (m, 9H, CHCH2CH of Ad group, CHCH2 of iBu group), 1.38 (s, 9H), 1.36 (s, 9H),
0.90–0.80 (m, 6H); 13C NMR (100 MHz, CDCl3)
δ 196.2, 196.1, 172.9, 172.7, 170.6, 170.5,
155.5, 155.3, 141.9, 141.8, 141.7, 141.6, 137.6, 137.6, 137.5, 137.5, 136.3, 136.2, 136.1, 132.4,
132.4, 132.3, 130.5, 130.4, 130.4, 130.0, 129.9, 129.9, 129.3, 129.3, 129.2, 128.3, 128.2, 83.4,
1
82.7, 80.4, 80.3, 55.6, 55.5, 52.0, 51.9, 45.9 (d, JPC = 105.8 Hz), 44.5, 44.4, 38.0, 37.6, 36.5,
2
1
36.0, 35.6, 31.1, 28.2, 27.1 (d, JPC = 27.2 Hz), 24.4, 24.3, 23.7 (d, JPC = 90.0 Hz), 23.6 (d,
1JPC = 89.7 Hz), 23.4, 21.1, 21.0; 31P NMR (160 MHz, CDCl3)
δ 48.5, 48.4; ESI-HRMS m/z
calcd. for C40H56N2O8P ([M+H]+) 723.3774, found 723.3783.
3.1.6. General Procedure for Removal of the C-Terminal Methyl Ester Group
In phosphinotripeptides 20 and 21, the C-terminal methyl ester was removed by
saponification. A 4 M NaOH aqueous solution (2 equiv) was added dropwise to a solution
of compound 20 or 21 dissolved in MeOH. The final concentration of the NaOH was 0.4 M
The mixture was stirred overnight at room temperature. Then, methanol was removed
under reduced pressure, and the residue was diluted with water and acidified with 0.5 M
HCl to pH 1 in an ice-water bath. The aqueous solution was extracted 3 times with AcOEt,
and the organic layer was washed twice with water and brine. The organic layer was dried
and concentrated in vacuo to give the crude product.
.
3-[(Adamantan-1-yloxy)(1-((S)-2-(tert-butoxycarbonylamino)-4-
(methylthio)butanamido)-3-phenylpropyl)phosphinyl]propanoic acid (24)
(403 mg, 91%) obtained as a white solid from phosphinotripeptide 20 (453 mg,
0.697 mmol), 4 M NaOH (0.35 mL, 1.394 mmol), and MeOH (3.15 mL) as described in
the general procedure. mp: 171–172 ◦C; 1H NMR (400 MHz, CD3OD)
δ
7.29–7.16 (m, 5H),
4.25–4.19 (m, 2H, CH-P, NHCHCO), 2.81–2.47 (m, 6H, PCH2CH2, CH2CH2S, CH2CH2Ph),

Int. J. Mol. Sci. 2021, 22, 5090
20 of 27
2.19–1.89 (m, 18H, CCH2 and CHCH2 of Ad group, CH2CH2Ph, PCH2CH2, CH2CH2SCH3),
1.66 (s, 6H, CHCH2CH of Ad group), 1.45 (s, 9H)major, 1.44 (s, 9H)minor; ,
13C NMR (100 MHz
3
3
CD3OD)
δ 175.8 (d, JPC = 13.5 Hz), 175.1 (d, JPC = 21.8 Hz), 158.0, 142.2, 142.1, 130.0,
129.8, 129.7, 129.6, 127.4, 127.3, 85.0 (d, 2JPC = 10.0 Hz), 80.8, 55.8minor, 55.5major, 49.4 (d,
)major, 49.1 (d, JPC = 107.4 Hz)minor, 45.6 (d, JPC = 2.6 Hz)minor, 45.4 (d,
3JPC = 2.9 Hz)major, 36.9major, 36.8minor, 33.2, 33.0 (d, 2JPC = 12.5 Hz), 32.8, 31.4, 31.3, 28.9,
1JPC = 100.8 Hz
1
3
1
1
28.0minor, 27.7major, 23.8 (d, JPC = 93.1 Hz)minor, 23.6 (d, JPC = 93.1 Hz)major, 15.5minor
,
15.4major; δ 51.8minor, 51.6major; ESI-HRMS m/z calcd. for
31P NMR (160 MHz, CD3OD)
C32H50N2O7PS ([M+H]+) 637.3071, found 637.3071.
3-[(Adamantan-1-yloxy)(1-((S)-2-(tert-butoxycarbonylamino)-4-
(methylthio)butanamido)-3-methylbutyl)phosphinyl]propanoic acid (25)
(160 mg, 80%) obtained as a white solid from phosphinotripeptide 21 (204 mg,
0.34 mmol), 4 M NaOH (0.17 mL, 0.68 mmol), and MeOH (1.53 mL) as described in the
general procedure. This compound showed to be very unstable, and it was subjected to
1
deprotection of Boc and adamantyl groups without further purification steps. H NMR
(400 MHz, CD3OD)
δ 4.35–4.07 (m, 2H, CH-P, NHCHCO), 2.66–2.41 (m, 4H, PCH2CH2,
CH2CH2S), 2.20–1.76 (m, 16H, CCH2 and CHCH2 of Ad group, CH2CH2SCH3, PCH2CH2),
1.76–1.56 (m, 9H, CHCH2CH of Ad group, CHCH2 of iBu group), 1.40 (s, 9H), 0.92 (bs, 3H),
0.84 (bs, 3H); 31P NMR (160 MHz, CD3OD) δ 52.1, 51.9.
3.1.7. General Procedure for Removal of Boc and Ad Protecting Groups
In phosphinotripeptides 20, 21, 23–29, the Boc and Ad protecting groups were re-
moved in acidic conditions. Then, N-Boc and P-Ad protected phosphinotripeptide deriva-
tives were treated with a mixture of 50% TFA/CH2Cl2 and stirred for 3 h at room tem-
perature. Then, the mixture was concentrated in vacuo. CH2Cl2 was added to the
residue, and the solvent was removed under reduced pressure (3 times). A mixture
of Et2O/hexanes 1:1 was added to the residue. The white precipitate was filtered and
washed with Et2O/hexanes 1:1.
Methyl
3-[(1-((S)-2-amino-4-(methylthio)butanamido)-3-phenylpropyl)phosphinyl]propanoate (1)
(512 mg, 99%) obtained as a white solid from phosphinotripeptide 20 (807 mg,
1.24 mmol), and a mixture of 50% TFA/CH2Cl2 (5.6 mL) as described in the general
◦
procedure. mp: 161–164 C; 1H NMR (400 MHz, DMSO-d6)
δ 8.37 (bs, 2H, NH2), 7.29–7.08
(m, 5H), 3.99–3.89 (m, 2H, NHCHCO, CH-P), 3.56 (s, 3H), 2.85–2.37 (m, 6H, PCH2CH2,
CH2CH2Ph, CH2CH2S), 2.12–1.59 (m, 6H, CH2CH2Ph, PCH2CH2, CH2CH2SCH3), 2.03 (s,
3H, SCH3); 13C NMR (100 MHz, DMSO-d6)
δ
173.1, 172.9, 172.7, 141.5, 141.3, 128.5, 128.4,
128.3, 128.3, 125.9, 125.8, 52.0minor, 51.7major, 51.6major, 51.5minor, 47.5 (d, 1JPC = 103.1 Hz minor
47.4 (d, 1JPC = 103.5 Hz)major, 32.0minor, 31.8major, 31.3minor, 31.3major, 29.4, 28.4, 28.2, 22.7
)
,
1
1
(d, JPC = 91.6 Hz)major, 22.5 (d, JPC = 91.0 Hz)minor, 14.5minor, 14.4major
;
31P NMR (160
MHz, DMSO-d6)
δ
40.5, 40.3; ESI-HRMS m/z calcd. for C18H30N2O5PS ([M+H]+) 417.1613,
found 417.1609.
Methyl
3-[(1-((S)-2-amino-4-(methylthio)butanamido)-3-methylbutyl)phosphinyl]propanoate (2)
(245 mg, 99%) obtained as a white solid from phosphinotripeptide 21 (405 mg,
0.673 mmol), and a mixture of 50% TFA/CH2Cl2 (3 mL) as described in the general pro-
cedure. mp: 183–184 ◦C; 1H NMR (400 MHz, D2O)
δ 4.21–4.11 (m, 2H, CH-P, NHCHCO),
3.68 (s, 3H), 2.63–2.51 (m, 4H, PCH2CH2, CH2S), 2.22–2.12 (m, 2H, CH2CH2S), 2.09 (s,
i
3H), 1.95–1.88 (m, 2H, PCH2CH2), 1.65–1.49 (m, 3H, CH2CH of Bu group), 0.90 (d,
3
3JHH = 5.9 Hz, 3H), 0.83 (d, JHH = 4.8 Hz, 3H); 13C NMR (100 MHz, D2O + CD3OD)
δ
176.8 (d, 3JPC = 15.7 Hz minor, 176.4 (d, 3JPC = 15.3 Hz)major, 169.9major, 169.8minor, 53.8,
)

Int. J. Mol. Sci. 2021, 22, 5090
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1
1
53.6major, 53.5minor, 48.4 (d, JPC = 102.5 Hz)minor, 48.2 (d, JPC = 103.4 Hz)major, 36.5,
31.5major, 31.2minor, 29.3major, 29.3minor, 27.6minor, 27.5major, 23.8minor, 23.7major, 23.0 (d,
1
1JPC = 91.1 Hz
D2O)
)
minor, 22.8 (d, JPC = 90.3 Hz)major, 21.2, 21.0, 15.1; 31P NMR (160 MHz,
δ
45.6major, 44.3minor; ESI-HRMS m/z calcd. for C14H30N2O5PS ([M+H]+) 369.1608,
found 369.1618.
Methyl
3-[(1-((S)-2-amino-4-(hydroxybutanamido)-3-methylbutyl)phosphinyl]propanoate (3)
(179 mg, 85%) obtained as a white solid from phosphinotripeptide 23 (357 mg,
0.623 mmol), and a mixture of 50% TFA/CH2Cl2 (4 mL) as described in the general pro-
cedure. mp: 174–176 ◦C; 1H NMR (400 MHz, D2O)
δ 4.16–4.08 (m, 2H, CH-P, NHCHCO),
3.79–3.72 (m, 2H, CH2-OH), 3.31 (s, 3H), 2.62–2.53 (m, 2H, PCH2CH2), 2.21–1.80 (m,
4H, PCH2CH2, CH2CH2OH), 1.65–1.52 (m, 3H, CHCH2 of iBu group), 0.96–0.81 (m 6H,
3
CH2CH(CH3)2); 13C NMR (100 MHz, D2O)
δ
177.3 (d, JPC = 15.9 Hz)minor, 177.1 (d,
3JPC = 15.4 Hz
1JPC = 102.4 Hz
)
major, 168.9minor, 168.9major, 67.3, 48.8major, 48.4minor, 47.9, 47.3 (d,
1
)
minor, 47.2 (d, JPC = 103.3 Hz)major, 36.2, 35.5minor, 35.4major, 26.7major
,
1
1
26.6minor, 22.7, 22.6, 22.6 (d, JPC = 95.1 Hz)major, 22.0 (d, JPC = 91.1 Hz)minor, 20.1; 31P
NMR (160 MHz, D2O)
46.1, 45.3; ESI-HRMS m/z calcd. for C13H28N2O6P ([M+H]+)
339.1685, found 339.1680.
δ
3-[(1-((S)-2-Amino)-4-(methylthio)butanamido)-3-phenylpropyl)-phosphinyl]propanoic
acid (4)
(173 mg, 99%) obtained as a white solid from phosphinotripeptide 24 (276 mg, 0.434
mmol), and a mixture of 50% TFA/CH2Cl2 (4 mL) as described in the general procedure.
◦
mp: 180–185 C; 1H NMR (400 MHz, D2O + CD3OD)
δ
7.31–7.24 (m, 5H), 3.92–3.87 (m, 1H,
CH-P), 2.87–2.53 (m, 6H, PCH2CH2, CH2CH2S, CH2CH2Ph), 2.37–1.56 (m, 6H, CH2CH2Ph,
PCH2CH2, CH2CH2SCH3), 2.06 (s, 3H, SCH3); 13C NMR (100 MHz, D2O + CD3OD)
183.8 (d, 3JPC = 14.3 Hz), 183.7 (d, 3JPC = 13.9 Hz), 164.9, 164.6, 143.5, 143.4, 130.5, 128.1,
δ
1
1
128.0, 55.8, 55.7, 50.4 (d, JPC = 100.4 Hz), 50.3 (d, JPC = 100.0 Hz), 35.2, 35.2, 34.1 (d,
2JPC = 11.1 Hz), 31.3, 31.4, 30.8, 30.6, 26.6 (d, JPC = 91.9 Hz), 26.5 (d, JPC = 91.6 Hz),
1
1
15.9; 31P NMR (160 MHz, CD3OD)
([M+H]+) 403.1457, found 403.1452.
δ 40.4, 40.3; ESI-HRMS m/z calcd. for C17H28N2O5PS
3-[(1-((S)-2-Amino-4-(methylthio)butanamido)-3-methylbutyl)phosphinyl]propanoic
acid (5)
(95 mg, 99%) obtained as a white solid from phosphinotripeptide 25 (159 mg, 0.27 mmol),
and a mixture of 50% TFA/CH2Cl2 (1 mL) as described in the general procedure. mp:
218–222 ◦C; 1H NMR (400 MHz, D2O)
δ 4.25–3.96 (m, 2H, CH-P, NHCHCO), 2.73–2.40 (m,
4H, PCH2CH2, CH2S), 2.28–1.99 (m, 5H, CH2CH2SCH3), 1.95–1.75 (m, 2H, PCH2CH2), 1.70–
1.38 (m, 3H, CH2CH of iBu group), 0.90 (bs, 3H), 0.82 (bs, 3H); 13C NMR (100 MHz, D2O
+ CD3OD)
δ
178.4 (d, 3JPC = 14.8 Hz)minor, 178.0 (d, 3JPC = 16.3 Hz)major, 169.9, 53.8minor
,
,
,
;
53.5major, 48.2 (d, 1JPC = 101.6 Hz)minor, 48.0 (d, 1JPC = 103.8 Hz)major, 36.6minor, 36.5major
31.4major, 31.2minor, 29.3, 27.9minor, 27.8major, 23.9minor, 23.8major, 23.3 (d, 1JPC = 86.8 Hz)minor
23.2 (d, 1JPC = 92.1 Hz)major, 21.3, 21.0, 15.1; 31P NMR (160 MHz, D2O)
δ 43.9major, 42.8minor
ESI-HRMS m/z calcd. for C13H28N2O5PS ([M+H]+) 355.1457, found 355.1461.
Methyl 3-[(1-((S)-3-([1,10-biphenyl]-4-yl)-2-aminopropanamido)-3-
phenylpropyl)phosphinyl]-propanoate (6)
(345 mg, 99%) obtained as a white solid from phosphinotripeptide 26 (509 mg,
0.686 mmol), and a mixture of 50% TFA/CH2Cl2 (5 mL) as described in the general pro-
◦
1
cedure. mp: 126–128 C; H NMR (400 MHz, CD3OD)
δ 7.64–6.94 (m, 14H), 4.28–4.02
(m, 2H, CH-P, NH2CHCO), 3.65 (s, 3H), 3.60 (s, 3H), 3.43–3.06 (m, 2H, CH2-biphenyl),
2.86–2.47 (m, 4H, PCH2CH2, CH2CH2Ph), 2.25–1.65 (m, 4H, PCH23CH2, CH2CH2Ph); 13
C
3
NMR (100 MHz, CD3OD)
δ
175.6 (d, JPC = 16.1 Hz), 175.1 (d, JPC = 17.1 Hz), 169.8,

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169.3, 142.8, 142.5, 142.2, 141.9, 141.7, 134.9, 131.2, 131.2, 130.1, 129.9, 129.8, 129.7, 129.6,
129.5, 129.0, 128.8, 128.7, 128.5, 128.1, 128.0, 127.2, 127.0, 56.3, 55.8, 52.5, 52.5, 38.5, 38.2,
33.9, 33.8, 31.6, 30.9, 27.9, 27.8, 24.2 24.2 (d, 1JPC = 91.6 Hz), 24.0 (d, 1JPC = 91.4 Hz); 31
NMR (160 MHz, CD3OD)
P
δ
42.9, 40.7; ESI-HRMS m/z calcd. for C28H34N2O5P ([M+H]+)
509.2205, found 509.2210.
Methyl 3-[(1-((S)-3-([1,10-biphenyl]-4-yl)-2-aminopropanamido)-3-
methylbutyl)phosphinyl]-propanoate (7)
(209 mg, 78%) obtained as a white solid from phosphinotripeptide 27 (405 mg,
0.583 mmol), and a mixture of 50% TFA/CH2Cl2 (4 mL) as described in the general proce-
dure. mp: 150–152 ◦C; 1H NMR (400 MHz, CD3OD)
δ 7.81–7.32 (m, 9H), 4.64–4.13 (m, 2H,
CH-P, NH2CHCO), 3.67 (s, 3H), 3.61 (s, 3H), 3.53–3.05 (m, 2H, CH2-biphenyl), 2.75–2.57 (m,
2H, PCH2CH2), 1.93–1.22 (m, 5H, PCH2CH2, CH2CH of iBu group), 0.98 (d, 3JHH = 5.9 Hz,
3H), 0.92 (d, 3JHH = 5.9 Hz, 3H), 0.71 (d, 3JHH = 6.4 Hz, 3H), 0.68 (d, 3JHH = 6.3 Hz, 3H);
13C NMR (100 MHz, CD3OD) 175.6 (d, 3JPC = 16.3 Hz), 175.1 (d, 3JPC = 17.1 Hz), 169.6,
δ
169.1, 142.2, 141.9, 141.8, 134.9, 134.8, 131.2, 130.1, 128.9, 128.7, 128.6, 128.1, 128.0, 56.2, 55.7,
52.6, 52.5, 38.5, 38.1, 37.8, 37.3, 27.9, 27.8, 25.8 (d, 3JPC = 10.8 Hz), 25.3 (d, 3JPC = 10.7 Hz),
24.1, 23.3, 23.1, 21.6, 21.4; 31P NMR (160 MHz, CD3OD)
for C24H34N2O5P ([M+H]+) 461.2205, found 461.2215.
δ 43.8, 41.6; ESI-HRMS m/z calcd.
Methyl 3-[(1-((S)-2-amino-3-(4-benzoylphenyl)propanamido)-3-
phenylpropyl)phosphinyl]-propanoate (8)
(377 mg, 99%) obtained as a white solid from phosphinotripeptide 28 (547 mg,
0.710 mmol), and a mixture of 50% TFA/CH2Cl2 (5 mL) as described in the general
◦
1
procedure. mp: 134–140 C (dec); H NMR (400 MHz, CD3OD)
δ 7.79–6.92 (m, 14H),
4.33–4.02 (m, 2H, CH-P, NH2CHCO), 3.65 (s, 3H), 3.61 (s, 3H), 3.59–3.15 (m, 2H, CH2-4-
benzoylphenyl), 2.83–2.53 (m, 4H, PCH2CH2, CH2CH2Ph), 2.20–1.73 (m, 4H, PCH2CH2,
CH2CH2Ph); 13C NMR (100 MHz, CD3OD) 198.2, 197.9, 175.6 (d, 3JPC = 16.8 Hz), 175.1 (d,
δ
3JPC = 14.7 Hz), 169.6, 169.1, 142.7, 142.4, 141.3, 141.0, 138.8, 138.7, 138.3, 138.0, 133.8, 132.8,
132.1, 132.0, 131.2, 131.0, 130.9, 129.7, 129.6, 129.5, 127.2, 127.1, 55.8, 55.5, 52.6, 52.6, 38.6,
38.3, 33.7, 33.6, 31.5, 31.0, 27.8, 27.7, 24.1 (d, 1JPC = 103.4 Hz), 24.0 (d, 1JPC = 96.0 Hz); 31
NMR (160 MHz, CD3OD)
P
δ
40.7, 38.9; ESI-HRMS m/z calcd. for C29H34N2O6P ([M+H]+)
537.2154, found 537.2166.
Methyl 3-[(1-((S)-2-amino-3-(4-benzoylphenyl)propanamido)-3-
methylbutyl)phosphinyl]-propanoate (9)
(334 mg, 99%) obtained as a white solid from phosphinotripeptide 29 (500 mg,
0.692 mmol), and a mixture of 50% TFA/CH2Cl2 (5 mL) as described in the general pro-
cedure. mp: 124–126 ◦C; 1H NMR (400 MHz, CD3OD)
δ 7.81–7.45 (m, 9H), 4.26–4.13 (m,
2H, CH-P, NH2CHCO), 3.68 (s, 3H), 3.65 (s, 3H), 3.46–3.12 (m, 2H, CH2-4-benzoylphenyl),
i
2.63–2.57 (m, 2H, PCH2CH2), 1.95–1.29 (m, 5H, PCH2CH2, CH2CH of Bu group), 1.00
3
3
3
(d, JHH = 6.4 Hz, 3H), 0.93 (d, JHH = 6.3 Hz, 3H), 0.77 (d, JHH = 6.6 Hz, 3H), 0.74 (d,
3JHH = 6.6 Hz, 3H); 13C NMR (100 MHz, CD3OD) 198.2, 197.9, 175.7 (d, 3JPC = 16.2 Hz),
δ
175.1 (d, 3JPC = 16.5 Hz), 169.3, 168.8, 141.1, 140.9, 138.8, 138.8, 138.5, 138.2, 134.1, 134.1,
132.0, 131.9, 131.2, 131.0, 130.8, 130.8, 129.7, 55.9, 55.4, 52.6, 52.5, 38.7, 38.3, 37.3, 37.0, 27.8,
27.7, 25.8 (d, 3JPC = 11.0 Hz), 25.5 (d, 3JPC = 11.1 Hz), 24.1, 24.0 (d, 1JPC = 91.1 Hz), 23.8 (d,
1JPC = 92.1 Hz), 21.5, 21.3, 31P NMR (160 MHz, CD3OD)
δ 45.3, 43.1; ESI-HRMS m/z calcd.
for C25H34N2O6P ([M+H]+) 489.2154, found 489.2170.

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3.2. Inhibitor Activity
3.2.1. Enzyme Preparation
Leucyl aminopeptidase (SsLAP) was isolated from the porcine kidney modified
method described by Spackman [56] and Ledeme [57]. KM of the isolated enzyme on
the synthetic substrate L-leucine-p-nitroanilide (L-Leu-pNA) was 0.86 mM, and Vmax was
0.0012
µmol/min. KM values correlate well with those given in the works 0.77 mM [58,59].
The purity of the enzyme was confirmed by electrophoresis.
The aminopeptidase from barley seeds (HvLAP) was isolated and purified accord-
ing to the procedure described by Oszywa [46]. KM of the isolated enzyme on the
synthetic substrate -leucine-p-nitroanilide (L-Leu-pNA) was 0.55 mM, and Vmax was
L
0.0146 µmol/min [46,48]. The purity of the enzyme was confirmed by electrophoresis.
3.2.2. Kinetic Assays
The enzymatic reaction in the presence of SsLAP was carried out at 37 ◦C in 7.5 mM
TEA-HCl buffer (pH 8.5), containing 5 mM NaCl. The progress of the reaction was
monitored spectrophotometrically (UV-VIS Spectrophotometer Cintra 303, GBC Scien-
tific Equipment Ltd., Victoria, Australia) at a wavelength of 405 nm against a control
sample containing no enzyme. The attempting mixture contained: Solution of the synthetic
substrate -Leu-p-nitroanilide in DMSO (substrate concentration: 1.5 to 0.2 mM—final
L
concentration), 7.5 mM TEA-HCl buffer (pH 8.5), containing 5 mM NaCl, the solution of
the potential inhibitor in reaction buffer (concentration of compound depending on the
braking force), and enzyme (0.005 mg of the sample).
The enzymatic reaction in the presence of aminopeptidase from barley seeds (HvLAP)
was carried out at 37 ◦C in 50 mM Tris-HCl buffer (pH 7.5), containing 50 mM NaCl, and
10 mM 2-mercaptoethanol. The progress of the reaction was monitored spectrophotomet-
rically (UV-VIS Spectrophotometer Cintra 303, GBC Scientific Equipment Ltd., Victoria,
Australia) at a wavelength of 405 nm against a control sample containing no enzyme. The
attempting mixture contained: Solution of the synthetic substrate -Leu-p-nitroanilide
L
in DMSO (substrate concentration: 1.5 to 0.2 mM—final concentration), 50 mM Tris-HCl
buffer (pH 7.5), containing 50 mM NaCl, and 10 mM 2-mercaptoethanol, the solution of
the potential inhibitor in reaction buffer (concentration of compound depending on the
braking force), and enzyme (0.028 mg of the sample).
3.2.3. Kinetic Calculation
Kinetic constants KM, Vmax, and KI and type of inhibition was determined by methods
Lineweaver-Burk, Dixon, Hanes-Woolf, and method half-inhibitory concentration. In the
calculation, linearization methods used weighted regression. The values (Ki) given in the
tables are the values calculated by the Dixon method. The Ki values calculated with all the
above-mentioned methods close to each other. All measurements were made in three-fold
repetitions. The graphs show the average values of the three measurements and Sb.
The used weight factors have been calculated as follow:
wi = 1/(σi2), σ2 = (dy/dx)2, where y = 1/V, or σ2 = (dy/dx)2 which gives the formula
for weight I = V4; wi—statistical weight of the i-th measurement, σi2—variance of the i-th
experimental point.
3.3. Molecular Modeling
Due to synthetic route designed inhibitors contain S isomers of amino acids in the P2
position, whereas in the P1 position, both R and S stereoisomers are possible. Because of
that, all combinations of R and S stereoisomers in the P1 position with stereoisomer S in
the P2 position for all phosphinic inhibitors were considered during molecular modeling
studies. The structures of all the ligands were optimized in program Gaussian09 at the
B3LYP/6-311g (d,p) level of theory [60] with the PCM solvent model using water as the
solvent. This protocol was applied for the designed phosphinic inhibitors, as well as for

Int. J. Mol. Sci. 2021, 22, 5090
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the phosphonic analog of leucine, a well-known inhibitor of LAP, which was considered as
the reference compound, serving to verify the docking methodology.
Next, ligands were prepared with the Discover Studio Visualizer 5 (Dassault Sys-
tems BIOVIA, Waltham, MA, USA). For each ligand, 10 of the most possible conformers
have been generated and use in docking protocol. For further analysis, five of them with
the best value of GoldScore function have been selected. As a model of mammalian
leucine aminopeptidase, the crystal structures from bovine lens LAP, whereas as the plant
aminopeptidase model, the structure of tomato aminopeptidase has been taken. The struc-
tures were obtained from the RCSB Protein Data Bank ID: 1LCP [51] and 4KSI [52]. Lacking
protons and charges were added to the protein using the H++ server [54] according to the
experimental pH value. The ligand, besides metal ions, was removed from the structure
of the enzymes. The active site sphere was selected based on the location of zinc Zn408
and magnesium Mg601 ions for SsLAP and HvLAP, respectively, with the radius 15Ang.
During the docking protocol, the protein molecules have been rigid. Molecular docking in
the defined active sites was carried out using GOLD Algorithm (2010, 1 version, CCDC,
Cambridge, UK) [53] with applied Genetic Algorithm protocol. Analysis of inhibitor-
enzyme interactions of docked molecules was performed with Discovery Studio Visualizer
5 (Dassault Systems BIOVIA, Waltham, MA, USA).
4. Conclusions
It is known that phosphinic moiety is a good analog of the transition state during
peptide bond hydrolysis. It is possible thanks to imitating the tetrahedral geometry, electron
distribution, and the ability to metal complexation. Application of the phosphonic moiety
to construct pseudopeptides, which are effective inhibitors of therapeutically significant
metalloproteases, including porcine and barley leucine aminopeptidase, obtain one of the
most potent inhibitors of these enzymes. Because both enzymes are responsible for the
consecutive removal of the N-terminal residue from the peptidic substrates, elongation
of the N-terminal parts of the inhibitor is not suggested. However, within this work, we
showed that elongation of the phosphinatedipeptides by adding one more amino acid
residue in the P2 position will not deprive their biological properties. Obtained compounds
are micromolar inhibitors of SsLAP and HvLAP. The best inhibitor activity has been
found for
3 toward SsLAP (Ki = 14 µM) and 1 toward HvLAP (Ki = 38 µM). Moreover,
the introduction of additional amino acid residue in the P2 position induced the higher
selectivity of the final tripeptides.
Additionally, based on the retrosynthesis analysis, we proposed a synthetic route
to obtain phosphinate pseudotripeptide
satisfactory overall yield.
1–9 within a reasonable number of steps with a
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10
.3390/ijms22105090/s1.
Author Contributions: Conceptualization, R.L.; methodology, J.M.d.l.S., M.P. and M.J.; software—
molecular modeling, K.H., M.J.; investigation: B.O. and M.P. isolated the enzyme and together
with K.H. performed kinetic studies; J.M.d.l.S. and Z.E.S. performed synthesis; writing—original
draft preparation, J.M.d.l.S. and M.J.; writing—review and editing, J.M.d.l.S., M.P., F.P. and R.L.;
visualization, M.J.; supervision, R.L. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the Polish Ministry of Science and Higher Education (PMSHE)
for the Faculty of Chemistry of Wrocław University of Science and Technology and by Wroclaw
Research Center EIT+ under the project “Biotechnologies and advanced medical technologies—
BioMed”, grant number POIG 01.01.02-02-003/08-00, 00 financed from the European Regional
Development Fund (Operational Programme Innovative Economy, 1.1.2. B. O. is recipient of a Ph.D.
fellowship from a project funded by the European Social Found. Financial support by the Ministerio
de Ciencia, Innovación y Universidades (MCIU), Agencia Estatal de Investigación (AEI) y Fondo
Europeo de Desarrollo Regional (FEDER) (RTI2018-101818-B-I00, UE), and Gobierno Vasco (GV), (IT
992-16) is gratefully acknowledged.

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Institutional Review Board Statement: Not applicable.
Acknowledgments: The molecular modeling was carried out using hardware and software resources
of The Supercomputing and Networking Center in Wroclaw (grant no. 197). The authors thank
technical and human support provided by SGIker (UPV/EHU/ERDF, EU).
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
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