Phosphinotripeptidic inhibitors of leucylaminopeptidases

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 ²and 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

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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:

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://

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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://ww w .mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 5090

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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 P1⁰in 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

Int. J. Mol. Sci. 2021, 22, 5090

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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

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

+

Ar, R

O

R

O

BocHN

CO₂H

O

+

OH

O

c

NH₂

+

P

PG

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

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

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 H₂in 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).

Int. J. Mol. Sci. 2021, 22, 5090

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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 hPhePO₃H₂is more than two times more active than LeuPO₂H₃[48].

Figure 3. The designation of type inhibition and the inhibition constant K_Iby Dixon method for compound 3 in the presence

of SsLAP.

Figure 4. The designation of type inhibition and the inhibition constant K_Iby 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.

K_I[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

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 P1⁰and 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 LeuPO₃H₂to 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

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 NH₃of 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 S1⁰direction. It

results in the interaction of Leu residue from the P1 position with the S1⁰binding 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

NH₃of Lys262 (2.60 Å). Moreover, in this case, the inhibitor molecule is

shifted into S1⁰direction, 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 (δ_H) are reported in parts per million (ppm).

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All coupling constants (J) values are given in Hz. Chemical shifts (δ_C) 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 F₂₅₄plates, and spot

visualization was accomplished by UV light (254 nm) or KMnO₄solution.

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, J_PH= 8.8 Hz, 1H,

OH), 7.39–7.16 (m, 10H), 6.77 (d, ¹J_PH= 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, ³J_PC= 3.2 Hz), 141.1, 137.0, 128.4, 128.4, 128.3, 127.9, 127.7,

125.9, 65.6, 49.9 (d, ¹J_PC= 105.1 Hz), 31.4 (d, ²J_PC= 12.4 Hz), 28.3 (d, ³J_PC= 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, ²J_PH= 9.0 Hz, 1H, OH), 7.39–7.15 (m, 5H), 6.74

(d, ¹J_PH= 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, ³J_HH= 6.7 Hz

,

3H), 0.83 (d, ³J_HH= 6.6 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d6)

δ

156.2 (d, ³J_PC= 3.4 Hz),

1

2

137.0, 128.3, 127.8, 127.6, 65.6, 48.7 (d, J_PC= 105.5 Hz), 34.9 (d, J_PC= 3.6 Hz), 23.9 (d,

³J_PC= 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, ²J_PH= 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, ³J_PC= 15.8 Hz), 156.3 (d, ³J_PC= 3.6 Hz), 141.2, 137.2, 128.4,

;

¹³C NMR

2

(

δ

128.4, 128.3, 127.8, 127.6, 125.9, 65.6, 51.6, 49.4 (d, ¹J_PC= 106.2 Hz), 31.6 (d, ²J_PC= 11.8 Hz),

29.1, 26.1, 21.7 (d, ¹J_PC= 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,

²J_PH= 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, CHCH₂of ⁱBu group), 0.89 (d, ³J_HH= 6.5 Hz, 3H), 0.81 (d, ³J_HH= 6.4 Hz,

3H); ¹³C NMR (100 MHz, DMSO-d6)

δ

172.6 (d, ³J_PC= 15.7 Hz), 156.3 (d, ³J_PC= 3.8 Hz),

137.2, 128.3, 127.8, 127.5, 65.5, 51.6, 48.2 (d, ¹J_PC= 106.9 Hz), 35.6, 26.2 (d, ²J_PC= 2.3 Hz),

24.0 (d, ³J_PC= 11.3 Hz), 23.3, 21.5 (d, ¹J_PC= 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, ³J_PH= 10.3 Hz, 1H, NH)_major, 5.10

δ

(AB system, J = 12.3 Hz, 2H, PhCH₂O), 4.96 (d, ³J_PH= 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 CHCH₂of Ad group, CH₂CH₂Ph, PCH₂CH₂), 1.63–1.57 (m, 6H, CHCH₂CH of Ad

group); ¹³C NMR (100 MHz, CDCl₃) 172.9 (d, ³J_PC= 16.9 Hz), 172.8 (d, ³J_PC= 17.7 Hz),

δ

156.4 (d, ³J_PC= 5.6 Hz)_major, 156.1 (d, ³J_PC= 4.8 Hz)_minor, 141.1_major, 140.9_minor, 136.4_major

,

136.1_minor, 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, ²J_PC= 9.9 Hz)_major, 83.0 (d, ²J_PC= 10.0 Hz)_minor, 67.2_minor, 67.0_major, 51.9_minor, 51.8_major

49.5 (d, ¹J_PC= 110.8 Hz), 44.4 (d, ³J_PC= 3.3 Hz)_minor, 44.3 (d, ³J_PC= 3.3 Hz)_major, 35.6, 32.1 (d,

,

²J_PC= 11.2 Hz), 31.1, 29.6, 27.2, 23.6 (d, ¹J_PC= 90.0 Hz)_minor, 23.5 (d, ¹J_PC= 89.9 Hz)_major³¹P

;

NMR (160 MHz, CDCl₃) δ 48.4_minor, 47.7_major; 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, CCH₂and CHCH₂of Ad group), 1.73–1.37 (m, 11H,

CHCH₂CH of Ad group, CHCH₂of ⁱBu group, PCH₂CH₂), 0.91 (d, ³J_HH= 6.4 Hz, 3H),

0.89 (d, ³J_HH= 6.8 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃)

172.8 (d, J_PC= 17.8 Hz)_minor, 156.2 (d, J_PC= 5.1 Hz)_major, 156.0 (d, J_PC= 4.5 Hz)_minor

δ

172.9 (d, ³J_PC= 16.8 Hz)_major

,

3

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136.4_major, 136.2_minor, 128.5_major, 128.4_minor, 128.2_minor, 128.1_major, 128.0_minor, 127.9_major, 83.0

(d, ²J_PC= 10.0 Hz _major, 82.7 (d, ²J_PC= 10.0 Hz)_minor, 67.1_minor, 67.0_major, 51.9_minor, 51.8_major

) ,

48.1 (d, ¹J_PC= 110.9 Hz)_major, 48.0 (d, ¹J_PC= 105.6 Hz)_minor, 44.5 (d, ³J_PC= 3.3 Hz)_minor, 44.3

(d, ³J_PC= 3.2 Hz)_major, 37.1_minor, 36.7_major, 35.6_major, 35.6_minor, 31.1, 27.2 (d, ²J_PC= 1.1 Hz),

24.5 (d, ³J_PC= 11.7 Hz)_minor, 24.4 (d, ³J_PC= 10.9 Hz)_major, 23.7 (d, ¹J_PC= 89.5 Hz)_minor, 23.5,

23.4, 23.4 (d, ¹J_PC= 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, CCH₂and CHCH₂of 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, ³J_PC= 16.5 Hz

)

,

minor

172.6 (d, ³J_PC= 17.3 Hz)_major, 172.3 (d, ³J_PC= 11.2 Hz)_minor, 172.3 (d, ³J_PC= 11.7 Hz)_major

155.4_minor, 155.3_major, 141.0_minor, 140.8_major, 128.4, 128.4, 128.3, 128.3, 126.0_major, 125.9_minor

83.5 (d, ²J_PC= 10.2 Hz)_minor, 83.4 (d, ²J_PC= 10.3 Hz)_major, 79.8, 54.4_minor, 54.0_major, 51.9_major

51.8_minor, 47.1 (d, ¹J_PC= 111.1 Hz)_minor, 47.0 (d, ¹J_PC= 111.2 Hz

)

_major, 44.3 (d, ³J_PC= 3.5 Hz),

2

1

35.5, 32.2, 32.0 (d, J_PC= 11.4 Hz), 31.1, 30.1, 29.4, 28.2, 27.3, 23.6 (d, J_PC= 90.4 Hz),

15.3_minor, 15.2_major; 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

.

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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, CCH₂and CHCH₂

of Ad group, CH₂CH₂SCH₃), 1.87–1.84 (m, 2H, PCH₂CH₂), 1.60–1.40 (m, 9H, CHCH₂CH

i

3

of Ad group, CHCH₂of Bu group), 1.38 (s, 9H), 0.90 (d, J_HH= 6.1 Hz, 3H), 0.84 (d,

³J_HH= 6.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃)

δ

172.7 (d, ³J_PC= 17.4 Hz), 171.9, 155.3,

83.3 (d, ²J_PC= 10.1 Hz), 79.8, 53.9, 51.9, 45.6 (d, ¹J_PC= 111.5 Hz), 44.3, (d, ³J_PC= 3.6 Hz),

36.3, 35.6, 32.1, 31.1, 30.1, 28.2, 27.3, (d, ²J_PC= 4.1 Hz), 24.4 (d, ³J_PC= 10.9 Hz), 23.4, 23.3 (d,

¹J_PC= 90.5 Hz), 21.1, 15.3; ³¹P NMR (160 MHz, CDCl₃)

δ 49.3_minor, 49.0_major; 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, CCH₂and CHCH₂of Ad group, PCH₂CH₂,

i

t

CH₂CH₂OSi), 1.63–1.21 (m, 18H, CHCH₂CH of Ad group, CHCH₂of Bu group, Bu

of Boc protecting group), 0.92–0.82 (m, 6H, CH₂CH(CH₃)₂), 0.86 (s, 9H, Si^tBu), 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)

³J_PC= 16.6 Hz), 172.2_minor, 172.0_major, 155.8_major, 155.4_minor, 83.0 (d, ²J_PC= 9.9 Hz)_minor, 82.9

(d, ²J_PC= 9.8 Hz)_major, 80.5_minor, 79.9_major, 65.7, 55.2_minor, 54.8_major, 51.9_major, 50.1_minor, 45.6

(d, ¹J_PC= 109.9 Hz), 44.3_major, 44.3_minor, 41.6_minor, 41.5_major, 36.2_major, 36.1_minor, 35.7, 31.1,

30.8, 28.2_major, 28.1_minor, 27.2_minor, 27.1_major, 25.8_minor, 25.8_major, 24.4 (d, ³J_PC= 10.9 Hz

)

,

major

3

1

24.1 (d, J_PC= 10.9 Hz)_minor, 23.5, 23.3 (d, J_PC= 90.1 Hz), 21.0_major, 20.9_minor, –5.6_minor

–5.7_major;³¹P NMR (160 MHz, CDCl₃)

δ 49.0_major, 48.8_minor; 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, CCH₂and CHCH₂of Ad group, PCH₂CH₂, CH₂CH₂OH), 1.67–1.32 (m,

18H, CHCH₂CH of Ad group, CHCH₂of 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

18 of 27

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, CCH₂and CHCH₂of 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, J_PC= 17.0 Hz)_minor, 172.7

3

(d, J_PC= 16.8 Hz

)

_major, 171.0 (d, J_PC= 11.2 Hz)_minor, 170.9 (d, J_PC= 11.4 Hz)_major

,

155.5_minor, 155.3_major, 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,

²J_PC= 10.1 Hz)_major, 83.0 (d, ²J_PC= 10.1 Hz

)

_minor, 80.4_minor, 80.2_major, 56.0_major, 55.7_minor

,

1

52.0_minor, 51.9_major, 47.3 (d, J_PC= 110.0 Hz)_minor, 47.2 (d, J_PC= 110.0 Hz)_major, 44.4 (d,

³J_PC= 4.1 Hz)_minor, 44.3 (d, ³J_PC= 3.2 Hz

(d, ²J_PC= 11.6 Hz _major, 32.0 (d, ²J_PC= 11.5 Hz

)_major, 37.7_minor, 37.3_major, 35.5_major, 35.4_minor, 32.2

)

_minor, 31.1_major, 31.0_minor, 29.4, 28.2_minor,

1

28.1_major, 23.6 (d, J_PC= 90.7 Hz

)_minor, 23.4 (d, J_PC= 90.0 Hz)_major;

³¹P NMR (160 MHz,

CDCl₃)

δ

48.0_major, 47.9_minor; 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, CCH₂and CHCH₂of Ad group, PCH₂CH₂), 1.63–1.43 (m,

i

9H, CHCH₂CH of Ad group, CHCH₂of Bu group), 1.40 (s, 9H)_minor, 1.38 (s, 9H)_major

,

0.88 (d, J_HH= 6.3 Hz, 3H), 0.81 (d, J_HH= 6.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃)

172.8 (d, J_PC= 18.5 Hz)_minor, 172.8 (d, J_PC= 17.0 Hz)_major, 170.8_major, 170.7_minor, 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, ²J_PC= 10.2 Hz)_major, 82.8

δ

3

(d, ²J_PC= 10.1 Hz

)

_minor, 80.1_major, 79.9_minor, 55.8_major, 55.6_minor, 51.9_major, 51.8_minor, 45.7 (d,

¹J_PC= 110.7 Hz)_minor, 45.7 (d, ¹J_PC= 110.7 Hz)_major, 44.4_minor, 44.3_major, 37.6_minor, 37.3_major

36.2_minor, 35.9_major, 35.6_major, 35.5_minor, 31.1_major, 31.0_minor, 28.2_major, 28.1_minor, 27.1 (d,

,

²J_PC= 30.7 Hz)_major, 27.0 (d, ²J_PC= 28.2 Hz)_minor, 24.3, 24.1, 23.4 (d, ¹J_PC= 89.6 Hz

)

,

minor

23.4_major, 23.2 (d, ¹J_PC= 89.1 Hz)_major, 21.1_minor³¹P NMR (160 MHz, CDCl₃)

; δ 48.6_minor

48.5_major; 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, CCH₂and CHCH₂of 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

196.2_minor, 196.1_major, 173.0 (d, J_PC= 14.1 Hz)_minor

,

172.7 (d, ³J_PC= 16.6 Hz)_major, 171.4_major, 170.7_minor, 155.4_minor, 155.3_major, 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, ²J_PC= 93.8 Hz), 83.4 (d, ²J_PC= 10.4 Hz), 80.3_major

,

1

80.2_minor, 55.6_major, 55.5_minor, 52.0_minor, 52.0_major, 47.5 (d, J_PC= 104.4 Hz)_minor, 47.4 (d,

¹J_PC= 109.8 Hz

)

_major, 44.4 (d, ³J_PC= 3.0 Hz)_minor, 44.3 (d, ³J_PC= 3.0 Hz)_major, 37.9, 37.6, 35.5,

35.5, 32.0 (d, ²J_PC= 11.6 Hz), 31.1_minor, 31.1_major, 29.5, 28.2_minor, 28.1_major, 27.1_major, 26.8_minor

,

23.6 (d, ¹J_PC= 90.8 Hz)_minor, 23.4 (d, ¹J_PC= 90.7 Hz)_major³¹P 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, CCH₂and CHCH₂of Ad group, PCH₂CH₂),

1.62–1.41 (m, 9H, CHCH₂CH of Ad group, CHCH₂of 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, J_PC= 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, J_PC= 27.2 Hz), 24.4, 24.3, 23.7 (d, J_PC= 90.0 Hz), 23.6 (d,

¹J_PC= 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, CCH₂and CHCH₂of 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

CD₃OD)

δ 175.8 (d, J_PC= 13.5 Hz), 175.1 (d, J_PC= 21.8 Hz), 158.0, 142.2, 142.1, 130.0,

129.8, 129.7, 129.6, 127.4, 127.3, 85.0 (d, ²J_PC= 10.0 Hz), 80.8, 55.8_minor, 55.5_major, 49.4 (d,

)_major, 49.1 (d, J_PC= 107.4 Hz)_minor, 45.6 (d, J_PC= 2.6 Hz)_minor, 45.4 (d,

³J_PC= 2.9 Hz)_major, 36.9_major, 36.8_minor, 33.2, 33.0 (d, ²J_PC= 12.5 Hz), 32.8, 31.4, 31.3, 28.9,

¹J_PC= 100.8 Hz

1

3

1

28.0_minor, 27.7_major, 23.8 (d, J_PC= 93.1 Hz)_minor, 23.6 (d, J_PC= 93.1 Hz)_major, 15.5_minor

,

15.4_major; δ 51.8_minor, 51.6_major; 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, CCH₂and CHCH₂of Ad group, CH₂CH₂SCH₃, PCH₂CH₂),

1.76–1.56 (m, 9H, CHCH₂CH of Ad group, CHCH₂of 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/CH₂Cl₂and stirred for 3 h at room tem-

perature. Then, the mixture was concentrated in vacuo. CH₂Cl₂was 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, 52.0_minor, 51.7_major, 51.6_major, 51.5_minor, 47.5 (d, ¹J_PC= 103.1 Hz _minor

47.4 (d, ¹J_PC= 103.5 Hz)_major, 32.0_minor, 31.8_major, 31.3_minor, 31.3_major, 29.4, 28.4, 28.2, 22.7

)

,

1

(d, J_PC= 91.6 Hz)_major, 22.5 (d, J_PC= 91.0 Hz)_minor, 14.5_minor, 14.4_major

;

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

³J_HH= 5.9 Hz, 3H), 0.83 (d, J_HH= 4.8 Hz, 3H); ¹³C NMR (100 MHz, D₂O + CD₃OD)

δ

176.8 (d, ³J_PC= 15.7 Hz _minor, 176.4 (d, ³J_PC= 15.3 Hz)_major, 169.9_major, 169.8_minor, 53.8,

)

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1

53.6_major, 53.5_minor, 48.4 (d, J_PC= 102.5 Hz)_minor, 48.2 (d, J_PC= 103.4 Hz)_major, 36.5,

31.5_major, 31.2_minor, 29.3_major, 29.3_minor, 27.6_minor, 27.5_major, 23.8_minor, 23.7_major, 23.0 (d,

1

¹J_PC= 91.1 Hz

D₂O)

)

_minor, 22.8 (d, J_PC= 90.3 Hz)_major, 21.2, 21.0, 15.1; ³¹P NMR (160 MHz,

δ

45.6_major, 44.3_minor; 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, CHCH₂of ⁱBu group), 0.96–0.81 (m 6H,

3

CH₂CH(CH₃)₂); ¹³C NMR (100 MHz, D₂O)

δ

177.3 (d, J_PC= 15.9 Hz)_minor, 177.1 (d,

³J_PC= 15.4 Hz

¹J_PC= 102.4 Hz

)

_major, 168.9_minor, 168.9_major, 67.3, 48.8_major, 48.4_minor, 47.9, 47.3 (d,

1

)

_minor, 47.2 (d, J_PC= 103.3 Hz)_major, 36.2, 35.5_minor, 35.4_major, 26.7_major

,

1

26.6_minor, 22.7, 22.6, 22.6 (d, J_PC= 95.1 Hz)_major, 22.0 (d, J_PC= 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, ³J_PC= 14.3 Hz), 183.7 (d, ³J_PC= 13.9 Hz), 164.9, 164.6, 143.5, 143.4, 130.5, 128.1,

δ

1

128.0, 55.8, 55.7, 50.4 (d, J_PC= 100.4 Hz), 50.3 (d, J_PC= 100.0 Hz), 35.2, 35.2, 34.1 (d,

²J_PC= 11.1 Hz), 31.3, 31.4, 30.8, 30.6, 26.6 (d, J_PC= 91.9 Hz), 26.5 (d, J_PC= 91.6 Hz),

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, ³J_PC= 14.8 Hz)_minor, 178.0 (d, ³J_PC= 16.3 Hz)_major, 169.9, 53.8_minor

,

;

53.5_major, 48.2 (d, ¹J_PC= 101.6 Hz)_minor, 48.0 (d, ¹J_PC= 103.8 Hz)_major, 36.6_minor, 36.5_major

31.4_major, 31.2_minor, 29.3, 27.9_minor, 27.8_major, 23.9_minor, 23.8_major, 23.3 (d, ¹J_PC= 86.8 Hz)_minor

23.2 (d, ¹J_PC= 92.1 Hz)_major, 21.3, 21.0, 15.1; ³¹P NMR (160 MHz, D₂O)

δ 43.9_major, 42.8_minor

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, J_PC= 16.1 Hz), 175.1 (d, J_PC= 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, ¹J_PC= 91.6 Hz), 24.0 (d, ¹J_PC= 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, ³J_HH= 5.9 Hz,

3H), 0.92 (d, ³J_HH= 5.9 Hz, 3H), 0.71 (d, ³J_HH= 6.4 Hz, 3H), 0.68 (d, ³J_HH= 6.3 Hz, 3H);

¹³C NMR (100 MHz, CD₃OD) 175.6 (d, ³J_PC= 16.3 Hz), 175.1 (d, ³J_PC= 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, ³J_PC= 10.8 Hz), 25.3 (d, ³J_PC= 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, ³J_PC= 16.8 Hz), 175.1 (d,

δ

³J_PC= 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, ¹J_PC= 103.4 Hz), 24.0 (d, ¹J_PC= 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

(d, J_HH= 6.4 Hz, 3H), 0.93 (d, J_HH= 6.3 Hz, 3H), 0.77 (d, J_HH= 6.6 Hz, 3H), 0.74 (d,

³J_HH= 6.6 Hz, 3H); ¹³C NMR (100 MHz, CD₃OD) 198.2, 197.9, 175.7 (d, ³J_PC= 16.2 Hz),

δ

175.1 (d, ³J_PC= 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, ³J_PC= 11.0 Hz), 25.5 (d, ³J_PC= 11.1 Hz), 24.1, 24.0 (d, ¹J_PC= 91.1 Hz), 23.8 (d,

¹J_PC= 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]. K_Mof the isolated enzyme on

the synthetic substrate _L-leucine-p-nitroanilide (_L-Leu-pNA) was 0.86 mM, and V_maxwas

0.0012

µmol/min. K_Mvalues 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]. K_Mof the isolated enzyme on the

synthetic substrate -leucine-p-nitroanilide (_L-Leu-pNA) was 0.55 mM, and V_maxwas

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 K_M, V_max, and K_Iand 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 S_b.

The used weight factors have been calculated as follow:

w_i= 1/(σ_i²), σ²= (dy/dx)², where y = 1/V, or σ²= (dy/dx)²which gives the formula

for weight I = V⁴; w_i—statistical weight of the i-th measurement, σ_i²—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 (K_i= 14 µM) and 1 toward HvLAP (K_i= 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://ww w .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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25 of 27

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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Phosphinotripeptidic inhibitors of leucylaminopeptidases

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