functional characterization ... - ACS Publications

Subscriber access provided by MIDWESTERN UNIVERSITY

Article

Synthesis and structural/functional characterization of selective M14 metallo-carboxypeptidase inhibitors based on phosphinic pseudo peptide scaffold: Implications on the design of specific optical probes Giovanni Covaleda, Pablo Gallego, Josep Vendrell, Dimitris Georgiadis, Julia Lorenzo, Vincent Dive, Francesc X. Aviles, David Reverter, and Laurent Devel J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01465 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on January 29, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

Synthesis and structural/functional characterization of selective M14 metallocarboxypeptidase inhibitors based on phosphinic pseudo peptide scaffold: Implications on the design of specific optical probes

Giovanni Covaleda†, Pablo Gallego†, Josep Vendrell†, Dimitris Georgiadis§, Julia Lorenzo†, Vincent Dive‡, Francesc Xavier Aviles*†, David Reverter*† and Laurent Devel*‡

†Institut

de Biotecnologia i de Biomedicina and Departament de Bioquímica i de

Biologia Molecular, UniversitatAutònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain. ‡CEA,

Institut des Sciences du Vivant Frédéric Joliot, Service d’Ingénierie Moléculaire

des Protéines (SIMOPRO), Université Paris-Saclay, Gif-sur-Yvette 91190, France. §Department

of Chemistry, Laboratory of Organic Chemistry, University of Athens,

Panepistimiopolis Zografou, 15771, Athens, Greece.

Corresponding authors Laurent Devel. Phone: +33 169089565; Fax: +33 169089071; E-mail: [email protected]. David Reverter. Phone: +34 935868955; Fax: +34 935812011; E-mail: [email protected]. Francesc Xavier Aviles. Phone +34 606873290; Fax, +34 935812011; E-mail: [email protected]

ACS Paragon Plus Environment

1

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Metallo-carboxypeptidases of the M14 family (MCPs) are Zn2+ dependent exoproteases present in almost every tissue or fluid in mammals. These enzymes perform a large variety of physiological functions and are involved in several pathologies such as pancreatic diseases, inflammation, fibrinolysis and cancer. Here we describe the synthesis and functional/structural characterization of a series of reversible tightbinding phosphinic pseudo peptide inhibitors that show high specificity and potency towards these proteases. Characterization of their inhibitory potential against a large variety of MCPs, combined with high-resolution crystal structures of three selected candidates in complex with human CPA1, allowed to decipher the structural determinants governing selectivity for type-A of the M14A MCP family. Further, the phosphinic pseudo peptide framework was exploited to generate an optical probe selectively targeting human CPAs. The phosphinic pseudo peptides presented here constitute the first example of chemical probes useful to selectively report on type-A MCPs activity in complex media.


2

Page 2 of 51

Page 3 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Metallo-carboxypeptidases are exoproteases involved in a great variety of processes, from digestion to blood coagulation/fibrinolysis, inflammation, pro-hormone and neuropeptide processing among others, and are also reportedly involved in the progress of certain cancers and other pathologies 1,2. M14 metallo-carboxypeptidases (MCPs), one of the largest families of the metallopeptidase clan

3

including many variants from

archaea, bacteria and eukaryotes, have been divided into three main subfamilies based on structural similarity and sequence homology. The first one, which includes the digestive enzymes CPA1, CPA2 and CPB1, mast cell CPA3, as well as CPA4, CPA5, CPA6 and CPO from distinct localizations, has been termed subfamily M14A or A/B 3,4. The second subfamily, including the bioactive peptide-processing or regulatory enzymes CPN, CPE, CPM, CPD among others, has been termed M14B or N/E 5. In addition, a novel subfamily termed M14D, Nna-like or CCP comprising enzymes of larger size with predominant cytosolic location, has also been described

6,7.

An

additional classification into three main classes can be established according to substrate specificity against C-terminal aromatic/hydrophobic residues (A-like), basic residues (B-like) and acidic residues (O-like)

4,8.

With some relevant exceptions due to structural

similarities, this latter classification based on specificities grossly correlates with M14A, M14B and M14D subfamilies. Overall, differences in classification are also related to differences in cellular or tissular localization, a fact that is relevant for biotechnological or biomedical applications. A consensus sketch of the mutual recognition regions in peptide/protein substrates and MCPs considers that the essential structures are constituted by five subsites: P4, P3, P2, P1 and P1’ and the corresponding S4, S3, S2, S1 and S1’, in substrates and enzymes, respectively. Specificity of recognition is mainly governed by the substrate residue located C-terminally to the scissile peptide bond (P1’ position, Figure 1A), facing the S1’ specificity pocket of the enzyme. In addition, the substrate unprimed positions (P1 to P3) also contribute to the tuning of the selectivity profile towards different members of the MCP family, but to a lesser extent 8–10.


3


Page 4 of 51

Protease inhibitors, both of natural or synthetic origin, are major elements in the control of peptidase activity

11,12.

Besides the wide distribution of the natural ones,

protease inhibitors display a significant variety of structures, sizes and functionalities, particularly for the serine- cysteine- and metallo-endoproteases

13–16.

The relatively

recent attribution of metallo-exoproteases to diseases, as well as the more difficult chemistry involved, somewhat delayed the finding of inhibitors for this class of enzymes 1,2. An exception to this has been the development of synthetic small inhibitors for the angiotensin-converting enzyme (ACE), a dipeptidyl metallo-carboxypeptidase belonging to the M2 family, in what was an early and successful attempt to generate drugs to treat hypertension and some types of renal and cardiac dysfunction17,18. More recent reports on new inhibitors of proteases, including MCPs, describe both synthetic or natural proteinaceous molecules frequently associated to biomedical interest, of which hirudin, serpins and cystatins are some of the most relevant examples for the latter type 19–21. Most of the synthetic metallo-protease inhibitors that have been designed to date are competitive inhibitors that incorporate a zinc-chelating moiety targeting the zinc ion within the enzyme active site. Different families of peptides or pseudo peptides displaying a thiol, carboxylate, hydroxamate or phosphonate function as zinc-binding group have been described as potent metallo-protease inhibitors

22–24.

Fairly similar to

the latter, the phosphinic pseudo peptides with a zinc-chelating phosphinate moiety (PO2-CH2-) represent a unique and valuable class of compounds that share structural features with the transition-state intermediates generated during the hydrolysis of the peptide bond

25

resulting in competitive inhibitors with high affinity for their target.

More importantly, due to their capacity to probe the whole active site across primed and unprimed enzyme subsites

26

the phosphinic pseudo peptides offer further

opportunities for selective interactions compared to inhibitors with a hydroxamate or carboxylate zinc-binding moiety that probe a limited number of subsites. In addition, the phosphinate function itself seems to play a critical role in the inhibitor selectivity 27.


4

Page 5 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Due to their stability and their absence of toxicity, the phosphinic pseudopeptides are also ideal chemical tools to selectively target metalloproteases activity in vivo 28–32. In the frame of this study, we have explored an original series of phosphinic pseudopeptides enabling to better understand the molecular determinants governing selectivity between different members of the M14A family, particularly between A-type and B-type family members. To this purpose, we have designed a small library of 11 tripeptide phosphinic inhibitors differing in residues facing S1’, S1 and S2 subsites within MCPs catalytic cleft (Figure 1A). The affinity and selectivity profile of these pseudo peptides have been characterized against relevant recombinant human MCPs (i.e., CPA1, 2, 3, 4, B1, TAFI and D). Three pseudo peptides (1, 3 and 8) have been crystallized in complexes with human CPA1 (hCPA1). In addition, pseudo peptide 8 displaying a high potency toward hCPA1, 2 and 3 and sparing hCPB1 and D has been declined into three additional analogues with a N-terminal extension consisting in a PEG linker with a free amino function, a N-acyl moiety or an amino group conjugated to a fluorescent reporter (Figure 1B, compounds 9, 10 and 11 respectively). Such fluorescent probe, keeps a restricted selectivity for A-type M14A MCPs, displaying a null or very weak affinity for the B- or O- type forms as well as for a variety of proteases from different clans. Overall, the impact of these structural modifications on the binding properties of the inhibitors towards human MCPs has been systematically examined in vitro and structurally characterized.


5


Figure 1. Chemical structure of phosphinic pseudo peptides 1 to 11. A) Structure of pseudo peptide 1 to 7 differing in their P1’ to P2 residues facing subsites S1’ to S2 subsites. B) Chemical structure phosphinic pseudo peptides 8 to 11 with a simple acyl function (8), with a PEG extension (9 and 10) and a PEG extension conjugated to a fluorescent reporter (11).


6

Page 6 of 51

Page 7 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


RESULTS AND DISCUSSION Design and synthesis of phosphinic pseudo peptides 1 to 11: To generate an exploratory set of phosphinic pseudopeptides we selected basic units with three residues, covering the S2/S1/S1’ subsites of MCPs (Figure 1A). It is worth mentioning that the nature of the different P2, P1 and P1’ side chains in the phosphinic pseudo peptides were chosen according to previous knowledge from complexes between MCPs and natural protein inhibitors, such as the LCI-hCPA2 (PDB code 1DTD), ACI-hCPA1 (PDB code 3FJU), SmCI-hCPA4 (PDB code 4BD9), NvCI-hCPA4 (PDB code 4A94), TCIbCPA1 (PDB code 1ZLH) and PCI-bCPA (PDB code 4CPA). These natural compounds, respectively isolated from leeches, intestinal worms, marine worms, marine snails, tick and potato

1,33–38

harbor substrate-like properties with Ki values in the low

nanomolar/sub nanomolar range. In their P1’ position, the pseudo peptides harbored either a phenylalanine (1) or a homophenylalanine residue (2 to 11), coincident with the preference for hydrophobic side chains at A-like MCPs S1’ subsite, as derived from studies with synthetic substrates/inhibitors

2

or from proteome derived-peptide libraries

10,39.

Among the 11

compounds selected for synthesis, eight were simple pseudo tripeptides with chemical variations both in their P1 and P2 position. Since the P1 position has only moderate impact on substrate recognition, either a phenylalanine (1 and 2) or an alanine (3 to 11) was indifferently incorporated. Regarding the P2 position, the impact of aliphatic (3), acidic (4), basic (5), constrained (7), and polar (6 and 8) side chains on inhibitors binding was explored. Three additional pseudo tripeptides with either an N-terminal PEG spacer (9 and 10) or the same extension plus a 6SIDCC fluorophore (11) were also synthesized. A short PEG spacer was incorporated between the targeting motif and the bulky fluorescent group to limit steric clashes during probes/MCPs interaction. In the perspective of cells assay, 6SIDCC fluorophore as a highly negatively charged fluorescent tag was selected as it displays reduced unspecific binding by comparison with standard cyanine derivatives 40,41. The phosphinic pseudo peptide 1 was obtained as previously described


7

42.

The


Page 8 of 51

phosphinic pseudo peptides 2 and 3-11 were synthesized on solid support from FmocPhe-POOAd-hPhe-OH

43

and

Fmoc-Ala-POOAd-hPhe-OH

17

building

blocks

respectively (Scheme 2). Building block 17 was first synthesized in solution according to Scheme 1.

Scheme 1: Chemical pathway to access phosphinic building block 17. Briefly, diethylmalonate was first converted into the acrylate 12 in three steps (65% yield), after alkylation in presence of 2-phenylethyl bromide, mono saponification, and Knoevenagel condensation with formaldehyde. This acrylate was then esterified with benzyl alcohol to yield protected acrylate 13 (70% yield). This electrophile then participated in a P-Michael addition reaction with phosphinic acid 15 under mild, silylating conditions

43

to give compound 16 with a yield of 59%. Compound 15 was

generated in two steps from derivative 14 prepared as previously reported

44,45.

Finally,

17 was obtained in two steps after protection of the hydroxyphosphinyl moiety with an adamantyl protecting group followed by a saponification step (85% yield). The phosphinic building block 17 as a mixture of diastereomers, was thus synthesized in seven steps from the commercially available diethylmalonate and precursor 15 in an overall yield of 23%.


8

Page 9 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 2: Solid phase synthesis of phosphinic pseudo peptides 3 to 11. The two Fmoc-phosphinic building blocks Fmoc-Phe-POOAd-hPhe-OH

43

and

Fmoc-Ala-POOAd-hPhe-OH 17 were first activated in presence of DIC followed by the addition of DMAP. This solution was immediately added to the resin and the coupling reaction was carried out under microwave irradiation at 60°C for 10 min. Elongation of the pseudo peptide sequences was performed using standard Fmoc strategy under microwave irradiation. For compounds 2-8 and 10, the acetylation reaction was performed at room temperature in presence of 1-acetylimidazole. In the case of pseudo peptides 9, 10 and 11, 8-(Fmoc-amino)-3,6-dioxaoctanoic acid was coupled to the peptide sequence in conditions comparable to those used for amino acid incorporation. Each resulting pseudo peptide was then cleaved from the solid support and analyzed by RP-HPLC. Analysis of the crude showed two peaks corresponding to two diastereomers that possess a S configuration at P2, a R configuration at P1 and a R or S configuration at P1’. Regardless of the P2 position within the pseudo peptide structure, the major peak observed in RP-HPLC systematically corresponded to the most potent inhibitor towards


9


Page 10 of 51

CPs. This is a known trend in the chemistry of phosphinic peptides, initially reported by B.P Roques and co-workers

45

and verified by others later on

27,46,47

with no exception

reported to our knowledge. Since in transition-state analogues maximum potency is directly related to mimicry of natural peptides, we postulated that the most potent isomer possessed a S configuration within P1’ position. Furthermore, given that the major isomer of the starting block (16) leads to the major isomer of the pseudo peptides, we deduced that the major diastereomer (0.8) of 16 possess the RS configuration and the minor diastereomer (0.2) the RR configuration. To access fluorescent derivative 11, compound 9 was coupled to 6S-IDCC-NHS ester

48.

The phosphinic pseudo peptides 1

to 11, as well as the building blocks 6 and 8, were stored at −20 °C and protected from light.


10

Page 11 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Inhibitory potency and selectivity of phosphinic pseudo peptides. The Ki values for compounds 1 to 11 were determined on a set of five relevant human MCPs (Table 1). These phosphinic compounds act as tight-binding inhibitors of MCPs, with Ki values in the nanomolar to sub nanomolar range. In particular, they displayed the strongest affinity towards hCPA1, with Ki values 10-to-25-fold lower than those for other A/Btype MCPs (Table 1). The most significant difference appears when P1’ side chain length is increased. Thus, elongation by a single methylene group leads to an inhibitor that displays almost no affinity for B-type enzymes. Regarding the P1 position, its simplification (2 with a methylbenzene vs 3 with a methyl group) has no significant impact on the inhibitor potency and selectivity towards the different A-type MCPs (Table 1). Table 1. Potency of Phosphinic Peptide Inhibitors (Ki) toward several MCPs.

Ki (nM) P3

P2

P1

P1’

hCPA

hCPA

hCPA

hCPB

1

2

4

1

hCPD

1

CH3

0.04 ± 0.01

1.0 ± 0.2

0.39 ± 0.06

0.12 ± 0.01

NI

2

CH3

0.08 ± 0.02

0.2 ± 0.1

1.9 ± 0.2

NI

NI

3

CH3

CH3

0.087 ± 0.002

0.7 ± 0.1

3.3 ± 0.3

NI

NI

4

CH3

CH3

3.1 ± 0.2

4.6 ± 0.7

38.6 ± 3.2

NI

NI


11


Page 12 of 51

5

CH3

CH3

1.2 ± 0.2

2.4 ± 0.3

25.4 ± 1.9

NI

NI

6

CH3

CH3

5.9 ± 0.4

13.8 ± 1.4

60.6 ± 7.2

NI

NI

7

CH3

CH3

1.7 ± 0.1

3.2 ± 0.6

7.5 ± 0.5

NI

NI

8

CH3

CH3

0.107 ± 0.002

1.7 ± 0.3

2.6 ± 0.2

NI

NI

9

CH3

0.3 ± 0.1

7.8 ± 0.5

5.4 ± 0.6

NI

NI

10

CH3

0.1287 ± 0.0004

5.7 ± 0.4

3.5 ± 0.2

NI

NI

11

CH3

0.089 ± 0.005

5.3 ± 0.7

5.7 ± 0.9

NI

NI

Data are means (n=3) ± SD. Ki in nM. NI: No inhibitory activity detected for the [I]0 range from 1 nM to 1 mM.

Interestingly, when an acidic (4), a basic (5) or a polar side chain (6) is present in the P2 position a drop of affinity ranging from 10 to 20 is observed toward the three hCPA 1, 2 and 3. Conversely, P2 aliphatic or aromatic side chains (3, 7 and 8) restore the binding capacity of the inhibitors towards those enzymes. Additionally, none of these compounds are able to inhibit human CPD, a representative member of the M14B subfamily. The synthesized series is therefore addressed to the M14A subfamily of MCPs. Based on these results, compound 8 displaying a high potency and selectivity


12

Page 13 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


towards hCPA 1, 2 and 3, was selected as a relevant starting scaffold for the development of an optical probe 11. Inhibitory activity of 11 as well as that of its precursors 9 and 10 was assessed on MCPs. Incorporation of the linker (with two different ends) and fluorophore does not substantially affect the enzymatic inhibitory potency and selectivity of the constructs, as indicated by Ki values in the nanomolar range for these three molecules (Table 1). All the above kinetic data was collected in steady state conditions after 2h of pre incubation with inhibitors 1-11 since in certain cases inhibitors might act as slow binders, besides tight binders 49. In addition, pre-steady state data was also collected to derive kon and koff, and dependent Ki, from the action of compounds 3, 8, 10 and 11 on human CPA1. The results are presented in Table 2 (see also Figure S1, S2 and S3 for details). The derived constants indicate that the four constructs can be considered as a slow tight binding inhibitors, with values equivalent to those of well-known cases of proteases 50, and with derived pre-steady state Ki values very similar to the ones found in steady state conditions. These experiments also confirm that the addition of the PEG bridge and fluorescent head resulting in optical probe 11 does not significantly modify such constants Table 2. Kinetic parameters of phosphinic inhibitors on human carboxypeptidase A1. Pre-steady-state

Steady-state

koff

kon

Kiapp=koff/kon

Ki

Ki

(x10-5 s-1)

(x105 M-1.s-1)

(x10-9 M)

(x10-9 M)

(x10-9 M)

3

6.0 ± 0.2

1.27 ± 0.02

0.47 ± 0.02

0.14 ± 0.01

0.087 ± 0.002

8

7.6 ± 0.3

1.44 ± 0.04

0.53 ± 0.04

0.16 ± 0.01

0.107 ± 0.002

10

7.0 ± 0.2

1.33 ± 0.04

0.53 ± 0.02

0.16 ± 0.01

0.1287 ± 0.0004

Compound


13


11

4.8 ± 0.5

2.03 ± 0.04

0.24 ± 0.03

Page 14 of 51

0.07 ± 0.01

0.089 ± 0.005

[hCPA1] =0.5 nM. 100 mM of AAFP substrate. T=37ºC. Data are means (n=3) ± SD.

Interestingly, probe 11 not only strongly inhibits other A-type M14A-MCPs like secretory mast cells CPA3, but did not display any inhibitory capacity against other Btype M14B-MCPs (like blood hCPU/TAFI and hCPN), cytosolic M14D-MCPs (like hCCP1 and hCCP6), or other peptidases of different catalytic type such as aspartic, serine or cysteine peptidases (Table 3). Moreover, it displayed only modest inhibitory activity in the micromolar range against other metallo-peptidases of distinct families, such as matrix metallo-proteases 9, 10, 11 and 13 (Table 3). Table 3. Potency of Phosphinic-based probe (compound 11) (Ki) toward several proteases. Enzyme

Ki (nM)

rat CPA3

0.017 ± 0.008

hCPU (TAFI)

NI

hCPN

NI

hCCP1

NI

hCCP6

NI

hMMP9

2588 ± 560

hMMP10

983 ± 198

hMMP12

1340 ± 195

hMMP13

562 ± 169

pPepsin

NI

Papain

NI

bTrypsin

NI

hThrombin

NI

hNElastase

NI

Data are means (n=3) ± SD. Ki in nM. NI: No inhibitory activity detected for the [I]0 range from 1 nm to 1 mM

Structural characterization of compounds 1, 3 and 8/hCPA1 complexes. In order to


14

Page 15 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


structurally-functionally analyze the above phosphinic compounds in a detailed and integrated way, three of them (compounds 1, 3 and 8) were selected and characterized by protein X-ray crystallography in complex with hCPA1. The crystal structure of 1, 3 and 8 in complex with this enzyme was solved at 2.29 Å, 2.27 Å and 1.72 Å resolution respectively. The quality of the electron density maps allows the perfect trace of 1, 3 and 8 side chains within the enzyme subsites (Figure 2). This allowed us to confirm the three stereogenic centers relative configuration of compounds 3 and 8. Indeed, the electron density maps clearly display only one configuration in P1’ position and no hint of an electron density for another configuration was observed.


15


A)

Page 16 of 51

P1

H N

O

O N H

O

P2

P

OH O

HO

1

B)

P1 ’

P1 H N

O

O N H

O

P2

P

OH O

HO

3

P1 ’

C)

P2 Y

P1 H N

R145

Y248

O

O N H

O

P

OH

R145

O

HO

R71

OH

P1’ hF

M203

H69

I255 T268

P1’ hF

8

R71

I243

P1 A

P2

P2 Y

Y248

E270

E72 H196

E270

P1 ’

Figure 2. Structural details of the complexes between hCPA1 and the phosphinic compounds 1 (A), 3 (B) and 8 (C). Left column: Chemical structure of compounds 1, 3 and 8. Central column: 2Fo-Fcomit map (contour sigma level is 1.2) of 1, 3 and 8 in complex with hCPA1 with the respective following PDB IDs: 4UEZ, 4UEE and 6I6Z. Right column: close-up view of the primary binding regions within the active site of human hCPA1. The phosphinic compounds are represented in yellow sticks and hCPA1 residues are shown in cyan. The functionally essential zinc is visualized as a blue sphere.

Inhibitor 1 displayed smaller B-factors around its P1’ side chain and the phosphinate


16

Page 17 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


chelating moiety, indicating a low degree of flexibility around this binding region. The zinc atom is located at close distance to one oxygen atom of the phosphoryl group (2.19 Å), and also coordinates with the two histidine (at 2.04 and 2.03 Å, respectively) and the glutamic acid (at 2.05 Å) of the carboxypeptidase zinc-binding residues (Figure 2A). Additionally, the zinc-coordinating oxygen of the phosphoryl group is also at 2.77 Å to the active-site Glu270 of the enzyme, the general base residue in the water-mediated nucleophilic catalysis of metallo-carboxypeptidases. The second oxygen of the phosphoryl group is at 2.69 Å to the active-site Arg127, which is responsible for the polarization of the carbonyl group of the scissile peptide bond during the catalysis and shaping of the tetrahedral intermediate. Thus, the most important residues in the active site and catalytic machinery of CPA1 are trapped in a net of close interactions with the inhibitor. The geometry of this complex perfectly resembles the tetrahedral intermediate formed during the transition state of the catalytic mechanism and, therefore, these ligands can be considered as transition-state analogues. In the most accepted mechanism of MCPs activity, the zinc-bound water molecule, polarized by Glu270, is added to the substrate carbonyl group of the scissile bond, forming an unstable tetrahedral structure that will lead to the cleavage of the peptide bond 8. In the case of the complexes of other metallopeptidases with phosphinic peptide inhibitors, the phosphinate-methylene group of the inhibitor mirrors the tetrahedral intermediate of the transition state

26,27

as

its tetrahedral configuration is shaped by the zinc-coordinating oxygen, next to the active-site nucleophile (equivalent to Glu270 in MCPs) that mimics the polarized catalytic water, and by the second oxygen mimicking the carbonyl oxygen polarized by a positive group (equivalent to Arg127 in MCPs). The observed quasi-planar geometry of the bonding between the side-chain atoms of Glu270 and Arg127 of hCPA1 with the zinc atom and with the two oxygen atoms of the compound 1 phosphoryl group confirms previous hypotheses about the catalytic mechanism of carboxypeptidases

1,8.

The same geometry is observed in the other complex structures between phosphinic compounds 3 and 8 with hCPA1 (Figure 2B and 2C). The P1’ subsite of the phosphinic peptide 1 is formed by both the side-chain of a


17


Page 18 of 51

phenylalanine and the carboxylate group, which resembles the typical C-terminal carboxylate binding with Arg145 and Asn144. Tyr248, normally forming a hydrogen bond with the amide nitrogen of the scissile peptide bond, also displays a “down” conformation but interacts with one of the two carboxylate oxygens of the P1’ subsite (Figure 2A). Considering that A-type carboxypeptidases selectively hydrolyze bulky and aliphatic C-terminal side-chains, and that even tryptophan residues can be accommodated in the large hydrophobic pocket 4. The presence of a Phenyl residue in the hydrophobic pocket shaped by Met203, Ile243, Ile247, Ile255, Thr268 and Asn144 simply confirms this preference, which is reinforced by the fact that compounds bearing a bulkier homophenyl at the P1’ position also behave as potent CPA1 inhibitors (Table 1 and Figures 2B-2C). Phosphinic compound 1 displays nano- or sub nanomolar inhibitory constants against several A-type carboxypeptidases of the M14A family and is also unexpectedly able to inhibit B-type carboxypeptidases (see Table 1), despite the preference

of

the

latter

for

basic

C-terminal

residues.

However,

B-type

carboxypeptidases are not inhibited by compounds containing a homophenyl at the P1’ position (as happens with compounds 3 and 8; see Figures 2B-2C). As shown in the comparison between the S1’ pockets of A-type and B-type carboxypeptidases in Figure 3, hCPA1 is able to accommodate both Phenyl and homophenyl at the P1’ position. However, a model of our phosphinic inhibitors docked into human CPB1 active site displays steric hindrances within the cavity when homophenyl is located at P1’. This is in accordance with the observed lack of inhibition against B-type carboxypeptidases. This result reveals the phosphinic inhibitors containing a homophenyl Alanine residue at the P1’ position as optimal MCPs inhibitors able to distinguish between A-type and Btype classes of M14A metallo-carboxypeptidases.


18

Page 19 of 51


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


19


Figure 3. Comparison of the hydrophobic binding pockets of CPA1 and CPB in complex with phosphinic compounds 1 and 8. A) close-up stereo view of the surface representation of the cavity of the substrate binding-site of hCPA1 in complex with phosphinic compounds 1 and 8. B) close-up stereo view of the surface representation of the cavity of the substrate binding-site of hCPB modelled in complex with the same compounds. Compounds 1 and 8 are shown in stick representation in green and yellow, respectively. Major active site residues of hCPA1 and hCPB are labelled and shown in stick representation.

The electron density maps of the P1 and P2 sub-sites of the phosphinic peptides are well defined in all structures, and in compound 1 is formed by the side chains of phenylalanine and leucine, respectively. The peptide bond between the P1 and the P2 is also forming hydrogen bond interactions, in particular between the P2 carbonyl oxygen of the inhibitor with Arg71 (at 2.81 Å). The major differences between 1 and 3 are found in the P1 subsite, with the substitution of a phenylalanine by alanine in compound 3. However, kinetic analysis indicates that the side chain of the P1 position does not affect the inhibitory properties of the compound, as shown by comparison of inhibitory constants of compounds 2 and 3 (Table 1). As commented upon earlier, the P2 Leucine (1 and 3) or Tyrosine residue (8) seems to play a major role in the activity of the inhibitor, as its substitution by either Glutamate, Lysine, Proline or Serine (compounds 4 to 7) reduces the inhibitory constant for hCPA1 by two orders of magnitude (Table 1). This may be explained by a favorable interaction between aromatic or aliphatic P2 side chains and CPA-active-site Tyr248 at 3.5 Å distance (Figure 2A and C). In solution, such an interaction may induce the movement of Tyr248 to the “down” orientation described during catalysis. However, in crystal structures, this favorable interaction could compete with additional hydrophobic interactions from its neighboring symmetric molecule and might explain both a weaker electron density map and higher B-factors at P2 site by comparison to other sites. No particular structural effect due to the substitution of P2 Leucine by Tyrosine (compounds 3 and 8, respectively) was observed, coincident with the similarity observed in the inhibitory constants of those compounds against hCPA1.


20

Page 20 of 51

Page 21 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Inhibition on a peptide degradative assay. The capability of the new inhibitors in an orthogonal assay mimicking real conditions was evaluated with construct 8 on the neurotensin peptide used as model. Samples taken at different intervals from 0-7h degradative experiment by human CPA1 (20nM) in the absence and presence of inhibitor 8 at two different concentrations (5 and 500nM), were analyzed by MALDITOF MS. As shown in figure S4, the phosphinic pseudo peptide 8 counteracts the hCPA1 action over the substrate. Cytotoxicity and cellular uptake. To start evaluating the potential utility of the herein synthesized phosphinic inhibitors and derived fluorescent probe under biological conditions, cytotoxicity/cellular viability and initial uptake studies were performed on both HeLa and HEK293T human cells. Importantly, Phosphinic inhibitor 8 at 1 and 5 µM applied to cell cultures during 24h showed no sign of cell cytotoxicity using the XTT assay (Figure S5). A similar behavior was observed by microscopy when the treatment was performed with fluorescent probe 11 (data not shown). After determining that inhibitors have no effect on the cell viability, we investigated the probe cellular uptake in the first cell line type. When using HeLa cells with abundant human CPA3 expression in cytoplasm (see experimental section), and after a 6h treatment with probe 11 (1 or 5 µM) followed by cell washings, cell analysis by fluorescence confocal microscopy showed that only a small part of the fluorescent probe internalized into cells (Figure S6), the rest being washed away. This poor internalization is consistent with the negative global charge of probe 11. In this respect, a similar behavior has been observed for another optical probe whose structure was derived from a phosphinic peptide

40.

Interestingly, internalized probe 11 was found to co-localize with human CPA3 as shown in Figure S6, proving its capacity to detect CPA-type enzymes in biological assays


21


Page 22 of 51

New chemical tools for studying human CPAs. The pioneer peptide-based phosphonic or phosphinic metallo-carboxypeptidase inhibitors

23,51,52

were shown to behave as transition state

analogues and to display inhibition constants in the pico-femtomolar range for A-like enzymes. Detailed crystal structures and enzymatic parameters were obtained in complex with bovine CPA, but the specificity of those molecules on proteolytic enzymes from the same or related families or clans was not explored. Later reports related to the present work 26,53 describe peptide phosphinic derivatives addressed to the M2 metallo-carboxypeptidase angiotensin-converting-enzyme (ACE) 42,

to zinc peptidases

54,55

and to TAFI, a circulatory plasma carboxypeptidase B

56.

Here we

report an original series of phosphinic pseudo peptides affording for both potency of inhibition and high selectivity towards human carboxypeptidases. Specifically, we have demonstrated that a P1’ homo phenylalanine within the phosphinic motif allowed to restrict selectivity to A-type enzymes. Importantly, we also showed that such a scaffold could be exploited for the design of optical probes targeting these enzymes. Therein, the close similarity of Ki values between parent compound 8, compounds 9-10 and optical probe 11 indicates that additional N-ter extensions, even combined with a bulky fluorophore, do not compromise the binding strength. Noteworthy, crystals of probe 11 in complex with human CPA1 were obtained as indicated by the fluorescent green color of these latter. Despite the flexibility of the PEG fluorophore extension resulting in a choppy-defined electron density component, the positioning of the phosphinic moiety within the CPA1 catalytic cleft has been unambiguously confirmed and was similar to that of compound 8. Overall, compound 11 constitutes to our knowledge the first example of specific optical probe for human CPA-like enzymes within the M14A subfamily, with no detectable activity against human M14B and M14D carboxypeptidase subfamily variants and only a very weak one against metallo-endopeptidases of the MMPs family. Many of the reports commented upon before had an indirect or direct association with biomedical goals, like the recent ones on phosphinic inhibitors of the Helicobacter pylori Zn carboxypeptidases Csd4

57.

While only a limited number of CPA-like forms are located within

the acinar cells of the pancreas, in the mast cells granules or in a few cases in the cytoplasm 1,5,14, the majority of them act extracellularly and, in some cases, have been related with diseases, like the Duane syndrome and epilepsy5. With their negatively charged phosphinate function,


22

Page 23 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


phosphinic pseudo peptides can be considered as ideal tools to preferentially bind to extracellular targets40. This property combined with the excellent selectivity displayed by compounds 2, 3, 8 towards the M14A family encourages further work focused on chemical modifications and structural polishing in order to subsequently validate and adapt these new molecules to in vivo action for biomedical applications. To this extent, the design of a first fluorescent probe 11 for Atype MCPs and its validation in a human cell model in culture constitute an important step and opens possibilities to study these proteases in vivo through fluorescent imaging.

CONCLUSIONS The combination of the proper size of a pseudo-tripeptide with an uncleavable phosphinic unit harboring a phenylalanine side chain at the C-terminus/P1’ subsite, results in very tight binding inhibitors for human CPA1-type forms. The developed compounds also show an excellent inhibitory capability against CPB-like enzymes, which abruptly fades when their P1’ side chain is elongated. Importantly, such a P1’ modification has almost no impact on the inhibitory constants against a number of M14 MCPAs variants, and only a moderate impact when combined with variations at P1 and P2 positions. Additionally, a A-type M14 MCPs selective fluorescent probe derived from a phosphinic pseudo peptide with a P1’ homo phenylalanine, a P1 alanine and a P2 tyrosine has been developed and its capacity to co-localize with a CPA enzyme in human cell cultures has been demonstrated. These results both validate our structure-based design and suggest that this optical probe, or other ligand-derived probes with a similar phosphinic scaffold, may be developed for targeting CPA in vivo activity through different imaging modalities.


23


Page 24 of 51

EXPERIMENTAL SECTION 1. Synthetic Chemistry. 1.1. Chemical reagent and procedures. All Commercially available reagents and solvents were used as received without further purification. Diisopropylcarbodiimide (DIC) and Trifluoroacetic acid (TFA), 8-(Fmoc-amino)-3,6-dioxaoctanoic acid were purchased from Aldrich (Saint Louis, MO, USA). Wang resin® (100-200 Mesh, 0,9 mmol/g) and Fmoc-AA-OH were purchased from Merck (Darmstadt, Germany). 6Chloro-1-Hydroxybenzotriazole (ClHOBt) was purchased from Molekula (Newcastle, United Kingdom). 6S-IDCC-NHS ester was purchased from IC Discovery (Berlin, Germany). Pseudo peptide syntheses were performed manually in polypropylene syringe (Supelco) equipped with a polyethylene frit and a stopper. Microwave experiments were performed with a Discover apparatus (CEM µWave, Matthews, NC, USA) using the open vessel mode with a SPS kit. Reactions were monitored by thinlayer chromatography (Merck TLC aluminum sheets coated with silica gel 60F254). Compounds were purified by Flash chromatography (Silica gel Si 60, 40-43mm). 1H (200 MHz),

13C

(50 MHz) and

31P

(81 MHz) NMR spectra were recorded on either a Varian

200 MHz Mercury (compounds 12 to 17) or a Bruker 500Mz Avance (compounds 3, 8, 10 and 11). 1H and 13C spectra are referenced according to the residual peak of the solvent based on literature data 58. 31P NMR chemical shifts are reported in ppm downfield from 85% H3PO4 (external standard).

13C

and

31P

NMR spectra are fully proton decoupled.

Suggested 1H NMR peak assignments of reported compounds are based on 2D COSY experiments. Data are reported as follows: chemical shift, multiplicity (s= singlet, d= doublet, t= triplet, q= quartet, br= broad, m= multiplet), coupling constants (Hz) and integration. Optical rotation data were acquired on a Perkin-Elmer 343 polarimeter at 25oC. DO measurements were performed on a Shimadzu UV spectrophotometer (UV1800). Analytical RP-HPLC separations were performed on a LC-20AB Prominence apparatus (Shimadzu Corp., Japan) using a solvent system consisting of (A) 0.1% v/v aqueous TFA, and (B) 0.1% v/v TFA in CH3CN on three different columns :


24

a

Page 25 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Hypersil C18 column (4.6 x 250 mm, 5 µm), flow rate=1 ml.min-1, linear gradient: 0-30 min / 0-100% B : conditions 1, a Kromasil® C8 column (4.6 x 150 mm, 5 µm), flow rate=1 mL.min-1, linear gradient: 0-30 min / 0-100% B : conditions 2, or a XBridge BEH300 C18 column (2.1 x 150 mm, 5 µm), flow rate=1 mL.min-1, linear gradient: 0-30 min / 0-100% B: conditions 3.Preparative RP-HPLC separations were performed using the solvent system on Kromasil® C18 (10 x 250 mm, 10 µm, flow rate=4 mL.min-1), linear gradient: 030 min / 0-100% B: conditions 4, or a Kromasil® C8 column (10 x 250 mm, 10 µm), flow rate=4 mL.min-1, linear gradient: 0-30 min / 0-100% B: conditions 5. Retention times (Rt) were reported in minutes. Mass spectrometry data were registered using a 4800 MALDITOF mass spectrometer (Applied Biosystems, Foster City, CA, USA) or an ion trap Esquire HCT spectrometer (Bruker Daltonics, Billerica, MA, USA). Amino acid compositions were performed on an aminoTac JLC-500/V amino acids analyzer (JEOL, Tokyo, Japan). The identity and purity (> 95% pure) of each newly synthesized compound is assessed by analytical RP-HPLC, NMR, and mass spectrometry. 1. 2. Compound synthesis and characterization. Solid phase synthesis of pseudo peptides (2-10). Wang resin® (1 equivalent) was first swelled in DCM for 15 min at room temperature. The appropriate Fmoc-phosphinic building block (3 equivalents) was dissolved in DMF/DCM: 1/1 and activated with DIC (3 equivalents, RT, 5 min) followed by the addition of DMAP (0.5 equivalent). The solution was immediately added to the resin and the coupling reaction was carried out under microwave irradiation (60°C, 25 W, 10 min). Fmoc removal was performed with piperidine 20% in DMF under microwave irradiation (3 x 2min, 60°C, 25 W) and the resin was then washed (2 x DMF, 2 x DCM). Fmoc-AA(OtBu)-OH (0.27 g, 0.64 mmol, 5 eq) was pre-activated during 5 min in presence of DIC (5 equivalents) and Cl-HOBt (5 equivalents) in DMF and added to resin. The coupling reaction was performed under microwave irradiation (10 min, 60°C, 25 W). To access compounds 2 to 8, after Fmoc removal and washing steps, their N-terminal extremity was acetylated with 1acetylimidizole (100 equivalents) in DMF (45 min, RT). To access compounds 9 and 10 incorporating a PEG linker, an additional elongation step on the N-terminal extremity


25


was implemented. In this respect, 8-(Fmoc-amino)-3,6-dioxaoctanoic acid (5 equivalents) was first activated for 5 minutes in the presence of DIC (10 equivalents) and HOBt (10 equivalents) and then incorporated onto the solid support. The coupling reaction was run under microwave irradiation (60°C, 25 W, 20 min) followed by a Fmoc removal step. In the case of compound 10, this step was completed by an acetylation step as described above. For each individual pseudo peptide, the resin was then washed as described and suspended in TFA/ H2O: 95/5 for 45 min at room temperature under gentle stirring. The resin was removed by filtration and washed with TFA/DCM: 1/1 (30 min, RT). This cycle was repeated twice. Combined filtrates were evaporated under light flow of argon and the crude material purified by RP-HPLC. (2S)-2-({[(1R)-1-(N-acetyl-L-leucylamido)-2phenylethyl](hydroxy)phosphoryl}methyl)-4-phenylbutanoic acid (2). Obtained as a foamy white solid (14 %) after semi preparative RP-HPLC using conditions 4. Rt=16.25 min in conditions 1, Rt=20.0 min in conditions 2. MS m/z for [C27H36N2O6P]-, calc. 515.6, found 515.3. (2S)-2-({[(1R)-1-(N-acetyl-L-leucylamido)ethyl](hydroxy)phosphoryl}methyl)-4phenylbutanoic acid (3). Obtained as a foamy white solid (42 %) after RP-HPLC purification using conditions 4, Rt=14.2 min in conditions 1, Rt=13.8 min in conditions 2, Rt=19.7 min in conditions 3. 1H NMR (500 MHz, D2O) δ 8.09 (t, J= 6.0 Hz, 1H), 7.62 (d, J= 10.4 Hz, 1H), 7.28 – 6.87 (m, 5H), 4.22 – 4.13 (m, 1H), 3.98 – 3.77 (m, 1H), 2.63 –2.47 (m, 3H), 1.86 – 1.71 (m, 3H), 1.63 – 1.40 (m, 4H), 1.13 (dd, J= 13.1, 7.6 Hz, 3H), 0.81 (d, J = 6.6 Hz, 3H), 0.77 (d, J = 6.6 Hz, 3H). HRMS m/z for [C21H32N2O6P-], calc. 439.2003, found 439.2108. (2S)-2-({[(1R)-1-(N-acetyl-L-glutamyl-1-amido)ethyl](hydroxy)phosphoryl}methyl)-4phenylbutanoic acid (4). Obtained as a foamy white solid (35 %) after semi preparative RP-HPLC in conditions 4. Rt=11.3 min in conditions 1, Rt=14.7 min in conditions 3. MS m/z for [C20H28N2O8P]-, calc. 455.4, found 455.2. (2S)-2-({[(1R)-1-(Nα-acetyl-L-lysylamido)ethyl](hydroxy)phosphoryl}methyl)-4phenylbutanoic acid (5). Obtained as a foamy white solid (28 %) after semi preparative


26

Page 26 of 51

Page 27 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


RP-HPLC in conditions 4. Rt=10.9 min in conditions 1, Rt=14.1 min in conditions 3. MS m/z for [C21H33N3O6P]-, calc. 454.5, found 454.3. (2S)-2-({[(1R)-1-(N-acetyl-L-serylamido)ethyl](hydroxy)phosphoryl}methyl)-4phenylbutanoic acid (6). Obtained as a foamy white solid (41 %) after semi preparative RP-HPLC in conditions 4. Rt=10.9 min in conditions 1, Rt=14.5 min in conditions 3. MS m/z for [C18H26N2O7P]-, calc. 413.3, found 413.2. (2S)-2-({[(1R)-1-(N-acetyl-L-prolylamido)ethyl](hydroxy)phosphoryl}methyl)-4phenylbutanoic acid (7). Obtained as a foamy white solid (38 %) after semi preparative RP-HPLC in conditions 4. Rt=12.3 min in conditions 1, Rt=16.3 min in conditions 3. MS m/z for [C20H28N2O6P]-, calc. 423.4, found 423.2. (2S)-2-({[(1R)-1-(N-acetyl-L-tyrosylamido)ethyl](hydroxy)phosphoryl}methyl)-4phenylbutanoic acid (8). Obtained as a foamy white solid (42 %) after semi preparative RP-HPLC in conditions 4. Rt=12.7 min in conditions 2, Rt=12.5 min in conditions 3. 1H NMR (500 MHz, D2O) δ8.04 (d, J= 7.8 Hz, 1H), 7.68 (d, J= 10.4 Hz, 1H), 7.28 – 7.09 (m, 5H), 7.08 – 6.99 (m, 2H), 6.78 – 6.69 (m, 2H), 3.89 – 3.77 (m, 1H), 3.03 – 2.91 (m, 1H), 2.79 – 2.65 (m, 1H), 2.53 – 2.34 (m, 3H), 1.92 – 1.81 (m, 1H), 1.75 – 1.59 (m, 2H), 1.53 – 1.37 (m, 1H), 1.14 – 1.06 (m, 3H). HRMS m/z for [C24H30N2O7P]-, calc. 489.4780, found 489. 4801. (2S)-2-({[(1R)-1-(N-{2-[2-(2-aminoethoxy)ethoxy]acetyl}-Ltyrosylamido)ethyl](hydroxy)phosphoryl}methyl)-4-phenylbutanoic

acid

(9).

Obtained as a foamy white solid (27%) after semi preparative RP-HPLC in conditions 4. Rt=11.3 min in conditions 2, Rt=18.3 min in conditions 3. MS m/z for [C28H39N3O9P]-, calc. 592.6, found 592.3. (2S)-2-({[(1R)-1-(N-{2-[2-(2-acetamidoethoxy)ethoxy]acetyl}-Ltyrosylamido)ethyl](hydroxy)phosphoryl}methyl)-4-phenylbutanoic

acid

(10).

Obtained as a foamy white solid (57%) after semi preparative RP-HPLC in conditions 4. Rt=12.6 min in conditions 2, Rt=19.2 min in conditions 3. 1H NMR (500 MHz, D2O) δ7.94 (d, J= 8.2 Hz, 1H), 7.90 (br s, 1H), 7.80 (d, J= 10.4 Hz, 1H), 7.27 – 7.09 (m, 4H),7.08 – 7.00 (m, 2H), 6.78 – 6.68 (m, 2H), 3.95 – 3.76 (m, 2H), 3.56 (s, 1H), 3.51 – 3.41 (m, 4H), 3.41 – 3.31 (m, 2H), 3.27 – 3.19 (m, 2H), 3.12 – 3.01 (m, 1H), 2.82 – 2.68 (m, 1H), 2.53 – 2.39 (m,


27


3H), 1.86 (s, 3H), 1.96 – 1.81 (m, 1H), 1.79 – 1.61 (m, 2H), 1.54 – 1.42 (m, 1H), 1.17 – 1.07 (m, 3H). HRMS m/z for [C30H41N3O10P]-, calc. 634.6344 and found 634.6389. Synthesis of fluorescent probe (11). To a solution of pseudo peptide 9 in DMF (C = 2 mM) were added successively N,N-diisopropylethylamine (10 equivalents) and a solution of the 6-SIDCC-NHS ester in DMF (1.5 equivalents, C = 3 mM). The resulting solution was stirred at room temperature and the progress of the reaction was monitored by RP- HPLC (Supelco Ascentis® Express C18 column, 0 to 100% B in 10 min). The reaction mixture was quenched with water and the crude material was purified by RP-HPLC. 2-((1E,3Z,5Z)-3-(4-(2-((2-carboxy-4-phenylbutyl)(hydroxy)phosphoryl)-5-(4hydroxybenzyl)-4,7,16,29-tetraoxo-9,12,19,22,25-pentaoxa-3,6,15,28tetraazahentriacontan-31-yl)phenyl)-5-(1,1-dimethyl-6,8-disulfo-3-(4-sulfobutyl)-1,3dihydro-2H-benzo[e]indol-2-ylidene)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-3(4-sulfobutyl)-1H-benzo[e]indol-3-ium (11). Obtained as a foamy blue solid (78 %) after semi preparative RP-HPLC in conditions 5. Rt=9.2 min in conditions 1. ε680 nm= 233,200 M-1. cm-1. 1H NMR (500 MHz, D2O/d6-DMSO) δ 8.74 – 8.63 (m, 3H), 8.31 – 8.15 (m, 3H), 7.97 – 6.78 (m, 19H), 6.56 – 6.48 (m, 2H), 5.77 – 5.66 (m, 2H), 3.99 – 2.94 (m, 30H), 2,86 2.43 (m, overlapping with solvent), 2.36 – 2.18 (m, 9H), 1.90 (s, 12H), 1.79 – 1.43 (m, 9H), 1.15 – 1.08 (m, 3H). HRMS m/z for [C87H110N6O32S6P]+, calc. 1973.5221, found 1973.5322. 2-Methylene-4-phenylbutanoic acid (12). Sodium (2.88 g, 125 mmol) was slowly added to abs. EtOH (162 mL) over a period of 1 h. To the resulting solution, diethyl malonate (20 g, 125 mmol) was added drop wise over a period of 30 min. The solution was then stirred at room temperature for 30 min and 2-phenylethyl bromide was added drop wise during 1 h. After the addition was completed, the mixture was refluxed for 6 h, the solvent was then removed under vacuum. H2O (200 mL) and Et2O (100 mL) were added to the residue. The aqueous layer was extracted with Et2O (2 × 100 mL) and the combined organic layers were washed with brine (50 ml), dried over Na2SO4 and concentrated. The oily residue was then dissolved in abs. EtOH (50 ml) and a solution of KOH (28 g, 500 mmol) in abs. EtOH (250 mL) was slowly added over 2 h. After the


28

Page 28 of 51

Page 29 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


addition was completed, the resulting mixture was stirred at room temperature for 5 h. After evaporation, the residue was dissolved in H2O (100 ml) and Et2O (100 mL). The aqueous phase was washed with Et2O (3 × 50 ml), acidified with 3M HCl and extracted with Et2O (3 × 50 ml). The latter extracts were washed with brine (50 mL), dried over Na2SO4 and concentrated. The residue (18.8 g) was dissolved in AcOEt (360 mL) and the mixture was cooled at 0oC. Et2NH (10.8 mL, 104 mmol) and paraformaldehyde (3.8 g, 126 mmol) were then added and the resulting mixture was refluxed for 3 h. After the completion of the reaction, volatiles were evaporated and the crude residue was dissolved in Na2CO3 5% (100 mL) and Et2O (100 mL). The aqueous phases were washed with Et2O (2 × 50 mL), acidified with 3M HCl and extracted with Et2O (3 × 50 mL). The latter extracts were washed with brine (50 mL), dried with Na2SO4 and concentrated under vacuum to afford 14.3 g (65% for 3 steps) of compound 12 as a hygroscopic colorless solid. The obtained spectral data for acid 12 were found identical to literature data 59. Benzyl 2-methylene-4-phenylbutanoate (13). To an ice-cooled solution of acid 12 (5.0 g, 28 mmol) in dry CH2Cl2 (140 mL), DIPEA (5.45 ml, 31.2 mmol), benzyl alcohol (3.37 g, 31.2 mmol), DMAP (346 mg, 2.84 mmol) and EDC.HCl (5.96 g, 31.2 mmol) were successively added. The resulting solution was stirred at room temperature for 6 h. After the completion of the reaction, the volatiles were evaporated and the crude residue was partitioned between H2O (20 mL) and Et2O (50 mL). The aqueous layer was removed and the organic layer were successively washed with 1M HCl (2 × 20 mL), H2O (20 mL), 5% NaHCO3 (2 × 20 mL) and brine (20 mL), dried over Na2SO4 and concentrated under vacuum. The oily residue was further purified by silica gel column chromatography using PE (40-60 ºC)/AcOEt 9:1 as eluent solvent system to afford 5.23 g (70%) of compound 13 as a colorless oil. 1H NMR (200 MHz, CDCl3) δ 7.46-7.13 (m, 10H), 6.23 (s, 1H), 5.55 (t, J = 1.1 Hz), 5.23 (s, 2H), 2.87-2.76 (m, 2H), 2.71-2.60 (m, 2H); 13C NMR (50 MHz, CDCl3) δ 167.02, 141.45, 139.87, 136.12, 128.69, 128.61, 128.46, 128.32, 128.21, 126.08, 125.98, 66.58, 34.98, 34.06. MS m/z for C18H19O2+ (M+H+), calcd 267.1, found 267.2. (R)-1-[N-(9-fluorenylmethoxycarbonyl)amino]ethyl phosphinic acid (15). A solution of


29


Page 30 of 51

Cbz-protected aminophosphinic acid 14 (1.0 g, 4.1 mmol) 44,45,60, in 33% HBr/AcOH was stirred during 2 h at room temperature. The reaction solution was then concentrated and the crude residue was dissolved in 10% aqueous solution of Na2CO3 (10.8 mL, 10.2 mmol) and dioxane (8.6 mL). The mixture was cooled at 0oC and FmocCl (1.48 g, 5.74 mmol) was added portion wise over a period of 1 h. After the end of the addition, the mixture was vigorously stirred for 1 h at 0o C and for 5 h at room temperature. The volatiles were the evaporated and the crude residue was dissolved in 2.5% NaHCO3 (100 mL) and Et2O (20 mL). The aqueous phases were acidified with 3M HCl and the precipitate was filtrated and washed with small portions of H2O and Et2O. The obtained white solid was dried over P2O5 to afford 1.32 g (97%) of compound 15. The obtained spectral data were found identical to literature data. [α]D20 = -23 (c = 1.0, DMSO). Benzyl

2-[({(1R)-1-[N-(9-

fluorenylmethoxycarbonyl)amino]ethyl}(hydroxy)phosphoryl)methyl]-4phenylbutanoate (16). A solution of phosphinic acid 15 (700 mg, 2.11 mmol), ester 13 (844 mg, 3.17 mmol) and DIPEA (2.58 mL, 14.8 mmol) in CH2Cl2 (25 ml) were added in a Schlenk flask and the mixture was degassed by applying three freeze-pump-thaw cycles. The mixture was then cooled to -78ºC and purged with argon for 15 min. Precooled at -78 ºC freshly distilled TMSCl (1.86 mL, 14.8 mmol) was then added at once to the reaction vessel. The temperature was slowly raised to 25ºC and the clear solution was stirred at room temperature for 48 h. The mixture was then cooled to 0 ºC, abs. EtOH (2 mL) was added drop wise and stirred at room temperature for 30 min follows. After removal of the volatiles, 2M HCl (20 mL) and AcOEt (40 mL) were added to the residue, the aqueous phase was removed and the milky organic layer was concentrated. 16 (741 mg, 59%) was obtained as a white, highly insoluble solid after silica gel column chromatography, using CHCl3/MeOH/AcOH 7:0.1:0:1 to 7:0.4:0:4 as eluent solvent system.1H NMR (200 MHz, d6-DMSO+1%TFA) δ 7.93-6.90 (2×d + m, 18H), 5.08 (dd, J = 12.3, 19.5 Hz, 2H), 4.33-4.12 (m, 3H), 3.86-3.61 (m, 1H), 2.90-2.65 (m, 1H), 2.62-2.25 (overlapping with d6-DMSO, m, 2H), 2.18-1.67 (m, 4H), 1.69-1.16 (dd, J = 7.3, 14.1 Hz, 3H);

13C

NMR (50 MHz, d6-DMSO+1%TFA) δ 174.21, 155.99, 155.91, 141.23, 141.16,


30

Page 31 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


140.82, 128.47, 128.28, 127.75, 127.17, 125.97, 125.41, 120.22, 66.04, 65.92, 47.21, 46.75, 45.22, 35.34, 35.25, 32.46, 32.42, 29.01, 27.22, 14.16, 13.82;

31P

NMR (81 MHz, d6-

DMSO+1%TFA) δ 46.66, 46.35 (diastereoisomeric ratio: 0.2:0.8). HRMS m/z for C35H37NO6P+ (M+H+), calcd 598.2358, found 598.2317. 2-{[((1R)-1-[N-(9-fluorenylmethoxycarbonyl)amino]ethyl)(adamant-1yloxy)phosphoryl]methyl}-4-phenylbutanoic acid (17). To a refluxing solution of phosphinic acid 16 (650 mg, 1.09 mmol) and 1-adamantylbromide (351 mg, 1.63 mmol) in CHCl3 (30 mL), silver (I) oxide (378 mg, 1.63 mmol) was added portion wise over 1 h. After 12 h at reflux, the solvent was evaporated and the crude residue was treated with Et2O (10 mL). The resulting mixture was filtered through celite and the filtrates were evaporated. The residue was purified by silica gel column chromatography, using PE (40-60 ºC)/AcOEt 2:1→0:1 and the resulting adamantylester (680 mg) was dissolved in abs. EtOH (10 mL). To this solution, Pd/C 10% (100 mg) was carefully added and H2 was introduced at a pressure of 1 Atm. After 2 h, the catalyst was removed by filtration through celite and the filtrates were concentrated to dryness. The residue was treated with a cold mixture of PE (40-60ºC)/Et2O 3:1. The resulting white precipitate was filtrated and washed with PE (40-60ºC) to afford 595 mg (85% for 2 steps) of compound 17 as a white solid (mixture of 4 diastereoisomers). 1H NMR ((200 MHz, CDCl3) δ 7.896.93 (m, 13H), 6.92-6.70 + 6.32-6.09 + 5.89-5.53 (m, 1H), 4.49-3.94 (m, 4H), 3.04-2.34 (m, 4H), 2.26-1.79 (m, 12H), 1.69-1.16 (m, 9H);

13C

NMR (50 MHz, CDCl3) δ 177.24, 177.03,

156.86, 156.72, 156.56, 156.41, 156.29, 156.15, 156.00, 155.90, 155.85, 155.31, 155.16, 144.13, 144.01, 143.86, 141.48, 141.24, 141.03, 140.94, 128.56, 128.50, 128.45, 128.40, 128.32, 127.74, 127.23, 127.12, 126.07, 126.00, 125.91, 125.52, 125.27, 125.08, 119.92, 84.85, 84.64, 84.32, 84.22, 84.11, 84.01, 83.89, 67.57, 67.26, 48.04, 47.17, 47.02, 45.80, 44.36, 44.30, 41.65, 40.82, 36.10, 36.01, 35.63, 35.52, 33.18, 33.06, 31.55, 31.28, 31.18, 30.84, 30.73, 29.76, 15.32, 14.80, 14.29, 13.85;

31P

NMR (81 MHz, CDCl3) δ 52.18, 51.66, 51.03, 49.49 (diastereoisomeric

ratio: 0.15:0.2:0.12:0.52). HRMS m/z for C38H44NO6PNa+ (M+Na+), calcd 664.2803, found 664.2804. 2. Enzyme Assays.


31


2. 1. Metalloproteases assay. For 96-well assays, a multiplate reader Wallac 1420 VICTOR2 (PerkinElmer Corp., USA) was used in a final reaction volume of 250 l. For cuvette assays a UV-1800 UV-VIS spectrophotometer (Shimadzu Corp., Japan) was used with a final reaction volume of 1 ml. The reactions were followed at 30 sec intervals for 15 min, and measured in terms of initial velocities. Mixtures of activity buffer, inhibitor and enzyme were placed in a 96-well microplate and pre-incubated at 37ºC for 45 min. A fixed volume of substrate was then added to initiate the reaction. All assays were performed in triplicate. Inhibition constants (Ki) were determined according to the method described for tight-binding inhibitors 61. A-type MCPs. 1.0x10-8 M hCPA1, 3.0x10-8 M hCPA2, 4.3x10-8 M hCPA4, 7.5x10-9 M rat CPA3, 1.0x10-4 M AAFP substrate, activity buffer of 20 mM Tris-HCl pH 7.5, 500 mM NaCl, 1% v/v DMSO, 0.05% w/v Brij-35 and =355 nm were used 62. B-type MCPs. 6.0x10-9 M hCPB1, 2.0x10-8 M hCPD, 1.0x10-4 M AAFA substrate, activity buffer of 20 mM Tris-HCl pH 7.5, 100 mM NaCl, 1% v/v DMSO, 0.05% w/v Brij-35 and =355 nm were used 63. hTAFI. 7.5x10-9 M hTAFI, 1.0x10-4 M AAFA substrate, activity buffer of 20 mM Tris-HCl pH 7.5, 100 mM NaCl, 1% v/v DMSO, 0.05% w/v Brij-35 and =355 nm were used34,63. hCPN. 2.0x10-9 M hCPN, 2.0x10-4 M FAAK substrate, activity buffer of 20 mM Tris-HCl pH 7.5, 100 mM NaCl and =330 nm were used 64. hCCPs. 1.0x10-6 g hCCP1, 1.0x10-6 g hCCP6, 12.5x10-5 g tubulin as substrate, activity buffer of 20 mM Tris-HCl pH 7.5, 100 mM NaCl (unpublished method). hMMPs. 1.3x10-2 M Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 fluorogenic substrate, activity buffer of 50 mM Tris-HCl pH 6.8, 10 mM CaCl2 and hMMPs in the nanomolar range 65. 2. 2. Other proteases assays. pPepsin1.0x10-8 M pepsin, 2.0x10-4 M LSPNP substrate, activity buffer of 100 mM acetate pH 4.4 was and =310 nm were used66.


32

Page 32 of 51

Page 33 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Papain. 4.5x10-8 M papain, 4.0x10-4 M PFLNA substrate, activity buffer of 100 mM phosphate pH 6.5, 100 mM KCl, 0.1 mM EDTA, 3mM DTT, 0.05% w/v BRIJ-35 and =414 nm were used67. bTrypsin. 2.8x10-7 M trypsin, 1.0x10-3 M BAPNA substrate, 20 mM Tris-HCl pH 8.0, 20 mM CaCl2, 150 mM NaCl, 0.05% v/v Triton X-100 as activity buffer and =414 nm were used 68. Subtilisin. 2.0x10-7 M subtilisin, 4.0x10-4 M GLPNA substrate, 50 mM TrisHCl pH 8.5, 10% v/v DMSO as activity buffer and =414 nm were used 69. hNElastase. 2.0x10-7 M hNElastase, 1.0x10-3 M Suc-AAPVPNA substrate, 100 mM TrisHCl pH 8.0 as activity buffer and =414 nm were used 70,71. hThrombin. 2.0x10-8 M hThrombin, 1.0x10-3 M BzFVRPNA substrate, 100 mM Tris-HCl pH 8.0 as activity buffer and =414 nm were used 72. 2. 3. Determination of inhibition constants (Ki). The Ki values of phosphinic pseudo peptides 1 to 11 against hCPA1, hCPA2, hCPA4, hCPB1 and hCPD were determined by measuring the enzymatic residual activity (vi/v0=a) at different inhibitor concentrations and using a fixed enzyme and substrate concentrations as described above. The determination of Ki values was carried out on equilibrium conditions ([E0]/Ki≤ 10), using a fixed preincubation time of 45 min, as described

73

for this kind of inhibitors.

The best estimates of apparent Ki values (Kiapp) were obtained by fitting the experimental data to the equation for tight-binding inhibitors

49

by non-linear regression using the

Graphadprisma 5 software (GraphPad Software, Inc., USA) at p

functional characterization ... - ACS Publications

Recommend Documents