Monocyclic beta-lactams revive the research field on antibiotics, which are threatened by the emergence of resistant bacteria. A six-step synthetic route was developed, providing easy access to new 3-amino-1-carboxymethyl-4-phenyl-beta-lactams, of which the penicillin-binding protein (PBP) inhibitory potency was demonstrated biochemically

Amoroso, Ana Maria

D'hooghe, Matthias

Decuyper, Lena

Gobec, Stanislav

Joris, Bernard

Jukič, Marko

Sosič, Izidor

Verlaine, Olivier

English

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Synthesis and Penicillin‐binding Protein Inhibitory Assessment of Dipeptidic 4‐Phenyl‐β‐lactams from α‐Amino Acid‐derived Imines

Ghent University Academic Bibliography

Synthesis and Penicillin-binding Protein Inhibitory Assessment ofDipeptidic 4-Phenyl-b-lactams from a-Amino Acid-derived IminesLena Decuyper,[a] Marko Jukicˇ,[b] Izidor Sosicˇ,[b] Ana Maria Amoroso,[c] Olivier Verlaine,[c]Bernard Joris,[c] Stanislav Gobec,[b] and Matthias D’hooghe*[a]Abstract: Monocyclic b-lactams revive the research fieldon antibiotics, which are threatened by the emergence ofresistant bacteria. A six-step synthetic route was devel-oped, providing easy access to new 3-amino-1-carboxy-methyl-4-phenyl-b-lactams, of which the penicillin-bindingprotein (PBP) inhibitory potency was demonstrated bio-chemically.In light of the rapidly escalating bacterial resistance problem,antibiotic discovery remains a highly important drug discoveryendeavour.[1] To break away from traditional bicyclic b-lactamscaffolds, the research on monocyclic b-lactam antibioticsgains in importance, as we previously demonstrated through athree-decade literature overview.[2] Taking into account the as-sembled state-of-the-art, and combining the structural proper-ties of aztreonam (the only FDA-approved monobactam), thenocardicins (the first described, natural monocyclic b-lactams)and late-generation cephalosporins, our group recently studieda new pharmacophore 1 to mimic d-alanyl-d-alanine, the natu-ral substrate of the penicillin-binding proteins, playing a crucialrole in the bacterial cell wall biosynthesis (Figure 1). Using avirtual screening approach as an onset, a ten-step syntheticpathway was developed towards a model library of 4-unsubsti-tuted b-lactams 1, characterised by diverse side chains ’R’. a-Benzylidene-substituted analogue 1A was promoted tobecome our first hit, favoured with promising PBP inhibitoryactivity and high b-lactamase stability. In order to gain addi-tional insights in the chemistry and structure–activity relation-ships of these complex b-lactams, an explorative study on syn-thetic routes towards C4-substituted analogues 2 was now ini-tiated, holding on to the same design objectives (Figure 1). Inlight of synthetic feasibility, 4-phenyl-b-lactams 2 were target-ed first to provide a proof of concept. Their synthesis was per-ceived to be achievable through functionalisation, in a similarway as previously described,[3] of 3-amino-1-carboxymethyl-b-lactams. As opposed to C4-unsubstituted b-lactams, C4-substi-tuted variants can be easily prepared via Staudinger’s ketene-imine [2+2]-cyclocondensation.[4] To date, this is one of themost renowned and versatile methods for the construction ofthe four-membered amide ring system and is often selectedfor its simplicity in reaction procedures and predictability ofthe stereochemical outcome.[5] For the synthesis of 3-amino-b-lactams specifically, phthalimido- (FtN) and azido-containingketenes are typically employed.[6] As a means to shorten thesynthetic pathway as much as possible, the variable side chain’R’ could be introduced directly in the amine part of the imine.Indeed, when protected a-amino acids would be used ascheap and easily available substrates and condensed with ben-zaldehyde, the a-functionalised carboxymethyl moiety couldbe installed quite easily onto the resulting b-lactam N1-posi-tion. Based on the in silico design criteria regarding the a-sidechain ’R’ (Figure 1c),[3] several divergent a-amino acids were se-lected to i. optimise the synthetic procedures (l-/dl-Phe); ii. in-troduce terminal hydrogen bond donors or acceptors (l-Glu, l-Trp and l-/d-Met, for oxidation to sulphones); and iii. changethe linker type to aromatic nocardicin-like motifs (d-Phg).[2]Taking all the above into account, l-phenylalanine 3A (R=Bn) as a model substrate was converted to the correspondingmethyl ester 4A by means of 2,2-dimethoxypropane and hy-drochloric acid (Method A, Scheme 1, Table 1). The resultingamine HCl salt was converted to the free base and subse-quently condensed with benzaldehyde, affording aldimine 5Ain quantitative yield (Method C). Also the racemic starting ma-terial 3A’ (dl-Phe) was converted in this way in view of HPLCanalysis using a chiral stationary phase (’chiral HPLC’) at a laterstage. Meanwhile, N-phthaloylglycine 6 was converted to thecorresponding glycyl chloride 7. After dehydrochlorination ofacid chloride 7 using triethylamine, the in situ generatedketene was coupled with imines 5A-A’ to yield 4-phenyl-3-phthalimidoazetidin-2-ones 8A-A’. Profound variation of theStaudinger reaction conditions was necessary to enhance theconversion rate and yield. The optimised set of reaction param-eters and corresponding reaction outcomes are displayed inTable 1. As expected, comparison of the results of Methods Eand F, showed that a higher amount of trans-isomer was ob-[a] Dr. L. Decuyper, Prof. Dr. M. D’hoogheSynBioC Research GroupDepartment of Green Chemistry and TechnologyFaculty of Bioscience EngineeringGhent UniversityCoupure Links 653, 9000 Ghent (Belgium)E-mail : Matthias.Dhooghe@UGent.be[b] Dr. M. Jukicˇ, Dr. I. Sosicˇ, Prof. Dr. S. GobecDepartment of Pharmaceutical ChemistryFaculty of PharmacyUniversity of LjubljanaAsˇkercˇeva 7, 1000 Ljubljana (Slovenia)[c] Dr. A. M. Amoroso, O. Verlaine, Prof. Dr. B. JorisCentre for Protein EngineeringFaculty of SciencesUniversity of LiHgeQuartier Agora, All8e du 6 Ao0t 13, B.t B6a4000 LiHge Sart-Tilman (Belgium)Supporting information and the ORCID identification number(s) for the au-thor(s) of this article can be found under:https://doi.org/10.1002/asia.201901470.Chem. Asian J. 2020, 15, 51 – 55 T 2020 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim51CommunicationDOI: 10.1002/asia.201901470tained when 2,6-lutidine was used as a base in boiling tolu-ene.[5] The accessibility of both cis- and trans-isomers was con-sidered an asset with respect to future evaluation of the bio-logical potency of the corresponding target products 2.Next, phthaloyl deprotection of compounds 8A-A’ using hy-drazine hydrate furnished 3-aminoazetidin-2-ones 9A-A’(Table 2, Scheme 2).[7] In the next step, the obtained freeamines were coupled with 2-(2-aminothiazol-4-yl)-2-(methoxy-imino)acetic acid (2-ATMO, predominantly syn) in alkaline envi-ronment using O-(benzotriazol-1-yl)-N,N,N’,N’-tetramethyluroni-um tetrafluoroborate (TBTU) as a coupling reagent. The result-ing cyclic amides 10A-A’ were obtained in variable yields ofFigure 1. Design strategy. [a] 3-Amino-1-carboxymethyl-b-lactam, mimicking the natural PBP substrate, d-alanyl-d-alanine, and enabling crucial molecular in-teractions with the enzyme’s active site; [b] Optimised 2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido substituent;[2] [c] Variable side chain embedding alipophilic linker and terminal hydrogen bond-donating or -accepting functionalities, enabling additional non-covalent interactions with the key active site resi-dues.Scheme 1. Staudinger synthesis of Ft-protected 3-amino-1-methoxycarbonylmethyl-4-phenyl-b-lactams 8.Table 1. Optimised synthesis of a-substituted 3-amino-1-methoxycarbonylmethyl-4-phenylazetidin-2-ones 8.Compound(Amino acid)Esterification Imination Staudinger synthesisMethod 4 [%] (ee)[a] Method 5 [%] (ee)[a] Method(Eq. base; Eq. 7)dr before purif. 8 [%] (dr)3A (l-Phe) A 99 (99) CD46-99 (6)99 (86)E (1.5; 0.8) 56/44/0/0 8Acis : 5 (58/42)[b]3A’ (dl-Phe) A 99 (0) C 64 (2) F (4; 1.5) 3/2/53/42 8A’cis : 6 (59/41); 8A’trans : 64 (55/45)[b]3B (l-Glu) B 79 D 31 F (2; 1.5) 2/1/51/46 8Btrans : 89 (52/48)[c]3C (l-Trp) –[d] –[d] D 77 F (2; 1.5) 0/0/63/37 8Ctrans : 85 (59/41)[c]3D (l-Met) A 96 D 99 F (2; 1.5) 4/4/53/39 8Dtrans : 64 (56/44)[c]3D’ (d-Met) –[d] –[d] D 87 F (2; 1.5) 4/4/53/39 8D’trans : 60 (57/43)[c]3E (d-Phg) A 99 D 69 F (3; 2.25) –[b,e] –[b,e][a] Chromatographic conditions: see Supporting information. [b] Purified via automated column chromatography (C18). [c] Purified via automated columnchromatography (SiO2). [d] Commercially available. [e] Complex mixture, containing 29% of coupling product of 4E and 7, 4% 8Ecis (dr=62/38) and 11%8Etrans (dr=51/49).Chem. Asian J. 2020, 15, 51 – 55 www.chemasianj.org T 2020 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim52Communication35–69% after conventional purification techniques. Additionalseparation of the obtained diastereomeric mixtures via prepa-rative HPLC permitted to isolate the individual diastereomers10A1, 10A2 and 10A’1, 10A’2 (all trans). The isolated isomerswere used to assess the optical purity of the compounds withrespect to the a-position after the different reaction steps viachiral HPLC. This technique, however, demonstrated a lowenantiomeric excess (6%) for compounds 10A1 and 10A2,which implied epimerisation of the a-position of the methylester derived from optically pure l-phenylalanine. Retrospec-tive analysis of all intermediates revealed that epimerisationtook place already during imination (Scheme 1, Table 1,Method C), which is not in accordance with preceding literaturereports, stating conservation of the chirality of similar aminoacid-derived imines using comparable or even harsher reactionconditions (alkaline environment, elevated temperatures).[8] Forthis reason, a new imination procedure was tested (Method D),which did give rise to imine 5A with a considerably higher eeof 86%.[9] Consequently, this method was applied for the syn-thesis of the other derivatives 5,8-10B-E as well. As such, ourtarget library was enlarged by converting the above-men-tioned a-amino acids 3B–E to b-lactams 10B-E via similarchemical transformations (Scheme 1, Scheme 2, Table 1,Table 2). Despite the (presumed) use of chiral imines, a moreor less equimolar amount of the two trans-isomers 8B-E wasobtained in each case, which is in good agreement with thestatement that a chiral imine derived from a chiral amine andan achiral aldehyde is not an appropriate chiral inductor in theStaudinger synthesis.[10] Both l- and d-methionine 3D-D’ wereemployed individually to be able to use the enriched mixturesof compounds 10D-D’ for chiral HPLC method optimisationand determine if the configuration of the chiral centre in a-po-sition of the carboxylic acid was now conserved throughoutthe pathway (Table 2). When compared with l-Phe-derived b-lactams 10A (synthesised using imination Method C, Table 1,ee=6%), the epimerisation issue of most other derivatives wasfar more limited (e.g. , ee >76% for 10D’, Method D). As ex-pected, only d-Phg-derived compounds 5,8-10E were com-pletely racemised as a result of the formation of an extensiveconjugated system after deprotonation at the benzylic posi-tion.[11] In previous work,[3] it was shown that epimerisation atthe a-position can also take place during subsequent hydroly-sis, depending on the reagent used. The deprotection reactionof diastereomerically pure trans ester 10A1 using LiOH·H2Oindeed caused slight epimerisation (Method A, Table 2). SN2-type dealkylation with LiI resulted in high stereoselectivity butTable 2. Yields and diastereomeric ratios of intermediate and target products 9–11.Compound AA Deprotection Acylation Oxidation Hydrolysis9 [%] (dr) 10A-E [%] (dr) 10F-F’ [%] (dr) Method 11 [%] (dr)8Atrans[a] l-Phe 61 (53/47) 35 (53/47)[b,c] – A (on 10A1) ; B (on 10A1) 99 (83/17); 50 (96/4)8A’cis dl-Phe 90 (59/41) 36 (60/40)[b] – A 99 (67/33)8A’trans 90 (58/42) 69 (54/46)[b,c] – A (on 10A’1-2) ; A (on 10A’2) 99 (69/31); 99 (58/42)8Btrans l-Glu 70 (53/47) 16 (51/49)[b] – A[d] 99 (62/38)8Ctrans l-Trp 89 (51/49) 22 (56/44)[e] – A 99 (54/46)8Dtrans l-Met 67 (59/41) 35 (55/45)[f] – A (on 10D) 99 (66/34)10F : 30 (62/38)[g,h] A (on 10F) 99 (61/39)8D’trans d-Met 84 (55/45) 69 (54/46)[f,h] – A (on 10D’) 99 (58/42)10F’: 35 (53/47)[f] A (on 10F’) 99 (57/43)8Ecis d-Phg 38 (66/34) 99 (60/40)[g,h] – A 99 (74/26)8Etrans 79 (62/38) 38 (52/48)[g,h] – A 99 (70/30)[a] dr=68/32. [b] Purified via automated column chromatography (C18). [c] Additional purification via prep. HPLC permitted to separate the two diastereo-mers 10Atrans-1 and 10Atrans-2 (6% ee each) and 10A’trans-1 and 10A’trans-2 (0% ee each). [d] 2 equiv. LiOH·H2O were used. [e] Purified via prep. TLC. [f] Purifiedvia automated column chromatography (SiO2). [g] Purified via prep. TLC. [h] Chiral HPLC analysis was performed on compounds 10D’trans (ee>76%),10Ecis,trans (ee=0%) and 10Ftrans (ee=85%).Scheme 2. Functionalisation of key intermediates 8 towards target products 11.Chem. Asian J. 2020, 15, 51 – 55 www.chemasianj.org T 2020 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim53Communicationlower conversion (Method B). Similar results were obtained forthe racemic analogues 10A’. In either way, the loss of enantio-purity at the a-position was not a major issue as long as thebiological potency of these compounds had not been proven.Analogues 10B-E were therefore hydrolysed using the LiOH-mediated procedure.As mentioned before, in case of methionine-derived ana-logues 10D-D’, sulphide oxidation was performed prior to hy-drolysis using meta-chloroperbenzoic acid to bring along theinsertion of two additional hydrogen bond acceptors in a ter-minal position, which is one of the premised design require-ments (Figure 1).[3,12] Sulphones 10F-F’ were obtained in thisway without significant loss of enantiopurity at the a-position(ee(10Ftrans)=85%, Table 2), and could subsequently be hydro-lysed in quantitative yields relying on LiOH·H2O. As such, thispathway enabled the preparation of a set of novel cis- and/ortrans-3-acetamido-1-carboxymethyl-4-phenylazetidin-2-ones 11in a limited amount of six (or seven for 11F-F’) steps in up to30% overall yield, remarkably more efficient compared withthe synthesis of their C4-unsubstituted analogues (ten steps,up to 2% overall yield).[3]The reactant scope of the synthetic pathway can be expand-ed towards other aromatic or aliphatic aldehydes, combinedwith readily available (un)natural a-amino acids, if desired.In order to provide some preliminary data on the biologicalpotential of this series of functionalised 3-amino-1-carboxy-methyl-b-lactams 2 (after neutralisation of 11 with HCl to pro-vide the corresponding carboxylic acids), the compounds weresubjected to a PBP binding and competition assay. The purifiedPBP3 of E. coli K12, PBP5fm of E. faecium D63r and R39 DD-car-boxypeptidase of Actinomadura spp.[13] were incubated withacids 2, after which their residual activity (%RA) was deter-mined by labelling the free enzymes with Bocillin FL, a fluores-cent reporter molecule and penicillin V analogue (Table 3).[14]The results revealed the remarkably low residual activity ofPBP3 (10% RA), a lethal target of E. coli, provoked by com-pound 2Etrans. The inhibitory potency is higher than that of ourinitial hit 1A (28% RA),[3] confirming the potential of C4-substi-tuted, a-aromatic–and therefore nocardicin-like–3-amino-1-car-boxymethyl b-lactam scaffolds for further PBP inhibitor design.The unprecedented and highly straightforward six-step syn-thetic route described in this work, converting simple sub-strates into highly functionalised C4-(phenyl-)substituted no-cardicin analogues, might therefore be used to further guideexpansion studies in the future starting from 1-carboxymethyl-b-lactam 2Etrans as a promising new hit structure (Figure 2).AcknowledgementsThe authors gratefully acknowledge the Research FoundationFlanders (FWO), the Slovenian Research Agency (research pro-gram P1-0208) and Belspo IAP-Project P44-iPros for financialsupport.Conflict of interestThe authors declare no conflict of interest.Keywords: amino acids · antibiotics · biologicalactivity · Staudinger synthesis · b-lactams[1] K. Bush, Future Med. Chem. 2016, 8, 921–924.[2] L. Decuyper, M. Jukicˇ, I. Sosicˇ, A. Zˇula, M. D’hooghe, S.Gobec, Med. Res. Rev. 2018, 38, 426–503.[3] L. Decuyper, S. Deketelaere, L. Vanparys, M. Jukicˇ, I.Sosicˇ, E. Sauvage, A. M. Amoroso, O. Verlaine, B. Joris, S.Gobec, M. D’hooghe, Chem. Eur. J. 2018, 24, 15254–15266.[4] H. Staudinger, Justus Liebigs Ann. Chem. 1907, 356, 51–123.[5] a) Y. Wang, Y. Liang, L. Jiao, D. M. Du, J. Xu, J. Org. Chem.2006, 71, 6983–6990; b) L. Jiao, Y. Liang, J. Xu, J. Am.Chem. Soc. 2006, 128, 6060–6069.[6] a) J. C. Sheehan, J. J. Ryan, J. Am. Chem. Soc. 1951, 73,1204–1206; b) A. K. Bose, B. Anjaneyulu, S. K. Bhatta-charya, M. S. Manhas, Tetrahedron Lett. 1967, 23, 4769–4776.[7] M. A. Sierra, M. Rodr&guez-Fern#ndez, M. J. MancheÇo, L.Casarrubios, M. Gjmez-Gallego, Tetrahedron 2008, 64,9592–9598.[8] a) I. Ojima, H.-J. C. Chen, X. Qui, Tetrahedron 1988, 44,5307–5318; b) G. Hughes, P. N. Devine, J. R. Naber, P. D.O’Shea, B. S. Foster, D. J. McKay, R. P. Volante, Angew.Table 3. Residual enzymatic activities after incubation of PBP3, PBP5fm and R39 (2.5 mm)with compounds 2 (1 mm, pH 7, 30 8C, 3 h) and IC50 values.Compound %RAPBP3 (IC50 [mM])%RAPBP5 fm%RAR39 (IC50 [mM])Aztreonam 0 (0.004–0.022)[a] –[b] 0 (4)[c]Blank 100 100 1002A’cis ; 2A’trans 87; 77 – –2Btrans 88 – –2Ctrans 79 – –2D’trans 89 – –2Ecis ; 2Etrans 73; 10 (720)[d] –; 100 –; 1002Ftrans 83 78 99[a] Computed from literature data.[15] [b] Aztreonam is not active against Gram-positivebacteria. [c] Computed from literature data.[16] [d] Indicatory value due to low solubilityat >0.8 mm.Figure 2. Structure of 2Etrans.Chem. Asian J. 2020, 15, 51 – 55 www.chemasianj.org T 2020 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim54CommunicationChem. Int. Ed. 2007, 46, 1839–1842; Angew. Chem. 2007, 119, 1871–1874; c) J. Lewkowski, R. Karpowicz, Heteroat. Chem. 2010, 21, 326–331;d) A. Hess, J. Sehnert, T. Weyhermuller, N. Metzler-Nolte, Inorg. Chem.2000, 39, 5437–5443; e) T. C. Maier, J. Podlech, Eur. J. Org. 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Lakaye, W. Zorzi, J.Grandchamps, J.-M. Wilkin, C. Fraipont, B. Joris, C. Duez, M. Nguyen-Dis-tHche, J. Coyette, M. Leyh-Bouille, J. Dusart, L. Christiaens, J.-M. FrHre, J.-M. Ghuysen, Methods Enzymol. 1994, 244, 249–266.[14] a) G. Zhao, T. I. Meier, S. D. Kahl, K. R. Gee, L. C. Blaszczak, Antimicrob.Agents Chemother. 1999, 43, 1124–1128; b) B. Lakaye, C. Damblon, M.Jamin, M. Galleni, S. Lepage, B. Joris, J. Marchand-Brynaert, C. Frydrych,J.-M. FrHre, Biochem. J. 1994, 300, 141–145; c) S. Turk, O. Verlaine, T.Gerards, M. Zivec, J. Humljan, I. Sosic, A. M. Amoroso, A. Zervosen, A.Luxen, B. Joris, S. Gobec, PLoS One 2011, 6, e19418.[15] a) R. L. Then, I. Kohl, Chemotherapy 1985, 31, 246–254; b) T. A. Davies,W. Shang, K. Bush, R. K. Flamm, Antimicrob. Agents Chemother. 2008, 52,1510–1512.[16] J.-M. FrHre, B. Joris, G. D. Shockman, Crit. Rev. Microbiol. 1984, 11, 299–396.Manuscript received: October 18, 2019Revised manuscript received: October 31, 2019Accepted manuscript online: November 5, 2019Version of record online: November 28, 2019Chem. Asian J. 2020, 15, 51 – 55 www.chemasianj.org T 2020 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim55Communication

Synthesis and penicillin‐binding protein inhibitory assessment of dipeptidic 4‐phenyl‐β‐lactams from α‐amino acid‐derived imines

peer reviewedMonocyclic β-lactams revive the research field on antibiotics, which are threatened by the emergence of resistant bacteria. A six-step synthetic route was developed, providing easy access to new 3-amino-1-carboxymethyl-4-phenyl-β-lactams, of which the penicillin-binding protein (PBP) inhibitory potency was demonstrated biochemically

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Synthesis and Penicillin-binding Protein Inhibitory Assessment of Dipeptidic 4-Phenyl-β-lactams from α-Amino Acid-derived Imines.

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Synthesis and penicillin‐binding protein inhibitory assessment of dipeptidic 4‐phenyl‐β‐lactams from α‐amino acid‐derived imines

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