Saidbakhrom Saidjalolov

Emmanuelle Braud

Zainab Edoo

Laura Iannazzo

Filippo Rusconi

Margaux Riomet

Antoine Sallustrau

Frédéric Taran

Michel Arthur

Matthieu Fonvielle

Mélanie Etheve-Quelquejeu

Click and release chemistry for activity-based purification of -lactam targets

teaching and research institutions in France or abroad, or from public or private research centers.

Bacterial resistance is a major threat to the efficacy of antibiotics requiring innovative strategies for maintaining the efficacy of antimicrobial chemotherapy. Identification and characterization of the biological targets of drugs is a key step for their development and the study of the mechanisms of resistance. This is particularly the case for antibiotics belonging to the β-lactam family since these drugs have multiple targets in bacterial cells with partially overlapping functions. [START_REF] Zapun | Penicillin-binding proteins and beta-lactam resistance[END_REF][START_REF] Gold | Novel Cephalosporins Selectively Active on Nonreplicating Mycobacterium tuberculosis[END_REF] The essential targets of β-lactams are the high-molecular weight (HMW) penicillin-binding proteins (PBPs) that catalyze the last cross-linking step of peptidoglycan synthesis. This cell wall polymer is essential for sustaining the osmotic pressure of the bacterial cytoplasm. It consists of glycan chains linked to each other by short stem peptides (Figure 1A). The cross-linking reaction catalyzed by HMW PBPs, the D,D-transpeptidases, proceeds by the cleavage of the C-terminal D-Ala-D-Ala peptide bond of a stem pentapeptide and linkage of the penultimate D-Ala to the third residue of a stem peptide carried by an adjacent glycan chain (4→ 3 cross-links) (Figure 1B). β-lactams are structure analogues of the D-Ala-D-Ala extremity of peptidoglycan precursors and act as suicide substrates of the HMW PBPs 3 (Figure 1C). Acylation of the active site Ser of the PBPs results in a long-lasting covalent adduct and irreversible inactivation of the target at the time scale of a bacterial generation.

The cross-linking reaction is performed by multiple HMW PBPs in most bacteria, typically 5 to 6 in the main human pathogens. [START_REF] Zapun | Penicillin-binding proteins and beta-lactam resistance[END_REF] In addition, β-lactams similarly inactivate low-molecular weight (LMW) PBPs that hydrolyze the D-Ala-D-Ala peptide bond of stem pentapeptides (D,D-carboxypeptidases) or the cross-links formed by the HMW PBPs (endopeptidases). A particular class of β-lactams, the carbapenems, also inactivate L,D-transpeptidases (Ldts) that form unusual cross-links connecting the third position on both stem peptides (3→ 3 cross-links) [START_REF] Mainardi | Unexpected inhibition of peptidoglycan LDtranspeptidase from Enterococcus faecium by the beta-lactam imipenem[END_REF] (Figure 1B). Ldt inactivation results from acylation of their active-site Cys residue by the β-lactam ring of carbapenems (Figure 1C). Ldts are the main cross-linking enzyme in Mycobacterium tu-berculosis [START_REF] Lavollay | The peptidoglycan of stationary-phase Mycobacterium tuberculosis predominantly contains cross-links generated by L,D-transpeptidation[END_REF] and can be responsible for in vitro acquired resistance to β-lactams of the penam (penicillin) and cephem (cephalosporin) classes in other bacteria. [START_REF] Hugonnet | Factors essential for L,Dtranspeptidase-mediated peptidoglycan cross-linking and betalactam resistance in Escherichia coli[END_REF][START_REF] Mainardi | Novel mechanism of beta-lactam resistance due to bypass of DD-transpeptidation in Enterococcus faecium[END_REF] Historically, the targets of β-lactams have been identified by incubating membrane preparations with radiolabeled drugs followed by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. [START_REF] Preston | Biological Characterization of a New Radioactive Labeling Reagent for Bacterial Penicillin-Binding Proteins[END_REF] This method typically resolves a pattern of ca. 10 bands corresponding to proteins covalently labeled by the drug. Competitive binding with unlabeled drugs has been largely used to compare the reactivity of various β-lactams for the PBPs. For this approach, fluorescent β-lactams, such as bocillin, were developed to avoid the use of radioactive reagents. [START_REF] Zhao | BOCILLIN FL, a sensitive and commercially available reagent for detection of penicillin-binding proteins[END_REF][START_REF] June | A fluorescent carbapenem for structure function studies of penicillin-binding proteins, beta-lactamases, and betalactam sensors[END_REF][START_REF] Beatty | Fluorescent probes for investigating peptidoglycan biosynthesis in mycobacteria[END_REF] Further refinement of the PBP labeling methods afforded single cell imaging of bacteria exposed to the drugs. [START_REF] Wright | Chemical proteomics approaches for identifying the cellular targets of natural products[END_REF] The key element of this application is the incorporation of a bioorthogonal active group in the β-lactam with minimal impact on drug penetration and on interaction of the drug with its multiple targets. Following labeling of the targets in the bacterium, the drug-target adducts were revealed by a bioorthogonal reaction with a functionalized dye. [START_REF] Staub | Beta-lactams as selective chemical probes for the in vivo labeling of bacterial enzymes involved in cell wall biosynthesis, antibiotic resistance, and virulence[END_REF][START_REF] Staub | Beta-lactam probes as selective chemicalproteomic tools for the identification and functional characterization of resistance associated enzymes in MRSA[END_REF] The most popular approach is the incorporation of an alkyne group on the drug allowing copper-catalyzed cycloaddition (CuAAC) with an azido-containing dye. [START_REF] Staub | Beta-lactams as selective chemical probes for the in vivo labeling of bacterial enzymes involved in cell wall biosynthesis, antibiotic resistance, and virulence[END_REF][START_REF] Staub | Beta-lactam probes as selective chemicalproteomic tools for the identification and functional characterization of resistance associated enzymes in MRSA[END_REF] This approach enables detection of known PBPs but present limitations concerning the identification of unknown targets since it only provides information on the electrophoretic mobility of the protein-dye adduct. This limitation has been addressed by the use of biotinylated azidocontaining dyes for streptavidin affinity pull-down purification followed by mass spectrometry analysis. [START_REF] Romeo | Activity-Based Probe for N-Acylethanolamine Acid Amidase[END_REF][START_REF] Chen | Target identification of natural medicine with chemical proteomics approach: probe synthesis, target fishing and protein identification[END_REF] Selective penicillin-binding protein imaging probes have also been described using fluorescent β-lactams, 17 such as fluorescent cephalosporins. [START_REF] Kocaoglu | Selective penicillin-binding protein imaging probes reveal substructure in bacterial cell division[END_REF] Of note, fluorescent -lactams were also used as fluorogenic molecular probes for the detection of -lactamases in vitro or in living bacteria. [START_REF] Ding | Fluorogenic Probes/Inhibitors of beta-Lactamase and their Applications in Drug-Resistant Bacteria[END_REF][START_REF] Das | Chemiluminescent Carbapenem-Based Molecular Probe for Detection of Carbapenemase Activity in Live Bacteria[END_REF][START_REF] Wang | A minor structure modification serendipitously leads to a highly carbapenemase-specific fluorogenic probe[END_REF] In this study, we explore a strategy for combined affinity purification and fluorescence labeling of carbapenem targets. We report synthetic routes to alkynylated carbapenems that effectively labeled a model L,D-transpeptidase (Ldtfm) both with respect to the kinetics of acylation

N O CO 2 H R 1 R 2 H H HN O CO 2 H R 1 R 2 H H S

I nhibition

Hydrol ysis

HN O CO 2 H R 1 R 2 H H OH SH + Carbapenem SH H 2 O
and the stability of the resulting acylenzymes. The alkyne group of the enzyme-bound carbapenem was subsequently exploited for CuAAC attachment of a biotin-containing iminosydnone linker, recently developed as a bioorthogonal cleavable spacer (Figure 2). [START_REF] Bernard | Bioorthogonal Click and Release Reaction of Iminosydnones with Cycloalkynes[END_REF][START_REF] Decuypere | Sydnonealkyne cycloaddition: applications in synthesis and bioconjugation[END_REF] After binding to strep-tavidin magnetic beads and purification, the iminosydnone mesoionic compound enabled the release and labeling of the captured carbapenem targets from the streptavidin support through a bioorthogonal click and release reaction, [START_REF] Bernard | Bioorthogonal Click and Release Reaction of Iminosydnones with Cycloalkynes[END_REF] affording pure and fluorescent targets in a single step.

RESULTS AND DISCUSSION

Synthesis of alkynylated carbapenems. We started our synthesis by using a post-functionalization strategy of the fully deprotected methyl-thienamycin (Route A, Scheme 1). Methyl-thienamycin was obtained in two steps (21% yield) from commercially available methyl vinylphosphate. [START_REF] Iannazzo | Routes of Synthesis of Carbapenems for Optimizing Both the Inactivation of L,D-Transpeptidase LdtMt1 of Mycobacterium tuberculosis and the Stability toward Hydrolysis by beta-Lactamase BlaC[END_REF] Methyl-thienamycin is renowned for its unstable chemical nature. [START_REF] Papp-Wallace | Carbapenems: past, present, and future[END_REF] Indeed, strong acidic, basic, or nucleophilic conditions as well as exposure to light, high temperature, or prolonged storage in solution lead to lactam ring opening. Thus, the reaction conditions for functionalizing methyl-thienamycin should be mild (6.0 < pH < 8.5; < 25˚C) and should not involve prolonged reaction time (< 1 hour). Despite these limitations, three alkyne-carbapenems were obtained, CBA-1, CBA-2, and CBA-3, using methyl-thienamycin in the presence of alkyne linkers, which were commercially available (for CBA-1 and CBA-2) or synthesized in this study (Compound 1, Scheme 1). Alkyne-carbapenems CBA-1, CBA-2, and CBA-3 were obtained with variable yields (49%, 67%, and 14%, respectively; Scheme 1, Route A).

Ring opening could not be fully prevented and subsequent purification of the desired compounds free of their hydrolyzed products was cumbersome. For this reason, we explored an alternative approach starting from a protected carbapenem for direct addition of a thiol-alkyne onto the β-methyl vinyl-phosphate (Scheme 1, route B). Syntheses of the thiol alkyne linkers 3, 6, and 10 are described in the supporting information (SI, Section 2). This first step provided the intermediate carbapenems 11a, b, c, d in 97%, 77%, 78% [START_REF] Iannazzo | Routes of Synthesis of Carbapenems for Optimizing Both the Inactivation of L,D-Transpeptidase LdtMt1 of Mycobacterium tuberculosis and the Stability toward Hydrolysis by beta-Lactamase BlaC[END_REF] and 54% yields, respectively. The critical step of the latter strategy was the final selective deprotection of the para-nitrobenzyl group (PNB), which was successfully achieved using zinc dust. [START_REF] Kumagai | Mild and Chemoselective Cleavage of P-Nitrobenzyl Esters and p-Nitrobenzyloxycarbonyl Amines with Zinc Dust[END_REF] Minimal β-lactam ring opening provided by this approach facilitated purification of the final compounds (CBA-1, CBA-2, CBA-4, and CBA-5 in 11%, 77%, 40%, and 43% yields, respectively). Comparison of the NMR spectra of CBA-1 and CBA-2 obtained by routes A and B (Supporting Scheme S5) supported the superiority of route B with respect to the purity of the final compound. Acylation of L,D-transpeptidase Ldtfm by alkyne-carbapenems. The alkyne function of compounds CBA-1 to CBA-5 is connected to the C2 position of the carbapenem core via variable links, including an amide (CBA-1), a urea (CBA-2), a squaramide (CBA-3), an alkyl (CBA-4), or a benzamide (CBA-5). These compounds were synthesized to explore the impact of the linkers on the kinetics of target acylation and the stability of the resulting covalent adduct. This was first investigated by mass spectrometry to detect Ldtfm-carbapenems adducts (Figure 3). Incubation of Ldtfm (6.5 µM) with alkyne carbapenems CBA-1 to CBA-5 (39 µM) for 15 min at 25 °C in 20 mM ammonium acetate (pH 7.2) led to full enzyme acylation, except for CBA-3 (ca. 30% acylation). Stability of the Ldtfm-carbapenems adducts was also monitored by mass spectrometry. Scheme 1. Two strategies for alkyne carbapenems synthesis. Reaction conditions for route A: i. 4-pentynoic acid succinimidyl ester or N-(prop-2-yn-1-yl)-1H-imidazole-1-carboxamide [START_REF] Rillahan | On-chip synthesis and screening of a sialoside library yields a high affinity ligand for Siglec-7[END_REF] or compound 1. Reaction condition for route B: i. thiol alkyne-linkers 3, 6, 3-butynyl ethanethioate or 10 and DIPEA in DMF, ii. Zn dust in THF:PB (1:2). Compounds were obtained by route A (CBA-3), route B (CBA-4 and CBA-5), or both routes (CBA-1 and CBA-2). For this purpose, the Ldtfm-carbapenem adducts were purified by gel filtration to remove the excess of unreacted carbapenem and incubated for 1 h and 3 h at 25 °C. This analysis showed that acylenzymes resulting from acylation of Ldtfm by CBA-2, CBA-4, CBA-5, and the clinically-used carbapenem meropenem (Figure 1C) were stable (Figure 3). In addition, native Ldtfm resulting from hydrolysis of the carbapenem-Ldtfm thioester was not detected in the mass spectra. In contrast, partial hydrolysis ca. 20% was detected for CBA-1 since the ratio of peak intensities was in the order of 0.2 and 0.8 for native Ldtfm and the Ldtfm-CBA-1 acylenzyme, respectively. Since Ldtfm was only partially acylated by CBA-3 this alkyne carbapenem was not selected for further studies.

Reactivity of alkyne-carbapenems in the Cu I -catalyzed cycloaddition. The success of our click and release strategy relies on both the stability of the acylenzyme (above) and on the efficiency of the cycloaddition reaction (step 2; Figure 2). Kinetics of CuAAC were performed with a fluorogenic azido partner (3-azido-7-hydroxycoumarin) [START_REF] Kislukhin | Relative performance of alkynes in copper-catalyzed azide-alkyne cycloaddition[END_REF][START_REF] Bevilacqua | Copper-chelating azides for efficient click conjugation reactions in complex media[END_REF] to compare the impact of the linkers present in the four alkyne carbapenems (Figure 4). The rate of the cycloaddition reaction in the presence of CuSO4 (at 25 M), was higher for CBA-2 (2.00 ± 0.02 µM min -1 ) than for CBA-1, CBA-4, and CBA-5 (0.56 to 0.90 µM min -1 ). Fluorescence reached a maximum after a 30 min reaction. To evaluate the yield of the reaction for each alkyne carbapenem, 7-hydroxy-3-azidocoumarin was allowed to react with alkyne carbapenems for 30 min and the progress of the reaction was followed by HPLC. The yield was the highest for CBA-2 (85%), intermediate for CBA-5 (59%), and the lowest for CBA-1 and CBA-4 (21%). The high reactivity of CBA-2 might be accounted for by the chelating effect of the urea linker on the copper catalyst as observed for other chelating functions. [START_REF] Bevilacqua | Copper-chelating azides for efficient click conjugation reactions in complex media[END_REF] This alkyne carbapenem was selected for further experiments as the best candidate based on stability of the acylenzyme (Figure 3) and its efficiency in the cycloaddition reaction (Figure 4).

Kinetics of Ldtfm acylation by alkyne carbapenem

CBA-2. Our next objective was to evaluate the impact of the incorporation of a urea-linked alkyne function in CBA-2 on the inactivation of Ldtfm by this carbapenem. Kinetics based on variation in the intrinsic fluorescence of tryptophane residues of Ldtfm upon acylation were monitored for the reference carbapenem meropenem and CBA-2 (Figure 5). CBA-2 proved to be superior to meropenem both with respect to the rate constant k1 for formation of the amine anion (0.426 ± 0.009 versus 0.168 ± 0.001 µM -1 min -1 ) and the rate constant k2 for formation of the acylenzyme from this intermediate (3.30 ± 0.05 versus 2.24 ± 0.03 min -1 ) (Figure 5). In agreement with mass spectrometry analysis (Figure 3), hydrolysis kinetics obtained by spectrophotometry revealed that the acylenzymes formed with CBA-2 and meropenem display a similar stability (half-lives 323 ± 2 and 504 ± 4 min, respectively). These values are higher than expected from the mass spectrometry analysis reported in Figure 3, which revealed that Ldtfm remained fully acylated after 3 h of incubation. This discrepancy may indicate that the free unreacted carbapenems were not fully eliminated by the gel filtration step used in the mass spectrometry analysis (Figure 3) enabling re-acylation after acylenzyme hydrolysis. These results indicate that we have successfully functionalized the carbapenem core without impairing the efficiency of Ldtfm acylation or the stability of the resulting adduct. [START_REF] Bhattacharjee | Negative Impact of Carbapenem Methylation on the Reactivity of beta-Lactams for Cysteine Acylation as Revealed by Quantum Calculations and Kinetic Analyses[END_REF][START_REF] Triboulet | Tryptophan Fluorescence Quenching in beta-Lactam-Interacting Proteins Is Modulated by the Structure of Intermediates and Final Products of the Acylation Reaction[END_REF] Capture of Ldtfm and release of the fluorescent protein adduct. CBA-2 was used to explore the efficacy of step 3 (capture) and step 4 (release) of our click and release strategy (Figure 2). Ldtfm (0.65 nmole) was acylated with 6 equivalents of CBA-2 (Figure 2, step 1). Excess alkyne-carbapenem was removed by gel filtration (G25 microspin). The CuAAC reaction was performed with 10.8 equivalents of the azido-platform [START_REF] Bernard | Bioorthogonal Click and Release Reaction of Iminosydnones with Cycloalkynes[END_REF][START_REF] Decuypere | Sydnonealkyne cycloaddition: applications in synthesis and bioconjugation[END_REF] leading to the CBA-2-Ldtfm adduct (Figure 2, step 2). The platform provided a biotin handle for capture of the Ldtfm-CBA-2 adduct by streptavidincoated magnetic beads (Figure 2, step 3). Following extensive washing, the click and release reaction was performed with 60 equivalents of the TAMRA fluorescent cyclooctyne (Figure 2, step 4). The latter reaction enabled elution and labeling of the Ldtfm adduct in a single step. SDS-PAGE analysis (Figure 6A) showed that TAMRA-labeled Ldtfm was successfully eluted from the streptavidin beads.

Assessing the specificity of the capture and release reactions. As a negative control, we used FemX, a tRNAdependent aminocacyl transferase that does not interact with carbapenems. This protein was not recovered from streptavidin beads indicating that the procedure was specific to the Ldtfm carbapenem-target (Figure 6B). The procedure was also specific in recovering Ldtfm from a reconstituted mixture containing a whole protein extract from E. faecium (18.6 g of protein) and 0.65 nmole of Ldtfm (18.8 g) (Figure S5 in Supporting information).

Specificity was further evaluated by preincubating Ldtfm with meropenem to block the active-site Cys and prevent acylation by the CBA-2 alkyne carbapenem. This pretreatment almost completely abolished recovery of fluorescent Ldtfm from streptavidin beads (Figure 6C). In addition, we showed that recovery of TAMRA-labeled Ldtfm required the native carbapenem core of CBA-2 since preincubation of this compound with carbapenemase KPC-2, a hydrolytic enzyme of the ß-lactamase family, abolished detection of the fluorescent protein band at 35 kDa (Figure 6D). Recovery of the TAMRA-labeled-Ldtfm was also dependent upon acylation of the active-site Cys of the enzyme since no fluorescent band was observed for an Ldtfm derivative devoid Bi-functionalization of the carbapenem scaffold with photoactivable and alkyne groups to improve acylenzyme stability. One possible limitation of the click and release procedure is the instability of the link connecting the drug to the target, a thioester bond in the case of Ldtfm-carbapenem adducts. Indeed, the link is exposed to multiple conditions during the four steps of the click and release procedure (Figure 2), which lasts for ca. 2 hours in its current version. Thus, our next objective was to add a photoactivable group to the alkyne carbapenem in order to stabilize the drug-target adduct (Figure 7). Diazirines have been successfully used for photoaffinity labeling [START_REF] Blencowe | Development and application of diazirines in biological and synthetic macromolecular systems[END_REF] and for studies aimed at detecting a variety of interactions, such as ligand-receptor, [START_REF] Dubinsky | Synthesis and validation of a probe to identify quorum sensing receptors[END_REF] inhibitor-enzyme, [START_REF] Dubinsky | Diazirine based photoaffinity labeling[END_REF] and protein-protein, [START_REF] Durek | Synthesis of photoactive analogues of a cystine knot trypsin inhibitor protein[END_REF] or for fishing of drug targets. [START_REF] Hill | Fishing for Drug Targets: A Focus on Diazirine Photoaffinity Probe Synthesis[END_REF][START_REF] Hulce | Proteome-wide mapping of cholesterol-interacting proteins in mammalian cells[END_REF] The combined advantages of diazirines over other photoactivable cross-linking agents include their small size, long irradiation wavelength, short period of irradiation, and high stability in the presence of various nucleophiles. [START_REF] Dubinsky | Diazirine based photoaffinity labeling[END_REF][START_REF] Hatanaka | Photoaffinity labeling in drug discovery and developments: chemical gateway for entering proteomic frontier[END_REF] Upon irradiation in the 330 to 380 nm range, diazirines form reactive carbenes, which can insert into C-H, N-H, and O-H bonds. [START_REF] Brunner | New Photolabeling and Cross-Linking Methods[END_REF] In our approach, synthesis of the bi-functionalized carbapenem started from the protected methyl-thienamycin, [START_REF] Iannazzo | Routes of Synthesis of Carbapenems for Optimizing Both the Inactivation of L,D-Transpeptidase LdtMt1 of Mycobacterium tuberculosis and the Stability toward Hydrolysis by beta-Lactamase BlaC[END_REF] which reacted with activated 3-(3-methyl-3H-diazirin-3-yl) propanoic acid [START_REF] Ahad | A traceless Staudinger reagent to deliver diazirines[END_REF] to give compound 14 in 92% yield (Figure 7A). Removal of the PNB group using hydrogenolysis catalyzed by platinum on carbon, followed by the functionalization of compound 15 by N-(prop-2-yn-1-yl)-1H-imidazole-1-carboxamide, afforded compound CBA-6.

Capture and release of labeled Ldtfm was performed as described above (Figure 2) with or without an additional photoactivation step between steps 1 and 2. Mass spectrometry analysis showed formation of the additional cross-link upon irradiation that resulted in the expected loss of two nitrogen atoms (-28.0 Da, see Table S1). CBA-6 was functional in the click and release procedure (step 1 to 4 in Figure 2) but the irradiation did not improve recovery of labeled Ldtfm (Figure 7C). This result suggests that hydrolysis of the thioester bond connecting CBA-6 to Ldtfm is not limiting to the recovery of fluorescent Ldtfm after the final release step.

Conclusion

The targets of β-lactams are highly diverse and complex since they include both essential biosynthetic enzymes, the transpeptidases, as well as unessential hydrolases that participate in peptidoglycan metabolism. [START_REF] Zapun | Penicillin-binding proteins and beta-lactam resistance[END_REF] Assessing the essential nature of these transpeptidases is critical for drug development since it determines the killing activity of the drug. This task remains highly challenging despite of decades of analyses of the mode of action and of the mechanisms of resistance to β-lactams. Since the enzymes have partially redundant functions, essentiality of the targets is defined by combinations of proteins rather than a single enzyme complement. These combinations differ both between and within bacterial species following the acquisition of the so-called "low-affinity" PBPs responsible for βlactam resistance. In addition, the complexity of these combinations includes evolutionary unrelated enzymes, 41- 44 including unidentified ones, [START_REF] Lopez Quezada | Activity-Based Protein Profiling Reveals That Cephalosporins Selectively Active on Non-replicating Mycobacterium tuberculosis Bind Multiple Protein Families and Spare Peptidoglycan Transpeptidases[END_REF] in particular in M. tuberculosis. [START_REF] Gupta | The Mycobacterium tuberculosis protein Ldt(Mt2) is a nonclassical transpeptidase required for virulence and resistance to amoxicillin[END_REF][START_REF] Kumar | 3rd, Meropenem inhibits D,Dcarboxypeptidase activity in Mycobacterium tuberculosis[END_REF] This context prompted us to develop here a new strategy for affinity purification of β-lactam targets. We functionalized the carbapenem core for affinity purification of β-lactam targets using a biotin-containing iminosydnone probe. First, we introduced at the carbapenem C2 position an alkyne function connected by a series of five linkers. A urea linker was identified as the best candidate because (i) it did not impair the kinetics of target acylation and the stability of the resulting acylenzyme and (ii) it displayed the highest reactivity for copper-catalyzed cycloaddition of a fluorogenic probe. Second, we showed that the reaction of biotinylated Ldtfm adducts captured on streptavidin magnetic beads with a TAMRA-cyclooctyne + enables release and fluorescence labeling of Ldtfm in a single step. Third, we synthesized bi-functionalized carbapenems containing both an alkyne and a photoactivable diazirine to introduce a second stabilizing covalent bond between Ldtfm and the carbapenem. The strategy was compatible with mass-spectrometry detection of protein adducts in addition to fluorescent labeling. These tools should facilitate the characterization of the complex set of carbapenem targets, including the detection of unknown enzymes. Access to purification strategies based on acylation of active-site nucleophiles and photoactivation, alone or in combination offers specific advantages. Photoactivation provides very stable links but may decrease the specificity. Acylation of the nucleophile active-site is highly specific but the half-life of the adduct may compromise purification of certain targets. Present Addresses †If an author's address is different than the one given in the affiliation line, this information may be included here.

Ministère de l'Enseignement supérieur, de la Recherche et de l'Innovation" for S. S. PhD grant.

Notes

Any additional relevant notes should be placed here.

ACKNOWLEDGMENT ABBREVIATIONS

Figure 1 .

 1 Figure 1. (a) Peptidoglycan structure. (b) Formation of peptidoglycan cross-links by PBPs and Ldts. (c) Inhibition of Ldts by carbapenems. The efficacy of Ldt inhibition depends upon slow hydrolysis of the acylenzyme. The inset shows the structure of carbapenems discussed in the text.

Figure 2 .

 2 Figure 2. General strategy for activity-based purification of carbapenem targets.

Figure 3 .

 3 Figure 3. (a) Acylation of Ldtfm by carbapenems. (b) Stability of Ldtfm-carbapenem adducts determined by mass spectrometry (SI, Section 7.3).

Figure 4 .

 4 Figure 4. Impact of the linker of alkyne carbapenems on the efficiency of CuAAC. (a) CuAAC reaction with 3-azido-7-hydroxycoumarin. CuAAC performed with CBA-1, CBA-2, CBA-4, and CBA-5 provided compounds 13a, b, c, and d, respectively. (b) Upper panel: Fluorescence kinetics obtained with alkyne carbapenems CBA-1, CBA-2, CBA-4, and CBA-5. Lower panel: Calibration curves obtained with purified triazole compounds, 13a to 13d. Synthesis and characterization of these triazole compounds are reported

Figure 5 .

 5 Figure 5. Kinetic parameters for Ldtfm acylation by carbapenems. R2 = H for CBA-2 and a diazirine moiety for CBA-6. (a) Reaction scheme for the two-step acylation reaction. R2 = H for CBA-2 and a diazirine moiety for CBA-6. (b) Fluorescent kinetics obtained with three concentrations of carbapenem. (c) Kinetic parameters for the acylation reaction. (d) Relative fluorescence of the three forms of Ldtfm. Biphasic fluorescence kinetics are accounted for by the low fluorescence intensity of the amine anion intermediate.[START_REF] Bhattacharjee | Negative Impact of Carbapenem Methylation on the Reactivity of beta-Lactams for Cysteine Acylation as Revealed by Quantum Calculations and Kinetic Analyses[END_REF][START_REF] Triboulet | Tryptophan Fluorescence Quenching in beta-Lactam-Interacting Proteins Is Modulated by the Structure of Intermediates and Final Products of the Acylation Reaction[END_REF]

  nucleophile following substitution of this residue by Ala.

Figure 6 .

 6 Figure 6. SDS-PAGE analyses of CBA-2-Ldtfm adducts. (a) Click and release reactions applied to the Ldtfm L,D-transpeptidase. Lane 1, 2 µg of native Ldtfm; Lane 2, protein recovered from streptavidin beads. Proteins were detected by fluorescence (top panel) and Coomassie blue staining (bottom panel). (b) Assessing specificity by applying the click and release reactions to a mixture of equimolar amounts of Ldtfm and FemX (Lane 1) and to FemX alone (Lane 3). Lane 2, native Ldtfm and FemX proteins. (c) Assessing specificity by applying the click and release reactions to Ldtfm (Lane 2) and to Ldtfm preincubated with meropenem (Lane 3) to prevent acylation of the protein by the CBA-2 alkyne carbapenem. Lane 1, native Ldtfm. (d) Assessing specificity by applying the click and release reactions to native Ldtfm (Lane 1), to CBA-2 preincubated with carbapenemase KPC-2 (Lane 2) and to a derivative of Ldtfm that cannot be acylated following replacement of the catalytic cysteine by alanine (Lane 4). Lane 3, click and release reactions applied to the Ldtfm L,D-transpeptidase (as performed for panel A).

Figure 7 .

 7 Figure 7. Synthesis and use of photoactivable carbapenem CBA-6. (a) Synthesis of CBA-6. i) 3-(3-methyl-3H-diazirin-3-yl) propanoic acid, DMAP, EDC, -20 ˚C, 92%. ii) H2, 10% wt. Pt/C, THF/Buffer, 1h, 25%. iii) N-(prop-2-yn-1-yl)-1H-imidazole-1-carboxamide, DMF/H2O, NaHCO3, 1h, 25 ˚C, 63%. (b) Acylation of Ldtfm by CBA-6 and photoactivation leading to formation of a second covalent bond stabilizing the adduct. The Ldtfm-CBA-6 interaction remains covalent even after hydrolysis of the Cys-CBA-6 thioester bond. (c) SDS-PAGE of the click and release procedure applied to the Ldtfm-CBA-6 acylenzyme.

Filippo

  Rusconi, PAPPSO, Université Paris-Saclay, INRAE, CNRS, AgroParisTech, GQE -Le Moulon, 91190, Gif-sur-Yvette, France. Margaux Riomet, Université Paris Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SCBM, F-91191 Gif-sur-Yvette, France. Antoine Sallustrau, Université Paris Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SCBM, F-91191 Gif-sur-Yvette, France. Frédéric Taran, Université Paris Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SCBM, F-91191 Gif-sur-Yvette, France.

  CuAAC, copper-catalyzed alkyne-azide cycloaddition; DIPEA, N,N-diisopropylethylamine; DMAP, N-dimethylaminopyridine; DMF, N-dimethylformamide; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; HPLC, high performance liquid chromatography; KPC-2, Klebsiella pneumoniae carbapenemases; Ldt, L,D-transpeptidases; PB, phosphate buffer;PBP, penicillin-binding proteins; TAMRA, 5-Carboxytetramethylrhodamine; THF, tetrahydrofuran; THPTA, tris(3hyrdoxypropyltriazolylmethyl)amine. Authors are required to submit a graphic entry for the Table of Contents (TOC) Click and release chemistry for activity-based purification of -lactam targets

Hydrol yzed car bapenem Ldt fm Ldt fm Ldt fm Ldt fm

	SH	+	OR 2 O H	N H	CO 2 H SR 1 Me	k -1 k 1	O	S OR 2 H	N H	CO 2 H SR 1 Me	k 2	O	S OR 2 HN H H	CO 2 H SR 1 Me	H 2 O k 3	SH	+	O	OH HN OR 2 H H	CO 2 H SR 1 Me
	E							EI An			EI *
					Ldt

fm acylation Acylenzyme hydrolysis Carbapenem k 1 (µM -1 min -1 ) k -1 (min -1 ) k 2 (min -1 ) k 3 (min -1 ) Half-life (min)

	Meropenem	0.168 ± 0.001	< 0.1	2.24 ± 0.03 0.00137 ± 0.00001	504 ± 4
	CBA-2	0.426 ± 0.009	< 0.1	3.30 ± 0.05 0.00215 ± 0.00001	323 ± 2
	CBA-6	0.0410 ± 0.0010	< 0.1	2.37 ± 0.03 0.00307 ± 0.00001	226 ± 1

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ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION

Author Contributions

The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript. / ‡These authors contributed equally.


François Stoffelbach

Bertrand Rebière

Rinaldo Poli
email: poli@u-bourgogne.fr

A bis(diazadiene) adduct of MoCl2: mononuclear octahedral, undistorted and diamagnetic

Keywords: molybdenum, diazadiene ligands, spin state, X-ray diffraction

Complex MoCl2( i Pr2dad)2 ( i Pr2dad = i PrN=CH-CH=N-i Pr) is obtained in one step by reduction of MoCl3(THF)3 in the presence of i Pr2dad. The X-ray structure reveals a relatively undistorted octahedral coordination geometry with a relative cis configuration and points to a more appropriate description of the ligands as enediamides. The NMR investigation is in agreement with the same cis structure in solution and underlines the diamagnetism of the compound, at odds with previously reported very similar complexes. A bulk magnetic susceptibility measurement further confirms the compound diamagnetism. No equilibrium with a dinuclear, metal-metal bonded species is apparent from the solution studies. A DFT calculation on the real molecule and on two R2dad model systems with R = Ph and 4-HOC6H4 reveal how the ligand periphery delicately controls the magnetic and structural properties of this system.

Introduction

The coordination chemistry of diazadiene ligands (RN=CH-CH=NR; R2dad) has attracted attention for quite some time, [1,2] because a high electronic and coordination mode flexibility (i.e. see A, B and C) imparts unusual electron donor and acceptor properties to these ligands, combining the useful features of N-based donors such as bipy and unsaturated acceptors such as dienes. Consequently, these ligands are generally compatible with metal centers in both high and low oxidation states. For some time, we have developed the coordination chemistry and the organometallic chemistry of molybdenum in intermediate oxidation states, mostly II, III and IV. Ligands with mild -acidic properties such as phosphines are quite compatible with these systems, but a wide range of ligands with extreme  and  donor/acceptor properties also afford isolable and stable compounds. It was therefore of interest to probe the coordination of diazadiene ligands to these ions. We have recently shown [3] that a variety of R2dad ligands easily add to CpMoCl2 to yield CpMoCl2(R2dad) complexes and a crystallographic study indicates that the dad ligand adopts a coordination mode closest to B. The metal center, therefore, is most appropriately described as formally Mo(V). In this contribution, we report the synthesis, the structural and physical characterization, and a DFT study of a compound resulting from the formal addition of two i Pr2dad ligands to MoCl2. The product presents several unusual features when compared with other compounds that are very closely related to it. [Bu4N]PF6 (ca. 0.1 M) was used as supporting electrolyte in THF. All potentials are reported relative to ferrocene standard, which was added to the solution and measured at the end of the experiment and measured at 100 mV/s. MoCl3(THF)3 [4] and the i Pr2dad ligand [5] were prepared as described in the literarure. The elemental analyses were carried out by the analytical service of the laboratory with a Fisons EA 1108 apparatus. NMR spectra were recorded in C6D6 at 25°C on a Brucker DRX 500 spectrometer. The peaks positions are reported with positive shifts in ppm downfield of TMS, as calculated from residual solvent peaks. Synthesis of complex MoCl2( i Pr2dad)2. MoCl3(THF)3 (1.25 g, 2.99 mmol), Zn (0.2 g, 3.05 mmol) and i Pr2-DAD (0.903 g, 6.44 mmol) were suspended in 15 mL of toluene at room temperature. The mixture was then stirred for 3 days at room temperature. The solution was filtered through Celite to remove an insoluble residue, evaporated to dryness, and warmed to 70°C under vacuum in order to sublime the residual i Pr2dad. A crude product was obtained as a red dark microcrystalline solid. Yield: 0.92 g (69.2 %). Recrystallization by cooling from Et2O at -80°C afforded X-ray quality crystals, one of which was used for the X-Ray structural study. Anal.: Calc. for C16H32Cl2MoN4 (447.3): C 42.96, H 7.21, N 12.53 %.

Found: C 43.19, H 7.30, N 12.21 %. Cyclic voltammetry (THF): reversible oxidation at E1/2 = -0.24 V (Ep = 185 mV) and reversible reduction at E1/2 = -1.27 V (Ep= 177 mV) (the ferrocene peak shows Ep = 248 mV). IR data (Golden gate, cm -1 ): 2968m, 2926m, 2867m, 1490s, 1457m, 1363m, 1229s, 1219s, 1172s, 1121m, 1097m, 1024m, 795s. 1 H NMR (C6D6, 25°C):  0.32 (d, J = 6.6 Hz, CH3),  0.47 (d, J = 6.7 Hz, CH3),  1.12 (d, J = 6.7 Hz, CH3),  1.54 (d, J = 6.6 Hz, CH3),  4.18 (sept J = 6.6 Hz, CH), 5.15 (sept, J = 6.7 Hz, CH), 6.02 (d, J = 2.0 Hz, CH=N), 6.52 (d, J = 2.0 Hz, CH=N). M = -143.2x10 -6 cgs units (calculated correction for the ligand diamagnetism: M = -259.6x10 -6 cgs units).

X-ray analysis of MoCl2( i Pr2dad)2. Intensity data were collected on a Nonius Kappa CCD at 110K. The structure was solved via a Patterson search program [6] and refined with full-matrix least-sqares methods based on F² (SHELX-97) [6] with the aid of the WINGX program. [START_REF] Farrugia | [END_REF] All non-hydrogen atoms were refined with anisotropic thermal parameters. H atoms were found in a difference Fourier map and refined freely with their isotropic displacement parameters fixed at 1.2 or 1.5 (for CH3) times the Ueq of their parent atoms.

Refinement of the Flack parameter [8] showed that the crystal is an inversion twin [9] with a twin fraction of 0.54 (3). The crystal data and refinement parameters are collected in Table 1, whereas selected bond distances and angles are listed in Table 2. CCDC-206713 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge at www.ccdc.cam.ac.uk/conts/retrieving.html [or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (internat.) + 44-1223/336-033; Email: deposit@ccdc.cam.ac.uk].

<Insert Tables 1 and2 here> Computational details. All calculations were performed using the Gaussian98 program package. [START_REF] Frisch | Gaussian 98[END_REF] The three-parameter form of the Becke, Lee, Yang and Parr functional (B3LYP), [START_REF] Becke | [END_REF] was employed, in combination with the LanL2DZ library of basis functions, which includes both Dunning and Hay's D95 sets for H, C, O and N [START_REF] Dunning | III[END_REF] and the relativistic Electron Core Potential (ECP) sets of Hay and Wadt for the 10 innner electrons of Cl and the 28 inner electron of Mo. [START_REF] Hay | [END_REF][14][15] All geometries were fully optimized without symmetry restrictions. For the open-shell species, the calculations used unrestricted open-shell methods.

The mean value of the spin over the electronic density in an unrestricted calculations does not reproduce exactly the assigned spin multiplicity. In all our cases, though, it was considered to be suitable to identify unambiguously the spin state. Mean values of <S 2 > were in the narrow 2.097-2.174 range for triplets. In all cases, relative energies are given in kcal mol -1 and do not include a correction for zero-point energy.

Results and Discussion

Complex MoCl2( i Pr2dad)2 ( i Pr2dad = i PrN=CH-CH=N i Pr) was prepared in one step from MoCl3(THF)3 by reduction with Zn in the presence of the diazadiene ligand. A similar strategy was previously used for the preparation of the related R2dad complex with R = C6H4OMe-4, [16] and for the preparation of related MoCl2(R2dad)(CNMe)2 when conducted in the presence of methyl isocyanide, the main difference being the use of sodium as the reducing agent. [17] These other derivatives of MoCl2, however, were not structurally characterized and no details of their stereochemistry (e.g. cis vs. trans) were evident from the spectroscopic studies.

The NMR properties unambiguously establish the diamagnetic nature of the compound and establish the cis stereochemistry of the molecule in solution. In fact, the four isopropyl methyl groups of an individual dad ligand are all inequivalent as expected for the C2 molecular symmetry, whereas they would all be equivalent for the C2v-symmetric trans isomer. Since the two dad ligands are symmetry-related, four methyl peaks are observed overall in the 1 H NMR spectrum, each being split to a doublet by coupling with the corresponding methyne proton. The methyne protons correspondingly give rise to two different septets and the inner CH=N protons to two mutually coupled signals with a very small coupling constant (2.0 Hz). Suitable crystals of the MoCl2( i Pr2dad)2 derivative for an X-ray structural study could be grown from diethyl ether at low temperature. The structural analysis confirms the identity of the molecule and establishes the cis stereochemistry, see Figure 1 and also with those of complexes to which the bonding mode A can be clearly assigned (N-C: 1.26-1.30 Å; C-C: 1.40-1.46 Å). [1] The observed structural parameters are more consistent with a bonding description where the arrangement B plays a significant role and are very similar to those of our recently reported dad adduct of CpMoCl2. [3] Compounds such as the formally d 6 Mo(CO)4(R2dad) (R = i Pr or C6H3-i Pr-2,6), [18] d 4 Mo(allyl)Br(CO)2(R2dad) (R = t Bu, Ph), [19,20] and d 0 MoO2Cl2( t Bu2dad), [21] on the other hand, show N-C and C-C parameters much closer to those of the free dad ligands, in better agreement with coordination mode A.

Other Mo complexes with dad or dad-related ligands are also known. [22][23][24][25] We can advance the hypothesis that the dad ligand cannot operate as an effective -acid in the above mentioned d 6 satisfy the maximum valence of the molybdenum center and therefore the dad ligand can only bind via the coordination mode A. [21] This is further supporting evidence for the description of the coordination geometry as B in the title compound. The Mo-Cl distances average 2.404(1)

Å, these also being shorter than those found in other cis-dichloro complexes of Mo(II) with strong trans ligands, e.g. 2.52(1) Å in MoCl2(dppe)2. [26] and closer to typical Mo(VI)-Cl distances, such as 2.356(1) in MoO2Cl2(Bu t 2dad) [21] and 2.3251(7) Å in (C5HPr i 4)MoO2Cl, [27] in further agreement with the assignment of a higher formal oxidation state to the metal in compound 3.

The compound diamagnetism is the most surprising result concerning this compound, especially when compared with the paramagnetism (two unpaired electrons) measured for the very similar MoCl2(R2dad)2 and MoCl2(R2dad)(CNMe)2 (R = C6H4-OMe-4) molecules [16,17] and which is reasonably expected for low-spin octahedral d 4 Mo(II) complexes, an example of which is MoCl2(PMe3)4. [28] Related W(II) complexes with the same dad ligand are also paramagnetic. [16] The bulk diamagnetism of the title compound was confirmed by a magnetic susceptibility study. The striking difference between the two MoCl2(R2dad)2 compounds (diamagnetic for R = i Pr, paramagnetic for R = C6H4-OMe-4), under the assumption that the aryl derivative has a similar structure, leads to the hypothesis that the i Pr-substituted ligand is a better  acceptor, delocalizing more effectively the metal electrons onto the ligand by adopting a binding mode closer to type B (as verified by the X-ray structural analysis), whereas the p-MeO-phenyl-substituted ligand leads to a molecule where the ligand adopts a binding mode closer to type A and which can be described more appropriately as a coordination compound of Mo(II). It seems rather surprising, however, that the 4-MeOC6H4 substitution would render the dad ligand a poorer  acceptor than substitution by i Pr. This question has been addressed more in detail by a DFT investigation, vide infra.

Another peculiar feature of the title compound, given its diamagnetism, is the fact that no major distortion is revealed by the X-ray structural analysis. In fact, the large family of diamagnetic octahedral complexes of Mo(II) (d 4 ) typically show rather severe distortions from the ideal octahedral geometry, with angles between bonds in relative cis positions as low as 72° and as high as 120°. [29] In the title compound, on the other hand, the only evident structural deformation is the straightforward consequence of the small bite of the two dad ligands. This phenomenon can again be rationalized on the basis of the extensive backbonding to the dad ligands which is revealed by the solid state structural data, so that the compound can no longer be assimilated as an octahedral d 4 dichlorobis(dad)-complex of Mo(II). An electrochemical investigation of the title compound in THF shows two reversible one-electron processes, a reduction at E1/2 = -1.27 V and an oxidation at E1/2 = -0.24 V, see Figure 2. Evidently, the orbitals involved in the electron addition and removal processes are more or less delocalized between the metal center and the ligands. In order to explore the details of the electronic structure for this compound and to understand the effect of the peripheral groups on the dad coordination mode, we have carried out DFT calculations on three different MoCl2(R2dad)2 systems with R = i Pr (the "real" system), Ph and 4-HOC6H4. The pairwise comparison between these three systems allows us to assess the electronic effect of aryl vs. alkyl groups on the dad ligand, as well as the effect of a para substituent with a mesomeric donating effect on the aryl groups. The computationally simpler OH substituent was used instead of the OMe substituent which is present in the literature compound, [16] since we only wish to probe the electronic effect of the OR conjugation with the aryl group. No major steric effect is expected to differentiate the electronic action of the p-OH and OMe groups.

All three systems have been fully optimized in both singlet and triplet states. The relative energies of the two spin states are reported in Table 3 together with relevant optimized geometrical parameters. First, it can be noted that the DFT optimized structure for the real molecule in the spin singlet compares very closely with the experimentally determined structure (cf. first column of Table 3 with the data in Table 2). In particular, the <Insert Table 3 here>

The most interesting feature is the effect of R on the relative energy. For R = i Pr and Ph, the singlet state is more stable than the triplet by approximately the same amount (2-3 kcal mol -1 ). Therefore, a change from an alkyl to an aryl group does not affect very much the electronic structure at the metal center. On the other hand, the addition of an OH substituent in the para position of the aryl group inverts the stability order in favor of the triplet state, in perfect agreement with the experimental results reported by Pombeiro et al. We have attempted to pinpoint the reason for this unexpected results by inspecting the orbital interactions that are responsible for the Mo-dad bonding. As it turns out, the observed trend results from the combination of minor energy changes caused by the p-OH group on several bonding molecular orbitals in the deep energy region. From the qualitative point of view, we can argue that the p-OMe group, through its  donating effect, makes the aryl group less capable of accepting electron density from the dad system and consequently renders the dad ligand a poorer  acceptor, the ultimate effect being a relative destabilization of the singlet state. Some support for this statement comes from an analysis of the Mulliken charges (see Table 4). In the singlet state, the metal atom has a lower positive charge in the order 4-HOC6H4 < Ph < i Pr and, significantly, the HOC6H4 group has a greater positive charge relative to Ph.

<Insert Table 4 here>

The energies of the relevant frontier orbitals for the three calculated systems in both spin states are shown in Figure 3. The shape of the five orbitals for the singlet i Pr system, i.e.

the title compound, are also shown. It can be noted that the overall trend of the MO energies is identical in the three cases, in particular on going from the Ph to the 4-HOC6H4 substituent.

The Mo-dad back bonding interaction is present with an approximately identical intensity in the HOMO and in the second highest occupied MO (SHOMO), as revealed by the contribution from the N atomic orbitals. For the SHOMO, this is quite evident from the contour plot included in Figure 3, whereas the HOMO maximizes its interaction with the dad ligand on the opposite side of the molecule (not visible). The LUMO is essentially a pure metal orbital, whereas the two higher energy empty orbitals are slightly Mo-dad antibonding (*). The three lowest orbitals (SHOMO, HOMO and LUMO) also exhibit a small contribution from the Cl atoms which is Mo-Cl * in nature. The two Mo-dad  interactions are responsible for the large splitting of the pseudooctahedral "t2g" set of orbitals and for the consequent adoption by the molecule of the spin singlet state. It is to be noted that one of these two Mo-dad  bonding orbitals remains doubly occupied also in the triplet configuration, whereas one electron from the other orbital becomes Mo-dad nonbonding in the triplet state. Therefore, the overall Mo-dad  back-bonding interaction is only slightly reduced upon going from the singlet to the triplet. The MO analysis also shows that, in spite of the Mo-dad  interaction, the major contribution to the HOMO remains that of the Mo atomic orbitals, while the Mulliken analysis shows that the majority of the spin density is localized on the Mo atom. Therefore, the one-electron oxidation process which is observed in the cyclic voltammetric study may be considered to be essentially a metal-based process. This observation serves to illustrate, once again, that the attribution of formal oxidation states has only a limited bookkeeping value when the ligands are able to adapt to their electronic environment via tunable  interactions. This problem is commonplace in organometallic chemistry, for instance for M-olefin, M-diene, M-carbene complexes, and so forth. [START_REF] Collman | Principles and Applications of Organotransition Metal Chemistry[END_REF] The title compound may be described by the two limiting forms as a bis(diazadiene) Mo(II) complex (bonding of type A), or as a bis(enediamido) Mo(VI) complex (bonding of type B), or as anything in between. The real situation can only be revealed by the experimental observations (structural parameters, redox potentials, and so forth) and interpreted on the basis of the MO calculations.

A final interesting observation concerns the absence of any dinuclear product of formula Mo2Cl4(dad)2, containing a quadruple Mo-Mo bond. There are numerous examples of Mo2Cl4L4 compounds where the neutral ligands are N-based donors, mostly saturated amines [START_REF] Cotton | [END_REF][32][33][34][35][36] , but also nitriles, [37] pyridines, [38,39] bipyridines and other aromatic amines. [40] This observation may once again be rationalized by considering the strong -accepting properties of the dad ligand. It is known, in fact, that strongly -acidic ligands disfavor metal-metal bonds [41,42] and favor the establishment of mononuclear structures.

Conclusions

Compound MoCl2( i Pr2dad)2 is a remarkable coordination compound because of its diamagnetism, at odds with very similar complexes. The X-ray structure shows a relatively undistorted mononuclear octahedral coordination geometry for the complex, with bond distances that are in better agreement with a bis(enediamido) Mo(VI) model than with a bis(diazadiene) Mo(II) model. The greater propensity of the di-i Pr-substituted dad ligand to adopt an enediamido coordination relative to the di-(C6H4OMe-4)-substituted dad ligands was not anticipated. The theoretical investigation shows the propensity of the dad ligands to accept electron density from the metal center in the HOMO and SHOMO and to reorganize to a coordination mode closer to that of enediamido ligands. These orbitals, however, retain a substantial metal contribution.

  All manipulations were carried out under an atmosphere of dry and oxygen-free argon with standard Schlenk techniques. Toluene and ether were purified by reflux over sodium benzophenone ketyl and distilled under argon prior to use. The cyclic voltammogram was obtained with an EG&G 362 potentiostat connected to a Macintosh computer through MacLab hardware/sofware. The electrochemical cell was fitted with a Ag-AgCl reference electrode, a platinum disk working electrode and a platinum wire counterelectrode.

  . The geometry is essentially regular octahedral, the only slight deviations being attributable to the small bite of the chelating dad ligands [N(1)-Mo-N(2) = 75.55(8)° and N(3)-Mo-N(4) = 75.52(8)°]. The N(1) and N(3) ligands are essentially perfectly opposite to each other [N(1)-Mo-N(3) = 177.93(8)°] and at nearly right angles from both Cl atoms. The Mo-dad five-member rings are essentially planar (RMS deviation of fitted atoms = 0.0433 and 0.0256 for the MoN(1)C(1)C(2)N(2) and MoN(3)C(9)C(10)N(4) cycles, respectively; highest deviations from the plane: 0.0553(14) Å for atom N(2) and 0.0329(14) Å for atom N(3)). The N-C distances average 1.338(8) Å and the C-C distances average 1.372(4) Å. These are considerably lengthened and shortened, respectively, when compared with those of free dad molecules such as s-trans-Cy2dad (N-C: 1.258(2) Å; C-C: 1.457(2) Å)

Figure 1 .

 1 Figure 1. An ORTEP view of the geometry of compound MoCl2( i Pr2-dad)2.

Figure 2 .

 2 Figure 2. Cyclic voltammogram of a THF solution of compound MoCl2( i Pr2dad)2.

C

  -N and C-C distances for the dad ligand are in close agreement with the experimental structure and with a description where the enediamido form B plays a significant role, whereas the triplet molecule shows longer C-N and shorter C-C distances in agreement with reduced Mo-dad back-bonding. The angular parameters are not dramatically different in the two spin states. The opposite Cl-Mo-Cl and N-Mo-N (cis) angles become closer to the ideal octahedral values in the triplet state, whereas the N-Mo-N (trans) angle is less distorted in the singlet state. The optimized molecules with R = Ph and 4-HOC6H4 have geometrical features in close proximity to the R = i Pr system for both spin states.

Figure 3 .

 3 Figure 3. Energy diagram of the five 4d-derived frontier orbitals for singlet and triplet MoCl2(R2dad)2 (R = i Pr, Ph, and 4-HOC6H4). On the left, MOLDEN views of the five orbitals for the singlet R = i Pr compound are also shown.

  diff. peak and hole; e.Å -3 0.544 and -0.692 a R1=(||Fo|-|Fc||)/|Fo|. b wR2=[w(Fo 2 -Fc 2 ) 2 /[w(Fo 2 ) 2 ] 1/2 where w=1/[ 2 (Fo 2 )+ 0.00*P+ (0.0212*P) 2 ] where P=(Max(Fo 2 ,0)+2*Fc 2 )/3 c Goodness of fit =[w(Fo 2 -Fc 2 ) 2 /(No-Nv)] 1/2 .

Table 1 .

 1 Crystal data and structure refinement for compound MoCl2( i Pr2dad)2.

	Formula

Table 2 .

 2 Bond lengths [Å] and angles [deg] for compound MoCl2( i Pr2dad)2.

	Bond	Distance	Bond	Distance

Acknowledgement. We thank the CNRS and the French Ministry of Research for funding and Dr. P. Richard for assistance. FS thanks the CNRS and the Conseil Régional de Bourgogne for a BDI fellowship.